New HCV Therapies on the Horizon

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REVIEW 10.1111/j.1469-0691.2010.03430.x

New HCV therapies on the horizon

J. Vermehren and C. Sarrazin Medizinische Klinik 1, Klinikum der J. W. Goethe-Universita¨t, Frankfurt am Main, Germany

Abstract

Improved understanding of the hepatitis C virus (HCV) life cycle has led to the discovery of numerous potential targets for antiviral therapy. HCV polyprotein processing and replication have been identified as the most promising viral targets. However, viral entry and fusion, RNA translation, virus assembly and release and several host cell factors may provide alternative attractive targets for future anti-HCV therapies. Inhibitors of the HCV NS3/4A protease are currently the most advanced in clinical development. Monotherapy with protease inhibitors has shown high antiviral activity, but is associated with frequent selection of resistant HCV variants, often resulting in viral breakthrough. However, there is encouraging evidence from phase 2/3 trials indicating that the addition of a protease inhibitor (e.g. telaprevir and boceprevir) to pegylated interferon-a/ribavirin substantially improves sustained virological response rates in both treatment-naı¨ve and treatment-experienced patients with HCV genotype 1. Nucleos(t)ide inhibitors of the HCV NS5B polymerase have shown variable antiviral activity against different HCV genotypes, but seem to have a higher genetic barrier to resistance than protease inhibitors. In addition, several allosteric binding sites have been identified for non-nucleoside inhibitors of the NS5B polymerase. How- ever, the development of a substance with high antiviral activity and a high genetic barrier to resistance seems to be difficult. Among the different host cell-targeting compounds in early clinical development, cyclophilin inhibitors have shown the most promising results. Although advances have also been made in improving interferons, combinations of antiviral agents with different mechanisms of action may lead to the eventual possibility of interferon-free regimens.

Keywords: Chronic hepatitis C, direct-acting antiviral, protease inhibitor, polymerase inhibitor, NS5A inhibitor, cyclophilin inhibitor, review Article published online: 18 November 2010 Clin Microbiol Infect 2011; 17: 122–134

Corresponding author: C. Sarrazin, Medizinische Klinik 1, Klinikum der J.W. Goethe-Universita¨t, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany E-mail: [email protected]

antiviral (DAA) agents that specifically target post-transla- Introduction tional processing and HCV replication [7–10]. Inhibitors of HCV entry, HCV RNA translation and virus assembly and Chronic hepatitis C represents a major health burden, affect- release are mostly still in preclinical development, whereas ing approximately 170 million people worldwide [1]. With several host cell-targeting compounds rate among the the current standard of care (SOC), a combination of pegy- more promising therapeutic approaches in recent anti-HCV lated interferon (PEG-IFN)-a plus weight-based ribavirin drug discoveries. Finally, improvements have also been (RBV), viral eradication can be achieved in fewer than half of made in the development of new, long-acting interferons all patients with hepatitis C virus (HCV) genotype 1 and in with the potential for more favourable safety and efficacy approximately 80% of patients with HCV genotype 2 or 3 profiles. [2–5]. Treatment may take up to 72 weeks [6], but therapy- This review will give an overview of recent developments associated side effects such as pancytopenia, flu-like symp- in the HCV drug pipeline. DAA agents, host cell-targeting toms or depression are commonly observed, and may lead compounds and new interferons that have already been eval- to early treatment discontinuation. uated in clinical trials will be discussed. Other treatment Improved understanding of the HCV life cycle in recent approaches, such as toll-like receptor agonists and therapeu- years has supported the development of direct-acting tic vaccines, are beyond the scope of this article.

ª2011 The Authors Clinical Microbiology and Infection ª2011 European Society of Clinical Microbiology and Infectious Diseases CMI Vermehren and Sarrazin New HCV therapies 123

The clinical proof-of-concept for NS3/4A protease inhibi- Antivirals Targeting HCV Polyprotein tors was obtained with ciluprevir, a macrocyclic NS3/4A pro- Processing tease inhibitor that showed substantial antiviral activity in patients with HCV genotype 1 [11]. However, the develop- NS3/4A protease inhibitors ment of ciluprevir was stopped because of serious cardiotox- The NS3/4A protease has been recognized as an important icity in a monkey model. target for anti-HCV therapy because of its key role within the HCV life cycle (e.g. cleavage of the genome-encoded Telaprevir polyprotein and inactivation of cellular proteins required for The orally bioavailable, linear, ketoamide protease inhibitor innate immunity). NS3/4A protease inhibitors have shown telaprevir was initially investigated in a 14-day randomized, strong antiviral efﬁcacy but a low genetic barrier to resis- placebo-controlled monotherapy study in patients with HCV tance in early clinical studies. They can be divided into two genotype 1 [12]. In this study, the greatest reduction in HCV chemical classes: macrocyclic inhibitors, and linear, tetrapep- RNA was achieved in patients who received 750 mg of tela- tide a-ketoamide derivates (Fig. 1). previr every 8 h, with a median viral load decline of

(a) NS3 protease inhibitors (b) Nucleos(t)ide polymerase inhibitors 4 6 5.4

5 4.6 4.4 4.3 4.2 3 2.7 3.8 3.9 3.9 4 3.6 3.3 3.3 2.9 1.9 3 2 2.1 decline decline decline decline 1.8 2 1 0.7 1 Mean or median HCV RNA Mean or median HCV RNA 0 0

