How to prevent, diagnose and overcome resistance to nucleoside analogs ? Fabien Zoulim Liver department, Hôtel Dieu Hospital & Hepatitis research laboratory,

How to prevent, diagnose and overcome resistance to nucleoside

analogs ?

Fabien Zoulim

Liver department, Hôtel Dieu Hospital

& Hepatitis research laboratory, INSERM U871

Lyon, France

Clinical definitions

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: <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 responsePartial response

Secondary treatment failureAntiviral drug resistance

Host factorsDrug metabolismPatient’s compliance

Drug factorsAntiviral potency

Drug factorsBarrier to resistance

Viral factorsResistant mutants

Zoulim & Perrillo J Hepatol 2008; EASL CPG J Hepatol 2009

Incidence of Resistance in Nucleoside Naive Patients

% o

f pa

tien

ts w

ith

resi

stan

ce m

utat

ions

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

0

10

20

30

40

50

60

70

80

Lamivudine Adefovir Entecavir Telbivudine Tenofovir

year 1year 2

year 3year 4

year 5

Incidence of Resistance in Lamivudine Refractory Patients

% o

f pa

tien

ts w

ith

resi

stan

ce m

utat

ions

0

10

20

30

40

Adefovirswitch

Adefoviradd-on

Entecavirswitch

Tenofovir +FTC/3TC

baseline

Year 1

Year 2

Year 3

Year 4

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

Time

Antiviral drug

HB

V D

NA

(lo

g1

0 I

U/m

L)

AL

T (IU

/L)

4

6

5

3

2 ULN

1

0

Detection of Genotypic Resistance

Nadir1 log10

VirologicBreakthrough

BiochemicalBreakthrough

Genotypic resistance

VirologicalBreakthrough

BiochemicalBreakthrough

Kinetics of drug resistance emergence

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

0

5

10

15

20

25

0 6 12 18 24 30 36

Time after randomization (Months)

% W

ith

dis

ea

se

pro

gre

ss

ion

Placebo (N=215)

YMDDm (N=209) (49%)

Wild Type (N=221)

YMDDm

WT

Placebo

5%

13%

21%

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

Polymerase gene mutations reponsible for drug resistance

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

RNaseH

845 a.a.

Terminal protein Spacer Pol/RTPol/RT

AA BB CC EED D

1 183 349 692

YMDD

V173L

L180M M204I/V

GVGLSPFLLA

I(G)I(G) II(F) II(F)

(rt1) (rt 344)

LAM / FTC

ETV I169T T184G S202G/I M250V

ADV A181V/T N236TI233V ?

LdT M204I

TDF A194T ?

M204I/V

Tools for the diagnosis of resistance

Dynamic ranges of quantificationof HBV DNA assays

Amplicor HBV Monitor v2.0 (Roche)

HBV Hybrid-Capture II (Digene)

Ultra-sensitive HBVHybrid-Capture II

Versant HBV DNA3.0 (bDNA, Siemens)

Cobas Taqman HBV(Roche)

Abbot Real-time HBV(Abbott)

Versant HBV DNA 1.0(kPCR, Siemens)*

*in development

1010 101022 101033 101044 101055 101066 101077 101088 101099

RealArt HBV LC PCR(Artus Biotech)

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

DNA probe

Nitrocellulose stripI555

M555L555

V555

I552

V552

M528

M552

L528

Amp. Cont.Conj. Cont.

Marker line

AlkalinePhosphatase

Streptavidin

Chromogen(NBT/BCIP)

Purpleprecipitate

BiotinAmplified

target

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 assaysIn

hib

itio

n o

f vi

ral

rep

lica

tio

n

100%

0%

50%

Concentration of drug

Wild-type virus

Patient’s virus

IC50IC50

IC50 wild type

IC50 patient= Fold change

IC50 = Drug susceptibility

Choice of drug for treatment adaptation

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).

Cross-resistance data for the most frequent resistant mutants

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

Lamivudine / Telbivudine resistance

Add ADV Add TDFSwitch to TVD: TDF+FTC

Switch to ETVNot validLAMFTCLdT

Management of HBV Drug Resistance

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

Adefovir resistance

Add LamivudineAdd ETVAdd Telbivudine

Switch to TVD: TDF+FTC


Switch toTDFTVDETVLdT

Adefovir non response


Entecavir resistance

Add ADVAdd TDF

Switch to TVD: TDF+FTCNot validLAMLdT



Add-on or switch ?

