Original Article Multiplex Phenotyping for Systems Medicine: A One-Point Optimized Practical Sampling Strategy for Simultaneous Estimation of CYP1A2, CYP2C9, CYP2C19, and CYP2D6 Activities Using a Cocktail Approach Fernando de Andre ´ s, 1 Santiago Tera ´n, 2 Marcela Bovera, 3 Humberto Farin ˜ as, 1 Enrique Tera ´ n, 2 * and Adria ´ n LLerena 1 * Abstract Phenotyping of the CYP450 enzyme activities contributes to personalized medicine, but the past phenotyping approaches have followed a piecemeal strategy measuring single enzyme activities in vivo. A barrier to phenotyping of populations in rural and remote areas is the limited time and resources for sample collection. The CEIBA cocktail approach allows metabolic capacity estimation of multiple CYP450 enzymes in a single sample analysis, but the attendant sample collection schemes for applications in diverse global settings are yet to be optimized. The present study aimed to select an optimal matrix to simultaneously analyze CYP450 enzyme activities so as to simplify the sampling schemes in the phenotyping protocol to enhance its throughput and feasibility in native populations or in remote and underserviced geographies and social contexts. We evaluated 13 Ecuadorians healthy volunteers for CYP1A2, CYP2C9, CYP2C19, and CYP2D6 genotypes and their metabolic phenotypes, including CYP3A4, in plasma and urine after administering one reduced dose of caffeine, losartan, omeprazole, and dextromethorphan. Pharmacokinetic analyses were performed, and the correlation between AUC parent/AUC metabolite and the ratio between concentrations of probe drugs and their corresponding metabolites at timepoints ranging from 0 to 12 hours post-dose were analyzed. A single sampling timepoint, 4 hours post-dose in plasma, was identified as optimal to reflect the metabolic activity of the attendant CYP450 enzymes. This study optimizes the CEIBA multiplexed phenotyping approach and offers new ways forward for integrated drug metabolism analyses, in the pursuit of global personalized medicine applications in resource-limited regions, be they in developed or developing countries. Introduction I nterindividual variability in drug metabolism leads to differences in drug responses and the occurrence of adverse drug reactions, and is a major concern for rational drug development and clinical practice. Indeed, the Ameri- can Food and Drug Agency (FDA) and the European Medicines Agency (EMA) recommend the application of pharmaco- genetics principles to caution individuals at risk of either developing drug-induced adverse side reactions or to detect those who will not benefit from standard doses. Biomarker science and personalized medicine continue to grow in popularity worldwide (Tutton and Prainsack, 2011; Tutton, 2012), but advances in rural or resource-limited regions demand consideration of the local context (Bosch et al., 2014; Dandara et al., 2014; Mhandire et al., 2014). In a context of personalized medicine, although the hydrox- ylation capacity of CYP450 enzymes is frequently assumed from genotyping (i.e., phenotype predicted from genotype), the actual enzyme hydroxylation capacity (‘‘metabolic phenotype’’) is not always correlated with the ‘‘predicted phenotype’’. This phenomenon has been demonstrated by the large degree of variability observed within any particular genotype group, es- pecially for the so-called ultrarapid metabolizers (LLerena et al., 2009; 2012). Moreover, drug-drug interactions, various genes that influence pharmacokinetics, environmental factors, endo- genous metabolism and co-morbidity can all influence enzyme activity. Consequently, the gene–environment interaction results 1 CICAB Clinical Research Centre, Extremadura University Hospital and Medical School, Badajoz, Spain. 2 Colegio de Ciencias de la Salud, Universidad San Francisco de Quito, Quito, Ecuador. 3 Servicio de Laboratorio, Hospital de los Valles, Quito, Ecuador. *These authors made an equal contribution. OMICS A Journal of Integrative Biology Volume 19, Number 00, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/omi.2015.0131 1