A New Bio-Mathematical Model to Describe the Dynamics of HBV-DNA and Infected Hepatocytes in HBEAG-Negative/Anti-HBE-Positive Chronic Hepatitis B Patients Treated with Peginterferon Alfa-2A (40KD) (Pegasys), Lamivudine, or Pegasys Plus Lamivudine Combination Therapy

P. Colombatto ¹, R. Bizzarri ², L. Civitano ¹, F. Oliveri ¹, F. Bonino ³, G. Germanidis ⁴, P. Farci ⁵, G. Kitis ⁶, S. Hadziyannis ⁷, S. Choudhury ⁸, R. Gieschke ⁹, F. Zahm ⁹, M. Brunetto ¹

¹ UO Gastroenterologia Ed Epatologia, Azienda Ospedialiera Pisana, Pisa, Italy; ² Scuola Normale Superiore Of Pisa, Pisa, Italy; ³ Scientific Direction, IRCCS Ospedale Maggiore, Milano, Italy; ⁴ Papageorgiou General Hospital, Pathology Clinic, Thessalonika, Greece; ⁵ Division Of Clinical Medicine, University Of Cagliari, Cagliari, Italy; ⁶ Gastroenterology Department, G. Papanikolaou Hospital, Thessaloniki, Greece; ⁷ Department Of Medicine And Hepatology, Henry Dunant Hospital, Athens, Greece; ⁸ Roche, Nutley NJ, USA; 9 Roche, Basel, Switzerland

Introduction

Biochemical hepatitis B disease profiles correlate well with the time course of viral load and one major determinant of viral load is the number of infected hepatocytes. A bio-mathematical model was recently developed to assess viral dynamics and infected-hepatocyte numbers in a sub-study of the PEGASYS Phase III trial.

Methods

Patients included in this viral kinetic sub-analysis received 48 weeks of either lamivudine (Group A; n=25); PEGASYS 180 μg qw plus lamivudine (Group B; n=23) or PEGASYS 180 μg qw plus placebo (Group C; n=24).

The model calculated decay constants for viral kinetic profiles and baseline infected-cell numbers (Io) using HBV-DNA and ALT measured on days -30,-15,0,0.3,1,2,4,5,7,14 and 28.

Results

Fifty-three patients showed low pre-treatment HBV-DNA and ALT fluctuations, whereas 19 patients with high spontaneous fluctuations were excluded from the analysis.

Triphasic HBV-DNA profiles were observed in Groups A and B, and biphasic in all but one patient in Group C, who had a monophasic profile. Early ALT flares (>=2xULN) and/or persistent ALT increases impaired calculation of Io in seven patients from Group B and nine patients from Group C. The mean ± SD of HBV-DNA decay constants (day) and Io are shown below.

Conclusions

HBV dynamics with PEGASYS monotherapy are significantly different from those seen with lamivudine monotherapy. PEGASYS may increase early-phase HBV-DNA decay when combined with lamivudine.

Peginterferon Alfa-2A (40KD) (Pegasys) Monotherapy is More Effective Than Lamivudine Monotherapy in the Treatment of HBEAG-Negative Chronic Hepatitis B: 72 Week Results From a Phase III, Partially Double-Blind Study of Pegasys Alone vs. Pegasys Plus Lamivudine vs. Lamivudine

P. Marcellin ¹, G.K.K. Lau ², F. Bonino ³, P. Farci ⁴, S. Hadziyannis ⁵, R. Jin ⁶, Z-M. Lu ⁷, T. Piratvisuth ⁸, G. Germanidis ⁹, N. Pluck ¹⁰

¹ Service DHepatologie, INSERM Unite 481 And Centre De Recherches Claude Bernard Sur Les Hepatitis Virales, Hopital Beaujon, Clichy, France; ² Queen Mary Hospital, The University Of Hong Kong, Hong Kong, China; ³ Scientific Direction, IRCCS Ospedale Maggiore, Milano, Italy; ⁴ Division Of Clinical Medicine, University Of Cagliari, Cagliari, Italy; ⁵ Department Of Medicine And Hepatology, Henry Dunant Hospital, Athens, Greece; ⁶ Digestive Disease Department, Beijing You An Hospital, Beijing, China; ⁷ Department Of Infectious Diseases, Ruijin Hospital, Shanghai, China; ⁸ Songklanakarin Hospital, Songkla, Thailand; ⁹ Papageorgiou General Hospital, Pathology Clinic, Thessalonika, Greece; ¹⁰ Roche, Welwyn

Introduction

Conventional IFNα and lamivudine are first-line treatments for chronic hepatitis B (CHB). Recent data have shown that peginterferon alfa-2a (40KD) (PEGASYS) is superior to conventional IFNα in HBeAg-positive CHB (Cooksley et al, J Viral Hepat 2003).

Objective

To compare PEGASYS with and without lamivudine to lamivudine alone in the treatment of HBeAg-negative CHB.

Methods

Adults (n=537) with HBeAg-negative CHB received PEGASYS 180 μg once-weekly plus placebo, PEGASYS 180 μg once-weekly plus lamivudine 100 mg daily, or lamivudine 100 mg daily, for 48 weeks, with 24-weeks follow-up.

Results

Baseline characteristics were comparable in all treatment groups. At end-of-follow-up (week 72), using the intent-to-treat analysis, the percentage of patients with normalised ALT or HBV-DNA <20,000 copies/mL was significantly higher in the PEGASYS plus placebo and PEGASYS plus lamivudine groups than the lamivudine group (see table).

Histological response (>=2 point reduction in HAI) was reported in 47% of patients receiving PEGASYS plus placebo, 37% receiving PEGASYS plus lamivudine, and 39% receiving lamivudine. HBsAg response (HBsAg loss ± detection of HBsAb at end-of-follow-up) was reported in 12 patients receiving PEGASYS (± lamivudine) compared with none receiving lamivudine alone.

Conclusions

Significantly higher sustained response rates were observed with PEGASYS monotherapy than with lamivudine monotherapy in the treatment of HBeAg-negative CHB. The combination of lamivudine and PEGASYS did not improve end-of-follow-up response rates over PEGASYS monotherapy.

