title: Cytochrome P450 11A1 bioactivation of a kinase inhibitor in rats: use of radioprofiling, modulation of metabolism, and adrenocortical cell lines to evaluate adrenal toxicity.
abstract: A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.
authors.affiliation: Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research & Development, Princeton, New Jersey 08543, USA.
authors.forename: Donglu
authors.initials: D
authors.lastname: Zhang
authors.forename: Oliver
authors.initials: O
authors.lastname: Flint
authors.forename: Lifei
authors.initials: L
authors.lastname: Wang
authors.forename: Ashok
authors.initials: A
authors.lastname: Gupta
authors.forename: Richard A
authors.initials: RA
authors.lastname: Westhouse
authors.forename: Weiping
authors.initials: W
authors.lastname: Zhao
authors.forename: Nirmala
authors.initials: N
authors.lastname: Raghavan
authors.forename: Janet
authors.initials: J
authors.lastname: Caceres-Cortes
authors.forename: Punit
authors.initials: P
authors.lastname: Marathe
authors.forename: Guoxiang
authors.initials: G
authors.lastname: Shen
authors.forename: Yueping
authors.initials: Y
authors.lastname: Zhang
authors.forename: Alban
authors.initials: A
authors.lastname: Allentoff
authors.forename: Jonathan
authors.initials: J
authors.lastname: Josephs
authors.forename: Jinping
authors.initials: J
authors.lastname: Gan
authors.forename: Robert
authors.initials: R
authors.lastname: Borzilleri
authors.forename: W Griffith
authors.initials: WG
authors.lastname: Humphreys
mesh_terms: D000311:Adrenal Glands / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D000818:Animals; D002250:Carbon Radioisotopes / Q000493:pharmacokinetics / Q000633:toxicity; D002460:Cell Line; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D047428:Protein Kinase Inhibitors / Q000097:blood / Q000493:pharmacokinetics* / Q000633:toxicity*; D011725:Pyridines / Q000097:blood / Q000493:pharmacokinetics* / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution
chemical_list: D002250:Carbon Radioisotopes; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D002786:Cholesterol Side-Chain Cleavage Enzyme