Readout: % remaining at time point, t1/2 determination, and intrinsic clearance (Clint) estimation
Controls: w/o NADPH; Dextromethorphan (CYP2D6), Midazolam (CYP3A4), Phenacetin (CYP1A2), Diclofenac (CYP2C9), Omeprazole (CYP2C19)
Working solutions of each compound are prepared from 10 mM stock solution in DMSO diluted to a final concentration of 100 μM in 0.05 M phosphate buffer (pH 7.4).
Aliquots of Liver Microsome working solution are transferred into 1.1 mL tubes using a multichannel pipette. Positive control (5 mixed) and test compound working solutions are transferred into the tubes. The mixtures are vortexed gently and then pre-incubated at 37 °C. 5 mM NADPH or LM buffer (no NADPH buffer) is aliquoted into the tubes using a multichannel pipette and vortexed gently.
At each time point of 0 min, 5 min, 15 min, 30 min and 60 min with NADPH or 0, 30 min and 60 min without NADPH, an aliquot is removed from each tube. Terfenadine/tolbutamide in ACN/MeOH (1:1, v/v) is added to quench and precipitate the microsomal incubations. Samples are capped and vigorously vortexed and then centrifuged at 4 °C.
An aliquot of each supernatant is transferred for LC-MS/MSC analysis.
The MS detection is performed by using a SCIEX API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6μ C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – Solvent A: water with 0.1% formic acid, solvent B: ACN with 0.1% formic acid. The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area) for each time point.
The estimation of Clint (in uL/min/mg protein) is calculated using the following equation:
CLint (uL/min/mg protein) = ln(2) * 1000 / t1/2 / protein conc.
Unit of t1/2= min
Unit of Protein Conc. = mg/mL
Final DMSO concentration: 1%
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry
NADPH Nicotinamide adenine dinucleotide phosphate