Durability of HBeAg Seroconversion after Adefovir Dipivoxil Treatment for Chronic Hepatitis B

¹T. Chang, ²M. Shiffman, ³M. Tong, ⁴P. Marcellin, ⁵Y. Liaw, ⁶P. Luengrojanakul, ⁷G. Choy, ⁷C. James, ⁷J. Ma, ⁷S. Xiong, ⁷G. Currie, and ⁷C. Brosgart, for the International Adefovir Dipivoxil 481 Study Group

¹National Cheng Kung University Hospital, Tainan, Taiwan; ²McGuire VAMC, Richmond, VA, USA; ³Huntington Memorial, Pasadena, CA, USA; ⁴Hopital Beaujon, Clichy, France; ⁵Chang- Gung Memorial Hospital, Taipei, Taiwan; ⁶Siriraj Hospital, Bangkok, Thailand; ⁷Gilead Sciences, Inc, Foster City, CA, USA

Aim

To evaluate the durability of hepatitis B e antigen (HBeAg) seroconversion after adefovir dipivoxil (ADV) treatment in patients with Chronic hepatitis B (CHB)

Methods

CHB patients with compensated liver function who had undergone HBeAg seroconversion defined as loss of HBeAg and appearance of antibodies against HBeAg (anti-HBe) confirmed on 2 consecutive visits at least 4 weeks apart, were enrolled in a long-term off treatment follow-up study.

Results

Seventy-six patients were enrolled; 63% male, 22% Caucasian, 76% Asian, median age 34 (range, 21–63) years at ADV discontinuation. Median duration on ADV therapy was 80 weeks (range, 30–193) and patients have been off treatment for a median of 55 weeks postseroconversion (range, 5–125). At time of ADV discontinuation, 87% (66 of 76) achieved undetectable HBV DNA as measured by Roche Amplicor MonitorTM PCR assay (LLQ < 1,000 copies/mL); 89% had normalized ALT (median 23 IU/L). Results at the last available observation are summarized below.

	Last Available Observation (n=75)
HBeAg-/Ab+ (seroconversion)	69 (92%)
HBeAg+Ab- /HBV DNA > / = 1000 copies/mL	4 (6%)
HBeAg-/Ab-/HBV DNA >/= 1000 copies/mL	1 (1%)
HBeAg-/Ab-/HBV DNA < 1000 copies/mL	1 (1%)

Conclusions

HBeAg seroconversion achieved during ADV treatment is durable in greater than 90% of patients with a median follow-up of more than 1 year.

Models to Predict Inflammation and Fibrosis in Patients with Chronic Hepatitis B

¹A. Lok, ²Z. Goodman, ³P. Marcellin, ⁴S. Hadziyannis, ⁵S. Hudson, ⁵G. Currie and ⁵C. Brosgart, On behalf of the Adefovir Dipivoxil 437 and 438 Study Groups

¹University of Michigan, Ann Arbor, MI, USA, ²Armed Forces Institute of Pathology, Washington, D.C., USA, ³Hopital Beaujon, Clichy, France, ⁴Henry Dunant Hospital, Athens, Greece, ⁵Gilead Sciences, Inc., Foster City, CA, USA

Introduction

Histologic activity index (HAI) has been shown to correlate with antiviral response in patients with chronic hepatitis B (CHB) and fibrosis determines the urgency of treatment.

Aim

To develop models using readily available objective data to predict inflammation and fibrosis in patients with CHB.

Methods

Baseline data and liver histology from CHB patients who participated in two trials of adefovir dipivoxil for HBeAg+ (study 437) and HBeAg- (study 438) patients were analyzed. Univariate analyses and logistic regression models were developed to predict inflammation (Knodell necroinflammatory score >8), and fibrosis (Ishak fibrosis score >3).

Results

Inflammation could be predicted using the same model for HBeAg+ and HBeAg- patients consisting of 2 variables, albumin and AST, with an Area Under the Receiver Operating Characteristic Curve (AUROC) of 0.74. Models using more variables had marginal improvement in the accuracy of predicting inflammation. The best model for predicting fibrosis in HBeAg+ patients comprised ALT, age at entry, SAP, PT, AST and bilirubin with an AUROC of 0.77; and, in HBeAg- patients, the best model consisted of platelet, PT, AST/ALT ratio and SAP with an AUROC of 0.83. Work is ongoing to develop a single model with fewer variables for predicting fibrosis in both HBeAg+ and HBeAg- patients.

Conclusion

Models using readily available laboratory and demographic data (complete blood count and serum chemistry) can predict inflammation and fibrosis in patients with CHB and may obviate the need for liver biopsies in some patients.

The Pharmacokinetics (PK) and Safety of A Single Dose of Adefovir Dipivoxil in Children and Adolescents (Aged 2-17) with Chronic Hepatitis B

¹E. Sokal, ²D. Kelly, ³S. Wirth, ⁴J. Mizerski, ⁵B. Lu, ⁵K. Kleber, ⁵J. Shah, ⁵G. Currie, ⁵C. Brosgart

¹Cliniques Universitaries, St. Luc, Burxelles, Belgium; ²University of Birmingham, Birmingham Children's Hopsital, Birmingham, UK; ³Zentrum für Kinder- und Jugendmedizin der Klinikum Wuppertal GmbH, Wuppertal, Germany; ⁴Oddział Chorób Infekcyjnych Dzieci, Krakowski Szpital Specjalistyczny im. Jana Pawła II, Kraków, Poland; ⁵Gilead Sciences Inc., Foster City, California, USA

Background

Adefovir Dipivoxil (ADV) 10 mg is approved for the treatment of chronic hepatitis B (CHB) in adults. ADV as an oral suspension is in development for the treatment of CHB in pediatrics.

Objective

To evaluate the PK of adefovir in pediatrics and establish dosing for patients aged 2-17 years.

Methods

47 HBeAg+ CHB patients with serum HBV DNA ≥5 log₁₀ copies/mL (Roche Amplicor Monitor™ PCR), and ALT ≥1.2xULN enrolled. Subjects 2-11 years received single doses of 0.14 mg/kg and 0.3 mg/kg (2x adult dose) in a cross over design (7-day washout). Subjects 12-17 years received a single dose of 10 mg ADV. Drug levels were assessed at: 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours post-dose.

Results

45 subjects dosed. PK parameters of adefovir in subjects ≥12 years were similar to adults. In subjects 2-6 and 7-11 years, 0.14mg/kg resulted in relative underexposure; 0.3 mg/kg resulted in a higher C_max but a comparable AUC0-_∞ to adults. No clinically significant adverse events except one SAE unrelated to treatment were observed.

Conclusion

The pediatric dose for phase 3 studies is 0.3 mg/kg for patients 2-6, 0.25 mg/kg for patients 7-11 and 10 mg for patients 12-17 years. ADV was well tolerated at all doses.

Three Year Study of Adefovir Dipivoxil Demonstrates Sustained Efficacy in Presumed Precore Mutant Chronic Hepatitis B Patients in A Long Term Safety and Efficacy Study

¹S. Hadziyannis, ²N. Tassopoulos, ³T. Chang, ⁴J. Heathcote, ⁵G. Kitis, ⁶M. Rizzetto, ⁷P. Marcellin, ⁸S. Lim, ⁹S. Chen, ⁹S. Arterburn, ⁹J. Ma, ⁹S. Xiong, ⁹M.Wollman, ⁹C. James, ⁹G. Currie, ⁹C. Brosgart for the Adefovir Dipivoxil 438 Study Group

¹Henry Dunant Hospital, Athens, Greece, ²Western Attica Hospital, Athens, Greece, ³National Cheng Kung Hospital, Taiwan, ⁴Toronto Western Hospital, Toronto, Canada ⁵Georgios Papanikolaou Hospital, Thessaloniki, Greece ⁶Azienda Ospedaliera San Giovanni Battista, Torino, Italy, ⁷Hopital Beaujon, Clichy, ⁸National University, Singapore, ⁹Gilead Sciences, Inc., Foster City. CA, USA

Introduction

HBeAg-Chronic Hepatitis B (CHB) patients were previously enrolled in a randomized, placebo controlled trial of Adefovir Dipivoxil (ADV) for 96 weeks. Patients receiving ADV in the second 48 weeks were offered up to 3 additional years of ADV.

Objective

To evaluate safety and efficacy of ADV over 96 and 144 weeks.

Results

Baseline (in original study) for patients entering LTSES: 81% male; 70% Caucasian; 26% Asian; median age 47 years; median HBV DNA 7.08 log₁₀ copies/mL; median ALT: 99 (2.3 x ULN).

The rtN236T and A181V mutations were associated with resistance to ADV. The rate of either mutation at weeks 48, 96, and 144 is 0 %, 3.0% and 5.9%, respectively. Three patients had confirmed elevations in serum creatinine ≥ 0.5 mg/dL over 144 weeks. All resolved, one on treatment and two following discontinuation.

Conclusions

Treatment with ADV 10mg over 144 weeks resulted in significant and continued reductions in HBV DNA levels with increasing proportion of patients achieving HBV DNA undetectability. ALT change and normalization was sustained. The safety profile of ADV during three years of treatment was similar to that seen in the first and second year. Resistance is delayed and infrequent.

Long-Term Incidence of Adefovir Dipivoxil Resistance in Chronic Hepatitis B Patients after 144 Weeks of Therapy

¹S. Xiong, ¹X. Qi, ¹A. Snow, ²V. Thibault, ¹Y. Zhu, ¹M. Curtis, ³S. Hadziyannis, ¹C. Brosgart, ¹G. Currie, ¹S. Arterburn, ¹C. Gibbs, and ¹M. Miller

¹Gilead Sciences, Inc., Foster City, CA, USA, ²Hospital Pitie-Salpetriere, Paris, France, ³Henry Dunant Hospital, Athens, Greece

Background

Lamivudine resistance occurs in 24% and 70% of Chronic hepatitis B (CHB) patients after 1 and 4 years. Adefovir (ADV) resistance mutations, rtN236T and rtA181V, were observed in 0% of patients after 48 weeks and 2% of patients after 96 weeks.

Aim

To determine the incidence of ADV resistance after 144 weeks.

Methods

This analysis included 629, 293, and 167 patients who received ADV through 48, 96 and 144 weeks, respectively. At baseline, patients had either wild-type or YMDDmutant HBV. The majority received ADV monotherapy. Most (>90%) patients with YMDDmutant HBV continued on lamivudine after addition of ADV. HBV RT was sequenced for all samples with detectable HBV DNA by PCR (≥1000 copies/mL). The cumulative probability of ADV resistance was calculated using the Life Table method.

Results

In addition to the patients who developed rtN236T (n=4) or rtA181V (n=2) at week 96, three additional patients developed rtN236T (n=2) or rtA181V (n=1) at week 144. All rtN236T and rtA181V mutants emerged during ADV monotherapy. All patients with the rtN236T and 2 of 3 patients with the rtA181V experienced confirmed serum HBV DNA rebound (≥1 log₁₀ increase from nadir). Two novel conserved site mutations (rtA211P, rtS202G) were observed once each in single patients at week 144 but were not associated with HBV DNA rebound; in vitro phenotypic analysis of these mutations is ongoing.

Conclusion

ADV resistance mutations (rtN236T and rtA181V) emerged at a delayed rate and with a low frequency (cumulative probability of 3.9%) after 144 weeks of therapy in all patients in this analysis.

The Immunomodulatory Effect of Steroids in Chronic Hepatitis B

J. Gotto²,G.J.M. Webster¹, S. Reignat¹, J. Jenkins³ ,D. Mesogiti³ ,D. Brown² ,R. Williams¹,G.M. Dusheiko² ,A. Bertoletti¹.

¹Institute of Hepatology, UCL, London, UK; ² of Hepatology, Royal Free, London, UK; ³ London, UK

Introduction

Short courses of steroids have been used in an attempt to enhance the efficacy of subsequent antiviral therapy in chronic hepatitis B patients. Serum ALT elevation after steroid withdrawal has been interpreted as evidence of immunological rebound against HBV, which might explain the increased efficacy of the combined steroid + antiviral therapy in some studies.

Methods

The dynamics of the HBV-specific CD8+ T-cells was analysed in 6 patients (HBeAg+, ALT < 80U/L, HBV-DNA> 107 copies/mL) treated with steroid and lamivudine (2 weeks 30mg/daily + 2 weeks 15mg/daily Prednisolone 2 weeks treatment free 12 months lamivudine 100mg daily).

The frequencies and functions of CD8+ T-cells specific for 11 different HLA-A2 restricted HBV peptides were analysed longitudinally (before and during steroid and lamivudine therapy) directly ex vivo and after in vitro stimulation using HLA-tetramers and intracellular cytokine staining.

