Chronic Hepatitis B - Antiviral Resistance in Korea -

Chronic Hepatitis B - Antiviral Resistance in Korea - Young-Suk Lim University of Ulsan College of Medicine Asan Medical Center Seoul, Korea

HBV Genome partially double-stranded DNA genome about 3200 nucleotides HBV polymerase lacks proof-reading function Estimated mutation frequency: 1.4-3.2 x 10-5 (approximately 10-fold higher than other DNA viruses) HBV production rate: 10 11 /day dynamic quasispecies

Evolution of HBV Resistance Mutations to Antiviral Drugs Primary resistance mutations mutations directly responsible for the associated drug-resistance Compensatory (Secondary) mutations - mutations that can restore replication fitness of primary resistant mutants - fix the primary drug-resistant mutations as a genetic archive with quasispecies memory Cross Resistance mutants selected by one agent may also confer resistance to other antiviral agents

Terminal Protein Lamivudine-Resistance Mutations at HBV polymerase/rt domain Spacer POL/RT RNaseH 1 183 349 (rt1) 692 (rt 344) 845 a.a. I(G) II(F) F V LLAQ YMDD A B C D E LMV Resistance rtl80v/i rtv173l rtm204v/i/s rtl180m primary resistance mutation compensatory mutations rtm204 mutation confers cross resistance to entecavir, clevudine, telbivudine, emtricitabine

Mechanisms of Resistance S S Sensitive S Sensitive R S Sensitive S Sensitive R Sensitive (wild type) R Resistant (rtm204v) Resistant (rtm204v+ rtl180m) R Resistant (rtm204v+ rtl180m) Resistant (rtm204v+ rtl180m) Lamivudine Discontinuation Lamivudine Clevudine Entecavir Telbivudine Emtricitabine

Clinical Definitions - HBV Resistance to Antiviral Drugs - Genotypic Resistance Detection of mutations in the HBV genome, known to confer resistance Virologic Breakthrough Rebound in serum HBV DNA levels, more than 1 log10 cpm (10 fold) Biochemical (Clinical) Breakthrough Increase of ALT levels (or worsening histology) with virologic breakthrough

Development of Antiviral Resistance HBV DNA (Log) Antiviral Drug ALT (IU/L) 6 5 Virologic Breakthrough 80 4 3 nadir 1 log 40 (ULN) 2 Development of Genotypic Resistance Biochemical Breakthrough

Higher Retention of Mutant Virus Go with More Subsequent Occurrence of Viral Breakthrough 180 165 (2) 160 140 120 (1) Incidence 120 100 80 103 (2) 60 40 20 25 (5) 10 (9) 3 (3) 3 (3) 0 0 1 2.5 5 7.5 10 Mutant / Wild type Lee CH et. al. Gastroenterology. 2006;130(4)1144

Genotypic Assays for antiviral drug resistance of HBV Sequencing (direct or after cloning) Hybridization Assay (Line probe assay, LiPA) Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Mass Polymorphism (RFMP) Allele-specific PCR DNA Chip

Genotypic Assays for antiviral drug resistance of HBV Sequencing (direct or after cloning) - the best approach to identify new mutations - unable to detect mixed populations of two or more HBV genotypes - expensive and time-consuming Hybridization Assay (Line probe assay, LiPA) Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Mass Polymorphism (RFMP) Allele-specific PCR DNA Chip

Genotypic Assays for antiviral drug resistance of HBV Sequencing (direct or after cloning) Hybridization Assay (Line probe assay, LiPA) - more sensitive than direct sequencing - most commonly used method in Western countries Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Mass Polymorphism (RFMP) Allele-specific PCR DNA Chip

Genotypic Assays for antiviral drug resistance of HBV Sequencing (direct or after cloning) Hybridization Assay (Line probe assay, LiPA) Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Mass Polymorphism (RFMP) Allele-specific PCR - can detect minor HBV populations comprising up to 5% of the total viral population (about 10 3 cpm) - robust high throughput manner - most commonly used methods in Korea DNA Chip

Advances in Management of HBV Infection Lamivudine Adefovir Interferon Entecavir Clevudine Peg-IFN α-2a Tenofovir Telbivudine 1992 1998 2003 2005 2006 - - -

