miR-122-based therapies select for three distinct resistance mechanisms based on alterations in RNA structure.

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5' untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor-based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

Variants associated with Bedaquiline (BDQ) resistance identified in Rv0678 and efflux pump genes in Mycobacterium tuberculosis isolates from BDQ naïve TB patients in Pakistan.

BACKGROUND: Mutations in the Rv0678, pepQ and atpE genes of Mycobacterium tuberculosis (MTB) have been reported to be associated with reduced antimycobacterial susceptibility to bedaquiline (BDQ). Resistance conferring mutations in treatment naïve MTB strains is likely to have implications for BDQ based new drug regimen that aim to shorten treatment duration. We therefore investigated the genetic basis of resistance to BDQ in MTB clinical isolates from BDQ naïve TB patients from Pakistan. In addition, mutations in genes associated with efflux pumps were investigated as an alternate mechanism of resistance. METHODS: Based on convenience sampling, we studied 48 MTB clinical isolates from BDQ naïve TB patients. These isolates (from our strain bank) included 38 MDR/pre-XDR/XDR (10 BDQ resistant, 8 BDQ intermediate and 20 BDQ susceptible) and 10 pan drug susceptible MTB isolates. All strains were subjected to whole genome sequencing and genomes were analysed to identify variants in Rv0678, pepQ, atpE, Rv1979c, mmpLS and mmpL5 and drug resistance associated efflux pump genes. RESULTS: Of the BDQ resistant and intermediate strains 44% (8/18) had variants in Rv0678 including; two reported mutations S63R/G, six previously unreported variants; L40F, R50Q and R107C and three frameshift mutations; G25fs, D64fs and D109fs. Variants in efflux pumps; Rv1273c (G462K), Rv0507c (R426H) and Rv1634c (E198R) were found to be present in drug resistant isolates including BDQ resistant and intermediate isolates. E198R in efflux pump gene Rv1634c was the most frequently occurring variant in BDQ resistant and intermediate isolates (n = 10). CONCLUSION: We found RAVs in Rv0678 to be commonly associated with BDQ resistance. Further confirmation of the role of variants in efflux pump genes in resistance is required so that they may be incorporated in genome-based diagnostics for drug resistant MTB.

Treatment failure with DAA therapy: Importance of resistance.

Viral resistance is a major reason for virological failure in patients being treated with direct-acting antivirals (DAAs) for chronic HCV infection. However, the importance of viral resistance mainly depends on the DAA regimen and HCV genotype. For first-line therapy with glecaprevir/pibrentasvir (G/P) or velpatasvir/sofosbuvir (VEL/SOF) no general baseline resistance analysis is required because of the high antiviral activity and high barrier to resistance. If available, resistance testing may help to optimise therapy in certain subgroups of patients with HCV genotype 3 and other rare HCV geno/subtypes. Voxilaprevir/velpatasvir/sofosbuvir (VOX/VEL/SOF) is the first choice for the second-line treatment of patients following a previous DAA failure, with rates of viral eradication above 90% irrespective of the presence of resistance-associated substitutions (RASs). However, in resource-limited settings, only first-generation DAAs may be available for second-line therapy. Here, RASs selected during initial antiviral therapy should be considered if testing is available and rescue treatment should include a switch to a regimen with a new DAA class to optimise treatment response. Patients with HCV genotype 3 are overrepresented in the group who experience DAA treatment failure. Limited data are available for third-line therapies, but promising results have been achieved with G/P plus SOF or VOX/VEL/SOF with or without ribavirin for 12 to 24 weeks; these regimens should be administered irrespective of a patient's RAS profile.

Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy.

