Compared with genotypes 2 and 3, patients with

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1 R1626 Plus Peginterferon Alfa-2a Provides Potent Suppression of Hepatitis C Virus RNA and Significant Antiviral Synergy in Combination with Ribavirin Paul J. Pockros, 1 David Nelson, 2 Eliot Godofsky, 3 Maribel Rodriguez-Torres, 4 Gregory T. Everson, 5 Michael W. Fried, 6 Reem Ghalib, 7 Stephen Harrison, 8 Lisa Nyberg, 9 Mitchell L. Shiffman, 10 Isabel Najera, 11 Anna Chan, 12 and George Hill 11 R1626, a prodrug of the hepatitis C virus (HCV) RNA polymerase inhibitor R1479, showed time-dependent and dose-dependent reduction of HCV RNA levels in a previous study. The present study evaluated the efficacy and safety of R1626 administered for 4 weeks in combination with peginterferon alfa-2a ribavirin in HCV genotype 1-infected treatment-naive patients. Patients were randomized to: DUAL 1500 (1500 mg R1626 twice daily [bid] peginterferon alfa-2a; n 21); DUAL 3000 (3000 mg R1626 bid peginterferon alfa-2a; n 32); TRIPLE 1500 (1500 mg R1626 bid peginterferon alfa-2a ribavirin; n 31); or standard of care (SOC) (peginterferon alfa-2a ribavirin; n 20). At 4 weeks HCV RNA was undetectable (<15 IU/mL) in 29%, 69%, and 74% of patients in the DUAL 1500, DUAL 3000, and TRIPLE 1500 arms, respectively, compared with 5% of patients receiving SOC, with respective mean reductions in HCV RNA from baseline to week 4 of 3.6, 4.5, 5.2, and 2.4 log 10 IU/mL. Synergy was observed between R1626 and peginterferon alfa-2a and between R1626 and ribavirin. There was no evidence of development of viral resistance. Adverse events (AEs) were mainly mild or moderate; seven patients had nine serious AEs (including one patient with one serious AE in SOC). The incidence of Grade 4 neutropenia was 48%, 78%, 39%, and 10% in DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC, respectively, and was the main reason for dose reductions. Conclusion: A synergistic antiviral effect was observed when R1626 was combined with peginterferon alfa-2a ribavirin; up to 74% of patients had undetectable HCV RNA at week 4. Dosing of R1626 was limited by neutropenia; a study of different dosages of R1626 in combination with peginterferon alfa-2a and ribavirin is underway. (HEPATOLOGY 2008;48: ) Compared with genotypes 2 and 3, patients with hepatitis C virus (HCV) genotype 1 typically have a lower response to combination therapy of pegylated interferon and ribavirin. 1,2 Approximately half of patients with chronic HCV genotype 1 infection achieve a sustained viral response with the current standard of care (SOC) and response rates are even lower in patients coinfected with human immunodeficiency virus, Abbreviations: bid, twice daily; AE, adverse event; ALT, alanine aminotransferase; C, plasma concentration; HCV, hepatitis C virus; po, orally; qd, once daily; qw, once weekly; sc, subcutaneous(ly); SOC, standard of care. From the 1 Division of Gastroenterology and Hepatology, Scripps Clinic, La Jolla, CA; 2 Division of Gastroenterology, Hepatology, and Nutrition, University of Florida, Gainesville, FL; 3 University Hepatitis Center at Bach & Godofsky, Bradenton, FL; 4 Fundacion de Investigacion de Diego, San Juan, Puerto Rico; 5 Section of Hepatology, Division of Gastroenterology, University of Colorado Health Sciences Center, Denver, CO; 6 Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; 7 The Liver Institute at Dallas Methodist Hospital, Dallas, TX; 8 Brooke Army Medical Center, Fort Sam Houston, TX; 9 Kaiser Permanente, San Diego, CA; 10 Hepatology Section, Virginia Commonwealth University Medical Center, Richmond, VA; 11 Roche Pharmaceuticals LLC, Palo Alto, CA; 12 Hoffmann-La Roche Inc., Nutley, NJ. Received December 4, 2007; accepted March 31, Address reprint requests to: Dr. Paul Pockros, Division of Gastroenterology and Hepatology, Scripps Clinic, North Torrey Pines Road, La Jolla, CA pockros.paul@scrippshealth.org; fax: Copyright 2008 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Dr. Shiffman is a consultant for, advises, is on the speakers bureau of, and received grants from Roche. He also advises and received grants from Vertex. He received grants from Schering-Plough. Dr. Pockros is a consultant for, advises, is on the speaker s bureau, and received grants from Roche. He is a consultant for, advises, and received grants from Wyeth and Idenix. Drs. Nelson and Everson are consultants for, advise, are on the speakers bureau of, and received grants from Roche. Dr. Godofsky is on the speakers bureau of, and received grants from Roche. Dr. Rodriguez-Torres advises, is on the speakers bureau of, and received grants from Roche. Dr. Fried is a consultant for, is on the speakers bureau of, and received grants from Roche. Dr. Ghalib is on the speakers bureau of, and received grants from Schering-Plough and Roche. He is also on the speakers bureau of Valeant. He received grants from Vertex and Coley. Dr. Harrison advises, is on the speakers bureau of, and received grants from Roche. He also received grants from Schering-Plough and Valeant. Dr. Nyberg is on the speakers bureau of, and received grants from Schering-Plough. She also received grants from Roche, Vertex, Novartis, Idenix, and Human Genome Sciences. 385

