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

Abstract

[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.