Identification of cholecystokinin tetrapeptide amide metabolites in liver microsomes of human, Rhesus Monkey, Sprague-Dawley rat and CD1 mouse using ultra-high performance liquid chromatography coupled to high resolution mass spectrometer

Endogenous cholecystokinin tetrapeptide (CCK-4, Trp-Met-Asp-Phe-NH2) is a fragment derived from a larger peptide hormone, cholecystokinin (or gastrin). As a panicogenic agent, CCK-4 is commonly used in clinic settings to induce panic attacks for the study of new anxiolytic drugs. However, few studies on CCK-4 metabolism have been published to date. In the present study, we investigate the metabolism of CCK-4 in liver microsomes of human (HLM), Rhesus Monkey (RMLM), Sprague-Dawley rat (RLM) and CD1 mouse (MLM) using ultra-high performance liquid chromatography coupled to a high resolution mass spetrometer. Ten metabolites, inlcuding tryptophan (M1), tryptophan amide (M2), hydroxy metabolites (M3-M5), truncated peptides (M6-M9), and CCK-4 acid (M10), were identified and 8 of them were reported for the first time. The metabolic pattern of CCK-4 in HLM was distinctly different from these in RMLM, RLM, and MLM. M2 and M9 were the major metabolites in HLM and accounted for 19.8% and 13.4% of initial CCK-4, respectively. In contrast, M2 was the major metabolite in RMLM and accounted for 41.4%, whereas M6 was the major metabolite in RLM and account for 39.1%. Three major metabolites M2, M7 and M8 in MLM accounted for 22.6%, 17.9% and 17.8% of initial CCK-4, respectively. Chemical inhibition experiment showed that aminopeptidase and/or endopeptidase hydrolysis were the major metabolic pathways in human to generate these metabolites. We further showed that cytochrome P450 were also involved in the metabolism of CCK-4 via hydroxylation, but to a less extend. These findings provide valuable information for the metabolic processes of CCK-4 among various species and an important reference basis for its safety evaluation and rational clinical application.