2-甲氧基雌二醇磷脂复合物研究

发布时间：2018-04-26 03:05

本文选题：2-甲氧基雌二醇 + 磷脂复合物　；参考：《郑州大学》2014年硕士论文

【摘要】：2-甲氧基雌二醇（2-methoxyestradiol，简称2-ME）是20世纪70年代，在研究雌二醇体内代谢过程时，尿液中发现的主要雌激素的天然代谢产物，已研究证实，该化合物对细胞增殖和血管新生具有潜在抑制作用，且具有高效低毒、不良反应少、不易产生交叉耐药性等优点，目前国外正处于Ⅱ期临床研究阶段。2-ME作为一种浓度时间依赖性的新型广谱抗肿瘤药，但由于它难溶于水，口服血药浓度和剂量之间缺乏相关性，肝首过效应强，，代谢速率快，导致体内生物半衰期短和口服生物利用度低，而且常规给药剂型很难满足其浓度时间依赖性药理学特征。因此，如何提高2-ME的口服吸收效率，提高生物利用度，以维持其有效的血药浓度是临床应用的关键。本研究是欲以2-ME为主药，大豆卵磷脂为载体，利用二者相互作用形成磷脂复合物，希望该磷脂复合物能够解决2-ME生物药剂学方面存在的不足，以达到临床口服给药目的。本研究首先建立了高效液相色谱测定2-ME磷脂复合物中2-ME含量的分析方法，选用Akasil C18色谱柱，甲醇：水(70:30)为流动相，流速为1ml/min，柱温为30℃，紫外检测波长为288nm。该方法的线性回归方程A=0.2876C+0.1537(R2=0.9999)，日内和日间精密度分别为1.35%和2.02%，平均回收率达到0.88%，符合样品分析要求。2-ME溶解度测定结果显示：2-ME难溶于水(约0.225±0.016μg·ml-1)，略溶于二氯甲烷和乙醚，易溶于四氢呋喃和甲醇等有机溶剂，表观油水分配系数：log P=3.61。其次采用溶剂挥发法制备了2-ME磷脂复合物，以2-ME与大豆卵磷脂的复合率为评价指标，综合考察了处方因素(反应溶剂、反应药物浓度、药物与磷脂投料比)及工艺因素(反应温度、反应时间)对复合率的影响，最终确定了最优处方及工艺为：以四氢呋喃为反应溶剂，2-ME质量浓度为5mg·ml-1，2-ME与磷脂投料摩尔比为1:1.2，反应温度40℃，反应时间3h，制得复合率可达92.4%的淡黄色黏性固体物。再次对2-ME磷脂复合物进行表征，SEM和XRD法分析结果显示2-ME晶体特征消失，磷脂复合物表现出无定型态；DSC法测定显示复合物中2-ME熔点峰消失；UV法测定显示复合物中2-ME的发色基团未发生改变；IR和1H-NMR表征结果显示2-ME磷脂复合物是磷脂分子的极性端与2-ME分子之间发生相互作用的结果。复合物水中溶解度(0.477±0.021μg·ml-1)和表观油水分配系数(log P4.02)分别提高了2.12倍和1.11倍。桨法考察了复合物体外溶出度，结果显示2-ME磷脂复合物累积溶出度高于其物理混合物，2-ME原料药溶出最慢。初步稳定性试验考察结果显示2-ME磷脂复合物在高温、光照和湿度条件下稳定性会降低，应当贮藏在低温干燥避光处。此外，以MCF-7细胞株为研究对象，SRB法评价了2-ME磷脂复合物的体外细胞生长抑制作用，结果显示2-ME磷脂复合物对MCF-7细胞的增殖抑制作用呈时间浓度依赖性。2-ME磷脂复合物在48h和72h的IC50值分别为3.26μmol·l-1和0.98μmol·l-1，而2-ME原料药的为5.28μmol·l-1和2.72μmol·l-1。最后以大鼠为给药模型，建立了荧光高效液相色谱测定复合物生物样品中2-ME浓度的分析方法。大鼠灌胃给药，体内药代动力学测定结果显示：2-ME磷脂复合物的AUC、Tmax、Cmax和t1/2β分别为(37.367±4.212) μg·ml-1·min、(8.865±0.187)min、(0.535±0.077)μg·ml-1、(121.912±10.812)min，相对应的2-ME原料药AUC、Tmax、Cmax和t1/2β依次为(25.099±3.004) μg·ml-1·min、(8.872±0.886) min、(0.405±0.031)μg·ml-1、(73.693±4.579)min，2-ME磷脂复合物的口服生物利用度约是2-ME的1.49倍，生物半衰期延长近50min，说明将2-ME制成磷脂复合物可提高其口服吸收效率。
[Abstract]:2- methoxy estradiol (2-methoxyestradiol, 2-ME) is a natural metabolite of the main estrogen found in the urine during the metabolic process of estradiol in 1970s. It has been confirmed that the compound has a potential inhibition effect on cell proliferation and angiogenesis, with high toxicity and low toxicity, and is not easy to produce. At present,.2-ME is a new type of broad-spectrum antitumor drug with time dependence in phase II clinical study. However, because it is difficult to dissolve in water, there is a lack of correlation between the concentration and dose of oral blood, strong head over effect and fast metabolic rate, resulting in short biological half-life in the body and oral bioavailability. It is low, and it is difficult to meet the time dependent pharmacological characteristics of the conventional drug type. Therefore, how to improve the oral absorption efficiency of 2-ME, improve the bioavailability and maintain its effective blood concentration is the key to the clinical application. This study is intended to use 2-ME as the main drug, soybean lecithin as the carrier, and use the interaction of the two to form phospholipids. It is hoped that the phospholipid complex can solve the deficiency of 2-ME Biopharmaceutics, so as to achieve the goal of oral administration.
In this study, a high performance liquid chromatography (HPLC) method for the determination of 2-ME in 2-ME phospholipid complex was established by using Akasil C18 chromatographic column, methanol: water (70:30) as the mobile phase, the flow velocity of 1ml/min, the column temperature of 30 C, and the linear regression square A=0.2876C+0.1537 (R2=0.9999) of the UV detection wavelength for 288nm., and the intra day and day precision points. The average recovery rate of 1.35% and 2.02% is 0.88%. The results show that the solubility of.2-ME shows that 2-ME is difficult to dissolve in water (about 0.225 + 0.016 mu g. ML-1), slightly dissolved in dichloromethane and ether, easily dissolved in organic solvents such as tetrahydrofuran and methanol, and the apparent oil water distribution coefficient: log P=3.61.
Secondly, 2-ME phosphatide complex was prepared by solvent evaporation, and the compound ratio of 2-ME and soybean lecithin was used as evaluation index. The effect of prescription factors (reaction solvent, reaction drug concentration, drug and phospholipid ratio) and process factors (reaction temperature and reaction time) on the compound rate were investigated, and the optimal formulation and process were finally determined. With tetrahydrofuran as the reaction solvent, the mass concentration of 2-ME is 5mg / ml-1,2-ME and the molar ratio of phospholipid is 1:1.2, the reaction temperature is 40 and the reaction time is 3h, and the light yellow sticky solid with the compound rate of up to 92.4% is obtained.
The 2-ME phospholipid complex was characterized again. The results of SEM and XRD analysis showed that the characteristics of 2-ME crystal disappeared, the phospholipid complex showed the amorphous state, and the DSC method showed that the peak of 2-ME melting point in the compound disappeared, and the UV method showed that the chromophore group of 2-ME in the complex was not changed; IR and 1H-NMR characterization showed 2-ME phospholipid complex. It is the result of interaction between the polar end of phospholipid molecule and 2-ME molecule. The solubility of the composite water (0.477 + 0.021 G. ML-1) and the apparent oil and water distribution coefficient (log P4.02) increased by 2.12 times and 1.11 times respectively. The paddle method examined the exsolution degree of the compound object, and the results showed that the cumulative dissolution of the 2-ME phospholipid complex was higher than that of its physical mixture. The initial stability test results show that the stability of the 2-ME phospholipid complex will decrease under the conditions of high temperature, light and humidity, and should be stored at the low temperature drying and light avoiding place. The preliminary stability test results show that the stability of 2-ME phospholipid complex will decrease.
In addition, the inhibitory effect of 2-ME phospholipid complex on the proliferation of 2-ME phospholipid complex was evaluated by SRB method. The results showed that the inhibitory effect of 2-ME phospholipid complex on the proliferation of MCF-7 cells was time dependent.2-ME phospholipid complex at 48h and 72h in IC50 value of 3.26 Mu mol. L-1 and 0.98 micron mol. 5.28 mol. L-1 and 2.72 Mu mol. L-1.
At last, a method for the determination of the concentration of 2-ME in the biological samples of the compound was established with the rat as the drug delivery model. The rats were administered by GC and the pharmacokinetics of 2-ME phospholipid complex showed that the AUC, Tmax, Cmax and t1/2 beta of the phospholipid complex were (37.367 + 4.212) mu g. Ml-1. Min, (8.865 + 0.187) min, (0.535 + 0.077) micron. G. Ml-1, (121.912 + 10.812) min, the corresponding 2-ME AUC, Tmax, Cmax and t1/2 beta are (25.099 + 3.004) mu g ml-1 min, (8.872 + 0.886) min, (0.405 + 0.031) micron, (73.693 + 4.579), and the bioavailability of the phospholipid complex is about 1.49 times that of that, and the biological half-life is prolonged, indicating that the phospholipid will be made into phospholipid. The compound can improve its oral absorption efficiency.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

【共引文献】