Restoration of the activated Rig-I pathway in hepatitis C virus (HCV) replicon cells by HCV protease, polymerase, and NS5A inhibitors in vitro at clinically relevant concentrations.

Development of persistent hepatitis C virus (HCV) infection may be mediated by HCV NS3 · 4A protease-dependent inhibition of host innate immunity. When double-stranded RNA (dsRNA) is detected in virus-infected cells, host innate immunity mounts an antiviral response by upregulating production of type I interferons (α/ß interferon [IFN-α/ß]); HCV counters by cleaving the IFN-ß stimulator 1 (IPS-1) adaptor protein, decreasing synthesis of IFN-α/ß. We evaluated HCV protease (telaprevir, boceprevir, and TMC435350), polymerase (HCV-796 and VX-222), and NS5A (BMS-790052) inhibitors for the ability to restore IPS-1-mediated Rig-I signaling by measuring Sendai virus-induced IFN-ß promoter activation in HCV replicon cells after various exposure durations. All direct-acting HCV antivirals tested restored mitochondrial localization of IPS-1 and rescued Sendai virus-induced IRF3 signaling after 7 days by inhibiting HCV replication, thereby reducing the abundance of HCV NS3 · 4A protease. With 4-day treatment, HCV protease inhibitors, but not polymerase inhibitors, restored mitochondrial localization of IPS-1 and rescued IFN-ß promoter activation in the presence of equivalent levels of NS3 protein in protease or polymerase inhibitor-treated cells. The concentrations of HCV protease and polymerase inhibitors needed to rescue IRF3-mediated signaling in vitro were in the range of those observed in vivo in the plasma of treated HCV patients. These findings suggest that (i) HCV protease, polymerase, and NS5A inhibitors can restore virus-induced IRF3 signaling by inhibiting viral replication, thereby reducing NS3 protease levels, and (ii) HCV protease inhibitors can restore innate immunity by directly inhibiting NS3 protease-mediated cleavage of IPS-1 at clinically achievable concentrations.

Development and validation of a multi-dose neutropenic rat thigh infection model using real-time monitoring of Staphylococcus aureus growth in vivo.

BACKGROUND: Several animal models have been described using luminescent bacteria for non-invasive, real-time monitoring of infection in mice. In this study, a multidose rat thigh infection model with luminescent Staphylococcus aureus was developed for the evaluation of antibiotic efficacy. MATERIALS AND METHODS: Bioluminescent imaging and bacterial loads of S. aureus infected rat thighs with or without vancomycin treatment at different time-points post-infection were compared. RESULTS: Correlation between luminescence and bacterial load was observed based on the dose- and time-dependent activity of vancomycin in the model. CONCLUSION: While luminescence detection offered the advantage of monitoring an infection in live animals, limitations to this method included reduced sensitivity and a narrow dynamic range, as compared to a traditional tissue culturing method. Real-time luminescence monitoring of infection may be most appropriate for experiments where rapid in vivo assessment of compound efficacy is desired and absolute quantitation of colony forming units in infected tissue is not required.

Preclinical profile of VX-950, a potent, selective, and orally bioavailable inhibitor of hepatitis C virus NS3-4A serine protease.

VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (K(i)*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.