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NS5A inhibitors in the treatment of hepatitis C

  • Jean-Michel Pawlotsky
    Correspondence
    Corresponding author. Address: Department of Virology, Hôpital Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France. Tel.: +33 1 4981 2827; fax: +33 1 4981 4831.
    Affiliations
    National Reference Centre for Viral Hepatitis B, C and Delta, Department of Virology, Henri Mondor Hospital, University of Paris-Est, Créteil, France
    INSERM U955, Créteil, France
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Open AccessPublished:April 08, 2013DOI:https://doi.org/10.1016/j.jhep.2013.03.030

      Summary

      Hepatitis C virus infection is a major health problem worldwide and no vaccine has yet been developed against this virus. In addition, currently approved pharmacotherapies achieve suboptimal cure rates and have side effects that result in non-compliance and premature treatment discontinuation. Significant research has been devoted to developing direct-acting antiviral agents that inhibit key viral functions. In particular, several novel drug candidates that inhibit the viral non-structural protein 5A (NS5A) have been demonstrated to possess high potency, pan-genotypic activity, and a high barrier to resistance. Clinical trials using combination therapies containing NS5A inhibitors have reported results that promise high cure rates and raise the possibility of developing interferon-free, all-oral regimens.

      Abbreviations:

      HCV (hepatitis C virus), DAA (direct acting antiviral), NS (nonstructural), RdRp (RNA-dependent RNA polymerase), IFN (interferon), UTR (untranslated region), IRES (internal ribosome entry site), SVR (sustained virological response), RVR (rapid virologic response), cEVR (complete early virologic response)

      Keywords

      Introduction

      Recent estimates indicate that there are more than 120–130 million chronic hepatitis C virus (HCV) carriers worldwide [
      Global Burden of Hepatitis C Working Group
      Global burden of disease (GBD) for hepatitis C.
      ], who are at risk of developing cirrhosis and/or hepatocellular carcinoma (primary liver cancer). As many as 4 million persons are thought to be chronically infected in the US [

      World Health Organization. Global alert and response, Hepatitis C. <http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html>.

      ], 5–10 million in Europe [

      World Health Organization. Global alert and response, Hepatitis C. <http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html>.

      ], 12 million in India [

      World Health Organization. Global alert and response, Hepatitis C. <http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html>.

      ], and 1.2 million in Japan (2004 figure) [
      • Toyoda H.
      • Kumada T.
      • Kiriyama S.
      • Sone Y.
      • Tanikawa M.
      • Hisanaga Y.
      • et al.
      Changes in hepatitis C virus (HCV) antibody status in patients with chronic hepatitis C after eradication of HCV infection by interferon therapy.
      ]. Most of these individuals are not aware of their infection. The incidence of acute infection in the US has declined from 7.4/100,000 in 1982 to 0.7/100,000 in recent years, primarily due to screening of blood in transfusion centres and improved safety of intravenous drug use [
      • Poordad F.
      • Dieterich D.
      Treating hepatitis C: current standard of care and emerging direct-acting antiviral agents.
      ]. It is estimated that approximately 150,000 new cases occur annually in the United States and in Western Europe, and about 350,000 in Japan. Only 25% of acute cases are symptomatic, but up to 80% of these acute cases progress to chronic infection and liver disease, and up to 20% of chronic infections progress to cirrhosis [

      World Health Organization. Global alert and response, Hepatitis C. <http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html>.

      ]. Every year, 4–5% of cirrhotic patients develop hepatocellular carcinoma [
      • Fattovich G.
      • Stroffolini T.
      • Zagni I.
      • Donato F.
      Hepatocellular carcinoma in cirrhosis: incidence and risk factors.
      ]. Despite the decrease in HCV incidence, the number of patients with chronic HCV-related complications is increasing in those aging patients who have been infected for many years, and chronic hepatitis C infection will continue to be a significant cause of premature mortality, causing at least 200,000–300,000 deaths per year worldwide [
      • Poordad F.
      • Dieterich D.
      Treating hepatitis C: current standard of care and emerging direct-acting antiviral agents.
      ].
      A number of direct-acting antiviral agents (DAAs) are under development for the treatment of chronic HCV infection. These agents block viral production by directly inhibiting one of several steps of the HCV lifecycle. As shown in Fig. 1, the genomic organization of HCV has been elucidated, and several viral proteins involved in the HCV lifecycle, such as the non-structural (NS) 3/4A serine protease, the NS5B RNA-dependent RNA polymerase (RdRp), and the NS5A protein, have been targeted for drug development [
      • Poordad F.
      • Dieterich D.
      Treating hepatitis C: current standard of care and emerging direct-acting antiviral agents.
      ]. Two NS3/4A protease inhibitors, telaprevir and boceprevir, which inhibit post-translational processing of the HCV polyprotein into individual non-structural proteins, have been approved by the US Food and Drug Administration, the European Medicines Agency, and several other regulatory agencies for the treatment of chronic HCV genotype 1 infection in combination with pegylated interferon (IFN)-α and ribavirin [

      Victrelis (boceprevir). Package insert 2005. Merck Sharp & Dohme Corp., Whitehouse Station, NJ, USA.

      ,

      Incivek (telaprevir). Package insert 2012. Vertex Pharmaceuticals, Cambridge, MA, USA.

      ].
      Figure thumbnail gr1
      Fig. 1Structural organization of HCV RNA and viral proteins. NS, non-structural; UTR, untranslated region.

