The protease inhibitor, GS-9256, and non-nucleoside polymerase inhibitor tegobuvir alone, with ribavirin, or pegylated interferon plus ribavirin in hepatitis C


  • Potential conflict of interest: The authors disclose the following financial relationships. S.Z. has been a clinical investigator and/or consultant for Abbott Laboratories, Achillion Pharmaceuticals, Anadys Pharmaceuticals, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, iTherX, Merck & Co., Novartis Pharmaceuticals, Pharmasset, Roche/Genentech, Santaris Pharma A/S, Tibotec Pharmaceuticals, Transgene, and Vertex Pharmaceuticals. P.B. has been a consultant and invited speaker for Janssen Pharmaceuticals, Roche, Schering-Plough Corporation, Merck & Co., Gilead Sciences, and Novartis Pharmaceuticals. K.A. has received grant support from Gilead Sciences, Roche, and Astellas Pharma and has been a consultant and speaker for Gilead Sciences, Merck Sharp & Dohme, Janssen Pharmaceuticals, Abbott Pharmaceuticals, GlaxoSmithKline, Roche, Astellas Pharma, Novartis Pharmaceuticals, and Bristol-Myers Squibb. P.M. has received grant support, served as a speaker, and/or participated as an investigator for Roche, Schering Plough, Gilead, Bristol-Myers Squibb, Vertex Pharmaceuticals, Novartis Pharmaceuticals, Pharmasset, Tibotec Pharmaceuticals, MSD, Boehringer Ingelheim, Abbott Laboratories, Pfizer, and Echosens. D.S. has been a clinical investigator for Gilead Sciences. H.K. has been a consultant for and/or received honoraria for speaking engagements from Abbott Laboratories, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck Sharp & Dohme, Roche, and Tibotec Pharmaceuticals. C.M. has been a consultant for Janssen Pharmaceuticals, adviser for Janssen Pharmaceuticals and Merck Sharp & Dohme, speaker for Bristol-Myers Squibb, Janssen Pharmaceuticals, and Merck Sharp & Dohme, and received research grants from Roche and Merck Sharp & Dohme. J.-P.Z. has been a consultant and speaker for Merck & Co., Roche, Janssen Pharmaceuticals, Bristol-Myers Squibb, and Gilead Sciences. Y.H. has served as a consultant for Gilead Sciences, Roche, Merck & Co., Ipsen, Helsinn, and Johnson & Johnson Pharmaceuticals, a clinical investigator for Gilead Sciences, Novartis, Merck & Co., Tibotec Pharmaceuticals, Boehringer Ingelheim, Roche, and GlaxoSmithKline, and received grants from GlaxoSmithKline, Roche, Merck & Co., and AstraZeneca. M.P.M, has received grant support, consulting fees or honoraria, and support for travel from Merck & Co., consultant fees from Roche, Bristol-Myers Squibb, Gilead Sciences, Boehringer-Ingelheim, Novartis Pharmaceuticals, Tibotec Pharmaceuticals, Vertex Pharmaceuticals, GlaxoSmithKline, and Merck & Co., grants from Roche, Gilead Sciences, Novartis Pharmaceuticals, Boehringer-Ingelheim, Bristol-Myers Squibb, and Merck & Co., and payment for development of educational presentations from Merck & Co., Roche, Bristol-Myers Squibb, GlaxoSmithKline, and Gilead Sciences. G.F. has received personal and institutional funding from Gilead Sciences, Roche, Bristol-Myers Squibb, Merck & Co., Chugai Pharma, and GlaxoSmithKline. H.M., S.A., S.K., D.O., and J.G.M. are current or former employees of Gilead Sciences.

  • This trial was supported by Gilead Sciences.


Tegobuvir (GS-9190), a non-nucleoside nonstructural protein (NS)5B polymerase inhibitor, and GS-9256, an NS3 serine protease inhibitor, individually have activity against hepatitis C virus (HCV) genotype 1. The antiviral activity of tegobuvir and GS-9256 as oral combination therapy, or together with ribavirin (RBV) or pegylated interferon (Peg-IFN) alpha-2a and RBV, was assessed in a phase II, randomized, open-label trial. Treatment-naïve patients with genotype 1 HCV were assigned 28 days of tegobuvir 40 mg twice-daily (BID) and GS-9256 75 mg BID (n = 16), tegobuvir and GS-9256 plus RBV 1,000-1,200 mg daily (n = 15), or tegobuvir and GS-9256 plus Peg-IFN alpha-2a (180 μg once-weekly)/RBV (n = 15). The primary efficacy endpoint was rapid virologic response (RVR), with HCV RNA <25 IU/mL at day 28. After 28 days, all patients received Peg-IFN/RBV. All patients with viral rebound or nonresponse, defined as >0.5-log10 increase in HCV RNA from nadir or <2-log decrease at day 5, initiated Peg-IFN/RBV immediately. Median maximal reductions in HCV RNA were −4.1 log10 IU/mL for tegobuvir/GS-9256, −5.1 log10 IU/mL for tegobuvir/GS-9256/RBV, and −5.7 log10 IU/mL for tegobuvir/9256/Peg-IFN/RBV. RVR was observed in 7% (1 of 15) of patients receiving tegobuvir/GS-9256, 38% (5 of 13) receiving tegobuvir/GS-9256/RBV, and 100% (14 of 14) receiving tegobuvir/9256/PEG-IFN/RBV. The addition of Peg-IFN/RBV at day 28 or earlier resulted in HCV RNA <25 IU/mL at week 24 in 67% (10 of 15), 100% (13 of 13), and 94% (13 of 14) of patients in the three treatment groups. Transient elevations in serum bilirubin occurred in all treatment groups. Conclusion: In genotype 1 HCV, adding RBV or RBV with Peg-IFN provides additive antiviral activity to combination therapy with tegobuvir and GS-9256. (HEPATOLOGY 2012)

For the past decade, the standard of care for patients with chronic infection with genotype 1 hepatitis C virus (HCV) has been 48 weeks of pegylated interferon (Peg-IFN) alpha and ribavirin (RBV). Observed rates of sustained virologic response (SVR) with Peg-IFN and RBV therapy are 40%-52%. 1-4 However, the addition of the HCV nonstructural protein (NS)3 serine protease inhibitors, telaprevir or boceprevir, results in higher rates of SVR (67%-75%), leading to the recent approval of these two drugs in the United States and the European Union. 5-10 Because triple therapy can result in higher rates of rapid virologic response (RVR; HCV RNA < lower limit of quantification at week 4) in the range of 60%-70%, 5, 6, 9, 10 shortened treatment duration, from 48 to 24 weeks, is possible in a significant proportion of patients.

Several novel inhibitors of viral replication, including those targeting NS3 serine protease and NS5B RNA-dependent RNA polymerase, are in clinical development. 11 Although many of these direct-acting antiviral agents (DAAs) can cause rapid, substantial reductions in viral load (VL), their use as monotherapies has been limited by inadequate suppression of replication and/or the development of resistance. 12, 13 In the context of polymerase- or protease-inhibitor therapy, Peg-IFN and RBV have repeatedly demonstrated their importance in reducing VL and suppressing viral breakthrough. 14-16 In studies of regimens containing telaprevir or boceprevir, excluding RBV or using a reduced dose results in higher rates of viral breakthrough and relapse. 5, 7, 17

Several recent studies have explored the combining of two DAAs to enhance early antiviral activity and to theoretically minimize the development of resistance. In a study of treatment-naïve patients with HCV genotype 1, 14 days of combination therapy with the nucleoside analog, RG7128, and the NS3 protease inhibitor, danoprevir, resulted in 5-log10 IU/mL HCV RNA reductions from baseline. 18 More recently, the combination of the non-nucleoside NS5B polymerase inhibitor, VX-222, with telaprevir improved early antiviral response, but was associated with high rates of viral breakthrough. 19

Tegobuvir (GS-9190) is a novel, non-nucleoside inhibitor of NS5B polymerase. Studies to elucidate tegobuvir's mechanism of action are ongoing; however, current data indicate that the inhibitory effect may be exerted via an interaction with the β-hairpin in the NS5B thumb subdomain. 20 Tegobuvir and the NS3 protease inhibitor, GS-9256, each have demonstrated antiviral activity in HCV-infected patients. 21-23 Tegobuvir demonstrated median reductions in HCV RNA of 1.5 log10 IU/mL for individual patients with 8 days of monotherapy 21 and enhanced rates of RVR (HCV RNA <25 IU/mL at week 4), when combined with Peg-IFN and RBV. 22 At 200 mg twice-daily (BID) for 3 days, GS-9256 monotherapy demonstrated a median HCV RNA reduction of 2.7 log10 IU/mL. 22 Both tegobuvir and GS-9256 were well tolerated in these short-term monotherapy studies. We, therefore, evaluated the antiviral activity of tegobuvir and GS-9256 dual therapy, tegobuvir and GS-9256 plus RBV, and tegobuvir and GS-9256 plus Peg-IFN and RBV for 28 days. After 28 days of treatment, patients then continued treatment with Peg-IFN and RBV for 48 weeks.


