TECHNICAL FIELD
`
`The present invention relates to a method for detectin,q patho,qenic microor,qanism,
`method for evaluating an effect of an antimicrobial agent on pathogenic
`microorganism ~ .... +~,., ,~or ~+ r~,~o+~,~ +,-, and a method for eva!uatJng o~’ ~’~’"+ ’-’~
`a,q agent whereindetectinq an antimicrobial agent. The present invention also relates
`to an antimicrobial aqent ex4st4,qg-~and a therapeutic aqent for onychomycosis,
`which are obtained accordinq to the above-mentioned method for evaluatinq the
`
`inf~nf~rl elf~ ~^,ifh fh~ n~fhr~n~nln r~inrr~r~rn~nler~ ie rl~f~nf~rl ~nrl rl~f~rmln~rl ~^,ifh
`
`dru,q effect¯
`
`BACKGROUND ART
`
`A method for evaluating a drug effect with an animal model is needed in order to
`explore a novel antimicrobial agent (also hereinafter "referred to "drug")¯ Further, a
`method ~enablinq a drug effect to be evaluated with accuracy is needed
`because of gr-eat:,qrate importance in view of predicting a clinical therapeutic efficiency
`thereof¯
`
`Historically, an experimental dermatophytosis model that back, planta and interdigital
`I of a ~,quniea pig have been infected 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 Js carried out by applying the
`antifungal agent to the infected animal, by excising the skin after the certain period of
`time to cut into plural small pieces, by cultivating the skin pieces on the medium, and
`by counting the number of pieces wherein no growth of fungus is seen or the number
`of animals or feet wherein no growth of fungus is seen in 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 effect is
`referred to as "the conventional method"¯
`
`Although the drug having a potent activity against Trichophyton in vitro such as
`
`ACRUX DDS PTY LTD. et al.
`EXHIBIT 1009
`IPR Petition for
`U.S. Patent No. 7,214,506
`
`1 of 25
`
`

`

`lanoconazole or amorolfine has been marketed in these days, an improvement of cure
`rate in a clinical use is hardly seen. As a main reason thereof, a relapse that since
`fungus in the skin is not completely killed after a treatment, the fungus grow again is
`pointed.
`
`In also animal experiments, when an effect of lanoconazole on ~.quniea pig
`models of tinea pedis was evaluated using the conventional method, though "fungus-
`negative" was observed in all feet out of 20 feet 2 days after the last treatment, a
`relapse was observed in 11 out of 20 feet 30 days after the last treatment, and no
`correlation was seen between the 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, Leuisi~n~,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 days after 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 presence of
`viable fungus in the excised skin. On the other hand, since the concentration of the
`drug remained in the skin is reduced 30 days after the last treatment, fungus in 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 became t_oo clear that the conventional method has the problem that the
`drug effect can not be accurately evaluated, because the drug effect is e,-a!,-ated so
`
`i iviviiviii~ iii ~iiv v~v vi vv~i~iii:~ ~ ~i~:~ viivv~ vii ~ii~iiiiiviv~i~i ~:~vii~ v~r~vvi~ii~,
`
`2 of 25
`
`

