RESEARCH ARTICLE
`
`Efficacy Coefficients Determined Using Nail
`Permeability and Antifungal Activity in
`Keratin-Containing Media Are Useful for
`Predicting Clinical Efficacies of Topical Drugs
`for Onychomycosis
`Yoshiki Matsuda☯, Keita Sugiura☯, Takashi Hashimoto☯, Akane Ueda☯, Yoshihiro Konno☯,
`Yoshiyuki Tatsumi*☯
`
`a11111
`
`Drug Research Center, Kaken Pharmaceutical Co., Ltd., Kyoto, Japan
`
`☯ These authors contributed equally to this work.
`* tatsumi_yoshiyuki@kaken.co.jp
`
`Abstract
`
`Onychomycosis is difficult to treat topically due to the deep location of the infection under
`the densely keratinized nail plate. In order to obtain an in vitro index that is relevant to the
`clinical efficacy of topical anti-onychomycosis drugs, we profiled five topical drugs: amorol-
`fine, ciclopirox, efinaconazole, luliconazole, and terbinafine, for their nail permeabilities, ker-
`atin affinities, and anti-dermatophytic activities in the presence of keratin. Efinaconazole
`and ciclopirox permeated full-thickness human nails more deeply than luliconazole. Amorol-
`fine and terbinafine did not show any detectable permeation. The free-drug concentration of
`efinaconazole in a 5% human nail keratin suspension was 24.9%, which was significantly
`higher than those of the other drugs (1.1–3.9%). Additionally, efinaconazole was released
`from human nail keratin at a greater proportion than the other drugs. The MICs of the five
`drugs for Trichophyton rubrum were determined at various concentrations of keratin (0–
`20%) in RPMI 1640 medium. The MICs of ciclopirox were not affected by keratin, whereas
`those of efinaconazole were slightly increased and those of luliconazole and terbinafine
`were markedly increased in the presence of 20% keratin. Efficacy coefficients were calcu-
`lated using the nail permeation flux and MIC in media without or with keratin. Efinaconazole
`showed the highest efficacy coefficient, which was determined using MIC in media with ker-
`atin. The order of efficacy coefficients determined using MIC in keratin-containing media
`rather than keratin-free media was consistent with that of complete cure rates in previously
`reported clinical trials. The present study revealed that efficacy coefficients determined
`using MIC in keratin-containing media are useful for predicting the clinical efficacies of topi-
`cal drugs. In order to be more effective, topical drugs have to possess higher efficacy
`coefficients.
`
`OPEN ACCESS
`
`Citation: Matsuda Y, Sugiura K, Hashimoto T, Ueda
`A, Konno Y, Tatsumi Y (2016) Efficacy Coefficients
`Determined Using Nail Permeability and Antifungal
`Activity in Keratin-Containing Media Are Useful for
`Predicting Clinical Efficacies of Topical Drugs for
`Onychomycosis. PLoS ONE 11(7): e0159661.
`doi:10.1371/journal.pone.0159661
`
`Editor: Joy Sturtevant, Louisiana State University,
`UNITED STATES
`
`Received: February 8, 2016
`
`Accepted: July 5, 2016
`
`Published: July 21, 2016
`
`Copyright: © 2016 Matsuda et al. This is an open
`access article distributed under the terms of the
`Creative Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any
`medium, provided the original author and source are
`credited.
`
`Data Availability Statement: All relevant data are
`within the paper.
`
`Funding: Kaken Pharmaceutical Co., Ltd. provided
`support in the form of salaries for authors YM, KS,
`TH, AU, YK and YT, but did not have any additional
`role in the study design, data collection and analysis,
`decision to publish, or preparation of the manuscript.
`The specific roles of these authors are articulated in
`the 'author contributions' section.
`
`PLOS ONE | DOI:10.1371/journal.pone.0159661 July 21, 2016
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`1 / 12
`
`

`

`Competing Interests: This study was funded by
`Kaken Pharmaceutical Co., Ltd. YM, KS, TH, AU, YK
`and YT are employees and stockholders of Kaken
`Pharmaceutical Co., Ltd. This does not alter the
`authors' adherence to PLOS ONE policies on sharing
`data and materials.
