Female C57BL/6 mice with average weight of 19 g were purchased from Charles River Laboratories. Food and water were provided ad libitum. Fifteen mice were divided into three dosage groups: 0, 10, and 20 mg/kg. For each mouse, 0.1 ml of compound suspension in formulation (0.5% O-carboxymethylcellulose and 0.4% Tween 80) was given by intraperitoneal injection. Blood (0.1 ml) was collected retroorbitally from a different mouse within each dosage group at 5 min, 15 min, 30 min, 1 hour, 4 hours, and 24 hours. Animals were euthanized by cardiac puncture at 48 hours after injection. Blood samples were treated with 10 μl of EDTA sodium solution to prevent coagulation. Blood was kept on ice and centrifuged for 3 min at 13,000 rpm in a desktop centrifuge to collect plasma. Twenty-five microliters of plasma samples were combined with 75 μl of internal standard (2 μM warfarin) in acetonitrile in a 96-well plate and centrifuged at 4000 rpm for 20 min at 4°C. The supernatant (40 μl) was collected, mixed with two parts of Milli-Q water (EMD Millipore), and centrifuged again at 3000 rpm for 20 min at 4°C. Plasma concentration was determined with partially validated LC/MS-MS assay with multiple reaction monitoring (MRM) detection (AB Sciex). The assay limit of quantification (LLOQ) was 1.5 nM in plasma. The processed plasma concentration–time data were analyzed using noncompartmental analysis in WinNonlin 6.0 with the plasma (200-202) model type.

The area under the concentration-time curve was calculated with the linear trapezoidal, linear interpolation rule using mean concentrations and nominal times. The terminal elimination rate (Lambda_z) and half-life (HL_Lambda_z) were determined using the default “Best Fit” method. The predicted AUC from the last time point to infinity (AUCINF_pred) was calculated as AUClast plus Clast(pred)/Lambda_z.

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