CHARACTERIZATION OF IN VITRO METABOLITES OF TROLEANDOMYCIN, A METABOLISM-DEPENDENT INHIBITOR OF CYP3A4, BY UPLC~(TM) QTOF MASS SPECTROMETRY

摘要

Troleandomycin was confirmed as a metabolism-dependent inhibitor of CYP3A4 under the conditions of this study using a traditional IC_(50) approach monitoring activity toward marker substrates. Analysis of human liver microsomes incubated with troleandomycin by UV/visible spectrophotometry yielded evidence of MIC formation involving the ferrous heme iron. Eighteen in vitro metabolites of troleandomycin were determined and characterized by UPLC/MS~(E) following incubation under conditions shown to promote CYP3A4 inhibition for elucidation of biotransformation routes of troleandomycin in HLM. Based on metabolite structural elucidation by MS~(E), four biotransformation routes were proposed: ester hydrolysis, N-demethylation, O-dealkylation and oxidation. Data were scrutinized for evidence of metabolites associated with the postulated mechanism of nitroso metabolite formation. No components consistent with the expected m/z of free (non-complexed) di-N-demethylated nitroso troleandomycin, the historically postulated CYP3A4 inhibitory metabolite, were detected. However, a late eluting metabolite, with an elemental composition corresponding to its proposed precursor, namely di-N-demethylated hydroxylamine troleandomycin, was observed, although attempts at structural elucidation were unsuccessful. A nitrosoalkane species may have formed but remain bound to the ferrous iron of the heme group of CYP3A4, rendering it undetectable. Attempts to isolate and characterize the inhibitory metabolite by UPLC/MS~(E) analysis are currently underway.