`
`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
`
`Introduction
`Onychomycosis is a chronic superficial mycosis of the nails that is difficult to treat. Its preva-
`lence is reported to be 23% across Europe [1], 13.8% in North America [2] and approximately
`10% in Japan [3]. Patients with onychomycosis develop physical and psychological issues,
`including pain, difficulty wearing shoes, secondary infection, and difficulties performing every-
`day functions due to the resulting nail dystrophy or unacceptable cosmetic appearance [4].
`The current preferred therapy for onychomycosis is the oral antifungals, terbinafine and
`itraconazole. However, their use is limited by hepatotoxicity and drug-drug interactions (espe-
`cially for itraconazole), which represent a safety concern, particularly in elderly patients, in
`whom underlying diseases and polypharmacy are common. Routine liver function testing is
`recommended for patients being treated with oral terbinafine and itraconazole, and their use is
`contraindicated in patients with impaired liver function [5]. Due to its localized effects, topical
`drug delivery is desirable for treating nail disorders, which results in minimal adverse systemic
`events and possibly improved adherence [6]. Thus, safe and efficacious topical therapies
`against onychomycosis remain an unmet medical need.
`Distal lateral subungual onychomycosis (DLSO) is the most common form of fungal nail
`infection. Trichophyton rubrum, the most frequent causative fungus, invades the nail bed
`under the nail plate in DLSO. Therefore, topical antifungal drugs need to permeate through the
`nail plate to the nail bed in order to be effective.
`The nail plate is composed of keratin proteins that are held together by a disulfide linkage.
`The nail’s unique properties, particularly its thickness and relatively compact construction,
`make it a formidable barrier to the permeation of topically applied agents. Moreover, the bind-
`ing of the drug to keratin in the nail plate reduces the free (active) drug, thereby diminishing
`the concentration gradient and limiting permeation into deeper tissues [7]. Furthermore, kera-
`tin binding would reduce the antifungal potency of a drug in the nail bed. Therefore, the con-
`centration of a topically applied drug fails to reach a therapeutically effective concentration in
`the nail bed, in which fungi reside.
`To date, the number of approved topical anti-onychomycosis drugs is markedly lower than
`topical anti-tinea pedis drugs. In the topical treatment of onychomycosis, 5% amorolfine nail
`lacquer (not approved in North America) and 8% ciclopirox nail lacquer have been widely
`used in Europe and North America, 10% efinaconazole solution was recently launched in
`North America, Canada, and Japan [8], 5% tavaborole solution was recently launched in North
`America [9], and 5% luliconazole solution was launched in Japan in April 2016 [10]. Terbina-
`fine formulations are currently being developed for the additional indication of a topical anti-
`onychomycosis drug [11, 12].
`The in vitro activities of the above antifungals (excluding luliconazole) have been investi-
`gated in media containing a keratin powder concentration of 5% [13, 14, 15]. However, since
`the nail plate and nail bed have higher keratin concentrations (approximately 80–90%) [16],
`further studies at higher keratin concentrations than those examined in previous studies are
`needed in order to more accurately predict the potency of antifungal activity in nails.
`We previously reported that efinaconazole permeated the human nail more deeply than
`amorolfine and to a similar extent to ciclopirox using commercial products with different vehi-
`cle compositions [17]. It is important to assess the nail permeation of active pharmaceutical
`ingredients using the same vehicle composition in order to accurately compare nail permeation
`between drugs. Furthermore, comparative studies have yet to be conducted on the nail perme-
`ation of efinaconazole, luliconazole, and terbinafine.
`In an attempt to clarify the favorable properties of topical anti-onychomycosis drugs, we
`herein evaluated five topical drugs with different physical properties (Table 1) for their in vitro
`
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`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
`
`Table 1. Physical properties of five test drugs.
`
`Molecular weight
`ClogP
`
`Amorolfine
`318
`6.44
`
`Ciclopirox
`207
`2.03
`
`ClogP is the logP calculated using ChemBioDraw Ultra software (version 12.0).
