The addition of the recently approved protease

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1 All-Oral Combination of Ledipasvir, Vedroprevir, Tegobuvir, and Ribavirin in Treatment-Na ıve Patients With Genotype 1 HCV Infection David L. Wyles, 1 Maribel Rodriguez-Torres, 2 Eric Lawitz, 3 Mitchell L. Shiffman, 4 Stanislas Pol, 5 Robert W. Herring, 6 Benedetta Massetto, 7 Bittoo Kanwar, 7 James D. Trenkle, 7 Phil S. Pang, 7 Yanni Zhu, 7 Hongmei Mo, 7 Diana M. Brainard, 7 G. Mani Subramanian, 7 John G. McHutchison, 7 François Habersetzer, 8 and Mark S. Sulkowski 9 This phase II trial assessed the efficacy and safety of a combination regimen of the nonstructural protein (NS)5A inhibitor ledipasvir (LDV), NS3 protease inhibitor vedroprevir (VDV), non-nucleoside NS5B inhibitor tegobuvir (TGV), and ribavirin (RBV) in treatment-na ıve patients with chronic hepatitis C virus (HCV) genotype 1 without cirrhosis. Patients were randomized 1:2 to LDV 30 mg once daily (QD; Arm 1; n 5 46) or LDV 90 mg QD (Arm 2; n 5 94); patients in both arms also received VDV 200 mg QD, TGV 30 mg twice-daily, and RBV 1,000-1,200 mg/day. Patients in Arm 2 with vrvr, defined as HCV RNA below the lower limit of quantification (LLOQ) from treatment weeks 2 to 10, were randomized 1:1 to stop treatment at 12 weeks or continue for 24 weeks. Sustained virologic response 12 weeks after treatment (SVR12) was higher in patients receiving 90 mg of LDV for 24 weeks (63%), compared with LDV 90 mg for 12 weeks (54%) and LDV 30 mg for 24 weeks (48%). In patients with very rapid virologic response (vrvr) in Arm 2, SVR12 was achieved by 68% and 81% of patients treated for 12 and 24 weeks, respectively. Virologic breakthrough was more common in patients with HCV genotype 1a and was associated with resistance-associated variants for all three direct-acting antiviral agents (DAAs); however, in all but 1 patient who relapsed, resistance-associated variants directed against only one or two of the DAAs were detected. The most common adverse events were fatigue, headache, nausea, rash, and diarrhea. Conclusion: In patients with HCV genotype 1, an interferon-free regimen containing LDV/VDV/TGV/RBV was well tolerated and led to SVR12 in up to 63% of patients. LDV 90 mg is currently being investigated in combination with the nucleotide polymerase inhibitor, sofosbuvir. (HEPATOLOGY 2014;60:56-64) The addition of the recently approved protease inhibitors, boceprevir or telaprevir, to pegylated interferon (Peg-IFN) and ribavirin (RBV) has led to significant improvement in treatment efficacy for patients chronically infected with hepatitis C virus (HCV), with cure rates of up to 75% in treatmentna ıve patients with HCV genotype 1. 1,2 However, these regimens have several disadvantages, including lower rates of sustained virologic response (SVR) among patients who are poorly responsive to Abbreviations: AEs, adverse events; CI, confidence interval; DAAs, direct-acting antiviral agents; ECG, electrocardiogram; HCV, hepatitis C virus; IFN, interferon; IL, interleukin; LDV, ledipasvir; LLOQ, lower limit of quantification; Peg-IFN, pegylated interferon; NS, nonstructural protein; QD, once-daily; RAVs, resistance-associated variants; RBV, ribavirin; SAE, serious AE; SVR, sustained virologic response; SVR12, sustained virologic response 12 weeks after treatment; SVR24, sustained virologic response 24 weeks after treatment; TGV, tegobuvir; ULN, upper limit of normal; VDV, vedroprevir; vrvr, very rapid virologic response. From the 1 University of California, San Diego, La Jolla, CA; 2 Fundacion de Investigacion de Diego, Santurce, Puerto Rico; 3 Texas Liver Institute, San Antonio, TX; 4 Liver Institute of Virginia, Richmond, VA; 5 H^opital Cochin, Paris, France; 6 Quality Medical Research, PLLC, Nashville, TN; 7 Gilead Sciences, Inc., Foster City, CA; 8 Inserm 1110, Universite de Strasbourg, H^opitaux Universitaires de Strasbourg, Strasbourg, France; and 9 Johns Hopkins University School of Medicine, Baltimore, MD. Received December 12, 2013; accepted January 31, This trial was supported by Gilead Sciences, Inc. 56

2 HEPATOLOGY, Vol. 60, No. 1, 2014 WYLES ET AL. 57 interferon (IFN), regimen complexity, and poor tolerability, which represent a barrier to achieving a cure for many patients. Moreover, no treatment options are currently available for patients with HCV infection who are ineligible for or intolerant of interferon. 3,4 Efforts to optimize outcomes of HCV treatment have focused on combinations of investigational direct-acting antiviral agents (DAAs) with unique mechanisms of action. Studies investigating DAA combinations have shown that SVR can be achieved with shorter treatment durations and in the absence of IFN. 5,6 In this phase II trial, we assessed the safety and efficacy of a combination regimen consisting of ledipasvir (LDV), an inhibitor of the nonstructural (NS)5A protein, vedroprevir (VDV; previously known as GS-9451), a reversible noncovalent inhibitor of the HCV NS3/4A serine protease, tegobuvir (TGV), a non-nucleoside inhibitor of the HCV NS5B polymerase, and RBV. Individually, these agents have demonstrated antiviral activity against genotype 1 HCV. 7-9 In a 3-day monotherapy study, LDV dosed at 90 mg demonstrated significant antiviral activity within the first 12 hours of therapy. At doses of 3 mg or greater, the median maximal reduction in HCV RNA from baseline was >3 log 10 IU/mL in patients infected with HCV genotype 1a; LDV dosed at 30 mg or higher provided >95% of maximal antiviral response. 