Journa! of pharmaceutical scnences‘UM)
`v. 96. no‘ 10 (Oct 2007)
`Genet‘ai Collection
`W'I J0829
`2007-10—1 8 M5
`
`: Pharm
`Sci
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`Volume 96 Number 1 0 October 2007
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`
`
`American Pharmacists Asflcjatifl'
`Humid...‘ .n ..
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`..‘
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`APhA
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`Articles Published Onllne In Wiley InterScience, 11 May 2007 - 14 August 2007
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`Page 1 of 17
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`Acrux V. Kaken
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`IPR2017-0019O
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`Kaken Exhibit 2048
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`

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`JOURNAL OF
`
`Pharmaceutical
`Seances
`
`as?
`,KSJ A Publication of the American Pharmacists Association
`AN‘IA
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`«65-.“my.”41
`1'
`Egg A Publication of the Board of Pharmaceutical
`'3
`e
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`Federation
`
`9) sans
`mnerican Attenuation of
`Pharmate utical Scientists
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`Published in Cooperation with the American
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`Page 2 of 17
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`

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`JOURNAL OF
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`Pharmaceutical
`Suences
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`VOLUME 96, NUMBER 10
`OCTOBER 2007
`
`Editorial
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`Ronald T. Borchardt .
`Published onlinc 13 August 2007
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`2507
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`MINIREVIEWS
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`Enaminones: Exploring Additional Therapeutic Activities
`[van 0. Edafiogho, Samuel B. Kornbian, KcihireddyV. V. Ananthalakshmi, Noha N. Salama,
`Nalalic I). Eddington, Tiffany L Wilson, Mariano 5. Alexander, Patrice L. Jackson, Clive D. Hanson,
`and K.R. Scott" .
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`Published onlan 9 july 2007
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`2509
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`Effects of Cyclodextrins on Drug Delivery Through Biological Membranes
`Thorsteinn Lofissoni‘, Stine Byslcov Vogcnsen, Marcus E. Brewster, and Pita Konrjbsrlottir.
`Published online 13 july 2007
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`2532
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`REVIEWS
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`2547
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`Role of Nanotechnology in Pharmaceutical Product Development
`Harikrishna Dcvalapally, Ananthsrinivas Chakilam and Mansoor M. Amiji“ .
`a
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`Published onlfnc 9 August 2007
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`Evaluation of the Chemical Compatibility of Plastic Contact Materials and
`Pharmaceutical Products; Safety Considerations Related to Extractables and
`Lcachables
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`Dennis Jenke .
`Published onlinc 13 August 2007
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`2566
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`Effect of Placental Fatty Acid Metabolism and Regulation by Peroxisome Proliferator
`Activated Receptor on Pregnancy and Fetal Outcomes
`Yan Xu, Qing Wang, Thomas]. Cook, and Gregory T. Knipp“ _
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`Published online 4 june 2007
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`2582
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`(continued)
`
`Journal of Pharmaceutical Sciences
`VOL. 96, NO. 10, OCTOBER 2007
`
`This journal is online
`*LJWILEY
`lnterS' elence'“
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`WWW-intersaence Wiley-com
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`Volume 96, Number 10 was mailed the week ofSeptclnber 24, 2007.
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`Page 3 of 17
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`

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`BIOTECHNOLOGY
`
`RESEARCH ARTICLES
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`The LCJ'MS Analysis of Glycation of IgG Molecules in Sucrose Containing Formulations
`Himanshu S. Gadgil*, Pavel V. Bondarenko, Cary Pipes, Doug Rehder, Arnold Mcauley, Natalie Perico,
`Tom Dillon, Margaret Ricci, and Michael Treuheil .
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`Published onlinc 9 July 2007
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`2607
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`DRUG DISCOVERY INTERFACE
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`In. Vitro Penetration of A Novel Oxaborole Antifungal (AN2690) into the Human Nail
`Plate
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`Xiaoying Hui*, Stephen J. Baker, Ronald C. Wester, Sherry Barbadillo, Anne K. Cashmere, Virginia Sanders,
`Karin M. Hold, Tsutomu Akama, Yong-Kang Zhang, iacob J. Planner, and Howard I. Mailmch .
