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`ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2002, p. 3797–3801
`0066-4804/02/$04.00⫹0 DOI: 10.1128/AAC.46.12.3797–3801.2002
`Copyright © 2002, American Society for Microbiology. All Rights Reserved.
`
`Vol. 46, No. 12
`
`Therapeutic Efficacy of Topically Applied KP-103 against Experimental
`Tinea Unguium in Guinea Pigs in Comparison with
`Amorolfine and Terbinafine
`Yoshiyuki Tatsumi,1* Mamoru Yokoo,1 Hisato Senda,1 and Kazuaki Kakehi2
`Central Research Laboratories, Kaken Pharmaceutical Co., Ltd., Shinomiya, Yamashina-ku, Kyoto-city, Kyoto
`607-8042,1 and Faculty of Pharmaceutical Sciences of Kinki University, Kowakae, Higashi-Osaka-shi,
`Osaka 557-0818,2 Japan
`
`Received 30 May 2002/Returned for modification 28 June 2002/Accepted 19 August 2002
`
`The therapeutic efficacy of KP-103, a novel topical triazole, in a guinea pig tinea unguium model was
`investigated. Experimental tinea unguium and tinea pedis were produced by inoculation of Trichophyton
`mentagrophytes SM-110 between the toes of the hind paw of guinea pigs. One percent solution (0.1 ml) of
`KP-103, amorolfine, or terbinafine was topically applied to the nails and whole sole of an infected foot once
`daily for 30 consecutive days, and terbinafine was also orally administered at a daily dose of 40 mg/kg of body
`weight for 30 consecutive days, starting on day 60 postinfection. The fungal burdens of nails and plantar skin
`were assessed using a new method, which makes it possible to recover infecting fungi by removing a carryover
`of the drug remaining in the treated tissues into the culture medium. Topically applied KP-103 inhibited the
`development of nail collapse, significantly reduced the fungal burden of the nails, and sterilized the infected
`plantar skin. On the other hand, topical amorolfine and topical or oral terbinafine were ineffective for tinea
`unguium, although these drugs eradicated or reduced the fungal burden of plantar skin. The in vitro activities
`of amorolfine and terbinafine against T. mentagrophytes SM-110 were 8- and 32-fold, respectively, decreased by
`the addition of 5% keratin to Sabouraud dextrose broth medium. In contrast, the activity of KP-103 was not
`affected by keratin because its keratin affinity is lower than those of the reference drugs, suggesting that KP-103
`largely exists in the nails as an active form that was not bound to keratin and diffuses in the nail without being
`trapped by keratin. The effectiveness of KP-103 against tinea unguium is probably due to its favorable
`pharmacokinetic properties in the nails together with its potent antifungal activity.
`
`Among dermatophytoses, tinea unguium is the most resis-
`tant to the treatment of antifungal drugs. At present, the oral
`antifungal agents griseofulvin (10), terbinafine (12), and itra-
`conazole (9) are available for the treatment of tinea unguium
`because antifungal agents are generally ineffective when ap-
`plied topically (13, 23). The treatment of tinea unguium has
`improved following the introduction of terbinafine and itra-
`conazole. However, some 20% of patients fail to benefit from
`therapy (20). The main reason for the treatment failure was
`suggested to be inadequate compliance for the usual long-
`period treatment of 6 to 12 months with these oral drugs (20).
`In addition, the long-period oral treatment can lead to severe
`systemic adverse effects or interactions with other systemic
`drugs being taken by the patients (5, 6), which interrupt the
`therapy. Therefore, there is a clear need for new antifungal
`drugs that can be administered topically to reduce the risk of
`side effects or that achieve complete cure in a shorter treat-
`ment period than the existing oral antifungal drugs to improve
`compliance. Two topical antifungal drugs, amorolfine (8) and
`ciclopirox (7), are available in nail lacquer formulations con-
`taining concentrations of 5 and 8%, respectively, and are used
`
`* Corresponding author. Mailing address: Central Research Labo-
`ratories, Kaken Pharmaceutical Co., Ltd., 14 Minamikawara-cho, Shi-
`nomiya, Yamashina-ku, Kyoto-city, Kyoto 607-8042, Japan. Phone:
`81-75-594-0787. Fax: 81-75-594-0790. E-mail:
`tatsumi_yoshiyuki
`@kaken.co.jp.
`
`3797
`
`for the treatment of tinea unguium. However, complete cure
`cannot be expected in every case (13).
`When terbinafine was orally administered to healthy indi-
`viduals at a daily dose of 250 mg for 28 days, a concentration
`10- to 1,000-fold higher than MICs for dermatophytes was
`achieved in nails and persisted for up to 4 months after stop-
`ping treatment (3). Nevertheless, a long-period treatment over
`3 months does not lead to cure rates above 80% for toenails
`(31), and relapse often occurs after treatment (4, 21, 30).
`Presumably because oral antifungal drugs cannot remove fungi
`from nails before nails are turned over, the pace of response of
`tinea unguium seems to be a function of the nail growth rate.
`It is likely that the low fungicidal effect for tinea unguium is
`related to pharmacokinetic problems of oral antifungal drugs
`in the nails (20). Indeed, many antifungal agents are strongly
`bound to keratin, which not only reduces their antifungal po-
`tency but also may restrict their penetration into the nails (22,
`24, 25).
`We reported that KP-103, a novel topical triazole antimy-
`cotic, is highly effective in treating and preventing relapse in
`guinea pig models of tinea pedis and tinea corporis (16, 24–26),
`and its effectiveness is presumably because KP-103 has lower
`keratin affinity than the existing antifungal drugs and is largely
`retained as an active form that is not bound to keratin in the
`horny layer (25).
`In the present study, to predict the potency of antifungal
`activity of KP-103 in the nails, we examined the influence of
`keratin on its in vitro antidermatophyte activity and its affinity
`
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`TATSUMI ET AL.
`
`ANTIMICROB. AGENTS CHEMOTHER.
`
`to keratin compared with those of amorolfine and terbinafine.
`Moreover, we evaluated the therapeutic effect of KP-103 for
`guinea pigs with tinea unguium and plantar tinea pedis caused
`by Trichophyton mentagrophytes in comparison with the two
`reference drugs. Their therapeutic efficacies were assessed us-
`ing the new method that we recently reported (26), which
`permits the precise recovery of fungi from the treated tissues
`by removal of a carryover of the drug remaining in the skin
`tissues into the culture media.
`(This work was presented at the 40th Interscience Confer-
`ence on Antimicrobial Agents and Chemotherapy, Toronto,
`Ontario, Canada, 17 to 20 September 2000 [Y. Tatsumi, M.
`Yokoo, and H. Senda, Abstr. 40th Intersci. Conf. Antimicrob.
`Agents Chemother., abstr. 384, 2000].)
`
`MATERIALS AND METHODS
`
`Antifungal agents. KP-103, amorolfine, and terbinafine hydrochloride were
`synthesized at the Central Research Laboratories, Kaken Pharmaceutical Co.,
`Ltd., Kyoto, Japan.
`Media. Sabouraud dextrose broth (SDB) and Sabouraud dextrose agar (SDA)
`were purchased from Difco Laboratories, Detroit, Mich. Potato dextrose agar
`was purchased from Nissui Seiyaku, Tokyo, Japan. The morpholinepropanesul-
`fonic acid (MOPS)-buffered RPMI 1640 agar medium (pH 7.0), which contained
`liquid RPMI 1640 medium supplemented with L-glutamine (Nissui), 2% sodium
`bicarbonate, 0.165 M MOPS, and 1.6% agar, was used.
`Test organism. Clinical isolates of T. mentagrophytes SM-110 and KD-04 were
`supplied by S. Fujita, Niigata University School of Medicine, Niigata, Japan, and
`H. Takahashi, Teikyo University School of Medicine, Tokyo, Japan, respectively.
`Candida kefyr JCM 1167 was purchased from The Institute of Physical and
`Chemical Research, Saitama, Japan.
`In vitro study. (i) In vitro antidermatophyte activity in the presence of keratin.
`MICs for T. mentagrophytes KD-04 were determined by the following method.
`Ten microliters of twofold serial dilutions of the drugs solubilized in dimethyl
`sulfoxide (DMSO) was dispensed into a tube containing 1 ml of SDB medium
`containing 50 mg of defatted-keratin powder. Each tube was seeded with 106
`microconidia of T. mentagrophytes SM-110 and then incubated at 30°C for 7 days.
`For reference, the same test was also run in SDB without keratin powder. The
`MICs were defined as the lowest drug concentrations that inhibited visible
`growth of the fungi.
