Entecavir for treatment of hepatitis B virus displays no in vitro mitochondrial toxicity or DNA polymerase gamma inhibition

Abstract

Therapy with nucleoside reverse transcriptase inhibitors (NRTIs) can be associated with mitochondrial toxicity. In vitro studies have been used to predict the predisposition for and characterize the mechanisms causing mitochondrial toxicity. Entecavir (ETV) is an approved deoxyguanosine nucleoside for the treatment of chronic hepatitis B virus (HBV) infection that exhibits potent activity against viral reverse transcriptase. We assessed the potential for mitochondrial toxicity of ETV in long-term cultures of HepG2 hepatoma cells by measuring mitochondrial function (through lactate secretion), levels of mitochondrial DNA (mtDNA), and levels of mitochondrial proteins COX II and COX IV. Furthermore, we tested the activity of ETV-triphosphate (ETV-TP) against mitochondrial DNA polymerase gamma (Pol gamma) in vitro. ETV concentrations as high as 100 times the maximal clinical exposure (C(max)) did not affect cell proliferation, levels of lactate, mitochondrial DNA, or mitochondrial proteins throughout the 15-day culture. The lack of mitochondrial toxicity was consistent with the finding that ETV-TP was not recognized by mitochondrial DNA Pol gamma and failed to be incorporated into DNA or inhibit the polymerase assay at the highest levels tested, 300 microM. Combinations of ETV with each of the other HBV NRTI antivirals, adefovir, tenofovir, and lamivudine at 10 times their respective C(max) levels also failed to result in cellular or mitochondrial toxicity. In summary, cell culture and enzymatic studies yielded no evidence that would predict mitochondrial toxicity of ETV at exposure levels in excess of those expected to be achieved clinically.

FIG. 1.

Effect of NRTI exposures on HepG2 cell numbers. Cell cultures were exposed to 10-fold or 100-fold the C_max levels of the NRTIs listed, and cell numbers were determined after 5, 10, or 15 days. Values represent the percentage of 0.1% DMSO vehicle-only control cultures and represent the means ± the SD of three to six independent experiments. The duration of culture was 5 days (▪), 10 days (░⃞), and 15 days (□). *, P < 0.05; **, P < 0.001 (Dunnett's multiple-comparison test).

FIG. 2.

Effect of NRTI exposures on extracellular lactate levels. HepG2 cell cultures were exposed to concentrations 10- or 100-fold higher than the C_max levels of the NRTIs listed, and the levels of lactate in the media were determined after 5, 10, or 15 days. Lactate levels were normalized according to cell numbers. Values represent the percentage of 0.1% DMSO vehicle-only control cultures and represent the means ± the SEM of three to six independent experiments. The duration of culture was 5 days (▪), 10 days (░⃞), and 15 days (□). *, P < 0.05; **, P < 0.001 (Dunnett's multiple-comparison test). Levels greater than 300% were truncated to enhance presentation and were for, ddC-treated cells, 824 ± 136% and 875 ± 116% of the vehicle-treated control values for 10 and 100 times the C_max, respectively, and for 100-fold the C_max (for LVD) was 335% ± 28% of the vehicle-treated control.

FIG. 3.

Effect of NRTI exposures on mtDNA levels. HepG2 cell cultures were exposed to 10- or 100-fold the C_max levels of the NRTIs listed, and the levels of intracellular mtDNA and nDNA were determined by real-time PCR. The ratios of mtDNA to nDNA were determined, and values representing the percentage of 0.1% DMSO vehicle-only control cultures are presented. Values represent the means ± the SEM of three to six independent experiments performed in duplicate. *, P < 0.05; **, P < 0.001 (Dunnett's multiple-comparison test).

FIG. 4.

Effect of ETV-NRTI combinations. HepG2 cells were exposed to ETV at a concentration equivalent to 10-fold the C_max either alone or in combination with ADV, LVD, or TFV also at 10-fold their respective C_max. HepG2 cells exposed to 100-fold the C_max of ddC was included as a positive control for mitochondrial toxicity. (A) Effect ETV-NRTI combinations on HepG2 cell numbers after exposure for 5 days (▪), 10 days (░⃞), and 15 days (□). The data represent the mean percentage of vehicle-treated control ± the SD of three independent experiments. (B) Levels of extracellular lactate normalized to cell number and compared to levels derived from vehicle-treated control. The data represent the means ± the SEM of three independent experiments. (C) Comparison of the effects of ETV-NRTI combinations on mtDNA levels in HepG2 cells exposed for 5, 10, and 15 days. The data represent the means ± the SEM of three independent experiments of mtDNA normalized to nDNA and compared as a percentage of vehicle-treated controls. **, P < 0.001 (Dunnett's multiple-comparison test).

FIG. 5.

Western blot analysis of mitochondrial proteins. Cells were exposed to the 100-fold the C_max values of ETV or LVD or 10-fold the C_max of ddC for 10 days. Cells were processed and analyzed as described in Materials and Methods. (A) Western blot detection of mitochondrially encoded COX II or nuclear encoded COX IV and the mitochondrial porin protein. (B) The levels of COX II and COX IV proteins were normalized according to the levels of porin protein and plotted with respect to the levels in DMSO vehicle-treated cells. A representative Western blot is shown, and the average results of three independent experiments, performed in duplicate, are plotted as means ± the SEM.