Studies On Tacrolimus In Situ Thermosensitive Gel For Ophthalmic Use

In situ thermosensitive gels refer to the polymer solutions can be administrated as liquid, which undergo a phase transition to a non-crosslinked gel upon exposure to physiological environments.Most of the applications of the in situ thermosensitive gels are concerned with ophthalmic disease treatment for its convenient administration combined with the favorable ocular residence time.We selected tacrolimus（FK506）as a model drug to develop a kind of in situ thermosensitive gels for ophthalmic use.HPLC methods was used to determine the tacrolimus concentration and drug release in situ thermosensitive gels.Agilent Zorbax SB-C₈ column was used as analytical column with a temperature of 50℃.The mobile phase consisted of acetonitrile and water in the ratio of 55:45（v/v） at the flow rate of 1.0 ml·min^-1.The UV detector wavelength was at 215 nm.The recovery rate was 99%-101%.The method was simple,accurate and reproducible.The properties of tacrolimus such as solubility and oil/water partition coefficient.The solubility is 2.04μg·ml^-1,3.16μg.ml^-1and 4.84μg·ml^-1in water,pH7.4 phosphate buffer and simulated tear fluid（STF）respectively.The apparent partition coefficient of water,in 1-octanol/water,phosphate buffer and simulated tear fluid system is 1.33,1.18 and 1.17.FK506/HP-β-CD complex was prepared to improve the solubility of tacrolimus and proved by DSC and X-ray.The optimized formulation was prepared by microwave method.The mass ratio of FK506 and HP-β-CD was 1:15,the quality concentration of HP-β-CD was 30%,microwave intensity was 50W and microwave time was 10min.The inclusion rate of inclusion complex was 91.47%.The results of initial stability indicated the inclusion complex was more stable than tacrolimus.Appling criterion of gelling capacity,the formulation was optimized by signal factor studies. The reversible gelation behaviors of poloxamer 407（P407）solutions showed strong concentration dependency.The reversible gelation behaviors could not occur when the concentration process of poloxamer 407 less 15%.The gelation temperature（GT）was adjusted by incorporating P188.The phase transition process of poloxamer solution was completed in 3-5℃and exhibited exponential increase of viscosity with temperature;the gelation mechanism was not changed by incorporating P188.The fitted equation was established for the GT with the concentration of poloxamer solutions.GT was enhanced about 3-8℃after being diluted by STF.An optimized formulation was freely flowing liquid at 26.1℃and converted to a film gel at 33.8℃after diluted by STF.According to the in vivo characteristic of ocular administration,a membraneless model was used to study the gel erosion and drug release simultaneously.Correlation analysis demonstrated that drug release from poloxamer vehicle was completely controlled by gel erosion.The increase of release area and shaking frequency could improve the rate of gel erosion and drug release.The results of influential factors and accelerated experiment showed that FK506 in situ thermosensitive gel were sensitive to light and temperature.It should be kept at low temperature with protection from light.The long-time experiment results indicated the formulation of FK506 was good stability at 6℃±2℃.The results of rabbit ocular irritation tests of single dose and multiple dose suggested that FK506 in situ thermosensitive gel was non-erritant.To evaluate the immunosuppressive effect of FK506 in situ thermosensitive gel,the allogeneic corneal transplantation models in rabbit were established.FK506 in situ thermosensitive gel,FK506 eye drop and Cyclosporin A（CsA）eye drop were applied in the corneal transplantation models.The results indicated that FK506 in situ thermosensitive gel,FK506 eye drop and CsA eye drop group could relieve the edema of corneal implant.The three groups could marldy prolong the survival time of corneal grafts in comeal transplantation.