ME1111, a New Antifungal Agent for Topical Treatment of Onychomycosis: Characterization of Antifungal Activity and Nail Penetration

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1 AAC Accepted Manuscript Posted Online 7 December 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 ME1111, a New Antifungal Agent for Topical Treatment of Onychomycosis: Characterization of Antifungal Activity and Nail Penetration Yuji Tabata* $, Naomi Takei-Masuda $, Natsuki Kubota, Sho Takahata, Makoto Ohyama, Kaori Kaneda, Maiko Iida, Kazunori Maebashi Meiji Seika Pharma Co., Ltd., Pharmaceutical Research Center, Yokohama, Japan, $ These authors contributed equally to the manuscript *Corresponding author: Yuji Tabata, Ph.D. Meiji Seika Pharma Co., Ltd., Pharmaceutical Research Center 760 Morooka-cho, Kohoku-ku, Yokohama , Japan Tel: Fax: yuuji.tabata@meiji.com Running title: ME1111, a New Topical Antifungal for Onychomycosis Key words: ME1111, antifungal agent, onychomycosis, dermatophytes, minimum inhibitory concentration 23

2 Abstract Fungal nail infection (onychomycosis) is a prevalent disease in many areas of the world with a high incidence approaching 23%. Available antifungals to treat this disease suffer from a number of disadvantages, necessitating the discovery of new efficacious and safe antifungals. Here we evaluated the in vitro antifungal activity and nail penetration ability of ME1111, a novel antifungal agent, along with comparator drugs including ciclopirox, amorolfine, terbinafine and itraconazole. ME1111 showed potent antifungal activity against Trichophyton rubrum and Trichophyton mentagrophytes (the major etiologic agents of onychomycosis) strains isolated in Japan and reference fungal strains with an minimum inhibitory concentration (MIC) range of 0.12 to 0.5 mg/l, and an MIC 50 and MIC 90 of 0.5 mg/l for both. Importantly, none of the tested isolates showed elevated MIC to ME1111. Moreover, the antifungal activity of ME1111 was minimally affected by 5% wool keratin powder, in comparison to the other antifungals tested. The ME1111 solution was able to penetrate human nails and inhibit fungal growth in a dose-dependent manner using the TurChub assay. In contrast, 8% ciclopirox and 5% amorolfine nail lacquers showed no activity under the same conditions. ME1111 demonstrated approximately 100-fold greater selectivity in inhibition of Trichophyton spp. compared to human cell lines. Our findings demonstrate that ME1111 possesses potent anti-dermatophyte activity, maintains this activity in the presence of keratin, and possesses excellent human nail permeability. These results suggest that ME1111 is a promising topical medication for the treatment of onychomycosis, and therefore warrants further clinical evaluation. 46

3 Introduction Onychomycosis is a progressive fungal infection of the nails, which, if left untreated, can cause nail destruction and deformity. This disease affects up to 23% of adults worldwide (1-4). The prevalence of this disease increases in the elderly, affecting approximately 28% of people over the age of 60 (5). Onychomycosis is primarily caused by dermatophytes, particularly Trichophyton rubrum and Trichophyton mentagrophytes (1). Although both oral (terbinafine and itraconazole) and topical (ciclopirox, amorolfine, efinaconazole, and tavaborole) medications are available for the treatment of fungal nail infection, these agents suffer from a number of disadvantages: 1) Oral onychomycosis drugs can cause liver toxicity issues and/or drug-drug interaction concerns (6), making them difficult to prescribe to elderly patients, especially those taking multiple medications. 2) The efficacy of topical therapeutics is relatively low, presumably due to poor nail permeability and high keratin binding. Additionally, the recently approved topical drugs efinaconazole (7-9) and tavaborole (6, 10) have low efficacy and local side effects issues. As a result of the various drawbacks of currently available therapeutics, there is an opportunity for the development of new topical agents with greater efficacy and fewer side effects. In order to be effective against onychomycosis, a topical antifungal should have low molecular weight (an important factor for nail penetration) (6, 11) and low affinity to keratin (a major component of the nail) (12). Therefore, a topical antifungal having both a low molecular weight and low affinity to keratin would be considered a promising therapeutic for onychomycosis. 2-(3,5-Dimethyl-1H-pyrazol-1-yl)-5-methylphenol (ME1111) is a novel anti-dermatophytic drug (Fig. 1) discovered by Meiji Seika Pharma Co., Ltd. (Meiji; Tokyo, Japan) through an optimization process directed at selecting compounds with: 1) potent anti-dermatophyte activity, 2) favourable physicochemical

