Resistance characterization of ledipasvir and velpatasvir in hepatitis C virus genotype 4.

HCV genotype 4 (GT4) has often been overlooked in drug development, even though it infects ~20 million people worldwide. Ledipasvir/sofosbuvir and sofosbuvir/velpatasvir were highly efficacious in GT4 HCV-infected patients from GS-US-337-1119 and GS-US-342-1138. Here, we characterize the resistance profile of ledipasvir (LDV) and velpatasvir (VEL) in patients with GT4 HCV infection. NS5A deep-sequencing was performed for 454 patients infected with HCV GT4 at baseline, including 44 patients enrolled in GS-US-337-1119 and 116 patients enrolled in GS-US-342-1138, and at relapse for patients with virologic failure. LDV and VEL susceptibilities of 56 patient isolates were determined. In GS-US-337-1119, SVR12 rates were 100% for all subtypes except 4b and 4r. Phenotypic assessment of 56 HCV NS5A patient isolates from various GT4 subtypes indicated that LDV had high potency for the common subtypes 4a/d, and subtypes 4c/f/k/l/m/n/o/p/r/t despite the presence of resistance-associated substitutions (RASs). For the rare GT4b, LDV median EC50 was higher, but with a broad range of individual values. Importantly, all GT4b isolates tested had 2-4 NS5A RASs, some including Y93H. Similarly, the 2 GT4r infected patients who had virologic relapse had rare triple RASs. Reversion of these substitutions to the consensus residue significantly increased LDV susceptibility. In GS-US-342-1138, all patients achieved SVR12, regardless of their subtype or presence of RASs. In vitro data confirmed that VEL is potent against all GT4 isolates tested. LDV and VEL are potent antiviral drugs, estimated to be effective against >95% and >99%, respectively, of GT4 HCV isolates.

Resistance Analyses of Japanese Hepatitis C-Infected Patients Receiving Sofosbuvir or Ledipasvir/Sofosbuvir Containing Regimens in Phase 3 Studies.

High rates of sustained virologic response (SVR) has been achieved in Japanese patients with chronic hepatitis C virus (HCV) genotype (GT)1 and GT2 infection treated with ledipasvir/sofosbuvir (LDV/SOF) ±ribavirin (RBV) and SOF+RBV, respectively. We evaluated the effect of baseline HCV NS5A and NS5B resistance-associated variants (RAVs) on treatment outcome and characterized variants at virologic failure. Baseline deep sequencing for NS5A and NS5B genes was performed for all GT1 patients. Deep sequencing of NS5A (GT1 only) and NS5B (GT1 and GT2) was performed for patients who failed treatment or discontinued early with detectable HCV RNA (i.e., >25 IU/mL). In patients with HCV GT1 infection, 22.3% (GT1a: 2/11; GT1b: 74/330) had ≥1 baseline NS5A RAV. The most frequent NS5A RAVs in GT1b were Y93H (17.9%, 59/330) and L31M (2.4%, 8/330). Despite the presence of NS5A RAVs at baseline, 100% and 97% of patients achieved SVR12, compared with 100% and 99% for those with no NS5A RAVs with LDV/SOF and LDV/SOF+RBV, respectively. All patients with NS5B RAVs at baseline achieved SVR12. Of the 153 patients with GT2 infection (GT2a 60.1%, GT2b 39.9%), 3.3% (5/153) experienced viral relapse. No S282T or other NS5B RAVs were detected at baseline or relapse; no change in susceptibility to SOF or RBV was observed at relapse. In conclusion, LDV/SOF and SOF+RBV demonstrate a high barrier to resistance in Japanese patients with HCV GT1 and GT2 infection. The presence of baseline NS5A RAVs did not impact treatment outcome in GT1 Japanese patients treated with LDV/SOF for 12 weeks.

GS-9857 in patients with chronic hepatitis C virus genotype 1-4 infection: a randomized, double-blind, dose-ranging phase 1 study.

