In Vivo Mouse Pharmacokinetics

All animal experiments were approved by the Institutional Animal Care and Use Committee of Hackensack University Medical Center for Discovery and Innovation, and were conducted in compliance with their guidelines. Female CD-1 mice were weighed and received a single dose of Kang A, J4, or KZ via IV (5 mg/kg), PO (5 mg/kg), and IP (5 mg/kg) dosing routes. Compound was formulated as a solution in 5% DMA/95% (4% Cremophor in water). Sequential bleeds were collected at 0.017, 0.25, 1, 3, 5, and 7 h post dose via the tail snip method for IV dosing and 0.5, 1, 3, and 5 h post dose for PO and IP dosing. Blood (50 μL) was collected in capillary microvette EDTA blood tubes and kept on ice prior to centrifugation at 1500g for 5 min. The supernatant (plasma) was transferred into a 96-well plate and stored at −80 °C. LC-MS/MS analysis was performed on a Sciex Applied Biosystems Qtrap 6500+ triple-quadrupole mass spectrometer coupled to a Shimadzu Nexera 2 HPLC system to quantify each drug in plasma. Neat 1 mg/mL DMSO stocks of each compound were serial diluted in 50/50 acetonitrile/water to create standard curves and quality control spiking solutions. Standards and QCs were created by adding 10 μL of spiking solutions to 90 μL of drug free plasma (CD-1 K2EDTA Mouse, Bioreclamation IVT). Ten μL of control, standard, QC, or study sample were added to 100 μL of acetonitrile protein precipitation solvent containing internal standard (10 ng/mL Verpamil). Extracts were vortexed for 5 min and centrifuged at 4000 rpm for 5 min. 75 μL of supernatant were transferred for HPLC-MS/MS analysis and diluted with 75 μL of Milli-Q deionized water. Chromatography was performed on an Agilent Zorbax SB-C8 column (2.1 × 30 mm; particle size, 3.5 μm) using a reverse phase gradient. Milli-Q deionized water with 0.1% formic acid was used for the aqueous mobile phase and 0.1% formic acid in acetonitrile for the organic mobile phase. Multiple-reaction monitoring of precursor/product transitions in electrospray positive-ionization mode was used to quantify the analytes. The following MRM precursor/product transitions were used for Kang A (982.26/822.20), J4 (1071.35/911.20), KZ (1227.43/1067.30) and Verapamil (455.4/165.2). Data processing was performed using Analyst software (version 1.6.2; Applied Biosystems Sciex).