ADVANCES IN
Pharmacology and Chemotherapy
VOLUME 13
ADVISORY BOARD
D. BOVET
J. F. DANIELLI
Zstituto Superiore di...
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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
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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.
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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
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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
. . . . .
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360 361 366 370 384 385 . . 388 . . 393 . . 397 . . 402 . . 403
. . . . . . . . . . . . . . . . . . . . . .
409
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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
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ADVANCES IN
Pharmacology and Chemotherapy
VOLUME 13
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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
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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 . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
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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
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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.
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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
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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
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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
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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).
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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
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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
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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
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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
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.
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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
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