Transmucosal Permeation of Topically
`Applied Diclofenac and Piroxicam
`
`Pieter van der Bijl, BChD, BSc (Hons) (Pharmacol), PhD, DSc
`Armorel D. van Eyk, PhD
`Heiner L Seifart, Dr rer nat
`landa Viljoen, BSc (Hons) (Pharmacol)
`Marli Jooste, BSc (Hons) (Pharmacol)
`
`Department of Pharmacology, Faculty of Health Sciences, University of Stellenbosch, Tygerberg, South Africa
`
`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 buccal
`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 ±
`16 years (mean ± 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 diffusion
`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 of Applied Research • Vol. 3, No. 4, Fall 2003
`
`  
`
`

 
`
`505
`MYLAN - EXHIBIT 1025
`
`

`

`gastrointestinal tract bleeding 5-fold,4 is ele­
`vated in the elderly,5 and might be even
`higher for certain NSAIDs.6,7 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.2,9 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
`mucosal membranes for a variety of condi­
`tions ranging from pain to inflammation.10"15
`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 pemieability
`studies of a wide variety of chemical com­
`pounds.16-21 FurthemDre, 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
`In view of the fact that the therapeutic
`efficacy of NSAIDs depends on their pene­
`tration into the mucosal and underlying tis­
`sues, it was the objective of the present study
`to investigate the permeability of human
`vaginal mucosa, as a model of buccal
`506
`
`mucosa, to aqueous and commercially avail­
`able gel forms of diclofenac and piroxicam.
`
`MATERIALS AND METHODS
`Vaginal Mucosa
`Specimens were obtained from excess tissue
`removed from 8 postmenopausal patients
`aged 57 ± 16 years (mean ± standard devia­
`tion [SD]; range, 32-76 y) after vaginal hys­
`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,1"1 and trans-
`fened 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 -850C 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.
`Permeability Experiments
`Before each permeability experiment tissue
`specimens were thawed at room temperature
`in phosphate-bu ffered sahne (PBS, pH 7.4).
`Thereafter the specimens were cut into
`5-mm2 sections and mounted in flow-
`through diffusion cells (exposed areas 0.039
`cm2) as previously described,1"1 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 200C 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 EmulgeF, Novartis SA [Pty] Ltd,
`Rivonia, South Africa). Alternatively, 1 mL
`of 5 mg/mL, 10 mg/mL of piroxicam solu­
`tion, or 0.5 mL of the 5 mg/g piroxicam gel
`
`Vol. 3, No. 4, Fall 2003 • The Journal of Applied Research
`
`

`

`(Rheugesic gel0, 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 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
`(Feldene0 IM, Pfizer Laboratories [Pty] Ltd,
`Sandton, South Africa) was used. Each mL
`of Feldene0 IM solution contains 20 mg of
`piroxicam, 2% m/v benzyl alcohol, and 10%
`m/v ethanol.
`HPLC Detection of Permeants
`Permeant-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 Technolcgies,
`Waldbronn, Germany) with a Zorbax XDB
`C18, 150 mm [] 4.6 mm (ID), 5-[]ni 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
`KH2P04, 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 were filtered through a 0.45 Gm filter.
`Aliquots of 50 QL 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
`
`The Journal of Applied Research • Vol. 3, No. 4, Fall 2003
`
`an Agilent Chem Station (Agilent
`Technologes, Waldbronn, Germany).
`Spiked standards over the expected concen­
`tration range (0.5-20 •g/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 01 (|4,g x cm 2 x min1)
`
`where Q = quantity of substance crossing
`vaginal tissue (in Og), 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 coirection
`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 ver­
`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 ±0.164
`(standard error of mean [SEM]) Qg/cmVmin
`and 3.969 ± 0.292 (SEM) Qg/cm2/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) Qg/cmVmin,
`which is found to be on average 6.3 times
`lower than the 10 mg/mL diclofenac solu-
`507
`
`

`

`£ 3-
`
`s
`
`Bar represents SEM
`
`• 10 mg/ml
`• 5 mg/ml
`• Gel
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Time (h)
`
`5
`
`Figure 1. Mean flux values for diclofenac solutions (5 mg/mL and 10
`mg/mL) and gel (10 mg/g) across human vaginal mucosa.
`
`5000 n
`
`4000
`
`cp
`E
`
`3000
`
`li.
`D
`S5 2000 -
`5
`
`1000
`
`0 ^
`
`• 10 mg/ml
`• 5 mg/ml
`• Gel
`
`3
`
`4
`Time(h1/2)
`
`5
`
`6
`
`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 approxi­
`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 (Qg/cm2) versus
`square root of time
`(h1/2).23-25 For diclofenac
`5% and 10% solutions
`and gel, apparent
`release constants were
`751.9 ±34.8, 1826 ±
`74.9, and 288.6 ± 9.7
`[]g/cm2/h1/2, lag times
`were 2.91,2.87, and
`2.66 h1/2, and revalues
`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"2, were occluded
`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
`508
`
`Figure 2. Cumulative flux values for diclofenac solutions (5 mg/mL
`and 10 mg/mL) and gel (10 mg/g) across human vaginal mucosa,
`
`£ 3-
`
`|2 s
`
`u.
`
`Bar represents SEM
`
`• 10 mg/ml
`• 5 mg/ml
`• Gel
`
`* *
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Time (h)
`
`Figure 3. Mean flux values for piroxlcam 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
`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
`
`Vol. 3, No. 4, Fall 2003 • The Journal of Applied Research
`
`