Ribavirin (RBV) induces nucleotide (nt) substitutions in hepatitis C virus (HCV) genome nonstructural (NS) regions. Although emergence of drug resistance-associated variants is associated with direct-acting antiviral treatment failure, the effect of RBV on genome substitutions in such patients is unknown. Genotype 1b HCV subgenomic replicon cells were treated with RBV for 120 hours. Six patients with chronic genotype 1b with HCV-infected patients who failed to respond to prior daclatasvir plus asunaprevir (DCV/ASV) therapy were treated with 12 weeks of sofosbuvir and ledipasvir plus RBV after 4 weeks of RBV monotherapy. RBV-induced genome mutations in the HCV NS region (nt3493-9301) in replicon cells and in patients during 4 weeks of RBV monotherapy were analyzed by deep sequencing. RBV-associated G-to-A and C-to-U transitions increased in a dose-dependent manner in HCV replicon cells after the RBV treatment. In patients with prior DCV/ASV treatment failures, the median serum HCV RNA level was 6.25 ± 0.31 log IU/mL at the start of RBV therapy and decreased significantly to 5.95 ± 0.4 log IU/mL (P = .03) after 4 weeks of RBV monotherapy. Although predominant HCV genome substitutions rates were similar between nontreatment and RBV-treatment periods (0.042 and 0.031 per base pair, respectively; P = .248), the frequencies of G-to-A and C-to-U transitions significantly increased after RBV monotherapy. These transitions were enriched, particularly within the HCV NS3 region in all patients. RBV treatment induces G-to-A and C-to-U transitions in the HCV genome even in chronic patients with hepatitis C with prior DCV/ASV treatment failures.

Favorable efficacy of glecaprevir plus pibrentasvir as salvage therapy for HCV failures to prior direct-acting antivirals regimens.

In real-world settings, not much information is probably available on the treatment efficacy of the combination of glecaprevir and pibrentasvir (G/P) as a salvage therapy in failures of prior direct-acting antiviral (DAA) regimens. Especially, the evolution of NS5A resistance-associated variants (RAVs) and treatment efficacy in patients who received G/P for failures of prior treatment more than once is unknown. Twenty patients, who were exposed to glecaprevir 300 mg/d and pibrentasvir 120 mg/d for 12 weeks in failures of prior DAAs regimens were evaluated for sustained virological response at 12 weeks after the end of treatment (SVR12). The overall rate of SVR12 was 100%, based on intention-to-treat analysis. Five patients infected with genotype 1b, who received G/P for failures of prior treatment more than once, were analyzed for the evolution of RAVs in NS5A region. All of the five patients exhibited SVR12, regardless of the numbers of times of prior treatment (more than once), prior treatment response (nonresponse), and fibrosis stage (FIB-4 index ≥ 3.25). At the commencement of G/P, all five patients were detected with NS5A RAVs at the position of aa 93. Four patients, except for one, were detected with RAVs at both positions of aa 31 and aa 93 (double mutation). All patients could achieve SVR12 with G/P, regardless of the emergence of NS5A RAVs, accompanied by failure to prior NS5A regimens more than once. In conclusion, our study indicated that G/P was a potentially useful salvage treatment for patients who failed prior DAAs regimens more than once.

Comparison of direct sequencing and Invader assay for Y93H mutation and response to interferon-free therapy in hepatitis C virus genotype 1b.

BACKGROUND AND AIM: Virologic failure of interferon-free therapy has been associated with Y93H mutation in the non-structure 5A region in hepatitis C virus (HCV) genotype 1b, and screening is recommended. A simple assay based on Q-Invader technology was developed for Y93H mutant screening to reduce cost and effort. The present study sought to compare two methods of detection of Y93H mutation and to evaluate the effect of Y93H mutation on response to interferon-free therapy. METHODS: Y93H mutation was examined in 258 patients with HCV genotype 1b using both direct sequencing analysis and the polymerase chain reaction (PCR)-Invader assay. Daclatasvir and asunaprevir or ledipasvir and sofosbuvir therapy was administered to 205 patients whose sustained virological responses (SVR) were checked. RESULTS: Hepatitis C virus was detected in 232 of 258 patients by direct sequencing and in 236 of 258 patients by the PCR-Invader assay. Forty of 231 cases were defined as Y93 mutation by direct sequencing, and 46 of 236 cases were defined as Y93 mutation by the PCR-Invader assay. SVR of patients who were Y93H by direct sequencing, Y93H by the PCR-Invader assay, and Y93H by both methods was 62.5%, 82.4%, and 50%, respectively. CONCLUSIONS: The sensitivity of the PCR-Invader assay was similar to that of direct sequencing analysis; however, the PCR-Invader assay had a better ability to detect minor strains. Combination of the two assays would improve prediction of the response to daclatasvir and asunaprevir, but Y93H mutation had little effect on SVR in ledipasvir and sofosbuvir therapy.

