`mproving medication use. Advancing patient care.
`
`Page 1
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`.IOU NALOF
`
`A Publication of the Arnerican Pharrnacists Association
`
`APhA
`
`A Publication of the Board of Pharrnaceutical
`Sciences of the International Pharrnaceutical
`·
`Federation
`
`American Association of
`Pharmaceutical Scientists
`
`Published in Coo
`Association of
`
`eration with the Arnerican
`armaceutical Scientists
`
`The Journal of Pharmaceutical Sciences (Print ISSN 0022-3549; Online
`ISSN 1520-6017 at Wiley Interscience, www.interscience.wiley.com)
`is published monthly (one volume per year), by Wiley-Liss, Inc., through
`Wiley Subscription Services, Inc., a Wiley Company, 111 River Street,
`Hoboken, NJ 07030, and the American Pharmacists Association, 2215
`Constitution Avenue NW, Washington, DC 20037.
`Copyright © 2006 Wiley-Liss, Inc., a Wiley Company, and the
`American Pharmacists Association. All rights reserved. No part of this
`publication may be reproduced in any form or by any means, except as
`permitted under section 107 or 108 of the 1976 United States Copyright
`Act, without either the prior written permission of the publisher, or
`authorization through the Copyright Clearance Center, 222 Rosewood
`Drive, Danvers, MA 01923, Tel.: (978) 750-8400, Fax: (978) 750-4470.
`Periodicals postage paid at Hoboken, NJ and at additional mailing
`offices.
`The copyright notice appearing at the bottom of the first page of an
`article in this journal indicates the copyright holder's consent that 2
`copies may be made for personal or internal use, or for personal or
`internal use of specific clients, on the condition that the copier pay for
`copying beyond that permitted by law. This consent does not extend to
`other kinds of copying such as copying for general distribution, for
`advertising or promotional purposes, for creating new collective works, or
`for resale. Such permission requests and other permission inquiries
`should be addressed to the Permissions Department, c/o John Wiley &
`Sons, Inc., 111 River Street, Hoboken, NJ 07030; Tel.: (201) 748-6011;
`Fax: (201) 748-6008; or visit http://www.wiley.com/go/permissions.
`Subscription Price (Volume 96, 2007): Print only: $1,295.00 in U.S.,
`Canada, and Mexico, $1,352.00 outside North America. For all other
`prices please consult the journal's website at www.interscience.wiley.
`com/jpharmsci. Subscriptions at the personal rate are available to
`individuals. All subscriptions containing a print element, shipped outside
`U.S., will be sent by air. Payment must be made in U.S. dollars drawn on
`U.S. bank. Claims for undelivered copies will be accepted only after the
`following issue has been delivered. Please enclose a copy of the mailing
`label. Missing copies will be supplied when losses have been sustained in
`transit and where reserve stock permits. Please allow four weeks for
`processing a change of address. For subscription inquiries, please call
`(201) 748-6645; E-mail: SUBINFO@ wiley.com.
`
`Postmaster: Send address changes to Journal of Pharmaceutical
`Sciences, Subscription Distribution, c/o John Wiley & Sons, Inc., 111 ,
`River Street, Hoboken, NJ 07030. For subscription inquiries, please call
`customer service at (201) 748-6645 or write to the above address.
`Advertising Sales: Inquiries concerning advertising should be
`forwarded to Advertising Sales, c/o John Wiley & Sons, Inc., 111 River
`Street, Hoboken, NJ 07030, (201) 748-8832.
`Manuscript Submission: Five copies of the manuscript must be 1
`submitted together with a transmittal letter and a signed Copyright
`Transfer Agreement to the Editor: Dr. Ronald T. Borchardt, Department
`of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant
`Ave., Room 121A, Lawrence, KS 66047. [Tel: (785) 864-5919; Fax: (785)
`864-5875; E-mail: rborchardt@ku.edu or tdunning@ku.edu]
`Reprints: Reprint sales and inquiries should be directed to the
`Customer Service Department, c/o John Wiley & Sons, Inc., 111 River
`Street, Hoboken, NJ 07030, Tel.: (201) 748-8789.
