*2.1. Materials*

LOX (2-(4-((2-Oxocyclopentyl)methyl)phenyl)propanoic acid, C15 H18 O3, CAS ID 68767-14-5) was procured from Tokyo Chemicals Industry (Tokyo, Japan). LOX was used to synthesize cis-LOX and trans-LOX ((2*S*)-2-[4-[[(1 *<sup>R</sup>*,2*S*)-2-hydroxycyclopentyl]methyl]phenyl]propanoic acid, C15 H20 O3, CAS ID 83648-76-4) with purities of 96.5% and 97.9%, respectively [18]. DEX, KTC, dextromethorphan, and phenacetin were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA) while midazolam was procured from Bukwang Pharmaceutical Co., Ltd. (Seoul, Korea). Mass spectrometry (MS) grade water and acetonitrile (ACN) were obtained from Fischer Scientific (Pittsburgh, PA, USA).

#### *2.2. Animal Treatments and Sample Preparations*

Male ICR mice (36 mice of 5 weeks) were purchased from Orient Co. (Seongnam, Korea) andwere randomly divided into 4–6 mice per cage. Then, the mice were acclimatized for 1 week in a controlled environment (relative humidity: 60%, temperature: 25 ◦C) under a 12-h/12-h light/dark cycle and supplied standard rodent chow and tap water freely. All animal handling procedures followed protocols issued by the Society of Toxicology (USA, 1989) and were approved on March 21, 2019 by the Institutional Review Board of Kyungpook National University (project ID # 2019-41).

The acclimatized animals were divided into eight groups, with each group containing 3 mice: groups I and II, vehicle (corn oil); groups III and IV, DEX-treated; groups V and VI, vehicle (10% Ethanol); and groups VII and VIII, KTC-treated group. Briefly, groups I and II were intraperitoneally (*i.p*.) treated with corn oil for 3 days while DEX (dissolved in corn oil and administered at 40 mg/kg) was given to groups III and IV for 3 consecutive days [19]. Then, animals were fasted for 12 hours with free access

to water before starting the experiment. After fasting, LOX (20 mg/kg) was administered orally to groups I, II, III, and IV. Ethanol (10%) and KTC (60 mg/kg) were administered *i.p.* to the vehicle-treated groups (V and VI) and KTC-treated groups (VII and VIII), respectively [20], and after 3 min, LOX was given orally (20 mg/kg).

After the oral administration of LOX, blood from groups I, III, V, and VII was collected from the tail at 0, 5, 10, 15, 30, 60, 120, and 240 min post-administration and placed into sodium heparin-containing tubes. After the last blood collection, the mice were sacrificed by cervical dislocation. Plasma was then prepared by centrifuging the blood at 4000× *g* for 15 min at 4 ◦C and stored at −80 ◦C until analysis. For analysis, each sample was prepared by mixing plasma (10 μL) and 90 μL of ACN containing 0.1% formic acid and 5 μM of tolbutamide as an internal standard (IS). The samples were then vortexed and centrifuged at 13,000× *g* for 10 min at 4 ◦C. Finally, 10 μL of sample supernatant was injected into the LC-MS/MS system. The PK parameters (the maximum plasma concentration [*C*max], time to reach the maximum plasma concentration [*T*max], elimination half-life [*T*1/2], and area under the plasma concentrations [AUC]) were analyzed by WinNonlin software (Version 2.1, Scientific Consulting, Louisville, KY, USA).

Similarly, blood from groups II, IV, VI, and VIII was collected from the hepatic portal vein 10 min post-LOX administration to identify metabolites of LOX and to analyze drug–drug interactions. Plasma from these samples was prepared as explained above. Then, 300 μL of ACN having 0.1% formic acid and 5 μM of the IS were mixed with 100 μL of each plasma sample. Next, the samples were vortexed and centrifuged at 13,000× *g* for 10 min at 4 ◦C. Supernatants were transferred into tubes and dried using a Labconco Speed Vac (Labconco Corporation, Kansas City, MO, USA). The dried samples were reconstituted using 100 μL of 50% methanol and centrifuged at 13,000× *g* for 10 min at 4 ◦C. Each supernatant was transferred into an LCMS vial, and 5 μL were injected into a high-resolution mass spectrometer (HRMS).