IDX320 400 mg once daily IDX184 100 mg once daily TMC435 200 mg once daily GS-9256GS-9451 200MK-5172 mg 400 twice mg 400daily once mg daily once daily VaniprevirBI 201335700 mg 240twicePHX mg 1766daily once 800 daily mg twice dailyACH-1625 600 mg twice daily RG 7128 1500 mg twice daily BMS-650032 400 mg twice daily Telaprevir 750 mg three times daily Boceprevir 400Danoprevir mg three 200 times mg daily three times daily ABT-450/ritonavir 200/100 mg once daily PSI-7977 (PSI-7851) 400 mg once daily

4 3.7 5 3.1 2.9 3.9 3 4 3.6 Cyclophilin inhibitors 2.5 3 2.1 1.9 3

decline decline 2.3 2 2.1 2 1.7 decline decline 1.5 1 0.6 Mean or median HCV RNA 1 Mean or median HCV RNA 0 0

Alinia 500 mg twiceSilibinin daily* 15 mg/ kg BW IV VX-222 750 mg twice dailyANA598 800 mg twice daily ABT-333 600Tegobuvir mg twice 120daily mg twice daily Alisporivir 1000 mg once daily Filibuvir 300 mg three timesVX-759 daily 800 mg three times daily BMS-790052 100 mg once daily BI 207127 800 mg three times daily AZD7295 233 SCY-635mg three 300times mg daily three times daily

BMS-824393 50 mg once daily in GT1 patients FIG. 1. Antiviral activity of (a) NS3/4A protease inhibitors, (b) nucleos(t)ide analogue NS5B polymerase inhibitors, (c) non-nucleoside analogue NS5B polymerase inhibitors and (d) NS5A and other inhibitors during monotherapy for 3–14 days in patients with chronic hepatitis C, modiﬁed from [9]. No head-to-head studies were conducted. *Alinia was administered at a dose of of 500 mg twice daily for 24 weeks in genotype 4 patients only. BW, body weight; HCV, hepatitis C virus; IV, intravenous.

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4.4 log10 IU/mL HCV RNA from baseline. Viral rebound investigated in patients infected with HCV genotypes 2, 3 caused by selected drug-resistant mutants occurred in the and 4, with varying success [22,23]. majority of patients during treatment [13]. Mutations associ- The two pivotal phase 3 studies, known as ADVANCE ated with clinical resistance to telaprevir were identified at and ILLUMINATE, were conducted in more than 1500 treat- four positions of the NS3 catalytic domain — V36A/M/L, ment-naı¨ve patients infected with HCV genotype 1, and T54A, R155K/M/S/T and A156S (all three conferring low- showed SVR rates of 75% and 72% following triple therapy level to medium-level resistance) and A156T/V (conferring for 12 weeks followed by PEG-IFN-a-2a/RBV for an addi- high-level resistance) [13–16]. tional 12 or 36 weeks, respectively. Thus, SVR could be In two short-term studies of telaprevir, given in combina- increased by approximately 30% as compared with control tion with PEG-IFN-a-2a ± RBV for 2–4 weeks, an even more patients treated with SOC for 48 weeks (44%). Patients who pronounced viral decline, with a reduced frequency of resis- received triple combination therapy for 8 weeks, followed by tance mutations and no viral breakthrough, was observed SOC for 16 or 40 weeks, still achieved an SVR rate of 69% [17,18]. (ADVANCE). Precise plans for the management of adverse Subsequently, two large phase 2b placebo-controlled trials events, especially the frequently observed rashes, were of telaprevir in combination with PEG-IFN-a ± RBV were developed for the phase 3 studies, and rates of discontinua- conducted in treatment-naı¨ve patients with HCV genotype 1 tion attributable to adverse events were somewhat lower in the USA (PROVE 1; n = 250) [19] and in Europe than in the PROVE trials (7–8% vs. 4% in patients receiving (PROVE 2; n = 323) [20]. Overall, sustained virologic SOC) [24,25]. In the ADVANCE study, a response-guided response (SVR) rates of up to 67–69% were achieved in the therapy approach was applied, with a shortened treatment telaprevir-containing study arms vs. 41–46% in patients who period of 24 weeks, in patients who achieved an eRVR, received SOC. In PROVE 1, only patients who achieved an whereas all other patients were treated for 48 weeks. In the extended rapid virological response (eRVR) (HCV RNA neg- ILLUMINATE study, no benefit of a longer treatment dura- ative at week 4 and thereafter) were eligible for a shortened tion (48 weeks vs. 24 weeks) in eRVR patients could be treatment duration. The exploratory study arms with demonstrated (Fig. 2a). 12 weeks of telaprevir plus PEG-IFN-a ± RBV were associ- Together, these findings show that an eRVR was achieved ated with high relapse rates, especially in patients without in 57–65% of treatment-naı¨ve patients with HCV genotype 1. RBV (up to 48%), indicating that 12 weeks of overall therapy Thus, the majority of patients were eligible for shortened was insufficient to achieve favourable treatment outcomes, treatment duration, with equally high SVR rates as those and highlighting the importance of adding RBV, because of obtained with the standard therapy duration of 48 weeks. improved antiviral activity, reduced relapse rates, and a smal- Telaprevir in combination with PEG-IFN-a-2a ± RBV was ler number of patients with selection of resistant variants also investigated in treatment-experienced HCV genotype 1 and viral breakthrough. Rates of treatment discontinuation patients (PROVE 3, n = 453; and study 107, n = 117) [26]. attributable to adverse events were higher in the telaprevir- Overall, significantly higher SVR rates were achieved in prior containing study arms than in patients treated with SOC relapsers who received triple combination therapy than in alone (12–21% vs. 7–11%). Moreover, discontinuation rates those receiving SOC (up to 69–97% vs. 20%). Patients with increased to nearly 30% in patients who received telaprevir prior breakthrough or non-response achieved SVR rates in for 24 weeks. Thus, in subsequent studies, telaprevir dosing the ranges of 57–86% and 38–39%, respectively, as compared was restricted to a maximum of 12 weeks. with 20% and 9% when given SOC. Prior non-responders,

