1. Hépatite C: Résistance
aux Traitements
Prof. Jean-Michel Pawlotsky
CNR des Hépatites B, C et delta
Laboratoire de Virologie & INSERM U635
Hôpital Henri Mondor
Université Paris XII
Créteil

2. HCV Resistance
α
• HCV resistance to IFN-α therapy
• HCV resistance to ribavirin ?
• HCV resistance to specific antiviral
molecules

3. I
HCV resistance to
α
IFN-α Therapy

4. α
Incidence of Peg-IFNα-Ribavirin
Treatment Failures
60 58%
54% PEG-IFN-α2a+ribavirin (Fried et al)
PEG- IFN- 2a+ribavirin
48% PEG-IFN-α2a+ribavirin (Hadziyannis et al)
PEG- IFN- 2a+ribavirin
PEG-IFN-α2b+ribavirin (Manns et al)
PEG- IFN- 2b+ribavirin
45
30
24%
16% 18%
15 2%
0
Genotype 1 Genotypes 2/3
(Manns et al, Lancet 2001 ; Fried et al, N Engl J Med 2002 ; Hadziyannis et al, Ann Intern Med 2004)

5. Treatment
Failure

6. Treatment
Schedule
Treatment
Failure

7. Treatment Host
Schedule Factors
Treatment
Failure

8. Treatment Host
Schedule Factors
Treatment
Failure
Disease
Characteristics

9. Treatment Host
Schedule Factors
Treatment
Failure
Disease
Viral Factors
Characteristics

10. Treatment Host
Schedule Factors
Treatment
Failure
Disease
Viral Factors
Characteristics

11. SNP and SVR in the IDEAL Trial
2 4 6 8 10 12 14 16 18 20 22 Y
1 3 5 7 9 11 13 15 17 19 21 X M
30.0
IL28B
-log10(P)
rs12979860
15.0 P=1.37×10-28
0.0
Chromosome 19 ideogram
0M 10 M 20 M 30 M 40 M 50 M 60 M
39,623 K 39,666 K 39,708 K 39,750 K 39,793 K 39,835K
30.0
-log10(P)
15.0
0.0
PAK4 NCCRP1 SYCN IL28B AC011445.6
IL28A IL29 LRFN1 GMFG
39,711 K 39,721 K 39,732 K 39,743 K 39,753 K 39,764K
30.0
-log10(P)
15.0
0.0
IL28B AC011445.6 IL28A
(Ge et al, Nature, 2009;461:399-401)
2009;461:399-

12. rs12979860 Allele and SVR
(Ge et al, Nature, 2009;461:399-401)
2009;461:399-

13. rs12979860 Allele Frequency
Caucasian African American
ancestry ancestry
n=871 n=191
12%
16%
39% 37%
49% 47%
C/C C/T T/T
(Ge et al, Nature, 2009;461:399-401)
2009;461:399-

14. Geographic Distribution
(Thomas et al, Nature, 2009;461:798-801)
2009;461:798-

15. Effect on HCV Kinetics
(Caucasians)
0
∆ HCV RNA (Log10 IU/mL)
-1.0
-2.0
-3.0 TT
-4.0
CT
-5.0
-6.0 CC
0 2 4 12
Weeks
(Thompson et al, AASLD 2009)

16. Effect on HCV Kinetics
(African Americans)
0
∆ HCV RNA (Log10 IU/mL)
-1.0
TT
-2.0
CT
-3.0
-4.0
-5.0 CC
-6.0
0 2 4 12
Weeks
(Thompson et al, AASLD 2009)

17. SVR Predictors
Odds Ratio 95% CI p-value
rs12979860 CC vs non-CC 5.2 4.1 6.7 <0.0001
HCV RNA ≤ 600,000 IU/mL 3.1 2.3 4.1 <0.0001
Caucasian vs African American 2.8 2.0 4.0 <0.0001
Hispanic vs African American 2.1 1.3 3.6 0.004
METAVIR score ≤F2 2.7 1.8 4.0 <0.0001
Fasting blood sugar < 5.6 mmol/L 1.7 1.3 2.2 <0.0001
(Thompson et al, AASLD 2009)

18. Summary
• In patients infected with HCV genotype 1,
the rs12979860 genotype:
• Is strongly associated with the SVR
• Explains 60% of the ethnic influence on SVR
• Influences HCV kinetics on therapy
• Is probably a marker of patient cell “resistance“
α
to the effect of IFN-α through mechanisms that
remain to be elucidated

19. Viral Resistance
• Intrinsic properties of viral strains
that counteract the antiviral action of
antiviral drugs

20. α
Incidence of Peg-IFNα-Ribavirin
Treatment Failures
60 58%
54% PEG-IFN-α2a+ribavirin (Fried et al)
PEG- IFN- 2a+ribavirin
48% PEG-IFN-α2a+ribavirin (Hadziyannis et al)
PEG- IFN- 2a+ribavirin
PEG-IFN-α2b+ribavirin (Manns et al)
PEG- IFN- 2b+ribavirin
45
30
24%
16% 18%
15 2%
0
Genotype 1 Genotypes 2/3
(Manns et al, Lancet 2001 ; Fried et al, N Engl J Med 2002 ; Hadziyannis et al, Ann Intern Med 2004)

21. HCV Kinetics by Genotype
EC-sponsored DITTO-Trial
7 HCV RNA (log IU/ml)
* = significant difference,, 4 and 1 vs 3
difference
6
5
4
*
3 Quantitative assay cutoff * Genotype 4
Genotype 1
*
2 Qualitative assay cutoff *
* * Genotype 3
1
*
0
-28 -7 01 4 7 8 15 22 29
(Pawlotsky et al., manuscript in preparation)

22. HCV Kinetics by Genotype
EC-sponsored DITTO-Trial
7 HCV RNA (log IU/ml)
* = significant difference,, 4 and 1 vs 3
difference
6
5
4
*
3 Quantitative assay cutoff * Genotype 4
Genotype 1
*
2 Qualitative assay cutoff *
* * Genotype 3
1
*
0
-28 -7 01 4 7 8 15 22 29
(Pawlotsky et al., manuscript in preparation)

23. HCV Kinetics by Genotype
EC-sponsored DITTO-Trial
7 HCV RNA (log IU/ml)
* = significant difference,, 4 and 1 vs 3
difference
6
5
4
*
3 Quantitative assay cutoff * Genotype 4
Genotype 1
*
2 Qualitative assay cutoff *
* * Genotype 3
1
*
0
-28 -7 01 4 7 8 15 22 29
(Pawlotsky et al., manuscript in preparation)

24. HCV Kinetics by Genotype
EC-sponsored DITTO-Trial
7 HCV RNA (log IU/ml)
* = significant difference,, 4 and 1 vs 3
difference
6
5
4
*
3 Quantitative assay cutoff * Genotype 4
Genotype 1
*
2 Qualitative assay cutoff *
* * Genotype 3
1
*
0
-28 -7 01 4 7 8 15 22 29
(Pawlotsky et al., manuscript in preparation)

25. Antiviral Delayed
resistance clearance
Longer
half-life of Viral Factors
infected cells

26. Summary
α
• HCV resistance to IFN-α antiviral effect
exists
• Its molecular mechanisms are unknown
and probably complex
• It accounts for only a small part of IFN-
α-based treatment failures

27. II
HCV Resistance to
Ribavirin ?

28. Ribavirin’s Antiviral Mechanisms
• Direct inhibition of HCV RNA-dependent RNA
polymerase ?
• Depletion of intracellular GTP pools via IMPDH
inhibition ?
• RNA mutagenesis leading to "error catastrophe" ?
• Enhancement of IFN-induced responses in the
liver ?

29. Ribavirin’s Antiviral Effect
Mean HCV RNA decrease (log IU/ml)
0.5
0.0 Controls
Ribavirin
monotherapy
-0.5
-1.0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (days)
(Pawlotsky et al., Gastroenterology 2004;126:703-14)

30. Ribavirin’s Antiviral Effect
HCV RNA changes (log IU/ml) HCV RNA changes (log IU/ml)
+0.5 +0.5
0.0 0.0
-0.5 -0.5
-1.0 -1.0
-1.5 -1.5
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Days Days
HCV RNA changes (log IU/ml) HCV RNA changes (log IU/ml)
+0.5 +0.5
0.0 0.0
-0.5 -0.5
-1.0 -1.0
-1.5 -1.5
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Days Days
(Pawlotsky et al., Gastroenterology 2004;126:703-14)

31. Summary
• Ribavirin mechanisms of action in chronic
hepatitis C remain unknown
• Ribavirin direct antiviral effect is modest
and transient
• Long-term ribavirin administration does
not select for specific resistance
substitutions

32. III
HCV Resistance to Specific
Inhibitors

33. HCV Resistance
• Definition
Selection of viral variants bearing amino acid
substitutions that alter the drug target and
thereby confer reduced susceptibility to the drug
• Resistant variants are pre-existing at
baseline as minor viral populations
All single mutants
~10% of double mutants
(Pawlotsky JM, Ther Adv Gastroenterol 2009;2:205-219; Perelson AS, unpublished data)
2009;2:205-

34. Mechanisms of Resistance
sensitive
resistant

35. Mechanisms of Resistance
Drug
sensitive
resistant

36. Mechanisms of Resistance
Drug
sensitive
sensitive
resistant resistant

37. Mechanisms of Resistance
Drug Stop drug
sensitive
sensitive
resistant resistant

38. Mechanisms of Resistance
Drug Stop drug
sensitive
sensitive sensitive
resistant resistant resistant

39. Mechanisms of Resistance
Drug Stop drug
sensitive
sensitive
sensitive
resistant resistant
resistant + fit

40. Mechanisms of Resistance
Drug Stop drug
sensitive sensitive
sensitive
resistant resistant resistant + very fit

41. DAA Drugs Targets
Receptor binding
and endocytosis Transport
and
release
Fusion and
uncoating
(+) RNA
Virion
assembly
RNA
replication
Translation and
polyprotein
processing

42. HCV DAA Drug Development
Phase of Development
Viral entry inhibitors Preclinical I II III IV
Hepatitis C immunoglobulin HCIg)
HCIg)
HCV-Ab 68 and Ab 65 (monoclonal Ab)
HCV-
HCV RNA translation inhibitors
ISIS 14803 (antisense) *
AVI – 4065 (antisense)
Heptazyme (ribozyme) *
VGX-410C (small molecule IRES inhibitor)
VGX-
TT 033 (siRNA)
(siRNA)
Posttranslational processing inhibitors
NS3-4A serine proteinase inhibitors
NS3-
BILN 2061 *
ITMN 191
VX-950
VX-
SCH 503034
ACH-806/GS-9132
ACH- 806/GS- *
HCV replication inhibitors
NS5B polymerase inhibitors
MK-0608
MK-
HCV-796
HCV- *
R1626
JTK-003
JTK- *
NM-283
NM- *
XTL 2125
Cyclophilin B inhibitors
DEBIO-025
DEBIO-
NIM 811
NS5A inhibitors
A-831, A-689
A-
Helicase inhibitors
QU663
Recombinant Ab fragments
Virus assembly and release inhibitors
UT-231B (iminosugar-glucosidase inhibitor)
UT- (iminosugar-
Celgosivir (glucosidase inhibitor)
(Pawlotsky JM, Chevaliez S, McHutchison JG, Gastroenterology 2007;132:1979-98)

43. Targets for New HCV Inhibitors
IRES
NS3 Protease RNA-Dependent
RNA Polymerase

44. Resistance to NS3/4A
Protease Inhibitors

45. Antiviral Efficacy of NS3/4A
Protease Inhibitors
Median/mean log
Drug Phase Dose Duration
HCV RNA reduction
Telaprevir III 750 mg q8h 14 days -4.4
Boceprevir III 400 mg tid 7 days -1.6
TMC435 II 200 mg qd 7 days -4.1
ITMN-191/R7227 II 200 mg q8h 14 days -3.8
MK-7009 II 700 mg bid 8 days -4.7
BI201335 II 240 mg qd 14 days -4.0
Narlaprevir II 400 mg bid 7 days -4.2
BMS-650032 I 300 mg bid 3 days -3.3

46. Amino Acid Substitutions
Associated with Resistance
Macrocyclic Linear
BILN 2061 R7227 VX-950 SCH503034
(Ciluprevir) (ITMN-191) (Telaprevir) (Boceprevir)
A156S/V,
In vitro
A156V/T,
D168A, Q41R, A156S/T,
D168A/V, A156S/T/V
F43S, S138T, T54A, V170A
R155Q
S489L
In vivo
A156S/T/V,
V36M/A,
No data No data R155K/T,
T54A/S, R155K
T54A, V36A/M
(Kwong et al., Curr Opin Pharmacol, 2008;8:522-531; Susser et al., J Hepatol 2009;50(Suppl.1):S7)
Pharmacol, 2008;8:522- 2009;50(Suppl.1):S7)

47. Amino Acid Substitutions
Associated with Resistance
Arg155
Asp168
Ala156
Thr54
Val36
(Pawlotsky J-M, Ther Adv Gastroenterol 2009;2: 205-219)
J- 205-

48. Telaprevir (Vertex & Tibotec)

50. Paramètres Associés
à la Résistance
• Niveau de résistance au médicament
conféré par la ou les substitutions
amino acidiques
• In vivo “fitness“ des variants viraux
sélectionnés

54. PROVE 2 Trial (Phase II)
Naïve, Genotype 1, Europe
PR48 α
Placebo + Peg-IFNα2a + Ribavirin (RBV) Follow-up
Follow-
Telaprevir 750 mg q8h
T12/PR24 α
+ Peg-IFNα2a α
Peg-IFNα2a + RBV Follow-up
Follow-
+ RBV
Telaprevir 750 mg q8h
T12/PR12 α
+ Peg-IFNα2a Follow-up
Follow-
+ RBV
Telaprevir 750 mg q8h
T12/P12 Follow-up
Follow-
α
+ Peg-IFNα2a
0 12 24 48 72
Weeks on therapy
(Hézode et al., N Engl J Med 2009;360:1839-50)
2009;360:1839-

55. PROVE 2 Trial (Phase II)
Breakthoughs through week 12
100
80
12-week SVR rates (%)
60
40
24%
20
3%
0
T12/P12 (no RBV) T12/PR12
+ T12/PR24
(Hézode et al., N Engl J Med 2009;360:1839-50)
2009;360:1839-

56. PROVE 2 Trial (Phase II)
Relapses after treatment
100
80
SVR rates (%)
60
48%
40
30%
22%
20 14%
0
PR48 T12/P12 (no RBV) T12/PR12 T12/PR24
(n=82) (n=78) (n=82) (n=81)
(Hézode et al., N Engl J Med 2009;360:1839-50)
2009;360:1839-

62. Boceprevir (SP/Merck)

63. Patterns of Response
to Boceprevir
N=22 non-responders to previous standard treatment who were dosed for 14 days with BOC (Phase 1b)
(Susser et al., Hepatology 2009;50:1709-18)
2009;50:1709-

64. In vitro Resistance to
In
Boceprevir (replicon)
(replicon)
(Susser et al., Hepatology 2009;50:1709-18)
2009;50:1709-

65. Relative in vivo Fitness of
Boceprevir-Resistant Variants
Boceprevir-Resistant
(Susser et al., Hepatology 2009;50:1709-18)
2009;50:1709-

66. Dynamics of NS3
Protease Variants
(Susser et al., Hepatology 2009;50:1709-18)
2009;50:1709-

67. Summary
• During telaprevir or boceprevir monotherapy, pre-
existing resistant HCV variants are rapidly selected in
virtually all treated patients
• Both single mutants (36, 54, 155 and 156) and double
mutants (36/155, 26/156) can be selected
• The corresponding viral variants generally have
reduced fitness compared to wild-type HCV
• Most of these amino acid substitutions confer cross-
resistance to all of the NS3/4A protease inhibitors in
development

68. Summary (cont’d)
• Failure of the triple combination of pegylated IFN-α,
ribavirin and telaprevir to clear HCV is associated with
the selection of telaprevir-resistant variants
• These variants may persist after therapy or be
progressively replaced by wild-type virus
• Telaprevir resistant variants remain sensitive to
pegylated interferon and ribavirin

69. Resistance to Inhibitors of
HCV Replication

70. Inhibitors of HCV Replication
• RNA-dependent RNA polymerase (RdRp)
inhibitors
• Nucleoside analogues
• Non-nucleoside inhibitors (NNIs)
• NS5A inhibitors
• Cyclophilin inhibitors
• miR122 antagonists

71. In vitro Resistance
4’ azido-cytidine
azido-
Ser 96
2’-methyl nucleosides
Ser 282
Copyright © 2006 Merck Co., Inc., Whitehouse Station, New Jersey, USA All rights reserved

72. HCV Resistance to 2’-C-Methyl
Nucleoside Inhibitors
2’C-Me-ATP in the catalytic site
(Migliaccio et al., J Biol Chem 2003;278:49164-70)

73. 7-Deaza-2’-C-Methyl-Adenosine
(MK-0608) in Chimpanzees
wt S282R/T/I wt S282
MK-0608 at 1mg.kg-1 orally
7
6
HCV RNA (Log IU/mL)
5
4
3
2 - 4,6 - 4,1
LOQ
1 (20 IU/mL)
TMA + + + + - - - - - - - - + +
0
-15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Days
(Carroll et al., Antimicrob Agents Chemother 2009;53:926-934)
2009;53:926-

74. Valopicitabine (NM283)
Phase IIb, IFN Nonresponders
Week 24
0 n=7
-0.27
-0.5
HCV RNA reduction
-1 NM283 800 mg monotherapy
-1.5
-2
-2.5
-3
-3.5
0 4 8 12 16 20 24 28 32 36 40 44 48
Weeks
(Afdhal et al, EASL 2006)

75. Antiviral Efficacy of NNIs
Median/mean log
Drug Phase Dose Duration
HCV RNA reduction
GS-9190 II 40 mg bid 8 days -1.4
Filibuvir II 300 mg bid 8 days -2.1
ANA598 II 800 mg bid 3 days -2.9
BI207127 II 800 mg q8h 3 days -3.1
VCH-759 II 400 mg tid 10 days -1.7
ABT-333 II 600 mg bid 2 days -1.5
VX-222 Ib 750 mg bid 3 days -3.7
MK-3281 Ib 800 mg bid 7 days -1.3 (1a), -3.8 (1b)

76. In vitro Resistance
NNI site C
Benzothiadiazine
NNI site A
Asn 411, Met 414, Tyr 448
Indoles, Benzimidazoles
Indoles,
Pro 495, Pro 496, Val 499
A
Allosteric GTP
B E
C
D
C
NNI site B
Thiophene-COOH
Thiophene-
Met 423
NNI site D
Benzofurans NNI site E (hypothetical)
Cys 316, Val 201 Cys 445, Tyr 448, Tyr 452
Copyright © 2006 Merck Co., Inc., Whitehouse Station, New Jersey, USA All rights reserved

77. HCV 796 (ViroPharma Wyeth)
Antiviral efficacy
1
Treatment Follow-up
Log HCV RNA change
0
-1 Placebo
50 mg
100 mg
250 mg
-2 500 mg
1000 mg
1500 mg
-3
-1 2 5 8 11 14 17 20 23 26 29
(Chandra et al, DDW 2006)
Study Day

78. Prevention of Resistance

79. Prevention of Resistance to
HCV Inhibitors
• Exclude use as a monotherapy
• Combine with one or several other
antivirals with:
• At least additive antiviral effect
• No cross-resistance

80. R7128/R7227 Combination
INFORM Trial
1
Mean log10 HCV RNA change from
Placebo
0
-1 R7128 500b D1-7/R7227 100t D4-7-Na
-2 R7227 100t D1-7/R7128 500b D4-7-Na
-3
-4 500b/100t
1000b/200t
-5 500b/200t
1000b/100t
-6
-7
0 2 4 6 8 10 12 14
Day
(Gane et al., EASL 2009)

81. R7128/R7227 Combination
INFORM Trial
1
Mean log10 HCV RNA change from
Placebo
0
-1
-2
-3
-4 1000b/600b-TE
-5 1000b/900b-TE
1000b/900b-Na
-6
-7
0 2 4 6 8 10 12 14
Day
(Gane et al., AASLD 2009)

82. Cure with IFN-Free Regimens ?

83. Combination of MK-7009 and
MK-608 (Merck) in vitro
10
5-10
5
Synergy in the genotype 1b 0-5
replicon at low MK-0608,
high MK-7009 doses 0
-5-0
-5
0.3
0.02
0.15 MK-7009 NS3
12
1.20 inhibitor (nM)
MK-608 pol
inhibitor (mM)
(Olsen et al., APASL 2008)

84. Combination of MK-7009 and
MK-608 (Merck) in vivo
6 MK 7009 MK 7009
+ MK-608 monotherapy Chimp A
5 Chimp B
Chimp C
log IU/mL
4
3 SVR
2
LOQ
1
0
0 50 90 130 170 210 250
Day
(Olsen et al., APASL 2008)