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 log 10 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: <1log 10 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 Patient’s 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. Time Antiviral drug HBV DNA (log 10 IU/mL) ALT (IU/L) 4 6 5 3 2 ULN 1 0 Detection of Genotypic Resistance Nadir 1 log 10 Virologic Breakthrough Biochemical Breakthrough Genotypic resistance Virological Breakthrough Biochemical Breakthrough 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

8. Lamivudine Resistance Accelerates Progression of Liver Disease YMDDm WT Placebo 5% 13% 21% Liaw YF et al. N Engl J Med. 2004;351:1521-1531

9. 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/RT ABCED 1183 349692 YMDD V173L L180MM204I/V GVGLSPFLLA I(G)II(F) (rt1)(rt 344) LAM / FTC ETV I169T T184G S202G/I M250V ADV A181V/TN236T I233V ? LdT M204I TDF A194T ? M204I/V

10. Tools for the diagnosis of resistance

11. Dynamic ranges of quantification of HBV DNA assays 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) Abbot Real-time HBV (Abbott) Versant HBV DNA 1.0 (kPCR, Siemens)* *in development 10 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 RealArt HBV LC PCR (Artus Biotech)

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 DNA probe Nitrocellulose strip I555 M555 L555 V555 I552 V552 M528 M552 L528 Amp. Cont. Conj. Cont. Marker line Alkaline Phosphatase Streptavidin Biotin Amplified target 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 Inhibition of viral replication 100% 0% 50% Concentration of drug Wild-type virus Patient’s virus IC 50 IC 50 wild type IC 50 patient = Fold change IC 50 = Drug susceptibility

16. Choice of drug for treatment adaptation

17. LamivudineTelbivudineEntecavirAdefovirTenofovir Wild-typeSSSSS M204lRRISS L180M + M204V RRISS A181 T/VISSRS N236TSSSRI I169T + V173L + M250V* RRRSS T184G + S202lI/G * RRR 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

18. Lamivudine / Telbivudine resistance Add ADV Add TDF Switch to TVD: TDF+FTC Switch to ETVNot valid LAM FTC LdT Management of HBV Drug Resistance Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

19. Adefovir resistance Add Lamivudine Add ETV Add Telbivudine Switch to TVD: TDF+FTC Management of HBV Drug Resistance Switch to TDF TVD ETV LdT Adefovir non response Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

20. Entecavir resistance Add ADV Add TDF Switch to TVD: TDF+FTCNot valid LAM LdT Management of HBV Drug Resistance Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

21. Add-on or switch ?

22. 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 ADV LAM

23.  HBV DNA  ALT The problem of sequential therapy and switching strategy Villeneuve et al, J Hepatol 2003 N236T Serum HBV DNA (Log 10 copies/mL) 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-98janv-99janv-00janv-01janv-02janv-03janv-04janv-05 LAM

24. Months ADV mono Patients with virological breakthrough 27326825622520115861 30% 6% P<0.001 ADV+LAM 255238223213200177 103 Patients with ADV-R 22922521719417914657 16% 0% P<0.001 ADV mono ADV+LAM 24222721420520017492 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 0369121518212427303336 0 20 40 60 80 100 0369121518212427303336 Patients with lamivudine resistance: adefovir add-on strategy

25. When changing treatment ?

26. Month of therapy Rescue therapy in patients with clinical breakthrough Drug A Drug B Serum HBV DNA (Log10 copies/mL) and ALT (x ULN)

27. Month of therapy Rescue therapy in patients at the time of virologic breakthrough Drug A Drug B Serum HBV DNA (Log10 copies/mL) and ALT (x ULN) 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) TelbivudineLamivudine HBeAg Positive, n=921 HBeAg Negative, n=446 Lai et al, NEJM, 2007

29. Month of therapy Early add-on therapy to prevent drug resistance Drug A Drug B Serum HBV DNA (Log10 copies/mL) and ALT (x ULN) Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

30. Secondary Treatment Preferences Based on Virologic Monitoring Partial virologic responseVirologic breakthrough Nucleoside analog treatment Add a more potent agent* or switch to a more potent combination (emtricitabine/tenofovir *) * Choice based on cross-resistance data Monitor at 12-24 weeks Early non reponse Monitor every 12 weeks Switch to more potent agent* 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 Drug A + Drug B Month of therapy 1. Start with a drug having high potency and low rate of resistance 2. 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 ?

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