In vitro, telaprevir selects subtype-specific resistance pathways for hepatitis C virus GT-1a and GT-1b, as described to have occurred in patients. In GT-1a, the HCV-796 resistance mutation C316Y has low replication capacity (7%) that can be compensated for by the emergence of the mutation L392F or M414T, resulting in an increase in replication levels of >10-fold.The current standard of care for hepatitis C virus (HCV)-infected patients involves a treatment regimen of pegylated alpha interferon in combination with ribavirin, which results in a sustained viral response of approximately 50% for genotype 1 (GT-1)-infected patients (1, 11). There is a clear medical need for more efficacious therapies, and to this effect, a number of novel specific antiviral compounds are currently in preclinical and clinical development. A majority of these compounds inhibit the enzymatic activity of either the NS3/4A serine protease or the NS5B RNA-dependent RNA polymerase.One factor that may limit the clinical efficacy of specific HCV antiviral drugs is the development of resistance. HCV presents a number of features that make drug resistance likely to occur upon treatment, such as the following: (i) the NS5B polymerase lacks proofreading activity, which results in the introduction of random mutations during the replication of the genomic RNA; (ii) HCV replicates as a genetic population known as a quasispecies that allows quick adaptation of the viral population upon changes in the environment (12); (iii) HCV produces a large number of infectious particles (up to 10 12 ) per day, which means that each genetic variant made during RNA replication may be packaged into an infectious viral particle and can quickly spread (15); and (iv) the short half-life of the HCV genome, as estimated for the circulating virus (14) and calculated for the HCV replicon (3), is such that a variant present at low prevalence within the quasispecies can quickly become the dominant sequence if it offers a selective advantage. Resistance to specific HCV inhibitors in vitro has been well characterized through the use of the HCV GT-1b replicon system, and these studies have been predictive of the amino acid substitution(s) selected in HCV-infected patients upon drug treatment (4, 7-10, 13). For example, for the NS3/4A protease inhibitor telaprevir and the nonnucleoside polymerase inhibitor HCV-796, the resistance mutations identified in vitro (NS3 substitutions at residues T54 and A156 for telaprevir and an NS5B substitution at residue C316 for HCV-796) were also identified in GT-1b-treated patients (4, 5, 16).One limitation of the majority of the replicon resistance studies reported to date is that only a single HCV subtype, GT-1b, has been used. HCV subtypes can vary by up to 25% at the nucleotide level, and this variability may lead to subtypespecific differences in the resistance profiles. In fact, subtypespecific resistance profiles for HCV-infected patients treated with telaprevir have been described previously. Substitutions at NS3 residues V36 and R155...

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-a) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-a, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-a-specific effect, which was not observed with IFN-c, correlated with an increase in expression of the IFN-a-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-a-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-a after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-a-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-a treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-a pathway and its antiviral effect.

Pure naproxen particles and mixed naproxen-polylactic acid particles were formed by pulsed rapid expansion of supercritical CO2 solutions (RESS). The characterization of the particles was carried out in the aerosol phase as a function of time after particle formation with rapid-scan infrared spectroscopy and with an aerosol particle sizer. After collection of the particles from the aerosol phase, the particles were characterized with scanning electron microscopy and powder diffraction. The present study shows clear evidence that RESS is capable of forming sub-micron naproxen particles that are coated with polylactic acid. Furthermore, it is demonstrated that coating with polylactic acid stabilizes the naproxen particles against agglomeration and coagulation.

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