Fechrical
`
`Field
`
`of
`
`TECHNICAL FIELD
`
`The present invention relates to a method for detecting pathogenic microorganism
`method for evaluating an effect of an antimicrobial agent on pathogenic
`
`microorganism.tp-particujarrelates_te—anda method for evaluating-an-effect-oftt
`
`an-agentwhereindetecting an antimicrobial agent. The present invention also relates
`to_an antimicrobial agent existiagin-and a therapeutic agent for onychomycosis
`
`which areobtained according,to the above-mentioned method foror evaluating Be
`
`drug effect.
`
`BACKGROUND ART
`
`A method for evaluating a drug effect with an animal model is needed in orderto
`explore a novel antimicrobial agent (also hereinafter “referred to drug"). Further, a
`method forevaluating-enabling a drug effect to be evaluated with accuracy is needed
`because of greatgrate importance in view of predicting a clinical therapeutic efficiency
`thereof.
`
`Historically, an experimental dermatophytosis model that back, planta and interdigital
`| of a guineaguniea pig have beeninfected with Trichophyton mentagrophytes has
`been used in order to evaluate an effect of an antifungal agent on dermatophytosis.
`Such animal models have been already employed to develop some antifungal agent.
`The evaluation of the effect of such antifungal agent is—carried out by applying the
`antifungal agent to the infected animal, byexcising the skin after the certain period of
`time to cut into plural small pieces, bycultivating the skin pieces on the medium, and
`‘by counting the numberof pieces wherein no growth of fungus is seen or the number
`of animals or feet wherein no growth of fungus is seenin all skin pieces, as an
`indicator (Antimicrobial Agents and Chemotherapy, 36: 2523-2525, 1992, 39: 2353-
`2355, 1995). Hereinafter, the conventional method for evaluating the drug effectis
`referred to as "the conventional method".
`
`Although the drug having a potent activity against Trichophyton in vitro such as
`
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`lanoconazole or amorolfine has been marketed in these days, an improvementof cure
`rate in a clinical use is hardly seen, As a main reasonthereof, a relapse that since
`fungusin the skin is not completely killed after a treatment, the fungus grow againis
`pointed.
`
`In also animal experiments, when aneffect of lanoconazole on guipeaguniea pig
`models of tinea pedis was evaluated using the conventional method, though "fungus-
`negative” was observedin all feet out of 20 feet 2 days after the last treatment, a
`relapse was observedin 11 out of 20 feet 30 daysafter the last treatment, and no
`correlation was seen betweenthe effect 2 days after the last treatment and the effect
`30 days after the last treatment (36th Interscience Conference on Antimicrobial
`Agents and Chemotherapy, New Orleans, Levisiana-La., 1996, Abstr. F80).
`
`As a reason thereof, there were followings. Since lanoconazole have very potent
`antitrichophyton activity in vitro, lanoconazole persisted in the skin 2 daysafter the
`last treatment in the concentration wherein the sterilization effect was shown.
`Therefore, when the skin is excised and cultivated on the medium to detect fungus,
`the lanoconazole remaining in the skin is diffused in the medium, and therefore, no
`fungus was detected due to prevention of the growth regardless of the presenceof
`viable fungus in the excised skin. On the other hand, since the concentration of the
`drug remainedin the skin is reduced 30 daysafter the last treatment, fungusin the
`skin can grow again and can be detected by culture study.
`
`According to this hypothesis,it is ascertained —-that- the drug remain in the skin
`through the inhibition of the growth of fungus around the skin blocks completely, when
`the lanoconazole-treated skin blocks were located and cultivated on the medium
`which contains dermatophytes.
