Antiviral Therapy 2016; 21: (doi: /IMP3062)

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1 Antiviral Therapy 2016; 21: (doi: /IMP3062) Original article Ledipasvir/sofosbuvir-based treatment of patients with chronic genotype-1 HCV infection and cirrhosis: results from two Phase II studies Eric Lawitz 1 *, Fred Poordad 1, Robert H Hyland 2, Jing Wang 2, Lin Liu 2, Hadas Dvory-Sobol 2, Diana M Brainard 2, John G McHutchison 2, Julio A Gutierrez 1 1 Texas Liver Institute and the University of Texas Health Science Center, San Antonio, TX, USA 2 Gilead Sciences, Inc., Foster City, CA, USA *Corresponding author lawitz@txliver.com Background: Ledipasvir/sofosbuvir ± ribavirin administered for 12 weeks to patients with genotype-1 HCV infection and compensated cirrhosis is effective and well-tolerated. The Phase II and studies investigated whether ledipasvir/sofosbuvir plus the non-nucleotide NS5B inhibitor GS-9669 or the NS3/4A protease inhibitor vedroprevir could reduce treatment duration and/or eliminate the need for ribavirin in genotype-1 HCV-infected patients with compensated cirrhosis. Methods: In, 100 cirrhotic patients were randomized (1:1:1) to 8 weeks of ledipasvir/sofosbuvir plus ribavirin, ledipasvir/sofosbuvir plus GS mg or ledipasvir/sofosbuvir plus GS mg. In TRIL- OGY-2, 46 previously treated cirrhotic patients were randomized (1:1) to 8 weeks of ledipasvir/sofosbuvir plus vedroprevir ± ribavirin. The primary end points were the proportion of patients with sustained virological response 12 weeks after treatment discontinuation (SVR12) and safety. Results: In both studies, most patients were male (each 65%) and white (92 96%), infected with HCV genotype- 1a (62 70%) and had IL28B non-cc genotypes (82 87%). In total, 37 39% of patients were Hispanic or Latino. SVR12 rates were similar across treatment arms in TRIL- OGY-1 (82 91%) and (88 95%); no patient had on-treatment virological failure. Two serious adverse events (acute myocardial infarction and cardiomyopathy) were reported in two patients participating in, both of whom had pre-existing cardiac conditions. Laboratory abnormalities were infrequent. Conclusions: All ledipasvir/sofosbuvir-based regimens were well-tolerated. To shorten therapy and eliminate ribavirin, use of a more potent third agent or a third agent with a different mechanism of action may be required. Introduction At least 150 million people worldwide are chronically infected with HCV [1]. Of the six main genotypes of HCV, genotype-1 is the most common, accounting for an estimated 46% of all HCV infections [2]. Chronic HCV infection causes progressive liver fibrosis, which can lead to cirrhosis, hepatic decompensation and hepatocellular carcinoma [3]. At the time that the studies described in this report were initiated, pegylated interferon plus ribavirin in combination with a protease inhibitor (telaprevir or boceprevir) was the standard-of-care regimen for patients with chronic genotype-1 HCV infection. Since then, a number of new agents have been approved, greatly expanding treatment options for patients with HCV. However, a scientific question remains regarding the possibility of an effective and safe short-duration regimen for patients with cirrhosis. Sofosbuvir is a nucleotide analogue inhibitor of HCV NS5B polymerase approved for the treatment of chronic HCV in combination with other antiviral agents. Ledipasvir is an HCV NS5A inhibitor with potent activity against HCV genotype-1. The combination of ledipasvir and sofosbuvir was found to be highly effective in Phase II and III trials in patients with genotype-1 HCV with and without cirrhosis [4 6]. A fixeddose combination tablet of ledipasvir and sofosbuvir has been approved in the United States for patients with chronic genotype-1, -4, -5 or -6 HCV infection [7]. Current treatment guidelines recommend that treatmentnaive patients with chronic genotype-1 HCV infection, 2016 International Medical Press (print) (online) 679

