FORMULATION DEVELOPMENT AND EVALUATION OF IN SITU OPHTHALMIC GEL OF EPINASTINE HYDROCHLORIDE

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1 Page3071 Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: FORMULATION DEVELOPMENT AND EVALUATION OF IN SITU OPHTHALMIC GEL OF EPINASTINE HYDROCHLORIDE Azmat Shaikh 1 *, Talat Farheen 1, Sadhana Shahi 2, Zahid Zaheer 1 1 Y.B. Chavan College of Pharmacy, Department of Pharmaceutics, Aurangabad , Maharashtra, India. 2 Government College of Pharmacy, Department of Pharmaceutics, Aurangabad , Maharashtra, India. ARTICLE INFO Article history Received 21/09/2015 Available online 31/10/2015 Keywords In Situ Ophthalmic Gel, Epinastine Hydrochloride, Gelrite, HPMC E-50LV, Factorial Design. ABSTRACT The poor bioavailability of ophthalmic solutions caused by dilution and drainage from the eye can be overcome by using in situ forming ophthalmic drug delivery system prepared from polymer that exhibit reversible liquid gel phase transition. The objective of the study was to develop optimized formulation of in situophthalmic gel of Epinastine hydrochloride using ion activated polymer, Gelrite (gellan gum) as a gelling polymer, HPMC E-50LV (hydroxyl propyl methyl cellulose) as release retardant and benzalkonium chloride as a preservative. The 3 2 full factorial design was employed to optimize the formulation wherein HPMC E- 50LV (X 1 ) and Gelrite (X 2 ) were taken as independent variables & the dependent variables were viscosity (Y 1 ) and release of drug (Y 2 ). The formulations were assessed for appearance, gelation ability, sterility, ph, drug content, viscosity, release through cellophane membrane & corneal membrane of goat, ocular irritation study & stability study.the developed formulations F4 and F5 werefound to be stable, non-irritant to rabbit eyes and in vitro drug release were found to be 95.56% and 95.13% over the period of 12 hr through cellophane membrane and 91.24% and 91.22% through corneal membrane of goat respectively.the stable in situ gelling system of Epinastine hydrochloridewas successfully developed using Gelrite and HPMC E-50LV. Corresponding author Azmat Shaikh Y.B. Chavan College of Pharmacy, Department of Pharmaceutics, Aurangabad azmat.pharma@gmail.com Please cite this article in press as Azmat Shaikh et al. Formulation Development and Evaluation of In Situ Ophthalmic Gel of Epinastine Hydrochloride. Indo American Journal of Pharmaceutical Research.2015:5(10). Copy right 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page3072 INTRODUCTION As per USPXII ophthalmic solutions are sterile solutions, essentially free from foreign particles, suitably compounded and packed for instillation into the eye [1].Ophthalmic preparations can be in the form of oily solutions, suspensions or ointments and gels. They are sterile products and free from particulate matter. For ailments of the eye, topical administration is usually preferred over systemic administration. Before reaching the anatomical barrier of the cornea, any drug molecule administered by the topical route has to cross the precorneal barriers. The medication, upon instillation, stimulates the protective physiological mechanisms, i.e., blinking and tears production, which exert a formidable defense against ophthalmic drug delivery. Another serious elimination route of topically applied drugs from the precorneal area is the Nasolachrymal drainage. Because of this drug loss, very little drug is available to enter the cornea and inner tissue of the eye. Drug delivery to the eye is restricted due to these limitations imposed by the efficient protective mechanism. Ideal ophthalmic drug delivery must be able to sustain the drug release and to remain in the vicinity of front of the eye for prolonged period of time. Consequently, it is imperative to optimize ophthalmic drug delivery; some of the ways to do so is by addition of polymers of various grades, development of viscous gel, development of colloidal suspension or using erodible or nonerodible insert to prolong the precorneal drug retention. A significant increase in precorneal residence time of drugs and consequently increase in bioavailability can be achieved by using delivery systems based on concept of in situ gel formation. These systems consist of polymers, which undergo reversible sol to gel phase transition in response to physiological stimuli. The sol-gel transition can be induced by a shift in ph, temperature or ion activated systems. This type of gel combines the advantage of a solution (accurate and reproducible administration of drug) and gels (prolonged residence time) for enhancing ocular bioavailability [2,3]. Epinastine hydrochloride is an antihistaminic drug with potent anti-allergic and modest anti-inflammatory activity. It is indicated for the prevention of itching associated with allergic conjunctivitis. In the present study, an attempt is made to develop ion induced in situ gelling ophthalmic delivery system of Epinastine hydrochloride using ion sensitive polymer, Gelrite and viscosity enhancing agent HPMC E50LV for sustained drug delivery by enhancing residence time of drug in eye. The topical Epinastine hydrochloridemay be useful for inflammatory conditions in the eye and is approved for the treatment of seasonal allergic conjunctivitis and ocular inflammation. MATERIALS AND METHODS Materials The Epinastine hydrochloride and Gelrite was gifted by Cipla Research Center, Mumbai, India. HPMC E-50LV was gifted by Colorcon Asia Pvt. Ltd., Goa. All the chemicals used were of analytical grade. UV method development The method development was done by determining λ max, precision, recovery, LOD, LOQ, linearity and by preparing calibration curve[4]. Preliminary batches The preliminary batches of 0.05% Epinastine hydrochloride were formulated using Gelrite, HPMC E-50LV, Benzalkonium chloride, buffering agent and disodium edetate[5].the batches were evaluated for in vitro in situ gelation in Artificial Tear Fluid (ATF) and for viscosity to optimize the concentration of Gelrite and HPMC E-50LV for final formulation as shown intable 1. Table 1: Formulation of Preliminary Batches and its Evaluation. Batch Code Gelrite HPMC E-50LV Gelling capacity Viscosity (cps) (% concentration) (%concentration) E1-0.6 No Gelation 17 E2-0.8 No Gelation 30 E3-1.0 No Gelation 38 E4-2.0 No Gelation 50 E5-3.0 No Gelation 68 E E E E E Viscous Liquid 111 E Viscous Liquid 212 E Viscous Liquid 356 E Direct Gelling 487 E Direct Gelling 555 E Direct Gelling 828 ( ): The solutions which did not undergo phase transition at all. (+):The solutions which exhibited phase transition only after 60 seconds and the formed gels which collapsed within 1-2 hr.