4 and nail permeability properties, and 3) small molecular size. Further research demonstrated that ME1111 is a first-in-class, low molecular weight compound with antifungal activity, mediated by the inhibition of succinate dehydrogenase (complex II), a critical enzyme involved in mitochondrial respiratory electron transfer (13). Thus, ME1111 has desirable properties as a candidate compound for the topical treatment of onychomycosis. The aim of this study was to: 1) characterize the antifungal activity of ME1111 against clinical and reference isolates of Trichophyton species, 2) evaluate whether keratin reduces ME1111 s antifungal activity, and 3) determine ME1111 s ability to penetrate human nails MATERIALS AND METHODS Test strains. Japanese clinical isolates of T. rubrum isolated between 1999 and 2011 (n = 62 strains) and T. mentagrophytes isolated between 1999 and 2011 (n = 47 strains) were obtained from Teikyo University Institute of Medical Mycology (Tokyo, Japan) and the National BioResource Project-Pathogenic Microbes (Chiba, Japan). In addition, two reference dermatophyte strains (T. rubrum ATCC MYA-4438 and T. mentagrophytes ATCC MYA-4439) and two other dermatophyte strains representing other species (T. tonsurans ATCC 56186, Epidermophyton floccosum ATCC 26072), obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) were also evaluated. Antifungal agents. The following agents were tested in this study (Fig. 1): ME1111 was provided by Meiji. Amorolfine hydrochloride was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Ciclopirox olamine, terbinafine hydrochloride, and itraconazole were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). All test compounds were supplied in powder form and reconstituted in dimethyl sulfoxide (DMSO). Antifungal susceptibility testing. MIC testing was performed according to the CLSI

5 M38-A2 standard microbroth dilution methodology for the susceptibility testing of dermatophytes (14). Briefly, serial dilutions of test compounds were prepared in RPMI1640 (Life Technologies, Grand Island, NY, USA) buffered with 3-(N-morpholino) propanesulfonic acid (MOPS; Nakarai Tesque, Ltd., Kyoto, Japan) in a range of: mg/l for ME1111; mg/l for amorolfine, and mg/l for ciclopirox. Microtiter plates were incubated at 35 C for 4 days. MICs were read visually, and the MIC endpoint was defined as the minimum concentration of the test compounds that prevented visible growth relative to the growth control. MIC 50 and MIC 90 were defined as the lowest concentration to inhibit 50% and 90% of the strains tested, respectively. Measurement of anti-dermatophytic activity of ME1111 in the presence and absence of keratin. For a topical drug to penetrate the nail plate it should have low keratin-binding properties. To determine the binding potential of ME1111 to keratin, we measured the susceptibility of dermatophytes to ME1111 and comparators in the presence and absence of keratin. Briefly, MICs against T. rubrum and T. mentagrophytes in RPMI M MOPS, ph 7.0 with or without 5% wool keratin powder (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were measured by colorimetric assay using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Wool keratin was defatted according to the method reported by Uchida et al (15). MIC was defined as the lowest concentration at which the inhibition was > 80% compared to that of the respective growth control. Geometric mean MICs were calculated from MICs of 7 strains for T. rubrum and 6 strains for T. mentagrophytes. Measurement of anti-dermatophytic activity after human nail penetration. To determine the ability of ME1111 and comparators to penetrate human nail plates, antifungal activity after human nail penetration was measured in the TurChub assay as 121 described earlier (6, 7, 16). Briefly, the TurChub cells were filled with Sabouraud

