*3.3. Pharmacokinetic Analysis*

The validated method was applied to determine the concentration of LOX, cis-LOX, and trans-LOX in mice pretreated with DEX or KTC. After pretreatment, LOX was orally administered to mice (20 mg/kg) after a 12-h fasting period. The plasma concentrations of LOX, cis-LOX, and trans-LOX were significantly decreased in the DEX pretreated group (Figure 1). In the KTC pretreated group, the plasma concentrations of cis-LOX, and trans-LOX were significantly increased and the plasma concentration of LOX was also increased but not significantly (Figure 1).

The PK parameters of LOX, cis-LOX, and trans-Lox in the VH- (corn oil) and DEX-treated groups are shown in Table 1. Although blood was collected for up to 240 min, LOX and its metabolites were not detected after 60 min. The *C*max, AUC(0–60), and AUC(0– ∞) of all three compounds were significantly lowered in the DEX-pretreated group as opposed to the VH group. In the DEX-treated group, the *C*max of LOX, cis-LOX, and trans-LOX was 2.5 ± 0.2, 1.1 ± 0.2, and 2.1 ± 0.2 μg/mL, respectively, and the AUC(0–60) of the three compounds was 53.5 ± 6.1, 29.9 ± 4.4, and 67.6 ± 5.7 <sup>μ</sup>g·min/mL, respectively. However, the *T*max from all three compounds did not show any statistical di fference when compared to the VH group. In contrast to the VH group, AUC(0– ∞) of LOX, cis-LOX, and trans-Lox in the DEX-treated group indicated a lower area under plasma concentration (56.2 ± 6.9, 31.5 ± 4.4, and 85.8 ± 5.0, respectively) over an extended time period. Moreover, the elimination *<sup>T</sup>*1/2 of LOX, cis-LOX, and trans-Lox in the VH group was 14.9 ± 0.6, 12.3 ± 0.3, and 18.2 ± 0.6 min, respectively, which is significantly di fferent from the respective elimination *<sup>T</sup>*1/2 (12.0 ± 0.7, 13.9 ± 0.6, and 26.4 ± 1.6 min) generated by the DEX-pretreated group.

**Figure 1.** Mean plasma concentration versus time profiles of LOX, cis-LOX, and trans-LOX in either the presence of a CYP3A4 inducer (DEX) or inhibitor (KTC) with their respective vehicle. (**A**) Mean plasma concentration versus time profiles after *i.p.* administration of either VH (corn oil) or DEX (40 mg/kg) for 3 consecutive days. The plasma concentrations of all the compounds in the VH and DEX groups showed significant decrement up to 60 min in the DEX-treated group as compared to its VH group. The bars represent standard error (SE) (*n* = 3). (**B**) Mean plasma concentration versus time profiles after a single dose *i.p.* administration of either VH (10% ethanol) or KTC (60 mg/kg). In the KTC group, LOX, cis-LOX, and trans-LOX showed increased mean plasma concentrations as opposed to the VH group. The bars indicate standard error (SE) (*n* = 3).

Furthermore, the PK parameters for LOX, cis-LOX, and trans-LOX in the VH and KTC groups are represented in Table 2. The *C*max of cis-LOX, and trans-LOX in the VH group (1.2 ± 0.1, and 2.1 ± 0.2 μg/mL, respectively) were significantly lower than those in the KTC-treated group (1.6 ± 0.1, and 3.1 ± 0.3 μg/mL, respectively). However, the *T*max of these three compounds did not show significant variation between the VH and KTC groups. In contrast to the *T*max, the elimination *<sup>T</sup>*1/2 of trans-LOX in the VH group (26.0 ± 0.5 min) was statistically di fferent from that in the KTC-treated group (19.8 ± 0.7 min). Moreover, The AUC(0–60) for, cis-LOX, and trans-LOX in the KTC-treated group was 49.0 ± 5.9, and 80.4 ± 9.6 <sup>μ</sup>g·min/mL, respectively, which were higher than the respective values generated by the VH group. Altogether, the PK data generated by the DEX- and KTC-treated groups and their respective VH indicate that DEX and KTC significantly a ffected the PK of cis-LOX, and trans-LOX but PK of LOX was only a ffected by DEX, even though the formation of cis-LOX and trans-LOX was regulated by CR.


**Table 1.** Pharmacokinetic parameters of loxoprofen (LOX), cis-LOX, and trans-LOX in VH- (corn oil) and dexamethasone (DEX)-treated groups.

All data are expressed as the mean ± standard error (SE) (*n* = 3). *C*max: maximum plasma concentration; AUC(0*–*60): area under the plasma concentration-time curve (AUC) from 0 to 60 min; *T*max: time to reach maximum plasma concentration; *<sup>T</sup>*1/2: elimination half-life; AUC(0*–*<sup>∞</sup>): area under the plasma concentration-time curve from 0 to infinite time. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01 and \*\*\* *p* ≤ 0.001.

**Table 2.** Pharmacokinetic parameters of LOX and its metabolites in 10% ethanol (VH) and KTCtreated groups.


All data are expressed as the mean ± standard error (SE) (*n* = 3). *C*max: maximum plasma concentration; AUC(0*–*60): area under the plasma concentration-time curve (AUC) from 0 to 60 min; *T*max: time to reach maximum plasma concentration; *<sup>T</sup>*1/2: elimination half-life; AUC(0*–*<sup>∞</sup>): area under the plasma concentration-time curve from 0 to infinite time. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01 and \*\*\* *p* ≤ 0.001.