HBV Therapies Approved in Korea For Naïve CHB Interferon Peginterferon α-2a Lamivudine* Entecavir* For LAM-Refractory CHB Interferon Peginterferon α-2a Adefovir dipivoxil* Entecavir* Clevudine* *Approved only as monotherapy and after ALT flare or biochemical breakthrough (ALT>80 IU/L)

New Oral Agents Future agents Telbivudine (LdT) Tenofovir Emtricitabine New experimental agents LB80380 Pradefovir Alamifovir Valtorcitabine Torcitabine

High potency in reducing HBV DNA level High rate of HBeAg seroconversion Few adverse events or toxicity Cost-effective Ideal anti-viral agent Long-term, low rate of drug resistance

Lamivudine-Resistance

Terminal Protein HBV polymerase/rt domain Spacer POL/RT RNaseH 1 183 349 (rt1) 692 (rt 344) 845 a.a. I(G) II(F) F V LLAQ YMDD A B C D E LMV Resistance rtl80v/i rtv173l rtm204v/i/s rtl180m L-dT Resistance rtm204i ADV Resistance rta181t/v rtn236t ETV Resistance rtt184g rts202i rtm250v TFV Resistance rta194t/rtv214a/rtq215s

Long-term LAM therapy can lead to the emergence of resistant viruses 90 Patients with YMDD mutants (%) 80 70 60 50 40 30 20 10 0 15% 40% 55% 66% 69% 1 2 3 4 5 6 7 8 Years 1. Leung NW, et al. Hepatology. 2001;33:1527-1532. 2. Yuen et al AASLD 2005

High lamivudine resistance with poor early HBV suppression Patients (%) 100 80 60 Serum DNA at month 6 vs. LAM resistance by month 61 64% 40 32% 20 8% 13% 0 < 200 (n = 12) < 3 log 10 (n = 23) < 4 log 10 (n = 41) > 4 log 10 (n = 118) Serum HBV DNA Level at 6 months (copies/ml) Yuen et al. Hepatology. 2001; 34:785-791

Disease Progression by YMDD Status 25 20 Placebo (n=215) YMDDm (n=209) (49%) Wild Type (n=221) Placebo 21% % with 15 disease progression 10 YMDDm 13% 5 WT 5% 0 0 6 12 18 24 30 36 Liaw YF, et al. N Engl J Med. 2004;351:1521-1531. Time after randomization (months)

Hepatitis flares and Serious Adverse Events with Lamivudine resistance mutations hepatitis flare Liver Disease Related Serious Adverse Events Lok AS, et al. Gastroenterology. 2003;125:1714.

Clinical consequences of Lamivudine resistance It is now clear that drug-resistant HBV is not a benign or attenuated virus. Disease progression, loss of initial benefit, fulminant hepatic failure and death can occur.

Management of Lamivudine-Resistant HBV

Continuation or Discontinuation of Lamivudine 75 % * 50 Events within 12 mo. after emergence of YMDD mutations * P > 0.05 * Continued (n=66) Discontinued (n=68) 25 * 11% 7% 0 ALT flare (>5x ULN) Decompensation HBeAg seroconversion Liaw YF, et al. Antivir Ther. 2004;9:257.

Continuation or Discontinuation of Lamivudine Hepatitis flares and decompensation frequently occur after emergence of YMDD mutations regardless of continuation or discontinuation of lamivudine therapy. Compensatory mutations will be selected during continued lamivudine treatment leading to subsequent viral rebound and possibly hepatitis flares. Patients with confirmed lamivudine-resistance should receive effective rescue therapy.

Peg-Interferon alfa-2a in Asian patients with NA resistance 12 wks PEGASYS + LAM 12 wks of PEGASYS monotherapy HBeAg seroconversion (with HBV DNA <10 5 copies/ml) 50 43.7% Patients (%) 40 30 20 10 31.2% HBsAg seroconversion In 13% of patients at week 72 0 7/16 5/16 End of treatment 24 weeks post-treatment Shi et al. APASL 2007

Adefovir alone or in combination with lamivudine in lamivudine-resistant HBV (small, short-term study) Change in HBV DNA 1 0 LAM (n=19) 0.0 (log 10 copies/ml) -1-2 -3-4 -5 ADV+LAM* (n=20) 37% frequency of grade 3 ALT flare in switchover to ADV alone Weeks of Therapy ADV (n= 19)* - 3.6-4.0 0 8 16 24 32 40 48 * p<0.001 compared to lamivudine Peters MG et al, Gastroenterology 2004; 126: 91-101

