Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus

Abstract

Tenofovir is an acyclic nucleotide analog with activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Tenofovir disoproxil fumarate (tenofovir DF), a bis-alkoxyester prodrug of tenofovir, is approved for the treatment of HIV and is currently being developed to treat chronic hepatitis B. In this report, we further characterize the in vitro activity of tenofovir against HBV as well as its metabolism in hepatic cells. We show that tenofovir is efficiently phosphorylated to tenofovir diphosphate (TFV-DP) in both HepG2 cells and primary human hepatocytes. TFV-DP has a long intracellular half-life (95 h) and is a potent and competitive inhibitor of HBV polymerase (Ki = 0.18 microM). Tenofovir has a 50% effective concentration of 1.1 microM against HBV in cell-based assays, and potency is improved > 50-fold by the addition of bis-isoproxil progroups. Tenofovir has previously demonstrated full activity against lamivudine-resistant HBV in vitro and clinically. Here we show that the major adefovir resistance mutation, rtN236T, confers three- to fourfold-reduced susceptibility to tenofovir in cell culture; the clinical significance of this susceptibility shift has not yet been determined. The rtA194T HBV polymerase mutation recently identified in tenofovir DF-treated HIV/HBV-coinfected patients did not confer in vitro resistance to tenofovir as a single mutation or in a lamivudine-resistant viral background. Overall, the antiviral and metabolic profile of tenofovir supports its development for the treatment of chronic hepatitis B.

FIG. 1.

Competitive inhibition of HBV polymerase by TFV-DP. HBV polymerase was incubated with template (activated calf thymus DNA) deoxynucleoside triphosphates and α-³³P-labeled dATP. Inhibition of labeled dATP incorporation was measured in the presence of the indicated concentrations of TFV-DP. Data are presented as a Lineweaver-Burk plot.

FIG. 2.

Phosphorylation kinetics of tenofovir to mono- and diphosphate species in HepG2 cells and primary human hepatocytes. Primary human hepatocytes (A) and HepG2 cells (B) were treated with 10 μM tenofovir. At the indicated time points, cells were washed and lysed and the amounts of intracellular tenofovir, TFV-MP, and TFV-DP were quantified by LC/MS/MS. Parallel experiments were performed with 10 μM adefovir in primary hepatocytes (C) and HepG2 cells (D) to determine levels of adefovir, AFV-MP, and AFV-DP.

FIG. 3.

Intracellular half-life of TFV-DP in primary human hepatocytes. Primary human hepatocytes were incubated with 10 μM tenofovir (A) or 10 μM adefovir (B) for 24 h to accumulate diphosphate. Cells were then washed, fed fresh medium without drug, and then lysed at the indicated times for quantification of intracellular diphosphate levels by LC/MS/MS. Using a single exponential decay model, intracellular diphosphate half-lives of 95 ± 6 and 75 ± 1 h were calculated for tenofovir and adefovir, respectively.