Efficacy, safety and pharmacokinetics of simeprevir and TMC647055/ritonavir with or without ribavirin and JNJ-56914845 in HCV genotype 1 infection

Background A Phase 2a, open-label study (NCT01724086) was conducted to assess the efficacy and safety of a once-daily, 2-direct-acting-antiviral-agent (2-DAA) combination of simeprevir + TMC647055/ritonavir ± ribavirin and of the 3-DAA combination of simeprevir + TMC647055/ritonavir + JNJ-56914845 in chronic hepatitis C virus genotype (GT)1-infected treatment-naïve and prior-relapse patients. Methods The study comprised four 12-week treatment panels: Panel 1 (n = 10; GT1a) and Panel 2-Arm 1 (n = 12; GT1b): simeprevir 75 mg once daily + TMC647055 450 mg once daily/ritonavir 30 mg once daily + ribavirin 1000–1200 mg/day; Panel 2-Arm 2 (n = 9; GT1b): simeprevir 75 mg + TMC647055 450 mg/ritonavir 30 mg without ribavirin; Panel 3: simeprevir 75 mg + TMC647055 600 mg/ritonavir 50 mg with (Arm 1: GT1a; n = 7) or without (Arm 2: GT1b; n = 8) ribavirin; Panel 4: simeprevir 75 mg + TMC647055 450 mg/ritonavir 30 mg + JNJ-56914845 30 mg once daily (Arm 1: n = 22; GT1a/GT1b) or 60 mg once daily (Arm 2: n = 22; GT1a/GT1b). Primary endpoint was sustained virologic response 12 weeks after end of treatment (12 weeks of combination treatment; SVR12). Results In Panel 1 and Panel 2-Arm 1, 5/10 and 6/12 (50%) GT1a/GT1b + ribavirin patients achieved SVR12, versus 3/9 (33%) GT1b without ribavirin patients in Panel 2-Arm 2. In Panel 3-Arm 1 and Panel 3-Arm 2, 6/7 (86%) GT1a + ribavirin and 4/8 (50%) GT1b without ribavirin patients, respectively, achieved SVR12. In Panel 4, 10/14 (71%) and 14/15 (93%) GT1a patients in Arms 1 and 2 achieved SVR12 compared with 8/8 and 7/7 (100%) GT1b patients in each arm, respectively. No deaths, serious adverse events (AEs), Grade 4 AEs or AEs leading to treatment discontinuation occurred. Conclusions The 2- and 3-DAA combinations were well tolerated. High SVR rates of 93% and 100% in GT1a- and GT1b-infected patients, respectively, were achieved in this study by combining simeprevir with JNJ-56914845 60 mg and TMC647055/ritonavir. Trial registration NCT01724086 (date of registration: September 26, 2012) Electronic supplementary material The online version of this article (doi:10.1186/s12876-017-0580-2) contains supplementary material, which is available to authorized users.


Background
Interferon (IFN)-free regimens comprising direct-acting antivirals (DAA) with different mechanisms of action can result in high sustained virologic response (SVR) rates in patients chronically infected with hepatitis C virus (HCV). Combination regimens comprising an HCV NS3/4A protease inhibitor (PI), a non-nucleoside inhibitor (NNI) of the HCV NS5B polymerase and/or an HCV NS5A replication complex inhibitor with/without ribavirin have been shown to be successful in the treatment of chronic HCV infection [1]. For example, the combination of the PI paritaprevir, the NNI dasabuvir and the NS5A inhibitor ombitasvir, with ritonavir included as a pharmacologic booster for paritaprevir, is approved for the treatment of chronic HCV genotype (GT) 1 infection. In the PEARL-III (HCV GT1b) and PEARL-IV (HCV GT1a) Phase 3 studies, treatment of previously untreated patients with paritaprevir/ritonavir, dasabuvir and ombitasvir for 12 weeks resulted in SVR 12 weeks after end of treatment (SVR12) rates of 99.5% and 97.0% in the presence of ribavirin, and 99.0% and 90.2% in the absence of ribavirin, respectively [2].
Simeprevir is a once-daily, HCV NS3/4A PI approved as part of an IFN-free combination with sofosbuvir for HCV GT1 infection. In addition, simeprevir with sofosbuvir is approved for HCV GT4 infection and HCV/human immunodeficiency virus (HIV) co-infection in the European Union (EU). Simeprevir is also approved in combination with pegylated IFN (pegIFN)/ribavirin for chronic HCV GT1 and GT4 infection in the United States and EU [3]. Simeprevir is a cytochrome P450 (CYP) 3A substrate and mild inhibitor (intestinal only). Simeprevir also inhibits organic anion transporting polypeptide (OATP) 1B1/3 and P-glycoprotein [4].
TMC647055 is an NNI of the HCV NS5B polymerase, binding in the NNI-1 pocket on the polymerase. TMC647055 has in vitro antiviral activity against HCV GT1, 3, 4, 5 and 6 [5]. In a Phase 1 study (NCT01202825), TMC647055 1000 mg twice daily in combination with simeprevir 150 mg once daily for 10 days demonstrated good antiviral activity and was well tolerated in HCV GT1a-and GT1b-infected patients [6]. However, the systemic exposure to both compounds decreased during treatment to levels potentially lower than required for complete viral suppression. Coadministering TMC647055 with the potent CYP3A4 inhibitor ritonavir was expected to increase exposure by overcoming CYP3A4 induction [5].
JNJ-56914845 (previously known as GSK-2336805) is a potent HCV NS5A replication complex inhibitor, with in vitro antiviral activity against HCV GT1, 4, 5 and 6a/6b [7,8]. It is a CYP3A and P-glycoprotein substrate and, in vitro, it inhibits P-glycoprotein, OATP1B1/3 and breast cancer resistance protein (data on file). This Phase 2a, open-label study (NCT01724086) assessed the efficacy, pharmacokinetics, safety and tolerability of the 2-DAA combination of simeprevir with TMC647055 and a low dose of ritonavir with or without ribavirin for 12 weeks in chronic HCV GT1-infected treatment-naïve and prior-relapse patients. The study also included the addition of a third DAA, JNJ-56914845, to the simeprevir and TMC647055 combination to assess the potential of further increasing the efficacy of the regimen by combining three DAAs with different mechanisms of action. Results from the final analysis, when all patients in all panels had completed the study, are presented here.

