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`The Low Keratin Affinity of Efinaconazole Contributes to Its Nail
`Penetration and Fungicidal Activity in Topical Onychomycosis
`Treatment
`
`Keita Sugiura,a Noriaki Sugimoto,b Shinya Hosaka,b Maria Katafuchi-Nagashima,a Yoshio Arakawa,a Yoshiyuki Tatsumi,a
`William Jo Siu,c Radhakrishnan Pillaic
`Kaken Pharmaceutical Co. Ltd., Kyoto, Japana; Kaken Pharmaceutical Co. Ltd., Shizuoka, Japanb; Dow Pharmaceutical Sciences, a division of Valeant Pharmaceuticals,
`Petaluma, California, USAc
`
`Onychomycosis is a common fungal nail disease that is difficult to treat topically due to the deep location of the infection under
`the densely keratinized nail plate. Keratin affinity of topical drugs is an important physicochemical property impacting thera-
`peutic efficacy. To be effective, topical drugs must penetrate the nail bed and retain their antifungal activity within the nail ma-
`trix, both of which are adversely affected by keratin binding. We investigated these properties for efinaconazole, a new topical
`antifungal for onychomycosis, compared with those of the existing topical drugs ciclopirox and amorolfine. The efinaconazole
`free-drug concentration in keratin suspensions was 14.3%, significantly higher than the concentrations of ciclopirox and amo-
`rolfine, which were 0.7% and 1.9%, respectively (P < 0.001). Efinaconazole was released from keratin at a higher proportion than
`in the reference drugs, with about half of the remaining keratin-bound efinaconazole removed after washing. In single-dose in
`vitro studies, efinaconazole penetrated full-thickness human nails into the receptor phase and also inhibited the growth of
`Trichophyton rubrum under the nail. In the presence of keratin, efinaconazole exhibited fungicidal activity against Trichophyton
`mentagrophytes comparable to that of amorolfine and superior to that of ciclopirox. In a guinea pig onychomycosis model with
`T. mentagrophytes infection, an efinaconazole solution significantly decreased nail fungal burden compared to that of ciclopirox
`and amorolfine lacquers (P < 0.01). These results suggest that the high nail permeability of efinaconazole and its potent fungi-
`cidal activity in the presence of keratin are related to its low keratin affinity, which may contribute to its efficacy in
`onychomycosis.
`
`Onychomycosis is a chronic superficial mycosis of the nails
`
`that is difficult to treat. It has a prevalence of about 10% in
`Japan (1) and 13.8% in North America (2), and it is more com-
`mon in older persons, affecting as many as one in three individuals
`in that population (1, 3). The most common form of the disease is
`distal lateral subungual onychomycosis (DLSO), and the causative
`pathogens include Trichophyton rubrum, Trichophyton mentagro-
`phytes, nondermatophyte molds (e.g., Scopulariopsis brevicaulis
`and Fusarium species), and Candida albicans.
`Oral terbinafine and itraconazole are currently the preferred
`treatments for onychomycosis (4, 5). However, their use is limited
`by hepatotoxicity and drug-drug interactions (especially with itra-
`conazole), which represent a safety concern, particularly in older
`persons, in whom underlying disease and polypharmacy are com-
`mon. Routine liver function testing is recommended for those
`being treated with oral terbinafine and itraconazole, and their use
`is contraindicated in patients with abnormal liver function (6). In
`contrast, topical ciclopirox and amorolfine nail lacquers have a
`favorable safety profile, but their cure rates are considerably lower
`(4, 6). Safe and efficacious topical therapies against onychomyco-
`sis remain an unmet medical need.
