title: Involvement of CYP2A6 in the formation of a novel metabolite, 3-hydroxypilocarpine, from pilocarpine in human liver microsomes.
abstract: Pilocarpine is a cholinergic agonist that is metabolized to pilocarpic acid by serum esterase. In this study, we discovered a novel metabolite in human urine after the oral administration of pilocarpine hydrochloride, and we investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by liquid chromatography-tandem mass spectrometry and NMR analyses and by comparing to the authentic metabolite. To clarify the human cytochrome P450 (P450) responsible for the metabolite formation, in vitro experiments using P450 isoform-selective inhibitors, cDNA-expressed human P450s (Supersomes; CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4), and liver microsomes from different donors were conducted. The formation of 3-hydroxypilocarpine in human liver microsomes was strongly inhibited (>90%) by 200 microM coumarin. Other selective inhibitors of CYP1A2 (furafylline and alpha-naphthoflavone), CYP2C9 (sulfaphenazole), CYP2C19 [(S)-mephenytoin], CYP2E1 (4-methylpyrazole), CYP2D6 (quinidine), and CYP3A4 (troleandomycin) had a weak inhibitory effect (<20%) on the formation. The highest formation activity was expressed by recombinant CYP2A6. The K(m) value for recombinant CYP2A6 was 3.1 microM, and this value is comparable with that of human liver microsomes (1.5 microM). The pilocarpine 3-hydroxylation activity was correlated with coumarin 7-hydroxylation activity in 16 human liver microsomes (r = 0.98). These data indicated that CYP2A6 is the main enzyme responsible for the 3-hydroxylation of pilocarpine. In conclusion, we identified a novel metabolite of pilocarpine, 3-hydroxypilocarpine, and we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.
authors.affiliation: Pharmacokinetics Research, Kissei Pharmaceutical Co., Ltd., 19-48 Yoshino Matsumoto-city, Nagano 399-8710, Japan. takuro_endo@pharm.kissei.co.jp
authors.forename: Takuro
authors.initials: T
authors.lastname: Endo
authors.forename: Masaaki
authors.initials: M
authors.lastname: Ban
authors.forename: Kazuma
authors.initials: K
authors.lastname: Hirata
authors.forename: Akitoshi
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Hara
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Momose
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000493:pharmacokinetics* / Q000652:urine; D010862:Pilocarpine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics* / Q000652:urine
chemical_list: C520141:3-hydroxypilocarpine; D018721:Muscarinic Agonists; D010862:Pilocarpine; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6