Stephanos HADZIYANNIS

Following HBeAg seroconversion a proportion of patients retains or redevelops significant HBV replication associated with persistent or intermittent elevations in ALT activity and histological liver changes of chronic hepatitis referred as: HBeAg-negative/anti-HBe-positive/precore mutant chronic hepatitis B (CHB).

Why Do I Treat my Patients with Nucleos(t)ide Analogues Stephanos J. Hadziyannis Department of Medicine and Hepatology, Henry Dunant Hospital and Hepatitis Research Laboratory of Athens University

Flares without normalization Without flares Flares with normalization ALT DNA % patients 44 % 20 % 36 % Brunetto, J Hepatol 2002; 36: ●Asymptomatic hepatitis flares: 90% of patients Biochemical and virological patterns in HBeAg - CHB 164 untreated pts monitored monthly for a median of 21 months (12-36) ●Annual frequency of flares: - < Once 23% - Once 57% - Twice 20%

ALT (U/L) HBV DNA (pg/mL) ALT HBV DNA TIME (days) In HBeAg-negative chronic HBV infection HBV replication precedes and induces ALT increases and liver necroinflammation

Goals and Aims of Therapy in HBeAg-Negative CHB The most ambitious (and closest to “cure”) goal is HBsAg seroconversion but unfortunately this can actually be achieved only in a minority of patients Efficient, long term suppression of HBV replication currently remains the most realistic goal. It results in return of ALT to normal, reduction of liver necroinflammation and improvement of hepatic fibrosis but its impact on prevention of HCC remains uncertain. Problems of cost, toxicity, long term safety and of HBV resistance still remain unsolved.

Two Treatment Strategies in HBeAg-Negative Chronic Hepatitis B A.Virologic/Biochemical Remission Sustained after stopping a finite course of treatment. B. Virologic/Biochemical Remission Maintained under long term/indefinite treatment.

A Reminder A. Treatment courses of finite (1-2 yrs) duration: Can be applied with all approved drugs but sustained responses are practically limited to interferon-alfa. B. Long-term / indefinite antiviral treatment: Is currently applicable only with nucleos(t)ide analogues in mono- and combination therapies

Finite treatment courses of 48w duration with pegylated interferon alfa monotherapy in HBeAg(-) CHB have better chances for sustained response than nucl. analogues courses of the same duration and in some patients achieve the important goal of HBsAg seroconversion ( Marcellin et al NEJM 2004, Marcellin et al EASl 2005 and 2006, Hadziyannis et al EASL 2005 ) However, even with the best results, the majority of treated HBeAg-negative patients are not expected to achieve a sustained virologic and biochemical response. Why and When to Treat HBeAg-Negative CHB with Nucleos(t)ide Analoques

Currently Approved Nucleos(t)ide Analogues for Chronic Hepatitis B DRUGS LamivudineAdefovirEntecavirTelbivudine

Nucleoside Analogues in HBeAg- Negative Chronic Hepatitis B Sufficient Worldwide 2-Year Data Both on Efficacy and Resistance With all 4 Approved Nucleos(t)ide Analogues. Long Term Prospective Data (5yrs) on Efficacy and Resistance Limited to Lamivudine (LAM) and Adefovir Dipivoxil (ADV).

Hadziyannis SJ et al. Hepatology 2000;32: Long-term LAM therapy in HBeAg(-) CHB: ALT and HBV DNA responses over time with development of LAM-R Lamivudine treatment duration (months) Normal ALT Undetectable HBV DNA (%) ALT HBV DNA YMDD 0% 20% 40% 60% 80% 100% >= 30 0% 10% 20% 30% 40% 50% 60% Patients YMDD + (%)

Risk for HBV Resistance to LAM Increases With Level of HBV DNA at Week 24 Yuen MF, et al. Hepatology. 2001;34: Lamivudine Resistance at Follow-up* (%) Serum HBV DNA Level at 6 Months (copies/mL) % 13% 32% 64% < 200 (n = 12) < 3 log 10 (n = 23) < 4 log 10 (n = 41) > 4 log 10 (n = 118) *Median follow-up: 29.6 months.

Prediction of loss of the response under LAM therapy and early diagnosis of HBV resistance in HBeAg(-) CHB Monitoring of HBV DNA levels by sensitive PCR techniques, preferably by the real time TaqMan PCR assay Continuation of LAM treatment in those achieving non-detectability of HBV by month 6 and adapting frequency of subsequent HBV DNA monitoring according to disease severity. Add Adefovir on Lamivudine in those not achieving the above end point whether with or without selected LAM-resistant HBV mutants.

Long-term Entecavir in HBeAg(-) CHB Entecavir Lamivudine P <.0001 Patients (%) HBV DNA < 300 copies/mL Through Week 96* (n = 325) (n = 313) P =.01 (n = 296) (n = 287) Histologic Improvement Through Week Shouval D, et al. EASL Abstract 45. Lai C, et al. N Engl J Med. 2006;354: *Cumulative confirmed data: 2 data points or last observation on therapy.

