합성 Thiocarbanilide 유도체 L-4에 의한 나병치료에 관한 연구

Abstract

[한글]

[영문]

Though Chaulmoogra oil(Mouat 1854) had been used empirically in the treatment of leprosy from the ancient time, the very beginning of modern chemotherapy of leprosy was initiated by the work of Faget et al.(1943) who originated the Promin treatment of leprosy. Promin was less toxic derivative of the parent sulphone, ie., DDS(Diaminodiphenyl sulphone) with was synthesized by Fromm and Shittmann(1908). However, Promin itself was too toxic to be given orally or subcutaneoulsy, and therefore, it had only to be given intravenously. By the introduction of other derivatives of DDS such as Sulfoxone sdium, U.S.P. (Diasone sodium), Solapsone, B.P.(Sulphetrone), Acetosulphone(Promacetin) and Thiazolsulphone that could be given orally in similar doses with safety, Promin was eventually replaced by those drugs.

Meanwhile, the pioneering works done by Cochrane et al.(1949), Lowe and Smith(1949) and Lowe(1950) firmly established that DDS, the parent sulphone of those derivatives, did possess acrivity against Mycobacteria, and DDS has become the choice of drug in the treatment of leprosy since then on.

Today, it is well recognized that DDS is the drug having the virtues of low cost, general efficacy, suitability of outpatient treatment, rarity of drug resistance, and its prophylactic effectiveness. However, as Davey(1964) pointed out, DDS has still many limitations such as 1) its rather strong toxicity, 2) the existence of idiosyncrasy, 3) the precipitation of hypersensitive state, 4) the development of leprosy reactions, 5) the prolonged time of medication, 6) occasional occurrence of non-responding patients and etc. Those limitations DDS indicate that it could not be a sole and the most ideal antileprosy chemotherapeutic.

Efforts (Buu-Hoi 1954, 1955a, 1955b, Buu-Hoi et al. 1955a, Buu-Hoi et al. 1966b, Buu-Hoi et al. 1955c, Mayer 1941, 1954, Mayer et al. 1953, Mayer et al. 1959) have been made to produce alternative, ideal drugs that would exert lesser toxicity, speedier action and smoother progress during the treatment in addition to the virtues of DDS Following representatives of several classes of various organic compounds have been discovered and have been shown to possess activities against Mycobacterium leprae at more than one institute throughout the world;

Diaminodiphenylsulfoxide(Buu-Hoi et al. 1955a) Sulfamethoxypyrazine (Lederkyn; Schneider et al. 1958, Schneide et al. 1960), Sulfamethoxydiazine (Barclay and Wilkinson 1963), Acetylsulphomethxypyrazine (Schneider and Laguillon 1963), P-actamidobenzaldehyde thiosemicarbazone (Amithozone or TBI/689;Ryrie 1950, Lowe 1952, Lowe 1954, Dharmendra and chatterjee 1952), 4-butoxy-4'-dimethylaminodiphenyl thiourea (DPT or Ciba 1906; Davey and Currie 1956, Davey et al. 1958, Davey 1960)

and Diethylditiolisophthalate (Ditophal or Etisul; Devey and Hogerzeil 1959).

All of those chemical compounds have been developed in the hope that they could be administered singly or in combination with DDS to surpass or th improve the limitations of DDS. Recently, there have been some reports which provides additional data in support of the general efficacy and advantages of DDS administration for the treatment of patients and for the control of leprosy. Such

reports include the effectiveness of low dose DDS treatment and its advantages (Browne 1965, Leiker and Carling 1965, Ramu and Ramanujam 195), and the sensitivity of Mycobacterium leprae to low levels of DDS in mice (Shepard et al. 1966).

Choi and Lew(1965) synthesized a series of thiocarbanilide derivatives in the efforts of developing new antileprosy and antituberculosis chemotherapeutics. Among the synthesized derivatives, L-4 was shown to possess a significant activity

against murine leprosy(Choi and Lew 1965). Following their original study, Kim and Lew(1967) demonstrated that L-1 and L-4 exerted potent antifungal activitiy in in-vitro experiment, and Chang and Lew(1967) alos reported that activities of L-1 and L-4 against mycobacteria and superficial mycoses were specific and selective ones.