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`Published onllne 9 july 2007
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`2622
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`PHARMACEUTICS, PREFORMULATION AND DRUG DELIVERY
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`An Examination of the Rheological and Mueoadhesive Properties of Poly(AeryIic
`Acid) Organogcls Designed as Platforms for Local Drug Delivery to the Oral Cavity
`David S. Jones“, Brendan C.O. Muldoon, A. David Woolfson, and F. Dominic Sanderson .
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`Publlshed onllne 13 August 2007
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`2632
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`Critical Assessment of Inverse Gas Chromatography as Means of Assessing Surface
`Free Energy and Acid—Base Interaction of Pharmaceutical Powders
`Martin J. Tcll<o and Anthony J. Hickey“ .
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`Published online 22 May 2007
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`2647
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`Effect of Polymer Molecular Weight on The Production of Drug Nanoparticles
`S. Sepassi, DJ. Goodwin, A.F. Drake, 5. Holland, G. Leonard, L. Martini, and MJ. Lawrence“ .
`Published onllne 14 August 2007
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`2655
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`2667
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`The Role of the Cyclic Imide in Alternate Degradation Pathways for
`Asparagine-containing Peptides and Proteins
`Michael P. Dehart and Bradley D. Anderson*‘ .
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`Published onllne 22 May 2007
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`Combined Use of Crystalline Salt Forms and Precipitation Inhibitors to Improve
`Oral Absorption of Celecoxib from Solid Oral Formulations
`Hector R. Guzmén*, Mark Tawa, Zhong Zhang, Pasut Ratanabanangkoon, Paul Shaw,
`Colin R. Gardner, Hongming Chen, Jean-Pierre Moreau, Om Almarsson, and Julius F. Remenar.
`Published onllne 22 May 2007
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`2686
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`Relaxation and Crystallization of Amorphous Carbamazepine Studied by Terahertz
`Pulsed Spectroscopy
`J. Axel Zeitlcr*, Philip F. Today, Michael Pepper, and Thomas Rades .
`Published online M August 2007
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`2703
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`PHARMACEUTICAL TECHNOLOGY
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`Modulation of Ganciclovir Intestinal Absorption in Presence of Absorption Enhancers
`Pranav Shah, Viral Jogani, Pushpa Mishra, Anil Kurnar Mishra, Tamishraha Bagchi,
`and Amhilranandan Misre" .
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`Published onllne 6 August 2007
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`2710
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`Page 4 of 17
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`

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`Utilization of a Modified Special-Cubic Design and an Electronic Tongue for
`Sitterness Masking Formulation Optimization
`Llanii Li*, Venkatesh Naini, and Salah U. Ahmed .
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`Published aniline 16 May 2007
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`2723
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`2735
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`Application of Direct Crystallization for Racemie Compound Propranolol
`Hydrochloride
`>giuiufm Wang“. iie Lu and Chi Bun Ching .
`l ublishecl onllne 4 june 2007
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`Investigation of the Influence of Particle Size on the Excipient Percolation
`Thresholds of HPMC Hydrophilic Matrix Tablets
`Antonia Miranda, Monica Millzin*, and isidoro Carabailo .
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`Published onlino M May 2007
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`2746
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`2757
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`Fluorescence Studies of the Dehydration of Cefadroxil Monohydrate
`Harry G. Brittain .
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`Published onllno 9 july 2007
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`Solid State Chemistry of Proteins: I- Glass Transition Behavior in Freeze Dried
`Disaccharide Formulations of Human Growth Hormone (hGH)
`Michael L Pikai“, DR. Rigshee, and MJ. Roy .
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`Published online 9 luly 2007
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`2765
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`pH Indicator Titration: A Novel Fast pKa Determination Method
`Xiaoie Kong, Tao Zhou, Zudong Liu, and Robert C. Hider“ .
