Susan Obeid

Statins potentiate the in vitro anti‐hepatitis C virus activity of selective hepatitis C virus inhibitors and delay or prevent resistance development

Statins are 3‐hydroxyl‐3‐methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication. We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti‐HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect. A combination of statins with interferon‐alpha (IFN‐α) or HCV nonstructural (NS)5B polymerase or NS3 protease inhibitors results in an additive antiviral activity in short‐term (3 days) antiviral assays. Neither statins, at a concentration of five‐fold their median effective concentration (EC50) value, nor polymerase, protease inhibitors, or IFN‐α, at concentrations 10‐ or 20‐fold their EC50 value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure. However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon. In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV‐796. Conclusion: A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors. (HEPATOLOGY 2009.)

Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus

BACKGROUND & AIMS Worldwide, about 180 million people are chronically infected with the hepatitis C virus (HCV). Current in vitro culture systems for HCV depend chiefly on human hepatoma cell lines. Although primary human hepatocytes support HCV infection in vitro, and immunodeficient mice repopulated with human hepatocytes support HCV infection in vivo, these models are limited because of shortage of human livers to isolate hepatocytes. Therefore, there is significant interest in the establishment from of a HCV culture system in human stem cell-derived hepatocyte-like cells. METHODS Human embryonic stem cell (hESC)-derived hepatocytes were infected with HCV in the presence or absence of direct acting antivirals. After inoculation, replication of HCV was analyzed extensively. RESULTS We demonstrate that hESC-derived hepatocytes can be infected with the HCV JFH1 genotype 2a, resulting in the production of viral RNA in the stem cell progeny. Viral replication is inhibited by a non-nucleoside HCV polymerase-inhibitor (HCV-796), a cyclophilin binding molecule (Debio 025-Alisporivir) and the protease inhibitor VX-950 (Telaprevir). Stem cell-derived hepatocytes produced, for more than 10 days, the HCV core protein as well as virions that were capable of re-infecting hepatoma cells. CONCLUSIONS Hepatocytes derived from hESC support the complete HCV replication cycle (including the production of infectious virus), and viral replication in these cells is efficiently inhibited by selective inhibitors of HCV replication.

Preclinical Characterization of Naturally Occurring Polyketide Cyclophilin Inhibitors from the Sanglifehrin Family

ABSTRACT Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F < 4%) and low solubility (<25 μM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2 > 5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

Inhibition of hepatitis C virus replication by semi-synthetic derivatives of glycopeptide antibiotics

Abstract Objectives Some semi-synthetic derivatives of glycopeptide antibiotics have been shown to exert in vitro antiviral activity against HIV and coronaviruses. Here we report and characterize the in vitro anti-hepatitis C virus (HCV) activity of several semi-synthetic derivatives of teicoplanin aglycone. Methods Anti-HCV activity was analysed in: (i) three different subgenomic HCV replicon systems using a luciferase or quantitative RT–PCR (qRT–PCR) assay; and (ii) an infectious HCV cell culture system by means of qRT–PCR and immunofluorescence assays. Results Several teicoplanin aglycone derivatives elicited selective anti-HCV activity in replicons as well as infectious cell culture systems, with LCTA-949 being the most potent derivative. LCTA-949 proved, in contrast to several directly acting antivirals for HCV, efficient in clearing cells of their replicons. When LCTA-949 was combined with HCV protease or polymerase inhibitors an overall additive effect was observed. Likewise, LCTA-949 was equipotent against wild-type replicons as well as against replicons resistant to polymerase and protease inhibitors. Following up to 4 months of selective pressure, no drug-resistant replicons were selected. When combined with the HCV NS3 protease inhibitor VX-950, LCTA-949 prevented the development of VX-950-resistant variants. Conclusions Semi-synthetic derivatives of teicoplanin aglycone constitute a novel class of HCV replication inhibitors that are not cross-resistant with various HCV protease and polymerase inhibitors and in particular are potent in clearing hepatoma cells of their replicons. This class of molecules also provides a good tool to obtain novel insights into the replication cycle of HCV and into cellular factors/processes that are crucial for viral replication.