For more convenient application and to increase treat- including partial non-responders (‡2 log10 decline but detect- ment adherence, telaprevir every 12 h at a dose of 1125 mg able HCV RNA at treatment week 12) and null-responders was investigated in a small open-label phase 2 study, con- (<2 log10 decline in HCV RNA at week 12), experienced the ducted in treatment-naı¨ve patients with HCV genotype 1 highest rates of viral breakthrough during triple combination (study C208). In this study, telaprevir was also given in com- therapy. bination with PEG-IFN-a-2b and RBV. Equally high SVR rates A phase 3 study (REALIZE) of telaprevir evaluated the effi- were observed with telaprevir every 12 h vs. every 8 h, and cacy of a 12-week triple combination treatment regimen fol- with PEG-IFN-a-2b vs. PEG-IFN-2a [21]. However, differ- lowed by 36 weeks of SOC, or a 4-week lead-in with SOC, ences between the two PEG-IFN formulations may only followed by 12 weeks of triple combination therapy, fol- become visible in a more difficult-to-treat patient population lowed by 32 weeks of SOC in treatment-experienced geno- (i.e. patients with high viral load, advanced fibrosis or prior type 1 patients (partial non-responders, null-responders and non-response to PEG-IFN-a therapy). Telaprevir was also relapsers) vs. SOC for 48 weeks. SVR rates for the three

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(a) Telaprevir (b) Boceprevir 100 92 100 90 87.5 ADVANCE REALIZE 90 SPRINT-2 RESPOND-2 80 75 ILLUMINATE* 80 70 69 66 64 70 66 63 66 59 60 60 50 44 50 40 40 38 SVR (%) 30 SVR (%) 30 21 20 17 20 10 10 0 0

PR48 PR48 PR48 PR48 T12PR24T12PR48 T12PR48 T8PR24/48 T12PR24/48 PR4/PRB44 PR4/PRB44 PR4/T12PR44 *SVR data are shown for patients with eRVR only PR4/PRB24 ± PR20 PR4/PRB32 ± PR12 FIG. 2. Sustained viral response (SVR) results of recent telaprevir (ADVANCE, ILLUMINATE and REALIZE) and boceprevir (SPRINT-2 and RESPOND-2) trials. No head-to-head studies were conducted. (a) ADVANCE and ILLUMINATE were conducted in treatment-naı¨ve patients with hepatitis C virus (HCV) genotype 1. The ADVANCE treatment arms were as follows: (i) 12 weeks of telaprevir (T) plus PEG-IFN-a-2a and ribavirin (PR) followed by PR for 12 or 36 weeks, based on treatment response at weeks 4 and 12; (ii) 8 weeks of T plus PR followed by PR for 16 or 40 weeks, based on treatment response at weeks 4 and 12; and (iii) PR alone for 48 weeks. In the ILLUMINATE trial, patients who achieved an extended rapid virological response (eRVR) were randomized to receive 12 weeks of T plus PR followed by PR for (i) 12 weeks or (ii) 24 weeks. Treatment-experienced patients with HCV genotype 1 (REALIZE) received: (i) 12 weeks of T plus PR followed by PR for 36 weeks; (ii) a 4-week PR lead-in followed by T plus PR for 12 weeks, followed by PR alone for 32 weeks; or (iii) PR alone for 48 weeks. In the REALIZE study, relapsers and non-responder (null-responder and partial non-responder) patients were enrolled. (b) The SPRINT-2 treatment arms in treatment-naı¨ve patients with HCV genotype 1 were as follows: (i) 4-week lead-in with PEG-IFN-a-2b plus ribavirin (PR) followed by 44 weeks of PR plus boceprevir (B); (ii) 4-week lead-in with PR followed by PR plus B for 24 weeks or followed by PR plus B for 24 weeks and PR for an additional 20 weeks, based on treatment response at weeks 8 and 12; and (iii) PR alone for 48 weeks. A similar treatment schedule was used in treatment-experienced genotype 1 patients (RESPOND-2): (i) 4-week lead-in with PR followed by PR plus B for 44 weeks; (ii) 4-week lead in with PR followed by PR plus B for 32 weeks or followed by PR plus B for 32 weeks and PR for an additional 12 weeks, based on treatment response at weeks 8 and 12; and (iii) PR alone for 48 weeks. treatment arms were 64%, 66% and 17%, respectively. Prior a-ketoamide HCV protease inhibitor, with or without PEG- relapsers achieved SVR rates of 83–88%, whereas prior null- IFN-a-2b, resulted in mean maximum reductions of HCV responders achieved SVR rates in the range of 29–33%. Dis- RNA of up to 1.61 log10 and 2.88 log10 IU/mL, respectively, continuation because of adverse events occurred in 4% of in treatment-experienced patients infected with HCV geno- the combined telaprevir study arms and in 3% of the control type 1 [29]. Subsequently, a number of mutations conferring group. [27]. boceprevir resistance at low to medium levels were discov- During triple therapy with telaprevir plus PEG-IFN-a/RBV, ered in vivo: V36M/A, T54A/S, V55A, R155K/T, A156S and viral breakthrough associated with the selection of resistant V170A [30–33]. As for telaprevir, reduced antiviral activities variants was observed in 1–5% of treatment-naı¨ve patients of boceprevir in HCV genotype 2/3-infected patients were and in up to 25% of previous non-responders. Moreover, reported. variants conferring resistance to NS3 protease inhibitors In a phase 2 clinical trial (SPRINT-1), boceprevir (800 mg were also detected in patients with viral relapse after the every 8 h) in combination with PEG-IFN-a-2b and RBV was end of treatment. The long-term signiﬁcance of these resis- investigated in treatment-naı¨ve patients with HCV geno- tant variants is unknown but, generally, a decline in fre- type 1 (n = 595) [34]. Patients received either all three drugs quency over time was observed. However, in single patients, in combination for 28 or 48 weeks or for 24 or 44 weeks low to medium levels of the V36 and A156 variants were after a previous 4-week lead-in phase of PEG-IFN-a-2b/RBV, observed for up to 4 years after telaprevir therapy [28]. or SOC for 48 weeks. SVR rates ranged from 54% for 28 weeks of triple therapy to 75% after a 4-week lead-in Boceprevir with PEG-IFN-a-2b/RBV followed by 44 weeks of triple ther- In a phase 1 study, administration of boceprevir (200 apy, as compared with 38% in the control group. Treatment or 400 mg every 8 h), a peptidomimetic orally bioavailable discontinuations attributable to adverse events occurred in

