A ciclofosfamida (CPA) é um agente alquilante da classe das oxazafosforinas amplamente usada no tratamento de múltiplas formas de câncer e de doenças autoimunes em adultos e crianças. A CPA está disponível na clínica como racemato, no entanto, dados pré-clínicos demonstraram diferenças na eficácia e toxicidade dos seus enantiômeros, sendo o enantiômero (S)-(-)-CPA o de maior índice terapêutico. O presente estudo investigou a enantiosseletividade e a influência do CYP2B6, CYP2C9, CYP2C19 e CYP3A na disposição cinética da ciclofosfamida (CPA) em pacientes portadoras de câncer de mama. Foram incluídas na investigação 15 pacientes previamente submetidas ao procedimento de retirada do tumor e tratadas com CPA racêmica (900-1000 mg) e epirrubicina. A atividade in vivo do CYP3A foi avaliada empregando o midazolam como fármaco marcador. As amostras seriadas de sangue foram coletadas até 24 horas após a administração do primeiro ciclo da CPA. Os enantiômeros da CPA foram extraídos do plasma, usando mistura de acetato de etila:clorofórmio (75:25, v/v) e separados na coluna Chiralcel® OD-R com fase móvel constituída por acetonitrila:água (25:75, v/v), contendo 0,2% de ácido fórmico. Os enantiômeros da CPA foram analisados por LC-MS-MS, sendo que os íons protonados e seus respectivos íons-produtos foram monitorados nas transições 261>141 para a CPA e 189>104 para o padrão interno (antipirina). A recuperação foi maior que 95% para ambos os enantiômeros da CPA e o limite de quantificação foi de 2,5 ng/mL de plasma para cada enantiômero. Os coeficientes de variação e os erros relativos obtidos na avaliação da precisão e exatidão intra e inter-ensaios foram menores que 10%. Os parâmetros farmacocinéticos foram calculados empregando o programa WinNonlin e utilizando modelo monocompartimental e cinética de primeira ordem. Os parâmetros farmacocinéticos com razões enantioméricas diferentes da unidade foram avaliados com base no teste de Wilcoxon (P0,05). A disposição cinética da CPA é enantiosseletiva em pacientes com câncer de mama, com acúmulo plasmático do enantiômero (S)-(-)-CPA (AUC 195,00 vs 174,80 g.h/mL) em função do clearance preferencial do enantiômero (R)- (+)-CPA (5,13 vs 5,99 L/h). Os clearances de ambos os enantiômeros da ciclofosfamida não diferem em função dos genótipos CYP2B6, CYP2C9 e CYP2C19 e da atividade in vivo do CYP3A avaliada pelo clearance do midazolam. / Cyclophosphamide (CPA) is an alkylating oxazaphosphorine agent widely used in the treatment of multiple forms of cancer and autoimmunes disesases in adults and children. CPA is used as a racemic mixture, although preclinical data have demonstrated differences in the efficacy and toxicity of its enantiomers, with the (S)-(- )-CPA exhibiting a higher therapeutic index. The present study investigated the enantioselectivity and influence of CYP2B6, CYP2C9, CYP2C19 and CYP3A on the kinetic disposition of cyclophosphamide (CPA) in patients with breast cancer. Fifteen patients previously submitted to removal of the tumor and treated with racemic CPA (900-1000 mg) and epirubicin were included in the study. The in vivo activity of CYP3A was evaluated using midazolam as a marker drug. Serial blood samples were collected up to 24 h after administration of the first cycle of CPA. The CPA enantiomers were extracted from plasma using a mixture of ethyl acetate:chloroform (75:25, v/v) and separated on a Chiralcel® OD-R column, with the mobile phase consisting of acetonitrile:water (25:75, v/v) and 0.2% formic acid. The CPA enantiomers were analyzed by LC-MS-MS, and the protonated ions and their respective ion products were monitored at transitions of 261>141 for CPA and of 189>104 for the internal standard (antipyrine). Recovery was higher than 95% for both CPA enantiomers and the quantification limit was 2.5 ng/mL plasma for each enantiomer. The coefficients of variation and relative errors obtained for the evaluation of the intra- and interassay precision and accuracy were less than 10%. The pharmacokinetic parameters were calculated with the WinNonlin program using a monocompartmental model and first-order kinetics. The pharmacokinetic parameters presenting enantiomer ratios different from one were evaluated using the Wilcoxon test (P0.05). The kinetic disposition of CPA was enantioselective in patients with breast cancer, with plasma accumulation of the (S)-(-)-CPA enantiomer (AUC 195.00 vs 174.80 g.h/mL) due to the preferential clearance of the (R)-(+)-CPA enantiomer (5.13 vs 5.99 L/h). Clearances of both CPA enantiomers did not differ between the CYP2B6, CYP2C9 and CYP2C19 genotypes or as a function of in vivo activity of CYP3A evaluated by the midazolam clearance.

