Cytochrome P450 11A1 Bioactivation
of a Kinase Inhibitor
in Rats: Use of Radioprofiling, Modulation of Metabolism, and Adrenocortical
Cell Lines to Evaluate Adrenal Toxicity
posted on 2016-02-21, 17:22authored byDonglu Zhang, Oliver Flint, Lifei Wang, Ashok Gupta, Richard A. Westhouse, Weiping Zhao, Nirmala Raghavan, Janet Caceres-Cortes, Punit Marathe, Guoxiang Shen, Yueping Zhang, Alban Allentoff, Jonathan Josephs, Jinping Gan, Robert Borzilleri, W. Griffith Humphreys
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.