齐多夫定细胞内磷酸化过程的动力学研究

发布时间：2018-01-13 22:28

本文关键词：齐多夫定细胞内磷酸化过程的动力学研究　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：在细胞培养体系中研究细胞内核苷类药物磷酸化的动力学过程,以期对细胞内药物活性成分浓度变化情况有更为直接的理解。在健康人中给药,探究血浆内原药浓度与单个核细胞中原药及磷酸化产物浓度的变化规律及磷酸化动力学过程,为核苷类药物的临床用药提供参考信息。方法：①建立化学合成齐多夫定一磷酸、二磷酸化物及三磷酸化物的方法,并用制备液相色谱方法分离纯化。②建立LC-MS/MS测定血浆内AZT及细胞内AZT、AZT-MP、AZT-DP和AZT-TP浓度的方法。③利用cck-8法考察齐多夫定对Molt-4和HUVCEs的细胞毒性作用,并进行Molt-4及HUVCEs细胞培养体系中齐多夫定细胞内磷酸化过程的动力学研究。④健康志愿者单次给药600mg,用Ficoll密度梯度离心法分离血浆及外周血单个核细胞,测定给药后不同时间受试者血浆及单核细胞内齐多夫定及其磷酸化代谢物的浓度,用DAS2.0软件计算药动学参数,研究齐多夫定及其磷酸化代谢物在血浆及单个核细胞中的动力学过程。结果：①利用化学合成法成功合成齐多夫定一、二、三磷酸化物。②经方法学考察,本研究中建立的测定细胞中AZT、AZT-MP、AZT-DP及AZT-TP浓度的LC-MS/MS法符合相关指导原则对生物样本测试方法的要求。③细胞培养体系中磷酸化产物的生成主要是以一磷酸化物为主,二磷酸及三磷酸化物的细胞内浓度远低于一磷酸化物。④人体试验中,血浆中齐多夫定均以原型存在,细胞内磷酸化产物以一磷酸化物为主,二磷酸及三磷酸化物的浓度很低,与体外实验结果一致；依据血浆与细胞内齐多夫定浓度计算所得的药动学参数,以及细胞内不同磷酸化产物之间的药动学参数存在差异。血浆内齐多夫定的Tmax为0.583h,t1/2为2.022h；细胞内齐多夫定的Tmax为1.083h,t1/2为2.493h；细胞内齐多夫定一磷酸化物的Tmax为1.5h,t1/2为13.428h；细胞内齐多夫定二磷酸化物的Tmax为1.417h,t1/2为8.285h；细胞内齐多夫定三磷酸化物的Tmax为1.583h,t1/2为4.24h。结论：齐多夫定在细胞内生成三种磷酸化代谢产物,其中一磷酸化物浓度最高,半衰期最长,是体内主要的贮存形式。三磷酸化物作为主要的效应成分,浓度低,半衰期较血浆齐多夫定有明显延长,消除速度减慢。细胞内外药动学结果可以解释临床给药时间点的差异。
[Abstract]:Objective: To study the dynamic process of intracellular nucleoside phosphorylation in cultured cells, in order to have a more direct understanding of the changes of the active pharmaceutical ingredient concentration within the cell. Dosing in healthy people, explore the regularity and phosphorylation dynamics process in plasma drug concentration and mononuclear cells and phosphorylation of Zhongyuan medicine the concentration of product, to provide the reference information for the clinical use of nucleoside drugs.
Method: to establish a chemical synthesis method of Zidorf acid, two phosphorylation and phosphorylation of three compounds, and prepared by liquid chromatography method for separation and purification. The establishment of LC-MS/MS and AZT were measured in plasma cells in AZT, AZT-MP, AZT-DP and AZT-TP concentration. The Caci Dov test cell toxicity to Molt-4 and using HUVCEs CCK-8 method, and kinetics of phosphorylation of intracellular zidovudine Molt-4 and HUVCEs cell culture system. The healthy volunteers after a single dose of 600mg by Ficoll density gradient centrifugation separation of plasma and peripheral blood mononuclear cells, measured by different time after drug plasma concentration test monocytes and Nezi Dov and phosphorylated metabolites, the pharmacokinetic parameters were calculated by DAS2.0 software, the research of zidovudine phosphorylation and its metabolites in plasma and mononuclear cells in the dynamic process.
Results: by using chemical synthesis method successfully synthesized Zidorf one, two, three phosphorylation. The methodology investigation, in AZT cells, this study established the determination method of LC-MS/MS in AZT-MP, AZT-DP and AZT-TP concentration to meet the requirements of relevant guidelines for biological sample test method. The cultured cells generate phosphorylation product system the main is a phosphate based, two phosphoric acid and three phosphorylation of intracellular concentration is much lower than a phosphate compound. The experiment, plasma Zidorf in prototype exists, intracellular phosphorylation product with a phosphate, phosphoric acid concentration of two and three phosphorylation. Very low, consistent with in vitro results; based on the kinetic parameters of plasma cells and Nezi Dov concentration calculated between different intracellular drug and phosphorylation product pharmacokinetic parameters are different. In the plasma of zidovudine. Tmax 0.583h, t1/2 2.022h; Nezi Dov Tmax 1.083h cells, t1/2 2.493h cells; Nezi Dov a phosphate Tmax 1.5h, t1/2 13.428h; cell Nezi Dov two phosphorylation of Tmax 1.417h, t1/2 8.285h; cell Nezi Dov three phosphorylation of Tmax is 1.583h. T1/2 4.24h.
Conclusion: Zidovudine generated three phosphorylated metabolites in cells, one of the highest concentration of phosphate, the longest half-life, is the main storage form of in vivo phosphorylation. Three compounds as the main effective components, low concentration of plasma half-life is zidovudine has significantly prolonged the elimination of cells slows down. Inside and outside the difference in the pharmacokinetic results can explain clinical dosing time points.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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