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`Yoshiyuki Tatsumi
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`Yoshiyuki Tatsumi
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`Practitioner’s Docket No. 700938-052220-DIV
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`PATENT
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`IN TFIE UNITED STATES PATENT AND TRADEMARK OFFICE
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`In re application of."
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`Tatsumi et al.
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`Application No.:
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`TBA (divisional of 10/031,929)
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`Group No.:
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`To be Assigned (1651)
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`Filed:
`
`For:
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`Herewith
`
`Examiner:
`
`To be assigned (Srivastava,
`
`METHOD FOR DETECTING PATHOGENIC MICROORGANISM AND
`ANTIMICROBIAL AGENT, METHOD FOR EVALUATING EFFECT OF
`ANTIMICROBIAL AGENT, AND ANTIMICROBIAL AGENT
`
`Kailash C.)
`
`Mail Stop Patent Application
`Commissioner for Patents
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`EXPRESS MAH, CERTH~’ICATE
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`"Express Mail" Label Number: EL 934997148 US
`Date of Deposit: October 14, 2003
`
`I hereby state that the following attached papers and fees:
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`°
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`2.
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`Check: $770.00;
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`Linda M. Ginsberg
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`Bi317341.1
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`Page 3
`
`
`
`ADDENDUM
`
`Title:
`
`METHOD FOR DETECTING PATHOGENIC MICROORGANISM AND ANTIMICROBIAL
`AGENT, METHOD FOR EVALUATING EFFECT OF ANTIMICROBIAL AGENT, AND
`ANTIMICROBIAL AGENT
`
`BOS1317472.1
`
`Page 4
`
`
`
`_
`
`DESCRIPTION
`
`METHOD FOR DETECTING PATHOGENIC MICROORGANISM AND
`
`ANTIMICROBIAL AGENT, METHOD FOR EVALUATING EFFECT OF
`
`ANTIMICROBIAL AGENT, AND ANTIMICROBIAL AGENT
`
`TECHNICAL FIELD
`
`The present invention relates to a method for detecting
`
`pathogenic microorganism, method for evaluating an effect of an
`
`10
`
`antimicrobial agent on pathogenic microorganism and a method for
`
`detecting an antimicrobial agent. The present invention also relates to
`
`an antimicrobial agent and a therapeutic agent for onychomycosis,
`
`which are obtained according to the above-mentioned method for
`
`evaluating the drug effect.
`
`15
`
`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 enabling a drug effect to be
`
`2O
`
`evaluated with accuracy is needed because of grate importance in view
`
`of predicting a clinical therapeutic efficiency thereof.
`
`Historically, an experimental dermatophytosis model that
`
`back, planta and interdigital of a guniea pig have been infected with
`
`Trichophyton mentagrophytes has been used in order to evaluate an
`
`25
`
`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 carried out by applying
`
`Page 5
`
`
`
`¯
`
`_
`
`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
`
`10
`
`Trichophyton in vitro such as 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.
`
`15
`
`In also animal experiments, when an effect of lanoconazole
`
`on guniea 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, Louisiana, 1996,
`
`Abstr. F80).
`
`As
`
`a reason thereof, there were followings.
`
`Since
`
`25
`
`lanoconazole
`
`have very potent antitrichophyton activit~y in vitro,
`
`lanoconazole persisted in the skin 2 clays after the last treatment in the
`
`concentration wherein the sterilization effect was shown. Therefore,
`
`Page 6
`
`
`
`3
`
`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
`
`5
`
`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 to clear that the conventional method
`
`has the problem that the drug effect can not be accurately evaluated,
`
`15
`
`because the apparent therapeutic effect need to be evaluated after
`
`removing the drug remaining in the skin.
`
`Meanwhile, a kind of mycosis, delmatophytosis, is the
`
`superficial dermatosis which is caused by dermatophyte parasitizing the
`
`keratin such as skin (stratum corneum), the nail and the hair. In
`
`9.0
`
`particular, tinea unguium forined 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
`
`25
`
`unguium. However, there are many cases where the 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
`
`Page 7
`
`
`
`4 -
`
`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
`
`5
`
`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 unguium 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
`
`10
`
`the current antifungal agent for 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
`
`15
`
`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 model of tinea
`
`unguium has been hardly reported.
