| Medication is applied to the surface of the eye for two purposes: to treat the outside of the eye for such infections as conjunctivitis,blepharitis, keratitis sicca, or to provide intra-ocular treatment through the cornea for diseases such as glaucoma or uveitis. Most ocular diseases are treated with topical application of solutions administered as eye-drops. These conventional dosage forms account for nearly 90% of the currently accessible marketed formulations[1] .One of the major problems encountered with the topical delivery of ophthalmic drugs is the rapid and extensive precorneal loss caused by drainage and high tear fluid turnover. After instillation of an eyedrop, typically less than 5% of the applied drug penetrates the cornea and reaches intraocular tissues, while a major fraction of the instilled dose is often absorbed systemically via the conjunctiva and nasolacrimal duct[2]. After topical administration of an ophthalmic drug solution, the drug is firstly mixed with the lacrimal fluid. The contact time of drug with ocular tissues is relatively short (1-2 min) because of the permanent production of lacrymal fluid (0.5-2.2/l/min). (0.5-2.2/l/min). Then, approximately half of the drug flows through the upper canaliculus and the other half, through the lower canaliculus into the lacrimal sac, which opens into the nasolacrimal duct. Drainage of lacrymal fluid during blinking (every 12 s) towards the nasolacrimal duct induces a rapid elimination of conventional dosage forms . [3,4]Drug delivery, as it pertains to the eye, is a generic term which is defined broadly as representing an approach to controlling and ultimately optimizing delivery of the drug to its target tissue in the eye. An optimum ocular drug delivery system would be one which can be delivered in eye-drop form with no creation of blurred vision or irritancy and which would need no more than one to two instillations each day [5].Objective: This test selects puerarin as model drug ,by studying to increase the resistance time before cornea ,prepare temperature sensitive and mucosa membrane attachment in-situ gelling for ophthalmos with suitable phase transition temperature . Utilize rheology method to explain gel process, study drug release in gelling , pharmacokinetics and bioavailability in aqueous fluid and in tears after administration.Method s: (1) Methodology consideration: according to Chinese Pharmacopeia method , determine the assay method of puerarin , on the same time evaluate precision and stability and so on.(2) formulation select:on the basis of literatures, the formulation screening was performed by taking sol-gel phase transition temperature as index before and after diluted with artificial tears , in which poloxamer F-127,poloxamer 188, sodium hyaluronate and carbopol 1342 were used as the main base materials. And then ,the usage amount of poloxamer F-127,poloxamer 188, carbopol 1342,and pH were taken as the four factors and there are three levels in each factors. To arrange experiments according to L9(4)3,the sol-gel phase transition temperature before and after diluted with artificial tears were regarded as a target to choose the best,meanwhile,the better formulation obtained from single- factor investigation was retained to following experiments.(3) Vitro delivery study: the release media was artificial tears ,with nulli-membrane dissolution model,ex-vivo cornea penetrate diffusion method ,oar-method dissolution at the temperature of 34.5±1℃,samples were obtained at set time, then accumulated drug release data were analyzed with three models : Higuichi equation, zero order equation ,first order equation , inspected the gelatinous corrosion and drug release kinetics, and release mechanism .(4)The chemical and physical stability of formulation was investigated under following circumstances: strong light and high temperature.(5)The irritant effects and toxic reactions of puerarin in-situ gelling on New Zealand white rabbits eyes:The irritant effects: chosen isotonic Na chloride as control group, adopted left and right of homobody as subject contrast , at set time single dosing eye irritation and multiple dosing eye irritation were observed ,recorded and evaluated . The toxic reactions :the puerarin in-situ gelling or eye drop were topically administered to the right eyes of 6 New Zealand white rabbits and isotonic Na chloride was administered to the left eyes .All eyes were excised for histological section to evaluate the possible toxic reactions.(6) Pharmacokinetics study in vivo : New Zealand white rabbits were selected as laboratory animals and divided into two groups in random .One group was administered puerarin in-situ gelling and the other was administered puerarin eye drop. The samples were obtained and quantified at set times .The puerarin contents in tears and in aqueous fluids were surveyed by being injected into high performance liquid chromatography (HPLC) with Ultraviolet detector ,then evaluated the bioavailability of the formulation .Results: (1) Methodology consideration: adopt the Pharmacopeia method as the assay method, by evaluating precision and stability of the assay method of puerarin.(2) formulation select: the best formulation was obtained by taking sol-gel phase transition temperature as index before and after diluted by artificial tears, which includes 1.0% puerarin, 32% poloxamer F-127,0.8%poloxamer 188, 0.1%sodium hyaluronate , 0.1%carbopol 1342 and buffer phosphate with pH6.5. The sol-gel phase transition temperature of preparation before and after diluted by artificial tears are 28±1℃and 32±1℃(3) Vitro delivery study: the accumulated drug release data obtained from dissolution experiments, such as nulli-membrane dissolution model,ex-vivo cornea penetrate diffusion method ,oar-method dissolution. The accumulated drug releases from ex-vivo cornea penetrate diffusion method all fit zero order equation:drop: Q=0.0511t-1.1356,r=0.9988;gel 1:Q=0.0343t-0.0814 , r=0.9963; gel 2:Q=0.0306t-0.0337 ,r=0.9964. The accumulated drug releases and drug dissolutions from nulli-membrane dissolution model fit zero order equation, they are Q=3010.6t+2567.6,r=0.9989 and Q=0.429t-0.0559,r=0.9984. The accumulated drug releases from oar-method dissolution fit zero order equation, which is .Q=1.2944t+6.0242,r=0.9896.(4)Study of stabilities: the chemical and physical stability tests of formulation were investigated under strong light and high temperature. The results showed that formulation was stable to light and heat.(5) The irritant effects and toxic reactions of puerarin in-situ gelling on New Zealand white rabbits eyes:The irritant effects: the results of single dosing eye irritation test and multiple dosing eye irritation test showed that there were not visible influences on subjects, eyes, both corneas are transparent without turbid phenomenon, and iris are clear ,there were no conjunctiva hyperemia ,no edema and no secretion;Electron microscope examinations exhibited that there was no toxic effect , and there was no significant difference between control group and experimental group. The results showed that the gel is safe for application .(6) Pharmacokinetics study in vivo: the pharmacokinetic parameters of two formulations in tears were calculated by DAS pharmacokinetic software and respectively as following: Eye drop:t1/2 was 27.986 min,AUC(0-t) was 86250.7μg/L*min,AUC(0-∞) was 90458.295μg/L*min;In-situ gel:t1/2 was 6931.472 min,AUC(0-t) was 145537.205μg/L*min,AUC(0-∞) was 150263.21μg/L*min.The values of t1/2, AUC(0-t), AUC(0-∞) in experimental group showed significantly higher compared with control group.Conclusion : The results for in vitro and in vivo confirmed that the in-situ gel could prolong the residence time of drug before cornea .It showed better toleration and low irritation after administer ,and there was no obvious toxic effect on ocular tissue . Therefore, the puerain in-situ gel for ocular application could significantly prolong precorneal residence time of drug after administration ,enhance bioavailability ,and showed there would be a great potential application.
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