`

`2000 |
`
`C 1500-
`E
`I
`3 E 1000 -
`
`3 500 -
`
`• 5% mg/ml
`• 10% mg/ml
`•Gel
`
`0
`
`2
`
`3
`
`4
`Time (h")
`Figure 4. Cumulative flux values for piroxicam solutions (5 mg/mL and
`10 mg/mL) and gel (5 mg/g) across human vaginal mucosa.
`
`5
`
`6
`
`partition coefficient,
`and degree of ioniza­
`tion of the permeant
`molecules are impor­
`tant.26 The transmem­
`brane diffusion process
`is passive in nature and
`depends on a concen­
`tration differential as
`the driving force, each
`molecule requiring
`kinetic energy to effect
`a net movement down
`this gradient. Permeant
`molecules must there­
`fore diffuse through the vehicle in which
`they are contained to the mucosal interface
`and have to partition from the famulation
`into the upper layers of the tissue. From
`here molecules must diffuse 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 peimeation.
`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.
`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
`sparingly soluble in water, hence their
`release is favored from aqueous solutions
`and hydrophilic bases such as gels, which
`are poor solvents for these drugs. As a
`result, there are less drug-vehicle interac-
`
`the gel. Mean steady-state flux rates of 1.665
`±0.160 (SEM) Qg/cmVminand 1.578 ± 0.128
`(SEM) Qg/cm2/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 of0.589 ±
`0.038 (SEM) •g/cmVmin, 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 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 Dg/cm2/h1/2, lag
`times were 2.59, 2.59, and 2.61 h1/2, and reval­
`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
`ease by achieving systemically active levels
`of the agent. To achieve the required degree
`of penetration, not only the properties of the
`membranes involved, but also the chemical
`nature, size and conformation, lipid/water
`
`The Journal of Applied Research • Vol. 3, No. 4, Fall 2003
`
`509
`
`

`

`26,28
`
`tions in these solvent systems and this leads
`to improved partitioning into the mucosa.
`The pKa-value of diclofenac is 4, whereas
`that of piroxicam is 6.3 (dioxane:water
`2:1).27 At the pH values of the PBS buffer
`system used in this study, ie, 7.4, approxi­
`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.
`It is clear that the flux rates across vagi­
`nal mucosa of the aqueous solutions of
`diclofenac are statistically significant^
`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).
`23-25
`This relationship exists for formulations in
`which the drug is fully 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.29
`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 and ethyl alcohol,
`10% m/v). Both of these compounds can be
`510
`
`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
`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 piroxi­
`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 diffusion 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
`Council for supporting this work.
`
`Vol. 3, No. 4, Fall 2003 • The Journal of Applied Research
`
`

