International Journal of Pharmaceutics 453 (2013) 569578

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Microemulsion and poloxamer microemulsion-based gel for sustained transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation
Shahinaze A. Fouad a , Emad B. Basalious b, , Mohamed A. El-Nabarawi b , Saadia A. Tayel b
a b

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ahram Canadian University, Cairo, Egypt Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-aini Street, Cairo 11562, Egypt

a r t i c l e

i n f o

a b s t r a c t
Microemulsion (ME) and poloxamer microemulsion-based gel (PMBG) were developed and optimized to enhance transport of diclofenac epolamine (DE) into the skin forming in-skin drug depot for sustained transdermal delivery of drug. D-optimal mixture experimental design was applied to optimize ME that contains maximum amount of oil, minimum globule size and optimum drug solubility. Three formulation variables; the oil phase X1 (Capryol ), Smix X2 (a mixture of Labrasol /Transcutol , 1:2 w/w) and water X3 were included in the design. The systems were assessed for drug solubility, globule size and light absorbance. Following optimization, the values of formulation components (X1 , X2 , and X3 ) were 30%, 50% and 20%, respectively. The optimized ME and PMBG were assessed for pH, drug content, skin irritation, stability studies and ex vivo transport in rat skin. Contrary to PMBG and Flector gel, the optimized ME showed the highest cumulative amount of DE permeated after 8 h and the in vivo anti-inammatory efcacy in rat paw edema was sustained to 12 h after removal of ME applied to the skin conrming the formation of in-skin drug depot. Our results proposed that topical ME formulation, containing higher fraction of oil solubilized drug, could be promising for sustained transdermal delivery of drug. 2013 Elsevier B.V. All rights reserved.

Article history: Received 17 April 2013 Accepted 1 June 2013 Available online 18 June 2013 Keywords: Diclofenac epolamine In-skin depot Sustained transdermal delivery D-optimal design Microemulsion

1. Introduction Musculoskeletal pain is a common problem often treated with topical NSAIDs. Topical NSAIDs have a reduced risk of upper GI complications such as gastric and peptic ulcers, dyspepsia as well as a lack of drugdrug interactions (McCarberg and Argoff, 2010). Diclofenac epolamine is a NSAID, known as diclofenac-N-(2-hydroxyethyl)-pyrrolidine) (DHEP) (Conte et al., 2002). The diclofenac molecule, in its acidic form, is hydrophobic with very low solubility in water. The epolamine salt of diclofenac has greater solubility in water and non-polar solvents (n-octanol) than other diclofenac salts. High concentrations of aqueous diclofenac epolamine solutions exhibit surfactant behavior (McCarberg and Argoff, 2010). The solubility and the surfactant properties of diclofenac epolamine enhance its membrane permeability (OConnor and Corrigan, 2001). DE is currently available as topical gel and patch marketed under the brand name of Flector. Flector patch (10 cm 14 cm) contains an adhesive material containing 1.3% DE which is applied to a

Corresponding author. Tel.: +20 1200010002. E-mail addresses: dremadbasalious@cu.edu.eg, dremadbasalious@gmail.com (E.B. Basalious). 0378-5173/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.06.009

non-woven polyester felt backing and covered with a polypropylene lm release liner under patch (Petersen and Rovati, 2009). The use of external drug reservoir (topical patch) is the common technique used to sustain the transdermal delivery of water soluble drugs. The major disadvantages of transdermal patches are their sophisticated method of manufacture and the possibility that a local irritation will develop at the site of application. Erythema and itching can be caused by the drug and the adhesive in the patch formulation. Topical NSAID gels or creams are applied up to four times daily. Moreover, patches and gels are inconvenient to patients regarding discrepancy with cleaning and washing of skin. The combination of all advantages of gels (simple method of manufacture and ease of application by patients) with that of patches (sustained delivery) is the goal of this study. Our hypothesis was to increase the penetration of DE through epithelial tissue for loading of the drug into the skin forming in-skin drug depot where skin acts as in situ skin patch. Effective penetration of drugs through the stratum corneum is a major challenge in transdermal drug delivery. The presence of lipid of the stratum corneum represents a lipophilic barrier that restricts the permeation of molecules. Several approaches have been proposed to increase skin permeation. Microemulsions (MEs) which are clear, thermodynamically stable mixtures of oil, water and surfactant, have been shown to be able to deliver drugs through

