Advertisement

Safety and efficacy of vebicorvir administered with entecavir in treatment-naïve patients with chronic hepatitis B virus infection

Open AccessPublished:June 10, 2022DOI:https://doi.org/10.1016/j.jhep.2022.05.027

      Highlights

      • Complete suppression of HBV replication is essential for finite treatment regimens
      • Vebicorvir (VBR) is an HBV core protein inhibitor developed to treat chronic HBV
      • VBR interferes with 2 additional steps in HBV replication and thus complements NrtIs
      • VBR+ETV led to a significantly deeper reduction in HBV DNA and pregenomic RNA than ETV alone
      • VBR+ETV was associated with favorable safety and tolerability during 24 weeks of treatment

      Background & Aims

      Nucleos(t)ide reverse transcriptase inhibitors do not completely suppress HBV DNA in chronic HBV infection (cHBV). Vebicorvir (VBR) is an investigational core inhibitor that interferes with multiple aspects of HBV replication. This phase II trial evaluated the safety and efficacy of VBR in combination with entecavir (ETV) in treatment-naïve patients with cHBV.

      Methods

      HBeAg-positive, treatment-naïve patients without cirrhosis were randomised 1:1 in a double-blind manner to once-daily VBR 300 mg+ETV 0.5 mg or placebo (PBO)+ETV 0.5 mg for 24 weeks. The primary endpoint was change in mean log10 HBV DNA from Baseline to Week 12 and 24.

      Results

      All patients in both treatment groups (PBO+ETV: 12/12; VBR+ETV: 13/13) completed the study. At Week 12, VBR+ETV led to a greater mean (SD) reduction from Baseline in log10 IU/ml HBV DNA (–4.45 [1.03]) vs. PBO+ETV (–3.30 [1.18]; p = 0.0077). At Week 24, VBR+ETV led to a greater reduction from Baseline in log10 IU/ml HBV DNA (–5.33 [1.59]) vs. PBO+ETV (–4.20 [0.98]; p = 0.0084). Greater mean reductions in pregenomic RNA were observed at Week 12 and 24 in patients receiving VBR+ETV vs. PBO+ETV (p <0.0001 and p <0.0001). Changes in viral antigens were similar in both groups. No drug interaction between VBR and ETV was observed. Two patients experienced HBV DNA rebound during treatment, with no resistance breakthrough detected. The safety of VBR+ETV was similar to PBO+ETV. All treatment-emergent adverse events and laboratory abnormalities were Grade 1/2. There were no deaths, serious adverse events, or evidence of drug-induced liver injury.

      Conclusions

      In this 24-week study, VBR+ETV provided additive antiviral activity over PBO+ETV in treatment-naïve patients with cHBV, with a favourable safety and tolerability profile.

      Clinical trial number

      NCT03577171

      Lay summary

      Hepatitis B is a long-lasting viral infection of the liver. Current treatments can suppress hepatitis B virus but do not offer the opportunity of cure, hence, new treatment approaches are required. Herein, we show that the combination of the novel core inhibitor vebicorvir with an existing antiviral (entecavir) in treatment-naïve patients chronically infected with hepatitis B virus demonstrated greater antiviral activity than entecavir alone. Additionally, vebicorvir was safe and well tolerated. Thus, further studies evaluating its potential role in the treatment of chronic hepatitis B are warranted.

      Graphical abstract

      Keywords

      Introduction

      Worldwide, over 250 million individuals are estimated to be chronically infected with HBV, and an estimated 800,000 people die from HBV-related cirrhosis or hepatocellular carcinoma each year.
      Organization WH
      Global hepatitis report 2017.
      Nucleos(t)ide reverse transcriptase inhibitors (NrtIs) are administered orally and can reduce virion production through several mechanisms, including blocking the reverse transcription of pregenomic (pg)RNA to HBV DNA, preventing DNA elongation, and inhibiting HBV protein priming.
      Organization WH
      Guidelines for the prevention care and treatment of persons with chronic hepatitis B infection.
      • Fanning G.C.
      • Zoulim F.
      • Hou J.
      • Bertoletti A.
      Therapeutic strategies for hepatitis B virus infection: towards a cure.
      • Lok A.S.
      • Zoulim F.
      • Dusheiko G.
      • Ghany M.G.
      Hepatitis B cure: from discovery to regulatory approval.
      • Ohsaki E.
      • Suwanmanee Y.
      • Ueda K.
      Chronic hepatitis B treatment strategies using polymerase inhibitor-based combination therapy.
      • Jones S.A.
      • Murakami E.
      • Delaney W.
      • Furman P.
      • Hu J.
      Noncompetitive inhibition of hepatitis B virus reverse transcriptase protein priming and DNA synthesis by the nucleoside analog clevudine.
      They are generally well tolerated, and second-generation NrtIs (i.e., entecavir [ETV] and tenofovir) have high barriers to resistance.
      Organization WH
      Guidelines for the prevention care and treatment of persons with chronic hepatitis B infection.
      However, they do not completely suppress viral replication, with studies demonstrating that approximately 40% of HBeAg-positive patients and 10% of HBeAg-negative patients continue to have detectable levels of HBV DNA 1 year after treatment initiation.
      • Chan H.L.
      • Fung S.
      • Seto W.K.
      • Chuang W.-L.
      • Chen C.-Y.
      • Kim H.J.
      • et al.
      Tenofovir alafenamide versus tenofovir disoproxil fumarate for the treatment of HBeAg-positive chronic hepatitis B virus infection: a randomised, double-blind, phase 3, non-inferiority trial.
      • Chang T.T.
      • Gish R.G.
      • de Man R.
      • Gadano A.
      • Sollano J.
      • Chao Y.C.
      • et al.
      A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B.
      • Terrault N.A.
      • Bzowej N.H.
      • Chang K.M.
      • Hwang J.P.
      • Jonas M.M.
      • Murad M.H.
      AASLD guidelines for treatment of chronic hepatitis B.
      • Marcellin P.
      • Gane E.
      • Flisiak R.
      • Manns M.
      • Kaita K.
      • Gaggar A.
      • et al.
      Evidence for ongoing low-level viremia in patients with chronic hepatitis B receiving long-term nucleos(t)ide analog therapy: 1861.
      • Mak L.Y.
      • Huang Q.
      • Wong D.K.
      • Stamm L.
      • Cheung K.S.
      • Ko K.L.
      • et al.
      Residual HBV DNA and pgRNA viraemia is associated with hepatocellular carcinoma in chronic hepatitis B patients on antiviral therapy.
      Residual viraemia remains due to the inability of NrtIs to fully suppress HBV replication and eliminate HBV covalently closed circular (ccc)DNA, the primary template for HBV replication.
      • Lok A.S.
      • Zoulim F.
      • Dusheiko G.
      • Ghany M.G.
      Hepatitis B cure: from discovery to regulatory approval.
      ,
      • Wong D.K.-H.
      • Seto W.-K.
      • Fung J.
      • Ip P.
      • Huang F.-Y.
      • Lai C.-L.
      • et al.
      Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos (t) ide analogues of different potency.
      Therefore, long-term NrtI treatment is necessary for most patients.
      Organization WH
      Guidelines for the prevention care and treatment of persons with chronic hepatitis B infection.
      Core inhibitors are a novel class of small molecules being evaluated for the treatment of chronic HBV (cHBV).
      • Yan Z.
      • Wu D.
      • Hu H.
      • Zeng J.
      • Yu X.
      • Xu Z.
      • et al.
      Direct inhibition of hepatitis B e antigen by core protein allosteric modulator.
      Core inhibitors interfere with multiple aspects of the HBV replication cycle, including the capsid assembly process, pgRNA encapsidation, and the formation of new covalently closed circular DNA (cccDNA).
      • Diab A.
      • Foca A.
      • Zoulim F.
      • Durantel D.
      • Andrisani O.
      The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: implications for the development of HBc-targeting antivirals.
      • Mak L.-Y.
      • Wong D.K.-H.
      • Seto W.-K.
      • Lai C.-L.
      • Yuen M.F.
      Hepatitis B core protein as a therapeutic target.
      • Yuen M.-F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      Core inhibitors have demonstrated activity against known HBV genotypes, and the Baseline prevalence of resistant variants is low.
      • Zhou Z.
      • Hu T.
      • Zhou X.
      • Wildum S.
      • Garcia-Alcalde F.
      • Xu Z.
      • et al.
      Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) inhibit hepatitis B virus replication by different molecular mechanisms.
      ,
      • Berke J.M.
      • Tan Y.
      • Verbinnen T.
      • Dehertogh P.
      • Vergauwen K.
      • Vos A.
      • et al.
      Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro.
      Safety has been a concern for some experimental core inhibitors, which have been associated with gastrointestinal adverse events (AEs) and alanine aminotransferase (ALT) elevations.
      • Janssen H.
      • Hou J.
      • Asselah T.
      • Chan H.
      • Zoulim F.
      • Tanaka Y.
      • et al.
      Efficacy and safety results of the phase 2 JNJ-56136379 JADE study in patients with chronic hepatitis B: interim week 24 data.
      • Zhang H.
      • Zhu X.
      • Chen H.
      • Li X.
      • Hu Y.
      • Wu M.
      • et al.
      Safety, pharmacokinetics and anti-viral efficacy of novel core protein allosteric modifier GLS4 in patients with chronic hepatitis B: interim results from a 48 weeks phase 2a study.
      • Yuen M.F.
      • Zhou X.
      • Gane E.
      • Schwabe C.
      • Tanwandee T.
      • Feng S.
      • et al.
      Safety, pharmacokinetics, and antiviral activity of RO7049389, a core protein allosteric modulator, in patients with chronic hepatitis B virus infection: a multicentre, randomised, placebo-controlled, phase 1 trial.
      • Feng S.
      • Gane E.
      • Schwabe C.
      • Zhu M.
      • Triyatni M.
      • Zhou J.
      • et al.
      A five-in-one first-in-human study to assess safety, tolerability, and pharmacokinetics of RO7049389, an inhibitor of hepatitis B virus capsid assembly, after single and multiple ascending doses in healthy participants.
      Vebicorvir (VBR) is an orally administered, potent, selective, and pan-genotypic core inhibitor that induces altered, non-functional core protein assembly.
      • Yuen M.-F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      ,
      • Huang Q.
      • Cai D.
      • Yan R.
      • Li L.
      • Zong Y.
      • Guo L.
      • et al.
      Preclinical profile and characterization of the hepatitis B virus core protein inhibitor ABI-H0731.
      In a randomised, placebo (PBO)-controlled phase Ib study in patients with cHBV, VBR at doses up to 300 mg/day for 28 days was well tolerated, with no pattern of treatment-emergent AEs (TEAEs). VBR 100, 200, and 300 mg administered once daily demonstrated dose-dependent decreases in serum HBV DNA and HBV RNA, consistent with its mechanism of action.
      • Yuen M.-F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      By interfering with 2 additional steps in HBV replication, the addition of VBR to NrtI therapy has the potential to enhance antiviral activity in patients with cHBV while maintaining a high barrier to resistance.
      • Yuen M.-F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      The objective of this phase II study was to evaluate the safety and efficacy of VBR plus NrtI in treatment-naïve patients with HBeAg-positive cHBV and to test the hypothesis that the addition of VBR would lead to greater suppression of HBV viraemia.

