In Vitro Metabolic Stability Assay

Tested compounds at a 10 μM concentration (1 μL of 5 mM DMSO stock solution) were incubated with rat liver S9 fraction (2 mg protein/mL), Vivid^® regenerating system (2 μL) and NADPH (1mM) in ABIC 50 mM buffer pH 7.4, for a total incubation volume of 500 μL. Incubations were run in duplicate at 37 °C. Additional incubation was run using the same conditions in the presence of glutathione (1 mM). Incubations were run in duplicate at 37 °C. Aliquots were collected at 0, 5, 10, 20, 30, 40, 50, 60, 75, 90, 120, 180 min and reactions were terminated by adding an ice-cold solution of reserpine (2.5 μM) in acetonitrile. Following centrifugation (10,000 rpm, 15 min, RT) the clear supernatants were collected and analyzed LC-ESI-HRMS/MS. Control incubations were conducted in the same conditions: (1) using DMSO as negative control, in absence of the test drug; (2) using heat-denatured HLM (90 °C, 15 min); (3) using nevirapine, as a positive control, instead of the tested compound; and (4) in absence of NADPH co-factor