sent problems in Southern Europe. In China and other parts of the Far East, eventual relief and rehabilitation efforts will have to deal not only with these diseases but also with filariasis, paragonimiasis, and clonorchiasis. . First attention must be given to the prevention of epidemic diseases such as typhus, cholera, typhoid, dysentery, influenza, measles, meningitis, and others. While it is the aim of U N R R A to turn back relief problems as soon as possible to newly constituted local governments, treatment of disease will have t o be carried out until local authorities are able to assume responsibility. Consequently, rehabilitation will involve problems in medical care as well as preventive medicine. In the financing of the relief program, the United States will be expected t o con-
tribute in proportion to ats resources, and a large proportion, of the medical supplies and equipment will probably come from our domestic production. Under these conditions, there "will b e need for amebicidal drugs, antimonials for t h e treatment of leishmaniasis a n d schistosomiasis, and anthelmintics for u s e in intestinal parasitisms. A n y new a n d more effective compounds which can be developed for these or other tropical parasitic diseases will undoubtedly find widespread application.
Literature Cited (1) Ameel, Am. J. Hyg., 19,279 (1034). (2) Brady, F. J., and Jones, Myrna, unpublished experimental work in the Zoology Laboratory, National Institute of Health., in cooperation with military authorities.
(3) Brady, Jones, and Newton, War Med,, 3,409(1943). (4) Brown, H. W., paper before Am. Soc. Trop. Med., Cincinnati, Ohio* Nov. 16, 1943. (5) Chopra and Sundar Rao, Indian J. Med. Res., 27, 549 (1939). (6) Figueroa Ortiz, Luis, personal communication. (7) Guiteras, Med. News, 48, 399 (1886). (8) Indian Med. Gaz., 78, 97 (1943). (9) Lynch, Kenneth, personal communication. (10) Mazza, Basso, and Basso, Univ. Buenos Aires, Mision Estudios Pat. Reg. Argentina (Jujuy), Publ. 61 (1942). (11) Miller and Wilbur, Naval Med. Bull.. 42, 108 (1944). (12) Strong, "Stitt's Diagnosis, Prevention and Treatment of Tropical Diseases", Philadelphia, Blakiston Co., 1942. (13) Wayson, Pub. Health Repts., 42, 3129 (1927). (14) Yokogawa, Wakisaka, and So. Acta Japoniea Med. Trop., 2, 23 (1940).
Experimental Methods for the Evaluation of Antiparasitic Agents H A M I L T O N H. A N D E R S O N Pharmacology Laboratory. University of California Medical School, San Francisco, Calif. T H E chance observation that a given chemical produces a desirable pharmacologic action has been demonstrated repeatedly to be the first step in a series of studies leading to t h e development of important chemotherapeutic agents. Properly presented, this first step m a y serve to stimulate the preparation of more effective chemically related compounds. There is no one group of scientific workers competent t o cover the entire field of research in chemotherapy. Actually all fields of science that can contribute to the problem should be included. Such endeavors require coordination. Properly integrated studies have led to real advances in the past and there is every hope that these advances will continue. The drug hazard must not exceed the disease hazard; this is the first principle of chemotherapy. F e w parasitic diseases of man pursue identical courses in animals. Similar, though not identical, infections in animals may serve as models and yield important information for future application of chemicals to man.
