复方2-甲氧基雌二醇缓释植入剂的研究

发布时间：2018-02-28 03:05

本文关键词： 2-甲氧基雌二醇 PLGA 复方药效学植入剂　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：2-甲氧基雌二醇（2-methoxyestradiol，2-ME）最早是在孕妇的尿液中被发现的雌激素代谢产物，但其几乎不发挥内雌激素样作用。近几年来，很多研究表明该化合物是一种特异性高毒性低的新型抗肿瘤药物，对多种恶性肿瘤有抑制作用，其在国外的研究处于Ⅱ期临床阶段。由于它水溶性差，口服吸收无规律，静脉注射半衰期短（约为12min），国内外研究将其制备成固体纳米脂质体、磷脂复合物、混悬剂等克服2-ME的缺点，但效果仍不佳。本研究以聚乳酸-羟基乙酸共聚物（PLGA）为载体，以2-ME和吉非替尼为主药，制备成复方2-ME缓释植入剂，通过皮下瘤旁局部给药方式给药，使药物直接发挥作用，并能达到缓释长效的作用。本研究用SRB法检测了2-ME和吉非替尼单独以及二者联合对MCF-7、A549、4T-1细胞的增殖抑制作用，2-ME对三种细胞作用72h的IC50值分别为0.96μmol·L-1、0.866μmol·L-1、0.692μmol·L-1，可知不同细胞系对药物的敏感性不同。在0.625μmol·L-1~10.0μmol·L-1浓度范围内，2-ME对A549的抑制率随着时间的增加而增大，作用48h的IC50为1.993μmol·L-1，可知2-ME和吉非替尼的抗肿瘤作用具有明显的时间浓度依赖性。2-ME和吉非替尼在低浓度时联合对A549作用的联合指数低至0.17（CI1），二者在一定浓度范围内联合具有协同作用。以药物对A549细胞的联合指数为指标，筛选出2-ME和吉非替尼联合抗肿瘤活性最佳的配比为2.2:1（w/w）。确立了检测复方制剂中2-ME和吉非替尼含量的分析方法----高效液相色谱法。色谱条件为：流动相是甲醇：水（75:25），柱温25℃，，流速是0.1ml·min-1，紫外检测器波长为285nm。建立了2-ME和吉非替尼的线性回归方程，分别为：A=0.2599C+0.0488（r=0.9998）， A=0.4004C－0.1305（r=0.9999）。考察了在该色谱条件下，2-ME的日间、日内相对标准偏差分别为1.57%和1.38%，吉非替尼的日内、日间相对标准偏差为0.93%和1.08%；2-ME的平均回收率为99.87%（RSD=0.90%，n=9），吉非替尼的平均回收率为99.55%（RSD=0.67%，n=9）。该色谱条件和方法完全满足实验要求。通过测定2-ME和吉非替尼在不同释放介质中的溶解度，最终确定用于体外释放的介质为1%SDS-PBS溶液。本实验采用单因素考察法，考察了不同型号的PLGA、不同载药量、不同的制备工艺和方法对制剂释放行为的影响，确定了作为制剂载体的PLGA的型号为PLGA（75:25）；制剂的载药量为60%；最终采用制剂的制备方法为溶剂挥发法。体外释放第6天、16天、30天时，2-ME分别累计释放33.9%、56.6%、73.1%，制剂有缓释作用。对体外释放结果进行拟合，显示制剂中2-ME和吉非替尼的释放均与Ritger-Peppas方程拟合较好，二者释放行为一致。考察了制剂的体内释放，药物第13天、45天、54天的累计释放百分数达到30%、50%、72.8%，表明该植入剂具有缓释作用。将植入剂的体内释放结果进行数学模型拟合，结果显示2-ME和吉非替尼的释放与Ritger-Peppas方程拟合较好，相关系数r分别为0.9957和0.9924，两药的体内释放行为一致。根据Ritger-Peppas，其释药机理是骨架溶蚀和药物扩散的共同作用。且制剂中2-ME和吉非替尼体内外释放相关性良好。本研究采用在小鼠右前肢背侧皮下的部位接种S180细胞悬液的方法建立肿瘤模型，以相对肿瘤增殖率为考察指标，研究复方2-ME缓释植入剂高、中、低剂量组对小鼠癌症的抑制效果。复方2-ME缓释植入剂高、中、剂量组的相对肿瘤增殖率分别为：38.55%、38.96%、49.13%，空白PLGA组为82.29%。结果显示，复方制剂的高、中剂量组的相对肿瘤增殖率40%，能抑制肿瘤的增殖；空白PLGA组和植入剂低剂量组对S180肿瘤无效。
[Abstract]:2- methoxyestradiol (2-methoxyestradiol, 2-ME) is the earliest estrogen metabolites were found in the urine of pregnant women, but it almost does not play in estrogenic effects. In recent years, many studies show that this compound is a novel antitumor drug with high specificity and low toxicity, has inhibitory effect on a variety of malignant tumors in the Foreign Studies in phase II clinical stage. Due to its poor water solubility, absorption of oral, intravenous injection of short half-life (about 12min), the domestic and foreign research to prepare a solid nano liposome, phospholipid complex, mixed suspensions and other shortcomings overcome 2-ME, but the effect is still poor. In this study, poly lactic acid glycolic acid copolymer (PLGA) as the carrier, with 2-ME and gefitinib based medicine, prepared compound 2-ME sustained-release implants, administered by subcutaneous tumor by topical administration, the drug directly play a role, and can achieve sustained release A long effect.
This study used SRB method to detect the 2-ME and gefitinib alone and the combination of the two MCF-7, inhibited the proliferation of A549,4T-1 cells, 2-ME cells of three 72h IC50 = 0.96 mol - L-1,0.866 mol - L-1,0.692 mol - L-1, susceptibility to drugs of different show different cell lines in 0.625 ~ mol ~ L-1~10.0 ~ mol ~ L-1 concentration range, 2-ME inhibition rate of A549 increases with the increase of time, the effect of 48h IC50 1.993 mol, L-1, 2-ME and gefitinib antitumor effect has obvious concentration dependent.2-ME and gefitinib in when combined with low concentration index on the role of A549 as low as 0.17 (CI1), the combination of the two has a synergistic effect in a certain concentration range. The drug combination index of A549 cells were selected as indexes, 2-ME and gefitinib combined with the ratio of antitumor activity for the best 2.2:1 (w/w).