Les cytochromes P450s (CYPs) sont des hémoprotéines intervenant dans la fonction de détoxication cellulaire. Le site actif des CYPs est enfoui dans la protéine, mais accessible aux ligands par des canaux. A l'aide d'une méthode récente basée sur la triangulation de Delaunay de la protéine, et implémentée dans le logiciel CCCPP, nous avons modélisé géométriquement ces canaux pour plusieurs isoformes humaines, dont le 3A4, présent au niveau du foie humain et responsable de la métabolisation d'un nombre important de médicaments, afin de constituer un filtre stérique destiné au criblage virtuel rapide de chimiothèques. Cette approche nous a permis d'obtenir des informations sur les mécanismes d'ouverture et de fermeture des canaux, permettant d'expliquer comment des ligands volumineux peuvent accéder au site actif. Ces résultats confirment et étendent ceux de la littérature, et peuvent contribuer à l'élaboration de médicaments nouveaux ou ayant moins d'effets secondaires. / The cytochromes P450s (CYPs) are hemoproteins involved in the cellular detoxification function. The CYPs active site is buried inside the protein, but it can be accessed by the ligands through channels. With a recent method based upon the Delaunay triangulation of the protein, and implemented in the CCCPP software, we modelized geometrically these channels for several human isoforms, including the 3A4, located in the human liver and responsible of the metabolization of an important number of drugs, in order to build a sterical filter devoted to high throughput virtual screening of chemical libraries. This approach let us to get information on mechanisms of opening and closing of the channels, allowing to explain how large ligands can access to the active site. These results are in agreement and extend those found in the literature, and can contribute to the design of new drugs or of drugs having less side effects.

La bio-transformation naturelle des médicaments peut produire des métabolites toxiques; l’identification de ces métabolites est essentielle dans la stratégie de choix de molécules thérapeutiques. En appliquant les technologies de fermentation en bioréacteur des cellules hétérologues (souches d’E. coli recombinantes exprimant une iso-enzyme de cytochrome P450 humain avec la réductase humain), la bioconversion du substrat (principe actif) en ses métabolites de dégradation, a été réalisée à grande échelle (g-g). Notre choix s’est porté sur le complexe hCYP3A4/HR fonctionnel produit par un hôte E. coli. Les cellules intactes ou les membranes cellulaires peuvent être exploitées comme biocatalyseur dans un système bioréacteur. Cependant, la faible solubilité des principes actifs dans des milieux de bioconversion aqueuse limitent le rendement. Un bioréacteur biphasique a été étudié. En solution, plusieurs combinaisons eau/solvants organiques conciliant la viabilité des cellules, la solubilité des principes actifs et produits de réaction et la catalyse des complexes enzymatiques ont conduit à l’établissement d’un mélange approprié. Cependant, ces combinaisons présentent toujours une inhibition importante du pouvoir catalytique des complexes enzymatiques. Pour minimiser un effet dénaturant possible des solvants sur le système enzymatique, ce dernier a été maintenu dans un environnement aqueux en immobilisant les cellules et/ou les membranes cellulaires dans une matrice hydrophile. L’alginate de calcium apparaît être une matrice d’immobilisation idéale pour les membranes assurant la fonctionnalité du complexe CYP/HR et permettant en outre un stockage à long terme des préparations. Par contre, l’immobilisation des cellules dans diverses matrices, si elle permet une viabilité et une conservation à long terme des souches recombinantes, ne permet aucune expression de l’activité enzymatique présente dans les cellules. La combinaison d’une localisation du complexe hCYP/HR fonctionnel dans la membrane interne et d’une perméabilité réduite des cellules d’E. coli (immobilisées) en est une explication possible mais non-démontrée. Entre-temps, cette technologie de bioréacteur homogène biphasique ou par immobilisation des membranes cellulaires a été utilisée plusieurs reprises pour produire des métabolites humains à partir de divers principes actifs. Ces métabolites ont été purifiés avec succès, démontrant que cette approche technologique est compétitive comparée aux procédures conventionnelles. Néanmoins, de nouvelles pistes de recherche seraient extrêmement intéressantes. La localisation des complexes enzymatiques recombinants en surface des cellules permettrait de concilier les propriétés hydrophobes des principes actifs et l’environnement hydrophile nécessaire aux enzymes. D’autre part une investigation de complexes enzymatiques résistant aux solvants pourrait remplacer avantageusement l’immobilisation.