`One aspect contributing to the difficulty in developing an ef-
`fective topical agent against onychomycosis is the location of the
`fungal infection deep in the nail bed. To be effective, an adequate
`amount of drug must penetrate the nail bed (7) and remain active
`within the keratin matrix of the nail and nail bed (8–10). The
`unique properties of the nail, particularly its thickness and rela-
`tively compact construction, make it a formidable barrier to the
`entry of topically applied agents (11). The upper dorsal layer is
`
`only a few cell layers thick but consists of hard keratin, constitut-
`ing the main barrier for drug diffusion into and through the nail
`plate (12). Further, many antifungal agents bind strongly to ker-
`atin, which not only restricts their penetration through the nail
`but may also reduce their antifungal activity (8–10). Keratin-
`bound drugs do not contribute to a concentration gradient, re-
`sulting in the accumulation of topically applied drug in the surface
`nail layers and decreased penetration to the deeper layers and nail
`bed (8). Thus, low keratin affinity is considered a desirable phys-
`icochemical property of drugs for topical treatment of onychomy-
`cosis and possibly of other superficial mycoses.
`Efinaconazole (Fig. 1) is a new triazole antifungal agent devel-
`oped as a low-surface-tension 10% (wt/wt) topical solution for the
`treatment of mild to moderate DLSO. It was originally identified
`as a potent antifungal with in vitro activity minimally affected by
`keratin (13). The methylene-piperidine group at the C-4 position
`of the efinaconazole molecule may be responsible for its relatively
`low keratin binding. Efinaconazole possesses broader-spectrum
`antifungal activity than existing antifungals against dermatophyte
`and nondermatophyte molds and yeasts (14). It has potent in vitro
`
`Received 16 January 2014 Returned for modification 19 February 2014
`Accepted 17 April 2014
`Published ahead of print 21 April 2014
`Address correspondence to Keita Sugiura, sugiura_keita@kaken.co.jp.
`Copyright © 2014, American Society for Microbiology. All Rights Reserved.
`doi:10.1128/AAC.00111-14
`
`July 2014 Volume 58 Number 7
`
`Antimicrobial Agents and Chemotherapy p. 3837–3842
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`at 50 ␮g/ml, and gentamicin (Merck & Co., Inc., Whitehouse Station, NJ,
`USA) at 75 ␮g/ml. The keratin medium was prepared by mixing a 0.2%
`(wt/vol) K2HPO4, 0.005% (wt/vol) CaCl2, and 0.005% (wt/vol) MgSO4
`aqueous solution with 200 mg/ml defatted keratin powder (MP Biomedi-
`cals, LLC, Solon, OH, USA).
`Test organisms. T. rubrum and T. mentagrophytes strains were kindly
`supplied by Cardiff University (Cardiff, United Kingdom) and Niigata
`University School of Medicine (Niigata, Japan), respectively. Both strains
`were susceptible to the test drugs used in the experiments (our unpub-
`lished data).
`Affinity to keratin. The affinities of the test drugs to keratin were
`determined as described previously (10), with a slight modification.
`Briefly, 100 ␮l of drug solution in dimethyl sulfoxide (DMSO) (50 ␮g/ml
`efinaconazole and 100 ␮g/ml ciclopirox or amorolfine) was mixed with
`9.9 ml of 0.2 mol/liter Tris-HCl buffer (pH 7.4) containing 0.5 g of defat-
`ted keratin powder. After shaking at 37°C for 1 h (75 rpm), the suspension
`was centrifuged. The drug concentration in the supernatant was deter-
`mined by liquid chromatography-tandem mass spectrometry (LC-MS/
`MS) (TSQ Quantum Ultra; Thermo Fisher Scientific, Inc., Waltham, MA,
`USA). The lower limit of quantification of the assay was 1 ng/ml for all
`analytes. The percentage of free drug was calculated for each drug, and
`the mean values were compared by Tukey’s multiple-comparison test
`(EXSUS version 7.7.1; CAC Exicare Corporation). P values of ⬍0.05 were
`regarded as significant. The keratin pellet (containing bound drug) was
`resuspended in 10 ml of 0.2 mol/liter Tris-HCl buffer and washed by
`shaking for 10 min. The suspension was centrifuged and the washing
`procedure repeated 5 times. The drug concentration of the supernatant
`obtained after each wash was measured by LC-MS/MS, and the cumula-
`tive percent release of drug was calculated.
