**3. Results**

#### *3.1. Identification of Loxoprofen and Its Metabolites*

To determine the plasma concentration of LOX and its metabolites via LC-MS/MS analysis, the produced product ions were checked and optimized for each compound. The ion intensities of LOX, cis-LOX, and trans-LOX were high in the negative mode of ionization; therefore, all the conditions

for analysis used the negative mode for LC-MS/MS analysis. The MRM transitions chosen for LOX, cis-LOX, and trans-LOX were *m*/*z* 245.0 → 83.1, 247.1 → 202.2, and 247.1 → 203.1, respectively [15]. Representative MRM chromatograms of LOX, cis-LOX, trans-LOX, and the IS in mouse plasma are presented in Figure S1. LOX, cis-LOX, trans-LOX, and the IS were eluted at 8.5, 8.0, 8.2, and 8.9 min, respectively. No endogenous sources of interference were observed. LOX and its two metabolites were evaluated for linearity, precision, and accuracy. The calibration curves calculated within the range of 0.1–40.0 μg/mL for LOX and within the range of 0.2–40.0 μg/mL for cis-LOX and trans-LOX were linear. The precision (RSD %) range of LOX, cis-LOX, and trans-LOX was 1.8–12.9%, and the accuracy (RE %) range was less than 14.7% (Table S1). Thus, the values were within the acceptable range and the method was accurate and precise.

#### *3.2. Evaluation Model for Determination of LOX–Drug Interaction*

To evaluate the CYP3A-induced LOX interaction, we prepared an experimental model that regulated CYP3A activity by administering an inducer (DEX) and an inhibitor (KTC) of CYP3A into mice. Briefly, DEX in corn oil was administered up to 3 consecutive days to induce CYP3A. Inhibition of CYP3A was induced by a single dose of KTC in 10% ethanol. Only vehicle groups were administered with their respective solvents without the addition of a CYP3A inducer or inhibitor. The induction and inhibitionofCYP3AwereconfirmedwithCYPassayusingfivedi fferentprobesubstrates(FigureS2).

DEX increased the metabolism of CYP3A substrate (midazolam) by approximately 10-fold when compared to the vehicle (VH) group. KTC significantly decreased the metabolism of CYP3A4 substrate (midazolam) when compared to the VH group (Figure S2). However, the metabolic activities of other CYP enzymes were una ffected between VH and treated groups. Thus, we validated our method of specifically regulating CYP3A activity via DEX and KTC.
