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Long-term efficacy of interferon alpha-2B and lamivudine in combination compared to lamivudine monotherapy in patients with chronic hepatitis B. An Italian multicenter, randomized trial
318A
AASLD ABSTRACTS
HEPATOLOGY October 2001
583
584
THERAPY W I T H CLEVUDINE FOLLOWED BY VACCINE DELAYS THE PROGRESSION OF WHV-INDUCED HEPATITIS AND HEPATOCELLULAR CARCINOMA IN CHRONICALLY-INFECTED WOODCHUCKS. Brent E Korba, Paul J Cote, John L Gerin, Georgetown University Medical Center, Rockville, MD; Stephan Menne, Ilia A Toshkov, Bud C Tennant, Cornell University, College of Veterinary Medicine, Ithaca, NY
ME3738 PROTECTS FROM CON A INDUCED LIVER FAILURE VIA AN IL6-DEPENDENT MECHANISM. Christian Klein, Michael P Manns, Christian Trautwein, Medical school Hannover, Hannover Germany
Previous studies in the woodchuck model of HBV infection demonstrated that the nucleoside analogue Clevudine (CLV, L-FMAU) has potent and persistent antiviral activity in woodchucks chronicaUy-infected with the woodchuck hepatitis virus (WHV). However, these animals still harbor low levels of actively replicating virus and do not develop neutralizing antibody (anti-WHs). We sought to determine whether CLV treatment followed by conventional WHsAg vaccination was capable of establishing additional antiviral benefit by inducing cellular and humoral responses that are suppressed during chronic WHV infection. Groups of 8 chronic carrier woodchucks received (1) 10rag/ kg/day of CLV orally for 32 weeks followed by vaccine (alum-adsorbed, serumderived formalin-inactivated WHsAg) 50 ug/dose, s.c. at wks. 32, 36, 40 and 48, (2) CLV for 32 wks. and 4 saline injections, (3) placebo for 32 wks. and 4 doses of vaccine, and (4) placebo and 4 saline injections. CLV treatment induced remarkable (>107) reductions in viremia and antigenemia (102 to >.104), and hepatic WHV antigen expression (>102) within 12 weeks of drug treatment. Viral markers remained suppressed upon drug withdrawal in 75% of animals in both the vaccinated and non-vaccinated groups. Virus markers were stable in both the control and vaccine only groups. Analysis of host immune response to vaccine demonstrated a broad response to multiple WHV antigens at both the cellular and humoral levels in the combined treatment group, which resembled that typical of recovery from acute infection, including the development of anti-WHs antibody that persisted for up to one year. Combined therapy significantly affected biochemical and histological markers of hepatitis progression and the development of HCC. Combined therapy resulted in a significant ( > 3 2 wks) delay in the development of HCC versus the control group. Portal hepatitis was observed in only 1 of 8 animals receiving the combined therapy over the 80 week study period (vs. all animals in the control group). This study demonstrates (1) the enhanced benefit of a combination of chemotherapeutic and immunotherapeutic approaches and provides a platform for further development of this type of treatment regimen, and (2) that cellular and humoral responses which are suppressed during chronic hepadnaviral infection can be recovered following therapy, leading to delays in the progression of viral-induced disease.
Chronic infection with the hepatitis B and C virus induce inflammatory and necrotic changes in the liver and are one of the main causes to trigger liver cirrhosis. The actual treatment options aim to clear the virus. However antiviral therapy is not always successful and therefore treatment options to block disease progression are attractive. ME3738 (Meiji-Seika Company) is a new compound that attenuates liver disease in several models of acute and chronic liver inflammation. Here we used the Concanavalin A (Con A) model to elucidate the molecular mechanisms of ME3738 to block liver cell damage. Pretreatment of Balb C mice with 40mg/kg ME3738 2 and 14 hours before Con A injection results in a significant reduction in liver cell damage compared to animals treated with the carrier control alone. The protective effect of ME3738 before Con A injection was associated with a reduction in IL6 serum levels and NF-kB DNA binding in liver nuclear extracts. However no major impact was observed on STAT3 activation. As ME3738 had a direct effect on IL6 serum levels after Con A injection we used IL6 knock out mice to further elucidate the effect of ME3738 on Con A induced liver damage. No significant difference in the sensitivity of wt and IL6 knock out mice was evident after Con A injection. In contrast, while ME3738 was protective in wt animals no protection was found in IL6 knockout animals indicating that IL6-dependent pathways are involved in the protective effect of ME3738. Therefore we studied the possibility that ME3738 has an impact on IL6 expression. Directly after ME3738 stimulation there was an increase in IL6 serum levels and maximal expression was found 3 hours after injection in wt mice. This effect was not observed in animals treated with the carrier solution alone. In ME3738 treated wt animals higher IL6 serum levels resulted in an increase concentration of phosphorylated nuclear STAT3 in the liver and STAT3-DNA complex formation as evidenced by gel shift experiments. A dramatic increase in mRNA expression of acute phase genes (haptoglobin, hemopexin and SAA) in the liver was found after ME3738 stimulation. For example the concentration of SAA transcripts rises up to 1000 foId compared with the pretreatment level. In parallel these experiments were performed in IL6 knockout mice. In these animals there was no IL6 induction and the effect on STAT3 activation and the increase in acute phase gene expression was blunted. In summary, we describe ME3738 as a new liver protective compound. ME3738 protects from Con A induced liver cell damage via an IL6-dependent mechanism. Further clinical and basic research will aim to elucidate the exact molecular mechanism that is important to prevent disease progression by ME3738 and to evaluate its benefit in humans.
585
586
REBOUND OF HBV REPLICATION FOLLOWING WITHDRAWAL OF ANTIVIRAL TREATMENT. Ayman M Abdelhamed, Penn State College of Medicine, Hershey, PA; Phillip A Furman, Triangle Pharmaceuticals, Durham, NC; Harriet C Isom, Penn State College of Medicine, Hershey, PA
LONG-TERM EFFICACY OF INTERFERON ALPHA-2B AND LAMIVUDINE IN COMBINATION COMPARED TO LAMIVUDINE MONOTHERAPY IN PATIENTS W I T H CHRONIC HEPATITIS B. AN ITALIAN MULTICENTER,RANDOM1ZED TRIAL. Giorgio Barbarini, 1RCCS S Matteo Univ of Pavia Italy; Franco Zechini, Adriano Pellicelli, Lazzaro Spaflanzani Hosp, Rome Italy; Ruggiero Francavilla, General Hosp, Bisceglie Italy; Gaetano Scotto, Infectious Diseases, Foggia Italy; Donato Bacca, Gastroenterolocy Div, Casarano Italy; Marcello Bruno, Infectious Diseases, Guadagna Hosp, Palermo Italy; Sergio Babudieri, Univ of Sassari Italy; Filippo Matarazzo, Infectious Diseases, Formia Hosp Italy; Mauro Annese, Gastroenterology Svc, Matera Hosp Italy; Gabriele Di Stefano, Infectious Diseases, Latina Hosp Italy; Giuseppe Barbaro, Univ La Sapienza, Rome Italy
A number of nucleoside analogues and other drugs are in use in clinical trials or under evaluation at the research level as antivirals for HBV. Lamivudine is a nucleoside analogue that has been approved for treatment of HBV. Treatment of patients with lamivudine results in loss of detectable levels of HBV DNA from serum, but the relapse rate both with regard to reappearance of virns in the bloodstream arid hepatic inflammation is high when therapy is stopped. In this study, we used the HBV recombinant baculovirus/HepG2 system to measure the time course of antiviral mediated loss of HBV replication as well as the relative time course and magnitude of reappearance or rebound of HBV production after release from antiviral treatment. Because of the sensitivity of the system, it was possible to measure both disappearance and reappearance of virions (extracellular HBV DNA), intracellular replicative intermediates (RI), and nuclear non-protein bound HBV DNA. This system is particularly unique in that individual species of DNA such as single stranded (SS) HBV DNA compared to the double stranded (DS) form and relaxed circular (RC) compared to covalentiy closed circular (CCC) HBV DNA were able to be analyzed separately. To measure relapse, we first determined whether it was possible to use the HBV recombinant/HepG2 system to study HBV replication for a longer period of time than the 11 days initially published. We determined that when HepG2 ceils were infected at 100pfu recombinant baculovirus/cell, HBV replication reached a plateau by 4 to 6 days post infection that was maintained for at least 30 days post infection. HBV extracellular DNA, RI, and nuclear CCC DNA were readily detectable by Southern blot analysis of DNA isolated from cells 30 days post infection. To measure the effect of drug treatment followed by cessation of treatment, HepG2 ceils 5 days post infection with HBV recombinant baculovirus were treated with antiviral for 14 days. At 6 days after treatment, (11 days post infection), cells were washed free of drug and maintained drug free for an additional 8 days. Ceils were fed fresh medium daily with or without drug. Control cells, drug treated cells, and cells treated with drug and released were analyzed at various time points for HBV replication. Southern blots were analyzed using a phosphorimager system to generate quantitative data. We conclude the following from these studies. (1) HBV DNA species decrease when treated with antivirals in a time dependent fashion. (2) Extracellular HBV DNA and intracellular RI can be markedly reduced with time while CCC DNA remains highly resistant to drug therapy. (3) HBV DNA species rapidly reappear after drug treatment ceases. (4) Following removal of drug treatment, extracellular HBV DNA rapidly returns to the same level as in control untreated cultures. (5) With regard to the intracellular HBV RI, the SS RI returns more rapidly than the DS form. (6) Ahhough the nuclear HBV DNA is highly resistant to antiviral therapy, it is possible to measure differential rebound of the nuclear RC and CCC forms of HBV DNA. We conclude that the HBV recombinant baculovirus/HepG2 system lends itself to evaluating the efficacy of antivirals for therapeutic use including the potential for rebound that is observed in patients upon cessation of antiviral therapy.