Patients and study design
This was a Phase 2a, open-label study, conducted between 12 September 2012 and 16 December 2014 at 12 sites in Belgium and Germany. The study was approved by the Institutional Review Board or Independent Ethics Committee at each participating centre, and met the ethical principles of the Declaration of Helsinki and Good Clinical Practice guidelines. All patients provided written, informed consent.
Adults (18-70 years of age) with chronic HCV GT1a/ GT1b infection and screening plasma HCV RNA >10,000 IU/mL who were treatment-naïve or had relapsed following previous treatment with pegIFN/ribavirin were eligible for inclusion. Patients had to have a documented liver biopsy within 3 years of the screening visit, or have an elastography prior to first dosing.
Exclusion criteria included liver cirrhosis, hepatic decompensation, liver disease of non-HCV aetiology, infection/co-infection with non-GT1a/GT1b HCV, hepatitis A or B, or HIV-1/-2, and significant laboratory abnormalities, including total bilirubin ≥1.5 x upper limit of normal and platelet count <90,000/mm 3 . Patients who had received prior HCV-specific DAA treatment or a liver transplant were also excluded.
The study comprised four 12-week treatment panels (Fig. 1). Panels 1 and 2, which ran in parallel, comprised the first part of the study and were sequentially followed by Panels 3 and 4. Panels 1-3 evaluated simeprevir in combination with TMC647055/ritonavir (at two different doses of TMC647055) with ribavirin in GT1a-infected patients and with or without ribavirin in GT1b-infected patients. Panel 4 evaluated the triple combination of simeprevir with TMC647055/ritonavir plus the NS5A inhibitor JNJ-56914845 at a low and high dose in GT1a/b-infected patients.
More specifically, patients in Panels 1 and 2 received simeprevir 75 mg once daily, TMC647055 450 mg once daily, ritonavir 30 mg once daily and ribavirin 1000-1200 mg (patients in Panel 2-Arm 2 did not receive ribavirin). Patients in Panel 3 received simeprevir 75 mg once daily, TMC647055 at a higher dose of 600 mg once daily, ritonavir 50 mg once daily and ribavirin 1000-1200 mg (patients in Panel 3-Arm 2 did not receive ribavirin). Patients in Panel 4 received simeprevir 75 mg once daily, TMC647055 450 mg once daily, ritonavir 30 mg once daily and JNJ-56914845 30 (Arm 1) or 60 mg (Arm 2) once daily. Simeprevir was dosed at 75 mg once daily (rather than the 150 mg dose approved) to account for CYP3A inhibition by ritonavir, which is not a part of the approved regimen for simeprevir. Further details on the dosing rationale are provided in Additional file 1. For ethical reasons, patients in Panels 1-3 received follow-up treatment with pegIFN/ribavirin for an additional 12 or 36 weeks if their Week-4 HCV RNA levels did not comply with predefined criteria (Fig. 1). For patients in Panel 4 receiving the 3-DAA regimen, no follow-up treatment was planned.
Central randomisation was implemented in Panel 2, with patients randomly assigned 1:1 to Arm 1 or Arm 2 based on a computer-generated schedule prepared before the study, balanced by using randomly permuted blocks, and stratified by IL28B genotype (CC and non-CC). Patients in Panel 3 were not randomised as their treatment was dependent on their HCV geno/subtype (1a or 1b) (Fig. 1). In Panel 4, patients were randomised in a 1:1 ratio between Arm 1 or Arm 2, stratified by HCV geno/subtype.
To avoid unnecessary drug exposure, the following virologic stopping rules were adopted: all study drugs were discontinued for patients with viral breakthrough (confirmed on-treatment increase of >1 log 10 IU/mL in HCV RNA from the lowest level reached, or confirmed HCV RNA >100 IU/mL in patients whose HCV RNA had previously been <25 IU/mL), or with inadequate virologic response (confirmed HCV RNA >100 IU/mL at Week 4 or afterwards until Week 11).
Details on protocol deviations are provided in Additional file 1.

Outcomes
The primary efficacy endpoint was SVR12. Patients achieved SVR12 if they received no pegIFN/ribavirin follow-up treatment after 12 weeks of combination treatment (Panels 1-3 only) and achieved HCV RNA <25 IU/mL undetectable/detectable 12 weeks after actual end of treatment (Panels 1-4). Patients in Panels 1-3 who received follow-up therapy and had HCV RNA <25 IU/mL at 12 weeks after end of treatment were also classed as having achieved SVR12; however, they were considered as failures with regards to the primary endpoint of the study, which focused on the 12-week DAA therapy. The primary safety endpoints included the proportion of patients with adverse events (AEs), serious AEs (SAEs) or abnormal changes in safety-related  Week-4 pharmacokinetics of the study drugs; on-treatment virologic failure, including patients with viral breakthrough (defined in the previous section); viral relapse (defined as HCV RNA <25 IU/mL undetectable at the actual end of treatment and confirmed HCV RNA ≥25 IU/mL during post-treatment follow-up); and the presence of HCV NS3/4A, NS5A and/or NS5B variants at baseline and at time of failure in patients not achieving SVR.

