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新合成 화학제 thiocarbanilides의 실험적 결핵증 및 鼠癩에 대한 화학요법에 관한 연구

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 Studies on the newly synthesized thiocarbanilides for chemotherapeutic activity against mycobacterium tuberculosis and mycobacterium leprae murium 
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[영문] [한글] Studies on the Newly Synthesized Thiocarbanilides for Chemotherapeutic Activity against Mycobacterium tuberculosis and Mycobacterium leprae murium Tae Kyung Choi, M.D. Department of Microbiology, College of Medicine, Yonsei University Directed by Professor Joon New. M.D., D.M. Sc., Ph.D. The agents which have been used in the chemotherapy of leprosy are closely related to those used against tuberculosis. The present knowledge of Mycobacterium leprae and Mycobacterium leprae murium is heavily in debt to the study of Mycobacterium tuberculosis because of the impossibility of Myco. leprae in artificial cultivation and animal transmission. The similarity between Myco, leprae and Myco. tuberculosis in morphology, acidfastness and other biological characteristics, suggest the possibility that a therapeutic agent for leprosy may also be effective against tuberculosis. Myco. leprae murium was discovered by Stefansky 30 years after the discovery of Myco. leprae. However animal inoculation of Myco. leprae murium is successful. Ten years earlier, in 1872, Myco. leprae was discovered by A. Hansen as the first microorganism to be pathogenic to the human host. Brilliant progress has been achieved in the field of tuberculosis, i.e. in its artificial culture, animal transmission and chemotherapy, while it is far from success in those fields of Myco. leprae, though it is proceeded to the tubercle bacilli by 10 years in it's discovery. Both of these organisms belong to Actinomycetales and there are many similarities in their host-parasite relationship, immunological and biological properties. Study of leprosy is heavily dependent on that of tuberculosis at the present time. This is the reason why workers investigate both leprosy and tuberculosis at the same time. Modern chemotherapy of tuberculosis has been developed since the late 1930's. Domagk recognized the chemotherapeutic effect of prontosil(2',4'-diaminoazobenzene 4-sulfonamide hydroxide) on experimental streptococcal infection in mice. Later, Prontosil failed in the chemotherapy of tuberculosis. The introduction of Prontosil, however, encouraged workers to bring many sulfa drugs in to the field of modern chemotherapy. Meanwhile, almost at the same time Streptomycin by Waksman and Isoniazid by the workers at Farbenfabriken Bayer, at Hoffman-LaRoche and at E.R. Squibb and Sons Company were introduced. In 1942 Fagot reported the chemotherapy of leprosy by Promin, a derivative of DDS. This was the first successful use of a synthetic compound for modern chemotherapy of leprosy. Since that time a variety of DDS derivatives (Promin, Diason sodium, Promizole and Sulphetrone etc.) or DDS itself have been used as chemotherapeutic drugs for leprosy. Now DDS has become the standard chemotherapeutic agent for leprosy. However, DDS is not the final drug in the treatment of leprosy and remains far from satisfactory because it requires a long period of treatment (it takes 3-5 years to bring negative bacteriology), and its strong toxicity with many side reactions. These defects have stimulated the desire to have a better antileprosy agent. Even though DDS in leprosy, and INH, Streptomycin and PAS in tuberculosis have been considered as the most effective and the standard drugs at present moment, shortages of these drugs and many dissatisfactions have led workers to develop new and more effective compounds for the chemotherapy of tuberculosis and leprosy. Mayor developed a series of the derivatives of thiocarbanilides at Ciba Company, and among these compounds SU-1906 has been widely known as a therapeutic drug for leprosy. Buu-Hoi et al. also reported using a series of synthetic compounds of thiocarbanilides as antileprosy agents. In his clinical evaluation Davey reported SU-1906 and DDSO (Diaminodiphenyl sulfoxide) and ETIP (diphenyl-dithiol isophthalate) as effective antileprosy drugs. Attention was paid to the thiocarbanilide derivatives and the author synthesized same new compounds