      HCV structure and lifecycle, and physiological role of the NS5A protein

      HCV is an enveloped virus with a single-stranded positive RNA genome of approximately 9.6 kb. At the flanking ends of the genome are 2 highly conserved untranslated regions (UTRs). The 5′ UTR is highly structured and contains the internal ribosome entry site (IRES), which is important for the initiation of the cap-independent translation of the polyprotein [
      • Reynolds J.E.
      • Kaminski A.
      • Kettinen H.J.
      • Grace K.
      • Clarke B.E.
      • Carroll A.R.
      • et al.
      Unique features of internal initiation of hepatitis C virus RNA translation.
      ]. The 3′ UTR consists of a short genotype-specific variable region, a tract consisting solely of pyrimidine residues (predominantly uridine) and a conserved 98-nucleotide sequence, known as X region, containing 3 stem-loops [
      • Kolykhalov A.A.
      • Feinstone S.M.
      • Rice C.M.
      Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA.
      ,
      • Foster T.L.
      • Belyaeva T.
      • Stonehouse N.J.
      • Pearson A.R.
      • Harris M.
      All three domains of the hepatitis C virus nonstructural NS5A protein contribute to RNA binding.
      ]. The HCV open reading frame is situated between the two UTRs.
      After entering the bloodstream, HCV binds to a receptor complex at the surface of its target cells, the hepatocytes. The envelope glycoproteins E1 and E2 are essential for target cell recognition, binding, and internalization [
      • Andre P.
      • Perlemuter G.
      • Budkowska A.
      • Brechot C.
      • Lotteau V.
      Hepatitis C virus particles and lipoprotein metabolism.
      ]. The bound virus then undergoes clathrin-mediated endocytosis [
      • Blanchard E.
      • Belouzard S.
      • Goueslain L.
      • Wakita T.
      • Dubuisson J.
      • Wychowski C.
      • et al.
      Hepatitis C virus entry depends on clathrin-mediated endocytosis.
      ]. Acidification of the endocytosis vesicle frees the genomic RNA from the nucleocapsid for release into the cytoplasm. Along with host RNA molecules, the viral RNA migrates to the endoplasmic reticulum (ER). Binding of the 40S ribosomal subunit to the HCV IRES produces a stable pre-initiation complex that begins translation of the viral open reading frame to generate an approximately 3000 amino acid polyprotein. Following translation, the polyprotein is cleaved by both cellular and viral proteases to produce at least 10 viral proteins, including structural proteins (core, E1 and E2) and non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [
      • Choo Q.L.
      • Richman K.H.
      • Han J.H.
      • Berger K.
      • Lee C.
      • Dong C.
      • et al.
      Genetic organization and diversity of the hepatitis C virus.
      ,
      • Lindenbach B.D.
      • Rice C.M.
      Unravelling hepatitis C virus replication from genome to function.
      ].
      Viral replication (i.e., the synthesis of new positive RNA genomes that may also serve as messenger RNAs for viral protein synthesis) is catalyzed by the viral RdRp, or NS5B protein. A negative-strand intermediate of replication is initially produced, which then serves as a template for the synthesis of numerous positive strands. The NS5A viral protein has been shown to play an important role in the regulation of replication. In addition, host cell proteins, such as cyclophilin A, act as necessary co-factors of HCV replication through their interactions with both NS5A and the RdRp in the replication complex [
      • Hanoulle X.
      • Badillo A.
      • Wieruszeski J.M.
      • Verdegem D.
      • Landrieu I.
      • Bartenschlager R.
      • et al.
      Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.
      ,
      • Chatterji U.
      • Bobardt M.D.
      • Lim P.
      • Gallay P.A.
      Cyclophilin A-independent recruitment of NS5A and NS5B into hepatitis C virus replication complexes.
      ].
      The non-structural NS5A protein bears pleiotropic functions, including roles in viral replication and assembly, and complex interactions with cellular functions. The latter include inhibition of apoptosis and promotion of tumorigenesis, both of which may play a role in the triggering of the hepatocarcinogenic process [
      • Ghosh A.K.
      • Steele R.
      • Meyer K.
      • Ray R.
      • Ray R.B.
      Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth.
      ,
      • Lan K.H.
      • Sheu M.L.
      • Hwang S.J.
      • Yen S.H.
      • Chen S.Y.
      • Wu J.C.
      • et al.
      HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis.
      ,
      • Arima N.
      • Kao C.Y.
      • Licht T.
      • Padmanabhan R.
      • Sasaguri Y.
      Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A.
      ,

      Higgs MR, Lerat H, Pawlotsky JM. Hepatitis C virus-induced activation of beta-catenin promotes c-Myc expression and a cascade of pro-carcinogenetic events. Oncogene, in press.

      ]. The protein is comprised of approximately 447 amino acids and localizes to ER-derived membranes. It basally exists in phosphorylated (p56) and hyperphosphorylated (p58) forms that are implicated in different functions [
      • Evans M.J.
      • Rice C.M.
      • Goff S.P.
      Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.
      ,
      • Neddermann P.
      • Quintavalle M.
      • Di Pietro C.
      • Clementi A.
      • Cerretani M.
      • Altamura S.
      • et al.
      Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture.
      ,
      • Fridell R.A.
      • Qiu D.
      • Valera L.
      • Wang C.
      • Rose R.E.
      • Gao M.
      Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052.
      ]. Its cytoplasmic moiety contains 3 domains, of which Domain I is the most conserved [
      • Tellinghuisen T.L.
      • Foss K.L.
      • Treadaway J.C.
      • Rice C.M.
      Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein.
      ]. The mechanism by which NS5A regulates replication regardless of the HCV genotype is still unclear [
      • Scheel T.K.
      • Prentoe J.
      • Carlsen T.H.
      • Mikkelsen L.S.
      • Gottwein J.M.
      • Bukh J.
      Analysis of functional differences between hepatitis C virus NS5A of genotypes 1–7 in infectious cell culture systems.
      ]. Considerable information has been gathered on its molecular interactions and role in the viral lifecycle. NS5A and the RdRp directly interact, both in vivo and in vitro [
      • Shirota Y.
      • Luo H.
      • Qin W.
      • Kaneko S.
      • Yamashita T.
      • Kobayashi K.
      • et al.
      Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.
      ]. In vitro, this interaction stimulates RdRp-catalyzed synthesis of the negative RNA strand [
      • Quezada E.M.
      • Kane C.M.
      The hepatitis C virus NS5A stimulates NS5B during in vitro RNA synthesis in a template specific manner.
      ]. It was shown that all 3 domains of NS5A bind to RNA [
      • Kolykhalov A.A.
      • Feinstone S.M.
      • Rice C.M.
      Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA.
      ]. The interactions of Domain I with the polypyrimidine tract of 3′ UTR suggest it may affect the efficiency of RNA replication by the RdRp; however, these results also suggested the binding of RdRp and NS5A to RNA are mutually exclusive. In addition, Domain II of NS5A interacts with cyclophilin A, a host cell protein required for replication, and this interaction is vital for RNA binding [
      • Foster T.L.
      • Gallay P.
      • Stonehouse N.J.
      • Harris M.
      Cyclophilin A interacts with domain II of hepatitis C virus NS5A and stimulates RNA binding in an isomerase-dependent manner.
      ]. NS5A also plays a role in viral packaging and assembly. Domain III appears to be essential for this function [
      • Cun W.
      • Jiang J.
      • Luo G.
      The C-terminal alpha-helix domain of apolipoprotein E is required for interaction with nonstructural protein 5A and assembly of hepatitis C virus.
      ,
      • Appel N.
      • Zayas M.
      • Miller S.
      • Krijnse-Locker J.
      • Schaller T.
      • Friebe P.
      • et al.
      Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly.
      ]. This may be due, at least in part, to NS5A recruiting apolipoprotein E, a component of the HCV production process [
      • Cun W.
      • Jiang J.
      • Luo G.
      The C-terminal alpha-helix domain of apolipoprotein E is required for interaction with nonstructural protein 5A and assembly of hepatitis C virus.
      ,
      • Benga W.J.
      • Krieger S.E.
      • Dimitrova M.
      • Zeisel M.B.
      • Parnot M.
      • Lupberger J.
      • et al.
      Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles.
      ]. Indeed, inhibiting apolipoprotein E expression results in marked reduction of infectious particle production without affecting viral entry and replication [
      • Benga W.J.
      • Krieger S.E.
      • Dimitrova M.
      • Zeisel M.B.
      • Parnot M.
      • Lupberger J.
      • et al.
      Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles.
      ].

