ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1990, p. 2250-2253 0066-4804/90/112250-04$02.

00/0 Copyright X) 1990, American Society for Microbiology

Vol. 34, No. 11

Effects of Butenafine Hydrochloride, a New Benzylamine Derivative, on Experimental Dermatophytosis in Guinea Pigs
TADASHI

ARIKA,1* MAMORU YOKOO,1 TOYOJI HASE,1 TETSUYA MAEDA,' KOUJI AMEMIYA,'

AND

HIDEYO YAMAGUCHI2

Central Research Laboratories, Kaken Pharmaceutical Co., Ltd., 2-28-8, Honkomagome, Bunkyo-ku, Tokyo 1133' and Research Center for Medical Mycology, Teikyo University, 359 Otsuka, Hachioji-shi, Tokyo 192 03,2 Japan
Received 15 March 1990/Accepted 15 August 1990

Butenafine hydrochloride, N-4-tert-butylbenzyl-N-methyl-l-naphthalenemethylamine hydrochloride (butenafine), is a novel antifungal agent of the class of benzylamine derivatives. Butenafine was investigated for its activity against guinea pig dermatophytosis caused by Trichophyton mentagrophytes or Microsporum canis in comparison with those of naftifine, tolnaftate, clotrimazole, and bifonazole. Topical butenafine showed excellent efficacy against dermatophytosis when it was applied once daily, and the effect was superior to those of all four reference drugs. When applied once at 24 or 48 h before infection, the drug exhibited excellent prophylactic efficacy against experimental T. mentagrophytes infection. The concentrations of butenafine in animal skin at 24 and 48 h after application of 0.2 ml of a 1% solution were several hundred times higher than those required to kill T. mentagrophytes and M. canis. The good efficacy of butenafine against dermatophytosis may be attributable to its fungicidal activity and long retention in the skin after topical application.

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Imidazole antifungal agents such as clotrimazole, miconazole, econazole, oxiconazole, and bifonazole have been widely used in the topical treatment of superficial fungal infections. Recently, naftifine, an antifungal agent of the allylamine class, has been developed. Naftifine has a broad spectrum of antifungal activity in vitro and exhibits high efficacy against experimental guinea pig dermatophytosis (1, 4). It interferes with fungal ergosterol biosynthesis by inhibiting the conversion of squalene into squalene epoxide which is catalyzed by squalene epoxidase. Thus, the mechanism of action of naftifine is distinct from that of imidazole antifungal agents which block a later process in the ergosterol biosynthesis pathway involving 14-a-demethylation of lanosterol (3, 7, 9). More recently, it has been clarified that the mode of action of a thiocarbamate antifungal agent, tolnaftate, against dermatophytosis is similar to that of naftifine (2, 8). Butenafine (N-4-tert-butylbenzyl-N-methyl- 1-naphthalenemethylamine hydrochloride) belongs to the class of benzylamine derivatives and was synthesized by Maeda et al. (T. Maeda, T. Arika, K. Amemiya, and K. Sasaki, Chem. Pharm. Bull., in press). Butenafine is easily soluble in methanol, ethanol, dichloromethane, and chloroform; and is only slightly soluble in water; shows a broad spectrum of antifungal activity; and is particularly active against dermatophytes, aspergilli, dimorphic fungi, and dematiaceous fungi (Maeda et al., in press). Like allylamine and thiocarbamate antifungal agents, butenafine inhibits sterol synthesis by blocking the squalene epoxidation step in fungi (T. Hiratani and H. Yamaguchi, unpublished data). In this report, we address the effect of butenafine against guinea pig dermatophytosis caused by Trichophyton mentagrophytes or Microsporum canis. (These data were presented in part at the 27th Interscience Conference on Antimicrobial Agents and Chemotherapy, 4 to 7 October 1987, New York [T. Arika, M. Yokoo, T. Maeda, K. Amemiya, and K. Sasaki, Program Abstr. 27th

Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 980, 1987].)

MATERIALS AND METHODS Animals. Male Hartley strain guinea pigs (weight, 650 to 900 g) were used in this study. Experiments were done with groups of five animals. Test organisms. Clinical isolates of T. mentagrophytes KD-04 and M. canis KD-305 were kindly supplied by H.

Correponding author.
2250

Takahashi, Teikyo University School of Medicine, Tokyo, Japan, and T. Horie, Horie Dermatological Clinic, Tokyo, Japan, respectively. Preparation of inocula. Conidia were harvested from T. mentagrophytes KD-04 cultures grown on potato sucrose agar slants at 27C for 3 weeks. The culture was flooded with sterile saline containing 0.1% Tween 80 and scraped off with the aid of a loop. The conidial suspension was then pipetted into a test tube. After shaking, the suspension was filtered through gauze to remove hyphal fragments and agar blocks. The suspension was adjusted to make a conidial concentration of 2 x 10 cells per ml by counting with a hemacytometer. Conidia and mycelia of M. canis KD-305 were harvested from cultures grown on Sabouraud glucose agar slants at 27C for 3 weeks. The experimental cell suspension was prepared as described above, except that the filtration step was not done. Determination of MIC and MFC. The in vitro fungistatic concentrations were determined by a serial dilution method by using Sabouraud glucose broth. Concentrations of the drugs ranged from 0.003 to 100 ,ug/ml. All tubes containing 3 ml of the assay medium were inoculated with approximately 3 x 104 cells and incubated at 30C for 7 days. The MIC was estimated as the lowest concentration that completely inhibited the growth of dermatophytes. The minimal fungicidal concentration (MFC) was estimated as the lowest concentration that prevented visible growth when subcultured onto Sabouraud glucose agar plates at 30C for 7 days. Infection. Infection was established by the procedure of Sakai et al. (10). Briefly, hair was plucked by hand from an area (3 by 3 cm) on the backs of the guinea pigs to make a

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EFFECTS OF BUTENAFINE ON DERMATOPHYTOSIS

2251

TABLE 1. Fungistatic and fungicidal activities of butenafine, naftifine, tolnaftate, clotrimazole, and bifonazole against T. mentagrophytes KD-04 and M. canis KD-305
Strain
Butenafine MIC MFC MIC Naftifine MFC Tolnaftate MIC MFC Clotrimazole MIC MFC

Bifonazole MIC MFC

(>Lg/ml)

(~Lg/ml)

(tLg/ml)
0.05 0.10

(tLg/ml)
0.05 0.20

(Lg/ml) 0.20 0.39

(,ug/ml) 0.39 0.39

(,ug/ml)
0.39 0.78

T. mentagrophytes KD-04 M. canis KD-305

0.012 0.025

0.012 0.05

(>Lg/ml) 0.78 3.13

44gml)
0.78

(,ug/ml)

