Transmucosal Permeation of Topically
`Applied Diclofenac and Piroxicam
`
`Pieter van der Bijl, BChD, BSc (Hons) (Pharmacol), PhD, DSc
`Armorel D. van Eyk, PhD
`Heiner I. Seifart, Dr rer nat
`
`Ianda Viljoen, BSc (Hons) (Pharmacol)
`Marli Jooste, BSc (Hons) (Pharmacol)
`
`Department ofPharmacology, Faculty ofHealth Sciences, University ofStellenbosch, ingerberg, South Afiica
`
`KEY WORDS: Diclofenac, piroxicam,
`
`human mucosa, permeability studies
`
`ABSTRACT
`
`Nonsteroidal antiinflammatory drugs
`(NSAIDs) are frequently used for the treat-
`ment of acute myalgias, orthopedic injuries,
`postoperative pain, chronic rheumatoid
`arthritis, and osteoarthritis. This study
`involves the permeation of 2 NSAIDs,
`diclofenac and piroxicam, from topically
`applied solutions and gels through human
`vaginal mucosa as a model of hiccal
`mucosa. Permeation of diclofenac and
`
`piroxicam from the solutions and gels
`through human vaginal mucosa was deter-
`mined using a flow-through diffusion appa-
`ratus. Vaginal specimens were obtained
`from 8 postmenopausal patients aged 57 d:
`16 years (mean i standard deviation [SD];
`range, 32—76 y) after vaginal hysterec—
`tomies. Experiments were conducted at
`20°C and over a time period of 24 hours.
`High-pressure liguid chromatography
`(HPLC) analysis was used as a detection
`method. Statistical tests used included
`
`analysis of variance (ANOVA) and Duncan’s
`multiple range test for determination of
`steady state and an unpaired t test with
`Welch’s correction for comparing differ-
`ences between the mean flux values at each
`
`time point. Flux rates of both diclofenac and
`piroxicam from aqueous solutions were sig-
`
`nificantlyhigher than those from the gels.
`Steady-state flux rates of the 5 mg/mL
`diclofenac solution were approximately half
`that of the 10 mg/mL solution, whereas max-
`imal partitioning to the mucosa from the
`vehicle and/or saturable diffusion kinetics
`
`was achieved at concentrations of 5 mg/mL
`or less for the piroxicam solutions under the
`experimental conditions used. The diffiision
`
`of diclofenac and piroxicam across mucosal
`surfaces appears to be more efficient when
`aqueous solutions of these compounds are
`used as opposed to gel formulations. This
`must be considered when vehicles for both
`
`these NSAIDs are chosen. There also
`
`appeared to be a limit to the release of
`piroxicam from the vehicle and/or its flux
`rate across mucosal tissues.
`
`INTRODUCTION
`
`Nonsteroidal antiinflammatory drugs
`(NSAIDs) belong to the most frequently
`used drugs worldwide, with as many as 8%
`of the global adult population taking pre-
`scribed forms of these agents at any given
`time.1 However, it is estimated that they are
`also responsible for approximately 25% of
`all adverse drug reaction reports.2 In this
`respect, NSAID—related gastrointestinal toxi-
`city is the most frequently observed adverse
`event, and it is a significant cause of mor-
`bidity and mortality.3 The ingestion of
`NSAIDs increases the relative risk of upper
`
`The Journal oprplied Research ° Vol. 3, N0. 4, Fall 2003
`
`  
`
`

 
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`505
`MYLAN - EXHIBIT 1025
`
`

