BRENDA K. SEAT, Esq].
`
`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
`
`ACRUX DDS PTY LTD. et al.
`EXHIBIT 1002
`IPR Petition for
`U.S. Patent No. 7,214,506
`
`1 of 61
`
`
`
`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
`
`ACRUX DDS PTY LTD. et al.
`EXHIBIT 1002
`IPR Petition for
`U.S. Patent No. 7,214,506
`
`1 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`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
`
`2 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`[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
`
`3 of 61
`
`
`
`[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.
`
`2 -
`
`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
`
`1
`
`1
`
`4 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`[Document Name]
`
`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.
`
`5 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`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.
`
`6 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`- 3
`
`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
`
`7 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`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
`
`8 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`5 -
`
`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.
`
`9 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`6 -
`
`Herefrom, in the case of evaluating a drug effect on
`
`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]
`
`10 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`7 -
`
`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
`
`11 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`8 -
`
`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
`
`12 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`9 -
`
`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
`
`13 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`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.
`
`14 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`11
`
`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
`
`15 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`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
`
`16 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`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
`
`17 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`14 -
`
`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
`
`Sabouraud medium, modified Sabouraud medium, Czapek agar
`
`medium, Potato dextrose agar medium or the like. On the other hand,
`
`in the case of bacteria, example of the medium is Mueller Hinton
`
`medium, modified Mueller Hinton medium, Heart Infusion agar
`
`medium, Brain Heart Infusion agar medium, normal agar medium or
`
`the like.
`
`A reacting temperature is 10 to 40°C, preferably 20 to 40°C.
`
`A microorganism may be cultivated with standing during a sufficient
`
`time when the microorganism can be grown, for example, 1 to 20 days
`
`for fungi, 1 to 5 days for bacteria.
`
`The present evaluation method can be utilized as an
`
`evaluation method of a drug effect in exo vivo which comprises
`
`infecting a skin, a nail excised from an animal body with a
`
`microorganism, thereafter administering an antimicrobial agent as a
`
`test compound, then removing the antimicrobial agent and detecting
`
`and determining quantity of the microorganism inthe 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
`
`18 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`15 -
`
`effect of a therapeutic agent for superficial mycosis. That is to say, it
`
`is possible to evaluate an effect of a therapeutic agent for deep mycosis
`
`or an antibacterial agent by means of administering an antimicrobial
`
`agent to an animal infected with a microorganism such as a fungus or
`
`a bacterium by inoculating percutaneously, orally, intravenously,
`
`transbronchially, transnasally, intraperitoneally, then obtaining
`
`biosample such as skin, kidney, lung or brain, and detecting the viable
`
`microorganism in the biosample in which 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 test of significance is carried out about the
`
`number of microorganisms in the infected site with the microorganism
`
`for the treated group with drug and for the reference infected group
`
`using a statistical method such as Kruskal-Wallis Test, and thereby a
`
`quantitative comparison between the groups can be done.
`
`As the antimicrobial agent obtained by the present
`
`evaluation method, there is an antimicrobial agent comprising a
`
`compound having an eradication effect for microorganism in vivo or a
`
`composition for therapy of the superficial mycosis, deep mycosis or
`
`bacterial infection containing the compound; an antimicrobial agent
`
`having the true effect selected by means of showing a statistically
`
`significant effect; furthermore, an antimicrobial agent having an
`
`excellent eradication effect for microorganism in vivo, which is selected
`
`by means of clarifying the pure antimicrobial activity thereof; or an
`
`19 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`antimicrobial agent of the complete cure type without relapse. A
`
`concrete example is a compound which shows a therapeutic effect like
`
`KP- 103 mentioned below.
`
`The antimicrobial agent obtained in such manner can be
`
`used as a drug composition, the drug composition in order to sterilize a
`
`microorganism. In other words, it comes to be a drug composition
`
`which cures disease such as mycosis completely, and prevents a
`
`relapse.
`
`EXAMPLE
`
`The present invention is further explained in details based
`
`on the Examples hereinafter, but is not limited thereto.
`
`Millipore Filter (made by Millipore Corporation, HA,
`
`diameter 47 mm, 0.45 p m) was placed on Brain-Heart-infusion agar
`
`medium (available from Nissui Pharmaceutical Co., LTD.), and 10~ cells
`
`of microcondium of Trichophyton mentagrophytes KD-04 strain were
`
`applied thereon. The cultivation was carried out at 30°C under 17 %
`
`of C02 for 7 days. After the cultivation, an appropriate amount of
`
`physiological saline containing 0.05 % of Tween 80 was dropped on the
`
`filter and arthroconidia were collected using a platinum loop. After a
`
`hyphal mass was removed with a sterile gauze, the number of
`
`arthroconidia in the arthroconidia suspension was calculated by
`
`hemocytometer to adjust in concentration of 1 x 108 arthroconidia I ml
`
`to obtain a fungal inocula.
