Classes Of Direct-acting Antivirals Research Articles

An Increase in the Prevalence of Clinically Relevant Resistance-Associated Substitutions in Four Direct-Acting Antiviral Regimens: A Study Using GenBank HCV Sequences.

Direct-acting antivirals (DAAs) revolutionized the therapeutics of chronic hepatitis C. The emergence and transmission of HCV variants with resistance-associated substitutions (RASs) can undermine HCV treatment. This study aimed to assess the prevalence and temporal trends of RASs in HCV, with a particular focus on clinically relevant RASs (cr-RASs). Near-complete HCV GenBank sequences archived in the Los Alamos HCV Database were analyzed. The study period was divided into two phases: before 2011 and from 2011 onward. Identification of RASs across three DAA classes (NS3, NS5A, and NS5B inhibitors) was based on the 2020 EASL guidelines. The AASLD-IDSA recommendations were used to identify cr-RASs for three HCV genotypes/subtypes (1a, 1b, and 3) and four DAA regimens: ledipasvir/sofosbuvir; elbasvir/grazoprevir; sofosbuvir/velpatasvir; and glecaprevir/pibrentasvir. The final HCV dataset comprised 3443 sequences, and the prevalence of RASs was 50.4%, 60.2%, and 25.3% in NS3, NS5A, and NS5B, respectively. In subtype 1a, resistance to ledipasvir/sofosbuvir was 32.8%, while resistance to elbasvir/grazoprevir was 33.0%. For genotype 3, resistance to sofosbuvir/velpatasvir and glecaprevir/pibrentasvir was 4.2% and 24.9%, respectively. A significant increase in cr-RASs was observed across the two study phases as follows: for ledipasvir/sofosbuvir in subtype 1a, cr-RASs increased from 30.2% to 35.8% (p = 0.019); for elbasvir/grazoprevir in subtype 1a, cr-RASs increased from 30.4% to 36.1% (p = 0.018); In subtype 1b, neither ledipasvir/sofosbuvir nor elbasvir/grazoprevir showed any cr-RASs in the first phase, but both were present at a prevalence of 6.5% in the second phase (p < 0.001); for sofosbuvir/velpatasvir in genotype 3, cr-RASs increased from 0.9% to 5.2% (p = 0.006); and for glecaprevir/pibrentasvir, cr-RASs increased from 12.0% to 29.1% (p < 0.001). The rising prevalence of HCV RASs and cr-RASs was discernible. This highlights the necessity for ongoing surveillance and adaptation of novel therapeutics to manage HCV resistance effectively. Updating the clinical guidelines and treatment regimens is recommended to counteract the evolving HCV resistance to DAAs.

Metabolism of Direct-acting Antiviral Agents (DAAs) in Hepatitis C Therapy: A Review of the Literature.

Hepatitis C virus (HCV) infection is still one of the leading causes of chronic liver disease, with chronically infected making up approximately 1% of the global population. Of those infected, 70% (55-85%) will develop chronic HCV infection. Chronic HCV infection causes substantial morbidity and mortality, with complications including cirrhosis, end-stage liver disease, hepatocellular carcinoma, and eventually death. Therapeutic options for chronic HCV infection have evolved dramatically since 2014, with a translation from pegylated interferon and ribavirin (associated with suboptimal cure and high treatment-related toxicity) to oral direct-acting antiviral treatment. There are four classes of direct-acting antivirals which differ by their mechanism of action and therapeutic target. They are all pointed to proteins that form the cytoplasmic viral replication complex. Multiple studies have demonstrated that direct-acting antiviral therapy is extremely well tolerated, highly efficacious, with few side effects. We performed an indexed MEDLINE search with keywords regarding specific direct-acting antiviral regimes and their pharmacokinetics, drug-drug interactions, and metabolism in specific settings of pregnancy, lactation, liver cirrhosis, liver transplantation and HCC risk, kidney failure and kidney transplantation. We present a comprehensive overview of specific direct-acting antiviral metabolism and drug-drug interaction issues in different settings. Despite its complex pharmacokinetics and the possibility of drug-drug interactions, direct-acting antivirals are highly efficacious in providing viral clearance, which is an obvious advantage compared to possible interactions or side effects. They should be administered cautiously in patients with other comorbidities, and with tight control of immunosuppressive therapy.

Molecular design, synthesis and biological evaluation of cage compound-based inhibitors of hepatitis C virus p7 ion channels

The hepatitis C caused by the hepatitis C virus (HCV) is an acute and/or chronic liver disease ranging in severity from a mild brief ailment to a serious lifelong illness that affects up to 3% of the world population and imposes significant and increasing social, economic, and humanistic burden. Over the past decade, its treatment was revolutionized by the development and introduction into clinical practice of the direct acting antiviral (DAA) agents targeting the non-structural viral proteins NS3/4A, NS5A, and NS5B. However, the current treatment options still have important limitations, thus, the development of new classes of DAAs acting on different viral targets and having better pharmacological profile is highly desirable. The hepatitis C virus p7 viroporin is a relatively small hydrophobic oligomeric viral ion channel that plays a critical role during virus assembly and maturation, making it an attractive and validated target for the development of the cage compound-based inhibitors. Using the homology modeling, molecular dynamics, and molecular docking techniques, we have built a representative set of models of the hepatitis C virus p7 ion channels (Gt1a, Gt1b, Gt1b_L20F, Gt2a, and Gt2b), analyzed the inhibitor binding sites, and identified a number of potential broad-spectrum inhibitor structures targeting them. For one promising compound, the binding to these targets was additionally confirmed and the binding modes and probable mechanisms of action were clarified by the molecular dynamics simulations. A number of compounds were synthesized, and the tests of their antiviral activity (using the BVDV model) and cytotoxicity demonstrate their potential therapeutic usefulness and encourage further more detailed studies. The proposed approach is also suitable for the design of broad-spectrum ligands interacting with other multiple labile targets including various viroporins.

A PDE multiscale model of hepatitis C virus infection can be transformed to a system of ODEs

Direct-acting antivirals (DAAs) treat hepatitis C virus (HCV) by targeting its intracellular viral replication. DAAs are effective and deliver high clinical performance against HCV infection, but optimization of the DAA treatment regimen is ongoing. Different classes of DAAs are currently under development, and HCV treatments that combine two or three DAAs with different action mechanisms are being improved. To accurately quantify the antiviral effect of these DAA treatments and optimize multi-drug combinations, we must describe the intracellular viral replication processes corresponding to the action mechanisms by multiscale mathematical models. Previous multiscale models of HCV treatment have been formulated by partial differential equations (PDEs). However, estimating the parameters from clinical datasets requires comprehensive numerical PDE computations that are time consuming and often converge poorly. Here, we propose a user-friendly approach that transforms a standard PDE multiscale model of HCV infection (Guedj J et al., Proc. Natl. Acad. Sci. USA 2013; 110(10):3991–6) to mathematically identical ordinary differential equations (ODEs) without any assumptions. We also confirm consistency between the numerical solutions of our transformed ODE model and the original PDE model. This relationship between a detailed structured model and a simple model is called ``model aggregation problem'' and a fundamental important in theoretical biology. In particular, as the parameters of ODEs can be estimated by already established methods, our transformed ODE model and its modified version avoid the time-consuming computations and are broadly available for further data analysis.

Urgency to treat patients with chronic hepatitis C in Asia.

Chronic hepatitis C (CHC) infection poses a global healthcare burden, being associated with serious complications if untreated. The prevalence of hepatitis C virus (HCV) infection is highest in areas of Central, South, and East Asia; over 50% of HCV patients worldwide live in the region, where HCV genotypes 1b, 2, 3, and 6 are the most prevalent. Treatment outcomes for chronic hepatitis C vary by ethnicity, and Asian patients achieve higher sustained virologic response rates following interferon (IFN)-based therapy than non-Asians. However, low efficacy, poor safety profile, and subcutaneous administration limit the use of IFN-based therapies. Superior virologic outcomes have been observed with different classes of direct-acting antivirals (DAAs) alone or in combination, and several all-oral DAA regimens are available in Asia. These regimens have shown excellent efficacy and favorable tolerability in clinical trials, yet there is a need for further studies of DAAs in a real world context, particularly in Asia. Furthermore, IFN-free treatment may not be accessible for many patients in the region, and IFN-based regimens remain an option in some countries. There is a need to improve current clinical practices for HCV management in Asia, including effective screening, disease awareness, and prevention programs, and to further understand the cost-effectiveness of IFN-free regimens. The evolution of potent treatments makes HCV eradication a possibility that should be available to all patients. However, access to these therapies in Asian countries has been slow, primarily because of economic barriers that continue to present a hurdle to optimal treatment.

NS5A inhibitors for the treatment of hepatitis C infection.

