DEVELOPMENT AND ESTIMATION OF ANTI-INFLAMMATORY ACTIVITY OF TOPICAL ETORICOXIB EMULGEL BY CARRAGEENAN INDUCED PAW OEDEMA METHOD

Emulgel is one of the emerging topical drug delivery system for the delivery of hydrophobic drugs which overcome various disadvantages of ointments and creams such as greasiness and phase inversion. Etoricoxib is poorly aqueous soluble Non steroidal Anti-Inflammatory Drug (NSAID). It is used in osteoarthritis, rheumatoid arthritis, acute gouty arthritis, ankylosing spondylitis, low back pain, acute postoperative pain, and primary dysmenorrheal. Its oral delivery is associated with greater chance of adverse effects or therapeutic failure and large amount of drug is lost in the vicinity of the target organ. Also oral administration of etoricoxib causes gastro-intestinal irritation. The aim of the present study was to develop an emulgel formulations using the gelling agent like Carbopol 934 and HPMCK4M with emulsifiers like Span 20 and Tween 20. Six emulgel formulations were developed and evaluated on different parameters like physical appearance, pH, viscosity, extrudability, drug content, spreadability, In-vitro diffusion studies, and skin irritation test. Best formulation of batch EG1 was further evaluated for stability study and anti-inflammatory activity in carrageenan induced rat paw edema. Anti-inflammatory effect of formulation EG1 was compared with standard market product Indomethacin.


INTRODUCTION
Drug delivery via skin has a potential view for a long time because skin is easy to access with large surface area and the route is non invasive. This route of drug delivery has gained popularity due to some advantages like, it avoids first pass effects, gastrointestinal irritation, and metabolic degradation associated with oral administration 1,2 . Gels are a relatively newer class of drug delivery systems constituting large amounts of aqueous or hydro alcoholic liquid in a complex network of solid particles. Topical gel formulations provide suitable delivery system for drugs because they are less greasy and can be easily removed from the skin 3 . They are intended to be applied to the skin or certain mucous membranes for protective, prophylactic or therapeutic purposes. They embrace a higher aqueous component that assists in dissolution of drugs, and facilitate easy drug release through the vehicle that is essentially a liquid 4 . Despite many advantages some limitations are associated with gels, major drawback remains, which is its ability to deliver hydrophobic drugs. To overcome this limitation, emulgel have been emerged as an effective delivery system aiding the incorporation of hydrophobic drugs in gel dosage forms. When gels and emulsions are utilized in combination, the dosage forms are referred as emulgel 5 . Emulgel is emerging field for the topical drug delivery, and till date it has less marketed product, so it is interesting and challenging to focus on emulgel 6 . Emulgel is the approach using the benefits of both emulsion and gels, gaining the dual controlled release effect where the emulsion either oil in water or water in oil is gelled by incorporation in the gel base, simply the emulgel are emulsion in gel. Emulgel possess the properties as thixotropic, grease less, easily spreadable, easily removable, emollient, non staining, water soluble, long shelf life, bio friendly and pleasing appearance that improves the patient acceptability 7 .
Etoricoxib is a Non steroidal Anti-Inflammatory Drug (NSAID) belonging to the class of cyclooxygenase 2 (COX-2) inhibitors used in the treatment of relieving moderate pain and swelling of joints associated with different forms of arthritis 8 . Clinical trials have established the efficacy of etoricoxib in osteoarthritis, rheumatoid arthritis, acute gouty arthritis, low back pain, acute postoperative pain, and primary dysmenorrheal 9 .  It's very low aqueous solubility and poor dissolution can cause formulation problems and limit its therapeutic application by delaying the rate of absorption and the onset of action by oral route. Moreover, oral treatment is associated with greater chance of adverse effects or therapeutic failure and large amount of drug is lost in the vicinity of the target organ. Also oral administration of etoricoxib causes gastro-intestinal irritation 10 . In the present study, an attempt has been made to develop emulgel formulations of etoricoxib so as to reduce the side effects of oral administration and to enhance percutaneous absorption. The selected formulations were further characterized for antiinflammatory activity and compared with standard market product Indomethacin.

