title: Studies on the metabolism of the novel, selective cyclooxygenase-2 inhibitor indomethacin phenethylamide in rat, mouse, and human liver microsomes: identification of active metabolites.
abstract: The metabolism of 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]-N-phenethyl-acetamide (indomethacin phenethylamide, LM-4108), a highly selective cyclooxygenase-2 inhibitor, was studied in rat, mouse, and human liver microsomes. The primary site of oxidation in all species examined was on the methylene carbons of the phenethyl side chain to form the 1'- and 2'-hydroxy and 2'-oxo metabolites as determined by electrospray ionization liquid chromatography-tandem mass spectrometry. Half-lives for the disappearance of 10 microM LM-4108 in rat, human, and mouse liver microsomes (0.15 pmol P450/ml) were 11 min, 21 min, and 51 min, respectively. Indomethacin formation was not observed in incubations with rat, mouse, or human liver microsomes. Both the 2'-hydroxy-LM-4108 and 2'-oxo-LM-4108 metabolites were synthesized and found to be equipotent to the parent compound with regard to COX-2 inhibitory potency and selectivity [2'-hydroxy-LM-4108: IC(50)(COX-2) = 0.06 microM, IC(50)(COX-1) >66 microM; 2'-oxo-LM-4108: IC(50)(COX-2) = 0.05 microM, IC(50)(COX-1) >66 microM]. The formation of the metabolites was strongly inhibited by specific CYP3A4 inhibitors ketoconazole and troleandomycin but not by other isoform-selective inhibitors. These findings were confirmed by demonstrating that cloned, expressed CYP3A4 catalyzed side chain oxidation. O-Demethylation was a minor oxidative pathway in contrast to the metabolism of indomethacin and was catalyzed by CYP2D6. Upon intravenous administration of LM-4108 to Sprague-Dawley rats, oxidative metabolism on the phenethyl side chain constituted the rate-limiting steps in its clearance. The active metabolites, 2'-oxo- and 2'-hydroxy-LM-4108, as well as 1'-hydroxy-LM-4108, were all observed in rat plasma and thus may contribute to COX-2 inhibition in vivo. The glucuronides of 2'hydroxy-LM-4108 and O-desmethyl-2'-hydroxy-LM-4108 were also identified in rat bile.
authors.affiliation: Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Rory P
authors.initials: RP
authors.lastname: Remmel
authors.forename: Brenda C
authors.initials: BC
authors.lastname: Crews
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Kozak
authors.forename: Amit S
authors.initials: AS
authors.lastname: Kalgutkar
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
authors.forename: Amit S
authors.initials: AS
authors.lastname: Algutkar
mesh_terms: D000818:Animals; D001646:Bile / Q000378:metabolism; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000097:blood / Q000493:pharmacokinetics*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007213:Indomethacin / Q000031:analogs & derivatives / Q000097:blood / Q000493:pharmacokinetics*; D007275:Injections, Intravenous; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D011994:Recombinant Proteins; C489645:indomethacin phenethylamide; D003577:Cytochrome P-450 Enzyme System; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin