HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 6 3 3 A

1.843

I N H I B I T I O N O F I N O S I N E M O N O P H O S P t t A T E D E H Y D R O G E N A S E (IM- P D H ) A S A T H E R A P E U T I C A P P R O A C H F O R H E P A T I T I S B. Robert Hamatake, Lauro Minimo, J o h n s o n Y Lau, Anneke Raney, ICN Pharmaceuti- cals, Costa Mesa, CA

Background: Ribavirin is a b road spec t rum antiviral nucleoside analog. It is current ly approved for t reatment against respiratory syncytial virus and, in combinat ion wi th interferon-c~, against hepatitis C virus. Ribavirin affects the Th l /Th2 balance of the host immune system in favor of an antiviral response. Ribavirin monophospha te is also k n o w n to inhibit host IMPDH. Aim: To determine the potential of inhibit ion of IMPDH as a therapeutic approach to hepatitis B virus (HBV) based on an in vitro cell-based model. Methods: Riba- virin, levovirin (L-sugar of ribavirin k n o w n not to undergo phosphorylat ion) , mycophenol ic acid (MPA, k n o w n to inhibit IMPDH), t iazofurin (another IM- PDH inhibitor) , and 3TC (positive control) were tested at different concentra- tions (ranging from 0.01-500 uM) against HBV replication using the HepG2 2.2.15 cell line. Cells were treated wi th c o m p o u n d for 10 days wi th media changes every 2 days. Product ion of HBV particles was measured in media supernatants after 8 and 10 days of t reatment by Southern blot analysis. Rep- lication intermediates were isolated from cell lysates after 10 days of t reatment and analyzed by Southern blots. The amounts of HBV DNA produced dur ing treatment were quanti tated by phosphor imager analysis and compared to the no t reatment controls. Results: Ribavirin has no detectable anti-HBV activity. At concentra t ions above 5 uM, there was a dose-dependent increase in HBV replicative intermediates up to 40 uM (200% of control) with decreasing levels at higher concentrat ions, coinciding with a CC50 of 75 uM. MPA also showed some proviral effect at 1 uM (125% of control) but the amoun t of replicative intermediates d ropped wi th concentra t ion above 3 uM, not ing that the CC50 was 8 uM. Tiazofurin also showed a similar pa t te rn as ribavirin with a proviral effect (150% of control) from 12-125 uM which dropped again with higher concentrat ion, wi th CC50 being 40 uM. Levovirin has no antiviral/proviral effect or toxicity and 3TC was the positive control wi th IC50 being 0.03 uM. Conclusions: ( i ) Inhibit ion of IMPDH is not a good therapeutic approach to inhibit HBV replication. (2) At concentrat ions close to toxic levels, inhibit ion of IMPDH may allow HBV replication to occur at a h igher level.

1 8 4 4

N A T U R A L H I S T O R Y OF EXTENDED LAMIVUDINE (LAM) T R E A T M E N T IN P A T I E N T S W I T H H B E A G N E G A T I V E C H R O N I C H E P A T I T I S B (CHB). Nicholas C Tassopoulos, Western Attica General Hospital, Athens Greece; Johanna K Delladetsima, Laikon General Hospital, Athens Greece; George D Anagnostopoulos , Western Attica General Hospital, Athens Greece

