Bilayered Nail Lacquer of Terbinafine Hydrochloride for
`Treatment of Onychomycosis
`
`H.N. SHIVAKUMAR,1 SIVA RAM KIRAN VAKA,1 N.V. SATHEESH. MADHAV,2 HARISH CHANDRA,2
`S. NARASIMHA MURTHY1
`
`1Department of Pharmaceutics, The University of Mississippi, University, Mississippi 38677
`
`2Department of Pharmaceutics, Dehradun Institute of Technology, Dehradun, India
`
`Received 7 October 2009; revised 1 February 2010; accepted 20 February 2010
`
`Published online 13 April 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22150
`
`ABSTRACT: The present study aimed to develop bilayered nail lacquer of terbinafine hydro-
`chloride (TH) for treatment of onychomycosis. The composite nail lacquer formed an underlying
`drug-loaded hydrophilic layer and overlying hydrophobic vinyl layer. The hydrophilic lacquer
`made of hydroxylpropyl methylcellulose E-15 contained polyethylene glycol 400 (PEG 400) as a
`drug permeation enhancer. The vinyl lacquer was composed of poly (4-vinyl phenol) as a water-
`resistant film former. In vitro permeation studies in Franz diffusion cells indicated that the
`amount of TH permeated across the human cadaver nail in 6 days was 0.32 0.14, 1.12 0.42,
`and 1.42 0.53 mg/cm2 from control (hydrophilic lacquer devoid of PEG 400), monolayer (hydro-
`philic lacquer alone), and bilayered nail lacquers, respectively. A higher nail drug load was seen
`in vitro with the bilayered lacquer (0.59 0.13 mg/mg) as compared to monolayer (0.36 0.09 mg/
`mg) and control (0.28 0.07 mg/mg) lacquers. The drug loss despite multiple washing was
`significantly low ( p < 0.001) for the bilayered lacquer owing to the protective vinyl coating.
`Clinical studies demonstrated the efficacy of bilayered lacquer to achieve better drug load in
`the nail plate (1.27 0.184 mg/mg) compared to monolayer (0.67 0.18 mg/mg) and control
`(0.21 0.04 mg/mg) lacquers. ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association
`J Pharm Sci 99:4267–4276, 2010
`Keywords:
`onychomycosis; nail; microscopy; calorimetry (DSC); bilayer
`
`INTRODUCTION
`
`Onychomycosis is the fungal infection of the human
`nail affecting 19% of the global population.1 It
`accounts for 50% of the nail diseases in diabetic
`and elderly patients. Onychomycosis is commonly
`caused by dermatophytes though yeasts and candida
`may be the other causative organisms.2 The disease is
`difficult to manage as it is chronic, hard to eradicate,
`and tends to relapse. The infected nails appear ugly,
`discolored, thickened, and dystrophic which makes a
`significant negative impact on the social life of the
`patient.
`In the past, the treatment modality for onychomy-
`cosis was surgical extraction of the diseased nail
`which would be extremely traumatic and painful.3
`Currently, the disease is treated with oral or topical
`antifungal agents. Following oral administration the
`drug is absorbed into systemic circulation following
`which it eventually diffuses into the nail plate
`
`Correspondence to: S. Narasimha Murthy (Telephone: 662-915-
`5164; Fax: 662-915-1177; E-mail: murthy@olemiss.edu)
`
`Journal of Pharmaceutical Sciences, Vol. 99, 4267–4276 (2010)
`ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association
`
`through the nail bed. Unfortunately about 20% of
`the patients fail to respond to the oral treatment in
`which the relapse of the disease is quite common.4
`Moreover, the oral therapy has been associated with
`systemic adverse effects and drug interactions. On
`the contrary, topical therapy based on nail lacquers
`circumvents most of the limitations of oral adminis-
`tration. But because of poor drug diffusion into the
`highly keratinized nail plate and the long duration of
`treatment, the topical monotherapy has been cur-
`rently recommended only in the early stages of the
`disease or when the systemic therapy is contra-
`dicted.5 However,
`the combination therapy has
`proved to achieve a higher success rate and demon-
`strated a better cost per cure rate compared to oral
`therapy alone.6
`Considering these facts persistent attempts have
`been made to improve the efficacy of topical nail
`preparations.7–9 Therefore, there is an urgent need to
`develop a topical formulation, which can be effectively
`used in topical monotherapy in the initial stages of
`onychomycosis or in combination with the oral
`therapy in the advanced stages.
