Tra N S Mucosal Permeation of to P I C a L Ly Applied Diclofenac and Pirox I C a M

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Tra n s mucosal Permeation of To p i c a l ly Applied Diclofenac and Pirox i c a m

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 of Pharmacology, Faculty of Health Sciences, University of Stellenbosch, Ty ge r b e rg, South A f r i c a

KEY WO R D S : Diclofenac, pirox i c a m , n i f i c a n t ly higher than those from the gels. human mucosa, permeability studies Steady-state flux rates of the 5 mg/mL diclofenac solution were approx i m a t e ly half ABSTRACT that of the 10 mg/mL solution, whereas max- Nonsteroidal antiinflammatory dru g s imal p a rtitioning to the mucosa from the (NSAIDs) are frequently used for the treat- vehicle and/or saturable diffusion kinetics ment of acute myalgias, orthopedic injuries, was achieved at concentrations of 5 mg/mL p o s t o p e r a t ive pain, chronic rheumatoid or less for the piroxicam solutions under the a r thritis, and osteoarthritis. This study experimental conditions used. The diff u s i o n i nvo l ves the permeation of 2 NSAIDs, of diclofenac and piroxicam across mucosal diclofenac and piroxicam, from topically s u r f aces appears to be more efficient wh e n applied solutions and gels through human aqueous solutions of these compounds are vaginal mucosa as a model of bu c c a l used as opposed to gel formulations. T h i s mucosa. Pe r meation of diclofenac and must be considered when vehicles for both p i r oxicam from the solutions and gels these NSAIDs are chosen. There also through human vaginal mucosa was deter- appeared to be a limit to the release of mined using a flow-through diffusion appa- p i r oxicam from the vehicle and/or its flux ratus. Vaginal specimens were obtained rate across mucosal tissues. from 8 postmenopausal patients aged 57 ± 16 years (mean ± standard deviation [SD]; INTRODUCTION range, 32–76 y) after vaginal hy s t e r e c- Nonsteroidal antiinflammatory dru g s tomies. Experiments were conducted at (NSAIDs) belong to the most frequently 20˚C and over a time period of 24 hours. used drugs worldwide, with as many as 8% High-pressure liguid chromatogr a p hy of the global adult population taking pre- (HPLC) analysis was used as a detection scribed forms of these agents at any give n method. Statistical tests used included t i m e . 1 H oweve r, it is estimated that they are a n a lysis of variance (ANOVA) and Duncan’s also responsible for approx i m a t e ly 25% of multiple range test for determination of all adverse drug reaction report s . 2 In this steady state and an unpaired t test with respect, NSAID-related gastrointestinal tox i- We l c h ’s correction for comparing diff e r- city is the most frequently observed adve r s e ences between the mean flux values at each event, and it is a significant cause of mor- time point. Flux rates of both diclofenac and bidity and mort a l i t y.3 The ingestion of p i r oxicam from aqueous solutions were sig- NSAIDs increases the relative risk of upper

