Implication du CYP2D6 dans la pharmacodynamie et la pharmacogénomique de l’oxycodone

La variabilité interindividuelle dans la réponse aux médicaments constitue une problématique importante pouvant causer des effets indésirables ou l’échec d’un traitement. Ces variabilités peuvent être causées par une diminution de l’activité de l’enzyme responsable du métabolisme de certains médicaments, fréquemment les cytochromes P450, un système enzymatique majeur dans le métabolisme de ces derniers. Ces enzymes sont sujets à des mutations génétiques appelées polymorphismes, qui altèrent l’activité métabolique. Il est donc important d’évaluer le rôle de ces enzymes dans le métabolisme des médicaments afin d’identifier leur responsabilité dans la variabilité interindividuelle de la réponse au traitement. Parmi l’important système enzymatique que représentent les cytochromes P450, l’isoenzyme CYP2D6 est particulièrement étudiée, ses variations métaboliques revêtant une haute importance clinique. L’un des substrats du CYP2D6 est l’oxycodone, un analgésique narcotique largement prescrit en clinique. Une grande variabilité est observée dans la réponse analgésique à l’oxycodone, variabilité pouvant être causée par un polymorphisme génétique. Il est connu que des variations génétiques dans le CYP2D6 compromettent la réponse analgésique à la codéine en rendant moins importante la formation de son métabolite actif, la morphine. Par analogie, plusieurs études supportent l’hypothèse selon laquelle le métabolite oxymorphone, formée par l’isoenzyme CYP2D6, serait responsable de l’analgésie de l’oxycodone. Une déficience génétique de l’enzyme compromettrait la réponse analgésique au médicament. Les travaux effectués dans le cadre de ce mémoire ont démontré que l’inhibition du CYP2D6 chez des sujets volontaires réduit de moitié la production d’oxymorphone, confirmant l’importante implication de l’enzyme dans le métabolisme de l’oxycodone. Ces résultats démontrent une forte ressemblance avec le métabolisme de la codéine, suggérant que l’oxymorphone pourrait être responsable de l’analgésie. Cependant, les travaux effectués n’ont pu établir de relation entre la concentration plasmatique d’oxymorphone et le niveau d’analgésie ressenti par les sujets. La continuation des études sur le mécanisme d’action de l’oxycodone dans la réponse analgésique est essentielle afin d’établir la source des variabilités interindividuelles expérimentées par les patients et ainsi d’éviter des effets secondaires ou lacunes dans le traitement.

L’inhibition des cytochromes P450 dans les cas d’insuffisance rénale chronique : rôle de l’hormone parathyroïdienne.

L’insuffisance rénale chronique (IRC) est associée à une réduction du métabolisme de plusieurs médicaments, due à une diminution du cytochrome P450 (CYP450) hépatique. Nos études précédentes ont montré que l’IRC affecte l’activité in vivo et in vitro, de même que l’expression protéique et génique des différents isoformes du CYP450, via la présence du sérum urémique et de l’hormone parathyroïdienne (PTH). Ce projet de doctorat se divise en quatre parties. Premièrement, nous avons développé une méthode d’analyse de l’activité du CYP450, à l’aide de la production du 3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl] furan-2(5H)-one (DFH) à partir du 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one (DFB). Cette méthode nous a permis de mieux quantifier l’activité dans les études subséquentes. Deuxièmement, l’activité du CYP450 3A est diminuée chez les patients atteints d’IRC. De plus, il a déjà été démontré que des toxines urémiques dialysables seraient impliquées puisque l’hémodialyse prévient cette inhibition du CYP450. Par contre, le mécanisme expliquant l’amélioration transitoire la composition du sérum de patients atteints d’IRC par l’hémodialyse n’est pas connu. L’objectif du projet est d’évaluer l’effet de l’hémodialyse sur l’expression protéique et génique, de même que sur l’activité du CYP450 3A2 dans un modèle d’hépatocytes de rat en culture. Troisièmement, la déficience en calcidiol est fréquente dans les cas d’IRC et l’étiologie est peu connue. Nous avons récemment montré que l’IRC est associée à une diminution du métabolisme des médicaments par le foie suite à une réduction des différents isoformes du CYP450 en partie médiée par l’hormone parathyroïdienne (PTH). La 25-hydroxylation de la vitamine D, au niveau du foie, permet la formation du calcidiol par différents isoformes du CYP450 (CYP2C11, 27A1, 2R1, 3A2 et 2J3) et pourrait être ainsi altérée en présence d’IRC. Les objectifs de cette étude sont de a) confirmer la diminution de synthèse de calcidiol en présence d’IRC et b) évaluer le rôle de la PTH dans la déficience en calcidiol. Finalement, afin de mieux comprendre les inhibitions du CYP450, nous avons étudié les voies de signalisation impliquées dans la régulation du CYP450 en présence d’IRC et avec la PTH puisque les mécanismes d’action demeurent imprécis. La contribution des facteurs de transcription et des récepteurs nucléaires suivants est étudiée ; le récepteur pregnane X (PXR), le récepteur constitutif androstane (CAR) et le facteur nucléaire kappa B (NF-κB), puisqu’ils sont potentiellement activés par le récepteur de la PTH et ces molécules ont été précédemment impliqués dans la régulation du CYP450. Les résultats obtenus montrent que l’hémodialyse des patients atteints d’IRC améliore transitoirement l’expression du CYP450 lorsque des hépatocytes sont mis en culture avec du sérum provenant de ces patients. Aussi, la 25-hydroxylation de la vitamine D est affectée par l’IRC. Les voies de signalisation du NF-κB et les facteurs nucléaires PXR et CAR sont impliqués dans l’inhibition du CYP450. En conclusion, l’IRC affecte, non seulement le métabolisme des médicaments mais aussi l’hydroxylation de la vitamine D, un des rôles endogènes effectués par le CYP450. Ces études nous permettent de mieux comprendre les effets de l’IRC afin de mieux cibler les traitements de choix pour les patients qui en sont atteints.