`Terbinafine hydrochloride (TH) is one of the most
`potent antifungal agent used to treat onychomycosis.
`
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`The drug is known to have low minimum inhibitory
`concentrations (0.001–0.01 mg/mL) and low mini-
`mum fungicidal concentrations (0.003–0.006 mg/
`mL).10 The oral therapy with TH has been associated
`with severe side effects, toxicity, drug interaction,
`and high rate of recurrence.11 In this context there is
`a strong need for an effective, safe, and patient
`compliant topical formulation capable of delivering
`and maintaining therapeutic concentrations of terbi-
`nafine in the nail bed.
`The key in the treatment of onychomycosis and other
`nail diseases is the delivery and maintenance of
`effective drug concentrations in deeper nail layers.12
`But most of the conventional nail lacquer preparations
`based on water-insoluble resins have limited potential
`to enhance the transungual drug delivery. Considering
`this, aqueous-based nail lacquers have been preferred
`for treatment of onychomycosis as they promote the
`nail hydration and the drug diffusion across the nail
`plate.8 However, the water-soluble preparations suffer
`from the limitation of being easily wiped off or washed
`off the nail surface. Therefore, the present investiga-
`tion aimed to develop a bilayered nail
`lacquer
`comprising of an underlying drug-loaded hydrophilic
`layer and overlying water-resistant film. The novel
`bilayered nail lacquer proposed for TH would be the
`first of its kind for the treatment of onychomycosis. The
`unique formulation is expected to overcome most of
`the limitations of the conventional lacquer formula-
`tions and ensure effective local therapy against fungi,
`dermatophytes, and molds.
`
`MATERIALS AND METHODS
`
`Materials
`
`TH (MW 327.90 Da) was procured from Uquifa
`(Jiutepac, Mexico). Human cadaver fingernails, both
`male and female with varying thicknesses of 0.4–
`0.7 mm were procured from Science Care (Phoenix,
`AZ). Methocel E 15 Premium LV-Hydroxypropyl
`methylcellulose (HPMC E-15 LV) was the gift sample
`from Dow Chemical Company (Midland, MI). Poly (4-
`vinyl phenol) [average molecular weight 25,000],
`polyethylene glycol 400 (PEG 400), and dibutyl
`phthalate were purchased from Sigma–Aldrich (St.
`Louis, MO). All other chemicals and reagents used
`were of analytical grade.
`
`Methods
`
`Analytical Method
`
`2487). Elution was performed isocratically using
`Phenomenex C18 (2) 100 R analytical column
`(4.6 mm  150 mm, Luna, Torrance, CA 5.0 mm) at a
`temperature of 328C. The mobile phase consisting of
`aqueous solution (0.096 M triethyl amine, 0.183 M
`orthophosphoric acid) and acetonitrile (60:40) was set
`to pH 2.0 and a flow rate of 1.0 mL/min. The sample
`injection volume was 20 mL and the column effluent
`was monitored at 224 nm. The method developed was
`validated for the linearity, precision, and accuracy.
`The range of calibration curve was found to be 2–
`1000 ng/mL (R2¼ 0.99) with the coefficient of var-
`iance and accuracy ranging from 1.03% to 6.08% and
`0.54% to 6.96%, respectively.
`
`Preparation of Nail Lacquers
`
`The nail lacquers formulated in the present study were
`the drug-loaded hydrophilic lacquer (HPMC lacquer
`containing PEG 400) and hydrophobic vinyl lacquer.