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`Published onlino Qiuly .2007
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`2777
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`PHARMACOKINETICS, PHARMACODYNAMICS AND DRUG METABOLISM
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`2784
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`High Pressure Differential Scanning Calorimetry Investigations on the Pressure
`Dependence of the Melting of Paracetamol Polymorphs I and II
`J. Ledru“, C.T. imrie, (LR. Pulham, R. Céoiin, and LM. Hutchinson .
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`Published online 31 May 2007
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`Role of Nitric Oxide in Downregulation of Cytochrome P450 lal and NADPH:
`Quinone Oxidoreductase 1 by Tumor Necrosis Factor-o: and Lipopolysaccharide
`Negar Charm/i and Ayman 0.5. Ei-Kadi* .
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`Published online 22 lune 2007
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`2795
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`Grapefruit Juice-Drug Interactions: Grapefruit Juice and Its Components Inhibit
`P—Glyeoprotein (ABCBI) Mediated Transport of Talinolol in Caeo-2 Cells
`Whocely Victor De Castro, Susanne Mertens-Talcott, Hartmut Derenclorl, and Veronika Butterweck“. .
`Published online 3i May 2007
`
`.
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`2808
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`Relative in vitro Efficacy of the Phosphate Binders Lanthanum Carbonate and
`Sevelamer Hydrochloride
`Valerie Autissier, Stephen J.P. Damment, and Richard A i-ienderson* .
`Published onllne H May 2007
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`2818
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`In papers with more than one author, an asterisk (ii) in the byline indicates the author to whom
`inquiries should be directed.
`
`Page 5 of 17
`
`

`

`DRUG DISCOVERY INTERFACE
`
`In Vitro Penetration of A Novel Oxaborole Antifungal
`(AN2690) into the Human Nail Plate
`
`XIAOYING HUI,‘ STEPHEN J. BAKE-2,2 RONALD c. wrsrsc,‘ SHERRY sacsamuo,‘ ANNE K. CASHMORE,‘
`VIRGINIA SANDERS} KARIN M. HOLD,2 TSUTOMU AKAMA,2 YONG-KANG ZHANG,’
`JACOB J. PLATTNERE HOWARD I. MAIBACH‘
`
`'Deparlment of Dermatology, University of California San Francisco, Box 0989,
`90 Medical Center Way, San Francisco, California 94143
`
`lAnacor Pharmaceuticals, Inc., 1060 East Meadow Circle, Palo Alto, California 94303
`
`Received 11 August 2006; revised 2 November 2006; accepted 20 December 2006
`
`Published online 9 July 2007 in Wiley InterScience (www.mterscience.ioiieycom}. D01r 10.1002/jp3.20901
`
`ABSTRACT: Onychomycosis is a challenging fungal infection to treat topically, likely
`due to the unique properties of the nail plate. This seemingly impenetrable barrier has
`high resistance to the passage of antifungal drugs in sufficient concentrations to kill the
`causative fungi deep in the nail bed. Recently, a new class of antifungal agent was
`described, termed oxahoroles, which have broad-spectrum activity. These oxaboroles
`were designed with properties believed to be required to allow for easier transit through
`the nail plate. Herein, we report (i) the nail penetration results of four oxaboroles that
`led to the selection ofAN 2690, (ii) the results ot'the nail penetration ofANZSQO from four
`vehicles, and (iii) the nail penetration of AN2690 in its chosen vehicle compared to a
`commercial control, ciclopirox. AN2690 has superior penetration compared to ciclopirox,
`and achieves levels within and under the nail plate that suggest it has the potential to be
`an effective topical treatment for onychomycosis. (c) 2007 Wiley-Liss, Inc. and the American
`Pharmacists Association J Pharm Sci 96:2622—2631, 2007
`
`absorption; solubility;
`Keywords:
`AN2690; nail penetration
`
`log p; drug effects; diffusion; onychomycosis;
`
`INTRODUCTION
`
`Onychomycosis is a common and complex fungal
`infection, affecting 13—14% of the US popula-
`tion.l'2 In diabetic and elderly patients,
`the
`incidents range from 33% to 50%..“ Onychomy-
`cosis is typically caused by dermatophytes.1‘5"3
`Onychomycosis treatment is usually difficult.