Assessments
Blood samples for HCV RNA level determination were collected at screening and at predefined time points throughout the treatment phase and follow-up period. HCV RNA was measured using the COBAS® TaqMan® HCV Test version 2.0 (Roche Molecular Diagnostics, Pleasanton, CA, USA) for use with the High Pure System assay (lower limit of quantification: 25 IU/mL; limit of detection: 10-15 IU/mL). HCV geno/subtypes were determined pre-treatment based on sequencing of a part of the NS5B gene (at baseline) when available or by the VERSANT® HCV Genotype 2.0 assay (LiPA) or Trugene HCV Genotyping assay (at screening) (Siemens Healthcare Diagnostics, Erlangen, Germany).
Standard population-based sequencing of the HCV NS3/4A and NS5B regions in Panels 1-3 and of the NS3/4A, NS5A and NS5B regions in Panel 4 was performed at baseline for all patients and post-baseline for patients not achieving SVR12 based on the HCV RNA changes observed in each individual patient and the limits of the sequencing assay.
AEs were monitored throughout the treatment phase and follow-up period and up to 24 weeks after actual end of treatment. AEs were coded using the Medical Dictionary for Regulatory Activities (version 16.0 for Panels 1 and 2 and version 17.0 for Panels 3 and 4).

Statistical analyses
Statistical analyses were performed using SAS® version 9.1 or higher (SAS Institute Inc, Cary, NC, USA).
No formal sample size calculations were performed as this was a proof-of-concept study. For each efficacy endpoint, the proportion of patients was summarised descriptively together with a 95% confidence interval by treatment group and population. The change in log 10 HCV RNA from baseline at all time points was calculated using descriptive statistics. Pharmacokinetic parameters were calculated using non-compartmental analysis (Phoenix WinNonlin® 6.2.1). All safety and tolerability data were summarised descriptively.
All analyses were conducted using data from the intent-to-treat population, which comprised all patients who received at least one dose of study drug.

Patient disposition and baseline characteristics
In total, 125 patients were screened, and 90 (n = 10, 21, 15 and 44 in Panels 1, 2, 3 and 4, respectively) were treated (intent-to-treat population) (Fig. 2 ). All treatment discontinuations were due to viral breakthrough, as detailed in the next section.

Virologic response
All patients who achieved SVR12 also went on to achieve SVR24, with the exception of one patient in Panel 1 and one patient in the GT1b/without ribavirin group in Panel 3 who had HCV RNA ≥25 IU/mL 24 weeks after end of treatment (1,830,000 IU/mL and 2770 IU/mL, respectively).
An overview of treatment outcome, including reasons for not achieving SVR12, is shown in Fig. 3. A total of 5/10 (50%), 1/12 (8%) and 1/9 (11%) patients in Panel 1 and in the GT1b/with ribavirin and GT1b/without ribavirin groups of Panel 2, respectively, experienced viral breakthrough. In Panel 3, 1/8 (13%) patients in the GT1b/without ribavirin group had viral breakthrough compared with 1/22 (5%) patients in the 60-mg group of Panel 4 (Fig. 3). Among the patients with undetectable HCV RNA at end of treatment and with at least one post-treatment follow-up HCV RNA measurement available, 2/6 patients (33%) in each of the Panel 2 and Panel 3 GT1b/without ribavirin groups and 4/14 (18%) GT1a-infected patients in the Panel 4 30mg group experienced viral relapse. At the time of the final analysis, one patient in Panel 1 and one patient in the Panel 3 GT1b/without ribavirin group achieved SVR12 but had HCV RNA ≥25 IU/mL 24 weeks after end of treatment.