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gastrointestinal tract bleeding 5-fold,4 is ele- mucosa, to aqueous and commercially ava i l- vated in the elderly,5 and might be eve n a ble gel forms of diclofenac and pirox i c a m . higher for certain NSAIDs.6 , 7 The NSAIDs are often the first choice of treatment for MATERIALS AND METHODS patients with acute myalgias, ort h o p e d i c Vaginal Mucosa injuries, postoperative pain, chronic rheuma- Specimens were obtained from excess tissue toid arthritis, and osteoart h r i t i s . 8 r e m oved from 8 postmenopausal patients In an attempt to reduce the relative ly aged 57 ± 16 years (mean ± standard dev i a- high incidence of serious adverse eff e c t s tion [SD]; range, 32–76 y) after vaginal hy s- associated with the systemic use of terectomies at the Louis Leipoldt and NSAIDs, a gr owing number of topical for- Panorama Mediclinic Hospitals, Bellville, mulations of these drugs have become com- South Africa. Surgical specimens we r e m e r c i a l ly ava i l a ble. These topical i m m e d i a t e ly placed in a transport fluid, pre- f o r mulations, either on their own or as pared as prev i o u s ly described,1 6 – 2 1 and trans- adjuncts to reduced dosages of systemic f e rre d to our laboratory within 1 hour. Excess agents, have proven to be useful in the man- c o n n e c t ive tissue was trimmed away and all agement of a variety of musculoskeletal and specimens were snap-frozen in liquid nitro- rheumatic diseases.2 , 9 Although the topical gen and stored at -85˚C for periods up to 6 NSAIDs have mainly been studied rega r d i n g m o n t h s . 2 2 No specimens were obtained in their transdermal diffusion kinetics, these which there was clinical evidence of any dis- agents might also have useful applications ease that might have influenced the perm e- when topically applied to mucous mem- ability characteristics of the vaginal mucosa. branes. In this respect, piroxicam, benzy- The study was approved by the Ethics damine, ketoprofen, flunoxaprofen, and Committee of the University of Stellenbosch diclofenac have all been topically applied to and the Ty g e r b e rg Hospital. mucosal membranes for a variety of conditions ranging from pain to inflammation.1 0 – 1 5 Permeability Experiments H oweve r, the majority of the topically ava i l- Before each permeability experiment tissue a ble NSAIDs on the market have been for- specimens were thawed at room temperature mulated for cutaneous application and hence in phosphate-bu ffered saline (PBS, pH 7.4). contain components and enhancers suitabl e Thereafter the specimens were cut into for improving skin, but not necessarily 5 - m m 2 sections and mounted in flow - mucosal, absorp t i o n . through diffusion cells (exposed areas 0.039 We have prev i o u s ly shown that human c m 2) as prev i o u s ly described,1 6 – 2 1 and perm e- vaginal mucosa can be used as a model for ation studies performed on 7 tissue repli- the buccal mucosa for in vitro perm e a b i l i t y cates for each patient. Before beg i n n i n g studies of a wide variety of chemical com- each permeability experiment, tissue disks p o u n d s . 1 6 – 2 1 F u rt h e rmo re, we have demon- were equilibrated for 10 minutes with PBS strated that both these tissues can be (pH 7.4) at 200C in both the donor and snap-frozen in liquid nitrogen and stored at r e c e iver compartments of the diffusion cells. –85˚C for many months and thereafter used After equilibration, the PBS was remove d for permeability experiments without signifi- from the donor compartment and replaced cant changes in permeability characteristics.2 2 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% e ffi c a cy of NSAIDs depends on their pene- ethanol or 0.5 mL of 10 mg/g diclofenac gel tration into the mucosal and underlying tis- ( Voltaren Emulgel‚, Nova r tis SA [Pty] Ltd, sues, it was the objective of the present study R ivonia, South Africa). A l t e rn a t ive ly, 1 mL to inve s t i gate the permeability of human of 5 mg/mL, 10 mg/mL of piroxicam solu- vaginal mucosa, as a model of bu c c a l tion, or 0.5 mL of the 5 mg/g piroxicam gel

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(Rheugesic gel‚, Cipla-Medro [Pty] Ltd, an Agilent Chem Station (Agilent Bellville, South Africa) was used. The gels Te c h n o l ogies, Waldbronn, Germ a ny ) . were covered with a Teflon disk and 1 mL S p i ked standards over the expected concen- of PBS. PBS at 37˚C was pumped through tration range (0.5–20 mg/mL) were random- the receiving chambers at a rate of 1.5 mL/h ly included in each batch. and collected, by means of a fraction collec- Calculation of Flux Values t o r, at 2-hour intervals for 24 hours. T h e Flux (J) values across vaginal tissue we r e p e r meability study was performed under calculated by means of the relationship sink conditions, ie, at the completion of each run the concentration of diclofenac or J = Q / A ¥ t (µg x cm- 2 x min- 1 ) p i r oxicam in the acceptor chamber neve r reached 10% of that in the donor compart- where Q = quantity of substance crossing ment. The permeant was detected by means vaginal tissue (in mg), A = tissue area of HPLC analysis. Crystalline diclofenac exposed (in cm2) and t = time of ex p o s u r e was obtained from Sigma Chemical (in min). C o m p a ny (St. Louis, MO). For the pirox i- cam solution, a readily ava i l a ble IM solution Steady-State Kinetics ( Fe l d e n e ‚ IM, Pfizer Laboratories [Pty] Ltd, When no statistically significant diff e r e n c e s Sandton, South Africa) was used. Each mL (P <0.05) (ANOVA and Duncan’s multiple of Fe l d e n e ‚ IM solution contains 20 mg of range test) between flux values were obtained p i r oxicam, 2% m/v benzyl alcohol, and 10% over at least 2 consecutive time intervals, a m/v ethanol. steady-state (equilibrium kinetics) wa s assumed to have been reached for a part i c u l a r HPLC Detection of Permeants specimen and diclofenac or pirox i c a m . Pe rmeant–containing effluent samples, collected from the acceptor compartments of Statistical Analysis the perfusion apparatus over the 2-hour An unpaired t test with We l c h ’s corr e c t i o n sampling intervals, were analyzed using an was used to inve s t i gate possible diff e r e n c e s Agilent 1100 series high-performance liquid b e t ween flux means of vaginal tissues at 2- c h r o m a t ograph (Agilent Te c h n o l og i e s , hour intervals. A significance level of P Waldbronn, Germ a ny) with a Zorbax XDB <0.05 was used for all tests and comparisons.