`
`20
`
`DISCLOSURE OF INVENTION
`
`The present invention has been accomplished based on
`
`findings that it is desirable that an effect of antimicrobial agent such as
`
`particularly antifungal agent is evaluated after removing a drug
`
`remaining in the infected site after treatment of an animal or a
`
`25
`
`biosample such as skin with the pathogenic microorganism. An object
`
`of the present invention is to provide a novel method for evaluating the
`
`effect of the antimicrobial agent and the antimicrobial agent obtained
`
`Page 8
`
`
`
`5 -
`
`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 biosarnple, 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 a
`
`10
`
`pathogenic microorganism. In addition, the present invention provides
`
`the antimicrobial agent obtained according to the above-mentioned the
`
`method for evaluating the drug effect, and the detecting method of the
`
`antimicrobial agent which comprises detecting the existing
`
`antimicrobial agent in the infected site of the animal or the biosample
`
`15
`
`with the pathogenic microorganism, to which the antimicrobial agent is
`
`administered.
`
`In more detail, according to the present invention a detection
`
`of a pathogenic microorganism and an evaluation of an effect of an
`
`antimicrobial agent can be carried out by infecting an animal or a
`
`2O
`
`biosarnple with the pathogenic microorganism, administering the
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`antimicrobial agent comprising a compound having an antimicrobial
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`effect or a composition thereof before or after the infection, then
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`removing the antimicrobial agent, and thereafter detecting the viable
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`pathogenic microorganism in the infected site with the pathogenic
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`25
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`microorganism.
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`According to the present invention a detection of an existing
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`antimicrobial agent can be carried out by infecting an animal or a
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`Page 9
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`6 -
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`biosample with a pathogenic microorganism, administering the
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`antimicrobial agent comprising a compound having an antimicrobial
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`effect or a composition thereof before or after the infection, then excising
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`the infected site with the pathogenic microorganism, placing and
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`cultivating it on a medium containing the pathogenic microorganism,
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`and thereafter observing a growth inhibition of the pathogenic
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`microorganism around the infected site with the pathogenic
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`microorganism.
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`Additionally, an object of the present invention is to provide
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`10
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`the evaluation method of a drug which enables the effect of an antifungal
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`agent to accurately evaluate in a guinea pig model of tinea unguium.
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`Another object of present invention is to provide a therapeutic agent for
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`onychomycosis which exhibits the effect on tinea unguium by topical
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`applicartion and which is capable of Curing tinea unguium shorter
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`16
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`period than that of the marketed oral preparation due to good
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`permeability, good retention capacity and conservation of high activity in
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`nail plate as well as the potent antifungal activity thereof based on the
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`present invention. Another object of the present invention is to provide
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`the effective therapeutic agent for onychomycosis exhibiting no side
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`effect even if therapeutically effective amounts of it are administered
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`sufficiently.
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`More concretely, the present invention provides a therapeutic
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`agent for onychomycosis containing a compound having a formula (1):
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`25
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`/ (CH2)m~~=~R1
`--N
`R2
`
`(CH2).
`
`(I)
`
`Page 10
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`
`
`O
`
`_
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`¯ Wherein RI and R2 are the same or different and are hydrogen atom, CI_6
`
`alkyl group, a non-substituted aryl group, an aryl group substituted
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`with 1 to 3 substituents selected from a halogen atom, trifluoromethyl
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`group, nitro group and CI.6 alkyl group, C2_a alkenyl group, C2_6 alkinyl
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`group, or Cv_12 aralkyl group,
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`m is 2 or 3,
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`n is 1 or 2,
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`or a salt thereof as active ingredient.
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`In addition, "presence" includes the mean of "remaining~.
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`10
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`BRIEF DESCRIPTION OF DRAWINGS
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`Fig. 1 is a color copy of a photograph to identify the agent
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`remaining in the skin which is previously evaluated by the conventional
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`method in the detecting method of the antimicrobial agent five days after
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`15
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`last treatment in the present invention. The note (a) shows the infected
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`control group, (b) the KP-103-treated group, (c) the lanoconazole-treated
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`group.
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`Fig. 2 is a color copy of photograph to identify agent
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`remaining in the skin which is previously evaluated by the detecting
`
`2O
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`method of the antimicrobial agent five days after last treatment in the
`
`present invention. The note (a) shows the infected control group, (b) the
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`KP-103-treated group, (c) the lanoconazole-treated group.
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`Fig. 3 is a graph showing a distribution of the number of
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`fungal cells in the nail of a guinea pig model of tinea unguium in each
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`25
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`treated group according to the evaluation method of the drug effect in
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`the present invention.
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`Fig. 4 is a graph showing a distribution of the number of
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`Page 11
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`
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`8
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`fungal cells in the skin of a guinea pig model of tinea pedis in each.
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`treated group according to the evaluation method of the drug effect in
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`the present invention.
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`5
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`BEST MODE FOR CARRYING OUT THE INVENTION
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`As an animal employed in the present invention, there
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`includes mammal such as mice, rat, guinea pig or rabbit. As a
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`biosample, there includes a skin of back or planta, a nail or the like,
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`which is taken from such animal.
