Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β-L-2′,3′-Didehydro-2′,3′-Dideoxy-N4-Hydroxycytidine: Relevance for Activation in HepG2 Cells

beta-L-2',3'-Didehydro-2',3'-dideoxy-N-4-hydroxycytidine (L-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED50] = 0.03 mu M; 50% cytotoxic dose [CD50] = 2,500 mu M). In the present study, we investigated the intracellular pharmacology of tritiated L-Hyd4C in HepG2 cells. L-[H-3] Hyd4C was shown to be phosphorylated extensively and rapidly to the 5'-mono-, 5'-di-, and 5'-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of L-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer beta-D-N-4-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5'-diphosphocholine derivative of L-Hyd4C, as described for some L-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 mu M L-[H-3] Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 +/- 2.7 pmol/10(6) cells. The predominant peak belonged to 5-diphosphate, with 43.5 +/- 4.3 pmol/10(6) cells. The intracellular half-life of the 5'-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5'-phosphorylated L-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5'-diphosphate during a drug-free incubation. The high 5'-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of L-Hyd4C.