C18, 150 mm ¥ 4.6 mm (ID), 5-mm column. The temperature was maintained at 40˚C RESULTS and flow rates of 1.0 mL/min and 1.2 Mean flux values for solubilized diclofenac mL/min were used for diclofenac and pirox- (5 mg/mL and 10 mg/mL) and gel icam, respective ly. The mobile phase con- diclofenac (Voltaren Emulgel®, 10 mg/g) sisted of a mixture of 2 solvents, A (50 mM through frozen/thawed vaginal mucosa ve r-

K H 2P O 4, pH 5.42) and B (acetonitrile–iso- sus time are shown in Figure 1. Steady-state propanol; 4:1 v/v). Isocratic mixtures of A : B flux conditions were reached after approx i- were 65:35 and 58:42 for diclofenac and m a t e ly 9 hours across vaginal mucosa for p i r oxicam, respective ly. Solvents were “pro both the diclofenac solutions and gel. Mean analisi” (E Merck, Darmstadt, Germ a ny ) steady-state flux values of 1.682 ± 0 . 1 6 4 and were filtered through a 0.45 mm fi l t e r. (standard error of mean [SEM]) mg / c m 2/m i n Aliquots of 50 mL from each sample we r e and 3.969 ± 0.292 (SEM) mg / c m 2/min we r e injected directly into the column. Diclofenac found for the 5 mg/mL and 10 mg/mL solu- and piroxicam were detected at 273 nm tions, respective ly. The diclofenac gel (10 (retention time 2.3 min) and 354 nm (reten- mg/g) yielded a mean steady-state flux tion time 2.2 min), respective ly. Total ru n value of 0.626 ± 0.058 (SEM) mg / c m 2/ m i n , time was 3.5 minutes. Recording and inte- which is found to be on average 6.3 times gration of peaks was performed by means of l ower than the 10 mg/mL diclofenac solu-

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tion used in the perm e- ability ex p e r i m e n t s . S t a t i s t i c a l ly signifi c a n t d i fferences (P < 0 . 0 5 ) b e t ween flux rates of both diclofenac concentrations (5 mg/mL and 10 mg/mL) and diclofenac gel (10 mg/g) across va g i n a l mucosa were found after approx i m a t e ly 6 hours. The diclofenac Figure 1. solution had higher flux rates than the gel f o r m, whereas the flux rates of the 5 mg/mL diclofenac solution we r e Mean flux values for diclofenac solutions (5 mg/mL and 10 mg/mL) and gel (10 mg/g) across human vaginal mucosa. found to be approx i- m a t e ly half that of the 10 mg/mL diclofenac solution (Fig. 1). Mean apparent release constants (slopes) and lag times (x-axis inter- cepts) were obtained by linear regression analy- Figure 2. sis of plots of cumula- t ive amount of released d r ug (mg / c m 2) ve r s u s square root of time ( h 1 / 2) . 23–25 For diclofenac Cumulative flux values for diclofenac solutions (5 mg/mL 5% and 10% solutions and 10 mg/mL) and gel (10 mg/g) across human vaginal mucosa. and gel, apparent release constants we r e 751.9 ± 34.8, 1826 ± 74.9, and 288.6 ± 9.7 mg / c m 2/h1 / 2 , lag times were 2.91, 2.87, and 2.66 h1 / 2, and r2- va l u e s were 0.9873, 0.9900, Figure 3. and 0.9931, respective- ly (Fig. 2). The non-lin- e a r ly related port i o n s of the data curves, ie, <3 h1 / 2, were ex c l u d e d mg/mL and 10 mg/mL) and piroxicam gel from the linear regression plots for Meanboth flux values (forRh piroxicameugesic gel solutions®, 5 mg/ g(5) acmg/mLross v aand g i n a 10 l diclofenac and piroxicam andmg/mL) hence and are gel not (5 mg/g) muacrosscosa human over ti mvaginale are s hmucosa.own in Figure 3. s h own in the figures (Figs. 2 and 4). Steady-state flux values were achieved after O verall mean flux values for pirox i c a m a p p r ox i m a t e ly 6 hours across the va g i n a l intramuscular injectable solution (Fe l d e n e ®, 5 mucosa for both the piroxicam solutions and