`

`onlr~ol r~r ~ hlr~eor~nl~ fh~n r~r~r~,in~ fh~ onflr~ir-rr~hiol o~nf on~1 fh~r~off~r
`
`therapeutic effect need to be evaluated after removinq the druq remaininq in the skin.
`
`Meanwhile, a kind of mycosis, dermatophytosis, is the superficial dermatosis which is
`caused by dermatophyte parasitizin,q the keratin such as skin (stratum corneum), the
`nail and the hair. In particular, tinea un,quium formed in the nail is known as the
`intractable disease amonq 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 ,qriseofulvin or terbinafine) is used for the
`treatment of such tinea unquium. However, there are many cases where the patient
`stops takinq the druq or that takes the druq irre,qularly, since they have to take the
`dru,q for a Ionq period, for example at least a half a year in order to completely cure
`tinea un,quium. It is thouqht that this is a main cause of difficulty of curin,q tinea
`un,quium completely. Furthermore, by takin,q the dru,q for a Ion,q period, ,qriseofulvin
`has the problem of side effects on internal orqan (,qastrointestinal disorder,
`hepatotoxicity) and hepatotoxicity is reported as the side effect in terbinafine.
`Therefore, in order to improve the compliance of the patient it is desired to develop a
`topical preparation which cure tinea unquium for a short period and has less the
`systemic side effect than the oral preparation.
`
`However, in case of the simple application on nail plate with the current antifunqal
`a,qent for topical use, the antifun,qal effect on funqus in the nail was not seen, because
`the druq could not sufficiently permeate the thick keratin in nail plate (Markus
`Niewerth and Hans C. Kortinq, Mana,qement of Onychomycoses, Dru,qs, 58: 283-296,
`1999).
`
`In addition, the therapeutic effect of a topical preparation of antifunqal a,qent on the
`experiment model of trichophytosis can not be evaluated usinq the conventional
`method as mentioned above. This may be a reason why the druq effect on the ,quniea
`pi,q model of tinea unquium has been hardly reported.
`
`DISCLOSURE OF INVENTION
`
`The present invention has been accomplished based on findinqs that it is desirable
`that an effect of antimicrobial aqent such as particularly antifunqal a,qent is evaluated
`after removinq a dru,q remainin,q in the infected site after treatment of an animal or a
`biosample such as skin with the patho,qenic microorqanism. An obiect of the present
`
`3 of 25
`
`

`

`invention is to provide a novel method for evaluatinq the effect of the antimicrobial
`a,qent and the antimicrobial aqent obtained accordinq to the method for evaluatinq the
`dru,q effect. In detail, the present invention provides the method for detectinq the
`viable patho,qenic microorqanism in the above-mentioned infected site of the animal
`or the biosample with the patho,qenic microorqanism after removinq the antimicrobial
`a,qent which has been administered to the animal or the biosample, and the method
`for evaluatinq the effect of antimicrobial aqent 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 t-hea pathogenic
`microorganism; a,’q,. In addition, the present invention provides the antimicrobial
`agent obtained according to the above-mentioned the method for evaluating the dru,q
`effect-~Pthe-age,~, and a--me&he4~the detecting a,’q, method of the antimicrobial
`agent wher~p~p,-which comprises detectinq the existinq antimicrobial agent is
`,~ ~ ÷k~ ~÷~k~, agent existing in the
`infected site of the animal or the biosample with athe pathogenic microorganism-is
`~;kteet-e~.
`
`|11 ~l~lvii7 ~,lv~viivv iiivl~v~ ~llv iiiv~lllll~ vi iviil~llllll~ ,
`
`{4Vleap~, to which the antimicrobial a,qent is
`administered.
`
`In more detail, accordin,q to Selve4he
`
`order to seek for a method to accurately e,-a!,-ate the the present invention a
`detection of a patho,qenic microorqanism and an evaluation of an effect of an
`antimicrobial agent witho,-t the inf!,-ence of the antimicrobia! agent remaining in the
`
`r~e,,if ÷h~ in~,~nfnre h~,~ Tn,,nr4 ~ m~fhnr4 ÷n ~nn,,r~f~h, ~,~I,,~÷~ ÷h~ ~ff~n÷ n’F
`
`~nflr~inrnhi~l ~n~nf r~r~ininn in fh~ inToNer4 eif~ ,-’,,.F fh~ ~nir~l nr fh~ hlne~r~nl~
`
`e..r.k
`
`.-~e elzin fkn .e ,~,~mnlz~fin,~ ÷hz~ nrz~ez~n÷ ;n~,z~nfi,~n
`
`microorganism which comprises can be carried out by infecting an animal or a
`biosample with the pathogenic microorganism, administering a4qthe 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 with the pathogenic
`microorganism/r-,~;~ ~ ~ ÷k~ ~÷k~ *~. detecting " ~÷k .... ;,-. ,-,.,; ........ ;o,-,.,
`
`Accordin,q to the present invention a detection of C’’~;’’ ~ ; .... ki~-k fk~ n-~fk .... h",
`
`4 of 25
`
`

