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No. of bitstreams: 2 Tese - Stela Ramirez de Oliveira - 2015.pdf: 1513277 bytes, checksum: 674fd5339cc57193fd2c8d8e2c093272 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-28 / The objective of this study is to evaluate the inhibitory effects of derivatives of medicinal plants on CYP450 activity in rat liver microsomes by HPLC-PDA (high-performance liquid chromatography - photodiode array detection). Cerrado’s four plants were chosen for the inhibition test, because of the wide popular and traditional use: Stryphnodendron adstringens Mart (barbatimão), Copaifera langsdorffii Desf (copaíba), Lafoensia pacari A.St.-Hil. (pacari) and Pterodon emarginatus Vogel (sucupira), in addition to ten herbal medicines highly commercialized in Brazilian drugstores. To evaluate the inhibition of CYP3A, CYP2D6, CYP2C9, CYP1A2 four analytical methods were developed and validated by HPLC-PDA to quantify the substrates and metabolites. Afterward dried extracts of stem bark of Stryphnodendron adstringens and Lafoensia pacari, Copaifera langsdorffii oleoresin, Pterodon emarginatus essential oil and herbal medicines were incubated for one hour with rats liver microsomes to evaluate CYP3A inhibition (2.23 mg protein / mL liver microsome). All samples were dissolved in DMSO 5% and evaluated in three concentrations (high, medium, low). The analytical methods for quantification of biomarkers was appropriate after the validation for being linear, it did not show residual effect, it showed post-processing stability, and matrix effect within the ranges specified in the brazilian regulations. The method used to prepare the microsomal incubation and the method were suitable to assess the in vitro inhibition of CYP 3A. After analyzing the dried extracts and oil and oleoresin, was observed that they did not inhibit CYP3A. The absence of inhibitory activity of the extracts in oil and oleoresin isoenzyme CYP3A, although in vitro, adds relevant information about possible interference from non-traditional use of these plants on the metabolism of a large amount of drugs biotransformed by it. Among the tested herbal medicines, which showed greater inhibition of CYP3A was Monaless (O. sativa fermented by M. purpureus), which in higher concentration inhibited 31.94%, indicating that this product may lead to a possible interference when co-administered with drugs metabolized by CYP3A and may lead to drug interactions and adverse reactions. / O objetivo deste trabalho foi avaliar os efeitos inibitórios de derivados de plantas medicinais sobre a atividade de CYP450 em microssomas de fígado de rato por HPLC-PDA (cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodos). Foram escolhidas quatro plantas do Cerrado para realizar o teste de inibição, pela ampla utilização popular e tradicional: Stryphnodendron adstringens Mart (barbatimão), Copaifera langsdorffii Desf (copaíba), Lafoensia pacari A.St.-Hil. (pacari) e Pterodon emarginatus Vogel (sucupira), além de outros dez fitoterápicos bastante comercializados em drogarias brasileiras. Para avaliar a inibição de CYP3A, CYP2D6, CYP2C9, CYP1A2 foram desenvolvidos e validados quatro métodos analíticos por HPLC-PDA para quantificar os substratos (marcador) e seus metabólitos. Posteriormente, extratos secos de cascas dos caules de Stryphnodendron adstringens e Lafoensia pacari, oleorresina de Copaifera langsdorfii, óleo essencial de Pterodon emarginatus e os fitoterápicos foram incubados por uma hora com microssomas hepáticos de ratos para avaliação da inibição de CYP3A (2,23 mg de proteína/mL de lisado). Todas as amostras foram dissolvidas em DMSO 5% e avaliadas em três concentrações (alta, média e baixa). Os métodos analíticos para os biomarcadores de metabolismo se mostraram adequados após a validação por serem lineares, não demonstrarem efeito residual, apresentarem estabilidade pós-processamento e efeito matriz dentro dos limites especificados em regulamentação brasileira. O método utilizado para preparar os microssomas e o método de incubação se mostraram adequados para avaliar a inibição in vitro de CYP3A. Após analisar os extratos secos, oleorresina e o óleo, observou-se que eles não inibiram significativamente a atividade da CYP3A nessas condições. A ausência de atividade inibidora dos extratos, óleo e oleorresina na isoenzima CYP3A, apesar de ser in vitro, acrescenta uma informação relevante acerca de possível não interferência do uso tradicional dessas plantas sobre o metabolismo de uma quantidade elevada de fármacos biotransformados pela mesma. Dos dez fitoterápicos testados, o que apresentou maior percentual inibitório de CYP3A foi um produto denominado Monaless® (O. sativa fermentado por M. purpureus), que na maior concentração inibiu 31,94%, indicando que que esse produto pode levar a uma possível interferência quando co-administrado com fármacos metabolizados por CYP3A, podendo levar a interações medicamentosas e reações adversas.

