Characterization of metabolites in infiltrating gliomas using ex vivo &supl;H high-resolution magic angle spinning spectroscopy.

Gliomas are routinely graded according to histopathological criteria established by the World Health Organization. Although this classification can be used to understand some of the variance in the clinical outcome of patients, there is still substantial heterogeneity within and between lesions of the same grade. This study evaluated image-guided tissue samples acquired from a large cohort of patients presenting with either new or recurrent gliomas of grades II-IV using ex vivo proton high-resolution magic angle spinning spectroscopy. The quantification of metabolite levels revealed several discrete profiles associated with primary glioma subtypes, as well as secondary subtypes that had undergone transformation to a higher grade at the time of recurrence. Statistical modeling further demonstrated that these metabolomic profiles could be differentially classified with respect to pathological grading and inter-grade conversions. Importantly, the myo-inositol to total choline index allowed for a separation of recurrent low-grade gliomas on different pathological trajectories, the heightened ratio of phosphocholine to glycerophosphocholine uniformly characterized several forms of glioblastoma multiforme, and the onco-metabolite D-2-hydroxyglutarate was shown to help distinguish secondary from primary grade IV glioma, as well as grade II and III from grade IV glioma. These data provide evidence that metabolite levels are of interest in the assessment of both intra-grade and intra-lesional malignancy. Such information could be used to enhance the diagnostic specificity of in vivo spectroscopy and to aid in the selection of the most appropriate therapy for individual patients.

Protein synthesis in jejunum and liver of neonatal calves fed vitamin A and lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.

Cell-cell communication in the biocontrol strain Pseudomonas fluorescens CHA0

Summary Pseudomonas fluorescens CHAO is a soil bacterium which was isolated near Morens (Switzerland) and which protects plants from root-pathogenic fungi. This protection is due to extracellular secondary metabolites whose synthesis is regulated by the two-component system GacS/GacA in strain CHAO. Extracellular signals of bacterial origin activate this regulatory system. These signals are different from N-acyl-homoserine lactones, are extracted by dichloromethane and appear to have a low molecular weight. Preliminary evidence was obtained from a small molecule m/z 278 produced by strain CHAO. Similar signals capable of activating GacS/GacA-dependent regulation in strain CHAO were found in a large number of different Gram-negative bacteria. Once activated by signal(s), the sensor GacS is assumed to phosphorylate the response regulator GacA, which positively influences a regulatory cascade, resulting in the synthesis of secondary metabolites. This cascade includes three GacA-controlled small regulatory RNAs and two translational repressor proteins. The regulatory RNAs titrate the repressor proteins; this allows translation of target genes and the synthesis of exoenzymes and secondary metabolites such as antibiotics and hydrogen cyanide. A GFP-based sensor for signal detection was constructed in strain CHAO by fusing the gfp reporter gene to the rsmZ small RNA gene. CHAO mutants defective for signal production were isolated following transposon insertion mutagenesis. In one class of mutants obtained, the gacS gene was inactivated, indicating that GacS/GacA positively controls signal production. In a second class, the thiC gene required for thiamine (vitamin B1) biosynthesis was disrupted. Addition of excess (> 10E-6 M) thiamine to the medium restored signal production. By contrast, when the thiamine concentration was just sufficient to allow normal growth, no production of signal(s) was observed. The mechanism by which thiamine activates signal production remains to be elucidated. Résumé Pseudomonas fluorescens CHAO est une bactérie du sol, isolée près de Morens (Suisse), qui a la capacité de protéger les plantes contre des champignons pathogènes de la racine. Cette protection provient de métabolites secondaires excrétés par la bactérie, dont la synthèse est régulée par le système à deux composants GacS/GacA. Des signaux extracellulaires d'origine bactérienne activent ce système de régulation. Ces signaux, différents des N-acyl¬homosérines lactones, sont extraits par le dichlorométhane et semblent avoir une petite masse moléculaire. Une molécule (masse m/z 278) a été mise en évidence par des expériences préliminaires chez la souche CHAO. Des signaux similaires, capables d'activer la régulation dépendante de GacS/GacA chez la souche CHAO, ont été trouvés chez un grand nombre de bactéries à Gram négative. Une fois activé par le(s) signal(aux), le senseur GacS est supposé phosphoryler le régulateur de réponse GacA, qui influence positivement la cascade de régulation menant à la synthèse des métabolites secondaires. Cette cascade inclut trois petits ARNs régulateurs contrôlés par GacA et deux protéines répresseurs de la traduction. Les ARNs régulateurs titrent les protéines répresseurs, ce qui permet la traduction des gènes cibles et la synthèse d'exoenzymes et de métabolites secondaires tel les antibiotiques et le cyanure d'hydrogène. Un senseur basé sur la GFP pour la détection de signaux a été construit dans la souche CHAO en fusionnant le gène rapporteur gfp au gène de petit ARN rsmZ. Des mutants de CHAO déficients pour la production de signaux ont été isolés au moyen d'une mutagenèse par insertion de transposon. Chez une classe de mutants obtenus, le gène gacS a été inactivé, indiquant que GacS/GacA contrôle positivement la production de signaux. Dans une seconde classe, le gène thiC nécessaire à la biosynthèse de thiamine (vitamine B1) a été interrompu. L'addition en excès (> 10E-6 M) de thiamine au milieu restaure la production de signaux. A l'opposé, quand la concentration de thiamine est juste suffisante pour permettre une croissance normale, aucune production de signaux n'a été observée. Le mécanisme par lequel la thiamine active la production de signaux reste à élucider.