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the skin better than conventional systems such as gel, creams and ointment (Kreilgaard, 2002). ME and ME based gels were prepared in an attempt to increase the transdermal drug delivery of both hydrophilic and lipophilic drugs (Barot et al., 2012; Kreilgaard et al., 2000; Trotta et al., 1997). Moreover, NSAIDs are one of the most important drug classes that have been formulated as microemulsion-based hydrogels for both topical and transdermal use such as ibuprofen (Chen et al., 2006), ketoprofen (Rhee et al., 2001) and diclofenac (Kweon et al., 2004). MEs and ME based gels were found to have favorable solvent properties due to the potential incorporation of large fraction of lipophilic and/or hydrophilic phases (Malcolmson and Lawrence, 1993; Malcolmson et al., 1998). Only the dissolved fraction of a drug in a vehicle can enter the skin. The small globule size of MEs makes them a suitable vehicle to penetrate epithelial tissue and use skin as a depot for sustained drug delivery (Yuan and Acosta, 2009). Development of a pharmaceutical formulation consumes a lot of time and is considered as a complex process. Thus, D-optimal mixture design is applied to develop pharmaceutical formulation because it was demonstrated to be an efcient method for optimization of the formulation and to understand the relationship between independent variables and dependent variables in a formulation (Basalious et al., 2010; Gao et al., 2004). Literature lacks any data about the use of ME for loading of drug into skin to form in-skin depot for sustained transdermal delivery of DE (a water soluble drug). Thus, the aim of this study was the formulation and optimization of ME and PMBG. D-optimal design was applied to optimize formulation that contains a maximum amount of lipid, small globule size (<100 nm) and possess enhanced skin transport of the drug forming in-skin drug depot. In vivo study of the anti-inammatory efcacy and sustained delivery of in-skin depot of DE was carried out by carrageenan induced rat paw edema method. 2. Materials and methods 2.1. Materials Diclofenac epolamine was kindly supplied by Marcyrl for Pharmaceutical industries, El-Obour (Egypt), Tween 80 and Isopropyl Myristate were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA.), Miglyol 812 and Miglyol 840 (was obtained from Sasol (Witten, Germany). Polyethylene glycol 400 was from El-Nasr Pharmaceutical Chemicals Co. (Egypt), Labrasol , Dipalmitoylphosphatidylglycerol (DPPG ), Capryol 90, Cocoate cc, Labrafac cc, and Gatuline Intense and Transcutol HP were supplied by Gattefoss (France.), Poloxamer 407 was obtained from BASF Corporation, Chemical Division (USA). Distilled water was used throughout the study. All other chemical reagents and solvents were of analytical grade and used as received. 2.2. Screening of oils, surfactants and co-solvents for ME formation For selecting solvents with good solubilizing capacity for DE, the saturated solubility of DE in various oils such as Capryol 90, Cocoate cc, isopropyl myristate , Gatuline in-Tense , Miglyol 812 and Miglyol 840, surfactants (Labrasol , Labrafac cc, Tween 80 and DPPG ) and co-solvents (Transcutol HP and polyethylene glycol 400) was determined. Excess amount of DE was added in 5 g of oils, surfactants and co-solvents in 10-ml-capacity stoppered glass vials and shaken on a shaker for 48 hours at ambient temperature. Suspension was centrifuged at 4000 rpm for 10 min and the concentration of DE in the supernatant was determined by UV spectrophotometer

(Spectrophotometer (UV 1601, PC UVVisible, Shimadzu, Japan) after appropriate dilution with methanol at max about 276 nm. Appropriate diluted solutions of oils, surfactants and co-solvents in methanol were taken as blank. Components which showed the highest solubility of DE were used for further studies (Barot et al., 2012). 2.3. Construction of pseudo-ternary phase diagrams The pseudo-ternary phase diagrams were constructed using titration method to determine the ME region and to obtain the concentration range of components for the existing range of MEs with different possible compositions of oil, surfactant/co-solvents, and water (Barot et al., 2012). The ratio of surfactant to co-solvent (Smix) was altered at 1:1, 1:2 and 2:1 and such mixtures were prepared. These mixtures (Smix) were mixed with the oil phase to give the weight ratios of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. Applying the aqueous titration method, distilled water was titrated drop by drop to the oil and Smix mixture under magnetic stirring at ambient temperature. After each addition, the mixture was examined for the appearance. The end point of the titration was the point where the solution becomes cloudy or turbid. The quantity of the aqueous phase required to make the mixture turbid was noted. The percentages of the different incorporated components were then calculated and the same procedure was followed for the other Smix ratios to plot the pseudo-ternary phase diagram. Pseudoternary phase diagrams were constructed with Tri-plot software Version 4.1.2. (Todd Thompson software). The clear ME zones were identied and marked. 2.4. Formulation optimization of MEs D-optimal mixture experimental study was designed based on a three component system: the oil phase X1 (Capryol 90), Smix X2 (a mixture of Labrasol /Transcutol , 1:2 w/w) and aqueous phase X3 (water). The total concentration of the three components summed to 100%. Based on the previous results obtained from phase diagram, the range of each component was selected as follows: The amount of oil was chosen in the range 1030%. The Smix ranged from 50% to 80%. Since hydration of the stratum corneum significantly affects penetration of drug into the skin, water range was selected to be 1030%. The solubility of drug in ME, mg/ml (Y1 ), mean globule size (Y2 ) and absorbance of ME (Y3 ) were used as the responses (dependent variables). The responses of all model formulations were treated by Design-Expert software (version 7; Stat-Ease, Inc., Minneapolis, MN). Suitable models for mixture designs consisting of three components include linear, quadratic and special cubic models. The best tting mathematical model was selected based on the comparisons of several statistical parameters including the multiple correlation coefcient (R2 ), adjusted multiple correlation coefcient (adjusted R2 ) and the predicted residual sum of square (PRESS), proved by Design-Expert software. Among them, PRESS indicates how well the model ts the data, and for the chosen model it should be small relative to the other models under consideration (Huang et al., 2004). D-optimal design was selected since it minimizes the variance associated with the estimates of the coefcients in the model (Holm et al., 2006). The software selected a set of candidate points as a base design. These included factorial points (high and low level from the constraints on each factor, centers of edges, constraint plane centroids, axial check point, and an overall center point). The base design consisted of 16 runs (Table 1). The optimum formulation was selected, which had the highest oil content, minimum content

