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`Attorney Docket No. 700938-052220-DIV
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`First Inventor
`
`Yoshiyuki Tatsumi
`
`Title
`
`i See Addendum
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`Express Mail Label No.
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`EL 934997148 US
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`0
`P" ¢o
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`f I~"~ ’.a,
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`, 1--~ ~)
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`od"- ~ ~--
`c~J
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`O
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`Reduction Act of 1!
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`ACRUX DDS PTY LTD. et el.
`EXHIBIT 1006
`IPR Petition for
`U.S. Patent No. 7,214,506
`
`1 of 396
`
`

`
`Under the Paperwork Reduction Act of 1995t no persons are required to,
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`FEE TRANSMITTAL
`for FY 2003
`
`Effective 01/01/2003. Patent fees are subject to annual news/on.
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`--I Applicant claims small entity status. See 37 CFR 1.27
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`~,TOTAL AMOUNT OF PAYMENT
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`($) 770.00
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`PTO/SB/17 (08-03)
`Approved for use through 07/3112006. OMB 0651-0032
`U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE
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`Complete if Known
`TBA
`Herewith
`Yoshiyuki Tatsumi
`TBA
`TBA
`700938-052220-DIV
`
`Application Number
`
`Filing Date
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`First Named Inventor
`
`Examiner Name
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`Art Unit
`
`Attorney Docket No.
`
`¯
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`METHOD OF PAYMENT (check all that apply)
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`FEE CALCULATION (continued)
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`Order
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`Account
`Number
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`Name
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`50-0850
`
`NIXON PEABODY LLP
`
`[ ~_je Commlssioner is authorized to: (check all that apply)
`
`Charge fee(s) indicated below [] Credit any overpayments
`
`3. ADDITIONAL FEES
`Large Entity Small Entity
`Fee Fee Fee Fee Description
`Fee
`Code (S)
`Code ($)
`1051 130
`
`2051 65
`
`Surcharge - late filing fee or oath
`
`1052 50
`
`1053 130
`
`1812 2,520
`
`2052 25
`
`Surcharge - late provisional filing fee or
`cover sheet
`1053 130 Non-English specification
`1812 2,520 For filing a request for ex parte reexamination
`
`Fee Paid
`
`1804 920* Requesting publication of SIR prior to
`1804 920*
`Charge any additional fee(s) ...... ...... ~ "_% ; ;-~:~:~,~~_. "_’ :r --rr "" ~--
`Examiner action
`
`]Charge fee(s)
`
`indicated
`
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`
`for the
`
`filing fee
`
`to the above-identified deposit account.
`FEE CALCULATION
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`Code ($)
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`Fee Description
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`1001 750
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`2001 375
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`1002 330
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`2002 165
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`Utility filing fee j
`Design filing fee
`
`1003 520
`
`2003 260
`
`Plant filing fee
`
`1004 750
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`2004 375
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`Fee Paid
`
`1805 1,840"
`
`1805
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`1,840*
`
`Requesting publication of SIR after
`Examiner action
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`1251
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`110
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`1252
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`410
`
`1253
`
`930
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`1254 1,450
`
`1255 1,970
`
`Extension for reply within first month
`2251
`55
`2252 205 Extension for reply within second month
`2253 465 Extension for reply within third month
`2254 725 Extension for reply within fourth month
`Extension for reply within fifth month
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`2255 985
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`1401
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`320
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`2401 160 Notice of Appeat
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`1402 320
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`2402 160
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`Filing brief in support of an appeal
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`1403 280
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`2403 140 Request for oral hearing
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`Provisional filing fee
`SUBTOTAL(I) J($)770.00 J
`2. EXTRA CLAIM FEES FOR UTILITY AND REISSUE
`Fee from
`T ots, C,aims
`Fee Paid 1
`r
`1 20..=
`4
`1
`I 3"° Iml ×1
`1=1
`1
`I 1=1
`
`1005 160
`
`2005 80
`
`Extra Claims
`
`Independent
`[
`Claims
`Multiple Dependent
`
`-
`
`=
`
`Large Entity Small Entity
`Fee Fee
`Fee Fee
`Code ($)
`Code ($)
`2202 9
`
`1202 18
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`Fee Descdption
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`Claims in excess of 20
`
`1451 1,510
`
`1451 1,510 Petition to institute a public use proceeding
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`1452
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`110
`
`2452
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`55 Petition to revive - unavoidable
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`1453 1,300
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`2453 650 Petition to revive - unintentional
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`1501 1,300
`1502 470
`
`650 Utility issue fee (or reissue)
`2501
`2502 235 Design issue fee
`
`1503 630
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`2503 315 Plant issue fee
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`1460
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`130
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`1460
`
`130 Petitions to the Commissioner
`
`1807
`
`50
`
`1807
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`50 Processing fee under 37 CFR 1.17(q)
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`1806 180
`
`1806
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`180 Submission of Information Disclosura Strut
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`8021
`
`40
`
`8021
`
`Recording each patent assignment per
`40 property (times number of properties)
`
`1809 750
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`2809 375 Filing a submission after final rejection
`(37 CFR 1.129(a))
`
`.
