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Perspectives and challenges of interferon-free therapy for chronic hepatitis C

Open AccessPublished:October 25, 2012DOI:https://doi.org/10.1016/j.jhep.2012.10.019

      Summary

      Recent data have clearly shown that a sustained virologic response can be achieved in different HCV infected patient populations with various interferon-free treatment regimens. Despite the successful implementation of telaprevir- and boceprevir-based triple therapies, all-oral regimens will certainly become a first choice for a number of HCV-infected patients in the very near future, as triple therapy approaches are burdened with significant side-effects and limited success in patients with advanced liver fibrosis and prior null-response to pegylated interferon-α (pegIFN-α)/ribavirin therapy. However, available data from phase I and II clinical trials evaluating interferon-free regimens have not yet revealed a clearly outstanding all-oral combination, and numerous challenges remain to be addressed by intensive ongoing and future research. In particular, thus far evaluated all-oral regimens did not cure a satisfactory percentage of patients with unfavorable baseline characteristics, namely patients infected with HCV genotype 1a, previous null-response to pegIFN-α/ribavirin, or liver cirrhosis. In this review, we summarize available data of interferon-free regimens for the treatment of chronic hepatitis C and assess implications for perspectives and challenges in the further development of all-oral therapies.

      Abbreviations:

      HCV (hepatitis C virus), HIV (human immunodeficiency virus), SVR (sustained virologic response), RVR (rapid virologic response), eRVR (extended rapid virologic response), EVR (early virologic response), peg (pegylated), IFN (interferon), IL28B (interleukin 28B), DAA (directly acting antiviral agent)

      Keywords

      A need for interferon-free treatment regimens for chronic hepatitis C

      Figure thumbnail fx3

      The current repertoire of DAA agents for all-oral combination therapies

      HCV NS3-4A protease inhibitors

      NS3-4A inhibitors target the shallow enzymatic groove of the HCV protease and thereby inhibit HCV polyprotein procession, a crucial step in the early HCV life cycle [
      • Morikawa K.
      • Lange C.M.
      • Gouttenoire J.
      • Meylan E.
      • Brass V.
      • Penin F.
      • et al.
      Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.
      ]. In the meanwhile, numerous NS3-4A protease inhibitors have been developed which can be divided into two molecular classes, the macrocyclic inhibitors and linear tetra-peptide α-ketoamide derivatives [
      • Morikawa K.
      • Lange C.M.
      • Gouttenoire J.
      • Meylan E.
      • Brass V.
      • Penin F.
      • et al.
      Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.
      ] (Table 1). In general, NS3-4A inhibitors are characterized by a remarkable antiviral activity, but also by a low barrier to resistance. Hence, as it was shown for example for the approved α-ketoamide derivatives telaprevir and boceprevir, monotherapy with NS3-4A inhibitors results in an approximately 4 log10 decrease of serum HCV RNA within days, but also in a rapid selection of resistant variants and viral breakthrough [
      • Forestier N.
      • Reesink H.W.
      • Weegink C.J.
      • McNair L.
      • Kieffer T.L.
      • Chu H.M.
      • et al.
      Antiviral activity of telaprevir (VX-950) and peginterferon alfa-2a in patients with hepatitis C.
      ,
      • Sarrazin C.
      • Kieffer T.L.
      • Bartels D.
      • Hanzelka B.
      • Muh U.
      • Welker M.
      • et al.
      Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir.
      ,
      • Sarrazin C.
      • Rouzier R.
      • Wagner F.
      • Forestier N.
      • Larrey D.
      • Gupta S.K.
      • et al.
      SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders.
      ,
      • Susser S.
      • Welsch C.
      • Wang Y.
      • Zettler M.
      • Domingues F.S.
      • Karey U.
      • et al.
      Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients.
      ]. The risk of resistance development can be significantly reduced by the addition of pegIFN-α and ribavirin, and telaprevir or boceprevir-based triple therapies result in SVR rates of approximately 70–80%, 80–90%, and 30–40% in treatment-naïve HCV genotype 1 patients, previous relapsers, and null responders to pegIFN-α and ribavirin, respectively [
      • Bacon B.R.
      • Gordon S.C.
      • Lawitz E.
      • Marcellin P.
      • Vierling J.M.
      • Zeuzem S.
      • et al.
      Boceprevir for previously treated chronic HCV genotype 1 infection.
      ,
      • Jacobson I.M.
      • McHutchison J.G.
      • Dusheiko G.
      • Di Bisceglie A.M.
      • Reddy K.R.
      • Bzowej N.H.
      • et al.
      Telaprevir for previously untreated chronic hepatitis C virus infection.
      ,
      • Poordad F.
      • McCone Jr., J.
      • Bacon B.R.
      • Bruno S.
      • Manns M.P.
      • Sulkowski M.S.
      • et al.
      Boceprevir for untreated chronic HCV genotype 1 infection.
      ,
      • Zeuzem S.
      • Andreone P.
      • Pol S.
      • Lawitz E.
      • Diago M.
      • Roberts S.
      • et al.
      Telaprevir for retreatment of HCV infection.
      ].
      Table 1Selected DAAs and host-targeting agents (HTAs) in the pipeline.
      Another important feature of most NS3-4A protease inhibitors is the selective activity against distinct HCV genotypes, which is explained by sequence differences in important parts of the protease domain between HCV genotypes [
      • Lange C.M.
      • Sarrazin C.
      • Zeuzem S.
      Review article: specifically targeted anti-viral therapy for hepatitis C – a new era in therapy.
      ]. Thus far, most NS3-4A inhibitors have been developed predominantly to target HCV genotype 1. Newer NS3-4A protease inhibitors than telaprevir and boceprevir, which are currently in phase 1–3 development, include for example simeprevir (TMC435), danoprevir (R7227/ITMN191), vaniprevir (MK-7009), asunaprevir (BMS-650032), BI201335, ACH-1625, ABT-450, MK-5172, GS-9256, and GS-9451. Potential advantages of these second and third generation protease inhibitors might be improved tolerability, broader genotypic activity (e.g., MK-5172), different resistance profiles (e.g., MK-5172), and/or improved pharmacokinetics, which allow a once daily dosage (e.g., TMC435, BI201335) [
      • Brainard D.M.
      • Petry A.
      • Van Dyck K.
      • Nachbar R.
      • De Lepeleire I.M.
      • Caro L.
      • et al.
      Safety and antiviral activity of MK-5172, a novel HCV NS3/4A protease inhibitor with potent activity against known resistance mutants, in genotype 1 and 3 HCV-infected patients.
      ,
      • Reesink H.W.
      • Fanning G.C.
      • Farha K.A.
      • Weegink C.
      • Van Vliet A.
      • Van ’t Klooster G.
      • et al.
      Rapid HCV-RNA decline with once daily TMC435: a phase I study in healthy volunteers and hepatitis C patients.
      ,
      • Summa V.
      • Ludmerer S.W.
      • McCauley J.A.
      • Fandozzi C.
      • Burlein C.
      • Claudio G.
      • et al.
      MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants.
      ,
      • Manns M.P.
      • Bourliere M.
      • Benhamou Y.
      • Pol S.
      • Bonacini M.
      • Trepo C.
      • et al.
      Potency, safety, and pharmacokinetics of the NS3/4A protease inhibitor BI201335 in patients with chronic HCV genotype-1 infection.
      ,
      • Moreno C.
      • Berg T.
      • Tanwandee T.
      • Thongsawat S.
      • Van Vlierberghe H.
      • Zeuzem S.
      • et al.
      Antiviral activity of TMC435 monotherapy in patients infected with HCV genotypes 2–6: TMC435-C202, a phase IIa, open-label study.
      ].
      Unfortunately, the resistance profiles of linear tetrapeptide and macrocyclic inhibitors are overlapping. Amino acid position R155 in NS3 constitutes the central position for resistance development [
      • Sarrazin C.
      • Zeuzem S.
      Resistance to direct antiviral agents in patients with hepatitis C virus infection.
      ]. Mutations at this amino acid site confer resistance to nearly all protease inhibitors which are currently in advanced clinical development. Consequently, combining different NS3-4A inhibitors is not a logical strategy for interferon-free regimens. A possible exception is MK-5172, which exhibits potent antiviral activity against variants carrying mutations at position R155 [
      • Summa V.
      • Ludmerer S.W.
      • McCauley J.A.
      • Fandozzi C.
      • Burlein C.
      • Claudio G.
      • et al.
      MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants.
      ].
      Importantly, the genetic barrier to resistance against telaprevir (and other NS3-4A inhibitors) differs significantly between HCV genotype 1 subtypes. In all clinical studies of telaprevir alone or in combination with pegIFN-alfa and ribavirin, viral resistance and breakthrough occurred much more frequently in patients infected with HCV genotype 1a compared to HCV genotype 1b [
      • Jacobson I.M.
      • McHutchison J.G.
      • Dusheiko G.
      • Di Bisceglie A.M.
      • Reddy K.R.
      • Bzowej N.H.
      • et al.
      Telaprevir for previously untreated chronic hepatitis C virus infection.
      ,
      • Zeuzem S.
      • Andreone P.
      • Pol S.
      • Lawitz E.
      • Diago M.
      • Roberts S.
      • et al.
      Telaprevir for retreatment of HCV infection.
      ]. This difference was shown to result from nucleotide differences at position 155 in HCV subtype 1a (AGA, encodes R) vs. 1b (CGA, also encodes R). The mutation most frequently associated with resistance to telaprevir is R155K; changing R to K at position 155 requires 1 nucleotide change in HCV subtype 1a and 2 nucleotide changes in subtype 1b isolates [
      • McCown M.F.
      • Rajyaguru S.
      • Kular S.
      • Cammack N.
      • Najera I.
      GT-1a or GT-1b subtype-specific resistance profiles for hepatitis C virus inhibitors telaprevir and HCV-796.
      ]. Consequently, HCV genotype 1a may be a problematic subtype for successful all-oral therapy based on NS3-4A inhibitors.
      An additional possible limitation of most NS3-4A inhibitors is the interaction with CYP3A4, resulting in numerous drug–drug interactions including tacrolimus, cyclosporine, antiretroviral agents, statins, antifungals, and many more [
      • Wilby K.J.
      • Greanya E.D.
      • Ford J.A.
      • Yoshida E.M.
      • Partovi N.
      A review of drug interactions with boceprevir and telaprevir: implications for HIV and transplant patients.
      ]. This complicates their use in distinct patient populations with a high need for interferon-free regimens, such as liver transplanted patients or patients co-infected with human immunodeficiency virus (HIV).
      In summary, the high antiviral activity of NS3-4A protease inhibitors predestines these agents as favorable partners for all-oral regimens. However, as discussed below in detail, adequate combination partners are required to overcome the high risk for resistance development against NS3-4A inhibitors, and their usage in patients infected with HCV genotypes 2–6 is still limited.