ALT, alanine aminotransferase; AST, aspartate aminotransferase; BID, twice-daily; BMI, body mass index; DAA, direct-acting antiviral agent; ECG, electrocardiogram; HCV, hepatitis C virus; IL, interleukin; NS, nonstructural protein; Peg-IFN, pegylated interferon; QW, once weekly; RBV, ribavirin; RT-PCR, reverse-transcriptase polymerase chain reaction; RVR, rapid virologic response; SNP, single-nucleotide polymorphism; SVR, sustained virologic response; VL, viral load.

Patients and Methods


Eligible patients were adults 18-70 years of age with chronic HCV infection who had not been previously treated. Patients had HCV genotype 1 infection and absence of cirrhosis, as judged by liver biopsy within 2 years before 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 had any of the following conditions or characteristics: elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), or gamma-glutamyl transferase levels to >5 times the upper limit of normal; autoimmune diseases; decompensated liver disease; cirrhosis; severe psychiatric illness; severe chronic obstructive pulmonary disease; coinfection with human immunodeficiency virus or hepatitis B virus; or history of clinically significant cardiac disease or relevant electrocardiogram (ECG) abnormalities during screening.

Interleukin (IL)28B genotyping was retrospectively performed at the Duke Center for Human Genome Variation, utilizing a custom Taqman assay (Applied Biosystems, Foster City, CA) for the rs12979860 IL28B single-nucleotide polymorphism (SNP). 24

All patients provided written informed consent before undertaking any study-related procedures.

Study Design.

This was a phase II, randomized, open-label trial of tegobuvir plus GS-9256, both administered orally in combination for 28 days, or both in combination with RBV, or both in combination with Peg-IFN alfa-2a and RBV. Patients were randomly assigned (1:1) to 40 mg of tegobuvir taken BID plus 75 mg of GS-9256 BID or 40 mg of tegobuvir BID plus 75 mg of GS-9256 BID plus RBV for 28 days. RBV (Copegus; Roche, Nutley, NJ) was administered in a divided total daily oral dose of 1,000-1,200 mg (1,000 mg for patients weighing <75 kg and 1,200 mg for patients weighing ≥75 kg). Randomization was stratified by plasma HCV RNA level (< or ≥ 2,000,000 IU/mL) at screening (15 blocks of size 2). The sponsor's biometrics group generated the randomization schedule by using SAS software (SAS Institute Inc., Cary, NC). In assigning patients to treatment in this open-label study, individual study sites sent randomization worksheets to the sponsor, who assigned subject numbers on the basis of the randomization schedule.

After the first two treatment arms were completed, the protocol was amended and a third arm was added to the study to evaluate antiviral response with quadruple therapy. Patients received 40 mg of tegobuvir BID plus 75 mg of GS-9256 BID in combination with Peg-IFN alpha-2a (Pegasys; Roche) and RBV for 28 days. Subcutaneous injections of 180 μg of Peg-IFN alpha-2a were given once-weekly (QW).

After the first 28 days of treatment, all patients received continued treatment with Peg-IFN and RBV and were asked to return for follow-up visits 12, 24, 48, and 72 weeks after the last study drug dose. The selection of the specific Peg-IFN (alpha-2a or alpha-2b) and RBV products and regimen during this phase of the study was at the discretion of the investigator. Peg-IFN and RBV standard of care was initiated earlier than 28 days in the following circumstances: lack of early response, as defined by a <2-log10 IU/mL HCV RNA reduction from baseline by day 5, or rebound, as defined by an HCV RNA increase of >0.5 log10 IU/mL from nadir confirmed over two time points occurring after day 5 with an absolute value >1,000 IU/mL.

The study protocol (EudraCT identifier: 2009-013690-18) was approved by each institution's independent ethics committee before study initiation.

Efficacy Assessments.


The primary efficacy endpoint was VL suppression at day 28, as measured by the proportion of patients achieving RVR, defined as plasma HCV RNA <25 IU/mL at day 28. Plasma HCV RNA reduction from baseline was also evaluated. Plasma for HCV RNA measurements was collected at screening, on day 1 predose (baseline), and on days 3, 5, 7, 14, 21, and 28. HCV RNA levels were measured with the Roche COBAS TaqMan reverse-transcriptase polymerase chain reaction (RT-PCR) assay, with lower limit of quantification at 25 IU/mL.