of thiocarbanilides for experimental studies on tuberculosis and leprosy. The method of chemical synthesis and detailed data will be published elsewhere in the future. The results of the treatment of experimental tuberculosis and murine leprosy with these newly synthesized thiocarbanilides are included in this paper. MATERIALS AND METHODS A. Experiments on antituberculous activities 1. The newly synthesized and control drugs in the experiment. For newly synthesized compounds of thiocarbanilides (or thioureas); L-1, L-2, L-3 and L-4 were used in the experiment Dialide, an already known derivative of thiocarbanilide, INH, PAS, Streptomycin and DDS were used as controls. 2. The media and strains in the in-vitro test. Myco. tuberculosis (H^^37 Rv, Ravenel and BCG) and Myco. phlei, the strains from National Institute of Health, Republic of Korea, were used. Fresh, vigorously growing, organisms which had been cultured in Dubos' tween albumin media at 37℃ for 2 weeks, were used for the examination of sensitivity to the newly synthesized compounds, Myco. phlei in the experiment were from one week cultures using the same media. 3. In-vitro tests for newly synthesized drugs and the method of observation. Okawa egg media containing 1γ/ml, 10γ/ml , 50γ/ml, 100γ/ml concentration of L-1, L-2, L-3 and L-4 (newly synthesized compounds): 1γ/ml, 10γ/ml, 50γ/ml and 100γ/ml concentration of Dialide; 1γ/ml, 10γ/ml,50γ/ml and 100γ/ml of PAS; 0.1γ/ml, 1γ/ml and 10γ/ml of INH; and 1γ/ml, 5γ/ml and 10γ/ml of DDS were prepared for the experimentation. Because the newly synthesized drugs were insoluble a liquid media could not be used. Therefore Okawa's solid medium was selected and a 380γ/ml initial solution in polyethylene glycol was prepared for the further dilution in subsequent media and varying concentrations of the drugs were used. The LD^^50 of PAS was 4,000 mg/kg while those of the newly synthesized thiocarbanilides were around 1,000 mg/kg on the average. To each prepared solid medium containing the selected concentration of the drugs, 0.2ml of mycobacterial strains previously cultured in Dubo's tween albumin media was added using accurate pipettes and the inoculated tubes were placed in the horizontal position for 2 hours. After the bacterial suspension had evenly spread over the surface of the media, the inoculated tubes were kept in an incubator at 37℃ for observation. From the time when colonies were identified macroscopically, observations were made at 2~3 day intervals and the colonies were counted for comparison and control. 4. In-vivo test for the newly synthesized drugs and the method of observation. For in-vivo test two experiments were performed concurrently. One experiment checked the acute toxicity and the other evaluated the antituberculous activity of compounds in mice. 1) The acute toxicity test for the newly synthesized drugs, L-1 and L-4. Approximately 40-day old white male mice of the CFW strain(body weight; 18 gm ± 1gm), which had been obtained originally from the National Institute of Health in U.S.A. and under continued breeding at the National Institude of Health in Republic of Korea, were used. The experimental datae were obtained according to the Reed and Muench method; the total number was divided into of 5 groups each containing 5 mice; the dosage of LD^^50 of the tested drugs were calculated by the mortality ratio. 2) Evaluation of the chemotherapeutic effect of L-1, L-2, L-3, L-4, INH, PAS and Dialide. a. The animals: Male white mice of the CFW strain (body weight;18gm±1gm), as previously described, were used. b. the baterial strain and the inoculating dosage: The ravenel strain of Myco tuberculosis was used in the experiment which followed the modification of the Geoffray's standardized test. The Ravenel strain of young, vigoourously growing, Myco. tuberculosis, two week old culture in solid egg medium, was thorously ground with a fine agate mortor and pestle, and suspended it in 2 mg per ml of distilled water. The 0.1ml suspension of tubercle bacilli was inoculated into the tail vein of each mouse. c. Method of drug administration: Each drug under study was thoroughly mixed with 3 grams of standard feed which contained the proper amount per day per mouse. Since the daily food consumption per mouse is about 5 grams, each mouse was fed 3 grams of the previously mixed