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

Fabien Zoulim Liver department, Htel Dieu Hospital & Hepatitis research laboratory, INSERM U871 Lyon, France

Clinical definitions

Dfinition 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: <1log10 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

Treatment failure

Primary non response Partial response

Secondary treatment failure Antiviral drug resistance

Host factors
Drug metabolism Patients compliance

Drug factors
Barrier to resistance

Viral factors Drug factors

Antiviral potency Resistant mutants

Incidence of Resistance in Nucleoside Naive Patients

80 70 60 50 40 30 20 10 0 Lamivudine Adefovir Entecavir Telbivudine Tenofovir year 5 year 4 year 3 year 2 year 1

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

Incidence of Resistance in Lamivudine Refractory Patients

40 30 20 10 0
Adefovir switch Adefovir add-on Entecavir Tenofovir + switch FTC/3TC Year 2 Year 1 baseline Year 4 Year 3

Lampertico et al Hepatology 2005 & Gastroenterology 2007; Colonno et al AASLD 2007; Lacombe et al AIDS 2006

Kinetics of drug resistance emergence

Genotypic resistance Virological Breakthrough Biochemical Breakthrough
Biochemical Breakthrough

6
HBV DNA (log10 IU/mL)

Virologic Breakthrough Detection of Genotypic Resistance 1 log10

5 4 3 2 1

ALT (IU/L)

Nadir

ULN

Antiviral drug

Time

Si Ahmed et al. Hepatology. 2000;32:1078-1088; Zoulim Antivir Chem Chemother 2001;12: 131-142; Yuen et al Hepatology 2001; 34:784-791; Locarnini et al Antiviral Therapy 2004;9:679-693

Lamivudine Resistance Accelerates Progression of Liver Disease

25
% With disease progression

Placebo (N=215) YMDDm (N=209) (49%) Wild Type (N=221)

Placebo

20 15 10 5 0 0

21%

YMDDm

13%

WT

5%

12

18

24

30

36

Time after random ization (Months)

Liaw YF et al. N Engl J Med. 2004;351:1521-1531

Polymerase gene mutations reponsible for drug resistance

Terminal protein 1 183 Spacer 349 (rt1) GVGLSPFLLA I(G) II(F) A B

Pol/RT
692 (rt 344)

RNaseH 845 a.a.

YMDD
C D E

LAM / FTC

V173L L180M A181V/T M204I/V N236T I233V ? M204I/V S202G/I M204I A194T ? M250V

ADV

ETV
LdT TDF

I169T T184G

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

Tools for the diagnosis of resistance

Dynamic ranges of quantification of HBV DNA assays

10
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)

102

103

104

105

106

107

108

109

RealArt HBV LC PCR (Artus Biotech)

Abbot Real-time HBV (Abbott) Versant HBV DNA 1.0 (kPCR, Siemens)*
*in development

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)

The INNO-LiPA principle

Chromogen (NBT/BCIP)
Marker line Conj. Cont. Amp. Cont. L528

Purple precipitate

Alkaline Phosphatase
M528

Streptavidin
M552 V552 I552

Biotin

Amplified target

V555 L555 M555

DNA probe Nitrocellulose strip

I555

Stuyver et al. J. Clin. Microbiol. 2000; 38: 702; Sablon E. et al Int. J. Med. Sci. 2005; 2: 8-16

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

Results of phenotypic assays

100%

Inhibition of viral replication

Wild-type virus

Patients virus

IC50 =

Drug susceptibility

50%

IC50 patient IC50 wild type

= Fold change

0% IC50 IC50

Concentration of drug

Choice of drug for treatment adaptation

Cross-resistance data for the most frequent resistant mutants

Lamivudine
Wild-type M204l L180M + M204V A181 T/V N236T I169T + V173L + M250V* T184G + S202lI/G * S R R

