Abstract
In vivo rat kidney tissue metabolites of an anticancer drug, cisplatin (cis-diamminedichloroplatinum [II]) (CP) which is used for the treatment of testicular, ovarian, bladder, cervical, esophageal, small cell lung, head and neck cancers, have been identified and characterized by using liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with on line hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, kidney tissues were collected after intravenous administration of CP to adult male Sprague-Dawley rats (n = 3 per group). The tissue samples were homogenized and extracted using newly optimized metabolite extraction procedure which involves liquid extraction with phosphate buffer containing ethyl acetate and protein precipitation with mixed solvents of methanol-water-chloroform followed by solid-phase clean-up procedure on Oasis HLB 3cc cartridges and then subjected to LC/ESI-HRMS analysis. A total of thirty one unknown in vivo metabolites have been identified and the structures of metabolites were elucidated using LC-MS/MS experiments combined with accurate mass measurements. Online HDX experiments have been used to further support the structural characterization of metabolites. The results showed that CP undergoes a series of ligand exchange biotransformation reactions with water and other nucleophiles like thio groups of methionine, cysteine, acetylcysteine, glutathione and thioether. This is the first research approach focused on the structure elucidation of biotransformation products of CP in rats, and the identification of metabolites provides essential information for further pharmacological and clinical studies of CP, and may also be useful to develop various effective new anticancer agents.
Highlights
Identification of metabolites is crucial in the drug discovery and development process to optimize lead compounds for further development
As the glutathione-conjugates pass through the kidney, they cleaved to cysteinyl-glycine-conjugates by gamma glutamyl transpeptidase (GGT) expressed on the surface of the proximal tubule cells [60, 61]
To elucidate the structures of metabolites, we examined the fragmentation of the [M+H]+ ions of CP and its metabolites by using online LC/electrospray ionization mass spectrometry (ESI-MS)/MS experiments in combination with accurate mass measurements
Summary
Identification of metabolites is crucial in the drug discovery and development process to optimize lead compounds for further development. The information generated in the early discovery phase of metabolic identification can be used to identify lead compounds and undesirable metabolic products followed by optimization of pharmacokinetic and safety profiles. Identification of reactive or toxic metabolites is essential to avoid the toxicity and helps to modify the structure by means of chemical transformations. These studies are usually carried out by employing in vitro and in vivo systems followed by using modern analytical instruments. Accurate and reproducible identification of metabolites in tissue homogenate samples is of high importance to characterize animal models and to identify metabolic changes that occur in different tissue types in specific diseases. The extraction of metabolites from tissue homogenates is one of the most labor intensive steps for metabolomic studies to yield reproducible results between repeated samples
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