The Present State of the Chemotherapy of Tuberculosis
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ALFRED BURGER University of Virginia, Charlottesville, Virginia
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HEMOTHERAPEUTIC agents for infections caused by pathogenic mycobacteria, such as the human and bovine tubercle bacilli and the so-called leprosy bacillus, may be divided into three groups: compounds prepared by pure empiricism, drugs conceived from considerations of the drug-versus-metabolite antagonist theory (I), and compounds designed to penetrate the lipid or waxy layer of the acid-fast bacteria and so get a t the protoplasm of the organisms. Only a few of the thousands of random chemicals tested in vitro were found to be strongly tuberculocidal. Among these were certain metallic derivatives such as gold thiocyanate, gold sodium thiosulfate, and gold diacetyl: certain alkaloids and alkaloidal drugs such as harmine, conessine, ethylapoquinine, and aminohydroquinine (2); vitamins (cod-liver oils) : and hormones (diethylstilbestrol) (3). Halogenated aromatic ketones (4) and basic ethers of halogenated pheuols (6, 6) were tried, as well as basic dyes (7) ; the latter were supposed to be tuberculocidal because of the neeative charge on the bacterial cell when suspended in serum. Guaiacol and some of its derivatives which act only by alleviating some external symptoms in pulmonary tuberculosis barely deserve passing mention. Some of these compounds have given occasional relief from clinical symptoms but no cbre. This is partly due to the chronic character of tuberculous lesions which cannot be healed readily by drugs. The goal of antitubercular chemotherapy is the disappearance of bacilli from the infected host; walling off the bacteria with the aid of the natural defense mechanisms of restrng patients is a poor substitute for this. Since 1940 (1)the planning of new chemotherapeutic agents has often been based on the intake of pathogenic organisms. Information concerning the intake of tubercle bacilli can be obtained from metabolic studies using the Long or Prokauer and Beck synthetic media (8) but only a limited amount of work has been done in this field. In its natural surroundings the tubercle bacillus chooses a typical cell diet of uitrogenous materials and salts; its sole source of carbon is glycerol but it is adaptable to unrelated carbon compounds such as benzoic or salicylic acid (9). Not much is known about specific growth factors needed by the bacilli but, like other bacteria, they can produce their own essential metabolites. Several B-vitamins have been isolated from cultures, and the presence of phthiocol, 2-hydroxy3-methyl-1,4-naphthoquinone, suggests the use by the bacilli of a vitarnin-K factor, since phthiocol may be formed from antihemorrhagic vitamins by alkaline
cleavage. The bacilli need oxygen; indeed, as measured in the Warburg apparatus, oxygen uptake usually serves as a corollary to estimating inhibition of bacillary growth in vitro. Typical antioxidants, such as diphenylamine, are known to inhibit the growth of cultures. Based on these observations, a number of aminosubstituted aromatic sulfones were tested whose structure classifies them as metabolite antagonists to paminobenzoic acid (10). Several of the most promising compounds which may eventually yield a clinicnlly useful drug hake been found in this group. The parent compound of the series, 4,4'-diaminodiphenyl sulfone, is still used as a reference compound (8). In spite of its high antitubercular activity, its toxicity and insolnbility have prevented significant clinical tests. Promin, its bis-(glucose sulfonate), and diasone, its bis-N-methylene sodium sulfoxylate derivative (11), are much more soluble and exhibit such a low toxicity that both drugs have been subjected to extensive clinical trials (12). However, their curative effects are too erratic to make their introduction into therapy advisable. An even greater promise is held out by promizole, 4,2'-diaminophenyl-5'-thiazole sulfone (13), which shows a particularly low toxicity, and a high antitubercular activity in uivo (14). Preliminary clinical reports have confirmed these properties in man. The output of lipids and waxes by the mycobacteria has influenced chemothefapeutic thought for over 20 years, probably because certain fatty acids can both cause irritations or lesions like those produced in acidfast infections, and damage the bacilli as true chemotherapeutic~. This is especially true of branched fatty acids, such as tuberculostearic acid (10-methylstearic acid) and phthioic acid (probably d-3,13,19-trimethyltricosanoic acid) (15) which were isolated by Anderson (16) from their trehalose esters produced by the bacilli. Other naturally occurring, and synthetic branched long-chain acids, such as the acids from chaulmoogra oil, and 2,2-dimethyl-dodecanoic acid (17), also cause lesions in the tissues of animals resembling those produced by the bacilli. The antileprotic studies of Adams and his coworkers (18) in the series of branched fatty acids and amines containing long forked alkyl groups led to the observation that the action of such compounds is due largely to their physical character, their ,activity in vitro paralleling their surface-tension-depressingproperties. I t may be that the bacilli are attacked physically by such compounds by a weakening of their protective
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lipid capsules and then more easily disposed of by unfavorable conditions i n yitro, or, i n Gwo, by antibodies or phagocytosis. This view is supported both by electron microscope studies which show the existence of an envelope around the bacilli (19), and by pharmacological data: since treatment must be continued long after the symptoms have disappeared, and relapses are frequent. Several compounds prepared by Adams' group were highly active against tubercle bacilli i n uitro. 1,5-dicyclohexylbntan-2-oic acid was active in dilutions of 1:50000. Other acids with 16 to 18 carbon atoms, and some related bases in which a diethylamino group takes the d lace of carboxvl. were tuberculocidal while homologiwith 19 to 20 ;arbon atoms showed no activity. No definite success was achieved by combining lipid or fatty acid groups with molecules of known bacteriostatic activity. Thus, the introduction of sultanilamido groups into alkyl naphthoquinones, or of lipophilic constituents into sulfathiazole (20) did not give satisfactory results. It is doubtful, however, how much significance can be attributed to any in eritro evaluation of antitubercular drugs. For instance, 2,3,5-triiodobenzoic acid which is highly tuberculocidal i n yitro has no chemotherapeutic action i n yivo (21). The lack of a rapid and economical animal test has retarded prowess in this field more than any other factor. A typical procedure has recently been recommended by Feldman and Hinshaw (8). In their screening test, six to ten guinea pixs are treated with the drug for two to four days before inoculation with a virulent tubercular culture. The administration of the drug is continued for 60 days after the infection when a thorough necropsy determines the activity of the drug by comparison with the condition of untreated controls. The screening test is followed by the crucial test in which 20 tuberculous guinea pigs are treated with the drug over a period of six months. The authors of this test argue that any screening test of shorter duration, directed against more acute infection, cannot give a true picture of t f e antitubercular activity of a drug. Such tests should not be used because the pathological and serological character of a virulent infection requires properties in a chemical entirely d i e r e n t from those a clinically useful drug would need in chronic conditions. Whiie these arguments may be sound from a pharmacologist's point of view, they raise a barrier against some important practical aspects of antitubercular testing. Few chemists will be able to furnish 50 g. of a complicated compound for screening tests, and the Feldman and Hinshaw test may well consume 180 to 300 g. of a moderately active drug before the crucial test is undertaken. A chemist should be able to get some preliminary, even if inconclusive, information about a compound from a sample not exceeding 5 or 10 g. Any compound which shows real promise in a less exacting test might well be subjected later to the screening test of Feldman and Hinshaw. Three i n vier0 screening tests are used for this preliminary trial.
The omental weight test (5) may be carried out with a few grams of a drug, and does not take over 12 days. Several infected guinea pigs are divided into a control and a test group, and the latter treated with the drug. Part of the control group is inoculated with dead tubercle bacilli in order to avoid errors arising from the reaction to dead organisms. After 12 days, the animals are killed and their omenta weighed. The weight of the omentum in the average normal guinea pig per 100 g. of body weight is known. Any increase in the omental weight after infection is due almost entirely to tubercle formation. The average omental weight of infected animals after a given period depends on the amount and virulence of the infection, and is readily determined in the control group. The decrease in omental weight of the