Stephanos HADZIYANNIS

1. Stephanos HADZIYANNIS

2. 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).

3. 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

4. Biochemical and virological patterns in HBeAg - CHB 164 untreated pts monitored monthly for a median of 21 months (12-36) % patients Flares with normalization ALT DNA 44 % • Asymptomatic hepatitis flares: 90% of patients Flares without normalization 20 % • Annual frequency of flares: - < Once 23% - Once 57% - Twice 20% Without flares 36 % Brunetto, J Hepatol 2002; 36: 263-70

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

6. Goals and Aims of Therapy in HBeAg-Negative CHB The most ambitious (and closest to “cure”) goal is HBsAg seroconversion but unfortunately this can actuallybe achieved only in a minority of patients Efficient, long term suppression of HBV replicationcurrently 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.

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

8. 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:Iscurrently applicable only with nucleos(t)ide analogues in mono- and combination therapies

9. Why and When to Treat HBeAg-Negative CHB with Nucleos(t)ide Analoques 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.

10. Currently Approved Nucleos(t)ide Analogues for Chronic Hepatitis B

11. Nucleoside Analogues in HBeAg-Negative Chronic Hepatitis B • Sufficient Worldwide 2-YearData 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).

12. 100% 60% 50% 80% 40% 60% Normal ALT Undetectable HBV DNA (%) 30% Patients YMDD + (%) 40% 20% 20% 10% 0% 0% 6 12 18 24 >= 30 YMDD HBV DNA ALT Lamivudine treatment duration (months) Long-term LAM therapy in HBeAg(-) CHB: ALT and HBV DNA responses over time with development of LAM-R Hadziyannis SJ et al. Hepatology 2000;32:847-51.

13. Risk for HBV Resistance to LAM Increases With Level of HBV DNA at Week 24 100 80 64% 60 Lamivudine Resistance at Follow-up* (%) 40 32% 20 13% 8% 0 < 200 (n = 12) < 3 log10 (n = 23) < 4 log10 (n = 41) > 4 log10 (n = 118) Serum HBV DNA Level at 6 Months (copies/mL) *Median follow-up: 29.6 months. Yuen MF, et al. Hepatology. 2001;34:785-791.

14. 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.

15. Long-term Entecavir in HBeAg(-) CHB Entecavir Lamivudine Histologic Improvement Through Week 48 HBV DNA < 300 copies/mL Through Week 96* P < .0001 100 100 94 P = .01 77 80 80 70 61 60 60 Patients (%) 40 40 20 20 (n = 287) (n = 313) (n = 296) (n = 325) 0 0 *Cumulative confirmed data: 2 data points or last observation on therapy. Shouval D, et al. EASL 2006. Abstract 45. Lai C, et al. N Engl J Med. 2006;354:1011-1020.

16. Clinical Efficacy 2-Year Results:Telbivudine vs Lamivudine *P ≤ .05 vs lamivudine. Lai C, et al. AASLD 2006. Abstract 91.

17. Virologic Response to ADV in LAM-R Patients Detectable HBV-DNA* (by baseline levels) Detectable HBV-DNA* (all patients) p<0.0001 >6 log 5-6 log <5 log Undetectable HBV-DNA *LLQ of HBV-DNA assay: 2-3 log10 copies/mL Lampertico P, et al. 56th Annual AASLD, San Francisco, CA, 2005, Poster #983.

18. Adefovir Resistance in Patients Receiving ADV + LAM* ADV monotherapy (Study 438) 60 ADV + LAM Study 435 - pre and post OLT (LAM-r) ADV + LAM Study 460i - HIV/HBV (LAM-r) 40 29% Incidence of Resistance 18% 20 11% 3% 0% 0% 0% 0% 0% 0% N/A N/A 0% N/A 0% 0 Year 1 Year 2 Year 3 Year 4 Year 5 *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. Hadziyannis S, et al. Hepatology. 2005;42(suppl 1):754A. Lampertico P. EASL 2006. Abstract 499.

