`
`Certification
`
`We, the undersigned, do hereby certify the following: We are fluent in the
`
`English and Japanese languages. We have translated and!or reviewed the
`
`translation of the Japanese document identified as: JPOO/04617 (Priority
`
`Document for the ’506 Patent), and find it to be a true and accurate
`
`translation to the best of my knowledge and ability.
`
`We certify under penalty of peijury that the foregoing is true and correct.
`
`Signature
`
`Miki Yanai Hernandez
`
`Signature
`
`Brenda K. Seat, Esq.
`
`Date: September 26, 2016
`
`SIX WHITEHALL COURT, SILVER SPRING, MARYLAND 2090 I TEL.: 30 ]-593-39B0 FAX: 301-68 ]-9243
`
`B S EAT(,~)S HIN SH U S ERVICE S. B OH W’+~’W. S H IN S H LI S ERVII3 ES. CO M
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`ARGENTUM EX1002
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`Page 1
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`Certified Translation by Shinshu Services, Inc.
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`PATENT OFFICE
`JAPANESE GOVERNMENT
`
`PCT/JP00/04617
`11.07.00
`
`REC’D 25 AUG 2000
`WIPO
`PCT
`
`l\
`
`This is to certify that the annexed is a true copy of the following application as filed with this
`Office.
`
`Date of Application: July 28, 1999
`
`Application Number: Heisei 11 [ 1999] Patent Application Number 214369
`
`Applicant(s): Kaken Pharmaceutical Co., Ltd.
`
`PRIORITY DOCUMENT
`SUBMITTED OR TRANSMITTED IN
`COMPLIANCE WITH
`RULE !7.1 (a) OR (b)
`
`August 11, 2000
`
`Commissioner,
`Patent Office
`
`Kozo Oikawa [signature with the official seal]
`
`Application Number: Priority Certificate 2000-3062601
`
`Page 2
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`
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`Certified Translation by Shinshu Services, Inc.
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`[Document Name]
`
`[Reference Number]
`[Filing Date]
`[To Whom]
`[International Patent
`
`Classification]
`[Inventor]
`[Residence or
`Address]
`
`[Name]
`
`[Inventor]
`[Residence or
`Address]
`
`[Name]
`[Inventor]
`[Residence
`or Address]
`[Name]
`
`PETITION FOR PATENT APPLICATION JP-11531
`July 28, 1999
`
`Commissioner of Patent Office, Takeshi
`Isayama
`
`C12Q 1/18
`
`c/o DEVELOPMENT Research Laboratories,
`KAKEN PHARMACEUTICAL CO., LTD.,
`14 Shinomiyaminamigawara -cho, Yamashina-ku,
`Kyoto-shi, Kyoto- fu
`Yoshiyuki Tatsumi
`
`c/o DEVELOPMENT Research Laboratories,
`
`KAKEN PHARMACEUTICAL CO., LTD.,
`
`14 Shinomiyaminamigawara-cho, Yamashina-ku,
`Kyoto-shi, Kyoto-fu
`
`Mamoru Yokoo
`
`c/o KAKEN PHARMACEUTICAL CO., LTD.,
`28-8 Honkomagome 2-chome, Bunkyo-ku, Tokyo-to
`Kosho Nakamura
`
`Page 3
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`
`
`[Inventor]
`
`[Residence
`or Address]
`
`[Name]
`[Patent Applicant]
`[Identification No.]
`[Name]
`[Patent Representative]
`[Identification No.]
`[Patent Attorney]
`[Name]
`[Telephone No.]
`[Patent Representative
`Appointed]
`[Identification No. ]
`[Patent Attorney]
`[Name]
`[Indication of Charge]
`[Number in
`Prepayment File]
`[Sum of Payment]
`[List of Filing Materials]
`
`[Material Name]
`[Material Name]
`[Material Name]
`[Number of General
`Power of Attorney]
`[Necessity of Proof]
`
`Certified Translation by Shinshu Services, Inc.
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`2 -
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`c/o DEVELOPMENT Research Laboratories,
`KAKEN PHARMACEUTICAL CO., LTD.,
`301 Gensuke, Fujieda-shi, Shizuoka-ken
`Tadashi ARIKA
`
`000124269
`
`KAKEN PHARMACEUTICAL CO., LTD.
`100065226
`
`SohtaAsahina
`06-6943-8922
`
`100098257
`
`Keiji Saki
`
`001627
`
`¥21,000
`
`Specification
`
`Drawing(s)
`
`Abstract
`
`9002226
`
`Necessary
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`1
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`1
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`Page 4
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`Certified Translation by Shinshu Services, Inc.