`

`Additionally, an obiect of the present invention is to provide the evaluation method of
`a dru,q which enables the effect of an antifunqal a,qent to accurately evaluate in a
`.quinea pi.q model of tinea unquium. Another obiect of present invention is to provide a
`therapeutic aqent for onychomycosis which exhibits the effect on tinea un.quium by
`topical applicartion and which is capable of curinq tinea unquium shorter period than
`that of the marketed oral preparation due to .qood permeability, .qood retention
`capacity and conservation of hiqh activity in nail plate as well as the potent antifunqal
`activity thereof based on the present invention. Another obiect of the present
`invention is to provide the effective therapeutic a,qent for onychomycosis exhibitinq no
`
`5 of 25
`
`

`

`side effect even if therapeutically effective amounts of it are administered sufficiently.
`
`More concretely, the present invention provides a therapeutic aqent for
`onychomycosis containinq a compound havinq a formula (1): ##STRI## Wherein
`R.sup.1 and R.sup.2 are the same or different and are hydro,qen atom, C.sub.l-6
`alkyl ,qroup, a non-substituted aryl ,qroup, an aryl ,qroup substituted with 1 to 3
`substituents selected from a haloqen atom, trifluoromethyl ,qroup, nitro ,qroup and
`C.sub.l-6 alkyl ,qroup, C.sub.2-8 alkenyl ,qroup, C.sub.2-6 alkinyl ,qroup, or C.sub.7-12
`aralkyl ,qroup, m is 2 or 3, n is 1 or 2, [0019] or a salt thereof as active inqredient.
`
`In addition, "presence" includes the mean of "remaininq".
`
`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 method for infecting such animal or biosample with a pathogenic microorganism
`includes an inoculation ~. ............. jpercutaneouslly, 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 inj~cticn cr th~ !ik.~.
`
`injection or the like. In case of the nail, there includes a method for inoculatin,q on nail,
`a method in which a skin of the animals’ foot is infected by the above-mentioned
`infectinq method to the skin, and thereafter the infection is moved into the nail by
`leavinq it for several months.
`
`The term "skin" means a tissue including the three layers being epidermis, demis and
`subcutaneous tissue, accompanied by pilus (hair), nail, glandulae sebaceae,
`glandulae sudoriferae and glandulae mammaria as appendages.
`
`The epidermmis is separated five layers beinq stratum corneum, stratum lucidum,
`,qranulosum epidermidis, stratum spinosum, and stratum basale from surface in order.
`
`6 of 25
`
`