Transfer of drugs into milk and the clearance of drugs in neonates are critical determinants of the exposure of infants to drugs in breast milk. Models predicting both parameters have been proposed. The objective of this dissertation is to test two models predicting milk to serum ratio and an ontogeny clearance model predicting clearance in the neonate. Predicted milk to serum ratio (M/S) values were generated according to the Atkinson and Begg model. The model did not adequately predict M/S when comparing the predicted values to observed values in the literature. The Fleishaker model was also tested. The model was able to predict whether the drugs appeared in milk by passive diffusion only or whether active transport processes were involved. This model, together with appropriate animal models, is useful in understanding the mechanism of drug transfer into milk. An ontogeny model that predicts clearance was proposed earlier by our laboratory. In order to test the model prediction and assumptions of constant microsomal protein and constant Km for an enzyme-substrate system with age, the male rat was used as an animal model. The ontogeny of Cyp3a1, Cyp3a2, Mdr1a and Mdr1b mRNA was examined in the male rat liver and intestine. The ontogeny pattern of Cyp3a2 mRNA, protein and in vitro Cyp3a activity were found to be similar in male rat liver. The microsomal protein content was found to vary with age in the liver. Km was found to be constant with age for the midazolam 4-hydroxylation by male rat liver microsomes. Scaling factors that extrapolate adult clearance to infant clearance were calculated from in vitro data. The model did not predict the in vivo oral clearance of midazolam for day 7 and 21 age groups from the 112 day age group (adult). The assumption that intestinal availability in the rat pups and adults was equal to unity might not be true resulting in overprediction of rat pup clearance when compared to the adult. Intestinal first pass effect for midazolam in adult rats might be significant. More experiments are needed to further test the model adequacy in clearance prediction.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 114-127).

No description available.