2 386 POCKROS ET AL. HEPATOLOGY, August 2008 in older patients, those with cirrhosis, or of African American origin. 3-5 The development of novel therapeutic approaches is a priority for these patient populations. Viral polymerase enzymes are essential for viral replication, and polymerase inhibitors form the largest class of antiviral drugs with proven efficacy in hepatitis B virus, herpes simplex virus, and human immunodeficiency virus infections. A large proportion of polymerase inhibitors are nucleoside analogs, which, following enzymatic conversion to the corresponding nucleoside triphosphate, competitively inhibit viral nucleic acid synthesis. R1626 is the tri-isobutyl ester prodrug of the nucleoside analog R1479, which gets phosphorylated to its active moiety R1479-TP, a potent and highly selective inhibitor of NS5B, the viral RNA-dependent RNA polymerase that synthesizes and elongates viral RNA. 6 R1479-TP is a chain terminator, which inhibits RNA chain extension following incorporation into nascent HCV RNA. 6 Orally administered R1626 is rapidly converted to R1479 during uptake in the gastrointestinal tract. R1626 was developed to increase the bioavailability, and hence maximize the efficacy, of R1479; animal studies have demonstrated that transport across the gut wall is 10-fold greater with R1626 than with R R1626 was well tolerated up to doses of 12,000 mg in healthy subjects, with no serious adverse events (AEs) reported. 8 In a Phase 1b multicenter, randomized, placebo-controlled multiple ascending dose study to evaluate the antiviral activity, pharmacokinetics, and safety of R1626, a potent antiviral effect was reported with time-dependent reductions in viral load. Mean (median; range) reductions in HCV RNA of 0.3 (0.2; 0-0.7), 1.2 (0.8; ), 2.6 (2.7; ), and 3.7 (4.1; ) log 10 were observed at doses of 500, 1500, 3000 and 4500 mg, respectively, after 14 days of monotherapy. 9 Doses up to and including 3000 mg twice daily (bid) were generally well tolerated. However, mean reductions of 0.9 g/dl in hemoglobin (placebo-adjusted) were noted with the 4500 mg bid dosage. Neutropenia was not observed in any of the dosage groups. This Phase 2a study is the first to assess the safety and efficacy of R1626 in combination with peginterferon alfa- 2a, with or without ribavirin, versus SOC in treatmentnaive patients with chronic HCV genotype 1 infection. We report here the primary safety, pharmacokinetic, and pharmacodynamic analyses on the total study population after all study patients had completed the 4-week experimental treatment, as well as the data generated to assess the potential for development of viral resistance to R1626. Patients and Methods Patients Patients were eligible for inclusion if they were aged 18 to 65 years and had chronic HCV genotype 1 infection with HCV RNA levels 50,000 IU/mL. Only treatment-naive patients were enrolled in the study. Other inclusion criteria included chronic liver disease consistent with chronic HCV infection on biopsy, and compensated liver disease (Child-Turcotte-Pugh grade A). Women of childbearing potential were required to have a negative blood pregnancy test within the 24-hour period prior to the first dose of study medication. All fertile patients, male and female, were required to use two forms of effective contraception during treatment and for 6 months afterward. Patients were excluded from the study if they had infection with any HCV genotype other than genotype 1, or an indeterminate or mixed genotype; hepatic cirrhosis (Knodell score of 4, Metavir score of 4, or Ishak modified histological activity index score of 5 or 6) or incomplete/ transition to cirrhosis (Knodell score of 3, Metavir score of 3, or an Ishak modified histological activity index score of 4 with nodules or 3 bridges); a low absolute neutrophil count ( 1500 cells/mm 3 ); a low platelet count ( 120,000 cells/mm 3 ); or a low hemoglobin concentration ( 13 g/dl in females or 14 g/dl in males). Study Design This was a multicenter, Phase 2a, randomized, doubleblind, parallel group study in which SOC was used as the active control. All patients provided informed consent, and the study protocol (PV18369/B) conformed to the 1975 Declaration of Helsinki. Patients were recruited into the study between October and November A 35-day screening phase preceded a 4-week treatment period with the experimental regimens. At the end of the 4-week treatment period, all patients received SOC for 44 weeks. The study was double-blinded until all patients finished week 8 (that is, 4 weeks of experimental treatment plus the first 4 weeks of the SOC follow-up phase). In accordance with the study protocol, the primary analysis was defined as the safety, pharmacokinetic, and pharmacodynamic analyses on the total study population after all study patients had completed the 4-week experimental treatment. A planned secondary analysis evaluated safety and pharmacodynamics for the total study population after all study patients had completed the 4 week experimental period followed by 4 weeks of treatment with SOC, to ensure that any hematological abnormalities were reversible once treatment with R1626 was completed. At the clinical cutoff date for each analysis, the

3 HEPATOLOGY, Vol. 48, No. 2, 2008 POCKROS ET AL. 387 Fig. 1. Study treatments and number of patients initially randomized to each group. safety data also included results for those patients who were beyond weeks 4 or 8, respectively. Reported here are the complete results of the primary analyses (week 4) and efficacy and safety data based on the secondary analyses ascertained at week 8. Study Treatments At the end of the 35-day screening period, 107 patients were randomized in a 2:3:3:2 ratio to one of four treatment groups (Fig. 1): DUAL 1500 received R1626 (1500 mg orally [po] bid), R1626 placebo (po bid), peginterferon alfa-2a (180 g subcutaneously [sc] once weekly [qw]), and ribavirin placebo (po once daily [qd]) for 4 weeks (Fig. 1A); DUAL 3000 (n 34) received R1626 (3000 mg po bid), peginterferon alfa-2a (180 g sc qw), and ribavirin placebo (po qd) for 4 weeks (Fig. 1B); TRIPLE 1500 (n 31) received R1626 (1500 mg po bid), R1626 placebo (po bid), peginterferon alfa-2a (180 g sc qw), and ribavirin (1000 mg po qd for those weighing 75 kg; 1200 mg po qd if 75 kg) for 4 weeks (Fig. 1C); SOC received R1626 placebo (po bid), peginterferon alfa-2a (180 g sc qw), ribavirin (1000 mg po qd for those weighing 75 kg; 1200 mg po qd if 75 kg) for 4 weeks (Fig. 1D). The patients randomized to each arm were then stratified into two cohorts according to the intensity of sampling for pharmacokinetic evaluation: the first group underwent intensive evaluation whereas the second group underwent sparse evaluation. R1626 was provided as a 500-mg tablet, peginterferon alfa-2a (PEGASYS) was provided as 180 g in1.0ml solution in vials, and ribavirin (COPEGUS) was provided as a 200-mg tablet. R1626 and ribavirin were administered daily with food in divided doses (morning and evening). R1626 