Combination therapy is crucial to the management of HIV infection. Although there are important viral and pathogenic differences between HBV and HIV infections, it is hoped that combination therapy will provide similar benefits for patients with chronic hepatitis B infection. Ideally, combination therapy for chronic hepatitis B would significantly enhance HBeAg seroconversion rates. Unfortunately, seroconversion may not be an achievable endpoint for all chronic hepatitis B patients, particularly those who lack significant immune activity against the virus. However, even in the absence of seroconversion, antiviral therapy could aim to achieve long-term suppression of viral load, which has proven to translate into a remission of liver disease. Thus, for patients that do not undergo HBeAg seroconversion, the combination of two or more antiviral agents with additive and/or synergistic antiviral activity and distinct resistance profiles may represent the best option for long-term suppression of viral load and disease management.
Few data on the in vitro activity of drug combinations against HBV are currently available. Korba et al. previously showed that combinations of lamivudine and penciclovir had synergistic activity against HBV in HepG2 2.2.15 cells (21
) and that the combination of lamivudine and famciclovir produced additive to synergistic effects against woodchuck hepatitis virus in vivo (22
). Colledge et al. reported additive or synergistic effects of adefovir, lamivudine, and penciclovir when used in combination against the duck HBV in primary duck hepatocytes (5
). Seigneres et al. provided enzymatic, cell culture, and in vivo data indicating that combinations of emtricitabine, amdoxovir, and clevudine (l
-FMAU) provided efficacy superior to treatment with any of the drugs as a single agent (36
Our results, which were generated with a novel stable cell line expressing high levels of HBV, indicate that combinations of adefovir with other anti-HBV drugs produce greater in vitro antiviral effects than any of the agents used alone. We did not observe any evidence of cytotoxicity during these assays at the highest tested combination doses. Our observations are in agreement with the reported 50% cytotoxic concentrations for all compounds, which are orders of magnitude higher than the antiviral doses used here. Further experiments will be needed to determine whether additive, synergistic, or antagonistic in vitro cytotoxicity is observed when cells are treated with high doses of these drugs.
Interpretation of the antiviral results with the Bliss independence model indicated that none of the tested combinations deviated significantly from additivity. Analysis using the Loewe additivity model indicated that the activity of adefovir in combination with l
-nucleosides (lamivudine, FTC, and L-dT) was additive while combinations of adefovir with entecavir or tenofovir were synergistic. As noted previously, it is not uncommon for the Bliss independence and Loewe additivity models to disagree (10
). The underlying basis for the discrepancy between these two models is generally due to the shape of the individual drugs dose-response curves. When both drugs have identical exponential dose responses, both models will be in agreement (3
). However, for compounds with steeper dose responses, greater combination effects may be observed with the Loewe additivity model. For compounds with flatter dose responses, greater combination effects may be observed with the Bliss model (10
It is unclear why combinations of adefovir with entecavir or tenofovir resulted in Loewe synergy, while the l
-nucleoside combinations produced Loewe additivity. Interestingly, adefovir, tenofovir, and entecavir are purine analogs, whereas all of the tested l
-nucleosides are pyrimidines. One explanation is that combinations of adefovir with entecavir or tenofovir affect purine metabolism in a way that enhances the anabolic and antiviral efficacy of these drugs. Alternatively, tenofovir and entecavir may have better in vitro activity in a subpopulation of cells (e.g., those in a distinct stage of the cell cycle and with differential nucleoside and/or nucleotide kinase expression) than the tested l
nucleosides. It should be noted that differential activation of nucleoside and nucleotide analogs in distinct cell types has previously been observed in vivo with the duck HBV model (26
). Activity of different agents in distinct cell compartments provides another theoretical argument in favor of using combination therapy in a clinical setting.
The observed Loewe synergy could also theoretically result from combining multiple agents that act as inhibitors of the HBV-priming reaction. Indeed, previous in vitro studies have demonstrated that entecavir inhibits HBV priming in addition to DNA synthesis (34
); similar studies performed with the duck HBV system have indicated that adefovir can also inhibit hepadnaviral priming (35
). In contrast, deoxycytidine analogs (including lamivudine and FTC) are theoretically unable to act as substrates for the HBV priming reaction, since the priming sequence (UUC or UUCA) does not allow for base pairing with these nucleotides (25
). It is currently unclear if L-dT can act as an inhibitor of HBV priming. Base pairing with adenosine does not appear to be required, and there are no in vitro studies addressing L-dT's ability to inhibit the hepadnaviral priming reaction.
To date, there are relatively few controlled clinical studies that have examined the use of two drugs to treat chronic hepatitis B. The majority of these have been combinations of IFN-α and lamivudine (reviewed by Schalm) (33
); however, the use of adefovir in combination with lamivudine and other agents is now being explored. Preliminary clinical studies using IFN-α plus lamivudine have provided evidence of greater antiviral effect and a delay in lamivudine resistance; however, these studies have not demonstrated a conclusive improvement in serocoversion. Furthermore, the drawbacks of IFN-α therapy (side effects, the difficulties of parenteral administration, and contraindications to using the drug with several patient populations) limit its utility as a combination agent, especially for long-term patient management. The treatment of lamivudine-resistant HBV with the combination of lamivudine and adefovir dipivoxil was shown to suppress viremia in patients but more than with adefovir dipivoxil alone (30
). Preliminary results from a study comparing the combination of adefovir dipivoxil plus lamivudine versus lamivudine monotherapy in naïve patients have recently been presented (J. Sung, Lai, J. S. Zeuzem, W. Chow, E. Heathcote, R. Perrillo, C. Brosgart, M. Woessner, S. Scott, and E. Campbell, Abstr. 38th Annu. Meet. Eur. Assoc. Study Liver Dis. 2003, abstr. 69, 2003). These results indicated that the two therapies produced similar initial viral load reductions, but the combination reduced the emergence of lamivudine resistance and produced better virologic response late in the study. Seroconversion rates were not significantly different between patients receiving combination therapy and those receiving lamivudine monotherapy. Analyses from the second year of this study are ongoing and may reveal a greater difference between the monotherapy and combination arms; these data will have strong implications for the treatment of chronic infection.
In conclusion, we used a novel HBV-expressing cell line to investigate the antiviral activity of activity of adefovir in combination with other approved or phase III investigational anti-HBV drugs. Our results indicated that two-drug combinations that included adefovir produced greater in vitro antiviral effects than those of single agents. The observed antiviral combination effects were characterized as additive with the Bliss independence model or additive to synergistic with the Loewe additivity model. Importantly, there was no evidence of in vitro cytotoxicity and no evidence of antiviral antagonism with any of the drug combinations at the tested doses. Combination therapy should be explored further clinically, especially for patients who are unable to achieve full serum HBV DNA suppression during monotherapy. The activity of adefovir against lamivudine-resistant HBV, the infrequent emergence of adefovir resistance, and the data provided here suggest that adefovir dipivoxil may form an important component of future combination regimens.