S. De Meyer

Mycophenolic acid, an immunosuppressive agent, inhibits HBV replication in vitro

Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA), is currently used as an immunosuppressive agent in kidney transplant recipients. After oral administration, MMF is hydrolysed to MPA, the active compound, which is a potent inhibitor of inosine monophosphate dehydrogenase (IMP‐DH). Inhibition of this enzyme results in a depletion of the intracellular GTP and dGTP pools. MPA has been shown to inhibit the replication of a number of viruses, including arena viruses (Junin and Tacaribe), yellow fever virus, reovirus‐1, parainfluenza‐3 virus, Coxsackie B4 virus, Epstein–Barr virus and human immunodeficiency virus. To examine whether MPA also has an inhibitory effect on HBV replication, experiments were performed using cultures of primary human hepatocytes and HBV‐transfected, HepG2 2.2.15 cells. After in vitro infection with HBV in human hepatocytes, HBV covalently‐closed‐circular (ccc) DNA and HBV mRNAs were detectable in the cells during the 10 days following infection. HBV DNA and hepatitis B surface antigen (HBsAg) were also secreted into the culture medium. In the presence of 10 μg ml–1 MPA (the therapeutic serum level of MPA as an immunosuppressive agent) in culture medium, HBV ccc DNA and HBV mRNAs became undetectable 5 days after treatment was started. The secretion of HBV DNA and HBsAg into the medium was also markedly reduced. No cytotoxic effect of the drug was noted during the experiments. The effect of MPA on HBV replication was abolished by the presence of guanosine (50 μg ml–1). In HepG2 2.2.15 cells (which contain an integrated tandem dimer of the HBV genome), MPA treatment had no significant inhibitory effect on the secretion of HBV DNA and HBsAg into the culture medium. HBV ccc DNA and HBV mRNAs in HepG2 2.2.15 cells were also not affected. The observed effect of MPA on HBV replication in primary human hepatocyte cultures may involve only episomal replication and may have clinical implications, especially before integration of HBV DNA into the host genome.

5 REALIZE TRIAL FINAL RESULTS: TELAPREVIR-BASED REGIMEN FOR GENOTYPE 1 HEPATITIS C VIRUS INFECTION IN PATIENTS WITH PRIOR NULL RESPONSE, PARTIAL RESPONSE OR RELAPSE TO PEGINTERFERON/RIBAVIRIN

5 REALIZE TRIAL FINAL RESULTS: TELAPREVIR-BASED REGIMEN FOR GENOTYPE 1 HEPATITIS C VIRUS INFECTION IN PATIENTS WITH PRIOR NULL RESPONSE, PARTIAL RESPONSE OR RELAPSE TO PEGINTERFERON/RIBAVIRIN S. Zeuzem, P. Andreone, S. Pol, E.J. Lawitz, M. Diago, S. Roberts, R. Focaccia, Z.M. Younossi, G.R. Foster, A. Horban, P.J. Pockros, R. Van Heeswijk, S. de Meyer, D. Luo, G. Picchio, M. Beumont. Johann Wolfgang Goethe University Medical Center, Frankfurt am Main, Germany; Universita di Bologna, Bologna, Italy; Universite Paris Descartes, INSERM Unite 567, and Assistance Publique-Hopitaux de Paris, Cochin Hospital, Paris, France; Alamo Medical Resarch, San Antonio, TX, USA; Hospital General de Valencia, Valencia, Spain; Department of Gasteroenterology, Alfred Hospital, Melbourne, VIC, Australia; Emilio Ribas Infectious Diseases Institute, Sao Paulo, Brazil; Center for Liver Disease, Inova Fairfax Hospital, Falls Church, VA, USA; Institute of Cell and Molecular Science, Queen Mary University of London, London, UK; Medical University of Warsaw, Warsaw, Poland; Scripps Clinic and The Scripps Research Institute, La Jolla, CA, USA; Tibotec BVBA, Beerse, Belgium; Tibotec Inc., Titusville, NJ, USA E-mail: zeuzem@em.uni-frankfurt.de Background: The Phase 3 study REALIZE evaluated telaprevir (T) in combination with pegylated-IFN alfa-2a (P) and ribavirin (R) in well-characterised prior PR treatment-failure patients. Methods: REALIZE was a randomised, international, multicentre, double-blind, placebo-controlled trial to evaluate the efficacy, safety and tolerability of T (750mg q8h) plus P (180mg/w) and R (1000– 1200mg/d) compared with PR alone in G1 HCV-infected patients with prior PR treatment failure including non-responders (nulland partial-responders) and relapsers. The treatment arms (randomised 2:2:1, stratified by viral load and type of prior response) were: 1) T/PR for 12 weeks, followed by PR for 36 weeks (T12/PR48); 2) PR for 4 weeks followed by T/PR for 12 weeks, then PR for 32 weeks (Lead-in T12/PR48); 3) PR for 48 weeks (Pbo/PR48). The primary objective was to evaluate the superior efficacy of the T/PR arms for non-responders and relapsers. Secondary objectives included the evaluation of a lead-in and efficacy in prior nulland partialresponders separately. HCVRNA was quantified with the COBAS TaqMan v2.0 assay (LLOQ=25 IU/mL). ESAs were not allowed for anaemia management. Results: 833 patients were screened, 663 randomised, and 662 treated. 70% of patients were male, 93% were Caucasian, 26% had cirrhosis, and 89% had baseline HCVRNA ≥800,000 IU/mL.