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9–19% of patients receiving boceprevir as compared with 8% recently reported phase 2 results showed SVR rates compa- in the control group. rable to those achieved with telaprevir and boceprevir [48– In patients with prior non-response to SOC therapy, the 50]. addition of boceprevir to PEG-IFN-a and RBV gave only Potential advantages of the newer antiviral compounds slightly increased SVR rates as compared with standard ther- include improved pharmacokinetics (once-daily to twice-daily apy in a phase 2b study with suboptimal boceprevir/RBV dos- dosing), broader genotypic activity and better tolerability. In ing (14% vs. 2%) [35]. addition, ritonavir boosting is being investigated for a number The 4-week lead-in phase with PEG-IFN-a/RBV was intro- of protease inhibitors, including narlaprevir and danoprevir, duced into boceprevir phase 2 trials to decrease the proba- to reduce side effects and to enhance patient exposure to bility of resistance development. During the lead-in, a viral the latter agents, thereby potentially overcoming resistance decline of >2 log10 IU/mL was induced in the majority of and allowing less frequent dosing. treatment-naive patients, resulting in undetectable HCV The resistance profiles of all NS3 protease inhibitors cur- RNA levels in 60–64% of patients after an additional 4 weeks rently in clinical development overlap (Table 2) [9]. of triple therapy (week 8 of overall treatment), whereas only 37–39% of patients without lead-in therapy achieved unde- Antivirals Targeting HCV Replication tectable HCV RNA levels after 4 weeks of triple therapy. Numerically, lower breakthrough rates and higher SVR rates were observed in patients with a lead-in period than in those NS5B polymerase inhibitors without induction therapy. The lead-in period may help in Two classes of NS5B polymerase inhibitors can be distin- the selection of the optimal triple-therapy duration for both guished: nucleoside or nucleotide analogue inhibitors, which treatment-naı¨ve and treatment-experienced patients. How- mimic the natural substrates of the RNA-dependent RNA ever, in virological non-sustained responders treated with polymerase and are incorporated into the elongated RNA, telaprevir-based triple therapy, no benefit of a lead-in-phase where they act as chain terminators; and non-nucleoside ana- was observed [27]. logue inhibitors, representing a heterogeneous group of In subsequent phase 3 studies, a larger number of treat- compounds that bind to different allosteric enzyme sites, ment-naı¨ve (SPRINT-2; n = 1097) and treatment-experienced resulting in a conformational protein change before the elon- (RESPOND-2; n = 403; partial non-responders and relapsers gation complex is formed. only) patients with HCV genotype 1 infection were enrolled to receive a 4-week lead-in treatment with PEG-IFN-a-2b/ Nucleos(t)ide inhibitors RBV alone, followed by either 44 weeks of triple combina- Nucleos(t)ide inhibitors potentially show similar efficacies tion therapy or a response-guided schedule with the possibil- against all HCV genotypes, because the active centre of ity of stopping therapy at week 28 or week 36 of overall NS5B is a highly conserved region. The first nucleoside ana- treatment duration, respectively. SVR rates were 63–66% in logue in development was valopicitabine (NM283, 2¢-C-meth- treatment-naı¨ve patients and 59–66% in treatment-experi- ylcytidine/NM107). However, valopicitabine showed only enced patients, as compared with 38% and 21% in the weak antiviral activity, and further drug development was respective control groups (Fig. 2b). Treatment discontinua- stopped because of gastrointestinal side effects [53]. The tions attributable to adverse events occurred in 12–16% of nucleoside analogue R1626 (4¢-azidocytidine/PSI-6130) treatment-naı¨ve patients and in 8–12% of treatment-experi- showed marked antiviral activity when given at high doses enced patients, as compared with 16% and 3% in the respec- alone or in combination with PEG-IFN-a-2a/RBV. No viral tive control groups [36,37]. breakthrough was reported for either monotherapy or combination therapy. However, further development was Other protease inhibitors stopped because of severe adverse events, including severe A large number of NS3/4A protease active site inhibitors are lymphocytopenia with lethal outcomes [54,55]. currently in early clinical development (Table 1a): danoprevir Among the nucleos(t)ide analogues still in development, (RG 7227/ITMN191), TMC435, vaniprevir (MK-7009), RG 7128 is the most advanced. Week 12 interim results of a BI 201335, narlaprevir (SCH900518), BMS-650032, phase 2 trial of RG 7128 in combination with PEG-IFN-a-2a/ PHX 1766, ACH-1625, IDX320, ABT-450, MK-5172, GS- RBV in treatment-naı¨ve genotype 1 or 4 patients have shown 9256 and GS-9451 have all been tested in phase 1 and/or early virological response rates of >80% and no resistance- phase 2 clinical trials [38–52]. Overall, these compounds related viral breakthrough [56]. SVR rates have to be exhibit high antiviral activity in HCV genotype 1 patients, and awaited. Other nucleotide analogue inhibitors that are

