合成 thiocarbanilide 劑 (L-1 및 L-4)의 試驗管內 抗眞菌作用에 關한 實驗的 硏究

Abstract

[한글]

[영문]

In nature there are many kinds of fungi, which have close relations with animals and plants in various ways. Among these fungi, some cause diseases in man. for a lone time the biological and biochemical characteristics of fungi and diseases caused by them have been studied by many workers.

At present, superficial fungal infection is widely prevalent, covering all of races and areas, and has been recognized as a serious problem among all nations.

Especially soldiersserving in moist and hot areas mostly suffer from athlete's foot. Despite these circum-stances, to our regret, there are no ideal chemotherapeutic agents, but some used no ware effective.

Emmons(1933) studied antifungal activities against superficial mycoses with several disinfectants, and Loewenthal (1961, 1964) made a similar study with alcohol.

Griseofulvin, which Oxford et al. (1939) isolated from Penicillium griseofulvum, is widely used as a chemotherapeutic agent for superficial mycoses, and its activities were studied by many workers. (Gentles, 1958; William et al., 1958;

Bedford et al., 1960;Gentles and Barnes, 1960; Aytoun et al., 1960; Desai, 1960; Harrell et al., 1960; Kaplanand Ajello, 1960; Kirk and Miles, 1960; Lazar, 1960; Maibach and Kligman, 1960; Pipen,1960; Prazak, 1960; Blank, 1965; El-Nakeeb et al., 1965; and Woo and Chang 1965). Other compounds tested for their antifungal activities were Candicidin isolated from Streptomyces griseus by Lechevalier et al.

(1953), Fungicidin from Actinomyces by Hazen et al (1950), Mycobacillin from Bacillus subtilis by Majumdar and Bose(1958) ,Hamycin from Streptomyces pimprina by Bennett(1964), and Amphotericin A and B from the other strains of Streptomyces by

Fraser and Knox(1959).

Noguchi et al. (1960) of Japan synthesized Tolnaftate and claimed that it has excellent antifungal activity against superficial fungal infections. Robinson and Rasm (1964) ,and Robinson and Raskin (1965) carried out clinical trials with

1'olnaftate and reportedthe results indicated that Tolnaftate was highly effective against superficial fungal infections .

Robinson et al. (1964), Fleischmajer (1965), Blank (1965) and Stone et al.

(1966)studied antifungal activities with Thiabendazole, Merges (1964) with Candeptin, Olansky(1965) with Nafcillin, Hazen et al, (1953) and Wright et al.

(1957) with Nystatin, eraser and Knox (1959), Kravetz et at. (1961), Rosenthal (1963) and Spinner et al. (1965) with Amphotericin B, and Uts (1965) with various chemical compounds. These investigators presented data showing that some of those

chemicals possessed antifungal activities against deep mycotic infections.

Antifungal activities of ten different chemicals were compared by Mayer (1949), andsimilar studies were made with Imidazole derivatives by Ellis (1964), with severalantileprosy and antituberculosis chemotherapeutic agents by Woo et al. (1965), and withUndecylenic acid and Tolnaftate by Lubowe et al. (1965).

For a long time it has been known that fungi have similar characteristics and closerelations to mycobacteria from the point of view of phylogenetic taxonomy. It has beenspeculated that compounds which have suppressive effects on mycoses would be similarlysuppressive to Mycobacterium tuberculosis and other mycobacteria such as Mycobacteriumlepraemurium and Mycobacterium leprae. Mayer (1942, 1964), Lorincz and Pearson (1962), Woo et al. (1965), and Albright and Hitch (1966) reported that antimycobacterial agents were effective in treating fungal infections.

Choi and Lew (1965) resorted that a series of newly synthetic thiocarbanilides, L-1 and L-4, had remarkably suppressive activities against Mycobacterium lepraemurium andMycobacterium tuberculosis. It was also shown that the same thiocarbanilides in vitrotests exerted remarkable growth-inhibiting activities

against the drug-resistant strains of Mycobacterium tuberculosis (Chang et al. 1965 and Oh 1966), but not so effective sup-pressive activities against non-mycobacterial pyogenic bacteria (Chang et al. 1965). They suggested that the antibacterial activities against mycobacteria and fungi of

syntheticthiocarbanilides L-1 and L-4 were specific and selective ones.

In the hope of finding a superior chemotherapeutic agent against fungal infections, in this investigation L-1 and L-4, synthetic thiocarbanilides which were originally developedas chemotherapeutic compounds for Mycobacterium tuberculosis and Mycobacterium leprae(Choi and Lew 1965), were tested in vitro for

their antifungal activities against superficial and deep mycoses.