Assessment of Tandem Mass Spectrometry and High Resolution Mass Spectrometry for the Analysis of Bupivacaine in Plasma

Triple quadrupole mass spectrometers coupled with high performance liquid chromatography are workhorses in quantitative bioanalyses. It provides substantial benefits including reproducibility, sensitivity and selectivity for trace analysis. Selected Reaction Monitoring allows targeted assay development but data sets generated contain very limited information. Data mining and analysis of non-targeted high-resolution mass spectrometry profiles of biological samples offer the opportunity to perform more exhaustive assessments, including quantitative and qualitative analysis. The objectives of this study was to test method precision and accuracy, statistically compare bupivacaine drug concentration in real study samples and verify if high resolution and accurate mass data collected in scan mode can actually permit retrospective data analysis, more specifically, extract metabolite related information. The precision and accuracy data presented using both instruments provided equivalent results. Overall, the accuracy was ranging from 106.2 to 113.2% and the precision observed was from 1.0 to 3.7%. Statistical comparisons using a linear regression between both methods reveal a coefficient of determination (R2) of 0.9996 and a slope of 1.02 demonstrating a very strong correlation between both methods. Individual sample comparison showed differences from -4.5% to 1.6% well within the accepted analytical error. Moreover, post acquisition extracted ion chromatograms at m/z 233.1648 ± 5 ppm (M-56) and m/z 305.2224 ± 5 ppm (M+16) revealed the presence of desbutyl-bupivacaine and three distinct hydroxylated bupivacaine metabolites. Post acquisition analysis allowed us to produce semiquantitative evaluations of the concentration-time profiles for bupicavaine metabolites.

Genotipificación de HLA-B en pacientes colombianos afectados por el síndrome Stevens-Johnson y la Necrólisis Epidérmica Tóxica

Las reacciones alérgicas a medicamentos cutáneas severas (RAM) como el Síndrome Stevens Johnson (SJS) y la Necrólisis Epidérmica Tóxica (NET),caracterizadas por exantema, erosión de la piel y las membranas mucosas, flictenas, desprendimiento de la piel secundario a la muerte de queratinocitos y compromiso ocular. Son infrecuentes en la población pero con elevada morbi-mortalidad, se presentan luego de la administración de diferentes fármacos. En Asia se ha asociado el alelo HLA-B*15:02 como marcador genético para SJS. En Colombia no hay datos de la incidencia de estas RAM, ni de la relación con medicamentos específicos o potenciales y tampoco estudios de aproximación genómica de genes de susceptibilidad.