Parasitic Diseases I n man the parasitic diseases which present chemotherapeutic problems are essentially chronic in their clinical course. I t is not difficult to control the acute phase of such diseases as amebiasis and leishmaniasis, and with persistent, properly supervised therapy relapses can be eliminated, but only b y vigorous, specific treatment in the early stages of disease. Chronicity is not the only peculiarity of parasitic in-
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fections. I t is uncommon to find patients with clear-cut, classical disease entities. It is more common to find parasites invading a patient who already suffers from malnutrition, a deficiency disease, or other parasitic or bacterial invaders, or more commonly from & comibinatnon of these factors. T h e problem then resolves itself into one i n which w e have not a parasite in an uncontaminated environment readily exposed t o destruction, b u t rather a parasite whose tissues d o not differ markedly in their biologic requirements from those of man. T h e power to resist chemical and physical agents roughly may parallel or exceed man's ability to d o likewise. Parasitic disease differs in various parts of the world and i s probably conditioned by the diverse environments in which man finds himself. In the Philippines, China, and other tropical areas, amebiasis differs clinically in being more prone to liver abscess and more resistant t o therapy; patients i n such areas m a y be less tolerant to chemicals which are potentially harmful. I t is possible that pathogenic amebae differ in virulence, and small and large races h a v e been described. Actually massive infection or repeated exposure t o pathogenic amebae would seem to play a more important role in resistant cases. Leishmaniasis differs in response t o therapy also, not only geographically, but with respect t o t h e parts of the body involved, as well a s species o f animal infected. While s o m e parasitologists contend that infections caused b y L. donovani and L. infantum are identical, actually
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animals which harbor either strain differ in their response to the aromatic diamidines (1). L. infanlum-infected Syrian hamsters appear to be more resistant t o these agents t h a n do animals infected with L. donovani. Chung and his associates have presented considerable evidence (JO t o show that canine and human kala-azar in North China are the same disease. Soong and H o u (15) have found that experimental infections with the canine strain in Chinese hamsters (Cricetulus griseus) are resistant to antimonials which promptly clear hamsters infected with leishmania obtained from patients. T h e canine disease in North China involves chiefly the skin, whereas human infections are confined largely to the liver and spleen. Arrest of symptoms and signs or the absence of parasites in tissues or tissue fluids which lend themselves to clinical pathologic study should not lead investigators t o assume that "clinical cure" has been effected. This is one of the most difficult aspects of chemotherapeutic investigations and is further complicated by the natural tendency of some parasitic diseases t o become arrested spontaneously. The specific characteristics of individual parasites in man and other animals must be given primary consideration. Onchocerciasis is a filarial infection in which nodules are present in the subcutaneous tissues containing the adult worms commonly about the head, neck, and trunk of man. Thus far the only other mammalian hosts in which a parasite morphologically identical to Onchocerca volvulus have been
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reported are the eland, antelope, and cattle in West Africa where the disease in m a n is endemic. A similar parasite, O. gibsoni, affects cattle in Australia, Ceylon, and MaLaya (.9). Since no laboratory animal has been found as y e t to serve as a test subject, we must depend upon studies in dogs infected with Dimfilaria immitia for preliminary information, despite apparent biologic differences. Schistosomiasis is placed second only t o the intestinal nematode infections in the number of people affected in Africa, the Far East, and Tropical America. Trivalent antimonials have been used extensively, but because of the great numbers of patients mass therapy with agents which require many intravenous or intramuscular injections has not been entirely satisfactory. Ideally, an agent that can be administered by mouth or subcutaneously would offer a real advance. Lithium antimony thiomalate can be given subcutaneously and smaller concentrations of antimony are effective in this form. E x periences with Fuadin (sodium-antimony III bis-catechol-2,4 disulfonate, N . N . R . ) differ, but relapses occur not infrequently after its use. Potassium antimonyl tartrate or the sodium salt is believed t o be more efficient. Lee and Khaw (10) successfully e s t a b lished Schistosoma japonicum infections in rabbits. This infection killed control animals by the production of cirrhosis of the liver within 2 t o 3 months. Studies of the tissues of rabbits indicated t h a t as many as half the ova apparently died spontaneously. S. mansoni infections are less responsive t o specific therapy t h a n are infections by S. haematobium. In vitro tests suggest- a direct action of b o t h emetine and antimony against adult flukes. It is likely that current studies will yield results in the chemotherapy of schistosomiasis, since a satisfactory laboratory infection is available. Trypanosomiasis in Africa has been the subject of considerable investigation both in the laboratory and clinically for m a n y years. The results of these studies and their application t o the field control of
- mm '-$
Photomicrograph of Leishmania grown in culture
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sleeping sickness are well known. Less well known, perhaps, is the therapeutic problem that American trypanosomiasis, caused by Trypanosoma cruzi, presents. Despite repeated efforts with a great variety of chemicals, this disease remains in the class of incurables. Mice serve as suitable test subjects and inoculations into mice are 9 4 % successful. Kofoid, Wood, and McNeil (11) have followed the development of Trypanosoma cruzi in tissue cultures of heart muscle and believe that the cyclic stages comparable t o those observed in mammals can be seen with the aid of the microscope. Embryonic hearts of rats and mice were grown on slides and inoculated with trypanosomes. Multiplication of parasites occurred and macrophages, fibroblasts, and extracellular parasites could be seen easily. Mature trypanosome forms appeared on the fifth day, which agrees with the time required for infective stages t o enter tissues, multiply, and appear in the blood. S u c h a technique might well be utilized in experimental chemotherapy.
