Christoph Sarrazin J. W. Goethe-University Hospital Frankfurt am Main, Germany

1. Resistance is important? No 5th Paris Hepatitis Conference Paris, 31.01.2012 Christoph Sarrazin J. W. Goethe-University Hospital Frankfurt am Main, Germany

2. Hepatitis C virus is an RNA virus

3. 155 36 36/155 WT 36 54 WT 156 155 155 WT 54 36 36/155 1 3.5 6.8 12 46 466 781 Selected resistant variants are not archived Long-term follow-up VX-950 Dosing Period Post-Dosing 7 7 7 7 6 6 6 6 5 5 5 5 4 4 4 4 Median Log HCV RNA Long-term follow-up 3-7 months post-dosing 3 3 3 3 Follow-Up 7/10 days post-dosing 2 2 2 2 EOD 14 days Baseline 1 1 1 1 WT IC50 fold change WT T54A 36/155 36/156 V36 M/A/L R155 K/T/S/M A156 V/T Sarrazin et al. Gastroenterology 2007

4. Selected resistant variants are not archived during long-term follow-up 100 95 Boceprevir (Phase 3) 100 Telaprevir (Phase 2/3) (median FU 29 months) 91 85 84 82 80 60 Percent of patients with no detectable variants 20% 40 53% 20 0 Patients (n) 0 n/N = 107/126 74/81 20/21 69/82 0/1 49/60 11/11 36+155 Overall 168 36 156 54 155 Population based sequencing Barnard RJO, et al. Hepatology 2011;54 (Suppl. S1): Abstract 164 Sherman KE, et al. Hepatology 2011;54 (Suppl. S1): Abstract 248

5. Resistant variants at long-term FU are rarely observed: clonal / deep sequencing Deep sequencing (Illumina) 5.7y after Telaprevir (Phase 1) [4.5 years] Shape by position Colour by variant V36 T54 R155 A156 V36A V36M T54A T54S R155K R155T A156S A156T A156V 100% 10% Data below this line represents assay ‘noise’ based on calibration of deep sequencing analysis Frequency [4.5 years] 1% Cut off 0.1% Patient # 1 2 3 4 5 6 7 8 Susser et al., J ClinVirol 2011 Sarrazin et al., AASLD 2011

6. Re-Treatment of patients is not commonly associated with treatment failure 108 107 106 105 104 103 102 101 100 1 Patient had a confirmed viral breakthrough at Week 8 (V36M/V + R155K/R) Genotype 1a Genotype 1b Baseline 1 2468 Time (weeks) Sarrazin et al., AASLD 2011

7. Re-Exposure of patients is not commonly associated with re-selection of same variants #1, GT 1a #2, GT 1a #3, GT 1b #4, GT 1b Vermehren et al., J Viral Hepatitis 2012

8. Theoretically all possible single and double variants exist - but… Hepatitis C virus: ~9600 nucleotides Error rate during replication: ~10-4 – 10-5 per copied nucleotide Viral turnover: ~1012virions produced every day • Not all variants survive • Dead mutations (variants that can not replicate) • Immune sensitive mutations (variants that are eliminated by the immune system) Rong L, et al, SciTranslMed 2010;2:30ra32; Neumann AU, et al. Science 1998;282:103–7Domingo E, et al. Viral Hepatitis Rev 1996;2:247–61; Cuevas JM, et al. J Virol 2009;83:5760–4

9. Study 1 (n=570)0.9% V36M, 0.7% R155K, 0.2% V170A, 0.2% R109K Study 2 (n=507)0.3–2.2% dominant PI resistant mutations within NS3 protease Study 3 (n=662)1.8% T54S, 0.6% R155K, 0.3% V36M Study 4 (n=595)4% dominant PI resistant mutations within NS3 protease Presence of resistant variants at baseline is rare (TVR/BOC) and at low frequencies has no impact on SVR Deep sequencing* (454) Population based sequencing *cut-off: 0.1% according to statistical test based on Poisson’s law; SVR: sustained virologic response; NR: non-responder; RR responder-relapser Bartels DJ, et al. J Infect Dis 2008;198:800–7Kuntzen T, et al. Hepatology 2008;48:1769–78; Vierling JM, et al. Hepatology 2010;52(Suppl.):702A De Meyer S, et al. J Hepatol 2011;54(Suppl. 1):S475; ChevaliezS, et al. J Hepatol 2011;54(Suppl. 1):S30

10. Presence of resistant variants at baseline at high frequencies requires combination therapies Cyclophilin inhibitors Interferon alfa NS5A inhibitors Ribavirin NS3/4A-protease- inhibitors polymerase- inhibitors 3 C p7 E2 3 5B E1 2 4B 5A 4A RNA-dependent RNA-polymerase NS3-protease cofactor metallo-/ cysteine-protease membrane anchor capsid regulation of replication envelope NTPase/ helicase serine- protease

11. Barrier to resistance depend on number and probability of nucleotide exchanges Main first generation NS3 Protease-Inhibitor Resistance site (R155K) NS3, NS5A and non-nuc resistance mutations typically require transitions while nuc resistance site requires transversion (S282T) One nucleotide exchange for subtype 1a versus two nucleotide exchanges for subtype 1b NS5B polymerase in favor of transitions (A ↔ G and C ↔ T) over tranversions (A/G ↔ C/T and C/T ↔ A/G NS3 proteasesequence 155 HCV-1a GIFRAAV HCV-1b GIFRAAV HCV-1a AGG → AAG (R155K) HCV-1b CGG →AAG (R155K) NS3 proteasesequence 155 HCV-1a GIFRAAV HCV-1b GIFRAAV Transition HCV-1a AGG → AAG (R155K) Tranversion HCV-1b CGG →AAG (R155K) Kieffer et al., Hepatology 2007 Sarrazin et al., Gastroenterology 2010 Powdrill et al., PNAS 2011

12. New Triple- / Quadruple-Therapies and all-oral DAA therapies are highly effective Null-respondergenotype 1 patients, n=10 (NS5A InhDaclatasvir + PI Asunaprevirwithandwithout PEG2a + R for 24 weeks) Treatment-naive non-cirrhotic genotype 2/3 patients, n=50 (Nuc PSI-7977 +/- PEG2a +/- R for a total duration of 12 weeks) NS5A-Inh + PI 36% SVR [2/2, GT 1b] Quadruple 90% SVR Lok et al., NEJM 2012 Gane et al., AASLD 2011, #34

13. Summary • HCV is an RNA virus- treatment induced eradication of virus is possible- resistant variants can not be archived • Frequency of resistant variants at baseline is- low for many DAA- has no importance for SVR at low levels • Barrier to resistance is high for certain subtypes / isolates, and in general for nucleoside analogues • Combination therapies are effective to avoid resistance- many DAAs / HTA without cross resistance in development- quadruple therapies shown to highly effective- proof of principle to achieve SVR with all oral treatment