      HCV NS5A inhibitors

      The HCV NS5A protein plays a manifold role in HCV replication, assembly and release [
      • Moradpour D.
      • Penin F.
      • Rice C.M.
      Replication of hepatitis C virus.
      ]. Hence, pharmacological inhibition of NS5A can tackle the HCV life cycle effectively at various stages. It was shown that doses of NS5A inhibitors in a picomolar range can sufficiently suppress HCV replication in vitro [
      • Gao M.
      • Nettles R.E.
      • Belema M.
      • Snyder L.B.
      • Nguyen V.N.
      • Fridell R.A.
      • et al.
      Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.
      ]. Furthermore, due to conserved structural features of domain I of NS5A, currently developed NS5A inhibitors are highly effective against all HCV genotypes. Despite these conserved structural features, NS5A inhibitors display a low genetic barrier to resistance, which resulted in rapid selection of RAVs during monotherapy [
      • Nettles R.E.
      • Gao M.
      • Bifano M.
      • Chung E.
      • Persson A.
      • Marbury T.C.
      • et al.
      Multiple ascending dose study of BMS-790052, an NS5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1.
      ].
      Daclatasvir (BMS-790052) was the first NS5A inhibitor reaching clinical evaluation. Daclatasvir monotherapy resulted in an approximately 4 log10 HCV RNA decline [
      • Nettles R.E.
      • Gao M.
      • Bifano M.
      • Chung E.
      • Persson A.
      • Marbury T.C.
      • et al.
      Multiple ascending dose study of BMS-790052, an NS5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1.
      ]. Like for NS3-4A protease inhibitors, RAVs during treatment with daclatasvir monotherapy were observed more frequently in HCV genotype 1a patients (main residues M28, Q30, L31, and Y93 of NS5A) compared to HCV genotype 1b patients (main residues L31 and Y93) [
      • Nettles R.E.
      • Gao M.
      • Bifano M.
      • Chung E.
      • Persson A.
      • Marbury T.C.
      • et al.
      Multiple ascending dose study of BMS-790052, an NS5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1.
      ,
      • Fridell R.A.
      • Wang C.
      • Sun J.H.
      • O’Boyle 2nd, D.R.
      • Nower P.
      • Valera L.
      • et al.
      Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in humans: in vitro and in vivo correlations.
      ]. In a phase IIa clinical trial in treatment-naïve HCV genotype 1 patients, Daclatasvir-based triple therapy resulted in extended rapid virologic response (eRVR) in up to 83% of patients, compared to 9% in the control group treated with pegIFN-α and ribavirin only [
      • Pol S.
      • Ghalib R.H.
      • Rustgi V.K.
      • Martorell C.
      • Everson G.T.
      • Tatum H.A.
      • et al.
      Daclatasvir for previously untreated chronic hepatitis C genotype-1 infection: a randomised, parallel-group, double-blind, placebo-controlled, dose-finding, phase 2a trial.
      ]. Other NS5A inhibitors (e.g., BMS-824393, ABT-267, PPI-461, GS-5885) are in earlier clinical development.
      Due to their high antiviral activity against all HCV genotypes, NS5A inhibitors are highly promising combination therapy partners for interferon-free therapy regimens with broad genotypic coverage. Furthermore, thus far available data indicate a good tolerability of NS5A inhibitors. However, like for NS3-4A protease inhibitors, adequate combination partners are required to overcome the low barrier to resistance against NS5A inhibitors.

