In vitro assays
4-MAA (50 µM) or control substrates (CYP1A2: 10 µM tizanidine, CYP2B6: 1 µM efavirenz, CYP2C8: 10 µM paclitaxel, CYP2C9: 1 µM flurbiprofen, CYP2C19: 1 µM omeprazole, CYP2D6: 10 µM metoprolol, CYP2E1: 1 µM chlorzoxazone, CYP3A4: 1 µM midazolam) were preincubated for 15 minutes in 100 mM phosphate buffer containing 1.5% BSA, NADPH-regenerating solution A and B (1:20 dilution and 1:100 dilution, respectively) in the presence or absence of specific CYP inhibitors (CYP1A2: 10 µM furaphylline, CYP2B6: 1 µM ticlopidine, 2C8: 20 µM montelukast, CYP2C9: 10 µM sulfaphenazole, CYP2C19: 10 µM (1)-N-3-benzylnirvanol, CYP2D6: 1 µM quinidine, CYP2E1: 20 µM methylpyrazole, CYP3A4: 1 µM ketoconazole). The final volume was 500 µL. The reaction was started by the addition of recombinant supersomes (20 pmol/mL final concentration) and the mixture was incubated on a Thermomixer 5436 (Eppendorf AG, Hamburg, Germany) at 37°C and 600 rounds per minute. After 15 minutes, 30 minutes, 1 hour and 2 hours, a sample (CYP substrates: 50 µL, 4-MAA: 20 µL) was removed and transferred into a autosampler tube containing ice-cold methanol spiked with internal standards (CYP substrates: 150 µL methanol containing 25 ng/mL chlorzoxazone-d3, 50 ng/mL efavirenz-d5, 50 ng/mL flurbiprofen-d3, 5 ng/mL metoprolol-d6, 10 ng/mL midazolam-d6, 10 ng/mL omeprazole-d3, 200 ng/mL paclitaxel-d5, and 10 ng/mL tizanidine-d4; 4-MAA: 400 µL methanol containing 20 ng/mL 4-MAA-d3, 30 ng/mL 4-AA-d3 and 60 ng/mL 4-AAA-d3). The tubes were vigorously shaken for 30 seconds and stored at -20°C until analysis.