7 LDV has been shown to be well tolerated in more than 1,000 patients across six phase II studies. 10 VDV dosed at 200 or 400 mg for 3 days suppressed HCV RNA levels by a median of log 10 IU/mL from baseline in treatment-na ıve patients infected with HCV genotype 1a or 1b. 8 In another study, 8 days of TGV treatment resulted in a mean maximal decline in HCV RNA of >1.5 log 10 IU/mL in patients with HCV genotype 1. 9 Resistance mutations detected in these studies did not confer cross-resistance to other classes of DAAs. 7-9 Given the positive results of combining two DAAs with RBV in previous trials, we hypothesized that the addition of a third DAA with a distinct mechanism of action might result in improved response with a shortened treatment duration. Although the results from the 3-day monotherapy study with LDV suggested that the 30-mg dose or higher resulted in robust viral suppression, the optimal dose of LDV given for a longer period has not been determined. 7 Therefore, two doses of LDV were selected for this study. This phase II trial evaluated the antiviral activity and safety of an all-oral regimen consisting of LDV (30 or 90 mg), VDV, TGV, and RBV for 12 or 24 weeks based on early on-treatment response. Patients and Methods Patients. Eligible patients were years of age with chronic HCV genotype 1a or 1b infection. Patients had not been previously treated and did not have cirrhosis based on liver biopsy performed within 2 years of screening or by FibroTest (BioPredictive, Paris, France) or FibroScan (Echosens, Paris, France) within the previous 6 months. Patients were excluded from the study if they were coinfected with human immunodeficiency virus or Address reprint requests to: Mark S. Sulkowski, M.D., Johns Hopkins University School of Medicine, 600 North Wolfe Street, 1830 Building, Room 445, Baltimore, MD msulkowski@jhmi.edu; fax: Copyright VC 2014 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Dr. Herring received grants from Gilead. Dr. Pol consults for and received grants and lecture fees from Bristol-Myers Squibb, Gilead, Roche, and MSD. He consults for and received lecture fees from Boehringer Ingelheim, Tibotec, Vertex, Novartis, Abbott/AbbVie, Sanofi, and GlaxoSmithKline. Dr. Rodriguez-Torres consults for and received grants from Akros, Bristol-Myers Squibb, Genentech, Hoffman-La Roche, Inhibitex, Merck, Pharmasset, Santaris, and Vertex. She consults for Janssen. She received grants from Abbott, Anadys, Beckman, Boehringer Ingelheim, Gilead, GlaxoSmithKline, Human Genome Sciences, Idenix, Idera, Johnson & Johnson, Mochida, Novartis, Pfizer, Scynexis, Siemens, and Zymogenetics. Dr. Pang owns stock in and is employed by Gilead. Dr. McHutchison owns stock in and is employed by Gilead. Dr. Brainard owns stock in and is employed by Gilead. Dr. Trenkle owns stock in and is employed by Gilead. Dr. Subramanian owns stock in and is employed by Gilead. Dr. Massetto owns stock in and is employed by Gilead. Dr. Mo is employed by Gilead. Dr. Shiffman advises, is on the speakers bureau for, and received grants from Gilead, Merck, and Roche/Genentech. He advises, consults for, and is on the speakers bureau for Janssen. He advises and received grants from Achillion, Bristol-Myers Squibb, Boehringer Ingelheim, Globeimmune, and Novartis. He advises and is on the speakers bureau for Bayer, Salix, and Vertex. He advises and consults for Gen-Probe and GlaxoSmithKline. He received grants from AbbVie, Beckman-Colter, Idenix, Intercept, Lumena, and Mochida. Dr. Lawitz advises, is on the speakers bureau for, and received grants from Merck and Vertex. He is on the speakers bureau for and received grants from Gilead and GlaxoSmithKline. He advises and received grants from AbbVie, Achillion, Idenix, Janssen, Novartis, and Santaris. He advises BioCryst, Biotica, Enanta, and Theravance. He is on the speakers bureau for Kadmon. He received grants from Boehringer Ingelheim, Bristol-Myers Squibb, Intercept, Medtronic, Presidio, and Roche. Dr. Wyles consults for and received grants from Gilead, AbbVie, and Bristol-Myers Squibb. He received grants from Vertex and Janssen. Dr. Habersetzer consults for and advises Gilead. He consults for Transgene and Boehringer Ingelheim. Dr. Zhu is employed by and owns stock in Gilead. Dr. Kanwar is employed by and owns stock in Gilead. Dr. Sulkowski consults for and received grants from Gilead, Merck, AbbVie, Bristol-Myers Squibb, Boehringer Ingelheim, Janssen, Vertex, and Idenix. He consults for Pfizer.

