ABSTRACT:Ebastine undergoes extensive metabolism to form desalkylebastine and hydroxyebastine. Hydroxyebastine is subsequently metabolized to carebastine. Although CYP3A4 and CYP2J2 have been implicated in ebastine N-dealkylation and hydroxylation, the enzyme catalyzing the subsequent metabolic steps (conversion of hydroxyebastine to desalkylebastine and carebastine) have not been identified. Therefore, we used human liver microsomes (HLMs) and expressed cytochromes P450 (P450s) to characterize the metabolism of ebastine and that of its metabolites, hydroxyebastine and carebastine. In HLMs, ebastine was metabolized to desalkyl-, hydroxy-, and carebastine; hydroxyebastine to desalkyland carebastine; and carebastine to desalkylebastine. Of the 11 cDNA-expressed P450s, CYP3A4 was the main enzyme catalyzing the N-dealkylation of ebastine, hydroxyebastine, and carebastine to desalkylebastine [intrinsic clearance (CL int ) ‫؍‬ 0.44, 1.05, and 0.16 l/min/pmol P450, respectively]. Ebastine and hydroxyebastine were also dealkylated to desalkylebastine to some extent by CYP3A5. Ebastine hydroxylation to hydroxyebastine is mainly mediated by CYP2J2 (0.45 l/min/pmol P450; 22.5-and 7.5-fold higher than that for CYP3A4 and CYP3A5, respectively), whereas CYP2J2 and CYP3A4 contributed to the formation of carebastine from hydroxyebastine. These findings were supported by chemical inhibition and kinetic analysis studies in human liver microsomes. The CL int of hydroxyebastine was much higher than that of ebastine and carebastine, and carebastine was metabolically more stable than ebastine and hydroxyebastine. In conclusion, our data for the first time, to our knowledge, suggest that both CYP2J2 and CYP3A play important roles in ebastine sequential metabolism: dealkylation of ebastine and its metabolites is mainly catalyzed by CYP3A4, whereas the hydroxylation reactions are preferentially catalyzed by CYP2J2. The present data will be very useful to understand the pharmacokinetics and drug interaction of ebastine in vivo.Ebastine, a potent and selective histamine H 1 -receptor antagonist, belongs to a second generation of nonsedating antihistamines but with negligible anticholinergic and antiserotonergic properties (Llupia et al., 2003). Ebastine undergoes extensively sequential metabolism in the liver (Hashizume et al., 1998(Hashizume et al., , 2001). The major primary metabolites identified in humans are hydroxy-and desalkylebastine, and hydroxyebastine is further metabolized to carebastine. In vitro studies indicate that the formation of desalkyl-and hydroxyebastine from ebastine is catalyzed by CYP3A4 and CYP2J2, respectively (Hashizume et al., 2002). The specific hepatic cytochrome P450 (P450) enzymes involved in hydroxy-and carebastine metabolism have not been identified so far, despite some information which could be obtained from the previously published pharmacokinetics of ebastine. After oral administration to experimental animals and humans, ebastine is almost completely metabolized to the pharmacologically active princi...