Le cytochrome P450 3A4 hépatique humain (CYP3A4) est le cytochrome P450 le plus abondant. Celui-ci est impliqué dans la dégradation de plus de 50% des médicaments que nous absorbons. Les cytochromes P450 forment une grande famille d'enzymes, caractérisée par la présence d'un hème au site actif, que l'on retrouve chez tous les organismes vivants. La réaction générale catalysée par les cytochromes P450 est une réaction d'oxygénation de substrats qui consomme de l'oxygène et du NADPH. L'étude du CYP3A4 nous a permis de constater que le site actif de cette enzyme est très sensible à la pression et qu'il était essentiel de privilégier un mode de lyse des cellules exprimant le CYP3A4 aux ultra-sons. L'expression en grande quantité du polypeptide a été réussie par l'utilisation en combinaison du vecteur d'expression pET-30a et de la souche Escherichia coli C41 (DE3). Les premières études de spectroscopie de résonance Raman du CYP3A4 purifié nous ont permis d'assigner le mode de vibration VFe-co comme étant situé à 476 cm"1 . De plus les spectres de résonance Raman dans la région des basses fréquences n'ont démontré qu'un très léger déplacement du mode VFeco lors de l'ajout des substrats imipramine et nifédipine. Ce résultat est compatible avec le fait que le site actif du CYP3A4 est flexible et relativement grand de sorte que la molécule de CO liée à Thème n'est presque pas été affectée par l'ajout de ces substrats.

1. The induction of CYP3A enzymes was investigated using a range of structurally unrelated drugs using in vivo and in vitro models. Hepatic microsomal testosterone 6(3-hydroxylation, anti-CYP3A immunoblot analysis, and molecular biology approaches were utilised in the investigation. 2. Using the rat as an in vivo model, potent induction of CYP3A enzymes was observed after administration of the synthetic glucocorticoid dexamethasone (at 150mg.kg.day for 4 days) and pregnenolone 16?-carbonitrile (at 150mg.kg-1.day-1 for four days). However, no induction was observed after administration of rifampicin (at 50 g.kg-1.day-1 for 4 days, a dose which causes potent induction in the rabbit). 3. Investigations into the effects of drug exposure on testosterone 6?- hydroxylation in cultured female rat hepatocytes revealed a positive in vivo/in vitro correlation. Cultured cells were treated with the same drugs (at 50 M concentration) for 72hrs. Dexamethasone was shown to be more potent than PCN, and rifampicin again had no effect. Dexamethasone-mediated induction of testosterone 6?- hydroxylation was dose-dependent and was shown to be maximal after 72hrs exposure. 4. The presence of the differentiating agent dimethylsulphoxide at 2% (v/v) in the cultiure medium enhanced CYP3A induction by the synthetic steroids by approximately 100% (p 0.05). 5. The potent glucococorticoid antagonist RU 486 induced testosterone 60- hydroxylation 5-fold when administered at 50mg.kg-1.day-1 for 4 days. Induction of the CYP3A protein was confirmed by immunoblot analysis of liver microsomes. Administration of RU 486 at 50mg.kg-1.day-1 did not antagonise the induction of testosterone 6?-hydroxylatiomn by dexamethasone at 150mg.kg-1.day-1. 6. Dexamethasone (0.1 to 10 M) -mediated induction of testosterone 6(3- hydroxylation in cultured rat hepatocytes was attenuated in the presence of RU 486. It is not known whether this was due to effects on CYP3 A gene expression or inhibition of enzyme mediated activity at the active site of the enzyme. 7. The lipid lowering drug SK F 98016 (150mg.kg-1.day-1) induced testosterone 6?-hydroxylation 10-fold when administered at 150mg.kg-1.day-1 for 4 days. This was confirmed by immunoblot analysis. Co-administration of RU 486 with SK F 98016 attenuated induction of CYP3A-mediated enzyme activity. The mechanism of induction of the CYP3A genes by SK F 98016 may therefore involve 'steroidal' compounds, the action of which is antagonised by RU 486. The dexamethasone- mediated increase in spectrally determined cytochrome P450 levels was also attenuated after co-administration with RU 486. As CYP3A induction was not affected by co-administration of dexamethasone with the anti-glucocorticoid RU 486, this result suggests that the glucocorticoid receptor may be involved in the induction of other P450 genes. 