The research presented in this thesis focuses on the study of the reaction between boronic acids and diols and its evaluation as a possible "click" reaction, possibly applicable in bioconjugation and drug delivery. A key feature of this reaction is its reversibility at acidic pH, which could allow the release of a diol-containing drug from a bioconjugate in the acidic environment of late endosome/lysosome, possibly after undergoing receptor mediated endocytosis. Over the last two decades various studies have focused on the study of the conjugation of boronic acids to diols using Alizarin Red S as a fluorescence reporter. In this research we have presented an alternative method based on the batochromic shifts of Alizarin Red S absorbance; this method is particularly advantageous in complex systems with an elevated scattering, such as colloidal dispersions or for binding to complexed active compounds. We have therefore demonstrated that this method allows the determination of equilibrium constants between diols (e.g. catecholamines) and boronic acids. We have also demonstrated that the method allows to follow the kinetics of enzymatic reactions involving catechols; in particular, we have focused on cytochrome P450-mediated reactions such as the conversion of estradiol to 2-hydroxyestradiol using CYP1A2, or the demethylation of 3-methoxytyramine to dopamine using CYP2D6. Once we have established a reliable method for following this reaction on low molecular weight compounds, we have applied it to polymeric bioconjugates. Specifically, we have selected hyaluronic acid (HA) as a biocompatible and biodegradable polymeric backbone and produced derivatives containing boronic acids, catechols and dimethylated catechols (as negative controls). The resulting polymers where characterised via UV-Vis, 1H NMR and SLS, also qualitatively evaluating their cytotoxicity and enzymatic degradability. The conjugates with boronic acids showed the lowest cytotoxicity, and the highest degradability. The complexation of HA-boronic derivatives was then studied; using the same library of diols previously used with low molecular weight compounds, evaluating the effect of the presence of the polysaccharidic macromolecular chain.

Yes / Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700. / The authors would like to thank Bradford Institute for Health Research for funding a PhD studentship to DP through a competitive scheme and Yorkshire Cancer Research programme Grant (B381PA) for supporting our cytochrome P450-focused drug discovery research.

Orientador: Edson Luis Maistro / Resumo: Das sementes de Pentaclethra macroloba (Willd.) Kuntze, popularmente conhecida como Pracaxi, é possível a extração de um óleo que vem sendo utilizado no Brasil e em outros países para fins terapêuticos e cosméticos. Este vegetal é endêmico da região amazônica e frequentemente utilizado pela população ribeirinha como agente cicatrizante tópico, aplicado principalmente em parturientes e em picadas de serpentes, devido à sua ação antiofídica. Apesar do uso popular desse óleo, na literatura encontram-se poucos estudos avaliando seu potencial citotóxico e genotóxico. Frente a esta lacuna científica, o objetivo deste estudo foi investigar os efeitos deste óleo em células humanas HepG2/C3A in vitro, sob os aspectos de citotoxicidade, genotoxicidade, influência sobre o ciclo celular, apoptose e expressão de genes do metabolismo de xenobióticos e outras vias de sinalização celulares. Os testes do cometa e do micronúcleo foram utilizados na avaliação da genotoxicidade e mutagênese, citometria de fluxo na avaliação dos efeitos sobre o ciclo celular e apoptose, bem como a avaliação da expressão de alguns genes envolvidos nesses processos. Os resultados obtidos revelaram que o óleo não reduz a viabilidade celular nas concentrações de 31, 125 e 500 µg/mL. Nos ensaios do cometa e do micronúcleo, o óleo não apresentou efeitos genotóxico nas concentrações testadas. Além disso, a citometria de fluxo revelou que o óleo não induz apoptose nas células. A análise da expressão gênica revelou que, d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: From the Pentaclethra macroloba (Willd.) Kuntze seeds, popularly known as Pracaxi, it is possible to extract an oil that is being used in Brazil and in other