3 Page3073 (++): The solutions which formed the gels after 60 seconds however, the gels formed did not remain stable for more than 3 hr. (+++): The solutions which exhibited phase transition within 60 seconds and the gels formedremained stable for more than 7-8 hr. The above preliminary batches indicated that; HPMC alone does not possess any in situ gelling properties (E1 to E5). Gelrite above the conc. of 0.4 % forms in situ gel in artificial tear fluid (ATF) (E7 to E9). Gelrite above 1.0 % conc. produce formulation of high viscosity that is not suitable for instillation into the eyes (E10 to E12). Gelrite above the conc. of 1.6% produced direct gel formulation (E13 to E15). Hence the level of Gelrite was decided based on in situ gelling capacity. Optimization by 3 2 factorial design Some polymers which are known to undergo transition from sol to gel in the presence of cations present in tear fluid (Ca +2, Na + ) were selected to form in situ gelling ophthalmic solutions of Epinastine hydrochloride. The 0.05% w/v solutions of Epinastine hydrochloridewere prepared using different concentrations of Gelrite as per Table 2. The two independent variables selected were HPMC E-50LV (X 1 ) and Gelrite (X 2 ), and the dependent variables were viscosity (Y 1 ) and release of drug (Y 2 ). The factorial designed batches are shown in Table 2. Table 2: Formulation of Factorial Batches of Epinastine hydrochloride. Ingredients (%w/v) Formulation Code F1 F2 F3 F4 F5 F6 F7 F8 F9 Epinastine hydrochloride HPMC E-50LV (X 1 ) Gelrite (X 2 ) Monobasic sodium phosphate Disodium edentate Benzalkonium chloride (BKC)* Sodium hydroxide/hydrochloric acid q.s. to adjust ph Purified water q.s *The Benzalkonium chloride solution to be added as 50%w/v solution. Preparation of in situ Gelling Ophthalmic Formulation Accurately weighed quantities of monobasic sodium phosphate and disodium editate were dissolved in a part quantity of purified water. The ph of this solution was adjusted to 7.0±0.1 with 0.1N sodium hydroxide or hydrochloric acid. To this solution a weighed quantity of epinastine hydrochloride was added and dissolved, then benzalkonium chloride solution was added under slow stirring (Solution A). The Gelrite was sprinkled over a part quantity of hot water (temperature C) and was allowed to hydrate for 15 min to produce a clear solution (Solution B). The solution B was mixed slowly to solution A with continuous mechanical stirring at 100 rpm to produce clear and transparent solution. The volume was made upto 100 ml with distilled water. The final formulation was sterilized by membrane filtration technique. The solution was filtered through 0.22 m polyether sulphone (PES) membrane filter(make: Pall). The formulation was filled into LDPE bottle with translucent droptainer and white opaque cap (Make: Rexam). Evaluation of in situ Gelling Formulations The ophthalmic formulations were evaluated for various physicochemical characteristics i.e. appearance, clarity, ph and viscosity (Brookfield LVDV-II). The test for sterility was confirmed by Method B described in USP and the end point was judged visually noting the presence of turbidity in the inoculated media. Both positive and negative controls were also maintained simultaneously. The method of detection was visual inspection of turbidity. The test for gelling ability was conducted using ATF[6]. The transition of solution to viscous gel was observed visually and numerical scores were assigned depending upon the quickness of gel formation and time taken for collapse of gel structure on shaking the vials. The drug content was determined by UV spectrophotometer. In vitro Drug Release Study In vitro release was performed through cellophane membrane (pore size 0.45μm) using modified dissolution testing apparatus. The USP type-i apparatus (Basket type) was modified by replacing the basket with glass cylinder of same size. The glass cylinder was attached to the shaft of USP apparatus-1 (Basket type) instead of basket (as shown in Figure.1).The dissolution media was ATF (50 ml) maintained at 37 ± 0.5 C. The sample (1 ml) was withdrawn at regular interval of 1 hr till 12 hr and it was replaced immediately with the same volume of ATF. The samples withdrawn were analyzed for drug content[7].