`(ii) Affinity to keratin. The affinities of the test drugs to keratin powder were
`determined by a slight modification of the method of Uchida et al. (29). A 100-␮l
`sample of the drug solution prepared in DMSO at a concentration of 1 mg/ml
`was dispensed into 9.9 ml of saline containing 5% defatted-keratin powder. After
`shaking at 37°C for 1 h (75 rpm), the mixture was centrifuged at 3,000 ⫻ g for 5
`min, and two 100-␮l portions of the supernatant were taken to determine the rate
`of binding of drug to keratin. The precipitate (the drug-bound keratin) was
`washed 10 times by shaking (75 rpm) in 10 ml of saline at 37°C for 10 min. After
`each washing, the mixture was then centrifuged at 3,000 ⫻ g for 5 min, and two
`100-␮l samples were taken from the supernatant to determine the rate of release
`of the drug from keratin.
`To determine the drug concentration in the sample solution using bioassay,
`standard curves were constructed. All drugs tested were dissolved in DMSO at a
`concentration of 1 mg/ml, serially diluted twofold with DMSO, and then diluted
`1:100 with saline. Bioassay was performed in MOPS-buffered RPMI 1640 agar
`medium containing C. kefyr JCM 1167 (2 ⫻ 104 cells/ml) for KP-103 and SDA
`and potato dextrose agar containing T. mentagrophytes KD-04 (1 ⫻ 104 conidia/
`ml) for amorolfine and terbinafine, respectively. Assay plates were prepared in
`duplicate. Wells (8 mm in diameter) were cut in the agar. Aliquots (100 ␮l) of
`samples and standards were dispensed into the wells and incubated at 30°C for
`3 days. Diameters of zones of inhibition were measured with a vernier caliper to
`the nearest 0.01 mm. Standard curves relating the zone diameter to the concen-
`tration of the drugs were prepared on a semilogarithmic graph and were linear
`over the range of 0.039 to 10 ␮g/ml for KP-103 and terbinafine and 0.156 to 10
`␮g/ml for amorolfine, with ␥2 being ⬎0.99.
`In vivo efficacy. (i) Animals. Male Hartley strain guinea pigs weighing 370 to
`420 g were used in the study. The experiments were performed with groups of
`five animals.
`(ii) Preparation of inocula. Arthrospores of T. mentagrophytes SM-110 were
`prepared by the method described previously (24–26). The arthrospores were
`
`collected and suspended in saline containing 0.05% Tween 80. The suspension
`was homogenized with a glass homogenizer, filtered through gauze, and then
`adjusted to give a concentration of 108 spores/ml by counting with a hemocy-
`tometer.
`(iii) Production of tinea unguium and tinea pedis. Guinea pigs were infected
`by a slight modification of the method described previously (25, 26). In brief, two
`paper disks (4 by 8 mm; AA disk; Whatman Japan KK) were immersed in the
`fungal suspension, applied between the toes of the hind paw (between the second
`and third toes and between the third and fourth toes) with a foam pad (Reston
`self-adhering foam pads [catalog no. 1560]; 3M Co.), and fixed with an adhesive
`elastic tape (Elastopore; Nichiban, Tokyo, Japan) (day 0 postinfection). The
`disks were removed on day 21 postinfection.
`(iv) Histological examination of nail tissue. Nails were taken from infected
`animals on day 60 postinfection. The nails were fixed in 10% (vol/vol) buffered
`neutral formalin solution, decalcified in 10% (vol/vol) buffered formic acid for 1
`month, fixed again in the formalin solution, and embedded in paraffin. Thin
`paraffin sections of the nails were examined by light microscopy after staining
`with periodic acid-Schiff stain.
`(v) Drugs and treatment. KP-103, amorolfine, or terbinafine was dissolved in
`a mixture of polyethylene glycol 400-ethanol (75:25, vol/vol) (1, 17, 24–26) at 1%
`for topical application. Terbinafine hydrochloride tablets (Lamisil) were pur-
`chased from Novartis Pharma KK and suspended in coconut oil with a mortar,
`and the suspension was dispensed into capsules for oral administration. A 1%
`solution (0.1 ml) of KP-103, amorolfine, or terbinafine was topically applied to
`the nails and whole sole of the foot of guinea pigs once daily, or a capsule of
`terbinafine was orally administered to guinea pigs at a once-daily dose of 40
`mg/kg of body weight. Each treatment was started on day 60 postinfection and
`continued for 30 consecutive days. The control group of animals was infected and
`received vehicle therapy.