1.56

1.56 6.25

hairless square. On the following day, the skin was lightly abraded with sandpaper, and 50 RI of the inoculum (106 cells) was applied with a glass rod (day 0). Drugs and treatment. Butenafine (lot 840317), naftifine (lot 831011), clotrimazole (lot 840614), and bifonazole (lot S11858) were synthesized in our laboratories. Tolnaftate (lot 8509) was purchased from Sagami Kasei Kogyo Co., Ltd., Machida, Japan. Butenafine, naftifine, clotrimazole, and bifonazole were dissolved in a mixture of polyethylene glycol 400 (PEG)-ethanol (75:25; vol/vol); and tolnaftate was dissolved in a mixture of PEG-acetone (75:25; vol/vol) (4). Each guinea pig was topically treated with 0.2 ml of the solution of the test compound. The treatment was started on day 2, 3, or 4 postinfection and was continued for 4 or 10 days. Evaluation. Culture studies were done to assess the efficacy of treatment. Two days after the last treatment, all animals were sacrificed under ether anesthesia, and 10 skin sections were obtained from each treated site. Each section was implanted onto a Sabouraud glucose agar plate containing 500 ,ug of cycloheximide per ml, 50 ptg of kanamycin per ml, and 5 ,ug of gentamicin per ml; and all plates were incubated at 27C for 10 days. The treatment was assessed as effective if no fungal growth was detected. Prophylactic effect. A hairless area (3 by 3 cm) was produced on the back of guinea pigs as described above. On the following day, the skin was treated with 0.2 ml of a 1% solution of butenafine or bifonazole. At 24, 48, or 72 h after drug application, 50 ,ul (106 cells) of the T. mentagrophytes suspension was rubbed onto the pretreated area of the skin with a glass rod. Before infection, the unabsorbed drug was wiped off with a cotton swab containing 70% ethanol. All infected sites were visually examined daily throughout the experimental period to assess skin lesions. The intensity of the lesions was graded 1+ to 4+ by the scoring system of Weinstein et al. (12). On day 17 postinfection, culture studies were done. Concentrations of butenafine in the skin. (i) Extraction of butenafine from the skin. An area (3 by 3 cm) of the backs of the guinea pigs was clipped and treated with 0.2 ml of a 1% butenafine solution. At 24, 48, or 72 h after-butenafine treatment, the unabsorbed drug was wiped off with a cotton swab containing 70% ethanol and the treated skin was removed. After a specimen of the skin was chopped into small pieces (<1 mm) and alkalinized with 1 N NaOH (2 ml), butenafine was extracted twice with 10 ml of methanol. All methanol extracts were combined and concentrated under reduced pressure, D7-butenafine (N-4-tert-butylbenzyl-Nmethyl-i- [2,3, 4,5, 6,7, 8- 2H7]naphthalenemethylamine hydrochloride) was added as an internal standard, and the solution was applied to a glass column of Extrelut (3 g; Merck) and eluted with 30 ml of n-hexane. The eluate was evaporated under nitrogen gas, and the residue was dissolved in CH3CN and N,O-bis(trimethylsilyl)trifluoacetamide (50 ,ul each). The sample was submitted to gas chromatography-mass spectrometry.

(ii) Gas chromatography-mass spectrometry. A JEOL model JMS-D300 mass spectrometer connected with a Shimadzu GC-7A gas chromatograph was used for gas chromatography-mass spectrometry. The flow rate of the helium carrier gas was 50 ml/min. Separation was performed by using a glass column (1 m by 3 mm) packed with 4% OV-101 on 100-120 mesh Gaschrom Q. The column was heated to 270C. Injector and enricher temperatures were 300 and 250C, respectively. Other operating conditions were as follows: electron energy, 70 eV; electron beam current, 200 v.A; and selected monitoring ion, m/z 317 (butenafine), m/z 324 (D7-butenafine). Statistical analysis. The percentage of negative cultures was analyzed by the x2 test. P values of less than 0.05 were regarded as significant.

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RESULTS In vitro fungistatic and fungicidal activities. The fungistatic (MIC) and fungicidal (MFC) activities of butenafine against T. mentagrophytes KD-04 and M. canis KD-305 were compared with those of the reference drugs. MICs and MFCs of butenafine for the two dermatophyte strains were in the range of 0.012 to 0.025 and 0.012 to 0.05 ,ug/ml, respectively (Table 1), showing that butenafine exhibits potent fungicidal activity. The antidermatophyte activity of butenafine in vitro was 4 times greater than that of naftifine and 8 to 128 times greater than those of the other reference drugs. Therapeutic efficacy against T. mentagrophytes infection. In the first experiment, once daily topical treatment with 0.01, 0.1, and 1.0% solutions of butenafine and the reference drugs was started on day 2 postinfection and was continued for 10 days. As shown in Table 2 (experiment 1), 1.0% solutions of butenafine, naftifine, and tolnaftate were satisfactorily effective, with complete eradication of fungi from the focus, while the negative culture rate obtained with a 1.0% solution of clotrimazole was only 28%. The negative rates with 0.1 and 0.01% solutions of butenafine were 94 and 60%, respectively. Comparable concentrations of naftifine and toinaftate were less effective. In another study, the animals were treated for 4 consecutive days. As shown in Table 2 (experiment 2), treatment with a 1% solution of butenafine completely cured the T. mentagrophytes infection, while the rates of cure with naftifine and tolnaftate were 82 and 64%,

respectively.
In the next experiment, once daily treatment was started on day 3 or 4 after infection and was continued for 4 or 10 days. Butenafine was again effective against the experimental T. mentagrophytes infection, although 10 days was necessary to eradicate fungi from the skin lesions (Table 3). The third experiment was performed to compare the therapeutic efficacies of the two different butenafine treatment regimens. Once or twice daily treatment with 0.125, 0.25, 0.5, and 1.0% solutions of butenafine was started on day 4 postinfection and was continued for 10 days. The therapeutic efficacy of twice daily treatment with a 0.125 or

