IN VITRO TESTING FOR AGENTS THAT INHIBIT HBV REPLICATION

HEPATITIS B VIRUS (HBV) IS A MAJOR HUMAN PATHOGEN WHICH INFECTS APPROXIMATELY 200 MILLION INDIVIDUALS WORLDWIDE AND CAUSES PROGRESSIVE LIVER DAMAGE, CIRRHOSIS AND PRIMARY HEPATOCELLULAR CARCINOMA. VACCINATION OFFERS A MEANS FOR DISEASE PREVENTION BUT THERE REMAINS AN IMMEDIATE NEED TO TREAT THOSE WHO ARE CHRONICALLY INFECTED. THE IDENTIFICATION OF NEW ANTIVIRAL AGENTS TO TREAT HBV-INFECTEDINDIVIDUALS HAS BEEN HAMPERED BY HIGH COST AND INJURIOUS DRUG TOXICITY ENTAILED WITH CURRENT IN VIVO TESTING PROTOCOLS. THE GOAL OF THE PROPOSED RESEARCH IS TO EXAMINE IN VITRO ASSAYS WHICH COULD BE USED TO EVALUATE COMPOUNDS WHICH MAY BLOCK HBV REPLICATION. WE HAVE RECENTLY DESCRIBED THE ISOLATION OF A HUMAN HEPATOBLASTOMA CELL LINE WHICH CONSTITUTIVELY PRODUCES HBV IN CONFLUENT CULTURES. ASSAYS WHICH MEASURE THE CAPACITY OFAGENTS TO INHIBIT HBV PRODUCTION IN THESE CELLS MAY PROVE VALUABLE FOR RAPID SCREENING OF POTENTIAL ANTI-HBV COMPOUNDS. IN PHASE I, THREE TYPES OF ASSAYS WILL BE EVALUATED FOR THEIR ABILITY TO ASSESS INHIBITION OF HBV PRODUCTION AND SECRETION BY QUANTITATION OF (1) HBV DNA IN VIRIONS, (2) HBV VIRAL POLYMERASE ACTIVITY, AND (3) TWO HBV PROTEINS (HBSAG AND HBEAG). EACH ASSAY WILL BE EXAMINED FOR EASE OF USE AND EFFECTIVENESS USING THREE TEST DRUGS (AZT, ACYCLOVIR, AND ARA-A) THAT HAVE BEEN SHOWN TO INHIBIT HBV PRODUCTION. IN PHASE II, THE ASSAY(S) AND CONDITIONS DETERMINED TO BE OPTIMAL FOR IDENTIFICATION OF INHIBITORS OF HBV PRODUCTION IN THE PHASE I STUDIES WILL BE USED TO SCREEN THE EFFECTIVENESS OF POTENTIAL AGENTS FOR TREATMENT OF CHRONIC HEPATITIS B.