6 dextrose agar, inoculated with T. rubrum, and placed under human full thickness nails. Two microliters each of ME1111 solution (2, 5, 10 and 15% w/v), 8% ciclopirox nail lacquer (Penlac ), or 5% amorolfine nail lacquer (Loceryl ) were applied to the human nails, and the cells were occluded and incubated for 7 days. The efficacy of each test solution was determined by measuring the zone-of-inhibition (ZOI) against T. rubrum in the TurChub cell. Effect of ME1111 against host cells. To show whether ME1111 is toxic to human cells, we used anti-cell proliferation assays (17). Briefly, the concentration that caused 50% inhibition of cell proliferation (IC 50 ) was measured in four different human cell lines obtained from the ATCC: K562 (leukaemia, ATCC CCL-243), HepG2 (liver tumour, ATCC HB-8065), U937 (lymphoma, ATCC CRL-1593), and A431 (skin, ATCC CRL-1555). Cell suspension in growth medium was placed in 96-well microtiter plates in an atmosphere of 5% CO 2 at 37 C. After 24 hours, test substances were added respectively for an additional 72-hour incubation period. At the end of the incubation period, AlamarBlue reagent (AbD Serotec, Kidlington, Oxford, UK) was added to evaluate cell proliferation RESULTS ME1111 demonstrated broad spectrum anti-dermatophyte activity. To confirm that ME1111 is a compound with potent anti-dermatophyte activity for onychomycosis, we evaluated its in vitro antifungal activity against the major etiologic agents of onychomycosis, T. rubrum (n = 62 strains) and T. mentagrophytes (n = 47 strains), clinical strains isolated in Japan and reference strains (Table 1). Our data showed that the MIC of ME1111 against dermatophytes ranged from 0.12 to 0.5 mg/l, with an MIC 50 and MIC 90 of 0.5 mg/l (Table 1). Of note, none of the isolates tested showed an elevated MIC. ME1111 inhibited a clinical isolate of T. mentagrophytes with an elevated terbinafine

7 MIC (>0.5 mg/l) where the MIC of ME1111 was also 0.5 mg/l. MICs of amorolfine, ciclopirox and itraconazole against this strain were 0.12 mg/l, 0.25 mg/l and mg/l, respectively. In addition to inhibiting T. rubrum and T. mentagrophytes, ME1111 showed potent antifungal activity against other dermatophytes, E. floccosum, and T. tonsurans with an MIC of 0.25 mg/l. These results demonstrate that ME1111 possesses broad-spectrum anti-dermatophyte activity. Anti-dermatophytic activity of ME1111 was minimally affected by keratin. To evaluate the antifungal activity of ME1111 in the presence of keratin, the MICs of ME1111 and other antifungal agents were determined against 7 strains of T. rubrum and 6 strains of T. mentagrophytes in RPMI1640 with or without 5% keratin (Table2). Geometric mean MICs of ME1111 did not change or were slightly increased (1.5-fold) in the presence of 5% keratin. In contrast, the geometric mean MICs in the presence of 5% keratin against T. rubrum and T. mentagrophytes increased 512-fold for ciclopirox, 8 and 16-fold for amorolfine, 3.3 and 4.5-fold for terbinafine, and, 71 and 228-fold for itraconazole, respectively. These results indicate that the anti-dermatophytic activity of ME1111 was minimally affected by keratin compared to other antifungals. ME1111 was able to penetrate the human nail and inhibited the growth of T. rubrum. The ability of a topical antifungal agent to penetrate the nail plate is critical to its efficacy in the treatment of onychomycosis. Thus, the human nail penetration of ME1111 and those of comparators (8% ciclopirox nail lacquer and 5% amorolfine nail lacquer) were measured using the TurChub model. Our data showed that ME1111 solutions (2, 5, 10 and 15% w/v) were able to penetrate the nail plate and inhibit the growth of T. rubrum in a dose-dependent manner. In contrast, 8% ciclopirox and 5% amorolfine nail lacquers did not show antifungal activity under the same conditions (Table 3; Fig. 2) indicating that

