HCV-796

2 years -20°C Powder, 2 weeks 4°C in DMSO, 2 months -80°C in DMSO

Administrated orally

Clone A cells which are derived from Huh-7 cells

0-100 nM

HCV-796, is dissolved in DMSO.

Besides HCV RNA, treatment of HCV-796 is associated with a concomitant decrease in the level of HCV protein (EC50 for genotype 1a = 19 nM, and for genotype 1b = 14 nM). Multiple treatments with HCV-796 in replicon-containing cells result in a 3 to 4 log10 reduction in HCV RNA levels, as compared with 2 to 3, log10 reduction with interferon. No adverse impact is observed for cellular functions at effective antiviral concentrations, a therapeutic index of greater than 1100 is demonstrated. [1], HCV-796 is a nonnucleoside inhibitor which binds to a site in the palm domain of the HCV polymerase. In vitro replicon resistance studies indentifies substitutions at NS5B residues C316Y/F/S, S365T/A/L, and M414I, which are located near the inhibitor binding site and confer a loss of sensitivity to HCV-796. [2] HCV-796 shows slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 +/-2 nM. Slow binding kinetics of HCV-796 is driven by slow dissociation from NS5B with a, Koff of 4.9 +/- 0.5, 10-4/s. [3]

3 days

Clone A cells (licensed from APATH, LLC) are derived from Huh-7 cells, a human hepatoma cell line. The Clone A cells contain approximately 1000 genome copies of HCV genotype 1b replicon per cell when maintained in a subconfluent monolayer in the presence of 1 mg/ml GeneticinTM (G418 sulfate). The sequence of the replicon in Clone A cells is similar to that of the Genotype 1b, Con 1 strain of HCV (GenBank accession no. AJ238799) with the exception of two mutations in NS3 (Q1112R) and NS5A (S2204I). Clone A cells are propagated in Dulbecco's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with 1% penicillin/streptomycin, 1% non-essential amino acids, 1 mg/ml G418, and 0.66 mM HEPES buffer, pH 7.5. The plasmid pBB7 containing the HCV genotype 1b, BB7 replicon complementary DNA is also licensed from APATH, LLC (GenBank accession no. for 1b, BB7 or con 1 is AJ238799). The coding sequence of pBB7 is similar to that of the genotype 1b, Con 1 strain of HCV except one nucleotide mutation resulting in an amino acid change of S2204I within NS5A. Drug susceptibility of the replicon-containing Huh7 cells to HCV-796 is evaluated as described following. Briefly, cells are treated with increasing concentrations of HCV-796, in medium containing 2% FCS and no G418 for three days at 37 C and 5% CO2. After incubation, total RNA from the replicon-containing cells is isolated. The levels of HCV, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ribosomal (rRNA) RNAs are quantified using TaqMan reverse transcriptase PCR assays. The amounts of HCV, 18S ribosomal, and GAPDH RNAs in each sample are estimated by comparing the number of cycles during the exponential phase of the PCR amplification with those in the corresponding standard curves. HCV RNA standards used for the standard curve are prepared by extracting the total RNA from the replicon-containing (Clone A) cells. The RNA sample is sent to National Genetics Institute to quantify HCV RNA. Total RNA extracted from the Clone A cells is quantified by O.D.260 measurement and used for construction of the standard curves of rRNA and GAPDH. The concentrations of the HCV-796 that inhibit 50% of the HCV RNA level (EC50) are determined using the MDL LSW Data AnalysisTM software in Microsoft Excel TM. The amounts of HCV or GAPDH RNAs in the samples are expressed as HCV RNA (copies) or GAPDH (ng), respectively, per ng of total RNA using rRNA as a marker for total RNA measurement.

5-cyclopropyl-2-(4-fluorophenyl)-6-(N-(2-hydroxyethyl)methan-4-ylsulfonamido)-N-methylbenzofuran-3-carboxamide