      NS5A inhibitor mechanism of action

      Several viral proteins have generated interest as potential targets for specific inhibitory drugs. In addition to the two NS3/4A protease inhibitors already approved for clinical use, numerous other protease inhibitors are being developed as well as inhibitors of viral replication, including nucleoside/nucleotide analogue inhibitors of HCV RdRp, non-nucleoside inhibitors of RdRp, cyclophilin inhibitors, and NS5A inhibitors.
      Because of its critical involvement in viral replication and assembly [
      • Guedj J.
      • Dahari H.
      • Rong L.
      • Sansone N.D.
      • Nettles R.E.
      • Cotler S.J.
      • et al.
      Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life.
      ], NS5A has been identified as a target for viral inhibition, leading to development of therapeutic agents. In HCV replicon-containing cells, inhibition of NS5A, but not other HCV proteins, resulted in redistribution of NS5A from the ER to lipid droplets. NS5A-targeting agents did not cause similar alterations in the localization of other HCV-encoded proteins, and the transfer of NS5A to lipid droplets coincided with the onset of inhibition of replication [
      • Targett-Adams P.
      • Graham E.J.
      • Middleton J.
      • Palmer A.
      • Shaw S.M.
      • Lavender H.
      • et al.
      Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.
      ]. Inhibition of NS5A at picomolar concentrations has been associated with significant reductions in HCV RNA levels in cell culture-based models, which makes these agents among the most potent antiviral molecules yet developed [

      Targett-Adams P. NS5A inhibitors: picomolar power to combat hepatitis C virus? <http://www.hepatitiscnewdrugresearch.com/what-are-ns5a-inhibitors.html>.

      ,
      • 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.
      ,
      • Lemm J.A.
      • O’Boyle 2nd, D.
      • Liu M.
      • Nower P.T.
      • Colonno R.
      • Deshpande M.S.
      • et al.
      Identification of hepatitis C virus NS5A inhibitors.
      ]. NS5A inhibitors have pan-genotypic activity, i.e., they suppress replication of all HCV genotypes, but their antiviral effectiveness against genotypes other than 1 may vary from one molecule to another [
      • 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.
      ]. Use of multiple DAAs including an NS5A inhibitor in replicon systems in cell culture has resulted in additive/synergistic inhibition of viral production and an increased barrier to resistance [
      • Pelosi L.A.
      • Voss S.
      • Liu M.
      • Gao M.
      • Lemm J.A.
      Effect on HCV replication by combinations of direct acting antivirals including NS5A inhibitor aaclatasvir.
      ].
      The exact mechanism of antiviral action of NS5A inhibitors is unknown. Available evidence suggests that they have multiple effects, which contribute to their potency [
      • Guedj J.
      • Dahari H.
      • Rong L.
      • Sansone N.D.
      • Nettles R.E.
      • Cotler S.J.
      • et al.
      Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life.
      ]. One putative mechanism is the inhibition of hyperphosphorylation. Phosphorylation of NS5A seems required for viral production [
      • Masaki T.
      • Suzuki R.
      • Murakami K.
      • Aizaki H.
      • Ishii K.
      • Murayama A.
      • et al.
      Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles.
      ], but the relative roles of the phosphorylated and hyperphosphorylated forms are unclear, and conflicting results have been reported suggesting that reduced hyperphosphorylation may either enhance or reduce replication [
      • Evans M.J.
      • Rice C.M.
      • Goff S.P.
      Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.
      ,
      • Quintavalle M.
      • Sambucini S.
      • Summa V.
      • Orsatti L.
      • Talamo F.
      • De Francesco R.
      • et al.
      Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors.
      ]. It is thought that a tightly regulated control of phosphorylation vs. hyperphosphorylation is required for efficient viral function. It was also shown that NS5A acts in two different pathways in RNA replication, and one of them likely requires hyperphosphorylation [
      • Fridell R.A.
      • Qiu D.
      • Valera L.
      • Wang C.
      • Rose R.E.
      • Gao M.
      Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052.
      ]. However, other mechanisms may also play a role. For instance, NS5A inhibitors alter the subcellular localization of NS5A, which may cause faulty viral assembly [
      • Targett-Adams P.
      • Graham E.J.
      • Middleton J.
      • Palmer A.
      • Shaw S.M.
      • Lavender H.
      • et al.
      Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.
      ,
      • Lee C.
      • Ma H.
      • Hang J.Q.
      • Leveque V.
      • Sklan E.H.
      • Elazar M.
      • et al.
      The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein.
      ].

