Abstract
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. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-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 mum, 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 recombinant HCV polymerase 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.
Highlights
Titis C virus (HCV),2 the most predominant genotype in the United States and Europe (2– 4)
The incorporation of the nucleotide analogs into nascent HCV RNA strongly reduces the efficiency of further RNA elongation by NS5B, 2 The abbreviations used are: HCV, hepatitis C virus; MP, 5Ј-monophosphate; DP, 5Ј-diphosphate; TP, 5Ј-triphosphate; High Performance Liquid Chromatography (HPLC), high performance liquid chromatography
Metabolic Profile of PSI-6130—Cellular extracts from primary human hepatocytes incubated with tritium-labeled -D-2Ј-deoxy-2Ј-fluoro-2Ј-C-methylcytidine (PSI-6130) were resolved by ion exchange HPLC
Summary
Compounds—-D-2Ј-Deoxy-2Ј-fluoro-2Ј-C-methylcytidine (PSI-6130) was provided by Pharmasset, Inc. (18). The inhibition of the RNA synthesis activity of the HCV replicases by PSI-6130-TP was determined as described (6) except that 5 l of cytoplasmic replicase complex (2.5 ϫ 106 replicon cell equivalent) was added to a 20-l reaction for 60 min. HCV Polymerase Assay—The inhibition potency of PSI6130-TP on the RNA-dependent RNA polymerase activity of recombinant NS5B570-Con (genotype 1b, GenBankTM accession number AJ242654) was measured as the incorporation of radiolabeled nucleotide monophosphate into acid-insoluble RNA products as described (6) with the following modifications; IC50 determinations were carried out using 200 nM in vitro transcribed complementary internal ribosome entry site RNA template, 1 Ci of tritiated UTP (42 Ci/mmol), 500 M ATP, 500 M GTP, 1 M CTP, 1ϫ TMDN buffer (40 mM TrisHCl, pH 8.0, 4 mM MgCl2, 4 mM dithiothreitol, 40 mM NaCl) and 200 nM NS5B570-Con. The radiolabeled RNA products were separated on a TBE-urea acrylamide gel and analyzed using phosphorimaging (GE Healthcare)
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