Advances in Pharmacology and Chemotherapy Volume 13

ADVANCES IN Pharmacology and Chemotherapy VOLUME 13 ADVISORY BOARD D. BOVET J. F. DANIELLI Zstituto Superiore di...

Author: Silvio Garattini

17 downloads 1079 Views 20MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN

Pharmacology and Chemotherapy

VOLUME 13

ADVISORY BOARD

D. BOVET

J. F. DANIELLI

Zstituto Superiore di Sanita Rome, Ztaly

Worcester Polytechnic Institute Worcester, Massachusetts

B. B. BRODIE National Heart Institute Bethesda, Maryland

J. H. BURN Oxford University Oxford, England

A. CARLSSON Department of Pharmacology University of Goteborg Goteborg, Sweden

K. K. CHEN Department of Pharmacology University of Zndiana Indianapolis, Zndiana

R. DOMENJOZ Pharmakologisches Znstitut Universitat Bonn Bonn, Germany

B. N. HALPERN De'partement de Me'decine Expe'rimentale Collgge de France Paris, France

A. D. WELCH Squibb Institute for Medical Research New Brunswick, New Jersey

ADVANCES IN

Pharmacology and Chemotherapy EDITED BY Silvio Garattini

A. Goldin

lstituto di Ricerche Farmacologiche “Mario Negri” Milano, Italy

National Cancer Institute Bethesda, Maryland

F. Hawking

1. J. Kopin

Clinical Research Centre Harrow, Middlesex, England

National Institute of Mental Health Bethesda, Maryland

Consulting Editor

R. J. Schnitzer Mount Sinai School of Medicine New York, New York

VOLUME 13-1975

ACADEMIC PRESS

New York

San Francisco

London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1975, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. N O PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED I N ANY F O R M OR BY ANY MEANS. ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION I N WRITING FROM T H E 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 N W I

LIBRARYOF CONGRESS CATALOG CARD NUMBER: 61-18298 ISBN 0-12-032913-1 PRINTED I N THE UNITED STATES OF AMERICA

CONTENTS CONTRIBUTORS TO THIS VOLUME

. . . . . . . . . . . . . . . .

ix

Chemotherapy of Trypanosoma cruzi Infections Z . BRENER

I . Introduction . . . . . . . . . . . . . . . . . . . . I1. I11. IV . V. VI . VI .

In Yivo Drug Testing . . . . . . . . . . . . . . . . . In Vitro Drug Testing . . . . . . . . . . . . . . . . . Compounds Active Against Trypanosoma cruzi Infections . . . . . Mode of Action . . . . . . . . . . . . . . . . . . . Clinical Trials . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

2 3 12 15 24 34 39 40

Enzyme Study As a Source of Strategy in Drug Design CORWINHANSCH

I . Introduction

. . . . . . . . . . . . . . . . . . . .

I1. Problem of Drug Modification . . . . . . . . . . . . . 111. Type of Enzyme-Ligand Interactions . . . . . . . . . . . IV . Formulation of Quantitative Structure-Activity Relationships . . . V . Examples of Enzymic Quantitative Structure-Activity Relationships VI . Nature of Receptors . . . . . . . . . . . . . . . . VII . Therapeutic Index Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Conclusion References . . . . . . . . . . . . . . . . . . . .

. .

.

.

. .

45 47 48 51 52 75 76 77 78

The Cephalosporin Group of Antibiotics D . R . OWENS.D . K . LUSCOMBE.A . D . RUSSELL.AND P . J . NICHOLLS

I . Introduction . . . . . . . . . . . . . . . . . . . . I1. I11. IV . V. VI .

Chemical Aspects . . . . . . . . Antibacterial Activity . . . . . . . Pharmacology and Toxicology . . . . Clinical Aspects . . . . . . . . . Hypersensitivity and Allergenicity . . . References . . . . . . . . . . . Addendum . . . . . . . . . . . References to Addendum . . . . . . V

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . . . . . . .

. . . . . . . . . .

83 85 89 111 132 145 147 164 170

CONTENTS

vi

Sex As a Factor in Metabolism. Toxicity. and Efficacy of Pharmacodynamic and Chemotherapeutic Agents FUNS C . GOBLE Introduction . . . . . . . . . . . . . . . . . . . . Central Nervous System Depressants . . . . . . . . . . . . Central Nervous System Stimulants . . . . . . . . . . . . Psychotropic Agents . . . . . . . . . . . . . . . . . Local Anesthetics . . . . . . . . . . . . . . . . . . Myotropics . . . . . . . . . . . . . . . . . . . . Cardiac Glycosides . . . . . . . . . . . . . . . . . . Anti-inflammatory Compounds . . . . . . . . . . . . . . Antihistamines . . . . . . . . . . . . . . . . . . . Hypoglycemic Agents . . . . . . . . . . . . . . . . . Anticoagulants . . . . . . . . . . . . . . . . . . . Diuretics . . . . . . . . . . . . . . . . . . . . . Laxatives . . . . . . . . . . . . . . . . . . . . . XIV . Antitussive Compounds . . . . . . . . . . . . . . . . . Anti-infective Compounds . . . . . . . . . . . . . . . XVI . Antineoplastic Agents . . . . . . . . . . . . . . . . . XVII . General Considerations . . . . . . . . . . . . . . . . XVIII . Final Remarks . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

I. I1 . I11. IV . V. VI . VII . VIII . IX . X. XI . XI1. XI11.

xv

174 176 198 202 207 208 209 211 211 212 213 214 214 215 215 225 230 232 233

L-Dopa and the Treatment of Extrapyramidal Disease E . WILLIAMSPELTON 11 AND THOMAS N . CHASE I. I1. 111. IV . V.

Introduction . . . . . . . . . . . . . . . . . . . . Metabolism . . . . . . . . . . . . . . . . . . . . Pharmacology . . . . . . . . . . . . . . . . . . . Therapeutic Applications . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

253 254 263 272

293 294

Effect of Amphetamine-Type Psychostimulants on Brain Metabolism C.-J. ESTLER

I . Introduction . . . . . . . . . . . . . . . . . . . . I1 . Behavioral Changes, Electroencephalogram, and Amphetamine Levels in the Central Nervous System

. . . . . . . . . . . . .

I11. Cerebral Function and Brain Metabolism IV . Concluding Remarks References . . .

. . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

305 307 311 349 349

vii

CONTENTS

Biological Inhibitors of Lymphoid Cell Division DAVIDF. RANNEY

. . Classic Lymphocyte Chalones . . . . . . . . . . . . . Low Molecular Weight Inhibitors Released by Lymphoid Tissues . Macrophage Factors . . . . . . . . . . . . . . . . Suppression Due to Cell-Cell Interaction . . . . . . . . .

I . Introduction . . . . . . . . . . . . . . . . . . I1 . Assay Systems . . . . . . . . . . . . . . . . .

111.

IV . V. VI . VII . VIII . IX . X.

Other Factors . . . . . Possible Mechanisms of Action Clinical Implications . . . Conclusion . . . . . . References . . . . . .

SUBJECT INDEX

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . . .

360 361 366 370 384 385 . . 388 . . 393 . . 397 . . 402 . . 403

. . . . . . . . . . . . . . . . . . . . . .

409

This Page Intentionally Left Blank

CONTRIBUTORS TO THIS VOLUME Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Z. BRENER(l),Department of Parasitology, I . C. B., University of Minas Gerais and Instituto de Endemias Rurais, B. Horizonte, Brazil THOMAS N. CHASE (253), Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland

C.-J. ESTLER(305), Pharmakologisches Institut der Universitat ErlangenNurnberg, Erlangen, West Germany

FRANS C. GOBLE (173), Research and Development Division, Cooper Laboratories, Inc., Cedar Knolls, New Jersey

CORWIN HANSCH (45), Department of Chemistry, Pomona College, Claremont, California

D. K. LUSCOMBE(83), Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cardiff, Great Britain

P. J. NICHOLLS(83), Welsh School of Pharmacy, University of Wales Institute of Science and Technology, CardifA Great Britain

D. R. OWENS(83), Department of Medicine, Welsh National School of Medicine, University of Wales, Cardiff, Great Britain

E. WILLIAMPELTON I1 (253), Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland

DAVIDF. RANNEY(359), Department of Surgery, Northwestern University Medical School and Veterans Administration Research Hospital, Chicago, Illinois

A. D. RUSSELL(83), Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cardiff, Great Britain

ix

This Page Intentionally Left Blank

ADVANCES IN

Pharmacology and Chemotherapy

VOLUME 13

This Page Intentionally Left Blank

Chemotherapy of Trypanosoma cruzi Infections* Z . BRENER Department of Parasitology

I . C . B . , University of Minas Gerais and Instituto de Endemias Rurais B . Horizonte. Brazil

I . Introduction I1.

111.

IV.

V.

V1.

. . . . . . . . . . . . . . . . . . . . .

Trypanosornu cruzi Life Cycle . . . . . . . . . In Vivo Drug Testing . . . . . . . . . . . . . . A. Experimental Methods . . . . . . . . . . . B. Long-Term Schedules of Drug Administration . . . . C . Immunity in Treated Animals . . . . . . . . . D. Treatment in the Chronic Phase . . . . . . . . In Vitro Drug Testing . . . . . . . . . . . . . A. Culture Forms . . . . . . . . . . . . . . B . Tissue Cultures . . . . . . . . . . . . . . C . Screening of Drugs to Be Added to Banked Blood . . Compounds Active Against Trypanosoma cruzi Infections . A . Antibiotics . . . . . . . . . . . . . . . . B . 8-Aminoquinoliues . . . . . . . . . . . . . C . Bisquinaldines . . . . . . . . . . . . . . D . Arsenicals . . . . . . . . . . . . . . . . E . Phenauthridinium Compounds . . . . . . . . . F . Emetine and Derivatives . . . . . . . . . . . G . Mtrofuran Derivatives . . . . . . . . . . . H . Nitroimidazole Derivatives . . . . . . . . . . I . 2.Acetamido-Snitrothiazole . . . . . . . . . . J . Nitrothiazole Derivative (Niridazole) . . . . . . . K . Piperazine Derivatives . . . . . . . . . . . L . Triphenylmethane Dyes . . . . . . . . . . . M . l'riaminoquinazolines . . . . . . . . . . . . N . Thioisonicotinic Acid Amide . . . . . . . . . 0. Thiabendazole [2-(4'-lhiazolyl)benzimidazole] . . . . P . Other Compounds . . . . . . . . . . . . . Mode of Action . . . . . . . . . . . . . . . . A. Studies of Metabolic Inhibitors . . . . . . . . . B. Studies in Tissue Cultures . . . . . . . . . . C. Studies in the Living Host . . . . . . . . . . Clinical Trials . . . . . . . . . . . . . . . . . A. Parasitological Methods . . . . . . . . . . . B. Serological Methods . . . . . . . . . . . .

* Work

supported by the National Research Council. Brazil . 1

. . . . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . .

. . . .

. . . . . . . . . . . . . .

. . . . .

. . . . .

. . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

. . . . . . . . . .

. . . . .

. . . . .

. . . . . . . . . . . . .

. . . . . . . . . .

2 2 3 3 9 10 12 12 12 13 14 15

15 17 18 18 19 19 20 21 21 22 22 23 23 23 23 23 24 24 28 32 34 36 36

2

Z. BRENER

C. Treatment in the Acute Phase . D. Treatment in the Chronic Phase VII.

Concluding Remarks . References . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

36 37 39 40

I. Introduction Human Chagas’ disease has been affecting people from nearly all countries of the American continent, from the South of the United States to Argentina and Chile. The prevalence of the disease in the different countries has been roughly estimated as occurring in 20% of the whole population, which means that, at least, 7 million people are infected with Trypanosoma cruzi (World Health Organization, 1960). Vectors have been found in large areas of the neotropical region, from the 43rd parallel north to the 49th south latitude. Trypanosoma cruzi-like trypanosomes have been detected in more than 100 mammalian wild species, belonging to several orders, in endemic regions and in areas apparently free of human Chagas’ disease. About 35 millions inhabitants are probably exposed to the risks of infection in all endemic area. Although no complete data on morbidity and mortality are so far available, the impact caused by Chagas’ disease may be drawn from its high prevalence in rural areas, the physical disability provoked by clinical cardiac forms affecting especially young people in the second half of life, the occurrence of a relatively high proportion of sudden death, the cost of hospitalization, and the psychological burden imposed by such potentially harmful disease on a large number of asymptomatic patients. Control of Chagas’ disease may be carried out by housing improvement (depending on usually slow-moving economic and social factors) and spraying of residual insecticides in human dwellings (a long-term expensive program). To date no drugs are known to cure Chagas’ disease effectively. A one-shot inexpensive, nontoxic drug to be used in individual cases as well as for preventing Chagas’ disease transmission is still a vague dream. There is, then, plenty of room for new active compounds against Trypanosoma cruzi. Trypanosoma cruzi LIFE CYCLE Trypanosoma cruzi is usually transmitted by hematophagous insects (Hemiptera, Reduviidae) which, after a blood meal, eliminate feces containing infective metacyclic trypomastigotes. These metacyclic forms penetrate the vertebrate host either by skin lesions or normal mucous

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

3

membranes and then undergo discontinuous multiplication: the flagellates, after entering into a wide range of cells, change into amastigote forms that multiply by binary fission and, after 4 to 5 days, differentiate into trypomastigotes. These newly formed flagellates are released from the parasitized cells into the bloodstream and, after circulating for a certain period of time without multiplying, penetrate different host cells to accomplish again the tissue cycle. The acute phase, characterized by the presence of large numbers of trypomastigotes in the bloodstream and tissue stages, is commonly followed by a chronic phase with subpatent parasitemia, scarce tissue forms, and a steady balance between host and parasite. Spontaneous cure has not been reported so far. The vectors become infected by ingesting bloodstream forms which develop all along the insect’s digestive tract; numerous dividing epimastigote stages are found in the midgut; most metacyclic infective forms apparently are formed in the rectum where they tend to accumulate until their elimination with the feces. Trypanosoma cruzi grows in a number of undefined media (reviewed by Taylor and Baker, 1968) but has not been so far cultivated in completely defined media. Differentiation of epimastigotes into the infective metacyclic trypomastigotes begins at the end of the growth period and proceeds during the stationary phase. Only the metacyclic forms are infective for the vertebrate host and their infectivity seems to depend on the parasite strain, number of inoculated trypomastigotes, and length of cultivation in artificial media (Chiari, 1971).

II. In Vivo Drug Testing A. EXPERIMENTAL METHODS 1. Hosts Trypanosoma cruzi, having broad host spectra, infects mammalian species of several orders, including many of the common laboratory animals. Infected mice, usually presenting a characteristic acute phase with large numbers of bloodstream and tissue forms as well as high mortality rates, are widely used for screening purposes. Nevertheless, variations in susceptibility of different mouse strains to T. cruzi have been reported (Pizzi, 1957; Brener et al., 1974); some other factors, such as the host sex or age and environmental temperature, may influence the course of infection and should, therefore, be under control (reviewed by Brener, 1973). Only limited experience in preclinical trials, with compounds active in preliminary screening tests, can be offered.

4

2. BRENER

Dogs are generally used (Goble, 1952a; Haberkorn and Gonnert, 1972), and, although the adult animals may present spontaneous recovery, infections in puppies are usually fatal. Serological tests [complement fixation test (CFT), immunofluorescence, and hemagglutination] become positive a few months after inoculation, which may be used as important criteria in drug testing (Haberkorn, 1971). A nitrofuran compound (Nifurtimox) when regularly tested on different species of animals infected with T . cruzi displayed similar activity in mice, rats, guinea pigs, hamsters, cats, and dogs (Haberkorn and Gonnert, 1972). Monkeys are likely to be suitable animals for such preclinical studies but have not been so far investigated for this purpose. Cebus and rhesus monkeys survive acute infections and develop tissue lesions (Marsden et a l . , 1970; Torres and Tavares, 1958). Erythrocebus patas is also susceptible to T . cruzi and presents heavy heart muscle infection (Neal et a l . , 1973). 2. Trypanosoma cruzi Strains Different degrees of susceptibility of the B and WBH T . cruzi strains to treatment with an active bisquinaldine compound have been reported (Hauschka, 1949). Brener and Chiari (1967) studied the susceptibility of seven different T . cruzi strains to four known suppressive drugs (Cruzon I.C.I., carbidium sulfate, nitrofurazone, and primaquine). Although suppressive action could be detected in all cases, two of the strains seemed to be less susceptible to the phenanthridinium derivative. Among seven strains studied, the Tulahuen was seen to be less sensitive to a nitrofurfurylidene derivative (Bock et a l . , 1969). In groups of mice inoculated with four different strains of T. cruzi and treated with an active nitrofuran, cure rates between 6.4 and 93.3% were found; with one of the strains, the percentages of cure obtained after administration of two different nitrofurans and one 2-nitroimidazole derivative were significantly lower than with other strains (Brener and Costa, 1974). The occurrence of mutants lacking a nucleotide-dependent reductase, essential for the reduction of the nitro group, as reported in bacteria (reviewed by McCalla and Voutsinos, 1974), has never been described in protozoa and cannot be invoked to explain those discrepancies, nor has the presumable existence of specific drug receptors been demonstrated. Naturally occurring strains strongly resistant to the usual chemotherapeutic agents have so far not been detected. Drug resistance has emerged in animals within a year of treatment with the nitrofuran derivatives, Nifurtimox and nitrofurazone (Haberkorn and Gonnert, 1972). A 10-fold increase of the minimum effective concentration of

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

5

nitrofurazone and primaquine has been observed in experiments with culture forms (Amrein, 1965) but the persistence of such resistance after vertebrate host passage has not been investigated. The choice of a suitable strain will basically depend on the criteria to be selected for drug testing since different patterns of parasitemia and mortality rates are observed in animals inoculated with different T. cruzi strains (Hewitt et al., 1953; Brener, 1965).

3. Inocula Trypanosoma cruzi culture forms have been used to infect mice for chemotherapeutic testing (Goble, 1951). A steady virulence, however, is difficult to attain with this material since the morphogenesis of trypomastigote metacyclic forms and their infectivity are under the influence of erratic factors not yet fully understood. There is some evidence that gradual decrease in the virulence of culture forms is related to the length of cultivation in growth media (Bice and Zeledon, 1970; Chiari, 1971), which implies a need for continuous evaluation of the inoculum infectivity. In mice inoculated with metacyclic trypomastigotes from experimentally infected Triatoma bugs, treatment with a nitrofuran derivative provided results similar to those obtained with conventional bloodstream inocula (Haberkorn and Gonnert, 1972). A satisfactory degree of standardization may be achieved by the inoculation of bloodstream trypomastigotes, provided that quantitative methods are used for estimating levels of parasitemia in the donor mice and inoculum. Nevertheless, repeated passages in mice with the same amount of blood containing a progressively lower number of parasites causes deterioration of the strain virulence, as demonstrated by gradual decrease of parasitemia and mortality (Phillips, 1960). Passing through hazards not faced by other pathogenic trypanosomes, such as the need for an intracellular cycle, T. cruzi probably demands greater degree of uniformity in experimental infections. Under standardized conditions, when a previously determined number of parasites is inoculated, predictable mortality periods and parasitemia patterns are regularly obtained in mice (Hewitt et al., 1963; Brener et al., 1974). Routinely we have been using the following method based on Pizzi’s technique (Pizzi, 1957) for counting trypomastigote bloodstream forms. Five cubic millimeters of blood, taken from mouse tails with a hemoglobin pipette, is compressed between slide and a 22 x 22 mm cover slip, so that a monolayer of blood cells is obtained. The number of microscopic fields of the cover slip, previously determined with a 45x objective and a 1 0 ~ eyepiece, is known to be 3500. The number of

6

2. BRENER

trypomastigotes in 50 unselected microscopic fields is then scored and the number of parasites in the 5 mm3 is estimated by multiplying that number by 70. For the inoculation of a large number of animals, mice with high parasitemia are anesthetized and their blood is collected from the severed axillary vessels in a syringe containing 3.8% citrate solution. The counting of parasites is carried out in the same way as described above but the number of samples is accordingly increased and two or three hemoglobin pipettes with the pooled blood are examined (Brener, 1961a, 1962b).

4. Assessment of Drug Activity Goble (1951) pointed out that it is much easier to count mice than trypanosomes and established a screening routine based on the percentage of survival, to 60 days, of mice inoculated with T. cruzi culture forms. Hewitt et al. (1963) used quantitative determinations of critical mortality periods for assessing therapeutic activity in highly standardized infections. After studying the influence of the inoculation on the degrees of virulence obtained in mice infected with B-strain trypomastigotes, the authors reported that mortality patterns may detect minor differences in the survival periods of treated and control animals. In our routine experiments, we have been using quantitative methods based on the counts of bloodstream trypomastigotes. In order to prevent tedious daily counts, we previously studied the course of parasitemia in mice inoculated with Y strain (Silva and Nussenzweig, 1953). Figure 1 shows the curves of parasitemia from mice inoculated, by intraperitoneal route, with 100,000 bloodstream forms. An 8-year (1964-1972) period of investigation on inoculated mice showed that the parasitemia curves were quite similar during all this time (Brener et al., 1974). For drug screening, male albino mice weighing 18-20 gm are inoculated intraperitoneally with 50,000 to 100,000 bloodstream forms; at least 5 animals are used for each drug. Treatment begins on the day after inoculation, and doses corresponding to 0.2 or 0.1 of the LDSo are administered for 6 consecutive days. Counting of parasites is performed only on the fifth day after infection when the first appearance of parasites generally occurs and on the seventh day when the number of parasites is usually higher. Drug activity is readily assessed by comparing the curves of parasitemia of control and treated animals. It is difficult to ascertain whether survival time data or parasitemia counts are the more sensitive or practical method for screening. Comparing the two methods, Hewitt et al. (1963) concluded that both may provide evidence of slight increases in the responses to different

CHEMOTHERAPY OF Trypanosoma

24000

cruzi INFECTIONS

7

A

21000

18000

"

E

q

15000

a YI l c

0 0

c 12000 0

E,p.

;9ooc z 6000

3ooc

5

6 NP

7 8 9 days ofter inoculation

10

FIG. 1. Curves of parasitemia in mice inoculated intraperitoneally bloodstream forms of Trypanosoma cruzi (1 strain).

with 150,000

drug doses and that estimates of parasitemia become very important when a drug increases the survival time since the ultimate goal of treatment is the elimination of the parasites. Trypanosoma cruzi infections are not usually suppressed by single doses of active drugs, which makes it difficult to determine reliable end points and comparative drug activity. Hewitt et a l . (1963) studied a series of related compounds using a drug-diet method and, in this way, he was able to select the more active derivative through quantitative evaluation of critical mortality periods.

5. Criteria of Cure Mice treated with active drugs often present repeated negative fresh blood examinations for long periods. These animals may either be parasitologically cured or may be undergoing the usually nonpatent chronic phase of the disease; in the latter case, a number of laboratory

8

2. BRENER

1ABLE I RESULTS OF SUBINOCULATION OF MICE EXPERIMENTALLY INFECTEDWITH Trypanosornu cruzi, ~ R E A T E DWITH VARIOUSACTIVEDRUGS,A N D PRESENTING REPEATEDLY~ E G A T I V E FRESHBLOODEXAMINATION

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Treatment (Compound)

3024, 50 mg/kg, l o x , 3024, 50 mg/kg, l o x , 3024, 50 mdkg, 10x 3024, 50 mg/kg, l o x , 3024, 50 mg/kg, l o x , Carbidium sulfate, 15 mg/kg, 10x Carbidium sulfate, 15mg/kg, 10x Carbidium sulfate, 15mg/kg, 10x Carbidium sulfate, 15 mdkg, l o x Nitrofurazone, 100 rng/kg, 2 0 ~ Nitrofurazone, 100 rng/kg, 2 0 ~ Nitrofurazone, 100 rng/kg, 20x Nitrofurazone. 100 rng/kg, 20x Nitrofurazone, 100 rng/kg, 50X

Negative blood examination (No. of days)

40 40 40 40 31 20 18 25 25 30 60 90 90 105

Prepatent period in the Results of subinoculated subinoculation animals (days) Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

10 12 12 12 11 11 11 5 9 10 11 10 12 14

methods should be used in order to establish a dependable criterion of cure. Some of these methods are rather fastidious or time-consuming and, therefore, they are not used for routine procedure; they are performed only in few instances when active drugs with presumptive curative action are detected. a. Subinoculation. Animals kept in the laboratory for 1 or 2 months after treatment are killed and 0.4-0.6 ml of citrated blood from their severed axillary vessels is intraperitoneally inoculated into 2 normal mice weighing 14-16 gm. From the seventh day after inoculation, blood examinations are carried out for about 4 weeks. The number of parasites detected in the subinoculated mice is often extremely low and a typical acute phase does not generally occur. b. Hemoculture. Blood from treated animals, also kept in the laboratory for 1 to 2 months after treatment, is collected in the same way and inoculated into agar-blood or LIT media (Camargo, 19@) to be examined 15 and 30 days later. c . Xenodiagnosis. The treated animals are anesthetized with Tionembutal and four fifth-instar Triatoma infestam are allowed to feed on

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

9

them for 40 minutes; the bugs are then kept at 26°C for 30 to 60 days and their feces are microscopically examined for living flagellates. d . Histological Examinations. Sections of the hearts and other viscera of the mice are examined for parasitic tissue stages. Table I shows that long periods of repeated negative fresh blood examination in treated mice are not evidence of parasite eradication and that subinoculation may disclose the presence of bloodstream forms in many of these animals. A comparative study of xenodiagnosis and subinoculation, carried out in groups of mice treated with active compounds, demonstrated no significant differences between both methods (Brener, 196213). The following results were obtained when different methods for detecting parasites were used in the same group of animals treated with several suppressive drugs and presenting negative blood examination: subinoculation (63.6%), hemoculture in agar-blood medium (39.3%), histological sections (9.0%) (Brener, 196213). Liquid media, such as the “LIT” (liver-infusion tryptose) described by Camargo (1964) provide far better results than the usual biphasic media, and there is an increasing tendency to rely on hemoculture as a basic criterion for demonstrating parasite eradication (Brener and Costa, 1974). According to Neal (1973), as few as 10 bloodstream trypomastigotes may start growth in liquid medium whereas at least 600 forms are necessary to give a positive xenodiagnosis. Reinoculation plays an important role in assessment of parasitological cure and is discussed in detail in the section dealing with the relationship between treatment and immunity (Section 11, C).

B. LONG-TERM SCHEDULES OF DRUGADMINISTRATION Repeated failures in curing experimental Chagas’ disease with the available suppressive drugs led to an attempt to provoke exhaustion of T. cruzi infection through a long-term schedule of drug administration (Brener, 1961a). Among several compounds tested, nitrofurazone (5nitro-2-furaldehyde semicarbazone) was selected because it provides high blood concentration and is well tolerated by mice. A group of 65 mice, inoculated with Y strain and presenting parasites in the bloodstream on the fourth day after inoculation, were given orally 53 consecutive daily doses (100 mg/kg); a group of 10 mice received only 20 consecutive doses. After the usual control procedures (fresh blood examination, subinoculation, hemoculture, and xenodiagnosis), it was found that in 95.6% of the mice treated according to the long-term schedule, no parasites could be detected, whereas in those treated with

10

Z. BRENER

20 doses, 8 animals out of 10 presented parasites in their bloodstream (Brener, 1961a). As far as we know, this was the first time that presumptive cures in T. cruzi experimental infection have been obtained in such high proportion. When groups of mice have been treated for 50 consecutive days, with four different nitrofuran compounds (nitrofurazone, furazolidone, furaltadone, and Furadantin) the percentages of animals apparently cleared from T. cruzi infection were, respectively, 95.6, 86.7, 7.2, and 50% (Brener, 1961b). Experiments on mice performed with another nitrofuran derivative (Nifurtimox) showed that, as regards cures of infected animals, the duration of treatment is apparently more important than the total dose administered, provided that individual dosages are not lower than 25-50 mg/kg; no parasitological cures were obtained when treatment was interrupted even for short periods of time (Haberkorn and Gonnert, 1972). Such schedules are recommended for active drugs that are not curative when given for short periods of time and present low cumulative toxicity. Despite obvious limitations, such as the impossibility of being used in mass treatment, successful prolonged drug administration could be extensively used in the treatment of clinical Chagas’ disease. A large number of clinical experiments with nitrofurans, administered according to such prolonged schedules, have followed these findings and are discussed in Section V I .

c. IMMUNITY I N TREATEDANIMALS Animals inoculated with T. cruzi and having survived the acute stage of the disease, display strong immunity to challenge infection (Pizzi, 1957; Norman and Kagan, 1960; Brener, 1967) and do not undergo a new acute phase after reinoculation with either homologous or heterologous strains. On the other hand, spontaneous cure is not likely to occur: parasites have been recovered after 1 year in inoculated mice (Brener and Chiari, 1963a). These facts were used to devise a complementary method in the assessment of cure in treated animals: it may be assumed that animals cured from their original infection, lose their acquired immunity and present, when reinoculated, new outbreaks of parasitemia, characteristic of the acute phase. After a series of experiments on mice receiving long-term treatment with nitrofurazone, the following results were reported (Brener, 1962a): in mice treated from the day after inoculation, a challenge infection given 1 month after treatment, induced a typical acute phase similar to that presented by normal controls; in mice treated from the fifth day

CHEMOTHERAPY OF Trypanosoma

cruzi INFECTIONS

9ooc

11

b

800C

7000

6000 10

E

B

5000 \

-

"73 a

.-

4000

0 .n

E, :3000 c z 2000

loo0

5

7

8

10

12

7

5 Doys

ofter

8

10

12

5

7

8

1 0

12

reinoculotion

0

Treoted on the doy ofter inoculotion

0

Treoted on the 5 t h doy ofter inoculotion

0

Untreated

controls

FIG. 2. Curves of parasitemia in mice treated with nitrofurazone for 53 days (100 mg/ kg, p.0.) and reinoculated with 4000 bloodstream forms of l'rypanosoma cruzi (Y strain) per gram. A, B, and C: mice reinoculated 1, 3, and 5 months, respectively, after treatment.

after inoculation, challenge infections, performed 1, 3 , and 5 months after treatment, produced gradually increasing parasitemia, indicating a slow decrease of the acquired immunity (Fig. 2). However, mice treated with an active drug for 53 days, and still presenting bloodstream parasites, proved to be highly resistant to a challenge infection performed 7 months after treatment (Brener, 1962a). Reinoculation may, therefore, be recommended as a further method in the assessment of parasitological cure in treated animals.

12

Z. BRENER

D. TREATMENT IN THE CHRONICPHASE Most chemotherapeutic testing is done in the acute phase of T. cruzi infection, which is a peculiar stage characterized by large numbers of parasites and low immunity. It has been suggested that, in certain instances, the drug activity is probably reinforced by the host's immune mechanisms, which might carry out the removal of killed or damaged parasites (Goble and Singer, 1960). A routine procedure for drug testing in the chronic phase of Chagas' disease can not be easily established because of the high mortality rates caused by usual T. cruzi strains. Chronic infections with light or subpatent parasitemia may, however, be regularly obtained by treating inoculated mice with a suppressive drug for some days and then keeping the animals under observation for different periods until the chronic phase is established. The following results were obtained in a group of 275 mice handled in this way: 96% of the animals presented positive fresh blood examination shortly after treatment; 25% of them survived for 1 year; parasites were recovered up to 1 year after treatment; and strong immunity against challenge infections was observed 6 and 9 months after treatment (Brener, 1963). Animals kept for 9 months after inoculation and treated with different inactive compounds presented positive subinoculation, thus showing persistence of infection. A detailed study with active compounds had not, nevertheless, been reported. Bock et al. (1969) found that a nitrofurfurylidene derivative can apparently eradidate T. cruzi in acute infection of mice, whereas no cures could be obtained in the chronic disease.

Ill. In Vitro Drug Testing

A. CULTURE FORMS I n vitro screening tests using T. cruzi culture forms are restricted by the lack of a defined medium and the impossibility of growing the parasite at the host's blood or tissue pH and temperature. The parasite is usually cultivated in extremely complex media at 25" to 28"C, and only under special conditions has it been grown at 35.5"C (Pan, 1971). Moreover, the possible physiological similarities between infective metacyclic trypomastigotes from cultures and trypomastigotes bloodstream forms have not yet been investigated. Thus, only limited information arises from this kind of experiment, which has been chiefly used to investigate the action of isolated compounds. Beside different chemicals that proved to be active in vitro, such as

CHEMOTHERAPY OF Trypanosoma cruzi INFECTIONS

13

biotin concentrate (Adler and Bichowsky, 1946) or menadione (Lopetegui and Miatello, 1958), various antibiotics have been demonstrated to be effective against T. cruzi culture forms: tyrocidine (Amrein, 1951); Rimocidin (Seneca et al., 1952); Achromycin (Hewitt et al., 1953); polymyxin B, prodigiosine (McRary et al., 1953); Magnamycin (Seneca and Ides, 1953); amphotericin B (Abithol et al., 1960); mitomycin C, actinomycin D (Fernandes et al., 1965); actinospectacin (Apt et al., 1967); rubiflavin, porfiromycin (Ebringer and Foltinova, 1971). Correlation of in vitro with in vivo activity has not been established, but some of these antibiotics showed suppressive effect on T. cruzi experimental infections (Achromycin, amphotericin B, rubiflavin, porfiromycin).

B. TISSUECULTURES Trypanosoma cruzi infects a wide range of cells in monolayer tissue culture and primary explants of embryonic or adult cells (reviewed by Pipkin, 1960). Extra- and intracellular parasites are thus readily available for in vitro studies of drug activity. When using this method, only small amounts of drug (-2 mg) are usually needed, which may be of some advantage in screening programs; selective action against extraor intracellular forms may be detected; activity against intracellular parasites may be directly demonstrated by using floating cover slips, which are removed and stained accordingly; quantitative data are obtained by using different drug concentrations. The disadvantages are that compounds hard to dissolve cannot be tested, active drug metabolites are not likely to be detected in tissue culture, and, finally, drugs are tested in a system lacking immunity mechanisms and drug excretion. Despite the foregoing restrictions, some attempts to establish a screening routine using T. cruzi-infected tissue cultures have been reported (Bayles et al., 1966; Mieth and Seidenhat, 1967; Gutteridge and Knowler, 1968). Different tissues (chick embryo, human heart, or HeLa cells) are infected with cultured metacyclic trypomastigotes and then kept at 33°C in order to prevent excessive cell propagation. At this temperature, depending on the inocula, extracellular parasites increase 2-14 times on the first 2 or 3 days, whereas 8-22% of the cells become infected within the first 5 days after inoculation (Bayles et al., 1966; Gutteridge et al., 1969). The maintenance medium is then replaced, 2 or 3 days after cell infection, by the drug-containing medium. After 3 or 4 days, drug activity is assessed by (a) counting the number of living extracellular parasites and (6) staining the cover slips of treated and control tubes and determining the percentage of parasitized cells as well

14

Z. BRENER

as the mean number of amastigote intracellular forms in a number of unselected cells. A clear dose-response effect was detected with graded. dilutions of furazolidone and tris(p-aminophenyl) carbonium chloride (Bayles et a l . , 1966), aminonucleoside of Stylomycin, and trypacidin (Gutteridge et al., 1969). Nevertheless, compound 7602 Ac, which is active against T. cruzi in the living host, showed no activity in tissue culture against both intraand extracellular forms (Mieth and Seidenhat, 1%7), thus leading the authors to consider tissue culture of limited value in the screening of new compounds. Conversely, promising compounds against T. cruzi in tissue cultures were not active in mice (Bayles et al., 1966). A systematic comparative study between tissue culture-infected cells and living host, as experimental models for screening work, has not yet been conducted. It is, however, quite clear that any drug showing, in preliminary surveys employing infected tissue culture, effects on extraor intracellular growth and on cell invasion should encourage further in vivo tests .

C. SCREENING OF DRUGSTO BE ADDEDTO BANKEDBLOOD Transmission of Chagas’ disease by banked blood in endemic and nonendemic areas has often been reported. Surveys among candidates for blood donors revealed a high percentage of positive serological tests in different countries of Latin America (reviewed by Salgado and Pellegrino, 1968). Nussenzweig et al. (1953) described the action of triphenylmethane dyes, especially gentian violet, on T. cruzi bloodstream forms and suggested its use as an additive to banked blood in order to prevent transmission through transfusion. In the experiments performed by Nussenzweig et al. (1953), blood from heavily infected mice was kept in the refrigerator for different periods of time, then examined, and inoculated into healthy mice. Trypanocidal activity was detected through repeated fresh blood examination and examination of histological sections. Beside gentian violet, other triphenylmethane dyes, such as malachite green, methyl violet, rosaniline, and basic fuchsin, have been tested and proved inactive. Gentian violet has since been widely used in many Brazilian endemic areas: in Goiania, Central Brazil, 2973 blood transfusions with gentian violet added to a final concentration of 1:4000 have been performed, and no Chagas’ disease transmission or side effects were reported (Rezende, 1965). Despite such good results, the need is still felt for a new soluble, colorless, nontoxic, stable compound which can be routinely added to bottles used for storing blood.

CHEMOTHERAPY OF

Trypanosoma cruzi

INFECTIONS

15

A screening test for such kind of compound has not so far been devised. In vitro testing of soluble compounds added to blood containing T. cruzi bloodstream trypomastigotes would be an easy procedure since this would not involve the probably less vulnerable tissue stages. A screening procedure should be devised including in vitro evaluation of drugs added to blood infected with T. cruzi trypomastigote forms, inoculation of “cleared” blood in susceptible animals, and further study of these animals through the usual methods employed for detecting subpatent infections (repeated fresh blood examinations, subinoculation, hemoculture, xenodiagnosis, reinoculation). The next step would be to repeat the crucial experiment of Amato Neto and Mellone (1959) who, in order to confirm the prophylactic activity of gentian violet, injected in a volunteer (probably one of the authors), 420 ml of treated blood from an acute case of Chagas’ disease with patent parasitemia. Chagas’ disease was not transmitted in this case either.

IV. Compounds Active Against Trypanosoma cruzi Infections Compounds proven effective against T. cruzi have been previously listed by Goble (1961) and Hawking (1963). More recently, Steck (1972) surveyed the chemical groups of interest in T. cruzi therapy and discussed the action of most drugs tried in vivo and/or in vitro against T. cruzi. An appraisal of those reports shows T. cruzi to be often resistant to compounds active against apparently related parasitic diseases, for example, metal organic compounds (antimonials) and aromatic diamidines used in human leishmaniasis and aromatic arsenicals usually active against Salivaria trypanosomes. On the other hand, a large number of active compounds are found among nitrogen heterocyclic-type derivatives (quinolines, phenanthridines, piperazines, and purines), but as suggested by Steck (1972), this is the case because there are a larger number of evaluated compounds presenting this general structure. The following list includes only compounds clearly effective in T. cruzi infections; if available, pertinent data on clinical use of these compounds are mentioned.

A. ANTIBIOTICS Hewitt et al. (1953) described, the in vivo activity of Achromycin @uromycin, stylomycin), an antibiotic produced by Streptomyces alboniger. This substance is a 6-dimethylamino-9-[3’-(p-methoxy-~-phenylalanylamino)-3’-deoxy-P, D-nbofuranosyl]purine (I). The suppressive action

16

Z. BRENER

in early infections has been confirmed by Sonntag and Kloetzel (1953) and by Pizzi et al. (1953) who, however, reported failures of treatment in delayed and already established experimental infections. CH,-

N-CH,

I

I

CH-CHOH-&H-CH-CH,OH

L=/

Fernandes and Castellani (1958, 1959) showed the synthesis of purine nucleotides in T. cruzi culture forms to be inhibited by the aminonucleoside of stylomycin but not by the whole antibiotic itself; the flagellates were, nevertheless, permeable to both substances. Administered to infected mice, the aminonucleoside of stylomycin showed a clear suppressive action (Fernandes et al., 1959). Since the aminonucleoside of stylomycin is active against intracellular T. cruzi stages (Silva et al., 19591, whereas primaquine seems to be active only against extracellular forms (Pizzi, 1951), Moraes et al. (1960) administered to infected mice a combination of both drugs. The results strongly suggested that each drug alone is less active than in the combined form. No effect was detected with a 6-diethylamino analog of stylomycin. No cure was obtained in 5 human acute cases treated with puromycin (Amato Neto, 1958). Abithol et al. (1964) described the suppressive action of amphotericin B in rats inoculated with T. cruzi. This drug has been since used, for 1 to 3 months, in 8 human cases who, apparently, recovered more rapidly than placebo-treated patients; 2 cases presented, for years, repeated negative serological and parasitological examinations. Some tetracycline-type antibiotics (tetracycline, chlortetracycline, and oxytetracycline) showed no efficacy in Chagas' disease (Jarpa et al., 1949, 1950). An antibiotic isolated from Aspergillus fumigatus, trypacidin, was active against T. cruzi in vitro but was of no value in experimental infections in the vertebrate host (Ebringer et al., 1964). The structure of this compound was determined by Balan et al. (1965) to be a Pmethoxy-6-methylcoumaran-3-one-2-spiro-l'-(2'-carboxymethyl-6'dimethoxycyclohexa-2',5'-dien-4'-one), similar to the antifungal griseofulvin. An extensive study has been reported by Ebringer and Foltinova

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

17

(1971), who tested, in vitro and in vivo, forty-five new antibiotics that were divided, according to their mode of action in other systems, into four groups: inhibitors of DNA synthesis; inhibitors of RNA synthesis; inhibitors of protein synthesis; and, finally, inhibitors of purine or pyrimidine synthesis. Only two antibiotics, belonging to the group of DNA inhibitors-rubiflavin and porfiromycin-exerted action against T. cruzi culture forms and showed suppression in infected mice.

B.

8-AMINOQUINOLINES

Although no direct relationship could be established between antimalarial drug activity and chemotherapeutic action against T. cruzi (quinine, atebrin, chloroquine, and proguanil are not active in Chagas’ disease), some 8-aminoquinolines used on Plasmodium infections proved to be active in experimental Chagas’ disease. Goble (1949, 1952a) demonstrated the action of pentaquine [8-(5-isopropylaminoamylamino)6-methoxyquinoline] in T. cruzi experimental infections in dogs and reported that some puppies had been apparently cured after the oral drug administration. In further experiments, Goble (1952b) showed isopentaquine [6-methoxy-8-(l-methyl-4-isopropylaminobutylamino)quinoline] to be about twice as active as pentaquine. Pentaquine has been used associated with quinine (Christen et al., 1951) and the combination was apparently more effective than either compound alone. As quinine itself is not active, the apparently synergistic action was considered to be caused by a decrease of pentaquine excretion. Primaquine [6methoxy-8-(l-methyl-4-aminobutylamino)quinoline]has also marked suppressive activity in T. cruzi experimental infections (Pizzi, 1951; Goble, 1952 ) A systematic study of 6-methoxy-8-aminoquinolines demonstrated that the 6-methoxy group is essential for the activity of this series of compounds; 6-methoxy analogs of pentaquine are inactive despite the marked activity of the parent compound (Goble, 1961) Because of their toxicity, pentaquine and isopentaquine could not be tried in clinical cases but primaquine has been used in human acute and congenital cases of Chagas’ disease (Howard et al., 1957; Amato Neto, 1958); no parasitological cures were reported. Three cases of accidental laboratory infections were also treated with this compound, which has been considered as helpful in the suppression of clinical symptoms (Pizzi et al., 1963). Another quinoline derivative [6-methoxy-8-(5-propylaminopentylamino)quinoline] demonstrated good activity against T. cruzi infection in rats and was subjected to limited clinical trials (Lucena et a l . , 1962). A

18

2. BRENER

single chronic case has been successfully treated with a compound whose chemical structure has not yet been uncovered but may be recognized as an 6-methoxy-8-aminoquinoline connected to a piperazine moiety attached to an alkylamino chain (Goble, 1961; Steck, 1972). This compound belongs to a series of compounds active in experimental leishmaniasis (Beveridge et al., 1958).

C. BISQUINALDINES Quinaldine derivatives were the first to show activity in T. cruzi infections, and a derivative, described by Jensch (1937),[diallylmalonyl(4-amino-2-methylquinolyl-6-amide) acetate or Bayer 7602 (Ac)] (11) had

FHa

YH

(n) for a time some prominence as a suppressive agent in the human disease. Owing to war-time conditions, this compound was later synthesized in England, where it was known as Cruzon I.C.I. Fulton (1943) compared, in infected mice, the original compound and its corresponding synthetic product and was unable to find any significant biological difference between both compounds. Pratt and Archer (1948)synthesized 23 compounds related to Bayer 7602, which have been tested in mice and dogs (Goble, 1950, 1952 ). The most active compounds in T. cruzi infections were branched-chain derivatives, which showed no activity against the African trypanosomes, whereas straight-chain compounds were most active against Trypanosoma brucei. Bayer 7602 has been extensively used, in humans, in the acute phase, including in the severe meningoencephalic forms (Mazza et al., 1942), but only suppressive activity has been reported.

D. ARSENICALS Spirotrypan (Bayer 10557), which has some suppressive action in experimental T. cruzi infections, is a 2-di-(P, ydihydroxypropyl) aminophenol-(4-arseno-5)-~-[benzoxazolyl-2(2’)-mercapto]propionic acid (Wagner and Schultz, 1952). This drug has been used in acute cases

CHEMOTHERAPY OF Trypanosoma

cruzi

INFECTIONS

19

with discouraging results (Romafia, 1953). Two other tervalent arsenical compounds, Bayer 9736 (a derivative of compound 10557) and butarsen (a butyric acid derivative of phenylarsenoxide) showed low activity against T. cruzi (reviewed by Goble, 1961). Pentavalent arsenicals, such as acetarsone and tryparsamide, are not effective in T . cruzi infections.

E. PHENANTHRIDINIUM COMPOUNDS Browning et al. (1946) were the first to demonstrate phenanthridinium derivatives containing carbethoxyamino groups to be active against T. cruzi in infected mice. Such compounds have been widely used for trypanosomiasis of cattle in Africa and in patients with Trypanosoma garnbiense (reviewed by Hawking, 1963). A large number of related compounds have been tested in T . cruzi infections and some 9phenylphenanthridinium salts with urethan substituents showed activity. Two compounds had been investigated in more detail by Goodwin et al., (1950): 2-amino-9-p-carbethoxyaminophenyl-lO-methylphenanthridinium bromide (3C47) and 3-amino-9-p-carbethoxyaminophenyl-10-methylphenanthridinium ethanesulfonate (74C48). The pharmacological and chemotherapeutic properties of 74C48 (carbidium ethanesulfonate) (111), the

most active compound of this series, was investigated by Goodwin et al.,

(1950), who reported a marked suppressive action. A few human acute cases treated with carbidium ethanesulfonate have not been cured (Barros and Nogueira, 1951; Amato Neto, 1958).

F. EMETINEAND DERIVATIVES Among single-nitrogen heterocyclic products, emetine increased survival time of mice infected with T . cruzi, whereas 2-dehydroemetine and other related compounds were inactive (Konopka et al., 1964).

20

2. BRENER

G. NITROFURAN DERIVATIVES This group probably includes some of the most active compounds against T. cruzi infections. Packchanian (1952) was the first to report, in a short note, suppressive action of seven nitrofuran derivatives among 52 tested in infected mice. Mintzer et aZ. (1953) described in uivo activity of N-(5-nitro-2-furfurylidene)-l-aminohydantoin(Furadantin). Packchanian (1957) again tested 47 nitrofuran derivatives and found four with marked activity in experimental infections: 5-nitro-2-furaldehyde semicarbazone (nitrofurazone), 5-nitro-2-furfurylidene aminobiuret, 5nitro-2-furaldehyde trimethylammonium acethydrazone chloride, and 5nitro-2-furanacrolein semicarbazone. Brener (1961a) has since demonstrated that nitrofurazone is apparently able to eradicate T. cruzi infections when given to mice in long-term schedules. Prolonged treatment was also tried with N-(5-nitro-2-furfury1idine)-3-amino-5-(N’morpholinylmethyl-2-oxazolidinone (furaltadone), Furadantin and N-(5nitrofurfurylidene)-3-amino-2-oxazolidone(furazolidone) (Brener, 1961b). Further experiments showed the furaltadone Zeuo isomer to be significantly more active in infected mice than the dextro and racemic compounds (Moon and Coleman, 1962). The dextro isomer, on the other hand, is more active against the salivarian group of trypanosomes (T. gumbiense, T. rhodesiense) (Steck, 1972). Clinical trials with Z-furaltadone showed this drug to be highly toxic and unable to cure patients in the chronic phase (Marra, 1965). The trypanocidal action of 5-nitro-2furaldehyde-2-(2-hydroxyethyl)semicarbazone was reported by Costa and Corrado (1963). Foster et ul. (1969) tested, in experimental T. cruzi infections, several compounds that were condensation products from 5nitrofurfuraldehyde; one of these compounds, obtained from the aldehyde by condensation with 2-hydroxyethyl carbazate, displayed suppressive activity. A systematic survey of nitrofurfurylidene derivatives showed that hydrazones of 5-nitro-2-furaldehyde and 4-aminotetrahydro-W-1,4-thiazine-1,l-dioxides were very active in experimental infections. One of these compounds, 3-methyl-4-(5’-nitrofurfurylidene-amino)tetrahydro~1,4-thiazine-l,l-dioxide(Nifurtimox) (IV) exerted a high suppressive action in T. cruzi-infected animals (Bock et aZ., 1969, 1972; Haberkorn

n

=NO2

CHEMOTHERAPY OF

Trypanosoma cruzi

INFECTIONS

21

and Gonnert, 1972). In detailed experiments, Nifurtimox has been demonstrated to be very efficient against eight T . cruzi strains inoculated in six different animal species and to produce definite cures when administered for long periods in tolerated doses (Haberkorn and Gonnert, 1972). This compound has been extensively used in human chronic and acute cases in different countries of South America and the results are discussed in Section VI. The chemotherapeutic properties of nitrofuran compounds have been reviewed by Paul and Paul (1966). Some of the side effects produced by this series of compounds often interfere with their prolonged administration which is at present considered essential for T . cruzi eradication in the vertebrate host. Peripheral neuritis is frequently associated with nitrofuran therapy and has been suggested to be caused by a disturbance in pyruvate metabolism. Sensitivity reactions (urticaria), loss of weight, and digestive symptoms may also appear. Investigations on acute, subchronic, and chronic toxicity of Nifurtimox have been recently compiled by Hoffman (1972).

H. NITROIMIDAZOLE DERIVATIVES Pizzi (1961) reported a strong but transient suppressive action of an imidazole derivative [ 1- p - hydroxyethyl)-2-methyl-5-nitroimidazole], known as an effective agent in Trichomonas vaginalis infections. Grunberg et al. (1968) investigated a series of 2-nitroimidazole derivatives presenting a broad spectrum of chemotherapeutic activity against protozoa and bacteria. One of the tested compounds [3-(2-nitro-1imidazolyl)-1,2-propanediol] showed prophylactic and therapeutic effect in experimental T . cruzi infections. A high suppressive activity was also reported with 2-amino-5-(l-methyl-5-nitro-2-imidazolyl)-1,3,4,-thiadiazole given by the drug-diet method (Burden and Racette, 1969).

1.

2-ACETAMIDO-5-NITROTHIAZOLE

A decrease of parasitemia and mortality rates was detected in mice experimentally infected with T . cruzi and treated orally with 2acetamido-5-nitrothiazole (Brener and Pellegrino, 1958). Like nitroimidazole derivatives, this compound has also an activity in Trichomonas infections (Cuckler et al., 1955).

22

Z. BRENER

J. NITROTHIAZOLE DERIVATIVE (NIRIDAZOLE) Compound 1-(5-nitro-2-thiazolyl)-2-imidazolidinone(V), a potent schistosomacidal agent (Lambert and Stauffer, 19M) showed strong suppres-

sive action when given, by oral route, to mice with T. cruzi infection, some animals having apparently been cured (Velisques-Antich, 1970). No effect on circulating trypanosomes was observed, however, in patients presenting acute Chagas’ disease and treated with this nitrothiazole derivative (Prata et al., 1966). This interesting relationship between schistosomacidal agents and therapeutic activity against T. cruzi infections has again been confirmed with some nitrofuran compounds that possess prophylatic and curative activity against Schistosoma mansoni and Schistosoma japonicum (reviewed by Archer and Yarinsky, 1972).

K. PIPERAZINE DERIVATIVES Tomcufcik et a l . (1965) reported high activity, in mice infected with

T. cruzi, of some N4-substituted N1-(3-dimethylaminopropyl) piperazines with the general structure (VI).

Compound “E” (R=4-acetamidophenyl) was apparently the most active of the series and showed a higher suppressive activity than primaquine and furaltadone. Good results have also been obtained in experimentally infected mice and dogs (Tomcufcik et a l . , 1965)’ but no cures have been reported. A new derivative of this series [ 1-(3-dimethylaminopropyl)-4-(p-methoxyphenyl) piperazine dihydrochloride] has been experimentally tested (Brener, 1971; Andrade et al., 1972). Parasites disappeared from the bloodstream in most animals after prolonged treatment, but only a few were apparently cured.

CHEMOTHERAPY OF Trypanosoma

cruzi INFECTIONS

23

L. TRIPHENYLMETHANE DYES The in vitro action of crystal violet, gentian violet, and methyl violet has been described by Nussenzweig et al. (1953).Although not active in classic chemotherapeutic experiments, a number of triphenylmethane dyes (basic and triamino compounds) proved to be active in experimental infections when administered by the drug-diet method (Goble and Konopka, 1963). Bayles et al. (1966)showed that tris-(p-aminophenyl) carbonium chloride was active in tissue culture forms and prevented death of mice in usually lethal infections produced by T. cruzi. M. TRIAMINOQUINAZOLINES Among many compounds tested in mice, three compounds showed encouraging results: 2,4-diamino-6-(3,4-dichlorobenzylamino)quinazoline; 2,4-diamino-6-[(3,4-dichlorobenzyl)nitroamino]quinazoline (CI-679base), and CI-679 acetate (Thompson et al., 1969; Thompson and Bayles, 1970). The three derivatives protected mice from lethal infections but were unable to cure T. cruzi parasitism.

N. THIOISONICOTINIC ACID AMIDE In a series of related compounds, thioisonicotinic acid proved to be active against T . cruzi infections in mice. Long-term treatments (220mg/ kg, per os, during 31 days) produced, apparently, parasitological cures. Good results were also obtained with large, single subcutaneous doses (1250mg/kg) (Raether et al., 1972).

0. THIABENDAZOLE [2-(4’-THIAZOLYL)BENZIMIDAZOLE] This agent, known as an antihelminthic, slightly increased survival time of mice inoculated with T . cruzi when fed in powdered diets containing 0.1% of the drug. In higher doses, however, this compound showed adverse effects on survival mice, probably by a immunosuppressive action (Shoemaker and Hoffman, 1971).

P. OTHERCOMPOUNDS Some drugs that interfere with the host-parasite balance rather than with the parasite itself may enhance the parasitemia and significantly decrease the survival time Gf infected animals. They are mentioned here because they are often used drugs and potentially harmful agents which may alter Chagas’ disease natural development. Cortisone increases the parasitemia of the acute phase in experimen-

24

Z. BRENER

tally infected mice and rats (Jarpa et a l . , 1951; Pizzi and Chemke, 1955); latent infections in monkeys became patent after cortisone administration (Goble, 1961). The chronic infection course in mice was not, however, apparently changed by cortisone treatment (Brener, 1963). Aureomycin also exacerbated acute infections in mice (Thiermann and Christen, 1952). Administration of chlorpromazine resulted in higher mortality, probably because of its hypothermic action (lower environmental temperature increases parasitemia and mortality) (Friebel and Kastner, 1955). Cyclophosphamide, a strong immunosuppressive agent, enhanced acute phase and myocarditis in mice (Kumar et al., 1970). A similar effect, was noticed with 8-azaguanine (Shoemaker and Hoffman, 1969). Administration of cyclophosphamide to mice in the chronic phase induced, in a certain percentage of the animals, a new acute phase; other immunosuppressive drugs, such as azathioprine and 6-mercaptopurine did not to affect T . cruzi chronic infection (Brener and Chiari, 1971).

V. Mode of Action The mode of action of some compounds active against T . cruzi has been investigated at different levels, such as their role as metabolic inhibitors of culture forms, their activity against extra- and intracellular forms present in infected tissue cultures, and their selective action against bloodstream and/or tissue forms in the living host.

A. STUDIES OF METABOLIC INHIBITORS Some drugs whose action had already been analyzed in vitro and had been found to be strong inhibitors of the metabolism in T. cruzi culture forms, proved inactive against established infections in the vertebrate host. For example, drugs interfering with the metabolism of T . cruzi nucleic acids were inactive i n vivo either because they could not damage the nondividing bloodstream forms or were not able to reach the actively dividing amastigote forms sequestered into cells harboring the parasites. Despite these disappointing discrepancies, these observations help to uncover the mechanism of selective activity of some compounds effective against T . cruzi.

1. Drugs Interfering with Carbohydrate Metabolism Trypanosoma cruzi probably utilizes more fat and protein than carbohydrates in order to support its endogenous metabolism (Ryley,

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

25

1967), although utilization of glucose by culture and bloodstream forms has already been referred to (Ryley, 1956). A number of anaerobic fermentation end-products as well as of glycolytic and of pentosephosphate pathway enzymes have been detected in T . cruzi (reviewed by von Brand, 1966; Raw, 1959). Trypanosoma cruzi seems to keep an electrontransfer system mediated by cytochromes during all stages of development. In this respect, terminal respiration of T . cruzi would differ from the Trypanosoma brucei which presents cyclical changes from a cytochrome- to a noncytochrome-mediated metabolism (Ryley, 1956; Fulton and Spooner, 1959). Jaffe (1968) suggested that, on grounds of circumstantial evidence, at least two groups of active compounds could act as inhibitors of T . cruzi carbohydrate metabolism. Based on an analogy with the antiplasmodial activity, the author proposes that 8-aminoquinolines are probably converted into redox intermediate derivatives that accelerate the transfer hydrogen from reduced nicotinamide adenine dinucleotide phosphate (NADPH) and, thus, interfere with the process dependent on this coenzyme in T . cruzi bloodstream forms. This might be a selective action, since 8-aminoquinolines do not hinder nucleic acid synthesis in plasmodia (Schellenberg and Coatney, 1961). The second group of compounds likely to exhibit similar activity would be the 5-nitrofurans which, at least in bacteria, are good electron acceptors and inhibit a number of dehydrogenases (Jaffe, 1968). Santos (1962) demonstrated, by polarographic studies, inhibition of the respiration of T . cruzi culture forms by different nitrofuran compounds and suggested these drugs to be strong inhibitors of glucose active transport. It should be remembered, however, that nitrofuran compounds are able to disturb DNA synthesis and cell permeability in Euglena gracialis (Paul and Paul, 1966). Beside this indirect evidence, there are no further substantial data demonstrating drug action against T. cruzi by inhibition of specific enzymes of carbohydrate metabolism either in intact cells or in enzymes from parasite extracts. 2. Drugs Interfering with Protein Metabolism

Trypanosoma cruzi culture forms have a high protein content (43-53%) (von Brand et a l . , 1959), and some amino acids are probably formed by transamination (Wiliamson and Desowitz, 1961). The metabolism of protein and amino acids in bloodstream forms is not yet quite understood; it has been suggested that oxidation of amino acids take place only in trypomastigotes presenting an active tricarboxylic acid cycle (Honigberg, 1967).

26

Z. BRENER

Drugs that inhibit DNA synthesis may subsequently interfere with the buildup of the parasite’s proteins, as has been demonstrated in culture forms in vitro, with mytomycin C, actinomycin D, and puromycin (Fernandes and Castellani, 1966; Fernandes et al., 1965). Puromycin bears some resemblance to the adenosine group in the transferring RNA molecule, suggesting that it inhibits protein synthesis as an analog of the aminoacyl transfer RNA (Fernandes and Castellani, 1966). The intact molecule of puromycin strongly inhibits protein synthesis, whereas its aminonucleoside moiety, which is active in infected animals, does not display any inhibitory action, even at a ten-fold higher concentration (Fernandes and Castellani, 1966). These experiments have been performed with T . cruzi culture forms; data regarding bloodstream or tissue forms are not available so far.

3 . Drugs Interfering with Lipid Metabolism Cholesterol has been detected in flagellates growing only in cholesterol- or serum-containing media (von Brand et al., 1959; Korn et al., 1969). Ergosterol compounds, however, were present in culture forms cultivated in serum-free medium (Korn et al., 1969). The presence of ergosterol may account for the activity of the antifungal polyene antibiotic amphotericin B against T . cruzi (Abithol et al., 1964). Interaction between this antibiotic and cell membrane sterols, specially ergosterol, is supposed to increase the membrane permeability and cause the loss of low molecular components such as amino acids, ions, and soluble-fraction nucleotides (Ghosh and Chatterjee, 1961, 1962; Jaffe, 1968). The antiparasitic action would be predominantly physical since no metabolic disturbances could be detected.

4. Drugs Interfering with Nucleic Acids Metabolism Trypanosoma cruzi culture forms are not able to synthetize de novo their purine nucleotides, and a supply of exogenous preformed purines is needed for the synthesis of these nucleotides (“salvage” pathway) (Fernandes and Castellani, 1958). Trypanosoma cruzi also seems to depend on exogenous pyrimidine bases for its nucleotide biosynthesis (Rey and Fernandes, 1962). It has been suggested that uptake of purine and pyrimidine bases may play an additional role as energy source for anabolic reactions (Honigberg, 1967). The biosynthesis of T. cruzi intracellular forms has been investigated by Yoneda (1971) in tissue culture, using infected monkey heart cells, labeled precursors, and metabolic inhibitors. Radioautographs showed de novo synthesis to be

CHEMOTHERAPY OF

Trypanosoma cruzi

INFECTIONS

27

the preferential pathway for the buildup of purine nucleotides in T. cruzi intracellular forms, whereas extracellular stages still used the salvage pathway. The apparent dependence of T. cruzi on exogenous bases has suggested the chemotherapeutic uses of purine and pyrimidine analogs to inhibit the normal biosynthesis of nucleotides and nucleic acids. The aminonucleoside of stylomycin, for instance, works this way, inhibiting the incorporation of adenine into purine nucleotides (Fernandes and Castellani, 1959, 1968). This drug has been found to be active in the living host (Fernandes et al., 1959) and is able to destroy intracellular forms in tissue culture (Silva et al., 1959). Synthesis of DNA is inhibited by 5-fluorouracil and 5-fluorouracil deoxyriboside, which interfere with the incorporation of uracil or uridine into pyrimidine nucleotides and nucleic acids (Fernandes and Castellani, 1968; Fernandes et al., 1965; Castellani and Fernandes, 1965). A study of the effects of several purine and pyrimidine analogs on growth rate and nucleic acid synthesis in T. cruzi culture forms has been published by Castellani and Fernandes (1965). Among the pyrimidine analogs, only 5-hydroxyuracil, 5-bromouracil, and 6-azauracil inhibited the incorporation of uracil into nucleic acid pyrimidines. The six purine analogs tested were unable to disturb the rate of adenine incorporation into nucleic acid purines. The administration of 6azauracil, however, did not increase the survival time of mice experimentally inoculated with T. cruzi (Jaffe, 1968). Some drugs are known to combine with nucleic acids and, thereby, inhibit the parasite’s growth. Mitomycin inhibits T. cruzi DNA and protein synthesis and, at a later stage, RNA as well; by combining with DNA, the drug hinders the synthesis of DNA polymerase (Fernandes and Castellani, 1966). Actinomycin D strongly blocks both the multiplication and the differentiation of culture forms by disturbing the synthesis of the DNA-dependent RNA polymerase (Fernandes et al., 1965). This irreversible inhibition, which can be demonstrated by nonincorporation of thymidine, determines a loss of infectivity to tissue cultures and living hosts. As discussed by Jaffe (1968), the activity of nucleic acid antagonists as chemotherapeutic agents has been rather disappointing in Chagas’ disease: again, mitomycin C and actinomycin D were not active against experimental T. cruzi infections. Porfiromycin, a methyl congener of mitomycin C, however, increased the survival of infected mice, probably because it can be administered in higher doses (Ebringer and Foltinova, 1971).

28

Z . BRENER

5. Inhibitors of Dihydrofolate Reductase Dihydrofolate reductase plays an important role in the synthesis of the metabolic active form of folic acid; moreover, inhibitors of this enzyme are toxic for cancer cells. As a first step toward further use of such inhibitors against trypanosome infections, the activity of dihydrofolic acid reductase on flagellates has been investigated (Gutteridge and Senior, 1968). Enzyme activity was demonstrated on intracellular parasites isolated from T. cruzi-infected human heart cells. Aminopterin, a 4amino analog of folic acid, which inhibits dihydrofolate reductase, displayed activity in tissue culture against extra- and intracellular forms of T. cruzi (Gutteridge et al., 1969). Aminopterin, however, did not prove active against T. cruzi infections in mice. The T. cruzi enzyme is sensitive to 4-amino analog and to 2,4-diaminopyrimidines, thus suggesting the possible use of selective inhibitors in the chemotherapy of Chagas’ disease.

B. STUDIES IN TISSUECULTURES Trypanosoma cruzi readily grows in different tissue culture systems, such as plasma clot, hanging-drop cultures and monolayer tissue cultures (reviewed by Pipkin, 1960; Neva et al., 1961). Infected tissue cultures may, therefore, be a suitable tool for studying drug action against extra- and intracellular forms. Infected tissue fragments (Lock, 1950; Silva et al., 1959) present some inconveniences such as the impossibility of using quantitative methods for estimating the number of intracellular parasites and the difficulty in having homogenous drug concentration. Monolayer tissue cultures provide a far better method for investigating drug action: (1) cells may be cultivated over floating cover slips and, after being infected and submitted to drug action, they can be stained and examined in order to observe the drug’s direct action against tissue forms (Fig. 3); (2) some quantitative data such as the percentage of parasitized cells, the number of intracellular parasites, and the relative proportion of cells with trypomastigote or amastigote forms, may be easily obtained; (3) the number of extracellular flagellates in the nutrient medium may be determined, thus providing data on selective drug action against these forms; (4) drug toxicity to culture cells may be investigated along with its action on the parasites. Despite the foregoing advantages, the results should be cautiously interpreted, especially those related to the drug’s specific action against extracellular forms. The presence of flagellates in the fluid medium results from a supply of parasites emerging from parasitized cells; the

CHEMOTHERAPY OF Trypanosoma

cruzi INFECTIONS

29

FIG. 3. Effect of a nitrofuran derivative hF-902 on Trypanosoma cruzi intracellular forms in tissue culture (trypsinized embryo chicken cells). (A) Untreated control; (B) parasites 24 hours after treatment, showing marked decrease in the number of intracellular amastigotes (magnification: ~ 4 5 0 ) ; (C) untreated control; (D) disintegrated parasites 48 hours after treatment (magnification: x 1125).

effect of drugs that specifically act against extracellular forms may be underrated by the flow of intracellular parasites (Silva and Kirchner, 1962). On the other hand, physiological identity of bloodstream trypomastigotes with those obtained in tissue culture has not so far been demonstrated: drug action against parasites that grow in vitro does not

30

Z. BRENER

necessarily imply corresponding action against the living host parasitic forms.

1. Aminonucleoside of Stylomycin Silva et al. (1959) investigated the action of this compound using T. cruzi-infected brain pieces of young chicken kept according to Carrel's hanging-drop technique. The drug proved almost inactive against extracellular forms but strongly affected the intracellular stages which were often destroyed after 48 to 96 hours. This action could not be counteracted by a number of precursors and cofactors of purine nucleotide synthesis. In infected monolayer tissue cultures treated with this compound, intracellular forms were rather rare and almost exclusively formed by amastigote forms, providing evidence of some disturbance in the normal intracellular parasite development (Silva and Kirchner, 1962). Intracellular stages are apparently 200 times more sensitive to the drug than extracellular forms: amastigote forms are damaged at a 10 pg/ml concentration, whereas, in flagellate culture forms, the incorporation of I4C into nucleic acids is inhibited at a 2200 pg/ml concentration (Fernandes and Castellani, 1959; Gutteridge et al., 1969). This has been explained either by differences between purine metabolism in both forms or by a presumptive metabolite derivative from the parasitized cells. When aminonu~leoside-~H of stylomycin has been incubated with human cells, two radioactive compounds could be separated by paper chromatography, which, according to Gutteridge et al. (1969), suggests the presence of an active metabolite.

2. Primaquine No effect on T. cruzi intracellular stages could be detected with this drug. A great number of parasitized cells were observed in tissue culture preparations, the ratio of trypomastigote-harboring cells to amastigote-harboring cells being similar to that of the controls, suggesting that the drug has no effect on the intracellular morphogenesis (Silva and Kirchner, 1962). Primaquine is apparently effective only against extracellular forms. 3. Phenanthridinium Compounds Lock (1950) tested the action of 2-amino-9-p-carbethoxyaminophenyl10-methylphenanthridinium bromide on T. cruzi-infected heart explants from embryo rats. Direct toxic effect on intracellular forms was demonstrated, no motile flagellates being detected a few days after drug

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

31

removal. Apparently, this drug blocks intracellular parasite differentiation, as demonstrated by the gradual disappearance of intracellular trypomastigotes and the persistence of a relatively high number of amastigote-harboring cells (Silva and Kirchner, 1962; Brener, 1966). 4. Bisquinaldine (7602 Ac) This compound, markedly active against in vivo established infections, was tested in infected HeLa cells, and found to be inactive against intra- and extracellular forms (Mieth and Seidenhat, 1967). The possibility of metabolic transformation of the drug in the living vertebrate host has not been investigated.

5. Spirotrypan An arsenical compound that has only limited value in the treatment of

T . cruzi infections in vivo, spirotrypan showed activity only against extracellular forms and was unable to hinder the development of intracellular stages in infected HeLa cells (Mieth and Seidenhat, 1967).

6. Trypacidin An antibiotic isolated from Aspergillus fumigatus , trypacidin kills epimastigotes in LIT medium but is not very active against trypomastigote metacyclic forms. In human heart-tissue cells infected with T. cruzi, it kills extracellular forms and prevents infection of new cells; however, trypacidin does not disturb the development of parasites in established cell infections (Gutteridge et a1 ., 1969).

7 . Tris-(paminophenyl) Carbonium Chloride Tested on monolayers of trypsinized chick cells, this derivative showed little evidence of action against intracellular forms and moderate activity on extracellular flagellates (Bayles et a1., 1966).

8. Nitrofurans Furazolidone [3-(5-nitrofurfurylideneamino)-2-oxazolidinone]proved effective against extracellular parasites, preventing cell invasion by T. cruzi. Although slightly harmful to tissue culture cells, it clearly damaged intracellular stages at suitable concentrations. Two soluble nitrofuran compounds were tested on T. cruzi-infected chicken embryo cells (Brener, 1966): ~-5-morpholinomethyl-3-(5-nitrofurfurylideneamino)2-oxazolidinone hydrochloride (NF 902) and ~-(5-nitrofurfurylideneami-

32

Z . BRENER

no)hydantoin sodium (Furadantin sodium). An early decrease in the number of intracellular forms soon followed by complete destruction of the parasites was observed with both compounds. Nitrofurazone strongly affected intra- and extracellular stages at concentrations quite harmless to infected HeLa cells (Mieth and Seidenhat, 1967); Nifurtimox damaged intracellular stages of T. cruzi in HeLa cells as well as extracellular epimastigotes and trypomastigotes (Gonnert and Bock, 1972). The fine structure of parasites growing in HeLa cells and submitted to the action of Nifurtimox was studied by Voigt et al. (1972). Early changes, such as vacuolization and mitochondria1 swelling were observed 8-10 hours after treatment; within 72 hours the parasites were irreversibly damaged, presenting significant decrease of ribosomes, vacuolization, swollen mitochondria, and enlargement of the perinuclear space.

C. STUDIES IN

THE

LIVINGHOST

In an attempt to confirm in vivo the activity of nitrofuran compounds against intracellular stages of T. cruzi in tissue culture, a progressive histopathological study was carried out in infected mice experimentally treated with nitrofurazone (Andrade and Brener, 1969). Amastigote forms were seen to be actually destroyed within the cytoplasm of parasitized cells in the living host; the parasite-harboring cells were also affected by the parasite’s disintegration, but neighboring cells remained normal. An electron-microscopic study of intracellular forms in mice experimentally treated with nitrofurazone was also conducted (Brener et al., 1969). Normal and degenerated amastigotes were observed in treated and control mice; however, the number of degenerated parasites in the two groups of animals was 86.5 and 17.5%, respectively, showing a direct relationship between treatment and proportion of degenerated parasites. Progressive parasite lesions could be detected as early as 24 and 48 hours after treatment, which is in agreement with data obtained in tissue culture (Brener, 1966). In a similar study by Velasquez-Antich and Aleman (1971), using l-(5-nitro-2-thiazolyl)-2-imidazolidinone (niridazole), changes of the fine structure in 97.0% of amastigote forms in the tissues of treated mice were described. Drug action limited to bloodstream forms is hardly detected in the living host: decrease of parasites in treated animals is likely to be caused by direct action of the drug on circulating trypomastigotes and/or intracellular amastigotes. Haberkorn and Gijnnert (1972), after observations with a reflex microscope, described morphological changes in living bloodstream forms of trypomastigote from mice treated with Nifurtimox. As soon as 10-12 hours after treatment, enlargement of the

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

33

40000

30000

20000

--

-,”

10000

m

E

;5000 \

-

Y a l)

0

?

5 5 <

2000

0 h

o,

z

1000

500

Hours a f t e r inoculation

FIG. 4. Curves of parasitemia in mice previously treated with five daily doses of piperamide (100 mglkg, p.0.) (A) and nitrofurazone (100 mglkg, p.0.) ( 0 ) , and then intravenously inoculated with 2,000,000 bloodstream forms of Trypanosoma cruzi (MR strain). 0 , Normal mice.

nucleus and kinetoplast as well as cytoplasm vacuolization could be detected; further decay of the trypomastigotes was characterized by splits in the parasite cell membrane. The occurrence of T. cruzi strains presenting a high proportion of “ stout” trypomastigotes was described by Brener and Chiari (1963a). Brener (1965) demonstrated that these stout forms, intravenously inoculated into normal mice, persist for some days in the animal’s bloodstream without penetrating the tissues. This finding was used as a tool to study drug action against circulating T. cruzi forms (Brener, 1971). Mice were pretreated, for 5 consecutive days, with active compounds and, afterward, intravenously inoculated with populations of trypomastigotes presenting a high predominance of stout forms (MR strain). The rapid decrease in the number of inoculated parasites, as compared with the data from untreated controls, is considered as evidence of drug action against the circulating parasites. Figure 4 shows the course of parasitemia in groups of mice previously treated with

34

Z . BRENER

nitrofurazone and with piperamide, as well as in untreated controls, after intravenous inoculation of about 2,000,000 bloodstream trypomastigotes of T. cruzi MR strain. In the animals treated with nitrofurazone, a rapid decrease in the number of parasites was observed, showing the strong action of the compound against the newly inoculated parasites. Such decrease was not, nevertheless, observed in the mice treated with piperamide, a compound that is active in the living host only after an unusual delay of 2 weeks following drug administration (Brener, 1971). This method appears to have some advantage over in vitro techniques used to detect drug action against the bloodstream form of T. crzui, since it is carried out in a system providing the natural requisites of drug metabolism and excretion. It should be remembered, however, that these experiments were performed using a peculiarly stout trypomastigote form likely to be physiologically different from other T . cruzi stages (Brener, 1969); this might limit generalization of these findings.

VI. Clinical Trials Human Chagas’ disease is usually acquired by contact with Triatoma bug feces containing metacyclic trypomastigotes that infect patients either through skin lesions or whole normal mucous membrane. Congenital (Howard and Rubio, 1968) and blood transfusion transmission (Amato Neto, 1968) have often been reported in the literature. Accidental laboratory infections are becoming an increasing hazard in institutions engaged in T. cruzi research. The acute phase of Chagas’ disease in humans, which occurs soon after parasite penetration, is characterized by a primary focus of infection (T. cruzi portal of entry), followed by clinical signs and symptoms caused by the hematogenic spreading of the parasites: fever, edema, lymphadenopathy, hepatosplenomegaly, cutaneous rash, variable electrocardiographic alterations, and high parasitemia. Death, usually due to myocarditis or to meningoencephalitis in children, seems to occur in less than 10% of the acute cases. After 1 to 2 months, the parasitemia gradually decreases and the patients enter the chronic phase. At this stage, the disease is asymptomatic in a high percentage of patients, the remaining cases usually presenting two symptomatic clinical forms: (a) chronic Chagasic cardiomyopathy (which is observed especially in young people after an asymptomatic stage of unpredictable duration) characterized by a wide range of electrocardiographic changes (ventricular extrasystoles, right bundle branch block, complete A-V block, etc.), heart failure and, often, sudden death; (b) digestive f o r m ,

CHEMOTHERAPY OF Trypanosoma

cruzi INFECTIONS

35

caused by dilatation of hollow muscular organs such as megacolon and megaesophagus (Koeberle, 1968). The pathogenesis of both clinical forms is not yet completely understood; it is probably related to a decrease in the number of ganglion cells of the heart autonomic nervous system and myenteric plexuses (Koeberle, 1968; Tafuri, 1970). Spontaneous cures have not been reported so far. Evaluation of drug activity in man may, therefore, be performed either in the acute or the chronic phase. The acute phase would seem to be better suited for such experiments because of the high parasitemia and the common occurrence of many physical signs that could be used as criteria for suppressive drug activity. Most cases at this stage, however, present a marked tendency to spontaneous clinical remission which takes place regardless of drug administration and often misleads chemotherapists. In the chronic phase, parasites are rather scarce in the bloodstream and not detectable by fresh blood examination; most clinical manifestations are probably an expression of irreversible organic damage not likely to revert after treatment. Chronic cases, nevertheless, constitute the bulk of Chagasic human infections in endemic areas and, therefore, any presumptive curative drug will have to be extensively administered to those patients, either to prevent clinical manifestations in the asymptomatic cases or to reinforce Chagas’ disease control campaigns. In the literature there are a large number of papers dealing with treatment, using different compounds, of single patients or of small groups of acute or chronic cases.* In most cases, follow-up has been limited to a short period of time, clinically unreliable criteria have been used, and cures are not positively asserted. A comprehensive critical retrospect of these numerous cases is beyond the scope of this review and, therefore, only some selected trials providing significant data on the methodology of evaluating drug activity in the human disease is discussed. Most such papers, recently published, deal with groups of patients treated with nitrofuran derivatives on long-term schedules. From the above description we gather that clinical data are often elusive criteria for drug activity in Chagas’ disease. The following laboratory methods are recommended for evaluation of drug activity in humans.

* References covering the subject may he found in “Chagas’ Disease-A Bibliography” (Miles and Rouse, 1970), which includes 2000 references compiled from the Tropical Diseases Bulletin, and “A Bibliography on Chagas’ Disease (1909-1969)” (Olivier et al., 1972), a special publication of the Index Catalogue of Medical and Veterinary Zoology, which quotes about 4000 papers.

36

Z. BRENER

A. PARASITOLOGICAL METHODS In the acute phase, the parasites are usually detected by fresh blood examination. In the chronic phase, xenodiagnosis is still used as the most dependable method: clean (laboratory-bred) triatomid bugs are allowed to feed on the patients and are examined, after 30 to 90 days, for living T. cruzi flagellates in their feces. Conventional xenodiagnosis, using ten fourth-to-fifth stage nymphs, is positive in about 25% of the chronic cases. Schenone et al. (1968) described serial xenodiagnosis, which is carried out using 42 insects applied for 3 consecutive days; this method proovides about 70% positive results among unselected chronic patients and has recently been extensively used in human therapeutic experiments (Schenone et al., 1969; Wegner and Rohwedder, 1972).

B. SEROLOGICAL METHODS Complement fixation test using culture form antigen is a highly specific reaction, giving over 90% positive results in the chronic phase (Freitas and Almeida, 1949; Cerisola et a l . , 1970). The specific reactive capacity of the antigen-antibody complex may be estimated by quantitative techniques (Freitas and Almeida, 1949). Indirect fluorescent antibody test (FAT) using formalin-preserved T. cruzi culture forms shows close agreement with results obtained by CFT and is useful in the diagnosis of both acute and chronic phases (Camargo, 1966; Cerisola et al., 1970). Hemagglutination test (HAT) using either tannic acid (Neal and Miles, 1970) or chromium chloride (Camargo et al., 1971) for coating erythrocytes with T. cruzi antigen, has also been employed in the diagnosis of Chagas’ disease and in the assessment of drug action (Cerisola et al., 1970). C. TREATMENT IN THE ACUTEPHASE The most extensive study of treatment of patients in the acute phase was carried out by Cerisola et al. (1970) in Argentina. A serological follow-up by CFT, HAT, and FAT was conducted on 550 patients treated with Nifurtimox for 3 consecutive months and on 55 patients who received placebo. The serological tests were performed monthly for 6 months and, afterward, every 3 months for 24 months. Figure 5 shows the serological development in both groups of patients after a 2-year follow-up. In the group treated with the nitrofuran derivative, 81% of patients showed persistent and repeated negative serology, whereas in the untreated group 100% of the patients turned positive. In treated patients with negative serology, xenodiagnosis was also negative in 100%

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

37

treatment with

.......................................... .......................... 0

. .

!...

1 2 3 4 5 6

months drvclopmsnt

9

12

18

24

FIG. 5. Serological data on groups of patients with acute Chagas' disease, treated with Nifurtimox (Lampit) and with placebo. CFR (complement fixation reaction), HAR (hemagglutination reaction), FAT (fluorescent antibody test). (Data from Cerisola et al.,

1972).

of the cases. The patients with prolonged negative serology were therefore considered cured. Such remarkable results with nitrofuran compounds have not, however, been entirely confirmed in Brazil by Rassi and Ferreira (1971), who treated a small group of 13 patients in the acute phase with Nifurtimox, according to the same schedule. When treatment was terminated, CFT and xenodiagnosis remained positive in 53.9 and 61.5%, respectively, of the patients. Although persistence of infection could be observed in a high proportion of patients, suppressive action of the drug was evidenced by the low percentage of positive xenodiagnosis observed during treatment (18.1%).

D. TREATMENT IN THE CHRONIC PHASE Contrary to what occurs in the acute phase, serological reactions are not affected by treatment with nitrofuran derivatives. Titers of CFT and HAT remained unchanged in sera from groups of patients treated with Nifurtimox for 120 days and who presented positive serological tests (CFT, HAT, immunofluorescent antibody test) even 3 years after treatment (Cerisola et al., 1972). An overall survey of 54 chronic patients treated in Argentina, Chile, and Brazil provided similar results with regard to serological reactions (Wegner and Rohwedder, 1972).

38

Z. BRENER

TABLE I1 GROUPSOF PATIENTS WITH CHRONIC CHAGAS’ DISEASETREATED WITH NIFURTIMOX AND WITH PLACEBO

POSITIVITY OF SERIAL XENODIAGNOSIS IN

No. of Test

xenodiagnoses

Positive

% Positive

Placebo Treated Before treatment During treatment After treatment

84

46

57.7

10 39 63

10 0 0

100.0 0 0

Placebo Treated Before treatment During treatment After treatment

285

168

65.1

106 109 395

95 0

89.6 0

0

0

Source Schenone et al. (1969)

Cerisola et al. (1972)

The steady positivity of serological reactions in treated patients persuaded the authors to use, on a large scale, in spite of its limitations and disadvantages, xenodiagnosis as the main method of treatment evaluation in Chagas’ disease chronic phase. Table I1 shows results of serial xenodiagnosis from groups of patients treated with Nifurtimox and placebo by Schenone et al. (1969) and Cerisola et al. (1972), respectively, and followed for 12 and 36 months after treatment. The results, which, according to the above-mentioned authors suggest parasitological cures, have not, however, been confirmed by Brazilian workers. In a group of 10 patients who received high doses of Nifurtimox for over 2 months (4 of them having been treated for 3 months), 5 cases presented positive xenodiagnosis and CFT soon after treatment (CanCado et al., 1969). Despite discrepancies in the results obtained in different endemic areas, clinical researchers have now come to an agreement as to some general principles concerning the selection of patients for clinical trials and their follow-up: 1. For the chronic phase, only patients with positive parasitological tests should be used; patients showing a tendency to present repeated positive xenodiagnosis before onset of treatment are most suitable for such experiments. A 3-year study performed monthly in an unselected group of cases in the chronic phase showed a wide variation in the percentage of positive xenodiagnosis in different patients (one patient

CHEMOTHERAPY OF Trypanosoma cruzi INFECTIONS

39

had 29 consecutive negative examinations, whereas in another 16 out 17 were positive) (Cancado, 1973). 2. Serial xenodiagnosis, which provides high positivity rates in the chronic patients, should replace conventional xenodiagnosis. 3. Serology is quite useful as a complementary method for the assessment of drug activity, especially in the acute phase (Cerisola et al., 1970), but must not be the only criterion used, since patients with active infection, as demonstrated by positive xenodiagnosis, may present prolonged negative serology (Marra, 1965; Rassi and Ferreira, 1971). During treatment, serological tests may be influenced by drug administration: Warmbrand et al. (1963) demonstrated that two drugs active against T . cruzi infections, primaquine and aminonucleoside of stylomycin, induced a significant decrease of antibody levels in rabbits immunized with crystalline egg albumin, probably through inhibition of nucleic acid synthesis. 4. Assessment of drug activity by laboratory methods must be carried out for at least a few years. A chronic patient treated with Nifurtimox by Schenone et al. (1972) exhibited a positive xenodiagnosis after 34 months of repeated negative tests. The difficulty in establishing a dependable criterion of cure in Chagas’ disease is exemplified by Prata and Ferreira’s (1969) report, in which a single case treated with nitrofurazone, presented 129 negative xenodiagnosis over a period of 3 years and was therefore considered as “apparently” cured.

VI. Concluding Remarks Chagas’ disease treatment will remain a research challenge even after successful prevention programs based on vector control or housing improvement have been achieved in endemic areas: millions of young people are already irreversibly infected by T. cruzi. The needs in the field of Chagas’ disease chemotherapy are quite clear: extensive in uivo screening programs as well as in vitro tests for drugs to be used in banked blood; data on the parasite’s nutritional requirements and growth factors, on growth in defined media at the vertebrate host’s temperature, and on the physiology of culture forms (which are essential for the development of reliable in uitro screening tests); further data on mode of action especially at enzymic and molecular levels; better criteria for clinical drug evaluation, which implies the development of new parasitological methods for demonstrating persistence of T . cruzi infection as well as additional knowledge on the mechanisms involved in

40

Z . BRENER

the host’s response to T. cruzi parasitism. Prospects of finding a cure for Chagas’ disease depend largely on joint study of these fundamental and practical problems. REFERENCES Abithol, H., Pattini, R. E. and Salvador, J. (1%0). Rev. SOC.Argent. Biol. 36, 41-44. Abithol, H., Boris, E., Piulats, E., Podesta, D., Maranon, J., and Gori, C. (1964). Bol. Chil. Parasitol. 19, 132-134. Adler, S., and Bichowsky, L. (1946). Harefuah 31, 57-58. Amato Neto, V. (1958). Thesis, 332 pp. Univ. of Sao Paulo, Sao Paulo. Amato Neto, V. (1968). In “Doenga de Chagas” (J. R. Cancado, ed.), pp. 130-142. B. Horizonte, Brazil. Amato Neto, V., and Mellone, 0. (1959). Hospital (Rio de Janeiro) 5 5 , 343-346. Amrein, Y. U. L. (1951). Proc. SOC.Exp. Biol. Med. 76, 744-746. Amrein, Y. U. L. (1965). Exp. Parasitol. 17, 261-263. Andrade, S. G., Figueira, R. M., and Carvalho, M. L. (1972). Rev. Inst. Med. Trop. Sao Paulo 14, 135-145. Andrade, Z., and Brener, Z. (1969). Rev. Inst. Med. Trop. Sao Paulo 11, 222-228. Apt, W., Lanas, C., and Mancilla, R. (1967). Bol. Chil. Parasitol. 22, 96-99. Archer, S. and Yarinsky, A. (1972). Fortschr. Arzneirnittelforsch. 16, 12-66. Balan, J., Kjaer, A., Kovak, S., and Shapiro, R. H. (1965). Acta Chern. Scand. 19, 528530. Barros, 0. M., and Nogueira, D. P. (1951). Rev. Clin. Sao Paulo 27, 11-18. Bayles, A., Waitz, J. A., and Thompson, E. P. (1966). J . Prototool. 13, 110-114. Beveridge, E., Goodwin, L. G., and Walls, L. P. (1958). Nature (London) 182, 316-317. Bice, D. E., and Zeledon, R. (1970). J. Parasitol. 56, 663-670. Bock, M., Giinnert, R., and Haberkorn, A. (1969). Bol. Chil. Parasitol. 24, 13-19. Bock, M., Haberkorn, A., Herlinger, H., Mayer, K. H., and Petersen, S. (1972). Arzneirn.Forsch. 22, 1564-1569. Brener, Z. (1961a). Rev. Inst. Med. Trop. Sao Paulo 3, 4349. Brener, Z. (1961b). Hospital (Rio de Janeiro) 60, 947-952. Brener, Z. (1962a). Rev. Inst. Med. Trop. Sao Paulo 4, 119-123. Brener, Z. (196213). Rev. Inst. Med. Trop. Sao Paulo 4, 38%396. Brener, 2. (1%3). Rev. Inst. Med. l‘rop. Sao Paulo 5 , 12a132. Brener, Z. (1965). Ann. Trop. Med. Parasitol. 59, 19-26. Brener, Z. (1966). Ann. Trop. Med. Parasitol. 60, 445-451. Brener, Z. (1967). Rev. Inst. Med. Trop. Sao Paulo 9, 233-238. Brener, Z. (1969). Ann. Trop. Med. Parasitol. 63, 215-220. Brener, Z. (1971). Rev. Inst. Med. Trop. Sao Paulo 13, 302-306. Brener, 2. (1973). Annu. Rev. Microbiol. 27, 347-382. Brener, Z., and Chiari, E. (1963a). Rev. Inst. Med. Trop. Sao Paulo 5 , 128-132. Brener, Z., and Chiari, E. (196313). Rev. Inst. Med. Trop. Sao Paulo 5 , 220-224. Brener, Z., and Chiari, E. (1967). Rev. Inst. Med. Trop. Sao Paulo 9, 197-207. Brener, Z., and Chiari, E. (1971). Trans. Roy. Soc. Trop. Med. Hyg. 65, 629-636. Brener, Z., and Costa, C. A. G. (1974). Proc. Int. Cong. Parasitol. 3rd 3, 1292-1293. Brener, Z., and Pellegrino, J. (1958). Rev. Brasil. Malariol. Doencas Trop. 10, 327-330. Brener, Z., Tafuri, W. L., and Maria, T. A. (1969). Rev. Inst. Med. Trop. Sao Paulo 11, 245-249.

CHEMOTHERAPY OF

Trypanosoma cruzi INFECTIONS

41

Brener, Z.,Chiari, E., and Alvarenga, N. J. (1974). Rev. Inst. Med. l’rop. Sao Paulo 16, 32-38. Browning, C. H., Calver, k. M., Leckie, M., and Walls, L. P. (1946). Nature (London) 157,263-264. Burden, E., and Racette, E. (1%9). Antimicrob. Ag. Chemother., p. 54.5-547. Camargo, E. P . (1964). Rev. Inst. Med. Trop. Sao Paulo 6, 93-100. Camargo, M. E. (1966). Rev. Inst. Med. Trop. Sao Paulo 8, 227-234. Camargo, M. E., Hoshino, S., Correa, N. S., and Peres, B. A. (1971). Rev. Inst. Med. Trop. Sao Paul0 13, 45-50. Cansado, J. R. (1973). Personal communication. Cansado, J. R., Marra, U. D., Lopes, M., Mourio, O., Faria, C. A. F., Alvares, J. M., and Salgado, A. A. (1969). Bol. Chil. Parasitol. 24, 2%32. Castellani, O., and Fernandes, J. F. (1965). Rev. Inst. Med. Trop. Sao Paulo 7, 275-282. Cerisola, J. A., Alvares, M., and Di Rissio, A. M. (1970). Rev. Inst. Med. Trop. Sao Paulo 12,403-411. Cerisola, J. A,, Lugones, H., and Rabinovich, L. B. (1972). “Tratamiento de la Enfermedad de Chagas,” 75 pp. Ed. Fundaciou Rizzuto, Buenos Aires. Chiari, E. (1971). Thesis, 72 pp. Univ. of Minas Gerais, B. Horizonte, Brasil. Christen, R., Agosin, M., Jarpa, A. and Atias, A. V. (1951). Bol. Inform. Parasit. Chil. 6, 23-24. Costa, R. B., and Corrado, A. P. (1963). Rev. Inst. Med. Trop. Sao Paulo 5, 243-248. Cuckler, A. C., Kupferberg, A. B., and Millman, N. (1955). Antibiot. Chemother. (Washington, D.C.) 5 , 540-550. Ebringer, L., and Foltinova, P. (1971). In “Advances in Antimicrobial and Anti-neoplastic Chemotherapy” , pp. 417418. Ebringer. L., Balan, J., and h e m e c , P. (1964). J. Protozool. 11, 153-156. Fernandes, J. F., and Castellani, 0. (1958). Exp. Parasitol. 7, 224-235. Fernandes, J. F., and Castellani, 0. (1959). Exp. Parasitol. 8, 480485. Fernandes, J. F., and Castellani, 0. (1966). Exp. Parasitol. 18, 195-202. Fernandes, J. F., and Castellani, 0. (1968). In “Mode of Action of Anti-Parasitic Drugs” (J. Rodrigues da Silva and M. J. Ferreira, eds.), pp. 93-104. Pergamon, Oxford. Fernandes, J. F., Pereira, J. P. M., and Silva, L. H. P. (1959). Exp. Parasitol. 8, 496501. Fernandes, J. F., Halsrnan. M., and Castellani, 0. (1965). Nature (London) 207, 10041005. Foster, R., Pringle, G., and Paris, J. (1969). Ann. Trop. Med. Parasitol. 63, 9C107. Freitas, J . L. P., and Almeida, J. 0. (1949). Hospital (Rio de Janeiro) 35, 787-800. Friebel, H., and Kastner, H. (1955). NaunynSchmiedebergs Arch. E x p . Pathol. Pharamkol. 225, 210-236. Fulton, J. D. (1943). Ann. Trop. Med. Parasitol. 37, 164-173. Fulton, J. D., and Spooner, D. F. (1959). Exp. Parasitol. 8, 137-162. Ghosh, B. K., and Chatterjee, A. N. (1961). Ann. Biochem. Exp. Med. 21, 343-354. Ghosh, B. K . , and Chatterjee, A. N. (1962). Antibiot. and Chemother. (Washington, D . C . ) 12,204-208. Goble, F. C. (1949). J . Parasitol. 35,375-378. Goble, F. C. (1950). J . Pharmacol. E x p . Ther. 98, 49-61. Goble, F. C. (1951). J. Parasitol. 37,408-414. Goble, F. C. (1952a). Amer. J . Trop. Med. Hyg. 1, 189-204. Goble, F. C. (1952b). Antibiot. Chemother. (Washington, D.C.) 2, 265-270.

42

Z . BRENER

Goble, F. C. (1961). Bol. 05 Sanit. Panamer. 51, 439-449. Goble, F. C., and Konopka, E. A. (1963). J . Parasitol. 49, Sect. 2, 17. Goble, F. C., and Singer, I. (1960). Ann. N. Y. Acad. Sci. 88, 149-171. Gonnert, R., and Bock, M. (1972). Arzneim.-Forsch. 22, 1582-1586. Goodwin, L. G., Goss, M. D., Lock, J. A., and Walls, L. P. (1950). Brit. J. Pharmacol. Chemother. 5, 277-286. Grunberg, E., Beskid, G., Cleeland, R., DeLorenzo, W. F., Titsworth, E., Scholer, H. J., Richle, R., and Brener, Z. (1968). Antimicrob. Ag. Chemother. pp. 513-519. Gutteridge, W. E., and Knowler, J, (1%8). Trans. Roy. Soc. Trop. Med. Hyg. 62, 134135. Gutteridge, W. E., and Senior, D. S. (1968). Trans. Roy. SOC. Trop. Med. Hyg. 62, 13% 136. Gutteridge, W. E., Knowler, J., and Coombes, J. D. (1969). J . Protozool. 16, 521-525. Haherkorn, A. (1971). In “Advances in Antimicrobial and Anti-neoplastic Chemotherapy,” pp. 419421. Prague. Haberkorn, A., and Giinnert, R. (1972). Arzneim.-Forsch. 22, 1570-1582. Hauschka, T. S. (1949). J. Parasitol, 35, 593-599. Hawking, F. (1963). In “Experimental Chemotherapy” (R. J. Schnitzer and F. Hawking, eds.), pp. 131-238. Academic Press, New York. Hewitt, R., Wallace, W. S., Gumhle, A. R., Gill, E., and Williams, J. H. (1953). Amer. J . l’rop. Med. Hyg. 2, 254-266. Hewitt, R., Entwistle, J., and Gill, E. (1963). J . Parasitol. 49, 22-30. Hoffmann, K. (1972). Arzneim.-Forsch. 22, 1590-1603. Honigberg, B. M. (1967). In “Protozoa” (M. Florkin, B. T. Scheer, and G. W. Kidder, eds.), Chemical Zoology, pp. 695-814. Academic Press, New York. Howard, J., and Ruhio, M. (1968). Bol. Chil. Parasitol. 23, 107-112. Howard, J., Rios, C., Ebensperger, I., and Olivos, P. (1957). Bol. Chil. Parasitol. 12, 4245. Jaffe, J. J. (1968). In “Mode of Action of Anti-Parasitic Drugs” (J. Rodrigues da Silva and M. J. Ferreira, eds.), pp. 107-114. Pergamon, Oxford. Jarpa, A., Christen, R., and Agosin, M. (1949). Bol. Inform. Parasitol. Chil. 4, 49-50. Jarpa, A., Christen, R., Agosin, M., and Pizzi, T. (1950). Bol. Inform. Parasitol. Chil. 5, 5-6. Jarpa, A., Agosin, M., Christen, R., and Atias, V. (1951). Bol. Inform. Parasit. Chil. 6, 2S27. Jensch, H. (1937). Angau. Chem. 50, 891-895. Koeberle, F. (1968). Aduan. Parasitol. 6, 63-116. Konopka, E. A., Goble, F. C., and Prins, D. A. (1964). Antimicrob. Ag. Chemother. 4, 772-776. Korn, E. D., von Brand, T., and Tobie, E. J. (1969). Comp. Biochem. Physiol. 30, 601610. Kumar, R., Kline, 1. K., and Abelmann, W. H. (1970). Amer. J . Trop. Med. Hyg. 19, 932939. Lambert, C. R., and Stauffer, P. (1964). Ann. Trop. Med. Parasitol. 58, 292-303. Lock, J. A. (1950). Brit. J . Pharmacol. Chemother. 5, 398408. Lopetegui, R., and Miatello, C. S. (1958). MEPRA 86/88, S 1 6 . Lucena, D. T., Carvalho, J. A. M., Abath, E. C., and Amorim, N. (1962). Hospital (Rio de Janeiro) 62,1277-1296. McCalla, D. R., and Voutsinos, Z. (1974). Mutation Res. 26,3-16.

CHEMOTHERAPY OF Trypanosoma

cruzi

INFECTIONS

43

McRary, W. L., Berver, E. L., and Noble, E. R. (1953). Exp. Parasitol. 2, 125-128. Marra, U. D. (1965). Thesis, 143 pp. Univ. of Minas Gerais, B. Horizonte, Brazil. Marsden. P. D., Voller, A., Seah, S. K. K., Hawkey, C., and Green, D. (1970). Rev. Soc. Brasil. Med. Trop. 4, 177-182. Mazza, S., Basso, G., and Basso, R. (1942). MEPRA 61, 3-7. Mieth, H., and Seidenhat, H. (1967). Z . Tropenrned. Parasitol. 18, 53-60. Miles, M. A., and Rouse, J. E. (1970). “Chagas’ Disease-A Bibliography,” 209 pp. London. Mintzer, S., Kadison, E. R., Shalaes, W. H., and Felsenfeld, 0. (1953). Antibiot. Chernother. (Washington, D.C.) 3, 151-157. Moon, A. P., and Coleman, J. F. (1962). Bol. Chil. Parasitol. 17, 63-66. Moraes, G. E. S., Faria, J. I,., and Fernandes, J. F. (1960). Rev. Inst. Med. Trop. Sao Paul0 2, 147-154. Neal, R. A. (1973). Proc. Znt. Cong. Trop. Med. Malaria 9th 1, 56. Neal, R. A., and Miles, R. A. (1970). Rev. Znst. Med. Trop. Sao Paulo 12, 325-332. Neal, R. A., Richards, W. H. G., and Fareobrother, D. A. (1973). Trans. Roy. Soc. Trop. Med. Hyg. 67, 277-278. Neva, F. A , , Malone, M. F., and Myers, B. R. (1961). Arner. J. Trop. Med. Hyg. 10, 140154. Norman, L., and Kagan, I. G. (1960). J. Znfec. Dis. 107, 168-174. Nussenzweig, V., Sonntag, R., Biancalana, A., Freitas, J. L. P., Nussenzweig, R. S., and Kloetzel, J. (1953). Hospital (Rio de Janeiro) 44, 731-744. Olivier, M. C., Olivier, L. J., and Segal, D. B. (1972). “A Bibliography on Chagas’ Disease. Index Catalogue of Medical and Veterinary Zoology,” 633 pp. Washington, D.C. Packchanian, A. (1952). J . Parasitol. 38, Sect. 2, 30. Packchanian, A. (1957). Antibiot. Chernother. (Washington, D.C.) 7, 13-23. Pan, C. T. (1971). J . Protozool. 18, 556-560. Paul, H. H., and Paul, M. F. (1966). In “Experimental Chemotherapy” (R. J. Schnitzer and F. Hawking, eds.), Vol. 4, pp. 521-531. Academic Press, New York. Phillips, N. R. (1960). Ann. Trop. Med. Parasitol. 54, 60-70. Pipkin, A. C. (1960). E z p . Parasitol. 9, 167-203. Pizzi, T. (1951). Proc. Sac. Exp. Biol. Med. 78, 643444. Pizzi, T. (1957). “Immunologia de la Enfermedad de Chagas,” 183 pp. Univ. de Chile, Santiago. Pizzi, T. (1961). Bol. Chil. Parasitol. 16, 3 5 3 6 . Pizzi, T., and Chemke, S. (1955). Biologica 21, 31-58. Pizzi, T., Prager, R. S., and Knierim, T. F. (1953). Bol. Inform. Parasitol. Chil. 8 , 77-79. Pizzi, T., Niedmann, G., and Jarpa, A. (1963). Bol. Chil. Parasitol. 18, 32-36. Prata, A,, and Ferreira, H. (1969). Gaz. Med. Bahia 69, 25-29. Prata, A., Machado, R., and Macedo, V. (1966). Acta Trop., Suppl. 9, 180-186. Pratt, M. G., and Archer, S. (1948). J . Arner. Chern. Soc. 70, 80654069. Raether, W . , Schorr, M., and Seidenhat, H. (1972). 2. Tropenrned. Parasitol. 2 3 , 227240. Rassi, A., and Ferreira, H. (1971). Rev. SOC.Brasil. Med. Trop. 5 , 235-262. Raw, I. (1959). Rev. Znst. Med. Trop. Sao Paulo 1, 192-194. Rey, L., and Fernandes, J. F. (1962). Ezp. Parasitol. 12, 55-60. Rezende, J. M. (1965). Rev. Goiana Med. 11, 35-47. Romaiia, C. (1953). An. Znst. Med. Reg. 3 , 255263.

44

Z . BRENER

Ryley, J. F. (1956). Biochem. 1. 62, 215-222. Ryley, J. F. (1967). I n “Protozoa” (M. Florkin, B. J. Scheer, and G. W. Kidder, eds.), Chemical Zoology, Vol. 1, pp. 55-92. Academic Press, New York. Salgado, A. A,, and Pellegrino, J. (1%8). In “DoenCa d e C h a w ’ ’ (J. R. C a n d o , ed.1, PP 143-162. Santos, U. M. (1962). lhesis. 28 pp. Univ. of Minas Gerais, B. Horizonte, Brasil. Schellenberg, K. A., and Coatney, G. R. (1961). Biochem. Pharmacol. 6, 143-152. Schenone, H., Alfaro, R., Reyes, H., and Taucher, E. (1968). Bol. Chil. Parasitol. 23, 149-154. Schenone, H., Concha, L., Arauda, R., Rojas, A , , and Alfaro, E. (1969). Bol. Chil. Parusitol. 24, 66-69. Schenone, H., Concha, L., Arauda, R., Rojas, A., Knierim, F., and Rojo, M. (1972). Bol. Chil. Parusitol. 27, 11-14. Seneca, H., and Ides, D. (1953). Antibiot. Chemother. (Washington, D.C.) 3, 117-121. Seneca, H., Kane, J. H., and Rockenbach, J. (1952). Antibiot. Chemother. (Washington, D.C.) 2,435-437. Shoemaker, J. P., and Hoffman, R. V. (1969). J. Parasitol. 5 5 , 6 5 4 4 5 9 . Shoemaker, J. P . , and Hoffman, R. V. (1971). Proc. W . V. Acad. Sci. 43, 87-92. Silva, L. H. P., and Kirchner, E. (1962). Rev. Znst. Med. Trop. Sao Paulo 4, 16-28. Silva, L. H. P., and Nussenzweig, V. (1953). Folia Clin. Biol. 20, 191-207. Silva, L. H. P., Yoneda, S., and Fernandes, J. F. (1959). Exp. Parasitol. 8, 486495. Sonntag, R., and Kloetzel, J. (1953). Folia Clin. Biol. 20, 133-138. Steck, E. A. (1972). “The Chemotherapy of Protozoan Diseases.” Walter Reed Army Inst. Res., Washington, D.C.. Tafuri, W. L. (1970). Amer. J. Trop. Med. Hyg. 19, 405, 417. Taylor, A. E. R., and Baker, J. R. (1968). “The Cultivation of Parasites I n Vitro.” Blackwell, Oxford. Thiermann, I. E., and Christen, A. R. (1952). Bol. Inform. Parasitol. Chil. 7, 53-55. Thompson, P. E., and Bayles, A. (1970). J . Parasitol. 56, 61fi-617. Thompson, P. E., Bayles, A., and Olszewski, B. (1969). Exp. Parasitol. 25, 32-49. Tomcufcik, A. S., Hewitt, R. S., Fabio, P. F., Hoffman, A. M., and Entwistle, J. (1965). Nature (London) 205, 605-606. Torres, C. M., and Tavares, B. M. (1958). Mem. Inst. Oswaldo Cruz 56, 85-119. Velisquez-Antich, A. (1970). Rev. Inst. Med. Trop. Suo Paulo 12, 347-353. Velisquez-Antich, A., and Aleman, C. (1971). Acta Cient. Venez. 22, 112-115. Voigt, W. H., Bock, M., and GSnnert, R. (1972). Arzneim.-Forsch. 22, 1586-1589. von Brand, T. (1966). “Biochemistry of Parasites,” 429 pp. Academic Press, New York. von Brand, T.. McMahon, P., Tobie, E. J., Thompson, M. J., and Mosettig, E. (1959). Exp. Parasitol. 8, 171-181. Wagner, W. H., and Schultz, W. (1952). Z . Gesamte Exp. Med. 119, 304-228. Warmbrand, M., Nussenzweig, V., and Fernandes, J. F. (1963). Rev. Just. Med. Trop. Sao Paulo 5, 294-296. Wegner, D. H. G., and Rohwedder, R. W. (1972). Arzneim.-Forsch. 22, 1624-1635. Williamson, J., and Desowitz, R. S. (1961). E z p . Parasitol. 11, 161-175. World Health Organization (1960). World Health Organ., Tech. Rep. Ser. No. 202, 21 pp. Yoneda, S. (1971). Rev. Brasil. Pesq. Med. Biol. 4, 205-218.

Enzyme Study As a Source of Strategy in Drug Design* CORWINHANSCH Department of Chemistry Pomona College Claremont. California

. . . . . . . . . . . . . . . . . . . . I . Introduction I1 . Problem of Drug Modification . . . . . . . . . . . . . . . 111. Types of Enzyme-Ligand Interactions . . . . . . . . . . . .

45 47 48 49 49 49 50 50 51

IV .

51

V.

VI . VII . VIII .

A . No Interaction . . . . . . . . . . . . . . . . . . B. Hydrophobic Interactions . . . . . . . . . . . . . . . C . Polarizability and Dispersion Forces . . . . . . . . . . . D. Electronic Interactions . . . . . . . . . . . . . . . E . Steric Interactions . . . . . . . . . . . . . . . . . F. Indeterminate Interactions . . . . . . . . . . . . . . Formulation of Quantitative Structure-Activity Relationships . . . . Examples of Enzymic Quantitative Structure-Activity Relationships . . A . No Interaction . . . . . . . . . . . . . . . . . . B. Hydrophobic Interactions . . . . . . . . . . . . . . . C . Polarizability and Dispersion Forces . . . . . . . . . . . D . Electronic Interactions . . . . . . . . . . . . . . . E . Steric Interactions . . . . . . . . . . . . . . . . . F. Indeterminate Interactions . . . . . . . . . . . . . . Nature of Receptors . . . . . . . . . . . . . . . . . . Therapeutic Index Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . References . . . .

52 53 57 60 69 70 72 75 76 77 78

I . Introduction Ultimately. the control of the enzymic systems of host and pathogen will constitute the most important weapon of the medicinal chemist in the battle against disease. This goal is a long way in the future. However. the study of enzymes as a source of understanding the interaction of substrates and inhibitors with macromolecular systems constitutes a great opportunity for sorting out the fundamentals of structure-activity relationships on the best models we have of drug * The author’s work was supported by Grant CA 11110 from the National Institutes of Health . 45

46

CORWIN HANSCH

receptor sites. Not only are enzymes good models of receptors, they are often the receptor sites. As the science of drug development matures, researchers are becoming more introspective. Discussion continues to develop about how new drugs are actually found and what objective techniques can be employed most efficiently to modify a new lead compound so that one can have some degree of assurance that important structural features have not been left unexplored. A recent symposium has produced an interesting volume on “Drug Discovery” in which a number of eminent medicinal chemists have analyzed the routes by which new drugs arise (Gould, 1971). Questions about the prospects for new drugs obtained by screening of new compounds, extracting plants or microorganisms, etc., were examined. In their chapter in the Gould volume, Biel and Martin (1971) advance the interesting proposal that “biological concepts beget synthetic drug discoveries which, in turn, give rise to new biological discoveries and concepts and these exert a positive feedback on the creation of new structural drug prototypes.” In the end, this leads back to the fundamental enzymic processes of living systems. Our concern in this chapter will not be with broad policy decisions but, instead, attention will be directed to the problem of molecular modifications once a lead compound has been uncovered. The problem of how to modify a parent molecule with a minimum of redundancy is extremely vexing to contemplate. Another problem that produces much nail biting and fidgeting is that of when to stop seeking better modifications. It is only recently that such questions have commenced to open up public discussion of what promises to be a long and interesting conversation among medicinal chemists (Topliss, 1972; Martin and Dunn, 1973; Hansch, 1972a; Hansch et al., 1973a). Probably no scientist is condemned to work with more variables than the medicinal chemist. The number of molecules open for consideration, the number of cellular systems that may be simultaneously perturbed by a given drug, and the number of different ways of evaluating drugs have no end. The swift advance of analytical chemistry constantly opens up new possibilities for clearer assessment of the interactions of a drug with the vast number of molecular constellations of the central nervous system, immune response system, enzyme systems, membranes, etc. The outpouring of new compounds and test data from thousands of laboratories around the world is relentlessly driving medicinal chemists to the use of large computers. Already many complex but exciting new aids to the drug designer are clearly in view. Cluster analysis (Hansch et al., 1973a), pattern recognition (Kowalski and Bender, 1974; Schiffman, 1974), discriminate analysis (Martin et al., 1974), factor analysis (Weiner

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

47

and Weiner, 1973), and, of course, the explosive growth of molecular orbital calculations (Kier, 1971) are placing the computer squarely in the laboratory of the medicinal chemist. The development of computerized techniques for manipulation of chemical structures (Lynch et al., 1971; Wipke et al., 1974) and chemical synthesis as well as the management of structure-activity data (Hansch et al., 1974; Campey et al., 1970) are all in an expanding state of development. Although luck will continue to play its important role in drug research (Gould, 1972), the lucky breaks will occur at higher and higher levels of sophistication (Hansch, 1974). The saying, “chance favors the prepared mind” might be emended to “chance favors the prepared mind coupled with the prepared computer.”

II. Problem of Drug Modification The different permutations and combinations of atoms that modern synthetic organic chemistry allows one to consider are truly wonderful. For example, consider the relatively simple structure (I):

ii

Assume (I) has some feeble anti-inflammatory activity that is to be enhanced through molecular modification. First, consider the five ringcarbon positions; for substituents at these positions one might choose from a recently published list of 236 aromatic substituents for which a variety of physicochemical constants are known (Hansch et al., 1973b). If we elect to explore 30 substituents well disposed in substituent space (Hansch et al., 1973a), the number of possible derivatives considering all combinations of 30 substituents in five positions is given by X ” , where X is the number of substituents and n is the number of positions assuming no elements of symmetry in the system. This leads to 305 or 24,300,000 possibilities. Now assume that 10 different groups can be attached to the CH, moiety; 10 different ester functions and 10 substituents on the nitrogen can also be varied. The possibilities now go up to 305 x 10 x 10 x 10. Let us say that one of the functions attached we might consider 10 different functions in to the N is C,H,(C=O)-; the ortho, meta, and para positions yielding lo3 variations on this single

48

CORWIN HANSCH

function without even considering heterocyclic functions such as

and the like. These same possibilities exist at any of the positions on (I). How is one to react to this problem? It is all too easy to imagine one billion or one trillion or, in fact, any number of variations of (I). Synthetic organic chemistry has reached the point where there would be little argument that most if not all of these possibilities could be made. How many molecules constitute a reasonable sample of a population of one billion? What are the best 100 or 1000 representatives that should be made? Until the last two decades this answer was largely dictated by the cost of synthesis. This restriction is continually being relaxed; first, by more expeditious synthetic techniques and, second, by an increasingly affluent society willing to pay almost any price for modern drugs that without any doubt can greatly increase one’s chances of survival. Budgets for drug research will continue to rise the world over because drugs have become central to man’s basic fight for life. As possibilities expand for making more derivatives of a problem molecule such as (I), the development of more systematic techniques for deciding how many and what kinds of derivatives should be prepared is imperative.

Ill. Types of Enzyme-Ligand Interactions For many years the molecularly concerned pharmacologist has been thinking in terms of drugs reacting at receptor sites. Although the isolation of such sites is being accomplished, suitable alternatives are needed to study the parameters of drug interactions with the constellations of atoms that constitute receptor sites. Enzymes are not only suitable models for receptor sites, they must often constitute the very sites on which drugs act. The interaction of a ligand with an enzyme is a multiparameter problem on which all of the above-mentioned computer techniques can be brought to bear. This report is limited to the consideration of computerized regression analysis. What are the kinds of interactions one can expect from the

ENZYME STUDY AS STRATEGY I N DRUG DESIGN

49

components of a molecule such as (I) with a complex protein such as an enzyme? At least a partial answer can be given in the following way.

A. No INTERACTION Part of molecule (I) might be embedded in an enzyme or held to the surface of the enzyme, yet other parts of the molecule could remain in the solution surrounding the enzyme. Substituents on these parts could only affect interaction in an indirect fashion.

B. HYDROPHOBIC INTERACTIONS This is a most important area in enzymic studies (Jencks, 1969; Tanford, 1973), and Baker has discussed many examples of the importance of this type of interaction in enzymic processes (Baker, 1967). Kuntz (1972) has made graphic illustrations of hydrophobic pockets from an analysis of X-ray crystallographic data. We have found that partition coefficients of organic compounds serve as suitable references for hydrophobic interactions of enzymes (Hansch and Coats, 1970).

C. POLARIZABILITY AND DISPERSIONFORCES Broadly speaking, there must be two kinds of space in and on enzymes: the hydrophobic pockets, which have been of greatest interest, and the “other space,” which, in a general sense, might be termed polar space. There are, of course, many kinds of specific interactions that can occur with ligands in polar space. There also appear to be nonspecific interactions that do not parallel hydrophobic interactions in their properties. For lack of understanding, we tend to think of these as nonspecific polarizability and dispersion forces, which are relatively independent of the desolvation process characteristic of hydrophobic binding. Of course, it is hard to imagine that there are any completely hydrophobic pockets of significant size. Polar space is also nonhomogeneous. To model nonspecific binding in what we think is nonhydrophobic space, we have found molar refractivity to be of value. If, for a set of congeners, it is shown that the hydrophobic constants (T)are not significantly collinear with molar refractivity ( M R ) and that MR yields a better quantitative structure-activity relationship (QSAR), then nonspecific binding is occurring in nonhydrophobic space (i.e., polar space). We have followed the suggestion of Agin et al. (1965) and used the

50

CORWIN HANSCH

relationship log 1IC or log k a M R , where k is a rate or equilibrium constant and C is the molar concentration of the compound producing a standard response.

D. ELECTRONIC INTERACTIONS Although quantum chemical techniques hold out promise for the future, for a set of congeners that react with a drug receptor through a specific electronically modulated reaction such as covalent bond formation, hydrogen bonding, or charge transfer, we have found that the substituent effects on such processes are best correlated by HammettTaft u constants (Chapman and Shorter, 1972; Shorter, 1973; Johnson, 1973). Since the time of the pioneering work of Taft and Lewis (1958), it has been evident that it is profitable to factor u into ur and uR in order to assess the polar and resonance components of the electronic effect of substituents separately. Taft and his colleagues have recently published an extension (Dayal et al., 1972) of his initial work in which they recommend using several different types of uR in conjunction with U I to account for special resonance interactions between substituents and reaction center. At present the number of these substituent constants is limited to about two dozen substituents. Swain and Lupton have factored 9 and % in such a way that it has been possible to calculate 9 and % values for many substituents (Hansch et al., 1973b). The 9 and % should be quite useful for exploratory work in QSAR studies; however, it is now clear that % will not work well in situations where u+ or u- give much better correlations than u.Thus, 9 and % will be most useful where direct resonance interactions between substituents and reaction center are absent.

E. STERIC INTERACTIONS Ever since the formulation of Fischer’s famous “lock and key” analogy for enzyme-substrate interactions, chemists have approached the analysis of such problems with great trepidation. However, it has become increasingly evident in recent years that the active sites on enzymes are often flexible and that specific steric effects can be rationalized using Taft’s steric parameter E , (Taft, 1956; Unger and Hansch, 1975; Kutter and Hansch, 1969); E , has been defined using the acid hydrolysis of esters, X-CH,COOC,H, (11). There is evidence to show that the electronic effects of X can be neglected as a first approximation although they undoubtedly play a secondary role (Unger and Hansch, 1975). The steric effect in the hydrolysis of the above

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

51

esters is due to the interference of X in the formation of the transition state with the nucleophilic reagent water:

+

X-CH,CP

KO--

H+

L

kC,H,

OH

I

X-CH,C-OC,& I+

dO'H

(JI)

Parameter E, is defined as log kxlkH = E,, where k , represen s the ra e of hydrolysis of the various derivatives and k H that of the parent compound. One might expect that E, constants derived for this intramolecular effect would not be of value for intermolecular effects. The evidence in hand does indicate that E, can be used to model intermolecular effects (Kutter and Hansch, 1969).

F. INDETERMINATE INTERACTIONS Many instances are known where certain groups of atoms produce strong effects in biochemical systems for reasons which are not clear. For example, the barbiturate function, the steroid system, and L-amino acids all show a special kind of activity. The use of indicator variables discussed below helps one to treat such problems in numerical terms.

IV. For m u Iat ion of Quantitative St r uct u re-Ac t iv it y Re1ati onsh ips With the above outline of the interaction parameters in mind, the present state of the art of formulation of QSAR can be considered. Although there are many ways to attack such a complex puzzle, one way is to consider the influence on activity of functions in one or possibly two positions of a parent compound at a time. Referring back to structure (I), the problem of disubstitution in the 5- and 6-positions might be approached as follows. Assuming we can infer little or nothing from inspection of the data, we can explore all linear combinations of the appropriate variables as log k = av,

+ b v z + c 9 , + d% + e B , + fG& + gMR, + hMRz + iE,, + jE,, + k

(1)

where k is some kind of rate-related constant. Equation (1) contains 10 variables and a minimum of 30 to 40 data points (tested derivatives) are necessary to evaluate coefficients a-j. This would be enough to

52

CORWIN HANSCH

eliminate those variables that are not significant. Generation of all possible linear combinations of the 10 variables would produce (assuming no singular matrices) 2" -1 or 1023 equations. The "best" equation from this set would be that having the lowest standard deviation with all terms justified by the F test.* From inspection of the coefficients in the equations in this first pass, those terms having similar coefficients can be merged in certain cases. For example, if the hydrophobic effect is the same from both the 5- and 6-positions7 the coefficients with T and T' will be essentially the same . the field effect is not and one can use the single term C T ~ , ~Since highly position-dependent, sometimes nothing is lost by merging these terms and, of course, MR is comparable to T in this respect. With this simpler equation one can then test exponential terms such as (ZT)', ( ~ J V R ) 'or , cross-product terms, such as n l . s 1and E,,-%?z to explore second-order effects (Daniel and Wood, 1971a). In cases where few data points are in hand, a further simplification in the number of variables 2 or a,and uR. can be achieved by using u constants instead of 9and 9 Of course, if one is carrying out such a QSAR from the start, it is most important to select substituents with well-defined physicochemical constants. One also wants to include the widest possible range of u,T , etc., values. Over and above these obvious factors, correlation matrices of the parameters of the potential substituents should be studied carefully in advance of the synthesis of any derivatives so that one can minimize collinearity among the various vectors (Hansch et al., 1973a).

V. Examples of Enzymic Quantitative Structure-Activity Relationships There are definite advantages in studying enzymic reactions as a means of developing our techniques for the formulation of QSAR. One does not have to face the difficult problems of metabolism and elimination that tests in whole animals involve. Enzyme homogeneity is much less a problem than the homogeneity of test animals. In general, one is dealing with a single receptor site. Experience gained in the relatively simple enzyme systems can then be extended to more complicated biological systems. Of course, as large numbers of good QSAR studies with enzymes accumulate, they provide the medicinal chemist with a stock of information about which kind of compounds

* The reader unfamiliar with regression analysis should consult texts by Daniel and Wood (1971a) or Draper and Smith (1966).

53

ENZYME STUDY A S STRATEGY I N DRUG DESIGN

perturb which kind of enzymes. In the long run, this kind of information will enable us to design more specific drugs.

A. N o

~NTER AC TI O N

As pointed out above, one should expect to find instances where substituents in certain positions of a parent compound do not contact the macromolecules with which the substrate or inhibitor is reacting. An illustration of this situation can be cited in the hydrolysis of phenylglucosides (111) by emulsin.

In the analysis of the experimental results of Nath and Rydon (1954), the following equations were obtained (Hansch et al., 1965). Para derivatives:

+

+

log kl/kz = 0 . 3 3 ~ 0 . 6 1 ~ - 1.80 log k3 = - 0 . 4 6 ~ 0 . 8 7 ~ -- 6.32

+

n

r

S

8 8

0.921 0.964

0.189 0.221

(2) (3)

Meta derivatives:

+

log k,lkz = 0 . 9 5 ~ 1.63 log k3 = 1 . 5 2 ~- 6.28

n 6 6

r 0.949 0.922

S

0.120 0.242

(4) (5)

In these equations k , and k , refer to the adsorption-desorption of substrate by enzyme and constitute a binding constant; k3 is a rate constant for the hydrolysis step. In the case of para substituents correlated by Eq. (2) and (3), it is necessary to include a term in T along with u in order to obtain a good correlation. Neither a term in T nor M R improves the correlation in the case of the meta derivatives. Hence one concludes that meta substituents do not make direct contact with the enzyme; however, their electronic effect on the reaction is important and well correlated by C. It is interesting to compare the equations correlating binding and hydrolysis. In Eq. (2), the positive coefficient with T indicates that the more lipophilic the substituent (the larger its T value), the tighter the binding. The negative coefficient with n in Eq. (3) shows the opposite

54

CORWIN HANSCH

effect. Lipophilic substituents retard desorption of products in the hydrolysis step. The overall effect is that the hydrophobic role of substituents is canceled. Equation (6) log k,.(k,lk,) = 1 . 6 0 ~ -- 4.38

n

r

S

13

0.947

0.318

(6)

correlates the overall rate for both meta and para derivatives. Meta functions have been assigned a IT value of 0 in the development of Eq. (6). Addition of a term in IT to Eq. (6) does not result in a significant reduction in variance as judged by the F test with a = 0.1. An almost universal difficulty with studies of the above type which have been reported so far is that the investigators have paid little or no attention to possible collinearity between variables in selecting the derivatives to be made. This collinearity is a difficult problem to avoid unless considerable attention is given to experimental design (Hansch et al., 1973a; Farrar and Glauber, 1967). In the present case there is high collinearity between 77-4 and MR-4. The correlation coefficient for the linear relationship between these two vectors is 0.849. While IT gives a somewhat better correlation in Eq. (3,MR gives a better correlation in Eq. (2). There is much less collinearity between IT and M R for the set of meta substituents; however, neither of these parameters plays a significant role in this situation. Another example of this type of lack of substituent effect is found with alcohol dehydrogenase (Hansch et al., 1973~). The following correlation was obtained in a study of the ternary complex between alcohol dehydrogenase, reduced nicotinamide adenine dinucleotide (NADH), and benzamides: log l/KER,I= 0 . 4 5 ~ - 4- 0 . 8 0 ~ - 0.23E8-4 - 2.37

n

r

S

14

0.953

0.170

(7)

The equilibrium constant K,,,, is defined as

where E = enzyme concentration, I = inhibitor concentration (benzamide), and R = NADH concentration. In the formulation of Eq. (7), over 1000 regression equations were considered and no role could be established for hydrophobic or dispersion types of interaction of substituents in the 3-position. Although this type of effect is quite clear with aromatic inhibitors of alcohol dehydrogenase, a similar effect could

55

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

not be found with the more flexible aliphatic amides (Hansch et al.,

1972a). An additional example of this type of noninteraction of substituents has been found with phenethanolamine inhibitors of N-methyltransferase (Hansch and Glave, 1972). In this instance it is the para substituents that do not appear to establish contact with the enzyme. Examples such as the above indicate the high importance of factoring substituent effects in the study of structure-activity relationships. In the case of tissue or whole animal experiments, it is highly unlikely that one would find no hydrophobic effect for any class of substituents because the movement of drugs in living tissue is so heavily dependent on their lipophilic character (Hansch and Clayton, 1973). However, an effect similar to that seen with enzymes can be observed from a study of antimalarials acting in mice. Phenanthrene carbinols of type (IV) were tested for their ED,, against Plasmodium berghei in mice in a program of the Walter Reed Army Institute of Research. The QSAR is contained in the following equations: y-LNRlR2 CHOH

>=;

f

\o-B-'\?

0-0

'.?! L$ Y

X

(N)

+ +

+

log 1/C = 0.33nx+y 0.83~x+y 2.52 log 1/C = 0.31n~+y 0.79~x+y

+

+

- 0 . 0 1 5 ( ~ ~ ) *0 . 1 3 2 ~ 2.35

n 102

r

S

0.894

0.278

102

0.908

0.263 (10)

(9)

Only substituent effects of X and Y were used in the formulation (Craig and Hansch, 1973) of Eq. (9). The X and Y represent multiple substitution with, in some cases, as many as four substituents on these two rings. Despite large variation in R (i.e., groups as large as dioctyl were studied), Eq. (9) rationalizes 80% of the variance in log 1/C, where C is the moles/kilogram necessary to produce a 50% cure of the mice. Only the electronic and hydrophobic properties of X and Y are necessary to explain most of the variance. In Eq. (lo), n values for X, Y, and R are lumped to obtain %. Only large or small R groups have much effect on log 1/C. For moderate changes in R, one can neglect the

56

CORWIN HANSCH

I

.90

-0.47

-0.011

0.39

0.w

1.25

1.68

-

2.11

2.5'4

2.97

3.40

r x+Y ~ ux+y for 102 antimalarials on which Eqs. (9) and (10) are FIG. 1. Plot of ? T ~ +vs. based. There are, of course, far less than 102 points shown in the plot because many of the points are identical. Circled points are compounds that were synthesized later to break up the high collinearity between mand u.

hydrophobic character of these two substituents. Obviously, the R groups are not interacting with the receptor in the way X and Y are. Exactly why R, and Rz play such a small role in the antimalarial activity of the phenanthrene carbinols is not clear; nevertheless, this information is most important in the design of new drugs. The above antimalarial study is an interesting example of the problems of collinearity in drug modification. In the first analysis (Craig and Hansch, 1973) of about 60 drugs, essentially the same quality

ENZYME STUDY A S STRATEGY I N DRUG DESIGN

57

correlation was obtained using either T ~ or+u ~ ~ +The ~ .reason for this ambivalence was extremely high cofinearity between rr and u for the set of substituents selected. This can be seen in Fig. 1. The circled points off the main line were compounds synthesized after the collinearity problem was brought to light. These few well-placed points broke the collinearity (at least enough) so that the separate roles for 7~ and u could be delineated. There is still significant correlation between the two vectors: r2 = 0.320.