`Other Correspondence: Address all other correspondence to
`Journal of Pharmaceutical Sciences, Publisher, STM, c/o John Wiley &
`Sons, Inc., 111 River Street, Hoboken, NJ 07030 or jpsprod@wiley.com.
`The contents of this journal are indexed in the following: Analytical
`Abstracts (RSC), CAB Abstracts'il' (CAB!), Cambridge Scientific Abstracts
`(CIG), CCR Database (Thomson lSI), Chemical Abstracts Service/
`SciFinder (ACS), Chemistry Server Compound Center (Th,Qlnson IS]},
`Chemistry Server Reaction Center (Thomson ISI), ChemPrepTM (Thom(cid:173)
`son lSI), Chimica Database (Elsevier), Chromatography Abstracts (RSC), ~
`Current Chemical Reactions 11 (Thomson lSI), Current Contents n !Life
`Sciences (Thomson IS!), ·EMBASE I Excerpta Medica (Elsevier), Index
`Chemicus 11 (Thomson lSI), Index Medicus I MEDLINE I PubMed (NLM),
`International Pharmaceutical Abstracts (Thomson lSI), MDL Beilstein
`(Elsevier), Reaction Citation Index™ (Thomson lSI), Reference Update
`(Thomson lSI), Science Citation Index 11 (Thomson ISI), Science Citation
`Index Expanded™ (Thomson lSI), SCOPUS (Elsevier), and SIIC
`Databases (Sociedad Iberoamericana de Informacion Cientifica).
`
`This paper meets the requirements of ANSI/NISO
`Z39.48-1992 (Permanence of Paper).
`
`Page 2
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`I
`
`VOlUME 96, NUMBER 2
`FEBRUARY 2007
`
`REVIEW
`
`Lipid Formulation Strategies for Enhancing Intestinal Transport and Absorption of
`P-Glycoprotein (P-gp) Substrate Drugs: In vitro/In vivo Case Studies
`Panayiotis P. Constantinides* and Kishor M. Wasan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 18 October 2006
`
`235
`
`COMMENTARY
`
`On the Calculation of the Concentration Dependence of Drug Binding to Plasma
`Proteins with Multiple Binding Sites of Different Mfinities: Determination of the
`Possible Variation of the Unbound Drug Fraction and Calculation of the Number of
`Binding Sites of the Protein
`Leonid M. Berezhkovskiy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 18 October 2006
`
`COMMUNICATIONS
`
`Diffusion of Nutrients Molecules and Model Drug Carriers Through Mucin Layer
`Investigated by Magnetic Resonance Imaging with Chemical Shift Resolution
`Geraldine Lafitte,* Krister Thuresson, and Olle Soderman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 12 October 2006
`
`Quantitation of Crystalline Material within a Liquid Vehicle using
`1H/19F CP!MAS NMR
`BrianT. Farrer,* Andrey Peresypkin, and Robert M. Wenslow
`Published online 18 October 2006
`
`RESEARCH ARTIClES
`
`BIOTECHNOlOGY
`
`Quantitation of Aggregate Levels in a Recombinant Humanized Monoclonal
`Antibody Formulation by Size-Exclusion Chromatography, Asymmetrical Flow
`Field Flow Fractionation, and Sedimentation Velocity
`John P. Gabrielson, Mark L. Brader, Allen H. Pekar, Kathrin B. Mathis, Gerhard Winter,
`John F. Carpenter, and Theodore W. Randolph* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 1 November 2006
`
`249
`
`258
`
`264
`
`268
`
`Volume 96, Number 2 was mailed the week of January 25, 2007.
`
`(continued)
`
`journal of Pharmaceutical Sciences
`VOL. 96, NO. 2, FEBRUARY 2007
`
`&!!WILEY
`
`In r
`
`www.interscience.wiley.com
`
`Page 3
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`DRUG DISCOVERY INTERFACE
`
`Impact of Cremophor-EL and Polysorbate-SO on Digoxin Permeability across Rat
`Jejunum: Delineation of Thermodynamic and Transporter Related Events Using
`the Reciprocal Permeability Approach
`Kasiram Katneni, Susan A. Charman, and Christopher J.H. Porter*
`Published online 18 October 2006
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`PHARMACEUTICS PREFORMULATION AND DRUG DELIVERY
`
`Factors Mfecting the Formation of Eutectic Solid Dispersions and Their
`Dissolution Behavior
`Sudha R. Vippagunta,* Zeren Wang, Stefanie Hornung, and Steven L. Krill . . . . . . . . . . . . . . . . . . . . .