Increased Risk of Adefovir Resistance in Patients with Lamivudine-Resistant CHB after Adefovir Monotherapy % 20 Incidence of ADV-Resistance at 48 wks of ADV monotherapy * * P < 0.01 naive (n=38) LMV-resistant (n=57) 10 18% * Genotypic assay 7% ; RFMP 0 0% Genotypic mutation 0% Virologic breakthrough Lee YS, et al. Hepatology 2006;43:1385.

Cumulative Incidence of Adefovir-Resistance in Lamivudine-Resistant CHB patients treated with Adefovir Monotherapy Genotypic Resistance Virologic Breakthrough Genotypic assay ; RFMP 25.4% Yeon JE, et al. Gut 2006;55:1488.

Adding-on vs. Switching-to Adefovir in Lamivudine-Resistant HBeAg(-) CHB Incidence of Genotypic Resistance LAM + ADV ADV Genotypic assay ; direct sequencing Rapti I, et al. Hepatology 2007;45:307.

When should we add Adefovir on Lamivudine? Lampertico P, et al. Hepatology 2005;42:1414.

Achievement of PCR Undetectability on Entecavir % Patients w/hbv DNA <300 Copies/mL 100 90 80 70 60 50 40 30 20 10 HBeAg - Naïve HBeAg + LVD Refractory N = 319 0 0 50 100 150 Treatment (Weeks) N = 345 N = 178 94% 40%

Incidence of Entecavir Resistance Genotypic resistance in naive patients Genotypic resistance in LAM-R R patients Genotypic resistance plus viral rebound in LAM-R R patients % 40 30 Genotypic assay ; direct sequencing 32 25 20 14 10 6 10 0 0.1 1 0.4 1.1 1 2 3 Year Colonno R, et al. Hepatology. 2006;44:229A-230A.

ETV Resistance Profile ETV-R was only observed in patients with preexisting LAM-R virus (M204 V/I and/or L180M). Resistance to ETV appears to occur through a two-hit mechanism with initial selection of M204V/I mutation followed by amino acid substitutions at rti169, rtt184, rts202, or rtm250. LAM should be discontinued to decrease the risk of entecavir resistance. Lamivudine Entecavir T184, S202 or M250 M204V ± L180M M204V ± L180M

Tenofovir for Lamivudine-Resistant CHB Nucleotide analogue, structurally similar to adefovir, equipotent in wild-type and LAM-R HBV (In vitro). Because tenofovir appears to be less nephrotoxic, the approved dose is much higher than that of adefovir, 300 mg versus 10 mg daily. Thus, tenofovir has more potent antiviral activity than adefovir. No randomized controlled trials in HBV.

Tenofovir vs Adefovir in Patients with Lamivudine Resistance (not randomized) P < 0.001 HBV DNA (log 10 copies/ml) Adefovir (10 mg/day, n=18) Tenofovir* (300 mg/day, n=35) Weeks * including HIV/HBV coinfection (n=21), and HBV/kidney Tpl (n=5). Van Bommel et al. Hepatology. 2004;40:1421-1425.

Recommendations for the Tx of LAM-R CHB It is better to start rescue therapy as soon as genotypic resistance is detected before the development of virologic breakthrough. Therefore, careful monitoring of genotypic resistance is needed during antiviral therapy for early detection and early rescue of drug resistance. Adefovir add-on is preferred over switch (increased rate of adefovir resistance with switch) and preferred over entecavir (high rate of novel mutations in patients with lamivudine resistance). If entecavir is used, lamivudine should be stopped as continued presence of lamivudine-resistant mutations will increase the risk of entecavir resistance. Tenofovir, 300 mg daily, appears to be superior to ADV, 10 mg daily, in suppressing LAM-resistant strain HBV.