B. HYDROPHOBIC INTERACTIONS There are now many examples where T or log P can be used to model the hydrophobic interaction of enzymic processes to formulate QSAR (Hansch and Coats, 1970; Kutter and Hansch, 1969; Hansch and Deutsch, 1966; Schaeffer et al., 1970; Coats et al., 1970; Hansch, 1971a, 1972b; Hansch and Dunn, 1972; Hansch and Silipo, 1974; Fujita, 1972; Silipo and Hansch, 1974; Hayashi and Penniston, 1973; Hulbert, 1974; Cohen and Mannering, 1973; Anderson and Graves, 1973; Dupaix et al., 1973; Unkovskii et al., 1972; Bartinek et al., 1972; Kakeya et al.,

1969). Hein and Niemann (1962) formulated a convenient way of discussing the interaction of asymmetric molecules (V) with enzyme receptors (Hamilton et al., 1966). Two views of binding are shown. Ps

NHCOR, PH

H-c4% \COR,

COR,

PI

I

PZ

Ps

,

R(*'NHCOR, Pz

PI

(B)

(A)

(V)

Substituent space (space in or on the enzyme where substituents fall) around the asymmetric carbon atom is labeled p H , pl, pz, and p3 in these two projections. Hein and Niemann were concerned with natural amino acids. In such cases an a-hydrogen is always present and the symbol pH is reasonable. However, in medicinal chemistry, a hydrogen may be lacking at this position. A better general approach would be to use the symbol ps, where S stands for the smallest group in the CahnIngold-Prelog system of nomenclature (Cahn et al., 1966; Hanson, 1966). In molecule V,B, the smallest group (H) is understood to be below the plane of the page. Employing this type of nomenclature, the inhibitors and substrates of chymotrypsin have been used to formulate QSAR

58

CORWIN HANSCH

(Hansch and Coats, 1970). It was found in these studies that the interaction of substituents with p1 space was not well-correlated with the hydrophobic constant but was well-correlated by M R . The interaction of substituents with p2 space was well-correlated by IT constants, indicating the hydrophobic nature of p2 space. Not enough data are available to characterize p3 space with assurance. Once pl and p2 space were characterized in preliminary studies with single sets of substrates, it was found possible to include two sets of the following substrates in a single equation: CH,O,

C

$0

I

Rf C‘NHCOR, L Configuration (VI)

+

log 1/K, = 1.381~w 0.082MRRI - 3.88

n

21

r 0.934

S

0.331 (11)

Equation (11) “explains” 87% of the variance in the binding of 21 substrates to chymotrypsin. The Michaelis constant (K,) varies over a concentration range of 3000, and there is a good deal of variety in R, and R2. This brings out the great flexibility of the receptor site. It is becoming more and more evident that the lock and key analogy for interaction of enzyme and receptor is, for perhaps the majority of receptors, much too rigid a concept. There are shortcomings to Eq. (11) that merit further study. The standard deviation is higher than that simply due to experimental error. One would think that at least half of the unexplained 13% of variance could be accounted for by a better model. Although there is a good deal of evidence from other sources (Hansch and Coats, 1970) to establish that p2 space is hydrophobic, one cannot be certain from the R2 substituents studied in Table I. There is is justified in high collinearity between nb and MRR,. The fact that nRP this case rather than M R R , is supported by QSAR for eight sets of substrates and inhibitors that appear to interact in this area. The mean and standard deviation for the eight coefficients of the IT terms was found (Hansch and Coats, 1970) to be 1.21 ? 0.23. These results support the idea of a common area of binding for substrates and inhibitors. It seems likely that by using well-designed sets of congeners and the interaction models V, A or B, one should be able to formulate QSAR for different types of stereoisomers. An excellent example of an enzymic study in which there is very little

59

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

TABLE I PARAMETERS FOR BINDING OF AMIDESTO CHYMOTRYPSIN

R,

R2

Benzoyl 2-Quinolinyl 2-Furoyl 2-Theophenoyl Nicotinyl Isonicotinyl Picolinyl Acetyl o-Aminobenzoyl Chloroacetyl Benzoyl Acetyl Chloroacetyl Benzoyl Acetyl Acetyl Acetyl Acetyl Acetyl Acetyl Acetyl

Methyl Methyl Methyl Methyl Methyl Methyl Methyl Methyl Methyl Propyl Propyl Isopropyl Isopropyl Isopropyl Propyl Benzyl Isobutyl Ethyl Butyl Pentyl Cyclohexylmethyl

0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 1.50 1.50 1.30 1.30 1.30 1.50 2.03 1.80 1.00 2.00 2.50 2.89

-0.99

25.10 41.00 17.00 22.71 23.00 23.00 23.00 5.72 29.40 10.58 25.10 5.72 10.58 25.10 5.72 5.72 5.72 5.72 5.72 5.72 5.72

-1.12 0.19 -1.78 -1.31 -1.29 -1.29 -1.29 -2.71 -0.76 - 0.93 0.27 -1.61 -1.21 -0.01 - 1.33 -0.60 -0.92 -2.02 -0.64 0.05 0.59

0.66 -1.69 -1.18 -1.57 -1.46 -1.25 -2.87 -0.67 -0.70 0.07 -2.05 - 1.64 -0.66 - 1.01 -0.10 -0.58 -1.72 -0.83 -0.21 0.72

collinearity between T and MR s o that a clear case can be made for hydrophobic bonding can be seen in the work of Hulbert (1974). Equations (12)-( 15) correlate the inhibition of L-methionine S-adenosyltransferase by 0-phenyl-DL-homoserines:

log log log log

l/C l/C l/C l/C

= = = =

+

0 . 7 2 ~ 1.71 0.66MR + 1.60 0 . 6 2 ~+ 0 . 6 1 ~+ 1.62 0.42MR + 0.64~+ 1.61

n 11 11 11 11

r

S

0.807 0.533 0.989 0.787

0.215 0.308 0.056 0.239

(12) (13) (14) (15)

60

CORWIN HANSCH

TABLE I1 CORRELATION MATRIX:r2 FOR CORRELATION BETWEEN VARIABLESUSED TO DERIVEEQS. (12~15)

U

U

P

MR

1.00

0.04

0.10 0.18 1.00

1.00

57

MR

The set of vectors used in this study was very well chosen and remarkably orthogonal (see Table 11). Variable M R is a much poorer parameter than T ;compare Eqs. (12) and (13) and Eqs. (14) and (15). Equation (14) correlates more than 97% of the variance in the data! Another example where log P and M R are completely orthogonal and a high correlation with hydrophobicity is found is in the binding of neutral ligands to bovine serum albumin. In this QSAR, log 1lC = 0.75 log P

+ 2.30

n 42

r 0.960

S

0.159

(16)

C is the molar concentration which produces a 1: 1 complex of the ligand with albumin via equilibrium dialysis (Helmer et al., 1968). Using MR instead of log P, an extremely poor correlation is found (Leo et al., 1969) with r = 0.31. In this example, log P and MR are quite orthogonal, and hydrophobicity is the process involved.

C. POLARIZABILITY AND DISPERSION FORCES Physical organic chemists have long been concerned with the importance of polarizability in organic reactions (Ingold, 1969). It was, of course, recognized that this property of organic compounds is important for their interaction with proteins (Pauling and Pressman, 1945), but few systematic attempts were made to explore this molecular property until the study of Agin et al. (1965). These workers advanced evidence to support the idea that the interaction of organic ligands with proteins could be correlated by the relationship: log 1lC

=

kcrl

+ constant

(17)

where a, the polarizability, was modeled using electronic polarizability

ENZYME STUDY AS STRATEGY I N DRUG DESIGN

61

obtained from the Lorentz-Lorenz equation:

a=MR=

l)*M (n’ + 2)sd

(n’ -

In this expression, n represents the index of refraction, M the molecular weight, and d the density. The MR has long been recognized as an additive-constitutive property of organic compounds (Ingold, 1969). In Eq. (17), I is the ionization potential, and k is a proportionality constant obtained via the method of least squares. In testing their equation, Agin et al. (1965) studied the narcotic action of a group of miscellaneous compounds on frog muscle. This was an unfortunate choice since nerve membranes have a very high lipid content and ligand interactions in these systems are much better correlated (Leo et al., 1969) with log P or P. That the ionization potential (I) in Eq. (17) is of little importance has been shown (Hansch, 1971b) by means of the following equations: -log MBC = 0.010cJ - 3.99 -log MBC = 0 . 0 8 2 ~-~ 3.67

n

r

S

39 39

0.994 0.987

0.238 0.342

(19) (20)

Equation (19) “explains” only 1.4% more of the variance in MBC (minimum nerve-blocking concentration) than Eq. (20). Further studies with I must be made before its true importance can be assessed. The first comparison (Leo et al., 1969) of MR and other physical constants such as parachor suggested that MR was simply mimicking log P or T in an inferior way. More recent systematic studies comparing hydrophobic constants with M R strongly indicate that MR does represent a different type of interaction which is best seen with purified proteins (Hansch and Coats, 1970; Hansch and Silipo, 1974; Silipo and Hansch, 1974, 1975; Hansch and Yoshimoto, 1974; Yoshimoto et al.,

1975). A recent study (Loontiens et al., 1973) of the binding of 19 4-Xphenyl-P-D-glucosides by the protein concanavalin A yields the following correlation equations: n r S (21) log M50 = 0 . 9 7 1 ~+ 2.37 19 0.664 0.095 (22) log M5,, = 0.0188MR + 2.23 19 0.954 0.038 The hydrophobic parameter P in Eq. (21) gives a very poor correlation, whereas MR in Eq. (22) gives a good correlation. Although the two parameters are significantly collinear (r2 = 0.5), the vectors are divergent enough to show that MR is far more significant. The F1,17

PARAMETERS FOR INHIBITION

Log 1IC

No. 10 2 30 4 5 6' 7 8 9 10 11 12 13 14 15 16 17" 18 19 20 21 22 23 24 25

Substituent 4-CGH5 3-OCHzCO-N(CHzCHz)zO CCN 3-OCHzCONMez 3-OCH3 4-OCHzCON(Me)C,H5 3-COCHZCI 4-OCHzCONMez 4-COCHZCI 3-CHzNHCOCHzBr 4-CHzCONMez 4-OCHZCO-N(CHz), 3-OCHzCON(Me)C6H, 4-OCHzCONEtZ 4-CHZCONEtz 3-OCHZCONHCGHs 3-CsH5 4-CHZCN H ~-OCH~CGH,-~'-NHCOCH~BI 4-CHZCON(Me)CBH5 C(CH& C ONMe2 3-NOZ 3 4 CHz)zC0CHzC1 3 4 CHz)4C0CH, C1

QI

TABLE 111 OF DIHYDROFOLATE REDUCTASEBY TRIAZINES

Obsd.

Calcd.

4.70 4.85 5.14 5.44 6.17 6.17 6.21 6.26 6.45 6.58 6.63 6.66 6.68 6.72 6.77 6.85 6.85 6.92 6.92 6.92 7.00 7.05 7.07 7.10 7.10

6.998 5.149 6.713 5.187 6.615 7.300 6.487 7.005 6.860 6.136 6.972 7.114 6.738 7.144 7.111 7.122 7.889 6.769 6.633 7.570 7.268 7.042 6.374 6.806 7.518

1 A log

1

2.30 0.30 1.57 0.25 0.45 I. 13 0.28 0.74 0.41 0.44 0.34 0.45 0.06 0.42 0.34 0.27 1.04 0.15 0.29 0.65 0.27 0.01 0.70 0.29 0.42

N

a3

r-4

0.0 -1.39 0.0 -1.36 -0.02 0.0

1.96 0.0 -0.57 0.0 0.0 0.12

-0.16

0.0

0.0 0.0 -0.52 0.0 0.0 0.12 0.0 0.0 0.60 1.96 0.0 0.0

1.29 0.0 0.0 -0.28 0.20 1.20

-1.36 -0.16 0.0 -1.70 -0.72 0.0 -0.36 -0.70 0.0 0.0 -0.57 U.0 0.0

-0.19 -1.20 0.0

0.0 0.0

MR-3 0.103 3.488 0.103 2.583 0.787 0.103 1.618 0.103 0.103 2.743 0.103 0.103 4.554 0.103 0.103 4.092 2.536 0.103 0.103 5.394 0.103 0.103 0.736 2.471 3.430

MR-4 2.536 0.103 0.633 0.103 0.103 4.554 0.103 2.583

1.618 0.103 2.365 3.312 0.103 3.513 3.294 0.103 0.103 1.011 0.103 0.103 4.336 2.830 0.103 0.103 0.103

n

B

52

F 5n

3:

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

4-OCH2CO-N( CHz), 4-CHzC 0-N( CHz C Hz)z0 3-C1, 4-OCHZCONMez 4-(CHZ),CONEt 3-C1, 4-OCHZCO-N(CHz), 4-OCHzCO-N(CHzCHz)zO 4-CHzCON(Me)CHzCfiH5 4-(CHz)zCON(Me)CHzC,H, 4-(CHz),C 0-N(C Hz CHJz 0 ~-(CHZ)ZCON(C~H,)Z 3-(CHz),C,H3-2’,4’-Clz 3-C1, 4-OCHZC O-N(CHz), 3-C1, 4-OCHzC~H4-4’-SOZNMez 3-C1, 4-OCHzCCH4-3’-CN 3-C1, 4-OCHZCsHs 3-O(CHz)30CfiH4-4’-NHCOCHzBr 4-(CHz),C0N(Me)CfiH5 3-C1,4-OCHzCONEtz 3-O(CHz)zOCfiH4-3’-NHCOCHzBr 3-O(CHz)zOC6H4-2’-NHCOCHzBr

~-O(CHZ)ZOC,H~-~’-NHCOCH~B~ 4-O(CHz)zOCfiH4-4‘-NHCOCHzBr 3-C1, 4-OCHzCfiH4-3’-CONMez 3-C1, 4-OCHzC~H4-4‘-S03CsH4-3”-CI 3x1 3-CF3 3-C1, 4-OCH,C,jH,-4’-SO3CfiH4-4”-Cl 3-C1,4-OCHzCO-N(CHzCHz)zO 3-C1, ~-OCH~C,H~-~’-CON(CHZCHZ)ZO 3-Cl, 4-OCHzC+jH4-3’-CO-N(CH,), 3-C1, 4-OCH,CON(Me)C,H,

7.12 7.12 7.16 7.28 7.29 7.29 7.30 7.31 7.32 7.35 7.45 7.47 7.48 7.51 7.52 7.55 7.56 7.64 7.64 7.66 7.66 7.70 7.72 7.72 7.76 7.76 7.77 7.85 7.85 7.85 7.89

7.184 7.108 7.573 7.181 7.682 7.141 7.337 7.407 7.178 7.320 7.648 7.752 7.975 7.748 7.668 7.810 7.337 7.712 7.810 7.558 7.558 7.530 7.938 8.278 7.201 7.318 8.278 7.708 8.074 8.047 7.868

0.06

0.0

0.01 0.41 0.10 0.39 0.15 0.04 0.10 0.14 0.03 0.20 0.28 0.49 0.24 0.15 0.26 0.22 0.07 0.17 0.10 0.10 0.17 0.22 0.56 0.56 0.44 0.51 0.14 0.22 0.20 0.02

0.0

0.71 0.0 0.71 0.0 0.0 0.0 0.0 0.0 5.55 0.71 0.71 0.71 0.71 1.77 0.0 0.71 1.77 1.27 1.27 0.0 0.71 0.71 0.71 0.88 0.71 0.71 0.71 0.71 0.71

-0.32 -1.70 -1.36 -0.21 -0.72 -1.39 0.43 0.93 -1.20 0.80 0.0 -0.32 0.88 1.09 1.66 0.0 0.31 -0.36 0.0 0.0 0.0 1.27 0.15 3.92 0.0 0.0 3.92 -1.39 0.13 0.80 0.12

0.103 0.103 0.603 0.103 0.603 0.103 0.103 0.103 0.103 0.103 5.394 0.603 0.603 0.603 0.603 6.555 0.103 0.603 6.555 6.090 6.090 0.103 0.603 0.603 0.603 0.502 0.603 0.603 0.603 0.603 0.603

3.777 3.270 2.583 3.759 3.312 3.488 4.801 5.266 3.735 4.688 0.103 3.777 5.268 3.749 3.219 0.103 4.801 3.513 0.103 0.103 0.103 6.090 5.021 7.286 0.103 0.103 7.286 3.488 5.926 5.750 4.554 (continued)

TABLE 111-Continued Log 1IC

1

No.

Substituent

Obsd.

Calcd.

IA log

57 58 59 60 61 62 63 64” 65 66 67 68 69 70 71 72 73 74 75 76 77 78

4-OCHzCONHC6H, 4-(CHz)ZCOCHzCl 3-OC6H4-4’-NHCOCHzBr 3-C1, 4-(CH&C,H, 3-C1, 4-OCHzC6H4-3’-CONHC6H, 3-CHzC6H, 3-C1, 4-OCHzC6H4-3’-CO-N(CH& 4-CHzC6HS 3-C1, 4-OCHzC6H4-4‘-SO3C6H4-3”-CF3 3-C1, 4-0CHzC6H4-3’-CON(Me)C6H, ~-O(CHZ)ZOC,H~-~‘-NHCOCH,B~ 3-C1, 4-OCH,C6H4-3’-CONEtz 3 4 , 4-OCHzC6H4-4’-SO3C6H, 3-C1, 4-OCHzC6H4-4’-SO3C6H4-3”-CN 3-C1, 40CH2CcH4-4S03C6-3”,4”-C1, 3-(CHz)zC6H4-4’-NHCOCHzBr 3-C1, 4-OCHzCtjH4-4’-SO,C6H4-P”-CF3 3-(CHz),C6H4-4’-NHC0CHzBr 3-C1, 4-OCH,C,H4-4‘-SO,C6H4-4“-CN 3-C1, 4-OCH~C6H4-4’-SO~C6H,-4”-OCH3 3-C1, 4-OCHzC6H4-4’-SO3C6H4-4”-F 3-C1, 4-0CH2CcH4-4‘-S03C6H4-2”-OCH3

7.89 7.92 7.92 7.% 8.00 8.00 8.02 8.05 8.09 8.12 8.13 8.14 8.20 8.24 8.25 8.26 8.33 8.38 8.39 8.40 8.40 8.40

7.231 6.988 7.783 7.844 8.164 7.908 8.117 7.068 8.262 8.234 7.558 8.077 8.203 8.282 8.353 8.004 8.262 8.184 8.282 8.305 8.201 8.305

0.66 0.93 0.14 0.12 0.16 0.09 0.10 0.98 0.17 0.11 0.57 0.06 0.00 0.04 0.10 0.26 0.07 0.20 0.11 0.10 0.20 0.10

c/

T-3 0.0 0.0 1.71 0.71 0.71 2.01 0.71 0.0 0.71 0.71 1.27 0.71 0.71 0.71 0.71 2.29 0.71 3.67 0.71 0.71 0.71 0.71

T-4 0.60 0.20 0.0 4.13 2.15 0.0 1.20 2.01 4.09 2.15 0.0 1.15 3.21 2.64 4.63 0.0 4.09 0.0 2.64 3.19 3.35 3.19

MR-3 0.103 0.103 4.943 0.603 0.603 3.001 0.603 0.103 0.603 0.603 6.090 0.603 0.603 0.603 0.603 5.640 0.603 6.632 0.603 0.603 0.603 0.603

MR-4 4.092 2.471 0.103 4.394 6.530 0.103 6.214 3.001 7.185 6.992 0.103 5.950 6.786 7.316 7.786 0.103 7.185 0.103 7.316 7.470 6.775 7.470

n 0

m

3z F 5 2

79 80 81 82 83“ 84 85 86 87 88 89 90“

3-(CHz)4C+jH4-3’-NHCOCH2Br 8.41 3-C1, 4-OCHzC6H4-4’-SO3C8H4-3’‘-CH3 8.44 3-C1, 4-0CHzCsH4-4’-S03CfiH4-3’’-F 8.46 3-CL ~-OCHZC~H~-~’-SO~C,H,-~”-OCH~ 8.52 3, 4C12 8.54 3-C1, 4-OCHZC~H4-4’-SO3CGH4-2”-Cl 8.62 3-C1, 4-0CHzCsH4-4‘-S03CfiH4-4’’-CON(CH3)2 8.62 3-CL 4-OCHzC6H4-4’-SO3C,jH,-2”-CON(CH3)28.63 3-C1, 40CH~C,H4-4’-SO~C,H4-Z”-CN 8.70 3-C1, 4-OCHzCsH4-4’-S03CsH4-2’‘-F 8.74 3-CL 4-OCHzC6H4-4’-S03C,H4-3”-CON(CH3)z 8.76 4-(CH2)4CGH3-2’,4’-C12 9.21

These points not used in deriving Eq. (23).

8.184 8.272 8.201 8.305 7.276 8.278 8.473 8.473 8.282 8.201 8.473 7.426

0.23 0.17 0.26 0.21 1.26 0.34 0.15 0.16 0.42 0.54 0.29 1.78

3.67 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.0

0.0 3.77 3.35 3.19 0.71 3.92 1.70 1.70 2.64 3.35 1.70 5.55

6.632 0.603 0.603 0.603 0.603 0.603 0.603 0.603 0.603 0.603 0.603 0.103

0.103 7.248 6.775 7.470 0.603 7.286 8.588 8.588 7.316 6.775 8.588 5.394

M

5 id

E (fl

c3

2

Y

66

CORWIN HANSCH

value for Eq. (21) is only 13.4, whereas for Eq. (22) it is 172. In an earlier study of phenylglucoside binding to concanavalin A, Poretz and Goldstein (1971) reported a good correlation between binding and T ; however, for the set of substituents they studied, T and MR are so collinear that essentially the same correlation is obtained with either parameter. Concanavalin A has two binding sites for carbohydrates; because these are highly polar moieties, it is not surprising that the region around the sites is apparently not hydrophobic as was earlier concluded (Poretz and Goldstein, 1971). A good example to illustrate the utility of T and MR in the formulation of QSAR comes from the work of B. R. Baker and his students on the inhibition of dihydrofolate reductase by the type of diaminotriazenes shown in structure VIII (Hansch and Silipo, 1974). Many regression equations were formulated (Hansch and Silipo, 1974) from the data in Table I11 from which

n

r

83

0.905

S

log 1/C = 0 . 8 9 ~ 3- 0 . 1 3 ~ 3 ~

+ 0.15MR-4 + 6.61

0.328 (23)

emerged as the best equation. By “best” equation is meant that equation having the lowest standard deviation, all terms of which are justified by the F test. In Eq. (23) C is the molar concentration of triazine which caused 5wo inhibition of the enzyme, T-3 is the hydrophobic parameter for substituents in :he meta position of the phenyl ring, and MR-4 refers to the group molar refractivity of substituents in the para position of the benzene moiety. Equation (23) was formulated using 83 of the 90 data points of Table 111. This table is included to give the reader a view of the enormous variation in the substituents utilized by Baker; in fact, many of the “substituents” are larger than the “parent” molecule. Considering the gross structural changes in the derivatives, the quality of the fit of the data by Eq. (23) is indeed surprisingly good. Unfortunately, despite the great variation, there is still considerable collinearity between the “independent”

67

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

variables employed in this study as can be seen in Table IV. It is seen from Table IV that r2 for correlation between T-3 and MR-3 is 0.37 and that between u-4 and MR-4 is 0.50. Also, there is a high correlation between MR-4 and a-4. Although this lack of orthogonality among the vectors does introduce a certain amount of ambivalence into the analysis, there is little doubt about the difference in meta and para space (the space in or on the enzyme near the 3- and Cpositions of the phenyl ring where the substituents fall). Meta space appears to be hydrophobic since it is much better correlated by T than by MR. The “other” nature of para space leads us to suggest that it is polar in character. Desolvation of substituents does not appear to be the primary cause of binding in para space. Two processes may be involved in inhibiting the activity of the enzyme. The hydrophobic binding in meta space and binding via dispersion forces and polarization in para space serve to attach the inhibitor to dihydrofolate reductase. This simple occupation of the active site causes inhibition. In addition, binding in these two areas could easily produce deleterious conformational changes that could cause inhibition. These two processes are likely to be highly collinear and, in fact, this has been demonstrated for 7r in the binding of ligands by albumin (Helmer et al., 1968). The high correlation between MR-4 and u-4 was not anticipated and again emphasizes the supreme importance of experimental design before undertaking a structure-activity study. Although there is relatively little collinearity between the global sets (Hansch et al., 1973a) of u and MR constants, any given two subsets may be highly collinear. In the present instance, the electronic effects of substituents related to u are deemed to be of little importance. The reasoning behind this assumption is that xu for substituents in the 3- and 4-positions is not a significant variable. TABLE IV MATRIXFOR PARAMETERS FORMULATION OF EQ. (23)

SQUARED CORRELATION

lr-3 ~~~~

lr-3 w-4 MR-3 MR-4 0-3 a-4

STUDIED IN THE

7r-4

MR-3

MR-4

a-3

a-4

0.01 1.00

0.37 0.04 1.00

0.02 0.50 0.38 1.00

0.00 0.31 0.09 0.28 1.00

0.02 0.17 0.32 0.63 0.21 1.00

~~

1.00

68

CORWIN HANSCH

Since u is not a position-dependent parameter in the sense that m and MR are, and since it was established that xu is orthogonal with respect to W R as well as to MR-3 and MR-4, it is safe to conclude that MR-4 and not u-4 is significant. Baker’s interest in studying dihydrofolate reductase was to find inhibitors of this enzyme that could be used for cancer chemotherapy. He was successful in this in that compound 55 of Table I11 was found to be highly active against leukemia and is now undergoing clinical trials. Many of the molecules of Table I11 showed very low activity against leukemia in mice; it seems most likely that this is often due to their excessive lipophilicity. Overall lipophilicity of drugs is of the highest importance in determining their movement through an organism (Hansch and Clayton, 1973); this has been confirmed for antitumor drugs as well (Hansch et al., 1972b; Montgomery et al., 1974). The QSAR of Eq. (23) can be employed to circumvent this problem. Taking the partial derivative of Eq. (23) with respect to m-3, setting this to zero and solving, we obtain the optimum value of 3.5 for m-3. Hence, suitable substituents (X) with constants near this value should be placed in the 3-position. Since functions in the 4-position do not have to interact with hydrophobic space, quite hydrophilic functions can be. placed in this position to maintain a low overall lipophilicity to expedite the randomwalk process as well as to minimize drug destruction by microsomal oxidation (Martin and Hansch, 1971; Hansch, 1972b). Compounds of the following type should be explored:

Substituent Y in this structure should be an ionic function such as -COO-, OSOp-, or -N+(CH3),, to provide overall low lipophilicity. The bridge could be constructed of functions such as -(CHz)x-, -O(CH,)X-, -O(CH,),O-, or NH(CH,),-. The length of the bridge could be adjusted to modulate lipophilicity. However, one should keep Baker’s “bridge principle” (Baker, 1967) in mind. The thought behind this principle is that, given sufficient maneuverability, a reactive function on the end of a bridge has the possibility of finding a binding site that may vary

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

69

between isozymes and, hence, provide the possibility of selectively inhibiting enzymes from the pathogen. The role of MR in the formulation of a QSAR is still uncertain and on somewhat shaky ground. The uncertainty pertains to the meaning one should attach to M R . Since MR is, to a considerable extent, a measure of volume, a negative coefficient with this term would suggest steric hindrance. Actually, positive coefficients are usually found, suggesting that M R is involved in the binding of ligand and macromolecule. Although this binding can be shown in certain instances to differ from that modeled by qr, its true nature is still unclear.

D. ELECTRONIC INTERACTIONS Although there are many examples where electronic effects of substituents have been shown to be important in ligand interaction with enzymes (Hansch et al., 1965, 1973c; Hansch, 1971b, 1972c; Fujita, 1972; Hulbert, 1974; Dupaix et al., 1973; Cammarata and Rogers, 1972; Kirsch, 1972), our concern in this section is with the use of factored u constants. Factoring the electronic effect of substituents into an inductive and resonance component provides more insight into the role of the electronic interaction (Unger and Hansch, 1973; Swain and Lopton, 1968). There are almost no suitable sets of data in the literature to illustrate this point, but the excellent study of Kakeya et al. (1969) can be employed. These workers made a study of the inhibition of carbonic anhydrase by sulfonamides:

I

II

By using regression equations, they established the importance of the hydrophobic role of substituents. We have rederived their equation in qr and u by using their data at 15": n r S log 1/Ki = 0 . 2 h 0 . 8 3 ~ 0.48 16 0.959 0.188 (24)

+

+

The slight difference between their constants and those of Eq. (24) is due to rounding errors. Our constants were rounded to two places. The

70

CORWIN HANSCH

u term in Eq. (24) can be factored as follows: n

r

S

16

0.974

0.173

log l/Ki = 0.17(+-0.21)~

+1.05(*0.56)9-3 - 0.36(+ 1.2)3-3 1.22(+0.50)9-4 0.53(+0.40)3-4

+ +

(25)

It is clear from the confidence intervals with the 3 - 3 coefficient that this term is not significant. Also, the coefficients with 9 - 3 and 9 - 4 are essentially the same so that by merging these two variables and dropping 3-3, we obtain

n

r

16

0.971

S

log l/Ki = 0.18(40.19)~

+ 0.57(+0.36)3-4 + 1.07(+0.35)9-3’4 + 0.36(+0.16)

0.165

(26)

Equation (26) gives a clearer view of the substituent effects. Hydrophobicity is of relatively little importance although the T term in Eq. (26) is justified by the F test (Fl,lz= 4.3). Variable 3 - 4 is the next most important term, but the most important term is 9. The inductive effect of the substituents is no doubt influencing the protons of the NHz function, and it is likely that this is one of the rate-controlling interactions of these inhibitors. The fact that the coefficient with 9 - 4 is much smaller and has greater confidence limits is not surprising if it is the electronic effect on the NH, which is important. The intervening SOz dampens the resonance effect. Factoring electronic effects into inductive and resonance components gives one additional insight into the mechanism. Incidentally, T gives a better correlation in Eq. (26) than M R .

E. STERIC INTERACTIONS Whereas MR can be used to study the problem of bulk tolerance, Taft’s E, parameter can be used to explore more specific steric effects. There are now a number of cases where E, has been shown to give good correlations (Hansch and Coats, 1970; Kutter and Hansch, 1969; Hansch et al., 1965; Hansch, 1972c; Silipo and Hansch, 1974; Cohen and Mannering, 1973; Dupaix et al., 1973; Hansch, 1970). Although not involving an enzymic system, a most instructive example of the use of E, comes from Pauling and Pressman’s studies of hapten-antibody

71

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

TABLE V 0-0

/ \o--.m-

.=.

BINDINGAFFINITY FOR

xA

Log Kre,

X

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

2-NO, 2, Cdi-NO, 2-BR 2-Me 2-OH, 3-NO2 2-OH, 5-N02 2-c1 3-NHZ 3-NO, 3-OH 2, Cdi-OH 3-Me 4-NH2 H 3-1 4-OH 4-F 3-Br

4ME 4-C1 CBr 4-N02

Obsd.

Calcd.

-2.26

-2.288 - 1.070 -1.133 -1.201 -0.615 -0.615 -0.972 -0.148 -0.148 -0.148 -0.349 -0.148 0.147 - 0.148 -0.148 0.118 0.074 -0.148 0.450 0.320 0.410 1.070

- 1.22

-1.13 -1.08 -0.77 -0.72 -0.70 -0.48 -0.40 -0.37 -0.29 -0.18 -0.06 0.00 0.00 0.06 0.06 0.10 0.41 0.57 0.70 1.06

HAPTENS WITH ANTIBODIES

/

1 lAlogc1

E," 0.03 0.15 0.00 0.12 0.15 0.10 0.27 0.33 0.25 0.22 0.06 0.03 0.21 0.15 0.15 0.06 0.01 0.25 0.04 0.25 0.29 0.01

- 1.28 -1.28 0.08 0.00 0.69 0.069 0.27 1.24 1.24 1.24 0.69 1.24 1.24 1.24 1.24 1.24 1.24 1.24 1.24 1.24 1.24 1.24

ESP

1.24 -1.28 1.24 1.24 1.24 1.24 1.24 1.24 1.24 1.24 0.69 1.24 0.63 1.24 1.24 0.69 0.78 1.24 0.00 0.27 0.08 -1.28

interactions (Pressman and Grossberg, 1968). In their classic study, diazotized p-aminobenzoic acid was allowed to react with albumin. The resulting protein, when injected into rabbits, produced antibodies that were isolated in partially purified form. Allowing the antibodies to react with the antigen [alb~min-(N=N-C~H~Coo-)~] resulted in the formation of a precipitate; however, if an X-c6H4COO- hapten was first added to the antibody, prevention of union of antibody and antigen could be achieved. Pauling and Pressman calculated a Krel for the affinity of hapten and antibody for a number of benzoate ions (Table V). This parameter is in part a measure of the affinity between hapten and antibody, but it is also a measure of the hapten's ability to prevent union of antibody and antigen. In an analysis of Pauling and Pressman's

72

CORWIN HANSCH

results (Kutter and Hansch, 1969), it was found that Krel depended almost entirely on the E,-defined characteristics of the substituents on the hapten. The essential results are contained in Eqs. (27) and log Krel = 0.86E," + O.O8E," - 0.45ESp - 0.69 log Krel = 0.85E," - O.48ESp- 0.60

n

r

22 22

0.974 0.970

S

0.177 (27) 0.188 (28)

(28). Superscripts 0, m, and p in these equations refer to substituents in the ortho, meta, and para positions of the benzoate ions. Parameters T , u,and M R were also studied and found to be of little or no significance. Comparison of Eqs. (27) and (28) shows that meta substituents do not interact with the antibody. The situation is like that discussed above for enzymes. Equation (28) accounts for 94% of the variance in log Krel. The coefficients with the E, terms are particularly interesting; the positive value with E," indicates that large groups in this position give poor K,,, constants. This is probably the result of two types of interactions of ortho substituents. Intramolecularly, these groups can twist the carboxylate group out of the plane of the benzene ring and thus, apparently, lower affinity of the haptens (Pauling and Pressman, 1945; Pressman et al., 1944). Bulky ortho substituents may also interact with the antibody to prevent the hapten from binding to the antibody. It should be recalled that the larger the substituent, the more negative is its E, value (Taft, 1956). Hence the negative coefficient with ESP indicates that large groups in the para position are effective in preventing union of antibody and antigen. This could be viewed as a kind of "corking" action of para substituents of the hapten. Antibody interactions are considered to be among the most specific in biochemical systems; hence the high correlation using E, constants is a good test of their utility for other sterically demanding systems.

F. INDETERMINATE INTERACTIONS Bruice et al. (1956) first suggested deriving de novo substituent constants to show the additive character of substituent effects and to aid in rationalizing chemical structure with biological activity. Later, Free and Wilson (1964) generalized this approach (Craig, 1972). The FreeWilson philosophy is embodied in the following equation: biological activity

=

&Xi

+ constant

(29)

In this expression Xirepresents a given substituent such as 3-C1, 4-C1,

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

73

3-CH3, or 2-CH3, whereas ai represents the numerical contribution of each substituent to the total biological activity of a given compound. The constant refers to the invariant portion of the molecule. The chief limitation of this approach is the necessary assumption of complete additivity of substituent effects. For example, a 4-C1 function is assumed to always make the same contribution to activity, regardless of how one might be varying substituents in, say, the 2- and 3-positions. Since it is known that activity depends on lipophilicity in a parabolic way, the Free-Wilson technique can yield misleading results in the hands of inexperienced workers (Hansch, 1973). When one is studying a set of congeners for which substituent constants are not available, the Free-Wilson method is a valuable technique. The Free-Wilson approach carries the use of indicator variables to the limit where all terms in the QSAR are, in essence, indicator variables (Daniel and Wood, 1971b). We have found that the use of indicator variables, along with physicochemical constants, greatly extends one's reach in the formulation of QSAR (Hansch, 1971a; Silipo and Hansch, 1974; Hansch and Yoshimoto, 1974; Yoshimoto et al., 1975; Martin, 1970; Hansch and Lien, 1968). The QSAR

+ 0.69MR-3 + 1.1OEs-2 + 1.341 + 3.75 0 . 4 6 ~ 4+ 0.5WR-3

n

r

32

0.928

0.363 (30)

18

0.961

0.297 (31)

S

log 1IC = 0 . 3 8 ~ - 4

log 1/C

=

+ 1.33 + 5.12

were formulated (Silipo and Hansch, 1974) in a study of 9-Xphenylguanines inhibiting guanine deaminase. Equation (30) was derived with data obtained from rat liver enzyme, and Eq. (31) with data from Walker tumor enzyme. In the case of Eq. (30), a number of congeners were substituted in the ortho position; hence the need for E,-2 in this equation. No such data were available for Eq. (31); hence there is no E, term in this result. The indicator variable Z is given a value of 1 for four

74

CORWIN HANSCH

cases where an OR group is present in the 4-position [R = H, CH3, C2H5, (CH,),C,H,]. Oxygen in the 4-position produces a twenty-fold increase in activity over what one would expect from hydrophobicity alone (T-4). It was not possible to explain this activating effect in terms of c,c+,or steric effects; it seems most likely that a special interaction of the lone-pair electrons on oxygen is involved. In the case of Eq. (31), only two such congeners were tested; nevertheless, the same coefficient is found with Z. The equations are remarkably similar, indicating similarity of enzyme from the two sources. Equation (32) correlates (Hansch and Glave, 1972) phenethanolamine inhibitors of N-methyl transferase. In Eq. (32) Z has the value of 1.00 for four cases where 3-OMe is present. In Eq. (30), OR enhances inhibitory power whereas it reduces this power in Eq. (32). Methoxy is a common function in many natural products (for example, mescaline) and it may play some special role which as yet we do not understand.

+ 0.99~-2,3 + 1 . 4 1 h - 1.011 + 2.55

n

r

32

0.940

S

log 1/C = 0.48E8-2

0.288 (32)

A more complex example of the use of indicator variables comes from a study of the inhibition of complement with benzamidines of the type

(XII).

- Parameterized by I- 1

Equation 33 correlates data from 108 variations on structure XI1 (Hansch and Yoshimoto, 1974):

+ 1.071-1 + 0.521-2 + 0.431-3 + 2.42

n

r

108

0.935

S

log 1/C = 0.15MR-1,2

0.258 (33)

In Eq. (33), 1-1 is given a value of 1.00 for all derivatives in which the following bridges were used: -O(CH,),O-, -(CH2),, -0(CH2),0-,

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

75

and -O(CH&. The MR-1,2 refers to X and Y; 1-2 is an indicator variable for the presence of a pyridine moiety as the second ring instead of phenyl, and I-3 was used to account for the special activating ability of functions of the type NHC=O(X)C,H, when attached to the second ring. X stands for zero, NH, CH,, NHCH,CH,, or CHZO. These functions, when placed in the 3-position, increased activity by about 2.5 times that expected by MR alone. An enormous amount of structural variation in 108 molecules along with the biological activity can be reduced to a single line of information [Eq. (33)] by using these indicator variables. New derivatives can be tested against this equation and the equation can be further developed to keep score in a growing structure-activity study. Molecular properties that have not been considered in the above discussion are hydrogen bonding, charge transfer, and dipole moments. Present experience suggests that variation in these properties may parallel the electronic effects of substituents. Tute (1970) has formulated a very interesting enzymic QSAR for the inhibitor of viral neuraminidase by X-l-phenoxymethyl-3,4-dihydroisoquinolines in which the group dipole moment plays an important role. The wide variety of substituent effects expected to occur between ligand and enzyme means that separating the different effects is not an easy problem and that proper experimental design is crucial for success in such an undertaking (Hansch et al., 1973a; Unger and Hansch, 1973).

-O(CH&-, -O(CH,),-O-,

VI. Nature of Receptors Our concept of the receptor sites keeps developing (Rang, 1971, 1973). Fischer’s early analogy of enzyme and ligand as lock and key has largely ceased to dominate thinking in structure-activity studies. The many examples discussed and cited above clearly indicate that enzymic receptors have a large amount of flexibility; as a crude analogy, one might think of them as being held together by rubber bands. However, the situation is more complex than this as demonstrated recently by Smith (Smith and Hansch, 1973; Smith et al., 1974). He has shown that hydrophobic ionic inhibitors of chymotrypsin cause a rapid initial inhibition of enzyme activity and then a slow continual decrease in activity until eventually all activity is destroyed. What is most interesting, however, is that the slow destruction of activity is brought about by a concentration of inhibitor which produces less than one molecule of bound inhibitor per enzyme molecule. In the case of chymotrypsin, evidence was presented to show that this process occurs at a

76

CORWIN HANSCH

TABLE VI INHIBITION OF VARIOUS TYPESOF DIHYDROFOLATE REDUCTASE BY TRIMETHOPRIM

Enzyme source Escherichia coli Staphylococcus aureus Proteus vulgaris Rat liver Guinea pig liver Rabbit liver Human liver

Molar concentration causing 50% inhibition ( X 10s)

0.5 1.5

0.5 26,000 2,000 37,000 30,000

hydrophobic control site outside of the active site. This is not a necessary condition for the process; deactivation could just as well occur at the active site. The mechanism whereby less than one bound mole of inhibitor per mole of enzyme can in time bring about complete inactivation of an active site has great importance for the study of enzymes for drug design. The process is best explained by considering th'at, when a ligand is adsorbed at a control site or a receptor site, it produces a small conformational change after desorption. If the relaxation time of the macromolecule is slow, it will not completely return to the original state before a second molecule is adsorbed and an added increment in conformational distortion produced. This gradual change in activity can continue until all activity is destroyed. This means that in instances where this process can occur, the Michaelis constant K , or the inhibition constant K i are not really constant but time-dependent. The implications of this for receptor theory have been developed (Smith and Hansch, 1975). If this type of time-dependent deactivation of a receptor occurs in the whole animal in a manner similar to that in vitro, then in vitro data will be applicable to in vivo work. If, on the other hand, the enzyme is more stabilized against such time-dependent deactivation by the constellations of macromolecules surrounding it in living tissue, then results obtained in vitro could be misleading.

VII. Therapeutic Index Problem The above discussion has centered on a single receptor site. Not always, but in general, one is faced with inhibiting a receptor site in the

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

77

pathogen without equivalent inhibition of the same receptor in the host. That such selective enzyme inhibition can be achieved has been elegantly demonstrated by Hitchings and his colleagues (Burchall, 1973). The selective inhibition of dihydrofolate reductase from different sources by trimethoprim is shown in Table VI. The enzyme from Escherichia coli is 60,000 times more sensitive than the human enzyme. This provides an enormous therapeutic index and trimethoprim (XIII), especially when used with a sulfa drug, is one of

the most important antibacterial drugs to be developed in recent years (Hitchings, 1972, 1973; Roth et al., 1962).

VIII. Conclusion The work of Hitchings (1969a,b) and Baker (1967) has brilliantly illustrated the importance of the study of enzymes as starting points in drug research. The advances in biochemistry and molecular biology will continue to be constant sources of new critical points where chemotherapy can be applied to inhibit selectively the molecular processes of the pathogen invading a host. The concern of this chapter has been to outline the type of molecular interactions expected between ligand and enzyme and the means for differentiating them. Our thesis is that through the careful formulation of QSAR for inhibitors of enzymes from both host and pathogen, one can expect to work out the maximum therapeutic index for selective inhibition of critical molecular processes. A clear understanding of the in vitro situation will be of great help in designing drugs for in vivo work. Of course the work of Baker, Hitchings, and others (Rando, 1974) systematically studying the inhibition of enzymes has not gone unnoticed by drug researchers. Many attempts to transfer molecules from in vitro studies to in vivo drugs have been most disappointing; it is highly important to learn about the underlying reasons for these failures. Although metabolism will always be a problem, our increasing understanding of which kind of moieties are sensitive and which are not sensitive to metabolism will do much to help us cope with this problem.

78

CORWIN HANSCH

A point that is often overlooked in making in vitro inhibitors is that increasing hydrophobicity will often greatly increase inhibitory power. In in vitro work the inhibitor simply makes a one-step partitioning from the solution onto the enzyme, whereas in the whole animal the enzyme inhibitor must make a very complex random walk from the site of introduction to the sites of action. Optimum lipophilic character for this process is crucial; there is little point in superoptimal lipophilic character (Hansch and Clayton, 1973). One should attempt to get some idea of what ideal lipophilicity is for the system early in an enzyme study. It is clear from a perusal of the work considered in Section V,F that quite potent in vitro inhibitors can be made by simply adding very large moieties; it is highly unlikely that such an effect will carry over to in vivo situations when the activity of such moieties is simply correlated by M R . The activity correlated by MR appears to be of the most nonspecific type. Every macromolecule encountered by an inhibitor in a living system will have large polar areas (especially on its surfaces), and there is little or no chance for selectivity. Highly specific polarizability could be used to enhance selectivity, but this would not correlate with M R . Its role might be established by the use of indicator variables. Although there have been many failures in the transfer of in vitro studies to in vivo systems in the past, the author is most optimistic about the future of such work. A great deal of experience from the past decade is now at our disposal. The use of regression analysis will enable us to delineate the causes for failure, and steps can be rationally taken to overcome these difficulties. REFERENCES Agin, D., Hersh, L., and Holtzman, D. (1965). Proc. Nut. Acad. Sci. U.S. 5 3 , 952-958. Anderson, R. A., and Graves, D. J. (1973). Biochemistry 12, 1895-1900. Baker, B. R. (1967). “Design of Active-Site-Directed Irreversible Enzyme Inhibitors.”Wiley, New York. Bartinek, K., Dorovska, V. N., and Varfolomeev, S.D. (1972). Biokhimiya 37, 1245-1250. Biel, J. H., and Martin, Y. C. Advan. Chem. Ser. 1 0 8 , 8 1 (1971). Bruice, T. C., Kharasch, N., and Winzler, R. J. (1956). Arch. Biochem. Biophys. 62, 305317. Burchall, J. J. (1973). J . Znfec. Dis. 128, Suppl. S4374439. Cahn, R. S.,Ingold, C. K . , and Prelog, V. (1966). Angezu. Chem., Znt. Ed. Engl. 5 , 38% 415. Cammarata, A., and Rogers, K. (1972). I n “Advances in Linear Free Energy Relationships” (N. B. Chapman and J. Shorter, eds.), pp. 421425. Plenum, New York. Campey, L. H., Hyde, E., and Jackson, A. (1970). Chem. Brit. 6 , 4 2 7 4 3 0 .

ENZYME STUDY AS STRATEGY I N DRUG DESIGN

79

Chapman, N. B., and Shorter, J., eds. (1972). “Advances in Linear Free Energy Relationships.” Plenum, New York. Coats, E., Glave, W. R., and Hansch, C. (1970). J. Med. Chem. 13, 913-919. Cohen, G. M., and Mannering, G. J. (1973). Mol. Pharmacol. 9, 383-397. Craig, P. N. (1972). Aduan. Chem. Ser. 114, 115-129. Craig, P. N., and Hansch, C. (1973). J. Med. Chem. 16, 661-667. Daniel, C., and Wood, F. S. (1971a). “Fitting Equations to Data.” Wiley (Interscience), New York. Daniel, C., and Wood, F. S. (1971b). “Fitting Equations to Data,” pp. 55, 169, 203. Wiley (Interscience), New York. Dayal, S. K., Ehrenson, S., and Taft, R. W. (1972). J. Amer. Chem. SOC. 94, 9113-9122. Draper, N. R., and Smith, H. (1966). “Applied Regression Analysis.” Wiley, New York. Dupaix, A., Bichet, J. J., and Roucous, C. (1973). Biochemistry 12, 2559-2565. Farrar, D. E., and Glauber, R. R. (1967). Rev. Econ. Stat. 49, 92-107. Free, S. M., Jr., and Wilson, J. W. (1964). J. Med. Chem. 7, 395-399. Fujita, T. (1972). Advan. Chem. Ser. 114, 1-19. Gould, R. F., ed. (1971). Aduan. Chem. Ser. 108. Hamilton, C. L., Niemann, C., and Hammond, G. (1966). Proc. Nat. Acad. Sci. U.S. 55, 664-670. Hansch, C. (1970). J. Org. Chem. 35, 620-621. Hansch, C. (1971a). Ann. N.Y. Acad. Sci. 186, 235-247. Hansch, C. (1971b). I n “Drug Design” (E. J. Ariens, ed.), Vol. 1, p. 333. Academic Press, New York. Hansch, C. (1972a). Cancer Chemother. Rep. 56, 4 3 U 1 . Hansch, C. (1972b). Drug Metab. Rev. 1, 1-13. Hansch, C. (1972~).J. Org. Chem. 37, 92-95. Hansch, C. (1973). I n “Structure-Activity Relationships” (C. J. Cavaillito, ed.), Vol. I, p. 146. Encyclopedia of Pharmacology and Therapeutics, Section 5. Pergamon, Oxford. Hansch, C. (1974). J. Chem. Ed. 51, 360-365. Hansch, C., and Clayton, J. M. (1973). J. Pharm. Sci. 62, 1-21. Hansch, C., and Coats, E. (1970). J. Pharm. Sci. 59, 731-743. Hansch, C., and Deutsch, E. W-.(1966). Biochim. Biophys. Acta 126, 117-128. Hansch, C., and Dunn, W. J., 111 (1972). J. Pharm. Sci. 61, 1-19. Hansch, C., and Glave, W. R. (1972). J. Med. Chem. 15, 112-113. Hansch, C., and Lien, E. J. (1968). Biochem. Pharmacol. 17, 709-720. Hansch, C., and Silipo, C. (1974). J. Med. Chem. 17, 661-667. Hansch, C., and Yoshimoto, M. (1974). J. Med. Chem. 17, 1160-1167. Hansch, C., Deutsch, E. W., and Smith, R. N. (1965). J. Amer. Chem. SOC. 87, 27382 742. Hansch, C., Schaeffer, J., and Kerley, R. (1972a). J. Biol. Chem. 247, 47034710. Hansch, C., Smith, R. N., Engle, R., and Wood, H. (1972b). Cancer Chemother. Rep. 56, 443-456. Hansch, C., Unger, S. H., and Forsythe, A. B. (1973a). J. Med. Chem. 16, 1217-1222. Hansch, C., Leo, A., Unger, S . H., Kim, K. H., Nikaitani, D., and Lien, E. J. (1973b). J. Med. Chem. 16, 1207-1216. Hansch, C., Kim, K. H., and Sarma, R. H. (1973~).J. Amer. Chem. Soc. 95, 6447-6449. Hansch, C., Leo, A., and Elkins, D. (1974). J. Chem. Doc. 14, 57-61. Hanson, K. R. (1966). J. Amer. Chem. SOC. 88, 2731-2745. Hayashi, H., and Penniston, J. T. (1973). Arch. Biochem. Biophys. 159, 563-569. Hein, G. E., and Niemann, C. (1962). J. Amer. Chem. SOC.84, 44874503.

80

CORWIN HANSCH

Helmer, F., Kiehs, K., and Hansch, C. (1968). Biochemistry 7, 2858-2863. Hitchings, G. H. (1969a). Cancer Res. 29, 1895-1903. Hitchings, G. H. (1%9b). J. Amer. Med. Ass. 209, 1339-1342. Hitchings, G. H. (1972). Annu. Rep. Med. Chem. 7, 1-5. Hitchings, G. H. (1973). J. Infec. Dis. 128, Suppl. S433-S436. Hulbert, P. B. (1974). Mol. Pharmacol. 10, 315-318. Ingold, C. K. (1969). “Structure and Mechanism in Organic Chemistry,” 2nd Ed., pp. 142, 1216. Cornell Univ. Press, Ithaca, New York. Jencks, W. P. (1969). “Catalysis in Chemistry and Enzymology,” Ch. 8. McGraw-Hill, New York. Johnson, C. D. (1973). “The Hammett Equation.” Cambridge Univ. Press, Cambridge, England. Kakeya, N., Yata, N., Kamada, A., and Aoki, M. (1969). Chem. Pharm. Bu.11. 17, 25582564.

Kier, L. B. (1971). “Molecular Orbital Theory in Drug Research.” Academic Press, New York. Kirsch, J. F. (1972). In “Advances in Linear Free Energy Relationships” (N. B. Chapman and J. Shorter, eds.), pp. 369-398. Plenum, New York. Kowalski, B. R., and Bender, C. F. (1974). J. Amer. Chem. Soc. 96, 916-918. Kuntz, I. D. (1972). J. Amer. Chem. SOC.94, 856M572. Kutter, E., and Hansch, C. (1969). J. Med. Chem. 12,647-652. Leo, A., Hansch, C., and Church, C. (1969). J. Med. Chem. 12, 766-771. Loontiens, F. G., Van Wauwe, J. P., De Gussem, R., and De Bruyne, C. K. (1973). Carbohyd. Res. 30, 51-62. Lynch, M. F., Harrison, J. M., Town, W. G., and Ash, J. E. (1971). “Computer Handling of Chemical Structural Information.” MacDonald, London. Martin, Y. C. (1970). J. Med. Chem. 13, 145-148. Martin, Y. C., and Dunn, W. J., 111 (1973). J. Med. Chem. 16, 578-579. Martin, Y. C., and Hansch, C. (1971). J. Med. Chem. 14, 777-779. Martin, Y. C., Holland, J. B., Jarboe, C. H., and Plotnikoff, N. (1974). J. Med. Chem. 17, 409-413.

Montgomery, J. A., Mayo, J. G., and Hansch, C. (1974). J. Med. Chem. 17,477-480. Nath, R. L., and Rydon, H. N. (1954). Biochem. J. 5 7, 1-10. Pauling, L., and Pressman, D. (1945). J. Amer. Chem. SOC. 67, 100%1012. Poretz, R. D., and Goldstein, I. J. (1971). Biochem. Pharmacol. 20, 2727-2739. Pressman, D., and Grossberg, A. L. (1968). “The Structural Basis of Antibody Specificity.” Benjamin, New York. Pressman, D., Swingle, S. M., Grossberg, A. L., and Pauling, L. (1944). J. Amer. Chem. Soc. 66, 1731-1738. Rando, R. R. (1974). Annu. Rep. Med. Chem. 9, 234-243. Rang, H. P. (1971). Nature (London) 2 3 1 , 91-97. Rang, H. P. (1973). Brit. J. Pharmacol. 48,475-495. Roth, B., Falco, E. A., Hitchings, G. H., and Bushby, S. R. M. (1962). J. Med. Chem. 5 , 1103-1108.

Schaeffer, H. J., Johnson, R. N., Odin, E., and Hansch, C. (1970). J. Med. Chem. 13, 452-455.

Schiffman, S. S. (1974). Science 18 5, 112-117. Shorter, J. (1973). “Correlation Analysis in Organic Chemistry: An Introduction to Linear Free-Energy Relationships.” Oxford Univ. Press (Clarendon), London and New York.

ENZYME STUDY AS STRATEGY IN DRUG DESIGN

81

Silipo, C., and Hansch, C. (1974). Mol. Pharmacol. 10, 954-962 Silipo, C., and Hansch, C. (1975). Farmaco 3 0 , 3 5 4 0 . Smith, R. N., and Hansch, C. (1973). Biochemistry 12, 49244937. Smith, R. N., and Hansch, C. (1975). (submitted for publication). Smith, R. N., Poindexter, T. P., and Hansch, C. (1974). Physiol. Chem. Phys. 6, 323-331. Swain, C. G., and Lupton, E. C. (1968).J. Amer. Chem. SOC. 90, 432W333. Taft, R. W. (1956). I n “Steric Effects in Organic Chemistry” (M. S. Newman, ed.), pp. 556-675. Wiley, New York. Taft, R. W., and Lewis, I. C. (1958). J. Amer. Chem. SOC.80, 2436-2443. Tanford, C. (1973). “The Hydrophobic Effect: Formation of Micelles and Biological Membranes.”Wiley, New York. Topliss, J. G. (1972). J. M e d . Chem. 1 5 , 1006-1011. ’ h e , M. S. (1970). J. M e d . Chen. 13, 48-51. Unger, S. H., and Hansch, C. (1973). J. M e d . Chem. 16, 745-749. Unger, S. H., and Hansch, C. (1975). Progr. Phys. Org. Chem. (submitted for publication). Unkovskii, B. U., Cherkasova, E. M., Lutsyk, A. I., Bogatkov, S. V., Sokolova, T. D., and Malina, Y. F. (1972). Biokhimiya 3 7 , 1156-1160. Weiner, M. L., and Weiner, P. H. (1973). J. M e d . Chem. 16, 655-661. Wipke, W. T., Heller, S. R., Feldmann, R. J., and Hyde, E., eds. (1974). “Computer Representation and Manipulation of Chemical Information.”Wiley (Interscience), New York. Yoshimoto, M., Hansch, C., and Jow, P. Y. C. (1975). Chem. Pharm. Bull. 23,437-444.

This Page Intentionally Left Blank

The Cephalosporin Group of Antibiotics D . R. OWENS.D. K . LUSCOMBE. A . D . RUSSELL.AND P . J . NICHOLLS Department of Medicine. Welsh National School of Medicine. Cardiff and Welsh School of Pharmacy. University of Wales Institute of Science and Technology Cardifj Great Britain

I . Introduction . I1 . Chemical Aspects A . Production

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B . Structure-Activity

Relationships

. . . . . . . . . . . . .

I11. Antibacterial Activity . . . . . . . . . . . . . . . . . . A . Spectrum of Activity . . . . . . . . . . . . . . . . . B . Bacterial Resistance

. . . . . . . . . . . . . . . . .

C . Mode of Action . . . . . . . . . . . . . . . . . . .

IV. Pharmacology and Toxicology

V.

VI .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . Cephaloridine . . . . . B. Cephalothin . . . . . . C . Cephalexin . . . . . . D . Cephaloglycin . . . . . E . Other Cephalosporins . . . Clinical Aspects . . . . . . A . Urinary Tract Infections . . B. Respiratory Tract Infections . C . Venereal Disease . . . . D. Obstetrics and Gynecology . E . Pediatrics . . . . . . . F. Dermatology . . . . . . G . Ophthalmology . . . . . H . Meningitis . . . . . . I . Endocarditis . . . . . . J . Bones and Joints . . . . Hypersensitivity and Allergenicity A . General Considerations . . B. Conclusions . . . . . . References . . . . . . . . Addendum . . . . . . . . References to Addendum . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . . . . . . . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . . . . . . . . . . .

83 85 a5 87 89 89 96 106 111 112 116 121 124 126 132 132 135 137 139 140 141 142 142 143 144 145 145 147 147 164 170

I . Introduction In 1945. an antibiotic-producing organism was isolated (Brotzu. 1948) from the sea near a sewage outfall off the Sardinian coast . This was 83

84

D. R. OWENS ET AL.

H$Jc(,.HA

H P p ?

0

COOH

0

’

CGOCOCH,

COOH (1)

FIG. 1. 6-Aminopenicillanic acid (I) and 7-aminocephalosporanic acid (11).

later identified as a species of Cephalosporium, which secreted material that inhibited the growth of several gram-positive bacteria but not of yeasts and molds. Subsequent studies on the Cephalosporium sp. were carried out at Oxford and at Clevedon, Somerset, England (see Abraham, 1962). An acidic antibiotic, termed cephalosporin P because it was active mainly against gram-positive bacteria, was isolated from culture fluids of this mold; however, this antibiotic differed from that described by Brotzu (Burton and Abraham, 1951), and a second, chemically unrelated acidic antibiotic was found in culture fluids from which cephalosporin P had been extracted (Crawford et a l . , 1952). This compound showed activity against gram-negative as well as against gram-positive bacteria and was thus named cephalosporin N. It was, however, subsequently found to be a new type of penicillin, since its structure was based on 6-aminopenicillanic acid (6-APA) [Fig. 1 (I)] and not on 7-aminocephalosporanic acid (7-ACA) [Fig. 1 (II)]. Cephalosporin N (penicillin N) was identical with synnematin B (Abraham et a l . , 1955), which was produced by Cephalosporium charticola and by a member of the genus Tilachlidium, the latter species subsequently appearing to be a new species of Cephalosporium salmosynnematum (Gottshall et a l . , 1951; Roberts, 1952; Olson et al., 1953). A third antibiotic, cephalosporin C (Fig. 2) was obtained during the purification of cephalosporin N (Newton and Abraham, 1955, 1956). Cephalosporin C is a true cephalosporin; its antibacterial activity was low, but it was found to be resistant to the enzyme P-lactamase (penicillinase; see Section II1,B) produced by some strains of Staphylococcus aureus, and hence to be of potential clinical importance. In addition, 7-ACA was obtained in low yield by mild acid hydrolysis of cephalosporin C, and this has been an important starting point in the production of new cephalosporins (Hale et a l . , 1961; Loder et a l . , 1961). Further chemical details are provided in Section 11. Extensive investigations by leading pharmaceutical companies in the United States and in Great Britain have resulted in the marketing of some clinically important cephalosporins; many other cephalosporins have been isolated, some of which may be added to this list. The more important cephalosporins are

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

Cephalosporin Cephalosporin C

R, HO,CCH(NH,)(CH,),C~ -

R, - OCOCH,

-&El

Cephaloridine QCHzCOCephalothin

- OCOCH,

Cephalexin

-H

Cephaloglycin

- OCOCH,

Cephacet rile

NGC-CH2CO-

Cefazolin

-OCOCH,

-H

Cephradine

Cephapirin

85

N>

S-CH2CO-

NzN h-CH2CON-=j

I

-OCOCH,

-S

l7ICH3 N-N

Cefamandole

-s:

N-N

II

H3C

FIG. 2. C h e m i c a l structures of t h e m o s t important cephalosporins.

considered in this paper, which deals with the chemical, microbiological, pharmacokinetic, pharmacodynamic, and clinical aspects of these drugs.

II. Chemical Aspects A. PRODUCTION The cephalosporin nucleus [7-ACA; Fig. 1 (11)] shows a close relationship to the penicillin nucleus [6-APA; Fig. 1 (I)], the main

86

D. R. OWENS ET AL.

difference being the six-membered dihydrothiazine ring of the former in place of the five-membered thiazolidine ring of the latter, although both possess the characteristic p-lactam ring. Loder et al. (1961) succeeded in producing 7-ACA in small amounts by removing a-aminoadipic acid from the cephalosporin C molecule (Fig. 2). Later, Morin et al. (1962) described a method of obtaining better yields of 7-ACA from cephalosporin C; their procedure involved the formation of a readily hydrolyzed iminolactone by an intramolecular cyclization of cephalosporin C. Countless cephalosporins have been prepared from 7-ACA and examined for antibacterial activity. Details of the chemical evolution of the cephalosporin antibiotics are well documentated in the papers of O’Callaghan and Kirby (1970) and Flynn (1971). The first significant finding was the introduction of cephalothin (Fig. 2) (Boniece et al., 1962; Chauvette et al., 1963; Godzeski et al., 1963), which is produced by reaction of thiophene-2-acetic acid with 7-ACA. The second derivative of clinical importance, cephaloridine (Fig. 2) (Muggleton et al., 1964; Murdoch et al., 1964) is produced chemically from cephalosporin C by replacing the a-aminoadipic side-chain at position 7 by 2-thienylacetic acid, and the acetoxy group at position 3 by pyridine. Two other cephalosporins which are being used clinically are cephaloglycin and cephalexin (Fig. 2). Cephaloglycin (Wick and Boniece, 1965; Wick et al., 1971) is a synthetic analog of cephalosporin C which is well absorbed after oral administration. Cephalexin (Wick, 1967; Muggleton et al., 1969) is a semisynthetic analog of cephalosporin C in which the a-aminoadipic acid is replaced by phenylglycine, and esterlinked acetic acid is condensed to a simple methyl group. The chemical structure of cephalexin resembles that of ampicillin. More recently, other new cephalosporins have been described: cefazolin (Fig. 2) (Kariyone et al., 1970; Nishida et al., 1970a,b,c; Mine et a l . , 1970a,b; Wick and Preston, 1972) and other heterocyclic cephalosporins (Wick and Preston, 1972) are prepared from 7-ACA by substitution of heterocyclic groups; cephacetrile (CIBA 36,278-Ba), the sodium salt of 7-cyanacetamidocephalosporanic acid, is prepared from 7-ACA and the mixed anhydride of cyanacetic acid and trichloroacetic acid (Knusel et al., 1971). Further details of the chemistry of cephalosporin antibiotics may be found in the reviews of Abraham (1967), Van Heyningen (1967), Manhas and Bose (1969), Barton and Sammes (1971), and Sassiver and Lewis (1970). Enzymic synthesis of cephalexin and cephaloglycin from their corre-

THE C E P H A L O S P O R I N G R O U P OF A N T I B I O T I C S

87

sponding organic acid esters and 7-aminocephem compounds in a single step by means of bacteria of the family Pseudomonadaceae has recently been demonstrated (Takahashi et al., 1972). The preparation of 7-ACA is described by Chauvette et al. (1972).

B. STRUCTURE-ACTIVITY RELATIONSHIPS Space does not permit a comprehensive review of the research carried out on this aspect of the cephalosporins, and what follows summarizes the excellent accounts of structure-activity relationships in the cephalosporins group provided by Abraham (1967), Van Heyningen (1967), and Sassiver and Lewis (1970). Two sites in the cephalosporin molecule [Fig. 1 (11)] have attracted particular interest among chemists. These are (1) the 7-acyl side chain, leading to the production of 7-acylaminocephalosporanic acids, e.g., ring-substituted phenylacetylcephalosporanic acids (analogous to benzylpenicillin) and ring-substituted phenoxyacetylcephalosporanic acids, members of both types being active mainly against gram-positive bacteria; and (2) the 3-acetoxymethyl side chain leading to the production of, e.g., deacetylcephalosporins and deacetoxylcephalosporins. The former have about half the activity of the parent compounds against gram-positive bacteria, such as staphylococci, but much reduced activity against gram-negative bacteria. Removal of the acetoxy group gives 3-hydroxyl derivatives that are less antibacterial than the parent acetoxy compounds (O’Callaghan and Muggleton, 1963). Cephalosporin C, cephalothin, and cephaloglycin (see Fig. 2) all lose antibacterial activity rapidly when incubated with rat liver homogenate, because of removal of the acetoxy group; however, cephaloridine and cephalexin do not possess these labile ester linkages and are, thus, not inactivated (O’Callaghan and Kirby, 1970). Flynn (1971) discusses those portions of the 7-ACA molecule [Fig. 1 (11)] which, when changed, will modify antibacterial activity. Some examples are listed in the following. a. The double bond of the dihydrothiazine ring: reduction of this bond destroys activity. Similarly, activity is lost if there is a migration of the double bond from the 3 4 position from normal A3-cephalosponns to the 2-3 position in A’-cephalosporins. b. C-7: simple epimerization of the hydrogen atom at C-7 destroys activity. c. S atom: oxidation of this, to give a sulfoxide or sulfone, destroys activity.

88

D. R. OWENS ET AL.

d. The dihydrothiazine ring: opening of this gives an inactive molecule. At least three major components of the 7-ACA molecule [Fig. 1 (II)] are necessary for antibacterial activity (Benner, 1971; Flynn, 1971): (I) the p-lactam ring must remain chemically reactive [Van Heyningen and Aherne (1968) have ascribed the lack of antibacterial activity of A'cephalosporins to their much greater stability toward basic hydrolysis than A3-cephalosporins, since it is likely (see Fig. 3 in Section III,c1) that a covalent bond is formed by p-lactam acylation of an active site in the bacterial cell wall]; (2) definite chemical binding sites on the molecule are required; (3) the three-dimensional shape of the ring system must be maintained intact. A fourth factor, the transport (permeability ?) of a cephalosporin antibiotic to its active site in the cell is also mentioned by Flynn and Benner; this will be considered in Section III,B and C). The above has dealt very briefly with some of the structural changes that influence antibacterial activity in the cephalosporin group. The cephalosporins are not normally considered a s possessing antifungal or antimycoplasmal activity, because their mode of action is believed to involve an inhibition of bacterial cell wall synthesis (Section 111,C). There have, nevertheless, been some recent reports indicating that modification of the molecule may result in a cephalosporin with antifungal and/or antimycoplasmal activity. The introduction of a dimethyldithiocarbamate grouping at position 3 gives a cephalosporin [7(S-benzylthioacetamido)cephem-3-ylmethyl-N-dimethyldithiocarbamate-4carboxylic acid] with both activities i n vitro (Fallon, 1970; Russell and Fountain, 1971), and another cephalosporin with marked antifungal activity [sodium (N-benzyldithiocarbamoylacetamido)cephalosporanate] has been described recently by Gottstein et al. (1971); the minimum inhibitory concentration of this drug necessary against a strain of Cryptococcus neoformans was 8 &ml. The in vivo significance of these findings is difficult to assess; it is, however, a logical conclusion that, although at present such compounds may not give sufficiently high in vivo levels for systemic use, further studies should be made along these lines. Resistance to the various types of p-lactamases (Section II1,B) can be associated with the cephalosporin molecule. The resistance of cephalosporin C to staphylococcal f?-lactamase is associated with the ring system of the molecule and not with the nature of the N-acyl side chain, because cephalosporin N (penicillin N) is rapidly inactivated by the enzyme althoug!i also containing the 6-(D-a-aminoadipoyl) side chain

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

89

(Abraham, 1967). Cephalosporins with N-acyl side chains have a high activity against most gram-positive bacteria including p-lactamaseproducing staphylococci. Cephalosporins with a 2,6-dimethoxybenzoyl side chain have a high affinity for, and are resistant to hydrolysis by, plactamases from some gram-negative bacteria, but have only a low order of activity against these organisms (Hamilton-Miller et a l . , 1965).

Ill. Antibacterial Activity A. SPECTRUM OF ACTIVITY 1. Cephaloridine Cephaloridine { 7-[(2-thienyl)acetamido1-3-(l-pyridylmethyl)-3-cephem4-carboxylic acid betaine} has a broad antibacterial spectrum, is bactericidal, and is highly active against a and P-hemolytic streptococci, pneumococci, Corynebacterium sp., penicillin-sensitive strains of Staphylococcus aureus, and various Clostridia sp. and Neisseria sp. (although N . gonorrhoeae is less sensitive than N. catarrhalis and N . menigitidis) (Barber and Waterworth, 1964; Murdoch et al., 1964; Muggleton et al., 1964; Stewart and Holt, 1964; Benner et al., 1965a; Vymola and Hejzlar, 1966; Muggleton and O’Callaghan, 1967; Newton and Hamilton-Miller, 1967; Gonnella et al., 1967; Perkins et a l . , 1967b; Turck et al., 1967; Hewitt and Parker, 1968; Stratford and Dixson, 1968; Acar, 1971; Cox and Montgomery, 1971; Kayser, 1971). Enterococci show a low degree of sensitivity (Kislak et a l . , 1966; Naumann, 1967), and penicillinase (p1actamase)-producing strains of S . aureus are less sensitive to cephaloridine than are non-p-lactamase producers; much higher concentrations of the drug are needed to inhibit the growth of large inocula of the p lactamase producers although small inocula are very sensitive (Benner et al., 1965a; Ridley and Phillips, 1965; Kislak et al., 1966; Editorial, 1967; Hewitt and Parker, 1968; Eykyn, 1971; Kayser, 1971; Russell, 1972a,b,). Cephaloridine and cephalothin are some 2 4 times more active than cloxacillin against Actinomyces israelii strains, but Actinomyces bovis is much less sensitive (Lerner, 1968). Methicillin-resistant strains of staphylococci frequently pose a serious clinical problem in hospitals (Finland, 1972; Kayser et al., 1972). The sensitivity of methicillin-resistant S. aureus to p-lactam antibiotics depends markedly on the test conditions, e.g., incubation at 30°, or the

90

D. R. OWENS ET AL.

presence of 5% w/v sodium chloride in the culture medium (see Russell et a l . , 1973). Methicillin-resistant staphylococci are resistant to cephalosporins (Chabbert, 1967a,b), although cephaloridine is considered to be more active than other cephalosporins against such strains (Hallander and Laurell, 1972). It is, however, highly unlikely that cephaloridine is a practical in vivo proposition because of the high blood levels which would have to be attained. Activity of cephaloridine against some strains of Mycoplasma has also been described (Fallon and Hutchinson, 1967; Stewart et a l . , 1969) although generally concentrations of at least 100 pg/ml may be needed to achieve an inhibitory effect. Cephaloridine also shows a high degree of activity against many different species of gram-negative bacteria, and bactericidal levels are generally not much higher than minimum inhibitory concentrations (MIC’s) (Murdoch et a l . , 1964; Muggleton et a l . , 19M; Thornton and Andriole, 1966; Muggleton and O’Callaghan, 1967; Perkins et a l . , 1967b; Turck et a l . , 1967) although minimum bactericidal concentrations (MBC’s) of 16 to 64 times the MIC’s have been recorded in some cases (Gonnella et a l . , 1967). Indole-positive Proteus sp. are resistant to cephaloridine and to cephalexin and cephalothin, whereas the majority of Proteus mirabilis strains are sensitive. Pseudomonas aeruginosa is highly resistant to all cephalosporins, but Escherichia coli, Salmonella sp. (Adams and Nelson, 1968), and Shigella sp. (Nelson and Haltalin, 1972) are sensitive to cephaloridine, and Neisseria meningitidis and Neisseria catarrhalis are highly sensitive (Muggleton and O’Callaghan, 1967; Eykyn, 1971). P-Lactamase-producing gram-negative bacteria may inactivate cephaloridine (Muggleton et a l . , 1964; Hamilton-Miller et a l . , 1965; O’Callaghan and Muggleton, 1967) and this aspect is considered later in this section.

2 . Cephalothin The sodium salt of 7-(2-thienylacetamido)cephalosporanic acid, cephalothin is an important member of the cephalosporin group of antibiotics, with a fairly broad spectrum of activity (Godzeski et al., 1963). It was used clinically before cephaloridine had been discovered, but its relatively greater resistance to staphylococcal p-lactamase was not observed until some time later. It is less active than cephaloridine but rather more active than cephaloglycin or cephalexin against non+ -

T H E CZPHALOSPORIN GROUP OF ANTIBIOTICS

91

lactamase-producing Staphylococcus aureus strains, pneumococci, and a- and P-hemolytic streptococci (Kayser, 1971); cephalothin is bactericidal in its action against gram-positive bacteria, with the MBC generally being about twice the MIC. Low concentrations of cephalothin are inhibitory to both p-lactamase and non-p-lactamase-producing strains of S. aureus (Boniece et al., 1962). Cephaloridine is more active than cephalothin against penicillin-sensitive S. aureus strains and against small inocula of p-lactamase-producing S. aureus and has a greater bactericidal effect (Perkins et al., 1967b), but the MIC’s of cephalothin are less affected by changes in inoculum size of the latter organisms than are ampicillin (Sherris et a l . , 1967) or cephaloridine (Benner et a l . , 1965a; Hewitt and Parker, 1968; Russell, 1972a,b). However, inoculum size has a very marked effect on the MBC’s of cephalothin (Turck et a l . , 1965). Enterococci are resistant to cephalothin (Walters et al., 1964; Naumann, 1967; Eykyn, 1971). A close association between methicillin resistance and resistance to various cephalosporins. including cephalothin, with heteroresistance (Sutherland and Rolinson, 1964) a typical trait, has recently been observed (Hallander and Laurell, 1972). Methicillin-resistant strains are less sensitive to cephalothin than to cephaloridine (Barber and Waterworth, 1964; Benner and Morthland, 1967). Cephalothin is inhibitory and bactericidal to many types of gramnegative bacteria, but these are less sensitive and more variable than gram-positive bacteria (Walters et al., 1964; Turck et al., 1965). Barber and Waterworth (1964) suggested that there was little difference between cephalothin and cephaloridine in their activity against coliforms, but cephaloridine is rather more inhibitory against Escherichia coli and P . mirabilis (Turck et a l . , 1965, 1967; Perkins et al., 196713). Pseudomonas aeruginosa and indole-positive Proteus strains are highly resistant to cephalothin (Turck et al., 1965; Naumann, 1967). Some authorities consider cephalothin as having a low degree of activity against Klebsiella-Aerobacter strains (Turck et al., 1965; Steigbeigel et al., 1967); however, there is evidence that nearly all the motile strains (Aerobacter) in this group are highly resistant to cephalothin and cephaloridine, whereas nonmotile strains (Klebsiella) are sensitive to cephalothin and cephaloridine especially the former (Benner et ul., 1965b; Lerner and Weinstein, 1967; Kunin and Brandt, 1968). Cephalothin is inhibitory to Shigella strains (Nelson and Haltalin, 1972). The effects of some types of p-lactamases from gram-negative bacteria on cephalothin are considered in Section II1,B.

92

D. R. OWENS ET AL.

3. Cephalexin The monohydrate of 7-(Diu-aminophenylacetamido)-3-methyl-3-~ephem-4-carboxylic acid, cephalexin is one of the newer group of cephalosporin antibiotics, and it is of particular interest because it is given orally. Details of its absorption, excretion, and tissue distribution and of its clinical efficacy are described later (Sections IV, V, and VI). Cephalexin is considered to be a broad-spectrum antibiotic (Griffith and Black, 1968), although its in vitro activity may be less than that of cephaloglycin (Wick, 1967; Braun et al., 1968; Kayser, 1971); however, cephaloglycin is incompletely absorbed after oral administration (Applestein et al., 1968) and higher blood and urine levels are obtained after orally given cephalexin (Wick, 1967). The majority of S. aureus strains are considered by Leigh et al. (1970) to be highly sensitive to cephalexin, and Thornhill et al. (1969) found that 88% of such strains were inhibited by 6.3 pg/ml and 100% inhibited by 12.5 pg/ml of cephalexin. Strains of a- and P-hemolytic streptococci, Streptococcus pneumoniae, gonococci, and meningococci are moderately to highly susceptible to cephalexin (Braun et al., 1968; Muggleton et al., 1969; Kayser, 1971). Despite being less active than cephaloridine against most bacteria (Hoeprich, 1968; Levison et al., 1969; Muggleton et al., 1969; Bond et a l . , 1970; Eykyn, 1971; James and Walker, 1971), cephalexin is more active than cephaloridine against N. gonorrhoeae (Muggleton et al., 1969). Cephalexin has a bactericidal action, and is considered to be equally effective against penicillin-sensitive and p-lactamase-producing strains of S. aureus (Wick, 1967). However, although it is more active than ampicillin against the latter strains and although it is more resistant than cephaloridine to destruction by p-lactamase-producing staphylococci and gram-negative bacteria, Muggleton et al. (1969) do not consider this is of a sufficient extent to make cephalexin effective against these gram-negative organisms. Some inoculum effect occurs with p-lactamase-producing staphylococci but to a much smaller extent than with cephaloridine (Eykyn, 1971). Methicillin-resistant strains of staphylococci are resistant to cephalexin (Kayser, 1971; Russell, 1972a,b). Gram-negative bacteria are considerably less sensitive to cephalexin than is S. aureus (Leigh et al., 1970), and strains of Haemophilus influenzae and many common gram-negative bacilli are moderately to highly resistant (Braun et a l . , 1968). Of Proteus strains, P . mirabilis is the most sensitive (Levison et al., 1969; Thornhill et al., 1969; Leigh et al., 1970); Thornhill et al. (1969) reported that in their studies, 56% of P . mirabilis strains, 80% of E. coli strains, and 72% of Aerobacter-

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

93

Klebsiella strains were inhibited by 12.5 pglml of cephalexin. Pseudomonas aeruginosa and indole-positive Proteus strains are resistant (Thornhill et a l . , 1969; Russell, 1972a,b). A marked inoculum effect is noted with the activity of cephalexin against various types of gramnegative bacteria (Fountain and Russell, 1969; Muggleton et a l . , 1969; Bond et al., 1970; Russell, 1972a,b) but this does not appear to be associated with /I-lactamase production and drug destruction. Subinhibitory concentrations of cephalexin induce very long filamentous forms in various types of gram-negative bacteria (Fujii et al., 1969; Muggleton et a l . , 1969; Nakazawa et a l . , 1969; Russell and Fountain, 1970).

4. Cephaloglycin The first oral cephalosporin, cephaloglycin [7-(DaYaminopheny1acetamido)-3-acetoxymethylcephem-4-carboxylicacid] was described by Wick and Boniece (1965). It is a bactericidal antibiotic, the MBC generally being about twice the MIC for most groups of gram-positive and gram-negative bacteria (Johnson et a l . , 1968; Kayser, 1971). It is about twice as active against penicillin-sensitive staphylococci as against p-lactamase producers (Kayser, 1971; however, cf. Pitt et al., 1968). Cephaloglycin is inhibitory to a- and P-hemolytic streptococci, staphylococci, and pneumococci (Applestein et al., 1968; Johnson et a l . , 1968; Kayser, 1971). Cephaloglycin is also inhibitory and bactericidal to E. coli and P . mirabilis, with rather higher drug concentrations necessary for activity against the Klebsiella-Aerobacter group (Applestein et al., 1968; Pitt et al., 1968). Johnson et al. (1968) found cephaloglycin to be more active than tetracycline, ampicillin, and cephalothin, and of equal activity to chloramphenicol and colistin, against E. coli, whereas it was less active than ampicillin, cephalothin, and kanamycin against P . mirabilis. Waterworth (1971) showed that cephaloglycin was more active than other cephalosporins against some types of gram-negative bacteria. Pseudomonas aeruginosa is resistant to cephaloglycin (Pitt et al.,, 1968). It must be added that the activity of cephaloglycin is markedly pHdependent, with a much greater bactericidal effect at acid pH (Kunin and Brandt, 1968). It is unstable at alkaline pH (Wick and Boniece, 1965), and the rates of degradation in trypticase soy broth and in nutrient broth have been stated to be 14% per hour and 3.5% per hour, respectively (Applestein et al., 1968). Differences in MlC ranging from two- to eightfold occur when incubation at 37" is extended from 12 to 18 hours (Pitt et al., 1968) and this fact must be taken into account in considering MIC values. After oral administration, cephaloglycin is

94

D. R. OWENS ET AL.

partially converted in man to a biologically active metabolite, deacetylcephaloglycin, which is as active as cephaloglycin against gram-positive bacteria but less active against gram-negative bacteria (Wick et al., 1971).

5 . Other Cephalosporins New cephalosporins continue to be developed. Of these, the following are worthy of comment. a . Cephacetrile (CIBA 36,278-Ba; CAA). The sodium salt of 7cyanacetamidocephalosporanic acid, this cephalosporin derivative is less active than cephaloridine against benzylpenicillin-sensitive staphylococci, but is more active than cephaloridine against p-lactamaseproducing strains; it is less active than cephaloridine and ampicillin against non+-lactamase-producing strains of E. coli, but is more active against E. coli R+ TEM (Kniisel et al., 1971; Russell, 1972a,b). Gramnegative p-lactamase-producing strains produce a red color when incubated in broth containing cephacetrile and this has been suggested as being of possible use in the detection of such organisms (Russell, 1972~).Cephacetrile is without effect on P. aeruginosa. b. Cephradine [Velosef (Squibb)].* There is, as yet, insufficient evidence on which to base an assessment of the usefulness of this new oral antibiotic. Limson et al. (1972) have listed the MIC values of cephradine against various gram-positive and gram-negative bacteria; the MIC against a penicillin-sensitive strain of S. aureus (inoculum size lo6 viable cellslml) was 3.1 and 18.7 p d m l against a comparable inoculum of a @-lactamase producer. Low inocula (lo3 viable cells/ml) of some gram-negative strains were sensitive to about 10 pg/ml or less of cephradine, but P . aeruginosa was resistant. According to Klastersky et al. (1973), cephradine inhibits most strains of Pneumococcus and Staphylococcus, as well as S. pyogenes. Landa (1972) has shown that oral administration to patients with infections caused by salmonellas and shigallae gave an 82% cure rate with no side effects. c. Cephapirin. A new cephalosporin antibiotic, cephapirin is administered parenterally (Gordon et al., 1971; McCloskey et al., 1972). As is usual with cephalosporins, it is more active against gram-positive bacteria than gram-negative bacteria (Wiesner et al., 1972), with an activity equivalent to that of cephalothin (Bran et al., 1972). Wiesner et al. (1972) found that all of their test strains of penicillin-sensitive and p -

* Further details are listed in the technical information published by E. R. Squibb & Sons, Ltd.

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

95

g lactamase-producing staphylococci were inhibited and killed by 5 ~ r or less of cephapiridml against an inoculum of l o 4 or lo6 viable organisms/ ml, although an inoculum effect with S. aureus and various strains of gram-negative bacteria was also observed. Cephapirin is bactericidal to E. coli, pneumococci, and P . mirabilis but has negligible activity against P . aeruginosa and indole-positive Proteus strains. d . Cefazolin. Also a new cephalosporin antibiotic (Kariyone et al., 1970; Mine et al., 1970a,b; Nishida et a l . , 1970a,b), cefazolin shows promise clinically (Shibata and Fujii, 1971). It is more active against gram-positive than gram-negative bacteria, and its antibacterial spectrum includes @-lactamase-producing strains of S. aureus, although Group D streptococci are resistant, as are P . aeruginosa, indole-positive Proteus strains, and nonpigmented Serratia strains (Wick and Preston, 1972). Cefazolin is inhibitory to N . gonorrhoeae and N . meningitidis, but methicillin-resistant strains of S. aureus require high concentrations of cefazolin for inhibition, especially when sodium chloride is included in the medium. Cefazolin is less active than ampicillin or cefamandole (see below) against strains of Haemophilus influenzae. Overall, cefazolin could well prove to be a useful addition to the cephalosporin group of antibiotics. e . Cefamandole. This antibiotic was described by Wick and Preston (1972) as CMT. It shows activity against S. aurem, streptococci, Neisseria, Clostridia, and Corynebacterium and is more active than cefazolin against gram-negative bacteria including E. coli, Enterobacter sp., indole-positive Proteus, Salmonella sp., and Shigella sp. although P . aeruginosa and Serratia marcescens strains are resistant. The antiProteus activity is particularly interesting, and evidence has been presented to show that cefamandole is considerably more stable to cephalosporinase than is cephalothin. Cefamandole is very active against H . influenzae. f. Comments. Continuing research in the cephalosporin field has led to the development of some new and interesting antibiotics. Provided that it is shown to be clinically effective (see Wick and Preston, 1972), and if the preliminary findings are confirmed, cefamandole may prove to be a valuable addition to the cephalosporin range. Further research is, however, needed to produce a cephalosporin that combines high activity against gram-positive bacteria with potent bactericidal action against gram-negative bacteria, including P . aeruginosa and S . marcescens. It must also be pointed out that the type of assay medium used in determining the sensitivity of bacteria to cephalosporins may greatly influence the observed results (Pursiano et a l . , 1973). This finding could

96

D. R. OWENS ET AL.

be one reason in explaining the variable results which may occur from laboratory to laboratory.

B. BACTERIAL RESISTANCE 1. Development of Resistance When bacteria are, repeatedly subcultured in media containing gradually increasing concentrations of a cephalosporin, they frequently show an increase in resistance to that particular drug and to other p lactam antibiotics. Penicillin-resistant staphylococci may be selected in this manner in the laboratory, but the lack of emergence of such strains (as opposed to p-lactamase-producing ones) may be the result of the relatively low mutation rates in such organisms, because the levels of penicillin in the body will greatly exceed the sensitivity of both the original bacteria and first-step mutants as they arise (Rolinson, 1971). Generally, the type of resistance acquired to cephalosporins is of a similar, stepwise type (Jago, 1964; Bernard and Lambin, 1965; Ott and Godzeski, 1967; Fountain and Russell, 1970). Godzeski et al. (1963) found that gram-negative bacteria developed a stepwise resistance to cephalothin, whereas there was no in vitro development of resistance with S. aureus; gram-negative bacteria passaged in cephaloridine or benzylpenicillin have been reported to have a considerable increase in resistance to the antibiotic through which they were passaged and a moderate increase to the other antibiotic (Barber and Waterworth, 1964). Emergence of cephalexin-resistant S. aureus by repeated subculture has been reported (Bond et al., 1970) with as few as three steps giving variants resistant to high cephalexin concentrations (Kayser et al., 1970; Kayser, 1971), although Muggleton et a l . (1969) have proposed that there is no rapid emergence of organisms resistant to this drug. The manner in which these organisms with acquired resistance differ from the original bacterial population is by no means clear, although there are two distinct possibilities. (1) Alterations in the amount of p-lactamase produced. This could be applicable to a strain that originally produces no p-lactamase but does so when resistance has developed, or to a strain that produces an increased amount of fI-lactamase when resistance has been acquired. There is little evidence in support of this hypothesis (also see below) since cultures that have developed resistance to a penicillin or cephalosporin may not inactivate that drug (Ott and Godzeski, 1967; Park et a l . , 1971). Barber and Waterworth (1964) found that E. coli and

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

97

P . mirabilis passaged in cephaloridine or benzylpenicillin showed no change in B-lactamase activity, whereas Klebsiella aerogenes, passaged similarly, possessed an increased p-lactamase activity. (2) Changes in cell wall composition. There is, at present, insufficient information available on this topic for sound conclusions to be made. However, it has been found (Fountain and Russell, 1970) that E . coli made resistant to cephaloridine showed cross-resistance to other plactam antibiotics, that the resistant strain produced no P-lactamase, but that, significantly, ethylenediaminetetraacetic acid (EDTA) potentiated the effect of cephaloridine against this, but not the parent, strain. These findings suggest that changes in outer lipid layers might have occurred, a conclusion similar to that reached with carbenicillinresistant strains of P . aeruginosa (Thomas and Broadridge, 1972). Inaccessibility of the total murein transpeptidase to penicillin is one possible reason for the development of penicillin-resistant mutants of aureus strain H (Park et al., 1971).

s.

2. Cross-resistance An organism that has acquired resistance to one cephalosporin may be expected also to show cross-resistance to other members of this group. This need not be, necessarily so, however. S. aureus strains (Blactamase producers or not) made resistant to cephalexin also showed some cross-resistance with methicillin, whereas their susceptibility to cephalothin and cephaloridine was only decreased slightly (Kind et al., 1969a,b). In contrast, Kayser et al. (1970) showed that S. aureus could be induced to acquire intrinsic resistance to cephalexin with a concomitant increase in resistance to cephalothin and to the two Blactamase-stable penicillins, oxacillin and dicloxacillin. A marked crossresistance between cephalexin and methicillin in some naturally occurring methicillin-resistant strains has been observed (Kind et al., 1969b). Ott and Godzeski (1967) found extensive cross-resistance when S. aureus was cultured to higher levels of resistance with oxacillin, cephalothin, or cephaloglycin, but there was a marked lack of consistency among the various cultures in their response to the related antibiotics; the least cross-resistance was shown to cephaloridine, which was also the antibiotic with the least ability to give an increase in resistance level by serial passage. Staphylococci that had acquired in vitro resistance to one derivative of 7-ACA also showed strong cross-resistance to the other derivatives (Jago, 1964). In the case of gram-negative bacteria, some degree of cross-resistance is shown in a culture made resistant to one p-lactam antibiotic (Barber

98

D. R. OWENS ET AL.

and Waterworth, 1964; Ott and Godzeski, 1967; Fountain and Russell, 1970; Russell and Fountain, 1970). 3. Effect of P-Lactamases on Cephalosporins P-Lactamase (penicillinase, cephalosporinase, penicillin amidoj3-lactam hydrolase; E.C.3.5.2.6.) is widely distributed among bacteria. It is extracellular with gram-positive bacteria and it is inducible in grampositive, but not gram-negative, bacteria (an exception occurs with P. aeruginosa, as described later; see also Mildvan et a l . , 1973). In gramnegative bacteria, p-lactamase is cell-bound, and these organisms may possess an “accessibility barrier” to many, but not all, p-lactam drugs so that /3 -1actamase activities in disrupted cell preparations may be several times greater than in intact cells (Hamilton-Miller et a l . , 1965; Smith et a l . , 1969). Most varieties of p-lactamases will hydrolyze most p-lactam antibiotics, but this hydrolysis proceeds at widely divergent rates, depending on the enzyme and the drug. p-Lactamase production may be either constitutive or it may be induced by the presence of a p-lactam antibiotic, and the enzyme activity may be transduced by phage in S. aureus (Richmond, 1965). Sabath et al. (1965), Hamilton-Miller et al. (1970a,b), and O’Callaghan et a l . (1972a) have shown that enzymic hydrolysis of the cephalosporins results in an opening of the p-lactam ring accompanied by expulsion of R’ (Fig. 2) as acetate (from cephalothin) or pyridine (from cephaloridine) with a concomitant disappearance of the characteristic UV absorption. This is replaced with an absorption band (A,, 230 nm) which itself disappears in turn after several hours (Sabath et al., 1965). However, opening of the p-lactam ring without these other changes occurs with deacetylcephalosporin C lactone, and the resulting product has a A,,, of 265 nm. In contrast to the relatively stable D-a-penicilloates produced by the action of p lactamases on penicillins, cleavage of the p-lactam rings of cephalosporins thus involves rapid initial changes in UV absorption spectra with further extensive degradation of cephaloridine and cephalosporin C (Newton et al., 1968). Hamilton-Miller et a l . (1970b) used proton magnetic resonance to study the nature of the compound with ,,,A of 230 nm and showed that the same compound with this Amax was formed from both cephalosporin C and deacetylcephalosporin C on enzyme hydrolysis. The /3 -1actamases from gram-positive and gram-negative bacteria differ to a considerable extent (Pollock, 1965, 1967, 1971; Citri and

T H E C E P H A L O S P O R I N GROUP O F A N T I B I O T I C S

99

Pollock, 1966; Citri, 1971), and their effects on cephalosporins will thus be considered separately. a. p-Lactamases from Gram-Positive Bacteria. (i) Staphylococcal plactamases. In one of the original papers on cephaloridine, Murdoch et a l . (1964) reported that this compound was 100-1000 times more resistant than phenoxymethylpenicillin (penicillin V) to staphylococcal plactamase and, thus, that, for practical purposes, cephaloridine was unaffected by this enzyme. However, as has been pointed out earlier, cephaloridine is more sensitive than cephalothin or cephalexin to changes in inoculum size of P-lactamase-producing strains of S. aureus (Barber and Waterworth, 1964; Benner et a l . , 1965a; Ridley and Phillips, 1965; Kislak et al., 1966; Seligman and Hewitt, 1966; Newton and Hamilton-Miller, 1967; Thompson et al., 1967; Hewitt and Parker, 1968; Knusel et al., 1971; Russell, 1972b). Benner et a l . (1965a) found that many p-lactamase S. aureus producers required much larger amounts of cephaloridine than cephalothin for inhibition when large inocula were used; this has been claimed to be the most important reason for the failure of the former antibiotic in some staphylococcal infections (Seligman and Hewitt, 1966; Editorial, 1967; Hewitt and Parker, 1968). However, an alternative reason could be an increase in intrinsic resistance of the cells (Hamilton-Miller, 1967a,b). In support of this is the finding that, although cephaloridine is much more rapidly hydrolyzed by staphylococcal p-lactamase than cephalothin (HamiltonMiller and Ramsey, 1967; Muggleton and O’Callaghan, 1967) or cephalexin (Eykyn, 1971), it is, in fact, hydrolyzed at a rate of only 0.10.2% of that of benzylpenicillin (Hamilton-Miller, 1967a,b; Naumann, 1967; Knusel et al., 1971) (Table I). Cefazolin appears to be rather more, and cefamandole (CMT) less, susceptible than cephaloridine to staphylococcal P-lactamase (Wick and Preston, 1972). Several derivatives of 7-ACA behave as competitive inhibitors of the action of staphylococcal p-lactamase on penicillins (Crompton et al., 1962) although this does not apply to cephalosporin C and deacetylcephalosporin C (K,IK, values being >lo0 and >50, respectively); however, N-phenylacetyl-7-ACA, with a K J K , value of 1.7 x lo-’, is a powerful inhibitor (Abraham and Newton, 1956, 1961; Crompton et a l . , 1962). Methicillin ( 0 . 5 2 pglml) is a good inducer of staphylococcal p lactamase; negligible induction occurs with cephalosporin C at concentrations of 1 d m l although considerable induction occurs with higher concentrations (5 pg/ml or more) (Swallow and Sneath, 1962). All compounds tested that induce p-lactamase formation in Bacillus cereus

c,

0 0

TABLE I SUBSTRATE PROFILES OF SOME ~ L A C T A M A S E S " . ~ PLactamase source

Pen

Amp

Meth

Clox

Carb

Cer

Cet

Cex

CAA

Staphylococcus aureus 8325 i+p+

100 100

B. cereus 569 i+p+

100

Escherichia coli R+ TEM E. coli 53 Salmonella typhi 152, E. coli 1 E M or E . coli K-12 RTEMR-factor E. coli K-12 R,,,,R-factor E . coli K-12 R,,,,R-factor Enterobacter cloacae 214 (P99) Pseudomonas aeruginosa Enzyme Type I (inducible) Enzyme Type I1 (R-factor) Enzyme Type I11 (Dalgleish) Proteus morganii (2 strains)

100 100 100

313 220 134 128 120 150

0.7 0.23 3.8 1 1

-

-

0.4 0.22 4.6 158 150 175

0.1 0.1 3.3 15.6

-

S.aureus 2999 i+p+

0.3 0.26 4.4 25

+

+ +

100 100 100 100 100 100 100

91 100 1

10 160 100

-

2

-

21 0.1 1

126 1.5 -

-

-

-

-

-

0 10 150 -

-

-

-

-

15

-

-

86 145 8600 400 125 40 c.325

46 17 1250

-

-

c.220

-

-

780 140 0 0

-

-

-

' I Data of Datta and Kontomichalou (1%5), Hamilton-Miller (1967a,b), Hennessey and Richmond (1%8), Jack and Richmond (19701, newsom et al. (1970),Sykes and Richmond (1971),Kniisel et a l . (1971). " Abbreviations: Pen, benzylpenicillin; Amp, ampicillin; Meth, methicillin; Clox, cloxacillin; Carb, carbenicillin; Cer, cephaloridine; Cet, cephalothin; Cex, cephalexin; CAA, 7-cyanacetamidocephalosporanic acid.

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

101

(Pollock, 1957; see below) will induce p-lactamase production in S. aureus. Nevertheless, there are striking differences in that the relative efficiencies of the inducer vary greatly from one organism to the other (Crompton et al., 1962). (ii) Bacillus cereus p-lactamase. Abraham and Newton (1956) showed that a crude p-lactamase preparation from B. cereus contained only 1/ 100 as much cephalosporinase activity as penicillinase activity; crude supernatant fluids from cultures of this organism, strain 569, had only about 1/20 as much activity against cephalosporin C as against benzylpenicillin, and Zn was found to be a cofactor for the cephalosporinase, but not for the penicillinase, activity of crude p-lactamase from B . cereus 569 and 569H (Sabath and Abraham, 1966; Sabath and Finland, 1968). The maximal rate of hydrolysis of several derivatives of 7-ACA by B. cereus p-lactamase is very low, with the exception of deacetylcephalosporin C lactone for which the rate of hydrolysis is of the same order as for benzylpenicillin (Crompton et al., 1962). The y-type p-lactamase from B. cerem 569H has been extracted and purified by Citri and Kalkstein (1967), who compared the substrate profiles of the y - and atype p-lactamases. The relative rates of hydrolysis of cephaloridine and methicillin by the y-type enzyme were very high, but were much lower with the y-type. Cephalothin and cephalosporin C were not hydrolyzed to any significant extent by either enzyme. Pollock (1956) had earlier shown that two types of cell-bound p-lactamases, the p - and y-types, were produced by this organism. The p-kype is thought to be identical with the a-type and with the p-lactamase Type I of Kuwabara and Abraham (1967, 1969); p-lactamase Type I1 of these authors is a Znrequiring enzyme that is active against a variety of cephalosporins and penicillins. Cephaloridine is more susceptible to B. cereus p-lactamase than cefazolin, cephalothin, and cephamandole (Wick and Preston,

1972). Unlike the case with the crude extracellular S. aureus enzyme described in the foregoing, cephalosporin C is a competitive inhibitor of B. cereus on benzylpenicillin (Abraham and Newton, 1956, 1961), and the general pattern of values for KiIK, with this enzyme and cephalosporin C, deacetylcephalosporin C, cephalosporin CA (pyridine), and N phenylacetyl 7-ACA falls within the range of 1 to 5. Cephalosporin C in optimal concentration is a better inducer of plactamase in B. cereus than is benzylpenicillin, although a higher concentration of the former compound is needed to produce a maximal effect (Pollock, 1967). Deacetylcephalosporin C behaves similarly to

102

D. R. OWENS ET AL.

cephalosporin C, but cephalosporin C A (pyridine) has a higher maximum inducing activity than cephalosporin C (Crompton et al., 1962). Some practical laboratory uses of these p -1actamases have recently been described; various types of p-lactamases may be used for inactivating cephaloridine and cephalothin, e.g., in assays of cephalosporin-noncephalosporin mixtures (Newson and Walsingham, 1973), and a highly purified p-lactamase (Neutrapen) from B. cereus may be employed (1) as an inactivating agent for cephaloridine in carrying out viable counts of bacteria treated with this antibiotic and (2) a s a means of carrying out sterility tests on cephaloridine and possibly cephalothin (Russell and Furr, 1973). Cephalexin is highly resistant to inactivation by this enzyme (Russell and Furr, 1973). b. p-Lactamases from Gram-Negative Bacteria. The situation with p lactamases from gram-negative bacteria is exceedingly complex (Richmond and Sykes, 1973) and at least eight distinct types of @-lactamase from gram-negative bacteria, apart from P. aeruginosa, are known (Jack and Richmond, 1970). High concentrations (5-10 mg/ml) of benzylpenicillin or cephalosporin C are needed to induce the enzyme in P. aeruginosa (Sabath et al., 1965; Sykes and Richmond, 1971). The best inducer of p-lactamase in strains of this organism appears to be 6-APA, and cephalothin the worst (Garber and Friedman, 1970). Significantly, carbenicillin appears to be resistant to the inducible p-lactamase enzyme in these organisms and, in fact, inhibits their activity against cephalosporins (Bobrowski and Borowski, 1971). A small number of P. aeruginosa strains produce a constitutive p lactamase, i.e., in the absence of inducer, and three types of plactamase have been identified (Sykes and Richmond, 1971) in these organisms . Type I: the p-lactamase produced is inducible and is common among p . aeruginosa strains. It is predominantly a cephalosporinase and rapidly hydrolyzes cephaloridine and cephalexin, whereas carbenicillin is resistant (also see above). Type 11: a constitutive p-lactamase of the R-factor-mediated type found in enteric bacteria. Cephalexin is not inactivated and carbenicillin only slightly so. Strains of P. aeruginosa producing this enzyme also produce Type I enzyme. Type 111: one strain (Dalgleish) produces a p-lactamase that is constitutive, but which is quite distinct from Type I1 and markedly inactivates carbenicillin. Cephaloridine is less labile, and cephalexin is resistant. This strain also produces an inducible Type I p-lactamase. The substrate profiles of these enzymes against various p-lactam

103

T H E CEPHALOSPORIN G R O U P OF ANTIBIOTICS

TABLE I1 SENSITIVITY TO SOME8-LACTAM ANTIBIOTICS OF Escherichia coli K-12 INFECTED WITH DIFFERENTR-FACTORS~ MIC (pglml) of

Escherichia coli

K-12 + K-12 + K-12 + K-12 + K-12 +

TEM

1818 1818 (cured) 7268 7268 (cured) Original K-12

+ R-factor

Pen

Amp

Cer

4000 250 200 1000 1000 30

4000 250 200 1000 1000 2

60 4 4 8 8 1

Data of Datta and Kontomichalou (1965). Inoculum size throughout, 10’ viable c e l l s h l . MIC, minimum inhibitory concentration; Pen, benzylpenicillin; Amp, ampicillin: Cer, cephaloridine. a

drugs, relative to benzylpenicillin, are shown in Table 11. The plactamase activity against cephaloridine of sonically disrupted P . aeruginosa cells is the same as whole cells, suggesting that this antibiotic readily penetrates all strains (Bobrowski and Borowski, 1971). The b-lactamases of the Enterobacteriaceae are not inducible but present an even more complex picture (Fleming et al., 1963, 1970; Jack and Richmond, 1970; Richmond, 1972; Richmond and Sykes, 1973). Extensive studies of the effects of these p-lactamases on penicillins and cephalosporins have been made (Jago et al., 1963; Ayliffe, 1964, 1965; Hamilton-Miller and Smith, 1964; Hamilton-Miller et al., 1965; Datta and Kontomichalou, 1965; Datta and Richmond, 1966; Hamilton-Miller, 1967a,b; Muggleton and O’Callaghan, 1967; O’Callaghan and Muggleton, 1967; O’Callaghan et al., 1967, 1968, 1969, 1972a,b; Hennessey and Richmond, 1968; Sykes and Richmond, 1970; Kniisel et al., 1971; Marshall et al., 1972; O’Callaghan and Morris, 1972; Wick and Preston, 1972; Russell, 1972c; Sykes and Nordstrom, 1972; Anderson and Sykes, 1973). Examples of the substrate profiles of the various types of p lactamases on cephalosporins and penicillins, relative to benzylpenicillin, are provided in Table I. The p-lactamase from Enterobacter cloacae strain 214 (believed to be identical with strain P99) has been isolated and purified; this enzyme hydrolyzes cephaloridine, cephalothin, and cephalosporin C at rates 80, 10, and 70 times, respectively, as rapidly as

104

D. R. OWENS ET AL.

benzylpenicillin, but ampicillin is not inactivated (Hennessey and Richmond, 1968). Nevertheless, this strain is highly resistant to ampicillin in terms of MIC values (Russell, 1974), although changes in morphology are induced over a short incubation period (Russell, 1973). There is no transfer of p-lactamase in this organism, and the gene specifying this type of enzyme is chromosomal (Jack and Richmond, 1970), in contrast to E. coli R+ TEM (Datta and Kontomichalou, 1965; Datta and Richmond, 1966; Jack and Richmond, 1970; see also Matsuhashi, 1971). Enterobacter cloacae P99 and Klebsiella aerogenes strain 1082E (also known as K1) are considered to represent extremes of cephalosporinase and penicillinase activity, respectively (Marshall et a l . , 1972). E . coli strain K-12 is sensitive to ampicillin and cephaloridine, and fairly sensitive to benzylpenicillin. However, when R-factors from 3 donors are transferred to K-12, a different sensitivity pattern emerges (Table 11) (Datta and Kontomichalou, 1965). The MIC values of cephaloridine in this table for K-12 infected with R-factors 1818 and 7268 are not very high, however, being some 4-8 times than for the original K-12 strain, whereas the substrate profiles in Table I indicate that this antibiotic is hydrolyzed at least as rapidly as ampicillin or benzylpenicillin. Medeiros and OBrien (1968) have shown that R-factors from different isolates mediate different levels of enzymic activity in the same recipient strains. Interestingly, their experimental findings also illustrate that increases in resistance of recipients to cephalosporins (and especially cephaloridine) are not necessarily high. Cephaloridine is believed to penetrate gram-negative bacteria readily, the p-lactamase activity of disrupted cell penetrations against this antibiotic being the same as that of whole cell suspensions (HamiltonMiller et a l . , 1965). This ratio, termed the permeability factor by Hamilton-Miller et a l . (1%5), is thus 1 for cephaloridine, in contrast to values of 1-18, 1-14, and 1-3 for benzylpenicillin, ampicillin, and 6APA, respectively, as found by these authors. Staphylococcal p-lactamase-stable penicillins, such as methicillin, cloxacillin, quinacillin, and nafcillin, may act as competitive inhibitors of p-lactamases produced by gram-negative bacteria on readily hydrolyzable substrates (Sabath et at!., 1965; Hamilton-Miller et a l . , 1965; O’Callaghan and Muggleton, 1967; O’Callaghan and Morns, 1972). Cloxacillin, methicillin, and nafcillin inhibit a cell-free P99 enzyme against cephaloridine, whereas only the first two penicillins “protect” cephaloridine from a cell-free R+ TEM P-lactamase, with only nafcillin inhibitory to a cell-free K. aerogenes K-1 enzyme (O’Callaghan and

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

105

Morris, 1972). The situation may, nevertheless, be rather different with whole cells, the degree of synergism being less than might be expected (O’Callaghan and Morris, 1972) and varying with difference in inoculum size (O’Callaghan and Morris, 1972; Russell, 1974). Destruction by plactamase, although an important factor in the resistance of gramnegative bacteria to cephalosporins, is most certainly not the only reason for their resistance (Sabath and Finland, 1967; Madeiros and O’Brien, 1968; Garber and Friedman, 1970) and accessibility of the sensitive transpeptidase is possibly of equal importance (as discussed in Section 111,C).

4. Antibiotic Combinations a. Cephalosporin + P-Lactam Antibiotic. In an attempt to increase the sensitivity of a strain (NCTC 8203) of P. aeruginosa to cephalosporins, Sabath and Abraham (1964) used a combination of a p-lactamaseresistant penicillin (methicillin or cloxacillin) with low intrinsic activity against this organism, with cephalosporin C, cephalothin, or cephaloridine and found that the synergistic response noted appeared to be due to the delay in the destruction of the hydrolyzable cephalosporin. Methicillin and, especially, cloxacillin considerably increase the activity of cephaloridine against several types of p-lactamase-producing gramnegative bacteria by preventing its destruction in vitro and in experimental animals (O’Callaghan and Muggleton, 1967; O’Callaghan et a l . , 1967). Cephalosporins that are p-lactamase inhibitors still protect plactamase-sensitive cephalosporins from inactivation (O’Callaghan et a l . , 1968, 1969). For synergism to occur between two p-lactam compounds against a certain bacterial strain, the following general criteria must be obeyed (Sabath, 1968; Hamilton-Miller, 1971a,b): (a) the strain must produce a p-lactamase; (b) the first p-lactam antibiotic must be hydrolyzable (hydrolyzable compound); (c) the second p-lactam compound (inhibitor) must be poorly, or not, hydrolyzed; ( d j the inhibitor must have a markedly greater affinity for the active center of the enzyme than the hydrolyzable antibiotic being protected and must be a stable competitive inhibitor of the p-lactamase in the presence of whole bacterial cells; and (ej the inhibitor must show no, or little, antibacterial activity at the concentration used. The p-lactamases from gram-negative bacteria may be distinguishable on the basis of their susceptibility to inhibition, because none of the potential inhibitors tested by O’Callaghan and Morns (1972) was a

D. R. OWENS ET AL.

106

potent inhibitor of all three enzymes tested, although nafcillin possessed the broadest activity. In addition, it must be added that an inhibitor may potentiate the activity of one p-lactam hydrolyzable antibiotic against a particular gram-negative strain but not of another p-lactam hydrolyzable compound against the same strain (Russell, 1974). Obviously, against intact cells, intrinsic resistance to a particular compound as well as drug inactivation must play a role here. The clinical significance of antibiotic combinations, including plactam combinations, has been discussed by Jawetz (1968). b. Cephalosporin Non- p-lactam Antibiotic. Inevitably, cephalosporins have been tested in the presence of other, no@-lactam antibiotics, kanamyin attempts to obtain a synergistic response. A cephalothin cin combination has been found to be synergistic against methicillinresistant, multiple antibiotic-resistant strains of S. aureus (Bulger, 1967a), Klebsiella strains (Bulger, 1967b), and against strains of E. coli (Kaplan and Koch, 1968), with bactericidal effects noted. Unfortunately, the reasons for this synergism remain unexplored. Enterococci are resistant to cephalosporins (see earlier); however, synergism is detectable i n vitro over a wide range of antibiotic concentrations with mixtures of streptomycin and benzylpenicillin, cephaloridine, cephalothin, or ampicillin. As a result of these findings, Fekety and Weiss (1967) have proposed that treatment with streptomycin plus one of these cephalosporins is worthy of consideration in patients with enterococcal endocarditis who are allergic to penicillin. The mechanism of synergism is again unknown, although the authors state that penicillin (and presumably, therefore, a cephalosporin also) rapidly enhances the intracellular uptake of streptomycin. Other examples of antibiotic combinations are provided later (see Section V).

+

+

c. MODEOF ACTION 1. Inhibition of Cell Wall Synthesis Cephalothin inhibits the incorporation of l y ~ i n e - ' ~ into C the cell wall, but not the cell protein fraction of S. aureus (Chang and Weinstein, 1964a). Cephalothin, cephaloridine, and cephalexin induce a considerable increase in the intracellular accumulation of N-acetylglucosamine in this organism (Chang and Weinstein, 1964a; Russell and Fountain, 1971). Both these findings suggest that the cephalosporins have a similar mode of action as the penicillins, involving an inhibition of cell wall

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

107

synthesis. In brief, mucopeptide (murein, peptidoglycan) synthesis involves the formation and utilization of two uridine mucleotides, uridine diphosphate (UDP) N-acetylmuramic acid (MurNAc) pentapeptide and UDP N-acetylglucosamine (GlcNAc) to give a linear polymer, involving the participation of a membrane-bound phospholipid ca.l-rier; a complete disaccharide pentapeptide subunit is formed, which is detached from the carrier. In S . aureus there is the addition of ammonia to the acarboxyl group of glutamic acid to form isoglutamine, and five glycine residues are added to form a chain substituted on the €-amino group of lysine. A transpeptidation reaction then takes place, whereby cross-links are formed. The entire process is summarized in Fig. 3, which also shows the proposed site of action of cephalosporins (Strominger et al., 1967). However, Hartmann et al. (1972) have proposed that penicillin blocks the hydrolysis of two murein hydrolases from E. coli, i.e., ( a ) an endopeptidase, which has the properties of a transpeptidase and which may be predominantly involved in cell division, and (b) a glycosidase, which acts on the polysaccharide chains of murein, and which may be predominantly involved in cell elongation. Interestingly, it was found that penicillin concentrations affecting cell elongation and glycosidase activity were of the same order of magnitude, and likewise that there was a close similarity between penicillin concentrations stopping cell division and those blocking endopeptidase activity. In this context, it is thus important to note that a new penicillin, FL 1060, appears to have novel properties in that it does not inhibit D-Ala carboxypeptidase, transpeptidase or endopeptidase (Park and Burman, 1973). Although FL 1060 induces spherical forms, it does not induce filaments at any concentration (Greenwood and O’Grady, 1973a). By contrast, cephalexin induces filaments over a wide range of concentrations, and it has thus been proposed (Greenwood and O’Grady, 1973b) that, whereas FL 1060 might act by inhibiting the glycosidase, the filament formation, i.e., an inhibition of septum formation, induced by cephalexin results from an inhibition of endopeptidase.

2 . Uptake and Cellular Permeability The binding of radioactive penicillin to bacterial cells (S. aureus) has been extensively studied (see the review by Russell, 1969). Radioactive cephalosporins are not often available, and thus their binding to bacteria has to be measured by an indirect method, which involves preexposure of the cells to ordinary e2C) cephalosporin drugs, followed by exposure to benzylpenici1lin-l4C. If the cephalosporin is irreversibly bound to the

1. F o rm a t i on of Linear Po ly m e r UDP- MurNAc

I

pentapeptide

MurNAc-P-P-lipid

attachment to membranebound phospholipid carrier

I

Pentapeptide z-GlcNAc

Lipid P

LLipid-p-p GlcNAc-MurNAc- P- P-lipid Pentakptide

(In S .aureus, stepwise addition of G5)

GlcNAc-MurNAc-acceptor (in wall)

I

Pentapeptide (+G 5 in S. aureus )

2. Cross-LWring (a) Staphylococcus aureus

Glyco ptide

Glycopeptide

MurNAc

M ~ N A

'I"

L-Ala

L-Ala

D - G ~

-. '.

~-Lys-G5 D-Ala

Cephalosporin---

I

\,

tTranspeptidase o-; Glycopeptide

MurNAc

D-G~u ~-Lys-G5 o-Ala

--=La Gly copeptide MU~NAC

L-Ala

L-Ala

D-G~u

D-G~u

I

--- D - A l a - ~ - L y s - G 5-

D-

Na-L-hys-G 5

+ Po- A la (b) Escherichia coli

I

GlcNAc-MurNAc-L- Ala-o-Glu- meso-DAP-o- Ala-o- Ala

/

/

-

+

GLcNAC- MurNAc- L- Ma-D -Glu- m es o - DAP-0- Ala- D- Ala

/

Cephalosporin---

/

t t

- - transpeptidase

GlcNAc-MurNAc-L-Ala-o-Glu-meso -DAP-D-Ala

/ I / GLcNAc-MurNAc-L-Ala-o-Glu-meso-DAP-o-Ala-D-Ala / Cephalosparin---

/

-- D-Ala

+ D- Ala

carboxypeptidase

GlcNAc-MurNAc-L- Ala-o-Glu-weso -DAP-D- Ala

I / / GlcNAc-MurNAc-L-Ala-o-Glu-meso-DAP-o-Ala+ o-Ala /

FIG. 3. Peptidoglycan (murein, mucopeptide) synthesis in Staphylococcus aureus and Escherichia coli and sites of inhibition by cephalosporins. Abbreviations: UDP, uridine diphosphate; UMP, uridine monophosphate; GlcNAc, N-acetylglucosamine; MurNAc, N acetylmuramic acid; D-G~u, D-glutamic acid; L-LYS, L-lysine, D-Ala, D-alanine; G5, pentaglycine chain; meso-DAP, meso-diaminopimelic acid.

T H E C E P H A L O S P O R I N GROUP O F A N T I B I O T I C S

109

binding site, the subsequent uptake of penicillin-14C will be prevented. Edwards and Park (1969) have shown that the concentration of a test cephalosporin which caused a 50% inhibition of penicillin-14C uptake correlated very closely with the MIC of that cephalosporin. A similar technique has been used by Retsena and Ray (1972), who found that the extent of penicillin-14C binding by a competing cephalosporin-12C (or other penicillin) was a function of pH and that it could be correlated both with the net charge of the competing lZC molecule and the net charge of the S. aureus cell at a given pH. The ability of a cephalosporin or penicillin to compete for ben~ylpenicillin-'~Cbinding sites on the bacterial cell appears to be correlated with the hydrophobic nature of the molecule (Retsena and Ray, 1972), the more lipophilic penicillins exerting the greatest effect against S. aureus (Biagi et a l . , 1970). Hamilton-Miller et al. (1965) proposed the existence of permeability (accessibility) barriers in gram-negative bacteria to various p-lactam antibiotics except cephaloridine, and in this context it is of interest to note that the MIC of cephaloridine against a strain of E. coli was virtually the same in the presence and absence of EDTA (Fountain and Russell, 1970), a substance that is believed to increase the permeability of these cells (Russell, 1971).

3. Induction of Morphological Variants Penicillins have, for some considerable time, been known to induce morphological variants in gram-negative bacteria (for a review, see Russell, 1969). Likewise, long filaments, spheroplasts and L-forms are produced on exposure of gram-negative bacteria to cephalosporin C, cephalexin, cephalothin, cephaloridine, and cephaloglycin (Bond et d . , 1962; Chang and Weinstein, 1964b, 1966; Kagan et al., 1964; Kagan, 1965; Burdash et a l . , 1968; Russell, 1968; Fountain and Russell, 1969; Muggleton et al., 1969; D. G. Smith, 1969; Lorian and Sabath, 1972). It must also be noted, however, that studies with the scanning electron microscope have indicated a spectrum of morphological changes induced by inhibitors of protein synthesis similar to those induced by cephalothin (Klainer and Perkins, 1970, 1972; Fass et a l . , 1970a,b; Perkins and Miller, 1973). Naturally resistant organisms are unaffected, and organisms made cephalosporin-resistant may undergo changes in cellular form which are qualitatively the same but less intense than those induced in the parent cells (Chang and Weinstein, 1964b). Certainly, much higher concentrations of a cephalosporin are needed to induce spheroplasts in laboratory-induced cephalosporin-resistant cells (Fountain and Russell, 1970).

110

D. R. OWENS ET AL.

Electron micrographs of cephalothin-induced long forms of P. vulgaris indicate that the only major difference between them and normal cells is in the length (Burdash et al., 1968), although Muggleton et al. (1969) consider that cephalexin modifies the cell surface of E. coli and inhibits the formation of fimbriae. Electron micrographs reveal that the cephalosporins inhibit cell division and the formation of transverse cell walls (Burdash et al., 1968; Muggleton et al., 1969; Russell and Fountain,

1970). Gram-negative bacteria that produce large amounts of p-lactamase would, by the nature of the high MIC values of ampicillin and some cephalosporins, be expected to be resistant to these p-lactam drugs; nevertheless, depending on the organism and the p-lactam drug under test, morphological variants similar to those described above may be produced (Russell, 1973a). Cephalosporins are not inhibitory to the growth of methicillin-induced or lysostaphin-induced spheroplasts of S. aureus (Watanakunakorn et al., 1969), of L-forms of staphylococci (Kagan et al., 1964; Kagan, 1965) or of gonococci (Roberts, 1966), or of protoplasts of B. megaterium (Fountain and Russell, 1969). 4. Lysis

Concentrations of a cephalosporin above its MIC will induce lysis of actively growing gram-negative bacteria. This has been observed with cephaloridine for E. coli by Lambin and Bernard (1967) and with cephaloridine, cephalexin, and cephaloglycin for E. coli and other Enterobacteriaceae by Fountain and Russell (1969) and Russell and (1970). The presence of a sufficient concentration of a Fountain stabilizing agent, such as sucrose, prevents lysis leading to the formation of osmotically sensitive spheroplasts, as described above.

5. Conclusions Current theories on the mode of action of penicillin consider that this antibiotic binds to, and inactivates, cell wall murein transpeptidase (Park et al., 1971). The findings described in the foregoing suggest that cephalosporins have a similar mechanism of action. Nevertheless, differences in morphological response in ten strains of P. mirabilis exposed to benzylpenicillin (very long forms up to 93 pm) as compared to cephaloridine, cephalothin, and cephaloglycin (maximum long form, 14 pm) have been reported by Lorian and Sabath (1972), who suggest

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

111

that long filamentous forms could be a consequence of the selective inhibition of D-Ala-carboxypeptidase, the cephalosporin then specifically inhibiting cell septation at concentrations well below the level needed to inhibit transpeptidase.

IV. Pharmacology and Toxicology The biological activity of an antibiotic is characterized by selectivity; ideally such a compound has no effect on the host but exerts a profoundly toxic effect upon the foreign organism. It is not surprising, therefore, that a successful antibiotic possesses few pharmacological (pharmacodynamic) and toxicological properties in animals and man and that these are observed normally only at high doses or concentrations. An important aspect of the pharmacology of antibiotics is their fate in the body. In this, a correlation of the rate and extent of absorption, distribution, metabolism, and excretion processes (pharmacokinetics) to the therapeutic utility of the antibiotics in animals and man is attempted. Such knowledge allows for the intelligent planning of suitable dosage regimens and routes of administration. As well as the antibacterial spectrum, the characteristics of distribution and excretion are important for the selection of an antibiotic for a particular therapeutic purpose. Thus in cases of biliary tract infection, it would be logical to employ, ceteris paribus, an antibiotic that is extensively excreted by this route in an active form. The binding of a drug to plasma protein, in addition to being a factor potentially affecting the availability of the drug to its site of action, may also be a site of interaction with other drugs by competition or displacement from the binding. Information on tissue localization is useful in alerting investigators during the preliminary evaluation of a new antibiotic to possible therapeutic applications or to potential sites of toxicity, e . g . , penetration into the eye and the fetus. Knowledge of the penetration of an antibiotic into soft and dense tissue is of obvious importance for the successful treatment of infection at these locations. Whereas a complete study of drug distribution can be readily made in animals, this is not so for man. However, a useful general guide for the distribution of a drug in the various body compartments is given by the apparent volume of distribution. It is to be expected that greater use of this and other pharmacokinetic parameters will be made in clinical studies of the newer antibiotics.

112

D. R. OWENS ET AL.

A. CEPHALORIDINE 1. Absorption, Distribution, and Excretion Although cephaloridine is better absorbed than cephalothin from the gastrointestinal tract, only low levels (about 0.9 &ml) of this antibiotic are found in the plasma after a 500-mg dose (Griffith and Black, 1971). Because of this a n d . the existence of orally active cephalosporins, cephaloridine is administered only parenterally. Following intramuscular injection of 500 mg cephaloridine, peak plasma levels of 15 &ml are obtained (Kislak et al., 1966). After 1 gm intramuscularly, peak cephaloridine concentrations in plasma of 10-30 &ml occur in 0.5 to 1 hour (Apicella et al., 1966b) and significant amounts of the antibiotic can be detected for up to 6 hours. Peak concentrations of the drug in the plasma of pregnant women appear to be reached slightly more slowly (Barr and Graham, 1967b). After equivalent doses, plasma levels of cephaloridine are about twice those of cephalothin measured at several times (Benner et al., 1966) and are more sustained. When administered by intravenous infusion at 0.5 gm/hour, the plasma concentration of cephaloridine continues to rise beyond 3 hours. However, a constant plasma level of 24.7 pg/ml is obtained when an initial loading dose (0.35 gm) is followed by an infusion rate of 0.25 gm/ hour (Kirby et al., 1971). Plasma levels of cephaloridine are slightly increased and antibacterial activity persists much longer when probenecid is administered concurrently with the drug (Kaplan et al., 1967; Tuano et al., 1967). In patients with chronic uremia, plasma levels are elevated above the normal range and persist at higher levels for a prolonged time following cephaloridine therapy (Perkins et al., 1969b). In normal volunteers, the plasma half-life of cephaloridine, after a single intramuscular dose, is 1-1.5 hours (Kunin and Atuk, 1966; Pryor et al., 1967). It is possible that the half-life of this antibiotic may be longer in patients over the age of 50 years than in the under-50 age group (Apicella et al., 1966b). When determined following a steady state achieved by continuous intravenous infusion of cephaloridine, the halflife in plasma has been found to be 1.12 hours (Kirby et al., 1971). Early investigators (Barber and Waterworth, 1964; Muggleton et al., 1964; Kunin and Atuk, 1966) concluded that the binding of cephaloridine to serum protein was very low because the i n vitro activity of this antibiotic was not affected by serum. However, more recently, using ultrafiltration techniques it has been shown that cephaloridine is 20% bound to serum protein (Kind et al., 1969a).

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

113

From the limited studies made, it appears that cephaloridine is fairly widely distributed in human body tissues and fluid compartments. Substantial concentrations of the drug have been found in liver, spleen, stomach wall, and lung (Apicella et al., 1966b). A smaller amount was detected in the cerebral cortex. Cephaloridine is detected in pleural effusions in fairly high concentrations (Murdoch et al., 1964). After a dose of 1 gm cephaloridine, no antibacterial activity is detected in the aqueous humor. However, up to 28 d r n l is found in the secondary aqueous humor that refills the eye following aspiration of primary fluid (Records, 1969). Cephaloridine is capable of penetrating fibrin clots in higher concentrations than cephalothin because of the higher plasma concentrations and longer half-life of cephaloridine (O’Connell, 1971). Penetration of cephaloridine into the cerebrospinal fluid (CSF), in the absence of meningeal inflammation, is negligible (Brayton et al., 1967; Steigbeigel et al., 1968). In the presence of severe renal impairment and, therefore, of high plasma levels, cephaloridine is found in concentrations of 6 to 12% of the serum levels in the CSF of patients with normal meninges (Kabins and Cohen, 1966; Gonnella et al., 1967; Gabriel et al., 1970); in patients with meningeal inflammation, there is a greater penetration of the drug into the CSF (Lerner, 1971). However, penetration of cephaloridine into CSF is not so consistently related to CSF protein content as it is with cephalothin. When cephaloridine is given to pregnant women in active labor, the drug crosses the placenta and is found in the serum of the newborn at concentrations up to 54% of the level in the maternal serum (Barr and Graham, 1967b; Arthur and Burland, 1969). Levels are measurable in neonatal serum up to 22 hours postpartum. Following a 1-gm intramuscular dose to pregnant women, a peak of cephaloridine in cord blood occurs at about 4 hours. The concentration of cephaloridine in amniotic fluid also rises slowly, taking about 3 hours to reach an effective antibacterial concentration after this dose (Barr and Graham, 196733). Cephaloridine is widely distributed in various tissues of the rat and rabbit including brain and spinal fluid, aqueous humor, fetus, and milk (Welles et al., 1966). The highest concentrations occur in the kidney and 4 hours after administration the kidney/serum concentration ratio for cephaloridine is 30. In the rabbit, cephaloridine penetrates into the cornea, aqueous and vitreous humors, and optic nerve following its subconjunctival injection (Moll, 1970). Following an intramuscular injection of cephaloridine, 40-100% of the dose can be recovered in human urine (Benner et al., 1966; Kislak et al., 1966; Kaplan et al., 1967; Griffith and Black, 1971). The major

114

D. R. OWENS ET AL.

portion of this is excreted in 6 hours (Benner et al., 1966) and, after a 1gm dose, urine concentrations of this cephalosporin are 286-1024 &ml in this time period (Kaplan et al., 1967). Excretion occurs primarily through the glomeruli, and renal clearance is approximately the same as the creatinine clearance (Kirby et a l . , 1971) but some renal tubular secretion is also present because probenecid slightly increased plasma levels and slightly decreased renal clearance of this antibiotic (Kaplan et al., 1967; Tuano et a l . , 1967). Normal kidney can excrete 4-6 gm total dose of cephaloridine daily, but this amount decreases over the age of 50 years (Apicella et a l . , 196613). Elimination of cephaloridine is very dependent on renal function, the plasma half-life becoming more and more prolonged as creatinine clearance decreases below 20 &minute. Thus, in patients with little or no kidney function, the half-life is 20-25 hours (Kabins and Cohen, 1966; Kunin and Atuk, 1966; Pryor et a l . , 1967). The bile is a minor pathway of excretion for this cephalosporin, concentrations of cephaloridine in bile being at most 50% of serum levels (Apicella et a l . , 1966b; Acocella et al., 1968; Brogard et a l . ,

1972). In animals, renal excretion of cephaloridine is solely by glomerular filtration, except in the hen where a small but significant tubular secretion occurs (Child and Dodds, 1966; Welles et a l . , 1966). In perfused rabbit liver, the concentrations of cephaloridine in the bile are less than the concurrent serum levels (Brogard et al., 1972).

2. Metabolism The greatly increased plasma half-life of cephaloridine in patients with renal failure (Kabins and Cohen, 1966) suggests that this antibiotic is stable in the body. In fact, no degradation products of the drug have, as yet, been identified in human urine. This apparent resistance to metabolic transformation may be related to the replacement of acetyl (as in cephalothin) by pyridyl on C-3 of the cephalosporin nucleus. However, Kunin and Atuk (1966) observe that, although cephaloridine is retained in the presence of renal failure, this is not for nearly a s long a period of time as are drugs that depend almost entirely on the kidney for excretion, e.g., streptomycin and kanamycin. These authors suggest that a significant proportion of cephaloridine is inactivated by nonrenal mechanisms. Although results from perfusion experiments with rabbit liver suggest that cephaloridine may be fixed and inactivated in this organ (Brogard et

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

115

al., 1972), it is possible to recover 92% of an intravenous dose as unchanged drug in dog urine (Welles et al., 1966).

3. Toxicology Cephaloridine is painless when administered intramuscularly, and it may be given by this route over long periods of time thus avoiding the general hazards of continuous therapy (Lerner, 1971). This may be related, in part, to the greater solubility of cephaloridine allowing for a small injection volume (Turck et al., 1967). This cephalosporin is an internal salt, a betaine, containing neither Na+ nor K+. Thus it has the advantage of not overloading patients with cations if this is contraindicated clinically. Most patients receiving cephaloridine experience no adverse effects (Murdoch et al., 1964; Stewart and Holt, 1964; Benner et al., 1966; Cohen et al., 1966; Kislak et al., 1966) and the side effects noted are usually quite minor. One case of the appearance of an extrapyramidal syndrome after cephaloridine has been reported (Mintz et al., 1971); this is not considered to be an allergic reaction. In this connection it is interesting to note that intrathecal administration of cephaloridine can give rise to hallucination, confusion, and nystagmus (Murdoch et al., 1964). Cephaloridine is well tolerated when given to mothers prior to amniotomy (Barr and Graham, 1971). A transient rise of SGOT has been observed in some patients receiving this antibiotic (Dennis et al., 1966; Hermans et al., 1966). Cephaloridine may also elevate prothrombin time, but this effect is readily reversible and does not lead to hemorrhage (Hermans et al., 1966). In cases of reduced renal function, it has been observed that treatment with cephaloridine is followed by further deterioration of kidney function in a small proportion of patients (Cohen et al., 1966; Holloway and Scott, 1966; Kislak et al., 1966). It was not possible to determine whether these changes were due to the antibiotic or to the progression of the disease. However, there is accumulating evidence that cephaloridine has a nephrotoxic action particularly when the drug is given in doses producing high plasma levels, i.e., 15(1-200 &ml (Lawson et al., 1970) or where kidney function is already impaired (Benner, 1970; Foord, 1971). In elderly patients with an apparently normal renal function, there is an increase in hyaline casts in urine when large daily doses (6 gm) of cephaloridine are administered (Linsell et al., 1967). The production of casts returns to normal on withdrawal of the drug. Fleming and Jaffe (1967) have also reported the presence of hyaline casts without accompanying proteinuria in children receiving

116

D. R. OWENS ET AL.

high doses of cephaloridine. These authors find that cephaloridine (200 mg/kg) administered to rabbits produces proximal tubular necrosis and shedding of casts into the tubules. The nephrotoxic activity of cephaloridine is well-documented in animals (Atkinson et al., 1966a; Child and Dodds, 1966, 1967; Perkins et al., 1968), but the dose required to induce proximal tubular necrosis of the kidney varies with species, the rabbit being the most sensitive. From the results of a detailed study in rabbits by light and electron microscopy (Silverblatt et al., 1970), it appears that the first changes to be seen in the kidney are in the brush border of the outermost proximal tubules In a review of cases of renal failure associated with administration of cephaloridine (Foord, 1969), it was observed that in several patients there had been coincidental administration of frusemide, and it was suggested that this diuretic might enhance the nephrotoxicity of the antibiotic. Studies in rats and rabbits have confirmed that such an interaction occurs (Dodds and Foord, 1970; Lawson et al., 1970, 1972). In these experiments, nephrotoxicity developed at plasma levels of cephaloridine previously regarded as nontoxic. The mechanism of this interaction has not yet been elucidated, although involvement of the renin-angiotensin system has been suggested (Lawson et al., 1972). It has also been proposed that frusemide may aggravate the precipitation of cephaloridine from concentrated solution and the polymerization of the drug in the renal tubules, both of which events could be responsible for damage to the brush borders (Boyd et al., 1971). In the light of these findings it is now the clinical practice to reduce the dosage of cephaloridine adequately in the presence of preexisting renal impairment and also in the elderly and to avoid concurrent administration of frusemide. As determined in animals, the pharmacodynamic effects of cephaloridine are negligible. At high doses there is a transient fall of blood pressure related to a ganglion blocking action (Atkinson et al., 1966b).