`
`Therefore, it becameto clear that the conventional method has the problem that the
`
`drug effectcan not se aeelaesa a thenellyIyaa
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`ar
`ie
`5
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`therapeutic effect need to be evaluated after removing the drug remaining in the skin,
`
`is the superficial dermatosis whichis
`Meanwhile, a kind of mycosis. dermatophytosis,
`caused by dermatophyte parasitizing the keratin such as skin (stratum corneum), the
`nail and the hair. In particular, tinea unguium formed in the nail is known as the
`intractable disease among dermatomycoses based on dermatophytoses, and is
`accompanied by symptom such as opacity, tylosis, destruction and deformation of nail
`plate. Now the oral preparation (such as griseofulvin or terbinafine) is used for the
`treatment of such tinea unguium. However, there are many cases wherethe patient
`stops taking the drug or that takes the drug irregularly, since they have to take the
`drug for a long period, for example at least.a half a year in order to completely cure
`tinea unguium. It is thought that this is a main cause of difficulty of curing tinea
`unguium completely. Furthermore, by taking the drug for a long period, griseofulvin
`has the problem of side effects on internal organ (gastrointestinal disorder,
`hepatotoxicity)
`and hepatotoxicity is reported as the side effect in terbinafine.
`Therefore, in order to improve the complianceof the patientit is desired to develop a
`topical preparation which cure tinea unguium for a short period and hasless the
`systemic side effect than the oral preparation.
`
`in case of the simple application on nail plate with the current antifungal
`However,
`agentfor topical use, the antifungal effect on fungus in the nail was not seen, because
`the drug could not sufficiently permeate the thick keratin in nail plate (Markus
`Niewerth and Hans C. Korting, Management of Onychomycoses, Drugs, 58: 283-296,
`1999).
`
`In addition, the therapeutic effect of a topical preparation of antifungal agent on the
`experiment model of trichophytosis can not be evaluated using the conventional
`method as mentioned above. This may be a reason why the drug effect on the guniea
`pig modelof tinea unguium has been hardly reported.
`
`DISCLOSURE OF INVENTION
`
`The present invention has been accomplished based onfindings thatit is desirable
`that an effect of antimicrobial agent such as particularly antifungal agent is evaluated
`after removing a drug remainingin the infected site after treatment of an animal ora
`biosample such as skin with the pathogenic microorganism. An object of the present
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`invention is to provide a novel method for evaluating the effect of the antimicrobial
`agent and the antimicrobial agent obtained according to the method for evaluating the
`drug effect. In detail, the present invention provides the method for detecting the
`viable pathogenic microorganism in the above-mentioned infected site of the animal
`or the biosample with the pathogenic microorganism after removing the antimicrobial
`agent which has been administered to the animal or the biosample, and the method
`for evaluating the effect of antimicrobial agent which can accurately evaluate the
`effect of the antimicrobial agent without the influence of the antimicrobial agent
`remaining in the infected site of the animal or the biosample with thea pathogenic
`microorganism—ar-. In addition,
`the present invention provides the antimicrobial
`agent obtained according to the above-mentioned the method for evaluating the drug
`effect-efthe—agent, and a-methed-ferthe detecting aa—method of the antimicrobial
`
`seewhereip-an-which sormers2s detecting.the Sxisting satteropial =is.
`
`to which the antimicrobial agentis
`{Mears,
`administered.