Materials and Methods

The synthetic chemical rompounds of thiocarbanilides, namely L-1 and L-4, and Und ecylenic acid, Zinc undecylenate and Whitfield's ointment as controls were used in the experiments .

A. Structural formula of L-1, L-4 and control drugs.

1. Synthetic chemical compounds.

1) L-1

CH^^3 CH^^2 O (??) NH-C-NH (??) SCN

S

N- (p-ethoxyphenyl) -N'- (thiocyanophenyl) thiocarbamide

2) L-4

CH^^3 CH^^2 O (??) NH-C-NH (??) (??) NH^^2

S

N- (p-ethoxyphenyl) -N'- (p-aminodiphenyl) thiocarbamide

2. Control drugs.

1) Undecylenic acid.

CH^^2 = CH-(CH^^2)^^8·COOH

2) Zinc undecylenate.

CH^^2 = CH·(CH^^2)^^8·COO Zn

CH^^2 = CH·(CH^^2)^^8·COO/

3) Whitfield's ointment. *1

COOH COOH - OH

(??) (??)

(Salicylic acid plus Benzoic acid)

B. Strains of fungi used.

The following 26 strains of superficial and deep mycoses were used.

1.Superficial mycoses:

Trichophyton rubrum; 4 strains, Trichophyton gypseum, 4 strains, Trichophyton mentagrophytes; 2 strains, Trichophyton ferrugineum; 3 strains, Epidermophyton floccosum; 5 strains, Microsporum cams; 3 strains, and Microsporum gypseum; 1

strain were incluled .

2. Beep mycoses:

One strain each of Candida albicans, Penicillium nntatum, Sporotricum schenckii andFusarium species were Included.

C. The media used.

Sabouraud media 72 containing 10γ, 20γ, 40γ, 80γ, 100γ, 200γ, 400γ, 800γ, and 1,600γ of L-1, L-4 and control drubs per ml were prepared for the experiments. For this purpose Polyethylene glycol 400 was used as a solvent in making stock

solutions of L-1and L-4 because the comvounds were insoluble in water. Occasionally precipitation occurredin higher concentration during the preparation of stock solutions, but there was no such precipitation when L-1 and L-4 were contained in the media used.

D. The methods of culture and observation in vitro.

1. Tube culture method.

After growing the strains of fungi in Sabouraud broth media at 37℃ for 2 weeks the growths were ground with mortar and pestle. The well-ground fungal suspensions were used as inocula both for tube culture and slide culture methods. One loopful of the ground material was inoculated in the center of slant media containing

appropriate concentrations of L-1, L-4 and control drugs. After they were kept horizontally for two hours, inoculated tubes were transferred to a dark place and cultured at room temperature for 20 days. During incubation, every other day the inoculated tubes were examined macroscopically for the presence of a growth. The

absence or presence of a growth was regarded as the indication of growth inhibition or positive growth of fungi in a given concentration of L-1, L-4 and the control drugs respectively, and was recorded as negative or positive in the tables.

2. Slide culture method.

The same media used above and containing various concentrations of the drugs were applied in this method. Onto the sterilized cover glass, 0.05ml of media was poured an done loopful of well-ground fungal suspension was inoculated. A ring (inside

diameter; 1.5cm and depth, 0.5cm) of polyvinyl tube was placed between the cover glass and slide glass, and the ring was sealed to the cover and slide glass by a mixture of paraffin and wax. It was then placed in a moist petri dish in an inverted position and cultured at the same conditions as the al) one tube culture

method. During incubation from 10 to 14 days the inoculated slides were observed microscopically, and the presence or absence of formation of hyphae, macroconidia, microconidia and chlamydospore was regarded as the indicationof growth or inhibition of Positive growth of fungi in a given concentration of L-1, L-4 and control drags respectively, and recorded as positive or negative in the tables.

Results

A. Results of the tube culture methods.

1. Antifungal activities of L-1.

In general L-1 showed remarkable suppressive effects on and Microsporum species tested.

a) Trichophyton species.

The growth of he most of Trichophyton species were completely inhibited in media containing 20γ per ml. However, T.rubrum (No. 63, 66), T.ferrugineum and T.gypseum (No.85) were resistant to 20γ per ml, and T.ferrugineum (No. 103) to 40γ per ml respectively (Table 1 a).

b) Epidermophyton species.

Trichophyton, Epidermophyton

With most of the Epidermophyton species tested, remarkable suppressive effects were observed in media with 40γ per ml concentration. In cases of E. floccosum (No. 35, 79),the growths were suppressed even in media with only 20γ per ml concentration (Table 1 b).

c) Microsporum species.