`(vi) Evaluation of therapeutic efficacy. The therapeutic efficacy for tinea un-
`guium was evaluated by the method reported previously (26) with a slight mod-
`ification. Two days after the last treatment, all animals were sacrificed and nails
`were taken from the treated feet, which were wiped with a cotton swab contain-
`ing 70% ethanol. The length of each nail was measured with a vernier caliper.
`The nail sample was minced with scissors, homogenized in phosphate-buffered
`saline (PBS) (pH 7.4) containing 2% trypsin, and digested at 37°C for 1 h. After
`centrifugation, the precipitate was washed with 10 ml of PBS three times by
`centrifugation at 3,000 ⫻ g for 10 min to remove trypsin and the remaining drug
`and was suspended in 4 ml of sterilized water. The suspension was dialyzed with
`a cellulose membrane tube in distilled water at 4°C for 14 days to eliminate
`completely the drugs in the nails. The suspension was centrifuged at 3,000 ⫻ g for
`10 min, and the precipitate was suspended in 1 ml of PBS. One hundred micro-
`liters of the sample and serial 10-fold dilutions thereof were spread onto an SDA
`plate containing 1 g ofcycloheximide, 100 mg of gentamicin, 50 mg of chloram-
`phenicol, and 50 mg of flucytosine, per liter, to select the fungi. The plates were
`incubated for 10 days at 30°C, and fungal colonies were counted. The CFU were
`enumerated, and the log10 of CFU in the nails per foot was calculated (assay
`limit, 10 CFU per foot). Nail specimens yielding more than one fungal colony
`were regarded as fungus positive. The therapeutic efficacy against plantar tinea
`pedis was evaluated by the same method reported previously (26), on the basis
`of fungus-positive feet (percentage) and the log10 of CFU in the treated plantar
`skin per foot (assay limit, 20 CFU per foot).
`(vii) Statistical analysis. Nail and skin samples with negative culture results
`were considered to contain 10 and 20 CFU, respectively, for numerical and
`statistical purposes. The frequency of fungus-positive nails or plantar skin per
`foot was analyzed by Fisher’s exact probability test. The log10 of CFU in nails and
`plantar skin and the length of the nails were analyzed by the Kruskal-Wallis test
`(Tukey-type comparison test). P values of less than 0.05 were regarded as sig-
`nificant.
`
`RESULTS
`
`In vitro antidermatophyte activity in the presence of keratin
`powder. It is known that many antifungal agents are inactivated
`when bound to keratin (21, 24, 25). To predict the potency of
`antifungal activity of KP-103 in infected nails, we investigated
`the influence of keratin on its in vitro antidermatophyte activ-
`ity. Table 1 shows the MICs of KP-l03, amorolfine, and terbi-
`nafine for T. mentagrophytes SM-110 in SDB with and without
`5% keratin powder. Amorolfine and terbinafine were 8- and
`
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`VOL. 46, 2002
`
`EFFICACY OF TOPICAL KP-103 AGAINST TINEA UNGUIUM 3799
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`FIG. 2. Histological examination of the nails of guinea pig hind
`paws, 60 days after T. mentagrophytes SM-110 inoculation. The fungi
`invaded the nail plate. Bar ⫽ 40 ␮m.
`
`guinea pigs. T. mentagrophytes SM-110 invaded the nails of
`guinea pigs at day 60 postinfection (Fig. 2). Once-daily topical
`treatment with 1% solutions (0.1 ml) of KP-103, amorolfine,
`and terbinafine or oral treatment with a capsule of terbinafine
`at a daily dose of 40 mg/kg was started on day 60 postinfection
`and continued for 30 consecutive days. Table 2 shows the
`length and fungal burden of infected nails that were taken 2
`days after the last treatment. In the vehicle-treated control
`animals, the nails collapsed and the average length of the nails
`was shorter than that of intact nails (11.82 ⫾ 1.24 mm). Sim-
`ilarly, in the topical amorolfine-, topical or oral terbinafine-
`treated animal, nails collapsed and the length of nails did not
`significantly differ from those of the vehicle-treated animals.
`On the other hand, topically applied KP-103 was highly effec-
`tive in inhibiting the development of nail collapse, and the
`length of KP-103-treated nails did not differ from that of intact
`nails and was significantly longer than that of nails of oral
`terbinafine-treated animals. The viable dermatophytes were
`recovered from all nails of the vehicle-treated control animals.