`
`doi:10.1371/journal.pone.0159661.t001
`
`Drugs
`Efinaconazole
`348
`2.15
`
`Luliconazole
`354
`3.49
`
`Terbinafine
`291
`5.96
`
`human nail permeabilities, in vitro human keratin affinity, and in vitro anti-dermatophytic
`activities in the presence of keratin at various concentrations. The aim of the present study is to
`establish an in vitro index that is relevant to clinical efficacy by measuring nail permeation flux
`and MIC in media without or with keratin. This index will provide a strategy to develop more
`effective topical anti-onychomycosis drugs in the future.
`
`Materials and Methods
`Antifungals, fungal strains, media, and keratin
`Amorolfine hydrochloride and terbinafine hydrochloride were purchased from Tokyo Chemi-
`cal Industry Co., Ltd. (Tokyo, Japan). Ciclopirox was purchased from Sigma-Aldrich Corpora-
`tion (St. Louis, MO, USA). Efinaconazole was synthesized at Kaken Pharmaceutical Co., Ltd.
`(Tokyo, Japan). Luliconazole was purchased from Toronto Research Chemicals (Toronto,
`Canada).
`T. rubrum NBRC 6203 and T. rubrum (IFM 46615, IFM 47622, IFM 47627, IFM 46157, and
`IFM 46204) were obtained from the Biological Resource Center, National Institute of Technol-
`ogy and Evaluation (Tokyo, Japan) and Medical Mycology Research Center, Chiba University
`(Chiba, Japan), respectively.
`RPMI 1640 medium (Nissui Seiyaku, Tokyo, Japan) buffered with 0.165 M morpholinopro-
`panesulphonic acid (MOPS) at pH 7.0 was used in drug susceptibility tests.
`Human nails were purchased from Science Care (Phoenix, AZ, USA), which was accredited
`by the American Association of Tissue Banks. These were powdered using a miller (Wonder
`Blender, Osaka Chemical Co. Ltd., Osaka, Japan) and defatted with a mixture of ethanol-
`diethyl ether (1:1, vol/vol). Defatted keratin powder was also made from porcine hooves (O.C.
`farm, Okayama, Japan) using a similar method to that for human nail keratin powder.
`
`Affinity of drugs to keratin
`The affinities of the test drugs to human nail keratin were determined as described previously
`[17], with a slight modification. In order to examine the relationship between drug-keratin
`affinity and increases in the rate of MIC with the addition of keratin to medium, dimethyl sulf-
`oxide (DMSO), which is the same solvent in the two tests, was used. We confirmed that the
`final concentration of DMSO (1%) did not affect drug-keratin affinity. One hundred microli-
`ters of the drug solution in DMSO (50 μg/mL amorolfine, ciclopirox, efinaconazole, lulicona-
`zole, or terbinafine) was mixed with 9.9 mL of 0.2 mol/L Tris-HCl buffer (pH 7.4) containing
`0.5 g of defatted keratin powder. After shaking at 37°C for 1 h (75 rpm), the suspension was
`centrifuged. The drug concentration in the supernatant was determined by liquid chromatog-
`raphy-tandem mass spectrometry (LC-MS/MS) (TSQ Quantum Ultra; Thermo Fisher Scien-
`tific, Inc., Waltham, MA, USA). The lower limit of quantification by the assay was 0.5 ng/mL
`for amorolfine, ciclopirox, and efinaconazole and 1 ng/mL for luliconazole and terbinafine.
`The percentage of the free drug was calculated for each drug, and mean values were compared
`
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`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
`
`by Tukey’s multiple-comparison test (EXSUS version 8.0.0; CAC Exicare Corporation). P val-
`ues of less than 0.05 were regarded as significant. The keratin pellet (containing bound drug)
`was subsequently resuspended in 10 mL of 0.2 mol/L Tris-HCl buffer and washed by shaking
`for 10 min. The suspension was centrifuged and the washing procedure repeated 5 times. All
`procedures were performed in triplicate on the same day. The drug concentration of the super-
`natant obtained after each wash was measured by LC-MS/MS, and the cumulative percent
`release of the drug was calculated. The cumulative release of each drug after 5 washes was com-
`pared using Tukey’s multiple-comparison test. P values of less than 0.05 were regarded as
`significant.
