發(fā)布時(shí)間：2018-01-13 22:28

  本文關(guān)鍵詞：齊多夫定細(xì)胞內(nèi)磷酸化過(guò)程的動(dòng)力學(xué)研究　出處：《中南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 齊多夫定 磷酸化 化學(xué)合成 LC-MS/MS 細(xì)胞內(nèi)動(dòng)力學(xué)

【摘要】：目的：在細(xì)胞培養(yǎng)體系中研究細(xì)胞內(nèi)核苷類藥物磷酸化的動(dòng)力學(xué)過(guò)程,以期對(duì)細(xì)胞內(nèi)藥物活性成分濃度變化情況有更為直接的理解。在健康人中給藥,探究血漿內(nèi)原藥濃度與單個(gè)核細(xì)胞中原藥及磷酸化產(chǎn)物濃度的變化規(guī)律及磷酸化動(dòng)力學(xué)過(guò)程,為核苷類藥物的臨床用藥提供參考信息。 方法：①建立化學(xué)合成齊多夫定一磷酸、二磷酸化物及三磷酸化物的方法,并用制備液相色譜方法分離純化。②建立LC-MS/MS測(cè)定血漿內(nèi)AZT及細(xì)胞內(nèi)AZT、AZT-MP、AZT-DP和AZT-TP濃度的方法。③利用cck-8法考察齊多夫定對(duì)Molt-4和HUVCEs的細(xì)胞毒性作用,并進(jìn)行Molt-4及HUVCEs細(xì)胞培養(yǎng)體系中齊多夫定細(xì)胞內(nèi)磷酸化過(guò)程的動(dòng)力學(xué)研究。④健康志愿者單次給藥600mg,用Ficoll密度梯度離心法分離血漿及外周血單個(gè)核細(xì)胞,測(cè)定給藥后不同時(shí)間受試者血漿及單核細(xì)胞內(nèi)齊多夫定及其磷酸化代謝物的濃度,用DAS2.0軟件計(jì)算藥動(dòng)學(xué)參數(shù),研究齊多夫定及其磷酸化代謝物在血漿及單個(gè)核細(xì)胞中的動(dòng)力學(xué)過(guò)程。 結(jié)果：①利用化學(xué)合成法成功合成齊多夫定一、二、三磷酸化物。②經(jīng)方法學(xué)考察,本研究中建立的測(cè)定細(xì)胞中AZT、AZT-MP、AZT-DP及AZT-TP濃度的LC-MS/MS法符合相關(guān)指導(dǎo)原則對(duì)生物樣本測(cè)試方法的要求。③細(xì)胞培養(yǎng)體系中磷酸化產(chǎn)物的生成主要是以一磷酸化物為主,二磷酸及三磷酸化物的細(xì)胞內(nèi)濃度遠(yuǎn)低于一磷酸化物。④人體試驗(yàn)中,血漿中齊多夫定均以原型存在,細(xì)胞內(nèi)磷酸化產(chǎn)物以一磷酸化物為主,二磷酸及三磷酸化物的濃度很低,與體外實(shí)驗(yàn)結(jié)果一致；依據(jù)血漿與細(xì)胞內(nèi)齊多夫定濃度計(jì)算所得的藥動(dòng)學(xué)參數(shù),以及細(xì)胞內(nèi)不同磷酸化產(chǎn)物之間的藥動(dòng)學(xué)參數(shù)存在差異。血漿內(nèi)齊多夫定的Tmax為0.583h,t1/2為2.022h；細(xì)胞內(nèi)齊多夫定的Tmax為1.083h,t1/2為2.493h；細(xì)胞內(nèi)齊多夫定一磷酸化物的Tmax為1.5h,t1/2為13.428h；細(xì)胞內(nèi)齊多夫定二磷酸化物的Tmax為1.417h,t1/2為8.285h；細(xì)胞內(nèi)齊多夫定三磷酸化物的Tmax為1.583h,t1/2為4.24h。 結(jié)論：齊多夫定在細(xì)胞內(nèi)生成三種磷酸化代謝產(chǎn)物,其中一磷酸化物濃度最高,半衰期最長(zhǎng),是體內(nèi)主要的貯存形式。三磷酸化物作為主要的效應(yīng)成分,濃度低,半衰期較血漿齊多夫定有明顯延長(zhǎng),消除速度減慢。細(xì)胞內(nèi)外藥動(dòng)學(xué)結(jié)果可以解釋臨床給藥時(shí)間點(diǎn)的差異。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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本文編號(hào)：1420841