`All drugs tested failed to sterilize all of the 10 infected nails.
`Topical amorolfine and topical or oral terbinafine were inef-
`fective even in terms of reducing the fungal burden. By con-
`trast, topically applied KP-103 significantly reduced fungal bur-
`den in the infected nails compared with the burdens found in
`vehicle- and oral-terbinafine-treated groups.
`Therapeutic efficacy against experimental plantar tinea pe-
`dis in guinea pigs. Table 3 shows the therapeutic effect of
`KP-103, amorolfine, and terbinafine on the fungal burden of
`the plantar skin in the same animals that were used for eval-
`uation of their therapeutic effect on tinea unguium. Topical
`KP-103 and topical or oral terbinafine achieved mycological
`eradication for all of the 10 infected plantar skin sites. Topical
`amorolfine significantly reduced fungal counts in the infected
`plantar sites and sterilized 4 of the 10 infected plantar skin
`sites.
`
`DISCUSSION
`
`TABLE 1. Influences of keratin powder on the antifungal activities
`of KP-103 and reference antifungal drugs against
`T. mentagrophytes SM-110a
`
`Compound
`
`MIC (␮g/ml) for T.
`mentagrophytes SM-110
`
`SDB alone (A)
`
`SDB with 5%
`keratin (B)
`
`Reduction in activity
`(B/A ratio)
`
`KP-103
`Amorolfine
`Terbinafine
`
`0.2
`0.025
`0.006
`
`0.2
`0.2
`0.2
`
`1
`8
`32
`
`a Inoculum size, 104 microconidia/ml. The MIC was read after incubation at
`30°C for 7 days in each assay medium.
`
`32-fold, respectively, less active against the dermatophytes in
`SDB containing 5% keratin than in SDB alone. However, the
`antifungal activity of KP-l03 was not affected by keratin. Thus,
`KP-103 was as active as amorolfine and terbinafine in the
`presence of 5% keratin, although it was less active than the
`reference drugs in SDB alone.
`Affinity to keratin powder. To clarify the high antifungal
`activity of KP-103 in the presence of keratin, we examined its
`affinity to keratin powder in comparison with those of amo-
`rolfine and terbinafine. Amorolfine and terbinafine showed
`extremely high rates of binding to keratin of 91.8 and 96.0%,
`respectively. On the other hand, the rate of binding of KP-103
`to keratin was at the lower level of 62.8% compared to the
`reference drugs. Figure 1 shows the rates of release of KP-103,
`amorolfine, and terbinafine from each drug-preloaded keratin
`powder. Even though KP-103 was bound to keratin in saline
`containing 5% keratin, it was readily released from keratin by
`the washings with saline. The 10-times washings of KP-103-
`preloaded keratin yielded the cumulative rate of release of
`KP-103 from keratin of 66.8%. On the other hand, under the
`same experimental conditions, the cumulative rates of release
`of amorolfine and terbinafine from keratin were 39.1 and
`21.6%, respectively, and the reference drugs showed a lower
`level of release from keratin than KP-103.
`Therapeutic efficacy against experimental tinea unguium in
`
`FIG. 1. Release of KP-103 or two reference antifungal drugs from
`the drug-preloaded keratin powder. The drug-bound keratin was
`washed in saline at 37°C for 10 min with shaking (75 rpm), and this
`procedure was repeated 10 times.
`
`Antifungal drugs such as lanoconazole, butenafine, terbin-
`afine, and amorolfine developed in recent years have potent in
`vitro antidermatophyte activity (11, 14, 18, 28) and accumulate
`
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`3800
`
`TATSUMI ET AL.
`
`ANTIMICROB. AGENTS CHEMOTHER.