Virologic resistance testing
At baseline, the NS3 Q80K polymorphism was observed in 2/10 (20%) GT1a-infected patients in Panel 1 and in 2/13 (15%) GT1a-infected patients in the 30-mg group  Fig. 2 Patient disposition. a In Panel 2, one patient in the GT1b/without RBV group had <25 IU/mL detectable HCV RNA at Week 4 and, therefore, met the criterion for a 12-week follow-up treatment with pegIFN/RBV. This patient refused to receive follow-up treatment. b In Panel 4, the results of the Trugene or LiPA test performed at screening were used to randomise the patients to the 30-mg or 60-mg dose of JNJ-56914845. For analyses purposes, NS5B sequencing was used to determine the HCV geno/subtype. Based on this method, the geno/subtype for two patients in the 30-mg group was found to be non-1a, non-1b (1c, n = 1; 1l, n = 1) based on geno/subtyping using NS5B sequencing. These two patients were analysed together with the GT1a-infected patients in the category 'GT1a/other'. DAA direct-acting antiviral agent, FU follow-up, GT genotype, HCV hepatitis C virus, pegIFN pegylated interferon α-2a, RBV ribavirin  Virologic failure (including the two patients who achieved SVR12 but had HCV RNA ≥25 IU/mL 24 weeks after end of treatment) was, in the majority of patients, associated with the emergence of simeprevir and TMC647055 resistance-associated variants (RAVs) in Panels 1-3 and simeprevir and JNJ-56914845 RAVs with or without TMC647055 RAVs in Panel 4 at time of failure. In line with the previously characterised simeprevir resistance profile, most GT1b-infected patients harboured an emerging mutation at NS3 position 168, while emerging mutations in GT1a-infected patients were mostly observed at NS3 position 155 (R155K alone or in combination with a Q80R; R155S) at time of failure. TMC647055 RAVs were only observed at NS5B position 495 (mainly P495L), consistent with the resistance profile of NNI-1 polymerase inhibitors. In all patients with virologic failure in Panel 4, emerging JNJ-56914845 RAVs at NS5A positions 30 and/or 31 were detected at time of failure. Treatment-emergent RAVs in NS3 and NS5B became undetectable in many of the patients after treatment was stopped, while emerging RAVs in NS5A could still be detected by population sequencing at the end of the study in the five patients who failed the 3-DAA treatment.
No differences were observed in simeprevir, TMC647055 or ritonavir pharmacokinetics based on ribavirin use  o  t  t  s  o  L  e  s  p  a  l  e  R  T  B  V  U  F  V  B  R  /  N  F  I  g  e  P  2  1  R  V  S EOT HCV RNA DET Overall GT1

GT1a GT1b
Overall GT1 GT1a GT1b shown represent SVR12 achieved after treatment with only the combination of SMV + TMC647055/RTV ± RBV. In the Panel 2 GT1b/with RBV and GT1b/without RBV arms, four and two patients, respectively, had follow-up therapy with pegIFN/RBV and are, therefore, not included in the SVR12 category that focuses on SVR12 after 12 weeks of DAA treatment. Among them, five patients achieved SVR12 after completing follow-up therapy; one patient refused to receive follow-up treatment and was lost to follow-up. One additional patient in the Panel 2 GT1b/ without RBV group had <25 IU/mL detectable HCV RNA at Week 4 and, therefore, met the criterion for the 12-week follow-up treatment with pegIFN/RBV; however, this patient refused to receive followup treatment. DAA direct-acting antiviral agent, DET detectable, EOT end of treatment, FU follow-up, GT genotype, HCV hepatitis C virus, pegIFN pegylated interferon α-2a, RBV ribavirin, RTV ritonavir, SMV simeprevir, SVR12 sustained virologic response 12 weeks after end of treatment, VBT viral breakthrough (Additional file 4: Table S2, Additional file 5: Table S3 and Additional file 6: Table S4).

Safety
No patients discontinued any of the study drugs due to an AE, and there were no SAEs or deaths. The incidence of AEs was similar between Panels 1 and 2, and Panel 3 (Table 2). In Panels 1 and 2, all reported AEs were Grade 1 or 2, and there were no clinically relevant differences among patients treated with ribavirin and those who did not receive ribavirin in terms of the incidence or severity of AEs. In Panel 3, all AEs were Grade 1 or 2, with the exception of one patient (GT1b/without ribavirin group) who experienced a Grade 3 AE (hypercholesterolaemia). This AE was not serious and was not considered related to simeprevir, TMC647055 or ritonavir by the investigator; the AE was ongoing at the time of the final analysis. A higher incidence of patients in Panel 3 treated with ribavirin experienced a Grade 1 or 2 AE compared with those who did not receive ribavirin. All AEs in Panel 4 were Grade 1 or 2, with the exception of one patient who experienced a Grade 3 increase in white blood cell count (not related to any study drug). There were no clinically relevant differences in the incidence of AEs between patients treated with JNJ-56914845 at either dose ( Table 2).
The most frequently (in >20% of patients) reported AEs during the treatment phase were as follows; Panels 1 and 2: headache ( The majority of graded laboratory abnormalities were Grade 1 or 2 and were not clinically significant. Grade 3 or 4 laboratory abnormalities were observed in <10% of patients in all panels (further details are provided in Additional file 1). Hyperbilirubinaemia was reported as a laboratory-related AE in 1 (3%) patient in Panels 1 and 2 (considered possibly related to TMC647055 and simeprevir) and 1 (2%) patient in the 60-mg group in Panel 4 (considered possibly related to TMC647055, ritonavir and JNJ-56914845, and probably related to simeprevir). Both hyperbilirubinaemia AEs were Grade 1 or 2 in severity.