L-1 also showed a remarkable suppressive effects on most of the Microsporum species in media with 20γ Per ml concentration. However, M. gypseum (No. 50) was resistant to 40γ per ml concentration (Table 1 c).

d) Deep mycoses.

L-1 showed ineffectiveness on deep mycoses. These showed resistance in media with 200γ per ml or 800γ per ml concentration (Table 1 d).

2. Antifungal activities of L-4.

a) Trichophyton species.

Antifungal activities of L-4 against the Trichophyton species were not as marked as with L.1, and it showed great variations in suppressive effectiveness (Table 2 a)

b) Epidermophyton species.

Only growth of E. floccosum (No. 12) was suppressed in media with 80γ per ml concentration and the rest of Epidermophyton species tested were resistant to media with 80γ per ml concentration. Antifungal activities of L-4 against Epidermophyton speciesdid not show such a great differences in suppressive effects as in Trichophyton species(Table 2 b).

c) Microsporum species.

Microsporum canis (No. 28) was resistant to 80γ per ml, and other Microsporum species to 200γ per ml concentration of L-4 (Table 2 c).

d) Deep mycoses.

L-4 showed great differences in suppressive effects on deep mycoses. These fungi showed resistance In media with 400γ per ml concentration (Table 2 d).

3. Antifungal activities of Whitfield's ointment.

a) Trichophyton species.

Whitfield's ointment showed suppressive effects on most of the Trichophyton species in media with 80γ per ml concentration, although, some strains were resistant in media with 80γ-100γ per ml concentration respectively (Table 3 a).

b) Epidermophyton species.

Whitfield's ointment also showed suppressive effects on most of the Epidermophyton species in media 80r per ml. However, E. floccosum (No. 12) was resistant to 100γ perml concentration (Table 3 b) .

c) Miclosporum species .

Whitfield's ointment showed suppressive effects on most of the Microsporum species in media containing 100γ per ml concentration (Table 3 c),

d) Deep rnycoses.

Antifungal activities of Whitfield's ointment against deep mycoses were not so significant as those observed in other chemicals (Table 3 d)

4. Antifungal activities of Zinc undecylenate.

a) Trichophyton species.

Suppressive effects of Zinc undecylenate on most of the Trichophyton species were observed in media with 20γ-4Oγ Per ml concentration. However, T. gypseum (No. 85,110) and T. ferrugineum were resistant to 40γ per ml concentration (Table 4 a)

b) Epidermophyton species.

Zinc undecylenate also showed suppressive effects on all of the Epidermophyton species in media with 80γ per ml concentration (Table 4 b).

c) Microsporum species.

The growths of all of Microsporum species were suppressed in media with 20γ-4Oγ per ml concentration (Table 4 c)

d) Deep mycosis.

Antifungal activities of Zinc undecylenate were negligible against all of the deep mycoses (Table 4 d).

5. Antifungal activities of Undecylenic acid.

a) Trichophyton species.

Suppressive effects of Undecylenic acid on most of the Trichophyton species were apparent in media with 40γ-8Oγ Per ml concentration. However, T. gypseum (No. 110)was resistant to 80γ per ml concentration (Table 5 a).

b) Epidermophyton species.

Undecylenic acid showed suppressive effects on most of the Epidermophyton species in media with 40γ per ml concentration. However, E. floccosum (No. 69) was resistant to 40γ per ml concentration (Table 5 b)

c) Microsporum species.

Undecylenic acid in the concentration of 80γ per ml was suppressive on all of the Microsporum species (Table 5 c) .

d) Deep mycoses.

Antifungal activities of Undecylenic acid against all of the deep mycoses were negligible as compared with other chemicals (Table 5 d).

B. Results of the slide culture methods.

1. Antifungal activities of L-1,

The results obtained by slide culture methods of L-1 were the same as those in tube culture methods for Trichophyton, Epidermophyton, Microsporum species and deep mycoses. No mycelium formation were observed In the growth-suppressed slices under

microscopic observation, whereas formations of numerous microconidia, chlamydospore or racquet hyphae were observed in resistant and control slides.

2. Antifungal activities of L-4.

The results of slide culture methods of L.4 were the same as in tube culture methodsfor Trichophyton, Epidermophyton, Microsporum species and deep mycoses.

3. Antifungal activities of control drugs.

The results obtained with slide culture methods of control drugs were the same as in tube culture methods for all species of fungi experimented on and there was no ,significant difference between the results of control drugs and those of synthetic

chemicals.

Discussion

For a long time, many workers have devoted numerous efforts to develope better and more effective antifungal agents. As the result of continued studies Griseofulvin was isolated by Oxford et al. (1939), Fungicidin by Hazen et al. (1950), Candicidin by Lechevalier (1953), Mycobacillin by Majumdar and Bose (1958),

Amphotericin B by eraser and Knox (1959), and these investigators presented data showing that those chemicals possessed effective antifungal activities.

