A Long-Standing Mystery Solved: The Formation of 3-Hydroxydesloratadine Is Catalyzed by CYP2C8 But Prior Glucuronidation of Desloratadine by UDP-Glucuronosyltransferase 2B10 Is an Obligatory Requirement

Abstract

Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating long-lasting antihistamine that is widely used for the treatment of allergic rhinitis and chronic idiopathic urticaria. For over 20 years, it has remained a mystery as to which enzymes are responsible for the formation of 3-hydroxydesloratadine, the major active human metabolite, largely due to the inability of any in vitro system tested thus far to generate this metabolite. In this study, we demonstrated that cryopreserved human hepatocytes (CHHs) form 3-hydroxydesloratadine and its corresponding O-glucuronide. CHHs catalyzed the formation of 3-hydroxydesloratadine with a K m of 1.6 mM and a V max of 1.3 pmol/min per million cells. Chemical inhibition of cytochrome P450 (P450) enzymes in CHHs demonstrated that gemfibrozil glucuronide (CYP2C8 inhibitor) and 1-aminobenzotriazole (general P450 inhibitor) inhibited 3-hydroxydesloratadine formation by 91% and 98%, respectively. Other inhibitors of CYP2C8 (gemfibrozil, montelukast, clopidogrel glucuronide, repaglinide, and cerivastatin) also caused extensive inhibition of 3-hydroxydesloratadine formation (73%-100%). Assessment of desloratadine, amodiaquine, and paclitaxel metabolism by a panel of individual CHHs demonstrated that CYP2C8 marker activity robustly correlated with 3-hydroxydesloratadine formation (r 2 of 0.70-0.90). Detailed mechanistic studies with sonicated or saponin-treated CHHs, human liver microsomes, and S9 fractions showed that both NADPH and UDP-glucuronic acid are required for 3-hydroxydesloratadine formation, and studies with recombinant UDP-glucuronosyltransferase (UGT) and P450 enzymes implicated the specific involvement of UGT2B10 in addition to CYP2C8. Overall, our results demonstrate for the first time that desloratadine glucuronidation by UGT2B10 followed by CYP2C8 oxidation and a deconjugation event are responsible for the formation of 3-hydroxydesloratadine.

Further Characterization of the Metabolism of Desloratadine and Its Cytochrome P450 and UDP-glucuronosyltransferase Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor

“…The N-glucuronide of desloratadine is highly unstable (both before and after 3-hydroxylation by CYP2C8). We were unable to detect desloratadine N-glucuronide or 3-hydroxydesloratadine N-glucuronide by LC-MS/MS in our previous study (Kazmi et al, 2015). Instability is a characteristic property of certain N-glucuronides (Ciotti et al, 1999).…”

“…Metabolic scheme for the conversion of desloratadine to 3-hydroxydesloratadine. Desloratadine is converted to an unstable N-glucuronide by UGT2B10 followed by CYP2C8-dependent hydroxylation to 3-hydroxydesloratadine as described previously (Kazmi et al, 2015).…”

“…Samples were analyzed by LC-MS/MS for CYP2C8 activity toward amodiaquine and paclitaxel and 3-hydroxydesloratadine formation as described previously (Kazmi et al, 2015). Briefly, samples were analyzed by LC-MS/MS with a Shimadzu LC system (Columbia, MD) interfaced by electrospray ionization (ESI) with an AB Sciex API4000 QTrap mass spectrometer (Foster City, CA) in positive mode with the multiple reaction monitoring (MRM) scan type.…”

“…Both 3-hydroxydesloratadine and its O-glucuronide conjugate are major in vivo metabolites excreted in approximately equal amounts in urine and feces (Ramanathan et al, 2007).Conventional in vitro systems such as recombinant P450 enzymes, human liver microsomes (HLM), and human liver S9 fractions do not convert desloratadine to 3-hydroxydesloratadine; hence, its enzymology and the basis for certain individuals being identified as poor metabolizers of desloratadine have remained a mystery for many years (Prenner et al, 2006;Ghosal et al, 2009). Previously, we demonstrated that cryopreserved human hepatocytes (CHH) can form 3-hydroxydesloratadine, as can HLM provided they are supplemented with both NADPH and UDPglucuronic acid (UDP-GlcUA) (Kazmi et al, 2015). As shown in Fig.…”

Human UDP-Glucuronosyltransferase (UGT) 2B10: Validation of Cotinine as a Selective Probe Substrate, Inhibition by UGT Enzyme-Selective Inhibitors and Antidepressant and Antipsychotic Drugs, and Structural Determinants of Enzyme Inhibition

“…Although initially considered an orphan enzyme, more recent studies have shown that UGT2B10, like UGT1A4, catalyzes the N-glucuronidation of a number of xenobiotics that incorporate an aliphatic tertiary amine or aromatic N-heterocyclic group (Kaivosaari et al, 2011). Known substrates are nicotine and its oxidation product cotinine (Chen et al, 2007;Kaivosaari et al, 2007); desloratadine (Kazmi et al, 2015a); medetomidine (Kaivosaari et al, 2008); the tricyclic antidepressants (TCAs) amitriptyline, clomipramine, imipramine, and trimipramine (Chen et al, 2007;Zhou et al, 2010;Kato et al, 2013); several tobacco-specific nitrosamines (Chen et al, 2008); RO5263397 (Fowler et al, 2015); and miscellaneous drugs that include diphenhydramine, ketoconazole, ketotifen, midazolam, olanzapine, pizotifen, and tamoxifen (Erickson-Ridout et al, 2011;Kato et al, 2013). Consistent with the known selectivity of UGT1A4 for N-glucuronidation (Kubota et al, 2007), most, if not all, UGT2B10 substrates are additionally glucuronidated by UGT1A4 and biphasic kinetics are frequently observed when human liver microsomes (HLM) are used as the enzyme source (Kaivosaari et al, 2011;Kato et al, 2013).…”