`
`In more detail, according to Selve-the
`
`
`
`
`
`erderte-seektoramethodteaccuratelyevaluatethethe present invention a
`detection of a pathogenic microorganism 2and an evaluation of an Steckor an
`
`pilcrogudaciate:absichs-ciereenassont be artied outqe infectingan1 animal ora
`
`biosample with the pathogenic microorganism, administering aathe antimicrobial
`agent comprising a compound having an antimicrobial effect or a- composition thereof
`before or after the infection, then removing the antimicrobial agent, and thereafter
`
`detecting the viable‘pathogenic microorganism in the infected site win the ralfogents
`
`
`‘
`5
`FEE
`delen
`microorganism-+Gta
`
`According to the present invention a detection of Glaira—t+—iriwhichthe—pathegenis
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`Page 4
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`antinlaeelal agar
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`
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`

` “:
`
`raprsesby infecting 2an1 animal
`ora biosample with a mathgerié microorganism, admteeaie aathe antimicrobial
`agent comprising -a compound -having an antimicrobial effect or a composition
`thereof before or after the infection, —then— excising —the —infected —site -with —the
`pathogenic microorganism, placing and cultivating it on aa-agara medium containing
`ee eaelal EHIeECOECanonY, ang elarCC Otee seeeeeeee:
`
`
`:
`observing a
`arewininhibition ofthe pathoaanie microorganism ebserved-around the infected site
`with the pathogenic microorganism+Claim—++4-
`
`Embed
`
`be =
`
`Additionally, an object of the present invention is to provide the evaluation method of
`a_ drug which enables the effect of an antifungal agent to accurately evaluate in a
`guinea pig model of tinea unquium. Another object of present invention is to provide a
`therapeutic agent for onychomycosis which exhibits the effect on tinea unguium by
`topical applicartion and which is capable of curing tinea unquium shorter period than
`that of the marketed oral preparation due to good permeability, qood retention
`capacity and conservation of high activity in nail plate as well as the potent antifungal
`activity thereof based on the present invention. Another object of the present
`invention is to provide the effective therapeutic agent for onychomycosis exhibiting no
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`side effect even if therapeutically effective amounts of it are administered sufficiently.
`
`More concretely, the present invention provides a therapeutic agent for
`onychomycosis containing a compound having a formula (1): ##STR1## Wherein
`R.sup.1 and R.sup.2 are the same or different and are hydrogen atom, C.sub.1-6
`alkyl group, a non-substituted aryl group, an aryl group substituted with 1 to 3
`substituents selected from a halogen atom, trifluoromethy! group, nitro group and
`C.sub.1-6 alkyl group, C.sub.2-8 alkenyl group, C.sub.2-6 alkinyl group, or C.sub.7-12
`aralkyl group, m is 2 or 3, nis 1 or 2, [0019] or a salt thereof as active ingredient.
`
`In addition, "presence"includes the mean of "remaining".
`
`BEST MODE FOR CARRYING OUT THE INVENTION
`
`As an animal employed in the present invention, there includes mammal such as
`mice, rat, guinea pig or rabbit. As a biosample, there includes a skin of back or planta,
`a nail or the like, which is taken from such animal.
`
`A methodfor infecting such animal or biosample with a pathogenic microorganism
`includes an inoculation percutaneoustypercutaneouslly, orally, intravenously,
`transbronchially, -transnasally- or intraperitoneally. Especially in case of the skin,
`there includes a method for inoculating it on the skin, a method for inoculating on the
`exposed demis, the closed patch method, intracutaneous -injection—orthe-ike-
`
`injection or the like. In case of the nail, there includes a method for inoculating on nail,
`a method in which a skin of the animals’ foot is infected by the above-mentioned
`infecting method to the skin, and thereafter the infection is moved into the nail by
`leaving it for several months.
`
`The term "skin" meansa tissue including the three layers being epidermis, demis and
`subcutaneoustissue, accompanied bypilus (hair), nail, glandulae sebaceae,
`glandulae sudoriferae and glandulae mammaria as appendages.
`
`The epidermmis is separated five layers being stratum corneum, stratum lucidum
`granulosum epidermidis, stratum spinosum, and stratum basale from surface in order.
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`the stratum lucidum and the stratum granulosum epidermidis is
`The stratum corneum,
`
`referred to as a stratum corneum in a broad sense. Herein. keratin sbustance means
`a part of the above-mentioned stratum corneum.
`
`The term "nail" includes nail plate, nail bed, nail matrix, further side nail wall, posterial
`nail wall, eponychium and hyponychium which make upa tissue around thereof.
