Antiviral Activity, Safety, and Tolerability of Multiple Ascending Doses of Elbasvir or Grazoprevir in Participants Infected With Hepatitis C Virus Genotype-1 or -3

Clinical Therapeutics/Volume ], Number ], 2018

Antiviral Activity, Safety, and Tolerability of Multiple Ascending Doses of Elbasvir or Grazoprevir in Participants Infected With Hepatitis C Virus Genotype-1 or -3 Wendy W. Yeh, MD1; Iain P. Fraser, MD, DPhil1,*; Patricia Jumes, MS1; Amelia Petry, BS1,†; Inge De Lepeleire, PharmD2; Martine Robberechts, MS2; Christina Reitmann, MS1,‡; Kristien Van Dyck, PhD2; Xiaobi Huang, PhD1,§; Zifang Guo, PhD1; Deborah Panebianco, MS1; Robert B. Nachbar, PhD1,||; Edward O’Mara, MS1,¶; John A. Wagner, MD, PhD1,#; Joan R. Butterton, MD1; Frank J. Dutko, PhD1; Valentin Moiseev, MD3; Zhanna Kobalava, MD, PhD3; Andreas Hu ¨ser, MBA4; Sorin Visan, MD5; Christian Schwabe, MD6; Edward Gane, MBCHB, MD, FRACP, MNZM7; Serghei Popa, MD, PhD5; Nelea Ghicavii, MD5; Markus Uhle, MD4; and Frank Wagner, MD, PhD4 1

Merck & Co, Inc, Kenilworth, New Jersey; 2MSD, Brussels, Belgium; 3Center of Applied Clinical Pharmacology at Russian Peoples’ Friendship University, Moscow, Russia; 4Charite´ Research Organisation GmbH, Berlin, Germany; 5ARENSIA Exploratory Medicine, Republican Clinical Hospital, Chisinau, Moldova; 6Auckland Clinical Studies, Auckland, New Zealand; and 7Auckland City Hospital, Auckland, New Zealand ABSTRACT Purpose: Elbasvir (MK-8742) and grazoprevir (MK-5172; Merck & Co, Inc, Kenilworth, New Jersey) are hepatitis C virus (HCV)-specific inhibitors of the nonstructural protein 5A phosphoprotein and the nonstructural protein 3/4A protease, respectively. The aims of these studies were to evaluate the antiviral activity and safety of different doses of elbasvir or grazoprevir each administered as monotherapy to participants infected with either HCV genotype (GT) 1 or GT3.

*

Current affiliation: Abide Therapeutics, Inc, Princeton, New Jersey. † Current affiliation: Bristol-Myers Squibb, Wallingford, Connecticut. ‡ Current affiliation: Novartis, East Hanover, New Jersey. § Current affiliation: Sanofi-Aventis, Bridgewater, New Jersey. || Current affiliation: Wolfram Research, Inc., Champaign, Illinois. ¶ Current affiliation: PTC Therapeutics Inc, South Plainfield, New Jersey. # Current affiliation: Takeda Pharmaceuticals, Cambridge, Massachusetts.

] 2018

Methods: These 2 double-blind, randomized, placebo-controlled, sequential-panel, multiple ascending dose studies were conducted to assess the safety and pharmacodynamics of 5 days of once-daily elbasvir or 7 days of once-daily grazoprevir in adult male participants chronically infected with either HCV GT1 or GT3. Findings: Oral administration of elbasvir or grazoprevir once daily exhibited potent antiviral activity in participants with chronic GT1 or GT3 HCV infections. HCV RNA levels declined rapidly (within 1 day for elbasvir and 2 days for grazoprevir). At 50 mg of elbasvir once daily, the mean maximum reductions in HCV RNA from baseline were 5.21, 4.17, and 3.12 log10 IU/mL for GT1b-, GT1a-, and GT3-infected participants, respectively. At 100 mg of grazoprevir once daily, the mean maximum reductions in HCV

Accepted for publication March 5, 2018. https://doi.org/10.1016/j.clinthera.2018.03.002 0149-2918/$ - see front matter & 2018 Elsevier HS Journals, Inc. All rights reserved.

1

Clinical Therapeutics RNA from baseline were 4.74 and 2.64 log10 IU/mL for GT1- and GT3-infected participants. Implications: The results in the elbasvir monotherapy study showed that 10 to 50 mg of elbasvir was associated with a rapid decline in HCV viral load; the results in the grazoprevir monotherapy study suggest that doses of 50 mg of grazoprevir and higher are on the maximum response plateau of the dose–response curve for GT1-infected participants. The results of these proof-of-concept studies provided preliminary data for the selection of the dosages of elbasvir and grazoprevir to test in Phase II and III clinical studies. ClinicalTrials.gov identifiers: NCT00998985 (Protocol 5172-004) and NCT01532973 (Protocol 8742002). (Clin Ther. 2018;]:]]]–]]]) & 2018 Elsevier HS Journals, Inc. All rights reserved. Key words: elbasvir, genotype 1, genotype 3, grazoprevir, hepatitis C virus, monotherapy.

INTRODUCTION According to current estimates, 71 million people worldwide and 2.7 to 3.9 million people in the United States are chronically infected with hepatitis C virus (HCV).1,2 Sixteen percent of people with chronic HCV infection will develop liver cirrhosis within 20 years after infection without treatment, and this percentage rises to 41% within 30 years after HCV infection.3 Approximately 500,000 deaths in 2010 worldwide were attributable to liver cirrhosis and hepatocellular carcinoma due to HCV infection.4 Efficacious anti-HCV therapy and virologic cure can reduce long-term complications of HCV infection, including hepatic decompensation and hepatocellular carcinoma.5,6 Because HCV is an RNA virus that is capable of substantial mutation in response to targeted antiviral agents, highly efficacious treatments for HCV infection require a combination of antiviral agents that attack multiple points of the viral replication cycle simultaneously. The development of direct-acting antiviral (DAA) agents with unique mechanisms of action has revolutionized the treatment of HCV infection.7 Three classes of DAA agents acting at distinct points in the HCV life cycle have been developed: nonstructural protein (NS)3/4A protease inhibitors, NS5A replication complex inhibitors, and NS5B polymerase inhibitors. New DAA agents that

2

are available as single entities or as part of a fixeddose combination—including daclatasvir, ledipasvir, ombitasvir, and velpatasvir (NS5A inhibitors); asunaprevir, paritaprevir, and simeprevir (NS3/4A protease inhibitors); sofosbuvir (NS5B inhibitor); and dasabuvir (nonnucleoside NS5B inhibitor)— offer improved efficacy and safety over older treatment options when administered as components of a combination therapy.8 Elbasvir (EBR; MK-8742; Merck & Co, Inc, Kenilworth, New Jersey) and grazoprevir (GZR; MK-5172; Merck & Co, Inc) are highly potent, virus-specific inhibitors of the HCV NS5A protein and the HCV NS3/4A protease, respectively.9,10 The combination of EBR and GZR with or without ribavirin has been approved by the US Food and Drug Administration, the European Medicines Agency, and other countries for the treatment of HCV-infected, treatment-naive, or peginterferon/ribavirin–experienced individuals infected with HCV genotype (GT) 1 or GT4.11–13 In clinical trials, the combination of EBR and GZR has been shown to be efficacious across a broad cross-section of individuals with HCV infection, including challenging treatment populations such as those with cirrhosis, chronic kidney disease, HIV/HCV coinfection, and individuals receiving opioid agonist therapy.14–20 EBR and GZR were identified as potent and highly selective inhibitors of HCV based on data that showed pan-genotypic in vitro activity with EC50 values in the low picomolar range for most GTs.9,10 EBR and GZR were previously found to be well tolerated in healthy non–HCV-infected participants when administered in single doses (EBR, 5–400 mg; GZR, 2–1600 mg) or multiple doses (EBR, 10–200 mg; GZR, 100–1000 mg).21,22 Short-duration monotherapy trials in HCVinfected participants have been used to establish proof-of-concept and for the initial dose exploration of novel DAA agents before their evaluation in larger, longer duration Phase II treatment clinical trials. Accordingly, 2 Phase Ib proof-of-concept studies were conducted to assess the pharmacodynamics of EBR and GZR in HCV-infected participants. Here we report the results of the placebo-controlled, multiple ascending dose clinical studies to evaluate the HCV viral load dynamics, safety, and tolerability of EBR and GZR when administered as short-term monotherapy in noncirrhotic participants chronically infected with HCV GT1 or GT3.

Volume ] Number ]

] 2018

HCV ¼ hepatitis C virus; GT ¼ genotype. ⁎ One participant who was randomized to receive GT3 placebo was mistyped and found confirmed to be GT1b. This participant was included in the overall safety analysis and was counted as a GT1b-infected participant in the pharmacodynamic analysis.

6.69 6.44 6.86 6.82 6.55 6.54 6.83 6.45 6.97 6.14 6.67

6.54

5 0 5 0 5 0 2 0 5 0 5 0 1 0 3 0 5 0 3 1 3 0

5 0

5 GT3 34.6 5 GT3 35.8 4 GT1a 50.3 3 GT1a 58.0

No. of participants HCV GT and subtype Mean age, y Race, no. White Black or AfricanAmerican Mean baseline HCV RNA, log10 IU/mL

5 GT1a 51.0

5 GT1a 53.2

3 GT1b 43.0

1 GT1b 57.0

5 GT1b 38.8

5 GT1b 36.6

5 GT3 34.8 2* GT3 39.5

EBR 100 mg/d EBR 50 mg/d EBR 50 EBR 10 mg/d Placebo mg/d EBR 10 mg/d EBR 50 EBR 5 mg/d Placebo mg/d EBR 10 mg/d

The placebos were manufactured to match the images of the active drugs. Participants were randomly assigned an allocation number based on a computer-generated allocation schedule. Participants, clinical study personnel, and sponsor personnel were blinded to treatment assignment. For EBR, there were 8 groups in the study: 2 groups of GT1a-infected participants; 2 groups of GT1b-infected participants; 1 group of either GT1a- or GT1b-infected participants; and 3 groups of GT3-infected participants. Each dose group consisted of 6 participants who were administered oral doses of EBR or placebo at a ratio of 5:1 once daily in the morning in the fasted state for 5 consecutive days. The doses of EBR were 5, 10, or 50 mg once daily in participants infected with HCV GT1a or GT1b, and 10, 50, or 100 mg once daily in participants infected with HCV GT3. Table I presents the number and demographic characteristics of the participants in each group. All doses of EBR or placebo were given in the fasted state to minimize potential confounding factors. All doses were administered under direct observation.

EBR 5 mg/d

Randomization and Masking

Placebo

These 2 double-blind, randomized, placebo-controlled, multiple dose studies were conducted to assess the safety and pharmacodynamics of EBR (Protocol 8742-002; NCT01532973; See Supplement, © 2018 Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.) or GZR (Protocol 5172-004; NCT00998985; See Supplement, © 2018 Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.) in adult male participants chronically infected with either HCV GT1 or GT3. Inclusion criteria were as follows: minimum level of HCV RNA of at least 105 IU/mL at screening; male between 18 and 65 years of age; body mass index of 18 to ≤37 kg/m2; alanine aminotransferase (ALT) levels ≤6 × upper limit of normal (ULN); aspartate aminotransferase (AST) levels ≤6 × ULN; total bilirubin ≤2.4 mg/dL; direct bilirubin ≤1.0 mg/dL; albumin ≥3.5 g/dL; alkaline phosphatase ≤260 U/L; hemoglobin ≥12.0 g/dL; white blood cell count ≥3.5 × 103/μL; and platelet count ≥100 × 103/μL. Exclusion criteria included the following: estimated creatinine clearance ≤70 mL/min based on the Cockcroft-Gault equation; history of neoplastic disease; positive hepatitis B surface antigen; or history of documented HIV infection or positive HIV serologic test results.

Variable

PATIENTS AND METHODS Study Design and Participants

Table I. Participant demographic and baseline characteristics in the monotherapy study according to study group with elbasvir (EBR) or placebo.

W.W. Yeh et al.

3

Clinical Therapeutics For GZR, 13 groups of participants were administered oral doses of GZR or matching placebo once daily in the morning in the fasted state for 7 consecutive days (Table II). Each dose group enrolled 6 participants who were randomized to receive GZR or placebo at a 5:1 ratio, except for the 800-mg GZR group, which enrolled 18 participants to receive GZR: placebo in a 5:1 ratio. The increase to 18 participants receiving the dose of 800 mg of GZR was chosen to increase the clinical safety experience at the highest dose administered in the study. The dose levels of GZR were 10, 30, 50, 100, 200, 400, 600, and 800 mg once daily in participants infected with HCV GT1, and 100, 200, 400, 600, and 800 mg once daily in participants infected with HCV GT3. All doses of GZR or placebo were given in the fasted state to minimize potential confounding factors. All doses were administered under direct observation. All participants provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki and guidelines for Good Clinical Practice. Independent ethics committees reviewed and approved the protocol and applicable amendments for each institution.

Viral Kinetics and Statistical Analysis Viral load quantitation was performed by using the Roche Cobas TaqMANs 2.0 assay, with a lower limit of quantitation (LOQ) of 25 IU/mL and a lower limit of detection (LOD) of 3.8 IU/mL (Roche Molecular Diagnostics, Pleasanton, California). Descriptive statistics for HCV RNA according to treatment and time point were calculated. The mean maximal reduction of HCV RNA in a group was computed from the nadir for each participant in the group. Numerical values for viral load below the LOQ and below the LOD were imputed as the average of LOQ and LOD and 1/2*LOD, respectively. The former is midway between LOQ and LOD, and the latter is one half of the LOD. For each active dose of study drug for each GT, and for the placebo results from all groups combined, summary statistics for the change from baseline in HCV RNA (log10) at each postdose time point were calculated. In addition, the profiles of the change from baseline in log10 HCV RNA are displayed graphically. For GZR, the maximum reduction in log10 HCV RNA for the pooled (across GT1 and GT3) placebo group and GT1- or GT3-infected participants who received active dose were analyzed

4

separately by using an ANOVA model with a fixed effect for treatment. At each dose, a 2-sided 90% CI for the difference in mean maximum viral RNA reduction in log10 HCV RNA (active dose minus placebo) was constructed. For EBR, similarly separate models were run for GT1-, GT3-, and GT1a-infected participants who received active dose, and the same set of placebo data, pooled across all panels and GTs, was included in each model.

Resistance Analyses The methods for resistance analyses have been reported previously.23 Briefly, the target genes (NS5A and NS3) were amplified by using reverse transcription-polymerase chain reaction followed by population and selective clonal sequencing. Due to the sensitivity of the assay, resistance analysis was performed only on samples from participants with HCV viral loads 41000 IU/mL. The limit of variant detection in population sequencing was 425% of the viral population. For clonal sequencing, polymerase chain reaction amplification was performed only on amino acids 1–448 of NS5A and the resultant amplicons cloned into a TOPO TA vector (Invitrogen, Carlsbad, California). Approximately 40 clones were sequenced at each time point. Resultant amino acid sequences from population and clonal sequencing were compared with wild-type HCV GT1a, H77 (GenBank NC_004102), GT1b, Con1 (GenBank AJ238799), or GT3, S52 (GU814263) reference sequences to detect amino acid substitutions.

Clinical Safety The safety and tolerability of EBR and GZR were monitored daily by clinical assessment of adverse experiences (AEs) and by repeated measurements of vital signs, physical examinations, 12-lead ECGs, and standard laboratory safety tests (plasma biochemical panel, hematology, and urinalysis). Recorded AEs were listed and tabulated according to system organ class, preferred term, and treatment. Selected nonserious and serious AEs were also known as events of clinical interest. These events included an overdose of EBR or GZR that was not associated with clinical symptoms or abnormal laboratory results; they also included an elevated AST or ALT laboratory value that was ≥3 × the ULN and an elevated total bilirubin laboratory value that was ≥2 × the ULN and, at the same time, an alkaline phosphatase value that was o2 × the ULN determined by way of Volume ] Number ]

] 2018 Table II. Participant demographic and baseline characteristics in the monotherapy study according to study group with grazoprevir (GZR) or placebo.

Variable No. of participants HCV GT and subtype GT1a GT1b Mean age, y Race, no. White Black or African American Mean baseline HCV RNA, log10 IU/mL

GZR GZR GZR GZR 10 30 50 100 Placebo mg/d mg/d mg/d mg/d

GZR 200 mg/d

GZR 400 mg/d

GZR 600 mg/d

GZR GZR 800 100 mg/d Placebo mg/d

GZR 200 mg/d

GZR 400 mg/d

GZR 600 mg/d

GZR 800 mg/d

10 GT1 4 6 46.2

5 GT1 4 1 48.4

5 GT1 3 2 55.8

5* GT1 1 4 35.0

5 GT1 3 2 48.4

5 GT1 2 3 51.0

5 GT1 3 2 42.4

5 GT1 4 1 47.2

15 GT1 7 8 46.7

5 GT3 NA NA 44.4

5 GT3 NA NA 31.4

5 GT3 NA NA 40.0

5 GT3 NA NA 42.8

5 GT3 NA NA 38.0

4* GT3 NA NA 44.8

9 1 6.71

5 0 6.08

5 0 6.61

5 0 6.70

4 1 5.90

5 0 6.74

5 0 6.30

5 0 6.90

15 0 6.53

5 0 6.58

5 0 6.03

5 0 5.93

5 0 6.28

5 0 6.73

4 0 6.31

NA ¼ Not applicable. * One HCV GT1-infected participant who received GZR 50 mg and one HCV GT3-infected participant who received GZR 800 mg were discontinued early from the study and therefore were excluded from these analyses.

