R. Dalton,1 B. Phillips,2 L. Risler,2 D. D. Shen,2 E. L. Woodahl1; 1University of Montana, Missoula, MT, 2University of Washington, Seattle, WA
BACKGROUND: CYP2D6 is a highly polymorphic gene and within the same genotype class, there are often individuals who are phenotypic outliers. We hypothesize that rare, uncharacterized variants may explain some of the CYP2D6 genotype-phenotype variability. Our goal was to characterize CYP2D6 activity in human liver microsomes (HLMs) from a panel of human liver samples (n=347) and correlate to known CYP2D6 genotype and novel variants.
METHODS: HLMs were incubated with two probe drugs, dextromethorphan and metoprolol, to look for drug-specific changes to CYP2D6 activity. Incubation conditions were validated in pooled HLMs. Substrate depletion and metabolite formation (dextrorphan and α-hydroxymetoprolol) was quantitated by liquid chromatography-mass spectrometry (LC-MS). Intrinsic clearance was calculated as rate of metabolism divided by substrate concentration in the linear range of metabolite formation. Phenotypes will be correlated to common genotypes and rare variants identified by PGRNSeq.
RESULTS: We have refined our incubation conditions to ensure linearity over parent drug concentration and incubation time. We established incubation conditions of 1.5 µM dextromethorphan for 20 min and 4 µM metoprolol for 30 min. HLM concentration was 0.2 mg/mL for both drugs. Using these conditions in pooled HLMs, we have measured consistent metabolite formation of about 0.65 ng α-hydroxymetoprolol and 2.8 ng dextrorphan in the reaction volume, both of which are significantly higher than the limits of quantitation.
CONCLUSION: Our detection method is sensitive to detect variability in CYP2D6 due to common and rare variation. Our study will help increase understanding of the contribution of rare variants to CYP2D6 phenotype.