pH INDUCED IN-SITU GEL FORMULATION OF LOMEFLOXACIN HCL S. J. SHANKAR*, ANUDEEP KALIKONDA Department of Pharmaceutical Technology, PES College of Pharmacy *PES College of Pharmacy, Department of Pharmaceutical Technology, 50 feet road, Hanumantha nagar, BSK Ist stage, Bangalore-560050, karnataka, India. e-mail: shankarsj@rediffmail.com ABSTRACT The effective dose administered ophthalmically may be altered by increasing the retention time of medication into the eye by using an in situ gel forming system. The aim of this work was to prepare and evaluate an ophthalmic delivery system of an antibacterial drug Lomefloxacin HCl based on the concept of pH induced in situ gellation. Carbopol 940 was used as the gelling agent in combination with HPMC E50LV and HPMC E4M as viscosity enhancing agent and formulations were evaluated for in vitro gelling capacity, rheological study and percentage drug release. The in vitro gelling capacity was carried out using simulated tear fluid showing a higher gelling capacity for Carbopol 940. All the formulations were found to exhibit pseudo plastic behaviour for both the solution and gel. The developed formulations provide sustained release of drug from formulation at the site of action over 8 hrs. Drug release was followed by zero-order kinetic with increased time of residence of the formulation in precornea. The optimized formulations were tested in albino rabbits (male) using the draize test protocol with cross over studies and found to be nonirritant to the rabbit eye. The selected formulations also subjected to stability studies, which show that no changes observed in pH, drug content and clarity during 6 weeks of studies. Thus, the developed system may be valuable alternative to the conventional system. KEY WORDS pH induced insitu gel, Lomefloxacin HCl, Carbopol 940, HPMC E50LV, HPMC E4M INTRODUCTION Ophthalmic drug delivery is one of the most interesting and challenging endeavors facing the pharmaceutical scientist. The anatomy, physiology, and biochemistry of the eye render this organ exquisitely impervious to foreign substances. The challenge to the formulator is to circumvent the protective barriers of the eye without causing permanent tissue damage. The development of newer, more sensitive diagnostic techniques and therapeutic agents render urgency to the development of more successful ocular delivery system.1Drugs may be delivered to treat the precorneal region for such infections as conjuctivitis, and blepharitis, or to provide intra ocular treatment via the cornea for diseases such as glaucoma and uveitis.2 The eye drops is easy to instil but suffers from the inherent drawback that the majority of the medication is immediately diluted in the tear as soon as the eye drop is instilled into the cul-de-sac and is rapidly drained away from the precorneal cavity by constant tear flow. The higher the drug concentration in the eye drop solution, the greater the amount of drug lost through lacrimal-nasal drainage system. Subsequent absorption of this drained drug, if it is high enough, may result in undesirable systemic side effects.3 It is widely accepted that increasing the viscosity of a drug formulation in the precorneal region leads to an increased bioavailability, due to slower drainage from the cornea, several concepts for the in-situ gelling systems have been investigated. These systems can be triggered by pH, temperature and ion activation.6 In the present work, an attempt has been made to formulate pH-induced in-situ gel as an ophthalmic drug delivery system. In situ gels are made from polymers that exhibit phase transition due to physicochemical change in the environment. They can be conveniently dropped as a solution into the conjuctival sac in the eye. Upon contact with the lacrimal fluid, the polymer changes its conformation to form a gel. This delivery system has the ease of administration similar to an opthalmic solution and has a long retention time because of the gel formation. Different polymers for preparing pH induced in-situ gels have been evaluated. MATERIALS AND METHODS Lomefloxacin HCl (IPCA Limited, Ratlam, India), HPMC E50LV &HPMC E4M (Color cone Asia Ltd., Verna, India), Carbopol 940 and all other excipients used are from S.D. Fine Chemicals Ltd., Mumbai and were HPLC grade. Preparation of pH Induced In-Situ Gelling System: The pH sensitive polymers like Carbomer derivatives, Carbopol, forms aqueous solution in the unionized form. These polymers undergo reversible ionization at pH 7.4, physiological condition, to form a stiff gel network, which swells and forms large aqueous pores. Hence, Carbopol 940 was selected for preparation of pH induced insitu gelling system. Preparation of Gelling System: The buffer salts were dissolved in 50 ml of purified water; HPMC (E50LV / E4M) was added to hydrate. Carbopol 940 was sprinkled over this solution and allowed to hydrate overnight. The solution was stirred with an overhead stirrer. Lomefloxacin Hydrochloride was dissolved in small quantity of water, Benzalkonium Chloride (Preservative) and Sodium chloride (Isotonocity adjusting agent) was added to this solution; the drug solution was added to the polymer solution under constant stirring until a uniform solution was obtained. Purified water was then added to make up the volume to 100ml. Formulation pH was adjusted to pH 5 with the help of 0.5 M Sodium Hydroxide. This solution was filtered through 0.2m filter paper. The optimized formulations were sterilized in an autoclave at 121º C and 15 psi for 15 minutes. Evaluation of Prepared Gelling System: Drug Content Analysis: Drug content analysis of prepared in-situ gelling systems was carried out using Spectrophotometric method. The assay of these formulations was carried out by pipetting 0.1 ml of all four optimized formulations, and it was diluted up to 100 ml of Simulated Tear Fluid (pH 7.4). The absorbance was measured at 281.5 nm using UVVisible spectrophotometer. In-Vitro Gellation: The Gelling capacity of the formulations containing different ratio of Carbopol 940 and HPMC (E50LV / E4M) was evaluated. It was performed by placing a drop of polymeric solution in vials containing 1 ml of Simulated Tear Fluid, freshly prepared and equilibrated at 34C, and visually assessed the gel formed and time for gellation as well as time taken for the gel formed to dissolve. Sterility Testing: Sterility testing were intended for detecting the presence of viable form of microorganisms and were performed for aerobic and anaerobic bacteria and fungi by using Fluid Thioglycolate Medium and Soyabean Casein Digest medium, respectively as per the Indian Pharmacopoeia. Rheological Studies: Rheological properties of the prepared gelling systems under the different Shear rates (2, 4, 6, 10, 20, and 30 rpm) were measured at non-physiological (pH 5 and 25C) and physiological condition (pH 7.4 and 34C), respectively. The hierarchy of the shear rate was reversed, and the average of two reading was used to calculate the viscosity. In-Vitro Release Studies: In-vitro drug release from the formulations was studied by the diffusion process. Here the pH of the Lacrimal fluid and the blinking rate of the eye were taken into consideration and were simulated. The in-vitro release of Lomefloxacin HCl was studied through cellophane membrane using diffusion cell. The cellophane membrane was soaked overnight in the receptor medium (Simulated Tear Fluid, pH 7.4). It was tied to one end of a glass diffusion cell. The diffusion cell was filled with 2 ml of the formulation and suspended in 100 ml of receptor containing beaker by assuring that the membrane was just touched the receptor medium surface.The whole assembly was transferred on magnetic stirrer and was maintained at 34C 1C and 22 rpm. The drug samples (1ml) were withdrawn at the interval of 30 minutes from receptor medium and replaced by equal volumes of the receptor medium. The samples were diluted with appropriate receptor medium and analyzed by a UV-Visible spectrophotometer at 281.5 nm using receptor medium as a blank. Comparative evaluations of prepared formulations were compared with that of marketed formulations. Pharmacokinetic Release Studies: All the optimized