and ribavirin were double-blinded, with appropriate and visually similar placebos administered, while peginterferon alfa-2a was open-label. All medicines and placebos were provided by F. Hoffmann-La Roche (Basel, Switzerland). Protocol Amendment for Dose Modification and Use of Erythropoietins or Growth Factors During the first 4 weeks, investigators were required to follow the protocol-defined guidelines for dose modification. When approximately 50% of the patients had completed 4 weeks of treatment, the protocol was amended, as apparent time-related changes in hematological parameters (mostly neutrophil counts) were observed in the blinded safety analysis. Based on these safety data the protocol was modified to mandate earlier modification of the R1626 (or placebo) dose based on specifically defined laboratory parameters. In addition, prior to the protocol modification the definition of a one level dose reduction was 500 mg/day. This was changed to a 50% reduction in the daily dose. The original and amended instructions are summarized in Table 1. The adjustments for peginterferon alfa-2a and ribavirin remained unchanged after the protocol amendment. The use of erythropoietins or granulocyte-colony stimulating factors was discouraged prior to the protocol amendment. In the protocol amendment, physicians were advised to manage and treat treatment-related AEs according to their best medical judgment which may have involved the use of erythropoietins or granulocytecolony stimulating factors.

4 388 POCKROS ET AL. HEPATOLOGY, August 2008 Table 1. Original and Amended Dose Modification Instructions for Neutropenia, Thrombocytopenia, and Anemia Original Protocol Amendment Absolute neutrophil count 1,000 cells/mm 3 No dose modification Reduce daily R1626 dose by 50% 750 cells/mm 3 Reduce R1626 by 500 mg/day (8%-17% reduction in daily dose) Hold R1626 dose Platelets 50,000 cells/mm 3 Reduce R1626 by 500 mg/day (8%-17% reduction in daily dose) Reduce daily R1626 dose by 50% Hemoglobin 10 g/dl Reduce R1626 by 500 mg/day (8%-17% reduction in daily dose) Reduce daily R1626 dose by 50% Dose reduction for R1626 based on severity of AEs was done as follows: for moderate/persistent/severe limited AE s, 0 to 1 level of dose reduction was recommended; for severe persistent 1 to 2 levels of reduction; for life-threatening AEs drug was stopped. Prior to protocol modification the definition of a 1 level dose reduction was 500 mg/day. This was changed to a 50% reduction of the daily dose. General dose reduction instructions provided for clinical AEs were based on severity of the AE. For moderate/ persistent/severe limited AEs 0 to 1 levels of dose reduction was recommended; for severe persistent AEs, 1 to 2 levels of reduction were recommended; and for lifethreatening AEs the drug was stopped. These instructions remained unchanged after the protocol amendment. At the end of the 4-week experimental treatment period, all patients received peginterferon alfa-2a (180 gsc qw) plus ribavirin (1000 mg po qd for those weighing 75 kg; 1200 mg po qd if 75 kg) for an additional 44 weeks. Efficacy Analyses Serum HCV RNA levels were quantified using the Roche COBAS Taqman HCV assay (Roche Molecular Systems Inc., Branchburg, NJ). Virological response is presented as HCV RNA 50 IU/mL and 15 IU/mL. The baseline viral load was defined as the HCV RNA level in the day 1 predose sample. HCV RNA was measured on days 1, 8, 15, 22, and 29 during the 4-week experimental period. Similarly, serum alanine aminotransferase (ALT) activity was also measured on days 1, 8, 15, 22, and 29 during the 4-week experimental period and is summarized as the number and percentage of patients who had elevated ALT levels at baseline (day 1 predose sample) but became ALT normal at week 4. Pharmacokinetics Plasma samples for the evaluation of R1479 and ribavirin pharmacokinetics were collected at baseline (day 1) and on days 15 and 29 in all patients. Plasma samples for population pharmacokinetic analysis were collected predose and 2, 4, and 12 hours postdose in approximately 30% of patients. For the remaining approximately 66 patients, plasma samples were collected predose and at 1 hour postdose. Nonlinear mixed effects modeling (using NONMEM VI) was used to analyze R1479 plasma concentration-time data following R1626 administration in this study. Detailed results of the population pharmacokinetic analysis of R1479 will be presented elsewhere. Safety Analysis AEs, laboratory tests (including hematology, chemistry, and urinalysis), electrocardiography, and vital signs were evaluated at each assessment. The primary data reported are cumulative data for all patients up to week 4 (when all patients had completed the 4-week experimental treatment period). In addition, secondary safety data are reported for week 8 (when all patients had completed the 4-week experimental treatment followed by 4 weeks of SOC). As the database containing the week 8 safety data will only be locked when all patients have completed the entire study (72 weeks), the week 8 data included in this analysis should be considered as preliminary according to the rules of good clinical practice. Assessment of Viral Resistance Assessment of the potential development of resistance to R1479, the active compound derived from R1626 in vivo, was performed for those patients who experienced rebound in HCV RNA, defined as a 0.5 log 10 IU/mL decrease in viral load followed by a subsequent on-treatment increase of 0.5 log 10 IU/mL above nadir. The sensitivity of the clinical isolates to R1479 was determined using the HCV NS5B phenotypic assay. 10 Doseresponse curves and median inhibitory concentration values were generated using the XLfit3 program (ID Business Solutions Ltd., Guildford, UK). Decreased sensitivity to R1479 was defined as a three-fold increase in median inhibitory concentration. The entire region of the HCV genome encoding NS5B protein, the target of R1479 inhibitory activity, was sequenced using an ABI 3730 xl DNA Analyzer and chromatograms analyzed using Sequencher (Gene Codes Corp., Ann Arbor, MI) and VNTI (InforMax Inc., Rockville, MD).