AIMS: To evaluate the therapeutic efficacy of Interferon alpha-2b and lamivudine in combination compared to lamivudine monotherapy in patients with chronic hepatitis B HBeAg positive. METHODS: One hundred and fifty-one patients were randomly assigned to receive either recombinant Interferon alpha-2b(9 MU three times weekly)and lamivudine(100mg/daily p.o.)for 24 weeks or lamivudine alone(100mg/daily p.o.)for 52 weeks.Patients were followed for a further 48 weeks. RESULTS: Sustained HBeAg seroconversion with undetectable serum levels of HBVDNA was observed in 25 of 76 patients(33%)treated with the combination therapy and in 1 of 75 patients(15%)treated with monotherapy(p=0.014).Histological improvement defined as a reduction of at least 2 points in the inflammation score as compared with pretreatment score,was observed in 35 of 76 patients(46%)treated with combination therapy and in 20 of 75 patients(27%)treated with monotherapy(p= 0,021).Both therapeutic regimens were well tolerated. CONCLUSIONS: Six months treatment with Interferon alpha-2b and lamivudine in combination appeared to increase the rate of sustained HBeAg seroconversion compared to one-year lamivudine monotherapy.However,the potential benefit of combining lamivudine and Interferon should be investigated further in studies with different regimens of combination therapy.
AASLD ABSTRACTS
HEPATOLOGY October 2001
583
584
THERAPY W I T H CLEVUDINE FOLLOWED BY VACCINE DELAYS THE PROGRESSION OF WHV-INDUCED HEPATITIS AND HEPATOCELLULAR CARCINOMA IN CHRONICALLY-INFECTED WOODCHUCKS. Brent E Korba, Paul J Cote, John L Gerin, Georgetown University Medical Center, Rockville, MD; Stephan Menne, Ilia A Toshkov, Bud C Tennant, Cornell University, College of Veterinary Medicine, Ithaca, NY
ME3738 PROTECTS FROM CON A INDUCED LIVER FAILURE VIA AN IL6-DEPENDENT MECHANISM. Christian Klein, Michael P Manns, Christian Trautwein, Medical school Hannover, Hannover Germany
Previous studies in the woodchuck model of HBV infection demonstrated that the nucleoside analogue Clevudine (CLV, L-FMAU) has potent and persistent antiviral activity in woodchucks chronicaUy-infected with the woodchuck hepatitis virus (WHV). However, these animals still harbor low levels of actively replicating virus and do not develop neutralizing antibody (anti-WHs). We sought to determine whether CLV treatment followed by conventional WHsAg vaccination was capable of establishing additional antiviral benefit by inducing cellular and humoral responses that are suppressed during chronic WHV infection. Groups of 8 chronic carrier woodchucks received (1) 10rag/ kg/day of CLV orally for 32 weeks followed by vaccine (alum-adsorbed, serumderived formalin-inactivated WHsAg) 50 ug/dose, s.c. at wks. 32, 36, 40 and 48, (2) CLV for 32 wks. and 4 saline injections, (3) placebo for 32 wks. and 4 doses of vaccine, and (4) placebo and 4 saline injections. CLV treatment induced remarkable (>107) reductions in viremia and antigenemia (102 to >.104), and hepatic WHV antigen expression (>102) within 12 weeks of drug treatment. Viral markers remained suppressed upon drug withdrawal in 75% of animals in both the vaccinated and non-vaccinated groups. Virus markers were stable in both the control and vaccine only groups. Analysis of host immune response to vaccine demonstrated a broad response to multiple WHV antigens at both the cellular and humoral levels in the combined treatment group, which resembled that typical of recovery from acute infection, including the development of anti-WHs antibody that persisted for up to one year. Combined therapy significantly affected biochemical and histological markers of hepatitis progression and the development of HCC. Combined therapy resulted in a significant ( > 3 2 wks) delay in the development of HCC versus the control group. Portal hepatitis was observed in only 1 of 8 animals receiving the combined therapy over the 80 week study period (vs. all animals in the control group). This study demonstrates (1) the enhanced benefit of a combination of chemotherapeutic and immunotherapeutic approaches and provides a platform for further development of this type of treatment regimen, and (2) that cellular and humoral responses which are suppressed during chronic hepadnaviral infection can be recovered following therapy, leading to delays in the progression of viral-induced disease.