      Resistance to NS5A inhibitors

      HCV displays a large degree of genomic variability, resulting in its quasispecies distribution [
      • Pawlotsky J.M.
      • Hepatitis C.
      Virus population dynamics during infection.
      ]. Variants that confer resistance to NS5A inhibitors pre-exist within HCV quasispecies populations in the absence of any previous exposure to these drugs. These variants generally replicate at low levels and are thus undetectable by currently available techniques. However, they can be selected if an NS5A inhibitor is administered and may be grown to high levels. Clinically significant resistance is usually associated with an escape pattern whereby viral replication returns to pretreatment levels and the dominant virus harbours amino acid substitutions that confer high levels of drug resistance without impairing fitness of the virus. Very high levels of the drug may be required to suppress highly resistant viruses, which may not be achievable without compromising safety [
      • Strahotin C.S.
      • Babich M.
      Hepatitis C variability, patterns of resistance, and impact on therapy.
      ].
      At present, only genotype 1, the most prevalent HCV genotype, has been studied in detail for resistant variants. Table 1, adapted from Fridell et al. [
      • Fridell R.A.
      • Wang C.
      • Sun J.H.
      • O’Boyle 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.
      ], describes the resistance profile of the NS5A inhibitor daclatasvir in genotype 1a and 1b replicons. The barrier to resistance is lower for genotype 1a than for genotype 1b. Substitutions at positions L31 and Y93 have the greatest ability to confer resistance to daclatasvir, and double mutations may increase the EC50 to a far greater extent (Table 1). These substitutions also confer resistance to other first-generation NS5A inhibitors. In addition, studies with daclatasvir have shown that double and triple inhibitor combinations in replicon systems can generate resistance pathways that differ from those observed during NS5A inhibitor monotherapy [
      • Pelosi L.A.
      • Voss S.
      • Liu M.
      • Gao M.
      • Lemm J.A.
      Effect on HCV replication by combinations of direct acting antivirals including NS5A inhibitor aaclatasvir.
      ]. Agents without cross-resistance with NS5A inhibitors should thus be used in combination with this class of drugs.
      Table 1Resistance profile of daclatasvir in the in vitro genotype 1a and 1b replicon systems. Adapted from Fridell et al.
      • Fridell R.A.
      • Wang C.
      • Sun J.H.
      • O’Boyle 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.
      .

      NS5A inhibitors undergoing clinical trials

      Although no NS5A inhibitor has yet been approved for therapeutic use, these agents are viewed with optimism due to their favourable characteristics, including the requirement for low dosing to inhibit HCV replication; pan-genotypic activity; once-daily dosing; resistance profiles that do not overlap with those of other DAAs in development; and successful suppression of HCV replication with an acceptable safety profile in early clinical trials [

      Targett-Adams P. NS5A inhibitors: picomolar power to combat hepatitis C virus? <http://www.hepatitiscnewdrugresearch.com/what-are-ns5a-inhibitors.html>.

      ].

       Daclatasvir (BMS790052)

      Daclatasvir is an oral, once-daily, highly selective NS5A inhibitor with broad coverage of HCV genotypes in vitro developed by Bristol-Myers Squibb. Daclatasvir currently is in Phase III clinical trials. Its inhibitory target maps to Domain I, and it has been shown to block hyperphosphorylation of NS5A [
      • Fridell R.A.
      • Qiu D.
      • Valera L.
      • Wang C.
      • Rose R.E.
      • Gao M.
      Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052.
      ], as well as alter the subcellular localization of the viral protein [
      • Targett-Adams P.
      • Graham E.J.
      • Middleton J.
      • Palmer A.
      • Shaw S.M.
      • Lavender H.
      • et al.
      Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.
      ,
      • Lee C.
      • Ma H.
      • Hang J.Q.
      • Leveque V.
      • Sklan E.H.
      • Elazar M.
      • et al.
      The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein.
      ]. Daclatasvir has an EC50 of 50 pM against genotype 1a, 9 pM against genotype 1b, and 28 pM against genotype 2a [
      • 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.
      ]. Daclatasvir has been tested in Phase II clinical trials in combination with pegylated IFN-α and ribavirin; in quadruple combination with asunaprevir, an NS3/4A protease inhibitor, and pegylated IFN-α/ribavirin; and with asunaprevir, the nucleotide analogue sofosbuvir and the non-nucleoside inhibitor of HCV RdRp BMS-791325 in IFN-free regimens.
      In a randomized, parallel-group, double-blind, placebo-controlled, dose-finding Phase IIa trial of treatment-naïve patients infected with HCV genotype 1, 5 of 12 patients who received 3 mg daclatasvir with pegylated IFN-α and ribavirin for 48 weeks achieved extended rapid virologic response (eRVR), compared with 10 of 12 who received 10 mg daclatasvir, 9 of 12 who received 60 mg daclatasvir, and 1 of 12 who received placebo. Adverse events and discontinuations as a result of adverse events occurred with similar frequency across treatment groups [
      • 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.
      ]. In another Phase IIa trial in genotype 1-infected patients who were non-responders to a prior course of pegylated IFN-α and ribavirin, all 10 patients who received quadruple therapy with daclatasvir, asunaprevir, and pegylated IFN-α/ribavirin showed a sustained virologic response (SVR) after 12 weeks, as opposed to 4 of 11 who received daclatasvir and asunaprevir only [
      • 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.
      ]. A higher incidence of viral breakthrough due to resistance was observed in genotype 1a patients who were given only the 2 DAAs without pegylated IFN-α/ribavirin vs. genotype 1b patients receiving the same treatment regimen, as a result of the lower barrier to resistance in genotype 1a [
      • 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.
      ]. Among patients who experienced virologic failure, the most common variants harboured Y93H and L31M, two substitutions well known for conferring resistance to daclatasvir.
      In a 24-week dual-oral Phase II trial with daclatasvir and asunaprevir in genotype 1b-infected patients, 90.5% of null responders and 63.6% of patients ineligible for or intolerant to pegylated IFN-α/ribavirin achieved SVR 24 weeks after the end of treatment (SVR24) [

      Suzuki Y, Ikeda K, Suzuki F, Toyota J, Karino Y, Chayama K, et al. Dual oral therapy with daclatasvir and asunaprevir for patients with HCV genotype 1b infection and limited treatment options. J Hepatol, 2013; 58: 655-662.