`The hydrophilic nail lacquer was composed of TH (5%,
`w/v), HPMC E-15 (6%, w/v), PEG 400 (10%, v/v),
`ethanol (60%, v/v), and purified water (q.s.). TH was
`dissolved in a mixture of water and ethanol (pH 3.0) on
`a bath sonicator. HPMC E-15 was soaked overnight in
`the hydroalcoholic mixture (pH 3.0) and sonicated to
`ensure complete polymer dissolution. The two hydro-
`alcoholic solutions were mixed thoroughly on a
`magnetic stirrer to obtain a clear homogeneous solution
`to which PEG 400 was added and the stirring
`continued. The pH, viscosity, drying time, and TH
`content of the hydrophilic nail lacquers was found to be
`4.0, 500 cps, and 300 75 s, respectively. A ‘‘control’’
`hydrophilic nail lacquer of HPMC E-15 containing TH
`but devoid of PEG 400 and a drug free ‘‘placebo’’
`hydrophilic lacquer of HPMC E-15 containing PEG 400
`were similarly prepared for comparison.
`The hydrophobic nail lacquer was prepared by
`dissolving poly (4-vinyl phenol) in ethyl acetate at
`concentration of 10% (w/v). Dibutyl phthalate was
`used as a plasticizer in the lacquer at a concentration
`of 4% (v/v).
`lacquer formulations repeatedly
`The different
`referred throughout the article are ‘‘control lacquer’’
`(TH-loaded HPMC lacquer devoid of PEG 400),
`‘‘monolayer lacquer’’
`(TH-loaded HPMC lacquer
`containing PEG 400), ‘‘bilayered lacquer’’ (monolayer
`lacquer overlaid with the hydrophobic lacquer), and
`‘‘placebo lacquer’’ (drug-free HPMC lacquer contain-
`ing PEG 400).
`
`Microscopy Studies
`
`The amount of TH in the samples was quantified by
`high-performance liquid chromatography (HPLC)
`system (Waters, Hunlingdon Vly, PA, 1525) equipped
`with an autosampler (Waters 717 Plus) and a variable
`wavelength dual l absorbance detector (Waters,
`
`Sulforhodamine B was dissolved in the hydrophilic
`nail lacquer at a concentration of 1 mg/mL while
`methylene blue was solubilized in the hydrophobic
`vinyl lacquer at similar concentration. The hydro-
`philic lacquer was applied to a human cadaver nail
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 10, OCTOBER 2010
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`BILAYERED NAIL LACQUER OF TERBINAFINE HYDROCHLORIDE
`
`4269
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`plate and allowed to dry (for about 5 min). The vinyl
`lacquer was applied on top of the drug-loaded
`hydrophilic layer and dried to form a bilayer.
`Cross-sections of the nail with the adhering lacquer
`films were taken perpendicular to the surface of the
`nail using a microtome (Rotary Microtome, Reichert-
`820, Buffalo, NY). The sections were mounted on a
`gelatinous slide, viewed, and photographed under an
`optical microscope (Zeiss, MicroImaging, with Axio-
`cam, Thornwood, NY).
`
`Differential Scanning Calorimetry
`
`Differential scanning calorimetry (DSC) was used to
`characterize the physical state of TH in the casted
`hydrophilic lacquer film. DSC thermograms of TH,
`casted HPMC lacquer film, and physical mixture of
`TH and HPMC E-15 (in ratio of 5:6) were recorded in
`differential scanning calorimeter (Perkin Elmer Pyris
`1 DSC, Waltham, MA). Samples weighing about 2 mg
`were heated from 25 to 2258C in flat-bottomed
`hermetically sealed aluminum pans at a linear
`heating rate of 108C/min. Ultra pure nitrogen was
`purged at a flow rate of 30 mL/min and the data
`recorded was interpreted using Pyris ManagerTM
`software.
`
`Bioadhesivity Studies
`
`Bioadhesion is one of the key factors to be considered
`in development of a successful transnail drug delivery
`system in onychomycosis.13 The adhesion of HPMC
`lacquer films to the cadaver nails was determined
`using Texture Analyzer1 equipped with a 50-kg load
`cell (TA.XT2i, Technologies Corp., Scarsdale, NY/
`Stable Micro Systems, Godalming, Surrey, UK)
`(Fig. 1). The TH loaded or the placebo HPMC lacquer
`was spread on a glass slide that was secured on the
`base of Texture analyzer1. The cadaver nail plate of
`6 mm diameter was mounted on TA-96 probe. The
`
`Figure 1. TA.XT2i Texture Analyzer (from Texture Ana-
`lyzer Corp., Scarsdale, NY) used for bioadhesivity testing of
`the nail lacquer formulations.
