Formulation and Evaluation of Diclofenac Sodium as a Topical Preparation

The aim of this study was to evaluate and compare the in vitro and in vivo transdermal potential of w/o microemulsion (M) and gel (G) bases for diclofenac sodium (DS). The effect of dimethyl sulfoxide (DMSO) as a penetration enhancer was also examined when it was added to the M formulation. To study the in vitro potential of these formulations, permeation studies were performed with Franz diffusion cells using excised dorsal rat skin. To investigate their in vivo perfoniranc e, a car-rageenan - induced rat paw edema model was used. The commercial formulation of DS (C) was used as a reference formulation. The results of the in vitro permeation studies and the paw edema tests were analyzed by repeated - measures analysis of variance. The in vitro permeation studies found that M was superior to G and C and that adding DMSO to M increased permeation rate. The permeability coefficient (Kp) of DS from M and M + DMSO were higher (Kp :4.9 X 10 -3 +3.6 X l0 -4 cm lhand 5.3 X 10 -3 + l.2X 10 -3 cm / h, respectively) than the ($p) of nS from C (Kp: 2.7 xl0-3 +7.3 X 10 -4 cm /h) and G (Kp:4.5 X i0 -3 x+.s X 10 -s cm / h). In the paw edema test, M showed the best permeation and effectiveness, and M + DMSO had nearly the same effect as M. The in vitro and in vivo studies showed that M could be a new, altemative dosage form for effective therapy.


Introduction
Transdermal drug delivery offers many important advantages.For instance, it is easy and painless, it protects the active compound from gastric enzymes, and it avoidi the hepatic firstpass effect.Also, it is simple to terminate the therapy if any adverse or undesired effect occurs.But skin is a natural barrier, and only a few drugs can penetrate the skin easily and in sufficient quantities to be effective.Therefore, in recent years, numerous studies have been conducted in the area of penetration enhancement (1' 2).
Penetration enhancers such as hydrogenated soybean phospholipidr '',, ethanol, alcohols with long carbon chains (Cs -Cr+), noctanol and cyclic-monoterpenes @' t), nonionic surfactants (u ),_ p_ropylene glycol, and isopropyl myristate (4, 5' /) have been used in many studies to increase the percutaneous absorption of drugs.Membranes from rats, mice, pigs, guinea pigs, snakes, rabbits, and huma,ns as well as synthetic membranes have been used for these drug diffusion studies.
Although human cadaver skin may be the first choice as a skin model for a study of a final product to be used in humans, it is not always basy to obtain, and rat ,kin i, u commonly used substitute (8'e).Al - Saidan et al showed that in vitro permeation studies using rat skin could provide information useful for manipulating the design of transdermal therapeutic system (TTS) patches so that the desired permeation of the drug across human skin would be achieved (10).
Therefore, we used rat skin as a model membrane for our permeation studies.Microemulsions containing the oil and aqueous phase, surfactant and cosurfactant (cos) are optically transparent mixtures with very small droplet size (<.140 nm) (ll, 12).Microemulsions have been increasing in popularity and garnering more attention in recent years, because they may enhance the transdermal absorption of drug molecules by increasing drug solubilities and modifiring their partition coefficients (r3).A hydrogel !qs.e-.is used very often in topical formulations (14' 1s).The hydrogel formulition was prepared and studied as a vehicle for its permeation potential.DS is a nonsteriodal antirheumatic agent that has a potent antiinflammatory effect, but it does not penetrate well through skin and cannot reach the effective concentration at the site of action dfter transdermal application (a).For this reason, it had been suggest new, alternative dosage forms for transdermal application of DS.M and G formulations were developed and in vitro trnasdermal penetration of these formulations was compared with that of C.
Furthermore, a pharmacodynamic study of DS was evaluated for its antiinflammatory activity on a canageenaninduced rat paw  drop to mixture I by stiming with a mixer (IKA Labortechnik, Breen, Germany).The gelling process was completed, and G was obtained.M (w/o) was prepared using soybean oil as the oily phase, Brij 58 and span 80 as the surfactants, isopropyl alcohol as the cosurfactant, and distilled water as the aqueous phase (Table D. For the M preparation, the surfactants were mixed and melted at 60'C, then added to soybean oil.Cos and distilled water were added to this mixture by stirring using a magnetic stirrer (IKA Labortechnik, Staufen, Germany).The I Tilrrit Journal of Pharmaceutical Sciences 2006, 2 (2); 1 22-1 3I surfactanttocosurfactant weight ratio was 5:1.Then, a transparent microemulsion was obtained.Droplet sizes of M were determined using a Zetasizer (Malvern HPPS, Malvern, UK).DMSO l0% (w/w) was added as an enhancer at the last stage (M + DMSO).One gram of Voltaren@ Emulgel [(Ir{ovartis)], the commercial topical formulation, contains I 1.6 mg of diclofenac diethyl ammonium (equivalent to 10 mg / gm DS), isopropyl alcohol, propylene glycol, perfume, Cream 45, and other additives.All prepared formulations and the C dosage form contained l% (wlw) DS.4ssay of DS A spectrophotometric analysis was used to determine DS permeation.First 25 to 500 pl of stock solution (10 mg / 10 ml, adjusted with phosphate buffer pld7.4) was transferred by microasyringe into the 10 ml volumetric flasks.Then the volume was adjusted with phosphate buffer pH7.4.The absorbances of the solutions were determined against a blank spectrophotometrically (Shimadzu W 160,4.) at277 nm.The aliquots of permeated formulations without DS were used as a blank.A calibration curve was then obtained (Y ; 3a.1 X -0.603, in whioh Y was concentration [pg / ml], X was absorbance, and 12 was 0.999).The sensitivity was 2.5 to 50 pg / ml.The limit of detection was 7.5 pg I mLIn vitro permeation studies Vertical Franztype diffusion cells (PermeGear, Bethlehem, PA) with a diffusional surface area of 1.76 cm2 were used to study the permeability of DS.Skin samples were obtained from male albino rats weighing 140 to 160 gm.After hair was shaven using a mechanical hair clipper, without damaging skin, a 5 X 5 cm patch of skin was excised from the dorsal region of each sacrificed rat.
The excised rat skins were stored at -80"C.
The skin membranes were first hydrated for 30 minutes in the buffer solution (pH7.4) at room temperature (23 C) to remove extraneous debris and leachable enzymes (7).
They were then placed between the donor and receptor compartments of the cells, with the dermal side in direct contact with the receptor medium.Approximately /0, ml of ptrosphate buffer (pH 7.$ was placed in the receptor compartment.Its temperature was maintained al37 r 0.5 C using a thermostatic water bath (Variomag, Munich, Germany), and it was stirred at 600 rpm throughout the experiment.
The donor compartment contained 1 gm of the sample.The aliquots (0.7 ml) " yyel's withdrawn at predetermined time intervals and then immediately analyzed spectrophotometrically at 277 nm against a blank prepared with the permeated formulation (M, M + DMSO, or G) without the drug 06' t7).The same amount of fresh buffer was added to the receptor compartment to replace what had been removed.Three replicates of each experiment were performed.Determination of drug solubilifiTo determine the drug solubility, an excess amount of DS was added to distilled water.This suspension was stirred at room temperature for 24 hours with a magnetic stirrer.The sample was then filtered through a 0.45pm cellulose acetate filter (Sartorius AG, Goeffingen, Germany).The concentration of DS was determined spectrophotometrically at277 nm (18' 1?).
Determinstion of noctanoldistilled water partition cofficient n-Octanol phases were saturated with distilled water for at least 24 hours before the experiment.A solution of DS (10 -4 M) *ir pr"puted with distilled water.Then, 2 ml of this solution was transferred to 10 -ml assay tubes containing 2 ml of the organic phase.The tubes were stoppered and agitated for 24 hours at room temperature.After centrifugation at 3500 U / min for 15 minutes, the concentration of the drug in the water phase was analyzed spectrophotometrically; the concentration of the drug in noctanol was calculated from the difference between the initial and final concentrations in the water phase.Six replicates were used for the concentrations of noctanoldistilled water solutions for partition coefficient calculations Q0).The same experiment was repeated using soybean oil as an organic phase (tn) Arti inJlummatory effict test The formulations of DS were evaluated for their anti inflammatory activity on a carrageenaninduced rat paw edema model Ql' 22).
Inflammation was produced in the rats (Male, Wistar, weighing 200 -250 gm) using 100 pL of 1o/o carcageenan (w/v) in saline.This was injected into the plantar surface of the rats' left hind paw.To evaluate the topical anti inflammatory activity of the formulations G, M, M + DMSO, and C, 4 groups of animals (n : 3) with carrageenaninduced paw edema were examined.Thirty minutes later, 100 pI-of G, M, M + DMSO, or C was applied topically on the edematous paw.A fifth group of rats was used as a control (untreated).The increase in paw thickness was measured with the help of dial calipers before (time 0) and 1, 2,3, 4,5, and 6 hours after carrageenan administration.The percentage of paw thickness increase from time 0 was calculated.Statisticul anu.lysisThe results of permeation studies through rat skin were analyzed by repeated measures analysis of variance (ANOVA).Two different factors were examined.Factor 1 was the time repeated and was composed of 8 levels.
Factor 2 was formed by 4 different formulations (M, M + DMSO, G, C).The results of the paw edema test were evaluated according to repeatedmeasures ANOVA.
Two different factors were compared.Factor 1 was the time and was composed of 6 levels (repeated factor).Factor 2 was the formulations and was composed of 5 levels (M, M i IMSO, G, C, control).One -way ANOVA was used for further analysis, because there was significant interaction between 2 factors;0.05wbs taken as the level of significance.The Duncan test was also used as post hoc analysis in this study.Each value represents the mean + SD (n: 3).