`

`gastrointestinal tract bleeding 5—fold,4 is ele—
`
`mucosa, to aqueous and commercially avail—
`
`vated in the elderly,5 and might be even
`
`able gel forms of diclofenac and piroxicam.
`
`higher for certain NSAIDsF’7 The NSAIDs
`are often the first choice of treatment for
`
`patients with acute myalgias, orthopedic
`
`injuries, postoperative pain, chronic rheuma-
`toid arthritis, and osteoarthritis.8
`
`In an attempt to reduce the relatively
`
`high incidence of serious adverse effects
`
`associated with the systemic use of
`
`NSAIDs, a growing number of topical for-
`
`mulations of these drugs have become com-
`
`mercially available. These topical
`formulations, either on their own or as
`
`adjuncts to reduced dosages of systemic
`
`agents, have proven to be useful in the man-
`
`agement of a variety of musculoskeletal and
`
`rheumatic diseases}9 Although the topical
`
`NSAIDs have mainly been studied regarding
`their transdermal diffusion kinetics, these
`
`agents might also have useful applications
`
`when topically applied to mucous mem—
`
`branes. In this respect, piroxicam, benzy—
`
`damine, ketoprofen, flunoxaprofen, and
`
`diclofenac have all been topically applied to
`
`mucosa] membranes for a variety of condi-
`
`MATERIALS AND METHODS
`
`Vaginal Mucosa
`
`Specimens were obtained from excess tissue
`
`removed from 8 postmenopausal patients
`
`aged 57 i 16 years (mean i standard devia-
`
`tion [SD]; range, 32—76 y) after vaginal hy s—
`
`terectomies at the Louis Leipoldt and
`
`Panorama Mediclinic Hospitals, Bellville,
`
`South Africa. Surgical specimens were
`
`immediately placed in a transport fluid, pre-
`
`pared as previously described,‘“‘ and trans-
`
`fe ned to our laboratory within 1 hour. Excess
`
`connective tissue was trimmed away and all
`
`specimens were snap-frozen in liquid nitro-
`
`gen and stored at -85°C for periods up to 6
`
`months.22 No specimens were obtained in
`
`which there was clinical evidence of any dis-
`
`ease that might have influenced the perm e-
`
`ability characteristics of the vaginal mucosa.
`
`The study was approved by the Ethics
`Committee of the University of Stellenbosch
`and the Tygerberg Hospital.
`
`tions ranging from pain to inflammation.“HS
`
`Permeability Experiments
`
`However, the majority of the topically avail-
`able NSAIDs on the market have been for-
`
`mulated for cutaneous application and hence
`
`contain components and enhancers suitable
`
`for improving skin, but not necessarily
`
`mucosal, absorption.
`
`We have previously shown that human
`
`vaginal mucosa can be used as a model for
`
`the buccal mucosa for in vitro permeability
`
`studies of a wide variety of chemical com-
`
`pounds.”21 Furth e more, we have demon-
`strated that both these tissues can be
`
`snap-frozen in liquid nitrogen and stored at
`
`—85°C for many months and thereafter used
`
`for permeability experiments without signifi-
`
`cant changes in permeability characteristics.22
`
`Before each permeability experiment tissue
`
`specimens were thawed at room temperature
`
`in phosphate-bu ffered saline (PBS, pH 7.4).
`
`Thereafter the specimens were cut into
`5-mm2 sections and mounted in flow-
`
`through diffusion cells (exposed areas 0.039
`
`cmz) as previously described,‘”‘ and perm e-
`
`ation studies performed on 7 tissue repli-
`
`cates for each patient. Before beginning
`
`each permeability experiment, tissue disks
`
`were equilibrated for 10 minutes with PBS
`
`(pH 7.4) at 20°C in both the donor and
`
`receiver compartments of the diffusion cells.
`
`After equilibration, the PBS was removed
`
`from the donor compartment and replaced
`
`with either 1 mL of 10 mg/mL or 5 mg/mL
`
`In view of the fact that the therapeutic
`
`diclofenac solution in PBS containing 10%
`
`efficacy of NSAIDs depends on their pene-
`
`tration into the mucosa] and underlying tis-
`
`ethanol or 0.5 mL of 10 mg/g diclofenac gel
`(Voltaren Emulgel”, Novartis SA [Pty] Ltd,
`
`sues, it was the objective of the present study
`
`Rivonia, South Africa). Alternatively, 1 mL
`
`to investigate the permeability of human
`
`of 5 mg/mL, 10 mg/mL of piroxicam solu-
`
`vaginal mucosa, as a model of buccal
`
`tion, or 0.5 mL of the 5 mg/g piroxicam gel
`
`506
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`
`

