In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir

Abstract

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC 50 ) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC 50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC 50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC 50 values equivalent to those for the wild type.

The emergence of NS5B resistance associated substitution S282T after sofosbuvir‐based treatment

“…1D). The frequency of NS5A RASs is high in untreated patients with non‐GT1 genotypes, which is consistent with the observed reduced antiviral activity of LDV against genotypes 2, 3, and 6 22. Patient 7, a 45‐year‐old white man with GT3a infection, had no NS5A RASs detected at baseline or posttreatment.…”

“…The prevalence rates of treatment‐emergent NS5B S282T, L159F, and V321A were assessed at the time of virologic failure. For patients receiving NS5A inhibitors, NS5A RASs at positions 24, 28, 30, 31, 32, 58, and 93 that conferred >2.5‐fold reduced susceptibility to LDV in vitro were included in the analysis; K24G/N/R, M28A/G/T, Q30E/G/H/L/K/R/T, L31I/F/M/V, P32L, S38F, H58D, A92K, and Y93C/F/H/N/S for patients with GT1a HCV infection and L31I/F/M/V, P32L, P58D, A92K, and Y93C/H/N/S for patients with GT1b infection 21, 22. A genotype‐specific reference was used for each HCV genotype (HCV1a_H77_ NC_004102, HCV1b_Con1_AJ238799, HCV2a_JFH1_AB047639, HCV2b_MD2b10_AY232748, HCV3a_S52_GU814263, HCV4a_ED43_GU814265, HCV5a_SA13_AF064490, HCV6a_EUHK2_Y12083).…”

Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor

“…Administration of a single dose of EDP-239 resulted in rapid reductions in GT1a and GT1b HCV RNA levels in patients receiving 10, 30, 100, and 200 mg (14). Deep sequencing of the NS5A gene from GT1a and GT1b clinical viral isolates at baseline discovered many known NS5A RAMs similar to those reported here or elsewhere (15). NS5A amino acid mutations at a frequency of Ն1% were analyzed from all patient plasma samples with HCV RNA levels of Ͼ1,000 IU/ml.…”

Preclinical Profile and Clinical Efficacy of a Novel Hepatitis C Virus NS5A Inhibitor, EDP-239

“…EDP-239 inhibits HCV subgenomic replicon replication, with activity at picomolar concentrations against genotypes 1a, 1b, 4a, 5a, and 6a and potency at nanomolar concentrations against genotypes 2a and 3a. This genotypic potency profile is not dissimilar to reports of another NS5A inhibitor, ledipasvir, which has comparable potencies against similar genotypes (32). However, in a phase I clinical study of ledipasvir, there were no significant differences in HCV RNA reduction between GT1a and GT1b patients receiving three daily 10-mg doses of study drug nor were there discernible dose effects among GT1a patients receiving 10, 30, or 90 mg despite clear dose-dependent drug exposures (15).…”