Daclatasvir and asunaprevir plus peginterferon alfa and ribavirin in HCV genotype 1 or 4 non-responders

Accepted Manuscript Daclatasvir and asunaprevir plus peginterferon alfa and ribavirin in HCV genotype 1 or 4 non-responders Donald Jensen, Kenneth E Sherman, Christophe Hézode, Stanislas Pol, Stefan Zeuzem, Victor de Ledinghen, Albert Tran, Magdy Elkhashab, Ziad H Younes, Marcelo Kugelmas, Stefan Mauss, Gregory Everson, Velimir Luketic, John Vierling, Lawrence Serfaty, Maurizia Brunetto, Jeong Heo, David Bernstein, Fiona McPhee, Delphine Hennicken, Patricia Mendez, Eric Hughes, Stephanie Noviello, on behalf of the HALLMARK-QUAD Study Team PII: DOI: Reference:

S0168-8278(15)00125-7 http://dx.doi.org/10.1016/j.jhep.2015.02.018 JHEPAT 5563

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

7 November 2014 3 February 2015 9 February 2015

Please cite this article as: Jensen, D., Sherman, K.E., Hézode, C., Pol, S., Zeuzem, S., Ledinghen, V.d., Tran, A., Elkhashab, M., Younes, Z.H., Kugelmas, M., Mauss, S., Everson, G., Luketic, V., Vierling, J., Serfaty, L., Brunetto, M., Heo, J., Bernstein, D., McPhee, F., Hennicken, D., Mendez, P., Hughes, E., Noviello, S., on behalf of the HALLMARK-QUAD Study Team, Daclatasvir and asunaprevir plus peginterferon alfa and ribavirin in HCV genotype 1 or 4 non-responders, Journal of Hepatology (2015), doi: http://dx.doi.org/10.1016/j.jhep.2015.02.018

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Daclatasvir and asunaprevir plus peginterferon alfa and ribavirin in HCV genotype 1 or 4 non-responders

Donald Jensen1, Kenneth E Sherman2, Christophe Hézode3, Stanislas Pol4, Stefan Zeuzem5, Victor de Ledinghen6, Albert Tran7, Magdy Elkhashab8, Ziad H Younes9, Marcelo Kugelmas10, Stefan Mauss11, Gregory Everson12, Velimir Luketic13, John Vierling14, Lawrence Serfaty15, Maurizia Brunetto16, Jeong Heo17, David Bernstein18, Fiona McPhee19, Delphine Hennicken20, Patricia Mendez21, Eric Hughes21, Stephanie Noviello21, on behalf of the HALLMARK-QUAD Study Team

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University of Chicago Medical Center, Chicago, Illinois, USA; 2University of Cincinnati, Cincinnati,

Ohio, USA; 3Department of Hepatology and Gastroenterology, Hôpital Henri Mondor, AP-HP, Université Paris-Est, INSERM U955, Créteil, France; 4APHP, Université Paris Descartes, INSERM U1016, Paris, France; 5Johann Wolfgang Goethe Universitaet, Frankfurt, Germany; 6Hepatology Unit, Hôpital Haut-Leveque, CHU Bordeaux and INSERM U1053, Bordeaux University, France; 7INSERM U1065, Team 8: Hepatic Complications in Obesity and CHU of Nice Digestive Center, Nice, France; 8

Toronto Liver Center, Toronto, Canada; 9Gastro One, Germantown, Tennessee, USA; 10South Denver

Gastroenterology, Denver, Colorado, USA; 11Center for HIV and Hepatogastroenterology, Duesseldorf, Germany; 12University of Colorado Denver, Aurora, Colorado, USA; 13Virginia Commonwealth University School of Medicine and McGuire Research Institute, McGuire DVAMC, Richmond, Virginia, USA; 14Baylor College of Medicine, Houston, Texas, USA; 15Hôpital Saint Antoine, Paris, France; 16Hepatology Unit, Reference Center of the Tuscany Region for Chronic Liver Disease and Cancer, University Hospital of Pisa, Pisa, Italy; 17Department of Internal Medicine, Pusan National University School of Medicine and Medical Research Institute, Pusan National University Hospital, Busan, Republic of Korea; 18North Shore-Long Island Jewish Health System, Manhasset, New York, USA; 19Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, USA;

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Bristol-Myers Squibb Research and Development, Braine-l'Alleud, Belgium; 21Bristol-Myers Squibb

Research and Development, Princeton, New Jersey, USA.

Corresponding Author: Prof Donald Jensen, MD Section of Gastroenterology, Center for Liver Diseases, The University of Chicago Biological Sciences, 5841 S. Maryland Ave., Chicago, IL 60637, USA Telephone: +1 773 795 7624 Fax: +1 773 834 1288 E-mail: [email protected]

Word count (including abstract, references, tables and figure legends): 5341 Figures and tables: 4 tables, 1 figure Trial registration: ClinicalTrials.gov identifier NCT01573351

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Abbreviations HCV, hepatitis C virus SVR, sustained virological response LLOQ, lower limit of quantification DAA, direct-acting antiviral ALT, alanine aminotransferase ULN, upper limit of normal AE, adverse event RVR, rapid virological response cEVR, complete early virological response eRVR, extended rapid virological response CI, confidence interval mITT, modified intent-to-treat AST, aspartate aminotransferase

Conflicts of Interest D. Jensen has received research funding from AbbVie, Boehringer-Ingelheim, Bristol-Myers Squibb, Genentech and Janssen. K. Sherman has received research funding from AbbVie, Bristol-Myers Squibb, Genentech, Gilead, Janssen, Merck and Janssen; and served as a board member for Janssen, Merck, MedPace and Synteract. C. Hézode has served as a speaker or advisor for AbbVie, BristolMyers Squibb, Gilead, Janssem, Merck, and Roche. S. Pol has received research funding from BristolMyers Squibb, Gilead, Roche, and Merck Sharp & Dohme; and served as a speaker and board member for Bristol-Myers Squibb, GlaxoSmithKline, Boehringer Ingelheim, Janssen, Gilead, Roche, Merck, Sanofi, Novartis, Vertex Pharmaceuticals, and AbbVie. S. Zeuzem has served as a speaker and/or consultant for AbbVie, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead, Idenix, Janssen, Merck, Novartis, Roche, Santaris, and Vertex. V. de Ledinghen has served a consultant for AbbVie,

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Bristol-Myers Squibb, Gilead, Janssen and Merck. Z. Younes has received research grants from AbbVie, Bristol-Myers Squibb, Idenix, Janssen, Merck, and Vertex; and personal fees from Gilead and Vertex. M. Kugelmas has received research funding from Bristol-Myers Squibb and served as a consultant for Bristol-Myers Squibb. S. Mauss has served a board member and consultant for AbbVie, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead, Janssen, Merck and Roche. G. Everson has received research funding from AbbVie, Bristol-Myers Squibb, Eisai, Gilead, Janssen, Merck, and Roche/Genentech; and is a board member for AbbVie, BioTest, Boehringer Ingelheim, Bristol-Myers Squibb, Eisai, Galectin, Gilead, Idenix, and Janssen; and has an issued patent (United States) and a pending patent (United States, European Union, Canada, and Australia) for liver function testing. V. Luketic has received research funding from AbbVie, Bristol-Myers Squibb, Conatus, Idenix, Merck, and Vertex. J Vierling has research funding and/or been a consultant for AbbVie, Bristol-Myers Squibb, Conatus, Gilead, GlaxoSmithKline, Globeimmune, Idenix, Janssen, Merck, Novartis, Pfizer, Pharmasset, Roche, and Vertex. M. Brunetto has served as a speaker for Abbott, Bristol-Myers Squibb, Gilead, Janssen, Merck, Novartis and Roche. J. Heo has received research funding from Bristol-Myers Squibb, GlaxoSmithKline, and Roche; and has served as an advisor for AbbVie, BristolMyers Squibb, Gilead, Jennerex, and Johnson & Johnson. D. Bernstein has received research funding from AbbVie, Bristol-Myers Squibb, Gilead, Janssen, Merck and Vertex; and has served as a board member/consultant for AbbVie, Bristol-Myers Squibb, Gilead, and Merck. F. McPhee, D. Hennicken and P. Mendez are employees of Bristol-Myers Squibb. E.Hughes and Stephanie Noviello are employees and stockholders of Bristol-Myers Squibb. A Tran, M. Elkhashab and L. Serfaty have no conflicts of interest to disclose.

Financial Support This study was funded by Bristol-Myers Squibb.

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Author’s Contributions P. Mendez and E. Hughes designed the study. D. Jensen, K. Sherman, C. Hézode, S. Pol, S. Zeuzem, V. de Ledinghen, A. Tran, M. Elkhashab, Z. Younes, M. Kugelmas, S. Mauss, G. Everson, V. Luketic, J. Vierling, L. Serfaty, M. Brunetto, J. Heo, and D. Bernstein recruited patients and obtained data. F. McPhee, D. Hennicken, E. Hughes and S. Noviello analysed the data. All authors interpreted the data, participated in writing the manuscript, and approved the final version of the manuscript.

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ABSTRACT (word count: 249; limit 250) Background and aims: Improved therapies for peginterferon/ribavirin null or partial responders are needed. This study evaluated daclatasvir (NS5A inhibitor) and asunaprevir (NS3 protease inhibitor) plus peginterferon-alfa-2a and ribavirin in this patient population. Methods: This open-label, phase 3 study (HALLMARK-QUAD; NCT01573351) treated patients with chronic hepatitis C virus (HCV) genotype 1 (n=354) or 4 (n=44) infection who had a prior null or partial response to peginterferon/ribavirin. Patients received daclatasvir 60 mg once-daily plus asunaprevir 100 mg twice-daily with weekly peginterferon-alfa-2a and weight-based ribavirin for 24 weeks. The primary endpoint was sustained virological response at post-treatment week 12 (SVR12) among genotype 1-infected patients. Results: Daclatasvir plus asunaprevir and peginterferon/ribavirin demonstrated SVR12 rates of 93% (95% CI 90-96) in prior non-responders infected with HCV genotype 1. SVR12 rates among genotype 4-infected patients were 98% (95% CI 93-100); one patient had a missing post-treatment week 12 HCV-RNA measurement, but achieved an SVR at post-treatment week 24, yielding a 100% SVR rate in genotype 4 patients. Prior peginterferon/ribavirin response, sex, age, IL28B genotype, or cirrhosis status did not influence SVR12 rates. Serious adverse events occurred in 6% of patients; 5% discontinued treatment due to an adverse event. Grade 3/4 laboratory abnormalities included neutropenia (22%), lymphopenia (16%), anemia (6%), thrombocytopenia (4%), and ALT/AST elevations (3% each). Conclusions: Daclatasvir plus asunaprevir and peginterferon/ribavirin demonstrated high rates of SVR12 in genotype 1- or 4-infected prior null or partial responders. The combination was well tolerated and no additional safety and tolerability concerns were observed compared with peginterferon/ribavirin regimens.

Keywords: daclatasvir, asunaprevir, direct-acting antiviral, peginterferon alfa, null, partial, NS5A

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INTRODUCTION Chronic hepatitis C virus (HCV) infection is associated with a progressive liver disease that can lead to cirrhosis, and hepatocellular carcinoma. Current estimates indicate that 130–150 million people worldwide are chronically infected with HCV, resulting in up to 350,000 deaths annually [1, 2]. Seven HCV genotypes have been identified, with genotype 1 being the most prevalent worldwide [3].

Current treatment options for HCV genotype 1 or 4 infection include the NS5B polymerase inhibitor sofosbuvir or the NS3 protease inhibitor simeprevir (approved for genotype 4 in Europe) in combination with peginterferon/ribavirin [4, 5]. In addition, daclatasvir plus peginterferon/ribavirin has been recently approved for the treatment of genotype 4 HCV infection in Europe, along with the all-oral combinations of daclatasvir plus sofosbuvir ± ribavirin and simeprevir plus sofosbuvir ± ribavirin for genotype 1- or 4-infected patients. Patients with a prior null or partial response to peginterferon/ribavirin therapy typically represent some of the more difficult patients to treat successfully. Simeprevir plus peginterferon/ribavirin achieved sustained virological response (SVR) rates of 44% and 70% in genotype 1 null and partial responders, respectively, and 40% and 60% in genotype 4 null and partial responders, respectively [6, 7]. Sofosbuvir and peginterferon/ribavirin was not evaluated in peginterferon/ribavirin failures, but it is estimated that SVR rates will approximate those in patients with poor response indicators (71%) [8]. Higher SVR rates of 94% were achieved in the small numbers of genotype 1 null responders treated with simeprevir and sofosbuvir, with or without ribavirin for 12–24 weeks [9]. Similarly, the combination of daclatasvir plus sofosbuvir has demonstrated SVR rates of 95–100% among 41 genotype 1 patients who had failed prior protease inhibitor plus peginterferon/ribavirin therapy [10].

Daclatasvir is a potent, pan-genotypic inhibitor of the HCV NS5A protein with activity against genotypes 1 to 6 in vitro [11]. Asunaprevir is an NS3 protease inhibitor with activity against genotypes 1 and 4 [12]. The efficacy and safety of the combination of daclatasvir and asunaprevir

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with or without peginterferon/ribavirin in genotype 1 null responders was evaluated in a phase 2 study (AI447011). In a sentinel cohort, SVR was achieved in all 10 patients treated with daclatasvir plus asunaprevir and peginterferon/ribavirin [13], with SVR rates of greater than 90% achieved in an expansion cohort of 41 genotype 1 null responders [14].

This phase 3 study (HALLMARK-QUAD) evaluated the efficacy and safety of daclatasvir plus asunaprevir combined with peginterferon/ribavirin in patients infected with HCV genotype 1 or 4 who were prior null or partial responders to peginterferon/ribavirin.

PATIENTS AND METHODS Study design and participants This was a single-arm, open-label, phase 3 study in patients infected with HCV genotype 1 or 4 who were null or partial responders to peginterferon alfa-2a or -2b plus ribavirin (Study AI447029; ClinicalTrials.gov number NCT01573351). Patients received daclatasvir 60 mg once-daily, asunaprevir 100 mg softgel capsule twice-daily and 180 µg peginterferon alfa-2a weekly and twice-daily ribavirin dosed according to bodyweight (<75 kg, 1000 mg daily; ≥75 kg, 1200 mg daily) for 24 weeks and were subsequently followed for 24 weeks post-treatment. Patients discontinued therapy for futility (any confirmed HCV-RNA greater than or equal to the lower limit of quantification [LLOQ] at week 8) or virological breakthrough (confirmed >1 log10 increase in HCV-RNA over nadir or confirmed HCVRNA ≥LLOQ after confirmed undetectable HCV-RNA on-treatment). Patients who required permanent discontinuation of peginterferon and/or ribavirin in accordance with the package inserts were allowed to continue on daclatasvir plus asunaprevir until week 24.

Eligible patients were prior null or partial responders to peginterferon/ribavirin with HCV genotype 1 or 4 infection. Patients were aged at least 18 years with HCV-RNA ≥10,000 IU/mL. A minimum of 40%

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of either subtype 1a or non-1a were enrolled; genotype 4-infected patients were capped at a maximum of 10%. Null response to peginterferon/ribavirin was defined as a <2 log10 decline in HCVRNA after ≥12 weeks of therapy, or a <1 log10 decline after ≥4 weeks of therapy (limited to 10%). Partial responders had achieved a ≥2 log10 decline, but never achieved undetectable HCV-RNA after ≥12 weeks of peginterferon/ribavirin therapy, or became undetectable and subsequently had detectable HCV-RNA on-treatment (limited to 10%). Patients with compensated cirrhosis were eligible but were capped at a maximum of 25% of the treated population. Patients were considered ineligible if they had received prior direct-acting antiviral (DAA) therapy, were co-infected with HIV or HBV, had alanine aminotransferase (ALT) levels ≥5× upper limit of normal (ULN), evidence of hepatic decompensation, platelets <90×109/L, or albumin <3.5 g/dL.

Assessments and endpoints HCV-RNA was assayed using the Roche HCV COBAS® TaqMan® Test v2.0 (LLOQ 25 IU/mL). HCV genotype/subtype was determined by the VERSANT HCV genotype 2.0 assay (LIPA), and IL28B genotype (rs12979860 single nucleotide polymorphism) by the Applied Biosystems® TaqMan assay. Resistance testing was performed by population sequencing at baseline and on samples from patients with virological failure or relapse with HCV-RNA ≥1000 IU/mL. Safety monitoring assessed the incidence of adverse events (AEs) and abnormalities in clinical laboratory assessments, vital signs, and physical examinations.

The primary endpoint was the proportion of genotype 1-infected patients with SVR at posttreatment week 12 (SVR12; HCV-RNA <25 IU/mL, detectable or undetectable). Secondary efficacy endpoints included the proportion of genotype 1-infected patients with undetectable HCV-RNA at week 4 (rapid virological response; RVR) or week 12 (complete early virological response; cEVR); at weeks 4 and 12 (extended rapid virological response; eRVR); end of treatment; and with HCV-RNA <25 IU/mL, detectable or undetectable at post-treatment week 24 (SVR24); the proportions of

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genotype 1-infected patients with SVR12 by IL28B status; and genotype 4-infected patients with SVR12. On-treatment safety endpoints included the incidence of serious AEs and discontinuations due to AEs.

Statistical analyses A target sample size of 390 patients was selected to detect, with 90% probability, a safety event occurring at an incidence of 0.6%. For the primary analysis, a sample size of approximately 350 genotype 1-infected patients ensures a 95% CI for the SVR12 rate with a width <11%.

The primary endpoint and other efficacy analyses were based on a modified intent-to-treat (mITT) analysis of all treated patients where patients with missing data were counted as failures. For the primary endpoint, an analysis based on SVR12 documented on or after post-treatment week 12 was also conducted. Two-sided 95% CIs for response rates were computed using normal approximations to the binomial distribution. Safety analyses included all patients who had received at least one dose of study medication.

Role of the funding source The sponsor, in collaboration with the authors, participated in study design; data collection, analysis, and interpretation; and drafting of the manuscript. All authors had full access to the data and vouch for the integrity and accuracy of the data reported. The corresponding author had final responsibility for the decision to submit for publication.

RESULTS Patient disposition and baseline characteristics Of the 496 patients who were screened for the study, 398 were treated between May 2012 and December 2013 (Fig. 1). The primary reasons for patients not being treated were failure to meet the

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protocol criteria or withdrawn consent. The majority of patients (379/398; 95.2%) completed the 24week treatment period.

Baseline demographics and disease characteristics are presented in Table 1. The majority of patients were male (68.6%) and white (76.4%) with a median age of 52.7 years. Of note, the genotype-4 arm contained fewer Asian patients and patients were of younger age; baseline HCV-RNA levels were also lower (86.7% of genotype 1-infected patients had HCV-RNA ≥800,000 IU/mL vs. 65.9% of genotype 4-infected patients). Per protocol, the proportions of genotype 1-infected patients with subtype 1a and 1b were similar. Approximately two-thirds of the study population were null responders and as expected for such a patient population, a high proportion of patients had a nonCC IL28B genotype. Overall, 23.4% of patients enrolled in the study had compensated cirrhosis; 45.5% of genotype 4-infected patients had compensated cirrhosis compared with 20.6% of genotype 1-infected patients.

Virological response Daclatasvir plus asunaprevir and peginterferon/ribavirin achieved SVR12 rates of 92.9% (329/354) in genotype 1-infected patients (Table 2). Among genotype 4-infected patients, the SVR12 rate was 97.7% (43/44). One patient had a missing post-treatment week 12 HCV-RNA value, but subsequently achieved SVR24, yielding a 100% SVR rate in genotype 4-infected patients. This regimen demonstrated rapid early antiviral activity, with mean HCV-RNA reductions from baseline of -5.0 and -4.7 log10 IU/mL at week 2 among genotype 1- and 4-infected patients, respectively; RVR was observed among 82.5% (292/354) and 81.8% (36/44) of patients, respectively. High proportions of patients achieved cEVR, consistent with the high rates of SVR12 observed (Table 2). SVR12 rates in genotype 1-infected patients who achieved RVR were 94.9% compared with 83.1% among those who did not achieve RVR.

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SVR12 rates in genotype 1-infected patients with cirrhosis at baseline (90.4%; 66/73) were comparable to those without cirrhosis (93.6%; 263/281). Response rates were also similar among those who were null or partial responders (Table 3). Additionally, baseline factors previously associated with a suboptimal response to peginterferon/ribavirin-based regimens, such as sex, age, body mass index, and baseline HCV-RNA did not appear to affect response, as SVR12 rates were high across all groups. SVR12 rates were slightly lower among the small number of black patients (87.9%; 29/33) compared with white patients (92.6%; 251/271). SVR12 was higher among patients infected with subtype 1b (98.9%; 176/178) compared with subtype 1a (86.9%; 153/176).

Virological failure Virological failure was observed infrequently among genotype 1-infected patients: 3.1% (11/354) experienced virological breakthrough, 0.6% (2/354) had detectable HCV-RNA at the end of treatment, and 2.4% (8/337) experienced relapse post-treatment (Table 2). Nineteen patients met the criteria for resistance testing (HCV-RNA ≥1000 IU/mL). The most frequently observed resistanceassociated variants detected at failure in genotype 1a patients were NS5A-Q30 plus NS3-R155 (11 patients) or NS5A-Q30 plus NS3-D168 (5 patients) (Supplementary Tables 1 and 2). In the single genotype 1b patient with virological failure, NS5A-L31M-P58S polymorphisms were present at baseline and NS5A-L31I/M-Y93H and NS3-D168V were detected together at failure. At baseline, NS5A polymorphisms known to impact daclatasvir activity ≥2-fold in vitro were detected in 35/372 patients with available sequences (Supplementary Table 3): 31 of these 35 patients achieved SVR at post-treatment week 12 or later, while one achieved SVR4 and was lost to follow-up, and three experienced virological breakthrough. NS3 polymorphisms known to impact asunaprevir activity ≥2fold in vitro were detected in 58/378 patients with available NS3 baseline sequences (Supplementary Table 3): 47 achieved SVR12; one of the remaining genotype 1a patients achieved SVR4 and was lost to follow-up, and three had pre-existing NS3-R155 polymorphisms and experienced virological

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failure. Two of these three patients also had other baseline polymorphisms associated with protease inhibitor resistance, including one patient with a linked R155K-D168N polymorphism.

No genotype 4-infected patients experienced virological failure. One patient with a missing HCV-RNA measurement at post-treatment week 12 was classified as a failure in the SVR12 mITT analysis; however, this patient had undetectable HCV-RNA at post-treatment week 24.

Safety Serious AEs were reported in 5.5% (22/398) of patients during treatment, with nine events considered by the investigator to be related to study therapy: anemia, anemia/dehydration, encephalopathy, pneumonia, traumatic ulcer, lymphadenopathy, sepsis, hepatic enzymes increased, and dry skin in one patient each. Discontinuations due to AEs occurred in 4.5% (18/398) of patients; the most frequent events leading to discontinuation were rash, malaise, neutropenia, and vertigo, occurring in two patients each. Three patients discontinued peginterferon alone and three patients discontinued peginterferon/ribavirin; these patients continued therapy with daclatasvir plus asunaprevir, with or without ribavirin, and all achieved SVR12. One death occurred during posttreatment week 12 due to pneumonia, and this was not considered to be related to therapy.