Viral resistance

The complete NS3/4A and NS5B genes from plasma samples were PCR amplified, and population sequencing was performed from samples with HCV RNA ≥1,000 IU/mL by Virco BVA (Beerse, Belgium). The detection limit with this assay for detecting a drug-resistant variant was approximately 25%. Viral sequence analysis was performed for baseline (day 1 predose) and day 28 samples and in the events of viral plateau or rebound. Because results from the baseline (day 1) sample were not available at patient enrollment, HCV genotyping for study eligibility was performed in parallel, according to Versant INNO-LiPA HCV 2.0 (Innogenetics, Gent, Belgium).

Safety Assessments.

Safety was evaluated on the basis of adverse events, vital signs, ECG findings, and laboratory abnormalities. Concomitant medication intake was also recorded. Prohibited medications included atypical antipsychotic agents, systemic chemotherapeutics, immunosuppressants, immunomodulators, H2-receptor antagonists, agents potentially causing QT prolongation, and alternative medicines (e.g., St. John's wort and milk thistle).

Endpoints and Statistical Analyses.

A sample size of 15 patients per treatment arm was determined on the basis of experience with other proof-of-concept studies; no formal power or sample-size calculations were planned or undertaken.

The full efficacy analysis set included patients who had HCV genotype 1a or 1b, as evaluated by NS5B sequencing/phylogenetic analysis, not Versant INNO-LiPA HCV 2.0 (Innogenetics) alone. The primary endpoint was the proportion of patients achieving an RVR. Patients who added or switched to standard of care early were counted as failures and were characterized as censored patients. The analysis set for safety included all patients who received at least 1 dose of study drug.

All statistical summaries and analyses were performed using SAS software (SAS Institute).


Patient Population.

Between February and October 2010, a total of 46 patients were randomized and treated in four European countries (Belgium, France, Germany, and United Kingdom). Among the treatment arms, patients were predominately male (73%-88%) and white (80%-93%), and mean age ranged from 45 to 54 years (Table 1). Of the 46 patients treated, 45 patients completed week 6 of the study (Table 2), and 42 were still on Peg-IFN/RBV at week 24. Treatment with Peg-IFN/RBV is ongoing at the time of this report. As evaluated at baseline with the LiPA 2.0 assay, 15 (33%) patients were HCV genotype 1a, 30 (65%) were genotype 1b, and 1 (2%) was unable to be genotyped. Upon subsequent NS5B sequencing/phylogenetic analysis, 4 patients were identified as having HCV genotypes 1e, 1l, 1e/m, and 4r (refer to Supporting Table for virologic outcomes). These patients were, therefore, excluded from the primary efficacy analysis.

Table 1. Patient Demographic and Baseline Disease Characteristics
 Tegobuvir/GS-9256 (n = 16)Tegobuvir/GS-9256/RBV (n = 15)Tegobuvir/GS-9256/Peg-IFN/RBV (n = 15)
  • Abbreviation: SD, standard deviation.

  • *

    Four patients were later identified by NS5B sequencing as having HCV genotypes 1e, 1l, 1e/m, and 4r.

Age, mean (SD), years48 (10.0)45 (12.7)54 (9.0)
Male sex (%)14 (87.5)11 (73.3)12 (80.0)
Race (%)   
 White13 (81.3)12 (80.0)14 (93.3)
 Black2 (12.5)2 (13.3)1 (6.7)
 Asian1 (6.3)00
 Other01 (6.7)0
Body mass index (kg/m2)   
 Mean (SD)27.0 (3.13)26.3 (4.58)25.6 (3.42)
Baseline HCV RNA   
 Mean (SD) log10 IU/mL6.18 (0.685)6.29 (0.440)6.50 (0.779)
 Median (Q1, Q3) log10 IU/mL6.17 (5.57, 6.71)6.34 (5.96, 6.50)6.68 (6.45, 6.88)
 ≥800,000 IU/mL, n (%)10 (62.5)13 (86.7)13 (86.7)
Baseline HCV genotype (LipA 2.0)* (%)   
 1a8 (50.0)3 (20.0)4 (26.7)
 1b8 (50.0)12 (80.0)10 (66.7)
 Unable to genotype001 (6.7)
IL28B genotype (%)   
 CC2 (12.5)6 (40.0)4 (26.7)
 CT10 (62.5)8 (53.3)9 (60.0)
 TT4 (25.0)1 (6.7)2 (13.3)
ALT IU/mL   
 Mean (SD)88.4 (51.13)66.7 (25.11)73.1 (38.28)
Metavir fibrosis score, n (%)   
 F00 (0)2 (13.3)0 (0)
 F19 (56.3)6 (40)7 (46.7)
 F23 (18.8)5 (33.3)6 (40)
 F34 (25)2 (13.3)2 (13.3)
Table 2. Patient Disposition Throughout the Study
 Randomized (n = 46)
Treated (n = 46)
Tegobuvir/GS-9256 (n = 16)Tegobuvir/GS-9256/RBV (n = 15)Tegobuvir/GS-9256/ Peg-IFN/RBV (n = 15)
Day 28
 Completed assigned treatment3915
 Completed assigned treatment with overlapping  Peg-IFN/RBV or Peg-IFN940
 Discontinued tegobuvir and GS-9256 early420
Week 6
 Completed Peg-IFN/RBV151515
 Discontinued Peg-IFN/RBV100
Week 12
 Completed Peg-IFN/RBV151515
 Discontinued Peg-IFN/RBV100
Week 24
 Completed Peg-IFN/RBV131514
 Discontinued Peg-IFN/RBV301