4 Page3074 Figure1: Modified dissolution test apparatus. In vitro Release test through corneal membrane All the test conditions of in vitro drug release were followed except the cellophane membrane was replaced with the biological membrane i.e. freshly excised cornea of goat. The study was carried out upto 12 hr for the selected formulation F4 and F5, respectively. Preparation of corneal membrane (Goat) The goat eyeballs was procured from a nearby slaughter house and was carefully transported to the laboratory in an airtight container containing cold saline solution (0-2 C). The cornea was separated carefully from eye ball. The surrounding sclera tissue (5-6 mm) was washed with cold saline and corneal membrane was preserved in freshly prepared ATF (maintained at 0-2 C). Ocularirritation study in rabbits The selected formulationsf4 and F5were instilled (0.1ml) into the eye of rabbit. The eyeball was observed for acute toxicity symptoms i.e. redness, inflammation & tear flux at an interval of 1, 4, 24, 28 and 72 hr. The ocular irritation study was measured on a scale of 0 to 4[8]. Stability study The formulations F4 and F5were subjected to stability studies as per ICH guidelines. The formulations were assessed for appearance, gelation, sterility, ph, drug content and viscosity[12]. Population bioequivalence study Population bioequivalence study was done by measurement of drop weights of formulation. The weight of each drop was measured for 10 drops each at 45 & 90 angle as per ASTM guideline [15]. The study was conducted on 10 different bottles and mean drop weight of 10 bottles was compared with marketed formulation to establish the population bioequivalence. RESULTS AND DISCUSSION The formulation of preliminary batches revealed in vitro gelation above the concentration of 0.4% Gelrite and no gelation was observed below 0.4% of Gelrite. The increase in concentration of Gelrite increased the viscosity of the formulation making it difficult to instill into the eyes. The formulated ophthalmic formulations were evaluated for various physical and performance characteristics. The ophthalmic formulations were observed carefully for color, odor and presence of suspended particulate matter if any. The ph of each of prepared ophthalmic formulation was recorded using previously calibrated digital ph meter. The ph of all the formulations was found to be in the range of 6.5 to 7.5. Viscosity was found to be increased with the increase in the concentration of Gelrite from 0.4 to 0.6%. Similarly viscosity increased with the increase in the concentration of HPMC E-50LV for concentration from 0.6 to 1.0%. Increase in viscosity of ophthalmic solutions after instillation in eye was a desired feature for the purpose of sustaining therapeutics actions of the antihistaminic agent, epinastine hydrochloride,by providing increased precorneal residence time. The increase in viscosity was achieved due to the inclusion of Gelrite which undergoes gelation when it comes in contact of calcium or sodium ions of tear fluid (Table 3).

