Establishing a cell-based screening workflow for determining the efficiency of CYP2C9 metabolism: moving towards the use of breath volatiles in personalised medicine.

The use of volatile biomarkers in exhaled breath as predictors to individual drug response would advance the field of personalized medicine by providing direct information on enzyme activity. This would result in enormous benefits, both for patients and the healthcare sector. Non-invasive breath tests would also gain a high acceptance by patients. Towards this goal, differences in metabolism resulting from extensive polymorphisms in a major group of drug-metabolizing enzymes, the cytochrome P450 (CYP) family, need to be determined and quantified. CYP2C9 is responsible for metabolising many crucial drugs (e.g., diclofenac) and food ingredients (e.g., limonene). In this paper, we provide a proof-of-concept study that illustrates the in vitro bioconversion of diclofenac in recombinant HEK293T cells overexpressing CYP2C9 to 4'-hydroxydiclofenac. This in vitro approach is a necessary and important first step in the development of breath tests to determine and monitor metabolic processes in the human body. By focusing on the metabolic conversion of diclofenac, we have established a workflow using a cell-based system for CYP2C9 activity. Using limonene, we illustrate how the bioconversion of diclofenac can be limited in the presence of another CYP2C9 metabolising substrate, with increasing limonene levels continuously reducing the formation of 4'-hydroxydiclofenac. Michaelis-Menten kinetics were performed for the diclofenac 4'-hydroxylation, with and without limonene, giving a kinetic constant of the reaction, KM, of 103 µM and 94.1 µM, respectively, and a maximum reaction rate, Vmax, of 46.8 pmol min-1 106 cells-1 and 56.0 pmol min-1 106 cells-1 with and without the inhibitor, respectively, suggesting a non-competitive or mixed inhibition type. The half maximal inhibitory concentration value (IC50) for the inhibition of the formation of 4'-hydroxydiclofenace by limonene is determined to be 1413 µM.