`Systemic therapy, including oral terbinafine and
`oral itraconazole, requires a relatively long-term
`
`
`Correspondence to: Xiaoying l-lui (Telephone: 415-502-7761;
`Fax: 415-?53-5304; E-mail: huixfljdermmcsfedu)
`Journal of Pharmaceutical Sciences, Vol. 96, 262%2631 (200'?)
`I!) 200'? Wiley-Lise, Inc. and the American Pharmacists Association
`
`that may cause side effects and
`treatment
`frequently has a high level of recurrence. An
`attractive alternative is topical application of an
`antifungal directly to the nail? However, the only
`topical
`treatment approved for use against
`onychomycosis in the USA is ciclopirox (see
`Tab. 1), formulated as Penlac'"’. Current topical
`treatments have poor clinical efficacy due to the
`nail’s unique properties such as its thickness and
`compact construction, and the infection’s deep-
`seated nature. Consequently, most antifungal
`drugs that successfully treat skin fungal infection
`do not effectively penetrate throughout
`the
`nail plate and nail bed. To achieve an effective
`chemical concentration into and through the
`sawurv
`ir- ———"—— _
`“g. InterscienCen
`é",
`uIiroun SOMtINInu u-In
`i»
`
`2622
`
`IOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. It}, OCTOBER 2007
`
`Page 6 of 17
`
`

`

`1N vmao PENETRATION or A NOVEL OXABOROLE‘. ANTIFUNGAL {M2690} INTO THE HUMAN NAIL PLATE
`
`2823
`
`Table 1. Penetration Results of Four Oxaboroles into the Nail Plate, Compared to Their
`Physicochemlcal Parameters
`
`s
`t ‘
`DorsalfInte
`d'
`Ventral/'Intermediate
`MIC'I-b-B
`Layer (“wagon
`Layer (ugfmg nail)
`cLogP
`MW
`(ngme)
`Structure
`Compound
`
`
`Physicochemical
`parametergb
`
`Absolute Amount in the Nail Plate
`After Single Dose Left for 3 Days“
`
`
`M2690“
`
`F
`
`OH
`8'
`
`1
`
`152.0
`
`1.24
`
`2.47 i 3.79
`
`2.07 e 0.77
`
`O H
`1 m 1
`CI
`
`168.4
`
`1.81
`
`0.78 i063
`
`2.27:1: 1.66
`
`2
`
`3
`
`1
`
`4
`
`HO
`
`05::
`
`F
`
`;
`£260
`
`F
`
`Me
`
`242,0
`
`2.51
`
`0.4312067
`
`2.01i0.96
`
`212.0
`
`3.55
`
`0.00i0.00
`
`1.91i LSD
`
`Ciclopirnx"
`
`0.5
`
`207.0
`
`2.03
`
`I ‘\
`N O
`OH
`
`
`0.00 :t 0.00
`
`1.47 a; 0.34
`
`nrIV’Iii-iimum inhibitory concentration against T. rubrunL
`bData provrded by Anacor Pharmaceuticals, Inc.
`LEach number represents the mean :l: SD of three samples.
`“Test article was formulated at. 10% wfv (100 glL) in ethanol.
`“Test article was formulated at 8"“ who (80 gflrg) in commercial lvauer.
`
`human nail plate, a successful local therapy is
`dependent upon an appropriate antifungal drug
`that overcomes the nail’s unique barrier behavior.
`In a recent
`report, a new class of boron
`containing compounds, called oxaboroles, was
`described that demonstrated broad-spectrum
`antifungal activity against yeast, molds, and
`dermatophytes. Further evaluation of the anti-
`fungal ability of
`this new oxaborole family
`concluded that 5-fluoro-1,3-dihydro—1-hydroxy-
`2,1-benzoxaborole (AN2690) was the most effec-
`tive, especially against
`the dermatophytes T.
`
`rubrum and T. mentagrophytes, fungal pathogens
`that are the primary cause of onychomycosis.8
`The aim of this study was threefold: first, to
`select the one oxaborole that showed the best
`ability to penetrate the nail plate from a panel of
`four candidates; second, to couple this oxaborole
`with a delivery vehicle that should maximize it’s
`delivery into the nail bed;
`third,
`to compare
`the in vitro nail penetration efficacy of the final
`oxaborolefvehicle combination to the topical ony-
`chomycosis drug, ciclopirox,
`formulated in its
`commercial nail lacquer.
`
`DOI l 0. 100 2!] [15
`
`woman OF PHARMACEUTICAL SCIENCES, VOL. 05. NO. 10, OCTOBER 2007
`
`Page 7 of 17
`
`

`

`2624
`
`IIUI ET AL.
`
`MATERIALS AND METHODS
`
`Test Article and Dosage Formulation
`
`AN2690 (5-fluoro-1,3-dibydro-1-hydroxy-2,1-bcn-
`zoxaborole),
`1 (5-chloro-1,3-dihydro—1-hydroxy~
`2,1—benzoxaborole), 2 [5-fluorc-1,3-dihydro-1-(3-
`hydroxymethylphenyllllibenzoxaborolc), and 3
`(5-fluoro-1,3-dihydro-l- phenyl-2,1-benzoxaborole)
`were obtained from Anacor Pharmaceuticals, Inc.
`(Palo Alto, CA). [MCI—ANZGQO was synthesized by
`Amersham Biosciences UK Limited (Bucking-
`bamshire HP7 QNA, UK) radiochemical purity
`and specific activity of >99.3% and 55 mCi/mmol,
`respectively.
`(ciclopirox 8% topical
`lacquer
`Penlac'” nail
`solution) was manufactured by Dermik [Berwyn,
`PA).
`[1'4C]-Ciclopirox
`(pyridinone-G-fMC)-ciclo-
`pirox) was synthesized by Perkin—Elmer Life and
`Analytical Sciences (Boston, MA). The radiochem-
`ical purity and specific activity of the chemical was
`>95% and 12.5 mCii'mmol, respectively.
`
`Nail Penetration Procedure
`
`Details of the nail penetration study have been
`previously described?‘10 Briefly, a cadaver finger
`nail plate with no obvious signs of infection or
`damage was mounted in a oneichamber diffusion
`cell (Fig. l; Permegear, Inc., Hellcrtown, PA) with
`the outer dorsal surface (top center) open to the air
`and the inner ventral surface in contact with a
`
`small cotton ball acting as a supporting nail bed.
`The supporting cotton ball under the nail was
`wetted with normal saline to provide moisture to
`the nail plate, and the degree of hydration was
`monitored and controlled during the experiment.
`The mounted nails were incubated at 32C and
`
`44 :t 8% relative humidity starting 24 h prior to
`the first dose, to allow the nail plate to stabilize to
`the approximate natural hydration level, and for
`the duration of the experiment. The exposed
`dosing area was 0.78 cm2 and aliquots (10 uL)
`were applied to this surface once daily.
`Dosed surface area washing was conducted at
`the end of the experiment for the single dose
`study, or each morning before the next dose for
`the multiple dose study. The desed surface area of
`Dow reservoir
`Dorsaifinlcnnediaio section
`
`Vcnlrnlfintmncdiatc sccii on
`Nail plate
`Remainder nail
`Walled cotton hall supponing bed
`
`
`
`Receiving chamber
`
`Figure 1. Cross-section of one-cell diffusion chamber
`showing sections of nail sampled.