`In vitro drug penetration in the human nail plate. Healthy human
`nails at full thickness were purchased from KAC Co., Ltd. (Kyoto, Japan)
`and cut in squares of approximately 16 mm2. The nails were fixed between
`two acrylic plates with a 2-mm-diameter hole and mounted in Franz
`diffusion cells (Japan Glass Industry Co., Ltd., Tokyo, Japan). The recep-
`tor compartment was filled with phosphate-buffered saline (PBS) (pH
`7.4) containing 4% (wt/vol) bovine serum albumin (BSA) and 0.01%
`(wt/vol) sodium azide and placed in an incubator at 32°C. Next, 2 ␮l of
`efinaconazole 10% solution, ciclopirox 8% nail lacquer, or amorolfine 5%
`nail lacquer was singly applied on the dorsal nail surface in the donor
`compartment. The receptor solution was continuously stirred by a spin-
`ning magnetic bar. The receptor fluid was sampled once daily for 14 days,
`drug concentrations were determined by LC-MS/MS, and the cumulative
`drug amount permeated per nail unit area (1 cm2) was calculated.
`In vitro antifungal activity under the human nail plate. The drug
`growth inhibition of T. rubrum under the human nail plate was deter-
`mined as reported previously (21). Briefly, distal nail clippings of ⱖ9 mm2
`obtained from volunteer toenails were mounted in TurChub cells. SDA
`containing T. rubrum microconidia (5 ⫻ 104 cells) was loaded into the
`lower chamber of the cells. Ten microliters of efinaconazole 5% solution,
`ciclopirox 8% nail lacquer, or amorolfine 5% nail lacquer was applied
`once on the dorsal nail side, dried for 1 h, and incubated for 7 days at 25°C.
`After incubation, the lengths (cm) of the growth inhibition zones pro-
`duced in the agar under the nail were measured with a caliper.
`Fungicidal activity in the presence of keratin. Forty microliters of
`4-fold serial dilutions of the drugs in DMSO were added into tubes con-
`taining 4 ml of keratin medium and T. mentagrophytes microconidia at
`2 ⫻ 104 cells/ml and incubated at 30°C. The final drug concentrations in
`the medium were 0 (DMSO), 0.313, 1.25, 5, and 20 ␮g/ml. After incuba-
`tion for 2, 4, 7, 10, and 14 days, 0.5 ml of culture suspension was taken
`from the tube, homogenized with a glass homogenizer, and spread onto
`GPLP plates. After incubation for 10 days, viable cell counts (CFU/ml)
`were determined (detection limit, 10 CFU/ml).
`Therapeutic efficacy in guinea pig model of onychomycosis. All ex-
`perimental procedures were approved by the Institutional Animal Care
`and Use Committee of Kaken Pharmaceutical Co., Ltd. Arthrospores of T.
`
`Sugiura et al.
`
`FIG 1 Chemical structures of test substances. (a) Efinaconazole; (b) ciclopi-
`rox; (c) amorolfine.
`
`antifungal activity against nondermatophytes, such as Scopulari-
`opsis brevicaulis and Fusarium species, which cause nail infections
`that respond poorly to oral drugs (15). Thus, efinaconazole may
`be effective in treating nondermatophyte onychomycosis.
`In a guinea pig onychomycosis model, topically applied efina-
`conazole was more effective in reducing toenail fungal burden
`than were amorolfine and terbinafine (10). Further, the therapeu-
`tic efficacy of efinaconazole has been established in mild to mod-
`erate DLSO patients in two phase III clinical trials (16). Its myco-
`logical cure rate is comparable to that reported with oral
`itraconazole, and the complete cure rate is 2- to 3-fold higher than
`that of ciclopirox nail lacquer (17, 18).
`To further understand the efficacy of efinaconazole in onych-
`omycosis, its keratin affinity was investigated in relation to its nail
`penetration and antifungal activity in the keratin matrix. In vitro
`and in vivo models of nail drug delivery and onychomycosis were
`used to characterize the properties of efinaconazole, which were
`compared with two topical antifungals currently available in the
`United States and/or Europe, ciclopirox and amorolfine (Fig. 1).