Adefovir-Resistance

Terminal Protein HBV polymerase/rt domain Spacer POL/RT RNaseH 1 183 349 (rt1) 692 (rt 344) 845 a.a. I(G) II(F) F V LLAQ YMDD A B C D E LMV Resistance rtl80v/i rtv173l rtm204v/i/s rtl180m L-dT Resistance rtm204i ADV Resistance rta181t/v rtn236t ETV Resistance rts184g rts202i rtm250v TFV Resistance rta194t/rtv214a/rtq215s

Incidence of Adefovir Resistance with Adefovir Monotherapy in HBeAg-Negative NA naïve CHB Patients 100 Patients (%) 80 60 40 20 0 Genotypic resistance Virologic rebound Clinical breakthrough 29 18 11 13 16 8 10 11 6 0 0 0 3 3 2 Year 1 Year 2 Year 3 Year 4 Year 5 *Cumulative probabilities calculated by life-table analysis. Presence of genotypic resistance plus HBV DNA rebound; confirmed 1 log 10 copies/ml increase in HBV DNA from nadir and/or having never achieved HBV DNA suppression 4 log 10 copies/ml. Borroto-Esoda K. EASL 2006. Abstract 483.

Higher HBV DNA at Year 1 Predictive of Year 3 ADV Resistance Genotypic Resistance at Year 3 (%) 100 80 67% 60 40 26% 20 0 4% < 3 (n = 80) 3-6 (n = 31) > 6 (n = 3) HBV DNA at Year 1 (log 10 copies/ml) Locarnini S, et al. EASL 2005. Abstract 36.

Risk factors for the development of adefovir resistance Old age High baseline HBV DNA load Suboptimal early viral suppression Short-duration of LAM overlap in LAM-R HBV (adding-on is better than switching to ADV) Presence of LAM-R HBV mutants ; The main LMV resistance mutations rtm204v/i do not confer cross-resistance to adefovir, but the minor LMV resistance mutations rta181t as well as the rtq215s are partially cross-resistant to lamivudine in vitro.

Management of Adefovir-Resistant HBV

In Vitro Cross-Resistance Analysis Drugs Efficacy against various HBV strains Wild LAM-R ADF-R type (L180M+M204V) (N236T) Lamivudine + - + Clevudine + - + Telbivudine + - + Emtricitabine + - + Entecavir + +/- + Adefovir + + - Tenofovir + + +/- 1. Zoulim F. Antiviral Res. 2004;64(1):1-15. 2. Brunelle et al. Hepatology. 2005;41:1391-1398. 3. Locarnini et al. EASL 2005. Abstract 36.

Practical Options for the Tx of ADR-R CHB In patients with no prior exposure to other NA add lamivudine add (> or switch to) entecavir In patients with prior LAM-R, switched to ADV add lamivudine add (> or switch to) entecavir switch to tenofovir (when approved) in combination with lamivudine or entecavir

Case: Adding on LMV for ADV-R Marcellin P and Asselah T. J Hepatol 2005;43:920. Yeon JE, et al., Gut 2006;55:1488-1495. Villeneuve JP, et al. J Hepatol 2003;39:1085 1089.

Case: Switching to ETV for ADV-R S.K. Fung SK, et al., J Hepatol 2006;44:283 290.

Case: Switching to Tenofovir for ADV-R Ratziu V, et al., Comp Hepatol 2006;5:1.

Prevention of Antiviral Drug Resistance in Patients with CHB Current therapy of CHB has limited long-term efficacy. Once antiviral-resistant HBV mutants have been selected, they are archived (retained in the virus population) persistently even if treatment is stopped. Thus, the best way to reduce the emergence of drug resistance is to select the right patients, right time to start treatment and the right antiviral agent(s).

Prevention of Antiviral Drug Resistance in Patients with CHB Select the right patients Patients with minimal disease and those who are unlikely to achieve sustained response should not be treated with NA Select right time to start treatment Select the right antiviral agent(s)

Prevention of Antiviral Drug Resistance in Patients with CHB Select the right patients Select right time to start treatment Start therapy at right time with clear indication to maximize antiviral activity Select the right antiviral agent(s)

Prevention of Antiviral Drug Resistance in Patients with CHB Select the right patients Select right time to start treatment Select the right antiviral agent(s) The most potent NA with the lowest rate of genotypic resistance should be administered.

APASL Seoul 2008 2008. 3. 23-26 26 See you in Seoul, Korea ありがとうございました. Thank you for your attention!!