多西他赛纳米胶束和雷公藤有效部位口服制剂的研究

发布时间：2018-05-07 23:28

本文选题：多西他赛 + mPEG-b-PDLLA　；参考：《延边大学》2014年硕士论文

【摘要】：目的：多西他赛(docetaxel, DTX)为一种临床常用抗癌药,但其强疏水性给临床使用带来不便。为了克服多西他赛在水中溶解度低的缺点,合成了一种新的二嵌段聚合物作为药物传送载体。mPEG作为亲水段,PLA作为疏水段,在水溶液中自组装成具有壳-核结构的聚合物胶束,多西他赛为模型药物被包裹入疏水核心,达到增加其在水中溶解度的目的。方法：实验利用mPEG2ooo和DL-丙交酯在辛酸亚锡的催化作用下开环聚合成mPEG-b-PDLLA二嵌段聚合物,通过核磁共振、MALDI-TOF-TOF-MS.临界胶束浓度对聚合物进行表征。采用薄膜水化法制备载多西他赛胶束(DTX-PM),通过单因素考察确定最优处方,并将最优处方进行工艺重现性考察；利用透射电镜观察最优条件下制备的载多西他赛纳米胶束形态；分别用PSS·NICOMP粒径测定仪和Malvern NanoZS激光粒度分析仪测定其粒径和分布；利用PSS·NICOMP粒径测定仪测定Zeta电位；高效液相色谱法(HPLC)测定包封率及载药量；以累积释药百分率为指标,通过拟合释药曲线,考察并比较市售多帕菲(?)和DTX-PM的体外释药特性；通过眼眶取血的方法取豚鼠血液,比较市售制剂多帕菲(?)和DTX-PM的溶血情况。载多西他赛纳米胶束的体外评价：通过细胞迁移试验观察空白胶束、市售多帕菲(?)和DTX-PM对4T1细胞的迁移能力；采用MTT法分别检测空白胶束、市售多帕菲(?)和DTX-PM对4T1细胞的体外细胞毒性做了评价；通过流式细胞仪分别检测空白胶束、市售多帕菲(?)和DTX-PM对4T1细胞的凋亡情况；利用激光共聚焦显微镜通过对香豆素-6的包载观察DTX-PM进入4T1细胞的入胞情况。结果；1H-NMR显示其平均分子量在3300左右,临界胶束浓度为4.677μg/mL, MALDI-TOF-TOF-MS显示检测峰主要为3300Da,但含有少量2000Da以下的杂质。采用薄膜水化法制备载药胶束,通过单因素考察确定的最佳制备条件为：药物与材料比为1：5,乙腈量为1mL,超声时间为30s,旋蒸时间为30min,旋转速度为80rpm,水化体积为1mL；在最佳制备条件下制备的DTX-PM测定其平均粒径为(16.2±0.28)nm, PDI值为0.107+0.026,平均Zeta电位为-1.35my；通过HPLC检测,得出载药胶束的包封率为(89.6±0.22)%,载药量为(14.01±0.16)%；体外释放显示DTX-PM释放平稳,充分,而市售多帕菲(?)出现突释现象；溶血试验表明市售多帕菲(?)在浓度为0.1、0.25、0.5、0.75和1mg/mL时,对豚鼠红血球的溶血率分别达到了0%、32.45%、34.24%、39.52%、41.74%,而DTX-PM制剂在上述浓度中均不存在溶血现象。载多西他赛纳米胶束的体外评价：细胞迁移试验显示,市售多帕菲(?)和DTX-PM都抑制了细胞的增殖,并且市售多帕菲(?)比DTX-PM抑制细胞增殖的能力更强；MTT法结果显示,不同浓度药物的细胞增殖抑制率显示,低浓度载药胶束的细胞增殖抑制率高于市售多帕菲,具有较好的体外活性；细胞凋亡试验显示,说明了胶束化药物更能显著的诱导4T1细胞的晚期凋亡；入胞试验表明,胶束在60mmin内充分入胞,速度较快。结论：多西他赛嵌段共聚物胶束呈球形,粒径分布均匀,性质稳定。载多西他赛纳米胶束能够更有效地抑制小鼠4T1细胞的增长,是一种有前景的新型纳米给药技术,为乳腺癌肿瘤的治疗提供重要参考依据。目的：为了制备TZT-9片剂,本部分系统的研究了TZT-9片剂的成型工艺及质量标准,研制出安全、有效、稳定、可控的片剂。方法：我们系统研究了TZT-9制剂的成型工艺和质量标准。为了提高工艺适应性,采用粉末直接压片的方法,并对影响TZT-9片的成型工艺的主药含量、吸附剂、填充剂、崩解剂等方面因素进行筛选与优化。为防止TZT-9片出现严重的吸湿问题,所以制剂进行了防潮薄膜包衣；为考察TZT-9片剂的工艺重现性,所以制备了三批中试规模的TZT-9片剂,并对其质量进行检测；同时为了长期保存,对TZT-9薄膜包衣片进行了铝塑包装。