Metabolic studies on BMS-200475, a new antiviral compound active against hepatitis B virus

Abstract

BMS-200475 was recently shown to have potent antiviral activity against hepatitis B virus (50% effective concentration = 3.7 nM; 50% cytotoxic concentration = 30 microM). In metabolic studies in both HepG2 and hepatitis B virus-transfected 2.2.15 human hepatoma cell lines, the metabolism was similar, the primary products being the di- and triphosphates. The accumulation of triphosphate was rapid and detectable down to a 5 nM concentration of added drug. When cells were labeled at 25 microM, the intracellular triphosphate concentration attained 30 pmol/10(6) cells ( approximately 30 microM). The intracellular half-life of the triphosphate was about 15 h. Compared with five other nucleoside analogs of medical interest (lamivudine, penciclovir, ganciclovir, acyclovir, and lobucavir), BMS-200475 was most efficiently phosphorylated to the triphosphate in HepG2 cells.

FIG. 1

Structure of BMS-200475.

FIG. 2

Concentration dependence of the accumulation of BMS-200475 nucleotides in HepG2 cells. (A) Cells were labeled for 3 days in the presence of 1 to 25 μM [³H]BMS-200475 (2.5 to 0.5 Ci/mmol). The graph shows the levels of intracellular nucleoside, diphosphate, and triphosphate. The monophosphate did not accumulate to appreciable levels. (B and C) Intracellular BMS-200475 triphosphate accumulation at submicromolar levels of BMS-200475 in the medium. HepG2 cells were labeled for 3 days in the presence of 50 to 1,000 nM (B) or 5 to 100 nM (C) [³H]BMS-200475 (13.9 Ci/mmol). At the lowest concentrations of [³H]BMS-200475, the triphosphate was the major detectable [³H]BMS-200475 metabolite. All data points are the average of triplicate determinations.

FIG. 3

Kinetics of [³H]BMS-200475 uptake and phosphorylation by HepG2 cells. (A) Cells were labeled with 1 μM [³H]BMS-200475 (2.5 Ci/mmol) for up to 4 days and then analyzed for intracellular nucleotides. The data are presented as counts per minute per well for each of the intracellular solutes. (B) Empirical and normalized data for the phosphorylation of 50 nM [³H]BMS-200475 (13.9 Ci/mmol) to the triphosphate (475-TP) during a 36-h incubation. The dotted line corresponds to the radioactivity in the triphosphate present at each time point (counts per minute per well). The solid circles correspond to these data following conversion to picomoles per 10⁶ cells.