`(This work was presented in part at the 71st Annual Meeting,
`American Academy of Dermatology, Miami Beach, FL, 1 to 5
`March 2013 [abstract P-6546] [19], and International Investiga-
`tive Dermatology, Edinburgh, Scotland, 8 to 11 May 2013 [ab-
`stract 1151] [20].)
`
`MATERIALS AND METHODS
`Antifungal agents. Efinaconazole was synthesized by Kaken Pharmaceu-
`tical Co., Ltd. (Tokyo, Japan). Ciclopirox olamine and amorolfine hydro-
`chloride were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA)
`and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively. The
`efinaconazole solutions (5% and 10% [wt/wt]) were manufactured by
`Dow Pharmaceutical Sciences, Inc. (Petaluma, CA, USA) and Kaken
`Pharmaceutical Co., Ltd., respectively. Ciclopirox 8% nail lacquer (Pen-
`lac) and amorolfine 5% nail lacquer (Loceryl) were purchased from
`Sanofi-Aventis (Paris, France) and Galderma Laboratories, Inc. (Laus-
`anne, Switzerland), respectively.
`Media. Sabouraud dextrose agar (SDA) was purchased from Becton,
`Dickinson and Company (Franklin Lakes, NJ, USA) or Oxoid Ltd. (Bas-
`ingstoke, United Kingdom). Glucose peptone agar with lecithin and poly-
`sorbate 80 (GPLP) was purchased from Wako Pure Chemical Industries,
`Ltd. (Tokyo, Japan). A modified GPLP medium containing 1% lecithin
`and antibiotics was used to grow the dermatophytes from guinea pig nails.
`The antibiotics used were chloramphenicol (Wako Pure Chemical Indus-
`tries, Ltd.) at 10 ␮g/ml, cycloheximide (Wako Pure Chemical Industries,
`Ltd.) at 500 ␮g/ml, 5-fluorocytosine (Tokyo Chemical Industry Co., Ltd.)
`
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`Properties of Efinaconazole in Onychomycosis Treatment
`
`FIG 2 Affinity to keratin. Efinaconazole (0.5 ␮g/ml), ciclopirox (1 ␮g/ml), or amorolfine (1 ␮g/ml) was incubated in Tris-HCl buffer (pH 7.4) with keratin
`powder (0.5 g/10 ml). (a) The percentage of free drug in buffer after shaking at 37°C for 1 h. The graphs represent the mean ⫾ SD of three replicates. Tukey
`multiple-comparison test: **, P ⬍ 0.01 versus ciclopirox; ***, P ⬍ 0.001 versus ciclopirox; †††, P ⬍ 0.001 versus amorolfine. (b) The cumulative release of
`efinaconazole (Œ), ciclopirox (o), and amorolfine (〫). Each plot represents the mean value of triplicates.
`
`zole was detected in 4/6 receptor-phase samples, and the cumula-
`tive permeated amounts were 2.94 ⫾ 3.91 ␮g/cm2 and 6.53 ⫾ 8.15
`␮g/cm2 (mean ⫾ SD), respectively (Table 1). Ciclopirox was de-
`tected in 2 to 3 of 6 samples, and the cumulative permeated
`amounts were 0.326 ⫾ 0.590 ␮g/cm2 and 4.57 ⫾ 6.89 ␮g/cm2
`(mean ⫾ SD), respectively. On the other hand, amorolfine was
`not detectable in any receptor-phase sample.
`In vitro antifungal activity under the human nail plate. To
`confirm drug penetration through the nail and further evaluate
`the antifungal activity of the permeated drug, growth inhibition
`was studied using TurChub cells (21). A prototype formulation
`with a lower efinaconazole concentration (5%) was used in the
`experiment. The efinaconazole 5% solution produced a region of
`growth inhibition under the nail (mean ⫾ SD, 2.52 ⫾ 0.42 cm),
`whereas ciclopirox 8% and amorolfine 5% nail lacquers did not
`(Fig. 3).