.

B. CEPHALOTHIN 1. Absorption, Distribution, and Excretion

Cephalothin is available as a water-soluble sodium salt for parenteral administration. It is poorly absorbed by mouth and, after a dose of 500 mg, no or just assayable levels of the drug are detected in the serum

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

117

(Griffith and Black, 1971). When given intramuscularly, 500 mg of cephalothin is rapidly absorbed, peak concentrations of 7.6 to 10 &ml of serum being obtained in 0.5 hour (Griffith and Black, 1964, 1971). A doubling of the dose increases the peak concentration by the same factor, and measurable serum levels of the drug persist for 4 hours after this higher dose. When infused intravenously at 0.5 gm/hour, constant plasma levels of 10 to 30 pg/ml are reached after 0.5-2.5 hours (Tuano et al., 1967; Griffith and Black, 1971). Plasma concentrations of cephalothin are elevated above the normal range when probenecid is also administered (Griffith and Black, 1968) and in the presence of renal insufficiency (Kabins and Cohen, 1965). After a dose of 12.5 mg/kg, distinctly higher and more sustained blood levels of cephalothin are observed in full-term newborn and premature infants than in older infants and children (Sheng et al., 1965). In the healthy adult, after a single intramuscular or intravenous dose, the plasma half-life of cephalothin vanes between 0.5 and 0.85 hour (Kabins and Cohen, 1965; Kunin and Atuk, 1966; Naumann, 1971). The half-life is somewhat shorter (0.47 hour) when determined after a steadystate plasma level has been reached by means of a constant intravenous infusion (Kirby et al., 1971). As determined by ultrafiltration, cephalothin is 65% bound to plasma protein (Kirby et al., 1971). The apparent volume of distribution appears to be slightly larger for cephalothin than for either cephaloridine or cephalexin (Kirby et al., 1971). The differences in plasma level between these three antibiotics is greater than can be accounted for by the differences in apparent volume of distribution and probably relate to differences in renal and nonrenal clearance rates. Cephalothin is widely distributed in the body, high concentrations being found in renal cortex, pleural fluid, skin, striated muscle, myocardium, and stomach wall (Perkins and Saslaw, 1966). A lower concentration was detected in the liver. Bactericidal amounts of the drug are found within a short time of administration in ascitic (Kabins and Cohen, 1965), pericardial, and synovial fluids (Gump and Lipson, 1968). The drug does not cross the normal blood-brain barrier as no cephalothin is detectable in the CSF of patients without meningitis. However, where the meninges are inflammed, cephalothin is present in the CSF at concentrations of 0.16 to 0.31 pg/ml with concomitant plasma levels of 10-80 &ml (Vianna and Kaye, 1967). Drug concentrations in CSF up to 5 &ml have been observed in patients with CSF protein in excess of 50 mg/100 ml (Lerner, 1969). Cephalothin traverses the placenta, and significant bactericidal concentrations are found in cord serum and amniotic fluid within 15

118

D. R. OWENS ET AL.

minutes of administration (MacAulay and Charles, 1968; Paterson et al., 1970; Hirsch, 1971). The peak concentration of cephalothin in cord blood occurs later than in the maternal blood (Sheng et al., 1965). At the time of peak concentration in cord blood the levels are 2550% of those in the maternal circulation. Cephalothin has been found in fetal urine in concentrations up to 100 d m l (Morrow et al., 1968). A similar pattern of distribution of cephalothin has been found in animals (Lee and Anderson, 1963; Perkins et al., 1968). In addition, this antibiotic has been shown to penetrate into the aqueous humor in low concentration (Uwaydah and Faris, 1970). In rats, using 14C-labeled cephalothin, radioactivity was found in bone soon after subcutaneous administration (Kanyuck et al., 1971); this had a similar half-life to the cephalothin in serum. Cephalothin is rapidly cleared by the human kidney (Griffith and Black, 1971), and there is no evidence of accumulation when intravenous doses of 4 gm are given every 8 hours for 32 hours. Even in cases of severe impairment of creatinine clearance, there is little accumulation of the drug (Kabins and Cohen, 1965). However, in patients with serious impairment of the renal clearance mechanism, Benner (1970) has found that high serum concentrations of cephalothin occur when large doses of the antibiotic are administered for several days. In the newborn infant, cephalothin is quickly excreted (Sheng et al., 1965). In adults, following 500 mg intramuscularly, 60-90% of the administered dose appears in the urine within 6 hours (Griffith and Black, 1971). A mean recovery of 75.6% in 24-hour urine has been reported (Naumann, 1971). The secretion of this antibiotic involves a significant renal tubular component as may be seen from the clearance rate which is over twice that of creatinine (Kirby et al., 1971) and from the marked reduction of clearance when probenecid is administered (Tuano et al., 1967). Although the bile is only a minor excretory pathway for this cephalosporin, cephalothin is concentrated in gall bladder bile to the extent of 5 to 100 times the serum concentration (Rain, 1971; Brogard et al., 1973b). A pattern and mode of urinary excretion similar to that in man has been shown for the drug in animals (Lee et al., 1963).

2 . Metabolism The major route of metabolism following parenteral administration of cephalothin to man is by hydrolysis to 0-deacetylcephalothin (Lee et al., 1963). This metabolite is considerably less active in vitro than cephalothin, particularly against gram-negative bacteria (Kirby et al., 1971).

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

119

The esterase capable of hydrolyzing cephalothin is present in human tissues, principally the liver, but insignificant amounts occur in the serum (Lee et al., 1963). The metabolite is excreted into urine in varying proportions together with unchanged cephalothin (Lee et al., 1963). In the 6-hour urine, after a single intramuscular dose of 1 gm cephalothin, approximately 65% of the excreted cephalosporin is as the parent compound and 35% as deacetylcephalothin (Griffith and Black, 1971). In uremic patients receiving. cephalothin, the antibacterial activity of the serum is characterized by two half-lives (Kabins and Cohen, 1965)-a rapid early decline in serum levels with a half-life of about 3 hours followed after 8 hours by a slower decline with a half-life of 12 to 18 hours. One explanation of this is that the first phase represents the clearance of the parent compound, whereas the second phase represents chiefly the deacetyl metabolite. Recently, the plasma levels of cephalothin and deacetylcephalothin were measured in an uremic patient receiving 1 gm of the drug intravenously (Kirby et al., 1971). While cephalothin levels fell rapidly with a half-life of 2 hours, concentrations of deacetylcephalothin increased over the first 12 hours and then declined slowly with a half-life of approximately 8 hours. It has also been observed that repeated dosage with cephalothin leads to an accumulation of deacetylcephalothin in the blood of an uremic patient (Kirby et al., 1971). In rabbits with reduced renal function, 45-90% of the antibacterial activity of plasma is in the deacetylated form 1 hour after cephalothin administration (Venuto et al., 1972). The metabolism of 14C-labeled cephalothin has been studied in the rat. After oral administration, the drug is degraded in the gut and the products are absorbed (Sullivan and McMahon, 1967). The urine contains only a trace of deacetylcephalothin and no parent compound. However, 32% of urinary radioactivity is present as thienylacetylglycine and 13% as thienylacetamidoethanol. By contrast, when cephalothin-14C is given intraperitoneally the major urinary metabolite is the deacetylated compound (Sullivan et al., 1971). Only a small proportion of urinary radioactivity is present as thienylacetylglycine, a metabolite that is not found in human urine. A long-lived minor metabolite occurs in rat blood and appears to be an albumin-cephalothin complex.

3. Toxicology Because of poor oral absorption, cephalothin must be given parenterally. However, intramuscular administration is painful and in some

120

D. R. OWENS ET AL.

cases the local irritation may progress to induration or, rarely, sterile abcess or slough (Weinstein and Kaplan, 1970). Hence, the intravenous route is often preferred for large doses. By this latter route, thrombophlebitis may occur with continuous infusions of doses greater than 6 gm daily (Perkins and Saslaw, 1966). Cephalothin is administered as its sodium salt which contains 55 mg Na+/gm. Thus, if large doses are given to patients with severe renal impairment, there is the possibility of overloading the patient with this cation. The incidence of adverse reactions in patients treated with cephalothin is low. These appear to result largely from mild-to-moderate hypersensitization (Davis et al., 1964; Heitler et al., 1964; Weinstein et al., 1964; Kabins and Cohen, 1965; Molthan et al., 1967). Cephalothin has been extensively investigated clinically since 1963, and it is generally concluded that this antibiotic does not possess a nephrotoxic action in the doses employed therapeutically (Maynard, 1969; Benner, 1970) or at least that the risk of renal injury is very much less likely than it is from cephaloridine therapy (Perkins et al., 1968; Rahal et al., 1968; Turcotte et al., 1970). It has been observed that, in certain patients with impaired renal function and with high serum concentrations of cephalothin, deterioration of renal function occurs (Maynard, 1969; Benner, 1970 ). However, the renal necrosis in these patients could well occur from other factors such as congestive heart failure, low cardiac output, or unrecognized hypertension. There is as yet, no evidence suggesting an interaction between cephalothin and diuretics such as frusemide (Lawson et al., 1970). However, cases of deterioration in renal function after treatment with cephalothin in combination with other potentially nephrotoxic antibiotics has been observed (Thoburn and Fekety, 1970). Rabbits receiving large daily doses (500 mg/kg) of cephalothin for 3 weeks showed only slight histological changes in the kidney (Perkins et al., 1968). These consisted of minimal swelling or hydropic changes of the tubular epithelium. No renal histological changes were seen in monkeys similarly treated. Even in rabbits with an extensive reduction in glomerular filtration rate, subacute administration of cephalothin (300 mg/kg, daily) produced no further deterioration in renal function (Venuto et al., 1972). In rats with mild transient renal impairment induced by glycerol, cephalothin, at concentrations in plasma of 113 pg/ml, produced extensive acute tubular necrosis when combined with frusemide (Lawson et al., 1972). Until further evidence is produced it would appear to be prudent to regard cephalothn as being potentially nephrotoxic in

THE CEPHALOSPORIN GROUP O F ANTIBIOTICS

121

man when at high plasma concentrations andfor in combination with diuretics. In animals cephalothin has minimal pharmacodynamic properties. Perrelli and Tempesta (1968) have demonstrated a slight transient decrease in diastolic and systolic pressures after injection of the drug.

C. CEPHALEXIN 1. Absorption, Distribution, and Excretion In cephalexin the substituent at C-7 of the cephalosporin nucleus is the same as that at C-6 in ampicillin. Cephalexin is a zwitterion and at pH 3.8 exists as an inner salt. It is used clinically as the monohydrate. The drug is stable in gastric acid, and, following oral administration to normal fasting subjects, absorption is rapid (O’Callaghan et al., 1971), peak plasma levels being obtained at about 1 hour. The magnitude of the peak is dose-related, and, after 500 mg cephalexin, peak levels of 13 to 18 pg/ml are reached (Braun et al., 1968; Griffith and Black, 1968, 1971; Levison et al., 1969; Meyers et al., 1969). Measurable concentrations persist in plasma for 4 to 6 hours. Absorption takes place primarily from the upper portions of the gastrointestinal tract (Griffith and Black, 1970). However, in animals, absorption from the rectum is also possible (Welles et al., 1969). The drug is well absorbed from the gastrointestinal tract following partial gastrectomy, but poor absorption may occur in patients with obstructive jaundice and pernicious anemia (Davies et al., 1970). Absorption of cephalexin is delayed when the drug is given with or shortly after food (Griffith and Black, 1968, 1971; Gower and Dash, 1969; O’Callaghan et al., 1971). The peak level, which is about 50% of that observed in the fasting state, is reached after 2 hours. However, there is a prolongation of plasma levels, and total absorption of cephalexin is not decreased. The presence of very small amounts of food does not interfere with the absorption of this cephalosporin (Thornhill et al., 1969). Delayed absorption of cephalexin is also observed in some anephric patients (Reisberg and Mandelbaum, 1971). Following a constant intravenous infusion at a rate of 0.5 gmthour, a steady-state cephalexin concentration of 27 pg/ml in plasma is reached in 2 hours (De Maine and Kirby, 1971). There is no accumulation of cephalexin on repeated oral administration (Griffith and Black, 1968, 1971) unless renal insufficiency is present (Levison et a l . , 1969). Administration of probenecid increases the height and duration of antibacterial activity in the plasma after a dose of cephalexin (Braun et al. 1968; Griffith and Black, 1968; Gower and Dash, 1969).

122

D. R. OWENS ET AL.

In newborns and infants up to the age of 6 months, absorption of cephalexin is delayed, and peak plasma levels are lower compared with older children (Marget and Daschner, 1969; Marget, 1971). When administered after bottle feeding, cephalexin is more slowly absorbed by infants compared with fasting infants (Fujii et al., 1969). In subjects with normal renal function, the plasma half-life of cephalexin is 0.9-1.2 hours after oral administration (Bergan et al., 1970; Kabins et al., 1970; Kunin and Finkelberg, 1970). When given by intravenous infusion to achieve a steady state, the half-life is 0.61 hour (Kirby et al., 1971). The longer half-life after oral administration is probably related to absorption of the drug still occurring while the halflife is being determined. The half-life of orally administered cephalexin in neonates and infants up to 12 months of age is prolonged in patients with impaired renal function (Clark and Turck, 1969; Kabins et al., 1970; Kirby et al., 1971; Regamey and Humair, 1971). In patients with a creatinine clearance below 2-5 muminute, the half-life of cephalexin is about 22 hours (Kabins et al., 1970). This may be reduced considerably by either hemodialysis (Linquist et al., 1970; Kirby et al., 1971) or peritoneal dialysis (Yamasaku et al., 1970). Cephalexin is bound to plasma protein to the extent of 15% (Kirby et al., 1971). The value for the volume of distribution of cephalexin in man suggests that the drug is well distributed in total body water (Orsolini, 1970) and, considered with the low plasma binding, it indicates a good accessibility to tissues of the body. This is confirmed by results of tissue assays, the drug being found in lung, spleen, liver, adrenal,pancreas, kidney, myocardium, stomach, appendix, omentum, vein, genital organs, and tumor tissue (Mizuno et a l , 1969; Orsolini, 1970; Griffith and Black, 1971). Concentrations of cephalexin in these tissues are lower than the plasma level, except for the kidney which is higher. Cephalexin is well distributed in the tissues of children (Simon et al., 1970). The drug is also found in breast milk (Mizuno et al., 1969), aqueous humor (Gager et al., 1969; Boyle et al., 1970), and pus (Shibata and Kato, 1969). In the latter, relatively high peak concentrations of the antibiotic are reached more slowly than those in plasma. No (or very small amounts of) cephalexin has been found in the CSF of patients with normal meninges receiving this cephalosporin (Bergan et al., 1970; Davies et al., 1970). The passage of cephalexin into the CSF of patients with meningeal inflammation has not, as yet, been reported. Cephalexin traverses the placenta rapidly to pass into the fetus (Mizuno et al., 1969; Paterson et al., 1972). Five hours after

T H E C E P H A L O S P O R I N GROUP O F ANTIBIOTICS

123

administration the antibiotic concentration in cord blood is higher than in the maternal circulation. The level of cephalexin in amniotic fluid rises gradually up to 7 hours. A similar pattern of distribution to that in man has been found in animals (Welles et al., 1969, 1970; Kanyuck et al., 1971; Berte et al., 1972). A detailed review of the absorption and distribution of cephalexin has recently been published (Speight et al., 1972). From the plasma profile of cephalexin levels in patients with renal failure, it is evident that the antibiotic depends on the kidney for excretion. After oral doses, 75-100% of the drug is excreted in the urine within 8 hours (Braun et al., 1968; Clark and Turck, 1969; Kind et al., 1969a; Meyers et al., 1969). This high recovery is evidence of the efficiency of absorption of orally administered cephalexin. Thus, the drug is excreted in high concentration; after an oral dose of 250 mg, mean urine concentrations are 830 &ml over 6 hours (Thornhill et al., 1969). In patients with poor renal function, excretion is reduced to 3.6% in 8 hours (Meyers et al., 1969), but even in such patients the urinary concentrations of cephalexin are adequate for the treatment of most urinary tract infections (Reisberg and Mandelbaum, 1971). After intravenous administration to patients with normal renal function, 80% of the dose is excreted in urine within 2 hours (Davies and Holt, 1972). In normal subjects the renal clearance ratio of cephalexin to creatinine is 1.7 (Kirby et al., 1971). The urinary excretion of cephalexin occurs partly through glomerular filtration and partly by tubular secretion (Foord et al., 1969b). Concurrent administration of probenecid delays the renal tubular secretion of cephalexin (Braun et al., 1968; Thornhill et al., 1969). The renal clearance of this antibiotic is increased by 15% when urinary pH is reduced to 4.8 (Asscher et al., 1970). However, this is considered to have no therapeutic importance. After doses of 500 mg and 1 gm cephalexin, antibacterial activity is detected in the bile at 2 and 4 hours in concentrations that are lower than the concurrent plasma levels, except in a case of obstructive jaundice where the biliary concentration was significantly higher (Sales et al., 1969). In animal experiments (Welles et al., 1969, 1970), it is found that recovery of cephalexin in urine is slightly less complete than in man. In the species studied renal clearance is both by glomerular filtration and tubular secretion. In the dog and rabbit, a significant tubular reabsorption of antibiotic occurs. Biliary excretion is low in the dog. Up to 15% of the dose of cephalexin administered is found in rat feces in 24 hours and this is largely the result of biliary excretion (Sullivan et al., 1969b).

124

D. R. OWENS ET AL.

2. Metabolism The antibacterial activity in human urine after administration of cephalexin is unchanged drug (Chou, 1969), and it is assumed that this cephalosporin is not metabolized. However, for similar reasoning applied to cephaloridine, because in cases of renal failure cephalexin is not retained in the body for nearly as long as antibiotics such as kanamycin, it is possible that metabolic inactivation of the compound may occur to some degree when raised plasma levels of the drug are prolonged. No evidence for the metabolism of cephalexin has been obtained from animal experiments (Welles et a l . , 1969; Sullivan et a l . , 1969b). 3. Toxicology The most frequent side effects (up to 2%) reported during cephalexin therapy are gastrointestinal disturbances such as diarrhea, nausea, vomiting, and abdominal cramp (Braun et a l . , 1968; Griffith and Black, 1968; Kind et a l . , 1969a; Levison et a l . , 1969; Page et a l . , 1970; Fass et al., 1970b). The incidence of allergic reactions, mainly rash or urticaria, is low (Griffith and Black, 1970; Landes et a l . , 1972) Although no nephrotoxicity from cephalexin has- been reported clinically, a slight vacuolar nephrosis has been found in some rabbits receiving a high dose of the antibiotic (4 gm/kg) (Welles et a l . , 1969, 1970). In 1 of 5 patients receiving a 1-week course of cephalexin, there was a fall in the creatinine clearance 2 weeks after the last dose of the antibiotic. However, renal function improved and the effect cannot be proved to be drug-related (Kunin and Finkelberg, 1970).

D. CEPHALOGLYCIN 1. Absorption, Distribution, and Excretion

An orally active cephalosporin, cephaloglycin (see Fig. 2) has a spectrum of antibacterial activity comparable to that of cephalothin and cephaloridine (Wick and Boniece, 1965). Unfortunately, cephaloglycin is not extensively absorbed from the gastrointestinal tract, only about onequarter of an orally administered dose being absorbed. This is in contrast to the nearly complete absorption of cephalexin (Griffith and Black, 1971) which is the deacetoxy analog of cephaloglycin. After an oral dose of 500 mg cephaloglycin, maximal concentrations of antibacterial activity in the blood occur at 2 hours, but these levels are

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

125

extremely low and variable. For example, levels that range from 0.2-0.9 p d m l (Perkins et al., 1969a) to 1.0-3.0 pg/ml have been reported (Applestein et al., 1968; Boyer and Andriole, 1968; Braun et al., 1968; Johnson et al., 1968; Pitt et al., 1968; Griffith and Black, 1971). In some cases no antimicrobial activity has been detected in the blood (Kunin and Brandt, 1968). Serum concentrations have been shown by Braun et al. (1968) to be significantly elevated and prolonged when probenecid is concurrently administered with cephaloglycin. However, other workers using probenecid have failed to observe any increase in the serum levels, although antimicrobial activity in the serum was prolonged (Pitt et al., 1968). Because of the low blood levels achieved with oral cephaloglycin, many workers have indicated that this drug should not be administered to patients with suspected bacteremia or with infections located outside the urinary tract (Ronald and Turck, 1967; Boyer and Andriole, 1968; Johnson et al., 1968; Kunin and Brandt, 1968; Ronald et al., 1968). Little is known about the distribution of cephaloglycin in man. However, in studies carried out in mice, following an oral dose of 200 mdkg, the concentrations of antibacterial activity are highest in the liver and kidney, being 1.9 and 3.2 times, respectively, greater than in the serum. Other tissues such as spleen, heart, lung, and skeletal muscle possess approximately one-half the activity of the serum (Welles, 1972). Cephaloglycin is bound to human plasma to the extent of 67% (Pruitt and Dayton, 1971). Urine collected from human volunteers over the first 8 hours after a single oral dose of 500 mg cephaloglycin shows concentrations of antibacterial activity ranging from 76 to 1330 pg/ml. Between one-fifth and one-third of the administered dose is accounted for in the urine in 8 hours (Braun et al., 1968; Pitt et al., 1968; Griffith and Black, 1971). 2. Metabolism The metabolism of 14C-labeled cephaloglycin has been studied extensively in rats by Sullivan et al., (1969a). Following oral administration of labeled material, the total recovery of radioactivity in 24 hours was 90%, of which 20% was in the urine and 70% in the feces. The urine contained one microbiologically active substance, identified as deacetylcephaloglycin. The biologically inactive compound D-2-phenylglycine was also present. Thus, cephaloglycin is metabolized in the rat by two pathways; deacetylation to form deacetylcephaloglycin and hydrolysis of the amide linkage to form D-2-phenylglycine. When administered in large doses, some unchanged material is also recovered in the urine.

126

D. R. OWENS ET AL.

These results, which indicate that cephaloglycin is substantially metabolized, contrast with results obtained for cephalexin which is not metabolized but is eliminated by the kidney in an unchanged form (Sullivan et al., 1969b).

3. Toxicology Toxicity does not constitute a major problem in therapy with cephaloglycin. No consistent alterations in blood cell counts, urinalysis, blood urea nitrogen, SGOT, and SGPT have been attributed to the drug (Ronald et al., 1968; Perkins et al., 1969a). However, Boyer and Andriole (1968) recommend that cephaloglycin should be used cautiously in patients with renal insufficiency. The most frequent adverse effects reported during cephaloglycin therapy have been gastrointestinal disturbances such as diarrhea, nausea, and vomiting (Ronald and Turck, 1967; Boyer and Andriole, 1968; Johnson et al., 1968; Ronald et al., 1968). A comparatively high incidence (in the order of 30%) of gastrointestinal tract reactions have been recorded when patients were administered oral cephaloglycin in daily doses of 2 gm for 10 days. Less common side effects associated with oral cephaloglycin therapy include skin rashes and eosinophilia (Boyer and Andriole, 1968; Johnson et al., 1968; Ronald et al., 1968).

E. OTHERCEPHALOSPORINS 1. Cephacetrile Like many other cephalosporin derivatives, cephacetrile (see Fig. 2) is poorly absorbed from the gastrointestinal tract. Therefore, to be therapeutically effective, the drug must be administered parenterally. In a study carried out in human volunteers by Luscombe et a l . (1972, 1973), a single intravenous dose of 500 mg cephacetrile produced mean serum levels of 30.7 &ml, 15 minutes after administration. Antibacterial activity rapidly disappeared from the blood and the serum half-life was calculated to be 33.2 2 4.2 minutes which is comparable with the half-life of cephalothin. A similar value (33.0 minutes) has been determined by other workers using intravenous doses of 1 gm cephacetrile (Brogard et al., 1973~).When determined following a steady state achieved with a continuous intravenous infusion of the drug, the serum half-life has been found to be 1.3 f 0.3 hours (Nissenson et a l . , 1972). Cephacetrile is readily absorbed from the intramuscular site, a peak serum concentration of 14.5 Fg/ml being observed 30 minutes after a 1-

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

127

gm dose. The serum half-life by the intramuscular route has been calculated to be 1.5 hours (Brogard et al., 1973~).Blood levels can be slightly increased and prolonged by administering probenecid concurrently with cephacetrile. In animal studies, Luscombe et al. (1972) showed that in rabbits receiving probenecid (30 mg/kg intravenously) immediately prior to the antibiotic, the serum half-life of antibacterial activity is increased by 79% over the half-life achieved with the same dose of cephacetrile but without probenecid (i.e., an increase from 22.1 to 38.6 minutes). Cephacetrile is 33-36% bound to human serum proteins (Luscombe et al., 1973) compared with 15-20% for cephalexin and cephaloridine and with 65% for cephalothin (Kind et al., 1969a; Kirby et al., 1971). Following intravenous and intramuscular injections of cephacetrile, between 70 and 85% of the dose is recovered in the urine in 10 hours (Luscombe et al., 1972; Brogard et al., 1973~).The phase of maximum urinary excretion of antibacterial activity occurs in the first 2 hours of the drug being administered, but small amounts of the active substance continue to be excreted for up to 10 hours. From renal and creatinine clearance studies performed by Nissenson et al. (1972), cephacetrile appears to be excreted by both glomerular filtration and tubular secretion in normal subjects, On the other hand, in patients with renal failure, there is little evidence of tubular excretion. Animal experiments using probenecid-treated rabbits confirm that under normal conditions, renal excretion of cephacetrile is accomplished by both glomerular filtration and active tubular excretion (Luscombe et al., 1972). Antibacterial activity in human urine has been determined to be due largely to unchanged material (Luscombe et al., 1973). Studies in normal subjects and rabbits show that the excretion of cephacetrile in bile is low (Luscombe et al., 1972; Brogard et al., 1973a,c). Nevertheless, in patients with renal failure, biliary excretion of antibacterial activity is considerably increased (Brogard et ul., 1973a). Following intravenous and intramuscular injections of 25 mdkg cephacetrile in rabbits, antibacterial activity can be detected in the CSF. However, the ratio of antibacterial concentration in the CSF to the concurrent concentration unbound in the serum is less than unity. Thus, cephacetrile does not appear to penetrate readily the blood-brain barrier (Luscombe et al., 1973). In toxicological studies in laboratory animals carried out by Kradolfer et al. (1971), cephacetrile exhibited a low degree of toxicity. The acute intravenous LD,, in mice was 4.5 gm/kg although in dogs a 6-gm/kg dose by the same route was lethal. In rabbits, intravenous doses of up to 6

128

D. R. OWENS ET AL.

g d k g had no detectable effects on renal function or kidney structure. When compared with cephaloridine and cephalothin, cephacetrile lacked nephrotoxicity in a similar manner to cephalothin, whereas cephaloridine even at 0.1 gm/kg was found to produce both functional and structural impairment of the proximal tubules of the kidney. The concurrent administration of frusemide and either cephalothin or cephaloridine (Dodds and Foord, 1970; Lawson et al., 1972; Linton et al., 1972) has been shown in animal studies to increase the incidence and extent of nephrotoxicity produced by large doses of cephalothin and cephaloridine. However, in a recent investigation carried out by Luscombe and Nicholls (1975), no biochemical or histological evidence of increased nephrotoxicity was observed when frusemide was administered concurrently with 500-mg and 1-gm doses of cephacetrile in rats and rabbits. In a similar study carried out in dogs, no adverse acute effects of cephacetrile on glomerular filtration rate and effective renal plasma flow was found either in the presence or absence of concurrently administered doses of frusemide (Naber and Madsen, 1973). This evidence, therefore, suggests that cephacetrile, unlike cephaloridine, is relatively free from any nephrotoxicity. Little is known about the possible side effects of continuous cephacetrile therapy. However, in volunteer studies the drug is well tolerated after intravenous injection, no discomfort being experienced either locally or systemically after dosing. In addition, no changes in hematological parameters and hepatic or renal function have been observed (Luscombe et al., 1972).

2. Cephradine Chemically, cephradine is 7-[D-%amino-%(1,4-cyclohexadien-l-yl)acetamido]-3-methyl-3-cephen-4 carboxylic acid (Fig. 2). It is of particular interest since it is rapidly absorbed from the gastrointestinal tract and can, therefore, be administered orally. In a recent human volunteer study carried out in Belgium (Harvengt et al., 1973), a single oral dose of 500 mg in fasted individuals was rapidly absorbed giving peak serum levels of 18.3 ? 2 d m l 1 hour after ingestion. In nonfasted persons, cephradine was less rapidly absorbed although peak serum concentrations also occurred at 1 hour and were similar (19.2 4.1 pg/ml) to those observed in fasted volunteers. Antibacterial activity is rapidly excreted in the urine in high concentrations, more than 85% of the administered dose being recovered in the initial 6-hour period following ingestion. This is a further

*

THE C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

129

indication of the high degree of gastrointestinal absorption following oral doses of cephradine. At present it is not known whether the antibacterial activity in urine samples is due to unchanged cephradine or to a metabolite. Limson et al. (1972) have recently carried out a trial demonstrating that cephradine is highly effective in patients suffering from moderateto-severe, acute infective disease. In addition, these workers found that orally administered cephradine is relatively safe since no toxic effects were observed on the renal, hepatic, or hemopoietic systems although mild adverse reactions involving the gastrointestinal tract were noted in 2 of the treated patients. Landa (1972) has also demonstrated the absence of side effects attributable to the drug in a trial in which the usefulness of cephradine in the treatment of intestinal infections caused by Shigella or Salmonella organisms was examined.

3. Cephapirin Chemically, cephapirin is the sodium salt of 7-[~-(4-pyridylthio)acetamido]cephalosporanic acid (Fig. 2). It is effective only when administered parenterally and possesses antibacterial and pharmacological properties similar to those of cephalothin (Chisholm et a l . , 1970; Gordon et a l . , 1971; Axelrod et al., 1972; Bodner and Koenig, 1972). In pharmacological studies carried out in human volunteers by Axelrod et a l . (1972), 1-gm doses of cephapirin administered intravenously and intramuscularly produced mean serum levels after 1 hour of 6.1 and 18.6 pg/ml, respectively. These levels are similar to those occurring in patients with bacterial infections after the same dose regimen (Bran et al., 1972). Following intramuscular injection, Axelrod et a l . (1972) found a peak serum level after 30 minutes of 24.2 CLglml. Serum half-lives were calculated to be 21 and 47 minutes for the intravenous and intramuscular routes, respectively. Within 6 hours of administration, antibacterial activity in the urine represented 72% of the intravenous and 53% of the intramuscular dose of cephapirin. Gordon et al. (1971) examined the serum levels of cephapirin in pediatric patients. After a 20-mg/kg intramuscular dose, mean peak serum concentrations of 14.5 &ml occurred at 30 minutes. Following rapid intravenous administration of the same dose, levels as high as 54 pg/ml were recorded at 15 minutes. However, antibacterial activity was absent from the serum 4 hours after both intravenous and intramuscular administration. The recovery in the urine of children was comparable to that found in adults (Khan and Pryles, 1973).

130

9. R. OWENS ET AL.

With regards to protein binding, cephapirin is bound to human serum proteins to the extent of 44 to 50% (Chisholm et al., 1970). Cephapirin is fairly well tolerated although pain is produced at the site of intramuscular injection (Bran et al., 1972; Wiesner et al., 1972), and some workers have even advocated that the drug should not be given by this route for this reason (Axelrod et al., 1972). Phlebitis is an additional problem with cephapirin following repeated intravenous administration (Jameson et al., 1971; Bodner and Koenig, 1972; Wiesner et al., 1972) although many workers have stated that the incidence and severity of phlebitis is milder than that which occurs with cephalothin (Lane et al., 1972; Carrizosa et al., 1973; Inagaki and Bodey, 1973). There is no evidence of renal or hepatic toxicity in animals or humans (Wiesner et al., 1972) even after prolonged therapy at high dosage (Bodner and Koenig, 1972). However, eosinophilia is a side effect that has been definitely attributed to cephapirin (Gordon et al., 1971; Bodner and Koenig, 1972; Khan and Pryles, 1973). Bone marrow depression with leukopenia, neutropenia, and anemia has also been observed in isolated cases (Bran et al., 1972).

4. Cefazolin Cefazolin, 7-[ 1-(lH)-tetrazolylacetamido]-3-[2-(5-methyl-1,3,4thiadiazoly1)-thiomethyll-A3-cephem-4-carboxylicacid (Fig. 2), was first described by Kariyone et al. (1970). It is readily absorbed from the intramuscular site, a 500-mg dose in human volunteers giving mean peak serum concentrations ranging from 34.9 (De Schepper et al., 1973) to 44.6 &ml (Nishida et a l . , 1970a). This is 2-3 times as high as serum levels obtained after an equivalent dose of cephaloridine. Serum levels of cefazolin steadily decline after 1 hour, but therapeutically effective concentrations are maintained for as long as 6-10 hours after intramuscular administration (Nishida et al., 1970a,c; Shibata and Fugii, 1971; Phair et al., 1972). Although there are no data available for the serum half-life of cefazolin in humans, in the mouse the half-life after subcutaneous injection of a 20-mg/kg dose has been calculated to be 36.2 k 3.5 minutes which is similar to that found with cephaloridine (Wick and Preston, 1972). Nishida et al. (1970a) examined the degree of binding of cefazolin to human serum protein and found that the drug is bound to the extent of 74%. However, more recently Wick and Preston (1972) have arrived at the somewhat lower figure of 48%. The distribution of I4C-labeled cefazolin has been extensively studied

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

131

in rats and mice by Kozatani et a l . (197233) using whole-body radioautography and liquid scintillation counting. Following intravenous and intramuscular injections of 20-mglkg doses of labeled drug, radioactivity was rapidly distributed throughout the whole body except for the brain. Its absence from the brain indicates that cefazolin does not cross the blood-brain barrier. Radioactivity was almost completely removed from all organs and tissues at 4 hours except from the kidney, liver, and gastrointestinal tract which are responsible for the elimination of the drug. In pregnant mice after intravenous administration, the placenta showed a considerable amount of radioactivity but the fetuses showed a much lower activity than that occurring in the maternal circulation. Placental transfer of cefazolin, therefore, appears to be of a low order. Antibacterial activity is rapidly excreted in the urine following an intramuscular dose of 500 mg and maximal concentrations in excess of 1 mg/ml have been recorded in an initial 3-hour sample (Nishida et a l . , 1970a). Therapeutically effective urine levels of antibacterial activity are maintained for at least 8 to 10 hours after dosing. A total of about 80% of the dose is recovered in a 24-hour urine sample which is a little greater than the recovery of cephaloridine after a comparable dose (Nishida et a l . , 1970a; De Schepper et a l . , 1973). From human (Nishida et a l . , 1970a) and animal studies (Nishida et a l . , 1970c; Ishiyama et a l . , 1971; Kozatani et al., 1972a) it appears that cefazolin is metabolically stable when administered parenterally and is excreted in the urine as unchanged compound. In rats and dogs, cefazolin is excreted in the bile in levels higher than observed with other cephalosporins such as cephaloridine and cephalothin (Nishida et a l . , 1970~).For example, after an intramuscular dose of 20 mg/kg, the 24-hour biliary recovery of cefazolin in rats was 17% compared with 0.6% for cephaloridine and 1.0% for cephalothin. In the dog, 3.3% of cefazolin was recovered in 24 hours but only 0.1 and 0.2% of cephaloridine and cephalothin, respectively. In metabolism and excretion studies using 14C-labeled cefazolin in the rat, Kozatani et al. (1972a) demonstrated that antibacterial activity in the bile is due almost entirely to unchanged drug, providing further evidence that cefazolin is a metabolically stable compound. 5. Cephanone

Chemically, cephanone is 3-(5-methyl-l, 3,4-thiadiazol-2-yl-thiomethyl)-7-[2-(3-sydnone) acetamido]-3-cephem-4-carboxylic acid. It is not absorbed after oral administration and must, therefore, be administered

132

D. R. OWENS ET AL.

parenterally. In human volunteer studies carried out by Meyers et al. (1972b), 1-gm doses of cephanone produced peak serum concentrations of 38.0 and 81.2 d m l following intramuscular and intravenous administration, respectively. Cephanone is only slowly removed from the serum and even at 6 hours the serum concentration of antibacterial activity was found to be in excess of the MIC’s for most of the gram-positive organisms which these authors investigated. A recovery of 96 and 50% of antibacterial activity was found in the urine after intravenous and intramuscular doses, respectively, during an initial 8-hour period following drug administration. The total 24-hour recovery was 100% for the intravenous and 60% for the intramuscular route of administration. Cephanone was well tolerated in these volunteer studies, no untoward side effects or changes in hematological parameters, nor hepatic and renal function being noted. In animal studies, Wick and Preston (1972) administered 20 mg/kg cephanone subcutaneously to mice and found peak serum concentrations of 10.9 d m l at 30 minutes. The calculated serum half-life (29.5 & 2.2 minutes) was similar to that of cephaloridine (33.8 2 4.1 minutes) and cefazolin (36.2 f 3.5 minutes). Serum protein binding was found to be concentration-dependent, increasing from 32 to 50% as the serum concentration of cephanone decreased from 10 to 2.5 ,ug/ml. High concentrations of antibacterial activity were found in the urine and this was shown to be due largely to unchanged cephanone.

V. Clinical Aspects A. URINARY TRACTINFECTIONS The complexity of urinary tract infection (Kass and Zinner, 1969; Cox and Montgomery, 1971) is such, that the determination of efficacy and comparative efficacy of antimicrobial agents is very difficult. The most commonly encountered organisms include E. coli, P. mirabilis, Klebsiella, and S . aureus which cause between 60 and 85% of all urinary tract infections (Cox and Montgomery, 1971). Of the cephalosporins, cephalothin appears to be slightly more effective against P. mirabilis isolates, cephaloglycin against the Enterobacter species, and cephaloridine against enterococci, cephalexin being generally slightly less active than the others (Cox and Montgomery, 1971); all are ineffective against the pseudomonads (see Section II1,A). The success or failure of antimicrobial therapy in urinary tract

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

133

infections is dependent on many factors (Hodson, 1959; McCabe and Jackson, 1965; Condie et al., 1968; Norden and Kass, 1968; Smellie and Normand, 1968; Brumfitt et a l . , 1970), not solely the antimicrobial potency of the compound.

1. Cephalothin Cephalothin has been shown to be valuable in the treatment of patients with urinary tract infection both in adults (Anderson and Petersdorf, 1963; Philson et a l . , 1964; Walters et a l . , 1964; Turck et al., 1965; Wilson et al., 1966; Cox and Montgomery, 1971) and in children (Heitler et a l . , 1964; Hallberg and Svenningsen, 1970). It is well tolerated in patients with penicillin allergy (Griffith and Black, 1964) and those with renal insufficency (Holloway and Scott, 1965; Bulger and Petersdorf, 1970; Lang and Levin, 1971). Urologists have used cephalothin prophylactically in irrigation solutions of the bladder during cytoscopy and retrograde pyelography (Seneca and Lattimer, 1965) and in urological surgery (Wilson et a l . , 1971). By combining cephalothin with other antibacterial agents, its success rate in patients with resistant urinary tract infection is increased (Thoburn and Fekety, 1970; Weissbach et a l . , 1971).

2 . Cephaloridine The value of cephaloridine in patients with urinary tract infections is well documented (Murdoch et a l . , 1964; Stewart and Holt, 1964; Gherardi et a l . , 1965; Ishigami et a l . , 1965; Lenti et a l . , 1965; Apicella et a l . , 1966b; Dennis et al., 1966; MacLean et al., 1967; Foord, 1967; McAllister and Mack, 1967), a very high success rate being recorded in acute urinary tract infections (Rocca-Rossetti et a l . , 1965; Foord, 1967; Landes et al., 1967), but a slightly less impressive result being observed in those cases with acute on chronic infections. In resistant gram-negative infections, cloxacillin and methicillin (Ohkoshi et a l . , 1967) have been added to cephaloridine with encouraging results. Chronic urinary tract infections are invariably difficult to treat irrespective of antimicrobial agent employed (Garrod et a l . , 1955). Cephaloridine is no exception, reinfection occurring in the majority of patients after an initial favorable response (Murdock et a l . , 1964; McAllister and Mack, 1967). A high failure rate with respect to cephaloridine may be related to the development of L-forms of E . coli in patients with chronic urinary tract infection. Better results are obtained

134

D. R. OWENS ET AL.

by increasing the duration of treatment (Seneca et al., 1967; Stratford and Dixson, 1967; Ueda et al., 1967; Burgess et al., 1968). Cephaloridine has also been used prophylactically in patients undergoing gynecological operations (Matthews et al., 1967). It is also considered of value in patients undergoing prostatectomy (Lacy et al., 1971). Drach et al. (1971) showed that the addition of a closed neomycin-polyrnixin-irrigated catheter system, in addition to systemic cephaloridine, further reduced the rate of acquired bacteriuria. In very severe urinary tract infections, cephaloridine alone in a high dose or in combination with gentamicin, or a combination of polymixin B or colistimethate and kanamycin may be necessary (Cox and Montgomery, 1971).

3 . Cephalexin Acute urinary tract infections respond extremely well to cephalexin (Griffith, 1969; S. Ishigami et al., 1969; Brumfitt et al., 1970; Daikos et al., 1970; Erikssen et al., 1970; Kawamura, 1970; Leigh et al., 1970; Wise and Schwartz, 1971; Seneca et al., 1972). Long-term results in patients with chronic urinary tract infections are poor and essentially similar to all the other antimicrobial agents in this indication (Griffiths, 1969; Kind et al., 196913; Levison et a l . , 1969; Ohkoshi et al., 1969; Richards, 1969; Spiers et al., 1969; Fass et al., 1970b; Rohner, 1970; Mohring et al., 1971; Speight et al., 1972). However, good results have been recorded by Kaye et al. (1970) and Smith and Williams (1970). Eyckmans (1970), Fairley (1970), Ohkoshi et a l . (1970), and Acar (1971) found that the rate of eradication of bacteriuria is dependent on .the site of the infection. Gruneberg and Brumfitt (1967) and Davies et al. (1971) found cephalexin and ampicillin equally effective in the treatment of urinary tract infections. In bacteriuria of pregnancy, Brumfitt and Pursell (1972) found ampicillin and cephalexin equally effective in their hospital patients, whereas ampicillin was clearly superior in general practice. Acar (1971) compared cephalothin, cephaloridine, cephaloglycin, and cephalexin, and found no difference in the rate of cure, but failures were more common with cephaloglycin. 4. Cephaloglycin A variable response has been seen with cephaloglycin in urinary tract infections (Eyckmans et al., 1968; Hogan et al., 1968; Cox and Montgomery, 1971; Wick et al., 1971). It is also more effective in

THE C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

135

patients with lower urinary tract infections (Ronald et al., 1968), and its effectiveness is likewise dependent on certain host factors (Boyer and Andriole, 1968; Johnson et al., 1968). The treatment of chronic urinary tract infections has been essentially unsatisfactory (Seneca et al., 1970). Cephaloglycin sometimes is poorly tolerated (Eyckmans et al., 1968; Hogan et al., 1968; Ronald et al., 1968), but others have found the drug well tolerated even in patients with chronic urinary tract infections on long-term therapy, with reduced renal function (Landes et al., 1969; Lowentritt et al., 1971).

5. Others Cephapirin (Bodner and Koenig, 1972), cephradine (Elkins et al., 1972), cephacetrile (Benner, 1972; Opitz et al., 1972; Maurice et al., 1973), and cefazolin (Benner, 1972; Cox et al., 1972) are also valuable in the treatment of urinary tract infections.

B. RESPIRATORYTRACTINFECTIONS 1. Cephaloridine This drug has been used successfully in the treatment of various lower respiratory tract infections (Verney, 1967; Eyckmans, 1970). Numerous authors have noted the effectiveness of cephaloridine in pneumococcal pneumonia (Katsu et al., 1965; Foord, 1967; Galbraith, 1967; MacLean et al., 1967; Brayton et al., 1967). In such infections, cephaloridine compares favorably with penicillin G (Cohen et al., 1966; Thornton and Andriole, 1966; Tempest and Austrian, 1967) and ampicillin (Matts, 1967), although it is inferior to erythromycin in cases with Mycoplasma infections (Sterner et al., 1967). The response in staphylococcal infections has also been most encouraging (Holloway and Scott, 1965; Apicella et al., 1966b; Foord, 1967; Galbraith, 1967; Merchant, 1967). Cephaloridine has a similar high clinical and bacteriological rate of success (Galbraith, 1967) in streptococcal infections. However, it is not as effective against Haemophilus influenzae respiratory infections-not an unexpected finding (May, 1967). Also, certain Klebsiella organisms are equally resistant (Hinman and Wolinsky, 1967). However, H . influenzae and Klebsiella pneumoniae lung infections cleared in 68 and 60% of cases, respectively, in a multicenter trial with cephaloridine (Foord, 1967). Even better clinical results have been achieved by

136

D. R. OWENS ET AL.

Galbraith (1967) and Eyckmans (1970). Coliform pneumonia or bronchopneumonia, on the other hand, responds very poorly to cephaloridine (Foord, 1967). Superinfection by P. aeruginosa has been occasionally reported in patients with pneumonia who were treated with cephaloridine (Apicella et a l . , 1966b; Dennis et al., 1966). This is not a problem specific to cephaloridine. Cephaloridine compares well with ampicillin (Matts, 1967; Bailey, 1970; Pines, 1970), penicillin, and streptomycin (Pines et al., 1967; Pines and Raafat, 1967; Citron et a l . , 1968) in acute and acute on chronic bronchitis. Pines et al. (1971) also have shown cephaloridine to be effective in very troublesome purulent bronchitis. Cephaloridine has also been administered by intravenous infusion (Matsumoto et al., 1970) and as inhalations (Kennedy, 1967). In the treatment of lung abscess, cephaloridine compares favorably with the combination of penicillin and streptomycin (Seftel et a l . , 1970). Also, Le Roux (1970) has demonstrated its value in chronic suppurative cavitated lung lesions. Merchant (1967) also successfully treated children with lung abscesses.

2. Cephalothin

This drug has been used with success in patients with respiratory tract infections (Griffith and Black, 1964; Klein et al., 1964; Weinstein et al., 1964). Cephalothin and cephaloridine are equally effective in respiratory tract infections (Smith, 1971), cephalothin being usually restricted to the patient with severe life-threatening infections (Merrill et al., 1966). 3 . Cephalexin This antibiotic has been found valuable in various infections of the upper respiratory tract (Busca, 1969; Seftel et a l . , 1969; Fass et al., 1970a; Marks and Garrett, 1970). A large number of studies indicate its value in acute pneumonia or bronchopneumonia (Bieder, 1969; Daikos, 1969; Fujii et a l . , 1969; Hedlund, 1969; Jacquot, 1969; Lidman et al., 1969; Stratford, 1969; Bailey et a l . , 1970; Daikos et al., 1970; Macquet and Lafitte, 1970; Poggiolini, 1970; Rohner, 1970; Solberg et a l . , 1972). Gould (1970) showed cephalexin to be slightly inferior to doxycycline in acute Haemophilus and S. pyogenes respiratory tract infections in general practice. Better results are obtained in acute bronchitis (Seftel et a l . , 1969; Bailey et al., 1970; Fass et a l . , 1970b) than in chronic

THE CEPHALOSPORIN GROUP OF ANTIBIOTICS

137

bronchitis (Bailey et al., 1970; Eyckmans, 1970; Macquet and Lafitte, 1970; Pines, 1970; Rohner, 1970) or bronchiectasis (Bieder, 1970; Marks and Garrett, 1970). Increasing the dosage and duration of treatment improves the response (Pines, 1972). A varying response has been observed with cephalexin in the treatment of lung abscess (Seftel et al., 1969; Daikos et al., 1970). Oral plus intrapleural injections have been employed with success by Macquet and Lafitte (1970) in subjects with empyema.

4. Cephaloglycin

This drug has been used with success in upper respiratory tract infections (Leiderman et al., 1970; Stillerman, 1970; Matsen, 1971). A poorer response is seen in cases with lower respiratory tract infections (Rohner, 1970), although Hogan et al. (1968) have obtained a satisfactory response. 5. Others Cephapirin (Benner, 1972; Bran et al., 1972; Quintiliani et al., 1972) and cephacetrile are effective in cases with respiratory tract infection (Hodson and Holloway, 1973; Maurice et al., 1973), the latter having comparable efficacy to ampicillin in acute exacerbations of chronic bronchitis (Eckolt et al., 1972).

C. VENEREALDISEASE New chemotherapeutic agents have to be compared to penicillin with respect to efficacy and tolerability in venereal disease (Fiumara, 1972).

1. Gonorrhea Penicillin remains the drug of choice in the treatment of gonorrhea (Meyer-Rhon, 1972). The cephalosporin analogs have an advantage in penicillin-sensitive patients although cross-sensitization has been occasionally reported (Meyler and Herxheimer, 1968) (see Section VI). a. Cephaloridine. This antibiotic has been successfully employed in the treatment of gonorrhea (Seftel et al., 1966), following encouraging in vitro sensitivity of Neisseria gonorrhoeae to cephaloridine (Barber and Waterworth, 1964; Muggleton et al., 1964). A single 1-gm dose has been found to be inadequate (Marshall and Curtis, 1967; Oller, 1967a), better results being obtained with higher doses (Oller, 1967b; Jouhar and Fowler, 1968; Haberman et al., 1969; Shapiro and Lentz, 1970). Despite

138

D. R. OWENS ET AL.

higher doses the failure rate may still be high (Lucas et a l . , 1966; McClone et a l . , 1968; Keyes and Halverson, 1969; Molin and Nystrom, 1970; Pariser and Marino, 1970), penicillin-resistant strains of gonococci being an ever increasing problem (Fowler, 1969). b. Cephalexin. This drug is more active against N. gonorrhoeae than cephaloridine (Muggleton et al., 1969). Other advantages are ease of administration and high urinary excretion (Foord et al., 1969b). The sensitivity of gonococci to cephalexin runs parallel to their penicillin sensitivity (Oller and Smith, 1969). The cure rate is seen to be related to total dosage and frequency of administration (Oller et a l . , 1970; Csonka, 1971; Taylor and Holloway, 1972). Unfortunately, patient default rate with repeated administrations is high (Willcox and Woodcock, 1970) as seen previously with kanamycin (Wilkinson et al., 1967). The addition of probenecid further improves the response rate (Fowler, 1970; Landes et

al., 1972). Wilcox (1971) compared cephalexin with demethylchlortetracycline and found the latter achieved a lower failure rate. 2. Nongonococcal Urethritis (N.G.U.) This is probably among the commonest of the sexually communicable diseases (Csonka, 1971; King, 1971). The treatment of nongonococcal urethritis with cephaloridine has been unimpressive (Csonka and Murray, 1967); tetracyclines remain the treatment of choice (Csonka,

1967). 3. Ophthalmia Neonatorum and Lymphogranuloma Venerum Cephaloridine has been successful in the treatment of these two conditions (Vegas, 1965; Oller et al., 1970).

4. Syphilis Penicillin is still the most valuable treatment of syphilis, since its first application to man by Mahoney, Arnold, and Harris in 1943. Success can be expected in more than 95% of cases (Jefferiss and Willcox, 1963; King, 1971). No evidence of true resistance of Treponema pallidum to penicillin has been yet demonstrated (Guthe, 1965). Penicillin hypersensitivity reactions present a real problem (Frank et a l . , 1965; Minkin and Lynch, 1968), especially in pregnant patients when alternative treatments have also well-recognized hazards (Kline et a l . , 1964; South et a l . , 1964; Davis and Kaufman, 1966). Also Hardy (Hardy et a l . , 1970)

T H E C E P H A L O S P O R I N GROUP O F A N T I B I O T I C S

139

reported the failure of penicillin to prevent congenital syphilis in an adequately treated mother with syphilis. a . Cephaloridine. The effectiveness of cephaloridine against syphilis was initially demonstrated by Galla et a l . (1965), although the antisyphilitic activity of cephaloridine was shown to be less than that of penicillin G (Flarer, 1967). Several encouraging reports have been published on its use in early syphilis (Flarer et al., 1965; Ochoa and Cravioto, 1965; Seftel et a l . , 1966; Oller, 1967b; Glicksman et a l . , 1968; Duncan and Knox, 1971). Pregnant patients with syphilis respond very satisfactorily to cephaloridine (Flarer, 1967; Oller, 1967a). b. Cephalexin. Unimpressive results have been seen with cephalexin in the treatment of syphilis (Duncan and Knox, 1971).

AND GYNECOLOGY D. OBSTETRICS

The cephalosporins are readily transferred across the placenta to the fetal blood and later into the amniotic fluid, as evidenced by the following reports: cephalothin (Lee and Anderson, 1963; Reichelderfer and Reichelderfer, 19M; Sheng et a l . , 1965; MacAulay and Charles, 1968; Morrow et a l . , 1968; Hirsch, 1970; Paterson et a l . , 1970; Stephen and Bolognese, 1970; cephaloridine (Barr and Graham, 1967a; Arthur and Burland, 1969a; cephalexin (Mizuno et a l . , 1969; Bert6 et a l . , 1972; Paterson et al., 1972.) If they are to be of value in the treatment of infections in the pregnant patient, these drugs should satisfy certain basic criteria (Atkinson et a l . , 1966a; Hirsch, 1970, 1971). The antibiotic to be employed must be capable of attaining therapeutically effective concentration both in the mother, fetus, and amniotic fluid, while at the same time being free of any unwanted effects.

1. Cephalothin

This drug has been used with success in obstetric practice (Holloway and Scott, 1965; Hirsch, 1968; Paterson et al., 1970; Soto et al., 1970). Urinary tract infections associated with pregnancy respond well to cephalothin therapy (Conti and Iurlaro, 1967). Allen et a l . (1972) showed quite clearly that, in gynecological use, cephalothin reduced morbidity following hysterectomy.

140

D. R. OWENS ET AL.

2. Cephaloridine This antibiotic is valuable in a variety of obstetric problems (Josey and Farrar, 1970; Ostergard, 1970; Soto et a l . , 1970), amniotitis and postpartum endometritis (Holloway and Scott, 1965; Conti and Iurlaro, 1967), various pelvic infections, postabortal sepsis (Tully and Smith, 1968), and mastitis. Cephaloridine has also been used successfully in pregnant women with urinary tract infections (Landes et a l . , 1967; Brumfitt et a l . , 1970). It has also been used prophylactically in patients with premature rupture of the membranes, but Barr and Graham (1967a) and Charles and MacAulay (1970) feel that neither cephaloridine or any other antibiotic (Davies, 1972) should be given in all cases with premature rupture of the membranes. Cephaloridine used prophylactically in patients undergoing gynecological operations reduced significantly the incidence of postoperative urinary tract infection (Matthews et a l . , 1967).

3. Cephalexin This drug has been successfully used in treatment of pelvic infections, puerperal mastitis, bartholinitis (Mizuno et a l . , 1969; Poggiolini, 1970), and in pregnant patients with associated urinary .tract infections (Hirsch, 1971). Good results have also been obtained with cephalexin in cases of gynecological infections (Daikos et a l . , 1970).

E. PEDIATRICS It is difficult to evaluate new antibiotic agents in newborn (Butler, 1967), infection being a serious problem (Butler and Bonham, 1963) yet amenable to prevention and treatment (Keay et a l . , 1967). A significant change has also been noticed in the type of organism involved (Nyham and Fousek, 1958; Forfar et al., 1966; Burland and Simpson, 1967; Keay et a l . , 1967).

1. Cephalothin This is a valuable drug for a variety of infections in infants and children (Riley et a l . , 1963; Flux et al., 1964; Hallberg and Svenningsen, 1970). Davies (1970) regards it as the treatment of choice in penicillinallergic children with serious infections.

THE CEPHALOSPORIN G R O U P OF ANTIBIOTICS

141

2. Cephaloridine This drug has been used successfully in pediatric practice (Flux et a l . , 1966; Sweetman et a l . , 1966; Albores et a l . , 1967). It has also been used effectively in combination with streptomycin (Fine et a l . , 1966; Fekety and Weiss, 1967; Burland et a l . , 1970; Gould, 1970; Keay and Fleming, 1970) and polymixin B or E (Marget, 1967). Cephaloridine is of undoubted value in children with septicemia, meningitis, and respiratory and urinary tract infections (Corda and Scane, 1965; Flux et a l . , 1966; Merchant, 1967). It has been shown to have little affinity for albumin binding, as apposed to cephalothin, which may be of importance in the treatment of premature infants (Malaka-Zafiriu and Strates, 1969).

3. Cephalexin This is also a valuable antibiotic in the treatment of the severe infections of the newborn (MacMillan, 1971). Respiratory tract infections in older infants and children respond well to cephalexin (Bamatter and Hazeghi, 1969; Marget and Daschner, 1969; Donnison and Davison, 1970; Grislain, 1970; Leiderman et a l . , 1970; Stillerman and Isenburg, 1971; Azimi et a l . , 1972; Disney et a l . , 1971; Gau et a l . , 1972; Paschetta and De Biasi, 1971; Riley, 1971).

4. Cephaloglycin This drug is also effective in children with mild infections (Leiderman et a l . , 1970; Matsen, 1971). F. DERMATOLOGY The cephalosporins, cephalothin (Limson and Santos, 1968; Watermann et a l . , 1968; Bernard et a l . , 1969; Smith, 1971; Tong, 1972),

cephaloridine (Dennis et a l . , 1966; Holloway and Scott, 1965; Thornton and Andriole, 1966; Foord, 1967; Ishiyama et a l . , 1967; Da Silva et a l . , 1967; Smith, 1971; Kaplan et a l . , 1968a; Brown et a l . , 1970; Polk and Lopez-Mayor, 1969; Dillon et a l . , 1972), cephalexin (Goto, 1969; Hedlund, 1969; Kienitz and Mann, 1969; Lidman et a l . , 1969; Shibata and Kato, 1969; Page et a l . , 1970; Grislain, 1971; Lyons and Andriole, 1971; Lautre and Baker, 1972), cephapirin (Bodner and Koenig, l972), and cephacetrile (Benner, 1972; Hodges et a l . , 1973; Maurice et a l . , 1973), have all been shown to be most valuable in the treatment of skin and soft tissue infections.

142

D. R. OWENS ET AL.

G. OPHTHALMOLOGY Excellent intraocular penetration of cephaloridine occurs following both i.m. and i.v. administration in man (Records, 1968a,b; Riley et a l . , 1968; Richards et a l . , 1972) and is superior to that following cephalothin (Mizukawa et a l . , 1965; Hatano et a l . , 1966; Records, 1969a). Even higher concentrations are achieved following local subconjunctival injection (Records and Ellis, 1969; Moll, 1970; Moll et a l . , 1971). Cephaloridine has been used for severe occular infections (Rosa, 1967; Richards et al., 1972) and prophylactically in patients undergoing intraocular surgery (Ikui et a l . , 1966; Records and Ellis, 1969; Moll,

1971). Cephalexin penetrates the eye both in animal and in man (Gager et a l . , 1969; Boyle et a l . , 1970) and has been used with success in the treatment of various eye infections and also as an antibiotic cover during surgery (Constantindes, 1971). Cephalosporins may also be found to be useful in eradicating Treponema pallidum from the eyes of syphilitic patients (J. L. Smith, 1969).

H. MENINGITIS Cephalothin and cephaloridine are able to reach adequate concentrations in the CSF (Klein et a l . , 1964; Perkins and Saslaw, 1966; Ruedy, 1967; Vianna and Kaye, 1967; Lerner, 1969; Oppenheimer and Beaty, 1969; Walker and Gonzales, 1969; Brown et a l . , 1970), both in experimental meningitis and true clinical meningitis in man.

1. Cephalothin Satisfactory response has been obtained in the treatment of pneumococcal meningitis (Griffith and Black, 1964; Perkins and Saslaw, 1966; Herrell, 1968) and staphylococcal meningitis (Weinstein et al., 1964); a less favorable and variable response is observed in meningococcal and H . influenzae meningitis (Binns and Pankey, 1966; Almond, 1969; Southern and Sanford, 1969; Brown et a l . , 1970). The combination of cephalothin and gentamicin may be necessary in gram-negative meningitis (Rahal, 1972).

2. Cephaloridine Murdock et a l . (1964), Katsu et a l . 1965), Apicella et a l . (1966b), McKenzie et a l . (1967), Love et a l . (1970), and Lerner (1971) have all

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

143

shown cephaloridine to be valuable for the treatment of pneumococcal meningitis. Impressive results have also been seen in staphylococcal (Toscano et a l . , 1967; Walker and Gonzales, 1969), streptococcal, and H. influenzae meningitis (McKenzie et a l . , 1967; Lerner, 1971). Meningococcal meningitis is a more serious infection with the increasing occurrence of sulfonamide-resistant strains (Eickhoff and Finland, 1965), and cephaloridine may or may not (Walker and Gonzales, 1969) prove to be effective.

I. ENDOCARDITIS Treatment with the cephalosporin analogs should be considered particularly in patients allergic to penicillin (Koya and Misawa, 1970) (see Section VI).

1. Cephalothin The value of cephalothin in endocarditis is well documented (Flux et al., 1964; Walters et al., 1964; Merrill et al., 1966; Siguier et al., 1966; Tuano et a l . , 1967; Rahal et al., 1968). Both streptococcal and staphylococcal endocarditis respond well to cephalothin (Saslaw and Perkins, 1966; Limson and Santos, 1968; Weinstein, 1971; Meyers et al., 1972a). Also aerobic diphtheroid endocarditis (Kaplan and Weinstein, 1969; Goldsweig et a l . , 1972) responds well to this drug. In the treatment of severe gram-positive and gram-negative endocarditis, combination with methicillin, oxacillin, streptomycin (Herrell et al., 1965, 1967), polymixin B (Pressman et al., 1966), or kanamycin (Foster, 1969) may be necessary. Cephalothin has also been used prophylactically in patients undergoing cardiac surgery (Okies, Vitoslav and Williams, 1971). 2 . Cephaloridine

This drug has also been used with success in cases ol' endocarditis (Ueda et al., 1965; Apicella et al., 1966a; Parker et al., 1968; Kump et al., 1969). The response of staphylococcal endocarditis has been less encouraging (Rowntree and Bullen, 1967), despite adequate serum concentrations (Burgess and Evans, 1966). Therapy may need to be precise and prolonged (Foord and Snell, 1966; Vacek and MolikoviWagnerova, 1967) to achieve better results. Similar combinations to those mentioned for cephalothin may be valuable (Ehrenkranz, 1971). Cephaloridine may be employed prophylactically in patients at risk

144

D. R. OWENS ET AL.

undergoing surgery, as it can kill strains of Streptococcus viridans more rapidly than erythromycin or vancomycin (Tozer et al., 1966).

3. Cephalexin The value of cephalexin in the treatment of endocarditis remains unclear (Zabransky et al., 1969; Stratford, 1970). J. BONESAND JOINTS The main causative organism in acute osteomyelitis is S. aureus; less frequent offenders include streptococci, E. coli, Proteus sp., and H. influenzae (Smith, 1958; Blockey and Watson, 1970). The expected effectiveness of the cephalosporin analogs is in the region of 80 to 90% (Smith, 1971), equivalent to the combination of dicloxacillin, ampicillin, and benzylpenicillin (Kienitz, 1971). Treatment should be commenced with a high dosage of the antibiotic, in order to prevent a recurrence rate of up to 30% observed in chronic osteomyelitis by Waldvogel et a l . (1970). 1. Cephalothin Success with cephalothin in bone infection has been well-documented (Griffith and Black, 1964; Barrett and Ehrenkranz, 1965; Hess and Martin, 1971). Norden (1971) found that cephalothin penetrates well into the diseased bone, and parenteral administration, therefore, should be adequate (Argen et al., 1966; Nelson, 1971).

2 . Cephaloridine It has been shown that cephaloridine is of value in treating both acute and chronic osteomyelitis and septic arthritis (Cabitza, 1965; Taylor and Fallon, 1966; Lai, 1967; Azimi and Cramblett, 1968; Kaplan et a l . , 1968b; Steigbeigel et al., 1968; Fleming et al., 1970; Hermans, 1971; Kienitz, 1971).

3 . Cephalexin Cephalexin has also been used successfully in patients with acute osteomyelitis (Daikos et a l . , 1969, 1970; Hedlund, 1969, 1970), due mainly to staphylococcal infection. In chronic osteomyelitis, results have been encouraging (Cheng et al., 1970; Herrell, 1971), oral medication having undoubted value in the long-term management of chronic bone infection.

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

145

4. Other Cephalosporins Benner (1972) has shown cephacetrile and cefazolin valuable in osteomyelitis.

VI. Hypersensitivity and Allergenicity A. GENERALCONSIDERATIONS It is a well-known fact that, although they are not toxic in the pharmacological sense, the penicillins may elicit allergic responses in a small percentage of patients. The immunological properties of penicillins are discussed in detail by Shaltiel et al. (1971), who point out that commercially available benzylpenicillin and 6-APA (the latter being implicated as a major antigenic determinant in penicillin allergy) may contain trace amounts of penicilloylated protein impurities and that the removal of these impurities markedly reduces the immunological manifestations induced by these antibiotics. The position with cephalosporins is, in many respects, less clear-cut. The Coombs test is widely used to detect the presence of blood-group antigens absorbed on the surface of red blood corpuscles. Coombspositive hemolytic anemia is known to occur in patients receiving large doses of intravenous benzylpenicillin, cephaloridine, cephalothin, or cefazolin (Gralnick et al., 1967; Molthan et al., 1967; Perkins et al., 1967a; Kaplan et al., 1968a,b; York and Landes, 1968; Kosakai and Miyakawa, 1970; Mine et al., 1970a; Gralnick, 1971; Petz, 1971). Much higher concentrations of cefazolin are needed to produce a positive direct Coombs reaction than penicillin or other cephalosporins, and the intensity of the positive reaction appears to be related to the lytic action of the antibiotics on the red blood cells (Mine et al., 1970a). However, the minimum concentration of a cephalosporin needed for a positive reaction varies markedly with the antiglobulin serum used (Gralnick, 1971). Stewart (1962) found that cephalosporin C was not cross-allergenic with the penicillin series in skin tests in human volunteers. However, subsequent studies have indicated that the cephalosporins have crossreactivity with benzylpenicillin (Brandriss et al., 1964; Batchelor et al., 1966; Shibata et at!., 1966; Mine et at!., 1970b; Mashimo, 1971). Brandiss et al. (1964) proposed that the benzylpenicilloyl group was an antigenic determinant in benzylpenicillin hypersensitivity and that it was formed by conjugation of the highly reactive intermediate, benzylpenicillenic

146

D. R. OWENS ET AL.

acid, to protein. However, cephaloridine was also antigenic in rabbits despite its inability to undergo a penicillanic acid-type rearrangement in vitro ; in addition, both the hemagglutinating and passive cutaneous anaphylaxis reacting antibodies formed in response to injection with this antigen cross-reacted with the benzylpenicillin antigen. Batchelor et al. (1966) showed that cephaloridine and cephalothin formed protein conjugates that stimulated the production of hemagglutinating, precipitating, and guinea pig-sensitizing antibodies in the rabbit and that there was cross-reaction between benzylpenicillin and cephalosporins; they suggested that this cross-reaction was caused by an immunologically similar side chain. Cephaloridine and cephalothin were found by Mashimo (1971) to have a higher affinity to antibenzylpenicilloyl antibody than cephalexin, ampicillin, or oxacillin; it was proposed that similarities in the stereochemical structure of the side chain of cephaloridine, cephalothin, and benzylpenicillin are associated with the degree of hapten inhibition. Ky et al. (1970) described a very sensitive lymphoblastic transformation test for studying cross-reactions between benzylpenicillin and cephaloridine and found that such reactions were rare (circa 12%); they suggested that the p-lactam nucleus is, in some patients, a common hapten responsible for cross-reactions. Cefazolin gives a minimal cross-reactivity with benzylpenicillin, ampicillin, and cephaloridine (Mine et al., 1970b), and Mine et al. (1970b) agreed with other authors cited above that the cross-reactivity between cephalosporins and related penicillins against benzylpenicillin appears to be mediated mainly by the acyl side chain. Reaction of cephalosporins with ammonia, amino acids, and other simple amino compounds in weakly alkaline solutions gives labile compounds with a A,,, of 230 nm (Hamilton-Miller et al., 1970a,b). Such compounds obtained from cephalosporin C are less stable in concentrated than in dilute solution, and their breakdown is followed by the appearance of new chromophores with A,,, 270 and 278 nm. These are attributable to the fragmentation of the molecule with the formation of penaldates and penamaldates from the side chain and the carbon atoms of the p-lactam ring. Derivatives similar to those obtained with simple amino compounds, but which are virtually unaffected by dilution, may be formed when cephalosporins react with lysine polymers such as serum). Interestingly, Hamilton-Miller et al. polylysine or poly (lysine (1970a,b) concluded that “haptens resulting from the primary reaction of cephalosporins, deacetylcephalosporins and deacetoxycephalosporins with the ammonia groups of proteins will differ from each other and from the penicilloyl haptens derived from the penicillins in several respects other than in the nature of their N-acyl side-chains.’’

+

THE C E P H A L O S P O R I N G R O U P O F A N T I B I O T I C S

147

Traces of macromolecular protein or peptide complexes that possess immunogenic and allergenic properties are found in natural penicillins and cephalosporins (Stewart, 1968). Cross-immunization and crosssensitization between the two types of p-lactam antibiotics occurs especially when macromolecular conjugates are used (Stewart et al.,

1970).

B. CONCLUSIONS James and Walker (1971) consider that cephaloridine is an effective alternative for the penicillin-allergic subject, and that, although crosssensitization has occurred between penicillin and this antibiotic, such reactions are rare clinically, so that cephalosporins can safely be prescribed for those who are allergic to penicillins. Cross-hypersensitivity in patients is not, in fact, a complete one (Mashimo, 1971) although an increased incidence of hypersensitivity reactions to cephalosporins occurs in patients with a history of hypersensitivity to penicillins (Thoburn et al., 1966; Petz, 1971). Sensitization in human subjects appears to be caused primarily by penicillins. Although allergy to cephalosporins is much less frequent, however, there may be a higher incidence of anaphylaxis among the reactants (Stewart et al., 1970). Stewart et al. (1970) are of the opinion that cephalosporins can replace penicillins in allergic subjects, but only with caution and not if an unrelated antibiotic is equally effective. This is a very reasonable statement, since hypersensitivity to cephalosporins may be manifested in about 10% or so of penicillin-hypersensitive patients. REFERENCES Abraham, E. P. (1962). Pharmacol. Rev. 1 4 , 4 7 3 . Abraham, E. P.(1%7). Quart. Rev.. Chem. SOC. 21, 231. Abraham, E. P., and Newton, G. G. F. (1956). Biochem. J . 6 3 , 6 2 8 Abraham, E. P., and Newton, G . G. F. (1961). Biochem. J . 7 9 , 3 7 7 . Abraham, E. P . , Schenck, J. R., Hargie, M. P . , Olson, B. H., Schuurmans, D. M., Fischer, M. W., and Fusari, S. A. (1955). Nature (London) 176, 551. Acar, J. F. (1971). Postgrad. Med. J . , Suppl. 47, 103. Acocella, G., Mattussi, R., Nicolis, F. B., Pallanza, R., and Tenconi, L. T. (1968). Gut 9, 536. Adams, R., and Nelson, J. D. (1968). Appl. Microbiol. 16, 1570. Albores, J. M., Meyer, C. A. M., Ots, T. P., Guilhem, A., Scavuzzo, F. C., and Cedrato, A. E. (1%7). Prensa Med. Argent. 54, 247. Allen, J. L., Rampone, J. F., and Wheeless, C. R. (1972). Obstet. Gynecol. 39, 218. Almond, M. R. (1969). New Engl. J . Med. 281, 1163. Anderson, K. N., and Petersdorf, R. G. (1963). Antimicrob. Ag. Chemother. p. 724.

148

D. R. OWENS ET AL.

Anderson, J. D., and Sykes, R. B. (1973). J. Med. Microbiol. 6, 201. Apicella, M. A., Perkins, R. L., and Saslaw, S. (1966a). N e w Engl. J. Med. 274, 1002. Apicella, M. A., Perkins, R. L., and Saslaw, S. (1966b). Amer. J. Med. Sci. 251, 266. Applestein, J. M., Crosby, E. B., Johnson, W. D., and Kaye, D. (1968). Appl. Microbiol. 16, 1006. Argen, R. T., Wilson, C. H., and Wood, P . (1966). Arch. Intern. Med. 117, 661. Arthur, L. J. H., and Burland, W. L. (1%9). Arch. Dis. Childhood 44, 82. Asscher, A. W., Johnson, S. E., and Simon, D. A. (1970). Postgrad. Med. J., Suppl. 46,

55. Atkinson, R. M., Caisey, J. D., Currie, J. P., Middleton, T. R., Pratt, D. A. H., Sharpe, H. M., and Tomich, E. G. (1966a). Toxicol. Appl. Pharmacol. 8, 407. Atkinson, R. M., Currie, J. P., Davis, B., Pratt, D. A. H., Sharpe, H. M., and Tomich, E. G. (1966b). Toxicol. Appl. Pharmacol. 8,398. Axelrod, J., Meyers, B. R., and Hirschman, S. Z. (1972). J. Clin. Pharmacol. J. New Drugs 12,84. Ayliffe, G. A. J. (1964). Nature (London) 201, 1032. Ayliffe, G. A. J. (1965). J. Gen. Microbiol. 40, 119. Azimi, P. H., and Cramblett, H. G. (1968). J. Pediat. 73, 255. Azimi, P. H., Cramblett, H. G., Del Rosario, A. J., Kronfol, H., Haynes, R. E., and Hilty, M. D. (1972). J. Pediat. 80, 1042. Bailey, A. J. (1970). Brit. J. Dis. Chest 64, 232. Bailey, A., Walker, A., Hadley, A., and James, D. G. (1970). Postgrad. Med. J. 46, 157. Bamatter, F., and Hazaghi, P. (1969). Advan. Clin. Pharmacol., Oracef Symp., Frankfurt; Int. Z . Klin. Pharmakol. Ther. Toxikol. Suppl., p 92. Barber, M., and Waterworth, P. M. (1964). Brit. Med. J. ii, 344. Barr, W., and Graham, R. M. (1967a). Postgrad. Med. J., Suppl. 46, 101. Barr, W., and Graham, R. M. (1967b). J. Obstet. Gynaecol. Brit. Commonw. 74, 739. Barrett, E. L., and Ehrenkranz, I\r. J. (1965). Bull. Univ. Miami Sch. Med. Jackson Mem. Hosp. 19, 2 . Barton, D. H. R., and Sammes, P. G. (1971). Proc. Roy. SOC.,Ser. B 179, 345. Batchelor, F. R., Dewdrrey, J. M., Weston, R. D., and Wheeler, A. W. (1%6). Immunology 1 0 , 2 1 . Benner, E. J. (1970). J. Infec. Dis. 122, 104. Benner, E. J. (1971). Postgrad. Med. J . , Suppl. 47, 135. Benner, E. J. (1972). Zntersci. Conf., 12th, Antimicrob. Ag. Chemother. Abstr. No. 150. Benner, E. J., and Morthland, V. (1967). New Engl. J. Med. 277, 678. Benner, E. J., Bennett, J. V., Brodie, J. L., and Kirby, W. M. M. (1965a). J . Bacteriol. 90, 1599. Benner, E. J., Micklewait, J. S., Brodie, J. L., and Kirby, W. M. M. (1965b). Proc. Soc. Exp. Biol. Med. 119, 536. Benner, E. J., Brodie, J. S., and Kirby, W. M. M. (1966). Antimicrob. Ag. Chemother. p. 888. Bergan, T., Midtvedt, l., and Erikssen, J. (1970). Pharmacology 4, 264. Bernard, H. R., Clark, W. R., Leather, R. P., and Gray, V. C. (1969). Arch. Surg. (Chicago) 99, 388. Bernard, J., and Lambin, S. (1965). Ann. Znst. Pasteur, Paris 109, 907. Berte, F., De Bernardi, M., Manzo, L., and Benzi, G. (1972). Chemotherapy 17, 344. Biagi, G. L., Guerra, M. C., Barbaro, A. M., and Gamba, M. F. (1970). J. Med. Chem. 13, 511. Bieder, L. (1969). Proc. Roy. Soc. Med. (London)Symp. Clin. Eval. Cephalexin p. 99.

T H E C E P H A L O S P O R I N GROUP O F A N T I B I O T I C S

149

Bieder, L. (1970). Postgrad. Med. J., Suppl. 46, 141. Binns, J. O., and Pankey, G. A. (1966). J. La. State Med. Soc. 118, 493. Blockey. h. J., and Watson, J. T. (1970). J . Bone Joint Surg., Brit. Vol. 52, 77. Bobrowski, M.,and Borowski, E. (1971). J. Gen. Microbiol. 68,263. Bodner, S. J., and Koenig, M. G. (1972). Amer. J . Med. Sci. 263, 43. Bond, C. M., Lees, K. A., and Packington, J. L. (1970). Pharm. J. 2 0 5 , 2 1 0 . Bond, J. M., Brimblecombe, R. W., and Codner, R. C. (1962). J . Gen. Microbiol. 27, 11. Boniece, W. S., Wick, W. E., Holmes, D. E., and Redman, C. E. (1962). J. Bacteriol. 84, 1292. Boyd, J. F., Butcher, B. T., and Stewart, G. T. (1971). Brit. J. Exp. Pathol. 52, 503. Boyer, J. L., and Andriole, V. I’. (1968). Yale J . Biol. Med. 40, 284. Boyle, G. L., Hein, H. F., and Leopold, I. H. (1970). Amer. J . Opthalmol. 69,868. Bran, J. L., Levinson, M. E., and Kaye, D. (1972). Antimicrob. Ag. Chemother. 1,35. Brandriss, M. W., Smith, J. W., and Steinman, H. G. (1964). Postgrad. Med. J . , Suppl. 40, 157. Braun, P., Tillotson, J. R., Wilcox, C., and Finland, M. (1968). Appl. Microbiol. 16, 1684. Brayton, R. G., MacLean, R. A,, and Louria, D. B. (1967). Antimicrob. Ag. Chemother. p. 96. Brogard, J. M., Haegele, P., Dorner, M., and Lavillaureix, J. (1972). Ann. Biol. Clin. (Paris) 30, 593. Brogard, J. M., Haegele, P., Dorner, M., and Lavillaureix, J. (1973a). Antimicrob. Ag. Chemother. 3, 19. Brogard, J. M., Haegele, P., Kohler, J. J., Dorner, M., Lavillaureix, J., and Stahl, J. (1973b). Chemotherapy 18, 212. Brogard, J. M., Kuntzmann, F., and Lavillaureix, J. ( 1 9 7 3 ~ )Schweiz. . Med. Wochenschr.

103,100. Brotzu, G . (1948). Lavori. dell’istituto D’lgiene di Cagliari Brown, 1. D., Mathias, A. W., Ivler, D., Warren, W. S. and Leedom, J. M. (1970). Antimicrob. Ag. Chemother. p. 432. Brumfitt, W., Faiers, M. C., and Franklin, I. N. S. (1970). Postgrad. Med. J., Suppl. 46, 65. Brumfitt, W., and Pursell, R. (1972). Brit. Med. J. ii, 673. Bulger, R. J. (1967a). Lancet i , 17. Bulger, R. J. (1967b). Ann. Intern. Med. 67,523. Bulger, R. J., and Petersdorf, R. G. (1970). Postgrad. Med. J. 47, 160. Burdash, N. M., Erhlich, M. A., Erhlich, H. G., and Paris, J. T. (1%8). J . Bacteriol. 95, 1956.

Burgess, M. A., and Evans, R. J. (1966). Brit. Med. J . ii, 1244. Burgess, M. A., Fairley, K. F., and Habersburger, P. (1968). Med. J. Aust. 1, 947. Burland, W. L., and Simpson, K. (1967). Postgrad Med. J . , Suppl. 43, 112. Burland, W. L., Simpson, K., and Samuel, P. D. (1970). Postgrad. Med. J., Suppl. 46, 85. Burton, H. S., and Abraham, E. P . (1951). Biochem. J. 62,315. Busca, G. (1969). Proc. Roy. Soc. Med. (London) Symp. Clin. Eval. Cephalexin p. 57. Butler, N. R. (1967). Postgrad. Med. J . , Suppl. 43, 97. Butler, N. R., and Bonham, D. G. (1963). “British Perinatal Mortality Survey,” First Report. Livingstone, Edinburgh. Cabitza, .4. (1965). Rass. Med. Sarda 68,697. Carrizosa, J., Levinson, M. E., and Kaye, D. (1973). Antimicrob. Ag. Chemother. 3, 306. Chabbert, Y. A. (1967). Postgrad Med. J., Suppl. 43, 40.

150

D. R. OWENS ET AL.

Chang, T.-W., and Weinstein, L. (1964a). Science 143, 807. Chang, T.-W., and Weinstein, L. (1964b). J. Bacteriol. 88, 1790. Chang, T.-W., and Weinstein, L. (1%6). Nature (London) 211, 763. Charles, D., and MacAuIey, M. A. (1970). Clin. Obstet. Gynecol. 106, 237. Chauvette, R. R., Flynn, E. H., Jackson, B. G., Lavagnino, E. R., Morin, R. B., Mueller, R. A., Pioch, R. P., Roeske, R. W., Ryan, C. W., Spencer, J. L., and Van Heyningen, E. (1963). Antimicrob. Ag. Chemother. p. 687. Chauvette, R. R., Hayes, H. B., Huff, G. L., and Pennington, P. A. (1972). J. Antibiot. 25, 248. Cheng, D. L., Voth, D. W. and Lin, C. (1970). Intersci. Con$ Antimicrob. Ag. Chemother., 10th Abstr. No. 25. Child, K. J., and Dodds, M. G. (1966). Brit. J. Pharmacol. Chemother. 26, 108. Child, K. J., and Dodds, M. G. (1967). Brit. J. Pharmacol. Chemother. 30,354. Chisholm, D. R., Leitner, F., Misiek, M., Wright, G. E., and Price, K. E. (1970). Antimicrob. Ag. Chemother. p. 244. Chou, T. S. (1969). J . Med. Chem. 12,925. Citri, N. (1971). In “The Enzymes” (P. D. Boyer, ed.), 3rd Ed., Vol. 4, p. 23. Academic Press, New York. Citri, N., and Kalkstein, A. (1967). Arch. Biochem. Biophys. 121, 720. Citri, N., and Pollock, M. R. (1966). Advan. Enzymol. 28,237. Citron, K. M., Emerson, P . A., Joyce, C. R. B., May, J. R., Selkon, J. B., Sommer, A. R., and Spriggs, E. A. (1968). Lancet ii, 592. Clark, H., and Turck, M. (1969). Antirnicrob. Ag. Chemother. p. 2%. Cohen, P. G., Romansky, M. J., and Johnson, A. C. (1%6). Antimicrob. Ag. Chemother. p. 894. Condie, A. P., Williams, J. D., Reeves, D. S., and Brumfitt, W. (1968). In “Urinary Tract Infections” (F. O‘Grady and W. Brumfitt, Eds.), p. 148. Oxford Univ. Press, London and New York. Constantindes, G. (1971). Lille Med. 8, 1178. Conti, M., and Iurlaro, F. (1967). Ann. Ostet. Ginecol. 89, 637. Corda, R. and Scane, V. (1965). Rass. Med. Sarda 68, 669. Cox, C. E., and Montgomery, W. G. (1971). Postgrad. Med. J., Suppl. 47, 107. Cox, R., Modhavan, T., Hwang, C., Pohlod, D., and Quinn, E. (1972). Intersci. Con$, 12th, Antimicrob. Ag. Chemother. Abstr. No. 142. Crawford, K., Heatley, N. G., Boyd, P. F., Hale, C. W., Kelly, B. K., Miller, G. A , , and Smith, N . (1952). J . Gen. Microbiol. 6, 47. Crompton, B., Jago, M., Crawford, K., hewton, G. G. F., and Abraham, E. P. (1962). Biochem. .J. 83, 52. Csonka, G. W. (1%7). Postgrad. Med. J . , Suppl. 43, 63. Csonka, G. W. (1971). Postgrad. Med. J., Suppl. 47, 122. Csonka, G. W., and Murray, M. (1967). Postgrad. Med. J . , Suppl. 43, 123. Daikos, G. K., Kontomichalou, P., Roussos, C., Papachristou, E., and Kosmidis, J. (1969). Proc. Roy. SOC.Med. (London) Symp. Clin. Eval. Cephalexin p. 74. Daikos, G. K., Kontomichalou, P., Roussos, C., Papachristou, E., and Giamazelou, E. (1970). Postgrad. Med. J., Suppl. 46, 149. da Silva, J. R., Coura, J. R., and Lopes, P. A. (1967). J . B r a d Med. I’rop. 1, 51. Datta, N., and Kontomichalou, P. (1965). Nature (London) 208, 239. Datta, N., and Richmond, M. H. (1%6). Biochem. J . 98, 2M. Davies, J. A., and Holt, J. M. (1972). J. Clm. Pathol. 2 5 , 518.

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

151

Davies, J. A., Strangeways, J. E. M., and Holt, J. M. (1970). Postgrad. Med. J., Suppl. 46, 16. Davies, J. A , , Strangeways, J. E. M., Mitchell, R. G., Beilin, L. J., Ledingham, J. G. G., and Holt, J. M. (1971). Brit. Med. J. iii, 215. Davies, P. A. (1972). Brit. J. Hosp. Med. 8, 13. Davis, A., Seligman, S. J., Hewitt, W. L., and Finegold, S. M. (1964). Antimicrob. Ag. Chemother. p. 272. Davis, J. R. (1970). Med. J . A w . , Suppl. 1, 15. Davis, J. S., and Kaufman, R. M. (1966). Amer. j . Obstet. Gynecol. 95, 523. De Maine, J. B., and Kirby, W. M. M. (1971). Antimicrob. Ag. Chemother. p. 190. Dennis, M., Rasch, J. R., and Hastings, H. K. (1966). Antimicrob. Ag. Chemother. p. 724. De Schepper, P., Harvengt, C., Vranckx C . , Boon, B., and Lamy, F. (1973). J. Clin. Pharmacol. J . New Drugs 13,83. Dillon, M. L., Bowling, K. A.. and Posthethwait, R. W. (1972). Surg., Gynecol. Obstet. 134,83. Disney, F. A., Breese, B. B., Green, J. L., Talpey, W. B., and Tobin, J. R. (1971). Postgrad. Med. J., Suppl. 47, 47. Dodds, M. G., and Foord, R. D. (1970). Brit. J. Pharmacol. 40, 227. Donnison, A. B., and Davison, C . E. (1970). Postgrad. Med. J., Suppl. 46, 93. Duncan, W. C., and Knox, J. M. (1971). Postgrad. Med. J., Suppl. 47, 49. Drach, G. W., Lacy, S. S., and Cox, C. E. (1971). J. Urol. 105, 840. Eckolt, R., Biirgi, H., and Regli, J. (1972). In “Advances in Antimicrobial and Antineoplastic Chemotherapy” (M. Hejzlar, ed.), Vol. 7, p. 1245. Urban & Schwarzenberg, Munich. Editorial. (1%7). Brit. Med. J. i, 515. Edwards, J. R., and Park, J. T. (1969). J . Bacteriol. 99, 459. Ehrenkranz, N. J. (1971). Postgrad. Med. J., Suppl. 47, 94. Eickhoff, T. C., and Finland, M. (1%5). New Engl. J . Med. 272, 395. Elkins, I., Montgomery, W. G . , and Cox, C . E. (1972). Intersci. Conf., 12th, Antimicrob. Ag. Chemother. Abstr. No. 33. Erikssen, J., Midtvedt, l., and Bergan, T. (1970). Scand. J . Infec. Dis. 2, 53. Eyckmans, L. (1970). Chemotherapy 15, 322. Eyckmans, L., Van Landuyt, M., and Verberckmoe, S. R. (1968). Chemotherapy 13, 193. Eykyn, S. (1971). J. Clin. Pathol. 24, 419. Fairley, K. F. (1970). Postgrad. Wed. J., Suppl. 46, 21. Fallon, R. J. (1970). J . Appl. Bacteriol. 33, 744. Fallon, R. J . , and Hutchinson, D. (1967). Lancet ii, 417. Fass, R. J., Carleton, J., Watanakunakorn, C., Mainer, A. S., and Hamburger, M. (1970a). Infect. Immunol. 2, 504. Fass, R. J., Perkins, R. L., and Saslaw, S. (1970b). Amer. J . Med. Sci. 259, 187. Fekety, F. R., and Weiss, P. (1%7). Antimicrob. Ag. Chemother. p. 156. Fine, R. N., Davison, J. H., Volle, 1’. N., and Medina, D. A. (1%6). J. Pediat. 69, 1079. Finland, M. (1972). Amer. J. Med. Sci. 263, 207. Fiumara, N . J. (1972). Med. Clin. N . Amer. 56, 1105. Flarer, F. (1967). Postgrad. Med. J., Suppl. 47, 133. Flarer, F., Galla, F., Pagnes, P., and Ferrari, M. (1965). Antibioticu, 3, 216. Fleming, P. C., and Jaffe, D. (1%7). Postgrad. Med. J . , Suppl. 43, 89. Fleming, P . C., Goldner, M., and Glass, D. G . (1%3). Lancet i, 1399. Fleming, P. C . , Huda, S. S., and Bubechko, W. P. (1970). Postgrad. Med. J., Suppl. 46, 89.

152

D. R . OWENS ET AL.

Flux, M., Riley, H. D., and Bracken, E. C. (1964).Antimicrob. Ag. Chemother. p . 254. Flux, M., Nunnery, A. W., and Riley, H. D. (1966). Antimicrob. Ag. Chemother. p . 933. Flynn, E. M. (1971). Postgrad. Med. J., Suppl. 47, 9. Foord, R. D. (1967). Postgrad. Med. J., Suppl. 43,63. Foord, R. D. (1969).Progr. Antimicrob. Anticancer Chemother. 1, 597. Foord, R. D. (1971).Brit. Med. J. ii, 493. Foord, R. D. and Snell, E. S. (1966).Brit. Med. J. ii, 1956. Foord, R. D. Dash, C. M., and Johnson, S. E. (1969a). Progr. Antimicrob. Anticancer Chemother. 1, 705. Foord, R. D., O'Callaghan, Cynthia, H., Muggleton, P. W., and Currie, J. P . (1969b). Proc. Roy. Soc. Med. (London) Symp. Clin. Eval. Cephalexin p . 3. Forfar, J. O., Keay, A. .I.Maccabe, , A. F., Gould, J. C., and Bain, A. D. (1966). Lancet ii, 295. Foster, F. P. (1969). Med. Clin. N. Amer. 53, 437. Fountain, R. H., and Russell, A. D. (1969). J. Appl. Bacteriol. 32, 312. Fountain, R. H., and Russell, A. D. (1970).Microbios 2, 933. Fowler, W. (1969).Proc. Roy. Soc. Med. (London) Symp. Clin. Eval. Cephalexin p . 54. Fowler, W. (1970). Postgrad. Med. J., Suppl. 46, 106. Frank, P. F., Stollerman, G. H., and Miller, L. F. (1965).J. Amer. Med. Ass. 193, 775. Fujii, R., Konno, M., and Ubukata, K. (1969). Proc. Roy. Soc. Med. Symp. Clin. Eval. Cephalexin p . 79. Gabriel, R., Foord, R. D., and Joekes, A. M. (1970).Brit. Med. J. iv, 283. Gager, W. E., Elsas, F. J., and Smith, J. L. (1969).Brit. J. Ophthalmol. 53, 403. Galbraith, H. J. B. (1967).Postgrad. Med. J., Suppl. 43, 58. Galla, F., Pagnes, P., and Ferrari, M. (1%5). Chemotherapia 10, 24. Garber, N., and Friedman, J. (1970). J. Gen. Microbiol. 64, 343. Garrod, L. P., Shooter, R. A., and Curwen, M. P . (1955).Brit. Med. J. ii, 1013. Gau, D. W., Horn, R. F. H., Solomon, R. M., Johnson, P. and Leigh, D. A. (1972). Practitioner 208, 276. Gherardi, C. R., Mazzei, C. M., Diez, E., and Rechniewki, C. (1965). Prensa Med. Argent. 52, 1916. Glicksman, J. M., Short, D. H., and Knox, J. M. (1968). Arch. Intern. Med. 1 2 1 , 342. Godzeski, C. W., Brier, G., and Pavey, D. E. (1963).Appl. Microbiol. 11, 122. Goldsweig, H. G., Matsen, J. M., and Castaneda, A. R. (1972). J . l'horac. Cardiovasc. Surg. 63,408. I Gonnella, J. S., Olexy, V. M., and Jackson, G. G. (1967).Amer. J. Med. Sci. 254, 93. Gordon, R. C., Barrett, F. F., Clark, D. J., and Yow, M. D. (1971). Curr. Zher. Res., Clin. Exp. 13,398. Goto, M. (1969). Chemotherapy 17, 1619. Gottshall, R. Y., Roberts, J. M., Portwood, L. M., and Jennings, J. C. (1951). Proc. Soc. Exp. Biol. Med. 76, 307. Gottstein, W . J., Eachus, A. H., Misco, P. F., Cheney, L. C., Misick, M. and Price, K. E. (1971).J. Med. Chem. 14, 770. Gould, J. C. (1970). Postgrad. Med. J., Suppl. 46, 83. Gower, P. E., and Dash, C. H. (1969). Brit. J. Pharmacol. 37, 738. Gralnick, H. R. (1971). Postgrad. Med. J., Suppl. 47, 67. Gralnick, H. R., Wright, L. D., and McGinniss, M. (1967). J. Amer. Med. Ass. 199, 725. Greenwood, D., and O'Grady, F. (1973a).J. Clin. Pathol. 26, 1. Greenwood, D., and O'Grady, F. (1973b).J. Infec. Dis. 1 2 8 , 791.

THE CEPHALOSPORIN GROUP O F ANTIBIOTICS

153

Griffith, R. S. (1969). Aduan. Clin. Pharmacol., Oracef Symp., Frankfurt; Int. Z . Klin. Pharmakol. l’her. loxikol. Suppl., p. 92. Griffith, R. S., and Black, H. R. (1964). J. Amer. Med. Ass. 189, 823. Griffith, R. S., and Black, H. R. (1968). Clin. Med. 7 5 , 14. Griffith, R. S., and Black, H. R. (1970). Med. Clin. N . Amer. 54, 1229. Griffith, R. S., and Black, H. R. (1971). Postgrad. Med. J., Suppl. 47, 32. Grislain, J. R. (1970). Postgrad Med. J., Suppl. 46, 95. Grislain, J. R. (1971). Gaz. Med. Fr., Suppl. 78, No. 4, 40. Gruneberg, R. N., and Brumfitt, W. (1967). Brit. Med. J . iii, 64.9. Gump, D. W., and Lipson, R. L. (1968). Curr. Ther. Res., Clin. Exp. 10, 583. Guthe, T. E. (1965). “Research Reviews and Clinical Trials, 1964-1%5,“ p. 162. Medical News, London. Haberman, H., Thomas, J., Weiss, M., Cook, G., and Hari, C. H. (1969). Cutis 5 , 945. Hale, C. W., Newton, G. G. F., and Abraham, E. P. (1961). Biochem. J . 79, 403. Hallander, H. O., and Laurell, G. (1972). Antimicrob. Ag. Chemother. 1, 422. Hallberg, l., and Svenningsen, N. W. (1970). Acta Paediat. Scand. 206, 110. Hamilton-Miller, J. M. 1. (1967a). Nature (London) 214, 1333. Hamilton-Miller, J. M. T. (196713). [Discussion of paper by Newton and Hamilton-Miller (1%7). Postgrad. Med. J., Suppl. 43, 101. Hamilton-Miller, J. M. T. (1971a). J. Med. Microbial. 4, 227. Hamilton-Miller, J. M. T.(1971b). J. Theor. Biol. 31, 171. Hamilton-Miller, J. M. T., and Ramsey, J. (1967). J. Gen. Microbial. 49, 491. Hamilton-Miller, J. M. T., and Smith, J. 1.(1964). Nature (London) 201, 999. Hamilton-Miller, J. M. l., Smith, J. T., and Knox, R. (1965). Nature (London) 208, 235. Hamilton-Miller, J. M. T., Newton, G. G. F., and Abraham, E. P. (1970a). Biochem. J. 116, 371. Hamilton-Miller, J. M. T., Richards, E. and Abraham, E. P. (1970b). Biochem. J., 116, 385. Hardy, J. B., Hardy, P. H., Oppenheimer, E. H., Ryan, S. J., and Sheff, C. N. (1970). J . Amer. Med. Ass. 121, 1345. Hartmann, R., Holtje, J.-V., and Schwartz, U. (1972). Nature (London) 235, 426. Harvengt, C., De Schepper, P., Lamy, F., and Hansen, J. (1973). J. Clin. Pharmacol. J . New Drugs 13, 36. Hatano, H., Kayaba, T., and Shiga, N . (1966). Rinsho Ganka 20, 805. Hedlund, P. (1969). Proc. Roy. SOC. Med. (London) Symp. Clin. Eval. Cephalexin p. 68. Hedlund, P. (1970). Postgrad. Med. J., Suppl. 46, 152. Heitler, M. S., Isenberg, H. D., Karelitz, S., Acs, H., and Driller, M. (1964). Antimicrob. Ag. Chemother. p. 261. Hennesey, T. D., and Richmond, M. H. (1968). Biochem. J. 1 0 9 , 4 6 9 . Hermans, P. E. (1971). Postgrad. Med. J . , Suppl. 47, 99. Hermans, P. E., Martin, J. K., Needham, G. M., and Nichols, D. R. (1966). Antimicrob. Ag. Chemother. p. 879. Herrell, W. E. (1%8). Clin. Med. 75, 21. Herrell, W. E. (1971). Clin. Med. 78, 15. Herrell, W. E., Balows, A,, and Becker, J. (1965). Antimicrob. Ag. Chemother. p. 350. Herrell, W. E., Balows, A., and Becker, J. (1967). Int. Med. Dig. 2, 23. Hess, R. .I. and Mastin, J. H. (1971). J. Amer. Med. Ass. 218, 592. Hewitt, J. H., and Parker, M. T. (1968). J . Clin. Pathol. 21, 75. Hinman, A. R., and Wolinsky, E. (1967). J. Aner. Med. Ass. 200, 724.

154

D. R. OWENS ET AL.

Hirsch, H. A. (1968). Int. J. Clin. Pharmacol. Sondescheft Cephalosporine, 76. Hirsch, H. A. (1970). Progr. Antimicrob. Anticancer Chemother. 1,815. Hirsch, H. A. (1971). Postgrad. Med. J. 47,90. Hodges, G. R., Scholand, J. F. and Perkins, R. L. (1973). Antimicrob. Ag. Chemother. 3, 228. Hodson, C. J. (1959). Proc. Roy. SOC. Med. 52,669. Hodson, A. H., and Holloway, W. J. (1973). Int. Z. Klin. Pharmakol. lher. loxikol. 7, 89. Hoeprich, P. D. (1968). Antimicrob. Ag. Chemother. p. 697. Hogan, L. B., Holloway, W. J., and Jakubowitch, R. A. (1968). Antimicrob. Ag. Chemother. p. 624. Holloway, W. J., and Scott, E. G. (1%5). Antimicrob. Ag. Chemother. p . 215. Holloway, W. J., and Scott, E. G. (1966). Antimicrob. Ag. Chemother. p . 915. Ikui, H., Oniki, S., and Nonaka, Y. (1966).Rinsho Ganka 20, 1339. Inagaki, J., and Bodey, G. P. (1973). Curr. Ther. Res. 15, 37. Ishigami, J., Hara, S., and Shafi. T. (1965).J. Antibiot., Ser. A 18, 292. Ishigami, J., Hara, S., and Shafi, T. (1969). Proc. Roy. SOC.Med. (London) Symp. Clin. Eval. Cephalexin p. 44. Ishiama, S., Kawakami, I., and Diakayama, I. (1967).Postgrad. Med. J., Suppl. 43, 148. Ishiama, S.. Diakayama, I., Iwamoto, H., Iwai, S., Okui, M., and Matsubara, T. (1971). Antimicrob. Ag. Chemother. p. 476. Jack, G. W., and Richmond, M. H. (1970).J . Gen. Microbiol. 61,43. Jacquot, A. (1%9). Proc. Roy. SOC.Med. Symp. Clin. Eval. Cephalexin p. 102. Jago, M. (1964). Brit. J . Pharmacol. Chemother. 22,22. Jago, M., Migliacci, A., and Abraham, E. P. (1963).Nature (London)l99,375. James, D. G., and Walker, A. (1971). Brit. J. Hosp. Med. 7, 795. Jameson, S., McHenry, M. C., Gavan, 1’. L., Vanommen, R. A., Vidt, D. G., and Butler, D. A. (1971).Del. Med. J . 43, 384. Jawetz, E. (1968). Annu. Rev. Pharmacol. 8, 151. Jefferiss, F. J. G., and Willcox, R. R. (1963). Brit. J. Vener. Dis. 39, 143. Johnson, W. D., Applestein, J. M., and Kaye, D. (1968). J. Amer. Med. Ass. 206, 2698. Josey, W. E., and Farrar, W. E. (1970). Amer. J. Obstet. Gynecol. 106, 237. Jouhar, A. J., and Fowler, W. (1968).Brit. J. Vener. Dis. 44, 223. Kabins, S. A., and Cohen, S. (1965). Antimicrob. Ag. Chemother. p. 207. Kabins, S. A., and Cohen, S. (1966).Antimicrob. Ag. Chemother. p . 922. Kabins, S. A., Kelner, B., Walton, E., and Goldestein, E. (1970).Amer. J. Med. Sci. 2 5 9 , 133. Kagan, B. M. (1965).Ann. N.Y. Acad. Sci. 128, 81. Kagan, B. M., Molander, C. W., Zolla, S., Heimlich, E. M., Weinberger, H. J., Busser, R., and Liepnieks, S. (1964).Antimicrob. Ag. Chemother. p. 517. Kanyuck, D. O., Welles, J. S., Emmerson, J. L., and Anderson, R. C. (1971). Proc. SOC. Exp. Biol. Med. 136, 997. Kaplan, D., and Koch, W. (1968).Nature (London) 218, 1165. Kaplan, K., and Weinstein, L. (1969).Ann. Intern. Med. 7 0 , 919. Kaplan, K., Reisberg, B. E., and Weinstein, L. (1967).Amer. J. Med. Sci. 253, 667. Kaplan, K., Reisberg, B. E., and Weinstein, L. (1%8a), Arch. Intern. Med. 121, 168. Kaplan, K., Reisberg, B. E., and Weinstein, L. (1968b). Arch. Intern. Med. 121, 17. Kariyone, K., Harade, H., Kurita, M., and Takano, T. (1970). J. Antibiot. 23, 131. Kass, E. H., and Zinner, S. H. (1969).J. Infec. Dis. 120, 27. Katsu, M., Fujimori, I., Ogawa, J., Itoh, S., and Shimada, S. (1965). J. Antibiot. Ser. A 18, 261.

T H E C E P H A L O S P O R I N GROUP OF ANTIBIOTICS

155

Kawamura, J. (1970). Postgrad. Med. J., Suppl. 46, 28. Kaye, D., Levison, M. E., lhornhill, 1. S., and Johnson, W. D. (1970). Progr. Antimicrob. Anticancer Chemother. 1, 913. Kayser, F. H. (1971). Postgrad. Med. J., Suppl. 47, 14. Kayser, F. H., Benner, E. J., and Hoeprich, P. D. (1970). Appl. Microbiol. 20, 1. Kayser, F. H., Wusy, J. and Corrodi, P. (1972). Antimicrob. Ag. Chemother. 2, 217. Keay, A. J., and Fleming, J. G. (1970). Postgrad. Med. J., Suppl. 46, 81. Keay, A. J., Syme, J., and Barnes, P. M. (1%7). Postgrad. Med. J., Suppl. 43, 105. Kennedy, R. S. (1967). Postgrad. Med. J., Suppl. 43, 51. Keyes, T. F., and Halverson, C. W. (1969). J. Amer. Med. Ass. 210, 857. Khan, A. J., and Pryles, C. V. (1973). Cum. "her. Res. 15, 198. Kienitz, M. (1971). Postgrad. Med. J., Suppl. 47, 87. Kienitz, M., and Mann, G. (1%9). Advan. Clin. Pharmacol., Oracef Symp., Frankfurt; Int. Z . Klin. Pharmakol. l'her. l'oxikol. Suppl., p. 97. Kind, A. C., Kestle, D. G., Standiford, H. C., and Kirby, W. M. M. (1969a). Antimicrob. Ag. Chemother. p . 361. Kind, A. C., Kestle, D. G., Standiford, H. C., Freeman, P., and Kirby, W. M. M. (1%9b). Antimicrob. Ag. Chemother. p. 405. King, A. J. (1971). Brit. J . Clin. Pract. 25, 295. Kirby, W. M. M., De Maine, J. B., and Serrill, W. S. (1971). Postgrad. Med. J., Suppl. 47,41. Kislak, J. W., Steinhauser, B. W., and Finland, M. (1966). Amer. J . Med. Sci. 251, 433. Klainer, A. S., and Perkins, R. L. (1970). J . Znfec. Dis. 122, 323. Klainer, A. S., and Perkins, R. L. (1972). Antimicrob. Ag. Chemother. 1, 164. Klastersky, J., Daneau, D., and Weerts, D. (1973). Chemotherapy 18, 191. Klein, J. O., Eickhoff, J. C., Tilles, J. G., and Finland, M. (1964). Amer. J . Med. Sci. 248,640. Kline, A. H., Blattner, R. J., and Lunin, M. (1964). J. Amer. Med. Ass. 188, 178. Kniisel, F., Konopka, E. A., Gelzer, J., and Rosselet, A. (1971). Antimicrob. Ag. Chemother. p. 140. Kosakai, N., and Miyakawa, C. (1970). Postgrad. Med. J., Suppl. 46, 107. Koya, G., and Misawa, H. (1970). Progr. Antimicrob. Anticancer Chemother. 1, 747. Kozatani. J . , Okui, M., Matsubara, l.,and Nishida, M. (1972a). J. Antibiot. 25, 86. Kozatani. J., Okui, M., Noda, K., Ogina, l.,and Noguchi, H. (1972b). Chem. Pharm. Bull. 20, 1105. Kradolfer, F., Sackmann, W., Zak, O., Brunner, H., Hess, R., Konopka, E. A., and Gelzer, J. (1971). Antimicrob. Ag. Chemother. p. 1.50. Kump, J., Moskowitz, J., Ehrenkranz, N. J., and Panunzio, Y. (1%9). S. Med. J. 62, 461. Kunin, C. M., and Atuk, N. (1966). New Engl. J . Med. 274, 654. Kunin, C. M., and Brandt, D. (1968). Amer. J. Med. Sci. 255, 1%. Kunin, C. M., and Finkelberg, Z. (1970). Ann. Intern. Med. 72, 349. Kuwahara, S., and Abraham, E. P. (1%7). Biochem. J. 103, 27C. Kuwabara, S., and Abraham, E. P. (1969). Biochem. J . 115, 859. Ky, N. T., Chauvin, M. T., Pinon, C., and Halpern, B. N. (1970). Postgrad. Med. J., Suppl. 46, 109. Lacy, S. S., Drach, G. W., and Cox, C. E. (1971). J. Urol. 105, 836. Lai, G. (1%7). Minerva Ortop. 18, 190. Larnbin, S., and Bernard, J. (1967). Postgrad. Med. J., Suppl. 43,42. Landa, L. (1972). Curr. Ther. Res. 14, 496.

156

D. R. OWENS ET AL.

Landes, R. R., McCormick, B. H., Graham, R. H., and Melnick, I.(1967). J. Urol. 97, 147. Landes, R. R., Melnick, I., Fletcher, A., and McCormick, B. H. (1969). J. Urol. 102, 246. Landes, R. R., Melnick, I., Hoffman, A. A., Fehrenbaker, L. G., and Oakes, A. L. (1972). Clin. Med. 79, 23. Lane, A. Z., Taggart, J. G., and Iles, R. L. (1972). Antimicrob. Ag. Chemother. 2, 234. Lang, G. R., and Levin, S. (1971). Med. Clin. N . Amer. 55, 1439. Lautre, G., and Baker, L. W. (1972). S. Afr. Med. J. 47, 837. Lawson, D. H., Macadam, R. F., Singh, H., Gavras, H., and Linton, A. L. (1970). Postgrad Med. J., Suppl. 46, 36. Lawson, D. H., Macadam, R. F., Singh, H., Gavras, H., Hartz, S., Turnhull, D., and Linton, A. L. (1972). J. Infec. Dis. 126, 593. Lee, C.-C., and Anderson, R. C. (1963). Antimicrob. Ag. Chemother. p. 695. Lee, C.-C., Herr, E. B., and Anderson, R. C. (1963). Clin. Med. 70, 1123. Leiderman, E., Stowe, F. R., and Mogahgah, W. J. (1970). Clin. Med. 77, 27. Leigh, D. A., Faiers, M. C., and Brumfitt, W. (1970). Postgrad. Med. J., Suppl. 46, 69. Lenti, G., Pellegrini A., Pagano, G., Pisano, E., and Brotzu, M. V. (1965). Rass. Med. Sarda 68, 621. Lerner, P. I.(1968). Antimicrob. Ag. Chemother. p. 730. Lerner, P. I. (1969). Amer. J. Med. Sci. 257, 125 Lerner, P. I. (1971). Amer. J . Med. Sci. 262, 321. Lerner, P. I., and Weinstein, L. (1967). Amer. J. Med. Sci. 254, 63. Le Roux, B. T. (1970). Postgrad. Med. J., Suppl. 46, 119. Levison, M. E., Johnson, W. D., ThornhiU, 1’. S., and Kaye, D. (1969). J. Amer. Med. Ass. 209, 1331. Lidman, K., Sterner, G., and Henning, C. (1969). Proc. Roy. Soc. Med. (London) Symp. Clin. Eval. Cephalexin p. 63. Limson, B. M., and Santos, R. J. (1968). Clin. Med. 75, 36. Limson, B. M., Siasoco, R. E., and Dial, F. P. (1972). Cum. Ther. Res. 14, 101. Linquist, J. A., Siddiqui, J. Y., and Smith, I. M. (1970). New Engl. J. Med. 283, 720. Linsell, W. D., Pines, A., and Hayden, J. W. (1967). Postgrad Med. J., Suppl. 43, 90 Linton, A. L., Bailey, R. R., and Turnbull, D. I. (1972). Can. Med. Ass. J. 107, 414. Loder, B., hewton, G. G. F., and Abraham, E. P. (1961).Biochem. J . 79, 408. Lorian, V., and Sabath, L. D. (1972). J. Infec. Dis. 125, 560. Love, W. C., McKenzie, P., Lawson, J. H., Pinkerton, I. W., Jamieson, W. M., Stevenson, J., Roberts, W., and Christie, A. B. (1970). Postgrad. Med. J., Suppl. 46, 155. Lowentritt, L., Schlegel, J. U., and Dominque, G. J. (1971). J . Urol. 106, 615. Lucas, J. B., Thayer, J. D., Utley, P. M., Billings, 1’. E., and Hackney, J.E(l966). J. Amer. Med. Ass. 195, 919. Luscombe, D. K., and Nicholls, P. J. (1975). J . Antimicr-ob. Chemother. 1, 67. Luscombe, D. K., Ilicholls, P. J., Owens, D. R., and Russell, A. D. (1972). Brit. J . Pharmacol. 46, 552p. Luscombe, D. K., hicholls, P. J., Owens, D. R., and Russell, A. D. (1973). Antimicrob. Ag. Chemother. 3, 677. Lyons, R. W., and Andriole, V. 7’. (1971). Yale J . Biol. Med. 44, 187. McAllister, 1’. A., and Mack, W. S. (1967). Postgrad. Med. J . , Suppl. 43, 71. MacAulay, M. A., and Charles, D. (1968).Amer. Obstet. Gynecol. 100, 940. McCabe, W. R., and Jackson, G. G. (1965).New Engl. J . Med. 272, 1037.

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

157

McClone, D. G., Scott, A., Mackey, D. M., and Hackney, J. F. (1%8). Brit. J. Vener. Dis. 44,220. McCloskey, R. V., Terry, E. E., McCracken, A. W., Sweeney, M. J., and Forland, M. F. (1972). Antimicrob. A s . Chemother. 1, 90. McKenzie, P., Love, W. C., Lawson, J. H., Pinkerton, I. W., Jamieson, M., and Stevenson, J. (1967). Postgrad. J . , Suppl. 43, 142. MacLean, R., Brayton, R. G., and Louria, D. B. (1967). Antimicrob. Ag. Chemother. p. 61. MacMillan, M. G. (1971). Arch. Dis.childhood 46, 739. Macquet, V., and Lafitte, P. L. (1970). Postgrad. Med. J . , Suppl. 46, 128. Malaka-Zafiriu, K., and Strates, B. S. (1969). Actu Paediat. Scand. 58, 281. Manhas, M. S., and Bose, A. K. (1969). “Synthesis of Penicillin, Cephalosporin C and Analogues.” Dekker, New York. Marget, W. M. (1967). Postgrad. Med. J . , Suppl. 43, il5. Marget, W. M. (1971). Postgrad. Med. J., Suppl. 47, 54. Marget, W. M., and Daschner, F. (1969). Arzneim.-Forsch. 19, 1956. Marks, J . G., and Garrett, R. T. (1970). Postgrad. Med. J . , Suppl. 46, 113. Marshall. J. H., and Curtis, F. R. (1967). Postgrad. Med. J . , Suppl. 43, 121. Marshall. J. H., Ross, G. V., Ghanter, K. V., and Harris, A. M. (1972). Appl. Microbiol. 23, 765. Mashimo, K. (1971). Postgrad. Med. J . , Suppl. 47, 67. Matsen, J. M. (1971). Amer. J. Dis. Child 121, 38. Matsuhashi, S., ed. (1971). “l’ransferable Drug Resistance Factor R.“ Univ. of Tokyo Press, Tokyo. Matsumoto, K., Yokoyama, K., and Nakamura, T. (1970). Postgrad. Med. J . , Suppl. 46, 123. Matthews, A. E. B., Harding, J. W., and Jauhar, A. J. (1967). Postgrad. Med. J., Suppl. 43, 116. Matts, S. G. F. (1967). Postgrad. Med. J., Suppl. 43, 55. Maurice, P. N . , Riess, W., Welke, A., and Amsou, K. (1973). Schweiz. Med. Wochenschr. 103, 718. May, J. R. (1967). Postgrad. Med. J . , Suppl. 43, 68. Maynard, E. P. (1969). New Engl. J . Med. 280, 505. Medeiros. A. A., and O‘Brien, T. F. (1968). Antimicrob. Ag. Chemother. p. 271. Merchant, S. M. (1967). Postgrad. Med. J . , Suppl. 43, 56. Merrill, S. L., Davis, A., Smolens, B., and Finegold, S. M. (1966). Ann. Intern. Med. 64, 1. Meyer-Rhon, J. (1972). Postgrad. Med. J . 48, 58. Meyers, B. R., Kaplan, K., and Weinstein, L. (1969). Clin. Pharmacol. Ther. 10, 810. Meyers, B. R., Bottone, E., Hirschman, S. Z., and Schneierson, S. S. (1972a). Ann. Intern. Med. 76, 9. Meyers, B. R., Hirschman, S. Z., and Nicholas, P. (197213). Antimicrob. Ag. Chemother. 2,250. Meyler, L., and Herxheimer, A. (1968). “Side Effects of Drugs,”Vol. 6, p. 277. Excerpta Med. Found., Amsterdam. Mildvan, D., Hirschman, S. Z., Meyers, B. R., and Keusch, G. T. (1973). Can. J . Microbiol. 18, 1039. Mine, Y . , Nishida, M., Goto, S., and Kuwahara, S. (1970a). J. Antzbiot. 23, 575. Mine, Y., Nishida, M., Goto, S., and Kuwahara, S. (1970h). J. Antzbiot. 23, 195. Minkin, W., and Lynch, P. J. (1968). Mil. Med. 133, 557. Mintz, U.. Liberman, U. A., and De Vries, A. (1971). J. Amer. Med. Ass., 216, 1200.

158

D. R. OWENS ET AL.

Mizukawa, T., Azuma, I., and Kawaqughi, S. (1965). J. Antibiot., Ser. A 18, 525. Mizuno, S., Matsuda, S., and Mori, S. (1969). Proc. ROY. SOC.Med. (London) sump. C h Eval. Cephalexin p. 49. Mohring, K., Genster, H. G., and Madsen, P. 0.(1971).J. Urol. 106, 757. Molin, L., and Nystrom, B. (1970). Chemothzrapy 15,384. Moll, T. F. (1970). Invest. Opthalmol. 9, 157. Moll, T. F. (1971). Surg. Forum 22,466. Moll, T. F., Crawford, J. R., and McPherson, S. D. (1971). Amer. J. Opthalmol. 71, 992. Molthan, L., Reidenberg, M. M., and Eichman, M. F. (1%7). New Engl. J. Med. 277, 123. Morin, R. B., Jackson, B., Flynn, E., and Roesice, R. W. (1%2). J. Amer. Chem. SOC.84, 3400. Morrow, S., Palmisano, P., and Cassady, G. (1968). J. Pediat. 73, 262. Muggleton, P. W., and O'Callaghan, C. H. (1%7). Postgrad. Med. J., Suppl. 43, 17. Muggleton, P. W., O'CAaghan, C. H., and Stevens, W. K. (1%). Brit. Med. J. ii, 1234. Muggleton, P. W., OCallaghan, C. H., Foord, R. D., Kirby, S. M., and Ryan, D. M. (1969). Antimicrob. Ag. Chemother. p. 353. Murdoch, J.McC., Speirs, C. F., Geddes, A. M., and Wallace, E. T. (1964). Brit. Med. J. ii, 1238. Naber, K. G., and Madsen, P. 0.(1973).Antimicrob. Ag. Chemother. 3,81. Nakazawa, S., Ono, H., Nishino, T., and Kawabe, H. (1%9). Proc. Roy. SOC. Med. (London) Symp. Clin. Eval. Cephalexin p. Naumann, P. (1%7). Postgrad. Med. J., Suppl. 43, 26. Naumann, P. (1971).Postgrad. Med. J., Suppl. 47, 38. Nelson, J. D. (1971). New Engl. J. Med. 284, 349. Nelson, J. D., and Haltalin, K. C. (1972). Chemotherapy 17, 40. Newsom, S. W. B., and Walsingham, B. M. (1973). J . Med. Microbiol. 6, 59. Newsom, S. W. B., Sykes, R. B., and Richmond, M. H. (1970). J. Bacteriol. 101, 1079. Newton, G. G. F., and Abraham, E. P. (1955).Nature (London) 175, 548. Newton, G. G. F., and Abraham, E. P. (1956). Biochem. J. 62, 651. Newton, G. G. F., and Hamilton-Miller, J. M. T. (1967).Postgrad. Med. J., Suppl. 43, 10. Newton, G. G. F., Abraham, E. P., and Kuwahara, S. (1%8).Antimicrob. Ag. Chemother. p. 449. Nishia, M., Matsuhara, l., Murakawa, l., Mine, Y., Yokota, Y., Kuwahara, Y., Kuwakara, S., and Goto, S. (1970a).Antimicrob. Ag. Chemother. p. 236. Nishida, M., Matsubara, T., Murakawa, T., Mine, 'k ., Yokota, Y ., Goto, S., and Kuwahara, S. (1970h).J. Antibiot. 23, 137. Nishida, M., Matsubara, T., Murakawa, T., Mine, Y., Yokota, Y., Goto, S., and Kuwahara, S. (1970~). J. Antibiot. 23, 184. Nissenson, A. R., Levin, N . W., and Parker, R. H. (1972). Clin. Pharmacol. Zher. 13, 887. Norden, C. W. (1971).J. Infec. Dis. 124, 565. Norden, C. W., and Kass, E. H. (1968). Annu. Rev. Med. 19, 431. Ilyham, W. L., and Fousek, M. D. (1958). Pediatrics 22,268. O'Callaghan, C. H., and Kirby, S . M. (1970). Postgrad. Med. J., Suppl. 46, 9. OCaUaghan, C. H., and Morris, A. (1972). Antimicrob. Ag. Chemother. 2, 442. O'Callaghan, C. H., and Muggleton, P. W. (1%). Biochem. J. 89, 304. O'Callaghan, C. H., and Muggleton, P. W. (1967). J. Gen. Microbiol. 48,449. O'Callaghan, C. H., Muggleton, P. W., Kirby, S. M., and Ryan, D. M. (1967). Antimicrob. Ag. Chemother. p. 337.

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

159

O’Callaghan, C. H., Kirby, S. M., and Wishart, D. R. (1968). Antimicrob. Ag. Chemother. p. 716. OCallaghan, c. H., Muggleton, P. W., and Ross, G. W. (1969). Antimicrob. Ag. Chemother. p. 57. O’Callaghan, C. H., Tootill, J. P. R., and Robinson, W. D. (1971). J. Pharm. Pharmacol. 23, 50. O’Callaghan, C. H., Kirby, S. M., Morris, A., Waller, R. E., and Duncombe, R. E. (1972a). J . Bacteriol. 110,998. OCallaghan, C. H., Morris, A., Kirby, S. M., and Shingler, A. H. (1972b). Antimicrob. Ag. Chemother. 1, 283. Ochoa, A. G., and Cravioto, J. M. (1965). Rev. Inst. Salubr. Enferm. l i o p . (Mexico) 2 5 , 47. O‘Connell, C. J. (1971). J. Med. (Basel) 2, 211. Ohkoshi. M., Suzuki, K., haide, Y., Kawamura, l.,Kawakami, l., and Nagakubo, I. (1%7). Postgrad. Med. J. 43, 76. Ohkoshi, M., Naide, Y.. Kawamura, l.,Suzuki, K., Kawakami, l.,and Nagakubo, I. (1%9). Proc. Roy. Soc. Med. (London) Symp. Clin. Eval. Cephalexin p. 26. Ohkoshi, M., Takayasu, H., Kato, T., Ishigami, J., hijima, H., Kurokawa, K., Momose, S., Shigematsu, S., Okamato, K., and Iki, Y. (1970). Progr. Antimicrob. Anticancer Chemother. 1, 917. Okies, J. E., Vitoslav, J., and Williams, 1’. W. (1971). Chest 5 9 , 198. Oller, L. Z. (1967a). Brit. J. Vener. Dis. 43, 39. Oller, L. Z. (1%7b). Postgrad. Med. J., Suppl. 43, 124. Oller, L. Z., and Smith, H. C. (1%9). Proc. Roy. Sac. Med. (London) Symp. Clin. Eval. Cephaleuin p. 51. Oller, L. Z., Smith, H. C., and Marshall, M. J. (1970). Postgrad. Med. J. 46, 99. Olson, B. H., Jennings, J. C.. and Jnnek, A. J. (1953). Science 117, 76. Opitz, A., Knoop, H., Kraft, D., Offermann, G., Schaefer, K., and Herrath, D. V. (1972). I n “Advances in Antimicrobial and Antineoplastic Chemotherapy” (H. Hejzlar, ed.), Vol. 7, p. 1345. Urban & Schwarzenberg, Munich. Oppenheimer, S., and Beaty, H. N. (1969). J. Lab. Clin. Med. 73, 535. Orsolini, P. (1970). Postgrad. Med. J., Suppl. 46, 13. Ostergard, D. R. (1970). Obstet. Gynecol. 36,473. Ott, J . L., and Godzeski, C. W. (1967). Antimicrob. Ag. Chemother. p. 75. Page, J., Levison, M. E., lhornhill, 1. S., and Kaye, D. (1970). J. Amer. Med. A S S . 211, 1837. Pariser, H., and Marino, A. F. (1970). S. Med. J. 63, 384. Park, J. ’l., and Burman, L. (1973). Biochem. Biophys. Res. Commun. 51, 863. Park, J. T., Griffith, M. E., and Stevenson, 1. (1971). J . Bacteriol. 108, 1154. Parker, R. H., Kalinske, R. W., and Hoeprich, P. D. (1968). Antimicrob. Ag. Chemother. p. 150. Paschetta, G., and De Biasi, V. (1971). Minerua Pediat. 23, 2065. Paterson, M. L., Henderson, A., Lunan, C . B., and McGurk, S. (1970). J. Obstet. Gynaecol. Brit. Commonw. 7 7 , 565. Paterson, M. L., Henderson, A., Lunan, C. B., and McGurk, S. (1972). Clin. Med. 79, 22. Perkins, R. L., and Miller, M. A. (1973). Can. J. Microbiol. 19, 251. Perkins, R. L., and Saslaw, S. (1966). Ann. Intern. Med. 64, 13. Perkins, R. L., Saslaw, S., and Billmaier, D. (1%7a). Clin. Res. 15, 426. Perkins, R. L., Saslaw. S., and Hackett, J. (1%7b). Amer. J. Med. Sci. 253, 69.

160

D. R. OWENS ET AL.

Perkins, R. L., Apicella, M. A., Lee, I.-S., Cuppage, F. E., and Saslaw, S. (1968). J. Lab. Clin. Med. 71, 75. Perkins, R. L., Glontz, G. E., and Saslaw, S. (1969a). Clin. Pharmacol. l'her. 10, 244. Perkins, R. L., Smith, E. J., and Saslaw, S. (1969b.) Amer. J. Med. Sci. 257, 116. Perrelli, L., and l'empesta, E. (1968). Gazz. Int. Med. Chir. 73, 5283. Petz, L. D. (1971). Postgrad. Med. J., Suppl. 47, 64. Phair, J. P., Carleton, J., and l a n , J. S. (1972). Antimicrob. Ag. Chemother. 2, 329. Philson, J. R., Clancy, C. F,, and Alexander, J. D. (1964). Antimicrob. Ag. Chernother. p. 267. Pines, A. (1970). Progr. Antimicrob. Anticancer Chemother. 1, 731. Pines, A. (1972). Brit. J. Clin. Pract. 26, 209. Pines, A., and Raafat, H. (1967). Postgrad. Med. J., Suppl. 43, 61. Pines, A,, Raafat, H., Pencinski, K., Greenfield, J. S. B., and Linsell, W. D. (1967). Brit. J . Dis. Chest 61, 101. Pines, A., Raafat, H., Greenfield, J. S. B., Marshall, M. J., and Solari, M. (1971). Brit. J. Dis. Chest 65, 91. Pitt, J . , Siasoco, R., Kaplan, K., and Weinstein, L. (1968). Antimicrob. Ag. Chemother. p. 630. Poggiolini, D. (1970). Postgrad. Med. J., Suppl. 46, 117. Polk, H. C., and Lopez-Mayor, J. F. (1%9). Surgery 66, 97. Pollock, M. R. (1956).J. Gen. Microbiol. 15, 154. Pollock, M. R. (1957). Biochem. J. 66, 419. Pollock, M. R. (1965).Antimicrob. Ag. Chemother. p. 292. Pollock, M. R. (1967). Brit. Med. J. 4 , 71. Pollock, M. R. (1971). Proc. R o y . SOC.,Ser. B 179, 385. Pressman, R. S., Geczy, M., Maranhao, V., and Goldberg, H. (1966). Amer. J . Cardiol. 17, 97. Pruitt, A. W., and Dayton, P. G . (1971). Eur. J. Clin. Pharmacol. 4 , 59. Pryor, J. S., Joekes, A. M., and Foord, R. D. (1967). Postgrad. Med. J . , Suppl. 43, 82. Pursiano, l. A., Misiek, M., Leitner, F., and Price, Ic. E. (1973). Antimicrob. Ag. Chemother. 3, 33. Quintiliani, R. A., Leutnik, A., Campos, M., and Di Meola, H. (1972). Clin. Med. 79, 17. Rahal, J. J. (1972). Ann. Intern. Med. 77, 295. Rahal, J. J., Meyers, B. R., and Weinstein, L. (1968). h e w Engl. J. Med. 279, 1305. Rain, M. D. (1971). Clin. Res. 19, 401. Records, R. E. (1968a). Amer. J. Opthalmol. 66, 436. Records, R. E. (1968b). Amer. J. Opthalmol. 66, 441. Records, R. E. (1969). Arch. Opthalmol. 81,331. Records, R. E., and Ellis, P. P. (1969). Zrans. Lect. Coast Oto. Opthalmol. Soc. 21, 179. Regamey, C., and Humair, L. (1971). Postgrad. Med. J., Suppl. 47, 69. Reichelderfer, L. C., and Reichelderfer, T. E. (1964). Clin. Med. 71, 339. Reisberg, B. E., and Mandelbaum, J. M. (1971). Infections Immunity 3, 540. Retsema, J. A., and Ray, V. A. (1972). Antimicrob. Ag. Chernother. 2, 173. Richards, A. B. (1969). Proc. R o y . Soc. Med. (London) Clin. Eval. Cephalexin p. 31. Richards, A. B., Bron, A. T., Rice, N. S. C., Fells, P., Marshall, M. J., and Jones, B. R. (1972). Brit. J.Opthalmo1. 56, 531. Richmond, M. H. (1965). J. Bacteriol. 90, 370. Richmond, M. H. (1972). J. Appl. Bacteriol. 35, 155. Richmond, M. H., and Sykes, R. B. (1973). In "Advances in Microbial Physiology" (A. H. Rose and D. W. Tempest, eds.), Vol. 9, pp. 31-88. Academic Press, New 7york.