drug in the forenoon and remaining 2 grams or more of standard food was given in the afternoon. This feeding program was started immediately after the bacterial inculation and was continued until the termination of the experiment. d. Experiment to evaluate the antituberculous activity of drugs using the mortality ratio: Experiments each evaluating drug were done in groups if two-one, the small dosage group, the other, the large dosage group. Each group contained 10 mice: L-1, 0.8mg per day per mouse and 1.6mg per day per mouse; L-3, 0.8mg per day per mouse and 1.6mg per day per mouse; INH, 0.1 mg per day per mouse and 0.5mg per day per mouse; Dialide,0.8 mg per day per mouse and 1.6 mg per day per mouse; PAS, 4 mg per day per mouse and 8 mg per day per mouse, and the control received no drugs. Mice dying before 20 days, the average 50 per cent survival time, were not reported. e. Experiment to evaluate the antituberculous activity of the drugs using autopsy findings: The mice of control of group which had no drugs died of tuberculosis with in 35 days after inoculation. The mice in experimental groups were observed for 75 days, and died within 40 days more than the time that all of the control group lived. By this time only 5 mice were left in the group getting PAS, 4mg per day per mouse. Among all the experimental groups the mortality of the group getting PAS, 4 mg per day per mouse was the highest. At 75 days, all surviving mice of each experimental group were sacrificed, and from each group 5 mice were selected at random, and autopsied. The results were compared to those of the control group. The mice which died before the 37th day of the experiment were not included in the results. At the autopsy after macroscopic study, each visceral organ was weighed and later emulsified. The bacterial index according to Gaffky scale was made for each organ and compared that of to the control. The groups of mice were L-1, 1 mg per day per mouse, 4 mg per day per day per mouse, 10mg per day per mouse; L-4, 1 mg per day per mouse,4 mg par day per mouse: IHH,0.1 mg per day per mouse, 1 mg per day per mouse and PAS, 4 mg per day per mouse, 16 mg per dry per mouse respectively. 5. Experiments on the suppressive effect of the drugs on murine leprosy Mice, CFW strain (18 gm ± 1 gm), as used in previous experiments, were used throughout these experiments. Myco. leprae murium, Hawaian strain, which had multiplied in the rat testis for five months, were inoculated into the left lower abdominal region subcutaneously with 0.5 ml of a 50 times dilution of emulsified leproma suspension containing numerous bacteria and tissue particles. The size and weight of leproma at the site of inoculation, weight of organs including spleen, liver, kidneys, lungs and large lymph nodes, were measured and compared with the control. And also the distribution of bacteria in these organs were compared that found the controls. RESULTS A. Results of the antituberculous activity of the newly synthesized drugs 1. Results of in-vitro test. In order to know the antibacterial activity of the newly synthesized thiocarbanilides, the sensitivities of Myco. tuberculosis (H^^37Rv, Ravenel and BCG) and Myco. phlei against these drugs were compared in-vitro with the control drugs. Myco. tuberculosis roar. hominis (H^^37Rv strain), L-1 and L-4 of the thiocarbanilides showed no growth of the organism in 50γ/ml concentration in 20 days indicating a strong suppressive effect, while Dialide, L-2 and L-3 permitted the organism growth in 100γ/ml in 15 days. There was no growth of the bacteria on PAS if 50r1m1 throughout the 25 days observation period, while L-1 and L-4 permitted growth of the organism after 20 days. L-1 showed no growth of Myco. tuberculosis var. bovid (Ravenel strain) in 50γ/ml in 15 days nor in 100γ/ml throughout the 25 days of observation. L-2, L-3, L-4, Dialide and DDS permitted the growth of the organism in 100γ/ml in 10 days. PAS in 10γ/ml and INH in 1γ/ml showed no growth of the organism throughout the 25 day, observation. In 18 days L-1, L-2, L-3, L-4 and Dialide showed no significant suppressive effect on BCG growth as compared to the control while INH in 1γ/ml and PAS in 10γ/ml showed remarkable suppressive effects. All the drugs tested above did not show any suppressive effect on the growth of Myco. phlei, non-pathogenic mycobacteria, except for Streptomycin in 10γ/ml. In summary L-1 among the newly synthesized thiocarbanilides showed the most promising suppressive effect against pathogenic Myco. tuberculosis. 