La kétamine est un antagoniste des récepteurs NMDA fréquemment utilisée pour des procédures anesthésiques et moins fréquemment pour traiter la douleur chronique. Des travaux de notre laboratoire ont montré que l’utilisation de la kétamine en combinaison avec la xylazine chez des rats âgés d’environ deux ans pouvait prolonger la durée d’anesthésie. Considérant que les patients gériatriques présentent des caractéristiques physiologiques demandant des adaptations au niveau de la pharmacothérapie, que cette classe de patient requiert fréquemment des anesthésies afin de réaliser des interventions chirurgicales et qu’elle représente la sous-population qui consomme le plus de médicaments pour traiter des problèmes de douleur aiguë et chronique, il convient alors d’investiguer l’impact que l’âge peut avoir sur le métabolisme des médicaments. Le but de ce projet est donc d’évaluer l’impact de l’âge sur le métabolisme de la kétamine à l’aide de fractions cellulaires (in vitro) et d’une technique de chromatographie en phase liquide couplée à la spectrométrie de masse. En premier lieu, nous avons dû confirmer la voie métabolique principale de formation de la norkétamine en utilisant le substrat et l’inhibiteur de référence de la voie métabolique du CYP3A qui sont respectivement le midazolam et le kétoconazole. De plus, nous avons caractérisé l’interaction métabolique de la kétamine fréquemment combinée au midazolam. Ensuite, nous avons comparé les profils de dégradation de la kétamine par des fractions S9 hépatiques de rats âgés de 3, 6, 12 et 18 mois. Les valeurs (±ÉT) de Vmax obtenues étaient respectivement de 2.39 (±0.23), 2.61 (±0.18), 2.07 (±0.07) pour les plus jeunes comparé à 0.68 (±0.02) pour le groupe de 18 mois (p < 0.001). Ces résultats suggèrent donc que le CYP3A est rapidement saturé par la présence de kétamine. De plus, la valeur de Km a diminué de 6 à 7 fois pour le groupe de 18 mois comparé aux autres groupes (p < 0.05). Ainsi, on peut émettre l’hypothèse que l’enzyme subit une modification de sa conformation tridimentionnelle, ce qui résulte en une diminution de formation de norkétamine. Finalement, nous avons tenté d’évaluer la contribution du métabolisme cérébral au métabolisme de la kétamine. Toutefois, nous n’avons pas trouvé de différence significative dans les concentrations de kétamine ou de midazolam entre le début de la réaction et 60 minutes plus tard. Le même constat a été observé pour d’autres substrats du CYP3A et CYP2D (dextromethorphan et codéine). D’autres expériences sont nécessaires afin d’arriver à des conclusions définitives. Les différences observées entre les différents groupes d’âge suggèrent que la voie métabolique hépatique du CYP3A est détériorée de façon importante. Les protocoles anesthésiques pour patients âgés devraient donc tenir compte de cette importante diminution d’activité métabolique étant donné que l’exposition aux concentrations systémiques risque d’être significativement augmentée. / Ketamine is a NMDAR antagonist widely used for anesthetic procedures and less often to treat chronic pain. Work in our laboratory showed that the use of ketamine combined to xylazine is a poor anesthetic choice for aged rats of approximately 2 years. Considering that geriatric patients present physiological characteristics demanding adaptations of the pharmacotherapy, that this class of patients frequently needs anesthesia to perform surgeries and that they represent the subpopulation that uses many drugs to treat acute and chronic pain, it is relevant to investigate the impact of aging on drug metabolism. The goal of this project was to assess the impact of aging on ketamine metabolism using cellular fractions (in vitro) and liquid chromatography tandem mass spectrometry. First, we needed to confirm the main metabolic pathway of norketamine formation using well-established reference substrate and inhibitor of CYP3A metabolic pathway which are respectively midazolam and ketoconazole. Furthermore, we also characterized the metabolic interaction of ketamine frequently combined with midazolam. Second, we compared the degradation profiles of ketamine in liver S9 fractions of rats aged of 3, 6, 12 and 18 months. Vmax values (±SD) were respectively of 2.39 (±0.23), 2.61 (±0.18), 2.07 (±0.07) for the younger groups compared to 0.68 (±0.02) for the 18 month older group (p < 0.001). Those results suggest that CYP3A is quickly saturated by the presence of ketamine. Moreover, Km value showed a 6 to 7 fold decrease for the oldest group compared to the younger one (p < 0.05). Therefore, we hypothesized that the enzyme undergoes conformational changes, which results in a decrease of norketamine formation. Finally, we intend to assess the contribution of brain metabolism to the overall ketamine metabolism. Unfortunately, we did not find any significative difference in ketamine or midazolam concentrations between the beginning of the reaction and 60 minutes later. The same ascertainment has been observed with other susbstrates of CYP3A and CYP2D (dextromethorphan and codeine). Other experiences are needed to definetely conlude that brain metabolism is negligible. The differences observed between age groups suggest that the hepatic metabolic pathway of CYP3A is severely impaired. Anesthetic protocols for aging patients should take into consideration the diminished metabolic activity since it may lead to a significant increase of exposition to systemic drug concentrations.

The purpose of the project was to investigate the impact of IV and PO routes difference for MDZ, a prototypical CYP3A substrate, and two CYP3A inhibitors (CYP3AI) -FLZ and ERY-, on the magnitude and time course of their inhibitory metabolic DDI. Individual semi-PBPK models for MDZ, FLZ and ERY were developed and validated separately, using pharmacokinetic (PK) parameters from clinical/in-vitro studies and published physiological parameters. Subsequently, DDI sub-models between MDZ and CYP3AIs incorporated non-competitive and mechanism-based inhibition (MBI) for FLZ and ERY, respectively, on hepatic and gut wall (GW) CYP3A metabolism of MDZ, using available in-vitro/in-vivo information. Model-simulated MDZ PK profiles were compared with observed data from available clinical PK and DDI studies, by visual predictive check and exposure metrics comparison. DDI magnitude and time course for CYP3AI (IV vs. PO) followed by MDZ (IV vs. PO) at various time points were predicted by the validated semi-PBPK-DDI models. Two hypothetical CYP3A substrates and four CYP3AI (derived from MDZ, FLZ and ERY, with GW metabolism removed, hepatic metabolism reduced, or oral bioavailability (Foral) and/or elimination half-life (t1/2) modified) were also simulated to generalize conclusions. The final semi-PBPK-DDI models predict well the PK profiles for IV/PO MDZ in absence/presence of IV/PO CYP3AI, with deviations between model-predicted and observed exposure metrics within 30%. Prospective simulations demonstrate that: 1) CYP3A substrates, e.g., MDZ, are consistently more sensitive to metabolic inhibition after PO than after IV administration, due to pre-systemic hepatic and/or GW metabolism. For substrates without GW metabolism and limited hepatic metabolism, only a marginal route difference for substrate administration is observed. 2) For high-Foral CYP3AIs, e.g., FLZ, no inhibitor IV-PO route DDI differences are expected, unless they are given simultaneously with PO MDZ. 3) For low-Foral CYP3AIs, e.g., ERY, greater inhibition is expected after IV than after PO administration for IV MDZ, but is difficult to predict for PO MDZ. 4) In addition to Foral and plasma t1/2 of CYP3AIs, the DDI onset, peak and duration are determined by their oral absorption rate and by the resulting hepatic and/or GW concentration profiles relative to Ki for noncompetitive CYP3AIs, but by CYP3A kinetics (synthesis, degradation rate) for MBI CYP3AIs.