formulations were subjected to study the release kinetics of it. So the best fit kinetic model was determined for the optimized formulations using analysis software. Antimicrobial Efficacy Studies: The antimicrobial efficacy studies were carried out to ascertain the biological activity of the optimized formulations. Staphylococcus aureus, Pseudomonas aeruginosa and E.coli were used as the test organisms. These were determined by Agar diffusion test employing Cup-Plate method. Ocular Irritancy Studies: It is measured according to Draize test.According to this test, the amount of the test substance applied to the eye is normally 100 l placed into the lower cul-de-sac with observation of the various criteria made at a designated required time interval of 1hr, 24 hrs, 48 hrs, 72 hrs, and 1 week after administration.A total four albino rabbits (male) weighing 1.5-2 kg was used for the present study. The sterile formulations were instilled twice a day for a period of 7 days. Rabbits were observed periodically for redness, swelling, watering of the eye. The evaluation was made according to the Draize test protocol. Accelerated Stabilities Studies: Stability studies were carried out on optimized formulations according to International Conference on Harmonization (ICH) guidelines.A sufficient quantity of formulations in previously sterilized vials was stored in desiccators containing a saturated solution of sodium chloride, which gives a relative humidity of 75±5 %. The desiccators were placed in a hot air oven maintained at a temperature 40C0.5C and at room temperature. Samples were withdrawn at 7, 14, 21 and 30days followed by 30 days till 90 days. The logarithms of percent drug remaining were calculated and plotted against time in days. RESULTS AND DISCUSSION Preparation of pH Induced In-Situ Gelling System: The prepared formulations were evaluated for their viscosity using a Rheometer. 3.2. Evaluation of Prepared In-Situ Gelling Systems: Evaluation of Visual appearance, Clarity, pH, and Drug Content: All the prepared in-situ gelling systems were evaluated for preliminary steps such as Visual appearance, Clarity, pH, and Drug content. These formulations were transparent and clear. The pH of the formulations were found to be 50.5, and drug content was in between 99.38 % to 99.71% shown in table 2. Interaction Studies: The result of these studies reveals that there were no definite changes obtained in the bands of drug with respect to pure drug. Shown in Figures 1 & 2 Sterility Testing: All the prepared in-situ gelling systems were evaluated for the sterility. After 7 days of incubation the results showed that the no microbial growth was found in all formulations. Rheological Studies: Results obtained reveals that the viscosity of all formulations decreased as the shear rate increased, which showed the character of pseudoplastic fluid. The results were shown in table 3. In-Vitro Release Studies: Results (shown in table 4) reveal that all formulations exhibited sustained release of the drug (above 88 %) from the Carbopol 940 and HPMC E50LV /E4M network over 8-hours. Cellulose derivatives like, HPMC E50LV / E4M dissolve in water and yield much more viscous solution compared to Carbopol 940 solution. Thus, the increase in viscosity might have contributed to the decrease in rate of drug release from these formulations. Pharmacokinetic Release Studies: The best fit kinetic model for the optimized formulation were the zero order and peppas model which suggest that the drug release was independent of the concentration and occurred by diffusion mechanism. The polymer can absorb a significant amount of water to form to elastic gel and at the same time, release the dissolved entrapped drug by diffusion through swollen regions of the gel. With a hydrophilic Carbopol-HPMC gel, tear fluid would except to diffuse into the gel interior and leach out water soluble drug such as Lomefloxacin HCl. Antimicrobial Efficacy Studies: Clear zones of inhibition were obtained in the case of all formulations. The diameter of zone of inhibition produced by formulations against