*2.4. Induction of Mertansine on Human Major CYPs and UGTs in Human Hepatocytes*

Plateable cryopreserved human hepatocytes (lots 319, 321, and 361) were thawed in cryopreserved hepatocyte recovery medium according to the manufacturer's protocol.

#### 2.4.1. Cytotoxicity of Mertansine in Human Hepatocytes

To estimate the cytotoxicity of mertansine, viable hepatocytes (lot 319) cells were seeded in a collagen type 1 precoated 96-well plate in 100 µL of hepatocyte plating medium (6 × 10<sup>4</sup> cells/well) and incubated for 4 h at 37 ◦C in 5% CO2. Next, the plating medium was removed, and a matrigel medium containing 0.25 mg/mL of Matrigel™ matrix was applied to each cell prior to incubation for 24 h at 37 ◦C in 5% CO2. The hepatocytes were incubated with 0.0125, 0.0625, 0.125, 0.250, 0.625, 1.25, 2.5, and 6.25 µM mertansine in triplicate for 48 h at 37 ◦C in 5% CO2. The medium was exchanged with fresh medium containing mertansine every 24 h. Then, 20 µL of MTS solution was added to each well and the plate was incubated for 1 h at 37 ◦C in 5% CO2. The absorbance of the reaction mixture was measured at 492 nm.

#### 2.4.2. Treatment of Mertansine in Human Hepatocytes

To evaluate the induction effect of mertansine on drug-metabolizing enzymes, three different cryopreserved human hepatocytes (lots 319, 321, and 361) were thawed in cryopreserved hepatocyte recovery medium, and viable cells were seeded in collagen type 1 precoated 48-well plates in 250 µL of hepatocyte plating medium (6 × 10<sup>5</sup> cells/well) and incubated for 4 h at 37 ◦C in 5% CO2. Next, the plating medium was removed and replaced with matrigel medium containing 0.25 mg/mL of Matrigel™ matrix prior to incubation at 37 ◦C for 24 h. The hepatocytes were incubated with 1.25, 12.5, 125, 625, 1250, and 2500 nM mertansine, vehicle (0.1% DMSO in hepatocyte culture media), and prototypical inducers including 50 µM omeprazole, 10 nM CITCO, and 10 µM rifampicin in triplicate. Samples were incubated for 48 h at 37 ◦C in 5% CO2, and the medium was exchanged with 250 µL of fresh medium containing drugs or the vehicle every 24 h.

#### 2.4.3. CYP1A2, CYP2B6, and CYP3A4 Activity Measurement

The effects of mertansine on CYP1A2, CYP2B6, and CYP3A4 activities were evaluated. Plates were prepared with a vehicle, omeprazole, CITCO, rifampin, and mertansine, and incubated for 48 h. Next, 150 µL of a CYP cocktail solution containing 40 µM phenacetin (CYP1A2 substrate), 20 µM bupropion (CYP2B6 substrate), and 20 µM midazolam (CYP3A4 substrate) in William's E buffer was added to each well and incubated for 30 min, and then 100 µL aliquots of the incubate from each well were stored at −80 ◦C until LC-MS/MS analysis. <sup>13</sup>C2, <sup>15</sup>N-acetaminophen (0.1 µg/mL, IS for acetaminophen), and d9-1′ -hydroxybufuralol (0.01 µg/mL, IS for hydroxybupropion and 1 ′ -hydroxymidazolam) in methanol were added to 50 µL of the medium obtained from each well. Mixtures were vortexed for 2 min and then centrifuged at 13,000 g for 4 min at 4 ◦C. The supernatant (40 µL) was diluted with 60 µL of deionized water and then mixed for 2 min by vortexing. An aliquot (5 µL) was analyzed using LC-MS/MS [37], and CYP1A2, CYP2B6, and CYP3A4 enzyme activities were expressed as formation rates (pmol/million cells/min).

#### 2.4.4. RNA Purification and RT-PCR Analysis

At the end point of the experiment, total RNA was immediately isolated using an RNeasy Micro Kit, and RNA concentration and purity were determined using an absorbance test at 260 nm/280 nm using a NanoVue Plus spectrophotometer (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). Samples were stored at −80 ◦C until RT-PCR analysis.

RT-PCR analysis was performed using an RT-PCR detection system (Bio-Rad, Hercules, CA, USA) with a TaqMan® RNA-to-CT™ 1-Step Kit and TaqMan® Gene Expression Assay Kits (CYP1A2, Hs01070369\_m1; CYP2B6, Hs03044634\_m1; CYP3A4, Hs00430021\_m1; CYP2C8, Hs00426387\_m1; CYP2C9, Hs00426397\_m1; CYP2C19, Hs00559368\_m1; UGT1A1, Hs02511055\_s1; UGT1A4, Hs01655285\_s1; UGT1A9, Hs02516855\_sH) according to the manufacturer's protocol. Total RNA (15 ng) in each reaction sample was used for RT-PCR: 25 min for reverse transcription at 48 ◦C, 15 min for enzyme activation at 95 ◦C, 44 cycles of denaturation (each 15 s) at 95 ◦C, and 1 min annealing/extension at 60 ◦C. The relative threshold cycle (∆Ct) values of all samples, including CYP1A2, CYP2B6, CYP3A4, CYP2C8, CYP2C9, CYP2C19, UGT1A1, UGT1A4, and UGT1A9, were normalized to the ∆C<sup>t</sup> value of glyceraladehyde 3-phosphate dehydrogenase (GAPDH). The relative mRNA abundance was then calculated with the normalized relative C<sup>t</sup> value (∆∆Ct) of each sample using the formula: 2−(∆∆Ct) .

#### *2.5. Data Analysis*

The percentage changes in enzymatic activities were calculated as (CYP activity with test compound treatment/CYP activity with vehicle control treatment) × 100. The IC<sup>50</sup> (the concentration of the inhibitor needed for half-maximal inhibition) values were calculated using SigmaPlot ver. 12.5 (Systat Software, Inc.; San Jose, CA, USA). *K*<sup>i</sup> (the inhibition constant) and the inhibition mode of UGT1A1, UGT1A3, and UGT1A4 activities were determined using Enzyme Kinetics ver. 1.1 (Systat Software, Inc.).