      ]. Interestingly, many patients in this study with pre-existing resistance-associated NS5A polymorphisms were cured of their chronic HCV infection.
      In a Phase IIb study with daclatasvir, pegylated IFN-α, and ribavirin, 100% of genotype 4-infected patients achieved SVR at 12 weeks post-treatment (SVR12) [
      • Hezode C.
      • HIrschfield G.M.
      • Ghesquiere W.
      • Sievert W.
      • Rodriguez-Torres M.
      • Shafran S.D.
      • et al.
      Daclatasvir, an NS5A replication complex inhibitor, combined with peginterferon alfa-2a and ribavirin in treatment-naive HCV genotype 1 or 4 subjects: phase 2b COMMAND-1 SVR12 results.
      ]. A combination of daclatasvir and sofosbuvir (formerly GS-7977), a nucleotide analogue inhibitor of HCV RdRp developed by Gilead Sciences, given for 24 weeks achieved SVR in 100% (44/44) of treatment-naïve patients infected with HCV genotype 1, and in 91% (40/44) of patients infected with HCV genotypes 2 and 3 at 4 weeks post-treatment. Addition of ribavirin had no effect on SVR rates [
      • Sulkowski M.
      • Gardiner D.
      • Lawitz E.
      • Hinestrosa F.
      • Nelson D.
      • Thuluvath P.
      • et al.
      Potent viral suppression with the all-oral combination of daclatasvir (NS5A inhibitor) and GS-7977 (nucleotide NS5B inhibitor), +/− ribavirin, in treatment-naive patients with chronic HCV GT1, 2, or 3 (100% SVR gt1, 91% gt2).
      ]. Finally, the triple combination of daclatasvir, asunaprevir and BMS-791325, a non-nucleoside inhibitor of HCV RdRp, resulted in an SVR12 in 15 of 16 patients (94%) treated for 12 weeks (data missing in the remaining patient) [
      • Everson G.T.
      • Sims K.D.
      • Rodriguez-Torres M.
      • Hezode C.
      • Lawitz E.
      • Bourliere M.
      • et al.
      An interferon-free, ribavirin-free 12-week regimen of daclatasvir (DCV), asunaprevir (ASV) and BMS-791325 yielded SVR4 of 94% in treatment-naive patients with genotype (GT) 1 chronic hepatitis C virus (HCV) infection.
      ].

       ABT-267

      This drug candidate, developed by AbbVie, is in Phase II clinical trials. It is an oral, once-daily NS5A inhibitor that significantly reduces HCV RNA levels in vitro and in vivo. In a study of treatment-naïve genotype 1-infected patients, ABT-267 in combination with pegylated IFN-α and ribavirin produced a rapid virologic response (RVR) at 4 weeks in 22 of 28 patients as compared with 2 of 22 who received placebo; after 12 weeks, 25 of 28 patients receiving the NS5A inhibitor in combination with pegylated IFN-α and ribavirin showed complete early virologic response (cEVR) compared with 6 of 9 patients in the placebo group. A recently presented Phase IIb clinical trial, which used a 4-drug combination of ABT-267, ritonavir-boosted ABT-450 (a protease inhibitor), ABT-333 (a non-nucleoside inhibitor of HCV RdRp), and ribavirin achieved SVR12 in 97.5% of treatment-naïve patients and in 93.3% of prior null-responders infected with genotype 1 [
      • Kowdley K.V.
      • Lawitz E.
      • Poordad F.
      • Cohen D.E.
      • Nelson D.R.
      • Zeuzem S.
      • et al.
      A 12-week interferon-free treatment regimen with ABT-450/r, ABT-267, ABT-333 and ribavirin achieves SVR12 rates (observed data) of 99% in treatment-naïve patients and 93% in prior null responders with HCV genotype 1 infection.
      ,

      Abbott Laboratories. Abbott’s investigational interferon-free hepatitis C treatment regimen achieved SVR12 (observed data) rates in 99 percent of treatment-naive and 93 percent in prior null responders for genotype 1 patients in Phase 2b study. <http://www.abbott.com/news-media/press-releases/abbotts-investigational-interferonfree-hepatitis-c-treatment-regimen-achieved-svr12-observed-data.htm>.

      ]. In treatment-naïve patients, the SVR rates were 87.5% when the three drugs and ribavirin were administered for 8 weeks, 89.9% when ABT-267 was administered with ABT-450 and ribavirin for 12 weeks, and 87.3% when the three DAAs were administered without ribavirin for 12 weeks. In null responders, the SVR rate was 88.9% with the combination of ABT-450, ABT-267, and ribavirin. Based on these results, Phase III trials with the 3 DAAs with and without ribavirin are planned [

      Abbott Laboratories. Abbott’s investigational interferon-free hepatitis C treatment regimen achieved SVR12 (observed data) rates in 99 percent of treatment-naive and 93 percent in prior null responders for genotype 1 patients in Phase 2b study. <http://www.abbott.com/news-media/press-releases/abbotts-investigational-interferonfree-hepatitis-c-treatment-regimen-achieved-svr12-observed-data.htm>.

      ].

       Ledispasvir (GS-5885)

      This oral, once-daily drug candidate, developed by Gilead Sciences, is a potent NS5A inhibitor against genotypes 1a, 1b, 4a, and 5a in vitro, but has lower activity against genotypes 2a and 3a [
      • Cheng G.
      • Peng B.
      • Corsa A.
      • Yu M.
      • Nash M.
      • Lee Y.J.
      • et al.
      Antiviral activity and resistance profile of the novel HCV NS5A inhibitor GS-5885.
      ]. In a randomized, placebo-controlled study of 14 days of ledipasvir monotherapy in genotype 1-infected patients, significant HCV RNA reductions (up to 1000-fold) were observed. Several resistance-associated substitutions were selected, including the aforementioned Y93H and L31M. In patients infected with HCV genotype 1b, daclatasvir has been reported to be more active than ledipasvir, whereas ledipasvir has been found to be 4–5 times more active than daclatasvir for the M28T and Q30H substitutions in HCV genotype 1a infection. In addition, daclatasvir has been demonstrated to be 2-fold more active against the L31M substitution as compared with ledipasvir [
      • Lawitz E.J.
      • Gruener D.
      • Hill J.M.
      • Marbury T.
      • Moorehead L.
      • Mathias A.
      • et al.
      A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C.
      ]. Ledipasvir is now in a Phase II trial as a component of a 4-drug regimen with tegobuvir (a non-nucleoside inhibitor of HCV RdRp), GS-9451 (an NS3/4A protease inhibitor), and ribavirin [
      • Cheng G.
      • Peng B.
      • Corsa A.
      • Yu M.
      • Nash M.
      • Lee Y.J.
      • et al.
      Antiviral activity and resistance profile of the novel HCV NS5A inhibitor GS-5885.
      ]. Recent results from the ELECTRON Phase II trial have shown SVR rates 12 weeks after the end of treatment of 100% in 25 treatment-naïve and 10 null responder patients infected with HCV genotype 1 with the combination of sofosbuvir, ledipasvir and ribavirin [

      Gane EJ, Stedman CA, Hyland RH, Ding X, Pang PS, Symonds WT. ELECTRON: 100% SVR rate for once-daily sofosbuvir plus ledipasvir plus ribavirin given for 12 weeks in treatment-naïve and previously treated patients with HCV GT 1. Presented at the 20th conference on retroviruses and opportunistic infections. Atlanta, Georgia, March 3–6, 2013.