treated group is a measure of the drugs activity. The chick-embryo test (22, 23) is based on the fact that tubercle formation occurs in the chorio-allantoic membrane six days after implantation of the bacilli. This makes possible observation of the inhibition of tubercle formation by a given drug. In the piscine infection test (24) gold fish, infected with Mycobacterium marinurn, absorb the drug from the water so that the tissues come to an equilibrium as regards the drug. The therapeutic effect so produced is measured by comparing the number of acid-fast organisms in smears from fish, infected three weeks previously and maintamed in normal water, with those from infected fish kept in water containing the drug. All these tests will need further improvements, but they may well be the basis for a reliable rapid trial of large numbers of potentially antitubercular compounds. They may, perhaps, also prove useful in the evaluation of two highly antitubercular mold products discovered during the past three years. One of these antibiotics, streptothricin, was isolated from Actinomyces laoendulac by Waksman (1942). It has a low acute toxicity but a high delayed and cumulative lethal effect in animals. The second one, streptomycin, was obtained from Actinomyces griseus by extraction of the mold with neutral solvents, adsorption on activated carbon, and elution with dilute hydrochloric acid. Like streptothricin, it is bacteriostatic for many Gram-positive and Gram-negative bacteria. It is toxic to animals only in huge doses, and is often curative in tuberculous guinea pigs. Its solubility in dilute acids has given rise to speculations that streptomycin is an amine-type base of low molecular weight but no further details about its composition have been published. The construction of a large extraction plant for streptomycin just announced in the daily press (August 23, 1945) by a leading pharmaceutical manufacturer lends emphasis to the importance of this antibiotic. The difficulties of the chemotherapeutic approach to tuberculosis have stimulated interest in serological studies of this infection. When a culture of tubercle bacilli is filtered, the filtrate contains polysaccharideprotein conjugates which are the source of tuberculin, the tubercular antigen used in diagnostic tests (25). (Continued on page 597)
T H E PRESENT STATE OF THE CHEMOTHERAPY OF TUBERCULOSIS (Continuedfrom page 587) C
When dead tubercle bacilli a r e injected i n t o a n animal, antibacterial antibodies a r e formed i n its tissues, b u t only sensitizing and anaphylactic antibodies h a v e been produced. None of these antibodies h a s shown immunizing properties (26)
(10) STEENKRN, W..AND F. H. HBISE,P m . SOC.Exptl. Biol. Med.. 52. 180 (1943). (11) RAIZIS~ G.'*.I $.&I-& 98,351) (191S); F F. T . CALLOMON m n I.. GnosKlN, Am. K'?. T u b e r r . , 47, 97 (1943,. (12) HISSHAW, 11. C..K. H.PPUI(TZI+, AN" W. H FELDMAN, ibid., 50.52 - ~ , 110M .-.--, -~
(13) B A ~ A L. S ,L.,J.Am. Chcm. Soc., 67,671 (1945). (14) FELDMAN, W. H.. H. C. HINSHAW, AND F. C. M A N N , A ~ . R ~ . Tuberc.. 50, 418 (1944). LITERATURE CITED J. Chem. Soc., 389 (1945). (15) PoLoAR, N., AND R. ROBINSON, (1) WOODS, D. D., AND P. FILDES,C h m . b I d . , 59,133 (1940). (16) ANDERSON, R. J., Chem. Rm.,29, 225 (1941). (2) MEISSNER, G., AND E. HESSE.Arch. exp. Path. Phermakol.. (17) HALLER:A . L ~E. ~BAUER, ~ Ann. Chim. [91. 1, 6 (1914); 147,339 (1930). B., ret. (13). S ,W.. J. Endocrinol., 2,444 (1941); 3, 168 (1942). (18) STANLEY, (3) E M ~ N C. W . M.. G. H. COLEMAN, C. M. GREER.J. SACKS. (4) FRIEDLANDER, S., Calif. and Westem Med., 61, 85 (1944). AND R. ADAMS, J . Phemacol., 45, 121 (1932). A. BURGER, AND F. BERNHEIM, (19) ROSENBLAT~, (5) SAZ,A. K., F. R. JOHNSTON, M. B., E. F. FULLAM,AND A. E. G ~ s s ~ s a , Am. Rev. Tuberc., 48, 40 (1943). Am. Rev. Tuberc., 4 4 587 (1942). E. BERN- (20) S J O G ~ B., (6) Bmcen. A.. E. L. WILSON,C. 0.BRINDLEY,AND N , Nature, 150,431 (1942). HEIM,J. Am. Chem. Soc., 67, 1416 (1945). MPhe?maCol., , 73,78 (1941). (21) S * Z , A. K . , AND F. B E R N ~ I J. (7) SWTH,M. I., J. Phermacol.. 20, 419 (1923); E. Hesss, (22) MOORE,M., Am. J.Path., 18,827 (1942). ANO G. QUAST, A~ch.ex$. Path. Pherme- (23) EMMART, G. MEISSNER, E. W., AND M. I. SXITH.Am. Rev. Tzrberc., 47, kol.. 135, 82 (1928). d9F. 11 ,A"047) .",. (8) FELDMAN, W. H., AND H. C. HINSHAW,A~. Rev. Tubwc..51, (24) FEINSTONE, W. H.. ibid..46,101 (1942). 582 (1945);cf. also, F. F. T. CALLOMON, aid., 47,97 (1943). (25) BOYD, W . C., "Fundamentals of Immunology." Interscience F., J. Bad., 41, 387 (1941); J. BBi. Chem., (9) BERNHEIM, Publishers, Inc., New York, 1945, p. 411. 143, 383 (1942). (26) Ibid., p. 311. Am"