5 HEPATOLOGY, Vol. 48, No. 2, 2008 POCKROS ET AL. 389 Table 2. Baseline Patient Demographics (Safety Population) DUAL 1500 DUAL 3000 (n 32) TRIPLE 1500 (n 31) SOC (n 20) Mean age, years (range) 48.0 (30 63) 46.5 (23 66) 48.3 (24 62) 45.2 (23 65) Male (%) Mean height (cm) Mean body weight (kg) Mean BMI (kg/m 2 ) Race, n (%) Caucasian 16 (76) 31 (97) 29 (94) 17 (85) Black 5 (24) 1 (3) 3 (15) Other 1 (3) 1 (3) HCV genotype, n (%) (3) 0 1a 15 (71) 23 (72) 26 (84) 16 (80) 1a/1b 0 1 (3) 0 0 1b 6 (29) 8 (25) 4 (13) 4 (20) HCV RNA titer (log 10 IU/mL) ALT (U/L) Fibrosis score F0-F1 (%) F2 (%) Statistical Methods The intent-to-treat population included those patients receiving at least one dose of study medication. HCV RNA levels are reported using descriptive statistics for each treatment group, and data are presented as mean standard error and median (with range) unless otherwise stated. Rates of virological response are presented with 95% confidence intervals. The safety population included all patients who received at least one dose of study medication and had at least one postbaseline safety assessment. Descriptive statistics are reported for clinical laboratory data. AEs are presented as categories. Results Patient Disposition A total of 107 patients were randomized. A total of three patients withdrew consent prior to receiving any study medication. The remaining 104 patients were evaluable in both intent-to-treat and safety populations. Patient Demographics The majority of patients were Caucasian (89%), male (64%), and infected with HCV genotype 1a (77%; Table 2). The study arms were balanced with respect to mean age, body weight, height, body mass index, race, ethnicity, and hepatic fibrosis score. The mean baseline HCV RNA levels across the four treatment arms were similar ( log 10 IU/mL), as were the mean baseline ALT concentrations (45-59 U/L) (Table 2). Drug Exposure The mean percent of expected cumulative dose of R1626 taken by week 4 was similar across the three arms; 82.7%, 75.1%, and 86.0% for the DUAL 1500, DUAL 3000, and TRIPLE 1500 arms, respectively. The mean percent of expected cumulative dose of peginterferon alfa-2a taken during the first 4 weeks was similar for DUAL 1500, TRIPLE 1500, and SOC (90.8%, 95.8%, and 94.1%, respectively), but slightly lower, at 82.4%, in the DUAL 3000 arm. The percent of expected cumulative dose of ribavirin in the TRIPLE 1500 arm (95.4%) was similar to that in the SOC arm (96.9%). The percent of expected cumulative dose of peginterferon alfa-2a taken by week 8 was 85.6%, 76.0%, 86.6%, and 93.4% for DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC arms, respectively. The percent of expected cumulative dose of ribavirin taken by week 8 in the TRIPLE 1500 arm (81.3%) was slightly lower than that taken in the SOC arm (96.6%). Pharmacokinetics R1479 plasma concentrations were similar to those following single (conducted in healthy subjects) and multiple (conducted in treatment-naive HCV patients) dose R1626 monotherapy studies (Fig. 2). Plasma was collected up to 72 hours for R1479 plasma concentration (C) determination in the single-dose and multiple-dose monotherapy studies. Following the administration of TRIPLE 1500, median estimated R1479 maximum plasma concentration (C max ) values on day 14 of dosing were similar to observed R1479 C max on day 14 of multiple dose R1626 monotherapy: 9.4 mg/l (n 26) versus 9.9 mg/l (n 9), respectively. Following the administration of TRIPLE 1500, median estimated R1479 minimum plasma concentration (C min ) values on day 14 of dosing were similar to observed R hour plasma

6 390 POCKROS ET AL. HEPATOLOGY, August 2008 Fig. 2. R1479 Plasma concentration as a function of time following R1626 administration. (A) R mg bid in Phase 1a, 8 1b, 9 and 2a studies; (B) R mg bid in Phase 1a, 8 1b, 9 and 2a studies. concentration (C 12h ) on day 14 of multiple dose R1626 monotherapy: 2.7 mg/l (n 26) versus 2.5 mg/l (n 9), respectively. Following the administration of TRIPLE 1500, the median estimated R1479 AUC at steady state over 12 hours were similar to the median estimated values following single-dose and multiple-dose R1626 monotherapy: 69.3 mg.hour/l (n 31) versus 63.3 mg.hour/l (n 15), respectively. Antiviral Efficacy Reduction in HCV RNA Level. The mean reductions in viral load after 2 weeks of treatment were 3.1, 4.3, 4.6, and 1.6 log 10 IU/mL for the DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC arms, respectively (Table 3). All four treatment arms achieved a mean reduction in HCV RNA level from baseline of 2.4 log 10 IU/mL at week 4. The greatest reduction in HCV RNA at week 4 was observed in the TRIPLE 1500 arm (Fig. 3). The 95% confidence intervals between the TRIPLE 1500 and DUAL 1500 arms, and between the TRIPLE 1500 and SOC arms were nonoverlapping, indicating a superior antiviral effect in the TRIPLE 1500 arm compared with the DUAL 1500 mg and SOC arms. Individual profiles of HCV RNA decline are shown for all treatment groups (Fig. 4A-D). At week 8, after all patients who had received the experimental treatment for 4 weeks had received an additional 4 weeks of SOC, the mean (median; range) reduction in HCV RNA from baseline to week 8 in the DUAL 1500 arm was 3.6 (4.0; ) log 10 compared to 3.6 (4.2; ) log 10 at week 4. The respective reductions at weeks 8 and 4 in the other study arms were 4.2 (4.9; ) log 10 and 4.5 (4.9; ) log 10 in the DUAL 3000 arm, 4.5 (5.2; ) log 10 and 5.2 (5.3; ) log 10 in the TRIPLE 1500 arm, and 4.0 (4.6; ) log 10 and 2.4 (2.5; ) log 10 in the SOC arm. Virological Response. At week 4, the proportion of patients achieving virological response (defined as HCV RNA 50 IU/mL or 15 IU/mL) was higher for all Table 3. Reduction in Viral Load (log 10 [median; range] IU/mL) from Baseline Mean Reduction in HCV RNA Level from Baseline log 10 (median; range) IU/mL Treatment Arm Week 1 Week 2 Week 3 Week 4 DUAL (2.1; ); 3.1 (3.1; ); 3.9 (4.2; ); 3.6 (4.2; ); n 21 n 21 n 19 n 21 DUAL (3.6; ); 4.3 (4.2; ); 4.7 (5.0; ); 4.5 (4.9; ); n 31 n 32 n 30 n 30 TRIPLE (3.0; ); 4.6 (4.7; ); 5.1 (5.2; ); 5.2 (5.3; ); n 31 n 31 n 31 n 31 SOC 1.1 (0.8; ); 1.6 (1.1; ); 2.1 (1.7; ); 2.4 (2.5; ); n 20 n 20 n 18 n 18

