In vitro Microsomal Stability Assay

The stability of IEM-1460 in the presence of guinea pig liver microsomes (BioIVT, PN: X008070) or IEM-1925 in the presence of human (BioIVT, PN: M00101), mouse, or guinea pig liver microsomes was evaluated (Paraza Pharma, Inc., Montreal, Quebec, Canada). Both IEM-1925 and IEM-1460 were incubated with microsomes at 37°C for a total of 45 min. Working solutions were prepared in 100 mM potassium phosphate buffer and the final reaction was performed at pH 7.4 in 100 mM potassium phosphate buffer containing 0.5 mg/mL liver microsomal protein. Phase I metabolism was assessed by adding NADPH to a final concentration of 1 mM and collecting samples at time points 0, 5, 15, 30, and 45 min. All collected samples were quenched 1:1 with ice-cold stop solution (1 μM labetalol and 1 μM glyburide in acetonitrile) and centrifuged to remove precipitated protein. Resulting supernatants were further diluted 5-fold with water. Samples were analyzed by LC-MS/MS using a Thermo Vanquish UPLC and a Thermo Quantis triple quadrupole mass spectrometer. Analytes were injected into a Phenyl column and chromatographed using a reverse phase gradient with 0.1% formic acid in water and 0.1% formic acid in 20/80 Isopropanol/Acetonitrile mobile phases. Reference compounds were also assessed to ensure accuracy and precision of experimental protocol: imipramine and verapamil for human liver microsomes, diphenhydramine and verapamil for mouse liver microsomes, and warfarin and verapamil for guinea pig liver microsomes. Calculations for half-life, in-vitro clearance, and percentage of hepatic blood flow were performed using Microsoft Excel. Half-life was determined from a plot of the natural logarithm of the peak area ratio (remaining compound peak area/internal standard peak area) against time.