4.7. Human liver microsome metabolic stability assay

Testosterone, diclofenac and propafenone were tested as control drugs in this assay as all of them are substrates of cytochrome P450 enzymes. A DMSO solution of the tested compound (10 mM, 10 μl per well) and a solution of microsome (80 μl per well) were added to a 96-well plate and the mixture was first incubated at 37 °C for 10 min. Then, potassium phosphate buffer (100 mM, 10 μl per well) was added and no co-factor (NCF) remaining was evaluated after a further incubation for 60 min. After this pre-warming process, an NADPH regenerating system (10 μl per well) was added, and each compound was tested at 5 time points (5 min, 10 min, 20 min, 30 min and 60 min). At each time point, stop solution (including 100 ng ml^–1 tolbutamide and 100 ng ml^–1 labetalol, cold in 4 °C, 300 μl per well) was added to terminate the reaction. The sampling plates were shaken for approximately 10 min, and then the samples were centrifuged at 4000 rpm for 20 min at 4 °C to afford the supernatant (100 μl) for LC/MS test. Intrinsic clearance (CL_int) and half-life (T_1/2) values were then calculated: CL_int(mic) = 0.693/half-life/mg microsome protein per ml; CL_int(liver) = CL_int(mic) × (45 mg microsomal protein per g liver weight) × (20 g liver weight per kg body weight).