[Preparation of an ophthalmic formulation of TPGS-modified insulin-loaded liposomes and its corneal permeation and pharmacokinetics in rabbit eyes].

抄録

OBJECTIVE: To prepare vitamin E polyethylene glycol 1000 succinate (TPGS)-modified insulin-loaded liposomes (T-LPs/INS) and evaluate its safety, corneal permeability, ocular surface retention and pharmacokinetics in rabbit eyes.

METHODS: The safety of the preparation was investigated in human corneal endothelial cells (HCECs) using CCK8 assay and live/dead cell staining. In the ocular surface retention study, 6 rabbits were randomized into 2 equal groups for application of fluorescein sodium dilution or T-LPs/INS labeled with fluorescein in both eyes, which were photographed under cobalt blue light at different time points. In the cornea penetration test, another 6 rabbits divided into 2 groups for application of Nile red diluent or T-LPs/INS labeled with Nile red in both eyes, after which the corneas were harvested for microscopic observation. In the pharmacokinetic study, 2 groups of rabbits (=24) were treated with eye drops of T-LPs/INS or insulin, and the aqueous humor and cornea were collected at different time points for measurement of insulin concentrations using enzyme linked immunosorbent assay. DAS2 software was used to analyze the pharmacokinetic parameters.

RESULTS: The prepared T-LPs/INS showed good safety in cultured HCECs. Corneal permeability assay and fluorescence tracer ocular surface retention assay demonstrated a significantly higher corneal permeability of T-LPs/INS with a prolonged drug residence in the cornea. In the pharmacokinetic study, insulin concentrations in the cornea at 6, 15, 45, 60, and 120 min ( < 0.01) and in the aqueous humor at 15, 45, 60, and 120 min after dosing were significantly higher in T-LPs/INS group. The changes in insulin concentrations in the cornea and aqueous humor were consistent with a two-compartment model in T-LPs/INS group and with the one-compartment model in the insulin group.

CONCLUSION: The prepared T-LPs/INS shows an improved corneal permeability, ocular surface retention capacity and eye tissue concentration of insulin in rabbits.

目的: 制备维生素E聚乙二醇1000琥珀酸酯（TPGS）修饰的胰岛素脂质体（T-LPs/INS），探讨其表征，生物安全性及在兔眼的角膜渗透性、眼表滞留能力与药物代谢动力学。

方法: 采用细胞毒性实验（CCK8）、活/死细胞染色考察T-LPs/INS的安全性。眼表滞留研究中采用随机数字表法将实验兔分为2组，每组3只兔6眼。对照组：以荧光素钠稀释液滴眼，实验组：以荧光素钠标记的T-LPs/INS滴眼。各组滴眼后在不同时间点进行钴蓝光下观察与拍摄。角膜渗透实验中以随机数字表法将实验兔分为2组，每组3只兔6眼。对照组：尼罗红稀释液滴眼，实验组：尼罗红标记的T-LPs/INS滴眼。各组滴眼后在适当时间取下角膜进行染色、切片观察，评估药物在角膜组织各层的渗透性。药代动力学研究中将实验兔按随机数字表法分为实验组和对照组，每组24只兔48眼。分别以T-LPs/INS、单纯胰岛素滴眼液（INS）50 μL单次点眼，并于点眼后0.1、0.25、0.75、1、2、2.5、4.5、6 h采集各组兔眼房水、角膜等组织标本，利用酶联免疫吸附试验法测量各个标本中胰岛素浓度，应用DAS2软件拟合分析各组兔眼角膜与房水中胰岛素的药物浓度-时间曲线下面积（AUC）、半衰期（T½）等药代动力学参数。

结果: CCK8实验、活/死细胞染色表明T-LPs/INS安全性良好。角膜渗透性实验证明T-LPs/INS在角膜渗透性显著增加，眼表滞留实验研究证明T-LPs/INS延缓了药物在角膜的驻停时间。药代动力学研究中，点药后0.1、0.25、0.75、1、2 h两组兔眼角膜组织胰岛素浓度相比较，差异均有统计学意义（ < 0.01）；点眼后0.25、0.75、1、2 h两组兔眼房水中INS浓度相比较，差异均有统计学意义（ < 0.01）。实验组兔眼角膜与房水中胰岛素浓度变化符合二室模型，对照组兔眼角膜与房水中胰岛素浓度变化符合一室模型。

结论: 成功制备出T-LPs/INS，有效的提高了药物的角膜渗透性、眼表滞留能力与眼组织的药物浓度。