Seventeen Cree anti-diabetic herbal medicines and eight Traditional Chinese Medicines have been examined for their potential to cause interactions with drugs, which is considered as a major reason for adverse drug effects. Specifically, the effect of these natural health products was examined on major Phase I drug metabolism enzymes including cytochrome P450, human carboxylesterase-1 and flavin-containing monooxygenases. Several of these natural health products have the potential to cause adverse drug effect through the inhibition of major drug metabolism enzymes. The results indicated that 7 Cree medicines plant extracts inhibited CYP3A4 activity, and 3 of them have been proven to cause potent mechanism-based inactivation of CYP3A4. Seven of eight Traditional Chinese Medicines have been identified as strong CYP3A4 inhibitors; the ethanol extract of Goji has identified as a potent inhibitor for CYP2C9 and 2C19. Goji juice showed universal inhibitory effects on most of the tested enzymes except flavin-containing monooxygenases 3.

Le traitement des patients avec une coronarographie après un SCA est l'aspirine et les thiénopyridines. La réponse aux thiénopyridines est variable, cette variabilité, multifactorielle, a des répercutions cliniques. Leur efficacité a été évaluée sur la réduction de la survenue d'évènements cliniques et peu sur le risque d'hémorragie qui est un effet indésirable majeur. Les plaquettes, jouent un rôle dans l'athérosclérose et les SCA notamment par le CD40L.J'ai étudié les facteurs plaquettaires conditionnant le risque hémorragique chez ces patients et apporté un éclairage sur des fonctions plaquettaires peu connues comme l'inflammation. Les génotypes du cytochrome P450 CYP2C19*2 et *17 ont une influence sur la réponse plaquettaire aux thiénopyridines et il existe une relation entre les complications hémorragiques et la réactivité plaquettaire.Une très faible réactivité plaquettaire (VASP<10%) est un facteur prédictif du risque hémorragique et les valeurs de VASP < 10 % sont plus fréquentes chez les patients traités par prasugrel. Nous avons ensuite ciblé un marqueur de l'état inflammatoire plaquettaire, le CD40L. Sa libération plaquettaire dépend de la voie du P2Y12, son expression, elle, dépend moins de cette voie. Une faible expression du CD40L est associée à des évènements hémorragiques chez les patients traités par thiénopyridines.Ainsi le déterminisme génétique de l'efficacité du traitement par thiénopyridines a un impact sur le risque hémorragique et d'autres paramètres plaquettaires influencent ce risque indépendamment de l'inhibition de l'agrégation plaquettaire. Le CD40L, serait un lien entre l'inflammation et l'équilibre saignement/thrombose. / Aspirin and thienopyridine are the therapy for patients with percutaneous coronary intervention after ACS. The level of platelet inhibition by thienopyridine varies between patients, this variability, multifactorial, is associated with adverse clinical outcomes. Treatment efficacy was evaluated mainly on the association between poor thienopyridine response and thrombotic events but less on the principal side effect: bleeding complications. Platelet play a key role in atherosclerosis and thrombosis, notably via CD40L.I studied platelet factors that influence the bleeding risk in these patients and brought a new highlight on platelet function less known such as inflammation.P450 cytochrome genetic variants (2C19*2 and 2C19*17) influence platelet response to thienopyridines. There is a relation between platelet reactivity and bleeding events. A very low on-treatment platelet reactivity (VASP<10 %) is a predictor of bleeding and is mainly observed with prasugrel treatment. We then focussed on a marker of platelet inflammatory status, CD40L. Its release by platelets depends on P2Y12 signalling, whereas its surface expression is less dependent on this signalling pathway. A low platelet-CD40L surface expression is associated with bleeding events in these patients We show that genetic background on thienopyridine treatment efficacy is related to bleeding risk and that other platelet parameters influence the bleeding risk independently of platelet aggregation inhibition. Thus, a molecule of inflammation, CD40L, would be a link between inflammation and bleeding/thrombosis equilibrium.