W.W. Yeh et al.

5

Clinical Therapeutics

Log 10 HCV RNA Change From Baseline (IU/mL)

A

1 0

Placebo GT1a and GT1b: 5 mg

−1

GT1a: 10 mg −2

GT1a: 50 mg GT1b: 10 mg

−3

GT1b: 50 mg −4 −5 −6 0

1

2

3

4

5

6

7

8

9

10 11 12 13

Day

Log 10 HCV RNA Change From Baseline (IU/mL)

B

Wk 3

Mo 1

Mo 2

1 0

Placebo GT3: 10 mg

−1

GT3: 50 mg −2

GT3: 100 mg

−3 −4 −5 −6 0

1

2

3

4

5

6

7

8

9

10 11 12 13

Day

Wk 3

Mo 1

Mo 2

Figure 1. Mean reduction in hepatitis C virus (HCV) RNA from baseline after 5 days of different dosages of elbasvir (EBR) or matching placebo in HCV genotype (GT) 1- or GT3-infected participants. Serial blood samples after administration of EBR were obtained daily on treatment days 1 through 5; at 2, 8, and 12 hours after dosing on days 1 and 5 (“on-treatment”); and then on days 6, 6.5, 7, 8, 9, 10, 13, 26, 33, and 61 (“posttreatment”). Study days 1 and 5 are the first and last days of drug or placebo administration, respectively. Error bars are SEs. (A) HCV GT1a- or GT1b-infected participants; (B) mean reduction in HCV RNA from baseline after 5 days of different dosages of EBR or matching placebo in HCV GT3-infected participants. protocol-specified laboratory testing or unscheduled laboratory testing in the study with EBR. Vital signs, ECG parameters, and clinical laboratory tests were summarized according to dose.

RESULTS Efficacy of EBR Profiles of the mean change from baseline in log10 HCV RNA for each EBR dosage group in participants with chronic GT1a HCV infections are presented

6

graphically in Figure 1A. Mean viral load reductions in GT1a-infected participants were similar among all dose groups from 5 to 50 mg of EBR once daily. HCV GT1a RNA levels declined rapidly after the first dose of EBR and returned to baseline after EBR was stopped on day 5. At 50 mg of EBR once daily, the changes in HCV RNA from baseline for GT1a were –3.46 log10 at day 2, –3.90 log10 at day 3, –4.00 log10 at day 4, and –3.97 log10 at day 5 (see Supplemental Table I in the online version at https://doi.org/10. 1016/j.clinthera.2018.03.002). At day 13, the HCV

Volume ] Number ]

W.W. Yeh et al.

Table III. Summary statistics for hepatitis C virus (HCV) viral load decreases according to HCV genotype (GT) and dosage of grazoprevir (GZR) or elbasvir (EBR). Drug

GZR

GZR

EBR

EBR

EBR

Mean Maximum log10 HCV RNA Reduction (95% CI)†

Difference From Placebo (90% CI)†

HCV GT

Dose (mg)

N*

GT1

Placebo‡ 10 30 50 100 200 400 600 800

15 5 5 5 5 5 5 5 15

0.39 3.84 5.06 5.26 4.74 5.53 5.14 5.32 5.72

(0.04 to 0.73) (3.25 to 4.43) (4.47 to 5.66) (4.67 to 5.85) (4.15 to 5.33) (4.94 to 6.12) (4.55 to 5.73) (4.73 to 5.91) (5.38 to 6.07)

3.46 4.68 4.87 4.35 5.15 4.76 4.93 5.34

– (2.89 to 4.03) (4.11 to 5.25) (4.30 to 5.44) (3.78 to 4.92) (4.58 to 5.72) (4.19 to 5.33) (4.36 to 5.50) (4.93 to 5.74)

GT3

Placebo‡ 100 200 400 600 800

15 5 5 5 5 4

0.39 2.64 3.32 4.23 5.36 4.60

(0.05 (2.06 (2.75 (3.65 (4.79 (3.95

to 0.72) to 3.22) to 3.90) to 4.81) to 5.94) to 5.24)

2.25 2.94 3.84 4.98 4.21

– (1.70 to (2.38 to (3.29 to (4.42 to (3.60 to

GT1

Placebo‡ 5 (GT1a and GT1b) 10 (GT1b) 50 (GT1b)

8 5 5 5

0.54 4.15 4.44 5.21

(0.03 (3.50 (3.78 (4.56

to to to to

1.06) 4.81) 5.09) 5.87)

– 3.61 (2.92 to 4.30) 3.89 (3.20 to 4.58) 4.67 (3.98 to 5.36)

G1a

Placebo‡ 10 50

8 5 5

0.54 (0.07 to 1.02) 3.72 (3.12 to 4.31) 4.17 (3.57 to 4.77)

– 3.17 (2.55 to 3.80) 3.63 (3.00 to 4.26)

GT3

Placebo‡ 10 50 100

8 5 5 5

0.54 1.27 3.12 3.38

– 0.72 (–0.09 to 1.54) 2.57 (1.75 to 3.39) 2.84 (2.02 to 3.66)

(–0.07 to 1.16) (0.49 to 2.04) (2.34 to 3.89) (2.61 to 4.16)

2.81) 3.49) 4.40) 5.53) 4.81)

Note: Viral load quantitation was performed by using the Roche Cobas TaqMANs 2.0 assay, with a lower limit of quantitation (LOQ) of 25 IU/mL and a lower limit of detection (LOD) of 3.8 IU/mL (Roche Molecular Diagnostics, Pleasanton, California). Values below the LOQ (HCV RNA o25 IU/mL) were imputed as 0.5*(LOQ þ LOD). Values below the LOD (HCV RNA o3.8 IU/mL) were imputed as 0.5*LOD. ⁎ One GT1-infected participant who received GZR 50 mg and one GT3-infected participant who received GZR 800 mg were discontinued early from the study and were therefore excluded from these analyses. The GT1-infected participant who received 50 mg of GZR and discontinued early was replaced with another participant who received 50 mg of GZR. † Least-squares means (mean differences) and CIs obtained from the ANOVA model with maximum log10 HCV RNA reduction as response and a fixed effect for treatment. ‡ Placebo data from GT1- and GT3-infected participants were pooled for the analyses.

] 2018

7

Clinical Therapeutics RNA levels in GT1a-infected participants treated with 50 mg of EBR per day had rebounded to –1.29 log10 from baseline. At 50 mg of EBR per day, the mean maximum reductions in HCV RNA from baseline and the reduction in HCV RNA versus placebo were 4.17 and 3.63 log10 IU/mL, respectively, for GT1a-infected participants (Table III). No on-treatment viral breakthrough was observed. As reported previously, postbaseline NS5A resistance-associated substitutions (RASs) selected by different doses of EBR in GT1ainfected participants included M28A/T/V, Q30H/R, L31V/F/M, and Y93H/N/C/R. All of the NS5A RASs observed in GT1a-infected participants conferred a 45-fold potency reduction to EBR except Y93R.23 In general, there were no notable differences in terms of the types of RASs or the prevalence of RASs selected by using different dose levels of EBR. Profiles of the mean change from baseline in log10 HCV RNA for each EBR dosage group in participants with chronic GT1b HCV infections are presented graphically in Figure 1A. Mean maximum viral load reductions in GT1b-infected participants were similar among all dose groups from 5 to 50 mg of EBR once daily (Table III). HCV GT1b RNA levels declined rapidly after the first dose of 50 mg of EBR per day and remained low after the dosing of EBR stopped on day 5 (see Supplemental Table I in the online version at https://doi.org/10.1016/j.clinthera.2018.03.002). At 50 mg/d of EBR, the changes in HCV RNA from baseline for GT1b were –3.49 log10 at day 2, –3.88 log10 at day 3, –4.00 log10 at day 4, –4.19 log10 at day 5, and –4.81 log10 at day 13. At 10 mg of EBR per day, the change in HCV RNA in GT1b-infected participants had rebounded to –2.17 log10 from baseline at day 13 (Figure 1A). At 50 mg/d of EBR, the mean maximum reductions in HCV RNA from baseline and the reduction in HCV RNA versus placebo were 5.21 and 4.67 log10 IU/mL for GT1binfected participants, respectively. No on-treatment viral rebound was observed. As reported previously, postbaseline NS5A RASs selected by different doses of EBR in GT1b-infected participants included L28M, L31V/M/I, and Y93H.24 In general, there were no notable differences in terms of the types of RASs or the prevalence of RASs selected by using different dose levels of EBR. Profiles of the mean change from baseline in log10 HCV RNA for each EBR dosage group in participants with chronic GT3 HCV infections are presented