`
`TABLE 2. Therapeutic efficacies of KP-103 and reference drugs in a guinea pig model of tinea unguiuma
`
`Treatment
`
`No. of feet with culture-positive
`nails/total no. of feet (%)
`
`Topical vehicle
`1.0% KP-103 solution
`1.0% amorolfine solution
`1.0% terbinafine solution
`Oral vehicle
`Terbinafine (40 mg/kg/day, p.oe)
`
`10/10 (100)
`10/10 (100)
`10/10 (100)
`10/10 (100)
`10/10 (100)
`10/10 (100)
`
`Log CFU in nails/foot
`(mean ⫾ SD)
`3.70 ⫾ 0.44
`2.20 ⫾ 0.56b
`3.26 ⫾ 0.54
`3.21 ⫾ 0.47
`3.76 ⫾ 0.67
`3.80 ⫾ 0.44
`
`Length (mm) of nails
`(mean ⫾ SD)
`9.47 ⫾ 1.02
`11.64 ⫾ 1.29c,d
`9.66 ⫾ 1.65
`10.33 ⫾ 1.49
`9.41 ⫾ 1.78
`9.46 ⫾ 1.69
`
`a Treatment was started on day 60 postinfection and continued for 30 days.
`b P ⬍ 0.01 versus the topical-vehicle-, oral-vehicle-, and oral-terbinafine-treated groups.
`c P ⬍ 0.01 versus the topical-vehicle-, oral-vehicle-, topical-amorolfine, and oral-terbinafine-treated groups.
`d P ⬍ 0.05 versus the topical-terbinafine-treated group.
`e p.o., orally.
`
`in the horny layer and nails at high levels when topically ap-
`plied (1, 15, 19, 29). In our previous study with a guinea pig
`tinea pedis model (26), we demonstrated that the therapeutic
`efficacy of lanoconazole was not correctly assessed by the con-
`ventional culture method because of a carryover of the drug
`remaining in the treated skin tissues to the culture media for
`detecting fungi. Similarly, even when lanoconazole, butenafine,
`terbinafine, and amorolfine were administered topically to
`guinea pigs with tinea unguium once a day for 20 days and
`culture studies were performed on day 2 posttreatment, these
`drugs yielded all negative cultures for all nail specimens from
`the guinea pigs because of their carryover effects to culture
`medium (data not shown). We, therefore, found that the per-
`sisting drug should be eliminated from the treated nails before
`the culture study to evaluate correctly their therapeutic effects.
`To our knowledge, there has been no study of the therapeutic
`effects of antifungal agents against animal models of tinea
`unguium. This is probably because the sterility of the treated
`nails determined by the conventional method does not corre-
`late with the finding that the existing antifungal drugs are
`almost entirely ineffective for human tinea unguium by topical
`application (13, 23). The present study is the first to report on
`the therapeutic efficacies of antifungal agents in an animal
`model of tinea unguium.
`In the present study, we successfully eliminated drugs from
`all treated nails using the new technique with trypsin treatment
`and the dialysis procedure of treated tissues that we reported
`previously (26), with resultant detection of infecting fungi in all
`
`TABLE 3. Therapeutic efficacies of KP-103 and reference drugs in
`a guinea pig model of tinea pedisa
`
`Treatment
`
`Topical vehicle
`1.0% KP-103 solution
`1.0% amorolfine solution
`1.0% terbinafine solution
`Oral vehicle
`Terbinafine
`(40 mg/kg/day, p.o.d)
`
`No. of feet with
`culture-positive
`skin/total no. of
`feet (%)
`
`10/10 (100)
`0/10 (0)b
`6/10 (60)
`0/10 (0)b
`10/10 (100)
`0/10 (0)b
`
`Log CFU in
`skin/foot
`(mean ⫾ SD)
`
`4.37 ⫾ 0.33
`⬍1.3b
`1.74 ⫾ 0.45c
`⬍1.3b
`3.85 ⫾ 0.68
`⬍1.3b
`
`a Treatment was started on day 60 postinfection and continued for 30 days.
`b P ⬍ 0.01 versus the topical-vehicle- and oral-vehicle-treated groups.
`c P ⬍ 0.05 versus the topical-vehicle-treated group.
`d p.o., orally.
`
`treated nails. The dialysis period of 14 days was necessary for
`removing all drugs tested from the treated nails, which was
`longer than the 3 days used for plantar skin tissues previously
`(26). This is presumably because antifungal agents have a high
`binding affinity to keratin (24, 28) and more accumulated in the
`nails than in the skin tissues. It is suggested that the method is
`useful for evaluating the therapeutic efficacy of antifungal
`agents against the tinea unguium model of animals because it
`permits the complete recovery of viable fungi from all drug-
`treated nails, whereas the conventional method does not re-
`cover fungi because of the drug carryover effects. Moreover,
`the results obtained by the new method in the present study
`correlated well with clinical knowledge that tinea unguium
`responds more poorly to antifungal chemotherapy than does
`tinea pedis (13, 23).