`
`In the present invention, the term "pathogenic microorganism" means a
`microorganism which causes human and animal disease in one way or another. An
`example of the pathogenic microorganism (hereinafter referred to "microorganism"is
`bacteria including —aerobic —Gram-negative —bacillus —and —coccus —such— as
`Pseudomonas and Neisseriaceae species; facultative anaerobic Gram-negative
`bacillus such as Eschrichia, Salmonella and Enterobacter species; Gram-positive
`coccus such as Staphylococcus and Streptococcus species. The other examples of
`microorganism are fungi including Hyphomycetes such as Trichophyton, Microsporum
`and Epidermophyton species; Blastomycetes such as Candida and Malassezia;
`Ascomycetes such as Aspergillus species; Zygomycetes such as Mucor species; and
`variants thereof. Examples of such variants are resistant strain which naturally obtains
`drug resistance; auxotrophic mutation strain which comes to have nutritious
`dependency;artificial mutation strain whichis artificially mutated by treatment with
`mutagenic agent; and thelike.
`
`Mycosis means a disease which is caused by invading andproliferating in the tissue
`of human or animal. Usually, mycosis is broadly divided into superficial mycosis and
`deep mycosis. A seat of the diseaselie in the skin or visible mucosa in case of the
`former, in viscus, central nervous system, subcutaneous tissue, muscle, born or
`articulation in case of the latter. Chief example of superficial mycosis is
`dermatophytosis which is caused by infecting with dermatophyte such as
`Trichophyton, Microsporum and &pidermephyterEpidernophyton species, including
`three disease,tinea, tinea favosa and tinea imbricata. Tinea may be conventionally
`employed a synonymouswith dermatophytosis.
`
`In addition, dermatophyte belonging to Trichophyton species is referred usually to as
`trichophytosis.
`
`In the present invention, an antimicrobial agent means a compound having an
`antimicrobial effect or a composition containing the compound. The composition
`includes a preparation form being artificial composition and a natural composition
`such as a natural product.
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`A methodfor administration of the antimicrobial agent in the present invention
`depends onthe kind thereof and includes topical application, subcutaneous
`administration, oral administration, intravenous administration or the like.
`
`When the method for detecting the pathogenic microorganism, the method for
`evaluating the drug effect and the method for detecting the antimicrobial agent
`according to the present invention is carried out, either an infection with
`microorganism or an administration of the antimicrobial agent may be carried outfirst.
`Especially, in the method for evaluating the drug effect of the present invention
`(hereinafter referred to "the present -evaluation method"), a therapeutic effect of the
`antimicrobial agent can be evaluated in case where the antimicrobial agentis
`administered after the infection with microorganism, meanwhile, thea effect of the
`antimicrobial agent protecting from the infection and the retention capacity thereof can
`be evaluated in case wherethe infection with microorganism is carried out after the
`administration of the antimicrobial agent. In order to evaluateevaluating the retention
`capacity of the antimicrobial agent, the evaluation can be carried out with varying the
`period until infection with microorganism from the administration of the antimicrobial
`agent.
`
`In the present invention, it is preferable to use dialysis orultra filtration for removing
`the antimicrobial agent in view point of simplernessthe usefulness, but notlimited
`thereto as long as a microorganism to be a detecting target or a microorganism used
`in the present evaluation method and thelike is not affected byit.
`
`In dialysis, a marketed dialysis membrane made of cellulose is convenient. A
`membrane made of theother material can be used without -problem, —as -long -as -the
`microarganism -to be the detecting target or the microorganism used -in- the present
`evaluation method and the like can not be passed, and the antimicrobial agent can be
`passed throughit. -Since sizes of most fungi and bacteria are at least 0.2 yra.mu.m, it is
`preferable to use the membrane having less than 0.2 yr.mu.m of the pore size,
`particularly it is suitable to use dialysis membrane having fractional molecular weight of
`1,000 to 50,000._
`
`As eutsideout side solutions usedin dialysis, there include physiological saline,
`distilled water, phosphate buffered physiological saline, theother buffer and thelike.