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`[Document Name]
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`SPECIFICATION
`
`[Title of the Invention]
`
`METHOD FOR DETECTING PATHOGENIC
`
`MICROORGANISM AND ANTIMICROBIAL AGENT,
`
`METHOD FOR EVALUATING EFFECT OF
`
`ANTIMICROBIAL AGENT, AND ANTIMICROBIAL AGENT
`
`OBTAINED THEREBY
`
`[CLAIMS]
`
`1. A method for detecting a pathogenic microorganism
`
`which comprises infecting an animal or a biosample with the
`
`pathogenic microorganism, administering an 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.
`
`2. The method for detecting a pathogenic microorganism of
`
`Claim 1,
`
`in which the pathogenic microorganism is a bacterium or a
`
`fungus.
`
`3. The method for detecting a pathogenic microorganism of
`
`Claim 2, in which the fungus is a pathogenic fungus causing superficial
`
`mycosis or deep mycosis.
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`Page 5
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`4. The method for detecting a pathogenic microorganism of
`
`Claim 1, in which the antimicrobial agent is a therapeutic agent for
`
`superficial mycosis, a therapeutic agent for deep mycosis and an
`
`antibacterial agent.
`
`5. The method for detecting a pathogenic microorganism of
`
`Claim 1, which comprises removing the antimicrobial agent using
`
`dialysis or ultra filtration.
`
`6. The method for detecting a pathogenic microorganism of
`
`Claim 1, in which the infected site with the pathogenic microorganism
`
`is a skin or a nail.
`
`7. The method for detecting a pathogenic microorganism of
`
`Claim 1, in which the administration of the antimicrobial agent is
`
`carried out percutaneously, orally or intravenously.
`
`8. The method for detecting a pathogenic microorganism of
`
`Claim 1, which comprises treating the infected site with the pathogenic
`
`microorganism with a digestive enzyme to detect the pathogenic
`
`microorganism.
`
`9. A method for evaluating an effect of an antimicrobial
`
`agent which comprises detecting a pathogenic microorganism
`
`according to the method for detecting the pathogenic microorganism of
`
`Claim 1,2, 3, 4, 5, 6,7or 8.
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`Page 6
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`- 3
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`10. An antimicrobial agent obtained according to
`
`the
`
`method for evaluating an effect of an antimicrobial agent of Claim 9.
`
`11. A method for detecting an antimicrobial agent which
`
`comprises infecting an animal or a biosample with a pathogenic
`
`microorganism, administering an antimicrobial agent comprising a
`
`compound having an antimicrobial effect or a composition thereof
`
`before or after the infection, then excising the infected site with the
`
`pathogenic microorganism, placing and cultivating it on an agar
`
`medium containing the pathogenic microorganism, and thereafter
`
`detecting the antimicrobial agent existing in the infected site with the
`
`pathogenic microorganism through a growth inhibition of the
`
`pathogenic microorganism observed around the infected site with the
`
`pathogenic microorganism.
`
`[DETAILED EXPLANATION OF THE INVENTION]
`
`[Technical Field of the Invention]
`
`The present invention relates to a method for evaluating an
`
`effect of an antimicrobial agent on pathogenic microorganism. In
`
`particular, it relates to a method for evaluating an effect of an agent
`
`wherein an antimicrobial agent existing in the infected site of an
`
`animal or a biosample with a pathogenic microorganism after therapy
`
`is removed and then the viable pathogenic microorganism remaining in
`
`the infected site with the pathogenic microorganism is detected and
`
`determined with accuracy.
`
`[Prior Art]
`
`A method for evaluating a drug effect with an animal model
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`Page 7
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`Certified Translation by Shinshu Services, Inc.
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`is needed in order to explore a novel antimicrobial agent (also
`
`hereinafter referred to "drug"). Further, a method for evaluating a drug
`
`effect with accuracy is needed because of great importance in view of
`
`predicting a clinical therapeutic efficiency thereof.
`
`Historically, an experimental dermatophytosis model that
`
`back, planta and interdigital of a guinea 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 is carried out by
`
`applying the antifungal agent to the infected animal, excising the skin
`
`after the certain period of time to cut into plural small pieces,
`
`cultivating the skin pieces on the medium, and 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 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 guinea pig models of tinea pedis was evaluated using the
`
`conventional method, though "fungus-negative" was observed in all feet
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`5 -
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`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
`
`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 clear that the conventional method
`
`has the problem that the drug effect can not be accurately evaluated,
`
`because the drug effect is evaluated so that there is apparent
`
`therapeutic effect due to the drug remaining in the skin.