Analysis of genotype 2 and 3 hepatitis C virus variants in patients treated with telaprevir demonstrates a consistent resistance profile across genotypes

Study C209 evaluated the activity of telaprevir in treatment‐naïve patients with genotypes 2 or 3 (G2, G3) hepatitis C virus (HCV) infection. Telaprevir monotherapy showed potent activity against HCV G2, but limited activity against G3. This analysis was performed to characterize HCV viral variants emerging during telaprevir‐based treatment of G2/G3 HCV‐infected patients. Patients were randomized to receive 2 weeks of treatment with telaprevir (telaprevir monotherapy), telaprevir plus peginterferon alfa‐2a and ribavirin (triple therapy), or placebo plus peginterferon alfa‐2a and ribavirin (control), followed by 22–24 weeks of peginterferon/ribavirin alone. Viral breakthrough was defined as an increase >1 log10 in HCV RNA from nadir, or HCV RNA >100 IU/mL in patients previously reaching <25 IU/mL. Twenty‐three patients (47%) had G2 and 26 (53%) had G3 HCV. Viral breakthrough occurred during the initial 2‐week treatment phase in six G2 patients (66.7%; subtypes 2, 2a and 2b) and three G3 patients (37.5%; all subtype 3a), all in the telaprevir monotherapy arm. Four breakthrough patients (three G2, one G3) subsequently achieved sustained virologic response (SVR). In all patients with breakthrough and available sequence data, mutations associated with reduced susceptibility to telaprevir in genotype 1 (G1) HCV were observed. No novel G2/G3‐specific mutations were associated with telaprevir resistance. The telaprevir resistance profile appeared consistent across HCV genotypes 1, 2 and 3. Although viral breakthrough with resistance occurred in patients receiving telaprevir monotherapy, half of these patients achieved an SVR upon addition of peginterferon/ribavirin highlighting the importance of combination therapy.

Hepatitis B virus infection in microcarrier‐attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long‐term episomal replication of HBV

Studies on the pathobiology of chronic (long‐term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome‐transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an invitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier‐attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12000–14000) membrane (dialysis) bags for a duration of 2 months. HBV covalently‐closed‐circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86‐ and 3.28‐fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long‐term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.

Incidence of virological failure and emergence of resistance with twice-daily vs every 8-h administration of telaprevir in the OPTIMIZE study

The OPTIMIZE study demonstrated noninferior efficacy between telaprevir (TVR) twice daily (bid) vs every 8‐h (q8h) administration. This analysis compared the selective pressure of both dosing regimens by characterisation of the hepatitis C virus (HCV) variants emerging in genotype 1 (G1) HCV‐infected patients who did not achieve sustained virological response (SVR). HCV NS3•4A population sequencing was performed at baseline and time of failure (viral breakthrough, stopping rule or relapse). TVR‐resistant variants were classified by fold change in inhibitory concentration (IC50). Baseline TVR‐resistance was low (<5%) and did not preclude achieving SVR in either arm. The proportion of patients with TVR‐resistant variants at time of failure was similar in the bid (15%) and q8h (17%) dosing arms. The majority of variants and virological failures occurred in G1a patients, and mutations V36M, R155K and R155T (G1a), and V36A, T54A and A156S (G1b) were significantly enriched in both treatment arms. The number and type of emerging TVR‐resistant variants in non‐SVR patients were comparable between treatment arms and were consistent with previous observations. No differences in viral resistance profiles were observed between TVR‐based treatment arms in non‐SVR patients, indicating a similar selective pressure of TVR bid and q8h dosing.

798 EFFICACY OF TELAPREVIR DOSED TWICE DAILY VERSUS EVERY 8 HOURS BY IL28B GENOTYPE: RESULTS FROM THE PHASE III OPTIMIZE STUDY

798 EFFICACY OF TELAPREVIR DOSED TWICE DAILY VERSUS EVERY 8 HOURS BY IL28B GENOTYPE: RESULTS FROM THE PHASE III OPTIMIZE STUDY M. Buti, K. Agarwal, Y. Horsmans, W. Sievert, E. Janczewska, S. Zeuzem, L. Nyberg, R.S. Brown Jr., C. Hezode, M. Rizzetto, R. Parana, S. De Meyer, R. DeMasi, D. Luo, J. Witek. Hospital Valle Hebron and Ciberehd del Instituto Carlos III, Barcelona, Spain; Kings College Hospital, London, UK; Cliniques Universitaires Saint-Luc, Universite Catholique de Louvain, Brussels, Belgium; Monash Medical Centre and Monash University, Melbourne, VIC, Australia; Outpatients Clinic for Hepatology, Myslowice, Poland; Johann Wolfgang Goethe University Medical Center, Frankfurt am Main, Germany; Kaiser Permanente, San Diego, CA, Columbia University College of Physicians and Surgeons, New York, NY, USA; Hopital Henri Mondor, Creteil, France; University of Torino, Torino, Italy; Medical School, Federal University of Bahia, Bahia, Brazil; Janssen Infectious Diseases BVBA, Beerse, Belgium; Janssen Research & Development LLC, Titusville, NJ, USA E-mail: 14100abf@comb.cat

P221 VIROLOGY ANALYSES OF SIMEPREVIR IN PHASE 2B AND 3 STUDIES

P219 FINE MAPPING OF THE BUTYROPHILIN GENOMICS REGION: ROLE IN HEPATITIS C VIRUS INFECTION (HCV) L. Rojas, J. Ampuero, J.A. Del Campo, R.J. Garcia-Lozano, R. Solá, X. Forns, R. Moreno-Otero, R. Andrade, M. Diago, J. Salmeron, L. Rodrigo, J.A. Pons, J.M. Navarro, J.L. Calleja, J. GarciaSamaniego, M. Buti, J. Crespo, C. Fernandez-Rodriguez, R. Millan, M.F. Gonzalez-Escribano, M. Romero-Gomez. Valme University Hospital & CIBERehd, Virgen del Rocio University Hospital, Seville, Del Mar Hospital, Clinic Hospital, Barcelona, Hospital de la Princesa, Madrid, Virgen de la Victoria University Hospital, Malaga, Valencia General Hospital, Valencia, San Cecilio Hospital, Granada, Asturias Central Hospital, Oviedo, Virgen Arrixaca Hospital, Murcia, Costa del Sol Hospital, Malaga, Puerta de Hierro Hospital, Carlos III Hospital, Madrid, Vall d’Hebron University Hospital, Barcelona, Marques de Valdecilla University Hospital, Santander, Alcorcon Hospital, Madrid, Spain E-mail: lurraits@gmail.com

1167 PRE-TREATMENT IP-10 LEVELS AND IL28B GENOTYPE IN PREDICTION OF SVR IN PRIOR TREATMENT-EXPERIENCED GENOTYPE 1 HCV PATIENTS TREATED WITH TELAPREVIR/PEGINTERFERON/RIBAVIRIN IN THE REALIZE STUDY

1167 PRE-TREATMENT IP-10 LEVELS AND IL28B GENOTYPE IN PREDICTION OF SVR IN PRIOR TREATMENT-EXPERIENCED GENOTYPE 1 HCV PATIENTS TREATED WITH TELAPREVIR/PEGINTERFERON/RIBAVIRIN IN THE REALIZE STUDY L. Vijgen, W. Talloen, A. Scholliers, S. Johansson, M. Tuefferd, S. De Meyer, J. Witek, G. Fanning, G. Picchio, S. Pol, S. Zeuzem, J. Aerssens. Tibotec BVBA, Beerse, Belgium; Janssen Research and Development, Beerse, Belgium; Janssen Research and Development, LLC, Titusville, NJ, USA; Universite Paris Descartes, INSERM Unite 1016, and Assistance Publique-Hopitaux de Paris, Cochin Hospital, Paris, France; Johann Wolfgang Goethe University Medical Center, Frankfurt am Main, Germany E-mail: lvijgen@its.jnj.com

1174 DEEP SEQUENCING SCREENING FOR TELAPREVIR-RESISTANT VIRAL VARIANTS IN PREVIOUS NULL RESPONDERS FAILS TO IDENTIFY THOSE PATIENTS AT RISK OF FAILING TELAPREVIR PLUS PEGINTERFERON/RIBAVIRIN THERAPY

Background and Aims: Naturally-occurring telaprevir-resistant viral variants likely exist in all hepatitis C (HCV) patients at low frequencies. Based on population sequencing, few patients (<5%) have resistant variants as the dominant species before treatment, thus the utility of baseline resistance testing with population sequencing to identify patients at risk of treatment failure is low. The impact of baseline resistance could be higher in patients with a poor interferon response and with the use of more sensitive sequencing techniques. Methods: Previous null-responder patients (10 genotype 1a and 5 genotype 1b) with diverse treatment outcomes on telaprevir-based treatment (5 sustained virologic response [SVR], 9 on-treatment virologic failure, 1 relapser) in the Phase 3 REALIZE study were analyzed at baseline, during treatment, and follow-up. Paired-end deep sequencing of a fragment spanning the HCV NS3–4A regions was performed using Illumina technology. Telaprevir-resistant variants (V36A/M, T54A/S, R155K/T, A156S/T/V) were reported when present in ≥1% of sequence reads. Results: No telaprevir-resistant variants were detected by deep sequencing at baseline in 12/15 patients; 5 of these 12 patients achieved an SVR. The 3 remaining patients had a baseline variation at R155K (67%), T54S (2%), or T54A (1%). All 3 patients had ontreatment virologic failure. Of the 10 patients who did not achieve an SVR, 8 had the V36M and R155K variants present at failure. For 5 of these 8 patients with follow-up data available, the frequency of these mutations decreased gradually after treatment. At Week 72, V36M remained detectable in 3/5 patients (at 3–8% frequency), whereas R155K was no longer detectable by deep sequencing (<1%) in any of these 5 patients. Conclusions: In this small subset of prior null-responder patients with poor peginterferon/ribavirin response, baseline telaprevirresistant variants were detected by deep sequencing in only 3 of 10 patients without SVR. Hence, deep sequencing only identified a small fraction of patients at risk for treatment failure. The frequency of telaprevir resistance-associated mutations decreased over time after treatment in patients without SVR.

Virology analyses of HCV genotype 4 isolates from patients treated with simeprevir and peginterferon/ribavirin in the Phase III RESTORE study

Simeprevir is a hepatitis C virus NS3/4A protease inhibitor. Hepatitis C virus baseline NS3/4A polymorphisms and emerging mutations were characterized in treatment‐naїve and treatment‐experienced genotype 4‐infected patients treated with simeprevir+peginterferon/ribavirin in the RESTORE study. Population sequencing of the NS3/4A region was performed and in vitro simeprevir activity against site‐directed mutants or chimeric replicons with patient‐derived NS3 protease sequences was assessed in a transient replicon assay. Simeprevir remained active against most (83/91 [91%]) baseline isolates tested in the chimeric replicon assay. Eight baseline isolates reduced simeprevir activity; these carried I132L or D168E substitutions reducing simeprevir median activity by 4.6‐ and 39‐fold, respectively. Six of these eight isolates were from patients achieving sustained virologic response. Baseline NS3 Q80K polymorphism was not observed in the genotype 4‐infected patients. Of the 107 simeprevir‐treated patients, 37 did not achieve sustained virologic response for any reason. Of the 32 patients who failed treatment and had sequencing information, 28 (88%) had emerging mutations at NS3 positions 80, 122, 155, 156 and/or 168 at time of failure, similar to those in genotype 1. Emerging mutations were mainly D168V and D168E alone or combined with mutations at position 80. In general, isolates obtained at time of failure displayed high‐level in vitro resistance to simeprevir (fold change ≥50) in a chimeric replicon assay with a median simeprevir fold change value of 440, consistent with observed mutations. In conclusion, emerging mutations in genotype 4 patients failing simeprevir+peginterferon/ribavirin treatment were similar to those in genotype 1 and conferred high‐level resistance to simeprevir.