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`In removing the antimicrobial agent according to the present invention, even though
`the infected site with the microorganism is the nail, organ or the like as well as the
`skin, the antimicrobial agent can be efficiently removed. Usually, since there is the
`case whereit takes longertime fer-dialysis to remove the antimicrobial agent from nail
`than, skin, the following treatment with digestive enzyme may be carried out before’
`removingit in order to enhance the removaleffect.
`
`Dialysis conditions depend on variety, dose concentration, dose term and the drug
`holidays (the term until evaluation from last day of treatment) of an antimicrobial
`agent. Therefore, it is preferable to previously investigate the dialysis conditions
`enabling the antimicrobial agent to be removed from the treated skin about individual
`cases using the following detecting method of the existing antimicrobial agent in the
`infected site with a microorganism in the present invention (hereinafter referred to "the
`present method for detecting an agent”) to adjust the conditions appropriately.
`
`Whether an antimicrobial agent has been removed can be easily determined using
`the following method.
`
`The present method for detecting an agent is carried out by placing and cultivating
`the infected site with a microorganism which is subjected to the removing method of
`the antimicrobial agent (e.g. an skin piece) or a suspension obtained accordingto the
`following extraction procedure of the microorganism from the above skin piece on an
`agar medium containing the microorganism, and observing a growth inhibition of the
`microorganism found aroundit. -When there is the remaining antimicrobial agent, the
`growth inhibition of the microorganism is observed.
`
`The present evaluation method can be carried out by locating and cultivating, on a
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`medium, the skin piece in which a removal of an antimicrobial agent has been
`determined using the above--mentioned present method for detecting the agent after
`carrying out the appropriate- removal of the antimicrobial agent and observing
`whether there is a growth of microorganism or not, or by smearing and cultivating a
`suspension obtained -according —to -the- extraction procedure of the microorganism
`from the skin piece on an agar medium and observing whetherthere is the growth of
`microorganism or not or counting colonies emerging on those medium.
`
`A treatment with trypsin can be carried out in order to extract a microorganism
`efficiently from a biosample such as a skin or a nail. Other digestive enzyme than
`trypsin such as pronase orkeratinase, or a keratin resolvent such as urea also can be
`used withoutlimitation to trypsin as long as they have an extraction effect.It is
`necessary to adjust concentrations of the digestive enzyme such astrypsin and
`keratin resolventin a treating solution, and reaction time to no affect range to a
`microorganism. The treatment with digestive enzyme such as trypsin may be carried
`out either before or after dialysis. When the treatment with trypsin is -carried -out
`before dialysis, it is necessary to remove the digestive enzyme suficiently so that the
`microorganism is not affected on dialysis.
`
`As a medium usedfor a cultivation of a microorganism in the present invention, any
`medium can be used aslong asit can be conventionally used for the cultivation and a
`separation of the microorganism. In the-case of fungi, +e-example -of -the -medium
`is Sebouraud medium, modified Sabouraud medium, Czapek agar medium, Potato
`dextrose agar medium orthe like. -On the other hand, inthe case of bacteria,
`example of the medium is Mueller Hinton medium, modified Mueller Hinton medium,
`Heart Infusion agar medium, Brain Heart Infusion agar medium, normal agar medium
`or the like.
`
`A reacting temperature is 10 to 40°.degree. C;., preferably-.20 to 40°.degree. C. A
`microorganism may be cultivated with standing during a sufficient time when the
`microorganism can be grewrAgrowth, for example, 1 to 20 days ferin case of fungi, 1 to
`5 days ferin bacteria.
`
`The present evaluation method car—be utiizedutilizable as ana evaluation method of
`a drug effect in exo vivo which comprisesinfecting a skin, a nail excised from an
`animal body with a microorganism, thereafter administering an antimicrobial agent as
`a test sempeuAdcompound, then removing the antimicrobial agent and detecting and
`determining quantity of the microorganism in the sample.