      HCV N5B polymerase inhibitors

      NS5B RNA polymerase inhibitors can be divided into two distinct categories. Nucleoside analog inhibitors (NIs), like mericitabine (R7128) or sofosbuvir (GS-7977, former PSI-7977), mimic the natural substrates of the polymerase and are incorporated into the growing RNA chain, thus causing direct chain termination by tackling the active site of NS5B [
      • Koch U.
      • Narjes F.
      Allosteric inhibition of the hepatitis C virus NS5B RNA dependent RNA polymerase.
      ]. Because the active centre of NS5B is a highly conserved region of the HCV genome, NIs are usually effective against different HCV genotypes [
      • Lange C.M.
      • Sarrazin C.
      • Zeuzem S.
      Review article: specifically targeted anti-viral therapy for hepatitis C – a new era in therapy.
      ]. Single amino acid substitutions in every position of the active centre may result in loss of function or in extremely impaired replicative fitness. Thus, there is a high barrier to resistances for NIs.
      The development of several NIs (e.g., valopicitabine, R1626, PSI-938, BMS-986094) has been stopped due to toxicity issues. Of the earlier NIs, mericitabine (RG7128) is still in clinical development. Like most early NIs, mericitabine has a moderate antiviral activity and thus far RAVs against mericitabine have been observed very rarely in clinical studies [

      Le Pogam S, Yan J, Chhabra M, Ilnicka M, Kang H, Kosaka A, et al. Characterization of HCV quasispecies dynamics upon short term dual-therapy with the HCV NS5B nucleoside polymerase inhibitor mericitabine and the NS3/4 protease inhibitor danoprevir. Antimicrob Agents Chemother 2012 [epub].

      ,
      • Pawlotsky J.M.
      • Najera I.
      • Jacobson I.
      Resistance to mericitabine, a nucleoside analogue inhibitor of HCV RNA-dependent RNA polymerase.
      ,
      • Gane E.
      • Pockros P.
      • Zeuzem S.
      • Marcellin P.
      • Shikhman A.
      • Bernaards C.
      • et al.
      Interferon-free treatment with a combination of mericitabine and danoprevir with or without ribavirin in treatment-naive HCV genotype 1-infected patients.
      ]. Mericitabine-based triple therapy in HCV genotype 1, 2, and 3 infected patients revealed superior SVR rates compared to peg-IFN-α and ribavirin alone, though the increased chance of cure was lower as compared for example to NS3-4A inhibitor-based triple therapies [
      • Pockros P.
      • Jensen D.M.
      • Tsai N.
      First SVR data with the nucleoside analogue polymerase inhibitor mericitabine (RG7128) combined with peginterferon/ribavirin in treatment-naive HCV G1/4 patients: interim analysis of the JUMP-C trial.
      ]. Very promising clinical data have been recently published for sofosbuvir (GS-7977), another NI effective against all HCV genotypes. In HCV genotype 1, 2, and 3 infected patients, short durations of sofosbuvir-based triple therapy resulted in SVR rates of 90–100% [
      • Lawitz E.
      • Lalezari J.P.
      • Hassanein T.
      • Kowdley K.V.
      • Poordad F.
      • Sheikh A.M.
      • et al.
      Once-daily PSI-7977 plus peg/RBV in treatment-naive patients with HCV GT1: robust end of treatment response rates are sustained post-treatment.
      ,
      • Kowdley K.V.
      • Lawitz E.
      • Crespo I.
      • Hassanein T.
      • Davis M.
      • De Mico D.R.
      • et al.
      ATOMIC: 97% RVR for PSI-7977 + PEG/RBV × 12 week regimen in HCV GT1: an end to response-guided therapy?.
      ]. These data indicate that some newer NIs like sofosbuvir are also characterized by high antiviral activities. In addition, their high barrier to resistance development suggests that they are optimal candidates for interferon-free combination therapies.
      In contrast to NIs, the heterogeneous class of non-nucleoside inhibitors (NNIs) achieves NS5B inhibition by binding to different allosteric enzyme sites, which results in a conformational protein change before the elongation complex is formed [
      • Beaulieu P.L.
      Non-nucleoside inhibitors of the HCV NS5B polymerase: progress in the discovery and development of novel agents for the treatment of HCV infections.
      ]. For allosteric NS5B inhibition, high chemical affinity is required. NS5B is structurally organized in a characteristic “right hand motif”, containing finger, palm and thumb domains, and offers at least four NNI-binding sites, a benzimidazole-(thumb 1)-, thiophene-(thumb 2)-, benzothiadiazine-(palm 1)- and benzofuran-(palm 2)-binding site [
      • Lesburg C.A.
      • Cable M.B.
      • Ferrari E.
      • Hong Z.
      • Mannarino A.F.
      • Weber P.C.
      Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site.
      ]. Because NNIs bind distantly to the active centre of NS5B, their barrier to resistance is significantly lower as for NIs, and viral breakthrough has frequently been observed in monotherapy studies evaluating NNIs [
      • Wagner F.
      • Thompson R.
      • Kantaridis C.
      • Simpson P.
      • Troke P.J.
      • Jagannatha S.
      • et al.
      Antiviral activity of the hepatitis C virus polymerase inhibitor filibuvir in genotype 1-infected patients.
      ,
      • Larrey D.
      • Lohse A.W.
      • de Ledinghen V.
      • Trepo C.
      • Gerlach T.
      • Zarski J.P.
      • et al.
      Rapid and strong antiviral activity of the non-nucleosidic NS5B polymerase inhibitor BI 207127 in combination with peginterferon alfa 2a and ribavirin.
      ]. Currently, numerous non-nucleoside inhibitors are in phase I and II clinical evaluation (e. g., thumb 1 inhibitor BI207127; thumb 2 inhibitors filibuvir (PF-00868554), and VX-222; palm I inhibitors ANA598 and ABT-333; Palm II inhibitors tegobuvir (GS-9256) and IDX-375). In general, these non-nucleoside analogues display a low to medium antiviral activity as well as a low barrier to resistance [
      • Troke P.J.
      • Lewis M.
      • Simpson P.
      • Gore K.
      • Hammond J.
      • Craig C.
      • et al.
      Characterization of resistance to the nonnucleoside NS5B inhibitor filibuvir in hepatitis C virus-infected patients.
      ,
      • Afdhal N.
      • O’Brien C.
      • Godofsky E.
      Valopicitabine alone or with PEG-interferon/ribavirin retreatment in patients with HCV-1 infection and prior non-response to PEGIFN/RBV: One-year results.
      ,
      • Kneteman N.M.
      • Howe A.Y.
      • Gao T.
      • Lewis J.
      • Pevear D.
      • Lund G.
      • et al.
      HCV796: a selective nonstructural protein 5B polymerase inhibitor with potent anti-hepatitis C virus activity in vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus.
      ,
      • Villano S.
      • Raible D.
      • Harper D.
      • Speth J.
      • Chandra P.
      • Shaw P.
      • et al.
      Antiviral activity of the non-nucleoside polymerase inhibitor, HCV-796, in combination with pegylated interferon alfa-2b in treatment-naive patients with chronic HCV.
      ]. In contrast to NIs, NNIs in general do not display antiviral activity against different HCV genotypes [
      • Pockros P.J.
      Drugs in development for chronic hepatitis C: a promising future.
      ]. In view of these characteristics, NNIs will probably not be developed as triple therapy but rather as components of quadruple or all-oral regimens (see below).