Today, we are witnessing a new era for the treatment of hepatitis C with excellent rates of virologic response and very good safety profiles. Among the many classes of direct-acting antivirals, the inhibitors of nonstructural protein 5A are particularly interesting. NS5A is a phosphorylated protein with a relevant role in viral replication. HCV-NS5A inhibitors show high potency, very good safety profile and high barrier to resistance. The amazing in vitro effectiveness of this class is associated with great efficacy in clinical trials in combination protocols with antivirals of other classes, with sustained virological response (SVR) obtained in more than 90% of patients. Herein, we sought to review the current knowledge regarding the NS5A protease complex inhibitors with special emphasis on clinical efficacy and development of viral resistance.

Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies.

Despite the excellent efficacy of direct-acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance-associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real-life DAA failures. Of the 200 virological failures that were analyzed in 197 DAA-treated patients, 89 with pegylated-interferon+ribavirin (PegIFN+RBV) and 111 without (HCV-1a/1b/1g/2/3/4=58/83/1/6/24/25; 56.8% treatment experienced; 65.5% cirrhotic) were observed. Sanger sequencing of NS3/NS5A/NS5B was performed by home-made protocols, at failure (N=200) and whenever possible at baseline (N=70). The majority of the virological failures were relapsers (57.0%), 22.5% breakthroughs, 20.5% non-responders. RAS prevalence varied according to IFN/RBV use, DAA class, failure type and HCV genotype/subtype. It was 73.0% in IFN group vs 49.5% in IFN free, with the highest prevalence of NS5A-RASs (96.1%), compared to NS3-RASs (75.9% with IFN, 70.5% without) and NS5B-RASs (66.6% with IFN, 20.4% without, in sofosbuvir failures). In the IFN-free group, RASs were higher in breakthrough/non-responders than in relapsers (90.5% vs 40.0%, P<.001). Interestingly, 57.1% of DAA IFN-free non-responders had a misclassified genotype, and 3/4 sofosbuvir breakthroughs showed the major-RAS-S282T, while RAS-L159F was frequently found in sofosbuvir relapsers (18.2%). Notably, 9.0% of patients showed also extra target RASs, and 47.4% of patients treated with ≥2 DAA classes showed multiclass resistance, including 11/11 NS3+NS5A failures. Furthermore, 20.0% of patients had baseline-RASs, which were always confirmed at failure. In our failure setting, RAS prevalence was remarkably high in all genes, with a partial exception for NS5B, whose limited resistance is still higher than previously reported. This multiclass resistance advocates for HCV resistance testing at failure, in all three genes for the best second-line therapeutic tailoring.

New prospects for the treatment and prevention of hepatitis C in children.

Combined pegylated interferon-α and ribavirin remains the standard therapy for pediatric hepatitis C virus (HCV) infections in 2016, but direct-acting antivirals (DAAs) with greatly improved efficacy and safety are now approved for adults. Here we review the major classes of DAAs and their anticipated use for treatment and potentially prevention of HCV in children. Currently approved DAAs target the viral protease, polymerase, and NS5A, a protein involved in viral replication and assembly. In combination, DAAs have lifted sustained virologic response rates in adults to more than 90% for multiple HCV genotypes, and the rich DAA pipeline promises further improvements. Clinical trials of interferon-free DAA regimens have been initiated for children ages 3-17 years. In 2016, the first efficacy trial of a preventive HCV vaccine is also underway. While awaiting a vaccine, there is hope that increased DAA utilization may prevent pediatric HCV infections by shrinking the pool of infectious persons. Interferon-free DAA regimens have revolutionized therapy for HCV-infected adults and, pending results of pediatric trials, will likely do the same for HCV-infected children. If widely deployed, DAA therapies may also help to reduce the number of new vertically and horizontally acquired pediatric infections.

Evaluation of antiviral drug synergy in an infectious HCV system.