The detection of 2-ME in compound preparation and gefitinib imatinib content analysis method -- high performance liquid chromatography. The chromatography conditions were: mobile phase is methanol: water (75:25), the column temperature was 25 degrees, the flow rate is 0.1ml / min-1, the UV detector wavelength was 285nm. to 2-ME and gefitinib line the regression equation, respectively: A=0.2599C+0.0488 (r=0.9998), A=0.4004C 0.1305 (r=0.9999) were investigated. Under the chromatographic conditions, 2-ME day, days of the relative standard deviation is 1.57% and 1.38% respectively, gefitinib days during the day, the relative standard deviation is 0.93% and 1.08%; the average recovery rate of 2-ME 99.87% (RSD=0.90%, n=9), gefitinib, the average recovery rate was 99.55% (RSD=0.67%, n=9). The chromatographic conditions and the method can meet the experimental requirements.
Through the determination of 2-ME and non Kyrgyzstan solubility of imatinib in different release media, and ultimately determine the medium for in vitro release of 1%SDS-PBS solution. The experimental study using single factor method, the effects of different types of PLGA and different drug loading, preparation process and different methods to influence the drug release behavior, identified as preparation of vector PLGA model PLGA (75:25); preparation of the drug loading was 60%; the final preparations for the preparation method using solvent evaporation method. The in vitro release of sixth days, 16 days, 30 days, 2-ME were 56.6%, 73.1% and 33.9% cumulative release, sustained release preparation, in vitro release to fit. The results show, in the preparation of 2-ME and gefitinib release with Ritger-Peppas equation, the release behavior of two is consistent.
Effects of in vivo drug release formulations, thirteenth days, 45 days, 54 days, the cumulative release percentage reached 30%, 50%, 72.8%, showed that the implant with sustained-release effect. The implants in vivo release results were fitted to a mathematical model, the results show that 2-ME and gefitinib release and the Ritger-Peppas equation, the better correlation the coefficient of R were 0.9957 and 0.9924, two drug release behavior in vivo. According to Ritger-Peppas, the release mechanism is the interaction of skeleton dissolution and drug diffusion. And in the preparation of 2-ME and gefitinib in vivo release good correlation.
Based on the method of subcutaneous in mouse right forelimb dorsal parts of the cell suspension were inoculated with S180 tumor model, the relative tumor growth rate as the study index, study of compound 2-ME sustained-release implants in high, low dose group, the inhibitory effect on mouse cancer. Compound 2-ME sustained-release implantation agent in high dose group. The relative tumor growth rates were: 38.55%, 38.96%, 49.13%, blank group PLGA 82.29%. showed that the compound high, middle dose group relative tumor growth rate of 40%, can inhibit the proliferation of tumor cells; blank PLGA group and low dose group implants ineffective on S180 tumor.

【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

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