      Drugs targeting host factors

      HCV depends on various host factors such as cyclophilin A throughout its life cycle [
      • Kaul A.
      • Stauffer S.
      • Berger C.
      • Pertel T.
      • Schmitt J.
      • Kallis S.
      • et al.
      Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.
      ]. Alisporivir (former Debio-025) is an orally bioavailable cyclophilin A inhibitor exerting an antiviral impact on both HCV and HIV replication. Alisporivir is characterized by a relatively high antiviral activity against all HCV genotypes and by a high barrier to resistance development [
      • Coelmont L.
      • Kaptein S.
      • Paeshuyse J.
      • Vliegen I.
      • Dumont J.M.
      • Vuagniaux G.
      • et al.
      Debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis C virus (HCV) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for HCV (STAT-C) inhibitors.
      ]. In treatment-naïve HCV genotype 1 patients, combination therapy with alisporivir, pegIFN-α-2a and ribavirin for 24–48 weeks resulted in SVR rates of 69–76% compared to 55% in the control group [
      • Flisiak R.
      • Pawlotsky J.M.
      • Crabbe R.
      • Callistru P.I.
      • Kryczka W.
      • Häussinger D.
      Once daily alisporivir (Debio025) plus pegIFNalfa2a/ribavirin results in superior sustained virologic response (SVR) in chronic hepatitis C genotype 1 treatment naive patients.
      ,
      • Flisiak R.
      • Feinman S.V.
      • Jablkowski M.
      • Horban A.
      • Kryczka W.
      • Pawlowska M.
      • et al.
      The cyclophilin inhibitor Debio 025 combined with PEG IFN alpha 2a significantly reduces viral load in treatment-naive hepatitis C patients.
      ]. Despite these promising data, the development of alisporivir is currently on hold due to rare cases of severe pancreatitis during combination therapy with alisporivir and pegIFN-α-2a. During alisporivir monotherapy, pancreatitis was not observed. Nevertheless, the pan-genotypic activity of alisporivir together with the low risk of alisporivir-resistance has proven the high potential of drugs targeting host factors for interferon-free treatment regimens.

      Other compounds

      Numerous additional approaches to inhibit the HCV life cycle are in preclinical or early clinical development. These include viral entry or assembly inhibitors, NS4B inhibitors, the micro-RNA-122 inhibitor miravirsen, silibinin (a flavonolignans of milk thistle, targeting the HCV life cycle at various steps), or immune-modulatory agents like toll-like receptor agonists [
      • Lange C.M.
      • Sarrazin C.
      • Zeuzem S.
      Review article: specifically targeted anti-viral therapy for hepatitis C – a new era in therapy.
      ,
      • Ferenci P.
      • Scherzer T.M.
      • Kerschner H.
      • Rutter K.
      • Beinhardt S.
      • Hofer H.
      • et al.
      Silibinin is a potent antiviral agent in patients with chronic hepatitis C not responding to pegylated interferon/ribavirin therapy.
      ,
      • Janssen H.L.
      • Reesink H.W.
      • Zeuzem S.
      • Lawitz E.
      • Rodriguez-Torres M.
      • Chen A.
      • et al.
      A randomized, double-blind, placebo controlled safety and anti-viral proof of concept study of miravirsen, an oligonucleotide targeting mir-122, in treatment naive patients with genotype 1 chronic HCV infection.
      ]. Notably, the development of miravirsen has proven for the first time that targeting a micro-RNA might be a suitable therapeutic option for viral (or other) diseases [
      • Janssen H.L.
      • Reesink H.W.
      • Zeuzem S.
      • Lawitz E.
      • Rodriguez-Torres M.
      • Chen A.
      • et al.
      A randomized, double-blind, placebo controlled safety and anti-viral proof of concept study of miravirsen, an oligonucleotide targeting mir-122, in treatment naive patients with genotype 1 chronic HCV infection.
      ]. A possible role of these agents in the treatment of chronic hepatitis C remains to be defined.

      Clinical trials evaluating interferon-free treatment regimens

      As it is well established for the treatment of HIV infection, combining DAA agents with different antiviral resistance profiles should result in a substantially decreased risk of viral breakthrough of RAVs [
      • Sarrazin C.
      • Zeuzem S.
      Resistance to direct antiviral agents in patients with hepatitis C virus infection.
      ]. Nucleoside analogue NS5B inhibitors, but also drugs targeting host factors such as the cyclophilin inhibitor alisporivir, display a high barrier to resistance and may therefore be key agents for effective DAA combination therapies. In contrast, NS3-4A and NS5A inhibitors display a low barrier to resistance development, but in view of their high antiviral efficacy they appear to be promising combination partners for NIs or cyclophilin inhibitors. Due to their low antiviral efficacy and low barrier to resistance development, the benefit of using non-nucleoside analogue NS5B inhibitors appears to be less pronounced compared to the above indicated drug classes. As described in the following, an increasing number of studies evaluating interferon-free treatment regimens have generally proven these theoretical considerations, but also yielded surprising results, e.g., with respect to the possible role of some NNIs (Table 2).
      Table 2Summary of presented interferon-free trials.
      These studies include quadruple therapies resulting in 100% SVR
      • Lok A.S.
      • Gardiner D.F.
      • Lawitz E.
      • Martorell C.
      • Everson G.T.
      • Ghalib R.
      • et al.
      Preliminary study of two antiviral agents for hepatitis C genotype 1.
      and 100% RVR
      • Zeuzem S.
      • Buggisch P.
      • Agarwal K.
      • Marcellin P.
      • Sereni D.
      • Klinker H.
      • et al.
      The protease inhibitor GS-9256 and non-nucleoside polymerase inhibitor tegobuvir alone, with RBV or peginterferon plus RBV in hepatitis C.
      , respectively.
      ∗∗SVR rates for all-oral arms have not yet been presented for this study. Quadruple-therapy arms in ZENITH resulted in high SVR rates.