`Published online 18 October 2006
`
`Iontophoretic Drug Delivery across Human Nail
`S. Narasimha Murthy,* Dora E. Wiskirchen, and Christopher Paul Bowers
`Published online 1 November 2006
`
`Precipitation Complexation Method Produces Cannabidiol/p-Cyclodextrin Inclusion
`Complex Suitable for Sublingual Administration of Cannabidiol
`Janne Manni Ia, * Tomi Jarvinen, Kristiina Jarvinen, and Pekka Jarho . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 18 October 2006
`
`Selectivity of Similar Compounds Identification Using IR Spectroscopy: Steroids
`Nina Sadlej-Sosnowska* and Agnieszka Ocios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 18 October 2006
`
`Analysis of Coating Structures and Interfaces in Solid Oral Dosage Forms by
`Three Dimensional Terahertz Pulsed Imaging
`J. Axel Zeitler, Yaochun Shen, Colin Baker, Philip F. Taday,* Michael Pepper, and Thomas Rades . . . .
`Published online 30 October 2006
`
`Air-Liquid Interface (ALI) Culture of Human Bronchial Epithelial Cell Monolayers as
`an In Vitro Model for Airway Drug Transport Studies
`Hongxia Lin, Hong Li, Hyun-Jong Cho, Shengjie Bian, Hwan-Jung Roh, Min-Ki Lee, Jung Sun Kim,
`Suk-Jae Chung, Chang-Koo Shim, and Dae-Duk Kim*
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 1 November 2006
`
`Effect of Moisture and Pressure on Tablet Compaction Studied With FTIR
`Spectroscopic Imaging
`Noha Elkhider, K.L. Andrew Chan, and Sergei G. Kazarian* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 1 November 2006
`
`Hot-Melt Extrusion for Enhanced Delivery of Drug Particles
`Dave A. Miller, Jason T. McConville, Wei Yang, Robert 0. Williams Ill,* and James W. McGinity*
`Published online 30 October 2006
`
`The Relationship Between Water Vapor Absorption and Desorption by Phospholipids
`and Bilayer Phase Transitions
`Heidi M. Mansour* and George Zografi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 1 November 2006
`
`280
`
`294
`
`305
`
`312
`
`320
`
`330
`
`341
`
`351
`
`361
`
`377
`
`PHARMACEUTICAl NANOTECHNOLOGY
`
`Biodistribution and Pharmacokinetic Analysis of Long-Circulating Thiolated Gelatin
`Nanoparticles Following Systemic Administration in Breast Cancer-Bearing Mice
`Sushma Kommareddy and Mansoor Amiji* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 30 October 2006
`
`397
`
`Page 4
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`PHARMACEUTICAl TECHNOlOGY
`
`Modified Cellulose II Powder: Preparation, Characterization, and
`Tableting Properties
`Maria de Ia Luz Reus Medina and Vijay Kumar*
`Published online 30 October 2006
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Preparation of Insulin Loaded PLGA-Hp55 Nanoparticles for Oral Delivery
`Fu-de Cui,* An-jin Tao, Dong-mei Cun, Li-qiang Zhang, and Kai Shi
`. . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 18 October 2006
`
`In Vivo Drug Metabolism Model for Human Cytochrome P450 Enzyme Using
`Chimeric Mice with Humanized Liver
`Miki Katoh, Tashiro Sawada, Yoshinori Soeno, Miki Nakajima, Chise Tateno, Katsutoshi Yoshizato,
`and Tsuyoshi Yokoi* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 18 October 2006
`
`Modifying Drug Release and Tablet Properties of Starch Acetate Tablets by
`Dry Powder Agglomeration
`Riikka Maki, * Eero Suihko, Susanne Rost, Minna Heiskanen, Matti Murtomaa, Vesa-Pekka Lehto,
`and Jarkko Ketolainen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 30 October 2006
`
`Determination of the Hydrolysis Kinetics of ct-Naphthyl Acetate in Micellar Systems
`and the Effect of HPMC (Catalyst Present)
`Pornpen Werawatganone and Dale Eric Wurster*
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 30 October 2006
`
`PHARMACOKINETICS, PHARMACODYNAMICS AND DRUG METABOliSM
`
`Development and Validation of a Preclinical Food Effect Model
`Kimberley A. Lentz,* Megan Quitko, Daniel G. Morgan, James E. Grace Jr., Carol Gleason,
`and Punit H. Marathe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Published online 30 October 2006
`
`408
`
`421
`
`428
`
`438
`
`448
`
`459
`
`In papers with more than one author, an asterisk(*) in the byline indicates the author to whom
`inquiries should be directed.