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TABLE 1. New hepatitis C virus therapies in the pipeline

(a)

Drug name Company Target/active drug Study phase

NS3/4A protease inhibitors Ciluprevir (BILN 2061) Boehringer Ingelheim Active site/macrocyclic Stopped Boceprevir (SCH503034) Merck Active site/linear Phase 3 Telaprevir (VX-950) Vertex Active site/linear Phase 3 Danoprevir (RG 7227) InterMune/Roche Active site/macrocyclic Phase 2 TMC435 Tibotec/Medivir Active site/macrocyclic Phase 2 Vaniprevir (MK-7009) Merck Active site/macrocyclic Phase 2 BI 201335 Boehringer Ingelheim Active site/linear Phase 2 BMS-650032 Bristol-Myers Squibb Active site Phase 2 GS-9256 Gilead Active site Phase 2 ABT-450 Abbott/Enanta Active site Phase 2 Narlaprevir (SCH900518) Merck Active site/linear On hold PHX 1766 Phenomix Active site Phase 1 ACH-1625 Achillion Active site Phase 1 IDX320 Idenix Active site/macrocyclic On hold MK-5172 Merck Active site/macrocyclic Phase 1 VX-985 Vertex Active site Phase 1 GS-9451 Gilead Active site Phase 1 Nucleos(t)ide NS5B polymerase inhibitors Valopicitabine (NM283) Idenix/Novartis Active site/NM107 Stopped RG 7128 Roche/Pharmasset Active site/PSI-6130 Phase 2 IDX184 Idenix Active site On hold R1626 Roche Active site/R1479 Stopped PSI-7977 Pharmasset Active site Phase 2 Non-nucleoside NS5B polymerase inhibitors BILB 1941 Boehringer Ingelheim NNI site 1/thumb 1 Stopped BI 207127 Boehringer Ingelheim NNI site 1/thumb 1 Phase 2 MK-3281 Merck NNI site 1/thumb 1 Stopped Filibuvir (PF-00868554) Pﬁzer NNI site 2/thumb 2 Phase 2 VX-916 Vertex NNI site 2/thumb 2 On hold VX-222 Vertex NNI site 2/thumb 2 Phase 2 VX-759 Vertex NNI site 2/thumb 2 Phase 1 ANA598 Anadys NNI site 3/palm 1 Phase 2 ABT-333 Abbott NNI site 3/palm 1 Phase 2 ABT-072 Abbott NNI site 3/palm 1 Phase 2 Nesbuvir (HCV-796) ViroPharma/Wyeth NNI site 4/palm 2 Stopped Tegobuvir (GS-9190) Gilead NNI site 4/palm 2 Phase 2 IDX375 Idenix NNI site 4/palm 2 Phase 1 NS5A inhibitors BMS-790052 Bristol-Myers Squibb NS5A domain 1 inhibitor Phase 2 BMS-824393 Bristol-Myers Squibb NS5A inhibitor Phase 1 PPI-461 Presidio NS5A inhibitor Phase 1 AZD7295 AstraZeneca NS5A inhibitor Phase 1

(b)

Indirect inhibitors/unknown mechanism of action NIM811 Novartis Cyclophilin inhibitor Stopped SCY-635 Scynexis Cyclophilin inhibitor Phase 1 Alisporivir (Debio 025) Debiopharm/Novartis Cyclophilin inhibitor Phase 2 Nitazoxanide Romark PKR induction? Phase 2 Celgosivir Migenix a-Glucosidase inhibitor Phase 2 New interferons Locteron (BLX-883) Biolex Interferon receptor type 1 Phase 2 PEG-rIL-29 (pegylated interferon-k) ZymoGenetics/BMS Interferon receptor type 3 Phase 2 Joulferon (albinterferon-a-2b) HGS/Novartis Interferon receptor type 1 Stopped

Double-stranded-RNA-activated protein kinase. PKR.