Chronic infection with the hepatitis B and C virus induce inflammatory and necrotic changes in the liver and are one of the main causes to trigger liver cirrhosis. The actual treatment options aim to clear the virus. However antiviral therapy is not always successful and therefore treatment options to block disease progression are attractive. ME3738 (Meiji-Seika Company) is a new compound that attenuates liver disease in several models of acute and chronic liver inflammation. Here we used the Concanavalin A (Con A) model to elucidate the molecular mechanisms of ME3738 to block liver cell damage. Pretreatment of Balb C mice with 40mg/kg ME3738 2 and 14 hours before Con A injection results in a significant reduction in liver cell damage compared to animals treated with the carrier control alone. The protective effect of ME3738 before Con A injection was associated with a reduction in IL6 serum levels and NF-kB DNA binding in liver nuclear extracts. However no major impact was observed on STAT3 activation. As ME3738 had a direct effect on IL6 serum levels after Con A injection we used IL6 knock out mice to further elucidate the effect of ME3738 on Con A induced liver damage. No significant difference in the sensitivity of wt and IL6 knock out mice was evident after Con A injection. In contrast, while ME3738 was protective in wt animals no protection was found in IL6 knockout animals indicating that IL6-dependent pathways are involved in the protective effect of ME3738. Therefore we studied the possibility that ME3738 has an impact on IL6 expression. Directly after ME3738 stimulation there was an increase in IL6 serum levels and maximal expression was found 3 hours after injection in wt mice. This effect was not observed in animals treated with the carrier solution alone. In ME3738 treated wt animals higher IL6 serum levels resulted in an increase concentration of phosphorylated nuclear STAT3 in the liver and STAT3-DNA complex formation as evidenced by gel shift experiments. A dramatic increase in mRNA expression of acute phase genes (haptoglobin, hemopexin and SAA) in the liver was found after ME3738 stimulation. For example the concentration of SAA transcripts rises up to 1000 foId compared with the pretreatment level. In parallel these experiments were performed in IL6 knockout mice. In these animals there was no IL6 induction and the effect on STAT3 activation and the increase in acute phase gene expression was blunted. In summary, we describe ME3738 as a new liver protective compound. ME3738 protects from Con A induced liver cell damage via an IL6-dependent mechanism. Further clinical and basic research will aim to elucidate the exact molecular mechanism that is important to prevent disease progression by ME3738 and to evaluate its benefit in humans.
