. One approach to increase the antiviral efficacy of 2-C-methylcytidine is to increase the concentration of the active inhibitory species, the 5-triphosphate, in infected hepatocytes. HepDirect prodrug technology can increase intracellular concentrations of a nucleoside triphosphate in hepatocytes by introducing the nucleoside monophosphate into the cell, bypassing the initial kinase step that is often rate limiting. Screening for 2-C-methylcytidine triphosphate levels in rat liver after oral dosing identified 1-[3,5-difluorophenyl]-1,3-propandiol as an efficient prodrug modification. To determine antiviral efficacy in vivo, the prodrug was administered separately via oral and intravenous dosing to two HCV-infected chimpanzees. Circulating viral loads declined by ϳ1.4 log 10 IU/ml and by >3.6 log 10 IU/ml after oral and intravenous dosing, respectively. The viral loads rebounded after the end of dosing to predose levels. The results indicate that a robust antiviral response can be achieved upon administration of the prodrug.Chronic hepatitis C virus (HCV) infection is associated with increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation in the United States (13). The current standard for treatment is a combination of pegylated interferon alpha and ribavirin, which results in sustained viral response (SVR) (undetectable virus 6 months after the end of treatment) in 50% or 80% of treated patients harboring a genotype 1 or a genotype 2 or 3 infection, respectively (25). Thus, novel therapies resulting in higher SVR rates, particularly in the treatment of genotype 1 infections, are needed.Nucleoside analogs inhibiting the HCV RNA polymerase have been investigated as potential therapeutics to treat HCV infections. 2Ј-C-Methylcytidine inhibits HCV RNA replication in the replicon assay (consensus 1 50% effective concentration [EC 50 ], ϳ1 M) via inhibition of the HCV RNA polymerase, which is inhibited by the 2Ј-C-methylcytidine triphosphate in vitro in cell-free biochemical assays (50% inhibitory concentration [IC 50 ], ϳ0.2 M) (15). NM283, the 3Ј-O-valinyl ester prodrug of 2Ј-C-methylcytidine, has improved oral bioavailability compared to 2Ј-C-methylcytidine (21) and has been investigated in HCV-infected patients in clinical studies. In a phase IIb study, patients receiving NM283 in combination with pegylated interferon alpha and ribavirin at 24 weeks of therapy experienced mean viral load reductions of up to 3.3 log 10 IU/ml compared to 2.3 log 10 IU/ml reductions for patients receiving pegylated interferon-ribavirin alone (2). Due to the high incidence of gastrointestinal side effects, which required reduction of the highest daily dose of 800 mg to 200 or 400 mg, further development of NM283 was placed on hold.The HepDirect prodrug modifications of nucleoside 5Ј-monophosphates (NMP) are substituted cyclic 1,3-propanyl esters that offer an opportunity to bypass the initial phosphorylation step that is often rate limiting for the formation of...