      Combinations of NS3-4A inhibitors and nucleoside analogue NS5B inhibitors, with or without ribavirin

      The first interferon-free clinical trial (INFORM-1 study) evaluated the combination of an NI (mericitabine, R7128) and an NS3-4A inhibitor (danoprevir, R7227). In this proof-of-principle study, patients were treated with both compounds for up to 2 weeks [
      • Gane E.J.
      • Roberts S.K.
      • Stedman C.A.
      • Angus P.W.
      • Ritchie B.
      • Elston R.
      • et al.
      Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial.
      ]. HCV RNA concentrations decreased up to 5.2 log10IU/ml, viral breakthrough was observed in only one patient (but no RAVs were identified), and HCV RNA was undetectable at the end of dosing, in up to 63% of treatment-naïve patients. However, the fundamental question whether an SVR can be achieved with combination therapies of different DAA compounds without pegIFN-α and ribavirin had to be answered by subsequent trials. In the meanwhile, the INFORM-SVR study provided SVR data for the combination of mericitabine and danoprevir (ritonavir-boosted) with or without ribavirin for 12–24 weeks [
      • Gane E.
      • Pockros P.
      • Zeuzem S.
      • Marcellin P.
      • Shikhman A.
      • Bernaards C.
      • et al.
      Interferon-free treatment with a combination of mericitabine and danoprevir with or without ribavirin in treatment-naive HCV genotype 1-infected patients.
      ]. SVR rates in HCV genotype 1a and 1b patients were 26% and 71% in treatment arms including ribavirin (total n = 83), respectively, but significantly lower in all ribavirin-free treatment groups (total n = 86). Importantly, RAVs in patients with viral breakthrough were predominantly identified within NS3-4A while a resistance mutation in NS5B was discovered only in a single patient (S282T).
      Several phase II clinical trials assessed the combination of the NI sofosbuvir (GS-7977) with or without ribavirin. In the QUANTUM trial, treatment-naïve HCV genotype 1, 2, and 3 patients were treated for 12–24 weeks with sofosbuvir and ribavirin. In a recent interims analysis, 59% of HCV genotype 1 patients who completed the 12-week course of therapy achieved SVR at week 4 post treatment []. In this preliminary analysis, IL28B genotype appeared to be a predictor of treatment outcome. In ELECTRON, the same 12-week regimen resulted in SVR in only 11% (1/9) of HCV genotype 1a null responders, but in 88% (22/25) of treatment-naïve HCV genotype 1 patients, and in 80% (12/15) of treatment-experienced HCV genotype 2 and 3 patients, and in 100% (10/10) of treatment-naïve HCV genotype 2 and 3 patients [
      • Gane E.J.
      • Stedman C.A.
      • Hyland R.H.
      • Sorensen R.D.
      • Symonds W.T.
      • Hindes R.
      • et al.
      Once daily PSI-7977 plus RBV: pegylated interferon-alpha not required for complete rapid viral response in treatment-naive patients with HCV Gt2 or Gt3.
      ,
      • Gane E.
      • Stedman C.A.
      • Hyland R.H.
      • Sorensen R.D.
      • Symonds W.T.
      • Hindes R.
      • et al.
      Electron: once daily PSI-7977 plus RBV in HCV Gt1/2/3.
      ]. In addition, the ELECTRON study evaluated sofosbuvir monotherapy in treatment-naïve HCV genotype 2 and 3 patients, which, however, resulted in SVR in only 60% (6/10) of the cases, highlighting again the important role of ribavirin in interferon-free treatment regimens [
      • Gane E.J.
      • Stedman C.A.
      • Hyland R.H.
      • Sorensen R.D.
      • Symonds W.T.
      • Hindes R.
      • et al.
      Once daily PSI-7977 plus RBV: pegylated interferon-alpha not required for complete rapid viral response in treatment-naive patients with HCV Gt2 or Gt3.
      ]. In the meanwhile, phase III approval trials (FISSION, POSITRON, FUSION, and NEUTRINO) have been initiated to evaluate sofosbuvir + ribavirin for 12–16 weeks in HCV genotype 2 and 3 patients.

      Combinations of NS3-4A inhibitors and non-nucleoside analogue NS5B inhibitors, with or without ribavirin

      The SOUND-C1 trial assessed the combination therapy of the NS3-4A inhibitor BI-201335, the NNI BI-207127 (400 or 600 mg q8h) and ribavirin for 4 weeks [
      • Zeuzem S.
      • Asselah T.
      • Angus P.
      • Zarski J.P.
      • Larrey D.
      • Mullhaupt B.
      • et al.
      Efficacy of the protease inhibitor BI 201335, polymerase inhibitor BI 207127, and ribavirin in patients with chronic HCV infection.
      ]. Virologic response rates in patients treated with 600 mg q8h of BI-207127 were 82%, 100%, and 100% at treatment days 15, 22, and 29, respectively. In patients who received the lower dose of BI-207127, virologic response rates were significantly lower, and in these patients lower virologic response rates were observed for patients infected with HCV subtype 1a compared to HCV subtype 1b. SOUND-C1 provided no SVR rates for this all-oral regimen, since patients received pegIFN-α-based therapy from treatment week five. However, the larger SOUND-C2 trial provided SVR rates for BI-201335 in combination with BI-207127 with or without ribavirin, administered for 16–40 weeks in approximately 360 treatment-naïve HCV genotype 1 patients [
      • Zeuzem S.
      • Soriano A.
      • Asselah T.
      • Bronowicki J.P.
      • Lohse A.
      • Müllhaupt B.
      • et al.
      SVR4 and SVR12 with an interferon-free regimen of BI201335 and BI207127, +/-ribavirin, in treatment-naive patients with chronic genotype-1 HCV infection: interim results of Sound-C2.
      ]. Overall, SVR12 rates ranged from 56% to 68% in treatment arms including ribavirin, compared to 39% in a single ribavirin-free treatment arm. Within all treatment arms, SVR rates were consistently higher in HCV genotype 1b vs. 1a patients or in patients with a good-response IL28B genotype.
      The combination of tegobuvir (GS-9190), another NNI, with GS-9256, an NS3-4A inhibitor, ± ribavirin was assessed in another trial in treatment-naïve HCV genotype 1 patients [
      • Zeuzem S.
      • Buggisch P.
      • Agarwal K.
      • Marcellin P.
      • Sereni D.
      • Klinker H.
      • et al.
      The protease inhibitor GS-9256 and non-nucleoside polymerase inhibitor tegobuvir alone, with RBV or peginterferon plus RBV in hepatitis C.
      ]. Importantly, tegobuvir + GS-9256 + ribavirin led to a higher RVR rates compared to tegobuvir + GS-9256 alone (38% vs. 7%, respectively), further proving the possible benefit of ribavirin in distinct interferon-free DAA combination therapies. However, with tegobuvir and GS-9256-based quadruple therapy (pegIFN-α, and ribavirin), RVR was achieved in 100% of patients.
      A comparable approach is followed in the ZENITH trial, assessing the antiviral activity of the NS3-4A inhibitor telaprevir and the NNI VX-222 alone, in combination with ribavirin, or in combination with pegIFN-α and ribavirin (quadruple therapy), in treatment-naive HCV genotype 1 patients. Again, quadruple therapy led to high SVR rates. However, in the all-oral treatment arms, high rates of viral breakthrough were observed [
      • Nelson D.R.
      • Gane E.J.
      • Jacobson I.M.
      • Di Bisceglie A.M.
      • Alves K.
      • Koziel M.J.
      VX-222/telaprevir in combination with peginterferon-alfa-2a and ribavirin in treatment-naive genotype 1 HCV patients treated for 12 weeks: Zenith study, SVR 12 interim analyses.
      ].
      Compared to the above described data for all-oral combinations based on NS3-4A inhibitors plus NNIs, results of the recently presented Co-Pilot study were strongly encouraging. In Co-Pilot, 12 weeks of combination therapy with the NS3-4A inhibitor ABT-450 (ritonavir-boosted), the NNI ABT-333, and ribavirin resulted in 93% and 47% SVR in treatment-naïve HCV genotype 1 patients and in previous null responders to pegIFN-α and ribavirin alone, respectively [
      • Poordad F.
      • Lawitz E.
      • Kowdley K.V.
      • Everson G.T.
      • Freilich B.
      • Cohen D.
      • et al.
      12-Week interferon-free regimen of ABT-450/R +ABT-333 +ribavirin achieved SVR12 in more than 90% of treatment-naive HCV genotype-1-infected subjects and in 47% of previous non-responders.
      ]. In addition, in the single arm of the so-called Pilot study evaluating ABT-450 in combination with the NNI ABT-072 and ribavirin, SVR was achieved in 91% (10/11) of treatment-naïve HCV genotype 1 patients, who, however, all had a good-response IL28B genotype [
      • Lawitz E.
      • Poordad F.
      • Kowdley K.V.
      • Jensen D.M.
      • Cohen D.
      • Siggelkow S.
      • et al.
      12-Week interferon-free regimen of ABT-450/R, ABT-072, and ribavirin was well tolerated and achieved sustained virologic response in 91% treatment-naive HCVIL28B-CC genotype-1-infected subjects.
      ]. Importantly, a single patient in Pilot, who had achieved SVR 24 weeks after treatment completion, experienced a late viral relapse between week 24 and 36 post treatment. In this patient, a resistant mutant in NS5B has been discovered, and the question remains whether interferon-free treatment regimens require longer follow-up times than they have been established for pegIFN-α and ribavirin therapy.