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`10
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`A method for infecting such animal or biosample with a
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`pathogenic microorganism includes an inoculation percutaneouslly,
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`orally, intravenously, transbronchially, transnasally or intraperitoneally.
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`Especially in case of the skin, there includes a method for inoculating it
`
`0
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`on the skin, a method for inoculating on the exposed demis, the closed
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`patch method, intracutaneous injection or the like. Incase of the nail,
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`there includes a method for inoculating on nail, a method in which a
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`skin of the animals’ foot is infected by the above-mentioned infecting
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`method to the skin, and thereafter the infection is moved into the nail by
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`leaving it for several months.
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`20
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`The term "skin" means a tissue including the three layers
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`being epidermis, demis and subcutaneous tissue, accompanied by pilus
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`(hair), nail, glandulae sebaceae, glandulae sudoriferae and glandulae
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`mammaria as appendages. The epidermmis is separated five layers
`
`being stratum corneum, stratum lucidum, granulosum epidermidis,
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`25
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`stratum spinosum, and stratum basale from surface in order. The
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`stratum comeum, the stratum lucidum and the stratum granulosum
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`epidermidis is referred to as a stratum corneum in a broad sense.
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`Page 12
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`
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`_
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`Herein, keratin sbustance means a part of the above-mentioned stratum
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`corneum.
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`The term "nail" includes nail plate, nail bed, nail matrix,
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`further side nail wall, posterial nail wall, eponychium and hyponychium
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`which make up a tissue around thereof.
`
`In the present invention,
`
`the tei m "pathogenic
`
`microorganism" means a microorganism
`
`which causes human and
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`animal disease in one way or another. An example of the pathogenic
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`microorganism (hereinafter referred to "microorganism") is bacteria
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`10
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`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
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`15
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`including Hyphomycetes such as Trichophyton, Microsporum and
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`Epidermophyton species; Blastomycetes such as Candida and
`
`MalasSezia; Ascomycetes such as AspergiIIus species; Zygomycetes such
`
`as Mucor species; and variants thereof. Examples of such variants are
`
`resistant strain which naturally obtains drug resistance; auxotrophic
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`2O
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`mutation strain which comes to have nutritious dependency; artificial
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`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
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`25
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`broadly divided into superficial mycosis and deep mycosis. A seat of
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`the disease lie in the skin. or visible mucosa in case of the former, in
`
`viscus, central nervous system, subcutaneous tissue, muscle, born or
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`Page 13
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`10 -
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`articulation in case of the latter. Chief example of superficial mycosis is
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`dermatophytosis which is caused by infecting with dermatophyte such
`
`as Trichophyton, Microsporum and Epidermophyton species, including
`
`three disease, tinea, tinea favosa and tinea imbricata. Tinea may be
`
`conventionally employed a synonymous with dei-matophytosis. In
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`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
`
`10
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`the compound. The composition includes a preparation form being
`
`artificial composition and a natural composition such as a natural
`
`product.
`
`A method for administration of the antimicrobial agent in the
`
`present invention depends on the kind thereof and includes topical
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`15
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`application, subcutaneous administration, oral administration,
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`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
`
`E5
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`where the antimicrobial agent is administered after the infection with
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`microorganism, meanwhile, a effect of the antimicrobial agent protecting
`
`from the infection and the retention capacity thereof can be evaluated in
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`Page 14
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`11 -
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`case where the infection with microorganism is carried out after the
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`administration of the antimicrobial agent. In order to evaluating the
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`retention capacity of the antimicrobial agent, the evaluation can be
`
`carried out with varying the period until infection with microorganism
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`,5
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`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
`
`detecting target or a microorganism used in the present evaluation
`
`10
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`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 detecting target
`
`or the microorganism used in the present evaluation method and the
`
`15
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`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 l~m, it
`
`is preferable to use the membrane having less than 0.2 l~m of the pore
`
`size, particularly it is suitable to use dialysis membrane having
`
`fractional molecular weight of 1,000 to 50,000.
`
`2O
`
`As out side solutions used in dialysis, there include
`
`physiological saline, distilled water, phosphate buffered physiological
`
`saline, the other buffer and the like.
`
`In removing the antimicrobial agent according to the present
`
`invention, even though the infected site with the microorganism is the
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`25
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`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 dialysis to remove the antimicrobial agent from nai! than.
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`Page 15
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`- 12
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`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
`
`10
`
`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
`
`15
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`placing and cultivating the infected site with a microorganism which is
`
`subjected to the removing method of the antimicrobial agent (e.g. an
`
`