S.A. Fouad et al. / International Journal of Pharmaceutics 453 (2013) 569578 Table 1 The formulations of mixture design and their characterization results. Formulation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A: Capryol 20 10 30 30 25 15 10 30 10 30 20 22.5 15 10 20 10 B: Smix 70 70 50 50 60 65 60 60 80 60 50 55 55 60 50 80 C: Water 10 20 20 20 15 20 30 10 10 10 30 22.5 30 30 30 10 Solubility (mg/ml) 292.27 268.61 374.70 277.04 188.29 248.00 304.44 262.29 171.19 260.42 358.31 273.77 480.09 359.09 370.02 248.71 Globule size (nm) 62.93 49.15 77.2 28.19 48.9 37.55 54.8 50.4 151.6 60.5 43.45 54.6 51.6 59.6 54.68 140.6

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Absorbance 0.075 0.023 0.045 0.01 0.032 0.012 0.034 0.036 0.089 0.027 0.059 0.022 0.023 0.024 0.025 0.091

of Smix, intermediate drug solubility (Y1 ) and the globule size (Y2 ) less than 100 nm. 2.5. Preparation of MEs From the pseudo-ternary phase diagrams, Smix ratio with maximum ME region was selected. Different proportions of oil and Smix were mixed based on the ratios presented in Table 1. The mixture of oil and Smix was mixed using vortex (VSM-3 model, PRO Scientic Inc., Oxford, England) at ambient temperature. The measured amount of distilled water was added drop wise to the oily mixture until clear and transparent liquid was obtained. All MEs were then stored at ambient temperature. 2.6. Evaluation of the prepared MEs 2.6.1. Determination of drug solubility in the prepared MEs The solubility of DE in MEs (in mg/ml: Y1 ) was determined. Excess amount of DE was added in 5 g of each of the previously prepared ME in 10-ml-capacity stoppered vials. The resultant mixture was mixed initially by vortex mixer then, all the vials were shaken in the shaker for 24 h at 25 C. Afterwards, centrifugation was done at 4000 rpm for 10 min and the concentration of DE in the supernatant was determined by UV spectrophotometer after appropriate dilution with methanol at its respective max . The plain ME without drug with the same composition was taken as blank after appropriate dilution with methanol. 2.6.2. Determination of globule size by photon correlation spectroscopy The globule size (in nm: Y2 ), was determined using photon correlation spectroscopy that analyzes the uctuations in light scattering due to the Brownian motion of particles using Malvern Zetasizer Nano-ZS (Ver.6.20, Malvern Instruments Ltd., Worcestershire, England). All measurements were done at room temperature (25 C) and at 90 C to the incident beam. 2.6.3. Measurement of spectroscopic absorbance at 400 nm The optical clarity of aqueous dispersions of SNEDD formulations was measured spectroscopically. The absorbance of each formulation was measured at 400 nm, using distilled water as a blank. 2.6.4. Transmission electron microscopy (TEM) of the optimized DE loaded MEs The morphology of the optimized ME systems was observed using transmission electron microscopy. A drop of each ME was