The majority of patients were genotype CT (ranging from 53% to 63%) at the IL28B polymorphism, rs12979860. A higher percentage of patients were IL28B genotype CC in the tegobuvir/GS-9256/RBV arm (40%) versus the tegobuvir/GS-9256 arm (12.5%) or tegobuvir/GS-9256/Peg-IFN/RBV arm (26.7%).

Efficacy Assessments.


Patients in all treatment arms had an initial sharp decline in plasma HCV RNA levels during the first 48 hours of therapy (Fig. 1). In the tegobuvir/GS-9256 arm, this decrease was generally maintained through day 7, after which HCV RNA levels began to rebound, associated with the emergence (i.e., detection) of resistance-associated variants. The addition of RBV to the treatment regimen increased the magnitude, extent, and duration of viral reduction; in the tegobuvir/GS-9256/RBV arm, reductions in HCV RNA levels were observed through day 14 and were generally maintained through day 28. The addition of Peg-IFN alpha-2a had a similar additive effect; in the tegobuvir/GS-9256/Peg-IFN/RBV arm, reductions in HCV RNA levels were observed through day 28. The association of IL28B genotype and initial antiviral response was variable, with a trend toward a greater magnitude of HCV RNA reductions in IL28B CC patients. No differences in mean maximal HCV RNA reduction by HCV subtype (i.e., 1a or 1b) were observed. Virologic responses in the 4 patients infected with other HCV-1 subtypes are presented in the Supporting Table. In each case, HCV RNA reductions from baseline during randomized therapy ranged from −0.75 to −2.84 log10 IU/mL. After the switch to Peg-IFN/RBV, continued VL reductions were observed, ranging from −2.98 to −5.23 log10 IU/mL from baseline by week 6.

Figure 1.

HCV RNA levels in individual patients with confirmed genotype 1a or 1b HCV through day 28. (A) Tegobuvir 40 mg BID and GS-9256 75 mg BID. (B) Tegobuvir 40 mg BID and GS-9256 75 mg BID plus RBV. (C) Tegobuvir 40 mg BID and GS-9256 75 mg BID plus Peg-IFN and RBV. Antiviral activity was expressed as the log10 change from baseline in HCV RNA. Plots ending before 28 days indicate the early addition of Peg-IFN alpha-2a or RBV therapy or early cessation of GS-9256 and tegobuvir. Color indicates IL28B genotype: red is CC, black is CT, and blue is TT. Dotted line indicates HCV RNA 25 IU/mL.

In the primary efficacy analysis, a greater percentage of patients achieved RVR after receiving tegobuvir/GS-9256 in combination with RBV (38%), compared with tegobuvir/GS-9256 alone (7%) (Table 3). All patients (14 of 14) receiving tegobuvir/GS-9256 in combination with Peg-IFN/RBV achieved RVR.

Table 3. Virologic Response Among Patients With HCV Genotype 1a or 1b
 Tegobuvir/ GS-9256 (n = 15)Tegobuvir/GS-9256/ RBV (n = 13)Tegobuvir/GS-9256/ Peg-IFN/RBV (n = 14)
  • *

    Includes patients who initiated standard of care with Peg-IFN/RBV before 28 days.

  • One patient experienced breakthrough after week 12.