`

`The stratum corneum, the stratum lucidum and the stratum .qranulosum epidermidis is
`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 up a 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 which is artificially mutated by treatment with
`mutagenic agent; and the like.
`
`Mycosis means a disease which is caused by invading and proliferating in the tissue
`of human or animal. Usually, mycosis is broadly divided into superficial mycosis and
`deep mycosis. A seat of the disease lie 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 _~, ........ ~,,,, .... Epldernophyton species, including
`three disease, tinea, tinea favosa and tinea imbricata. Tinea may be conventionally
`employed a synonymous with dermatophytosis.
`
`In addition, dermatophyte belon.qin.q 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 method for administration of the antimicrobial agent in the present invention
`depends on the 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 out first.
`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 agent is
`administered after the infection with microorganism, meanwhile, t-hea effect of the
`antimicrobial agent protecting from the infection and the retention capacity thereof can
`be evaluated in case where the infection with microorganism is carried out after the
`administration of the antimicrobial agent. In order to ~evaluatin,q 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 or ultra filtration for removing
`the antimicrobial agent in view point of ~the usefulness, but not limited
`thereto as long as a microorganism to be a detectin,q tar,qet or a microorqanism used
`in the present evaluation method and the like is not affected by it.
`
`In dialysis, a marketed dialysis membrane made of cellulose is convenient. A
`membrane made of the other material can be used without -problem, -as -long -as -the
`microorganism -to be the detectinq tar,qet or the microorqanism used -in- the present
`evaluation method and the like can not be passed, and the antimicrobial agent can be
`passed through it. -Since sizes of most fungi and bacteria are at least 0.2 #m.mu.m, it is
`preferable to use the membrane having less than 0.2 pm.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 eut-si4~out side solutions used in dialysis, there include physiological saline,
`distilled water, phosphate buffered physiological saline, the other buffer and the like.
`
`8 of 25
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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 where it takes longer time fcr dialysis to remove the antimicrobial agent from nail
`than~ skin, the following treatment with digestive enzyme may be carried out before
`removing it in order to enhance the removal effect.
`
`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 according to 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 around it. -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 observinq whether there is the ,qrowth of
`microorqanism 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 or keratinase, or a keratin resolvent such as urea also can be
`used without limitation to trypsin as long as they have an extraction effect. It is
`necessary to adjust concentrations of the digestive enzyme such as trypsin and
`keratin resolvent in 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 used for a cultivation of a microorganism in the present invention, any
`medium can be used as long as it can be conventionally used for the cultivation and a
`separation of the microorganism. In t-he-case of fungi, t-he-example-of-the-medium
`is Sabouraud medium, modified Sabouraud medium, Czapek agar medium, Potato
`dextrose agar medium or the like. -On the other hand, in-t-he 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°-.de,qree. CT= preferably--20 to 40-t.de,qree. C. A
`microorganism may be cultivated with standing during a sufficient time when the
`
`microorganism can be ....... ~, ~,,, ,,,l,~rowth, for example, 1 to 20 days fcnn case of fungi, 1 to
`5 days ferJn bacteria.
`
`The present evaluation method can be ut&qzedutilizable as a4qa evaluation method of
`a drug effect in exo vivo which comprises infecting a skin, a nail excised from an
`animal body with a microorganism, thereafter administering an antimicrobial agent as
`a test ccmpcundcompound, then removing the antimicrobial agent and detecting and
`determining quantity of the microorganism in the sample.
`
`10 of 25
`
`

`

`The present evaluation method also can be applied to an evaluation of an
`antimicrobial agent such as a therapeutic agent for deep mycosisinycosis or an
`antibacterial agent as well as an evaluation of an effect of a therapeutic agent for
`superficial mycosis._That is to say, it is possible to evaluate an effect of a therapeutic
`agent for deep mycosis or an antibacterial agent by means of administering an
`antimicrobial agent to an animal infected with a microorganism such as a fungus or a
`bacterium _by inoculating-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 number of viable microorganisms in
`the treated biosample.
`
`That is to say, a si,qnificant deference test of significance is carried out about the
`number of 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:
`
`^- *~’~, --,-*~,’,<’--"~’~--~ ~gent ..~.,..~,.~,,4 ~.,, ,~,~, present .... ~’ ’"*~"" methcd, by usin,q a multiple
`test such as Tukey method.
`
`The present invention is useful as either a method for evaluatin,q a dru,q effect or a
`method for detectinq the antimicotics in keratin substance or nail, after administerin,q the
`antifunqus to the patient infected with funqus. For example, accordin,q to the present
`invention, an effect of an antifun,qal a,qent can be evaluated by administerinq it to the
`patient-whose skin or nail is infected with funqus, obtainin,q the keratin substance or
`nail, then removin,q the above-mentioned antifunqal a,qent, and thereafter detectin,q the
`viable funqus in the keratin substance or nail. Additionally, accordin,q to the present
`invention, a detection of an antifun,qal a,qent can be carried out by administerinq it to the
`patient whose skin or nail is infected with funqus, then obtainin,q the keratin substance
`or nail, cultivatin,q it on aqar medium containinq fun,qus, and thereafter detectin,q the
`existinq antifunqal a,qent in the keratin substance or nail throuqh a ,qrowth inhibition of
`fun,qus observed around the keratin substance or nail. Such evaluation of an antifunqal
`
`11 of 25
`
`