8

graphically in Figure 1B. HCV GT3 RNA levels declined rapidly after the first dose of EBR. At 50 mg of EBR per day, the changes in HCV RNA from baseline for GT3 were –2.48 log10 at day 2, –2.86 log10 at day 3, –2.78 log10 at day 4, and –2.41 log10 at day 53 (see Supplemental Table I in the online version at https://doi.org/10.1016/j.clinthera.2018.03.002). The change in HCV RNA in GT3-infected participants had rebounded to –0.63 log10 from baseline at day 13. At 50 mg of EBR per day, the mean maximum reductions in HCV RNA from baseline and the reduction in HCV RNA versus placebo were 3.12 and 2.57 log10 IU/mL (Table III). In GT3-infected participants, the magnitude of HCV RNA suppression was dose responsive from 10 to 50 mg of EBR once daily. Postbaseline NS5A RASs selected by different doses of EBR that were observed in GT3-infected participants included A30K, L31F/I, and Y93H. In general, there were no notable differences in terms of the types of RASs or the prevalence of RASs selected by different doses of EBR.23

Efficacy of GZR Profiles of the mean change from baseline in log10 HCV RNA for each GZR dosage group in participants with chronic GT1 HCV infections are presented graphically in Figure 2A. HCV RNA levels declined rapidly after the first few doses of GZR and remained low after dosing on day 7. Mean maximum viral load reductions in GT1-infected participants were similar among all dose groups from 30 to 800 mg of GZR once daily. After the initial rapid and profound decline from baseline, HCV RNA remained suppressed below the baseline concentration for 1 month beyond the treatment period in HCV GT1 groups. No ontreatment viral rebound was observed. At 100 mg of GZR per day, the predose changes in HCV RNA from baseline were –2.71 log10 at day 2, –3.65 log10 at day 3, –3.80 log10 at day 4, –4.10 log10 at day 5, –4.27 log10 at day 15, and –1.77 log10 at 1 month (see Supplemental Table II in the online version at https:// doi.org/10.1016/j.clinthera.2018.03.002). At 100 mg of GZR per day, the mean maximum reductions in HCV RNA from baseline and the reduction in HCV RNA versus placebo were 4.74 and 4.35 log10 IU/mL for GT1-infected participants, respectively (Table III). The most frequently observed postbaseline NS3 RASs occurring in 410% of the participants treated with GZR were at amino acid positions 168, 156, 56, and

Volume ] Number ]

W.W. Yeh et al.

Log 10 HCV RNA Change From Baseline (IU/mL)

A

1 0

Placebo GT1: 10 mg

−1

GT1: 30 mg −2

GT1: 50 mg GT1: 100 mg

−3

GT1: 200 mg −4

GT1: 400 mg GT1: 600 mg

−5

GT1: 800 mg −6

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15

Day

Log 10 HCV RNA Change From Baseline (IU/mL)

B

Wk 3

Mo 1

Mo 2

1 0

Placebo GT3: 100mg

−1

GT3: 200mg −2

GT3: 400 mg GT3: 600 mg

−3

GT3: 800 mg −4 −5 −6

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15

Day

Wk 3

Mo 1

Mo 2

Figure 2. Mean reduction in hepatitis C virus (HCV) RNA from baseline after 7 days of different dosages of grazoprevir (GZR) or matching placebo in HCV genotype (GT) 1- or GT3-infected participants. Serial blood samples for quantitation of plasma HCV RNA were obtained at screening and daily before each dose of GZR on treatment days 1 through 7; at 2, 8, and 12 hours after dosing on days 1 and 7 (“on-treatment”); and then on days 8, 9, 10, 12, 15, 21, 28, and 56 (“posttreatment”). Study days 1 and 7 are the first and last days of drug or placebo administration, respectively. Error bars are SEs. (A) GT1-infected participants; (B) GT3-infected participants.

155. Although these RASs occurred in both GT1aand GT1b-infected participants, they occurred in a higher percentage of the GT1a group compared with the GT1b group (eg, substitutions at amino acid 168, 65% GT1a-infected participants and 52% GT1binfected participants; amino acid 156, 27% GT1a and 19% GT1b; amino acid 56, 27% GT1a and 9.5% GT1b; amino acid 155, 23% GT1a and 19% GT1b).24 Postbaseline substitutions with a 45-fold decreased susceptibility to GZR (Y56H, R155W/T,

] 2018

A156T/G, and D168A/E/K/V) were detected in participants dosed with GZR.25 Profiles of the mean change from baseline in log10 HCV RNA for each GZR dosage group in participants with chronic GT3 HCV infections are presented graphically in Figure 2B. During dosing with GZR (4200 mg) in these participants, HCV GT3 RNA levels declined less quickly and to a lesser extent compared with GT1-infected participants. In GT3-infected participants, the magnitude of HCV

9

Clinical Therapeutics RNA suppression was dose dependent in the range of 100 to 600 mg of GZR once daily. No on-treatment viral rebound was observed. At 100 mg of GZR per day, the predose changes in HCV RNA from baseline were –0.30 log10 at day 2, –0.80 log10 at day 3, –1.46 log10 at day 4, –1.55 log10 at day 5, –1.73 at day 7, and –0.31 log10 at day 15 (see Supplemental Table III in the online version at https://doi.org/10.1016/j. clinthera.2018.03.002). The mean maximum reductions in HCV RNA from baseline and the reduction in HCV RNA versus placebo were 2.64 and 2.25 log10 IU/mL for GT3-infected participants, respectively (Table III). Only 1 GT3-infected patient had a postbaseline NS3 RAS (Q168Q/R) that does not affect the susceptibility to GZR (o5-fold decrease in EC50 to GZR).24,26

Safety Administration of EBR (5–100 mg) for 5 days was generally well tolerated in HCV-infected male participants. There were no discontinuations during this study. Eighteen (45%) participants reported adverse events after administration of EBR compared with 4 (50%) after administration of placebo (see Supplemental Table IV in the online version at https://doi.org/10.1016/j.clinthera.2018.03.002). Thirteen (33%) participants reported drug-related adverse events after administration of EBR compared with 2 (25%) after administration of placebo. There were no serious AEs, events of clinical interest, or deaths reported during the study. All adverse events reported were mild to moderate in intensity and resolved by the end of the study. The most common adverse event reported in the study was headache, which was reported by 12 (30%) participants who received EBR and 3 (38%) participants who received placebo. Three (3) participants experienced vital sign adverse events in this study, including 1 event of blood pressure increase after administration of 10-mg EBR and 1 event of pyrexia after administration of 50-mg EBR; these events were considered drug related. No clinically meaningful relationships were observed for differences between clinical laboratory values, vital signs, orthostatic vital signs, or ECG safety parameter values as a function of dose or treatment. Administration of GZR (10-800 mg) for 7 days was generally well tolerated in HCV-infected male participants. One GT1-infected participant who received GZR 50 mg withdrew consent on day 1 and

10

was replaced, and another GT3-infected participant who received GZR 800 mg was discontinued from the study for a protocol violation relating to alcohol intoxication before day 5 dosing. Thirty-eight (42%) participants who received GZR reported adverse events compared with 3 (20%) participants who received placebo (see Supplemental Table IV). The most common adverse event reported in the study was headache, which was reported by 18 (24%) participants who received GZR and 1 (7%) participant who received placebo. There were no serious adverse events, events of clinical interest, or deaths reported during the study. All adverse events reported were mild to moderate in intensity, and all resolved by the end of the study with the exception of 1 participant reporting ongoing pain in the extremities and abdominal pain, both of which were considered unrelated to study treatment by the study investigator. No trends were observed between adverse event incidence and increasing dose levels. No clinically meaningful relationships were observed for differences between clinical laboratory values, vital signs, orthostatic vital signs, or ECG safety parameter values as a function of dose or treatment. No laboratory AEs were reported during the course of the GZR study. Mean serum total bilirubin concentrations showed a trend toward an increase from pretreatment baseline to the end of the 7-day treatment period, but it did not exceed the upper limit of normal and subsequently declined to or below mean pretreatment baseline values. Review of laboratory data for individual participants showed that in no cases were the increases from baseline in total serum bilirubin preceded or accompanied by elevations in serum transaminase or alkaline phosphatase concentrations, suggesting that the observed bilirubin elevations were isolated and not associated with hepatocyte injury or with other markers of cholestasis.

