Lamivir, a French cohort of patients with chronic hepatitis B starting lamivudine treatment: initial clinical and virological results

I-tEPATOLOGYVol. 34, No. 4, Pt. 2, 2001

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AASLD ABSTRACTS

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L A M M R , A FRENCH COHORT OF PATIENTS W I T H CHRONIC HEPATITIS B STARTING LAMIVUDINE TREATMENT : INITIAL CLINICAL AND VIROLOGICAL RESULTS. Fabien Zoulim, INSERM, Lyon France; Thierry Poynard, Hopital Piti4 Salpetri~re, Paris France; Fran~oise Degos, Hopital Beaujon, Clichy France; Paul Deny, Hopital Avicenne, Bobigny France; Paul Landais, Hopital Necker, Paris France; Patrick Blin, Icare, Massy France; Abdelkader E1 Hasnaoui, Alain Slama, GlaxoSmithKline, Marly-Le-Roi France; Parviz Parvaz, Christian Tr4po, INSERM, Lyon France

LAMIVUDINE RESTORES HBEAG-SPECIFIC SYNTHESIS OF INTERLEUKIN-18 IN PBMC OF PATIENTS W I T H CHRONIC HEPATITIS B VIRUS INFECTION. Tobias Manigold, Jingsan Chen, Ulrich Bgcker, Jutta Gundt, Petra Traber, Manfred V Singer, Siegbert Rossol, University Hospital Mannheim, Mannheim Germany

Both clinical and virological aspects of chronic hepatitis B (CHB) are under-evaluated in France, as are the incidence and clinical significance of viral polymerase sequence variation during lamivudine treatment. The objectives of the study are to determine the incidence, the kinetics and the predicitive factors of emergence of viral polymerase gene variant. Methods : Lamivir is a 2-year cohort study of 297 patients with chronic hepatitis B who have recently started lamivudine treatment (100 rag/d), being carried out in 52 French centres of Hepatology since October I999. Six monthly clinical data and centralised virology samples are collected. Viral polymerase gone sequence, including domains A to E, are analyzed after direct sequencing of PCR amplification products. HBV genotype was determined by phylogenic analysis of the viral polymerase sequence. Population data at inclusion and preliminary results at 6 and 12 months are presented. Results: M/F sex ratio was 3.4; mean age 43.1 ~ 14.1 years; BMI (body mass index) -> 25 in 36.3% of patients; known duration of the disease 6.3-+ 7.0 years. Asthenia was recorded in 43% of patients at i11dusion. Contamination risk factors were absent in I8.5% of cases. Serum ALT levels were > IxULN in 85.3% and > 3xULN in 32.8% of patients at inclusion. Serology profiles consistent with a pro-C(-) mutant (HBeAg-, anti-HBe Ab +, HBV DNA+ ) were found in 48.7% of patients. Lamivudine was prescribed as a first intention treatment for 55.9% of patients. The prevalence of viral genotypes was : 23% A, 6.5% B, 14% C, 37.5% D, 14% E, 4% F, suggesting a change in this prevalence in France, compared to previous studies. Liver biopsies were performed in 284 patients and histology analysis using the METAVIRscore prior to inclusion, available on 244 patients, showed that 20% of the patients had minimal hepatitis (-< A1F1), 61% had moderate to severe hepatitis (> A1F1, 400copies/ml) in I47/190 (77%) and 60/81 (74%) patients at 6 and 12 months, respectively. The incidence of polymerase variants was 7% and 27% at M6 and M12, respectively. Viral persistence without detection of polymerase variants in the dominant viral species as well as the progressive selection of the variants during treatment, suggest that emergence of these variants may be impeded by pre-existing wild type virus. In patients with genotypic lamivudine resistance, M5521 variant was observed in 11 cases, M552V variant in 2 cases, and the double variant L528M + M552V in 22 cases. This indicates that the double variant may have a higher replication capacityin vivo compared to the single C domain variant, and that the B domain mutation may represent a compensatory mutation for the C domain mutation. Conclusions: These results of the Lamivir cohort provide new epidemiological and clinical information concerning chronic hepatitisB in France, especially a higher prevalence of infection by pre-C(-) mutants in comparison with previous epidemiological data. In the setting of routine clinical practice, the interim analysis of incidence of polymerase variants in the YMDD motif in France after 1 year of Iamivudine therapy (100rag daily) is in the range of previous phase III studies. These results also provide new insight in the in vivo dynamic of replication of these variants.

