- Email: [email protected]
4A PROTEASE
POSTERS essentially hepatotropic, recent studies suggest that it can also infect peripheral blood mononuclear cells (PBMC). HCV infection has been clinically associated with serum lipid abnormalities. The aim of this study is to analyze the possible roles of genes involved in HCV-mediated lipid metabolism in PBMC from HCV infected patients. Materials and Methods: Using The Human Adipogenesis RT2 Profiler™ PCR Array, we have analyzed the expression profile of 84 genes in chronic HCV PBMCs patients normalized with PBMCs from heathy donors. To compared the results obtained from HCV+ PBMCs with the expression in the hepatocytes, we have used a JFH1/HCV in vitro system after 48h post infection normalized with uninfected Huh 7.5.1 cell line. Results: Both in vivo and in vitro models HCV was able to upregulate the same genes (14% in both of them) involved in lipid metabolism such as SREBP1c, FASN, FABP4, ACACB and in signal transduction (29% and 27% respectively) resulted modulated both in HCV PBMCs and J6/JFH1-infection system. In both models the Kruppel-like ¨ transcription factor 15 (KLF15). KLF15 results significantly upregulated in HCV+ PBMCs as well as the J6/JFH1 in vitro infection. In addition, in PBMCs, KLF15 upregulation is statistically and positively correlated to the Body Weight, the BMI and the ALT levels. Furthermore, in PBMCs from HCV+ donors, KLF15 expression correlates with the HCV Viral Load and the antibody titer (anti-NS3 anti-NS4 and anti-capsid), suggesting a possible intriguing role of this factor in HCV infection. Conclusions: Our results confirm the ability of HCV to affect lipid metabolism; we have highlighted a similar modulation both in HCV patients lymphoid cells and in J6/JFH1 HCV infection model. In particular, the virus-related KLF15 enhanced expression, in both in vivo and in vitro models, suggests this protein as new possible marker of HCV infection and as a potential therapeutic target useful to counteract HCV infection. 1168 BL-8030: A NOVEL, POTENT, SELECTIVE, ORALLY AVAILABLE INHIBITOR OF HEPATITIS C VIRUS NS3/4A PROTEASE P. Halfon1 , J. Courcambeck1 , T. Whitaker2 , P.M. Tharnish2 , T.R. McBrayer2 , S.J. Coats2 , Y. Pereg3 , R. Tabakman3 , J. Schumann3 , M. Matto3 , R.F. Schinazi4 . 1 Genoscience, Marseille, France; 2 RFS Pharma LLC, Tucker, GA, USA; 3 BioLineRx Ltd., Jerusalem, Israel; 4 Department of Pediatrics, Center for AIDS Research, Laboratory of Biochemical Pharmacology, Emory University School of Medicine, and Veterans Affairs Medical Center, Decatur, GA, USA E-mail: [email protected] Background and Aims: Hepatitis C Virus (HCV) NS3/4A protease inhibitors (PI) are likely to be a key component of future combination therapies for patients with chronic HCV infections. These agents demonstrate dramatic antiviral effects, but tend to be genotype 1 specific and associated with unpleasant adverse effects. Furthermore, PIs have demonstrated a high risk for development of resistant virus. Based on structure-based drug design and NS3 molecular resistance mechanisms, a set of peptidomimetic NS3/4A protease inhibitors were synthesized. Here, we report the pharmacodynamic and pharmacokinetic profile of BL-8030 (formerly GNS-227), a low-nanomolar inhibitor of HCV. Methods: The antiviral activity and selectivity of BL-8030 was assessed versus: 1. a Huh-7 cell based HCV replicon assay; 2. wild type and PI mutant enzymes and 3. a panel of human proteases to demonstrate specificity. Human cytochrome P450 enzyme inhibition and induction were also evaluated. The plasma pharmacokinetics (PK) and plasma to liver partition of BL-8030 following intravenous or oral administration in rats were evaluated.
