本文選題：多西他賽 + mPEG-b-PDLLA��； 參考：《延邊大學(xué)》2014年碩士論文

【摘要】：目的：多西他賽(docetaxel, DTX)為一種臨床常用抗癌藥,但其強(qiáng)疏水性給臨床使用帶來不便。為了克服多西他賽在水中溶解度低的缺點(diǎn),合成了一種新的二嵌段聚合物作為藥物傳送載體。mPEG作為親水段,PLA作為疏水段,在水溶液中自組裝成具有殼-核結(jié)構(gòu)的聚合物膠束,多西他賽為模型藥物被包裹入疏水核心,達(dá)到增加其在水中溶解度的目的。 方法：實(shí)驗(yàn)利用mPEG2ooo和DL-丙交酯在辛酸亞錫的催化作用下開環(huán)聚合成mPEG-b-PDLLA二嵌段聚合物,通過核磁共振、MALDI-TOF-TOF-MS.臨界膠束濃度對(duì)聚合物進(jìn)行表征。 采用薄膜水化法制備載多西他賽膠束(DTX-PM),通過單因素考察確定最優(yōu)處方,并將最優(yōu)處方進(jìn)行工藝重現(xiàn)性考察；利用透射電鏡觀察最優(yōu)條件下制備的載多西他賽納米膠束形態(tài)；分別用PSS·NICOMP粒徑測(cè)定儀和Malvern NanoZS激光粒度分析儀測(cè)定其粒徑和分布；利用PSS·NICOMP粒徑測(cè)定儀測(cè)定Zeta電位；高效液相色譜法(HPLC)測(cè)定包封率及載藥量；以累積釋藥百分率為指標(biāo),通過擬合釋藥曲線,考察并比較市售多帕菲(?)和DTX-PM的體外釋藥特性；通過眼眶取血的方法取豚鼠血液,比較市售制劑多帕菲(?)和DTX-PM的溶血情況。 載多西他賽納米膠束的體外評(píng)價(jià)：通過細(xì)胞遷移試驗(yàn)觀察空白膠束、市售多帕菲(?)和DTX-PM對(duì)4T1細(xì)胞的遷移能力；采用MTT法分別檢測(cè)空白膠束、市售多帕菲(?)和DTX-PM對(duì)4T1細(xì)胞的體外細(xì)胞毒性做了評(píng)價(jià)；通過流式細(xì)胞儀分別檢測(cè)空白膠束、市售多帕菲(?)和DTX-PM對(duì)4T1細(xì)胞的凋亡情況；利用激光共聚焦顯微鏡通過對(duì)香豆素-6的包載觀察DTX-PM進(jìn)入4T1細(xì)胞的入胞情況。 結(jié)果；1H-NMR顯示其平均分子量在3300左右,臨界膠束濃度為4.677μg/mL, MALDI-TOF-TOF-MS顯示檢測(cè)峰主要為3300Da,但含有少量2000Da以下的雜質(zhì)。采用薄膜水化法制備載藥膠束,通過單因素考察確定的最佳制備條件為：藥物與材料比為1：5,乙腈量為1mL,超聲時(shí)間為30s,旋蒸時(shí)間為30min,旋轉(zhuǎn)速度為80rpm,水化體積為1mL；在最佳制備條件下制備的DTX-PM測(cè)定其平均粒徑為(16.2±0.28)nm, PDI值為0.107+0.026,平均Zeta電位為-1.35my；通過HPLC檢測(cè),得出載藥膠束的包封率為(89.6±0.22)%,載藥量為(14.01±0.16)%；體外釋放顯示DTX-PM釋放平穩(wěn),充分,而市售多帕菲(?)出現(xiàn)突釋現(xiàn)象；溶血試驗(yàn)表明市售多帕菲(?)在濃度為0.1、0.25、0.5、0.75和1mg/mL時(shí),對(duì)豚鼠紅血球的溶血率分別達(dá)到了0%、32.45%、34.24%、39.52%、41.74%,而DTX-PM制劑在上述濃度中均不存在溶血現(xiàn)象。 載多西他賽納米膠束的體外評(píng)價(jià)：細(xì)胞遷移試驗(yàn)顯示,市售多帕菲(?)和DTX-PM都抑制了細(xì)胞的增殖,并且市售多帕菲(?)比DTX-PM抑制細(xì)胞增殖的能力更強(qiáng)；MTT法結(jié)果顯示,不同濃度藥物的細(xì)胞增殖抑制率顯示,低濃度載藥膠束的細(xì)胞增殖抑制率高于市售多帕菲,具有較好的體外活性；細(xì)胞凋亡試驗(yàn)顯示,說明了膠束化藥物更能顯著的誘導(dǎo)4T1細(xì)胞的晚期凋亡；入胞試驗(yàn)表明,膠束在60mmin內(nèi)充分入胞,速度較快。 結(jié)論：多西他賽嵌段共聚物膠束呈球形,粒徑分布均勻,性質(zhì)穩(wěn)定。載多西他賽納米膠束能夠更有效地抑制小鼠4T1細(xì)胞的增長,是一種有前景的新型納米給藥技術(shù),為乳腺癌腫瘤的治療提供重要參考依據(jù)。 目的：為了制備TZT-9片劑,本部分系統(tǒng)的研究了TZT-9片劑的成型工藝及質(zhì)量標(biāo)準(zhǔn),研制出安全、有效、穩(wěn)定、可控的片劑。 方法：我們系統(tǒng)研究了TZT-9制劑的成型工藝和質(zhì)量標(biāo)準(zhǔn)。