I Illll Illlllll Ill Illll Illll Illll Illll Illll Illll Illll M Illlll Illl Illl Ill
`
`US007214506B2
`
`(12) United States Patent
`Tatsumi et al.
`
`(10) Patent No.: US 7,214,506 B2
`(45) Date of Patent: May 8, 2007
`
`(54)
`
`METHOD FOR TREATING
`ONYCHOMYCOSIS
`
`(75)
`
`Inventors:
`
`Yoshiyuki Tatsumi, Otsu (JP);
`Mamoru Yokoo, Otsu (JP); Kosho
`Nakamura, Moriyama (JP); Tadashi
`Arika, Suita (JP)
`
`(73) Assignee:
`
`Kaken Pharmaceutical Co., Ltd.,
`Bunkyo-ku, Tokyo (JP)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 451 days.
`
`(21) Appl. No.: 10/685,266
`
`(22) Filed:
`
`Oct. 14, 2003
`
`(65)
`
`Prior Publication Data
`
`US 2007/0082375 A1 Apr. 12, 2007
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4/1997 Naito et al .................. 514/326
`5,620,994 A *
`2/1998 Naito et al .................. 514/326
`5,716,969 A *
`5,962,476 A 10/1999 Naito et al.
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`JP
`JP
`WO
`WO
`
`0 402 989 A2
`8 - 103291
`10-28597
`WO 94/26734
`WO 99/39680 A1
`
`12/1990
`4/1996
`2/1998
`11/1994
`8/1999
`
`OTHER PUBLICATIONS
`
`Kitazaki Tomoyuki et al., Chem. Pharm. Bull, vol. 44 (No. 2), p.
`314-327, (Feb. 1996).
`Ogura, Hironobu et al., Chem. Pharm. Bull, vol. 47 (No. 10), p.
`1417-1425, (Oct. 1999).
`
`Related U.S. Application Data
`
`* cited by examiner
`
`(62)
`
`Division of application No. 10/031,929, filed as appli-
`cation No. PCT/JP00/04617 on Jul. 11, 2000, now
`abandoned.
`
`Primary Examiner Jon Weber
`Assistant Examiner Kailash C. Srivastava
`(74) Attorney, Agent, or Firm~ixon Peabody LLP
`
`(30)
`
`Foreign Application Priority Data
`
`(57)
`
`ABSTRACT
`
`Jul.
`
`28, 1999 (JP)
`
`................................. 11/214369
`
`(51)
`
`(52)
`(58)
`
`Int. CI.
`(2006.01)
`C12Q 1/18
`A O1N 43/26
`(2006.01)
`A O1N 43/34
`(2006.01)
`A 61K 31/445
`(2006.01)
`U.S. Cl .......................................... 435/32; 514/326
`Field of Classification Search .................. 435/32;
`514/326
`See application file for complete search history.
`
`A novel method for evaluating an effect of an antimicrobial
`agent which comprises removing the antimicrobial agent
`remaining in a biological sample or the like to thereby
`accurately evaluate the effect of the antimicrobial agent
`without being affected by the remaining antimicrobial agent.
`A therapeutic agent for onychomycosis which can be
`obtained according to the evaluation method of the &rug
`effect.
`
`2 Claims, 4 Drawing Sheets
`
`ACRUX DDS PTY LTD. et al.
`EXHIBIT 1001
`IPR Petition for
`U.S. Patent No. 7,214,506
`
`1 of 14
`
`

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`U.S. Patent
`U.S. Patent
`
`May 8, 2007
`May 8, 2007
`
`Sheet 1 of 4
`Sheet 1 of 4
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`US 7,214,506 B2
`US 7,214,506 B2
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`F I G. I (a)
`FIG. 1(6)
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`F I G. I (b)
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`FI G. 1 (c)
`
`$
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`M
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`$
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`M
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`2 of 14
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`

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`U.S. Patent
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`May 8, 2007
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`Sheet 2 of 4
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`US 7,214,506 B2
`
`FIG. 2(a)
`
`FIG. 2(b)
`
`FI G. 2 (c)
`
`M
`
`E
`
`E
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`M
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`E
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`3 of 14
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`U.S. Patent
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`May 8, 2007
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`Sheet 3 of 4
`
`US 7,214,506 B2
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`The number of fungal cells in the nail
`(Log CFU/foot)
`
`4 of 14
`
`

`
`U.S. Patent
`
`May 8, 2007
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`Sheet 4 of 4
`
`US 7,214,506 B2
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`(Log CFU/foot)
`
`5 of 14
`
`

`
`US 7,214,506 B2
`
`1
`METHOD FOR TREATING
`ONYCHOMYCOSIS
`
`This application is a divisional under 35 U.S.C. § 120 of
`U.S. Non-Provisional application Ser. No. 10/031,929 filed 5
`25 Jan. 2002, now abandoned, which was a National Stage
`filing under 35 U.S.C. § 371 of PCT/JP00/04617 filed 11 Jul.
`2000, which claimed priority to Japanese patent application
`Ser. No. 11/214,369 filed 28 Jul. 1999.
`
`TECHNICAL FIELD
`
`10
`
`The present invention relates to a method for detecting
`pathogenic microorganism, method for evaluating an effect
`of an antimicrobial agent on pathogenic microorganism and 15
`a method for detecting an antimicrobial agent. The present
`invention also relates to an antimicrobial agent and a thera-
`peutic agent for onychomycosis, which are obtained accord-
`ing to the above-mentioned method for evaluating the drug
`effect. 20
`
`BACKGROUND ART
`
`A method for evaluating a &rug effect with an animal
`model is needed in order to explore a novel antimicrobial 25
`agent (also hereinafter referred to "drug"). Further, a method
`enabling a drug effect to be evaluated with accuracy is
`needed because of grate importance in view of predicting a
`clinical therapeutic efficiency thereof.
`Historically, an experimental dermatophytosis model that 3o
`back, planta and interdigital of a guniea pig have been
`infected with Trichophyton mentagrophytes has been used in
`order to evaluate an effect of an antifungal agent on der-
`matophytosis. Such animal models have been already
`employed to develop some antifungal agent. The evaluation 35
`of the effect of such antifungal agent 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 4o
`is seen or the number of animals or feet wherein no growth
`of fungus is seen in all skin pieces, as an indicator (Anti-
`microbial 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 conven- 45
`tional method".
`Although the drug having a potent activity against Tri-
`chophyton 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 50
`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 lanocona-
`zole on guniea pig models oftinea 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, La., 1996, Abstr. F80).
`As a reason thereof, there were followings. Since lano-
`conazole 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 culti-
`
`2
`vated on the medium to detect fungus, the lanoconazole
`remaining in the skin is diffused in the medium, and there-
`fore, 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 culti-
`vated 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, because the apparent therapeutic effect need to be
`evaluated after removing the drug remaining in the skin.
`Meanwhile, a kind of mycosis, dermatophytosis, is the
`superficial dermatosis which is 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 dermatomy-
`coses based on dermatophytoses, and is accompanied by
`symptom such as opacity, tylosis, destruction and deforma-
`tion of nail plate. Now the oral preparation (such as griseof-
`ulvin or terbinafine) is used for the treatment of such tinea
`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 halfa 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 terbin-
`afine. 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 the current antifungal agent for topical use, the anti-
`fungal 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 trichophy-
`tosis 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.
`
`DISCLOSURE OF INVENTION
`
`55 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 treat-
`meut of an animal or a biosample such as skin with the
`6o pathogenic microorganism. An object of the present inven-
`tion 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 detect-
`65 ing the viable pathogenic microorganism in the above-
`mentioned infected site of the animal or the biosample with
`the pathogenic microorganism after removing the autimi-
`
`6 of 14
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`

`
`US 7,214,506 B2
`
`3
`crobial 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 a 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 detect-
`ing the existing antimicrobial agent in the infected site of the
`animal or the biosample 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 biosample with the pathogenic
`microorganism, administering the antimicrobial agent com-
`prising 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.
`
`According to the present invention a detection of an
`existing antimicrobial agent can be carried out by infecting
`an animal or a biosample with a pathogenic microorganism,
`administering the antimicrobial agent comprising a com-
`pound 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 a medium containing the pathogenic
`microorganism, and thereafter observing a growth inhibition
`of the pathogenic microorganism around the infected site
`with the pathogenic microorganism.
`
`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 unguium. Another object of
`present invention is to provide a therapeutic agent for
`onychomycosis which exhibits the effect on tinea unguium
`by topical application and which is capable of curing tinea
`unguium shorter period than that of the marketed oral
`preparation due to good permeability, good retention capac-
`ity 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 side effect even if therapeutically effective
`amounts of it are administered sufficiently.
`
`More concretely, the present invention provides a thera-
`peutic agent for onychomycosis containing a compound
`having a formula (1):
`
`(I)
`
`/(CH2)m R1
`
`-\ )==(2
`
`(CH2)n R
`
`4
`group and C1_6 alkyl group, C2_, alkenyl group, C2_6 alkinyl
`group, or C7_12 aralkyl group,
`
`m is 2 or 3,
`
`5 nis 1 or2,
`
`or a salt thereof as active ingredient.
`In addition, "presence" includes the mean of "remaining".
`
`10
`
`BRIEF DESCRIPTION OF DRAWINGS
`
`FIG. 1 is a color copy of a photograph to identify the agent
`remaining in the skin which is previously evaluated by the
`conventional method in the detecting method of the antimi-
`15 crobial agent five days after last treatment in the present
`invention. The note (a) shows the infected control group, (b)
`the KP-103-treated group, (c) the lanoconazole-treated
`group.
`FIG. 2 is a color copy of photograph to identify agent
`2o remaining in the skin which is previously evaluated by the
`detecting method of the antimicrobial agent five days after
`last treatment in the present invention. The note (a) shows
`the infected control group, (b) the KP-103-treated group, (c)
`the lanoconazole-treated group.
`25 FIG. 3 is a graph showing a distribution of the number of
`fungal cells in the nail of a guinea pig model of tinea
`unguium in each treated group according to the evaluation
`method of the drug effect in the present invention.
`FIG. 4 is a graph showing a distribution of the number of
`3o fungal cells in the skin of a guinea pig model of tinea pedis
`in each treated group according to the evaluation method of
`the drug effect in the present invention.
`
`35
`
`BEST MODE FOR CARRYING OUT THE
`INVENTION
`
`4o
`
`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 percuta-
`neously, orally, intravenously, transbronchially, transnasally
`45 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 or the like. Incase 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" means a tissue including the three layers
`
`50
`
`55 being epidermis, demis and subcutaneous tissue, accompa-
`nied by pilus (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 spino-
`
`Wherein R1 and R2 are the same or different and are
`hy&ogen atom, C1_6 alkyl group, a non-substituted aryl 65
`group, an aryl group substituted with 1 to 3 substituents
`selected from a halogen atom, trifluoromethyl group, nitro
`
`60 sum, and stratum basale from surface in order. The stratum
`corneum, the stratum lucidum and the stratum granulosum
`epidermidis is referred to as a stratum corneum in a broad
`sense. Herein, keratin substance 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.
`
`7 of 14
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`US 7,214,506 B2
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`5
`In the present invention, the term "pathogenic microor-
`ganism" means a microorganism which causes human and
`animal disease in one way or another. An example of the
`pathogenic microorganism (hereinafter referred to "micro-
`organism") 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 dermato-
`phyte such as Trichophyton, Microsporum and Epidermo-
`phyton species, including three disease, tinea, tinea favosa
`and tinea imbricata. Tinea may be conventionally employed
`a synonymous with dermatophytosis. In addition, dermato-
`phyte 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.
`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 microor-
`ganism, 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 (herein-
`after referred to "the present evaluation method"), a thera-
`peutic effect of the antimicrobial agent can be evaluated in
`case where the antimicrobial agent is administered after the
`infection with microorganism, meanwhile, a 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 admin-
`istration of the antimicrobial agent. In order to evaluating 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 antimi-
`crobial 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 method and the like is not
`affected by it.
`
`15
`
`6
`In dialysis, a marketed dialysis membrane made of cel-
`lulose 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
`5 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 ~tm, it is
`preferable to use the membrane having less than 0.2 grn of
`the pore size, particularly it is suitable to use dialysis
`10 membrane having fractional molecular weight of 1,000 to
`50,000.
`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 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
`20 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 concentra-
`25 tion, dose term and the drug holidays (the term until evalu-
`ation 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
`30 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
`35 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 microor-
`ganism which is subjected to the removing method of the
`antimicrobial agent (e.g. an skin piece) or a suspension
`40 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
`45 inhibition of the microorganism is observed.
`The present evaluation method can be carried out by
`locating and cultivating, on a medium, the skin piece in
`which a removal of an antimicrobial agent has been deter-
`mined using the above-mentioned present method for detect-
`50 ing 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 culti-
`vating a suspension obtained according to the extraction
`procedure of the microorganism from the skin piece on an
`55 agar medium and observing whether there 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
`60 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
`65 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.
`
`8 of 14
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`US 7,214,506 B2
`
`7
`When the treatment with trypsin is carried out before
`dialysis, it is necessary to remove the digestive enzyme
`sufficiently 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 sepa-
`ration of the microorganism. In case of fungi, 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 case of bacteria,
`example of the medium is Mueller Hinton medium, modi-
`fied 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° C., preferably 20 to
`40° C. A microorganism may be cultivated with standing
`during a sufficient time when the microorganism can be
`growth, for example, 1 to 20 days in case of fungi, 1 to 5
`days in bacteria.
`The present evaluation method be utilizable as a evalua-
`tion method of a &rug 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 compound, then removing the antimicrobial
`agent and detecting and determining quantity of the micro-
`organism in the sample.
`The present evaluation method also can be applied to an
`evaluation of an antimicrobial agent such as a therapeutic
`agent for deep mycosis or an antibacterial agent as well as
`an evaluation of an effect of a therapeutic agent for super-
`ficial mycosis. That is to say, it is possible to evaluate an
`effect of a therapeutic agent for deep mycosis or an anti-
`bacterial 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, intraperito-
`neally, 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 significant deference test 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 compari-
`son between the groups can be done by using a multiple test
`such as Tukey method.
`The present invention is useful as either a method for
`evaluating a drug effect or a method for detecting the
`antimicotics in keratin substance or nail, after administering
`the anti fungus to the patient infected with fungus. For
`example, according to the present invention, an effect of an
`antifungal agent can be evaluated by administering it 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 anti-
`fungal agent can be carried out by administering it 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
`
`10
`
`8
`existing antifungal agent in the keratin substance or nail
`through a growth inhibition of fungus observed around the
`keratin substance or nail. Such evaluation of an antifungal
`agent administered to a patient with fungus and detection of
`s the antifungal agent from the keratin substance or nail can be
`carried out in the same manner as in the above-mentioned
`evaluation method of a &rug effect and detecting method of
`the antimicrobial agent administered to an animal or a
`biosample.
`Furthermore, the present invention provides various use-
`ful antimicrobial agents according to the present evaluation
`method. As the antimicrobial agent obtained by the present
`evaluation method, there is an antimicrobial agent compris-
`ing a compound having an eradication effect for microor-
`is ganism in vivo or a composition for therapy of the super-
`ficial 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 hav-
`2o ing an excellent eradication effect for microorganism in
`vivo, which is selected by means 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
`2s compound having the group represented by the above-
`mentioned formula (I). Among them, more preferable con-
`crete example is a therapeutic agent for onychomycosis
`comprising the compound represented by the formula (II):
`
`30
`
`35
`
`(II)
`
`40 wherein, Ar is a non-substituted phenyl group or a phenyl
`group substituted with 1 to 3 substituents selected from a
`halogen atom and trifluoromethyl group,
`
`R1 and R2 are the same or different and are hy&rogen atom,
`Cl_6 alkyl group, a non-substituted aryl group, an aryl group
`4s substituted with 1 to 3 substituents selected from a halogen
`
`atom, trifluoromethyl group, nitro group and Cl_6 all group,
`C2_s alkenyl group, C2_6 alkinyl group, or
`
`C7_12 aralkyl group,
`
`50
`
`m is 2 or 3,
`
`nis 1 or2,
`
`X is nitrogen atom or CH, and
`
`ss *1 and *2 mean an asymmetric carbon atom.
`In the above-mentioned formula (I) or (II), the substituted
`phenyl group is a phenyl group having 1 to 3 substituents
`selected from a halogen atom and trifluoromethyl, and
`includes, for instance, 2,4-difluorophenyl, 2,4-dichlorophe-
`6o nyl, 4-fluorophenyl, 4-chlorophenyl, 2-chlorophenyl, 4-trif-
`luoromethylphenyl, 2-chloro-4-fluorophenyl, 4-bromophe-
`nyl or the like. Cl_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, iso-
`6s propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, iso-
`pentyl, neopentyl, tert-pentyl and cyclohexyl. The non-
`substituted aryl group includes, for example, phenyl,
`
`9 of 14
`
`

`
`US 7,214,506 B2
`
`9
`naphthyl, biphenyl or the like. The substituted aryl group
`includes, for example, 2,4-difluorophenyl, 2,4-dichlorophe-
`nyl, 4-fluorophenyl, 4-chlorophenyl, 2-chlorophenyl, 4-trif-
`luoromethylphenyl, 2-chloro-4-fluorophenyl, 4-bromophe-
`nyl, 4-tert-butylphenyl, 4-nitrophenyl or the like. C2_s
`alkenyl group includes, for example, vinyl, 1-propenyl,
`styryl or the like. C2_6 alkynyl group includes, for example,
`ethynyl or the like. C7_12 aralkyl group includes, for
`example, benzyl, naphthylmethyl, 4-nitrobenzyl or the like.
`
`In addition, the most preferable compound among the
`above-mentioned antimicrobial agent includes the com-
`pound which shows the therapeutic efficiency like the fol-
`lowing KP-103.
`
`The above-mentioned KP-103 means an antifungal indi-
`cated by (2R,3R)-2-(2,4-difluorophenyl)-3-(4-methylenepi-
`peridine- 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-
`difluorophenyl)-3-methyl-2-[(1 H-1,2,4-triazole-1 -yl)me-
`thyl]oxirane 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 con-
`firmed, but its antifungal activity has bee