DISCUSSION The results of monotherapy proof-of-concept studies with EBR and GZR for short durations in HCVinfected participants found that the in vitro picomolar potencies of EBR and GZR translate to substantial clinical antiviral activities in humans. In addition, these results provided preliminary data for the selection of the dosages of drugs to test in Phase II and III clinical studies that investigated cure rates for HCV

Volume ] Number ]

W.W. Yeh et al. infection. Both EBR and GZR are generally well tolerated and have pharmacokinetic profiles that are supportive of once-daily dosing.27 These studies were placebo controlled to allow for a critical examination of the safety of multiple daily dosing of EBR or GZR and also to ensure that any declines in HCV RNA in participants administered EBR or GZR were not due to random fluctuations in viral replication. In addition, the inclusion of HCV-infected participants who received placebo was helpful to determine if any adverse events were drug specific in the intended patient population. The number of participants in each group was chosen to expose a limited cohort of HCV-infected participants to these investigational medicines; thus, the risk of potential emergence of viral resistance would be minimized in the event that the compounds were not effective. The limited treatment durations of 5 or 7 days of monotherapy with EBR or GZR, respectively, were selected to provide similar clinical proof-of-concept data compared with other HCV DAA agents while minimizing the risk of the emergence of RASs that could reduce the efficacy of future treatment with an NS5A inhibitor– and protease inhibitor–containing DAA regimen.28–35 In the current studies, exposure to either EBR or GZR resulted in a rapid decline in HCV RNA in most participants, followed by viral rebound, as typically observed for short courses of DAA agents when administered as monotherapy in proof-of-concept studies. The EBR study showed that doses of 10 to 50 mg of EBR were associated with similar antiviral efficacy for the initial viral decline. However, it appears that participants infected with HCV GT1b virus responded better, with a more marked and sustained viral RNA decline than participants infected with HCV GT1a virus. This scenario is consistent with both the difference in the intrinsic potency of EBR for GT1a and GT1b replicons, and the higher level of resistance observed in vitro for GT1a variants.25 The treatmentemergent NS5A RASs observed in this study with EBR administered as monotherapy may explain the observed rebound in HCV RNA after dosing of EBR was stopped in GT1a-infected, but not in GT1binfected, participants. NS5A RASs were observed in GT1a-infected participants at M28A/T/V, Q30H/R, L31V/F/M, and Y93H/N/C/R. The majority of these GT1a RASs (M28A/T/V, Q30H/R, L31V/F/M, and Y93H/N/C) exhibited a 45-fold shift in EC50 to

] 2018

EBR.23 In contrast, NS5A RASs were observed in GT1b-infected participants in the EBR monotherapy study at L28M, L31V/M/I, and Y93H, but only the Y93H RAS in GT1b exhibited a 45-fold shift in EC50 to EBR. These results suggest that variants bearing high-impact (45-fold shift in EC50 to EBR) NS5A RASs in GT1a-infected participants are less susceptible to EBR and likely contributed to the rapid viral rebound to baseline levels after cessation of dosing. In contrast, the majority of NS5A RASs in GT1b-infected participants remain susceptible to EBR and likely accounted for the persistent low HCV RNA levels even after cessation of EBR dosing. In addition, 50 mg of EBR may provide better sustained suppression of GT1a compared with 10 mg of EBR based on the viral load rebound. In particular, the 10-mg dose of EBR was associated with a slight increase in viral load at the end of the active treatment period in GT1a-infected participants, whereas the 50-mg dose was not. Although 20 and 50 mg of EBR once daily for 12 weeks in combination with 100 mg GZR ± RBV resulted in similar efficacy in noncirrhotic, treatment-naive, GT1-infected participants in a Phase II dose-ranging study,15,19 50 mg of EBR once daily was selected as the dosage level in Phase III to ensure a margin for efficacy in more-difficult-to-treat patient populations (eg, GT1ainfected individuals with cirrhosis and/or baseline NS5A RAS). The data presented here suggest that doses of 50 mg of GZR per day and higher were on the maximum response plateau of the dose–response curve for GT1infected participants. Although comparisons across compounds and across studies are complicated by differences in dose, duration, and baseline viral load among other factors, the viral load–lowering effects observed with GZR seemed to be greater in magnitude and duration than those reported with other HCV protease inhibitors.29,31,36–38 In addition, the viral load suppression associated with GZR was prolonged after cessation of dosing and notable for the lack of on-treatment viral breakthrough. Although the largest mean maximum reductions in viral load were similar for GT1- and GT3-infected participants and there were reductions in GT3 HCV viral load, the GZR dose–response was different for these 2 GTs. In GT3infected participants, the GZR dose–response for viral load lowering was apparent at higher daily dose

11

Clinical Therapeutics levels, with decreased viral load–lowering activity at daily doses o400 mg. Because the mean viral dynamic effects in GT3-infected participants were reduced at daily GZR doses of 200 and 100 mg, daily doses o100 mg in GT3-infected participants were not studied further. Differences in the timing to viral load nadir and in the time to return to pretreatment baseline viral load were also observed between GT1and GT3-infected participants. For GT1-infected participants, mean viral load continued to decline even after cessation of dosing on day 7 at all daily dose levels of GZR except for the lowest dose level studied (10 mg once daily). For GT3-infected participants at all daily dose levels studied, mean viral load profiles started to rebound soon after cessation of GZR dosing. These clinical data are consistent with the  8- to 13-fold less potency of GZR against GT3 versus GT1 HCV in the in vitro replicon assay.25 In a Phase II study in GT1-infected participants, 25- to 800-mg doses of GZR coadministered with pegylated interferon and ribavirin demonstrated high efficacy rates.39,40 However, late elevations of ALT and/or AST levels were observed after treatment week 4 among a proportion of participants who received 200, 400, or 800 mg of GZR þ pegylated interferon and ribavirin but not in those who received 100 mg of GZR.40 The results from the Phase III study combined with the results from the GZR proof-of-concept study described in the present article suggest that 100 mg of GZR was associated with maximal antiviral efficacy in GT1-infected participants and provided a safety margin to a clinically acceptable risk of late ALT/AST elevations.39,40 Therefore, 100 mg of GZR was selected to be further evaluated in additional Phase II and III trials in combination with EBR. The reductions in HCV RNA in the GZR monotherapy study informed the differential efficacy rates in Phase II and III clinical trials with GZR. One hundred milligrams of GZR per day, which led to a robust reduction in HCV GT1 RNA in the present study (mean maximum log10 HCV RNA reduction of 4.74), was sufficient to achieve efficacy rates of 92% to 98% in Phase III clinical trials with EBR/GZR in GT1infected participants.14,17,18,20,41 However, the modest reduction in HCV RNA in GT3-infected participants with 100 mg of GZR per day in this study (mean maximum log10 HCV RNA reduction of 2.64) likely contributed to the lower efficacy rates of 45%

12

and 57% in a clinical trial of 12 and 18 weeks of EBR/GZR þ RBV in GT3-infected individuals, respectively.26 Overall, once-daily administration of EBR and GZR was generally safe and well tolerated. Of note, rapid declines from baseline in mean serum transaminase concentrations were observed during treatment with EBR and GZR. In participants who received GZR, there was a trend toward an increase from baseline in total serum bilirubin concentrations that peaked at the end of the 7-day treatment period but remained within normal limits and returned rapidly to or below pretreatment baseline during the first week off treatment. Total bilirubin was fractionated only when requested by the investigator, and elevations were predominantly in the indirect (ie, unconjugated) fraction in these samples. During the GZR treatment period, no changes in serum alkaline phosphatase concentrations occurred, and there were no clinical or laboratory indications of hemolysis. Taking all the data into consideration, these small elevations in serum bilirubin concentrations were likely caused by drug-mediated inhibition of transporter proteins and/or enzymes involved in the hepatic uptake and metabolism of bilirubin42 and were not associated with signs of hepatocellular damage or injury. This phenomenon has been reported for other HCV protease inhibitors in clinical development.43 Limitations of the current studies include a lack of ethnic and sex diversity in that the subjects who enrolled were predominantly males of white race. In addition, because the sample size for each dosing group was relatively small, the safety data are limited, and the estimates and the associated CIs for antiviral activity must be interpreted with caution. For example, it appears that the 600-mg dose of GZR produced a greater reduction in HCV RNA than the 800-mg dose of GZR in GT3-infected participants. The small sample size along with high interindividual variability in the GZR pharmacodynamics, particularly at the highest doses, may have contributed to these results. The SE for the 4 participants in the GT3 800-mg group (1 participant was discontinued from the study due to protocol violation) is the highest of all the dose cohorts in the GZR study. Lastly, the potent antiviral activities of EBR and GZR observed in these proof-ofconcept studies over a short duration of dosing may not necessarily translate into clinical cure for HCV after longer durations of treatment.

Volume ] Number ]

W.W. Yeh et al.