placed on a copper grid and the excess was removed with a lter paper. One drop of 2% aqueous solution of phosphotungistic acid (PTA) was added onto the grid and left for 3060 s to allow staining. The excess was removed with a lter paper. The grid was nally examined under the transmission electron microscope (JEOL (JEM-1400), Tokyo, Japan). 2.7. Formulation of DE-loaded ME and PMBG As MEs have low viscosity, their retention at the affected parts is quiet less. Therefore, their viscosity was required to be increased by the addition of a suitable gelling agent. Poloxamer was used as a gelling agent for the optimized ME formulation to formulate thermosensitive microemulsion-based gel of DE. Plain poloxamer gel (25%) was rstly prepared according to the cold technique (Chang et al., 2002; Shin et al., 1999). ME containing the drug was added portion-wise onto the plain gel in a ratio of gel:ME (2:1) with continuous stirring. The nal microemulsionbased gel formulation contained 1.3% w/w DE. DE was dissolved directly in the optimized ME to prepare drug loaded ME containing 1.3% w/w DE. 2.8. Evaluation of DE microemulsion and PMBG 2.8.1. pH measurements and drug content The apparent pH of the formulations was measured by a pH meter in triplicate at 25 C. For determination of drug content, one gram of ME formulations was diluted with appropriate amount of methanol. The concentration of DE was determined by UV spectrophotometer at its respective max . The plain ME formulations without drug with the same composition was taken as blank after appropriate dilution with methanol. 2.8.2. Stability study The optimized DE loaded ME and PMBGl were stored at 40 C/75% RH for three months. Optical clarity and drug content were performed for the stored drug loaded ME and microemulsionbased gel using the same procedures adopted for the fresh samples. Morphology of the stored drug-loaded ME was determined using transmission electron microscopy. 2.8.3. Skin irritation test Three male albino Wistar rats (130150 g) were kept under standard laboratory conditions and housed in cages with free access to a standard laboratory diet and water ad libitum. A single dose of 100 L of the optimized drug-loaded ME, optimized drug-loaded PMBG and the market formulation (Flector gel) was applied to the

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left ear of the rat, with the right ear as a control. The development of erythema was monitored for 24 h then the gel was removed, and the application sites were graded according to a visual scoring scale from no erythma, mild, moderate, high and severe erythema (Azeem et al., 2009; Shakeel et al., 2007). 2.8.4. Study of ex vivo transport of DE from optimized formulations into rat skin and ability to form in situ drug depot Ex vivo skin transport studies were performed using newly born rat skin (Azeem et al., 2009; Sarigullu Ozguney et al., 2006). Newly born albino Wistar rats were sacriced and skin samples obtained was inspected for the presence of any holes or irregularities. Fresh skin used in the study was preserved in 10% glycerin solution at 20 C. The study performed in this section was approved by Research Ethics Committee, Faculty of Pharmacy, Cairo University. Skin was slowly thawed and was cut into small circular pieces. The lower surface of the skin was allowed to hydrate for 1 h at 37 C prior to experimentation. The Ex vivo skin transport studies of DE from the optimized ME, PMBG and the market product (Flector gel) were performed in a USP dissolution apparatus tester (USP apparatus II) at 37 0.1 C. One gram of drug loaded ME, PMBG and the market formulation, all containing 1.3% drug w/w were placed in double open-sided glass cylindrical tubes (2.5 cm in diameter and 5 cm in length, with area = 4.9 cm2 ) tightly covered from one side with rat skin. The loaded tubes were attached from the second side to the shafts of the USP dissolution tester apparatus. This assembly represents the donor compartment. The shafts rotated at a speed of 50 rpm in phosphate buffer pH 7.4. The dissolution vessels (receptor compartment) were lled with 300 ml of phosphate buffer pH 7.4. Four milliliter samples were withdrawn periodically at predetermined time intervals of 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7 and 8 h and replaced instantly by equal amount of fresh phosphate buffer pH 7.4 in order to maintain the same volume. The drug concentration was determined by UV spectrophotometer at 276 nm. The skin transport studies were done in duplicates and the average percentage drug permeated was plotted versus time. Cumulative amount of drug in receptor chamber for the three formulations was plotted as a function of time. To study the ability of the three formulations to form in situ depot in the skin, transport of DE in the skin was observed after removal of the formulations from the donor compartment. Rat skin was removed after 3 h. The formulations were wiped off with wetted cotton pieces then the rat skin was mounted again on glass cylinder to continue the skin transport study. 2.8.5. In vivo study of the anti-inammatory efcacy and sustained delivery of in-skin depot of DE The sustained anti-inammatory efcacy and the ability of the optimized ME and PMBG to form in-skin depot were compared in vivo using carrageenan induced rat paw edema test. Flector gel is the market formulation and it is used as a reference product. Also, plain PMBG is prepared to be used as a control. Each formulation except the prepared plain gel contains 1.3% w/w DE. Thirty two adult male albino Wistar rats, weighing 130150 g were used in this study. They were purchased from Helwans Farm of experimental animals (Cairo, Egypt). The animals were acclimatized to environment for one week, they were housed under controlled environment at 25 1 C with a 12 hur light/dark cycle. All animals had free access to standard rodent pellet food consisting of vitamin mixture (1%), mineral mixture (4%), corn oil (10%), sucrose (20%), cellulose (0.2%), casein 95% (10.5%), starch (54.3%) and water. Animals were divided into four groups of eight rats each. The plain PMBG was assigned to the rst group, the optimized ME was