8 they failed to penetrate the nail plate. Thus, ME1111 solutions showed greater efficacy against T. rubrum than 8% ciclopirox nail lacquer and 5% amorolfine nail lacquer after penetrating human nail plates. ME1111 demonstrated selective toxicity. The effects of ME1111 and comparators on host cell proliferation were evaluated using four human cell lines. The IC 50 values of ME1111 in K562, HepG2, U937, and A431 cells were 47, 37, 33, and 40 mg/l, respectively. In contrast, MIC 50 s and MIC 90 s of ME1111 against Trichophyton spp. were 0.5 mg/l (Table 4). IC 50 values of amorolfine in these cell lines were less than 10 mg/l, and those of ciclopirox were less than 1 mg/l. The MIC 90 s of amorolfine and ciclopirox against T. rubrum were 0.06 mg/l and 0.25 mg/l, respectively (Table 4). ME1111 demonstrated good therapeutic index (TI; IC 50 for human cell proliferation / MIC 90 for Trichophyton spp.). All TIs of ME1111 were more than DISCUSSION In this study we demonstrated that ME1111 possesses broad-spectrum activity against dermatophytes that cause nail infections. Moreover, this activity was not reduced in the presence of keratin. Additionally, we showed that ME1111 was able to penetrate human nail plates and inhibit fungal growth. Among the antifungals tested, terbinafine, amorolfine, and itraconazole showed stronger in vitro antifungal activity than ME1111. ME1111 demonstrated as potent antifungal activity as ciclopirox against clinical dermatophyte strains isolated from Japanese patients, including T. rubrum and T. mentagrophytes, which are common etiologic agents of onychomycosis (Table 1). These results are in line with the activity of ME1111 against clinical dermatophyte isolates obtained from patients in the United States (18), where the MIC 90 against the U.S. isolates was 0.25 mg/l. The finding that

9 susceptibility of dermatophyte isolates obtained from the U.S. sites to ME1111 was similar to that of non-u.s. sites, indicates that there is no difference in ME1111 susceptibility within the dermatophyte species obtained worldwide. To date, susceptibility to ME1111 has been measured against more than 500 strains of dermatophytes (400 strains were tested in reference 18 and 109 strains were tested in this study), and the results show that none of the tested isolates have elevated MICs to ME1111. Moreover, it was reported that MICs of ME1111 against 7 strains of terbinafine-resistant T. rubrum were not elevated (18). Our study also demonstrated the potent activity of ME1111 against a terbinafine-resistant T. mentagrophytes strain. These results suggest that ME1111 demonstrates a potent activity against terbinafine-resistant strains, but further tests with strains presenting high terbinafine MICs are needed. In this study, the antifungal activity of ME1111 was unaffected or slightly increased (1- to 1.5-fold) in the presence of 5% keratin. In contrast, the antifungal activity of ciclopirox, amorolfine, terbinafine and itraconazole was diminished in the presence of 5% keratin. It has been reported that the antifungal activity of some antifungal agents diminishes in the presence of keratin, a main component of nails (12). In this regard, Osborne et al. showed that incubation of terbinafine with nail powder increased the minimum fungicidal concentration of this drug from 0.03 mg/l to 4 mg/l (19). This result may explain why topical solutions of terbinafine failed to meet their primary endpoint for the treatment of onychomycosis (20). The antifungal activity of ME1111 is minimally affected by keratin, as compared to other topical antifungals tested. This result indicates that keratin binding affinity of ME1111 is lower than those of other topical antifungals, which may be due to its unique physicochemical properties (i.e., LogP, water solubility, molecular weight etc.). Our findings show that ME1111 permeated across the human nail plate and exhibited dose-dependent antifungal activity. In contrast, 8% ciclopirox and 5% amorolfine showed