`
`Page 5
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`Iontophoretic
`
`rug
`
`G 1very across uman Nail
`
`S. NARASIMHA MURTHY,1 DORA E. WISKIRCHEN/ CHRISTOPHER PAUl BOWERS3
`
`1 Department of Pharmaceutics, The University of Mississippi, Oxford, Mississippi 38677
`
`2Department of Pharmaceutical and Biomedical Sciences, Raabe College of Pharmacy,
`Ohio Northern University, Ada, Ohio 45810
`
`3 Department of Chemistry and Biochemistry, Ohio Northern University, Ada, Ohio 45810
`
`Received 14 March 2006; revised 26 June 2006; accepted 14 July 2006
`
`Published online 1 November 2006 in Wiley InterScience (www.interscience.wiley.com). DOl 10.1002 /jps.20757
`
`ABSTRACT: Topical trans-nail delivery of antifungal drugs is limited by several physico(cid:173)
`chemical and physiological factors. Use of chemical permeation enhancers has been a
`common approach for enhancing trans-nail delivery of drugs. The potential of physical
`permeation enhancement techniques has been found to be higher than the potential
`of chemical permeation enhancers in transdermal delivery of hydrophilic drugs and
`macromolecular therapeutic agents. However, application of physical permeation
`enhancement techniques has not been explored for trans-nail drug delivery. In the
`current work, iontophoresis was applied across human nail in vitro to assess its efficiency
`in enhancing drug delivery. Salicylic acid (SA) was used as test diffusant. The influence of
`pH, ionic strength, and current density was studied. Obviously, increase in current
`density increased the trans-nail transport flux. It appears that about 50-100 mM ionic
`strength is required for optimal conduction of electric current across nail. The flux
`enhancement factor (iontophoretic flux/passive flux) also increased with increase in pH
`due to increased ionization of SA. This study demonstrates the efficacy of iontophoresis in
`enhancing the trans-nail delivery of drugs. © 2006 Wiley-Liss, Inc. and the A1nerican
`Pharmacists Association J Pharm Sci 96:305-311, 2007
`Keywords:
`trans-nail; drug delivery; iontophoresis; salicylic acid
`
`INTRODUCTION
`
`Onychomycosis is fungal infection of the nail in
`which the infection is often deep seated in the nail
`stratums. Currently, it is treated by oral admin(cid:173)
`istration of antifungal drugs. The major task in
`treatment is to accumulate and maintain effective
`concentrations (above the minimum inhibitory
`concentrations (MIC)) of the drug in the deeper
`nail stratums during the treatment period. This
`requires large doses and frequent administration
`of drugs. Oral administration of antifungal ther(cid:173)
`apy is inherently associated with GI and systemic
`side effects. 1 Obviously, topical delivery is the
`most desired therapy due to relatively less severe
`
`Correspondence to: S. Narasimha Murthy (Telephone: 662-
`915-5164, Fax: 662-915-1177; E-mail: murthy@olemiss.edu)
`Journal of Pharmaceutical Sciences, Vol. 96, 305-311 (2007)
`© 2006 Wiley-Liss, Inc. and the American Pharmacists Association
`
`side effects and better patient compliance parti(cid:173)
`cularly in case of pediatric patients.