3 58 WYLES ET AL. HEPATOLOGY, July 2014 hepatitis B virus, had contraindications to treatment with IFN and/or RBV, decompensated liver disease, severe psychiatric illness, severe chronic obstructive pulmonary disease, or a history of clinically significant cardiac disease or relevant electrocardiogram (ECG) abnormalities during screening. Patients were also excluded if they had any of the following laboratory abnormalities: markedly elevated alanine aminotransferase or aspartate aminotransferase (>10 times the upper limit of normal [ULN]), hemoglobin <12 g/ dl, or direct (conjugated) bilirubin ULN. This study was designed in accord with the International Conference on Harmonization Guidelines, with applicable local regulations and the ethical principles of the Declaration of Helsinki. The study protocol was approved by the institutional review board or independent ethics committee, and written informed consent was obtained from all patients. Study Design. Patients were randomized in a 1:2 ratio to LDV 30 mg once-daily (QD), VDV 200 mg QD, TGV 30 mg twice-daily, and weight-based RBV (Copegus; Hoffman La-Roche, Nutley, NJ) for 24 weeks (Arm 1) or LDV 90 mg QD, VDV 200 mg QD, TGV 30 mg twice-daily, and weight-based RBV for 12 or 24 weeks based on response at week 2 (Arm 2). RBV was administered in a divided total daily oral dose of 1,000 mg for patients weighing <75 kg or 1,200 mg for patients weighing 75 kg. Patients in Arm 2 who achieved a very rapid virologic response (vrvr; defined as HCV RNA <25 IU/mL at treatment week 2) and maintained an HCV RNA level below the lower limit of quantification (LLOQ) through week 10 were rerandomized in a 1:1 ratio to stop treatment at week 12 or continue therapy to week 24. Before randomization and rerandomization, patients were stratified according to plasma HCV RNA viral load ( or >800,000 IU/mL) and HCV genotype (1a or 1b) at screening. Patients who failed to achieve vrvr, had a confirmed virologic breakthrough on treatment (HCV RNA >LLOQ after two consecutive visits in which the HCV RNA viral load was <LLOQ) or demonstrated relapse during the 24 weeks after stopping therapy at week 12 (Arm 2) were given the option to receive LDV (at their original assigned dose), VDV, RBV, and 180 mg of Peg-IFN-a-2a (Pegasys; Hoffman- La Roche) once-weekly by subcutaneous injection for an additional 24 or 48 weeks in a rescue substudy. Study Endpoints. The original primary antiviral efficacy endpoint was the percentage of patients with SVR (defined as plasma HCV RNA <25 IU/mL) 24 weeks after treatment (SVR24). However, after the initiation of this trial, use of SVR 12 weeks after treatment (SVR12) as the primary efficacy endpoint for HCV clinical trials was endorsed by the U.S. Food and Drug Administration. 11 Accordingly, SVR12 is reported as the primary efficacy endpoint. Secondary efficacy assessments included the percentage of patients with virologic response during treatment, virologic breakthrough (defined as a confirmed HCV RNA 25 IU/mL after achieving HCV RNA <25 IU/mL on treatment), and relapse (defined as a confirmed HCV RNA 25 IU/mL during the 24 weeks after treatment after achieving HCV RNA <25 IU/mL at the end of treatment). Safety endpoints included frequency and documentation of adverse events (AEs) that led to discontinuation of study treatment. HCV RNA Measurement. HCV RNA levels were measured with the COBAS TaqMan HCV Test (Roche Diagnostics, Basel, Switzerland) with an LLOQ of 25 IU/mL and a lower limit of detection of 10 IU/mL. Plasma HCV RNA levels were measured at screening, at baseline, during treatment (at weeks 1 and 2, every 2 weeks from week 2 through 12, and every 4 weeks from weeks 12 through 24), and during follow-up (4, 12, and 24 weeks after the end of treatment). Sequencing Analysis. Plasma samples for genotypic monitoring were collected at each visit before the dose was administered on the dosing days. Population sequencing of the HCV NS3/4A protease, NS5B polymerase, and NS5A-encoding region was performed using standard population-sequencing technology (Monogram Biosciences, South San Francisco, CA) for patients who experienced virologic breakthrough during treatment or viral relapse posttreatment. Presence of previously identified VDV resistance-associated variants (RAVs) at the NS3 amino acid positions R155, A156, and D168, LDV RAVs at the NS5A amino acid positions M28, Q30, L31, H58, and Y93 in genotype 1a and L31 and Y93 in genotype 1b, and TGV RAVs at the NS5B amino acid positions C445, Y452, and Y448 were examined. Safety Assessments. Study investigators evaluated safety by documenting AEs, assessing clinical laboratory tests, conducting physical examinations, and measuring vital signs throughout treatment and during the 24-week follow-up period. As part of routine safety monitoring, ECG data including QTcF was collected at baseline, week 1, and week 12. Clinical and laboratory AEs were coded using the Medical Dictionary for Regulatory Activities. Statistical Analyses. Efficacy and safety analyses were performed on the full analysis set, defined as