`
`probe was lowered from a height of 25 mm at a speed
`of 1 mm/s with a force of 3.5 N till the nail plate
`touched the surface of the lacquer film. When the nail
`plate detected the surface of the lacquer film, a trigger
`force of 0.5 N was applied for 30, 60, or 120 s. After the
`contact time was lapsed the probe was withdrawn
`from the surface film at a preset speed of 0.5 mm/s
`and the force required to withdraw the nail from the
`lacquer film was recorded as peak adhesive force
`(PAF). The area under the curve (AUC)
`that
`represents the work of adhesion was determined
`from the force deflection profiles. The two parameters
`for the placebo and drug-loaded lacquer films were
`recorded in triplicate and interpreted using Texture
`ExpertTM software.
`
`In Vitro Drug Permeation Studies
`
`Cadaver nails were cleaned and the adherent tissue
`was removed with a pair of scissors and a scalpel.
`Each nail plate having an average thickness of
`0.5 mm was washed with water, soaked in normal
`saline for 1 h prior to use and mounted on a nail
`adapter having a active diffusion area of 0.2 cm2
`(Permegear, Hellertown, PA). The whole assembly
`was sandwiched between the two chambers of a Franz
`diffusion cell (Logan Instruments Ltd, Somerset, NJ).
`About 20 mL of the TH-loaded hydrophilic nail lacquer
`was applied onto the nail plate in the donor
`compartment and allowed to dry for 5 min. On drying,
`20 mL of the vinyl nail lacquer was applied on top of
`the drug-loaded hydrophilic film to form a bilayered
`lacquer film on the surface of the nail plate. The
`receiver chamber was filled with 5 mL of saline set to
`pH 3.0, maintained at a temperature of 378C and
`stirred at 600 rpm with a 3-mm magnetic bead.
`Samples were withdrawn from the receiver compart-
`ment at regular time interval for a period of 6 days
`and analyzed for TH by HPLC. The second set of
`experiment was performed by application of 20 mL of
`the drug-loaded hydrophilic nail lacquer alone on the
`nail plate that formed monolayer lacquer film. The
`third set of trails performed by application of 20 mL of
`control nail lacquer alone was run in parallel for
`comparison.
`
`Drug Load in the Nail Plate
`
`The amount of TH loaded in the nail plate following the
`permeation studies was determined following the
`validated extraction procedure reported earlier.14
`The active diffusion area on the nail plates measuring
`0.2 cm2 were marked (using permanent marker) and
`cut using metric punch, washed by standardized
`procedure with water and ethanol (95%) until the
`surface was free from drug. The active diffusion area
`was cut into small pieces, weighed, and taken in screw
`cap pyrex vials. The vials were incubated with 1.5 mL
`
`DOI 10.1002/jps
`
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`4270
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`SHIVAKUMAR ET AL.
`
`of sodium hydroxide (1 M) on a shaker water bath at
`room temperature for 24 h to allow the nail to
`completely dissolve. The solutions obtained were
`neutralized with 200 mL of 5 M hydrochloric acid and
`extracted with hexane (3 mL) by vigorous agitation for
`30 min. The resulting mixture was transferred into
`tubes and centrifuged at 4000 rpm for 10 min to
`separate the organic phase. The hexane layer was
`separated and treated with 1 mL of 0.5 M sulphuric
`acid/isopropyl alcohol (85:15) and shaken vigorously for
`30 min. The lower acidic aqueous layer containing the
`drug was collected and assayed for the drug content.
`The extraction procedure was validated by spiking
`different drug concentrations (2–20 mg/mL) in sodium
`hydroxide in which the nail was previously dissolved.
`The amount of TH laterally diffused into the
`peripheral regions was determined by dissecting
`the peripheral nail area (4–5 mm surrounding the
`active diffusional area) which is further washed,
`dried, and weighed. The drug content
`in the
`peripheral region was determined following the
`extraction procedure described above.
