To investigate the potential of BI 425809 to reversibly inhibit the major human CYPs, CYP-selective substrates (phenacetin 60 μM [CYP1A2], bupropion 80 μM [CYP2B6], amodiaquine 2 μM [CYP2C8], diclofenac 5 μM [CYP2C9], S-mephenytoin 80 μM [CYP2C19], dextromethorphan 5 μM [CYP2D6], midazolam 2 μM, and testosterone 50 μM [CYP3A4/5]) were incubated with human liver microsomes and BI 425809 (0.015, 0.046, 0.137, 0.411, 1.23, 3.70, 11.1, 33.3, and 100 μM). For positive control reactions, BI 425809 was replaced with a CYP-selective inhibitor (α-naphthoflavone [CYP1A2], ticlopidine [CYP2B6], montelukast [CYP2C8], sulfaphenazole [CYP2C9], benzylnirvanol [CYP2C19], quinidine [CYP2D6], and itraconazole [CYP3A4/5]). Substrate metabolites were quantified with liquid chromatography–tandem mass spectrometry using gradient elution (mobile phase for amodiaquine metabolite—A, 5 mM ammonium formate in water/formic acid [100:0.1, v/v]; B, acetonitrile/formic acid [100:0.1, v/v]; mobile phase for all other substrate metabolites—A, water/formic acid [100:0.1, v/v]; B, acetonitrile/formic acid [100:0.1, v/v]) on a Synergi Hydro RP column (50 × 2.0 mm, 4 μm; Phenomenex) with positive electrospray ionization.
IC50 values were obtained using a 3-parameter dose-response, 4-parameter dose-response, or normalized dose-response model; model comparisons were performed in Prism 6 (GraphPad Inc) to determine the optimal model for each data set. A least-squares fitting approach was used, and the Hill slope was not constrained for the 4-parameter model.
IC50 values were obtained using a 3-parameter dose-response, 4-parameter dose-response, or normalized dose-response model; model comparisons were performed in Prism 6 (GraphPad Inc) to determine the optimal model for each data set. A least-squares fitting approach was used, and the Hill slope was not constrained for the 4-parameter model.