2 E Lawitz et al. regardless of cirrhosis status, receive 12 weeks of ledipasvir/sofosbuvir, while treatment-experienced patients with cirrhosis receive either 12 weeks of ledipasvir/ sofosbuvir plus weight-based ribavirin or 24 weeks of ledipasvir/sofosbuvir only [8,9]. It has been hypothesized that the addition of a third direct-acting antiviral with a mechanism of action complementary to that of ledipasvir/sofosbuvir could further reduce treatment duration in patients with chronic genotype-1 HCV infection and cirrhosis. GS-9669 is a non-nucleoside antiviral that inhibits HCV NS5B polymerase by binding to thumb site II [10,11] and vedroprevir is an inhibitor of HCV NS3/4A protease [11,12]. In a Phase IIa proof-of-concept study, the combination of ledipasvir/sofosbuvir plus GS-9669 or ledipasvir/ sofosbuvir plus vedroprevir was successful in shortening treatment duration to 6 weeks in treatmentnaive patients without cirrhosis (sustained virological response 12 weeks after treatment discontinuation [SVR12] rates, 95% [19/20] in both groups) [11]. The present report describes outcomes from two Phase II studies, which investigated whether ledipasvir/sofosbuvir plus GS-9669 () or ledipasvir/sofosbuvir plus vedroprevir ± ribavirin () could reduce treatment duration to 8 weeks and/or eliminate the need for ribavirin in genotype-1 HCV-infected patients with cirrhosis. Methods Patients To be eligible for or, patients had to be aged 18 years, with a body mass index 18 kg/m 2. Patients were required to have chronic genotype-1 HCV infection documented by either liver biopsy or prior medical history, with HCV RNA levels 10 4 IU/ml at screening. We only enrolled patients with compensated cirrhosis, which was defined as a liver biopsy showing cirrhosis (Metavir score 4 or Ishak score 5), Fibroscan indicating cirrhosis (value >12.5 kpa) or a FibroTest score >0.75 plus an aspartate aminotransferase:platelet ratio index >2 at screening. study participants could have been treatment-naive or failed prior treatment with a pegylated interferon plus ribavirin-containing regimen. All participants in had received 4 weeks of prior treatment with a pegylated interferon plus ribavirin-containing regimen that did not include a protease inhibitor. In both studies, prior use of NS5A or NS5B polymerase inhibitors was prohibited. See Additional file 1 for comprehensive inclusion/exclusion criteria. Study design and treatment was an open-label, Phase II study (NCT ) conducted at the Texas Liver Institute/ American Research Corp, Inc. (San Antonio, TX, USA). Patients were randomized (1:1:1) to receive 8 weeks of once-daily ledipasvir/sofosbuvir (90 mg/400 mg) fixeddose combination tablet plus twice-daily weight-based ribavirin (1,000 or 1,200 mg/day divided), once-daily ledipasvir/sofosbuvir (90 mg/400 mg) plus once-daily GS mg or once-daily ledipasvir/sofosbuvir (90 mg/400 mg) plus once-daily GS mg. Dose modifications were not permitted for ledipasvir/ sofosbuvir and GS If ledipasvir/sofosbuvir was discontinued in patients randomized to ledipasvir/ sofosbuvir plus ribavirin, so was ribavirin. Dose reductions and discontinuation of ribavirin were permitted in accordance with the prescribing information [13]. Randomization was stratified by prior treatment experience (treatment-naive, treatment-experience of <12 weeks duration or treatment-experience of 12 weeks duration) and genotype-1 HCV subtype (1a or 1b). Patients with mixed genotype-1 HCV infection were stratified as genotype-1a. was also an open-label, Phase II study (NCT ) conducted at the Texas Liver Institute/ American Research Corp, Inc. Patients were randomized (1:1) to receive 8 weeks of once-daily ledipasvir/sofosbuvir (90 mg/400 mg) fixed-dose combination tablet plus oncedaily vedroprevir 80 mg either with or without twice-daily weight-based ribavirin (1,000 or 1,200 mg/day divided). Dose modifications were not permitted for ledipasvir/ sofosbuvir or vedroprevir. If ledipasvir/sofosbuvir was discontinued, so was vedroprevir and ribavirin. Dose reductions and discontinuation of ribavirin were permitted in accordance with the prescribing information [13]. Randomization was stratified by genotype-1 HCV subtype (1a or 1b). Patients with mixed genotype-1 HCV infection were stratified as genotype-1a. The protocols for and were approved by the central institutional review board at the Texas Liver Institute. These studies conformed to Good Clinical Practice guidelines and Declaration of Helsinki principles and all patients provided written informed consent. Assessments and end points The assessments performed at each study visit in and are summarized in Assessment schedule for and Assessment schedule for in Additional file 1, respectively. The primary efficacy end point in both studies was the proportion of patients with SVR12, which was defined as HCV RNA levels (COBAS AmpliPrep/ COBAS TaqMan V2.0 assay) below the lower limit of quantitation (LLOQ; <15 IU/ml) 12 weeks after treatment discontinuation. The presence of resistance-associated variants (RAVs) was monitored by deep sequencing of reverse transcription (RT)-PCR International Medical Press