assigned to the second group, PMBG was assigned to the third group and the Flector gel was assigned to the fourth group. In order to induce inammation, animals were rst injected with 0.1 ml of 1% carrageenan solution in saline in the plantar region of the right hind paw. The initial paw thickness (Ti ) was measured using a Micrometer Caliper, one hour after carrageenan injection. Then, 1 g of each formulation was applied to the right hind paw of the rats. After 3 h of formulation application (sufcient time for skin loading and formation of in-skin drug depot), formulations remaining on the surface of the paw were wiped off with cotton then, the paw thickness (Tf ) was measured again using a Micrometer Caliper at different time intervals (3, 4, 5, 6, 7, 8 and 12 h). The edema % was calculated from the mean effect in treated animals according to the following equation: % edema = Tf Ti 100 Ti

where Tf is the thickness measured following administration of the formulae at different time intervals. Ti is the thickness measured 1 h after carrageenan sodium injection. Data were analyzed statistically by Students t-test at 5% signicance level using GraphPad Prism 5 program (GraphPad Inc., USA). 3. Results and discussion 3.1. Screening of components for ME The saturated solubility of DE in various oils, surfactants and cosolvents was estimated as shown in Fig. 1. Amongst the various oily phases that were screened, Capryol 90 provided the highest solubility of DE so was chosen for further investigations. Solubility of DE in Labrasol was the highest among the surfactants. Labrasol was selected for further studies due to its solubility prole and its low toxicity level as a non-ionic surfactant (Shaq-un-Nabi et al., 2007). Transcutol HP, which is a solubilizer and absorption enhancer (Basalious et al., 2010), was found to be a very efcient solubilizer for DE, and so was chosen as a co-solvent in the development of DE loaded ME formulations aiming to improve the drug loading capabilities. 3.2. Construction of pseudo-ternary phase diagrams To obtain the appropriate components and their concentration ranges for MEs, pseudo-ternary phase diagrams were constructed for different Smix ratios 1:1, 1:2 and 2:1, so that o/w ME regions could be identied and ME formulations could be optimized. The three ratios gave stable and clear MEs but the ratio which gave the largest ME region was found to be 1:2 and therefore it was selected for further studies. This is clearly shown in Fig. 2. The phase study clearly reveals that with a decrease in the weight ratio of Labrasol from 1 to 0.5, the ME region is expanded. This observation conforms to the results obtained from the study of Barot et al. (2012). It is obvious also that an increase of the weight ratio of Labrasol from 1 to 2 resulted also in expansion of the ME region. This observation is in agreement with Shakeel et al. stating that as the surfactant concentration was increased in the Smix ratio, a higher ME region was observed, perhaps because of further reduction of the interfacial tension, increasing the uidity of the interface, thereby increasing the entropy of the system (Shakeel et al., 2007). Thus, the effect of Labrasol on ME area depends the other components of ME especially co-solvent. This is because the reduction of o/w interface is not achieved by single-chain surfactants alone. The combination of short to medium chain length alcohols (such as Transcutol HP) with single chain surfactants could result in lowering the interfacial tension due to increased uidity at the interface (Binks et al., 1989). Miscibility of aqueous and oily phases could

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Fig. 1. Solubility (mg/ml) of diclofenac epolamine in various microemulsion components.

Fig. 2. Pseudo-ternary phase diagrams of microemulsions composed of oil (Capryol 90), Smix (surfactant: Labrasol , co-solvent: Transcutol ) and water at various oil/Smix ratios 1:2 (a), 1:1 (b) and 2:1 (c).

