Studying the mechanism of protein covalent binding of fipexide via reactive metabolites by mass spectrometry

摘要

Covalent binding of drug metabolites to proteins is thought to be an important cause of idiosyncratic reactions. In order to assess the involvement of reactive metabolites, trapping experiments are performed with small nucleophilic molecules. Drug bioactivation can often be studied using such in vitro incubations, using mass spectrometry as a method for characterizing metabolites and trapped species. The metabolism of the nootropic drug fipexide was studied for the formation of reactive metabolites and possible routes of bioactivation were found from in vitro incubations using LC-MS. Furthermore, protein covalent binding was specifically investigated using non-radiolabelled drug. Microsomal and hepatocyte incubations were performed to characterize in vitro metabolites. Fipexide, MDBP and 4-CPA were incubated under oxidative conditions, with and without trapping agents, such as glutathione, N-acetyl cysteine, UDPGA and coenzyme A. A QTRAP 4000 instrument was employed for detecting metabolites formed in these incubations with accurate mass measurements obtained from a QSTAR XL. The in vitro assessment of protein binding was performed on protein pellets following microsomal incubations. Proteins were digested into individual amino acids to detect the covalent binding to cysteine residues in microsomes.