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`6 -
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`Herefrom, in the case of evaluating a drug effect on
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`antimicrobial agent, especially antifungus agent, it is necessary to
`
`evaluate the drug effect after removing the agent remaining in the
`
`infected site of the animal or the biosample with a pathogenic
`
`microorganism, such as a skin after treatment.
`
`[Problems to be Solved by the Invention]
`
`The present invention aims to provide a novel method for
`
`evaluating an effect of an antimicrobial agent and an antimicrobial
`
`agent obtained according to the method for evaluating an effect of an
`
`antimicrobial agent. In particular, the invention provides a method for
`
`detecting a viable pathogenic microorganism which comprises
`
`administering an antimicrobial agent to an animal or a biosample,
`
`then removing the antimicrobial agent, and thereafter detecting the
`
`viable pathogenic microorganism in the infected site with the
`
`pathogenic microorganism; a method for evaluating effect of an
`
`antimicrobial agent comprising the method for detecting a pathogenic
`
`microorganism 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 the
`
`pathogenic microorganism; an antimicrobial agent obtained according
`
`to the method for evaluating the effect of the agent; and a method
`
`for detecting an antimicrobial agent wherein an antimicrobial agent
`
`is administered to an animal or biosample and the antimicrobial
`
`agent existing in the infected site of the animal or the biosample
`
`with a pathogenic microorganism is detected.
`
`In addition, "presence" includes the meaning of "remaining".
`
`[Means to Solve the Problems]
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`Page 10
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`7 -
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`To solve the above problems, the inventors have
`
`researched intensively in order to seek for a method to accurately
`
`evaluate the effect of an antimicrobial agent without the influence
`
`of the antimicrobial agent remaining in the infected site of the animal
`
`or the biosample with a pathogenic microorganism. As a result, the
`
`inventors have found a method to accurately evaluate the effect of
`
`antimicrobial agent such as antifungal agent, comprising: fully
`
`removing an antimicrobial agent remaining in the infected site of the
`
`animal or the biosample with a pathogenic microorganism, and
`
`detecting and determining a viable pathogenic microorganism in the
`
`infected site with the pathogenic microorganism such as skin, thus
`
`completing the present invention.
`
`That is, the present invention relates to a method for
`
`detecting a pathogenic microorganism which comprises infecting an
`
`animal or a biosample with the pathogenic microorganism,
`
`administering an 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 (Claim 1),
`
`the method for detecting a pathogenic microorganism of Claim 1, in
`
`which the pathogenic microorganism is a bacterium or a fungus (Claim
`
`2),
`
`the method for detecting a pathogenic microorganism of Claim 2, in
`
`which the fungus is a pathogenic fungus causing superficial mycosis
`
`or deep mycosis (Claim 3),
`
`the method for detecting a pathogenic microorganism of Claim 1, in
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`8 -
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`which the antimicrobial agent is a therapeutic agent for superficial
`
`mycosis, a therapeutic agent for deep mycosis and an antibacterial
`
`agent (Claim 4),
`
`the method for detecting a pathogenic microorganism of Claim 1,
`
`which comprises removing the antimicrobial agent using dialysis or
`
`ultra filtration (Claim 5),
`
`the method for detecting a pathogenic microorganism of Claim 1, in
`
`which the infected site with the pathogenic microorganism is a skin or
`
`a nail (Claim 6),
`
`the method for detecting a pathogenic microorganism of Claim 1, in
`
`which the administration of the antimicrobial agent is carried out
`
`percutaneously, orally or intravenously (Claim 7),
`
`the method for detecting a pathogenic microorganism of Claim 1,
`
`which comprises treating the infected site with the pathogenic
`
`microorganism with a digestive enzyme to detect the pathogenic
`
`microorganism (Claim 8),
`
`a method for evaluating an effect of an antimicrobial agent which
`
`comprises detecting a pathogenic microorganism according to the
`
`method for detecting the pathogenic microorganism of Claim 1, 2, 3, 4,
`
`5, 6, 7 or 8 (Claim 9),
`
`an antimicrobial agent obtained according to the method for evaluating
`
`an effect of an antimicrobial agent of Claim 9 (Claim 10), and
`
`a method for detecting an antimicrobial agent which comprises
`
`infecting an animal or a biosample with a pathogenic microorganism,
`
`administering an antimicrobial agent comprising a compound having
`
`an antimicrobial effect or a composition thereof before or after the
`
`infection, then excising the infected site with the pathogenic
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`9 -
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`microorganism, placing and cultivating it on an agar medium
`
`containing the pathogenic microorganism, and thereafter detecting the
`
`antimicrobial agent existing in the infected site with the pathogenic
`
`microorganism through a growth inhibition of the pathogenic
`
`microorganism observed around the infected site with the pathogenic
`
`microorganism (Claim 11).