also be increased by medium chain length alcohols due to their partitioning behavior between the two phases (Lawrence and Rees, 2000; Shaq-un-Nabi et al., 2007). 3.3. Formulation optimization of ME using D-optimal design In order to rapidly obtain the optimal ME, D-optimal mixture experimental design was applied in this study. The oil phase X1 (Capryol 90), Smix X2 (a mixture of Labrasol /Transcutol , 1:2 w/w) and aqueous phase X3 (water) were chosen as formulation variables and the solubility of drug in ME, mg/ml (Y1 ), mean globule size (Y2 ) and absorbance of ME (Y3 ) were used as the responses (dependent variables). The responses of these formulations are summarized in Table 1. The independent and response variables were related using polynomial equation with statistical analysis through Design-Expert software. As shown in Table 2, the approximation of response values of Y1 based on linear model was the most suitable because its PRESS was smallest. The values of the
Table 2 Reduced Regression results of the measured responses. Response Y1 Y2 Y3 Model Linear Quadratic Quadratic R2 0.547 0.8193 0.7951 Adjusted R2 0.4774 0.7537 0.7439 Predicted R2 0.342 0.5469 0.6327

coefcients X1 , X2 and X3 are related to the effect of these variables on the response. A positive sign of coefcient indicates a synergistic effect while a negative term indicates an antagonistic effect upon the response (Huang et al., 2005). The larger coefcient means the independent variable has more potent inuence on the response. As shown in Table 1, solubility of DE in the different ME formulation varied between 171.1 and 480 mg/ml. It can be inferred that the three independent factors have a profound effect on drug solubility. As illustrated in Table 3, a p-value of 0.05 for any factor in analysis of variance (ANOVA) indicates a signicant effect of the corresponding factors on the solubility of drug in ME (Y1 ). It can be inferred that the terms X1 , X2 , and X3 have a signicant effect on the drug solubility (p < 0.05). This result could be conrmed by the positive value of these coefcients (Table 2). Fig. 3 shows the contour diagrams illustrating the effect of varying ratios of (X1 ), (X2 ) and (X3 ) on the solubility of drug in ME (Y1 ). It is obvious that the water content in ME formulation has the highest positive effect on the solubility of DE in ME. This means that increasing the water

PRESS 58,715.09 7792.269 0.0036

Regression equation for the responses Y1 = +307.68X1 + 117.74X2 + 832.50X3 Y2 = +526.53X1 + 384.48X2 + 830.97X3 1631.31X1 X2 2172.43X2 X3 Y3 = 0.29X1 + 0.26X2 + 1.25X3 2.70X2 X3

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Table 3 ANOVA of the solubility of drug in microemulsion formulations (Y1 ). Source Model Linear Mixture Residual Lack of Fit Pure Error Cor Total Sum of squares 48,819.5 48,819.5 40,416.4 31,080 9336.38 89,235.8 dF 2 2 13 8 5 15 Mean square 24,409.7 24,409.7 3108.95 3885 1867.28 F 7.85143 7.85143 2.08057 p-Value 0.0058 0.0058 0.2179

Fig. 3. Contour plot of the effect of variables on the solubility of drug in microemulsion formulations (Y1 ).

content in ME formulation increases the fraction of DE that is soluble in the aqueous phase of ME. As shown in Table 1, the globule size of the different ME formulation varied between 28.19 and 151.6 nm. As shown in Table 2, the approximation of response values of Y2 and Y3 based on the quadratic model was the most suitable. ANOVA of the effect of variables on globule size (nm) of ME (Y2 ) and spectroscopic absorbance of ME (Y3 ) shows that the terms X1 , X2 , and X3 have a signicant effect on both responses (p < 0.05). The coefcient of X2 X3 for both responses was largest, showing the

negative effect of combination of Smix and water content on the globule size of the MEs and thus, their absorbance. Fig. 4a and b shows the contour diagrams illustrating the effect of varying ratios of (X1 ), (X2 ) and (X3 ) on the globule size (nm) of ME (Y2 ) and spectroscopic absorbance of ME (Y3 ), respectively. It is obvious that there is an optimum ratio of all the mixture components for ME formulation having small globule size and absorbance. Sufcient concentration of water is needed for maximal effect of Smix on emulsication of Capryol . The aim of the optimization of pharmaceutical formulations is generally to determine the levels of the variable from which a robust product with high quality characteristics may be produced (Basalious et al., 2010). Some of the measured responses have to be minimized. In this case, these responses comprise the globule size (<100 nm) and the absorbance (<0.09). The small globule size of oil phase of ME with high fraction of drug entrapped allows better penetration of these oil globules into the skin to act as in situ depot releasing the drug in controlled rate. Some responses, such as the solubility of drug in ME should have an optimal intermediate range (200350 mg/ml) to increase the fraction of drug incorporated into the oil globules allowing the controlled release of drug from ME globules. The very high solubility of drug in ME systems (about 480 mg/ml in case of formulation 13) is not required for formulation of in-skin depot MEs as most of the drug is entrapped in the aqueous phase of ME systems showing poor absorption into the lipid of stratum corneum. Under these conditions, these three responses were then combined to determine an all over optimum region. Fig. 5 shows an acceptable region met the requirement of these responses. According to the selection criteria, those ME compositions with high oil content, minimum content of Smix, optimal DE solubility and smallest globule size were chosen. A ME formulation satisfying these criteria was prepared and evaluated. An optimum response was found with Y1 , Y2 , and Y3 of 317.6 mg/ml, 54.4 nm and 0.024 at X1 , X2 and X3 values of 30%, 50% and 20%, respectively. DE was dissolved directly in the optimized ME to prepare drug loaded ME containing 1.3% w/w DE. PMBG (containing 1.3% w/w DE) was prepared by mixing plain poloxamer gel with the optimized ME containing the drug in a ratio of gel:ME (2:1) with continuous stirring. Our goal was to prepare thermosensitive PMBG that undergoes gelling at skin temperature. The low viscosity is required at the application site to allow better penetration of oil globules of the optimized ME into skin. However, Poloxamer gel had lost its thermosensitive properties upon mixing with ME. PMBG and ME were subjected to further studies to investigate their ability to enhance DE transport into skin and formation of in-skin depot.