為了提高工藝適應(yīng)性,采用粉末直接壓片的方法,并對(duì)影響TZT-9片的成型工藝的主藥含量、吸附劑、填充劑、崩解劑等方面因素進(jìn)行篩選與優(yōu)化。為防止TZT-9片出現(xiàn)嚴(yán)重的吸濕問題,所以制劑進(jìn)行了防潮薄膜包衣；為考察TZT-9片劑的工藝重現(xiàn)性,所以制備了三批中試規(guī)模的TZT-9片劑,并對(duì)其質(zhì)量進(jìn)行檢測(cè)；同時(shí)為了長期保存,對(duì)TZT-9薄膜包衣片進(jìn)行了鋁塑包裝。 質(zhì)量標(biāo)準(zhǔn)研究中按《中國藥典》2010版一部附錄I[)項(xiàng)下片劑的有關(guān)規(guī)定對(duì)TZT-9片的性狀、鑒別、檢查、含量測(cè)定等方面進(jìn)行系統(tǒng)地研究,同時(shí)建立TZT-9片中15-羥基內(nèi)酯醇(TL-1)、地芰普內(nèi)酯(TL-2)和雷公藤內(nèi)酯醇(TL-3)的含量測(cè)定方法；并分別測(cè)定了TL-1、TL-2和TL-3的日內(nèi)精密度、日間精密度、溶液穩(wěn)定性、回收率、最低定量限與最低檢出限的測(cè)定。 結(jié)果：根據(jù)試驗(yàn)結(jié)果最終確定TZT-9片的最優(yōu)處方為(每1000片用量)：TZT-9提取物0.25g,微晶纖維素37.66g,直壓輔料75.34g,羧甲淀粉鈉4.8g,二氧化硅0.75g,硬脂酸鎂1.2g。TZT-9提取物先與二氧化硅充分混合,再加入其他輔料攪拌混合均勻,調(diào)節(jié)壓片機(jī)壓力,壓制成重約120mg的片劑,其硬度達(dá)到80N,崩解時(shí)間1.44min,且片面光滑無缺,成形性好。對(duì)三批中試放大的片劑進(jìn)行包衣,每片3%的包衣增重。 采用高效液相色譜法測(cè)定TZT-9片劑中三種主要成分TL-1、TL-2和TL-3的含量,結(jié)果表明TL-1在0.196-2.94μg范圍內(nèi),與相應(yīng)峰面積呈良好線性關(guān)系(r2=0.9998)；回收率高濃度RSD=102.39%,中濃度RSD=100.34%,低濃度RSD=100.45%；日內(nèi)精密度RSD=1.42%(n=6),溶液穩(wěn)定性RSD=1.80%(n=4)；日間精密度高濃度RSD=8.60%(n=5),中濃度RSD=1.75%,低濃度RSD=4.24%；最低檢出限為3.27ng；最低定量限為1.31ng。 TL-2在0.194-3.88μg范圍內(nèi),與相應(yīng)峰面積呈良好線性關(guān)系(r2=0.9996)；回收率高濃度RSD=100.15%,中濃度RSD=98.66%,低濃度RSD=98.65%；日內(nèi)精密度RSD=0.38%(n=6),溶液穩(wěn)定性RSD=6.94%(n=4)；日間精密度高濃度RSD=1.48%(n=5),中濃度RSD=6.44%,低濃度RSD=2.17%；最低檢出限為1.94ng；最低定量限為0.97ng。 TL-3在0.36-3.28μg范圍內(nèi),與相應(yīng)峰面積呈良好線性關(guān)系(r2=0.9997)；回收率高濃度RSD=101.86%,中濃度RSD=98.72%,低濃度RSD=100.28%；日內(nèi)精密度RSD=1.02%(n=6),溶液穩(wěn)定性RSD=5.84%(n=4)；日間精密度高濃度RSD=0.68%(n=5),中濃度RSD=0.30%,低濃度RSD=1.05%；最低檢出限為6.07ng；最低定量限為2ng。 三批中試放大的含量測(cè)定結(jié)果為：20130801批TL-1含量為35.88μg,TL-2為69.7μg,TL-3為31.22μg：20130802批TL-1含量為37.39μg,TL-2為74.19μg,TL-3為30.08μg；20130803批TL-1含量為35.13μg,TL-2為74.04μg,TL-3為31.95μg。且三批含量均勻度均合格,片重差異20130801批RSD=1.15%(n=20),20130802批RSD=1.54%,20130803批RSD=1.55%,均符合標(biāo)準(zhǔn)。 結(jié)論：TZT-9片劑粉末直接壓片,通過成型工藝條件的考察制備出了安全、有效、質(zhì)量穩(wěn)定的片劑,在保證療效的同時(shí)減少服用量,提高患者依從性。質(zhì)量標(biāo)準(zhǔn)中制定的含量測(cè)定方法簡便、準(zhǔn)確、靈敏度高、重現(xiàn)性好,可用于該制劑質(zhì)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.

