39 Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 39-45. ISSN 0976 – 1047 IJB International Journal of Biopharmaceutics Journal homepage: www.ijbonline.com DEVELOPMENT AND EVALUATION OF THERMOREVERSIBLE OCULAR GELS OF KETOROLAC TROMETHAMINE Sirish Vodithala *, Sadhna Khatry 1, Nalini Shastri 1, Dr. M. Sadanandam 1 *,1 Department of Pharmaceutics, Sri Venkateshwara College of Pharmacy, Madhapur, Hyderabad -500081 Abstract: In-situ gelling systems are viscous polymer-based liquids that exhibit sol-to-gel phase transition on the ocular surface due to change in a specific physico-chemical parameter like temperature, ionic strength, or pH. A major problem in ocular therapeutics is the attainment of optimal drug concentration at the site of action, which is compromised mainly due to pre-corneal loss resulting in only a small fraction of the drug being ocularly absorbed. The effective dose administered can be altered by increasing the retention time of medication into the eye by using in situ gel forming systems, thereby preventing the tear drainage. The aim of the present investigation is to prepare and evaluate novel in situ ocular gelling system (Thermo-reversible gelling systems) of Ketorolac tromethamine. These gelling systems involve the use of Poloxamer (Pluronic F 127) as thermo reversible polymer and Methyl cellulose as release retardant. The formulations were evaluated for clarity, pH measurement, gelling capacity, drug content estimation, rheological study, in vitro drug release, ocular irritation studies (as per draize test) and ex-vivo corneal permeation studies using isolated goats cornea. The developed formulations showed sustained release of drug for upto 5 hrs. The formulations were found to be non-irritating with no ocular damage. Keywords: Ketorolac tromethamine, in situ gels, Draize test, ex-vivo studies, Pluronic F 127 Introduction Ophthalmic drug delivery is one of the interesting and of actions. Approaches to improve the ocular bioavailability challenging endeavors facing the pharmaceutical scientist aim at increasing the corneal permeability by using today. The anatomy, physiology, and biochemistry of the penetration enhancers or prodrugs, and prolonging the eye render this organ highly impervious to foreign contact time with the ocular surface by using viscosity- substances. enhancing or in-situ gelling polymers. The in-situ gelling A significant challenge to the formulator is to circumvent polymers undergo sol-to-gel phase transition on exposure to the protective barriers of the eye without causing permanent the physiological conditions present in the eye. In-situ tissue damage. The major problem encountered with topical gelling systems are viscous polymer-based liquids that administration is the rapid pre-corneal loss caused by exhibit sol-to-gel phase transition on the ocular surface due nasolacrimal drainage and high tear fluid turnover which to change in a specific physico-chemical parameter leads to only 10% drug concentrations available at the site (temperature, ionic strength, or pH) (Ashim K. Mitra., 2003., M. Gibaldi et al 1982). In situ gel forming systems * Corresponding Author can be classified as temperature dependent systems (e.g Pluronics [El-Kamel, 2002; Cho et al., 2003; Cho et al., 2005; Qi et al., 2007]), Tetronics ([Spancake et al,. 1991]) Sirish Vodithala and polymethacrylates), pH triggered systems (e.g Carbopol Email: email@example.com (Aggarwal et al ,. 2005; Sultana et al,. 2006; Wu et al., 2007) and cellulose acetate phthalate ([Gurny et al,. 1985]) and ion activated systems (e.g Gelrite [Balasubramaniam et 40 Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 39-45. al,. 2003, 2005]) and sodium alginate[Liu et al., 2006]). The simulated tear fluid (STF) is sodium chloride 0.670 g, principal advantage of in-situ gelling systems is the easy, sodium bicarbonate 0.200 g, calcium chloride dihydrate accurate and reproducible administration of a dose 0.008 g, purified water upto 100.0 g. The optimized compared to the application of preformed gels. They have formulation for thermo-reversible ocular gels is shown in an advantage over preformed gels that they can be easily Table 3. instilled in liquid form, and are capable of prolonging the Characterization of Thermo reversible ocular gels residence time of the formulation on the surface of the eye a. Clarity: due to gelling. The clarity of the formulations before and after gelling was Ketorolac tromethamine is a nonsteroidal anti-inflammatory determined by visual examination of the formulations under drug, used to treat seasonal allergic conjunctivitis. light alternatively against white and black backgrounds. Temperature induced insitu ocular gels of Ketorolac b. pH: tromethamine were prepared by using polymers like Formulation was taken in a beaker and 0.1M NaOH was Pluronic F 127 and methyl cellulose. Pluronic F 127 (Wen- added dropwise with continuous stirring. pH was checked Di Ma et al ., 2008) was used as a thermo-reversible using pH meter ( µ pH Systronics digital pH meter) polymer and methyl cellulose was used to sustain the drug c. Assay: release. The present investigation deals with development Accurately weighed amount KT insitu gel equivalent to 5mg and evaluation of temperature induced insitu ocular gels of of drug was taken in a 100ml volumetric flask. Simulated ketorolac tromethamine. The dosage regimens prepared Tear Fluid (STF pH 7.4) was added to it and kept on provide ease in application and provides sustained drug magnetic stirrer to dissolve the drug. The volume was made release with reduced frequency of administration. to 100ml with STF (pH 7.4).and filtered using Whatmann Materials and Methods: filter paper (No 42). 10ml aliquot of the above solution was Ketorolac tromethamine was obtained as a gift sample from taken and diluted to 100ml with STF (pH 7.4). The Symed labs; Hyderabad. Pluronic F 127 was obtained as gift absorbance of sample solution was determined at 322nm sample from Dr. Reddy’s Laboratories, Hyderabad. Methyl against STF (pH 7.4) as blank after suitable dilution with cellulose was obtained as gift sample from Hychem STF. laboratories, Hyderabad. Glass cylinders were fabricated by d. In-vitro Dissolution studies: Murthy labs works, Hyderabad. A.P. Rabbits used for the In-vitro drug release studies of samples were carried out by Draize eye irritancy test are from the college animal house using modified USP apparatus II paddle method with STF and the Goat’s cornea for the ex-vivo studies was obtained (pH 7.4) as dissolution medium. A glass cylinder of 2.5 cm from the local slaughter house. in diameter open at both ends was designed for the purpose Preliminary studies for optimum amount of Pluronic F of the study. Dialysis membrane previously soaked in STF 127 and Methyl cellulose for insitu gellation (pH 7.4) was taken, dried, and tied on to one end of the Preliminary studies were conducted using different glass cylinder and to this one ml of the formulation was concentrations of Pluronic F127 (Eve Ruel-Garie py et al,. accurately pipetted (Satish kumar P et al., 2008). The glass 2004 ) and Methyl cellulose. Based on the gellation studies, cylinder was attached to the shaft of USP apparatus II, in Pluronic F127 and Methyl cellulose were selected as place of basket as shown in Figure 1. The cylinder was then polymers for thermo-reversible gels. Placebo systems using suspended in 50 mL of dissolution medium maintained at 34 different amounts of Pluronic F 127 and methyl cellulose ± 0.5°C such that the membrane just touched the dissolution were taken as shown in Table 1. For optimizing the amount medium. The speed of the metallic device shaft was set at of Pluronic F127 and Methyl cellulose, gellation studies 50 rpm. Aliquots were withdrawn at intervals of 1, 2, 3, 4, were carried at 34 º ±. 0.5º C. Preliminary studies revealed and 5 hours and replaced by equal volumes of dissolution optimum results with 15 % Pluronic F127 and 2% Methyl medium. Aliquots were suitably diluted with STF (pH 7.4) cellulose. and analyzed by UV Spectrophotometer at 322 nm. The Preparation of the formulations percent release of the drug was computed as shown on Preparation of in-situ gelling systems: Table 4 and the graph of percent drug release versus time Aqueous solutions of various concentrations of pluronic F were plotted as shown in Fig 2. 