8. Treatment of rat hepatocytes with SK F 98016 (50 M) for 72 hours did not result in an increase in testosterone 6?-hydroxylation. In fact testosterone 6?-, 16?- and 17-oxidation activities were reduced to 50% of the activities measured in untreated hepatocytes. This pointed to some P450 inhibitory potential of SK F 98016. Investigation of the inhibitory potential of SK F 98016 on testosterone 60- hydroxylation in hepatic microsomes from PCN-treated rats showed an inhibitory effect with an IC50 of 50 M. The inhibitory effect seen in hepatocytes is similar to the effects of exposure to clotrimazole (50 .M) for 72 hours where testosterone metabolism at the 60 and 17 positions were inhibited by >90%. 9. To investigate whether the lack of inducing effect of SK F 98016 was due to the very high lipophilicity and extensive partitioning into the cultured hepatocyte, therefore resulting in a non-physiological state, cultured hepatocytes were exposed to the same drugs with albumin (from bovine serum, at the concentration present in human blood-36g/litre) in the medium in attempt to encourage an equilibrium of drug concentration between the medium and the inside of the hepatocyte. No significant induction of testosterone 60-hydroxylation was observed in the presence of albumin.

A maioria dos agentes terapêuticos, frequentemente prescritos são formulados e comercializados sob a forma racêmica, embora para alguns deles, já tenha sido demonstrado que os efeitos farmacológicos e ou tóxicos estejam relacionados apenas a um dos enantiômeros. Além disso, é conhecido o fato de que os enantiômeros podem apresentar perfis farmacocinéticos e farmacodinâmicos diferentes. O estudo avaliou a influência da exposição inalatória ao vapor de gasolina e ao etanol combustível na farmacocinética enantiosseletiva dos fármacos verapamil, ibuprofeno e fluoxetina. Ratos machos Wistar foram divididos em 09 grupos: controle, gasolina, etanol combustível. A exposição aos solventes foi realizada em câmara de exposição do tipo apenas pelo nariz, durante 6 horas/dia, cinco dias por semana, durante 6 semanas. A análise das AUCs foram calculadas diretamente no intervalo de zero a infinito com base na Quadratura de Gauss- Laguerre. As concentrações correspondentes aos tempos foram estimadas por interpolação polinomial. A comparação dos valores de AUC e Cl/f obtidos para cada fármaco e para cada Grupo exposto e seu respectivo Controle, foi realizada através da construção de Intervalos de Confiança, ao nível de 95%. A farmacocinética do verapamil, do ibuprofeno e da fluoxetina é enantiosseletiva. Os dados mostram que a exposição inalatória de ratos ao etanol combustível na concentração de 2 LEOSTEL mostrou indução do CYP2C através da redução do AUC e do aumento do clearance aparente do enantiômero (+)-(S)-ibuprofeno, inibição do CYP2D indicada pelo aumento da AUC e redução do clearance aparente do enantiômero (-)-(R)- fluoxetina e indução do CYP3A evidenciada por redução dos valores de AUC e aumento dos valores de clearance aparente de ambos os enantiômeros do verapamil. A exposição inalatória de ratos à gasolina na concentração de 2-LEOTWA também mostrou indução do CYP2C denotada pela redução do AUC e do aumento do clearance aparente de ambos os enantiômeros do ibuprofeno, inibição do CYP2D indicada pelo aumento dos valores de AUC e redução dos valores de clearance aparente de ambos enantiômeros da fluoxetina e, em não alteração do CYP3A evidenciada pela obtenção de valores de AUC e clearance aparente do verapamil similares aos do grupo controle. / Most therapeutic agents frequently used are formulated and sold under the racemic form, although for some of them, it has been demonstrated that the pharmacological or toxic and are associated only with one of the enantiomers. The study evaluated the influence of inhalation exposure to vapor of gasoline and ethanol in the enantioselective pharmacokinetics of the drug verapamil, ibuprofen and fluoxetine. Male Wistar rats were divided into 09 groups: control, gasoline, ethanol. The exposure was carried out in solvent exposure chamber by nose only exposure system for 6 hours / day, five days per week for six weeks. The analysis of the AUC were calculated directly in the