#### *2.3. CYP Activities in the Mouse Liver*

Male ICR mice were divided into 4 groups with each group containing 3 mice: group I, vehicle (corn oil); group II, DEX-treated; group III, vehicle (10% Ethanol); and group IV, KTC-treated group. Group I was treated with corn oil for 3 days while DEX (dissolved in corn oil and administered at 40 mg/kg) was administered *i.p.* to group II for 3 consecutive days [19]. Similarly, group III and group IV were treated once with 10% ethanol or KTC (60 mg/kg), respectively [20]. Twenty-four hours after the last treatment, the liver was excised and homogenized with three volumes of ice-cold 0.1 M potassium phosphate buffer (pH 7.4). The supernatant fraction was then separated as the S9 fraction from the mixture by centrifugation at 9000× *g* at 4 ◦C and stored at −80 ◦C until use.

To characterize the CYP activities in the harvested livers, phenacetin *O*-demethylation (using 80 μM of phenacetin) for CYP1A, dextromethorphan *O*-demethylation (using 5 μM of dextromethorphan) for CYP2D, and midazolam 1'-hydroxylation (using 5 μM of midazolam) for CYP3A were used as cocktail probe reactions [21,22]. The level of protein in the S9 fraction was determined by Bradford assay [23]. The cocktail probes were then incubated with 10 μL of each S9 fraction, potassium phosphate buffer (pH 7.4), and an NADPH generating system (NGS) in a final volume of 100 μL. After incubation for 30 min at 37 ◦C, ice-cold ACN containing 0.1% formic acid and IS (5 μM) was added to stop the reaction. Samples were then vortexed and centrifuged at 13,000× *g* for 10 min at 4 ◦C. Finally, the supernatants were transferred to LC-MS/MS vials for analysis.

#### *2.4. Instrument and Data Acquisition*

A Shimadzu Prominence UFLC system (Kyoto, Japan) connected to a TSQ vantage triple quadrupole mass spectrometer with a HESI-II spray source incorporated with a DGU-20A5 degasser, an LC-20AD pump, a SIL-20A autosampler, and a CTO-20A column oven was used for the analyses. A shim-pack GIS C18 column (150 × 3.0 mm, 3 μM) was used to separate analytes in the samples. Mobile phases A and B were composed of water with formic acid and ACN with 0.1% formic acid, respectively, and the flow rate was 0.50 mL/min at 40 ◦C. The gradient conditions were as follows: 20% of B between 0 and 0.25 min, 20–80% of B between 0.25 and 9.75 min, 80–20% of B between 9.75 and 10 min, and 20% of B between 10 and 13 min. The MS was operated under the following conditions: electrospray ionization in negative mode at 3.0 kV, capillary temperature at 350 ◦C, vaporizer temperature at 300 ◦C, sheath gas pressure at 35 Arb, and auxiliary gas pressure at 10 Arb. Finally, Xcalibur software (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for data analysis.

HRMS coupled with ultrahigh performance liquid chromatography (UHPLC) was used to detect hydroxy-LOX and other metabolites of LOX. The UHPLC system, Dionex Ultimate 3000 (Dionex Softron GmbH, Germering, Germany) consisted of an HPG-3200SD Standard binary pump, a WPS 3000 TRS analytical autosampler, and a TCC-3000 SD column compartment. In this experiment, the HRMS was a Q Exactive Focus quadrupole-Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization (HESI-II) ion probe.

LOX was detected as a deprotonated ion [M-H]− at *m*/*z* 245.1175 in negative ion mode. Therefore, negative ion mode was used with the following optimized conditions for LOX: spray voltage of 2.5 kV, capillary temperature of 320 ◦C, auxiliary gas at 12 aux unit, aux gas heater temperature of 200 ◦C, sheath gas at 35 aux units, and S-lens RF level of 50. LOX and its metabolites were separated using a 150 mm × 2.1 mm, 2.6-μm reverse-phase liquid chromatography column, Kinetex® C18 column (Phenomenex, CA, USA) at 40 ◦C. Furthermore, MS-grade solvents were used as the mobile phase in gradient elution mode: 0.1% aqueous formic acid as Solvent A and 0.1% formic acid in ACN as Solvent B. The flow was set to 0.22 mL/min with a gradient condition of Solvent B as 10% between 0 and 0.5 min, 10–50% between 0.5 and 21.5 min, 50–95% between 21.5 and 22.5 min, 95% between 22.5 and 25.5 min, 95–10% between 25.5 and 25.6 min, and 10% between 25.6 and 30 min.