`Fungicidal activity in the presence of keratin. The fungicidal
`activity of efinaconazole was examined in keratin medium, mim-
`
`TABLE 1 Cumulative drug amount permeated after a single application
`of efinaconazole 10% solution, ciclopirox 8% nail lacquer, and
`amorolfine 5% nail lacquer to human nailsa
`Cumulative amt permeated (␮g/cm2) for:
`
`mentagrophytes were prepared by a previously reported method (22, 23).
`Six-week-old male Hartley guinea pigs (Japan SLC, Inc., Hamamatsu,
`Japan) were randomly assigned to one of four treatment groups consisting
`of six animals each. The nails of guinea pigs were infected with the arthro-
`spores (2 ⫻ 107 cells/foot) as previously described (10), with a slight
`modification. Treatment was initiated on day 29 after fungal inoculation
`and continued for 4 weeks. Thirty microliters of each drug was individu-
`ally applied topically to all three nails/foot once daily (efinaconazole 10%
`solution or ciclopirox 8% nail lacquer) or once weekly (amorolfine 5%
`nail lacquer). The dosing frequency was based on typical clinical use. Nails
`treated with ciclopirox nail lacquer or amorolfine nail lacquer were wiped
`with 70% isopropyl alcohol once a week before applying the drugs to
`remove any residual material. One group was left untreated as the infected
`control. The therapeutic efficacy was evaluated by a previously described
`method (10), with a slight modification. Following a rest period of 7 days
`after the last treatment, each animal was euthanized and the nails were
`wiped with 70% ethanol and removed from the feet. The nails from each
`foot were pooled, minced thoroughly, homogenized with a glass homog-
`enizer in PBS (pH 7.4) containing 0.25% trypsin and 10 mmol/liter FeCl3,
`and digested at 37°C for about 1 h. The samples were spread onto modi-
`fied GPLP plates containing 1% lecithin and antibiotics. The plates were
`incubated for 14 days at 30°C, and the fungal colonies were counted (de-
`tection limit, 1.11 log10 CFU/foot). The mean log10 CFU/foot values for all
`treatments were analyzed by Tukey’s multiple-comparison test. P values
`of ⬍0.05 were regarded as significant.
`
`RESULTS
`Affinity to keratin. The efinaconazole, ciclopirox, and amorolfine
`free-drug levels after incubation in keratin suspensions were
`14.3% ⫾ 0.4%, 0.7% ⫾ 0.0%, and 1.9% ⫾ 0.2% (mean ⫾ stan-
`dard deviation [SD]), respectively; the cumulative drug release
`levels after five washes were 46.0% ⫾ 0.6%, 2.4% ⫾ 0.2%, and
`6.9% ⫾ 0.1% (mean ⫾ SD), respectively (Fig. 2). The efinacona-
`zole free-drug and keratin release levels were ⱖ6-fold higher than
`those of amorolfine and ciclopirox. The amorolfine levels were
`about 2-fold higher than those of ciclopirox.
`In vitro drug penetration in the human nail plate. Drug nail
`penetration was investigated in Franz diffusion cells after a single
`topical application of efinaconazole 10% solution, ciclopirox 8%
`nail lacquer, or amorolfine 5% nail lacquer to human nails for 14
`days (n ⫽ 6 cells/group). Efinaconazole was detected in the recep-
`tor-phase samples as early as day 1, whereas ciclopirox was first
`detected on day 6 (data not shown). On days 7 and 14, efinacona-
`
`Time (day)
`
`Ciclopirox
`NCb
`NC
`NC
`NC
`NC
`0.0998 ⫾ 0.2445
`0.326 ⫾ 0.590
`0.535 ⫾ 0.908
`0.895 ⫾ 1.495
`1.32 ⫾ 2.18
`2.03 ⫾ 3.32
`2.82 ⫾ 4.34
`3.56 ⫾ 5.43
`4.57 ⫾ 6.89
`
`Efinaconazole
`0.401 ⫾ 0.672
`1
`0.742 ⫾ 1.242
`2
`1.29 ⫾ 1.91
`3
`1.59 ⫾ 2.37
`4
`2.16 ⫾ 3.11
`5
`2.57 ⫾ 3.49
`6
`2.94 ⫾ 3.91
`7
`3.45 ⫾ 4.46
`8
`3.98 ⫾ 5.20
`9
`4.49 ⫾ 5.70
`10
`4.84 ⫾ 6.12
`11
`5.42 ⫾ 6.97
`12
`5.90 ⫾ 7.39
`13
`6.53 ⫾ 8.15
`14
`a Data represent mean ⫾ SD (n ⫽ 6).