CONCLUSION Data from these clinical proof-of-concept monotherapy studies show that EBR and GZR administered once daily are potent inhibitors of HCV replication in humans and are well tolerated in treatment-naive GT1- and GT3-infected participants. These results provided strong rationale for the selection of clinical doses for further investigation in additional Phase II and III studies of EBR/GZR. The fixed-dose combination of EBR (50 mg/d) and GZR (100 mg/d) have been approved globally to treat HCV GT1- or GT4infected treatment-naive and treatment-experienced individuals, including those with comorbidities of cirrhosis, HIV infection, and chronic kidney disease, and those receiving opioid agonist therapy.11–13

ACKNOWLEDGMENTS The authors extend their gratitude to the participants, their families, investigators, and site personnel who participated in these studies. Michele McColgan of Merck & Co, Inc, provided editorial support. Drs. Yeh and Guo contributed to the conception, design, or planning of the study; acquisition and analysis of the data; interpretation of the results; and the drafting, critical review, or revision of the manuscript. Drs. Fraser and Butterton contributed to the conception, design, or planning of the study; interpretation of the results; and the critical review or revision of the manuscript. Drs. Van Dyck, Schwabe, and Wagner, and Ms. Jumes, Ms. Petry, and Mr. O’Mara contributed to the conception, design, or planning of the study; acquisition of data; and the critical review or revision of the manuscript. Dr. De Lepeleire and Ms. Panebianco contributed to the conception, design, or planning of the study; interpretation of the results; and the critical review or revision of the manuscript. Ms. Robberechts contributed to the conception, design, or planning of the study; acquisition of the data; interpretation of the results; and the critical review or revision of the manuscript. Drs. Moiseev, Kobalava, Visan, Ghicavii, and Wagner, and Ms. Reitmann contributed to the conception, design, or planning of the study; and the critical review or revision of the manuscript. Dr. Huang contributed to the conception, design, or planning of the study; analysis of the data; interpretation of the results; and the critical review or revision of the manuscript. Dr. Nachbar contributed to the analysis of the data, interpretation of the results,

] 2018

and the critical review or revision of the manuscript. Dr. Dutko contributed to the analysis of data, interpretation of results, and the drafting and critical review or revision of the manuscript. Dr. Gane contributed to the acquisition of data, interpretation of results, and the drafting of the manuscript. Dr. Popa contributed to the acquisition of data, interpretation of results, and the critical review or revision of the manuscript. Dr. Uhle and Mr. Hüser contributed to the acquisition of data and the critical review or revision of the manuscript.

DATA AVAILABILITY Merck & Co., Inc.’s data sharing policy, including restrictions, is available at http://engagezone.merck. com/ds_documentation.php. Requests for access to the study data can be submitted through the EngageZone site or via email to [email protected].

CONFLICTS OF INTEREST Funding for this research was provided by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc. Relevant employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., are authors. Details of their contributions are in the Acknowledgments. Drs. Yeh, Fraser, Huang, Guo, Nachbar, Wagner, Butterton, and Dutko and Ms. Jumes, Ms. Petry, Ms. Reitmann, Ms. Panebianco, and Mr. O'Mara are current or former employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., who may own stock and/or hold stock options in the company. Drs. De Lepeleire and Van Dyck and Ms. Robberrechts are current or former employees of MSD Brussels. Dr. Nachbar also reports being a consultant for Merck & Co, Inc., outside the submitted work. Dr. Schwabe reports grants from Merck & Co., Inc., during the conduct of the study; and grants from AbbVie, Gilead, and Janssen, outside the submitted work. Dr. Gane reports personal fees from Merck & Co., Inc., Gilead, AbbVie, and Alios, outside the submitted work. The authors have indicated that they have no other conflicts of interest regarding the content of this article.

SUPPLEMENTARY MATERIAL Supplemental tables accompanying this article can be found in the online version at https://doi.org/10.1016/ j.clinthera.2018.03.002.

13

Clinical Therapeutics

REFERENCES 1. World Health Organization (WHO). Global HCV Report. 2017. http://apps.who.int/iris/bitstream/ 10665/255016/1/9789241565455eng.pdf?ua=1. Accessed June 30, 2017. 2. Centers for Disease Control and Prevention. Hepatitis C FAQ's for health professionals. Centers for Disease Control and Prevention Web site. https://www.cdc.gov/ hepatitis/hcv/hcvfaq.htm. Accessed January 27, 2017. 3. Thein HH, Yi Q, Dore GJ, Krahn MD. Estimation of stage-specific fibrosis progression rates in chronic hepatitis C virus infection: a metaanalysis and meta-regression. Hepatology. 2008;48:418–431. 4. Lozano R, Naghavi M, Foreman K, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2095–2128. 5. Pearlman BL, Traub N. Sustained virologic response to antiviral therapy for chronic hepatitis C virus infection: a cure and so much more. Clin Infect Dis. 2011;52: 889–900. 6. van der Meer AJ, Veldt BJ, Feld JJ, et al. Association between sustained virological response and all-cause mortality among patients with chronic hepatitis C and advanced hepatic fibrosis. JAMA. 2012;308:2584–2593. 7. D'Ambrosio R, Degasperi E, Colombo M, Aghemo A. Direct-acting antivirals: the endgame for hepatitis C? Curr Opin Virol. 2017; 24:31–37. 8. American Association for the Study of Liver Diseases, Infectious Diseases Society of America. HCV guidance: recommendations for testing, managing, and treating hepatitis C. https://www.hcvguide lines.org. Updated September 21, 2017.

14

9. Summa V, Ludmerer SW, McCauley JA, et al. MK-5172, a selective inhibitor of hepatitis C virus NS3/ 4a protease with broad activity across genotypes and resistant variants. Antimicrob Agents Chemother. 2012;56:4161–4167. 10. Coburn CA, Meinke PT, Chang W, et al. Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity. ChemMedChem. 2013;8:1930–1940. European Medicines Agency. Zepat11. ier Summary of Product Characteristics. 2016. 12. Health Canada. Product monograph including patient medication information: Zepatier. Kirkland, QC, Canada: Merck Canada Inc; 2017. 13. Zepatier [package insert]. Kenilworth, NJ: Merck & Co, Inc; 2017. 14. Dore GJ, Altice F, Litwin AH, et al. Elbasvir-grazoprevir to treat hepatitis C virus infection in persons receiving opioid agonist therapy: a randomized trial. Ann Intern Med. 2016;165: 625–634. 15. Lawitz E, Gane E, Pearlman B, et al. Efficacy and safety of 12 weeks versus 18 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin for hepatitis C virus genotype 1 infection in previously untreated patients with cirrhosis and patients with previous null response with or without cirrhosis (C-WORTHY): a randomised, open-label phase 2 trial. Lancet. 2015;385:1075–1086. 16. Lawitz E, Poordad F, Gutierrez JA, et al. Short-duration treatment with elbasvir/grazoprevir and sofosbuvir for hepatitis C: a randomized trial. Hepatology. 2017;65: 439–450. 17. Rockstroh JK, Nelson M, Katlama C, et al. Efficacy and safety of grazoprevir (MK-5172) and elbasvir (MK-8742) in patients with hepatitis C virus and HIV co-infection (C-EDGE CO-INFECTION): a

18.

19.

20.

21.

22.

23.

24.

non-randomised, open-label trial. Lancet HIV. 2015;2:e319–e327. Roth D, Nelson DR, Bruchfeld A, et al. Grazoprevir plus elbasvir in treatment-naive and treatment-experienced patients with hepatitis C virus genotype 1 infection and stage 4-5 chronic kidney disease (the C-SURFER study): a combination phase 3 study. Lancet. 2015;386:1537–1545. Sulkowski M, Hezode C, Gerstoft J, et al. Efficacy and safety of 8 weeks versus 12 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin in patients with hepatitis C virus genotype 1 monoinfection and HIV/hepatitis C virus co-infection (C-WORTHY): a randomised, open-label phase 2 trial. Lancet. 2015;385:1087–1097. Zeuzem S, Ghalib R, Reddy KR, et al. Grazoprevir-elbasvir combination therapy for treatment-naive cirrhotic and noncirrhotic patients with chronic HCV genotype 1, 4, or 6 infection: a randomized trial. Ann Intern Med. 2015;163:1–13. Mangin E, Yeh WW, Caro L, et al. Safety and pharmacokinetics of single and multiple oral doses of MK-8742, an HCV NS5A inhibitor, in healthy subjects. Hepatology. 2017;58:444A. Brainard DM, Patry A, Anderson MS, et al. Safety, tolerability, and pharmacokinetics after single and multiple doses of MK-5172, a novel HCV NS3/4 protease inhibitor with potent activity against know resistance mutants, in healthy subjects. Hepatology. 2010;52:1216A–1217A. Liu R, Curry S, McMonagle P, et al. Susceptibilities of genotype 1a, 1b, and 3 hepatitis C virus variants to the NS5A inhibitor elbasvir. Antimicrob Agents Chemother. 2015;59:6922–6929. Strizki JM, Barnard RJO, Cheney C, et al. Evaluation of N53 amino acid variants in a phase 1B study

Volume ] Number ]

W.W. Yeh et al.

25.

26.

27.

28.

29.

30.