 Day 14   
   <25 (%)1/15 (7)6/13 (46)10/14 (71)
 Day 28 (RVR)   
   <25 (%)1/15 (7)5/13 (38)14/14 (100)
   <25 (uncensored population*) (%)3/15 (20)8/13 (62)14/14 (100)
 Week 12   
   <25 (%)12/15 (80)13/13 (100)14/14 (100)
 Week 24 (%)   
   <2510/15 (67)13/13 (100)13/14 (94)
Median (Q1, Q3) maximal HCV RNA change from baseline, log10 IU/mL−4.1 (−4.4, −2.9)−5.1 (−5.3, −4.4)−5.7 (−5.9, −5.5)

Excluding data points after the early introduction of Peg-IFN/RBV, the median (i.e., Q1 and Q3) maximal reduction in HCV RNA was highest for patients receiving tegobuvir/GS-9256/Peg-IFN/RBV, −5.7 (−5.9, −5.5) log10 IU/mL, versus −5.1 (−5.3, −4.4) for tegobuvir/GS-9256/RBV, and −4.1 (−4.4, −2.9) for tegobuvir/GS-9256 alone.

Viral breakthrough was most common in the tegobuvir/GS-9256 arm, where the majority of patients (80%) started standard of care with Peg-IFN and RBV before day 28. Although RBV decreased and delayed breakthrough, in the tegobuvir/GS-9256/RBV arm, 31% started standard of care early because of the observed increases in HCV RNA at or before day 28. None of the patients receiving tegobuvir/GS-9256/Peg-IFN/RBV experienced viral plateau or rebound through day 28. For patients in the tegobuvir/GS-9256 arm who had an increase in HCV RNA levels observed at days 14 or 21, HCV RNA levels declined again by day 28 after initiating Peg-IFN and RBV.

Among the patients who either did not experience early response or had viral rebound, several achieved RVR after starting either Peg-IFN or Peg-IFN and RBV early. Two patients in the tegobuvir/GS-9256 arm who started Peg-IFN and RBV early achieved RVR, as did 3 patients in the tegobuvir/GS-9256/RBV arm who started Peg-IFN early (Table 3).

Viral suppression continued through 24 weeks for many patients, especially those initially assigned to therapy with RBV (arm 2) or Peg-IFN/RBV (arm 3). All patients (13 of 13) receiving tegobuvir/GS-9256/RBV initially and continuing on Peg-IFN/RBV had HCV RNA <25 IU/mL at week 24; 13 of 14 (94%) patients assigned to tegobuvir/GS-9256/Peg-IFN/RBV and continuing on Peg-IFN/RBV maintained HCV RNA <25 IU/mL at week 24.

Resistance Mutants.

Population sequence analysis was performed in 15 rebound patients whose HCV RNA was ≥1,000 IU/mL at the time of rebound. In 14 of 15 of these patients, mutations were detected in both the NS3 and NS5B genes (Table 4), and the mutations are known to cause lowered antiviral susceptibility to GS-9256 and tegobuvir in vitro. The remaining patient had only the NS3 R155K mutation detected. The dual-therapy arm with tegobuvir/GS-9256 had the highest rate of detected mutations. In HCV genotype 1a patients, NS3 R155K and NS5B Y448H were the most common mutations selected; in HCV genotype 1b patients, NS3 D168E/V and NS5B Y448H were the most common. In 4 of 5 patients with HCV genotype 1b with either NS5B C316N or C445F at baseline, viral rebound was associated with the emergence of NS3 D168E/V/H/L mutations without the selection of additional NS5B mutations.

Table 4. Resistance Mutation Detection
HCV genotype1a (n = 8)1b (n = 7)1a (n = 3)1b(n = 10)
  • *

    NS3 mutations: R155K/Q and D168H/L/E/V.

  • NS5B mutations: Y448H, C316N, and C/F445F.

  • C316N (baseline): C316N mutation in NS5B that occurred at baseline and during treatment.

Mutations in NS3*+NS5B    
 D168E/V+Y448H 2  
 D168H/L/V+C/F445F 1  
 R155K/Q, D168E/D+ Y448H, C316N (BL)   1
 D168E/V+ C316N (BL) 2 1
 Total, n (%)8 (100)5 (71)02 (20)

Safety Assessments.

Tegobuvir/GS-9256 was well tolerated, and most adverse events were mild to moderate in severity. Adverse events were more common in the tegobuvir/GS-9256/Peg-IFN/RBV treatment arm, with events consistent with those reported for IFNs (Table 5). Two serious adverse events were reported during the study: infective bursitis and vasovagal collapse. Both were considered by the investigator to be unrelated to study drug. One patient, in the tegobuvir/GS-9256 arm, discontinued tegobuvir and GS-9256 on day 22 because of fatigue. This patient had initiated Peg-IFN and RBV on day 19, but continued with Peg-IFN/RBV after discontinuing tegobuvir and GS-9256. The patient completed study participation to week 6, but was later lost to follow-up.