countries for therapeutic and cosmetic purposes. This plant is endemic to the Amazon region and is frequently used by the riverine population as a topical healing agent, applied mainly to parturients and snake bites due to its antiofidic action. Despite the popular use of this oil, there are few studies in the literature evaluating its cytotoxic and genotoxic potential. The objective of this study was to investigate the effects of this oil on human HepG2 / C3A cells in vitro, under the aspects of cytotoxicity, genotoxicity, influence on the cell cycle, apoptosis and expression of xenobiotic and other metabolism genes cellular signaling pathways. The comet and micronucleus tests were used in the evaluation of genotoxicity and mutagenesis, flow cytometry in the evaluation of effects on the cell cycle and apoptosis, as well as the evaluation of the expression of some genes involved in these processes. The results showed that the oil did not reduce cell viability at concentrations of 31, 125 and 500 μg / mL. In the comet and micronucleus tests, the oil had no genotoxic effects at the concentrations tested. In addition, flow cytometry revealed that the oil does not induce apoptosis in cells. Analysis of gene expression revealed that of all genes tested, those that underwent stimulation were responsible for the metabolism of x... (Complete abstract click electronic access below) / Doutor

Os antagonistas adrenérgicos dos receptores ?, tais como o nebivolol, podem reduzir a mortalidade dos pacientes na fase crônica da doença de Chagas causada pelo Trypanossoma cruzi. O nebivolol está disponível na clínica como mistura racêmica dos isómeros d e l com duplo mecanismo de ação. O d-nebivolol é antagonista do receptor adrenérgico ?1, enquanto o l-nebivolol é responsável pelas propriedades vasodilatadoras do fármaco. O nebivolol é metabolizado principalmente por glicuronidação e metabolismo oxidativo dependente do CYP2D6, formando os glicuronídeos do nebivolol e os metabólitos hidroxilados do nebivolol, os quais contribuem para o antagonismo do receptor ?1 adrenérgico. O objetivo do presente estudo foi avaliar a influência da doença de Chagas na farmacocinética-farmacodinâmica dos isômeros do nebivolol e seus metabólitos em pacientes idosos metabolizadores rápidos para o CYP2D6. Foram investigados pacientes idosos portadores da doença de Chagas (n = 11) e idosos hipertensos (n = 11) previamente fenotipados como metabolizadores extensivos (EM) ou metabolizadores lentos (PM) para o CYP2D6, usando o metoprolol como fármaco marcador (21 EM e 1 PM). As coletas seriadas de sangue foram realizadas até 48 h após a administração de dose única oral de 10 mg de nebivolol racêmico. As concentrações plasmáticas dos isômeros individuais do nebivolol e glicuronídeos do nebivolol foram avaliadas por LC-MS/MS. O método mostrou linearidade nas concentrações de 15-3000 pg de cada isômero do nebivolol/mL de plasma e de 0,2-125 ng de cada isômero do glicuronídeo do nebivolol/mL de plasma. Os parâmetros farmacocinéticos foram avaliados usando o programa Phoenix (WinNonlin) e expressos em mediana, média e intervalo de confiança 95%. Os testes estatísticos foram utilizados para comparar os parâmetros farmacocinéticos entre os isômeros (teste de Wilcoxon) e entre os grupos (teste de Mann-Whithey); p < 0,05. A farmacocinética do nebivolol é estereosseletiva em pacientes idosos hipertensos portadores (9,7 vs.. 6,1 ng.h/mL) ou não (10,1 vs.. 5,4 ng.h/mL) da doença de Chagas forma crônica fenotipados como metabolizadores rápidos, com observação de maiores valores de AUC para o isômero l-nebivolol. A glicuronidação do nebivolol também é estereosseletiva em pacientes idosos hipertensos portadores (72,9 vs.. 311,6 ng.h/mL) ou não (65,3 vs.. 335,2 ng.h/mL) da doença de Chagas forma crônica fenotipados como metabolizadores rápidos, com observação de maiores valores de AUC para o isômero d-glicuronídeo. A doença de Chagas forma crônica não altera a farmacocinética e a capacidade de glicuronidação de ambos os isômeros do nebivolol em pacientes fenotipados como metabolizadores rápidos. Os valores de clearance do l-nebivolol (48,6 vs.. 14,3 L/h) e do d-nebivolol (48,4 vs.. 20,4 L/h) estimados pelo modelo populacional foram menores para os indivíduos fenotipados como metabolizadores lentos