<p>The subject of this thesis was to study two intestinal barriers to oral drug bioavailability, drug efflux proteins of the ABC-transporter family, and in particular ABCB1/P-glycoprotein (Pgp), and the drug metabolizing enzyme cytochrome P450 (CYP) 3A4. At the onset of this thesis, similarities between CYP3A4 and Pgp in terms of their tissue distribution and gene regulation, along with overlapping substrate specificities, had generated the hypothesis that CYP3A4 and Pgp may have a complementary function and thus form a coordinated intestinal barrier to drug absorption and gut wall metabolism.</p><p>In the first part of this thesis, a cell culture model of the intestinal epithelium that expressed both functional Pgp and CYP3A4 was developed. This model was then used to investigate the steroselective drug efflux and metabolism of R/S-verapamil. In summary, the results indicated that the two barriers in the cell culture model were in agreement with those in the human intestine.</p><p>Both ABC-transporters and CYPs are regulated by drugs that interact with nuclear receptors. However, while the regulation of CYPs is quite well understood, less is known about how repeated drug administration regulates the most abundantly expressed ABC-transporters. Therefore, in the second part of this thesis, the effects of repeated drug administration on the gene regulation of four ABC-transporters and CYP3A4 were studied in intestinal epithelial cell lines in vitro and in the perfused human jejunum in vivo. The in vitro studies revealed that the ABC-transporters are induced by drugs that interact with slightly different sets of nuclear receptors. The in vivo study showed that repeated oral administration of St John’s wort decreased the bioavailability of verapamil, predominantly by induction of intestinal CYP3A4. This part of the thesis provides new information about the regulation of ABC-transporters, shows that the intestinal metabolism is the most significant barrier to oral bioavailability of verapamil and provides evidence for a clinically significant interaction between verapamil and St John’s wort in vivo.</p>

The subject of this thesis was to study two intestinal barriers to oral drug bioavailability, drug efflux proteins of the ABC-transporter family, and in particular ABCB1/P-glycoprotein (Pgp), and the drug metabolizing enzyme cytochrome P450 (CYP) 3A4. At the onset of this thesis, similarities between CYP3A4 and Pgp in terms of their tissue distribution and gene regulation, along with overlapping substrate specificities, had generated the hypothesis that CYP3A4 and Pgp may have a complementary function and thus form a coordinated intestinal barrier to drug absorption and gut wall metabolism. In the first part of this thesis, a cell culture model of the intestinal epithelium that expressed both functional Pgp and CYP3A4 was developed. This model was then used to investigate the steroselective drug efflux and metabolism of R/S-verapamil. In summary, the results indicated that the two barriers in the cell culture model were in agreement with those in the human intestine. Both ABC-transporters and CYPs are regulated by drugs that interact with nuclear receptors. However, while the regulation of CYPs is quite well understood, less is known about how repeated drug administration regulates the most abundantly expressed ABC-transporters. Therefore, in the second part of this thesis, the effects of repeated drug administration on the gene regulation of four ABC-transporters and CYP3A4 were studied in intestinal epithelial cell lines in vitro and in the perfused human jejunum in vivo. The in vitro studies revealed that the ABC-transporters are induced by drugs that interact with slightly different sets of nuclear receptors. The in vivo study showed that repeated oral administration of St John’s wort decreased the bioavailability of verapamil, predominantly by induction of intestinal CYP3A4. This part of the thesis provides new information about the regulation of ABC-transporters, shows that the intestinal metabolism is the most significant barrier to oral bioavailability of verapamil and provides evidence for a clinically significant interaction between verapamil and St John’s wort in vivo.

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.