`The nail apparatus consists of nail folds, nail
`matrix, nail bed, hypochonium, and nail plate. The
`nail plate lies under the proximal nail fold and over
`the matrix, nail bed, and hypochoniium. The nail
`plate (0.25-0.6 mm) consists of tightly packed dead
`cells that are highly keratinized and less than 1%
`lipids. 2 For the topical therapy to be successful, the
`drug is required to penetrate across the nail plate
`and distribute in the nail stratums at therapeuti(cid:173)
`cally effective amounts (>MIC). Unfortunately,
`there are at least two factors that could limit the
`accumulation and activity of drugs in the nail on
`topical application. First, the physicochemical prop(cid:173)
`erties of the drug need to be favorable for absorption
`through nail matrix. The nail matrix is reported to
`be relatively more permeable to polar compounds
`4
`than nonpolar compounds. 3
`,
`
`6i?WILEY
`lnterScience®
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 2, FEBRUARY 2007
`
`305
`
`Page 6
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`306 ' MURTHY, WISKIRCHEN, AND BOWERS
`
`Second, binding of the drug to keratin reduces
`the availability of the free drug. Antifungal drugs
`are reported to possess high-binding affinity to
`keratin. 5 The drug, to elicit biological activity,
`should be present in the free form because the
`bound drug is not active. Therefore, the total
`· amount of drug present in the nail matrix does not
`represent the actual active levels relative to MIC.
`This, most likely, is one of the reasons for pro(cid:173)
`longed durations of treatment of nail disorders.
`Moreover, the bound form of the drug does not
`contribute to the concentration gradient due to the
`lack of thermodynamic activity. This decreases the
`amount of drug penetrating into the deeper nail
`layers. In agreement with this, Hui and coworkers
`found that a large portion of topically applied
`drugs were retained in the surface layers of the
`nail6 due to binding which reduced the depth of
`drug penetration in the nail.
`Nail drug delivery has been a topic of research
`interest for the past two decades. The common
`approach for enhancing nail drug delivery has been
`to use keratolytic and thiolytic agents. These agents
`are known to increase the permeability of nail
`matrix by chemical modification of keratin. How(cid:173)
`ever, their permeability enhancement potential is
`limited by the factors like penetrability of enhancer
`and the duration of its presence in the nail matrix
`might significantly influence the chemical modifi(cid:173)
`cation of keratin. 7 Physical permeation enhance(cid:173)
`ment techniques have been successfully utilized
`in the transdermal delivery of drugs. 8 They are
`known to possess greater permeability enhance(cid:173)
`ment potential compared to chemical enhancers in
`delivering hydrophilic and macromolecular thera(cid:173)
`peutic agents. However, up until now, there has
`been few reports regarding possible use of physical
`permeation enhancement techniques for nail drug
`delivery. James and coworkers9 carried out ionto(cid:173)
`phoresis of prednisolone sodium phosphate across
`thumb nail and determined the time course of
`prednisolone in plasma. They also discussed the
`possibilities of using iontophoresis for treating nail
`diseases. However, there is need for systematic
`preliminary studies to assess the efficacy and
`resolve the mechanistic aspects of iontophoresis
`across nail. In this direction, the current work was
`planned to assess the efficiency of iontophoresis in
`enhancing trans-nail delivery of drugs. It was
`hypothesized that the diffusion rate of drug
`through hydrated keratin (which acts like a hydro(cid:173)
`gel membrane) could be enhanced by iontophoretic
`driving force. The hypothesis was assessed by
`studying the transport of SA across the whole
`
`human nail plate. SA was chosen as a test permeant
`to assess the influence of various factors on ionto(cid:173)
`phoretic delivery.
`
`MATERIAlS AND METHODS
`
`Chemicals
`Salicylic acid (SA, Mol wt. 138.12, pKa 3.1),
`sodium chloride, sodium hydroxide, hydrochloric
`acid, potassium chloride, monobasic potassium
`phosphate (KH2P04), and dibasic sodium phos(cid:173)
`phate (Na2HP04·7H20) phosphoric acid were
`purchased from Sigma-Aldrich (St. Louis, MO).
`The buffers prepared were of 10 mM ionic
`strength. Additional sodium chloride was incor(cid:173)
`porated to increase the buffer to desired ionic
`strengths. In this paper, the mM value given in
`the parenthesis indicates the total ionic strength
`of the buffer.