      Materials and methods

      Study population and design

      This phase II multicentre, double-blind, PBO-controlled study (NCT03577171) evaluated VBR+ETV in treatment-naïve patients with cHBV. The study enrolled patients from 13 sites in 5 regions (USA, Canada, UK, New Zealand, and Hong Kong) between 21 June 2018 and 21 June 2019.
      Patients aged 18 to 70 years with cHBV infection were eligible if they were HBeAg positive, with HBV DNA ≥2 × 105 IU/ml and HBsAg >1,000 IU/ml at Screening. Patients were treatment-naïve (no prior treatment with standard of care or investigational agents for >4 weeks). Patients were without evidence of bridging fibrosis, cirrhosis, or advanced liver disease (meta-analysis of histological data in viral hepatitis score ≤2 by liver biopsy within 1 year of Screening or FibroScan ≤8 kPa within 3 months of Screening). Key inclusion and exclusion criteria appear in the supplementary information.
      Patients were randomly assigned to receive VBR 300 mg (Assembly Biosciences, South San Francisco, CA) or matching placebo plus ETV 0.5 mg (Accord Healthcare Inc., Durham, NC) (VBR+ETV or PBO+ETV) administered orally once daily for up to 24 weeks. Patients were randomised in a 1:1 ratio (VBR+ETV:PBO+ETV) by an Interactive Phone/Web Response System. Three patients were enrolled in the study with deviations to eligibility criteria (supplementary information). Efficacy and safety evaluations were conducted at Day 1 (Baseline) and treatment Weeks 2, 4, 8, 12, 16, 20, and 24.
      The study was conducted in compliance with the principles of the Declaration of Helsinki, International Council for Harmonisation guidelines, and all applicable US FDA regulations. Approval of the study protocol by the Institutional Review Board/Independent Ethics Committee was given, and patients gave written informed consent before initiating study procedures.

      Assays

      HBV genotyping was performed using the INNO-LiPa line probe assay (Covance laboratory; Innogenetics). Serum HBV DNA was quantified using the COBAS AmpliPrep–COBAS TaqMan HBV Test, version 2.0 (Roche Diagnostics, Pleasanton, CA, USA), with a lower limit of quantification (LLOQ) of 20 IU/ml and limit of detection (LOD) of 10 IU/ml. Serum HBV pgRNA was quantified using a quantitative reverse transcription-PCR method developed at Assembly Biosciences (Assembly Biosciences, South San Francisco, CA, USA), with an LLOQ of 135 U/ml.
      • Huang Q.
      • Yan R.
      • Cai D.
      • Tang X.
      • Xu X.
      • Colonno R.
      Development of a highly sensitive multiplex platform assay to monitor low levels of HBV DNA and pgRNA in samples from patients with chronic hepatitis B.
      This newly developed HBV pgRNA assay provides greater sensitivity than commercial assays (supplementary information).
      • Huang Q.
      • Yan R.
      • Cai D.
      • Tang X.
      • Xu X.
      • Colonno R.
      Development of a highly sensitive multiplex platform assay to monitor low levels of HBV DNA and pgRNA in samples from patients with chronic hepatitis B.
      HBeAg and HBsAg were quantified using the Architect i2000SR HBeAg and HBsAg assays, with an LLOQ of 0.11 IU/ml and 0.05 IU/ml, respectively (Abbott Diagnostics, Lake Forest, IL, USA). Hepatitis B core-related antigen (HBcrAg) was quantified using the Lumipulse G HBcrAg assay (Fujirebio, Malvern, PA, USA) with an LLOQ of 1 kU/ml. Resistance was monitored by population sequencing of the HBV core protein and polymerase reverse transcriptase regions (mutant detection limit ≥15%). The upper limit of normal (ULN) for ALT is 34 U/L for females and 43 U/L for males.

      Efficacy endpoints

      The co-primary efficacy endpoints were change in mean log10 HBV DNA from Baseline (Day 1) to Week 12 and Week 24. Secondary efficacy endpoints included the proportions of patients with abnormal ALT at Baseline who had normal ALT at Week 24; decline in HBV DNA to below the LLOQ at end of treatment; emergence of resistant HBV variants (per protocol, patients with persistently detectable HBV DNA on study were subject to resistance sequencing of the reverse transcriptase and core protein regions); change from Baseline at each time point for pgRNA, HBsAg, HBeAg, and HBcrAg; loss (defined as below LLOQ) or decline of HBsAg and HBeAg; HBsAg and HBeAg seroconversion (defined as loss of s/e antigen and appearance of s/e antibody); and achievement of “target not detected” for HBV DNA.