TABLE 2. Mutations conferring resistance to hepatitis C virus NS3 protease inhibitors; modiﬁed from [9]

V36A/M T54S/A V55A Q80R/K R155K/T/Q A156S A156T/V D168A/V/T/H V170A/T

Telaprevir (linear) a a Boceprevir (linear) a Narlaprevir (linear) Ciluprevir (macrocyclic) Danoprevir (macrocyclic) a a Vaniprevir (macrocyclic) TMC435 (macrocyclic) BI-201335 (linear) aMutations associated with resistance in vitro but not described in patients

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currently in clinical development include PSI-7977 (a chirally inhibitors include ABT-072 and ABT-333, both of which have pure isomer form of PSI-7851) and IDX184 [57,58]. entered phase 2 clinical trials [70].

Allosteric, non-nucleoside inhibitors Non-nucleoside site 4 inhibitors (palm 2/benzofuran site) The structure of the NS5B polymerase resembles a character- Monotherapy with HCV-796 (nesbuvir) showed low antiviral istic ‘right-hand motif’, consisting of finger, palm and thumb activity in HCV genotype 1-infected patients, and resulted in domains. At least four different allosteric binding sites have selection of resistant variants and viral breakthrough in sev- been identified for the inhibition of the NS5B polymerase by eral patients. Further drug development was suspended non-nucleoside inhibitors: a benzimidazole (thumb 1)-binding because of abnormal liver enzyme elevations in a subsequent site, a thiophene (thumb 2)-binding site, a benzothiadiazine phase 2 study [71]. GS-9190 (tegobuvir) displayed low antivi- (palm 1)-binding site, and a benzofuran (palm 2)-binding site ral activity in a phase 1 clinical study, and drug-resistant vari- [59,60]. As non-nucleoside inhibitors bind more distantly to ants were observed [72]. However, GS-9190 has entered the active site of NS5B, resistant mutations occur more fre- phase 2 clinical trials, both in combination with SOC and quently than for nucleos(t)ide analogues. with the protease inhibitor GS-9256 ± RBV. Finally, IDX375 also binds to the palm pocket, and is currently in phase 1 Non-nucleoside site 1 inhibitors (thumb 1/benzimidazole clinical trials [73]. site) BI 207127, BILB 1941 and MK-3281 are non-nucleoside NS5A inhibitors site 1 inhibitors that have been shown to exhibit low to Inhibitors of NS5A are potentially active against all HCV medium antiviral activity in phase 1 clinical trials [61–63]. genotypes. BMS-790052 binds to domain I of the NS5A pro- Four weeks of BI 207127 (600 mg three times daily) in com- tein, which is crucial for the regulation of HCV replication, bination with PEG-IFN-a-2a/RBV resulted in a median reduc- assembly and release. Following promising results from a tion of 5.6 log10 HCV RNA in treatment-naı¨ve genotype 1 phase 1 study, BMS-790052 is currently under investigation patients, and no viral breakthrough was observed [64]. in a number of phase 2 clinical trials. Recently, interim data Results of further studies have to be awaited. The further were released from a phase 2 study investigating different development of BILB 1941 and MK-3281 was halted because doses of BMS-790052 in combination with SOC. Rapid viro- of gastrointestinal adverse events. logical response rates were 83% and 92% in patients who received 10 and 60 mg BMS-790052 once daily, respectively. Non-nucleoside site 2 inhibitors (thumb 2/thiophene site) In addition, undetectable HCV RNA levels at week 12 The non-nucleoside site 2 inhibitor filibuvir (PF-00868554) (cEVR) were observed in 83% of patients [74]. has shown medium antiviral activity in phase 1 clinical trials. Other NS5A inhibitors that are in early clinical develop- Interim results from a phase 1 trial investigating different ment include BMS-824393, AZD7295 and PPI-461 [75–77]. doses of filibuvir in combination with PEG-IFN-a-2a/RBV for 4 weeks followed by SOC for 44 weeks have shown DAA Combination Therapies similar SVR (at 12 weeks of follow-up) results for the different filibuvir groups and for the SOC group, indicating that longer dosing with filibuvir is needed [65]. Other site 2 Interferon-associated side effects may limit treatment success inhibitors include VX-759, VX-916 and VX-222 [66–68]. in a large number of patients. Therefore, one of the primary However, only VX-222 has progressed to phase 2 develop- future goals of DAA therapy is to control viral replication or ment. even to achieve SVR in interferon-free regimens. In the INFORM-1 study, combinations of different doses of a poly- Non-nucleoside site 3 inhibitors (palm 1/benzothiadiazine merase inhibitor (RG 7128) and the NS3/4A protease inhibi- site) tor danoprevir (RG 7227/ITMN191) were tested in both A phase 2 trial of ANA598 in combination with PEG-IFN-a- treatment-naı¨ve and treatment-experienced patients with 2a/RBV in treatment-naı¨ve patients with HCV genotype 1 is HCV genotype 1 for up to 2 weeks. Up to 63% of treat- currently ongoing. It was recently reported that 75% of ment-naı¨ve patients achieved undetectable HCV RNA levels patients who received 400 mg of ANA598 twice daily after 2 weeks of combination therapy, and only one patient achieved undetectable HCV RNA levels at treatment experienced viral rebound without exhibiting resistance week 12 (complete early virological response (cEVR)) [69]. mutations [78]. HCV RNA negativity rates were somewhat However, SVR rates have to be awaited. Other site 3 palm 1 lower in treatment-experienced patients (25%).