4 HEPATOLOGY, Vol. 60, No. 1, 2014 WYLES ET AL. 59 Table 1. Patient Demographics and Baseline Disease Characteristics Arm 2 LDV 90 mg/vdv/tgv/rbv for 12 or 24 Weeks (n 5 94) Arm 1 LDV 30 mg/vdv/tgv/rbv for 24 Weeks (n 5 46) 12 Weeks (n 5 33) 24 Weeks (n 5 31) Not Rerandomized (n 5 30) Male, n (%) 25 (54) 17 (52) 16 (52) 22 (73) Race, n (%) White 40 (87) 28 (85) 26 (84) 26 (87) Black 6 (13) 4 (12) 5 (16) 2 (7) Ethnicity (%) Hispanic or Latino 2 (4) 8 (24) 4 (13) 2 (7) Not Hispanic or Latino 44 (96) 25 (76)* 27 (87) 27 (90) Mean age, years (range) 47 (21-67) 47 (20-66) 47 (18-63) 54 (23-66) Mean BMI (range) 26.9 ( ) 27.0 ( ) 27.2 ( ) 25.8 ( ) IL28B CC genotype, n (%) CC 16 (35) 11 (33) 15 (48) 11 (37) CT 21 (46) 14 (42) 10 (32) 17 (57) TT 9 (20) 8 (24) 6 (19) 2 (7) Mean HCV RNA, log 10 IU/mL (range) 6.5 ( ) 6.3 ( ) 6.4 ( ) 6.9 ( ) HCV genotype 1a, n (%) 35 (76) 22 (67) 23 (74) 23 (77) *One patient did not declare ethnicity. Abbreviations: BMI, body mass index; F, Metavir fibrosis score. patients who were randomized into the study and received 1 dose of any study drug. The difference in SVR12 rates between Arm 1 and Arm 2 was tested using the Cochran-Mantel-Haenszel test stratified by randomization stratification factors. The two-sided 95% confidence interval (CI) for the rate difference between Arm 1 and Arm 2 was constructed based on the stratum-adjusted Mantel-Haenszel proportions. Similarly, a one-sided 90% CI (or a two-sided 80% CI) for the difference in SVR12 rates was constructed using the same method to compare rerandomized patients in Arm 2 who received 12 weeks of treatment and patients rerandomized in Arm 2 to continue treatment through week 24. Because differential SVR12 rates were observed between the two rerandomized subarms of Arm 2, comparisons of each subarm to Arm 1 were performed, adjusting for the rerandomization by using an inverse probabilityweighting approach. This approach was used to evenly distribute patients in Arm 2 who were not rerandomized across the two rerandomization subarms. The inverse probability-weighted SVR rates were as follows: [X 1 1 (k/2)] 4 [N 1 1 (m/2)] for patients rerandomized to 12 weeks of therapy and [X 2 1 (k/2)] 4 [N 2 1 (m/ 2)] for patients rerandomized to 24 weeks of therapy. The variable m was defined as the number of patients initially randomized to Arm 2 who terminated the study before rerandomization, and k was defined as the number of responders, X 1 was defined as the number of responders rerandomized to 12 weeks of therapy, and X 2 was defined as the number of responders rerandomized to 24 weeks of therapy. Furthermore, N 1 was defined as the total number of patients rerandomized to 12 weeks of therapy, and N 2 was defined as the total number of patients rerandomized to 24 weeks of therapy. All statistical summaries and analyses were performed using SAS software (SAS Institute Inc, Cary, NC). Point estimates and two-sided 95% exact CIs, based on Clopper-Pearson s method, were also calculated for the proportion of patients with HCV RNA <25 IU/ ml at the end of treatment and at follow-up week 12 (i.e., SVR12 rate) for each treatment arm. The number and percentage of patients who had vrvr, breakthrough, or relapse were calculated by treatment arm and by HCV genotype or interleukin (IL)28B genotype, as appropriate. Results Patient Population. Between June 2011 and November 2012, a total of 140 patients (46 in Arm 1 and 94 in Arm 2) were randomized and treated at 34 sites in the United States, France, and Germany. Patient demographics and baseline disease characteristics are shown in Table 1. Of the randomized and treated patients, 57% were male, 12% were black, 74% were infected with HCV genotype 1a, and 38% carried the IL28B CC genotype (35% in Arm 1 and 39% in Arm 2). Likewise, HCV genotype 1 subtype was balanced between treatment arms: 76% of patients in Arm 1 and 72% of patients in Arm 2 were infected with HCV genotype 1a (Table 1). Patient disposition throughout the study is depicted in Fig. 1. vrvr and SVR12 Rates. A similar percentage of patients treated with the 90-mg dose of LDV achieved

