Resistance to DAAs: is it a real issue ? Today yes Tomorrow may be no

O. Lada

PHD

Service d’Hépatologie et INSERM CRB3, AP-HP Hopital Beaujon, Paris, France. [email protected] Progress in the Treatment of

HBV HIV HCV Genome DNA RNA RNA Mutation rate + +++ +++ Daily viral production 1013 1010 1012 Viral Reservoir cccDNA Integrated c DNA None Therapeutic strategy Single Multiple Multiple recovery No No Yes Targets for DAAs

5’NTR Structural proteins Nonstructural proteins 3’NTR Metalloprotease Envelope Serine protease Glycoproteins RNA Capsid RNA helicase Cofactors Polymerase

C E1 E2 NS1 NS2 NS3 NS4A NS4B NS5A NS5B

Protease Inhibitors NS5A Inhibitors Polymerase Inhibitors

Telaprevir Nucs Non-Nucs ABT-267 ABT-333 GS-5816 VX-135 ACH-3102 IDX-20963 BMS-791325 ABT-450 PPI-668 ACH-3422 PPI-383 MK-5172 GSK-2336805 GS-9669 TMC-647055 ACH-2684 MK-8742 Asselah T et al. Liver International 2013 Resistance to specific HCV inhibitors

Selection of viral variants bearing amino acid substitutions alters the drug target and thereby confers reduced susceptibility to the drug Resistance to specific HCV inhibitors

Null-ResponseNull-Response l e v

e Breakthrough

l Breakthrough

RelapseRelapse

A PartialPartial responderresponder N R

V C H

Detection limit

Treatment including DAA

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 Time

Resistance mutants

DAA – Direct acting antiviral agents Adapted from Zoulim F, PHC 2012 Factors influencing viral resistance with DAA

Viral Factors • Level of viral replication (1012/day) • Low fidelity of polymerase • Impact of mutations on fitness • Viral quasi-species • Half-life of infected hepatocytes

Resistance

Pharmacological Factors Host Factors • Drug potency • Compliance • Genetic barrier • Immune system • Pharmacokinetic • Replication space • Activity of protein kinase • Nuceos(t)ide transporters Emergence of resistance during antiviral therapy

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Time Emergence of resistance during antiviral therapy Treatment Sensitive variants

H Resistant variants

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Time Emergence of resistance during antiviral therapy Treatment Sensitive variants

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Time Resistant-associated viruses (RAVs) are rapidly selected by alone ) L 7 m / U I 6 0 1 g

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n 2 a i d

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M 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Study Time (days)

Placebo Telaprevir 450 mg q8h Telaprevir 750 mg q8h Telaprevir 1250 mg q12h

Reesink HW, et al. Hepatology. 2005;42:234A. Subtype impacts the genetic barrier to resistance to Protease Inhibitors (PI)

Number of nucleotide substitutions RAVs in GT1 patients who failed to needed according subtype BOC PR treatment (Sprint-2 and Respond-2) Genotype 1b n=246

R155K Genotype 1a 97 CGG-AAG 2 step n ,

R155K R155K s

R155K t 1 step n e i AGG-AAG CGG-CAG t N=96 CGG-CAG a

P 117 55 %* 43

38 47 %* 32 15

Patients with HCV GT1a showed a higher rate of RAVs than GT1b

Thibault-GEMHEP_Nov.11 Ogert et al. AASLD 2013, Abst. 927 Different patterns of Resistance to BOC in GT1a vs GT1b

Frequency of the most common RAVs in GT1a In vitro Characterization of Boceprevir and GT1b infected patients RAVs in HCV replicons s t n

e n=14

i n=71 t a P

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n=16 t V f

n=14 i S h

- n=12 ) S n 0

n=10 n=9 o 5

n=23 d l n C

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Differential patterns of RAVs were observed in RAVs found in GT1b appear to have GT1a vs GT1b patients who dis not achieve SVR. higher level of resistance than those in GT1a

Ogert et al. AASLD 2013, Abst. 927 The subgroup of Prior null responders: Selection of RAVs

REALIZE trial (Telaprevir)

Only 14% of Prior Null 80% responders with cirrhosis achieved SVR

% o f S V R

Prior relapsers Prior partial Prior null responders responders

Thibault-GEMHEP_Nov.11Zeuzem et al. NEJM 2011 The subgroup of Prior null responders: Selection of RAVs

REALIZE trial (Telaprevir) Characterization of Viral Variants in prior null responders without SVR.

Rt-variants

80% Wild-type (no-Rt variants)

% o f S V R Not availalble

Prior relapsers Prior partial Prior null responders responders

 TreatmentSo, the poor failure response with thefavors triple the combination growth of resistant of Peg-IFN, virus selected and by atelaprevir 1st generation or ofboceprevir. PI is principally due to an insufficient antiviral response to Peg-IFN and ribavirin.