3 LDV/SOF-based treatment in patients with HCV and cirrhosis products (Illumina MiSeq Platform) acquired at baseline and at the time of virological failure (if applicable); the threshold for reporting RAVs was 1%. See Additional file 1 for the definitions of NS5A, NS5B and NS3 RAVs. In, adverse events (AEs) were coded using the Medical Dictionary for Regulatory Activities (MedDRA), Version AEs were coded in using MedDRA, Version Statistics The efficacy and safety analyses in both studies included all randomized patients who received 1 dose of study drug. Exact two-sided 95% CIs were constructed for the primary end point using the Clopper-Pearson method [14]. For, a sample size of 30 patients in each treatment group was calculated to provide a two-sided 95% exact CI that would extend a maximum of 37% in length. For, a sample size of 25 patients in each treatment group was estimated to provide a two-sided 95% exact CI that would extend a maximum of 42% in length. Data are presented using descriptive statistics; no statistical hypothesis testing was performed. Results Patients A total of 101 patients with cirrhosis were randomized to between 8 November 2013 and 6 December However, one patient randomized to ledipasvir/sofosbuvir plus GS mg did not receive study drug. Of the 100 treated patients, 99 (99%) completed study treatment. One treated patient prematurely discontinued ledipasvir/sofosbuvir plus GS mg at week 4 (Figure 1A). Baseline demographic and disease characteristics are summarized in Table 1. The majority of patients were male (65%) and white (92%); 39% of patients were ethnically Hispanic or Latino. The mean age was 57 years (range years). Most patients were infected with genotype-1a HCV (62%), had viral loads 800,000 IU/ml (65%) and had a non-cc IL28B genotype (82%). Overall, 74 (74%) patients were treatmentexperienced, with 11 (11%) having received <12 weeks of prior treatment and 63 (63%) having received 12 weeks of prior treatment. In total, 25 of 74 treatmentexperienced patients were reported to have received a protease inhibitor in combination with pegylated interferon plus ribavirin. The response to prior HCV treatment in these 25 patients was as follows: 13 (52%) had experienced relapse/breakthrough and 12 (48%) were non-responders. It was subsequently discovered that a patient who had previously received treatment with an NS5B polymerase inhibitor was mistakenly enrolled in group 3 of. The other 49 treatmentexperienced patients had received pegylated interferon plus ribavirin only. Of these 49 patients, 18 (37%) had experienced relapse/breakthrough, and 31 (63%) were non-responders. Efficacy In total, 89% (31/35; 95% CI 73, 97%) of patients randomized to ledipasvir/sofosbuvir plus ribavirin achieved SVR12. The corresponding values for patients randomized to ledipasvir/sofosbuvir plus GS mg and ledipasvir/sofosbuvir plus GS mg were 91% (29/32; 95% CI 75, 98%) and 82% (27/33; 95% CI 65, 93%), respectively (Table 2). All 13 patients who did not achieve SVR12 relapsed (that is, no on-treatment breakthrough or non-response). Except for the one patient who prematurely discontinued treatment at week 4, all relapsed patients completed 8 weeks of study treatment. Selected baseline characteristics of the 13 patients who relapsed are presented in Table 3. As shown in Table 2, no relationship between on-treatment viral kinetics and achieving SVR12 was apparent. Although of limited value given the high SVR12 rates, SVR12 rates in clinically relevant patient subgroups in can be found in Additional file 1. Virology A total of 23 study participants had NS5A RAVs present at baseline (ledipasvir/sofosbuvir plus ribavirin, n=5; ledipasvir/sofosbuvir plus GS mg, n=9; ledipasvir/sofosbuvir plus GS mg, n=9). Of these, 18 achieved SVR12 (four of five in the ledipasvir/sofosbuvir plus ribavirin group; eight of nine in the ledipasvir/sofosbuvir plus GS mg group; six of nine in the ledipasvir/sofosbuvir plus GS mg group). The other eight relapsed patients did not have baseline NS5A RAVs. The NS5B RAV S282T was not detected in any patient at baseline and no patient had other nucleoside inhibitor RAVs. One treatment-experienced patient, who was randomized to ledipasvir/sofosbuvir plus GS mg, had a GS-9669 RAV (M423I) present at baseline and achieved SVR12. Baseline NS3 RAVs were detected in 11% (8/71) of protease inhibitornaive patients and 25% (6/24) of protease inhibitorexperienced patients. 