      Pharmacokinetic endpoints

      Pharmacokinetic samples for VBR and ETV were collected predose on Day 1 and at Weeks 2, 4, 12, and 24, and at Week 28 follow-up, with optional samples collected at 4 (±2) hours after dosing on Day 1, Week 2, and Week 4. Pharmacokinetic analyses included trough levels (where available) and assessment of ETV mean trough plasma concentrations for the VBR+ETV vs. PBO+ETV regimens.

      Safety endpoints

      Safety endpoints included TEAEs, premature discontinuations, and laboratory abnormalities. AEs were recorded from the time written informed consent was obtained until completion of the Week 36 follow-up visit or until 30 days after the last dose of study drug for patients who discontinued from follow-up. Verbatim descriptions of AEs were coded using the Medical Dictionary for Regulatory Affairs version 21.0. Safety was monitored by a data-monitoring committee, including a safety analysis after all patients completed Week 12.

      Statistical analysis

      This study used an intent-to treat (ITT) population to analyse efficacy-related endpoints between patients in different groups and a safety analysis set to analyse safety-related endpoints between treatment arms. The ITT population consisted of all randomised patients; the safety population included all randomised patients who received at least 1 dose of study drug.
      A sample size of 24 patients, randomised 1:1, allowed the study to have a 2-sided significance level of 0.05 and a power of 87.7% to detect a difference of at least 0.4 log10 (IU/ml) in the change from Baseline in HBV DNA level at Week 12 or Week 24 between the VBR+ETV and PBO+ETV groups. Power calculations under various mean differences in the change from Baseline in HBV DNA levels are presented in the supplementary information.
      For the co-primary endpoints, descriptive statistics were provided by treatment for change from Baseline in mean log10 HBV DNA, as well as observed values. Changes from Baseline to Week 12 and Week 24 were analysed with a linear mixed-effects repeated-measure model, which included fixed effects for treatment, visit, treatment-by-visit interaction, and Baseline value. At each post-Baseline time point, the number and percentage of patients with detectable (≥LLOQ [20 IU/ml] and <LLOQ target detected) vs. undetectable (<LOD [10 IU/ml]) HBV DNA was tabulated for each treatment group. For numerical summaries, HBV DNA <LLOQ was imputed to 19 IU/ml, <LOD was coded to 9 IU/ml, and HBV pgRNA <LLOQ (Assembly Biosciences) was coded to 134 U/ml. For HBV pgRNA and levels of HBV antigens, a repeated-measures analysis using observed data from all scheduled visits for change from Baseline was performed using the same linear mixed-effects repeated-measure model as the primary endpoint. Where applicable, efficacy endpoints are described with the difference in least squares mean (LSM) between treatment groups and 95% CIs. The number and percentage of patients with loss (defined as below LLOQ) or decline in HBsAg or HBeAg (≤0.5 log10 decrease from Baseline) was tabulated for all measured visits in the ITT population.
      Safety data were summarised using descriptive statistics and are presented as total number and percentage of patients experiencing an event. For ALT normalisation, the American Association for the Study of Liver Diseases ULN was used (25 U/L for females and 35 U/L for males).
      • Terrault N.A.
      • Lok A.S.
      • McMahon B.J.
      • Chang K.M.
      • Hwang J.P.
      • Jonas M.M.
      • et al.
      Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance.
      All CIs are 2-sided and use the Clopper-Pearson (exact binomial method) at 95% CI. Adjustments for multiplicity were not made in any analysis. Statistical analyses were performed using the SAS® software package version 9.4 or higher, unless otherwise specified.

      Results

      Baseline demographics and disease characteristics

      Thirty-three patients were screened, and 25 enrolled between July 2018 and June 2019. Reasons for exclusion and screening failure are summarised in the supplementary information. Of these, 13 received VBR+ETV, and 12 received PBO+ETV. All patients completed the 24-week study (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Patient disposition (ITT population).
      aTreatment-naïve patients with HBeAg-positive or -negative cHBV. cHBV, chronic HBV; ETV, entecavir; ITT, intent-to-treat; PBO, placebo; VBR, vebicorvir.
      Overall, Baseline demographic characteristics (Table 1) were well balanced between treatment groups: most patients were Asian (24/25 [96%]) and female (17/25 [68%]); mean age was 34.9 (all but 1 aged <65 years [range 20–66 years]). Baseline HBV DNA, HBV pgRNA, HBsAg, HBeAg, and HBcrAg levels were similar between treatment groups. All but 1 patient, 24/25 (96%), were negative for anti-HBe, and 22/25 (88%) were negative for anti-HBs. Additional details on patients who were positive for anti-HBe and anti-HBs are described in the supplementary information.
      Table 1Baseline demographic and disease characteristics (ITT population).
      PBO + ETV

      (n = 12)
      VBR + ETV

      (n = 13)
      Demographic
      Age (years), mean ± SD34.1 ± 11.435.7 ± 14.1
      Sex (Female)7 (58)10 (77)
      Race,
       Asian11 (92)13 (100)
       Black1 (8)0
       White00
       Other00
      BMI, (kg/m2), mean ± SD23.6 ± 4.223.0 ± 3.0
      Disease
      Years positive for HBV, mean ± SD11.4 ± 9.410.1 ± 8.0
      HBV genotype,
       A2 (17)0
       B4 (33)7 (54)
       C6 (50)5 (38)
       Unable to genotype01 (8)
      ALT, U/L, mean ± SD46.8 ± 31.665.9 ± 87.1
      HBV DNA, log10 IU/ml, mean ± SD
      For the purpose of numerical summaries, HBV DNA <20 IU/ml was coded to 19 IU/ml and <10 IU/ml was coded to 9 IU/ml.
      8.0 ± 1.07.9 ± 0.9
      HBV pgRNA, log10 U/ml, mean ± SD
      For the purpose of numerical summaries, pgRNA <135 U/ml was coded to 134 U/ml.
      7.4 ± 1.17.1 ± 1.0
      HBsAg, log10 IU/ml, mean ± SD4.7 ± 0.44.5 ± 0.5
      HBeAg, log10 IU/ml, mean ± SD2.5 ± 1.22.5 ± 0.8
      HBcrAg, log10 kU/ml, mean ± SD5.4 ± 1.05.5 ± 0.7
      Data shown as n (%) unless otherwise specified.
      ALT, alanine aminotransferase; ETV, entecavir; HBcrAg, hepatitis B core-related antigen; ITT, intent-to-treat; PBO, placebo; pgRNA, pregenomic RNA; ULN, upper limit of normal; VBR, vebicorvir.
      a For the purpose of numerical summaries, HBV DNA <20 IU/ml was coded to 19 IU/ml and <10 IU/ml was coded to 9 IU/ml.
      b For the purpose of numerical summaries, pgRNA <135 U/ml was coded to 134 U/ml.

