This report describes parameters influencing the antiviral activity of ETV against wild-type and 3TC-resistant HBV. To examine the intrinsic activity of ETV-TP directly, the inhibition of wild-type and 3TC-resistant HBV Pol was measured using recombinant HBV nucleocapsids formed by coexpression of the polymerase and core proteins, in which the polymerase protein is specifically primed for replication of a pseudo-pregenomic HBV RNA in an epsilon-mediated fashion (29
). Assay modifications were made to further optimize the in vitro reaction conditions and to improve the sensitivity of detection using these nucleocapsids. The apparent Km
values reported here for wild-type HBV Pol are about 7- to 14-fold lower than values reported previously using similar recombinant HBV nucleocapsids (30
). One likely explanation for this difference is that reactions in the current study were carried out at 37°C, versus 30°C. This temperature increase would not only accelerate the enzyme reaction rate but would also increase the rate of diffusion of nucleotides into the interior of nucleocapsids where they are needed to act as substrates for HBV Pol. The resulting assay provided an optimal system to evaluate the effects of specific 3TC resistance mutations on DNA synthesis by HBV Pol. The measurements described here confirmed the excellent inhibitory potency of ETV-TP against wild-type HBV Pol and demonstrated reduced but still potent inhibition of 3TC-resistant HBV Pol. A comparison of the relative change in binding kinetics caused by YMDD mutations clearly demonstrated that the recognition of ETV-TP by HBV Pol was impacted to a lesser degree than that of 3TC-TP.
A key to understanding the potential for in vivo efficacy is to know the level of active drug that is present in HBV-infected hepatocytes. In the case of 3TC, in vitro phosphorylation measurements have been a very useful indicator of the in vivo phosphorylation profile in the peripheral blood mononuclear cells of patients infected with human immunodeficiency virus (27
). In this report, we examined the phosphorylation of ETV in liver-derived cell lines exposed to clinically relevant concentrations of ETV. Human pharmacokinetic and pharmacodynamic data for both ETV and 3TC are the benchmark by which the in vitro results must be compared. The results of recent clinical trials have demonstrated the efficacy of a once-daily dose of 0.5 mg of ETV for the treatment of chronic HBV infections (7
), including those that are resistant to 3TC (Tassopoulos et al., Hepatology 34:
340A, 2001). Patients treated with this dose achieve an average steady-state concentration in blood of ~2.4 nM (unpublished results). The in vitro phosphorylation data reported here (Table ) confirm an important aspect of the activation of ETV inside cells. The results showed that the efficiency of phosphorylation to the TP form increases as the concentration of extracellular ETV is lowered. On the basis of these in vitro data, 2.4 nM ETV in blood could produce an average intracellular concentration of ETV-TP in the range of 53 to 67 nM. If so, this would be well above the Ki
measured for inhibition of wild-type HBV DNA synthesis by ETV-TP, and strong inhibition of wild-type HBV replication would be expected at this exposure level. The inhibition kinetics of ETV-TP, in this respect, are consistent with the significant reduction of HBV DNA levels observed in patients treated with a 0.5-mg daily dose (7
). In patients treated with a once-daily dose of 100 mg of 3TC, the average steady-state concentration in blood is estimated to be 0.85 μM (16
). This could produce an average intracellular TP concentration of 0.85 to 1.7 μM, based on reports that 3TC-TP levels in hepatoma cells are one- to twofold higher than the extracellular 3TC concentration (17
). At this level, the concentration of 3TC-TP would also be well above its Ki
for wild-type HBV Pol and consistent with the initial efficacy of 100 mg of 3TC against chronic HBV infection.
A very different picture begins to emerge when the inhibition kinetics of 3TC-resistant HBV Pol are evaluated. Given that resistance to 3TC occurs readily, it is not surprising that the inhibitory potency of 3TC-TP was reduced >1,000-fold against 3TC-resistant YMDD variants. It is notable that the Ki of 3TC-TP for the 3TC-resistant YMDD variant of HBV is above the level of intracellular 3TC-TP expected at clinically relevant exposure levels. This would be expected to result in incomplete inhibition and a consequent increase in HBV DNA replication relative to that of the wild-type HBV Pol, as occurs when resistance emerges. In contrast, the inhibition kinetics with ETV-TP clearly demonstrated potent inhibition of DNA synthesis by 3TC-resistant HBV Pol. While the potency of ETV-TP is reduced from that of the wild-type HBV Pol, the phosphorylation kinetics suggest that the intracellular concentration of ETV-TP expected in patients treated with 0.5 mg of ETV would remain above the Ki for 3TC-resistant HBV Pol (Ki = 22 nM). This leads to the expectation that clinically relevant ETV exposure levels would reduce the replication of 3TC-resistant HBV.
Another way to evaluate potential cross-resistance in vitro is to examine the complete replication cycle of HBV inside cells. It has been demonstrated that HBV Pol has a stronger preference for binding ETV-TP than its natural dGTP substrate (30
) and that ETV-TP can inhibit the priming function of HBV Pol (30
). Because these properties are unique relative to 3TC, it was important to determine the potential impact of YMDD mutations on ETV inhibition in an assay which involves all the replication functions of HBV Pol. For this reason, cell culture antiviral assays were used to gauge the potential for cross-resistance to ETV. Another fact to consider is that YMDD mutations have been reported to reduce the replicative fitness of HBV in cells (26
), and this may have an impact on the outcome of viral replication assays which was not evident in enzyme inhibition assays. Because antiviral potency data reported in the literature for HBV vary significantly, two different cell culture systems were used to determine the effects of YMDD mutations on the antiviral potency of ETV. Similar results were obtained with either system, demonstrating that YMDD mutations conferred full resistance to 3TC but conferred no more than 20- to 30-fold cross-resistance to ETV.
The in vitro data reported here are consistent with recent clinical studies demonstrating the efficacy of ETV for the treatment of chronic HBV infections (7
) and 3TC-resistant HBV infections (Tassopoulos et al., Hepatology 34:
340A, 2001). The combined results indicate that the presence of YMDD mutations have a limited impact on the antiviral activity of ETV. Because of its superior potency against these variants, the development of resistance during ETV therapy may take longer to emerge and require other, or additional, mutations in HBV Pol. As a result, ETV therapy in treatment-naive patients might be expected to suppress HBV DNA levels more than 3TC therapy, due to increased potency, and for a longer duration, due to the reduced emergence of viable YMDD variants. Greater suppression of HBV DNA levels by ETV was, in fact, evident in patients treated with a 0.5-mg daily dose, which led to a drop in HBV DNA of >4 log10
units at 22 weeks, a reduction of 1 to 1.5 log10
units more than that observed for 3TC (20
). The results presented here are encouraging and underscore the importance of monitoring HBV isolated from ETV-treated patients for the potential emergence of resistant HBV. They also point clearly to the development of ETV as a promising advance in the treatment of chronic HBV infections.