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p a rtition coeffi c i e n t , and degree of ioniza- tion of the perm e a n t molecules are impor- t a n t . 2 6 The transmem- brane diffusion process is passive in nature and depends on a concentration differential as the driving force, each molecule requiring kinetic energy to eff e c t Figure 4. a net movement dow n this gradient. Pe r m e a n t molecules must there- the gel. Mean steady-state flux rates of 1.665 fore diffuse through the vehicle in wh i c h ± 0.160 (SEM) mg / c m 2/ m i n and 1.578 ± 0.128 t h ey are contained to the mucosal interfa c e (SEM) mgCumulative / c m 2/ m i n w efluxre o valuesbtained for fo piroxicamr the 5 solutionsand (5ha mg/mLve to par andtition from the form u l a t i o n 10 mg/mL) and gel (5 mg/g) across human vaginal mucosa. mg/mL and 10 mg/mL piroxicam solutions, into the upper layers of the tissue. Fr o m r e s p e c t ive ly. The piroxicam gel (5 mg/g) yield- here molecules must diffuse within the ed a mean steady-state flux rate of 0.589 ± mucosa, equilibrating laterally, and must 0.038 (SEM) mg / c m 2/min, which was approx i- e m e rge, eve n t u a l ly under steady-state condi- m a t e ly 2.8 times lower than the flux rate tions, from the distal surface of the tissue. obtained for the 5 mg/mL piroxicam solution. A d s o r p t ive interaction might be ex t e n s ive in S i g n i f icant differences (P <0.05) between flux this laye r, forming a reservoir of the perm e- rates across vaginal mucosa of the gel and ant molecules. Further partitioning into either of the 2 solutions were found from neighboring tissue strata or into the receptor a p p r ox i m a t e ly 4 hours onward. Howeve r, no fluid then takes place under the influence of s t a t i s t i c a l ly significant differences in flux va l- the concentration gradient, and adsorp t i o n ues were obtained between the 2 pirox i c a m might occur once again. Diffusion through solutions (5 mg/mL and 10 mg/mL) used. Fo r a ny one of the layers or any of the part i t i o n- p i r oxicam 5% and 10% solutions and gel, ing events might form the rate-limiting step apparent release constants were 808.1 ± 21.6, controlling the overall rate of perm e a t i o n . 778.3 ± 18.0, and 286.2 ± 6.1 mg / c m 2/h1 / 2, lag I n i t i a l ly the concentration gradient across 1 / 2 2 times were 2.59, 2.59, and 2.61 h , and r - va l- the mucosa will not be linear as the perm e- ues were 0.9957, 0.9968, and 0.9973, respec- ant equilibrates within the tissue. Howeve r, t ive ly (Fig. 4). after sufficient time has elapsed, steady state will be achieved and the eff e c t ive perm e a n t DISCUSSION concentration at all points in the tissue will Mucosal delive r y of therapeutic agents remain constant. i nvo l ves the penetration of a drug into Penetration of drugs into tissues is mucosal surfaces either for the purpose of dependent on the influence of the vehicle on treating diseases or alleviating symptoms in the thermodynamic activity of the active deeper lying tissues, or to treat systemic dis- i n g redient. Diclofenac and piroxicam are ease by achieving systemically active leve l s s p a r i n g ly soluble in wa t e r, hence their of the agent. To achieve the required degr e e release is favored from aqueous solutions of penetration, not only the properties of the and hydrophilic bases such as gels, wh i c h membranes invo l ve d, but also the chemical are poor solvents for these drugs. As a nature, size and conformation, lipid/wa t e r result, there are less dru g – vehicle interac-