`
`In vitro nail permeation of drugs
`Healthy human nails at full thickness were purchased from Science Care (AZ, USA) and cut
`into squares of approximately 49 mm2. The nails were firmly fixed between two fluoro rubbers
`with a 5-mm diameter hole and mounted in Franz diffusion cells. The fluoro rubbers were
`used to prevent the leakage of the drug solutions. The receptor compartment was filled with
`phosphate-buffered saline (PBS) (pH 7.4) containing 4% (wt/vol) bovine serum albumin and
`0.01% (wt/vol) sodium azide and placed in an incubator at 32°C. Twelve microliters of cocktail
`solution (propylene glycol: ethanol = 1:4, vol/vol) including amorolfine, ciclopirox, efinacona-
`zole, luliconazole, and terbinafine at 5% was then singly applied to the dorsal nail surface in the
`donor compartment. The donor compartment in Franz cells was not occluded. The receptor
`solution was continuously stirred using a spinning magnetic bar. The receptor fluid was sam-
`pled once daily for 18 days, drug concentrations were determined by LC-MS/MS, and the flux
`(ng/cm2/day) was calculated from the cumulative drug amount permeated per nail.
`
`Effects of keratin concentrations on MIC for T. rubrum
`Two hundred and fifty microliters of MOPS buffered RPMI 1640 with 2-fold serial dilutions of
`the test drugs was added to the glass tubes without or with 6.25 mg, 25 mg, or 100 mg defatted
`porcine hoof keratin powder. Two hundred and fifty microliters of MOPS buffered RPMI 1640
`containing T. rubrum microconidia (final fungal concentration: 1×104 cells) was then added
`(Final keratin concentrations: 0, 1.25, 5 and 20%). It was not possible to set keratin concentra-
`tions to greater than 20% because keratin powder at such high concentrations absorbs the
`medium and forms clumps, thereby making it difficult to mix microconidia and the drugs to
`be tested. The glass tubes were incubated at 35°C for 7 days. After the incubation, the minimal
`inhibitory concentration (MIC) was read as the lowest concentration that prevented any dis-
`cernible growth (100% inhibition).
`
`Efficacy coefficient for the onychomycosis treatment
`Mertin and Lippold [18] introduced an efficacy coefficient to maximize the therapeutic effec-
`tiveness of antifungals. This simple equation allows for the estimation and comparison of rela-
`tive efficacies among various antifungal agents. The efficacy coefficient was calculated by the
`ratio of the flux of an antifungal drug through the nail plate to the MIC at each keratin concen-
`tration (Equation: Efficacy coefficient = flux/MIC).
`
`Results
`Affinity of drugs to keratin
`Amorolfine, ciclopirox, efinaconazole, luliconazole, and terbinafine free-drug levels after their
`incubation in 5% human nail keratin suspensions were 3.9 ± 0.1%, 1.1 ± 0.2%, 24.9 ± 1.5%,
`
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`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
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`Fig 1. Affinities of five test drugs to keratin. (a) The percentage of the free drug in buffer after shaking at 37°C for 1 h. The graphs represent the
`mean + SD of three replicates. Tukey’s multiple-comparison test: ***, P < 0.001 versus amorolfine, ciclopirox, luliconazole and terbinafine; †,
`P < 0.05 versus terbinafine; ‡‡, P < 0.01 versus ciclopirox. (b) The cumulative release of amorolfine, efinaconazole, luliconazole, and terbinafine.
`Each plot and bar represent the mean + SD of three replicates. Tukey’s multiple-comparison test: ***, P < 0.001 versus amorolfine, ciclopirox,
`luliconazole and terbinafine; †, P < 0.05 versus terbinafine; ‡‡, P < 0.01 versus ciclopirox.