`b NC, not calculated because drug concentration in the receptor phase was below the
`lower limit of quantification (⬍1 ng/ml) in all samples.
`
`Amorolfine
`
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`
`July 2014 Volume 58 Number 7
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`FIG 3 Fungal growth inhibition under the human nail plate. The inhibition of T. rubrum growth under the human nail plate was determined in TurChub cells
`after 7 days of a single application of efinaconazole 5% solution, ciclopirox 8% nail lacquer, or amorolfine 5% nail lacquer. (a) Representative results of TurChub
`cells. (b) Mean growth inhibition zone lengths (cm) for 5 or 6 replicates. N.D., not detectable.
`
`icking the keratin-rich environment of the nail plate and nail bed
`where the fungal infection resides. In the untreated growth con-
`trol cultures, viable cell counts were unchanged through day 4 but
`began increasing on day 7 of incubation, showing that dermato-
`phytes utilize keratin as an energy source (Fig. 4). Both efinacona-
`zole and amorolfine at 1.25, 5, and 20 ␮g/ml decreased viable cell
`counts by one log as early as day 2 and produced a near-complete
`kill by day 14. Exposure to these drugs at 0.313 ␮g/ml produced an
`initial transient decrease in viability, which recovered to at least
`baseline levels by day 14; efinaconazole was slightly more potent
`than amorolfine at this concentration. Ciclopirox decreased via-
`bility from day 7 only at 5 and 20 ␮g/ml. Thus, the fungicidal
`activity of efinaconazole was higher than that of ciclopirox and
`comparable to that of amorolfine in the presence of keratin.
`Therapeutic efficacy in guinea pig model of onychomycosis.
`The in vivo efficacy of the efinaconazole 10% solution was inves-
`tigated in a guinea pig onychomycosis model. After repeated top-
`ical treatment, the viable cell counts in the nails in the groups
`treated with efinaconazole 10% solution, ciclopirox 8% nail lac-
`quer, and 5% amorolfine nail lacquer were 2.41 ⫾ 0.48, 3.17 ⫾
`0.77, and 3.99 ⫾ 0.48 log10 CFU/foot (mean ⫾ SD), respectively
`(Fig. 5). The viable cell counts in these three groups were signifi-
`cantly lower (P ⬍ 0.001, P ⬍ 0.05, and P ⬍ 0.001, respectively)
`than those of the infected control group (mean ⫾ SD, 4.64 ⫾ 0.30
`log10 CFU/foot). Furthermore, the viable cell counts in the nails
`treated with the efinaconazole 10% solution were significantly
`
`lower than those treated with the ciclopirox 8% and amorolfine
`5% nail lacquers (P ⬍ 0.01 and P ⬍ 0.001, respectively).
`
`DISCUSSION
`Topical onychomycosis therapy is challenged by the location of
`the fungal infection deep within the nail, the unique physico-
`chemical properties of the nail (e.g., thickness and relatively com-
`pact construction), and low drug penetration (24). Enhanced
`drug delivery to the site of action may correlate with greater effi-
`cacy; thus, it is expected that drugs targeting the lower nail layers
`will be more effective in treating onychomycosis. Although the
`vehicle in which the drug is formulated may aid delivery, the in-
`trinsic properties of the drug molecule are considered to play an
`important role in its clinical efficacy. Keratin affinity is an impor-
`tant physicochemical property affecting the efficacy of antifungal
`drugs, many of which are reported to have high affinity to keratin
`(10, 12, 25).
