Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor β-D-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and identification of a novel active 5′-triphosphate species

beta- D- 2'- Deoxy- 2'- fluoro- 2'- C- methylcytidine ( PSI- 6130) is a potent inhibitor of hepatitis C virus ( HCV) replication in the subgenomic HCV replicon system, and its corresponding 5 '- triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI- 6130- triphosphate was characterized in primary human hepatocytes. PSI6130 and its 5 '- phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI- 6130, '- D- 2 '- deoxy- 2 '- fluoro2 '- C- methyluridine ( RO2433, PSI- 6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI- 6130. The formation of the 5 '- triphosphate ( TP) of PSI- 6130 ( PSI- 6130- TP) and RO2433 ( RO2433- TP) increased with time and reached steady state levels at 48 h. The formation of both PSI- 6130- TP and RO2433- TP demonstrated a linear relationship with the extracellular concentrations of PSI- 6130 up to 100 mu M, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half- lives of PSI- 6130- TP and RO2433- TP were 4.7 and 38 h, respectively. RO2433- TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinantHCVpolymerase NS5B with potencies comparable with those of PSI- 6130- TP. Incorporation of RO2433- 5 'monophosphate ( MP) into nascent RNA by NS5B led to chain termination similar to that of PSI- 6130- MP. These results demonstrate that PSI- 6130 is metabolized to two pharmacologically active species in primary human hepatocytes.