2252

ARIKA ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Therapeutic efficacies of butenafine, naftifine, tolnaftate, and clotrimazole solutions against T. mentagrophytes infection by once daily application for 4 or 10 days starting on day 2 after infection

TABLE 4. Therapeutic efficacy of butenafine solution against T. mentagrophytes infection by once or twice daily application for 10 days starting on day 4 after infection
Treatment
No. (%) of skin sections with negative cultures (n = 50)

Exptmno treatment
Expt 1 0.01% Butenafine 0.1% Butenafine 1.0% Butenafine 0.01% Naftifine 0.1% Naftifine 1.0% Naftifine 0.01% Tolnaftate 0.1% Tolnaftate 1.0% Tolnaftate 1.0% Clotrimazole Placebo (PEG-ethanol) Placebo (PEG-acetone) None Expt 2 1% Butenafine 1% Naftifine 1% Tolnaftate None

Duration of treatment (days)

No. (%) of skin sections with negative cultures (n = 50)

0.125% Butenafine
Once daily .................................. Twice daily ..................................
25 (50) 36 (72)a
30 (60) 40 (80)a

10 10 10 10 10 10 10 10 10 10 10 10

30 (60) 47 (94)" 50 (100)a 2 (4)a.b 29 (58)b 50 (100)a 5 (10)a"b 20 (40)b 50 (100)a 14 (28)b.c 0 (0) 0 (0) 0 (0)
50 41 32 0

0.25% Butenafine
Once daily .................................. Twice daily ..................................

0.5% Butenafine Once daily .................................. Twice daily .................................. 1.0% Butenafine
Once daily .................................. Twice daily ..................................

48 (96) 50 (100)b

50 (100) 50 (100)b 0 (0)

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None ..........
I

........................

4 4 4

(100) (82)d (64)e (0)

P < 0.05 versus the once daily treatment group. Not significant versus the once daily treatment group.

" P < 0.001 versus the 0.01% butenafine treatment group. b p < 0.001 versus the 0.1% butenafine treatment group. C P < 0.01 versus the 0.01% butenafine treatment group. d p < 0.01 versus the 1.0% butenafine treatment group. e p < 0.001 versus the 1% butenafine treatment group.

0.25% solution was better than that of once daily treatment. However, once daily treatments with 0.5 and 1% solutions of butenafine were sufficiently effective (Table 4). Therapeutic efficacy against M. canis infection. Once daily topical treatment with a 0.1 or 1.0% solution of butenafine and the reference drugs was started on day 2 or 4 postinfection and was continued for 10 days. Butenafine, naftifine, and tolnaftate showed almost the same activity against M. canis infections as they did against T. mentagrophytes
TABLE 3. Therapeutic efficacies of butenafine and naftifine solutions against T. mentagrophytes infection by once daily application for 4 or 10 days starting on day 3 or 4 after infection
TTreatment

infections (Table 5). Clotrimazole, which was the least potent against T. mentagrophytes infection, again had activity lower than those of the other drugs in this model. Prophylactic effects of butenafine and bifonazole. Guinea pigs were pretreated with 0.2 ml of 1% solutions of the drugs at 24, 48, or 72 h before T. mentagrophytes infection. Lesions in the untreated group were the most intense (score 4+) on day 12 (Fig. 1). In the group that was pretreated with bifonazole 24 h before infection, lesions developed in four of five animals on day 17 after infection. When a 1% solution of butenafine was applied once at 24 or 48 h before infection, no lesion developed, and mycological examination of the lesions performed on day 17 after infection indicated no viable
TABLE 5. Therapeutic efficacies of butenafine, naftifine, tolnaftate, and clotrimazole solutions against M. canis infection by once daily application for 10 days starting from day 2 or 4 after infection
Treatment

No. (%) of skin sections Treatment cultures negative rawith period (days) (n = 50)

Treatment Treatment period

atm

No.