`
`1201
`
`84
`
`1203 280
`
`2201 42
`
`independent daims in excess of 3
`
`2203 140
`
`Multiple dependent claim, if not paid
`
`1810 750
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`1204 84
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`2204 42
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`** Reissue independent claims
`over original patent
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`2810 375 For each additional invention to be
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`1801
`
`750
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`2801
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`375 Request for Continued Examination (RCE)
`
`1205
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`18
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`2205 9
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`"" Reissue claims in excess of 20
`and over original patent
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`1802 900
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`1802 900 Request for expedited examination
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`0.001
`SUBTOTAL (2) kS)
`*’or number ~viousl~ paid, if greater; For Reissues, see above
`
`Other fee (specify)
`*Reduced by Basic Filing Fee Paid
`
`~SUBMITI’ED BY
`
`Name (/:WnttType)
`
`Ronald I. Eisenstein
`
`RegistneUon No. J
`(Atlomey/A,qent) 30~628
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`SUBTOTAL (3) ($)
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`including gathering, preparing, and submitting the completed application form to the USPTO. Time will vary depending upon the individual case. Any comments on
`the amount of time you require to complete this form and/or Suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent and
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`2 of 396
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`

`
`Practitioner’s Docket No. 700938-052220-DIV
`
`PATENT
`
`IN TFIE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`In re application of."
`
`Tatsumi et al.
`
`Application No.:
`
`TBA (divisional of 10/031,929)
`
`Group No.:
`
`To be Assigned (1651)
`
`Filed:
`
`For:
`
`Herewith
`
`Examiner:
`
`To be assigned (Srivastava,
`
`METHOD FOR DETECTING PATHOGENIC MICROORGANISM AND
`ANTIMICROBIAL AGENT, METHOD FOR EVALUATING EFFECT OF
`ANTIMICROBIAL AGENT, AND ANTIMICROBIAL AGENT
`
`Kailash C.)
`
`Mail Stop Patent Application
`Commissioner for Patents
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`EXPRESS MAH, CERTH~’ICATE
`
`"Express Mail" Label Number: EL 934997148 US
`Date of Deposit: October 14, 2003
`
`I hereby state that the following attached papers and fees:
`
`°
`
`2.
`3.
`4.
`5.
`6.
`7.
`8.
`9.
`
`10.
`11.
`12.
`
`Check: $770.00;
`Return receipt postcard;
`Certificate of Express Mail (1 pg.) EL 934997148 US;
`Fee Transmittal for FY2003 (1 pg.);
`Utility Patent App. Transmittal (PTO/SB05) (2 pp.);
`Copy of Dec. & POA filed in parent case (4 pp.);
`Preliminary Amendment (6 pp.);
`Application Data Sheet (3 pp.);
`Application (44 pp.): Spec: 34 pp.; 5 of Cls (Cls 1-17) and 1 pg. abstract; 4 pp. of
`drawings;
`Information Disclosure Statement (3 pp.), including Refs. AA-AC and BA-BC
`Assi~ment Recordation on Cover Sheet (2 pp.);
`Executed Assignment (3 pp.)
`
`are being deposited with the United States Postal Service "Express Mail Post Office to Addressee" service
`under 37 C.F.R. section 1.10, on the date indicated above and is addressed to Mail Stop Patent Application,
`Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450.