Ultra-Deep Genomic Sequencing of HCV NS5A Resistance-Associated Substitutions in HCV/HIV Coinfected Patients.

BACKGROUND AND AIMS: The prevalence of naturally occurring HCV-NS5A resistance-associated substitutions (RAS) to DAA drugs might affect the response to treatment in HCV/HIV coinfected subjects. There are limited data on the frequency of HCV-NS5A naturally occurring drug-RAS at baseline in HCV/HIV coinfected patients when ultra-deep sequencing methodologies are applied. METHODS: HCV-NS5A-RAS were evaluated among 25 subjects in each group. Patients were matched by age, gender, and hepatic fibrosis stage category to control for selection bias. RESULTS: Within subtype 1a, RAS were observed in 28% of HCV monoinfected and 48% of HCV/HIV coinfected subjects. More patients in the HCV/HIV coinfected group had clinically relevant mutations to DAA directed at NS5A. CONCLUSION: While the clinical significance of this observation may be limited in highly drug adherent populations, some HCV/HIV coinfected persons may be at greater risk of viral resistance if suboptimal dosing occurs.

Ledipasvir plus sofosbuvir as salvage therapy for HCV genotype 1 failures to prior NS5A inhibitors regimens.

There is little information on retreatment efficacy and predictors of the combination of ledipasvir and sofosbuvir (ledipasvir/sofosbuvir) for patients who fail to respond to NS5A inhibitors. NS5A resistance variants are known to persist for long periods after such treatment. Here, we evaluated 54 patients with chronic HCV genotype 1b infection, free of decompensated cirrhosis, and hepatocellular carcinoma, for sustained virological response after 12 weeks (SVR12) of once-daily treatment with 90 mg ledipasvir and 400 mg sofosbuvir. Intention-to-treat analysis showed SVR12 of 70%. Using ultra-deep sequencing, non-responder to ledipasvir/sofosbuvir showed no change in the rates of detection of NS5A and NS5B resistant-variants at re-elevation of viral loads, relative to baseline. According to response to prior treatment, SVR12 rates were 18, 69, 94, and 100% in non response, viral breakthrough, relapse, and discontinuation due to adverse events, respectively. SVR12 rates in non response were significantly lower than those of the others. Multivariate analysis identified response to previous treatment (failure except for non response) and FIB4 index (<3.25) as significant determinants of SVR12. The SVR12 rates were significantly lower in patients with FIB4 index of ≥3.25 and had not responded to prior treatment, relative to others. The specificity, and positive- and negative-predictive values were high for prediction of poor response based on the combination of two predictors. In conclusion, our study indicated that ledipasvir/sofosbuvir is a potentially useful salvage treatment for patients who fail prior NS5A inhibitors-based therapy. Response to prior treatment was an important predictor of retreatment efficacy.

Retreatment efficacy and predictors of ledipasvir plus sofosbuvir to HCV genotype 1 in Japan.

