In the work described here the antiviral activity of β-l
-Fd4C was analyzed in vivo in the mammalian model of WHV infection and was compared with that of 3TC, another l
-deoxycytidine analog. Both short-term and long-term treatments with β-l
-Fd4C at a dose of 1 or 4 mg/kg administered intraperitoneally allowed inhibition of WHV replication. However, this was followed by a relapse of viral replication after drug withdrawal, as already described in this model with other nucleoside analogs (6
), but with a longer delay for the case of the 3TC group in our experiment. The inhibition of viremia assessed by WHV DNA detection and determination of EPA was, however, less pronounced than that observed with a recently developed compound, cyclopentyl guanosine (entecavir; BMS-200475), which allowed 107
- to 108
-fold decreases in viremia titers (12
). By comparison, 3TC was only moderately active against WHV replication, as already observed with a dosage of 5 mg/kg administered orally (12
). In a recently published study, 3TC had to be administered at an oral dose of 200 mg/kg to obtain a significant antiviral effect in woodchucks (29
). These results are consistent with the more potent inhibitory effect of β-l
-Fd4C on the DHBV reverse transcriptase in vitro and in vivo in comparison with that of 3TC in the duck model of HBV infection (26
). Therefore, it would be interesting to gain comparative information on the in vivo metabolisms of 3TC and β-l
-Fd4C in both the duck and woodchuck models by comparison with those in humans.
-Fd4C treatment for 15 weeks was not able to achieve clearance of intrahepatic viral CCC DNA, explaining the relapse of viral replication after the cessation of therapy. This is consistent with the results obtained with the duck model of HBV infection with antiviral therapy based on 2′-carbodeoxyguanosine or FTC (9
), as well as in the woodchuck model with 3TC (29
), FTC (6
), and cyclopentyl guanosine (12
). This was associated with the absence of a decline in the number of infected hepatocytes and the WHV surface antigen titer in serum, suggesting that the residual viral CCC DNA is an active template for viral genome expression, allowing the reinitiation of viral replication when therapy is stopped. Genome sequence analysis of the B and C domains of the polymerase gene of the dominant virus in the sera of the animals demonstrated the wild-type sequence, suggesting the absence of selection of drug-resistant mutants in β-l
-Fd4C- or 3TC-treated animals with this duration of treatment. However, selection of drug-resistant mutants has previously been observed after longer durations (>9 to 12 months) of 3TC treatment in the woodchuck model (44
). As the rise in the viremia titer associated with the selection of drug-resistant mutants was shown to depend on the clearance of hepatocytes infected with wild-type virus (44
), in our experiments we cannot rule out that this process was ongoing when therapy was stopped. As direct sequencing of the PCR product may allow detection of minor variants representing 10 to 20% of the circulating viral quasispecies, clonal analysis may already have revealed the presence of mutants as minor species.
It was recently shown in the woodchuck model of hepadnavirus infection that the recruitment of CD4+
T cells and the production of gamma interferon and tumor necrosis factor alpha in the infected liver, accompanied by a significant increase in apoptosis and regeneration of heptocytes, are critical events involved in viral clearance (15
). In light of these observations, the evaluation of combination treatment strategies based on the use of reverse transcriptase inhibitors and immune modulators such as the DNA vaccine approach (38
) to enhance the clearance of infected cells is warranted.
Interestingly, we observed a decrease in liver histology activity in β-l
-Fd4C-treated animals. Since the number of animals was limited in this study, further investigations are warranted to determine whether prolonged antiviral treatment may control the progression of the liver disease, as suggested in clinical trials with 3TC (7
). Since expression of WHV antigen in the liver induces an antiviral immune response that leads to chronic disease, the decrease of hepatitis activity during β-l
-Fd4C therapy could also be explained by an immunomodulatory action of this antiviral since the number of infected cells and the level of viral protein expression were apparently not significantly modified by our protocol. However, in our woodchuck cohort, which was obtained at 12 months of age and treated 4 months later in the long-term β-l
-Fd4C group 1 protocol, this beneficial effect on liver histology was not sufficient to prevent or delay the occurrence of liver cell dysplasia and subsequently the development of hepatocellular carcinoma. Accordingly, in another study, long-term 3TC therapy of chronically infected woodchucks did not prevent the occurrence of hepatocellular carcinoma (29
). WHV-induced liver cancer involves a cascade of complex events including early viral genome integration in the N-myc
proto-oncogenes as well as hepatocyte turnover (8
). As the rate of hepatocellular carcinoma reaches 25% of the infected animals each year (13
), our findings suggest that early antiviral intervention should be evaluated as prophylaxis for hepatocellular carcinoma.
Indeed, a study performed with younger animals showed that long-term 3TC treatment may delay the development of hepatocellular carcinoma in this model (S. F. Peek, I. A. Toshkov, H. N. Erb, R. F. Shinazi, B. E. Korba, P. J. Cote, J. L. Gerin, and B. C. Tennant, Abstr. Am. Assoc. Study Liver Dis., abstr. 957, 1997).
As fialuridine was responsible for major mitochondrial toxicity in humans as confirmed with the woodchuck model (42
), the potential toxicity of β-l
-Fd4C was another very important issue that was addressed with the woodchuck model. Whether the loss of weight that was observed during β-l
-Fd4C therapy could be related to the occurrence of hepatocellular carcinoma, the physiology of the woodchuck during hibernation, or side effects of β-l
-Fd4C needs to be determined by further toxicological studies. Indeed, careful electron microscopy analysis of liver samples during antiviral treatment did not reveal any significant ultrastructural modifications to hepatocyte mitochondria, biliary canaliculi, or bile ducts in β-l
-Fd4C- and 3TC-treated animals, suggesting an absence of liver cytotoxicity of these antivirals under our experimental conditions, as already observed with 3TC treatment in humans (17
). However, skin hyperpigmentation related to an accumulation of melanin pigment was observed during β-l
-Fd4C treatment and slowly disappeared after drug withdrawal. This skin modification may be related to the particular physiology of the woodchuck, especially since therapy was performed in part during the hibernation period, when the general metabolism of these animals is greatly modified. A similar hyperpigmentation phenomenon has been described in patients treated with zidovudine and was reproduced in mice experimentally receiving zidovudine (14
). The molecular mechanisms responsible for this increase in melanocyte activity as a result of nucleoside analog administration and their relation to hibernation remain to be elucidated.
In conclusion, the results of our study suggest that although β-l-Fd4C exhibits a more potent antiviral effect than 3TC in a mammalian model of HBV infection, clearance of viral CCC DNA and infected cells from the liver is difficult to achieve with monotherapy with a potent inhibitor of viral replication. Furthermore, effective prevention of hepatocellular carcinoma in this animal model may require an early therapeutic intervention, before the integration of viral genome sequences in the host genome. Future approaches that combine antivirals and immune modulators for the eradication of chronic hepadnavirus infection should be evaluated.