AAC Accepted Manuscript Posted Online 7 December 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.01739-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ME1111, a New Antifungal Agent for Topical Treatment of Onychomycosis:

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Characterization of Antifungal Activity and Nail Penetration

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Yuji Tabata*$, Naomi Takei-Masuda$, Natsuki Kubota, Sho Takahata, Makoto Ohyama,

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Kaori Kaneda, Maiko Iida, Kazunori Maebashi

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Meiji Seika Pharma Co., Ltd., Pharmaceutical Research Center, Yokohama, Japan,

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$

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*Corresponding author:

These authors contributed equally to the manuscript

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Yuji Tabata, Ph.D.

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Meiji Seika Pharma Co., Ltd.,

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Pharmaceutical Research Center

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760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan

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Tel: +81-45-541-2521

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Fax: +81-45-541-1768

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[email protected]

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Running title: ME1111, a New Topical Antifungal for Onychomycosis

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Key words: ME1111, antifungal agent, onychomycosis, dermatophytes, minimum

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inhibitory concentration

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Abstract

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Fungal nail infection (onychomycosis) is a prevalent disease in many areas of the world

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with a high incidence approaching 23%. Available antifungals to treat this disease suffer

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from a number of disadvantages, necessitating the discovery of new efficacious and safe

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antifungals. Here we evaluated the in vitro antifungal activity and nail penetration ability

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of ME1111, a novel antifungal agent, along with comparator drugs including ciclopirox,

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amorolfine, terbinafine and itraconazole. ME1111 showed potent antifungal activity

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against Trichophyton rubrum and Trichophyton mentagrophytes (the major etiologic

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agents of onychomycosis) strains isolated in Japan and reference fungal strains with an

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minimum inhibitory concentration (MIC) range of 0.12 to 0.5 mg/L, and an MIC50 and

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MIC90 of 0.5 mg/L for both. Importantly, none of the tested isolates showed elevated MIC

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to ME1111. Moreover, the antifungal activity of ME1111 was minimally affected by 5%

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wool keratin powder, in comparison to the other antifungals tested. The ME1111 solution

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was able to penetrate human nails and inhibit fungal growth in a dose-dependent

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manner using the TurChub® assay. In contrast, 8% ciclopirox and 5% amorolfine nail

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lacquers showed no activity under the same conditions. ME1111 demonstrated

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approximately 100-fold greater selectivity in inhibition of Trichophyton spp. compared to

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human cell lines. Our findings demonstrate that ME1111 possesses potent

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anti-dermatophyte activity, maintains this activity in the presence of keratin, and

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possesses excellent human nail permeability. These results suggest that ME1111 is a

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promising topical medication for the treatment of onychomycosis, and therefore warrants

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further clinical evaluation.

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Introduction

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Onychomycosis is a progressive fungal infection of the nails, which, if left untreated, can

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cause nail destruction and deformity. This disease affects up to 23% of adults worldwide

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(1-4). The prevalence of this disease increases in the elderly, affecting approximately

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28% of people over the age of 60 (5). Onychomycosis is primarily caused by

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dermatophytes, particularly Trichophyton rubrum and Trichophyton mentagrophytes (1).

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Although both oral (terbinafine and itraconazole) and topical (ciclopirox, amorolfine,

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efinaconazole, and tavaborole) medications are available for the treatment of fungal nail

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infection, these agents suffer from a number of disadvantages: 1) Oral onychomycosis

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drugs can cause liver toxicity issues and/or drug-drug interaction concerns (6), making

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them difficult to prescribe to elderly patients, especially those taking multiple medications.

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2) The efficacy of topical therapeutics is relatively low, presumably due to poor nail

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permeability and high keratin binding. Additionally, the recently approved topical drugs

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efinaconazole (7-9) and tavaborole (6, 10) have low efficacy and local side effects issues.

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As a result of the various drawbacks of currently available therapeutics, there is an

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opportunity for the development of new topical agents with greater efficacy and fewer

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side effects.

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In order to be effective against onychomycosis, a topical antifungal should have low

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molecular weight (an important factor for nail penetration) (6, 11) and low affinity to

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keratin (a major component of the nail) (12). Therefore, a topical antifungal having both a

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low molecular weight and low affinity to keratin would be considered a promising

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therapeutic

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(ME1111) is a novel anti-dermatophytic drug (Fig. 1) discovered by Meiji Seika Pharma

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Co., Ltd. (Meiji; Tokyo, Japan) through an optimization process directed at selecting

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compounds with: 1) potent anti-dermatophyte activity, 2) favourable physicochemical

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onychomycosis.