`
`JOURNAL OF Pl lARMACEUTlCAl. SCIENCES, VOL. ‘36, NO. 10, OCTOBER .2007
`
`Page 8 of 17
`
`the nail was washed with cotton tips in a cycle, as
`follows: two times with ethanol, then with 50%
`Ivory"" liquid soap (Procter & Gamble, Cincinnati,
`OH), then two times with distilled water. The
`washing samples from each cycle were pooled.
`Under the controlled humidity and temperature,
`we did not observe any abnormal situations such
`as the nail plate color change, hydration changes,
`or fungal growth during,r the 14-day dosing period.
`After completion of the experiment, the nail plate
`was transferred to a cutting holder for sampling.
`The nail plate was secured in position so that the
`outer dorsal-dosed surface faced the holder and
`
`the inner ventral layer faced the cutting drill. The
`cutting holder was moved to bring the ventral
`surface just barely in contact with the cutter tip.
`The drill was then started and a fine adjustment
`moved the mounted nail plate into the cutter tip,
`removing a powder sample from the layers of
`the nail plate. In this way, a core approximately
`0.3—0.4 mm in depth and 7.9 mm in diameter was
`drilled from the lower layers ofeach nail, and the
`powder was harvested. These powder samples are
`referred to as the “ventralfintermediate section”
`
`(Fig. 1). The dosed section of nail plate above the
`powder sampling area was cut out as one piece
`and saved as “the dorsalfintermediate section”
`
`(Fig. l). The remaining nail plate outside of the
`dosing area was then saved as the “remainder nail
`plate” (Fig. 1}. All the nail plate samples were
`individually collected into a glass scintillation vial
`and weighed.
`
`Pilot Study 1: Nail Penetration of Four
`Oxaborole Compounds
`
`AN2690, 1, 2, and 3 (Tab. 1), formulated at 10%
`(w/v) in ethanol, were compared for their ability to
`penetrate the nail plate. A single aliquot (10 nL) of
`each formulation was dosed to the dorsal layer of
`the human nail plates using the nail penetration
`procedure described above. After 3 days, the closed
`area was washed and the dorsal/intermediate
`and ventral/intermediate nail
`samples were
`collected, stored at 4”C and analyzed for drug
`using LCfMSMS.
`
`Pilot Study 2: Effect of Vehicle on
`AN2690 Nail Penetration
`
`formulations, all containing 10% (wfw)
`Four
`AN2690 were compared for their ability to deliver
`AN 2690 to the deep layers of the nail plate and into
`the nail bed. Formulation A: 70% ethanol, 20% poly
`
`DOI “3.1002,?an
`
`

`

`m VITRO PENETRATION OF A NOVEL OXABOROLE AN'I‘IFUNGAL (AN259U) INTO THE HUMAN NAIL PLATE
`
`2625
`
`(vinyl methyl ether alt maleie acid monobutyl
`ester) (wlwl; formulation B: 56% ethanol, 14%
`water, 15% poly {2-hydroxyethyl methacrylatel,
`5% dibutyl sebacate (wlwl‘, formulation C: 55%
`ethanol,
`15% ethyl acetate, 15% poly (vinyl
`acetate), 5% dibutyl sebacate (wx’wl; formulation
`D: 20% propylene glycol, 70% ethanol (w/w). Using
`the nail penetration procedure described above,
`aliquots (10 uL) of the dose formulations were
`applied to human nail plates once daily for 14 days.
`The cotton ball supporting bed was collected from
`each cell chamber and replaced with a new one at
`days 5, 10, and 15 after the first dose. The ventral!