*P ≤.05 vs lamivudine. Clinical Efficacy 2-Year Results: Telbivudine vs Lamivudine HBeAg(+)HBeAg(-) Response LdT (n = 458) LAM (n = 463) LdT (n = 222) LAM (n = 224) Mean HBV DNA reduction, log 10 copies/mL -5.7* *-4.2 HBV DNA undetectable, %56*3982*57 ALT normalization, %70* Therapeutic response, %64*4878*66 HBeAg loss, % HBeAg seroconversion, % Primary treatment failure, %4.0*12.30*2.7 Lai C, et al. AASLD Abstract 91.

p< >6 log Detectable HBV-DNA* (all patients) Detectable HBV-DNA* (by baseline levels) 5-6 log <5 log *LLQ of HBV-DNA assay: 2-3 log 10 copies/mL Undetectable HBV-DNA Virologic Response to ADV in LAM-R Patients Lampertico P, et al. 56 th Annual AASLD, San Francisco, CA, 2005, Poster #983.

Adefovir Resistance in Patients Receiving ADV + LAM* Hadziyannis S, et al. Hepatology. 2005;42(suppl 1):754A. Lampertico P. EASL Abstract 499. Incidence of Resistance *Based on experience in controlled clinical trials. † 2 patients enrolled in Study 435, initially on combination therapy with ADV + LAM, and subsequently selected ADV resistant mutation N236T. However, they were on ADV monotherapy when ADV resistance mutation was detected. 0% 3% 0% 11% 0% 18% Year 1Year 2Year 3Year 4 0% N/A Year 5 29% N/A ADV monotherapy (Study 438) ADV + LAM Study pre and post OLT (LAM-r) ADV + LAM Study 460i - HIV/HBV (LAM-r)

Probability of Developing Resistance to ADV in LAM Resistant Patients with Compensated HBeA-negative CHB (Rapti et al Hepatoloy Febr. 2007) ADV+LAM ADV only Patients at Risk n=32 n=14

Liver Biopsy (Y4 cohort ADV Baseline) Liver BiopsyLiver Biopsy † (Y4 cohort 1st yr ADV) Liver Biopsy † * Patients in ADV 10mg group re-randomized in a 2:1 fashion at week 48. ** Patients transferred to a general open-label study. † Optional liver biopsy 0Week 48Week 96 Week 144 ADV(n=55) ADV(n=70) ADV(n=60) ADV(n=80) PLB(n=62) ADV * (n=123) PLB** (n=40) Liver Biopsy † Week 240 ADV for 5 years (Y5 cohort) ADV for 4 years (Y4 cohort) Y5 cohort Y4 cohort ADV for HBeAg- CHB (0-240 weeks) GS 438 Study Design Hadziyannis S, et al. 56th AASLD, San Francisco,.2005

Weeks Log 10 copies/mL LLQ* *Lower limits of quantification (LLQ)=1000 copies/mL. Roche Amplicor PCR Assay. 24 Relapse after stop of 1y treatment with nucl. analogues (ADV) in HBeAg-negative CHB 2 ADV for 96 weeks ADV for 48 weeks + placebo for 48 weeks Hadziyannis et al. N Engl J Med 2005

Serum HBV DNA, ALT, and Serologic Responses at End of the Study in the 4- and 5- Year Cohorts Six patients (5%) had HBsAg loss, 5 with anti-HBs at the last available time point. Four lost HBsAg after > 3.5 years of ADV and 2 after 141 and 419 days of treatment. † * * Missing=failure for resistance or hepatocellular carcinoma. 38/5521/3026/4037/ %65%69%67% ALT normalization*HBV DNA < 3 log copies/mL* Patients (%) Year 4Year 5

A Note on the Potency and Timing of Optimal Virologic Response to ADV Slow, rather weak maximal suppression of HBV replication linked with the low anti-HBV potency of the approved “safe” 10mg daily dose of the dug Maximal initial HBV DNA suppression is achieved at month 12 not as with LAM, ENT and TEL at month 6 HBV DNA levels remaining detectable at month 12 are predictors of subsequent genotypic ADV resistance

Higher HBV DNA at Year 1 Predictive of Year 3 Genotypic ADV Resistance Locarnini S, et al. EASL HBV DNA at Year 1 (log 10 copies/mL) Genotypic Resistance at Year 3 (%) 4% 67% 26% (n = 80) < 3 (n = 31) 3-6 (n = 3) > 6

3 Cumulative Probabilities* of Virologic Outcomes With Adefovir Monotherapy Borroto-Esoda K. EASL Abstract Year 1Year 2Year 3Year 4Year 5 Patients (%) Genotypic resistanceVirologic rebound † Clinical breakthrough *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.