Table 5. Most Common Adverse Events in 28 Days of Therapy*
 Part APart B
 Tegobuvir/GS-9256 (n = 16)Tegobuvir/GS-9256/ RBV (n = 15)Tegobuvir/GS-9256/ Peg-IFN/RBV (n = 16)Tegobuvir/GS-9256/ Peg-IFN/RBV (n = 15)
  • *

    Events occurred in at least 2 patients per treatment regimen.

  • Patients who initiated Peg-IFN and RBV before 28 days of assigned therapy.

Number of patients experiencing any treatment-emergent adverse event (%)8 (50)14 (93)13 (81)15 (100)
Blood and lymphatic system disorders (%)    
 Anemia0002 (13)
Eye disorders (%)    
 Eye pain0002 (13)
Gastrointestinal disorders (%)    
 Diarrhea3 (19)3 (20)1 (6)6 (40)
 Nausea2 (13)3 (20)1 (6)6 (40)
 Abdominal pain01 (7)02 (13)
 Vomiting0002 (13)
General disorders (%)    
 Influenza-like illness007 (44)12 (80)
 Fatigue1 (6)5 (33)2 (13)5 (33)
 Asthenia02 (13)2 (13)2 (13)
 Chills01 (7)2 (13)1 (7)
 Irritability002 (13)0
Metabolism and nutrition disorders (%)    
 Anorexia0002 (13)
 Decreased appetite01 (7)02 (13)
Musculoskeletal and connective tissue disorders (%)    
 Myalgia01 (7)6 (38)3 (20)
Nervous system disorders (%)    
 Headache5 (31)7 (47)2 (13)6 (40)
 Disturbance in attention1 (6)2 (13)1 (6)0
Psychiatric disorders (%)    
 Insomnia03 (20)1 (6)2 (13)
 Sleep disorder0002 (13)
Respiratory, thoracic, and mediastinal disorders (%)    
 Dyspnea01 (7)2 (13)2 (13)
 Cough0003 (20)
Skin and subcutaneous tissue disorders (%)    
 Dry skin02 (13)00
 Pruritus1 (6)3 (20)01 (7)
 Erythema1 (6)002 (13)

No grade 4 adverse events or lab abnormalities were observed. Reductions in hemoglobin and neutrophils were consistent with those associated with RBV and Peg-IFN alpha-2a administration. Transient bilirubin elevations, primarily grades 1 and 2, occurred in all treatment groups, but were generally indirect and not associated with elevations in ALT or AST. Overall, while taking assigned therapy, 9 patients experienced grade 1 elevations in total bilirubin, 4 had grade 2 elevations, and 2 had grade 3 elevations (maximum, 3.2 mg/dL). Overall incidence of hyperbilirubinemia (grade 1 and above) in treated patients was 4 of 16 (25%), 5 of 15 (33%), and 6 of 15 (40%) in the tegobuvir/GS-9256, tegobuvir/GS-9256/RBV, and tegobuvir/GS-9256/Peg-IFN/RBV arms, respectively.

No clinically significant effect on cardiac repolarization (i.e., prolongation of the QTcF interval >60-msec change from baseline or increase to >500 msec) was observed for the tegobuvir/GS-9256 combination after multiple dosing.


This study of tegobuvir plus GS-9256 is the first to explore the additional contribution of RBV to a two-drug oral DAA regimen during a limited 4-week dosing period. The two oral DAAs exhibited additive antiviral activity: Tegobuvir 40 mg BID monotherapy induces median HCV RNA reductions of 1.5 log10, 21 whereas GS-9256 monotherapy induces median HCV RNA reductions of 2.7 log10, 22 and in this study, the combination of the two drugs resulted in median HCV RNA reductions of 4.1 log10. The additive antiviral effect we observed is consistent with the additive interaction of tegobuvir and GS-9256 in the replicon system (Gilead Sciences, unpublished data). Even with the additive antiviral activity of these two classes of HCV inhibitors, viral breakthrough was common, especially in patients with genotype 1a HCV infection. The addition of RBV enhanced antiviral activity, delayed the emergence/selection of resistance, and resulted in a greater proportion of patients achieving an RVR. Adding Peg-IFN plus RBV to the two antiviral agents further enhanced viral suppression, with 100% of patients reaching RVR. In the majority of patients, treatment with Peg-IFN plus RBV after 28 days maintained HCV RNA suppression to <25 IU/mL up to week 24. Virologic response data beyond week 24 are awaited. Four patients with non-1 HCV genotype were treated in the study. Virologic responses in these patients were suboptimal. Three patients discontinued randomized treatment and initiated Peg-IFN/RBV. The 4th patient, assigned to tegobuvir/GS-9256/RBV/Peg-IFN, remained on assigned therapy for 28 days per protocol. Virologic response rates observed in these patients are consistent with the specificity of tegobuvir and GS-9256 for HCV genotypes 1a and 1b.