Chronic hepatitis B virus (HBV) infection is characterised by a T helper 2 (TH2) predominance and it was shown recently that IFN-gamma derived by hepatic infiltrating peripheral ceils is capable to eradicate HBV from the liver by non-cytopathic mechanisms. Interleukin- 18 (IL- 18), a proinflammatory cytokine produced in monocytic ceils potentially induces IFN-gamma and shares further immune functions with IL-12. Therapy with lamivudineleads to effective suppression of HBV replication and reconstitutes the function of HBVspecific T-cells in patients with chronic HBV infection. The aim of this study was to investigate whether lamivudine influences in vitro synthesis of IL-18 and IFN-gamma in these patients. We therefore challenged PBMC of patients with chronic HBV infection either with unspecific (SAC/LPS) or specific (HBeAg/HBcAg) stimuliin vitro. 15 controls and 15 patients with chronic HBV (HBeAg+/HBV-DNA+, antiHDV-, antiHIV-) were included into the study. 11 patients receiving therapy with 100 mg lamivudine daily were screened longitudinally before (To) and after 48-52 weeks of therapy (TI). Isolated PBMC (3x106 cells/ml) were stimulated with either HBeAg or HBcAg (1/a.g/ml each) or SAC (10 -3 vol/vol)/LPS (1/zg/ml) for 22 hours. IL-18 and IFN-gamma were determined by ELISA's using different monoclonal antibodies. Bacterial incubation (SAC) of PBMC from patients with HBV infection resulted in a IL-18 production comparable to healthy controls at To and T1 demonstrating no substantial defect for this cytokine. Remarkably, after 12 months of lamivudine treatment IL-18 synthesis after SAC but not after LPS stimulation was signifcantly increased compared to To (p < 0.05). HBcAg induced significant increased levels of IFN-gamma but not of IL-18 in HBV infected patients compared to healthy controls (p < 0.01). Although lamivudine therapy did not result in significant differences of HBcAg-specific IL-18 and 1FN-gamma synthesis between To and TI, correlation between both was substantial and significant (r = 0.748, p < 0.01). In contrast to HBcAg, HBeAg-specific IFN-gamma synthesis was comparable to healthy controls at To, while IL-18 synthesis was significantly impaired (p < 0.05). In 10/11 patients lamivudine was capable to alter both HBeAg-specific synthesis of IL-18 (p < 0.05) and IFN-gamma (p < 0.01). The current study demonstrates that lamivudine in patients with chronic HBV infection is capable to alter HBeAg-specific 1L-18 and IFN-gamma response in vitro in patients with chronic HBV infection. Whether these mechanisms are involved in HBe/anti-HBe seroconversion and sustained response towards HBV, remain aims of further studies.

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I N VITRO STUDIES ON THE MECHANISM OF ACTION OF L-NUCLEOSIDE INHIBITORS OF HEPATITIS B VIRUS REPLICATION REVEAL DIFFERENCES BETWEEN LDT AND LAMIVUDINE, Maria Seller, Feng Zhou,

ETHNIC DIFFERENCES IN PATIENTS W I T H HEPATITIS C IN AN URBAN UNIVERSITY CLINIC. Maurizio Bonacini, Anne Ce|ona, Timothy Kuo, Manish Prakash, Keck School of Medicine, Los Angeles, CA

