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 μM 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: IC50(COX-2) = 0.06 μM, IC50(COX-1) >66 μM; 2′-oxo-LM-4108: IC50(COX-2) = 0.05 μM, IC50(COX-1) >66 μM]. 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.
Footnotes
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↵1 Abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; LM-4108, 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]-N-phenethyl-acetamide or indomethacin phenethylamide; 1′-hydroxy-LM-4108, 1′-hydroxy-indomethacin-phenethylamide; 2′-hydroxy-LM-4108, 2′-hydroxy-indomethacin-phenethylamide; 2′-oxo-LM-4108, 2′-oxo-indomethacinphenethylamide; LM-4106, 1′-methyl-indomethacin-(p-methyl)benzylamide; LM-5105, indomethacin-(p-chloro)-phenylamide; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; ESI, electrospray ionization; CID, collision-induced dissociation; P450, cytochrome P450; TLC, thin-layer chromatography; LC/MS/MS, liquid chromatography/tandem mass spectrometry; Rt, retention time.
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This work was supported by research and center grants from the National Institutes of Health (CA89450, ES00267). A preliminary account of this work was presented at the American Association of Cancer Research Meeting, San Francisco, CA, April 1–5, 2000.
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↵1 Research conducted while on sabbatical leave from the Department of Medicinal Chemistry, 308 Harvard St. SE, University of Minnesota, Minneapolis, MN 55455
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↵2 Current address: Pharmacokinetics, Dynamics, and Metabolism Department, Pfizer Global Research and Development, Groton, CT 06340
- Received August 14, 2003.
- Accepted August 20, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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