`

`(Rheugesic gelD, Cipla-Medro [Pty] Ltd,
`
`an Agilent Chem Station (Agilent
`
`Bellville, South Africa) was used. The gels
`were covered with a Teflon disk and 1 mL
`
`of PBS. PBS at 37°C was pumped through
`
`the receiving chambers at a rate of 1.5 mL/h
`
`and collected, by means of a fraction collec-
`tor, at 2-hour intervals for 24 hours. The
`
`permeability study was performed under
`
`sink conditions, ie, at the completion of
`each run the concentration of diclofenac or
`
`piroxicam in the acceptor chamber never
`
`reached 10% of that in the donor compart-
`
`ment. The permeant was detected by means
`
`of HPLC analysis. Crystalline diclofenac
`
`was obtained from Sigma Chemical
`
`Company (St. Louis, MO). For the piroxi-
`
`cam solution, a readily available IM solution
`
`(Feldenefl IM, Pfizer Laboratories [Pty] Ltd,
`
`Sandton, South Africa) was used. Each mL
`
`of Feldene” 1M solution contains 20 mg of
`piroxicam, 2% m/v benzyl alcohol, and 10%
`m/v ethanol.
`
`HPLC Detection of Permeants
`
`Pe meant—containing effluent samples, col-
`
`lected from the acceptor compartments of
`
`the perfusion apparatus over the 2-hour
`
`sampling intervals, were analyzed using an
`Agilent 1100 series high-performance liquid
`
`chromatograph (Agilent Technologies,
`Waldbronn, Germany) with a Zorbax XDB
`C18, 150 mm [I 4.6 mm (ID), 5-|]m column.
`The temperature was maintained at 40°C
`and flow rates of 1.0 mL/min and 1.2
`
`mL/min were used for diclofenac and pirox-
`icam, respectively. The mobile phase con-
`
`sisted of a mixture of 2 solvents, A (50 mM
`KHZPO4, pH 5.42) and B (acetonitrile—iso-
`
`propanol; 4:1 v/v). Isocratic mixtures of AB
`were 65:35 and 58:42 for diclofenac and
`
`piroxicam, respectively. Solvents were “pro
`
`analisi” (E Merck, Darmstadt, Germany)
`and were filtered through a 0.45 Elm filter.
`Aliquots of 50 [IL from each sample were
`
`injected directly into the column. Diclofenac
`and piroxicam were detected at 273 nm
`
`(retention time 2.3 min) and 354 nm (reten-
`tion time 2.2 min), respectively. Total run
`
`time was 3.5 minutes. Recording and inte-
`
`gration of peaks was performed by means of
`
`Technologies, Waldbronn, Germany).
`
`Spiked standards over the expected concen-
`tration range (0.5—20 [jg/mL) were random—
`ly included in each batch.
`
`Calculation of Flux Values
`
`Flux (J) values across vaginal tissue were
`
`calculated by means of the relationship
`
`J=Q/A|:|t(ugxcm'2xmin'1)
`
`where Q = quantity of substance crossing
`vaginal tissue (in fig), A = tissue area
`exposed (in cm2) and t = time of exposure
`
`(in min).
`
`Steady-State Kinetics
`
`When no statistically significant differences
`
`(P <0.05) (ANOVA and Duncan’s multiple
`
`range test) between flux values were obtained
`over at least 2 consecutive time intervals, a
`
`steady-state (equilibrium kinetics) was
`
`assumed to have been reached for a particular
`
`specimen and diclofenac or piroxicam.
`
`Statistical Analysis
`
`An unpaired t test with Welch’s c01rection
`
`was used to investigate possible differences
`
`between flux means of vaginal tissues at 2-
`
`hour intervals. A significance level of P
`
`<0.05 was used for all tests and comparisons.
`
`RESULTS
`
`Mean flux values for solubilized diclofenac
`
`(5 mg/mL and 10 mg/mL) and gel
`diclofenac (Voltaren Emulgel®, 10 mg/g)
`
`through frozen/thawed vaginal mucosa ve r-
`sus time are shown in Figure 1. Steady-state
`
`flux conditions were reached after approxi-
`
`mately 9 hours across vaginal mucosa for
`both the diclofenac solutions and gel. Mean
`steady-state flux values of 1.682 i 0.164
`(standard error of mean [SEM]) [lg/cmZ/min
`and 3.969 1 0.292 (SEM) [g/cmZ/min were
`found for the 5 mg/mL and 10 mg/mL solu-
`tions, respectively. The diclofenac gel (10
`
`mg/g) yielded a mean steady-state flux
`value of 0.626 r 0.058 (SEM) [g/cmZ/min,
`which is found to be on average 6.3 times
`lower than the 10 mg/mL diclofenac solu—
`
`The Journal oprplied Research ' Vol. 3, N0. 4, Fall 2003
`
`507
`
`