      Change from Baseline in HBV DNA and HBV pgRNA

      Changes from Baseline to Week 12 and Week 24 in mean HBV DNA (log10 IU/ml) were significantly greater for patients treated with VBR+ETV vs. PBO+ETV at Week 12 (LSM −4.454 [95% CI –5.029, –3.878] vs. −3.300 [95% CI −3.899, –2.701]; p = 0.0077; LSM difference = –1.154) and Week 24 (LSM −5.338 [95% CI –5.914, –4.762] vs. −4.197 [95% CI −4.797, –3.598]; p = 0.0084; LSM difference = –1.141) (Fig. 2; Table S1).
      Figure thumbnail gr2
      Fig. 2Mean HBV DNA (log10 IU/ml) change from Baseline over time and individual patient data (ITT population).
      (A) LS mean change from Baseline (95% CI) and (B) individual patient HBV DNA through 24 weeks (top panels). Levels of significance: ∗∗p <0.01. (linear mixed-effects repeated-measure model). Bottom panels show individual patient HBV DNA data. The differences in the number of samples tested per time point reflect missing samples and samples not tested due to haemolysis or receipt at the laboratory beyond stability. LS, least squares; ETV, entecavir; ITT, intent-to-treat; PBO, placebo; VBR, vebicorvir.
      A higher proportion of patients treated with VBR+ETV achieved HBV DNA <20 IU/ml at Week 24 than those receiving PBO+ETV: 3/13 (23%) vs. 1/12 (8%) (Fig. S1). Time to HBV DNA <LLOQ was shorter in the VBR+ETV group; the first VBR-treated patient to achieve HBV DNA <LLOQ did so at Week 8 vs. Week 20 for the first patient in the PBO+ETV group. No patient in either treatment group achieved HBV DNA target not detected (<10 IU/ml) at Week 24.
      Changes from Baseline in mean HBV pgRNA (log10 U/ml) were significantly greater for patients treated with VBR+ETV vs. PBO+ETV at all time points: Week 12 (LSM –2.280 [95% CI –2.643, –1.916] vs. –0.427 [95% CI –0.805, –0.048]; p <0.0001) and Week 24 (LSM −2.327 [95% CI –2.691, –1.964] vs. −0.631 [–1.009, –0.252]; p <0.0001) (Fig. 3; Table S1). Patients receiving VBR+ETV showed greater initial mechanism-based reductions in mean HBV pgRNA than those receiving PBO+ETV: Week 2, −1.940 for VBR+ETV vs. −0.152 for PBO+ETV (p <0.001). This initial reduction was followed by a slower second-phase decline over the remainder of the treatment period at each time point to Week 24 (Fig. 3). One patient (VBR+PBO) experienced undetectable pgRNA at Week 16.
      Figure thumbnail gr3
      Fig. 3Mean HBV pgRNA (log10 U/ml) change from Baseline over time and individual patient data (ITT population).
      (A) LS mean change from Baseline (95% CI) and (B) individual patient HBV pgRNA through 24 weeks. Level of significance: ∗∗∗p <0.0001 (linear mixed-effects repeated-measure model; top panels). Bottom panels show individual patient HBV DNA data. The differences in the number of samples tested per time point reflect missing samples and samples not tested due to haemolysis or receipt at the laboratory beyond stability. ETV, entecavir; ITT, intent-to-treat; LS, least squares; PBO, placebo; pgRNA, pregenomic RNA; VBR, vebicorvir.

      Change from Baseline in serum viral antigens

      Mean changes from Baseline to Week 24 were similar between treatment groups for all HBV antigens (Fig. 4; Table S1). Most patients experienced decreases from Baseline in HBsAg, HBeAg, HBcrAg, and pgRNA (Fig. S2). No patient achieved HBsAg or HBcrAg loss (<LLOQ) at Week 24. One patient (PBO+ETV) achieved HBeAg loss at Week 24; this patient entered the study with low HBeAg (0.24 IU/ml at Screening and 0.18 IU/ml on Day 1). No patient achieved HBsAg or HBeAg seroconversion.
      Figure thumbnail gr4
      Fig. 4HBsAg, HBeAg, and HBcrAg mean changes from Baseline to Week 24 (ITT population).
      HBsAg and HBeAg were not measured at Week 2 per protocol. HBcrAg was included as an exploratory assessment at Week 2 (linear mixed-effects repeated-measure model). Antigens were not different from each other at any timepoint in this study (p >0.05). The differences in the number of samples tested per time point reflect missing samples and samples not tested due to haemolysis or receipt at the laboratory beyond stability. ETV, entecavir; HBcrAg, hepatitis B core-related antigen; ITT, intent-to-treat; LS, least squares; PBO, placebo; VBR, vebicorvir.

      Normalisation of ALT

      At Baseline, 6/13 (46%) and 7/12 (58%) VBR+ETV and PBO+ETV patients had ALT ≤ULN; at Week 24, those proportions were 12/13 (92%) for VBR+ETV compared with 5/12 (42%) for PBO+ETV (Fig. 5). Outside of Baseline, the proportion of patients with ALT ≤ULN was greater at all timepoints in VBR+NrtI compared to PBO+NrtI patients (Fig. S3). In addition, most of the patients who received VBR+ETV experienced a numerical decrease in ALT from Baseline at Week 24 (Fig. S4).
      Figure thumbnail gr5
      Fig. 5Normalisation of ALT (ITT population).
      ULN (American Association for the Study of Liver Diseases) = 25 U/L for females and 35 U/L for males. ALT, alanine aminotransferase; BL, Baseline; ETV, entecavir; ITT, intent-to-treat; PBO, placebo; ULN, upper limit of normal; VBR, vebicorvir; Wk, week.

      Emergence of resistant HBV variants

      One patient in each treatment group experienced HBV DNA rebound (≥1 log10 increase in HBV DNA from nadir) during treatment and underwent sequencing. The patient receiving VBR+ETV experienced an increase in HBV DNA from 4.29 log10 IU/ml at Week 16 to 6.17 and 7.31 log10 IU/ml at Weeks 20 and 24. This patient continued to the follow-up period on ETV alone, exhibiting 4.82 and 4.08 log10 IU/ml HBV DNA reductions at Weeks 28 and 36, respectively. The PBO+ETV patient experienced an increase from 5.40 log10 IU/ml HBV DNA at Week 8 to 7.46, 7.09, and 7.01 log10 IU/ml at Weeks 12, 16, and 20, respectively. HBV DNA values returned to 5.95 log10 IU/ml at Week 24 in this patient. No known drug-resistant substitutions were found in the core protein or reverse transcriptase region of the polymerase gene for either patient. Additional information on these 2 patients is provided in the supplementary information.

      Pharmacokinetic plasma concentrations

      When administered with ETV, mean trough plasma concentrations of VBR remained consistent throughout the 24-week study (range: 1,270–1,480 ng/ml), with coefficient of variation (CV%) generally remaining constant (range: 31.0–37.3) (Fig. S5). Findings were consistent with patients receiving VBR 300 mg in the phase Ib study (NCT02908191): Week 2 mean [CV%]: 1,450 ng/ml [38.9]).
      • Yuen M.-F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      With few exceptions, mean trough plasma concentrations of ETV were similar between both treatment groups: VBR+ETV, 0.378–0.432 ng/ml; PBO+ETV, 0.408–0.666 ng/ml (Table S2; Fig. S6). These data indicate no clinically significant drug interactions between VBR and ETV at the doses studied.