`In the present study, to further understand the properties of
`efinaconazole in terms of efficacy for onychomycosis and nail per-
`meability, ciclopirox and amorolfine were compared using three
`unique models of onychomycosis (in vitro and in vivo).
`Efinaconazole had significantly lower binding to keratin, with
`an unbound fraction ⬎7-fold higher and a higher release rate
`from keratin than those of ciclopirox or amorolfine (Fig. 2). This
`low keratin affinity of efinaconazole correlated with faster nail
`penetration and fungicidal activity in the presence of keratin.
`
`FIG 4 Fungicidal activity of drugs against T. mentagrophytes in the presence of keratin. The drugs were incubated at 30°C in keratin medium containing T.
`mentagrophytes at 2 ⫻ 104 cells/ml for 14 days. The graphs represent the mean ⫾ SD of three cultures treated with efinaconazole, ciclopirox, and amorolfine (},
`0 ␮g/ml; Œ, 0.313 ␮g/ml; , 1.25 ␮g/ml; 䊐, 5␮g/ml; , 20␮g/ml).
`
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`Properties of Efinaconazole in Onychomycosis Treatment
`
`The high nail penetration and potent fungicidal activity of efi-
`naconazole in the presence of keratin observed in vitro and in vivo
`may not necessarily be predictive of clinical outcome. However,
`the mycological and complete cure rates observed clinically with
`the efinaconazole 10% solution were 2- to 3-fold greater than
`those previously reported with the ciclopirox nail lacquer (16, 18).
`Therefore, our results suggest that these important features of
`efinaconazole contribute to its clinical efficacy.
`
`ACKNOWLEDGMENTS
`We thank Brian Bulley, Jun Nakano, and Hisato Senda for manuscript
`discussions and review and Marc Brown and Rob Turner (MedPharm
`Ltd., Guildford, United Kingdom) for performing the antifungal activity
`study under the human nail plate using TurChub cells.
`This study was supported by Kaken Pharmaceutical Co., Ltd., and
`Valeant Pharmaceuticals North America LLC.
`Each of us is an employee and stockholder in Kaken Pharmaceutical
`Co., Ltd., or Valeant Pharmaceuticals North America LLC.
`
`REFERENCES
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`matol. 37:397– 406. http://dx.doi.org/10.1111/j.1346-8138.2009.00741.x.
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`A large-scale North American study of fungal isolates from nails: the fre-
`quency of onychomycosis, fungal distribution, and antifungal susceptibil-
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`.org/10.1016/S0065-7743(05)40021-4.
`5. Gupta AK, Konnikov N, Lynde CW. 2001. Single-blind, randomized,
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`6. Niewerth M, Korting HC. 1999. Management of onychomycoses. Drugs
`58:283–296. http://dx.doi.org/10.2165/00003495-199958020-00005.
`7. Murdan S. 2002. Drug delivery to the nail following topical application. Int. J.
`Pharm. 236:1–26. http://dx.doi.org/10.1016/S0378-5173(01)00989-9.
`8. Narasimha Murthy S, Wiskirchen DE, Bowers CP. 2007. Iontophoretic
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`.org/10.1002/jps.20757.
`9. Schaller M, Borelli C, Berger U, Walker B, Schmidt S, Weindl G,
`Jäckel A. 2009. Susceptibility testing of amorolfine, bifonazole and
`ciclopiroxolamine against Trichophyton rubrum in an in vitro model of
`dermatophyte nail infection. Med. Mycol. 47:753–758. http://dx.doi
`.org/10.3109/13693780802577892.
`10. Tatsumi Y, Yokoo M, Senda H, Kakehi K. 2002. Therapeutic efficacy of topically
`applied KP-103 against experimental Tinea unguium in guinea pigs in compari-
`son with amorolfine and terbinafine. Antimicrob. Agents Chemother. 46:3797–
`3801. http://dx.doi.org/10.1128/AAC.46.12.3797-3801.2002.