Results

Restoration of HBV-specific CD8+ T cell response was demonstrated in all the 4 patients who experienced ALT elevation after steroid withdrawal. CD8+ cells specific for different HBV epitopes were detectable in the periphery during prednisolone treatment.

HBV-specific CD8 response was maximal during steroid therapy and not after withdrawal. ALT flares were not associated with an increase frequency of peripheral HBV-specific CD8+ response.

Conclusions

Steroid priming can lead to an immunological rebound against HBV in HBeAg+ chronic hepatitis B patients, but the immune restoration is associated with steroid treatment and not with its withdrawal. These data demonstrated a new and unexpected immunomodulatory effect of corticosteroids in HBV infection.

Development of Hepavir B, a Prodrug of PMEA with Excellent Liver-Targeting Properties

C-C. Lin¹ ,A. Teng¹,L-T. Yeh¹, D. Vitarella¹, Z. Hong¹,M.Erion²

¹R&D Drug Development, ICN Pharmaceuticals, Inc., Costa Mesa CA, USA; ² Metabasis Therapeutics, San Diego CA, USA

Introduction

Hepsera was approved for the treatment of chronic hepatitis B. However, it has dose-limiting nephrotoxicity. Hepavir B is a prodrug of PMEA designed to increase the PMEA level in liver.

Aim

To evaluate (1) liver-targeting property, (2) animal safety profile, (3) safety and pharmacokinetics of Hepavir B in man.

Methods

Levels in rat liver and kidney following oral dosing of [ B (30 mg/kg). A 28-day toxicity study for Hepavir B in rats (5, 30, 100 or 300 mg/kg) and monkeys (3, 20, 60 or 180 mg/kg). Single oral dose study for hepavir B in healthy volunteers at 10, 30 or 60 mg.

Results

Hepavir B gave 4x higher liver level, but 1/10 kidney levels compared to Hepsera. Hepavir B toxicity study demonstrated renal and hepatic effects in male rats at 100 mg/kg and acceptable safety in monkeys at dose up to 60 mg/kg.

In man following oral dose of 10, 30 or 60 mg, AUC (0-96 hr) was 24.8, 110 and 180 ng-hr/mL, respectively for Hepavir B and 22.1, 145 and 278 ng-hr/mL, respectively for PMEA. T_max was 1.5 hr. There was no dose-related trend in incidences.

Conclusions:

1. Hepavir B has excellent liver-targeting properties and gives >40x higher liver to kidney ratio than Hepsera,
2. Hepavir B is safer than Hepsera in rats and monkeys,
3. Hepavir B is well tolerated in man, rapidly absorbed and converted to PMEA. Thus, Hepavir B has potential as a safe and effective drug for treatment of chronic hepatitis B.

HBV Genomes From Liver Tissue of Patients With Occult HBV Infection Show Wide Range of Sequence Divergence and Normally Replicate In Vivo

T. Pollicino¹, G. Raffa¹ L. Costantino¹, C. Saitta¹, G. Squadrito¹,M. Levrero¹ ,0. Raimondo¹

¹Unit of Clinical and Molecular Hepatology, Department of Internal Medicine, University of Messina, Messina, Italy; 5 Andrea Cesalpino, Roma, Italy

Background/Aims

Occult HBV infection is characterized by persistence of HBV-DNA in the liver of HBsAg-negative individuals. This peculiar infection significantly associates with cirrhosis and HCC development. Occult HBV may persist as free genome, although it is undefined whether it may carry genomic rearrangements inhibiting its replicative activity.

Aims of this study were to investigate the genomic variability of HBV isolates from patients with occult infection and to explore their capacity to replicate in vitro.

Methods

We studied liver HBV isolates from 15 patients with HCC (13 with occult infection and 2 HBsAg-positive). cccHBV-DNA was detected in all the cases by specific PCR amplification. Full-length HBV genomes from each case were amplified and directly sequenced. Additionally, full-length HBVs from four occult-HBV and two HB5Ag-positive cases were cloned and sequenced. Finally, four monomeric linear HBV genomes from occult cases were transiently transfected in HuH7 cells: one had wild-type sequence, three had in-frame deletions, respectively, in the preSl, preS2 and X regions.

Results

Direct sequencing showed that the major population of each case had no mutation interfering with viral replication and gene expression. Cloning experiments revealed highly divergent HBV strains both in HB5Ag-positive and HB5Ag-negative individual cases (range of divergence 1.4-7.1%). All the 4 above mentioned full-length HBV isolates from occult cases showed normal patterns of replication in vitro.

Conclusions

1. Multiple HBV variants accumulate in liver of patients with occult infection.
2. Occult HBV strains normally replicate in vitro demonstrating that host but not viral factors are responsible for cryptic HBV infection.

Results of a One-Year International Phase IIB Trial of LDT, and LDT, Plus Lamivudine, In Patients With Chronic Hepatitis B

S.H. Han¹ ,N.W.Y. Leung¹ ,E.K. Teo³ ,M. Tong⁴ ,F. Wong⁵,H.W. Hann⁶ ,T. Poynard⁷, N.A. Brown⁸,M. Myers⁸ ,G. Chao⁸ D. Lloyd⁸,C.L. Lai⁹

¹UCLA School of Medicine, Los Angeles CA, USA; ² University of Hong Kong, Hong Kong, Hong Kong; ³ General Hospital, Singapore, Chine; ⁴ Memorial Hospital, Pasadena CA, USA; ⁵ General Hospital, Toronto, Canada; ⁶Jefferson Medical College, Philadelphia PA, USA; ⁷Pitie-Salpetriere, Paris, France; ⁸ Idenix Pharmaceuticals, Cambridge MA, USA; University of Hong Kong, Hong Kong, Hong Kong

Introduction

L-deoxythymidine (LdT; telbivudine) is a potent inhibitor of HBV replication and has a favorable preclinical safety profile.

Methods

This randomized multicenter study compared the 1-year efficacy and safety of LdT 400 or 600 mg/day, and LdT 400 or 600 mg/day combined with lamivudine 100 mg/day, to lamivudine 100 mg/day in adults with HBeAg-positive CHB and baseline ALT >1.3 xULN.

The primary endpoint was serum HBV DNA reduction, assayed by PCR. Key secondary endpoints included serum ALT normalization. HBeAg response, and safety.

