3--fluoro-2--3--dideoxyguanosine and Carcinoma--Hepatocellular

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) was shown to inhibit wild-type (wt) hepatitis B virus (HBV) replication in a human hepatoma cell line permanently expressing HBV. Experiments performed in the duck model of HBV infection also showed its in vivo antiviral activity. In this study, we investigated the mechanism of inhibition of FLG on HBV replication and its profile of antiviral activity against different HBV or duck hepatitis B virus (DHBV) drug-resistant mutants. We found that FLG-triphosphate inhibits weakly the priming of the reverse transcription compared to adefovir-diphosphate in a cell-free system assay allowing the expression of an enzymatically active DHBV reverse transcriptase. It inhibits more potently wt DHBV minus-strand DNA synthesis compared to lamivudine-triphosphate and shows a similar activity compared to adefovir-diphosphate. FLG-triphosphate was most likely a competitive inhibitor of dGTP incorporation and a DNA chain terminator. In Huh7 cells transiently transfected with different HBV constructs, FLG inhibited similarly the replication of wt, lamivudine-resistant, adefovir-resistant, and lamivudine-plus-adefovir-resistant HBV mutants. These results were consistent with those obtained in the DHBV polymerase assay using the same drug-resistant polymerase mutants. In conclusion, our data provide new insights in the mechanism of action of FLG-triphosphate on HBV replication and demonstrate its inhibitory activity on drug-resistant mutant reverse transcriptases in vitro. Furthermore, our results provide the rationale for further clinical evaluation of FLG in the treatment of drug-resistant virus infection and in the setting of combination therapy to prevent or delay drug resistance.

Topics: Adenine; Animals; Antiviral Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Dideoxynucleosides; Dose-Response Relationship, Drug; Drug Resistance, Viral; Ducks; Filaggrin Proteins; Hepatitis B virus; Hepatitis B Virus, Duck; Hepatocytes; Humans; Lamivudine; Liver Neoplasms; Mutation; Organophosphonates; Reverse Transcriptase Inhibitors; Virus Replication

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) has been shown to have an effect on duck hepatitis B virus (DHBV) in vivo and in vitro. In this study the inhibitory effect of FLG on DHBV and human hepatitis B virus (HBV) was evaluated in vitro. Cell lines transfected either with DHBV or HBV DNA and primary duck hepatocyte cell cultures were used. Virus production was analysed by PCR and a quantitative PCR was established for DHBV for determination of the inhibitory concentrations of the drug. 50% inhibition was achieved with an FLG concentration of 0.2 microg/ml (0.7 microM) and 90% inhibition was observed with an FLG concentration of 1.0 microg/ml (3.7 microM) using the DHBV transfected cell line. FLG showed an effect on DHBV production in primary duck hepatocyte cell cultures at concentrations down to 0.1 microg/ml (0.4 microM). However, the DHBV production returned to pre-treatment levels within a few days after cessation of treatment. HBV production in transfected cell lines was also inhibited by FLG. Both DHBV and HBV DNA-polymerases were inhibited by FLG triphosphate and 50% inhibition was observed at a concentration of 0.05 microg/ml (0.1 microM) for DHBV and 0.03 microg/ml (0.05 microM) for HBV. FLG is an efficient inhibitor of DHBV replication both in vivo and in vitro and of HBV in vitro which makes it a good candidate for treatment of HBV infections. However, it does not completely eliminate the virus since a relapse in virus production was observed when treatment was withdrawn. Therefore it would be interesting to evaluate FLG in combination with other types of anti-HBV drugs.

Topics: Animals; Antiviral Agents; Carcinoma, Hepatocellular; Cells, Cultured; Dideoxynucleosides; Ducks; Electrophoresis, Agar Gel; Enzyme-Linked Immunosorbent Assay; Filaggrin Proteins; Hepatitis B virus; Hepatitis B Virus, Duck; Humans; Liver; Nucleic Acid Synthesis Inhibitors; Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured; Virus Replication