`intermediate nail samples were collected at the end
`of the 14-day dose period, stored at 4°C and
`analyzed for drug by LCfMSfMS,
`
`Pivotal Study: Nail Penetration of AN2690
`Compared to a Commercial Control
`
`AN2690, 10% (w/v) in propylene glycoli'ethanol
`(1:4, va) was compared to ciclopirox, 8% (wlw) in
`commercial nail lacquer were for their ability to
`
`penetrate nails. Trace amounts of carbon-l4
`radiolabeled AN2690 and ciclopirox were added
`to their respective formulations the day before
`the first dose. Using the nail penetration proce-
`dure described below, aliquots (10 pL) of the dose
`formulations were applied to human nail plates
`once daily for 14 days. The cotton ball bed
`supporting the nail was collected from each cell
`chamber and replaced with a new one every 72 h
`after the first dose (days 3, 6, 9, 12, and 15).
`The dorsal/intermediate, ventralfint‘ermediate
`and remaining nail samples were collected at
`the 14-day dose period. The radioactivity of all
`samples, including the washings, were analyzed
`and compared.
`
`Quantitative Analysis of Oxaboroles from Pilot
`Studies 1 and 2
`
`LCfMS/MS (APIBOOO, Applied Biosystems, Foster
`City, CA) was used to quantitate the amounts
`of nonradiolabeled oxaboroles, AN2690, 1, 2, and 3
`in samples from the nail penetration studies.
`For the cotton ball analysis eleven calibration
`standards were prepared fresh in normal saline.
`A volume of 100 p.L of each standard was spiked
`onto a fresh cotton ball with final calibration
`standard concentrations of O, 2.5, 5, 10, 20, 40, 80,
`
`160, 320, 640, 1280, and 2560 ugme. Acetonitrile
`(Burdick & Jackson, Muskegon, MI] containing
`
`the internal standard p»nitrophenol (PNP) was
`added to all cotton balls. The cotton ball sampleS
`and any residual solvent were transferred to
`centrifuge filter tubes. After centrifugation, the
`filtrate from the cotton ball samples was trans-
`ferred to autosampler vials and analyzed by
`LCKMSIMS. For the ciclopirox samples, the filtrate
`was first derivatized with dimethylsulfate accord-
`ing to a previously described method11 before
`analysis by LCIMSIMS. Samples with calculated
`concentrations above the highest calibration
`standard were diluted 10— or 20-fold with acet-
`onitrile containing internal standard PNP (TCI
`America, Portland, OR). For the nail analysis, two
`separate calibration curves were prepared, one for
`nail powder analysis and one for top of the nail
`analysis. Each curve contained eleven calibration
`standards. Standards were first prepared in
`dimethylsulfoxide. A. volume of 10 uL of each
`standard was spiked onto keratin powder (TCI,
`Tokyo Kasei Kogyo, Tokyo, Japan) (6.5 mg for
`nail powder curve and 17 mg for top of the nail
`curve). Nail samples were digested with 1 N
`NaOH overnight at 45'JC. The next morning,
`before extraction with methylenechloride, the pH
`of the samples was adjusted to pH 3. After
`extraction,
`the organic layer was transferred
`and evaporated. Samples were reconstituted in
`acetonitrile and analyzed by LC/MSJ'MS LISng an
`Eclipse XDB-CIB 5 am, 2.1 mm x 50 mm column
`(Agilent, Wilmington, DE) and a gradient mobile
`phase from 5 mM ammonium acetate and
`acetonitrile. The extraction efficiency reported
`for ciclopirox from hoof membranes was approxi-
`mately 80%.11 The extraction efficacy of AN2690
`recovered from spiked nail clippings was deter-
`mined to be 89d:2.7% (n .: 3).
`
`Radioactivity Measurement from the Pivotal Study
`
`All radioactivity measurements were conducted
`with a Model 1500 Liquid Scintillation Counter
`(Packard Instrument Company, DOWneI‘ Grove,
`IL). The counter was audited for accuracy using
`sealed samples of quenched and unquenched
`standards as detailed by the instrument manual.
`The 14C counting efficiency is equal to or greater
`than 95%. All nail samples pretreated with
`Packard soluene-350 were incubated at 40°C
`for 48 h followed by the addition of 10 mL
`scintillation cocktail (HIONICwFLUOR, Packard
`Instrument Company, Meriden, CT). Other sam-
`ples (standard dose, surface washing, and bedding
`material) were mixed directly with Universal ES
`
`DOI 10.1002ij5
`
`FOURNAL Ol' l‘l'lARMACLUTlCAL SCIENCFSr VOL. 00. ND. 10. OCTOBER 200?