      ]. A Phase III trial with a fixed-dose combination of sofosbuvir and ledipasvir, with or without ribavirin, is in progress in treatment-naïve patients infected with HCV genotype 1 [

      Safety and efficacy of sofosbuvir/GS-5885 fixed-dose combination (FDC) +/− ribavirin for the treatment of HCV. <http://clinicaltrials.gov/ct2/show/NCT01701401?term=hepatitisc&cond=%22HepatitisCVirus%22&lup_s=09%2F08%2F2012&lup_d=30>.

      ]. A recent presentation also showed that ledipasvir, in combination with GS-9451, pegylated IFN-α, and ribavirin achieved SVR at 4 weeks post-treatment in 100% of CC IL28B patients infected with HCV genotype 1 [
      • Thompson A.J.
      • Shiffman M.L.
      • Rossaro L.
      • Ghalib R.
      • Han S.-H.B.
      • Beavers K.L.
      • et al.
      Six weeks of a NS5A inhibitor (GS-5885), protease inhibitor (GS-9451) plus peginterferon/ribavirin (PR) achieves high SVR4 rates in genotype 1 IL28B CC treatment-naive HCV patients: interim results of a prospective, randomized trial.
      ].

       GSK-2336805

      This oral, once-daily drug candidate is being developed by GlaxoSmithKline. Preliminary studies show that GSK-2336805 is particularly effective against HCV genotype 1b, and has potent antiviral activity against other genotypes as well. A placebo-controlled Phase I study of treatment-naïve patients with chronic genotype 1 infection found a reduction in HCV RNA level of up to 1000-fold following 14 days of monotherapy. This NS5A inhibitor is currently in Phase II clinical trials in treatment-naïve patients infected with HCV genotype 1 in combination with pegylated IFN-α, ribavirin, and telaprevir [

      Dose ranging of GSK2336805 in combination therapy. <http://clinicaltrials.gov/ct2/show/NCT01648140>.

      ]. Resistance to GSK-2336805 maps to NS5A [
      • Spreen W.
      • Wilfret D.
      • Bechtel D.
      • Adkinson K.
      • Lou Y.
      • Jones L.H.
      • et al.
      GSK2336805 HCV NS5A inhibitor demonstrates potent antiviral activity in chronic hepatitis C (CHC) genotype 1 infection: results from a first time in human (FITH) single and repeat dose study.
      ].

       ACH-2928

      This oral, once-daily drug candidate, developed by Achillion Pharmaceuticals, displays highly potent activity in vitro against genotype 1a replicons as well as chimeric replicons of genotypes 2–6. ACH-2928 has demonstrated in vitro synergistic activity in combination with sovaprevir (formerly ACH-1625), an HCV NS3/4A protease inhibitor, which is further enhanced by ribavirin [
      • Zhao Y.
      • Yang G.
      • Fabrycki J.
      • Patel D.
      • Wiles J.
      • Wang X.
      • et al.
      In vitro combination studies of ACH-1625 (HCV NS3 protease inhibitor) & ACH-2928 (HCV NS5A inhibitor) in presence and absence of ribavirin.
      ]. In Phase I trials, ACH-2928 monotherapy for 3 days produced up to a 3.7 log10 reduction in HCV RNA levels in patients with chronic HCV genotype 1 infection [
      • Vince B.
      • Lawitz E.
      • Searle S.
      • Marbury T.
      • Robison H.
      • Robarge L.
      • et al.
      Novel NS5A inhibitor ACH-2928 phase 1 results in healthy volunteers and HCV GT-1 patients.
      ].

       BMS824393

      This NS5A inhibitor is being developed by Bristol-Myers Squibb. It has shown strong in vitro potency against genotypes 1a and 1b. In a Phase I study in which this agent was used as a monotherapy for 3 days in genotype 1-infected patients, a decline of up to 3.9 log10 was observed [
      • Nettles R.
      • Wang X.
      • Quadri S.
      • Wu Y.
      • Gao M.
      • Belema M.
      • et al.
      BMS-824393 is a potent hepatitis C virus NS5A inhibitor with substantial antiviral activity when given as monotherapy in subjects with chronic G1 HCV infection (Abstract 1858).
      ].

       IDX719

      This drug candidate, developed by Idenix Pharmaceuticals, has shown greater potency in vitro than daclatasvir against HCV genotypes 1a, 1b, 2a, 3a, 4a, and 5a [

      Bilello JP, La Colla M, Chapron C, et al. The preclinical profile of the pan-genotypic HCV NS5A inhibitor IDX719 demonstrates its potential for combination therapy. In: seventh international workshop on hepatitis C-resistance and new compounds, June 28–29, 2012. <http://www.idenix.com/hcv/Bilello%20HCV%20Resistance%20Workshop%202012.pdf>.

      ]. In Phase I studies, HCV RNA levels declined by more than 3 log10 in single-dose trials for all genotype 1, 2, and 3 patients after 24 hours. Similar reductions in HCV RNA levels (over 3 log10) were observed for genotype 1, 3, and 4 patients, and reductions of 2 log10 for genotype 2, in 3-day monotherapy studies [

      Zhou XJ, Vince B, Dubuc Patrick G, et al. Safety, pharmacokinetic and preliminary antiviral activity results from a first-in-human study of IDX719, a pan-genotypic novel HCV NS5A inhibitor. In: seventh international workshop on clinical pharmacology of hepatitis therapy, June 27, 2012. <http://www.idenix.com/hcv/IDX719_HCVClinPharmMtg_FINAL%206%2027%2012.pdf>.

      ,
      • Mayers D.L.
      • Vince B.
      • Hill J.M.
      • Lawitz E.
      • O’Riordan W.D.
      • Webster L.R.
      • et al.
      IDX719, HCV NS5A inhibitor, demonstrates pan-genotypic activity after three days of monotherapy in genotype 1, 2, 3 or 4 HCV-infected subjects.
      ]. However, evidence indicates that the Y93H substitution confers resistance to this NS5A inhibitor [

      Bilello JP, La Colla M, Chapron C, et al. The preclinical profile of the pan-genotypic HCV NS5A inhibitor IDX719 demonstrates its potential for combination therapy. In: seventh international workshop on hepatitis C-resistance and new compounds, June 28–29, 2012. <http://www.idenix.com/hcv/Bilello%20HCV%20Resistance%20Workshop%202012.pdf>.

