Received for publication October 20, 2006.
Revised November 15, 2006.
Accepted for publication December 15, 2006.

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 investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by LC-MS/MS and NMR analyses and by comparing to the authentic metabolite. In order 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 µM coumarin. Other selective inhibitors of CYP1A2 (furafylline, -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 Km value for recombinant CYP2A6 was 3.1 µM, and this value is comparable to that of human liver microsomes (1.5 µM). 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 clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.

Key words: CYP2A, cytochrome P450 catalyzed oxidations, cytochrome P450 isoforms, human CYP enzymes, liver microsomes, metabolite identification

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