quando comparados com os ii metabolizadores rápidos do CYP2D6. O cálculo da biodisponibilidade dos isômeros individuais do nebivolol para os indivíduos metabolizadores rápidos (9% para o l-nebivolol e 5% para o d-nebivolol) e metabolizadores lentos (42% para o l-nebivolol e 29% para o d-nebivolol) do CYP2D6 permitiu inferir que o clearance não difere entre os isômeros na administração oral. As concentrações plasmáticas de nebivolol obtidas no presente estudo seguindo a administração de dose única oral de 10 mg de nebivolol racêmico a pacientes idosos hipertensos portadores ou não doença de Chagas não foram suficientes para detectar alterações nos intervalos PR, RR e QT, oriundos dos eletrocardiogramas, realizados nos mesmos tempos de colheita das amostras de sangue. Em conclusão, a doença de Chagas na forma crônica não alterou a farmacocinética e a farmacodinâmica dos isômeros do nebivolol nos idosos investigados. / Adrenergic antagonists in ? receptors, such as nebivolol may reduce mortality of patients in the chronic phase of Chagas disease caused by Trypanosoma cruzi. Nebivolol is available as a racemic mixture of d and l isomers with dual mechanism of action. The d isomer is a ?1 adrenergic receptor antagonist, while the l isomer is responsible for the drug vasodilatory properties. Nebivolol is primarily metabolised by glucuronidation and oxidative metabolism dependent on CYP2D6 to form glucuronide and hydroxylated metabolites of nebivolol, which contribute to the antagonism of adrenergic receptor ?1. This study aims to evaluate the effect of Chagas disease on the pharmacokinetics-pharmacodynamics of nebivolol isomers and its metabolites in CYP2D6 extensive metabolisers elderly patients. Hypertensive elderly patients with (n = 11) and without Chagas disease (n = 11) were previously phenotyped as extensive metabolizers (EM) or poor metabolizers (PM) for CYP2D6, applying metoprolol as a probe drug (21 EM and 1 PM). Serial blood samples were collected within 48 hours after a single oral dose administration of 10 mg racemic nebivolol. Plasma concentrations of nebivolol individual isomers and its glucuronides were measured by LC-MS/MS. The assay was linear over the rage of 15-3000 pg of each isomer of nebivolol/mL plasma and 0.2 to 125 ng of each isomer of nebivolol glucuronide/mL plasma. Pharmacokinetic parameters were evaluated applying Phoenix (WinNonlin) software and expressed as median, mean and 95% confidence interval. Statistical tests compared the pharmacokinetic parameters between isomers (Wilcoxon test) and between groups (Mann-Whithey test); p < 0.05. Pharmacokinetics of nebivolol is stereoselective in hypertensive elderly patients with (9.7 vs. 6.1 ng.h/mL) and without (10.1 vs 5.4 ng·h/mL) the chronic form of Chagas disease and phenotyped as extensive metabolisers, with higher AUC values for l-nebivolol. Nebivolol glucuronidation is also stereoselective in hypertensive elderly patients with (311.6 vs 72.9 ng.h/mL) and without (335.2 vs 65.3 ng.h/mL) the chronic form of Chagas disease and phenotyped as extensive metabolisers, with higher AUC values for d-glucuronide. The chronic form of Chagas disease does not alter the pharmacokinetics and glucuronidation capacity of either nebivolol isomers in patients phenotyped as extensive metabolizers. Clearance values for l-nebivolol (48.6 vs 14.3 L/h) and d-nebivolol (48.4 vs 20.4 L/h) estimated by the population model were lower for individuals phenotyped as CYP2D6 poor metabolisers compared to extensive metabolizers. Bioavailability calculation of individual nebivolol isomers for CYP2D6 extensive metabolisers (l-nebivolol 9%, d-nebivolol 5%) and poor metabolizers (l-nebivolol 42%, d-nebivolol 29%) made it possible to infer that clearance does not differ between the isomers in oral administration. Plasma concentrations of nebivolol observed in the present study following a single oral dose of 10 mg of racemic nebivolol to hypertensive elderly patients with and without Chagas disease were not sufficient to detect alterations in the PR, RR, and QT intervals in the electrocardiograms performed at the same iv times of blood sampling. In conclusion, Chagas disease in the chronic form did not alter the pharmacokinetics or pharmacodynamics of nebivolol isomers in the investigated elderly patients.

