Comparison of Daclatasvir Resistance Barriers on NS5A from Hepatitis C Virus Genotypes 1 to 6: Implications for Cross-Genotype Activity

Abstract

A comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b > 4a > 5a > 6a Х 1a > 2a JFH > 3a > 2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC 50 s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC 50 s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC 50 s ranging from 2 to 250 nM). The in vitro data suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy. D aclatasvir (DCV [BMS-790052]) is a cross-genotypic NS5Ainhibitor with picomolar to low nanomolar potency in the replicon system (1, 2). The antiviral activity of DCV in vitro translated into clinical efficacy, with hepatitis C virus (HCV) RNA declines of ϳ3 to 4 log 10 observed in genotype 1a (GT-1a)-infected subjects treated once daily (QD) with 60 mg of DCV in a 14-day multiple ascending dose (MAD) monotherapy study (3, 4). Moreover, DCV was effective against GT-1b and -1a in combinations that include either pegylated interferon and ribavirin (PEG-IFN-RBV) or other direct-acting anti-HCV agents (DAAs) (5-8).There are large populations of viral quasispecies preexisting in infected individuals, and variants that confer resistance to antiviral agents can be rapidly enriched and/or selected during antiviral treatment (9-11). Since DCV resistance variants show no crossresistance to other DAAs, DCV should rapidly suppress wild-type virus and variants resistant to other DAAs, thereby enhancing the effectiveness of other DAAs in combination therapies (2, 3). This effect is predicted to lead to higher rates of sustained viral response (SVR) and/or shorten the duration of treatment necessary to achieve SVR. Recent clinical results with DCV plus asunaprevir (ASV ) in patients infected with GT-1b and with DCV plus sofosbuvir (SOF ) in patients infected with GT-1, -2, and 3 demonstrate the effectiveness of DCV in interferon-free DAA combination therapies (6,12).Prior studies using the in vitro replicon system in...

Synergistic Activity of Combined NS5A Inhibitors

“…DCV alone (250 nM) is efficient for replicon elimination on gts 4a, 5a, and 6a. The ability of DCV to eliminate replicons in this assay format (4a Х 5a Ͼ 6a Ͼ 1a Ͼ 3a Х 2a NIH) is in agreement with the results from previous experiments (3). Syn-690 alone (150 nM) appears most active on gt 3a but has poor activity overall.…”

“…NS5A-Syn activity enhances the pangenotype coverage of DCV. Colony elimination studies have been used to determine the relative resistance barrier of DCV on multiple genotypes: 1b Ͼ 4a Ն 5a Ͼ 6a Х 1a Ͼ 2a JFH Ͼ 3a Ͼ 2a NIH (3,25,26). DCV combined with a protease inhibitor (MK-5172) effectively eliminated the genotype with the lowest barrier to resistance (2a NIH), indicating that DCV used in combination with other DAAs can be highly effective on all genotypes.…”

“…All replicons and virus stocks contained a Renilla-based luciferase reporter incorporated in the genome and were described previously (1,3,23,24 Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) with 10% fetal bovine serum (FBS; Invitrogen) and 0.25 mg/ml to 1.0 mg/ml G418 and penicillin-streptomycin (pen-strep), as previously described (25,26). Cells were passed twice weekly in T175 flasks.…”

“…The nonstructural 5A replication complex inhibitor (NS5A RCI) class of compounds represented by daclatasvir (DCV) (BMS-790052, Daklinza; Bristol-Myers Squibb) has activity against genotypes (gts) 1a, 1b, 2a, 3a, 4a, 5a, and 6a and is the most potent class of HCV inhibitor yet described (1)(2)(3). NS3 protease inhibitors and NS5B polymerase inhibitors have thus far been the preferred DAA partners for DCV combination therapies (1).…”

Efficacy of daclatasvir plus peginterferon alfa and ribavirin for patients with chronic hepatitis C genotype 4 infection

“…Hezode et al (2014) reported that patients with CC IL28B genotype had a higher chance of achieving SVR, compared to those with a non-CC genotype, regardless of therapy . Also, as shown by in vivo and in vitro studies, the combination of L30 polymorphism and IL28B non-CC genotype can significantly increase daclatasvir resistance (Wang et al, 2012;Wang et al, 2014).…”