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tions in these solvent systems and this leads c l a s s i f ied as chemical penetration to improved partitioning into the mucosa. enhancers, ie, they might improve drug dif- The pKa-value of diclofenac is 4, wh e r e a s fusion by modifying the therm o d y n a m i c that of piroxicam is 6.3 (diox a n e : wa t e r p r o p e rties of the drug or by violating the 2 : 1 ) . 2 7 At the pH values of the PBS bu ff e r mucosal barrier making it more perm e a bl e . system used in this study, ie, 7.4, approx i- H oweve r, the point at which substances m a t e ly 99.9% of the diclofenac and approx i- become enhancers has not been we l l m a t e ly 92.6% of the piroxicam are present d e f ined. Both of these alcohols are also in their dissociated forms. Although this c o s o l vents and aid solubilization of the i m p r oves diffusion through the hy d r o p h i l i c almost wa t e r- i n s o l u ble piroxicam into the outer layers of the epithelium, it does not gel vehicle, thereby nega t ive ly affecting its facilitate penetration of the lipoidal laye r, p a rtitioning into the mucosa. The net eff e c t which is thought to constitute the major per- of these complex interactions between the meability barrier of vaginal and bu c c a l constituents of the mucosa, piroxicam, and mucosa and is located in the outer third of the components of the donor vehicle that the epithelium.2 6 , 2 8 might enhance drug permeability or retain it It is clear that the flux rates across va g i- in the formulation are difficult to predict. It nal mucosa of the aqueous solutions of is also interesting to note that notwithstand- diclofenac are statistically signifi c a n t ly ing the fact that the concentration of pirox i- higher than those of the gel (Fig. 1). This is cam in the 10 mg/mL solution was doubl e also reflected in the relative apparent release that of the other solution, no statistically sig- constants, showing that the release of n i f icant differences between the steady-state diclofenac from the gel formulation is we l l flux rates for these 2 solutions were found. described by the “Higuchi” model in wh i c h This appears to indicate that maximal part i- the rate-controlling step is the process of tioning to the mucosa from the ve h i c l e d i ffusion through the gel matrix (Fig. 2).2 3 – 2 5 and/or saturable diffusion kinetics for pirox- This relationship exists for formulations in icam across the tissue is achieved at concen- which the drug is fully dissolved or is in trations of 5 mg/mL or less under the suspension. Hence, the mucosa has no sig- experimental conditions used. n i f icant effects on drug release, the latter being controlled mainly by the properties of CONCLUSION the formulation. This might be attributed to We have demonstrated that diffusion of the higher viscosity of the gel matrix com- diclofenac and piroxicam into mucosa pared with the aqueous solutions as well as appears to be more efficient when aqueous the presence of a fatty emulsion base con- solutions of these compounds are used taining isopropanol and propylene glycol, all instead of gels formulated for transcuta- of which retard partitioning of the neous use. This aspect should be kept in diclofenac to the mucosal surface. T h e mind when vehicles for both these NSAIDs e ffect of these cosolvents on the penetrabili- intended for intraoral, or even intrava g i n a l , ty of diclofenac has been well documented.2 9 transmucosal use are chosen. Furt h e r m o r e , Similar observations for flux rates and there appears to be a limit to the release of apparent release constants were made for p i r oxicam from the vehicle and/or its flux the injectable piroxicam solutions and the rate across mucosal tissues. These aspects gel (Figs. 3 and 4). Although the pirox i c a m will, howeve r, require further inve s t i g a t i o n . gel contained 0.01% m/m benzyl alcohol as a preserva t ive, the injectable solutions had a ACKNOWLEDGMENTS c o n s i d e r a bly higher total alcohol content The authors thank the University of (benzyl alcohol, 2% m/v and ethyl alcohol, Stellenbosch and the SA Medical Research 10% m/v). Both of these compounds can be Council for supporting this wo r k .

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