Thibault-GEMHEP_Nov.11Zeuzem et al. NEJM 2011 De Meyer et al. Hepatology 2012 ASPIRE trial: n= 411 HCV GT 1 patients who had failed to respond or relapsed following following relapsed or respond to failed had who pegIFN/RBV therapy. patients 1 GT HCV 411 n= trial: ASPIRE Zeuzem et al. Abst 2998. EASL 2011

Patients achieving SVR24, % PI 2nd generation :TMC435 (ASPIREtrial): 100 100 10 10 20 30 40 50 60 70 80 90 n= 0 PR48 27 Pbo 37 100 mg* 100 TMC435 Relapsers PR48 79 85 Proportion ofpatientsachievingSVR24 150 mg* 150 TMC435 PR48 79 85 * Duration groups pooled; pooled; groups Duration * PR48 Pbo 23 9 Partialresponders 100 mg* 100 TMC435 PR48 68 57 SV R24, HCV RNAHCV R24, ttt of end the after weeks IU/mL <25 24 undetectable 150 mg* 150 TMC435 PR48 75 69 PR48 Pbo 19 16 Null responders Null 100 mg* 100 TMC435 PR48 46 50 150 mg* 150 TMC435 PR48 51 51 Zeuzem et al. Abst 2998. EASL 2011

Patients achieving SVR24, % 100 100 90 10 20 30 40 50 60 70 80 n= 0 12 PR48 33 33 Pbo 15 ASPIRE: SVR24 by prior response prior by SVR24 ASPIRE: 40 40 34 100 mg* 100 TMC435 Relapsers PR48 82 and HCVgenotypesubtypeand 44 89 33 150 mg* 150 TMC435 PR48 85 45 84 84 * Duration groups pooled; pooled; groups Duration * 8 PR48 13 13 Pbo 15 Partialresponders 7 7 100 mg* 100 TMC435 23 39 PR48 44 SV 68 68 R24, HCV RNAHCV R24, ttt of end the after weeks IU/mL <25 24 undetectable 150 mg* 150 TMC435 25 56 PR48 43 88 88 7 PR48 genotype 1a genotype 1b genotype 1a genotype 0 Pbo 33 9 Null responders Null 100 mg* 100 TMC435 24 33 33 PR48 25 56 56 150 mg* 150 TMC435 26 42 PR48 24 58 58 Q80KLower mutation SVR12 rates observed are observed in in NaïveTMC435 HCV treated GT1 subjects patients with baseline Q80Ktreated compared with to subjects TMC-435 without Q80K

100 Author of study ( y ear) Genoty pe 1a – Data source* Total prev alence of baseline Q80k 90 84,9 polymo82,3rphism 78,5 80 Bae 2010 45.1% Susceptibility testing on samples N=268 isolates (1a) 75 from Los Alamos and Gilead N=372 isolates (1b) Sciences databases 70 Kirst 2011 53%* Sequencing analysis N=22 (1a=15, 60 1b=7) subjects 53,3 R 51,7 50 Bartels 2013 38% Sequencing analysis on patients N=3447 patients V from Phase II-III trials of

S 50 46,7 44,2 42,5 telaprevir % 40 SMV C08/C216 pooled 48.1% TMC435 phase 3 naïve clinical North America analysis trials region (US, Canada, 30 30 26,5 Mexico) Author of study ( y ear) prevalence of Data source* Total 20 baseline Q80k polymorphism 10 Gaudieri 2011 22.6%* Sequencing analysis on patients N=500 subjects (Germany) within the Munich and ATAHC 0 cohorts

Paolucci 2012 (Italy 29% Direct sequencing of HCV NS3/4A in 156 HCV patients protease was performed naïve to PIs

Vicenti 2011 (Italy) 16% Sequencing analysis N=109 (1a=67, 1b=42) Plaza 2012 (Spain) 39.7% Sequencing analysis on patients N=5790 HCV from Los Alamos Database sequences (of which NS3 protease = 1612) Trevinio 2011 (Spain 17.1% All individuals newly diagnosed with HIV-1 at several clinics in Madrid between 2000 and 2010 were tested for serum HCV antibody and HCV RNA.

Lower SVR12 rates are observed in Prevalence of Q80K baseline polymorphism in patients with baseline Q80K HCV genotype 1a – in USA or HCV genotype compared to subjects without Q80K 1a /1b in Europe. Problem of Cross-Resistance to PI!

NS3/4A V36 T54 V55 Q80 R155 A156 A156 ND168 V170 IF Protease M S A R/K K/T/ S T/Vt A/E/ A/T u Inhibitors Q o G/H/ h it T/Y w Telaprevir    r   o r h o Boceprevir it it    w ib    y h   g In   te a se tr a s te   ic ro TMC435 t P     eu p a BI201335 a e    er d h lu t c in   ew n Asunaprivir y    an ABT-450M   GS-9451   Resistance Profile need to be determined after virological failure and before retreatment ! How to retreat the subgroup of experienced-patients with Prior Null response to PEG+ RIBA or/and resistance to Telaprevir and Boceprevir ? Quadruple Therapy With BMS-790052, BMS-650032 and Peg-IFN/RBV f…th only BMS-790052 (NS5A inhibitor) + BMS-650032 (protease inh) 05/02/12 15:51

Quadruple Therapy With BMS-790052, BMS-650032 and Peg-IFN/RBV f…th only BMS-790052 (NS5A inhibitor) + BMS-650032 (protease inh) 05/02/12 15:51

Quadruple-Therapies for null responders

Null-responders genotype 1 patients, n=20 (NS5A Inh Daclatasvir + PI Asunaprevir with PEG2a + R for 24 weeks)

is th r o f d e d n e m m o ts c n re e t ti o a n p s i y p ra e h it B

Quadruple 90% SVR NS5A-Inh + PI 36% SVR [2/2, GT 1b]

http://www.natap.org/2012/HCV/020312_02.htm Page 6 sur 9

http://www.nataLokp.org/ 2et012 al.,/HCV /NEJM020312_02 .2012htm Page 7 sur 9 IFN FREE : Sofosbuvir (anti-NS5B)+Ledipasvir (anti- NS5A) +/-ribavirin in HCV GT1: LONESTAR

Proportion of patients achieving SVR 12

• GT1 naïve patients (n = 60) 100 100 100 95 95 95 – SOF/LDV + RBV 8 weeks

– Or SOF/LDV 12 weeks 80 • Patients with prior non response to PI (n = 40) 60 – Cirrhosis, n = 22 40 – SOF/LDV + RBV during 12 weeks 20

19/20 21/21 18/19 18/19 21/21 0 RBV - + - - + Ttt duration 8 8 12 12 12 (week) Naïve patients PI experienced patients (50 % cirrhosis)

Lawitz E ,Etats-Unis, AASLD 2013, Abs. 215/1844, actualisés In vitro analysis of HCV NS5B 282T mutants

Replication capacity of 282T mutants to Susceptibility of 282T mutants to their corresponding wild type sofosbuvir and ribavirin

 S282T mutants across all HCV genotypes  The S282T mutant EC50 values were showed reduced replication capacity 2.4 to 16.2 fold higher than their compared to their corresponding WT HCV corresponding WT. strain.  S282T mutants in multiple HCV genotype show low level of reduced sensibility to sofosbuvir and no Han, Etats-Unis, AASLD 2013, Abs. 1078 resistance to ribavirin. In vitro cross-resistance assessment to other classes of DAA

Susceptibility of NS5 282T to Susceptibility of NS5 282T to protease nonnucleoside NS5B inhibitors (NNIs) ihnibitors (GS-9451) and NS5A inhibitors (GS- GS-9669 and tegobuvir (GS-9190) 5885)

 S282T mutants remained sensitive to other class of DAA including NS5A inhibitors , Nonnucleoside NS5B inhibitors, NS3 protease Inhibitors and Ribavirin.

Han, Etats-Unis, AASLD 2013, Abs. 1078, actualisés Re-treatment of patients who relapsed after 8 weeks of sofosbuvir + ledipasvir

Patient from LONESTAR study

NS5A : L31M (25,5 %) NS5A : Q30L (3,47 %) L31M (94,38 %) NS5B : no mutations L31V (4,67 %) Y93H (98,19 %) NS5B : 282T (8,00 %)

7 ) l 6 NS5A : Q30L (4,5 %) m / I L31M (> 99 %)

U 5 Y93H (96,74 %) 0 1 NS5B : S282T (91,24 %)

g 4 o l (

3 A

N 2 R < 25 UI/ml détectable V < 25 UI/ml non détectable C

H SVR12 0 SOF/LDV Post- Re-traitement Post- 8 sem. traitement SOF/LDV + RBV traitement 24 sem.

Lawitz E ,Etats-Unis, AASLD 2013, Abs. 215/1844, actualisés Conclusions: Resistance to protease inhibitors: is it a real issue ?

• Today: Yes – Importance of adherence – Impact of subtype (1a/1b) on genetic barrier – The subgroup of Null-responders (with or without cirrhosis). – Retreatment of patients with resistance to Telaprevir or Boceprevir – Determination of cross resistance profile • Tomorrow: may be no – Development of new combination therapy