Results and discussion
After addition of DS to M, no opalescence was observed and no significant changes were detected in the droplet size, indicating that these systems retained their stability when the drug was added.The viscosities of G and M were determined using a Brookfield digital viscometer III Rheometer V 3.3 HB (Middleboro, MA) (Spindle: SC4 -2I) atZ00 rpm and 25 + 0.1"C as 1872 cps and 80 cps, respectively.The pHs of G and M were adjusted ro 5.45 + 0.1 and 6.75+ 0.1, respectively, and physicochemical stability of the formulation was observed.The mean droplet diameter of M prepared with isopropyl alcohol without DS was 11.7 + 0.5 nm, and with DS the diameter was 9. l9 + 0.1 nm (1e).Park and Kim offered 2 explanations for the decrease in droplet size with the addition of the drug: (l) a certain portion of undissolved drug acts as an emulsifuing agent by the deposition of drug particles at the interface of M, or (2) the deposition of drug at the interface of M causes reduced surfactant mobility, which decreases the particle size of drugloaded microemulsions '"'.The solubility of DS in distilled water has been previously found to be > 9 mg I ml Qa).In this study, the solubility of DS in distilled water was 19.1 mg / ml.The partition coefficient of DS in noctanoldistilled water and soybean oildistilled water was calculated as 5.75 and 1.8, respectively.One gram of M in the donor compartment contains 10 mg DS.In the water phase (0.063 gm) of M, = 10Yo of DS might be dissolved in conformity with the solubility studies.
Judging from the partition coefficients of both noctanol / distilled water and soybean oil / distilled water, the remaining DS may exist largely in the oilsurfactant region of do M in a dissolved form because of the high concentration of surfactants.To calculate the permeation parameters of the Fick's law equation from the ploi of penetrated amounts vs. time, a graph was plotted (Figure 1).It is possible to calculate the steadystate flux (J) from the slope of the -lin-ear portion (2 -S hours) of the graph ("' 'o).The permeability coefficient (Kp) was calculated from the steady state flux and the applied concentration in the donor compartment (Caonor) as follows:Kp : J / C4oro, (Eq. 1) The flux and permeability coefficients of the formulations are given in (Table 2).
The results of permeation studies through rat skin were analyzed by repeatedmeasures ANOVA.According to the ANOVA, rhe interaction was significant between Factor 1 and Factor 2. There was a significant difference in the permeability rates among all formulations studies (P < 0.5).As to the results obtained using post hoc analysis (Duncan test),.the differences among all pairwise comparisons of formulations were found to be significant.There was a significant difference among the formulations studied from the first hour; the rank order for in vitro percutaneous absorption of DS from the bases was M + DMSO > M > G > C (Figure 1; Table 3).This rank order held throughout the 8 hours.Pairwise comparisons of these formulations were done at each hour.
At the second hour, the difference between C and G was not significant.All other pairs were found to be significantly different at all hours (Table 4).According to this order, the permeability rate of DS from M + DMSO was the highest (Kp : 5.3 X 10 -3) and from C was the lowest (Kp : 2.7 X 10 -3 cm / h) (Table 2).The higher permeability rate of DS from M is most probably due to the surfactants and the oily phase, whioh act as penetration enhancers to facilitate transdermal drug delivery (27).In this study, the flux values from M and M + DMSO were 4.9 X 10 -2 and 5.3 X 10 -' pg I cm' lh, respectively, yielding 1.8 and 2 times greater than that observed from C (2.7 X 10 -' pg I cm2 I h1. As shown in (Figure 1), addition of DMSO to M increased the in vitro permeation rate of DS, In a previous study, it was explained that DMSO interaction with stratum corneum lipid alkyl chains resulted in decreased diffusion resistance of the barrier and increased drug penetration into the skin (28).Many different theories concerning the mechanism of action of penetrants have appeared in the literature.One of them attributes the penetrant effects of'DMSO, dimethylformamide, and dimethylacetamide to their hygroscopic properties, which are said to increase the water content of the stratum corneum, thereby greatly increasing its permeability.Another attributes the effectiveness of penetration enhancers to their ability to lower the barrier properties of the stratum corneum by modifying its natural structure.Organic solvents like benzene, alcohol, and ether, which have been shown to enhance the penetration rate of both watersoluble and lipidsoluble substances, may act by removing the lipids from the stratum corneum.However, the action of hydrogenbonding solvents .likeDMSO, dimethylformamide, and dimethylacetamide is attributed to membrane expansion and uniform increase in media diffusivity.It is known that microemulsions have a great capacity to release drugs through the skin (2e).
In this study, M showed a higher release capacity for DS than did G.The M structure may have allowed high drug mobility in the vehicle, which would translate into faster drug diffusion through the skin surfase and thus a higher transdermal flux (30).It has been reported that carrageenaninduced edema can be divided into 2 phases.The first phase occurs throughout the first hour after carrageenan injection.It derives from the release of cytoplasmic enzymes and serotonin from mast cells and the increase of prostaglandin in the inflammatory area.The second phase occurs 3 to 5 hours after carrageenan injection.In this phase, the macrophages in carrageenan insulted dermal tissue release interleukin -1 to induce accumulation of polymorphic nuclear cells into the inflammatory area.This then releases the lysosomal enzymes and active oxygen to destroy connective tissues and induce paw swelling (31).In this study, the progress of the paw edema test was compatible with that found in the literature.Induction of acute inflammation in control rats resulted in a prominent increase in paw thickness throughout the first hour after intraplantar injection of carraggenan and reached a peak f inflammation after 4 hours (Figure 2).The results of the paw edema test were evaluated using repeatedmeasures ANOVA, and the interaction wai found to be significant between Factor 1 and Factor 2. This means that the paw edema differences among formulations for each hour were not similar (Table 5).The difference in the increase of paw thickness between hours was significant.
Because of interaction between Factor I and Factor 2, formulations were compared for each hour using 1 -way ANOVA.
Homogeneity of variance, analyzed using the Levene test, was observed at all hours.The Duncan test, used as post hoc analysis, found that when all formulations were compared with the control, a significant difference was found.The differences in paw thickness increase among formulations against time are shown in (Table 6) (Figure 2).In vitro and in vivo studies were compared.According to both in vivo and in vitro studies, M showed the best permeation and effectiveness (Figure I,2).In in vitro studies, the permeation of M increased when DMSO was added (Figure 1), but in in vivo studies, M and M + DMSO had nearly the same effect.In addition, in vitro studies demonstrated that G had better penetration than C, whereas in vivo studies showed that their effectiveness was nearly similar.

