Human Metabolism of Nebicapone (BIA 3-202), a Novel Catechol-O-Methyltransferase Inhibitor: Characterization of in Vitro Glucuronidation

Abstract

ABSTRACT:Nebicapone (BIA 3-202; 1-[3,4-dihydroxy-5-nitrophenyl]-2-phenylethanone), a novel catechol-O-methyltransferase inhibitor, is mainly metabolized by glucuronidation. The purpose of this study was to characterize the major plasma metabolites of nebicapone following p.o. administration of nebicapone to healthy volunteers, and to determine the human UDP-glucuronosyltransferase (UGT) enzymes involved in nebicapone glucuronidation. Plasma samples were collected as part of a clinical trial at different time points postdose and were analyzed for nebicapone and its metabolites using a validated method consisting of a solid-phase extraction, followed by high-performance liquid chromatography/mass spectrometry detection. The primary metabolic pathways of nebicapone in humans involve mainly 3-O-glucuronidation, the major early metabolite, and 3-O-methylation, the predominant late metabolite. Of the nine commercially available recombinant UGT enzymes studied (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15), only UGT1A9 exhibited high nebicapone glucuronosyltransferase specific activity (24.3 ؎ 1.3 nmol/mg protein/min). UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7, and UGT2B15 exhibited low activity (0.1-1.1 nmol/mg protein/min), and UGT1A1 and UGT1A3 showed extremely low activities (less than 0.03 nmol/mg protein/min). The results show that nebicapone is mainly glucuronidated in humans and that multiple UGT enzymes are involved in this reaction.L-3,4-dihydroxyphenylalanine (L-DOPA) therapy has revolutionized treatment of idiopathic Parkinson's disease (PD) by providing a p.o. administered source of dopamine precursor with ready access to the brain, and it remains the most widely used palliative drug for PD. However, L-DOPA is metabolized in the periphery by aromatic Lamino acid decarboxylase (AADC) and catechol-O-methyltransferase (COMT) enzymes, reducing brain availability. Therefore, an improvement in L-DOPA therapy is provided by a combination treatment of L-DOPA with an AADC inhibitor plus a COMT inhibitor, effectively increasing its availability to the brain (Backstrom et al., 1989).Second-generation COMT inhibitors entacapone and tolcapone ( Fig. 1) have proven beneficial when used together with L-DOPA and an AADC inhibitor, such as carbidopa or benserazide, in the medical treatment of patients with PD (Dingemanse et al., 1995;Dingemanse, 1997;Heikkinen et al., 2001Heikkinen et al., , 2002. When metabolized, these nitrocatechol derivatives are extensively conjugated, usually undergo direct glucuronidation catalyzed by UDP-glucuronosyltransferases (UGT), and are then rapidly excreted in the urine (Lautala et al., 1997). Additionally, both methylation and sulfation compete with glucuronidation for conjugation of the adjacent phenolic hydroxyls (Lautala et al., 1997). Major metabolites of tolcapone in human plasma are the 3-O-␤-glucuronic acid (ϳ18.6%) and the 3-O-methyl conjugate (ϳ2.1%). In urine, the 3-O-␤-glucuronic conjugates of tolcapone (ϳ13%) and its derivative N-acetyl amino (ϳ5.7...

UDP-Glucuronosyltransferases 1A6 and 1A9 are the Major Isozymes Responsible for the 7-O-Glucuronidation of Esculetin and 4-Methylesculetin in Human Liver Microsomes

“…These results demonstrated that the UGTs primarily responsible for the glucuronidation of ET and 4-ME in vitro were UGT1A6 and UGT1A9. It was demonstrated that UGT1A6, UGT1A9, and UGT2B7 can be involved in the glucuronidation of hydroxycoumarins (Antonio et al, 2003;Loureiro et al, 2006). However, UGT2B7 was not found to be involved in glucuronidating ET and 4-ME in our study.…”

Identification and Characterization of Human UDP-Glucuronosyltransferases Responsible for the In Vitro Glucuronidation of Daphnetin

“…For example, 4-methylumbelliferone has been widely used as a nonselective probe for evaluation of glucuronidation activities (UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17) because of its high affinity for UGTs and turnover for the formation of the metabolite (Uchaipichat et al, 2004). With respect to the catechol compounds, UGT1A6, UGT1A9, and UGT2B7 can potentially be involved in their glucuronidation (Antonio et al, 2003;Loureiro et al, 2006). Daphnetin is a hydroxycoumarin derivative as well as a catechol-containing compound.…”

“…Structurally, daphnetin contains a catechol group on its basic skeleton, which could be susceptible to metabolism by phase II enzymes, such as UDP-glucuronosyltransferases (UGTs), sulfotransferases, and catechol-O-methyltransferases (Antonio et al, 2002(Antonio et al, , 2003. Many glucuronidation derivatives of catechol-containing drugs have been detected both in vitro and in vivo, e.g., L-3,4-dihydroxyphenylalanine, a therapeutic drug for Parkinson's disease (Antonio et al, 2002(Antonio et al, , 2003; protocatechuic aldehyde, a antiasthmatic agent ; and 2-((2-(1H-imidazol-1-yl)-6-methylpyrimidin-4-yl) (3-((benzo-[d] [1,3]dioxol-5-ylmethyl)(methyl)amino)propyl)amino)acetamide , an inhibitor of nitric oxide synthase (Loureiro et al, 2006). On the other hand, it was noted in previous studies that 7-hydroxycoumarin and 4-methylumbelliferone, the structural analogs of daphnetin, are substrates for the UGTs with high affinity (Uchaipichat et al, 2004;Wang et al, 2006).…”

Pharmacokinetics, Disposition, and Metabolism of [14C]-Nebicapone in Humans

“…Within 12 hours nebicapone was mostly metabolised to 3-O-ß-glucuronic acid derivative in human plasma, with 3-O-methylation of parent accounting for initially lower but later more sustained plasma metabolite levels. Other minor nebicapone metabolites identified included 3-O-sulfate and acetamino derivatives, accounting for approximately 1% of the total nebicapone and metabolites AUC [17].…”

“…While metabolism studies with nebicapone in humans have suggested that glucuronide conjugation is the major route of elimination through the action of multiple UGT enzymes [17], a human metabolism study using [ 14 C]-labelled nebicapone was undertaken to further elucidate the routes of metabolism and the rate of excretion as further support for the clinical development of this compound. Plasma and blood drug and metabolite concentrations were estimated and additionally mass balance data was obtained for routes of elimination.…”