Cytochromes P450 (CYP) are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents. Several prodrug antitumor agents have been found as CYP substrates. Ellipticine, an alkaloid found in Apocynaceae plants, is an example of such type of pro-drug. Here, we investigate the efficiencies of human recombinant CYPs expressed in eukaryotic and prokaryotic expression systems, namely in SupersomesTM , microsomes isolated from insect cells transfected with baculovirus construct containing cDNA of human CYP1A1, 1A2 and 3A4 with NADPH:CYP reductase or in Bactosomes, the membrane fraction of E. coli transfected with cDNA of the same human CYP enzymes and NADPH:CYP reductase to oxidize their marker substrates and ellipticine. Cytochrome b5, an aditional component of the mixed function oxidase system, which metabolize xenobiotics was also expressed in some of the systems. The results found in this work demonstrate that human CYP1A1, 1A2 or 3A4 expressed in both eukaryotic and procaryotic systems oxidize their marker substrates (EROD for CYP1A1/2, MROD for CYP1A2 and testosterone 6β-hydroxylation for CYP3A4). They also oxidize...

55 Abstract Frequency of selected genetic polymorphisms of cytochrome P450 in the Czech population and the influence of CYP2C9 genotype on the hypolipidemic effect of fluvastatin Introduction: One of the main factors of genetically determined variability in response of humans to administered drugs are differences in catalytic activity of metabolizing enzymes, which are caused mainly by genetic polymorphisms in cytochrom P450 family enzymes. This thesis consists of two parts and it is presented as a commentary to the original papers. The first aim was to investigate the frequency of functionally important variant alleles of three main isoenzymes of cytochrome P450 gene: CYP2D6, CYP2C9, CYP2C19, throughout the Czech population, predict the prevalence of poor metabolizer phenotypes, and then to compare the results to the data from other populations. Secondly, we analysed the correlation between the CYP2C9 genotype and cholesterol-lowering effect of fluvastatin in human hypercholesterolemic patients. Methods: Genotypes were determined by PCR-RFLP. The presence of alleles CYP2D6*1, *6, *5, *4, *3, and gene duplication was analysed in 233 healthy volunteers, CYP2C9*1, *2 and*3 in 254 subjects and CYP2C19*1, *2 and *2 in 218 subjects. Eighty seven patients on fluvastatin therapy, and 48 patients on monotherapy...

Orientador: Roberta Okamoto / Resumo: Atualmente, com os avanços da Farmacogenética, estudos estão demonstrando que a resposta individual de medicamentos pode ser diretamente afetada pela alteração da farmacocinética induzida pela genética de cada paciente, e isto pode induzir à ausência, redução, alteração ou aumento da atividade enzimática associada. Esse fato pode modificar a eficácia clínica de determinados medicamentos e, nos casos de anti-inflamatórios não esteroidais (AINEs), alterar sua capacidade de lidar com a dor e até aumentar a frequência e a gravidade dos efeitos adversos. Este estudo teve como objetivo genotipar e fenotipar o gene CYP2C9 em 89 pacientes saudáveis submetidos à cirurgia de terceiro molar inferior, sob medicação de 20 mg de tenoxicam por dia durante 4 dias, comparando a influência do gene na dor pós-operatória, edema, trismo, quantidade de medicamentos de socorro consumidos pelos pacientes, avaliação global e satisfação do paciente em relação à ingestão do medicamento. Trata-se de um ensaio clínico randomizado, desenvolvido no Departamento de Cirurgia e Traumatologia Bucomaxilofacial da Faculdade de Odontologia de Araçatuba (FOA/UNESP) e na Disciplina de Farmacologia do Departamento Ciências Biológicas da Faculdade de Odontologia de Bauru (FOB/USP). Foi realizado o sequenciamento genético dos participantes do estudo, a fim de verificar polimorfismos do gene CYP2C9, e estes dados foram cruzados com as características pós-operatórias acima mencionadas. Oitenta e nove participantes foram... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