Introduction: One year LAM treatment in patients (pts) with HBeAg (-) CHB resulted in complete response in 65% of pta. Analysis of 3-year therapy with histological evaluation of CHB are now reported here. Methods: 35 patients (29 M, 6 F) with a median age of 54 years were followed up for 39±1I(12-54) months. Complete response was defined as ALT normalization and serum HB- VDNA negativity (HBVDNA < 2.5 pg/ml by bDNA). Histological improvement was also defined as a 2 point reduction in the necroinflammatory activity (N1A) (endpoim of treatment in this setting of pts) at 24 months after initiation of LAM treatment. Breakthrough was defined as ALT and HBVDNA flares. Results: At baseline, median ALT and HBVDNA were 118 IU/L and 133.8 pg/ml respectively. Liver histology showed mild in 7 (20%), moderate in 27 (77%) and severe CH in i (3°/0) pts. Cirhosia was found in (43%) pts. By Kaplan-Meier the cumulative probability of com- plete response to LAM progressively increased (74.3% at 12-too, 82.9% at 24-mo) and reached 86% at 30 too. 19 (54%) pts showed breakthrough and developed YMDD variant during the 39-mo of LAM therapy. The cumulative probability of YMMD variant was 10% at 12-too and 37% at 24-mo. Of the 19 pts, all (100%) had the 552 and 9 (47%) had also the 528 mutation ; in 3 (16°/0) of the pts ALT normalized and HBVDNA became negative during LAM therapy. At month 24, liver tissue specimens (ranked assessment) in 31 pts showed improvement of NIA score in 11 (73%) of 15 pts without and 7 (44%) of 16 pts with YMDD-variant. Development of YMDD variant was dependent only to Body Mass Index (BMI) and was significantly more frequent in patient with BMI>25 than in pts < 25 (17/23 or 73,9% vs 2/12 or 16,7%, p<0.001 ). A third liver biopsy in 10 pts with YMDD, 8-26 months later showed improvement or unchanged NIA in 45% (5 of 11) pts. 2 pts died of liver failure 34-46 rot, 2 pts with YMDD variant developed HCC and t pt had EVhemorrhage. Thus, 5 (14%) pts developed complications of end stage liver disease 12-46 mo during LAM treatment. The median HBVDNA is shown in the following figure. Conclusions : 1. The probability of complete response seems to be progressively increasing and reached to 86% at 30 rot. 2. A 3-year treatment seems to be necessary for complete response. 3.54% of the pts developed YMDD variants during the 39-mo of LAM treatment ; despite the appearance of YMDD variants the pts continue to derive benefit from LAM. 4. A 2-year treatment is necessary for histological improvement.

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1845

I N T E R F E R O N T R E A T M E N T I N C H I L D R E N W I T H C H R O N I C H E P A T I - TIS B. 5MU/M 2 VS. 10MU/M 2. Hanoch Hager, Pediatric Gastroenterology Unit HaEmek Medical Center, Afula Israel; Corina Har tman, Drora Berkowitz, Rambam Medical Center, Bruce Rappapor t school of medicine, Haifa Israel; Philippe Trougouboff, Depar tment of Pathology HaEmek Medical center, Afula Israel; Nurit Rimon, Laboratory of Virology, Rambam Medical Center, Haifa Israel; Raanan Shamir, Rambam medical Center, bruce Rappaport School Of Medicine, Haifa Israel

Background and aims: Interferon (INF) is the single therapeutic agent ap- proved for the t reatment of chronic hepatitis B in children. Overall, 10-40 % of chi ldren achieve complete response. However the dose and dura t ion of treat- ment remain to be established. Data on long-term outcome of INF treatment are scarce. The a im of this s tudy was to compare the efficacy, safety~ and long-term outcome of chi ldren with chronic hepatitis B treated with two In- terferon regimens at two Pediatric Gastroenterology centers from Nor thern Israel. Patients and Methods: Charts of 23 children, wi th biopsy proven chronic hepatitis B virus infection, were retrospectively reviewed. All patients were positive for hepatitis B virus DNA (HBV DNA). Transaminases levels were above twice the uppe r limit of normal in all children. Patients were given 1NF 10MU/M 2 ( n = 12), or 5MU/M 2 (n = 11) of body surface area administered subcutaneous , three times a week for 24 weeks. The chi ldren were followed month ly dur ing INF therapy and every 3 to 6 months thereafter for an average follow up of 3.9 years (range 0.5-8). Results: A sustained complete response (normal enzymes, negative HbeAg and HBV DNA at 12 months after the end of INF) occurred in 9/12 chi ldren (75%) treated with 10MU/m2 thrice a week as compared to 3/11 (27%) in the g roup given 5MU/m2 thrice a week (p =0.0315) . Long term follow up (4 years), demonst ra ted similar seroconver- sion rate in the 10MU and 5MU INF treatment groups (83% vs. 72%). Hepatitis B surface antigen clearance occurred in 2 chi ldren (20%), both of them from the 10MU/m2 group. Minor side effects were present in almost all chi ldren in both groups, but more serious adverse effects occurred only in the high dose group. All the side effects were transient. Conclusions: In this small cohor t of children, 10MU/m2 INF treatment achieves higher shor t - term complete re- sponse rate compared to 5MU/m2. However, after long-term follow-up there are no differences in response rates between the two regimens.