5 60 WYLES ET AL. HEPATOLOGY, July 2014 Fig. 1. Patient disposition. HCV RNA levels <25 IU/mL at week 2 (vrvr), compared with those treated with the 30-mg dose of LDV (79% vs. 72%). Patients who did not achieve vrvr (13 patients in Arm 1 and 20 patients in Arm 2) were offered Peg-IFN-based rescue therapy, as described in Patients and Methods. SVR rates are shown in Table 2. SVR12 rates for the full analysis set were 48% for patients treated with LDV 30 mg in Arm 1 and 59% for patients treated with LDV 90 mg in Arm 2. The difference in SVR12 rates between Arm 1 and Arm 2 was 12.2%, with an associated 95% CI of SVR12 rates were not significantly different across the two treatment arms (P ) according to the Cochran-Mantel- Haenszel test stratified by randomization stratification factors. SVR12 Rates According to Duration of Therapy in Patients Who Achieved vrvr in Arm 2. Of the 74 patients in the LDV 90-mg arm (Arm 2) who achieved vrvr, 64 were rerandomized at week 12 (31 to stop treatment immediately and 33 to continue treatment for a total of 24 weeks). Two patients who were rerandomized to continue treatment for 24 weeks terminated the study at week 12 and were summarized with the 31 patients who were rerandomized to stop treatment at week 12. Therefore, a total of 33 patients were analyzed in the 12-week subarm and a total of 31 patients were analyzed in the 24-week subarm. Ten patients with Table 2. End-of-Treatment and Posttreatment Response Arm 1 LDV 30 mg/ VDV/TGV/RBV for 24 Weeks (n 5 46) Arm 2 LDV 90 mg/vdv/tgv/rbv for 12 or 24 Weeks (n 5 94) 12 Weeks (n 5 48) 24 Weeks (n 5 46) Overall (n 5 94) vrvr (%) 33 (72) N/A N/A 74 (79) EOT (%) 25 (54) 33 (69) 30 (65) 63 (67) EOT (in patients with vrvr; %) 24/33 (73) 33/38 (87) 30/36 (83) 63/74 (85) SVR4 (%) 24 (52) 32 (67) 30 (65) 62 (66) SVR12 (%) 22 (48) 26 (54) 29 (63) 55 (59) SVR12 (patients with vrvr; %) 22/33 (67) 26/38 (68) 29/36 (81) 55/74 (74) Relapse* (%) 2/23 (9) 7/33 (21) 0 7/64 (11) *Among patients who completed treatment. Abbreviations: EOT, end of treatment; N/A, not applicable.

6 HEPATOLOGY, Vol. 60, No. 1, 2014 WYLES ET AL. 61 Table 3. vrvr and SRV12 by IL28B Status and Genotype Arm 1 LDV 30 mg/vdv/tgv/rbv for 24 Weeks Arm 2 LDV 90 mg/vdv/tgv/rbv for 12 or 24 Weeks Genotype 1a Genotype 1b Genotype 1a Genotype 1b Response CC (n 5 14) CT/TT (n 5 21) CC (n 5 2) CT/TT (n 5 9) CC (n 5 30) CT/TT (n 5 38) CC (n 5 7) CT/TT (n 5 19) vrvr (%) 13 (93) 11 (52) 2 (100) 7 (78) 23 (77) 32 (84) 5 (71) 14 (74) SVR12 (%) 9 (64) 6 (29) 2 (100) 5 (56) 19 (63) 20 (53) 4 (57) 13 (68) vrvr were not eligible for rerandomization because of virologic breakthrough (n 5 8) or early termination (n 5 2; 1 patient terminated the study early because of an AE and the other withdrew consent). SVR12 rates for patients in Arm 2 who achieved vrvr and were treated for 12 or 24 weeks were 68% and 81%, respectively. By contrast, patients in Arm 1 who achieved vrvr (all of whom received 30 mg of LDV and were treated for 24 weeks) had an SVR12 rate of 67%. Influence of Host and Viral Genotypes on Virologic Response. Table 3 shows response by HCV subtype (1a vs. 1b) and IL28B genotype (CC vs. non-cc). Across both arms, patients with HCV genotype 1b and the IL28B CC genotype had higher rates of SVR12 (68% and 64%, respectively) than those with HCV genotype 1a and IL28B non-cc genotype (52% and 51%, respectively). Among patients with non-cc genotype, 30 mg of LDV yielded lower rates of SVR12 (29% in HCV genotype 1a and 56% in HCV genotype 1b) than did 90 mg of LDV, which provided SVR12 rates of 53% in HCV genotype 1a and 74% in HCV genotype 1b. In patients with HCV genotype 1b, vrvr was predictive of SVR12(allbut4patientswhohadvRVRachieved SVR12), whereas of the 79 patients with HCV genotype 1a who achieved vrvr, only 54 (68%) achieved SVR12. Table 4. Resistance-Associated Variants in Patients With Virologic Failure Arm 1 LDV 30 mg 1 VDV 1 TGV 1 RBV (n 5 46) Arm 2 LDV 90 mg 1 VDV 1 TGV 1 RBV (n 5 94) Patients with breakthrough 9 (20) 10 (11) NS3/4A 7/7 (100) 10/10 (100) NS5A 9/9 (100) 10/10 (100) NS5B 7/9 (78) 9/10 (90) Patients with relapse 3 (7) 9*(10) NS3/4A 2/3 (67) 3/7 (43) NS5A 3/3 (100) 9/9 (100) NS5B 0 2/8 (25) Data are n (%) or number of patients with RAVs/number of patients with data available (%). *Includes 2 patients who did not complete treatment. Virologic Breakthrough, Relapse, and Resistance Characterization. Incidence of virologic breakthrough was nearly twice as high in the LDV 30-mg arm as in the LDV 90-mg arm: 20% vs. 11%. Nine