Preparation And Evaluation Of Cyclosporine Ophthalmic In Situ Organogel For Punctal Occlusion

Objective:（1）Cyclosporine in situ organogel was prepared using cyclosporine as model drug,stearic acid,soybean oil and N-methylpyrrolidone as raw materials,and its quality was evaluated.（2）To investigate the feasibility of occlusion of lacrimal passage by injection of cyclosporine in situ organogel through lacrimal puncta,and evaluate its embolization effect.（3）To investigate the ocular tissue distribution of cyclosporine in situ organogel system lacrimal plug after puncta injection to block the lacrimal passage.Methods:（1）The in vitro analysis method of cyclosporine was established by HPLC to fully understand the physical and chemical properties of cyclosporine and lay a foundation for the preparation of organogel.（2）The type and amount of organogel components were examined by the test tube inversion method using the gelation time and gelation temperature as indicators to determine the optimal prescription.A series of quality evaluations were carried out on the prepared cyclosporin in situ organogel.The in vitro release behavior of drug in organogel was investigated using dialysis bag method.The exact mechanism of organogel formation determined using Fourier transform infrared spectroscopy（FT-IR）.The morphology of organogel was observed using optical microscope and environmental scanning electron microscope.The rheology of organogel was studied using rheometer.The biocompatibility of organogel was investigated using Draize eye irritation test and histopathological study.（3）The injectability of organogel was evaluated using in vitro injection experiment,and the in vitro embolization model was established to evaluate the effect of organogel system lacrimal plug on tear flow.In order to explore the occlusion effect of cyclosporin in situ organogel as a lacrimal canaliculus plug,the excretion of sodium fluorescein was assessed in rabbits.（4）Rabbits were randomly divided into cyclosporine in situ organogel group（0.00545mg/kg）and cyclosporine eye drops group（0.00545 mg/kg）.A method for in vivo drug analysis of cyclosporine was established to determine the drug content of cyclosporine in eye tissue and calculate the related pharmacokinetic parameters.Results:（1）The established cyclosporin in vitro analysis method has high specificity and good repeatability,which meets the requirements of methodological investigation.The solubility of cyclosporine in castor oil,olive oil,soybean oil and medium chain triglyceride are 4.53 mg/g,6.93 mg/g,22.59 mg/g and 102.93 mg/g,respectively.Cyclosporine has a large solubility in the oil phase,which can ensure that the prepared organogels can fully contain drugs.（2）The optimal formulation of cyclosporine in situ organogel was stearic acid:soybean oil:N-methylpyrrolidone（1.25:10:0.6,w/v/v）.From the observation of the properties of the organogel,it can be seen that cyclosporin in situ organogel is a colorless transparent liquid in the soluble state and a white-like semi-solid state after gelling,with p H around 6.9 and good stability,but it is not suitable for storage at high temperature of60℃.Equation fitting of the in vitro release results showed that the highest fit of the Ritger-Peppas equation was 0.9886,with a diffusion index of n=0.3850,and the release behavior was more consistent with Fickian diffusion.FT-IR results show that the formation mechanism of organogel is non-covalent interaction（hydrogen bond）,and the structure of organogel is mainly fiberlike network structure,accompanied by some platelike structure.The results of the flow curve in the rheological study show that the organogel is a pseudoplastic non-Newtonian fluid in both sol and gel state,and the viscosity becomes thinner with the increase of shear rate.In the frequency sweep test,in the sol state,the loss modulus is greater than the storage modulus,and the organogel behaves as a liquid-like state,which is convenient for injection through a syringe.The storage modulus is greater than the loss modulus in the gel state,exhibiting a solid-like state,which can resist the mass loss of the organogel caused by shear stress during blinking and maintain the integrity of the gel structure over time.In addition,the Draize eye irritation test showed that the organogel has good biocompatibility and meets the requirements of ophthalmic preparations.（3）The in vitro injection experiment confirmed that the organogel solution could be injected through a 22G syringe and gelled rapidly by replacement with water on the surface of artificial tears.In vitro model evaluation results showed that the injected organogels could block microcatheters by in situ gelling and reduce tear flow to 52.8%within two minutes,which was feasible for in vivo experiments.The results of sodium fluorescein excretion experiments in rabbits showed that cyclosporin in situ organogel injected into the lacrimal duct could completely block the lacrimal passage,and then the fluorescent dye could be gradually observed in the nasal cavity of rabbits with the degradation of the organogel.The results of sodium fluorescein excretion experiments in rabbits showed that cyclosporin in situ organogel injected into the lacrimal passage could completely block the lacrimal passage,and then the fluorescent dye could be gradually observed in the nasal cavity of rabbits with the degradation of the organogel.（4）The established in vivo LC-MS/MS assay for cyclosporine is highly specific and sensitive,which meets the requirements of in vivo drug methodology investigation.The standard curves of LC-MS/MS in cornea,conjunctiva and sclera were Y=0.148X+0.062（R²=0.9993）,Y=0.0913X+0.092（R²=1）,Y=0.122X+0.066（R²=0.9969）.Cyclosporine has a good linear relationship in the range of 5 ng/m L～200 ng/m L.The results showed that the C_max of cyclosporine in situ organogel and cyclosporine eye drops in cornea,conjunctiva and sclera were（475.73±64.62 vs.244.27±60.10;3124±180 vs.1812±84;162.90±22.10 vs.82.50±6.50）,the difference was statistically significant compared with the control group.The concentration of cyclosporine in cornea,conjunctiva and sclera was significantly higher than that in cyclosporine eye drops,and AUC_0-72h was 2.49 times,2.27 times and 2.15 times of cyclosporine eye drops,respectively.Compared with cyclosporine eye drops,the retention time of cyclosporine in situ organogel on the cornea and conjunctiva was significantly longer,and the T_max was two and four times that of cyclosporine eye drops.Conclusion:In this paper,cyclosporin in situ organogel was prepared using heating-cooling method and a series of quality evaluations were performed,which showed that the ophthalmic formulation was safe,non-irritating and met the requirements of ophthalmic formulation.In vivo and in vitro lacrimal passage occlusion experiments showed that the organogel solution could be injected through a syringe（size:22G）,and the organogel system lacrimal passage plug was formed in situ,which significantly reduced the tear drainage flow.In vivo eye tissue distribution results showed that the pharmacokinetic parameters of cyclosporine in situ organogel were better than the reference preparation cyclosporine eye drops,and the difference was statistically significant.In summary,we developed a drug sustained-release lacrimal plug based on organogel system,which can not only reduce ocular tear loss to achieve individualized treatment,but also continuously release therapeutic drug cyclosporine,providing a dual mechanism for the treatment of dry eye.