Predictors of treatment efficacy with ledipasvir plus sofosbuvir as direct-acting antiviral (DAA) regimen for HCV infection are still unclear. Retreatment efficacy of ledipasvir plus sofosbuvir for failures to prior DAA regimens, including NS5A inhibitors, are also unknown because resistance-associated variants (RAVs) in NS5A have been shown to persist up to the long-term of post-treatment. One hundred seventy-five patients with chronic HCV genotype 1 infection, without decompensated liver cirrhosis and hepatocellular carcinoma, were evaluated SVR12 by ledipasvir 90 mg plus sofosbuvir 400 mg once-daily for 12 weeks. Overall, SVR12 were 92%, based on intention to treat analysis. In failures to daclatasvir plus asunaprevir, SVR12 were 71%. The study using ultra-deep sequencing showed that ledipasvir plus sofosbuvir was effective to one case of failures to daclatasvir plus asunaprevir with multidrug RAVs (triple mutation in NS3-D168/NS5A-L31/NS5A-Y93). Multivariate analysis identified FIB4 index (<3.25), IL28B rs8099917 (TT type), and NS5A-L31 (Wild type) as significant determinants of SVR12. SVR12 rates in patients with three factors of poor response (RAVs Positive, IL28B non-TT, and FIB4 index ≥3.25) simultaneously were significantly lower than those of the other patients. Prediction of response to therapy based on combination of three predictors had high sensitivity and positive predictive values. In conclusions, this study indicated the favorable efficacy of ledipasvir plus sofosbuvir for HCV genotype 1 infection, including multidrug RAVs in Japan. Treatment efficacy could be predicted by the combination of viral and host factors. J. Med. Virol. 89:284-290, 2017. © 2016 Wiley Periodicals, Inc.

Association between vitamin D deficiency and pre-existing resistance-associated hepatitis C virus NS5A variants.

AIM: Although interferon-free therapy with direct-acting antivirals has developed as a standard of care for chronic hepatitis C, the existence of resistance-associated variants (RAVs) has a negative impact on treatment results. Recently, several studies indicated a relationship between chronic hepatitis C and serum vitamin D levels. However, the relationship between RAVs at the hepatitis C virus non-structure 5A (NS5A) region and serum vitamin D level has not yet been examined. METHODS: Among patients with genotype 1 chronic hepatitis C who were enrolled in a multicenter cooperative study, our subjects comprised 247 patients in whom it was possible to measure RAVs at the NS5A region. These RAVs were measured using a direct sequencing method. RESULTS: The median age of patients was 70 years (range, 24-87 years), and the number of female patients was 135 (54.7%). The median serum 25(OH) D3 level was 22 ng/mL (range, 6-64 ng/mL). L31 and Y93 RAVs at the NS5A region were detected in 3.7% (9/247) and 13.4% (33/247) of patients, respectively. Multivariate analysis identified vitamin D deficiency (serum 25(OH) D3 ≤ 20 ng/mL) (P = 5.91 × 10⁻5 , odds ratio = 5.015) and elderly age (>70 years) (P = 1.85 × 10-3 , odds ratio = 3.364) as contributing independent factors associated with the presence of the L31 and/or Y93 RAVs. The Y93H RAV was detected in 25.9% (29/112) of patients with a vitamin D deficiency, and in 8.9% (12/135) of those with a serum 25(OH) D3 level >20 ng/mL (P = 4.90 × 10-3 ). CONCLUSION: We showed that RAVs at the NS5A region are associated with vitamin D deficiency and elderly age, which may have a negative influence on innate/adaptive immune responses to hepatitis C virus infection.

Clinical evaluation of sofosbuvir/ledipasvir in patients with chronic hepatitis C genotype 1 with and without prior daclatasvir/asunaprevir therapy.