`
`doi:10.1371/journal.pone.0159661.g001
`
`2.9 ± 0.1%, and 1.8 ± 0.0% (mean ± standard deviation [SD]), respectively. The cumulative
`drug release levels after five washes were 15.4 ± 1.2%, 5.0 ± 0.6%, 55.1 ± 6.1%, 10.3 ± 0.5%, and
`7.6 ± 0.5% (mean ± SD), respectively (Fig 1). The efinaconazole free-drug ratio and keratin
`release levels were markedly higher than those of the other drugs. The amorolfine free-drug
`ratio and keratin release levels were slightly higher than those of ciclopirox and terbinafine.
`
`In vitro nail permeation of drugs
`The human nail permeation of drugs was investigated in Franz diffusion cells after a single top-
`ical application of the cocktail solution including amorolfine, ciclopirox, efinaconazole, lulico-
`nazole, and terbinafine at 5% to human nails for 18 days (n = 6). Efinaconazole was detected in
`the receptor fluids as early as day 2, whereas ciclopirox and luliconazole were first detected on
`day 4. On day 18, drug concentrations were detected in the receptor fluids of 6/6 nails for efina-
`conazole, 3/6 nails for ciclopirox, and 2/6 nails for luliconazole. On the other hand, neither
`amorolfine nor terbinafine was detectable in any receptor fluids. The cumulative permeated
`amounts of ciclopirox, efinaconazole, and luliconazole on day 18 were 0.221 ± 0.464,
`0.351 ± 0.369, and 0.0131 ± 0.0238 μg/cm2, respectively. In addition, the fluxes of ciclopirox,
`efinaconazole, and luliconazole were 31.7 ± 59.2, 38.8 ± 36.0, and 2.23 ± 3.89 ng/cm2/day,
`respectively (Table 2). The order of nail permeability of the test drugs remained unchanged in
`all human nail used. These results clearly indicated that efinaconazole and ciclopirox perme-
`ated through the human nail more deeply than the other drugs.
`
`Effects of keratin concentrations on MIC for T. rubrum
`The influence of keratin concentrations (0, 1.25, 5, or 20%) on the anti-T. rubrum activities of
`the five drugs was investigated and compared. The geometric mean MICs of ciclopirox were
`almost unaltered at all keratin concentrations, but reached the highest values (16–40 μg/mL)
`among the drugs tested. In contrast, the geometric mean MICs of the four other drugs
`increased with elevations in the concentration of keratin (Table 3 and Fig 2). The geometric
`mean MICs in the presence of 20% keratin of amorolfine, efinaconazole, terbinafine, and luli-
`conazole were 20-fold, 8-fold, 72-fold, and 102-fold higher, respectively, than those under the
`
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`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
`
`Table 2. Cumulative drug amount permeated after a single application of five test drugs to human nails.
`Cumulative amount (μg/cm2)
`Efinaconazole
`NC
`0.00130 ± 0.00318
`0.00577 ± 0.01413
`0.0127 ± 0.0275
`0.0222 ± 0.0389
`0.0396 ± 0.0602
`0.0817 ± 0.1222
`0.107 ± 0.158
`0.140 ± 0.186
`0.182 ± 0.205
`0.235 ± 0.262
`0.258 ± 0.276
`0.300 ± 0.309
`0.351 ± 0.369
`38.8 ± 36.0
`
`Time (day)
`
`1
`2
`3
`4
`7
`8
`9
`10
`11
`14
`15
`16
`17
`18
`Flux (ng/cm2/day)
`
`Amorolfine
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`
`Ciclopirox
`NC
`NC
`NC
`0.00161 ± 0.00394
`0.00420 ± 0.01029
`0.0118 ± 0.0288
`0.0438 ± 0.1074
`0.0590 ± 0.1445
`0.0783 ± 0.1866
`0.100 ± 0.233
`0.130 ± 0.297
`0.145 ± 0.326
`0.191 ± 0.414
`0.221 ± 0.464
`31.7 ± 59.2
`
`Luliconazole
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`0.00163 ± 0.00398
`0.00252 ± 0.00616
`0.00327 ± 0.00800
`0.00668 ± 0.01282
`0.00745 ± 0.01376
`0.0106 ± 0.0194
`0.0131 ± 0.0238
`2.23 ± 3.89
`
`Terbinafine
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`
`Data represent the mean ± SD (n = 6).
