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 HB 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 μM range for 2′,3′-dideoxy-3′-thiacytidine and 9-2(-phosphonylmethoxyethyl) adenine and>10 μM 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 ID50ʹs were 10 and 3.3 μM for 2′,3′-dideoxy-3′-thiacytidine and>100 and 30 μM 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 HB 611 cells. Cytotoxicity measured by inhibition of [3H-methyl] deoxythymidine incorporation and expressed as CD50 on day 4 was in the 10–100 μM range for ara-arabinoside monophosphate; in the 100–1000 μM range for 9-2(-phosphonylmethoxyethyl) adenine, ganciclovir and dideoxy-cytidine; and>1000 μM 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 long-lasting effect. In view of the progressive toxicity with continuous administration, intermittent administration might be an alternative mode of therapy.