`

`REFERENCES
`1. Stiel D: Exploring the link between gastrointesti­
`nal complications and over-the-counter anal­
`gesics: current issues and considerations. Am J
`Therapeutics 7:91-98, 2000.
`2. Heyneman CA, Lawless-Liday C, Wall GC: Oral
`versus topical NSAIDs in rheumatic diseases: a
`comparison. Drugs 60:555-574, 2000.
`3. Singh G: Gastrointestinal complications of pre­
`scription and over-the-counter nonsteroidal anti­
`inflammatory drugs: a view from the ARAMIS
`database. Arthritis, Rheumatism, and Aging
`Medical Information System. Am J Therapeutics
`7:115-121,2000.
`4. Gabriel SE, Jaakkimainen L, Bombardier C: Risk
`for serious gastrointestinal complications related
`to the use of nonsteroidal anti-inflammatory
`drugs. Ann Intern Med 115:787-796, 1991.
`5. Laporte J-R, Came X, Vidal X, et al: Upper gas-
`trointestinal bleeding in relation to previous use
`of analgesics and nonsteroidal anti-inflammatory
`drugs. Lancet 337:85-89, 1991.
`6. Kaufman DW, Kelly JP, Sheehan JE, et al:
`Nonsteroidal anti-inflammatory drug use in rela­
`tion to major upper gastrointestinal bleeding.
`Clin Pharmacol Ther 53:485—494, 1993.
`7. Rodriguez LAG, Jick H: Risk of upper gastroin­
`testinal bleeding and perforation associated with
`individual non-steroidal anti-inflammatory drugs.
`Lancet 343:769-772, 1994.
`8. Urban MK: Cox-2 specific inhibitors offer
`improved advantages over traditional NSAIDs.
`Orthopedics 23:S761-S764, 2000.
`9. Vaile JH, Davis P: Topical NSAIDs for muscu­
`loskeletal conditions. Drugs 56:783-799, 1998.
`10. Yalcin S, Altundag K, Asil M, et al: Sublingual
`piroxicam for cancer pain. Med Oncol
`15:137-139, 1998.
`11. Maamer M, Aurousseau M, Colau JC:
`Concentration of benzydamine in vaginal mucosa
`following local application: an experimental and
`clinical study. Int J Tissue Reac 9:135-145, 1987.
`12. Sironi M, Milanese C, Vecchi A: Benzydamine
`inhibits the release of tumor necrosis factor-alpha
`and monocyte chemotactic protein-1 by Candida
`a/fe/cans-stimulated human peripheral blood
`cells. Int J Clin Res 27:118-122, 1997.
`13. Passali D, Volonte M, Passali GC, et al: Efficacy
`and safety of ketoprofen lysine salt mouth wash
`versus benzydamine hydrochloride mouthwash in
`acute pharyngeal inflammation: a randomized, sin-
`gle-blind study. Clin Ther 23:1508-1518, 2001.
`14. Cecchini G, Fe rutta P, Solda A, et al: Vagmiti asped-
`fiche e trattamento topico. Confronto tra flunoxapro-
`fene e benzidamina [Non-specific vaginitis and
`topical treatment. Comparison of flunoxaprofen and
`benzydamine]. Minerva- Ginecol 41:287-290, 1989.
`
`15. Saxen MA, Ambrosius WR, Rehemtula al-KF, et al:
`Sustained relief of oral aphthous ulcer pain from
`topical diclofenac in hyaluronan: a randomized.
`double-blind clinical trial. Oral Surg Oral Med Oral
`Pathol Oral Radiol Endod 84:356-361, 1997.
`16. Van der Bijl P, Thompson IOC, Squier CA:
`Comparative permeability of human vaginal and
`buccal mucosa to water. Eur J Oral Sci
`105:571-575, 1997.
`17. Van der Bijl P, Van Eyk AD, Thompson IOC:
`Permeation of 17[]-estradiol through human vagi­
`nal and buccal mucosa. Oral Surg Oral Med Oral
`Pathol Oral Radiol Endod 85:393-398, 1998.
`18. Van der Bijl P, Van Eyk AD, Thompson IOC:
`Penetration of human vaginal and buccal mucosa
`by 4.4 and 12 kDa FITC-labeled dextrans. Oral
`Surg Oral Med Oral Pathol Oral Radiol Endod
`85:686-691, 1998.
`19. Van der Bijl P, Van Eyk AD, Thompson IOC:
`Diffusion rates of vasopressin through human
`vaginal and buccal mucosa. Eur J Oral Sci
`106:958-962, 1998.
`20. Van der Bijl P, Penkler L, Van Eyk AD:
`Pe rmeation of sumatriptan through human vaginal
`and buccal mucosa. Headache 40:137-141, 2000.
`21. Van der Bijl P, Van Eyk AD: Penetration of
`benzo[a]pyrene through human buccal and vagi­
`nal mucosa. Oral Surg Oral Med Oral Pathol
`Oral Radiol Endod 87:452-455, 1999.
`22. Van der Bijl P, Van Eyk AD, Thompson IOC:
`Effect of freezing on the permeability of human
`buccal and vaginal mucosa. SAfr J Sci
`94:499-502,1998.
`23. Higuchi WI: Analysis of data on the medicament
`release from ointments. JPharm Sci 51:802-804,
`1962.
`24. Guy RH, Hadgraft J: On the determination of
`drug release rates from topical dosage forms. Int
`JPharm 60:R1-R3, 1990.
`25. Rahman MS, Babar A, Patel NK, et al:
`Medicament release from ointment bases. V
`Naproxen in vitro release and in vivo percuta­
`neous absorption in rabbits. Drug Dev Ind
`Pharm 16:651-672, 1990.
`26. Squier CA: The permeability of oral mucosa.
`CritRev Oral Biol Med 2:13-32, 1991.
`27. Budavari S, O'Neil MJO, Smith A, et al (Eds.):
`The Merck Index. Whitehouse Station, NJ: Merck
`Research Laboratories; 1996:3133, 7657.
`28. Thompson IOC, Van der Bijl P, Van Wyk CW, et
`al: A comparative light-microscopic, electron-
`microscopic and chemical study of human vagi­
`nal and buccal epithelium. Arch Oral Biol
`46:1091-1098, 2001.
`29. Ho HO, Huang FC, Sokoloski TD, et al: The influ­
`ence of cosolvents on the in vitro percutaneous
`penetration of Diclofenac sodium from a gel sys-
`tem. JPharm Pharmacol 46:636-642, 1994.
`511
`
`The Journal of Applied Research • Vol. 3, No. 4, Fall 2003
`
`