Animal Infections T h u s far the discussion has centered about human infections with parasites t h a t can be transferred to animals or to tissue cultures and thus produce experimental conditions suitable for laboratory studies. Amebiasis in macaques, m a n y of w h o m have natural infections, does not differ pathologically from the human disease, and because it is a chronic disease in monkeys, these animals provide most satisfactory subjects. Experimental hum a n infections transferred t o cats d o not give the symptom-complex nor the chronicity so common in human infections, but in dogs an extensive invasion of tissues m a y occur. D o g s with heartworm disease caused by Dirofilaria immitis offer the laboratory convenient test subjects for studies in filariasis. Unfortunately the adult filaria lodge in the heart cavities of dogs a n d not in the lymphatics as they do in human filariasis caused b y Wuchereria bancrofti and W. malayi. T h e absorption a n d distribution of a therapeutic agent for action against adult filaria lodged in the l y m phatics and against even biologically similar worms lodged in the heart may be quite different. For the study of anthelmintics i n animals, dogs naturally infected with ascarids are most suitable. As in monkey amebiasis, this is usually a long-standing infestation and pathologic conditions which simulate those in m a n are encountered. Egg counts can b e made before a n d after therapy, a n d also after treatment t h e number of worms passed can be compared with the number remaining in the animal's intestine, and thus give a quantitative measure of therapeutic activity.
Laboratory Studies
leishmaniasis m a y begin with test-tube cultures of pathogenic parasites, and in ascariasis pig ascarids m a y be used for in vitro tests to gain some impression*of the direct action of various agents Such studies by no means simulate the conditions under which drugs act in m a n or animals. Although drug action m a y be intended to b e direct, many agents are now believed t o act indirectly—that is, by stimulation of a favorable tissue response to parasitic infection. T h e vehicle or medium in which the drug is dissolved or suspended should be as much the concern of t h e chemist and the pharmacologist as the active principle itself. It may have an important bearing on the availability of the chemotherapeutic agent and in this manner affect its relative activity. The medium used must have an indifferent action on the host, so that competition of t h e drug with the vehicle for distribution and absorption by tissues or tissue fluids does not take place. It is important t o know the pathologic effects that m a y result from the administration of drugs in suitable vehicles and to know whether or not disease may interfere with detoxification or excretion.
Experimental Methods Trial and error methods and large-scale screening of great numbers of compounds without adequate plan have been used freely in experimental chemotherapy. A more rational approach is certain t o produce results. Such an approach,.in which the nutritional requirements of the tissues of parasites and man are determined beforehand, would be helpful. If differences in requirements could be detected, chemical modifications to poison rather than nourish the parasite might be employed. In the chemotherapy of bacterial infections where the poison is one of the sulfonamides which competes with t h e essential metabolite p-aminobenzoic acid, one finds a n analogous situation. Chagas (8) is conducting action potential studies in chick embryo cultures of Trypanosoma cruzi with this end in view. Another laboratory aid in detecting the requirements of parasites is the use of
Photomicrograph of Trypanosoma
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in
blood
Laboratory studies in amebiasis and
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Fibrous cellullar scirs in heart muscle of a rab bit receiving orally 5 mg. per kilogram of emetine hydrochloride every third day for three doses, totaling 15 mg. per kilogram. The animal died 1 0 days after the last dose. Note the resemblance to the pathologic picture in chronic stages of rheumatic fever. Reduced from a photomicrograph with a magnification of 175 diameters. Cour tesy, Chauncey D. Leake, J. Aim. Med. Assoc, 98, 195-98, (Jan. 1 9 3 2 ) .