`
`Page 9 of 17
`
`

`

`2626
`
`HUI ET AL.
`
`(ICN Biomedicals, Costa
`scintillation cocktail
`Mesa, CA). Background control and test samples
`were counted for radioactivity for 3 min each.
`
`mat,er 0.3—0.5 mm in depth. The area is beneath
`the dosed site of the nail plate and includes the
`dosed surface.
`
`Calculations and Data Analysis
`
`Quantitation of nonradioactive compounds was
`based on peak area ratios of compound to internal
`standard. The method of regression for
`the
`calibration curves was selected based on the best
`
`fit. Linear and quadratic regression was used with
`1/35 or 1/2: squared weighting. All integrations were
`performed using Analyst version 1.3 {Applied
`Biosystems). The concentrations of compound in
`the cotton balls were converted to absolute
`
`amounts by taking the sample volume of 100 itL
`into account. The amount of compound in the nail
`powder from the inner ventral/intermediate sec—
`tion and dorsal/intermediate section of the nail
`were adjusted for their respective weights and
`reported in ugfmg nail.
`The individual and mean (iSD) amount of test
`chemical equivalent in nail, bedding material, and
`wash samples are presented as dpm, uCi, percent
`administered dose, and mg equivalent at each
`time point. The concentrations of [Mm-labeled
`test chemicals were calculated from the value
`
`based on the specific activity of each I 14Cl-labeled
`test chemical. The information of concentration of
`
`nonlabeled test chemical in the topical formula-
`tion was obtained from the manufacturers. The
`
`total concentration of test chemical equivalent is
`the sum of the concentration of [MCJ-labeled test
`chemical and the concentration of nonlabeled
`test chemical. The value of the total amount of
`
`test chemical equivalent in each nail sample was
`calculated from those values based on the radio-
`
`activity ofthe sample and the ratio oftotal mg test
`chemical equivalent and radioactivity of the test
`chemical. The data was further normalized by
`dividing by the weight of the sample.
`
`Terminology
`
`Venom/intermediate Center
`
`Powdered nail sample drilled from the center of the
`inner surface (facing the nail bed) approximately
`0.3—0.5 mm in depth to the surface. The area is
`beneath the dosed site of the nail plate but does not
`include dosed surface (dorsal nail surface).
`
`Dorsal/intermediate Center
`
`Nail sample cut from the center of the outer
`surface and immediate area of closed site approxi~
`
`IOURNAL OF PHARMACEUTICAL SCIENCES, VOL. ‘16, NO. 10, OCTOBER 2007
`
`Page 10 of 17
`
`Remainder Naif
`
`The nail plate outside of the dosed area that
`remains after the ventral/intermediate section
`and dorsal/intermediate
`section
`have been
`removed.
`
`Supporting Bed
`
`The cotton ball placed within the Teflon chamber
`of the diffusion cell
`to support the nail plate,
`provide moisture, and receive chemicals penetrat-
`ing through the nail plate.
`
`Surfacing Washing
`
`(or other organic solvents) and soap;r
`Ethanol
`water washing on the surface of the dosed site.
`
`Ring
`
`A plastic ring placed on the top of the nail plate to
`prevent leakage from the Close site onto rest of the
`nail plate or inside of the cell chamber.
`
`Cc” Washing
`
`Ethanol (or other organic solvents) and soap!
`water wash of the inside of the diffusion cell.
`
`RESU LTS
`
`the nail permeation
`In the first pilot study,
`capacity of four oxaboroles, AN2690, AN1677,
`ANZ? 18, and AN2416, each 10% (w/v) in ethanol
`was compared. The chemical deposition of each
`compound in the dorsal/intermediat