Wide inter individual differences in expression or

Wide inter-individual differences in Astemizole mg or activity of CYP450 contribute to variations in both clearance and efficacy/toxicity of several drugs. Phenotyping measures provide information on the real-time activities of CYP450 and provide clinically relevant information as they reflect the combination of factors such as genetic, drug–drug interactions, environmental and endogenous factors including diseases [11], [12], [13], [14], [15], [16]. Thus, a phenotyping measure using selective CYP450 probes is a valuable tool to determine in vivo CYP450 activities. In vivo methods to characterize subject’s phenotype may involve either the administration of single probe drugs, one at a time, on numerous occasions or a mixed phenotyping approach based on a cocktail method where multiple CYP450-probe drugs are administered simultaneously. The major advantage of the cocktail approach is to provide information on several CYP450 activities in a single experiment. However, drugs used in these cocktails must be specific for individual CYP450 isoenzymes and should not interact with each other. Also, in order to use such cocktails in a clinical setting, i.e. in patients with several co-morbidities, probe drugs should be given at low oral doses to minimize drug exposure and ensure patient’s safety. Numerous cocktails composed of several CYP450-selective probe drugs have already been described in the literature to study in vivo drug-metabolism activities [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31]. Although previous published cocktails such as Inje, Karolinska, Cooperstown 5+1 or Pittsburg cocktail have shown value they also suffer from many limitations [24], [26], [27], [28], [29], [31]. For instance, they often comprise probe drugs which are not or no longer available in several countries. Furthermore, they have been validated using high performance liquid chromatography methods with limited detection sensitivity. This led to the utilization of high doses of probe substrates. In recent years, the use of liquid chromatography/tandem mass spectrometry (LC–MS/MS) has become a preferred approach to measure drugs and metabolites in biological fluids due to its very high selectivity and sensitivity [17], [18], [19], [20], [21], [22], [23], [25], [30]. The great sensitivity of LC–MS/MS permits the use of small blood volumes in patients while its specificity allows measurement of metabolites that often co-elute. We describe herein the development and validation of three sensitive and specific LC–MS/MS assays allowing the determination of major human CYP450 isoenzyme activities following administration of a drug cocktail in healthy volunteers and patients. This seven-drug cocktail is composed of caffeine (CAF), bupropion (BUP), tolbutamide (TOL), omeprazole (OME), dextromethorphan (DM), midazolam (MDZ) and chlorzoxazone (CZX) (which is administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively (Fig. 1). Our objective was to develop highly sensitive, robust and fast assays to determine CYP450 phenotypes in a real clinical setting using small blood volumes and low oral doses of mixed probe drugs in order to minimize risk of side effects.