`
`Resistance to Multiple Washing
`
`The ability of the nail lacquer to withstand multiple
`washings was assessed by employing a washing
`procedure developed and validated in-house. Nails
`were mounted on a nail adapter with an active
`diffusion area of 0.2 cm2 (Permegear). The whole
`assembly was sandwiched between the two chambers
`of a Franz diffusion cell (Logan Instruments Ltd).
`About 20 mL of the drug-loaded hydrophilic nail
`lacquer was applied onto the nail plate in the donor
`compartment. After complete drying of the hydro-
`philic layer (about 5 min.), about 20 mL of the vinyl
`lacquer was applied on top of the hydrophilic drug-
`loaded film to form a bilayered film. On drying of the
`vinyl film, about 500 mL of distilled water (pH 3.0) was
`loaded into the donor compartment. The water was
`allowed to stand for 1 min after which it was collected
`to estimate the amount of TH dissolved. Each
`washing step with distilled water is repeated 50
`times and the drug content in each washing was
`determined from which the cumulative amount of TH
`lost from the bilayer lacquer after 50 washings was
`computed. The second set of experiments was carried
`out by application of 20 mL of the drug-loaded
`hydrophilic nail lacquer alone. The resulting hydro-
`philic monolayer was subjected to multiple washings
`following the same protocol. The third set of trial was
`performed similarly on application of the control nail
`lacquer alone and the cumulative amount of TH lost
`after multiple washings was calculated. After sub-
`jecting the lacquers to multiple washings,
`the
`residual amount of TH present in the lacquers
`retained on the nail plate in each case was also
`
`determined by extracting the nail plate following the
`procedure described previously.
`
`Human Subject Study
`
`A clinical study was conducted in healthy human
`subjects to determine the efficacy of the developed
`nail lacquer formulations. The total study period
`spanned 6 weeks comprising of 2 weeks of treatment
`period continued by 4 weeks of follow-up. Written
`consent was obtained from each participating subject
`before the performance of the screening procedure.
`The study plan was considered adequate by the
`human ethical committee of Dehradun Institute of
`Technology, Faculty of Pharmacy, Dehradun, India
`(Ref. No. DITP #2009/01A). The inclusion criteria
`were healthy human subjects in the age group of 20–
`50 with a mean weight of 65 kg. Subjects with nail
`disease or those allergic to terbinafine were excluded
`from participation.
`The subjects were divided into three groups with
`six volunteers in each group: group I was applied
`with control nail
`lacquer. Group II was applied
`with hydrophilic nail lacquer alone that resulted in
`formation of a monolayer film. Group III was applied
`with hydrophilic nail lacquer and allowed to dry. On
`drying, the vinyl lacquer was applied on top of the
`hydrophilic lacquer film to obtain a bilayered lacquer
`film. The drying time of the hydrophilic lacquers in all
`cases was around 5 min. About 20 mL of lacquers were
`applied to the big toe of the either feet on daily basis
`for a period of 2 weeks. Volunteers were instructed to
`avoid exposure to water within 1 h of application and
`avoid activities like swimming and dish washing
`during the study period. The nail clippings were
`sampled at the beginning of the study (0 weeks), after
`the treatment period of 2 weeks and on follow-up (at
`the end of 2 and 4 weeks after stopping the application
`of the lacquer). The content of the TH in the clippings
`was estimated by HPLC following the validated
`extraction procedure outlined earlier. The volunteers
`are monitored for the local and systemic side effects
`throughout the study period.
`
`Data Analysis
`
`Statistical analysis was performed by one-way
`analysis of variance and t-test in Graph pad Instat
`5 software (GraphPad Software, Inc., La Jolla, CA).
`p-Value <0.05 was considered statistically signifi-
`cant. The data points presented in the graphs are an
`average of three/six trials. The error bars represent
`the standard deviation (SD).