Os antagonistas adrenérgicos dos receptores ?, tais como o nebivolol, podem reduzir a mortalidade dos pacientes na fase crônica da doença de Chagas causada pelo Trypanossoma cruzi. O nebivolol está disponível na clínica como mistura racêmica dos isómeros d e l com duplo mecanismo de ação. O d-nebivolol é antagonista do receptor adrenérgico ?1, enquanto o l-nebivolol é responsável pelas propriedades vasodilatadoras do fármaco. O nebivolol é metabolizado principalmente por glicuronidação e metabolismo oxidativo dependente do CYP2D6, formando os glicuronídeos do nebivolol e os metabólitos hidroxilados do nebivolol, os quais contribuem para o antagonismo do receptor ?1 adrenérgico. O objetivo do presente estudo foi avaliar a influência da doença de Chagas na farmacocinética-farmacodinâmica dos isômeros do nebivolol e seus metabólitos em pacientes idosos metabolizadores rápidos para o CYP2D6. Foram investigados pacientes idosos portadores da doença de Chagas (n = 11) e idosos hipertensos (n = 11) previamente fenotipados como metabolizadores extensivos (EM) ou metabolizadores lentos (PM) para o CYP2D6, usando o metoprolol como fármaco marcador (21 EM e 1 PM). As coletas seriadas de sangue foram realizadas até 48 h após a administração de dose única oral de 10 mg de nebivolol racêmico. As concentrações plasmáticas dos isômeros individuais do nebivolol e glicuronídeos do nebivolol foram avaliadas por LC-MS/MS. O método mostrou linearidade nas concentrações de 15-3000 pg de cada isômero do nebivolol/mL de plasma e de 0,2-125 ng de cada isômero do glicuronídeo do nebivolol/mL de plasma. Os parâmetros farmacocinéticos foram avaliados usando o programa Phoenix (WinNonlin) e expressos em mediana, média e intervalo de confiança 95%. Os testes estatísticos foram utilizados para comparar os parâmetros farmacocinéticos entre os isômeros (teste de Wilcoxon) e entre os grupos (teste de Mann-Whithey); p < 0,05. A farmacocinética do nebivolol é estereosseletiva em pacientes idosos hipertensos portadores (9,7 vs.. 6,1 ng.h/mL) ou não (10,1 vs.. 5,4 ng.h/mL) da doença de Chagas forma crônica fenotipados como metabolizadores rápidos, com observação de maiores valores de AUC para o isômero l-nebivolol. A glicuronidação do nebivolol também é estereosseletiva em pacientes idosos hipertensos portadores (72,9 vs.. 311,6 ng.h/mL) ou não (65,3 vs.. 335,2 ng.h/mL) da doença de Chagas forma crônica fenotipados como metabolizadores rápidos, com observação de maiores valores de AUC para o isômero d-glicuronídeo. A doença de Chagas forma crônica não altera a farmacocinética e a capacidade de glicuronidação de ambos os isômeros do nebivolol em pacientes fenotipados como metabolizadores rápidos. Os valores de clearance do l-nebivolol (48,6 vs.. 14,3 L/h) e do d-nebivolol (48,4 vs.. 20,4 L/h) estimados pelo modelo populacional foram menores para os indivíduos fenotipados como metabolizadores lentos quando comparados com os ii metabolizadores rápidos do CYP2D6. O cálculo da biodisponibilidade dos isômeros individuais do nebivolol para os indivíduos metabolizadores rápidos (9% para o l-nebivolol e 5% para o d-nebivolol) e metabolizadores lentos (42% para o l-nebivolol e 29% para o d-nebivolol) do CYP2D6 permitiu inferir que o clearance não difere entre os isômeros na administração oral. As concentrações plasmáticas de nebivolol obtidas no presente estudo seguindo a administração de dose única oral de 10 mg de nebivolol racêmico a pacientes idosos hipertensos portadores ou não doença de Chagas não foram suficientes para detectar alterações nos intervalos PR, RR e QT, oriundos dos eletrocardiogramas, realizados nos mesmos tempos de colheita das amostras de sangue. Em conclusão, a doença de Chagas na forma crônica não alterou a farmacocinética e a farmacodinâmica dos isômeros do nebivolol nos idosos investigados. / Adrenergic antagonists in ? receptors, such as nebivolol may reduce mortality of patients in the chronic phase of Chagas disease caused by Trypanosoma cruzi. Nebivolol is available as a racemic mixture of d and l isomers with dual mechanism of action. The d isomer is a ?1 adrenergic receptor antagonist, while the l isomer is responsible for the drug vasodilatory properties. Nebivolol is primarily metabolised by glucuronidation and oxidative metabolism dependent