      Combinations of NS3-4A inhibitors and NS5A inhibitors

      The first clinical trial which has reported SVR data for an interferon-free regimen investigated therapy with the NS5A inhibitor daclatasvir (BMS-790052) in combination with the NS3-4A protease inhibitor asunaprevir (BMS-60032) for 24 weeks in 10 HCV genotype 1 patients with prior null response to pegIFN-α and ribavirin [
      • Lok A.S.
      • Gardiner D.F.
      • Lawitz E.
      • Martorell C.
      • Everson G.T.
      • Ghalib R.
      • et al.
      Preliminary study of two antiviral agents for hepatitis C genotype 1.
      ]. Importantly, 36% of all patients achieved SVR 24 weeks after treatment completion. All patients with viral breakthrough were infected with HCV genotype 1a, and in all of them RAVs against both agents were detected. Importantly, a quadruple arm included in this study (daclatasvir, asunaprevir, pegIFN-α, and ribavirin) resulted in 100% SVR in both HCV genotype 1a and 1b patients, highlighting the perhaps still important role of pegIFN-α and ribavirin in preventing resistance in such highly difficult-to-cure patient populations. Nevertheless, this trial constituted a proof-of-principle that SVR can be achieved by all-oral regimens, especially in patients infected with HCV subtype 1b. This has been confirmed with a 100% SVR rate by a small study evaluating the same agents (daclatasvir and asunaprevir) in Japanese HCV genotype 1b infected previous null responder patients [
      • Chayama K.
      • Takahashi S.
      • Toyota J.
      • Karino Y.
      • Ikeda K.
      • Ishikawa H.
      • et al.
      Dual therapy with the nonstructural protein 5A inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis C virus genotype 1b-infected null responders.
      ], and by a subsequent Japanese study in HCV genotype 1b patients with prior null response (n = 21) or ineligibility to IFN-therapy (n = 22), in whom SVR rates of 91% and 64% have been achieved, respectively [
      • Suzuki F.
      • Ikeda K.
      • Toyota J.
      • Karino Y.
      • Ohmura T.
      • Chayama K.
      • et al.
      Dual oral therapy with the NS5A inhibitor daclatasvir (BMS-790052) and NS3 protease inhibitor asunaprevir (BMS-650032) in HCV genotype 1b-infected null responders or ineligible/intolerant to peginterferon/ribavirin.
      ].

      Combinations of NS5A inhibitors and nucleoside analogue NS5B inhibitors with ribavirin

      Impressive results have been shown for the combination of the NS5A inhibitor daclatasvir with the NI sofosbuvir, with or without ribavirin for 24 weeks [
      • Sulkowski M.S.
      • Gardiner D.F.
      • Lawitz E.
      • Hinestrosa F.
      • Nelson D.R.
      • Thuluvath P.J.
      • et al.
      Potent viral suppression with all-oral combination of daclatasvir (NS5A inhibitor) and GS-7977 (NS5B inhibitor), +/− ribavirin, in treatment-naive patients with chronic HCV GT1, 2, or 3.
      ]. In approximately 90 treatment-naïve patients, RVR and SVR rates were 100% and 100% in HCV genotype 1 patients, and 100% and 91% in HCV genotype 2 and 3 patients, respectively. In this study, the addition of ribavirin did not improve virologic response rates but resulted in anemia in a significant proportion of patients. Furthermore, an unfavorable IL28B genotype apparently did not decrease the chance of cure in this study. Due to different strategies in the pipelines of Bristol-Myers Squibb and Gilead, the development of this promising regimen has been halted.

      Combinations of multiple DAAs with ribavirin

      A preliminary analysis of a first study evaluating a regimen containing multiple DAAs, namely NS5A inhibitor GS-5885, NS3-4A inhibitor GS-9451, NNI tegobuvir, and ribavirin has been presented recently [
      • Sulkowski M.S.
      • Rodriguez-Torres M.
      • Lawitz E.
      • Shiffman M.
      • Pol S.
      • Herring R.
      • et al.
      High sustained virologic response rate in treatment-naive HCV genotype 1a and 1b patients treated for 12 weeks with an interferon-free all-oral quad regimen: interim results.
      ]. In this so called QUAD study, treatment-naïve HCV genotype 1 patients were treated for 12–24 weeks with this all-oral quadruple regimen. Patients were switched to a pegIFN-α-based rescue therapy if HCV RNA did not fall below the limit of detection until treatment week 2. Approximately 70% of all included patients were eligible for all-oral therapy in this study, among them, at least 77% and 89% of HCV genotype 1a and 1b patients achieved SVR, respectively.