`
`Human finger Nails
`
`The whole nail tips were obtained from the fingers
`and toes of healthy adults using nail clippers (IRB
`protocol# 1001). The nail plates having a thickness
`of about 400 11m were used for the studies. The
`thickness of nail plates used for a set of test and
`control experiments were within ±5%. The nail
`plates were washed, dried, and stored in desiccator
`until use. Prior to use, the nail plates were soaked
`for 6 h in the donor compartment buffer. This was
`done to hydrate the nail plate to impart flexibility
`and to avoid breaking of nails during mounting on
`to the diffusion cell set up.
`
`Transport Studies
`
`In vitro transport studies were carried out using
`Franz diffusion cells (Logan Instruments Ltd.,
`Somerset, NJ) fitted with a custom made Teflon
`nail holder (Fig. 1). Ag/AgCl electrodes (Select
`Engineering, Fitchburg, MA) were fixed at a
`distance of 2 mm from the nail in the donor and
`receiver chambers. Iomed Phoresor II dose con(cid:173)
`troller was used for appliGation of constant DC.
`The cathode was connected to the donor and the
`anode was connected to the receiver chamber
`electrodes. Drug solution (500 IlL) prepared in
`buffer was placed in the donor compartment. The
`receiver compartment volume was 5 mL. The
`active diffusion area was 0.25 cm2
`. The receiver
`compartment was stirred at 600 rpm with a 3-mm
`magnetic stir bar. Intermittent samples of 0.5 mL
`were drawn from the receiver compartment at 2 h
`
`jOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 2, FEBRUARY 2007
`
`DOl 1 0.1 002/jps
`
`
`
`Page 7
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`IONTOPHORETIC DRUG DELIVERY ACROSS HUMAN NAIL
`
`307
`
`Nail adapter __ _
`
`Nail
`
`I I - - - - Donor
`Chamber
`
`pH 7.1 buffer (50 mM) irrespective of the
`buffer medium present in the donor cham(cid:173)
`ber.
`d. Effect of current density: PBS (50 mM,
`pH 7.1) buffer containing 2 mg/mL SA was
`placed in the donor chamber. The plain
`buffer was used in the receiver compart(cid:173)
`ment. The current densities applied were
`0.16, 0.3, and 0.5 mNcm2
`.
`
`Receiver
`chamber
`
`Analytical Method
`
`Figure 1. Trans-nail diffusion experimental set up.
`The receiver chamber was of 5 mL and donor was 500 ~tL
`capacities. The custom made Teflon nail adapter holding
`the nail was sandwiched between the two chambers and
`secured with clamps.
`
`intervals for 24 h and the amount of SA
`transported was measured. 9 Equal volume of
`fresh buffer was replaced in the receiver compart(cid:173)
`ment followed by each sampling. In all except the
`experiments in which the influence of current
`density was examined, the applied current den(cid:173)
`sity was 0.5 mNcm2
`. Passive transport experi(cid:173)
`ments were run in parallel with iontophoretic
`experiments wherever necessary, and the data
`are shown in the figures for comparison. The
`concentration of chloride ion after the transport
`experiments was measured in the donor and the
`receiver compartment buffers to be sure that the
`buffers contained sufficient chloride ions till the
`end of the experiment.
`
`a. Effect of concentration of SA: Different
`concentration solutions of SA were prepared
`in PBS buffer (pH 7.1, 50 mM) and placed in
`the donor compartment.
`b. Effect of ionic strength: The phosphate
`buffer of 10 mM ionic strength (pH 7.1) was
`adjusted to desired total ionic strength by
`incorporation of additional sodium chloride
`(0-140 mM). The concentration of salicylic
`acid in these buffers was 2 mg/mL. The PBS
`(pH 7 .1) buffer with the same ionic strength
`as that of donor was present in the receiver
`compartment.
`c. Effect of pH: All buffers were phosphate
`buffers with additional sodium chloride
`incorporated to
`increase the total ionic
`strength to 50 mM. The concentration of
`salicylic acid in these buffers was 2 mg/mL.
