Transmucosal Permeation of Topically
`Applied Diclofenac and Piroxicam
`
`Pieter van der Bijl, BChD, BSc (Hons) (Pharmacol), PhD, DSc
`Armorel D. van Eyk, PhD
`HeinerI. Seifart, Dr rer nat
`Ianda Viljoen, BSc (Hons) (Pharmacol)
`Marli Jooste, BSc (Hons) (Pharmacol)
`
`Department ofPharmacology, Faculty ofHealth Sciences, University ofStellenbosch, Tygerberg, South Africa
`
`KEY WORDS: Diclofenac, piroxicam,
`human mucosa, permeability studies
`
`ABSTRACT
`
`Nonsteroidal antiinflammatory dugs
`(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 modelof buccal
`mucosa. Permeation of diclofenac and
`piroxicam from the solutions and gels
`through human vaginal mucosa wasdeter-
`mined using a flow-through diffusion appa-
`ratus. Vaginal specimens were obtained
`from 8 postmenopausal patients aged 57 +
`16 years (mean + standard deviation [SD];
`range, 32-76 y) after vaginal hysterec-
`tomies. Experiments were conducted at
`20°C and overa timeperiod of 24 hours.
`High-pressure liguid chromatography
`(HPLC)analysis was usedas 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 ¢ 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 weresig-
`
`nificantly higher than those from thegels.
`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 diffusion
`of diclofenac and piroxicam across mucosal
`surfaces appears to be more efficient when
`aqueous solutions of these compoundsare
`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 mucosaltissues.
`
`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.’ However,it is estimated that they are
`also responsible for approximately 25% of
`all adverse drug reaction reports.’ In this
`respect, NSAID-related gastrointestinal tox 1-
`city is the most frequently observed adverse
`event, and it is a significant cause of mor-
`bidity and mortality.’ The ingestion of
`NSAIDsincreasesthe relative risk of upper
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`gastrointestinal tract bleeding 5-fold,* is ele-
`vated in the elderly,’ and might be even
`higher for certain NSAIDs.°’ The NSAIDs
`are often the first choice of treatment for
`patients with acute myalgias, athopedic
`injuries, postoperative pain, chronic rheuma-
`toid arthritis, and osteoarthritis.*
`In an attempt to reducethe relatively
`high incidence of serious adverse effects
`associated with the systemic use of
`NSAIDs, a growing numberoftopical 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-
`agementof a variety of musculoskeletal and
`rheumatic diseases.”° Although the topical
`NSAIDshave mainly been studied regarding
`their transdermal diffusion kinetics, these
`agents might also have useful applications
`whentopically applied to mucous mem-
`branes. In this respect, piroxicam, benzy-
`damine, ketoprofen, flunoxaprofen, and
`diclofenac haveall been topically applied to
`mucosal membranesfor a variety of condi-
`tions ranging from pain to inflammation.'*"°
`However, the majority of the topically avail-
`able NSAIDs on the market have been for-
`mulated for cutaneousapplication and hence
`contain components and enhancerssuitable
`for improving skin, but not necessarily
`mucosal, absorption.
`Wehavepreviously shown that human
`vaginal mucosa can be used as a modelfor
`the buccal mucosafor in vitro pameability
`studies of a wide variety of chemical com-
`pounds.'**! Furthemore, we have demon-
`strated that both these tissues can be
`snap-frozen in liquid nitrogen and stored at
`—85°C for many monthsandthereafter used
`for permeability experiments without signifi-
`cant changes in permeability characteristics.”
`In view ofthe fact that the therapeutic
`efficacy of NSAIDs dependson their pene-
`tration into the mucosal and underlyingtis-
`sues, it was the objective of the present study
`to investigate the permeability of human
`vaginal mucosa, as a model of buccal
`
`mucosa, to aqueous and commercially avail-
`able gel forms of diclofenac and piroxicam.
`
`MATERIALS AND METHODS
`
`Vaginal Mucosa
`Specimens were obtained from excesstissue
`removed from 8 postmenopausalpatients
`aged 57 + 16 years (mean + standard devia-
`tion [SD]; range, 32—76 y) after vaginalhy 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-
`fened to our laboratory within 1 hour. Excess
`connective tissue was trimmed awayandall
`specimens were snap-frozenin liquid nitro-
`gen andstored at -85°C for periods up to 6
`months.” No specimens were obtained in
`whichthere wasclinical evidence of any dis-
`ease that might have influenced the perm e-
`ability characteristics of the vaginal mucosa.
`The study was approvedby the Ethics
`Committee of the University of Stellenbosch
`and the Tygerberg Hospital.
`
`Permeability Experiments
`Before each permeability experimenttissue
`specimens were thawed at room temperature
`in phosphate-bu ffered saline (PBS, pH 7.4).
`Thereafter the specimenswere cut into
`5-mm/sections and mountedin flow-
`through diffusion cells (exposed areas 0.039
`cm”) as previously described,'*” and perm e-
`ation studies performed on7 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
`diclofenac solution in PBS containing 10%
`ethanol or 0.5 mL of 10 mg/g diclofenac gel
`(Voltaren Emulgel’, Novartis SA [Pty] Ltd,
`Rivonia, South Africa). Altematively, 1 mL
`of 5 mg/mL, 10 mg/mLofpiroxicam solu-
`tion, or 0.5 mL of the 5 mg/g piroxicam gel
`
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`(Rheugesic gel", Cipla-Medro [Pty] Ltd,
`Bellville, South Africa) was used. The gels
`were covered with a Teflon disk and 1 mL
`of PBS. PBS at 37°C was pumpedthrough
`the receiving chambersat a rate of 1.5 mL/h
`and collected, by meansofa 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 chambernever
`reached 10% ofthat in the donor compart-
`ment. The permeant was detected by means
`of HPLCanalysis. Crystalline diclofenac
`was obtained from Sigma Chemical
`Company (St. Louis, MO). For the pirox i-
`cam solution, a readily available IM solution
`(Feldene! IM, Pfizer Laboratories [Pty] Ltd,
`Sandton, South Africa) was used. Each mL
`of Feldene! IM solution contains 20 mg of
`piroxicam, 2% m/v benzyl alcohol, and 10%
`m/v ethanol.
`
`HPLC Detection of Permeants
`
`Pe rmeant—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-performanceliquid
`chromatograph (Agilent Technologies,
`Waldbronn, Germany) with a Zorbax XDB
`Cys, 150 mm [] 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
`KH,PO,, pH 5.42) and B (acetonitrile—iso-
`propanol; 4:1 v/v). Isocratic mixtures of A:B
`were 65:35 and 58:42 for diclofenac and
`piroxicam, respectively. Solvents were “pro
`analisi” (E Merck, Darmstadt, Germany)
`and werefiltered through a 0.45 []m filter.
`Aliquots of 50 [JL 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 andinte-
`gration of peaks was performed by means of
`
`an Agilent Chem Station (Agilent
`Technologes, Waldbronn, Germany).
`Spiked standards over the expected concen-
`tration range (0.5—20 []g/mL) were random-
`ly includedin each batch.
`
`Calculation of Flux Values
`
`Flux (J) values across vaginal tissue were
`calculated by meansofthe relationship
`
`J=Q/A [Jt (ug x cm” x min")
`
`where Q = quantity of substance crossing
`vaginal tissue (in []g), A = tissue area
`exposed (in cm’) and t = time of exposure
`(in min).
`
`Steady-State Kinetics
`Whennostatistically significant differences
`(P <0.05) (ANOVA and Duncan’s multiple
`range test) between flux values were obtained
`over at least 2 consecutive timeintervals, a
`steady-state (equilibrium kinetics) was
`assumedto have been reachedfor a patticular
`specimen and diclofenac or piroxicam.
`
`Statistical Analysis
`An unpaired ¢ test with Welch’s correction
`wasused to investigate possible differences
`between flux means of vaginal tissues at 2-
`hourintervals. A significance level of P
`<0.05 wasusedforall tests and comparisons.
`
`RESULTS
`
`Meanflux values for solubilized diclofenac
`(5 mg/mL and 10 mg/mL)and gel
`diclofenac (Voltaren Emulgel®, 10 mg/g)
`through frozen/thawed vaginal mucosa vet-
`sus time are shown in Figure 1. Steady-state
`flux conditions were reachedafter approx i-
`mately 9 hours across vaginal mucosa for
`both the diclofenac solutions and gel. Mean
`steady-state flux values of 1.682 + 0.164
`(standard error of mean [SEM]) []g/cm?/min
`and 3.969 + 0.292 (SEM) [/cm?/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 + 0.058 (SEM)[/cm?/min,
`which is found to be on average 6.3 times
`lower than the 10 mg/mLdiclofenac solu-
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`The JournalofApplied Research « Vol. 3, No. 4, Fall 2003
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`

`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, whereasthe flux
`rates of the 5 mg/mL
`diclofenac solution were
`found to be approx i-
`mately half that of the
`10 mg/mLdiclofenac
`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 ([g/cm’) versus
`square root of time
`(h'”).?>*For diclofenac
`5% and 10% solutions
`and gel, apparent
`release constants were
`751.9 + 34.8, 1826 +
`74.9, and 288.6 + 9.7
`[e/cm7/h'”, lag times
`were 2.91, 2.87, and
`2.66 h'?, and r’-values
`were 0.9873, 0.9900,
`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 henceare not
`shownin the figures (Figs. 2 and 4).
`Overall mean flux values for piroxicam
`intramuscular injectable solution (Feldene®, 5
`
`
`
`
`
`
`Time (h)
`
`Figure 1. Mean flux values for diclofenac solutions (6 mg/mL and 10
`mg/mL) and gel (10 mg/g) across human vaginal mucosa.
`
`
`
`CumulativeFlux(yig.cm)
`
`
`B10 mg/ml
`
`
`05 mg/ml
`
`
`*@ Gel
`
`
`
`Time (h'”)
`
`Figure 2. Cumulative flux values for diclofenac solutions (6 mg/mL
`and 10 mg/mL) and gel (10 mg/g) across human vaginal mucosa.
`
`
`
`Flux(ug.cm™.min™)
`
`
`
`Bar represents SEM
`
`0 10 mg/ml
`=a S5mgiml
`Gel
`
`30
`
`Time(h)
`
`Figure 3. Mean flux values for piroxicam solutions (5 mg/mL and 10
`mg/mL) and gel (5 mg/g) across human vaginal mucosa.
`
`mg/mL and 10 mg/mL)and piroxicam gel
`(Rheugesic gel®, 5 mg/g) across vaginal
`mucosaovertime are shown in Figure 3.
`Steady-state flux values were achieved after
`approximately 6 hours across the vaginal
`mucosafor both the piroxicam solutions and
`
`508
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`Vol. 3, No. 4, Fall 2003 * The Journal ofApplied Research
`
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`
`
`Bar represents SEM
`
`
`0110 mg/ml
`a 5mg/ml
`@ Gel
`
`
`~ 5E
`
`4
`Eo
`a=
`h
`
`*=w
`
`

`

` molecule requiring
`
`
`ae ,
`*Gel maim
`
`
`partition coefficient,
`and degree of ioniza-
`tion of the permeant
`molecules are impor-
`tant.” The transmem-
`brane diffusion process
`is passive in nature and
`depends on a concen-
`tration differential as
`the driving force, each
`
`*)
`
`£8aSs=
`
`xar
`
`s 2=&3E3o
`
`kinetic energy to effect
`a net movement down
`this gradient. Permeant
`molecules must there-
`fore diffuse through the vehicle in which
`theyare contained to the mucosalinterface
`and haveto partition from the fomulation
`into the upperlayers ofthe tissue. From
`here molecules must diffuse within the
`mucosa, equilibrating laterally, and must
`emerge, eventually under steady-state condi-
`tions, from the distal surface ofthe tissue.
`Adsorptive interaction might be extensive in
`this layer, forming a reservoir of the perme-
`ant molecules. Further partitioning into
`neighboringtissue strata or into the receptor
`fluid then takes place underthe influence of
`the concentration gradient, and adsorption
`might occur once again. Diffusion through
`any one ofthe layers or any ofthe partition-
`ing events might form the rate-limiting step
`controlling the overall rate of pameation.
`Initiallythe concentration gradient across
`the mucosa will not be linear as the perme-
`ant equilibrates within the tissue. However,
`after sufficient time has elapsed, steady state
`will be achieved and the effective permeant
`concentration at all points in the tissue will
`remain constant.
