RT Journal Article
SR Electronic
T1 Involvement of CYP2A6 in the Formation of a Novel Metabolite, 3-Hydroxypilocarpine, from Pilocarpine in Human Liver Microsomes
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 476
OP 483
DO 10.1124/dmd.106.013425
VO 35
IS 3
A1 Takuro Endo
A1 Masaaki Ban
A1 Kazuma Hirata
A1 Akitoshi Yamamoto
A1 Yoshiki Hara
A1 Yasunori Momose
YR 2007
UL http://dmd.aspetjournals.org/content/35/3/476.abstract
AB 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 μM coumarin. Other selective inhibitors of CYP1A2 (furafylline and α-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 with 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 we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes. The American Society for Pharmacology and Experimental Therapeutics