10 no antifungal activity under the same conditions, indicating that these antifungals failed to penetrate human nails. The potent nail penetration activity of ME1111 was also shown in other assays (21, 22). Nail penetration is another important factor for the efficacy of topical onychomycosis drugs since the main type of nail disease (distal subungual onychomycosis) resides in the nail bed. Therefore, antifungals should be able to penetrate the nail in order to kill the disease-causing dermatophytes. The inability of antifungal agents to penetrate the nail plate could be explained by the complex anatomical structure of the nail. Due to the special chemical nature of the nail plate, drug transport through it is influenced by the structure and the physicochemical properties of the drug molecule. Low molecular weight is reported to be an important factor for nail penetration and onychomycosis treatment (6). During initial hit identification, we showed that nail penetration of ME1111 analogues of larger molecule did not penetrate as well as low molecular weight compounds (data not shown). As a result of these molecular weight findings, we selected ME1111 ( g/mol) (Fig. 1). The low molecular weight of ME1111 is thought to play an important role in its human nail penetration. Of note, although ciclopirox is as small as ME1111, it failed to efficiently penetrate the nail plate, which could be due to its higher affinity to keratin relative to ME1111 (data not shown). ME1111 demonstrated approximately 60-fold greater selectivity in growth inhibition of Trichophyton spp. compared to host cells (Table 4). This selectivity in growth inhibition is likely due to the selective inhibition of succinate dehydrogenase in Trichophyton spp. over the same enzyme in human cells (13). Compared to other antifungal agents tested, ME1111 demonstrated weak inhibitory effects on human cell line proliferation. TIs of ME1111 (>60) is better than that of ciclopirox (<3), and that of amorolfine (<60) with one exception (TI for HepG2 and T. rubrum was 127). Furthermore, reports suggest that cumulative skin irritation is of little concern using ME1111 solution (23).

11 In conclusion, we demonstrated that ME1111: 1) has potent antifungal activity against dermatophytes, even in the presence of keratin, 2) demonstrates excellent human nail plate penetration, 3) has a good toxicity profile against human cells. These properties, which differ from those of currently available topical agents and other investigational drugs, suggest that ME1111 should be a promising candidate for the topical treatment of onychomycosis. As a result, clinical trials to assess the safety and efficacy of ME1111 in the treatment of onychomycosis are warranted Acknowledgments We thank Professor Mahmoud Ghannoum (Case Western Reserve University) for critical review and suggestions on the manuscript. We thank Ms. Kazue Nagano and Ms. Naoko Watanabe for their technical assistance with the experiments, and Ms. Noriko Tagata for assisting in preparation of the manuscript. This study was supported by Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). All authors are employees of Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) and most of the authors are stockholders in Meiji Holdings (Tokyo, Japan). Japanese clinical isolates were provided by Teikyo University Institute of Medical Mycology and the National BioResource Project-Pathogenic Microbes in Japan ( References 1. Ghannoum MA, Hajjeh RA, Scher R, Konnikov N, Gupta AK, Summerbell R, Sullivan S, Daniel R, Krusinski P, Fleckman P, Rich P, Odom R, Aly R, Pariser D, Zaiac M, Rebell G, Lesher J, Gerlach B, Ponce-de-Leon GF, Ghannoum A, Warner J, Isham N, Elewski B A large-scale North American study of fungal isolates from nails: the frequency of onychomycosis,