Direct-acting antivirals (DAAs) have greatly improved the treatment of HCV infection. To improve response and prevent resistance, combination regimens have been the focus of clinical development. Regimens are often first assessed in vitro, with most combination studies to date using subgenomic replicon systems, which do not replicate the complete HCV life cycle and preclude study of entry and assembly inhibitors. Infectious full-length HCV systems have been developed and are being used to test drug efficacy. Using cell-based HCV Con1b replicon and an infectious full-length HCV (HCVcc-Luc) infection system, we systematically tested the synergy, additivity or antagonism of combinations of protease, NS5A and nucleotide NS5B inhibitor classes as well as the combination of these DAAs with host-targeting agent cyclosporin A or non-antibody entry inhibitor (S)-chlorcyclizine. Two computational software packages, MacSynergyII and CalcuSyn, were used for data analysis. Combinations between different classes showed good consistency across the two viral assay systems and two software platforms. Combinations between NS5A and nucleotide NS5B inhibitors were synergistic, while combinations of protease inhibitors with the other two classes were additive to slightly antagonistic. As expected, combinations of antivirals of the same class were additive. Combination studies between these DAA classes and cyclosporin A or (S)-chlorcyclizine demonstrated additive to synergistic effects and highly synergistic effects, respectively. Combinations of these drugs did not show any added or unexpected cytotoxicity. Our results show that in vitro combination studies of anti-HCV DAAs in the HCVcc system may provide useful guidance for drug combination designs in clinical studies. We also demonstrate that these DAAs in combination with host-targeting agents or entry inhibitors may improve HCV treatment response.

Resistance-associated variants in HCV subtypes 1a and 1b detected by Ion Torrent sequencing platform.

As a result of increased understanding of the HCV life cycle, a new generation of drugs known as direct-acting antivirals (DAAs) was developed and is constantly being improved. At baseline, HCV variants resistant to DAA therapy may pre-exist, increasing the likelihood of treatment failure. The aim of this study was to investigate the presence of resistance-associated variants (RAVs) in treatment-naive patients infected with HCV subtypes 1a and 1b. Next-generation sequencing was used to assess the frequencies of NS3-4A, NS5A and NS5B RAVs in 100 HCV monoinfected DAA-naive patients (HCV-1a: n=51; HCV-1b: n=49). Complete HCV sequence information was obtained for most samples. RAVs were detected in the NS3-4A (T54S, V55A, Q80K and R155K), NS5A (Q30H/R, H58P and Y93C/H/N) and NS5B (A421V) regions in 10%, 22% and 8%, respectively, of patients infected with HCV subtype-1a. Among the patients infected with HCV subtype-1b, mutations in the NS3-4A (F43I, T54S, Q80H, D168E and M175L), NS5A (L28M, R30Q, L31M, Q54H, A92T and Y93H) and NS5B (L159F, C316N, A421V and S556G) regions were observed in 12%, 53% and 31% of patients, respectively. High-throughput DNA sequencing allows an easier and more complete analysis of DAA RAVs, including mutations that represent only a minor variant of the whole viral population. RAVs to the three different classes of DAAs were found in our population. The characterization of their profile in the circulating virus is relevant to determine the better treatment option for infected individuals or to guide the implementation of treatment policies.

Interferon-free combination therapies for the treatment of hepatitis C: current insights.

The hepatitis C virus (HCV) treatment landscape has rapidly changed over the past 5 years. The development of direct-acting antiviral (DAA) agents that specifically target various steps in the HCV lifecycle has revolutionized therapeutic options for patients with HCV, with the development of highly effective and well-tolerated oral interferon-free regimens. There are many DAAs that are currently in development or have recently been approved, which target different nonstructural HCV proteins and host targets that are essential for HCV replication. This review will focus on the different classes of DAAs and the various combinations that are in advanced development for the treatment of chronic HCV infection and will focus on the different regimens in specific patient populations.

Optimal Management of HIV-HCV Coinfection

Around 25 % of HIV-positive individuals are HCV coinfected. HIV infection clearly worsened the natural history of chronic HCV infection, and despite the improved cares of comorbidities (chronic alcohol intake, metabolic syndrome, poor immune status by an earlier and better immune restoration with antiretrovirals (ARV), and finally by a decreasing hepatotoxicity of ARV), liver-related mortality remains one of the main causes of mortality in HIV/HCV-coinfected patients as compared to HIV monoinfected patients. Treatment options in HIV-HCV-coinfected patients have been greatly improved in the last few years. The treatment of acute HCV infection in HIV-positive individuals using the association of pegylated interferon/ribavirin (PEG-IFN/RBV) (with a first-generation protease inhibitor in genotype 1-infected patient) will allow a HCV virologic cure in around 80 %. In chronically infected patients, the PEG-IFN/RBV association has demonstrated an efficacy of 20–40 % in genotype 1 and higher in other genotypes, and increasing with optimization of dosing and duration, stopping rules and ART adjustment; the adjunction of telaprevir or boceprevir for genotype 1 increased the chance of HCV cure to around drug-70 % with drug interaction concerns. Physicians are today, in 2014, in a period of transition between the standard treatment combining PEG-IFN and RBV with or without first-generation HCV protease inhibitors according to HCV genotype and oral combinations of different classes of direct-acting antivirals (DAAs) with a pan-genotypic antiviral potency and a fair safety which will clearly change the prognosis of HIV/HCV-infected patients. The DAA combination removes HIV-infected patients (like cirrhotics or liver transplant recipients) as difficult-to-treat patients.