7 HEPATOLOGY, Vol. 48, No. 2, 2008 POCKROS ET AL. 391 Fig. 3. On-treatment reduction in HCV RNA level from baseline: mean (median; range). Fig. 4. HCV RNA responses to treatment for individual patients. (A) DUAL 1500; (B) DUAL 3000; (C) TRIPLE 1500; and (D) SOC. All patients with virological rebound (defined as: viral load reduction 0.5 log 10 IU/mL followed by a 0.5 log 10 increase of viral load from nadir during treatment with R1626) discontinued drug or had dose modification. Samples from these patients were analyzed for potential drug resistance

8 392 POCKROS ET AL. HEPATOLOGY, August 2008 Fig. 5. Viral response (proportion of patients with undetectable HCV RNA 50 IU/mL or 15 IU/mL) at week 4. experimental arms compared to SOC and highest in the TRIPLE 1500 arm (Fig. 4): 29%, 69%, 74%, and 5% for the DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC arms, respectively, achieved HCV RNA 15 IU/mL at week 4. The rates of virological response (HCV RNA 15 IU/mL) at week 8 were 38%, 59%, 65%, and 35% for the DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC arms, respectively. ALT Normalization. Time-related and treatment-related decreases in ALT were observed during the experimental treatment period (not shown). Mean and median decreases in ALT level from baseline were largest in the DUAL 3000 and TRIPLE 1500 therapy arms. In patients with high ALT levels at baseline, there were similar levels of ALT normalization in the DUAL 3000 (16/27 patients; 59%) and TRIPLE 1500 arms (14/25 patients; 56%). In the other arms, 6/13 patients (46%) with high baseline ALT in the DUAL 1500 arm, and 4/15 patients (27%) in the SOC arm had normalized their ALT levels at week 4. No clinically relevant increase in ALT from baseline was observed in any treatment arm. The mean (median; range) reductions in ALT from baseline to week 8 and to week 4, respectively, were: 15 (8; 88-73) IU/mL and 19 (13; 26-68) IU/mL; 39 (28; 4-163) IU/mL and 32 (24; ) IU/mL; 33 (19; 0-183) IU/mL and 32 (26; ) IU/mL; and 27 (21; ) IU/mL and 23 (17; 39-99) IU/mL for the DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC arms, respectively. Viral Resistance A total of eight patients with viral load rebound during the 4-week treatment period were identified (Fig. 5; Table 4); all eight patients had discontinued R1626 because of

9 HEPATOLOGY, Vol. 48, No. 2, 2008 POCKROS ET AL. 393 Table 4. Sensitivity to R1479 as Determined Using HCV Replicons Containing NS5B Sequences from Patients with a Rebound in HCV RNA Treatment Arm/Patient NS5B Origin Genotype IC 50 SEM ( M) R1479 (n)* IC 50 -fold/day 1 Con1 1a (11) H77 1b (22) DUAL 1500 Patient 1 Day 1 1a (2) Day 15 1a (2) 0.6 Day 29 1a (6) 0.8 Patient 2 Day 1 1a (4) Day 29 1a (2) 1.4 Patient 3 Day 1 1b (4) Day 15 1b (6) 0.7 Day 22 1b (6) 0.7 DUAL 3000 Patient 4 Day 1 1a (2) Day 29 1a (7) 1.3 Patient 5 Day 1 1a (4) Day 29 1a (7) 1.4 Patient 6 Day 1 1a (2) Day 29 1a (3) 0.7 Patient 7 Day 1 1a (2) Day 29 1a (6) 2.1 SOC Patient 8 Day 1 1a (3) Day 28 1a (7) 1.2 Rebound defined as: viral load reduction 0.5 log 10 IU/mL followed by a 0.5 log 10 increase of viral load from nadir during treatment with R1626. Abbreviations: n, number of independent experiments conducted in the replicon assay; SOC, standard of care. *Results are presented as mean values ( standard error of mean [SEM]) from the total number of experiments, in parentheses; a decrease in sensitivity to drug is indicated by a three-fold increase in IC 50. neutropenia prior to experiencing viral load rebound. Phenotypic 10 and genotypic characterization of a series of samples from these patients were assessed for potential selection of resistance to R1479. Samples showed no change in susceptibility to R1479 (phenotypic resistance) at any of the time points compared to their respective baseline or to the reference controls (Table 4) and within the 2.6-fold variability of the assay. Sequence analysis of the entire NS5B coding region (genotypic analysis) revealed no known R1479 resistance mutations (S96T or S96T/N142T), nor were any other common amino acid substitutions selected upon treatment when compared to baseline sequence as assessed by population sequence as well as by sequence analysis of a total of 1061 molecular clones. Safety Most AEs were mild (80%) or moderate (16%); 4% were rated as severe by the investigator. The most frequently reported AEs were as anticipated from peginterferon alfa containing regimens (Table 5). Of the seven patients reporting serious AEs, four were in the DUAL 3000 arm, two were in the TRIPLE 1500 arm, and one was in the SOC arm; these events included neutropenia, pancytopenia, thrombocytopenia, anemia, peritonsillar abscess, pneumonia, generalized rash, syncope, and appendicitis. All