2 of these 14 patients relapsed; both patients were treatment-experienced (pegylated interferon plus ribavirin, n=1; pegylated interferon plus ribavirin plus protease inhibitor, n=1). The other 12 patients with NS3 RAVs achieved SVR12. Of the 13 patients who relapsed, 5 had baseline NS5A RAVs (L31M, n=4; Q30R/L31M, n=1) that were also detectable at the time of relapse (Table 3). In three Antiviral Therapy

4 E Lawitz et al. Figure 1. Patient disposition in and A Randomized and received study drug (n=100) 8 weeks of ledipasvir/sofosbuvir + ribavirin (n=35) 8 weeks of ledipasvir/sofosbuvir + GS mg (n=32) 8 weeks of ledipasvir/sofosbuvir + GS mg (n=33) study treatment (n=35) study treatment (n=31) Discontinued study treatment (n=1) study treatment (n=33) assessment (n=35) assessment (n=31) assessment (n=1) assessment (n=33) assessment (n=35) assessment (n=31) assessment (n=1) assessment (n=33) B Randomized (n=47) Randomized and received study drug (n=46) 8 weeks of ledipasvir/sofosbuvir + vedroprevir (n=22) 8 weeks of ledipasvir/sofosbuvir + vedroprevir + ribavirin (n=24) study treatment (n=22) study treatment (n=23) Discontinued study treatment (n=1) assessment (n=22) assessment (n=23) assessment (n=22) assessment (n=23) (A) and (B) International Medical Press

5 LDV/SOF-based treatment in patients with HCV and cirrhosis Table 1. Baseline demographics and disease characteristics of and study participants LDV/SOF + RBV LDV/SOF mg LDV/SOF mg LDV/SOF + VDV LDV/SOF + VDV (n=35) GS-9669 (n=32) GS-9669 (n=33) (n=22) + RBV (n=24) Mean age, years (range) 58 (36 71) 57 (44 75) 57 (32 77) 58 (35-70) 56 (39-69) Male 20 (57) 24 (75) 21 (64) 12 (55) 18 (75) Race White 32 (91) 32 (100) 28 (85) 21 (95) 23 (96) Black 2 (6) 0 4 (12) 1 (5) 1 (4) Native American 1 (3) 0 1 (3) 0 0 Hispanic ethnicity 14 (40) 13 (41) 12 (36) 6 (27) 11 (46) Mean BMI, kg/m 2 (range) 31 (19 48) 29 (19 43) 30 (20 42) 31 (22-52) 31 (25-39) HCV genotype 1a 21 (60) 20 (63) 21 (64) 14 (64) 18 (75) 1b 14 (40) 12 (38) 12 (36) 7 (32) 6 (25) 1a/b (5) 0 Cirrhosis 35 (100) 32 (100) 33 (100) 22 (100) 24 (100) IL28B allele CC 5 (14) 6 (19) 7 (21) 4 (18) 1 (4) CT 16 (46) 23 (72) 19 (58) 12 (55) 16 (67) TT 14 (40) 3 (9) 7 (21) 6 (27) 6 (25) Missing (4) Mean HCV RNA, log 10 IU/ml (sd) 6.0 (0.50) 6.1 (0.63) 6.0 (0.57) 6.1 (0.54) 6.0 (0.51) Mean egfr, ml/min a (range) 121 (56 267) 119 (47 223) 117 (68 216) 107 (49 184) 133 (74 282) HCV treatment-naive 10 (29) 8 (25) 8 (24) 0 (0) 0 (0) HCV treatment-experienced 25 (71) 24 (75) 25 (76) 22 (100) 24 (100) PI + PEG + RBV 10 (29) 8 (25) 7 (21) b 0 0 Relapse/breakthrough 5 (50) 6 (75) 2 (29) 0 0 Non-responder 5 (50) 2 (25) 5 (71) 0 0 PEG + RBV 15 (43) 16 (50) 18 (55) 22 (100) 24 (100) Relapse/breakthrough 6 (40) 5 (31) 7 (39) 5 (23) 5 (21) Non-responder 9 (60) 11 (69) 11 (61) 12 (55) 11 (46) Not applicable c (23) 8 (33) Data are n (%) unless otherwise stated. a Per the Cockcroft Gault equation [15]. b One patient who had previously received an NS5B polymerase inhibitor was mistakenly enrolled. This patient s results are assessed in the assigned group. c Not applicable refers to patients who had no on-treatment measurements available. ALT, alanine aminotransferase; BMI, body mass index; egfr, estimated glomerular filtration rate; LDV/SOF, ledipasvir/sofosbuvir; PEG, pegylated interferon; PI, protease inhibitor; RBV, ribavirin; ULN, upper limit of normal; VDV, vedroprevir. Table 2. Response during and after treatment in and LDV/SOF + RBV LDV/SOF mg LDV/SOF mg LDV/SOF + VDV LDV/SOF + VDV (n=35) GS-9669 (n=32) GS-9669 (n=33) (n=22) + RBV (n=24) HCV RNA <15 IU/ml On-treatment week 4 33 (94) 26 (81) 30 (91) 19 (86) 22 (92) SVR4 32 (91) 29 (91) 28 (85) 21 (95) 23 (96) SVR12 31 (89) 29 (91) 27 (82) 21 (95) 21 (88) 95% CI 73, 97 75, 98 65, 93 77, , 97 Virological failure Relapse 4 (11) 3 (9) 6 (18) 1 (5) 2 (8) Other (4) a Data are n (%) unless otherwise stated. a Patient did not achieve SVR12 (HCV RNA <15 IU/ml) or meet virological failure criteria. LDV/SOF, ledipasvir/sofosbuvir; RBV, ribavirin; SVR4, sustained virological response 4 weeks after treatment discontinuation; SVR12, sustained virological response 12 weeks after treatment discontinuation; VDV, vedroprevir. Antiviral Therapy