patients in the LDV 30-mg arm had virologic breakthrough (8 HCV genotype 1a and 1 HCV genotype 1b; Table 4). NS5A and NS5B sequences were successfully generated for all 9 patients, whereas the NS3/4A sequence was obtained for only 7 of 9 patients because of assay failure for 2 patient samples. Of the 7 patients with NS3/4A, NS5A, and NS5B sequence data available, 5 had RAVs within the NS3 protease (VDV RAVs), NS5A (LDV RAVs), and NS5B (TGV RAV) and 2 had RAVs within the NS3 (VDV RAVs) and NS5A (LDV RAVs) genes. The remaining 2 patients with NS5A and NS5B sequence data available had RAVs in both NS5A (LDV RAVs) and NS5B (TGV RAVs) genes. Similarly, 10 patients (all HCV genotype 1a) in the LDV 90-mg arm had virologic breakthrough during treatment (9 patients before week 8 and 1 after week 16). All 10 patients were tested for resistance mutations: Nine of ten had RAVs in NS3 protease (VDV RAV), NS5A (LDV RAVs), and NS5B (TGV RAV) genes, with the remaining patient having RAVs in NS5A (LDV RAVs) and NS3 (VDV RAVs) genes. In total, 14 of 17 (82%) patients with genotype 1a who had virologic breakthrough displayed drug resistance to all three DAAs based on population sequencing after therapy. Relapse was more common in patients receiving 12 weeks of therapy in the LDV 90-mg arm. A total of 7 patients (21%) relapsed during follow-up. Five of seven patients had sequence data available for NS3/4A, NS5A, and NS5B. Of these 5 patients, 3 had only single-daa RAVs (all LDV RAVs), 2 had dual-daa RAVs, and none had triple-daa RAVs. Among the 2 patients with dual-daa RAVs, 1 had mutations in NS3 (VDV RAV) and NS5A (LDV RAV) coding regions and the other had mutations in NS5A (LDV RAV) and NS5B (TGV RAV) genes. With a longer duration of therapy in the LDV 90-mg arm, no patients experienced relapse during the first 12 weeks of follow-up. Comparatively, patients who completed 24 weeks of treatment with LDV 30 mg had a relapse

7 62 WYLES ET AL. HEPATOLOGY, July 2014 Table 5. Safety Summary Arm 1 LDV 30 mg 1 VDV 1 TGV 1 RBV (n 5 46) Arm 2 LDV 90 mg 1 VDV 1 TGV 1 RBV (n 5 94) Treatment-emergent AEs, n (%) SAE 1 (2) 0 Grade 3-4 AE 3 (7) 2 (2) Discontinuation because of AE 1 (2) 2 (2) Most common AEs occurring in 10% of patients, n (%) Fatigue 16 (35) 17 (18) Headache 9 (20) 20 (21) Nausea 8 (17) 13 (14) Rash 6 (13) 10 (11) Anemia 6 (13) 6 (6) Diarrhea 5 (11) 14 (15) Pruritus 5 (11) 11 (12) Grade 3-4 laboratory abnormalities, n (%) WBC <1,500 cells/mm Platelets <100,000 cells/mm Hemoglobin <9 g/dl 4 (9) 8 (9) Total bilirubin ULN 8 (17) 4 (4) Abbreviation: WBC, white blood count. rate of 9%; 2 patients relapsed before week 12 of follow-up. Among these 2 patients, 1 had dual-daa RAVs (VDV and LDV RAVs) and the other had a single-daa LDV RAV. Safety. The most common AEs occurring in 10% of patients are shown in Table 5. Fatigue, headache, and nausea were the most common treatmentemergent AEs. Most AEs were mild in severity. One patient in the LDV 30-mg arm experienced a serious AE (SAE) of pancreatitis that required hospitalization. Three patients discontinued treatment (1 in the LDV 30-mg arm and 2 in the LDV 90-mg arm). Reasons for permanent discontinuation of study treatment included eye complications (iritis/vitritis), dyspepsia, irritability, muscle atrophy, alcohol poisoning, and acute psychosis. Grade 3-4 treatment-emergent AEs occurred in 3 (7%) and 2 (2%) patients in the LDV 30-mg and 90-mg arms, respectively. Twelve patients had elevations (2.5 3 ULN) in total bilirubin: 8 (17%) in the LDV 30-mg arm and 4 (4%) in the LDV 90-mg arm. Rescue Therapy Substudy. Patients who did not achieve vrvr, had virologic breakthrough or who relapsed during the 24 weeks after stopping therapy at week 12 (Arm 2 only) were offered treatment in a rescue substudy of 24 weeks of therapy with LDV/VDV plus Peg-IFN/RBV, with an additional 24 weeks of Peg-IFN/RBV for patients who did not achieve HCV RNA <LLOQ by week 4 of rescue treatment. A total of 50 patients (20 from Arm 1 and 30 from Arm 2) were enrolled into the rescue substudy, with the majority qualifying for rescue therapy by not attaining a vrvr (30 of 50). Of the remainder, 17 of 50 subjects qualified for rescue because of on-treatment virologic failure, and 4 of 50 because of relapse. One subject qualified for retreatment because of both failure to achieve vrvr and virologic breakthrough. Among the 50 subjects who entered the rescue therapy substudy, 37 (74%) completed treatment. Among the 13 subjects who discontinued rescue therapy, 7 were because of AEs, 3 because of virologic failure, and 