Results

104 patients were enrolled. At week 52, median reductions in serum HBV DNA (10gb copies/mL) were: lamivudine, 4.66; LdT 400mg/day, 6.43; LdT 600mg/day, 6.09; LdT 400mg/day + lamivudine, 6.40; and LdT 600mg/day + lamivudine, 6.05.

All treatments were well tolerated. Residual HBV viremia at Week 52 was highest in lamivudine monotherapy recipients (median HBV DNA level at week 52 = 2.9 log 10 copies/mL), while the median HBV DNA level in LdT monotherapy recipients was non-detectable by PCR <2 log 10 copies/mL).

Key efficacy results at Week 52 for comparisons by treatment type are as follows:

Conclusion

After one year of treatment, viral suppression, PCR non-detectability of serum HBV DNA, and ALT normalization were significantly greater for LdT compared to lamivudine.

Absence of Entecavir Resistance Emergence in Lamivudine-Refractory Patients for at Least One Year With Entecavir Study AI463-014

R. Colonno ,R. Rose, A. Cross, R. Hindes, D. Tenney. Bristol-Myers Squibb Pharmaceutical Reseamh Institute, Wallingfonl CT USA

Background

Entecavir (ETV) is a highly potent and selective inhibitor of hepatitis B virus (HBV) currently in Phase III development. All three doses of ETV (1.0.0.5 and 0.1 mg) showed superiority to continued LVD in reducing HBV DNA over 48 wk in Phase II Study A1463-0l4

Methods

Patients isolates were monitored for ETV resistance by genotypic analysis of RT sequences amplified from serum HBV samples.

Results

At study entry, 132(87%) of 181 patients treated had isolates that harbored LVDR substitutions L18OM and M204V11. Subsequent genotypic analysis of patient HBV isolates showed no apparent correlation between virologic response on ETV and either LVD-resistant or novel HBV RT substitutions.

Analysis of HBV isolates with paired baseline and on treatment samples showed that most patients with LVDR HBV at study entry retained their LVDR genotypes over 48 wks, regardless of the treatment arm, with a small proportion of isolates showing shifts among their LVDR substitutions.

There was no apparent correlation between these observed changes and ETV dose level. Comparison of 132 paired isolates (108 ETV and 24 LVD) showed no significant difference in the number of other RT substitutions, either in naturally polymorphic positions or in highly con served residues. A small number of substitutions at conserved residues did emerge on ETV therapy, but none were correlated with virologic rebounds or reduced susceptibility to ETV.

Conclusion

There was no evidence of resistance emergence in patients treated with ETV for 48 wk despite the presence of LVDR mutations at study entry.

Evaluation of the RealQuant HBV-DNA Assay (Realtime PCR)

M. Martinot-Peignoux¹ ,V. Esrsault¹,C. Kumas¹, B.N. Pham³,P. Marceilin²

¹INSERM U481 et Unite Claude Bernard Hopital Beaujon,Clichy, France: ² D ‘Hepatologie,Hopital Beaujon, Clichy, France; ³ D ‘Hematologie-immunologie Biologiques Hdpital Beaujon, Clichy, France

Aims

The aim of our study was to assess the relevance of the with REALQUANT® HBV-DNA assay for a routine laboratory.

Methods

180 serum samples tested with Cobas Amplicor HBV MONI TOR, Roche (sensitivity 2x102 HBV DNA copies/mi) were quantified with REALQUANT® HBV-DNA, (DiaTech, Italy) on the Rotor-Gene® system (Corbett Research, Australia).

REALQUANT® allows the amplification of a short length of the gene coding for the Surface Antigen and utilizes a Taqman® probe for the detection. Twenty samples HB5Ag negative/anti HCV negative and 20 samples HB5Ag negative/HCV RNA positive were tested for the specificity. Reproducibility was calculated with 3 serum samples tested 10 times. Sensitivity was determined with 76 samples HBV DNA undetectable with COBAS (30 patients under therapy and 46 untreated) and 35 samples HBV DNA detectable with COBAS.

Results

All the samples HBsAg negative/anti-HCV negative were found negative; 3/20 samples HBsAg negative/HCV RNA positive were found positive.(7.4x103; 181; and 70 copies/mi) with REALQUANT®. The 3 samples tested 10 times showed CV ranging from 13% to 24%.

HBV DNA was detectable with REALQUANT® in 58% and 63% of the samples from under therapy and untreated patients, respectively. Among the 35 samples COBAS positive 94% had HBV DNA detectable with ReaiQuant® HBV DNA. The correlation between the two assays was good (r2 =0.695). The HBV DNA levels were higher with REALQUANT® than with COBAS (ratio=9).

Conclusion

REALQUANT® HBV-DNA assay showed a good specificity 93%, a high sensitivity (mainly for patients under therapy) suggesting that monitoring patients with a sensitive assay could be clinically relevant.

The Long-Term Outcome of HBeAg-Negative Patients With Cirrhosis Treated With Lamivudine Monotherapy: A 5-Year Prospective Cohort Study

P. Lampertico¹ ,M. Viganfi¹, M. Iavarone¹ ,G. Lunghi R. Romeo¹,G. Colucci³, A. Morabito⁴,E. Del Ninno¹, M. Cobombo¹.

¹Division of Hepatology, IRCCS Maggiore Hospital, University of Milan, Milan, Italy; ² Dept of Hygiene, JRCCS Maggiore Hospital, Milan, Italy; ³ Molecular Systems, Rotkreutz, Swizerland; ⁴ of Statistics, Dept. of Medicine, Surgery and Dentistry, University of Milan, Milan, Italy

Aim

To assess the impact of long-term continuous lamivudine treatment in HBeAg-negative cirrhosis, 44 consecutive patients were treated with 100 mg/daily for 52 (5-84) months (89% men. 82% genotype D. 39% decompensated cirrhosis and 36% oesophageal varices).

HBV-DNA was tested by bDNA (<700.000 eq/mL. Bayer) and Cobas Amplicor HBV Monitor (<200 cplml, Roche). Lamivudine resistance was confirmed by INNO LiPA assay (Innogenetics. Belgium).

Results

After 5 years of treatment, 21(48%) patients maintained undetectable serum HBV-DNA by non-PCR assay and normal ALT whereas 22 (52%) developed lamivudine resistance.