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

161

Ridley, M., and Phillips, I. (1965). Nature (London) 208, 1076. Riley, F. C., Boyle, G. L., and Loepold, I. H. (1968). Amer. J . Opthalmol. 6 6 , 1042. Riley, H . D. (1971). Postgrad. Med. J., Suppl. 47, 59. Riley, H. D., Bracken, E. C., and Flux, M. (1963). Antimicrob. Ag. Chemother. p. 716. Roberts, J. M. (1952). Mycologia 44, 292. Roberts, R. B. (1966). J. Bacteriol. 9 2 , 1609. Rocca-Rossetti, S., Binaghi, G., Usai, E., and Ciccu, M. (1%5). Rass. Med. Sarda 6 8 , 681. Rohner, E. J. (1970). Proc. Symp. Karolinska Hosp., Stockholm pp. 56, 69. Rolinson, G. N. (1971). Proc. Roy. Soc., Ser. B 179, 403. Ronald, A. R., and l u r c k , M. (1967). Antimicrob. Ag. Chemother. p. 82. Ronald, A. R., Kind, A. C.. and Turck, M. (196s).Arch. Intern. Med. 121, 39. Rosa, D. (1967). Minerva Oftalmal. 9 , 177, Rowntree, P. M., and Bullen, M. N. (1967). Brit. Med. J. ii, 373. Ruedy, J. (1967). Postgrad. Med. J., Suppl. 43, 146. Russell, A. D. (1968). Lab. Pract. 13, 114. Russell, -4. D. (1969). In “Progress in Medicinal Chemistry” (G. P. Ellis and G. B. West,eds.), Vol. 6, pp. 135-199. Butterworth, London. Russell, A. D. (1971). In “Inhibition and Destruction of the Microbial Cell” (W. B. Hugo, ed.), pp. 209-225. Academic Press, New York. Russell, A. D. (1972a). Proc. Int. Congr. Chemother., 7th, Prague, 1971 p. 1095. Russell, A. D. (1972b). Microbios 6 , 221. Russell, A. D. (1972~).Antimicrob. Ag. Chemother. 2 , 255. Russell, A. D. (1973). J. Appl. Bacteriol.36, 357. Russell, A. D. (1974). Microbios. 10, 29. Russell, A. D., and Fountain, R. H. (1970). Postgrad. Med. J., Suppl. 46, 43. Russell, A. D., and Fountain, R. H. (1971). J. Bacteriol. 106, 65. Russell, A. D., and Furr, R. J. (1973). J. Clin. Pathol. 2 6 , 599. Russell, A. D., Morris, A,, and Allwood, M. C. (1973). In “Methods in Microbiology” (J. R. Norris and D. W. Ribbons, eds.), Vol. 8, pp. 95-182. Academic Press, New York. Sabath, L. D. (1968). Antimicrob. Ag. Chemother. p. 210. Sabath, L. D., and Abraham, E. P. (1964). Nature (London) 204, 1066. Sabath, L. D., and Abraham, E. P (1966). Antimicrob. Ag. Chemother. p. 392. Sabath, L. D., and Finland, M. (1967). Ann. N.Y. Acad. Sci. 145, 237. Sabath, L. D., and Finland, M. (1968). J . Bacteriol. 95, 1513. Sabath, L. D., Jago, M., and Abraham, E. P. (1965). Biochem. J. 96, 739. Sales, J . E. L., Sutcliffe, M. B., and O’Grady, F. (1969). Proc. Roy. SOC. Med. (London) Symp. Clin. Eval. Cephalxin p. 42. Saslaw, S., and Perkins, R. L. (1966). Ann. Intern. Med. 6 4 , 13. Sassiver, M . L., and Lewis, A. (1970). Advan. Appl. Microbiol. 13, 163. Seftel, H . C., Sieff, B., and Richardson, N. J. (1966). Med. Proc. 12, 29. Seftel, H . C., Robinson, R., Kew, M. C . , and Goldin, A. R. (1%9). Proc. Roy. SOC.M e d . (London) Symp. Clin. Eval. Cephalexin. p. 70. Seftel, H. C., Kew, M. C., and Levy, J. I. (1970). Postgrad. Med. J., Suppl. 46, 121. Seligman, S . J., and Hewitt, W. L. (1966). Antimicrob. Ag. Chemother. p. 387. Seneca, H., and Lattimer, J. K. (1965). J. Urol. 94, 485. Seneca, H., Peer, P., and Warren, B. (1967). J. Urol. 97, 153. Seneca, H., Zinsser, H. H., and Peer, P. (1970). J. Amer. Geriat. Soc. 18, 433. Seneca, H., Uson, A., and Peer, P. (1972). J. Urol. 107, 832. Shaltiel, S., Mizrahi, R., and Sela, M. (1971). Proc. Roy. Soc., Ser. B. 179, 411.

162

D. R. OWENS ET AL.

Shapiro, L. H., and Lentz, J. W. (1970).Amer. J . Obstet. Gynecol. 108, 471. Sheng, K. T., Huang, N. N., and Promadhattavedi, V. (1965). Antimicrob. Ag. Chemother. p. 200. Sherris, J. C., Rashard, A. L., and Lighthart, G. A. (1967). Ann. N.Y. Acad. Sci. 145, 248. Shibata, K., and Fugii, M. (1971). Antimicrob. Ag. Chemother. p. 467. Shibata, K., and Kato, T. (1969). Proc. Roy. Soc. Med. (London) Symp. Clin. Eval. Cephalexin p. 39. Shibata, K., Atsumi, T., Horiuchi, I.,and Mashimo, K. (1966). Nature (London) 212, 419. Siguier, F., Godeau, P., Dorra, M., and Grnnfeld, J.-P. (1966). Presse Med. 74, 1269. Silverblatt, F., Turck, M., and Bulger, R. (1970).J. Infec. Dzs. 122, 33. Simon, C., Sievers, G., Rowedder, H. J., and Malerczyk, V. (1970). Deut. Med. Wochenschr. 95, 2103. Smellie, J. M., and Normand, I. C. S. (1968). I n “Urinary Tract Infections’. (F. O‘Grady and W. Brumfitt, eds.), p. 123. Oxford Univ. Press, London and New York. Smith, D. G. (1969). Sci. Progr. (London) 57, 169. Smith, E. K., and Williams, J. D. (1970). Brit. J . Urol. 42, 522. Smith, I. M. (1958). In “Staphylococcal Infections” (I. Smith, ed.), Yearbook Publ., Chicago, Illinois. Smith, I. M. (1971). Postgrad. Med. J., Suppl. 47, 78. Smith, J. L. (1969). Trans. Amer. Acad. Opthalmol. Otolaryngol. 73, 1113. Smith, J. T., Hamilton-Miller, J. M. T., and Knox, R. (1969). J. Pharm. Pharmacol. 21, 337. Solberg, C. O., Schreiner, A., and Digranes, A. (1972). Scand. J. Infec. Dis. 4, 241. Soto, R. F., Zschaeck, D., Padrbn, M., Perera, G., Curiel, R., Parra, F. P., Requejo, J. P., Linares, J., Aure, M., Pittaluga, J. R., and Cabrera, A. (1970). Rev. Obstet. Ginecol. Venez. 30, 509. South, M. A., Short, D. H., and Knox, J. M. (1964).J . Amer. Med. Ass. 190, 70. Southern, P. M., and Sanford, J. P. (1969).niav Engl. J . Med. 280, 1163. Speight, T. M., Brogden, R. N., and Avery, G. S. (1972). Drugs 3, 9. Spiers, C. F., Murdoch, J.McC., Wanvick, R. W. G., and Wallace, E. 1. (1969). Proc. Roy. Soc. Med. (London) Symp. Clin. Eval. Cephalexin p. 23. Steigbeigel, N. H., MacCall, C. E., Reed, C. W., and Finland, M. (1967). Ann. N . Y . Acad. Sci. 145, 224. Steigbeigei, N . H., Kislak, J. W., Tilles, J. G., and Finland, M. (1968). Arch. Intern. Med. 121,24. Stephen, L. C., and Bolognese, R. J. (1970).J . Reprod. Med. 4, 105. Sterner, G., Frensen, H., Tunevall, G., and Svedmyr, A. (1967).Postgrad. Med. J., Suppl. 43, 53. Stewart, G. 1’. (1962). Lancet i, 509. Stewart, G. T. (1968).Antimicrob. Ag. Chemother. p. 543, Stewart, G. T., and Holt, R. J. (1964). Lancet ii, 1305. Stewart, S. M., Burnet, M. E., and Young, J. E. (1969).J. Med. Microbiol. 2 , 287. Stewart, G. T., Boyd, J. F., and Butcher, B. T. (1970).Postgrad. Med. J., Suppl. 46, 133. Stillerman, M. (1970). Clin. Pharmacol. Ther. 11, 205. Stillerman, M., and Isenburg, H. D. (1971).Antimicrob. Ag. Chemother. p. 270. Stratford, B. C. (1969). Proc. Roy. SOC. Med. (London) Symp. Clin. Eval. Cephalexin p. 88. Stratford, B. C., and Dixson, S. (1967). Postgrad. Med. J . , Suppl. 43, 158.

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

163

Stratford, B. C., and Dixson, S. (1968). Med. J . A w t . 1, 1114. Strominger, J. L., Izaki, K., Matsuhashi, M., and Tipper, D. J. (1967). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 26, 9. Sullivan, H. R., and McMahon, R. E. (1967). Biochem. J. 102, 976. Sullivan, H. R., Billings, R. E., and McMahon, R. E. (1969a). J. Antibiot. 22, 27. Sullivan, H. R., Billings, R. E., and McMahon, R. E. (1969b). J. Antibiot. 22, 195. Sullivan, H. R., McMahon, R. E., and Heiney, R. E. (1971). J. Antibiot. 24, 375. Sutherland, R., and Rolinson, G. N. (1964). J . Bacteriol. 87, 887. Swallow, D. L., and Sneath, P. H. A. (1962). J. Gen. Bacteriol. 28, 461. Sweetman, W. P., Hamlin, N., and Sykes, C. G. W. (1966). Lancet i, 658. Sykes, R. B., and Nordstrom, K. (1972). Antimicrob. Ag. Chemother. 1, 94. Sykes, R. B., and Richmond, M. H. (1970). Nature (London) 2 2 6 , 9 5 2 . Sykes, R. B., and Richmond, M. H. (1971). Lancet iii, 342. Takahashi, T., Yamazaki, Y., Kato, K., and Isono, M. (1972). J. Amer. Chem. SOC. 94, 4035. Taylor, J. C., and Fallon, R. J. (1966). Lancet i, 715. Taylor, W. A., and Holloway, W. J. (1972). Int. Z . KLin. Pharmakol. Z’her. Pbxikol. 6, 7. Tempest, B., and Austrian, R. (1967). Ann. Intern. Med. 66, 1109. Thoburn, R., and Fekety, F. R. (1970). J. Chronic Dis. 22, 593. Thoburn, R., Johnson, J. E., and Cluff, L. E. (1966). J . Amer. Med. Ass. 198, 345. Thomas, A. H., and Broadridge, R. A. (1972). J. Gen. Microbiol. 70, 231. Thompson, M., Barrett, D., Madden, S., and Ridley, M. (1967). Postgrad. Med. J., Suppl. 43, 136. Thornhill. T. S.,Levison, M. E., Johnson, W. D., and Kaye, D. (1969). Appl. Microbiol. 17,457. Thornton. G. F., and Andriole, V. T. (1966). Yale J . Biol. Med. 39, 9. Tong, M. J. (1972). J. Amer. Med. Ass. 2 1 9 , 1044. Toscano, F., Ricciardiello, P. T., and Stangalini, A. (1967). Minema Med. 58, 509. Tozer, R. A., Bontflower, S., and Gillespie, W. A. (1966). Lancet i, 686. Tuano, S. B., Brodie, J. L., and Kirby, W. M. M. (1967). Antimicrob. Ag. Chemother. p. 101. Tully, J. G., and Smith, L. G. (1968). J. Amer. Med. Ass. 204, 827. Turck, M., Anderson, K. IL., Smith, R. H., Wallace, J. F., and Petersdorf, R. C. (1%5). Ann. Intern. Med. 63, 199. Turck, M., Belcher, D. W., Ronald, A., Smith, R. H., and Wallace, J. F. (1967). Ann. Intern. Med. 119, 50. Turcotte, J. G . , Hermann, T. J., Haig, O., O’Dell, C., Cerny, J., and Green, A. J. (1970). Surg., Gynecol. Obstet 1 3 0 , 29. Ueda, Y . (1969). Proc. Roy. Sor. Med. Symp. Clin. Eual. Cephalexin p. 65. Ueda, Y . , Matsumoto, F., Nakamura, N., Sairo, A., Noda, K., Furuya, C., and Omori, M. (1965). J . Antibiot., Ser. B . , 18, 241. Ueda, Y., Matsumoto, F., Saito, A., and Ohmori, M. (1967). Postgrad. Med. J., Suppl. 43,80. Uwaydah, M. M., and Faris, B. M. (1970). Arch. Opthalmol. 83, 349. Vacek, V., and Molikovi-Wagnerova, M. (1967). Chemotherapia 12, 172. Van Heyningen, E. (1967). Aduan. Drug Res. 4, 1. Van Heyningen, E., and Aherne, L. K. (1968). J . Med. Chem. 11, 933. Vegas, J . S. (1965). Rev. Venez. Urol. 17, 345. Venuto, R . C., Stein, J. H., and Ferris, 7’. F. (1972). Proc. SOC. Exp. B ~ o l Med. . 139, 1065.

164

D. R. OWENS ET AL.

Verney, R. E. (1967). Postgrad. Med. J., Suppl. 43, 166. Vianna, N. J., and Kaye, D. (1967). Amer. J. Med. Sci. 254, 216. Vymola, F., and Hejzlar, M. (1966). J. Hyg., Epidemiol., Microbiol., Zmmunol. 10, 180. Waldvogel, F. A., Bredoff, G., and Swartz, M. N. (1970). N e w Engl. J. Med. 2 8 2 , 198. Walker, S. H., and Gonzales, E. (1969). S. Med. J. 62, 1%. Walters, E. W., Romansky, M. J., and Johnson, A. C. (1964). Antimicrob. Ag. Chemother. p. 247. Watanakunakorn, C., Goldberg, L. M., Carleton, J., and Hamburger, M. (1969). Antimicrob. Ag. Chemother. p. 382. Waterman, N. G., Howell, R. S., and Babich, M. (1968). Arch. Surg. 97, 365. Waterworth, P. M. (1971). Postgrad. Med. J., Suppl. 47, 25. Weinstein, L. (1971). Postgrad. Med. J., Suppl. 47, 94. Weinstein, L., and Kaplan, K. (1970). Ann. Intern. Med. 72, 729. Weinstein, L., Kaplan, K., and Chang, T-W. (1964). J. Amer. Med. Ass. 189, 829. Weissbacb, L., Tiimmers, H., and Ritzerfeld, W. (1971). Muenchen. Med. Wochenschr. 113,265. Welles, J. S. (1972). I n “Cephalosporins and Penicillins. Chemistry and Biology” (E. H. Flynn, eds.), pp. 583-608. Academic Press, New York. Welles, J. S., Gibson, W. R., Harris, P. N., Small, R. M., and Anderson, R. C. (1966). Antimicrob. Ag. Chemother. p. 863. Welles, J. S., Froman, R. O., Gibson, W. R., Owen, N. V., and Anderson, R. C. (1969). Antimicrob. Ag. Chemother. p. 489. Welles, J. S., Froman, R. O., Gibson, W. R., Owen, N . V., Small, R. M., and Anderson, R. C. (1970). Znt. J. Clin. Pharmacol. 2 , Suppl., 36. Wick, W. E. (1%7). Appl. Microbiol. 15, 765. Wick, W. E., and Boniece, W. S. (1965). Appl. Microbiol. 13, 248. Wick, W. E., and Preston, D. A. (1972). Antimicrob. A s . Chemother. 1, 221. Wick, W. E., Wright, W. E., and Kuder, H. V. (1971). Appl. Microbiol. 21, 426. Wiesner, P., MacGregor, R., Bear, D., Berman, S., Holmes, K., and Turck, M. (1972). Antimicrob. Ag. Chemother. 1, 303. Wilkinson, A. T., Race, J. W., and Curtis, F. R. (1%7). Postgrad. Med. J., Suppl. 43, 65. Willcox, R. R. (1971). Brit. J. Vener. Dis. 47, 31. Willcox, R. R., and Woodcock, K. S. (1970). Postgrad. Med. J., Suppl. 46, 103. Wilson, F. M., Retan, J. W., and Lerner, A. M. (1966). J. Urol. 96, 534. Wilson, F. M., Shumaker, E. J., Fentress, V., and Lerner, A. M. (1971). J. Urol. 105, 295. Wise, H. A., and Schwartz, H. (1971). J . Urol. 105,440. Yamasaku, F., Tsuchida, R., and Usuda, Y. (1970). Postgrad. Med. J., Suppl. 46, 57. York, P. S., and Landes, R. (1968). J. Amer. Med. Ass. 206, 1086. Zabransky, R. J., Gardner, M. A., and Geraci, J. E. (1969). Mayo Clin. Proc. 44, 876.

Addendum Since the main body of this article was written, several important papers on cephalosporins have been published. This brief Addendum will thus consider these papers in order to bring this chapter as up-todate as possible. The works of Flynn (1972) and Garrod et al. (1973)

THE C E P H A L O S P O R I N G R O U P OF A N T I B I O T I C S

165

should also be consulted (as should the following brief review articles: Hewitt, 1973; Levine, 1973; Mandell, 1973; Anon, 1974; Garrod, 1974; Sanders et al., 1974).

A. ANTIBACTERIALASPECTS The new cephalosporin cefamandole, has been shown (Eykyn et al., 1973; Williams and Geddes, 1973; Williams and Andrews, 1974) to be more active than cephaloridine, cephalothin, cephradine, cephalexin, or cephacetrile against H. influenzae and against gram-negative bacilli susceptible to cephalosporins. Cefamandole has also been found to be active against many strains resistant to other cephalosporins, such as indole-positive Proteus sp., but minimum bactericidal concentrations are considerably higher than minimum inhibitory concentrations (MICs) (Eykyn et al., 1973; Russell, 1974), and there is a marked inoculum effect (Eykyn et al., 1973). Isenberg et al. (1973) have observed a great similarity in the response of gram-negative rods and gram-positive cocci (other than enterococci) to cephalothin and cephacetrile, with the latter antibiotic more active than cephalothin against enterococci. Misiek et a l . (1973) have described the structure-activity relationship of more than 600 cephalosporins against M. tuberculosis H 37Rv, and have shown that among the most active derivatives of cephalosporin C were those in which a pyridyl or an aminomethylphenyl moiety was present in the side chain. The cephamycins are not cephalosporins, but are closely related to this antibiotic group, and as such are worthy of mention here, especially as a new cephamycin, cefoxitin, has shown considerable promise as an antibacterial agent. The cephamycins are produced by various strains of Actinomycetes (Stapley et al., 1972) and their isolation and chemical characteristics have been described (Miller et al., 1972). Cephamycins have a broad antibacterial spectrum, including many strains resistant to cephalosporins and penicillins, and induce spheroplast formation in gram-negative bacteria (Stapley et al., 1972) suggesting a similarity in mode of action. Cefoxitin is more active than cephalothin against indolepositive Proteus sp. and is not destroyed by the p-lactamases produced by gram-negative bacteria; it is, however, considerably less active than cephalothin against gram-positive bacteria (Kosmidis et al., 1973). Further studies (Wallick and Hendlin, 1974; Miller et a l . , 1974) have confirmed these findings. Dusart et al. (1973) have presented evidence that the same enzyme performs DD-carboxypeptidase and transpeptidase activities in the

166

D. R. OWENS ET AL.

Streptomyces strains they examined and that this enzyme is the killing site of penicillins and cephalosporins. Nishino and Nakazawa (1973a) have used the scanning electron microscope to investigate the surface changes in cephalexin-treated S . aureus and E . coli and by means of viable counting and ultrathin sections in the electron microscope they have demonstrated an antagonistic effect between cephalexin and an inhibitor of protein synthesis, erythromycin, in S. aureus (Nishino and Nakazawa, 1973b). Topp and Christensen (1974) have considered, from a theoretical viewpoint, the structure-activity relationship of fi -1actam antibiotics, based upon a two-step mechanism of inhibition of bacterial growth: (a) reversible binding of drug to enzyme, followed by (b) irreversible acylation of transpeptidase enzyme via thiol attack at the p lactam carbonyl carbon. A rapid assay method for cephalosporins, based on their inhibition of glucose or inositol fermentation by a strain of gentamycin-resistant Providencia (group A) has been described by Noone (1973). Reller et al. (1973) have made an in vitro and in vivo evaluation of cefazolin, and Saslaw and Carlisle (1973) have compared the use of various cephalosporins in the therapy of staphylococcal infections in monkeys.

B. RESISTANCE Jackson et al. (1973) have compared the ability of p-lactamases from staphylococci, Enterobacteriaceae, and two types of P. aeruginosa to hydrolyze benzylpenicillin and five different cephalosporins. Medeiros and O’Brien (1973) tested the sensitivity of ampicillin-resistant strains of E. coli to cephaloridine, cephalothin, cephalexin, and cefazolin, and found three distinct classes of ampicillin-resistant strains possessing different P-lactamases. These classes were as follows: Class I. Hydrolysis of different cephalosporins occurred at widely differing rates. The MICs against large inocula correlated with the rates of hydrolysis, except for cephalexin; against small inocula, the MICs were low for all four cephalosporins. Class ZZ. Much more “cephalosporinase” than “ampicillinase” activity occurred in these strains. There was resistance to the four cephalosporins even when tested with small inocula. Cephalexin was again the most resistant cephalosporin, but ampicillin was destroyed even less. Class ZZZ. The p-lactamase of these strains had low levels of specific activity, and the strains were only slightly more resistant to cephalosporins than were ampicillin-sensitive E. coli strains. Hedges et al. (1974) examined the molecular specifities of R-factor-

T H E C E P H A L O S P O R I N GROUP O F A N T I B I O T I C S

167

determined p-lactamases, and showed that the p-lactamases determined by 29 resistance plasmids could be divided into two groups: (a) TEM-type (20 out of 29 cases). These were very uniform with respect to substrate specificity, e.g., taking the rate of hydrolysis of benzylpenicillin as 100 (see Table I in main chapter), the relative rates of hydrolysis of oxacillin, ampicillin, and cephaloridine were, respectively, low (about 5), 100-110, about 75. (b) Oxacillin-hydrolyzing type. These were less common (9 out of 29 cases), showed lower absolute levels of activity, but were heterogenous as regards substrate specificities, e.g., with benzylpenicillin as 100, rate for oxacillin was 184-646, ampicillin 133-408, and cephaloridine 30-62. Hewitt (1973) has stated that there is a high correlation between p lactamase production and bacterial resistance to cephalosporins, but points out (see the main part of the present article) that (a) intrinsic resistance and (b) cellular permeability are also important in this context. Del Bene and Farrar (1973) studied p-lactamase activity in 10 strains of Bacteroides fragilis and found that these caused low rates of hydrolysis of cephalosporins, whereas penicillin substrates appeared to be undetected. In two strains, this “cephalosporinase” activity was increased some 40 to 80-fold by growing the cells in the presence of penicillin. Of the four cephalosporins used as substrates, cephaloridine was the most, and cephalexin the least, labile. Farrar and Newsome (1973) present evidence in support of the hypothesis that the synergistic antibacterial effects of a combination of p-lactam antibiotics on gram-negative p-lactamase-producing bacteria is due to the inhibition of p-lactamase by one of the antibiotics and the lethal effect of the second, “protected” drug. The cephamycin, cefoxitin, is highly resistant to inactivation by several p-lactamases (Daoust et al., 1973; Onishi et al., 1974), although the drug is degraded by some strains, and this enzymatic destruction could be an important factor in their resistance (Onishi et al., 1974). Hou and Poole (1973) have studied the effect of competitive inhibitors on the p-lactamase activity of S. aureus and B . cereus, and showed that the degree of inhibition depended on (i) the structural characteristics of the inhibitor, (ii) the time of exposure of enzyme to inhibitor, and (iii) the ratio of amount of inhibitor to amount of substrate. Haque and Russell (1974) have studied the effect of some chelating agents on the subsequent susceptibility of some gram-negative plactamase and non- @-lactamase producers to some @-lactam antibiotics and other antibacterial agents. Transferable resistance associated with cellular impermeability to penicillins and cephalosporins, rather than

168

D. R. OWENS ET AL.

inactivation by a P-lactamase, has been described by Richmond and his colleagues (Curtis et al., 1973). With P. aeruginosa, a major factor conferring resistance appears to be impermeability rather than drug destruction (Mills and Russell, 1974). Richmond and Curtis (1973) have considered the resistance of gram-negative bacteria in relation to plactamase production and intrinsic factors. Vernon and Russell (1974) have described the effects of methicillin, cephaloridine, and cephalothin on the growth, lysis, and viability of methicillin-resistant strains of S. aureus, and have shown that it is the treatment temperature, rather than the pretreatment growth temperature, which is of importance in determining resistance to these p-lactam antibiotics. Further studies on the combined action of p-lactam antibiotics against gram-negative bacteria have been described (Bobrowski et al., 1973). C. PHARMACOLOGICAL AND CLINICAL ASPECTS The absorption, distribution, and excretion of cephradine has recently been investigated in mice, rats, and dogs by Weliky et al. (1974). The antibiotic was well absorbed after oral and subcutaneous administration and its plasma half-life was about 1.0 hour. Cephradine was widely distributed throughout the body tissues with the greatest concentrations being in the liver and kidneys. Virtually all of an administered dose was recovered during a 24-hour urine collection period, cephradine being excreted in an unchanged form (Miraglia et al., 1973; Weliky et al., 1974). Cephradine has been reported to be well tolerated in laboratory animals (Gadebusch et al., 1972) and has a low order of acute and chronic toxicity after oral and parenteral administration to rats, dogs, and monkeys (Hassert et al., 1973). In a clinical study conducted by Zaki et al. (1974), cephradine was observed to be rapidly absorbed after oral administration, peak serum concentrations being reached in 1.0 hour. After single doses, nearly all the antibiotic was excreted in the urine as unmetabolized material within 6.0 hours of dosing. Trujillo et al. (1974) have described the usefulness of parenterally administered cephapirin in acute lung diseases in children. Evidence suggests, however, that the continuous intravenous administration of large doses of cephapirin produces a high incidence of side reactions. For example, in a study conducted by Sanders et al. (1974), most subjects experienced profound malaise, weakness, fever, lymphadenopathy, and a pruritic skin rash following administration of a 2.0 gm dose of

T H E C E P H A L O S P O R I N GROUP O F A N T I B I O T I C S

169

cephapirin by rapid intravenous injection, four times a day for 2 to 4 weeks. The pharmacokinetic properties of cefazolin have recently been compared with other cephalosporins by a number of workers (Bergeron et al., 1973; Kirby and Regamey, 1973; Cahn et al., 1974). After intravenous and intramuscular administration, serum levels of cefazolin have been found to be about twice those following equal doses of cephaloridine and three to four times those after cephalothin. Peak serum levels of 3 2 4 2 pg/ml have been reached after an intramuscular injection of 500 mg cefazolin. Following this dose, the half-life of cefazolin in the serum of normal persons was approximately 2.0 hours, although this has been observed to be extended up to 12.0 hours in patients with severely compromised renal function (Levison et al., 1973) and up to 35.0 hours in severely uremic patients (Craig et al., 1973). The long half-life (2.0 hours) in serum compared with those of cephaloridine (1.1 hour) and cephalothin (0.5 hour) appears to be primarily due to the low serum and renal clearance of cefazolin (Regamey and Kirby, 1973). These factors, together with an apparent small volume of distribution, no doubt contribute to the high peak levels of the antibiotic in the blood after dosing. Cefazolin is bound to human plasma protein to the extent of 7686% (Nishida et al., 1970a; Kirby and Regamey, 1973). This is relatively high when compared with the values for cephaloridine (20%), cephalexin (15%), and cephalothin (65%), although the recently introduced cephanone is also highly bound (88%). In toxicological studies in laboratory animals carried out by Birkhead et al. (1973), cefazolin exhibited low acute toxicity in mice and rats and was not teratogenic for mice or rabbits. In subacute and chronic toxicity experiments in rats and dogs, the major finding was damage to muscles following intramuscular injection. In nephrotoxic studies, cefazolin was less nephrotoxic than cephaloridine (Birkhead et al., 1973; Silverblatt et al., 1973).

Madhaven et al. (1973) have compared the clinical usefulness of cefazolin with other cephalosporins. It has been found to be effective in the treatment of bacterial pneumonia (Turck et al., 1973), bronchitis (Gold et al., 1973), bacterial endocarditis (Quinn et al., 1973; Reinarz et al., 1973), soft tissue infections (Gold et al., 1973), urinary tract infections (Cox, 1973), and in a preliminary report by Pickering et al. (1973) cefazolin has been observed to be effective in a number of childhood infections. On the other hand, cefazolin has only a limited

170

D. R. OWENS ET AL.

usefulness in uncomplicated gonorrhea (Karney et al., 1973; Nelson, 1973). The only adverse clinical effect so far reported with cefazolin is pain at the site of injection following both intravenous and intramuscular administration. The incidence is greater with cefazolin than with cephaloridine (Cahn et al., 1974), however, like cephaloridine, cefazolin appears to cause less ,pain on parenteral injection than cephalothin. Furthermore, cefazolin does not cause thrombophlebitis despite extended intravenous administration (Reller et al., 1973). In a study carried out by Regamey and Kirby (1973), cephanone administered intramuscularly at a dose level of 7 mg/kg (approx. 500 mg) to five normal volunteers gave peak serum concentrations (36 pg/ml) approximately four times as high as with the same dose of cephalothin, twice as high as with cephaloridine, but slightly lower than with cefazolin. The serum half-life of cephanone was about 2.5 hours and over 90% of the dose administered was recovered in the urine within 24 hours of dosing. Cephanone has a small apparent volume of distribution, probably related to its high serum protein binding (88%). Jackson et al. (1974) have made a double-blind comparison of cephacetrile with cephalothin or cephaloridine and concluded that cephacetrile can be considered as being comparable to cephalothin in antimicrobial treatment and overall reactions. The possibility that the cause of cephalothin-induced phlebitis may be associated with the acid pH of cephalothin when in solution (pH 4.8-5.0) has recently been investigated by Carrizosa et al. (1974). These authors carried out a double-blind comparison of phlebitis produced by intravenous infusions of cephalothin at acid and neutral pH values (7.2-7.4). However, they found that neither the incidence, degree, nor time of onset of phlebitis was altered by a change in pH of the cephalothin infusion.

REFERENCES TO ADDENDUM Anon. (1974).Pharmaceut. J. 212, 181. Bergeron, M. G., Brusch, J. L., Barza, M., and Weinstein, L. (1973). Antimicrob. Ag. Chemother. 4, 396. Birkhead, H. A., Briggs, G. B., and Saunders, L. Z. (1973). J. Infec. Dis. Suppl. 128, s379. Bobrowski, M. M., Gbbicka, K., and Borowski, J. (1973). J. Hyg. Epidemiol. Microbiol. Immunol. 17, 129. Brogard, J. M., Kuntzmann, J. and Lavillaureix, J. (1973). Schweiz. Med. Wochenschr. 103, 110. Cahn, M. M., Levy, E. J., Actor, P. and P a d s , J. F. (1974).J. Clin. Pharmacol. 14, 61. Carrizosa, J., Levison, M. E., and Kaye, D. (1974). Antimicrob. Ag. Chemother. 5 , 192.

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

171

Cox, C. E. (1973). J. Znfec. Dis. Suppl. 128, S397. Craig, W. A., Welling, P. G., Jackson, T. C., and Kunin, C. M. (1973). J. Znfec. Dis. Suppl. 128, S347. Curtis, N. A. C., Richmond, M. H., and Stanisich, V. (1973). J. Gen. Microbiol. 79, 163. Daoust, D. R., Onishi, H. R., Wallick, H., Hendlin, D., and Stapley, E. 0. (1973). Antimicrob. Ag. Chemother. 3 , 254. Del Bene, V. E., and Farrar, W. E. (1973). Antimicrob. Ag. Chemother. 3 , 369. Dusart, J., Marquet, A., Ghuysen, J. M., Frkre, J. M., Morens, R., Leyh-Bouille, M., Johnson, M., Lucchi, C., Perkins, H. R., and Nieto, M. (1973). Antimicrob. Ag. Chemother. 3 , 181. Eykyn, S., Jenkins, C., King, A., and Phillips, I. (1973). Antimicrob. Ag. Chemother. 3 , 657. Farrar, W. E., and Newsome, J. K. (1973). Antimicrob. Ag. Chemother. 4, 109. Flynn, E. H. (1972). Ed., “Cephalosporins and Penicillins. Chemistry and Biology.” Academic Press, New York and London. Gadebusch, H. H., Miraglia, G. J., Basch, H., Goodwin, C., Pan, S., and Renz, K. J. (1972). Aduan. Antimicrob. Antineoplastic Chemother. 1, 1059. Garrod, L. P. (1974). Brit. Med. J. 3 , 96. Garrod, L. P., Lambert, H. P.. and O’Grady, F. (1973). “Antibiotic & Chemotherapy,” 3rd. Ed. Churchill Livingstone. Edinburgh and London. Gold, J. A., McKee, J. P., and Ziv, D. S.(1973). J. Znfec. Dis. Suppl. 128, S415. Haque, H., and Russell, A. D. (1974). Antimicrob. Ag. Chemother. 6, 200. Hassert, G. L., DeBaecke, P. J., Kulesza, J. S., Traina, V. M., Sinha, D. P., and Bernal, E. (1973). Antirnicrob. Ag. Chemother. 3 , 682. Hedges, G. R., Scholand, J. F., and Perkins, R. L. (1973). Antirnicrob. Ag. Chemother. 3 , 228. Hedges, R. W., Datta, N., Kontomichalou, P., and Smith, J. T. (1974). J. Bacteriol. 117, 56. Hewitt, W. L. (1973). J. Znfec. Dis. Suppl. 128, S312. Hou, J. P., and Poole, J. W. (1973). Chemotherapy 19, 129. Hughes, S. P. F., Hughes, L., and Dash, C. H. (1974). Brit. J. Clin. Pruct. 28, 51. Isenberg, H. D., Painter, B. G., Sampson-Scherer, J., and Siegel, M. (1973). Amer. J. Clin. Pathol. 59, 700. Jackson, G. G., Lolans, V. T., and Gallegos, B. G. (1973). J. Znfec. Dis. Suppl. 128, S327. Jackson, G. G . , Riff, L. J., Zimelis, V. M., Daood, M., and Youssuf, M. (1974). Antimicrob. A g . Chemother. 5 , 247. Karney, W. W., Turck, M., and Holmes, K. K. (1973). J. Znfec. Dis. Suppl. 128, S399. Kirby, M. M., and Regamey, C. (1973). J. Znfec. Dis. Suppl. 128, S341. Kosmidis, J., Hamilton-Miller, J. M. T., Gilchrist, J. N. G., Kerry, D. W., and Brumfitt, W. (1973). Brit. Med. J. 4, 653. Levine, B. B. (1973). J. Znfec. Dis. Suppl. 128, S364. Levison, M. E., Levison, S. P., Ries, K., and Kaye, D. (1973). J. Infec. Dis. Suppl. 128, s354. Madhaven, T.. Quinn, E. L., Freimer, E., Fisher, E. J., Cox, F., Burch, K., and Pohlod, D. (1973). Antirnicrob. Ag. Chemother. 4, 525. Mandell, G. L. (1973). Ann. Intern. Med. 79, 561. Medeiros, A. A. and O’Brien, T. F. (1973). J. Znfec. Dis.Suppl. 128, S335. Miller, A. K., Celozzi, E., Kong, Y., Pelak, B. A., Hendlin, D., and Stapley, E. 0. (1974). Antimicrob. Ag. Chemother. 5, 33.

172

D. R. OWENS ET AL.

Miller, T. W., Goegelman, R. T., Weston, R. G., Putter, I., and Wolf, F. J. (1972). Antimicrob. Ag. Chemother. 2, 132. Mills, A. P., and Russell, A. D. (1974). J . Pharm. Pharmacol. 26, 102p. Miraglia, G. J., Renz, K. J. and Gadebusch, H. H. (1973). Antimicrob. Ag. Chemother. 3 , 270. Misiek, M., Moses, A. J., Pursiano, T. A., Leitner, F., and Price, K. E. (1973). J. Antibiot. 26, 737. Nelson, M. (1973). J. Znfec. Dis. Suppl. 128, S404. Nishino, T., and Nakazawa (1973a). J a p . J. Microbiol. 17, 383. Nishino, T., and Nakazawa (197333). J. Antibiot. 26, 362. Noone, P. (1973). J. Clin. Pathol. 2 6 , 5 0 6 . Onishi, H. R., Daoust, D. R., Zimmerman, S. B., Hendlin, D., and Stapley, E. 0. (1974). Antimicrob. Ag. Chemother. 5 , 38. Pickering, L. K., O’Connor, D. M., Anderson, D., Bairan. A. C., Feigin, R. D., and Cherry, J. D. (1973). J. Znfec. Dis. Suppl. 128, S407. Quinn, E. L., Pohlod, D., Madhavan, T., Burch, K., Fisher, E., and Cox, F. (1973). J. Infec. Dis. Suppl. 128, S386. Regamey, C., and Kirby, W. M. M. (1973). Antimicrob. Ag. Chemother. 4, 589. Reinarz, J. A., Kier, C. M., and Guckian, J. C. (1973). J. Znfec. Dis. Suppl. 128, S392. Reller, L. B., Karney, W. W., Beaty, H. N., Holmes, K. K., and Turck, M. (1973). Antimicrob. Ag. Chemother. 5 , 488. Richmond, M. H., and Curtis, N. A. C. (1974). Ann. N.Y. Acad. Sci. 255, 553. Russell, A. D. (1974). Unpublished data. Sanders, W. E., Johnson, J. E., and Taggart, J. G. (1974). New Engl. J . Med. 2 9 8 , 4 2 4 . Saslow, S., and Carlisle, H. N. (1973). J . Znfec. Dis. Suppl. 128, S373. Silverblatt, F., Harrison, W. O., and Turck, M. (1973). J . Znfec. Dis. Suppl. 128, S367. Stapley, E. O., Jackson, M., Hernandez, S., Zimmerman, S. B., Currie, S. A., Mochales, S., Mata, J. M., Woodruff, H. B., and Hendlin, D. (1972). Antimicrob. Ag. Chemother. 2, 122. Topp, W. C., and Christensen, B. G. (1974). J. Med. Chem. 17, 342. Trujillo, H., Manotas, R., Salazar, C., Rodriguez, A., Uribe, A., Agudelo, N., and de Vidal, E. L. (1974). J . Intern. Med. Res. 2, 125. Turck, M., Clark, R. A., Beaty, H. N., Holmes, K. K., Karney, W. W., and Reller, L. B. (1973). J. Infec. Dis. Suppl. 128, S382. Vernon, G. N., and Russell, A. D. (1974). J. Pharm. Pharmacol. 2 6 , 102p. Wallick, H., and Hendlin, D. (1974). Antimicrob. Ag. Chemother. 5 , 25. Weliky, I . , Gadebusch, H. H., Kripalani, K., Arnow, P., and Schreiber, E. C. (1974). Antimicrob. Ag. Chemother. 5 , 49. Westenfelder, S. R., Naber, K. G., andMadsen, P. 0. (1973). Infection 1, 157. Williams, J. D., and Andrews, J. (1974). Brit. Med. J . 1, 134. Williams, J. D., and Geddes, A. M. (1973). Brit. Med. J . 2, 613. Zaki, A., Schreiber, E. C., Weliky, I., Knill, J. R., and Hubscher, J. A. (1974). J. Clin. Pharmacol. 14, 118.

Sex As a Factor in Metabolism. Toxicity. and Efficacy of Pharmacodynamic and Chemotherapeutic Agents FRANS C . GOBLE Research and Development Division Cooper Laboratories. Inc . Cedar Knolls. New Jersey

I. Introduction

. . . . . . . . . . . . . . . . . . .

I1. Central Nervous System Depressants . . . . . . . . . . . .

I11.

IV .

V. VI .

VII . VIII .

A . General Anesthetics . . . . . . . . . . . . . . . . B. Ethyl Alcohol . . . . . . . . . . . . . . . . . . . C . Narcotic Analgesics . . . . . . . . . . . . . . . . D . Barbiturates . . . . . . . . . . . . . . . . . . . E . Mild Analgesics . . . . . . . . . . . . . . . . . . F. Anticonvulsants . . . . . . . . . . . . . . . . . . Central Nervous System Stimulants . . . . . . . . . . . . . A . Strychnine . . . . . . . . . . . . . . . . . . . B. Caffeine . . . . . . . . . . . . . . . . . . . . C . Picrotoxin . . . . . . . . . . . . . . . . . . . . D. Sympathomimetic Amines . . . . . . . . . . . . . . E . Antiparkinson Drugs . . . . . . . . . . . . . . . . F. Methylphenidate . . . . . . . . . . . . . . . . . . Psychotropic Agents . . . . . . . . . . . . . . . . . . A . Phenothiazines . . . . . . . . . . . . . . . . . . B . Dibenzazepines . . . . . . . . . . . . . . . . . . C . Thioxanthenes . . . . . . . . . . . . . . . . . . D . Rauwolfia Compounds . . . . . . . . . . . . . . . . E . Diols . . . . . . . . . . . . . . . . . . . . . F. Lithium . . . . . . . . . . . . . . . . . . . . G . Diazepines . . . . . . . . . . . . . . . . . . . H . Butyrophenones . . . . . . . . . . . . . . . . . . I . Cannabinoids . . . . . . . . . . . . . . . . . . J . Chlorzoxazone . . . . . . . . . . . . . . . . . . K . Combinations . . . . . . . . . . . . . . . . . . Local Anesthetics . . . . . . . . . . . . . . . . . . . Myotropics . . . . . . . . . . . . . . . . . . . . . A . Papaverine . . . . . . . . . . . . . . . . . . . B. Curare . . . . . . . . . . . . . . . . . . . . . C . Glyceryl Guaiacolate . . . . . . . . . . . . . . . . Cardiac Glycosides . . . . . . . . . . . . . . . . . . A . Animal Studies . . . . . . . . . . . . . . . . . . B . Clinical Observations . . . . . . . . . . . . . . . . Anti-inflammatory Compounds . . . . . . . . . . . . . .

173

174 176 176 180 182 185 195 198 198 198 199 199 200 201 201 202 202 203 204 204 204 205 205 206 206 207 207 207 208 208 208 208 209 209 210 211

174

FRANS C . GOBLE

IX . Antihistamines . . . . . . . . . . . . . X . Hypoglycemic Agents . . . . . . . . . XI . Anticoagulants . . . . . . . . . . . . . A . Animal Studies . . . . . . . . . . B . Clinical Observations . . . . . . . . XI1. Diuretics . . . . . . . . . . . . . . . A . Animal Studies . . . . . . . . . . B . Clinical Observations . . . . . . . . XI11. Laxatives . . . . . . . . . . . . . . XIV . Antitussive Compounds . . . . . . . . . XV . Anti-infective Compounds . . . . . . . . A . Antibacterial Compounds . . . . . . . B . Tuberculostatic Compounds . . . . . . C . Antifungal Compounds . . . . . . . D . Antiparasitic Compounds . . . . . . . E . Antiviral Compounds . . . . . . . . XVI . Antineoplastic Agents . . . . . . . . . A . Animal Studies . . . . . . . . . . B . Clinical Observations . . . . . . . . XVII . General Considerations . . . . . . . . . A . Defaulting . . . . . . . . . . . . . B . Placebo and “Nocebo” Effects . . . . . C . Adverse Reactions . . . . . . . . . XVIII . Final Remarks . . . . . . . . . . . . . References . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

.

. .

. . . . . . . .

. . . . .

. . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . .

. . . . . . . . . . . . . . . . . . . . . .

211 212 213 213 213 214 214 214 214 215 215 215 220 222 223 224 225 225 229 230 230 230 230 232 233

I. Introduction Although differences between the sexes in propensity to constitutional or infectious diseases have been known for centuries (references in Goble and Konopka. 1973). the divergent responses of males and females to environmental and iatrogenic conditions have been recognized only within the last few decades . Hanzlik (1913) carefully tabulated and took into consideration the sex of patients in his study of salicylate toxicity. noting that the toxic doses for women were about 20% lower than those for men. which he pointed out corresponded well with the average difference hetween the sexes in weight of adults. By 1916. Storm van Leeuwen had noticed a difference between the response of male and female cats to ether. but concerted animal studies on differences between the sexes in pharmacological responses did not begin until about 10 years later when Fujii (1926) reported on morphine in mice. Takahashi (1926) on strychnine in rats. and Abderhalden and Wertheimer (1927) on alcohol and ether in mice. rats. and rabbits . In the meantime. Whipple (1918) and Cleveland (1919) had observed the

S E X AS A FACTOR I N DRUG EFFECTS

175

predominance of men with respiratory complications following surgery, which was confirmed by Featherstone (1924/1925), Ravdin and Kern (1926), Rucker (1927), and Sise et al. (1928). After some additional studies on alcohol in rabbits (Kask, 1929), neoarsphenamine in mice (Durham et al., 1929), morphine in rats (MacKay and MacKay, 1929, 1930; Glaubach and Pick, 1930), papaverine in frogs (Takase, 1932), and cocaine in rats (Downs and Eddy, 1932), investigations on differences between the sexes in response to therapeutic agents was dominated by work on the barbiturates in rats in which it was established that the anesthetic effect was much more prolonged in females than in males (Nicholas and Barron, 1932; Barron, 1933; Holck and Kanan, 1934, 1935; Holck and Cannon, 1936; Moir, 1936, 1937; Holck et al., 1937; Carmichael, 1938; Cameron, 1938/1939). This preoccupation with the differences between the sexes of rats in response to barbiturates continued throughout the forties (Kinsey , 1940; Holck and Fink, 1940; Horinaga, 1941; Holck et al., 1942, 1943; Holck and Mathieson, 1944; Homburger et al., 1947) and along with the metabolic studies, some of them in other species, pointing to the liver as the site of degradation of these drugs (Pratt et al., 1932; Pratt, 1933; Koppanyi et al., 1936; Cameron and de Saram, 1939; Martin et al., 1940; Scheifley and Higgins, 1940; Tatum et al., 1941; Masson and Beland, 1945; Dorfman and Goldbaum, 1947), formed the background for the eventual elucidation by Brodie and his colleagues of the role of liver microsomal enzymes in the oxidative transformation of the barbiturates and other drugs in the rat (Quinn et al., 1954; Cooper and Brodie, 1955; Brodie, 1956). These differences between the sexes in response to barbiturates, observed in rats, were not observed in other species, as evidenced by early studies of Kennedy (1933) in mice; Holck and Kanan (1935) in rabbits and dogs; Holck et al. (1937) in these species as well as in the cat, the guinea pig, and the turtle; Horinaga (1941) in mice and guinea pigs; and Homburger et al. (1947) in dogs. This limitation on the extrapolation of the findings in rats to other species including man was confirmed, to their “consternation,” by Brodie (1956) and his colleagues (Quinn et a l . , 1958; Brodie et al., 1958). The abundant literature on the differences between the sexes of rats in the metabolism of drugs which appeared thereafter has been recently reviewed by one of the most active workers in the field (Kato, 1974). He reemphasizes the special position of rats and suggests some of the possible explanations for the lack, in other species, of a similar androgen effect on the reduced nicotinamide adenine dinucleotide

176

FRANS C . GOBLE

phosphate (NADPH)-linked system and terminal oxidases which are involved in drug transformation. The studies reviewed by Kato involve a number of therapeutic entities including, in addition to barbiturates, morphine, aminopyrine, strychnine, and carisoprodol, all of which show in varying degrees more rapid degradation of the drugs by males, as a result of the higher activity of the liver microsomal enzymes in them. If other species were physiologically similar to the rat and all drugs were metabolized in the same way, there might be little interest in the present review. Much of what has been summarized for the rat is not considered in this review; instead other species and findings that have not yet been fitted into a pattern as well worked out as that involving the interaction of drugs, androgens, and rat liver microsomal enzymes are discussed. The order in which the various types of pharmacodynamic and chemotherapeutic compounds are considered here tends to be chronological, starting with CNS depressants, which were the first to receive attention, going then to the CNS stimulants, which were studied later. Subsequently, certain exceptions have been made to place preparations with similar pharmacological actions together (e.g., the psychotropics, which are relatively recent, are immediately after the other CNS active compounds; anti-infectives are with the antineoplastics). In this review endocrine and nutritional agents, as well as certain other naturally occurring materials such as autocoids, have not been included among the therapeutic agents considered.

II. Central Nervous System Depressants A. GENERAL ANESTHETICS

1. Specific Compounds in Animals Ether was one of the first pharmacodynamic compounds to be observed in relation to a difference in tolerance between the sexes. After ether administration, male cats reacted with initial excitement, whereas females went immediately into narcosis (Storm van Leeuwen, 1916). Orchiectomy did not alter the reaction. Differences between the sexes in response to ether are minimal in intact mice, rats, and rabbits (Abderhalden and Wertheimer, 1927; Stortebecker, 1937, 1939; Horinaga, 1941; Buchsbaum and Buchsbaum, 1962). Tolerance to chloroform was not associated with any difference between the sexes of rats (Horinaga, 1941). There was no difference

SEX A S A FACTOR I N DRUG EFFECTS

177

between the sexes of A-strain mice in susceptibility to liver necrosis when they were fed chloroform (Eschenbrenner and Miller, 1945a,b), but doses high enough to produce liver necrosis caused renal necrosis in males but not in females. The proclivity for necrosis in males was reduced by castration and restored by treatment of orchiectomized males with testosterone (Eschenbrenner and Miller, 1945b). Male mice that developed renal necrosis died early at the higher doses, and hepatomas, which took more time to develop, were thus observed only in females (Eschenbrenner and Miller, 1945a). Inhalation of chloroform also results in renal lesions in males only in several other strains of mice: DBA (Shubik and Ritchie, 1953; Russell, 1955); C3H (Deringer et al., 1953; Russell, 1955); C3Hf, A, HR (Deringer et al., 1953); CBA (Hewitt, 1956, 1957); WH (Culliford and Hewitt, 1957); BALB hybrids (Russell, 1955; Christensen et al., 1963); H (Jacobsen et al., 19H); C3WHe (Zaleska-Rutczynska and G u s , 1972a,b); and C57 BlacW6 (Ilett et al., 1973). A difference between the sexes was not observed in other C57 strains and substrains (Deringer et al., 1953; Russell, 1955; Zaleska-Rutczynska and Krus, 1972a) nor in ST (Deringer et al., 1953), A, C, CAF (Shubik and Ritchie, 1953), and albino (Hewitt, 1956) mouse strains. Controversy concerning the susceptibility of C3H males may be attributable to the existence of sublines of the strain (Christensen et al., 1963). Metabolic studies with radioactive chloroform in CF/LP, CBA, and C57BL mice indicated that a greater amount of the compound finds its way to the kidney in the males, whereas higher values were found in the liver of females (Taylor et al., 1974). Testosterone changed the pattern in females, but stilbestrol did not alter the distribution in males. Orchiectomy reduced the amounts in the kidneys in males, and testosterone restored it. Stilbestrol did not effect a change. Greater hepatic metabolism in female mice is suggested. Radioautographs from rats and monkeys revealed no differences between the sexes in the distribution of chloroform or its metabolites (Brown et al., 1974), and extrapolation of the metabolic findings in mice to man is discouraged. Inhalation of chloroform by pregnant rats over a 7-day period in subanesthetic concentrations indicated that it was not teratogenic but was highly embryotoxic. At the highest dose (300 ppm), there was a significant effect on the sex ratio of the embryos resulting in a male-tofemale proportion of 34:66, far from the normal 53:47 observed in controls (Schwetz et al., 1974). Some steroid hormones have an anesthetic effect when given intraperitoneally to rats. They are of both adrenal and sex hormone

178

F R A N S C. GOBLE

types (deoxycorticosterone acetate, androsterone, testosterone, and methyltestosterone), but progesterone has received the most attention (Winter and Selye, 1941; Selye, 1941). The effect is more pronounced in females than in males, and the importance of the liver in the metabolism of the compounds was suggested by observation of their effect in partially hepatectomized rats. Subsequent metabolic studies demonstrated high activity of hydroxylation and low activity of A4reduction of progesterone in liver microsomes of male rats (Kato et al., 1971). There is no difference between the sexes of dd mice in the anesthetic action of progesterone (Kato, 1959) nor in the metabolism of the drug by liver from these mice (Kato et al., 1971). The strain of mice used had, on a number of previous occasions, been shown to be one that does not show differences between the sexes in response to aminopyrine, hexobarbital, or morphine (Kato et al., 1968, 1969, 1970a,b). Hydroxydione (21-hydroxy-5/+pregnone-3,20-dione 21-hemisuccinate sodium), however, does effect longer sleeping time in female mice of the GFF strain as compared with males (Atkinson et al., 1962). There was no difference between the sexes in another strain (Swiss) of mice (P’An et al., 1955). 2. Postoperative Complications in Man Respiratory complications following surgery were observed early to be about 3 times more prevalent in men than in women (Whipple, 1918, and others listed in Section I; Brock, 1936; Burford, 1938; Dripps et al., 1961; Wightman, 1968; Herzog and Keller, 1971). The greater incidence of acute respiratory conditions (Stoneburner and Finland, 1941), chronic bronchitis, and emphysema (Marshall and Wyche, 1972) in men is possibly a more important factor than the type of anesthetic used. Another factor may be the greater compromise to the normal abdominal and diaphragmatic respiration in the male following laparotomy, whereas the female, with costal respiration, is less handicapped by the procedure (Eliason and McLaughlin, 1932; King, 1933a,b; Battle, 1936). Spirometric studies show a great reduction in vital capacity during laparotomy, the greater crippling being in men (Beecher, 1933). Greater incidence of smoking in men is also mentioned as a factor (Griffiths, 1934; Morton, 1944; Holmes, 1948). Much of this earlier work is reviewed by Gordh (1950, 1964). Postoperative nausea and emesis is more prevalent in females than in males (Gordh, 1950; Boulton, 1955; Burtles and Peckett, 1957; Bellville et al., 1960; Robbie, 1959). This was attributed by Gordh (1950, 1964) to

SEXASAFACTORINDRUGEFFECTS

179

the “well known fact that women are more sensitive to morphine and its derivatives than men,” an idea that is difficult to document. Emesis is not related to previous emetic history in females but it is in males (Robbie, 1959). Chlorpromazine is useful in controlling postoperative vomiting, and it is more effective in females than in males (Boulton, 1955). Cyclizine, perphenazine, and prochlorperazine all decrease the incidence of vomiting after anesthesia. Perphenazine is more effective in females, prochlorperazine in males (Robbie, 1959). Haloperidol also reduces the incidence of postoperative nausea and emesis in both sexes, but to a greater degree in males (Dyrberg, 1962). With droperidol, the reduction of incidence was more striking in females (Nikki and Pohjola, 1972). Acute hepatic necrosis was associated with halothane in 1958 and, although the mechanism by which the damage is imposed on the liver has not been elucidated, the condition is clearly more prevalent in females (Trey et al., 1969; Sharpstone et al., 1971; Reed and Williams, 1972; Gottlieb and Trey, 1974; Inman and Mushin, 1974). Patients with methoxyfluorane-associated hepatitis are also predominantly women (Joshi and Conn, 1974). By contrast, postoperative icterus, following a variety of other types of anesthetics including thiopental and nitrous oxide, is observed more often in males (Strasberg and Silver, 1971; Wasmer al., 1972). Central nervous system symptoms such as drowsiness and headache are the most common complaints following anesthesia and occur more frequently in women than in men (Bellville et al., 1960; Bodman et al., 1960; Edmonds-Seal and Eve, 1962; Thomas, 1963; Fahy and Marshall, 1969). Following emergence from ketamine anesthesia, delirium and dreams, both pleasant and unpleasant, are more frequent in women than in men. The incidence of these phenomena is greater in patients undergoing minor procedures and receiving atropine as premedication than it is in those receiving antidepressant pretreatment such as droperidol-fentanyl, but the difference between the sexes is evident in both situations (Bovill et al., 1971). Hyperthermia is a rare condition that is encountered during or immediately after general anesthesia and often accompanied by rigidity. Over 600/0 of the cases are in males (Britt and Kalow, 1968; Kalow et al., 1970), but there is no significant difference between the sexes in mortality (Gjengstii, 1971). It appears to have a genetic basis (Britt et al., 1969; King et al., 1972), the inheritance involving an autosomal dominant with reduced penetrance and variable expressivity (Britt and Kalow, 1970).

180

F R A N S C. GOBLE

B. ETHYLALCOHOL

1. Animal Studies a . Mice. When administered chronically to mice, ethanol is more toxic to females than males (Abderhalden and Wertheimer, 1927). Normally mating mice are more resistant than those isolated from sexual activity, the difference in resistance being greater in females (Agduhr, 1938). Toxicity increases with age in either sex, but in old age, females again are resistant (Chen and Robbins, 1944b). Females tend to consume more alcohol (in a 1W solution) than do males (Mirone, 1958). There are considerable differences between strains (McClearn and Rodgers, 1959; Rodgers and McClearn, 1962a) with the variance attributable to sex, approaching, but not reaching the 5% significance level (Rodgers and McClearn, 1962b). The C57BL strain, which has a naturally high alcohol preference, shows a highly significant difference between the sexes in ethanol consumption. The differences are correlated with alcohol dehydrogenase (ADH) activity in the livers of the animals, the females drinking more alcohol than males and having higher ADH activity (Eriksson and Pikkarainen, 1968). There is also ADH activity in mouse kidney, but whereas the enzyme is made continuously in the liver, in the kidney it is made only in the presence of an inducer, the most potent of which is testosterone. The level of ADH in the kidney is higher in the male than in the female, and castration decreases it without affecting the level of it in the liver. Estradiol is the inducer of kidney ADH in female mice, but it is much less potent than testosterone (Ohno et al., 1970a,b). Redmond and Cohen (1972) studied the exhalation of acetaldehyde following ethanol administration to C57BL mice finding that the levels in expired air from males averaged about 5 times higher than those from females. Castration lowered the mean acetaldehyde level for males about 85%, into the range observed for both intact and oophorectomized females. Hungerbuhler et a l . (1972) found the LDs0 values for two different concentrations of ethanol in mice were 4 4 % less in females than in males but that this difference was not significant. b. Rabbits. Castrated rabbits are affected by smaller doses of alcohol than are intact ones (Kask, 1929). In oophorectomized rabbits, estrone lessens the degree of intoxication; the effect is not attributed to the metabolism of the alcohol but to a site of action in the CNS (Goldberg and Stortebecker, 1943).

S E X AS A FACTOR I N DRUG E F F E C T S

181

c. Rats. Oophorectomy lowers the tolerance of rats to alcohol (Motz and Besancgn, 1937). Castration increases the sensitivity of the brain of rats to alcohol (Ijiri, 1939). The difference between the sexes of rats in tolerance to alcohol is much less than that observed with barbiturates (Horinaga, 1941). Male rats have been reported to have a greater preference for alcohol than do females (Schadewald et al., 1953), although Mardones (1960) and Khanna et al. (1967) reported no difference between the sexes. Eriksson and Malmstriim (1967; Eriksson, 1968) found females to be greater drinkers, although estrogens? both natural and synthetic? inhibit spontaneous consumption of alcohol (Aschkenasy-Lelu, 1958, 1960a,b). Female rats in acute ethanol intoxication show more severe fatty liver infiltration than do males (Mallov and Bloch, 1956). The higher tolerance of the female is not attributable to a difference in rate of alcohol oxidation according to Wallgren (1959), because no significant difference in blood alcohol level is found. Prolonged ethanol administration decreases the succinate-oxidizing capacity of liver mitochondria in male rats but not in females (Kiessling and Tilander, 1963). In Sprague-Dawley rats, the nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase activity in both liver and kidney is much higher in females than in males, but this difference does not occur in the Wistar strain (Biittner, 1965). With the latter strain, alcohol treatment increased the activity of phosphofructokinase in livers from males and that of lactic dehydrogenase in livers from females (Kiessling and Pilstriim, 1966). In the rat, ethanol administration stimulates the release of triglycerides from the liver to the plasma, and this effect is greater in males than in females (Homing and Knox, 1967). There is also a microsomal ethanol-oxidizing system distinguishable from alcohol dehydrogenase. This enzyme oxidizes alcohol to acetaldehyde and is capable of an adaptive increase in ethanol consumption. Its activity is lower in females than in males, but the capacity for adaptation is greater in females. Microsomal protein increases more in males than in females after consumption of ethanol (Lieber and De Carli, 1970). Hatfield et al. (1972) induced a tolerance to alcohol in rats by repeated intraperitoneal injections: females were tolerant after 10 days, males after 15 days. Ethanol treatment did not produce a crosstolerance to hexobarbital, meprobamate, or zoxazolamine in male rats although there was a cross-tolerance to phenobarbital. Female rats, however, had a cross tolerance to hexobarbital, pentobarbital, meproba-

182

FRANS C . GOBLE

mate, and zoxazolamine but not to barbital or phenobarbital. They believed that the basis for the cross-tolerance observed may be diminished CNS sensitivity to the drugs rather than enhanced drug metabolism by induced hepatic microsomal enzymes.

2. Clinical Observations With the exception of the hyperlipemic effect that predominates in males (de Gennes et a l . , 1972), differences between the sexes in response to alcohol are difficult to establish in man. Sociological factors strongly influence the incidence of alcoholism in both sexes and with treatment in the same proportion of men as women became abstinent or controlled drinkers (Gerard and Saenger, 1966). Acute alcoholic intoxication, such as that observed in infants (Varese, 1967), may be more common in males but is probably more a manifestation of behavior than susceptibility. Acute pancreatitis also seems to be associated with alcoholism in some devious way and is more common in males (Edlund, 1970; Sarles, 1970; Forell and Stahlheber, 1973). The incidence of alcoholism and tuberculosis is high in males (Milne, 1970), but there is no evidence that this is a constitutional rather than sociological phenomenon. Dupuytren’s contracture is another condition associated with alcoholism but also the anticonvulsants and epilepsy (Editorial, 1972b) so that the correlation is no easier to establish than that between cigarettes, alcohol, coffee and sex of the person as factors in the incidence of peptic ulcer (Friedman et al., 1974). Disulfiram (tetraethylthiurarn disulfide) was reported by Hoff and McKeown (1953) and by Hoff (1955) to be more successful in men than in women in the treatment of alcohol addiction. Gerard and Saenger (1966) did not believe sex of the patient to be related to the effect, but Lundwall and Baekeland (1971) consider the earlier findings to be probably more reliable, being based on a much larger sample.

C. NARCOTIC ANALGESICS 1. Animal Studies

As early as 1926, Fujii had observed that both an ovarial extract (ovaritin) and a testicular extract (spermatin) had the effect of decreasing the action of morphine in the mouse. It was later reported that castrated rabbits were more resistant to morphine (Ikonen et a l . , 1929). The MacKays (1929, 1930) noted that female rats could tolerate larger doses of morphine than could males of the same weight and age. They

SEXASAFACTORINDRUGEFFECTS

183

(1935) related this to the sexual dimorphism of the adrenal glands which in females have more cortical tissue than in males. Horinaga (1941), on the other hand, found no difference between the sexes in tolerance to morphine in rats. Meanwhile, Glaubach and Pick (1930) had observed that after administration of thyroxine to rabbits for 5 days, the drop in temperature produced by morphine administration was not as marked in the males as it had been without thyroxine but in females the drop in temperature was equivalent with or without thyroxine. Fantoni (1936) found that follicular hormone decreased the effect of morphine in young thymectomized female rabbits, and Bun (1937) reported the same effect in oophorectomized rabbits, also noting that luteinizing hormone had no similar action. Smith et al. (1943) found a twofold difference in degree of analgesia between male rabbits and oophorectomized females given the same dose. In 19%, during a study on the distribution and excretion of radioactivity after administration of N-methylrn~rphine-~~C, March and Elliott administered cyclopentyltestosterone to some female rats and found that the amount of l4CO2 liberated by them was similar to that excreted by males, whereas the amount from untreated females was minimal. They remarked, “It is most interesting to find a process in the liver which is stimulated by androgens. Very little is known regarding the influence of castration or androgen administration on liver enzyme content.” This observation somewhat preceded the discovery of most of the compounds now recognized as enzyme inducers, and it has strangely been omitted from a number of reviews dealing with induction (Brodie et al., 1958b; Conney and Burns, 1962; Gillette, 1963; Conney, 1967; Kato,

1974). The difference between the sexes of rats in the analgesic efficacy of morphine persisted following pretreatment with cocarboxylase (Angelucci, 1958). A systematic study of apparent K , and TI, values of liver microsomal ethylmorphine N-demethylase from rats of either sex of eight inbred strains and one outbred strain (Page and Vesell, 1968) was followed by an investigation of two additional strains of laboratory ratsa strain of wild trapped Norway rat and a strain of Kangaroo rat (Page and Vesell, 1969a,b). Males metabolized ethylmorphine more rapidly (50-300%) than did females in all strains of Norway rats, both laboratory and wild, and after phenobarbital pretreatment there was a two- to fourfold increase in the ethylmorphine metabolism in both sexes. Female Kangaroo rats metabolized ethylmorphine at about the same rate as female Norway rats, but the rate for males was much lower than that

184

F R A N S C. GOBLE

for the females. After pretreatment with phenobarbital, however, the male Kangaroo rats had a forty-fold increase in ethylmorphine metabolism, contrasting with a fourfold increase in the females. The Kangaroo rats, therefore, resembled mice (Vesell, 1968b) in the metabolism of this drug. The difference between the sexes in N-demethylation of ethylmorphine in rats was not parallel to the cytochrome P-450 levels which were nearly equivalent in males and females, although males had a much greater ability to N-demethylate. Thus cytochrome P-450 in male rats was more efficient than that in females, suggesting a qualitative difference in the microsomal hemoproteins in the two sexes (Sladek and Mannering, 1969). An alternative explanation would be that cytochrome P-450 is not the rate-limiting component in the N-demethylation system in the female rat. In mice, the ethylmorphine N-demethylase activity was not greater in the male than in the female, nor was there a significant difference in the cytochrome P-450 content. Ethylmorphine inhibits the in vitro hepatic microsomal metabolism of [ureyl-l4C1 tolbutamide; the liver preparations from adult female rats were less susceptible to inhibition than those from males (Darby, 1972). This difference between the sexes was also detectable in preparations from livers of immature animals but was not as marked as in those from adults (Darby, 1973). The ethylmorphine N-demethylase activity in fractions from livers of male rats increased with the maturity of the animals, from values of the same order as those in females at 21 days of age to values 2-3 times higher at 49 days, whereas the activity in fractions from females decreased to about half during the same period. The apparent Michaelis constant decreased in males during this period but remained unchanged in females (El Defrawy El Masry et a l . , 1974). The changes in drug metabolism resulting in the difference between the sexes in the demethylation of ethylmorphine were attributed to qualitative changes in cytochrome P-450 in males and females. Orchiectomy prevented the increase in ethylmorphine N-demethylase activity, the increases in the amount and specific activity of cytochrome P-450, and the changes in the ethylisocyanide difference spectrum that occur in the developing male rat (El Defrawy El Masry and Mannering, 1974). Neither the magnitude of the ethylmorphine binding spectrum nor the NADPH-cytochrome P-450 reductase activities relative to the cytochrome P-450 content of microsomes changed with age or were correlated with the changes and difference between the sexes in ethylmorphine N-demethylase activity (Cohen and Mannering, 1974). Recent studies support the earlier conclusions of Kato and Gillette

SEXASAFACTORINDRUGEFFECTS

185

(1965b) that morphine depresses the metabolism of Type 1 drugs in mature male rats by impairing androgen-induced stimulation of the hepatic monooxidase system (Sladek et a l . , 1974). The latter remark, “Although it is apparent that male sex hormones are implicated in the sex difference in drug metabolism in rats, little is known of the mechanism involved. ”

2. Clinical Observations Differences between the sexes in response to the strong analgesics in man and nonrodent mammals are not well documented. The idea, which has even found its way into textbooks, that cats and women are likely to be abnormally affected by morphine seems to have originated with a statement of Guinard (1890) who studied morphine in the cat, which he found to exhibit hyperexcitability rather than sedation, and who ended his report by comparing the behavior with that sometimes “observed in the human species, particularly in women, where one sometimes encounters patients for which morphine is never a calming agent.” This was reinforced by a Dr. Macnaughton Jones (1895a,b), a gynecologist whose observations naturally emphasized women. Pancoast and Hopkins (1915) noted that “moderate doses” of opium derivatives “especially in young, nervous females, caused a prolongation of the emptying time of the stomach.” Following the administration of the synthetic analgesic meperidine, pregnant women, women taking oral contraceptives, and neonates have been found to excrete more unchanged meperidine and normeperidine than nonpregnant women or normal men on the same regimen. Administration of stilbesterol or progesterone to the men changed their pattern of excretion to that associated with pregnancy as a result of diminished capacity to metabolize the drugs (Rudofsky and Crawford, 1966). In passing, it may be of interest to note that recent studies have shown that proper doses of morphine will produce narcosis and analgesia rather than excitation in the cat and that its previous proscription for analgesia in that species is not warranted if suitable dosages are used (Davis and Donnelly, 1968; Heavner, 1970; Jacobsen, 1970; Watts et al., 1973). D. BARBITURATES 1. Animal Studies a . Early Studies in Rats. Observations on differences between the sexes in response to barbiturates commenced with the work of Nicholas

186

F R A N S C . GOBLE

and Barron (1932) on amobarbital which they found to produce deeper hypnosis and more severe toxicity in female rats than in male. Barron (1933) subsequently reported that rats did not show a similar difference in response to pentobarbital, but Holck and Kanin (1935) did not confirm this and reported pentobarbital as well as butallylonal, hexethal, and hexobarbital to exert a similar greater effect on females than on males. Between 1936 and 1943, Holck and co-workers studied some two dozen barbiturates in rats. Most of the compounds that had a different effect in males and females were of the intermediate or short-acting varieties, with none of the longest-acting (e.g., barbital and phenobarbital) nor shortest-acting (e.g., thiopental) showing this characteristic. The difference with amobarbital appeared when the rats reached weights between 50 and 60 gm, the weight at which differences between the sexes begin to appear in the hypophysis and adrenal (Barron, 1933). Very young female rats were more resistant to pentobarbital than were males; as they became older the sensitivity of females approximated that of males, and mature females were more susceptible than males (Moir, 1936, 1937). The difference between the sexes in reaction to pentothal, in either young or old rats, was not confirmed by Carmichael (1938), but subsequent papers have left no question as to the greater susceptibility of adult females (Kinsey, 1940; Holck and Mathieson, 1944; Homburger et al., 1947; Jarcho et al., 1950). Pregnancy markedly increased the tolerance of rats to pentobarbital, and the effect persisted through the period of lactation (Holck and Fink, 1940). Male rats castrated before the sex difference in response to amobarbital appeared showed the same resistance as intact males, but those castrated after the sex difference appeared became more susceptible (Barron, 1933). Gonadectomized males became more sensitive to pentobarbital but were still more resistant than females (Moir, 1937). Castration of adult male rats increased their susceptibility to butallylonal, the increase with this drug being greater than any of five other barbiturates tested (Holck et al., 1937). Oophorectomized females slept longer and had greater mortality with phenobarbital than did intact females (Cameron, 1938/1939). Testosterone propionate treatment of female rats lowered the average sleeping time with pentobarbital as did spaying (Kinsey, 1940). Although testosterone propionate did not change the tolerance of intact male rats, it did change the tolerance of castrated males back to the levels of intact males (Holck et al., 1942). Prolonged treatment with the compound increased the tolerance of females. Estradiol lessened tolerance in either sex. A clear-cut difference between the sexes was reported with propallylonal and this could not be affected by administra-

S E X AS A FACTOR I N DRUG E F F E C T S

187

tion of testosterone propionate to either males or females. On the other hand, testosterone acetate treatment did shorten the recovery time of female rats treated with butallylonal (Holck et a1 ., 1942). Orchiectomy prolonged sleeping time (de Boer, 1948). Following testosterone treatment the sleeping times in females given pronarcon were decreased to values approximately those observed in males (Grewe, 1953a,b). b. Early Studies in Mice, Guinea Pigs, and Nonrodents. In 1933, Kennedy noted that there was no difference between the sexes in the response of mice to hexobarbital, and this was confirmed by Holck et al. (1937) and Horinaga (1941). There was likewise no difference between the sexes in response to this compound in the dog, cat, rabbit, guinea pig, or turtle (Holck et al., 1937); the observation on guinea pigs was also confirmed by Horinaga (1941). There was no difference between the sexes observed in the toxicity of amobarbital in rabbits or dogs (Holck and Kanin, 1939, and there was no difference in the response to that drug in mice (Holck et al., 1937). Compound 897 likewise showed no difference between the sexes in dogs (Homburger et ul., 1947). Guinea pigs that had been anesthetized by a combination of ether and butallylonal during oophorectomy had a postoperative narcosis usually resulting in death, whereas ether anesthesia produced no such complication. This was attributed to an antinarcotic effect of estrone (Stortebecker, 1939). c . Later Studies in Rats. Contemporaneously with the demonstration that an enzyme system that oxidizes hexobarbital to ketohexobarbital is localized in liver microsomes (Cooper and Brodie, 1955), the activity of this enzyme system was reported as being markedly lower in females than in males (Quinn et al., 1954). Administration of testosterone increased this enzyme activity in females, suggesting that it might play a part in synthesis of the system. The voluminous literature on sex-related differences in drug metabolism, which was inspired by the studies of Brodie and his colleagues, has recently been summarized by one of that group (Kato, 1974). To paraphrase the comments of that spokesman for the field did not seem desirable, so an attempt has been made to minimize repetition and include certain related observations not specifically reviewed previously. The role of P-diethylaminoethyl diphenylpropylacetate hydrochloride (SKF’-525A), an experimental drug that impairs liver detoxication, in the localization of the site of the metabolic transformation of barbiturates as well as a number of other compounds has been set forth by Axelrod et al. (1954), Cooper et al. (1954), Cook et al. (1954), Streicher and Garbus (1955), Brodie (1956), and Brodie et al. (1958b). In connection with studies on SKF-525A, Streicher and Garbus extended some of the

188

F R A N S C. GOBLE

earlier observations on the effect of age on the sex difference in response to barbiturates, using hexobarbital. They found that females and males at 1 month of age slept the same length of time, but that by 2 months, a striking difference that persisted up to 20 months and was lost again in old age was apparent. When SKF-525A was given prior to the hexobarbital, the sleeping time was increased but the age and sex relationship remained the same. This was also true when they used chlorpromazine, which produces the same extension of sleeping time by a different mechanism. Edgren (1957) administered sex hormones to gonadectomized rats and found that testosterone shortened the sleeping time of castrated males but estrone had no effect. In oophorectomized females, estrone produced a longer hypnosis but testosterone had no effect. He interpreted these findings to indicate that Brodie’s experiments might have measured pituitary blockade, by testosterone in the females and by estradiol in males, or peripheral estrogen-androgen antagonism. Remmer’s (1958a) interest in the differences between the sexes of rats of hexobarbital vs. thiopental led to his investigation on a number of other barbiturates (1958b; Remmer and Alsleben, 1958) which he found to accelerate the in uivo and in vitro degradation of hexobarbital by activating the liver microsomal enzymes (Remmer, 1959a,b). The longer-acting compounds, barbital and phenobarbital, had a stronger stimulating effect than did the shorter-acting ones, such as butallylonal, hexobarbital, and thiopental. Prior to this time inducers had been known but all were polycyclic hydrocarbons, some of which were carcinogens (reviewed by Brody et al., 1958b). Following Remmer’s reports, other investigators discovered similar activity in a variety of compounds (for review; see Conney and Burns, 1962; Conney, 1967). Barbital and phenobarbital, however, continued to be used extensively as agents for enzyme activation. It was soon found that the barbiturates induce microsomal enzymes that metabolize not only other barbiturates but also other types of drugs (Conney and Burns, 1960) including androgens such as testosterone, methyl testosterone, and androstenedione (Booth and Gillette, 1962) and A4-androstene-3,17-dione (Conney and Klutch, 1963), anabolic steroids such as 19-nortestosterone and 4-chloro19-nortestosterone acetate (Booth and Gillette, 1962), and progesterone (Kuntzman and Jacobson, 1965). The barbiturates, therefore, were the first therapeutic agents shown to be inducers of liver microsomal enzymes. The enzymes induced are capable of metabolizing not only compounds related to the inducers but also a variety of unrelated chemical types. Subsequently many other medicinal agents were found to be stimulators of drug metabolism, and

S E X AS A FACTOR I N DRUG EFFECTS

189

the discovery of their broad spectrum of activity has led to the recognition of inducers as important factors in the area of drug interaction in experimental and clinical studies. It is of particular interest in relation to differences between the sexes in response to drugs that the therapeutic agents can induce enzymes that metabolize the sex hormones and the sex hormones can induce enzymes that metabolize the drugs. In further studies on the oxidative metabolism of drugs, Kato et al. (1969b) emphasized the difference between the sexes in hydroxylation of pentobarbital, whereas certain other drugs showed no such difference. They also noted the inhibition of hydroxylation of barbiturates by SKF525A and 2,4dichlor0-6-phenylphenoxyethylamine H C1 (DPEA), whereas certain other compounds were not affected. They regarded this as possible evidence of involvement of more than two terminal oxidases in the metabolism of drugs by the microsomes. Furner et al. (1969) also suggested that the liver microsomes contained a number of NADPHdependent enzymes that differed in their rates of prepuberal development. Jon et al. (1969) tested for the effects of progestational compounds (norethynodrel, medroxyprogesterone acetate, and progesterone) alone and combined with estrogens (mestranol and ethynylestradiol) on the metabolism of pentobarbital in female rats and their tissue homogenates. Only norethyndiol, in high doses, increased sleeping time and brain concentration, presumably by blocking hepatic metabolism. 6-Aminonicotinic acid and 4-acetylpyridine are also suppressors of barbiturate metabolism, prolonging sleeping time in rats. Their effects can be antagonized by 7-[2-hydroxy-3-(N-2-hydroxyethyl-N-methylamino)propyl]-1,3-dimethylxanthine pyridine-3-carboxylate (Xantinol nicotinate). The last compound, when given intravenously, considerably shortens sleeping time in rats but only in males (Brenner and Trautmann, 1969). Owen et a l . (1971) gave 5-(3,4-dichlorophenyl)-5-ethylbarbituric acid (dichlorophenobarbital) to rats in the diet for 30 days and measured the increase in hepatic drug metabolism, observing also that the amount of microsomal protein was significantly increased in males at a dose level (0.006%) one-third that at which an increase was observed in females (0.018%). Feuer et al. (1971) administered phenobarbital per 0s and found that the liver was the only organ in which the enzymemetabolizing hexobarbital was induced (the others were brain, kidneys, intestines, and adrenals). From a study of several generations of Wistar rats for susceptibility to hexobarbital as evidenced by deviation of sleeping time, Gut and

190

F R A N S C . GOBLE

Balinova (1972) concluded that there was a genetic nonhomogeneity of hepatic microsome metabolism within this strain. Hadley and Miya (1972) and Hadley et al. (1974) found that cadmium potentiated the action of hexobarbital in rats of either sex, but the male was more sensitive, which they interpreted to indicate a possible testicular damage in addition to an inhibition of drug metabolism. The in vitro inhibitory effect of pentobarbital on tolbutamide metabolism by hepatic microsomal preparations from rats of either sex treated with phenobarbital is less in female rats than in males of both juvenile and adult populations (Darby, 1972, 1973). Neonatal treatment of rats with estradiol benzoate significantly lengthened the hexobarbital sleeping time in 39-day-old males (Pedersen et al., 1974). There were a few studies during the past 10 years not dealing with liver microsomal metabolism. Fujii et al. (1965) did a tolerance study in which they found the usual difference between the sexes in duration of ataxia and sleep with pentobarbital, but no remarkable development of tolerance in either sex. Coville and Telford (1970) found that thyroxine pretreatment prolonged hexobarbital sleeping time in males but shortened it in females, although thyroidectomy prolonged it in both sexes. Ethinylestradiol lengthened sleeping time in males as did castration, but when combined with thyroxine, potentiation was observed only with castration. Thyroxine shortened the sleeping time in females in those rats in which sleeping was included but did not affect the percentage of rats which slept following small doses of hexobarbital. Peters and Leeuwin (1971) reported that pregnancy had no influence in the sleeping time with pentobarbital but that the time was shortened during lactation. Gergely (1972) used carbon tetrachloride poisoning in conjunction with estrogen treatment in confirming the importance of the liver and the role of hormones in the difference between the sexes in response to hexobarbital. Beattie et al. (1973) studied the effect of pentobarbital on the luteinizing hormone (LH) secretion in gonadectomized rats of either sex and concluded that the drug reduced LH release independently of gonadal steroid action. Biliary and urinary excretion of pentobarbital and its metabolites in the rat proceeds at a higher rate in males than females, and four metabolites are found in males, three in females (Buttar et al., 1974). The criteria used in measuring differences between the sexes in response to barbiturates, other than liver enzyme activity, are usually sleeping time or mortality; although loss of righting reflex (Holck et al., 1943), absence of ear reflex (Homburger et al., 1947), eye blink, and

S E X A S A FACTOR I N DRUG E F F E C T S

191

foot withdrawal response (Collins and Lott, 1968) have occasionally been tested. Behavior in a maze revealed the mean exploratory activities to be greater in females given various dosages of pentobarbital than in males (Stretch, 1963). The values for untreated males were lower than those for females, and increasing doses of the barbiturate did not change this low level of activity; however, low doses of the drug in females was followed by a doubling of activity which did not increase until high levels were used. King and Becker (1963) observed respiratory rates, heart rates, and rectal temperatures as well as mortality, finding male rats to be least susceptible to change and nonpregnant females to be most susceptible; pregnancy produced an intermediate response. King et al. (1963a,b) and King (1964) made additional studies on female rats in pregnancy, pseudopregnancy, estrus, and diestrus, using the same criteria as well as measuring pentobarbital levels in blood, brain, and liver. d . Later Studies in Mice. In agreement with the findings of Holck et al. (1937) and Horinaga (1941), Brodie (1956), and Quinn et al. (1958) reported no sex difference in the duration of action of hexobarbital in mice (or guinea pigs), and added that hormones did not induce an appreciable change in the disappearance rate of the drug in mice. Brown (1961), however, found that with pentobarbital, adult male LAC mice had greater sleeping time than did females regardless of strain and that this difference between the sexes was dependent on age, being at least partially sex hormone-dependent as had previously been described for rats. Westfall et al. (1964) extended these observations on greater sensitivity of male Webster-Swiss mice and found that stilbestrol shortened their sleeping time and that testosterone lengthened the sleeping time of females. Meanwhile, Kuntzman et a l . (1964) and Conney et al. (1965) had been studying hexobarbital in CF1 mice and found that the steroid hydroxylase and hexobarbital oxidase activity in mouse microsomes was greater in females than in males. They did not make any point of this but, although not tested for significance, the data showed no overlapping between the ranges of values for the two sexes. Rumke (1966) also noted a difference between the sexes in response to hexobarbital, and Novick et al. (1966) found that testosterone and methyl testosterone given to male CF mice increased the sleeping time. Backus and Cohn (1%6), after giving both sexes of mice hexobarbital, found that females slept a shorter time and that liver homogenates from females metabolized a greater amount of the drug than those from males of the same strain. The importance of age and strain as factors influencing the sex

192

F R A N S C. GOBLE

difference in response of mice to hexobarbital was emphasized by a number of investigators. Catz and Yaffe (1967) and Yaffe et al. (1968) found consistent differences between the sexes in three strains they studied (CF5BL, BALB/C, 1298 as did Rumke (1968) with four other strains (CPB-N, CPB-S, CPB-H, CPB-N x CPB-S). Vesell (1968a,b) found that the difference between the sexes was maximum in inbred mice (AL/N, NBL/N) and was not significant in outbred animals (CFW, GP). Rumke and Noordhoek (1969) found two strains of mice (CPB-FT and CPB-V) which showed no difference between the sexes (in contrast to those previously studied by Rumke (1968), and they suggested the possibility of a correlation between this lack of difference and a lack of difference between the sexes in major urinary protein (MUP) complex which was coincidental in the two strains. The question of whether the rate of hexobarbital hydroxylation was correlated to cytochrome P-450 content of the liver was also unanswered (Noordhoek and Rumke, 1%9a,b). 4 Kato et a l . (1970d) found that three (dd, ICR, C57BL) of the four strains of mice they studied (the fourth was C3H) did not show a significant difference between the sexes in the hydroxylation of hexobarbital and its related difference in sleeping time. One of these strains (dd) had previously been shown (Kato et a l . , 1970a) to have no significant difference in the cytochrome P-450 content nor in the magnitude of the spectral change of P-450 induced by hexobarbital in liver microsomes. Other investigations, each in a single strain of mice, showed differences between the sexes in response to hexobarbital under various conditions. Gessner et a l . (1967) found it of interest that male ICR mice slept longer than did females, but Fujii et a l . (1968) found that ICR males slept for a shorter time than females in agreement with most earlier reports. Messerschmidt et al. (1969) found that the combination of whole-body radiation and skin wounds resulted in a marked modification in the sensitivity of NMRI mice to hexobarbital and that this was sex-dependent, because females became more sensitive and recovered much more slowly than males. The response of female mice to phenobarbital as an anticonvulsant preceding electroshock was enhanced by pretreatment with estrogen and lowered by pretreatment with progestin (Blackham and Spencer, 1969, 1970). e . Comparative Studies with Different Species. The similarities between the mechanism by which increases in activity of microsomal drugmetabolizing enzymes were stimulated by inducing drugs and steroids

SEXASAFACTORINDRUGEFFECTS

193

were emphasized by Kato et al. (1962) who noted that the anabolic steroids, in addition to taking longer to produce their effect than the inducing drugs, acted only in rats, whereas the inducing drugs were effective in mice and guinea pigs also. Kato et al. (1962) reported that inducing drugs, in contrast to anabolic steroids that were effective only in rats, could produce an increase in enzyme activity in mice and guinea pigs. Kato et al. (1968) found no difference between the sexes in the hydroxylation of hexobarbital in mice (dd strain) or rats and no difference in the NADPH-linked electron-transport system in liver microsomes of mice or rabbits, but that there was a difference in both of these activities in rats. They found no difference between the three species in relation to a sex dichtomy in actions of NADH oxidase, NADH-cytochrome c reductase, NADHneotetrazoline reductase, nor in content of cytochrome in the liver. Kato et al. (1970a) studied the magnitudes of spectral change induced by hexobarbital and the binding capacity of P-450 with hexobarbital in the above three species and found that in mice and rabbits there were no differences between the sexes in those factors, in agreement with the lack of difference in hexobarbital hydroxylation. The magnitude of spectral change induced by hexobarbital in liver microsomes was markedly greater in male rats than in females, as had previously been shown by Schenkman et al. (1967), and the binding capacity of cytochrome P-450 with hexobarbital was higher in male rats than in female. From studies in which they compared the drug metabolism enzyme systems in rats with those in rabbits and mice, Kato et al. (1968) suggested that, since the differences between the sexes in NADPHlinked electron-transport system and drug-oxidizing activity of liver microsomes were observed only in rats, it was probable that androgen stimulated the NADPH-linked system and terminal oxidase through a common factor that was genetically absent in other species. Buchel (1954, 1969), who studied both rats and mice (Swiss) with pentobarbital, confirmed the contrary response of mice (as opposed to rats), and Halevy and Frumin (1973) used two substrains of RF/J mice (Webster) with pentobarbital as well as hexobarbital. They found a difference between the sexes in response to either drug in each of the substrains of mice, one of which was sensitive to bacterial and viral stress and was more sensitive to the barbiturates, whereas the other strain was resistant to bacteria and viruses and less sensitive to the drugs. Hexobarbital hydroxylation in male rats was markedly decreased by

194

F R A N S C. GOBLE

thyroxine treatment in contrast to the process in females in which it was slightly increased (Kato et a l . , 1969a). This activity in both sexes of mice and rabbits was decreased. Additional studies on the relationship of sex on substrate-induced spectral changes of cytochrome P-450 with regard to drug oxidation were done by Kato et al. in rats (1970a,e) and in mice and rabbits by (Kato et a l . , 1970a). In alloxan diabetic male rats, the magnitude of spectral change per unit of protein induced by hexobarbital was decreased, but it was increased in diabetic females. There was no significant change in diabetic or starved mice, although fasting increased the change in female rats (Kato et al., 1970b,c). These authors postulated that the decrease in the binding capacity of cytochrome P-450 for hexobarbital in liver microsomes in diabetic and starved male rats involves an impairment of the ability of androgen to stimulate the binding capacity of cytochrome P-450 with hexobarbital. This did not occur in mice and rabbits. Kato et al. (1970d) also studied strain differences in metabolism of hexobarbital and several other drugs in rats and mice, finding differences between the strains of mice but not of rats. CoviUe and Telford (1970) reported that LAC albino mice did not show a difference in duration of action of hexobarbital but, when thyroxine was administered prior to the barbiturate, the sleeping time was increased in both sexes, in contrast to the findings in rats in which thyroxine prolongs sleeping time in males (Conney and Garren, 1961) but has the opposite effect in females (Kato et a l . , 1969~).Hadley and Miya (1972) and Hadley et a l . (1974) found that cadmium potentiated the hexobarbital sleeping time in both rats and Swiss mice. The percentage increases indicated an inverse relationship in mice as opposed to rats (male mice 28%, female mice 45%; male rats 131%, female rats 29%). Maines and Westfall (1971) added another rodent to those which show differences between the sexes in hexobarbital metabolism, namely the Mongolian gerbil (Meriones unguiculatus). This species resembles the mouse rather than the rat, the hexobarbital hydroxylating activity of the liver microsomal enzymes and the microsomal content of cytochrome P450 being higher in the females and correlating with shorter duration of hypnosis in the females. 2 . Clinical Observations

Although acute barbiturate poisonings are more common in women than in men (Laignel-Lavastine and d’Heucqueville, 1938), this differ-

S E X A S A FACTOR I N DRUG EFFECTS

195

ence is probably attributable to behavioral factors rather than to a greater sensitivity of women. Anesthesiologists have no clinical impression of any difference between the sexes in reaction to barbiturates (Brodie, 1956).

E. MILD ANALGESICS 1. Salicylates When aspirin or salicylamide was administered to rats for 13 weeks, the inhibition of weight gain was greater in females than in males but this cannot be attributed with certainty to toxic action of the compounds (Ichniowski and Hueper, 1946). Aspirin has been recognized as injurious to the stomach mucosa since

1938, and since 1960 (Billington, 1960) there have been a series of reports from eastern Australia linking aspirin abuse with chronic gastric ulcers particularly in females (Duggan, 1972, lists the most important references). The epidemiology of gastric ulcer, of course, involves many other factors including alcohol consumption, smoking, domestic stress, and apparently geography as is pointed out by Alp et al. (1970) whose studies in southern Australia differed considerably from Billington’s

(1963, 1965). Other adverse effects (tinnitus, dizziness, deafness) are more common in women (Green and Krueger, 1963). Death from asthma attributable to aspirin sensitivity is more prevalent in women than in men and it occurs at an earlier age (Walton and Randle, 1957; Giraldo et al., 1969). Among patients with aspirin intolerance with asthma and/or rhinitis, over 70% are females (Miller, 1967; Settipane et al., 1974). Aspirin esterase activity is significantly higher in men than in women (Menguy et al.,

1972). Aspirin is also known to be an aggravating factor in urticaria and angioedema (Bruun, 1950), conditions having a marked difference between the sexes in incidence among those below the age of 15. NO boys had angioedema alone, and comparatively few had both conditions. Angioedema with or without urticaria was frequent in girls (Champion et al., 1969).

2. Aminopyrine The toxic effects of aminopyrine in mice as measured by mortality were not affected by orchiectomy, but oophorectomy decreased resistance to the drug (Borglin and Mansson, 1951). Administration of estrogen reversed this change in resistance, and the authors suggest this

196

FRANS C . GOBLE

as an explanation of the predilection for agranulocytosis in women who are in or past the climacterium. Aminopyrine was one of the compounds that produced the greatest difference between the sexes in N-demethylation in rats; male rats normally metabolize the drug at a greater rate but have this faculty impaired by starvation, whereas starved females have an increased ability to metabolize the compound (Kato and Gillette,

1964, 1965a). Neonatal treatment of rats with estradiol benzoate slightly decreased the rate of aminopyrine metabolism in males (Pedersen et al., 1974). Agranulocytosis, which was pointed out to occur in the United States mainly in women of middle age, has been associated with aminopyrine medication since the early thirties (Kracke and Parker, 1935, 1939). By 1937, when Plum repoaed aminopyrine to be the most important cause of agranulocytosis in Denmark, it was established that this condition had a far higher incidence in women than in men. Seventy to eighty percent of cases was in females (Plum and Thomsen, 1940). The incidence of drug-induced agranulocytosis, chiefly from pyrazolone derivatives, reported by Palva and Mustala (1970) showed a sex ratio similar to that given by Plum, about 1:2 or 3. In allergic reactions, however, in which aminopyrine is second only to penicillin (Patriarca et al., 1973), there was not a great difference in the number of cases in males and females (2195). Aminopyrine demethylase activity in human liver enzyme preparations from trauma victims showed a wide range and no difference between the sexes (Nelson et al., 1971). 3. Acetophenetidin The renal effects of acetophenetidin (phenacetin) described in 1953 (Spuhler and Zollinger, 1953) have been the subject of numerous subsequent papers most of which recognized women to be the predominant victims because they were the more likely to be abusers (Tholen et al., 1956; Gsell et al., 1957a,b, 1961; Moeschlin, 1957; Larsen and Mdller, 1958, 1959; Kasanen and Salmi, 1961; Kasanen et al., 1962; Reynolds, 1963; Dubach et al., 1968; Gault et al., 1968; Royal Australasian College of Physicians, 1969; Lameire et al., 1970; Wilson, 1972; Linton, 1972; Hoffler et al., 1973; Burry et al., 1974; Murray, 1974). The first exception to these observations on female preponderance was the report of Nordenfelt and Ringertz (1961) who found that 20 of 23 cases of interstitial nephritis attributable to acetophenetidin abuse were men and calculated the mortality in men to be 6 times that in women. Subsequent investigations, also in Sweden, suggested association of acetophenetidin abuse and renal tumors (Hultengren et al.,

S E X AS A FACTOR I N DRUG E F F E C T S

197

1965). The pattern of sex distribution followed the pattern of abuse, predominating in males in some areas (Grimlund, 1963; Angervall et al., 1969; Bock and Hogrefe, 1972; Leistenschneider and Ehmann, 1973) and in females in others (Hultengren, 1961; Bengtsson et al., 1968; Taylor, 1972). Animal studies indicate that female rats are much more susceptible to subacute administration of acetophenetidin (Boyd and Hottenroth, 1968). Although the LD,, values may be the same for males and females, during the period when rats weigh between 250 and 400 gm the females are more sensitive (Boyd and Krijnen, 1969). Pregnancy greatly decreases sensitivity of rats to acetophenetidin, but lactation restores it (Boyd, 1970).

4. Antipyrine Although Vesell and Page (1968b, 1969) reported no difference between the sexes in the plasma half-life of antipyrine, another group (O’Malley et al., 1971) found the mean half-life to be 30% greater in males. The apparent volume of distribution of antipyrine was found to be significantly lower in females, in agreement with Soberman et al.

(1949).

5. Ergot and Related Compounds In rats, ergot is significantly more toxic to males, particularly during the growing period. By contrast, neurofibromas of the ears, which were associated with long-term ergot administration, predominated almost 2: 1 in female rats (Fitzhugh et al., 1944). Dihydroergotamine (D.H.E.-45) when used in the treatment of “typical” migraine, gives “excellent” results in about the same percentages in men and women but “good” results in a considerably greater percentage of women. In atypical migraine, the efficacy was better in males (Horton et aZ., 1945). In the treatment of pain in herpes zoster, D.H.E.-45 produced excellent or satisfactory results in males more frequently than in females, and there were more failures in males (Combes et a l . , 1950). Methysergide has been implicated in retroperitoneal fibrosis which occurs in the 40-60 year age group with a male predominance of about 2 : l (Wagenknecht and Auvert, 1971; Schrub et al., 1973).

F R A N S C. GOBLE

198

F. ANTICONVULSANTS 1. Animal Studies In 1943, Spiegel reported that the intraperitoneal injection of deoxycorticosterone acetate into rats diminished seizures induced by electric stimulation. The dosage required to reduce the convulsive reactivity in males was about 6 times that needed for females. Blackham and Spencer (1970) studied the effects of estrogen and progestin on the anticonvulsant activity of diphenylhydantoin, phenobarbital, diazepam, and chlordiazepoxide in female mice subjected to electroshock. They found progestin reduced the intensity and duration of action with all of the drugs tested, whereas estrogen had the reverse effect, increasing the activity. Estrogen had the same type of action as SKF-525A which interferes with liver enzyme metabolism.

2. Clinical Observations Beernink and Miller (1973) observed 4 children (2 boys and 2 girls) with syndromes suggesting systemic lupus erythematosus among children receiving anticonvulsants. They also studied 48 asymptomatic children who were receiving hydantoins, trimethadiorie, or ethosuximide, or combinations of these drugs. In this group there were antinuclear antibodies in 11, a significantly greater incidence than in a control group (4 of 87), the incidence in the girls being 8 of 21 compared with 3 of 27 for the boys. The authors noted that this difference between the sexes was similar to that seen in prepuberal children and juvenile rheumatic arthritis.

Ill. Central Nervous System Stimulants A. STRYCHNINE Although no longer considered a useful medicinal agent, strychnine has been receiving considerable attention from pharmacologists as a poison and as an indicator in studies on induction of drug-metabolizing enzymes. Takahashi (1926) observed its greater toxicity in female rats, and this was confirmed by Poe et a l . (1936), Ward and Crabtree (1942), Hazleton and Fortunato (1942), and Kato (1959). Toxic effects are increased equally in both sexes by thiamine deficiency (Poe and Suchy, 1951). The toxicity of strychnine in mice differs between the sexes in two out of eleven strains tested, C3WJax and C3WHe being the only ones to

SEX A S A FACTOR I N DRUG EFFECT’S

199

show a significant difference (Cheymol et a1 ., 1964). When strychnine oxidation is studied with liver microsomes. from mice, significant differences between the sexes are not found with dd, ICR, or C57BL strains, only C3H shows a moderate difference, with results paralleling those found with hexobarbital: females metabolize the drug at a greater rate than males, contrary to the findings in rats (Kato et al., 1970d).

B. CAFFEINE Boughton (1942) found that caffeine stimulated all phases of maze behavior in female rats, whereas the effect in males was very slight or completely lacking. Caffeine-treated males made more errors during both learning and relearning. Peters and Boyd (1967) observed that sublethal signs of toxicity in male rats surviving toxicity tests were greater than in females, although the mortality incidence was not greater in males. Anorexia and loss of body weight was greater in males, and diuresis was less evident. The dosage ratio for men and women that produces the same CNSstimulating effect was observed to be about 2:l (Horst and Willson, 1935). In prisoners receiving a single 400-mg dose of caffeine, the relative incidence of symptoms was greater in females than in males of approximately the same body weights: shakiness, 10% vs. 4%; nausea 25% vs. 12%; nervousness 25% vs. 2% (Green and Krueger, 1963). An interesting difference between the sexes was observed with human lymphocytes in vitro (Timson, 1970). An increase in rate of mitosis occurred when lymphocytes from females were exposed to caffeine, but samples from males almost always had a decrease or no change. The reason for this difference is a subject for speculation; it is, perhaps, related to the subsequent observation that the relative risk of cancer of the lower urinary tract in male coffee drinkers is about twice that in female coffee drinkers when controlled for age, cigarette smoking, and occupation (Cole, 1971; Timson, 1971). C. PICROTOXIN Although sex differences were not discussed as such in the report by Cole (1943) of an investigation in which picrotoxin was used in a study of anticonvulsants, it was apparent from the data therein that male rats had a greater resistance to the drug than did females. This sex difference was also noted by Holck (1949) who found that gonadectomy lowered resistance of rats of either sex. No difference between the sexes was observed in mice, guinea pigs, or hamsters.

200

FRANS C . GOBLE

D. SYMPATHOMIMETIC AMINES 1. Animal Studies Although Chance (1947) reported no difference between the sexes of mice in toxicity of amphetamine sulfate, and Weaver and Kerley (1962) did not regard their difference as “appreciable,” the data of the last authors showed a tendency for nonisolated females to be more resistant to the drug, Brown and Julian (1968) also found females of some strains to be slightly more tolerant than males and pointed out that the complications arising from differences between the sexes in response to handling, aggregation, and isolation, as well as environmental temperatures, are superimposed on strain of mouse and action of drug (Brown, 1%5). Amphetamine inhibited hepatic metabolism of some drugs in rats, apparently more in males than in females (Louis-Ferdinand et al., 1970). Epinephrine is better tolerated by female rats than by males. Oophorectomy reduced the resistance to an equivalent with that in males, but orchiectomy produced no change (Astarabadi and Essex, 1952). In female dogs the pressor response to epinephrine is greater than that in males (Ahlquist et a l . , 1954). Metestrus or oophorectomy reduced the response (Boxill and Brown, 1955). Female rats and guinea pigs are more resistant than males to the induction of cardiac arrythmias by epinephrine, and the resistance of males is increased by implantation of estradiol benzoate (Louwerens and Smelik, 1956). The contractile responses of rat mesenteric arterioles to epinephrine and norepinephrine are greater in females than in males given an equivalent dosage, but comparable sex differences do not appear with dopamine or phenylephrine (Altura, 1972). Isoproterinol greatly stimulates DNA synthesis and mitotic activities in the submaxillary gland of the rat, and the action is more pronounced in females than in males (sevenfold difference). Oophorectomy or testosterone treatment did not alter the effect in females, but orchiectomy increased the effect in males (Barka, 1967). When 6-hydroxydopamine is given intraventricularly to neonatal male rats, there is a 106% increase in pineal hydroxyindole-0-methyl transferase; no increase occurs in females. Orchiectomy abolishes this response, and treatment of females with testosterone causes an increase in the pineal enzyme activity. The 6-hydroxydopamine is thought to act in a selective manner on catecholamine-containing neurons in the brain causing changes in central control of pineal function (Hyyppa et al., 1973). When 6-hydroxydopamine is injected into the veins of rabbits, females tolerate from 2.5 to 5 times as much drug as males before succumbing (Salem, 1970).

SEXASAFACTORINDRUGEFFECTS

201

Spinal vagotomized male cats used for catecholamine assay are more sensitive than females or castrates (Burn et a l . , 1950). Male cats are twice as sensitive to the pressor effects of norepinephrine as females. The pressor response is enhanced by testosterone to a greater extent in female cats (Bhargava et a l . , 1967). When data from angiotensin and norepinephrine studies in cats were graphed, with mean spike frequency plotted against mean blood pressure, the results were in two groups that were sex associated. The difference between the sexes in the response of cervical sympathetic activity to the infusion of norepinephrine in dogs and cats is abolished by orchiectomy or treatment of males with estrogens (Morrison and Pickford, 1969, 1971).

2. CLinical Observations Epinephrine and norepinephrine are used for the treatment of acute hypotension in anesthetized patients and sometimes precipitate severe ventricular disturbances, the latter being the more potent activator. Females are more sensitive to this effect (Johnstone, 1953). 2-Amino-5-phenyl-2-oxazoline (aminorex) has been associated with a pulmonary hypertensive condition which has been consistently predominant in women (Wirz and Arbenz, 1970; Rivier, 1970; Rivier et a l . ,

1972). A few cases of phenmetrazine addiction with acute psychotic syndromes have been reported, all in females in a service which also treated males (Pilasiewicz and Tolwinski, 1972).

E. ANTIPARKINSON DRUGS Amantadine, which is used to treat not only ideopathic Parkinson’s disease but also the “extrapyramidal side effects” of psychotropic drugs, mainly phenothiazines, produces a difference between the sexes of rats in acute oral toxicity: males tolerate higher doses than do females (Vernier et a l . , 1969). Choreoathetoid dyskinesia is a side effect of L-dopa treatment of parkinsonism which predominates in men, the ratio between males and females being from 2:l (Markham, 1971) to 3:l (Voller et a l . , 1972). The livedo reticularis, which was reported to result from amantadine treatment of women (Shealy et a l . , 1970), occurs with equal frequency in untreated men and women, and among those receiving amantadine it occurred in 21 of 21 women and 15 of 19 men (Vollum et a l . , 1971). F. METHYLPHENIDATE In contrast to observations with amphetamine, there were no significant differences between the sexes in relation to exploratory behavior in

202

F R A N S C . GOBLE

rats given methylphenidate (Hughes, 1972). There was, however, a “near”-significant difference between the sexes in relation to rearing: an increase occurred in males given methylphenidate, but in females, there was a decrease with a lower dose and a slight increase at a higher dose.

IV. Psychotropic Agents A. PHENOTHIAZINES

1. Animal Studies The hypothermic effect of chlorpromazine was greater for males than for females in ground squirrels (Citellus tridecemlineatus), hamsters, and rats (Hoffman and Zarrow, 1958). Female rats were more responsive than males to the locomotor depressant actions of perphenazine or chlorpromazine but are more resistant than males to the avoidancesuppressant action of the same drugs (Irwin, 1960, 1964; Irwin et al.,

1958). Chlorpromazine was studied as an enzyme-inducing agent in rats by Feuer et a l . (1971), who found that it produced significant liver enlargement in male rats only and that it affected the hexobarbital oxidase level in male rats only, although it was able to induce a significant increase in coumarin-3-hydroxylase in both sexes. 2. Clinical Observations Toxic reactions to chlorpromazine are generally about twice as prevalent in women as in men and include gastrointestinal, CNS, blood, and dermal effects (Lomas et al., 1955). Long-term adverse reactions to phenothiazines have recently been summarized (Lehmann and Ban, 1974). Specific observations follow. Dryness of the mouth is more common in men than in women receiving mepazine (Werenberg, 1955). Central nervous system (extrapyramidal) side effects of neuroleptic medications are primarily akathisia, parkinsonism, and dyskinesia, of which parkinsonism is about twice as prevalent in women a s in men (Freyhan, 1958; Gratton, 1960; Ayd, 1961; Everett, 1967), whereas about twice as many men have dyskinesia as do women (Ayd, 1961). A facialoral dystonic syndrome, however, is more prevalent in women (Hunter et al., 1964a,b; Morphew and Barber, 1965; Evans, 1965; Schmidt and Jarcho, 1966; Gralewski, 1973). A condition called “tardive dyskinesia” or “terminal insufficiency syndrome” is usually reported to be more prevalent in females (Degkwitz et al., 1966; Duvoisin, 1967; Crane,

SEX A S A FACTOR I N DRUG EFFECTS

203

1968; Heinrich et al., 1968; Mehta and Itil, 1973; Schiele et al., 1973), although some do not find this impressive (Crane and Paulson, 1967; Fann et al., 1972; Crane, 1973). With chlorpromazine medication, agranulocytosis is more prevalent in women (Hollister, 1958;Pisciotta, 1969), various reports ranging from all females (reviewed by Schick and Virks, 1956) to a few males (Pollack, 1956), to ratios of 7.3 (Pisciotta, 1967) and 3:l (Pisciotta et al., 1958; Mandel and Gross, 1968). With mepazine the ratio is 6:l (Fiore and Noonan, 1959). Skin melanosis (Robins, 1972), sometimes associated with corneal and lens opacities (Greiner and Berry, 1964), during prolonged chlorpromazine therapy is also more frequent in females. Glaucoma has also been reported as an ocular change with chlorpromazine therapy found in women but not men (Edler, 1966) and increased pigment in the iris occurs in about one-half of treated women and one-third of men. Retinopathy associated with thioridazine is more frequent in females but may be associated with a weight difference so that women receive a greater dosage than men (Kjaer, 1968). There are differences of opinion on the efficacy of chlorpromazine in the two sexes. Hyvert (1956) observed much greater frequency of ameliorations in chronic psychoses in females, which inspired him to give synthetic estrogens to males in order to enhance the effect of the drug. On the other hand, Charlin (1956)reported better results in males with chlorpromazine. Dosage for males is less than for females (Taylor and Levine, 1971). In the treatment of Huntington’s chorea with fluphenazine, a higher dose was required for females than for males, and benefit from treatment is greater in males (Whittier and Korenyi, 1968). In treating chronic schizophrenics with the same drug, a higher dose was also needed in women (Keskiner, 1973), but when adequate regimens were established there was no difference between the sexes in the resulting improvement (Christodoulidis, 1974).

B. DIBENZAZEPINES After intraperitoneal injection of imipramine into rats, the brain concentration of desmethylimipramine at 1 hour was much greater than in females, but at 4 hours there was little difference (Pscheidt, 1962). Men treated for primary depression respond much more rapidly to imipramine than do women. The salutory effect of imipramine can be greatly enhanced in women by the concurrent administration of triiodothyronine, but there is no potentiation in men (Prange, 1972).

2 04

F R A N S C. GOBLE

Estrogen worsens the antidepressant response of women to imipramine (Editorial, 1972a). When the possibility that testosterone might accelerate the antidepressant effect of imipramine in men was explored, it was found that men treated with the combination develop a paranoid response necessitating discontinuance of the hormone (Wilson et al., 1974). Ketoimipramine, in a trial involving a small number of patients gave a satisfactory therapeutic response in 5 of 7 males and in 1 of 12 females (Kline and Winick, 1971).

C. THIOXANTHENES When used to neutralize the extrapyramidal effect of phenothiazines, methexine effected complete remission in 13 of 24 males, and partial remission in 11 others, whereas in females the corresponding numbers were 5 of 17 and 12 (Maller and Heller, 1971). D. RAUWOLFIA COMPOUNDS The gastric ulcers that develop in 7-week-old rats as a result of reserpine administration are more severe in males than in females. In immature (3-week-old) rats, the difference between the sexes was not apparent, and at 20 weeks it was not as pronounced as it was at 7 weeks (Reilly et al., 1969). Although the differential response of the sexes to reserpine was not specifically pointed out in reports of some of the early clinical trials, its recognition was apparent in remarks such as “initial use on the female admission service was sufficiently encouraging to discontinue entirely the use of electroconvulsive therapy” and “the male admission service will continue to use electroshock” (Kline, 1956). Later, it was clearly stated that results of treatment were much more favorable with females than with males (Maggs and Ellison, 1960; Luby, 1967). E. DIOLS Rats showed a slight difference between the sexes in susceptibility to meprobamate in their first day of life, and at 21 days the greater susceptibility of females was most marked; at 63 and 100 days the difference was insignificant (Weinberg et al., 1966). The effects of meprobamate on various tests of performance in men and women are devious if not contradictory. In performance in a college test, examination scores were improved more in women than in men, and anxiety was also reduced more in women (Marquis et al., 1956). In other tests involving sensomotor concentration and emotional reactions,

S E X A S A FACTOR I N D R U G EFFECTS

205

performance was lowered in women as compared with that in men. Under the influence of meprobamate combined with alcohol, women had even greater impairment of sensomotor reaction but showed a compensation for emotional disturbance, whereas men compensated for the impairment of sensomotor reactions and showed an increase in emotional disturbances (Munkelt et al., 1962).

F. LITHIUM The toxic effects of lithium carbonate in rats is more pronounced in males: the blood nonprotein nitrogen is more markedly increased in males than it is in females (Andreoli, 1968). The urinary output and the elimination of sodium and potassium are also greater in males, as is a loss of stomach muscle tone (Andreoli et al., 1968). A difference between the sexes in the development of hypothydroidism associated with lithium treatment for psychoses has been noted (Villeneuve et al., 1973; Lloyd et al., 1973). A difference in serum protein-bound iodine was found by Fyro et al. (1973) which had not been observed by Villeneuve et al. Both groups suggest that lithium triggers latent thyroid hypofunction and point to the predominance of hypothyroidism in women.

G. DIAZEPINES When diazepam and chlordiazepoxide were tested as anticonvulsants in female mice subjected to electroshock, their efficacy was enhanced by pretreatment with estrogen and decreased by pretreatment with progestin (Blackham and Spencer, 1970). Chlordiazepoxide reduced exploratory behavior in males but had virtually no effect on this activity in females (Hughes and Syme, 1972). N-Demethyldiazepam (NDD) in the diet greatly reduced the survival of male mice kept in groups in which their aggression becomes destructive. Grouped females were not similarly affected. The difference in toxicity and aggressiveness was not associated with brain levels of NDD nor its major metabolite, oxazepam (Guaitani et al., 1971). Clothiapine in the guinea pig produced a reduction in the weights of the thyroid glands of females given intermediate or higher dietary levels, but there were no histopathological changes observed (Schultz et al.,

1969). Diazepam decreased sociability in women, increasing depression and withdrawal, whereas it increased euphoria in men (Jaattela et al., 1971). When used in the elderly, it has been noted to be antidepressant for men but not for women (Goldman, 1974).

2 06

F R A N S C. GOBLE

H. BUTYROPHENONES Trifluperiodol seems to have a differential effect on the sexes according to Serafetinides et al. (1970) who found improvements in chronic psychotic men to be disappointing and considerably less than anticipated from a previous study in schizophrenic women (Clark et a l . , 1968). With haloperidol, male patients took longer to control than females, and the number not controlled after 96 hours was 8% in males and 4% in females (Oldham and Bott, 1971). That females responded more rapidly was also observed by Sangiovanni et al. (1973) who found the uncontrolled patients at 72 hours to be 17% of males and 5% of females. I. CANNABINOIDS The 7-hydroxylation of A'-tetrahydrocannabinol (THC) by liver microsomes of male rats is more pronounced than by those of females (Burstein and Kupfer, 1971). Performance levels in behavioral studies show female rats to be more susceptible to intoxication by marijuana extract distillates (Cohn et al., 1971). Compounds As-THC, A'-THC, and cannabis extract were all significantly more toxic per 0s to female rats (Thompson et al., 1973a,b). Intravenously and by inhalation, there is no difference between the sexes in rats (Rosenkrantz et al., 1974), but intraperitoneally in mice, males are more sensitive to As-THC (Mantilla-Plata and Harbison, 1974). Toward the end of 5-day inhalation tests, some female rats vocalized for varying lengths of time and sporadically jumped spontaneously; this was not reported for males (Rosenkrantz and Braude, 1974). In 23-day inhalation tests, only females were involved in fighting when given high doses. There are differences between the sexes in the response of certain brain enzymes during administration of A9-THC for 28 days. Brains of female rats show a greater decrease in acetylcholinesterase than do those of males. On the other hand, with a moderate dose of crude marijuana extract given to 91 days, the decrease in males is greater than in females, whereas succinic dehydrogenase increases in both sexes but more in females (Luthra and Rosenkrantz, 1974). In man there are no discernible differences between the sexes with respect to the effect of marijuana on mental processes (Klonoff et al., 1973).

SEXASAFACTORINDRUGEFFECTS

207

J. CHLORZOXAZONE Chlorzoxazone has a greater depressant effect.on female mice than on males. The sleeping time in males given this drug is considerably increased by treatment with diethylstilbestrol, but testosterone does not so strikingly change the effect in females (Schrogie, 1973).

K. COMBINATIONS In a study in healthy volunteers with three drug combinations, clidinium-chlordiazepoxide, methscopalamine-phenobarbital, and propantheline-phenobarbital, females had a greater tendency than males to be constipated and to have higher heart rates after treatment. Males had a greater tendency to have difficulty in initiating micturation (Taylor, 1970). In psychiatric patients multiple therapy resulted in significant difference between the sexes in therapeutic response, being more often less effective in men than in women (Merlis et al., 1970).

V. Local Anesthetics Cocaine, when given intraperitoneally to rats at equivalent doses, was more toxic to males than to females. The number of doses that could be given before convulsions occurred was fewer in males as was the total number of tolerated doses and the number of days to death (Downs and Eddy, 1932). This difference between the sexes was also observed when the drug was given by continuous intraperitoneal infusion (Guerrero et al., 1965). The difference did not occur in young animals or in gonadectomized rats of either sex. Testosterone restored the susceptibility to orchiectomized animals but not to oophorectomized ones. Estradiol did not change the susceptibility of gonadectomized rats of either sex. The NADPH/dependent liver enzyme systems responsible for N-demethylation were more active in males (Kato and Gillette, 1965b). Procaine, contrastingly, was more toxic to female rats (Muiioz et al., 1961). Gonadectomy decreased the resistance of males but did not change the sensitivity of females. Chronic treatment with carbon tetrachloride did not change the sensitivity of the females but did decrease the resistance of males to the level of that in the females. Rats treated with SKF-525A prior to the administration of procaine showed no difference between the sexes (Paeile et al., 1964). Only one of eleven strains of mice tested showed a difference between the sexes in LDSoof procaine (Cheymol et al., 1964).

208

F R A N S C . GOBLE

Lidocaine and dibucaine manifested a difference between the sexes of rats qualitatively similar to that with procaine. Cocaine was, therefore, exceptional and the difference between the sexes, presumably based on liver metabolism, was unrelated to anesthetic activity (Paeile et al.,

1965). When the local anesthetics are used clinically for spinal anesthesia, postural headache is one of the most common sequelae, occurring in 9% of males and 15% of females (Dripps and Vandam, 1954).

VI. Myotropics

A. PAPAVERINE The response of frogs to papaverine is influenced by both sex and season. In the winter, male frogs receiving papaverine developed an early excitation followed by convulsions before they eventually weakened and succumbed to the depressing effects of the drug, whereas females became flaccid immediately. In the summer, the males as well as the females went directly into the weakened state without any violent reflexes (Takase, 1932). Orchiectomy reduced the excitatory convulsive reaction of frogs in winter in a few days, and androgenic substances restored the physical male reaction (Takase and Suzuki, 1939).

B. CURARE Male rats were more resistant to the effects of curare than were females (Gallagher and Koch, 1962). Rats of either sex at 1 month of age and 3-month-old males had about the same sensitivity to d-tubocurarine, but 3-month-old females were significantly more sensitive than the other groups as evidenced by the righting reflex. Gonadectomy at the age of 1 week did not abolish the sex difference in curare sensitivity in animals tested at 3 months (Wolf et al., 1964).

C. GLYCERYL GUAIACOLATE A pronounced difference between the sexes in the duration of action of glyceryl guaiacolate was found in ponies, the males requiring a longer time to recover. This difference was associated with differences in the rate of disappearance of the drug from the plasma. There was, however, no difference in dosage or time of onset of action, and the sex difference in duration was reported to cause no clinical complications (Davis and Wolff, 1970).

S E X A S A FACTOR I N DRUG EFFECTS

209

In rats, on the other hand, the duration is greater in females. Male rats demethylate the drug 3 times more rapidly than females, and kinetic data on 0-demethylase from liver microsomes suggest a quantitative rather than qualitative difference between the enzyme of male and female rats (Gin, 1973).

VII. Cardiac Glycosides A. ANIMALSTUDIES Pregnant rats may be as much as 60% more susceptible to digitalis than nonpregnant rats (Boucek, 1936), and female guinea pigs, primarily because of their chances of pregnancy, are likely to demonstrate irregularities in response to cardiac glycosides that should preclude their use in assays of these materials (Reiser, 1940). Results for lethal doses of lanatoside C in male and female guinea pigs showed pregnant females to have somewhat greater standard deviations than the nonpregnant females or the males, but not out of line if allowance was made for the weight of the fetuses (Dybing and Dybing, 1947). Female rats were more resistant to ouabain than were males (Holck and Smith, 1938), and this difference appeared when the rats were between 1 and 2 months of age (Holck and Kimura, 1944). Oophorectomy reduced the resistance of the females to that of the males, whereas castration of males had no effect on their tolerance. Estrogen given to females did not change their resistance, and the difference between the sexes persisted after unilateral adrenalectomy. Resistance to ouabain was greater in young rabbits, but Chen and Robbins (1944a) did not look at the results from the sex viewpoint. Their data, however, suggested that, although there was no difference between the sexes at 6 weeks of age, males were a little more sensitive at 3 months, but the numbers of animals representing each age group were small. In a study in which data on 421 different preparations were pooled, female guinea pigs were less tolerant than males to cardiac glycosides (Goldberg, 1949). The greatest difference between the sexes of rats in toxicity occurred in 2- to 4-month-old animals with a decline from 8 to 11 months. Gonadectomy in either sex did not significantly alter the response to ouabain. When ouabain was given to rats anesthetized with urethane, there was no difference between the sexes in response of intact animals, whereas with pentobarbital anesthesia the higher resistance of females was apparent (Holck and Kimura, 1951). The difference between the

210

F R A N S C . GOBLE

sexes was not based on the difference in heart weight, and the authors suggested that the female heart itself is more resistant to ouabain than that of the male. This hypothesis is encouraged by a study in which the isolated heart from an adult female rat, under closed perfusion in a Langendorff preparation, required 50% more ouabain to be arrested than that needed to stop an isolated male heart. This difference did not occur with hearts of 3-week-old rats (Wollenberger and Karsh, 1951). In dogs, estrogenic hormones (estrone, estradiol, and diethylstilbestrol) protected against the toxic effects of digoxin on the myocardium, intact males and oophorectomized females being highly sensitive. Females in estrus were more highly resistant than those in anestrus (Grinnell and Smith, 1957). Oophorectomized females receiving 1,3,5 (lo), 16-oestratetraen-3-01 methyl ether, a nonestrogenic steroid, were also protected against the arrhythmia induced by digoxin (Grinnell et al., 1961). In intact rabbits the difference in digitoxin toxicity between the sexes was also significant (Rodensky and Wasserman, 1964b), females being more tolerant. In rats, however, the relative sensitivity of the two sexes to digitoxin was reversed according to Scott et al. (1971), females being more sensitive, whereas males were more sensitive to another glycoside, proscillaridin (3-rhamnosido-14-hydroxy-4,20,22-bufatrienolide).On the other hand, Talcott and Stohs (1973) found female rats to be slightly more resistant to digitoxin than were males, and this difference was much more pronounced after pretreatment with phenobarbital.

B. CLINICALOBSERVATIONS In men with rheumatic heart disease, about 21% experienced digitalis-induced arrhythmias in contrast to about 10% in women (Rodensky and Wasserman, 19Ma), but in 70 cases of acute digitalis self-poisoning, with 20% mortality, no noteworthy correlation of sex with patient was established (Gaultier et al., 1968). Another study was also unable to correlate sex with disposition to digitoxin poisoning although the number of toxic cases was small (Smith, 1970). Mean serum estrogen values are increased in both sexes during digoxin treatment but the increase is significantly greater in men. The predominate estrogen is estrone. Serum LH values are decreased in both men and postmenopausal women receiving digoxin, and plasma testosterone is reduced in treated men. These decreases may account for some of the side effects observed with digitalis therapy, such as gynecomastia in elderly men, cornification of the vaginal epithelium, and decreased urinary gonadotropin excretion (Stoffer et al., 1973).

S E X A S A FACTOR I N DRUG E F F E C T S

211

There were differences between the sexes in response to strophanthin injections (Michel and Hartleb, 1958). Regardless of the degree of compensation of the heart, there was a real difference in diastolic blood pressure (which decreased in women but increased in men), in the pulse frequency (more prolonged in women), the pressure (generally decreased in women, increased in men), and the relative cardiac output (strongly increased in women).

VIII. Anti-inflammatory Compounds The incidence of hypersensitivity to phenylcinchoninic acid (cinchophen) was higher in women than in men and the expectation of recovery was also greater in men (Bryce, 1938). It has been suggested that there is a slightly lower rate of phenylbutazone metabolism in men than in women (Vesell and Page, 1968a; O’Malley et al., 1971), although in a large group of normal subjects no difference between the sexes was found (Whittaker and Price Evans, 1970). Some nonsteroidal anti-inflammatory agents induce multiple intestinal ulcers in rats when administered intravenously. With phenylacetic acid derivatives, mature males were more likely to have ulcers and to die than were females. There was no difference between the sexes in immature rats. Orchiectomy reduced the susceptibility of males, but this could be reversed by testosterone (Walter et al., 1970). When given per 0s to one strain of rats, l-p-chlorobenzylidine-5methoxy-2-methyl-3-indeneacetic acid produced a dose-related lesion in the renal papilla leading to eventual necrosis, hemorrhage, and hematuria. This condition was frequent in males, rare in females, and did not occur in another strain of rat (Bokelman et al., 1971). Bumadizone calcium preferentially affects female rats, producing stomach ulcers, anemia, and kidney lesions more commonly in them than in males (Konig et a l . , 1973).

IX. Antihistamines Pyribenzamine in the rat was better tolerated by males than by females when given by either the oral or the subcutaneous route, and oophorectomy made females as tolerant as males (Mayer et al., 1946). There was a clear difference between the sexes in the N-demethylation of diphenhydramine in the rat: males of at least six strains had more active liver microsomes than did females (Kato et al., 1970d). In man, diphenhydramine, when used for its sedative effect, produces

212

F R A N S C. GOBLE

euphoria in men but dysphoria in women; the drug also causes a smaller decrease in the pulse rate of men than does a placebo, whereas in women it effects a decrease in pulse rate greater than that of placebo (Jaattela et al., 1971).

X. Hypoglycemic Agents When rats fasted for 2 days were given glucose or a combination of glucose and tolbutamide, the blood sugar levels of the females were higher than those of the males in both tolbutamide-treated and in untreated groups. After 2 days of tolbutamide treatment, the difference in blood sugar between males and females in response to the drug was quite marked (Janes et al., 1959). Unabsorbed glucose was higher in males, and the authors considered that the considerable variations they encountered in glucose and glucogen levels in the experiments were attributable to differences in absorption rates of glucose in males and females as well as to a difference in response between the sexes. The toxicities of four synthetic hypoglycemic agents-tolbutamide, terbuzole, isobuzole, and FWA-106 (2-p-methylbenzenesulfonamido-5-isobutyl1,3,LE-thiadiazoletwere found to be equivalent in the two sexes of rats (McColl and Sacra, 1962). Treatment of female rats with stilbestrol, however, made them strikingly more susceptible to all four synthetic compounds as well as to insulin, whereas treatment with testosterone made females more resistant to the synthetics but not to insulin. Gorman and Weaver (1959) found that the proportion of initial failures with sulfonylurea treatment (chiefly tolbutamide but some carbutamine) was 30% in males and 15% in females. They believed this might be expected in view of the studies on pancreas obtained at autopsy by Wrenshall and Hamilton (1957) who found that at equivalent ages and duration of diabetes, specimens from males had a lower content of insulin. Later clinical studies have been controversial particularly in reference to excess cardiovascular deaths in tolbutamide-treated patients, in which the risk was reported by the University Group Diabetes Program (1970) to be mainly for women. This finding was at variance with that of Keen and Jarett (1970) and Paasikivi (1970) as pointed out promptly by the British Medical Journal (Editorial, 1970) and later by Marble (1972), who analyzed data of Balodimos et al. (1971) according to sex and found a greater percentage of death from coronary heart disease among sulfonylurea-treated males.

SEXASAFACTORINDRUGEFFECTS

2 13

XI. Anticoagulants A. ANIMALSTUDIES In laboratory rats the oral toxicity of 3-(acetonylbenzyl)-4-hydroxycoumarin (warfarin) was from 3 to 5 times greater in females than in males (Hagan and Radomski, 1953; Pyorala, 1968). Female rats had higher clotting factor levels (Factor VII, Factor X, and prothrombin) than did males (Pyorala, 1965). In wild Norway rats, on the other hand, females are a little more resistant than males, taking a significantly longer time to die. At a given dose level in subacute testing, the females have better survival rates than males on each day, although 6-day feeding generally brings the mortality to 98% in each of the sexes (Brooks and Bowerman,

1973). Fluindarol in rats caused death sooner and more commonly in females than males (van Eeken et al., 1970).

B. CLINICALOBSERVATIONS In a study which involved phenylindanedione or dicoumarol, or both, “intensive” anticoagulant therapy in patients without myocardial infarction significantly reduced mortality and incidence of infarction compared with “moderate” treatment; the effect was more pronounced in men than in women (Borchgrevink, 1960). Complications of warfarin sodium therapy occurred more commonly in women than in men (Cluff et al., 1964). Adverse reactions to heparin occurred with about twice the frequency in women as in men, although efficacy was not related to sex (Kernohan and Todd, 1966; Jick et al., 1968a,b). On the other hand, paralytic ileus as a result of overdosage with anticoagulants was found more frequently in males (Brummelkamp,

1965). Bleeding in the CNS as a complication of anticoagulant therapy has been reported more frequently for males than females, but it seems likely that the sex ratio may be the same as that of the population receiving anticoagulants (LCvy and Stula, 1971; LCvy, 1971). With anticoagulant therapy after acute myocardial infarction, recurrence was reduced in treated women but not in treated men (Drapkin and Merskey, 1972).

2 14

F R A N S C. GOBLE

XII. Diuretics A. ANIMALSTUDIES Published observations on differences between the sexes in response to diuretics have been few. A sex-specific saluretic, N, N-diisopropy1-N’n-butyl-N’-diethylaminoethyl urea (P-275), exerted its effect only in male rats and dogs. Treatment of intact females with testosterone, however, did not change their lack of response (Gardier and Rohrer, 1968). Treatment of a male dog with estrogen made it refractory to the drug, and oophorectomy of a female established sensitivity to the diuretic. The biliary excretion of chlorothiazide by female rats was less than that by males. Pretreatment with phenobarbital almost doubled the biliary excretion of the drug by females, whereas pretreated males excreted less than controls (Hart et al., 1969). Another diuretic, acetazolamide, showed a preferential teratogenic effect for female embryos when given to pregnant rats, causing a distal postaxial deficiency of the right forelimb. The incidence in females exceeded that in males of from 6% to 48% in five different regimens (Scott et al., 1972). This compound is a carbonic anhydrase inhibitor, and the isoenzymes from male rat liver are 10,000 times less sensitive to inhibition than the isoenzymes from the female liver. Testosterone changes the sensitivity of enzymes from female livers to a pattern resembling that in male livers (Garg, 1974).

B. CLINICALOBSERVATIONS The total incidence of reported side effects of hydrofluomethiazide in patients in a clinical trial was 21% in females as opposed to 12% in males; the incidence of drowsiness was 11% in females, and in males (Green and Krueger, 1963).

XIII. Laxatives There was a small but significant difference between the sexes in the biliary secretion of phenolphthalein glucuronide in the rabbit, being higher in females than in males (Smith, 1970). The threshold laxative doses of both white and yellow phenolphthalein in man were greater in males than in females (Munch and Calesnick, 1960). Laxatives are reported to be a much more important and aggravating factor in irritable colon in females than in males (Loizeau, 1969). Oxyphenisatin has been associated with liver damage in a number

S E X A S A FACTOR I N D R U G EFFECTS

215

of countries and, of 72 suspicious cases recently reviewed, only 3 were men (Dietrichson et a l . , 1973).

XIV. Antitussive Compounds When cough is experimentally induced in guinea pigs by exposure to citric acid aerosols, or in dogs by the magnetic activation of an intratracheally implanted iron slug, the number of coughs per minute is greater in males than in females. Although certain antitussives (codeine and dextromethorphan) affected the cough response equally in both sexes of guinea pigs, others produced differences in responses in males and females. Pseudoephedrine and methaqualone were more active in females, and chlorpheniramine was more active in males. In dogs, dextromethorphan was more active in females, whereas pseudoephedrine and chlorpheniramine were more active in males (Salem and Aviado, 1974).