HBeAg(-) patients from our center achieving undetectable HBV DNA on ADV monotherapy, maintained for 4-5 years under treatment Median log ¹º HBV-DNA (c/mL)

Histological Observations at the End of Year 5 of the ADV Study. Improvement in necroinflammation by ranked assessment in 80% Proportion of patients with improvement in fibrosis (71%) Seven of 12 patients (58%) with bridging fibrosis or cirrhosis at baseline, improved by ≥2 points in Ishak Fibrosis Score, including 3 of 4 with cirrhosis at baseline. Hadziyannis et al Gastronterology Dec. 2006

SUSTAINED BIOCHEMICAL AND VIROLOGICAL REMISSION AFTER DISCONTIMUATION OF 4 TO 5 YEARS OF ADEFOVIR DIPIVOXIL (ADV) TREATMENT IN HBeAg-NEGATIVE CHRONIC HEPATITIS B S.J.Hadziyannis, V. Sevastianos, I. Rapti and N.Tassopoulos Department of Medicine and Hepatology, Henry Dunant Hospital and Hepatitis Research Laboratory of Athens University at Evgenidion Hospital, Athens, Greece.

HBV-DNA levels during follow-up in sustained biochemical responders after stopping ADV treatment 0% 21% 79% 33% 17% 50% 43% 14% 43% 44% 31% 25% 33% 34% 30% 40% 30% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% FOLLOW-UP MONTH >10,000 copies/mL Detectable <10,000 copies/mL HBV-DNA not detectable 100% 0 70% of patients <10,000c/mL

Median ALT levels (IU/L) during 22 months of follow- up after stopping 4 or 5 yrs of ADV therapy. Results among patients in sustained biochemical remission Months Follow-up Median ALT ULN

But what to do with the 1/3 of patients not achieving on ADV ‘early’ non-detectability of HBV DNA? Switch to a more potent compound with the same or similar resistance profile? Increase the dose of Adefovir? Switch to Entecavir or Lamivudine? Continue Adefovir adding Lamivudine or Entecavir ?

Concluding Comments on Adefovir Maintenance by ADV monotherapy of HBV suppression to serum HBV DNA levels non-detectable by the Taqman Real Time PCR assay for a 5-year period is possible in about 2/3 of HBeAg-negative patients; with a good percentage of them keeping in remission 18 months after stop of this long-term Rx (Hadziyannis et al 2006 Gastroenterology) In HBeAg-negative patients with lamivudine resistant HBV adding ADV is clearly superior and preferable than switching to ADV and the same approach is reasonable with addition of LAM or ETV in those with detectable HBV DNA at month 12 on ADV monotherapy.

Final conclusion and answer to the question of why to treat HBeAg-negative CHB with nucleos(t)ide analagues Because in patients non-responding or non-eligible to IFN-α therapy, proper drug selection from the available nucleos(t)ide analogue Armamentarium with careful monitoring of the response to therapy, can achieve very long maintenance of the virologic and biochemical response and A number of such patients may also achieve the goal of sustained response and even of HBsAg clearance and seroconversion to anti-HBe.

BACK UP SLIDES

Why Do I Treat my Patients with Nucleos(t)ide Analogues Stephanos J. Hadziyannis Department of Medicine and Hepatology, Henry Dunant Hospital and Hepatitis Research Laboratory of Athens University

Patients’ Characteristics Total n (%) 33 (100%) Males n (%) 27 (82%) Females n (%) 6 (18%) Mean Age yrs (range) 50 (29-69) 5 Year Cohort 22 (67%) 4 Year Cohort 11 (33%) Normal ALT 33 (100%) HBV DNA <1,000c/ml 33 (100%) HBV DNA TaqMan (-)* 28 (85%) *22/27M, 6/6F

Patients 37 HBeAg(-) patients from our center achieved undetectable HBV DNA on Adefovir therapy, maintained for 4-5 years under treatment 33 of them gave informed consent to be included in this post- treatment long-term follow-up study Median log ¹º HBV-DNA (c/mL)

Transient rise in HBV DNA during the first 2 months after stopping ADV therapy in patient keeping in sustained biochemical and virologic remission to follow-up month 23

HBV DNA and ALT during and after stopping long-term ADV therapy in an HBeAg-negative patient with HBsAg seroconversion under therapy FUP Mo1FUP Mo3FUP Mo5 FUP Mo10FUP Mo16FUP Mo ALT HBV-DNA ALT (IU/L) Log HBV-DNA ADV 10mg/day

Summary After stopping adefovir dipivoxil treatment of 4 or 5 years duration in HBeAg-negative chronic hepatitis B patients, who have remained persistently HBV DNA negative by PCR under Rx, approximately 2/3 of them may keep in sustained biochemical remission for a hitherto period of months. Serum HBV DNA levels rise transiently during the first few months of follow-up in all instances but in sustained biochemical responders they progressively decline over time and may even become undetectable in >30% of them

Higher HBV DNA at Year 1 Is Predictive of ADV-R Development by Year 3 27% (n=31) 3-6 log 10 70% (n=80) <3 log 10 3% (n=3) >6 log 10 Serum HBV DNA at year 1 (n=114) 8/31 (26%) ADV-R by yr 3 2/3 (67%) ADV-R by yr 3 3/80 (4%) ADV-R by yr 3

Histologic Ranked Assessment in 4- and 5- Year Cohorts Compared to ADV Baseline Median change from ADV Baseline in Knodell necroinflammatory score -4.5 and -5.0 at 4 and 5 yrs; median change in Ishak fibrosis score was -1.0 in both cohorts. Necroinflam.Y4Necroinflam.Y5Fibrosis Y5Fibrosis Y4 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% ImprovedSameWorse