质量标准研究中按《中国药典》2010版一部附录I[)项下片剂的有关规定对TZT-9片的性状、鉴别、检查、含量测定等方面进行系统地研究,同时建立TZT-9片中15-羟基内酯醇(TL-1)、地芰普内酯(TL-2)和雷公藤内酯醇(TL-3)的含量测定方法；并分别测定了TL-1、TL-2和TL-3的日内精密度、日间精密度、溶液稳定性、回收率、最低定量限与最低检出限的测定。结果：根据试验结果最终确定TZT-9片的最优处方为(每1000片用量)：TZT-9提取物0.25g,微晶纤维素37.66g,直压辅料75.34g,羧甲淀粉钠4.8g,二氧化硅0.75g,硬脂酸镁1.2g。TZT-9提取物先与二氧化硅充分混合,再加入其他辅料搅拌混合均匀,调节压片机压力,压制成重约120mg的片剂,其硬度达到80N,崩解时间1.44min,且片面光滑无缺,成形性好。对三批中试放大的片剂进行包衣,每片3%的包衣增重。采用高效液相色谱法测定TZT-9片剂中三种主要成分TL-1、TL-2和TL-3的含量,结果表明TL-1在0.196-2.94μg范围内,与相应峰面积呈良好线性关系(r2=0.9998)；回收率高浓度RSD=102.39%,中浓度RSD=100.34%,低浓度RSD=100.45%；日内精密度RSD=1.42%(n=6),溶液稳定性RSD=1.80%(n=4)；日间精密度高浓度RSD=8.60%(n=5),中浓度RSD=1.75%,低浓度RSD=4.24%；最低检出限为3.27ng；最低定量限为1.31ng。 TL-2在0.194-3.88μg范围内,与相应峰面积呈良好线性关系(r2=0.9996)；回收率高浓度RSD=100.15%,中浓度RSD=98.66%,低浓度RSD=98.65%；日内精密度RSD=0.38%(n=6),溶液稳定性RSD=6.94%(n=4)；日间精密度高浓度RSD=1.48%(n=5),中浓度RSD=6.44%,低浓度RSD=2.17%；最低检出限为1.94ng；最低定量限为0.97ng。 TL-3在0.36-3.28μg范围内,与相应峰面积呈良好线性关系(r2=0.9997)；回收率高浓度RSD=101.86%,中浓度RSD=98.72%,低浓度RSD=100.28%；日内精密度RSD=1.02%(n=6),溶液稳定性RSD=5.84%(n=4)；日间精密度高浓度RSD=0.68%(n=5),中浓度RSD=0.30%,低浓度RSD=1.05%；最低检出限为6.07ng；最低定量限为2ng。三批中试放大的含量测定结果为：20130801批TL-1含量为35.88μg,TL-2为69.7μg,TL-3为31.22μg：20130802批TL-1含量为37.39μg,TL-2为74.19μg,TL-3为30.08μg；20130803批TL-1含量为35.13μg,TL-2为74.04μg,TL-3为31.95μg。且三批含量均匀度均合格,片重差异20130801批RSD=1.15%(n=20),20130802批RSD=1.54%,20130803批RSD=1.55%,均符合标准。结论：TZT-9片剂粉末直接压片,通过成型工艺条件的考察制备出了安全、有效、质量稳定的片剂,在保证疗效的同时减少服用量,提高患者依从性。质量标准中制定的含量测定方法简便、准确、灵敏度高、重现性好,可用于该制剂质量控制。
[Abstract]:Objective: docetaxel (DTX) is a common anticancer drug, but its strong hydrophobicity brings inconvenience to clinical use. In order to overcome the disadvantage of low solubility of docetaxel in water, a new two block polymer is synthesized as a drug delivery carrier.MPEG as a hydrophilic segment, PLA as a hydrophobic segment, and self assembled in aqueous solution. The polymeric micelles with shell core structure are docetaxel encapsulated into the hydrophobic core as a model drug to increase their solubility in water.