      Cyclophilin inhibitor-based therapies

      The VITAL-1 phase IIb clinical study evaluated the cyclophilin inhibitor alisporivir with or without ribavirin for 24 weeks in treatment-naïve HCV genotype 2 and 3, complemented by the addition of pegIFN-α if no RVR was achieved [
      • Pawlotsky J.M.
      • Sarin S.K.
      • Foster G.
      • Peng C.Y.
      • Rasenack J.
      • Flisiak R.
      • et al.
      Alisporivir plus ribavirin is highly effective as interferon-free or interferon-add-on regimen in previously untreated HCV-GT2 or GT3 patients: SVR12 results from the VITAL-1 phase 2b study.
      ]. SVR rates in the per protocol analyses were approximately 90% in all treatment arms, but only 29–42% of all patients were eligible for treatment with all-oral therapy.

      Perspectives and challenges for the further development of IFN-free regimens

      The above described data have clearly shown that an SVR can be achieved in different HCV infected patient populations with various interferon-free DAA combination regimens. In view of the inconvenience and high rate of significant side-effects of IFN-based therapy, all-oral regimens will therefore certainly become a first choice for a number of patients in the very near future, a scenario which had appeared speculative until very recently. Nevertheless, available data from phase I and II clinical trials evaluating interferon-free regimens have not yet revealed a clearly outstanding all-oral combination, and numerous challenges remain to be addressed by intensive research.
      A key lesson from the body of available data is that those patients who do not respond well to IFN-α-based therapies, especially previous null responders to pegIFN-α and ribavirin therapy, do not respond well to many interferon-free regimens as well, independently of whether these regimens include ribavirin or not. Exemplarily, as described above in detail, the combination regimen of the NS3-4A inhibitor ABT-450/r, NNI ABT-333, and ribavirin resulted in SVR rates of 100% and 47% in HCV genotype 1 treatment-naïve patients and previous null responders, respectively [
      • Poordad F.
      • Lawitz E.
      • Kowdley K.V.
      • Everson G.T.
      • Freilich B.
      • Cohen D.
      • et al.
      12-Week interferon-free regimen of ABT-450/R +ABT-333 +ribavirin achieved SVR12 in more than 90% of treatment-naive HCV genotype-1-infected subjects and in 47% of previous non-responders.
      ]. In addition to previous treatment outcome, IL28B genotype appears to be a predictor of success of some (but not of all) interferon-free regimens, although its impact is significantly attenuated compared to pegIFN-α and ribavirin dual therapy [
      • Zeuzem S.
      • Soriano A.
      • Asselah T.
      • Bronowicki J.P.
      • Lohse A.
      • Müllhaupt B.
      • et al.
      SVR4 and SVR12 with an interferon-free regimen of BI201335 and BI207127, +/-ribavirin, in treatment-naive patients with chronic genotype-1 HCV infection: interim results of Sound-C2.
      ,
      • Sulkowski M.S.
      • Rodriguez-Torres M.
      • Lawitz E.
      • Shiffman M.
      • Pol S.
      • Herring R.
      • et al.
      High sustained virologic response rate in treatment-naive HCV genotype 1a and 1b patients treated for 12 weeks with an interferon-free all-oral quad regimen: interim results.
      ,
      • Chu T.W.
      • Kulkarni R.
      • Gane E.J.
      • Roberts S.K.
      • Stedman C.
      • Angus P.W.
      • et al.
      Effect of IL28B genotype on early viral kinetics during interferon-free treatment of patients with chronic hepatitis C.
      ]. Regarding the completely different mode of operation of immune-modulating IFN-α-based therapy in contrast to IFN-free DAA regimens, which directly tackle HCV, these findings are not self-evident. A nearby explanation of these results might be that an appropriate endogenous immune response against HCV is still required for the final clearance of residual, possibly drug-resistant virus escaping DAA combinations. In addition to these host-associated determinants of treatment outcome, there clearly exist virus-associated factors which obviously attain significant importance in the upcoming era of interferon-free therapy. For all-oral regimens (and for IFN-α-based triple therapy approaches) based on either NS3-4A inhibitors or NS5A inhibitors, lower SVR rates have consistently been observed in HCV genotype 1a vs. 1b patients, a difference which can be explained by the significantly lower genetic barrier of resistance mutations against these drugs at defined positions in the HCV genotype 1a vs. 1b genome [
      • Sarrazin C.
      • Zeuzem S.
      Resistance to direct antiviral agents in patients with hepatitis C virus infection.
      ,
      • McCown M.F.
      • Rajyaguru S.
      • Kular S.
      • Cammack N.
      • Najera I.
      GT-1a or GT-1b subtype-specific resistance profiles for hepatitis C virus inhibitors telaprevir and HCV-796.
      ,
      • Fridell R.A.
      • Wang C.
      • Sun J.H.
      • O’Boyle 2nd, D.R.
      • Nower P.
      • Valera L.
      • et al.
      Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in humans: in vitro and in vivo correlations.
      ]. Hence, HCV genotype 1a infected patients will require special attention in the era of DAA therapies.
      The high replication rate of HCV and the poor fidelity of its RNA-dependent RNA polymerase are reasons for numerous HCV variants (quasispecies) which are continuously produced during HCV replication [
      • Sarrazin C.
      • Zeuzem S.
      Resistance to direct antiviral agents in patients with hepatitis C virus infection.
      ]. Among them, variants carrying mutations altering the conformation of the binding sites of DAA compounds can develop. Whether such naturally existing HCV isolates, which may confer resistance to DAAs, have an impact on the success of IFN-free combination therapy is currently not well defined [
      • Gaudieri S.
      • Rauch A.
      • Pfafferott K.
      • Barnes E.
      • Cheng W.
      • McCaughan G.
      • et al.
      Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy.
      ,
      • Kuntzen T.
      • Timm J.
      • Berical A.
      • Lennon N.
      • Berlin A.M.
      • Young S.K.
      • et al.
      Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naive patients.
      ]. However, it appears plausible that such pre-existing HCV variants, which have been vastly neglected during the era of pegIFN-α and ribavirin therapy, can significantly influence the outcome of IFN-free treatment regimens [
      • Gaudieri S.
      • Rauch A.
      • Pfafferott K.
      • Barnes E.
      • Cheng W.
      • McCaughan G.
      • et al.
      Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy.
      ,
      • Kuntzen T.
      • Timm J.
      • Berical A.
      • Lennon N.
      • Berlin A.M.
      • Young S.K.
      • et al.
      Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naive patients.
      ]. Intensive research is required to clarify whether the presence of distinct HCV isolates at baseline may already predict a limited chance of cure with specific DAA combinations in a given patient. Furthermore, a detailed characterization of the HCV quasispecies in patients who failed to be cured by IFN-free therapies will be important, in particular with respect to the selection of appropriate second-line regimens.
