Hernandez-Santiago B, Placidi L, Cretton-Scott E, Faraj A, Bridges E G, Bryant M L, Rodriguez-Orengo J, Imbach J L, Gosselin G, Pierra C, Dukhan D, Sommadossi J P
Department of Pharmacology, University of Alabama at Birmingham, USA.
Antimicrob Agents Chemother. 2002 Jun;46(6):1728-33. doi: 10.1128/AAC.46.6.1728-1733.2002.
beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.
β-L-胸苷（L-dT）和β-L-2'-脱氧胞苷（L-dC）在体内和体外都是乙型肝炎病毒（HBV）复制的强效且高度特异性抑制剂（在2.2.15细胞中的50%有效浓度为0.19至0.24微摩尔）。在HepG2细胞和原代培养的人肝细胞中研究了L-dT和L-dC的细胞内代谢情况。L-dT和L-dC在这两种细胞类型中都被广泛磷酸化，5'-三磷酸衍生物是主要代谢产物。在HepG2细胞中，L-dT和L-dC的5'-三磷酸水平分别为27.7±12.1和72.4±1.8皮摩尔/10⁶个细胞。在原代人肝细胞中，L-dT和L-dC的5'-三磷酸水平分别为16.5±9.8和90.1±36.4皮摩尔/10⁶个细胞。此外，在人肝细胞和HepG2细胞中分别检测到L-dCDP的胆碱衍生物浓度为15.8±1.8和25.6±0.1皮摩尔/10⁶个细胞。在暴露于L-dC的HepG2细胞中，还观察到β-L-2'-脱氧尿苷的5'-单磷酸和5'-三磷酸衍生物（分别为L-dUMP和L-dUTP），其细胞内浓度分别达到6.7±0.4和18.2±1.0皮摩尔/10⁶个细胞。在人肝细胞中，L-dUMP和L-dUTP的检测浓度分别为5.7±2.4和43.5±26.8皮摩尔/10⁶个细胞。很可能是脱氧胞苷酸脱氨酶将L-dCMP脱氨导致形成L-dUMP，因为母体化合物L-dC不是脱氧胞苷脱氨酶的底物。L-dTTP、L-dCTP和L-dUTP的细胞内半衰期至少为15小时，在从细胞培养物中去除药物后长达24小时，每种代谢产物的细胞内浓度都保持在高于它们对土拨鼠肝炎病毒DNA聚合酶各自50%抑制浓度的水平。将HepG2细胞暴露于L-dT与L-dC的组合中，导致活化代谢产物的浓度与单独使用任何一种药物时达到的浓度相似。这些结果表明，L-dT和L-dC的强效抗HBV活性与其广泛磷酸化有关。