`NC, not calculated because the drug concentration in the receptor fluid was below the lower limit of quantification in all samples.
`Flux was calculated from the cumulative amount on days 15, 16, 17, and 18.
`NC entered as zero in calculations.
`
`doi:10.1371/journal.pone.0159661.t002
`
`keratin-free conditions. Consequently, the geometric mean MICs under the 20% keratin condi-
`tion of luliconazole (0.11 μg/mL), efinaconazole (0.11 μg/mL), and terbinafine (0.20 μg/mL)
`indicated almost the same values, while luliconazole showed the lowest geometric mean MIC
`(0.0011 μg/mL) under keratin-free conditions. Amorolfine was less active than efinaconazole,
`luliconazole, and terbinafine at all keratin concentrations.
`
`Efficacy coefficient for the onychomycosis treatment
`Since amorolfine and terbinafine were not detectable in any receptor fluids of Franz diffusion
`cells, it was not possible to calculate their efficacy coefficients. Thus, the efficacy coefficients of
`efinaconazole, ciclopirox, and luliconazole were calculated (Table 4). The efficacy coefficients
`of ciclopirox were markedly lower than those of efinaconazole and luliconazole at all keratin
`concentrations. Although the efficacy coefficients of efinaconazole and luliconazole were
`
`Table 3. MICs of five test drugs for T. rubrum in medium with and without keratin.
`Geometric mean MIC (μg/mL) for T. rubrum in the following keratin concentrations
`Drugs
`0%
`1.25%
`5%
`20%
`0.039
`0.099
`0.31
`0.79
`32
`16
`20
`40
`0.014
`0.025
`0.044
`0.11
`0.0011
`0.016
`0.050
`0.11
`0.0028
`0.022
`0.050
`0.20
`
`Amorolfine
`Ciclopirox
`Efinaconazole
`Luliconazole
`Terbinafine
`
`Data represent the geometric mean MIC for 6 T. rubrum strains.
`
`doi:10.1371/journal.pone.0159661.t003
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`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
`
`Fig 2. MIC increase rates of five test drugs for T. rubrum by the addition of keratin. Each plot represents
`the rates of geometric mean MIC increases (n = 6) to the MIC in medium without keratin.
`
`doi:10.1371/journal.pone.0159661.g002
`
`almost the same when calculated using MIC in keratin-free media, that of efinaconazole was
`approximately 10- to 20-fold higher than that of luliconazole when calculated using MIC in the
`presence of keratin.
`
`Discussion
`To date, difficulties have been associated with proposing a strategy to develop more effective
`topical anti-onychomycosis drugs because a method for predicting clinical efficacy has not yet
`been fully established. Mertin and Lippold [18] introduced an efficacy coefficient determined
`using nail permeability (flux) and antifungal activity (MIC) in keratin-free medium in order to
`predict the therapeutic effectiveness of antifungals. However, the relationship between efficacy
`
`Table 4. Efficacy coefficients of five test drugs determined using MICs in medium with/without
`keratin.
`
`Drugs
`
`Amorolfine
`Ciclopirox
`Efinaconazole
`Luliconazole
`Terbinafine
`
`Efficacy coefficient in the following keratin concentrations
`0%
`1.25%
`5%
`20%
`NC
`NC
`NC
`NC
`1
`2
`2
`1
`2787
`1564
`878
`348
`2034
`143
`45
`20
`NC
`NC
`NC
`NC
`
`NC was not calculated because it was not possible to determine the fluxes of the two drugs.
`
`doi:10.1371/journal.pone.0159661.t004
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`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
`
`coefficients and clinical efficacies remains unclear. In the present study, in order to establish an
`in vitro index that is relevant to clinical efficacy, we profiled five topical antifungal drugs using
`a novel system that directly compares the human nail permeabilities of antifungal drugs under
`physiological conditions and an in vitro anti-dermatophytic activity assay system using kera-
`tin-containing media.