`
`[Embodiment of 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 percutaneously,
`
`orally, intravenously,
`
`transbronchially,
`
`transnasally
`
`or
`
`intraperitoneally. Especially in case of the skin, there includes a method
`
`for inoculating it on the skin, a method for inoculating on the exposed
`
`demis, the closed patch method, intracutaneous injection or the like.
`
`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.
`
`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
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`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 Epidermophyton species,
`
`including three disease, tinea, tinea favosa and tinea imbricata. Tinea
`
`may be conventionally employed a synonymous with dermatophytosis.
`
`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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`11
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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, the 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
`
`evaluate 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 of
`
`simpleness, but not limited thereto as long as a microorganism 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 other material can be
`
`used without problem, as long as the microorganism used in the
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`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 ~z m, it is preferable to use the membrane
`
`having less than 0.2 ~z m of the pore size, particularly it is suitable to
`
`use dialysis membrane having fractional molecular weight of 1,000 to
`
`50,000.
`
`As outside solutions used in dialysis, there include
`
`physiological saline, distilled water, phosphate buffered physiological
`
`saline, 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 for 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
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`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 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 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
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`14 -
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`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 the case of fungi, the example of the medium is
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`Sabouraud medium, modified Sabouraud medium, Czapek agar
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`medium, Potato dextrose agar medium or the like. On the other hand,
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`in the case of bacteria, example of the medium is Mueller Hinton
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`medium, modified Mueller Hinton medium, Heart Infusion agar
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`medium, Brain Heart Infusion agar medium, normal agar medium or
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`the like.
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`A reacting temperature is 10 to 40°C, preferably 20 to 40°C.
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`A microorganism may be cultivated with standing during a sufficient
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`time when the microorganism can be grown, for example, 1 to 20 days
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`for fungi, 1 to 5 days for bacteria.
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`The present evaluation method can be utilized as an
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`evaluation method of a drug effect in exo vivo which comprises
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`infecting a skin, a nail excised from an animal body with a
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`microorganism, thereafter administering an antimicrobial agent as a
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`test compound, then removing the antimicrobial agent and detecting
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`and determining quantity of the microorganism inthe sample.
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`The present evaluation method also can be applied to an
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`evaluation of an antimicrobial agent such as a therapeutic agent for
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`deep mycosis or an antibacterial agent as well as an evaluation of an
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`15 -
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`effect of a therapeutic agent for superficial mycosis. That is to say, it
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`is possible to evaluate an effect of a therapeutic agent for deep mycosis
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`or an antibacterial agent by means of administering an antimicrobial
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`agent to an animal infected with a microorganism such as a fungus or
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`a bacterium by inoculating percutaneously, orally, intravenously,
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`transbronchially, transnasally, intraperitoneally, then obtaining
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`biosample such as skin, kidney, lung or brain, and detecting the viable
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`microorganism in the biosample in which the remaining
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`antimicrobial agent has been removed.
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`In addition, the present evaluation method enables a
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`quantitative
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`comparison of antimicrobial effects by means of
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`determining
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`the number of viable microorganisms in the treated
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`biosample.
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`That is to say, a test of significance is carried out about the
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`number of microorganisms in the infected site with the microorganism
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`for the treated group with drug and for the reference infected group
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`using a statistical method such as Kruskal-Wallis Test, and thereby a
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`quantitative comparison between the groups can be done.
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`As the antimicrobial agent obtained by the present
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`evaluation method, there is an antimicrobial agent comprising a
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`compound having an eradication effect for microorganism in vivo or a
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`composition for therapy of the superficial mycosis, deep mycosis or
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`bacterial infection containing the compound; an antimicrobial agent
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`having the true effect selected by means of showing a statistically
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`significant effect; furthermore, an antimicrobial agent having an
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`excellent eradication effect for microorganism in vivo, which is selected
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`by means of clarifying the pure antimicrobial activity thereof; or an
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`antimicrobial agent of the complete cure type without relapse. A
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`concrete example is a compound which shows a therapeutic effect like
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`KP- 103 mentioned below.