585
586
REBOUND OF HBV REPLICATION FOLLOWING WITHDRAWAL OF ANTIVIRAL TREATMENT. Ayman M Abdelhamed, Penn State College of Medicine, Hershey, PA; Phillip A Furman, Triangle Pharmaceuticals, Durham, NC; Harriet C Isom, Penn State College of Medicine, Hershey, PA
LONG-TERM EFFICACY OF INTERFERON ALPHA-2B AND LAMIVUDINE IN COMBINATION COMPARED TO LAMIVUDINE MONOTHERAPY IN PATIENTS W I T H CHRONIC HEPATITIS B. AN ITALIAN MULTICENTER,RANDOM1ZED TRIAL. Giorgio Barbarini, 1RCCS S Matteo Univ of Pavia Italy; Franco Zechini, Adriano Pellicelli, Lazzaro Spaflanzani Hosp, Rome Italy; Ruggiero Francavilla, General Hosp, Bisceglie Italy; Gaetano Scotto, Infectious Diseases, Foggia Italy; Donato Bacca, Gastroenterolocy Div, Casarano Italy; Marcello Bruno, Infectious Diseases, Guadagna Hosp, Palermo Italy; Sergio Babudieri, Univ of Sassari Italy; Filippo Matarazzo, Infectious Diseases, Formia Hosp Italy; Mauro Annese, Gastroenterology Svc, Matera Hosp Italy; Gabriele Di Stefano, Infectious Diseases, Latina Hosp Italy; Giuseppe Barbaro, Univ La Sapienza, Rome Italy
A number of nucleoside analogues and other drugs are in use in clinical trials or under evaluation at the research level as antivirals for HBV. Lamivudine is a nucleoside analogue that has been approved for treatment of HBV. Treatment of patients with lamivudine results in loss of detectable levels of HBV DNA from serum, but the relapse rate both with regard to reappearance of virns in the bloodstream arid hepatic inflammation is high when therapy is stopped. In this study, we used the HBV recombinant baculovirus/HepG2 system to measure the time course of antiviral mediated loss of HBV replication as well as the relative time course and magnitude of reappearance or rebound of HBV production after release from antiviral treatment. Because of the sensitivity of the system, it was possible to measure both disappearance and reappearance of virions (extracellular HBV DNA), intracellular replicative intermediates (RI), and nuclear non-protein bound HBV DNA. This system is particularly unique in that individual species of DNA such as single stranded (SS) HBV DNA compared to the double stranded (DS) form and relaxed circular (RC) compared to covalentiy closed circular (CCC) HBV DNA were able to be analyzed separately. To measure relapse, we first determined whether it was possible to use the HBV recombinant/HepG2 system to study HBV replication for a longer period of time than the 11 days initially published. We determined that when HepG2 ceils were infected at 100pfu recombinant baculovirus/cell, HBV replication reached a plateau by 4 to 6 days post infection that was maintained for at least 30 days post infection. HBV extracellular DNA, RI, and nuclear CCC DNA were readily detectable by Southern blot analysis of DNA isolated from cells 30 days post infection. To measure the effect of drug treatment followed by cessation of treatment, HepG2 ceils 5 days post infection with HBV recombinant baculovirus were treated with antiviral for 14 days. At 6 days after treatment, (11 days post infection), cells were washed free of drug and maintained drug free for an additional 8 days. Ceils were fed fresh medium daily with or without drug. Control cells, drug treated cells, and cells treated with drug and released were analyzed at various time points for HBV replication. Southern blots were analyzed using a phosphorimager system to generate quantitative data. We conclude the following from these studies. (1) HBV DNA species decrease when treated with antivirals in a time dependent fashion. (2) Extracellular HBV DNA and intracellular RI can be markedly reduced with time while CCC DNA remains highly resistant to drug therapy. (3) HBV DNA species rapidly reappear after drug treatment ceases. (4) Following removal of drug treatment, extracellular HBV DNA rapidly returns to the same level as in control untreated cultures. (5) With regard to the intracellular HBV RI, the SS RI returns more rapidly than the DS form. (6) Ahhough the nuclear HBV DNA is highly resistant to antiviral therapy, it is possible to measure differential rebound of the nuclear RC and CCC forms of HBV DNA. We conclude that the HBV recombinant baculovirus/HepG2 system lends itself to evaluating the efficacy of antivirals for therapeutic use including the potential for rebound that is observed in patients upon cessation of antiviral therapy.
AIMS: To evaluate the therapeutic efficacy of Interferon alpha-2b and lamivudine in combination compared to lamivudine monotherapy in patients with chronic hepatitis B HBeAg positive. METHODS: One hundred and fifty-one patients were randomly assigned to receive either recombinant Interferon alpha-2b(9 MU three times weekly)and lamivudine(100mg/daily p.o.)for 24 weeks or lamivudine alone(100mg/daily p.o.)for 52 weeks.Patients were followed for a further 48 weeks. RESULTS: Sustained HBeAg seroconversion with undetectable serum levels of HBVDNA was observed in 25 of 76 patients(33%)treated with the combination therapy and in 1 of 75 patients(15%)treated with monotherapy(p=0.014).Histological improvement defined as a reduction of at least 2 points in the inflammation score as compared with pretreatment score,was observed in 35 of 76 patients(46%)treated with combination therapy and in 20 of 75 patients(27%)treated with monotherapy(p= 0,021).Both therapeutic regimens were well tolerated. CONCLUSIONS: Six months treatment with Interferon alpha-2b and lamivudine in combination appeared to increase the rate of sustained HBeAg seroconversion compared to one-year lamivudine monotherapy.However,the potential benefit of combining lamivudine and Interferon should be investigated further in studies with different regimens of combination therapy.