19. 1,1 1,0 ,9 ,8 no ,7 0 10 20 30 40 50 60 Duration of ADV treatment in Months Probability of Developing Resistance to ADV in LAM Resistant Patients with Compensated HBeA-negative CHB (Rapti et al Hepatoloy Febr . 2007) Patients at Risk ADV+LAM n=32 ADV only n=14

20. ADV for HBeAg- CHB (0-240 weeks) GS 438 Study Design ADV * (n=123) PLB** (n=40) Y5 cohort ADV (n=80) ADV (n=70) ADV for 5 years (Y5 cohort) PLB (n=62) ADV (n=60) ADV (n=55) ADV for 4 years (Y4 cohort) Y4 cohort 0 Week 48 Week 96 Week 144 Week 240 Liver Biopsy† Liver Biopsy LiverBiopsy (Y4 cohort ADV Baseline) Liver 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 Hadziyannis S, et al. 56th AASLD, San Francisco,.2005

21. Relapse after stop of 1y treatment with nucl. analogues (ADV) in HBeAg-negative CHB ADV for 96 weeks ADV for 48 weeks + placebo for 48 weeks 8 7 6 Log10 copies/mL 5 4 3 LLQ* 2 24 96 0 12 36 48 60 72 84 Weeks *Lower limits of quantification (LLQ)=1000 copies/mL. Roche Amplicor PCR Assay. • Hadziyannis et al. N Engl J Med 2005

22. Serum HBV DNA, ALT, and Serologic Responses at End of the Study in the 4- and 5-Year Cohorts Year 4 Year 5 100 90 80 70% 69% 65% 67% 70 21/30 38/55 26/40 37/55 60 Patients (%) 50 40 30 20 10 0 † ALT normalization* HBV DNA < 3 log copies/mL* * 10 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.

23. 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 achievedat month12not as with LAM, ENT and TEL at month 6 • HBV DNA levels remaining detectable at month 12 are predictors of subsequent genotypic ADV resistance

24. < 3 3-6 > 6 (n = 31) (n = 80) (n = 3) Higher HBV DNA at Year 1 Predictive of Year 3 Genotypic ADV Resistance 100 80 67% 60 Genotypic Resistance at Year 3 (%) 40 26% 20 4% 0 HBV DNA at Year 1 (log10 copies/mL) Locarnini S, et al. EASL 2005.

25. Cumulative Probabilities* of Virologic Outcomes With Adefovir Monotherapy 100 Genotypic resistance Virologic rebound† Clinical breakthrough 80 60 Patients (%) 40 29 18 20 16 13 11 11 10 8 6 3 3 2 0 0 0 0 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 log10 copies/mL increase in HBV DNA from nadir and/or having never achieved HBV DNA suppression ≤ 4 log10 copies/mL. Borroto-Esoda K. EASL 2006. Abstract 483.

26. HBeAg(-) patients from our center achieving undetectable HBV DNA on ADV monotherapy, maintained for 4-5 years under treatment Median log¹º HBV-DNA (c/mL)

27. 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

28. 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.

29. HBV-DNA levels during follow-up in sustained biochemical responders after stopping ADV treatment 100% 90% 25% 30% 34% 43% 80% 50% >10,000 copies/mL 70% 100% 79% 31% Detectable <10,000 copies/mL 60% 14% 40% 33% 50% HBV-DNA not detectable 17% 40% 30% 70% of patients <10,000 c/mL 44% 43% 20% 33% 33% 30% 21% 10% 0% 0% 0 1 2 6 12 18 22 FOLLOW-UP MONTH

30. 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 100 80 60 ULN Median ALT 40 20 0 2 4 6 12 18 22 Months Follow-up

31. 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 ?

32. 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.

33. 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.

34. BACK UP SLIDES

35. 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

36. Patients’ Characteristics

37. 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 log¹º HBV-DNA (c/mL)

38. 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

39. ALT HBV-DNA 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 36 42 48 55 61 FUP Mo1 FUP Mo3 FUP Mo5 FUP Mo10 FUP Mo16 FUP Mo22 HBV DNA and ALT during and after stopping long-term ADV therapy in an HBeAg-negative patient with HBsAg seroconversion under therapy 140 1000000 ADV 10mg/day 120 100000 100 10000 80 1000 Log HBV-DNA ALT (IU/L) 60 100 40 10 20 0 1

40. 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 19-24 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

41. 8/31 (26%) ADV-R by yr 3 2/3 (67%) ADV-R by yr 3 3/80 (4%) ADV-R by yr 3 Higher HBV DNA at Year 1 Is Predictive of ADV-R Development by Year 3 Serum HBV DNA at year 1 (n=114) 27% (n=31) 3-6 log10 70% (n=80)<3 log10 3% (n=3)>6 log10

42. Histologic Ranked Assessment in 4- and 5-Year Cohorts Compared to ADV Baseline Improved Same Worse 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Necroinflam.Y4 Necroinflam.Y5 Fibrosis Y4 Fibrosis Y5 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.