XV. Anti-infective Compounds A. ANTIBACTERIALCOMPOUNDS 1. Antibiotics a. Penicillins. When monobasic penicillins (dicloxacillin, nafcillin, and penicillin G) were administered per 0s to dogs, females had consistently higher and more prolonged blood serum levels of the antibiotics than did males. By contrast, amphoteric penicillins (ampicillin and cyclocillin) did not show a similar sex difference (Poole, 1970). A difference in acidity of the gastrointestinal tract or a difference in gut wall metabolism of the monobasic penicillins in males and females has been suggested as an explanation of the findings. Cyclacillin, in chronic and subchronic dietary studies in rats, produces kidney toxicity in males but not in females. Cyclacillin and one of its metabolites (1-aminocyclohexane carboxylic acid) are not as readily excreted by males as by females. In the dog, the monkey, and man, however, no difference between the sexes in the disposition of cyclacillin is observed (Tucker et a l . , 1971, 1974; Janssen et a l . , 1974). Men have allergic reactions to penicillin more often than women (Smith et a l . , 1966a). In some cases, skin tests for penicillin allergy may themselves cause serious adverse reactions. Of 9 such patients in one study, 8 were males (Baer et a l . , 1970).

216

F R A N S C . GOBLE

The risk of drug rash or exanthema with ampicillin is greater than with other penicillins, and this side effect is consistently more common in females than in males (Shapiro et al., 1969; Kriinig and Dennig, 1970; Boston Collaborative Drug Surveillance Program, Boston University Medical Center, 1973; Collaborative Study Group, 1973; Wemmer, 1973). The blood levels of ampicillin in women during menstruation are strikingly lower than in nonmenstruating women (Tegeris and Panteleakis, 1973). When allopurinol is given concurrently with ampicillin, there seems to be an increased risk of rash in both sexes, the occurrence being about 2.5 times more common in males and 3.5 times in females (Boston Collaborative Drug Surveillance Program, Boston University Medical Center, 1972). b. Chloramphenicol. In mice infected intraperitoneally with Streptococcus agalactiae, there was no difference between the sexes in the course of the disease that kills untreated animals in 24 to 48 hours. Treatment with chloramphenicol reduced mortality and increased survival in both sexes, but the effects were greater in females than in males at all dose levels (Wheater and Hurst, 1961). The hematopoietic disturbances attributed to chloramphenicol (aplastic anemia, agranulocytosis, and thrombocytopenia) are usually reported to be more common in females (Lewis et al., 1952), both children (Welch et a l . , 1954; Yunis and Bloomberg, 1964; Holzel, 1965) and adults (van der Hem et a l . , 1961; Kahler, 1962; Meyler, 1962; Best, 1967; Wallerstein et a l . , 1969; Pisciotta, 1971), but some have raised doubts about this (Sharp, 1963, 1965; Polak et a l . , 1972; Gadner et a l . ,

1973). When chloramphenicol is given at 500 mg in a single dose and blood samples are taken at 1 and 2 hours thereafter, the mean blood levels are 5.76 and 6.27 pglml for women and 3.78 and 4.18 pg/ml for men, the values for the males being one-third less than females at both time intervals. The difference is highly significant and cannot be explained by difference in body weight (Scotti, 1973). c . Colistin. Neurological reactions to colistin occur at a significantly higher rate in women than in men (Koch-Weser et a l . , 1970). The majority of earlier published cases of colistin neurotoxicity occurred in females, and in the cited study the rate was 14 of 127 women and 9 of 190 men. d . Rifampicin. With equal doses of rifampicin administered to 13 males and 21 females, mean blood levels of the drugs after 2 and 4 hours are significantly lower in men (about one-third less). The values

S E X A S A FACTOR I N DRUG E F F E C T S

217

are 6.12 and 4.68 puglml for the two intervals in women, 3.93 and 3.10 p d m l (Scotti, 1973). e. Streptomycin. Male mice were very slightly, but not significantly, more sensitive to intravenous streptomycin than were females (Ott, 1947). When streptomycin was given to mice twice daily for 3 days beginning immediately after intraperitoneal infection with streptococci, there was a difference in survival dependent on sex, at two different dose levels: 2 of 12 females died at the higher level as compared with 5 males, and 3 of 12 females succumbed at the lower level compared with 12 of 12 males (Hurst, 1958). f. Tetracyclines. Male rats fed chlortetracycline (Aureomycin) for 2 years had an abnormal amount of stainable fat in their livers, whereas females given the same regimen had no increase over the normal small amount of sudanophilia (Dessau and Sullivan, 1961). Oophorectomized rats also had an increase in stainable fat, but not as great as that in orchiectomized males or in oophorectomized females treated with testosterone propionate. Orchiectomized animals given estradiol along with the antibiotic were protected against the excessive accumulation of fat in the liver (Sparano, 1965). In mice given chlortetracycline intraperitoneally, there were dramatic increases in the intestinal weights (particularly small intestine) of both conventional and germfree females, but no such change occcurred in males. The same regimen caused a decrease in intestinal wall histamine in both sexes and a decrease in serotonin concentration in germfree mice of either sex and conventional males, but conventional females had an increase in serotonin, suggesting that gastrointestinal upsets may be caused not only by changes in the intestinal flow but also by alterations in autocoid levels (Rosen et a l . , 1967). The hepatotoxic effect of oxytetracycline (Terramycin) given to rats in high doses intramuscularly was more marked in females than in males. Gonadectomy has no effect on the toxicity in either sex. Nandrolone phenpropionate was protective in both sexes (Bielawski and Michalik, 1969). In humans, tetracycline hepatotoxicity is also more prevalent in females, and the males reported are usually prepuberal, senile, or castrate (Robinson and Rywlin, 1970). Tetracycline given as a single dose of 370 mg, after which blood samples are taken at 2 and 6 hours, produces mean blood levels in women of 2.79 and 2.23 pg/ml in contrast to levels in males of 2.26 and 1.75 pg/ml, the difference being highly significant (Scotti, 1973). g. Treatment of Gonorrhea. Perhaps the most striking example of difference between the sexes in efficacy of antimicrobial agents is in the

218

F R A N S C. GOBLE

treatment of gonorrhea. The difference in the pathogenesis of the disease in women and men has been summarized and emphasized by Shapiro and Lentz (1963). Women have proved to be less effectively treated than men with a number of antibiotics (Wren, 1967; Shapiro and Lentz, 1967; Benenson, 1970; Willcox, 1970; Sim, 1972). The effective dose of cephaloridine in men is 2 gm in a single intramuscular injection; in women 2 gm, repeated in 1 or 2 days, is more effective than a single dose of 2 or 3 gm (Fletcher et al., 1969). The number of failures in the treatment of gonococcal blennorrhagia with thiamphenicol, spiramycin, or erythromycin stearate are slightly greater in females (Siboulet et a l . , 1970). The U.S. Public Health Service recommends for women twice the dose of procaine penicillin as that advised for men (Schroeter and Pazin, 1970; Caldwell et al., 1971). Addition of probenicid reduces the failure rates in both sexes, but a differential remains between males and females. Failure rates with spectinomycin and methacycline are also greater in females receiving respective equivalent doses to those given males (Holmes et al., 1971, 1973; Wiesner et al., 1973). When treating women, doubling the dose of spectinomycin recommended for men, although questioned by some (Cornelius and Domescik, 1970), lowers the failure rate (Platts, 1970; Pedersen, 1972; Pedersen et al., 1972; Smithurst, 1972; Sinanian et a l . , 1973). Treatment failures with pivampicillin hydrochloride are more frequent in women (Forstrom and Lassus, 1972). Some drugs do not show such great differences in failure rates between the sexes; ampicillin (Bro-Jorgensen and Jensen, 1971; Shapiro et al., 1971), minocycline (Duncan et al., 1971) cephalexin-probenecid (Landes et al., 1972), sulfamethoxazole-trimethoprim (Carroll and Nicol, 1970; Schofield et a l . , 1971). Amoxicillin at lower doses (0.5 gm ~3 or 1 gm x 1) has fewer failures in females than in males, whereas at 3 gm ( X 1) the cure rates were the same in both sexes (Wise and Neu, 1974). 2. Synthetics a. Arsenicals. Not only in the vanguard of chemotherapeutic compounds but also among the first of the medicinal agents to indicate a difference between the sexes in toxicity was neoarsphenamine (Neosalvarsan) in which the mortality in adult mice was found to be invariably greater in females than in males (Durham et al., 1929). In mice below 15 gm the difference was less pronounced; this was later confirmed by others (Levy and Meyer, 1946). b. Sulfonamides. When given intraperitoneally to rats, sulfanilamide

SEX A S A FACTOR I N DRUG EFFECTS

219

was more toxic to females than to males. Some tolerance could be developed in both sexes with a 2-week regimen of smaller doses, but on challenge thereafter approximately the same difference in mortality occurred between the sexes (Krems et al., 1941). Sulfanilamide acetylation in the adult rat was significantly greater in the male than in the female, and orchiectomy did not affect the capacity of the male for the process. Testosterone propionate did, however, enhance the acetylating ability of females (Franz and Lata, 1953, 1957). Immature rats of either sex acetylated to the same extent. Early orchiectomy and estradiol reduced the ability in males, and adrenalectomy superimposed on gonadectomy did not alter the effect from that of gonadectomy alone in either sex. Oophorectomy was ineffective, whereas hypophysectomy lowered the acetylating capacity in both sexes (Fishkin and Lata, 1957, 1958). Starvation of female rats increased the activity of reduced triphosphopyridine nucleotide (TPNH)-dependent enzymes that catalyze the metabolism of Neoprontosil (Kato and Gillette, 1964). Liver microsomal activity in the male was unchanged (Kato and Gillette, 1965a). Sulfadimidine was used as a prophylactic during an epizootic of salmonellosis in mink farms, and several thousand animals were given the drug in their feed. Severe urinary bleeding occurred in a number of males, but only of the Aleutian type, which has an abnormal lysosomal structure associated with the Chediak-Higashi syndrome. Experimental feeding was carried out with 11 Aleutian males and 6 standard males. The latter group received high doses of the sulfonamide without developing hematuria. Of the 11 Aleutian males, 3 had massive hematuria and 3 others died without vesical bleeding but with various degrees of hepatic and renal damage. A total of about 20,000 mink of various genotypes and both sexes were treated, but toxicity appeared only in Aleutian males (Nordstoga et al., 1970). In 1957, long-acting sulfonamides were introduced in the United States and by 1965 the Federal Drug Administration had collected reports of 116 cases of the Stevens-Johnson syndrome (erythema multiforme exudativum; Behset syndrome) associated with their use (Carroll et al., 1966). Although all of the data were not analyzed for sex, a table including 27 cases attributable to sulfadimethoxine or sulfamethoxypyridazine in which sex was given for 25 showed a ratio of 18 females to 8 males. A subsequent report on adverse reactions to two other sulfonamides, sulfisoxazole and sulfamethoxazole, noted no sex preponderance (Koch-Weser et al., 1971). In a study of toxic reactions to a combination of sulfamethoxazole with trimethoprim, there were slightly more females than males reporting cutaneous sensitivity disor-

220

F R A N S C . GOBLE

der, which might have been attributed earlier to the sulfonamide component, but recent reports of sensitivity to trimethoprim alone obviate this speculation (Salter, 1973). c. Nalidixic Acid. In the treatment of urinary tract infections of children with nalidixic acid, the prognosis is more favorable in boys (Lorber, 1967). Phototoxic bullous eruptions attributable to the drug are rare, but all reports in the literature in which the sex was reported were in women (Birkett et a l . , 1969). d . Nitrofurans. In a study of adverse reactions to nitrofurantoin therapy, allergic reactions were almost of equal incidence in both sexes, but toxic reactions were significantly more common in femalesanorexia, nausea, and vomiting being the most prevalent conditions (Koch-Weser et al., 1971). e. Clioquinol. In Japan in 1958 there appeared sporadic cases of a myelitis-like illness in which the neural disorders were preceded by abdominal pain or diarrhea or both. By 1965, numerous reports of sporadic cases and localized outbreaks had been made, and it was proposed to name the condition subacute myelo-opticoneuropathy. By 1970 the clinical diagnostic guidelines were well defined, and it was found that the greenish fur on the tongue and the greenish color of the feces were attributable to clioquinol, some of its chelates, or other derivatives (Kono, 1971). In the first nationwide epidemiologic survey (1971), there were 2 cases in women to every 1 in men. Another report the same year suggests the sex ratio was the same as that of individuals given clioquinol. A later study from Nagoya City Hospital reported 5 times as many women as men (Aoki et al., 1972), and still later a 1:2.4 ratio of men to women was recorded (Nakae et a l . , 1973).

B. TUBERCULOSTATIC COMPOUNDS 1. Synthetics a . p-Aminosalicyclic Acid (PAS). To prevent the emergence of resistant strains of Mycobacterium tuberculosis, PAS is often given in combination with other tuberculostats and to be effective must be administered in doses near the limits of tolerance. Gastrointestinal upset is the most disturbing side effect and is definitely related to defaulting which can be detected by tests for PAS in the urine of patients. Women are consistently more likely to be defaulters than men, only about 35% of women below 30 years of age being “regular takers” in some studies (Dixon et al., 1957; Wynn-Williams and Arris, 1958; Luntz and Austin, 1%0). Data from a large series indicates an intolerance of about 34% in women as opposed to about 16% in men (Bethge, 1968).

SEXASAFACTORINDRUGEFFECTS

22 1

b. Isoniazid. In a series of hospital employees receiving isoniazid as a prophylactic for 4 to 8 months, there was about 9% who had an increase in serum glutamic oxalacetic transaminase (SGOT) after starting the regimen among black men in the series. However, the percentage was significantly higher than the incidence in black women and whites of either sex, which were between 6.7% and 9.9% (Bailey et al., 1973). Neurological (including psychological) disturbances are more frequent in women (Aoki et a l . , 1969), as are disturbances of eye accommodation which depend on total dose of isoniazid but may be influenced by concomitant streptomycin and PAS (Honegger and Genee, 1969). Females were less reliable than males in self-medication with isoniazid (Morrow and Rabin, 1966). c. Ethambutol. One of the toxic clinical effects of ethambutol is an ocular change, retrobulbar neuritis, which results in blurred vision and loss of ability to see green. In one study this affected only men patients (Leibold, 1966). Others have found it to occur in women as well, and even to be more frequent in women less than 40 years of age, but to be predominant in males when all ages are considered (Yoshizawa et a l . , 1972). Another side effect of ethambutol is dyspnea, probably caused by increased viscosity of bronchial secretions (Tsukamura, 1968). This condition was found to be much more prevalent in women (12:l). d . Ethionamide and Prothionamide. Women are more likely to have side effects with ethionamide than are men, particularly gastric distress and vomiting. The same is true with prothionamide, which in addition to the gastric effects, produces more giddiness and headaches in women (Fox et al., 1969). The difference between the side effects of ethionamide and prothionamide is insignificant in women, whereas in men there are more reactions to ethionamide than to prothionamide. The maximum tolerated dose, however, for males and females does not differ significantly (Devadatta et a l . , 1970). 2 . Combinations with Antibiotics

a. With Cycloserine. Side effects with cycloserine are slightly more common in women than in men, convulsions and mental changes being the most serious and likely to lead to suspension of the drug. When cycloserine is given in combination with isoniazid, the mental disturbances are about 10 times more frequent in women than in men (Villar, 1970). Complete loss of appetite is the most serious digestive problem observed with cycloserine in combination with ethionarnide, probably

222

F R A N S C. GOBLE

attributable to the latter. This is more frequent in females, sometimes making it necessary to suspend therapy (Villar, 1970). Insomnia and headaches are symptoms resulting from medication with a combination of viomycin and cycloserine and are especially troublesome in men (Villar, 1970). b. With Streptomycin. With a combination of streptomycin (intramuscular), isoniazid, and ethionamide (per os), the incidence of nausea and vomiting is higher (11 of 14) among women as compared with that (10 of 32) among men (Verbist et al., 1967). Gastric disturbances are, of course, the most prominent effects of ethionamide alone (Fox et al.,

1969). In the treatment of African patients with pulmonary tuberculosis, who were failures of primary chemotherapy, a combination of streptomycin and pyrazinamide was used. There was an unfavorable response in 62% of 172 men as opposed to 36% in women. The more favorable response of women could be attributed only in part to weight differences between the sexes (East AfricadBritish Medical Research Council Pyrazinamide Investigation, 1969). A combination of streptomycin, isoniazid, and PAS provoked drug hypersensitivity (allergy) in 25% of women as compared with 18.5% of men (not significant according to Govindaraj and Grant, 1968), but a larger series shows a twofold difference in hypersensitivity in women compared with that in men (Ng et al., 1971). C. ANTIFUNGALCOMPOUNDS

1. Griseofulvin When griseofulvin was given to rats either parenterally or per o s , the blood levels in females were significantly higher than in males. Liver slices from male rats metabolized griseofulvin more rapidly than those from females. The difference was greater in adults than in weanlings and greater in the WAG strain than in the PVG strain (Busfield et al., 1960). No such difference between the sexes appeared in guinea pigs or humans. Prolonged administration of griseofulvin to mice by diet resulted in liver enlargement, prophyria, and hepatomata; all of these findings were more prominent in males than in females (Hurst and Paget, 1963; De Matteis et al., 1966; Epstein et al., 1967). In man, treatment of ringworm of the scalp (tinea capitis) was more effective in females than in males. This was apparent both with systemic treatment alone and with systemic treatment combined with topical (Grin, 1962).

SEXASAFACTORINDRUGEFFECTS

223

2. Polyenes When mice infected intravenously with Candida albicans are treated subcutaneously with either amphotericin B or nystatin, the efficacy of each drug is greater in females than in males (Goble and Konopka,

1973).

D. ANTIPARASITIC COMPOUNDS 1. Antiprotozoals a . Antimalarials. Perhaps the earliest record of a difference between the sexes in efficacy of a chemotherapeutic compound was that of Bennison and Coatney (1948) who found that in chicks infected with the avian malarial parasite Plasmodium gallinaceum, the females were less effectively protected by administration of quinine hydrochloride. In Plasmodium berghei infections of mice, quinine sulfate similarly is more effective in males than females. On the other hand, diaminodiphenylsulfone and primaquine are more effective in females. A number of compounds show no sex difference: chloroquine pentaquine, pyrimethamine, quinacrine, and sulfadiazine (Goble and Konopka, 1973). b. Trichomonacides. The incidence of lung tumors induced in mice by metronidazole at high dosage was significantly lower in females than in males and the mean number of nodules was higher in males. Malignant lymphomas, however, were observed only in females at the higher dose levels (Rustia and Shubik, 1972). When metronidazole is given to rats per 0s at doses of 600 and 800 mglkg, there is no difference in food consumption, weight increase, or other ordinary signs, but behavioral studies indicate C N S changes which can be confirmed histologically and which are more noticeable in females (von Rogulja et a l . , 1973). In experimental infections with Trichomonas vaginalis in mice, males are more responsive to treatment with metronidazole than are females (Goble and Konopka, 1973). c. Trypanocides. Treatment of mice infected with Trypanosoma cruzi is more effective in females than in males with a number of drugs: amphotericin B, emetine, metronidazole, niridazole, nitrofurazone, puromycin. Certain other compounds, however, are equally effective in both sexes, namely pentaquine and primaquine (Goble and Konopka, 1973). Treatment of Trypanosoma congolense infections in mice is more effective in females than in males when ethidium bromide is used, whereas no difference between the sexes is seen with puromycin, quinapyramine, or stilbamidine (Goble and Konopka, 1973). In Trypanosoma gambiense infections in mice, suramin is more effective in females, whereas other compounds, such as ethidium

224

FRANS C. GOBLE

bromide, pentamidine, puromycin, quinapyramine, and tryparsamide, show no difference between the sexes (Goble and Konopka, 1973). 2. Anthelminthics a. Schistosomacides. When mice infected with Schistosoma mansoni are treated per 0s with niridazole or tartar emetic, the reduction in number of worms following 12-day treatment is appreciably greater in females than in males given the same dosage (Goble and Konopka, 1973). Sodium antimony1 dimethylcysteinotartrate (NAP) is much better tolerated by male patients than by females, gastric side effects occurring in 21% and 68%, respectively (Pedrique et al., 1970). b. Other Anthelminthics. When mebendazole was given to rats by diet for 13 weeks, a level of 160 mg/100 gm of food inhibited the weight gain in both sexes, but more females succumbed, only 30% surviving at the end of the period, whereas 60% of the males lived (Marsboom, 1973). Treatment of sheep with 3,5-diiodo-3’-chloro-4’-Or,-chlorophenoxy) salicylanilide (Rafoxanide) in single doses between 70 and 200 mg/kg produced prostration diarrhea, visual disturbances, and collapse; in some animals the reactions occurred earlier and more intensely in males (Guilhon et al., 1971).

COMPOUNDS E. ANTIVIRAL 1. Quinacrine

In mice infected with the virus of equine encephalomyelitis, there is little difference in mortality between untreated males and females, but a dose level of quinacrine that protects about half of the females offers almost no protection to males. When the dose level is increased to a point where about one-third of the animals of each sex are protected, it takes a little longer for the females to succumb (Hurst, 1957). Estradiol abolishes the effect of quinacrine on the titers of virus in the adrenals of mice with equine encephalomyelitis (Hurst et a l . , 1960). 2. Thiosemicarbazones

Isatin P-thiosemicarbazone exerts an effect on neurovaccinia virus in mice (infected intracerebrally) which is greater in females than in males, delaying deaths and protecting 60% of the females until the eleventh day, when 90% of the males have succumbed (Goble and Konopka, 1973). Methisazone was tested for its prophylactic effect in an endemic

S E X AS A FACTOR I N DRUG E F F E C T S

225

smallpox area, being compared with a placebo that was given to a group of almost the same number of individuals, both groups having a similar number of males and females. Of the 262 persons given methisazone, 7 contracted smallpox; all were males. Of the 260 persons given placebo, 14 contracted the disease, 8 males (of 134) and 6 females (of 126) (Heiner et al., 1971). When all of the variables in the study are considered, the significance of these findings are not clear, neither in relation to the efficacy of methisazone against smallpox nor as regards the effect of the drug or of sex on the results of the vaccination program which was simultaneously in process.

3. Idoxuridine Idoxuridine was used in the treatment of 29 cases of herpes simplex encephalitis between 1966 and 1972. Of the 26 for which sex was reported, 12 were males and 8 of them (67%) recovered; 14 were females and 8 of them (57%) recovered (Illis and Merry, 1972).

XVI. Antineoplastic Agents A. ANIMALSTUDIES 1. Immunosuppressives Compound E (ll-dehydro-17-hydroxy-corticosterone), when tested against a transmissible tumor (originally thoracic) in the mouse, caused regression of the neoplasm in all females and young males but did not influence the tumors in adult males. The carcinolytic effect could be obtained in adult males if they were castrated or given estradiol (Heilman and Kendall, 1944). Pretreatment with hydrocortisone prolonged the life of female Ha/ICR Swiss mice with Ehrlich ascite tumor by 93%, whereas the prolongation in males w a s 55%. With a hypotetraploid clonal subline (E2), however, little effect was observed, and with the hyperdiploid Ehrlich ELD, life was shortened 18% for females and 29% for males (Kodama, 1962). When hydrocortisone was given to tumor-bearing mice after sham surgery, the tumor weight was reduced in males but increased in females (Kodama, 1964). No significant inhibitory effect was found with another strain (DD) of mice (Takeuchi et al., 1965). In SMA mice with the hypotetraploid Ehrlich 14N tumor, the inhibitive effect of hydrocortisone was seen only in males (Kodama and Kodama, 1970). The chronic toxicity in rats of azathioprine is dependent on both sex

226

F R A N S C. GOBLE

and strain of animal: Fisher rats are more sensitive than SpragueDawley, and male Fisher rats are more tolerant than females of that strain (Frankel et a1 ., 1970). Although an anticancer agent, azathioprine is carcinogenic in rodents, producing a generalized increase in all types of tumors in male rats and a 150% increase in lung tumors in male mice, as well as an increased incidence of hematopoietic tumors in female mice (Prejean et al., 1972). 2. Alkylating Agents Nitrogen mustard (p,p’-dichlorodiethyl methylamine hydrochloride) had a more pronounced effect on the lymphoid organs of female mice than on those of males (Robertson, 1949). The toxicity of nitrogen mustard and three of its derivatives (chloroquine mustard, phenylalanine mustard, and cyclophosphamide mustard) was greater for males than for females (Rutman et a l . , 1969, but only with the last two named compounds was the difference statistically significant (Rutman and Lewis, 1964). Certain other chloroethyl alkylating agents, which show no difference between the sexes in toxicity in mice or rats (Thompson and Larson, 1972), do have a carcinogenic effect which is partially sex dependent: BCNU [1,3-bis(2-chlorethyl)-l-nitrosourea]produces a 150% increase in lung tumors in male rats, as does CCNU [ 1-(2-chloroethyl)-3-cyclohexyl1-nitrosourea] (Prejean et a1 ., 1972). Cyclophosphamide (Cytoxan) causes a marked difference between the sexes of rats in response of the urinary bladder epithelium, males showing a more extensive necrosis, slower regeneration, and mcre persistent hyperplasia than do females (Koss and Lavin, 1970). The acute toxicity of cyclophosphamide in mice can be reduced in females by pretreatment with glucose, but in males this effect is seen only in those that have been castrated or given diethylstilbestrol (Osswald, 1972). Increased incidence of hematopoietic tumors in Swiss mice receiving cyclophosphamide was reported by Prejean et a l . (1972) to be noteworthy only in males, but, in NZB/NZW mice (animal models of systemic lupus erythematosus), tumors appeared in both sexes of treated animals, but earlier in the females. The high dose of cyclophosphamide that was associated with the high incidence of tumors prolonged the life-spans of the females, but not of the males, by suppressing the immune complex nephritis occurring in this strain of mice (Walker and Bole, 1973). When neonatal mice are treated with cyclophosphamide, a few

S E X A S A FACTOR I N DRUG E F F E C T S

227

pulmonary adenomas developed in both sexes at low doses but at the highest dose there were none found in males (Kelly et al., 1974). Compound BIC{imidazole-4(or 5)-carboxamide, 5(or 4)-[3,3-bis(2-chloroethy1)-1-triazenol} produced an increase in hematopoietic tumors in female mice, whereas PDA (phosphorodiamic acid), N,N-bis(2-chloroethyl)-N'-3-hydroxypropyl cyclohexamine salt, and CAB (chlorambucil) increased (100%) the incidence of all types of tumors in female mice. The last compound produced an increased incidence of lung tumors in male mice and hematopoietic tumors in male rats (Prejean et al., 1972). Phenestrin, when given at high doses in subacute tests to Wistarl Furth rats, produces deaths 2 days earlier in females than in males. In longer-term tests, females succumb at lower doses than those affecting males. In Lewis rats, lymphopenia occurs in females at lower doses than those in males (Vollmer et al., 1973). Triethylenemelamine is more effective in inhibiting tumor growth (Taper liver tumor) in female mice than in male, and procarbazine produces a more pronounced effect in males (Cappuccino and Balis, 1969).

3. Metabolic Antagonists Folic acid (pteroylglutamic acid) was tolerated better by male mice than by females, doses up to 1600 mg/kg causing no deaths in males, whereas 600 mg/kg killed all females. Aminopterin (4-aminopteroylglutamic acid), on the other hand, was more toxic to males than females. The difference between the sexes was not apparent in immature mice. Estrogen increased the tolerance of males, but testosterone did not influence toxicity in either sex. Adrenalectomy abolished the difference between the sexes (Goldin et al., 1950). Gonadectomy increased male tolerance (Weintraub et al., 1950). Methotrexate (4-amino-N'o-methylpteroylglutamic acid) also showed a sex difference in toxicity, male mice being more susceptible, particularly with repeated dosing (Ferguson et al., 1950; Bennette, 1952). The antitumor effect of ethionine was more marked in female rats than in male rats of both the Long-Evans (Levy et a l . , 1953) and Wistar strains (Murphy and Dunn, 1957). Cytosine arabinoside is more effective in female than in male mice with the Taper liver tumor (Cappuccino and Balis, 1969). The toxicity of 3-deazauridine is greater in female than in male mice, and orchiectomy decreases tolerance, whereas oophorectomy and testosterone increase it (Bloch et a / ., 1972). 6-Mercaptopurine produces an increased incidence of hematopoietic tumors in male rats (Prejean et al., 1972).

228

F R A N S C. GOBLE

There are marked differences in the acute toxicity of allopurinol in both rats and mice, females being more tolerant than males. After intraperitoneal administration of a l l o p ~ r i n o l - ~ ~more C , radioactive compounds accumulate in the kidneys of male mice than in those of females. The allopurinol-oxidizing enzyme activity in livers from adult mice is higher in those from males than in those from females. Gonadectomy in males reduces this activity, but it is not affected by gonadectomy of females. Testosterone propionate increases the activity of the allopurinol-oxidizing enzyme in gonadectomized mice of either sex, whereas estradiol-17P has no influence on it (Iwata et a l . , 1974). The toxic effects of 6-azauridine in mice are determined by a multiallelic genetic system which is additive in females, whereas interallelic interactions (epistasis) occur in males (Zidek and Janku, 1973). 4. Antibiotics

Acetoxycyclohexamide was more toxic to female rats and mice than to males, but no such difference was found in dogs (Pallotta et al., 1960). Two closely related compounds, cycloheximide (Actidione) and streptovaricin A did not show any sex difference in rats. Bleomycin-induced toxicity, as reflected by clinical conditions during 28-week tests, is more serious in female dogs than in males (Thompson et al., 1972). Female rats receiving mycophenolic acid have hematopoietic changes earlier and at lower doses than do males, and only females have leukopenia (Sweeney et a l . , 1972).

5. Alkaloids Male rats were slightly more susceptible to the toxicity of vinblastine than were females (Noble, 1961), and pretreatment of immature female rats with stilbestrol allowed them to tolerate higher doses than was possible without it (Lotz and Noble, 1962). Diethylstilbestrol increased the tolerance of both males and females, but orchiectomy had no effect (Cutts, 1968). Male mice and rats tolerate much less lapachol than do females, the difference being more pronounced in mice (Morrison et a l . , 1970). By a number of regimens, female monkeys and dogs are more sensitive to thalicarpine than are males (Palm et al., 1972). 6. Miscellaneous The inhibitory effect on six mouse tumors of pyruvaldehyde bis(thiosemicarbazone) was greater in males than in females. Experi-

S E X A S A FACTOR I N D R U G EFFECTS

229

ments with the Walker carcinosarcoma 256 indicated that orchiectomy reduced the advantage of males and estradiol did not alter the effect of castration. Testosterone restored the response of orchiectomized animals and enhanced the therapeutic response of females. It did not change the toxicity (Cappuccino et a l . , 1967). Hydroxyurea has a greater effect on the Taper liver tumor in female mice than in males (Cappuccino and Balis, 1969). The L-asparaginase activity in rat liver was higher in females than in males. It was enhanced by administration of estradiol or cortisone to males and decreased by gonadectomy in both sexes or testosterone in females. There was no difference between the sexes in mice (Bonetti et al., 1969). The asparaginase levels in guinea pig serum were the same for both sexes (Tower et al., 1963).

B. CLINICALOBSERVATIONS 1. Individual Drugs Cyclophosphamide treatment was associated with cytological atypia of the urinary bladder epithelium which was more commonly a minimal effect in females than in males and more commonly a moderate effect in males than in females (Forni et al., 1964). In multiple myeloma the median survival time for females treated with cyclophosphamide is about 3 months longer than that for males, but the difference is not significant (Salokannel et al., 1971). Arabinosylcytosine has greater activity in females with metastatic melanoma than in males (Hart et a l . , 1972). When medroxyprogesterone acetate (Provera) is used in the treatment of metastatic; renal cancer, a favorable response is seen more commonly in men (21%) than in women (8%). The rate in men is increased to 27% if deaths within 6 weeks are excluded (Bloom, 1971). 2. Combinations In acute lymphoblastic leukemia treated with a combination of BCNU and 1-P-D-arabinofuranosyl cytosine (Ara-C), the remission rates between males and females showed a highly significant difference: 17% and 52%, respectively. In patients over 60 years old, the corresponding rates were 22% and 75% (Vogler, 1971). A more complicated combination treatment (Protocol 06LA 66-Paris) for acute lymphoblastic leukemia involves daunorubicin, vincristine, prednisone, 6-mercaptopurine, and methotrexate. With this regimen complete remissions are achieved in females more commonly than in

230

F R A N S C . GOBLE

males, and among males more commonly in children than in adults (Jacquillat et al., 1973). The regimen known as “MOPP” includes mechlorethamine hydrochloride, vincristine sulfate (Oncovin), procarbazine hydrochloride, and prednisone. Sex is a significant factor in the response of Hodgkin’s disease to this treatment (Stutzman, 1973). Women have a significantly better duration of response, the median being 10 months compared with 7 months for men (Stutzman and Glidewell, 1973). The ultimate percentages of complete remission, however, do not differ significantly: females, 67% and males, 64% (Luce et al., 1973).

XVI I. General Considerations A. DEFAULTING Unreliable drug intake is considered to be one of the frequent causes of “ineffectiveness” of diphenylhydantoin occurring in three-fourths of patients in one study and predominating in women (Kutt et al., 1966). Defaulting is also common among patients taking PAS (mentioned in Section XV) with women again the common offenders. Some investigators, however, have not found sex to be associated with noncompliance (Willcox et al., 1965; Charney et al., 1967; Davis, 1968; Boyd et al.,

1974).

B.

PLACEBO AND “NOCEBO”

EFFECTS

The salutory effects observed following administration of materials without specific activity for the condition are well known. In a controlled trial of hypnotic drugs, the efficacy of the placebo was greater among women than among men, although there was no difference between the sexes in response to the active products (Jick et al., 1969). A contrary effect called “nocebo effect” is also observed sometimes in which a placebo of inert material produces side effects simulating those produced by active medicaments. These may produce any of the untoward signs and symptoms considered to be adverse reactions and are more common in women than in men (Herzhaft, 1969).

C. ADVERSEREACTIONS With the development of computer surveillance and epidemiological studies of adverse drug reactions, information has become available on general incidence of adverse reactions, primarily in hospital patients. At

SEXASAFACTORINDRUGEFFECTS

23 1

Johns Hopkins, where women make up 45% of the patient population, they account for 62% of the drug reactions detected (Seidl et al., 1965). This preponderance was attributable to a high rate of gastrointestinal effects in women (Smith et al., 1966b), which occurs with all the common therapeutic classes of drugs (Stewart and Cluff, 1964). In Montreal there was no significant difference observed between the sexes (Ogilvie and Ruedy, 1967). In Ottawa, females predominated, but the patient population was not given (Montgomery and Jackson, 1968). In Belfast the percentage of treated females with drug reactions is twice that of treated males (Hurwitz, 1969). In a study at Yale, women predominated in headaches, nasal congestion, rapid weight gain, and lack of energy, but young men more often had slurred speech. Older women exceeded men in stiffness, dizziness, blurred vision, nausea, rapid heart beat, increased thirst, and increased sweating (Kupfer and Detre, 1971). Little difference between the sexes was found in New Zealand (Smidt and McQueen, 1972). Females predominated in South Yorkshire (Mulroy, 1973), North Carolina (Gray et al., 1973), Boston (Miller, 1973), New Zealand (Kellaway and McCrae, 1973), Sweden (Bottiger, 1973), Florida (Caranasos et al., 1974). In a clinicopathologic survey of infants and children with adverse reactions, three-fourths of the cases were white girls, and females also predominated in the black and oriental groups (Mullick et al., 1973).

1. Blood Dyscrasias Agranulocytosis (described by Schulz in 1922) was soon recognized to be more prevalent in women than in men (Lichtenstein, 1932; Taussig and Schnoebelen, 1931) and associated with medications (Kracke and Parker, 1934; Plum, 1937). When the drugs responsible for a difference between the sexes in hematopoiesis have been identified, they have been noted in specific sections of the text above. Drug-induced anemias are usually more frequent in women (Best 1967; Pisciotta, 1971; Bottiger and Westerholm, 1973a,b; O’Gorman Hughes, 1973). The aplastic type has been treated by androgen therapy (Shahidi and Diamond, 1959), but results have been equivocal (SanchezMedal et al., 1969; Sanchez-Medal, 1971; Li et al., 1972) and prognosis is better for females than for males (Williams et al., 1973). In southwest Iran, aplastic anemia, probably caused by a number of agents (generally antibiotics) predominates in men but is not related to a higher hospital admission rate of males (Banihashemi et al., 1973). Males also predominate in the sensitivity to drugs resulting from a deficiency of glucose-6-phosphate dehydrogenase (G 6-PD) which makes

232

F R A N S C . GOBLE

red blood cells less efficient in generating NADPH, the enzyme concerned with the production of glutathione and needed for maintaining the integrity of the erythrocyte membrane. The inheritance of this deficiency is sex-linked without dominance, the gene determining the characteristics of the G 6-PD being carried on the X chromosome. In vitro tests on red cells can readily be performed to reveal sensitive individuals (predominantly males of African and Mediterranean origin) who may be susceptible to hemolytic anemia as a result of treatment with drugs of a number of categories, including sulfonamides, 8aminoquinolines, nitrofurans, antipyretic analgesics, and PAS (Kirkman, 1968; Beutler, 1970; Hochstein, 1971). Drug-induced thrombocytopenia has a greater incidence in females than in males, the predominance being highest with quinine/quinidine medications, but also noteworthy with diuretics, particularly thiazides (Bottiger and Westerholm, 1972).

2. Allergy Allergic drug reactions are more frequent in women than in men. They are both dermal (Grosfeld et a l . , 1963; Adam, 1972; Rudzki, 1972; Szarmach and Poniecka, 1972) and asthmatic (Seropian et al., 1971; Horak, 1971) or combinations of these (Muranaka et a l . , 1969; Ohela and Ahonen, 1972) including Stevens-Johnson syndrome (Claxton, 1963; Coursin, 1966; Bianchine et a l . , 1968; Stillman, 1968; Ostler et a l . , 1970; Calcaterra and Strahan, 1971; Monnat, 1972).

3. CNS Abnormalities There are certain cerebral conditions that are sex related and may be associated with medication. One is pseudotumor cerebri which may result from a number of causes, one group of which includes tetracycline, nalidixic acid, inorganic lead and arsenic, carbon disulfide, and tannin (Benini, 1973). Another is multiple progressive cranial arterial occlusion (Mastri et al., 1973). Both of these predominate in females, and an association of the latter with contraceptive therapy has been suggested.

XVIII. Final Remarks From the above survey, it is apparent that sex as a factor in pharmacology and therapy has been recognized in a limited way for a half a century and as a more general phenomenon for the last 20 years,

S E X A S A FACTOR IN DRUG E F F E C T S

233

but that much is still being learned and many more observations are needed to bring complete understanding. Investigations in the field might flag if the knowledge gained from the extensive studies in rats was applicable to all species and all compounds, and it could be assumed that males, under the influence of their testosterone, could usually be expected to have higher levels of liver microsome activity than females and thus more rapidly metabolize most compounds. However, some strains of mice do not show differences dependent on sex in response to drugs whereas others do, and when they do, it is more often the females that metabolize drugs more efficiently. This presents another aspect to the subject that has yet to be elucidated fully, as does the lack of difference between the sexes of other animals, including man, in response to drugs. In man, sex-dependent differences in adverse reactions to drugs, not predictable from studies of the same preparations in animals, accentuate the impossibility of generalization. The explanation of some of the heterogeneity of the phenomena of differences in response of the sexes may involve the very fundamental difference between the sexes which involves every cell of the body, i.e., the genetic difference resulting from the presence of two X-chromosomes in cells of females versus one in those of males. The importance of this difference has been emphasized by Burch (1963) and Burch and Rowel1 (1965) and has been recognized in both infectious (Washburn et al., 1965) and noninfectious diseases (Childs, 1965); its role as a factor affecting the responses of either sex to disease prepuberally, before the elaboration of significant quantities of sex hormones, has been noted. With the need for continued accumulation of information in this field to aid in the eventual elucidation of some of the unanswered questions, the importance of examining data from the standpoint of sex should be reemphasized. Many series of observations on “patients” or “rabbits” might have furnished interesting information if the data had been separated according to sex. REFERENCES Abderhalden, E., and Wertheimer, E. (1927). Biochern. Z . 186, 252. Adam, H. (1972). Allerg. Immunol. 1 8 , 73. Agduhr, E. (1938). Skand. Arch. Physiol. 78, 259. Ahlquist, R. P., Taylor, J. P., Rawson, C. W., Jr., and Sydow, V. L. (1954). J . Pharmacol. Exp. Ther. 1 1 0 , 3 5 2 . Alp, M. H., Hislop, I. G., and Grant, A. K. (1970). M e d . J . Aust. 2 , 1128. Altura, B. M. (1972). Eur. J . Pharmacol. 20, 261. Andreoli, V. M. (1968). Experientia 24, 1155. Andreoli, V. M., Brunelli, A , . and Napoli, P. A. (1968). Boll. Soc. I t a l . B i d . Sper. 44, 1900.

234

F R A N S C . GOBLE

Angelucci, L. (1958). Nature (London) 181, 967. Angenall, L., Bengtsson, U., Zetterlund, C. G., and Zsigmond, M. (1969). Brit. J. Urol. 41,401. Aoki, K., Otani, M., Sobue, I., and Ando, K. (1972). Jap. J. Pub. Health 19, 305. Aoki, M., Kihara, K., Murase, S., and Hirasawa, I. (1969). Kekkaku 44, 103. Aschkenasy-Lelu, P. (1958). C. R . Acad. Sci. 247, 1044. Aschkenasy-Lelu, P. (1960a). Rev. Fr. Etud. Clin. Biol. 5 , 132. Aschkenasy-Lelu, P. (1960h). Arch. Sci. Physiol. 14, 165. Astarabadi, T. M., and Essex, H. E. (1952). Amer. J. Physiol. 171, 75. Atkinson, R. M., Pratt, M. A , , and Tomick, E. G. (1962). J. Pharm. Pharmacol. 14,698. Axelrod, J., Reichenthal, J., and Brodie, B. B. (1954). J. Phannacol. Exp. Z’her. 112, 49. Ayd, F. J., Jr. (1961). J. Amer. Med. Ass. 175, 1054. Backus, B., and Cohn, V. H. (1966). Fed. Proc., Fed. Amer. SOC. E x p . B i d . 25,531. Baer, R. L., Fellner, M. J., and Sibulkin, D. (1970). Brit. J. Dermatol. 83, Suppl., 37. Bailey, W. C., Taylor, S. L., Dascomh, H. E., Greenberg, H. B., and Ziskind, M. M. (1973). Amer. Rev. Resp. Dis. 107, 523. Balodimos, M. C., Gleason, R. E., Bradley, R. F., and Marble, A. (1971). Excerpta Med. Found. Int. Congr. Ser. 149, 270-280. Banihashemi, A., Kohout, E., and Hedayatee, H. (1973). Blut 26, 20. Barka, T. (1967). Exp. Cell Res. 48, 53. Barron, D. H. (1933). Science 77, 372. Battle, R. (1936). St. Thom. H o s p . Gar. 35, 368. Beattie, C. W., Campbell, C. S., Nequin, L. G., Soyka, L. F., and Schwartz, N. (1973). Endocrinology 92, 1634. Beecher, H. K. (1933). J. Clin. Invest. 12, 639. Beernink, D. H., and Miller, J. J. (1973). J. Pediat. 82, 113. Bellville, J. W., Bross, I. D. J., and Howland, W. S.(1%0). Anesthesiology 21, 186. Benenson, A. S. (1970). In “Control of Communicable Diseases in Man,” pp. 96101. Amer. Pub. Health Ass., New York. Bengtsson, U., Angervall, L., Ekman, H., and Lehmann, L. (1968). Scand. J. U r o l . Nephrol. 2 , 145. Benini, A. (1973). Deat. Med. Wochenschr. 98, 17. Bennette, J. G. (1952). Brit. J. Cancer 6, 377. Bennison, B. E., and Coatney, G. R. (1948). Science 107, 147. Best, W. R. (1967). J. Amer. Med. A s s . 201, 181. Bethge, W. (1968). 2. Tuberk. 128, 258. Beutler, E. (1970). Brit. J. Haematol. 18, 117. Bhargava, K. P., Dhawan, K. N., and Saxena, R. C. (1967). Brit. J. Pharmacol. Chemother. 31, 26. Bianchine, J. R., Macaraeg, P. V. J., Jr., Lasagna, L., Azarnoff, D. L., Brunk, S. F., Hvidberg, E. F., and Owen, J. A , , Jr. (1968). Amer. J. Med. 44, 390. Bielawski, W., and Michalik, T. (1969). Pol. Arch. Med. Wewn. 43, 1099. Billington, B. P. (1960). Australas. Ann. Med. 9, 111. Billington, B. P. (1963). Australas. Ann. Med. 12, 153. Billington, B. P. (1965). Gut 6, 121. Birkett, D. A., Garretts, M., and Stevenson, C. J. (1969). Brit. J. Dermatol. 81, 342. Blackham, A., and Spencer, P. S. J. (1969). Brit. J . Pharmacol. 37, 129. Blackham, A., and Spencer, P. S. J. (1970). J. Pharm. Pharmacol. 22, 304. Bloch, A., Dutschman, G., and Grindey, G. (1972). Proc. Amer. Ass. Cancer Res. 13, 19. Bloom, H. J. G. (1971). Brit. J. Cancer 25, 250.

SEXASAFACTORINDRUGEFFECTS

235

Bock, K. D., and Hogrefe, J. (1972). Muenchen. Med. Wochenschr. 1 1 4 , 6 4 5 , Bodman, R. I., Morton, H. J. V., and Thomas, E. T. (1960). Brit. Med. J . 1, 1327. *Okelman, D. L.3 Bogdon, J., Mattis, P. A., and Stonier, P. F. (1971). Toxicol, ~ ~ Pharmacol. 19, 111. Bonetti, E., Abbondanza, A., Della Corte, E., and Stirpe, F. (1969). Biochem. J. 115, 597. Booth, J., and Gillette, J. R. (1962). J. Pharmacol. Exp. Ther. 137, 374. Borchgrevink, C. F. (1960). Acta Med. Scand., Suppl. 359, 1-51. Borglin, N. E., and Mansson, B. (1951). Acta Endocrinol. (Copenhagen) 8, 81. Boston Collaborative Drug Surveillance Program, Boston University Medical Center. (1972). N . Engl. J. Med. 286, 505. Boston Collaborative Drug Surveillance Program, Boston University Medical Center. (1973). Arch. Dermatol. 107, 74. Bottiger, L. E. (1973). J. Clin. Pharmacol. New Drugs 13, 373. Bottiger, L. E., and Westerholm, B. (1972). Acta Med. Scand. 191, 541. Bottiger, L. E., and Westerholm, B. (1973a). Acta Med. Scand. 193, 227. Bottiger, L. E., Westerholm, B. (1973b). Brit. Med. J. 3, 339. Boucek, B. (1936). J. Amer. Pharm. Ass. 25, 97. Boughton, L. L. (1942). J. Amer. Pharm. Ass. 31, 240. Boulton, T. B. (1955). Anaesthesia 10, 233. Bovill, J. G., Dundee, J. W., Coppell, D. L., and Moore, J. (1971). Lancet 1, 1285. Boxill, G. C., and Brown, R. V. (1955). J. Pharmacol. Exp. Ther. 115, 1. Boyd, E. M. (1970). A p p l . Ther. 12, 9. Boyd, E. M., and Hottenroth, S. M. H. (1968). Toxicol. Appl. Pharmacol. 12, 80. Boyd, E. M., and Krijnen, C. J. (1%9). Jap. J . Pharmacol. 19, 386. Boyd, J. R., Covington, T. R., Stanaszek, W. F., and Coussons, R. T. (1974). Amer. J . Hosp. Pharm. 31, 362. Brenner, G., and Trautmann, H. (1969). Arzneim.-Forsch. 19, 1974. Britt, B. A., and Kalow, W. (1968). Ann. N . Y. Acad. Sci. 151, 947. Britt, B. A.. and Kalow. W. (1970). Can. Anaesth. Soc. J. 17, 293. Britt, B. A., Locher, W. G., and Kalow, W. (1969). Can. Anaesth. SOC.J. 16, 89. Brock, R. C. (1936). Guy’s Hosp. Rep. 86, 191. Brodie, B. B. (1956). J . Pharm. Pharmacol. 8, 1. Brodie, B. B., Gillette, J. R., and La Du, B. N. (1958). Annu. Rev. Biochem. 27, 427. Bro-Jorgensen, A., and Jensen, T. (1971). Brit. J. Vener. Dis. 47, M3. Brooks, J. E., and Bowerman, A. M. (1973). J. Hyg. 71, 217. Brown, A. M. (1961). J. Pharm. Pharmacol. 13, 679. Brown, A. M. (1965). Lab. Anim. Care 15, 111. Brown, A. M., and Julian, T, (1968). Int. J. Neuropharmacol. 7, 531. Brown, D. M., Langley, P. F., Smith, D., and Taylor, D. C. (1974). Xenobiotica 4, 151. Brummelkamp, W. H. (1965). In “Drug-Induced Diseases” (L. Meyler and H. M. Peck, eds.), Int. Congr. Ser. N o . 85, Excerpta Med. Found., pp. 35-46. Amsterdam. Bruun, E. (1950). Acta Allergol. 3, 257. Bryce, D. A. (1938). J. Allergy 9 , 514. Buchel, L. (1954). Anesth. Analg. (Paris) 11, 268. Buchel, L. (1969). Arch. Sci. Physiol. 23, 109. Buchsbaum, M., and Buchsbaum, R. (1962). Proc. Soc. E x p . B i d . Med. 109, 68. Bun, C. (1937). Nagasaki Igakkai Zasshi 15, 2341. Burch, P. R. J. (1963). Lancet 1, 1253. Burch, P . R. J., and Rowell, N. R. (1965). Amer. J . Med. 38, 793.

w.

~

1

236

F R A N S C. GOBLE

Burford, G. E. (1938). J. Amer. Med. ASS. 110, 1087. B ~ J. ~H., Finney, , D. J., and Goodwin, L. G. (1950). In “Biological Standardization,” zud ed., p. 219. Oxford Univ. Press, London and New York. Buny, A. F., Axelsen, R. A., and Trolove, P. (1974). Med. J. Aust. 1, 31. Burstein, S., and Kupfer, D. (1971). Ann. N . Y. Acad. Sci. 191, 61. Burtles, R., and Peckett, B. W. (1957). Brit. J . Anaesth. 29, 114. Busfield, D., Child, K. J., Basil, B., and Tornich, E. G. (1960). J. Pharm. Pharmacol. 12, 539.

Buttar, H. A., Coldwell, B. B., and Thomas, B. H. (1974). Arch. Int. Pharrnacodyn. Ther. 208,279.

Biittner, H. (1965). Biochem. Z. 341, 300. Calcaterra, T. C., and Strahan, R. W. (1971). Arch. Otolaryngol. 93, 37. Caldwell, J. G., Wessler, S., and Avioli, L. V. (1971). J . Amer. Med. Ass. 218, 714. Cameron, G. R. (193W1939). Proc. Roy. SOC.Med. 32, 309. Cameron, G. R., and de Saram, G. S. W. (1939). J . Pathol. Bacteriol. 48, 49. Cappuccino, J. G., and Balis, M. E. (1969). Cancer Chernother. R e p . 53, 325. Cappuccino, J. G., Mountain, I. M., Tarnowski, G. S., and Balis, M. E. (1967). Cancer Res. 27, 377. Caranasos, G. J., Stewart, R. B., and Cluff, L. E. (1974). J. Amer. Med. Ass. 228, 713. Carmichael, E. B. (1938). J. Pharmacol. Exp. Ther. 62, 284. Carroll, B. R. T., and Nicol, C. S. (1970). Brit. J . Vener. Dis. 46, 31. Carroll, 0. M., Bryan, P. A., and Robinson, R. J. (1966). J. Amer. Med. Ass. 195, 691. Catz, C., and Yaffe, S. J. (1967). J . Pharmacol. Exp. Ther. 155, 152. Champion, R. H., Roberts, S. 0. B., Carpenter, R. G., and Roger, J. H. (1969). Brit. J . Dermatol. 8 1, 588. Chance, M. R. A. (1947). J. Pharmacol. Exp. Ther. 89, 289. Charlin, A. (1956). Encephale 45, 1099. Charney, E., Bynum, R., Eldredge, D., Frank, D., MacWhinney, J. B., McNabb, N., Scheiner, A., Surnpter, E. A., and Iker, H. (1967). Pediatrics 40, 188. Chen, K. K., and Robbins, E. B. (1944a). J. Arner. Pharm. Ass. 33,61. Chen, K. K., and Robbins, E. B. (1944b). J . Arner. Pharm. Ass. 33, 62. Cheymol, J., and Sabourdy, M., Porte, R., Beauvallet, M., and Tuffrau, H. (1964). B i d . Med. (Paris) 53, 169. Childs, B. (1965). Pediatrics 3 5 , 798. Christensen, L. R., Wolff, G. L., Matanic, B., Bond, E., and Wright, E. (1963). Z. Versuchstierk. 2 , 135. Christodoulidis, H. (1974). Curr. Ther. Res., Clin. Exp. 16, 311. Clark, M. L., Huber, W. K., Kyriakopoulos, A. A , , Ray, T. S., Colrnore, J. P., and Ramsey, H. R. (1968). Psychopharmacologia 12, 193. Claxton, R. C. (1963). Med. J. Aust. 1, 963. Cleveland, M. (1919). Surg., Gynecol. Obstet. 28, 282. Cluff, L. E., Thornton, G. F., and Seidl, L. G. (1964). J. Amer. Med. Ass. 188, 976. Cohen, G. M., and Mannering, G. J. (1974). Drug Metab. Disposition 2, 285. Cohn, R., Barnes, P., Barratt, E., and Pirch, J. (1971). Pharmacologist 13, 297. Cole, P. (1971). Lancet 1, 1335. Cole, V. V. (1943). J. Pharmacol. E x p . Ther. 78, 170. Collaborative Study Group. (1973). Brit. Med. J. 1, 7. Collins, T. B., and Lott, D. F. (1968). Lab. Anim. Care 18, 192. Cornbes, F. C., Canizares, O., and Sirnuango, S. (1950). J. Invest. Dermatol. 14, 53. Couney, A. H. (1967). Pharmacol. Rev. 19, 317 and 342.

S E X A S A FACTOR I N DRUG E F F E C T S

237

ConneY, A. H.7 and Burns, J. J. (1960). Ann. N . Y . Acad. Sci. 86, 167. ConneY, A. H., and Burns, J. J. (1962). Advan. Pharmacol. 1, 31, 47, and 54. ConneY, A. H., and Garren, L. (1961). Biochem. Pharmacol. 6, 257, ConneY, A. H., and Klutch, A. (1963). J. Biol. Chem. 238, 1611. ConneY, A. H., Schneidman, K., Jacobson, M., and Kuntzman, R. (1965). Ann, N . y , Acad. Sci. 123,98. Cook, L.3 M a c h E., and Fellows, E. J. (1954). J . Pharmacol. E z p . Ther. 112, 382. Cooper, J. R., and Brodie, B. B. (1955). J . Pharmacol. E x p . Ther. 114, 409. Cooper, J. R., Axelrod, J., and Brodie, B. B. (1954). J. Pharmacol. E z p . Ther. 112, 55. Cornelius, C. C., 111, and Dornescik, G. (1970). Brit. J . Vener. Dis. 46, 212. Coursin, D. B. (1966). J. Amer. Med. Ass. 198, 113. Coville, P. F., and Telford, J. M. (1970). Brit. J. Pharmacol. 40, 747. Crane, G. E. (1968). Amer. J. Psychiat. 124 (Suppl.), 40. Crane, G. E. (1973). Brit. J . Psychiat. 122, 395. Crane, G. E., and Paulson, G. (1967). Znt. J . Neuropsychiat. 3, 286. Culliford, D., and Hewitt, H. B. (1957). J. Endocrinol. 14, 381. Cutts, J. H. (1968). J . Nat. Cancer Inst. 41, 9!9. Darby, F. J. (1972). Biochem. Pharmacol. 21, 1649. Darby, J. (1973). Biochem. Pharmacol. 22, 989. Davis, L. E., and Donnelly, E. J. (1968). J . Amer. Vet. Med. Ass. 153, 1161. Davis, L. E., and Wolff, W. A. (1970). Amer. J . Vet. Res. 31, 469. Davis, M. S. (1968). Med. Care 6, 115. de Boer, B. (1948). J. Amer. Pharm. Ass. 37, 302. d e Gennes, J. L., Thomopoulos, P., Truffert, J., and d e Tregomain, B. L. (1972). Nutr. Metab. 14, 141. Degkwitz, R., Wenzel, W., Binsack, K. F., Herkert, H., and Luxenburger, 0.(1966). Arzneim.-Forsch. 16, 276. De Matteis, F., Donnelly, A. J., and Runge, W. J. (1966). Cancer Res. 26, 721. Deringer, M. K., Dunn, T. B., and Heston, W. E. (1953). Proc. Soc. Exp. B i d . Med. 83, 474. Dessau, F. I., and Sullivan, W. J. (1961). Toxicol. Appl. Pharmacol. 3, 654. Devadatta, S., Menon, N. K., Nazareth, O., Radhakrishna, S., Rarnakrishnan, C. V., Somasundaram, P . R., Usha, S. P., and Velu, S. (1970). Tubercle 51, 263. Dietrichson, O . , Oxlund, J. J., Juhl, E., and Nielsen, J. 0. (1973). Ugeskr. Laeger 135, 2733. Dixon, W. M., Stradling, P., and Woolton, 1. D. P. (1957). Lancet 2, 871. Dorfman, A., and Goldbaum, L. R. (1947). J . Pharmacol. Exp. Ther. 90, 330. Downs, A. W., and Eddy, N . B. (1932). J . Pharmacol. Exp. Ther. 46, 199. Drapkin, A,, and Merskey, C. (1972). J. Amer. Med. Ass. 222, 541. Dripps, R. D., and Vandam, L. D. (1954). J. Amer. Med. Ass. 156, 1486. Dripps, R. D., Lamont, A., and Eckenhoff, J. E. (1961). J. Amer. M e d . Ass. 178, 261. Dubach, U. C., Levy, P. J., and Minder, F. (1968). Helv. Med. Acta 34, 297. Duggan, J. M. (1972). Gut 13, 631. Duncan, W. C . , Glicksman, J. M., Knox, J. M., and Holder, W. R. (1971). Brit. J. Vener. Dis. 47, 364. Durham, F. M., Gaddum, J. H., and Marchal, J. E. (1929). Medical Res. Counc. (Gt. Brit.), Spec. Rep. Ser. 128, 19 and 40. Duvoisin, R. C . (1967). Proc. 6th Annu. Meet. Amer. Coll. Neuropsychopharmacol., 1967 pp. 561-563. Dyhing, F., and Dybing, 0. (1947). Acta Pharmacol. Toxicol., 3, 189.

238

F R A N S C . GOBLE

Dyrberg, v. (1962).Acta Anaesthesiol. Scand. 6,37. East Affican/British Medical Research Council Pyrazinamide Investigation. (1969). Tubercle 50, 81. Edgren, R. A. (1957).Experientia 13, 86. Editorial. (1970). Brit. Med. J . 4, 444. Editorial. (1972a).J. Amer. Med. Ass. 219, 143. Editorial. (1972a). Lancet 2, 170.

Edler, K. (1966).Acta Ophthalmol. 44, 405. Edlund, Y. (1970). Schweiz. Med. Wochenschr. 100, 1174. Edmonds-Seal, J., and Eve, N. H. (1962). Brit. J . Anaesthesiol. 34, 44. El Defrawy El Masry, S., and Mannering, G. J. (1974). Drug Metab. Disposition 2, 279. El Defrawy El Masry, S., Cohen, G. M., and Mannering, G. J. (1974). Drug Metab. Disposition 2, 267. Eliason, E. L., and McLaughlin, C. (1932). Surg., Gynecol. Obstet. 55, 716. Epstein, S. S., Andrea, J., Joshi, S., and Mantel, N. (1967). Cancer Res. 27, 1900. Eriksson, K. (1968). Science 159, 739. Eriksson, K.,and Malmstrom, K. K. (1967).Ann. Med. Ezp. Biol. Fenn. 45,389. Erikkson, K., and Pikkarainen, P. H. (1968). Metab., Clin. E x p . 17, 1037. Eschenbrenner, A. B., and Miller, E. (1945a).J . Nat. Cancer Znst. 5, 251. Eschenbrenner, A. B., and Miller, E. (194.533).Science 102,302. Evans, J. H. (1965). Lancet 1, 458. Everett, G. M. (1967). Proc. 6th Annu. Meet. Amer. Coll. Neuropsychopharmacol., 1967 pp. 985 and 987. Fahy, A., and Marshall, M. (1969). Brit. J. Anaesthesiol. 41, 433. Fann, W. E., Davis, J. M., and Janowsky, D. S. (1972). Dis. New. Syst. 33, 182. Fantoni, A. (1936). Ateneo Parmense 8, 285. Featherstone, H.(1924/1925). Brit. J . Surg. 12(45/48), 487, 504 and 522. Ferguson, F. C., Thiersch, J. B., and Philips, F. S. (1950). J. Pharmacol. E z p . Ther. 98, 293-299. Feuer, G., Sosa-Lucero, J. C., Lumb, G., and Moddel, G. (1971). Toxicol. A p p l . Pharmacol. 19,579. Fiore, J. M., and Noonan, F. M. (1959). N . Engl. J . Med. 2 6 0 , 375. Fishkin, A. F., and Lata, G. F. (1957). Fed. Proc., Fed. Amer. Soc. Exp. Biol. 16, 180. Fishkin, A. F., and Lata, G. F. (1958). Endocrinology 63, 162. Fitzhugh, 0. G., Nelson, A. A., and Calvery, H. 0. (1944).J . Pharmacol. E z p . “her. 8 2 , 364. Fletcher, A., York, P. S., Musser, E., and Landes, R. R. (1969). Clin. Med. 76, 15. Forell, M. M., and Stahlheber, H. (1973). Med. Klin. (Munich) 68,1497. Forni, A. M., Koss, L. G., and Geller, W. (1964). Cancer 17, 1348. Forstrom, L., and Lassus, A. (1972). Brit. J. Vener. Dis. 48, 510. Fox, W., Robinson, D. K., Tall, R., Mitchison, D. A., Kent, P. W., and Macfadyen, D. M. (1969). Tubercle 50, 125. Frankel, H. H., Yamamoto, R. S., Weisburger, E. K., and Weisburger, J. H. (1970). Toxicol. A p p l . Pharmacol. 17, 462. Franz, J. M., and Lata, G. F. (1953). Fed. Proc., Fed. Amer. Soc. E x p . B i d . 12, 204. Franz, J. M., and Lata, G. F. (1957). Endocrinology 60, 602. Freyhan, F. A. (1958). I n “Triflnoperazine-Clinical and Pharmacological Aspects” (H. Brill, ed.), pp. 195-205 and 214-219. Lea and Febiger, Philadelphia, Pennsylvania. Friedman, G. D., Siegelaub, A. B., and Seltzer, C. C. (1974). N . Engl. J . Med. 290, 469. Fujii, K.,Jaffe, H., and Epstein, S. S. (1968). Toxicol. A p p l . Pharmacol. 13, 431.

SEXASAFACTORINDRUGEFFECTS

239

Fujii, M. (1926). Nippon Yakurigaku Zasshi 3, 331. FuJii, T.9 Imai, M., Morohashi, c., Wakahara, E., Watanabe, M., and Koyama, R. (1965). J a p . J . Pharmacol. 15, 157. Furner, R. L., Gram, T. E., and Stitzel, R. E. (1969).Biochem. Pharmacol. 18, 1635. Fyro, B., Petterson, U., and Sedvall, G. (1973). Acta Psychiat. Scand. 49, 230. Gadner, H., Gethmann, U., Jessenberger, K., and Riehm, H. (1973). Monatsschr. Kinderheilk. 1 2 1 , 590. Gallagher, C. H . , and Koch, J. H. (1962).Aust. J . Exp. B i d . Med. Sci. 40, 515. Gardier, R. W., and Rohrer, B. (1968).Proc. Soc. Exp. Biol. Med. 127, 405. Garg, L. C. (1974).J . Pharmacol. Exp. Ther. 180, 557. Gault, H. M., Rudwal, C. T., Engles, D. W., and Dossetor, J. (1968). Ann. Intern. Med. 68,906. Gaultier, M., Fournier, E., Efthymion, M. L., Frejaville, J. P., Jouannot, P., and Dentan, M. (1968). Bull. Soc. Med. Hop. Paris 119, 247. Gerard, D. L.. and Saenger, G. (1966). “Out-Patient Treatment of Alcoholism. A Study of Outcome and its Determinants.” Univ. of Toronto Press, Toronto. Gergely, J. (1972).Acta Pharm. Hung. 42, 171. Gessner, T., Acara, M., Baker, J. A , , and Edelman, L. L. (1967). J . Pharm. Sci. 56, 405. Gillette, J. R. (1963).Fortschr. Arzneimittelforsch. 6, 11. Giraldo, B., Blumenthal, M. N., and Spink. W. W. (1969).Ann. Intern. Med. 71, 479. Giri, S. N. (1973). Toxicol. Appl. Pharmacol. 24, 513. Gjensto, H. (1971).Anaesthesist 20, 299. Glaubach, S., and Pick, E. P. (1930). NaunynSchmiedebergs Arch. Exp. Pathol. Pharmakol. 151,341. Goble, F. C., and Konopka, E. A. (1973). Trans. N. Y. Acad. Sci. [Z] 35, 325. Goldberg, L. (1949). Arch. Int. Pharmacodyn. Ther. 78, 17. Goldberg, L., and Stortebecker, T. P. (1943).Acta Physiol. Scand. 5 , 289. Goldin, A,, Greenspan, E. M., Goldberg, B., and Schoenhach, E. B. (1950). Cancer 3, 849. Goldman, P. (1974). Drug Ther. 13, 50-51 and 5658. Gordh, T., (1950).Proc. Roy. SOC.Med. 43, 367. Gordh, T. (1964). Anesthesiology 25, 466. Gorman, C. K., and Weaver, J. A. (1959).Brit. Med. J. 2, 1214. Gottlieb, L. S., and Trey, C. (1974).Annu. Rev. Med. 25, 411-414 and 427-429. Govindaraj, M., and Grant, L. J. (1968). Brit. J. Dis. Chest 6 2 , 27. Gralewski, Z. (1973). Neurol. Neurochir. P o l . 7, 393. Gratton, L. (1960). Union Med. Can. 89, 679. Gray, T. K.. Adarns, L. L., and Fallon, H. J. (1973). J. Clin. Pharmacol. J . New Drugs 13, 61. Green, D. M., and Krueger, R. F. (1963).Feu!. Proc., Fed. Amer. SOC.Exp. Biol. 2 2 , 543. Greiner, A. C., and Berry, K. (1964). Can. Med. Ass. J . 90, 663. Grewe, H. E. (1953a). Anaesthesist 2, 179. Grewe, H. E. (1953b).Z. Gesamte Exp. Med. 1 2 1 , 497. Griffiths, H. F. (1934). Brit. J. Anaesthesiol. 11,89. Grirnlund, K. (1963).Acta Med. Scand., Suppl. 405, 1-26. Grin, E. I. (1962). Bull. W. H. 0 . 26, 797. Grinnell, E. H., and Smith, P. W. (1957).Proc. Soc. Exp. B i d . Med. 94, 524. Grinnell, E. H., Johnson, J. R., Rhone, J. R., TiUotson, A., Noffsinger, J., and Huffman, M. N. (1961). Nature (London) 190, 1117.

240

FRANS C . GOBLE

Grosfeld, J. C. M., Voorhorst, R., De Vries, J., and Kuiper, J. P. (1963). Ned. Tijdschr. Geneesk. 2, 1218. Gsell, O . , Rechenberg, H. K., and Miescher, P. (1957a). Deut. M e d . Wochenschr. 82, 1673. Gsell, O., Rechenberg, H. K., and Miescher, P. (1957b). Deut. M e d . Wochenschr. 82, 1718, 1825, and 1830. v. Kielholz, P., and Hegg, J. J. (1961). Schweiz. M e d . wochenschr. 91, 1529. Guaitani, A., Marcucci, F., and Garattini, S. (1971). Psychopharmacologia 1 9 , 2 4 1 . Guerrero, S., Gallardo, A., and Mu~ioz,C. (1965). Arch. B i d . M e d . E x p . 2, 51. Guilhon, J., Jolivet, G., and Barnabe, R. (1971). Bull. Acad. Vet. Fr. [N.S.] 44, 33. Guinard, L. (1890). C. R . Acad. Sci. 111,981. Gut, I., and Balinova, B. (1972). 11th Annu. Meet. SOC.Toxicol. p. 45 (abstr.). Hadley, W. M., and Miya, T. S. (1972). 11th Annu. Meet. SOC. Tosicol. p. 71 (abstr.). Hadley, W. M., Miya, T. S., and Bousquet, W. F. (1974). Toxicol. Appl. Pharmacol. 28, 284. Hagan, E. C., and Radomski, J. L. (1953). J. Amer. Pharm. Ass. 42, 379. Halevy, S., and Frumin, M. J. (1973). Brit. J. Anaesthesiol. 45,999. Hanzlik, P.J. (1913). J. Amer. M e d . Ass. 60, 957. Hart, J. S., Ho, D. H., George, S. L., Salem, P., Gottlieb, J. A., and Frei, E., 111. (1972). Cancer Res. 32,2711. Hart, L., Guarino, A., and Adamson, R. (1969). Amer. J. Physiol. 2 17, 46. Hatfield, G., Miya, T. S., and Bousquet, W. F. (1972). Toxicol. Appl. Pharmacol. 23, 459. Hazleton, L. W., and Fortunato, F. (1942). J. Amer. Pharm. Ass. 31, 289. Heavner, J. E. (1970). J. Amer. Vet. Med. Ass. 156, 1018. Heilman, E. C., and Kendall, E. C. (1944).Endocrinology 34, 416. Heiner, G. G., Fatima, N., Russell, P. K., Haase, A. T., Ahmad, N., Mohammed, N., Thomas, D. B., Mack, T. M., Muzaffar Khan, M., Knatterud, G. L., Anthony, R. L., and McCrumb, F. R., Jr. (1971). Arner. J. Epidemiol. 94, 435. Heinrich, K., Wegener, I., and Bender, H. J. (1968). Pharmakopsychiat./Neuro-Psychopharmakol. 1, 169. Herzhaft, G. (1969). Encephale 58,486. Herzog, H.,and Keller, R. (1971). Chirurg 42, 156. Hewitt, H. B. (1956). Brit. J. Exp. Pathol. 3 7 , 32. Hewitt, H. B. (1957). J. Endocrinol. 14,394. Hochstein, P.(1971). Exp. Eye Res. 11, 389. Hoff, E. C. (1955). Conn. State Med. J. 1 9 , 793. Hoff, E. C., and McKeown, C. E. (1953). Amer. J. Psychiat. 1 0 9 , 6 7 0 . Hoffler, D., Bittner, B., Fiegel, P., and Kohler, H. (1973). Deut. Med. Wochenschr. 98, 2012. Hoffman, R. A., and Zarrow, M. X. (1958). Amer. J . Physiol. 1 9 3 , 547. Holck, H. G. 0. (1949). J . Amer. Pharm. Ass., Sci. E d . 38,604. Holck, H.G. O., and Cannon, P. R. (1936). J. Pharmacol. Exp. Ther. 5 7 , 289. Holck, H. G. O., and Fink, L. D. (1940). J . Amer. Pharm. Ass. 29, 475. Holck, H. G. O., and Kanin, M. A. (1934). J . Lab. Clin. Med. 19, 1191. Holck, H. G. O., and Kanin, M. A. (1935). Proc. SOC.E z p . Biol. Med. 32, 700. Holck, H. G. O., and Kimura, K. K. (1944). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 3, 75. Holck, H. G . O., and Kimura, K. (1951). J. Amer. Pharm. Ass., Sci. e d . 40, 327. Holck, H. 6. O., and Mathieson, D. R. (1944). J . Amer. Pharm. Ass. 33, 174. Holck, H. G. O., and Smith, E. L. (1938). Amer. J . Physiol. 1 2 3 , 104.

o.,

S E X AS A FACTOR I N DRUG EFFECTS

24 1

Holck, H. G. O., Kanin, M. A., Mills, L. M., and Smith, E. L. (1937). J . Pharmacol. Exp. Ther. 60, 323. Holck, H. G. O., Mathieson, D. R., Smith, E. L., and Fink, L. D. (1942). J. Amer. Pharm. Ass. 31, 116. Holck, H. G . O., Weeks, J. R., Mathieson, D. R., and Duis, B. (1943). J. Amer. Pharm. Ass. 32, 180. Hollister, L. E:. (1958). Ann. Intern. Med. 49, 17. Holmes, F. (1948). Anaesthesia 3, 67. Holmes, K. K.3 Pedersen, A. H. B., Wiesner, P. J., Gutman, L. T., and Bear, D. M. (1971). Abstr., 11th Intersci. Conf. Antimicrob. Ag. Chemother. p. 49. Holmes, K. K., Karney, W. W., Harnisch, J. P., Wiesner, P. J., Turck, M., and Pederson, A. H. B. (1973). J. Infec. Dis. 127, 455. Holzel, A. (1965). Practitioner 194, 98. Homburger, E., Etsten, B., and Himwich, H. E. (1947). J. Lab. Clin. Med. 32, 540. Honegger, H., and Genee, E. (1969). Klin. Monatsbl. Augenheilk. 155, 371. Horhk, J. (1971). Vnitr. Lek. 18, 274. Horinaga, T. (1941). Hukuoka Acta Med. 34, 2 and 17. Homing, M., and Knox, K. L. (1967). Progr. Biochem. Pharmacol. 3, 416. Horst, K., and Willson, J. R. (1935). Sci. Sew. Horton, B. T., Peters, G. A., Blumenthal, L. S. (1945). Proc. Staff Meet. Mayo Clin. 20, 241. Hughes, J. R., Cayaffa, J. J., Pruitt, B. A., Jr., Boswick, J. A., McManus, W. F., Bruck, H. M., and Borges, J. (1973). Dis. Nerv. Syst. 34, 347. Hughes, R. N. (1972). Life Sci., Part I 1 1 , 161. Hughes, R. N., and Syme, L. A. (1972). Psychopharmacologia 2 7 , 3 5 9 . Hultengren, N. (1961). Acta Chir. Scand., S u p p l . 277, 1-84. Hultengren, N., Lagergren, C., and Ljungqvist, A. (1965). Acta Chir. Scand. 130, 314. Hungerbiihler, H., Mallach, H. J., and Schwarz, M. (1972). Arzneim.-Forsch. 22, 1212. Hunter, R., Earl, C. J., and Thornicroft, S. (1964a). Proc. Roy. Sac. Med. 57, 758. Hunter, R., Earl, C. J., and Janz, D. (1964b). J . Neurol., Neurosurg. Psychiat. LN.S.1 2 7 , 219. Hurst, E. W. (1957). J. Pharrr. Pharmucol. 9, 273. Hurst, E. W. (1958). I n “The Evaluation of Drug Toxicity” (A. L. Walpole and A. Spinks, eds.), pp. 12-25. Little, Brown, Boston, Massachusetts. Hurst, E. W., and Paget, G. E. (1963). Brit. J . Derrnatol. 75, 105. Hurst, E. W., Melvin, P. A., and Thorp, J. M. (1960). J . Camp. Pathol. 7 0 , 346. Hurwitz, N. (1969). Brit. Med. J. 1, 536. Hyvert, M. (1956). Encephale 45, 617. Hyyppa, M. T., Cardinali, D. P., and Wurtman, R. J. (1973). Neuroendocrinology 11, 274. Ichniowski, C. T., and Hueper, W. C. (1946). J. Amer. Pharm. A s s . 35, 225. Ijiri, J. (1939). Mitt. Med. Akad. Kioto 26, 653 and 813. Ikonen, M. W., Konjutschenko, T. U., and Kusnetzowa, S. M. (1929). Z h . Eksp. Biol. Med. 1 3 , 86. Ilett, K. F., Reid, W. D., Sipes, I. G., and Krishna, G. (1973). Exp. Mol. Pnthol. 19, 215. Illis, L. S., and Merry, R. T. G. (1972). J. Roy. Coll. Physicians 7, 34. Innian, W. H. W., and Mushin, W. W. (1974). Brit. Med. J . 1, 5. Irwin, S. (1960). In “The Dynamics of Psychiatric Drug Therapy” (G. J. Sarwer-Foner, ed.), pp. 5-28. Thomas, Springfield, Illinois. Irwin, S. (1964). In “Animal and Clinical Pharmacologic Techniques in Drug Evaluation” (J. H. Nodine and P. E. Siegler, eds.), pp. 15-26. Yearbook Publ., Chicago, Illinois.

242

FRANS C . GOBLE

Irwin, S., Slabok, M., and Thomas, G. (1958). J. Pharmacol. E r p . Ther. 124, 206. lwata, H., Yamamoto, I., and Huh, K. (1974). Biochem. Pharmacol. 23, 1144. Jaattela, A , , Mannisto, P., Paatero, H., and Tuomisto, J. (1971). Psychopharrnacologia 2 1, 202. Jacobsen, C. E. (1970). M o d . Vet. Pract. 51, 29. Jacohsen, L., Andersen, E. K., and Thorborg, J. V. (1964). Acta Pathol. Microbiol. Scand. 61, 503. Jacquillat, C., Weil, M., Gemon, M. F., Auclerc, G., Loisel, J. P., Delobel, J., Flandrin, G., Schaison, G., Israel, V., Bussel, A., Dresch, C., Weisgerher, c . , Rain, D., Tanzer, J., Najean, Y., Seligmann, M., Boiron, M., and Bernard, J. (1973). Cancer Res. 33, 3278. Janes, R. G., Alert, H. A., and Paul, N. D. (1959). Diabetes 8 , 466. Janssen, F. W., Young, E. M., Kirkman, S. K., Agersborg, H. P. K., Jr., Tucker, W. E., Jr., and Ruelius, H. W. (1974). Toxicol. A p p l . Pharmacol. 29, 34. Jareho, L. W.,Eyzaguirre, C., and Lilienthal, J. L., Jr. (1950). Proc. Soc. Exp. Biol. M e d . 74, 332. Jick, H., Slone, D., Borda, I. T., and Shapiro, S. (1968a). N. Engl. J . M e d . 279, 284. Jick, H., Slone, D., Borda, I. T., and Shapiro, S. (1968b). N. Engl. J . Med. 279, 887. Jick, H., Slone, D., Shapiro, S., and Lewis, G. P. (1969). J. Amer. Med. Ass. 209, 2013. Johnstone, M. (1953). Anaesthesia 8, 32. Jori, A., Bianchetti, A , , and Prestini, P. E. (1969). Eur. J . Pharmacol. 7, 196. Joshi, P. H., and Conn, H. 0. (1974). Ann. Intern. M e d . 80, 395. Kahler, H. J. (1962). Internist 3, 437. Kalow, W., Britt, B. A., Terreau, M. E., and Haist, C. (1970). Lancet 2, 895. Kasanen, A., and Salmi, H. A. (1961). Ann. M e d . Intern. Fenn. 50, 195. Kasanen, A., Forsstrom, J., and Salmi, H. A. (1962). Acta M e d . Scand. 172, 15. Kask, M. (1929). Folia Neuropathol. Eston. 9, 187. Kato, R. (1959). Atti SOC.Lomb. Sci. Med. B i d . 14, 783. Kato, R. (1974). Drug Metab. Rev. 3, 1. Kato, R., and Gillette, J. (1964). Fed. Proc., F e d . Amer. Soc. Exp. Biol. 23, 538 (abstr.). Kato, R., and Gillette, J. R. (1965a). J. Pharmacol. Exp. Ther. 150, 279. Kato, R., and Gillette, J. R. (1965b). J . Pharmacol. Exp. Ther. 150, 285. Kato, R., Chiesara, E., and Frontini, G. (1962). Biochem. Pharmacol. 11, 221. Kato, R., Takanaka, A., and Takayanagi, M. (1968). Jap. J. Pharmacol. 18, 482. Kato, R., Takanaka, A., Takahashi, A., and Onoda, K. (1969a). Jap. J. Pharmacol. 19,5. Kato, R., Takayanagi, M., and Oshima, T. (1969b). Jap. J. Pharmacol. 19, 53. Kato, R., Onoda, K., and Takayanagi, M. (1970a). / u p . J . Pharmacol. 20, 157. Kato, R., Onoda, K., and Takanaka, A. (1970b). J a p . J . Pharmacol. 2 0 , 546. Jap. J . Pharmacol. 20, 554. Kato, R., Onoda, K., and Takanaka, A. (1970~). Kato, R., Onoda, K., and Takanaka, A. (1970d). Jap. J . Pharrnacol. 20, 562. J. Biochem. (Tokyo) 68, 395. Kato, R., Takanaka, A., and Takayanaghi, M. (1970~). Kato, R., Takahashi, A., and Omori, Y. (1971). Eur. J. Pharmacol. 13, 141. Keen, H., and Jarrett, R. J. (1970). In “Atherosclerosis” (R. J. Jones, ed.), pp. 435449. Springer-Verlag. Berlin and New York. Kellaway, G. S. M., and McRae, E. (1973). N. 2. M e d . J. 78, 525. Kelly, W. A., Nelson, L. W., Hawkins, H. C., and Weikel, J. H., Jr. (1974). Toxicol. Appl. Pharmacol. 27, 629. Kennedy, W. P. (1933). 1.Pharmacol. Exp. Ther. 5 0 , 347. Kernohan, R. J., and Todd, C. (1966). Lancet 1, 621. Keskiner, A. (1973). Curr. Ther. Res. 15, 305.

S E X A S A FACTOR I N DRUG E F F E C T S

243

Khanna, J. M.9 Kalant, H., and Bustos, G . (1967). Can. J . Physiol. Pharmacol. 45, 777, Kessfing, K. H., and Tilander, K. (1963). Exp. Cell Res. 30, 476. K i e s s k , K. H., and Pilstrom, L. (1966). Quart. J. Stud. Ale. 27, 189. King, D. S. (19334 Surg., Gynecol. Obstet. 56, 43. King, D. S. (1933b).J. Amer. Med. Ass. 100, 21. King, J. E. (1964).Amer. J. Obstet. Gynecol. 89, 1019. King, J. E., and Becker, R. F. (1963).Amer. J. Obstet. Gynecol. 86, 856. King, J. E., Becker, R. F., and Marsh, R. H. (1963a).Amer. J. Obstet. Gynecol. 86, 865. King, J. E., Becker, R. F., and James, R. T. (1963b). Amer. J. Obstet. Gynecol. 86, 869. King, J. O., Ilenborough, M. A., and Zapf, P . W. (1972). Lancet 1, 365. Kinsey, V. E. (1940).J . Amer. Phnrm. Ass. 29, 387. Kirkman, H. N. (1968). Ann. N.Y. Acad. Sci. 151, 753. Kjaer, G. C. (1968).Dis. Nerv. Syst. 29, 316. U n e , N. S. (1956). In “Psychopharmacology-A Symposium by the Section on Medical Sciences of the A.A.A.S. and the American Psychiatric Association” (N. S. Mine, ed.), A.A.A.S. l’ubl. 42 pp. 81, 101, 108, and 165. Mine, N. S., and Winick, L. (1971). Curr. Ther. Res. 13, 57. Klonoff, H., Low, M., and Marcus, A. (1973). Can. Med. Ass. J . 108, 150. Klotz, H. P., and BesanGon, L. J. (1937). C. R. SOC.Biol. 1 2 4 , 23. Koch-Weser, J., Sidel, V. W., Federman, E. B., Kanarek, P., Finer, D. C., and Eaton, A. E. (1970).Ann. Intern. Med. 72, 857. Koch-Weser, J.. Sidel, V. W., Dexter, M., Parish, C., Finer, D. C., and Kanarek, P. (1971). Arch. Intern. Med. 1 2 8 , 399. Kodama, M. (1962). Cancer Res. 22, 1212. Kodama, M. (1964).Proc. Soc. E x p . Biol. Med. 116, 1143. Kodama, M., and Kodama, T. (1970). Cancer Res. 30, 221. Konig, J., Fenzki, J. E., Meier-Dorzenbach, E. D., Padberg, G., and Schafer, H. (1973). Arzneim.-Forsch. 2 3 , 1237. Kono, R. (1971).J a p . J. Med. Sci. & B i d . 2 4 , 195. Koppanyi, T., Dille, J. M., and Linegar, C. R. (1936).J. Pharmacof. E x p . Ther. 58, 119. Koss, L. G., and Lavin, P. (1970).J. Nut. Cancer Inst. 44, 1195. Kracke, R. R., and Parker, F. P. (1934).J . Lab. Clin. Med. 19, 799. Kracke, R. R., and Parker, F. P. (1935).J. Amer. Med. Ass. 105,960. Kracke, R. R.. and Parker, F. P. (1939). S. Med. J. 28, 911. Krems, A. D., Martin, A. W., and Dille, J. M. (1941).J. Pharmacol. E x p . Ther. 71, 215. Kronig, B., and Dennig, H. (1970).Arzneim.-Forsch. 20, 1930. Kuntzman, R., and Jacobson, M. (1965).Fed. Proc., Fed. Amer. Soc. E x p . Biol. 24, 152. Kuntzman, R.. Jacobson, M., Schneidman, K., and Conney, A. H. (1964). J. Pharmacol. E x p . Ther. 146, 280. Kupfer, D. J., and Detre, T. P. (1971). Clin. Pharmacol. Ther. 1 2 , 575. Kutt, H., Haynes, J., and McDowell, F. (1966). Arch. Neurol. (Chicago) 14, 489. Laignel-Lavastine, and dHeucqueville, G. (1938). Clinipue 2 9 7 , 2. Lameire, N., Ringoir, S., De Broe, M., Daneels, R., and Lornoy, W. (1970). J . Urol. Nephrol. 76, 993. Landes, R. R., Melnick, I., Hoffman, A. A., Fehrenbaker, L. G., and Oakes, A. L. (1972).Clin. Med. 79, 23. Larsen, K., and M ~ l l e r ,C. E. (1958). Ugeskr. Laeger 1 2 0 , 114. Larsen, K., and MGller, C. E. (1959). Acta Med. Scand. 164, 53. Lehmann, H. E., and Ban, T. A. (1974).Advan. Biochem. Psychopharmacol. 9,24%254. Leibold, J. E. (1%6). Ann. N . Y . Acad. Sci. 135, 904.

244

F R A N S C . GOBLE

Leistenschneider, w., and Ehmann, R. (1973). Schweiz. M e d . Wochenschr. 1037 433. LCvy, A. (1971). Ther. Umsch. 2 8 , 8 2 1 . Ltvy, A., and Stula, D. (1971). Deut. M e d . Wochenschr. 96, 1043. Levy, H.M., Montanez, G., Murphy, E. A., and Dunn, M. S. (1953). Cancer Res. 13, 507. Levy, J., and Meyer, R. (1946). C . R. Soc. B i d . 140,444. Lewis, C. N., Putnam, L. E., Hendricks, F. D., Kerlan, I., and Welch, H. (1952). Antibiot. Chemother. (Wushington, D.C.) 2, 601. Li, F. P., Alter, B. P., and Nathan, D. G. (1972). Blood 40, 153. Lichtenstein, A. (1932). Acta M e d . Scand., Suppl. 49, 1, 20, 24, and 136. Lieber, C. S., and DeCarli, L. M. (1970). J . Biol. Chem. 245, 2505. Linton, A. L. (1972). Can. Med. Ass. J. 107, 749. Lloyd, G. G., Rosser, R. M., and Crowe, M. J. (1973). Lancet 2, 619. Loizeau, E. (1969). Rev. M e d . Suisse Romande 89, 1033. Lomas, J., Boardman, R. H., and Markowe, M. (1955). Lancet 1, 1144. Lorber, J. (1%7). Practitioner 198, 6W. Lotz, F., and Noble, R. L. (1962). 8th Int. Cancer Congr., (Abstr.) pp. 551-552. Louis-Ferdinand, R., Puri, S., Kowal, F., Fuller, G., and La], H. (1970). Abstr., 9th Annu. Meet. Soc. Toxicol. p. 31. Louwerens, B., and Smelik, P . G. (1956). Acta Endocrinol. (Copenhagen) 23, 331. Luby, E. (1967). Proc. 6th Annu. Meet. Amer. Coll. Neuropsychopharmacol., 1967 pp. 1077 and 1081. Luce, J. K., Frei, E., 111, Gehan, E. A., Coltman, C. A,, Jr., Talley, R., and Monto, R. W. (1973). Arch. Intern. Med. 131,391. Lundwall, L., and Baekeland, F. (1971). J. Nerv. Ment. Dis. 153, 381. Luntz, G. R. W. N., and Austin, R. (1960). Brit. M e d . J. 1, 1679. Luthra, Y. K., and Rosenkrantz, H. (1974). Toxicol. Appl. Pharmacol. 27, 158. MacKay, E. M., and MacKay, L. L. (1929). J. Pharmacol. Exp. Ther. 35,67. MacKay, E. M., and MacKay, L. L. (1930). J. Pharmacol. Exp. Ther. 40, 207. MacKay, E. M., and MacKay, L. L. (1935).Arch. Int. P h a r m a c o d p . Ther. 52, 363. Macnaughton Jones (1895a). Brit. M e d . J . 1, 756. Macnaughton Jones (1895b). Lancet 1, 749. McClearn, G. E., and Rodgers, D. A. (1959). Quart. J . Stud. Ale. 20, 691. McColl, J. D., and Sacra, P. (1962). Toxicol. Appl. Pharmacol. 4,631. Maggs, R., and Ellison, R. M. (1960). J . Ment. Sci. 106,590. Maines, M. D., and Westfall, B. A. (1971). Proc. Soc. Exp. Biol. M e d . 138, 820. Maller, O.,and Heller, S. (1971). Dis. N e w . Syst. 32,409. Mallov, S.,and Bloch, J. L. (1956). Amer. J . Physiol. 184, 29. Mandel, A., and Gross, M. (1968). Dis.Nerv. Syst. 29, 32. Mantilla-Plata, B., and Harbison, R. D. (1974). Toxicol. Appl. Pharmacol. 27, 123. Marble, A. (1972). N. Y. State J . Med. 72,2174. March, C. H., and Elliott, H. W. (1954). Proc. SOC.Exp. Biol. Med. 86, 494. Mardones, J. (1960). lnt. Rev. Neurobiol. 2, 41. Markham, C. H. (1971). Clin. Pharmacol. Ther. 12,340. Marquis, D. G., Kelly, E. L., Miller, J. G., Gerard, R. W., and Rapoport, A. (1956). Ann. N . Y. Acad. Sci. 67, 701. Marsboom, R. (1973). Toxicol. Appl. Pharmacol. 24, 371. Marshall, B. E., and Wyche, M. Q., Jr. (1972). Aneithesiology 37, 178. Martin, S. J., Herrlich, H. C., and Clark, B. B. (1940). Anesthesiology 1, 153. Masson, G. M. C., and Beland, E. (1945). Anesthesiology 6 , 4 8 3 . Mastri, A. R., Silverstein, P. M., Gold, L., and Eselius, E. P. (1973). Stroke 4,380.

SEXASAFACTORINDRUGEFFECTS

245

Mayer, R. L., Hays, H. W., Brousseau, D., Mathieson, D., Rennick, B., and Yonkman, F. F. (1946). J. Lab. Clin. Med. 31, 749. Mehta, D. B., and ltil, T. (1973). Brzt. J. Psychiat. 123, 491. Menguy, R., Desbaillets, L., Masters, Y. F., and Okabe, S.(1972). Nature (London) 239, 102. Merlis, S., Sheppard, C., Collins, L., and Fiorentino, D. (1970). Amer. J. Psychiat. 126, 1647. Messerschmldt, O . , Langendorff, H., Rossner, R., and Heppeler, G. (1969). Strahlentherapie 137, 626. Meyler, L. (1962). Geneesk. Gids 40, 87. Michel, D., and Hartleb, 0. (1958). 2. Kreislauflorsch. 47, 580. Miller, F. F. (1967). J. Okla. State Med. Ass. 60, 122. Miller, R. R. (1973). Arner. J . Hosp. Pharrn. 30, 584. Milne, R. C. (1970). Med. J. Aust. 2, 955. Mirone, L. (1958). Quart. J. Stud. Ale. 19, 388. Moeschlin, S. (1957). Schweiz. Med. Vochenschr. 87, 123. Moir, W. M. (1936). J. Pharrnacol. Exp. Ther. 5 7 , 135. Moir, W. M. (1937). J. Pharmacol. Exp. Ther. 5 9 , 68. Monnat, A. (1972). Schweir. Med. Wochenschr. 102, 1876. Montgomery, D. C., and Jackson, R. (1968). Can. Med. Ass. J. 99, 712. Morphew, J. A., and Barber, J. E. (1965). Lancet (London) 1, 650. Morrison, J. F. B., and Pickford, M. (1969). J. Physiol. (London) 202, 37. Morrison, J. F. B., and Pickford, M. (1971). J. Physiol. (London) 216, 69. Morrison, R. K., Brown, D. E., Oleson, J. J., and Cooney, D. A. (1970). Toxicol. Appl. Pharmacol. 17, 1. Morrow, R., and Rabin, D. L. (1966). Clin. Res. 14, 362 (abstr.). Morton, H. J. V. (1944). Lancet 1, 368. Mullick, F. G., Drake, R. M., and hey, N. S. (1973). J. Pediat. 82, 506. Mulroy, R. (1973). Brit. Med. J . 2, 407. Munch, J. C.. and Calesnick, B. (1960). Clin. Pharmacol. Ther. 1, 311. Munkelt, V. P., Lienert, G. A,, Frahm, M., and Soehring, K. (1962). Arzneim.-Forsch. 12, 1059. Mufioz, C., Guerrero, S., Paeile, C., and Campos, I. (1961). Toxicol. Appl. Pharmacol. 3, 445. Muranaka, M., Okumura, H., and Takeda, K. (1969). Jap. J . Allergy 18, 572. Murphy, E. A., and Dunn, M. S. (1957). Cancer Res. 17, 567. Murray, R. M. (1974). Psychol. Med. 4, 69. Nakae, K., Yamamoto, S., Shigematsu, I., and Kono, R. (1973). Lancet 1, 171. Nelson, E. B., Raj, P. P., Belfi, K. J., and Masters, B. S. S. (1971). J. Pharmacol. Ezp. Ther. 178, 580. Ng, K. C., Virabhak, N. K., and Poh, S. C. (1971). Singapore Med. J . 12, 101. Nicholas, J. S., and Barron, D. H. (1932). ./. Pharmacol. Exp. Ther. 46, 125. Nikki, P., and Pohjola, S. (1972). Acta Ophthalmol. 50, 525. Noble, R. L. (1961). Proc. Can. Cancer Res. Conf. 4, 333. Nordenfelt, O., and Ringertz, N. (1961). Acta Med. Scand. 170, 385. Noordhoek, J., and Rumke, C. L. (1969a). NaunynSchmiedebergs Arch. Pharmakol. Exp. Pathol. 264, 288. Noordhoek, J., and Rumke, C. L. (1969b). Arch. Int. Pharmacodyn. Ther. 182, 401. Nordstoga, K . , Helgehostad, A., Loftsgard, G., and Stormorken, H. (1970). Acta Vet. Scand. 11, 481.

246

FRANS C . GOBLE

Novick, W. J., Stohler, C. M., and Swagzdis, J. (1966). J. Pharmacol. E x p . Ther. 1 5 1 , 139. Ogilvie, R. I., and Ruedy, J. (1967). C a n . Med. Ass. J . 97, 1450. O’Gorman Hughes, D. W. (1973). Med. J . Aust. 2 , 361. Ohela, K., and Ahonen, A. (1972). Duodecirn 88, 1177. Ohno, S., Stenius, C., and Christian, L. C. (1970a). Clin. Genet. 1, 35. Ohno, S., Stenius, C., Christian, L., Harris, C., and Ivey, C. (1970b). Biochem. Genet. 4, 565. Oldham, A. J., and Bott, M. (1971). Acta Psychiat. Scand. 47, 369. O’Malley, K . , Crooks, J., Duke, E., and Stevenson, I. H. (1971). Brit. Med. J. 3, 607. Osswald, H. (1972). Experientia 28, 186. Ostler, H. B., Conant, M. A., and Groundwater, J. (1970). Trans. Amer. A c a d . Ophthalmol. Otolaryngol. 74, 1254. Ott, W. H. (1947). J. Amer. Pharm. Ass., Sci. E d . 36, 193. Owen, N. V., Griffing, W. J., Hoffman, D. G., Gibson, W. R., and Anderson, R. C. (1971). Toxicol. A p p l . Pharmacol. 18, 720. Paasikivi, J. (1970). Acta M e d . Scand., Suppl. 5 0 7 , 82. Paeile, C., Guerrero, S., Campos, I., Muiioz, E., Novoa, L., and Murioz, C. (1964). Arch. B i d . M e d . E x p . 1, 152. Paeile, C., Guerrero, S., Gallardo, A., and Mufioz, C. (1965). Arch. B i d . M e d . Exp. 2, 48. Page, J. G., and Vesell, E. S. (1968). Pharmacologist 10, 179. Page, J. G., and Vesell, E. S. (1969a). Proc. Soc. E x p . B i d . M e d . 131, 256. Page, J. G., and Vesell, E. S. (1969b). Pharmacology 2, 321. Pallotta, A. J., Kelly, M. G., Rall, D. P., and Ward, J. W. (1960). Pharmacologist 2, 84. Palm, P . E., Nick, M. S., and Arnold, E. P. (1972). Abstr., 11th Annu. Meet. SOC. Toxicol. p. 57. Palva, I. P., and Mustala, 0. 0. (1970). Acta M e d . Scand. 187, 109. P A n , S. Y . , Gardocki, J. F., Hutcheon, D. E., Rudel, H., Kodet, M. J., and Laubach, G. D. (1955). J. Pharrnacol. Exp. Ther. 1 1 5 , 432. Pancoast, H. K., and Hopkins, A. H. (1915). J. Amer. M e d . Ass. 6 5 , 2 2 2 0 . Patriarca, G., Venuti, A., and Bonini, W. (1973). Ann. Allergy 3 1 , 84. Pedersen, A. H. B. (1972). Pub. Health Rev. 1 , 92. Pedersen, A. H. B., Wiesner, P. J., Holmes, K. K., Johnson, C. J., and Turck, M. (1972). J . Amer. Med. Ass. 220, 205. Pedersen, M. G., Zachariah, P. K., and Juchau, M. R. (1974). Proc. V e s t . Pharmacol. Soc. 17, 262. Pedrique, M. R., Barbera, S., and Ercoli, N. (1970). Ann. Trop. Med. Parasitol. 6 4 , 255. Peters, J. M., and Boyd, E. M. (1967). C a n . J. Physiol. Pharrnacol. 45, 305. Peters, J. M., and Leeuwin, R. S. (1971). Arch. l n t . Pharmacodyn. Ther. 189, 370. Pilasiewicz, B., and Tolwinski, T. (1972). W i a d . Lek. 25, 199. Pisciotta, A. V. (1967). Proc. 6 t h Annu. Meet. Amer. Coll. Neuropsychopharmacol., 1967 pp. 597 and 604. Pisciotta, A. V. (1969). J. Amer. M e d . Ass. 2 0 8 , 1862. Pisciotta, A. V. (1971). Clin. Pharmacol. Ther. 3 2 , 13. Pisciotta, A. V., Ebbe, S., Lennon, E. J., Metzger, G. O., and Madison, F. W. (1958). Amer. J . M e d . 25, 210. Platts, W. M. (1970). M e d . J . Aust. 2 , 500. Plum, P. (1937). “Clinical and Experimental Investigations in Agranulocytosis with Special Reference to the Etiology.” Nyt Nordisk Forlag, Arnold Busck, Copenhagen. Plum, P., and Thomsen, S. (1940). Acta M e d . Scand. 1 0 5 , 301.

SEXASAFACTORINDRUGEFFECTS

247

Poe, C. F., and Suchy, J. F. (1951).Arch. Int. Pharmacodyn. Ther. 86, 449. Poe, C. F., Suchy, J. F., and Witt, N. F. (1936).J. Pharmacol. Exp. Ther. 58, 239. Polak, B. C., Wesseling, H., and Schut, D., Herxheimer, A., and Meyler, L. (1972).Acta Med. Scand. 192,409. Pollack, B. (1956).Amer. J. Psychiat. 113, 557. Poole, J. W. (1970).J. Pharm. Sci. 59, 1255. Prange, A. J., Jr. (1972). Amer. J. Psychiat. 128, 1235. Pratt, T. W. (1933).J. Pharmacol. Exp. Ther. 43, p. 285. Pratt, T. W., Vanlandingham, H. W., Talley, E. E., Nelson, J. M., and Johnson, E. 0. (1932).Amer. J . Physiol. 1 0 2 , 148. Prejean, J. D., Griswold, D. P., Casey, A. E., Peckham, J. C., Weisburger, J. H., Weisburger, E. K., and Wood, H. B., Jr. (1972). Proc. Amer. Ass. Cancer Res. 13, 112. Pscheidt, G. R. (1962). Biochem. Pharmacol. 11, 501. Pyorala, K. (1965). Ann. Med. Exp. B i d . Fenn. 43, Suppl. 3, 43, 48, and 94. Pyorala, K. (1968). Ann. Med. Exp. B i d . Fenn. 46, 23. Quinn, G. P., Axelrod, J., and Brodie, B. B. (1954). Fed. Proc., Fed. Amer. Soc. Exp. Biol. 13, 395. Quinn, G. P., Axelrod, J., and Brodie, B. B. (1958). Biochem. Pharmacol. 1, 152. Ravdin, I. S., and Kern, R. A. (1926). Arch. Surg. (Chicago) 13, 120. Redmond, G. P., and Cohen, G. (1972).Nature (London) 2 3 6 , 117. Reed, W. D., and Williams, R. (1972). Brit. J. Anaesthesiol. 44, 935. Reilly, J., Ahlstrom, A . P., and Watts, J. S., Cassidy, P. S., and Lusky, L. M. (1%9). Toxicol. Appl. Pharmacol. 15, 97. Reiser, M. 11940).NaunynSchmiedebergs Arch. Exp. Pathol. Pharmakol. 195, 199. Remmer, H. (1958a). NaunynSchmiedebergs Arch. Exp. Pathol. Pharmakol. 2 3 3 , 173. Remmer, H. (1958b).Naturwissenschaften 45, 189. Remmer, H. (1959a).NaunynSchmiedebergs Arch. Exp. Pathol. Pharmakol. 235,281. Remmer, H. (1959b). NaunynSchmiedebergs Arch. Exp. Pathol. Pharmakol. 2 3 7 , 296. Remmer, H., and Alsleben, B. (1958). Klin. Wochenschr. 36, 332. Reynolds, T. B. (1963). Med. Trib. p. 15. Rivier, J. L. (1970).Schweiz. Med. Wochenschr. 100, 143. Rivier, J. L., Jaeger, M., Reymond, C. and Desballets, P. (1972). Arch. Mal. Coeur. Vaiss. 65,787. Robbie, D. 5. (1959).Anaesthesia 14, 349. Robertson, J. S. (1949).J. Pathol. Bacteriol. 61, 619. Robins, A. H. (1972). Psychopharmacologia 2 7 , 327. Robinson, M. J., and Rywlin, A. M. (1970).Amer. J. Dig. Dis. 15, 857. Rodensky, P. L., and Wasserman, F. (1964a).Amer. Heart J. 68, 325. Rodensky, P. L., and Wasserrnan, F. (1964b). Amer. Heart J. 67, p. 845. Rodgers, D. A., and McClearn, G. E. (1962a). In “Roots of Behavior. Genetics, Instinct, and Socialization in Animal Behavior” (E. L. Bliss, ed.), Vol. 1, 68-95. Harper (Hoeber), New York. Rodgers, D. A., and McClearn, G. E. (196213). Quart. J. Stud. Ale. 23, 26. Rosen, H., Einheber, A., Brown, N . D., and Wren, R. E. (1967). Nature (London) 214, 671. Rosenkrantz, H., and Braude, M. C. (1974). Toxicol. Appl. Pharmacol. 2 8 , 428. Rosenkrantz, H., Heyman, I. A . , and Braude, M. C. (1974). Toxicol. Appl. Pharmacol. 2 8 , 18. Royal Australasian College of Physicians (1969).Med. J . Aust. 1, 1388. Rucker, M. P. (1927). Amer. J. Obstet. Gynecol. 13, 764.

248

F R A N S C . GOBLE

Rudofsky, S., and Crawford, J. S. (1966). Pharmacologist 8, 181 (abstr.). Rudzki, E. (1972). Przegl. Dermatol. 59, 79. Riimke, C. L. (1966). NaunynSchmiedebergs Arch. Exp. Pathol. Pharmakol. 255, 64. Riimke, C. L. (1968). Arzneim-Forsch. 18, 60. Rumke, C. L., and Noordhoek, J. (1969). Arch. Int. Pharmacodyn. Ther. 182, 2. Russell, E. S. (1955). Brit. Med. J. 1, 826. Rustia, M., and Shubik, P. (1972). J. Nut. Cancer Inst. 48, 721. Rutman, R. J., and Lewis, F. S. (1964). Biochem. Pharmacol. 13, 551. Rutman, R. J., Lewis, F. S., and Price, C. C. (1964). Cancer Res. 24, 626. Salem, H. (1970). Report in files of Smith, Miller and Patch, Inc. Salem, H., and Aviado, D. M. (1974). Drug Inform. 8 , 111. Salokannel, S. J., Palva, I. P., Timonen, T., and Kaipainen, W. J. (1971). Blut 23, 129. Salter, A. J. (1973). Med. J. Aust., Spec. Suppl. 1, 70. Sanchez-Medal, L. (1971). Muenchen. Med. Wochenschr. 1 1 3 , 9 6 0 . Sanchez-Medal, L., Gomez-Leal, A., Duarte, I., and Guadalupe, M. G. (1969). Blood 34, 283. Sangiovanni, F., Taylor, M. A., Abrams, R., and Gaztanaga, P . (1973). Amer. J. Psychiat. 130, 1155. Sarles, H. (1970). Schweiz. Med. Wochenschr. 100, 1190. Schadewald, M., Emerson, G. A., Moore, W. T., and Moore, B. M. (1953). Fed. Proc. 12, Fed. Amer. Soc. Ezp. Biol., Part 1, 364. Scheifley, C. H., and Higgins, G. M. (1940). Amer. J. Med. Sci. 200, 264. Schenkman, J. B., Frey, I., Remmer, H., and Estabrook, R. W. (1967). Mol. Pharmacol. 3, 516. Schick, G., and Virks, J. (1956). N. Engl. J. Med. 255, 798. Schiele, B. C., Gallant, D., Simpson, G., Gardner, E. A , , and Cole, J. 0. (1973). Ann. Intern. Med. 79, 99. Schmidt, W. R., and Jarcho, L. W. (1966). Arch. Neurol. (Chicago) 14, 369. Schofield, C. B. S., Masterton, G., Moffett, M., and McGill, M. I. (1971). J. In&. Dis. 124, 533. Schroeter, A. L., and Pazin, G. J. (1970). Ann. Intern. Med. 72, 553. Schrogie, J. J. (1973). In “Human Reproduction-Conception and Contraception” (E. S. E. Hafez and T. N. Evans, eds.), pp. 529-538. Harper, New York. Schrub, J. C., Wolf, L. M., Courtois, H., and Duhuisson, M. (1973). Nouu. Presse Med. 2, 487. Schultz, F. H., Kay, D. L., Cervenka, H., Kohn, F. E., and Kay, J. H. (1969). Abstr., 8th Annu. Meet. Soc. Tozicol. 91-93. Schultz, W. (1922). Deut. Med. Vochenschr. 48, 1494. Schwetz, B. A., Leong, B. K. J., and Gehring, P. J. (1974). Tozicol. Appl. Pharmacol. 28, 442. Scott, W. J., Beliles, R. P., and Silverman, H. I. (1971). Tozicol. Appl. Pharmacol. 20, 599. Scott, W. J., Butcher, R. E., Kindt, C. W., and Wilson, J. G. (1972). Teratology 6, 239. Scotti, R. (1973). Chemotherapy (Basel) 18, 205. Seidl, L. G., Thornton, G. F., and Cluff, L. E. (1965). Amer. J . Pub. Health 5 5 , 1170. Selye, H. (1941). Proc. SOC.E x p . B i d . Med. 46, 116. Serafetinides, E. A., Coemore, J. P., Rahhal, D. K., and Clark, M. L. (1970). Physicians Drug Manual 2(1/2), 34-36. Seropian, E., Matei, C., and Ranga, B. (1971). Allerg. Immunol. 17, 218. Settipane, G. A., Chafee, F. H., and Klein, D. E. (1974). J. Allergy Clin. Immunol. 53, 200.

SEXASAFACTORINDRUGEFFECTS

249

Shahidi, N. T., and Diamond, L. K. (1959). Amer. J . Dis. Child. 98,293. Shapiro, L. H., and Lentz, J. W . (1963). Amer. J . Obstet. Gynecol. 87, 87. Shapiro, L. H., and Lentz, J. W. (1%7). Amer. J . Obstet. Gynecol. 97,968. Shapiro, S., Siskind, V., Slone, D., Lewis, G. P., and Jick, H. (1%9). Lancet 2, 969. Shapiro, L. H., Lynn, D. R., DiGiacomo, A. M., and Adrian, D. C. (1971). Obstet. Gynecol. 37,414. Sharp, A. A. (1963). Brit. Med. J . 1, 735. Sharp, -4. A. (1965). In “Drug-Induced Diseases” (L. Meyler and H. M. Peck, eds.), Int. Congr. Ser. No. 85, pp. 84-88. Excerpta Med. Found., Amsterdam. Sharpstone, P., Medley, D. R. K., and Williams, R. (1971). Brit. Med. J. 1, 448. Shealy, C. N., Weeth, J. B., and Mercier, D. (1970). J. Amer. Med. Ass. 212, 1522. Shubik, P.,and Ritchie, A. C. (1953). Science 117,285. Siboulet, A., Bouattour, H., Egger, E., and Majewski, E. (1970). Bull. Soc. Fr. Dermatol. Syphiligr. 77, 870. Sim, S. K. (1972). Can. Med. Ass. J . 107,986. Sinanian, R., Atkinson, W. H., and Panzer, J. D. (1973). Curr. Ther. Res. 15, 815. Sise, L. F., Mason, R. L., and Bogan, I. K. (1928). Anesth. Analg. (Cleveland) 7, 187. Sladek, N. E., and Mannering, G. J. (1969). Mol. Pharmacol. 5, 186. Sladek, N. E., Chaplin, M. D., and Mannering, G. J. (1974). Drug. Metab. Disposition 2 , 293. Smidt, N. A,, and McQueen, E. G. (1972). N. Z.Med. J. 76, 397. Smith, D. L., DAmour, M. C., and DAmour, F. E. (1943). J . Pharmacol. Exp. Ther. 77, 184. Smith, J. W., Johnson, J. E., and Cluff, L. E. (1966a). N . Engl. J . M e d . 274, 998. Smith, J.W., Seidl, L. G . , and Cluff, L. E. (1966b). Ann. Intern. Med. 65, 629. Smith, T, W. (1970). J . Pharmacol. E z p . Ther. 175,352. Smithurst, B. A. (1972). N. Z. Med. J. 75, 82. Soberman, R., Brodie, B. B., Levy, B. B., Axelrod, J., and Hollander, V. (1949). J . Biol. Chem. 179, 31. Sparano, B. M. (1965). Lab. Invest. 14, 1931. Spiegel, E. (1943). Fed. Proc., Fed. Amer. Soc. Exp. Biol. 2, 47. Spuhler, O.,and Zollinger, H. U. (1953). Z . Klin. Med. 151, 1. Stewart, R. B., and Cluff, L. E. (1974). Amer. J. Dig. Dis. 19, 1. Stillman, M. T. (1968). Minn. Med. 51, 273. Stoffer, S. S., Hynes, K. M., Jiang, N. S., and Ryan, R. J. (1973). J . Amer. Med. Ass. 225, 1643. Stoneburner, L. T., 111, and Finland, M. (1941). J . Amer. Med. Ass. 116, 1497. Storm van Leeuwen, W. (1916). Pfluegers Arch. Gesamte Physiol. Menschen Tiere 165, 84. Stortebecker, T. P . (1937). Klin. Vochenschr. 16,302. Stortebecker, T.P. (1939). Acta Pathol. Microbiol. Scand., Suppl. 41,43. Strasburg, S. M., and Silver, M. D. (1971). Surg., Gynecol. Obstet. 132,81. Streicher, E., and Garhus, J. (1955). J. Gerontol. 10,441. Stretch, R. (1963). Nature (London) 199,787. Stutzman, L. (1973). Annu. Rev. Med. 24, 325-334. Stutzman, L., and Glidewell, 0. (1973). J. Amer. Med. Ass. 225, 1202. Sweeney, M. J., Gerzon, K., Harris, P. N., Holmes, R. E., Poore, G. A., and Williams, R. H. (1972). Cancer Res. 32, 1795. Szarmach, H., and Poniecka, H. (1972). Przegl. Dermatol. 59, 745. Takahashi, H. (1926). Ber. Gesamte Physiol. Exp. Pharmakol. 34, 591. Takase, T. (1932). Tohoku J . Exp. Med. 18, 443.

250

F R A N S C . GOBLE

Takase, T., and Suzuki, S. (1939). Tohoku J. Exp. Med. 35, 57. Takeuchi, J., Satoshi, K., and Tauchi, H. (1965). Brit. J. Cancer 19, 353. Talcott, R. E., and Stohs, S. J . (1973). Arch. Int. P h a r m a c o d p . Ther. 204, 86. Tatum, H. J., Nelson, D. E., and Kozelka, F. L. (1941). J. Pharmacol. Exp. Ther. 72, 123. Taussig, A. E., and Schnoebelen, P. C. (1931). J . Amer. Med. Ass. 97, 1757. Taylor, D. C., Brown, D. M., Keeble, R., and Langley, P. F. (1974). Xenobiotica 4, 165. Taylor, J. S. (1972). Brit. J. Urol. 44, 126. Taylor, M. A., and Levine, R. (1971). Dis. Neru. Syst. 32, 131. Taylor, W. J. R. (1970). I n t . ' Z . Klin. Pharmakol., Ther. Toxikol. 3, 1. Tegeris, A. S., and Panteleakis, P. N. (1973). Toxicol. Appl. Pharmacol. 25, 497. Tholen, H., Voegtli, J., Renschler, H., and Schaeffer, A. (1956). Schweiz. Med. Wochenschr. 86, 946. Thomas, E. (1963). Brit. J. Anaesthesiol. 35, 327. Thompson, G. R., and Larson, R. E. (1972). Toxicol. Appl. Pharmacol. 21, 405. Thompson, G. R., Baker, J. R., Fleischman, R. W., Rosenkrantz, H., Schaeppi, U. H., Cooney, D. A,, and Davis, R. D. (1972). Toxicol. Appl. Pharmacol. 2 2 , 544. Thompson, G. R., Rosenkrantz, H., Schaeppi, U. H., and Braude, M. C. (1973a). Toxicol. Appl. Pharmacol. 25, 363. Thompson, G. R., Mason, M. M., Rosenkrantz, H., and Braude, M. C. (197313). Toxicol. Appl. Pharmacol. 25, 373. Timson, J. (1970). Brit. J. Pharmacol. 38, 731. Timson, J. (1971). Lancet 2, 212. Tower, D. B., Peters, E. L., and Curtis, W. C. (1963). J. B i d . Chem. 238, 983. Trey, C., Lipworth, L., and Davidson, C. S. (1969). Anesth. Analg. (Cleveland) 48, 1033. Tsukamura, M. (1968). Kekkaku 43, 409. Tucker, W. E., Ruelius, H. W., Janssen, F. W., and Agersborg, H. P. K. (1971). Abstr., 10th Annu. Meet. SOC.Toxicol. p. 1. Tucker, W. E., Jr., Janssen, F. W., Agersborg, H. P . K., Jr., Young, E. M., and Ruelius, H. W. (1974). Toxicol. Appl. Pharmacol. 29, 1. University Group Diabetes Program. (1970). Diabetes 19, Suppl. 2, 747. van der Hem, G. K., Meyler, L., Israels, A. A., Sluiter, H. J., and Nieweg, H. 0. (1961). Ned. Tijdschr. Geneesk. 105, 1629. van Eeken, C. J., Birtwhistle, R. D. R., and Mulder, D. (1970). Acta Pharmacol. Toxicol. 28,425. Varese, L. A. (1967). Minerva Pediat. 19, 885. Verbist, L., Prignot, J., Cosemans, J., and Gyselen, A. (1967). Scand. J. Resp. Dis. 47, 225. Vernier, V. G., Harmon, J. B., Stump, J. M., Lynes, T . E., Marvel, J. P., and Smith, D. H. (1969). Toxicol. Appl. Pharmacol. 15, 642. Vesell, E. S. (1968a). Ann. N . Y . Acad. Sci. 151, Art. 2, 900. Vesell, E. S. (1968b). Pharmacology 1, 81. Vesell, E. S., and Page, J. G. (1968a). Science 159, 1479. Vesell, E. S., and Page, J. G. (1968b). Science 161, 72. Vesell, E. S., and Page, J. G. (1969). J. Clin. Invest. 48, 2202. Villar, T. G. (1970). Scand. J. Resp. Dis., Suppl. 71, 196. Villeneuve, A., Gautier, J., Jus, A., and Perron, D. (1973). Lancet 2, 502. Vogler, W. R. (1971). Cancer 27, 1081. Voller, G. W., Deze, J., Gundlach, U., and Muschard, F. (1972). Neruenarzt 43, 584.

SEXASAFACTORINDRUGEFFECTS

251

Vollmer, E. P., Taylor, D. J., Masnyk, I. J., Cooney, D., Levine, B., Piczak, C., and Trench, L. (1973). Cancer Chemother., Part 3 4, 103. Vollum, D. I., Parkes, J. D., and Doyle, D. (1971). Brit. Med. J. 2, 627. von Rogulja, P., Kovac, W., and Schmid, H. (1973). Acta Neuropathol. 25, 36. Wagenknecht, L. V., and Auvert, J. (1971). Urol. Int. 26, 185. Walker, S. E., and Bole, G. G., Jr. (1973). J. Lab. Clin. Med. 82, 619. Wallerstein, R. O . , Condit, P. K., Kasper, C. K., Brown, J. W., and Morrison, F. R. (1969). J. Amer. Med. Ass. 208, 2045. Wallgren, H. (1959). Nature (London) 184, 726. Walter, J. E., Diener, R. M., and Earl, A. E. (1970). Abstr., 9th Annu. Meet. Soc. Toxicol. p. 85. Walton, C. H. A., and Randle, D. L. (1957). Can. Med. Ass. J . 76, 1016. Ward, J. C., and Crabtree, D. G. (1942). J. Amer. Pharrn. Ass., Sci. E d . 31, 113. Washburn, T. C., Medearis, D. N., Jr., and Childs, B. (1965). Pediatrics 35, 57. Wasmer, J. M., Gauthier-Lafaye, J. P., and Otteni, J. C. (1972). Anesth., Analg., Reanirn. 29, 575. Watts, S. J., Slocombe, R. F., Harbison, W. D., and Stewart, G. A. (1973). Aust. Vet. J . 49, 525. Weaver, L. C., and Kerley, T. L. (1962). J. Pharmacol. Exp. Ther. 135, 240. Weinberg, M. S., Goldhamer, R. E., and Carson, S. (1966). Toxicol. Appl. Pharmacol. 9 , 234. Weintraub, S., Kraus, S., and Wright, L. T. (1950). Proc. Soc. Exp. B i d . Med. 74, 609. Welch, H., Lewis, C. N., and Kerlan, I. (1954). Antibiot. Chemother. (Washington, D.C.) 4, 607. Wemmer, U. (1973). Fortschr. Med. 91, 1232. Werenberg, H. (1955). Nord. med. 54, 1787. Westfall, B. A., Boulos, B. M., Shields, J. L., and Garb, S. (1964). Proc. Soc. Exp. Biol. Med. 115 , 509. Wheater, D. W. F., and Hurst, E. W. (1961). J. Pathol. Bacteriol. 82, 117. Whipple, A. 0. (1918). Surg., Gynecol. Obstet. 26, 29. Whittaker, J. A,, and Price Evans, D. A. (1970). Brit. Med. J. 4, 323. Whittier, J. R., and Korenyi, C. (1968). Int. J. Neuropsychiat. 4, 1. Wiesner. P. J., Holmes, K. K., Sparling, P. F., Maness, M. J., Bear, D. M., Gutman, L. T., and Karney, W. W. (1973). J. Infec. Dis. 127, 461. Wightman. J. A. K. (1968). Brit. J. Surg. 55, 85. Willcox, D. R. C., Gillan, R., and Hare, E. H. (1965). Brit. Med. J. 2, 790. Willcox, R. R. (1970). Brit. J. Vener. Dis. 46, 217. Williams, D. M., Lynch, R. E., and Cartwright, G. E. (1973). Semin. Hematol. 10, 195. Wilson, D. R. (1972). Can. Med. Ass. J. 107, 752. Wilson, I. C., Prange, A. J., Jr., and Lara, P. P. (1974). Amer. J. Psychiat. 131, 21. Winter, H., and Selye, H. (1941). Amer. J . Physiol. 133, 495. Wirz, P., and Arbenz, U. (1970). Schweiz. Med. Wochenschr. 100, 2147. Wise, P. J., and Neu, H. C. (1974). J. Infec. Dis. 129, Suppl., 266. Wolf, S., Simmons, R. L., and Nastuk, W. L. (1964). Proc. SOC.Exp. Biol. Med. 117, 1. Wollenberger, A., and Karsh, M. L. (1951). J. Amer. Pharrn. Ass., Sci. Ed. 40, 637. Wren, B. G. (1967). Med. J. Aust. 1, 847. Wrenshall, G. A , , and Hamilton, J. D. (1957). Ann. N.Y. Acad. Sci. 71, 154. Wynn-Williams, N., and Arris, M. (1958). Tubercle 39, 138. Yaffe, S. J., Krasner, J., and Catz, C. S. (1968). Ann. N.Y. Acad. Sci. 151, 887.

252

F R A N S C . GOBLE

Yoshizawa, H., Tada, Y., Kobayashi, H., and Yui, M. (1972). Kekkaku 47, 121 Yunis, A. A., and Bloomberg, G. R. (1964). Progr. Hematol. 4, 138. Zaleska-Rutczynska, Z., and Krus, S. (1972a). Patol. Pol. 23, 181. Zaleska-Rutczynska, Z., and Krus, S. (197213). Patol. Pol. 23, 185. Zidek, Z., and Janku, I. (1973). Pharmacology 10, 45.

L-Dopa and the Treatment of Extrapyramidal Disease E. WILLIAMSPELTON I1 AND THOMAS N. CHASE Laboratory of Clinical Science National Institute of Mental Health Bethesda, Maryland

I. Introduction . . . . . . . . . . . . . 11. Metabolism . . . . . . . . . . . . . . A. Catecholamine Biosynthesis . . . . . . . B. Catecholamine Degradation . . . . . . . C. Fate of Exogenous L-Dopa . . . . . . . D. Minor (or Potential) Metabolites of L-Dopa . . E. Regulation of Dopamine Metabolism . . . . 111. Pharmacology . . . . . . . . . . . . . A. L-Dopa Effects on Dopaminergic Mechanisms . B. Other Effects of L-Dopa . . . . . . . . IV. Therapeutic Applications . . . . . . . . . A. Idiopathic and Postencephalitic Parkinsonism . B. Other Disorders Having Parkinsonian Features C. Miscellaneous Extrapyramidal Disorders . . . D. L-Dopa Adjuvants . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . . . .

. . . .

. . . . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

253 254 254 257 259 260 262 263 263 269 272 272 280 284 286 293 294

I. introduction The discovery of the impressive ability of L-dopa to ameliorate parkinsonian signs ranks as one of the most important therapeutic results of the process of systematic scientific inquiry of our time. It has led to far-ranging attempts to use L-dopa in the treatment of other neurological, psychiatric, and endocrinological conditions, and to apply the concept of neurohumoral replacement therapy to various other disorders of nervous system function. Although recent experimental evidence has confirmed many of the assumptions and observations which led to the initial use of L-dopa, current information, nevertheless, casts doubt on certain of the earlier notions concerning the pathogenesis of Parkinson’s disease and the mechanism of action of L-dopa. L-Dopa will be considered only in relation to the treatment of extrapyramidal disease. The principal focus will be on the function of central dopamine (DA)-containing neural systems, in view of their 253

254

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

crucial role in mediating the effects of L-dopa on motor behavior. The extensive literature on this and related topics has been the subject of several recent reviews (Barbeau and McDowell, 1970; Brogden et al., 1971; McDowell and Markham, 1971; Sandler, 1972; Sjaastad and Oftedal, 1972; Calne, 1973; Hornykiewicz, 1973a; Yahr, 1973).

II. Metabolism A. CATECHOLAMINE BIOSYNTHESIS 1. Tyrosine Hydroxylase Although the systemic administration of L-dopa elevates intraneuronal concentrations of both dopa and DA, dietary tyrosine serves as the natural precursor of these catechols in the central nervous system (Fig. 1). L-Tyrosine is hydroxylated in the meta-position to L-dopa by tyrosine hydroxylase (E.C. 1.14.3.-) (Dairman et a l . , 1972; Costa and Meek, 1974). The enzyme requires molecular oxygen, ferrous iron, and a reduced pteridine. It is specific for the hydroxylation of L-tyrosine or Lphenylalanine, but ineffective for D isomers or rn-tyzosine and, thus, differs from the rather nonspecific phenol-oxidizing enzyme, tyrosinase. Tyrosine and phenylalanine each inhibits the hydroxylation by tyrosine hydroxylase of the other amino acid, the former 4 times more actively than the latter (Karobath and Baldessarini, 1972).

2 . Dopa Decarboxylase

Dopa decarboxylase (E.C. 4.1.1.26), which is found in the soluble portion of cell homogenates, catalyzes the conversion of dopa to DA. This pyridoxal phosphate-requiring enzyme has been thought to act on all aromatic 1-amino acids including phenylalanine, tryptophan, 5hydroxytryptophan, and histidine (Goldstein et al., 1972a). Recent work, however, suggests the presence of different decarboxylase isoenzymes, at least for dopa and 5-hydroxytryptophan (Sims and Bloom, 1973). The distribution of dopa decarboxylase is ubiquitous both outside and within the central nervous system, notably including high concentrations in the gut mucosa and the walls of brain capillaries (Langelier et a l . , 1972). Normally there is a great excess of dopa decarboxylase, so that inhibition of its activity by as much as 95% has no effect on cerebral catecholamine levels.

tyrosine

tyramine

octopamine

TH

i

I

II

II

P-DH

PNMT

F

I H

O

OH CH3 D CH I - CH2 - NH I

H

m-tyrosine

m- tyramine

epinephrine

FIG. 1. AADC, L-amino acid decarboxylase; DBH, dopamine-phydroxylase; p-DH, p-dehydroxylase; PNMT, phenylethanolamine-N-methyltransferase; and TH, tyrosine hydmxylase.

t~

UI UI

WPAL

P WPAC

J-MT

WPET

on

MOPET

MOPAl

J VLA

1

I

+

oxidation

N-alkylation

i -CI(Z-NHz

-cn>-r

no

NEURoMELANIN DOPAL + DA-------*

/ 6-OHDA

on

m R

-

cn,

01

cn2-cn,

tetrahydropapaveroline

L-DOPA AND EXTRAPYRAMIDAL DESEASE

257

3. Dopamine-P-hydroxylae Dopamine-P-hydroxylase (E.C. 1.14.2.1) mediates the conversion of DA to norepinephrine (NE). A relatively nonspecific enzyme, it catalyzes the side-chain hydroxylation of several phenylethylamine derivatives. Dopamine-/3-hydroxylase contains copper ions and requires molecular oxygen and ascorbic acid. This enzyme is confined to the storage vesicles of NE-containing neurons and is released extraneuronally with NE during nerve impulse activity (Weinshilboum et al., 1971). B. CATECHOLAMINE DEGRADATION Two enzymes are primarily responsible for the degradation of catecholamines, monoamine oxidase (MAO) and catecho1-O-methyltransferase (COMT) (Fig. 2). L-Dopa, although also liable to catabolism by COMT, may be deaminated by tyrosine aminotransferase (Fellman and Roth, 1971; Bartholini et al., 1972).

1. Monoamine Oxidase Monoamine oxidase (MAO) (E.C. 1.4.3.4) deaminates DA or NE to the corresponding aldehyde. These aldehydes can then either be oxidized to an acid by aldehyde dehydrogenase (E.C. 1.2.1.3) or reduced to an alcohol by alcohol dehydrogenase (E.C. 1.1.1.1). P-Hydroxylated phenylethylamines (e.g. , NE) are predominantly metabolized to alcohols (e.g., 3-methoxy-4-hydroxyphenylglycol), whereas non-/3-hydroxylated derivatives (e.g., DA) largely undergo further oxidation to the corresponding acid (e.g., 3,4-dihydroxyphenylacetic acid) (Breese et al., 1969). Monoamine oxidase is widely distributed throughout the body and in the central nervous system occurs in glial cells as well as neurons. The enzyme is present in the outer membranes of mitochondria and may occur postjunctionally, as well as in the presynaptic catecholaminergic FIG.2. AADC, L-amino acid decarhoxylase; AO, aldehyde oxidase (aldehyde dehydrogenase); AR, aldehyde reductase (alcohol dehydrogenase); COMT, catechola-methyltransferase; DA, dopamine; DBH, dopamine-phydroxylase; DHPLA, dihydroxyphenyllactic acid; DHPPA, dihydroxyphenylpyruvic acid; DOPAC, dihydroxyphenylacetic acid; DOPAL, dihydroxyphenylaldehyde; DOPET, dihydroxyphenylethanol; HVA, homovanillic acid; MAO, monoamine oxidase; m-HPAC, m-hydroxyphenylacetic acid; MHPPA, methoxyhydroxyphenylpyruvic acid; MOPAL, methoxyhydroxyphenylaldehyde; MOPET, methoxyhydroxyphenylethanol; 3-MT, 3-methoxytyramine; NE, norepinephrine; 6-OHDA, 6hydroxydopamine; 3-OMD, 34-methyldopa; p-DH, p-dehydroxylase; p-HPPH-ase, p hydroxyphenylpyruvate hydroxylase; TAT, tyrosine aminotransferase; TOPAC, trihydroxyphenylacetic acid; VLA, vanillactic acid.

258

E . WILLIAMS PELTON I1 AND THOMAS N. CHASE

neuron (Horita and Lowe, 1972). By contrast, aldehyde dehydrogenase appears to be primarily presynaptic in location (Duncan et al., 1972) since lesions of the nigrostriatal tract deplete this enzyme to a greater extent than MAO. Multiple isoenzymes of M A 0 have been found in both animal and human brain with differing activity against various monoamine substrates (Youdim et al., 1972). 2. Catechol-0-methyltransferase Catechol-0-methyhansferase (E.C. 2.1.1.6) mediates the transfer of the methyl group of S-adenosylmethionine to the 3-position of catecholamines. The enzyme requires the presence of a divalent cation such as M g + . 0-Methylation can also occur in the 4-position in vitro and has been reported to do so in vivo (Barrass et al., 1972). Catechol-Omethyltransferase acts on all naturally occurring catechols including DA, NE, and dopa, as well as on their deaminated acidic and alcoholic derivatives. Most COMT is found in the soluble fraction of cells, although in contrast to MAO, COMT does not appear to be located within the terminals of catecholamine-containing neurons.

