PSI-7851, a Pronucleotide of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methyluridine Monophosphate, Is a Potent and Pan-Genotype Inhibitor of Hepatitis C Virus Replication

Abstract

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of ␤-D-2-deoxy-2-fluoro-2-C-methyluridine-5-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075 ؎ 0.050 M (mean ؎ standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 M). Crossresistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.Hepatitis C virus (HCV) currently affects more than 170 million people worldwide. Approximately 70% of infected individuals develop chronic hepatitis, among whom about 20% will develop liver cirrhosis and fibrosis and up to 5% will progress to hepatocellular carcinoma (2). The current standard of care (SOC), which combines pegylated alpha interferon (PegIFN-␣) and ribavirin (RBV), has limited efficacy in providing a sustained virological response (SVR), especially in individuals with HCV genotype 1 (ϳ50%), the most prevalent genotype in Western countries (8,11,35). The impact of genetic diversity of HCV in patients receiving SOC therapy has been reviewed (26): SVR rates are higher in patients infected with genotype 2 or 3 (ϳ80%), patients infected with genotype 4 appear to have a slightly better SVR rate (ϳ60%) than patients infected with genotype 1, and patients infected with genotypes 5 and 6 may achieve an SVR at a level between those of genotypes 1 and 2/3. In addition to the variability in efficacy, the lengthy treatment (24 to 48 w...

Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus

“…The 5=-triphosphate metabolite of PSI-7977, PSI-7409, inhibits HCV replication by serving as a nonobligate chain terminator (30). Cross-resistance studies have shown that PSI-7977 and PSI-7409 had reduced activity against GT 1b replicons and NS5B polymerase containing the S282T amino acid alteration, respectively (18,38).In this study, we further characterized the anti-HCV activity of PSI-7977 across various genotypes and subtypes and expanded the cross-resistance studies to include a panel of replicons containing resistant mutations selected by other classes of HCV inhibitors. Selection was performed with GT 1b (Con1 strain), 1a (H77 strain), and 2a (JFH-1 strain) replicon cells in order to examine for the emergence of any genotype-and/or subtype-dependent resistant variants associated with PSI-7977.…”

“…Among the various NS5B mutations, we included representative amino acid changes (C316Y, M414T, M423T, P495L) that conferred resistance to the four main classes of NNIs (12), the S96T/N142T substitutions that conferred resistance to the 4=-azidocytidine nucleoside R1479 (1), the S282T substitution which conferred resistance to 2=-C-methylnucleosides/tides (29) and 2=-F-2=-C-methylpyrimidines (18,40), the S15G/C223H/V321I (JFH-1 GT 2a) changes that in combination conferred resistance to 2=-F-2=-C-methylguanosine analogs (15), and the F415Y (GT 1a) alteration that has previously been found in patients treated with ribavirin (47). As summarized in Table 2, the replicons with the C316Y, M414T, M423T, and P495L changes remained fully susceptible to the inhibition of PSI-7977, while showing a 26-to 70-fold reduction in sensitivity for the corresponding class of NNIs.…”

“…Compared to NNIs, nucleoside/tide inhibitors have broader genotype coverage and a higher barrier to viral resistance (9,24,28). Previously, we reported the anti-HCV activity of PSI-7851, a phosphoramidate nucleotide prodrug of 2=-F-2=-C-methyluridine monophosphate (18). PSI-7851 is a 1:1 mixture of two diastereoisomers, PSI-7976 and PSI-7977, at the phosphorus center of the phosphoramidate moiety.…”

“…The 5=-triphosphate metabolite of PSI-7977, PSI-7409, inhibits HCV replication by serving as a nonobligate chain terminator (30). Cross-resistance studies have shown that PSI-7977 and PSI-7409 had reduced activity against GT 1b replicons and NS5B polymerase containing the S282T amino acid alteration, respectively (18,38).…”

Ralf Bartenschlager, Charles Rice, and Michael Sofia are honored with the 2016 Lasker~DeBakey Clinical Medical Research Award

“…These efforts cumulated with the generation of PSI-7851, which showed specificity for HCV, pan-recognition of HCV genotypes, and low cytotoxicity (36). Moreover, PSI-7851 cleared HCV replicons from transfected cells, with no viral rebound after drug removal (37). In clinical safety and tolerability trials, PSI-7851 was shown to be safe and to have a pharmacokinetic profile favorable for once-a-day dosing and a decrease in plasma HCV RNA after 3 days of dosing in treatment-naive patients.…”