AIM: This study explored treatment outcomes of sofosbuvir (SOF)/ledipasvir (LDV) therapy for chronic hepatitis C patients with and without prior daclatasvir (DCV)/asunaprevir (ASV) therapy. METHODS: Overall, 530 Japanese patients who were infected with hepatitis C virus genotype 1 received SOF/LDV therapy for 12 weeks, and resistance-associated variants (RAVs) in the hepatitis C virus non-structural protein (NS)5A and NS5B regions were assessed at baseline and virological relapse by direct sequencing. RESULTS: Sustained virological response (SVR) rates did not significantly differ between patients with and without NS5A Y93H/N (94.2% [113/120] vs. 97.7% [345/353]), but the SVR rate was significantly lower in patients with prior DCV/ASV therapy compared to those without (69.2% [18/26] vs. 98.4% [496/504], P < 0.001). Among 26 patients with prior DCV/ASV therapy, the prevalence of NS5A multi-RAVs (≥2) was similar between responders and non-responders (61% [11/18] vs. 75% [5/8]), but all patients without RAVs achieved SVR. Multivariate analysis showed that prior DCV/ASV therapy and history of hepatocellular carcinoma were independently associated with treatment failure (odds ratio, 37.55; 95% confidence interval, 10.78-130.76; P < 0.001 for prior DCV/ASV therapy; odds ratio, 4.42; 95% confidence interval, 1.09-18.04; P = 0.03 for the history of HCC). All SOF/LDV failure patients (n = 8) with prior DCV/ASV treatment had two or more factors of cirrhosis, IL28B unfavorable genotype, and baseline NS5A multi-RAVs. The multiple NS5A RAVs had increased but NS5B substitutions, C316N/A207T/A218S or L159F, had not changed at the time of relapse. CONCLUSIONS: Prior DCV/ASV therapy is associated with failure of SOF/LDV therapy due to multiple RAVs.

Ultra-deep sequencing analysis of resistance-associated variants during retreatment with simeprevir-based triple therapy after failure of telaprevir-based triple therapy in patients with genotype 1 hepatitis C virus infection.

AIM: Simeprevir (SMV)-based triple therapy is an effective retreatment option following failure of telaprevir (TVR)-based triple therapy. However, it is unclear whether the persistence of resistance-associated variants (RAVs) induced by TVR-based therapy may reduce the treatment effect of SMV-based therapy. METHODS: The factors associated with the treatment effect, including RAVs in the NS3 region, were examined in 21 patients with genotype 1b HCV infection who were treated with SMV-based therapy after failure of TVR-based therapy. Ultra-deep sequencing was carried out to detect RAVs. RESULTS: With the exception of one patient who discontinued treatment owing to adverse events, the sustained virologic response (SVR) rate was 50% (10/20). Ultra-deep sequencing at the start of SMV-based therapy revealed that TVR-resistant variants were detected in six patients (29%), and no variants were observed at position 168. Cross-resistance between TVR and SMV with low frequency was detected in only one patient, and this patient achieved SVR. Higher SVRs for SMV-based therapy were attained in patients who discontinued treatment owing to the adverse effects of prior TVR-based therapy (discontinuation 100% vs. non-discontinuation 29%, P = 0.005), and patients who relapsed following prior pegylated interferon plus ribavirin therapy (relapse 100% vs. non-response 20%, P = 0.007). CONCLUSIONS: In this study, ultra-deep sequencing analysis revealed that TVR and/or SMV-resistant variants may have no influence on the effect of SMV-based therapy after failure of TVR-based therapy. Patients who discontinued treatment owing to adverse effects of TVR-based therapy and relapsers to previous pegylated interferon/ribavirin therapy would be good candidates for retreatment with SMV-based therapy.

L159F and V321A Sofosbuvir-Associated Hepatitis C Virus NS5B Substitutions.

