CYP2C8- and CYP3A-mediated C-demethylation of (3-{[(4-tert-butylbenzyl)-(pyridine-3-sulfonyl)-amino]-methyl}-phenoxy)-acetic acid (CP-533,536), an EP2 receptor-selective prostaglandin E2 agonist: characterization of metabolites by high-resolution liquid chromatography-tandem mass spectrometry and liquid chromatography/mass spectrometry-nuclear magnetic resonance.
CP-533,536, (3-{[(4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino]-methyl}-phenoxy)-acetic acid (1), an EP2 receptor-selective prostaglandin E2 agonist, is being developed to aid in the healing of bone fractures. To support the development of this program, in vitro metabolism of 1 was investigated in human liver microsomes and major recombinant human cytochrome P450 (P450) isoforms. 1 was metabolized in vitro by at least three recombinant human P450s: CYP3A4, CYP3A5, and CYP2C8. The turnover of 1 was NADPH-dependent and was completely inhibited by ketoconazole and quercetin in the CYP3A4/5 and CYP2C8 incubations, respectively. The major metabolic pathways were caused by oxidation of the tert-butyl moiety to form the omega-hydroxy metabolite (M4), oxidation of the pyridine moiety, and/or N-dealkylation of the methylphenoxy acetic acid moiety. The alcohol metabolite M4 was further oxidized to the corresponding carboxylic acid M3. In addition to these pathways, three unusual metabolites (M22, M23, and M26) resulting from C-demethylation of the tert-butyl group were identified using high-resolution liquid chromatography/tandem mass spectrometry and liquid chromatography/mass spectrometry/NMR. The C-demethylated metabolites were not detected on incubation of carboxylic acid metabolite M3 with either human liver microsomes or CYP3A/2C8 isoforms, suggesting that these metabolites were not derived from decarboxylation of M3. A possible mechanism for C-demethylation may involve the oxidation of M4 to form an aldehyde metabolite (M24), followed by P450-mediated deformylation, to give an unstable carbon-centered radical and formic acid. The carbon-centered radical intermediate then undergoes either oxygen rebound to form an alcohol metabolite M23 or hydrogen abstraction leading to an olefin metabolite M26.
Prakash
Chandra
C
Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, Connecticut, USA. chandra.prakash@biogenidec.com
Wang
Weiwei
W
O'Connell
Thomas
T
Johnson
Kim A
KA
D000293:Adolescent; D000328:Adult; D000368:Aged; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D065727:Cytochrome P-450 CYP2C8; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015232:Dinoprostone / Q000819:agonists*; D005260:Female; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D008745:Methylation; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008875:Middle Aged; D011725:Pyridines / Q000493:pharmacokinetics* / Q000494:pharmacology; D018078:Receptors, Prostaglandin E / Q000187:drug effects*; D058307:Receptors, Prostaglandin E, EP2 Subtype
C475539:CP533536; C545644:PTGER2 protein, human; D011725:Pyridines; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; C104464:CYP3A protein, human; D065727:Cytochrome P-450 CYP2C8; D051544:Cytochrome P-450 CYP3A; D015232:Dinoprostone