6 E Lawitz et al. Table 3. Selected baseline characteristics of patients who relapsed IL28B HCV RNA, HCV RAVs Treatment arm Gender Race genotype log 10 IU/ml genotype Prior HCV treatment experience Baseline Post-baseline LDV/SOF + RBV Male White TT 5.8 1a Treatment-naive None None Male White CT 6.2 1a 12 wks of PEG-IFN + RBV None None Male White CT 6.2 1a 12 wks of PEG-IFN + RBV + PI NS5A: L31M NS5A: L31M Female White TT 5.6 1a <12 wks of PEG-IFN + RBV + PI None NS5A: Q30R LDV/SOF + GS-9669 Male White CT 6.6 1a Treatment-naive None None 250 mg Male White CT 6.0 1b Treatment-naive NS5A: L31M NS5A: L31M Male White TT 6.1 1b 12 wks of PEG-IFN + RBV + PI None None LDV/SOF + GS-9669 Male Black CT 6.0 1b <12 wks of PEG-IFN + RBV NS5A: L31M NS5A: L31M 500 mg Male White TT 6.2 1a 12 wks of PEG-IFN + RBV None None Male White TT 6.1 1a 12 wks of PEG-IFN + RBV None NS5A: H58D Male Black CC 5.8 1a 12 wks of PEG-IFN + RBV NS5A: L31M NS5A: L31M Female Black CT 6.6 1a <12 wks of PEG-IFN + RBV + PI None NS5A: Q30R Male White CT 6.5 1a 12 wks of PEG-IFN + RBV NS5A: Q30R, L31M NS5A: Q30R, L31M LDV/SOF + VDV Male White TT 6.1 1a 4 wks of PEG-IFN + RBV NS5A: Q30R, L31M; NS5A: L31M; NS3: NS3: Q80L Q80L LDV/SOF + VDV Male White CT 5.4 1a 4 wks of PEG-IFN + RBV NS5A: L31M; NS3: NS5A: L31M; NS3: + RBV Q80K Q80K Female White TT 6.4 1a 4 wks of PEG-IFN + RBV None NS5A: Q30R LDV/SOF, ledipasvir/sofosbuvir; PEG-IFN, pegylated interferon; PI, protease inhibitor; RAV, resistance-associated variant; RBV, ribavirin; VDV, vedroprevir; wks, weeks. patients, NS5A RAVs (Q30R or H58D) emerged at relapse. No NS5A RAV was detected at baseline or at the time of virological failure in the other five relapsed patients. No NS5B or other nucleoside inhibitor RAVs, treatment-emergent variants or GS-9669 RAVs were detected in any of the 13 patients with virological relapse. Safety In total, 80% of patients in the ledipasvir/sofosbuvir plus ribavirin, 69% in the ledipasvir/sofosbuvir plus GS mg and 79% in ledipasvir/sofosbuvir plus GS mg treatment arm experienced 1 treatment-emergent AE, most commonly headache, diarrhoea and nausea (Table 4). Two patients experienced serious AEs, both of whom had received 8 weeks of ledipasvir/sofosbuvir plus GS mg. The first, a 61-year-old white male with a history of diabetes, hypertension, tobacco use and coronary artery disease with prior stent placements and bypass grafting, had a serious AE of acute myocardial infarction on day 11 of treatment. The event was considered by the investigator to be unrelated to study treatment. Baseline electrocardiogram demonstrated ST and T wave abnormalities consistent with prior ischaemia. The patient completed treatment with ledipasvir/sofosbuvir plus GS-9669 and achieved SVR12. The second patient, a 59-year-old white male with a history of hypertension and chest pressure with negative cardiac evaluation 2 years prior to screening, had a serious AE of cardiomyopathy on day 18 of treatment; the event was deemed by the investigator to be unrelated to study treatment and likely related to the patient s pre-existing condition. Baseline electrocardiogram demonstrated left bundle branch block and left axis deviation reported as non-clinically significant. One patient discontinued treatment with ledipasvir/sofosbuvir plus GS mg at week 4 due to cardiomyopathy and arrhythmia; neither event was considered related to study medication. No deaths were reported during. Ten patients had laboratory abnormalities (ledipasvir/sofosbuvir plus ribavirin, n=3; ledipasvir/sofosbuvir plus GS mg, n=4; ledipasvir/sofosbuvir plus GS mg, n=3). Hyperglycaemia >250 mg/dl was measured in six patients, all of whom had elevated glucose at screening or baseline. Two patients receiving ledipasvir/sofosbuvir plus ribavirin had haemoglobin levels <10 g/dl. Two patients randomized to ledipasvir/sofosbuvir plus ribavirin had hyperbilirubinaemia >2.5 the upper limit of normal (ULN); these two patients had hyperbilirubinaemia >1.0 ULN at screening or baseline. Elevations in lipase were reported in three patients. Two cases were >3.0 ULN (one in each of the GS-9669 treatment arms) and one case was >5.0 ULN (patient received ledipasvir/sofosbuvir plus GS mg). All three patients had elevations in lipase from screening through week 4. None of the lipase elevations were associated with International Medical Press

