Discovery and Assessment of Endogenous Biomarkers of CYP2D6 Activity for Application in Clinical Studies

Abstract

The activity of cytochrome P450 2D6 (CYP2D6) is highly variable due to genetic and environmental influences. Assessing CYP2D6 activity may aid in initial dosing of drugs metabolized by CYP2D6, help maximize therapeutic efficacy or minimize adverse events. Traditionally, probe drugs, such as dextromethorphan (DM), metoprolol and atomoxetine, are administered to determine CYP2D6 activity. The primary aim of this dissertation was the discovery and evaluation of endogenous biomarkers of CYP2D6 activity as a non-invasive means of phenotyping. We developed two liquid chromatography mass spectrometry methods to quantify selected endocannabinoids and indolethylamines, previously reported as in vitro substrates of CYP2D6. Preliminary results suggest no difference between plasma anandamide concentrations at baseline and following CYP2D6 inhibition in healthy adults (P=0.61), although a possible difference in 5-MT/serotonin ratio was observed between two urine samples. With further refinement, these assays will allow evaluation of the endocannabinoid and indolethylamine substrates and metabolites as biomarkers of CYP2D6 and potentially other cytochromes P450 in clinical samples. Using global metabolomics, we detected a novel ion (M1) with m/z 444.3 eluting at 6.5 min. M1 was absent in the urine of CYP2D6 poor metabolizers and present in all other CYP2D6 phenotypes. In adults, M1 decreased on average 4- and 9-fold in plasma and urine, respectively after potent CYP2D6 inhibition and was negatively correlated with the DM parent-to-metabolite metabolic ratio (P=0.012). Furthermore, urinary M1 was correlated with the oral clearance of metoprolol in women studied during pregnancy and postpartum, and atomoxetine in children with attention deficit hyperactivity disorder. Our data suggest that M1 may be a product of a reaction catalyzed by CYP2D6. Following development of large-scale semi-purification of M1 in urine, characterization of M1 by multiple stage mass spectrometry was performed. Though the structure of M1 remains unknown, future efforts should focus on the identification of M1 and its parent. Validation of the M1 biomarker alone or in combination with its parent may provide a useful endogenous biomarker of CYP2D6 activity. Clinical application of endogenous biomarkers may have benefits of safety, cost, time and convenience over traditional phenotyping methods, and may permit the assessment of CYP2D6 activity in population-based studies.