the chemical laboratory to the clinic. Careful pharmacologic techniques should be employed to complete the study of new drugs, which includes controlled clinical trials. M o d e of Action
radioactive "tagged" elements in nutrient materials. Elements may then be fol lowed within the parasite and quantitative studies in infected animals might include observations on the course of these ele ments throughout the tissues of the in fected host. Such methods offer con siderable promise. Chick embryos may also be used for the study of drug effects on tissues. Because
A satisfactory program for the evaluation of drugs has been outlined by Leake {12). This has been followed in subsequent studies by the same author (2) in the chemotherapy of amebiasis, by Ander son et al. (2) in the evaluation of drugs proposed for the treatment of leprosy, and by Soong et al. in the chemotherapy of experimental leishmaniasis (14). A simi lar effort has been emphasized by Geiling
Uncertainty as to the mode of action of chemotherapeutic agents active against parasitic disease is the greatest concern of research workers. Such concern might appear to be academic without application to the urgent problems of parasitic disease; but when we consider the modes of action as outlined by Findlay (6) and Daniels (5) we have a number of possibilities: adsorption of the chemical by the parasite causing alteration of permeability of cell mem branes, interference with essential metabo lites, and injury or death of the parasite either by enzyme interference or by altera tion of reproduction. More than one chemical type may be required to enhance the activity of the compound chiefly re sponsible for therapeutic action. Findlay has postulated that after adsorption of a chemical at the parasite-solution interface, the nature of the interference with the
antiparasitic agents must be employed in greater concentration against essentially chronic infections than antibacterial drugs against acute infections, there is a smaller margin of safety. It is possible to study the effects of chemicals on epithelium, mesothelium, fibroblasts, and nerves. Arsenicals in various amounts, for ex ample, may be added to saline solution at about pH 6.7. Exposure can be main tained for 48 hours or longer. Physical changes and actual death of tissues have been observed by Hogue, who employed this method in a critical survey of com monly used amebicides (8). Chronic toxicity studies in animals must not be neglected, since it is possible that agents not acutely toxic may produce dis astrous chronic effects. Conversely, some agents such as nicotine, which have rela tively high acute toxicity and low chronic toxicity, may be used with comparative safety over extensive periods.
and Cannon (7) in their correlation of the clinical and experimental data, related to the toxicity of diethylene glycol. In their comments, Geiling and Cannon reiterate the dictum that there is no short cut from
metabolism of the parasite might depend upon multipoint groupings in the molecule within the acceptor groups in the parasite. Dobell and Laidlaw have been able to produce evidence to support Ehrlich's
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Photomicrograph οf lar val form οf Onchocerca volvulus. Moist film preparation made from cut section οf tu mor. Courtesy, Colonel Richard P. Strong M.C., U.S.A.
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hypothesis that chemicals, particularly dyes, act by virtue of their interference with the reproductive power of parasites. Emetine placed in cultures of B. histolytica apparently interferes with reproduction. Dale has intimated that phenylarsine oxides, known to act directly,areless useful per se than compounds from which they may be derived by intermediary action in the body (6). On the contrary, experience with cultures of E. histolytica indicates that, properly used, phenylarsine oxides may offer another adjunct to the chemotherapy of amebiasis. The uncertain action of antimony compounds against parasitic disease has been emphasized by Goodwin, as an added difficulty in the determination of mode of action (6). As indicated earlier, kala-azar in the Sudan does not respond well to therapy with antimony. Slibine oxide groupings may be the most active form against trypanosome and schistosome infections, but in leishmaniasis pentavalent compounds are more useful. After use of the latter, excretion of antimony is more rapid than after potassium antimonyl tartrate. Goodwin believes that rapidly excreted antimony may pass through the body unchanged. Whether indirect action by stimulation of phagocytosis is more important than direct toxic action on leishmania is not known. Current studies may give us the answers t o some of these vital questions. Drug Resistance Another problem of importance in chemotherapy is drug resistance. Any significant observations in this connection would influence the development of a hypothesis to explain mode of action. Reinvestigation of some of the phenomena of drug resistance has shown that a strain of trypanosomes made resistant to one member of a series of organicarsenicalsor antimonials also acquires resistance to other commonly used aromatic compounds of these metals. Strains resistant to metal-free derivatives such as Bayer 205 are not resistant to organic metallic compounds. It is believed that the phenomenon of drug resistance is due in some
