Biochemical-pharmacology of two L-nucleoside, beta-L-thymidine and beta-L-deoxycytidine

β-L-thymidine (β-L-dT) and (β-L-2′ -deoxycytidine (β-L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (EC₅₀ 0.19–0.24 μM in 2.2.15 cells). The intracellular metabolism of β-L-dT and β-L-dC was investigated in HepG2 cells and primary cultured human hepatocytes. Both β-L-dT and β-L-dC were extensively phosphorylated to β-L-dTTP and β-L-dCTP being the predominant metabolites in both cell types. Furthermore, a choline derivative of β-L-dCDP was also detected. The 5′-mono-, di- and triphosphate derivatives of β-L-2′deoxyuridine (β-L-dUMP, β-L-dUDP and β-L-dUTP, respectively) were also formed intracellularly in HepG2 cells and human hepatocytes after the administration of β-L-dC. It is likely that deamination of β-L-dCMP leads to the formation of β-L-dUMP by deoxycytidylate deaminase since the parent compound β-L-dC was not a substrate for cytidine deaminase. The intracellular half-lives of the β-L-dTTP, β-L-dCTP and β-LdUTP were at least 15 h in HepG2 with intracellular concentrations of each remaining above their respective 50% inhibitory concentrations (IC₅₀) for HBV polymerase for as long as 24 h after removal of the drug. Exposure of HepG2 cells to the combination of β-L-dT and β-L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-hepatitis B activity of β-L-dT and β-L-dC may be the result of their extensive phosphorylation. Our studies with specific inhibitors of dCK and TK, as well as with TK− and dCK− cell lines indicate that both kinases are involved in the initial phosphorylation of β-L-dT to β-Ld-TMP with further phosphorylation to β-L-dTTP. The involvement of dCK and TK1 in β-L-dT may be advantageous for therapy as phosphorylation of β-L-dT by TK1 can occur in actively proliferating cells (S phase of the cell cycle) when its activity is the highest and by dCK in quiesent cells where TK activity is limited. In the case of β-L-dC, dCK appears to be the only enzyme involved in its phosphorylation to the 5 ′-monophosphate. β-L-dT and β-L-dC are the first two compounds reported with potent, selective and specific activity against HBV replication. At the present β-L-dT is in Phase I/II clinical trials. β-L-dC is in preclinical studies and is expected to enter Phase I/II clinical trials in 2001.