Artemisinin Analogues as Potent Inhibitors of In Vitro Hepatitis C Virus Replication

We reported previously that Artemisinin (ART), a widely used anti-malarial drug, is an inhibitor of in vitro HCV subgenomic replicon replication. We here demonstrate that ART exerts its antiviral activity also in hepatoma cells infected with full length infectious HCV JFH-1. We identified a number of ART analogues that are up to 10-fold more potent and selective as in vitro inhibitors of HCV replication than ART. The iron donor Hemin only marginally potentiates the anti-HCV activity of ART in HCV-infected cultures. Carbon-centered radicals have been shown to be critical for the anti-malarial activity of ART. We demonstrate that carbon-centered radicals-trapping (the so-called TEMPO) compounds only marginally affect the anti-HCV activity of ART. This provides evidence that carbon-centered radicals are not the main effectors of the anti-HCV activity of the Artemisinin. ART and analogues may possibly exert their anti-HCV activity by the induction of reactive oxygen species (ROS). The combined anti-HCV activity of ART or its analogues with L-N-Acetylcysteine (L-NAC) [a molecule that inhibits ROS generation] was studied. L-NAC significantly reduced the in vitro anti-HCV activity of ART and derivatives. Taken together, the in vitro anti-HCV activity of ART and analogues can, at least in part, be explained by the induction of ROS; carbon-centered radicals may not be important in the anti-HCV effect of these molecules.

789 PRECLINICAL CHARACTERIZATION OF NOVEL CYCLOPHILIN INHIBITORS BASED ON THE POLYKETIDE, SANGLIFEHRIN

Introduction: Progressive hepatic fibrosis is a common feature of chronic hepatitis C. Natural Killer (NK) cells represent a major component of intra-hepatic lymphocytes and have been shown to be important for the early control and natural course of HCV infection. Moreover, in mouse models it has been shown that natural killer cells can attenuate liver fibrosis via killing of activated hepatic stellate cells (HSC) in a NKG2D and TRAIL dependent manner. Here, we analyzed the interactions of human NK cells from HCVpositive patients with activated primary HSC and the potential effects of interferon-a (IFN-a) treatment. Material and methods: NK cells from untreated HCV-RNA(+) patients (n = 11), interferon-a (IFN-a) treated patients (n = 10) and healthy controls (n = 12) were co-incubated with activated primary HSC (ScienCell). NK cells from healthy persons were incubated in the presence or absence of IFN-a (25 IU/ml). Cytotoxic activity of NK cells was studied using the CD107a assay. In addition, INF-g and TNFa production of NK cells was measured by FACS analysis. Induction of HSC apoptosis (active Caspase-3) was analyzed flowcytometrically. Results: Analyzing cytolytic activity of NK cells following coincubation with HSC only discrete CD107a expression could be observed on both NK cells from HCV(+) patients and healthy controls ([mean±SEM: 5.7±0.7% vs. 4.7±0.8%; p =n.s.). Furthermore, only negligible secretion of IFN-g and TNF-a could be observed. However, NK cells from untreated HCV-infected patients were significantly more effective in induction of HSC apoptosis (20.8±2.1%) than NK cells from healthy controls (6.2±0.6%). Of note, NK cells from IFN-a/RBV treated HCV(+) patients displayed an even stronger capability to kill HSC (26.9±4.3%). In vitro stimulation of NK cells with IFN-a resulted in a significant up-regulation of TRAIL and was associated with increased killing of HSC as compared to un-stimulated NK cells. This effect could be blocked with both NKG2Dand TRAIL-specific antibodies. Conclusion: NK cells from HCV-infected patients are highly efficient in inducing apoptosis of activated hepatic stellate cells. This function of NK cell is increased following IFN-a treatment. Thus, NK cells may play an important anti-fibrotic role in chronic hepatitis C.