The anti-HIV properties of tenofovir and tenofovir DF have been well characterized both in vitro and clinically. While in vitro and preliminary clinical data have demonstrated that tenofovir is a potent anti-HBV agent, detailed studies on its mechanism of action, resistance profile against newer mutations, and metabolism in hepatic cells have not been reported. Furthermore, while tenofovir is structurally similar to adefovir (differing only by addition of a single methyl group in the acyclic linker region), it cannot be assumed that the two molecules will behave similarly with respect to antiviral activity, resistance profile, or metabolism. The aim of our studies was to provide a specific in vitro evaluation of the properties of tenofovir relevant to its development as an anti-HBV agent.
Our enzymatic studies confirmed tenofovir inhibits HBV replication through competitive inhibition of the viral polymerase. The Ki
of TFV-DP for HBV polymerase (0.18 μM) is similar to that of AFV-DP (0.10 μM) and slightly lower than the Km
of the natural substrate, dATP (0.38 μM) (33
). This is analogous to what was observed for HIV reverse transcriptase, where TFV-DP had a Ki
of 0.16 μM, AFV-DP had a Ki
of 0.07 μM, and the Km
of dATP was 0.33 μM (16
). Overall, tenofovir appears to inhibit HIV and HBV by the same mechanism (inhibition of viral DNA polymerization). However, it should be noted that assays to study inhibition in greater detail (e.g., individual contributions of nucleotide incorporation and excision) are not currently available for HBV (31
In cell culture, tenofovir had an EC50
of 1 μM against wild-type HBV using the stable cell line HepG2.2.15; this is similar to previous reports which have used either 2.2.15 cells or other stable cell lines (7
). Addition of the two soproxil progroups to tenofovir lowers the EC50
to 0.02 μM, a 50-fold increase in activity. This improvement in potency is likely due to significantly improved cellular permeability as the two hydroxyl groups of the phosphonic acid are covered with uncharged lipophilic promoieties. Indeed, analysis of permeability using a Caco-2 assay indicated that tenofovir DF had moderate cell permeability while tenofovir had low cell permeability (data not shown).
Clinical experience with famciclovir, lamivudine, ADV, and entecavir has indicated that resistance can be selected to all these compounds. Although resistant mutants emerge relatively slowly during ADV and entecavir therapy, it can be expected that resistance will become an increasing problem, since most patients require long-term therapy to adequately control disease. The cross-resistance profiles of all new agents should be carefully evaluated to guide the development of rational treatment regimens. Like adefovir, tenofovir retains activity against all of the major patterns of lamivudine resistance mutations in vitro and clinically (17
). Our results indicated that tenofovir showed a small but reproducible decrease in susceptibility (3- to 4.2-fold) to clinical HBV isolates bearing rtN236T, the most common adefovir-associated resistance mutation. These findings are in agreement with a recent report by Brunelle et al., who observed a 4.5-fold shift in the tenofovir EC50
after introduction of rtN236T into a laboratory strain of HBV (2
It is important to note that the in vitro rtN236T susceptibility shift observed for tenofovir is smaller than that of adefovir (7.3- to 13.8-fold) and that tenofovir DF is administered clinically at a dose 30 times higher than adefovir dipivoxil (300 mg versus 10 mg, respectively). The combination of a smaller susceptibility shift and a significantly higher dose may enable tenofovir DF to effectively suppress serum HBV DNA in patients with the rtN236T mutant. Indeed, two recent reports indicated that two patients with rtN236T had serum HBV DNA reductions of ≥4 log10
copies/ml when switched from ADV to tenofovir DF therapy (20
). Similarly, patients with rtN236T also respond to therapy with 100 mg of lamivudine despite a 2.1- to 3.5-fold in vitro decrease in lamivudine susceptibility (19
). Future studies are needed to determine the best treatment options for patients with rtN236T; however, early clinical data indicate that tenofovir DF and lamivudine should each be explored.
