La schizophrénie est une pathologie sévère et fréquente. Elle constitue un problème majeur de santé publique. Les traitements disponibles présentent des problèmes de tolérance non négligeables et leur efficacité reste modérée. La recherche en pharmacogénétique des antipsychotiques a pour objectif d’aider les prescripteurs à choisir les traitements de façon plus rationnelle. Les carences méthodologiques des premières études réalisées expliquent sans doute le peu de résultat répliqué à ce jour. Dans une cohorte de patients schizophrènes caucasiens traités par olanzapine ou rispéridone et évaluée prospectivement pour l’efficacité et la tolérance du traitement, nous avons d’abord recherché des critères cliniques permettant de prédire la réponse au traitement. L’age précoce de début des troubles et la durée de la maladie sont des prédicteurs individuels de la mauvaise réponse au traitement. Nous avons également étudié l’implication de variants génétiques du transporteur de la noradrénaline dans l’efficacité des traitements. Nous avons observé l’implication de deux polymorphismes dans la décroissance des symptômes positifs sous traitement. L’analyse de l’impact du variant C825T de la GNB3 dans la prise de poids sous antipsychotique n’a pas retrouvé d’association significative. Enfin, nous avons étudié l’ensemble des variants alléliques du cytochrome P450 2D6 dans cette cohorte de patients schizophrènes comparée à des témoins. L’allèle CYP2D6*2 était associé à un effet protecteur vis à vis de la schizophrénie. Les associations retrouvées devraient aider à mieux comprendre les mécanismes physiopathologiques impliqués dans la schizophrénie et la réponse au traitement / Schizophrenia is a frequent and severe disease. It constitutes a major public health problem. All the available treatments however, have significant adverse side-effects and their efficacy remains moderate. The aim of pharmacogenetic research is to help practitioners to choose treatments in a more rational way. The methodological limits of the first published studies probably explain the lack of replication of such studies. In a prospective study of a sample of Caucasian schizophrenic patients treated with olanzapine or risperidone, clinical criteria were assessed as factors that may predict drug response. Early onset and duration of the disease, individually predicted an unfavourable drug response. We also studied genetic variants of the norepinephrine transporter to see how they may affect antipsychotic drug efficacy. Two polymorphisms were associated with a reduction in positive symptoms in treated schizophrenic patients. No association between the GBN3 C825T variant and weight gain in patients treated by antispychotic drugs was observed. Finally, we genotyped all the cytochrome P450 2D6 allelic variants in the same Caucasian schizophrenic sample and a Caucasian origin control cohort. The CYP2D6*2 allele was strongly associated with protection towards schizophrenia. The two observed associations may help to better understand the still unwell known physiopathological mechanisms implicated in schizophrenia aetiology and antipsychotic drug response

Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

La pharmacogénomique (PGx) étudie le concept selon lequel les déterminants génétiques peuvent aider à prédire la réponse clinique d’un patient aux médicaments. Les concentrations plasmatiques de ces derniers sont essentielles pour déterminer l’exposition, les profils pharmacocinétiques (PK), les effets cliniques et éventuellement les doses des médicaments, dont la plupart sont métabolisés par des enzymes hépatiques, les cytochromes P450 (CYPs). Néanmoins, la plupart des découvertes en matière de PGx concernant la prédiction des profils de concentrations des médicaments ont généralement recours à des plans d’études PK traditionnels avec une approche fonctionnelle. Bien qu’utile, cette méthodologie comporte des limites pour les études PGx, notamment le nombre restreint de sujets inclus, qui réduit la puissance statistique des associations PGx et limite l’identification de nouveaux variants génétiques moins fréquents. À l’inverse, les grandes cohortes observationnelles sont largement utilisées pour identifier des marqueurs génétiques physiopathologiques. Cette thèse de doctorat visait donc à 1) synthétiser les données publiées concernant les effets cliniques des polymorphismes génétiques de l’enzyme CYP2D6 sur le traitement au métoprolol, un agent β-bloquant. Les concentrations plasmatiques de métoprolol ont montré à plusieurs reprises qu’elles étaient fortement influencées par la PGx du CYP2D6; 2) développer une nouvelle méthode bioanalytique capable de quantifier les concentrations chirales de métoprolol des patients dans un contexte clinique; 3) mener une étude clinique en utilisant une grande cohorte observationnelle, ou biobanque, comme preuve de concept pour recréer l’association précédemment établie entre les phénotypes inférés des génotypes du CYP2D6 et les concentrations plasmatiques de métoprolol. Ces projets sont présentés en tant que chapitres de thèse et sous forme de manuscrits publiés. Le premier projet consistait en une revue systématique qui a permis d’extraire toutes les études relatives à la PGx du métoprolol-CYP2D6. La synthèse qualitative a suggéré que les métaboliseurs lents du CYP2D6, dépourvus de capacité enzymatique, avaient des valeurs plus élevées concernant les réductions de la fréquence cardiaque et de tension artérielle, ainsi que la survenue d’épisodes bradycardiques relativement aux autres phénotypes. Une méta-analyse ultérieure a confirmé la significativité de ces associations. Le deuxième projet a combiné des techniques bioanalytiques telles que la dérivation, l’extraction en phase solide et la chromatographie liquide avec spectrométrie de masse en tandem. Une méthode permettant de surmonter les limites analytiques antérieures a été validée avec succès pour mesurer les concentrations plasmatiques de (S)-métoprolol, l’énantiomère pharmacologiquement actif, et de son métabolite spécifique au CYP2D6. L’applicabilité d’une telle méthode a ensuite été démontrée grâce aux échantillons d’un groupe de patients issus de la Cohorte Hospitalière de l’Institut de Cardiologie de Montréal (ICM). Puis, le troisième projet présente la réalisation de l’étude LEVEL-PGx (LEVEraging Large observational cohort studies to identify pharmacogenetic determinants of drug dosing : A proof-of-concept study in the Montreal Heart Institute Hospital Cohort). L’étude portait sur un échantillon de >1000 patients sélectionnés dans la cohorte hospitalière de l’ICM, incluant leur génotypage pour CYP2D6 et la quantification du métoprolol racémique et de son métabolite spécifique au CYP2D6 dans des échantillons provenant de la Biobanque de l’ICM. Un seul échantillon unique et aléatoire par patient a été utilisé. Le recours à des modèles multivariables a validé le concept selon lequel de grandes cohortes transversales recueillant des échantillons biologiques pouvaient être utilisées afin d’identifier des associations PGx de concentrations de médicaments et ce, à des valeurs satisfaisant les seuils de significativité d’essais pangénomiques. D’autres analyses de cette cohorte ont indiqué que cette méthodologie parvenait à identifier des associations PGx qui influençaient la fréquence cardiaque au repos et la posologie du métoprolol à-travers les phénotypes du CYP2D6 et pour les déterminants génétiques uniques, même en présence de co-médications. Cependant, ces associations PGx avec les paramètres cliniques n’ont pas atteint une significativité applicable aux seuils pangénomiques. En résumé, par la reproduction d’une association PGx préalablement démontrée, l’ensemble des travaux présentés dans cette thèse suggère que l’identification et la découverte de nouveaux déterminants génétiques prédictifs des concentrations et des doses des médicaments pourrait s’effectuer par le biais de grandes cohortes observationnelles à l’échelle du génome. Ces approches permettraient de développer des modèles prédictifs plus précis de l’exposition et de la réponse aux médicaments, ce qui pourrait favoriser les découvertes PGx et, dans certains cas, éventuellement développer le potentiel translationnel d’une approche thérapeutique personnalisée selon le profil génétique des patients. / Pharmacogenomics (PGx) studies the concept that genetic determinants can help predict a patient’s clinical response to therapies. Drug concentrations are an essential component to determining the exposure, pharmacokinetic (PK) profiles, clinical effects, and potentially drug doses, most of which are metabolized through the cytochrome P450 (CYPs) liver enzymes. Nevertheless, most PGx discoveries regarding the prediction of drug concentration profiles have generally resorted to traditional PK study designs with a functional approach. Though useful, this methodology contains limitations for gene-drug interaction studies, most notably the restricted number of subjects included, which reduces the statistical power for PGx associations and limits the identification of new, less frequent genetic variants. On the opposite, large observational cohorts have long been utilized for identifying genetic markers of disease. This doctoral thesis therefore aimed to 1) synthesize published data regarding the clinical effects of CYP2D6 genetic polymorphism on metoprolol therapy. A β-blocker, metoprolol plasma concentrations have shown repeatedly to be heavily influenced by the PGx of the CYP2D6 enzyme; 2) develop a new bioanalytical method able to quantify patients’ chiral concentrations of metoprolol in a clinical setting; 3) conduct a clinical study using a large observational cohort, or biobank, as a proof of concept to recreate the previously established association between CYP2D6 genotype-inferred phenotypes and metoprolol plasma concentrations. Those projects are presented as thesis chapters in the form of published manuscripts. The first project was a systematic review that allowed us to find all studies pertaining to the PGx of metoprolol. The qualitative synthesis suggested that CYP2D6 poor metabolizers (PMs), without enzymatic capacity, had greater values regarding reductions in heart rate, blood pressures, and occurrences in bradycardia relative to non-PMs. A subsequent meta-analysis confirmed the significance of those associations. The second project combined bioanalytical techniques such as derivatization, solid phase extraction, and liquid chromatography-tandem mass spectrometry. A method overcoming previous analytical shortcomings was successfully validated to measure (S)-metoprolol plasma concentrations and its CYP2D6-specific metabolite. Its application was later demonstrated in a group of patients from the Montreal Heart Institute (MHI) Hospital Cohort. Then, the third project presents the conduct of the LEVEL-PGx study (LEVEraging Large observational cohort studies to identify pharmacogenetic determinants of drug dosing: A proof-of-concept study in the Montreal Heart Institute Hospital Cohort). The study implicated a sample of >1000 selected patients selected from the MHI Hospital Cohort, along with the genotyping of CYP2D6, and the quantification of racemic metoprolol and its CYP2D6-specific metabolite in samples from the MHI Biobank. A single, random sample per patient was used. Multivariable modeling validated the concept that large observational cohorts collecting biospecimens could be utilized to identify PGx associations of drug concentrations with genome-wide significance. Further analyses in our cohort indicated that the tested PGx associations influenced resting heart rate and metoprolol daily drug dosage across CYP2D6 phenotypes and for single genetic determinants, regardless of interfering comedications. However, such PGx associations with clinical parameters could not achieve genome-wide significance. In summary, the body of work presented in this thesis suggested that, using a previously validated PGx association, the identification of novel genetic determinants predictive of drug concentrations and dosage could be discovered and identified at the genome-wide level with large observational cohorts. These approaches would help develop more accurate predictive models of drug exposure and response, which could favor PGx discoveries and the translational potential of a personalized approach to treatments according to a patient’s genetic profile.