`
`RESULTS AND DISCUSSION
`
`An ideal transungual formulation should require less
`frequent applications, must be easy and convenient to
`
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`BILAYERED NAIL LACQUER OF TERBINAFINE HYDROCHLORIDE
`
`4271
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`use and possess good adhesivity to the nail.15 The
`specific objectives for developing the bilayered nail
`lacquer were (i) to maximize the adhesion of the drug-
`loaded lacquer film to the nail surface employing a
`hydrophilic bioadhesive polymer, (ii) to retain the
`supersaturated hydrophilic lacquer film on the nail
`plate for prolonged period of time using a durable
`water resistant hydrophobic layer, and thereby (iii) to
`ensure delivery of effective amounts of TH across
`human nail by exploiting the combined effects of
`occlusion and transungual penetration enhancement.
`Aqueous-based nail lacquers are known to promote
`the nail hydration and the drug diffusion across the
`nail plate and hence play a major role in treatment
`of onychomycosis.8 Since the nail plate is known to
`behave like a hydrogel upon hydration, one can expect
`a better adhesion of the hydrophilic lacquer film with
`the contours of the nail plate. In addition, the water-
`based nail
`lacquers are free from the burning
`sensation when applied to the highly sensitive nail
`bed. Hydroxypropyl methylcellulose was selected as
`the film former as it forms a nonsticky, nonglossy
`bioadhesive film with good plasticity.16 The HPMC
`film formed has a matte and natural look that would
`be preferred by majority of the mycosis patients.
`PEG 400 was selected as a penetration enhancer
`based on the outcome of TranScreen-NTM, a high-
`throughput method developed in our laboratory to
`screen compounds for their ability to enhance the
`transungual delivery of TH across nail.17 The upper
`vinyl film was employed to shield the underlying
`hydrophilic film from being washed off during the
`routine day-to-day activities.
`The cross-section of the full thickness cadaver nail
`along with the applied bilayered nail lacquer has been
`shown in Figure 2. It is clearly evident from the
`photograph that the underlying hydrophilic lacquer
`film was able to establish a good contact with the
`contours of the nail plate. An intimate contact of
`
`Figure 2. Cross-section of the nail (A) with applied with
`the bilayered nail lacquer showing the drug-loaded hydro-
`philic lacquer (B) over laid with the hydrophobic vinyl
`lacquer (C).
`
`the drug-loaded film with the nail plate is a key for
`successful topical transungual delivery.8 The proxi-
`mity of the occlusive hydrophobic vinyl film and the
`underlying hydrophilic drug-loaded film is distinc-
`tively visible from the cross-section. The close contact
`of the occlusive vinyl film would be crucial
`in
`maintaining the nail in a hydrated state.
`DSC thermogram of TH, casted drug-loaded
`lacquer film and physical mixture of TH and HPMC
`E-15 are represented in Figure 3. The DSC scan of TH
`presented a sharp endothermic peak at 215.888C with
`the peak onset at 211.458C that corresponds to the
`melting point of the drug.18 The enthalpy of fusion
`(DHf) for TH in pure state was found to be 54.93 J/g. A
`broader endothermic peak at 209.338C with the peak
`onset at 203.058C was observed in the thermogram of
`the physical mixture composed of TH and HPMC E-15
`in a ratio of 5:6. The appearance of endothermic peak
`ruled out the physical incompatibility between TH
`and the polymer and indicated the crystalline nature
`of TH in the physical mixture as well. The enthalpy of
`fusion of the physical mixture was found to be 25.62 J/
`g that corresponded to the amount of TH in the
`physical mixture (45%, w/w). The complete absence
`of the endothermic peak in the DSC of the casted
`drug-loaded lacquer film indicated the chances of
`existence of TH in amorphous state as a solid solution
`in HPMC E-15 lacquer film. In addition, the absence
`of the endothermic peak ruled out the possibility of
`crystallization of TH in the casted lacquer films.