Preparation of Etoricoxib emulgel
The methods used to prepare etoricoxib emulgel was followed as used by Yadav et al. in a previous study 11 . By dispersing carbopol and HPMCK4M in purified water with constant stirring at a moderate speed gels in formulations were prepared. By the means of triethanol amine pH are adjusted to around 6. Aqueous phase was prepared by dissolving Tween 20 in purified water while the oil phase of the emulsion was prepared by dissolving Span 20 in light liquid paraffin. Methyl paraben was dissolved in propylene glycol whereas drug was dissolved in ethanol and both solutions were mixed with the aqueous phase. Both the oily and aqueous phases were separately heated to 70° to 80°C; then the oily phase were added to the aqueous phase with continuous stirring until cooled to room temperature. Finally the emulgel was prepared by mixing the both gel and emulsion in 1:1 ratio. The composition of different formulations is shown in Table 1.

Evaluation of emulgel formulations Physical Examination
The prepared etoricoxib emulgel formulations were inspected visually for their colour, homogeneity, consistency, grittiness and phase separation. The evaluation of the developed formulations for clarity observed visually with naked eye. Smears of gels were prepared on glass slides and observed under a compound microscope for the presence of any insoluble particles or grittiness 12 . pH The pH of the sample was measured by a digital pH meter (New Heights Pharmaceuticals ltd, Nigeria) at room temperature. The electrode was dipped in gel for 10 seconds, and the value was read on the digital interface 13 .

Extrudability
It is an empirical test to measure the force required to extrude the material from tube. In the current study, the method adopted for extrudability is based upon the quantity of etoricoxib emulgel and quantity extruded from lacquered aluminium collapsible tube on application of weight in grams required to extrude at least 0.5 cm ribbon of emulgel in 10 seconds. More the quantity extruded, better is extrudability 14, 15 .

Drug content
The drug content of etoricoxib emulgel was measured by dissolving a known weight of the emulgel formulation (one gram) in 100 ml methanol, appropriate dilutions were made and the resulting solution was then filtering using millipore filter (0.45 μm). Absorbance was measured at 275 nm using UV spectrophotometer (Shimadzu UV 1800). Drug content was calculated using the slope and the intercept obtained by linear regression analysis of standard calibration curve 17 .

Skin irritation test
A set of 4 rats was used in the study. A 0.5 gm of etoricoxib emulgel formulation was applied on the properly shaven area of skin approximately 2.54x2.54 cm 2 . When the undesirable changes like skin color; change in skin morphology was checked for a period of 24 hr 18 .

In-vitro diffusion study
The in-vitro diffusion study of prepared gel was carried out in Keshary Chein diffusion cell apparatus. In Keshary-Chein diffusion cell, 500 mg of etoricoxib emulgel was spread uniformly on the cellophane membrane which was previously soaked in phosphate buffer pH 5.5 for 24 h and was sandwiched between donor and receptor compartment. 6 ml of phosphate buffer was used as receptor compartment. The temperature was maintained at 37±0.5°C. The whole assembly was fixed on a magnetic stirrer and the solution in the receptor compartment was constantly stirred using magnetic bead at 450 rpm. Sample of 1 ml was withdrawn and replacement was done with 1 ml of fresh buffer. The drug concentration on the receptor fluid was determined spectrophotometrically against blank at 275 nm. The cumulative amount of drug released expressed in % was plotted for formulation 19,20 .

Figure 2: Stability study of etoricoxib emulgel formulation of batch EG1 Anti-inflammatory study
Healthy albino rats of either sex (Wistar strain) weighing 150-200 g were used in current study. The animals were kept in plastic cages with soft bedding, 6 per cage with free access to food and water and were maintained under controlled temperature (27±2 0 C) and 12 hrs: 12 hrs light and dark cycle. Prior to the experiment, food was withdrawn overnight but adequate water was provided to the rats 21 .

Figure 3: Mean % paw volume and thickness inhibition
The animals were divided into two groups of 6 animals each. Standard group was treated with indomethacin given by I.P., test group receive 2g etoricoxib emulgel formulations of batch EG1 were applied over 9 cm 2 as transdermal patch on the dorsal skin after removing the hair with a clipper. The area of application was occluded with bandages and it was left in place for 2 hrs. The dressing was then removed and the emulgel remaining on the surface was wiped off with cotton. The animals were then injected with 0.1 ml of 1% carrageenan solution in saline in plantar region of left hind paw and the paw volume was measured after 1, 2, 4, 6, 8 hr using water plethysmometer. The right hind paw served as a reference non inflamed paw for comparison. Paw volume: Initial Rat paw volume was measured using plethysmometer. The paw was marked with ink at the level of lateral malleolus and immersed in mercury up to the mark in the plethysmometer 22 . Mean changes in paw volume were calculated and % inhibition of paw edema was calculated using formula: Where, Vc is mean changes in paw volume of control group and Vt is mean changes in paw volume treated group.   Paw thickness: Paw thickness was measured by compressing the joint by rotating the screw of micrometer screw gauge till the pain elicited as indicated by squeaking or leg withdrawal 23 . The distance moved by the screw gauge was recorded and % inhibition of paw thickness was calculated using formula: Where, Tc is mean change in paw thickness of arthritis control group and Tt is mean changes in paw thickness of treated group. Statistical analysis: Data was expressed as mean± SD and analyzed for statistical significance using Student's t test, one-way analysis of variance (ANOVA) followed by Dunnett's test or two-way ANOVA followed by Bonferroni test. P<0.05, p<0.01 and p<0.001 was considered to be significant.