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`The present evaluation method also can be applied to an evaluation of an
`antimicrobial agent such as a therapeutic agent for deep mysesisinycosis or an
`antibacterial agent as well as an evaluation of an effect of a therapeutic agent for
`superficial mycosis.-_Thatis to say, it is possible to evaluate an effect of a therapeutic
`agent for deep mycosis or an antibacterial agent by meansof administering an
`antimicrobial agent to an animal infected with a microorganism such as a fungusor a
`bacterium——_byinoculating -percutaneously,-orally, -intravenously, transbronchially,
`
`_transnasally, intraperitoneally, then obtaining biosample such as skin, kidney,
`lung or brain, and detecting the viable microorganism -in the biosample -in which
`removed the remaining: antimicrobial agent -has been removed.
`
`In addition, the present evaluation method enables a quantitative comparison of
`antimicrobial effects by means of determining the numberof viable microorganisms in
`the treated biosample.
`
`That is to say, a significant deference testefsignificance is carried out about the
`numberof microorganisms in the infected site with the microorganism for the treated
`group with drug and for the: reference infected group using a statistical method such
`as Kruskal-Wallis Test, and thereby a quantitative comparison between the groups
`can be done-
`
`
`
` byJ. using a multiple
`
`
`
`test such as Tukey method.
`
`The present invention is useful as either a method for evaluating a drug effect ora
`method for detecting the antimicotics in keratin substance or nail, after administering the
`antifungus to the patient infected with fungus. For example, according to the present
`invention, an effect of an antifungal agent can be evaluated by administeringit to the
`patient-whose skin or nail is infected with fungus, obtaining the keratin substance or
`nail, then removing the above-mentioned antifungal agent, and thereafter detecting the
`viable fungus in the keratin substance or nail. Additionally, according to the present
`invention, a detection of an antifungal agent can be carried out by administeringit to the
`patient whose skin or nail is infected with fungus, then obtaining the keratin substance
`or nail, cultivating it on agar medium containing fungus, and thereafter detecting the
`existing antifungal agent in the keratin substance ornail through a growth inhibition of
`fungus observed around the keratin substance or nail. Such evaluation of an antifungal
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`agent administered to a patient with fungus and detection of the antifungal agent from
`the keratin substance or nail can be carried out in the same manneras in the above-
`mentioned evaluation method of a drug effect and detecting method of the antimicrobial
`agent administered to an animal or a biosample.
`
`Furthermore, the present invention provides various useful antimicrobial agents
`according to the present evaluation method. As the antimicrobial agent obtained by the
`present evaluation method. there is an antimicrobial agent comprising a compound
`having an eradication effect for microorganism in vivo or a composition for therapy of
`the superficial mycosis, deep mycosis or bacterial infection containing the compound;
`an antimicrobial agent having the true effect selected by means of showing a
`statistically significant effect; furthermore, an antimicrobial agent having an excellent
`eradication effect for microorganism in vivo, which is selected by means-of clarfyingthe
`
`
`
`O
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`
`A
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`
`KPR-103mentionedbelow.of appearing the pure antimicrobial activity thereof; or an
`
`antimicrobial agent of the complete cure type without relapse. A concrete example is a
`therapeutic agent for onychomycosis comprising a compound having the group
`represented by the above-mentioned formula (I). Among them, more preferable
`concrete example is a therapeutic agent for onychomycosis comprising the compound
`represented by the formula (Il): ##STR2## wherein, Ar is a non-substituted pheny!
`group or a phenyl group substituted with 1 to 3 substituents selected from a halogen
`atom andtrifluoromethyl group, R.sup.1 and R.sup.2 are the sameor different and are
`hydrogen atom, C.sub.1-6 alkyl group, a nori-substituted aryl group, an aryl group
`substituted with 1 to 25 3 substituents selected from a halogen atom, trifluoromethy!
`group, nitro group and C.sub.1-6 all group, C.sub.2-8 alkenyl group, C.sub.2-6 alkinyl
`group, or C.sub.7-12 aralkyl group, mis 2 or 3, nis 1 or 2, X is nitrogen atom or CH,
`and *1 and *2 mean an asymmetric carbon atom.