`HPMC E-15 is a well-known bioadhesive polymer
`containing a high proportion (7–12%) of hydroxyl-
`propyl groups that would render a high surface
`charge density to the lacquer film. The good
`bioadhesion of the casted lacquer films with the nail
`has been attributed to the hydrogen bonds formed
`between the carboxylic and hydroxyl group with the
`biological substrate.19 The PAF and AUC values for
`the drug loaded and placebo films were found to
`increase with increase in the contact time intervals as
`represented in Figure 4. The contact time is reported
`to affect the extent of hydration and swelling of
`polymeric films that in turn would influence the
`adhesion to the nail plate.20 Though the PAF and
`AUC at any contact time period were higher for the
`placebo lacquer films compared to TH-loaded films
`they were not significantly different from each other
`( p > 0.05, t-test). The values of PAF and AUC for the
`drug-loaded films were comparable to those obtained
`by Mididoddi and Repka19 with bioadhesive cellulose-
`based films for transungual applications. The results
`of the studies indicated that the drug-loaded hydro-
`philic lacquer film have sufficient adhesiveness to be
`retained on the surface of nail plate for prolonged
`time periods.
`The cumulative amount of TH permeated across
`the nail plate at the end of 144 h (6 days) was found to
`
`DOI 10.1002/jps
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`4272
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`SHIVAKUMAR ET AL.
`
`Figure 3. Differential scanning thermograms of terbinafine hydrochloride (A), casted
`TH-loaded lacquer films (B), physical mixture of terbinafine and HPMC E-15 (C) and
`HPMC E-15 (D).
`
`be 0.32 0.14, 1.12  0.42, and 1.42  0.53 mg/cm2 in
`the case of control, monolayer, and bilayered nail
`lacquers, respectively (Fig. 5). The amount of TH
`permeated across the nail plate in vitro, was
`significantly high with the monolayer nail lacquer
`when compared to the control nail lacquer at 48 h
`( p < 0.01), 96 h ( p < 0.05), and 144 h ( p < 0.05), which
`could be attributed to the permeation enhancer
`property of PEG 400. PEG being a plasticizer and a
`humectant, was found to swell the nail plate making
`it more soft and flexible. It has been proposed that
`transungual penetration enhancers that act by
`
`Figure 4. Peak adhesive force (A) and work of adhesion
`(B) of terbinafine hydrochloride loaded (filled columns) and
`placebo hydrophilic lacquer films (unfilled columns) at dif-
`ferent contact time measured in Texture analyzer using
`human cadaver nail plate as substrate. The data represent
`mean SD of three determinations.
`
`Figure 5. Cumulative amount of terbinafine hydrochlor-
`ide permeated across the nail plate from control (black filled
`columns), monolayer (gray filled columns), and bilayered
`(unfilled columns) nail lacquers at different time points. The
`data represent mean SD of three determinations.
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`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 10, OCTOBER 2010
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`DOI 10.1002/jps
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`Page 6
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`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
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`BILAYERED NAIL LACQUER OF TERBINAFINE HYDROCHLORIDE
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`swelling and hydrating the nail keratin do render the
`nail plate into a less dense structure with large
`pores.21 Hydration-induced structural expansion
`of the nail
`is said to facilitate the diffusion of
`permeants.22,23
`The in vitro permeation of TH from bilayered nail
`lacquer was further improved (1.26-fold) compared
`to monolayer nail
`lacquer. This increase in the
`permeation is most likely due to the occlusive nature
`of the hydrophobic vinyl film that tends to further
`augment the hydration levels of the keratin mole-
`cules. Spruit24 found that that a nail lacquer film of a
`thickness of 0.05 mm reduced the transungual water
`loss from 1.6 to 0.4 g/m2 h. Murdan et al.25 observed a
`similar decrease in transonychial water loss (TOWL)
`on application of different nail varnish formulations.
`Such a reduction in the TOWL is said to induce hyper-
`hydration of the nail plate that is thought to facilitate
`diffusion of hydrophilic compounds into the nail.26
`The drug transport into the nail is reported to be a
`function of physicochemical properties of the per-
`meant (size, charge, partition coefficient, and keratin
`binding), formulation characteristics (pH, drug, and
`water concentrations), and nail properties such as
`extent of hydration, disease condition.5 The amount of
`TH loaded in the active diffusional area on application
`of different nail lacquer formulations in vitro are
`shown in Figure 6A. The amount of TH loaded in the
`
`Figure 6. The amounts of
`terbinafine hydrochloride
`loaded in the active diffusional (A) and the peripheral areas
`(B) from different lacquers after in vitro permeation studies.
