How to prevent, diagnose and overcome resistance to nucleoside analogs ?

1
How to prevent, diagnose and overcome resistance
to nucleoside analogs ?

• Fabien Zoulim
• Liver department, Hôtel Dieu Hospital
• Hepatitis research laboratory, INSERM U871
• Lyon, France

2
Clinical definitions
3
Définition of resistance

• Virologic Breakthrough Rebound in serum HBV DNA
  levels (e.g. 1 log10 above nadir)
• Genotypic Resistance Detection of mutations
  known to confer resistance while on therapy
• Virologic Breakthrough with Genotypic Resistance
  Viral rebound associated with a mutation(s)
  known to cause resistance.
• Primary non response lt1log10 decrease of viral
  load after 3 months
• Partial response detectable HBV DNA levels
  during therapy

Zoulim Perrillo, J Hepatol 2008 EASL CPG, J
Hepatol 2009
4
Treatment failure
Primary non response Partial response
Secondary treatment failure Antiviral drug
resistance
Host factors Drug metabolism Patients
compliance Drug factors Antiviral potency
Drug factors Barrier to resistance Viral
factors Resistant mutants
Zoulim Perrillo J Hepatol 2008 EASL CPG J
Hepatol 2009
5
Incidence of Resistance in Nucleoside Naive
Patients
of patients with resistance mutations
Lai et al CID 2003 Hadzyiannis et al
Gastroenterology 2006 Colonno et al AASLD 2006
Di Bisceglie et al AASLD 2006 Lai et al NEJM
2007 Marcellin et al NEJM 2008
6
Incidence of Resistance in Lamivudine Refractory
Patients
of patients with resistance mutations
Lampertico et al Hepatology 2005
Gastroenterology 2007 Colonno et al AASLD 2007
Lacombe et al AIDS 2006
7
Kinetics of drug resistance emergence
BiochemicalBreakthrough
Genotypic resistance
VirologicalBreakthrough
6
5
ALT (IU/L)
4
HBV DNA (log10 IU/mL)
Nadir
3
2
ULN
1
Antiviral drug
0
Time
Si Ahmed et al. Hepatology. 2000321078-1088
Zoulim Antivir Chem Chemother 200112 131-142
Yuen et al Hepatology 2001 34784-791 Locarnini
et al Antiviral Therapy 20049679-693
8
Lamivudine Resistance Accelerates Progression of
Liver Disease
Placebo
21
YMDDm
13
WT
5
Liaw YF et al. N Engl J Med. 20043511521-1531
9
Polymerase gene mutations reponsible for drug
resistance
Spacer
Pol/RT
RNaseH
Terminal protein
845 a.a.
349
692
1
183
(rt1)
(rt 344)
YMDD
GVGLSPFLLA
A
B
C
E
D
I(G)
II(F)
V173L L180M M204I/V
LAM / FTC
A181V/T
N236T I233V ?
ADV
M204I/V
I169T T184G S202G/I M250V
ETV
LdT
M204I
TDF
A194T ?
Allen et al. Hepatology 1998 Gish et al. J
Hepatol 2005 Qi et al. J Hepatol 2004 Tenney
et al. AAC 2004 2007 Lai et al.
Gastroenterology 2005 Sheldon et al. Antivir
Ther 2005 Delaney et al. AAC 2006 Schildgen
et al NEJM 2006 Borroto-Esoda JID 2007
Durantel et al Antiviral Therapy 2008 Villet et
al Gastroenterology 2006, J Hepatol 2007 2008
Warner et al Hepatology 2008
10
Tools for the diagnosis of resistance
11
Dynamic ranges of quantificationof HBV DNA assays
10
102
103
104
105
106
107
108
109
Amplicor HBV Monitor v2.0 (Roche)
HBV Hybrid-Capture II (Digene)
Ultra-sensitive HBV Hybrid-Capture II
Versant HBV DNA 3.0 (bDNA, Siemens)
Cobas Taqman HBV (Roche)
RealArt HBV LC PCR (Artus Biotech)
Abbot Real-time HBV (Abbott)
Versant HBV DNA 1.0 (kPCR, Siemens)
in development
12
Methods to detect genotypic resistance

• Direct PCR sequence analysis
• Reverse hybridization assay (INNO-LiPA DR)
• Others
• Restriction fragment length polymorphism (RFLP)
  analyses
• Matrix-assisted laser desorption/ionization-Time
  of light Mass Spectrometry (MALDI-TOF MS)

13
The INNO-LiPA principle
Marker line
Conj. Cont.
Amp. Cont.
L528
M528
M552
V552
I552
V555
L555
M555
DNA probe
I555
Nitrocellulose strip
Stuyver et al. J. Clin. Microbiol. 2000 38 702
Sablon E. et al Int. J. Med. Sci. 2005 2 8-16
14
Phenotypic resistance testing

• Determines in vitro inhibitory concentrations
  (IC)/effective concentration (EC) of specific HBV
  inhibitors relative to a wild type or reference
  strain
• Allows the quantification of the magnitude of
  resistance to a drug without the need to know the
  responsible mutation(s)
• Confirms the drug susceptibility associated with
  a given amino-acid change in HBV polymerase (eg.
  M204V/I) and determines its cross-resistance
  profile

15
Results of phenotypic assays
100
Wild-type virus
Patients virus
IC50 Drug susceptibility
Inhibition of viral replication
50
IC50 patient
Fold change
IC50 wild type
0
IC50
IC50
Concentration of drug
16
Choice of drug for treatment adaptation
17
Cross-resistance data for the most frequent
resistant mutants
Lamivudine Telbivudine Entecavir Adefovir Tenofovir
Wild-type S S S S S
M204l R R I S S
L180M M204V R R I S S
A181 T/V I S S R S
N236T S S S R I
I169T V173L M250V R R R S S
T184G S202lI/G R R R S S
( L180M M204I/V).
Durantel et al Hepatology 2004 Brunelle et al
Hepatology 2005 Yang et al Antiviral Therapy
2005 Villet et al Gastroenterology 2006 2008
Delaney et al AAC 2006 Villet et al J Hepatol
2007 2008 Brunelle et al AAC 2007 Qi et al
Antiviral therapy 2007 Tenney et al AAC 2004
2007
18
Management of HBV Drug Resistance
Lamivudine / Telbivudine resistance
Add ADV Add TDF Switch to TVD TDFFTC
Switch to ETV
Not valid LAM FTC LdT
Zoulim and Perrillo, J Hepatol, 2008 EASL CPG J
Hepatol 2009
19
Management of HBV Drug Resistance
Adefovir resistance
Adefovir non response
Switch to TDF TVD ETV LdT
Add Lamivudine Add ETV Add Telbivudine
Switch to TVD TDFFTC
Zoulim and Perrillo, J Hepatol, 2008 EASL CPG J
Hepatol 2009
20
Management of HBV Drug Resistance
Entecavir resistance
Add ADV Add TDF
Switch to TVD TDFFTC
Not valid LAM LdT
Zoulim and Perrillo, J Hepatol, 2008 EASL CPG J
Hepatol 2009
21
Add-on or switch ?
22
Add-on therapy with drugs having a complementary
cross-resistance profile
Zoulim Antivir Res 2004Villeneuve et al J
Hepatol 2003 Lampertico et al Gastroenterology
2007
23
The problem of sequential therapy and switching
strategy
LAM
LAM
300 250 200 150 100 50
10
ADV
9
8
L180MM204V
7
N236T
Serum HBV DNA (Log10 copies/mL)
6
Reverted to wild type
ALT (IU/L)
5
4
3
2
janv-98
janv-99
janv-00
janv-01
janv-02
janv-03
janv-04
janv-05
? HBV DNA ? ALT
Villeneuve et al, J Hepatol 2003
24
Patients with lamivudine resistance adefovir
add-on strategy
3-yr cumulative probability
Virologic breakthrough and ADV resistance
Virologic breakthrough
100
100
80
80
ADV mono
ADV mono
ADVLAM
Patients with virological breakthrough
ADVLAM
Patients with ADV-R
60
60
40
40
30
Plt0.001
Plt0.001
20
20
16
6
0
0
0
Months
0
3
6
9
12
15
18
21
24
27
30
33
36
0
3
6
9
12
15
18
21
24
27
30
33
36
273
268
256
225
201
158
61
Patients still at risk
229
225
217
194
179
146
57
255
238
223
213
200
177
103
242
227
214
205
200
174
92
gt 1 log rebound of HBV DNA compared to
on-treatment nadir N236T or A181T-V in
patients with a virological breakthrough
Lampertico et al Gastroenterology 2007
25
When changing treatment ?
26
Rescue therapy in patients with clinical
breakthrough
Drug A
Drug B
Serum HBV DNA (Log10 copies/mL) and ALT (x ULN)
Month of therapy
27
Rescue therapy in patients at the time of
virologic breakthrough
Drug A
Drug B
Serum HBV DNA (Log10 copies/mL) and ALT (x ULN)
Month of therapy
Zoulim and Perrillo, J Hepatol, 2008 EASL CPG J
Hepatol 2009
28
Viral Load at Week 24 is a Predictor of
Resistance at Week 104 of Therapy (Telbivudine
vs. Lamivudine trial)
HBeAg Positive, n921
HBeAg Negative, n446
Lai et al , NEJM, 2007
29
Early add-on therapy to prevent drug resistance
Drug A
Drug B
Serum HBV DNA (Log10 copies/mL) and ALT (x ULN)
Month of therapy
Zoulim and Perrillo, J Hepatol, 2008 EASL CPG J
Hepatol 2009
30
Secondary Treatment Preferences Based on
Virologic Monitoring
Nucleoside analog treatment
Monitor at 12-24 weeks
Monitor every 12 weeks
Partial virologic response
Virologic breakthrough
Early non reponse
Add a more potent agent or switch to a more
potent combination (emtricitabine/tenofovir)
Switch to more potent agent
Choice based on cross-resistance data
Zoulim and Perrillo, J Hepatol, 2008 EASL CPG J
Hepatol 2009
31
Very Early Add-on Therapy to Keep Viral Load as
Low as Possible
1. Start with a drug having high potency and low
rate of resistance 2. Add a drug with a different
cross-resistance profile
Drug A
Drug A Drug B
Long-term viral load suppression, or risk of
selection of MDR mutants ?
Month of therapy
outgrowth of drug resistant mutant?
32
Prevention of drug resistance

• First line therapy
• Use of antivirals with high antiviral potency and
  high barrier to resistance
• Combination therapy with complementary drugs
• Second line treatment
• Add-on strategies with complementary drugs
  preferred to sequential monotherapies
• Early treatment adaptation to prevent
  accumulation of mutations
• Choice always based on cross-resistance data
• Its prime time for the next treatment objective
• Clearance of HBsAg !