BACKGROUND: Sofosbuvir (SOF) exhibits a high barrier to resistance, with no S282T NS5B substitution or phenotypic resistance detected in phase 3 registration studies. METHODS: Here, emergence of the NS5B variants L159F and V321A and possible association with resistance was evaluated in 8 studies of SOF (NEUTRINO, FISSION, POSITRON, FUSION, VALENCE, PHOTON-1, PHOTON-2, and P7977-2025) and 5 studies of combination ledipasvir (LDV) and SOF (LDV/SOF; LONESTAR, ELECTRON [LDV/SOF arms], ION1, ION2, and ION3), using deep sequencing. RESULTS: Deep sequencing detected L159F in 15% (53 of 353) and V321A in 5% (17 of 353) of patients with virologic failure in the SOF studies. Intensification of SOF treatment with LDV reduced the emergence of L159F or V321A to 2% (1 of 50 each) at virologic failure. L159F and V321A did not influence the outcome of retreatment with SOF, ribavirin, and pegylated interferon. At baseline, L159F was detected only in genotype 1-infected patients (1%) and was only associated with increased virologic failure in patients treated for short durations with SOF and ribavirin. CONCLUSIONS: Deep-sequencing analysis confirmed that NS5B variants L159F and V321A emerged in a subset of patients treated with SOF at virologic failure. These variants had no impact on retreatment outcome with SOF, ribavirin, and pegylated interferon. Baseline L159F in genotype 1 did not affect the treatment outcome with LDV/SOF.

Grazoprevir, Elbasvir, and Ribavirin for Chronic Hepatitis C Virus Genotype 1 Infection After Failure of Pegylated Interferon and Ribavirin With an Earlier-Generation Protease Inhibitor: Final 24-Week Results From C-SALVAGE.

BACKGROUND: The phase 2 C-SALVAGE study (Hepatitis C-Salvage Study for Patients who Failed DAA/PR Therapy) demonstrated a 96.2% sustained virologic response at 12 weeks (SVR12) rate using the NS3/4A protease inhibitor grazoprevir and the NS5A inhibitor elbasvir together with ribavirin in treatment-experienced patients with chronic hepatitis C virus (HCV) genotype 1 infection. METHODS: C-SALVAGE was a prospective open-label trial of grazoprevir 100 mg once daily and elbasvir 50 mg once daily coadministered with weight-based ribavirin twice daily for 12 weeks in genotype 1-infected cirrhotic and noncirrhotic patients who had failed treatment with ≥ 4 weeks of pegylated interferon and ribavirin plus either boceprevir, telaprevir, or simeprevir. Although the primary efficacy outcome was SVR12, patients were also evaluated 24 weeks after cessation of study therapy. Population sequencing was performed at baseline and periodically in virologic failures throughout the 24-week posttherapy follow-up period. RESULTS: SVR24 rates were 76 of 79 (96.2%) overall, with all 3 relapses occurring by posttherapy week 8. Every NS3 and NS5A variant detected at baseline reappeared at the time of relapse and persisted throughout the available follow-up period. NS3_A156T emerged in virus from each patient at relapse, but rapidly disappeared over the ensuing 2 weeks in 2 patients. NS5A_Y93H emerged in virus from 2 patients at relapse and persisted for the entire follow-up period. CONCLUSIONS: Grazoprevir and elbasvir with ribavirin for 12 weeks maintained HCV suppression for at least 24 weeks posttherapy without late relapses. Baseline resistance-associated variants (RAVs) stably reappeared at relapse in all 3 patients with virologic failure. NS5A_RAVs emerging at relapse persisted for the full 24-week follow-up period. If confirmed, this finding could complicate retreatment of the small number of patients failing regimens containing an NS5A inhibitor. CLINICAL TRIALS REGISTRATION: NCT02105454.

Hepatitis C virus whole genome sequencing: Current methods/issues and future challenges.

Therapy for hepatitis C is currently undergoing a revolution. The arrival of new antiviral agents targeting viral proteins reinforces the need for a better knowledge of the viral strains infecting each patient. Hepatitis C virus (HCV) whole genome sequencing provides essential information for precise typing, study of the viral natural history or identification of resistance-associated variants. First performed with Sanger sequencing, the arrival of next-generation sequencing (NGS) has simplified the technical process and provided more detailed data on the nature and evolution of viral quasi-species. We will review the different techniques used for HCV complete genome sequencing and their applications, both before and after the apparition of NGS. The progress brought by new and future technologies will also be discussed, as well as the remaining difficulties, largely due to the genomic variability.