31.

of genotype 1 (GT1) and GT3 infected patients with HCV protease inhibitor, MK-5172. J Hepatol. 2012;56(suppl 1):S479. Lahser FC, Bystol K, Curry S, et al. The combination of grazoprevir, a hepatitis C virus (HCV) NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor, demonstrates a high genetic barrier to resistance in HCV genotype 1a replicons. Antimicrob Agents Chemother. 2016;60:2954–2964. Gane E, Nahass R, Luketic V, et al. Efficacy of 12 or 18 weeks of elbasvir plus grazoprevir with ribavirin in treatment-naive, noncirrhotic HCV genotype 3-infected patients. J Viral Hepat. 2017;24: 895–899. Center for Drug Evaluation and Research. FDA Summary Review. Zepatier. Report No.: 3874428. May 28, 2015. https://www.access data.fda.gov/drugsatfda_docs/ n d a / 2 016 / 2 0 8 2 61 O r i g 1 s 0 0 0 SumR.pdf. Gane EJ, Roberts SK, Stedman CA, et al. Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebocontrolled, dose-escalation trial. Lancet. 2010;376:1467–1475. Reesink HW, Fanning GC, Farha KA, et al. Rapid HCV-RNA decline with once daily TMC435: a phase I study in healthy volunteers and hepatitis C patients. Gastroenterology. 2010;138:913–921. Nettles RE, Gao M, Bifano M, et al. Multiple ascending dose study of BMS-790052, a nonstructural protein 5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1. Hepatology. 2011;54:1956–1965. Reesink HW, Zeuzem S, Weegink CJ, et al. Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-

] 2018

32.

33.

34.

35.

36.

37.

controlled, randomized study. Gastroenterology. 2006;131:997–1002. Lawitz E, Yang JC, Stamm LM, et al. Characterization of HCV resistance from a 3-day monotherapy study of voxilaprevir, a novel pangenotypic NS3/4A protease inhibitor. Antivir Ther. 2017. Oct 24. http://dx.doi.org/10.3851/ IMP3202. [Epub ahead of print]. Rodriguez-Torres M, Glass S, Hill J, et al. GS-9857 in patients with chronic hepatitis C virus genotype 1-4 infection: a randomized, doubleblind, dose-ranging phase 1 study. J Viral Hepat. 2016;23:614–622. Lawitz E, Freilich B, Link J, et al. A phase 1, randomized, dose-ranging study of GS-5816, a once-daily NS5A inhibitor, in patients with genotype 1-4 hepatitis C virus. J Viral Hepat. 2015;22:1011–1019. Lawitz EJ, Gruener D, Hill JM, et al. A phase 1, randomized, placebo-controlled, 3-day, doseranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C. J Hepatol. 2012;57:24–31. Forestier N, Larrey D, Guyader D, et al. Treatment of chronic hepatitis C patients with the NS3/4A protease inhibitor danoprevir (ITMN-191/ RG7227) leads to robust reductions in viral RNA: a phase 1b multiple ascending dose study. J Hepatol. 2011;54:1130–1136. Manns MP, Bourliere M, Benhamou Y, et al. Potency, safety, and pharmacokinetics of the NS3/4A protease inhibitor BI201335 in patients with chronic HCV genotype-

38.

39.

40.

41.

42.

43.

1 infection. J Hepatol. 2011;54: 1114–1122. Mederacke I, Wedemeyer H, Manns MP. Boceprevir, an NS3 serine protease inhibitor of hepatitis C virus, for the treatment of HCV infection. Curr Opin Invest Drugs. 2009;10:181–189. Lagging M, Brown A, Mantry PS, et al. Grazoprevir plus peginterferon and ribavirin in treatmentnaive patients with hepatitis C virus genotype 1 infection: a randomized trial. J Viral Hepat. 2016; 23:80–88. Caro L, Du L, Huang S, et al. Relationship between transaminase levels and plasma pharmacokinetics following administration of MK-5172 with pegylated interferon alfa-2b and ribavirin to HCV genotype G1 treatment-naive patients. J Hepatol. 2013;58:S328. Jacobson IM, Lawitz E, Kwo PY, et al. Safety and efficacy of elbasvir/grazoprevir in patients with hepatitis C virus infection and compensated cirrhosis: an integrated analysis. Gastroenterology. 2017; 152:1371–1382. Korenblat KM, Berk PD. Hyperbilirubinemia in the setting of antiviral therapy. Clin Gastroenterol Hepatol. 2005;3:303–310. Sane RS, Steinmann GG, Huang Q, et al. Mechanisms underlying benign and reversible unconjugated hyperbilirubinemia observed with faldaprevir administration in hepatitis C virus patients. J Pharmacol Exp Ther. 2014;351: 403–412.

Address correspondence to: Wendy W. Yeh, Merck & Co., Inc., BMB3411, 33 Ave Louis Pasteur, Boston, MA 02115-5727. E-mail: wendy. [email protected]

15

Clinical Therapeutics

SUPPLEMENTARY MATERIAL Power With respect to the power for antiviral activity of EBR or GZR, assuming that the true mean difference (comparing to placebo) in mean reduction in HCV RNA (log10) was 3.0 log10 for GT1 and 2.0 log10 for

GT3, with 5 participants receiving active dose and 6 participants receiving placebo, there would be at least 99% probability that the lower limit of the 90% confidence interval for the difference in mean maximum viral load reduction would exceed 0. Supplemental Tables I–IV

Supplemental Table I. Statistical summary of log10 HCV RNA and change from baseline following the administration of multiple oral doses of 50 mg EBR QD on Days 1–5 in GT1b-, GT1a-, and GT3-infected male participants. Log10 HCV RNA (IU/ml) HCV genotype Dose (mg) GT1a

GT1b

50

50

Day

Time Point

Day 1 Pre-dose Day 1 2 Hrs post-dose Day 1 4 Hrs post-dose Day 1 8 Hrs post-dose Day 1 12 Hrs post-dose Day 1 24 Hrs post-dose Day 2 36 Hrs post-dose Day 3 Pre-dose Day 4 Pre-dose Day 5 Pre-dose Day 5 2 Hrs post-dose Day 5 4 Hrs post-dose Day 5 8 Hrs post-dose Day 5 12 Hrs post-dose Day 5 24 Hrs post-dose Day 6 36 Hrs post-dose Day 7 48 Hrs post-dose Day 8 72 Hrs post-dose Day 9 96 Hrs post-dose Day 10 120 Hrs post-dose Day 13 192 Hrs post-dose 21 Days Post D5 Dose 3 Weeks post-dose 28 Days Post D5 Dose 1 Month post-dose 56 Days Post D5 Dose 2 Months post-dose Day 1 Pre-dose Day 1 2 Hrs post-dose Day 1 4 Hrs post-dose Day 1 8 Hrs post-dose Day 1 12 Hrs post-dose Day 1 24 Hrs post-dose

N Mean 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

6.97 6.82 5.99 4.49 4.01 3.51 3.28 3.08 2.97 3.01 2.97 2.914 2.86 2.90 3.02 3.09 3.18 3.50 4.01 4.40 5.68 6.14 6.58 6.58 6.55 6.50 5.68 4.14 3.55 3.07

SE 0.13 0.12 0.14 0.12 0.11 0.09 0.12 0.13 0.16 0.15 0.16 0.16 0.19 0.20 0.21 0.26 0.26 0.41 0.52 0.63 0.77 0.362 0.162 0.219 0.27 0.28 0.37 0.36 0.33 0.29

Log10 HCV RNA change from baseline Mean

SE

0.15 0.98 2.48 2.96 3.46 3.69 3.90 4.00 3.97 4.00 4.06 4.11 4.07 3.95 3.88 3.79 3.47 2.97 2.57 1.29 0.832 0.392 0.392

0.04 0.09 0.14 0.16 0.13 0.13 0.16 0.17 0.15 0.15 0.15 0.16 0.17 0.18 0.23 0.20 0.36 0.47 0.59 0.75 0.355 0.135 0.139

0.05 0.87 2.41 3.00 3.49

0.04 0.15 0.15 0.15 0.14 (continued)

15.e1

Volume ] Number ]

W.W. Yeh et al.