3 because of investigator decision. SVR rates at posttreatment weeks 4 and 12 (SVR4 and SVR12, respectively) were 65% (13 of 20) and 40% (8 of 20) for subjects who entered rescue from Arm 1. For subjects who entered rescue from Arm 2, SVR4 was 70% (21 of 30) and SVR12 was 60% (18 of 30). Incidence of treatment-emergent AEs was similar in both arms (95% and 93 % for Arm 1 and Arm 2, respectively), with 20% and 23% of patients in Arm 1 and Arm 2, respectively, experiencing Grade 3-4 AEs. Most AEs were associated with Peg-IFN/ RBV therapy. Discussion In this phase II study, all-oral, IFN-free treatment with the NS5A inhibitor, LDV, NS3 protease inhibitor, VDV, non-nucleoside NS5B inhibitor, TGV, and RBV was effective against HCV genotype 1 infection in previously untreated patients without cirrhosis. The highest SVR12 rate of 63% was attained after 24 weeks of treatment with a regimen containing LDV dosed at 90 mg. The majority of patients treated in the 90-mg LDV arm had HCV RNA <25 IU/mL at treatment week 2 (vrvr) and were eligible for rerandomization to 12 or 24 weeks of therapy. Although 12 weeks of treatment with this regimen resulted in a majority of patients with vrvr attaining SVR12 (68%), rates increased to 81% when treatment was extended to 24 weeks. Host IL28B and viral genotypes did not greatly affect viral responses in patients treated with the more potent 90-mg LDV regimen. By contrast, in patients treated with the lower 30-mg LDV regimen, responses were more variable and appeared to be influenced by host and viral genotypes. Thus, this study suggests that in the absence of IFN, the effect of host and viral genotype on virologic response can be attenuated as long as the DAA-based regimen is sufficiently potent. Virologic breakthrough was common in patients who received the lower LDV dose of 30 mg (20%). Increasing the LDV dose to 90 mg reduced the incidence of virologic breakthrough by approximately half, providing greater efficacy, especially in patients with CT or TT

8 HEPATOLOGY, Vol. 60, No. 1, 2014 WYLES ET AL. 63 IL28B genotypes. Relapse was more common in patients who were treated for 12 weeks in the LDV 90- mg arm (21%), but was reduced with a longer duration of therapy (24 weeks). Patients treated with 30 mg LDV for 24 weeks also had a low rate of relapse (4%). Thus, these findings suggest that potency was the key factor in preventing virologic breakthrough, whereas treatment duration was the key factor for relapse risk. Most cases of breakthrough and relapse occurred in patients with HCV genotype 1a infection. Resistance mutations to all three targeted proteins (NS3, NS5A, and NS5B) were found in almost all patients who experienced virologic breakthrough. However, patients who experienced viral relapse had only single-daa LDV RAVs or dual-daa RAVs (LDV RAV together with VDV RAV or TGV RAV). Long-term follow-up of some of these patients is ongoing to determine the persistence of these RAVs. In vitro cross-resistance analysis demonstrated that these RAVs did not confer crossresistance to the NS5B polymerase inhibitor, sofosbuvir, or the NS5B polymerase inhibitor, GS The combination regimen of LDV/VDV/TGV/ RBV was well tolerated: Only 1 patient experienced an SAE. The safety profile observed with 12 weeks of treatment was similar to that of 24 weeks of treatment. Mild transient elevations in total bilirubin levels occurred as a result of interaction with hepatocyte bile transporters by the HCV protease inhibitor, VDV; these resolved after drug discontinuation. 12 Overall, the safety profile of this DAA combination regimen was favorable. Rescue therapy with a quadruple regimen, including Peg-IFN and RBV, was moderately successful (SVR12 of 40%-60%), but at the expense of dramatically increased treatment duration and toxicity. Selected resistant variants to LDV and VDV may have limited the effectiveness of rescue therapy, leading to SVR rates similar to those observed with Peg-IFN/RBV alone. Given the rapid progress on attaining highly efficacious, well-tolerated IFN-free HCV treatment regimens, direct rollover to an IFN-containing rescue therapy may not be the best approach for patients failing an initial IFNfree regimen, though more studies are needed. The results of this phase II study provide further evidence that HCV infection can be cured without Peg- IFN in a subset of patients. 5,6 The first trial to confirm achievement of SVR with an IFN-free regimen was a small study in which 11 previous null responders receiving the NS5A inhibitor, daclatasvir, and the NS3 protease inhibitor, asunaprevir, achieved an SVR rate of 36%. 