of but one of these events (syncope) occurred during the 4-week experimental treatment period with R1626. All but two of these events (syncope and appendicitis) were considered to be related to study medications. Gastrointestinal side effects were seen most commonly in the DUAL 3000 arm, suggesting this dosage was not well tolerated. Rash events, which have been associated with ribavirin treatment, were more common in the TRI- PLE 1500 arm compared with the DUAL 1500 arm. Decreases in white blood cells, red blood cells, and platelets were more pronounced in patients receiving R1626 (Table 6) and were the primary reason for dose modification and discontinuation in these patients (Table 7). The incidence of grade 4 neutropenia was highest in the DUAL 3000 arm (Table 7). Absolute neutrophil counts in patients with infectious events are shown in Table 8. A total of 30 infectious events in 25 patients were reported by investigators, of these only one was confirmed as bacterial infection by culture (Staphylococcus aureus), whereas 16, 12, and 1 events were considered by the investigator to be of bacterial, viral, and fungal origin, respectively. Suspected infections were treated empirically with antibiotics, antivirals, or antifungals. Of the 25 patients with infectious events, Table 5. Frequency of Adverse Events in the Safety Population during the Experimental Treatment Regimen (Week 4) Most Frequent Events* DUAL 1500 (n 21 DUAL 3000 (n 32 TRIPLE 1500 (n 31 SOC (n 20 Nausea 8 (38) 22 (69) 19 (61) 10 (50) Headache 13 (62) 16 (50) 18 (58) 11 (55) Fatigue 9 (43) 16 (50) 20 (65) 10 (50) Chills 14 (67) 15 (47) 10 (32) 9 (45) Diarrhea 8 (38) 24 (75) 10 (32) 5 (25) Neutropenia 10 (48) 21 (66) 12 (39) Pyrexia 8 (38) 10 (31) 11 (35) 8 (40) Myalgia 3 (14) 10 (31) 10 (32) 11 (55) Insomnia 6 (29) 8 (25) 13 (42) 6 (30) Vomiting 2 (10) 16 (50) 8 (26) 2 (10) Arthralgia 6 (29) 10 (31) 5 (16) 5 (25) Pain 8 (38) 7 (22) 7 (23) 2 (10) Rash 3 (14) 5 (16) 12 (39) 2 (10) Irritability 5 (24) 5 (16) 10 (32) 1 (5) Cough 2 (10) 8 (25) 5 (16) 4 (20) Dizziness 5 (24) 4 (13) 6 (19) 3 (15) Pruritus 2 (10) 4 (13) 5 (16) 6 (30) Injection site erythema 3 (14) 4 (13) 3 (10) 5 (25) *Experienced by 20% of patients. Includes serious adverse events, discontinuations from treatment, and those requiring treatment.

10 394 POCKROS ET AL. HEPATOLOGY, August 2008 Table 6. Incidence of Laboratory Abnormalities and Use of Growth Factors Up to Week 4 DUAL 1500 (n 21 DUAL 3000 (n 32 TRIPLE 1500 (n 31 SOC (n 20 Hemoglobin 10 g/dl 3 (9) 10 (32) 1 (5) 8.5 g/dl 3 (10) White blood cell counts /L 11 (52) 14 (44) 22 (71) 1 (5) /L 1 (5) 10 (31) 1 (3) Neutrophils 0.5 but /L 8 (38) 5 (16) 16 (52) 6 (30) /L 10 (48) 25 (78) 12 (39) 2 (10) Lymphocytes /L 5 (24) 4 (13) 6 (19) Use of growth factors Epoetin alfa 1 2 Filgrastim Table 8. Absolute Neutrophil Counts in Patients with Infectious Event As Reported by Investigator four, nine, 11, and one patients, were in the DUAL 1500, DUAL 3000, TRIPLE 1500, and SOC arms, respectively (Table 8). The largest decrease in hemoglobin at week 4 was observed in the TRIPLE 1500 arm (Table 6); one patient required transfusion. The use of growth factors for neutropenia or anemia is shown in Table 6. Preliminary analysis of the week 8 safety data suggests that all the hematological abnormalities observed during the initial 4-week treatment period were reversible (Table 9). Neutrophil, hemoglobin, and platelet counts in patients who received R1626 increased promptly once R1626 treatment was completed and patients switched to SOC. These hematological values reached levels which were comparable to or higher than those seen in the SOC comparator arm at week 8 (Table 9). No clinically significant changes in electrocardiogram assessments or vital signs were observed. Discussion DUAL 1500 DUAL 3000 (n 32) TRIPLE 1500 (n 31) SOC (n 20) Total number of patients with infection Total number of infectious events Lowest ANC (before/after infection onset)* Grade 4 ( 0.5) 5 4 Grade 3 ( ) 1 Grade 2 ( ) Grade 1 ( ) 1 5 Normal ( 2.0) 3 4 Unknown 2 *Based on ANC within 7 days before to 2 days after the day of onset of infectious event /L. ANC, absolute neutrophil count; SOC, standard of care. There is a clear medical need for new antiviral therapies for patients infected with HCV genotype 1. Based on the Table 7. Dose Modifications and Discontinuations of R1626 in the Safety Population (Week 4) as Well as Percentage of Expected Accumulated Dose of Study Drugs at Weeks 4 and 8 DUAL 1500 (n 21 DUAL 3000 (n 32 TRIPLE 1500 (n 31 SOC (n 20 Dose modifications Total patients with dose modification of R (67) 28 (88) 28 (90) 6 (30)* Discontinuations Total patients discontinuing R (48) 21 (62) 12 (39) 1 (5) Reasons for discontinuation Grade 4 neutropenia 10 (48) 17 (53) 12 (39) Thrombocytopenia 6 (19) 1 (3) Leukopenia 1 (5) 2 (6) Anemia 1 (3) Generalized rash 2 (6) Pneumonia 1 (3) Abnormal thinking 1 (5) Percent expected accumulated dose of study drugs % of Expected mean cumulative dose at week 4 R % 75.1% 86.0% N/A PEG 90.8% 82.4% 95.8% 94.1% RBV N/A N/A 95.4% 96.9% % of Expected mean cumulative dose at week 8 PEG 85.6% 76.0% 86.6% 93.4% RBV 81.3% 96.6% N/A, not available. *Dose modification for R1626 in SOC arm: blinding ensured that it was not known whether these patients were on R1626 or placebo and so, in these patients, dose modifications and discontinuations were made to placebo for side effects that were not related to R1626 but to SOC.