One patient (2%) never achieved a virological response. The 5-yr cumulative probability of developing lamivudine resistance was 63% but only 10% of patients had undetectable HBV DNA by PCR assay.

Clinical decompensation occurred in none of responders but in 7 lamivudine-resistant cirrhotics (0% vs 32%, p<O.00l) whereas hepatocellular carcinoma occurred in both groups at similar rates (28% and 41%).

Late complications (>12 months of treatment) included both HCC (n.=7) and liver decompensation (n.=7) and occurred only in lamivudine-resistant cirrhotics (13/14, 93%).

Overall, liver-related complications occurred less frequently in responders than in lamivudine-resistant cirrhotics (28% vs. 64%, p<0.0l).

The 5-year patient survival was 74%, similar in responders and lamivudine-resistant cirrhotics, but 5 patients with lamivudine resistance and clinical decompensation were rescued by another antiviral drug.

Conclusions

Five-year administration of lamivudine monotherapy led to prolonged control of viral replication in approximately one third of HBeAg-negative cirrhotics, but complete suppression in only 10%. Lamivudine resistance was associated to increased risk of liver-related complications, particularly HCC.

A Prospective Open Study of Long-Term Lamivudine Treatment in Children with Chronic Hepatitis B Unresponsive to Previous In To Previous Interferon Alpha Therapy

D.M. Lebensztejn¹,E. Skiba¹, 0. Kovalchuk² ,M.E. Sobaniec-Lotowska³. M. Kaczmarski¹, L. Chyczewski²

¹ III/rd Department of Pediatrics, Medical University, Bialystok, Poland; ² of Clinical Molecular Biology, Medical University, Bialystok, Poland; ³ of Clinical Pathomorphology, Medical University, Bialystok, Poland

Introduction / Aim

Data regarding the effect of extended Lamivudine treatment in children who failed to respond to previous IFN a therapy are scarce. Therefore, the effects of Lamivudine treatment on ALT activity, HBV DNA and HBeAg seroconversion as well as the occurrence of YMDD mutant and adverse effects were determined.

Methods

The observation was carried out on 52 children, aged 4-17 years, with biopsy-proven chronic hepatitis B (HEeAg+, HEY DNA+, wild-type virus+), who were nonresponders to previous IFN a therapy in the dose of 3MU 3 times weekly for 20 weeks. Lamivudine in the dose of 3-4mg/kg/day up to 100mg/day was given for 24 months.

Results

Fifty-one children completed 12 months and 25 of them — 24 months of therapy; one boy was excluded from this study due to severe thrombocytopenia. After a year of Lamivudine therapy 3 1.3% of children cleared HBV DNA, 78.3% normalized ALT activity and 7.8% lost HBeAg. After completion of 24-month therapy 52% of children cleared HBV DNA, 28% seroconverted to antiHBe and 66.6% normalized ALT activity. No adverse effects were noted. except thrombocytopenia.

YMDD mutant was present in 20% of children after one year of therapy, in 32% - 6 months later and in 52% after two years of treatment.

Conclusions

The 24-month treatment with Lamivudine in children with chronic hepatitis B who did not respond to IFN a normalized ALT activity in most children and caused seroconversion to antiHBe in 28%. However it was associated with a high risk for developing YMDD mutation.

Lowest Serum Hepatitis B Virus DNA Level is Predictable Factor for Viral Breakthrough During Lamivudine Therapy in Chronic Hepatitis B Patients

M.Cho¹, J. Heo¹, DY. Lyu¹, B.M. Cho² H.H. Kim³ G.H. Kim¹,D.H. Kang¹,G.A. Song³. U.S. Yang¹

¹Department of Internal Medicine, Pusan National University, College of Medicine, Busan, Korea; ² Department of Preventive Medicine, Pusan National University, College of Medicine, Busan, Korea; ³ Department of Diagnostic Medicine, Pusan National University, College of Medicine, Busan, Korea

Introduction

Lamivudine is safe and effective therapy in patients with chronic HBV infection, but the emergence of resistant HBV mutants is major concern.

Aim

This study was performed to show whether kinetics of serum viral DNA level predicts viral breakthrough during lamivudine therapy.

Methods / Patients

Of 146 patients who had been treated with lamivudine for 12 months or more, were included 97 men and 49 women aged 15 to 72 years. Mean duration of lamivudine therapy was 21.5 months(12~51 months).

Serum AST/ALT. HBeAg, and anti-HBe were tested every 3 months and serum HBV DNA levels(PCR-based assay) every 6 months. Patients were classified into 3 groups by HBV DNA level at 6th month of treatment and lowest level during treatment: patients with HBV DNA level less than 2 x 10² copies/mL (group 1), 2 x 10² copies/mL or more and less than 1 x l0⁵ copies/mL (group 2), 2 x 10² copies/mL or more(group 3).

Results

Viral breakthrough occured 35.9% (51/142) during the treatment and the cumulative rate was 16.2% and 33.3% at 1 and 2 years. It was higher in HBeAg-positive patients than HBeAg-negative ones (43.0% (37/86) vs. 25% (14/56), p=0.O29).

The rate of viral breakthrough was not different between each group when classified by HBV DNA level on 6th month of treatment. However, classified by lowest HBV DNA level during the therapy, it was 15.9% (11/69) in group 1, 50% (27/54) in group 2, and 62.5% (5/8) in group 3.

The rate of viral breakthrough in group 1 was significantly lower than others (p<0.00l). The lowest serum HBV DNA levels during the lamivudine therapy is predictable factor for the viral breakthrough in patients with chronic HBV infection.

Paired Measurement of Quantitative Hepatitis B Virus DNA in Saliva, Urine and Serum of Chronic Hepatitis B Patients

A.A. van der Eijk¹, H.G.M. Niesters² ,B.E. Hansen³,S.D. Pas² ,H.L.A. Janasen’, SW. Schalm¹, R.A. de Man¹

¹Gastroenrerology & Hepatology, University Medical Center; Rotterdam, The Netherlands; ²Virology, University Medical Center; Rotterdam, The Netherlands;³Epidemiology & Biostatistics, University Medical Center; Rotterdam, The Netherlands

Introduction

In spite of abundant epidemiological evidence for horizontal transmission of Hepatitis B Virus (HBV) the exact transmission routes have not been fully elucidated.

