ANTIVIRAL AGENTS IN HEPATITIS-B VIRUS TRANSFECTED CELL-LINES - INHIBITORY AND CYTOTOXIC EFFECT RELATED TO TIME OF TREATMENT

The antiviral and cytotoxic effects of ara-arabinoside monophosphate, 2',3', dideoxy-cytidine, ganciclovir, 9-2(-phosphonylmethoxyethyl) adenine, 2',3'-dideoxy-3'-thiacytidine and recombinant interferon-alpha were studied using two human hepatitis B virus transfected hepatoma cell lines, HepG2 2.2.15 and HE 611. After 9 days of exposure, starting on day 3 after seeding, inhibition of extracellular HBV-DNA expressed as ID50 was in the 0.1-1.0 mu M range for 2',3'-dideoxy-3'-thiacytidine and 9-2 (-phosphonyhmethoxyethyl) adenine and >10 mu M for dideoxy-cytidine, ara-arabinoside monophosphate and ganciclovir in both cell lines. At 2.500 U/ml recombinant interferon-alpha showed less than 20% inhibition in both cell lines. The HBV-DNA inhibitory effects of 2',3'-dideoxy-3'-thiacytidine and 9-2(-phosphonylmethoxyethyl) adenine were also investigated after 1 and 3 days of exposure. In that setting ID50's were 10 and 3.3 mu M for 2',3'-dideoxy-3'-thiacytidine and >100 and 30 mu M for 9-2(-phosphonylmethoxyethyl) adenine, respectively. No major inhibitory effect on hepatitis B surface antigen and hepatitis B e antigen secretion was observed for any agent in this study, except for 9-2(-phosphonylmethoxyethyl) adenine in HE 611 cells. Cytotoxicity measured by inhibition of [H-3-methyl] deoxythymidine incorporation and expressed as CD50 on day 4 was in the 10-100 mu M range for ara-arabinoside monophosphate; in the 100-1000 mu M range for 9-2(-phosphonylmethoxyethyl) adenine, ganciclovir and dideoxy-cytidine; and >1000 mu M for 2',3'-dideoxy-3'-thiacytidine. This CD50 decreased considerably (7-100 fold) when measured on day 12 for dideoxy-cytidine, ganciclovir, 9-2(-phosphonylmethoxyethyl) adenine and 2',3'-dideoxy-3'-thiacytidine, but was similar for ara-arabinoside monophosphate. Since the order of antiviral HBV activity and cytotoxicity of nucleoside analogues was similar in the two transfected hepatoma cell lines, we conclude that 2',3'-dideoxy-3'-thiacytidine and 9-2(-phosphonylmethoxyethyl) adenine are very potent inhibitors of HBV-DNA, with a longlasting effect. In view of the progressive toxicity with continuous administration, intermittent administration might be an alternative mode of therapy.