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 personalised medicine by providing direct information on enzyme activity. This would result in enormous benefits, both for patients and for 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 4MODIFIER LETTER PRIME-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 been able to establish a workflow using a cell-based system for CYP2C9 activity. Furthermore, we illustrate how the bioconversion of diclofenac is limited in the presence of limonene, which is another CYP2C9 metabolising substrate. We show that increasing limonene levels continuously reduce the production of 4MODIFIER LETTER PRIME-hydroxydiclofenac. Michaelis-Menten kinetics were performed for the diclofenac 4MODIFIER LETTER PRIME-hydroxylation with and without limonene, giving a kinetic constant of the reaction, K (M), of 103 & mu;M and 94.1 & mu;M, respectively, and a maximum reaction rate, V (max), of 46.8 pmol min(-1) 10(6) cells(-1) and 56.0 pmol min(-1) 10(6) 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 4MODIFIER LETTER PRIME-hydroxydiclofenace by limonene is determined to be 1413 & mu;M.