Influencia de la suplementación de antioxidantes y de la administración de enrofloxacina en la calidad y seguridad de la carne de ave

Los fenómenos oxidativos y en particular la oxidación lipídica son uno de los principales responsables de la pérdida de calidad en la carne y en los productos cárnicos. Como consecuencia de estos procesos se generan compuestos que pueden afectar el flavor, color y textura de la carne disminuyendo la aceptabilidad por parte del consumidor y reduciendo su valor nutritivo. Por otro lado, el estrés oxidativo está relacionado con la etiología de diversas enfermedades comunes en nuestra sociedad. Las carnes de pollo y de pavo son particularmente sensibles a los procesos oxidativos debido a su elevada proporción de ácidos grasos poliinsaturados en comparación con otros tipos de carne. La suplementación de antioxidantes en la dieta de determinados animales es una de las estrategias más eficaces para proteger la carne de la oxidación. Otro aspecto que afecta a la calidad y seguridad de la carne es la presencia de residuos en los tejidos animales destinados al consumo humano, una parte de los cuáles puede proceder de la administración de antibióticos. En este trabajo se estudió la eficacia de tres compuestos antioxidantes, alfa-tocoferol, beta-caroteno y licopeno, adicionados en distintas concentraciones y combinaciones a la dieta de pollos y pavos. Para ello se determinó la estabilidad oxidativa de los tejidos musculares de pechuga y muslo mediante el análisis de los valores de TBARS, de las actividades de los enzimas antioxidantes GSHPx, CAT y SOD y desde un punto de vista sensorial. Asimismo, se analizaron las concentraciones de vitamina E presentes en ambos músculos. Por otro lado, se investigó la presencia de residuos del antibiótico enrofloxacina y de su metabolito en los tejidos muscular y hepático de ambas especies después de la administración del fármaco con o sin periodo de retirada. Finalmente, y dada la aparente relación existente entre el metabolismo de determinados antibióticos y los fenómenos oxidativos, se valoró la posible interacción entre el fármaco y la vitamina E suplementada a la dieta. La vitamina E, a dosis de 100 ppm y 200 ppm en pollos y pavos respectivamente, se comportó como un antioxidante eficaz disminuyendo la rancidez de la carne tanto en pechuga como en muslo. La dosis de vitamina E necesaria para conseguir un incremento significativo de la estabilidad oxidativa de la carne varió en función de la especie y de las características bioquímicas del tejido analizado. El beta-caroteno, suplementado en la dieta de pollos y pavos conjuntamente con la vitamina E, no sólo no manifestó propiedades antioxidantes sino que enmascaró la efectividad de la vitamina E. El licopeno, de cuya utilización en nutrición animal no existían estudios publicados anteriormente, no mostró eficacia antioxidante en la carne de pollo a una dosis de 10 ppm. Respecto al análisis de residuos de antibiótico se observó que tras el periodo de retirada del fármaco los niveles residuales de enrofloxacina y su metabolito disminuyeron notablemente. Debe tenerse en cuenta que se apreciaron diferencias en función de la especie y del tejido considerados, estando los residuos en algunos casos por encima de los límites máximos permitidos. Por otro lado, se observó una relación entre la enrofloxacina y la vitamina E suplementada en la dieta que, parecía depender tanto de la dosis de antioxidante como del metabolismo del fármaco. Esta interacción afectó tanto a los niveles de vitamina E como a la presencia de residuos de enrofloxacina en el tejido muscular, resaltando la importancia de no subestimar posibles interacciones entre distintos compuestos presentes en la dieta animal.

Therapeutic modulation of microbiota-host metabolic interactions

The complex metabolic relationships between the host and its microbiota change throughout life and vary extensively between individuals, affecting disease risk factors and therapeutic responses through drug metabolism. Elucidating the biochemical mechanisms underlying this human supraorganism symbiosis is yielding new therapeutic insights to improve human health, treat disease, and potentially modify human disease risk factors. Therapeutic options include targeting drugs to microbial genes or co-regulated host pathways and modifying the gut microbiota through diet, probiotic and prebiotic interventions, bariatric surgery, fecal transplants, or ecological engineering. The age-associated co-development of the host and its microbiota provides a series of windows for therapeutic intervention from early life through old age

Chemoprotective effect of the tetrahydrofuran lignan grandisin in the in-vivo rodent micronucleus assay

Objectives The chemoprotective effect of the tetrahydrofuran lignan grandisin against DNA damage induced by cyclophosphamide (200 mg/kg) has been evaluated using the in vitro rodent micronucleus assay. Methods The effects of a daily oral administration of grandisin (2, 4, or 8 mg/kg) for five days before exposure to cyclophosphamide on the frequency of micronucleus in the bone marrow of normal mice exposed and unexposed to cyclophosphamide were investigated (n = 5 per group). Electrochemical measurements were applied to investigate whether the antimutagenic effects of grandisin could be, at least in part, a consequence of its or its metabolite`s antioxidant properties. Key findings Grandisin did not show mutagenic effects on the bone marrow cells of exposed mice. On the other hand, the oral administration of grandisin (2, 4, or 8 mg/kg) per day reduced dose-dependently the frequency of micronucleus, induced by cyclophosphamide, in all groups studied. Cyclic voltammograms showed two peaks for a grandisin metabolite, which were absent for grandisin. Conclusions Under the conditions tested herein, this study has shown that mice treated with grandisin presented, in a dose-dependent manner, a protective effect against cyclophosphamide-induced mutagenicity. This effect could be, at least in part, associated to grandisin bioactivation. These data open new perspectives for further investigation into the toxicology and applied pharmacology of grandisin.