11 HEPATOLOGY, Vol. 48, No. 2, 2008 POCKROS ET AL. 395 Table 9. Laboratory Parameters: Change From Baseline to Week 4 And 8 Mean Change From Baseline Hemoglobin (g/dl) White Blood Cell Count ( 10 9 /L) Neutrophils ( 10 9 /L) Lymphocytes ( 10 9 /L) Platelet Count ( 10 9 /L) Week 4 Week 8 Week 4 Week 8 Week 4 Week 8 Week 4 Week 8 Week 4 Week 8 DUAL 1500 DUAL 3000 TRIPLE 1500 SOC (n 18) (n 17) (n 31) 3.0 (n 18) (n 17) (n 31) 2.04 (n 18) (n 17) (n 28) 0.77 (n 31) 0.69 (n 18) (n 17) (n 18) (n 17) promising results of earlier studies with the polymerase inhibitor R1626, 8,9 which demonstrated its ability to potently inhibit HCV viral replication, we sought to evaluate its activity in combination with a peginterferon alfa-2a backbone, with and without ribavirin. The present study documents a strong antiviral effect of R1626, with potent HCV RNA suppression in combination with peginterferon alfa-2a with or without ribavirin. The pharmacokinetic data suggest that peginterferon alfa-2a, alone or in combination with ribavirin, does not adversely affect the pharmacokinetics of R1626. A dose-proportional increase in antiviral effect was seen with R1626 when administered with peginterferon alfa-2a in the dual combination arms (DUAL 1500 and DUAL 3000). Comparison of the data from the R1626 monotherapy study and this study suggests a possible synergistic effect between R1626 and peginterferon alfa-2a. In the monotherapy study, after 2 weeks of R1626 treatment at the dose of 1500 mg bid, patients experienced a mean HCV RNA decline of 1.2 log 10 IU/mL (median 0.8 log 10 IU/mL) compared to baseline. 9 Assuming that peginterferon alfa-2a monotherapy would lead to an HCV RNA decline in the magnitude of 0.5 log 10 IU/ ml), 11 the anticipated reduction in HCV RNA for the combination of R1626 and peginterferon alfa-2a, assuming an additive effect, would be 2 log 10 IU/mL. The observed mean (median; range) reduction in viral load in the DUAL 1500 arm of the present study after 2 weeks of treatment was higher, at 3.1 IU/mL (3.1; ), suggesting a possible synergistic effect. The expected HCV RNA reduction from baseline after 2 weeks of treatment with the 3000 mg bid dose would be 2.6 IU/mL log 10 (2.7; ) 9 with a corresponding additive effect with peginterferon alfa of 0.5 log 10 IU/mL. The observed mean (median; range) reduction in viral load in the DUAL 3000 arm was 4.3 log 10 IU/mL (4.2; ), again consistent with a possible synergistic effect. This observation supports the in vitro findings documenting moderate synergy between R1626 and interferon alfa, 12 and are in accordance with those reported for the combination of peginterferon alfa with other small molecules under development for treatment of chronic hepatitis C The triple drug regimen, which added R1626 at the dose of 1500 mg bid to peginterferon alfa-2a and ribavirin (that is, SOC), resulted in an additional mean HCV RNA reduction of 3.0 and 2.8 log 10 IU/mL after 2 and 4 weeks of treatment, respectively. This additional antiviral effect is in excess of that expected based on the antiviral efficacy of R1626 as monotherapy (that is, a mean reduction of 1.2 log 10 IU/mL after 2 weeks). 9 All three R1626-containing treatment arms achieved higher rates of rapid viral response compared with SOC. The triple regimen resulted in a considerably higher proportion of patients achieving undetectable HCV RNA at weeks 2 and 4 compared to SOC; only 5% of patients achieved a rapid viral response at week 4 with SOC compared to 74% in the TRIPLE 1500 arm. The rate of rapid virologic response (undetectable HCV RNA at week 4) in the SOC arm may appear low compared with published data, which report response rates of 12% to 20%. 16 The observed difference may be due to the small sample size (n 20). At week 8, 35% of the patients in the SOC group had a virologic response ( 15 IU/mL), a rate which can be regarded as more comparable with the reported data. While the additive or synergistic antiviral effects of new molecules in combination with peginterferon alfa with or without ribavirin are difficult to anticipate based on their antiviral effects in monotherapy studies synergistic or additive effects observed in vitro can provide, at least directionally, an indication of what could be expected in vivo. 12 Synergy between R1626 and ribavirin has been documented in vitro. 12 The results of the present study show a marked increase in antiviral effect in patients when ribavirin is added to the combination of R1626 and

12 396 POCKROS ET AL. HEPATOLOGY, August 2008 peginterferon alfa-2a. As the effects of ribavirin alone on HCV RNA levels have been described to be minimal ( 0.5 log 10 IU/mL over a 4-week period), the additional reduction in mean HCV RNA level at week 4 of 1.6 log 10 IU/mL, which was observed when comparing the TRIPLE 1500 and DUAL 1500 arms and therefore attributable to the addition of ribavirin, is suggestive of a synergistic effect in patients. There is no evident pharmacokinetic interaction between R1626 and ribavirin leading to increased R1626 plasma levels that could explain the observed synergy. More likely, ribavirin is providing an additional antiviral function in addition to R1626 and peginterferon alpha-2a, which results in combination antiviral synergy. The molecular mechanism of this antiviral synergy between R1479 and ribavirin remains