Treatment of HCV infection with the novel NS3/4A protease inhibitors

HCV NS3/4A serine protease inhibitors are the first class of direct acting antivirals (DAA) introduced in clinical practice. The first generation agents, selective against HCV genotype 1, are used in association with pegylated interferons and ribavirin allowing increased cure rates at the price of increased toxicity, significant drug interactions and high risk of selecting mutants conferring cross-resistance to the entire class. A large number of second-wave HCV protease inhibitors are currently in clinical development. Advancements include higher potency, activity against a wider number of genotypes, improved tolerability, easier dosing schedules, although their genetic barrier to resistance remains low, especially for subtype 1a, except for the most recent grazoprevir and ACH-2684. The most relevant progress regards the combination with other classes of DAA allowing construction of interferon-free regimens of short duration, good tolerability with exceptionally high cure rates.

Kinetic Analyses Reveal Potent and Early Blockade of Hepatitis C Virus Assembly by NS5A Inhibitors

All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis.

Sofosbuvir for the treatment of chronic hepatitis C: between current evidence and future perspectives.

In recent years, clinical research in the field of new treatments for chronic hepatitis C (HCV) has been devoted to developing regimens based on direct-acting antivirals (DAAs), with the goal of increasing treatment efficacy and improving tolerability and safety. This can be achieved by Peginterferon (PegIFN)-free anti-HCV regimens, as PegIFN is responsible for many side effects and limits treatment access due to contraindications in some patient categories. Sofosbuvir (SOF) is the first compound to enter the market with IFN-free combination regimens; it belongs to the nucleotide inhibitors of viral polymerase NS5B and acts as a chain terminator during the HCV replication process, exhibiting pan-genotypic antiviral activity with a high barrier to resistance. Clinical trials in HCV genotype 2/3 patients have demonstrated optimal efficacy in HCV-2, where the combination SOF/ribavirin (Rbv) for 12 weeks resulted in >90% sustained virological response (SVR) rates, while HCV-3 patients with advanced liver fibrosis and previous failure to PegIFN plus Rbv therapy still require individualized and optimized treatment strategies. Historically difficult-to-treat genotypes HCV-1, −4–6 can benefit from reduced duration of PegIFN plus SOF and Rbv, while IFN-free regimens in these patients will be based on SOF in combination with other DAA classes. Due to an optimal tolerability and safety profile with no significant drug-to-drug interactions, SOF is currently undergoing clinical trials in the setting of pre- and post-liver transplantation and HIV-coinfected patients, with the objective to address the until now unmet need for safe and efficient treatment in these populations. This article provides an overview of SOF features and the main clinical trials, discussing key results and potential future developments.

Antiviral therapy of hepatitis C in 2014: Do we need resistance testing?

The treatment of chronic hepatitis C has fundamentally changed since the approval of the first direct-acting antivirals (DAA) in 2011. In addition to telaprevir and boceprevir, in 2014 two new NS3 protease inhibitors (simeprevir and faldaprevir), one non-nucleoside polymerase inhibitor (sofosbuvir) and one NS5a replication complex inhibitor (daclatasvir) have expanded the treatment options for chronic hepatitis C. Resistance-associated variants (RAV) are naturally produced during the HCV life cycle. The frequency of RAVs within HCV quasispecies mainly depends on their replicational fitness. Variants conferring resistance to nucleos(t)ide analogues have not been detected, and the majority of NS3 protease-resistant variants are present at low frequencies (0.1–3%) before initiation of DAA-based therapies. However, the Q80K variant conferring resistance to simeprevir has been observed in 9–48% of untreated HCV genotype 1a-infected patients, leading to reduced SVR rates. Resistant variants are detectable in the majority of patients with treatment failure to NS3 protease inhibitor- or NS5a inhibitor-based antiviral therapy. Long-term follow-up studies by population-based sequence analysis have shown the disappearance of resistant variants in the majority of patients, with median times to loss of mutations of 4–64weeks. For the nucleotide analogue sofosbuvir, the emergence of the S282T resistant variant has been observed only in single patients, with reversion to wild-type within several weeks. Data are sparse on retreatment of patients with the same DAA or the same class of DAAs. However, retreatment with a different class of DAAs after failure of NS3 protease inhibitor-based therapy has been successful in small studies. This article forms part of a symposium in Antiviral Research on “Hepatitis C: next steps toward global eradication.”