12 fungal distribution, and antifungal susceptibility patterns. J Am Acad Dermatol 43: Gupta AK, Jain HC, Lynde CW, MacDonald P, Cooper EA, Summerbell RC Prevalence and epidemiology of onychomycosis in patients visiting physicians' offices: a multicenter Canadian survey of 15,000 patients. J Am Acad Dermatol 43: Heikkilä H, Stubb S The prevalence of onychomycosis in Finland. Br J Dermatol 133: Ghannoum M, Isham N Fungal Nail Infections (Onychomycosis): A Never-Ending Story? PLoS Pathog 10: e doi: /journal.ppat Elewski BE, Charif MA Prevalence of onychomycosis in patients attending a dermatology clinic in northeastern Ohio for other conditions. Arch Dermatol 133: Alley MRK, Baker SJ, Beutner KR, Plattner J Recent progress on the topical therapy of onychomycosis. Expert Opin Investig Drugs 16: Sugiura K, Sugimoto N, Hosaka S, Katafuchi-Nagashima M, Arakawa Y, Tatsumi Y, Jo Siu W, Pillai R The low keratin affinity of efinaconazole contributes to its nail penetration and fungicidal activity in topical onychomycosis treatment. Antimicrob Agents Chemother 58: Elewski BE, Rich P, Pollak R, Pariser DM, Watanabe S, Senda H, Ieda C, Smith K, Pillai R, Ramakrishna T, Olin JT Efinaconazole 10% solution

13 in the treatment of toenail onychomycosis: Two phase III multicenter, randomized, double-blind studies. J Am Acad Dermatol 68: Patel T, Dhillon S Efinaconazole: first global approval. Drugs 73: Toledo-Bahena ME, Bucko A, Ocampo-Candiani J, Herz-Ruelas ME, Jones TM, Jarratt MT, Pollak RA, Zane LT The efficacy and safety of tavaborole, a novel, boron-based pharmaceutical agent: phase 2 studies conducted for the topical treatment of toenail onychomycosis. J Drugs Dermatol 13: Mertin D, Lippold BC In-vitro permeability of the human nail and of a keratin membrane from bovine hooves: prediction of the penetration rate of antimycotics through the nail plate and their efficacy. J Pharm Pharmacol 49: Murdan S Drug delivery to the nail following topical application. Int J Pharm 236: Takahata S, Tabata Y, Takei-Masuda N, Kubota N, Maebashi K Mechanism of action of ME1111, a new antifungal agent for topical treatment of onychomycosis, abstr F Abstr 54th Intersci Conf Antimicrob Agents Chemother. American Society for Microbiology, Washington, DC Clinical and Laboratory Standards Institute Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, 2nd ed.

14 Approved standard M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA Uchida K, Yamaguchi H Studies on the affinity of terbinafine with keratine. Jpn J Med Mycol 34: Traynor MJ, Turner RB, Evans CRG, Khengar RH, Jones SA, Brown MB Effect of a novel penetration enhancer on the ungual permeation of two antifungal agents. J Pharm Pharmacol 62: Ahmed SA, Gogal Jr RM, Walsh JE A New Rapid and Simple non-radioactive Assay to Monitor and Determine the Proliferation of Lymphocytes: An Alternative to [ 3 H]thymidine Incorporation Assay. J Immunol Methods. 170: Ghannoum M, Isham N, Long L. 8 June 2015 In Vitro Antifungal Activity of ME1111, a New Topical Agent for Onychomycosis, against Clinical Isolates of Dermatophytes Antimicrob. Agents Chemother. doi: /aac Osborne CS, Leitner I, Favre B, Ryder NS Antifungal drug response in an in vitro model of dermatophyte nail infection. Med Mycol. 42: Elewski BE, Ghannoum MA, Mayser P, Gupta AK, Korting HC, Shouey RJ, Baker DR, Rich PA, Ling M, Hugot S, Damaj B, Nyirady J, Thangavelu K, Notter M, Parneix-Spake A, Sigurgeirsson B Efficacy, safety and tolerability of topical terbinafine nail solution in patients with mild-to-moderate toenail onychomycosis: results from three randomized studies using