Results: BL-8030 demonstrated excellent potency, in the low nanomolar range, in HCV replicon genotypes 1a, 1b and 2a. Its pan-genotypic activity was tested on a panel of NS3 enzymes using a biochemical fluorometric protease assay. BL-8030 demonstrated potent activity against genotypes 1a, 1b, 2a and 4. BL-8030 was also potent against the main, clinically relevant resistance mutations. BL-8030 showed several thousand-fold selectivity relative to a panel of human proteases. PK studies in rats indicated good oral bioavailability after single 10 and 100 mg/kg dosing (17.4 and 33.7%). Following oral administration, BL-8030 was distributed in the liver with a liver to plasma ratio ranging from 28.4 to 45.4 (24hr and 12hr post dosing). Importantly, BL-8030 levels in the liver 24 hours post dosing were above its EC50 suggesting the potential for once a day dosing in the clinic. Conclusions: BL-8030 is a novel, highly potent and specific HCV NS3/4A protease inhibitor as demonstrated by cellular and biochemical assays and has the potential to prevent and treat emerging resistance variants. BL-8030 has a favorable pharmacological and safety profile with good oral bioavailability, liver distribution and the potential for once daily dosing. 1169 GENERATION OF PERMISSIVE BCLC5 CELL LINES FOR THE STUDY OF HCV REPLICATION: ROLE OF miR122 IN REPLICATION ENHANCEMENT 2 M. Coto-Llerena1 , G. Koutsoudakis1 , L. Boix2 , J.M. Lopez-Oliva ´ , 1 1 2 1 C. Fernandez-Carrillo ´ , P. Gonzalez , J. Bruix , X. Forns , S. Perez-del-Pulgar1 . 1 Liver Unit, 2 BCLC Group, Liver Unit, IDIBAPS, Ciberehd, Hospital Clinic, Barcelona, Spain E-mail: sofi[email protected] Robust hepatitis C virus (HCV) cell-culture propagation is restricted almost exclusively to the human hepatoma cell line Huh7 and its selected subclones, which are highly permissive for HCV replication; e.g., Huh-7.5 cells. Recent studies suggest that host factors such as innate immunity, gene polymorphisms, and/or miR122 are associated with the permissiveness of cells for HCV infection. We have recently characterized the BCLC5 cell line, which is derived from human hepatocellular carcinoma, and which can support subgenomic (SGR) HCV RNA replication for prolonged periods of time. In order to obtain highly permissive cell lines for HCV replication, clonal BCLC5 cells-(designated BCLC5-C8 cells) harboring a selectable neomycin (neo)-SGR were cured of HCV RNA with IFN-alpha treatment (designated BCLC5-C8.1 cells). In vitro transcribed neo-SGR RNA was then electroporated into the cured BCLC5-C8.1 cells and transduction efficiencies were determined 21 days post-G418 selection by counting the resulting colonies. Colony formation efficiency increased in BCLC5-C8.1 cells compared to their parental BCLC5 or Huh-7.5 cells (6- and 3-fold, respectively). Similarly, short-term replication efficiency was increased in BCLC5C8.1 cells as deduced by the reporter activity of a SGR-carrying Gaussia luciferase (GLuc-SGR). Since miR122 has been shown to facilitate HCV replication, we assessed miR122 expression in the BCLC5 cell line and its derivatives by real time qRTPCR. Neither BCLC5, BCLC5-C8, nor BCLC5-C8.1 cells expressed endogenous miR122. Therefore, we examined whether ectopic miR122 expression could enhance HCV replication in BCLC5-C8.1 cells. We then selected one clone that expressed miR122 at comparable levels to Huh-7.5 cells and analyzed HCV replication in these cells. BCLC-5 C8.1/mir122 cells exhibited a 3- to 4fold increase in GLuc-SGR replication as deduced by reporter secretion and intracellular HCV RNA determination. Similar results were obtained upon electroporation of full-length HCV RNA. In conclusion, we have established two new cell lines, BCLC5-C8.1 and BCLC5-C8.1/miR122, which are permissive for HCV replication. Our results reveal that miR122 is not essential for HCV replication;