`It is considered important for topical anti-onychomycosis drugs to have high permeabilities
`in the human nail and exhibit antifungal activities in the nail bed. Several antifungal com-
`pounds have been investigated for their human nail permeabilities using various diffusion cells
`[19]. However, there are difficulties associated with correctly comparing nail permeabilities
`between antifungal drugs because human nails exhibit marked individual differences, e.g., in
`thickness and uniformity. Indeed, we experienced that a certain compound that highly perme-
`ates one human nail sometimes does not as easily permeate another, even if they have the same
`appearance. As a consequence, the results vary depending on nails used, thereby making diffi-
`cult to directly compare nail permeation between compounds. Therefore, it is important to
`compare permeation of compounds by using a single nail at a time. Thus, in the present study,
`we investigated nail permeation using a cocktail solution of the five test drugs at the same time
`and confirmed that the order of nail permeability of the test drugs remained unchanged in any
`single human nail used, which indicates that our assay system enables accurate comparisons of
`drug permeabilities even in nails obtained from various donors. We previously evaluated the
`nail permeabilities of a large number of compounds using a cocktail solution and confirmed
`the order of nail permeation remained unchanged in our background data.
`Previous studies have examined the human nail permeation of amorolfine, terbinafine, and
`luliconazole [18, 20, 21]. A receptor fluid containing ethanol or methanol at 42–60% was used
`in these studies to detect drug nail permeation. Drug permeation was greater than that
`observed in this study. A comparison of nail drug permeabilities in the previous studies and
`the present study is not reasonable because alcohol increases the distribution of test drugs to
`the receptor fluid. In the present study, considering the physiological condition of the nail bed,
`we used alcohol-free solution as the receptor cell fluid and compared the nail permeabilities of
`the test drugs. In this system, ciclopirox and efinaconazole permeated the human nail plate
`deeply, whereas the permeabilities of amorolfine, luliconazole, and terbinafine were extremely
`low. The order of the nail permeation of amorolfine, ciclopirox, and efinaconazole was consis-
`tent with that of our previous findings using commercial products [17].
`From a physiochemical viewpoint, nails behave more like a hydrophilic gel membrane as
`opposed to a lipophilic membrane, such as the stratum corneum [22]. Drug permeation into
`the nail is influenced by the physicochemical properties of the drug (e.g., hydrophilicity and
`molecular weight). The water solubility of efinaconazole (29 μg/mL, unpublished data) is
`higher than those of the other test drugs [23]. Furthermore, the keratin affinity of drugs is also
`considered to be an important factor affecting nail permeability [6, 18]. The present study dem-
`onstrated that efinaconazole showed a lower affinity to human nail keratin than the other
`drugs. Taken together, these results indicate that the high human nail permeation of efinacona-
`zole is due to two favorable physicochemical properties, its lower keratin affinity and higher
`hydrophilicity than those of the other drugs. Ciclopirox permeated the human nail in spite of
`its high keratin affinity. This result may be explained by it having the lowest molecular weight
`(207) among the drugs tested.
`Although the keratin affinity of luliconazole was similar to those of amorolfine and terbina-
`fine, its nail permeation flux was higher. Several previous studies have shown that the drug per-
`meation flux correlates with the degree of drug saturation in a formulation [24, 25]. The five
`drugs tested in the present study had different solubilities in the vehicle (propylene glycol: etha-
`nol = 1:4, vol/vol) used in the human nail permeation study (approximate maximum solubility;
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`amorolfine: 30%, ciclopirox: 20%, efinaconazole: 50%, luliconazole: 8%, and terbinafine: 40%)
`(unpublished data). Among the drugs tested, the maximal solubility of luliconazole was close
`to a concentration of 5% in the present study. Therefore, the high degree of saturation of lulico-
`nazole may have partly contributed to its higher nail permeation flux than those of amorolfine
`and terbinafine.
`Interpreting our results from a lipophilicity (ClogP: calculated octanol/water partition coef-
`ficient) viewpoint, efinaconazole had lower ClogP (2.15) than those of amorolfine, terbinafine,
`and luliconazole (ClogP: 6.44, 5.96, and 3.49, respectively), as described in Table 1. Hansen
`et al. previously reported a direct relationship between the binding rates of several drugs to ker-
`atins and logP (octanol/water partition coefficient) [26]. The lower keratin affinity of efinaco-
`nazole is considered to be partly due to its lower lipophilicity than those of the three drugs.