Telbivudine
S R R

Entecavir
S I I

Adefovir
S S S

Tenofovir
S S S

I S

S S

S S

R R

S I

*: (+ 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

Management of HBV Drug Resistance

Lamivudine / Telbivudine resistance

Not valid LAM FTC LdT

Switch to ETV

Add ADV Add TDF Switch to TVD: TDF+FTC

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

Management of HBV Drug Resistance

Adefovir non response

Adefovir resistance

Switch to TDF TVD ETV LdT

Add Lamivudine Add ETV Add Telbivudine

Switch to TVD: TDF+FTC

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

Management of HBV Drug Resistance

Entecavir resistance

Not valid LAM LdT

Add ADV Add TDF

Switch to TVD: TDF+FTC

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

Add-on or switch ?

Add-on therapy with drugs having a complementary cross-resistance profile

Wild type

LAM
LAM-R

ADV

ADV-R

LAM

+
Zoulim Antivir Res 2004;Villeneuve et al J Hepatol 2003; Lampertico et al Gastroenterology 2007

ADV

The problem of sequential therapy and switching strategy

LAM LAM ADV 300

10 9

250

8 7 6 5 4 3 2
janv-98 janv-99 janv-00 janv-01 janv-02 janv-03 janv-04 janv-05

L180M+ M204V Reverted to wild type

N236T

200

150

100

50

HBV DNA

ALT Villeneuve et al, J Hepatol 2003

Patients with lamivudine resistance: adefovir add-on strategy

3-yr cumulative probability
100

Virologic breakthrough*

100

Virologic breakthrough* and ADV resistance**

ADV mono

80 ADV mono 60 ADV+LAM

80 ADV+LAM

60

40 P<0.001 30%

40 P<0.001 20 6% 0 3 6
268 238

20

16% 0% 9 12 15 18 21 24 27 30 33 36
217 214 194 205 179 200 146 174 57 92

0
273 255 256 223 225 213 201 200 158 177

9 12 15 18 21 24 27 30 33 36
61 103

Months
Patients still at risk

0
229 242

6
225 227

* > 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

When changing treatment ?

Rescue therapy in patients with clinical breakthrough

Drug A
8 6 4 2 0

Drug B

M0

M6

M12

M18

M24

M30

ALT

HBV DNA

M36

Month ALT of therapy

Rescue therapy in patients at the time of virologic breakthrough

Drug A

8 6 4 2 0

Drug B

M0

M6

M12

M18

M24

Month of therapy

M30

ALT

ALT HBV DNA

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

M36

Viral Load at Week 24 is a Predictor of Resistance at Week 104 of Therapy (Telbivudine vs. Lamivudine trial)
100% 100%

HBeAg Positive, n=921

% of patients with resistance % of patients with resistance

HBeAg Negative, n=446

80%

80%

60% 45% 30%

60% 50%

60% 56%

41% 40% 29% 25% 24% 20% 9% 4% 0%

< QL, n=203,146 QL - 3, n=57,63 3 to 4, n=83,79

40%

20% 20% 2% 0% 5% 12% 6%

> 4, n=115,175

< QL, n=178,157

QL - 3, n=18,20

3 to 4, n=16,24

> 4, n=10,23

Telbivudine

Lamivudine
Lai et al , NEJM, 2007

Early add-on therapy to prevent drug resistance

Drug A Drug B

8 6 4 2 0

M0

M6

M12

M18

M24

M30

Month ALT of therapy

ALT HBV DNA

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

M36

Secondary Treatment Preferences Based on Virologic Monitoring

Nucleoside analog treatment
Monitor at 12-24 weeks Monitor every 12 weeks

Early non reponse

Partial virologic response

Virologic breakthrough

Switch to more potent agent*

Add a more potent agent* or switch to a more potent combination (emtricitabine/tenofovir*)

* Choice based on cross-resistance data

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

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
Serum HBV DNA (Log10 copies/mL)

8 7 6 5 4 3 2 M0 M3 M6 M9 M12 M15 M18 M21 M24

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?

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 !