3 . Tyrosine Aminotransferase This enzyme, shown to transaminate dopa as well as tyrosine, has substantial activity in liver and brain (Fellman and Roth, 1971; Fellman et al., 1972). Tyrosine aminotransferase is not pyridoxine-dependent, but is inhibited by dopa as well as by NE due to condensation product formation (Black and Axelrod, 1969). Pharmacological doses of dopa or of a dopa decarboxylase inhibitor might thus affect tyrosine metabolism, since tyrosine aminotransferase is the rate-limiting step in the major pathway for the metabolism of this amino acid (cf. Fellman and Roth, 1971; Sandler et al., 1974). In catecholamine catabolism, either M A 0 or COMT can act on the product of the other so that the sequence of these reactions depends on substrate location. Oxidative deamination is believed to be the preponderant initial fate of unbound intraneuronal catecholamines, whereas catecholamines released extraneuronally may be liable first to 0methylation. Based on this concept, levels of 0-methylated amines have been used as an index of catecholamines released into the synaptic cleft and having access to postsynaptic receptors prior to degradation by COMT, whereas dearninated compounds have been considered a measure of intraneuronal metabolism (Roffler-Tarlov et al., 1971). More recently, however, striatal levels of deaminated DA (3,4-dihydroxyphen-

L-DOPA AND EXTRAPYRAMIDAL DESEASE

259

ylacetic acid) have been found to correlate with firing rates of DA neurons (Bunney et al., 1973a; Roth et al., 1973); this correspondence may reflect the fact that most DA released into the synaptic cleft is recaptured by the presynaptic neuron and is thus exposed to the action of MAO.

C. FATEOF EXOGENOUS L-DOPA A large fraction of orally administered L-dopa is metabolized prior to absorption. Less than 10-20% of the ingested dose reaches the general circulation as intact L-dopa (Coutinho et al., 1971; Bianchine et al., 1971). Like other amino acids, L-dopa is absorbed mainly by the intestine, and therefore, factors that delay gastric emptying retard absorption and increase degradation. Accordingly, a decrease in the pH of gastric juice, anticholinergic drugs, or large protein meals tend to reduce absorption, whereas antacids augment it (Bianchine et a1 ., 1971). Orally administered inhibitors of dopa decarboxylase also diminish gastric degradation of L-dopa (Bianchine et al., 1972). Once absorbed the systemic half-life of L-dopa is short. In the whole mouse, 20 minutes after the intraperitoneal injection of dopa-14C, 3-0methyldopa and phenylcarboxylic acids account for more than 50% of the total radioactivity, and only 20% occurs as DA (peak level) (Wurtman et al., 1970). When a pharmacological dose of L-dopa is added to the tracer radioactive dose, the decarboxylation of dopa becomes saturated at lower doses than the overall metabolism of dopa. Consequently, the percentage of L-dopa-forming catecholamines (predominantly DA) declines as the dose of L-dopa increases. Dopamine-phydroxylase also appears to be saturable, so that at higher dopa dose levels the ratio of DA to NE is increased. Only a small fraction of systemically administered ~ - d o p a - ' ~ C enters the central nervous system, with less than 0.1% of a dose of L-dopa given intraperitoneally to rats being present in brain at any time (Wurtman et al., 1970). The pattern of metabolism of L-dopa is essentially the same in brain as in the periphery. Levels of dopa, DA, and their acid metabolites attain maximum levels after about 30 minutes and then decline rapidly. The principal dopa metabolite, 34-methyldopa, peaks later ( 2 4 hours) and persists at least 48 hours. Chronic dopa treatment or the use of peripheral decarboxylase inhibitors acts to increase further the formation of 3-0-methyldopa (Bartholini and Pletscher, 1968; Kuruma et al., 1970; Wurtman et al., 1970). In man, exogenous L-dopa is also metabolized rapidly by decarboxylation and/or 0-methylation; ordinarily only traces are metabolized by

260

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

transamination. Within 8 hours, 80% of orally ingested labeled dopa is recovered in the urine, with over half occurring as carboxylic acids (Calne et al., 1969a; Peaston and Bianchine, 1970; O’Gorman et al., 1970; Bianchine et a l . , 1972; Goodall and Alton, 1972). Over the remainder of a 5-day period, little additional dopa or its metabolites are excreted, the rest presumably being stored (probably in muscle; Romero et al., 1973) and released more slowly. Usually less than 2% is detectable in feces or expired air. The addition of a peripherally acting inhibitor of dopa decarboxylase to patients receiving L-dopa increases peak plasma dopa levels threefold, and halves the total radioactivity excreted in urine during the first 8 hours (Bianchine et a l . , 1972). Moreover, with a decarboxylase inhibitor, the previously minor transamination pathway becomes the principal route of peripheral degradation of L-dopa, with 3-methoxy-4-hydroxyphenyllactic acid rather than decarboxylated catabolites being the major urinary excretion product (Bianchine et a l . , 1972; Sandler et al., 1974). During L-dopa treatment, the urinary excretion pattern of dopa metabolites in parkinsonian patients does not appear to differ from that found in normal individuals. D. MINOR(OR POTENTIAL) METABOLITES OF L-DOPA In addition to the major routes of catecholamine degradation, several quantitatively minor transformations have been implicated in the pathogenesis of certain disorders or in the central mechanism of action of L-dopa

synthesis and of dopa or DA extrapyramidal (Fig. 2).

1. Methylated Derivatives

3-0-Methyldopa, the immediate methylated product of dopa, is the only dopa metabolite known to penetrate the blood-brain barrier easily. It has a biological half-life of about 12 hours compared to about 30 minutes for L-dopa (Bartholini et a l . , 1971). Although most of 3-0methyldopa in rat is ultimately degraded to homovanillic acid, a significant portion (16% over 24 hours) is demethylated to re-form dopa. For this reason, 3-0-methyldopa could serve as a reservoir for L-dopa and thus provide an explanation for the persistance of small amounts of this amino acid several hours after administration (Bartholini et al., 1972). Although demethylation might occur in liver, there is some evidence attributing it entirely to the action of gut flora (Chalmers et a l . , 1971a). Moreover, with pharmacological doses of L-dopa, substantial quantities of the amino acid are still present in skeletal muscle and spleen 1 hour after administration, suggesting that these tissues

L-DOPA AND EXTRAPYRAMIDAL DESEASE

261

(especially muscle) are the major sources for the delayed dopa release (Romero et a l . , 1973). Dopamine aberrantly methylated in the 4- rather than the 3-position has been considered an endogenous toxin because it occurs normally, produces parkinsonian signs in animals and promotes oxidation of DA and NE (Barrass et al., 1972). 4-0-Methylated products are said to account for a few percent of the 0-methylated DA derivatives in human urine (Sandler, 1972). Methylation of DA in both the 3- and the 4positions has been reported by one investigator (Friedhoff et a l . , 1972). The product, 3,?-dimethoxyphenylethylamine (DMPEA) induces akinesia and rigidity in rodents. This syndrome is however, affected neither by Ldopa nor by apomorphine (Shulgin et a l . , 1969; Prepas et a l . , 1973).

2. m-Tyrosine m-Hydroxyphenylacetic acid, another minor catecholamine metabolite found in animal and human urine, is of importance as a reflection of the compounds from which it presumably derives, m-tyrosine via mtyramine. The latter substances may arise from the p-dehydroxylation of L-dopa and DA by intestinal microorganisms (Goldin et a l . , 1973). Although not a substrate for tyrosine hydroxylase and thus not a precursor of dopa or DA, m-tyrosine has some potentially important pharmacological properties (Section 111, B).

3. Condensation Products Another noteworthy route of catecholamine metabolism involves the condensation of DA with an aldehyde to form a tetrahydroisoquinoline alkaloid (Sourkes, 1971; Sandler, 1973; Collins et a l . , 1973). Although condensation reactions of this type have been observed in vitro and in plants, evidence for their occurrence in the living animal remains equivocal, possibly because the products are themselves extensively metabolized (Collins et a l . , 1973). The presence of such substances has, however, been reported in patients receiving long-term L-dopa treatment (Sandler et a l . , 1973). 4. N-Alkylation Products N-Acetylation of DA reportedly occurs in the isolated, perfused rat liver (Tyce, 1970). Although N-acetyldopamine plays a specific function in insects, its significance in man is unknown. Other alkylated DA derivatives may be dopa-mimetic but are not known to occur naturally (Borgman et a l . , 1973). N-Methylation of several biogenic amines in

262

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

vitro has also been recorded (Laduron, 1972; Banerjee and Snyder, 1973); one of these products, N-methyldopamine (epinine), reportedly has antitremor activity (Goldstein et al., 1972b).

5. Neurotoxins

The catecholamine neurotoxin 6-hydroxydopamine has been suggested to be a metabolic product of dopa. Dopamine itself might be oxidized directly to 6-hydroxydopamine (Sandler, 1972), although no evidence in support of this possibility is yet available. However, there is evidence for the natural formation of a compound, 2,4,5-trihydroxyphenylacetic acid, which resembles 6-hydroxydopamine in structure and may have similar toxicity (Wada and Fellman, 1973). Dopa, after transamination, is hydroxylated by the enzyme p-hydroxyphenylpyruvate hydroxylase (E.C. 1.14.2.2), undergoing the “NIH-shift” to form trihydroxyphenylacetic acid (Calne et al., 1969a; Fellman et al., 1972). One in vitro study suggests that 14% of dopa may be metabolized by this pathway (Wada and Fellman, 1973).

E. REGULATION OF DOPAMINE METABOLISM Neuronal DA concentrations remain notably constant despite large and possibly fluctuating rates of amine release into the synaptic cleft. This situation reflects both the activity of neuronal reuptake mechanisms for DA as well as the close regulation of DA metabolism. Several partially interrelated factors are now known to influence DA synthesis and turnover. Short-term effects of these factors are on the activity of tyrosine hydroxylase, whereas longer term effects involve the quantity of enzyme protein. With alterations of DA concentrations, changes in the activity of enzymes mediating catecholamine degradation may also occur (Weiss et al., 1971a; Tryding et al., 1971; Spector et al., 1972; Baldessarini, 1972). Intraneuronal (presumably extravesicular) DA levels affect tyrosine hydroxylation apparently through competition for the enzyme’s pteridine cofactor (Molinoff and Axelrod, 1971; Javoy et al., 1972; Cheramy et al., 1973). Alterations in nerve impulse traffic also influence the rate of tyrosine hydroxylation (Carlsson et al., 1972; Nyback, 1972). Moreover, the regulation of DA synthesis may be affected by the activity of DA receptors at the synaptic cleft. Drugs such as apomorphine or piribedil, which appear to stimulate postsynaptic dopaminergic receptors, diminish DA formation (Anden et al., 1967; Nyback et al., 1970; Corrodi et al., 1972; Kehr et al., 1972), whereas psychotropic phenothiazine and

L-DOPA AND EXTRAPYRAMIDAL DESEASE

263

butyrophenone derivatives, which block DA receptors, exert the opposite effect on DA synthesis (Corrodi et al., 1967; Nyback and Sedvall, 1971; Nyback, 1972; Kehr et al., 1972). The influence of these receptor active drugs on DA synthesis may be secondary to their effect on firing rates in DA neurons (Bunney et al., 1973a,b), although drug-induced alteration in DA synthesis can occur following axotomy (Nyback, 1972; Kehr et al., 1972). It is not yet clear whether an interneuronal (possibly transsynaptic) or a purely intraneuronal feedback mechanism is involved.

Ill. Pharmacology

A. L-DOPAEFFECTSON DOPAMINERGIC MECHANISMS The antiparkinsonian efficacy of L-dopa has generally been attributed to its ability to restore function to the partially degenerated nigrostriatal dopaminergic pathway. This inference is based on observations suggesting that DA acts as a neurotransmitter at the terminals of the nigrostriatal system, that this pathway influences motor behavior, and that L-dopa loading modifies nigrostriatal tract function even in the presence of a major loss of presynaptic DA terminals.

1. Dopamine As a Neurotransmitter Current evidence supports the view that DA serves as a neurotransmitter in the mammalian nigrostriatal pathway. Dopamine is present in nerve terminals together with enzymes for its synthesis and degradation. At presynaptic sites, DA is concentrated within dense core storage vesicles (Hokfelt, 1968; Fuxe et al., 1970; Bloom, 1972). Release of DA by electrical stimulation has been demonstratated both i n vitro (Ng et al., 1971) and in vivo (Riddell and Szerb, 1971; Chiueh and Moore, 1973). A vigorous neuronal reuptake mechanism as well as enzymic catabolism serve as efficient means for inactivating DA discharged into the synaptic cleft. Finally, the response to the application of DA to striatal cells mimics the effects of normal transmission, whereas drugs that either enhance or diminish the availability of DA to interact with postsynaptic receptors have the same effect as increasing or reducing normal transmission (Ungerstedt et al., 1969; Fuxe and Ungerstedt, 1970; Connor, 1970; York, 1970; McKenzie et al., 1972; Cools, 1973). Three main DA-containing neuronal pathways have now been identified in rat brain (Fuxe et al., 1970; Ungerstedt, 1971a). Largest is the

264

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

nigrostriatal system which arises from cell bodies in the pars compacta of substantia nigra and terminates in the corpus striatum. This pathway affects extrapyramidal function and characteristically degenerates in Parkinson’s disease. Another DA-containing pathway, probably involved in neuroendocrine function, is the tuberoinfundibular system, with cell bodies in the arcuate and anterior periventricular nuclei of hypothalamus, and terminals in the capillary plexus supplying the pituitary. A third dopaminergic tract, the mesolimbic system, has its cell bodies dorsal to the interpeduncular nucleus and terminates in the nucleus accumbens and olfactory tubercle; this pathway may influence behavioral function. In addition, recent evidence suggests the presence of DA terminals in rat cerebral cortex (Thierry et a l . , 1973). The location of the cell bodies of these neurons is unknown. 2. Nigrostriatal Pathway and Motor Function A close relationship between the activity of the dopaminergic nigrostriatal system and motor function is suggested by studies involving surgically or chemically induced interruption of this pathway as well as by studies employing drugs that influence dopaminergic mechanisms. Surgical lesions that damage the nigrostriatal pathway lead to a reduction in striatal DA levels, retrograde degeneration of cells in the pars compacta of the substantia nigra, and characteristic alterations in motor function. Unilateral ventromedial brainstem lesions in monkeys or cats may produce hypokinesia (Sourkes and Poirier, 1966; Stern, 1966) or tremor (Goldstein et a l . , 1972b). The systemic administration of Ldopa tends to restore normal motor function (Bedard et a l . , 1970; Goldstein et al., 1973) but fails to normalize striatal DA levels completely on the side of the lesion (Poirier et al., 1967). The motor effects of L-dopa in these animals are mimicked by apomorphine or piribedil, which are suspected DA receptor agonists (Goldstein et al., 1972b, 1973). Reserpine, which depletes the brain of DA as well as other monoamines, produces a distinctive alteration in motor behavior (often called catalepsy), consisting mainly of hypokinesia and rigidity (Carlsson et a l . , 1957; Carlsson, 1959). This syndrome, which depends on an intact striatum (Arvidsson et a l . , 1967), is reversed by L-dopa but not by 5-hydroxytryptophan, the immediate precursor of serotonin (5-HT). The potent and relatively specific tyrosine hydroxylase inhibitor, cy-methyl-ptyrosine, induces rigidity in rats, and tremor and catatonia in monkeys, which are abolished by L-dopa (Bedard et a l . , 1970; Jurna et a l . , 1972). Neuroleptic agents, such as chlorpromazine and haloperidol, also

L-DOPA AND EXTRAPYRAMIDAL DESEASE

265

produce catalepsy when given in sufficiently high doses. This syndrome presumably results from a drug-induced blockade of striatal DA receptors and is inconsistently reversed by L-dopa treatment (Maj et al., 1972a; Shintomi and Yamamura, 1973). Drugs believed to enhance central DA-mediated transmission also produce characteristic changes in motor function, most notably stereotyped hyperkinesia. Bilateral injections of increasing amounts of L-dopa into the striatum of rats causes forward locomotion, then stereotyped behavior (sniffing), and finally compulsive gnawing (Fuxe and Ungerstedt, 1970). The instillation of L-dopa or DA into some areas of cat caudate nucleus can also induce choreiform movements (Cools, 1972). Similar implantations into the substantia nigra, the site of DA cell bodies but no postsynaptic DA receptors, have no effect on motor function. On the other hand, caudate placement of 3-methoxytyramine, but not of NE or 3,4-dihydroxyphenylacetic acid, has the same effect as DA. The unilateral striatal injection of L-dopa or DA into rats pretreated either with an M A 0 inhibitor (nialamide) alone or reserpine plus nialamide produces asymmetrical posturing and turning to the contralatera1 side (Ungerstedt et al., 1969). Similar effects are observed with the topical application of DA receptor agonists such as apomorphine, whereas systemically administered DA receptor blocking agents, such as chlorpromazine, diminish the response to DA or apomorphine (Ungerstedt et al., 1969). The systemic administration of L-dopa to mice attenuates motor activity at low doses but markedly stimulates it at high doses (Stromberg, 1970; Schoenfeld and Uretsky, 1973). At relatively high dose levels, L-dopa, as well as apomorphine, can induce stereotyped hyperkinetic behavior including sniffing, licking, and gnawing (Randrup and Munkvad, 1968). Centrally but not peripherally active inhibitors of dopa decarboxylase block these behavioral effects of L-dopa (Bartholini et al., 1969) thus indicating that the ability of L-dopa to influence motor function is mediated by DA or some other dopa decarboxylation product. Parenterally administered L-dopa in very high doses to normal monkeys also produces hyperkinesia and abnormal oral-facial movements (Sassin et al., 1972). Pretreatment with p-chlorophenylalanine, which depletes 5-HT by inhibiting tryptophan hydroxylase, has no effect on the response to L-dopa. 3. Mechanism of Action of L-Dopa

L-Dopa effects on motor function have generally been ascribed to an elevation of DA levels in dopaminergic neurons. Precisely how attempts

266

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

to force DA neurons to synthetize and store more DA augments dopaminergic transmission, nevertheless, remains unclear. In the intact rat, loading with exogenous dopa diminishes the spontaneous firing rate of DA cells (Bunney et al., 1973a,b); for enhanced dopaminergic function, an increase in the amount of DA released per impulse would then be required. Alternatively, excessive DA, formed during L-dopa loading by decarboxylation within the terminals of dopaminergic neurons as well as elsewhere, might overflow into the extraneuronal space and diffuse onto postsynaptic DA receptors. If this were to account for the motor effects of L-dopa, then one must assume that nerve-impulsemediated release directly into the synaptic cleft may not be necessary for effective DA-mediated transmission in the nigrostriatal system. Although difficulties arise during attempts to explain precisely how exogenous L-dopa augments DA transmission in the intact nervous system, even greater difficulties arise when the action of L-dopa is considered in the situation where there is a loss or dysfunction of DA cells. Consequences of a decrease in functional DA terminals include: ( 1 ) diminished dopa decarboxylase availability; (2) fewer sites for the storage of DA and for its neurally mediated release into the synaptic cleft; (3) compensatory metabolic changes within residual DA terminals; and (4) enhanced sensitivity of postsynaptic DA receptors. The effects of L-dopa on mammalian motor function appear largely if not entirely contingent upon its decarboxylation (Bartholini et al., 1968, 1%9). Lesions of the nigrostriatal pathway cause a substantial reduction in dopa decarboxylase activity in the corpus striatum (Anden et al., 1965; Goldstein et al., 1969, 1972b; Lancaster et al., 1970; Uretsky and Iverson, 1970). In parkinsonian patients the activity of striatal dopa decarboxylase reportedly declines to 5-10% of normal (Lloyd and Hornykiewicz, 1970; Goldstein et a l . , 1972b). Moreover, the chronic administration of L-dopa has no generalized inductive (or repressive) effect on dopa decarboxylase activity in the intact brain (Lancaster et al., 1970). In the absence of DA terminals, several sites remain for the decarboxylation of exogenous L-dopa. Cells comprising the capillary walls in brain are rich in dopa decarboxylase and accumulate considerable DA during L-dopa treatment. A portion of the DA thus formed might diffuse through the cerebral parenchyma to DA receptor sites (Anden et al., 1972). It is unlikely, however, that DA formed in the capillary walls significantly contributes to the ability of L-dopa to influence motor function in the experimental animal (Bartholini et al., 1969) or in parkinsonian patients (Section IV,D), since the coadministration of the amino acid with drugs that inactivate decarboxylation at this site, but not in the cerebral parenchyma, potentiates rather than diminishes the

L-DOPA AND EXTRAPYRAMIDAL DESEASE

267

centrally mediated effects of L-dopa. Alternatively, it has been suggested that dopa might undergo nonenzymic decarboxylation (Sandler, 1971), although no certain evidence for this possibility is yet available (Bianchine et al., 1973). More probable alternative sites for dopa decarboxylation are the NEand 5-HT-containing neurons. During L-dopa loading, a portion of this amino acid enters 5-HT (and to a lesser extent NE) neurons where decarboxylation to DA occurs; nerve impulse activity might then produce extraneuronal release (Barrett and Balch, 1971; Ng et al., 1972a; Butcher et al., 1972). Conceivably, L-dopa acts by partial conversion of serotonergic nerves to dopaminergic nerves which release DA onto nearby DA receptors during nerve terminal depolarization (Ng et al., 1972a). More likely, however, 5-HT nerves do not act as surrogate DA neurons but merely provide sites for dopa decarboxylation. Overflow of excess DA formed in 5-HT neurons could simply diffuse onto postsynaptic DA receptors. This possibility is supported by the histochemical observation that the stimulating effects of L-dopa on motor behavior do not occur until substantial extraneuronal DA levels are attained (Butcher et d.,1972). A reduction in the number of normally functioning DA terminals would also result in fewer vesicular storage sites where the amine is protected against enzymic degradation. Although the effects of this situation may be partially overcome by storage in other monoaminergic neurons, the functional significance of such cells maintaining an adequate reservoir of DA is uncertain. Moreover, if DA must be present in nerve terminal vesicles for its release during nerve impulse activity, and if normal dopaminergic transmission is contingent upon such release, then a loss of functional DA terminals might be expected to disrupt synaptic transmission in spite of continuing DA formation. Reserpine-induced depletion of DA is a model of vesicular dysfunction, being due to inhibition of vesicular uptake and storage mechanisms. Despite this, the generally accepted explanation for the ability of L-dopa to reverse reserpine catalepsy is that L-dopa loading replenishes DA stores. Although extragranular DA can accumulate in nerve terminals of L-dopa-treated reserpinized rats, provided that M A 0 is inhibited (Fuxe, 1965), histochemical observations in reserpinized animals not receiving an M A 0 inhibitor show that a large part of DA formed in brain from exogenous L-dopa is located extraneuronally (Corrodi and Fuxe, 1967; de la Torre, 1973). These findings support the contention that DA-mediated transmission in the nigrostriatal system can be achieved in the absence of the neurally regulated, pulsatile release of the amine. The dopaminergic system may be relatively unique in this regard.

268

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

Dopamine neurons appear to be tonically active (chronically firing) (Bunney et al., 1973a) and thus may ordinarily be required only to maintain a fairly constant amine level at postsynaptic receptor sites. If the DA system were phasically active (intermittently discharging), restoration of normal function might not be expected from a pharmacological approach providing levels of the neurotransmitter that are relatively fixed and independent of the firing rates of the presynaptic neuron. Dopamine synthesis responds to changes in transmitter concentration, impulse flow, and receptor activity (Section 11, E). Incomplete lesions of the nigrostriatal system also provoke compensatory alterations in the metabolism of residual dopaminergic neurons. For example, subtotal destruction of the substantia nigra results in a partial loss of DA terminals in the ipsilateral striatum. Nevertheless, DA synthesis and utilization in surviving neurons is accelerated (Agid et al., 1973). A similar increase in DA metabolism appears to occur in the striatum of parkinsonian patients (Hornykiewicz, 1972). Presumably these metabolic changes reflect increased nerve impulse activity in residual DA neurons. Such changes may have considerable functional significance since a marked loss of DA neurons occurs in parkinsonian patients before symptoms become clinically evident (Hornykiewicz, 1973a). As yet it is not known how L-dopa loading affects firing rates in these surviving, hyperfunctional DA neurons. An increase in the sensitivity of postsynaptic DA receptors following pharmacological or surgical inactivation of the nigrostriatal system constitutes another important factor affecting the response to L-dopa. Either reserpine treatment or selective destruction of catecholamine nerve terminals with 6-hydroxydopamine markedly potentiates the ability of L-dopa or suspected DA receptor agonists to induce hyperkinesia in rodents (Ungerstedt, 1971b; Schoenfeld and Uretsky, 1973) or primates (Ng et al., 1973). Although the motor effects of L-dopa are substantially enhanced, brain DA increments are less on the 6hydroxydopamine-treated side than contralaterally (Schoenfeld and Uretsky, 1973; Langelier et al., 1973). In fact, the DA increase on the side of the lesion is no more than in areas known to receive few DA terminals (Davidson et al., 1971; Langelier et al., 1973). These observations support the contention that despite an impairment in DA synthesis from exogenous L-dopa in lesioned animals, the efficacy of DA-mediated transmission is heightened. Since either the destruction of DA terminals or the disruption of vesicular storage mechanisms increases the response to apomorphine as well as to dopa, it would appear that an impairment in the ability of neuronal reuptake mecha-

L-DOPA AND EXTRAPYRAMIDAL DESEASE

269

nisms to dissipate the action of DA in the synaptic cleft is not necessary for the enhanced effect of L-dopa on motor function. Moreover, since NE as well as DA cells are damaged by 6-hydroxydopamine, noradrenergic neurons would not appear to play a critical role in the dopa response. In patients with Parkinson’s disease, the number of surviving dopaminergic neurons capable of decarboxylating dopa to DA and releasing it into the synaptic cleft diminishes as the disease progresses (Bernheimer et al., 1973). One might thus expect L-dopa to be least effective in patients with advanced disease. However, there appears to be no definite correlation between pretreatment severity of parkinsonian signs and the response to L-dopa (Hoffmann-LaRoche, Inc., 1971; Chase and Ng, 1972a). A compensatory increase in the sensitivity of striatal DA receptors as the presynaptic fibers degenerate might explain these results. This possibility is supported by the finding that L-dopa dyskinesias occur at a much higher frequency in parkinsonian patients in whom denervation occurs than in equivalently treated patients with amyotrophic lateral sclerosis, who have no known lesions involving the dopaminergic system (Chase et a l . , 1973). Moreover, the dose requirement for L-dopa is usually less in patients with postencephalitic parkinsonism, in whom degeneration of dopaminergic neurons is characteristically more advanced, than in those with the idiopathic form of this disorder (Duvoisin et al., 1972). Denervation supersensitivity may have important implications relative to the therapeutic success of L-dopa in parkinsonian patients, since it would tend to make DA replacement treatment most effective at those sites where it is most needed. In addition, L-dopa-induced increases in brain DA would be relatively less active at normal (nondenervated) DA receptor sites where the amine might produce unwanted clinical effects.

B. OTHEREFFECTSOF L-DOPA Although it now appears that the principal effects of L-dopa on mammalian motor function reflect the increased availability of DA at striatal receptor sites, several other potential mechanisms of action merit consideration. These include (I) effects on nondopaminergic neural systems, (2) formation of pharmacologically active metabolites other than DA, and (3)miscellaneous effects on brain metabolism.

1. Effects on Nondopaminergic Neurohumoral Mechanisms Currently available evidence suggests that central noradrenergic systems play some role in the regulation of motor function (Anden et al.,

270

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

1970, 1973; Ahlenius and Engel, 1971; Ungerstedt, 1971c; Offermeier and Potgieter, 1972) as well as in the ability of L-dopa to affect locomotor activity (Chrusciel and Herman, 1969; Maj et al., 1972b; Plech et al., 1972). L-Dopa might influence central adrenergic function by increasing NE formation in noradrenergic neurons, by partially converting NE neurons to DA neurons, or by the interaction of DA on NE receptors. L-Dopa loading has variable effects on brain NE levels (Everett and Borcherding, 1970; Chalmers et al., 1971b; Narotzky et al., 1973) but appears to accelerate NE turnover (Romero et al., 1972). These metabolic changes may, in part, reflect the displacement of stored NE by DA (Anden and Fuxe, 1971; Ng et al., 1972a). Histochemical observations suggest, however, that it is unlikely that large amounts of DA accumulate in NE neurons after L-dopa loading (Corrodi et al., 1970). Any significant direct effect of DA on noradrenergic receptors would seem improbable since its activity in this regard may be as much as 20-100 times weaker than that of NE (Anden and Fuxe, 1971). L-Dopa loading is attended by a decrease in brain 5-HT together with an initial rise and then a fall in its chief metabolite, 5-hydroxyindoleacetic acid (Bartholini et al., 1968; Everett and Borcherding, 1970; Langelier et al., 1973). This effect largely results from displacement of 5-HT from its binding sites in serotonergic neurons due to uptake of dopa or DA (Barrett and Balch, 1971; Ng et al., 1972b; Butcher et al., 1972; Chase, 1974a), although competition between L-dopa and 5-HT precursors for uptake and decarboxylation might be a contributing factor. Despite these changes and the close functional relationship between central DA and 5-HT systems (Goldstein et al., 1969; Cools, 1973), no convincing evidence is yet available to suggest that L-dopainduced alterations in serotonergic function contributes to its effects on motor function (Chase, 1974a). Abundant clinical and preclinical evidence also points to the interdependence of central dopaminergic and cholinergic systems (Anden and Bedard, 1971; Klawans and Rubovits, 1972; Bartholini et al., 1973) as well as to the possible role of y-aminobutyric acid (GABA)-containing pathways in the modulation of dopaminergic function (Kim et al., 1971). Chronic but not acute treatment with L-dopa produces a small but significant increase in brain acetylcholine levels possibly due to an inhibitory effect of DA on central cholinergic neurons (Sethy and Van Woert, 1973). In support of this possibility, the acute administration of DA into the spinal fluid pathways has been reported to decrease the activity of choline acetylase (an enzyme involved in the synthesis of acetylcholine), whereas chronic administration activates this enzyme (Ho

L-DOPA AND EXTRAPYRAMIDAL DESEASE

271

and Loh, 1972). Long-term L-dopa treatment of parkinsonian patients reportedly restores glutamic acid decarboxylase activity to the normal range (Lloyd and Hornykiewicz, 1973).

2. Pharmacologically Active Metabolites Other Than Dopamine Several noncatecholamine derivatives of L-dopa have been suggested as possibly contributing to the effects of this amino acid on motor activity. A major pathway of L-dopa metabolism both in the periphery and in brain is 0-methylation (Section 11, D). One product, 3-0methyldopa, could in theory serve as a continuing source of L-dopa in view of its relatively long half-life and susceptibility to demethylation (Chalmers et al., 1971a; Bartholini et al., 1972). Other methylated derivatives of dopa, such as 3-methoxytyramine or methylated alcohol and aldehyde products of DA, have demonstrated receptor agonist activity (Ericsson et al., 1971; Cools, 1972), but whether they contribute to dopa’s antiparkinsonian effects or its ability to induce dyskinesias remains uncertain. m-Tyramine, formed from m-tyrosine, also has DA mimicking effects, transiently relieving tremor induced in monkeys by unilateral ventromedial tegmental lesions (Ungerstedt et al., 1973). Although m-tyramine can be P-hydroxylated to octopamine, antitremor activity is not influenced by inhibition of this conversion. The action of m-tyrosine is weaker than that of dopa, but its effect on tremor and its resistance to metabolism by COMT make it of potential value in the treatment of parkinsonism. However, clinical trials of m-tyrosine (without a peripheral decarboxylase inhibitor) have revealed little or no antiparkinsonian activity (Barbeau et al., 1962; Birkmayer and Hornykiewicz, 1964; Shaw et al., 1973). As previously noted (Section 11, D,3), DA can form a number of condensation products in vitro. One product, tetrahydropapaveroline, possesses p-adrenergic agonist activity; another, salsolinol, has certain properties in common with catecholamines (Cohen et al., 1972). Oxidative rearrangement of these substances could result in compounds related structurally to apomorphine, a DA receptor agonist, or to bulbocapnine which can induce catatonia. Although doubt remains as to the natural occurrence of these condensation products, their activity has been ,postulated to influence both the therapeutic and toxic effects of Ldopa (Sourkes, 1971).

3. Miscellaneous Effects L-Dopa loading is attended by various metabolic effects that may modify central nervous system function. Sustained L-dopa administra-

272

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

tion depletes rat brain of S-adenosylmethionine and methionine (Ordonez and Wurtman, 1973). In addition to possibly producing a deficiency state, S-adenosylmethionine depletion might diminish 3-0-methylation of L-dopa or DA thus prolonging their pharmacological activity (Wurtman and Romero, 1972). L-Dopa treatment also reportedly inhibits at least three enzymes that are involved in catecholamine metabolism: dopa decarboxylase (Dairman et al., 1971; Tate et al., 1971; Pfeiffer and Ebadi, 1972), COMT (Chalmers et al., 1971b; Weiss et al., 1971a; Baldessarini, 1972; Sharpless et al., 1973), and tyrosine aminotransferase (Fellman and Roth, 1971). The motor effects of L-dopa might, thus, be augmented by decreasing its peripheral degradation or reducing the central inactivation of DA or by affecting the metabolism of other monoamines. Although L-dopa can compete with other amino acids for transport across biological membranes (Van Woert, 1971), there is no definite evidence of a functionally significant interference in either animals or humans (Liu et al., 1972; Grundig et al., 1972). Similarly, as yet there appears to be no practical consequence of L-dopa’s reported effects on protein synthesis (Bosmann et a l . , 1971; Weiss et al., 1971), glucose metabolism (Rivera-Calimlim, 1971; Rivera-Calimlirn et al. , 1973; Tyce and Owen, 1973), or cellular morphology (Reger et al., 1972; Rivera-Calimlim et a l . , 1973).

IV. Therapeutic Applications A. IDIOPATHICAND POSTENCEPHALITIC PARKINSONISM Parkinsonism is a clinical syndrome with three cardinal manifestations: tremor, rigidity, and akinesia. Onset is usually in late middle life, the course slowly progressive, and the outcome often quite disabling. Parkinsonism may be a consequence of antecedent encephalitis lethargica, manganese intoxication, or neuroleptic drugs. At present, however, the vast majority of cases are of idiopathic origin. 1. Pathology

The characteristic neuropathological feature of idiopathic or postencephalitic parkinsonism is the degeneration of the melanin-containing dopaminergic neurons having their cell bodies in the pars compacta of substantia nigra. Similar, although usually less severe degenerative changes involve nerve cells in other pigmented brainstem nuclei including the locus ceruleus and dorsal nucleus of the vagus. Neurons arising from the locus ceruleus are believed to contain NE. Intracyto-

L-DOPA AND EXTRAPYRAMIDAL DESEASE

2 73

plasmic inclusion bodies (Lewy type), neurofibrillary degeneration, and neurodial scarring are also present. The morphological features of postencephalitic parkinsonism differ slightly from those of idiopathic origin in that there is a relatively greater degree of neuronal loss, neurofibrillary tangles, and gliosis, but Lewy bodies appear somewhat less frequently. No reason for the apparently selective destruction of neuromelanin-containing neurons in parkinsonism is known. Conceivably, these changes may result from the accumulation of an autotoxin (e.g., 6-hydroxydopamine), formed by the oxidation of dopa or DA, and possibly being an intermediate in the neuromelanin synthetic pathway; the pathogenesis of the toxic changes may involve catalase deficiency (Binns et al., 1970; Sandler, 1972; Heikkila and Cohen, 1972a; Wada and Fellman, 1973; Ambani and Van Woert, 1973).

2. Biochemistry The reduction in DA and homovanillic acid levels in the basal ganglia constitutes the most striking biochemical finding in Parkinson’s disease (Hornykiewicz, 1966). Dopamine depletion is most profound in the corpus striatum (caudate and putamen), but substantial reductions also occur in the substantia nigra and globus pallidus. Homovanillic acid concentrations are also reduced, although to a lesser degree than DA. Analogous biochemical observations have also been made in the living patient through measurement of cerebrospinal fluid levels of homovanillic acid. The cerebrospinal fluid content of this DA metabolite appears to derive largely from central rather than peripheral metabolism, and concentrations in the ventricular or cisternal fluid of the experimental animal tend to correlate with levels in the adjacent cerebral tissues (Moir et al., 1970). In parkinsonian patients, numerous studies have shown a marked reduction in steady-state concentrations of homovanillic acid in ventricular, cisternal, or lumbar cerebrospinal fluid (Johansson and Roos, 1967; Guldberg et a l . , 1967). A close correlation has also been found between the overall severity of cardinal parkinsonian signs (particularly rigidity and akinesia) and the reduction in central DA turnover as estimated by the probenecid technique (Chase and Ng, 1972a; Rinne, 1972).

3. Pathogenesis The morphological basis for the cardinal features of Parkinson’s disease has generally been ascribed to the degeneration of the nigrostriatal dopaminergic system. The number of surviving cell bodies in the substantia nigra tends to correlate with the severity of parkinson-

274

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

ian signs (Hornykiewicz, 1973b; Bernheimer et a l . , 1973). A close association also seems to exist between nigral cell loss and the reduction in DA and homovanillic acid concentrations in the corpus striatum (Bernheimer et al., 1973). Presumably, the reductions in DA and homovanillic acid levels as well as in the enzymes mediating DA synthesis occur as a consequence of the degeneration of this neuronal system. Although the foregoing observations as well as the preclinical studies discussed previously (Section 111, A) indicate that the nigrostriatal pathway plays a major role in the pathogenesis of parkinsonism, precisely how much nigrostriatal system dysfunction accounts for the various clinical features of the parkinsonian syndrome remains to be determined. In this regard, several of the other characteristic biochemical changes occurring in parkinsonian patients deserve consideration. The 5-HT content of the striatum, substantia nigra, and several other regions of brain is reduced by about 50% (Fahn et al., 1971; Hornykiewicz, 1972). In the apparent absence of characteristic lesions in the raphe nuclei of the lower brainstem where most 5-HT cell bodies reside, it is tempting to speculate that the observed reductions in 5-HT reflect a secondary, compensatory attenuation of 5-HT-mediated function (Chase, 1974a). Norepinephrine levels in the hypothalamus are also diminished in parkinsonian patients (Hornykiewicz, 1966). The morphological basis for this reduction is uncertain, although it might relate to neuronal degenerative changes in the locus ceruleus. Diminished cerebral concentrations of the GABA-synthetizing enzyme, glutamic acid decarboxylase, have also been reported in autopsy specimens from parkinsonian patients (McGeer et al., 1971; Lloyd and Hornykiewicz, 1973). The functional significance of these changes remains to be elucidated. 4. Therapeutic Effects of L-Dopa There is general agreement that of the cardinal features of parkinsonism, hypokinesia, often the most disabling single component of the syndrome, responds best to L-dopa. Improvement may be manifested by an enhanced ability to walk, better control over fine finger movements, restoration of more normal facial expression, and diminished dysarthria and dysphagia. Rigidity is also usually substantially benefitted by L-dopa treatment, whereas tremor is generally the most resistant of cardinal parkinsonian signs. This latter observation supports biochemical findings in parkinsonian patients suggesting that neurohumoral mechanisms subserving tremor may differ from those relating to akinesia and rigidity (Chase and Ng, 1972a; Bernheimer et al., 1973). Other features of

L-DOPA AND EXTRAPYRAMIDAL DESEASE

275

parkinsonism including a tendency toward a flexed posture, loss of balance, drooling, and oculogyric crises, also tend to improve with Ldopa. As a result, the functional ability of the patient in every day activities is improved. At least half of parkinsonian patients obtain more than a 5wo improvement with L-dopa; included in this group are perhaps 10% of patients who experience a total remission of parkinsonian signs. Another 25%, of patients have a fair response to L-dopa (2049% improvement), which equals or exceeds the response to conventional anticholinergic agents (England and Schwab, 1961). The remaining 20-25% of patients either experience little (< 20%) or no benefit from relatively high doses of L-dopa, or more frequently are unable to tolerate ordinary therapeutic dose levels because of intolerable adverse effects. The degree of improvement with L-dopa does not appear related to the age or sex of patients, nor to the duration or severity of clinical signs. Postencephalitic cases, however, tend to respond to lower doses of L-dopa and to have a more limited tolerance for the drug (Calne et al., 1969b; Krasner and Cornelius, 1970; Duvoisin et al., 1972). Surgical procedures, such as thalamotomy for the relief of parkinsonian symptoms, do not significantly alter the antiparkinsonian response to L-dopa (Hughes et al., 1971; Rinne, 1972). Moreover, no correlation between basal or probenecid-induced accumulations of homovanillic acid in lumbar cerebrospinal fluid and improvement on L-dopa have been found (Chase and Ng, 1972a; Lakke et al., 1973; Weiner and Klawans, 1973). It would thus appear that neither clinical nor chemical criteria exist for predicting the response to L-dopa with confidence.

5. Adverse Effects Nearly all patients given L-dopa experience some adverse effects. Some are more common during the induction phase, whereas others appear only with chronic treatment. Most are controllable by careful adjustment of dosage. Anorexia, nausea, and vomiting are the most common adverse effects of L-dopa during the initial stages of treatment, occurring in as many as 60 or 70% of patients. This incidence falls markedly during maintenance therapy. L-Dopa-induced nausea may reflect stimulation of the brainstem emetic center as well as local gastrointestinal irritation (Lotti and Porter, 1970). Gastrointestinal toxicity can usually be controlled by reducing L-dopa dosage or slowing the rate of dose increase. Taking Ldopa with food also tends to lessen these symptoms. They are most effectively controlled by the addition of a peripheral decarboxylase

276

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

inhibitor to the therapeutic regimen (Section IV, D). Rarely the coadministration of an antiemetic may be necessary. Nonphenothiazine antiemetics, such as trimethobenzamide, are preferable, since phenothiazine derivatives may reduce the therapeutic response to L-dopa. Involuntary movements are the most common adverse effect of L-dopa during maintenance therapy. At least 70% of parkinsonian patients are affected, although the incidence would probably rise to nearly 100% if sufficiently high doses were administered. L-Dopa-induced dyskinesias appear dose-related and characteristically occur at a time when maximum relief of parkinsonian signs has been achieved (Barbeau, 1%9a; Mones et al., 1971; Barbeau et al., 1971a; Markham, 1971). Onset may be gradual or sudden. The abnormal movements may assume a variety of clinical patterns, although in any one patient they remain notably constant in appearance. Commonly they are choreoathetoid in character and may resemble the involuntary movements associated with Huntington’s chorea or those that occasionally complicate the administration of the antipsychotic phenothiazine or butyrophenone derivatives. Intermittent or sustained dystonic posturing such a s curling of the toes, internal rotation at the ankles, or torticollis may also occur. In some instances the postural contortions of dystonia musculorum deformans may be mimicked. The diaphragm and accessory respiratory muscles may also be involved producing an exaggerated panting or gasping type of respiration. L-Dopa dyskinesias may not be noticed by the patient or even denied by him when their existence is called to his attention. When relatively mild, patients often prefer dyskinesias to their condition on lower L-dopa dose levels when there is less relief of parkinsonian symptoms. No definite relationship has yet been established between L-dopa dyskinesias and factors such as the severity or duration of parkinsonian symptoms, or previous neurosurgical procedures. In patients with predominantly unilateral parkinsonism, the severity of abnormal movements has no clear correlation with the lateralization of parkinsonian signs (Kaeser et al., 1970; Mawdsley, 1970; Muenter and Tyce, 1971; Mones et al., 1971; Rinne, 1972). Antecedent stereotaxic thalamotomy may afford some protection against involuntary movements in limbs contralateral to the side of operation, although axial movements can occur even after bilateral thalamic lesions (Siegfried et al., 1970; Hughes et al., 1971; Rinne, 1972; Van Buren et al., 1973). The pathogenesis of L-dopa dyskinesias remains uncertain, although, on the basis of previously cited studies (Section 111, A), hyperfunction of DA-mediated transmission would appear to be involved. Parkinson’s

L-DOPA AND EXTRAPYRAMIDAL DESEASE

277

disease clearly predisposes to these involuntary movements (Barbeau, 1969a; Markham, 1971; Chase et al., 1973). Brain lesions that increase the susceptibility to L-dopa dyskinesias are not, however, limited to those that produce parkinsonism. L-Dopa has occasionally been observed to induce dyskinesias in patients with dystonia musculorum deformans, Wilson’s disease, and progressive supranuclear palsy (Yahr, 1970; Barbeau et al., 1971a). To our knowledge, however, dyskinesias have not been reported in L-dopa-treated patients with central nervous system disorders not involving extrapyramidal function (Yahr, 1970; Mones et al., 1971; Markham, 1971; Mendell et al., 1971), with the exception of individuals previously exposed to psychotropic drugs (Sathananthan et al., 1973) and of one patient with amyotrophic lateral sclerosis (Lieberman et al., 1972). If denervation supersensitivity of the nigrostriatal DA system is crucial to the production of L-dopa dyskinesias, then patients with the most advanced disease and thus the most profound loss of DA neurons would be expected to have the greatest degree of hypersensitivity and therefore the highest incidence of dyskinesias. Such a relationship remains to be rigorously documented. Although available evidence indicates that supersensitivity of denervated DA receptors plays some role in predisposing patients to L-dopa dyskinesias, other factors would appear to be operative. Whether DA receptors mediating the antiparkinsonian activity of L-dopa differ from those mediating its dyskinesiainducing effects also remains to be determined. At present there is no established treatment for dyskinesias that does not also reduce the therapeutic efficacy of L-dopa. Ordinarily they disappear rapidly upon dose reduction, although rarely they may persist for weeks or months following L-dopa withdrawal (Weiss et al., 1971b). Concomitant administration of pyridoxine or of neuroleptics, such as the psychotropic butyrophenone and phenothiazine derivatives, reduces both the antiparkinsonian efficacy and dyskinesia-inducing ability of L-dopa. Intravenous administration of pyridoxine (10 mg) may be used for the rapid control of L-dopa-induced movement disorders in patients who are not receiving a peripheral decarboxylase inhibitor (Jameson, 1970). Although there are preliminary reports suggesting that apomorphine (Duby et a l . , 1972), lithium (Dalen and Steg, 1973), physostigmine (Klawans, 1973), or deanol (Miller, 1974) may ameliorate L-dopa dyskinesias without significantly diminishing antiparkinsonian efficacy, judgment as to the usefulness of these adjuvants must await further clinical study. Mental disturbances constitute the third most common group of

278

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

adverse effects of L-dopa (Goodwin et al., 1971). Approximately 2040% of L-dopa-treated parkinsonian patients develop some alteration in behavioral state. Insomnia, irritability, restlessness, impulsivity, and agitation occur in some patients while others manifest lethargy or somnolence. Affect may shift toward euphoria or depression (Selby, 1973). Occasional suicidal attempts have been attributed to L-dopa therapy. An increase in sexual interest may also occur (Hyyppa et al., 1970; Bowers, 1971), presumably reflecting the overall enhancement in functional activity of patients who are responding to L-dopa rather than a specific aphrodisiac effect. Psychotic manifestations including delusions, paranoid behavior, and hallucinations occur in 3-4% of dopatreated parkinsonian patients (Celesia and Barr, 1970). Confusion or less frequently delirium, similar to that observed with other drug intoxications, also may appear. The mechanisms by which these behavioral effects occur are not understood, although numerous studies have linked the function of dopaminergic systems with affective state, psychotic behavior, aggression, motivation, and cognitive function (Snyder et al., 1970; Goodwin et al., 1971; Thoa et al., 1972; Friedhoff and Alpert, 1973; Donnelly and Chase, 1973). Conceivably, effects of L-dopa on the mesolimbic DA system may in part be responsible. Management of the psychiatric complications of L-dopa treatment depends on their nature and severity. Ordinarily these disorders are mild and can be controlled during the induction stage of L-dopa treatment by temporarily reducing dose increments. More florid reactions may require temporary withdrawal of L-dopa and rarely the administration of a psychotropic agent (e.g., imipramine, thioridazine). Not uncommonly tolerance to the behavioral effects of L-dopa develops with time, and adequate therapeutic doses can eventually be achieved without recurrence of mental disturbances. In some patients, however, these symptoms recur whenever dosage is raised to effective antiparkinsonian levels. Indeed, behavioral alterations constitute the principal reason for discontinuing L-dopa treatment (Langrall and Joseph, 1972; Rinne, 1972). Cardiovascular effects occur in about 20% of L-dopa-treated parkinsonian patients. Orthostatic hypotension is most commonly observed (Calne et al., 1970). The decline in blood pressure is usually asymptomatic, although dizziness or syncope occasionally develop, and, in rare instances, L-dopa-induced hypotension may have contributed to myocardial or cerebral infarction. Difficulties with hypotension are usually most troublesome during the induction phase of treatment, since tolerance to

L-DOPA AND EXTRAPYRAMIDAL DESEASE

279

this effect often develops with continuing L-dopa administration. Hypotension induced by L-dopa appears to relate to the central action of DA or NE as well as to the effects of these dopa metabolites on periphral adrenergic function (Parks et a l . , 1970; Watanabe et a l . , 1970; Henning et a l . , 1972; Hoyer and Van Zwieten, 1972; Yamori et a l . , 1972; Calne, 1973). Etilefrine, an a- and P-adrenergic stimulating agent, reportedly diminishes L-dopa-induced postural hypotension (Miller et a l . , 1973). Various cardiac arrhythmias occasionally complicate treatment with Ldopa. Episodic sinus tachycardia and an increased frequency of atrial or ventricular extrasystoles occur most frequently. Atrial fibrillation has been reported in only rare instances. In such circumstances withdrawal of L-dopa followed, if necessary, by digitalization usually restores normal function. Effects of L-dopa on cardiac rhythm largely arise from the peripheral accumulation of catecholamines since they are reduced by the coadministration of a peripheral decarboxylase inhibitor (Parks et a l . , 1970; Chase and Watanabe, 1972; Kopin, 1973). 6. Mode of Administration L-Dopa is usually administered by mouth although when this is not possible it may be given intravenously or by rectal suppository (Beasley et a l . , 1973). When L-dopa is used without a peripheral decarboxylase inhibitor (Section IV, D), it is necessary to begin treatment at low, therapeutically ineffective doses (Cotzias, 1969; Yahr et a l . , 1969). Over a period of weeks dosage may be gradually increased to maintenance levels. Adverse reactions can ordinarily be overcome through the use of small dose increments as tolerance tends to develop to many of the side effects of L-dopa. Optimal beneficial results generally occur when maximum tolerated dose levels have been achieved. Even then careful patient monitoring, with frequent adjustment of dosage, must be continued in order to maintain full benefit. Optimal dose levels of L-dopa vary considerably but average about 4 gmlday given in divided doses. Approximately 4% of patients achieve optimal benefit at doses less than 2 gmlday, whereas about 16% require L-dopa in excess of 6 gm/day. An arbitrary dose maximum of 10 gm/day is now generally observed, although rarely, patients have required as much as 14 gm/day for optimal results. In order to minimize dose-bydose fluctuations in response, it is advisable to give L-dopa frequently, sometimes as often as every 24 hours while awake. There is a tendency for optimal maintenance dose levels to decline slowly with time (Cotzias et al., 1967; Klawans and Garvin, 1969).

280

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

Possibly the slow accumulation of a minor metabolite of L-dopa (Calne et al., 1969a) or the gradual depletion of some substrate vital to brain

function may account for this effect. More likely explanations are the effects of chronic L-dopa treatment on the activity of several of the enzymes involved in DA catabolism or changes in DA receptor sensitivity (Section 111, A and B). It would appear unlikely that the relatively poor efficacy of L-dopa in some patients reflects an inability to convert L-dopa to DA, since there appears to be no correlation between the response to L-dopa and levels of homovanillic acid in lumbar cerebrospinal fluid (Weiner et al., 1969; Mones et al., 1971; Rinne, 1972) even in patients receiving a peripheral decarboxylase inhibitor (Chase and Watanabe, 1972). Moreover, plasma L-dopa levels are similar in responders and nonresponders to this agent (Mones et al., 1971). 7. Long-Term Effects Long-term results with L-dopa appear to bear out the initial impression of its efficacy and safety (Barbeau, 1973; Godwin-Austen, 1973). The duration of L-dopa treatment in large groups of parkinsonian patients in controlled studies is now approaching 5 years. Initial improvement on maintenance dose levels has been well maintained over this interval. It is yet too early to say whether L-dopa can alter the course of this illness, although one preliminary study indicates that chronic L-dopa treatment may prolong the life of parkinsonian patients (Birkmayer, 1974). On the other hand, some adverse effects of L-dopa, particularly rapid diurnal oscillations in response, appear only after long-term exposure (Damasio et al., 1973). The onset of this on-off response appears more closely related to the duration of L-dopa treatmeet than to the pretreatment severity of parkinsonian signs. This suggests a drug-related effect, possibly analogous to the tardive dyskinesias that occasionally complicate chronic administration of psychotropic drugs. In part, the on-off response reflects an increased sensitivity to fluctuations in plasma L-dopa levels, since it can be eliminated when circulating L-dopa concentrations are stabilized by means of a constant intravenous infusion (Woods et a l . , 1973).

B. OTHERDISORDERSHAVINGPARKINSONIAN FEATURES Reports of the efficacy of L-dopa in the symptomatic relief of parkinsonism have prompted attempts to use this agent in the treatment of related disorders of extrapyramidal function. In general, most

L-DOPA AND EXTRAPYRAMIDAL DESEASE

28 1

successful results have occurred in diseases where parkinsonian signs account for most of the clinical disability.

1. Parkinsonism-Dementia of Guam This degenerative disorder of unknown etiology, unique to the Chamoro-speaking people of Guam and the Mariana Islands, is manifested by parkinsonian signs plus an organic dementia. Unlike idiopathic parkinsonism, parkinsonism-dementia is a rapidly progressive illness, with fatal termination usually occurring within 4 years of onset. Neuropathological findings also differ from idiopathic parkinsonism in that neuronal degenerative changes are present in the cerebral cortex, diencephalon, and basal ganglia as well as in the substantia nigra. Like idiopathic parkinsonism, however, cerebrospinal fluid levels of homovanillic acid and cerebral DA turnover, as estimated by the probenecidloading technique, are characteristically diminished (Brody et al., 1970; Chase et al., 1971). Moreover, clinical trials of L-dopa either alone (Schnur et al., 1971) or in combination with a peripheral decarboxylase inhibitor (Holden et al., 1974) have shown an efficacy on extrapyramidal function similar to that found in patients with idiopathic parkinsonism. No discernible improvement in the cognitive defect occurs, however.

2. Striatonigral Degeneration This is a rare disorder characterized pathologically by neuronal degeneration predominantly in the striatum (especially putamen) as well as substantia nigra, and clinically by parkinsonism sometimes with atypical features. L-Dopa was ineffective in 4 of 5 necropsy proven cases of this disorder (Izumi et a l . , 1971; Rajput et a l . , 1972; Sharpe et al., 1973). Treatment of one individual transiently improved akinesia and rigidity. In this patient there was no detectable DA in the caudate nucleus and putamen (Sharpe et a l . , 1973). In view of the extensive neuronal degeneration in the putamen of this individual, the response to L-dopa might seem quite remarkable. On the other hand, cell loss in the caudate nucleus was only moderate and, thus, caudate neurons with DA receptors may have played an important role in mediating the clinical response to L-dopa.

3. Drug-Induced Parkinsonism Chronic administration of neuroleptic agents including the psychoactive phenothiazine and butyrophenone derivatives as well as reserpine and its congeners occasionally produce parkinsonian signs. Intrave-

282

E. WILLIAMS PELTON I1 AND THOMAS N . CHASE

nously administered L-dopa has been found to reduce drug-induced hypokinesia and rigidity in schizophrenic patients who had been given neuroleptic treatment (Bruno and Bruno, 1966; von Hippius and Logemann, 1970), although, as in the animal studies already noted (Section 111, A), considerable individual variation in response occurred. L-Dopa given orally has been found either to be ineffective (YaryuraTobias et al., 1970) or to diminish akinesia but not tremor or rigidity (Rego et al., 1971). A deterioration in mental function was noted in many of the L-dopa-treated individuals (Yaryura-Tobias et al., 1970). 4. Progressive Supranuclear Palsy

This disorder is manifested clinically by late life onset of supranuclear ophthalmoplegia, pseudobulbar palsy, dysarthria, dystonic hypertonicity of the axial musculature, dementia, and less constant cerebellar and pyramidal tract signs. Tremor may also be present. The illness resembles parkinsonism due to the characteristic masked facies, akinesia, and to some extent the gait difficulties. However, pathological changes are much more extensive, involving not only the substantia nigra but also areas of brainstem and diencephalon not known to contain DA terminals. Numerous case reports describing the effects of L-dopa given alone or with a peripheral decarboxylase inhibitor are now in the literature. Nine of 15 cases summarized by Klawans and Ringel (1971) showed some improvement. Akinesia, rigidity, and postural reflexes were characteristically benefitted, but nonparkinsonian features did not change significantly. Accordingly, there was little overall functional improvement. An increased range of volitional eye movements was noted in 4 of these patients (Dehaene and Bogaerts, 1970; Mendell et al., 1970, Parkes et al., 1971). On the other hand, in 3 other cases occular motility became worse during L-dopa treatment and failed to improve when L-dopa was withdrawn (Klawans and Ringel, 1971). Cerebrospinal fluid levels of homovanillic acid were diminished in 3 of the 4 cases of suspected progressive supranuclear palsy in which these measurements were made (Mendell et a l . , 1970; Klawans, 1973). Parkinsonian manifestations were minimal in the 1 case in whom homovanillic acid levels were in the normal range. 5. Olivopontocerebellar Degeneration This usually sporadic disorder with onset in late middle life is associated with pathological alterations in the pons, medulla, and

L-DOPA AND EXTRAPYRAMIDAL DESEASE

283

cerebellum. Degenerative changes in the substantia nigra and basal ganglia may also occur. Klawans (1973) has reported 3 patients with combined cerebellar and parkinsonian signs consistent with the diagnosis of olivopontocerebellar degeneration. All had diminished lumbar spinal fluid levels of homovanillic acid and all showed definite amelioration of parkinsonian signs with L-dopa. Cerebellar dysfunction was unaffected by this treatment.

6 . Chronic Manganese Zntoxication Clinical features of this disorder include Parkinson-like signs as well as athetoid movements or dystonic posturing. Personality changes may also occur. Pathological alterations in chronic manganese poisoning differ from those of Parkinson’s disease and consist of neuronal loss in the pallidum, striatum, and subthalamic nucleus (Jellinger, 1968). A marked reduction in striatal DA levels has been observed in 1 patient dying with this disorder (cf. Klawans, 1973). In a controlled study, Mena et al. (1970) reported the successful use of L-dopa in 8 disabled Chilean ex-miners with chronic manganese poisoning. Five of the 6 individuals in whom the disease was manifested primarily by bradykinesia showed definite improvement. The sixth patient developed weakness, hypotonia, and tremor during L-dopa treatment but subsequently improved with DL-5-hydroxytryptophan. Two patients with the dystonic form of the disorder also improved with Ldopa.

7. Shy-Drager Syndrome The Shy-Drager syndrome is a multisystem degenerative disorder associated with orthostatic hypotension and other evidence of autonomic dysfunction. Neurological manifestations may include signs of pyramidal, extrapyramidal, cerebellar, and lower motor neuron involvement. Parkinsonism, however, is often the most conspicuous and disabling feature of this illness. Of 5 patients with the Shy-Drager syndrome treated with L-dopa, 1 showed no improvement in neurological signs or in functional disability, and the remaining 4 showed a definite worsening (Aminoff et al., 1973). It may be that striatal or pallidal lesions rather than degeneration of the nigrostriatal DA pathway serve as the anatomic basis for the parkinsonian features of Shy-Drager syndrome. No exacerbation in orthostatic hypotension occurred in these patients; their blood pressures either increased or remained unchanged during L-dopa therapy.

284

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

C. MISCELLANEOUS EXTRAPYRAMIDAL DISORDERS 1. Wilson’s Diseuse Wilson’s disease is an inherited disorder of adolescence and early adult life characterized by choreoathetosis, tremor, and rigidity. Neuropathological changes consist of diffuse ganglion cell degeneration and neuroglial proliferation in the putamen and to a lesser degree in the caudate nucleus and globus pallidus. A failure to synthesize ceruloplasmin or the presence of an abnormal protein with an increased affinity for copper is generally believed to be responsible for the excessive copper deposition and tissue destruction occurring in brain and other organs (Evans et al., 1973). Treatment usually involves administration of chelating agents such as D-penicaamine in conjunction with measures to decrease copper absorption from the gastrointestinal tract. Barbeau and Friesen (1970) using L-dopa in combination with a peripheral decarboxylase inhibitor successfully treated a young girl who had failed to respond to D-penicaamine. When the dose of L-dopa was increased, however, she temporarily developed dysphagia, catatonia, and frequent oculogyric crises. Improvement in rigidity, hypokinesia, and overall motor performance was regained a month after resuming treatment at lower L-dopa doses. In another case report, however, Ldopa given alone was found to be ineffective in a patient who had previously responded to the penicillamine (Morgan et al., 1970). The foregoing observations suggest that it may be worthwhile to try L-dopa in the akinetic-rigid forms of Wilson’s disease, but only in those rare instances when there is an inadequate response to copper chelation therapy.

2. Huntington’s Chorea This autosomal dominant disorder is usually characterized by adult onset of choreiform movements and dementia. Rarely, when onset occurs in childhood, parkinsonian rigidity and akinesia may take the place of the involuntary movements. Pathological findings include widespread neuronal degeneration, especially in the putamen and caudate, as well as in the cerebral cortex. Degenerative changes may also occur in the substantia nigra. Reductions in striatal DA and homovanillic acid concentrations (Bernheimer and Hornykiewicz, 1973) as well as in central DA turnover, as estimated by the probenecid test, have been reported in some studies (Aquilonius and Sjostrom, 1971; La1 et al., 1973; Chase, 1973a). Several young patients with the rigid-akinetic form of Huntington’s

L-DOPA AND EXTRAPYRAMIDAL DESEASE

285

chorea appear to have been benefitted by chronic L-dopa therapy. In 2 cases reported by Barbeau (1969b), L-dopa completely reversed akinesia and rigidity in 1, and led to a reduction in rigidity in the other. In both individuals almost normal motility was regained; at slightly higher doses, however, choreatic movements either appeared or became more prominent. Bird and Paulson (1970) also observed modest improvement in bradykinesia and rigidity during L-dopa treatment of patients with the juvenile form of Huntington’s chorea. Although L-dopa reportedly improved choreatic movements in a single adult with Huntington’s disease (Tan et a l . , 1972), in the vast majority of patients L-dopa induces a marked increase in involuntary movements (La1 et a l . , 1973; Chase, 1973a). L-Dopa has been given to the offspring of patients with Huntington’s chorea in an attempt to identify those with presymptomatic disease (Klawans et a l . , 1972). Given alone or in combination with a peripheral decarboxylase inhibitor L-dopa induced transient chorea in 10 of 28 subjects genetically at risk for Huntington’s disease. None of 24 control subjects manifested chorea at equivalent doses of L-dopa. Although similar results have been reported by Cawein and Turney (1971), it remains to be proven that those who develop dyskinesias will go on to develop Huntington’s chorea. Moreover, a negative response in a person genetically at risk for Huntington’s chorea cannot yet be regarded as indicating the absence of the gene for this disorder. Long-term followup must be carried out to determine the accuracy of these predictions. The psychological impact of the clinical changes produced by L-dopa in patients at risk for Huntington’s chorea as well as the ethics of conducting such tests in the absence of an effective treatment must also be considered. 3 . Dystonia Musculorum Deformans

This is a genetically determined disorder characterized by bizarre involuntary movements and alterations in muscle tone. Onset of symptoms is usually in childhood or adolescence and progression may be slow or intermittent. Spontaneous arrests occur in some individuals. The alterations in motor behavior are generally attributed to a defect in extrapyramidal function, although no characteristic neuropathological changes have been found in patients with this disorder. Clinical trials of L-dopa in patients with dystonia musculorum deformans have yielded variable results (Eldridge et a l . , 1973). It is clear that a majority of patients are not benefitted by L-dopa (Barrett et a l . , 1970; Mandell, 1970). On the other hand, occasional individuals

286

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

have demonstrated striking reductions in muscular rigidity and dystonic movements, together with improvement in overall motor function (Coleman, 190; Chase, 1970; Rajput, 1973; Hongladarom, 1973). Beneficial results in several of these cases occurred at relatively small doses of L-dopa (Rajput, 1973; Hongladarom, 1973). No relationship between the response to L-dopa and mean age when treatment was begun, duration of symptoms prior to therapy, or the mode of inheritance of the disorder has yet been discerned. It also remains unclear how L-dopa acts to benefit some dystonic patients, especially in view of a report that apomorphine abolished dystonic manifestations in 2 patients with dystonia musculorum deformans who had failed to respond to L-dopa (Braham and Sarova-Pinhas, 1973). 4. Athetoid Cerebral Palsy

A controlled trial of L-dopa in 9 athetoid cerebral palsy patients yielded varying degrees of improvement in 8 of these individuals (Rosenthal et al., 1972). Although no patient obtained complete relief of athetosis, there was a sustained reduction in many of the motor manifestations of this disorder, ranging from 18 to 88%, which resulted in a significantly enhanced ability to conduct everyday activities. D. L-DOPA ADJUVANTS 1. Anticholinergic Drugs

Conventional, anticholinergic antiparkinsonian agents tend to potentiate the therapeutic effects of L-dopa (Cotzias, 1969; Yahr et a l . , 1969; McDowell et al., 1970; Markham, 1971). Although the synergistic effect in patients receiving optimal dose levels of L-dopa is usually small, combined therapy may be of practical value in individuals able to tolerate only low doses of L-dopa (Hughes et al., 1971). Hypersalivation and to a lesser extent akinesia and tremor are most often benefitted. Since certain of the adverse effects of cholinolytic drugs (especially behavioral changes) resemble those of L-dopa, it is important that these agents be added with caution to L-dopa-treated patients. Anticholinergic drugs presumably potentiate the antiparkinsonian action of L-dopa not only by their ability to block the central muscarinic action of acetylcholine, but also by prolonging the postsynaptic effects of DA by inhibiting reuptake (Coyle and Snyder, 1969). The importance of this latter effect may be minimal in parkinsonian patients with advanced degeneration of striatal DA terminals.

L-DOPA AND EXTRAPYRAMIDAL DESEASE

287

2. Amantadine Numerous controlled studies have now confirmed the observation by Schwab and co-workers (1969) that amantadine benefits parkinsonian patients (cf. Barbeau et al., 1971b). The therapeutic efficacy of this polycyclic amine, however, is clearly inferior to that of L-dopa and, in some cases may be poorly sustained (Schwab et a l . , 1969; Mawdsley et a l . , 1972; Rinne et al., 1972a; Cox et al., 1973). The mechanism by which amantadine ameliorates parkinsonian signs remains uncertain. It exerts only a relatively weak effect on DA uptake in neostnatal tissues (Heikkila and Cohen, 1972b; Baldessarini et al., 1972) and does not appear to interact directly with postsynaptic dopaminergic receptors (Farnebo et al., 1971) or to possess significant anticholinergic activity (Grelak et al., 1970). There is some evidence to suggest, however, that amantadine increases the amount of DA release during nerve terminal depolarization (Farnebo et al., 1971; Von Voigtlander and Moore, 1971). If it is true that amantadine acts to enhance DA release per impulse, then it might be predicted that the addition of amantadine to patients who are already optimally treated with L-dopa would result in little further clinical improvement without producing or aggravating L-dopa side effects. Such appears to be the case. A majority of clinical trials have shown that amantadine exerts only a minimal potentiating effect on the antiparkinsonian response to L-dopa and fails to mitigate its side effects (Godwin-Austen et al., 1970; Rinne et al., 1972a; Fehling, 1973). A definite synergistic action may occur, however, in patients able to tolerate only suboptimal do? levels of L-dopa. 3. Dopa Decarboxylase Znhibitors Several inhibitors of dopa decarboxylase have now been shown to potentiate the antiparkinsonian efficacy of L-dopa. The most widely tested decarboxylase inhibitors are ~~-serine-2-(2,3,4-trihydroxybenzylhydrazine (serazide, R04-4602) (cf. Barbeau, 1973) and L-a-methyldopahydrazine (carbidopa, MK 486) (Calne et al., 1971; Papavasiliou et al., 1972; Chase and Watanabe, 1972; Rinne et a l . , 197233; Marsden et al., 1973). Results with either drug have been similar. Each reduces the optimal therapeutic dose requirement for L-dopa by 70430%. This potentiation presumably reflects the ability of these drugs to block the extracerebral decarboxylation of systemically administered L-dopa thus allowing more of the unmetabolized amino acid to enter the central

288

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

nervous system. Since these decarboxylase inhibitors penetrate the blood-brain b a m e r poorly, they do not interfere with the central conversion of dopa to DA. When given alone, neither drug exerts any clinically evident effect on parkinsonian patients, since, in the absence of L-dopa loading, decarboxylation is not the rate-limiting step in catecholamine synthesis. An important advantage of the coadministration of either inhibitor with L-dopa is the substantial reduction in gastrointestinal side effects. This usually permits optimal therapeutic dose levels to be attained within a few days. Cardiac arrhythmias, which occasionally arise when L-dopa is given alone, are also decreased by combined therapy (Parks et al., 1970; Chase and Watanabe, 1972; Mars, 1973). The use of peripheral decarboxylase inhibitors in L-dopa-treated parkinsonian patients does not influence centrally mediated actions of L-dopa such as behavioral disturbances, postural hypotension, or involuntary movements. On the other hand, the diurnal intermittency of response to Ldopa occurring in some chronically treated patients may be diminished by the addition of an extracerebral decarboxylase inhibitor. This action may relate to the ability of these agents to prolong the plasma half-life of orally administered L-dopa (Bianchine et al., 1973). No adverse effects of combination therapy with either carbidopa or serazide are known to occur which have not previously been observed with L-dopa given alone (Rinne et al., 1972b; Barbeau, 1973; Jaffe, 1973). The antihypertensive drug, a-methyldopa, when used alone has unpredictable effects on parkinsonian signs. On the other hand, when given in combination with L-dopa, a-methyldopa markedly potentiates the antiparkinsonian action of L-dopa (Sweet et al., 1972; Kofman, 1973; Fermaglich and Chase, 1973). This effect, which may be due to a more complete inhibition of peripheral than central decarboxylation, is often poorly sustained. The diminishing potency of a-methyldopa in some Ldopa-treated patients might relate to a gradual accumulation of a amethyldopamine and/or a-methylnorepinephrine which act as partial DA or NE antagonists. Another inhibitor of aromatic L-amino acid decarboxylase, brocresine (4-bromo-3-hydroxybenzyloxyamine dihydrogen phosphate), has also been reported in an uncontrolled clinical trial to diminish substantially the requirement for L-dopa (Howse and Matthews, 1973). Both the antiparkinsonian and dyskinesia-inducing effect of L-dopa were potentiated. These results are of interest since brocresine, like methyldopa, appears to act centrally as well as peripherally as a decarboxylase inhibitor.

L-DOPA AND EXTRAPYRAMIDAL DESEASE

289

4. Dopamine-P-hydroxylae Inhibitors Two dopamine-P-hydroxylase inhibitors have now been tested in parkinsonian patients. Fusaric acid (5-butylpicolinic acid), when given alone, has little if any effect on parkinsonian signs (Herskovits and Gacitua, 1973; Birket-Smith and Andersen, 1973; Chase, 1974b). Moreover, neither fusaric acid nor disulfiram appear to modify significantly the antiparkinsonian efficacy of L-dopa (Mena et al., 1971; BirketSmith and Andersen, 1973; Herskovits and Gacitua, 1973; Chase, 1974b). On the other hand, with either inhibitor, several investigators have reported a reduction in the severity of L-dopa dyskinesias (Mena et al., 1971; Herskovits and Gacitua, 1973; Birket-Smith and Andersen, 1973).

5 . Catechol-0-methyltransferaseZnhibitors There have been few attempts to use COMT inhibitors in L-dopatreated parkinsonian patients because of the general toxicity of these agents. In one uncontrolled study, N-butylgallate (GPA-1714) was reported to potentiate the antiparkinsonian effects of L-dopa while reducing nausea and dyskinesias (Ericsson et al., 1971). Another preliminary clinical trial with butylgallate failed, however, to confirm these results (Kremzner et al., 1973). Moreover, no decrease in cerebrospinal fluid levels of 3-0-methyldopa was found, thus casting doubt on the ability of this drug to inhibit 0-methylation significantly. 6. Monoamine Oxidase Inhibitors Given alone or in combination with anticholinergic drugs, monoamine oxidase inhibitors exert only a modest beneficial effect on parkinsonian signs (Barbeau et al., 1962; Gerstenbrand and Prosenz, 1965). Attempts to use these agents in L-dopa-treated parkinsonian patients have been abandoned due to their relatively poor ability to potentiate the antiparkinsonian efficacy of L-dopa and the occurrence of severe side effects, especially hypertensive episodes (Birkmayer and Hornykiewicz, 1961; Barbeau et al., 1962; Gerstenbrand and Prosenz, 1965; Hunter et al., 1970). 7. 3-0-Methyldopa Although initial reports suggested that 0-methyldopa may benefit parkinsonian patients (de Ajuriaguerra et al., 1971), subsequent studies have failed to confirm any clinically useful effects of this agent when

290

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

given alone or in combination with a peripheral decarboxylase inhibitor (Geissbuhler et a l . , 1972; Chase and Ng, 197233; Muenter et a l . , 1973). 0-Methyldopa also appears to cause no substantial change in the response to L-dopa, although the optimal dose of L-dopa may increase in some individuals.

8. Dopamine Receptor Agonists Injected apomorphine ameliorates parkinsonian signs, especially tremor and rigidity (Cotzias et a l . , 1970; Braham et a l . , 1970; Castaigne et al., 1971). Apomorphine is also reported to diminish parkinsonism in some L-dopa-treated patients during periodic akinetic episodes (Duby et a l . , 1972). In such patients, apomorphine induces an additive therapeutic effect, while L-dopa dyskinesias are temporarily diminished (Duby et a l . , 1972). These results may indicate that the intermittency of L-dopa action in patients under chronic therapy may reflect a failure of the drug to reach doparninergic receptors and not that these receptors become intermittently refractory to the action of receptor agonists. The ability of a constant intravenous infusion of L-dopa to stabilize the on-off response supports this contention (Woods et a l . , 1973). Recent biochemical and histochemical studies suggest that 1-(2”pyrimidyl)-4-piperonal piperazine (piribedil, ET 495) also acts centrally to stimulate DA receptors (Corrodi et al., 1972; Goldstein et a l . , 1973). The antiparkinsonian efficacy of piribedil in otherwise untreated parkinsonian patients resembles that of conventional anticholinergic preparations and is only about half that of L-dopa (Chase et a l . , 1974b). In Ldopa-treated patients, orally administered piribedil usually provides relatively little additional benefit (Vakil et a l . , 1972). Adverse effects closely resemble those of L-dopa. As expected of a DA receptor agonist, piribedil treatment appears to reduce substantially the central turnover of this arnine (Chase, 197313; Chase et a l . , 1974b).

9. Hydroxytryptamine Precursors and Inhibitors Administration of L-5-hydroxytryptophan together with a peripheral decarboxylase inhibitor increases akinesia and rigidity in untreated parkinsonian patients (Chase et a l . , 1972), whereas L-tryptophan in combination with pyridoxine (although not alone) also exacerbates parkinsonian signs (Hall et al., 1972). Treatment with the tryptophan hydroxylase inhibitor, p-chlorophenylalanine, however, has no consistent effect on parkinsonism (Chase, 1972; Van Woert et a l . , 1972). In Ldopa-treated parkinsonian patients, neither L-tryptophan nor 5-hydroxy-

L-DOPA AND EXTRAPYRAMIDAL DESEASE

291

tryptophan significantly modifies parkinsonian signs or L-dopa dyskinesias (Yahr, 1970; Coppen et a l . , 1972; Duvoisin, 1973). Moreover, the addition of p-chlorophenylalanine to the therapeutic regimen of patients receiving optimal doses of L-dopa has no clinically obvious effects (Chase, unpublished observations). These findings support the contention that the tryptophan- and 5-hydroxytryptophan-induced exacerbation of parkinsonism in non-L-dopa-treated patients may reflect indirect effects on dopaminergic mechanisms rather than a direct action on 5HT-containing neural systems (Chase, 1974a). 10. Tricyclic Antidepressants Imipramine and desipramine have both been reported to ameliorate parkinsonian rigidity and tremor (Strang, 1965; Laitinen, 1969). In Ldopa-treated patients, however, imipramine has little, if any, effect on antiparkinsonian efficacy or dyskinesias (Yahr, 1973; Chase, 1974a). In rodents, imipramine potentiates the ability of L-dopa to increase spontaneous motor activity but does not influence L-dopa-induced stereotyped behavior (Ross et al., 1971; Friedman and Gershon, 1972). Moreover, pretreatment with imipramine reportedly prevents the reduction in brain 5-HT produced by L-dopa, without altering the rise in DA levels (Friedman and Gershon, 1972). These results may be attributable to imipramine’s relatively greater ability to block monoamine transport into serotonergic than into dopaminergic or noradrenergic neurons (Carlsson, 1970; Lindbrink et a l . , 1971). 11. Hypothalamic Tripeptides Both thyrotropin-releasing hormone (TRH) and melanocyte-stimulating hormone release-inhibiting factor (MIF) have been found to potentiate Ldopa-induced changes in motor behavior in mice pretreated with an M A 0 inhibitor (Plotnikoff et a l . , 1971, 1972). Recently, a stimulatory effect of MIF on striatal DA synthesis has been reported (Friedman et a l . , 1973). The intravenous injection of 20 mg of either TRH or MIF into untreated parkinsonian patients or to those receiving optimal doses of Ldopa failed to produce any consistent effect on cardinal parkinsonian signs or L-dopa dyskinesias (Woods and Chase, 1973; Chase et a l . , 1974~).Results of another preliminary study, however, suggest that the acute intravenous administration of MIF produces a slight reduction in rigidity and that orally administered MIF (50 mg/day) tends to improve parkinsonian signs (Kastin and Barbeau, 1972). In patients receiving Ldopa, the addition of 50 mg of MIF, given orally in divided doses,

292

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

reduced L-dopa dyskinesias but failed to improve overall motor performance beyond that occurring with L-dopa alone (Kastin and Barbeau, 1972). 12. Lithium Lithium salts are reported to have reduced L-dopa dyskinesias in 2 parkinsonian patients (Dalen and Steg, 1973). A concomitant increase in parkinsonian symptoms occurred in 1 of these individuals. These preliminary observations are of interest in view of recent findings that chronic lithium treatment inhibits striatal DA synthesis (Friedman and Gershon, 1973) and that lithium may ameliorate involuntary movements in patients with Huntington’s chorea or tardive dyskinesias (Dalen, 1973; Prange et al. , 1973). 13. Pyridoxine Treatment with pyridoxine rapidly reverses both the therapeutic and toxic effects of L-dopa (Duvoisin et al., 1969). Although this action may be useful in the occasional dopa-treated patient who suddenly develops severe dyskinesias, the major practical importance of this observation is the identification of one source of therapeutic failure with L-dopa-the ingestion of multiple vitamin or antiemetic preparations containing pyridoxine. Pyridoxine markedly reduces plasma L-dopa levels in patients receiving both drugs in combination (Leon et al., 1971). It would thus appear that pyridoxine diminishes the centrally mediated pharmacological effects of dopa by reducing the amount of this amino acid available for transport into brain (but see Pfeiffer and Ebadi, 1972). Since pyridoxine, given alone, does not significantly alter plasma dopa levels (Leon et al., 1971), this cofactor of dopa decarboxylase may be acting by accelerating the conversion of dopa to DA in the periphery rather than by removing dopa by complexing it with pyridoxine (e.g., by Schiff base formation; Evered, 1971) or by diverting it to some alternative metabolic pathway. This contention appears to be supported by recent clinical observations indicating that peripheral decarboxylase inhibitors block the pyridoxine reversal of the dopa effect in parkinsonian patients (Cotzias and Papavasiliou, 1971; Klawans et al., 1971; Yahr et al. , 1972; Duvoisin, 1973). Moreover, in carbidopa-pretreated patients, pyridoxine no longer depresses plasma dopa levels following oral administration of this amino acid (Cote et al., 1971). Patients under treatment with L-dopa in combination with a peripheral decarboxylase inhibitor may, thus, ingest foods rich in pyridoxine or take drugs containing this vitamin without fear of losing the therapeutic benefit of

L-DOPA AND EXTRAPYRAMIDAL DESEASE

293

L-dopa. Although it has been suggested that chronic L-dopa therapy may lead to iatrogenic pyridoxine deficiency, the weight of current evidence fails to support this contention (Yahr et al., 1972).

V. Conclusions Five years after its introduction, L-dopa, given orally at high doses, remains by far the most effective approach to the symptomatic relief of Parkinson’s disease. L-Dopa has also been found to benefit parkinsonian signs arising as a part of several other central nervous system disorders. Although some individuals with various nonparkinsonian extrapyramidal syndromes improve during the administration of this amino acid, significantly beneficial results as yet appear unpredictable and of uncertain clinical usefulness. With the notable exception of peripheral decarboxylase inhibitors, currently available L-dopa adjuvants usually confer little additional benefit to patients already able to tolerate optimal dopa dose levels. Extracerebral inhibitors of dopa decarboxylase markedly diminish L-dopa gastrointestinal and cardiac toxicity and, thus, permit the rapid attainment of effective antiparkinsonian dose levels. Alterations in the function of the nigrostriatal dopaminergic system are necessary, even if insufficient, to produce the cardinal signs of Parkinson’s disease and to account for their amelioration by L-dopa. Replenishment of central DA stores, the original rationale for the use of L-dopa in the treatment of parkinsonian patients, appears primarily responsible for the therapeutic efficacy of L-dopa. Nevertheless, the ability of L-dopa to improve parkinsonian signs may be contingent upon a series of remarkably fortuitous circumstances. 1. Systemically administered L-dopa, the immediate precursor of DA, readily penetrates the blood-brain barrier (unlike the immediate precursors of NE or GABA) and leads to increased DA formation in the cerebral parenchyma (in contrast to the effect of exogenous choline on brain acetylcholine). 2. Although dopaminergic neurons degenerate in Parkinson’s disease, abundant alternative sites for the decarboxylation of dopa to DA (e.g., within NE or 5-HT neurons) exist in brain (in contrast to the exclusive localization of the enzyme synthesizing NE within neurons normally containing this amine); excess DA formed at such alternative sites can overflow into the extraneuronal space and diffuse into the vicinity of postsynaptic DA receptors. 3. The DA system appears to be tonically rather than phasically active; instead of requiring neuronally mediated, pulsatile release of DA,

294

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

relatively normal synaptic function may be achieved merely by attaining sufficient ambient concentrations of the amine within the synaptic cleft. 4. Supersensitivity of denervated postsynaptic DA receptors may confer specificity to the clinical response to L-dopa treatment; thus, DA may be most effective at denervated sites where it can act to restore normal function and least active at normally innervated DA receptors, where increased stimulation might produce unwanted clinical effects. Although the precise mechanism by which L-dopa influences extrapyramidal function remains incompletely understood, available evidence suggests that the concept of neurohumoral replacement therapy may have relatively limited applicability to the treatment of brain disease. REFERENCES Agid, Y.,Javoy, F., and Glowinski, J. (1973). Nature (London), New Biol. 245, 150-151. Ahlenius, S., and Engel, J. (1971). NaunynSchmiedebergs Arch. Pharmakol. Exp. Pathol. 270,349-360. Amhani, L. M., and Van Woert, M. H. (1973). Trans. Amer. Neurol. Ass. 98, 7-10. Aminoff, M. J., Wilcox, C. S., Woakes, M. M., and Kremer, M. (1973). J. Neurol., Neurosurg. Psychiat. 36,35&353. Anden, N.-E., and Bedard, P. (1971). J. Pharm. Pharmacol. 23,460462. Anden, N.-E., and Fuxe, K. (1971). Brit. J. Pharmacol. 43,747-756. Anden, N.-E., Magnusson, T., and Rosengren, E. (1965). Acta Physiol. Scand. 64, 127135. Anden, N.-E., Ruhenson, K., Fuxe, K., and Hokfelt, T. (1967). J. Pharm. Pharmacol. 19, 627-629. Anden, N.-E., Corrodi, H., Fuxe, K., Hokfelt, B., Hokfelt, T., Rydin, C., and Svensson, T. (1970). Life Sci. 9, Part 1, 513-523. Anden, N.-E., Engel, J., and Ruhenson, A. (1972). NaunynSchmiedebergs Arch. Pharmakol. Exp. Pathol. 273, 11-26. Anden, N.-E., Stromberg, U., and Svensson, T. H. (1973). Psychopharmacologia 29, 289298. Aquilonius, S. M., and Sjostrom, R. (1971). Life Sci. 10, 405414. Arvidsson, J., Jurna, I., and Steg, G. (1967). Life Sci. 6, 2017-2020. Baldessarini, R. J. (1972). J. Pharm. Pharmacol. 24, 78-79. Baldessarini, R. J., Lipinski, J. F., and Chace, K. V. (1972). Biochem. Pharmacol. 21, 77-87. Banerjee, S. P., and Snyder, S. H. (1973). Science 182, 74-75. Barbeau, A. (1973). In “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 173-198. Raven, New York. Barbeau, A. (1969a). Can. Med. Ass. J . 101, 791-800. Barbeau, A. (1969b). Lancet ii, 1066. Barheau, A., and Friesen, H. (1970). Lancet i, 1180-1181. Barbeau, A., and McDowell, F. H. (1970). “L-dopa and Parkinsonism.” Davis, Philadelphia, Pennsylvania. Barheau, A., Sourkes, T. L., and Murphy, G. F. (1962). I n “Monoamines and the Central Nervous System” (J. de Ajuriaguerra, ed.), pp. 247-262. Masson. Paris.

L-DOPA AND EXTRAPYRAMIDAL DESEASE

295

Barbeau, A.3 Mars, H., and Gillo-Joffroy, L. (1971a). I n “Recent Advances in Parkinson’s Disease” (F. H. McDoweu and C. H. Markham, eds.), Vol. 8, pp. 203-237. Davis, Philadelphia, Pennsylvania. Barbeau, A., Mars, H., Botez, M. I., and Joubert, M. (1971b). Can. Med. Ass. J . 105, 42-

46. Barrass, B. C., Coult, D. B., and Pinder, R. M. (1972). J . Pharm. Pharmacol. 24, 49% 501. Barrett, R. E., and Balch, T. S. (1971). Ezpen’entia 27, 663-664. Barrett, R. E., Yahr, M. D., and Duvoisin, R. C. (1970). Neurology 20, Part 2, 107-113. Bartholini, G., and Pletscher, A. (1968). J. Pharmacol. E z p . Ther. 161, 14-20, Bartholini, G., Da Prada, M., and Pletscher, A. (1968). J. Pharm. Pharmacol. 20, 228229. Bartholini, G., Blum, J. E., and Pletscher, A. (1969). J. Pharm. Pharmacol. 21,297-301. Bartholini, G., Kuruma, J., and Pletscher, A. (1971). Nature (London) 230, 533-534. Bartholini, G., Kuruma, J., and Pletscher, A. (1972). J. Pharmacol. E z p . Ther. 183, 6572. Bartholini, G., Stadler, H., and Lloyd, K. G. (1973). I n “Advances in Neurology” (D. B. Calne, ed.), Val. 3, pp. 233-241. Raven, New York. Beasley, B. L., Hare, T. A., Vogel, W. H., and Desimone, S. (1973). New Engl. J. Med. 289,919. Bedard, P., Larochelle, L., Poirier, L., and Sourkes, T. L. (1970). Can. J . Physiol. Pharmacol. 4 8 , 8 2 4 4 . Bernheimer, H., and Hornykiewicz, 0. (1973). I n “Advances in Neurology” (A. Barbeau, T. N. Chase, and G. W. Paulson, eds.), Vol. 1, pp. 525-531. Raven, New York. Bernheirner, H., Birkmayer, W., Hornykiewicz, O., Jellinger, K., and Seitelberger, F. (1973). J. Neurol. Sci. 20, 415455. Bianchine, J. R., Calimlim, L. R., Morgan, J. P., Dujuvne, C., and Lasagna, L. (1971). Ann. N.Y. Acad. Sci. 1 7 9 , 126-140. Bianchine, J. R., Messiha, F. S., and Hsu, T. H. (1972). Clin. Pharmacol. Ther. 13, 584594. Bianchine, J. R., Messiha, F. S., and Preziosi, T. J. (1973). I n “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 101-135. Raven, New York. Binns, T., King, J. A. G., Mishra, S. N., Percival, A., Robson, N. C., Swan, G. A., and Waggott, A. (1970). J . Chem. SOC.C , 2063-2070. Bird, M. T., and Paulson, G. W. (1970). Neurology 2 0 , 4 0 0 . Birket-Smith, E., and Andersen, J. V. (1973). Lancet i, 431. Birkmayer, W. (1974). Abstr. Int. Congr. Neurol., loth, Barcelona; Excerpta Med. Found. Znt. Congr. Ser. No. 296. Birkmayer, W., and Hornykiewicz, 0. (1961). Wien. Klin. Wochenschr. 73, 787-788. Birkmayer, W., and Hornykiewicz, 0. (1964). Arch. Psychiat. Nervenkr. 206,367-381. Black, I. B., and Axelrod, J. (1969). J. Biol. Chem. 244, 6124-6129. Bloom, F. E. (1972). I n “Catecholamines, Handbook of Experimental Pharmacology” (J. Blaschko and E. Muscholl, eds.), Vol. 33, pp. 46-78. Springer-Verlag, Berlin and New York. Borgman, R. J., McPhillips, J. J., Stitzel, R. E., and Goodman, 1. J. (1973). J. Med. Chem. 16,630-633. Bosmann, H. B., Shea, M. B., Case, K. R., and Pike, G. 2. (1971). Physiol. Chem. Phys. 3, 226-234. Bowers, M. B. (1971). Neuropharmacology 11, 101-111. Braham, J., and Sarova-Pinhas, I. (1973). Lancet i, 432-433.

296

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

Braham, J., Sarova-Pinhas, I., and Goldhammer, Y. (1970). Brit. Med. J . iii, 768. Breese, G. R., Chase, T. N., and Kopin, I. J. (1969). J. Pharmacol. Exp. Ther. 165, 913. Brody, J. A., Chase, T. N., and Gordon, E. K. (1970). New Engl. J. Med. 282,947-950. Brogden, R. N., Speight, T. M., and Avery, G. S. (1971). Drugs 2, 262400. Bruno, A., and Bruno, S. C. (1966). Acta Psychiat. Scand. 42,264-271. Bunney, B. S., Walters, J. R., Roth, R. H., and Aghajanian, G. K. (1973a). J. Pharmacol. Erp. Ther. 185, 56CL571. Bunney, B. S., Aghajanian, G. K., and Roth, R. H. (1973b). Nature (London), New Biol. 245, 123-125. Butcher, L. L., Engel, J., and Fuxe, K. (1972). Brain Res. 41,387-412. Calne, D. B., ed. (1973). “Advances in Neurology,” Vol. 3, 326 pp. Raven, New York. Calne, D. B., Karoum, F., Ruthven, C. R. J., and Sandler, M. (1969a). Brit. J . Pharmacol. 3 7 , 5 7 4 8 . Calne, D. B., Stern, G. M., Laurence, D. R., Sharkey, J., and Armitage, P. (1%9b). Lancet ii, 744-746. Calne, D. B., Brennan, J., Spiers, A. S. D., and Stern, G. M. (1970). Brit. Med. J. i, 474475. Calne, D. B., Reid, J. L., Vakil, S. D., Sumant, R., Petrie, A., Pallis, C. A., Gawler, J., Thomas, P. K , and Hilson, A. (1971). Brit. Med. J . iii, 729-732. Carlsson, A. (1959). Pharmacol. Rev. 11,490-493. Carlsson, A. (1970). J. Pharrn. Pharmacol. 22, 729-732. Carlsson, A., Lindqvist, M., and Magnusson, T. (1957). Nature (London) 180, 1200. Carlsson, A., Kehr, W., Lindqvist, M., Magnusson, T., and Atack, C. V. (1972). Pharmacol. Rev. 24, 371-384. Castaigne, P., Laplane, D., and Dordain, G. (1971). Res. Commun. Chem. Pathol. Pharmacol. 2, 154-158. Cawein, M., and Turney, F. (1971). New Engl. J . Med. 284, 504. Celesia, G. G., and B a r , A. N. (1970). Arch. Neurol. (Chicago) 23, 193-200. Chalmers, J. P., Davies, D. S., Draffan, G. H., Reid, J. L., and Thorgeirsson, S. S. (1971a). Brit. J. Pharmacol. 4 3 , 4 5 5 ~ 4 5 6 ~ . Chalmers, J. P., Baldessarini, R. J., and Wurtman, R. J. (1971b). Proc. Nut. Acad. Sci. US. 68,662-666. Chase, T. N. (1970). Neurology 20, Part 2, 122-130. Chase, T. N. (1972). Arch. Neurol. (Chicago) 2 7 , 354-356. Chase, T. N. (1973a) In “Advances in Neurology” (A. Barbeau, T. N. Chase, and G. W. Paulson, eds.), Vol. 1, pp. 533-542. Raven, New York. Chase, T. N. (1973b). Arch. Neurol. (Chicago) 29, 349-351. Chase, T. N. (1974a). I n “Advances in Neurology” (F. McDowell and A. Barbeau, eds.), Vol. 5, pp. 31-39. Raven, New York. Chase, T. N. (197433). Neurology 24,637-639. Chase, T. N., and Ng, L. K. Y. (1972a). Arch. Neurol. (Chicago) 27,486492. Chase, T. N., and Ng, L. K. Y. (1972b). Neurology 22,417. Chase, T. N., and Watanabe, A. M. (1972). Neurology 22, 384-392. Chase, T. N., Schnur, J. A., Brody, J. A., and Gordon, E. K. (1971). Arch. Neurol. (Chicago) 25, 9-13. Chase, T. N., Ng, L. K. Y., and Watanabe, A. M. (1972). Neurology 22,479-481.. Chase, T. N., Holden, E. M., and Brody, J. A. (1973). Arch. Neurol. (Chicago) 29, 328330. Chase, T. N., Woods, A. C., and Glaubiger, G. A. (1974b). Arch. Neurol. (Chicago) 30, 383-386.

L-DOPA AND EXTRAPYRAMIDAL DESEASE

297

Chase, T. N., Woods, A. C., Lipton, M. A., and Morris, C. E. (1974~).Arch. Neurol. (Chicago) 3 1 , 55-56. Cheramy, A., Gauchy, G., Glowinski, J., and Besson, M. J. (1973). Eur. J . Pharmacol. 2 1.246-24%. CKueh, C. C . , and Moore, K. E. (1973).Brain Res. 50, 221-225. Chrusciel, T. L., and Herman, Z. S. (1969).Psychopharmacologia 14, 124-134. Cohen, G., Mytilineou, C., and Barrett, R. E. (1972). Science 175, 1269-1272. Coleman, M. (1970).Neurology 20, Part 2, 114-121. Collins, A. C., Cashaw, J. L., and Davis; V. E. (1973). Biochem. Pharmacol. 22, 23372348. Connor, J. R. (1970).J . Physiol. (London) 208, 691-703. Cools, A. R. (1972).Psychopharmacologia 25,229-237. Cools, A. R. (1973). “The Caudate Nucleus and Neurochemical Control of Behavior,” pp. 1-149, Drukkerij Brakkenstein, Nijmegen, The Netherlands. Coppen, A., Metcalfe, M., Carroll, J. D., and Morns, J. G. L. (1972).Lancet i, 654-657. Corrodi, H., and Fuxe, K. (1%7). Li$e Sci. 6, 1345-1350. Corrodi, H., Fuxe, K., and Hokfelt, T. (1967). Life Sci. 6, 767-774. Corrodi, H., Fuxe, K., Hamherger, B., and Ljungdahl, A. (1970). E m . J . Phannacol. 12, 145-155. Corrodi, H., Farnebo, L-O., Fuxe, K., Hamberger, B., and Ungerstedt, U. (1972). Eur. J. Pharrnacol. 20, 195-204. Costa, E., and Meek, J. L. (1974). Annu. Rev. Pharmacol. 14, 491-511. Cote, L. J., Cohen, G., and Yahr, M. D. (1971). Trans. Amer. Neurol. Ass. 96, 222-223. Cotzias, G. C. (1969).J. Amer. Med. Ass. 210, 1255-1262. Cotzias, G. C., Van Woert, M. H., and Schiffer, L. M. (1967). New Engl. J . Med. 267, 374-379. Cotzias, G. C., and Papavasiliou, P. S. (1971).J. Amer. Med. Ass. 215, 1504-1505. Cotzias, G. C., Papavasiliou, P. S., Fehling, C., Kaufman, B., and Mena, I. (1970). New Enel. J . Med. 282, 31-33. Coutinho, C. B., Spiegel, H. E., Kaplan, S. A., Yu, M., Christian, R. P., Carbone, J. J., Symington, J., Cheripko, J. A., Lewis, M., Tonchen, A., and Crews, T. (1971). J. Pharm. Sci. 60, 1014-1018. Cox, B., Danta, G., Schnieden, H., and Yuill, G. M. (1973). J. Neurol., Neurosurg. Psychiat. 36, 354-361. Coyle, J . T., and Snyder, S. H. (1969). Science 166,899-901. Dairman, W., Christenson, J. G., and Udenfriend, S. (1971). Proc. Nat. Acad. Sci. U.S. 6 8 , 2117-2120. Dairman, W., Christenson, J. G., and Udenfriend, S. (1972). Pharmocol. Rev. 24, 269289. Dalen, P. (1973). Lancet i, 107-108. Dalen, P., and Step, G. (1973). Lancet i, 936-937. Damasio, A. R., Castro-Caldas, A., and Levy, A. (1973). I n “Advances in Neurology” (D. B. Calne, ed.), Vol. 3, pp. 11-22. Raven, New York. Davidson, L., Lloyd, K., Dankova, J., and Hornykiewicz, 0. (1971). Ezperientia 27, 10481049. de Ajuriaguerra, J., Gauthier, G . , Geissbuhler, F., Simona, B., Constantinidis, J., Yanniotis, G., Krassoievitch, M., Eisenring, J. J., and Tissot, R. (1971). Presse Med. 79, 1396. Dehaene, I., and Bogaerts, M. (1970).Lancet ii, 470. de la Torre, J. C. (1973). In “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 5977. Raven, New York.

298

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

Donnelly, E. F., and Chase, T. N. (1973). Dis. Nerv. Syst. 3 4 9 119-123. H. (1972). Arch. Duby, S. E., Cotzias, G . C., Papavasiliou, P. s., and Lawrence, Neurol. (Chicago) 27, 474480. Duncan, R. J. S., Sourkes, T. L., Boucher, R., Poirier, L. J., and Roberge, A. (1972). J. Neurochem. 19, 2007-2010. Duvoisin, R. C. (1973). In “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 229247. Raven, New York. Duvoisin, R. C., Yahr, M. D., and Cote, L. D. (1969). Trans. Amer. Neurol. Ass. 94, 81-

w.

a4. Duvoisin, R. C., Lobo-Antunes, J., and Yahr, M. D. (1972). J. Neurol., Neurosurg. Psychiat. 3 5 , 4 8 7 4 9 5 . Eldridge, R., Kanter, W., and Koerber, T. (1973). Lancet ii, 1027-1028. England, A. C., and Schwab, R. S. (l%l). New Engl. J. Med. 265, 785-792. Ericsson, A. D., Wertman, B. G., and Duffy, K. M. (1971). Neurology 21, 1023-1029. Evans, G. W., Dubois, R. S., and Hambidge, K. M. (1973). Science 181, 1175-1176. Evered, D. F. (1971). Lancet i, 914. Everett, G. M., and Borcherding, J. W. (1970). Science 168,849-850. Fahn, S., Libsch, L. R., and Cutler, R. W. (1971). J. Neurol. Sci. 14, 427455. Farnebo, L-0.. Fuxe, K., Goldstein, M., Hamberger, B., and Ungerstedt, U. (1971). Eur. J . Pharmacol. 16, 27-38. Fehling, C. (1973). Acta Neurol. Scand. 49, 245251. Fellman, J. H., and Roth, E. S. (1971). Biochemistry 10,4Q8-414. Fellman, J. H., Fujita, T. S., and Roth, E. S. (1972). Biochirn. Biophys. Acta 268, 601604. Fermagiich, J., and Chase, T. N. (1973). Lancet i, 1261-1262. Friedhoff, A. J., and, Alpert, M. (1973). Biol. Psychiat. 6, 165-169. Friedhoff, A. J., Schweitzer, J. W., Miller, J., and Van Winkle, E. (1972). Ezperientia 28, 517-5 19. Friedman, E., and Gershon, S. (1972). Eur. J. Pharmacol. 18, 183-188. Friedman, E., and Gershon, S. (1973). Nature (London) 243, 520-521. Friedman, E., Friedman, J., and Gershon, S. (1973). Science 182, 831-832. Fuxe, K. (1%5). Acta Physiol. Scand., Suppl. 247, 37-85. Fuxe, K., and Ungerstedt, U. (1970). In “International Symposium on Amphetamine and Related Compounds” (E. Costa and S. Garattini, eds.), pp. 83-86. Raven, New York. Fuxe, K., Hokfelt, T., and Ungerstedt, U. (1970). In “Principles of Psychopharmacology” (W. G. Clark and J. del Giudice, eds.), pp. 87-96. Academic Press, New York. Geissbuhler, F., Gaillard, J. M., and Eisenring, J. J. (1972). Rev. Eur. Etud. Clin. Biol. 17,3844. Gerstenbrand, F., and Prosenz, P. (1965). Praxis 46, 1373-1377. Godwin-Austen, R. B. (1973). In “Advances in Neurology” (D. B. Calne, ed.), Vol. 3, pp. 23-27. Raven, New York. Godwin-Austen, R. B., Frears, C. C., Bergmann, S., Parkes, J. D., and Knill-Jones, R. P. (1970). Lancet ii, 383-385. Goldin, B. R., Peppercorn, M. A., and Goldman, P. (1973). J. Pharmacol. Exp. Ther. 186,160-166. Goldstein, M., Anagnoste, B., Battista, A. F., Owen, W. S., and Nakatani, S. (1969). J. Neurochem. 16,645-653. Goldstein, M., Fuxe, K., and Hokfelt, T. (1972a). Pharmacol. Rev. 24, 293-309. Goldstein, M., Anagnoste, B., Battista, A. F., Nakatani, S., and Ogawa, M. (1972b). Res. Publ., Ass. Res. New. Ment. Dis. 5 0 , 434-447.

L-DOPA AND EXTRAPYRAMIDAL DESEASE

299

Goldstein, M., Battista, A. F., Ohmoto, T., Anagnoste, B., and Fuxe, K. (1973). Science 179,81-17. Goodall, M., and Alton, H. (1972). Biochem. Pharmacol. 21, 2401-2408. Goodwin, F., Murphy, D. L., Brodie, H, K. H., and Bunney, W. E. (1971). Clin. Pharmacol. Ther. 12,383-396, Grelak, R. P., Clark, R., Stump, J. M., and Vernier, V. G. (1970). Science 169,203-204. Grundig, E., Gerstenbrand, F., Oberhummer, J., Simanyi, M., Schedl, R., and Weiss, J. (1972). 2. Neurol. 203, 73-90. Guldberg, H. C., Turner, J. W., Hanieh, A., Ashcroft, G. W., Crawford, T. B. B., Perry, W. L. M., and Gillingham, F. J. (1967). Confin. Neurol. 29, 73-77. Hall, C. D., Weiss, E. A., Moms, C. E., and Prange, A. J. (1972). Neurology 22, 231237. Heikkila, R., and Cohen, G. (1972a). Mol. Pharmacol. 8 , 241-248. Heikkila, R., and Cohen, G. (197213). Eur. J. Pharmacol. 20, 156-160. Henning, M., Rubenson, A., and Trolin, G. (1972). J. Pharm. Pharmacol. 24,447451. Herskovits, E., and Gacitua, E. F. (1973). Medicina (Buenos Aires) 33,51-58. Ho, A. K. S., and Loh, H. H. (1972). Eur. J . Pharmacol. 19, 145-150. Hoffmann-LaRoche, Inc. (1971). Data on file. Nutley, New Jersey. Hokfelt, T. (1%8). Z . Zellforsch. Mikrosk. Anat. 91, 1-74. Holden, E. M., Brody, J. A., and Chase, T. N. (1974). Neurology 24,263-265. Hongladarorn, T. (1973). Lancet i, 1114. Horita, A., and Lowe, M. C. (1972). In “Advances in Biochemical Psychopharmacology” (E. Costa, and M. Sandler, eds.), Vol. 5, pp. 227-242. Raven, New York. Hornykiewicz, 0. (1966). Pharmacol. Rev. 18,925-964. Hornykiewicz, 0. (1972). I n “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. 7, pp. 465-501. Plenum, New York. Hornykiewicz, 0. (1973a). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 32, 183-190. Hornykiewicz, 0. (1973b). Brit. Med. Bull. 29, 172-178. Howse, P. M.. and Matthews, W. B. (1973). J. Neurol., Neurosurg. Psychiat. 36,27-29. Hoyer, I., and Van Zwieten, P. A. (1972). J. Pharm. Pharmacol. 24, 452458. Hughes, R. C., Polgar, J. G., Weightman, B., and Walton, J. N. (1971). Brit. Med. J . i, 713. Hunter, K. R., Stern, G. M., Lawrence, D. R., and Armitage, P. (1970). Lancet i, 11271129. Hyyppa, M., Rinne, U. K., and Sonninen, V. (1970). Acta Neurol. Scand., Suppl. 43,223224. Izumi, K., Inoue, N., Shirabe, T., Miyazaki, T., and Kuroiwa, Y. (1971). Lancet i, 1355. Jaffe, M. E. (1973). I n “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 161-173. Raven, New York. Jameson, H. D. (1970). J . Amer. Med. Ass. 211, 1700. Javoy, F., Agid, Y., Bouvet, D., and Glowinski, J. (1972). J . Pharmacol. Exp. Ther. 182, 454463. Jellinger, K. (1968). I n “Handbook of Clinical Neurology” (P. J. Vinkon and G. W. Bruyn, eds.), Vol. 6, pp. 632-693. North-Holland Publ., Amsterdam. Johansson, B., and Roos, B-E. (1%7). Life Sci. 6,1449-1454. Jurna, I., Ruzdic, N., Nell, T., and Grossmann, W. (1972). Eur. J. Pharmacol. 20, 341350. Kaeser, H., Ferel, D., and Wurmser, P. (1970). Schweiz. Med. Wochenschr. 100,805-813. Karobath, M.., and Baldessarini, R. J. (1972). Nature (London), New Biol. 236,206-208. Kastin, A. J., and Barbeau, A. (1972). Can. Med. Ass. J. 107, 1079-1081.

300

E . WILLIAMS PELTON I1 AND THOMAS N. CHASE

Kehr, w., Carlsson, A., Lindqvist, M., Magnusson, T., and Atack, c. (1972). J . Pharm. Pharmacol. 24, 74.2-747. E m , J. S., Bak, I. J., Hassler, R., and Okada, Y. (1971). Exp. Brain Res. (Berlin) 14, 95-104. Klawans, H. L. (1973). “Monographs in Neural Sciences,” Vol. 2, 136 pp. Karger, Basel. Klawans, H. L., and Garvin, J. S. (1969). Dis. Nerv. Syst. 30, 727-746. Klawans, H. L., and Ringel, S. P. (1971). Eur. Neurol. 5, 107-116. Klawans, H. L., and Rubovits, R. (1972). Neurology 22, 107-116. Klawans, H. L., Ringel, S. P., and Shenker, D. M. (1971). J . Neurol., Neurosurg. Psychiat. 34,682-686. Klawans, H. L., Paulson, G. W., Ringel, S. P., and Barbeau, A. (1972). New Engl. J. Med. 286, 1332-1334. Kofman, 0. S. (1973). Arch. Neurol. (Chicago) 29, 120-121. Kopin, I. J. (1973). In “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 137-148. Raven, New York. Krasner, N., and Cornelius, J. M. (1970). Brit. Med. J . iv, 4%. Kremzner, L. T., Berl, S., Mendoza, M., and Yahr, M. D. (1973). In “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 79-90. Raven, New York. Kuruma, I., Bartholini, G., and Pletscher, A. (1970). Eur. J. Pharmacol. 10, 189-192. Ladnron, P. (1972). Nature (London), New Biol. 238,212-213. Laitinen, L. (1969). Actu Neurol. Scand. 45, 109-113. Lakke, J. P. W. F., Korf, J., Hoorntje, S., Schut, T., and van Pragg, H. M. (1973). Psychiat. Neurol. Neurochir. 76, 139-146. Lal, S., de la Vega, C. E., Garelis, E., and Sourkes, T. L. (1973). Psychiat. Neurol. Neurochir. 76, 113-117. Lancaster, G . , Larochelle, L., Bedard, P., Missala, K., Sourkes, T. L., and Poirier, L. J. (1970). J . Neurol. Sci. 11, 265-274. Langelier, P., Parent, A., and Poirier, L. J. (1972). Bruin Res. 45, 622429. Langelier, P., Roberge, A. G., Boucher, R., and Poirier, L. J. (1973). J . Pharmacol. Exp. Ther. 187, 15-26. Langrall, H. M., and Joseph, C. (1972). Neurology 22, Part 2, 3-16. Leon, A. S., Speigel, H. E., Thomas, G., and Abrams, W. B. (1971). J. Amer. Med. Ass. 218, 1924-1927. Lieberman, A. N., Goodgold, A. L., and Goldstein, M. (1972). Neurology 22, 1205-1210. Lindbrink, P., Jonsson, G., and Fuxe, K. (1971). Neuropharmacology 10, 521-536. Liu, Y. P., Ambani, L. M., and Van Woert, M. H. (1972). J. Neurochem. 19, 2237-2239. Lloyd, K. G., and Hornykiewicz, 0. (1970). Science 170, 1212-1213. Lloyd, K. G., and Hornykiewicz, 0. (1973). Nature (London) 243, 521-523. Lotti, V. J., and Porter, C. C. (1970). J. Pharmacol. Exp. Ther. 172, 406-415. McDowell, F. H., and Markham, C. H., eds. (1971). “Recent Advances in Parkinsonism,” 245 pp. Davis, Philadelphia, Pennsylvania. McDowell, F. H., Lee, J. E., Swift, T., Sweet, R. D., Ogsbury, J. S., and Kessler, J. T. (1970). Ann. Intern. Med. 72, 29-35. McGeer, P. L., McGeer, E. G., and Wada, J. A. (1971). Neurology 21, 1000-1007. McKenzie, G. M., Gordon, R. J., and Viik, K. (1972). Brain Res. 47, 4 3 e 4 5 6 . Maj, J., Kapturkiewicz, Z., and Sarnek, J. (1972a). J . Pharm. Pharmacol. 24, 735-737. Maj, J., Sowinska, H., Kapturkiewicz, Z., and Sarnek, J. (1972b). J. Pharm. Pharmacol. 24, 412441. Mandell, S. (1970). Neurology 20, Part 2, 103-106. Markham, C. H. (1971). Clin. Pharmacol. Ther. 1 2 , Part 2, 340-346.

L-DOPA AND EXTRAPYRAMIDAL DESEASE

301

Mars, H. (1973). Arch. Neurol. (Chicago) 28, 91-95. Marsden, c. D., Barry, p. E., Parkes, J. D., and Zilkha, K. J. (1973). J . Neural. Neurosurg. Psychiat. 36, 10-14. Mawdsley, C. (1970). Brit. Med. J . i, 331-337. Mawdsley, C., Williams, I. R., Pullar, I. A., Davidson, D. L., and Kinloch, N. E. (1972). Clin. Pharmacol. Ther. 1 3 , 575-583. Mena, I., Court, J., Fuenzalida, S., Papavasiliou, P. S., and Cotzias, G. C. (1970). New Engl. J . Med. 282, 5-10. Mena, I., Court, J., and Cotzias, G. C. (1971). J. Amer. Med. Ass. 218, 1829-1830. Mendell, J. R., Chase, T. N., and Engel, W. K. (1970). Lancet i, 593-594. Mendell, J. R., Chase, T. N., and Engel, W. K. (1971). Arch. Neurol. (Chicago) 25, 320325. Miller, E. (1974). Neurology 24, 116-119. Miller, E., Wiener, L., and Bloomfield, D. (1973). Arch. Neurol. (Chicago) 29, 99-103. Moir, A. T. B., Ashcroft, G. W., Crawford, T. B. B., Eccleston, D., and Guldberg, H. C. (1970). Brain 93,357-368. Molinoff, P. B., and Axelrod, J. (1971). Annu. Rev. Biochem. 40,465-500. Mones, R. J., Elizan, T. S., and Siege], G. (1971). J. Neurol., Neurosurg. Psychiat. 34, 668-673. Morgan, J. P., Prezoisi, T. J., and Bianchine, J. R. (1970). Lancet ii, 659. Muenter, M. D., and Tyce, G. M. (1971). Mayo Clin. Proc. 46, 231-239. Muenter, M. D., Dinapoli, R. P., Sharpless, N. S., and Tyce, G. M. (1973). Mayo Clin. Proc. 48, 173-183. Narotzky, R., Griffith, D., Stahl, S., Bondareff, W., and Zeller, E. A. (1973). Exp. Neurol. 38,218-230. Ng, L. K. Y., Chase, T. N., Colburn, R. W., and Kopin, I. J. (1971). Science 172, 487489. Ng, L. K. Y., Chase, T. N., Colburn, R. W., and Kopin, I. J. (1972a). Neurology 22, 688696. Ng, L. K. Y., Colburn, R. W., and Kopin, I. J. (1972b). J . Pharmacol. Ezp. Ther. 183, 316-325. Ng, L. K. Y., Gelhard, R. E., Chase, T. N., and MacLean, P. D. (1973). In “Advances in Neurology” (A. Barheau, T. N. Chase, and G. W. Paulson, eds.), Vol. 1, pp. 651-655. Raven, New York. Nyhack, H. (1972). Acta Physiol. Scand. 84, 54-64. Nyback, H., and Sedvall, G. (1971). J . Pharm. Pharmacol. 23,322-326. Nyback, H., Schubert, J., and Sedvall, G. (1970). J. Pharm. Pharmacol. 22,622-624. Offermeier, J., and Potgieter, B. (1972). Symp. Znt. Triuastal, Monastir, Tunisia. O’Gorman, L. P., Borud, O., Khan, J. A., and Gjessing, L. R. (1970). Clin. Chim. Acta 29, 111-119. Ordonez, L. A., and Wurtman, R. J. (1973). Biochem. Pharmacol. 22, 134-137. Papavasiliou, P. S., Cotzias, G. C., Duby, S. E., Steck, A. T., Fehling, C., and Bell, M. A. (1972). New Engl. J . Med. 286, 8-14. Parkes, J. D., Knill-Jones, R. P., and Clements, P. J. (1971). Postgrad. Med. J. 47, 116119. Parks, L. C., Watanabe, A. M., and Kopin, I. J. (1970). Lancet ii, 1014-1015. Peaston, M. J. T., and Bianchine, J. R. (1970). Brit. Med. J. i, 400-403. Pfeiffer, R., and Ebadi, M. (1972). J. Neurochem. 19, 2175-2181. Plech, A., Herman, Z. S., and Chrusciel, T. L. (1972). Diss. Pharm. Pharmacol. 24, 279282.

302

E . WILLIAMS PELTON 11 AND THOMAS N . CHASE

Plotnikoff, N. p., Kastin, A. J., Anderson, M. S., and Schally, A. v. (1971). L& SCi. lo, Part 1, 1279-1283. Plotnikoff, N. P., Prange, A. J., Breese, G. R., Anderson, M. S., and Wilson, I. c. (1972). Science 178, 417-418. Poirier, L. J., Singh, P., Sourkes, T. L., and Boucher, R. (1%7). Brain Res. 6,654-666. Prange, A. J., Wilson, I. C., Moms, C. E., and Hall, C. D. (1973). Psychopharmacol. Bull. 9, 36-37. Prepas, S., Menon, M. K., and Clark, W. G. (1973). J. Pharm. Pharmacol. 25,659-660. Rajput, A. H. (1973). Lancet i, 432433. Rajput, A. H., Kazi, K. H., and Rozdilsky, B. (1972). J. Neurol. Sci. 16,331-341. Randrup, A., and Munkvad, I. (1968).Pharmakopsychiat. Neuropsychopharm. 1, 18-26. Reger, J. F., Holbrook, J. R., and Pozos, R. S. (1972). Exp. Neurol. 3 5 , 4 8 0 4 9 1 . Rego, A., Gago, B., and Tissot, R. (1971). Arch. Neurobiol. 34,497-510. Riddell, D., and Szerb, J. C. (1971). J . Neurochem. 18,989-1006. Rinne, U. K. (1972). Acta Neurol. Scand., Suppl. 48, 59-103. Rinne, U. K., Sonninen, V., and Siirtola, T. (1972a). Eur. Neurol. 7, 228-240. Rinne, U. K., Sonninen, V., and Siirtola, T. (1972b). Z. Neurol. 202, 1-20. Rivera-Calimlim, L. (1971). Clin. Res. 19, 355. Rivera-Calimlim, L., Bosmann, H. B., Penney, D. P., and Lasagna, L. (1973). J. Pharmacol. Exp. Ther. 1 8 4 , 4 4 0 4 4 8 . Roffler-Tarlov, S., Sharman, D. F., and Tegerdine, P. (1971). Brit. J. Pharmacol. 42, 343-351. Romero, J. A., Chalmers, J. P., Cottman, K., Lytle, L. D., and Wurtman, R. J. (1972). J. Pharmacol. Exp. Ther. 180, 277-285. Romero, J. A., Lytle, L. D., Ordonez, L. A., and Wurtman, R. J. (1973). J. Pharmacol. Exp. Ther. 1 8 4 , 67-72. Rosenthal, R. K., McDowell, F. H., and Cooper, W. (1972). Neurology 22, 1-11. Ross, S. B., Renyi, A. L., and Orgen, S-0. (1971). Life Sci. 10, Part 1, 1267-1277. Roth, R. H., Walters, J. R., and Aghajanian, G. K. (1973). In “Frontiers in Catecholamine Research” (E. Usdin and S. H. Snyder, eds.), 1219 pp. Pergamon, Oxford. Sandler, M. (1971). Lancet i, 784-785. Sandler, M. (1972). ZR “Catecholamines” (H. Blaschko and E. Muscholl, eds.), Vol. 33, pp. 845-899. Springer-Verlag, Berlin and New York. Sandler, M. (1973). In “Advances in Neurology” (M. D. Yahr, ed.), Vol. 2, pp. 255-264. Raven, New York. Sandler, M., Carter, S. B., Hunter, K. R., and Stern, G. M. (1973). Nature (London) 241, 439-443. Sandler, M., Johnson, R.. Ruthven, C. R. J., Reid, J. L., and Calne, D. B. (1974). Nature (London) 247,364-366. Sassin, J. F., Taub, S., and Weitzman, E. D. (1972). Neurology 22, 1122-1125. Sathananthan, G., Angrist, B. M., and Gershon, S. (1973). Biol. Psychiat. 7, 139-146. Schnur, J. A., Chase, T. N., and Brody, J. A. (1971). Neurology 21, 1236-1242. Schoenfeld, R. I., and Uretsky, N. J. (1973). J. Pharmacol. Exp. Ther. 186,616-624. Schwab, R. S., England, A. C., Poskanzer, D. C., and Young, R. R. (1969). J. Amer. Med. Ass. 208, 116%1170. Selby, G. (1973). Med. J . Aust. 1, 577-585. Sethy, V. H., and Van Woert, M. H. (1973). Neuropharmacology 12,27-31. Sharpe, J. A., Rewcastle, N. B., Lloyd, K. G., Hornykiewicz, O., Hill, M., and Tasker, R. R. (1973). J . Neurol. Sci. 1 9 , 275-286.

L-DOPA AND EXTRAPYRAMIDAL DESEASE

303

Sharpless, N. s.7 TYce, G. M., and Owen, C. A., Jr. (1973). Life Sci. 12, part 1, 97-106. Shaw, K. M., Stern, G. M., and Sandler, M. (1973). In “Advances in ~ ~ (D. Cdne, 4,Vol. 3, pp. 115-119. Raven, New York. Shintomi, K., and Yamamura, M. (1973). J. Pharm. Pharmacol. 25, 6 6 ~ 6 7 . Shuldn, A. T., Sargent, T., and Naranjo, C. (1969). Nature (London) 221, 537-541. Siegfried, J., Klaiber, R., and Ziegler, W. H. (1970). In “L-dopa and Parkinsonism” (A. Barbeau and F. H. McDowell, eds.), pp. 114-123. Davis, Philadelphia, Pennsylvania. Sims, K. L., and Bloom, F. E. (1973). Brain Res. 49, 165-175. Sjaastad, O., and Oftedal, S-I., eds. (1972). Acta Neurol. Scand., Suppl. 51, 1-150. Snyder, S. H., Taylor, K. M., Coyle, J. T., and Meyerhoff, J. L. (1970). Amer. J . Psychiat. 127, 199-207. Sourkes, T. L. (1971). Nature (London) 229, 413-414. Sourkes, T. L., and Poirier, L. J. (1%6). In “Biochemistry and Pharmacology of the Basal Ganglia” (E. Costa, L. H. Cote, and M. D. Yahr, eds.), pp. 187-190. Raven, New York. Spector, S., Tarver, J., and Berkowitz, B. (1972). Pharmacol. Rev. 24, 191-202. Stern, G. (1966). Brain 89, 449478. Strang, R. R. (1965). Brit. Med. J . ii, 33-34. Stromberg, U. (1970). Psychopharmacologia 1 8 , 58-67. Sweet, R. D., Lee, J. E., and McDowell, F. H. (1972). Clin. Pharmacol. Ther. 13, 23-27. Tan, B. K., Leijnse-Ybema, H. J., and Brand, H. J. (1972). Lancet i, 903. Tate, S. S., Sweet, R., McDowell, F. H., and Meister, A. (1971). Proc. Nut. Acad. Sci. U.S. 68,2121-2123. Thierry, A, M., Blanc, G., Sobel, A., Stinus, L., and Glowinski, J. (1973). Science 182, 499-501. Thoa, N. B., Eichelman, B., and Ng, L. K. Y. (1972). Brain Res. 4 3 , 4 6 7 4 7 5 . Tryding, N., Tufvesson, G., and Nilsson, S. (1971). Lancet i, 859. Tyce, G. M. (1970). In “L-dopa and Parkinsonism” (A. Barbeau, and F. H. McDowell, eds.), pp. 86-88. Davis, Philadelphia, Pennsylvania. Tyce, G. M., and Owen, C. A. (1973). J . Neurochem. 20, 1563-1573. Ungerstedt, U. (1971a). Acta Physiol. Scand., Suppl. 367, 1 4 8 . Ungerstedt, U. (1971b). Acta Physiol. Scand., Suppl. 367, 69-93. Ungerstedt, U. (1971~).Acta Physiol. Scand., Suppl. 367, 49-68. Ungerstedt, U., Butcher, L. L., Butcher, S. G., Anden, N.-E., and Fuxe, K. (1969). Brain Res. 1 4 , 4 6 1 4 7 1 . Ungerstedt, U., Fuxe. K., Goldstein, M., Battista, A., Ogawa, M., and Anagnoste, B. (1973). Eur. J . Pharmacol. 21,230-237. Uretsky, N. J., and Iverson, L. L. (1970). J. Neurochem. 17, 269-278. Vakil, S. D., Calne, D. B., Reid, J. L., Jestico, J. V., and Petrie, A. (1972). Symp. Int. Trivastal, Monastir, Tunisia. Van Buren, J. M., Li, C-L., Shapiro, D. Y., Henderson, W. G., and Sadowsky, D. A. (1973). Confin. Neurol. 35, 202-235. Van Woert, M. H. (1971). Clin. Pharmacol. Ther. 12,368-375. Van Woert, M. H., Ambani, L. M., and Levine, R. J. (1972). Dis. Nerv. Syst. 33, 777780. von Hippius, H., and Logemann, G. (1970). Arzneim.-Forsch. 20, 894-896. Von Voigtlander, P. F., and Moore, K. E. (1971). Neuropharmacology 10, 733-741. Wada, G. H., and Fellman, J. H. (1973). Biochemistry 12, 5212-5217. Watanabe, A. M., Chase, T. N., and Cardon, P. V. (1970). Clin. Pharmacol. Ther. 11, 740-746.

~

304

E. WILLIAMS PELTON I1 AND THOMAS N. CHASE

Weiner, W. J., and Klawans, H. L. (1973).J . Neurol., Neurosurg. Psychiat. 36, 747-752. Weiner, W. J., Harrison, W., and Klawans, H. L. (1%9).Life Sci. 8,971-976. Weinshilboum, R. M., Thoa, N. B., Johnson, D. G., Kopin, I. J., and Axelrod, J. (1971). Science 1 7 4 , 1349-1351. Weiss, B. F., Munro, H. N., and Wurtman, R. J. (1971).Science 1 7 3 , 833-835. Weiss, J. L., Cohn, C. K., and Chase, T. N. (1971a).Nature (London) 23%218-219. Weiss, J. L., Ng, L. K. Y., and Chase, T. N. (1971b).Lancet i, 1016-1017. Woods, A.C., and Chase, T. N. (1973). Lancet ii, 513. Woods, A. C., Glaubiger, G. A., and Chase, T. N. (1973).Lancet i, 1391. Wurtman, R. J., and Romero, J. A. (1972). Neurology 22, Suppl., 72-81. Wurtman, R. J., Chou, C., and Rose, C. (1970).J. Pharmacol. E z p . Ther. 174,351-356. Yahr, M. D. (1970). In “L-dopa and Parkinsonism” (A. Barbeau and F. H. McDowell, eds.), pp. 101-108. Davis, Philadelphia, Pennsylvania. Yahr, M. D., ed. (1973). “Advances in Neurology,” Vol. 2, pp. 1-303. Raven, New York. Yahr, M. D., Duvoisin, R. C., Schear, M. J., Barrett, R. E., and Hoehn, M. M. (1969). Arch. Neurol. (Chicago) 2 1, 343354. Yahr, M. D., Duvoisin, R. C., Cote, L. J., and Cohen, G. (1972). In “Advances in Biochemical Psychopharmacology” (M. S. Ebadi, and E. Costa, eds.), Vol. 4, pp. 185194. Raven, New York. Yamori, Y., De Jong, W., Yamabe, H . , Lovenberg, W., and Sjoerdsma, A. (1972). J . Pharm. Pharmacol. 24,690495. Yaryura-Tobias, J. A., Wolpert, A., Dana, L., and Merlis, S. (1970). Dis. N e w . Syst. 31, 6043. York, D. H. (1970).Brain Res. 20,233-249. Youdim, M. B. H., Collins, G. G. S., Sandler, M., Bevan-Jones, A. B., Pare, C. M. B., and Nicholson, W. J. (1972).Nature (London) 236, 225-228.

Effect of Amphetamine-Type Psychostimulants on Brain Metabolism C.-J. ESTLER Pharmakologisches lnstitut der Universitat Erlangen-Niirnberg Erlangen, West Germany

I. Introduction . . . . . . . . . . . . . . . . . . . . . 11. Behavioral Changes, Electroencephalogram, and Amphetamine Levels in the Central Nervous System

. . . . . . . . . . . . . . .

111. Cerebral Function and Brain Metabolism . . . . . . . . . . . A. Effect on Neurotransmitters . . . . . . . . . . . . . .

307 311 312

B.

IV.

Behavioral Effects of Amphetamines in Animals Pretreated with Metabolic Inhibitors . . . . . . . . . . . . . . . . . C. Effects on Other Metabolic Parameters . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

305

332 339 349 349

I. Introduction Amphetamines are important representatives of the category of drugs that are commonly referred to as “psychostimulants,” “psychotonics,” or “psychoenergizers.” They are to be differentiated from analeptics in that they exert pronounced effects on mood, emotion, and behavioral functions-effects that are absent in analeptics such as pentetrazole and nicethamide. Therapeutically, amphetamines* are used primarily because of their favorable action on psychomotor performance. In man, after ingestion of low doses of these drugs, the most pronounced effects, based on central stimulation, are prevention or disappearance of fatigue and sleepiness, increased vigilance, alertness, prolonged ability to perform exhausting and boring work, enhancement of athletic performance, shortening of reaction time, improvement of motor coordination and learning, elevation of mood or even euphoria but also nervousness, irritability, agitation, and disturbed sleep. When administered in higher doses, psychostimulants have analeptic properties, and just as other central stimulants, in toxic doses, they can cause convulsions and death. A detailed review of these effects is given by Weiss and Laties

* The term amphetamines refers to several derivatives of amphetamine combined in one common group. 305

TABLE I SOME IMPORTANT DERIVATIVES OF

AMPHETAMINE H

H

CH,

n

p- Chloroamphetamine

Amphetamine

n

n @-CHpCH--NI

\

Clk-@

CH2-yH-< CH,

cn,

CH,

cn,

p - Chloromethamphetamine

Methamphetamine

Chlorphentermine

F,C

n

C- CH-N

I @?cn,

/

‘

(@-

H

CH,-yH-(

‘2%

cn,

Fenfluramine

Diethylpropion

%YNH N ‘H

I

\

n,c

n

Phenmetrazine

Pemoline

@-““a I

H

Pipradrol Fencamfamine

Methylphenidate

‘ZH5

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

307

(1962). Elucidation of the mechanism underlying the psychostimulatory effects of amphetamines has been the object of numerous investigations on experimental animals; a review of these studies is presented in the following. The most important representatives of amphetamine-type psychostimulants and some related compounds are listed in Table I. Structureactivity relationships of amphetamines are discussed by Biel (1970) and Van der Schoot et al. (1962). Methamphetamine seems to produce almost identical effects to amphetamine, and, on first sight, it might be assumed that many derivatives act via mechanisms similar to that for amphetamine itself. Quite obviously this cannot apply to fenfluramine, which is not a central stimulant but, on the contrary, acts as a central depressant (Alphin et al., 1964; Colmore and Moore, 1966; Gropetti et al., 1972; Ziance et al., 1972a). It may also not be true for chloroamphetamines, chlorophenoxyamphetamine, and other chlorinated analogs of amphetamine (Everett and Yellin, 1971; Frey, 1970; Frey and Magnussen, 1968; Kaergaard Nielsen et al., 1967; Pletscher et al., 1966, 1968; Van Praag et al., 1968) that share most pharmacological and toxicological properties with amphetamine (Frey, 1970; Simon et d.,1970) and for methylphenidate and pipradrol (see Section 111,B,4).