1846

I M M U N O T H E R A P Y O F C H R O N I C H E P A T I T I S B BY H E P A T I T I S B VAC- CINE: I N D U C T I O N OF T CELL P R O L I F E R A T I V E R E S P O N S E SPECIFIC FOR E N V E L O P E P R O T E I N S A N D P R E D O M I N A N T T H 1 P A T H W A Y . Fe- nyu Ren, Keisuke Hin t , Ynhki Yamaguchi, Yamaguchi University, School of Medicine, Ube Japan; Satoyoshi Yamashita, Keiko Korenaga, Shimonoseki Kohsei Hospital, ShimonosekiJapan; Tomomi Konishi, Shuhtoh General Hos- pital, YanaiJapan; Muneko Okazaki, Masaaki Korenaga, Yamaguchi Univer- sity, School of Medicine, Ube Japan; Hiroaki Ishiko, Mitsubishi Kagaku Bit- Clinical Laboratories, Tokyo Japan; Kiwamu Okita, Yamaguchi University, School of Medicine, Ube Japan

Background/Aim: Specific immunotherapeutic strategies have been proposed as pos- sible alternatives to interferon (IFN) or antiviral drugs to enhance or broaden the defective T cell responses in chronically hepatitis B virus (HBV) infected patients. The aim of this study is to prospectively investigate whether specific T cell responses are induced and whether Thl/Th2 cytokine balance is changed in vaccine therapy in patients with chronic hepatitis B. Methods: We enrolled 14 patients with biopsy- proven chronic hepatitis B in this study. All patients were positive for HBV DNA in serum and had an elevated alanine aminotransferase (ALT) level, except for one. The hepatitis B vaccine (MEI-NYU) used here is derived from the human hepatoma cell line, huGK-14, containing integrated HBV DNAs and contains both HBsAg and a very small quantity ofpreS2 antigen secreted from these cells. A 10/zg intramuscular dose of vaccine was given at 1-month intervals for 6 months. Peripheral blood mononuclear cells (PBMCs) were separated from heparinized blood by density gra- dient cemrifugation before vaccination, 3, 6, 9, 12 and 24 months after inclusion. T cells were immunomagnetically isolated from PBMCs, cultured for 72 hours in the presence or absence of HBsAg (0.3-3 /zg/mL) or preS2 synthetic peptides (1.0-3.0 /zg/mL) (generous gift from Meiji Cell Technology Center), and subjected to prolif- eration assay using [3H]thymidine. Culture supernatants were collected and con- centrations of IL-2, IFN-3', TNF-a, IL-4 and II_-10 were determined by ELISA kits. Results: Currently, 10 patients have completed the vaccination and one patient discontinued because of elevated ALT level. A significant T cell proliferation (SI-->3) specific for both HBsAg andpreS2 peptide was detected 3 to 9 months after inclusion in 4 patients who completetivaccination. This proliferative response was inhibited by anti-CD4 + antibody or immunomagnetical depletion of CD4+cells. Also a signif- icant increase in IFN-?and TNF-~production was detected in the presence of HBsAg or preS2 peptide in T cells from patients who showed proliferative response, whereas IL-4 or IL-10 production did not change. Both Thl and Th2 cytokines production did not change in T cells from patients who lacked detectable proliferative response. Conclusions: These results suggest that vaccine therapy enables some patients to restore CD4 + T cell responsiveness towards HBsAg or preS2 antigen and to induce a predominant Thl pathway in chronic hepatitis B. As we are constructing the autol- ogous EBV-transformed B-LCL expressing HBsAg for cytotoxity assay of cytotmoxic T Iymphocytes (CTL), whether HBsAg specific CTL can be induced in vaccine therapy is also reported.