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`The antimicrobial agent obtained in such manner can be
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`used as a drug composition, the drug composition in order to sterilize a
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`microorganism. In other words, it comes to be a drug composition
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`which cures disease such as mycosis completely, and prevents a
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`relapse.
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`EXAMPLE
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`The present invention is further explained in details based
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`on the Examples hereinafter, but is not limited thereto.
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`Millipore Filter (made by Millipore Corporation, HA,
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`diameter 47 mm, 0.45 p m) was placed on Brain-Heart-infusion agar
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`medium (available from Nissui Pharmaceutical Co., LTD.), and 10~ cells
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`of microcondium of Trichophyton mentagrophytes KD-04 strain were
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`applied thereon. The cultivation was carried out at 30°C under 17 %
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`of C02 for 7 days. After the cultivation, an appropriate amount of
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`physiological saline containing 0.05 % of Tween 80 was dropped on the
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`filter and arthroconidia were collected using a platinum loop. After a
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`hyphal mass was removed with a sterile gauze, the number of
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`arthroconidia in the arthroconidia suspension was calculated by
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`hemocytometer to adjust in concentration of 1 x 108 arthroconidia I ml
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`to obtain a fungal inocula.
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`A guinea pig model of interdigital type of tinea pedis was
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`prepared according to the method of Arika et al (Antimicrobial Agents
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`and Chemotherapy, 36: 2523-2525, 1992). Concretely, in two hind
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`foots of male Hartley strain guinea pigs of 7 weeks age, the interdigital
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`skin was lightly abraded with sandpaper. A paper disc (AAdisc
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`available from Whatmen International Ltd cut in 8 x 4 ram) moisten
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`with the above-mentioned solution of the inoculated organism was
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`applied onto the region between the interdigital toes of the hind feet
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`and fixed using Self-adhering-Foam Pad (Restone 1560M; available
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`from 3M) and adhesive stretch bandage (ELASTPORE; available from
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`Nichiban Co., Ltd). The paper disc and the bandage were removed
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`seven days after the infection.
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`Preparation of drug-solution and topical treatment for guinea pig model
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`of interdigital type of tinea pedis
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`A marketed 1% lanoconazole solution (commercial name:
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`Astat (trade name) solution)
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`and a solution in which KP-103
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`synthesized in Development
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`Research Laboratories of Kaken
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`Pharmaceutical Co., Ltd.
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`((2R, 3 R)-2-(2,4-difluorophenyl)-3-(4-
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`methylenepiperidine- 1-yl)- 1-( IH- 1,2,4-triazole- 1-yl) butane-2-ol, the
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`compound as described in Example 1 in WO 94/26734) was solved in
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`a concentration of 1% in polyethylene glycol #400 : ethanol (75 : 25
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`v/v) mixture were used as test substance. Each solution in an amount
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`of 0.1 ml was applied to the plantar skin once a day from 10 days after
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`the infection for 10 days.
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`COMPARATIVE EXAMPLE 1
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`Conventional method for evaluating drug effect
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`The conventional method was described as follows. For
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`the infected control group without an application of the drug, the KP-
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`103-treated group and the lanoconazole-treated group, 10 guinea pigs
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`(hereinafter referred to "animal") were employed, respectively. Animals
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`of each group were sacrificed two days after and 30 days after the last
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`treatment. Their two hind feet were excised and wiped with the
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`cotton swab containing alcohol sufficiently. A skin of whole sole was
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`excised and cut into 15 skin pieces in total including 12 skin pieces
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`from plantar parts and 3 skin pieces from an interdigital part. Each
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`skin pieces were placed on 20 ml of Sabouraud dextrose agar medium
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`(available from Difco laboratories) containing 50 /~ g of
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`chloramphenicol (available from Wako Pure Chemical Industries, Ltd .),
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`100 /~ g of gentamicin (available from Schering-Plough Corporation),
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`50 /~ g of 5-fluorocytosine (available from Wako Pure Chemical
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`Industries, Ltd.) and 1 mg of cycloheximide (available from Nacalai
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`Tesque, Inc.) per ml. An antibiotic substance added to the medium was
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`set to a condition which enables bacteria not to grow and which
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`enables fungi to grow without problem. After 10 days of cultivation at
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`30°C, the result is described as "fungus-neg