2. The results of in-vivo tests of the newly synthesized drugs. The result of the acute toxicity test for the newly synthesized drugs and the tests determining their therapeutic effects were as follows; 1) Acute toxicity test: The 50 per cent end-points (LD^^50) were calculated according to the method of Reed and Muench. The result obtained was as follows; LD^^50 of L-1: 1,054 mg/kg LD^^50 of L-4; 1,028 mg/kg On the average, the LD^^50 of L-1 and L-4 was 1,000 mg/kg. This result was similar to the LD^^50 of Isonicotinic acid hydrazid methanesulfonate which was introduced recently for the chemotherapy of tuberculosis. 2) Chemotherapeutic effect of L-1, L-2, L-3, L-4, INH, PAS and Dialide. a. The result an experiment to evaluate the antituberculous activity of drugs using the mortality ratio: The mortality ratio of each group was compared with that of the control group up to the date when all of the control group were dead. L-1 had shown the most promising antituberculous activity in the in-vivo tests almost similar to INH. The chemotherapeutic effects of the tested drugs were arranged in the following order of success, INH 0.5mg>L-1 1.6mg>PAS 4mg>Dialide 1.6mg>L-3 1.6mg>L-1 0.8mg=PAS 8 mg>Dialide 0.8 mg>INH 0.1 mg>L-3 0.8 mg>Control. Consequently it could be recognized that L-1 was as active as PAS and INH in its chemotherapeutic effectiveness. b. Result of the experiment to evaluate antituberculous activity of drugs using results of the autopsy; Results of the suppressive effect of antituberculous activity compiled from the Gaffky scale index for each of the tested drugs was as follows; L-1 10mg>L-4 4mg>INH 0.2mg>PAS 16mg>L-1 4mg> INH 0.1 mg>L-1 1 mg>L-4 1 mg)PAS 4mg>Control. The Gaffky scale index of L-1, 1mg per day per mouse group was 42.8 and in more than half of the experiments the mice did not develop tuberculosis. This results indicated antituberculous activity equivalent to that of the INH 1 mg per day per mouse group. The group getting L-1, 1 mg per day per mouse did show tuberculosis in only two-third of the group and the mice having L-1, 10 mg per day per mouse showed tuberculosis. This represents a most remarkable suppressive effect against tuberculosis among the tested drugs including INH and PAS. Mice getting L-4, 1 mg per day per mouse showed a suppressive effect similar to that found after PAS 4 mg per day per mouse. B. Results of the experiment on the suppressive effect of murine leprosy from the newly synthesized thiocarbanilides 1. The results on the size of leproma. The suppressive activity of leproma formation was as in the following order; L-4 10 mg=INH 1 mg>INH 0.2mg>L-4 2mg> SU-1906 10mg>L-2 10mg> PAS 16mg>L-3 10mg) SU-1906 2 mg>L-3 2 mg>L-2 2mg>Control. Among the tested drugs, L-4, 10 mg group and INH, 1 mg group showed the most encouraging results with nor the formation of leproma or identification of bacteria. The suppressive effects of leproma formation with L-2, L-3, SU-1906 and PAS were weak and did not show any distinct difference to the controls. 2. The results of the weight of spleen. When weight of the spleen of each mouse was compared, the most effective agents were INH 1 mg, PAS 8 mg and L-4 10 mg. The other drugs showed a week or slightly suppressive effect and were not significantly different from the weights found for the untreated control group. These results were very similar to these in the previous method of measuring the leproma size for evaluation of chemothereapeutic agents in murine leprosy. 3. The results of counting the number of bacteria found in each visceral organ. When the drug evaluation was made using the method of counting bacterial numbers in each visceral organ, the suppressive effects were in the following order; INH 1 mg>L-4 10 mg>L-4 2mg>SU-1906 10mg>PAS 16mg>L-3 10mg> L-3 2 mg) L-2 10 mg >Control. Among these tested drugs, L-4 1Omg, INH 1 mg, were the most remarkable for their suppressive effect. This method of evaluation was 4i77cu1t and time consumming and yet the results very closely parallelled those of previous two methods of evaluation. DISCUSSI0N Since Mayer and other workers had reported that thiocarbanilides were