M1000

The in vitro microsomal stability assay was carried out using the mouse liver microsomal system (Xenotech, M1000), which was enriched with nicotinamide adenine dinucleotide phosphate and NADPH (BD Biosciences, San Jose, USA). Compounds at 1 uM concentration were incubated with a reaction mixture containing liver microsomal proteins (0.15 mg/mL) in 100 mM potassium phosphate buffer (pH7.4). The reaction was initiated by adding NADPH, followed by incubation at 37 °C. At various time points, one volume of reaction aliquot was taken and quenched with three volumes of acetonitrile containing 0.05% formic acid. The samples were then vortexed and centrifuged at 16,000 rpm for 10 min at 4 °C. The supernatants were collected and analyzed by LC/MS/MS to determine the remaining percentage and the half-life (t1/2) by integrating compound peaks and fitting the resulting AUC values and time points to a one-phase decay equation. Imipramine was used as a positive control with t1/2 of 24 min to confirm the activity of the microsomes.

A microsomal stability assay was performed using the Corning Gentest NADPH Regenerating System (Corning, #451220/41200) (Corning, NY, USA). Mouse liver microsomes (MLM) (Xenotech, M1000) (Kansas City, KS, USA) and compounds were diluted to a final concentration of 0.15 mg/mL and 1 μM respectively. These were prepared into a 500 μL solution with 100 mM potassium phosphate buffer (pH = 7.4). Imipramine was used as a control. NADPH solutions A and B were mixed in a 5:1 ratio, and 24 μL of the NADPH was added to each of the MLM/compound samples at 30 s intervals. At time points 10, 20, and 45 min, 100 μL of reaction was added to the stopping solution. Samples were centrifuged at 18,000× g and incubated at 4 °C for 10 min. Samples were then loaded onto a LC-MS 96-well plate for analysis.

Metabolic stability was measured by Wuxi AppTech (Shanghai, China). Briefly, peptides (1 μM, 5% MeOH in potassium phosphate buffer) were incubated with human (catalogue no. 452161 from BD Gentest) and mouse (catalogue no. M1000, Xenotech) liver microsomes at 37°C for 10 min. Liver microsomes were at a final assay concentration of 0.7 mg protein/mL. The reaction was started by the addition of 90 μL of NADP cofactor solution and stopped by the addition of 300 μL of stop solution (acetonitrile at 4°C, including 100 ng/mL tolbutamide as an internal standard) after 20 min of incubation. The samples were shaken for 5 min and then centrifuged for 20 min at 1500 g. A 100 μL aliquot of the supernatant was transferred to eight new 96-well plates with 300 μL of HPLC water and centrifuged at 1500 g for LC-MS/MS analysis (Shimadzu LC 10-AD−API 4000). An injection volume of 10 μL was added to a Phenomenex Synergi C18 column eluting with formic acid in water or acetonitrile at a flow rate of 800 μL/min. The percent loss of parent compound was calculated from the peak area ratio of the analyte/internal standard. Compounds and positive controls were tested in duplicate.