In vitro metabolic stability in liver microsomes

Incubation of the test compounds in mouse or human liver microsomes (Xenotech, Lenexa, KS, USA) was done with a previously described method⁵⁵ with modifications. We tested the metabolic stability of PRE-084 and PGB, the latter of which was used as a control because of its poor hepatic metabolism³⁶. Briefly, the drugs were incubated in 96-well plates at a concentration of 1 μM and at 37 °C during 1 h under standard incubation conditions: sodium-potassium phosphate buffer (50 mM, pH 7.4), MgCl₂ (3 mM), the NADPH-regenerating system and cytochrome P450 content (0.3 nmol/mL). Aliquots of the reaction mixture were obtained at 0, 10, 20, 40 and 60 min with an equal volume of cold acetonitrile. The assay was done in a robotic liquid handling system (Freedom Evo Tecan, Männedorf, Switzerland). Upon centrifugation of the resulting mixture, supernatants were analyzed with a generic ultra-high performance liquid chromatography–tandem mass spectrometry method.