range of zero to infinity on the basis of Quadrature Gauss-Laguerre. The concentrations corresponding to the times were estimated by polynomial interpolation. The comparison of AUC and Cl/f obtained for each drug and for each exposed group and its respective control, was accomplished through the construction of confidence intervals, at 95%. In conclusion, the pharmacokinetics of verapamil, ibuprofen and fluoxetine is enantioselective. The data show that inhalation exposure of rats to ethanol at a concentration of 2-LEO STEL showed induction CYP2C by reducing of the AUC and increase the apparent clearance of the enantiomer (+)-(S)-ibuprofen, inhibition of CYP2D indicated AUC increase and the reduction in the apparent clearance of the enantiomer (-)-(R)-fluoxetine and CYP3A induction as evidenced by reduction in AUC and increase and the values of apparent clearance of both enantiomers of verapamil. Inhalation exposure of rats to gasoline in a concentration of 2-LEO-TWA also showed induction CYP2C denoted by the reduction of AUC and increase and the apparent clearance of both enantiomers of ibuprofen, inhibition of CYP2D indicated by the increase in AUC and reduction values of apparent clearance of both enantiomers of fluoxetine and does not change the CYP3A evidenced by obtaining AUC and apparent clearance of verapamil similar to the control group.

A maioria dos agentes terapêuticos, frequentemente prescritos são formulados e comercializados sob a forma racêmica, embora para alguns deles, já tenha sido demonstrado que os efeitos farmacológicos e ou tóxicos estejam relacionados apenas a um dos enantiômeros. Além disso, é conhecido o fato de que os enantiômeros podem apresentar perfis farmacocinéticos e farmacodinâmicos diferentes. O estudo avaliou a influência da exposição inalatória ao vapor de gasolina e ao etanol combustível na farmacocinética enantiosseletiva dos fármacos verapamil, ibuprofeno e fluoxetina. Ratos machos Wistar foram divididos em 09 grupos: controle, gasolina, etanol combustível. A exposição aos solventes foi realizada em câmara de exposição do tipo apenas pelo nariz, durante 6 horas/dia, cinco dias por semana, durante 6 semanas. A análise das AUCs foram calculadas diretamente no intervalo de zero a infinito com base na Quadratura de Gauss- Laguerre. As concentrações correspondentes aos tempos foram estimadas por interpolação polinomial. A comparação dos valores de AUC e Cl/f obtidos para cada fármaco e para cada Grupo exposto e seu respectivo Controle, foi realizada através da construção de Intervalos de Confiança, ao nível de 95%. A farmacocinética do verapamil, do ibuprofeno e da fluoxetina é enantiosseletiva. Os dados mostram que a exposição inalatória de ratos ao etanol combustível na concentração de 2 LEOSTEL mostrou indução do CYP2C através da redução do AUC e do aumento do clearance aparente do enantiômero (+)-(S)-ibuprofeno, inibição do CYP2D indicada pelo aumento da AUC e redução do clearance aparente do enantiômero (-)-(R)- fluoxetina e indução do CYP3A evidenciada por redução dos valores de AUC e aumento dos valores de clearance aparente de ambos os enantiômeros do verapamil. A exposição inalatória de ratos à gasolina na concentração de 2-LEOTWA também mostrou indução do CYP2C denotada pela redução do AUC e do aumento do clearance aparente de ambos os enantiômeros do ibuprofeno, inibição do CYP2D indicada pelo aumento dos valores de AUC e redução dos valores de clearance aparente de ambos enantiômeros da fluoxetina e, em não alteração do CYP3A evidenciada pela obtenção de valores de AUC e clearance aparente do verapamil similares aos do grupo controle. / Most therapeutic agents frequently used are formulated and sold under the racemic form, although for some of them, it has been demonstrated that the pharmacological or toxic and are associated only with one of the enantiomers. The study evaluated the influence of inhalation exposure to vapor of gasoline and ethanol in the enantioselective pharmacokinetics of the drug verapamil, ibuprofen and fluoxetine. Male Wistar rats were divided into 09 groups: control, gasoline, ethanol. The exposure was carried out in solvent exposure chamber by nose only exposure system for 6 hours / day, five days per week for six