`11. Hui X, Shainhouse Z, Tanojo H, Anigbogu A, Markus GE, Maibach HI,
`Wester RC. 2002. Enhanced human nail drug delivery: nail inner drug
`content assayed by new unique method. J. Pharm. Sci. 91:189 –195. http:
`//dx.doi.org/10.1002/jps.10003.
`12. Kobayashi Y, Miyamoto M, Sugibayashi K, Morimoto Y. 1999. Drug
`permeation through the three layers of the human nail plate. J. Pharm.
`Pharmacol. 51:271–278. http://dx.doi.org/10.1211/0022357991772448.
`13. Ogura H, Kobayashi H, Nagai K, Nishida T, Naito T, Tatsumi Y, Yokoo
`M, Arika T. 1999. Synthesis and antifungal activities of (2R,3R)-2-aryl-
`1-azolyl-3-(substituted amino)-2-butanol derivatives as topical antifun-
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`/10.1248/cpb.47.1417.
`
`FIG 5 Therapeutic efficacies of topical drugs in a guinea pig onychomycosis
`model. Viable cell counts in guinea pig nails (log10 CFU/foot) were determined
`as a measure of drug efficacy after 28 days of treatment with efinaconazole 10%
`solution (once daily), ciclopirox 8% nail lacquer (once daily), or amorolfine
`5% nail lacquer (once weekly). The bars represent the mean viable cell counts
`from 12 feet (6 animals). The mean values were compared using Tukey’s mul-
`tiple-comparison test (*, P ⬍ 0.05 versus infected control; ***, P ⬍ 0.001
`versus infected control; ††, P ⬍ 0.01 versus ciclopirox 8% nail lacquer; ‡‡‡,
`P ⬍ 0.001 versus amorolfine 5% nail lacquer).
`
`When the antifungal activity against T. rubrum was investi-
`gated in vitro under healthy human nails at full thickness, growth
`was inhibited after treatment with the efinaconazole solution but
`not with the ciclopirox and amorolfine nail lacquers (Fig. 3).
`These observations are in agreement with the high nail permea-
`bility of efinaconazole and its potent antidermatophytic activity
`(14). These in vitro onychomycosis models used a single applica-
`tion for better differentiation of nail absorption and antifungal
`activity profiles between the drugs; characterization following re-
`peated application, as intended clinically for these drugs, was not
`investigated.
`Although the fungicidal activities of azole antifungals are gen-
`erally weaker than those of amorolfine and ciclopirox in RPMI
`1640 (9), the fungicidal activity of efinaconazole against T. men-
`tagrophytes in keratin medium was similar to that of amorolfine
`and higher than that of ciclopirox (Fig. 4). Because keratin me-
`dium contains keratin as the sole nutrient source for energy and
`mimics the nail bed environment, these data more accurately re-
`flect in vivo activity. The high fungicidal activity seen with efina-
`conazole is reflective of its higher free (unbound) concentration
`relative to those of the comparator drugs.
`Both the efinaconazole 10% solution and the comparator nail
`lacquer drugs decreased viable dermatophyte cell counts in in-
`fected guinea pig nails. However, the in vivo antifungal activity of
`efinaconazole was significantly higher (Fig. 5), which may be ex-
`plained by its nail permeability profile and antifungal activity in
`the presence of keratin.
`The limited efficacies of currently marketed topical onycho-
`mycosis drugs may be due to keratin binding, which decreases nail
`drug penetration and antifungal activity. In developing new top-
`ical onychomycosis drugs, keratin affinity is an important factor
`affecting efficacy. For example, oral terbinafine, which is approved
`for onychomycosis treatment and considered the gold standard, is
`98.9% keratin bound under the same in vitro experimental condi-
`tions as those in this study (data not shown); however, it has been
`shown to have limited success in treating the disease when applied
`topically (26).
`
`July 2014 Volume 58 Number 7
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`continuous terbinafine compared with intermittent itraconazole in treat-
`ment of toenail onychomycosis. BMJ 318:1031–1035.
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