`

`a,qent administered to a patient with funqus and detection of the antifunqal a,qent from
`the keratin substance or nail can be carried out in the same manner as in the above-
`mentioned evaluation method of a druq effect and detectinq method of the antimicrobial
`a,qent administered to an animal or a biosample.
`
`Furthermore, the present invention provides various useful antimicrobial aqents
`accordinq to the present evaluation method. As the antimicrobial aqent 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 ,,~v, ,-’~-;~,,;,~,-v,~,,,,,,,~ ÷h~,.,,~
`
`KP !03mentiened be!ew.of appearin,q the pure antimicrobial activity thereof; or an
`antimicrobial aqent of the complete cure type without relapse. A concrete example is a
`therapeutic a,qent for onychomycosis comprisin,q a compound havin,q the ,qroup
`represented by the above-mentioned formula (1). Amon,q them, more preferable
`concrete example is a therapeutic a,qent for onychomycosis comprisin,q the compound
`represented by the formula (II): ##STR2## wherein, Ar is a non-substituted phenyl
`,qroup or a phenyl ,qroup substituted with I to 3 substituents selected from a haloqen
`atom and trifluoromethyl ,qroup, Rsup.1 and Rsup.2 are the same or different and are
`hydro,qen atom, O.sub.1-6 alkyl ,qroup, a non-substituted aryl ,qroup, an aryl ,qroup
`substituted with I to 25 3 substituents selected from a haloqen atom, trifluoromethyl
`,qroup, nitro ,qroup and O.sub.1-6 all ,qroup, O.sub.2-8 alkenyl ,qroup, O.sub.2-6 alkinyl
`,qroup, or O.sub.7-12 aralkyl ,qroup, m is 2 or 3, n is I or2, X is nitro,qen atom or OH,
`and "I and *2 mean an asymmetric carbon atom.
`
`In the above-mentioned formula (I) or (11), the substituted phenyl ,qroup is a phenyl
`,qroup havin,q 1 to 3 substituents selected from a haloqen atom and trifluoromethyl, and
`includes, for instance, 2,4-difluorophenyl, 2,4-dichlorophenyl, 4-fluorophenyl, 4-
`chlorophenyl, 2-chlorophenyl, 4-trifluoromethylphenyl, 2-chloro-4-fluorophenyl, 4-
`bromophenyl or the like. C.sub.l-6 alkyl ,qroup includes, for example, a strai,qht chain,
`branched chain or cyclic alkyl ,qroup havin,q 1 to 6 carbon atoms such as methyl, ethyl,
`n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl,
`tert-pentyl and cyclohexyl. The non-substituted aryl ,qroup includes, for example, phenyl,
`naphthyl, biphenyl or the like. The substituted aryl ,qroup 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 ,qroup includes, for example, vinyl, 1-propenyl,
`
`12 of 25
`
`