The importance of resistance to direct antiviral drugs in HCV infection in clinical practice.

Treatment of chronic hepatitis C virus (HCV) infection with direct-acting antiviral agents (DAA) is associated with high rates of sustained virologic response. Remaining factors associated with treatment failure include advanced stages of liver fibrosis, response to previous antiviral therapy and viral factors such as baseline viral load and suboptimal interaction of the DAA with the target based on viral variants. Heterogeneity within NS3, NS5A, and NS5B areas interacting with DAAs exist between HCV geno- and subtypes as well as HCV isolates of the same geno- and subtype and amino acid polymorphisms associated with suboptimal efficacy of DAAs are termed resistance-associated variants (RAVs). RAVs may be associated with virologic treatment failure. However, virologic treatment failure typically occurs only if other negative predictive host or viral factors are present at the same time, susceptibility to additional antiviral agents is reduced or duration of treatment is suboptimal. In this review geno- and phenotypic resistance testing as well as clinical data on the importance of RAVs for conventional triple therapies with sofosbuvir, simeprevir, and daclatasvir and available interferon-free DAA combinations are discussed.

Second generation direct-acting antivirals - Do we expect major improvements?

The rapid progress in the development of direct-acting antiviral agents for hepatitis C has allowed the vast majority of patients to receive all oral therapy that will eliminate their virus. The success of the new regimens has led many to question the need for further developments in this field. Major improvements in drugs for hepatitis C are unlikely but we predict incremental improvements in the next few years. We hope that the next generation of drugs will address the unresolved issues for patients with genotype 3 infection where current treatments are still not entirely satisfactory and we anticipate improvements in the management of patients with renal failure. Shorter duration treatments, perhaps with novel modes of action, may allow simplified 'one-dose' treatments that will greatly expand our ability to treat patients who have difficulty accessing current services and we anticipate that the clinical community will better define the patients with advanced disease who will benefit from therapy prior to liver transplantation.

Viral dynamic modelling of Hepatitis C and resistance-associated variants in haemophiliacs.

AIM: Chronic hepatitis C virus (HCV) infection is an important source of morbidity and mortality among haemophiliacs. Limited data are available regarding treatment intervention using direct-acting antivirals (DAAs) and theoretical concerns regarding accumulation of drug-associated resistance variants (RAVs) remain. We conducted a pilot study of treatment with telaprevir/pegylated interferon-alfa/ribavirin to evaluate treatment response and the role of lead-in DAA therapy on mutational selection of resistance variants. METHODS: Ultra-deep sequence analysis was performed at baseline, 48 hours and 168 hours after treatment initiation. RESULTS: No dominant RAVs were identified at baseline, but low-level RAVs were noted at baseline in all subjects. Viral dynamic models were used to assess treatment responses. The efficacy parameter (Ɛ) for lead-in ranged from 0 to 0.9745 (mean = 0.514). Subsequent addition of telaprevir resulted in a mean efficacy of more than 0.999. This was comparable to subjects who started all three medications simultaneously. A total of 80% achieved SVR. While rapid shifts in the RAV population following DAA initiation were observed, treatment failure associated with A156V was observed in only one patient. Adverse event profiles were similar to that observed in non-haemophilia cohorts. There was no evidence of factor inhibitor formation. There was no evidence that lead-in provided benefit in terms of response efficacy. CONCLUSION: These data support DAA-based therapy in those with inherited bleeding disorders.

Substituted tetracyclic indole core derivatives of HCV NS5A inhibitor MK-8742.

As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing substitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly. However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of the tetracyclic indole core had a positive impact on the potency against the resistance associated variants, such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified.

Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition.