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Accepted Manuscript ABT-450, Ritonavir, Ombitasvir, and Dasabuvir Achieves 97% and 100% Sustained Virologic Response With or Without Ribavirin in Treatment-experienced Patients with HCV Genotype 1b Infection Pietro Andreone , MD Massimo G. Colombo , MD Jeffrey V. Enejosa , MD Iftihar Koksal , MD Peter Ferenci , MD Andreas Maieron , MD Beat Müllhaupt , MD Yves Horsmans , MD Ola Weiland , MD Henk W. Reesink , MD Lino Rodrigues Jr., MD Yiran B. Hu , MS Thomas Podsadecki , MD Barry Bernstein , MD PII: DOI: Reference: S0016-5085(14)00603-9 10.1053/j.gastro.2014.04.045 YGAST 59121 To appear in: Gastroenterology Accepted Date: 29 April 2014 Please cite this article as: Andreone P, Colombo MG, Enejosa JV, Koksal I, Ferenci P, Maieron A, Müllhaupt B, Horsmans Y, Weiland O, Reesink HW, Rodrigues Jr L, Hu YB, Podsadecki T, Bernstein B, ABT-450, Ritonavir, Ombitasvir, and Dasabuvir Achieves 97% and 100% Sustained Virologic Response With or Without Ribavirin in Treatment-experienced Patients with HCV Genotype 1b Infection, Gastroenterology (2014), doi: 10.1053/j.gastro.2014.04.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs. ACCEPTED MANUSCRIPT ABT-450, Ritonavir, Ombitasvir, and Dasabuvir Achieves 97% and 100% Sustained Virologic Response With or Without Ribavirin in Treatment-experienced Patients with HCV Genotype 1b Infection Short Title: ABT-450/r/Ombitasvir and Dasabuvir for HCV Pietro Andreone MD1, Massimo G Colombo MD2, Jeffrey V Enejosa MD3, Iftihar Koksal MD4, Peter RI PT Ferenci MD5, Andreas Maieron MD6, Beat Müllhaupt MD7, Yves Horsmans MD8, Ola Weiland MD9, Henk W Reesink MD10, Lino Rodrigues-Jr MD3, Yiran B Hu MS3, Thomas Podsadecki MD3, Barry Bernstein MD3 1 University of Bologna, Bologna, Italy; 2Ospedale Maggiore Policlinico, University of Milan, Milan, Italy; AbbVie Inc., North Chicago, IL, USA; 4Karadeniz Technical University, Trabzon, Turkey; 5Medical SC 3 M AN U University of Vienna, Internal Medicine III, Vienna, Austria; 6Elisabeth Hospital, Linz, Austria; 7University Hospital, Zurich, Switzerland; 8Université Catholique de Louvain, Brussels, Belgium; 9Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden; 10Academic Medical Center, Amsterdam, The Netherlands TE D Abbreviations: AE, adverse event; DAA, direct-acting antiviral agent; LLN, lower limit of normal; pegIFN, peginterferon; RAV, resistance-associated variant; RBV, ribavirin; SVR, sustained virologic response; SVR12, sustained virologic response 12 weeks after treatment; TEAE, treatment-emergent adverse event; AC C Correspondence: EP ULN, upper limit of normal Pietro Andreone Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna Via Massarenti, 9 40138 Bologna, Italy Email: [email protected] Phone: +39 051 6363618 1 ACCEPTED MANUSCRIPT Fax: +39 051 345806 Conflicts of Interest: Pietro Andreone has received research support from Roche, Merck, and Gilead Sciences; served on RI PT advisory committees for Roche, Merck, Janssen Cilag, AbbVie, Boehringer Ingelheim, Gilead Sciences, and BMS; and been a consultant for Merck and BMS. Massimo G Colombo has received grant/research support from Merck, Roche, BMS, and Gilead Sciences; served on advisory committees for Merck, SC Roche, Novartis, Bayer, BMS, Gilead Sciences, Tibotec, Vertex, Janssen Cilag, Achillion, Lundbeck, Abbott, Boehringer Ingelheim, GSK, GenSpera, and AbbVie; and served on speakers bureaus for Tibotec, M AN U Roche, Novartis, Bayer, BMS, Gilead Sciences, and Vertex. Iftihar Koksal has served on advisory committees and speaker’s bureaus for Roche, MSD, Janssen Therapeutics, AbbVie, Gilead Sciences, and BMS. Peter Ferenci has served on advisory committees and speakers bureaus for Roche, RottapharmMadaus; been a consultant for AbbVie, Boehringer Ingelheim, Janssen, BMS Austria, Idenix, Achillion, TE D GSK, Gilead Sciences, and MSD; and received research grants from Roche Austria. Andreas Maieron has served on advisory committees for MSD, Janssen Therapeutics, AbbVie, Boehringer Ingelheim, Gilead Sciences, BMS, and Rottapharm-Madaus; and received research grants from Roche and MSD. Beat EP Müllhaupt has served on advisory committees for Roche, MSD, Janssen Therapeutics, AbbVie, Boehringer Ingelheim, Gilead Sciences, and BMS; been a consultant for Gilead Sciences and AbbVie; and AC C received research grants from Roche and Gilead Sciences. Yves Horsmans has been a consultant for Janssen Pharmaceuticals, BMS, Merck Sharp & Dohme, Roche, Gilead Sciences, AbbVie and Boehringer Ingelheim. Ola Weiland has served on speaker’s bureaus and advisory committees for AbbVie, Gilead Sciences, BMS, Medivir, Johnson & Johnson, and Merck. Henk W Reesink has been a consultant for AbbVie, Astex, BMS, Gilead Sciences, GSK, Janssen-Cilag, Merck, PRA-International, Roche, Tibotec, RPharm, and Regulus; received research support from AbbVie, BMS, Boehringer Ingelheim, Gilead Sciences, Janssen-Cilag, Merck, PRA-International, Roche, and Santaris. Jeffrey V Enejosa, Lino 2 ACCEPTED MANUSCRIPT Rodrigues-Jr, Yiran B Hu, Thomas Podsadecki, and Barry Bernstein are employees of AbbVie and may hold stock or options. RI PT Writing Assistance: Medical writing support was provided by Douglas E. Dylla, PhD, employee of AbbVie. Author Contributions: PA was the coordinating investigator of the trial, was involved in the study setup, SC and was responsible for the clinical supervision of patients and performance of the study. MGC, IK, PF, AM, BM, YH, OW, and HWR were investigators in the study, responsible for the treatment of patients M AN U and were involved in the acquisition, analysis, and interpretation of the data. YBH performed the statistical analysis and provided input to the analysis plans. TP and BB provided scientific input in the clinical study design, reviewed and provided input with regards to the analysis plans. JVE and LRJ were responsible for the conduct and overview of the trial, analysis of the data, and review of the Clinical AC C EP TE D Study Report. All authors provided critical input and revisions to the writing of the manuscript. 3 ACCEPTED MANUSCRIPT ABSTRACT Background & Aims: The interferon-free regimen of ABT-450 (a protease inhibitor), ritonavir, ombitasvir RI PT (an NS5A inhibitor), dasabuvir (a non-nucleoside polymerase inhibitor), and ribavirin has shown efficacy in patients with hepatitis C virus (HCV) genotype 1b infection—the most prevalent subgenotype worldwide. We evaluated whether ribavirin is necessary for ABT-450, ritonavir, ombitasvir, and SC dasabuvir to produce high rates of sustained virologic response (SVR) in these patients. Methods: We performed a multicenter, open-label phase 3 trial of 179 patients with HCV genotype 1b M AN U infection, without cirrhosis, previously treated with peginterferon and ribavirin. Patients were randomly assigned (1:1) to groups given ABT-450, ritonavir, ombitasvir, and dasabuvir, with ribavirin (Group 1) or without (Group 2) for 12 weeks. The primary endpoint was SVR 12 weeks after treatment (SVR12). We assessed the noninferiority of this regimen to the rate of response reported (64%) for a similar TE D population treated with telaprevir, peginterferon, and ribavirin. Results: Groups 1 and 2 each had high rates of SVR12, which were noninferior to the reported rate of response to the combination of telaprevir, peginterferon, and ribavirin (Group1: 96.6%; 95% confidence EP interval [CI], 92.8%–100% and Group 2: 100%; 95% CI, 95.9%–100%). The rate of response in Group 2 was noninferior to that of Group 1. No virologic failure occurred during the study. Two patients (1.1%) AC C discontinued the study due to adverse events, both in Group 1. The most common adverse events in Groups 1 and 2 were fatigue (31.9% vs 15.8%) and headache (24.2% vs 23.2%), respectively. Decreases in hemoglobin to below the lower limit of normal were more frequent in Group 1 (42.0% vs 5.5% in Group 2, P<.001), although only 2 patients had hemoglobin levels below 10 g/dL. Conclusions: The interferon-free regimen of ABT-450, ritonavir, ombitasvir, and dasabuvir, with or without RBV, produces a high rate of SVR12 in treatment-experienced patients with HCV genotype 1b 4 ACCEPTED MANUSCRIPT infection. Both regimens are well tolerated, evidenced by the low rate of discontinuations and generally mild adverse events. ClinicalTrials.gov number: NCT01674725 AC C EP TE D M AN U SC RI PT Keywords: PEARL-II; ribavirin-free; IFN; interferon-free therapy; clinical trial 5 ACCEPTED MANUSCRIPT INTRODUCTION Untreated chronic hepatitis C virus (HCV) infection is a leading cause of liver damage, cirrhosis, and RI PT hepatocellular carcinoma.1 Prevalence of HCV infection is estimated at 3% worldwide and results in approximately 350 000 deaths annually.2, 3 Genotype 1 accounts for approximately 70% of all HCV infections and subgenotype 1b is most predominant in Europe and Eastern Asia. Approved direct-acting antiviral agents (DAAs), telaprevir, boceprevir, sofosbuvir, and simeprevir, given with peginterferon SC (pegIFN) and ribavirin (RBV) have reported sustained virologic response (SVR) rates of 67% to 89% in HCV genotype 1-infected patients. Response rates with DAA regimens are generally lower in patients M AN U who have failed previous pegIFN-containing treatment regimens than in treatment-naïve patients, and noticeably lower among prior null responders.4-8 Additionally, the toxicity of pegIFN and long duration of therapy (up to 48 weeks with some regimens) are a hardship for patients.9 Notably, pegIFN-based treatment regimens have a well-documented adverse event (AE) profiles including influenza-like TE D symptoms and depression that have led to unfavorable discontinuation rates in clinical trials,6, 9-12 while RBV also has associated side effects including teratogenicity, hemolytic anemia, and rash.13, 14 All-oral and interferon-free HCV treatment regimens with DAAs provide wider treatment access to EP patients in need with chronic liver disease. ABT-450 is an NS3/4A protease inhibitor with in vitro nanomolar antiviral activity and is co-dosed with the CYP3A4 inhibitor, ritonavir, which significantly AC C increases peak and trough drug concentrations enabling once-daily dosing.15 The multi-targeted, all-oral combination of the three DAAs (3D regimen) ABT-450/ritonavir, ombitasvir (formerly ABT-267), an HCV NS5A inhibitor with pangenotypic picomolar antiviral activity,16 and dasabuvir (formerly ABT-333), an HCV NS5B RNA nonnucleoside polymerase inhibitor, with RBV has shown in a phase 2b trial to achieve high rates of sustained virologic response 12 weeks post-treatment (SVR12) in treatment-naïve and experienced genotype 1-infected patients. With this regimen, a 93% SVR12 rate was achieved in 6 ACCEPTED MANUSCRIPT genotype 1-infected non-cirrhotic patients with prior null response to pegIFN/RBV, and 100% SVR12 in the genotype 1b patient subset.17 These high response rates in prior null responders, considered difficult to treat, are promising and require confirmation in a large phase 3 trial. While ABT- RI PT 450/ritonavir/ombitasvir and dasabuvir with RBV may achieve high SVR12 rates, determining the benefit gained by including RBV in the regimen has not been assessed in these patients. This phase 3 study (PEARL-II) evaluated the efficacy and safety of 12-week treatment with coformulated ABT- AC C EP TE D M AN U treatment-experienced HCV genotype 1b-infected patients. SC 450/ritonavir/ombitasvir and dasabuvir with or without RBV exclusively in non-cirrhotic pegIFN/RBV 7 ACCEPTED MANUSCRIPT METHODS Patients. Adults were 18 to 70 years old at the time of screening from 43 sites in Austria, Belgium, Italy, RI PT The Netherlands, Portugal, Puerto Rico, Sweden, Switzerland, Turkey, and the United States. Patients were required to have documentation that they previously failed treatment with pegIFN/RBV. Eligible patients were required to be non-cirrhotic with chronic HCV genotype 1b-infection for at least 6 months with an HCV RNA level > 10 000 IU/mL at screening. Patients were excluded if they had evidence of co- SC infection with any HCV genotype other than 1b or tested positive for Hepatitis B surface antigen or anti- M AN U HIV antibody at screening. Detailed eligibility criteria are provided in the Supplementary Appendix. Study design. Patients were stratified by type of previous non-response to pegIFN/RBV treatment (null responders, partial responders, and relapsers) and randomized 1:1 to receive the 12-week regimen of coformulated ABT-450/ritonavir/ombitasvir (150mg/100mg/25mg once daily) and dasabuvir (250mg twice daily) with either weight-based RBV dosed twice daily (1000mg daily if body weight was less than TE D 75kg, 1200mg daily if body weight was greater than or equal to 75kg) for Group 1 or without RBV for Group 2. After 12 weeks of treatment, patients were followed for 48 additional weeks. Additional details EP on study design are in the Supplementary Appendix. The study was conducted in accordance with the International Conference of Harmonisation guidelines, AC C applicable regulations, and guidelines governing clinical study conduct and ethical principles that have their origin in the Declaration of Helsinki. All patients provided written informed consent. All authors had access to relevant data, critically reviewed, revised, and approved the manuscript. Safety Assessments. Adverse event assessments were reported from the time of study drug administration until 30 days after last dose and were judged mild, moderate, or severe; clinical laboratory testing was performed at each study visit. Serious AEs were collected throughout the study. 8 ACCEPTED MANUSCRIPT Efficacy Endpoints. Plasma samples were collected at screening and each study visit and HCV RNA levels determined using the Roche COBAS TaqMan real-time RT-PCR assay v2.0 at a central lab. A fixedsequence testing procedure was used to control Type I error at 0.05. The primary efficacy endpoint was RI PT noninferiority of the SVR12 rates (assessed by HCV RNA < 25 IU/mL) of Group 2 and Group 1 to the historical SVR12 rate for telaprevir plus pegIFN/RBV in HCV genotype 1b-infected patients who were relapsers, partial responders, or null-responders to previous pegIFN/RBV treatment,4 adjusted for non- SC cirrhotic patients in this study. Group 1 and Group 2 noninferiority could be claimed if the SVR12 lower bound of the 95% confidence interval (CI) was greater than the upper bound of the CI for the historical M AN U rate minus a 10.5% noninferiority margin (64%). Further details of historical noninferiority calculations are provided in the Supplementary Appendix. Secondary efficacy endpoints in the fixed-sequence included the following: (1) comparison of the percentage of patients with a decrease in hemoglobin to below the lower limit of normal (LLN) at the end of treatment; (2) superiority of Group 1 and Group 2 to TE D the historical rate for telaprevir plus pegIFN/RBV (75%); and (3) noninferiority of Group 2 to Group 1 using a 10.5% noninferiority margin for the SVR12 difference. The percentage of patients with ontreatment virologic failure and post-treatment relapse was also assessed. EP Virologic failure criteria. Virologic failure leading to discontinuation of study drug was determined if the following criteria occurred: confirmed increase from nadir in HCV RNA (defined as 2 consecutive HCV AC C RNA measurements greater than 1 log10 IU/mL above nadir) at any point during treatment; failure to achieve HCV RNA < 25 IU/mL by Week 6; and confirmed HCV RNA ≥ 25 IU/mL in two consecutive measurements at any point during treatment after HCV RNA < 25 IU/mL. Post-treatment relapse was confirmed in patients with HCV RNA < 25 IU/mL at the end of treatment and subsequent RNA ≥ 25 IU/mL in two consecutive measurements. 9 ACCEPTED MANUSCRIPT Statistical Analyses. Efficacy analyses were performed using the intent-to-treat population defined as all randomized HCV genotype 1b-infected patients who received at least one dose of coformulated ABT450/ritonavir/ombitasvir. The safety population included all patients that received at least one dose of RI PT study drug. A population of 90 patients per treatment arm was calculated to provide greater than 90% power to achieve noninferiority of the active regimen to the historical threshold (64%). SAS software for the UNIX operating system was used for all analyses. All statistical tests and all AC C EP TE D M AN U SC confidence intervals were two-sided with a significance level of .05. 10 ACCEPTED MANUSCRIPT RESULTS Baseline Patient Demographics and Characteristics. Patient screening began 14 August 2012 and the last RI PT SVR12 data were collected 16 January 2014. Of 324 patients screened, 187 were randomized and 186 received study drug (91 Group 1, 95 Group 2) (Figure 1). Null-responders, partial-responders, and relapsers to previous pegIFN/RBV treatment made up 34.9%, 28.5%, and 36.6% of the study population, respectively, evenly stratified between treatment arms (Table 1). Reasons for screen failures are SC provided in the Supplemental Appendix. Seven randomized patients, three in Group 1 and four in Group 2 were not included in the intent-to-treat efficacy population. Of these, six patients were enrolled prior M AN U to a protocol amendment and received non-coformulated ABT-450/ritonavir/ombitasvir, 3 of whom were genotype 1a; a seventh patient’s HCV subgenotype was not determined. Efficacy. After 12 weeks of treatment, 96.6% (85/88; 95% CI, 92.8 – 100) of Group 1 and 100% (91/91; 95% CI, 95.9 – 100) of Group 2 patients achieved SVR12 using the intent-to-treat population for both TE D groups (Table 2). For the primary endpoint, SVR12 rates in both treatment groups were noninferior to the historical SVR rate for telaprevir plus pegIFN/RBV in comparable treatment-experienced patients. Both treatment groups were also superior to the historical rate. Noninferiority of Group 2 to Group 1 EP was met as the treatment difference in SVR12 rates was 3.4% (95% CI, -0.4 – 7.2). AC C No patients from either treatment group experienced on-treatment virologic failure or post-treatment relapse. Of the three patients in Group 1 that did not achieve SVR12, two (2.3%) patients discontinued study drug due to AEs, and one patient was lost to follow-up after SVR4 (Table 3). Sustained virologic responses in both groups were not influenced by previous non-response, age, race, or IL28B genotype. Among Group 1 null-responders, partial-responders, and relapsers to previous pegIFN/RBV treatment, SVR12 rates were 93.5%, 96.0%, and 100%, respectively. Group 1 rates were 11 ACCEPTED MANUSCRIPT similarly high regardless of IL28B genotype (CC: 100%, CT: 96.4%, TT: 95.5%), or sex (male: 95.3%, female: 97.8%). Group 2 SVR12 rates were 100% in all subgroups. RI PT Lastly, the seven patients excluded from the efficacy subset due to receiving non-coformulated study drug, confirmed genotype 1a, or undetermined genotype all completed treatment and achieved SVR12. Safety. Treatment-emergent AEs (TEAE) were experienced by 79.1% of patients in Group 1 and 77.9% of SC patients in Group 2. Most TEAEs were mild with the most commonly reported events in Group 1 and Group 2 being fatigue (31.9% vs. 15.8%, P = .015), headache (24.2% vs. 23.2%, P = NS), and nausea M AN U (20.9% vs. 6.3%, P = .005), respectively (Table 3). Patients in Group 1 also experienced statistically significantly more events of insomnia, anemia, rash, and increased blood bilirubin, all known to be associated with RBV use; no patient discontinued study drug due to these events. Overall, two (1.1%) patients discontinued treatment due to AEs, both in Group 1. One patient TE D experienced two serious AEs of pancreatitis that were considered by the investigator not to be study drug related. This patient had elevated amylase on Day 1 prior to receiving study drug; on Day 11, the patient reported abdominal pain and was hospitalized on Day 13, at which point study drugs were EP discontinued. The patient experienced another mild episode of pancreatitis on Day 31 that resolved by Day 36. This patient had an HCV RNA level of 28 IU/mL on Day 8. Resistance analysis performed on AC C baseline and post-treatment samples revealed no NS3 or NS5B resistance-associated variants (RAVs) present at baseline. The NS5A R30Q variant was present at baseline, and R30Q and Y93H were present at post-treatment Week 12. Another patient reported anxiety, tachycardia, fever, and dyspnea on Day 36 that led to study discontinuation; HCV RNA on Day 32 prior to discontinuation was < 15 IU/mL. This patient had no RAVs in NS3 or NS5A at baseline; NS5B variants C316N and S556G were present at baseline and post-treatment Week 4. Excluding the event of pancreatitis, 3 other serious TEAEs 12 ACCEPTED MANUSCRIPT (cellulitis, nephrolithiasis, and osteoarthritis) were reported; none were judged to be study drug-related or led to study drug discontinuation. RI PT Hemoglobin levels below the LLN at end of treatment, a secondary endpoint, was experienced more often by patients in Group 1 compared to Group 2 (42.0% vs. 5.5%, respectively, P < .001), although clinically significant grade 2 hemoglobin declines to below 10 g/dL at the end of treatment occurred in only 2 (1.1%) of patients, both in Group 1. No patient required a blood transfusion or erythropoietin. SC Elevations in total bilirubin greater than 2 x the upper limit of normal (ULN) were reported in 15.4% of patients in Group 1 and 1.1% of patients in Group 2 (P < .001), with 8.8% of patients in Group 1 and 0% M AN U in Group 2 reporting greater than 3 x ULN. Mean levels of total bilirubin peaked at Week 1 (predominantly indirect bilirubin) and were reduced at Week 2 in both groups, though remained elevated throughout the treatment period only in Group 1 (Supplemental Figure S3). The mean total bilirubin at Week 1 was 1.6 mg/dL in Group 1 and 0.9 mg/dL in Group 2; by Week 2, the mean levels TE D were reduced to 1.2 and 0.7 mg/dL, respectively. Five (5.5%) patients in Group 1 and 2 (2.1%) patients in Group 2 reported hyperbilirubinaemia; three (3.3%) patients in Group 1 reported jaundice. One hyperbilirubinaemia and one jaundice event were moderate in severity and the remaining events were EP judged mild; none led to study drug discontinuation. Ribavirin dose modification occurred in five patients, three due to anemia, one due to hyperbilirubinaemia, and one was dose adjusted due to AC C decrease in weight; all achieved SVR12. The percentage of patients with post-baseline alanine aminotransferase (ALT) levels greater than 3 x ULN was similarly low for both treatment groups. No patient experienced post-baseline ALT level greater than 5 x ULN. One patient in Group 2 had an aspartate aminotransferase (AST) level greater than 5 x ULN at a single time study visit, with all subsequent values normal. Twelve weeks of treatment with these regimens normalized liver enzymes in almost all patients with elevated baseline liver enzymes: 13 ACCEPTED MANUSCRIPT 96.9% (63/65) and 100% (66/66) of Group 1 and Group 2 patients, respectively, with high baseline ALT levels reached normal values following treatment; AST levels were normalized in 98.4% (60/61) and 91.8% (56/61) of Group 1 and Group 2 patients, respectively. Median changes from baseline in AC C EP TE D M AN U SC -36.0 U/L; AST: -22.0 vs. -21.0 U/L for Group 1 and Group 2, respectively). RI PT aminotransferase values at final treatment visit were similar comparing treatment groups (ALT: -35.0 vs. 14 ACCEPTED MANUSCRIPT DISCUSSION PEARL-II examined an all-oral, interferon-free regimen with or without RBV exclusively in pegIFN/RBV RI PT treatment-experienced, non-cirrhotic patients with HCV genotype 1b infection. The intent-to-treat SVR12 rates of 96.6% to 100% in patients receiving the 12-week regimen of ABT- 450/ritonavir/ombitasvir and dasabuvir with or without RBV, respectively, were superior to the historical rate of telaprevir plus pegIFN/RBV. The SVR12 rates of this multi-targeted regimen with RBV SC confirm results of the Phase 2b AVIATOR study17 in prior null-responders, the most difficult to treat of pegIFN/RBV non-responders, and further expands efficacy conclusions to patients who were partial M AN U responders and relapsers to pegIFN/RBV treatment. In addition, PEARL-II demonstrated noninferiority of the RBV-free regimen to the RBV-containing regimen, supporting the use of ABT450/ritonavir/ombitasvir and dasabuvir without RBV for 12 weeks in the treatment of HCV genotype 1binfected pegIFN/RBV-experienced patients without cirrhosis. TE D The TEAEs associated with either group in this 12-week regimen were generally mild and manageable. Overall, only two (1.1%) treated patients discontinued treatment due to AEs, and the five serious TEAEs reported in four patients were considered to be unrelated to study drug by the investigators. As EP expected, known RBV AEs (fatigue, nausea, insomnia, rash, anemia, and increased bilirubin) were statistically more prevalent in Group1RBV, although frequency and severity appear reduced compared to AC C when RBV is combined with pegIFN.7, 18 Hemoglobin declines were also more frequent in Group 1 although few (2.2%) reached clinical significance, and AEs leading to RBV dose reduction occurred in only four patients. Elevated bilirubin levels in Group 1 were predominantly due to indirect bilirubinemia, consistent with the hemolysis associated with RBV and the known effect of ABT-450 on the bilirubin transporter OATP1B1, though lack of sustained bilirubin elevations in Group 2 suggest the predominant 15 ACCEPTED MANUSCRIPT cause was RBV-related hemolysis. Liver enzyme normalization was consistent with the high rate of virologic response. RI PT The SVR12 rates reported here compare favorably to published reports of other interferon-free regimens using the NS5B RNA polymerase inhibitor sofosbuvir in combination with NS5A inhibitors (daclatasvir or ledipasvir), or with an NS3/4A protease inhibitor (simeprevir). Combinations of sofosbuvir plus daclatasvir with or without RBV have shown greater than or equal to 95% SVR12 in 41 treatment- SC experienced genotype 1 patients, of which only eight patients were genotype 1b.19 Similar SVR12 rates have been reported in treatment-experienced genotype 1 patients with sofosbuvir plus ledipasvir with M AN U (21/21, 100%) or without RBV (18/19, 95%), although only six genotype 1b patients were included.20 In 13 genotype 1b-infected patients receiving the combination of simeprevir plus sofosbuvir with or without RBV, 100% SVR8 was reported.21 A larger study of daclatasvir in combination with asunaprevir in pegIFN/RBV treatment-experienced genotype 1b-infected patients showed SVR12 rates of 80% (70/87) TE D with patients not achieving an SVR primarily due to lack of efficacy and AEs.22 Together with the results from PEARL-II, these data support a multi-targeted approach to achieve SVR; however, no other studies have exclusively analyzed, nor had the statistical power to draw conclusions regarding efficacy, including EP the contribution of RBV, in treatment-experienced HCV genotype 1b patients as the PEARL-II study does. AC C One of the strengths of the PEARL-II study includes its large sample size in genotype 1b-infected patients, the most prevalent subgenotype worldwide, including patients with previous null response and relapse to pegIFN/RBV treatment. Treatment-experienced genotype 1b-infected patients have not been extensively studied with currently approved or investigational IFN-free regimens, hence this large patient population represents a group with unmet need. Study limitations include the open label study design, the exclusion of patients with cirrhosis, HBV or HIV coinfection, and that these findings may be specific to genotype 1b-infected patients. However, the efficacy and safety of this regimen has recently 16 ACCEPTED MANUSCRIPT been described from phase 3 studies in treatment-naïve patients infected with genotype 1a and 1b,23 and in patients with cirrhosis.24 RI PT In conclusion, a 12-week regimen of ABT 450/ritonavir/ombitasvir and dasabuvir with or without RBV was generally well-tolerated in pegIFN/RBV treatment experienced, non-cirrhotic, HCV genotype 1binfected adults, as evidenced by the low rate of treatment discontinuation and serious AEs. Additionally, the regimen without RBV was associated with fewer AEs of fatigue, nausea, insomnia, rash, and a lower SC rate of laboratory abnormalities including bilirubin elevation and hemoglobin decrease. Sustained virologic response rates of 96.6% and 100% were achieved, including 93.5% and 100% in the difficult to M AN U treat previous pegIFN/RBV null-responders, with or without RBV, respectively. Therefore, ABT450/ritonavir/ombitasvir and dasabuvir without RBV is sufficient to achieve optimal treatment of HCV AC C EP TE D genotype 1b infection in this population. 17 ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS The authors would like to express their gratitude to the trial participants, and coordinators who made RI PT this study possible, as well as Sara Siggelkow, Nela Hayes, Karmin Robinson-Morgan, Lisa Rhiner, Ruxandra-Maria Stanica, Lorena De Castillo, Mia Poteracki, Manal Abunimeh, Kristine Richards, Lois Larsen, Sailaja Settivari, Yan Xie, Xiangdong Zhou, Prajakta Badri and the M13-389 Study Team for their contributions to the study. Authors thank the study investigators including Avanish M. Aggarwal, SC Sanjeev Arora, David Bernstein MD, Bal Raj Bhandari, Maurizia Rossana Brunetto, Filipe Calinas, Nicola M AN U Caporaso, Andreas Cerny MD, J-F. Dufour, Francque Sven MA, MD, PhD, Giovanni B. Gaeta, W. Jeffrey Fessel MD, Michael Gschwantler MD, Gurel Selim MD PhD, Camilla Håkanård, Jason McNeese, Ivan Melendez-Rivera MD, Christophe Moreno MD PhD, Frederik Nevens, Gunnar Norkrans MD PhD, Resat Ozaras MD, Ronald Pruitt MD, Giovanni Raimondo MD, H. Reynaert MD PhD, Federico Rodriguez-Perez MD, Lorenzo Rossaro MD, Rui Tato Marinho MD PhD, Hans Van Vlierberghe, Wolfgang Vogel MD, Debra TE D Weinstein MD, Cihan Yurdaydin, Philippe J. Zamor. AbbVie sponsored the study (NCT01674725), contributed to its design, participated in the collection, analysis, and interpretation of the data, and in the writing, reviewing, and approval of the final manuscript. Medical writing support was provided by AC C EP Douglas E. Dylla, PhD, of AbbVie. 18 ACCEPTED MANUSCRIPT REFERENCES 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. RI PT SC 7. M AN U 6. TE D 5. EP 4. World Health Organization. Media Centre - Hepatitis C, 2014. Gravitz L. Introduction: a smouldering public-health crisis. Nature 2011;474:S2-4. Perz JF, Armstrong GL, Farrington LA, et al. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. Journal of Hepatology 2006;45:529-38. Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med 2011;364:2417-28. Bacon BR, Gordon SC, Lawitz E, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. New England Journal of Medicine 2011;364:1207-17. D'Ambrosio R, Aghemo A, Colombo M. Treatment of experienced and naive patients with hepatitis C: focus on telaprevir. Biologics 2012;6:363-70. Lawitz E, Mangia A, Wyles D, et al. Sofosbuvir for previously untreated chronic hepatitis C infection. New England Journal of Medicine 2013;368:1878-87. Zeuzem S, Berg T, Gane E, et al. Simeprevir Increases Rate of Sustained Virologic Response Among Treatment-Experienced Patients With HCV Genotype-1 Infection: A Phase IIb Trial. Gastroenterology 2013. Chung RT. A watershed moment in the treatment of hepatitis C. New England Journal of Medicine 2012;366:273-5. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. New England Journal of Medicine 2002;347:975-82. Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001;358:958-65. Hezode C, Fontaine H, Dorival C, et al. Triple therapy in treatment-experienced patients with HCV-cirrhosis in a multicentre cohort of the French Early Access Programme (ANRS CO20-CUPIC) - NCT01514890. J Hepatol 2013;59:434-41. Jacobson IM, McHutchison JG, Dusheiko G, et al. Telaprevir for previously untreated chronic hepatitis C virus infection. New England Journal of Medicine 2011;364:2405-16. Poordad F, McCone J, Jr., Bacon BR, et al. Boceprevir for untreated chronic HCV genotype 1 infection. New England Journal of Medicine 2011;364:1195-206. Menon RM, Klein CE, Lawal AA, et al. Pharmacokinetics and Tolerability of the HCV Protease Inhibitor ABT-450 Following Single Ascending Doses in Healthy Adult Volunteers With and Without Ritonavir. HepDART. Kohala Coast, HI, 2009. Pilot-Matias T, Koev G, Krishnan P, et al. In vitro combinatory effect of HCV NS3/4A protease inhibitor ABT-450, NS5A inhibitor ABT-267, and non-nucleoside NS5B polymerase inhibitor ABT333, In 47th Annual Meeting of the European Association for the Study of the Liver, Barcelona, Spain, 2012. Kowdley KV, Lawitz E, Poordad F, et al. Phase 2b Trial of Interferon-free Therapy for Hepatitis C Virus Genotype 1. New England Journal of Medicine 2014;370:222-232. Jacobson IM, Kowdley KV, Kwo PY. Anemia management in the era of triple combination therapy for chronic HCV. Gastroenterol Hepatol (N Y) 2012;8:1-16. Sulkowski MS, Gardiner DF, Rodriguez-Torres M, et al. Daclatasvir plus Sofosbuvir for Previously Treated or Untreated Chronic HCV Infection. New England Journal of Medicine 2014;370:211221. AC C 1. 2. 3. 19 ACCEPTED MANUSCRIPT 22. 23. 24. RI PT 21. Lawitz E, Poordad FF, Pang PS, et al. Sofosbuvir and ledipasvir fixed-dose combination with and without ribavirin in treatment-naive and previously treated patients with genotype 1 hepatitis C virus infection (LONESTAR): an open-label, randomised, phase 2 trial. Lancet 2014;383:515-23. Lawitz E, Ghalib R, Rodriguez-Torres M, et al. COSMOS Study: SVR4 results of a once daily regimen of simeprevir (TMC435) plus sofosbuvir (GS-7977) with or without ribavirin in HCV genotype 1 null responders, In 20th Conference of Retroviruses and Opportunistic Infections, Atlanta, GA, USA, 2013. Chayama K, Suzuki Y, Ikeda K, et al. All-oral Combination of Daclatasvir Plus Asunaprevir in Interferon Ineligible Naive/Intolerant and Nonresponder Japanese Patients Chronically Infected with HCV Genotype 1b: Results from a Phase 3 Trial. Hepatology 2013;58:313A. Feld JJ, Kowdley KV, Coakley E, et al. Treatment of HCV with ABT-450/r–Ombitasvir and Dasabuvir with Ribavirin. New England Journal of Medicine 2014;370:1594-1603. Poordad F, Hezode C, Trinh R, et al. ABT-450/r-Ombitasvir and Dasabuvir with Ribavirin for Hepatitis C with Cirrhosis. New England Journal of Medicine. DOI:10.1056/NEJMoa1402869. AC C EP TE D M AN U Author names in bold designate shared co-first authorship. SC 20. 20 ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Patient flow diagram. RI PT Figure 2. ITT genotype 1b efficacy subset treatment response over time. Percent of patients achieving virologic response (± 95% confidence intervals) are presented for Week 4 of treatment (RVR), end of treatment (EOTR), 4 weeks post-treatment (SVR4), and 12 weeks post-treatment (SVR12), as well as n/N AC C EP TE D M AN U SC within the bars graphs. 