These evidences clearly indicated the possibility of clinical treatent of leprosy with L-4, a synthesized thiocarbanilide derivative.

In this study, domiciliary leprosy patients who had registered at the World Vision Leprosy Research Center were included. The results of clinical trial of L-4 in the treatment of leprosy patients were assessed clinically and bacteriologically in comparison with patients under ordinary DDS treatment

Materials and Methods

A. The drugs used.

1. A synthesized thiocarbanilide derivative, L-4.

L-4 was synthesized at the Department and its tentative structural formula is shown below. A detailed procedures and data of the synthesis will be published elsewhere in future.

L-4: N-(p-ethoxyphenyl)-N'-(p-aminodiphenyl) thiocarbamide.

2. DDS.

Local products of DDS were purchased and the content of DDS in a given sample had been analysed by the methods of Pharmacopoeia of U.S.A.(1965) and other's(Lee 1964).

DDS: 4:4'-diaminodiphenyl sulphone

B. Patients.

1. The selection of the patients.

A. total of 62 patients have been included for clinical trial of L-4. They were consisted of fresh and old patients who had registered at the World Vision Leprosy Research Center. Table 1. shown the types of the disease included for trial with L-4, and Table 2, shown their age and sex distribution.

As shown in Table 1., 22 patients out of 47 lepromatous type had received none of antileprosy treatment prior to L-4 trial. Through the course of L-4 treatment, 8 cases out of 22 patients have been excluded from the trial; 6 cases due to causes other than leprosy(Water et al. 1967), 1 of suicide and 1 with side effect of L-4.

2. The first experiment.

A total of 16 patients were included for the first experiment, 15 cases of lepromatous type and 1 of reactional tuberculoid. Among them, three of lepromatous type(Patient No. 3358, 3397 and 3518 in Table 4.) and 1 reactional tuberculoid were the fresh cases who had no experience of previous antileprosy treatment and they were treated with L-4 from the beginning of this experiment. However, the remaining 12 cases included in the first experiment.

3. The second experiment.

In the second experiment, the fresh lepromatous cases were included exclusively for the accurate comparions of the activity of L-4 against Mycobacterium leprae with that of DDS. The patients had no previous treatment with any kind of antileprosy remedy and the bacteriological examination of the skin smears revealed strong positiveness in relation to Bacterial Index and SFG value(Ridley 1964).

C. L-4 administration.

1. Routine laboratory examinations.

Before the initiation of L-4 administrations following laboratory examinations were completed and the results were recorded.

a). Detaile clinical and photographic records of the cases.

b). Bacteriological examinations of the skin by skin scraping method and calculation of Bacterial Index and SFG values(Ridley 1964).

c). Lepromin skin test with the trypsin-purified Lepromin antigen(Lew and Cappenter 1955) and the result of 4 week reaction.

d). Routine hematological examinations including total counts of RBC and WBC, differential count and the determination of hemoglobin level.

E). Urinalysis including microscopic examination of the sediment and the test for sugar and protein in the urine.

Theses laboratory examinations except Lepromin test were repeated at regular intervals through the course of the experiment.

2. Administration of L-4

1). L-4

L-4 were contained in gelatin capsule in the amount of 50mg to 100mg, and were given by mouth to the patients. For the adult, the initial dose of L-4 were 50mg to 100mg per day and the therapeutic maintenance doses of 200 to 300mg per day were gradually reached through the slow induction course that became routine in antileprosy treatment, particularly with DDS.

b). DDS.

The initial dose DDS was 25mg to 50mg per week and the doses were gradually increased to the levels of the therapeutic maintenance, ie., 300mg to 350m per week.

D. Evaluation of treatment of leprsoy with L-4

1. The first experiment.

The bases for the evaluation of the first experiment were the clinical improvement and the changes of the Bacterial Indices after the period of L-4 administration. Table 3. summarises the criteria used for the evaluation of the experiment.

2. The second experiment.

In the second experiment, the bacteriological changes, ie., decrease in Bacterial Indices and changes of SFG values were carefully followed through the course of L-4 administration to the patients in association with the clinical improvement. For

the comparison, the similar follw-up study of bacteriological changes were made with the patients who were given DDS as control.