Transient replication of a hepatitis C virus genotype 1b replicon chimera encoding NS5A-5B from genotype 3a

Although hepatitis C virus (HCV) is a pathogen of global significance, experimental therapies in current clinical development include highly efficacious all-oral combinations of HCV direct-acting antivirals (DAAs). If approved for use, these new treatment regimens will impact dramatically upon our capacity to eradicate HCV in the majority of virus-infected patients. However, recent data from late-stage clinical evaluations demonstrated that individuals infected with HCV genotype (GT) 3 responded less well to all-oral DAA combinations than patients infected with other HCV GTs. In light of these observations, the present study sought to expand the number of molecular tools available to investigate small molecule-mediated inhibition of HCV GT3 NS5A and NS5B proteins in preclinical tissue-culture systems. Accordingly, a novel subgenomic HCV replicon chimera was created by utilizing a GT1b backbone modified to produce NS5A and NS5B proteins from a consensus sequence generated from HCV GT3a genomic sequences deposited online at the European Hepatitis C Virus database. This approach avoided the need to isolate and amplify HCV genomes from sera derived from HCV-infected patients. The replicon chimera, together with a version engineered to express NS5A encoding a Y93H mutation, demonstrated levels of replication in transient assays robust enough to assess accurate antiviral activities of inhibitors representing different HCV DAA classes. Thus, the replicon chimera represents a new simple molecular tool suitable for drug discovery programmes aimed at investigating, understanding, and improving GT3a activities of HCV DAAs targeting NS5A or NS5B.

Characterization of Hepatitis C Virus Resistance from a Multiple-Dose Clinical Trial of the Novel NS5A Inhibitor GS-5885

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.).

Boceprevir and personalized medicine in hepatitis C virus infection.

Boceprevir was the first agent, along with telaprevir, of a novel class of direct-acting antivirals that entered clinical practice for the treatment of chronic hepatitis C. Boceprevir is an antiprotease that directly blocks hepatitis C virus (HCV) replication. Two studies in patients with HCV genotype 1 infection have shown that addition of boceprevir to the standard of care, ie, pegylated interferon-alfa (PEG-IFN-α) and ribavirin, markedly increased the rate of sustained virological response. A sustained virological response was obtained in about 70% of patients who had never been treated, as well as in 69%–75% and 40% of previous relapsers and nonresponders to PEG-IFN-α-ribavirin, respectively. Side effects were observed in almost all treated patients. Anemia, the most frequent adverse event related to administration of boceprevir, occurred in about 50% of patients. The decision to add boceprevir to the standard of care is made on an individual basis, and takes into account the prognosis of the liver disease, the efficacy of therapy, as it could be at best predicted, and the side effects that may arise, taking into account the comorbidities of the patient. Ultimately, the treatment must be accepted by the patient, who should fully understand the benefits and risks. Boceprevir trials were designed with the concept of individualized and response-guided therapy which establishes treatment decisions on how rapidly patients respond to treatment. Individualized therapy for chronic hepatitis C is based on patient and viral characteristics to make the best choice about whether a person will benefit from therapy and to evaluate on-treatment predictors of response to shorten therapy in patients with a rapid response as well as in patients who did not respond sufficiently to expect HCV eradication. This review focuses on the main results obtained so far, their impact on the treatment of patients with chronic hepatitis C, and potential therapeutic perspectives.

Antiviral resistance and direct-acting antiviral agents for HCV

Direct-acting antiviral (DAA) agents specifically target viral proteins. Two DAAs have been already been approved for the treatment of HCV infection and many more are in development. DAA treatment of HCV infection, however, leads to the selection of viral variants (produced by the error-prone HCV polymerase) that are resistant to the DAA agent in use. The selection of DAA-resistant HCV variants has been studied extensively in vitro and in vivo. Common amino acid substitution sites in each of the non-structural proteins are associated with DAA-resistance: D168, A155, A156 and V36 in NS3 protease; L31 and Y93 in NS5A; S282, S96, P495, M423, M414 and C316 in NS5B. In this review we cover the basic principles of DAA resistance, summarise the available resistance data for the various classes of DAAs and discuss the potential of DAA combination therapy for overcoming DAA-resistance, resulting in major advances in the treatment of HCV.