Aim

We evaluated quantitative HBV DNA content in serum, saliva and urine as a first step to further explore possible modes of horizontal transmission. Paired serum, saliva and urine samples were collected from 150 chronically infected Hepatitis B virus patients.

A validated HBV DNA TaqMan assay was used for the quantitative measurement of HBV DNA. Mean HBV DNA in serum was 5.68 x l0E8 (geq/ml), 50% of the patients had a HBV DNA of 10E5 geq/ml in serum. Mean HBV DNA level in saliva was 4.00 x 10E5, 15% had a HBV DNA of lOE5 geq/ml in saliva.

Mean HBV DNA level in urine was 3.26 x 10E3 and 1% had a HBV DNA of 1OE5 geq/ml in urine. A high non-linear correlation was shown between HBV DNA in serum and saliva (Spearman’s rho 0.82) and between serum and urine (Spearman’s rho 0.74).

Conclusion

The significant amounts of HBV DNA found in saliva and urine in high viremic chronic HBV patients could have important implications for the under standing of hepatitis B epidemiology, as the origin of infections especially in horizontal transmission remains unknown in up to 20% of cases.

Multi-Center Study of Hepatitis B Virus (HBV) Genotypes in France: Geographic Distribution, Correlation with HBeAg-Positive Status and Relation to Liver Fibrosis

P. Halfo ¹M. Bourliere² S. Pol⁴,Ouzan¹ M.H. Tainturier C. Renou M. Rotily D. Saadoun H. Khiri¹, T. ¹ Poynard⁵,Thibault⁵ Y. Benhamou⁵ ,P. Cacoub⁸

¹Virological, Alphabio L Aboratory, Marseille, France; ² Hepatology Saint Joseph, Hospital, Marseille, France; ³ Institut Arnault Tzank, Saint Laurent Du Var; France; ⁴ Necker; Hospital, Paris, France; ⁵La Pitie Salpetriere Hospital, Paris, France; ⁶ Hyeres, Hospital, Paris, France; ⁷Clinsearh, Paris, Francis, ⁸Internal Medicine, La Pitie Salpetriere, Hospital, Paris, France

Introduction

HBV genotypes may be associated with progression of liver disease.

Aim

To assess the distribution and clinical implications of HBV genotypes in France

Methods

280 patients HBSAG-positive (mean age: 45.8 yr ± 14, male:78%). from 5 center were studied. The study population consisted of 66% Caucasians, Asians 16%, Africans 18%. 40% of our patients were found HBEAG-positive. The distribution of the fibrosis available for 244 patients was 45,4% for FO-Fi, 18,9% for F2 and 35,7% For F3-F4. HBV genotype were assessed by InoLipa and sequence analysis.

Results

The genotype distribution for 265 patients with chronic HBV infection was as follows: A 25.4%; B 6%; C 11.4%; D 25.7%; E 1.5%; F 0.4%; G 1.1% and mixed 16.8%.

• Caucasians were mainly infected by A and D (33.7% and 34.2% reap).
• Asians by B and C (24.4% and 44.4% respectively), and
• Africans by E (50%).

In Caucasian patients, Genotype A is significantly associated with HBEAG-positive rate (46,4% vs 22.5%. p=0.00l), and Genotype D is significantly associated with HBEAG-negative rate (42.9% vs 43.1%. p=0.00l). Using multivariate analysis, the significant risk factors for advanced liver disease for patients with chronic HBV infection were old age, male gender and genotype G.

Conclusions

These results suggest:

1) The distribution of the HBV genotypes differs by ethnic origin.

2) A and D genotypes were the major genotypes in France.

3) In Caucasians patients, Genotype A were associated with HBEAG-positive and Genotype D with HBEAG-negative.

4) Genotype G is associated with more severe liver disease.

Interferon Plus Lamivudine vs. Lamivudine Monotherapy in Patients with Chronic Hepatitis B Anti-HBe Positive

S. Karataøanis¹,I. Ketikoglou² ,L. Skorda²,S. Manolakopoulos²,K. Komnianides²,F. Lisgos² C. Psellas¹,Th. Kalderi¹. D. Kipraios¹,C. Kouvidou¹, E. Spanos¹,V. Artikis¹

¹General Hospital of Rhodes, Greece; ² General Hospital, Athens, Greece

Introduction

Combination therapy with interferon-a (IFN) has been suggested to reduce the development of resistance to LAM.

Aim

The aim of the present study was to evaluate response and development of resistance in CHB patients treated with IFN plus LAM versus LAM monotherapy.

Methods

Sixty-nine patients with biopsy proven CHB and elevated ALT (anti-Hbe+, mean age 52, M/F—42/27) were included in our study. Thirty-four patients assigned to the combination treatment (LAM: 100mg/day plus IFN: 4.5MUX3/week) and thirty-five to LAM (100mg/day). IFN was stopped after the completion of 12 mo of therapy and LAM was continued in both groups. HBV-DNA was tested at baseline and at 6 months intervals during treatment for 2 years.

Results

At 6 months all patients had undetectable serum HBV-DNA and normal ALT levels in both groups. At 12 months 3/34 (8.8%) pts presented positive HBV-DNA and 2/34(5.8%) had abnormal ALT levels in the combination group while in the group of LAM we observed 8/35(22.8%) and 5/35 (14.2%) respectively. At 24 months HBV-DNA was positive in 4/34(14.7%) pts in the combination group, where in the LAM group was11/35 (31.4%), P<0.05.

Conclusion

We conclude that LAM/IFN combination therapy in anti-Hbe-positive CHB appears to prevent or delay the emergence of resistance to LAM.

Lamivudine-Refractory Hepatitis B Patients Can Be Safely Switched Directly to Entecavir 1 MG Daily Therapy

R.G. Gish¹ T.T. Chang²,S. Hadziyannis³,J. Cianciara⁴,M. Rizzetto⁵,E. Schiff⁶,G. Pastore⁷, R. Jackson⁸,A. Thiry⁸,R. Hinde⁸

¹California Pacific Medical Center; San Francisco CA, USA; ²Cheng Kung Univ Hosp, Tainan, Taiwan; ³ Dunant Hosp, Athens, Greece; ⁴ Chomb Zakaznych Akademii Medycznej, Warsaw, Poland; ⁵ Molinette, Torino, Italy; ⁶Univ of Miami, Miami FL, USA; ⁷ Di Ban, Ban, Italy; ⁸ Squibb Pharmaceutical Research Institute, Wallingford CT USA

Background

Hepatic flares have been reported in patients who have been abruptly discontinued from anti-HBV therapy.

Methods

In a phase II, double-blind, randomized, 4-ann. dose-ranging trial, ALT elevations and hepatic adverse events were analyzed in 181 lamivudine (LVD)-refractory patients (87% with YMDD mutations) switched directly to entecavir (ETV) at one of three doses (0.1.0.5. 1.0mg daily) or continued on LVD therapy (100mg daily). Post-switch ALT flares were defined as ALT>2x baseline (BL) and > 10x upper limit of normal (ULN). Patients were treated for up to 85 weeks.

Results

Eleven patients (6%) developed ALT flares during study treatment: 3 on 1.0mg, 1 on 0.5mg, 2 on 0.1mg ETV and Son continued LVD. All 4 patients on either 0.5 or 1.0mg ETV had transient ALT flares which resolved while on continued blinded ETV therapy; all had declines in HBV bDNA <LOQ and no SAEs. Two patients on 0.1mg ETV and 5 patients on continued LVD had ALT flares associated with elevated or rising HBV DNA levels which did not resolve while on continued blinded therapy. Two of these patients (0.1mg ETV and LVD) experienced a hepatic SAE.

Conclusion

Continuation or overlap of LVD is not necessary when switching a LVD-refractory/ resistant patient to ETV at the therapeutic dose of 1.0mg daily. On-treatment ALT elevations accompanied by viral load reductions may reflect a favorable response to therapy.

Hepatitis B Virus Genotype Mixtures: Clinical Relevance and Molecular Characteristics

S. Le^{1, 2, 3},H. T. Nguyen L¹,D. Dinh²,N. B. Vu Q⁴,P. Kremsner^3,J. Torres⁶,S. Kaise⁵,C.T. Bock¹

¹Dept. of Molecular Pathology, Institute of Pathology, University Hospital of Tuebingen, Tuebingen, Germany; ² Hung Dao Hospital, Hanoi, Vietnam; ³ Department of Parasitology, Institute of Tropical Medicine, University of Tuebingen, Tuebingen, Germany; ⁴ of Prevention Medicine, Truong Dinh, Dong Da, Hanoi, Vietnam; ⁵ Internal Medicine I, Dept. of Gastroenterology, Hepatology and Infektiology, University Hospital, Tuebingen, Germany; ⁶ Dept. of Medicine, Royal Melbourne Hospital, The University of Melbourne, Melbourne, Australia

Introduction

Seven hepatitis B virus (HBV) genotypes (A-G) have been identified. The clinical significance of infection with specific HBV genotypes or genotype-mixtures is unclear.

Aims

We have investigated the frequency of HBV-genotypes and genotype-mixtures in HBV-infected patients and correlated this to the clinical outcome. Additionally, we have performed in vitro analysis to evaluate the replication of HBV-genotypes.

Methods

Four hundred and three HBV-infected patients (365 Asian. 38 European) with well- characterized clinical profiles were enrolled. HBV-genotypes were determined by RFLP-PCR and DNA-sequencing. To determine the interference of different genotypes HepG2-cells were co-transfected with HBV-clones. Replication competence of different HBV-genotypes was evaluated by real time PCR and Southern-Blot analysis.

Results

In Asian patients all seven genotypes could be detected however, genotype A (20.5%). C (35.4%) and D (11%) predominated. In European patients only genotypes A (15.8%), D (44.5%), and G (15.8%) could be identified. Genotype-mixtures were identified in a similar proportion of Asian (18.9%) and European (13.1%).

In Asians predominant mixtures of genotypes were of A/C (34.7%) and C/D (18.8%) whereas in Europeans a mixture of C/D (40%) was most frequent.

Interestingly, genotype-mixtures were found more frequently in patients without HCC (27.9%) compared to HCC-patients (10%). Cell culture experiments revealed higher levels of HBV-replication in cells co-transfected with genotype-mixtures compared to single-genotype transfections.

Conclusion

Our findings show that co/super-infection with different HBV-genotypes is frequent both in Asia and Europe. Differences in the replication-fitness of single HBV-genotypes compared to genotype- mixtures may suggest that co/super-infection with different HBV genotypes is associated with altered pathogenesis and clinical outcome compared to single-genotype infections.

Models to Predict Inflammation and Fibrosis in Patients with Chronic Hepatitis B

A.Lok¹,Z. Goodman²,P. Marcellin³,S. Hadziyannis⁴,S. Hudson⁵,G. Curie⁵,C.L. Brosgart⁵,

¹University of Michigan, Ann Arbor MI, USA; ² Armed Forces Institute of Pathology, Washington DC, USA; ³Hopital Beaujon, Clichy, France; 4 Dunant Hospital, Athens. Greece; ⁵ Sciences, Inc. Foster City CA, USA

Introduction

Histologic activity index (HAI) has been shown to correlate with antiviral response in patients with chronic hepatitis B (CHB) and fibrosis determines the urgency of treatment.

Aim

To develop models using readily available objective data to predict inflammation and fibrosis in patients with CHB.

Methods

Baseline data and liver histology from CHB patients who participated in two trials of adefovir dipivoxil for HBeAg+ (study 437) and HBeAg- (study 438) patients were analyzed.

Univariate analyses and logistic regression models were developed to predict inflammation (Knodell necroinflammatory score >8), and fibrosis (Ishak fibrosis score >3).

Results

Inflammation could be predicted using the same model for HBeAg+ and HBeAg- patients consisting of 2 variables, albumin and AST, with an Area Under the Receiver Operating Characteristic Curve (AU ROC) of 0.74. Models using more variables had marginal improvement in the accuracy of predicting inflammation. The best model for predicting fibrosis in HBeAg+ patients comprised ALT, age at entry, SAP. PT, AST and bilirubin with an AUROC of 0.77; and, in HBeAg- patients, the best model consisted of platelet, PT, AST/ALT ratio and SAP with an AUROC of 0.83.

Work is ongoing to develop a single model with fewer variables for predicting fibrosis in both HBeAg+ and HBeAg- patients.

Conclusion

Models using readily available laboratory and demographic data (complete blood count and serum chemistry) can predict inflammation and fibrosis in patients with CHB and may obviate the need for liver biopsies in some patients.