      How can these insights be translated into the development of an ideal IFN-free regimen, which should be a regimen with minimal pill burden and side-effects, limited drug–drug interactions, and the potency to cure almost all HCV-infected individuals, independently of previous treatment outcome or infection with distinct HCV genotypes? Currently, patients with a good responsiveness to IFN-α (e.g., previous relapsers, patients with good response IL28B genotype without significant liver fibrosis, or HCV genotype 2 and 3 infected patients) apparently have a good chance of cure with both triple therapy approaches as well as with selected all-oral regimens [
      • Zeuzem S.
      • Andreone P.
      • Pol S.
      • Lawitz E.
      • Diago M.
      • Roberts S.
      • et al.
      Telaprevir for retreatment of HCV infection.
      ,
      • Kowdley K.V.
      • Lawitz E.
      • Crespo I.
      • Hassanein T.
      • Davis M.
      • De Mico D.R.
      • et al.
      ATOMIC: 97% RVR for PSI-7977 + PEG/RBV × 12 week regimen in HCV GT1: an end to response-guided therapy?.
      , ,
      • Lawitz E.
      • Poordad F.
      • Kowdley K.V.
      • Jensen D.M.
      • Cohen D.
      • Siggelkow S.
      • et al.
      12-Week interferon-free regimen of ABT-450/R, ABT-072, and ribavirin was well tolerated and achieved sustained virologic response in 91% treatment-naive HCVIL28B-CC genotype-1-infected subjects.
      ,
      • Lange C.M.
      • Zeuzem S.
      IL28B single nucleotide polymorphisms in the treatment of hepatitis C.
      ]. All-oral regimens for such “easy to cure” patients may include combinations of two DAAs (NS3-4A inhibitors, NS5A inhbitors, NIs or even NNIs), with, or – in selected regimens – even without ribavirin. While a high chance of cure in general is realistic in these patient populations with favorable baseline characteristics, further improvement is still relevant in terms of side-effects and convenience of available treatment options. However, in difficult-to-cure patients, such as patients with liver cirrhosis or HCV genotype 1a patients with previous null-response to pegIFN-α/ribavirin, most thus far investigated combinations of two DAAs ± ribavirin cannot prevent viral breakthrough in a significant proportion of patients. The addition of pegIFN-α and ribavirin as quadruple therapy approach has been proven highly effective to cure even null responders infected with HCV subtype 1a, but obviously this approach has a high risk of significant side effects and it is no option for patients not tolerating IFN-α [
      • Lok A.S.
      • Gardiner D.F.
      • Lawitz E.
      • Martorell C.
      • Everson G.T.
      • Ghalib R.
      • et al.
      Preliminary study of two antiviral agents for hepatitis C genotype 1.
      ,
      • Zeuzem S.
      • Asselah T.
      • Angus P.
      • Zarski J.P.
      • Larrey D.
      • Mullhaupt B.
      • et al.
      Efficacy of the protease inhibitor BI 201335, polymerase inhibitor BI 207127, and ribavirin in patients with chronic HCV infection.
      ]. Hence, it will be key to define DAA combination partners which could replace IFN-α for a successful all-oral therapy of these patients. Perhaps, optimized dual DAA regimens combining compounds with a very high antiviral activity (e.g., potent NS3-4A or NS5A inhibitors) together with a very high barrier to resistance (e.g., potent NIs like sofosbuvir or cyclophilin inhibitors) with or without ribavirin might still be successful even in difficult-to-cure HCV-infected patients. For example, the combination of daclatasvir plus sofosbuvir resulted in 100% cure in treatment-naïve HCV genotype 1 patients, independent of IL28B genotype or HCV subtype [
      • Sulkowski M.S.
      • Gardiner D.F.
      • Lawitz E.
      • Hinestrosa F.
      • Nelson D.R.
      • Thuluvath P.J.
      • et al.
      Potent viral suppression with all-oral combination of daclatasvir (NS5A inhibitor) and GS-7977 (NS5B inhibitor), +/− ribavirin, in treatment-naive patients with chronic HCV GT1, 2, or 3.
      ]. However, this combination has not been evaluated in previous null responders, and thus far no all-oral regimen achieving satisfying SVR rates in HCV genotype 1a null responders has been presented. Whether longer treatment durations, the combination of more DAAs (e. g., three DAAs plus ribavirin), application of even more potent DAAs, or perhaps the addition of alternative immune-modulatory agents can cure these patients is one of the most important questions for research in hepatology. Furthermore, it will be important to confirm efficacy and safety of already presented all-oral combinations in larger phase III clinical trials, as most studies evaluating IFN-free combinations thus far included only small numbers of highly selected patients (with the notable exception of Sound-C2, including 10% of cirrhotic patients of approximately 360 patients total [
      • Zeuzem S.
      • Soriano A.
      • Asselah T.
      • Bronowicki J.P.
      • Lohse A.
      • Müllhaupt B.
      • et al.
      SVR4 and SVR12 with an interferon-free regimen of BI201335 and BI207127, +/-ribavirin, in treatment-naive patients with chronic genotype-1 HCV infection: interim results of Sound-C2.
      ]).
      Several additional, more practical issues require attention for the further development of all-oral regimens. Importantly, most NS3-4A as well as NNIs have been developed exclusively for HCV genotype 1. Hence, the choice of DAA agents for treating HCV genotype 2–6 is currently restricted to other compounds, especially NS5A inhibitors, NIs, cyclophilin inhibitors, and some second-wave NS3-4A inhibitors [
      • Lange C.M.
      • Sarrazin C.
      • Zeuzem S.
      Review article: specifically targeted anti-viral therapy for hepatitis C – a new era in therapy.
      ]. Furthermore, possible drug–drug interactions within IFN-free regimens as well as with concomitant medications require attention [
      • Wilby K.J.
      • Greanya E.D.
      • Ford J.A.
      • Yoshida E.M.
      • Partovi N.
      A review of drug interactions with boceprevir and telaprevir: implications for HIV and transplant patients.
      ]. Finally, a few cases of late relapses after all-oral as well as after triple-therapies are worrisome, possibly making a reassessment of the equitation of SVR at week 24 post treatment with cure from HCV infection necessary [
      • Lawitz E.
      • Poordad F.
      • Kowdley K.V.
      • Jensen D.M.
      • Cohen D.
      • Siggelkow S.
      • et al.
      12-Week interferon-free regimen of ABT-450/R, ABT-072, and ribavirin was well tolerated and achieved sustained virologic response in 91% treatment-naive HCVIL28B-CC genotype-1-infected subjects.
      ,
      • Hezode C.
      • Forestier N.
      • Dusheiko G.
      • Ferenci P.
      • Pol S.
      • Goeser T.
      • et al.
      Telaprevir and peginterferon with or without ribavirin for chronic HCV infection.
      ].

      Conflict of interest

      C.M.L.: Consultancy for Roche. S.Z.: Consultancy for Abbott, Achillion, Astrazeneca, BMS, Gilead, Idenix, Janssen, Merck, Novartis, Presidio, Roche, Santaris, and Vertex.

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