2-甲氧基雌二醇脂质体冻干粉的研究
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摘要
2-甲氧基雌二醇(2-methoxyestradiol,2-ME)是雌激素的天然代谢产物,因其具有广谱抗肿瘤活性,且毒性较低,近年来被广泛关注,口服胶囊已被FDA批准完成二期临床研究。然而,2-ME体内胃肠道吸收差,血浆消除速率快,口服或腹腔注射大剂量2-ME才能达到抑制肿瘤的作用。脂质体作为抗癌药物的载体,在某种程度上可提高化疗药物的靶向性和化疗药物的治疗指数,降低用药剂量并大幅降低药物的毒副作用。本研究拟将2-ME制成脂质体冻干粉,以改变体内的释放特性,提高疗效,降低毒副作用,本研究主要内容包括以下几方面：
1、对2-ME脂质体的处方和工艺进行考察,以包封率为指标,筛选出注入法为最佳制备方法。通过选择磷脂的种类、有机溶剂的种类、药脂比、冻干保护剂的种类和用量、磁力搅拌时间和超声功率,确定脂质体冻干粉的处方组成和制备工艺,并进行正交试验优化处方,最终制备的2-ME脂质体冻干粉包封率为75%,平均粒径在220nm左右。透射电镜下观察,复溶后脂质体的外观圆整,分布均匀。稳定性实验表明,2-ME脂质体冻干粉4℃保存6个月内稳定性良好。
2、建立了以来曲唑为内标,HPLC法测定大鼠血浆样品中2-ME浓度的方法,并对其进行方法学确证。结果显示,该方法线性关系良好,日内和日间精密度均小于11.30%,平均提取回收率为86.2%,生物样品在室温、冷藏、冰冻以及反复冻融情况下稳定性良好,该方法可用于2-ME在大鼠体内血浆中药物浓度的测定。
3、SD大鼠的药动学实验：分别按10mg/kg的剂量尾静脉注射2-ME溶液和2-ME脂质体,在设定的时间点眼眶后静脉丛取血,测定血药浓度。用3P97程序求算药动学参数,进行药物动力学模型拟合。结果表明,2-ME在大鼠体内的药动学符合二室模型,同时静脉注射2-ME脂质体和2-ME溶液,2-ME脂质体可以快速到达靶器官,提高治疗效果。
4、昆明种小鼠的组织损伤实验表明,2-ME脂质体组对实验动物的毒副作用和组织损伤均明显减轻,组织损伤实验结果验证2-ME脂质体达到降低药物毒性的预期目标。
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.
1、对2-ME脂质体的处方和工艺进行考察,以包封率为指标,筛选出注入法为最佳制备方法。通过选择磷脂的种类、有机溶剂的种类、药脂比、冻干保护剂的种类和用量、磁力搅拌时间和超声功率,确定脂质体冻干粉的处方组成和制备工艺,并进行正交试验优化处方,最终制备的2-ME脂质体冻干粉包封率为75%,平均粒径在220nm左右。透射电镜下观察,复溶后脂质体的外观圆整,分布均匀。稳定性实验表明,2-ME脂质体冻干粉4℃保存6个月内稳定性良好。
2、建立了以来曲唑为内标,HPLC法测定大鼠血浆样品中2-ME浓度的方法,并对其进行方法学确证。结果显示,该方法线性关系良好,日内和日间精密度均小于11.30%,平均提取回收率为86.2%,生物样品在室温、冷藏、冰冻以及反复冻融情况下稳定性良好,该方法可用于2-ME在大鼠体内血浆中药物浓度的测定。
3、SD大鼠的药动学实验：分别按10mg/kg的剂量尾静脉注射2-ME溶液和2-ME脂质体,在设定的时间点眼眶后静脉丛取血,测定血药浓度。用3P97程序求算药动学参数,进行药物动力学模型拟合。结果表明,2-ME在大鼠体内的药动学符合二室模型,同时静脉注射2-ME脂质体和2-ME溶液,2-ME脂质体可以快速到达靶器官,提高治疗效果。
4、昆明种小鼠的组织损伤实验表明,2-ME脂质体组对实验动物的毒副作用和组织损伤均明显减轻,组织损伤实验结果验证2-ME脂质体达到降低药物毒性的预期目标。
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.
引文
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