The Study On The Lyophilized Liposomes Containing 2-Methoxyestradiol

2-Methoxyestradiol(2-ME), formerly regarded as an inactive metabolite of estradiol, is now known to be an anti-angiogenic agent with anti-proliferative and cytotoxic activity that confers anti-tumour activity in some animal models of solid tumour.2-ME is currently being evaluated in PhaseⅡclinical trials for the treatment of solid tumors and is undergoing preclinical evaluation for inflammatory conditions. However, poor bioavailability, resultant lack of efficacy and the estrogenic actions of 2-ME show that 2-ME is a suboptimal therapeutic agent in its current formulation. Liposomes are well recognized as drug delivery vehicles that have target effents, delayed drug release and degraded drug toxicity. The purpose of the study is to prepare 2-ME liposome to improve its therapeutic efficacy.The effects of the methods of preparation, the type of phospholipid and organic solvent, the ratio of drug to lipid, the types and amount of lyprotectant, the time of magnetic stirring and ultrasonic power were studied. Then based on the results of single factor analysis, the formulation and the preparation techniques were optimized by orthogonal design.2-ME liposomes were prepared by injection method. The optimized formulation of 2-ME liposomes was as follows:The quality ratio of phosphotidylcholine and cholesterol was 10:1, The quality ratio of lipids and drug was 12:1, The volume ratio of H2O and oil was 3:1, The concentration of poloxamer 188 was 0.20%.To resolve the instability of liposome dispersion, freeze-dried technique was utilized to prepare freeze-dried liposomes. Its main advantage is of high stability because of its solid form during preservation. Mannitol and trehalose were selected as the optimum cryoprotectants due to the minimum change of the average particle sizes and the entrapment efficiency before and after freeze-drying process. The size of freeze-dried liposomes after hydration was about 220nm. The entrapment efficiency was about 75%. The transmission electron microscopy showed that most liposomes were spherical. The stability test was conducted, the results showed that 2-ME freeze-dried liposomes kept at 4℃for 6 months showed good stability.The HPLC method based on fluorescence detection for the quantitative determination of 2-ME in plasma samples using letrozole as the internal standard, has been developed and validated. There was good linear relationship for 2-ME with the ranges in plasma samples. The intra-day and inter-day precision of quality control samples were less than 11.30%. The average recovery of extraction was 86.2%. Biological samples were stable in room temperature or 4℃, and in freezing or after three freeze/thaw cycles.Pharmacokinetic parameter calculation and pharmacokinetic model were carried out using the 3P97 pharmacokinetics software. In vivo pharmacokinetic studies data indicated that the 2-ME solution followed a two-compartment model while 2-ME liposome suspension also followed two-compartment model with different pharmacokinetic parameters after i.v. administration of liposomes and solution at a dose of 10 mg/kg to rats. The liposomes have good biocompatibility and biodegradability, so it might promote its distribution in tissues, the drug could rapidly act on therapeutic effect.Kunming mice acute toxicity experiments showed that 2-ME liposomes significantly reduced toxicity and tissue injury compared to 2-ME solution. The results verified that 2-ME liposome was security.