`
`is a pheny!
`group
`the substituted pheny!
`In the above-mentioned formula(I) or(Il),
`group having 1 to 3 substituents selected from a halogen atom andtrifluoromethyl, and
`includes, for instance, 2,4-difluorophenyl, 2,4-dichlorophenyl, 4-fluorophenyl, 4-
`chlorophenyl, 2-chlorophenyl, 4-trifluoromethylphenyl, 2-chloro-4-fluorophenyl, 4-
`bromopheny| orthe like, C.sub.1-6 alkyl group includes, for example, a straight chain
`branched chain or cyclic alkyl group having 1 to 6 carbon atoms such as methyl, ethyl,
`n-propyl,
`isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopenty!
`tert-pentyl and cyclohexyl. The non-substituted aryl group includes, for example, phenyl.
`naphthyl, biphenyl or the like. The substituted aryl group includes, for example, 2.4-
`difluorophenyl, 2.4-dichlorophenyl, 4-fluorophenyl, 4-chlorophenyl, 2-chlorophenyl, 4-
`trifluoromethylphenyl, 2-chloro-4-fluorophenyl, 4-bromophenyl, 4-tert-butylphenyl, 4-
`nitrophenyl or the like. C.sub.2-8 alkenyl group includes, for example, vinyl, 1-propeny!
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`styryl or the like. C.sub.2-6 alkynyl group includes, for example, ethynyl or thelike.
`C.sub.7-12 aralkyl group includes, for example, benzyl, naphthylmethyl, 4-nitrobenzy! or
`the like.
`
`In addition, the most preferable compound among the above-mentioned antimicrobial
`agent includes the compound which showsthe therapeutic efficiency like the following
`KP-103.
`
`The above-mentioned KP-103 means an antifungal indicated by (2R,3R)-2-(2,4-
`
`
`difluorophenyl)-3-(4-methylenepiperidine-1-yl)-1-(1 H-1,2,4-triazole-1-yl)butane-2-ol.
`The compound can be prepared by allowing (2R,3S)-2-(2,4-difluoropheny!)-3-methyl-2-
`[(1H-1,2,4-triazole-1-— yl)methylloxirane to react with 4-methylenepiperidine based on
`Example 1 in WO94/26734.
`
`An effectiveness of the KP-103 used as an antifungal in the present invention for
`onychomycosis has not been confirmed, but its antifungal activity has been already
`known (W0O94/26734).
`
`The antimicrobial agent obtained in such mannercan belo used as a drug
`composition, the drug composition in orderto sterilize a microorganism. In other
`words, it comes to be a drug composition which cures disease such as mycosis
`completely, and prevents a relapse.
`
`EXAMPLE
`
`Onychomycosis means a kind of the above-mentioned superficial mycosis, in the
`other word a disease which is caused by invading andproliferating in the nail of
`human or an animal. Trichophyton rubrum and Trichophyton mentagrophytes mainly
`cause onychomycosis in human. In rare case, Microsporum, Epidernophyton,
`Candida, Aspergillus or Fusarium causesit.
`
`As a disease which is susceptible to treat with a therapeutic agents for
`onychomycosis of the present invention, there is included tinea unguium caused by
`Trichophyton species, Onychocandidasis caused by Candida species or
`onychomycosis (sensu stricto) caused by the other fungus.
`
`Whena therapeutic agent for onychomycosis being a kind of antimicrobial agent in
`the present invention is given as topical preparation,
`thereis liquid preparation
`cream, ointoment or manicure preparation as dosage form. In this case,
`it can be
`prepared using oil vehicle, emulsion vehicle or the like. The preferable amount of
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`

`active ingredientis in 0.1 to 10% by weight. A dose amount may be appropriately
`aligned depending on the width of affected area and condition of disease.
`
`tablet, granule, capsule or syrup.
`In case of an oral preparation,it is used as powder,
`In addition,
`it is used in form of injection such as subcutaneousinjection
`intramuscular injection or intravenousinjection.