      ]. A Phase II clinical trial using IDX719, simeprevir (a protease inhibitor developed by Janssen and Medivir), and TMC647055, a non-nucleoside polymerase inhibitor developed by Janssen, has been announced [

      Idenix Pharmaceuticals. Idenix pharmaceuticals announces collaboration with Janssen to initiate phase II all-oral combination studies including IDX719, simeprevir (TMC435) and TMC647055 for the treatment of hepatitis C virus (HCV). <http://files.shareholder.com/downloads/IDIX/2297933580x0x630925/c52eb4ea-905e-4dd5-99fa-6e2ffc1b1388/IDIX_News_2013_1_28_General_Releases.pdf>.

      ].

       PPI461

      This oral drug candidate is under development by Presidio Pharmaceuticals. A Phase Ib trial of monotherapy for 3 days in patients with HCV genotype 1 infection showed a decrease of HCV RNA level of up to 3.6 log10. However, widespread resistance emerged rapidly, mapping to amino acids 28, 30, 31, and 93 [
      • Huang Q.
      • Huang N.
      • Lau M.
      • Bencsik M.
      • Huq A.
      • Peng A.
      • et al.
      Resistance monitoring of HCV patients treated for three days with the NS5A inhibitor PPI-461 reveals rapid emergence of resistant HCV variants.
      ].

       PPI668

      Also under development by Presidio Pharmaceuticals, this NS5A inhibitor has been shown to possess high efficacy against HCV genotype 1, with up to 3.7 log10 mean HCV RNA reductions, in a Phase Ib clinical trial [

      Presidio Pharmaceuticals. Presidio pharmaceuticals successfully completes phase 1 proof-of-concept for PPI-668, its potent HCV N5SA inhibitor, in Hepatitis C patients with Genotype-1 infection. <http://www.presidiopharma.com/>.

      ,
      • Lalezari J.P.
      • Farrell G.C.
      • Shah P.S.
      • Schwab C.
      • Walsh D.
      • Vig P.
      • et al.
      PPI-668, a potent new pan-genotypic HCV NS5A inhibitor: phase 1 efficacy and safety.
      ]. Activity was demonstrated against variants harbouring the L31M substitution. In an added genotype-2/3 cohort, the first 2 patients achieved mean 3.0 log10 RNA level reductions [
      • Lalezari J.P.
      • Farrell G.C.
      • Shah P.S.
      • Schwab C.
      • Walsh D.
      • Vig P.
      • et al.
      PPI-668, a potent new pan-genotypic HCV NS5A inhibitor: phase 1 efficacy and safety.
      ]. PPI668 will be studied in combination with two DAAs developed by Boehringer-Ingelheim, faldaprevir, an NS3/4A protease inhibitor, and BI207127, a non-nucleoside inhibitor of HCV RdRp.

       ACH-3102

      This NS5A inhibitor, developed by Achillion Pharmaceuticals, has a modified structure designed to have a higher pharmacologic barrier to resistance. Pharmacokinetic studies support once-daily oral dosing with this agent. ACH-3102 has shown potent antiviral activity against all genotypes in preclinical studies. In replicon studies, ACH-3102 has shown the smallest difference in potency between genotype 1a and 1b replicons, compared with daclatasvir and ACH-2928 [
      • Yang G.
      • Wiles J.
      • Patel D.
      • Zhao Y.
      • Fabrycki J.
      • Weinheimer S.
      • et al.
      Preclinical characteristics of ACH-3102; a novel NS5A inhibitor with improved potency against genotype-1A virus and variants resistant to 1st generation NS5A inhibitors.
      ]. ACH-3102 is potent against mutants harbouring substitutions that confer resistance to first-generation NS5A inhibitors (Fig. 2), such as those at positions Y93 and L31 [
      • Yang G.
      • Wiles J.
      • Patel D.
      • Zhao Y.
      • Fabrycki J.
      • Weinheimer S.
      • et al.
      Preclinical characteristics of ACH-3102; a novel NS5A inhibitor with improved potency against genotype-1A virus and variants resistant to 1st generation NS5A inhibitors.
      ]. Antiviral efficacy is also strong against double mutants that are highly resistant to other NS5A inhibitors (unpublished data). In addition, this inhibitor has shown very low potential for emergence of resistant variants in genotype 1b replicons (unpublished data). For these reasons, ACH-3102 is considered a “second-generation” NS5A inhibitor.
      Figure thumbnail gr2
      Fig. 2Antiviral efficacy of ACH-3102 (second-generation NS5A inhibitor) compared with ACH-2928 and daclatasvir (first-generation NS5A inhibitors) on wild-type (parent) and mutated HCV replicons
      [
      • Yang G.
      • Wiles J.
      • Patel D.
      • Zhao Y.
      • Fabrycki J.
      • Weinheimer S.
      • et al.
      Preclinical characteristics of ACH-3102; a novel NS5A inhibitor with improved potency against genotype-1A virus and variants resistant to 1st generation NS5A inhibitors.
      ]
      .
      A recently reported preclinical study using ACH-3102 and ACH-2684 (an NS3/4A protease inhibitor) has shown an additive to synergistic antiviral effect against genotypes 1a and 1b without the emergence of resistance variants [
      • Zhao Y.
      • Patel D.
      • Fabrycki J.L.
      • Weinheimer S.
      • Yang G.
      • Yang W.
      • et al.
      In vitro studies demonstrate a high probability of curing genotype 1 hepatitis C patients with combination of a novel NS3 protease inhibitor ACH-2684 and a novel NS5A inhibitor ACH-3102.
      ]. Recently announced results from a Phase Ia trial in patients infected with HCV genotype 1 show that a single dose of ACH-3102 produces a mean HCV RNA level reduction of up to 3.9 log10, with an upper range of 4.6 log10, with inhibition lasting for 4 days after dosing. Moreover, ACH-3102 has a half-life of approximately 250 hours (unpublished data), compared with 13–15 hours for daclatasvir [
      • Nettles R.E.
      • Sevinksy H.
      • Chung E.
      • Burt D.
      • Xiao H.
      • Marbury T.
      • et al.
      BMS-790052, a first-in-class potent hepatitis C virus NS5A inhibitor, demonstrates multiple-dose proof-of-concept in subjects with chronic GT1 HCV infection (potent, mean maximal 5.7 log reduction).
      ], 22–50 hours for ledipasvir [
      • Lawitz E.J.
      • Gruener D.
      • Hill J.M.
      • Marbury T.
      • Moorehead L.
      • Mathias A.
      • et al.
      A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C.
      ], and 25–32 hours for ABT-267 [
      • Lawitz E.
      Safety and antiviral activity of ABT-267, a novel NS5A inhibitor, during 3-day monotherapy: first study in HCV genotype-1 (GT1)-infected treatment-naive subjects.
      ]. A single Phase II trial has been initiated in genotype 1b patients using ACH-3102 in combination with ribavirin [

