title: Identification of a novel glutathione conjugate of flutamide in incubations with human liver microsomes.
abstract: Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.
authors.affiliation: Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, San Diego, CA 92121, USA. ping.kang@pfizer.com
authors.forename: Ping
authors.initials: P
authors.lastname: Kang
authors.forename: Deepak
authors.initials: D
authors.lastname: Dalvie
authors.forename: Evan
authors.initials: E
authors.lastname: Smith
authors.forename: Sue
authors.initials: S
authors.lastname: Zhou
authors.forename: Alan
authors.initials: A
authors.lastname: Deese
mesh_terms: D000726:Androgen Antagonists / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D018931:Antineoplastic Agents, Hormonal / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001711:Biotransformation; D056486:Chemical and Drug Induced Liver Injury; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005485:Flutamide / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000726:Androgen Antagonists; D018931:Antineoplastic Agents, Hormonal; D011994:Recombinant Proteins; D009249:NADP; D005485:Flutamide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C045793:CYP2C19 protein, human; C510164:CYP3A5 protein, human; D019388:Cytochrome P-450 CYP1A2; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005978:Glutathione