15 double-blind vehicle-controlled and open-label active-controlled designs. J Eur Acad Dermatol Venereol. 27: Hui X, Maibach H In vitro human nail plate penetration of a novel antifungal agent ME1111, abstr F Abstr 54th Intersci Conf Antimicrob Agents Chemother. American Society for Microbiology, Washington, DC Tabata Y, Nomoto M, Chikada T, Kubota N, Takei-Masuda N, Takahata S, Maebashi K In vitro and in vivo nail penetration of ME1111, a novel antifungal agent for topical treatment of onychomycosis, abstr F Abstr 54th Intersci Conf Antimicrob Agents Chemother. American Society for Microbiology, Washington, DC Ago K, Nomoto M, Chikada T, Uchida M, Tsuchiya T, Shibasaki S, Maebashi K, Hiratsuka K Nonclinical safety assessment of ME1111, a novel antifungal agent for the topical treatment of onychomycosis, abstr F Abstr 54th Intersci Conf Antimicrob Agents Chemother. American Society for Microbiology, Washington, DC. 351

16 TABLE 1 In vitro antifungal activity of ME1111 and four comparators against clinical isolates of T. rubrum and T. mentagrophytes isolated in Japan Organism MIC (mg/l) Reference (Number of strains) Drug Range MIC 50 MIC 90 strain # T. rubrum ME (62) Ciclopirox Amorolfine Terbinafine >0.5 Itraconazole T. mentagrophytes ME (47) Ciclopirox Amorolfine Terbinafine > Itraconazole # Reference strains for T. rubrum and T. mentagrophytes are ATCC MYA-4438 and ATCC MYA-4439, respectively

17 TABLE 2 In vitro antifungal activity of ME1111 and four reference drugs against T. rubrum and T. mentagrophytes with or without 5% wool keratin powder Organism Geometric mean MICs (mg/l) a determined in RPMI1640: (Number of strains) Drug without keratin with 5% keratin Ratio T. rubrum ME (7) Ciclopirox Amorolfine Terbinafine Itraconazole T. mentagrophytes ME (6) Ciclopirox Amorolfine Terbinafine Itraconazole a: >64, >2, >1, >0.25 mg/l for ciclopirox, itraconazole, amorolfine and terbinafine were calculated as 128, 4, 2, 0.5 mg/l. 364

18 TABLE 3 Anti-dermatophytic activity of ME1111 after human nail penetration in the TurChub model Treatment Number of cells Zone-of-inhibition (cm) Mean ± SD Placebo solution ± % ME1111 solution ± % ME1111 solution ± % ME1111 solution ± % ME1111 solution ± % Ciclopirox lacquar (Penlac ) ± % Amorolfine lacquer (Loceryl ) ±

19 TABLE 4 Inhibitory effects of ME1111 and other antifungals on human cell proliferation and Trichophyton spp. growth Drug Human cells IC 50 (mg/l) Trichophyton spp. MIC 90 (mg/l) K562 HepG2 U937 A431 T. rubrum T. mentagrophytes ME Ciclopirox Amorolfine

20 373 N OH O 374 ME1111 Ciclopirox 375 O N 376 Amorolfine Terbinafine Itraconazole 379 FIG. 1 Chemical structures of ME1111 and other antifungals for dermatophytes. 380

21 (a) 10% ME1111 solution (b) 8% Ciclopirox lacquer (c) 5% Amorolfine lacquer FIG. 2 Anti-dermatophytic activity of ME1111, ciclopirox and amorolfine lacquers after human nail penetration. The drug permeation and efficacy of ME1111 solutions, 8% ciclopirox nail lacquer (Penlac ), and 5% amorolfine nail lacquer (Loceryl ) through human nails were measured using the TurChub model. ME1111 solutions 10% w/v (a) showed greater efficacy against T. rubrum than 8% ciclopirox nail lacquer (b) and 5% amorolfine nail lacquer (c) after penetrating human nail plates. 394