127 and methyl cellulose (formulation codes KT-T1, KT- Data treatment of dissolution studies T2, KT-T3, KT-T4, KT-T5 and KT-T6) were prepared,in Various models like zero order, first order, Higuchi models, order to optimize the compositions for suitable for use as and Korsemeyer & Peppas were tested for explaining the insitu gelling systems for ocular use as shown in Table 2. kinetics of drug release based on the release rate data as as The gelling capacity was determined by placing a drop of shown in Table 7. the gel in a vial containing 2 ml of simulated tear fluid e. FTIR spectroscopy freshly prepared and equilibrated at 34º ± 0.5 º C and FTIR spectra of drug, and formulation were obtained. visually assessing the gel formation. The composition of Sample is suspended between KBr plates, and examined in 41 Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 39-45. 0.1mm KBr sealed cell, and scan for 16 times. The SHIMADZU and the FTIR spectrum was recorded from instrument model used for FTIR was Prestige 21, 3800 cm-1 to 650 cm-1. f. Rheological Evaluation: h. Ex vivo corneal permeation studies using goat’s Viscosity of formulation was determined before and after cornea gellation by using Brookfield’s viscometer (DV II model) in Goat corneas were used to study the permeation across the the small volume adaptor and the angular velocity was corneal membrane. Whole eyeballs of goat were procured increased gradually from 10, 20, 50 and 100 rpm. The from a slaughter house and transported to laboratory in cold hierarchy of the angular velocity was reversed. The average condition in normal saline maintained at 4ºC. The cornea of two readings was taken to calculate the viscosity of the were carefully removed along with a 5–6 mm of gels. Gellation was induced in formulation by raising the surrounding scleral tissue and washed with cold saline. The temperature to 34 ºC ± 0.5 ºC (Katarina Edsman et washed corneas were kept in cold freshly prepared solution al,.1998). of tear buffer of pH 7.4. (Himanshu Gupta et al ,. g. Ocular Irritancy test 2007).The study was carried out by using Franz-diffusion The optimized formulation was evaluated for in vivo cell in such a way that corneum side is continuously performance in animal model (Rabbits). The protocol is remained in an intimate contact with formulation in the approved by college ethical committee (Ethical committee donor compartment. The receptor compartment was filled Registration number is CPCSEA/IAEC/Reg. No. with STF pH 7.4 at 34 º C ± 0.5 º C. The receptor medium 518/2009). was stirred on a magnetic stirred. The samples were Three rabbits (Albino rabbits) were used for this study. withdrawn at different time intervals and analyzed for drug They were housed and maintained in the animal house at content. Receptor phase were replenished with an equal room temperature (27ºC) during the period of the study. volume of STF (pH 7.4) at each time interval. The percent They were fed with standard diet and water. The animals drug released was plotted against time to get dissolution rate were placed in cages and the eyes were marked as test and curves. control. The control group received no sample and the test Results and Discussions eye received the formulation (0.5ml), and the eyes were Pluronic F 127 and Methyl cellulose in the concentrations of observed for the ocular irritancy (includes the macroscopic 15% and 2% were found to be better carrier system because observation of cornea, iris, and conjunctiva) (Satish kumar P it shows optimum gellation. With the increase in the et al., 2008, Himanshu Gupta et al., 2007) concentration of pluronic F 127 and methyl cellulose the gellation capacity increases. Table 1: Gellation studies with Pluronic F 127 and Methyl cellulose S.No Pluronic F 127 (% wt/vol) Methyl Cellulose (% wt/vol) Gellation Gellation Capacity 1 13 1 YES + 2 13 2 YES + 3 15 1 YES + 4 15 2 YES ++ 5 20 - YES +++ 6 25 - YES +++ - No gellation + Gels after a few minutes and dissolves rapidly. ++ Gelation immediate and remains for few hours. +++ Gelation immediate and remains for extended period of time 42 Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 39-45. Table 2: Formulation of Thermo-reversible ocular gels containing Pluronic F 127 and Methyl Cellulose Concentration of Methyl cellulose Concentration of Concentration of Pluronic F (%wt/vol) KT(%wt/vol) Formulation code 127 (%wt/vol) 13 & 1 1 KT-T1 13 & 2 2 KT-T2 15 & 1 1 0.5% KT-T3 15 & 2 2 KT-T4 20 -- KT-T5 25 -- KT-T6 Table 3: Optimized Formulation of Thermoreversible ocular Gels of KT (KT-T4) Ingredients Amount (% wt/vol) Pluronic F 127 15 Methyl Cellulose 2 Drug 0.5 Benzalkonium chloride 0.02 Water 20mL 0.1 N NaOH q.s for pH adjustment Table 4: Comparative Dissolution profiles of Thermo-reversible ocular gels