*2.3. Kinetic Analysis for the Inhibition of UGT1A1, UGT1A3, and UGT1A4 by Mertansine*

Kinetic analysis was conducted to determine the *K<sup>i</sup>* values and inhibition mode of mertansine for UGT1A1, UGT1A3, and UGT1A4 enzymes. Human liver microsomes (0.1 mg/mL) were incubated with various concentrations of SN-38 (0.2–2 µM) for UGT1A1, chenodeoxycholic acid (0.5–5 µM) for UGT1A3 or trifluoperazine (0.2–2 µM) for UGT1A4, 5 mM UDPGA, 25 µg/mL alamethicin, 10 mM MgCl2, and various concentrations of mertansine (2.5, 5, 10, 20 µM for UGT1A1; 1, 2, 5, 10, 20 µM for UGT1A3; and 2.5, 5, 10, 20, 40 µM for UGT1A4) in 50 mM Tris buffer (pH 7.4) to a total incubation volume of 100 µL. Reactions were initiated by addition of UDPGA at 37 ◦C and stopped after 30 min by placing the incubation tubes on ice and adding 100 µL of internal standard (500 ng/mL meloxicam for SN-38 glucuronide and trifluoperazine *N*-β-d-glucuronide or propofol glucuronide for chenodeoxycholic acid 24-acyl-β-glucuronide) in ice-cold acetonitrile. The incubation mixtures were then centrifuged at 13,000 g for 4 min, and 50 µL of the supernatant was diluted with 50 µL of water. Aliquots (5 µL) were then analyzed using LC-MS/MS.