Daclatasvir plus asunaprevir and peginterferon/ribavirin was well tolerated in this patient population. The most frequent AEs and laboratory abnormalities observed during treatment were those typically associated with peginterferon/ribavirin therapy. AEs occurring at a frequency >20% were fatigue, headache, pruritus, asthenia, influenza-like illness, insomnia, and rash (Table 4; Supplementary Table 4). No clinically relevant differences in hepatic laboratory abnormalities or AEs were noted between patients with or without cirrhosis (Supplementary Table 5). Grade 3/4 ALT and aspartate aminotransferase (AST) elevations occurred in 3.0% and 3.3% of patients, respectively. One patient discontinued asunaprevir therapy due to elevated ALT levels (Grade 4) and completed

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treatment with daclatasvir plus peginterferon/ribavirin. Among the 12 patients who had ontreatment Grade 3/4 ALT elevations, the median times to first ALT elevation and reversal were 57 days (range 16–86) and 22 days (range 9–85), respectively. Further details on the time course of these Grade 3/4 ALT elevations are provided in Supplementary Figure 1. For the 13 patients with Grade 3/4 AST elevations, the median time to first elevation was 57 days (range 27–169), and for AST reversal was 27 days (range 8–141). One non-cirrhotic patient experienced a 4.0 g/dL drop in haemoglobin within the first 2 weeks of therapy and then met the pre-specified laboratory criteria for potential drug-induced liver injury (concurrent ALT ≥ 5× baseline or nadir value and ≥ 10×ULN and total bilirubin ≥ 2×ULN) at week 8. Asunaprevir therapy was interrupted at week 9 and was resumed after 9 days. The patient remained asymptomatic without clinical decompensation, and the event of ‘increased hepatic enzyme’ was considered resolved at week 20. The patient completed 24 weeks of therapy and achieved SVR12.

DISCUSSION This global phase 3 study evaluated the efficacy, safety, and tolerability of 24 weeks of treatment with daclatasvir plus asunaprevir and peginterferon/ribavirin in prior null and partial responders. This regimen achieved a rapid, sustained suppression of HCV-RNA and high rates of SVR12 in both genotype 1- and 4-infected patients. Viral breakthrough and relapse were observed infrequently, which combined with the low rate of AE-related discontinuations, led to a high number of patients completing treatment in this study compared with historical peginterferon/ribavirin-based studies [15, 16].

As with prior non-responders to peginterferon/ribavirin, patients with compensated cirrhosis represent a difficult-to-treat patient population with an unmet need, especially as the number of patients with HCV-associated cirrhosis is predicted to increase rapidly as the HCV-infected

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population ages and undergoes more advanced disease progression [17]. In this study, which treated a large number of cirrhotic null and partial responders (23%), daclatasvir plus asunaprevir and peginterferon/ribavirin resulted in high SVR12 rates in both cirrhotic (90.4%) and non-cirrhotic genotype 1 patients (93.6%). Null and partial responders infected with HCV genotype 4 also have fewer current treatment options and fewer therapies in development. This regimen demonstrated 100% SVR rates (post-treatment week 12 or later) in genotype 4-infected patients, confirming the efficacy of daclatasvir and asunaprevir in genotype 4 patients and supporting the ongoing development of the all-oral triple DAA regimen of daclatasvir and asunaprevir plus beclabuvir (BMS791325), which has achieved high SVR rates in both genotype 1 (89–94%) and genotype 4 (100%) patients [18, 19]. Other potential treatment options in development for this population include the combination of ABT-450/ritonavir plus ombitasvir, which achieved high SVR rates in a small cohort of 50 genotype 4-infected null and partial responders [20].

Baseline polymorphisms associated with resistance to daclatasvir and asunaprevir were observed in this study. Three patients with baseline asunaprevir resistance polymorphisms did experience virological breakthrough. Multiple linked resistance variants were present in two of these patients, which are unusual in protease inhibitor-naive patients, suggesting these patients may have been previously exposed to protease inhibitor-based therapy, but this could not be verified after further investigation. No association between the presence of baseline daclatasvir resistance polymorphisms and virological failure was observed.

The limitations of this study include that this was an open-label, single-arm trial, with no active comparator or placebo arm, which was due to the low efficacy of the approved peginterferon/ribavirin-based regimens in this patient population when the study was initiated. The results of this study will also not be applicable to patient populations who are either interferonintolerant or -ineligible.

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The combination of daclatasvir plus asunaprevir and peginterferon/ribavirin was generally well tolerated, with low rates of serious AEs and safety-related discontinuations observed. In general, the types of AEs observed were comparable to those in historical studies of peginterferon/ribavirin; no additional safety or tolerability concerns were identified compared with peginterferon/ribavirin alone, and the safety profile was comparable to that observed in studies of daclatasvir or asunaprevir plus peginterferon/ribavirin. ALT and/or AST elevations have been reported in other studies of asunaprevir, particularly with the higher 600-mg dose [21]. In this study, with a lower asunaprevir dose of 100 mg twice daily, rates of Grade 3 or 4 aminotransferase elevations were low and reflective of other asunaprevir-containing regimens [13, 14, 21, 22]; and when they occurred they were reversible and easily managed, and no clinical signs of hepatic decompensation were noted.

Historically, studies of peginterferon/ribavirin therapy have demonstrated high rates of AEs due to the systemic side effects of interferon. However, in this study, rates of serious AEs and discontinuations due to AEs were lower at 5.5% and 4.5%, respectively. In large studies of prior nonresponders, serious AEs were reported in 9–11% and discontinuations due to AEs occurred in 4–7% [15, 16]. Rates of discontinuations due to AEs observed during the telaprevir and boceprevir registration studies of non-responders were 11–15% and 8–12%, respectively [23, 24]. These differences may be due to the shorter duration of therapy in this study (24 vs. 48 weeks) in line with the lower discontinuation rates observed with shorter treatment durations of simeprevir plus peginterferon/ribavirin (1–3%) or sofosbuvir plus peginterferon/ribavirin (2%) [25-27]. Additionally, this study enrolled patients with prior peginterferon/ribavirin exposure who are likely to be familiar with, and more tolerant of, the side effects of peginterferon/ribavirin.

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Future therapeutic options for patients with a partial or null response to peginterferon/ribavirin are likely to focus on all-oral regimens; daclatasvir in combination with other DAAs including asunaprevir and sofosbuvir, and simeprevir plus sofosbuvir, have recently been approved in Europe and/or Japan. Those currently being evaluated include a 12-week regimen of ABT-450/ritonavir, ombitasvir, dasabuvir, and ribavirin in genotype 1-infected patients, which demonstrated SVR rates of 92% and 96% in cirrhotic (n = 208) and non-cirrhotic patients (n = 297), respectively [28, 29]. In a phase 3 study, 12 to 24 weeks’ treatment with the fixed-dose combination of sofosbuvir plus ledipasvir, with or without ribavirin, provided SVR rates of 94–99% in genotype-1 peginterferon/ribavirin or NS3 protease inhibitor failures [30]. All these regimens were well tolerated and provided SVR rates comparable to those observed in this study. All-oral regimens are generally associated with low rates of virologic failure, but where this occurs, it is possible that a combination of DAAs and peginterferon/ribavirin may provide a viable treatment option for these patients.

In conclusion, 24 weeks of treatment with the quad regimen of daclatasvir plus asunaprevir and peginterferon/ribavirin was well tolerated and demonstrated high SVR12 rates among genotype 1or 4-infected non-responders, including patients with cirrhosis. These data support the efficacy and safety of daclatasvir and asunaprevir. Daclatasvir is approved in Japan and Europe for the treatment of chronic hepatitis C infection in combination with other antivirals and is being further evaluated as a component of multiple DAA-only combinations across several different patient populations. Daclatasvir plus asunaprevir will be developed as part of an all-oral, interferon-free, three-DAA fixeddose combination with the NS5B inhibitor beclabuvir for use without peginterferon/ribavirin.

17

ACKNOWLEDGEMENTS This study was supported by Bristol-Myers Squibb. Editorial support was provided by Andrew Street, PhD, of Articulate Science and was funded by Bristol-Myers Squibb.

18

REFERENCES [1]

World Health Organization. Hepatitis C key facts. WHO factsheet No 164. July 2013.

[2]

Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology 2013;57:1333-1342.

[3]

Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, et al. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment Web resource. Hepatology 2014;59:318-327.

[4]

American Association for the Study of Liver Diseases, Infectious Diseases Society of America. Recommendations for testing, managing, and treating hepatitis C. Available at http://www.hcvguidelines.org/. Accessed October 1, 2014.

[5]

European Association for the Study of the Liver. EASL Clinical Practice Guidelines: Management of hepatitis C virus infection. J Hepatol 2014;60:392-420.

[6]

Reddy KR, Zeuzem S, Zoulim F, Weiland O, Horban A, Stanciu C, et al. A Phase III randomised, double-blind study to evaluate the efficacy, safety and tolerability of simeprevir vs telaprevir in combination with pegylated interferon and ribavirin in chronic hepatitis C virus genotype 1 treatment-experienced patients: the ATTAIN study [abstract]. Hepatol Int 2014;8:S397.

[7]

Moreno C, Hezode C, Marcellin P, Bourgeois S, Francque S, Samuel D, et al. Once-daily simeprevir (TMC435) with peginterferon/ribavirin in treatment-naïve or treatmentexperienced chronic HCV genotype 4-infected patients: final results of a phase III trial [abstract]. J Hepatol 2014;60:S535.

[8]

Food and Drug Administration. SOVALDI™ (Sofosbuvir) Prescribing Information 2014.

[9]

Lawitz E, Sulkowski MS, Ghalib R, Rodriguez-Torres M, Younossi ZM, Corregidor A, et al. Simeprevir plus sofosbuvir, with or without ribavirin, to treat chronic infection with hepatitis C virus genotype 1 in non-responders to pegylated interferon and ribavirin and treatment-naive

19

patients: the COSMOS randomised study. Lancet 2014; DOI: 10.1016/S0140-6736(14)61036-9 (E-pub July 28, 2014). [10] Sulkowski MS, Gardiner DF, Rodriguez-Torres M, Reddy KR, Hassanein T, Jacobson I, et al. Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection. N Engl J Med 2014;370:211-221. [11] Gao M. Antiviral activity and resistance of HCV NS5A replication complex inhibitors. Curr Opin Virol 2013;3:514-520. [12] McPhee F, Sheaffer AK, Friborg J, Hernandez D, Falk P, Zhai G, et al. Preclinical profile and characterization of the hepatitis C virus NS3 protease inhibitor asunaprevir (BMS-650032). Antimicrob Agents Chemother 2012;56:5387-5396. [13] Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib R, et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N Engl J Med 2012;366:216-224. [14] Lok AS, Gardiner DF, Hezode C, Lawitz EJ, Bourliere M, Everson GT, et al. Randomized trial of daclatasvir and asunaprevir with or without PegIFN/RBV for hepatitis C virus genotype 1 null responders. J Hepatol 2014;60:490-499. [15] Poynard T, Colombo M, Bruix J, Schiff E, Terg R, Flamm S, et al. Peginterferon alfa-2b and ribavirin: effective in patients with hepatitis C who failed interferon alfa/ribavirin therapy. Gastroenterology 2009;136:1618-1628. [16] Jensen DM, Marcellin P, Freilich B, Andreone P, Di Bisceglie A, Brandao-Mello CE, et al. Retreatment of patients with chronic hepatitis C who do not respond to peginterferon-alpha2b: a randomized trial. Ann Intern Med 2009;150:528-540. [17] Davis GL, Alter MJ, El-Serag H, Poynard T, Jennings LW. Aging of hepatitis C virus (HCV)infected persons in the United States: a multiple cohort model of HCV prevalence and disease progression. Gastroenterology 2010;138:513-21, 521.e1-6.

20

[18] Everson GT, Sims KD, Rodriguez-Torres M, Hezode C, Lawitz E, Bourliere M, et al. Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection. Gastroenterology 2014;146:420-429. [19] Hassanein T, Sims K, Bennett M, Gitlin N, Lawitz E, Nguyen T, et al. All-oral therapy with daclatasvir in combination with asunaprevir and BMS-791325 in treatment-naive patients with chronic HCV genotype 4 infection [abstract]. J Hep 2014;60(suppl1):S472. [20] Hezode C, Marcellin P, Pol S, Hassanein T, Fleischer-Stepniewska K, Baykal T, et al. Results from the phase 2 PEARL-I study: interferon-free regimens of ABT-450/r + ABT-267 with or without ribavirin in patients with HCV genotype 4 infection [abstract]. J Hepatol 2014;60(suppl1):S24. [21] Bronowicki JP, Pol S, Thuluvath PJ, Larrey D, Martorell C, Rustgi V, et al. Randomized study of asunaprevir plus peginterferon alfa and ribavirin for previously untreated genotype 1 chronic hepatitis C. Antivir Ther 2013;18:885-893. [22] Kumada H, Suzuki Y, Ikeda K, Toyota J, Karino Y, Chayama K, et al. Daclatasvir plus asunaprevir for chronic HCV genotype 1b infection. Hepatology 2014;59:2083-2091. [23] Zeuzem S, Andreone P, Pol S, Lawitz E, Diago M, Roberts S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med 2011;364:2417-2428. [24] Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med 2011;364:1207-1217. [25] Jacobson IM, Dore GJ, Foster GR, Fried MW, Radu M, Rafalsky VV, et al. Simeprevir with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients with chronic hepatitis C virus genotype 1 infection (QUEST-1): a phase 3, randomised, double-blind, placebo-controlled trial. Lancet 2014;384:403-413. [26] Manns M, Marcellin P, Poordad F, de Araujo ES, Buti M, Horsmans Y, et al. Simeprevir with pegylated interferon alfa 2a or 2b plus ribavirin in treatment-naive patients with chronic

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hepatitis C virus genotype 1 infection (QUEST-2): a randomised, double-blind, placebocontrolled phase 3 trial. Lancet 2014;384:414-426. [27] Lawitz E, Mangia A, Wyles D, Rodriguez-Torres M, Hassanein T, Gordon SC, et al. Sofosbuvir for previously untreated chronic hepatitis C infection. N Engl J Med 2013;368:1878-1887. [28] Zeuzem S, Jacobson IM, Baykal T, Marinho RT, Poordad F, Bourliere M, et al. Retreatment of HCV with ABT-450/r-Ombitasvir and Dasabuvir with Ribavirin. N Engl J Med 2014;370:16041614. [29] Poordad F, Hezode C, Trinh R, Kowdley KV, Zeuzem S, Agarwal K, et al. ABT-450/r-Ombitasvir and dasabuvir with ribavirin for hepatitis C with cirrhosis. N Engl J Med 2014;370:1973-1982. [30] Afdhal N, Reddy KR, Nelson DR, Lawitz E, Gordon SC, Schiff E, et al. Ledipasvir and sofosbuvir for previously treated HCV genotype 1 infection. N Engl J Med 2014;370:1483-1493.

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TABLES Table 1. Baseline demographics and disease characteristics. Genotype 1

Genotype 4

(n = 354)

(n = 44)

54 (19–76)

52 (20–71)

<65 years

320 (90.4)

41 (93.2)

≥65 years

34 (9.6)

3 (6.8)

Male sex, n (%)

240 (67.8)

33 (75.0)

White

271 (76.6)

33 (75.0)

Black

33 (9.3)

4 (9.1)

Asian

47 (13.3)

1 (2.3)

Other

3 (0.8)

6 (13.6)

6.50 (0.528)

6.08 (0.549)

<800,000 IU/mL

47 (13.3)

15 (34.1)

≥800,000 IU/mL

307 (86.7)

29 (65.9)

73 (20.6)

20 (45.5)

1a

176 (49.7)

NA

1b

178 (50.3)

NA

4

NA

44 (100)*

CC

33 (9.3)

3 (6.8)

CT

231 (65.3)

31 (70.5)

TT

90 (25.4)

10 (22.7)

Null

234 (66.1)

34 (77.3)

Partial

120 (33.9)

10 (22.7)

Characteristic Median age, years (range) Age category, n (%)

Race, n (%)

Mean HCV-RNA, log10 IU/mL (SD) HCV-RNA distribution, n (%)

Cirrhosis, n (%) HCV genotype, n (%)

IL28B genotype, n (%)

Prior response to P/R, n (%)

HCV, hepatitis C virus; NA, not applicable; P/R, peginterferon alfa and ribavirin; SD, standard deviation. *20 patients were subtype a/c/d, three patients were subtype e, one patient was subtype h; genotype 4 subtype could not be determined for the remaining 20 patients.

23

Table 2. Virological response.

Genotype 1*

Genotype 4

(n = 354)

(n = 44)

329 (92.9)

43 (97.7)

[90.3–95.6]

[93.3–100]

SVR12 on or after post-treatment

330 (93.2)

44 (100)

Week 12†,‡

[90.6–95.8]

Patients, n (%) [95% CI] SVR12 (mITT)

SVR24

†

†,§

313 (88.4)

42 (95.5)

[85.1–91.8]

[89.3–100]

292 (82.5)

36 (81.8)

[78.5–86.4]

[70.4–93.2]

284 (80.2)

36 (81.8)

[76.1 –84.4]

[70.4 –93.2]

337 (95.2)

44 (100)

HCV-RNA undetectable at: Week 4 (RVR)

Weeks 4 and 12 (eRVR)

Week 12 (cEVR)

[93.0 –97.4]

End of treatment

337 (95.2)

43 (97.7)

[93.0 –97.4]

[93.3 –100]

25 (7.1)

1 (2.3)

Virological breakthrough

11 (3.1)

0

Detectable HCV-RNA at end of

2 (0.6)††

0

Relapse

8/337 (2.4)

0/43 (0)

Missing HCV-RNA at follow-up

4/337 (1.2)

1/43 (2.3)§§

Virological failures (non-SVR12), n (%) All On-treatment failures¶

treatment Post-treatment failures‡‡

Week 12 cEVR, complete early virological response; eRVR, extended rapid virological response; HCV, hepatitis C virus; LLOQ, lower limit of quantification; mITT, modified intent-to-treat; RVR, rapid virological response; SVR12, sustained virological response at post-treatment week 12.

*One patient was infected with genotype 4 as determined by NS3 and NS5A sequence alignment (indeterminate genotype) but was assigned in the Interactive Voice Response System to the

24

genotype 1a cohort. This patient completed 24 weeks of treatment and was an RVR, eRVR, cEVR, SVR12, and SVR24 responder. †

HCV-RNA undetectable or below assay LLOQ for SVR12 and SVR24 endpoints.

‡

For patients with missing post-treatment week 12 HCV-RNA, the first available measurement after

post-treatment week 12 was used. §

Thirteen genotype 1-infected patients who achieved SVR12 had missing HCV-RNA measurements at

post-treatment week 24; three genotype 1a patients experienced a confirmed relapse between post-treatment weeks 12 and 24 and one additional genotype 1a patient had a HCV-RNA level of 39 IU/mL at post-treatment week 24, but a subsequent HCV-RNA measurement 29 days later was undetectable. Among those in the genotype 1 group with available HCV-RNA measurements, the concordance of SVR24 with SVR12 was 98.8% (326/330). ¶

No patients met the futility rule (confirmed HCV-RNA ≥LLOQ at week 8).

††

Two patients had HCV-RNA
SVR12; one patient achieved SVR24 but had a missing follow-up week 12 HCV-RNA measurement. ‡‡

Post-treatment failure rates based on patients with undetectable HCV-RNA at end of treatment.

§§

One genotype 4 patient with a missing follow-up week 12 HCV-RNA level subsequently achieved

SVR24.

25

Table 3. SVR12 by subgroup. Genotype 1

Genotype 4

(n = 354)

(n = 44)

Male

223/240 (92.9)

32/33 (97.0)

Female

106/114 (93.0)

11/11 (100)

<65

297/320 (92.8)

40/41 (97.6)

≥65

32/34 (94.1)

3/3 (100)

White

251/271 (92.6)

32/33 (97.0)

Black

29/33 (87.9)

4/4 (100)

Asian

46/47 (97.9)

1/1 (100)

<25

125/133 (94.0)

17/18 (94.4)

25–29

133/149 (89.3)

21/21 (100)

71/72 (98.6)

5/5 (100)

<800,000

46/47 (97.9)

15/15 (100)

≥800,000

283/307 (92.2)

28/29 (96.6)

Absent

263/281 (93.6)

24/24 (100)

Present

66/73 (90.4)

19/20 (95.0)

1a

153/176 (86.9)

NA

1b

176/178 (98.9)

NA

4

NA

43/44 (97.7)

CC

31/33 (93.9)

3/3 (100)

CT

213/231 (92.2)

30/31 (96.8)

TT

85/90 (94.4)

10/10 (100)

Null

219/234 (93.6)

33/34 (97.1)

Partial

110/120 (91.7)

10/10 (100)

Patients, n/N (%)

Sex

Age, years

Race

Body mass index, kg/m2

≥30 Baseline HCV-RNA, IU/mL

Cirrhosis status

HCV genotype

IL28B genotype

Prior response to P/R

HCV, hepatitis C virus; NA, not applicable; P/R, peginterferon alfa and ribavirin.

26

Table 4. Summary of on-treatment safety. Patients, n (%)

N = 398

Serious adverse events

22 (5.5)

Adverse events leading to discontinuation

18 (4.5)

Any adverse event

393 (98.7)

Adverse events in ≥20% of patients: Fatigue

165 (41.5)

Headache

124 (31.2)

Pruritus

104 (26.1)

Asthenia

96 (24.1)

Influenza-like illness

89 (22.4)

Insomnia

89 (22.4)

Rash

82 (20.6)

Grade 3/4 laboratory abnormalities

Hemoglobin (<9.0 g/dL)

25 (6.3) 9

89 (22.4)

9

64 (16.1)*

Absolute neutrophils (<0.75 × 10 /L)

Absolute lymphocytes (<0.5 × 10 /L) 9

Platelet count (<50 × 10 /L)

15 (3.8)

ALT (>5 × ULN)

12 (3.0)†

AST (>5 × ULN)

13 (3.3)†

Total bilirubin (>2.5 × ULN)

4 (1.0)†

ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal. *N = 397. †Grade 4 hepatic laboratory abnormalities: ALT >10 × ULN, 1/398 (0.3%); AST >10 × ULN, 1/398 (0.3%); no Grade 4 total bilirubin elevations (>5 × ULN) were observed.

27

FIGURE LEGEND Fig. 1: Patient disposition. SVR12, sustained virological response at post-treatment week 12. Enrolled and screened (N = 496) Excluded (n= 98) – Ineligible for protocol (n = 81) – Withdrew consent (n = 13) – Administrave reasons (n = 1) – Other (n = 3) Treated (N = 398)

Genotype 1 (n = 354)

Genotype 4 (n = 44)

Completed treatment (n = 335) Discon!nued (n = 19) – Lack of efficacy (n = 11) – Adverse event (n = 7) – Lost to follow up (n = 1)

Analysed for SVR12 (n = 354)

Completed treatment (n= 44)

Analysed for SVR12 (n = 44)

28