7 LDV/SOF-based treatment in patients with HCV and cirrhosis Table 4. Treatment-emergent adverse events in and LDV/SOF + RBV LDV/SOF mg LDV/SOF mg LDV/SOF + VDV LDV/SOF + VDV (n=35) GS-9669 (n=32) GS-9669 (n=33) (n=22) + RBV (n=24) Any AE 28 (80) 22 (69) 26 (79) 14 (64) 13 (54) Serious AE 0 2 (6) a AE leading to permanent discontinuation 0 1 (3) b (8) c of any study drug Death AE occurring in >5% of patients in any treatment group Headache 5 (14) 2 (6) 7 (21) 3 (14) 5 (21) Diarrhoea 4 (11) 5 (16) 3 (9) 0 0 Nausea 3 (7) 2 (6) 7 (21) 0 1 (4) Upper respiratory tract infection 5 (14) 2 (6) 3 (9) 0 0 Rash 3 (9) 1 (3) 1 (3) 2 (9) 3 (13) Sinusitis 0 1 (3) 1 (3) 2 (9) 3 (13) Laboratory abnormalities Lipase >3.0 ULN 0 1 (3) 2 (3) 0 0 Hyperglycaemia >250 mg/dl 1 (3) 3 (9) 2 (6) 1 (5) 3 (13) Haemoglobin <10 g/dl 2 (6) 0 1 (3) 1 (5) 2 (8) Hyperbilirubinaemia >2.5 ULN 2 (6) (8) Lymphocytes <50 cells/mm (5) 0 Platelets <50,000/mm (5) 0 Creatinine <3.00 mg/dl (5) 0 Data are n (%). a Acute myocardial infarction occurred in a patient with pre-existing coronary artery disease, and cardiomyopathy occurred in a patient with preexisting cardiomyopathy. b Patient permanently discontinued treatment with ledipasvir/sofosbuvir (LDV/SOF) on day 29 (week 4) due to cardiomyopathy (serious) and arrhythmia (non-serious). c Ribavirin (RBV) was discontinued on day 19 in a patient with anaemia. Anaemia resolved on day 57. AE, adverse event; ULN, upper limit of normal; VDV, vedroprevir. pancreatitis. No study participant had haematological abnormalities. Patients A total of 47 patients with cirrhosis were randomized to between 7 August 2014 and 19 September One patient was lost to follow-up prior to the baseline visit. Thus, the efficacy and safety analysis populations were composed of 46 patients (ledipasvir/ sofosbuvir plus vedroprevir, n=22; ledipasvir/sofosbuvir plus vedroprevir plus ribavirin, n=24). Of the 46 treated patients, 45 (98%) completed study treatment. One patient prematurely discontinued treatment with ledipasvir/sofosbuvir plus vedroprevir plus ribavirin at week 4 (Figure 1B). As in, the majority of patients were male (65%), white (96%) and not Hispanic or Latino (61%). The mean age was also 57 years (range years). Most patients were infected with genotype-1a HCV (70%), had viral loads 800,000 IU/ml (61%) and had a non-cc IL28B genotype (87%; Table 1). All study participants had been previously treated with pegylated interferon plus ribavirin, with 10 (22%) having experienced relapse/breakthrough and 23 (50%) being non-responsive to prior treatment; treatment response was unknown in the remaining 13 (28%) patients. Efficacy SVR12 rates in the ledipasvir/sofosbuvir plus vedroprevir and ledipasvir/sofosbuvir plus vedroprevir plus ribavirin treatment groups were 95% (21/22; 95% CI 77, 100%) and 88% (21/24; 95% CI 68, 97%), respectively (Table 2). Three of the four patients who did not achieve SVR12 relapsed; all completed 8 weeks of study treatment. The fourth patient completed 29 days of treatment and was subsequently lost to follow-up; at the patient s last treatment visit, HCV RNA was below LLOQ. It is not known whether this patient achieved SVR. Key baseline characteristics of the three patients who relapsed are presented in Table 3. Although of limited value given the high SVR12 rates, SVR12 rates in clinically relevant patient subgroups in can be found in Additional file 1. Virology Three patients had NS5A RAVs present at baseline (ledipasvir/sofosbuvir plus vedroprevir, n=2; ledipasvir/ sofosbuvir plus vedroprevir plus ribavirin, n=1), two of Antiviral Therapy

