Study On Preparation And Characterization Of Tetrandrine Microemulsion Eye Drops And Ocular Pharmacokinetics In Rabbits After Topical Application

Purpose:Tetrandrine has a variety of pharmacological effects such as anti-inflammatory,immunosuppressive,anti-tumor and inhibition of neovascularization.Studies have showed that tetrandrine could inhibit the growth of corneal neovascularization,but the water-insoluble nature of TET limits its application in clinical ophthalmology.This purpose of this study was to prepare and optimize the formulation of Tetrandrine microemulsion（TET-ME）eye drops,to evaluate its physicochemical properties and the behavior in vitro release of TET-ME,and to investigate the ocular pharmacokinetics of TET-ME eye drops after topical administration of one single dose in rabbit.MethodsTetrandrine solubility in different excipients were tested,and suitable lipids,surfactants and co-surfactants were selected.The TET-ME were prepared by titration method.Drug/lipid（X1）,lipid/surfactant（X2）were taken as independent factors,the particle size（Y1）,encapsulation efficiency（Y2）were taken as the dependent factors.The possible optimum formulation was predicted by central composite design-response surface methodology（CCD-RSM）and validated.The drug release of Tetrandrine microemulsion was analyzed by positive dynamic dialysis.Choose healthy rabbit eyes without eye diseases as a model to evaluate ocular irritation.According to the self-control method,one single dose of 50μL of 0.2%TET-ME eye drop was applied in the right conjunctival sac,and the same dose of normal saline in the left eye as a control.The clinical and slit lamp observations were performed at a set time after administration,and evaluated the rabbit eye irritation of 0.2%TET-ME eye drop.The high performance liquid chromatography with ultraviolet-visible detector（HPLC-UV）was used to determine the levels of tetrandrine in tear,cornea and conjunctival.Forty-two healthy New Zealand white rabbits without eye diseases were randomly divided into experimental group and control group.The two groups continued to be divided into seven subgroups,represented different administration time points.Both eyes of the rabbits in the experimental group are given one single dose of 50μL of 0.2%TET-ME eye drops while each eye of the control group received a single administration of 50μL of 0.2%tetrandrine suspension（TET-SUSP）eye drops,respectively.The tear fluid was collected using pre-weighed filter paper at predetermined time points of 0.1h,0.5h,1h,2h,4h,8h and 12 hours following administration of the drug.The cornea and conjunctival specimen were collected at above-mentioned time points respectively.The drug levels in each sample were assayed by HPLC-UV.The data were statistically analyzed by SPSS 22.0 software,and the pharmacokinetic parameters were processed by DAS 2.1.1.Results:According to the solubility results,oleic acid was selected as the lipid phase,polyoxyethylene hydrogenated castor oil（RH40）was used as the surfactant,and glycerin as the co-surfactant.The optimized formulation was tetrandrine 0.08 g,oleic acid 0.288 g,RH40 2.88 g,glycerin 0.56g,drug-lipid ratio 0.278,and ratio of lipid to surfactant 0.10 and add water to 40m L.According to the optimized formulation,the final prepared tetrandrine microemulsion were clear and transparent,with light blue opalescence.The p H was 7.04±0.02 and the osmotic pressure was 312±3 m Osm/L.The average particle size was 8.94±0.10 nm,PDI were 0.338,the Zeta potential were-0.055±0.055 m V.encapsulation efficiency was（97.50±0.6）%,the drug loading was（0.86±0.18）%.The in vitro drug release presented the characteristics of fast release and then slow.And it was conformed to the first-order release kinetics equation.After a single dose administration,the TET-ME showed no obvious irritation to rabbit eyes in rabbit eyes.Samples did not interfere with tetrandrine in tear,cornea and conjunctiva by HPLC-UV analysis.The relative standard deviation（RSD）of TET in conjunctiva,tear and cornea for within-run and between-run were all<5%,and the recovery rates were in the range of 92%to 107%.The peak time of TET of the TET-ME and the TET-SUSP in rabbit tears,cornea and conjunctiva were 0.1h.The peak concentration(C_max)of tear in TET-ME and TET-SUSP groups were189.71±54.45μg/g and 113.49±35.34μg/g.The C_maxof TET-ME was 1.65 folds of the TET-SUSP.The tear fluid drug levels of the TET-ME group were significantly higher than the TET-SUSP group at each time points（P<0.05）.The AUC_0-12hof tear fluid in TET-ME group was 2.27 folds higher than that of TET-SUSP group.C_maxof cornea in TET-ME and TET-SUSP groups were 51.46±12.65μg/g and 16.53±5.61μg/g.The C_maxof TET-ME was 3.11 folds of the TET-SUSP group.The corne drug levels of the TET-ME group were significantly higher than the TET-SUSP group at each time points（P<0.05）.The AUC_0-12hof cornea in TET-ME group was 5.36 folds higher than that of TET-SUSP group.The peak concentration of conjunctiva in TET-ME and TET-SUSP groups were 43.8±8.66μg/g and 12.5±3.50μg/g.The C_maxof TET-ME was 3.50 folds of the TET-SUSP group.The conjunctiva drug levels of the TET-ME group were significantly higher than the TET-SUSP group at each time points（P<0.05）.The AUC_0-12hof conjunctiva in TET-ME group was 4.50 folds higher than that of TET-SUSP group.Conclusion:The TET-ME can be easily prepared,with small particle size,high encapsulation efficiency and can enhance the ocular bioavailability greatly.Tetrandrine microemulsion had no irritation to rabbit eyes and improved the concentration of the drug in the cornea significantly.Therefore,the TET-ME eye drops provide new methods and new ideas for the ocular application of Tetrandrine.