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A number of other drug combinations are currently being 4 weeks of Debio 025 1000 mg/day as monotherapy [84]. investigated. Dual, triple and quadruple therapy with GS- No viral breakthrough has been observed in clinical studies 9256 and GS-9190 alone or in combination with RBV or so far. However, selection of HCV variants resistant to De- PEG-IFN-a/RBV for up to 28 days revealed the importance bio 025 with mutations clustering in the NS5A gene was of RBV for the enhancement of antiviral activity and reduc- observed in the HCV replicon system [85,86]. tion of viral breakthrough [79]. Interferon-sparing triple com- SCY-635 is another non-immunosuppressive analogue of bination therapy with BI 201335 and BI 207127 (400 or cyclosporin A that exhibits potent suppression of HCV. Dif- 600 mg three times daily) plus RBV resulted in undetectable ferent doses of SCY-635 were investigated in patients HCV RNA after 4 weeks in all patients treated with the infected with HCV genotype 1, and a mean maximum decline higher dose of BI 207127 [80]. Finally, in an ongoing trial of of 2.3 log10 IU/mL in viral load was observed after 15 days of HCV genotype 1 null-responders treated with BMS-790052 900-mg SCY-635 monotherapy [87]. No viral rebound was plus BMS-650032 alone or in combination with PEG-IFN-a/ observed during SCY-635 therapy. However, minimal evi- RBV for 24 weeks, 14/21 patients achieved cEVR, whereas all dence of resistance selection was observed within NS5B [88]. patients with viral breakthrough (n = 6) were genotype 1a patients, indicating that not all DAAs may be equally effective Silibinin in both HCV genotype 1a and 1b patients [81]. Silymarin, an extract of milk thistle (Silybum marianum) with Overcoming drug resistance will be the primary challenge antioxidant activity, has been used as self-medication for liver in DAA combination therapy, and drug combinations that diseases for centuries [89]. Silibinin is one of the six major have a genetic barrier of four or more mutations may be flavonolignans in silymarin. Although its mechanism of action required [82]. Future trials need to address the tolerability and treatment efficacy are not yet fully understood, it was and safety of long-term DAA administration, and whether recently reported that silibinin inhibited HCV RNA-depen- SVR can be achieved without the addition of PEG-IFN-a/ dent RNA polymerase activity [90]. However, its antiviral RBV. action includes blocking of virus entry and transmission, pos- sibly by targeting the host cell [91]. Intravenous silibinin was investigated in non-responders to Emerging Mechanisms prior IFN-based antiviral therapy, and led to a significant

decline in HCV RNA of between 0.55 and 3.02 log10 IU/mL Emerging potential drug candidates in future anti-HCV thera- after 7 days, and a further decrease after an additional 7 days pies include inhibitors of HCV entry, internal ribosome entry in combination with PEG-IFN-a-2a/RBV, in the range of site inhibitors, inhibitors of HCV assembly and release, and 1.63–4.85 log10 IU/mL [92]. Studies in larger patient cohorts, a-glucosidase inhibitors. However, potential compounds are including resistance analyses, are underway. currently still in preclinical or very early clinical development. Nitazoxanide Host cells may provide additional targets for antiviral ther- Nitazoxanide (alinia) is a thiazolide that has been shown in vi- apies, and several host cell-targeting inhibitors have entered tro to have activity against both hepatitis B virus and HCV. clinical development, with promising results. The antiviral method of action of nitazoxanide is still unknown, but it is believed that it inhibits viral glycoproteins Cyclophilin inhibitors at the post-translational level (i.e. PKR induction). A phase 2 Cyclophilins are ubiquitous proteins in human cells, and are study of 500 mg of nitazoxanide twice daily for 12 weeks, involved in protein folding. Moreover, cyclophilins participate followed by nitazoxanide, PEG-IFN-a-2a ± RBV for in HCV replication as functional regulators of the HCV 36 weeks, yielded an SVR rate of 79% in treatment-naı¨ve NS5B polymerase. genotype 4 patients [93]. However, a similar regimen in The cyclophilin inhibitor Debio 025 (alisporivir) showed treatment-naı¨ve genotype 1 patients yielded a week 12 SVR antiviral activity in patients infected with different HCV geno- rate of only 44% [94]. Further studies are warranted. types (1–4) during monotherapy and in combination studies with PEG-IFN-a. Maximum log changes in HCV RNA of up 10 New Interferons to 4.75 were observed in HCV genotype 1 or 4 patients who received 1000 mg of Debio 025 in combination with PEG-IFN-a-2a/RBV for 4 weeks [83]. There is one case Albinterferon-a-2b (Joulferon) is a recombinant protein con- report of a genotype 3a patient who achieved SVR following sisting of interferon-a-2b fused to human albumin that can