`

`
`
`
`
`
`
`
`
`
`
`Bar represents SEEM
`
`
`Z 10 mglmi
`a 5mglmi
`9 Gel
`
`30
`
`5,
`
`EE
`
`.“J
`E0.
`=3at
`X
`.2LL
`
`Figure 1. Mean flux values for diclofenac solutions (5 mg/mL and 10
`mg/mL) and gel (10 mg/g) across human vaginal mucosa.
`
`(AOO0
`
`
`
`CumulativeFlux(figcm‘z)
`
`
`
`
`
`
`
`
`
`Time (PM?)
`
`Figure 2. Cumulative flux values for diclofenac solutions (5 mg/mL
`and 10 mg/mL) and gel (l0 mg/g) across human vaginal mucosa.
`
`tion used in the perme—
`
`ability experiments.
`Statistically significant
`differences (P <0.05)
`between flux rates of
`
`both diclofenac con-
`
`centrations (5 mg/mL
`and 10 mg/mL) and
`diclofenac gel (10
`mg/g) across vaginal
`mucosa were found
`
`after approximately 6
`hours. The diclofenac
`
`solution had higher
`flux rates than the gel
`form, whereas the flux
`
`rates of the 5 mg/mL
`diclofenac solution were
`
`found to be approx i-
`mately half that of the
`10 mg/mL diclofenac
`
`solution (Fig. 1). Mean
`
`apparent release con-
`stants (slopes) and lag
`times (X-axis inter-
`cepts) were obtained by
`linear regression analy-
`sis of plots of cumula-
`tive amount of released
`
`drug (fig/c1112) versus
`square root of time
`(hm)?25 For diclofenac
`5% and 10% solutions
`
`and gel, apparent
`release constants were
`
`751.9 i 34.8, 1826 :t
`
`74.9, and 288.6 d: 9.7
`
`[g/cmZ/hl’z, lag times
`were 2.91, 2.87, and
`
`2.66 h‘”, and rZ-values
`
`were 0.9873, 0.9900,
`
`
`
`
`
`
`
`
`Flux(gtg.cm‘z.min")
`
`
`
`Bar represents SEEM
`
`E 10 mgiml
`a 5mglml
`
`
`
`Figure 3. Mean flux values for piroxicam solutions (5 mg/mL and 10
`mg/mL) and gel (5 mg/g) across human vaginal mucosa.
`
`and 0.9931, respective-
`ly (Fig. 2). The non-lin-
`early related portions
`of the data curves, ie, <3 h”, were excluded
`
`from the linear regression plots for both
`
`diclofenac and piroxicam and hence are not
`shown in the figures (Figs. 2 and 4).
`
`Overall mean flux values for piroxicam
`intramuscular injectable solution (Feldene®, 5
`
`mg/mL and 10 mg/mL) and piroxicam gel
`(Rheugesic gel®, 5 mg/g) across vaginal
`
`mucosa over time are shown in Figure 3.
`Steady-state flux values were achieved after
`
`approximately 6 hours across the vaginal
`
`mucosa for both the piroxicam solutions and
`
`508
`
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`
`