8 E Lawitz et al. whom had virological relapse (one in each treatment arm). The other relapsed patient did not have baseline NS5A RAVs but had an emergent NS5A RAV (Q30R) at relapse. Two patients had NS5B RAVs present at baseline (one with L159F and one with S282G); both achieved SVR12. A total of 51% (23/45) of patients had NS3 RAVs present at baseline and all but two achieved SVR12. The two patients with baseline NS3 RAVs who experienced virological relapse also had NS5A variants present at baseline (Table 3). Safety In total, 64% of patients in the ledipasvir/sofosbuvir plus vedroprevir and 54% of patients in the ledipasvir/sofosbuvir plus vedroprevir plus ribavirin treatment arm experienced 1 treatment-emergent AE, most commonly headache, rash and sinusitis (Table 4). One patient receiving ledipasvir/sofosbuvir plus vedroprevir plus ribavirin developed a rash, which was attributed to ribavirin; this patient discontinued ribavirin on day 2 of treatment. The rash resolved on day 33 following antihistamine and corticosteroid treatment. Another patient reported anaemia on day 16 of treatment, which led to discontinuation of ribavirin. Both of these patients achieved SVR12. No serious AE was reported. As in, no patient died. Ten patients had laboratory abnormalities (ledipasvir/ sofosbuvir plus vedroprevir, n=3; ledipasvir/sofosbuvir plus vedroprevir plus ribavirin, n=7). Hyperglycaemia >250 mg/dl was measured in four patients, all of whom had a history of diabetes. Three patients receiving ribavirin experienced decreases in haemoglobin <9.0 g/dl. Haemoglobin values in these three patients had begun to return toward baseline values by week 4. No patient had post-baseline haemoglobin values <8.5 g/dl, but three had post-baseline values <10 g/dl (ledipasvir/sofosbuvir plus vedroprevir, n=1; ledipasvir/ sofosbuvir plus vedroprevir plus ribavirin, n=2). Hyperbilirubinaemia >2.5 ULN was measured in two ribavirintreated patients; total bilirubin levels in these two patients returned to normal by week 4. No other laboratory abnormality was reported in > one patient. Discussion In both and, patients with genotype-1 HCV infection and cirrhosis achieved high SVR12 rates following 8 weeks of treatment with a ledipasvir/sofosbuvir-containing regimen. However, coadministration of ledipasvir/sofosbuvir and GS-9669 (either dose) in did not result in higher rates of SVR12 than those seen in patients receiving ledipasvir/sofosbuvir plus ribavirin, with observed SVR12 rates of 82 91% and 89%, respectively. In TRIL- OGY-2, SVR12 rates in patients administered ledipasvir/ sofosbuvir plus vedroprevir were numerically similar with (88%) and without concomitant ribavirin (95%). Notably, the SVR12 rates observed in the and studies of patients with compensated cirrhosis compare favourably with those reported in a Phase IIa proof-of-concept study that examined 6 weeks of combination treatment with ledipasvir/sofosbuvir plus either GS-9669 or vedroprevir in treatment-naive, non-cirrhotic patients with genotype-1 HCV infection; in the Phase IIa proof-of-concept study, 95% of patients in each treatment arm achieved SVR12 [11]. No patient in or experienced on-treatment virological failure. This is consistent with the low rate of viral breakthrough observed in the Phase III ION-1 and ION-2 studies of patients with genotype-1 HCV infection administered ledipasvir/sofosbuvir. One patient each in ION-1 and ION-2 had on-treatment virological breakthrough; however, pharmacokinetic analyses indicated non-adherence to treatment [4,5]. A total of 13 patients in and three patients in TRIL- OGY-2 experienced virological relapse. Among those patients who experienced virological relapse in and, NS5A RAVs were detected in 44% (7/16) of patients at baseline and 69% (11/16) of patients at the time of virological failure. NS5A RAVs emerged in three patients in (Q30R or H58D) and one patient in (Q30R). Although the presence of NS5A RAVs at baseline did not preclude patients from achieving SVR12, SVR12 rates were lower in patients who had NS5A RAVs (73%, 19/23) present at baseline compared with those who did not (92%, 110/119). Importantly, ledipasvir/sofosbuvir-based treatment was not associated with the emergence of NS5B or NS3 RAVs in any relapsed or study participant. All of the regimens studied in and TRIL- OGY-2 were well-tolerated, with no deaths and few treatment discontinuations due to AEs, serious AEs or laboratory abnormalities. The serious AEs reported in occurred in patients with related pre-existing conditions, with one serious AE leading to