Prior to these studies, Emmons (1933) experimented with antifungal activities of disinfectants, and the results indicated that iodine among the halogen elements had significant antifungal effects. Mayer (1943) carried out similar experiments with

ten synthetic chemicals, and the results indicated that compounds of thiocarbanilides had significant antifungal effects. Kligman and Lewis (1953) reported effective antifungal activities by animal and in vitro tests with Candicidin, Wright et al. (1957) with Nystatin, and Kravetz et al. (1961), Rosenthal (1963) and Spinner et al. (1965) with Am-photericin B.

Since then, Griseofulvin has been used for superficial fungal infections, and at the same time studies of this compound were carried out by Prazak et al. (1960), Dillaha and Jansen (1960), Gentles and Bardes (1960), Pipen (1960), LaBar and Rattier (1960),and El-Nakeeb et al. (1965) , who asserted that Griseofulvin showed good effectiveness for superficial mycoses. but not for deep mycoses. On the other hand a gamma globulin fraction has been used for superficial fungal infection by

Lindsay et al. (1960), and vitamin K^^5 has been used for non-pathogenic fungi by Merrifield and Yang (1965), and these showed good effectiveness. Meanwhile, Finlayson (1764), Melees (1964), Bennett(1964),01ansky (1965), and Dick (1965)

experimented with various kinds of chemicals for deep mycoses and obtained a good effect.

It has been recognized that chemical compounds containing sulfur (S) showed anti-mycobacterial and antifungal activities, and that antimycobacterial com pounds showed distinct trends in antifungal activities (Mayer, 1942, 1964; Lorincz and

Pearson, 1962; Woo and Chang, 1965; and Albright and Hitch 1966). Choi and Lew (1965) reported that thiocarbanilides (L-1 and L-4) had excellent growth-inhibiting activities against mycobacteria including Mycobacterium tuberculosis (H^^37 , R^^v ), Ravenel, BCG, Mycobacterium phlei and Mycobacterium lepraemurium.

Based on the data reported by Choi and Lew (1965), In this study experimentation was carried out to evaluate the antifungal activities of L-1 and L-4 with various species of superficial and deep mycoses and as control experiments, Undecylenic acid, Zinc undecylenate and Whitfield's ointment, which are commonly used at

present as antifungal agents, were included.

As shown in the results of the tube and slide culture methods, the antifungal activitie sof L-1 and L-4 appeared somewhat different from each other L-1 showed the strongest antifungal activities against superficial mycoses, in comparison with

the results of L-4,Undecylenic acid, Zinc undecylenate and Whitfield's ointment (Table 6).

Both L-1 and 74 have shown no effectiveness against deep mycoses so fat as tested in this study (Table 1 d and 2 d).

Control drugs showed no significant differences in their antifungal activities. (Table 3, 4 and 5)

With the data obtained in this in vitro experimentation, a clinical trial of L-1 on as mall scale was carried out, and the results definitely indicated that clinical application of L-1 in the treatment of superficial fungal infections would

bring a most remarkable clearing effect on the diseases.

In these circumstances a well-planned clinical trial on a large scale of L-1 will be a highly promising project, and the study may firmly establish that L-1 is the most effective antifungal therapeutic compound so far developed against superficial mycotic infections.

Conclusion

synthetic chemical compounds of thiocarbanilides, namely L-1 and L-4 were studied for their antifungal activities In vitro, and the results were compared with the antifungal activities of certain commonly used antifungal drugs.

1. L-1 showed remarkable suppressive effects in media with a concentration of 20γ-4Oγ per ml on most of the superficial fungi in the tube method.

2. L-4 did not show such remarkable suppressive effects in the tube method for superficial fungi as did L-1. Also it showed great variation in its suppressive effectiveness.

3. Undecylenic acid showed suppressive effects in media with 40γ-80γ per ml concentration for most of superficial fungi in tube method.

4. Zlnc undecylenate showed suppressive effects in media with 20γ-8Oγ per ml concentration for most of superficial fungi in tube method.

5. Whitfield's ointment showed suppressive effects in media with 80γ-100γ per ml concentration on most of superficial fungi in tube method.

6. Neither L-1 nor L-4 had suppressive effects on deep mycoses.

7. The slide culture method is a simple and practical one to get rapider and more accurate results of experiment than tube culture method.

8. By in vitro study and a clinical trial on a small scale, L-1 proved to be excellently effective for superficial fungi. Through a well-planned clinical trial on a large scale it could be demonstrated in the future that L.1 is the most effective topical antisuperficial fungal agent.