Pharmacology of β-l-Thymidine and β-l-2′-Deoxycytidine in HepG2 Cells and Primary Human Hepatocytes: Relevance to Chemotherapeutic Efficacy against Hepatitis B Virus

β-l-Thymidine (l-dT) and β-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 μM 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/106 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/106 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/106 cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to l-dC, the 5′-monophosphate and 5′-triphosphate derivatives of β-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/106 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/106 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.