all test microorganisms are calculated. The antimicrobial effect of the Lomefloxacin in prepared in-situ gelling systems is probably due to a fairly rapid initial release of the drug into the viscous solution formed by dissolution of gel, followed by formation of a drug reservoir that permits the drug to be released to the target site relatively slowly. Ocular Irritancy Studies: All the formulations were found to be non-irritating with no ocular damage or abnormal clinical signs to the cornea, iris, and conjuctiva observed. Accelerated Stabilities Studies: All the Formulations were analyzed for Visual appearance, Clarity, pH and Drug Remaining. 90 days studies reveal that there were no changes observed in Visual changes and Clarity. All the formulations showed slight changes in pH, but it were in acceptable limits ( 0.5). Study of drug content remaining in all formulations reveals that there were no definite changes observed for drug CONCLUSION The developed formulations are a viable alternative to conventional Lomefloxacin HCl eye drop by virtue of its abilities that it cannot only be readily administered and decreases the frequency of administration, thus resulting in better patient acceptance, but also prolong the precorneal residence time to get higher bioavailability and reduce the systemic side effects caused by the drainage from the nasolacrimal duct. Hence, “pH Induced In-Situ Gelling System of an Anti-infective drug (Lomefloxacin HCl) for Sustained Ocular Delivery” was successful experiment. 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Pharm. 137,233-41. 28. Coquelet C., et al.1996. Association between Benzalkonium Chloride and Poly (Acrylic Acid) Gel Study by Microfiltration and Membrane Dialysis, J. Membr. Sci. 120, 287-93. 29. Furrer P., et al. 2000. Application of In Vivo Confocal Microscopy to the Objective Evaluation of Ocular Irritation Induced by Surfactants, Int. J. Pharm. 207, 89-98. Table 1: Formulation of in-situ gels: S. No. Ingredients 1 2 3 4 5 6 7 Lomefloxacin HCl Carbopol 940 HPMC E50LV HPMC E4M Sodium Chloride Benzalkonium Chloride Acetate Buffer (pH 5) up to Concentrations (% w/v) CHL 2 CHL 3 0.3 0.3 0.4 0.2 1.5 0.4 0.9 0.9 0.01 0.01 100 100 CHL 1 0.3 0.3 1.5 0.9 0.01 100 CHL 4 0.3 0.3 0.4 0.9 0.01 100 Table 2: Preliminary evaluation of Visual appearance, Clarity, pH, and Drug Content. Evaluation steps Visual Appearance Clarity pH Drug Content CHL 1 Transparent Clear 4.99 99.71 % CHL 2 Transparent Clear 4.97 99.38 % CHL 3 Transparent Clear 5.01 99.43 % CHL 4 Transparent Clear 4.98 99.67 % Table 3: Rheological studies of Formulations Shear Rate (rpm) 2 4 6 10 20 30 Viscosity (cps) of Formulations at Viscosity (cps) of Formulationsat Non physiological Condition (pH 5) Physiological Condition (pH 7.4) CHL 1 CHL 2 CHL 3 CHL 4 CHL 1 CHL 2 CHL 3 CHL 4 102.3 90.1 78.2 60.0 35.2 26.9 109.4 92.3 80.4 56.1 40.3 22.9 98.4 86.7 71.5 53.9 45.3 27.1 101.7 89.2 74.3 58.1 50.7 22.8 221.0 132.5 113.5 105.8 87.9 72.6 234 125.1 103 92.3 83.9 63.8 242.1 133.9 101.3 88.1 70.9 51.3 254.5 156 109.4 85.6 68.2 57.5 Table 4: In-Vitro Release Profile: S. No. Time (min.) Pure drug % release of CHL 1 % release % release of CHL 2 of CHL 3 % release of CHL 4 Marketed eye drops 1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 24.83 52.23 72.10 85.96 97.15 11.33 14.93 19.08 23.61 27.50 33.11 37.76 41.96 48.03 52.28 58.65 64.71 69.65 76.65 81.70 88.96 10.83 15.26 19.58 22.45 27.00 32.10 36.90 42.43 47.83 51.30 57.78 63.16 68.60 78.25 84.15 90.00 22.50 49.88 62.55 79.16 90.28 96.88 12.33 16.45 20.31 25.70 30.08 35.03 40.06 44.90 50.35 55.33 61.36 67.28 72.25 78.26 86.50 94.63 Figure 1: IR spectra of pure drug 11.66 15.45 21.60 25.65 31.18 35.76 41.08 44.31 50.9 54.56 60.91 68.65 72.96 79.81 87.56 95.55 Figure 2: IR spectra of Formulation CHL 4 Figure 3: Comparative In-Vitro Release of Marketed Eye Drop and Prepared In-Situ Gels In-vitro Drug Release 100 Pure Drug 90 CHL 1 70 CHL 2 60 50 CHL 3 40 CHL 4 30 Lomebact (Marketed Product) 20 10 Time in Minites 480 450 420 390 360 330 300 270 240 210 180 150 120 90 60 30 0 0 % Release of Drug 80