`

`styryl or the like. C.sub.2-6 alkynyl .qroup includes, for example, ethynyl or the like.
`C.sub.7-12 aralkyl .qroup includes, for example, benzyl, naphthylmethyl, 4-nitrobenzyl or
`the like.
`
`In addition, the most preferable compound amonq the above-mentioned antimicrobial
`a.qent includes the compound which shows the therapeutic efficiency like the followinq
`KP-103.
`
`The above-mentioned KP-103 means an antifunqal indicated by (2R,3R)-2-(2,4-
`difluorophenyl)-3-(4-methylenepiperidine-l-yl)-l-(1 H-1,2,4-triazole-l-yl)butane-2-ol.
`The compound can be prepared by allowin.q (2R,3S)-2-(2,4-difluorophenyl)-3-methyl-2-
`[(1H-1,2,4-triazole-1-- yl)methyl]oxirane to react with 4-methylenepiperidine based on
`Example 1 in WO94/26734.
`
`An effectiveness of the KP-103 used as an antifunqal in the present invention for
`onychomycosis has not been confirmed, but its antifun.qal activity has been already
`known (WO94/26734).
`
`The antimicrobial agent obtained in such manner can be I._9.o used as a drug
`composition, the drug composition in order to sterilize a microorganism. In other
`words, it comes to be a drug composition which cures disease such as mycosis
`completely, and prevents a relapse.
`
`Onychomycosis means a kind of the above-mentioned superficial mycosis, in the
`other word a disease which is caused by invadinq and proliferatinq in the nail of
`human or an animal. Trichophyton rubrum and Trichophyton mentaqrophytes mainly
`cause onychomycosis in human. In rare case, Microsporum, Epidernophyton,
`Candida, Asper.qillus or Fusarium causes it.
`
`As a disease which is susceptible to treat with a therapeutic a.qents for
`onychomycosis of the present invention, there is included tinea un.quium caused by
`Trichophyton species, Onychocandidasis caused by Candida species or
`onychomycosis (sensu stricto) caused by the other fun.qus.
`
`When a therapeutic aqent for onychomycosis beinq a kind of antimicrobial aqent in
`the present invention is .qiven as topical preparation, there is liquid preparation,
`cream, ointoment or manicure preparation as dosaqe form. In this case, it can be
`prepared usin.q oil vehicle, emulsion vehicle or the like. The preferable amount of
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`active inqredient is in 0.1 to 10% by wei,qht. A dose amount may be appropriately
`ali,qned dependin,q on the width of affected area and condition of disease.
`
`In case of an oral preparation, it is used as powder, tablet, ,qranule, capsule or syrup.
`In addition, it is used in form of iniection such as subcutaneous iniection,
`intramuscular iniection or intravenous iniection.
`
`In the present invention, althou,qh the dosa,qe amount of a therapeutic a,qent for
`onychomycosis depends on aqe, wei,qht and individual conditions of a patient, it is
`about 10 mq to about 10 ,q per day, preferably about 50 mq to about 5 ,q as amount of
`the active inqredient. The aqent was ,qiven 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 Funqal Solution and Production of a Guinea Piq Model of Interdiqital
`Type of Tinea Pedis.
`
`Millipore Filter (made by Millipore Corporation, HA, diameter 47 mm, 0.45 # .m.mu.m)
`was =placed on Brain-Heart-infusion agar medium (available from Nissui Pharmaceutical
`Co., LTD.), and 4-0610.sup.6 cells of microcondium of Trichophyton mentagrophytes KD-
`04 strain were applied thereon. The cultivation was carried out at 30°.de,qree. C. under
`17-% of Ge~CO.sub.2 for 7 d~ys7days. After the cultivation, a,,~at~re~r4a~iust amount of
`physiological saline containing 0.05-% of Tween 80 was dropped on the filter and
`arthroconidia were collected using a platinum loop. After a hyphal mass was removed
`by a filtration with a sterile gauze, the number of arthroconidia -in- the =rthrcccn!d!=
`su~filtrate was calculated--by hemocytometer to adjust in concentration of 1-~
`-1-O~.times.10.sup.8 arthroconidia-/-/ml to obtain a fungal inocula.
`
`A guinea pig model of 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 x .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
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`Nichiban Co., Ltd). The paper disc and the bandage were -removed seven days after of
`the infection.
`
`[2] Preparation of .4~. ...... ~ ~.~..~.h .*~,-.~’Drun-Solution. M and tepica! treatmentTopical Treatment
`
`~-.~" ...~.""-"4~’1 ,-.~. interdigita! .j ~.~the Guinea Piq Model of
`