`
`Figure 4. Cumulative flux values for piroxicam solutions (5 mg/mL and
`10 mg/mL) and gel (5 mg/g) across human vaginal mucosa.
`
`the gel. Mean steady-state flux rates of 1.665
`+ 0.160 (SEM)[]g/cm’/min and 1.578 + 0.128
`(SEM) []g/cm’/min were obtained for the 5
`mg/mL and 10 mg/mLpiroxicam solutions,
`respectively. The piroxicam gel (5 mg/g) yield-
`ed a mean steady-state flux rate of 0.589 +
`0.038 (SEM) []g/cm?/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 mucosaofthe 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 mg/mL) used. For
`piroxicam 5% and 10% solutions and gel,
`apparent release constants were 808.1 + 21.6,
`778.3 + 18.0, and 286.2 + 6.1 []g/cm’/h"”, lag
`times were 2.59, 2.59, and 2.61 h'”, and r-val-
`ues were 0.9957, 0.9968, and 0.9973, respec-
`tively (Fig. 4).
`
`DISCUSSION
`
`Mucosaldelivery of therapeutic agents
`involves the penetration of a drug into
`mucosalsurfaces either for the purpose of
`treating diseasesor alleviating symptomsin
`deeperlying tissues, or to treat systemic dis-
`ease by achieving systemically active levels
`of the agent. To achieve the required degree
`of penetration, not only the properties of the
`membranesinvolved, but also the chemical
`nature, size and conformation,lipid/water
`
`Penetration of drugsinto tissuesis
`dependent on the influence of the vehicle on
`the thermodynamic activity of the active
`ingredient. Diclofenac and piroxicam are
`sparingly soluble in water, hence their
`release is favored from aqueoussolutions
`and hydrophilic bases such as gels, which
`are poorsolvents for these drugs. As a
`result, there are less drug—vehicle interac-
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`The JournalofApplied Research « Vol. 3, No. 4, Fall 2003
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`tions in these solvent systems and this leads
`to improvedpartitioning into the mucosa.
`The pKa-value of diclofenac is 4, whereas
`that of piroxicam is 6.3 (dioxane:water
`2:1).’ At the pH values of the PBS buffer
`system usedin this study, ie, 7.4, approx i-
`mately 99.9% of the diclofenac and approx i-
`mately 92.6% of the piroxicam are present
`in their dissociated forms. Althoughthis
`improves diffusion through the hydrophilic
`outer layers of the epithelium,it does not
`facilitate penetration of the lipoidallayer,
`whichis thought to constitute the major per-
`meability barrier of vaginal and buccal
`mucosaandis located in the outer third of
`the epithelium.”**
`It is clear that the flux rates across vagi-
`nal mucosaof the aqueoussolutions of
`diclofenacare 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).”**°
`This relationship exists for formulations in
`which the drug is fully dissolved oris in
`suspension. Hence, the mucosahas no sig-
`nificant effects on drugrelease, 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 aqueoussolutions as well as
`the presence of a fatty emulsion base con-
`taining isopropanol andpropyleneglycol,all
`of whichretard 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 injectable solutions had a
`considerably higher total alcohol content
`(benzyl alcohol, 2% m/v andethyl alcohol,
`10% m/v). Both of these compounds can be
`
`classified as chemical penetration
`enhancers, ie, they might improve drug dif-
`fusion by modifying the thermodynamic
`properties of the drug or by violating the
`mucosal barrier making it more permeable.
`However, the point at which substances
`becomeenhancershasnot been well
`
`defined. Both of these alcohols are also
`
`cosolvents andaid solubilization of the
`almost water-insoluble piroxicam into the
`gel vehicle, thereby negatively affecting its
`partitioning into the mucosa. Thenet 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, nostatistically sig-
`nificant differences between the steady-state
`flux rates for these 2 solutions were found.
`This appears to indicate that maximalparti-
`tioning to the mucosa from the vehicle
`and/or saturable diffusion kinetics for pirox-
`icam across the tissue is achieved at concen-
`trations of 5 mg/mLorless 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 compoundsare 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/orits flux
`rate across mucosaltissues. These aspects
`will, however, require further investigation.
`
`ACKNOWLEDGMENTS
`
`The authors thank the University of
`Stellenbosch and the SA Medical Research
`Council for supporting this work.
`
`510
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