2-(3,5-Dimethyl-1H-pyrazol-1-yl)-5-methylphenol

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and nail permeability properties, and 3) small molecular size. Further research

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demonstrated that ME1111 is a first-in-class, low molecular weight compound with

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antifungal activity, mediated by the inhibition of succinate dehydrogenase (complex II), a

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critical enzyme involved in mitochondrial respiratory electron transfer (13). Thus, ME1111

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has desirable properties as a candidate compound for the topical treatment of

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onychomycosis. The aim of this study was to: 1) characterize the antifungal activity of

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ME1111 against clinical and reference isolates of Trichophyton species, 2) evaluate

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whether keratin reduces ME1111’s antifungal activity, and 3) determine ME1111’s ability

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to penetrate human nails.

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MATERIALS AND METHODS

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Test strains. Japanese clinical isolates of T. rubrum isolated between 1999 and 2011 (n

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= 62 strains) and T. mentagrophytes isolated between 1999 and 2011 (n = 47 strains)

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were obtained from Teikyo University Institute of Medical Mycology (Tokyo, Japan) and

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the National BioResource Project-Pathogenic Microbes (Chiba, Japan). In addition, two

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reference dermatophyte strains (T. rubrum ATCC MYA-4438 and T. mentagrophytes

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ATCC MYA-4439) and two other dermatophyte strains representing other species (T.

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tonsurans ATCC 56186, Epidermophyton floccosum ATCC 26072), obtained from the

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American Type Culture Collection (ATCC; Manassas, VA, USA) were also evaluated.

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Antifungal agents. The following agents were tested in this study (Fig. 1): ME1111

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was provided by Meiji. Amorolfine hydrochloride was purchased from LKT Laboratories,

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Inc. (St. Paul, MN, USA). Ciclopirox olamine, terbinafine hydrochloride, and itraconazole

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were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). All test compounds were

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supplied in powder form and reconstituted in dimethyl sulfoxide (DMSO).

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Antifungal susceptibility testing. MIC testing was performed according to the CLSI

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M38-A2 standard microbroth dilution methodology for the susceptibility testing of

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dermatophytes (14). Briefly, serial dilutions of test compounds were prepared in

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RPMI1640 (Life Technologies, Grand Island, NY, USA) buffered with 3-(N-morpholino)

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propanesulfonic acid (MOPS; Nakarai Tesque, Ltd., Kyoto, Japan) in a range of: 0.06-32

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mg/L for ME1111; 0.001-0.5 mg/L for amorolfine, and 0.008-4 mg/L for ciclopirox.

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Microtiter plates were incubated at 35°C for 4 days. MICs were read visually, and the

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MIC endpoint was defined as the minimum concentration of the test compounds that

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prevented visible growth relative to the growth control. MIC50 and MIC90 were defined as

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the lowest concentration to inhibit 50% and 90% of the strains tested, respectively.

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Measurement of anti-dermatophytic activity of ME1111 in the presence and

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absence of keratin. For a topical drug to penetrate the nail plate it should have low

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keratin-binding properties. To determine the binding potential of ME1111 to keratin, we

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measured the susceptibility of dermatophytes to ME1111 and comparators in the

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presence and absence of keratin. Briefly, MICs against T. rubrum and T. mentagrophytes

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in RPMI1640-0.165 M MOPS, pH 7.0 with or without 5% wool keratin powder (Tokyo

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Chemical Industry Co., Ltd., Tokyo, Japan) were measured by colorimetric assay using

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Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Wool

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keratin was defatted according to the method reported by Uchida et al (15). MIC was

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defined as the lowest concentration at which the inhibition was > 80% compared to that

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of the respective growth control. Geometric mean MICs were calculated from MICs of 7

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strains for T. rubrum and 6 strains for T. mentagrophytes.

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Measurement of anti-dermatophytic activity after human nail penetration. To

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determine the ability of ME1111 and comparators to penetrate human nail plates,

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antifungal activity after human nail penetration was measured in the TurChub® assay as

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described earlier (6, 7, 16). Briefly, the TurChub® cells were filled with Sabouraud

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dextrose agar, inoculated with T. rubrum, and placed under human full thickness nails.

