HCV resistance Understanding the mechanism and Prevention Fabien Zoulim Hepatology department, Hospices Civils de Lyon INSERM U1052, Viral Hepatitis Team Lyon University, France

Novel targets for antiviral intervention using the HCV life cycle IFN + RBV ER HCV Endosome Uncoating Translation Entry Receptor NS2 NS3/4A NS4B NS5B NS5A + - Replication Progeny genomes Golgi E1 E2 Core Assembly Release Exocytosis Nucleus Cytoplasm Polymerase inhibitors Protease inhibitors Other viral and cellular targets Racanelli V, et a., Trends Immunol 2003;24:456-64; Manns MP, et al. Nat Rev Drug Discov 2007;6:991-1000. IFN – Interferon, RBV - Ribavirin

Key concepts for HCV Viral persistence but a curable disease ! Viral genome heterogeneity Resistance to polymerase & protease inhibitors Selection of resistant mutants Key objective: suppress resistance

The main differences between HIV, HBV and HCV Host cell H Host cell H Host cell HCV RNA cccDNA Host DNA Host DNA Proviral DNA Integrated DNA Nucleus Nucleus Nucleus Life-long suppression of viral replication Long-term suppression of viral replication Definitive viral clearance and SVR SVR – Sustained viral response Kieffer TL, et al. J Antimicrob Chemother 2010;65:202-12; Garrido C, et al. J Antimicrob Chemother 2010;65;320-6; Clavel F, et al. New Engl J Med 2004;350:1023-5; Zoulim F, et al. Gastroenterology 2009;137:1593-608; Sarrazin C, et al. Gastroenterology 2010;138:447-62.

How does HCV resistance develop? High replication rate with errors during replication ~1012 viruses produced per day ~1 mistake per virus produced Continual development of viral variants Drug-resistant variants exist before antiviral therapy Antiviral therapy This describes the steps involved in the development of resistance. Suppression of wild-type and susceptible variants Selection and expansion of drug-resistant mutants

Definitions of treatment failure Null-Response Breakthrough Relapse Partial responder Detection limit This illustrates the response patterns and definitions for 4 groups of patients failing therapy. The yellow line describes patients achieving a relatively small change in HCV decline from baseline. These patients are labeled as having a Null-Response. The red line describes patients with detectable HCV RNA at any time during treatment after previous undetectable HCV RNA with qualitative PCR-test during antiviral therapy. These patients are labeled as having a Breakthrough. The grey line describes patients achieving a > 1 log HCV RNA decline from baseline during therapy but not achieving PCR negativity. These patients are labeled as being Partial Responders. The blue line describes patients achieving PCR negativity by a sensitive assay and maintaining this through Week 48 of therapy. These patients are labeled as having a Relapse because upon withdrawal of therapy, HCV levels will rise above the limit of normal. Treatment including DAA Resistance mutants DAA – Direct acting antiviral agents

The virus and its environment The infected hepatocytes Virus replication in hepatocytes Hepatocyte response to infection Antiviral drugs Potency of antivirals Potency shift to mutant Barrier to resistance The virus Fitness of the resistant mutants Accumulation of compensatory mutations Cross-resistance profile

Emergence of drug resistant mutants Wild-type All single mutations likely to be generated every day Resistant mutants less fit than wt HCV RNA Hepatocytes Viral infection Viral replication Resistant mutant

Emergence of drug resistant mutants All single mutations likely to be generated every day Hepatocytes Wild-type HCV RNA Viral infection Viral replication Resistant mutants more fit than wt Resistant mutant Antiviral treatment with DAA DAA – Direct acting antiviral agents, wt – wild-type

Antiviral drug resistance testing Genotypic (sequence analysis) assays Detection of emerging mutants Population sequencing Clonal analysis Ultradeep sequencing HCV viral fitness assays Determination of fitness, infectivity and evolution of mutants Difficult to apply to clinical isolates HCV phenotypic assays Determination of drug susceptibility and cross-resistance profile / Replicon system

Evolution of NS3 protease variants in patients treated with telaprevir monotherapy Sarrazin C, et al. Gastroenterology 2007;132:1767-77 NS3 – Non structural protein 3

Towards the suppression of resistance Factors affecting the selection of resistant variants during antiviral treatment Drug selective pressure: Antiviral potency Drug concentration at the site of replication Genetic barrier to resistance: Number of mutations required for loss of activity Viral fitness: Relative rate of replication of resistant strains IFN and ribavirin backbone to maintain antiviral pressure on resistant mutants IFN – Interferon

Peg-IFN & Ribavirin inhibit PI resistant mutant replication Kieffer TL, et al. AASLD 2006 Peg-IFN – Pegylated interferon, PI – Protease inhibitor

Failure of triple therapy is the result of the failure of IFN – Ribavirin to clear PI resistant mutants ! IFN – Interferon, PI – Protease inhibitor

Virologic monitoring of triple therapy

Example of virologic breakthrough DAA stopped + sequencing 10 106 105 104 103 102 101 HCV - RNA (UI/ml) N 2 N 4 Y 6 Y 8 Y 10 Y 12 Y vBT Confirmed v BT DAA – Direct acting antiviral agents, v BT – Virologic breakthrough

Example of virologic breakthrough Importance of the timing of virologic monitoring 10 106 105 104 103 102 101 HCV - RNA (UI/ml) N 2 N 4 Y 6 Y 8 Y 10 Y 12 Y vBT Confirmed v BT v BT – Virologic breakthrough

Example of virologic breakthrough Importance of sequencing and mutation detection 10 106 105 104 103 102 101 HCV - RNA (UI/ml) V36M R155K N 2 N 4 Y 6 Y 8 Y 10 Y 12 Y vBT Confirmed v BT v BT – Virologic breakthrough

Loss of telaprevir resistant variants after treatment cessation 1.0 0.9 Médiane de retour des variants “sauvages” en séquençage direct = 7 mois (IC95% : 5-8) 0.8 0.7 0.6 médiane Probabilité 0.5 0.4 0.3 0.2 0.1 0.0 2 4 6 8 10 12 14 16 18 Suivi après l’échec thérapeutique (mois) Sullivan J. et al. EASL 2011.

Loss of telaprevir resistant variants after treatment cessation 1.0 0.9 1a 0.8 0.7 0.6 médiane Probabilité 0.5 0.4 1b 0.3 0.2 0.1 0.0 2 4 6 8 10 12 14 16 18 Suivi après l’échec thérapeutique (mois) Sullivan J. et al. EASL 2011.

Optimizing anti-HCV regimens to maximize SVR and minimize resistance The drugs Antiviral potency Pharmacokinetics (intracellular concentration of the inhibitor) The virus Genetic barrier to resistance Fitness of resistant variants Cross-resistance The host Treatment adherence IFN response: Genetics / IL28B gene polymorphism & others ISGs expression The treatment regimen Treatment duration / Dosage of the drug /Dosing interval Side effects IFN – Interferon, IL28 – Interleukin 28B, ISGs – Interferon stimulated genes, SVR – Sustained virologic response

All-oral HCV treatment GT-1

Site of action of BI compounds NS3/4A Protease inhibitor BI 201335 NS5B pocket I Polymerase inhibitor BI 207127

Serine protease domain Cross-resistance between polymerase and protease inhibitors is unlikely NS2–NS3 protease NS3 protease C E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B Core Envelope Serine protease domain Telaprevir; SCH 503034 T54 R155 A156 D168 V36 R1479* (R1626) S96 N142 S282 C316 M414 M419 P495 T423 Telaprevir; BI 201335 NM283* Telaprevir; BI 201335; SCH 503034 HCV-796† BI 201335 Telaprevir *nucleoside; †non-nucleoside BI207127† Sarrazin C, et al. Gastroenterol. 2007;132:1767–77; Tong X, et al. Antiviral Res. 2006;70:28–38; De Francesco R, et al. Nat. 2005;436:953–60; Le Pogam S, et al. Virol. 2006;351:349–59; Villano S, et al. 57th AASLD 2006, Boston, MA, October 27–31, 2006

BI 207127 + BI 201335 Combination: suppresses emergence of resistance BI 207127 X EC50 Long term selection of resistance in vitro is effectively suppressed by BI 201335 and BI 207127 combination BI 201335 X EC50 EC50 – Median estimated concentration

400 mg TID BI 207127 + 120 mg BI 201335 + RBV (n=15) BI 201335 + BI 207127: SOUND-C1 400 mg TID BI 207127 + 120 mg BI 201335 + RBV (n=15) 100000000 10000000 1000000 100000 10000 1000 100 10 1 HCV RNA (IU/mL) GT-1a GT-1b GT-6e 0 4 8 12 16 20 24 28 32 36 40 44 Treatment day BI 207127 600 mg/ BI 201335/RBV BI 201335/ PegIFN/RBV LLOQ LLOD 600 mg TID BI 207127 + 120 mg BI 201335 + RBV (n=17) 100000000 10000000 1000000 100000 10000 1000 100 10 1 GT-1a GT-1b HCV RNA (IU/mL) LLOQ LLOD 0 4 8 12 16 20 24 28 32 36 40 44 Treatment day BI 207127 400 mg/ BI 201335/RBV BI 201335/ PegIFN/RBV Zeuzem S, et al. Gastroenterology 2011;141:2047-55 LLOQ, lower limit of quantification; LLOD, lower limit of detection, GT - genotype

SOUND-C1 trial Multi-centre, open-label, randomised phase Ib study in treatment-naïve, HCV genotype-1 (GT-1) patients 400 mg BI 207127 TID + 120 mg BI 201335 QD + RBV 120 mg BI 201335 QD + PegIFN alfa 2a/RBV PegIFN alfa 2a/ RBVa Follow-up n = 15 n = 17 600 mg BI 207127 TID + 120 mg BI 201335 QD + RBV 120 mg BI 201335 QD + PegIFN alfa 2a/RBV PegIFN alfa 2a/ RBVa Follow-up Day 1 Week 4 Week 24 Week 48 Week 72 SOUND-C1 study G1 naive exclude cirrosisis High sustained virologic response following interferon-free treatment of chronic HCV GT1 infection for four weeks with HCV protease inhibitor BI 201335, polymerase inhibitor BI 207127 and ribavirin, followed by BI 201335 and PegIFN/Ribavirin – Initial virological response decreased with lower dose 400 MG Note initial to week 4 IFN free then 335 + IFN; IFN sparing thus Futility rules: Virological breakthrough (increase in HCV RNA by > 1 log, or HCV RNA ≥ 100 IU/mL after < 25 IU/mL) Lack of EVR (> 2 log decrease at Week 12) Lack of undetectable HCV RNA at Week 24 aPatients without eRVR (HCV RNA ≤ 25 IU/mL Week 4, undetectable Weeks 5 to 18) continued PegIFN/RBV up to Week 48 TID, three times daily; QD, once daily Zeuzem S, et al. AASLD 2010. Abstract LB-7 EVR – Early virologic response, eRVR – extended Rapid virologic response, PegIFN/RBV – Pegylated interferon/ribavirin

SOUND-C1 Virologic response 400 mg TID 600 mg TID 100 80 60 40 20 Proportion of patients (%) RVR eRVR EOT and SVR 12 high; higher with 600 mg 11/15 17/17 4/15 12/17 14a/15 17/17 11/15 16b/17 RVR eRVR EoT SVR12 aIncludes patient #61102 switched to Peg/RBV; bPatient #41203 outstanding RVR, rapid virologic response (after 4 weeks of treatment); eRVR, HCV RNA < 25 IU/mL at Week 4 and undetected at Week 5 to 18; EoT, undetectable HCV RNA at the end of all treatment Zeuzem S, et al. AASLD 2011. Abstract 249 EOT – End of treatment, SVR – Sustained virologic response

SOUND-C2 trial Multi-centre, open-label, randomised phase IIb study in treatment-naïve, HCV genotype-1 (GT-1) patients; Including ~15% cirrhotics A (n=81) B (n=81) C (n=81) D (n=81) E (n=81) 1335 + 7127TID + RBV Follow-up 1335 + 7127TID + RBV Follow-up 1335 + 7127TID + RBV 1335 + 7127BID + RBV Follow-up 1335 + 7127TID no RBV Follow-up SOUND-C2is a 5-arm,open-label,randomized,phase Iibstudy evaluating efficacy and safety of several interferon-free all-oral combination regimens of these compounds for up to Weeks of treatment Methods:362 TN HCV GT-1 5 arms arms: Prespecified interim analysis is all patients completed 12 weeks. Randomization was stratified by HCV subtype (1a vs 1b) and IL28B genotype Day 1 Week 12 Week 16 Week 26 Week 40 Interim analysis Zeuzem S, et al. AASLD 2010. Abstract LB-15 GT – Genotype, RBV - Ribavirin

Results: Antiviral activity; antiviral response assessment up to week 12 (5) patients with HCV RNA <LLOD at Week 12 by IL28 GT and viral subtype (per protocol analysis) 100 80 60 40 20 Proportion of patients with HCV RNA <LLOD at Week 12 (%) Interim analysis by two variables 60% group A SVR 12) 69% 1b 49% 1a effect of host immune response C/C Non-C/C C/C Non-C/C GT-1a GT-1b 1335 + 7127TID + RBV 22/25 39/61 24/26 79/92 1335 + 7127BID + RBV 6/7 10/22 10/11 32/36 1335 + 7127TID no RBV 3/3 2/9 7/7 13/20 LLOD – Lower limit of detection, IL28b Interleukin 28b, GT - genotype Zeuzem S, et al. AASLD 2010. Abstract LB-15

All-oral HCV treatment GT-1 BMS-790052 + BMS-650032

BMS-790052 + BMS-650032 Phase IIa study of BMS-790052 plus BMS-650032 for 24 weeks HCV GT-1b Japanese null responders BMS-790052 60 mg QD + BMS-650032 200 mg BID Follow-up n=10 (GT-1b) Week 24 48 72 Daclastavir and Asunaprevir to be mentioned Japanese non response (non serotic) Chayama K, et al. Hepatology. 2011;54(Suppl. S1): Abstract LB-4 GT – Genotype

BMS-650032/BMS-790052: virologic response (in GT1b only) One patient discontinued at Week 2; HCV RNA was undetectable after 24 weeks’ follow-up Proportion of patients with undetectable HCV RNA (%) 9 patients completed 24 weeks of treatment; 1 patient discontinued after 2 weeks, however, this patient achieved SVR24 In all 9 patients who completed treatment, HCV RNA was undetectable from treatment Week 8 through post-treatment Week 24 (SVR24) In the patient who discontinued, HCV RNA remained undetectable for >24 weeks after discontinuation No viral breakthrough was observed 2 serious AEs: grade 3 pyrexia and grade 4 hyperbilirubinemia 4/10 9/10 9/10 9/10 Week 4 Week 12 EOT SVR24 EOT – End of treatment, SVR – Sustained virologic response Chayama K, et al. Hepatology. 2011;54(Suppl. S1): Abstract LB-4

All-oral HCV treatment GT2/3 PSI-7977

PSI-7977 + RBV (GT-2/3) (n=10 per arm) phase II, randomized, open-label trial of PSI-7977 plus RBV, with or without PegIFN, in treatment-naïve HCV GT-2 and GT-3 patients; cirrhotics excluded PSI + R 12 weeks n=10 PSI-7977 400 mg + RBV PSI + PR 4 weeks n=10 PSI-7977 400 mg + PegIFN/RBV PSI-7977 400 mg + RBV PSI + PR 8 weeks n=10 PSI-7977 400 mg + PegIFN/RBV PSI-7977 400 mg + RBV PSI + PR 12 weeks n=10 PSI-7977 400 mg + PegIFN/RBV Week 4 8 12 Gane EJ, et al. AASLD 2011. Abstract 34

PSI-7977 + RBV (GT-2/3) (n=10 per arm) No viral breakthrough observed PSI-7977 very well tolerated, with no attributable safety signal, no treatment discontinuations and no treatment emergent laboratory abnormalities 100 100 100 100 100 100 100 100 Gane EJ, et al. AASLD 2011. Abstract 34

PSI-7977 + RBV (GT-2/3) (n=10 per arm) 100 100 100 100 60 Gane EJ, et al. AASLD 2011. Abstract 34 RBV - Ribavirin

Conclusions: Current and future HCV treatment regimens PegIFN Ribavirin HCV Protease Inhibitor* Polymerase Inhibitor** Inhibitor‘ Inhibitor **1 Inhibitor 2*** SOC Standard of Care NSOC New Standard of Care Dual-oral / QUAD 2 direct acting antivirals quadruple Triple-oral 3 direct acting antivirals +/ - HCV NS5A inhibitor HCV NS5A inhibitor OR HCV NS5A inhibitor