`The data represent mean SD of three determinations.
`
`diffusional area on application of the control, mono-
`layer, and the bilayer nail lacquer was found to
`be 0.28  0.07, 0.36  0.09, and 0.59 0.13 mg/mg,
`respectively. The potential of PEG 400 as a ‘‘drug
`load enhancer’’ has been already identified and
`reported in our earlier studies.17 Generally, an
`irreversible binding of drug with the nail keratin
`increases drug load in the nail plate and prevent the
`drug from reaching the deeper tissues in the nail bed.
`However, TH loaded in the nail plate during
`permeation is known to form a depot from which
`the drug would be released subsequently. Our earlier
`studies revealed that about 36.45  6.80% of TH
`loaded in the nail plate (by equilibrating with aqueous
`TH solution for 1 week) was released subsequently
`over a span of 18 days while the remaining was bound
`irreversibly to keratin.14 The drug load achieved by
`the bilayered nail lacquer was found to be 1.6-fold
`more than that attained by the monolayer nail
`lacquer.
`The active diffusion area is the area of nail that
`would be in direct contact with the formulation
`applied. Onychomycosis is known to affect the whole
`nail apparatus including the part of the nail that may
`not be accessible due to overlapping of the nail folds.
`Thus, the drug loaded in the active diffusional area
`must diffuse laterally into the peripheral area that
`has no direct access to the formulation. The drug load
`in the peripheral area is considered to play a crucial
`role in the success of topical onychomycosis mono-
`therapy.14 The amount of TH loaded in the peripheral
`area following application of the control, monolayer,
`and bilayered lacquers was found to be 0.03 0.01,
`0.03 0.02,
`0.07 0.02 mg/mg,
`and
`respectively
`(Fig. 6B). There was no significant difference in the
`drug load in the peripheral nail area between control
`and monolayer lacquers. However, the bilayered nail
`lacquer resulted in twofold higher drug load in the
`peripheral nail than others ( p < 0.05).
`An in vitro test was developed and validated to
`evaluate the resistance of the bilayered lacquer to
`multiple washings. Distilled water with pH 3.0 was
`used as a wash liquid considering the good solubility
`of TH in water in acidic conditions. The cumulative
`amount of TH lost on 50 washings was found to be
`62.34  3.31%, 61.32 2.62%, and 0.66 0.24% from
`the control lacquer, monolayer, and bilayered nail
`lacquer as represented in Figure 7.
`Negligible amount of TH (<1%) was lost during
`multiple washings from the bilayered nail lacquer.
`The drug loss was observed during the first five
`washings while no TH was detected from the
`subsequent washings. The residual amounts of TH
`present in the lacquer retained on the nail plate after
`the test was found to be 39%, 38%, and 99% for
`the control lacquer, monolayer, and bilayered nail
`lacquer, respectively. The study clearly demonstrated
`
`DOI 10.1002/jps
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`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 10, OCTOBER 2010
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`Page 7
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`Anacor Exhibit 2006
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00168
`
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`4274
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`SHIVAKUMAR ET AL.
`
`Figure 7. The cumulative amounts of terbinafine hydro-
`chloride lost from the control (gray filled column), mono-
`layer (unfilled columns), and bilayered (black filled column)
`lacquers during multiple washings. The data represent
`mean SD of three determinations.
`
`Figure 9. Content of terbinafine hydrochloride in the
`nail clippings collected at the end of 2 weeks (unfilled
`columns) and 4 weeks (filled columns) after stopping the
`application of the nail lacquers in human subjects. The data
`represent mean SD of six determinations.
`
`the ability of the hydrophobic vinyl lacquer film to
`effectively resist multiple washings in vitro and
`protect the underlying drug-loaded hydrophilic lac-
`quer film. The results obtained were supported by the
`report which indicates that poly (4-vinyl phenol)
`forms a durable film lasting over a period of 24–48 h.27
`The clinical study involving healthy human volun-
`teers spanned a treatment period of 2 weeks and a
`follow-up period of 4 weeks. In spite of the routine
`activ