O EPP-AF® é um extrato padronizado de própolis quimicamente caracterizado e com eficácia e segurança pré-clínica estabelecidas. O objetivo principal deste trabalho foi realizar um ensaio clínico de segurança para avaliar a influência do EPP-AF® na atividade da P-gp e das principais isoformas CYP, através de um teste in vivo tipo coquetel de fármacos marcadores administrados em doses subterapêuticas. Foram investigados 16 voluntários adultos sadios antes e após a exposição a 375 mg de EPP-AF® por via oral durante 15 dias. As amostras seriadas de sangue foram colhidas até 12 h após a administração do coquetel contendo midazolam (0,2 mg), cafeína (10 mg), omeprazol (2 mg), metoprolol (10 mg), losartana (2 mg) e fexofenadina (10 mg). Foram desenvolvidos e validados três métodos analíticos empregando LC-MS/MS para quantificar as concentrações plasmáticas de fexofenadina, losartana, E-3174 (método 1), omeprazol, 5-OH-omeprazol, midazolam, metoprolol, ?-OHmetoprolol (método 2) e cafeína (método 3). Os métodos não apresentaram efeito matriz ou efeito residual e mostraram-se lineares para os analitos nos intervalos de 0,05-20 ng/mL (fexofenadina); 0,03 - 5 ng/mL (losartana e E31-74); 0,1 - 50 ng/mL (omeprazol), 0,3 - 50 ng/mL (5-OH-omeprazol), 0,01 - 10 ng/mL (midazolam), 0,05 - 50 ng/mL (metoprolol e ?- OH-metoprolol) e 5 - 1000 ng/mL (cafeína). Os parâmetros farmacocinéticos dos compostos foram calculados com base nas curvas de concentração plasmática versus tempo (AUC) empregando o programa Phoenix® WinNonlin®. Os valores das razões das AUC0-t e Cmax após e antes da exposição ao EPP-AF®, apresentados como média geométrica (IC90%) foram de 0,74 (0,62 - 0,89) e 0,90 (0,76 - 1,07) para fexofenadina; 0,88 (0,80 - 0,97) e 0,86 (0,76 - 0,98) para losartana; 0,96 (0,83 - 1,11) e 0,91 (0,79 - 1,04) para E-3174; 1,18 (0,91 - 1,54) e 1,21 (0,87- 1,70) para omeprazol; 1,12 (0,95 - 1,31) e 1,22 (0,95 - 1,67) para 5-OHomeprazol; 1,14 (1,03 - 1,28) e 1,21 (1,00 - 1,46) para o midazolam; 1,04 (0,92 - 1,18) e 0,94 (0,80 - 1,12) para o metoprolol; 1,05 (0,99 - 1,12) e 0,99 (0,88 - 1,12) para ?-OH-metoprolol; 0,97 (0,77 - 1,21) e 0,87 (0,69 - 1,11) para a cafeína. Quando observadas as razões metabólicas das AUC0-t E3174/losartana, 5-OH-omeprazol/omeprazol e ?-OHmetoprolol/ metoprolol encontramos, respectivamente, 1,11 (0,98 - 1,25); 0,94 (0,81 - 1,10) e 1,01 (0,88 - 1,16), indicando que, com exceção do CYP2D6, a administração de EPP-AF® nas condições estudadas apresenta potencial para inibição das isoformas CYP2C19 e CYP3A4 e indução das enzimas CYP1A2, CYP2C9 e do transportador de efluxo P-gp, embora as suas magnitudes encontram-se abaixo dos limites definidos pelos órgãos reguladores e portanto não apresentam relevância clínica / EPP-AF® is a standardized extract of propolis chemically characterized and with established pre-clinical efficacy and safety. The main objective of this work was to perform a clinical trial to evaluate the effect of EPP-AF® on P-gp and the major CYP isoforms activity, through an in vivo assay using the cocktail approach with sub-therapeutic doses. Sixteen healthy adult volunteers were investigated before and after exposure to orally administered 375 mg/day of EPP-AF® for 15 days. Serum blood samples were collected up to 12 h after the administration of midazolam (0.2 mg), caffeine (10 mg), omeprazole (2 mg), metoprolol (10 mg), losartan (2 mg) and fexofenadine (10 mg). Three analytical methods were developed and validated applying LC-MS/MS to quantify plasma concentrations of fexofenadine, losartan, E-3174 (method 1), omeprazole, 5-OH-omeprazole, midazolam, metoprolol, ?-OH-metoprolol (method 2), and caffeine (Method 3). Neither matrix effect nor carryover effect were observed. The methods were linear for the analytes in the ranges of 0.05 - 20 ng/mL (fexofenadine); 0.03 - 5 ng/ml (losartan and E-3174); 0.1 - 50 ng/mL (omeprazole), 0.3 - 50 ng/mL (5-OH-omeprazole), 0.01 - 10 ng/mL (midazolam), 0.05 - 50 ng/mL (metoprolol and ?-OH-metoprolol) and 5 - 1000 ng/mL (caffeine). The pharmacokinetic parameters of the compounds were calculated based on plasma concentration versus time (AUC) curves applying Phoenix® WinNonlin® software. AUC0-t and Cmax ratios after and before the EPPAF ® exposure, presented as geometric mean (CI 90%) were 0.74 (0.62 - 0.89) and 0.90 (0.76 - 1.07) for fexofenadine, 0.88 (0.80 - 0.97) and 0.86 (0.76 - 0.98) for losartan, 0.96 (0.83 - 1.11) and 0.91 (0.79 - 1.04) for E-3174, 1.18 (0.91 - 1.54) and 1.21 (0.87 - 1.70) for omeprazole; 1.12 (0.95 - 1.31) and 1.22 (0.95 - 1.67) for 5-OH-omeprazole, 1.14 (1.03 - 1.28) and 1.21 (1.00 - 1.46) for midazolam, 1.04 (0.92 - 1.18) and 0.94 (0.80 - 1.12) for metoprolol, 1.05 (0.99 - 1.12) and 0.99 (0.88 - 1.12) for ?-OH-metoprolol, 0.97 (0.77 - 1.21) and 0.87 (0.69 - 1.11) for caffeine. AUC0-t metabolic ratios of E3174/losartan, 5-OH-omeprazole/omeprazole and ?-OH-metoprolol/metoprolol we found to be, respectively, 1.11 (0.98 - 1.25), 0.94 (0.81 - 1.10 ) and 1.01 (0.88 - 1.16), indicating that, with the exception of CYP2D6, the administration of EPP-AF® under the conditions studied shows potential for CYP2C19 and CYP3A4 inhibition and CYP1A2, CYP2C9 and P-gp induction, although their magnitudes are below the limits defined by the regulatory agencies and therefore exhibit no clinical relevance