      Achillion Pharmaceuticals. Achillion announces positive proof-of-concept data with ACH-3102. <http://ir.achillion.com/releasedetail.cfm?ReleaseID=709768>.

      ].

      Progress toward all-oral combination therapies for HCV and the role of NS5A inhibitors

      Currently, the standard of care for chronic HCV genotype 1 infection is a combination of pegylated IFN-α, ribavirin, and an NS3/4A protease inhibitor (i.e., boceprevir or telaprevir), whereas patients infected with other HCV genotypes continue to be treated with pegylated IFN-α and ribavirin. The SVR rates observed with the triple combination in patients infected with HCV genotype 1 range from 67% to 75% in clinical trials [
      • 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.
      ,
      • 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.
      ]. They are probably lower in the real-life setting, indicating that a significant proportion of patients will still experience virologic failure and that improved therapeutic regimens are needed. In addition, patients receiving pegylated IFN-α and ribavirin experience a plethora of adverse effects, some of which are aggravated by the protease inhibitor [

      Victrelis (boceprevir). Package insert 2005. Merck Sharp & Dohme Corp., Whitehouse Station, NJ, USA.

      ,

      Incivek (telaprevir). Package insert 2012. Vertex Pharmaceuticals, Cambridge, MA, USA.

      ,
      • Gao X.
      • Stephens J.M.
      • Carter J.A.
      • Haider S.
      • Rustgi V.K.
      Impact of adverse events on costs and quality of life in protease inhibitor-based combination therapy for hepatitis C.
      ]. Clinical trials of NS5A inhibitors in combination with pegylated IFN-α and ribavirin have shown promising results. However, the trials conducted thus far have included only small numbers of patients, and more studies are needed before the efficacy of such 3-drug combinations can be fully ascertained. In this respect, the results of a Phase III trial with daclatasvir, pegylated IFN-α, and ribavirin are awaited. Quadruple therapies including an NS5A inhibitor, pegylated IFN-α, ribavirin, and another DAA also appear promising. However, recent reports of very high SVR rates, over 90%, in patients treated with all-oral, IFN-free regimens with or without ribavirin clearly indicate that the IFN era is coming to an end. It is also noteworthy that NS5A inhibitors developed by one company have been used with different classes of DAAs developed by other companies [
      • Sulkowski M.
      • Gardiner D.
      • Lawitz E.
      • Hinestrosa F.
      • Nelson D.
      • Thuluvath P.
      • et al.
      Potent viral suppression with the all-oral combination of daclatasvir (NS5A inhibitor) and GS-7977 (nucleotide NS5B inhibitor), +/− ribavirin, in treatment-naive patients with chronic HCV GT1, 2, or 3 (100% SVR gt1, 91% gt2).
      ]; as such, a highly potent NS5A inhibitor may find uses in combinations with various other DAAs to achieve high cure rates.
      Due to their specificity, potency, and low EC50, NS5A inhibitors will likely be a critical component of future all-oral, IFN-free combinations. It is interesting to note that the most attractive all-oral combinations presented at the last annual meetings of the American and European liver societies all contained an NS5A inhibitor, combined either with a nucleotide analogue or a protease inhibitor and a non-nucleoside inhibitor of HCV RdRp, with or without ribavirin. Fixed-dose combinations (i.e., 2-drug combinations in 1 pill) including an NS5A inhibitor are already available in Phase II and III clinical trials. The advent of second-generation NS5A inhibitors, with a modified structure and near-equal efficacy against variants known to resist first-generation NS5A inhibitors, is also promising.

      Conclusions

      Although blood screening and other preventive measures have reduced the incidence of HCV in some parts of the world, infection with this virus remains a significant worldwide health concern. The multiple genotypes of HCV, as well as rapid development of mutations, have complicated the development of effective drugs. Until recently, a non-specific antiviral combination, pegylated IFN-α and ribavirin, was the mainstay of HCV therapy. The approval of two NS3/4A protease inhibitors has allowed the addition of a DAA to this treatment regimen. Although the first-generation protease inhibitors, telaprevir and boceprevir, in combination with pegylated IFN-α and ribavirin, have improved treatment of chronic HCV genotype 1 infection, response rates remain suboptimal. In addition, many patients are unable to tolerate this therapy and, among those who can, adverse events associated with the drugs can compromise patient compliance and lead to premature treatment discontinuations. Thus, there has been a strong desire to develop all-oral, IFN-free therapies with high efficacy. The discovery of the multiple roles of the NS5A protein in viral replication has been paralleled by the development of specific NS5A inhibitors. Evidence gathered thus far indicates that these agents are potent and possess antiviral activity against multiple HCV genotypes with acceptable safety profiles. In addition, clinical trial data support the efficacy of NS5A inhibitors with and without pegylated IFN-α and ribavirin, suggesting an important role for these agents as a component of all-oral therapeutic regimens for the treatment of HCV.

      Financial support

      Editorial assistance from ACCESS Medical was funded by Achillion Pharmaceuticals .

      Conflict of interest

      The author has received research grants from Gilead. He has served as an advisor for Abbott, Abbvie, Achillion, Boehringer-Ingelheim, Bristol-Myers Squibb, Gilead, Idenix, Janssen-Cilag, Madaus-Rottapharm, Merck, Novartis, and Roche.

      Acknowledgments

      The author would like to thank Amlan RayChaudhury, PhD, of ACCESS Medical, LLC, for editorial assistance in preparing the manuscript.

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