weeks. The analysis of the AUC were calculated directly in the range of zero to infinity on the basis of Quadrature Gauss-Laguerre. The concentrations corresponding to the times were estimated by polynomial interpolation. The comparison of AUC and Cl/f obtained for each drug and for each exposed group and its respective control, was accomplished through the construction of confidence intervals, at 95%. In conclusion, the pharmacokinetics of verapamil, ibuprofen and fluoxetine is enantioselective. The data show that inhalation exposure of rats to ethanol at a concentration of 2-LEO STEL showed induction CYP2C by reducing of the AUC and increase the apparent clearance of the enantiomer (+)-(S)-ibuprofen, inhibition of CYP2D indicated AUC increase and the reduction in the apparent clearance of the enantiomer (-)-(R)-fluoxetine and CYP3A induction as evidenced by reduction in AUC and increase and the values of apparent clearance of both enantiomers of verapamil. Inhalation exposure of rats to gasoline in a concentration of 2-LEO-TWA also showed induction CYP2C denoted by the reduction of AUC and increase and the apparent clearance of both enantiomers of ibuprofen, inhibition of CYP2D indicated by the increase in AUC and reduction values of apparent clearance of both enantiomers of fluoxetine and does not change the CYP3A evidenced by obtaining AUC and apparent clearance of verapamil similar to the control group.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Des facteurs génétiques, un régime riche en gras et en sucre, l'obésité additionnée d'une sédentarité accrue sont des facteurs de risque majeurs pour l'installation progressive du diabète de type IL Ce dernier est caractérisé par une résistance à l'insuline au niveau des tissus cibles (hépatiques, musculaires et adipeux) et un défaut dans la sécrétion d'insuline par les cellules bêta du pancréas. En plus des combinaisons de médicaments augmentant la sensibilité et la sécrétion de l'insuline, les patients diabétiques vont être traités pour leurs nombreuses et diverses complications. La polypharmacie, soit la thérapie médicamenteuse multiple, observée chez les patients souffrant de maladies chroniques, tel le diabète de type II, est donc une pratique commune et une fraction importante de ces médicaments peut être biotransformée par le CYP3 A4 aux niveaux intestinal et hépatique. Des études suggèrent que la variabilité au niveau de la biotransformation des médicaments peut entre autres être causée par certains états pathologiques comme le diabète de type IL Toutefois, les altérations de la biotransformation induites par le diabète de type II sont encore mal connues. Ce projet vise donc à évaluer l'hypothèse selon laquelle le diabète de type II perturbe la biotransformation des médicaments modulant ainsi à la fois leurs effets thérapeutiques et toxiques. L'objectif est de démontrer que cet état pathologique peut influencer l'expression ainsi que l'activité de la sous-famille CYP3a aux niveaux hépatique et intestinal chez un modèle de souris diabétiques de type II (C51BLKSI}-db/db). Des études fonctionnelles ont été réalisées grâce à des incubations de microsomes hépatiques et intestinaux avec un substrat spécifique au CYP3a. Suite aux incubations hépatiques, il y eut la formation de 5 metabolites (Ml, M2, M3, M4 et M5) et seulement 4 au niveau intestinal, M5 étant indétectable. Les résultats d'analyses fonctionnelles suggèrent une diminution de l'activité hépatique et une augmentation de l'activité intestinale du CYP3a. La technique de PCR en temps réel a été utilisée afin de quantifier l'expression de CYP3a et de facteurs de transcription nucléaires chez la souris. Au niveau hépatique, la transcription de CYP3all, CYP3al3, PXR et CAR a été augmentée significativement chez les souris db/db. Au niveau intestinal, seule la transcription de PXR a été augmentée chez les souris db/db. Dépendant de l'activité pharmacologique de la molécule mère et de ses metabolites, une variation de l'activité du CYP3A4 aux niveaux hépatique et intestinal chez les diabétiques de type II pourrait donc mener au niveau clinique à une efficacité thérapeutique diminuée et/ou à l'apparition d'effets toxiques.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.