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Two microliters each of ME1111 solution (2, 5, 10 and 15% w/v), 8% ciclopirox nail

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lacquer (Penlac®), or 5% amorolfine nail lacquer (Loceryl®) were applied to the human

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nails, and the cells were occluded and incubated for 7 days. The efficacy of each test

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solution was determined by measuring the zone-of-inhibition (ZOI) against T. rubrum in

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the TurChub® cell. Effect of ME1111 against host cells. To show whether ME1111 is toxic to human cells,

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we used anti-cell proliferation assays (17). Briefly, the concentration that caused 50%

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inhibition of cell proliferation (IC50) was measured in four different human cell lines

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obtained from the ATCC: K562 (leukaemia, ATCC CCL-243), HepG2 (liver tumour, ATCC

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HB-8065), U937 (lymphoma, ATCC CRL-1593), and A431 (skin, ATCC CRL-1555). Cell

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suspension in growth medium was placed in 96-well microtiter plates in an atmosphere

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of 5% CO2 at 37°C. After 24 hours, test substances were added respectively for an

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additional 72-hour incubation period. At the end of the incubation period, AlamarBlue

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reagent (AbD Serotec, Kidlington, Oxford, UK) was added to evaluate cell proliferation.

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RESULTS

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ME1111 demonstrated broad spectrum anti-dermatophyte activity. To confirm that

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ME1111 is a compound with potent anti-dermatophyte activity for onychomycosis, we

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evaluated its in vitro antifungal activity against the major etiologic agents of

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onychomycosis, T. rubrum (n = 62 strains) and T. mentagrophytes (n = 47 strains),

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clinical strains isolated in Japan and reference strains (Table 1). Our data showed that

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the MIC of ME1111 against dermatophytes ranged from 0.12 to 0.5 mg/L, with an MIC50

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and MIC90 of 0.5 mg/L (Table 1). Of note, none of the isolates tested showed an elevated

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MIC. ME1111 inhibited a clinical isolate of T. mentagrophytes with an elevated terbinafine

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MIC (>0.5 mg/L) where the MIC of ME1111 was also 0.5 mg/L. MICs of amorolfine,

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ciclopirox and itraconazole against this strain were 0.12 mg/L, 0.25 mg/L and 0.016 mg/L,

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respectively. In addition to inhibiting T. rubrum and T. mentagrophytes, ME1111 showed potent

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antifungal activity against other dermatophytes, E. floccosum, and T. tonsurans with an

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MIC of 0.25 mg/L. These results demonstrate that ME1111 possesses broad-spectrum

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anti-dermatophyte activity.

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Anti-dermatophytic activity of ME1111 was minimally affected by keratin. To

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evaluate the antifungal activity of ME1111 in the presence of keratin, the MICs of ME1111

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and other antifungal agents were determined against 7 strains of T. rubrum and 6 strains

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of T. mentagrophytes in RPMI1640 with or without 5% keratin (Table2). Geometric mean

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MICs of ME1111 did not change or were slightly increased (1.5-fold) in the presence of

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5% keratin. In contrast, the geometric mean MICs in the presence of 5% keratin against

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T. rubrum and T. mentagrophytes increased 512-fold for ciclopirox, 8 and 16-fold for

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amorolfine, 3.3 and 4.5-fold for terbinafine, and, 71 and 228-fold for itraconazole,

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respectively. These results indicate that the anti-dermatophytic activity of ME1111 was

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minimally affected by keratin compared to other antifungals.

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ME1111 was able to penetrate the human nail and inhibited the growth of T.

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rubrum. The ability of a topical antifungal agent to penetrate the nail plate is critical to its

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efficacy in the treatment of onychomycosis. Thus, the human nail penetration of ME1111

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and those of comparators (8% ciclopirox nail lacquer and 5% amorolfine nail lacquer)

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were measured using the TurChub® model. Our data showed that ME1111 solutions (2, 5,

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10 and 15% w/v) were able to penetrate the nail plate and inhibit the growth of T. rubrum

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in a dose-dependent manner. In contrast, 8% ciclopirox and 5% amorolfine nail lacquers

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did not show antifungal activity under the same conditions (Table 3; Fig. 2) indicating that

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they failed to penetrate the nail plate. Thus, ME1111 solutions showed greater efficacy

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against T. rubrum than 8% ciclopirox nail lacquer and 5% amorolfine nail lacquer after

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penetrating human nail plates. ME1111 demonstrated selective toxicity. The effects of ME1111 and comparators on

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host cell proliferation were evaluated using four human cell lines. The IC50 values of

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ME1111 in K562, HepG2, U937, and A431 cells were 47, 37, 33, and 40 mg/L,

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respectively. In contrast, MIC50s and MIC90s of ME1111 against Trichophyton spp. were

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0.5 mg/L (Table 4). IC50 values of amorolfine in these cell lines were less than 10 mg/L,

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and those of ciclopirox were less than 1 mg/L. The MIC90s of amorolfine and ciclopirox

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against T. rubrum were 0.06 mg/L and 0.25 mg/L, respectively (Table 4). ME1111

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demonstrated good therapeutic index (TI; IC50 for human cell proliferation / MIC90 for

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Trichophyton spp.). All TIs of ME1111 were more than 60.