II. Behavioral Changes, Electroencephalogram, and Amphetamine Levels in the Central Nervous System Important behavioral changes produced by amphetamines in human beings have been mentioned in the introductory section. It may be expected that similar effects occur also in animals. Reactions of greater complexity are, of course, dependent on the existence of a very highly developed CNS and are, therefore, best detectable in higher animal species, e.g., in primates. Their substantiation requires sophisticated psychological tests. In lower mammals, for instance in mice and rats, which are most frequently used for investigational purposes, the most impressive response to the administration of amphetamines is hyperkinesia, which appears as increased exploratory activity (rearing, etc.) and general hypermotility. Of the optic isomers of amphetamine, the dextrorotatory form [d-amphetamine, (+)-amphetamine, dexamphetamine] has greatest motor activity-stimulating effect; this is apparent particularly at lower-dose levels (Costa et aZ., 1972; Maj et al., 1972a; Snyder, 1970; Svensson, 1971; Taylor and Snyder, 1970, 1971). The phenomenon of general motor excitation is familiar to every experimentalist working with amphetamine compounds and is described in almost

308

C.-J. ESTLER

every paper dealing with behavioral effects of these drugs. Therefore, it Seems unnecessary to give a bibliography referring to this point. In some species (e.g., especially in rats) the general hyperactivity may become overshadowed by certain stereotyped movements such as gnawing, biting, licking, and sniffing (Ellinwood, 1971; Hauschild, 1939; Janssen et al., 1965; Randrup et al., 1963; Randrup and Munkvad, 1967). These apparently purposeless but compulsive activities have been interpreted as rudiments of exploring behavior (Ellinwood, 1971). In evoking these movements &hetamine is only slightly less potent than 1-amphetamine (Taylor and Snyder, 1970, 1971). Besides, amphetamines influence conditioned behavior (Brady, 1956; Cappell et al., 1972; Evans, 1971; Hanson, 1967; Hearst and Whalen, 1963; Kelleher and Morse, 1968; Kriekhaus et al., 1965; Phillips and Fibiger, 1973; Roffman and Lal, 1972; Stein, 1964). [Reviews of the earlier literature are given by Dews and Morse (1961) and Kelleher and Morse (1968).1 In general, amphetamines increase avoidance behavior. Larger doses, however, frequently lead to a reduction in conditioned behavior. This may explain controversial results from different laboratories. When performance is maintained by aversive stimuli, it seems that the stimulant effect may be elicited at higher doses than when it is maintained by positive reinforcement. In addition, amphetamines improve discriminative behavior. The behavioral effects of methylphenidate and pipradrol seem to be similar to those of amphetamine (Dews and Morse, 1961; KeUeher and Morse, 1968). Furthermore, amphetamine possesses an anticataleptic activity (Maj et al., 1972a; Papeschi and Randrup, 1973; Zetler and Thorner, 1973). Other effects of amphetamines, such as increase of basal metabolism, sympathomimetic effects on extracerebral organs, alteration of body temperature (mostly hyperthermia), and reduction of food and water consumption, shall not be discussed in this article. They are not related to the psychostimulatory actions and can be dissociated from them by modifications of the amphetamine molecule. Amphetamines are generally supposed to exert their psychotonic effects mainly by an action on the cerebral cortex and the reticular activating system (Campbell et al., 1969; Innes and Nickerson, 1970; Izquierdo and Izquierdo, 1971). Action on the medial septical nucleus is also suggested by Izquierdo and Izquierdo (1971), on the basis of EEG studies. Amphetamine produces an arousal-type of EEG (Bradley and Elkes, 1957; Hiebel et al., 1954; Longo and Silvestri, 1957). In chickens, alertness after dexamphetamine was associated with increased cortical activity on the EEG (Dewhurst and Marley, 1965). In cats treated with

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

309

amphetamine, the EEG became desynchronized, and simultaneously with the onset of stereotyped behavior the desynchronization was broken by spikes and waves. The activity of the reticular formation was increased (Wallach and Gershon, 1971). More detailed investigations on the effect of psychostimulants on the electrical activity of the CNS have been made by Monnier and Krupp (1960) in guinea pigs. After administration of amphetamine, methamphetamine, and methylphenidate, these authors also observed desynchronization of the cortical activity but synchronization in subcortical structures. Moreover, EEG arousal provoked by acoustical stimuli or by stimulation of the hypothalamus or the reticular formation was accentuated. Similarly, evoked potentials of the cerebral cortex were enhanced when the thalamus or the reticular formation were stimulated. Methylphenidate was the least effective of the three drugs. Destruction of the mesencephalic reticular formation abolished the desynchronizing action of amphetamine (Killam et al., 1959). Stereotyped behavior, on the other hand, obviously has its origin in the effect of amphetamines on the striatum and the substantia nigra (Cools and van Rossum, 1970; Ernst, 1967; Fog et al., 1970), which are part of the extrapyramidal motor system. Lesions of these brain areas markedly abolish amphetamine-induced stereotypies but do not likewise impair locomotor stimulation (Costal1 and Naylor, 1973; Creese and Iversen, 1972; Fibiger et al., 1973). Attempts have been made to correlate amphetamine levels in the brain with behavioral changes (Consolo et al., 1965; Maickel et al., 1969; Ziem et al., 1970) or amphetamine toxicity (Fuller and Hines, 1967). It became obvious that aggregated mice in which amphetamine effectivity and toxicity are greater than in isolated animals (Chance, 1946, 1947; Gunn and Gurd, 1940; Moore, 1963) have higher and longerpersisting amphetamine levels in their brains. Still it is very unlikely that the greater toxicity of amphetamine is only a function of the higher amphetamine content of the brain, because threshold concentrations for toxicity are different in isolated and grouped animals (Consolo et al., 1965; Fuller and Hines, 1967). Ziem et al. (1970) found no correlation between brain amphetamine levels and the degree of stereotypies. The study of Maickel et al. (1969) gives minimum brain concentrations of amphetamine for a number of behavioral effects. Unfortunately only the drug concentration in the whole brain has been determined, and thus, it is not possible to correlate amphetamine levels in different CNS structures with particular functional activities and behavioral effects of the drugs. On the other hand, Glowinski et al. (1966a) showed that amphetamine is distributed nearly uniformly over many brain

C.-J. ESTLER

310

Amphetamine

Norephedrine

-

Ho

HO-(Q--C~--~H-N~ CH,

p -Hydroxyamphetamine

p -Hydroxynorephedrine

Glucuronide of p-hydroxyamphetamine

FIG. 1. Pathways of amphetamine hydroxylation.

regions. Somewhat lower levels were found in cerebellum and somewhat higher concentrations in the hippocampus. Since amphetamine is metabolized in the body the possibility exists that some of the amphetamine effects are brought about by a metabolite. This assumption was supported by the fact that metabolic effects (e.g., catecholamine depletion) outlast the presence of amphetamine in brain (Brodie et al., 1970; Cook and Schanberg, 1970; Costa and Gropetti, 1970). Methamphetamine and other N-alkylated derivatives may be dealkylated to form amphetamine (Smith and Dring, 1970). The pathways of

@CH2-,,,0H

Benzylmethylcarbinol

t

Glucuronide of benzylmethylcarbinol

FIG. 2.

Phenylacetone

Benzoic acid

J

Hippuric acid

Sulfuric acid conjugate of phenylacetone

Pathways of amphetamine deamination.

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

311

TABLE I1 METABOLIC FATEOF AMPHETAMINE IN DIFFERENT SPECIES= Approximate percent dose excreted in urine

Species Man Rhesus monkey Dog Mouse Rat Guinea pig Rabbit

Unchanged

As hydroxylated metabolites

As deaminated metabolites

34 30-40 30 30-40 14 4 18

3 2-6 20 14 60 0 6

24 20-34 20-34 32 2 62 53

Data from Smith and Dring (1970).

amphetamine degradation (Figs. 1 and 2) differ in different animal species (Table 11). As Axelrod (1954) pointed out, p-hydroxylation is the major reaction in the dog and the rat but not in rabbits and guinea pigs. Ellison et al. (1966) contend that in beagles p-hydroxylation and deamination are equally important, whereas Dring et al. (1966) state that deamination is the principal route of metabolism in greyhounds. Also, in the rabbit and in man, deamination is believed to be the most important reaction (Axelrod, 1955; Dring et al., 1966; Ellison et al., 1966). In the mouse and the guinea pig, on the other hand, Hucker (1970) states that amphetamine seems not to be metabolized by deamination. This view is, however, not consistent with the data reported by Smith and Dring (1970). The literature concerning this point has been extensively reviewed by Smith and Dring (1970). With these facts in mind, some investigators also studied the effect of amphetamine metabolites on cerebral metabolism. The conclusions drawn from such studies are included in the following sections.

Ill. Cerebral Function and Brain Metabolism It is a challenging task to elucidate the mechanism of action of amphetamines on CNS function and to investigate the metabolic alterations underlying the functional changes. The latter has been tried in two ways: (1) by studying directly the metabolism of the CNS and (2) by studying the effect of amphetamines on the behavior of animals in

C.-J. ESTLER

312

which selected metabolic reactions have been blocked by means of pharmacological agents.

A. EFFECTON NEUROTRANSMITTERS It is the general opinion that CNS effects of amphetamines are the result of an interference of these drugs with intracerebral, neurohumoral synaptic transmission. Among the neurotransmitters known to be effective in the CNS (acetylcholine, norepinephrine, dopamine, serotonin, y-aminobutyric acid, and other amino acids; for references, see Lajtha, 1970), norepinephrine and dopamine are at first sight the most likely ones to be affected by amphetamines. Since amphetamines are derivatives of epinephrine and act peripherally as sympathomimetic drugs, the conclusion seems justified that the central effects of these agents are also brought about by some adrenergic mechanism. Thus the interest of most investigators has centered primarily on the interference of amphetamines with catecholamines within the CNS. 1. Catecholamines The most important catecholamines in the brain are norepinephrine and dopamine. The epinephrine content seems to be negligible. Norepinephrine is located mainly in the hypothalamus and the brainstem. Concentrations of dopamine and its major degradation product homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid) are highest in parts of the extrapyramidal motor system. Fundamentals of the role of neurotransmitters in the brain and their metabolism are reviewed by Baldessarini (1972), Bloom and Giarman (1968), Glowinski (1970), Glowinski and Baldessarini (1966), Kopin (1968), Shore (1972), and Vogt (1973). a. Catecholamine Content. In the majority of experiments, amphetamine and methamphetamine lowered the norepinephrine content of the brain. This was observed in mice, in which 10 mg/kg or more of

amphetamine decreased the norepinephrine content of whole brain (Beauvallet et al., 1963, 1964, 1967; Everett and Yellin, 1971; Hitzemann et a l . , 1971; Kaergaard Nielsen et al., 1967; Smith, 1965), in rats treated with amphetamine or methamphetamine (Carlsson et a1 ., 1965; Clay et al., 1971; Gunne and Lewander, 1968; Leonard, 1972; Lewander, 1968a; McLean and McCartney, 1961; Morgan et a l . , 1972a; Moore and Lariviere, 1963), and rabbits (Sanan and Vogt, 1962). Leonard and Shallice (1971a) and Welch and Welch (1970) showed that the effect is dose- and time-dependent. Higher doses only reduced the norepinephrine content, whereas lower doses first increased and then decreased

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

313

the cerebral norepinephrine. Furthermore, the norepinephrine-depleting effect became more accentuated after repeated doses of amphetamine (Lewander, 1968a; McLean and McCartney, 1961). From studies by Baird and Lewis (1963, 1964) and Clay et al. (1971), it appears that damphetamine is more effective than 1-amphetamine in lowering brain norepinephrine. Not all brain regions are equally sensitive to amphetamines. Amphetamine caused the norepinephrine content to fall in the cerebellum, the medulla oblongata, the hypothalamus, and the striatum of rats; the greatest effect occurred in the cerebellum (Glowinski et al., 1966a). A reduction of hypothalamic norepinephrine content was also observed in dogs, cats, and rabbits (Laverty and Sharman, 1965). Leonard (1972) described a depletion of norepinephrine in cortex, midbrain, and caudal brain after amphetamine and methamphetamine. On the other hand, a lower dose of amphetamine did not alter the steady-state level of norepinephrine in the forebrain (Salama and Goldberg, 1970). Again damphetamine turned out to be more potent in reducing the norepinephrine content in telencephalon, hypothalamus, and brainstem than the 1isomer (Morgan et d., 1972b). The amphetamine-produced decrease of the norepinephrine content occurred mainly in a particulate cell fraction, probably identical with the so-called varicosities. In the supernatant, the decrease of norepinephrine was not significant (Glowinski et al., 1966b). The effect of amphetamines on the cerebral norepinephrine content is dependent on the physiological state of the animals. For instance, damphetamine is more toxic and lowered brain norepinephrine to a greater extent in aggregated mice than in isolated mice. I-Amphetamine was equally effective under both conditions (Lal and Chessick, 1964; Moore, 1963). By contrast tobnormal animals in which &hetamine was more effective, in mice pretreated with nialamide, a monoamineoxidase inhibitor, the norepinephrine content of the brain was lowered by d- and Z-amphetamine to the same degree (Svensson, 1971). Syrosingopine and a-methyl-m-tyrosine did not influence the depletion of norepinephrine evoked by amphetamine (Moore, 1964), and amphetamines did not influence the catecholamine-depleting action of a-methyl-m-tyrosine. On the other hand, methamphetamine but not amphetamine antagonized the depletion of norepinephrine caused by a-methyl-m-tyrosine (Leonard, 1972). Breese et al. (1970) found no depletion of cerebral norepinephrine when amphetamine was given intracisternally to rats. They discuss whether p-hydroxylation of amphetamine in the liver, which is the major

3 14

C.-J.

ESTLER

metabolic pathway of d-amphetamine in rats (Axelrod, 1954; Smith and Dring, 1970) (see also Table I1 and Section 11), is necessary for the catecholamine-depleting effect of this drug. In fact, p-hydroxyamphetamine (a-methyltyramine) and p-hydroxynorephedrine (a-methyloctopamine), the degradation products of amphetamine (Fig. l), are able to act as false transmitters and to deplete the cerebral norepinephrine stores (Breese et al., 1970; Brodie et al., 1970; Clay et al., 1971; Fischer et al., 1965; Gropetti and Costa, 1969). However, in brain they are less potent than amphetamine, and their cerebral concentrations are very low (Brodie et al., 1970; Clay et a ) . , 1971; Gropetti and Costa, 1969). Important arguments against an essential role for p-hydroxyamphetamine and p-hydroxynorephedrine are that ( 1 ) the d- and l-isomers of amphetamine are hydroxylated equally (Gunne and Galland, 1967) but that, unlike d-amphetamine, l-amphetamine is not a substrate of dopamine-P-hydroxylase and, therefore, cannot be converted to phydroxynorephedrine in vivo (Goldstein and Anagnoste, 1965), and yet 1amphetamine depletes norepinephrine and causes, although to a lesser degree, central stimulation, stereotypies, and other functional changes typical of amphetamines; (2) amphetamine is also active in guinea pigs which do not at all p-hydroxylate amphetamine; (3) amphetamine continues to deplete brain norepinephrine after p- and P-hydroxylation of amphetamine is inhibited by SKF 525A, desmethylimipramine, or disulfram (Brodie et al., 1970; Clay et al., 1971; Lewander, 196813). Thus it becomes unlikely that the effect of amphetamine on cerebral catecholamines is mediated by the hydroxylated metabolites of amphetamine. Lewander (1968b) comes to the same conclusion. The question whether or not norephedrine, which can be formed from amphetamine by dopamine-fl-hydroxylase without previous p-hydroxylation (Clay et al., 1971; Dring et al., 1970), is involved in the cerebral effects of amphetamine remains to be settled. Reports on the effects of amphetamines on cerebral dopamine are more conflicting than those on amphetamines and norepinephrine. An early study by Vogt (1954) revealed no effect of amphetamine on brain dopamine. Also, Baird and Lewis (1963, 1964) using up to 20-mg/kg doses of d-amphetamine and 1-amphetamine found no significant effect on the dopamine content of the whole rat brain. DL-Methamphetamine, 90 pmoles/kg, was likewise ineffective in a study by Morgan et al. (1972a). &Amphetamine, 30 mg/kg, given to rats had only a transient depletory effect (Moore and Lariviere, 1963). On the other hand, Leonard (1972) observed already a decrease of dopamine in rat brain at low doses of d-amphetamine and d-methamphetamine, and 10 mg/kg amphetamine was able to reduce the dopamine content of whole mouse

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

315

brain (Everett and Yellin, 1971; Hitzemann et al., 1971; Smith, 1965). These findings could not be confirmed by Beauvallet et al. (1963, 1964). Lewander (1968a) even reported an increase in brain dopamine after administration of 20 mg/kg dI-amphetamine in rats. As in the case of norepinephrine, the result obtained seems to depend on the dose of amphetamine applied and the duration of action (Welch and Welch, 1970). For instance, 1 mg/kg amphetamine caused an increase of brain dopamine; the same dose of methamphetamine first increased and then decreased the dopamine content (Leonard and Shallice, 1971a). According to the same authors, low doses of amphetamine elevated and doses of 5 mg/kg or more amphetamine lowered the dopamine content after 2 hours. In nialamide-pretreated mice, I-amphetamine decreased and damphetamine increased brain dopamine (Svensson, 1971). The amphetamine effects became more pronounced when animals were treated repeatedly with the drug (Gunne and Lewander, 1968; Lewander, 1968a). In studies of separate brain areas instead of whole brain, the results still remained equivocal. The dopamine content was lowered by 10 mg/ kg amphetamine and methamphetamine in cortex and midbrain (Leonard, 1972). However, 15 mg/kg amphetamine, which significantly reduced the norepinephrine content, failed to alter the dopamine content of cerebellum, medulla oblongata, hypothalamus, and striatum of rats (Glowinski et al., 1966a). Amphetamine, 10 mg/kg, lowered the dopamine content of the caudate nucleus in cats but not in dogs and rabbits (Laverty and Sharman, 1965). In rats, the dopamine content of the striatum was reduced by repeated doses of methamphetamine (Koda and Gibb, 1973). L-Ephedrine (20 mg/kg) reduced the amount of norepinephrine in whole rat brain (Baird and Lewis, 1963, 1964). In the hypothalamus of cats, 50 mg/kg ephedrine caused no alteration of the norepinephrine content (Laverty and Sharman, 1965). The dopamine content of whole rat brain was unaltered by a similar dose of ephedrine (Baird and Lewis, 1964). Conversely, dl-phenmetrazine and I-phendimetrazine were able to increase the norepinephrine content of whole brain (Baird and Lewis, 1964). After prolonged treatment with similar doses, no change in brain norepinephrine content was found (Gunne and Lewander, 1968). The dopamine content of whole rat brain was unaltered by phenmetrazine and phendimetrazine in acute (Baird and Lewis, 1963, 1964) and chronic experiments (Gunne and Lewander, 1968) even if high doses of the drugs were used. Mephentermine caused a significant decrease of norepinephrine only

316

C.-J.

ESTLER

in the telencephalon but not in brainstem and hypothalamus (Morgan et al., 1972b); chlorphentermine was effective in telencephdon and brainstem (Morgan et al., 1972b); and pipradol reduced only slightly the cerebral contents of norepinephrine and dopamine (Smith, 1965). Among the chlorinated analogs of the amphetamines, only p-chlorophenoxyamphetamine reduced the norepinephrine content of whole brain (Everett and Yellin, 1971), whereas p-chloroamphetamine and pchloromethamphetamine were ineffective in this respect (Kaergaard Nielsen et al., 1967; Pletscher et al., 1963, 1968). However, Morgan et al. (1972b) reported that p-chloroamphetamine lowered norepinephrine in telencephalon and brainstem, in contrast to results by Costa et al. (1971b). A similar effect for p-chloromethamphetamine could not be observed (Morgan et al., 1972b). p-Chloromethamphetamine had also no effect on the dopamine content of rat brain (Pletscher et al., 1968), whereas p-chloroamphetamine increased dopamine turnover (Costa et al., 1971b). On the other hand, p-chlorophenoxyamphetamine markedly reduced the dopamine content in brains of mice (Everett and Yellin, 1971). p-Bromomethylamphetamine and p-nitromethylamphetamine reduced or enhanced the amount of norepinephrine present in the brains of rats depending on dose and time schedule (Leonard, 1972; Leonard and Shallice, 1971a,b). p-Methoxyamphetamine produced a significant decrease of norepinephrine and dopamine in whole mouse brain (Hitzemann et al., 1971). Fenfluramine when given in small doses did not alter the norepinephrine concentration of the brain (Costa et al., 1971a). At high doses, fenfluramine reduced cerebral norepinephrine content (Duce and Gessa, 1967; Ziance et al., 197213). This effect was seen in cerebral cortex, cerebellum, medulla oblongata, hypothalamus, and midbrain plus striatum (Ziance et al., 1972b) and referred to all cellular fractions tested (Ziance et al., 1972a). The dopamine content of mouse brain was also considerably reduced by dl-fenfluramine (Ziance et al., 1972b). In summary, the effects of amphetamine and methamphetamine on steady-state levels of catecholamines in the brain are variable. They differ, for instance, with the compound or isomer used, the dose applied, and the time interval between drug application and measurement of the catecholamine content. Besides, the effects vary from brain region to brain region, and species differences seem to be also involved. Repeated attempts have been made to correlate amphetamine-evoked behavioral changes with the alterations of the norepinephrine or dopamine content of whole brain or brain regions, which were supposed to be target areas for amphetamine. In some well-defined experiments,

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

317

a parallelism between functional changes and changes in the catecholamine content could, in fact, be detected. But, if one considers the conflicting experimental data, reviewed in this section, from the various laboratories, it seems impossible at the present time to correlate behavioral effects with any distinct alteration of the norepinephrine or dopamine level in the whole brain or in some particular brain area and in this way to get evidence for the biochemical mechanism underlying the amphetamine effect. This is by no means surprising because a priori it is not to be expected that the effect of amphetamine is closely related to the steadystate level of cerebral catecholamines. This is especially true for the catecholamine content of whole brain, since the effect of nontoxic doses of amphetamine probably focuses only on some particular brain areas. Not even a close relationship between certain behavioral effects and the catecholamine content in specific brain regions is likely to exist because, in neuronal tissue, catecholamines are present in more than one pool, which differ in their functional significance. Moreover, the steady-state level of the catecholamines results from a number of determinants (e.g., synthesis, release, degradation, and re-uptake), each of which can be modified by amphetamine (see the following sections). If amphetamine, indeed, should act as an indirect sympathomimetic within the CNS, then only the small amount of catecholamines that has been released from the pool and has gained access to the adrenergic receptor has functional relevance. This portion is, however, hardly measurable. Nevertheless, a dependence of amphetamine action on cerebral catecholamines must exist at least as far as amphetamines, if they are indirect sympathomimetics, are dependent on the availability of catecholamines within a functional catecholamine store from which they can be released. This explains why depletion of catecholamines will impair the behavioral activity of amphetamines (see Section III,B,l,b and c). What has been discussed about amphetamine itself also applies to its derivatives and halogenated analogs. It appears that the latter have less pronounced effects on the cerebral catecholamines than the parent compounds, but even fenfluramine, which is devoid of stimulatory properties, is able to deplete brain catecholamines. In order to circumvent the difficulties just mentioned and to obtain more conclusive data, attempts have been made to confirm by more specific investigations, the significance of catecholamines for the behavioral effects of amphetamines; for instance, by the estimation of catecholamine turnover, i.e., biosynthesis, metabolism, release, and uptake. The results are discussed in the following sections.

C.-J. ESTLER

318

HO COOH hydroxylase 3,4- Dihydroxyphenylalanine (dopa)

Tyrosine

HO

HO

'

lase Norepinephrine

3-Hydroxytyrarnine (dopamine)

FIG. 3. Norepinephrine biosynthesis.

b. Catecholamine Synthesis. The pathway of catecholamine synthesis is depicted in Fig. 3. Koda and Gibb (1971) and Harris and Baldessarini (1973) reported a decreased activity of tyrosine hydroxylase in the corpus striatum of rats treated with methamphet'amine. In a more recent paper, Koda and Gibb (1973) showed that this effect was only a temporary one. Maximum inhibition was found after 36 hours; later on the activity of the enzyme increased and even exceeded the control values. The inhibitory effect of amphetamine on the striatal tyrosine hydroxylase was antagonized by chlorpromazine and bicuculline, an assumed blocking agent of yaminobutyric acid receptors (Harris and Baldessarini, 1973). The authors suppose that descending neurons may be involved in a neurophysiological feedback regulation of activity. Inhibition of tyrosine hydroxylation was also observed in the caudate nucleus, when methamphetamine was given repeatedly to rats. A single dose caused only an insignificant inhibition of the enzyme. Tyrosine hydroxylase of brainstem and hypothalamus was affected neither by single nor by repeated doses of methamphetamine (Fibiger and McGeer, 1971). Since tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis in the brain (Udenfriend, 1968), an inhibition of this enzyme might result in a decreased rate of catecholamine synthesis and lead to reduced levels of catecholamines in certain brain areas, as described in Section III,A,l,a. In fact, in some experiments on whole brain, amphetamine reduced the accumulation of dopamine (Hitzemann et al., 1971) and norepinephrine (Hitzemann et al., 1971; Lewander, 1971) from tyrosine. Amphe-

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

319

tamine also caused a marked decrease of dopamine synthesis from labeled tyrosine in the caudate nucleus, whereas in the substantia nigra, accumulation of d ~ p a m i n e - ~ Hwas enhanced and accumulation of n~repinephrine-~H diminished (Javoy et al., 1970). In the striatum of rats treated with a small dose (0.3 mg/kg) of d-amphetamine, Costa et al. (1972) found enhanced conversion of tyrosine to dopamine. The conversion of tel-diencephalic tyrosine to norepinephrine was not changed by d-amphetamine. 1-Amphetamine (1 mg/kg) failed to increase the tyrosine conversion in both structures (Costa et a l . , 1972; Gropetti et al., 1972). In these investigations by Costa et a l. (1972) and Gropetti et al. (1972) a parallelism between increased tyrosine-to-dopamine conversion and locomotor stimulation was observed. In whole brain the formation of dopamine and norepinephrine from dopa was not inhibited by 10 mg/kg d-amphetamine (Hitzemann et al., 1971). This finding contrasts with earlier findings by Carlsson et al. (1966a,b) that amphetamines inhibit accumulation of norepinephrine but not of dopamine when dopa is given as precursor. This effect was especially pronounced in the brain hemispheres and striatum. Glowinski et al. (1966a) found that in four brain regions (cerebellum, medulla oblongata, hypothalamus, and striatum) the accumulation of norepinephrine from dopamine as precursor was markedly reduced by d-amphetamine (15 mg/kg). In accordance with the latter results, d-amphetamine-and also e p h e d r i n d e c r e a s e d the norepinephrine-to-dopamine ratio in whole rat brain (Baird and Lewis, 1963; Beauvallet et a l . , 1964). Glowinski et al. (1966a) explain this effect by the inhibition of dopamineP-hydroxylase by amphetamine as described by Goldstein and Contrera (1961). Similarly, in rats under chronic treatment with amphetamine, the accumulation of norepinephrine was reduced while the accumulation of dopamine remained unimpaired (Lewander, 1971). Other conflicting data come from Salama and Goldberg (1970) who report that 5 mg/kg amphetamine increase the turnover of norepinephrine. p-Methoxyamphetamine, which is a weak psychomotor stimulant, markedly inhibited the accumulation of dopamine and norepinephrine when tyrosine was given as precursor. When dopa was given as precursor the accumulation of dopamine and norepinephrine was doubled at higher (30 mg/kg) doses of p-methoxyamphetamine (Hitzemann et a l . , 1971). From the experiments by Fibiger and McGeer (1971), Koda and Gibb (1971, 1973), and Goldstein and Contrera (1961) a diminished rate of catecholamine formation from tyrosine and dopa could be expected at least in the striatum and the caudate nucleus. The enhanced accumulation of dopamine from tyrosine as precursor as well as the observations

320

C.-J.

ESTLER

that catecholamine depletion occurs also in the hypothalamus (Fuxe and Ungerstedt, 1970; Moore and Lariviere, 1963; Vogt, 19%), where, according to Fibiger and McGeer (19711, tyrosine hydroxylase is not inhibited, seems to be inconsistent with this expectation. It must be kept in mind, however, that the levels of catecholamines found after the administration of drugs (amphetamines) and a labeled precursor of catecholamine biosynthesis are not controlled only by tyrosine hydroxylase or dopamine-P-hydroxylase and do not solely reflect the conversion rate of tyrosine to catecholamines. They are also influenced by the rates of tyrosine uptake, catecholamine metabolism, catecholamine release, and reuptake of released catecholamines. Whereas the uptake of tyrosine into the tissue seems not to be modified by amphetamines, the other parameters may be changed markedly by amphetamines depending on the brain region investigated and the dose of amphetamine applied. These considerations are extensively discussed by Glowinski et al. (1966a) and Javoy et a l . (1970). Thus, the experiments reviewed in this section do not give conclusive evidence regarding the effects of amphetamine compounds on the biosynthesis of catecholamines within different regions of the CNS. However, from the majority of experiments, it. appears that amphetamines inhibit rather than increase catecholamine biosynthesis in the brain. But on the basis of our knowledge on the function of catecholamines within the CNS (Baldessarini, 1972; Glowinski and Baldessarini, 1966; Snyder, 1967; Vogt, 1973), it is difficult to understand that such an effect should be the basis of the central stimulatory actions of amphetamines. At least the inhibitory action of amphetamines on catecholamine synthesis is hard to reconcile with the concept that amphetamines act as indirect sympathomimetic agents unless one supposes that the catecholamines released by amphetamines cause a feedback inhibition of catecholamine synthesis. C . Catecholamine Release. Catecholamines are stored in the brain in more than one area. Whereas one storage site is characterized by a slow turnover, another site has a rapid turnover, and apparently from this site catecholamines can be readily released (Glowinski et al., 1965; Shore, 1972). Histochemical fluorescence studies by Carlsson et a l . (1967) indicate a catecholamine-releasing action of amphetamines on noradrenergic and dopaminergic nerve endings. This is confirmed by biochemical investigations. In acute experiments, amphetamine releases norepinephrine from brain in situ (Carr and Moore, 1969, 1970; Glowinski and Axelrod, 1965; Glowinski et a l . , 1966a; Salama and Goldberg, 1970; Sparber and Tilson, 1972; Stein and Wise, 1969) or from in vitro preparations (Azzaro

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

32 1

and Rutledge, 1973; Enna et al., 1973; Ng et al., 1970; Ziance et al., 1972a). The experiments by Glowinski et al. (1966b) and Ziance and Rutledge (1972) indicate that amphetamine releases norepinephrine preferentially from a particulate fraction perhaps corresponding to the varicosities and has only a small effect on norepinephrine in cell bodies and axons. d-Amphetamine was more effective than 1-amphetamine in releasing norepinephrine (Carr and Moore, 1970; Ziance et al., 1972a). The releasing effect of amphetamine was not lessened in amphetaminetolerant rats (Sparber and Tilson, 1972). Similar to the release of norepinephrine after electrical stimulation the amphetamine-induced norepinephrine release shows a dependency on the availability of calcium (Ziance et al., 1972b). a-Methyltyrosine inhibited the releasing action (Enna et al., 1973). The releasing effect of amphetamines affects not only norepinephrine. Experiments by several investigators show that dopamine is also released (Besson et al., 1969; McKenzie and Szerb, 1968; Voigtlander and Moore, 1973; Ziance et al., 1972a). d-Amphetamine was generally more effective than 1-amphetamine (Voigtlander and Moore, 1973). However, in the experiments by McKenzie and Szerb (1968) and Ziance et al. (1972a), higher doses of amphetamine were required for releasing dopamine from chopped cerebral cortex than for the release of norepinephrine. By contrast, high amphetamine concentrations released more dopamine than norepinephrine (Ziance et a l . , 1972a). Azzaro and Rutledge (1973) studied the effect of amphetamine on the release of catecholamines from minced tissue of various brain regions. Very low concentrations of amphetamine were needed to release catecholamines. It appears that the portion of amines released is a function of the localization of the neurons rather than a function of the amount of amines accumulated by the tissues or the density of the innervation. Cerebral cortex is most sensitive to the norepinephrinereleasing action: threshold concentration is lo-' M , whereas M is required to release norepinephrine from medulla oblongata and striatum. Conversely, the striatum is most sensitive to the dopamine-releasing activity of amphetamine. The threshold concentration is M compared to lop5M in cerebral cortex. These findings are in agreement with the observation by Taylor and Snyder (1971) that higher doses of amphetamine are required to initiate stereotyped behavior, which supposedly originates from the action of amphetamine on the extrapyramidal motor system, than are necessary for increasing locomotor activity, which supposedly arises from stimulation of cortical areas. In other studies, Ebstein et al. (1972) and Stein and Wise (1969)

322

H&-

C.-J. ESTLER

Dopamine

Norepinephrine

3-Methoxytyramine

Normetanephrine

0

Ho&c%-cOOH 3-Methoxy-4-hydroxyphenylacetic acid (homovanillic acid)

3,4-Dihydroxymandelic acid

3-Methoxy-4-hydroxymandelic acid (vanillylmandelic acid)

H,C-0 HO-&J--EP,.~OH 3-Methoxy-4-hydroxyphenylglycol

FIG. 4. Metabolism of dopamine and norepinephrine. AO, aldehyde oxidase; COMT, catechola-methyltransferase; DO, dopamine-p-hydroxylase; MAO, monoamine oxidase.

showed that amphetamine was more potent in releasing norepinephrine in the amygdala than in the hypothalamus. The ability of amphetamines to release catecholamines from cerebral structures is not unique to the parent compounds. Methylphenidate and deoxypipradrol were also effective as releasers of norepinephrine and dopamine from striatum and to a lesser degree from cerebral cortex and hypothalamus (Ferris et al., 1972; Moore et al., 1970). In the same way, fenfluramine can release norepinephrine from brain tissue (Ziance and Rutledge, 1972). d . Catecholamine Metabolism. Catecholamines can be degraded in the brain by catecholaminea-methyltransferase (0-methylation) or by monoamine oxidase (deamination) (Fig. 4) (Glowinski and Axelrod, 1965; Milhaud and Glowinski, 1963). The latter process takes place mainly inside, and the former outside the cells. The experiments by Glowinski and Axelrod (1965) and Ziance and Rutledge (1972) indicate that amphetamine releases norepinephrine as unchanged amine, which may, however, be metabolized outside the cells. Obviously, the degradation of catecholamines is changed by amphetamines from deamination to 0-methylation, because the amount of normetanephrine found in brains of amphetamine-treated rats was

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

323

increased, whereas the deaminated degradation products were diminished (Glowinski and Axelrod, 1965). This applies to many brain regions, such as cerebellum, medulla oblongata, hypothalamus, striatum, midbrain, hippocampus, and cerebral cortex (Glowinski et al., 1966a), where the amount of normetanephrine markedly increases due to the deaminated metabolites of norepinephrine. An increase of normetanephrine was also found by Carr and Moore (1970), and a decrease of dihydroxymandelic acid by Hitzemann et al. (1971). In a similar fashion, amphetamine increased the amount of 3-methoxytyramine, the 0methylated metabolite of dopamine (Carlsson et al., 1966a,b). Furthermore, amphetamine enhanced the formation of homovanillic acid, which is the major metabolite of dopamine in the striatum (Jori et al., 1973; Laverty and Sharman, 1965). The d-isomer was more active than the 1form (Jon et al., 1973). It has been discussed, whether the reason for these shifts in the catecholamine metabolic pathways might be an inhibitory effect of amphetamine on monoamine oxidase, which can be demonstrated to occur not only in the liver but also in brain tissue. Interestingly, damphetamine is again more potent than the I-isomer (Grana and Lilla, 1959; Mgller Nielsen and Dubnick, 1970). The argument against the results obtained by Grana and Lilla (1959) that very high concentrations of amphetamine were required to achieve only partial inhibition of monoamine oxidase (e.g., M for 40% inhibition) could be refuted by later experiments by Glowinski et al. (1966a). These authors pointed out that monoamine oxidase inhibition was small (30% with 2 X W 4 M amphetamine) when tryptamine was used as substrate for monoamine oxidase and was still incomplete with concentrations of amphetamine 20 times greater than those found to occur in the brain. If, however, norepinephrine was used as substrate, almost complete inhibition could be achieved with only 5 x lop4 M concentrations of amphetamine. Concentrations of this order of magnitude are actually found in the brains of rats treated with a 15-mg/kg dose of amphetamine. In view of these findings, the suggestion by Mann and Quastel (1940) long ago that the central stimulatory effect of amphetamine might be due to an inhibition of amine oxidase appears in a new light. Chloroamphetamine also inhibited monoamine oxidase. It was more potent when serotonin instead of tyramine or tryptamine was used as substrate (Mgller Nielsen and Dubnick, 1970). Methoxyamphetamine increased the level of normetanephrine and methoxytyramine in the brain. The amount of dihydroxymandelic acid was decreased (Hitzemann et al., 1971). Fenfluramine, unlike amphetamine, increased the release of deami-

324

C.-J.

ESTLER

nated degradation products of norepinephrine from brain (Ziance and Rutlege, 1972). An inhibition of monoamine oxidase was found in vitro with 2 ~ l O M - ~ concentrations of d-, I-, and dl-fenfluramine. In vivo 30 mg/kg dl-fenfluramine were ineffective (Duhault and Verdavainne, 1967). Fenfluramine as well as its metabolite norfenfluramine elevated the striatal concentrations of homovanillic acid. In this case the 1isomers were more potent than the d-isomers (Jori et a l . , 1973). In summary, the data reviewed in the last two sections show that amphetamine and some of its derivatives release norepinephrine and dopamine from storage sites within various regions of the CNS. In this way the data are well suited to establish the role of amphetamines as indirectly acting sympathomimetic drugs. The shift in the metabolism of the catecholamines released from the nerve cells from deamination to 0methylation is in agreement with this concept, since, as consequence of the releasing action of amphetamines, a greater amount of catecholamines will come in contact with the extraneuronally located catecholamine&-methyltransferase. The data indicate that possibly norepinephrine and/or dopamine are involved in the behavior effects of amphetamines, but they do not allow to decide clearly whether release of norepinephrine or release of dopamine is the basic mechanism for stimulating effects of amphetamines. It may well be that both catecholamines are involved in the behavioral effects. One must also be careful to infer from the regions where catecholamines are released to the primary locus of action of the amphetamines, since nerve endings and their cell bodies may be located in distant brain nuclei, and neuronal excitation may spread over wide distances from area to area. It is noteworthy to mention that the nonstimulating derivative, fenfluramine, behaves differently from the amphetamines themselves. e. Catecholamine Uptake. Nerve tissue including brain is capable to take up biogenic amines (norepinephrine, dopamine, and serotonin) from the surrounding medium. The uptake mechanisms seem to have some specificity for the different amines. Norepinephrine has a high affinity for noradrenergic neurons, whereas dopamine has a 5 times higher affinity than norepinephrine for the dopaminergic neurons of the striatum (Snyder and Coyle, 1969). Similar observations were made by Azzaro and Rutledge (1973) who stated that the accumulation of amines in various brain regions is related to the amount and type of neurons predominating in these areas. Thus, norepinephrine accumulates mostly in the medulla oblongata, which contains cell bodies and nerve endings from noradrenergic neurons, and to a lesser degree in the cerebral cortex, which has a lower density of noradrenergic nerve endings.

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

325

Norepinephrine is also taken up into the striatum, where dopaminergic neurons predominate, but the uptake of norepinephrine in this region is exceeded by far by the uptake of dopamine. On the other hand, the uptake of dopamine into cerebral cortex and medulla oblongata, which contain fewer dopaminergic neurons, is comparatively small. Histochemical studies by Arbuthnott (1969) and Carlsson et al. (1966b) show that amphetamine blocks the uptake of norepinephrine into fibers and cell bodies of cells from the cerebral cortex. This is confirmed by quantitative biochemical determinations of norepinephrine uptake. Glowinski and Axelrod (1965) and Strada et al. (1970) found an inhibition of norepinephrine uptake into the whole brain in situ. Studies by Glowinski et aI. (1966b) indicate that this effect concerns mainly a particulate fraction of brain cells, which may be identical with the socalled varicosities. Other investigations were concerned with the effect of amphetamines on different brain regions. Glowinski et al. (1966a) found that 10-20 mg/ kg amphetamine inhibited the uptake of norepinephrine into cerebellum, medulla oblongata, and hypothalamus but not the uptake into the cerebral cortex i n vivo. Amphetamine, 5 mg/kg, was ineffective (Glowinski et al., 1966a). In midbrain, brainstem, medulla oblongata, and cerebellum, d-amphetamine (10 mg/kg) likewise reduced the uptake of norepinephrine, whereas the same dose of 1-amphetamine was practically devoid of action. In the striatum, both isomers lowered the uptake of norepinephrine and dopamine to a similar degree (Snyder, 1970; Taylor and Snyder, 1970, 1971). These findings correlated well with the functional effects on locomotor and stereotyped behavior. Snyder (1970) concludes from the comparison between the described metabolic and the behavioral effects of the amphetamine isomers that inhibition of norepinephrine uptake might be a major mechanism of action and that metabolic effects on the striatum are not involved in the locomotor-stimulating effects of amphetamines. I n vitro amphetamine blocked the uptake of norepinephrine into the hypothalamus and the amygdala to the same extent (Ebstein et al., 1972). Amphetamine also inhibited the uptake of norepinephrine into cerebral cortex and synaptosomes of cortex, hypothalamus, and striatum (Ferris et al., 1972; Ziance and Rutledge, 1972). In this case the disomer was more potent than the I-isomer (Ferris et al., 1972). The reuptake of catecholamines released from stimulated neurons plays an important role in the termination of catecholamine action. Therefore, if the uptake of norepinephrine is inhibited, more norepinephrine is available for the reaction with the adrenoreceptors, and any noradrenergic or dopaminergic action should be enhanced in such case.

326

C.-J. ESTLER

In accordance with this concept seems to be the finding that desipramine, which also inhibits norepinephrine uptake into nerve endings, enhances both the diminution of norepinephrine uptake and the central stimulating action of amphetamine in rats (Strada et al., 1970). However, it is very likely that the true reason for this synergism between amphetamine and desipramine is not the similar action of amphetamines and desipramine on the neuronal membrane pump for catecholamines but the fact that imipramine-like compounds inhibit the p-hydroxylation of amphetamine and its degradation to p-hydroxyamphetamine and p-hydroxynorephedrine (Consolo et al., 1967; Sulser et al., 1%6), which is the major pathway of amphetamine metabolism in rats (Axelrod, 1954). This conclusion arises from the fact that iprindole, another tricyclic antidepressant, which also inhibits p-hydroxylation of amphetamine in rats (Freeman and Sulser, 1972; Lemberger et al., 1970) but does not interfere with the uptake of norepinephrine into nerve tissue (Freeman and Sulser, 1972; Lemberger et al., 1970; Miller et al., 1970) also enhances the stimulatory action of amphetamine in rats (Freeman and Sulser, 1972; Miller et al., 1970). On the other hand, the above-mentioned concept of Snyder (1970) is not in agreement with a number of other investigations indicating that dopaminergic rather than adrenergic mechanisms are responsible for the stimulating effect of amphetamine. There is, however, evidence that amphetamine is also able to inhibit the uptake of dopamine into dopaminergic neurons. This notion is further supported by experiments by Ferris et al. (1972) who demonstrated that amphetamine at low concentrations can inhibit the uptake of dopamine into synaptosomes of cerebral cortex, hypothalamus, and striatum in vitro. In these experiments once more the dextrorotatory isomer was more potent. On the other hand, Glowinski et al. (1966a) reported that in vivo 15 mg/kg amphetamine did not reduce the uptake of dopamine into cerebellum, medulla oblongata, and hypothalamus. Coyle and Snyder (1969) observed a reduction of dopamine uptake into the striatum. In later experiments by Taylor and Snyder (1970, 1971), d- and 1-amphetamine were almost equally effective in inhibiting the dopamine uptake into the striatum. Methylphenidate and deoxypipradrol had an effect similar to amphetamine on the uptake of norepinephrine, with the exception that in case of deoxypipradrol the R-(-)-form was the more potent isomer (Ferris et al.,

1972). According to the experiments by Strada et al. (1970) the chloroamphetamines seem to differ from the parent compounds, since p-chloroamphetamine did not significantly inhibit the uptake of norepinephrine into

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

327

rat cerebral tissue, but in other experiments p-chloroamphetamine inhibited the uptake of norepinephrine into brainstem and striatum (Morgan et al., 1972b). Chlorphentermine diminished the uptake of norepinephrine into brainstem, hypothalamus, and striatum (Morgan et al., 1972b). Fenfluramine depressed the uptake of norepinephrine into the hypothalamus (Morgan et al., 197233) and the cortex (Ziance and Rutledge, 1972). Taking into account the available biochemical data on catecholamine turnover, it appears that amphetamines may exert indirect adrenergic and dopaminergic effects in at least two ways: by the release of preformed catecholamines and by the inhibition of reuptake of released catecholamine molecules. Further effects such as inhibition of catecholamine biosynthesis and catecholamine metabolism cannot be precluded. These effects have been observed in many brain regions, and it seems that dopaminergic actions predominate in the extrapyramidal motor system, whereas noradrenergic actions cannot easily be restricted to any particular brain area. It may well be, but it cannot be proven from the presently available biochemical data, that the above-mentioned interference with adrenergic and dopaminergic neurotransmission is the basic mechanism of the behavioral effects of the amphetamines. Further evidence for these hypotheses was hoped to be gained by experiments with amphetamines in combination with pharmacological agents that inhibit or enhance catecholamine turnover within the CNS. The results of such investigations are discussed in Section 111, B. 2. Serotonin The role of serotonin in brain function is described by Page and Carlsson (1970). The pathways of its synthesis and degradation are shown in Fig. 5. Investigations on the action of amphetamines on brain serotonin furnished divergent results. In rats, 10-20 mglkg amphetamine had no influence on the serotonin content of whole brain (Pletscher and Gey, 1959; Pletscher et al., 1964); 30 mg/kg caused only a small increase (Ferri et al., 1963); and 90 mg/kg dl-amphetamine produced an insignificant decrease (Morgan et al., 1972a). In mice, d-amphetamine increased brain serotonin significantly already at doses of 10 mg/kg (Everett and Yellin, 1971; Smith, 1965). On the other hand, Leonard (1972) found that d-amphetamine and &methamphetamine (2X 10 mg/kg) significantly decreased the cerebral serotonin content. La1 and Chessick (1964) observed a reduction of the serotonin content of the brains of mice treated with 25 mg/kg amphetamine. The magnitude of this effect was comparable in isolated and aggregated animals. In the thalamus of

C.-J. ESTLER

328

COOH

c%- CH-NH,

Tryptophan

J

tryptophanhydroxylase

FOOH

H

O

m CH,-

CH-NH, I

tryptophan HO ~ C I Z - M , - - N % c

decarboxylase 5-Hydroxytryptamine (serotonin)

5- Hydroxytryptophan

monoamine oxidase

H o ~ C H 2 - c m H

-HoaQJ-aldehyde oxidase

5-Hydroxyindoleacetic acid

5- Hydroxyindoleacetaldehyde

FIG. 5. Biosynthesis and metabolism of serotonin.

cats and in the caudate nucleus of dogs, 10 mgkg d-amphetamine also caused a significant decrease of the serotonin content (Laverty and Sharman, 1965), whereas the 5-hydroxyindoleacetic acid content of these areas remained unchanged. The former result confirms earlier observations by Paasonen and Vogt (1956). In experiments by Leonard (1972), Morgan et al. (1972a), and Pletscher et al. (1964), methamphetamine (20-90 mg/kg) also decreased the serotonin content of whole brain of rats. Both optical isomers were equally effective (Morgan et al., 1972a). The serotonin-increasing effect of amphetamine was more pronounced after repeated doses (McLean and McCartney, 1961). Also Utena (1966) found an increase of serotonin in animals after extended treatment with methamphetamine. Given once a day for 1 month, doses of only 1 mg/kg d-amphetamine increased the cerebral serotonin content of rats (Diaz and Huttunen, 1972). The serotonin depletion produced by p-chlorophenylalanine, which is an inhibitor of tryptophan hydroxylase, an enzyme involved in serotonin

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

329

synthesis, is potentiated by amphetamine and methamphetamine (Leonard, 1972). Welch and Welch (1970) assume that amphetamine has a similar biphasic effect on serotonin as on norepinephrine and dopamine. Thus, the discrepancies observed by the various investigators may in part arise from the fact that the effect of amphetamine on brain serotonin varies with the duration of the experiment: depletion of serotonin may be followed by an increase (Leonard and Shallice, 1971a). Furthermore, as in the case of catecholamines, the steady-state content of serotonin measured in the above-mentioned experiments is the result of serotonin synthesis, release, and metabolism, parameters that may be influenced in different ways by amphetamines under different experimental conditions. Therefore, the measurement of serotonin levels is not very well suited to furnish information on the action of a drug on serotonin metabolism. More systematic investigations indicate that the synthesis of serotonin in brain seems to be increased by amphetamine in acute (Pletscher and Gey, 1959; Reid, 1970) and chronic experiments (Diaz and Huttunen, 1972). The release of serotonin from the brain in vivo appears not to be affected in nontolerant mice treated with 2.5 mg/kg d-amphetamine. If tolerance develops the release of serotonin and its metabolites is increased (Sparber and Tilson, 1972). In vitro, lop5 M amphetamine enhanced the release of serotonin from cerebral cortex (Azzaro and Rutledge, 1973) and striatum (Ng et al., 1970). The same concentration of amphetamine is required to stimulate the serotonin release from the medulla oblongata (Azzaro and Rutledge, 1973). Among the two uptake mechanisms for serotonin in cerebral tissue, amphetamine inhibits only the low-affinity component (Wong et al., 1973). The concentration of 5hydroxyindoleacetic acid was increased by methamphetamine in whole brain and in caudal brain (Leonard, 1972; Reid, 1970). It cannot be precluded that some of these changes are due to amphetamine-induced hyperthermia (Reid, 1970). Pipradrol had no effect on the serotonin content of whole mouse brain when it was given at doses up to 30 mg/kg (Smith, 1965). The same is true for chlorphentermine (55-90 mg/kg) (M@llerNielsen and Dubnick, 1970). Mephentermine reduced the serotonin content of brainstem and telencephalon (Morgan et a l . , 1972b). Fenfluramine also caused a depletion of brain serotonin (Duhault and Verdavainne, 1967; Morgan et a l . , 1972a; Opitz, 1967) and 5-hydroxyindoleacetic acid (Duhault and Verdavainne, 1967; Opitz, 1967). The d-form was more potent (Duhault and Verdavainne, 1967). Norfenfluramine had a similar effect in the

330

C.-J.

ESTLER

hypothalamus, in the brainstem, and especially in the telencephalon (Morgan et al., 1972b). There is evidence that fenfluramine does not inhibit the conversion of tryptophan to serotonin (Costa et al., 1971a) and that it does not inhibit tryptophan hydroxylase and 5-hydroxytryptophan decarboxylase (Duhault and Verdavainne, 1967). Monoamine oxidase is inhibited only in vitro but not in vivo (Duhault and Verdavainne, 1967). p-Chloroamphetamine greatly reduced the serotonin content of whole brain (Everett and Yellin, 1971; Opitz, 1967; Pletscher et al., 1968; Sanders-Bush and Sulser, 1970), telencephalon, hypothalamus, and brainstem (Costa et al., 1971b; Kaergaard Nielsen et al., 1967; Morgan et al., 1972b). The greatest effect is seen in the telencephalon. The restoration of the serotonin content seems to be an extremely slow process lasting for several weeks (Frey, 1970). Chloroamphetamines and chloromethamphetamine inhibit the uptake of serotonin (Carlsson, 1970a; Pletscher et al., 1964); this applies both to the high-affinity and to the low-affinity uptake mechanisms (Wong et al., 1973). For the highaffinity uptake, 4-chloroamphetamine was more potent than 2-chloroamphetamine and 3-chloroamphetamine. The low-affinity uptake, on the other hand, was inhibited to the same degree by 2-~hloroamphetamine, 3-chloroamphetamine, and 4-chloroamphetamine (Wong et al., 1973). In addition, 4-chloroamphetamine releases serotonin from brain in vitro (Wong et al., 1973) and diminishes serotonin synthesis, probably by inhibiting tryptophan hydroxylase (Costa and Revuelta, 1972; SandersBush and Sulser, 1970, 1972). It is an inhibitor of monoamine oxidase, especially when serotonin serves as substrate (Mgiller Nielsen and Dubnick, 1970). p-Chloromethamphetamine was less effective in reducing the brain serotonin content; nevertheless, the serotonin content was lowered in whole brain and telencephalon (Morgan et al., 1972b; Pletscher et al., 1963, 1964, 1968). 5-Hydroxyindoleacetic acid content of whole brain showed a parallel decrease (Pletscher et al., 1963, 1968); 5-hydroxytryptophan decarboxylase was not inhibited. Inhibition of 5-hydroxytryptophan uptake or tryptophan hydroxylation may occur (Pletscher et al., 1963). Chlorophenoxyamphetamine had a dual effect: after 1 hour the serotonin content was increased, and after 4 hours, high doses decreased cerebral serotonin (Everett and Yellin, 1971). p-Bromomethylamphetamine depleted serotonin in whole brain. The 5-hydroxyindoleacetic acid content rose in middle and caudal brain (Leonard, 1972). Other agents that deplete brain serotonin include ethylamphetamine, phentermine, chlorphentermine, dichlorphentermine, and fenfluramine (Opitz, 1967). p-Methoxyamphetamine raised the serotonin content and

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

33 1

lowered the 5-hydroxyindoleacetic acid content of mouse brain (Hitzemann et al., 1971). p-Nitromethylamphetamine caused serotonin to increase in middle brain and caudal brain (Leonard. 1972): the 5,, hydroxyindoleacetic acid content rose only in caudal brain (Leonard, 1972). Enhanced turnover of 5-hydroxyindoles may be related to an amphetamine-induced increase of cerebral tryptophan, the amino acid precursor of serotonin, (Leonard and Shallice, 1971a; Schubert et al., 1970; Schubert and Sedvall, 1972; Tagliamonte et al., 1971), which may be the result of an enhanced uptake of this amino acid from the plasma (see also Section III,C,6). A similar effect was observed in chronic experiments in which 1 mg/kg d-amphetamine per day also influenced the uptake of tryptophan into the brain of rats (Diaz and Huttunen, 1972). The tryptophan levels in brain remained, however, unchanged under these special conditions. p-Chloroamphetamine and p-hydroxyamphetamine were less effective in increasing the tryptophan content of brain (Schubert and Sedvall, 1972). It appears from the data reviewed in this section that the effects of amphetamines on cerebral serotonin turnover are small compared to those exerted by these drugs on catecholamine turnover. An exception are the chlorinated analogs of amphetamine. Their influence on brain serotonin cannot be ignored and prompted some authors to postulate that these compounds act mainly through interference with serotonergic mechanisms (Frey and Magnussen, 1968; Kaergaard Nielsen et al., 1967; Pletscher et a l . , 1966, 1968).

3. Acetylcholine Cerebral acetylcholine content fell considerably after treatment of rats with 2 mg/kg d-amphetamine. In rats pretreated with hemicholinium depletion of acetylcholine was even more pronounced (Domino and Wilson, 1972). These results can be explained by an increased turnover of acetylcholine induced by amphetamine. In accordance with these findings, a very high dose (100 mg/kg) of d-amphetamine enhanced the release of acetylcholine from brain cortex of anesthetized rats. Similar results were obtained with a much lower dose (5 mg/kg) of damphetamine in rabbits (Hemsworth and Neal, 1968) and in cats (Deffenu et al., 1970). These few data are not sufficient to allow conclusions on the significance of cholinergic mechanisms for amphetamine action, especially if one bears in mind the methodological difficulties that impede the exact measurement of acetylcholine in tissues. Release of acetylcholine may also be the result of a secondary excitation of cholinergic neurons.

332

C.-J.

ESTLER

4. y-Aminobutyric Acid Data on the effect of amphetamines on y-aminobutyric acid are still more erratic and inconclusive. d-Amphetamine and d-methamphetamine produced an increase of the cerebral content of y-aminobutyric acid (Leonard and Shallice, 1971a). The release of y-aminobutyric acid from rabbit brain in vivo was doubled by 2-3 mg/kg amphetamine (Blagoeva et al., 1972). p-Nitromethylamphetamine caused only a slight increase of the y-aminobutyric acid in the brain even when the drug was given at a high dose (60 mg/kg) (Leonard and Shallice, 1971b). p-Bromomethamphetamine reduced the y-aminobutyric acid content of the brain (Leonard and Shallice, 1971a).

5. Histamine The histamine content of rat brain was not changed by 8 mg/kg damphetamine (Green and Erickson, 1964).

B. BEHAVIORAL EFFECTSOF AMPHETAMINES I N ANIMALS PRETREATED WITH METABOLIC INHIBITORS As discussed in Section III,A,l, the measurement of the effect of amphetamines on various parameters of the turnover of catecholamines and other neurotransmitters made it very likely that alterations of catecholamine turnover are causally related to the behavioral effects of amphetamine. However, conclusive evidence for this assumption could not be furnished by these experiments and, thus, the necessity arose to pursue another route of investigation. One possibly successful way is to block some reactions that are considered essential for the drug effect and to observe whether or not functional effects are also inhibited. Such experiments are reviewed in the following sections. 1. Locomotor Activity a. Blockade of Adrenergic Receptors. Treatment of mice with the preceptor-blocking agent propranolol (2.5-20 mg/kg) partially inhibited the motor activation induced by amphetamine (Mantegazza et al., 1970) and methamphetamine (Estler and Ammon, 1967; Estler, 1974); 1 mg/kg or less propranolol was ineffective in this respect (Fischer et al., 1968; Hutchins and Rogers, 1971). This antagonism was not due to the inherent central depressant activity of propranolol since 1-(4’-nitrophenyl)-2-isopropylaminoethanol (INPEA), a P-sympatholytic drug with centrally stimulating properties, showed essentially the same effects (Estler and Ammon, 1971). Pronethalol (2.5-10 mg/kg) was ineffective

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

333

(Hutchins and Rogers, 1971). The rearing activity of rats was not antagonized by propranolol (Cahn and Herold, 1970). The a-sympatholytic drug phenoxybenzamine (20 mg/kg) had an antagonistic effect on methamphetamine in rats and even more pronounced in mice when phenoxybenzamine was given 2 hours before amphetamine (Maj et al., 1972b). Similar observations were made by Rolinski and Scheel-Kruger (1973), Cahn and Herold (1970), and Galambos et al. (1967) and by Rech and Stolk (1970) only when small doses of amphetamine were used. When phenoxybenzamine and methamphetamine were administered simultaneously to mice, only a partial inhibition of the methamphetamine-induced motor excitation could be achieved (Estler, 1974). Phentolamine had no antagonistic effect (Hutchins and Rogers, 1971). The increased motor activity could be blocked completely by pretreating the animals with dihydroergotamine (Fischer et al., 1968) or yohimbine (Fahse et al., 1971), compounds with aadrenergic receptor-blocking properties. In rats, however, dihydroergotamine had only a weak antagonistic effect against amphetamine (Rolinski and Scheel-Kruger, 1973). Chlorpromazine inhibited the amphetamine hyperactivity only at high doses; lower doses accentuated the effect (Sulser and Dingell, 1968). A combination of propranolol and phenoxybenzamine did not completely block the locomotor stimulation evoked by methamphetamine (Estler, 1975). Most of these results indicate that adrenergic receptors, especially the a-type, seem to be essential for elicitation of the full central stimulating effect of amphetamines, and in this way they support the hypothesis of a central sympathetic mechanism of action. But they do not allow to decide whether amphetamines act by combining directly with the receptor or indirectly by releasing catecholamines from intracerebral storage sites. The ineffectiveness of the adrenoreceptor blocking drugs observed by some investigators points to the involvement of other mechanisms, possibly an interaction with dopamine receptors. b. Depletion of Catecholamines. In experiments on animals pretreated with reserpine in order to deplete their CNS from preformed norepinephrine, conflicting results were obtained. There may be several reasons for this. Since amphetamines produce complex changes in animal behavior, the results obtained will be dependent on the method of measuring motor activity (e.g., Motimeter, Rota rod, and Zitterkufig). Furthermore, it is conceivable that species differences play a role. In mice, Heim et al. (1958) found a significant reduction of methamphetamine-induced excitation. Kobinger (1968), Maj et al. (1973), van Rossum et al. (1962), and Tripod and Meier (1954) reported only a small or insignificant antagonism against amphetamine or methamphetamine.

334

C.-J. ESTLER

In experiments by Fahse et al. (1971), Galambos et al. (1967), and Smith (1%3), the amphetamine-induced hypermotility was even accentuated by pretreatment of the animals with reserpine. Fahse et al. (1971) and Smith (1963) showed that the result obtained is partly dependent on the time interval between the administration of reserpine and the amphetamines. At short intervals the effect of amphetamine or methamphetamine was accentuated, at intervals of intermediary length, amphetamine excitation was diminished, and at long intervals reserpine had no gross effect on the hypermotility. For reasons not clearly understood, the time intervals necessary to elicit the different effects were, however, not alike for amphetamine and methamphetamine. In rats, reserpine given ca. 18 hours before amphetamine generally enhanced the increase in the locomotor activity produced by amphetamine, methamphetamine, or phenmetrazine (Buus Lassen, 1973; Quinton and Halliwell, 1963; Scheel-Kriiger, 1971; Stolk and Rech, 1968). Other manifestations of hyperactivity were depressed or enhanced dependent on the time interval between reserpine and amphetamine application (Morpurgo and Theobald, 1966). Although accentuation of the amphetamine effect shortly after the administration of reserpine may be caused by a release of catecholamines from reserpine-sensitive catecholamine stores, the mechanism of the heighte'ned response to amphetamines as long as 18 hours after the reserpine administration remains unclear. a-Methyldopa had an effect similar to reserpine (Smith, 1963). Prenylamine, which according to Schijne and Lindner (1960) also depletes catecholamine stores, likewise diminished amphetamine hypermotility in mice (Galambos et al., 1967). In rats, a-methyl-rn-tyrosine, which mainly displaces norepinephrine from its storage sites (Carlsson, 1964; Shore et al., 1964), was ineffective in this respect (Sulser et al., 1968). Administration of L-dopa to animals pretreated with reserpine or reserpine plus methyldopa restores the amphetamine effect on locomotor activity (Fahse et al., 1971; Quinton and Halliwell, 1963; Randrup and Munkvad, 1966). Interestingly, the effect of some amphetamine-like drugs (ephedrine, methylphenidate, pipradrol) can be completely antagonized by reserpine even in rats (Plummer et al., 1957; Scheel-Kriiger, 1971; Smith, 1963; Tripod et al., 1957; van Rossum and Hurkmans, 1964). The decrease of spontaneous motor activity produced by fenfluramine is further accentuated by reserpine (Ziance et al., 1972b), which may be due to synergism of the central depressant effects of both drugs. Reserpine inhibited the locomotor excitation produced by p-chloroamphetamine (Buus Lassen, 1974).

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

335

c . Inhibition of Catecholamine Synthesis. In contrast to reserpine, drugs that interfere with norepinephrine synthesis in the CNS exhibit a marked antagonism against amphetamine. This was demonstrated by Beuthin et a l . (1972), Buus Lassen (1973), Carlsson et a l . (1967), Dingell et a l . (1967), Dominic and Moore (1969), Frey and Magnussen (1968), Maj et a l . (1973), Menon et a l . (1967), Moore and Dominic (1971), Moore and Rech (1967), Randrup and Munkvad (1966), Rech et a l . (1966), Rolinski and Scheel-Kruger (1973), Scheel-Kruger (1971), Rech and Stolk (1970), Sulser et a l . (1968), Svensson (1970), Thornburg and Moore (1972), and Weissman et a l . (1966) with a-methyltyrosine, which is an inhibitor of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis (for reference, see Bloom and Giarman, 1968). Similarly, an inhibitor of dopa decarboxylase, a-methyldopa, also inhibited the locomotor stimulation evoked by methamphetamine (Fahse et a l . , 1971; Quinton and Halliwell, 1963). Blockade of dopamine-P-hydroxylase by diethyldithiocarbamate, dimethyldithiocarbamate, disulfiram (tetraethylthiuram disulfide), and other thiocarbamates (D’Encarnaco et a l . , 1969; Pfeifer et a l . , 1966; Maj and Przegalinski, 1967; Maj and Vetulani, 1970; Mayer and Eybl, 1971, 1973; Randrup and Scheel-Kruger, 1966) or by bis(4-methyl-1-homopiperazinyl-thiocarbonyl disulfide (FLA 63) (Carlsson, 1970b; Rolinski and Scheel-Kruger, 1973) caused, if at all, only a partial reduction of amphetamine-produced hypermotility, which may partly be due to the sedative action of the dithiocarbamates. A partial inhibition of amphetamine-elicited motor stimulation was also achieved by selective destruction of catecholaminergic nerve endings by means of 6-hydroxydopamine (Fibiger et a l . , 1973). The amphetamine antagonism of a-methyltyrosine is reversed by dopa (Randrup and Scheel-Kruger, 1966) and monoamine oxidase inhibitors (Moore and Rech, 1967). It is noteworthy to mention that the stimulatory effects of methylphenidate and pipradrol are not affected by a-methyltyrosine (Scheel-Kruger, 1971). To antagonize the locomotor stimulation produced by chloroamphetamines, repeated doses of a-methyltyrosine were needed or doses that were about 10 times higher than those needed to inhibit the effect of amphetamine (Frey and Magnussen, 1968). Buus Lassen (1974) found no antagonism against p-chloroamphetamine-induced locomotor excitation with a-methyltyrosine and FLA 63. d. Blockage of Dopamine Receptors. The results reviewed in the foregoing sections leave open for discussion the question whether synthesis and availability of norepinephrine or dopamine are prerequisite for the stimulatory effect of amphetamines. The assumption that

336

C.-J.

ESTLER

dopamine is essential is supported by experiments showing that compounds blocking dopamine receptors, i.e., pimozide and related butyrophenones (Andhn et al., 1970), abolish the excitatory response of amphetamine and methamphetamine (Estler, 1974; Janssen et al., 1968; Maj et al., 1972b; Miele et al., 1972; Rolinski and Scheel-Kriiger, 1973; Schlechter and Butcher, 1972). Such results imply that dopaminergic reactions play an important role in the amphetamine-induced motor excitation. e. Interference with Serotonergic Mechanisms. Serotonergic effects seem to have, if at all, only minor importance for the locomotor stimulation of mice by amphetamines. Methysergide increased amphetamine excitation (Galambos et al., 1967), cyproheptadine, another antiserotonergic drug, inhibited only at high doses (0.5 mg/kg) (Frey and Magnussen, 1968), and p-chlorophenylalanine, which inhibits serotonin synthesis, did not abolish the hyperactivity evoked by amphetamine and methamphetamine (Estler, 1974; Frey and Magnussen, 1968; Sulser et al., 1968). The central stimulating effects of chloroamphetamines, on the other hand, were depressed by low doses of cyproheptadine (0.11 mg/kg) and virtually abolished by chlorophenylalanine (Buus Lassen, 1974; Frey and Magnussen, 1968). This indicates that the chloroanalogs of amphetamine-in contrast to amphetamines themselves-act through a serotonergic mechanism. +f.Interference with Cholinergic Mechanisms. Cholinergic drugs (e.g., tremorine) increased, and benactyzine (Galambos et al., 1967) but not atropine (Ferrendelli et al., 1972) decreased the motor response to amphetamine. The effect of methylphenidate was intensified by anticholinergic substances (Meier et al., 1954). These results may be explained by shifts in the balance of neurotransmitter systems and do not necessarily mean that amphetamines interfere directly with cholinergic neurotransmission.

2. Conditioned Behavior A number of reports based on studies with amphetamines in combination with catecholamine-depleting drugs or inhibitors of catecholamine synthesis or turnover within the CNS (reserpine, a-methyltyrosine, imipramine, etc.) indicate that adrenergic mechanisms are also important for the effects of amphetamines on conditioned behavior (Carlton, 1961; Davis and Smith, 1973; Evans, 1971; Hanson, 1967; Moore and Dominic, 1971; Oliverio, 1967; Roffman and Lal, 1972, van Rossum, 1970).

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

337

3. Stereotyped Behavior Reserpine did not diminish stereotyped behavior (Herman, 1967). The same is true for a-methyl-m-tyrosine (Herman, 1967; La1 and Sourkes, 1972). Among a number of dithiocarbamates, only dibutyldithiocarbamate and dicyclohexyldithiocarbamate suppressed stereotyped behavior (Maj and Vetulani, 1970). Diethyldithiocarbamate and disulfiram rather accentuated stereotypies (D’Encarnaco et a1 ., 1969; Mayer and Eybl, 1971, 1973; Randrup and Scheel-Kruger, 1966). Drugs blocking adrenergic a-receptors (phenoxybenzamine, dihydroergotamine, phentolamine, Hydergine) had no inhibitory effect on amphetamine-induced stereotypies (Randrup et al., 1963; Herman, 1967). Among the 8-sympatholytics, only propranolol, at high doses, reduced stereotyped movements, whereas dichloroisoproterenol (DCI) and nethalide were ineffective (Herman, 1967; Randrup et al., 1963). Also ineffective was a combination of nethalide and phenoxybenzamine (Randrup et al., 1963; Munkvad and Randrup, 1966). On the other hand, monoamine oxidase inhibitors (pargyline and nialamide) increased the stereotypies (Herman, 1967). Chlorpromazine but not the minor tranquilizers (meprobamate, dichlorazepoxide, etc.) was capable of antagonizing stereotyped behavior (Herman, 1967; La1 and Sourkes, 1972). This effect of chlorpromazine can certainly not be brought into a causal relationship with the aadrenergic blocking but rather with the neuroleptic properties of this drug, since other neuroleptics show similar activity (Klingenstein et al., 1973). The effect of chlorpromazine seems, however, not to be very consistent. As is noted by La1 and Sourkes (1972), this may be partially owing to the fact that phenothiazines do not only counteract centrally stimulating drugs on account of their neuroleptic properties, but may also enhance amphetamine effects due to the interference with amphetamine degradation. It must be mentioned in this context that pimozide, which is known to block dopamine receptors (Andin et al., 1970; Janssen et al., 1968), also effectively inhibits amphetamine-induced stereotypies (Klingenstein et al., 1973). Haloperidol is similarly effective in abolishing the amphetamine-produced stereotypies (Cools and van Rossum, 1970). This is in good agreement with the concept that stereotypies evoked by amphetamine are caused by dysfunction of the striatum and the substantia nigra in which dopamine plays a predominant role. Further support for this idea is lent by the observations that dopa and apomorphine, activators of dopaminergic receptors (AndCn et al., 1967), worsen the stereotypies evoked by amphetamine (Ayhan and Randrup, 1973).

338

C.-J. ESTLER

4. Conclusions The results of the experiments described in Sections 111, A and B indicate that obviously both a-adrenergic and dopaminergic receptors are essential for the locomotor-stimulating effect of amphetamine. According to Fuxe and Ungerstedt (1970), very high levels of exploratory activity are found when both dopamine and norepinephrine receptor activity is high. If these receptors are blocked, amphetamine no longer causes maximal motor excitation. The question whether amphetamines act directly on the receptor or indirectly via the released catecholamines is answered by the experiments with inhibitors of catecholamine synthesis. It is known that, in peripheral nerves, electrical stimulation results in the selective release of newly synthesized norepinephrine, whereas mobilization of stored norepinephrine only plays a minor role (Kopin et al., 1968). Many authors feel that the motor stimulation by amphetamine is likewise dependent on the availability of newly synthesized catecholamines (norepinephrine and dopamine) present in a store from which they can be easily released. In some reports, a parallelism between depletion of catecholamines in a high turnover pool by inhibitors of catecholamine synthesis and reduction of the stimulatory activity of amphetamine is described (Dominic and Moore, 1969; Maj and Vetulani, 1970; Scheel-Kruger, 1971; Sulser et al., 1968). The fact that inhibitors of dopamine-P-hydroxylase, which interfere only with the formation of norepinephrine but not with dopamine synthesis, could not completely abolish the amphetamine-induced hyperactivity points to an important role of dopamine. The inability of reserpine to impair the excitatory effect of amphetamine may be explained by the fact that this drug does not deplete the catecholamine store sufficiently and that reserpine does not inhibit dopamine synthesis (Andin et a l . , 1964; Carlsson et al., 1966a); it may actually increase the turnover rate of central catecholamines (Hillarp et a l . , 1966; Neff and Costa, 1968). However, because the effects of methylphenidate and pipradrol are affected by reserpine but not by a-methyltyrosine, their action must be dependent on a reserpine-sensitive catecholamine store and seems to be independent on the availability of freshly synthesized catecholamines. Serotonergic mechanisms seem to be essential only for the action of chlorinated amphetamine derivatives, which are less dependent on catecholamine metabolism in their action. Thus, it appears that there are three different classes of amphetamine derivatives that act through three different mechanisms to elicit excitatory responses in animals, which, in each case, are expressed as increased locomotor activity.

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

339

The stereotyped activity evoked by amphetamines is obviously most effectively inhibited by antidopaminergic agents, such as pimozide, whereas antiadrenergic drugs are ineffective, and inhibitors of dopamine-S-hydroxylase enhance rather than inhibit amphetamine-induced stereotype behavior (Randrup and Scheel-Kriiger, 1966; Mayer and Eybl, 1971, 1973; D’Encarnaco et al., 1969). These results point to the predominant role of a dopaminergic mediation of this effect. Further evidence comes from comparing the activity of optical isomers of amphetamines. Whereas for eliciting locomotor stimulation and influencing norepinephrine uptake d-amphetamine is much more potent than the I-isomer, the potency of d- and l-amphetamine to inhibit dopamine uptake into the striatum and to produce stereotypies is similar (Taylor and Snyder, 1970). If this is correct, it becomes unlikely that stereotypies are only rudimentary forms of exploratory behavior as was suggested by Ellinwood (1971). However, all conclusions drawn exclusively from the interference of a given drug with metabolic inhibitors or similar pharmacological tools are valid only so far as, under the particular experimental condition, the designed effect has been actually obtained and so far as specificity of this effect is warranted. The latter requirement is certainly not met with many of the drugs used in such experiments. Many agents (e.g., propranolol, phenoxybenzamine, reserpine, and tranquilizers) have nonspecific central depressant properties that may interfere with the excitatory effect of stimulants. Antianxiety drugs (tranquilizers, etc.) may improve exploratory behavior due to reduction of fear. Reserpine does not deplete exclusively catecholamines but also serotonin (Pletscher et a l . , 1955). Imipramine-like compounds do not inhibit only the uptake of catecholamines and of serotonin into storage vesicles, but they also inhibit amphetamine metabolism (Consolo et a l . , 1967; Lemberger et al., 1970; Page and Carlsson, 1970; Sulser et al., 1966). The latter applies also to diethyldithiocarbamate (Jonsson and Lewander, 1973), chlorpromazine (Sulser and Dingell, 1968), and reserpine (Stolk and Rech, 1969). Therefore, inferences from these type of experiments must be made very cautiously.

c. EFFECTSON OTHER METABOLICPARAMETERS 1. Cyclic Nucleotides

Cyclic adenosine 3’,5’-monophosphate (cyclic AMP) and cyclic guanosine 3’,5’-monophosphate (cyclic GMP) have been detected in brain. The activities of the enzymes involved in the formation of cyclic AMP

340

C.-J. ESTLER

(adenylate cyclase) and its degradation to adenosine 5’-monophosphate (phosphodiesterases) are higher in brain than in other tissues and are found especially in areas that are rich in nerve endings (DeRobertis et al., 1967; Drummond and Perrott-Yee, 1961; Schmidt et al., 1972; Sutherland et al., 1962). The proposed neurotransmitters in brain (e.g., norepinephrine, dopamine, serotonin, histamine) and adenosine are able to increase cyclic AMP levels in brain tissue (Huang and Daly, 1972; Huang et al., 1971; Kakiuchi and Rall, 1968a,b;Rall and Sattin, 1970; Schmidt et al., 1972; Shimizu et al., 1969, 1970a,b,c; von Hungen and Roberts, 1973). This points to a special importance of cyclic AMP in brain function and metabolism, but a particular role for cyclic AMP has not yet been established. The same applies to cyclic GMP, which is found in extraordinarily high amounts in murine cerebellum (Ferendelli et a l . , 1970, 1972; Kuo et al., 1972). One is, however, tempted to assume that cyclic AMP has similar effects, for instance on carbohydrate and nucleic acid metabolism, as in other tissues. Since norepinephrine and other neurotransmitters released by amphetamine can activate adenylate cyclase, most authors expected amphetamines to produce an accumulation of cyclic AMP. Surprisingly, Paul et al. (1970)found no alteration of the cyclic AMP level in the brains of mice treated with 2 mg/kg amphetamine. Similar negative results were obtained by Uzunov and Weiss (1971) and by Schmidt et al. (1972)in rats made hyperactive by means of amphetamine. When specific brain regions were studied, amphetamine also did not raise the cyclic AMP content of the cerebellum of mice (Ferendelli et al., 1972) and of the cerebellum, brainstem, midbrain, and cerebral cortex of rats (Schmidt et al., 1972). In vitro amphetamine likewise failed to cause an accumulation of cyclic AMP (Huang and Daly, 1972). Palmer (1973) found no increase of cyclic AMP in slices of cerebral cortex and hypothalamus incubated with lop4 M amphetamine. p-Hydroxyamphetamine (lop4 M ) increased after 60 minutes only the cyclic AMP level of the hypothalamus (Palmer, 1973). Remarkably, both amphetamine and p-hydroxamphetamine (IOp5-10-4 M), antagonized the increase of cyclic AMP induced by norepinephrine in these brain structures (Palmer, 1973). The reason for this unexpected effect is obscure; inhibition of adenylate cyclase may be involved. Pemoline, methylphenidate, and pipradrol were ineffective in enhancing the formation of cyclic AMP in slices of guinea pig brain (Huang and Daly, 1972). In contrast to cyclic AMP, the cyclic GMP content of mouse cerebellum was doubled by d-amphetamine 10 mg/kg (Ferendelli et al.,

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

34 1

1972). This increase was prevented by pretreatment of the animals with chlorpromazine, whereas pretreatment with atropine did not block the amphetamine-induced increase of cerebellar cyclic GMP (Ferendelli et al., 1972). The failure of amphetamine to raise the cerebral cyclic AMP content is somewhat unexpected, because, under proper experimental conditions, catecholamines, serotonin, histamine, methyl xanthines, and electrical stimulation are able to increase the amount of cyclic AMP in the brain (Kakiuchi and Rall, 1968a,b; Rall and Sattin, 1970). One can only speculate about the reasons for this surprising discrepancy. There is no doubt that the determination of cyclic AMP in brain tissue involves a number of methodological problems, especially the problem of rapid fixation of the tissue in order to prevent postmortem changes. It may be that even microwave irradiation has not yet solved this problem completely. Furthermore, it is possible that changes in the cyclic AMP content occur only in very distinct areas of the CNS and are overshadowed when cyclic AMP is estimated in whole brain or even in grossly dissected parts of the brain. Release of an inhibitor of adenylcyclase may also be incriminated, and, of course, there is also the possibility that cyclic AMP is not involved in the action of amphetamines. It should be worthwhile to find out the true reason for the puzzling observation. That the increase of the cyclic GMP content in the cerebellum is causally related to the psychostimulatory action of amphetamines is hard to imagine.

2 . Carbohydrate Metabolism a . Glucose. Under normal conditions the brain meets its energy requirements almost completely by oxidative metabolism of carbohydrates. Since the cerebral carbohydrate reserves, i.e., the glycogen stores, are low in comparison to other organs the brain depends on the availability of glucose, which is taken up into the brain tissue by an active transport mechanism and is then degraded via the EmbdenMeyerhof glycolytic pathway and the tricarboxylic acid (Krebs) cycle (Bachelard, 1970; Elliott et a l . , 1962; McIlwain, 1966; Schmidt, 1956). Amphetamines increase the blood glucose level by stimulating the glycogen breakdown in the liver (Estler et a l . , 1970; Moore et a l . , 1965) and in this way increase the glucose supply of the brain. The glycogendepleting effect on the liver is much stronger in aggregated than in individually housed mice, but, nevertheless, in grouped mice the blood glucose level does not rise but falls to 15% of the control level after

342

C.-J. ESTLER

administration of 10 mg/kg d-amphetamine, showing the higher need of these animals for combustible substrates (Moore et al. , 1965). The effect of amphetamine on liver glycogen was completely abolished by reserpine and a-methyl-rn-tyrosine (Moore et al., 1965). Chlorpromazine and the a-sympatholytic drugs, phenoxybenzamine and ergotarnine, reduced the glycogenolytic effect of amphetamine on the liver (Moore et al., 1965). In a similar way, the P-antiadrenergic compound, propranolol, transiently reduced the effect of methamphetamine on hepatic glycogen content (Estler et al., 1970). Propranolol also antagonized the hyperglycemic effect of methamphetamine (Estler et al. , 1970), whereas reserpine, a-methyl-rn-tyrosine, chlorpromazine, phenoxybenzamine, and ergotamine prevented the amphetamine-induced decrease of the blood glucose level in aggregated mice (Moore et al.,

1%5). There are no investigations on the influence of amphetamines on the cerebral uptake mechanism of glucose, but it has been shown that methamphetamine raises the glucose content of the brain (Estler and Ammon, 1967, 1971). No such effect was seen by Nahorski and Rogers (1973) with d-amphetamine. Propranolol prevented the methamphetamine-induced rise in cerebral glucose, probably by reducing the supply of glucose from the blood (Estler and Ammon, 1967, 1971). b. Glycogen. The increase in the glucose supply to the brain should enable the CNS to meet higher energy requirements in case of CNS excitation. From this point of view the hyperglycemic action of amphetamines appears to be appropriate. Considering the small amount of glycogen stored, the fall in glycogen content of the brain, which occurs in animals treated with amphetamines (Estler and Ammon, 1967, 1971; Estler and Mitznegg, 1971; Estler, 1974; Hutchins and Rogers, 1970, 1971, 1973; Nahorski and Rogers, 1973; Rogers and Hutchins, 1972), appears to be less efficient. The glycogen content falls not only after amphetamine and methamphetamine but also after hydroxyamphetamine, ephedrine, benzphetamine, fenfluramine, norfenfluramine, p chloroamphetamine, fencamfamine, and diethylpropion. However, diethylpropion, benzphetamine, ephedrine, and p-hydroxyamphetamine had only a very low glycogen-depleting potency (13-5% of the activity of amphetamine) (Hutchins and Rogers, 1970; Rogers and Hutchins, 1972). At first sight it is not apparent whether the lowering of the glycogen content has to be ascribed to a glycogenolytic action of the amphetamines or to a diminished synthesis of glycogen from glucose in order to make a greater part of the glucose available for energy-yielding processes. In later experiments, it was demonstrated that glycogen synthesis is not depressed and that glycogen breakdown is stimulated by

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

343

methamphetamine (Estler and Mitznegg, 1971). This is in agreement with results of Breckenridge and Norman (1965) and Drummond and Bellward (1970) that amphetamines under special experimental conditions may increase the activity of phosphorylase b kinase and the phosphorylase activity of the brain. No increase in the ratio of phosphorylase a to phosphorylase b has, however, been observed by Iriye and Simmonds (1971) and by Estler (unpublished observations). This need not be in contradiction to the observed glycogenolytic effect since under normal conditions the rate of glycogenolysis in brain is not regulated by the interconversion of phosphorylases a and b but rather by changes in the activity of phosphorylase b brought about by alterations of the concentrations of activators (5’-AMP) or inhibitors (ADP, ATP, glucose-6-phosphate) of this enzyme (Haugaard and Hess, 1965). In the experiments by Iriye and Simmonds (1971), amphetamine restored the phosphorylase a-to-phosphorylase b ratio that had been lowered by reserpine. It appears, however, that in some way catecholamines or adrenergic receptors must be involved in the glycogenolytic action of amphetamines. Estler and Ammon (1967) showed that propranolol abolished the glycogen-depleting effect of methamphetamine. This was obviously not due to the central depressing properties of this drug but to its sympatholytic action, since INPEA, another p-receptor-blocking agent with central stimulating properties, had the same effect as propranolol (Estler and Ammon, 1971). Similar results were obtained by Hutchins and Rogers (1971) with propranolol and pronethalol. The a-adrenoreceptor-blocking agents, phenoxybenzamine, phentolamine, and chlorpromazine antagonized, if at all, only at high doses the glycogenolytic effects of amphetamine and methamphetamine (Estler, 1975; Hutchins and Rogers, 1971). This effect may not be specifically caused by a-receptor blockage. Pretreatment of mice with reserpine reduced somewhat the fall of the cerebral glycogen content produced by amphetamine (Hutchins and Rogers, 1973). The inhibitor of tyrosine hydroxylase, a-methyltyrosine, partially antagonized the glycogenolytic action. Diethyldithiocarbamate, an inhibitor of dopamine-P-hydroxylase, was without effect (Hutchins and Rogers, 1973). Pimozide, which is supposed to block dopamine receptors, effectively abolished the glycogenolytic effect of methamphetamine (Estler, 1974). Pretreatment of mice with p-chlorophenylalanine, a specific depletor of brain serotonin, has no effect on the reduction of the cerebral glycogen content induced by amphetamine (Hutchins and Rogers, 1973). The methamphetamine-induced lowering of the brain glycogen was

344

C.-J.

ESTLER

diminished (Estler, 1974). Methysergide, a serotonin antagonist, was without effect on amphetamine-produced glycogenolysis (Hutchins and Rogers, 1971). Atropine also failed to antagonize the glycogenolytic action of amphetamine (Hutchins and Rogers, 1971). The same applies to the antihistaminic drug mepyramine (Hutchins and Rogers, 1971). The results suggest that at least receptors that can be blocked by psympatholytic agents are essential for the action of amphetamines on brain glycogen. But there are still many open questions. For instance, the exact mode of action of the amphetamines is not yet elucidated. It remains uncertain, whether the drugs act directly on the receptors or indirectly via catecholamines released from their storage sites. Both views are compatible with the results reviewed in this section. From experiments with antiadrenergic drugs and pimozide, one would suspect that dopamine and norepinephrine may both mediate the action of amphetamine. It remains unclear by what mechanism the stimulation of the receptor causes activation of the phosphorylase system and of glycogen breakdown if cyclic AMP is not involved, as evidenced by the investigations reviewed in Section III,C, 1. Acetylcholine, histamine, and probably serotonin, too, seem not to be essential for the glycogenolytic effect of amphetamine. Since locomotor stimulation and glycogenolysis produced by methamphetamine can be blocked independently, these effects appear not to be causally related to each other (Estler, 1974). c. Glycolysis. Similar to catecholamines (Harrison and Gray, 1971), methamphetamine increases the hexokinase activity in the brains of mice (Estler, 1962). The effect may, therefore, be an indirect one. Nevertheless, the glucose-6-phosphate and fructose-1,6-diphosphate contents were not increased by d-amphetamine (Nahorski and Rogers, 1973). The pyruvate content of the brain was unchanged in the experiments by Bachelard and Lindsay (1966) with 10 mg/kg dlamphetamine and increased in experiments by Estler and Ammon (1967, 1971) with methamphetamine 3 mg/kg. Maximum levels were found after 60 minutes. In the experiments by Nahorski and Rogers (1973) the lactate content of the brain increased considerably after d-amphetamine, 5 mglkg, whereas Estler and Ammon (1967, 1971) using methamphetamine, 3 mglkg, found the lactate content to decrease significantly after 30 minutes. The results of most of these experiments together with the decrease of the cerebral glycogen content point to an enhanced rate of glycolytic carbohydrate metabolism in the brain. The decline of the cerebral lactate content observed by Estler and Ammon (1967, 1971) suggests

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

345

that glycolysis is depressed rather than enhanced. In fact, calculations by Nahorski and Rogers (1973) led to the surprising result that amphetamine initially reduces the metabolic rate of mouse brain by as much as 35%. This initial decrease of the cerebral metabolism coincides with the fall of the lactate content observed by Estler and Ammon (1967). After 60 minutes the metabolic rate increases by about 25% (Nahorski and Rogers, 1973). The decline of the lactate content observed by Estler and Ammon (1967, 1971) may, however, have still another reason. Under similar experimental conditions, coenzyme A in the brain was considerably increased (Heim et al., 1957). This could mean that under the influence of methamphetamine, pyruvate is preferentially channeled into the tricarboxylic acid cycle and, thus, made available for oxidative processes that yield much higher chemical bond energy than the reduction of pyruvate to lactate. Direct evidence for the assumption that amphetamine increases the oxidative metabolism in vivo is, however, still lacking, since turnover rates of the tricarboxylic acid cycle have not been measured in vivo, and since determinations of single constituents of the cycle (e.g., oxoglutarate by Bachelard and Lindsay, 1966) are not very conclusive. In vitro experiments by Mann and Quastel (1940) showed that amphetamine increases the respiration of cerebral cortex slices. No effect of amphetamine on the respiration of cerebral cortex in vitro was seen in experiments by Quastel (1943). High concentrations may even suppress cerebral respiration in vitro (Lewis, 1963). But such results are not easily transferable to in vivo conditions. Unlike the effects of amphetamines on neurotransmitters, which supposedly constitute the basis for the behavioral changes, the alterations of carbohydrate metabolism are by no means fundamental for the psychomotor stimulating action of these drugs. It appears that they are not even causally connected to the amphetamine-produced hyperactivity, since many of these changes can be prevented without abolishing hyperactivity (Estler, 1974; Estler and Ammon, 1967, 1971).

3. High-Energy Phosphates and Mononucleotides In context with the hypothesis that amphetamines improve the energy yield within the CNS are the early observations by Palladin (1952), Palladin and Rybina (1953), Palladin et a l . (1952), and Heim et a l . (1957) showing that methamphetamine elevates the ATP content of rabbit and mouse brain. This was confirmed by Lewis and van Petten (1962) using rats treated with amphetamine, methamphetamine, ephedrine, phenme-

346

C.-J.

ESTLER

trazine and phendimetrazine and by Estler and Ammon (1967, 1971) in mice. On the other hand, Edel (1967), Nahorski and Rogers (1973), Nichols and Walaszek (1964), Nichols et al. (1970) and Wilson (1969) found no significant change in the ATP content of the brains of rats and mice treated with amphetamine or methamphetamine. DiCarlo et a l . (1972) reported a small decrease of ATP after amphetamine treatment. Other free mononucleotides [cytidine triphosphate (CTP), guanosine diphosphate (GDP), uridine triphosphate (UTP), uridine diphosphate (UDP), uridine mon'ophosphate (UMP), inosine monophosphate (IMP)] were not affected in this study. The ADP and AMP contents showed a tendency to decrease after amphetamine, methamphetamine, ephedrine, and phenmetrazine (Lewis and van Petten, 1962). In the experiments by Estler and Ammon (1967) and DiCarlo et al. (1972), ADP tended rather to increase. An increase in the turnover of ATP phosphorus was described by Albaum and Milch (1961-1962) and DiCarlo et al. (1972). Such measurements reflect, however, not only ATP turnover but also the uptake of phosphate into the cells of the CNS and must, therefore, be interpreted cautiously. The authors suggest that these findings indicate adaptation of oxidative phosphorylation to the increased functional activity and probably higher ATP consumption. The phosphocreatine content was only transiently reduced by 0.9 mg/ kg methamphetamine (Lewis and van Petten, 1962). It was significantly decreased by methamphetamine, 3 mg/kg (Estler and Ammon, 1967) or d-amphetamine, 5 mg/kg (Nahorski and Rogers, 1973). The long-lasting effect observed by Estler and Ammon (1967) indicates that energy production is not sufficient to meet energy demands in animals treated with amphetamines. The incorporation of phosphate into phosphocreatine was diminished (DiCarlo et a l . , 1972). Obviously, there is no surplus of chemical energy that can be stored as phosphocreatine. The brain content of inorganic orthophosphate rises during methamphetamine-induced excitation (Estler and Ammon, 1967). Probably changes in the high-energy phosphate content are the consequence of increased energy requirements of the amphetaminestimulated brain tissue. An increase in ATP, which is the immediate donor of chemical energy, on account of phosphocreatine, the storage form of the high-energy phosphates, could serve to make chemical bond energy more easily available. 4. Coenzymes The cerebral nicotinamide adenine dinucleotide (NAD) content declines 2 hours after administration of 10 mg/kg d-amphetamine, whereas

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

347

the reduced NAD (NADH) content shows no corresponding increase (DiCarlo et a l . , 1972; Lewis and Pollock, 1965). The pyridoxal phosphate content was decreased by about 75% for 3 hours by the same dose of amphetamine (Bilodeau, 1965). Coenzyme A increases in the brains of mice treated with methamphetamine (Heim et al., 1957). The biological significance of these changes remains to be settled. 5. Nucleic Acids The metabolism and function of nucleic acids in the brain are reviewed by Mandel and Jacob (1971). Amphetamine, if at all, caused only a very slight increase of the total RNA content of the brain (Dewar and Winterburn, 1973; DiCarlo et al., 1972). There were no significant changes in the RNA-to-DNA ratio (DiCarlo et a l . , 1972). Incorporation of uridine in different fractions of cerebral RNA is, however, higher in amphetamine-treated mice. Obviously both biosynthesis and degradation are enhanced in parallel by amphetamine (DiCarlo et al., 1972). Using orotic acid as precursor, Dewar and Winterburn (1973) found no increase of RNA synthesis in rats treated with amphetamine doses ranging from 0.1 to 25 mg/kg. The rate of RNA transfer from cell nucleus to cytoplasm was also not affected by amphetamine (Dewar and Winterburn, 1973). Similar negative results were seen in rats under chronic treatment with amphetamine (Dewar and Winterburn, 1973). On the other hand, Nasello and Izquierdo (1969) reported that in rats chronic amphetamine treatment lowered hippocampal RNA but left unaltered that of several other brain regions. Although it appears that generally excitation, irrespective of the excitatory agent, causes acceleration of the turnover of cerebral RNA (DiCarlo et al., 1972), it is not yet possible to decide whether this is only a secondary effect due to the changes occurring in the mononucleotide metabolism, or if it is directly related to the alteration of CNS function. It is likewise impossible to establish the role of RNA in the functional effects of amphetamines. 6. Amino Acids, Proteins, and Lipids The amino acid metabolism is closely related to the metabolism of glucose in brain. During short-time experiments in vivo, a large fraction of glucose is converted to a-keto acids and amino acids (Gaitonde et a l . , 1964, 1965; Lindsay and Bachelard, 1966; Vrba, 1962). Amphetamine (10 mg/kg) had no influence on the incorporation of 14C from glucose into aspartate, glutamate, glutamine, and y-aminobutyrate. The incorporation into alanine was markedly reduced after 30 minutes (Bachelard

348

C.-J. ESTLER

and Lindsay, 1966). The cerebral contents of free and bound glutamate, aspartate, and N-acetyl-1-aspartate increased after 20-40 mg/kg methamphetamine (Bruns et al., 1967). Still smaller doses of amphetamine caused a marked increase in the rate of release of alanine, glycine, glutamic acid, and glutamine from rabbit brain in vivo (Blagoeva et al., 1972). Even if some of these amino acids may play a role in neuronal excitation or inhibition within the CNS (Curtis and Johnston, 1970), the significance of the changes described here for brain function and animal behavior can hardly be ascertained from the data presently available. Probably more important are the effects of amphetamines on tyrosine and tryptophan, the precursors of catecholamine and serotonin synthesis. At a dose of 5 mg/kg, amphetamine, methamphetamine, and p bromomethamphetamine reduced the tyrosine content of whole brain. Minimum levels were found after 2 hours. The effect may be related to a parallel decrease of the tyrosine concentration in the serum (Leonard and Shallice, 1971a). p-Nitromethylamphetamine was less effective in reducing serum tyrosine and had no effect on the tyrosine content of the brain (Leonard and Shallice, 1971b). Since only a minute fraction of brain tyrosine is used for the synthesis of catecholamines (Schubert et a1., 1970), the approximately 25-30% reduction of cerebral tyrosine observed by Leonard and Shallice (1971a) should not severely impair catecholamine synthesis. In contrast to tyrosine, the tryptophan content of brain was increased by amphetamine, methamphetamine, p-bromomethamphetamine (Leonard and Shallice, 1971a; Schubert et al., 1970; Schubert and Sedvall, 1972), p-nitromethylamphetamine (Leonard and Shallice, 1971b), p chloroamphetamine, and p-hydroxyamphetamine (Schubert and Sedvall, 1972). The latter drugs are, however, less potent than the parent compounds (Schubert and Sedvall, 1972). Obviously, amphetamines facilitate the uptake of tryptophan from the blood into the brain. Since the availability of tryptophan is of crucial importance for the rate of serotonin synthesis in brain (Page and Carlsson, 1970), an increase in intracellular tryptophan concentration could theoretically improve the synthesis of serotonin. The significance of such an effect of amphetamine is, however, difficult to establish from the available results. &Amphetamine has an inhibitory effect on protein synthesis (Loh et al., 1973). Norepinephrine and dopamine affect the synthesis of complex lipids in brain. In a similar way, prolonged treatment of rats with damphetamine inhibited the synthesis of phosphatidylcholine in cerebral cortex and cerebellum and decreased the turnover of phosphatidylcholine in cortex, diencephalon, brainstem, hypothalamus, caudate nucleus,

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

349

and cerebellum. The incorporation of choline into phosphatidylcholine was reduced by lop2M amphetamine in cortex slices in vitro; lop3M damphetamine had no effect (Hitzemann and Loh, 1973).

IV. Concluding Remarks Inspite of the great number of investigations concerned with this matter, the mode of the psychostimulatory action of the amphetaminetype psychostimulants is still far from being elucidated. Nevertheless, the majority of observations favors the view that amphetamine and methamphetamine act through an indirect sympathomimetic mechanism in which adrenergic and/or dopaminergic receptors are involved. The hyperkinetic effect of the drugs seems to be dependent on the availability of newly synthesized norepinephrine and/or dopamine. The relative importance of these two catecholamines has not yet been fully established. It appears, however, that dopaminergic mechanisms are involved in the stereotyped movements that supposedly originate from stimulation of the extrapyramidal motor system. In parallel to their stimulating effect on motor activity, amphetamine and methamphetamine enhance cerebral carbohydrate metabolism. Thus the substrate supply of the brain is improved. It appears, however, that the energy production in the brain does not meet the cerebral energy requirements of the hyperactive animals. As consequence of the close connection between the metabolism of glucose and amino acids, it seems possible that the enhanced carbohydrate metabolism serves also the purpose of providing amino acids important for brain function. Interestingly, it seems that not all amphetamine derivatives act through the same mechanism. For instance, chloroamphetamine and related chlorinated analogs of the amphetamines seem to exert their central stimulating activity, which closely resembles that of the parent compounds, through a serotonergic rather than through a catecholaminergic mechanism. REFERENCES Albaum, H. G., and Milch, L. J. (1961-1962). Ann. NY. Acad. Sci. 96, 190. Alphin, R. S., Funderburk, W. H., and Ward, J. W. (1964). Toxicol. Appl. Pharmacol. 6, 340. AndBn, N-E., Roos, B-E., and Werdenius, B. (1964). Life Sci. 3, 149. AndBn, N-E., Rubenson, A., Fuxe, K., and Hiikfelt, T. (1967). J . Pharm. Pharmacol. 19, 627. And& N-E., Butcher, S. G., Corrodi, C., Fuxe, K., and Ungerstedt, U. (1970). Eur. J . Pharmacol. 11, 303. Arbuthnott, G. W. (1969). J. Neurochem. 16, 1599.

350

C.-J.

ESTLER

Axelrod, J. (1954). J. Pharmacol. E z p . Ther. 1 1 0 , 315. Axelrod, J. (1955). J. Biol. Chem. 214, 753. Ayhan, I. H., and Randrup, A. (1973). Arch. Int. Pharmacodyn. Ther. 2 0 4 , 2 8 3 . Azzaro, A. J., and Rutledge, C. 0. (1973). Biochem. Pharrnacol. 2 2 , 2 8 0 1 . Bachelard, H. S. (1970). In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. IV, p. 1. Plenum, New York. Bachelard, H. S., and Lindsay, J. R. (1966). Biochem. Pharmacol. 1 5 , 1053. Baird, J. R. C., and Lewis, J. J. (1%3). Biochem. Pharmacol. 12, 579. Baird, J. R. C., and Lewis, J. J. (1964). Biochem. Pharmacol. 13, 1475. Baldessarini, R. J. (1972). Annu. Rev. Med. 23, 343. Beanvallet, M., Fugazza, J., and Solier, M. (1963). C. R. Acad. Sci. 257, 3251. Beauvallet, M., Fugazza, J., Godefroy, F., and Solier, M. (1964). J. Physiol. (Paris) 56, 532. Beauvallet, M., Fugazza, J., and Legrand, M. (1967). C. R . SOC.Biol. 161, 2161. Besson, M. J., Cheramy, A., Feltz, P., and Glowinski, J. (1969). Proc. Nat. Acad. Sci. US. 62, 741. Benthin, F. C., Miya, T. S., Blake, D. E., and Bosquet, W. F. (1972). J . Pharmacol. Exp. Ther. 1 8 1 , 4 4 6 . Biel, J. H. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 3. Raven, New York. Bilodeau, F. (1965). J. Neurochem. 1 2 , 6 7 1 . Blagoeva, P., Masi, I., Scotti de Carolis, A., and Longo, V. G. (1972). Physiol. Behau. 9, 307. Bloom, F. E., and Giarman, N. J. (1968). Annu. Rev. Pharrnacol. 8, 229. Bradley, P. B., and Elkes, J. (1957). Brain 80, 77. Brady, J. V. (1956). Science 1 2 3 , 1033. Breckenridge, B. M., and Norman, J. H. (1965). J. Neurochem. 12, 51. Breese, G. R., Kopin, I. J., and Weise, V. K. (1970). Brit. J. Pharmacol. 38, 537. Brodie, B. B., Cho, A. K., and Gessa, G. L. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 217. Raven, New York. Bruns, F. H., Reinauer, H., and Stork, W. (1967). HoppeSeyler’s 2. Physiol. Chem. 348, 512. Buus Lassen, J. (1973). Psychopharmacologia 29, 55. Buus Lassen, J. (1974). Psychopharmacologia 34, 243. Cahn, J., and Herold, M. (1970). In “Amphetamines and Related Compounds“ (E. Costa and S. Garattini, eds.), p. 493. Raven, New York. Campbell, B. A., Lytle, L. D., and Fibiger, H. C. (1969). Science 1 6 6 , 635. Cappell, H., Ginsberg, R., and Webster, C. D. (1972). Brit. J. Pharmacol. 45, 525. Carlsson, A. (1964). Progr. Brain Res. 8, 9. Carlsson, A. (1970a). J. Pharm. Pharmacol. 22, 729. Carlsson, A. (1970b). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 289. Raven, New York. Carlsson, A., Lindquist, M., Dahlstrom, A,, Fuxe, K., and Masuoka, D. (1965). J. Pharm. Pharmacol. 17, 521. Carlsson, A., Fuxe, K., Hamberger, B., and Lindquist, M. (1966a). Acta Physiol. Scand. 67,481. Carlsson, A., Lindquist, M., Fuxe, K., and Hamberger, B. (1966b). J. Pharm. Pharmacol. 18, 128. Carlsson, A., Fuxe, K., and Hiikfelt, T. (1967). Eur. J . Pharmacol. 2 , 196.

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

351

Carlton, P. L. (1961). Psychopharmacologia 2, 364. Carr, L. A.; and Moore, K. E. (1969). Science 164, 322. Carr, L. A., and Moore, K. E. (1970). Biochem. Pharmacol. 19, 2361. Chance, M. R. A. (1946). J. Pharmacol. Exp. Ther. 87, 214. Chance, M. R. A. (1947). J. Pharmacol. Exp. Ther. 89, 289. Clay, G. A., Cho, A. K., and Roberfroid, M. (1971). Biochem. Pharmacol. 20, 1821. Colmore, J. P., and Moore, J. P. (1966). J. New Drugs 6, 123. Consolo, S., Garattini, S., Ghielmetti, R., and Valzelli, L. (1965). J. Pharm. Pharmacol. 17, 666. Consolo, S., Dolfini, E., Garattini, S., and Valzelli, L. (1967). J. Pharm. Pharmacol. 19, 253. Cook, J. D.. and Schanberg, S. M. (1970). Biochem. Pharmacol. 19, 1165. Cools, A. R., and van Rossum, J. M. (1970). Arch. Int. Pharmacodyn. Ther. 187, 163. Costa, E., and Gropetti, A. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 231. Raven, New York. Costa, E., and Revuelta, A. (1972). Neuropharmacology 11, 291. Costa, E., Gropetti, A., and Revuelta, A. (1971a). Brit. J. Pharmacol. 41, 57. Costa, E., Naimzada, M. K., and Revuelta, A. (1971b). Brit. J. Pharmacol. 43, 570. Costa, E., Gropetti, A., and Naimzada, M. K. (1972). Brit. J. Pharmacol. 44, 72. Costall, B., and Naylor, R. J. (1973). Brit. J. Pharmacol. 49, 29. Coyle, J., and Snyder, S. (1969). J. Pharmacol. Exp. Ther. 170, 221. Creese, I., and Iversen, S. D. (1972). Nature (London) 238, 247. Curtis, D. R., and Johnston, G. A. R. (1970). In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. IV, p. 115. Plenum, New York. Davis, W. M., and Smith, S. G. (1973). J. Pharm. Pharmacol. 25, 174. Deffenu, G., Bartholini, A., and Pepeu, G. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 357. Raven, New York. D’Encarnaco, P. S., D’Encarnaco, P., and Tapp, J. T. (1969). Arch. Znt. Pharmacodyn. Ther. 182, 186. DeRobertis, E., Rodriguez de Lores Arnaiz, G., Alberici, M., Butcher, R. W., and Sutherland, E. W. (1967). J. Biol. Chem. 242, 3487. Dewar, A. J., and Winterburn, A. K. (1973). Brain Res. 59, 359. Dewhurst, W. G., and Marley, E. (1965). Brit. J. Pharmacol. 25, 682. Dews, P . B., and Morse, W. H. (1961). Annu. Rev. Pharmacol. 1, 145. Diaz, J-L., and Huttunen, M. 0. (1972). Psychopharmacologia 23, 365. DiCarlo, R., Edel, S., Randrianarsoa, H., and Mandel, P. (1972). Pharmacol. Res. Commun. 4, 275. Dingell, J. V., Owens, M. L., Norvich, M. R., and Sulser, F. (1967). Life Sci. 6, 1155. Dominic, J. A,, and Moore, K. E. (1969). Arch. Int. Pharmacodyn. Ther. 178, 166. Domino, E. F., and Wilson, A. E. (1972). Psychopharmacologia 25, 291. Dring, L. G., Smith, R. L., and Williams, R. T. (1966). J. Pharm. Pharmacol. 18,402. Dring, L. G., Smith, R. L., and Williams, R. T. (1970). Biochem. J. 116, 425. Drummond, G. I., and Bellward, G. (1970). J. Neurochem. 17, 475. Drummond, G. I., and Perrott-Yee, S. (1961). J. B i d . Chem. 236, 1126. Duce, M., and Gessa, G. L. (1967). Boll. Soc. Ital. B i d . Sper. 42, 1631. Duhault, J., and Verdavainne, C. (1967). Arch. Int. Pharmacodyn. Ther. 170, 276. Ebstein, R. P., Ebstein, B. S., Samuel, D., and Berger, B. D. (1972). J. Neurochem. 19, 2703. Edel, S. (1967). J. Physiol. (Paris) 59, 234.

352

C.-J. ESTLER

Ellinwood, E. H., Jr. (1971). Pharmakopsychiatrie 4, 351. Elliott, K. A. C., Page, I. H., and Quastel, J. H. (1%2). “Neurochemistry,” 2nd Ed. Thomas, Springfield, Illinois. Ellison, T., Gutzait, L., and van Loon, E. J. (1966). J. Pharmacol. Exp. Ther. 1 5 2 , 3 8 3 . Enna, S. J., Dorris, R. L., and Shore, P. A. (1973). J. Pharmacol. E z p . Ther. 184, 576. Ernst, A. M. (1967). Psychopharmacologia 1 0 , 3 1 6 . Estler, C.-J. (1962). Natumissenschaften 49, 472. Estler,C.-J. (1974). Anneim.-Forsch.24, 1087. Estler, C.-J. (1975). Neuropharmacology 14, in press. Estler, C.-J., and Ammon, H. P. T. (1%7). J. Neurochem. 14, 799. Estler, C.-J., and Ammon, H. P. T. (1971). J. Neurochem. 18, 777. Estler, C.-J., and Mitznegg, P. (1971). Biochem. Pharmacol. 20, 1331. Estler, C.-J., Ammon, H. P. T., Fickl, W., and Frohlich, H.-N. (1970). Biochem. Pharmacol. 19,2957. Evans, H. L. (1971). J. Pharmacol. E z p . Ther. 176, 244. Everett, G. M. and Yellin, 0. (1971). Res. Commun. Chem. Pathol. Pharmacol. 2 , 4 0 7 . Fahse, C., Lietz, W., and Matthies, H. (1971). Acta Biol. Med. Ger. 26, 1223. Ferrendelli, J. A., Steiner, A., McDougal, D. B., and Kipnis, D. M. (1970). Biochem. Biophys. Res. Commun. 41, 1061. Ferrendelli, J. A., Kinscherf, D. A., and Kipnis, D. M. (1972). Biochem. Biophys. Res. Commun. 46, 2114. Fern, S., Maffei-Faccioli, A., Ornesi, A., and Scamazzo, T. (1963). Boll. SOC. Ital. Biol. Sper. 39, 1381. Ferris, R. M., Tang, F. L. M., and Maxwell, R. A. (1972). J. Pharmacol. E z p . Ther. 1 8 1 , 407. Fibiger, H. C., and McGeer, F. G. (1971). Eur. J. Pharmacol. 16, 176. Fibiger, H. C., Fibiger, H. P., and Zis, A. P. (1973). Brit. J. Pharmacol. 47, 683. Fischer, E., Saavedra, J. M., and Heller, B. (1968). Arzneim.-Forsch. 18, 780. Fischer, J. E., Horst, W. D., and Kopin, I. J. (1965). Brit. J . Pharmacol. 24, 477. Fog, R., Randrup, A., and Pakkenberg, H. (1970). Psychopharmacologia 1 8 , 3 4 6 . Freeman, J. J., and Sulser, F. (1972). J. Pharmacol. E z p . Ther. 1 8 3 , 307. Frey, H.-H. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 343. Raven, New York. Frey, H.-H., and Magnussen, M. P. (1%8). Biochem. Pharmacol. 17, 1299. Fuller, R. W., and Hines, C. W. (1967). Biochem. Pharmacol. 16, 11. Fuxe, K., and Ungerstedt, U. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 257. Raven, New York. Gaitonde, M. K., Marchi, S. A., and Richter, D. (1964). Proc. Roy. Soc. Ser. B 160, 214. Gaitonde, M. K., Dahl, D. R., and Elliott, K. A. C. (1965). Biochem. J. 9 4 , 3 4 5 . Galambos, E., Pfeifer, A. K., GySrgy, L., and MolnL, J. (1967). Psychopharmacologia 1 1 , 122. Glowinski, J. (1970). In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. IV, p. 91. Plenum, New York. Glowinski, J., and Axelrod, J. (1965). J. Pharmacol. Ezp. Ther. 149, 43. Glowinski, J., and Baldessarini, R. J. (1966). Pharmacol. Rev. 18, 1201. Glowinski, J., Kopin, I. J., and Axelrod, J. (1965). J. Neurochem. 1 2 , 25. Glowinski, J., Axelrod, J., and Iversen, L. L. (1966a). 1. Pharmacol. Ezp. Ther. 153, 30. Glowinski, J., Snyder, S. H., and Axelrod, J. (196633). J. Pharmacol. Exp. Ther. 152, 282. Goldstein, M., and Anagnoste, B. (1965). Biochim. Biophys. Acta 107, 166. Goldstein, M., and Contrera, J. F. (1961). Biochem. Pharmacol. 7, 77.

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

353

Grana, E., and Lilla, L. (1959). Brit. J. Pharmacol. 14, 501. Green, H., and Erickson, R. W. (1964).Int. J. Neuropharmacol. 3, 315. Gropetti, A., and Costa, E. (1969). Life Sci. 8, Part 1, 653. Gropetti, A., Misher, A., Naimzada, M., Revuelta, A., and Costa, E. (1972). J. Pharmacol. Exp. Ther. 182,464. Gunn, J. A., and Gurd, M. R. (1940). J. Physiol. (London) 9 7 , 4 5 3 . Gunne, L. M., and Galland, L. (1%7). Biochem. Pharmacol. 16, 1374. Gunne, L.-M., and Lewander, T. (1968). Res. Publ., Ass. Res. Nerv. Ment. Dis. 46, 106. Hanson, L. C. F. (1967). Psychopharmacologia 1 0 , 2 8 9 . Harris, J. E., and Baldessarini, R. J. (1973). J. Pharm. Pharmacol. 25, 755. Harrison, W. H., and Gray, R. M. (1971). Biochim. Biophys. Acta 237, 391. Haugaard, N., and Hess, M. E. (1%5). Pharmacol. Rev. 17, 27. Hauschild, F. (1939). NaunynSchmiedebergs Arch. Exp. Pathol. Pharmakol. 191, 465. Hearst, E., and Whalen, R. E. (1%3). J . Comp. Physiol. Psychol. 56, 124. Heim, F., Leuschner, F., and Estler, C.-J. (1957). Ezperientia 13, 462. Heim, F., Leuschner, F., Estler, C.-J., and Friedrich, W. (1958). Anneim.-Forsch. 8, 216. Hemsworth, B. A., and Neal, M. J. (1968). Brit. J . Pharmacol. 34, 543. Herman, Z. (1967). Psychopharmacologia 1 1 , 136. Hiebel, G., Bonvallet, M., Huve, P., and Dell, P. (1954). Sem. Hop. 30, 1880. Hillarp, N.-A., Fuxe, K., and Dahlstrom, A. (1966). Pharmacol. Rev. 18, 727. Hitzemann, R. J., and Loh, H. H. (1973). Biochem. Pharmacol. 2 2 , 2 7 3 1 . Hitzemann, R. J., Loh, H. H., and Domino, E. F. (1971). Life Sci. 1 0 , Part 1, 1087. Huang, M., and Daly, J. (1972). J. Med. Chem. 15, 458. Huang, M., Shimizu, H., and Daly, J. (1971). Mol. Pharmacol. 7, 155. Hucker, H. B. (1970). Annu. Rev. Pharmacol. 1 0 , 9 9 . Hutchins, D. A., and Rogers, K. J. (1970). Brit. J. Pharmacol. 39, 9. Hutchins, D. A., and Rogers, K. J. (1971). Brit. J. Pharmacol. 43, 504. Hutchins, D. A., and Rogers, K. J. (1973). Brit. J . Pharmacol. 48, 19. Innes, I. R., and Nickerson, M. (1970). In “The Pharmacological Basis of Therapeutics” (L. S. Goodman and A. Gilman, eds.), 4th Ed., p. 478. Macmillan, New York. Iriye, T. T., and Simmonds, F. A. (1971). Znt. Pharmacopsychiat. 6, 98. Izquierdo, I., and Izquierdo, J. A. (1971). Annu. Rev. Pharmacol. 1 1 , 189. Janssen, P. A J., Niemegeers, C. J. E., and Schelekens, K. H. L. (1965). Arzneim.-Forsch. 15,104. Janssen, P. A. J., Niemegeers, C. J. E., Schelekens, K. H. L., Dresse, A., Lenaerts, F. M., Pinchard, A., Schaper, W. K. A., van Nueten, J. M., and Verbruggen, F. J. (1%8). Arzneim.-Forsch. 18, 261. Javoy, F., Hamon, M., and Glowinski, J. (1970). Eur. J . Pharmacol. 10, 178. Jonsson, J., and Lewander, T. (1973). J. Pharm. Pharmacol. 25, 589. Jori, A., Dolfini, E., Tognoni, G., and Garattini, S. (1973). J. Pharm. Pharmacol. 2 5 , 3 1 5 . Kaergaard Nielsen, C., Magnussen, M. P., Kampman, E., and Frey, H.-H. (1967). Arch. Int. Pharmacodyn. Ther. 1 7 0 , 4 2 8 . Kakiuchi, S., and Rall, T. W. (1968a). Mol. Pharmacol. 4, 367. Kakiuchi, S., and Rall, T. W. (1968b). Mol. Pharmacol. 4, 379. Kelleher, R. T., and Morse, W. H. (1968). Ergebn. Physiol. Biol. Chem. Exp. Pharmakol., 60, 1. Killam, E. K., Gangloff, H., Konigsmark, B., and Killam, K. F. (1959). I n “Biological Psychiatry,“ p. 53. Grune & Stratton, New York. Klingenstein, R. J., Wallach, M. B., and Gershon, S. (1973). Arch. Int. Pharmacodyn. Ther. 2 0 3 , 6 7 .

354

C.-J. ESTLER

Kobinger, W. (1958). Acta Pharmacol. Tozicol. 14, 138. Koda, L. Y., and Gibb, J. W. (1971). Pharmacologist 13,253. Koda, L. Y . , and Gibb, J. W. (1973). J. Pharmacol. Exp. Ther. 1 8 5 , 42. Kopin, I. J. (1968). Annu. Rev. Pharmacol. 8 , 377. Kopin, I. J., Breese, G. R., Krauss, K. R., and Weise, V. K. (1968). J. Pharmacol. Exp. Ther. 1 6 1 , 271. Kriekbaus, E. E., Miller, N. E., and Zimmerman, P. (1965). J. Comp. Physiol. Psychol. 60,35. Kuo, J.-F., Lee, T.-P., Reyes, P. L., Walton, K. G., Donelly, T. E., and Greengard, P. (1972). J . Biol. Chem. 247, 16. Lajtha, A. (1970). “Handbook of Neurochemistry,” Vol. IV. Plenum, New York. Lal, H., and Chessick, D. (1964). Life Sci. 3, 381. Lal, S., and Sourkes, T. L. (1972). Arch. Int. Pharmacodyn. Ther. 1 9 9 , 289. Laverty, R., and Sharman, D. F. (1965). Brit. J . Pharmacol. Chemother. 24, 759. Lemberger, L., Sernatinger, E., and Kuntzman, R. (1970). Biochem. Pharmacol. 1 9 , 3021. Leonard, B. E. (1972). Biochem. Pharmacol. 21, 1289. Leonard, B. E., and Shallice, S. A. (1971a). Brit. J . Pharmacol. 41, 198. Leonard, B. E., and Shallice, S. A. (1971b). Brit. J . Pharmacol. 43, 732. Lewander, T. (1968a). Psychopharmacologia 13, 394. Lewander, T. (196833). Eur. J . Pharmacol. 5 , 1. Lewander, T. (1971). NaunynSchmiedebergs Arch. Pharmakol. Exp. Pathol. 2 7 1 , 211. Lewis, J. J. (1963) Sci. Progr. (London) 51, 551. Lewis, J. J., and Pollock, D. (1965). Biochem. Pharmacol. 1 4 , 6 3 6 . Lewis, J. J., and van Petten, G. R. (1962). J. Pharmacol. Exp. Ther. 1 3 6 , 372. Lindsay, J. R., and Bachelard, H. B. (1966). Biochem. Pharmacol. 15, 1045. Loh, H., Hitzemann, R., and Stolman, S. (1973). l’oxicol. Appl. Pharmacol. 26, 476. Longo, V. G., and Silvestrini, B. (1957). J. Pharmacol. Exp. Ther. 120, 160. McIlwain, H. (1966). “Biochemistry and the Central Nervous System,” 3rd Ed. Little, Brown, Boston, Massachusetts. McKenzie, G. M., and Szerb, J. C. (1968). J. Phannacol. E z p . Ther. 162, 302. McLean, J. R., and McCartney, M. (l%l). Proc. SOC.Exp. B i d . Med. 107, 77. Maickel, R. P., Cox, R. H., Jr., Miller, F. P., Segal, D. S., and Russell, R. W. (1969). J . Pharmacol. Exp. Ther. 165, 216. Maj, J., and Przegalinski, E. (1967). J. Pharm. Pharmacol. 1 9 , 341. Maj, J., and Vetulani, J. (1970). Eur. J. Pharmacol. 9, 183. Ma;, J., Grabowska, M., Gajda, L., and Michaluk, J. (1972a). Diss. Pharm. Pharmacol. 24, 7. Maj, J., Sowinska, H., Kapturkiewicz, Z., and Sarnek, J. (1972b). J. Pharm. Pharmacol. 24,412. Maj, J., Sowinska, H., and Baran, L. (1973). Life Sci. 12, Part 1, 511. Mandel, P., and Jacob, M. (1971). I n “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. V, Part A, p. 165. Plenum, New York. Mann, P. J. G., and Quastel, J. H. (1940). Biochem. J. 3 4 , 4 1 4 . Mantegazza, P., Miiller, E. E., Naimzada, M. K., and Riva, M. (1970). I n “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 559. Raven, New York. Mayer, O., and Eybl, V. (1971). J . Pharm. Pharmacol. 2 3 , 8 9 4 . Mayer, O . , and Eybl, V. (1973). J. Pharm. Pharmacol. 2 5 , 6 7 2 . Meier, R., Gross, F., and Tripod, J. (1954). Klin. Wochenschr. 32, 445. Menon, M. K., Dandiya, P. C., and Bapna, J. S. (1967). Psychopharmacologia 10,437. Miele, E., Satta, M., and Anania, V. (1972). Pharmacol. Res. Commun. 4, 305.

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

355

Milhaud, G., and Glowinski, J. (1%3). C. R . Acad. Sci. 256, 1033. Miller, K. W., Freeman, J. J., Dingell, J. V., and Sulser, F. (1970). Experientia 26, 863. M d l e r Nielsen, I., and Dubnick, B. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 63. Raven, New York. Monnier, M., and Krupp, P. (1960).Arch. Int. Pharmacodyn. Ther. 1 2 7 , 3 3 7 . Moore, K. E. (1963). J . Pharmacol. Exp. Ther. 1 4 2 , 6 . Moore, K. E. (1964). J . Pharmacol. Exp. Ther. 1 4 4 , 4 5 . Moore, K. E., and Dominic, J. A. (1971). Fed. Proc., Fed. Amer. SOC.Ezp. Biol. 3 0 , 8 5 9 . Moore, K. E., and Lariviere, E. W. (1963). Biochem. Pharmacol. 12, 1283. Moore, K. E., and Rech, R. H. (1%7). J. Pharmacol. Exp. Ther. 156, 70. Moore, K. E., Sawdy, L. C., and Shaul, S. R. (1%5). Biochem. Pharmacol. 14, 197. Moore, K. E., Carr, L. A., and Dominic, J. A. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 371. Raven, New York. Morgan, C. D., Cattabeni, F., and Costa, E. (1972a). J. Pharmacol. Exp. Ther. 180, 127. Morgan, D., Liifstrandh, S., and Costa, E. (1972b). Life Sci. 11, Part 1, 83. Morpurgo, L., and Theobald, W. (1966). Int. J . Neurophannacol. 5, 375. Munkvad, I., and Randrup, A. (1966). Acta Psychiat. Scand., Suppl. 191, 178. Nahorski, S. R., and Rogers, K. J. (1973). J . Neurochem. 2 1 , 6 7 9 . Nasello, A. G., and Izquierdo, I. (1969). Exp. Neurol. 23, 521. Neff, N. H., and Costa, E. (1968). J. Pharmacol. Exp. Ther. 160,40. Ng, K. Y., Chase, T. N., and Kopin, I. J. (1970). Nature (London) 228, 468. Nichols, R. E., and Walaszek, E. J. (1964). Pharmacologist 6, 179. Nichols, R. E., Patterson, C. S., and Walaszek, E. J. (1970). Arch. Int. Pharmacodyn. Ther. 1 8 4 , 19. Oliverio, A. (1967). Farmaco, Ed. Sci. 22, 441. Opitz, K. (1%7). NaunynSchmiedebergs Arch. Pharmakol. Exp. Pathol. 259, 56. Paasonen, M. K., and Vogt, M. (1956). J. Physiol. (London) 131, 617. Page, I. H., and Carlsson, A. (1970). In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. IV, p. 251. Plenum, New York. Palladin, A. V. (1952). Biochimia 17, 456. Palladin, A. V., and Rybina, A. A. (1953). Dokl. Akad. Nauk. SSSR 91, 903. Palladin, A. V., Chaikina, B. I., and Polyakova, N. M. (1952). Dokl. Akad. Nauk SSSR 84, 777. Palmer, G. C. (1973). Life Sci. 12, Part 2, 345. Papeschi, R., and Randrup, A. (1973). Pharmakopsychiatrie 6, 137. Paul, M. I., Pauk, G. L., and Ditzion, B. R. (1970). Pharmacology 3, 148. Pfeifer, A. K . , Galambos, E., and Gyorgy, L. (1966). J. Pharm. Pharmacol. 1 8 , 2 5 4 . Phillips, A. G., and Fibiger, H. C. (1973). Science 179, 575. Pletscher, A,, and Gey, K. F. (1959). Helu. Physiol. Pharmacol. Acta 17, C35. Pletscher, A,, Shore, P . A., and Brodie, B. B. (1955). J. Pharmacol. Ezp. Ther. 116, 84. Pletscher, A., Burkard, W. P., Bruderer, H., and Gey, K. F. (1963). Life Sci. 2, 828. Pletscher, A., Bartholini, G., Bruderer, H., Burkard, W. P., and Gey, K. F. (1964). J . Pharmacol. Exp. Ther. 1 4 5 , 3 4 4 . Pletscher, A., DaPrada, M., Burkard, W. P., Bartholini, G., Steiner, F. A., Bruderer, H., and Bigler, F. (1966). J. Pharmacol. Exp. Ther. 1 5 4 , 6 4 . Pletscher, A., DaPrada, M., Burkard, W. P., and Tranzer, J. P. (1968). Aduan. Pharmacol. 6B, 55. Plummer, A. J., Maxwell, R. A., and Earl, E. A. (1957). Schweiz. Med. Wochenschr. 87, 370. Quastel, J. H. (1943). Trans. Faraday SOC.34, 348.

356

C.-J.

ESTLER

Quinton, R. M., and Halliwell, G. (1963). Nature (London) 200, 178. Rall, T. W., and Sattin, A. (1970). In “The Role of Cyclic AMP in Cell Function” (E. Costa and P. Greengard, eds.), p. 113. Raven, New York. Randrup, A., and Munkvad, I. (1966). Nature (London) 211, 540. Randrup, A., and Munkvad, I. (1%7). Psychopharmacologia 11, 300. Randrup, A., and Scheel-Kriiger, J. (1966). J. Pharm. Pharmacol. 18, 752. Randrup, A., Munkvad, I., and Udsen, P. (1%3).Acta Pharmacol. Toxicol. 20, 145. Rech, R. H., and Stolk, J. M. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 385. Raven, New York. Rech, R. H., Borys, H. K., and Moore, K. E. (1966). J. Pharmacol. Exp. Ther. 153,412. Reid, W. D. (1970). Brit. J. Pharmacol. 40, 483. Roffman, M., and Lal, H. (1972). Psychopharmacologia 25, 195. Rogers, K. J., and Hutchins, D. A. (1972). Eur. J. Pharmacol. 2 0 , 97. Rolinski, Z., and Scheel-Kriiger, J. (1973). Acta Pharmacol. Toxicol. 33, 385. Salama, A. I., and Goldberg, M. E. (1970). Biochem. Pharmacol. 19, 2023. Sanan, S., and Vogt, M . (1962). Brit. J. Pharmacol. Chemother. 18, 109. Sanders-Bush, E., and Sulser, F. (1970). J. Pharmacol. Exp. Ther. 175,419. Sanders-Bush, E., and Sulser, F. (1972). Biochem. Pharmacol. 21, 1501. Scheel-Kriiger, J. (1971). Eur. J. Pharmacol. 14, 47. Schlechter, J. M., and Butcher, L. L. (1972). J. Pharm. Phannacol. 24, 408. Schmidt, C. G. (1956). In “Physiologische Chemie” (B. Flaschentrager and E. Lennartz, eds.), Vol. 2,II,a, p. 613. Springer-Verlag, Berlin and New York. Schmidt, M. J., Hopkins, J. T., Schmidt, D. E., and Robison, G. A. (1972). Brain Res. 42,465. Schone, H., and Lindner, E. (1960). Arzneim.-Forsch. 10, 583. Schubert, J., and Sedvall, G. (1972). J. Pharm. Pharmacol. 24,53. Schubert, J., Fyro, B., Nyback, H., and Sedvall, G. (1970). J. Pharm. Pharmacol. 22, 136. Shimizu, H., Daly, J. W., and Creveling, C. R. (1969). J. Neurochem. 16, 1609. Shimizu, H., Daly, J. W., and Creveling, C. R. (1970a). Mol. Pharmacol. 6, 184. Shimizu, H., Creveling, C. R., and Daly, J. W. (1970b). J. Neurochem. 17, 41. Shimizu, H., Creveling, C. R., and Daly, J. (1970~). Proc. Nat. Acad. Sci. US. 65, 1033. Shore, P. A. (1972). Annu. Rev. Pharmacol. 12, 209. Shore, P. A., Busfield, D., and Alpers, H. S. (1964). J. Pharmacol. Exp. Ther. 146, 194. Simon, P., Larousse, C., and Boissier, J.-R. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 729. Raven, New York. Smith, C. B. (1963). J. Pharmacol. Exp. Ther. 142, 343. Smith, C. B. (1965). J. Pharmacol. Exp. Ther. 147, 96. Smith, R. L., and Dring, G. L. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 121. Raven, New York. Snyder, S. H. (1967). Amer. J. Orthopsychiat. 37, 864. Snyder, S. H. (1970). B i d . Psychiat. 2,367. Pharmacol. . Exp. Ther. 165, 78. Snyder, S. H., and Coyle, J. T. (1969). .I Sparber, S. B., and Tilson, H. A. (1972). Life Sci. 11, Part 1, 1059. Stein, L. (1964). Fed. Proc., Fed. Amer. SOC.Exp. B i d . 23, 836. Stein, L., and Wise, C. D. (1969). J. Comp. Physiol. Psychol. 67, 189. Stolk, J. M., and Rech, R. H. (1968). J. Pharmacol. Exp. Ther. 163, 75. Stolk, J. M., and Rech, R. H. (1969). Biochem. Pharmacol. i8, 2786. Strada, S. J., Sanders-Bush, E., and Sulser, F. (1970). Biochem. Pharmacol. 19,2621. Sulser, F., and Dingell, J. V. (1968). Biochem. Pharmacol. 17, 634.