Supplemental Table I. (continued). Log10 HCV RNA (IU/ml) HCV genotype Dose (mg)

GT3

50

Day Day 2 Day 3 Day 4 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 13 21 Days 28 Days 56 Days Day 1 Day 1 Day 1 Day 1 Day 1 Day 1 Day 2 Day 3 Day 4 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 13 21 Days

Post D5 Dose Post D5 Dose Post D5 Dose

Post D5 Dose

Time Point 36 Hrs post-dose Pre-dose Pre-dose Pre-dose 2 Hrs post-dose 4 Hrs post-dose 8 Hrs post-dose 12 hours post-dose 24 hours post-dose 36 hours post-dose 48 hours post-dose 72 Hrs post-dose 96 Hrs post-dose 120 Hrs post-dose 192 Hrs post-dose 3 Weeks post-dose 1 Month post-dose 2 Months post-dose Pre-dose 2 Hrs post-dose 4 Hrs post-dose 8 Hrs post-dose 12 Hrs post-dose 24 Hrs post-dose 36 Hrs post-dose Pre-dose Pre-dose Pre-dose 2 Hrs post-dose 4 Hrs post-dose 8 Hrs post-dose 12 Hrs post-dose 24 Hrs post-dose 36 Hrs post-dose 48 Hrs post-dose 72 Hrs post-dose 96 Hrs post-dose 120 Hrs post-dose 192 Hrs post-dose 3 Weeks post-dose

N Mean 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

2.90 2.67 2.55 2.36 2.27 2.13 2.05 2.07 2.15 2.04 1.84 1.74 1.72 1.56 1.74 4.84 5.16 5.61 6.44 6.29 5.68 4.50 4.03 3.96 3.53 3.58 3.66 4.04 4.00 4.05 4.04 4.13 4.38 4.43 4.70 5.10 5.52 5.60 5.81 5.90

Log10 HCV RNA change from baseline

SE

Mean

SE

0.28 0.35 0.28 0.28 0.31 0.39 0.36 0.32 0.30 0.38 0.42 0.36 0.34 0.42 0.57 1.01 1.05 0.89 0.44 0.49 0.60 0.60 0.57 0.60 0.52 0.61 0.62 0.68 0.69 0.69 0.72 0.71 0.76 0.87 0.88 0.90 0.98 0.87 0.90 0.76

3.65 3.88 4.00 4.19 4.28 4.42 4.50 4.48 4.40 4.51 4.71 4.81 4.84 4.99 4.81 1.71 1.39 0.95

0.19 0.17 0.21 0.22 0.19 0.22 0.21 0.24 0.22 0.25 0.27 0.22 0.21 0.27 0.47 0.81 0.88 0.72

0.15 0.76 1.94 2.41 2.48 2.91 2.86 2.78 2.41 2.44 2.39 2.40 2.31 2.06 2.01 1.74 1.3 0.92 0.84 0.63 0.54

0.06 0.17 0.16 0.10 0.18 0.10 0.17 0.27 0.32 0.34 0.37 0.31 0.38 0.44 0.57 0.59 0.64 0.75 0.69 0.65 0.49 (continued)

] 2018

15.e2

Clinical Therapeutics

Supplemental Table I. (continued). Log10 HCV RNA (IU/ml) HCV genotype Dose (mg)

Day

Time Point

N Mean

28 Days Post D5 Dose 1 Month post-dose 5 6.16 56 Days Post D5 Dose 2 Months post-dose 5 6.34

Log10 HCV RNA change from baseline

SE

Mean

SE

0.59 0.49

0.29 0.10

0.33 0.12

Legend: Viral load quantitation was performed by the Roche Cobas TaqMAN® 2.0 assay, with a lower limit of quantitation (LOQ) of 25 IU/mL and a lower limit of detection (LOD) of 3.8 IU/mL (Roche Molecular Diagnostics, Pleasanton, CA). Results below the LOQ (HCV RNA o25 IU/mL) were imputed as 0.5*(LOQþLOD). Results below the LOD (HCV RNA o3.8 IU/mL) were imputed as 0.5*LOD.

15.e3

Volume ] Number ]

W.W. Yeh et al.

Supplemental Table II. Statistical summary of log10 HCV RNA and change from baseline following the administration of multiple oral doses of 100 mg GZR QD on Days 1–7 in GT1infected male participants. Baseline log10 HCV RNA (IU/mL) Log10 HCV RNA change from baseline Day

Time point

N

Mean

SE

Mean

SE

Day 1 Day 1 Day 1 Day 1 Day 2 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 7 Day 7 Day 7 Day 8 Day 8 Day 9 Day 10 Day 12 Day 15 Week 3 Month 1 Month 2

Predose 2 hrs postdose 8 hrs postdose 12 hrs postdose Predose 12 hrs postdose Predose Predose Predose Predose Pre-dose 2 hrs postdose 8 hrs postdose 12 Hrs post-dose 24 hrs postday 7 dose 36 hrs postday 7 dose 48 hrs postday 7 dose 72 hrs postday 7 dose 120 hrs postday 7 dose 192 hrs postday 7 dose

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5.90 5.89 4.98 4.38 3.19 2.59 2.24 2.09 1.79 1.80 1.62 1.72 1.58 1.58 1.53 1.56 1.32 1.34 1.44 1.63 2.72 4.13 5.71

0.22 0.24 0.29 0.32 0.27 0.24 0.32 0.22 0.28 0.27 0.24 0.26 0.21 0.26 0.23 0.26 0.33 0.38 0.29 0.47 1.06 0.95 0.47

0.00 0.01 0.92 1.52 2.71 3.31 3.65 3.80 4.10 4.10 4.27 4.17 4.31 4.32 4.37 4.33 4.58 4.56 4.45 4.27 3.17 1.77 0.19

0.00 0.03 0.09 0.13 0.17 0.09 0.24 0.02 0.12 0.13 0.09 0.10 0.08 0.11 0.09 0.12 0.13 0.21 0.24 0.38 0.87 0.77 0.27

Legend: Viral load quantitation was performed by the Roche Cobas TaqMAN® 2.0 assay, with a lower limit of quantitation (LOQ) of 25 IU/mL and a lower limit of detection (LOD) of 3.8 IU/mL (Roche Molecular Diagnostics, Pleasanton, CA). Results below the LOQ (HCV RNA o25 IU/mL) were imputed as 0.5*(LOQþLOD). Results below the LOD (HCV RNA o3.8 IU/mL) were imputed as 0.5*LOD.

] 2018

15.e4

Clinical Therapeutics

Supplemental Table III. Statistical summary of log10 HCV RNA and change from baseline following the administration of multiple oral doses of 100 mg GZR QD on days 1–7 in GT3infected male participants. Baseline Log10 HCV RNA (IU/mL) Log10 HCV RNA change from baseline Day

Time point

N

Mean

SE

Mean

SE

Day 1 Day 1 Day 1 Day 1 Day 2 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 7 Day 7 Day 7 Day 8 Day 8 Day 9 Day 10 Day 12 Day 15 Week 3 Month 1 Month 2

Predose 2 hrs postdose 8 hrs postdose 12 hrs postdose Predose 12 hrs postdose Predose Predose Predose Predose Predose 2 hrs postdose 8 hrs postdose 12 hrs postdose 24 hrs postday 7 dose 36 hrs postday 7 dose 48 hrs postday 7 dose 72 hrs postday 7 dose 120 hrs postday 7 dose 192 hrs postday 7 dose

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

6.03 6.00 5.85 5.32 5.73 5.27 5.23 4.56 4.48 4.25 4.29 4.02 3.81 3.70 4.33 4.38 4.49 4.85 5.45 5.71 6.16 6.30 6.03

0.37 0.51 0.67 0.63 0.56 0.63 0.74 0.67 0.74 0.61 0.64 1.01 0.84 0.81 0.79 0.81 0.73 0.80 0.77 0.73 0.41 0.42 0.32

0.00 0.03 0.17 0.71 0.30 0.75 0.80 1.46 1.55 1.78 1.73 2.01 2.21 2.32 1.70 1.65 1.54 1.17 0.58 0.31 0.13 0.27 0.00

0.00 0.20 0.39 0.31 0.28 0.31 0.40 0.34 0.39 0.28 0.33 0.69 0.50 0.47 0.48 0.46 0.41 0.48 0.53 0.37 0.24 0.17 0.31

Legend: Viral load quantitation was performed by the Roche Cobas TaqMAN® 2.0 assay, with a lower limit of quantitation (LOQ) of 25 IU/mL and a lower limit of detection (LOD) of 3.8 IU/mL (Roche Molecular Diagnostics, Pleasanton, CA). Results below the LOQ (HCV RNA o25 IU/mL) were imputed as 0.5*(LOQþLOD). Results below the LOD (HCV RNA o3.8 IU/mL) were imputed as 0.5*LOD.

15.e5

Volume ] Number ]

W.W. Yeh et al.

Supplemental Table IV. Safety of GZR, EBR versus placebo. EBR (5 days of dosing) N¼40 Adverse events, n* (%) Drug-related adverse events, n (%) Common adverse events Headache, n (%) Diarrhea, n (%) Flatulence, n (%) ⁎

Placebo (5 days of dosing) N¼8

GZR (7 days of dosing) N¼76

Placebo (7 days of dosing) N¼15

18 (45%) 13 (33%)

4 (50%) 2 (25%)

38 (42%) 21 (28%)

3 (20%) 2 (13%)

12 (30%) 1 (3%) 0 (0%)

3 (38%) 0 (0%) 0 (0%)

18 (24%) 9 (12%) 5 (7%)

1 (7%) 0 (0%) 1 (7%)

n¼number of participants who experienced the adverse event.

] 2018

15.e6