`
`Linda M. Ginsberg
`
`person mailing paper or
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`Bi317341.1
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`3 of 396
`
`

`
`ADDENDUM
`
`Title:
`
`METHOD FOR DETECTING PATHOGENIC MICROORGANISM AND ANTIMICROBIAL
`AGENT, METHOD FOR EVALUATING EFFECT OF ANTIMICROBIAL AGENT, AND
`ANTIMICROBIAL AGENT
`
`BOS1317472.1
`
`4 of 396
`
`

`
`_
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`DESCRIPTION
`
`METHOD FOR DETECTING PATHOGENIC MICROORGANISM AND
`
`ANTIMICROBIAL AGENT, METHOD FOR EVALUATING EFFECT OF
`
`ANTIMICROBIAL AGENT, AND ANTIMICROBIAL AGENT
`
`TECHNICAL FIELD
`
`The present invention relates to a method for detecting
`
`pathogenic microorganism, method for evaluating an effect of an
`
`10
`
`antimicrobial agent on pathogenic microorganism and a method for
`
`detecting an antimicrobial agent. The present invention also relates to
`
`an antimicrobial agent and a therapeutic agent for onychomycosis,
`
`which are obtained according to the above-mentioned method for
`
`evaluating the drug effect.
`
`15
`
`BACKGROUND ART
`
`A method for evaluating a drug effect with an animal model is
`
`needed in order to explore a novel antimicrobial agent (also hereinafter
`
`referred to "drug~). Further, a method enabling a drug effect to be
`
`2O
`
`evaluated with accuracy is needed because of grate importance in view
`
`of predicting a clinical therapeutic efficiency thereof.
`
`Historically, an experimental dermatophytosis model that
`
`back, planta and interdigital of a guniea pig have been infected with
`
`Trichophyton mentagrophytes has been used in order to evaluate an
`
`25
`
`effect of an antifungal agent on dermatophytosis. Such animal models
`
`have been already employed to develop some antifungal agent. The
`
`evaluation of the effect of such antifungal agent carried out by applying
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`5 of 396
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`

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`¯
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`_
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`the antifungal agent to the infected animal, by excising the skin after the
`
`certain period of time to cut into plural small pieces, by cultivating the
`
`skin pieces on the medium, and by counting the number of pieces
`
`wherein no growth of fungus is seen or the number of animals or feet
`
`wherein no growth of fungus is seen in all skin pieces, as an indicator
`
`(Antimicrobial Agents and Chemotherapy, 36: 2523-2525, 1992, 39:
`
`2353-2355, 1995). Hereinafter, the conventional method for evaluating
`
`the drug effect is referred to as ~the conventional method".
`
`Although the drug having a potent activity against
`
`10
`
`Trichophyton in vitro such as lanoconazole or amorolfine has been
`
`marketed in these days, an improvement of cure rate in a clinical use is
`
`hardly seen. As a main reason thereof, a relapse that since fungus in
`
`the skin is not completely killed after a treatment, the fungus grow again
`
`is pointed.
`
`15
`
`In also animal experiments, when an effect of lanoconazole
`
`on guniea pig models of tinea pedis was evaluated using the
`
`conventional method, though ~fungus-negative" was observed in all feet
`
`out of 20 feet 2 days after the last treatment, a relapse was observed in
`
`11 out of 20 feet 30 days after the last treatment, and no correlation was
`
`seen between the effect 2 days after the last treatment and the effect 30
`
`days after the last treatment (36th Interscience Conference on
`
`Antimicrobial Agents and Chemotherapy, New Orleans, Louisiana, 1996,
`
`Abstr. F80).
`
`As
`
`a reason thereof, there were followings.
`
`Since
`
`25
`
`lanoconazole
`
`have very potent antitrichophyton activit~y in vitro,
`
`lanoconazole persisted in the skin 2 clays after the last treatment in the
`
`concentration wherein the sterilization effect was shown. Therefore,
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`6 of 396
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`
`3
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`when the skin is excised and cultivated on the medium to detect fungus,
`
`the lanoconazole remaining in the skin is diffused in the medium, and
`
`therefore, no fungus was detected due to prevention of the growth
`
`regardless of the presence of viable fungus in the excised skin. On the
`
`5
`
`other hand, since the concentration of the drug remained in the skin is
`
`reduced 30 days after the last treatment, fungus in the skin can grow
`
`again and can be detected by culture study.
`
`According to this hypothesis, it is ascertained that the drug
`
`remain in the skin through the inhibition of the growth of fungus around
`
`the skin blocks completely, when the lanoconazole-treated skin blocks
`
`were located and cultivated on the medium which contains
`
`dermatophytes.
`
`Therefore, it became to clear that the conventional method
`
`has the problem that the drug effect can not be accurately evaluated,
`
`15
`
`because the apparent therapeutic effect need to be evaluated after
`
`removing the drug remaining in the skin.
