| Cox-2 is a important adaptive enzyme in the program of inflammation. It is not express in the normal condition except induced by hypoxia, grows factors, mediators of inflammation, tumor, oncogene and so on. Cox-2 could induce the great grows of VEGF which is a strong chemoattractants of vascular endothelial cells by catalysis arachidonic acid chang into PG2 prostaglandin. Meanwhile VEGF also could induce the great express of COX-2. After keratoplasty, COX-2 will great grows by the stimulate of wound and inflammation,then VEGF would followed, at this time, corneal neovascularization grows. This corneal neovascularization has important effect on the development of rejection. Celecoxib as a great elective inhibitor of COX-2 would inhibit VEGF's grows by this way.PLGA is a high molecular compound polymerizated by glycolide and lactide monomers. It is a good material for prepare microparticle. PLGA have high biocompatibility and safety and was one of the two injectable polyments which approved by FDA. Now some drugs which carried by PLGA have been in the market.There were many reports about using celecoxib to inhibit corneal neovascularization. In this studies, celecoxib had a good effect on neovascularization no matter in tumor or ocular. Many studies administered celecoxib by system, drop or region inject drug solution, those administer ways had some disadvantages such as,drug would hard get into the eye by system administer because of the exist of blood-ocular barrier. It will be low efficiency and tedious by drop and damage the ocular tissue by region inject. In USA, Surya and his group have got a series of studies on the effect of celecoxib PLGA microparticls in eye's diseases which caused by diabetes. They found that celecoxib could inhibit VEGF which located in diabetes rat retina. When subconjunctival inject celecoxib, the drug concentration in rerina was 54 higher than oral the same drug. Microparticles had a better delayed release than nanoparticles, which could stay a longer time in the injection site. Subconjunctival inject celecoxib PLGA microparticles could retain concentration higher and longer than inject celecoxib solution in diabetes rat retina.14 days later after inject, the solution group could not detect drug concentration in retina, while the microparticle group still retain effective concentration. The same result was authenticate by cells experiment. When subconjunctival inject celecoxib-PLGA or placebo-PLGA microparticle, there were no effect on the rat's body weight or blood sugar. celecoxib-PLGA microparticles did not cause any changes in blood cell counts or chemistry and caused no histopathological damage to the retina or periocular tissus. Those result indicating that celecoxib microparticles were safe for animal.Surya's studies were research on the drug action and distribution of diabetes rat. When finish keratoplasty the pathoenvironment of eye is different from the diabetes rat. So how distribution and action of celecoxib PLGA microparticles to keratoplasty eye is don't know, our work is to find the phenomenon when subconjunct inject celecoxib PLGA microparticles in keratoplasty eye. We also want to give same help to solve this clinical puzzle throng our research results.Method and resultCelecoxib-PLGA microspheres were prepared by emulsification/solvent evaporation method, and optimize the preparation technology with orthogonality. The microspheres characteristics, such as the particle size, loading and entrapment efficiency were taken as indexes for evaluating the microspheres. It dissolution characteristics in vitro were also studied. Celecoxib-PLGA microspheres possessed a smooth and round appearance. Particle diameter was (1.5±0.2)μm, the encapsulation efficiency and drug loading was (8.7±0.3)% and (94.9±0.2)%, respectively. The in vitro release curves of celecoxib microspheres could be fitted with Weibull equation. The accumulated release percentage of which was 69.1% during 40 days. Among the numerous influential factors, the sequence is:stir-time >organic/liquid>PVA's conc> admin rate. The best prescription of preparation technology is stir-time:2 min, organic/liquid:1/10, PVA's conc:2%, and admin rate:1/10.Prepare same microparticles by the best prescription and subconjunctival inject microparticles or solution in allogenic penetrating keratoplasty rat.Got conjunctiva, aqua oculi and cornea on 1,7,15 and 30 day after inject. The drug concentration in tissue were detect by HPLC. Factorial analysis and one-way ANOVA were used to analysis the difference among the each groups. There is differernce between solution and microparticles group and different observation days(P<0.001). In each observation days, there are difference in each tissue between two groups(all P<0.001), and in the same observation days, each tissue also differernt between two groups. (all P<0.001). After injection, each tissue's concentration of solution group reduced fastly. On the 7th day, the concentration of conjunctiva, aqua oculi and cornea in microparticles group is 158.9,43.1 and 12.6 higher than solution group respectively. But on the first day there is no difference between solution and microparticles groups in conjunctiva(P>0.05) and drug concentration in aqua oculi and cornea in solution group were 3.9 and 1.3 higher than microparticle group.15 days later, the concentration in each tissue in solution group were under the detection limit, while microparticles group still retain a high concentration in each tissue.Compare with the inhibitory action to the corneal neovascularization after keratoplasty between celecoxib PLGA microparticles and blank PLGA microparticles on the 7th,15th and 30th day after keratoplasty. Evaluation inhibitory action by observe and calculate corneal neovessle areas with slit-lamp microscope, prepare corneal pathological sections and stained with HE and immunohistochemisty, test corneal PGE2 and VEGF content with ELISA. Preform factorial analysis and one-way ANOVA test.The result indicating that there is no difference between blank microparticle group and celecoxib microparticle group in cornea neovessels area (F=2.38, P=0.149). There is difference in cornea neovessels area on different observation days (F=10.43, P=0.002). In celecoxib group there is difference between different observation days (F=7.58, P=0.023), and there are differences between between 7 days and 15 days group and 15 days and 30 days group with multiple comparisons.From corneal pathological sections,wo found that cornea construct is complete, on the 7th day, a lot of vessels cavities are well distributed in the stroma in blank microparticle group. Neovessels concentrate on the epithelium side stroma in celecoxib group. Vessels on the endothelium side are significantly less than the blank group. On the 30th day, vessel cavities in blank group are mostly closed. Dark-stained nucleus of vascular endothelial cells only can be found in stroma on the epithelium side.Vessel cavities are more rare in celecoxib group. Collagen fibers stained steady pink decompose to strips. From the ELISA detection results, wo know there are differences between celecoxib microparticle group and blank microparticle group in corneal PGE2 and VEGF content. In each observation days, there are differerce between celecoxib microparticle group and blank microparticle group in corneal PGE2 and VEGF content. ConclusionThe technology of preparation is successful and the microspheres have a round appearance. Microspheres have a good prolonged release effect in vitro and the release equations is fit with the Weibull equation. Comparaed with the solution, celecoxib PLGA microspheres sustained drug levels longer in the ocular. Comparaed with the blank PLGA microparticle, celecoxib PLGA microspheres have inhibite activity to corneal neovascularization after keratoplasty. All the results indicating that celecoxib PLGA microspheres have potential usefulness in treating corneal neovascularization after keratoplasty.
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