Ce projet de recherche comporte deux parties principales qui possèdent comme lien unificateur l’amélioration des méthodes et techniques utilisées actuellement pour évaluer aussi bien l’activité que l’expression des cytochromes P450 (CYP450s) et menant par la suite à leur application en clinique. Le premier volet de ce projet de recherche porte sur le développement de méthodes LC-MS/MS pour un cocktail de 7 substrats marqueurs des CYP450s. Notre objectif est de développer et valider une méthode LC-MS/MS spécifique et sensible permettant l’évaluation des activités des CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 et 2E1 suivant l’administration orale et à faible dose d’un cocktail de substrats marqueurs chez des patients et sujets sains. Les méthodes développées peuvent être utilisées pour évaluer les mécanismes de variabilité interindividuelle comme l’impact de polymorphismes génétiques, de facteurs environnementaux et de maladies dans le processus de métabolisme et d’élimination des médicaments, mais également pour investiguer les interactions médicamenteuses. Ce cocktail a été appliqué avec succès, dans un projet clinique portant sur l’évaluation des effets du diabète sur la capacité métabolique par les CYP450s. Le deuxième volet de ce projet de recherche vise à développer des méthodes analytiques par LC-HRMS afin de caractériser et quantifier les CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 et 4F2 dans l’intestin grêle humain. Notre hypothèse suggère que les CYP450s retrouvés le long de l’intestin grêle peuvent affecter significativement l’effet de premier passage de certains médicaments administrés par voie orale et influencer leurs concentrations plasmatiques et conséquemment, leurs effets pharmacologiques et/ou toxiques. Ma participation à ce projet a permis d’identifier des peptides protéotypiques par digestion in silico et in vitro et de développer des méthodes de quantification absolue par LC-HRMS. Ce projet est une première étape dans la caractérisation des CYP450s majeurs le long de l'intestin grêle. Il permettra de mieux comprendre les mécanismes de variabilité interindividuelle dans la réponse aux médicaments associés au processus d'absorption intestinal et de mieux prédire la variabilité dans la biodisponibilité des médicaments et de développer des modèles pharmacocinétiques plus complexes. / This research project is divided into two sections, both aiming at the development of sensitive and specific LC-MS methods to evaluate activity and expression of CYP450 and finally, looking at their clinical application. The first section of this research project focuses on the development of analytical methods by LC-MS/MS for a seven CYP450 probe-drug cocktail. Although these cocktails have shown value they also suffer from many limitations. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1 activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. These methods can be used to phenotype CYP450 activities, evaluate inter-individual variabilities, study the impact of pathological conditions on drug metabolism and elimination, and evaluate drug-drug interactions. Our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in type 2 diabetic patients. The second section of this project aims at the development of a LC-HRMS method for the characterization and absolute quantification of CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2 in the human small intestine. Our hypothesis suggests that CYP450 isoenzymes found along the small intestine can significantly affect the first-pass effect of certain drugs administered orally and thus influence their pharmacological and/or toxic effects. My participation in this project allowed to identify proteotypic peptides by in silico and in vitro digestion and to develop LC-HRMS methods allowing the absolute quantification of CYP450. This project is a first step in the characterization of the main CYP450 along the small intestine. This project will allow a better understanding of inter-individual variability in drug response associated with intestinal absorption of drugs, a better prediction of variability in drug bioavailability and to develop more complex pharmacokinetic models.