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

+

LAM

ADV

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

Wild type

LAM-R

ADV-R

ADVLAM

HBV DNA ALT

The problem of sequential therapy and switching strategy

Villeneuve et al, J Hepatol 2003

N236T

Ser

um

HB

V D

NA

(L

og1

0 c

opi e

s/m

L)

ALT

(IU/L )

300

250

200

150

100

50

L180M+M204V

LAM

ADV

Reverted to wild type

2

3

4

5

6

7

8

9

10

janv-98 janv-99 janv-00 janv-01 janv-02 janv-03 janv-04 janv-05

LAM

Months

ADV mono

Pat

ient

s w

i th v

irolo

gica

l bre

a kth

roug

h

273 268 256 225 201 158 61

30%

6%

P<0.001

ADV+LAM

255 238 223 213 200 177 103

Pat

ient

s w

i th A

DV

-R

229 225 217 194 179 146 57

16%

0%

P<0.001

ADV mono

ADV+LAM

242 227 214 205 200 174 92

3-yr cumulative probability

* > 1 log rebound of HBV DNA compared to on-treatment nadir

** N236T or A181T-V in patients with a virological breakthrough

Patients

still at risk

Virologic breakthrough* Virologic breakthrough*

and ADV resistance**

Lampertico et al Gastroenterology 2007

0

20

40

60

80

100

0 3 6 9 12 15 18 21 24 27 30 33 36

0

20

40

60

80

100

0 3 6 9 12 15 18 21 24 27 30 33 36

Patients with lamivudine resistance: adefovir add-on strategy

When changing treatment ?

M0

M6

M1

2

M1

8

M2

4

M3

0

M3

6 ALT0

2

4

6

8

ALT HBV DNA

Month of therapy

Rescue therapy in patients with clinical breakthrough

Drug A

Drug B

Seru

m H

BV

DN

A (

L og1

0 c

opi e

s /m

L )

an

d A

LT (

x U

LN)

M0

M6

M1

2

M1

8

M2

4

M3

0

M3

6

ALT0

2

4

6

8

ALTHBV DNA

Month of therapy

Rescue therapy in patients at the time of virologic breakthrough

Drug A

Drug B

Seru

m H

BV

DN

A (

L og1

0

c op

i es/

mL )

an

d A

LT (

x U

LN)


Viral Load at Week 24 is a Predictor of Resistance at Week 104 of Therapy

(Telbivudine vs. Lamivudine trial)

4%

25%29%

30%

9%

24%

41%45%

0%

20%

40%

60%

80%

100%

< QL,n=203,146

QL - 3,n=57,63

3 to 4,n=83,79

> 4,n=115,175

% o

f pat

ient

s w

ith

resi

stan

ce

2%

12%

20%

60%

5% 6%

50%

56%

0%

20%

40%

60%

80%

100%

< QL,n=178,157

QL - 3,n=18,20

3 to 4,n=16,24

> 4,n=10,23

% o

f pat

ient

s w

ith

resi

stan

ce

Telbivudine Lamivudine

HBeAg Positive, HBeAg Positive, n=921n=921

HBeAg Negative, HBeAg Negative, n=446n=446

Lai et al , NEJM, 2007

M0

M6

M1

2

M1

8

M2

4

M3

0

M3

6

ALT0

2

4

6

8

ALTHBV DNA

Month of therapyMonth of therapy

Early add-on therapy to prevent drug resistance

Drug A

Drug B

Seru

m H

BV

DN

A (

L og1

0 c

opi e

s /m

L )

and A

LT (

x U

LN)


Secondary Treatment Preferences Based on Virologic Monitoring

Partial virologic response Virologic breakthrough

Nucleoside analog treatment

Add a more potent agent* or switch to a more potent combination

(emtricitabine/tenofovir*)

* Choice based on cross-resistance data

Monitorat 12-24 weeks

Early non reponse

Monitorevery 12 weeks

Switch to morepotent agent*


Very Early Add-on Therapy to Keep Viral Load as Low as Possible

2

3

4

5

6

7

8

M0 M3 M6 M9 M12 M15 M18 M21 M24

Se

rum

HB

V D

NA

(L

og

10

co

pie

s/m

L)

Drug ADrug ADrug ADrug A

++Drug BDrug B

Month of therapy

1. Start with a drug having high potency and low rate of resistance2. Add a drug with a different cross-resistance profile

outgrowth of drug resistant mutant?

Long-term viral load suppression, or risk of selection of MDR mutants ?

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

• It’s prime time for the next treatment objective– Clearance of HBsAg !

Documents

How to prevent, diagnose and overcome resistance to nucleoside analogs ? Fabien Zoulim Liver department, Hôtel Dieu Hospital & Hepatitis research laboratory,