Time Formulation code KT-T1 KT-T2 KT-T3 KT-T4 KT-T5 KT-T6 1 44.86 31.38 53.83 40.01 32.89 25.47 2 54.56 48.40 66.28 60.14 50.35 41.46 3 67.13 59.83 73.33 75.60 59.83 46.73 4 82.80 78.04 89.84 87.38 80.33 56.88 5 90.77 88.74 -- 96.30 90.77 65.37 Table 5: Comparative dissolution profiles of Formulation (KT-T4) and Marketed product (Acular 0.5%) Time (Hrs) Formulation (KT-T4) Marketed product (Acular 0.5%) 0 0 0 1 40.02 48.27 2 60.14 68.09 3 75.6 87.09 4 87.39 100.32 5 96.3 91.08 6 78.72 83.83 43 Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 39-45. Table 6: Shows the pH, viscosity (before and after gellation), gellation capacity, and the drug content of different thermo- reversible gels. Formulation code pH measurement Gelling capacity Drug content (%) KT-T1 7.4 + 79.53 KT-T2 7.4 + 85.23 KT-T3 7.4 + 88.61 KT-T4 7.4 ++ 99.69 KT-T5 7.4 +++ 98.76 KT-T6 7.4 +++ 100.15 VISCOSITY FOR FORMULATION CODE KT-T4 RPM Viscosity in cps (spindle no. S Viscosity in cps (spindle no. S 34) After 34) Before gellation gellation 10 1236 3012 20 954 2481 50 721 1842 100 594 1089 Table 7: Different Release Rate Constants of Thermo-reversible ocular gels of Ketorolac tromethamine (Formulation code KT- T4). Formulation Parameters Zero Order First order Higuchi Korsemeyer & Peppas K 0.072 0.010 1.168 1.828 KT-T4 r2 0.9921 0.9790 0.9902 0.9971 Table 8: Diffusion profile of formulation (KT-T4) on goat cornea Time (Hrs) % Drug Release 0 0 1 30.62 2 47 3 63.55 4 76.10 5 84.80 Figure 1: Dissolution apparatus 44 Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 39-45. Figure 2: Effect of polymers ratio on dissolution of Figure 4: FT-IR studies of Thermoreversible ocular gels of Ketorolac tromethamine Thermoreversible ocular gels Ketorolac tromethamine Figure 3 .Comparative Dissolution Profiles of formulation Figure 5: Viscosity before and after gellation (KT-T4) (KT-T4) and Marketed product (Acular 0.5%) Figure 6: Diffusion profile of Thermoreversible ocular gel of KT (on Goat cornea) Characterization of thermo-reversible insitu ocular gels official medium was selected for all dissolution studies. of ketorolac tromethamine Optimized formulation of Thermo-reversible insitu ocular Assay: The thermo-reversible insitu ocular gels of KT gels was subjected to in vitro dissolution testing in STF (pH prepared complied with the requirements of assay. The 7.4). Thermo-reversible insitu ocular gels KT-T4 showed results for assay were 99.69 %. highest dissolution rate as shown in Table 4 and Fig 2. In vitro dissolution: Simulated tear fluid (pH 7.4) the 45 Sirish Vodithala et al. / International Journal of Biopharmaceutics. 1; 2010: 39-45. FTIR spectroscopy curves. Table 8 shows the percent drug release and Fig. 6 It was performed by KBR pellet method. The principal shows the percent drug release. peaks of Ketorolac tromethamine were observed at 3353, Conclusions 3082, 2922, 2953cm-1, 1613.The characteristic peak for Rationale of the present study was to improve the pre- formulation were found at 2969 cm-1 as shown in Fig 4. corneal residence time, and sustain the drug release by Ocular Irritancy studies: utilizing the approach of in situ gelling systems using Ocular irritation studies indicate that KT-T4 was a various polymers. It was envisaged that this techniques nonirritant. The formulation was very well tolerated by the would prove successful in case of formulations prepared eye. No ocular damage or abnormal clinical signs to the with the drug (KT). cornea, iris, or conjunctivae were visible In situ gelling systems were prepared using Pluronic F 127 Ex vivo corneal permeation studies: and Methyl cellulose (15% pluronic F 127 and 2% methyl Corneal permeation studies were performed using isolated cellulose) as polymers for thermo reversible ocular gels. goat’s cornea on Franz-diffusion cell using STF (pH 7.4) at Acknowledgements: 34 ± 0.5 º C. The samples were withdrawn at regular The authors are thankful to Sri Venkateshwara college of intervals and analyzed for drug content. The percent drug Pharmacy, Madhapur, Hyderabad, for proving the facilities released was plotted against time to get dissolution rate for the present work. References: Aggarwal D, Kaur I P. Improved pharmacodynamics of timolol maleate from a mucoadhesive niosomal ophthalmic drug delivery system. Int. J. Pharm. 2005;290:155–159. Ashim K. 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