CYP2D6 metabolizes codeine to morphine, the active analgesic metabolite. Variation in brain CYP2D6 activity may affect brain morphine levels after codeine administration and thereby influence analgesia. We investigate the effect of inhibiting brain CYP2D on codeine-induced analgesia. METHODS: Rats received intracerebroventricular (i.c.v.) injections of CYP2D inhibitors or vehicle controls. Rats were then given subcutaneous codeine injections and analgesia was measured with the tail-flick test. Morphine and codeine concentrations in brain and plasma were measured. CYP2D activity in brain and liver were assessed in vitro. RESULTS: Compared to vehicle treatment, i.c.v. inhibitor treatments resulted in lower codeine-induced analgesia, lower morphine levels in brain but not in plasma after codeine injections, and lower CYP2D activity in brain membranes but not in liver microsomes. CONCLUSIONS: Inhibiting brain CYP2D reduces codeine’s metabolism to morphine, resulting in less analgesia. Variation in brain CYP2D6 activity may influence response to codeine and other CYP2D6 substrates.

CYP2D6 metabolizes codeine to morphine, the active analgesic metabolite. Variation in brain CYP2D6 activity may affect brain morphine levels after codeine administration and thereby influence analgesia. We investigate the effect of inhibiting brain CYP2D on codeine-induced analgesia. METHODS: Rats received intracerebroventricular (i.c.v.) injections of CYP2D inhibitors or vehicle controls. Rats were then given subcutaneous codeine injections and analgesia was measured with the tail-flick test. Morphine and codeine concentrations in brain and plasma were measured. CYP2D activity in brain and liver were assessed in vitro. RESULTS: Compared to vehicle treatment, i.c.v. inhibitor treatments resulted in lower codeine-induced analgesia, lower morphine levels in brain but not in plasma after codeine injections, and lower CYP2D activity in brain membranes but not in liver microsomes. CONCLUSIONS: Inhibiting brain CYP2D reduces codeine’s metabolism to morphine, resulting in less analgesia. Variation in brain CYP2D6 activity may influence response to codeine and other CYP2D6 substrates.

Gallic acid is a known antioxidant and has anti-inflammatory activity in addition to other biological activities, but GA efficiency is restricted due to low permeability and low oral bioavailability. This study was designed to investigate the solubility, permeability, oral bioavailability, enzymatic stability with cytochrome CYP2D6, antioxidant and anti-inflammatory activity of novel gallic acid sulfonamide derivatives; TMBS, and THBS. In addition, a novel in silico permeability model was designed to predict the permeability and bioavailability of eighty derivatives of GA. In sillico prediction of intestinal permeability of GA derivative indicated an increase in permeability with increased lipophilicity and decreased aqueous solubility, replacing the carboxylic group with sulfonamide group has increased intestinal permeability. A significant (P <0.01) increase was observed in the permeability of TMBS and THBS over GA, in both gastric fluids and HIEC cells. TMBS was O-demethylated by CYP2D6. TMBS had greater ROS scavenging activity than GA in HIEC-6 cells. There was a significant (P< 0.05) increase in anti-inflammatory activity of THBS, and TMBS compared to ibuprofen. TMBS, and THBS had better oral bioavailability than GA. This data suggests that the in silico permeability model can be used in the future to study new candidate of gallic acid, and further in vivo and clinical investigations are required to introduce TMBS and THBS as a new antioxidant and anti-inflammatory drugs.