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Genome sequencing has resulted in the identification of >55,000 cytochrome P450 enzymes, many of which have an unknown function and regulation. In mammals, CYP3 genes appear in only one subfamily (CYP3A), which metabolize >50% of pharmaceuticals and some steroids in humans. Unlike mammals, fish contain genes in the CYP3A, CYP3B, CYP3C and CYP3D subfamilies. While it is commonly assumed that fish and mammalian CYP3A are functional similar, the function and regulation of fish CYP3 remains largely unknown. In this thesis, the receptors and compounds that regulate CYP3C genes in zebrafish were assessed. The induction of CYP3C genes in response to the aryl hydrocarbon (AHR) and estrogen receptor (ER) ligands, β-naphthoflavone and 17β-estradiol, was measured using quantitative PCR in intestine, liver and gonads. Zebrafish CYP3C genes were inducible by β-naphthoflavone and 17β-estradiol, implicating the aryl hydrocarbon and estrogen receptor in CYP3C gene regulation and suggesting that regulation of CYP3 genes in fish differs from that in mammals. To define the function of zebrafish CYP3A65 and CYP3C1, fluorogenic compounds which are specific markers of CYP1 and CYP3A activity in humans, were screened for metabolism by CYP3A65 and CYP3C1. Both CYP3A65 and CYP3C1 had the capacity to metabolize several of these compounds and the substrate profile overlapped with zebrafish CYP1A, suggesting that these compounds are not specific in fish. A high throughput approach was employed to screen ~4000 small biologically and pharmacologically active compounds for metabolism by CYP3A65 and CYP3C1, using NADPH consumption to assess catalytic activity. The substrate profiles of CYP3A65 and CYP3C1 largely overlapped and were different than mammalian CYP3A4. CYP3A65 and CYP3C1 appeared to have a bias for quinone-based compounds but further studies are required to confirm quinones as substrates and to assess a strong structure-activity relationship. Overall, this study provides insight on the regulation, function and evolution on CYP3 genes in fish. / Dissertation / Doctor of Philosophy (PhD) / Cytochrome P450 (CYP) enzymes break down compounds such as hormones and pharmaceuticals. While mammals have genes in the CYP3A subfamily, fish have unique subfamilies not found in mammals. The function and regulation of the CYP3 family in fish is unknown, but commonly assumed to be like human CYP3. The purpose of this thesis was to identify what receptors and compounds regulate CYP3C enzymes in zebrafish. We found that regulation of CYP3C enzymes in zebrafish is different than humans. Zebrafish CYP3C genes are regulated by the aryl hydrocarbon receptor and estrogen receptor, while human CYP3A is regulated by the pregnane-x-receptor. I used a high throughput approach to screen thousands of compounds to identify the function of CYP3A65 and CYP3C1 from zebrafish. CYP3A65 and CYP3C1 metabolize several plant-based and pharmaceutical compounds. CYP3A65 and CYP3C1 are more functionally similar to each other than to CYP3A in humans.

No / Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.

No / Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

La lutte chimique est la principale méthode utilisée pour contrôler les maladies causées par les champignons phytopathogènes. Dans certains cas, desphénomènes de résistance envers les fongicides se développent au sein despopulations, altérant parfois l’efficacité des molécules. La compréhension du moded’action des fongicides et des mécanismes de résistance sous-jacents participe à élaboreret à adapter des stratégies de management anti résistance ; et ainsi permettre depérenniser la durée de vie des molécules. Le fenhexamid est un fongicide récent (BayerCropScience, 2000), avec un mode d’action unique. Il est le seul fongicide commercialisébloquant l’étape de C4-déméthylation de la biosynthèse de l’ergostérol. Plusieurs typesde résistance (naturelle et acquises) ont été détectées dans les vignobles européens chez lecomplexe d’espèces Botrytis spp. responsable de la pourriture grise de la vigne. Lestravaux développés durant la thèse s’inscrivent dans l’objectif de la caractérisation dumode d’action et de l’élucidation des mécanismes de résistance. Le premier axe s’estattaché à la caractérisation fonctionnelle de deux gènes impliqués dans la C-4déméthylation de la biosynthèse de l’ergostérol : le gène erg27 codant la 3-céto réductase,cible du fenhexamid, et le gène erg28 codant une protéine qui interagirait en partie avecla 3-céto réductase. Concernant la résistance au fenhexamid, il a été démontré que, pargénétique inverse, les mutations détectées dans le gène erg27 de différents types d'isolatsrésistants issus du vignoble (phénotypes de résistance HydR3- et HydR3+) conféraient larésistance. Par ailleurs, une analyse de fitness du phénotype le plus préoccupant(phénotype HydR3+) a été réalisée en conditions contrôlées sur des souches isogéniquesartificielles afin d’apporter une réponse sur la persistance possible de ces souches auvignoble. Une méthode fine de quantification moléculaire de ces mêmes isolats aégalement été mise au point pour faciliter le suivi de leur évolution et de la persistancedes populations naturelles à l’échelle des vignobles. Cette nouvelle méthode, nomméeASPPAA PCR, exploite le polymorphisme nucléotidique du gène erg27, à l’origine de larésistance. Enfin, la résistance naturelle au fenhexamid de l’espèce apparentée à Botrytiscinerea, appelée Botrytis pseudocinerea a été élucidée. La résistance au fongicide de cetteespèce a été expliquée par la combinaison de modifications de cible (mécanismeminoritaire) et d’une dégradation du fongicide par un cytochrome P450 nomméCyp68.4 (mécanisme majeur). Il s’agit de la première identification et caractérisationgénétique d’un mécanisme de résistance à un fongicide conférée par un processus dedétoxification chez un champignon phytopathogène. / Chemical control is the main method used to control diseases caused byphytopathogenic fungi. In some cases, the resistance phenomena towardfungicides occur within fungal populations, which might alter practicalefficiency of molecules. Understanding modes of action of fungicides andunderlying resistance mechanisms participate to the development and adaptationof management strategies against resistance, and thus help to sustain the life ofmolecules. Fenhexamid is a recent fungicide (Bayer CropScience, 2000), with aparticular mode of action. It is the only fungicide marketed blocking the C4-demethylation step of ergosterol biosynthesis. Several types of resistance (naturaland acquired) were detected in European vineyards in the Botrytis spp speciescomplex, causing grey mold disease. This work focused on the characterization ofthe mode of action and the elucidation of resistance mechanisms. The first aspectinvestigated the functional characterization of two genes involved in the C4-demethylation of ergosterol biosynthesis. The erg27 gene potentially encoding the3-keto reductase which is the fenhexamid target and the erg28 gene encoding aprotein that interact in part with the 3-ketoreductase. Concerning fenhexamidresistance, we shown by reverse genetics that mutations detected in the erg27 genefrom different resistant isolates from the vineyards (phenotypes HydR3- andHydR3+) confer resistance. Furthermore, a fitness analysis under controlledconditions on the most worrying resistant phenotype (HydR3+) was performed onisogenic artificial strains in order to predict the possible persistence of these strainsin vineyards. A fine molecular method to quantify these isolates was developed tofacilitate the follow-up of evolution and persistence of resistant populations in thevineyard. This new method, named ASPPAA PCR is based on the nucleotidepolymorphism of the erg27 gene, responsible for fenhexamid resistance. Finally,the natural resistance to fenhexamid of the related species to Botrytis cinerea, B.pseudocinerea, was elucidated. Fungicide resistance of this species is explained bythe combination of target site modifications (minor mechanism) and fungicidedegradation mediated by a cytochrome P450 named Cyp68.4 (major mechanism).This is the first characterization of a genetic resistance mechanism to a fungicideconferred by detoxification in a phytopathogenic fungus.