Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β- l -2′,3′-Didehydro-2′,3′-Dideoxy-N 4 -Hydroxycytidine: Relevance for Activation in HepG2 Cells

ß-l -2′,3′-Didehydro-2′,3′-dideoxy-N4 -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 μM; 50% cytotoxic dose [CD50 ] = 2,500 μM). In the present study, we investigated the intracellular pharmacology of tritiatedl -Hyd4C in HepG2 cells.l -[3 H]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 ofl -Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ß-d -N4 -hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5′-diphosphocholine derivative ofl -Hyd4C, as described for somel -deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 μMl -[3 H]Hyd4C for 24 h, the 5′-triphosphate accumulated to 19.4 ± 2.7 pmol/106 cells. The predominant peak belonged to 5-diphosphate, with 43.5 ± 4.3 pmol/106 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′-phosphorylatedl -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 ofl -Hyd4C.