Makino_2026_Drug.Metab.Dispos_54_100253

This study evaluated the properties of fatty acid amide hydrolase 2 (FAAH2) as a drug-metabolizing enzyme, specifically its substrate specificity and functionality. We used expression systems and various microsomes, with DS-8500a and its analogs as model substrates for xenobiotic hydrolysis. Among FAAH1, FAAH2, carboxylesterase 1, and carboxylesterase 2, FAAH2 exhibited significant hydrolytic activity, producing M20, a metabolite of DS-8500a. FAAH2 showed the highest affinity for the substrate DS-8500a with a Michaelis constant, which agreed with that observed in human liver microsomes. Furthermore, the FAAH2 protein levels in human liver microsomes showed a positive correlation with the amounts of M20 production. These findings suggested that FAAH2 is the primary enzyme responsible for metabolizing DS-8500a. We also found that human liver microsomes showed interindividual variability in FAAH2 activity with approximately a 15-fold maximal difference and a different extent of inhibitory effect by an FAAH2 inhibitor on M20 formation, possibly affecting the pharmacokinetic profile of FAAH2 substrates. Experiments using DS-8500 analogs suggested that structural modifications in the ethanolamide moiety influence the hydrolytic activity of FAAH2. Modifications to this moiety led to alterations not only in the hydrolytic activity of FAAH2 but also in the substrate specificity for FAAH1. In conclusion, we have demonstrated for the first time that FAAH2 catalyzes the hydrolysis of xenobiotics. This hydrolytic enzyme may affect the pharmacokinetics of FAAH2 substrates. SIGNIFICANCE STATEMENT: This study focuses on fatty acid amide hydrolase 2 (FAAH2) as a hydrolytic enzyme capable of metabolizing not only fatty acid amides but also xenobiotics, using DS-8500a as a model substrate. This work highlights the property of FAAH2 as a drug-metabolizing enzyme, emphasizing the need for evaluations with inhibitors such as cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester when xenobiotics are hydrolyzed. In addition, the quantification of FAAH2 protein levels and the assessment of individual variability provide insights into the influence of FAAH2 on drug metabolism across different individuals.