Fig. 4. Contour plots of the effect of variables on the globule size (Y2 ) (a) and the spectroscopic absorbance (Y3 ) (b) of the microemulsion formulations.

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3.5. Skin irritation test Various formulations, when applied topically, might cause skin irritation. Thus, rat skin irritation experiments were conducted in order to assess the potential irritant effects of the optimized drugloaded ME, PMBG and the market formulation (Flector gel). All the ME formulations and the market product show no erythema. Thus, the optimized ME formulations were safe to be used for transdermal drug delivery.

3.6. Ex vivo transport of DE from optimized formulations into rat skin and ability to form in situ drug depot Ex vivo skin transport from the optimized drug-loaded ME, PMBG and the market formulation (Flector gel) through newly born rat skin are illustrated in Fig. 7. One gram of each formulation was placed on skin of newly born rat attached to a cylindrical tube having surface area 4.9 cm2 . Amongst the formulations tested, the optimized drug-loaded ME showed the highest cumulative amount of DE permeated after 8 h (345.45 g/cm2 29.8) followed by PMBG (57.45 g/cm2 9.8) and nally the market formulation (9.45 g/cm2 2.9). The content of the surfactants mixture in MEs signicantly enhanced the transport of drug through skin. Moreover, the small globule size of the ME droplets also affects the percutaneous absorption of the drug. When the droplet size is very small, there is a chance that the number of vesicles that can interact with a xed area of stratum corneum to increase, thereby increasing the efciency in percutaneous uptake (Shah et al., 2010). Thus, the high skin transport of DE from ME is mainly due to the amount of drug solubilized in small oil globules that easily transport through the lipid of stratum corneum of the skin. Although containing the same surfactant mixture and globule size as ME, PMBG showed signicant reduction in drug transport in skin. An explanation for this observation may be due to the high water content of PMBG (about 75%). The major amount of DE is located in the aqueous phase interacting with Poloxamer micelles and consequently lower transport rate through the skin was observed (Xuan, 2011). The explanation is useful also for the poor skin transport of DE from Flector gel compared with ME formulations especially when we know that composition of Flector gel contains about 75% water with nonionic surfactant such as PEG 400 monostearate. The lack of oil globules in the market product

Fig. 5. Overlay plot for the effect of different variables on the three responses. The solubility of drug in microemulsion, mg/ml (Y1 ), mean globule size (Y2 ) and absorbance of microemulsion (Y3 ).

3.4. Stability studies of the optimized formulations The optimized formulations were stable when stored at 40 C/75% RH for three months where there was no obvious change in visual appearance. The drug content of the fresh and stored formulations of the optimized drug-loaded ME were 100.62% 2.12 and 98.54% 2.82, respectively. The drug content of the fresh and stored formulations of the optimized PMBG were 104.89% 3.22 and 101.98% 3.6, respectively. The pH values of the fresh and stored optimized formulations ranged from 6.5 to 7. The morphology of the optimized drug-loaded ME examined via TEM was not changed before and after storage. Transmission electron micrographs of the optimized formulation, Fig. 6 revealed that the globules of the developed MEs are spherical, discrete and have uniform droplet size distribution. Globules appear to have comparable size to the calculated values obtained by photon correlation spectroscopy (Table 1).

Fig. 6. Transmission electron micrographs of the fresh (a) and stored (b) optimized drug-loaded microemulsion taken at 30,000 magnication.

