Hepatitis B virus (HBV) infection is a major worldwide health problem. Infection with HBV not only causes hepatitis but also has a strong association with hepatocellular carcinoma (1
). A selective antiviral compound would be useful for the treatment of hepatitis caused by HBV and may even prevent or delay the onset of hepatocellular carcinoma associated with HBV.
Currently, the only drug approved for the treatment of HBV hepatitis is alpha interferon. However, only 25 to 50% of patients respond to this therapy. Furthermore, there are side effects associated with this treatment (19
). A more selective anti-HBV drug is needed. HBV is an incomplete double-stranded DNA virus. Its DNA replication process is quite unique and includes the step of reverse transcription catalyzed by HBV-specified DNA polymerase (23
). The fact that HBV DNA polymerase is quite different from human DNA polymerase raises the possibility of the discovery of compounds that could selectively inhibit HBV DNA replication.
Deoxynucleoside analogs were previously shown to be active against HBV and to have different degrees of toxicity (22
). Their mechanism of antiviral action is suggested to be mediated through a unique interaction of the triphosphate metabolites with HBV DNA polymerase. Although HBV DNA polymerase is essential for HBV DNA replication and virus propagation, it is not required for HBV supercoiled DNA or intergrated DNA synthesis. In order to be more effective in the treatment of chronic HBV infection, treatment with those anti-HBV nucleoside analogs which target HBV DNA polymerase will need to be long term. This prolonged treatment not only will inhibit HBV replication but also will deplete cells that harbor either supercoiled or integrated HBV DNA through natural turnover of virus-harboring cells. Thus, the safety of the drug upon long-term use is an important issue.
Several of the toxicities of nucleoside analogs can be associated with the incorporation of their triphosphate metabolites into nuclear DNA. Upon long-term treatment, these drugs can also interfere with mitochondrial DNA synthesis, resulting in delayed toxicity. In the search for new antiviral compounds with fewer short- and long-term toxicities, β-l
-SddC; 3TC) was found to have potent activity against HBV and human immunodeficiency virus (HIV) (3
). Nucleosides are found in nature only in the β-d
configuration, and this compound was the first nucleoside analog with the unnatural β-l
configuration shown to have biological activity. Subsequently, several other β-l
-deoxycytidine analogs were found to have similar activities against HBV and HIV (10
). However, none of these compounds is active against Epstein-Barr virus (EBV).
-SddC exerts its antiviral activity through the interaction of its triphosphate metabolite with HIV reverse transcriptase and HBV DNA polymerase. Unlike most previously studied antiviral dideoxynucleoside analogs including the anti-HBV compounds
-FMAU) and 1-(2′-deoxy-2′-fluoro-β-d
-SddC does not interfere with mitochondrial function. l
-SddC has already been approved for use for the treatment of patients with AIDS in combination with zidovudine. In addition, l
-SddC is currently showing impressive results in clinical trials for the treatment of chronic HBV infection (18
A problem with the use of l
-SddC for anti-HIV therapy is the development of resistance mediated by mutations in the HIV reverse transcriptase. Although l
-SddC-resistant viruses are likely to be cross-resistant to other deoxycytidine (dCyd) analogs, they may not be resistant to analogs with different base. In addition, the spectrums of toxicity and activity of these analogs might be different from those of analogs with a cytidine base. Therefore, several l
-nucleoside analogs with different bases were synthesized and examined for antiviral activity. One of these compounds, 2′-fluoro-5-methyl-β-l
-FMAU), was found to be active against HBV in 2.2.15 cells, with a 50% effective concentration of 0.1 μM. Unlike the β-l
-ddC) analogs, this compound was inactive against HIV. Interestingly, it was also active against EBV, with a 90% effective concentration of 5 μM (7
). This is the first example of an l
-thymidine analog with potent antiviral activity.
Although its spectrum of activity is different from those of other β-l
-nucleoside analogs, it shares their favorable toxicity profiles. Unlike its d
-FMAU triphosphate (l
-FMAUTP) could not be incorporated into nuclear DNA on the basis of cell culture studies and experiments done with purified human DNA polymerases (21
). In addition, treatment with l
-FMAU did not deplete cells of mitochondrial DNA. Therefore, delayed toxicities upon long-term treatments should not be a problem with this compound. Indeed, no toxicity was observed in mice after 30 days of continuous treatment with l
-FMAU at 50 mg/kg of body weight. In vivo, l
-FMAU was shown to have potent antiviral activity against duck HBV when it was administered at an oral dosage of 10 mg/kg for 5 days (30
). These results demonstrate the potential use of l
-FMAU for the treatment of human HBV infection.
Like other nucleoside analogs, l
-FMAU could be phosphorylated in cells to its mono-, di-, and triphosphate metabolites. l
-FMAUTP is the major metabolite in most cell lines examined except H1 cells, in which l
-FMAU monophosphate (l
-FMAUMP) is the major metabolite (27
). This unique metabolic feature of l
-FMAU in H1 cells could be due to the presence of EBV thymidine (dThd) kinase in cells. The metabolism of l
-FMAU in 2.2.15 cells was no different from its metabolism in HepG2 cells, suggesting that the activation of l
-FMAU is most likely carried out by cellular enzymes (21
). Given the unique structure of l
-FMAU, it was not clear which enzymes in human cells could be important in its metabolism. This report describes the results of our studies with respect to the enzymes with key roles in the phosphorylation of l
-FMAU to l