#### *3.4. Metabolism and Metabolite Identification of LOX During DEX or KTC Treatment*

The purpose of this study was to identify changes made by DEX and/or KTC on CR-mediated LOX metabolites (cis-LOX and trans-LOX) and also on CYP-mediated LOX metabolites (OH-LOX). During this PK study, OH-LOX could not be detected. Therefore, a full scan in Q Exactive Focus was used to identify all the metabolites present in plasma. A parallel reaction monitoring (PRM) mode was applied to confirm the metabolites through fragmentation patterns using collision induced dissociation (CID). Seven metabolites were confirmed after comparing the EICs of the test samples with blank plasma (Figures S3 and S4). LOX and all of its metabolites were detected in the negative ionization mode. To confirm the metabolites of LOX, their MS/MS fragmentation was checked (Figure S5). LOX (C15H17O3) was detected at a retention time of 18 min with only one major fragment ion 83.0492 (C5H7O). Trans-LOX (M1, C15H19O3) with an *m*/*z* ratio of 247.1339, eluted at 17.1 min with major fragment ions 233.1181 (C14H17O3, –CH2), 217.1230 (C14H17O2, –CH2O), 201.1279 (C14H17O), and 191.1071 (C12H15O2, –C3H4O). Cis-LOX (M2, C15H19O3), having the *m*/*z* ratio 247.1336, was detected at a retention time of 17.5 min. Its major fragments were 217.1230 (C14H17O2, –CH2O) and 191.1071 (C12H15O2, –C3H4O). M3 and M4 are OH-LOX (C15H17O4), having an *m*/*z* ratios of 261.1138 and 261.1133, and they were eluted at a retention time of 11.8 and 12.5 min, respectively. The MS/MS spectra of these metabolites were 99.0441 (C5H7O2, –C10H10O2) and 81.0335 (C5H5O, –C10H12O3) for M3 and only a single major product ion 99.0441 (C5H7O2) for M4. M5 is hydroxy trans-LOX (C15H19O4), having an *m*/*z* ratio of 263.1288, and it was detected at a retention time of 11 min. Its major fragment ions were 233.1181 (C14H17O3, –CH2O), 207.1022 (C12H15O3, –C3H4O), 133.0650 (C9H9O, –C6H10O3), and 99.0442 (C5H7O2, –C10H12O2). M6 was identified as a taurine conjugate (C17H24O5NS) whose *m*/*z* ratio was 354.1382, and it was detected at a retention time of 13.3 min. Its major fragments were 149.9859 (C3H4O4NS, –C14H20O), 124.0065 (C2H6O3NS, –C15H18O2), and 106.9798 (C2H3O3S, –C15H21O2N). M7 was a glucuronide conjugate (C21H25O9), having an *m*/*z* ratio 421.1514, and it eluted at a retention time of 14 min. Its major fragments were 245.1182 (C15H17O3), 193.0348 (C6H9O7), 175.0242 (C6H7O6), and 83.0492 (C5H7O). Parent compounds and their fragments detected during the studyarerepresentedinTable 3.


**Table 3.** Identified metabolites of LOX in mouse plasma using HRMS.

We identified the effects of CYP3A induction and inhibition on seven different known metabolites of LOX. The general characteristics of LOX, its metabolites, and their concentration in different groups are described in (Table S2). In this study, we found that the concentration of LOX, trans-LOX (M1), and cis-LOX (M2) significantly decreased in the DEX-treated group (74.1 ± 6.3%, 80.1 ± 1.2%, and 61.9 ± 3.9%, respectively) and increased in the KTC-treated group (178.2 ± 8.3%, 158.9 ± 11.9%, and 173.1 ± 5.8%, respectively). Furthermore, the concentrations of M3, M4, and M5 significantly increased in the DEX-treated group (160.5 ± 4.1%, 440.4 ± 8.3%, and 286.3 ± 11.4%, respectively), and only the concentrations of M4 and M5 decreased in the KTC-treated group (93.6 ± 1.9% and 90.2 ± 2.7%, respectively). The taurine conjugate (M6) decreased in both the DEX- (65.3 ± 2.84%) and KTC-treated (91.2 ± 2.0%) groups. In contrast, the glucuronide conjugate increased in both the DEX- (174.4 ± 6.5%) and KTC-treated (275.7 ± 14.1%) groups. M6 and M7 are phase 2 metabolites, which indicates that CYPs are not the main enzyme involved in the formation of these metabolites. The concentration of LOX and its metabolites in the VH, DEX-treated, and KTC-treated groups is represented graphically in Figure 2. DEX and KTC are well-known CYP3A modulators, and in this study, we found that the pretreatment of DEX or KTC had a significant effect on the concentration of both CYP-mediated and CR-mediated metabolites.

**Figure 2.** Comparison of loxoprofen (LOX) and its metabolites in male ICR mice treated with VH, DEX, or KTC. (**A**) The relative concentration of LOX and its metabolites after DEX administration (*i.p.* 40 mg/kg for consecutive 3 days, *n* = 3) compared to VH (*i.p.* corn oil for consecutive 3 days, *n* = 3). (**B**) The relative concentration of LOX and its metabolites after KTC administration (single dose *i.p.* 60 mg/kg, *n* = 3) compared to VH (10% ethanol, *n* = 3).

DEX and KTC affected the concentration and the PK of CYP-mediated metabolites, which, in turn, influenced the concentration and the PK of cis- and trans-LOX. The metabolites detected in this study are summarized in Figure 3.

**Figure 3.** Metabolic pathway of loxoprofen and its metabolites.