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Fig. 7. Permeation proles of DE from ME, PMBG and Flector gel through rat skin.

explains the signicant lowering of DE skin transport compared to PMBG. To conrm that DE containing oil globules transport through stratum corneum and act as in-skin drug depot for sustained delivery of drug, release of DE from the skin was observed after removal of formulations after 3 h of permeation from the donor compartment. As shown in Fig. 7, the cumulative amount of DE permeated from ME (removed 3 h after permeation from donor compartment) was continuously increased with lower rate compared with that of normal ME. This observation conrms that skin acts as drug reservoir (in-skin depot) after removal of ME. In case of PMBG and Flector gel, there was no remarkable increase of the cumulative amount permeated of DE after removal of these formulations from donor compartment. Thus, the gel matrix rather than skin acts as drug reservoir for these formulations. Contrary to ME, the availability of the gel on skin surface is very important to maintain anti-inammatory efcacy. To conrm the previous results, the three formulations were subjected to in vivo study for treating inamed rat skin.
Fig. 8. Anti-inammatory efcacy of drug-loaded ME and PMBG compared to the market product in rat paw edema.

3.7. In vivo study of the anti-inammatory efcacy and sustained delivery of in-skin depot of DE The anti-inammatory efcacy of DE was taken as a measure of in-skin depot formation and the extent of transport of drug through the skin from the medicated formulations (ME, PMBG and Flector gel). After 3 h of formulation application (sufcient time for skin loading and formation of in-skin drug depot), formulations remaining on the surface of the paw were wiped off with cotton. Rat hind paw edema was used as a model for inammation in this study (Winter et al., 1963). Results revealed that injection of carrageenan (selected as inammagen) produced a pronounced edema. Formulations applied to inamed area were removed after 3 h of application. Thus, the higher the amount of DE loaded into skin (in-skin drug depot), the extended transdermal drug delivery and the higher is the anti-inammatory efcacy as the skin itself acts as drug reservoir. The anti-inammatory efcacy of single dose application of DE-loaded ME and PMBG was tested compared to Flector gel containing the same concentration on the carrageenan induced rat hind paw edema at different time intervals up to 12 h using plain base as a control. As shown in Fig. 8, the inhibition of edema started 5 h

after formulation application (2 h after formulation removal). The highest inhibition of edema was observed in case of drug-loaded ME where the effect was sustained to 12 h and was signicantly different from that of plain base 6 h (p < 0.05). Fig. 9 shows photoimages of right hind rat paw showing edema before and six hours after application of drug-loaded microemulsion. These results correlate well with results previously obtained by ex vivo skin transport study conrming that oil globules of ME containing solubilized DE penetrate through stratum corneum and act as in-skin drug depot for the sustained delivery of drug in the skin. This is highly useful in dermal and transdermal deliver of drugs where the delivery system is applied onto skin for few hours (at night before sleeping) to load skin with drug then the in-skin depot continue the transdermal delivery of the drug. This is advantageous in case of skin where topical application of conventional systems for treatment of soft tissue injuries is highly frequent reaching up to four times daily. The inhibitory effect of PMBG and Flector gel was remarkably lower than that of ME conrming that these gels act as the

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Fig. 9. Photoimages of right hind rat paw showing edema before and 6 h after application of drug-loaded microemulsion.

drug reservoir and its availability on the skin surface is a must to sustain the delivery of the drug into the skin. 4. Conclusion In this study, ME and PMBG of DE were prepared and in vitro evaluated. D-optimal mixture experimental design was applied in order to rapidly obtain the optimal DE-loaded ME formulations containing maximum amount of oil having minimum globule size which allow transport of drug into skin forming in-skin depot for sustained transdermal delivery of the drug. The optimized ME formulation composed of 30% Capryol , 50% Smix (a mixture of Labrasol /Transcutol , 1:2 w/w) and 20% water. The stability of the optimized formulation was retained after storage at 40 C/75% RH for three months. The ME formulations showed no skin irritation and are safe to be used for transdermal drug delivery. Contrary to PMBG and Flector gel, the optimized ME showed the highest cumulative amount of DE permeated after 8 h and the release of DE from the skin was observed even after removal of ME applied to the skin. The high skin transport of DE from ME is mainly due to the amount of drug solubilized in small oil globules that easily transport through the lipid of stratum corneum of the skin. The in vivo anti-inammatory efcacy in rat paw edema was sustained after removal of ME applied to the skin conrming the formation of inskin drug depot. The signicant increase in DE transport through the skin and the formation of in-skin drug depot by the developed ME propose that the prepared system could be promising to sustain the transdermal delivery of DE for treatment of soft tissue injuries. The extended transdermal delivery of the optimized ME and clinical evaluation on human patients with musculoskeletal pain needs to be investigated. Acknowledgements We are very grateful for Marcyrl for Pharmaceutical Industries and Gattefosse for providing the required chemicals for research work. We are also grateful to Dr. Ayman El-Sahar (Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University), for his kind help in the in vivo study in this paper. References
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