Preclinical Characterization of PF-00868554, a Potent Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Abstract

PF-00868554 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, which exerts its inhibitory effect by binding to the thumb base domain of the protein. It is a potent and selective inhibitor, with a mean 50% inhibitory concentration of 0.019 M against genotype 1 polymerases and a mean 50% effective concentration (EC 50 ) of 0.075 M against the genotype 1b-Con1 replicon. To determine the in vitro antiviral activity of PF-00868554 against various HCV strains, a panel of chimeric replicons was generated, in which polymerase sequences derived from genotype 1a and 1b clinical isolates were cloned into the 1b-Con1 subgenomic reporter replicon. Our results indicate that PF-00868554 has potent in vitro antiviral activity against a majority (95.8%) of genotype 1a and 1b replicons, with an overall mean EC 50 of 0.059 M. PF-00868554 showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 M). Furthermore, the antiviral activity of PF-00868554 was retained in the presence of human serum proteins. An in vitro resistance study of PF-00868554 identified M423T as the predominant resistance mutation, resulting in a 761-fold reduction in susceptibility to PF-00868554 but no change in susceptibility to alpha interferon and a polymerase inhibitor that binds to a different region. PF-00868554 also showed good pharmacokinetic properties in preclinical animal species. Our results demonstrate that PF-00868554 has potent and broadspectrum antiviral activity against genotype 1 HCV strains, supporting its use as an oral antiviral agent in HCV-infected patients.

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

“…Baseline prevalence of NS5B allosteric thumb site inhibitor variants. Clinical candidates VCH-759, VX-222, and filibuvir (PF-00868554), bind allosterically in the thumb region of NS5B, and the resistance profiles, including early clinical resistance data, have been reported (10,49,50). Variants selected clinically during a 3-day dosing study of VX-222 were at NS5B 419, 422, 423, 482, 486, and 494 (51); however, in the ZENITH study in patients that had virologic breakthrough during treatment with VX-222 and telaprevir the NS5B variants observed were L419S and R422K (52).…”

“…Thus, it is likely that all patients have DAA-resistant variants that require one or two mutations present at some level prior to treatment, albeit usually transient and below the current detection limits, since they are typically less fit than drug-sensitive (wild-type [WT]) virus. Indeed, drug-resistant substitutions have been shown to emerge in vitro and in vivo with all classes of DAAs after inhibition of the drug-sensitive virus population or after treatment failure (7)(8)(9)(10).…”

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

“…Further assessment of these analogs, using enzyme isolates and intergenotypic chimeric replicons derived from clinical iso-lates, revealed that the genotypic coverage of the NNI-1 and -4 analogs extends beyond genotype 1, unlike the NNI-2 and -3 derivatives that typically inhibit genotype 1 only (16,38). An additional drawback stems from the lower genetic barrier of the NNI-2 and -3 analogs in genotype 1 (25) and the reduced susceptibility in subtype 1a of the NNI-3 series (7,16,34,38,43). This effect was mostly attributed to the Y415F polymorphism observed in the NNI-3 binding site in subtype 1a (38).…”

New developments in small molecular compounds for anti-hepatitis C virus (HCV) therapy

“…An in vitro resistance study of filibuvir identified M423T as the predominant resistance mutation, resulting in a 761-fold reduction in susceptibility to filibuvir, but no change in susceptibility to IFN-α and a polymerase inhibitor that binds to a different region. Filibuvir also showed good pharmacokinetic properties in preclinical animal species, revealing promising oral bioavailability in both rodent and nonrodent species (Shi et al, 2009). Also, filibuvir seemed not to affect any of the major CYP isoforms (IC 50 >30 µmol/L for 1A2, 2C8, 2D6, 3A4, 2C9, and 2C19) .…”