Results

A. The results of the first experiment.

For convenience of evaluating the results of the clinical trial with L-4, 16 patients included in the first experiment were divided into 3 groups as follow;

Group Ⅰ ; 6 cases of lepromantous type without any form of reaction or allergic manifestaion.

Group Ⅱ ; 5 cases of lepromatous type who were in a state of ENl or ENL with some other condition.

Group Ⅲ ; 4 cases of lepromatous and 1 of tuberculoid who were in a state of sulfone allergy, neuritis or reactional tuberculoid.

On the basis of the criteria shown in Table 3., the results of the first experiment were evaluated(Table 4,5 and 6). By the criteria, the majority of the patients responded to L-4 treatment with remarkable progress, ie., 6 cases of Excellent, 3 Better, and 6 Good through the course of the treatment with L-4, ranging from 4 months to 1 year and 2 months. Only one case (Pt. No. 3248) out of 16 patients included in the first experiment could be graded as Stationary.

1. Clinical improvement.

In general, clinical improvements observed after the initiation of L-4 treatment were much more evident than the decreases in Bacterial Indices. Clinical improvements included better general conditions, subsidence or decrease in the extent or number of skin lesions and nodules, and alleviation or disappearance of

reactional or allergic manifestations.

2. The decreases in bacterial Indices.

The decreases in Bacterial Indices were observed in 9 cases out of 15 lepromatous types included in the first experiment. Those changes of Bacterial Indices were most prominent in the Group Ⅱ and Ⅲ, and there observed (2+) decrease in Bacterial Indices in 6 cases out of 9 Lepromatous type included.

B. The results of the second experiment.

In the second experiment, the changes of Bacterial Indices and of SFG values were carefully followed during the course of L-4 administration in association with clinical improvement. Similar observations were made with the patients who given DDS as control drug for comparison.

1. Changes of Bacterial Indices and SFG values in patients who were given L-4 treatment.

As shown in Fig. 1,2,3,4,5 and 6, there occurred decreases in Bacterial Indices such as (6+) to (3+) in 1 case, (6+) to (+4) 2 cases, (5+) to (4+) 1 case, (4+) to (3+) 1 case and (3+) 1 case and (3+) to (3+) 1 case at the ends of relatively short period of L-4 administration, the shortest 4 months and the longest 9 months.

Meanshile, the changes of SFG values observed in the same patients through the period of L-4 administration were ,uch more significant. In patients No. 4067, 4100, 4139 and 4098 (Fig. 1,2,3 and 4), their SFG value were (2-2-1) or (2-0-0(before the L-4 administration, and those SFG values changed dramatically down to the level of (0-2-) or(0-1-2) at the ends of 5 to 9 months treatment with L-4. Similarly, SFG values of patients No. 4117 and 4045 (Fig. 5 and 6) whose SFG values were (1-2-1) before L-4 administration became (0-2-2) respectively at the ends of 2 months and 5 months treatment with L-4. Those drastic changes of SFG values strongly indicated that there occurred so rapid damage and destruction of Mycobacterium keprade by L-4 administration that the bacteriological examinations of the skin smears of the patients hardly revealed the presence of so-called solid-staining bacilli at the ends of such relatively short periods of L-4 treatment.

However, in some cases such as shown in Fig. 7,8 and 9, there observed practically no decrease in Bacterial Indices inpatients No. 3672, 3518 and 3358 though they had been treated with L-4 for more than a year. Of course, there observed clear signs of clinical improvement in those patients. Therefore, as a pilot trial, combined therapy of L-4 and DDS were initiated at the ends of 16 and 20 months of L-4 treatment in patients No. 3518 and 3358. Following these combined therapy, there observed rapid decreases in Bacterial Indices and significant changes of SFG values as shown in Fig. 8 and 9, particularly in patient No. 3518. These observations were interpreted to suggest the possible effective treatment of leprosy with combined therapy of L-4 and DDS.