Methods: mPEG2ooo and DL- lactide were used to synthesize mPEG-b-PDLLA two block copolymers under the catalysis of stannous octanate, and the polymer was characterized by NMR and MALDI-TOF-TOF-MS. critical micelle concentration.
A film hydration method was used to prepare docetaxel micelles (DTX-PM), the optimal prescription was determined by single factor investigation, and the optimal formulation was investigated. The morphology of docetaxel nano micelles was observed under the optimal conditions by transmission electron microscopy, and the particle size analyzer of PSS NICOMP and Malvern NanoZS laser particle size analysis respectively. The size and distribution of the particles were measured by the instrument. The Zeta potential was measured by PSS NICOMP particle size analyzer; the encapsulation efficiency and drug loading were measured by high performance liquid chromatography (HPLC). The release characteristics of Dopafi (?) and DTX-PM were compared and compared by the cumulative release percentage of the drug release curve, and the guinea pig was taken from the orbit by the method of taking the blood from the orbit. Blood, compare the hemolysis of Dopafi and DTX-PM.
In vitro evaluation of docetaxel nano micelles: the cell migration test was used to observe the blank micelles, the migration ability of Dopafi (?) and DTX-PM on 4T1 cells; the MTT method was used to detect the blank micelles respectively. The cytotoxicity of the 4T1 cells in the market was evaluated by the market Dopafi (?) and DTX-PM; the blank gel was detected by flow cytometry. The apoptotic status of Dopafi (?) and DTX-PM on 4T1 cells was sold, and the entry of DTX-PM into 4T1 cells was observed by laser confocal microscopy on the loading of coumarin -6.
The results showed that the average molecular weight of the 1H-NMR was about 3300, the critical micelle concentration was 4.677 g/mL, and the MALDI-TOF-TOF-MS showed that the detection peak was mainly 3300Da, but it contained a small amount of impurities below 2000Da. The best preparation conditions determined by the film hydration method were: the ratio of drug to material was 1:5, acetonitrile. The volume is 1mL, the ultrasonic time is 30s, the rotation time is 30min, the rotation speed is 80rpm, the hydration volume is 1mL. The average particle size is (16.2 + 0.28) nm, PDI is 0.107+0.026 and the average Zeta potential is -1.35my under the optimum preparation conditions. The encapsulation efficiency of the drug loading micelle is (89.6 + 0.22)% by HPLC detection, and the drug loading amount is (1). 4.01 + 0.16)%; in vitro release showed that the release of DTX-PM was stable and full, while the market Dopafi (?) appeared abrupt release. The hemolysis test showed that the hemolysis rate of Dopafi (?) to the guinea pig was 0%, 32.45%, 34.24%, 39.52%, 41.74% at the concentration of 1mg/mL and 1mg/mL, and the DTX-PM preparation did not exist in the above concentration. In the hemolysis phenomenon.
In vitro evaluation of docetaxel nanomicelles: cell migration tests showed that both Dopafi (?) and DTX-PM inhibited cell proliferation and marketed Dopafi (?) stronger ability than DTX-PM to inhibit cell proliferation. The MTT assay showed that the proliferation inhibition rate of different concentrations of drugs showed the cell proliferation of low concentration drug micelles. The inhibition rate was higher than that of the market Dopafi, which had better in vitro activity, and the apoptosis test showed that the micellization drugs could induce the advanced apoptosis of 4T1 cells more significantly, and the cell test showed that the micelles were fully cellular in 60mmin and the speed was faster.
Conclusion: the micelles of docetaxel block copolymer are spherical, the particle size distribution is uniform and the properties are stable. The loading of docetaxel nano micelles can more effectively inhibit the growth of mouse 4T1 cells. It is a promising new nano drug technology, which provides important reference for the treatment of breast cancer.
Objective: in order to prepare TZT-9 tablets, this part systematically studied the forming process and quality standard of TZT-9 tablets, and developed a safe, effective, stable and controllable tablet.