      Safety

      During the study, 7/13 (54%) patients in the VBR+ETV group and 5/12 (42%) in the PBO+ETV group experienced a TEAE. There were no serious TEAEs, TEAEs that led to treatment discontinuation, or deaths (Table 2). A total of 14 and 13 TEAEs were reported in the VBR+ETV and PBO+ETV groups, respectively; of these, all but 2 were Grade 1 (both Grade 2 TEAEs were ALT increases). TEAEs that occurred in ≥1 patient were upper respiratory tract infection, pruritis, ALT increase, and headache (Table 2). No TEAE occurred in more than 2 patients in either group. Skin and subcutaneous tissue disorder TEAEs were reported in 3/13 (23%) patients treated with VBR+ETV and 1/12 (8%) who received PBO+ETV. Each patient experienced 1 event of pruritis, acne, or skin irritation. No AE of rash was reported in either treatment group. Some TEAEs experienced by patients who received VBR+ETV were considered treatment-related—pruritis (2 patients) and ALT increase, dizziness, dyspepsia, and headache (each in 1 patient). In the patients with dizziness, dyspepsia, headache, and 1 patient with pruritis, TEAEs had resolved by the time of study conclusion. The ALT increase resolved with sequelae after study completion in the affected patient; pruritis was not resolved in the second affected patient by the end of study. No patients in the PBO+ETV treatment group experienced a treatment-related TEAE.
      Table 2Treatment-emergent AEs (safety population).
      Preferred termPBO+ETV (n = 12)VBR+ETV (n = 13)
      Patients with any AE5 (42)7 (54)
      Deaths00
      Patients with AE leading to study drug discontinuation00
      Patients with Grade 3 or 4 AEs00
      Patients with serious AEs00
      AEs occurring in ≥1 patient in either treatment group
       Rash00
       URTI1 (8)1 (8)
       Pruritus02 (15)
       ALT increased2 (17)1 (8)
       Headache02 (15)
      Grade 1Grade 2Grade 1Grade 2
      Patients with any laboratory abnormalities, n (%)5 (42)5 (42)5 (38)3 (23)
       AST (U/L) – High3 (25)2 (17)2 (15)1 (8)
       ALT (U/L) – High2 (17)3 (25)1 (8)1 (8)
       Amylase (U/L) – High1 (8)2 (17)1 (8)1 (8)
       Glucose (mmol/L) – High2 (17)01 (8)1 (8)
       Glucose (mmol/L) – Low1 (8)01 (8)0
       Urate (μmol/L) – High1 (8)01 (8)0
       Calcium (mmol/L) – Low001 (8)0
       Lipase (U/L) – High01 (8)00
       PT/INR – High1 (8)000
       Haemoglobin (g/L) – Low1 (8)000
      Data shown as n (%).
      AE, adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ETV, entecavir; PBO, placebo; PT/INR, prothrombin international normalisation ratio; URTI, upper respiratory tract infection; VBR, vebicorvir.
      All treatment-emergent laboratory abnormalities were Grade 1 or Grade 2 (Table 2). Among patients who had normal ALT levels at Baseline, ALT elevations were observed in 2 patients in the VBR+ETV group (1 Grade 1 and 1 Grade 2) and in 5 patients in the PBO+ETV group (2 Grade 1 and 3 Grade 2). High aspartate aminotransferase, amylase, and glucose were the only other results that were abnormal in more than 1 patient in either treatment group. No abnormal findings in vital signs or electrocardiogram were identified in either treatment group.

      Discussion

      Core inhibitors hold promise as a novel treatment in cHBV, as capsid assembly and disassembly are critical steps in virus production as well as generation of cccDNA.
      • Pei Y.
      • Wang C.
      • Yan S.F.
      • Liu G.
      Past, current, and future developments of therapeutic agents for treatment of chronic hepatitis B virus infection.
      VBR is a novel core inhibitor that induces altered, non-functional core protein assembly.
      • Yuen M.-F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      ,
      • Huang Q.
      • Cai D.
      • Yan R.
      • Li L.
      • Zong Y.
      • Guo L.
      • et al.
      Preclinical profile and characterization of the hepatitis B virus core protein inhibitor ABI-H0731.
      In this randomised, placebo-controlled phase II trial, we tested the hypothesis that blocking additional steps in the HBV replication cycle through the addition of VBR to ETV would lead to greater virological response than entecavir alone in treatment-naïve patients with HBeAg-positive cHBV. The co-primary endpoints were met, with VBR+ETV resulting in significantly greater reductions in HBV DNA from Baseline at Weeks 12 and 24 compared to PBO+ETV. Patients receiving VBR+ETV demonstrated faster and deeper levels of HBV DNA suppression than those receiving PBO+ETV. At Week 24, more patients receiving VBR+ETV achieved undetectable HBV DNA than those receiving PBO+ETV. Patients receiving VBR+ETV also demonstrated significantly greater decreases in pgRNA. The contribution of VBR to ETV was seen as early as Week 2, with decreases in HBV DNA and pgRNA at all subsequent visits through Week 24. The only source of pgRNA is cccDNA, and consequently, this marker represents the most direct measure of cccDNA transcriptional activity.
      Declines in HBV pgRNA observed with VBR appear to be biphasic. A similar profile has been seen with other core inhibitors in development. There appears to be an initial approximate 2-log10 reduction in HBV pgRNA, which is thought to represent the formation of empty capsids that do not contain pgRNA.
      • Zoulim F.
      • Lenz O.
      • Vandenbossche J.J.
      • Talloen W.
      • Verbinnen T.
      • Moscalu I.
      • et al.
      JNJ-56136379, an HBV capsid assembly modulator, is well-tolerated and has antiviral activity in a phase 1 study of patients with chronic infection.
      The first phase decline is considered related to the pharmacodynamic effect of the core inhibitor and is therefore considered mechanism-based. The second and more shallow phase decline is postulated to represent decreased cccDNA transcriptional activity.
      Most patients administered VBR+ETV experienced a numerical decrease in ALT from Baseline at Week 24. Furthermore, the percentage of VBR+ETV patients who had ALT ≤ULN doubled over the course of treatment, whereas this percentage decreased in PBO+ETV patients during the study (Fig. 5). Mean reductions in HBV antigens were similar between treatments and, at some time points, were numerically greater in patients receiving VBR+ETV vs. PBO+ETV. The depth of viral suppression and impact on antigens may be further increased with longer-term treatment.
      Safety and efficacy findings in the study are consistent with those from the phase Ib study.
      • Yuen M.-F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      In the current study, no pattern of TEAEs was associated with VBR 300 mg, all AEs were mild or moderate in intensity, and there were no deaths or serious AEs. The most common TEAEs in either treatment group were ALT increase, headache, and pruritus, with none occurring in more than 2 patients in either group. All TEAEs and treatment-emergent laboratory abnormalities were Grade 1 or 2. No patient discontinued the study due to a TEAE. Importantly, no pattern of hepatotoxicity occurred with VBR+ETV. Among patients who had normal ALT levels at Baseline, ALT elevations were Grade 1 or 2, similar between treatment groups, and consistent with the natural history of patients with cHBV with active viral replication. This is in contrast to Grade 3 or 4 ALT elevations reported with some other core inhibitors.
      • Sevinsky H.T.X.
      • Tang S.
      • Brown J.
      • Kunta J.
      • Dugyala R.
      • Picchio G.
      • et al.
      Evaluation or relationships between AB-506 related ALT elevations and AB-506 pharmacokinetics (PK), metabolite concentrations and plasma bile acids.
      ,
      • Yuen M.-F.
      • Schwabe C.
      • Tanwandee T.
      • Jin Y.
      • Gao L.
      • Zhou X.
      • et al.
      RO7049389, a core protein allosteric modulator, demonstrates robust decline in HBV DNA and HBV RNA in chronic HBV infected patients.
      Patients in a companion study examining virologically suppressed cHBV patients on NrtIs were also given VBR (NCT03576066). With the exception of rash, for which incidence was higher in virologically suppressed patients receiving VBR+NrtI (12 total events) compared to no reported events in the current study, safety and tolerability findings were similar in patients receiving VBR in both studies.
      Antiviral drug resistance to NrtIs results from mutations in the HBV polymerase gene.
      • Pawlotsky J.M.
      • Dusheiko G.
      • Hatzakis A.
      • Lau D.
      • Lau G.
      • Liang T.J.
      • et al.
      Virologic monitoring of hepatitis B virus therapy in clinical trials and practice: recommendations for a standardized approach.
      As agents with other mechanisms of action are developed, adaptive mutations may emerge in other gene sequences. Viruses that demonstrate amino acid substitutions may result in treatment failure and reduced susceptibility to the drug being used, a reflection of antiviral resistance.
      • Pawlotsky J.M.
      • Dusheiko G.
      • Hatzakis A.
      • Lau D.
      • Lau G.
      • Liang T.J.
      • et al.
      Virologic monitoring of hepatitis B virus therapy in clinical trials and practice: recommendations for a standardized approach.
      Resistance is associated with prolonged use of NrtIs and is influenced by several factors, including high pretreatment HBV DNA level, high serum levels of ALT, and inadequate viral suppression with therapy.
      • Papatheodoridis G.V.
      • Dimou E.
      • Papadimitropoulos V.
      Nucleoside analogues for chronic hepatitis B: antiviral efficacy and viral resistance.
      ,
      • Zoulim F.
      • Locarnini S.
      Hepatitis B virus resistance to nucleos (t) ide analogues.
      Because core inhibitors and NrtIs have distinct mechanisms of action, resistance to this treatment combination is also expected to be very low.
      • Lok A.S.
      • Zoulim F.
      • Dusheiko G.
      • Ghany M.G.
      Hepatitis B cure: from discovery to regulatory approval.
      ,
      • Mak L.-Y.
      • Wong D.K.-H.
      • Seto W.-K.
      • Lai C.-L.
      • Yuen M.F.
      Hepatitis B core protein as a therapeutic target.
      There were no cases of resistance breakthrough in VBR+ETV patients who were compliant with assigned treatment. The 2 cases of HBV DNA rebound observed in the study (1 in each treatment group) were likely the result of noncompliance rather than resistance, as low plasma concentrations of VBR were periodically observed (data not shown), and no known drug resistance variants were found in the core protein or reverse transcriptase region of the polymerase gene. Importantly, as VBR is being developed for concomitant use with NrtIs, no clinically meaningful drug interactions were observed between VBR and ETV at the dose levels studied.
      This study has several limitations. No stratification factors were utilised for treatment assignment. Consequently, there are numerical differences in Baseline disease characteristics between subgroups, with interpretation limited by the small sample size. Exploration of these potential differences with respect to treatment outcomes would need to be evaluated in future studies of larger sample size. Although viral suppression (assessed by both HBV DNA and pgRNA) and tolerability of VBR+ETV were demonstrated, a 24-week treatment duration may not be long enough to demonstrate the full extent of antiviral activity or to reveal potential AEs that may emerge after prolonged treatment. In particular, HBV-related biomarkers (such as HBcrAg, a surrogate biomarker for cccDNA transcriptional activity
      • Wong D.K.
      • Seto W.K.
      • Cheung K.S.
      • Chong C.K.
      • Huang F.Y.
      • Fung J.
      • et al.
      Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA.
      ,
      • Chen E.Q.
      • Feng S.
      • Wang M.L.
      • Liang L.B.
      • Zhou L.Y.
      • Du L.Y.
      • et al.
      Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B.
      ) may not show changes from Baseline until longer term changes in cccDNA are established. Previous studies have shown that although antiviral therapy can rapidly decrease HBV DNA to undetectable levels, HBcrAg decline may lag and take several months or years to reach a similar magnitude of decline.
      • Wong D.K.
      • Seto W.K.
      • Cheung K.S.
      • Chong C.K.
      • Huang F.Y.
      • Fung J.
      • et al.
      Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA.
      ,
      • Rokuhara A.
      • Tanaka E.
      • Matsumoto A.
      • Kimura T.
      • Yamaura T.
      • Orii K.
      • et al.
      Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core-related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment.
      ,
      • Wang L.
      • Cao X.
      • Wang Z.
      • Gao Y.
      • Deng J.
      • Liu X.
      • et al.
      Correlation of HBcrAg with intrahepatic hepatitis B virus total DNA and covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients.
      Changes in HBcrAg may also not be observed due to the small sample size in this study. Patients in this trial may enrol in an open-label extension study (NCT03780543) and receive VBR plus NrtI for up to 148 weeks. Finally, as patients had minimal hepatic fibrosis and almost all were of Asian descent with HBV genotype B or C, these findings cannot be normalised to a global population of patients with cHBV.
      In summary, once-daily VBR 300 mg plus ETV 0.5 mg resulted in no deaths or AEs leading to discontinuation. All TEAEs and laboratory abnormalities were Grade 2 or less in severity. There were no drug interactions observed between VBR and ETV, nor was there evidence of resistance breakthrough in treatment-compliant patients. When added to ETV, interference of the additional steps in HBV replication by VBR resulted in significant additive reductions in HBV DNA and pgRNA and increased rates of ALT normalisation over ETV monotherapy.