`

`p arlition coefficient,
`
`and degree of ioniza-
`
`tion of the permeant
`
`molecules are impor-
`tant.26 The transmem-
`
`fizz/my”!
`o 6610 mQIm
`
`
`brane diffusion process
`
`is passive in nature and
`depends on a concen-
`tration differential as
`the driving force, each
`
` molecule requiring
`
`
`2i
`
`EQm:1.H—
`
`xaL
`
`a.
`.3H
`.5!:1
`E:z
`0
`
`Figure 4. Cumulative flux values for piroxicam solutions (5 mg/mL and
`10 mg/mL) and gel (5 mg/g) across human vaginal mucosa.
`
`kinetic energy to effect
`a net movement down
`
`this gradient. Permeant
`molecules must there-
`
`the gel. Mean steady—state flux rates of 1.665
`d: 0.160 (SEM) [Ig/cmZ/minand 1.578 d: 0.128
`(SEM) [Ig/cmZ/minwere obtained for the 5
`mg/mL and 10 mg/mL piroxicam solutions,
`respectively. The piroxicam gel (5 mg/g) yield-
`
`ed a mean steady-state flux rate of 0.589 d:
`0.038 (SEM) [jg/cmZ/min, which was approxi-
`mately 2.8 times lower than the flux rate
`obtained for the 5 mg/mL piroxicam solution.
`
`Significant differences (P <0.05) between flux
`
`rates across vaginal mucosa of the gel and
`either of the 2 solutions were found from
`
`approximately 4 hours onward. However, no
`statistically significant differences in flux val-
`
`ues were obtained between the 2 piroxicam
`solutions (5 mg/mL and 10 mgmL) used. For
`
`piroxicam 5% and 10% solutions and gel,
`
`apparent release constants were 808.1 i 21.6,
`778.3 d: 18.0, and 286.2 i 6.1 [lg/cmZ/h“, lag
`times were 2.59, 2.59, and 2.61 h“, and rz—val-
`
`ues were 0.9957, 0.9968, and 0.9973, respec-
`
`tively (Fig. 4).
`
`DISCUSSION
`
`Mucosal delivery of therapeutic agents
`
`involves the penetration of a drug into
`
`mucosal surfaces either for the purpose of
`treating diseases or alleviating symptoms in
`deeper lying tissues, or to treat systemic dis-
`
`fore diffuse through the vehicle in which
`
`theyare contained to the mucosal interface
`
`and have to partition from the formulation
`
`into the upper layers of the tissue. From
`here molecules must diffiise within the
`
`mucosa, equilibrating laterally, and must
`emerge, eventually under steady-state condi-
`tions, from the distal surface of the tissue.
`
`Adsorptive interaction might be extensive in
`
`this layer, forming a reservoir of the perme-
`
`ant molecules. Further partitioning into
`
`neighboring tissue strata or into the receptor
`
`fluid then takes place under the influence of
`
`the concentration gradient, and adsorption
`
`might occur once again. Diffusion through
`
`any one of the layers or any of the partition-
`ing events might form the rate—limiting step
`controlling the overall rate of permeation.
`
`Initiallythe concentration gradient across
`
`the mucosa will not be linear as the perme-
`
`ant equilibrates within the tissue. However,
`
`afier sufficient time has elapsed, steady state
`
`will be achieved and the effective permeant
`
`concentration at all points in the tissue will
`remain constant.
`
`Penetration of drugs into tissues is
`
`dependent on the influence of the vehicle on
`
`the thermodynamic activity of the active
`
`ingredient. Diclofenac and piroxicam are
`
`ease by achieving systemically active levels
`
`sparingly soluble in water, hence their
`
`of the agent. To achieve the required degree
`
`release is favored from aqueous solutions
`
`of penetration, not only the properties of the
`membranes involved, but also the chemical
`
`and hydrophilic bases such as gels, which
`
`are poor solvents for these drugs. As a
`
`nature, size and conformation, lipid/water
`
`result, there are less drug—vehicle interac-
`
`The Journal oprplied Research ' Vol. 3, N0. 4, Fall 2003
`
`509
`
`