A small imbalance in the proportion of IL28B-CC patients was observed across groups (Fig. 1). The small sample size limited interpretation; however, it is possible that the apparent effect of RBV in reducing VL and suppressing resistance could be partially related to a relatively high proportion of IL28B CC patients in the tegobuvir/GS-9256/RBV arm.

Most adverse events occurring in the tegobuvir/GS-9256 arm were mild to moderate in severity. Although the number of adverse events was highest in the tegobuvir/GS-9256/Peg-IFN/RBV treatment arm, these events were consistent with those associated with IFNs. Transient bilirubin elevations were also observed, consistent with the known class effects of NS3 serine protease inhibitors on bilirubin transporters, such as organic anion transporting polypeptide 1B1, with resulting increase in unconjugated bilirubin. 25, 26

The emergence of resistance-associated variants with non-nucleoside NS5B or NS3 inhibitors has been described in other studies and is consistent with the lower genetic barrier against resistance for non-nucleoside analogs and NS3 protease inhibitors. 13 The high rate of emergence of the protease resistant variant, R155K, in genotype 1a–infected, but not in genotype 1b–infected, patients has also been described previously with this class of agent and is reflective of single-nucleotide change required for the development of resistance in genotype 1a patients, but two-nucleotide changes in the majority of genotype 1b patients. 27 It is of note that single-nucleotide change is required for both mutations at NS3 R155 and D168 in genotype 1a patients; however, a mutation at only R155, and not D168, was identified in genotype 1a patients by population sequencing. The R155 nucleotide sequence may be more susceptible to change than D168, or the R155K may be more fit than mutations at D168 in this genotype. Mutations at D168 were commonly selected in genotype 1b–infected patients, consistent with genotype 1b replicon data.

The Y448H mutation observed with tegobuvir has been observed frequently in monotherapy studies and is consistent with in vitro mutational data, indicating the tegobuvir interaction likely involves the β-hairpin in the thumb subdomain of the NS5B polymerase. 20 In the present study, 7 of 8 genotype 1a patients developed dual-class resistance: R155K against the NS3 protease inhibitor and Y448H for the NS5B polymerase inhibitor. However, with the addition of RBV, the incidence of resistance was significantly reduced, with none of genotype 1a patients (n = 3) exhibiting drug-resistant variants. Though RBV has been shown to have modest antiviral activity, 28 its ability to significantly reduce the development of resistance highlights a distinct mechanism of action. This may indicate a broader mutational effect of RBV on viral fitness, which renders a proportion of virus noninfectious, regardless of oral antiviral-resistance mutations. Although similar trials have been reported on, 29 the present study is the first report of an IFN-free NS5B polymerase/NS3 protease combination both with and without RBV, thus allowing for a prospective evaluation of the contribution of RBV to the antiviral effect of the regimen.

The emergence of various classes of DAAs for treating chronic HCV infection has enabled an evaluation of multiple combination approaches either with or without Peg-IFN and RBV. 19, 30, 31 Specifically, the strategy of quadruple therapy with a non-nucleoside analog, a protease inhibitor, and Peg-IFN and RBV has been supported by results from a recently reported study, in which the non-nucleoside NS5B polymerase inhibitor, VX-222, telaprevir, and peg-IFN/RBV resulted in RVR in 51 of 59 (86%) of treatment-naïve patients, 19 which is higher than those reported with telaprevir and Peg-IFN/RBV. 6, 9 In this study, 100% of patients receiving quadruple therapy achieved RVR at week 4, and a high proportion of patients (71%) had HCV RNA below 25 IU/mL at week 2. The rapidity of viral clearance in patients with quadruple therapy provides a basis for examining response-guided therapy, in which total duration of treatment could be fewer than 24 weeks. To explore this possibility, phase II combination studies of tegobuvir plus GS-9256 with Peg-IFN and RBV are under way.


The authors thank the patients for their participation in this study. The authors are also grateful to Caroline Lascoux-Combe, M.D., Hospital Saint-Louis (Paris, France) for her participation as an investigator. Alex McKenzie and Kevin V. Shianna, Ph.D., of the Duke Center for Human Genome Variation (Durham, NC), ran the Taqman assay on the IL28B SNP. Jennifer King, Ph.D., assisted in the preparation of the manuscript for this article.