      Abbreviations

      AEs, adverse events; ALT, alanine aminotransferase; cccDNA, covalently closed circular DNA; cHBV, chronic HBV; CV%, percent coefficient of variation; ETV, entecavir; HBcrAg, hepatitis B core-related antigen; ITT, intent-to-treat; LLOQ, lower limit of quantification; LOD, limit of detection; LSM, least squares mean; NrtI, nucleos(t)ide reverse transcriptase inhibitor; PBO, placebo; pgRNA, pregenomic RNA; TEAEs, treatment-emergent adverse events; ULN, upper limit of normal; VBR, vebicorvir.

      Financial support

      This study was sponsored and funded by Assembly Biosciences, South San Francisco, CA.

      Authors’ contributions

      All authors approved the final manuscript prior to submission. Mark S. Sulkowski contributed to study oversight, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript. Kosh Agarwal contributed to study oversight, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript. Xiaoli Ma contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Tuan T. Nguyen contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Eugene R. Schiff contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Hie-Won L. Hann contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Douglas T. Dieterich contributed study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Ronald G. Nahass contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. James S. Park contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Sing Chan contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Steven-Huy Han contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Edward J. Gane contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Michael Bennett contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Katia Alves contributed to data interpretation and critical revision of the manuscript. Marc Evanchik contributed to the study concept and design, experiments and procedures, data analysis, data interpretation, and critical revision of the manuscript. Ran Yan contributed to experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Qi Huang contributed to the study concept and design, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript. Uri Lopatin contributed to the study concept and design, study oversight, data analysis, data interpretation, and critical revision of the manuscript. Richard Colonno contributed to the study concept and design, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript. Julie Ma contributed to data acquisition data analysis, data interpretation, and critical revision of the manuscript. Steven J. Knox contributed to study oversight, data analysis, data interpretation, and critical revision of the manuscript. Luisa M. Stamm contributed to study oversight, data analysis, data interpretation, and critical revision of the manuscript. Maurizio Bonacini contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Ira M. Jacobson contributed to study oversight, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript. Walid S. Ayoub contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Frank Weilert contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Natarajan Ravendhran contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Alnoor Ramji contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Paul Yien Kwo contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Magdy Elkhashab contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Tarek Hassanein contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Ho S. Bae contributed to study oversight, experiments and procedures, data acquisition, data interpretation, and critical revision of the manuscript. Jacob P. Lalezari contributed to study oversight, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript. Scott K. Fung contributed to study oversight, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript. Man-Fung Yuen contributed to study oversight, experiments and procedures, data acquisition, data analysis, data interpretation, and critical revision of the manuscript.

      Data availability statement

      Data can be made available to researchers upon reasonable request.

