替尼泊苷亚微乳注射液的研究

【作者】 王静；

【导师】 唐星； 徐晖；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2009， 硕士

【摘要】 亚微乳剂凭借其生物相容性好、低毒低刺激性等优点被作为静脉给药的优良载体而广泛应用。替尼泊苷抗瘤谱广、疗效确切,是目前临床上应用较为广泛的化疗药之一。但由于其水难溶性,其市售注射剂卫萌(Vumon(?))以聚氧乙烯蓖麻油和无水乙醇等有机溶剂作为溶媒,在临床使用中存在发生过敏反应和稀释后析出替尼泊苷沉淀的风险。本研究的目的是制备并评价一种稳定的替尼泊苷亚微乳注射液(TSE)。本论文建立了替尼泊苷HPLC体外分析方法,该方法重现性好,准确度高。对替尼泊苷的溶解性能、降解速率常数等重要理化参数进行了考察,结果显示替尼泊苷在大豆油、中链油中溶解度均低于0.5mg·mL-1,因此可以选择加入增溶剂提高药物在油相中的溶解度制备替尼泊苷亚微乳注射液。对替尼泊苷在pH 5.0,6.0,6.5,7.0,8.0缓冲盐溶液中80℃加热条件下的降解速率常数进行考察,其中在pH6.0缓冲盐中的降解速率常数最小,半衰期最长为8.36h。本文通过高压均质法以直接载药方式制备了替尼泊苷亚微乳注射液,并利用HPLC、动态光散射技术、电泳激光散射技术和超速离心法等以亚微乳剂的理化性质(外观、粒径、ζ-电位、pH值、含量和包封率)和制剂灭菌前后稳定性作为评价指标,对主要工艺及处方因素进行了研究。最终确定处方和工艺为：按质量百分比,油相组成为替尼泊苷0.05%、MCT 10%、LCT 10%、VE 2%、PEG-40 02%、豆磷脂2.4%、注射用油酸0.06%,水相组成为甘油2.5%、Tween-80 0.4%、F-68 0.4%.依地酸二钠0.05%,加注射用水至全量；制备初乳的油相和水相温度均为70℃,高压均质参数为70MPa压力循环8次,均质后调节乳剂pH值至6.0-6.5,灭菌条件为100℃旋转水浴灭菌30min。本文以药物含量及制剂外观为评价指标,系统考察了“直接载药替尼泊苷亚微乳注射液”在不同pH值,不同灭菌条件下的稳定性。结果表明：调节TSE pH值为6.5,100℃旋转水浴灭菌30min,能保证制剂的理化稳定性。该制剂经室温留样1个月后,物理稳定性不佳。因此本文通过进一步深入的研究确定以制备磷脂复合物的方法载药来制备替尼泊苷亚微乳注射液。本研究以反应溶剂、磷脂比例与种类为考察项,确定了替尼泊苷磷脂复合物的制备方法为替尼泊苷：豆磷脂1：40(w/w),在无水乙醇中80℃反应2小时,蒸发除去溶剂,即得。利用DSC,红外光谱及X射线衍射技术确证分析了替尼泊苷磷脂复合物的形成以及药物和磷脂两者间可能存在的相互作用。采用与“直接载药替尼泊苷亚微乳注射液”相同的制备方法和检测技术,确定以磷脂复合物载药的TSE最终处方和工艺为：按质量百分比,油相组成为磷脂复合物(以替尼泊苷计)0.05%、MCT 20%、豆磷脂0.5%、注射用油酸0.06%,水相组成为甘油2.5%、Tween-800.4%、F-68 0.4%、依地酸二钠0.05%,加注射用水至全量；制备初乳的油相和水相温度分别为70℃和70℃,高压均质参数为70MPa压力循环8次,均质后调节乳剂pH值至6.0-6.5,灭菌条件为100℃旋转水浴灭菌30min。以磷脂复合物载药的TSE物理化性质测定结果显示TSE平均粒径162.4±51.14nm,ζ-电位-23.14mV,含量101.1%,包封率91.4%。稀释稳定性实验表明经氯化钠注射液和葡萄糖注射液稀释,替尼泊苷亚微乳注射液粒径无显著变化,无析出。可认为该替尼泊苷亚微乳注射液稀释稳定性良好。通过恒温加速试验预测TSE 4℃的有效期应为22天,而长期稳定性考察结果显示在4±2℃条件下放置2个月,本制剂物理化学性质仍然稳定。因此对TSE实测有效期延长的原因进行了考察,试验发现,非均相体系在低温状态下粘度变化剧烈,乳剂的流变学变化是导致低温下实测有效期大于推测有效期的主要原因之一。本文建立了UPLC/MS/MS法用于测定大鼠血浆中替尼泊苷的方法,该法测定时间短,准确性、重现性良好。以替尼泊苷溶液型注射液为参比制剂,以磷脂复合物载药的替尼泊苷亚微乳注射液和溶液型注射液的药时过程均符合三隔室模型,两者药代动力学行为相似。乳剂及溶液剂的AUC0-t,分别为(24065.096±6320.831)和(10590.352±2054.728)μg/L*h；t1/2分别(0.936±0.163)和(0.962±0.264)h。更多还原