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Results: BL-8030 demonstrated excellent potency, in the low nanomolar range, in HCV replicon genotypes 1a, 1b and 2a. Its pan-genotypic activity was tested on a panel of NS3 enzymes using a biochemical fluorometric protease assay. BL-8030 demonstrated potent activity against genotypes 1a, 1b, 2a and 4. BL-8030 was also potent against the main, clinically relevant resistance mutations. BL-8030 showed several thousand-fold selectivity relative to a panel of human proteases. PK studies in rats indicated good oral bioavailability after single 10 and 100 mg/kg dosing (17.4 and 33.7%). Following oral administration, BL-8030 was distributed in the liver with a liver to plasma ratio ranging from 28.4 to 45.4 (24hr and 12hr post dosing). Importantly, BL-8030 levels in the liver 24 hours post dosing were above its EC50 suggesting the potential for once a day dosing in the clinic. Conclusions: BL-8030 is a novel, highly potent and specific HCV NS3/4A protease inhibitor as demonstrated by cellular and biochemical assays and has the potential to prevent and treat emerging resistance variants. BL-8030 has a favorable pharmacological and safety profile with good oral bioavailability, liver distribution and the potential for once daily dosing. 1169 GENERATION OF PERMISSIVE BCLC5 CELL LINES FOR THE STUDY OF HCV REPLICATION: ROLE OF miR122 IN REPLICATION ENHANCEMENT 2 M. Coto-Llerena1 , G. Koutsoudakis1 , L. Boix2 , J.M. Lopez-Oliva ´ , 1 1 2 1 C. Fernandez-Carrillo ´ , P. Gonzalez , J. Bruix , X. Forns , S. Perez-del-Pulgar1 . 1 Liver Unit, 2 BCLC Group, Liver Unit, IDIBAPS, Ciberehd, Hospital Clinic, Barcelona, Spain E-mail: sofi[email protected] Robust hepatitis C virus (HCV) cell-culture propagation is restricted almost exclusively to the human hepatoma cell line Huh7 and its selected subclones, which are highly permissive for HCV replication; e.g., Huh-7.5 cells. Recent studies suggest that host factors such as innate immunity, gene polymorphisms, and/or miR122 are associated with the permissiveness of cells for HCV infection. We have recently characterized the BCLC5 cell line, which is derived from human hepatocellular carcinoma, and which can support subgenomic (SGR) HCV RNA replication for prolonged periods of time. In order to obtain highly permissive cell lines for HCV replication, clonal BCLC5 cells-(designated BCLC5-C8 cells) harboring a selectable neomycin (neo)-SGR were cured of HCV RNA with IFN-alpha treatment (designated BCLC5-C8.1 cells). In vitro transcribed neo-SGR RNA was then electroporated into the cured BCLC5-C8.1 cells and transduction efficiencies were determined 21 days post-G418 selection by counting the resulting colonies. Colony formation efficiency increased in BCLC5-C8.1 cells compared to their parental BCLC5 or Huh-7.5 cells (6- and 3-fold, respectively). Similarly, short-term replication efficiency was increased in BCLC5C8.1 cells as deduced by the reporter activity of a SGR-carrying Gaussia luciferase (GLuc-SGR). Since miR122 has been shown to facilitate HCV replication, we assessed miR122 expression in the BCLC5 cell line and its derivatives by real time qRTPCR. Neither BCLC5, BCLC5-C8, nor BCLC5-C8.1 cells expressed endogenous miR122. Therefore, we examined whether ectopic miR122 expression could enhance HCV replication in BCLC5-C8.1 cells. We then selected one clone that expressed miR122 at comparable levels to Huh-7.5 cells and analyzed HCV replication in these cells. BCLC-5 C8.1/mir122 cells exhibited a 3- to 4fold increase in GLuc-SGR replication as deduced by reporter secretion and intracellular HCV RNA determination. Similar results were obtained upon electroporation of full-length HCV RNA. In conclusion, we have established two new cell lines, BCLC5-C8.1 and BCLC5-C8.1/miR122, which are permissive for HCV replication. Our results reveal that miR122 is not essential for HCV replication;
Journal of Hepatology 2013 vol. 58 | S409–S566
S475