Background: The enzymatic biotransformations of drugs in living systems strongly affect their biological activity, often resulting in metabolites with decreased bioavailability and enhanced toxicity. The knowledge of specific sites of metabolic transformations is useful for guiding synthetic optimization of the lead compounds or drug candidates to overcome the stability and toxicity issues.
Service Details: To elucidate the main routes of hepatic metabolism the incubations of new chemical entities are carried out in liver microsomes (mouse, rat, human), that provide a major range of the metabolizing enzymes. Additionally, S9 microsomal fractions from mouse, rat, and human liver are used for more broad coverage of possible biotransformations (phases I and II metabolism).
The typical protocol includes incubation of compound (at concentration of 1 µM) with microsomes (0.5 mg/ml) or S9 (2.5 mg/ml) at the presence of co-factors at 37 °C, sampling at 2 time-points (0 and 60 minutes), the reaction quenching with acetonitrile, samples centrifugation. The analysis of the incubation sample and comparison to control (quenched at time zero) is performed using liquid chromatography/tandem mass spectrometry (LC-MS/MS, API3000, AB Sciex).
Our approach to drug metabolite identification integrates both software- and knowledge-based predictions of metabolic pathways allowing for list-dependent search of metabolites (1), as well as standard tandem mass spectrometry protocols (2) including MS/MS spectra of parent compound and metabolites, precursor ion and neutral loss scans to ensure the detection of unexpected metabolites formed by less-common metabolic reactions. The products of phase II metabolism such as glucuronide and sulfate conjugates are detected by constant neutral loss scans of 176 and 80 Da, respectively.