【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 應(yīng)鳳祥,楊式升,張敏,李豫喜,周宏燕;激光掃描共聚焦顯微鏡研究儲(chǔ)層孔隙結(jié)構(gòu)[J];沉積學(xué)報(bào);2002年01期

2 王朝虹,張吉林,何毅,侯小平;高效液相色譜法測(cè)定血漿中雷公藤甲素和雷公藤酮[J];中國法醫(yī)學(xué)雜志;2004年05期

3 陳坤成;馮思良;劉克良;;紫杉烷類抗腫瘤藥物臨床研究進(jìn)展[J];國際藥學(xué)研究雜志;2013年03期

4 程樹倉;龐鑫;翟光喜;;多烯紫杉醇納米制劑的研究進(jìn)展[J];藥學(xué)研究;2013年01期

5 吳瑤;曹俊;陳元維;羅祥林;;含聚乙二醇兩親性聚合物及其膠束用作藥物載體的理論研究及應(yīng)用潛力[J];中國組織工程研究與臨床康復(fù);2009年47期

6 季冬英;吳瓊珠;平其能;;PLA-mPEG的合成和多西紫杉醇聚合物膠束的制備[J];中國藥科大學(xué)學(xué)報(bào);2008年03期

7 景臨林;金瑛;張生勇;孫曉莉;;抗癌藥物多烯紫杉醇的合成[J];中國藥物化學(xué)雜志;2006年05期

8 湯致強(qiáng);抗腫瘤藥——多西他賽[J];中國藥學(xué)雜志;1999年02期

9 李平祝;楊卓理;楊可偉;王菲;劉艷;;多功能聚合物膠束的最新研究進(jìn)展[J];中國新藥雜志;2008年03期

，

本文編號(hào)：1858954