Conclusion
This study demonstrated that incorporating DS into M enhanced drug penetration through rat skin in vitro and in vivo (Figure 1, 2).M containing DS may offer promise as an arfii - Data are given as mean + SD (n :3).At all hours there was significant difference between the commercial formulation and the gel, except at hour 2.
Data are given as mean * SD (n:3).
edema model for all formulations.This study aimed to both suggest a new, alternative dosage form for enhancing topical penetration of DS and to compare the study's formulations with the commercial formulation available, evaluating the potential for penetration and transdermal absorption.940, Brij 58, Span 80, isopropyl alcohol, and DMSO {(SDDI.All chemicats used were analytical grade.preparation of topicul formulations G was prepared with Carbopol

Table 2 .
Permeation Parameters of Diclofenac Sodium from Different Bases through Rat Skin.

Table 3 .
Means of Perrneated Amounts of Diclofenac Sodium and Standard Deviations for All

Table 4 .
Differences among Formulations as to the Permeated Amount of DS for Each Hour Numbers show significant difference (P < 0.05) at hours befween 2 formulations crossed.

Table 6 .
Differences among Formulations as to the Paw Thickn -Numbers show significant difference (P < 0.05) at hours between the 2 formulations crossed.At first, second, and fifth hours there were significant differences between G and C.Irrurg rurrrurauors as [o rne rermeateo Amount ol DS lor llach Hour