2. Changes of Bacterial Indices and of SFG values in patients who were given DDS treatment

As shown in Fig. 10,11,12 and 13, the changes of Bacterial Indices in patients given DDS medication were rather insignificant, such as (6+) to (5+) in 1 case, (5+) to (4+) 1 case and (5+) to (3+) 2 cases. Meanwhile, in patients (NO. 3938 and 3914) whose SFG values were *2-1-0) before DDS administration, the SFG values became (0-2-20 of lower after 6 to 8 months of DDS treatment (Fig. 10 and 11). However, the SFG values of the patients (No. 4033 and 3962), whose SFG values before DDS treatment were (2-1-0), were still at the levels of (1-2-2) and (1-2-2) after 7 to 9 months of DDS treatment (Fig. 12 and 13). In general, there was tendency of paralleled changes of Bacterial Indices and SFG values in DDS given control group.

C. Side reactions and toxicity of L-4

Out of a total of 62 patients included for clinical trial of L-4, there observed side reactions in 6 cases; 1 case of neuritic symptom, 2 cases of gastrointestinal irritations such as nausea and vomiting, and 3 cases of possible toxic jaundice. The appearance of jaundice in such cases was preceeded by the signs of gastrointestinal upsets such an anorexia, nausea and vomiting. These jaundic conditions were easily controlled by the withdrawal of L-4 administration, bed rest and fluid therapy. The development of jaundic conditions were assumed to be due to the residual benzidine that was not completely removed through the process of L-4 synthesis during the early phase of this study.

Discussion

Though DDS was originally synthesized by Fromm and Whittmann in 1908, the recognition of its antileprosy activity followed the clinical studies conducted by Cochrane et al.(1949), Molesworth and Narayaswami(1949), Muir(1950) and Lowe(1950) Particularly, Lowe's work(1950) on the oral administration of DDS retains high

credit and the oral route has become the method of choice for widespread sulphone therapu. Dventually, the introduction of DDS as antileprosy drug has really brought the promise that 'leprosy in curable' and drastic changes have been taken place in the epidemiology of leprosy today in the world.

Through the experiences of clinical use of DDS in the treatment of leprosy, not only the virtues of DDS but also its limitations have been recognized as have been reviewed by Davey(1964). Therefore, the efforts have been made to develop compounds of kesser toxicity than DDS and of better antileprosy activity in order to replace DDS, and these were the very beginning of numerous studies involved in development of new antileprosy drugs.

Recently, Choi and Lew(1965) synthesized a series of thiocarbanilide derivatives in the hope of developing new compounds of antituberculosis and antileprosy activity. Their studies showed that 1) two of synthesized thiocarbanilide derivativew, ie., L-1 and L-4 possessed remarkable growth inhibiting activity against Mycobacterium tuberculosis in in-victro experiment and 2) L-4 exerted significant suppressive effect on the development of leproma in murine leprosy. More recently, Kim and Lew(1967) reported that both L-1 and L-4 showed remarkable suppressive effect on most of the superficial mycoses in in-vitro experiment, and Chang and Lew(1967), based on the data of nonspecific and insignificant antibacterial activities of antileprosy, antituberculosis and synthesized thiocarbanilides L-1 and L-4, concluded that potent antileprosy, antituberculosis and antifugal activities possessed by them were psecific and selective ones.

Furthermore, according to the calculations made by Choi and Lew(1965), LD ^^50 of L-1 was 1,054mg/kg and of L-4 was 1,028mg/kg of body weight in mouse. These data on L-4 clearly indicated the possible clinical trial of L-4 in the treatment of human leprosy, and clinical and acteriological assessment of L-4 administration in the treatment of leprosy were undertaken in this study.

In the first experiment of this study. L-4 treatment of 16 patients, including 15 lepromatous and 1 tuberculoid, for a period of 4 to 14 months were quite impressive. responses of 16 patients to L-4 medication could be graded, according to the criteria of evaluation (Table 3.), as follows; 6 cases of Excellent, 3 Better, 6 Good, and 1 Stationary(Table 4,5 and 6). The gradings used in the evaluation of L-4 treatment, ie., Excellent, Better, Good, Stationary and Worse(Table 3.) appears to be very much similar Moderate and Slight improvement, No change and Deterioration. Clinical improvements observed in lepromatous type patients having leprosy reaction or sulfone allergy(Table 1 and 6) were the most significant ones.