Methods: We systematically studied the forming process and quality standard of TZT-9 preparation. In order to improve the adaptability of the process, the method of direct powder pressing was adopted, and the factors such as the main drug content, the adsorbent, the filling agent and the disintegrating agent affecting the forming process of the TZT-9 sheet were screened and optimized. In order to prevent the serious moisture absorption of the TZT-9 tablets, The moisture proof film coating was carried out with the preparation. In order to investigate the reproducibility of the TZT-9 tablet, three batch of medium scale TZT-9 tablets were prepared, and the quality of the tablets was detected. At the same time, for the long-term preservation, the TZT-9 film coated sheet was packed with aluminum plastic.
In the study of quality standards, according to the relevant regulations of the tablets under Appendix I[of Chinese Pharmacopoeia >2010, the characteristics, identification, inspection and content determination of TZT-9 tablets were systematically studied. At the same time, the content determination method of 15- hydroxylactone (TL-1), TL-2 and triptolide (TL-3) in TZT-9 tablets was established and measured respectively. Determination of intraday precision, daytime precision, solution stability, recovery rate, minimum quantitation limit and minimum detection limit of TL-1, TL-2 and TL-3 were determined.
Results: according to the results of the test, the best prescription of TZT-9 was:TZT-9 extract 0.25g, microcrystalline cellulose 37.66g, 75.34g, sodium carboxymethyl starch, 4.8g, silica 0.75g, and magnesium stearate 1.2g.TZT-9 extract first mixed with silica, and then added to other auxiliary materials to mix and mix evenly to adjust the press. The mechanical pressure, which presses the weight of about 120mg, has a hardness of 80N, the disintegration time 1.44min, and the one side smooth and free, and the formability is good. The three batch of medium test tablets are coated with 3% coating weight per piece.
The content of three main components, TL-1, TL-2 and TL-3 in TZT-9 tablets was determined by high performance liquid chromatography. The results showed that TL-1 had a good linear relationship with the corresponding peak area (r2=0.9998) in the range of 0.196-2.94 mu g, and the recovery rate was high concentration RSD=102.39%, middle concentration RSD=100.34%, low concentration RSD=100.45%. Qualitative RSD=1.80% (n=4); day precision high concentration RSD=8.60% (n=5), medium concentration RSD=1.75%, low concentration RSD=4.24%; minimum detection limit of 3.27ng; minimum quantitative limit of 1.31ng.
In the range of 0.194-3.88 g, TL-2 has a good linear relationship with the corresponding peak area (r2=0.9996); the recovery is high concentration RSD=100.15%, medium concentration RSD=98.66%, low concentration RSD=98.65%, intra day precision RSD=0.38% (n=6), RSD=6.94% (n=4) for solution stability, high concentration RSD=1.48% in day, medium concentration, low concentration; The low detection limit is 1.94ng, and the minimum quantitative limit is 0.97ng.
In the range of 0.36-3.28 g, TL-3 has a good linear relationship with the corresponding peak area (r2=0.9997); the recovery is high concentration RSD=101.86%, medium concentration RSD=98.72%, low concentration RSD=100.28%, intra day precision RSD=1.02% (n=6), RSD=5.84% (n=4) for solution stability, high concentration RSD=0.68% in day, medium concentration, low concentration; The low detection limit is 6.07ng, and the minimum quantitative limit is 2ng.
The content determination results of the three batch of three batches were as follows: the content of 20130801 batches of TL-1 is 35.88 mu g, TL-2 is 69.7 mu g, TL-3 is 37.39 mu TL-1 content of 31.22 mu g:20130802 batch, TL-2 is 74.19 micron g, TL-3 is 30.08 mu g, 20130803 batch content is 35.13 Mu and 74.04 micron, and three batch content uniformity are all qualified, slice weight difference 20130801 Batch RSD=1.15% (n=20), 20130802 batches of RSD=1.54% and 20130803 batches of RSD=1.55% are all up to standard.
Conclusion: TZT-9 tablet powder directly pressed tablets, through the investigation of molding process conditions, prepared a safe, effective, stable quality tablet. It can reduce the dosage and improve patient compliance while ensuring the curative effect. The content determination method established in the quality standard is simple, accurate, high sensitivity and good reproducibility, which can be used in the quality control of the preparation.

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

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