Novel Application of 2D and 3D-Similarity Searches To Identify Substrates among Cytochrome P450 2C9, 2D6, and 3A4

Cytochrome P450 (CYP450) is a class of enzymes where the substrate identification is particularly important to know. It would help medicinal chemists to design drugs with lower side effects due to drug-drug interactions and to extensive genetic polymorphism. Herein, we discuss the application of the 2D and 3D-similarity searches in identifying reference Structures with higher capacity to retrieve Substrates of three important CYP enzymes (CYP2C9, CYP2D6, and CYP3A4). On the basis of the complementarities of multiple reference structures selected by different similarity search methods, we proposed the fusion of their individual Tanimoto scores into a consensus Tanimoto score (T(consensus)). Using this new score, true positive rates of 63% (CYP2C9) and 81% (CYP2D6) were achieved with false positive rates of 4% for the CYP2C9-CYP2D6 data Set. Extended similarity searches were carried out oil a validation data set, and the results showed that by using the T(consensus) score, not only the area of a ROC graph increased, but also more substrates were recovered at the beginning of a ranked list.

Substrate specificity, regulation, and polymorphism of human cytochrome P450 2B6

CYP2B6 is mainly expressed in the liver that has been thought historically to play an insignificant role in human drug metabolism. However, increased interest in this enzyme has been stimulated by the discovery of polymorphic and ethnic differences in CYP2B6 expression, identification of additional substrates for CYP2B6, and evidence for co-regulation with CYP3A4. This paper updates our knowledge about the structure, function, regulation and polymorphism of CYP2B6. CYP2B6 can metabolise approximately 8% of clinically used drugs (n > 60), including cyclophosphamide, ifosfamide, tamoxifen, ketamine, artemisinin, nevirapine, efavirenz, bupropion, sibutramine, and propofol. CYP2B6 is one of the CYP enzymes that bioactivate several procarcinogens and toxicants. This enzyme also metabolizes arachidonic acid, lauric acid, 17beta-estradiol, estrone, ethinylestradiol, and testosterone. Typical substrates of CYP2B6 are non-planar molecules, neutral or weakly basic, highly lipophilic with one or two hydrogen-bond acceptors. The crystal structure of CYP2B6 has not been resolved, while several pharmacophore and homology models of human CYP2B6 have been reported. Human CYP2B6 is closely regulated by constitutive androstane receptor (CAR/NR1I3) which can activate CYP2B6 expression upon ligand binding. Pregnane X receptor and glucocorticoid receptor also play a role in the regulation of CYP2B6. Induction of CYP2B6 may partially explain some clinical drug interactions observed. For example, coadministered carbamazepine decreases the systemic exposure of bupropion. There is a wide interindividual variability in the expression and activity of CYP2B6. Such a large variability is probably due to effects of genetic polymorphisms and exposure to drugs that are inducers or inhibitors of CYP2B6. To date, at least 28 allelic variants and some subvariants of CYP2B6 (*1B through *29) have been described and some of them have been shown to have important functional impact on drug clearance and drug response. For example, the efavirenz plasma levels in African-American subjects with the CYP2B6 homozygous 516T/T genotype are approximately 3-fold higher than individuals carrying the homozygous G/G genotype. The CYP2B6 516T/T genotype is associated with 1.7-fold greater plasma levels of nevirapine in HIV-infected patients. Smokers with the 1459C>T (R487C) variant of CYP2B6 may be more vulnerable to abstinence symptoms and relapse following treatment with bupropion as a smoking cessation agent. Further studies in the structure, function, regulation and polymorphism of CYP2B6 are warranted.