on CYP2D6 to form glucuronide and hydroxylated metabolites of nebivolol, which contribute to the antagonism of adrenergic receptor ?1. This study aims to evaluate the effect of Chagas disease on the pharmacokinetics-pharmacodynamics of nebivolol isomers and its metabolites in CYP2D6 extensive metabolisers elderly patients. Hypertensive elderly patients with (n = 11) and without Chagas disease (n = 11) were previously phenotyped as extensive metabolizers (EM) or poor metabolizers (PM) for CYP2D6, applying metoprolol as a probe drug (21 EM and 1 PM). Serial blood samples were collected within 48 hours after a single oral dose administration of 10 mg racemic nebivolol. Plasma concentrations of nebivolol individual isomers and its glucuronides were measured by LC-MS/MS. The assay was linear over the rage of 15-3000 pg of each isomer of nebivolol/mL plasma and 0.2 to 125 ng of each isomer of nebivolol glucuronide/mL plasma. Pharmacokinetic parameters were evaluated applying Phoenix (WinNonlin) software and expressed as median, mean and 95% confidence interval. Statistical tests compared the pharmacokinetic parameters between isomers (Wilcoxon test) and between groups (Mann-Whithey test); p < 0.05. Pharmacokinetics of nebivolol is stereoselective in hypertensive elderly patients with (9.7 vs. 6.1 ng.h/mL) and without (10.1 vs 5.4 ng·h/mL) the chronic form of Chagas disease and phenotyped as extensive metabolisers, with higher AUC values for l-nebivolol. Nebivolol glucuronidation is also stereoselective in hypertensive elderly patients with (311.6 vs 72.9 ng.h/mL) and without (335.2 vs 65.3 ng.h/mL) the chronic form of Chagas disease and phenotyped as extensive metabolisers, with higher AUC values for d-glucuronide. The chronic form of Chagas disease does not alter the pharmacokinetics and glucuronidation capacity of either nebivolol isomers in patients phenotyped as extensive metabolizers. Clearance values for l-nebivolol (48.6 vs 14.3 L/h) and d-nebivolol (48.4 vs 20.4 L/h) estimated by the population model were lower for individuals phenotyped as CYP2D6 poor metabolisers compared to extensive metabolizers. Bioavailability calculation of individual nebivolol isomers for CYP2D6 extensive metabolisers (l-nebivolol 9%, d-nebivolol 5%) and poor metabolizers (l-nebivolol 42%, d-nebivolol 29%) made it possible to infer that clearance does not differ between the isomers in oral administration. Plasma concentrations of nebivolol observed in the present study following a single oral dose of 10 mg of racemic nebivolol to hypertensive elderly patients with and without Chagas disease were not sufficient to detect alterations in the PR, RR, and QT intervals in the electrocardiograms performed at the same iv times of blood sampling. In conclusion, Chagas disease in the chronic form did not alter the pharmacokinetics or pharmacodynamics of nebivolol isomers in the investigated elderly patients.

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

NADPH-P450 oxidoreductase (POR) is a membrane bound flavoprotein that donates electrons to a wide spectrum of heme-containing proteins, among which are several steroidogenic and many xenobiotics-metabolizing enzymes. Given the important role of POR protein in drug metabolism and pharmacogenomics, there is a particular need to understand the contributions of POR genetic variants to these processes. Mutations in POR gene cause a disorder called POR deficiency, which manifests with a wide phenotypic spectrum ranging from disordered steroidogenesis to skeletal malformation, namely, Antley-Bixler syndrome (ABS). The aim of the present work was to investigate the POR gene in patients suspected to have POR deficiency syndrome from Czech Republic and to perform genotyping in Czech and Jewish control populations. We analyzed 644 alleles in unrelated individuals from the general Czech population and 1128 alleles in Jewish population, where 330 alleles were of Askhenazi and 798 of Sephardic Jews. We have also studied the impact of selected new genetic variants on POR activity and identified fourteen amino acid variations, two of which we have studied in detail to establish their influence on POR activity. Using the available human POR three-dimensional structure, we then modelled the newly identified variants...