`This low keratin affinity may contribute to a high concentration of the free drug and its con-
`centration gradient in the nail, resulting in permeation to the deeper layers and nail bed.
`Although ciclopirox has the lowest ClogP (2.03), it showed high keratin affinity. Ciclopirox
`chelates trivalent cations, such as Fe3+ and Al3+ [27], unlike the four other drugs. Therefore, its
`high keratin affinity may be due to binding to the protein and trivalent cations included in ker-
`atin powder.
`Furthermore, permeated antifungals must retain their antifungal activities in the nail bed in
`which dermatophyte resides. However, many antifungal agents are inactivated in the nail bed
`by their high affinities to keratin [13]. Therefore, in order to predict the potency of antifungal
`activity in the nail bed, the in vitro activities of the five test drugs were determined under vari-
`ous keratin concentrations. Conversely, the drug susceptibility of dermatophytes is commonly
`evaluated according to the CLSI M38-A2 method using keratin-free RPMI 1640 medium [28].
`The MICs of amorolfine, efinaconazole, luliconazole, and terbinafine increased with elevations
`in keratin concentrations (0, 1.25, 5, and 20%) in RPMI 1640 medium. In the presence of 20%
`keratin, the rates of MIC increases for luliconazole, terbinafine, amorolfine, and efinaconazole
`were 102, 72, 20, and 8, respectively. The rates of increases in MIC were largely associated with
`the degree of keratin affinity. The high rates of MIC increases for luliconazole and terbinafine
`were presumably due to their high keratin affinities. Since the concentration of keratin in the
`nail is approximately 80–90% [16], the antifungal activities of luliconazole and terbinafine
`would be further reduced in the infection site. Luliconazole was the most active in keratin-free
`RPMI 1640 medium, as previously reported by Koga et al. [29]. However, luliconazole was as
`active as efinaconazole and terbinafine in the presence of 20% keratin.
`Ciclopirox showed high affinity to keratin. Nevertheless, its antifungal activity was not
`affected by keratin concentrations. The geometric mean MICs (16–40 μg/mL) of ciclopirox in
`the presence of keratin were 200- to 1000-fold higher than those of the four other drugs. The
`ratios of the MICs of ciclopirox to keratin were 8- to 320-fold higher than that in the keratin
`affinity test. We confirmed that ciclopirox showed a high free-drug ratio in the keratin affinity
`test at a high ratio of the drug to keratin (unpublished data) presumably because of the satura-
`tion of drug-keratin binding, and, thus, it was considered that the activity of ciclopirox was not
`influenced by keratin.
`As described above, the success of topical therapy for onychomycosis depends on whether
`the permeated drug amount in the deep nail bed is retained above its MIC. In order to predict
`clinical efficacy, the efficacy coefficients of several antifungals have been calculated by an equa-
`tion of flux/MIC determined using keratin-free medium [10, 18, 30]. However, the binding of
`antifungals to keratin in nail bed reduces the availability of active (free) drugs and markedly
`weakens their antifungal potencies. Therefore, we introduced that the efficacy coefficient be
`determined using MIC values obtained in the presence of keratin.
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`Efficacy Coefficients of Topical Antifungals for Predicting Clinical Efficacies for Onychomycosis
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`The results of the present study suggest that amorolfine, ciclopirox, and terbinafine have
`lower efficacy coefficients than efinaconazole and luliconazole. In clinical trials with 5% amor-
`olfine lacquer, 8% ciclopirox lacquer, and 10% terbinafine solution, complete cure rates were
`low (0.96%, 5.5–8.5%, and 1.2%, respectively) [31, 32]. In contrast, in clinical trials with 10%
`efinaconazole solution and 5% luliconazole solution, complete cure rates in Japanese patients
`were high (28.8% and 14.9%, respectively) [33, 34]. The orde