Characterization of hepatitis B virus inhibition by novel 2'-fluoro-2',3'-unsaturated beta-D- and L-nucleosides.

The clinical emergence of lamivudine and adefovir resistance mutations on prolonged therapy further necessitates the development of additional drugs for the treatment of hepatitis B virus (HBV) infections. We have evaluated a number of novel 2'-fluoro-2',3'-unsaturated D- and L-nucleosides for their anti-HBV activity in the HepG2-2.2.15 cell system. The most potent nucleosides were beta-L-2'-fluoro-2',3'-dideoxy-2',3'-didehydrocy-tidine (L-2'-Fd4C) and beta-L-2'-fluoro-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (L-2'-Fd4FC) with median effective concentrations (EC50) of 0.002 microM and 0.004 microM, respectively. The D-enantiomers of the 2'-fluoro-substituted cytidine analogues in this series showed activity, with the 5-fluorocytidine (D-2'-Fd4FC) being the most potent (EC50 = 0.05 microM). The active compounds were not cytotoxic to a number of cell lines or to bone marrow progenitor cells. Furthermore, mitochondrial DNA synthesis and function were not affected by these nucleosides. L-2'-Fd4C did not affect viral transcription, implying that it does not inhibit cellular RNA polymerase II. Studies with the HBV polymerase in core particles revealed that the 5'-triphosphates of L-2'-Fd4C and D-2'-Fd4FC produced a dose-dependent inhibition of the incorporation of 32P-dCTP into the HBV DNA, indicating that the mechanism of action of these compounds is through specific inhibition of viral DNA synthesis. This class of nucleosides, which exhibit potent antiviral activity and a favourable safety profile, have potential for the treatment of HBV infections and warrant further development.

Emergence of a novel mutation in the FLLA region of hepatitis B virus during lamivudine therapy.

The emergence of resistance to lamivudine has been one of the major stumbling blocks to successful treatment and control of hepatitis B virus (HBV) infections. The major mechanism of resistance has been attributed to the alteration in the YMDD motif of the HBV polymerase due to an amino acid change of rtM204 to V/I and an accompanying rtL180M conversion. A novel mutation pattern in a patient having clinical breakthrough under lamivudine therapy was discovered. The mutant had a rtL180C/M204I genotype and was detected after 2 years of therapy with lamivudine. To characterize this novel variant, site-directed mutagenesis was performed using a vector construct containing the HBV genome. Transient transfection studies in human hepatoma cells with HBV carrying the new mutant demonstrated that the rtL180C/M204I mutant was resistant to lamivudine up to 10 microM. The resistance profile was comparable to that of the previously reported rtL180 M/M204I-containing virus. These observations were further confirmed by generation of stable cultures transfected with the mutant virus.