`
`Meanwhile, a kind of mycosis, delmatophytosis, is the
`
`superficial dermatosis which is caused by dermatophyte parasitizing the
`
`keratin such as skin (stratum corneum), the nail and the hair. In
`
`9.0
`
`particular, tinea unguium forined in the nail is known as the intractable
`
`disease among dermatomycoses based on dermatophytoses, and is
`
`accompanied by symptom such as opacity, tylosis, destruction and
`
`deformation of nail plate. Now the oral preparation (such as
`
`griseofulvin or terbinafine) is used for the treatment of such tinea
`
`25
`
`unguium. However, there are many cases where the patient stops
`
`taking the drug or that takes the drug irregularly, since they have to take
`
`the drug for a long period, for example at least a half a year in order to
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`7 of 396
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`4 -
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`completely cure tinea unguium. It is thought that this is a main cause
`
`of difficulty of curing tinea unguium completely. Furthermore, by
`
`taking the drug for a long period, griseofulvin has the problem of side
`
`effects on internal organ (gastrointestinal disorder, hepatotoxicity) and
`
`5
`
`hepatotoxicity is reported as the side effect in terbinafine. Therefore, in
`
`order to improve the compliance of the patient it is desired to develop a
`
`topical preparation which cure tinea unguium for a short period and has
`
`less the systemic side effect than the oral preparation.
`
`However, in case of the simple application on nail plate_with
`
`10
`
`the current antifungal agent for topical use, the antifungal effect on
`
`fungus in the nail was not seen, because the drug could not sufficiently
`
`permeate the thick keratin in nail plate (Markus Niewerth and Hans C.
`
`Korting, Management of Onychomycoses, Drugs, 58: 283-296, 1999).
`
`In addition, the therapeutic effect of a topical preparation of
`
`15
`
`antifungal agent on the experiment model of trichophytosis can not be
`
`evaluated using the conventional method as mentioned above. This
`
`may be a reason why the drug effect on the guniea pig model of tinea
`
`unguium has been hardly reported.
`
`20
`
`DISCLOSURE OF INVENTION
`
`The present invention has been accomplished based on
`
`findings that it is desirable that an effect of antimicrobial agent such as
`
`particularly antifungal agent is evaluated after removing a drug
`
`remaining in the infected site after treatment of an animal or a
`
`25
`
`biosample such as skin with the pathogenic microorganism. An object
`
`of the present invention is to provide a novel method for evaluating the
`
`effect of the antimicrobial agent and the antimicrobial agent obtained
`
`8 of 396
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`

`
`5 -
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`according to the method for evaluating the drug effect. In detail, the
`
`present invention provides the method for detecting the viable
`
`pathogenic microorganism in the above-mentioned infected site of the
`
`animal or the biosample with the pathogenic microorganism after
`
`removing the antimicrobial agent which has been administered to the
`
`animal or the biosarnple, and the method for evaluating the effect of
`
`antimicrobial agent which can accurately evaluate the effect of the
`
`antimicrobial agent without the influence of the antimicrobial agent
`
`remaining in the infected site of the animal or the biosample with a
`
`10
`
`pathogenic microorganism. In addition, the present invention provides
`
`the antimicrobial agent obtained according to the above-mentioned the
`
`method for evaluating the drug effect, and the detecting method of the
`
`antimicrobial agent which comprises detecting the existing
`
`antimicrobial agent in the infected site of the animal or the biosample
`
`15
`
`with the pathogenic microorganism, to which the antimicrobial agent is
`
`administered.
`
`In more detail, according to the present invention a detection
`
`of a pathogenic microorganism and an evaluation of an effect of an
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`antimicrobial agent can be carried out by infecting an animal or a
`
`2O
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`biosarnple with the pathogenic microorganism, administering the
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`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
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`pathogenic microorganism in the infected site with the pathogenic
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`25
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`microorganism.
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`According to the present invention a detection of an existing
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`antimicrobial agent can be carried out by infecting an animal or a
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`biosample with a pathogenic microorganism, administering the
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`antimicrobial agent comprising a compound having an antimicrobial
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`effect or a composition thereof before or after the infection, then excising
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`the infected site with the pathogenic microorganism, placing and
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`cultivating it on a medium containing the pathogenic microorganism,
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`and thereafter observing a growth inhibition of the pathogenic
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`microorganism around the infected site with the pathogenic
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`microorganism.