AMPHETAMINE-TYPE PSYCHOSTIMULANTS

357

Sulser, F., Owens, M. L., and Dingell, J. V. (1966). Life Sci. 5,2005. Sulser, F., Owens, M. L., Norvich, M. R., and Dingell, J. V. (1968). Psychopharmacologia 12, 322. Sntherland, E., Rall, T. W., and Menon, T. (1962). J . B i d . Chem. 237, 1220. Svensson, T. H. (1970). Eur. J. Pharmacol. 12, 161. Svensson, T. H. (1971). NaunynSchmiedebergs Arch. Pharmakol. Exp. Pathol. 271, 170. Tagliamonte, A., Tagliamonte, P., Pirez-Cruet, J., and Gessa, G. L. (1971). Nature (London), New Biol. 229, 125. Taylor, K. M., and Snyder, S. H. (1970). Science 168, 1487. Taylor, K. M., and Snyder, S. H. (1971). Brain Res. 28,295. Thornburg, J. E., and Moore, K. E. (1972). Neuropharmacology 11,675. Tripod, J., and Meier, R. (1954). Arch. Znt. Pharmacodyn. Ther. 97,251. Tripod, J., Studer, A., and Meier, R. (1957). Arch. Znt. Pharmacodyn. Ther. 62, 319. Udenfriend, S. (1968). In “Adrenergic Transmission” (G. E. W. Wolstenholme and M. O’Connor, eds.), p. 3. Little, Brown, Boston, Massachusetts. Utena, H. (1966). Progr. Brain Res. 21B, 192. Uzunov, P., and Weiss, B. (1971). Neuropharmacology 10,697. van der Schoot, J. B., Ariens, E. J., van Rossum, J. M., and Hurkmans, J. A. T. M. (1962). Arzneh-Forsch. 12, 902. van Praag, H. M., Korf, J., van Woudenberg, F., and Kits, T. P. (1968). Psychopharmacologia 13, 145. van Rossum, J. M. (1970). Znt. Rev. Neurobiol. 12,307. van Rossum, J. M., and Hurkmans, J. A. T. M. (1964). Znt. J. Neuropharmacol. 3 , 227. van Rossum, J. M., van der Schoot, J. B., and Hurkmans, J. A. T. M. (1962). Experientia 18,229. Vogt, M. (1954). J. Physiol. (London) 123,451. Vogt, M. (1973). Brit. Med. Bull. 29, 168. Voigtlander, P.F., and Moore, K. E. (1973). J. Pharmacol. Exp. Ther. 184, 542. von Hungen, K., and Roberts, S. (1973). Eur. J. Biochem. 3 6 , 3 9 1 . Vrba, R. (1%2). Nature (London) 195, 663. Wallach, M. B., and Gershon, S. (1971). Neuropharmacology 10,743. Weiss, B., and Laties, V. G. (1962). Pharmacol. Rev. 14, 1. Weissman, A., Koe, B. K., and Tenen, S. S. (1966). J. Pharmacol. Exp. Ther. 151, 339. Welch, B. L., and Welch, A. S. (1970). In “Amphetamines and Related Compounds” (E. Costa and S. Garattini, eds.), p. 415. Raven, New York. Wilson, W. S. (1969). Brit. J. Pharmacol. 36,448. Wong, D. T., Horng, J.-S., and Fuller, R. W. (1973). Biochem. Pharmacol. 22, 311. Zetler, G., and Thorner, R. (1973). Pharmacology 10,238. Ziance, R. J., and Rutledge, C. 0. (1972). J. Pharmacol. Exp. Ther. 180, 118. Ziance, R. J., Azzaro, A. J., and Rutledge, C. 0. (1972a). J. Pharmacol. Exp. Ther. 182, 284. Ziance, R. J., Sipes, I. G., Kinnard, W. J., Jr., and Buckley, J. P. (1972b). J . Pharmacol. Exp. Ther. 180, 110. Ziem, M., Coper, H., Broermann, I., and Strauss, S. (1970). NaunynSchmiedebergs Arch. Pharmakol. Exp. Pathol. 267,208.

This Page Intentionally Left Blank

Biological Inhibitors of Lymphoid Cell Division DAVIDF. RANNEY Department of Surgery Northwestern University Medical School and Veterans Administration Research Hospital Chicago. Illinois

I . Introduction

I1. Assay Systems

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . Classification of Inhibitors by Target Cell Specificity

. . . . .

B. Classification of Inhibitors by Functional Specificity . . . . . .

I11.

IV .

V. VI .

VII .

VIII .

C . Classification of Inhibitors by Their Effects on the Cell Cycle . . D. Standard Assays . . . . . . . . . . . . . . . . . Classic Lymphocyte Chalones . . . . . . . . . . . . . . . A . Isolation and Characterization . . . . . . . . . . . . . B . Effects . . . . . . . . . . . . . . . . . . . . . C . Mechanism of Action . . . . . . . . . . . . . . . . D. Specificity . . . . . . . . . . . . . . . . . . . E . Discussion . . . . . . . . . . . . . . . . . . . Low Molecular Weight Inhibitors Released by Lymphoid Tissues . . . A . Production and Assay of Inhibitor . . . . . . . . . . . . B . In Vitro Effects . . . . . . . . . . . . . . . . . . C . Distribution of Inhibitor-Releasing Cells . . . . . . . . . . D. Factors Affecting Production and Release of Inhibitor . . . . . E . In Vitro Regulation of Inhibitor Production . . . . . . . . . F. Specificity . . . . . . . . . . . . . . . . . . . . G. Partial Purification . . . . . . . . . . . . . . . . . H . Mechanism of Action . . . . . . . . . . . . . . . . I . Discussion . . . . . . . . . . . . . . . . . . . Macrophage Factors . . . . . . . . . . . . . . . . . . Suppression Due to Crll-Cell Interaction . . . . . . . . . . . A . Inhibitory Effects Produced by Unstimulated Cells . . . . . . B . Inhibitory Effects Produced by Stimulated Cells from Lymphoid Tissues . . . . . . . . . . . . . . . . . . . C . Potential Interaction of Suppressor Systems Following Lympho. . . . . . . . . . . . cyte Activation . . . . . Other Factors . . . . . . . . . . . . . . . . . . . A . Immunoregulatory a-Globulin . . . . . . . . . . . . B. a-Fetoprotein . . . . . . . . . . . . . . . . . . C . Interferon . . . . . . . . . . . . . . . . . . . D . Sea Star Factor . . . . . . . . . . . . . . . . . Possible Mechanisms of Action . . . . . . . . . . . . . A . Functional Activities . . . . . . . . . . . . . . . B . Sites of Action . . . . . . . . . . . . . . . . .

359

360 361 361 362 363 364 366 366 367 368 369 370 370 371 372 373 375 376 378 380 382 383 384 385 385 386 387 388 389 390 391 392 393 393 396

360

DAVID F. RANNEY

IX. X.

C. Specificity . . . . . . . . . . . . . Clinical Implications . . . . . . . . . . . . A. Association with Clinical Conditions . . . . . B. Potential Usefulness As Chemotherapeutic Agents Conclusion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

396 397 397 401 402 403

I. Introduction During the past 5 years there has been an expanding interest in the naturally occurring factors that regulate lymphoid cell division. This has occurred first, because of the recognition that they may participate in the regulation of normal immune responses, either by controlling the production of immunocompetent cells from lymphoid precursors or by regulating the immune response itself. Second, these factors are released by malignant cells (lymphoid and nonlymphoid) and may interfere with the processes of immune surveillance and tumor rejection. Third, some of these factors appear to be specific for lymphoid tissues. In the purified form, they may be useful as specific chemotherapeutic agents for the treatment of lymphoid malignancy, autoimmune disease, graft-versus-host (GVH) disease, and allograft rejection. The major naturally occurring inhibitors are listed in Table I, according to the present knowledge concerning their specificity of action. Regulation of the immune response by specific antibody has been the subject of several other reviews and will not be discussed in this chapter. A short discussion of the immunologically specific suppression produced by T cells and their soluble products is presented in Section VI. The remainder of this chapter is devoted to the nonspecific inhibitors of lymphoid cell division. These fall into three general categories: (I) the classic lymphocyte chalones, which are heat-labile, protease-sensitive, high molecular weight factors, isolated by extraction from homogenized lymphoid tissues; (2) the low molecular weight inhibitors, which are heat-stable, protease-resistant factors, released by metabolizing lymphoid cells into their culture supernatants; and (3) the inhibitors whose tissue of origin is either nonlymphoid, multiple, or as yet undetermined. The latter is a diverse group including macrophage factors, inhibitors present in normal serum, factors appearing in serum during various disease states, interferon, inhibitors isolated from the ascites fluid of tumor-bearing animals, and factors obtained from the coelomic fluid of invertebrates. In addition to reviewing the major factors in these groups, this chapter presents our recent studies of the low molecular weight inhibitors obtained from lymphoid tissues. The

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

361

TABLE I CLASSIFICATION OF

THE

BIOLOGICAL INHIBITORS BY

SOURCE AND

Lymphoid source Immunological specificity

1. Antibody 2. Specific T-cell suppressors

Lymphoid tissue specificity

TISSUESPECIFICITY honlymphoid source

Multiple or undetermined tissue specificity

1. Classic chalones" 1. Thymic Frac2. Low molecular tion C weight inhibi2. Interferon tor from lymphoid tissues 3. GanghosidesO

1. Serum factors a. Immunoregulatory a-globulin" b. a-fetoprotein 2. Nonspecific T-cell suppressors 3. Macrophage factors 4. Interferon 5. Sea star factor" 6. Tumor products

"

These factors inhibit T-cell but not B-cell functions.

"

G,, gangliosides obtained both in the lipid extracts of murine brain (Miller and Esselman, 1975), and in the culture supernatants of murine thymocytes (Miller and Esselman, personal communication), which cross-react serologically with K 3 H , nonspecifically suppress the in vitro responses of murine spleen and marrow cells to antigen stimulation. Preincubation studies implicate the B cell a s the target of this suppression. (This footnote was added in proof, and ganghosides ar e not further discussed in the text.)

problems of isolation and characterization, specificity as a function of structure and concentration of inhibitor, the possible interrelationships of these factors, and the potential mechanisms of action are discussed. Also preliminary data, suggesting their potential relationship to malignancy and autoimmunity, are included.

II. Assay Systems A wide variety of assays have been used to detect inhibitors of lymphocyte proliferation. In considering the following studies, it is important to recognize how these assays can be used to differentiate the target cell specificity and functional activity of a particular inhibitor.

A. CLASSIFICATION OF INHIBITORS BY TARGET CELLSPECIFICITY In mammals, the maturational cycle of lymphocytes involves (1) spontaneous division of lymphoid precursors within the primary lymph-

362

DAVID F. RANNEY

oid organs, (2) maturation and migration to the secondary lymphoid organs, (3) division and differentiation of these mature cells in response to specific antigens or nonspecific mitogens, and (4) release of effector substances (antibody or lymphokines) by mature lymphocytes following activation. The two major subpopulations of mammalian lymphoid precursors divide and mature in anatomically distinct primary lymphoid organs. The thymic, or T-cell precursors both divide and mature in the thymus (Stobo and Paul, 1972). Bursal, or B-cell precursors divide in the bursa of chickens, but in contrast to the T-cell precursors, they appear to mature (as determined by mitogen responsiveness) after leaving the bursa (Weber, 1967, 1973). The anatomical equivalent of the avian bursa has not yet been identified in primates. The secondary lymphoid organs (spleen, lymph nodes, and other peripheral lymphoid organs) and the peripheral blood contain mixtures of mature T and B cells which can be induced by specific antigens, allogeneic cells, or mitogens to undergo division and differentiation into antibody-secreting plasma cells (B cells), cytotoxic effector cells (T cells), or antigen-specific cooperator and suppressor cells (T cells). Also, these mature lymphocytes can be induced to release lymphokines such as migration inhibition factor (MIF) by the exposure of primed cells to the sensitizing antigen or unprimed cells to mitogens, usually phytohemagglutinin (PHA). Clearly, it can be determined whether an inhibitor affects the division of precursor vs. mature cells or B cells vs. T cells by the appropriate choice of lymphoid targets. For example, the former can be determined by comparing the relative inhibition of the whole thymocyte population (predominantly precursor cells) and the small subpopulation of medullary thymocytes (mature cells), obtained by treating the experimental animals with cortisone acetate prior to thymectomy (Stobo and Paul, 1972). The relative effects on B and T precursor cells can be determined by comparing the inhibition of unstimulated chicken bursa1 cells and thymocytes. The effects on mature B and T cells can be compared by determining the antibody responses of murine spleen cells following immunization with a thymic-dependent antigen, sheep red blood cells (SRBC) vs. a thymic-independent antigen, Escherichia coli lipopolysaccharide (LPS). B. CLASSIFICATION OF INHIBITORS BY FUNCTIONAL SPECIFICITY The biological inhibitors of lymphocyte responses can potentially affect several different processes, including the binding of antigen (recognition), the early steps of lymphocyte activation, or cell division itself. A factor can be considered to inhibit cell division directly only if

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

363

it decreases either the division of unstimulated lymphocytes or the division of stimulated lymphocytes subsequent to the full period of activation and following the removal of the activator. If an inhibitor must be present during activation in order to exert its effect, but has no effect when added later, it inhibits either recognition or early activation, but not cell division directly. Specific interference with recognition is suggested if the factor inhibits the spontaneous rosette formation between human T cells and SRBC, and is strongly indicated if it competitively inhibits the binding of radiolabeled antigens or mitogens.

C. CLASSIFICATION OF INHIBITORSBY THEIR EFFECTSON CYCLE

THE

CELL

Under ideal culture conditions, a commitment on the part of an activated lymphocyte to enter the replicative process involves its progression through the entire cycle of DNA synthesis (S), the second phase of growth (G2), and mitosis (M) as illustrated in Fig. 1. Inhibitors which interfere with the GI-S transition are of particular significance because they potentially interrupt the natural (but still unknown) signals that initiate cell division. By making use of the two phase markers, DNA synthesis and mitosis, one can determine whether a particular inhibitor is likely to exert its effect on the GI-S transition. Such an inhibitor will produce a gradual decrease in the incorporation of DNA

G,-

S

Inhibitor

to

+

s

FIG. 1. The effect of a GI-S inhibitor on DNA synthesis and mitosis. The bar with lines (So)depicts the number of cells synthesizing DNA when the inhibitor is added at t o . With time, the bar rotates and the number of cells synthesizing DNA decreases to a minimum at to + S. The mitotic rate decreases after this “bar” of cells has rotated through the approximate interval of S + Gz.

3 64

DAVID F. RANNEY

precursor, becoming maximal after an interval equivalent to the duration of S phase, which ranges from 3 to 4 hours for hormonally activated thymocytes (MacManus and Whitfield, 1974) to as long as 11-30 hours for PHA-activated peripheral lymphocytes (Bender and Prescott, 1962; Sasaki and Norman, 1966). Because no additional cells can enter S phase, the mitotic index also begins to decrease after a lag time equivalent to S G2. (The G2 interval is generally short relative to the S phase.)

+

D. STANDARD ASSAYS 1. In Vitro Assays The naturally occurring inhibitors have been studied using assays that measure the following lymphoid cell functions: immune recognition, DNA synthesis, cell division, and various immune responses including proliferation, antibody production, and lymphokine release. Several factors need to be considered when interpreting the results of these assays. 1. Immune recognition is evaluated by the binding of SRBC to human T cells (spontaneous rosette formation); the binding of antigen-treated, complement-activated SRBC (EAC-SRBC) to human B cells (Bianco et al., 1970; Papamichail et al., 1972); and the binding of radiolabeled antigens to lymphocytes (Glasgow et al., 1972). Inhibitors that affect the process of recognition may not have any direct effect on lymphocyte division. 2. DNA synthesis is evaluated by pulse-labeling with t h ~ m i d i n e - ~ H and by determining its incorporation into DNA either directly or by radioautography. Incorporation depends on at least three distinct processes: (a)transmembrane transport, (b) phosphorylation by intracellular nucleoside kinases, and (c) incorporation of radiolabeled thymidine triphosphate (TTP) into DNA. Factors that interfere with the early processes can reduce the incorporation of t h ~ m i d i n e - ~without H affecting DNA synthesis or cell division, as follows: cold thymidine at concentrations as low as lo-’ M can competitively inhibit the transmembrane transport of radiolabel, whereas concentrations of to lop4M are required to inhibit cell division (Cleaver, 1967); certain inhibitors present in various preparations of classic chalones appear to inhibit the incorporation of exogenous thymidine by blocking its intracellular phosphorylation (Voorhees, personal communication), but in mammalian cells, inhibition of the phosphorylating enzyme, thymidine kinase, does not appear to decrease DNA synthesis or cell division (Hauschka et al.,

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

365

1972); and factors that stimulate endogenous thymidine synthesis can produce a spurious suppression of exogenous nucleoside incorporation by decreasing its specific activity in the acid-soluble precursor pool. All of these potential artifacts can be excluded by determining the specific activities of the various precursor pools, or by heavy label radioautography. The latter method distinguishes between inhibitors that decrease the average labeling per synthesizing cell nucleus (producing no change in the number of cells entering S phase) and those that decrease the fraction of total cells with labeled nuclei (producing a decrease in the number of cells entering S phase). 3. Cell division is evaluated either by determining the mitotic index or by counting the number of viable cells present in culture. These direct measurements of division avoid the previous artifacts. However, in order for an inhibitor to produce a statistically significant reduction in cell number, the lymphoid targets must be previously stimulated and rapidly increasing in number, and the inhibitor must be present for a relatively long interval. First, in these assays that utilize stimulated target cells, the distinction between an antiactivator and an antiproliferative agent must be made, as discussed above. Secondly, because of their long duration, these assays are more susceptible to the addition of factors containing very low levels of cytotoxic activity. Also, because of this long duration, the possibility exists that certain factors in the fraction to be tested could induce the production and release of inhibitors by the target cells themselves. Although appearing to be an inhibitor, the active moiety in such a fraction could actually represent an activator or inducer. This is further discussed in Sections VI and VII, C. 4. The in vitro immune response assays are of two types. ( a ) The proliferative assays measure DNA synthesis or cell division following lymphocyte stimulation with specific antigens, or allogeneic cells, producing a mixed lymphocyte response (MLR). These assays embody all of the complexities mentioned above plus the additional problem that multiple processes (recognition, activation, or cell division) may be affected by an inhibitor when it is added at the time of stimulation, as is the usual situation. (b) The secretory assays measure the number of antibody-producing (plaque-forming) cells in a population of murine spleen cells that can respond to in vitro antigenic stimulation, usually by SRBC. This system is even more complex because the response is dependent not only on recognition, activation, and division but also on differentiation and secretion, any one of which could be inhibited separately. A second secretory assay measures the release of MIF in response to antigenic or mitogenic stimuli, as discussed above. This

366

DAVID F. RANNEY

unique assay does not involve cell division at all and, consequently, assesses the effects of an inhibitor on recognition, activation, or secretion.

2 . In Vivo Assays These assays include measurements of DNA synthesis (performed by in vivo labeling with t h ~ m i d i n e - ~ Hcell ) , division (performed by determining the increase in neonatal thymic weight), and the immune responses (allograft rejection, GVH, and antibody production). Allograft rejection, GVH, and the murine antibody response to SRBC constitute thymicdependent functions, whereas the murine antibody response to Escherichia coli LPS is thymic-independent and requires only functional B cells (Britton and Moller, 1968; Anderson and Blomgren, 1971). These assays embody all of the complexities discussed for the in vitro immune responses plus an important additional problem. Relatively large doses of foreign protein antigens can activate suppressor cells in the recipient animal and nonspecifically abrogate a variety of unrelated immune responses (Gershon et al., 1974). As discussed above, such a factor could be misconstrued as an inhibitor, but actually constitutes an activator. Rigorous exclusion of this potential mechanism of antigenic competition requires that the “inhibitor” be isolated from and tested in the same inbred strain. This is discussed further in Section VI.

Ill. Classic Lymphocyte Chalones The classic chalones were first described by Bullough and Laurence (1964) for epidermal cells. They represent a group of putative biological regulators of cell division, characterized as water-soluble, heat-labile, protease-sensitive factors whose action is reversible and tissue-specific but not species-specific. The first evidence for a lymphocyte chalone was reported by Moorhead et al. (1969) and Bullough and Laurence (1970), who demonstrated that aqueous extracts of porcine lymphocytes inhibited the mitosis of human peripheral blood lymphocytes and murine lymphoma cells. AND CHARACTERIZATION A. ISOLATION

Lymphoid chalones are obtained by extracting the homogenized lymphoid tissues of various species using distilled water (Moorhead et al., 1969) or saline solutions (Kiger, 1971; Houck et al., 1971). These crude extracts have been partially purified by three general methods:

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

367

molecular sieve chromatography (Jones et al., 1970), ethanol fractionation (Garcia-Giralt et al., 1970), and molecular (Diaflo) filtration (Houck et al., 1971). By these methods, one or more apparently noncytotoxic factors, which display inhibitory activity in uitro and in uiuo, can be isolated in the 75% ethanol-soluble fraction and in the 30,000-50,000 molecular weight range. This activity is heat-labile at 55°C and sensitive to trypsin (Houck et al., 1971). The ethanol-soluble fraction has been further purified by chromatography on diethylaminoethyl (DEAE) Sephadex A-50 followed by polyacrylamide gel electrophoresis (Kiger et al., 1972). The active fraction is weakly acidic and migrates with a rapidly moving band of glycoprotein-positive material on gel electrophoresis. The biological activity of this more purified fraction has been tested only in vitro. From these characteristics, the inhibitory activity of the lymphoid chalones appears to depend on tertiary protein structure. Its elution from DEAE Sephadex and migration on gel electrophoresis reportedly exclude the following proteins: histones, y-globulins, and a2globulins (Kiger et al., 1972). However, the potential presence of histocompatibility antigens and acidic nucleoproteins has not been excluded.

B. EFFECTS 1. Zn Vitro The crude lymphoid extracts and the Diaflo filtrates decrease the incorporation of t h ~ m i d i n e - ~(Moorhead H et al., 1969; Garcia-Giralt and Macieira-Coelho, 1974; Houck et al., 1971), the mitotic index (Moorhead et al., 1969), and the cell number (Garcia-Giralt and Macieira-Coelho, 1974; Houck, personal communication) in mitogen-stimulated normal human lymphocytes. The partially purified fractions have similar effects on the incorporation of radiolabel (Garcia-Giralt et al., 1970, 1972; Kiger et al., 1972); however, their effects on the direct assays of cell division have not been reported. Although certain of these preparations may affect unstimulated lymphocytes (Garcia-Giralt et al., 1972; Kiger et al., 1972), clearly others do not. The basis for this difference is uncertain, but the predominant effects appear to be those of an antiactivator rather than an inhibitor of spontaneous cell division (Garcia-Giralt et al., 1972). Lymphoid chalone blocks the release of MIF by PHA-stimulated lymphocytes if the chalone is present at the time of mitogen addition (Houck et al., 1973). It is presently uncertain whether lymphoid chalones inhibit the division of malignant lymphoid cells. Whereas early studies indicated that this was the case (Jones et al., 1970; Kiger, 1971; Houck and

368

DAVID F. RANNEY

Irasquin, 1973; Bullough and Laurence, 1970), recent investigations show that the suppression of t h ~ m i d i n e - ~ incorporation H in malignant lymphoid cell lines is not associated with a decrease in their rate of cell division (Garcia-Giralt and Macieira-Coelho, 1974). Moreover, the inhibitory effect on isotope incorporation by malignant cells appears to be less pronounced and more rapidly reversible than in normal lymphocytes (Houck and Irasquin, 1973). These important differences between normal and malignant lymphoid targets are currently under investigation. 2. I n Vivo

The in vivo administration of large doses of heterologous, crude, and partially purified chalones produce the following effects: (1) a reduction in lymphoid cell division, as determined by organ labeling with t h ~ m i d i n e - ~ (Garcia-Giralt H et al., 1970) and neonatal thymic growth (Chung and Hufnagel, 1973); (2) a decrease in the murine antibody responses to T-dependent antigens (Garcia-Giralt et al. , 1970, 1973a; Kiger, 1971) but not to T-independent antigens (Florentin et al., 1973); (3) prolongation of allograft and xenograft survival (Kiger et al., 1972; Houck et al., 1973; Chung and Hufnagel, 1973); and (4) a decrease in the GVH response. The last-mentioned effect is observed with treatment of the lymphocyte donor (Garcia-Giralt et al., 1970, 1972, 1973a,b; Kiger et al., 1972, 1973a,b), the recipient (Garcia-Giralt et al., 1972), or the donor lymphocytes themselves, during cell transfer (Garcia-Giralt et al. , 1973b; Kiger et al., 1973a). In these experiments, the injections of heterologous protein varied from 50 to 250 mg/kg/day (equivalent to 3500-17,500 mg/day in a 70-kg man). This constitutes several splenic or thymic equivalents of protein per animal each day. C. MECHANISMOF ACTION The most purified preparations of chalone appear to contain several inhibitory factors, some of which are effective only in vitro and others both in vitro and in vivo (Kiger, 1971). In the absence of a highly purified major fraction, it has been difficult to determine the single mechanism of action. Nevertheless, the fact that these preparations can suppress in vivo immune responses strongly indicates that they function either ( 1 ) by producing direct immunosuppression or (2) by activating nonspecific suppressor cells in the recipient. Several findings that are also characteristic of the nonspecific suppressor system require that this latter mechanism be considered. In vitro, the chalones appear to

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

369

represent antiactivators rather than antiproliferative agents. Nonspecific suppressor cells also appear to release antiactivators (as discussed in Section VI). I n vivo, the chalones inhibit T-dependent but not Tindependent antibody responses, and they cause a prolonged suppression of subsequent mitogen responses by the animal’s lymphoid cells (Garcia-Giralt et al., 1972). Both of these results are characteristic of activated nonspecific suppressor systems (Gershon, 1974). All of the in vivo tests have employed large doses of foreign (allogeneic or xenogeneic) proteins. These may contain T-dependent alloantigens that have the capacity to activate suppressor systems. Such a mechanism can best be excluded by utilizing chalone preparations and test animals from the same inbred strain. The following in vitro results suggest that the lymphoid chalones represent antiactivators: they inhibit predominantly stimulated targets and also the release of MIF which is dependent on recognition and activation but not on cell division. They appear to be noncytotoxic as determined by trypan blue viability and the sparing of RNA and protein synthesis. However, other data indicate that the in vitro inhibitory effects of lymphoid chalones on normal lymphocytes are not rapidly reversible (Houck and Irasquin, 1973). This could result from a high affinity for the lymphocyte membrane, the presence of subliminal cytotoxic activity in the preparations, or chalone-induced activation of nonspecific suppressor cells. In stimulated lymphocytes, the sparing of RNA and protein synthesis by an inhibitor that markedly decreases DNA synthesis and cell division constitutes a surprising result-one that would be expected to produce unusual morphological changes in the target cells. This also merits further investigation. It has not yet been established whether chalones have an immediate or delayed effect on the cell cycle (Lasalvia et al., 1970; Moorhead et al., 1969).

D. SPECIFICITY Contrasting results have been reported concerning the in vitro tissue specificity of lymphocyte chalones, some indicating that they are tissuespecific (Bullough and Laurence, 1970; Houck et al., 1971), and others indicating the absence of tissue specificity in a wide variety of cell lines (Garcia-Giralt et al., 1973a). In vivo, the lymphoid chalones appear to be relatively specific for lymphoid tissues (Garcia-Giralt et a l . , 1970, 1973a,b). Extracts from nonlymphoid tissues do not markedly affect the in vitro (Kiger, 1971; Houck et a l . , 1971) or in vivo lymphoid responses (Garcia-Giralt et al., 1972; Kiger et al., 1972). The question of tissue specificity has been an exceedingly difficult area to investigate because

370

DAVID F. RANNEY

of the constraints posed by multiple-target cell systems that have to be tested under differing conditions of activation. Therefore, although lymphoid tissue specificity has been suggested by the previous studies, the nature of this specificity is currently being reinvestigated using direct assays of cell division and dose-response studies.

E. DISCUSSION The lymphoid chalones represent important biological materials with established immunosuppressive activity in vivo. Whether the chalones produce this in vivo effect directly or indirectly by suppressor cell activation remains to be determined. Because many of their properties are similar to those produced by nonspecific suppressor cells, it has been suggested that the lymphoid chalones might represent a tissue form of immunoregulatory a-globulin (IRA), an antiactivator present in serum that is also capable of blocking the release of MIF and that appears to be involved in the nonspecific suppression of T-dependent immune responses (see Section VII, A). The different mobilities of partially purified chalone and the serum a2-globulins on gel electrophoresis suggest that these are different factors (Kiger et al., 1972). However, the recent discovery that the active moiety in IRA is a dissociable peptide, noncovalently bound to its a-globulin carrier (Occhino et al., 1973), reopens this question of possible identity. It has not yet been established whether or not the classic lymphoid chalones actually represent feedback regulators of immune activation. The proof of this functional role requires that these factors can be shown to be released by metabolizing cells and to exert reversible effects on stimulated normal lymphoid targets. Their relative lack of effect on spontaneously dividing lymphocytes suggests that they probably do not regulate the production of mature cells from their lymphoid precursors. Additional inhibitory extracts of lymphoid tissues have been prepared, but their methods of isolation differ significantly from those used for lymphoid chalones (Hand et al., 1970; Carpenter et al., 1971a,b). These extracts and the classic chalones also differ with respect to several functional activities (Phillips et al., 1975). The key to the structural and functional relationships among the factors present in these preparations lies in their further purification and characterization.

IV. Low Molecular Weight Inhibitors Released by Lymphoid Tissues Several previous studies have suggested that inhibitory factors might be released by cultured lymphoid tissues. This evidence includes (1) the

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

371

enhancement of mixed lymphocyte responses by daily changes in the culture medium (Weber, 1970), (2) the detection of inhibitory factors in the supernatants of control cultures during studies of the antigenstimulated release of mitogenic factors (Havemann and Burger, 1971; Dosch et al., 1971; Wolstencroft et al., 1971), and (3) the inhibition of mitogen-stimulated thymocyte division by the addition of bursal cells (Van Alten and Danielson, 1972), which has subsequently been shown to involve the release of soluble factors by the bursal cells (Danielson and Van Alten, 1974). Generally, these findings have not been vigorously pursued because it appeared they could result from the release of common metabolites or the depletion of nutrients in the culture medium. During our own studies of the interaction between neonatal and adult lymphocytes in rats [isologous lymphocyte interactions (ILI)], we observed that both neonatal spleen cells and a dialyzable factor in their culture supernatants produced a marked reduction in the incorporation of t h ~ r n i d i n e - ~by H unstimulated and stimulated thymocytes from the same inbred strain (Ranney and Oppenheim, 1972, 1973a). Two additional findings suggested that this effect was not due to either the release of common metabolites or the depletion of nutrients. First, this inhibitory activity was not released by the rapidly dividing L1210 murine lymphoid leukemia line (Ranney and Oppenheim, 1973a; Ranney et al., 1973). Second, the inhibitory activity could be recovered in the dialysates of these supernatants, indicating that their effect on the secondary targets did not result from the depletion of nutrients. This prompted the following studies to determine whether the low molecular weight inhibitor(s) could represent biological regulators of lymphoid cell division. A.

PRODUCTION AND

ASSAYOF

INHIBITOR

The usual method for producing and assaying low molecular weight inhibitor is shown in Fig. 2. Unstimulated spleen cells from inbred Brown Norway (BN) rats are incubated in RPMI 1640 culture medium containing low concentrations (0.5%) of heated (56°C) fetal calf serum. At various times up to 60 hours, the supernatants are harvested, Millipore filtered (0.22 pm), and assayed for their effects on DNA synthesis, protein synthesis, and the mitotic rate of unstimulated BN thymocytes (Ranney and Oppenheim, 1973a,b). These target cells were chosen because they have a high rate of spontaneous proliferation in short-term culture and do not themselves release the low molecular weight inhibitory factor(s). Also, the use of unstimulated targets represents the best available in vitro model for the in vivo regulation of

372

DAVID F. RANNEY

Assay

Production

8N

8N Rat

c u It u r e d

Spleen

( 0 - 6 0 h r )

Thymacytes

Supernatant

Cells

thymidine-jH

-

FIG. 2. The release and detection of low molecular weight inhibitor.

spontaneous lymphocyte division: it allows the comparison of precursor and mature cells under comparable conditions, it eliminates the problem of determining whether an inhibitor constitutes an antiactivator rather than an antiproliferative factor, and it avoids the other problems of stimulated cell assays discussed in Section 11.

B. In Vitro EFFECTS The 15-60 hour supernatants from unstimulated spleen cells produce a rapid, progressive decrease in t h ~ m i d i n e - ~ H incorporation which

becomes maximal 10-24 hours after exposure to the target cells (Fig. 3) (Ranney and Oppenheim, 1973a). They produce a slower but progressive inhibition of the mitotic index which reaches 40% (inhibition) by 5 hours and 60-75% by 10 hours (Ranney and Oppenheim, 1973b). The supernatants produce a minimal or insignificant decrease in the incorporation of ~-1eucine-l~C during the first 24 hours, and do not alter the exclusion of trypan blue dye by target cells. The effect on th~rnidine-~Hincorporation is reversible following the removal of supernatant (Ranney and Oppenheim, 1973a). These results suggest that the supernatants contain a reversible inhibitor of DNA synthesis and cell division that spares total protein synthesis (in unstimulated lymphocytes) and appears to be noncytotoxic within the limits of these methods.

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

3 73

100

E a

80

0

0 L

60

\

\ - \

\

t

C 0

V r

0

40

s 20

0

.

\

\

\ \ I 5 Hours

10

in

15

20

25

culture

FIG. 3. Effects of supernatants from cultured BN spleen cells on t h ~ m i d i n e - ~(H0 )and ~-leucine-'~C incorporation (A) by unstimulated BN thymic targets. Percent of control (cpm)=[incorporation with added spleen cell supernatanthcorporation with added control (thymocyte) supernatant] x 100. (Reproduced from Ranney and Oppenheim, 1973a, by permission from Academic Press.)

A comparison of the effects on normal and malignant lymphoid target cells reveal that some malignant lymphocytes are affected and others are not. For example, the dilutions of inhibitor required to produce a 50% inhibition (IDs0) of t h ~ r n i d i n e - ~ H incorporation by (morphologically) malignant spleen cells from a patient with acute lymphocytic leukemia, were equal to those required for normal lymphocytes. In contrast, the IDs0 dilutions for normal murine thymocytes and spleen cells produced no inhibition of murine L1210 cells, a malignant lymphoid cell line. The basis for these differences in target cell sensitivity is currently under investigation.

C. DISTRIBUTION OF INHIBITOR-RELEASING CELLS The release of low molecular weight inhibitory activity by cultured spleen cells is a generalized phenomenon, occurring in the following

374

DAVID F. RANNEY

species: the chicken, rat, mouse, guinea pig, rabbit, cow, and rhesus monkey.

1. Distribution b y Lymphoid Organ In the normal mouse and rat, the quantity of inhibitor released by various lymphoid organs is listed in descending order: bone marrow cells = spleen cells 3 lymph node cells.* No significant inhibitory activity is released by thymocytes. In the chicken, the order is as follows: bursa1 cells = bone marrow cells 2 spleen cells. Again, thymocytes release no inhibitory activity. This compartmentalization of production, which excludes the thymus but includes all other lymphoid organs, may have functional significance, but this remains to be determined. 2 . Distribution b y Cell Type Initial studies indicate that the release of inhibitor depends at least in part on a glass-adherent subpopulation of spleen cells. Its release is almost completely abrogated by the removal of these cells using glass bead columns. Surprisingly, the adherent cells by themselves also fail to release large amounts of the inhibitor. This suggests that its release may depend either on the interaction between glass-adherent and nonadherent cells, or on a critical cell density, which is reduced for the adherent cells when these subpopulations are separated. The effects of cell density on production are discussed below. The cell type releasing inhibitor is further elucidated by studies employing 3-week-old chickens that have been rendered agammaglobulinemic by treatment with cyclophosphamide, according to the method of Kincade et a l . (1971). These animals have no detectible B cells by fluoroescent anti-immunoglobulin techniques, and their levels of circulating y-globulin are less than 0.1% of normal. The spleen cells from these treated animals, which contain no detectible B lymphocytes, release normal amounts of inhibitor. This surprising result indicates that normal amounts of inhibitor are released in the absence of either T cells (as in the normal bursa) or B cells (as in the agammaglobulinemic spleen). This strongly implicates either null cells (lymphocytes lacking B or T surface markers) or macrophage-like cells as the producers of this inhibitor. In NZB/NZW mice, which exhibit an early deficiency in the release of inhibitor, an increase in the fraction of splenic null cells is

* Ficoll-hypaque purified peripheral blood cells (lymphocytes and mononuclear cells) release low but significant quantities of the inhibitor.

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

375

detectible from birth (Stobo et al., 1972), suggesting that null cells might be involved in its release. However, the possibility that these cells represent macrophages is favored by their removal with glass beads, their relative radioresistance (discussed below), and their organ distribution, macrophages being abundant in the medullary cords of lymph nodes and in B-cell-rich areas such as the splenic pulp and bone marrow (Dukor et a l . 1970). This hypothesis is also supported by the finding that similar soluble inhibitors are released by macrophage-like peritoneal exudate cells (Waldman and Gottlieb, 1973); however, further studies are required to provide a definitive answer to this interesting question.

D. FACTORS AFFECTINGPRODUCTION AND RELEASEOF INHIBITOR The production and release of inhibitor are functions of ongoing cellular metabolism rather than progressive cell death, with its associated release of intracellular breakdown products. This is supported by the observations that sonication and freeze-thawing of the spleen cells release insignificant amounts of inhibitory activity, and pretreatment of the producing cells with inhibitors of protein synthesis-puromycin or high concentrations of mitomycin C (100 pg/ml)-largely abrogates its release. Irradiation (2000-3000 R) results in a partial but lesser (20-30%) decrease. Based on these results, there does not appear to be a large pool of preformed active inhibitor present within the cell. Rather, it is either synthesized or generated from an inactive precursor during culture by a process that depends at least in part on protein synthesis. Increasing the packing density of the producing cells markedly increases the release of inhibitor. Also, its release is inversely related to the concentration of fetal calf serum, the maximal release occurring in serum-free medium. These findings suggest that optimal production is associated with maximal cell-to-cell contact and basal culture conditions. They also exclude the possibility that the inhibitor is generated from serum factors. In all of the species tested, the release of inhibitor by spleen cells is maximal during the neonatal period and decreases progressively with age, reaching low levels by 3 4 months in normal mice, 6-8 months in rats, and after longer intervals in guinea pigs, rabbits, cows, and rhesus monkeys. Therefore, in normal animals, this progressive decline is associated with aging of the immune system and may potentially be related to the progressive decrease in the rate of spontaneous lymphoid cell proliferation. In contrast to normal murine spleen cells, the malignant lymphoid cell

376

DAVID F. RANNEY

line, L1210, releases no detectable inhibitory activity. This suggests that the process of malignant transformation, the self-propagation of certain lymphoid cell lines, or the very rapid proliferation of this particular cell line may require the suppression of these low molecular weight inhibitory factors.

E. In. Vitro REGULATION OF INHIBITOR

PRODUCTION

In order for an inhibitor of cell division to represent a biological feedback regulator, several criteria must be met: (1) the factor must actually be released by metabolizing cells, (2) its effect must be reversible, and (3) either its rate of release or its effect must vary depending on the rate of cell division in the target tissue-in this case, the same tissue as the one producing the inhibitor. Its effect rather than its rate of release might be altered, for example, by the production of either a competitive or noncompetitive antagonist. To test the hypothesis that the low molecular weight inhibitor(s) from rat spleen constitutes such a regulator, these inhibitor-producing cells were stimulated by the addition of irradiated allogeneic spleen cells (Fig. 4). Irradiation (2000 R) prevented the stimulating cells from dividing, but only partially decreased their release of inhibitor, as discussed above. This residual release by the stimulating cells and the effects of cell density were controlled by adding equal numbers of irradiated autologous cells to the unstimulated cultures. Supernatants from the unstimulated and stimulated cultures were harvested daily and their effects on thymidine-3H incorporation by target thymocytes tested. Significant inhibitory activity was detected in the unstimulated spleen cell supernatant between days l and 2 and increased rapidly thereafter. By contrast, this activity was maintained at very low levels in the supernatants of the stimulated cells. The low level of inhibitory activity that is detectable following stimulation continues to reside entirely within the low molecular weight fraction (G 1000 mol wt, as determined by Diaflo filtration) (Fig. 4). This reduction of inhibitory activity following cell stimulation could result from (1) an actual suppression of inhibitor release or (2) the increased release of either a competitive antagonist or noncompetitive stimulatory factors. Recent evidence using murine spleen cells indicates that in resting cells, there is a balance between the release of low molecular weight inhibitory factors and high molecular weight stimulators, creating a net inhibitory effect in the unfractionated supernatant (Ranney, 1974). The change in this balance following cell stimulation is currently being investigated. The difference in the inhibitory activity of unstimulated and stimu-

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

100 r

i

ao -

60 -

15

-

10

40

5

0

2 Day

of

I

I

1

3

4

5

supernatant

0

harvest

FIG.4. Detection of inhibitor released by unstimulated and allogeneically stimulated BN spleen cells [using irradiated autologous (BN,) or allogeneic Lewis (L,) spleen cells]. Solid H by BN thymic targets in lines represent percent inhibition of t h ~ m i d i n e - ~incorporation the presence of supernatants from unstimulated BN BN, cultures (0)and stimulated BN L, cultures (A).The solitary symbols on the right represent the filterable inhibitory activity ( G 1,000 mol wt) present in the day-5 supernatants. The broken line represents the stimulation index for BN + L, production cultures. Similar results are obtained when the responding and stimulating cells are reversed.

+

+

lated cell supernatants can be quantitated by determining the respective dose-response curves and comparing the dilution of each supernatant required to inhibit t h ~ m i d i n e - ~incorporation H in the standard targets by 50%. This has been done for the day-5 supernatants shown in Fig. 4, as illustrated in Fig. 5. By this method, allogeneic stimulation produces a ninefold suppression of the inhibitory activity compared with that released by unstimulated controls. Similar results are obtained after stimulation with optimal mitogenic concentrations of PHA and concanavalin A (followed by separation of the low molecular weight factors from the mitogens by Diaflo filtration). These latter results must be interpreted with more caution because of the slight cytotoxic effects of these mitogens when used at optimal concentrations, which could result in the spurious release of a variety of intracellular products by the small fraction of dying spleen cells. Nevertheless, the suppression of superna-

378

DAVID F. RANNEY

\

\

\

\ \

, 25 S

Dilution

(fraction

o f

0.125 S

So)

FIG. 5. Dose-response (dilution) curves for the day-5 unstimulated ( 0 )and stimulated (A) spleen cell supernatants from Fig. 4. The supernatant dilutions are plotted logarithmically on the z axis, as decimal fractions of the highest concentration of supernatant added to the target cells (So). (So constitutes a 1:2 final dilution in the test cultures.) The plot for the stimulated cells is projected to the ID,, level of inhibition where the ID,, volumes of these respective supernatants are compared.

tant inhibitory activity following allogeneic and mitogenic cell stimulation strongly implicates this low molecular weight activity as a potential feedback regulator of lymphocyte DNA synthesis. Moreover, this reduction following lymphocyte activation represents the direction of change required for an excited lymphoid population to increase its rate of DNA synthesis and cell division. Also, this inverse relationship provides additional evidence that the inhibitory activity does not represent the accumulation of common toxic metabolites in the culture medium since, if the latter were true, the release of inhibitory activity would be expected to increase rather than to decrease following stimulation.

F. SPECIFICITY There are two aspects to the consideration of direct tissue specificity: the differential effects of inhibitor on various subpopulations of lymph-

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

379

oid cells and its relative effects on lymphoid vs. nonlymphoid targets. Dose-response studies using the t h ~ m i d i n e - ~assay H reveal the following effects on lymphoid targets: ( 1 ) inhibition of these targets is speciesindependent (Ranney and Oppenheim, 1973b); (2) B and T precursor cells (avian bursa and thymus) are affected equally (Ranney and Oppenheim, 1973b); (3) spontaneously dividing thymic precursor cells are inhibited at slightly lower concentrations than mature (cortisoneresistant) thymocytes; (4) spontaneously dividing cells from the primary lymphoid organs (rat and avian thymus and avian bursa) are inhibited at moderately lower concentrations than lymphocytes from the secondary organs (rat and avian spleen and lymph node); (5) unstimulated, MLRstimulated, and mitogen-stimulated mature cells are inhibited at comparable concentrations (Ranney and Qhattrone, 1974). Thus, it appears that this inhibitor has a slightly greater effect on precursor cells than mature cells, but within these two major categories, its effects are equal regardless of the subpopulation or state of activation. Nonlymphoid cell suspensions from neonatal rat liver, kidney, and intestinal mucosa are unaffected by inhibitor concentrations that produce a 50% inhibition of lymphoid targets. However, because of the

~ i l u t i o n

(

fraction of

So)

FIG. 6. Comparison of the dose-response curves for a standard spleen cell supernatant tested against murine thymocytes ( 0 )and murine L fibroblast target cells (A). so constitutes a 1:2 final dilutitm in the test cultures.)

380

DAVID F. RANNEY

difficulty in obtaining single-cell suspensions from these nonlymphoid organs, and their relatively low level of t h ~ m i d i n e - ~incorporation, H we have also compared the effects of inhibitor on normal murine thymocytes and murine L fibroblasts. The fibroblast line is tested during the log phase of growth, at least 24 hours after sparse plating, in order to allow the maximal time for recovery of surface antigens following trypsinization, and to avoid confluence and contact inhibition. A comparison of the dose-response curves and the ID5,, dilutions required for these respective targets is shown in Fig. 6. Approximately 4 times as much splenic supernatant factor is required to inhibit the L-fibroblast cell line. Although additional cell lines need to be tested before general conclusions can be drawn, these initial results suggest that the low molecular weight inhibitor may display a dose-dependent specificity for lymphoid cells. In studies of reciprocal specificity, we have not been able to detect factors capable of inhibiting lymphoid targets in the supernatants of nonlymphoid cells or the malignant HeLa and L-fibroblast cell lines. This evidence for direct and reciprocal tissue specificity should be interpreted with caution until confirmed by using the direct assays of cell division and additional cell lines, and until its underlying mechanism can be elucidated.

G. PARTIAL PURIFICATION The crude supernatant activity has been purified by UM-10 Diaflo filtration, lyophilization of the filtrate, and molecular sieve chromatography on Sephadex G-10. This results in two discrete peaks of inhibitory activity, both less than 1200 in molecular weight (Fig. 7). The lower molecular weight component contains the predominant activity but is also contaminated with tyrosine. Consequently, this fraction has been further purified by ion-exchange chromatography using AGl-X4. The active moiety is an anionic substance, eluted with 1.5 M acetic acid. This procedure increases the specific activity by a factor of 4 relative to the Sephadex fraction and removes the contaminating tyrosine. Compared with the crude supernatant, the specific activity of this purified fraction is increased by a factor of 3750. The resulting ID,,, for t h ~ m i d i n e - ~ incorporation H by rat thymocytes is 0.5% pg/ml.* In all of these steps, the introduction of inhibitory factors by the procedures themselves has been excluded by monitoring the effects of parallel fractions of a control supernatant.

* Expressed

as total weight per milliliter of test culture.

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

100

381

-

80 -

60-

n N 0

n

40 -

0

20 -

0

I

20

T Excluded

25

30 Peak

Fraction

T35

40

HO4C

number

FIG. 7. Elution of low molecular weight inhibitory activity from Sephadex G10. The solid line represents OD,,, and the broken line, the biological inhibitory activity.

The identity of this inhibitor has not yet been determined, but preliminary characterization indicates that it is anionic, less than 1000 in molecular weight, and resistant to trypsin digestion and boiling (lOO°C for 20 minutes) (Ranney and Oppenheim, 1973b; Ranney and Quattrone, 1974). These chemical characteristics differentiate this factor from the heat- and trypsin-sensitive classic lymphoid chalones. In addition, they exclude all proteins, such as histocompatibility antigens and the known lymphokines. Several low molecular weight factors, released by mammalian cells, constitute known inhibitors of lymphoid cell division, including cortisol and corticosterone (the major glucocorticoid in the rat) (Whitfield et a1 ., 1970), cyclic adenosine monophosphate (CAMP) (DeRubertis et al., 1974), and cold thymidine (Whittle, 1966). The presence of cortisol and corticosterone has been excluded using the standard fluorescent methods (Guillemin et a l . , 1959), and cyclic AMP has been excluded by protein-binding assay (Gilman, 1970). The presence of cold thymidine in concentrations sufficient to inhibit cell division has been excluded by three findings: ( 1 ) using both pyrimidine (th~midine-~H) and purine (deoxyguanosine-3H) precursors to evaluate DNA synthesis in the standard assay system, the ID,, concentration for purified inhibitor is approximately 1 log lower than the ID,, for cold thymidine itself; (2) this inhibition is not reversed by adding deoxycyti-

382

DAVID F. RANNEY

dine, which reverses thymidine-induced inhibition of DNA synthesis by bypassing cytidine diphosphate (CDP) deaminase, the enzyme blocked by thymidine (Morris and Fisher, 1963; Whittle, 1966); (3) L1210 cells and thymidine kinase+ murine L fibroblasts are unaffected by concentrations of inhibitor that suppress normal lymphocytes, essentially excluding the possibility of a thymidine block of DNA synthesis. Other substituted nucleosides, such as the “magic spot compounds,” ppGpp and pppGpp, are known to be released from bacteria following amino acid starvation (Cashel and Gallant, 1974); however, their existence in eukaryotic cells is at present questionable (Cashel, personal communication). The anionic nature of this inhibitor suggests that it may contain a free carboxyl or phosphate group. Therefore, it could potentially represent a compound such as the prostaglandin, PGE,, which has been reported to inhibit PHA-induced lymphocyte blastogenesis (Smith and Parker, 1971), but this is unlikely because PGE, also affects many nonlymphoid tissues. The factor might also fall into the class of substituted sugars, whose antimitogenic properties are presently being investigated by Dr. Paul Gordon and co-workers (Gordon, personal communication), or it could represent a blocked peptide similar to the known protease-resistant, informational transmitter, hypothalamic-releasing factor (Burgus et al., 1969). This interesting question remains to be solved. Finally, initial studies indicate that the inhibitor is either bound or inactivated by factors present in heated serum (Lopatin and Ranney, unpublished results). This distinction is important because the destruction of inhibitor by serum factors would limit its role to one of a local mediator, whereas its binding to serum would suggest the possibility that it could exert distant (hormonal) effects, for example on the thymus that does not produce inhibitor. H. MECHANISMOF ACTION Based on its equal inhibition of unstimulated and stimulated lymphocytes, this factor appears to modulate DNA synthesis and cell division directly, rather than inhibiting immune recognition or activation. Its reversibility and minimal effect on early protein synthesis in unstimulated targets suggest that it is nontoxic. The kinetics of its inhibition of DNA synthesis and mitosis appear to be consistent with those for a GIS inhibitor. Incorporation of t h ~ r n i d i n e - ~ H decreases progressively, reaching a minimum between 5 and 10 hours after addition of the factor. Heavy label radioautography reveals that the number of cells remaining in S phase decreases with approximately the same kinetics (Ranney,

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

383

unpublished results). As expected, the decrease in mitotic index lags behind the reduction in DNA synthesis, reaching 40% inhibition by 4 hours and 60-75% by 10 hours. These kinetics are consistent with the duration of DNA synthesis of 3 to 4 hours which has been reported for rat thymocytes stimulated with PGE, (MacManns and Whitfield, 1974). The dependence of the spleen cell inhibitor on the cyclic AMPcyclic guanosine monophosphate systems is currently under investigation.

1. DISCUSSION Cells that release low molecular weight inhibitor are present in all of the lymphoid organs that routinely come into contact with antigens, and in which B and T cells reside in close proximity. Thus, it seems possible that the entire peripheral lymphoid system may exist in an environment of immunologically nonspecific negative regulation, which decreases gradually with age and is withdrawn abruptly in response to either specific or nonspecific stimulation of neighboring lymphocytes. The maximal release of inhibitor appears to depend on close cell-to-cell contact. This may involve contact between adjacent lymphocytes or between lymphocytes and macrophage-like cells. Thus, a second “signal” for its release may consist of lymphocyte depletion within the stroma of the lymphoid organ. These two apparent signals for its decreased rate of release together with its functional capacity to modulate spontaneously dividing precursor and mature cells implicate this factor as a basic homeostatic regulator that has the potential to control the size of the mature lymphocyte population and its rate of generation from precursor cells. Following in vitro stimulation of young and old spleen cells, the residual release of inhibitor from young spleen cells is consistently greater than that from old cells. It is currently being investigated whether in very young or fetal animals, stimulation fails to suppress this release altogether. The potential implications of such a regulatory system in maternal-fetal rejection and autoimmune disease, and the development of various tumors is discussed in Section IX. Its potential interactions with the immunologically specific stimulators and inhibitors are outlined in Sections VI and X. The thymus is almost conspicuously excluded from this local regulatory effect. The implications of this compartmentalization of production remain to be determined. At the present, it would be overly speculative to postulate a hormonal role for this factor. This lymphoid factor appears to differ chemically and

384

DAVID F. RANNEY

functionally from all of the other inhibitors of lymphoid cells except possibly the macrophage factor, discussed next.

V. Macrophage Factors Macrophages and their soluble products have also been implicated as nonspecific regulators of lymphocyte division (Waldman and Gottlieb, 1973). The soluble inhibitor is obtained by culturing the glass-adherent cells from either spontaneous or oil-induced rat peritoneal exudates, followed by lyophilization of the resulting culture supernatant. This supernatant factor produces a rapid marked suppression of thymidine3H incorporation by unstimulated splenic lymphoid cells, but only a moderate decrease in the rate of protein synthesis. Both effects are reversible following the removal of the macrophages or their supernatants. The generation and release of this factor also appear to require ongoing metabolism, as evidenced by the abrogation of its release by heating the macrophages, and the inability to detect the inhibitor in macrophage extracts. The active factor is dialyzable, resistant to heating at 7VC, and has an estimated molecular weight of 500 to 1000 by elution from Sephadex G-10 and G-15.* Except for the two possibly minor differences mentioned as follows, both the initial chemical characteristics and effects of this factor are consistent with those for the low molecular weight inhibitor released by lymphoid organs. (1) Using lymphoid organs, we have been unable to detect the release of this inhibitor unless the adherent and nonadherent cells are in direct contact, whereas the glass-adherent peritoneal exudate cells appear to release the factor in the absence of nonadherent cells. This could simply represent a difference in macrophage cell density in culture or it might represent a qualitative difference between adherent lymphoid cells and adherent peritoneal exudate cells. (2) The supernatant from lymphoid organs produces a very minimal reduction in protein synthesis, whereas the macrophage factor results in a moderate decrease. This difference may be due to variations in the culture conditions during the assay for protein synthesis. Intact macrophages are capable of either enhancing or suppressing various immune responses, generally producing enhanced antibody responses at low ratios of macrophages to lymphocytes (1:2000-1:200) and suppressed responses at higher ratios (1:10-1:3) (Harris, 1965; Perkins and Makinodan, 1965; Feldman and Palmer, 1971). Waldman * T h e s e findings have recently been confirmed and extended by Unanue and co-workers (Calderon et al., 1974).

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

385

and Gottlieb (1973) have suggested that this ratio may be one of the critical factors in determining the nature of the macrophage effect. If macrophages are eventually identified as the source of low molecular weight inhibitor in lymphoid organs, it certainly would appear from the results discussed in Section IV that the naturally occurring ratios of macrophages to lymphocytes in the unstimulated bursa, bone marrow, spleen, and lymph node are sufficient for its abundant release. In this eventuality, it would also appear that lymphocyte stimulation as well as the ratio of adherent to nonadherent cells may be important in determining the resulting effects. This is discussed further in the next section.

VI. Suppression Due to Cell-Cell Interaction The regulation of various immune responses resulting from the interaction between lymphoid subpopulations is a complex problem because of the variety of potential interactions that may occur. (1) Regulation may be mediated by direct cell-to-cell contact or by the release of soluble mediators. (2) The regulatory effects produced by unstimulated and stimulated lymphoid populations are qualitatively different and appear to be mediated by different cell types. The regulation produced in the absence of stimulation appears to be immunologically nonspecific, whereas following stimulation there is evidence for both nonspecific and specific suppression. At least two different cell types, a subpopulation of T cells and a population of glassadherent, macrophage-like cells, are thought to function as suppressor cells, under appropriate conditions. At the risk of simplicity, this section provides a brief outline for the purpose of integrating the information on soluble inhibitors. For a more complete treatment of suppressor cell activities, the reader is referred to a thorough review by Gershon (1974). A. INHIBITORY EFFECTSPRODUCED BY UNSTIMULATEDCELLS

1. Lymphoid Cells Chicken bursa1 cells (Van Alten and Danielson, 1972) and untreated, irradiated or mitomycin C-treated spleen cells from inbred rats (Ranney and Oppenheim, 1973a) reversibly suppress the incorporation of thymidine-3H when added to proliferating autologous thymocytes. When this order is reversed, the irradiated thymocytes produce no inhibition of proliferating spleen cells. As previously discussed, soluble factors have

386

DAVID F. RANNEY

been implicated in this inhibition, and the release of these factors is dependent on a glass-adherent subpopulation. 2. Macrophages The glass-adherent cells from peritoneal exudates reversibly suppress the incorporation of t h ~ m i d i n e - ~ H by nonadherent lymphoid targets (Waldman and Gottlieb, 1973). Similar soluble factors have been implicated. Since all of the above cell types affect spontaneously dividing lymphoid targets, this suppression is immunologically nonspecific. B. INHIBITORY EFFECTSPRODUCED BY STIMULATED CELLS FROM

LYMPHOIDTISSUES Activated lymphocytes release a variety of soluble mediators, which, when tested under appropriate experimental conditions, can either stimulate (Anderson et a l . , 1972) or inhibit lymphoid cell division (Wheelock, 1965; Gresser et a l . , 1970), and either enhance (Katz and Benacerraf, 1972) or suppress the immune response (Rich and Pierce, 1973, 1974). In these studies, the suppressor effects that appear following cell stimulation have largely been investigated using whole cells and immune response assays. In practice, this means that both the suppressor cell and its target must exist in the stimulated environment. The effects produced by whole cells can be divided into two categories: immunologically specific suppression and nonspecific suppression.

1. Immunologically Specific Suppression Briefly, in vivo stimulation with either T-dependent or T-independent antigens activates a subpopulation of lymphoid cells that can specifically suppress the immune response to the sensitizing antigen (Gershon and Kondo, 1971a; Baker et a l . , 1970; Barthold et al., 1974; Kerbel and Eidinger, 1972), either during the initial response or on reexposure to the specific antigen. These effects appear to be mediated by a subpopulation of thymic-derived cells, as determined by their sensitivity to antilymphocyte serum (ALS) and irradiation (Baker et a l . , 1970; Elkins, 1972). Soluble nonantibody factors have been implicated in this process but have not yet been isolated (Gershon and Kondo, 1971a). 2. Nonspecific Suppression Under appropriate experimental conditions, lymphocyte stimulation activates a second subpopulation of suppressor cells whose effects are immunologically nonspecific. This type of suppression prevents the subsequent response to antigens other than the priming antigen, and has

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

387

been termed antigenic competition (Moller, 1971; Gershon and Kondo, 1971b). In contrast to specific suppression, which has been described for both T-dependent and T-independent antigens, nonspecific suppression occurs predominantly with T-dependent antigens (Moller, 1971). This has been demonstrated using the following two systems: (1) activation of lymphoid populations with specific antigens followed by challenge with nonspecific mitogens and (2) the reverse of method (1). In the first system, mice sensitized with a highly immunogenic form of bovine y-globulin (BGG) develop nonspecific suppressor activity in their spleen cells after 1 to 2 days, which inhibits the ability of these cells to respond to PHA (Gershon et al., 1974). The PHA response is suppressed even more if BGG is also present during the challenge with PHA, suggesting that the nonspecific suppressor cells, activated in vivo, are reactivated in vitro by the specific sensitizing antigen, BGG. The factor producing this suppression appears to block either recognition or activation rather than ongoing division. In the second system, mitogen stimulation of lymphoid populations abrogates a variety of subsequent specific immune responses (Rich and Pierce, 1974; Peavy and Pierce, 1974). This suppression appears to be mediated by a soluble factor that is released within 6 to 24 hours following mitogen stimulation, but produces a quite delayed effect on the immune response of the target cells (Rich and Pierce, 1974). The basis for this delayed suppression has not been determined, but mitogen activation is known to induce the release of lymphotoxin (Shacks and Granger, 1971; Coyne et al., 1973) and interferon (Wheelock, 1965), which can inhibit lymphocyte division (see Section VII, C). The nonspecific suppressor cells in these stimulated systems have not yet been identified. Macrophages have been implicated by the resistance of this effect to irradiation and anti-8 serum (Peavy and Pierce, 1974; Sjoberg, 1972), by its abrogation following treatment of suppressor animals with iron powder (Sjoberg, 1972), and by the production of similar suppressive effects by macrophages (Gery and Waksman, 1972). However, recent studies suggest that these cells may actually represent glass-adherent T cells (Folch and Waksman, 1974a,b). Isolation and purification of the soluble factors from defined cell populations will be required to clarify this problem.

INTERACTION OF SUPPRESSOR SYSTEMS FOLLOWING C. POTENTIAL LYMPHOCYTE ACTIVATION Immune stimulation appears to result in contradictory processes: it deactivates the low molecular weight inhibitor of lymphocyte division

388

DAVID F. RANNEY

(Section IV) while activating the nonspecific and specific suppressor systems just described. Although the decrease in low molecular weight inhibitor is quite compatible with the observed proliferative response, activation of suppressor systems is more difficult to interpret. However, since all of these changes are well documented, a reasonable synthesis should be possible. The key to this apparent dichotomy probably lies in the difference between the assay systems used to detect these respective inhibitors. The low molecular weight factors are measured by assays of spontaneous cell division, and the suppressors by assays of either stimulated celi division or the immune response, which do not differentiate between antiactivators and antiproliferative agents. With these differences in mind, the present data suggest that antiproliferative factors, antiactivators, and specific suppressors of the immune response may interact as follows. Lymphocyte activation by antigens or alloantigens stimulates a specific clone of cells, resulting in three changes, occurring prior to the onset of DNA synthesis (24-48 hours), which seem to regulate both the propensity for a proliferative response to occur as well as the specific cells that are permitted to respond. These changes consist of (1) a reduction in the high base-line release of low molecular weight antiproliferative factor, (2) the release of stirnulatory (blastogenic) factors, and (3) the release of nonspecific antiactivating factors. These respective changes would appear to place the whole lymphoid population in a division-ready condition, to facilitate division of the specifically activated clone, and to prevent spurious activation of the remainder of the division-vulnerable clones (primarily for T-dependent antigens). At the end of the proliferative response but before the immune effector responses have peaked, the low molecular weight antiproliferative factor begins to reappear (initial studies indicate that this occurs by days 6-7 in the rat MLR). Later, the release of specific suppressors of the immune response, T-cell factors, and antibody (Uhr and Moller, 1968) produces a regulatory feedback on the cell-mediated and humoral immune responses. In addition, it is possible that the low molecular weight inhibitor (whose release also depends on cell contact) remains at slightly reduced levels until repopulation of the lymphoid stroma by precursor cells has been completed. Various aspects of this hypothesis are currently being tested.

VII. Other Factors In addition to macrophage factor, there are a number of other soluble inhibitors of lymphocyte responses, which are released by nonlymphoid

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

389

tissues, multiple cell types, or as yet unknown sources. All of the endogenous factors in this group have potential access to the circulation. Several studies have indicated that the autologous, homologous, and heterologous sera from unstimulated animals can have a negative effect on both the mitogenic and immune responses of lymphocytes (Weber, 1970; Dosch et al., 1971; Veit and Michael, 1972). In general, these factors have not been extensively characterized, but two major immunosuppressive factors have been isolated from mammalian serum and partially purified: immunoregulatory a-globulin (IRA) and afetoprotein (AFP).

A. IMMUNOREGULATORY GLOBULIN The immunosuppressive properties of serum a-globulins were first demonstrated by Kamrin (1959). Subsequently, Mowbray (1963) isolated an immunosuppressive a2-globulin fraction from serum, which has been designated as Fraction C. This material has been isolated by eluting the serum fraction from DEAE cellulose using 0.5 M acetate buffer. IRA is prepared similarly, but the active fraction is eluted at a lower ionic strength (0.2 M acetate) (Mannick and Schmid, 1967) and may be qualitatively different from the original Fraction C. IRA specifically suppresses T-dependent immune responses. These include the i n vitro proliferation of lymphocytes in response to mitogens, specific antigens, and tumor cells (Cooperband et ul., 1972; Glaser and Herberman, 1974); the antigen-induced release of MIF (Davis et ul., 1971); and spontaneous T-rosette formation (Menzoian et al. , 1974). In addition, IRA inhibits the in vitro phagocytosis of bacteria by peritoneal leukocytes (Glaser et al., 1972). Its in vivo effects are similar, including the suppression of T-dependent but not T-independent antibody responses (Menzoian et al., 1973; Menzoian et al., 1974), prolongation of allograft survival (Glaser and Nelken, 1972), and (at higher doses) suppression of host resistance to bacterial infection, apparently by interfering with phagocytosis (Glaser et al., 1973). These effects are species-independent. IRA appears to represent a reversible inhibitor of lymphocyte recognition and activation, rather than cell division, since its addition following the activation interval produces no change in lymphocyte proliferation (Cooperband et al., 1972).* It inhibits the binding of specific antigens [bovine serum albumin (BSA)-1251]to lymphocytes at 4"C, but probably does not affect mitogen binding (Glasgow et al., 1972). Despite this apparent lack of effect on mitogen binding, IRA effectively

* Recent studies may qualify this conclusion (see Section VIII).

390

DAVID F. RANNEY

blocks lymphocyte activation by these mitogens. This suggests that, in addition to its effects on recognition (antigen binding and rosette formation), IRA may also inhibit one of the early steps of lymphocyte activation. Its ease of reversibility suggests that IRA may be loosely bound to the lymphocyte surface. Recent studies indicate that the active moiety probably represents a polypeptide, approximately 4+000-6000in molecular weight, which can be dissociated from its a-globulin carrier by acid dialysis at pH 3.8 (Occhino et al., 1973). This dissociable fraction contains at least ten distinct peptides but no significant amounts of DNA, RNA, lipid, neutral hexoses, cortisol, or ribonuclease activity. The purified fraction exhibits a fourfold increase in biological activity relative to undissociated IRA and displays continued activity against spontaneous rosette formation (Menzoian et al., 1974). Two properties of IRA, its predilection for T-dependent antigens and its capacity to inhibit activation without affecting ongoing proliferation, suggest that it might represent one of the soluble mediators of antigenic competition, released by activated nonspecific suppressor cells. Indeed, the serum levels of IRA-associated a2-globulin have been observed to rise in patients undergoing renal allograft rejection (Riggio et al., 1968). These considerations have led to the attempted extraction of similar immunosuppressive factors from thymic tissue, using the methods described by Mowbray (1963) for isolating serum Fraction C. The resulting thymic Fraction C has immunosuppressive properties both in vitro and in viva (Carpenter et al., 1971a,b), but it seems to differ significantly from IRA in its inability to suppress both the release of MIF and spontaneous rosette formation (Phillips et al., 1975). The relationship between these serum fractions (Fraction C and IRA) and the tissue Fraction C remains to be clarified.

B. a-FETOPROTEIN a-Fetoprotein is a specific fraction of mammalian serum protein, rich in al-globulins, which has been observed to reach peak levels during gestation (Gitlin and Boesman, 1966) and decrease to low levels during normal adult life. Higher levels recur in the presence of primary hepatomas (Sell et al., 1972), suggesting an hepatic origin. Its immunosuppressive properties have been studied by isolating AFP from the sera of rats bearing Morris hepatomas (Parmely and Thompson, 1974) and from cord blood (Caldwell et al., 1974). a-Fetoprotein is isolated by QAE Sephadex chromatography of the 50% ammonium sulfate supernatant fraction of serum. The peak activity is eluted using 0.25-0.35 M

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

391

NaCl and contains al- and a2-globulins and albumin. a-Fetoprotein suppresses the in vitro lymphocyte responses to PHA and allogenic cell stimulation (MLR), having a significantly greater effect on the MLR (Parmely and Thompson, 1974). Similarly prepared fractions of normal rat serum are as suppressive as the fractions obtained from tumorbearing animals. Therefore, initially it seemed that the suppressive activity might not be tumor-related. However, the AFP from tumorbearing animals produces effects that are considerably less reversible than the effects of AFP from normal animals, suggesting that there are tumor-related differences in these fractions. In contrast to the effects of IRA, the AFP does not inhibit spontaneous rosette formation (Caldwell et al., 1974). These initial results suggest that the active moiety in AFP may affect either activation or cell division rather than immune recognition. C. INTERFERON Animal interferons are proteins varying from 20,000 to 100,000 in molecular weight, released by lymphocytes, macrophages, reticuloendothelial cells (Kono and Ho, 1965; Cantell, 1973), and a variety of nonlymphoid cells (Sutton and Tyrrell, 1961; Ho, 1973) in response to a diverse group of inducers. In addition to RNA and DNA viruses, these inducers of its release from lymphoid tissues include PHA, pokeweed mitogen (PWM), heterologous double-stranded RNA, bacterial endotoxins, synthetic polynucleotides (poly I :C), and specific antigens (from sensitized lymphocytes) (Wheelock, 1965; Friedman and Cooper, 1967; Hilleman, 1969; Smith and Wagner, 1967; Kobayashi et al., 1969; Haber et al., 1972; Wallen et al., 1973; Merigan, 1973). The mitogen-induced release does not appear to correlate with the degree of subsequent transformation but is maximal when the lymphocytes and mitogen remain in continuous contact. Interferon blocks intracellular viral replication in a species-specific manner by preventing the ribosomal attachment of viral messenger RNA (mRNA) (Marcus and Salb, 1966). Recent studies indicate that purified interferon in antiviral concentrations can also inhibit the division of L1210 lymphoid cells (Gresser et al., 1970) and block the in vitro antibody response of murine spleen cells to SRBC (Gisler et al., 1973).*

* Recent work by Blomgren et al. (1974), indicates that low concentrations (10 unitslml) of partially purified viral-induced human interferon (Type I) will suppress the in vitro lymphocyte responses to PHA, concanavalin A, purified protein derivative (PPD), and allogeneic cell stimulation (MLR). Higher concentrations (1000 u n i t s h l ) are required to suppress stimulation by pokeweed mitogen (PWM). Interferon obtained from preparations of murine brain is also capable of suppressing the in vivo proliferation of allogeneic spleen cells and isogeneic marrow cells (Cerottini et al., 1973).

392

DAVID F. RANNEY

Moreover, the interferon inducer, poly I:C, also produces a complete suppression of the DNA synthetic response if added to cultured leukocytes 6 4 8 hours prior to stimulation with PHA (McIntyre et al., 1974). This is the appropriate time interval for interferon induction within the responding cell population. A second interferon inducer, PHA itself, abrogates the in vitro antibody response to SRBC, but only after a delay of 90 to 100 hours (Rich and Pierce, 1974). The kinetics of slower than interferon induction following PHA stimulation-somewhat for poly 1:C-are also consistent with such a mechanism. Indeed, PHA might terminate its own stimulation of lymphoid responses in part by this mechanism. The mitogen-induced release of interferon appears to require the presence of both T cells and macrophages, suggesting that interaction between these cells may be required for its induction (Epstein et al., 1974). The primary target in the inhibition of Tdependent antibody responses may represent the B cell (Gisler et a l . , 1973). However, interferon inducers also produce an immunologically nonspecific suppression of lymphocyte responses to the T-cell mitogens. I n vivo administration of poly I:C produces the same prolonged depression of PHA responsiveness in human peripheral blood cells, beginning 2 hours after injection of the inducer and persisting for 72 hours (McIntyre et al., 1974). The characteristics and kinetics of interferon induction both in vitro and i n vivo are very reminiscent of the nonspecific suppressor systems and provide one possible explanation for that effect. Whether or not this represents the actual mechanism, it probably should be considered when interpreting any delayed inhibition of a proliferative or immune response that occurs following exposure to a potential interferon inducer. It also emphasizes the problems that may arise when adding various column-purified materials to leukocyte cultures, because of the potential for contamination of these preparations with endotoxin from the columns. These associations of interferon with various aspects of the immune response are being vigorously pursued, and considerable information should become available in the near future.

D. SEASTARFACTOR An interesting immunosuppressive factor has been found associated with a 32,000-mol wt protein isolated from the coelomic fluid and homogenized coelomocytes of starfish, the invertebrate species Asterias forbesi (Prendergast and Suzuki, 1970). At milligram concentrations, sea star factor (SSF) specifically suppresses T-dependent proliferative and immune responses both in vitro and in vivo (Prendergast et al., 1974;

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

3 93

Willenborg and Prendergast, 1974). Its effects are noncytotoxic as evaluated by the strict criterion of in vivo immune responsiveness of T cells following prolonged in vitro exposure to the factor. Surprisingly, however, its in vitro effects are not rapidly reversible. In addition, it appears to be tightly bound to lymphocytes, requiring trypsinization to reverse the inhibitory effects. Despite this binding, SSF does not inhibit the formation of spontaneous T-cell rosettes with SRBC and, therefore, does not appear to block recognition. Also, it inhibits only the primary response and has no effect on the anamnestic response of primed cells. At lower (microgram) concentrations, SSF produces opposite effects, augmenting rather than suppressing T-cell responses. This dosedependent reversal in effect may be related to its ability to enhance macrophage activity at lower concentrations. Continuing interest in this factor has been engendered by its T-specific effects and by its potential significance in the evolution of immune regulation. A summary of the biological inhibitors and their effects is presented in Table 11.

VIII. Possible Mechanisms of Action A. FUNCTIONAL ACTIVITIES The precise mechanisms of action for the soluble inhibitors discussed in this chapter have not been determined. Functionally, these factors appear to be divided into three categories: (1) direct modulators of the cell cycle, including the low molecular weight lymphoid and macrophage factors (which can inhibit spontaneously dividing targets); (2) inhibitors of activation, including IRA (which also affects antigen recognition), and probably the classic lymphoid chalones and the factors released by nonspecific suppressor cells; and (3) other factors that cannot be classified due to uncertainty concerning their comparative in vitro effects on unstimulated vs. stimulated normal lymphoid targets. This latter group includes AFP, interferon,* SSF, thymic Fraction C, and the inhibitors released by specific suppressor cells. Elucidation of the specific action for all of these factors requires the determination of their effects on known cell regulatory mechanisms. These factors may act either directly to block precursor, DNA, RNA, or protein synthesis or indirectly by altering intracellular levels of cyclic

* Work now in progress indicates that interferon (from several sources) probably constitutes an inhibitor of activation, since lymphocyte DNA synthesis is reduced only if these interferon preparations are added prior to addition of the activating mitogens (D. 0. Lucas, personal communication).

394

DAVID F. RANNEY

TABLE

SUMMARY OF BIOLOGICAL

BY unstimulated

Factor"

1. Low mol wt lymphoid 2. Macrophage 3. IRA 4. Interferon 5. "Nonspecific" suppressors 6. Specific suppressors 7. AFP 8. SSF 9. Classic chalones 10. Thymic fraction C

Tissue Lymphoid

Cell type

-

Peritoneal exudate Adherent cells Serum Many Many Lymphoid Lymphoid Serum (liver) Starfish coelorn Lymphoid Thymus

Released (R) W) or or stimulated extracted (E) cells (S) R

U

R R

s>u

U

R

S S'

-

R

S'

Hepatic Coelomocytes

R R&E

-

E

U -

-

E

-

R

IRA = immunoregulatory a-globulin; AFP = a-fetoprotein: SSF = sea star factor. = thymocytes or T-derived lymphocytes; B = bursa1 cells (bone marrow cells) or Bderived lymphocytes: P = precursor cells; and M = mature cells. R = recognition; A = activation; D = division; and IR = immune responses. "

I/

T

AMP or cyclic guanosine monophosphate (cGMP) (Whitfield et al., 1971; Hadden et al., 1972). The effects of known lymphocyte activators, and inhibitors of activation, on intracellular levels of cyclic AMP are somewhat complex. For a thorough discussion of this evolving area, see the reviews by Parker (1974), Parker et al. (1974), and MacManus et al. (1974a,b). Basically, the following general picture is beginning to emerge. Some, and perhaps all, of the lymphocyte activators result in a rapid transient (30-minute) rise in intracellular cyclic AMP, followed by a depression to normal or subnormal levels (Parker, 1974; MacManus and Whitfield, 1974), although there may be some variation among lymphoid subpopulations (Parker et al., 1974). Activation itself appears to depend on changes in calcium flux, whereas the initiation of DNA synthesis may be contingent on the temporal spike in cyclic AMP activity (MacManus et al., 1974b). The hormonal inhibitors of lympho-

395

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

I1 INHIBITORS OF

LYMPHOIDRESPONSES

Molecular weight

Protein or peptide

Tissue specificity Probably lymphoid"

5W1,oOO 4,000-6,OOO 20,000-100,000

-

Specificity for lymphoid Target cell function subpopulationsb affected' T=B,P>M

D

-

D R and A

T Probably T and B

-

A Probably R or A

IR High 32,000 30,00CL50.000

Lymphoid

-

T T

High

"

A or D A or D R or A

A or D

This specificity is dose-dependent. Released by T-dependent antigens. Released by both T-independent and T-dependent antigens.

cyte activation (theophylline, cholera toxin, and PGE, at concentrations above k 1 5 puglml) appear to produce sustained high levels of cyclic AMP (De Rubertis et al., 1974; Parker, 1974). With these factors in mind, a biological inhibitor of lymphocyte division that acts indirectly via this system, might be expected to produce a sustained rise in intracellular cyclic AMP. Recent evidence indicates that one additional variable must be considered. The hormonal inhibitors of lymphocyte activation appear to block the initiation of DNA synthesis only when they are added concomitantly with the activator (De Rubertis et al., 1974). Exposure to these same inhibitors after full activation has occurred (36 hours) appears to have no effect on subsequent DNA synthesis, even though it produces similar elevations in cyclic AMP. This suggests that the fully activated lymphocyte may escape from the control of inhibitors that

396

DAVID F. RANNEY

function by elevating intracellular cyclic AMP. This is reminiscent of the effects of IRA (Section VII, A), which appears to function only when present during activation. It also raises the interesting possibility that the apparent capacity of certain inhibitors to permit the ongoing division of a stimulated clone while blocking the activation of unstimulated clones (antigenic competition) may merely be a function of their late temporal release plus this difference in the susceptibility of unstimulated and stimulated target cells, rather than any unique property of the inhibitor itself. This does not invalidate the distinction between inhibitors that affect only activation and those that have been shown to affect cell division directly. Indeed, it suggests, but does not prove, that the inhibitors affecting previously activated cells might, by necessity, bypass the cyclic AMP control mechanisms. It also indicates that a factor can be considered to represent a direct inhibitor of cell division rather than activation only if it affects either unstimulated cells or stimulated cells following the full activation interval and after removal of the activator.

B.

S I T E S OF ACTION

For two of these inhibitors, interferon and SSF, it is known whether the inhibitor acts at the cell membrane or intracellularly. Interferon preparations have access to the interior of the cell (Joklik and Merigan, 1966), whereas SSF functions at the level of the lymphocyte membrane, as determined by lysis of SSF-treated cells with anti-SSF antiserum plus complement and by the restoration of lymphocyte responsiveness following mild trypsinization of the SSF-treated cells (Prendergast et al., 1974). This indicates that the larger protein inhibitors (interferon) are not necessarily restricted to functioning at the cell surface. On the other hand, inhibitors that do function at this level (SSF) may be some of the most slowly reversible factors (see Section VII, D).

C. SPECIFICITY The basis for inhibitor specificity (lymphoid vs. nonlymphoid, precursor vs. mature, and T-cell vs. B-cell) has not been determined. In general, specificity would appear to require a matching of the transmitter (inhibitor) with an appropriate cell receptor, and in some cases also, an appropriate intracellular climate for its action. In future considerations of this problem, it is important to remember that the informational transmitter may be a complex protein (such as antibody or interferon) or a simple molecule (such as the steroid hormones, prostaglandins, and hypothalamic-releasing factor) which fits a more complex protein

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

397

receptor located either at the cell surface or intracellularly. The solution to this important problem will require highly purified fractions of these inhibitors.

IX. Clinical Implications There are two aspects to the clinical significance of these endogenous biological inhibitors: the possible association of abnormalities in their release with various diseases and their potential usefulness as specific chemotherapeutic agents. The studies of these clinical aspects are quite preliminary and essentially have utilized either in vitro models or the injection of relatively crude preparations of inhibitors in vivo . Nevertheless, several associations between altered production and various clinical conditions have become apparent and may point the way to fruitful investigations.

A. ASSOCIATION WITH CLINICALCONDITIONS Of the factors discussed in this chapter, the ones known to be released by metabolizing cells can be grouped as follows: (1)factors that are released continuously in the absence of immune stimulation (low molecular weight inhibitor from lymphoid tissues and macrophage factor), (2) nonspecific inhibitors released following immune stimulation (IRA, interferon, and nonspecific suppressor cell activity), and (3) the immunologically specific inhibitors released following stimulation (antibody and specific suppressor cell activity). Lymphoid chalones have not been included in this grouping because they are obtained by extraction from lymphoid homogenates. Interferon has been tentatively classified as a nonspecific inhibitor because its early release following immune stimulation does not appear to abrogate the ongoing specific immune response. Abnormalities in the production of any of these factors may contribute to the pathogenesis of disease states. A deficiency in their release could (a) facilitate maternal-fetal rejection, (b) permit the premature onset of immune responses, leading to the development of autoimmune disease, and (c) enhance the development of lymphoid tumors. An excessive release could impair the immune responses, leading to (a) the escape of nonlymphoid tumors from immune surveillance and (b) the development of multiple infections. The question of maternal-fetal survival is a multifaceted and intriguing problem. Recent data from our laboratory and others indicate that maternal lymphocytes (which can traverse the placental barrier) may be prevented from responding to fetal antigens by the fetal lymphocytes

398

DAVID F. RANNEY

themselves and by their soluble products. For example, the mononuclear cells in human cord blood markedly and nonspecifically reduce the mitotic rate of maternal and other adult lymphocytes, as determined by studies employing chromosomal markers (Olding and Oldstone, 1974). Also, in the rat system, we have shown that both intact neonatal spleen cells and their soluble products will nonspecifically block the maternal or other adult spleen cells from responding in the MLR (Ranney and Oppenheim, 1973a). As discussed in Section IV, this effect decreases with age. Since this type of regulation would appear to depend on the continuous release of immunologically nonspecific inhibitory factors, it could be mediated by the low molecular weight inhibitors released by lymphoid tissues and macrophages. The age-dependent decline in similar factors appears to be significantly altered in NZB/NZW mice. This abnormal strain develops a form of autoimmunity characterized by the production of antinuclear antibodies (Steinberg et al., 1969; Talal and Steinberg, 1974), with the female mice developing severe proteinuria by 7 months of age and dying of nephritis at about 10 months (Lambert and Dixon, 1968). Both their cellular (Gazdar et a l . , 1971) and humoral immune responses (Evans et al., 1968) as well as the responses to nonspecific mitogens (Stobo et al., 1972) develop prematurely. These responses appear within the first week of life, compared to several weeks in normal strains of mice (Evans et al., 1968; Talal and Steinberg, 1974). Humoral hyperresponsiveness continues during adult life (Talal and Steinberg, 1974). In contrast, by 2 to 3 months of age, thymic suppressor cell activity is lost (Steinberg et al., 1970), and, by 6 months of age, the ability of the thymic-derived cells to produce cellular immune responses has also become markedly deficient in an immunologically nonspecific fashion (Leventhal and Talal, 1970; Cantor et al., 1970; Gelfand and Steinberg, 1973). As NZB/NZW and NZB mice age, they develop widespread lymphoid hyperplasia which may involve the thymus, lungs, and salivary glands, developing into lymphoid malignancy in 1-20% of these mice (DeVries and Hijmans, 1967; Mellors, 1966). From birth, the NZB/NZW mice have an abnormally high fraction of splenic null cells (which bear neither B nor T surface markers). Because it appeared that the early nonspecific hyperresponsiveness in these abnormal mice might be due to the escape of their immune system from its normal negative regulation, we were prompted to compare the age-dependent release of low molecular weight inhibitor from the spleen cells of normal (C57B1/6 and Balb/c) and NZB/NZW mice (Ranney and Steinberg, unpublished results). Our initial results

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

399

reveal a moderate but significant early deficiency in the production of inhibitor by NZB/NZW spleen cells (between 1 week-the earliest point tested-and 2 months of age). Interestingly, this inhibitor began to reappear by 4 to 5 months of age and rose to very high levels by 7 to 10 months. (Normal spleen cells produce progressively less inhibitor from birth, releasing no detectable activity after 3 to 4 months.) Equally interesting was the observation that high levels of inhibitor could also be detected in the NZB/NZW thymic supernatants after 4 months of age, approximately 1-2 months before its reappearance in the spleen. This represents the only case in which we have observed inhibitor to be released by the thymus. It should be observed that these 10-month NZB/NZW thymuses were grossly abnormal and had probably been repopulated with hyperplastic, nonthymic lymphoid cells. These associated changes in immune function and inhibitor production suggest the possibility that the initial deficiency in low molecular weight inhibitor may be related to the premature onset of the humoral and cellular immune responses, and that its rise to high levels late during the course of disease may be related to the progressive deficiency in cellular immunity which has been described. In fact, this late increase in the production of inhibitor might represent either an attempt to regulate the semiautonomous proliferation of cells that produce autoantibody or an excessive release of inhibitor by the abnormal cells themselves. In either case, these cells are apparently no longer susceptible to such regulation. The murine system provides a good model for determining which cell types are responsible for the early deficiency and later excess in the release of inhibitor, and this work is currently in progress. As suggested by the data in Section IV, the inhibitor may preferentially affect the generation of mature cells from their precursors rather than having its maximal regulatory effect on the immune response itself. It remains undetermined whether the early deficiency in low molecular weight inhibitor represents a primary defect or a secondary suppression of its production due to immune stimulation of otherwise abnormal cells. This problem can be solved only when the etiology of autoimmune disease is more completely understood. Other inhibitors, such as antithymic antibody (NTA) (Shirai and Mellors, 1971), have been implicated in the pathogenesis of this disease. It is not unlikely that multiple factors are involved. However, the interesting aspect of the early deficiency in low molecular weight splenic inhibitor is that it could potentially represent either an early primary defect in the control of nonspecific immune hyperresponsiveness, or one manifestation of such a defect, capable of mediating further hyperresponsiveness.

400

DAVID F. RANNEY

The levels of serum a2-globulins have been found to rise in association with at least two pathological conditions-acute allograft rejection (Riggio et al., 1968) and ataxia telangectasis (McFarlin and Oppenheim, 1969). In the latter study, a significant correlation existed between the elevation of a2-globulins and the capacity of these sera to impair the in vitro proliferation of mitogen-stimulated normal lymphocytes. This suggests that factors associated with the a2-globulins (such as IRA) may be involved in the in vivo suppression of nonspecific immune responses during immunological (T-cell) activation. Interferon, which is induced by viruses and priming antigens (in sensitized cells), may function similarly, but this has not yet been established. In a number of other diseases, less well-defined serum factors can be detected, which produce either a cytotoxic or inhibitory effect on lymphocytes in vitro. These include multiple sclerosis (Stjernholm et al., 1970), hepatitis (Paronetto and Popper, 1970), lupus erythematosus, rheumatoid arthritis and rheumatic heart disease (Terasaki et al., 1970), tuberculosis (Knowles et al., 1968), chronic mucoid candidiasis (Canales et al., 1%9), syphilis (Levene et al., 1%9), and the state of immune activation following vaccination (Kreisler et al., 1970). The soluble products from various tumors can also inhibit the activation and division of lymphoid cells. By combining with specific antibody to form antigen-antibody complexes, these soluble products acquire the capacity to induce central immune tolerance (Sjogren et al., 1971, 1972). Although it is perhaps less widely recognized, other tumorassociated products can also inhibit lymphoid cells directly. For example, the ascites fluid from mice bearing JB-1 lymphoid tumors in the plateau stage of growth, contains inhibitors that slow the recurrent growth of this specific tumor (Bichel, 1972). This ascites fluid appears to contain both a GI inhibitor (10,000-50,000 mol wt) and a inhibitor ( 1 0 ~ 1 0 , 0 0 0mol wt) (Bichel, 1973). These factors also circulate in the blood, as determined by the slowing of recurrent tumor growth in parabiotic mice (Bichel, 1971). The best characterized inhibitor in this group has been obtained from the ascites fluid of a human ovarian carcinoma (Holmberg, 1968b) and constitutes an octapeptide (1900-2000 mol wt) (Holmberg, 1968a) that can decrease the in vitro division of multiple types of cells, including malignant murine lymphoblasts and HeLa cells (Holmberg, 1968~).In this latter cell line, it appears to inhibit the cell cycle only during S phase, an effect that can be counteracted by the addition of deoxyribonucleotides. For both the JB-1 and ovarian ascites tumors, it is likely but not proved that the factors originate from the tumor rather than the host. Their tumor origin is

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

401

clearer in the i n vitro systems, such as the lymphoid leukemia line, NC37. This line releases soluble factors that inhibit PHA-stimulated normal lymphocytes as well as the spontaneously dividing NC-37 cells themselves (Houck and Irasquin, 1973). This indicates that tumor products alone can produce nonspecific inhibition of normal and malignant lymphocytes. The relationship of these released products to the extracted chalones remains to be determined. Other workers have detected inhibitory factors in the serum of animals and humans bearing hepatomas (Sell et al., 1972), as well as various other lymphoid and nonlymphoid tumors (Trubowitz et al., 1966; Silk, 1967; Scheurlen et al., 1968; Gatti, 1971; Langer et al., 1971; Whittaker et al., 1971). The nature of these factors and their host or tumor origin remain largely unresolved.

B. POTENTIAL USEFULNESS As CHEMOTHERAPUTIC AGENTS The value of these biological agents lies in their potential ability to modulate cell division in a tissue or function-specific manner. However, the basis for these specificities and for the variable effects of lymphoid chalones and the low molecular weight inhibitors on hyperplastic (autoimmune) and malignant proliferation is not well understood. Tumors that remain susceptible to growth inhibition may retain some regulatory pathways that are missing in those that do not. This remains a fruitful area for investigation. With the availability of single-donor, HL-A-matched transfusions for the lymphohematopoietic support of immunosuppressed patients, the problems of therapeutic ratio for abnormal vs. normal lymphoid cells are diminished, allowing the administration of substantially larger doses of inhibitors, which generally seem to be required to reduce DNA synthesis in malignant cells (Houck and Irasquin, 1973). In addition, as suggested by Houck, the possibility exists for in vivo synchronization of malignant lymphoid cells using these biological inhibitors, followed by the administration of conventional chemotheraputic agents during the rebound from mitotic arrest. In planning these types of studies, it may be of value to consider the possibility that the ideal candidates for the control of malignant cell division or immune hyperresponsiveness may be the low molecular weight inhibitors, because these appear to (1) have an equal or preferential effect on unstimulated and precursor cells and (2) affect cell division directly. The antiactivators, on the other hand, may be at a disadvantage in attempts to control already initiated responses, because of their apparent propensity to spare the ongoing specific response.

402

DAVID F. RANNEY

Thus, the choice of inhibitor, the timing of its administration relative to the course of disease, its specificity, and the specific metabolic defects in the target cells will all be important factors to investigate. Hopefully further studies will prove these agents to be of clinical value and will elucidate the basic mechanisms that regulate lymphocyte division. Successful studies will be contingent on obtaining highly purified fractions of these inhibitors.

X. Conclusion In this chapter we have reviewed the major biologically derived transmitters of negative regulatory information affecting lymphoid cells, both those extracted from and released by lymphoid and nonlymphoid tissues. Because recent studies have fairly well established that lymphocyte stimulation both suppresses and induces the release of

TABLE I11 SUMMARY OF TEMPORAL RELEASEOF

FACTORS REGULATING IMMUNE PROLIFERATION RESPONSE

Factors released"

AND

Status of the immune system

1. Antiproliferative factors (-) a. Low molecular weight lymphoid inhibitor b. Macrophage factor

Resting

2. Proliferative factors (+) a. Blastogenic factor and others

Stimulated

3. Antiactivators (-) a. Nonspecific suppressor cell activity b. Immunoregulatory a-globulin and interferon

Proliferating

4. Reappearance of antiproliferative factors

Decreased proliferation. Onset of secretory and effector responses.

as in 1. (-)

5. Specific regulators of the immune response (-) a. Antibody b. Specific T-cell suppressor activity "

Cessation of the secretory response.

The direction of the effect is indicated in parentheses.

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

403

various inhibitors, a model has been proposed that attempts to accommodate not only the particular experimental results but also the available information concerning the temporal release of these inhibitors, the propensity of particular ones to spare the specific immune response in vivo, and the decrease in susceptibility of lymphoid target to inhibitors, which appears to occur following activation. The temporal release of these factors as a function of immune activation is summarized in Table 111. The basic concept of continuous negative regulation of proliferation during the resting state and withdrawal of this control following stimulation, with the concomitant appearance of antiactivators (which prevent spurious responses by the division-vulnerable, residual lymphoid clones), appears to be consistent with the majority of the available data. The release of nonspecific and specific inhibitors during states of chronic activation, in cases of lymphoid and nonlymphoid malignancy can be seen to disrupt this normal balance, facilitating the escape of nonlymphoid tumors (and perhaps also lymphoid tumors) from immune surveillance and leading to a deficiency in various immune responses. The opposite imbalance may potentially facilitate maternal-fetal rejection, the development of autoimmune disease, and the hyperplastic or malignant proliferation of lymphoid cells. The compartmentalization of production of the low molecular weight inhibitors, which includes all lymphoid organs except the thymus, may be developmentally and functionally significant. The low and high molecular weight inhibitors are biochemically and functionally distinct factors and do not appear to represent polymers or subunits of one another. Their usefulness as specific chemotheraputic agents and the elucidation of their mechanisms of action will depend on their being obtained in a highly purified form. Hopefully this will be possible within the foreseeable future. ACKNOWLEDGMENTS The author's research presented in this review has been supported in part by a grant from the National Cancer Institute (CA 15673). I would like to thank my colleagues and collaborators who have participated in various aspects of this research, and the many investigators who have kindly contributed their manuscripts in press, as well as valuable discussions which have allowed recent material to be incorporated in this review.

REFERENCES Andersson, B., and Blomgren, H. (1971). Cell. Immunol. 2, 411. Andersson, J., Sjiiherg, O., and Miiller, G. (1972). Transplant. Rev. 11, 131. Baker, P. J., Stashak, P . W., Amsbaugh, D. F., Prescott, B., and Barth, R . F. (1970). J . Immunol. 105, 1581.

404

DAVID F. RANNEY

Barthold, D. R., Stashak, P. W., Amsbaugh, D. F., Prescott, B., and Baker, P. J. (1974). J. Immunol. 112, 1042. Bender, M. A., and Prescott, D. M. (1962). E z p . Cell Res. 27, 221. Bianco, C., Patrick, R., and Nussenzweig, V. (1970). J. E z p . Med. 132, 702. Bichel, P. (1971). Nature (London) 231,449. Bichel, P. (1972). Eur. J. Cancer 8 , 167. Bichel, P. (1973). Eur. J. Cancer 9, 133. Blomgren, H.,Strandvr, H., and Cantrll, K. (1974). Srand. J . Immunol. 3, 697. Britton, S., and Miiller, G. (1968). J. Immunol. 100, 1326. Bullough, W. S., and Laurence, E. B. (1964). E z p . Cell Res. 33, 176. Bullough, W. S., and Laurence, E. B. (1970). Eur. J . Cancer 6, 525. Burgus, R., Dunn, T. F., Desiderio, D., Vale, W., and Guillemin, R. (1969). C . R. Acad. Sci., Ser. D 269, 226. Calderon, J., Williams, R. T., and Unanue, E. R. (1974). Proc. N at. Acad. Sci. U.S. 71, 4273. Caldwell, J. L., Goeken, N . E., Severson, C. D., and Thompson, J. D. (1974). Proc. Int. Symp. Alpha-Fetoprotein, Saint-Paulde-Vence, France p. 445. Canales, L., Middlemas, R. T., Louro, J. M., and South, M. A. (1969). Lancet ii, 567. Cantell, K. (1973). In “Interferons and Interferon Inducers“ (N. B. Finter, ed.), p. 6. Amer. Elsevier, New York. Cantor, H., Asofsky, R., and Talal, N. (1970). J. Exp. Med. 131, 223. Carpenter, C., Phillips, S., Boylston, A., and Merrill, J. (1971a). Transplant. Proc. 3, 929. Carpenter, C., Boylston, A., and Merrill, J. (1971b). Cell. Immunol. 2, 425. Cashel, M.,and Gallant, J. (1974). I n “The Ribosome” (M. Nomura, A. Tissieres and P. Lengyel, eds.), p. 733 Cold Spring Harbor Lab., Cold Spring Harbor, New York. Cerottini, J. C., Brunner, K. T., Lindahl, P., and Gresser, I. (1973). Nature (London), New Biol. 242, 152. Chung, A., and Hufnagel, C. (1973). Nat . Cancer Inst., Monogr. 38, 131. Cleaver, J. E. (1967). I n “Thymidine Metabolism and Cell Kinetics“ (A. Neuberger and E. L. Tatum, eds.), p. 93. Wiley, New York. Cooperband, S. R., Badger, A. M., Davis, R. C., Schmid, K., and Mannick, J. A. (1972). J. Immunol. 109, 154. Coyne, J. A., Remold, H. G., RoseLberg, S. A,, and David, J. R. (1973). J. Immunol.

110,1630.

Danielson, J. R., and Van Alten, P. J. (1974). Prog. Exp. Tumor Res. 19, 194. Davis, R. C., Cooperband, S. R., and Mannick, J. A. (1971). J. Zmmunol. 106, 755. DeRubertis, F. R., Zenser, T. V., Adler, W. H., and Hudson, T. (1974). J. Immunol. 113, 151. DeVries, M. J., and Hijmans, W. (1967). Immunology 12, 179. Dosch, H. M., Havemann, K., Malchow, H.: Sodoman, C. D., and Schmidt, M. (1971). In “The Role of Lymphocytes and Macrophages in the Immunological Response” (D. C. Dumonde, ed.), p. 62. Springer-Verlag, Berlin and N e w York. Dukor, P., Bianco, C., and Nussenzweig, V. (1970). Proc. Nat. Acad. Sci. U S . 6 7 , 991. Elkins, W. L. (1972). Progr. Allergy 15, 78. Epstein, L. B., Kreth, H. W., and Herzenberg, L. A. (1974). Proc. Leucocyte Cult. Conf.. 8th, Uniu. Uppsala, 1973 pp. 169-174. Evans, M. M., Williamson, W. G., and Irvine, W. J. (1968). Clin. Exp. Immunol. 3, 375. Feldman, M.,and Palmer, J. (1971). Immunology 21, 685. Florentin, I., Kiger, N., and Math6, G. (1973). Eur. J. Immunol. 3, 624. Folch, H., and Waksman, B. H. (1974a). J. Immunol. 113, 127.

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

405

Folch, H., and Waksman, B. H. (1974b). J. Zmmunol. 113, 140. Friedman, R. M., and Cooper, H. L. (1967). Proc. SOC. Exp. Biol. Med. 125, 901. Garcia-Giralt, E. (1973). Nut. Cancer. Znst., Monogr. 38, 123. Garcia-Giralt, E., and Macieira-Coelho, A. (1974). Proc. Leucocyte Cult. Conf., 8th, Univ. Uppsala, 1973 pp. 457-463. Garcia-Giralt, E., Lasalvia, E., Florentin, I., and Mathd, G. (1970). Rev. Etud. Clin. Biol. 15, 1012. Garcia-Giralt, E., Morales, V., Lasalvia, E., and Mathd, G. (1972). J. Zmmunol. 109, 878. Garcia-Giralt, E., Rella, W., Morales, V. H., Diaz-Rubio, E., and Richand, F. (1973a). Nut. Cancer Znst., Monogr. 38, 123-129. Garcia-Giralt, E., Morales, V., Bizzini, B., and Lasalvia, E. (1973b). Cell. Tissue Kinet. 6, 567. Gatti, R. A. (1971). Lancet i, 1351. Gazdar, A. F., Beitzel, W., and Talal, N. (1971). Clin. Exp. Zmmunol. 8 , 501. Gelfand, M. C., and Steinberg, A. D. (1973). J . Zmmunol. 110, 1652. Gershon, R. K. (1974). I n “Contemporary Topics in Immunobiology” (M. D. Cooper and N . L. Warner, eds.), pp. 1 4 0 . Plenum. New York. Gershon, R. K., and Kondo, K. (1971a). Immunology 21, 903. Gershon, R. K., and Kondo, K. (1971b). J. Zmmunol. 106, 1524. Gershon, R. K., Gery, I., and Waksman, B. (1974). J . Zmmunol. 112, 215. Gery, I., and Waksman, B. H. (1972). J. Exp. Med. 136, 143. Gilman, A. G. (1970). Proc. Nut. Acad. Sci. U S . 67, 1970. Gisler, R. H., Lindahl, P., and Gresser, I. (1973). Abstr., Leukocyte CuZt. Conf., 8th, liniv. Uppsala No. 73. Gitlin, D., and Boesman, M. (1966). J. Clin. Invest. 45, 1826. Glaser, M., and Herberman, R. B. (1974). J. Nut. Cancer Znst. 5 3 , 1767. Glaser, M., and Nelken, D. (1972). Proc. Soc. Exp. Biol. Med. 140, 996. Glaser, M., Ofek, I., and Nelken, D. (1972). Immunology 23, 205. Glaser, M., Nelken, D., Ofek, I., Berger-Rabinowitz, S., and Ginsburg, I. (1973). J. Znfec. Dis. 127, 303. Glasgow, A. H., Cooperband, S. R., Schmid, K., and Mannick, J. A. (1972). J. CZin. Invest. 51, 6, 36a. Gresser, I., Brouty-Boy&, D., Thomas, M-T., and Macieira-Coelho, A. (1970). Proc. Nut. Acad. Sci. U S . 66, 1052. Guillemin, R., Clayton, G. W., Lipscomb, H. S., and Smith, J. D. (1959). J . Lab. Clin. Med. 53, 830. Haber, J., Rosenau, W., and Goldberg, M. (1972). Nature (London), New Biol. 2 3 8 , 61. Hadden, J. W., Hadden, E. M., Haddox, M. K., and Goldberg, N. D. (1972). Proc. Nut. Acad. Sci. U S . 69, 3024. Hand, T., Ceglowski, W., Damrongsak, D., and Friedman, H. (1970). J . Zmmunol. 105, 442. Harris, G. (1965). Immunology 9, 529. Hauschka, P., Everhart, L., and Rubin, R. (1972). Proc. Nut. Acad. Sci. U.S. 69, 3542. Havemann, K., and Burger, S. (1971). Eur. J . Zmmunol. 1, 285. Hilleman, M. R. (1969). Science 164, 506. Ho, M. (1973). In “Interferons and Interferon Inducers” (N. B. Finter. ed.), p. 94. Amer. Elsevier, New York. Holmberg, B. (1968a). Eur. J. Cancer 4, 263. Holmberg, B. (1968b). Eur. J. Cancer 4, 271. Holmberg, B. (1968~).Eur. J. Cancer 4,475.

406

DAVID F. RANNE’Y

Houck, J., and Irasquin, H. (1973). Nat. Cancer Znst., Monogr. 38, 117. Houck, J., Irasquin, H., and Leikin, S. (1971). Science 173, 1139. Houck, J., Attallah, A., and Lilly, J. (1973). Nature (London) 245, 148. Joklik, W. K., and Merigan, T. C. (1%6). Proc. Nat. Acad. Sci. U.S. 56, 558. Jones, J., Paraskova-Tchernozenska, E., and Moorhead, J. F. (1970). Lancet i, 654. Kamrin, B. B. (1959).Proc. Soc. Exp. Biol. Med. 100, 58. Katz, D. H., and Benacerraf, B. (1972). Advan. Zmmunol. 15, 1. Kerbel, R. S., and Eidinger, D. (1972). Eur. J . Zmmunol. 2, 114. Kiger, N. (1971). Rev. Enr. Etud. Clin. Biol. 16, 566. Kiger, N., Florntin, I., and Math&, G. (1972). Transplantation 14, 448. Kiger, N., Florentin, I., and Math& G. (1973a). Transplantation 16, 393. Kiger, N., Florentin, I., and MathC, G. (1973b). Nut. CuncerZnst., Monogr. 38, 135. Kincade, P. W., Lawton, A. R., and Cooper, M. D. (1971). J. Zmmunol. 106, 1421. Knowles, M., Hughes, D., Caspary, E. A., and Field, E. J. (1968). Lancet ii, 1207. Kobayashi, S., Yasui, O., and Masuzumi, M. (1969). Proc. Soc. Exp. B i d . Med. 131,487. Kono, Y., and Ho, M. (1965). Virology 25, 163. Kreisler, M. J., Hirata, A. A., and Terasaki, P. I. (1970). Transplantation 10, 411. Lambert, P. H., and Dixon, F. J. (1968).J. Exp. Med. 127, 507. Langer, A., Pawinska-Proniewska, M., Glinski, W., and Maj, S. (1971). Brit. J. Dermatol. 85, 7. Lasalvia, E., Garcia-Giralt, E., and Macieira-Coelho, A. (1970). Rev. Eur. Etud. Clin. B i d . 15, 789. Levene, G. M., Turk, J. L., Wright, D. J. M., and Gimble, A. G. S. (1969). Lancet ii, 246. Leventhal, B. G., and Talal, N. (1970). J . Zmmunol. 104, 918. McFarlin, D. E., and Oppenheim, J. J. (1969). J. Zmmunol. 103, 1212. McIntyre, 0. R., Cornwell, G. G., and Smith, K. A. (1974). Proc. Leucocyte Cult. Conf., Bth, Univ. Uppsala, 1973 pp. 101-109. MacManus, J. P., and Whitfield, J. F. (1974). Prostaglandins 6, 475. MacManus, J. P., Whitfield, J. F., and Rixon, R. H. (1974a). In “Cyclic AMP, Cell Growth and the Immune Response” (W. Braun, L. Lichtenstein, and C . W. Parker, eds.), pp. 302-316. Springer-Verlag, Berlin and New York. MacManus, J. P., Whitfield, J. F., Boynton, A. L., and Rixon, R. H. (1974b). In “Advances in Cyclic Nucleotide Research’ (P. Greengard and G. A. Robison, eds.), VoI. 5, pp. 719-734. Raven, New York. Mannick, J. A., and Schmid, K. (1967). Transplantation 5 , 1231. Marcus, P. I., and Salh, J. M. (1966). Virology 30, 502. Mellors, R. C. (1966). J . Exp. Med. 123, 1025. Menzoian, J. O., Glasgow, A., Cooperband, S., Schmid, K., Saporoschetz, I., and Mannick, J. A. (1973). Transplant. Proc. 5 , 141. Menzoian, J. O., Glasgow, A. H., Nimberg, R. D., Cooperband, S. R., Schmid, K., Saporoschetz, I., and Mannick, J. A. (1974). J. Zmmunol. 113, 266. Merigan, T. C. (1973). I n “Interferons and Interferon Inducers” (N. B. Finter, ed.), pp. 45-72. Amer. Elsevier, New York. Miller, H. C., and Esselman, W. J. (1975). J. Zmmunol. (in press). Moller, G. (1971). J . Zmmunol. 106, 1566. Moorhead, J. F., Paraskova-Tchernozenska, E., Pirrie, A. J., and Hayes, C . (1969). Nature (London) 224, 1207. Morris, W. R., and Fisher, G. H. (1963). Biochim. Biophys. Acta 68, 85.

BIOLOGICAL INHIBITORS OF LYMPHOCYTE DIVISION

407

Mowbray. J. F. (1963). Trransplantation 1, 15. Occhino, J. C., Glasgow, A. H., Cooperband, S. R., Mannick, J. A., and Schmid, K. (1973). J . Immunol. 110, 685. Olding, L., and Oldstone, M. (1974). Nature (London) 249, 161. Papamichail, M.,Holborow, E. J., and Keith, H. I. (1972). Lancrt ii, 64. Parker, C. W. (1974). I n “Cyclic AMP, Cell Growth and the Immune Response” (W. Braun, L. Lichtenstein, and C. W. Parker, eds.), pp. 3 5 4 4 . Springer-Verlag, Berlin and New York. Parker, C. W., Sullivan, T., and Wadner, H. J. (1974). In “Advances in Cyclic Nucleotide Research’” (P. Greengard and G. A. Robison, eds.), Vol. 4, pp. 1-79. Raven, New York. Parmely, M. J., and Thompson, J. S. (1974). Proc. Int. Symp. Alpha-Fetoprotein, SaintPaulde-Vencr, France p. 467. Parnetto, F., and Popper, H. (1970). New Engl. J. Med. 283, 277. Peavy, D. L., and Pierre, C. W. (1974). Fed. Proc., Fed. Amrr. SOC.Exp. Biol. 33, 721. Perkins, E. H., and Makinodan, 1. (1965). J . Imrnunol. 94, 765. Phillips, S., Carpenter, C., and Lane, P. (1975). Ann. NY. Acad. Sci. 249, 236. Prendergast, R. A., and Suzuki, M. (1970). Nature (London) 227, 277. Prendergast, R. A,, Cole, G. A., and Henney, C. S. (1974). Ann. NY. Acad. Sci. 234, 7. Ranney, D. F. (1974). In “The Cell Surface: Chemical and Immunological Approaches” (B. D. Kahan and R. A. Reisfeld, eds.), pp. 239-241, Plenum, New York. Ranney, D. F. and Oppenheim, J. J. (1972). Abstr. Leucocyte Cult. Conf., 7th, Univ. Laval, Quebec No. 19. Ranney, D. F., and Oppenheim, J. J. (1973a). Proc. Leucocyte Cult. Conf., 7th, Univ. Laval, Quebec, 1972 p. 173. Ranney, D. F., and Oppenheim, J . J. (197313). Fed. Proc., Fed. Amer. Soc. Exp. Biol. 32, 4276. Ranney, D. F., and Quattrone, A. J. (1974). Fed. Proc., Fed. Amer. Soc. Exp. Biol. 33, 2896. Ranney, D. F., Quattrone, A. J., and Oppenheim, J. J. (1973). Abstr. Leucocyte Cult. Con$, 8th, Univ. Uppsala No. 157. Rich, R. R., and Pierce, C. W. (1973). J . Exp. Mrd. 137, 649. Rich, R. R., and Pierce, C. W. (1974). J. Immunol. 112, 1360. Riggio. R. R., Schwartz, G. H., Stenzel, K. H., and Rubin, A. L. (1968). Lancet i, 1218. Sasaki, M. S., and Norman, A. (1966). Nature (London) 210,913. Scheurlen, R. G., Pappas, A., and Ludwig, T. (1968). Klin. Wochenschr. 46,483. Sell, S., Jalowayski, I., Bellone, C., and Wepsic, H. T. (1972). Cancer Res. 32, 1184. Shacks, S. J., and Granger, G. A. (1971). J . Reticuloendothel. SOC.10, 28. Shirai, T.,and Mellors, R. C. (1971). Proc. Nut. Acad. Sci. U S . 68, 1412. Silk, M. (1967). Cancer (Philadelphia) 20, 2088. Sjoberg, 0.(1972). Clin. Exp. Immunol. 12,365. Sjogren, H. O., Hellstrom, I., Bansal, S. C., and Hellstrom, K. E. (1971). Proc. Nut. Acad. Sci. U.S. 68, 1372. Sjiigren, H. O., Hellstrom, I., Bansal, S. C., Warner, G. A., and Hellstrom, K. E. (1972). Int. J. Cancer 9, 274. Smith, J. W., and Parker, C. W. (1971). J. Clin. Invest. 5 0 , 422. Smith, T. J., and Wagner, R. R. (1967). J. Exp. Med. 125, 559. Steinberg, A. D., Pincus, T., and Talal, N. (1%9). J. Imrnunol. 102, 788.

408

DAVID F. RANNEY

Steinberg, A. D., Law, L. W., and Talal, N. (1970).Arthritis Rheum. 1 3 , 369. Stjernholm, R. L., Wheelock, E. F., and Van Den Noort, S. (1970). J. Reticuloendothel. Soc. 8,334. Stobo, J. D., and Paul, W. E. (1972). Cell. Immunol. 4, 367. Stobo, J. D., Talal, N., and Paul, W. E. (1972).J . Irnmunol. 109,692. Sutton, R. N., and Tyrrell, D. A. (1961).Brit. J . Exp. Pathol. 4 2 , 99. Talal, N., and Steinberg, A. D. (1974). Curr. Top. Microbiol. Immunol. 64, 79. Terasaki, P. I., Mottironi, V. D., and Barnett, E. V. (1970).New Engl. J . Med. 283, 724. Trubowitz, S., Masek, B., and DelRoasario, A. (1966). Cancer (Philadelphia) 1 9 , 2019. Uhr, J. W., and Moller, G. (1968).Advan. Immunol. 8 , 8 1 . Van Alten, P. J., and Danielson, J. R. (1972). Proc. Leucocyte Cult. Conf., 6th, Univ. of Washington p. 455. Veit, B. C., and Michael, J. G. (1972).Nature (London), New Biol. 235, 238. Waldman, S. R., and Gottlieb, A. A. (1973). Cell Immunol. 9, 142. Wallen, W. C., Dean, J. H., and Lucas, D. 0. (1973). Cell. Immunol. 6 , 110. Weber, W. T. (1%7). Exp. Cell Res. 46, 464. Weber, W. T. (1970).J. Reticuloendothel. Soc. 8 , 37. Weber, W. T. (1973). Fed. Proc., Fed. Amer. Soc. Exp. Biol. 32, 956. Wheelock, E. F. (1965). Science 1 4 9 , 310. Whitfield, J. F., MacManus, J. P., and Rixon, R. H. (1970). Proc. Soc. Exp. Biol. Med. 1 3 4 , 1170. Whitfield, J. F., MacManus, J. P., Franks, D. J., Gillan, D. J., and Youdale, T. (1971). Proc. Soc. Exp. Biol. Med. 1 3 7 , 453. Whittaker, M. G., Rees, K., and Clark, C. G. (1971). Lancet i, 892. Whittle, E. D. (1966). Biochim. Biophys. Acta 1 1 4 , 44. Willenborg, D. O., and Prendergast, R. A. (1974).J. Exp. Med. 1 3 9 , 820. Wolstencroft, R. A . , Matthew, M., Oates, C., Maini, R. N., and Dumonde, D. C. (1971). In “The Role of Lymphocytes and Macrophages in the Immunological Response” (D. C. Dumonde, ed.), p. 28. Springer-Verlag, Berlin and New New York.

SUBJECT INDEX A 2-Acetamido-Snitrothiazole, in T. cruzi therapy, 21 Acetophenetidin, sex factors in effects of, 196197 Acetylcholine, amphetamine effects on, 331-332 Alcohol, see Ethyl alcohol Alcohol dehydrogenase, in drug metabolism studies, 54-55 Alkaloids, sex factors in effects of, 228 Alkylating agents, sex factors in effects of, 226-227 Allergic drug reactions, sex differences in, 232 Amantadine, a s L-dopa adjuvant, 287 Amino acids, cerebral, amphetamine effects on, 347-348 y-Aminobutyric acid, amphetamine effects on, 332 7-Aminocephalosporanic acid, cepbalosporin relation to, 84 6-Aminopenicillanic acid, cephalosporin relation to, 84 Aminopyrine, sex factors in effects of, 195196 8-Aminoquinolines, in T. cruzi therapy, 1718 p-Aminosalicylic acid (PAS), sex factors in effects of, 220 Amphetamine, derivatives of, 306 Amphetamine-type psychostimulants, 305357 behavior changes from, 307-311 metabolic inhibitor pretreatment, 332339 EEG changes from, 307-311 effects on neurotransmitters, 312-332 Analgesics, sex factors in effects of, 182185, 195-198 Anesthetics, sex factors in effects of, 176179 409

Anthelminthics, sex factors in effects of, 224 Antibacterial compounds, sex factors in effects of, 215-220 Antibiotics sex factors in effects of, 215-218, 228 in T . cruzi therapy, 15-17 Antibodies, hapten affinity for, 71 Anticholinergic drugs, as L-dopa adjuvant, 286 Anticoagulants, sex factors in effects of, 2 13 Anticonvulsants, sex factors in effects of, 198 Antifungal compounds, sex factors in effects of, 222-223 Antihistamines, sex factors in effects of, 2 11-212 Anti-infective compounds, sex factors in effects of, 215-230 Anti-inflammatory compounds, sex factors in effects of, 211 Antimalarials enzyme metabolism of, 5 6 5 7 sex factors in effects of, 223 Antineoplastic agents, sex factors in effects of, 225-230 Antiparasitic compounds, sex factors in effects of, 22%224 Antiparkinson drugs, sex factors in effects of, 201 Antiprotozoals, sex factors in effects of, 223-224 Antipyrine, sex factors in effects of, 197 Antitussive compounds, sex factors in effects of, 215 Antiviral compounds, sex factors in effects of, 224-225 Arsenicals sex factors in therapy of, 218 in T. cruzi therapy, 18-19 Athetoid cerebral palsy, L-dopa treatment of, 286

410

SUBJECT INDEX

B Barbiturates, sex factors in effects of, 185195 Behavior, amphetamine effects on, 307311, 332-339 Bisquinaldines, in T. cruzi therapy, 18, 31 Blood dyscrasias, sex differences in, 231232 Brain, metabolism in, cerebral function and, 311-349 Butyrophenones, sex factors in effects of, 206

C Caffeine, sex factors in effects of, 199 Cannabinoids, sex factors in effects of, 206 Carbohydrate metabolism, amphetamine effects on, 341-345 Cardiac glycosides, sex factors in effects of, 209-210 Catechol-O-methyltransferase in catecholamine metabolism, 258 inhibitors of, as L-dopa adjuvants, 289 Catecholamines amphetamine effects on, 312-327 biosynthesis of, 254-257 degradation of, 257-259 Cefamandole, 85 antibacterial activity of, 95 Cefazolin, 85 antibacterial activity of, 95 pharmacology of, 13C131 Central nervous system depressants, sex factors in effects of, 176-198 Central nervous system stimulants, sex factors in effects of, 198-202 Cephacetriie, 85 antibaceriai activity of, 94 pharmacology of, 126-128 Cephalexin, 85 antibacterial activity of, 92-93 clinical uses of, 134, 136-141 metabolism of, 124, 144 pharmacology of, 121-123 toxicology of, 124 Cepbaloglycin, 85 antibacterial activity of, 93-94

clinical uses of, 134-135, 137, 141 metabolism of, 125-126 pharmacology of, 124-125 toxicology of, 126 Cephaloridine, 85 antibacterial activity of, 89-90 clinical uses of, 133-140, 142-144 metabolism of, 114-115 pharmacology of, 112-14 toxicology of, 11S116 Cephalosporins, 83-172 allergenicity to, 145-147 antibacterial activity of, 89-111 antibiotic combinations of, 105-106 bacterial resistance to, 96-106 cross-resistance, 97-98 development, 96-97 in bone and joint disease, 144-145 chemical properties of, 85-89 chemical structures of, 85 clinical aspects of, 132-145 dermatological use of, 141 effect on cell wall synthesis, 106-107 in endocarditis therapy, 143-144 hypersensitivity to, 145-147 lactamase effects on, 98-105 lysis by, 110 in meningitis therapy, 142-143 mode of action of, 107-111 morphological variants of, 109-110 in obstetrics and gynecology, 13%140 ophthalmological use of, 142 pediatric use of, 140-141 pharmacology of, 111-132 production of, 85-87 for respiratory infections, 135-137 spectrum of activity of, 89-96 structure-activity relationships of, 87-89 toxicology of, 111-132 uptake and cellular permeability of, 107109 for urinary infections, 132-135 for venereal infections, 137-139 Cephalothin, 85 antibacterial activity of, 90-91 clinical uses of, 133, 136, 139, 140, 142144 metabolism of, 118-119 pharmacology of, 116-118 toxicology of, 119-121

SUBJECT INDEX

Cephanone, antibacterial activity of, 131132 Cephapirin, 85 antibacterial activity of, 94-95 pharmacology of, 129-130 Cephradine, 85 antibacterial activity of, 94 pharmacology of, 128-129 Chagas’ disease. (See also Trypanosoma cruzi.) chemotherapy of, 1-81 Chemotherapy, sex factors in, 173-252 Chloramphenicol, sex factors in effects of, 2 16 Chlorzoxazone, sex factors in effects of, 207 Chymotrypsin, in drug metabolism, 59 Clioquinol, sex factors in effects of, 220 Coenzymes, amphetamine effects on, 346347 Colistin, sex factors in effects of, 216 Concanavalin A, in drug metabolism, 61, 66 Curare, sex factors in effects of, 208 Cyclic nucleotides, amphetamine effects on, 33941 Cycloserine, sex factors in effects of, 221222

D Defaulting, in drug intake, 230 Dermatology. cephalosporin use in, 141 Diazepines, sex factors in effects of, 205 Dibenzazepines, sex factors in effects of, 203-204 Dihydrofolate reductase, in drug metabolism, 76 Diols, sex factors in effects of, 204-205 Dispersion forces, in drug-enzyme metaholism, 60-69 Diuretics, sex factors in effects of, 214 L-Dopa adjuvants of, 286-293 adverse effects of, 2 7 5 2 7 9 catabolism of, 259-260 in extrapyramidal disease treatment, 253-304 long-term effects of, 280 mechanism of action nf, 2 6 5 2 6 9

41 1

metabolism of, 254-263 metabolites of, 2fjO-262 mode of administration of, 279-280 pharmacology of, 263-272 therapeutic effects of, 274-275 Dopa decarhoxylase, in L-dopa metabolism, 254 Dopa decarboxylase inhibitors, a s L-dopa adjuvants, 287-288 Dopamine metabolism of, regulation, 262-263 a s neurotransmitter, 263-264 Dopamine-D-hydroxylase inhibitors of, a s L-dopa adjuvants, 289 in L-dopa metabolism, 257 Dopamine receptor agonists, a s L-dopa adjuvants, 290 Drug design, enzymes in study of, 45-81 Drugs, sex factors in effects of, 173-252 Dystonia musculorum deformans, L-dopa treatment of. 285-286

E Electronic interactions, in drug-enzyme metabolism, 50, 6%70 Emetine and drrivatives, in T . cruzi therapy, 19 Emulsin, in drug metabolism studies, 5%54 Endocarditis, cephalosporin therapy of, 143-144 Enzymes in drug design studies, 45-81 examples, 52-75 ligand interactions in, 4%51 receptors in, 7 5 7 6 structure-activity relationships, 51-52 Ergot, sex factors in effects of, 197 Ethamhutol, sex factors in effects of, 221 Ethionamide, sex factors in effects of, 221 Ethyl alcohol, sex factors in effects of, 180182 Extrapyramidal disease, L-dopa in therapy of, 253-304

F a-Fetoprotein (AFP), as lymphocyte inhibitor, 390-391

412

SUBJECT INDEX

G Glucose metabolism, amphetamine effects on, 341-345 Glyceryl guaiacolate, sex factors in effects of, 208-209 Gonorrhea cephalosporin therapy of, 137-138 sex factors in therapy of, 217-218 Griseofulvin, sex factors in effects of, 223 Gynecology, cephalosporin use in, 139-141

H Histamine, amphetamine effects on, 332 Huntington’s chorea, dopamine metabolism in, 284-285 Hydrophobic reactions, in drug-enzyme studies, 49, 57-60 Hydroxytryptamine precursors and inhibitors, as L-dopa adjuvants, 290-291 Hypoglycemic agents, sex factors in effects of, 212 Hypothalamic tripeptides, as L-dopa adjuvants, 291-292

I Idoxuridine, sex factors in effects of, 225 Immunoregulatory a-globulin, as lymphocyte inhibitor, 389-390 Immunosuppressives, sex factors in effects of, 225-226 Interferon, as lymphocyte inhibitor, 391-

392 Isoniazid, sex factors in effects of, 221

L P-Lactamases effect on cephalosporins, 98-105 substrate profiles of, 100 Laxatives, sex factors in effects of, 214-215 Ligands, enzyme interaction with, in drug design, 48-51 Lipids, cerebral, amphetamine effects on,

347-348

Lithium as L-dopa adjuvant, 292 sex factors in effects of, 205 Local anesthetics, sex factors in effects of,

207-208 Locomotor activity, amphetamine effects on, 332-336 Lorentz-Lorenz equation, 61 Lymphocyte chalones effects of, 367-368 isolation and characterization of, 366-367 mechanism of action of, 368-369 specificity of, 369-370 Lymphogranuloma venerum, cephalosporin therapy of, 138 Lymphoid cell division biological inhibitors of, 35-08 assay of, 361-366 clinical aspects of, 397from lymphoid tissue, 370-375 macrophage factors, 384-385 mechanisms of action, 393-397

M Macrophage factors, as lymphocyte division regulators, 384-385 Manganese poisoning, L-dopa treatment of,

283 Meningitis, cephalosporin therapy of, 142-

143 Metabolic antagonists, sex factors in effects of, 227-228 L-Methionine S-adenosyltransferase, in drug metabolism, 59 3-Methyldopa, as L-dopa adjuvant, 289-290 Methylphenidate, sex factors in effects of,

201-202 Monoamine oxidase in catecholamine metabolism, 257-258 inhibitors of, as L-dopa adjuvants, 289 Myotropics, sex factors in effects of, 208-

209

N Nalidixic acid, sex factors in effects of, 220 Narcotic analgesics, sex factors in effects of, 182-185

413

SUBJECT INDEX

Neurotoxins, as dopa metabolites, 262 Neurotransmitters, amphetamine effects on, 312-332 Nigrostriatal pathway, L-dopa and, 264-265 Niridazole, in T. cruzi therapy, 22 Nitrofuran derivatives, in T. cruzi therapy,

20-21, 31-32 Nitrofurans, sex factors in effects of, 220 Nitroimidazole derivatives, in T. cruzi therapy, 21 Nocebo effect, sex differences in, 230 Nongonococcal urethritis (N.G.U.), cephalosporin therapy of, 138 Nucleic acids, amphetamine effects on, 347

0 Obstetrics. cephalosporin use in, 13%141 Olivoponto cerebellar degeneration, L-dopa treatment of, 282-283 Ophthalmia neouatorum, cephalosporin therapy of, 138 Ophthalmology, cephalosporin use in, 142

Phosphates, high-energy, amphetamine effects on, 345-346 Picrotoxin, sex factors in effects of, 199 Piperazine derivatives, in T. cruzi therapy,

22 Placebos, sex factors in effects of, 230 Polarizability, in drug-enzyme metabolism,

6049 Polyenes, sex factors in effects of, 223 Porfiromycin in T. cruzi therapy, 17 Postoperative complications, sex factors in,

178-179 Primaquine, in T. cruzi therapy, 30 Progressive supranuclear palsy, L-dopa treatment of, 282 Proteins, cerebral, amphetamine effects on, 347-348 Prothionamide, sex factors in effects of,

221 Psychotropic agents, sex factors in effects of, 202-207 Pyridoxine, as L-dopa adjuvant, 292-293

Q P Papaverine, sex factors in effects of, 208 Parkinsonism biochemistry of, 273 disorders resembling, 280-283 L-dopa in therapy of, 253-304 drug-induced, 281-282 pathogenesis of, 273-274 pathology of, 272-273 Parkinsonism-dementia of Guam, L-dopa treatment of, 281 Pediatrics, cephalosporin use in, 14&141 Penicillins, sex factors in effects of, 21%

2 16 Pharmacology, sex factors in, 173-252 Phenanthrene carbinols, enzyme metabolism of, 55-56 Phenanthridinium compounds, in T. cruzi therapy, 19, 30-31 Phenothiazines, sex factors in effects of, 202-203 Phenylglucosides, enzyme metabolism of, 53-54

Quinacrine, sex factors in effects of, 224 Quinaldine derivatives, in T. cruzi therapy,

18

R Rauwolfia compounds, sex factors in effects of, 204 Respiratory tract infections, cephalospirin therapy of, 135-137 Rifampicin, sex factors in effects of, 2 1 6

217 Rubiflavin, in

T.cruzi therapy, 17

S Salicylates, sex factors in effects of, 195 Schistosomatocides, sex factors in effects of, 224 Sea star factor, as lymphocyte inhibitor,

392-393

414

SUBJECT INDEX

Serotonin, amphetamine effects on, 327331 Sex factors in pharmacology and chemotherapy, 173-252 of anticoagulants, 213 of antihistamines, 211-212 of anti-infective compounds, 215-230 of anti-inflammatory compounds, 211 of antineoplastic agents, 225-230 of antitussive compounds, 215 of cardiac glycosides, 209-211 of CNS depressants, 176-198 of CNS stimulants, 198-202 of hypoglycemic agents, 212 of laxatives, 214-215 of local anesthetics, 207-208 of myotropics, 208-209 psychotropic agents, 202-207 Shy-Drager syndrome, L-dopa treatment of, 283 Spirotrypan, in T. cruzi therapy, 30 Steric interactions, in drug-enzyme metaholism, 50-52, 69-70 Streptomycin, sex factors in effects of, 217, 222 Striatonigral degeneration, L-dopa treatment of, 281 Strychnine, sex factors in effects of, 198199 Stylomycin aminonucleoside, in T. cruzi therapy, 30 Sulfonamides, sex factors in therapy of, 218-220 Sympathomimetic agents, sex factors in effects of, 20Cb201 Syphilis, cephalosporin therapy of, 138-139

T Tetracyclines, sex factors in effects of, 217 Therapeutic index, in studies of enzymedrug metabolism, 77 Thiahendazole, in T. cruzi therapy, 23 Thioisonicotinic acid amide, in T . cruzi therapy, 23 Thiosemicarbazones, sex factors in effects of, 224-225 Thioxanthenes, sex factors in effects of, 2M

Triaminoquinazolines, in 7'. cruzi therapy, 23 Triazines, enzyme inhibition by, 62-65 Trichomonacides, sex factors in effects of, 223 Tricyclic antidepressants, as L-dopa adjuvants, 291 Trimethoprim, enzyme inhibition by, 76 Triphenylmethane dyes, in T. cruzi therapy, 23 Tris-(p-aminophenyl) carbonium chloride, in T. cruzi therapy, 31 Trypacidin, in T. cruzi therapy, 31 Trypanocides, sex factors in effects of, 223-224 Trypanosoma cruzi chronic infections of, 12 chemotherapy of, 1-81 cure criteria, 7-9 in vitro drug testing, 12-15 in vivo drug testing, 3-12 clinical trials, 34-39 culture forms of, 12-13 hosts of, 3 4 immunity to, 10-11, 36 inocula of, 5-6 life cycle of, 2-3 strains of, 4-5 tissue cultures of, 13-14 compounds active against, 15-24 2-acetamido-5-nitrothiazole, 21 8-aminoquinolines, 17-18 antibiotics, 15-17 arsenicals, 1%19 bisquinaldines, 18, 31 emetine derivatives, 19 mode of action of, 24-34 niridazole, 22 nitrofuran derivatives, 20-21, 31-32 phenanthridinium compounds, 19 piperazine derivatives, 22 thiabendazole, 23 thioisonicotinic acid amide, 23 tissue culture studies on, 28-32 triaminoquinazolines, 23 triphenylmethane dyes, 23 Tuberculostatic compounds, sex factors in effects of, 220-221 Tyrosine aminotransferase, in catecholamine metabolism, 258-259

415

SUBJECT INDEX

V

Tyrosine hydroxylase, in L-dopa metabolism, 2%

Venereal disease, cephalosporin therapy of,

137-139

U Urinary tract infections, therapy of, 132-135

A

5

8 6

C l

D 8 E 9 F O

G I

H 2 1 3 J 4

W cephalosporin Wilson’s disease, L-dopa treatment of, 284

This Page Intentionally Left Blank