`
`A guinea pig model of interdigital type of tinea pedis was
`
`20 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`prepared according to the method of Arika et al (Antimicrobial Agents
`
`and Chemotherapy, 36: 2523-2525, 1992). Concretely, in two hind
`
`foots of male Hartley strain guinea pigs of 7 weeks age, the interdigital
`
`skin was lightly abraded with sandpaper. A paper disc (AAdisc
`
`available from Whatmen International Ltd cut in 8 x 4 ram) moisten
`
`with the above-mentioned solution of the inoculated organism was
`
`applied onto the region between the interdigital toes of the hind feet
`
`and fixed using Self-adhering-Foam Pad (Restone 1560M; available
`
`from 3M) and adhesive stretch bandage (ELASTPORE; available from
`
`Nichiban Co., Ltd). The paper disc and the bandage were removed
`
`seven days after the infection.
`
`Preparation of drug-solution and topical treatment for guinea pig model
`
`of interdigital type of tinea pedis
`
`A marketed 1% lanoconazole solution (commercial name:
`
`Astat (trade name) solution)
`
`and a solution in which KP-103
`
`synthesized in Development
`
`Research Laboratories of Kaken
`
`Pharmaceutical Co., Ltd.
`
`((2R, 3 R)-2-(2,4-difluorophenyl)-3-(4-
`
`methylenepiperidine- 1-yl)- 1-( IH- 1,2,4-triazole- 1-yl) butane-2-ol, the
`
`compound as described in Example 1 in WO 94/26734) was solved in
`
`a concentration of 1% in polyethylene glycol #400 : ethanol (75 : 25
`
`v/v) mixture were used as test substance. Each solution in an amount
`
`of 0.1 ml was applied to the plantar skin once a day from 10 days after
`
`the infection for 10 days.
`
`COMPARATIVE EXAMPLE 1
`
`Conventional method for evaluating drug effect
`
`21 of 61
`
`
`
`Certified Translation by Shinshu Services, Inc.
`
`The conventional method was described as follows. For
`
`the infected control group without an application of the drug, the KP-
`
`103-treated group and the lanoconazole-treated group, 10 guinea pigs
`
`(hereinafter referred to "animal") were employed, respectively. Animals
`
`of each group were sacrificed two days after and 30 days after the last
`
`treatment. Their two hind feet were excised and wiped with the
`
`cotton swab containing alcohol sufficiently. A skin of whole sole was
`
`excised and cut into 15 skin pieces in total including 12 skin pieces
`
`from plantar parts and 3 skin pieces from an interdigital part. Each
`
`skin pieces were placed on 20 ml of Sabouraud dextrose agar medium
`
`(available from Difco laboratories) containing 50 /~ g of
`
`chloramphenicol (available from Wako Pure Chemical Industries, Ltd .),
`
`100 /~ g of gentamicin (available from Schering-Plough Corporation),
`
`50 /~ g of 5-fluorocytosine (available from Wako Pure Chemical
`
`Industries, Ltd.) and 1 mg of cycloheximide (available from Nacalai
`
`Tesque, Inc.) per ml. An antibiotic substance added to the medium was
`
`set to a condition which enables bacteria not to grow and which
`
`enables fungi to grow without problem. After 10
Accessing this document will incur an additional charge of $.
After purchase, you can access this document again without charge.
Accept $ Charge
Still Working On It
This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.
Give it another minute or two to complete, and then try the refresh button.
A few More Minutes ... Still Working
It can take up to 5 minutes for us to download a document if the court servers are running slowly.
Thank you for your continued patience.
This document could not be displayed.
We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.
Your account does not support viewing this document.
You need a Paid Account to view this document. Click here to change your account type.
Your account does not support viewing this document.
Set your membership
status to view this document.
With a Docket Alarm membership, you'll
get a whole lot more, including:
- Up-to-date information for this case.
- Email alerts whenever there is an update.
- Full text search for other cases.
- Get email alerts whenever a new case matches your search.
One Moment Please
The filing “” is large (MB) and is being downloaded.
Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.
Your document is on its way!
If you do not receive the document in five minutes, contact support at support@docketalarm.com.
Sealed Document
We are unable to display this document, it may be under a court ordered seal.
If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.
Access Government Site
