L-FMAU has markedly less toxicity than its D-enantiomer, D-FMAU, and L-FMAU does not induce an increase in lactic acid production in the human hepatoblastoma cell line, HepG2, as observed following exposure to D-FMAU and the related nucleoside analogue, D-FIAU (fialuridine). [1][2][3][5][6][7][8][11][12][13][14][15] Fialuridine demonstrated potent anti-HBV activity in clinical trials for chronic HBV infection, but also induced severe delayed toxicity associated with lactic acidosis and hepatic failure that resulted in the death of several patients. 13,14 D-FMAU, D-FIAU, and D-FEAU have been shown to be potent inhibitors of woodchuck hepatitis virus (WHV) replication in chronically infected woodchucks, but all of these nucleoside analogues also demonstrated severe toxicity at daily doses greater than 2 mg/kg body weight. 11,12,15 WHV and its natural host, the Eastern woodchuck (M. monax), constitute a useful model of HBV-induced disease, including hepatocellular carcinoma (HCC). 16,17 A variety of published studies from several laboratories have reported the use of chronic WHV infection in woodchucks to investigate potential antiviral therapies for chronic HBV infection. 11,15,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] 26,31,32,34,35 ribavirin, 34 thymosin ␣1, 20 vidarabine (ara-A), 21,34 zidovudine (AZT), 34 combination therapy with either lamivudine/interferon alfa 35 or lamivudine/famciclovir 32 ) have shown similar relative antiviral activities against WHV and similar toxicity profiles in chronically infected woodchucks. These studies demonstrate that the WHV/woodchuck model of chronic HBV infection can be considered to be a predictive model for new, potential therapeutic applications against HBV infection in humans, especially nucleosides.

During long-term studies of the natural history of woodchuck hepatitis virus infection, five cases of histologically confirmed, primary hepatocellular carcinoma were observed in a total of 92 woodchucks which had recovered, by analysis of viral serologic markers (WHsAg-, anti-WHc+, anti-WHs+), from experimental acute woodchuck hepatitis virus infections 20 to 30 months prior to the detection of hepatocellular carcinoma. No hepatocellular carcinoma was observed in 167 uninfected controls at least 3 years of age and held in the same laboratory environment. Southern blot hybridization analysis of liver tissue taken from four of these recovered woodchucks revealed the presence of low levels (0.1 to 0.3 copies per cell) of integrated woodchuck hepatitis virus DNA in hepatocellular carcinoma (four of four animals) and nonneoplastic tissue (three of four animals). Similarly, hepatocellular carcinoma tissue obtained from two wild-caught, naturally infected and serologically recovered woodchucks also contained low levels of integrated woodchuck hepatitis virus DNA. Liver tissues from another 27 of these 92 recovered woodchucks (without hepatocellular carcinoma) were examined for woodchuck hepatitis virus nucleic acids 13 to 31 months following experimental woodchuck hepatitis virus infection. Nonreplicating woodchuck hepatitis virus DNA was present in the liver of eight (30%) and in the peripheral blood lymphocytes from eight (30%) of these 27 animals. These results were in marked contrast to the analysis of woodchuck hepatitis virus DNA in the liver tissue of chronic woodchuck hepatitis virus carriers (20 experimentally infected and nine naturally infected). In these animals, high levels of replicating woodchuck hepatitis virus DNA (up to 2,000 copies per cell) were observed in all hepatocellular carcinoma and nonneoplastic liver tissue. Integrated woodchuck hepatitis virus DNA was found in eight of 60 individual hepatocellular carcinomas detected in 29 chronic carriers, 15 to 40 months postinfection. Integrated woodchuck hepatitis virus DNA was present in the nonneoplastic tissue from four of these 29 chronic woodchuck hepatitis virus carriers.