Discussion
This was a proof-of-concept study performed to explore the efficacy and safety of the 2-DAA combination of the HCV NS3/4A PI simeprevir and the HCV NS5B NNI TMC647055 administered with ritonavir ± ribavirin, and of the 3-DAA combination of simeprevir, TMC647055 and the HCV NS5A inhibitor JNJ-56914845 administered with ritonavir in HCV GT1-infected patients. The results suggest that the addition of JNJ-56914845 (60 mg) to the 2-DAA regimen of simeprevir and TMC647055 was highly beneficial in improving the efficacy of the combination treatment in patients with HCV GT1 infection.
The 3-DAA combination resulted in an overall SVR12 rate of 95% with 60 mg JNJ-56914845. All GT1binfected patients (7/7; 100%) treated with the 3-DAA combination achieved SVR12, and the majority of GT1ainfected patients (14/15; 93%) also achieved SVR12 following treatment with this regimen. The SVR12 rates observed with this combination therapy are consistent with those reported in Phase 3 studies that assessed the combination of the PI paritaprevir with the NNI dasabuvir and the NS5A inhibitor ombitasvir in treatmentnaïve or treatment-experienced, non-cirrhotic, HCV GT1-infected patients [2,[10][11][12].
The simeprevir resistance profile observed in all panels in this study was similar to that observed in the simeprevir Phase 3 studies in the presence of pegIFN/ribavirin [13][14][15]. A genotypic and phenotypic analysis of baseline HCV isolates from all patients who participated in this study, as well as of isolates obtained at the time of failure and at the end of study from patients with virologic failure, are described in full in a separate paper (Leen Vijgen, Kim Thys, An Vandebosch, Pieter Van Remoortere, René Verloes, Sandra De Meyer. Virology analysis in HCV genotype 1-infected patients treated with the combination of simeprevir and TMC647055/ritonavir, with and without ribavirin, and JNJ-56914845, in preparation).
The interaction between the drugs assessed in this study had previously been evaluated in healthy volunteers [16]. In that study, co-administration of simeprevir increased exposure (AUC 0-24h ) of JNJ-56914845 2.6-fold with no additional effect when TMC647055/ritonavir was added. Co-administration with JNJ-56914845 resulted in small increases in the plasma concentrations of simeprevir and TMC647055. These increases were not considered to be clinically relevant for either DAA [16]. In the current study, administration of simeprevir 75 mg once daily in combination with TMC647055/ritonavir in HCV-infected patients resulted in simeprevir exposures within the range observed in historical data for simeprevir when dosed at 150 mg once daily (data on file). In addition, high exposure to TMC647055 was achieved when administered in combination with ritonavir.
Both the 2-DAA and the 3-DAA regimens were well tolerated. There were no deaths, SAEs, Grade 4 AEs or AEs leading to temporary/permanent treatment discontinuation in any of the four panels. In addition, there were no Grade 3 or 4 laboratory-related AEs that were considered related to any of the study drugs. Differences in AE incidence among patients who received ribavirin and those who did not in Panels 1 and 2 ( Table 2) were a result of ribavirin-related adverse drug reactions, as were the respective differences observed in the incidence of laboratory abnormalities in Panels 1-3 (see Additional file 1). A comparable safety profile was observed with TMC647055/ ritonavir 450/30 mg and TMC647055/ritonavir 600/50 mg (Panels 1-3) and also between the JNJ-56914845 30-mg and 60-mg doses (Panel 4).