5 Six patients had virologic breakthrough during therapy with the combination; all were infected with HCV genotype 1a, and resistance mutations to both targeted proteins were found in all cases. In a phase I trial employing a more potent three-drug regimen containing two DAAs (BI , an NS3 protease inhibitor, and BI , an NS5B polymerase inhibitor) and RBV, rapid and high rates of on-treatment virologic response (82%-100%) in both HCV genotype 1a and genotype 1b infected patients were achieved. 6 Likewise, in a small phase IIa exploratory study, ABT-450 (an HCV NS3 protease inhibitor) boosted with low-dose ritonavir, in addition to ABT-333 (a non-nucleoside NS5B polymerase inhibitor) and RBV, led to SVR12 rates of 93% to 95% in previously untreated patients with HCV genotype 1 infection. 13 Thus, as potency of the IFN-free regimen increased, so did virologic response. Treatment of HCV is a rapidly evolving field, and several investigational agents are in the latter stages of clinical development. More than 80% of the currently HCV-infected population is not being treated with the available IFN-based regimens because of intolerance or an unwillingness to receive IFN. 14 This study, along with smaller proof-of-concept studies and ongoing trials, demonstrate that all-oral, IFN-free regimens are effective across HCV genotypes, offer the potential of shortened treatment durations, and are easy to administer and well tolerated. Given the continued development and clinical progress of IFN-free regimens, they will most likely become part of the HCV treatment paradigm in the very near future. As demonstrated in this trial and others, potency of the DAA regimen plays an important role in preventing virologic breakthrough and attenuating the effect of baseline factors (viral and host genotypes) on treatment outcomes. References 1. Poordad F, McCone J, Jr., Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med 2011;364: Jacobson IM, McHutchison JG, Dusheiko G, Di Bisceglie AM, Reddy KR, Bzowej NH, et al. Telaprevir for previously untreated chronic hepatitis C virus infection. N Engl J Med 2011;364: Heim MH. 25 years of interferon-based treatment of chronic hepatitis C: an epoch coming to an end. Nat Rev Immunol 2013;13: Chen EY, Sclair SN, Czul F, Apica B, Dubin P, Martin P, et al. A small percentage of patients with hepatitis C receive triple therapy with boceprevir or telaprevir. Clin Gastroenterol Hepatol 2013;11: Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib R, et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N Engl J Med 2012;366: Zeuzem S, Asselah T, Angus P, Zarski JP, Larrey D, Mullhaupt B, et al. Efficacy of the protease inhibitor BI , polymerase inhibitor BI , and ribavirin in patients with chronic HCV infection. Gastroenterology 2011;141: Lawitz EJ, Gruener D, Hill JM, Marbury T, Moorehead L, Mathias A, et al. A phase 1, randomized, placebo-controlled, 3-day, dose-ranging

9 64 WYLES ET AL. HEPATOLOGY, July 2014 study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C. J Hepatol 2012;57: Lawitz E, Hill JM, Marbury TC, Rodriguez-Torres M, DeMicco MP, Quesada J, et al. Three-day, dose-ranging study of the HCV NS3 protease inhibitor GS-9451 [Abstract]. HEPATOLOGY 2010;52(Suppl):714A- 715A. 9. Harris J, Bae A, Sun S, Svarovskaia E, Miller M, Mo H. Antiviral response and resistance analysis of treatment-na ıve HCV infected subjects receiving single and multiple doses of GS-9190 [Abstract]. HEPA- TOLOGY 2010;52(Suppl):772A. 10. Everson GT, Lawitz E, Thompson A, Sulkowski M, Zhu Y, Brainard D, et al. The NS5A inhibitor GS-5885 is safe and well tolerated in more than 1000 patients treated in phase 2 studies [Abstract]. HEPATO- LOGY 2012;56(Suppl):572A. 11. Chen J, Florian J, Carter W, Fleischer RD, Hammerstrom TS, Jadhav PR, et al. Earlier sustained virologic response end points for regulatory approval and dose selection of hepatitis C therapies. Gastroenterology 2013;144: Tong L, Mwangi J, Roy A, Murray B, Delaney W, Yang C, et al. In vitro studies on the potential for the hepatitis c virus protease inhibitors GS-9256 and GS-9451 to affect bilirubin elimination [Abstract]. J Hepatol 2011;54(Suppl):S Poordad F, Lawitz E, Kowdley KV, Cohen DE, Podsadecki T, Siggelkow S, et al. Exploratory study of oral combination antiviral therapy for hepatitis C. N Engl J Med 2013;368: Butt AA, McGinnis KA, Skanderson M, Justice AC. Hepatitis C treatment completion rates in routine clinical care. Liver Int 2010;30: Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher s website.