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DISCUSSION

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In this study we demonstrated that ME1111 possesses broad-spectrum activity against

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dermatophytes that cause nail infections. Moreover, this activity was not reduced in the

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presence of keratin. Additionally, we showed that ME1111 was able to penetrate human

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nail plates and inhibit fungal growth.

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Among the antifungals tested, terbinafine, amorolfine, and itraconazole showed

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stronger in vitro antifungal activity than ME1111. ME1111 demonstrated as potent

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antifungal activity as ciclopirox against clinical dermatophyte strains isolated from

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Japanese patients, including T. rubrum and T. mentagrophytes, which are common

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etiologic agents of onychomycosis (Table 1). These results are in line with the activity of

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ME1111 against clinical dermatophyte isolates obtained from patients in the United

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States (18), where the MIC90 against the U.S. isolates was 0.25 mg/L. The finding that

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susceptibility of dermatophyte isolates obtained from the U.S. sites to ME1111 was

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similar to that of non-U.S. sites, indicates that there is no difference in ME1111

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susceptibility within the dermatophyte species obtained worldwide. To date, susceptibility to ME1111 has been measured against more than 500 strains of

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dermatophytes (400 strains were tested in reference 18 and 109 strains were tested in

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this study), and the results show that none of the tested isolates have elevated MICs to

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ME1111. Moreover, it was reported that MICs of ME1111 against 7 strains of

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terbinafine-resistant T. rubrum were not elevated (18). Our study also demonstrated the

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potent activity of ME1111 against a terbinafine-resistant T. mentagrophytes strain. These

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results suggest that ME1111 demonstrates a potent activity against terbinafine-resistant

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strains, but further tests with strains presenting high terbinafine MICs are needed.

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In this study, the antifungal activity of ME1111 was unaffected or slightly increased (1-

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to 1.5-fold) in the presence of 5% keratin. In contrast, the antifungal activity of ciclopirox,

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amorolfine, terbinafine and itraconazole was diminished in the presence of 5% keratin. It

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has been reported that the antifungal activity of some antifungal agents diminishes in the

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presence of keratin, a main component of nails (12). In this regard, Osborne et al.

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showed that incubation of terbinafine with nail powder increased the minimum fungicidal

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concentration of this drug from ≤0.03 mg/L to 4 mg/L (19). This result may explain why

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topical solutions of terbinafine failed to meet their primary endpoint for the treatment of

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onychomycosis (20). The antifungal activity of ME1111 is minimally affected by keratin,

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as compared to other topical antifungals tested. This result indicates that keratin binding

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affinity of ME1111 is lower than those of other topical antifungals, which may be due to its

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unique physicochemical properties (i.e., LogP, water solubility, molecular weight etc.).

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Our findings show that ME1111 permeated across the human nail plate and exhibited

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dose-dependent antifungal activity. In contrast, 8% ciclopirox and 5% amorolfine showed

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no antifungal activity under the same conditions, indicating that these antifungals failed

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to penetrate human nails. The potent nail penetration activity of ME1111 was also shown

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in other assays (21, 22). Nail penetration is another important factor for the efficacy of

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topical onychomycosis drugs since the main type of nail disease (distal subungual

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onychomycosis) resides in the nail bed. Therefore, antifungals should be able to

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penetrate the nail in order to kill the disease-causing dermatophytes. The inability of

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antifungal agents to penetrate the nail plate could be explained by the complex

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anatomical structure of the nail. Due to the special chemical nature of the nail plate, drug

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transport through it is influenced by the structure and the physicochemical properties of

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the drug molecule. Low molecular weight is reported to be an important factor for nail

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penetration and onychomycosis treatment (6). During initial hit identification, we showed

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that nail penetration of ME1111 analogues of larger molecule did not penetrate as well as

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low molecular weight compounds (data not shown). As a result of these molecular weight

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findings, we selected ME1111 (202.25 g/mol) (Fig. 1). The low molecular weight of

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ME1111 is thought to play an important role in its human nail penetration. Of note,

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although ciclopirox is as small as ME1111, it failed to efficiently penetrate the nail plate,

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which could be due to its higher affinity to keratin relative to ME1111 (data not shown).

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ME1111 demonstrated approximately 60-fold greater selectivity in growth inhibition of

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Trichophyton spp. compared to host cells (Table 4). This selectivity in growth inhibition is

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likely due to the selective inhibition of succinate dehydrogenase in Trichophyton spp.

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over the same enzyme in human cells (13). Compared to other antifungal agents tested,

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ME1111 demonstrated weak inhibitory effects on human cell line proliferation. TIs of

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ME1111 (>60) is better than that of ciclopirox (