GS-9857, an inhibitor of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A, demonstrates potent activity against HCV genotypes 1-6 and improved coverage against commonly encountered NS3 resistance-associated variants (RAVs). In this study, the safety, tolerability, antiviral activity and pharmacokinetics (PK) of GS-9857 were evaluated in patients with chronic HCV genotype 1-4 infection. Patients with genotype 1-4 infection received placebo or once-daily GS-9857 at doses ranging from 50 to 300 mg for 3 days under fasting conditions. GS-9857 was well tolerated; all reported adverse events (AEs) were mild or moderate in severity. Diarrhoea and headache were the most commonly reported AEs. Grade 3 or 4 laboratory abnormalities were observed in 17% of patients receiving GS-9857; there were no Grade 3 or 4 abnormalities in alanine aminotransferase, aspartate aminotransferase or alkaline phosphatase levels. GS-9857 demonstrated potent antiviral activity in patients with chronic HCV infection, achieving mean and median maximum reductions in HCV RNA of ≥3 log10 IU/mL following administration of a 100-mg dose in patients with HCV genotype 1a, 1b, 2, 3 or 4 infection. The antiviral activity of GS-9857 was unaffected by the presence of pretreatment NS3 RAVs. In patients with genotype 1-4 infection, GS-9857 exhibited linear PK and was associated with a median half-life of 29-42 h, supporting once-daily dosing. Thus, the tolerability, efficacy and pharmacokinetic profile of GS-9857 support its further evaluation for treatment of patients with chronic HCV infection.

Evolution of the HCV viral population from a patient with S282T detected at relapse after sofosbuvir monotherapy.

Clinical phase II/III studies of the nucleotide analogue HCV NS5B inhibitor sofosbuvir (SOF) have demonstrated high efficacy in HCV-infected patients in combination therapy. To date, resistance to SOF (S282T in NS5B) has rarely been detected in patients. In this study, we investigated the evolution of S282T viral variants detected in one HCV genotype 2b-infected patient who relapsed following 12 weeks of SOF monotherapy. Deep sequencing of the NS5B gene was performed on longitudinal plasma samples at baseline, days 2 and 3 on SOF, and longitudinal samples post-SOF treatment through week 48. Intrapatient HCV evolution was analysed by maximum-likelihood phylogenetic analysis. Deep sequencing analysis revealed a low level pre-existence of S282T at 0.05% of viral sequences (4/7755 reads) at baseline and 0.03% (6/23 415 reads) at day 2 on SOF. Viral relapse was detected at week 4 post-treatment where 99.8% of the viral population harboured S282T. Follow-up analysis determined that S282T levels diminished post-treatment reaching undetectable levels 24-48 weeks post-SOF. Phylogenetic analysis together with the persistence of unique post-treatment mutations in all post-SOF samples suggested that growth of wild type resulted from reversion of the S282T mutant to a wild type and not outgrowth of the baseline wild-type population. Our data suggest that a very low level of pre-existing S282T at baseline in this patient was enriched and transiently detected following SOF monotherapy. Despite relapse with drug resistance to SOF, this patient was successfully retreated with SOF plus ribavirin for 12 weeks and is now cured from HCV infection.

Gene targeting approach to selectively kill colon cancer cells, with hyperactive K-Ras pathway.

BACKGROUND: Ras mutations are present in approximately 50% of human colorectal tumors. We have previously shown that transfection of a non-tumorigenic rat intestinal epithelial cell line, IEC18, by the K-Ras oncogene (R1 cells), resulted in malignant cell transformation. Utilizing the constantly active Ras signaling pathway to selectively target transformed but not normal cells is a plausible goal. AIM: To selectively kill Ras transformed cells by over expressing a lethal gene using a Ras-responsive promoter. MATERIAL AND METHODS: IEC18, R1 and a number of colon cancer cell lines were transfected with luciferase (Luc) reporter gene under the control of different Ras-responsive elements. The Ras-responsive promoter Py2 contains two copies of adjacent Ets and AP I binding sites followed by a minimal promoter. Apoptotic genes (bax, caspase-8 and PKG) were cloned into the Py2 plasmids. RI cells co-transfected with expression constructs and a selected vector and then grown for 3 weeks under selection. RESULTS: R1, SW480 and HCT116 with mutated c-K-Ras expressed high level of Luc activity following transfection with the Py2 element. IEC18 cell lines that do not contain this mutation expressed negligible low Luc activity. Following transfection of SW480 and R1 cells with Py2-bax, caspase-8 and PKG, there was a significant reduction in the number of colony formation. CONCLUSIONS: 1. Selective over-expression of pro-apoptotic genes, inhibits the growth of Ras transformed cells, and not normal cells. 2. This gene approach therapy may become a useful, effective and safe to target Ras mutated tumor cells with sparing of the normal cells.