`Since dermatophytes parasitize the keratinized tissues of the
`horny layer of the epidermis, hair, and nails, the therapeutic
`efficacy of applied antifungal agents depends on not only their
`in vitro antifungal activities but also their pharmacokinetic
`properties in the keratinized tissues (27). KP-103 was 8- and
`32-fold, respectively, less active than amorolfine or terbinafine
`against T. mentagrophytes SM-110 in SDB medium. However,
`KP-103 was more effective than the reference drugs for inhib-
`iting nail collapse or reducing the fungal burden of the nails.
`These results suggest that KP-103 shows a better pharmacoki-
`netics in the nails than the reference drugs.
`To predict the pharmacokinetics of KP-103 in the nails, we
`examined the influence of keratin on its antifungal activity and
`its affinity to keratin compared with those of amorolfine and
`terbinafine. Terbinafine and amorolfine showed a greatly de-
`creased antifungal activity in the presence of 5% keratin. This
`was because the reference drugs have extremely high rates of
`binding to keratin of more than 90%. Uchida et al. (29) also
`reported that terbinafine showed a high rate of keratin binding,
`88.4%, when incubated in a buffer with 10% keratin. These
`results suggest that these reference drugs will be further inac-
`tivated in the nails, because about 90% of the constituent of
`nails is keratin. In contrast, the antifungal activity of KP-103
`was not affected by keratin because of its lower affinity to
`keratin, suggesting that KP-103 largely exists in the nails as an
`active form that is not bound to keratin. Antifungal agents
`bound to keratin need to be released readily from it to effec-
`tively diffuse into the deeper nail plate and sterilize infected
`nails. Even though KP-103 was bound to keratin suspended in
`saline, it was readily released from keratin by washing with
`
`Page 4
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`Anacor Exhibit 2037
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00169
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`Downloaded from
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`VOL. 46, 2002
`
`EFFICACY OF TOPICAL KP-103 AGAINST TINEA UNGUIUM 3801
`
`saline. Its release from keratin was higher than those of amo-
`rolfine and terbinafine. It is likely that KP-103 diffuses in the
`nails without being trapped by keratin.
`Topical amorolfine and topical or oral terbinafine were not
`effective in reducing the fungal burden of the nails, although
`the reference drugs reduced or eradicated the fungal burden in
`plantar skin tissues. These results suggest that the two refer-
`ence drugs were possibly inactivated by keratin in the nails,
`although they were absorbed into the nails as in plantar skins.
`On the other hand, topical KP-103 was effective for both re-
`ducing the fungal burden of nails and eradicating infection in
`plantar skins. The effectiveness of KP-103 for tinea unguium is
`presumably due to its high activity and good penetration into
`the nails, which are achieved by its lower keratin affinity.
`It was reported that orally administered terbinafine is effec-
`tive for the treatment of human tinea unguium (3, 4, 12, 30,
`31). However, in the present study, oral terbinafine was not
`effective for the tinea unguium in the guinea pig model. This
`discrepancy is possibly due to the fact that the treatment pe-
`riod of 30 days for guinea pigs was shorter than that necessary
`to show therapeutic effect against human tinea unguium, which
`is at least 3 months (4, 30, 31). It was reported that a concen-
`tration of terbinafine (0.1 to 1.53 ␮g/ml) higher than its MIC
`(0.001 to 0.02 ␮g/ml) for dermatophytes was achieved in the
`nails when it was orally administered to patients with tinea
`unguium at a daily dose of 250 mg for 28 days (2). In the
`present study, since guinea pigs were orally treated with terbi-
`nafine for 30 days at a daily dose of 40 mg/kg, which is about
`10-fold higher than that used in humans, a high concentration
`of terbinafine would be achieved in the nails. However, terbi-
`nafine failed to reduce the fungal burden in the affected nails.
`This finding also suggests that terbinafine would be inactivated
`by its strong binding to keratin in the nails.
`In conclusion, KP-103 is a very promising antifungal candi-
`date in the treatment of human tinea unguium because topi-
`cally applied KP-103 exhibits a better therapeutic effect than
`oral terbinafine and possibly allows shorter periods of treat-
`ment, which improves patient compliance and yields no risk of
`systemic adverse effects and drug interactions, unlike oral an-
`tifungal drugs.
`
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