to be determined. The virological response rates at week 8 for the DUAL 3000 (59%) and TRIPLE 1500 (65%) arms were approximately 10% lower than the antiviral responses observed at week 4 (69% and 74%, respectively). The difference observed is explained by the high incidence of dose modifications, for safety reasons, of both peginterferon alfa-2a and ribavirin between weeks 4 and 8. This can be seen from the lower percent of expected cumulative dose of peginterferon alfa-2a taken by week 8 in the DUAL 3000 (76.0%) and TRIPLE 1500 (86.6%) arms compared to week 4 (82.4% and 95.8% respectively) and a similarly reduced percent expected cumulative dose of ribavirin in the TRIPLE 1500 arm (81.3% by week 8 versus 95.4% by week 4). The most common AEs and laboratory abnormalities observed during this study were those commonly seen and attributable to peginterferon alfa-2a or ribavirin. The predominant hematological changes observed in this study in association with R1626 (neutropenia and anemia) appeared to be temporally related to treatment and reversible. The relatively high incidence of grade 4 neutropenia in the R1626 arms in this study was not anticipated from the R1626 monotherapy study. This observation is therefore considered to be mainly a consequence of combining R1626 and peginterferon alfa-2a and probably unrelated to ribavirin. In this study, 80% (84/104) of patients were found to have at least one absolute neutrophil count 1000/mm 3 ; however, only 3% (3/104) had serious infections and 21% (22/104) of patients had nonserious infectious events. A total of 25 patients experienced a total of 30 infectious events. Among the 28 events with an available absolute neutrophil count at the time of infection, 13 (46%) had a count of 1000/mm 3, 6 (21%) had counts between 500 and 1000/mm 3, and 9 (32%) had counts 500/mm 3. The study data therefore indicate that rates of infections were similar in patients with either mild or severe neutropenia. However, because of the relatively small sample size in this study, an association between degree of neutropenia and occurrence of infection cannot be ruled out with certainty, and this is something that will be carefully evaluated in the development program of the compound. Compared to the SOC arm, a higher incidence of grade 3 or 4 anemia was seen in patients receiving the triple combination regimen, the basis of which deserves further investigation. There was no evidence to suggest increased hemolysis with the triple combination regimen, consistent with the absence of hemolytic effects of R1479 in vitro. 12 Therefore, the combination of R1626 and ribavirin may be associated with reversible decreased formation of erythrocyte precursor cells from bone marrow stem cells. Phenotyping of samples before and during treatment from eight patients who experienced rebound in viral load showed similar susceptibility of HCV to inhibition by R1479 as baseline samples and reference replicons. In addition, there were no common amino acid substitution(s) selected in patients treated with R1626 that could be associated with resistance to R1479. Therefore, there was no evidence for the selection of viruses resistant to R1626 after 4 weeks of treatment. All 8 patients had discontinued R1626 prior to rebound. Therefore, the observed viral rebound was likely the consequence of R1626 treatment discontinuation and, as a result, reduced antiviral pressure. The absence of viral resistance after 28 days of treatment indicates that HCV exhibits a high genetic barrier to selection of R1626-resistant mutants. These results are consistent with previous observations from a 2-week monotherapy study with R1626, in which no resistant virus was detectable in any of the dose groups. 9,20 The apparent high barrier to R1626 resistance exhibited by HCV, alone or in combination, contrasts with the observations of rapid emergence of resistance (within 2 weeks) following administration of the protease inhibitor telaprevir. 21,22 In conclusion, this Phase 2a study has demonstrated a potent reduction in HCV RNA by R1626 and high viral responses with up to 74% rapid viral response after 4 weeks of treatment. The strong synergistic antiviral effect between R1626, peginterferon alfa-2a and ribavirin suggests that the dose of one or both of these agents could be lowered to improve tolerability without significantly compromising efficacy. An additional clinical study is underway to assess and optimize the dose of R1626 in combination with ribavirin and various doses of peginterferon alfa-2a. The promising results observed with R1626 in

13 HEPATOLOGY, Vol. 48, No. 2, 2008 POCKROS ET AL. 397 treatment-naive patients infected with HCV genotype 1 with regard to antiviral effect and absence of viral resistance compare favorably with those observed with other molecules in development for the treatment of chronic hepatitis C. Acknowledgment: We thank Karen Searle (medical writer), who provided editorial assistance. References 1. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL Jr, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347: Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, 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: Torriani FJ, Rodriguez-Torres M, Rockstroh JK, Lissen E, Gonzalez-Garcia J, Lazzarin A, et al. 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