Antigen otoxicity of Agaricus blazei mushroom organic and aqueous extracts in chromosomal aberration and cytokinesis block micronucleus assays in CHO-k1 and HTC cells

Agaricus blazei (Ab) has become popularly known for its medicinal properties. Scientifically, it has been tested with regard to its capacity to protect genetic material against damage. We examined different organic extracts (methanolic extract-ME, hexanic extract-HE and n-butanolic extract-BE) and an aqueous extract (AE) of Ab, for their capacity to induce DNA damage as well as for their protective effect. Genetic damage was determined by the chromosomal aberration assay (CA) in CHO-k1 cells for all extracts and the cytokinesis block micronucleus assay (CBMN) in non drug-metabolizing (CHO-k1) and drug-metabolizing (HTC) cell lines for extract BE only. The extracts did not show clastogenicity but showed anticlastogenicity. The greatest percent reduction obtained were with BE (105%) and AE (126%) treatments in CA. BE treatment did not display genotoxicity in CHO-k1, but was genotoxic in HTC. However, BE was shown to be antigenotoxic causing decreased micronucleus frequency in HTC and CHO-k1 cells. These results suggest that all the extracts contained protective substances, but in some cases they could show a genotoxic effect with regard to metabolism. Therefore, these findings warrant caution in the use of this mushroom by the population. (c) 2005 Elsevier Ltd. All rights reserved.

The Potential of p38 MAPK Inhibitors to Modulate Periodontal Infections

Periodontal disease initiation and progression occurs as a consequence of the host immune inflammatory response to oral pathogens. The innate and acquired immune systems are critical for the proper immune response. LPS, an outer membrane constituent of periodontal pathogenic bacteria, stimulates the production of inflammatory cytokines IL-1 beta TNF alpha IL-6 and RANKL either directly or indirectly. In LPS-stimulated cells, the induction of cytokine expression requires activation of several signaling pathways including the p38 MAPK pathway. This review will discuss the significance of the p38 MAPK pathway in periodontal disease progression and the potential therapeutic consequences of pharmacological antagonism of this pathway in the treatment of periodontal diseases.

ALTERED FOCI OF HEPATOCYTES IN RATS INITIATED WITH DIETHYLNITROSAMINE AFTER PROLONGED FASTING

The influence of fasting on the potential of diethylnitrosamine (DEN) to initiate liver cucinogenesis was tested in a medium-term assay using the development of putative preneoplastic altered foci of hepatocytes (AFH) as the endpoint. Male Wistar rats fasted for 48 hr were given a single ip injection of DEN (200 mg/kg body weight). Partial hepatectomies were carried out at wk 3 and the rats were killed at wk 8. Fasted rats exhibited a small increase in the numbers of AFH with glutathione S-transferase in the placental form and eosinophilic AFH when compared with non-fasted animals. However, after a 6-wk exposure to 0.05% sodium phenobarbital in the diet, there were no differences in the numbers of AFH between fasted and non-fasted animals. Fasting also increased DEN-dependent centrilobular cell necrosis and specifically drug metabolism as indicated in vivo by a decreased time of paralysis of the lower limbs induced by zoxazolamine (40 mg/kg body weight, ip) and by an unaltered sleeping time induced by sodium pentobarbital (40 mg/kg body weight, ip). The results indicate that although fasting during the initiation stage of carcinogenesis increases DEN hepatotoxicity, it does not interfere quantitatively with the development of liver preneoplastic lesions.

Protective effect of safranine on the mitochondrial damage induced by Fe(II)citrate: Comparative study with trifluoperazine

In this study, we show that safranine at the concentrations usually employed as a probe of mitochondrial membrane potential significantly protects against the oxidative damage of mitochondria induced by Fe(II)citrate. The effect of safranine was illustrated by experiments showing that this dye strongly inhibits both production of thiobarbituric acid-reactive substances and membrane potential decrease when energized mitochondria were exposed to Fe(II)citrate in the presence of Ca 2+ ions. Similar results were obtained with the lipophylic compound trifluoperazine. It is proposed that, like trifluoperazine, safranine decreases the rate of lipid peroxidation due to its insertion in the membrane altering the physical state of the lipid phase.

Can trifluoperazine protect mitochondria against reactive oxygen species-induced damage?

Trifluoperazine (TFP) (35 μM) prevents mitochondrial transmembrane potential (ΔΨ) collapse and swelling induced by 10 μM Ca2+ plus oxyradicals generated from δ-aminolevulinic acid autoxidation. In contrast with EGTA, TFP cannot restore the totally collapsed ΔΨ. So, TFP might not remove Ca2+ from its 'harmful site', but could impair the ROS-driven cross-linking between membrane -SH proteins. Our data are correlated with the protective uses of TFP against oxidative processes promoted by oxyradicals plus Ca2+.