Novirio Pharmaceuticals, Cambridge, MA; Abdesslem Faraj, David Dukhan, Gilles Gosselin, Jean-Louis Imbach, Claire Pierra, Samira Benzaria, Lab Coop4ratif Novirio, CNRS, Univ Montpellier II, Montpellier France; Anna G Loi, Novirio Pharmaceuticals SARL, Montpellier France; Paolo La Colla, Universita di Cagliari, Cagliari Italy; Jean-Pierre Somrnadossi, Martin Bryant, David N Standring, Novirio Pharmaceuticals, Cambridge, MA The L-nucleosides LdT and LdC have proven to be extremely specific and selective antiviral agents in vitro and have exhibited an exceptional safety profile in animals. LdT and LdC are invesdgational new drugs for the treatment of chronic hepatitis B virus (HBV) infection. LdT is currently being evaluated in a phase IYII dose-escalation clinical trial in HBV infected patients with oral drug administration at doses of between 25 and 400 rag. In that trial, LdT exhibited potent antiviraI activity, causing HBV viral titers to drop 3 logs or mere after 28 days of drug treatment in most tested doses. These L-nucleosides have been shown to target the HBV viral polymerase. Using a panel of hepadnaviral cell-based and enzymatic assays, LdT and LdC, despite their very close structural similarities, showed unexpected differences in their mode of action, and LdT also differed in its mechanism of action compared with lamivudine. Of note, LdT (along with the closely related molecule LdA) exerted a preferential effect on HBV second strand (DNA dependent) DNA synthesis compared to LdC or lamivudine, both of which strongly inhibited first strand (RNA dependent) DNA synthesis. Examination of the unique hepadnaviral priming reaction showed that the triphosphates of LdT, LdC and lamivudine did not significantly inhibit priming at concentrations greater than 100 mieromolar; interestingly, however, LdGTP and LdATP inhibited priming effectively (IC50s < 1 0 micromolar). These types of mechanistic variations may have implications for choosing which nudeosides to use in an optimal combination therapy approach, or for suppressing the emergence of drug-resistant HBV variants during the course of therapy.

The aim was to address ethnic differences in patients with antibodies to hepatitis C attending an urban outpatient Hepatitis Clinic. METHODS: We surveyed the clinic charts of patients seen from 1992 to the present, for demographics and laboratory data. RESULTS: 1271 patients were categorized into 4 major ethnic groups~ 95 patients were Asian (A), 232 African-American (AA), 323 Caucasian (C), 621 Latino (L). Latinos and AA were significantly overrepresented compared to LA County as a whole(p<0.0001). The A group was significantly older than all other groups (p= 0.0001) and were more commonly coinfected with HBV than AA or L. Caucasians were more likely to be HBV+ than L (p=0.02). The AA group had a lower percentage of female patients than either A (p<0.0001), C (p=0.0003), or L (p=0.01)(Table). Asian (26%) and Latino patients (24%) were significantly more likely to report transfusion as a risk factor than AA (11%) or C (11%). In all groups except C, men reported IDU more frequently than women. The L group had higher bilirubin and lower albumin levels compared to AA and C (p<0.01), and higher ALT levels compared to AA (p=0.02). CONCLUSIONS: 1: AA women are significantly under represented in our cohort, 2: Asians were more likely to have HBV coinfection than AA and L, and C were more likely to be HBV coinfected than L, 3: Significant differences existed in the reporting of risk factors for HCV, and 4: L had the lowest albumin levels and the highest bilirubin and ALT levels. Whether these differences represent referral biases or true biological differences related to race-ethnicity will require prospective evaluation. Variable % of group Age (mean) Female (%) Monthsf-up ALT < 40 lUlL MeanALT MeanTB MeanAlbumin % HBsAg÷re

95 Asian 7% 54.5 48 23.1 5% 1t0 0,9 4.1 11%

232 AA 18% 45.7 25 12.1 19% 118 0.9 4,1 4%

323 Cauc 25% 41.8 39 13.5 22% 144 0.9 4.1 6%

621 Latiao 49% 44.7 35 14.0 11% 166 1,3 4.2 2%