different from INH or DDS in their structural formula and might have antituberculous and antileprosy activities, many workers have synthesized a series of thiocarbanilides and studied them. Mayer et al. have cantinued to study thiocarbanilides and evaluated the results of their effectiveness. Eventually they had produced various newly synthesized compounds of thiocarbanilides as chemotherapeutic agents for mycobacterial infection. Simultaneously, Buu-Hoi studied the thiocarbanilides. Independently Dialide (4,4'-diisoamylioy thiocarbanilide), reported one of the thicarbanilides, and noted its excellent antituberculous activity. Mayer et al., Youman et al. have reported it's clinical effects in the chemotherapy of tuberculosis and leprosy as well. At present, Dialide is being used by clinicians as a chemotherapeutic drug for tuberculosis. In the Soviet Union, Etoxid, which is similar to Isoxyl and the same as Dialide in chemical Structure, is in the general use. In-vivo as compared to INH or PAS, L-1 and L-4 of the newly synthesized thiocarbanilides have a superior suppressive effect on experimental tuberculosis, although in-vitro they showed a inferior effect. The reason for this finding is thought to be the insolubility of the newly synthesized compounds. They are not soluble in water, but soluble in hydrochloric acid, glycerol and polyethylene glycol with in experimental concentrations. They are stable at boiling temperature. Steeken et al. also reported some thiocarbanilides to be insoluble in water and stated that they used such insoluble drugs after filtration. But with this method it was impossible to maintain homogenous suspension of accurate concentration of such kind of compound throughout the experiment. Therefore the author selected the solid media using mixtures of higher molecular weight. Among the newly synthesized thiocarbanilides L-4 suppressed murine leprosy remarkably. L-4 was as effective as INH in suppressing murine leprosy in mice. INH has been thoroughly investigated and is well known to be the most effective suppressive agent far murine leprosy. Because of its remarkably suppressive activity in murine leprosy and its rather low toxicity shown in LD^^50, L-4 is a strong candidate for trial on humans. As for the method of screening the chemotherapeutic agent in experimental murine leprosy, several techniques were used, compared and evaluated. The first technique evaluated chemotherapeutic agents by measuring the size and weight of leproma at the inoculated site; the second evaluated the agents using the weight of the spleen, and the third was counted numbers of bacteria in each visceral organ. Among these techniques, the first in evaluated the chemotherapeutic agent by measuring the size and weight of leproma was the most simple and convenient. CONCLUSION A series of newly synthesized chemical compounds of thiocarbanilides, namely L-1, L-2, L-3 and L-4 were studied for their antituberculous and anti-murine leprosy activities in-vitro and in-vivo experiments. 1) LD^^50 of L-1 was 1,054 mg/kg and that of L-4 was 1,028 mg/kg, while the LD^^50 of INH was 650 mg/kg and PAS was 4,000 mg/kg orally in the experimental animals. 2) L-1 and L-4 showed remarkable suppressive effects in-vitro using solid media with 100γ/ml concentration. This data was parallel to that found using 1γ/ml of INH and 50γ/ml of PAS. The inferiority of L-1 and L-4 to INH and PAS in-vitro experiment might have been due to the water insolubility of those compounds while INH and PAS were readily soluble in water. 3) In in-vivo experiments L-1 showed much a superior antituberculous effect than was found with INH and PAS. 4) A method of grading the bacterial count in a homogenized tissue suspension of visceral organs (lungs, liver, spleen and kidneys) with the simple technique of the Gaffky scale was the most accurate and time saving technique to screen the results of the chemotherapeutic agents in tuberculosis. 5) Among the newly synthesized compounds L-4 showed the most remarkable suppressive effect on murine leprosy. The suppressive results were similar to those of INH. 6) The method of measuring the size and the weight of leproma at the inoculated site was simple and is adequate a screening test for chemotherapeutic effect in murine leprosy.
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