We also studied the impact of the rtA194T mutation, which was recently reported to emerge in two HIV/HBV-coinfected patients receiving tenofovir DF plus lamivudine as part of their antiretroviral treatment regimen (26
). Our phenotypic analysis indicated that rtA194T did not cause a significant change in tenofovir susceptibility either alone or when expressed in combination with lamivudine resistance mutations (EC50
values changed 1.5- to 2.5-fold). These results do not agree with those reported by Sheldon et al., who observed a 7.6-fold change with the single rtA194T mutation and a >10-fold increase in the EC50
when rtA194T was expressed with lamivudine resistance mutations (26
). The discrepancy might be explained by differences between the EC50
assays used in the two labs. Our results were obtained using standard Southern blotting procedures to quantify intracellular HBV replication, whereas Sheldon et al. used a PCR assay to quantify extracellular HBV DNA following transient transfection. Examining the clinical data does not provide a clear association of rtA194T with viral load rebound: one patient had a transient viral load increase of 1.5 logs after the mutation emerged, while the second patient had continuous viral load decline after the emergence of rtA194T and a >9 log10
decline in serum HBV DNA after the initiation of tenofovir DF plus lamivudine therapy. We are not aware of any additional patients who have developed this mutation under tenofovir DF therapy. Nevertheless, this residue should be monitored closely during the clinical development of tenofovir DF for chronic hepatitis B.
Studies of the anabolism of tenofovir to its active diphosphate form were previously restricted to lymphoid cells to support the HIV indication of tenofovir DF (21
). Here we have shown that tenofovir phosphorylation occurs efficiently in a human hepatoblast cell line (HepG2) and in primary human hepatocytes. In the CEM (lymphoid) cell line, TFV-DP achieved levels of 5.2 μM, which is similar to the levels we observed in HepG2 cells (6.0 μM). However, TFV-DP formation in primary human hepatocytes was significantly greater (4.7 μM) than in primary lymphocytes (1.0 μM levels reached in peripheral blood mononuclear cells) (21
). In parallel experiments, the efficiency of TFV-DP formation was greater than that of adefovir in both cell types that we tested; however, the difference in primary human hepatocytes (<2-fold) was smaller than in HepG2 cells (2.5-fold).
Tenofovir diphosphate increased in a linear manner over the 24-hour incubation period. Linear increases over the same time period were also observed for adefovir diphosphate when run in parallel during these experiments as well as in previous studies (22
). The linear accumulation of adefovir and tenofovir diphosphates over 24 hours is in contrast to most nucleoside analogs, which usually reach a maximal intracellular concentration after 8 to 12 h. However, this result is consistent with the reduced permeability of adefovir and tenofovir due to the presence of the two negative charges on the phosphonate moiety. Similar studies conducted on T cells in our laboratory indicate that tenofovir diphosphate levels begin to reach maximal concentrations after 48 hours of incubation (data not shown). The diphosphates of both tenofovir and adefovir had very long intracellular half-lives, which is consistent with earlier studies demonstrating prolonged in vitro antiviral effects with tenofovir and adefovir after drug removal (38
). Overall, the efficient phosphorylation and long half-life we observed for tenofovir agree with the clinical results indicating that a single daily dose of tenofovir DF will be phosphorylated to levels sufficient to exert a potent antiviral effect in the liver.
Due to its favorable safety profile and efficacy, tenofovir DF is a recommended first-line treatment option for HIV infection (37
). Multiple small investigator studies have demonstrated that the 300-mg dose of tenofovir DF approved for HIV also results in a potent antiviral suppression of serum HBV DNA (>4 log10
reduction in serum HBV copies/ml) in coinfected patients (5
). The in vitro data presented here have confirmed that tenofovir inhibits HIV and HBV by similar mechanisms (competitive inhibition of DNA polymerization) and that tenofovir DF has potent cell-based anti-HBV activity (EC50
, 0.02 μM). Tenofovir also has a favorable metabolic profile in hepatic cells, the target compartment for antiviral activity in vivo. Accordingly, enrollment has recently begun for phase III studies of tenofovir DF in HBV e antigen-negative and e antigen-positive chronic hepatitis B patients. We have also shown that tenofovir is more efficacious than adefovir against the rtN236T adefovir resistance mutation in vitro. Since significantly higher doses of tenofovir DF are being used clinically, these data suggest tenofovir should also be explored for the treatment of adefovir-resistant HBV.