(%)

of skin sections
cultures

with

negative

~~~~(n

50)

1% Butenafine 1% Naftifine None 1% Butenafine 1% Naftifine None

1% Butenafine 1% Naftifine None 1% Butenafine 1% Naftifine None
p

3-6 3-6

39 (78)a 8 (16) 0 (0)

3-12 3-12
4-7 4-7 4-13 4-13
the naftifine treatment
group.

50 (100) 45 (90) 0 (0) 19 (38)b 8 (16) 0 (0)

50 (100)a 26 (52) 0 (0)

0.1% Butenafine 1.0% Butenafine 0.1% Naftifine 1.0% Naftifine 0.1% Tolnaftate 1.0% Tolnaftate 1.0% Clotrimazole Placebo (PEG-ethanol) Placebo (PEG-ethanol)
None

2-11 2-11 2-11 2-11 2-11 2-11 2-11 2-11 2-11

47 50 21 50

(94) (100) (42)a.b (100) 8 (16)a.b 42 (84)' 30 (60)a.b 0 (0) 0 (0) 0 (0)

1.0% Butenafine 1.0% Naftifine 1.0% Tolnaftate

None

4-13 4-13 4-13

49 (98) 39 (78)d 24 (48)e 0 (0)

<
<

bp

0.001

versus

o.05.

" P < 0.001 versus the 0.1% butenafine treatment group. b p <0.001 versus the 1.0% butenafine treatment group. C P < 0.01 versus the 1.0% butenafine treatment group. d p < 0.01 versus the 1.0% butenafine treatment group. I P < 0.001 versus the 1.0% butenafine treatment group.

VOL. 34, 1990

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EFFECTS OF BUTENAFINE ON DERMATOPHYTOSIS

2253

3
'3)

w
0

Un

2
0

uz

UL)

O
1

1 ~z
3 5

I-

11 13 15 17 9 Days after infection

FIG. 1. Prophylactic effects of butenafine and bifonazole against T. mentagrophytes infection. Symbols: 0, untreated control; *, 1% bifonazole (24 h before infection); A, 1% butenafine (72 h before infection); O, 1% butenafine (48 h before infection); x, 1% butenafine (24 h before infection).

shows a prophylactic effect against the infection. They suggested that this effect might be attributable to the long cutaneous retention of the drug after topical application. Butenafine exerted excellent prophylactic efficacy when applied even at 48 h before infection (Fig. 1). On the other hand, the concentration of butenafine in the skin at 24, 48, and 72 h after application of 0.2 ml of a 1% solution was several hundred times higher than the MFCs against T. mentagrophytes and M. canis (Table 1). Despite the high concentration of butenafine in the skin, lesions developed in the group that was pretreated with butenafine 72 h before infection (Fig. 1). Takahashi et al. (11) reported that many antifungal agents might have reduced potencies when they are bound to horny materials in the epidermis. Further pharmacokinetic studies are needed to elucidate whether the potency of butenafine decreases in the horny layer. From the results of this study, it appears that the efficacy of butenafine may be due to its fungicidal activity and long cutaneous retention after topical application and that once daily application of butenafine may have potential for the treatment of dermatophytosis in humans.
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fungi, while in the group that was pretreated with a 1% solution of butenafine 72 h before infection, lesions developed in two of five animals. Concentrations of butenafine in the skin. The concentration of butenafine in the skin of five guinea pigs was measured at 24, 48, or 72 h after application of 0.2 ml (2 mg of butenafine) standard of a 1% solution. The concentration (mean deviation) of butenafine in the skin at 24 h was 31.5 4.9 ,ug/g of tissue, and it gradually decreased to 18.3 6.9 ,ug/g at 48 h and to 8.8 5.1 ,ug/g at 72 h.

LITERATURE CITED 1. Georgopoulos, A., G. Petranyi, H. Mieth, and J. Drews. 1981. In vitro activity of naftifine, a new antifungal agent. Antimicrob.

