Intracellular Kinetic Study Of Zidovudine Phosphorylation

Aim:To develop a method which can quantify intracellular zidovudine and its phosphates simultaneously; To study the intracellular kinetics of Zidovudine (AZT) phosphorylation in chinese healthy people and in culcured cell lines.Methods:①Synthesize mono-di-and tri-phosphate of AZT.②Establish an anion exchange SPE(Solid-Phase Extraction) isolation of AZT phosphate coupled with dephosphorylation and desaltation LC-MS/MS method which can determin AZT and its mono-di-and tri-phosphate in plasma and mononuclear cells.③)12healthy male volunteers were taken part in the single-dose pharmacokinetic study. Dose was designed as600mg. Blood samples were collected from volunteers’ulnar vain about20mL at each point and centrifuged immediately accoding to the time schedule. Mononuclear cells were seperated by Ficoll-Hypaque density gradient centrifugation. Samples were determined by LC-MS/MS method.④Use cck-8method to investigate the cell toxicity of AZT to Molt-4and HUVECs cells in vitro and study the pharmacokinetics of AZT phosphorylation in cultured Molt-4and HUVECs cell lines.Results:①The chemical synthesis method can successfully synthetize zidovudine mono-, di-and tri-phosphate.②The LC-MS/MS method proved to be rapid, simple, specific and accurate for determination of zidovudine and its phosphates in human plasma and mononuclear cells and can be applied to pharmaceutical study.③In cultured Molt-4and HUVCEs cells, the main phosphorylated metabolite was zidovudine mono-phosphate. The amount of di-and tri-phosphate were far below that of mono-phosphate.④The main pharmacokinetic parameters of zidovudine in plasma were as follows:Tmax was0.583h, t1/2was2.022h, however Tmax was1.083h and t1/2was2.493h in cells. The main pharmacokinetic parameters of Zidovudine phosphate in cells were as follows:Tmax was1.5h and t1/2was13.428h for MP; Tmax was1.417h and t1/2was8.285h for DP; Tmax was1.583h and t1/2was4.24h for TP.Conclusions:Zidovudine was phosphorylated into mono-, di-and tri-phosphate after transportion into cells. Among these three metabolites, tri-phosphate was the active form and has longer t1/2than that of zidovudine in plasma. Thus zidovudine tri-phosphate can be used as the important indicators of therapeutic drug monitoring.