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be administered every 2 weeks, owing to a longer half-life HCV variants followed by viral breakthrough, convincing than that of the currently marketed pegylated interferons. results have been achieved for the combination of new anti- The efficacy and safety of albinterferon-a-2b administered viral agents with SOC. every 2 weeks in combination with RBV for 48 weeks in Results from phase 2 clinical studies of the currently most patients infected with HCV genotype 1, and for 24 weeks in advanced compounds, telaprevir and boceprevir, have indi- patients infected with HCV genotype 2/3, were recently cated that the addition of an HCV protease inhibitor to studied in two large phase 3 clinical trials (ACHIEVE 1 and PEG-IFN-a/RBV significantly improves SVR rates in both ACHIEVE 2/3) that were designed to demonstrate the non- treatment-naı¨ve and treatment-experienced patients. In addi- inferiority of these regimens as compared with SOC [95,96]. tion, response-guided treatment schedules have been suc- Both studies achieved the primary objective. In patients with cessfully applied [19,20,26]. Thus, two of the major goals of HCV genotype 1, intention-to-treat SVR rates of 51%, 48.2% anti-HCV therapy, i.e. higher SVR rates and shorter treat- and 47.3% were achieved for PEG-IFN-a-2a, albinterferon-a- ment duration based on RVR results, can be achieved. 2b (900 lg) and albinterferon-a-2b (1200 lg), respectively. The pivotal phase 3 trials of telaprevir and boceprevir in For genotype 2/3-infected patients, intention-to-treat SVR treatment-naı¨ve patients infected with HCV genotype 1 rates were 84.8%, 79.8% and 80%, respectively. The safety recently confirmed these promising results, showing SVR profile was similar between the different treatment regimens, rates ranging from 72% to 75% in telaprevir-treated patients indicating that albinterferon-a-2b was not better tolerated and from 63% to 66% in boceprevir-treated patients than PEG-IFN-a-2a. Owing to concerns expressed by the [24,25,37]. regulatory authorities regarding the benefit/risk ratio, further Most HCV polymerase inhibitors have shown low to med- development of albinterferon was recently suspended. ium antiviral activities during monotherapy studies. There- Locteron is a controlled-release formulation of interferon- fore, the future role of HCV polymerase inhibitors has yet a-2b that can be administered every 2 weeks. Week 12 to be determined, and SVR data from larger studies with tri- interim results from a phase 2b study have shown a similar ple combination therapies have to be awaited. Unlike prote- reduction in HCV RNA and a reduction of flu-like symptoms ase inhibitors, which are mostly active in patients with HCV by 57% for locteron at a dose of 480 lg as compared with genotype 1 only, polymerase inhibitors bind to the highly PEG-IFN-a-2b in treatment-naı¨ve patient with HCV geno- conserved centre of the HCV polymerase, with the possible type 1 [97]. SVR results have to be awaited. advantage of being equally effective for different HCV PEG-IFN-k (PEG-rIL-29) is a type III interferon that binds genotypes. to a unique receptor with a more limited distribution than With the addition of new antiviral compounds to standard the type I interferon receptor used by interferon-a. In a pha- therapy, a number of additional side effects unrelated to se 1b trial, 56 treatment-naı¨ve and SOC relapser patients SOC have been observed, and increased treatment discon- were enrolled to receive different doses of PEG-IFN-k (rang- tinuation rates have been reported. In addition, frequent ing from 0.5 to 3.0 lg/kg) administered every 2 weeks or dosing intervals (i.e. three times daily) may lead to increased weekly with or without daily RBV for 4 weeks. Antiviral non-adherence outside of clinical trials, and more patient- activity was seen at all dose levels, with a mean maximum friendly formulations need to be developed. change of HCV RNA from baseline of 3.27 log10 IU/mL in One of the major challenges of future anti-HCV treatment treatment-naı¨ve patients treated with weekly PEG-IFN-k regimens will be the emerging field of DAA drug resistance. (1.5 lg/kg) plus RBV [98]. Phase 2 trials are currently Relatively low genetic barriers to resistance have been ongoing. reported for HCV protease inhibitors and non-nucleoside polymerase inhibitors as compared with nucleos(t)ide HCV polymerase inhibitors, which appear to have a high genetic Conclusions barrier to resistance [9]. However, HCV eradication may not be achieved by monotherapy with either substance A number of advances have been made in the development group, and drug combinations with non-cross-resistance pat- of new therapeutic options for the treatment of chronic hep- terns have to be further evaluated. In addition, pre-existing atitis C. In particular, the challenges of treating patients with resistant variants and their potential long-term persistence HCV genotype 1 who achieve SVR rates in the range of 40– have to be taken into account for the selection of optimal 50% with the current SOC have been met by the develop- treatment and retreatment strategies. Although the addition ment of DAA agents. Although monotherapy with DAAs of PEG-IFN-a and RBV substantially reduces the frequency may not be suitable, owing to the selection of drug-resistant of viral breakthrough caused by resistance development,

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combinations of different classes of DAA agents may eventu- 8. Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat ally replace interferon-based regimens. Several trials address- Rev Microbiol 2007; 5: 453–463. 9. Sarrazin C, Zeuzem S. Resistance to direct antiviral agents in ing this issue are currently underway. In addition, the future patients with hepatitis C virus infection. Gastroenterology 2010; 138: of hepatitis C therapy will probably involve individually tai- 447–462. lored treatment durations, based on viral kinetics, IL28B 10. Wakita T, Pietschmann T, Kato T et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med polymorphisms and other parameters. 2005; 11: 791–796. Besides compounds that target HCV polyprotein process- 11. Lamarre D, Anderson PC, Bailey M et al. 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ª2011 The Authors Clinical Microbiology and Infection ª2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 17, 122–134