DISCUSSION Butenafine was investigated for its activity against guinea pig dermatophytosis in comparison with the activities of naftifine, tolnaftate, clotrimazole, and bifonazole. Because the experimental dermatophytosis produced in the dorsal skin of guinea pigs causes Kerion Celsi-like lesions at advanced stages of infection, topical treatment of the drugs was started on day 2, 3, or 4 postinfection, when lesions were barely detectable. Butenafine exhibited excellent therapeutic efficacy against T. mentagrophytes and M. canis infections when applied once daily for 10 days; its effect was significantly superior to those of naftifine, tolnaftate, and clotrimazole (Tables 2, 3, and 5). Unlike butenafine, clotrimazole was active against M. canis infection (60% cure rate) but was less active against T. mentagrophytes infection (28% cure rate) (Tables 2 and 5). As reported elsewhere (4, 5), the efficacy of imidazole antifungal agents such as econazole against experimental dermatophytosis is dependent on the strain of T. mentagrophytes used for challenge. The MICs of clotrimazole against T. mentagrophytes and M. canis were 0.39 and 0.78 g/ml, respectively, in the present study (Table 1). The difference in the efficacy of clotrimazole between T. mentagrophytes and M. canis infections might be associated with the virulence of the fungi or other pathogenic factors. As reported by Plempel et al. (6), when bifonazole is topically applied before infection with T. mentagrophytes, it

Agents Chemother. 19:386-389. 2. Morita, T., and Y. Nozawa. 1985. Effects of antifungal agents on ergosterol biosynthesis in Candida albicans and Trichophyton mentagrophytes: differential inhibitory site of naphthiomate and miconazole. J. Invest. Dermatol. 85:434-437. 3. Paltauf, F., G. Daum, G. Zuder, G. Hogenauer, G. Schulz, and G. Seidl. 1982. Squalene and ergosterol biosynthesis in fungi treated with naftifine, a new antimycotic agent. Biochim. Biophys. Acta 712:268-273. 4. Petranyi, G., A. Georgopoulos, and H. Mieth. 1981. In vivo antimycotic activity of naftifine. Antimicrob. Agents Chemother. 19:390-392. 5. Petranyi, G., J. G. Meingassner, and H. Mieth. 1987. Activity of terbinafine in experimental fungal infections of laboratory animals. Antimicrob. Agents Chemother. 31:1558-1561. 6. Plempel, M., E. Regal, and H. Buchel. 1983. Antimycotic efficacy of bifonazole in vitro and in vivo. Arzneim. Forsch. Drug Res. 33:517-524. 7. Ryder, N. S., and M. C. Dupont. 1985. Inhibition of squalene epoxidase by allylamine antimycotic compounds. Biochem. J. 230:765-770. 8. Ryder, N. S., I. Frank, and M. C. Dupont. 1986. Ergosterol biosynthesis inhibition by the thiocarbamate antifungal agents tolnaftate and tolciclate. Antimicrob. Agents Chemother. 29: 858-860. 9. Ryder, N. S., G. Seidl, and P. F. Troke. 1984. Effect of the antimycotic drug naftifine on growth of and sterol biosynthesis

in Candida albicans. Antimicrob. Agents Chemother. 25:483487. 10. Sakai, S., T. Kada, G. Saito, N. Muraoka, and Y. Takahashi. 1952. Studies on chemotherapy of Trichophyton infection. 1. Antifungal properties of halogen phenol esters. J. Sci. Res. Inst. 46:113-117. 11. Takahashi, H., A. Hasegawa, 0. Kaneko, A. Saito, and Y. Tanaka. 1986. Clinical studies of fungicidal agents with respect to their relationships in vitro and in vivo, p. 227-240. In K. Iwata and H. Vanden Bossche (ed.), In vitro and in vivo evaluation of antifungal agents. Elsevier Science Publishers, Amsterdam. 12. Weinstein, M. J., E. M. Oden, and E. Moss. 1965. Antifungal properties of tolnaftate in vitro and in vivo, p. 595-601. Antimicrob. Agents Chemother. 1964.