The importance of the selection of the patients in clinical trial of new antileprosy compounds has been emphasized, and Davey(1960) suggested that in such a trial only pure lipromatous cases should be included, and that the patients should be in an active, progressing condition, untreated by previous chemotherapy.

Water et al.(1967) also suggested that a patient whose MI(Morphologic Index, Water and Rees 1962) is less than 25 is unsuitable for inclusion in a standard trial of antileprosy drug. As the results of the first experiment of this study clearly showed that the oral administration of L-4 was quite acceptable to and well tolerated by patients, and the clinical improvements which followed L-4 administration were highly impressive, and the clinical improvements which followed L-4 administration were highly impressive, and therefore, in the second experiment of this study it was attempted to carry changes of SFG values in the patients whose bacteriology of skin smears could fulfill the requirement suggested by Water et al.(1967).

By L- administration, the changes of SF values were speedier and much more significant than the decreases in Bacterial Indices. As shown in Fig. 1,2,3,4,5 and 6, SFG values of the patients (No. 4067, 1040, 4139 and 4098) whose SFG values were

(2-2-1) or (2-0-0) prior to L-4 administration have been changed into (0-2-2) or )-(0-1-2) at the ends of relatively short period of treatment, ranging from the shortest 5 months to the longest 9 months (Fig. 1,2,3 and 4). Similarly, original SFG values of (1-2-1) of the patients (No. 4117 and 4045) have been changed to (0-2-2) at the ends of 2 to 5 months of L-4 administration. In comparison with, the changes of SFG values observed in L-4 given patients appeared to be much more significant than those observed in DDS given patients (Fig. 10,11, 12 and 13)

Though the SFG values instead of MI were followed in the second experiment of this study, the estimated Mi of the patients (No 4094, 4010, 4139 and 4098) appeared to be, at least, over 25% (Fig. 1,2,3 and 4).

Through the first and second experiment, it was assumed that the remarkable clinical improvements which followed L-4 administration were in good accordance with the changes of SFG values rather than with the decreases in Bacterial Indices of the patients.

In some instances such as seen in patients (No. 3672, 3518 and 3358) (Fig. 7,8 and 9), L-4 administration resulted in rather insignificant changes of bacteriology of the patients without any indication of correlation to clinical improvements of the patients. As shown in Fig. 8 and 9, administration of DDS in addition to L-4 did induce remarkable bacteriological changes in them, and the results are highly suggestive of combines effective therapy of leprosy with L-4 and DDS.

Finally, out of a total of 62 patients included in this study, 6 cases of nonacceptability due to side effects and toxicity of L-4 have been encountered. The cause of 3 incidences of jaundice appeared to be due to the incomplete removal of residual benzidine in the process of L-4 synthesis during the early phase of this study. Efforts were made to achieve the complete removal of residual benzidine from L-4 preparation and this precaution worked out effectively.

Conclusion,

A total of 62 leprosy patients, 47 lepromatous type, 9 tuberculoid, 5 borderline group and 1 indeterminate group, have been treated with a synthesized thiocarbanilide L-4 and the effectiveness of L-4 administraion in the treatment of leprosy in evaluated on the basis of clinical and bacteriological improvements. The results are summarized and concluded as follows;

1. L-4, contained in gelatin capsule, could be safely administered orally to the patients through slow induction, from initial dosages of 50mg to 100mg daily and to the therapeutic maintenance levels of 200mg to 300mg daily, for a long period of

time.

2. L-4 administration has brought apparent and remarkable improvements in clinical symptoms of the patients after the relatively short period of medication, compared with that of DDS administration.

3. Changes of SFG values caused by L-4 administration were much more speedier than, (or, at least, equivalent to) the effect caused by DDS. The changes of SFG values, in general, sychronized fairly well with the clinical improvements of the patients.

4. Lepromatous cases with lerposy reaction or sulfone allergy responded well to L-4 medication with remarkable clinical improvement, and prolonged administration of L-4 did not provoke such a precipitation action to leprosy reaction as did DDS.