`

`tions in these solvent systems and this leads
`
`classified as chemical penetration
`
`to improved partitioning into the mucosa.
`
`enhancers, ie, they might improve drug dif-
`
`The pKa-value of diclofenac is 4, whereas
`
`fusion by modifying the thermodynamic
`
`that of piroxicam is 6.3 (dioxanezwater
`
`properties of the drug or by violating the
`
`2: 1).27 At the pH values of the PBS buffer
`
`mucosal barrier making it more permeable.
`
`system used in this study, ie, 7.4, approx i-
`
`mately 99.9% of the diclofenac and approxi-
`
`mately 92.6% of the piroxicam are present
`
`in their dissociated forms. Although this
`
`improves diffusion through the hydrophilic
`
`outer layers of the epithelium, it does not
`
`facilitate penetration of the lipoidal layer,
`
`which is thought to constitute the major per-
`
`meability barrier of vaginal and buccal
`mucosa and is located in the outer third of
`
`the epithelium.”28
`
`It is clear that the flux rates across vagi-
`
`nal mucosa of the aqueous solutions of
`
`diclofenac are statistically significantly
`
`higher than those of the gel (Fig. 1). This is
`
`also reflected in the relative apparent release
`
`constants, showing that the release of
`
`diclofenac from the gel formulation is well
`
`described by the “Higuchi” model in which
`
`the rate-controlling step is the process of
`
`diffusion through the gel matrix (Fig. 2).”—25
`
`This relationship exists for formulations in
`
`which the drug is fiilly dissolved or is in
`
`suspension. Hence, the mucosa has no sig-
`
`nificant effects on drug release, the latter
`
`being controlled mainly by the properties of
`
`the formulation. This might be attributed to
`
`the higher viscosity of the gel matrix com-
`
`pared with the aqueous solutions as well as
`
`the presence of a fatty emulsion base con-
`
`taining isopropanol and propylene glycol, all
`
`of which retard partitioning of the
`diclofenac to the mucosal surface. The
`
`effect of these cosolvents on the penetrabili-
`ty of diclofenac has been well documented.”
`Similar observations for flux rates and
`
`apparent release constants were made for
`
`the injectable piroxicam solutions and the
`
`gel (Figs. 3 and 4). Although the piroxicam
`
`gel contained 0.01% m/m benzyl alcohol as
`a preservative, the inj ectable solutions had a
`considerably higher total alcohol content
`
`(benzyl alcohol, 2% m/V and ethyl alcohol,
`
`However, the point at which substances
`become enhancers has not been well
`
`defined. Both of these alcohols are also
`
`cosolvents and aid solubilization of the
`
`almost water-insoluble piroxicam into the
`
`gel vehicle, thereby negatively affecting its
`
`partitioning into the mucosa. The net effect
`
`of these complex interactions between the
`
`constituents of the mucosa, piroxicam, and
`
`the components of the donor vehicle that
`
`might enhance drug permeability or retain it
`
`in the formulation are difficult to predict. It
`
`is also interesting to note that notwithstand-
`
`ing the fact that the concentration of pirox i-
`
`cam in the 10 mg/mL solution was double
`that of the other solution, no statistically sig-
`nificant differences between the steady-state
`flux rates for these 2 solutions were found.
`
`This appears to indicate that maximal parti-
`
`tioning to the mucosa from the vehicle
`
`and/or saturable diffiision kinetics for pirox-
`icam across the tissue is achieved at concen-
`
`trations of 5 mg/mL or less under the
`
`experimental conditions used.
`
`CONCLUSION
`
`We have demonstrated that diffusion of
`
`diclofenac and piroxicam into mucosa
`appears to be more efficient when aqueous
`
`solutions of these compounds are used
`instead of gels formulated for transcuta-
`
`neous use. This aspect should be kept in
`mind when vehicles for both these NSAIDs
`
`intended for intraoral, or even intravaginal,
`transmucosal use are chosen. Furthermore,
`
`there appears to be a limit to the release of
`
`piroxicam from the vehicle and/or its flux
`rate across mucosal tissues. These aspects
`
`will, however, require further investigation.
`
`ACKNOWLEDGMENTS
`
`The authors thank the University of
`Stellenbosch and the SA Medical Research
`
`10% m/v). Both of these compounds can be
`
`Council for supporting this work.
`
`510
`
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`
`

`

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`
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`
`Stiel D: Exploring the link between gastrointesti-
`nal complications and over-the-counter anal-
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`
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