RESULTS AND DISCUSSION
The objective of the present study was to design and characterize an effective topical drug delivery of etoricoxib. Due to its low aqueous solubility and poor dissolution it is a poor candidate for oral route. Thus a unique approach of formulating hydrophobic drugs, emulgel, where emulsions could be easily incorporated into gels was attempted. The solubility of hydrophobic drugs can be improved by incorporating them into emulsion form, which further helps in enhancing their skin permeability. In current study 6 emulgel formulations of etoricoxib were were successfully formulated using the gelling agent like Carbopol 934 and HPMCK4M with emulsifiers like Span 20 and Tween 20 developed and evaluated on different parameters. The results of physicochemical parameters i.e pH, viscosity, extrudability and drug content of all batches are shown in Table 2. All trials of emulgel were found to be homogeneous and smears were transparent without grittiness or presence of any particles. The pH values of emulgel batches were found in the range of 6.8-7.1 which is similar to skin pH. Viscosity is an important parameter for characterizing the emulgels as it affects the extrudability and release of drug. The viscosity range observed for all formulation was 12208-12800 cps. Extrusion of the emulgel from the tube is an important during application and for the patient compliance. Emulgels with high consistency may not extrude from the tube easily, where as low viscous gel may show quickly extrudabilty of emulgels. The extrudability of all formulations was found to be satisfactory. The percentage drug content of all formulations was found to be satisfactory and in the range of 93.54±0.52-98.74±0.08%. The allergic symptoms like inflammation, redness, irritation, erythema and edema are not appeared on rats up to 24hr. The in vitro drug release profiles of etoricoxib emulgel formulations are shown in Figure 1. Drug release kinetics data of the emulgels is given in Table 3. Local injection of carrageenan into rat hind paw induces acute inflammatory responses such as edema. The development of the edema induced by carrageenan has been described as a biphasic event 22 . A rapid early phase (up to 2 h) is triggered by the concerted release of histamine, bradykinin, 5-hydroxytryptamine or cyclooxygenase products. And a more sustained late phase (2 to 5 h) is regulated by neutrophil infiltration and sustained production of arachidonic metabolites (prostanoids) (primarily by cyclooxygenase) or nitric oxide from inducible nitric oxide synthase 23 . Standard indomethacin shows 54.861% mean paw volume percentage inhibition, while emulgel formulation of batch EG1 shows 18.59% (Table 4). In a previous study Prakash et al. has evaluated topical etoricoxib gel for anti-inflammatory study for 6 hrs. Current study checked this study for 8 hrs to observe the changes for a longer duration 24 . Mean percentage inhibition of paw thickness by indomethacin was found to be 29.91%, while EG1 shows 18.986% (Table 5).

CONCLUSION
At present scenario, emulgel is one of the recent technologies used for dual control release of emulsion and gel for topical use. Moreover, they will become a solution for loading hydrophobic drugs in water soluble gel bases for the long term stability. The stability of emulsion is increased, when it is incorporated into gel. Etoricoxib is a hydrophobic drug. In current study Etoricoxib was successfully formulated in an emulgel drug delivery with gelling agent like Carbopol 934 and HPMCK4M with emulsifiers like Span 20 and Tween 20 developed with simple, commercial feasible manufacturing process. Etoricoxib emulgel formulation EG1 used in this study showed significant reduction of paw edema thickness and volume at 8 hrs or more after carrageenan injection, demonstrated that the emulgel possess fairly good anti-inflammatory activity. Based on different evaluation parameters, study concludes that EG1 emulgel formulation was the optimum formulations. However, in vivo investigation in relevant animal models may provide further insights into the efficiency of this drug delivery system and its relevance, which inevitably warrants further research.