treatment discontinuation. Two patients discontinued treatment in due to an AE; both AEs (anaemia and rash) were considered related to ribavirin. All of the laboratory abnormalities detected in occurred in patients with either pre-existing conditions or abnormal readings at screening and/or baseline. No patient had haematological abnormalities in. However, in, decreases in haemoglobin to <9.0 g/dl and hyperbilirubinaemia >2.5 ULN occurred in three and two patients, respectively; these laboratory abnormalities were consistent with the expected safety profile of ribavirin [13]. Overall, the safety profile observed in and is comparable to that reported in the Phase IIa proof-of-concept study, and International Medical Press

9 LDV/SOF-based treatment in patients with HCV and cirrhosis the most frequent treatment-emergent AE, headache, is consistent with the prescribing information for ledipasvir/sofosbuvir [7]. In conclusion, results from the Phase II and studies demonstrated that 8 weeks of treatment with a three-drug regimen that includes ledipasvir/sofosbuvir is effective and well-tolerated in patients with genotype-1 HCV infection and cirrhosis. To shorten therapy further, eliminate the need for ribavirin or achieve higher SVR12 rates, combination therapy with sofosbuvir and next generation NS5A and NS3 inhibitors with improved potency and resistance profiles may be required. Although a shortened treatment duration may allow for increased numbers of patients to be treated, the potential advantage of a three-drug regimen must be balanced with the safety of the additional agent. Acknowledgements The and studies were funded by Gilead Sciences, Inc. Third-party writing assistance was provided by Tiffany DeSimone of BlueMomentum, Lyndhurst, NJ, USA, an Ashfield Company, part of UDG Healthcare plc, and was supported and paid for by Gilead Sciences, Inc. These data were presented in part at the American Association for the Study of Liver Diseases in Boston, MA, USA on 7 11 November 2014 (Abstract 1948) and also in San Francisco, CA, USA on November 2015 (Abstract 247). Disclosure statement EL has received research/grant support from Abbott, Achillion, Anadys, Biolex, Boehringer Ingelheim, Bristol Myers Squibb, Gilead Sciences, Inc., GlaxoSmithKline, GlobeImmune, Idenix, Idera, Inhibitex, Intercept, Janssen, Medarex, Medtronic, Merck & Co., Novartis, Pharmasset, Roche, Schering Plough, Santaris, Scynexis, Vertex, ViroChem Pharma and ZymoGenetics; lecture fees from Gilead Sciences, Inc., Merck & Co. and Vertex; and consulting fees from Abbott, Achillion, Anadys, Biolex, Biotica, GlobeImmune, Inhibitex, Merck & Co., Pharmasset, Santaris, Tibotec and Theravance. FP has received research support and grants from AbbVie, Achillion, Boehringer Ingelheim, Bristol Myers Squibb, Gilead Sciences, Inc., GlaxoSmithKline, Idenix, Intercept, Janssen, Medtronic, Merck & Co., Novartis, Presidio, Roche, Santaris and Vertex; has served as a consultant or adviser to Gilead Sciences, Inc., Bristol Myers Squibb, AbbVie, Salix, Valeant, Merck & Co., Janssen, Novartis, Achillion and Intercept; and has served on a speakers bureau for Gilead Sciences, Inc., Bristol Myers Squibb, AbbVie, Salix, Valeant, Merck & Co., Janssen, Novartis, Achillion and Intercept. RHH, JW, LL, HD-S, DMB and JGM are salaried employees of and own stock in Gilead Sciences, Inc. JAG has served on speakers bureaus for Gilead Sciences, Inc., Merck & Co., Bristol Myers Squibb, AbbVie, Intercept and Synageva; has served as a consultant for AbbVie and Gilead Sciences, Inc.; and has received research support from AbbVie, Synageva, Bristol Myers Squibb, Gilead Sciences, Inc., Intercept, Janssen and Merck & Co. Additional file Additional file 1: Supplementary data can be found at Lawitz_Addfile1.pdf References Accepted 20 June 2016; published online 27 June Holmberg SD, Spradling PR, Moorman AC, Denniston MM. Hepatitis C in the United States. N Engl J Med 2013; 368: Shire NJ, Sherman KE. Epidemiology of hepatitis C virus: a battle on new frontiers. Gastroenterol Clin North Am 2015; 44: Messina JP, Humphreys I, Flaxman A, et al. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 2015; 61: Afdhal N, Zeuzem S, Kwo P, et al. Ledipasvir and sofosbuvir for untreated HCV genotype 1 infection. 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