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

Abstract

ABSTRACT 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/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 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/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.