cases to changes which occur in the cell membrane of trypanosomes themselves. Resistant strains do not absorb antimonials even when they are present in concentration lethal for nonresistant strains, owing presumably to frequent exposures to suitable concentrations. This acquired resistance apparently persists indefinitely. It might he well to reconsider the dosage and spacing of injections of drugs used against infections now believed to be drugresistant. In this connection, recent studies by Pak (13) are of interest. Pak has found that animals, such as rats and mice, given a dose equivalent to approximately one third of the lethal amount of an antimonial can, within 5 to 48 hours, be given a surely lethal dose of the same drug without harm. When smaller initial amounts of an antimonial are given, the animal is not usually able to survive the subsequent injection of a lethal amount. Perhaps we should reconsider our methods of administration of such compounds. As Warrington Yorke has indicated (6), drug resistance in trypanosomiasis has been extensively studied, and is known to exist in Babesia cants infections and in Plasmodium knowlesi infections of macaques, but little is known of this phenomenon in other infections. Drug resistance is an important factor in the therapy of parasitic disease. This factor might be minimized by using optimum dosage early in the course of chronic infections to avoid the production of drug-resistant strains. Alternative use of chemically unrelated compounds has proved effective against certain chronic parasitic diseases and may be a desirable approach to others. Summary In man the parasitic diseases which present chemotherapeutic problems are essentially chronic in their clinical course. It is not difficult to control the acute phase of such diseases as amebiasis and leishmaniasis, and with persistent, properly supervised therapy relapses can be eliminated, but only by vigorous, specific treatment in the early stages of the disease.
Amebiasis, helminthiasis, leishmaniasis, schistosomiasis, and trypanosomiasis may be given to or are found naturally in suitable experimental animals for laboratory study. This is not the case with onchocerciasis or filariasis, although heartworm infection in dogs and filarial infection in the Florida cotton rat offer convenient suggestive infections as a preliminary to studies in man. In vitro tests, where infected tissue slices from developing chick embryos are utilized, offer opportunities for the evaluation of new drugs which kill parasites directly or by interference with metabolism or reproduction without the intermediary action of the host's tissues. An ideal, arbitrary method of study of drug and vehicle toxicities, absorption, distribution, and excretion in various species of normal and infected animals is suggested as a prelude to critical evaluation in man. The mode of action and drug resistance of chemotherapeutic agents are discussed briefly. Literature Cited (1) Adler, S., and Tchernomoretz, I., Ann. Trop. Med. Parasil., 35, 9, No. 1 (1941). (2) Anderson, H. H., Emerson, G. A., and Leake, C. D., Univ. Calif. Publ. Pharmacol., 1, 31, No. 2 (1938). (3) Chagas, Carlos Filho, personal communication. (4) Chung, H. !»., personal communication. (5) Daniels, T. C , personal communication. (6) Findlay, G. M.( et al.t Biochem. J., 36. 1 (1942). (7) Geiling, E. M. K., Cannon, P., et at., J. Am. Med. Assoc, 109, 1531 (1937). (8) Hogue, M. J., Am. J. Trop. Med., 14, 443 (1934). ' r (9) Johnstone, H. G.t personal'communication. (10) Khaw, O. K., Proc. Soc. Exptl. Biol • Med., 32, 520 (1934). (11) Kofoid, C. A., Wood, F. D., and McNeil, E., Univ. Calif. Publ. Zool., 41, 23, No. 3(1935). (12) Leake, C. D., J. Am. Med. Assoc, 93, 1632 (1929); 98, 195 (1932). (13) Pak, C, personal communication. (14) Soong, H. Y., and Anderson, H. H., Am. J. Trop. Med., 21, 461 (1941). (15) Soong, H. Y., and Hou, E. A., personal communication.
Organometallic Compounds Used as Antiparasitic Agents C. KENNETH BANKS Parke, Davis * Co., Detroit, Mich. HEAVY metals and their compounds have been used as antiparasitic agents for several thousand years. That heavy metals should have been used by man in his first attempts at medication may be attributed to the availability of the 1368
metals and their compounds, but their continued utilization for many years is indicative of some therapeutic activity. It will probably never be ascertained whether kohl, a naturally occurring stibonite, was first a beauty aid and later found CHEMICAL
useful as a prophylactic against epidemic conjunctivitis (59), or if the reverse be true. In either event, its use dates at least to Egypt of the Middle Kingdoms (ca. 2100 B.C.) as evidenced by the contents of vanity cases unearthed by AND
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