【Abstract】 Parenteral submicro-emulsion, with high biocompatibility, low toxicity and irritation, has been widely used as an excellent drug delivery carrier. Teniposide (VM-26), one of the most broadly effective anticancer agents, is included in a wide variety of cancer chemotherapy protocols. However, because of its poor water solubility, teniposide is currently administered in the form of a nonaqueous formulation (Vumon(?)), which contains Cremophor(?)EL (polyethoxylated castor oil), dehydrated alcohol and other non-aqueous solvents. Vumon(?) therapy is associated with life-threatening hypersensitivity reactions and a danger that teniposide may crystallize during the process of dilution. Thus, the aim of this study was to prepare and evaluate a stable teniposide sub-microemulsion injection (TSE).An HPLC method was established for the analysis of teniposide in vitro. The method validations were carried out and approved. Several physicochemical parameters of teniposide were investigated, such as solubility, degradation parameter and so on. The solubility of teniposide in LCT and MCT were lower than 0.5mg·mL-1, so solubilizers would be used in the formulation of TSE. The study of degradation of teniposide in the buffers with the pH 5.0,6.0, 6.5,7.0,8.0 at 80℃shows that pH 6.5 was the most stable pH for teniposide with the half-life 8.36h.High pressure homogenization was used to prepare TSE. HPLC, dynamic light scattering and electrophoretic light scattering technology, and ultracentrifugation were also employed. Taking physical appearance, pH, particle size distribution (PSD),ξ-potential, content, entrapment efficiency and thermal sterile stability as index, the final formulation and preparation process for directly loaded TSE were as follows:as quality percentage, oil phase was composed of 0.05% teniposide,10% MCT,10% LCT,2% VE,2%PEG-400,2.4% soybean lecithin,0.06% oleic acid; the water phase was composed of 2.5% glycerol,0.4% F-68,0.4% Tween-80 and 0.05% EDTA-2Na; and the oil phase and water phase were both heated to 70℃; the homogenization pressure and cycles were 70MPa and 8 times; the pH was adjusted to 6.0-6.5 and then TSE was sterilized in a rotating water bath at 100℃for 30min.Through the study of different pH and sterile ways, the sterile stability of directly loaded TSE was investigated systematically. The results indicated that, with pH 6.5, the physicochemical characteristics of TSE were stable after sterilizing in a rotating water bath at 100℃for 30min. After 1 month room temperature storage, directly loaded TSE showed bad physical appearance. So the phospholipid complex TSE was investigated in the further study.The reaction solvents, the ratios and types of phospholipids were investigated, and the teniposide-phospholipid complex was finally prepared with teniposide and soybean lecithin at a ratio of 1:40 in dehydrated alcohol at 80℃for 2h, and then the reaction solvent was evaporated to obtain dry residue. DSC, IR, and X-ray diffraction were also employed to identify the formation of the complex and analyze the possible interaction between teniposide and phospholipids.The final formulation and preparation process for TSE using teniposide- phospholipid complex were as follows:as quality percentage, oil phase was composed of 0.05% teniposide in the form of the complex,20% MCT,2% VE,2%PEG-400,2.4% soybean lecithin,0.06% oleic acid; the water phase was composed of 2.5% glycerol,0.4% F-68,0.4% Tween-80 and 0.05% EDTA-2Na; and the oil phase and water phase were both heated to 70℃; the homogenization pressure and cycles were 70MPa and 8 times; the pH was adjusted to 6.0-6.5, and then TSE was sterilized in a rotating water bath at 100℃for 30min.The characteristics including PSD,ξ-potential, drug content and entrapment efficiency of TSE using teniposide-phospholipid complex were 162.4±51.14nm,-23.14mV,101.1%, and 91.4%. The dilution test of TSE with Sodium chloride injection solution (0.9%) and dextrose (5%) suggested that after diluting TSE was stable. The shelf-life of TSE was calculated to be 22 days by constant temperature accelerated test. However, the long term stability study showed that the physicochemical characteristics of TSE were still stable at 4±2℃after 2 months. The rheology change of the emulsion demonstrated to be the reason for the extension of the shelf-life detected compared with the shelf-life calculated through the viscosity test.An UPLC/MS/MS method was established to determine the concentration of teniposide in rat plasma. Both TSE using teniposide-phospholipid complex and teniposide solution (TS) fitted three-compartment model and their pharmacokinetic behaviors were similar. The AUC0-t and t1/2 of TSE and TS were (24065.096±6320.831) and (10590.352±2054.728)μg/L*h, and (0.936±0.163) and (0.962±0.264) h, respectively.更多还原