      Conflict of interest

      Mark S. Sulkowski reports receiving grants from AbbVie, Assembly Biosciences, Gilead Sciences, Janssen, and the National Institutes of Health; and receiving personal fees from AbbVie, Antios Therapeutics, Arbutus Biopharma, Atea Pharmaceuticals, Gilead Sciences, FH360, and Immunocore. Kosh Agarwal reports being on the advisory board, a consultant, and a speaker for AbbVie, Assembly Biosciences, Aligos, Arbutus, Bristol-Myers Squibb, Gilead Sciences, Immunocore, Janssen, Merck, Novartis, Roche, Sobi, Shinoigi, and Vir; and receiving grants from Bristol-Myers Squibb, Gilead Sciences, and Roche. Xiaoli Ma reports being a consultant and being on the speakers bureau for Gilead Sciences. Tuan T. Nguyen reports receiving research grant support from Gilead Sciences and Assembly Biosciences. Eugene R. Schiff reports receiving research and grant support from Assembly Biosciences, Celgene, and the University of Florida (TARGET) and receives royalties from the Schiff Diseases of the Liver, 12th edition. Hie-Won L. Hann reports serving on the National Advisory Board and receives research grant support from Gilead Sciences. Douglas T. Dieterich reports being a consultant for Gilead Sciences and Intercept Pharmaceuticals. Ronald G. Nahass reports having served on advisory boards and as a speaker for Gilead Sciences, Merck, and Janssen; and having conducted research for Gilead Sciences, Merck, Janssen, and AbbVie. James S. Park reports receiving research grants from Assembly Biosciences and GlaxoSmithKline and consulting fees from Gilead Sciences. Sing Chan reports receiving clinical trial–related payments from Assembly Biosciences. Steven-Huy Han reports being a consultant and being on the speakers bureau for Gilead Sciences. Edward J. Gane reports serving on advisory boards for AbbVie, Aligos Therapeutics, Arbutus Biopharma, Arrowhead Pharmaceuticals, Assembly Biosciences, Avilia Therapeutics, Clear B Therapeutics, Dicerna, Enanta Pharmaceuticals, Finch Therapeutics, Gilead Sciences, GlaxoSmithKline, Immunocore, Janssen, Roche, Silverback, and Vir Bio; and having served as a speaker for Gilead Sciences, AbbVie, and Roche. Michael Bennett reports having no conflicts of interest. Katia Alves reports being a former employee of and holding stock interest in Assembly Biosciences. Marc Evanchik reports having been an employee of and holding stock interest in Assembly Biosciences and is currently an employee of Edgewise Therapeutics. Ran Yan reports being an employee of and holding stock interest in Assembly Biosciences. Qi Huang reports being an employee of and holding stock interest in Assembly Biosciences. Uri Lopatin reports being a former employee and holding stock interest in Assembly Biosciences. Richard Colonno reports being an employee of and holding stock interest in Assembly Biosciences. Julie Ma reports being an employee of and holding stock interest in Assembly Biosciences. Steven J. Knox reports being an employee of and holding stock interest in Assembly Biosciences. Luisa M. Stamm reports being an employee of and holding stock interest in Assembly Biosciences. Maurizio Bonacini reports being a member of the speaking bureau for Intercept Pharmaceuticals, Gilead Sciences, and AbbVie and has received research support from Assembly Biosciences, Intercept Pharmaceuticals, Viking Therapeutics, and Boehringer Ingelheim. Ira M. Jacobson reports being a consultant or on advisory boards for AbbVie, Aligos Therapeutics, Arbutus Biopharma, Gilead Sciences, Janssen, Madrigal and Virion; having conducted research (all payments to institution) for Assembly Biosciences, Bristol-Myers Squib, Eli Lilly, Enanta Pharmaceuticals, Gilead Sciences, Janssen, Merck, and Novo Nordisk; receiving payment from the Chronic Liver Disease Foundation for manuscript preparation; and reports participation on a Data Safety Monitoring Board for GSK, Redhill, Galmed, NeuroBo, and Arrowhead Pharmaceuticals. Walid S. Ayoub reports being a member of the speaking bureau for both Gilead Sciences and Intercept Pharmaceuticals and has conducted research for Assembly Biosciences, Intercept Pharmaceuticals, Enanta Pharmaceuticals, and Gilead Sciences. Frank Weilert reports being a study investigator for AbbVie. Natarajan Ravendhran reports advising, being on the speakers’ bureau for, and receiving grants from Gilead Sciences and AbbVie; being on the speakers' bureau for Salix and Onyx; and having received grants from Bristol-Myers Squibb and Merck. Alnoor Ramji reports receiving grant support, lecture fees, and advisory board fees from AbbVie, Celgene, Gilead Sciences, Intercept Pharmaceuticals, Novartis, and Merck. Paul Yien Kwo reports being an advisor/consultant for AbbVie, Aligos Therapeutics, Antios Therapeutics, Enanta Pharmaceuticals, Gilead Sciences, Janssen, and receives grant/research supports from Assembly Biosciences, Arrowhead Pharmaceuticals, Eiger Biopharmaceuticals, Bristol-Myers Squibb, Altimmune, and Target Registries. Magdy Elkhashab reports receiving grants from AbbVie, Bristol-Myers Squibb, Eisai, Gilead Sciences, and Roche; and serving on advisory boards for AbbVie, Bristol-Myers Squibb, Gilead Sciences, and Merck. Tarek Hassanein reports being on the advisory committee, review panel, or consulting for AbbVie, Bristol-Myers Squibb, Gilead Sciences, Mallinckrodt Pharmaceuticals, Merck, and Organovo and receiving research support from AbbVie, Allergan, Cytodyn, Assembly Biosciences, Astra Zeneca, Boehringer Ingelheim, Bristol-Myers Squibb, CARA, DURECT Corporation, Enanta Pharmaceuticals, Galectin Therapeutics, Gilead Sciences, Grifols, Intercept Pharmaceuticals, Janssen, Merck, Mirum, Novartis, Novo Nordisk, Nucorion Pharmaceuticals, Pfizer, Salix Pharmaceuticals, Sonic Incytes, Terns Pharmaceuticals, and Valeant. Ho S. Bae reports having consultancy agreements with and receiving research support from Bristol-Myers Squibb and Gilead Sciences. Jacob P. Lalezari reports having no conflicts of interest. Scott K. Fung reports receiving fees for speaking and teaching and/or serving on advisory committees for AbbVie, Assembly Biosciences, Gilead Sciences, Janssen, and Springbank Pharma. Man-Fung Yuen reports being an advisor/consultant for and/or having received grant/research support from AbbVie, Aligos Therapeutics, Antios Therapeutics, Arbutus Biopharma, Arrowhead Pharmaceuticals, Assembly Biosciences, Bristol-Myers Squibb, Clear B Therapeutics, Dicerna Pharmaceuticals, Finch Therapeutics, Fujirebio Incorporation, GlaxoSmithKline, Gilead Sciences, Immunocore, Janssen, Merck Sharp and Dohme, Roche, Springbank Pharmaceuticals, Silverback Therapeutics, Sysmex Corporation and Vir Biotechnology.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      We would like to express our gratitude to all the patients, investigators, and site staff who participated in this study. Writing and editorial support was provided by Gregory Suess, PhD, of AlphaScientia, LLC, San Francisco, CA, USA, and was funded by Assembly Biosciences, South San Francisco, CA, USA.

      Supplementary data

      The following are the supplementary data to this article:

      References

        • Organization WH
        Global hepatitis report 2017.
        World Health Organization, 2017
        • Organization WH
        Guidelines for the prevention care and treatment of persons with chronic hepatitis B infection.
        World Health Organization, 2015
        • Fanning G.C.
        • Zoulim F.
        • Hou J.
        • Bertoletti A.
        Therapeutic strategies for hepatitis B virus infection: towards a cure.
        Nat Rev Drug Discov. 2019; 18: 827-844
        • Lok A.S.
        • Zoulim F.
        • Dusheiko G.
        • Ghany M.G.
        Hepatitis B cure: from discovery to regulatory approval.
        Hepatology. 2017; 66: 1296-1313
        • Ohsaki E.
        • Suwanmanee Y.
        • Ueda K.
        Chronic hepatitis B treatment strategies using polymerase inhibitor-based combination therapy.
        Viruses. 2021; 13
        • Jones S.A.
        • Murakami E.
        • Delaney W.
        • Furman P.
        • Hu J.
        Noncompetitive inhibition of hepatitis B virus reverse transcriptase protein priming and DNA synthesis by the nucleoside analog clevudine.
        Antimicrob Agents Chemother. 2013; 57: 4181-4189
        • Chan H.L.
        • Fung S.
        • Seto W.K.
        • Chuang W.-L.
        • Chen C.-Y.
        • Kim H.J.
        • et al.
        Tenofovir alafenamide versus tenofovir disoproxil fumarate for the treatment of HBeAg-positive chronic hepatitis B virus infection: a randomised, double-blind, phase 3, non-inferiority trial.
        Lancet Gastroenterol Hepatol. 2016; 1: 185-195
        • Chang T.T.
        • Gish R.G.
        • de Man R.
        • Gadano A.
        • Sollano J.
        • Chao Y.C.
        • et al.
        A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B.
        N Engl J Med. 2006; 354: 1001-1010
        • Terrault N.A.
        • Bzowej N.H.
        • Chang K.M.
        • Hwang J.P.
        • Jonas M.M.
        • Murad M.H.
        AASLD guidelines for treatment of chronic hepatitis B.
        Hepatology. 2016; 63: 261-283
        • Marcellin P.
        • Gane E.
        • Flisiak R.
        • Manns M.
        • Kaita K.
        • Gaggar A.
        • et al.
        Evidence for ongoing low-level viremia in patients with chronic hepatitis B receiving long-term nucleos(t)ide analog therapy: 1861.
        Hepatology. 2014; 60
        • Mak L.Y.
        • Huang Q.
        • Wong D.K.
        • Stamm L.
        • Cheung K.S.
        • Ko K.L.
        • et al.
        Residual HBV DNA and pgRNA viraemia is associated with hepatocellular carcinoma in chronic hepatitis B patients on antiviral therapy.
        J Gastroenterol. 2021; 56: 479-488
        • Wong D.K.-H.
        • Seto W.-K.
        • Fung J.
        • Ip P.
        • Huang F.-Y.
        • Lai C.-L.
        • et al.
        Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos (t) ide analogues of different potency.
        Clin Gastroenterol Hepatol. 2013; 11 (e1001): 1004-1010
        • Yan Z.
        • Wu D.
        • Hu H.
        • Zeng J.
        • Yu X.
        • Xu Z.
        • et al.
        Direct inhibition of hepatitis B e antigen by core protein allosteric modulator.
        Hepatology. 2019; 70: 11-24
        • Diab A.
        • Foca A.
        • Zoulim F.
        • Durantel D.
        • Andrisani O.
        The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: implications for the development of HBc-targeting antivirals.
        Antivir Res. 2018; 149: 211-220
        • Mak L.-Y.
        • Wong D.K.-H.
        • Seto W.-K.
        • Lai C.-L.
        • Yuen M.F.
        Hepatitis B core protein as a therapeutic target.
        Expert Opin Ther Targets. 2017; 21: 1153-1159
        • Yuen M.-F.
        • Agarwal K.
        • Gane E.J.
        • Schwabe C.
        • Ahn S.H.
        • Kim D.J.
        • et al.
        Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
        Lancet Gastroenterol Hepatol. 2020; 5: 152-166
        • Zhou Z.
        • Hu T.
        • Zhou X.
        • Wildum S.
        • Garcia-Alcalde F.
        • Xu Z.
        • et al.
        Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) inhibit hepatitis B virus replication by different molecular mechanisms.
        Sci Rep. 2017; 7: 1-12
        • Berke J.M.
        • Tan Y.
        • Verbinnen T.
        • Dehertogh P.
        • Vergauwen K.
        • Vos A.
        • et al.
        Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro.
        Antivir Res. 2017; 144: 205-215
        • Janssen H.
        • Hou J.
        • Asselah T.
        • Chan H.
        • Zoulim F.
        • Tanaka Y.
        • et al.
        Efficacy and safety results of the phase 2 JNJ-56136379 JADE study in patients with chronic hepatitis B: interim week 24 data.
        J Hepatol. 2020; 73: S129-S130
        • Zhang H.
        • Zhu X.
        • Chen H.
        • Li X.
        • Hu Y.
        • Wu M.
        • et al.
        Safety, pharmacokinetics and anti-viral efficacy of novel core protein allosteric modifier GLS4 in patients with chronic hepatitis B: interim results from a 48 weeks phase 2a study.
        Hepatology. 2018; 2018: 1454A-1455A
        • Yuen M.F.
        • Zhou X.
        • Gane E.
        • Schwabe C.
        • Tanwandee T.
        • Feng S.
        • et al.
        Safety, pharmacokinetics, and antiviral activity of RO7049389, a core protein allosteric modulator, in patients with chronic hepatitis B virus infection: a multicentre, randomised, placebo-controlled, phase 1 trial.
        Lancet Gastroenterol Hepatol. 2021;
        • Feng S.
        • Gane E.
        • Schwabe C.
        • Zhu M.
        • Triyatni M.
        • Zhou J.
        • et al.
        A five-in-one first-in-human study to assess safety, tolerability, and pharmacokinetics of RO7049389, an inhibitor of hepatitis B virus capsid assembly, after single and multiple ascending doses in healthy participants.
        Antimicrob Agents Chemother. 2020; 64 (e01323-1320)
        • Huang Q.
        • Cai D.
        • Yan R.
        • Li L.
        • Zong Y.
        • Guo L.
        • et al.
        Preclinical profile and characterization of the hepatitis B virus core protein inhibitor ABI-H0731.
        Antimicrob Agents Chemother. 2020; 64 (e01463-1420)
        • Huang Q.
        • Yan R.
        • Cai D.
        • Tang X.
        • Xu X.
        • Colonno R.
        Development of a highly sensitive multiplex platform assay to monitor low levels of HBV DNA and pgRNA in samples from patients with chronic hepatitis B.
        J Hepatol. 2020; 73: S596-S597
        • Terrault N.A.
        • Lok A.S.
        • McMahon B.J.
        • Chang K.M.
        • Hwang J.P.
        • Jonas M.M.
        • et al.
        Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance.
        Hepatology. 2018; 67: 1560-1599
        • Pei Y.
        • Wang C.
        • Yan S.F.
        • Liu G.
        Past, current, and future developments of therapeutic agents for treatment of chronic hepatitis B virus infection.
        J Med Chem. 2017; 60: 6461-6479
        • Zoulim F.
        • Lenz O.
        • Vandenbossche J.J.
        • Talloen W.
        • Verbinnen T.
        • Moscalu I.
        • et al.
        JNJ-56136379, an HBV capsid assembly modulator, is well-tolerated and has antiviral activity in a phase 1 study of patients with chronic infection.
        Gastroenterology. 2020; 159 (e529): 521-533
        • Sevinsky H.T.X.
        • Tang S.
        • Brown J.
        • Kunta J.
        • Dugyala R.
        • Picchio G.
        • et al.
        Evaluation or relationships between AB-506 related ALT elevations and AB-506 pharmacokinetics (PK), metabolite concentrations and plasma bile acids.
        in: The international workshop on clinical pharmacology of HIV, hepatitis, and other antiviral drugs. Virtual, 2020
        • Yuen M.-F.
        • Schwabe C.
        • Tanwandee T.
        • Jin Y.
        • Gao L.
        • Zhou X.
        • et al.
        RO7049389, a core protein allosteric modulator, demonstrates robust decline in HBV DNA and HBV RNA in chronic HBV infected patients.
        Sci HBV Cure. 2019;
        • Pawlotsky J.M.
        • Dusheiko G.
        • Hatzakis A.
        • Lau D.
        • Lau G.
        • Liang T.J.
        • et al.
        Virologic monitoring of hepatitis B virus therapy in clinical trials and practice: recommendations for a standardized approach.
        Gastroenterology. 2008; 134: 405-415
        • Papatheodoridis G.V.
        • Dimou E.
        • Papadimitropoulos V.
        Nucleoside analogues for chronic hepatitis B: antiviral efficacy and viral resistance.
        Am J Gastroenterol. 2002; 97: 1618-1628
        • Zoulim F.
        • Locarnini S.
        Hepatitis B virus resistance to nucleos (t) ide analogues.
        Gastroenterology. 2009; 137 (e1592): 1593-1608
        • Wong D.K.
        • Seto W.K.
        • Cheung K.S.
        • Chong C.K.
        • Huang F.Y.
        • Fung J.
        • et al.
        Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA.
        Liver Int. 2017; 37: 995-1001
        • Chen E.Q.
        • Feng S.
        • Wang M.L.
        • Liang L.B.
        • Zhou L.Y.
        • Du L.Y.
        • et al.
        Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B.
        Sci Rep. 2017; 7: 173
        • Rokuhara A.
        • Tanaka E.
        • Matsumoto A.
        • Kimura T.
        • Yamaura T.
        • Orii K.
        • et al.
        Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core-related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment.
        J Viral Hepat. 2003; 10: 324-330
        • Wang L.
        • Cao X.
        • Wang Z.
        • Gao Y.
        • Deng J.
        • Liu X.
        • et al.
        Correlation of HBcrAg with intrahepatic hepatitis B virus total DNA and covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients.
        J Clin Microbiol. 2019; 57