Identification of metabolites of meisoindigo in rat, pig and human liver microsomes by UFLC-MS/MS

Abstract

3-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-1,3-dihydro-1-methyl-2H-indol-2-one, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. To gain an understanding of the interspecies differences in the metabolism of meisoindigo, the relevant metabolism studies were carried out for the first time in rat, pig and human liver microsomes of different genders by ultra fast liquid chromatography/tandem mass spectrometry (UFLC-MS/MS). The qualitative metabolite identification was accomplished by multiple reaction monitoring (MRM) in combination with Enhanced Product Ion (EPI). The semi-quantitative metabolic stability and metabolite formation were simultaneously measured by MRM. The in vitro metabolic pathways of meisoindigo in three species were proposed as 3,3′ double bond reduction, followed by N-demethylation, and reduction followed by phenyl mono-oxidation. Two novel metabolic pathways involving direct phenyl mono-oxidation without reduction in the three species, and direct N-demethylation without reduction in only pig and human, were also proposed. It may be noted that the two metabolites formed after reduction followed by phenyl mono-oxidation at positions 4, 5, 6 or 7, as well as one metabolite formed from direct phenyl mono-oxidation at either of the two phenyl rings without reduction were found to be uniquely present in human. The in vitro t1/2 and in vitro CLint values of meisoindigo were calculated. Statistical analysis showed there were no significant differences in the metabolic stability profiles of meisoindigo among three species, and gender effect on the metabolic stability of meisoindigo was negligible. Formation profiles of the most significant reductive metabolites were obtained in the three species. © 2009 Elsevier Inc. All rights reserved.