5 Page3075 Table 3: Viscosity Values for Ophthalmic Formulation. Sr.no Batch Code Viscosity* 1 F F F F F F F F F *Brooke Field LVDVII + Viscometer- spindle no. S18 at 30 rpm The test for sterility for the selected formulations indicated no turbidity after incubation at specified conditions upto 14 days, while the positive controls revealed dense turbidity. The test for in vitro gelation ability was performed to assess the gel characteristics which would affect drug release in the ATF. The numerical scores for gelling ability of solutions were found to vary with change in the concentration of Gelrite (Table 4). Table 4: Gelling Capacity of Ophthalmic Formulations. Formulation Gelrite HPMC E-50 LV Gelling ability code (% w/v) (% w/v) F F F F F F F F F (-): No gelation (+): Gels after few seconds, dissolves rapidly (within 1-2 h) (++): Gelation immediately, remains for few h (3-4 h) (+++): Gelation immediately, remains for extended period (more than 6-8 h) The phase transition of the ophthalmic formulations containing Gelrite was found to be concentration dependent. Thus, the increased concentration of Gelrite caused decrease in the time taken for gelation. In vitro release through cellophane membrane revealed that with the increase in the concentration of HPMC E-50LV the release decreased due to the formation of gel structure. As the concentration of Gelrite increased from 0.4% to 0.6% there was further retardation in the release (Formulations F4-F9).This may be accounted for the reduction in number and dimensions of the channels in the gel structure due to enhanced viscosity of gel. The kinetics of drug release mechanism (PCP Disso version 2.08 software) revealed matrix model kinetics. Figure 2: Percentage drug release from factorial batches (F1, F2 &F3).

6 Page3076 Figure 3: Percentage drug release from factorial batches (F4, F5 &F6). Figure 4: Percentage drug release from factorial batches (F7, F8 & F9). The release of Epinastine hydrochloride through goat cornea was slower as compared to the release through cellophane membrane. Hence, the formulation containing Gelrite is most effective in sustaining the release of Epinastine hydrochloride over the period 12 hr (Figure 5). The ocular irritation study revealed that the numerical score for the formulation F4 and F5 were reading zero, an indication of safe and non-irritantformulation for administration into eye. The findings of the stability study suggested no significant change in the selected physical parameters for the formulations F4 and F5. Figure 5: Percentage drug release through cellophane and corneal membrane.

7 Page3077 F4 cel: Drug release through cellophane membrane F4 cor: Drug release through corneal membrane F5 cel: Drug release through cellophane membrane F5 cor: Drug release through corneal membrane The release profile of the F4 and F5 were compared with that of marketed preparation (EPINA Eye drops), as shown in figure 6. The formulation F4 & F5 indicated sustained release of drug upto 12hr as compared to marketed product (EPINA eye drops) which released 99.22% release within 6hrs. Figure 6: Comparison of release profile with marketed product. Population bioequivalence was carried out by comparing mean drop weight of developed formulation with marketed product of epinastine hydrochloride [EPINA Eye Drops manufactured by CIPLA].The results are as appended in Table 5. Table 5: Population bioequivalence study. Drop weight (in mg)* No of bottles F4 (Test) EPINA (CIPLA) (Reference) Bottle Bottle Bottle Bottle Bottle Bottle Bottle Bottle Bottle Bottle Mean Min Max Standard deviation % RSD No of drops (N) Population Bioequivalence Pass *Mean of drop weight of 10 drops each at 45 o and 90 o angle. CONCLUSION An attempt has been made to develop in situ gelling systems of Epinastine hydrochloride, anti-allergic & anti-inflammatory drug, to increase the ocular residence time. The variables Gelrite and HPMC E-50LV exhibited significant effect on the responses viscosity and release of the formulation. A stable in situ gelling system of Epinastine hydrochloride has been successfully developed using Gelrite and HPMC E-50LV and is a promising approach for the treatment seasonal allergic conjunctivitis to prevent the ocular itching.

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