21 ACCEPTED MANUSCRIPT Table 1. Baseline demographics and characteristics, n (%) Group 2 3D + RBV 3D Parameter (N = 91) (N = 95) Sex, male 45 (49.5) RI PT Group 1 57 (60.0) Race 84 (92.3) Black 3 (3.3) 4 (4.4) M AN U Ethnicity, Hispanic/Latino 86 (90.5) SC White Geographic region North America 6 (6.3) 2 (2.1) 14 (15.4) 19 (20.0) 77 (84.6) 76 (80.0) 54.2 ± 10.9 54.2 ± 10.5 26.2 ± 4.1 27.5 ± 4.3 10 (11.0) 7 (7.4) 81 (89.0) 88 (92.6) 6.56 ± 0.56 6.48 ± 0.53 Null responder 32 (35.2) 33 (34.7) Partial-responder 26 (28.6) 27 (28.4) Relapser 33 (36.3) 35 (36.8) 64 (70.3) 61 (64.2) Europe BMI (kg/m2), mean (±SD) IL28B genotype EP CC TE D Age (years), mean (±SD) Non-CC AC C HCV RNA (log10 IU/mL), mean (±SD) Previous pegIFN/RBV non-response Baseline fibrosis stage F0 – F1 ACCEPTED MANUSCRIPT F2 13 (14.3) 21 (22.1) F3 14 (15.4) 13 (13.7) Fibrosis scoring information is provided in the Supplemental Appendix. 3D, three direct-acting antivirals; AC C EP TE D M AN U SC RI PT RBV, ribavirin; BMI, body mass index; pegIFN, peginterferon. ACCEPTED MANUSCRIPT Table 2. Intent-to-treata virologic response, n/N (%) Group 2 Treatment difference 3D + RBV 3D (95% CI) 85/88 (96.6) 91/91 (100) 3.4 (-0.4, 7.2) Null responder 29/31 (93.5) 32/32 (100) 6.5 (-2.2, 15.1) Non/partial responder 24/25 (96.0) 26/26 (100) 4.0 (-3.7, 11.7) Relapser 32/32 (100) 33/33 (100) Parameter SVR12 RI PT Group 1 M AN U Sex 0 (N/A) Male 41/43 (95.3) 54/54 (100) 4.7 (-1.6, 10.9) Female 44/45 (97.8) 37/37 (100) 2.2 (-2.1, 6.5) 3/3 (100) 5/5 (100) 0 (N/A) 82/85 (96.5) 86/86 (100) 3.5 (-0.4, 7.5) 10/10 (100) 7/7 (100) 0 (N/A) 54/56 (96.4) 64/64 (100) 3.6 (-1.3, 8.4) 21/22 (95.5) 20/20 (100) 4.5 (-4.2, 13.2) Race TE D Black Non-black IL28B genotype TT AC C CT EP CC a SC Previous non-response Intent-to-treat genotype 1b efficacy population includes all patients with subgenotype 1b infection who were assigned to and treated with ABT-450/ritonavir/ombitasvir co-formulated drug. 3D, three direct-acting antivirals; RBV, ribavirin; SVR12, 12-week sustained virologic response. ACCEPTED MANUSCRIPT Table 3. Patients Reporting TEAEs, n (%) Group 2 3D + RBV 3D Parameter (N = 91) (N = 95) Any TEAE 72 (79.1) 74 (77.9) Any severe TEAE 0 (0) 1 (1.1) Any serious TEAE 2 (2.2) TEAE leading to discontinuation 2 (2.2) SC 2 (2.1) M AN U Common TEAEsa Fatigue P value RI PT Group 1 0 (0) 29 (31.9) 15 (15.8) 22 (24.2) 22 (23.2) 19 (20.9) 6 (6.3) 0.005 13 (14.3) 3 (3.2) 0.008 13 (14.3) 8 (8.4) 12 (13.2) 12 (12.6) 11 (12.1) 7 (7.4) 10 (11.0) 0 (0) < 0.001 8 (8.8) 0 (0) 0.003 8 (8.8) 1 (1.1) 0.017 37 (42.0) 5 (5.5) < 0.001 8 (8.8) 0 (0) 0.003 ALT > 5X ULN 0 (0) 0 (0) AST > 5X ULN 0 (0) 1 (1.1) Headache Nausea TE D Insomnia Pruritus Diarrhea EP Asthenia Anemia Rash AC C Blood bilirubin increased 0.015 Chemistry and Hematologic values of interest during treatment Hemoglobin below LLN at end of treatmentb Total bilirubin > 3X ULN ACCEPTED MANUSCRIPT a Investigator-reported TEAEs present in ≥ 10 % of either treatment group or with a statistically significant difference between treatment groups. b N’s = 88 and 91 for Group 1 and Group 2, respectively, using the intent-to-treat genotype 1b efficacy RI PT population. TEAE, treatment-emergent adverse event; 3D, three direct-acting antivirals; RBV, ribavirin; LLN, lower limit of normal; ULN, upper limit of normal; ALT, alanine aminotransferase; AST, aspartate AC C EP TE D M AN U SC aminotransferase. AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Table of Contents Investigators............................................................................................................................................ 3 RI PT PEARL-II Eligibility Criteria........................................................................................................................ 4 Randomization Methods ......................................................................................................................... 7 Blinding ................................................................................................................................................... 7 Collection of Samples for HCV RNA Measurement ................................................................................... 7 SC HCV RNA Measurement .......................................................................................................................... 7 Virologic Failure Criteria .......................................................................................................................... 7 M AN U Noninferiority and Superiority Analyses................................................................................................... 8 Sample Size Determination...................................................................................................................... 9 Ranked Efficacy Endpoint Analyses .......................................................................................................... 9 Figure S1. PEARL-II Study Design. ........................................................................................................... 10 Figure S2. HCV RNA Suppression Over Time. .......................................................................................... 11 Figure S3. PEARL-II Mean Total Bilirubin Values Over Time. ................................................................... 12 TE D Table S1. PEARL-II Primary Reasons for Not Meeting Study Eligibility ..................................................... 13 Table S2. Fibrosis Scoring....................................................................................................................... 15 Table S3. PEARL-II Adverse Events Occurring in > 5% of Patients in Either Treatment Group, n (%) ........ 16 Table S4. Grade 3 and 4 Laboratory Abnormalities, n (%) ....................................................................... 17 EP Table S5. Potentially Clinically Significant Laboratory Abnormalities, n/N (%) ........................................ 18 AC C References ............................................................................................................................................ 19 1 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ABT-450 was identified as a lead compound by AbbVie and Enanta Pharmaceuticals. 2 ACCEPTED MANUSCRIPT Investigators PEARL-II investigators included: AC C EP TE D M AN U SC RI PT Austria: Peter Ferenci, Michael Gschwantler, Andreas Maieron, Wolfgang Vogel Belgium: Sven Francque, Yves Horsmans, Christophe Moreno, Frederik Nevens, Hendrik Reynaert, Hans Van Vlierberghe Italy: Pietro Andreone, Maurizia Rossana Brunetto, Nicola Caporaso, Massimo Colombo, Giovanni B Gaeta, Giovanni Raimondo Netherlands: Henk W Reesink Portugal: Filipe Calinas, Rui Tato Marinho Puerto Rico: Ivan Melendez-Rivera, Federico Rodríguez-Pérez Sweden: Camilla Hakangard, Gunnar Norkrans, Ola Weiland Switzerland: Andreas Cerny, Jean-Francois Dufour, Beat Müllhaupt Turkey: Selim Gurel, Iftihar Koksal, Resat Ozaras, Cihan Yurdaydin United States: Avanish Aggarwal, Sanjeev Arora, David Bernstein, Bal Raj Bhandari, Jeffrey Fessel, Edward Galen, Jason McNeese, Ronald Pruitt, Lorenzo Rossaro, John Vierling, Debra Weinstein, and Philippe Zamor. 3 ACCEPTED MANUSCRIPT PEARL-II Eligibility Criteria Main Inclusion: 1. Male or female between 18 and 70 years of age, inclusive, at time of Screening. 2. Female who is: practicing total abstinence from sexual intercourse (minimum 1 complete menstrual cycle) • sexually active with female partners only • not of childbearing potential, defined as: postmenopausal for at least 2 years prior to screening (defined as amenorrheic for longer than 2 years, age appropriate, and confirmed by a follicle-stimulating hormone [FSH] level indicating a postmenopausal state), or o surgically sterile (defined as bilateral tubal ligation, bilateral oophorectomy or hysterectomy) or has a vasectomized partner(s). SC o of childbearing potential and sexually active with male partner(s): of childbearing potential and sexually active with male partner(s) currently using at least one effective method of birth control at the time of screening and two effective methods of birth control while receiving study drugs (as outlined in the patient informed consent or other patient information documents), starting with Study Day 1 and for 7 months after stopping study drug as directed by the local ribavirin label. (Note: Hormonal contraceptives, including oral, topical, injectable or implantable varieties, may not be used during study drug treatment.) TE D o M AN U • RI PT • 3. Females must have had negative results for pregnancy tests performed: at screening by serum specimen within 35 days prior to initial study drug administration; and at baseline (prior to dosing) by urine specimen. AC C EP 4. Sexually active males must have been surgically sterile or have had male partners only or if sexually active with female partner(s) of childbearing potential must have agreed to practice 2 effective forms of birth control (as outlined in the patient information and consent form or other patient information documents) throughout the course of the study, starting with Day 1 and for 7 months after stopping study drug or as directed by the local ribavirin (RBV) label. (Note: Contraceptives containing ethinyl estradiol or depo progesterone were considered effective if used by the female partners of male patients.) 5. Patient must have had documentation that they were adherent to prior pegIFN/RBV combination therapy and met 1 of the following categories: • • Null responders: received at least 12 weeks of pegIFN/RBV for the treatment of HCV and failed to achieve a 2 log10 IU/mL reduction in HCV RNA at Week 12. Patients were considered to have met this definition if the lack of treatment response was documented following 10 to 16 weeks of treatment; Nonresponders/partial responders: received at least 20 weeks of pegIFN/RBV for the treatment of HCV and achieved ≥ 2 log10 IU/mL reduction in HCV RNA at Week 12, but failed to achieve HCV RNA undetectable at the end of treatment. Patients were considered 4 ACCEPTED MANUSCRIPT to have met this definition if the lack of treatment response was documented following 10 to 16 weeks of treatment; or • Relapsers: received at least 36 weeks of pegIFN/RBV for the treatment of HCV and was undetectable at the end of treatment, but HCV RNA was detectable within 52 weeks of treatment follow-up. RI PT Viral loads documenting the type of prior nonresponse were to be obtained during the previous pegIFN/RBV treatment. PegIFN/RBV therapy must have been completed no less than 2 months prior to the Screening Visit. 6. Chronic HCV genotype 1b-infection for at least 6 months prior to Screening. Chronic HCV infection is defined as one of the following: positive for anti-HCV antibody (Ab) or HCV RNA at least 6 months before screening, and positive for HCV RNA and anti-HCV Ab at the time of screening or • positive for anti-HCV Ab and HCV RNA at the time of screening with a liver biopsy consistent with chronic HCV infection. SC • M AN U 7. Per local standard practice, documented results of 1 of the following: liver biopsy within 24 months prior to or during screening demonstrating the absence of cirrhosis, e.g., a Metavir score of 3 or less or an Ishak score of 4 or less; • screening FibroTest score of ≤ 0.72 and aspartate aminotransferase (AST) to platelet ratio index (APRI) ≤ 2; or • screening FibroScan® result of < 9.6 kPa; • patients with a nonqualifying FibroTest/APRI or FibroScan could have only been enrolled if they had a qualifying liver biopsy within 24 months prior to or during screening. TE D • 8. Patient had plasma HCV RNA level > 10,000 international units (IU)/mL at screening. Main Exclusion: EP 9. Patient's HCV genotype was subgenotype 1b at screening without coinfection with any other genotype/subgenotype. AC C 1. Recent (within 6 months prior to study drug administration) history of drug or alcohol abuse that could preclude adherence to the protocol. 2. Positive test result for Hepatitis B surface antigen (HBsAg) or anti-Human immunodeficiency virus antibody (HIV Ab). 3. Patient's HCV subgenotype at screening was not subgenotype 1b or indicated coinfection of 1b with any other genotype/subgenotype. 4. History of uncontrolled seizures, uncontrolled diabetes as defined by a glycated hemoglobin (hemoglobin A1C) level > 8.5% at the Screening visit, active or suspected malignancy or history of malignancy (other than basal cell skin cancer or cervical carcinoma in situ) in the past 5 years. 5. Any current or past clinical evidence of cirrhosis such as ascites or esophageal varices, or prior biopsy showing cirrhosis, e.g., a Metavir score of >3 or Ishak score of > 4. 5 ACCEPTED MANUSCRIPT • hemochromatosis; • alpha-1 antitrypsin deficiency; • Wilson's disease; • autoimmune hepatitis; • alcoholic liver disease; • drug-related liver disease. RI PT 6. Any cause of liver disease other than chronic HCV infection, including but not limited to the following: SC Steatosis and steatohepatitis on a liver biopsy coincident with HCV-related changes would not have been considered exclusionary unless the steatohepatitis was considered to be the primary cause of the liver disease. 7. Screening laboratory analyses showing any of the following abnormal laboratory results: Alanine aminotransferase (ALT) > 5 × upper limit of normal (ULN) • Aspartate aminotransferase (AST) > 5 × ULN • Calculated creatinine clearance (using Cockcroft-Gault method) < 60 mL/min • Albumin < Lower limit of normal (LLN) • Prothrombin time/International normalized ratio (INR) > 1.5. Patients with a known inherited blood disorder and INR > 1.5 may be enrolled with permission of the AbbVie Study Designated Physician, even if the INR > 1.5 • Hemoglobin < LLN • Platelets < 120,000 cells per mm3 • Absolute neutrophil count (ANC) < 1500 cells/μL (< 1200 cells/µL for patients of African descent who are black) • Indirect bilirubin > 1.5 × ULN and direct bilirubin > ULN EP TE D M AN U • AC C 8. Use of any medications listed below, as well as those that are contraindicated for ritonavir and ribavirin, within 2 weeks prior to study drug administration or 10 half-lives (if known), whichever is longer, including but not limited to: Alfuzosin Amiodarone Astemizole Bepridil Bosentan Buprenorphine Carbamazepine Cisapride Clarithromycin Conivaptan Lovastatin Methadone Midazolam (oral) Mifepristone Modafinil Montelukast Nefazodone Phenobarbital Phenytoin Pimozide Pioglitazone Propafenone Quercetin Quinidine Rifabutin Rifampin Rosiglitazone Salmeterol Simvastatin St. John's Wort Telithromycin Terfenadine Triazolam Trimethoprim Troglitazone Troleandomycin Voriconazole 6 ACCEPTED MANUSCRIPT Randomization Methods Patients who met the eligibility criteria were enrolled via the Interactive Response Technology (IRT) system on Study Day 1. Prior to the study, contact information and user guidelines for the IRT system were provided to each site. RI PT For enrollment of eligible patients on Day 1, the site contacted the IRT system in order to receive a unique randomization number and study drug kit numbers. The study drug kit numbers were assigned according to a randomization schedule computer generated before the start of the study by the AbbVie Statistics Department. SC Initial randomization occurred in a 1:1 ratio to the two treatment groups. Randomization of patients was stratified by the type of nonresponse to previous pegIFN/RBV treatment (null responders, nonresponders/partial responders and relapsers). The number of nonresponders/partial responders plus relapsers enrolled across Arms 1 and 2 was limited to 130 to ensure that at least 80 null responders were enrolled. In addition, the relapsers were limited to 60 patients (approximately 30% of all patients). Blinding M AN U This is an open-label study. There was no blinding between the arms. Collection of Samples for HCV RNA Measurement TE D Plasma samples for HCV RNA measurement were obtained at screening. Additional samples for HCV RNA measurement were obtained at each scheduled visits every 1-2 weeks through the final treatment visit or premature discontinuation. Following administration of the last dose of study drug, samples for HCV RNA measurement were collected at post-treatment weeks 2, 4, 8, 12, 24, 36, and 48 or at the time of premature discontinuation. Plasma samples were also collected at screening to assess HCV genotype and subtype using the Versant HCV Genotype Inno-LiPA Assay, version 2.0 or higher (LiPA; Siemens Healthcare Diagnostics, Tarrytown, NY), and for IL28B rs12979860 haplotype analysis. EP HCV RNA Measurement AC C Plasma HCV RNA levels were determined for each sample collected by the central laboratory using the ® Roche COBAS TaqMan real-time reverse transcriptase-PCR (RT-PCR) assay v2.0. The lower limit of detection (LLOD) is 15 IU/mL and the LLOQ (lower limit of quantification) is 25 IU/mL. Virologic Failure Criteria The following criteria were considered evidence of virologic failure leading to discontinuation of study drug for individual patients being treated with active drugs: • • • Confirmed increase from nadir in HCV RNA (defined as 2 consecutive HCV RNA measurements > 1 log10 IU/mL above nadir) at any time point during treatment; Failure to achieve HCV RNA < LLOQ by Week 6; Confirmed HCV RNA ≥ LLOQ (defined as two consecutive HCV RNA measurements ≥ LLOQ) at any point during treatment after HCV RNA < LLOQ. 7 ACCEPTED MANUSCRIPT If any of the above criteria were met, the patient was to discontinue study treatment. Patients who completed the treatment with HCV RNA < LLOQ at the end of treatment and who had a confirmed HCV RNA ≥ LLOQ (defined as 2 consecutive HCV RNA measurements ≥ LLOQ) at any point in the post-treatment period were considered to have relapsed. RI PT Noninferiority and Superiority Analyses SC Historical SVR rates for telaprevir plus pegIFN and RBV are not included in the US Prescribing Information (USPI) or SmPC on non-cirrhotic treatment-experienced patients by subgenotype and type of non-response, therefore, data from the REALIZE study1 were used, with an adjustment factor to account for the exclusion of cirrhotic patients from that study. REALIZE data are presented in the table below. M AN U For non-cirrhotic genotype 1b-infected patients in PEARL-II, the upper bound of the 95% confidence interval (CI) of SVR rate for telaprevir plus peg/IFN/RBV therapy was 75%, representing a threshold relevant to the overall population enrolled in PEARL-II. For the study regimen to be considered superior to the historical SVR rate for telaprevir, the lower bound of the SVR rate 95% CI must exceed the upper confidence bound of the historical SVR rate for telaprevir based therapy presented in the table below (i.e., 75%). To be considered noninferior to the historical SVR rate for telaprevir, a noninferiority margin of 10.5% was used. Thus, noninferiority to the historical SVR rate for telaprevir based therapy was met if the lower bound of the 95% CI for the regimen SVR rate was greater than the upper confidence bound of SVR rate for the telaprevir based therapy minus 10.5% (i.e., 64%). The 2-sided 95% confidence intervals are created using the normal approximation to the binomial. TE D Estimated SVR Rates for Telaprevir plus pegIFN/RBV Therapy in Treatment-Experienced, Non-cirrhotic Patients with HCV Subgenotype 1b, REALIZE study1 GT1b T12/PR48 n/N (%) Relapsers AC C Partial responders Null responders Projected Enrollment in PEARL-II (%) 123/140 (87.9) 88.4 30 27/40 (67.5) 79.5 30 22/59 (37.3) 46.5 30 EP Previous non-response With Increase for Excluding Cirrhotics (%) PopulationBased Weighted Average % [95% CI] 69 [62, 75] GT, genotype; T, telaprevir; PR, pegIFN/RBV To test noninferiority of the RBV-free group compared to the RBV-containing group, the 2-sided 95% confidence interval for the difference in SVR12 rates will be calculated using the normal approximation to the binomial distribution. If the lower bound of the 2-sided 95% confidence interval for the difference was above the noninferiority margin of -10.5%, noninferiority can be claimed for the regimen without RBV compared to regimen with RBV. 8 ACCEPTED MANUSCRIPT Sample Size Determination RI PT PEARL-II planned to enroll 210 patients in a 1:1 ratio to the DAA combination regimen with RBV or without RBV, so that at least 200 patients were dosed with the coformulated tablets of ABT450/ritonavir/ombitasvir. Based on a 2-sided significance level of 0.05, and an underlying SVR12 rate of 82% or higher in Group 1 and 82% or higher in Group 2, a sample size of 90 patients per group provides > 90% power to demonstrate noninferiority of the ABT-450/ritonavir/ombitasvir + dasabuvir regimen ± RBV to the historical SVR rate for telaprevir plus pegIFN and RBV therapy (64%). No adjustment for dropout was applicable because patients without data at Post-Treatment Week 12 (after imputing) are counted as failures for SVR12. SC Ranked Efficacy Endpoint Analyses In order to control the Type I error rate at 0.05, a fixed-sequence testing procedure2 was used to proceed through the primary and secondary efficacy endpoints in the order shown below. M AN U PEARL-II had the following primary efficacy endpoints: (1) non-inferiority of Group 2’s SVR12 rate to the historical rate for telaprevir plus pegylated interferon and RBV; and (2) non-inferiority of the SVR12 rate in RBV-containing treatment group (Group 1) to the historical rate for telaprevir plus pegylated interferon and RBV. TE D Secondary efficacy endpoints in the fixed sequence are: (3) comparison of the percentage of patients with a decrease in hemoglobin to below the lower limit of normal (LLN) at the end of treatment with ABT-450/ritonavir/ombitasvir and dasabuvir with RBV vs. without RBV; (4) superiority of SVR12 rate in RBV-containing treatment group to the historical rate for telaprevir plus pegIFN and RBV; (5) superiority of SVR12 rate in treatment group without RBV to the historical rate for telaprevir plus pegIFN and RBV; (6) noninferiority of Group 2 to Group 1 using a 10.5% noninferiority margin. AC C EP Other secondary endpoint analyses not included in the fixed-sequence testing procedure are the percentage of patients in each treatment group with on-treatment virologic failure (including failure to suppress and rebound) and post-treatment relapse. 9 SC RI PT ACCEPTED MANUSCRIPT Figure S1. PEARL-II Study Design. PEARL-II was a phase 3, open-label, randomised trial of 12-week AC C EP TE D M AN U treatment with ABT-450/r/ombitasvir, dasabuvir with or without RBV in HCV genotype 1b-infected patients with previous null response, partial response, or relapse to prior treatment with pegIFN/RBV. r, ritonavir; RBV, ribavirin; SVR12, sustained virologic response 12 weeks post-treatment. 10 ACCEPTED MANUSCRIPT 80 74.7 73.6 65/87 67/91 100 100 86/86 91/91 100 100 40 20.7 21.1 18/87 19/90 20 0 Week 1 Week 2 Week 4 Treatment Visit 83/83 87/87 Week 12 Group 2 M AN U Group 1 RI PT 60 SC % Patients with HCV RNA < LLOQ 100 Figure S2. HCV RNA Suppression Over Time. Percentage of patients in PEARL-II with HCV RNA < AC C EP TE D LLOQ at treatment visits, in patients with data at the treatment visit, during the treatment period (ITT genotype 1b efficacy subset). Numbers in bars are n/N for each time point. 11 ACCEPTED MANUSCRIPT RI PT 4 3 SC 2 1 0 1 2 4 6 M AN U Bilirubin mg/dL 5 8 10 12 PT4 Week Group 2 TE D Group 1 Figure S3. PEARL-II Mean Total Bilirubin Values Over Time. Mean total and indirect bilirubin AC C EP values of the safety population (all patients receiving at least one dose of study drug) are plotted over time during the treatment period and for the week-4 post-treatment follow-up (PT4). Solid lines depict total bilirubin; dashed lines depict indirect bilirubin. 12 ACCEPTED MANUSCRIPT Table S1. PEARL-II Primary Reasons for Not Meeting Study Eligibility # OF SCREEN FAILURES 7 1 24 Patient's HCV genotype is subgenotype 1b at Screening without co-infection with any other genotype/subgenotype. EXCLUSION CRITERIA Recent (within 6 months prior to study drug administration) history of drug or alcohol abuse that, in the opinion of the investigator, could preclude adherence to the protocol. Positive test result for hepatitis B surface antigen (HBsAg) or anti-HIV antibodies (anti-HIV Ab). Patient's HCV subgenotype at Screening is not subgenotype 1b or indicates co-infection of 1b with any other genotype/subgenotype. Positive result of a urine drug screen at the Screening Visit for opiates, barbiturates, amphetamines, cocaine, benzodiazepines, phencyclidine, propoxyphene, or alcohol with the exception of a positive result associated with documented short-term use or chronic stable use of a prescribed medication in that class. Clinically significant abnormalities, other than HCV infection, based upon the results of a medical history, physical examination, vital signs, laboratory profile and a 12-lead AC C # OF SCREEN FAILURES EP TE D 21 M AN U SC RI PT 22 INCLUSION CRITERIA Patient must have documentation that they were adherent to prior pegIFN/RBV combination therapy and meet one of the following categories: ● Null responders: received at least 12 weeks of pegIFN/RBV for the treatment of HCV and failed to achieve a 2 log10 IU/mL reduction in HCV RNA at Week 12. Patients will be considered to meet this definition if the lack of treatment response was documented following 10 to 16 weeks of treatment; ● Non-responders/partial responders: received at least 20 weeks of pegIFN/RBV for the treatment of HCV and achieved ≥ 2 log10 IU/mL reduction in HCV RNA at Week 12, but failed to achieve HCV RNA undetectable at the end of treatment. Patients will be considered to meet this definition if the lack of treatment response was documented following 10 to 16 weeks of treatment; or ● Relapsers: received at least 36 weeks of pegIFN/RBV for the treatment of HCV and was undetectable at the end of treatment, but HCV RNA was detectable within 52 weeks of treatment follow-up. ● Viral loads documenƟng the type of prior non-response should be obtained during the previous pegIFN/RBV treatment. PegIFN/RBV therapy must have been completed no less than 2 months prior to the Screening Visit. Chronic HCV genotype 1b-infection for at least 6 months prior to Screening. Chronic HCV infection is defined as one of the following: ● PosiƟve for anƟ-HCV antibody or HCV RNA at least 6 months before Screening, and positive for HCV RNA and anti-HCV antibody at the time of Screening; or ● PosiƟve for anƟ-HCV antibody and HCV RNA at the time of Screening with a liver biopsy consistent with chronic HCV infection. Per local standard practice, documented results of one of the following: ● Liver biopsy within the 24 months prior to or during screening demonstraZng the absence of cirrhosis, e.g., a Metavir score of 3 or less or an Ishak score of 4 or less, or ● A screening FibroTest score of ≤ 0.72 and an Aspartate Aminotransferase to Platelet Ratio Index (APRI) ≤ 2, or ● A screening FibroScan® result of < 9.6 kPa. ● Patients with a non-qualifying Fibrotest/APRI or Fibroscan result may only be enrolled if they have a qualifying liver biopsy performed within 24 months prior to or during screening. Patient has a plasma HCV RNA level > 10,000 International Units (IU)/mL at Screening. 1 2 4 3 3 13 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT electrocardiogram (ECG) that make the patient an unsuitable candidate for this study in the opinion of the Investigator. History of uncontrolled seizures, uncontrolled diabetes as defined by a glycated 6 hemoglobin A1c level > 8.5% at the Screening visit, active or suspected malignancy or history of malignancy (other than basal cell skin cancer or cervical carcinoma in situ) in the past 5 years. Any cause of liver disease other than chronic HCV infection, including but not limited to 3 the following: ● Hemochromatosis ● Alpha-1 antitrypsin deficiency ● Wilson's disease ● Autoimmune hepaƟƟs ● Alcoholic liver disease ● Drug-related liver disease Steatosis and steatohepatitis on a liver biopsy coincident with HCV-related changes would not be considered exclusionary unless the steatohepatitis is considered to be the primary cause of the liver disease. Screening laboratory analyses show any of the following abnormal laboratory 28 results: ● Alanine aminotransferase (ALT) > 5 × upper limit of normal (ULN), ● Aspartate aminotransferase (AST) > 5 × ULN, ● Calculated creaƟnine clearance (using CockcroŌ-Gault method) < 60 mL/min, ● Albumin < lower limit of normal (LLN), ● Prothrombin time/International normalized ration (INR) > 1.5. Patients with a known inherited blood disorder may be enrolled with permission of the AbbVie Study Designated Physician even if the INR > 1.5, ● Hemoglobin < LLN, ● Platelets < 120,000 cells per mm3, ● Absolute neutrophil count (ANC) < 1500 cells/μL (< 1200 cells/μL for patients of African descent who are black, ● Indirect bilirubin > 1.5 × ULN and direct bilirubin > ULN. Use of any medications that are contraindicated for ritonavir and ribavirin, within 2 0 weeks prior to study drug administration or 10 half-lives (if known), whichever is longer. Receipt of any investigational product within a time period equal to 10 half-lives of 1 the product, if known, or a minimum of 6 weeks prior to study drug administration. Consideration by the Investigator, for any reason, that the patient is an unsuitable 1 candidate to receive ABT-450, ombitasvir, dasabuvir, ritonavir, or RBV. Current enrollment in another clinical study, previous enrollment in this study, or 2 previous use of any investigational or commercially available anti-HCV therapy (other than interferon and/or pegIFN/RBV), including previous exposure to boceprevir, telaprevir, ABT-450, ombitasvir, or dasabuvir. Patients who previously participated in trials of investigational anti-HCV agents may be enrolled with the approval of the AbbVie Study Designated Physician if they can produce documentation that they received only placebo. Concurrent participation in a non-interventional, epidemiologic or registry trial may be permitted with approval by the AbbVie Study Designated Physician. The primary reasons for failing to meet inclusion or exclusion criteria are listed for each patient. 14 ACCEPTED MANUSCRIPT Table S2. Fibrosis Scoring Liver Biopsy Metavir or Batts-Ludwig or Knodell or IASL or Scheuer Score 0 or 1 2 3 (or higher) Liver Biopsy Ishak Score 0, 1, or 2 3 4 (or higher) FibroScan (kPa) <8.8 >8.8 to <9.6 >9.6 FibroTest <0.48 0.49 to 0.58 >0.59 RI PT Baseline Fibrosis Stage, Metavir Equivalents F0-F1 F2 F3 (or higher) AC C EP TE D M AN U SC Baseline fibrosis stage is defined based on available liver biopsy findings, FibroScan scores, or FibroTest scores. Fibrosis score was determined by a single score in patients with multiple scores available. If biopsy results were present, they were used to categorize the patient, regardless of the FibroScan/FibroTest score. Similarly, if a FibroScan score was present along with a FibroTest score, then the FibroScan score was used to categorize the patient. For patients who have only liver biopsy range available, the highest value for the range was used as their biopsy score. 15 ACCEPTED MANUSCRIPT Table S3. PEARL-II Adverse Events Occurring in > 5% of Patients in Either Treatment Group, n (%) TE D Hyperbilirubinaemia Hypertension Irritability Cough Influenza EP Palpitations 29 (31.9) 22 (24.2) 19 (20.9) 13 (14.3) 13 (14.3) 12 (13.2) 11 (12.1) 10 (11.0) 8 (8.8) 8 (8.8) 8 (8.8) 8 (8.8) 8 (8.8) 6 (6.6) 6 (6.6) 6 (6.6) 6 (6.6) 5 (5.5) 5 (5.5) 15 (15.8) 22 (23.2) 6 (6.3) 3 (3.2) 8 (8.4) 12 (12.6) 7 (7.4) 0 0 2 (2.1) 3 (3.2) 2 (2.1) 1 (1.1) 6 (6.3) 4 (4.2) 6 (6.3) 8 (8.4) 1 (1.1) 1 (1.1) P-value 0.015 0.005 0.008 RI PT Group 2 N = 95 M AN U Fatigue Headache Nausea Insomnia Pruritus Diarrhoea Asthenia Anaemia Blood bilirubin increased Decreased appetite Dizziness Dyspnoea Rash Arthralgia Myalgia Nasopharyngitis Pyrexia Constipation Dyspnoea exertional Group 1 N = 91 SC MEDDRA 16.0 PREFERRED TERM 5 (5.5) 2 (2.1) 5 (5.5) 4 (4.2) 5 (5.5) 1 (1.1) 5 (5.5) 2 (2.1) 3 (3.3) 7 (7.4) 2 (2.2) 6 (6.3) <0.001 0.003 0.017 AC C P-value for comparisons between treatment groups using fisher’s exact test. Only p-values ≤ 0.05 are presented. 16 ACCEPTED MANUSCRIPT Table S4. Grade 3 and 4 Laboratory Abnormalities, n (%) Treatment Group Post-baselineb Baseline ALT (U/L) Grade 3 0 1 (1.1) Grade 4 0 0 N = 90 0 0 Grade 4 0 0 1 (1.1) 2 (2.1) Total bilirubin (µmol/L) Grade 3 0 Grade 4 0 a 0 1 (1.1) 0 0 6 (6.6) 10 (10.5) 0 0 0 0 8 (8.8) 0 0 0 0 M AN U 0 0 0 Alkaline phosphatase (U/L) At least grade 2 0 N = 90 Grade 3 Grade 1 0 SC AST (U/L) Group 2 N = 95a RI PT Parameter (unit) Grade Group 1 N = 91a Group 2 N = 95a Group 1 N = 91a Number of patients with baseline and at least 1 post-baseline value. Values during treatment up to 2 days post-dosing are used. AC C EP TE D b 17 ACCEPTED MANUSCRIPT Table S5. Potentially Clinically Significant Laboratory Abnormalities, n/N (%) Group 1 N = 91 0/91 Group 2 N = 95 0/95 AST (> 5 × ULN and ≥ 2 × baseline) 0/90 1/95 (1.1) Alkaline phosphatase (> 1.5 × ULN) 0/91 1/95 (1.1) 14/91 (15.4) 1/95 (1.1) Creatinine (≥ 132.605 µmol/L) 0/91 2/95 (2.1)a Creatinine clearance, calculated (< 50 mL/m) 0/91 BUN (> 5 × ULN) 0/91 SC Bilirubin, total (≥ 2 × ULN) RI PT Variable (criteria) ALT (> 5 × ULN and ≥ 2 × baseline) Uric acid (> 713.817 µmol/L) Phosphate, inorganic (< 0.6 mmol/L) Calcium (< 1.75 mmol/L) 0/95 0/91 0/95 0/91 1/95 (1.1)c 0/91 0/95 M AN U Calcium (> 3.1 mmol/L) 2/95 (2.1)b 0/91 0/95 0/91 0/95 0/91 0/95 0/91 0/95 0/91 1/95 (1.1)b 0/91 0/95 0/91 0/95 0/91 1/95 (1.1)d 0/91 0/95 0/91 0/95 0/91 0/95 Cholesterol (> 10.34 mmol/L) 0/91 0/95 Triglycerides (> 5.7 mmol/L) 0/91 1/95 (1.1)b Magnesium (> 1.23 mmol/L) Magnesium (< 0.4 mmol/L) Sodium (> 155 mmol/L) Sodium (< 130 mmol/L) Potassium (> 6 mmol/L) Glucose (> 13.9 mmol/L) Glucose (< 2.2 mmol/L) Albumin (< 20 g/L) a EP Protein, total (< 50 g/L) TE D Potassium (< 3 mmol/L) AC C Patient had an elevation in creatinine on Day 44 while hospitalized for cellulitis which returned to normal 4 days later, then again had elevated creatinine at Post-Treatment Week 4. The other patient had an elevation in creatinine on Day 85 only. b Single time point abnormality only. c Decrease in phosphate occurred on Days 8 and 15, which subsequently returned to normal for the remainder of the study. d Glucose elevation noted at baseline and throughout the study in a patient with a history of diabetes. 18 ACCEPTED MANUSCRIPT References AC C EP TE D M AN U SC RI PT 1. Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med 2011;364:2417-28. 2. Westfall PH, Krishen A. Optimally weighted, fixed sequence and gatekeeper multiple testing procedures. J Stat Plan Inference 2001;99:25-40. 19