`The receiver compartment was filled with
`
`The amount of SA in the receiver compartment
`buffer was measured by fluorescence spectroscopy
`with a Hitachi FL2500 spectrofluorimeter. The
`sample drawn was diluted (1:4) with 0.05 M
`sodium hydroxide solution. The excitation wave(cid:173)
`length was 299 nm and the emission was at
`405 nm. Standard calibration curves were
`prepared in each buffer system. The sensitivity
`of the method was 10 ng/mL. The linearity was
`between 10 and 500 ng/mL (R 2 = 0.991). 9
`Control trials of iontophoresis across nail were
`run by placing plain buffers. The samples with(cid:173)
`drawn were treated the same way as described
`above and the fluorescence was measured at 2-h
`intervals for 24 h. No background fluorescence was
`observed. It is most likely that the no fluorescent
`components from the nail leached into the medium.
`Moreover, the emission wavelength of keratin
`( 450 nm) is out side the emission wavelength of
`SA.lO,ll
`
`Data Treatment
`
`The flux was calculated from the slope of the linear
`part of the cumulative transport-time profile. The
`permeability coefficient was calculated as the ratio
`of flux to the concentration of SA in donor. The
`curve fitting and statistical analysis was carried out
`using GraphPad Prism 3.03 software. An ANOVA
`was selected as the test for significance and a
`P-value less than 0.05 was considered statistically
`significant. The data points provided in the graphs
`are an average of three trials. The error bars repre(cid:173)
`sent the standard deviation.
`
`RESUlTS AND DISCUSSION
`
`Passive Transport of SA across Nail
`
`Effect of Concentration
`
`The trans-nail flux of SA increased with the
`concentration of SA in the donor compartment
`
`DOl 10.1 002/jps
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 2, FEBRUARY 2007
`
`Page 8
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`308
`
`MURTHY, WISKIRCHEN, AND BOWERS
`
`5
`
`5
`
`4.5 -:-l
`
`(,)
`
`4
`E
`2: tb 3.5
`..3-
`3
`<C
`(/)
`.....
`0
`)(
`j
`2
`li:
`•t;
`s;: 1.5
`CJ' c
`... 1-
`"'
`
`2.5
`
`1
`
`0.5
`
`0
`
`t
`
`~ 4.5
`E
`4
`::2
`~ 3.5
`3
`..... 2.5
`2
`
`(.)
`
`<(
`(/)
`
`0
`X
`:J
`16::
`"(ij
`£: u,
`«< ...
`1-
`
`£:
`
`1.5
`
`0.5
`
`0
`
`0
`
`0.5
`
`1
`
`1.5
`
`2
`
`Concentration of Salicylic acid {mglml)
`
`0
`
`2
`
`4
`pH
`
`6
`
`8
`
`Figure 2. Trans-nail flux of salicylic acid at different
`concentrations of SA. The filled triangles represent the
`passive transport and the filled circles represent
`iontophoretic transport. Donor chamber contained
`different concentration of SA prepared in PBS (pH 7.1,
`50m.M).
`
`Figure 3.
`Influence of pH on the passive transport
`flux (filled triangles) and iontophoretic transport flux
`(filled circles) of SA. All solutions were of 50 mM ionic
`strength. SA solution (500 ~LL of 2 mg/mL) in the
`respective buffers was placed in the donor. The receiver
`compartment was filled with PBS (50 m.M) in all cases.
`
`solution (Fig. 2). The lag time remained consistent
`(rv8 h). The permeability coefficient of human nail
`for salicylate was rvl.94 x 10-8 ± 0.31 cm/s which
`is comparable with the values reported for similar
`type of ionic drug substrates by Kobayashi and
`coworkers. 12
`
`Effect of pH
`Walters and coworkers13 studied the permeation
`of the weakly basic drug, miconazole, through
`hydrated human nail plate. They found that the
`permeability coefficient of the drug was essen(cid:173)
`tially the same at all pH values. Whereas, Soong
`and coworkers found that unionized benzoic acid
`was more permeable than ionized form due to the
`repulsion of benzoate ion by the negatively
`charged hoof membrane at pH>5. 13 In this paper,
`the influence of pH on the passive transport of SA
`was studied. At a fixed donor concentration of SA,
`the passive transport flux was pH dependent. As
`compared to pH at 1.2, the trans-nail flux at pH 3
`was about one half (Fig. 3). At pH 5 or 7, the trans(cid:173)
`nail flux was about one third the value seen at pH
`1.2. pH-dependent transport behavior of drugs
`across the nail plate has been argued generally on
`the basis ofpH-pKa relationship. One should note
`that these studies were carried out at gradient pH
`conditions across the nail plate. Although the nail
`was soaked in the donor compartment buffer, it is
`
`plausible that the charge density of the mem(cid:173)
`brane was altered due to its contact with the
`receiver compartment buffer. In the present
`context, assuming that the charge density on the
`membrane is primarily influenced by the donor
`compartment buffer, we can postulate four types
`of pH-pKa-dependent conditions to explain the
`transport of SA across the nail keratin matrix.
