1. Future Treatment: is resistance important? Future Treatment: is resistance important? Philippe Halfon Laboratoire Alphabio Hôpital Ambroise Paré, Marseille, France PHC 2012

3. Risk of selection of resistant virus MINIMALINCOMPLETE MAXIMAL Prevention of Resistance Key Dictum: No Replication = No Resistance

4. NS5A Inhibitors IC 50 nM 1-10 10-1000 10-100Potency0.01-0.1 KEY MESSAGE The genetic barrier is not link to the potency

5. Genetic Barrier for HCV Direct Antiviral Agents Low High Nucleos(t)ide Analog Inhibitors 1 st generation Protease ProteaseInhibitors n Non Nucleos(t)ide Analog Inhibitors Analog Inhibitors: NS5 A Inhibitors 2 st generation Protease ProteaseInhibitors Halfon P J Hepatol 2011

6. Resistance to DAA Monotherapy What we learned?

7. WT 1 T54A 6 V36A/M 7 R155K/T 7-29 36/155 57 A156V/T >74 36/156 > 74 CI 50 x fold 0102050100 0 2 4 6 8 Viral decrease > 5 000 000 fold Treatment regimen :PEG-IFNα-2a + RBV Telaprevir ARN-VHC LID (10 UI/ml) days Resistant virus is rapidly selected with Telaprevir alone 32 Log 10 ARN-VHC (UI/ml) Kieffer et al.Hepatology 2007 Detection limit (100 UI/ml)

8. Virologic Escape during Danoprevir (ITMN-191/RG7227) Monotherapy Lim S et al. AAC 2012

9. Genetic barrier can be overcome by combination with PEG+Riba Basal Status DAA Monotherapy DAA Monotherapy DAA + Peg-IFN+RBV Sensitive Virus Resistant Virus

10. Dynamics of the viral breakthrough during Telaprevir Monotherapy

11. Single resistant variants associated with telaprevir and boceprevir * 1. Kieffer T, et al. Hepatology 2007;46:631–9 2. Kieffer T, et al. Hepatology 2010;52(Suppl.):879A; 3. De Meyer S, et al. J Hepatol 2011;54(Suppl. 1): S475 4. Susser S, et al. Hepatology 2009;50;1709–18; 5. Zeuzem S, et al. J Hepatol 2011;54(Suppl. 1):S4 Telaprevir resistant variants 1–3 Increase in resistance † Boceprevir resistant variants 4,5 Low Medium High A156T V55A, R155K/T, V170A, T54A, A156S V36M/A, T54S A156V/T V36A/M, R155K/T, A156S T54A/S *Double mutants have also been reported with telaprevir and boceprevir; † Measured by fold change in IC 50 in the HCV replicon assay

12. Active Site Site Halfon P et al J Hepatol 2011 Location of NS3/4A protease amino acid substitutions conferring decreased sensitivity to Telaprevir Val36

13. Home messages from Resistance to DAA Monotherapy The HCV NS3,NS5A, Pol (NNI) mutations occurred quickly (less than 15 days) and longer for Nucleosides Inhbibitors in monotherapy. Genetic barrier can be overcome by combination with PEG+Riba There is a long term persistence of HCV NS3 Protease mutations after the end of Therapy Monotherapy will lead to selection of resistant variants that, in turn, could produce cross-resistance to whole class of drugs wit overlapping resistance profiles Telaprevir and Boceprevir has a well-characterized and consistent resistance profile –Subtype 1a was associated with V36M and R155K variants, while subtype 1b was associated with V36A, T54A/S, and A156S/T variants. Never use a Protease, NS5A, Non Nucleoside Inhibitors in monotherapy Nucleoside inhibitors ?

14. Resistance to Resistance to SOC+DAA (Triple Therapy)

15. Do we need a resistance test before treatment with direct-acting antiviral agents?

16. Emergence of Viral Resistance to DAA Agents Pre-existing Resistance Associated Variants within Quasispecies19 Viral Load Time on DAA Therapy Resistance Associated Variants (RAVs)= 0.3-2.8% Wild Type HCV Adapted from Lok ASF, McMahon BJ. Hepatology. 2009;50:1-36.

17. Resistance to DAA detection “Detection depends on how carefully you look for it”

18. Clinical significance of RA minority mutants detection Sequencing : 15-20% Pyrosequencing : 1-10 % Ultradeepsequencing : < 1%

19. Sarrazin C et al. AASLD 2011

20. Pyrosequencing assay Focus on R155 and A156 C/AGG Arginine R R155 AAG Lysine K R155K GCT Alanine A A156 ACT Threonin T A156T Wild-type cells Patient with 2 mutations Baseline mutations 1-10 % !

21. Clinical significance of baseline mutations on SVR using DAA 39 genotype 1 naïve patients (24 SVR and 15 NR) treated by a protease inhibitor (Boceprevir and/or Telaprevir). 24 Non responders : - 5/13 have one alone RAV at D0 - 9/13 have more one RAVs at D0 - 10/13 have multiples RAVs (2-4) None of the 15 complete responders have baseline mutations The presence of telaprevir/boceprevir-resistant variants was found at baseline in all patients who had not achieved an SVR. These preliminary data suggest that treatment failure can be predicted using a sensitive and standardized assay ( 1-10%) Halfon P, Oules V, Bourliere M et al Liver International 2012

22. Baseline R155K91% WT9% R155K59% WT41% Peg-Riba – Telaprevir Dosing Period Post-Dosing Week 4 Week 12 Week 24 Halfon P, Oules V, Bourliere M et al, Liver International 2012

23. Does resistance develop in all patients who do not achieve an SVR?

24. REALIZE: TVR-resistant variants associated with virologic failure in the TVR/Pbo treatment phase LI T12/PR48 (n=149) T12/PR48 (n=136) V36M+R155K A156T/V Combinations Not available Wild-type (no TVR-resistant variants) LI T12/PR48 (n=113) T12/PR48 (n=126) V36A/M T54A/S R155I/K/M/T A156S Combinations 20 25 15 10 5 0 20 25 15 10 5 0 Number of patients Genotype 1a Genotype 1b Lower-levelHigher-level Analysis used population-based sequencing On-treatment virologic failure: patients who had discontinued due to a virologic stopping rule and/or patients with viral breakthrough De Meyer S, et al. J Hepatol 2011;54(Suppl. 1):S475

25. Less fit than other variants Subtype Matter with Telaprevir Therapy Implication: subtype distinctions may become more important with DAA agents than with PEG IFN + RBV alone V36M R155K/T 1 nucleoside substitution in relevant codons of 1a 2 substitutions needed in 1b 1 nucleoside substitution in relevant codons of 1a 2 substitutions needed in 1b Genotype 1a (n = 6) A156V/T Genotype 1b (n = 14) Kieffer TL, et al. Hepatology. 2007;46:631.

26. Do resistance mutations persist?

27. EXTEND: 89% of patients no longer have detectable resistant variants Median follow-up time from end of prior study of 25 months (range 7–36) Overall36155156 36+155 variants 54 Variants at HCV NS3 position 50/5612/1234/37 22/26 patients 5/529/33 n/N = 0 20 40 60 80 100 Samples from patients with no detectable resistant variants (%) PopulationSequencing Variant categories are not mutually exclusive 89 92 100 88 100 85 Zeuzem S, et al. Hepatology 2010;52(Suppl.):436A

28. Telaprevir Phase III trials: loss of resistant variants according to NS3 position Analysis includes only patients with follow-up data and a resistant variant(s) at treatment failure (probability starts at 100%) Probability Time after treatment failure (months) V36MR155KV36AT54AA156S/T Median months to loss (95% CI) 9 (8,11)10 (9,11)4 (3,4)3 (2,4)4 (3,6) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 024681012141618 R155K V36M A156S/T V36A T54A Common 1a variants Common 1b variants the time taken for resistant HCV variant populations to return to WT is longer for patients with genotype 1a versus genotype 1b.

29. 174 patients who did not achieve SVR were followed for ≥2 years Cumulative rate of reversion to wild-type (%) Years after end of therapy 00.51.01.5 2.0 91% 71% 62% 59% 100 80 60 40 20 0 V36M T54S R155K Any mutation Vierling JM, et al. J Hepatol 2010;52(Suppl. 1):S470 Boceprevir long-term follow-up (2 years)* Analysis used population-based sequencing *Data from one Phase I study (N=22 previous non-responders), one Phase II dose-ranging study (P03659; N=357 previous non-responders), and the Phase II SPRINT-1 Study (P03523; N=520 treatment-naïve patients)

30. HCV Resistance and Polymerase Inhibitors? WARNING : RAVs will certainly occur with time !

31. Could the patient be re-treated with the same medication in the same regimen?

32. V36A/MT54AV55AQ80R/KR155K/T/QA156SA156V/T D168A/V/T/ H V170A Telaprevir (linear) ** Boceprevir (linear) * SCH900518 (linear) BILN-2061 (macrocyclic) ITMN191 (macrocyclic) ** MK7009 (macrocyclic) * TMC435350 (macrocyclic) ** BI-201335 (linear) MK5172 (macrocyclic) GS-9256 (macrocyclic) ABT 450 (macrocyclic) BMS-791325 (macrocyclic) * Mutations associated with resistance in vitro only Amino acid positions within the NS3/4A protease associated with resistance mutations to different NS3 protease inhibitors Halfon et al J Hepatol 2011

33. Peg-Riba – DAA Dosing Period Post-Dosing RAVs : 3-20 months DO NOT RE-TREATED WITH THE SAME MEDICATION IN THE SAME REGIMEN Could the patient be re-treated with the same medication in the same regimen?

34. Resistance to IFN-Free Regimen

37. Days 035791113 1 2 3 4 6 7 Mediane log 10 ARN VHC (log UI/ml) 5 RG7227 + PEG-IFN + RBV RG7227/RG7128 (naïfs) RG7227/RG7128 (NRC) DIL 1 RG7128 1000 mg x 2/j + RG7227 900 mg x 2/j DIL < 15 UI/ml Virological response at Day 14 Decrease median of Viral load INFORM-1 : Protease Inhibitor+Polymerase Inhibitor Gane EJ.,Lancet 2011 59 No emergeance of Resistance observed after 14 days of Treatment combination

38. for Hepatitis C Genotype 1 Lock A, NEJM N Engl J Med 2012;366:216-24. Breakthrough in 55% dual vs 0% Quad Developed as early as 3 weeks Both NS3 and NS5a Mutations All were GT1a

39. When to test for resistance mutation? Before starting any Protease, NS5A and NNPol inhibitors? at a level of 1-10 %? SO FAR UNKNOWN AND NEED TO BE EXPLORED At treatment failure? YES Before Re-starting any Protease, NS5A and NNPol inhibitors : YES

40. Boceprevir in non responders HCV RNA monitoring can predict HCV Protease resistance EASL 2008 – Schiff –Abstract 104 Non responders mutations = 49 % Virological breaktrough Mutations = 82 % Relapsers Mutations = 58 % Folow upWeeks of treatment LID SVR ARN VHC log Most frequent mutations : V36M, T54S, R155K, V36L, V170A, T54A. Partial virological respondersNon responders BreaktroughRelapsers 0 2 4 6

41. Treatment failure and resistance may be detected by frequent monitoring of HCV RNA levels Sensitive virusResistant virus Baseline HCV RNA HCV RNA Start treatment Before treatment Viral breakthrough Time on treatment

42. Li F JAMA 2011

43. Lessons learned from HIV Genotypic Resistance testing in standard of care in HIV Management HIV resistance testing recommended : - At Baseline: a diagnosis or prior initiation of HAART To detect transmitted drug resistance - When rebound occurs Unknown * Not for Nuc*

44. Is resistance important? YES? SOC+DAA : Protease, NNI and NS5A inihbitors : YES IFN-Free Regimen : MAYBE DAA resistance is Partially abrogated by addition of Peginterferon : in QUAD therapy ? Subtypes 1a and 1b are associated with different resistance profiling : for RAV 1a >1b: YES Issues on HCV archived mutations are not solved Effect of ribavirin important seems limit the DAA resistance Cross-resistance will probably occur for each target

45. Courtesy of Dr J Courcambeck