`
`In the present invention, although the dosage amountof a therapeutic agent for
`onychomycosis depends on age, weight and individual conditions of a patient, it is
`about 10 mg to about 10 g per day, preferably about 50 mg to about 5 g as amountof
`the active ingredient. The agent was given in the above-mentioned daily dose at once
`or several
`divided
`portions.
`
`The present invention is further explained in details based on the Examples
`hereinafter, but is not limited thereto.
`
`PRETREATMENT OF COMPARATIVE EXAMPLE 1 AND EXAMPLES 1 TO 3
`
`[1] Preparation of Fungal Solution and Production of a Guinea Pig Model of Interdigital
`Type of Tinea Pedis.
`
`Millipore Filter (made by Millipore Corporation, HA, diameter 47 mm, 0.45 ,A.mu.m)
`was-placed on Brain-Heart-infusion agar medium (available from Nissui Pharmaceutical
`Co., LTD.), and 46®10.sup.6 cells of microcondium of Trichophyton mentagrophytes KD-
`04 strain were applied thereon. The cultivation was carried out at 30°.degree. C. under
`17-% of SO,.CO.sub.2 for 7days/days. After the cultivation, ana—apprepriatejust amount of
`physiological saline containing 0.05-% of Tween 80 was dropped onthe filter and
`arthroconidia were collected using a platinum loop. After a hyphal mass was removed
`by a filtration with a sterile gauze, the numberof arthroconidia —in- the —arthreseridia
`
`suspension—filtrate was calculated —by hemocytometerto adjust in concentration of 1x
`40*times.10.sup.8 arthroconidia+/ml to obtain a fungal inocula._
`
`A guinea pig modelof interdigital type of tinea pedis was prepared according to the
`method of Arika et al (Antimicrobial Agents and Chemotherapy, 36: 2523-2525, 1992).
`Concretely, in -two- hind foots of male Hartley strain guinea pigs of 7 weeks age, the
`interdigital skin was lightly abraded -with sandpaper. A paper disc (AAdisc available
`
`from Whatmen International Ltd cut in 8<.times.4 mm) moisten with the above-
`mentioned solution of the inoculated organism was applied onto the region between the
`interdigital toes of the hind feet and fixed using Self-adhering-Foam Pad (Restone
`1560M; available from 3M) and adhesive stretch bandage (ELASTPORE; available from
`
`ee
`
`Page 14
`
`

`

`Nichiban Co., Ltd). The paper disc and the bandage were -removed seven daysafter of
`the infection.
`
`tepicaltreatmentTopical Treatment
`
`[2]Preparation of drug-selutioaDrug-Solution and
`
`
`for guinea-pigmodelcHnterdigitaltypethe Guinea Pig Modelof tinea-pedis
`
`Interdigital Type of Tinea Pedis
`
`A marketed 1-% lanoconazole solution (commercial name: Astat (trade name)
`
`oesaea oatralines ki103aietaeee
`
`
`deseribed_in-Example_tinWO-94/26734}was solved ina concentration af 1% in
`polyethylene glycelgrycole #400-: ethanol (75—:25 v/v) mixture were used astest
`substance. Each solution in-an amountof 0.1 ml was applied to the plantar skin once
`a day from 10 days after the infection for 10 days.
`
`COMPARATIVE EXAMPLE1
`
`Conventional methedMethod for evaluating-drug-effect
`
`Evaluating Drug Effect
`
`The conventional method wasdescribed asfollows.- For the infected control group
`without an application of the drug, the KP-103-treated group and the lanoconazole-
`treated group, 10 guinea pigs (hereinafter referred to "animal") were employed,
`respectively. Animals of each group were sacrificed two days after and 30 days after the
`last treatment. Their two hind feet were excised and wiped with the cotton swabsweb
`containing alcohol sufficiently. A skin of whole sole was excised and cut into 15 skin
`pieces in total inclu