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`Additionally, an object of the present invention is to provide
`
`10
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`the evaluation method of a drug which enables the effect of an antifungal
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`agent to accurately evaluate in a guinea pig model of tinea unguium.
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`Another object of present invention is to provide a therapeutic agent for
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`onychomycosis which exhibits the effect on tinea unguium by topical
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`applicartion and which is capable of Curing tinea unguium shorter
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`16
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`period than that of the marketed oral preparation due to good
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`permeability, good retention capacity and conservation of high activity in
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`nail plate as well as the potent antifungal activity thereof based on the
`
`present invention. Another object of the present invention is to provide
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`the effective therapeutic agent for onychomycosis exhibiting no side
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`effect even if therapeutically effective amounts of it are administered
`
`sufficiently.
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`More concretely, the present invention provides a therapeutic
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`agent for onychomycosis containing a compound having a formula (1):
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`25
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`/ (CH2)m~~=~R1
`--N
`R2
`
`(CH2).
`
`(I)
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`10 of 396
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`O
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`_
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`¯ Wherein RI and R2 are the same or different and are hydrogen atom, CI_6
`
`alkyl group, a non-substituted aryl group, an aryl group substituted
`
`with 1 to 3 substituents selected from a halogen atom, trifluoromethyl
`
`group, nitro group and CI.6 alkyl group, C2_a alkenyl group, C2_6 alkinyl
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`group, or Cv_12 aralkyl group,
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`m is 2 or 3,
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`n is 1 or 2,
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`or a salt thereof as active ingredient.
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`In addition, "presence" includes the mean of "remaining~.
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`10
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`BRIEF DESCRIPTION OF DRAWINGS
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`Fig. 1 is a color copy of a photograph to identify the agent
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`remaining in the skin which is previously evaluated by the conventional
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`method in the detecting method of the antimicrobial agent five days after
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`15
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`last treatment in the present invention. The note (a) shows the infected
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`control group, (b) the KP-103-treated group, (c) the lanoconazole-treated
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`group.
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`Fig. 2 is a color copy of photograph to identify agent
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`remaining in the skin which is previously evaluated by the detecting
`
`2O
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`method of the antimicrobial agent five days after last treatment in the
`
`present invention. The note (a) shows the infected control group, (b) the
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`KP-103-treated group, (c) the lanoconazole-treated group.
`
`Fig. 3 is a graph showing a distribution of the number of
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`fungal cells in the nail of a guinea pig model of tinea unguium in each
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`25
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`treated group according to the evaluation method of the drug effect in
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`the present invention.
`
`Fig. 4 is a graph showing a distribution of the number of
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`8
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`fungal cells in the skin of a guinea pig model of tinea pedis in each.
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`treated group according to the evaluation method of the drug effect in
`
`the present invention.
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`5
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`BEST MODE FOR CARRYING OUT THE INVENTION
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`As an animal employed in the present invention, there
`
`includes mammal such as mice, rat, guinea pig or rabbit. As a
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`biosample, there includes a skin of back or planta, a nail or the like,
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`which is taken from such animal.
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`10
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`A method for infecting such animal or biosample with a
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`pathogenic microorganism includes an inoculation percutaneouslly,
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`orally, intravenously, transbronchially, transnasally or intraperitoneally.
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`Especially in case of the skin, there includes a method for inoculating it
`
`0
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`on the skin, a method for inoculating on the exposed demis, the closed
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`patch method, intracutaneous injection or the like. Incase of the nail,
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`there includes a method for inoculating on nail, a method in which a
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`skin of the animals’ foot is infected by the above-mentioned infecting
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`method to the skin, and thereafter the infection is moved into the nail by
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`leaving it for several months.
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`20
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`The term "skin" means a tissue including the three layers
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`being epidermis, demis and subcutaneous tissue, accompanied by pilus
`
`(hair), nail, glandulae sebaceae, glandulae sudoriferae and glandulae
`
`mammaria as appendages. The epidermmis is separated five layers
`
`being stratum corneum, stratum lucidum, granulosum epidermidis,
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`25
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`stratum spinosum, and stratum basale from surface in order. The
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`stratum comeum, the stratum lucidum and the stratum granulosum
`
`epidermidis is referred to as a stratum corneum in a broad sense.
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`_
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`Herein, keratin sbustance means a part of the above-mentioned stratum
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`corneum.
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`The term "nail" includes nail plate, nail bed, nail matrix,
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`further side nail wall, posterial nail wall, eponychium and hyponychium
`
`which make up a tissue around thereof.