`First, at pH 1.2, the SA (pKa 3.1) is almost
`completely in the unionized form and the keratin
`membrane is positively charged (pi rv5). 13
`14 At
`,
`this pH, the transport that resulted could be
`attributed to the permeability of nail matrix to the
`unionized form of SA. Second, at pH 3, the
`membrane is positively charged and the drug is
`rv50% ionized. It is not clear if the decrease in the
`trans-nail flux of SA at pH 3 to one half of that at
`pH 1.2 is due to binding of the anionic drug
`to positively charged keratin membrane. It is
`also not clear to what extent the ionized fraction
`binds to the keratin as nothing is ~nown about the
`charge density of keratin matrix. Third and
`fourth conditions are pH 5 and pH 7 .1, respec(cid:173)
`tively, at which the drug is 99%-100% ionized.
`However, the keratin matrix is most likely to
`carry significantly less charge density at pH 5 as
`compared to pH 7.1 (pi of keratin is rv5). One
`would expect significantly less trans-nail flux of
`salicylate at pH 7.1, as opposed to pH 5, due to the
`likelihood of repulsion of salicylate ion by the
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 2, FEBRUARY 2007
`
`00110.1002/jps
`
`Page 9
`
`Anacor Exhibit 2008
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
`
`
`IONTOPHORETIC DRUG DELIVERY ACROSS HUMAN NAIL
`
`309
`
`negatively charged keratin membrane. In con(cid:173)
`trast, in the present work there was no significant
`difference in the trans-nail flux of SA at these two
`pH conditions (Fig. 3). These results indicate
`that the pH-dependent transport behavior of ion(cid:173)
`izable substances across the keratin matrix is
`primarily influenced by factors other than the
`charge. Apparently, the alternate factor that
`could influence the diffusion is the thermody(cid:173)
`namic activity of the drug in the donor compart(cid:173)
`ment.
`The thermodynamic activity of a drug solution
`is expressed as a fraction of the saturation
`solubility. Therefore, the thermodynamic activity
`of saturated solution of a drug is regarded as
`unity. 15 The solubility of SA at pH 1.2, 3, and at
`pH 5 or 7.1 is 2.74, 11.2, and rv > 894 mg/mL, res(cid:173)
`pectively, at the present experimental conditions.
`Relative to these solubility values, the donor solu(cid:173)
`tion of 2 mg/mL used in this study corresponds to
`thermodynamic activity of 0. 7' 0.2, and rv0.002
`at pH 1.2, 3, and 5 or 7.1, respectively. 16 This
`explains the difference in trans-nail flux of SA
`at different pH conditions. Walter's observation
`about pH-independent miconazole
`trans-nail
`transport is most likely due to the fact that the
`solubility or the thermodynamic activity of the
`drug did not differ significantly at different pH
`17
`buffers. 11
`18 However, in these explanations, the
`'
`,
`influence of pH of the donor solution on the nature
`and integrity of the membrane is not considered.
`Further investigation of the influence of pH on the
`permeability characteristics of keratin matrix
`could provide additional insight into this phenom(cid:173)
`enon.
`
`Iontophoretic Delivery of SA across Nail
`
`Cathodal iontophoresis was applied to effect
`electrorepulsion of salicylate anion. The ionto(cid:173)
`phoretic transport flux of SA increased with the
`increase in the SA concentration in the donor
`compartment buffer up to 2 mg/mL (Fig. 2). The
`lag time di