Investigating Tucatinib Metabolism and CYP3A and CYP2C8 Phenotypes in Human Liver Microsomes from African Americansin vitro

Abstract ID 99779 Poster Board 039 Tucatinib is a human epidermal growth factor receptor-2 (HER2) specific tyrosine kinase inhibitor for HER2-positive breast cancer metabolized by cytochrome P450 (CYP) 3A and CYP2C8. The goal of this study was to determine the relationship between CYP protein expression, enzyme activity, and tucatinib metabolic clearance in human liver microsomes (HLM) from individual donors. A total of 21 individual donors (n = 21) were included in this analysis: 14 European Americans, 7 African Americans. To determine CYP3A and CYP2C8 activity, midazolam (2.5 uM) and amodiaquine (1.9 uM), respectively, were used as probe substrates. Metabolite formation was measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The rate of 19-hydroxymidazolam formation (CYP3A activity) varied 45-fold between individual donors: the range was 145 – 6469 pmol/min/mg. The relative levels of N-desethylamodiaquine formation (CYP2C8 activity) varied 24-fold: the peak area ratio of N-desethylamodiaquine to internal standard ranged from 0.038–0.905. CYP2C8, CYP3A4, and CYP3A5 protein levels were also measured using quantitative targeted absolute proteomics by nanoLC-MS/MS analysis. For both CYP3A and CYP2C8, enzyme activity and protein concentration were highly correlated. To determine the tucatinib metabolic clearance, single-donor HLM were incubated with tucatinib (0.05-180 μM), and formation of the M1 metabolite was measured. The kinetic parameters were compared between two African American donors with low and high CYP2C8 activity. When fit to a substrate inhibition kinetic model (the best fit model), the high activity donor had a 2.5-fold higher Vmax/Km than the low activity donor. These data suggest that variable CYP2C8 activity impacts interindividual variability in tucatinib metabolic clearance.