`
`In the present invention,
`
`the tei m "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
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`10
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`including aerobic Gram-negative bacillus and coccus such as
`
`Pseudomonas and Neisseriaceae species; facultative anaerobic Gram-
`
`negative bacillus such as Eschrichia, Salmonella and Enterobacter
`
`species; Gram-positive coccus such as Staphylococcus and
`
`Streptococcus species. The other examples of microorganism are fungi
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`15
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`including Hyphomycetes such as Trichophyton, Microsporum and
`
`Epidermophyton species; Blastomycetes such as Candida and
`
`MalasSezia; Ascomycetes such as AspergiIIus species; Zygomycetes such
`
`as Mucor species; and variants thereof. Examples of such variants are
`
`resistant strain which naturally obtains drug resistance; auxotrophic
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`2O
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`mutation strain which comes to have nutritious dependency; artificial
`
`mutation strain which is artificially mutated by treatment with
`
`mutagenic agent; and the like.
`
`Mycosis means a disease which is caused by invading and
`
`proliferating in the tissue of human or animal. Usually, mycosis is
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`25
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`broadly divided into superficial mycosis and deep mycosis. A seat of
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`the disease lie in the skin. or visible mucosa in case of the former, in
`
`viscus, central nervous system, subcutaneous tissue, muscle, born or
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`10 -
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`articulation in case of the latter. Chief example of superficial mycosis is
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`dermatophytosis which is caused by infecting with dermatophyte such
`
`as Trichophyton, Microsporum and Epidermophyton species, including
`
`three disease, tinea, tinea favosa and tinea imbricata. Tinea may be
`
`conventionally employed a synonymous with dei-matophytosis. In
`
`addition, dermatophyte belonging to Trichophyton species is referred
`
`usually to as trichophytosis.
`
`In the present invention, an antimicrobial agent means a
`
`compound having an antimicrobial effect or a composition containing
`
`10
`
`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
`
`15
`
`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
`
`E5
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`where the antimicrobial agent is administered after the infection with
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`microorganism, meanwhile, a effect of the antimicrobial agent protecting
`
`from the infection and the retention capacity thereof can be evaluated in
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`case where the infection with microorganism is carried out after the
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`administration of the antimicrobial agent. In order to evaluating the
`
`retention capacity of the antimicrobial agent, the evaluation can be
`
`carried out with varying the period until infection with microorganism
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`,5
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`from the administration of the antimicrobial agent. .
`
`In the present invention, it is preferable to use dialysis or
`
`ultra filtration for removing the antimicrobial agent in view point of the
`
`usefulness, but not limited thereto as long as a microorganism to be a
`
`detecting target or a microorganism used in the present evaluation
`
`10
`
`method and the like is not affected by it.
`
`In dialysis, a marketed dialysis membrane made of cellulose
`
`is convenient. A membrane made of the other material can be used
`
`without problem, as long as the microorganism to be the detecting target
`
`or the microorganism used in the present evaluation method and the
`
`15
`
`like can not be passed, and the antimicrobial agent can be passed
`
`through it. Since sizes of most fungi and bacteria are at least 0.2 l~m, it
`
`is preferable to use the membrane having less than 0.2 l~m of the pore
`
`size, particularly it is suitable to use dialysis membrane having
`
`fractional molecular weight of 1,000 to 50,000.
`
`2O
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`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
`
`25
`
`nail,- organ or the like as well as the skin, the antimicrobial agent can be
`
`efficiently removed. Usually, since there is the case where it takes
`
`longer time dialysis to remove the antimicrobial agent from nai! than.
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`- 12
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`skin, the following treatment with digestive enzyme may be carried out
`
`before removing it in order to enhance the removal effect.
`
`Dialysis conditions depend on variety, dose concentration,
`
`dose term and the drug holidays (the term until evaluation from last day
`
`of treatment) of an antimicrobial agent. Therefore, it is preferable to
`
`previously investigate the dialysis conditions enabling the antimicrobial
`
`agent to be removed from the treated skin about individual cases_using
`
`the following detecting method of the existing antimicrobial agent in the
`
`infected site with a microorganism in the present invention (hereinafter
`
`10
`
`referred to "the present method for detecting an agent") to adjust the
`
`conditions appropriately.
`
`Whether an antimicrobial agent has been removed can be
`
`easily determined using the following method.
`
`The present method for detecting an agent is carried out by
`
`15
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`placing and cultivating the infected site with a microorgani