**4. Discussion**

LOX is a nonselective COX inhibitor that is administered for the managemen<sup>t</sup> of pain and inflammation, and it is well tolerated by patients [2,24,25]. Although the pharmacokinetics and metabolism of LOX are well defined, its interaction(s) with CYP enzymatic pathways have not been fully characterized [3,6–10,13,15,26–29]. However, CYP3A regulating commercial drugs are widely in clinical use [16,17] and there may be the chance to use CYP3A-regulating drugs and LOX together. Although CYP3A does not generate the LOX active metabolite, it may be possible to influence its generation by regulating CYP3A activity. To that end, we evaluated whether the rate of LOX active metabolite formation is influenced by CYP3A activity. We also investigated the e ffects of DEX and KTC, which are widely used CYP3A modulators, on the metabolism and PK of LOX, cis-LOX, and trans-LOX.

DEX is a steroidal drug used in the treatment of many conditions such as skin diseases, allergies, rheumatic disorder, asthma, and certain autoimmune diseases [30,31]. It is also e ffective in treating various cancers such as central nervous system tumors, brain metastases, advanced melanoma, leukemia, lymphoma, and multiple myeloma. Additionally, DEX is e ffective at combating the side effects of chemotherapy [32,33]. DEX is metabolized by CYP3A4 and CYP17A, but the CYP17A metabolic pathway plays no major role in its in vivo metabolism [34–36]. DEX has been used as a CYP3A4 inducer in clinical studies. Interestingly, rifampicin, rifabutin, phenytoin, phenobarbital, primidone, carbamazepine, etc., are strong CYP3A4 inducers [16]. It has also been reported that a single dose (50 mg/kg) of DEX can induce the activity of CYP3A. However, persistent administration of DEX could make stable induction of CYP3A activity, which was independent of inducer administration [37,38]. Moreover, chronic DEX administration likely leads to the autoinduction of CYP3A, which has a direct impact on the PK parameters of DEX [38]. Nevertheless, patients may su ffer from an increase in substrate concentration after they stop using the CYP3A inducer if they take both the substrate (LOX) and the inducer (DEX) of CYP3A [37].

KTC is a broad-spectrum antifungal drug used in the treatment of many fungal infections such as blastomycosis, coccidioidomycosis, histoplasmosis, etc. [39,40]. KTC is a known reversible inhibitor of CYP3A4 [16] and displays hepatotoxicity through immune-allergic mechanisms, which limits its therapeutic use [41,42]. Usually, CYP3A4 inhibitors are divided into reversible inhibitors (such as KTC, itraconazole, terfenadine, astemizole, and quinidine) [43] and irreversible inhibitors (such as gestodene and levonorgestrel) [44]. Many studies also reported the biotransformation of KTC through oxidation, O-dealkylation, hydroxylation, and FMO (via the UGT1A4 metabolic pathway) [42,45]. Furthermore, one study revealed that KTC showed dose-dependent kinetics, indicating that its metabolism occurs in the liver [46]. A recent study also revealed that KTC metabolism is similar in humans and mice, which may help to resolve the issue regarding drug metabolism and toxicology [42]. Nevertheless, a sudden increase or decrease in the *C*max of drugs and toxic metabolites could result from either the induction or inhibition of metabolizing enzymes [47].

CYP3A4 is the major CYP involved in LOX metabolism, but it does not form its active metabolite [15]. The active metabolite of LOX (trans-LOX) is formed via CR pathway [1,5]. Similarly to previous studies, we also found that DEX and KTC were strong CYP3A activity modulators [36,48,49]. In addition, we determined that the *C*max, AUC(0–60), and AUC(0–∞) of LOX, cis-LOX, and trans-LOX were significantly decreased in DEX-treated mice. However, the *C*max, AUC(0–60), and AUC(0–∞) of cis-LOX, and trans-LOX are significantly increased in KTC-treated mice. Furthermore, DEX increased the concentration of the OH-LOX metabolite and decreased the concentration of the active metabolite. This may lead to a decrease in the activity of trans-LOX during the subsequent administration of LOX and DEX. We also observed that changes in CYP pathway activity can modulate CR pathway activity regarding LOX, even though CYP3A4 does not participate in LOX active metabolite formation.

These results indicate that the co-administration of LOX and DEX may lead to a decrease in the pharmacological activity of LOX by decreasing the concentration of the active metabolite. We also observed that the concentration of active metabolites catalyzed by CR increased and that the concentration of inactive metabolites decreased during the co-administration of LOX and the CYP3A inhibitor, KTC. Therefore, if LOX and KTC are administered together, the pharmacological activity of LOX may be enhanced by increasing the active metabolite. Subsequently, this may also increase toxicity. In a previous study, LOX was associated with an increase in small bowel mucosal injury, erosive gastritis, gastroduodenal ulcers, etc., during concomitant use of a proton pump inhibitor such as lansoprazole [1,50,51]. Since lansoprazole is a known inhibitor of CYP3A and CYP2C19 [52,53], these side effects may have been caused by changes in the blood levels of LOX and its active metabolite caused by the concomitant use of LOX and lansoprazole. It has been reported that LOX slightly inhibited the metabolism of tacrolimus by interacting with CYP3A in human liver microsomes [54]. Similarly, tizanidine is typically used to manage muscle spasticity and pain [55]. However, it has been reported that combination therapy of tizanidine and LOX for neck pain increased the risk of irreversible symptomatic bradycardia via CYP inhibition [56].

These previous studies clearly show that LOX can interact with drugs that modulate CYP activity even though its active metabolite is not formed by CYP enzymes. Considering the pharmacokinetics and metabolism of LOX and its metabolites, the use of LOX with CYP modulators (e.g., DEX and KTC) may result in the decreased pharmacological action of LOX or may cause from minor toxicity to major toxicity by interacting with CYP3A.