Relationship between dopamine D2 receptor occupancy, clinical response, and drug and monoamine metabolites levels in plasma and cerebrospinal fluid. A pilot study in patients suffering from first-episode schizophrenia treated with quetiapine.

Combining measurements of the monoamine metabolites in the cerebrospinal fluid (CSF) and neuroimaging can increase efficiency of drug discovery for treatment of brain disorders. To address this question, we examined five drug-naïve patients suffering from schizophrenic disorder. Patients were assessed clinically, using the Positive and Negative Syndrome Scale (PANSS): at baseline and then at weekly intervals. Plasma and CSF levels of quetiapine and norquetiapine as well CSF 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-acetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were obtained at baseline and again after at least a 4 week medication trail with 600 mg/day quetiapine. CSF monoamine metabolites levels were compared with dopamine D(2) receptor occupancy (DA-D(2)) using [(18)F]fallypride and positron emission tomography (PET). Quetiapine produced preferential occupancy of parietal cortex vs. putamenal DA-D(2), 41.4% (p<0.05, corrected for multiple comparisons). DA-D(2) receptor occupancies in the occipital and parietal cortex were correlated with CSF quetiapine and norquetiapine levels (p<0.01 and p<0.05, respectively). CSF monoamine metabolites were significantly increased after treatment and correlated with regional receptor occupancies in the putamen [DOPAC: (p<0.01) and HVA: (p<0.05)], caudate nucleus [HVA: (p<0.01)], thalamus [MHPG: (p<0.05)] and in the temporal cortex [HVA: (p<0.05) and 5-HIAA: (p<0.05)]. This suggests that CSF monoamine metabolites levels reflect the effects of quetiapine treatment on neurotransmitters in vivo and indicates that monitoring plasma and CSF quetiapine and norquetiapine levels may be of clinical relevance.

Steady state plasma levels of the enantiomers of trimipramine and of its metabolites in CYP2D6-, CYP2C19- and CYP3A4/5-phenotyped patients.

Steady state plasma concentrations of the (L)- and (D)-enantiomers of trimipramine (TRI), desmethyltrimipramine (DTRI), 2-hydroxytrimipramine (TRIOH) and 2-hydroxydesmethyl-trimipramine (DTRIOH) were measured in 27 patients receiving between 300 and 400 mg/day racemic TRI. The patients were phenotyped with dextromethorphan and mephenytoin, and the 8-hour urinary ratios of dextromethorphan/dextrorphan, dextromethorphan/3-methoxymorphinan, and (S)-mephenytoin/(R)mephenytoin were used as markers of cytochrome P-450IID6 (CYP2D6), CYP3A4/5 and CYP2C19 activities, respectively. One patient was a CYP2D6 and one was a CYP2C19 poor metabolizer. A stereoselectivity in the metabolism of TRI has been found, with a preferential N-demethylation of (D)-TRI and a preferential hydroxylation of (L)-TRI. CYP2D6 appears to be involved in the 2-hydroxylation of (L)-TRI, (L)DTRI and (D)-DTRI, but not of (D)-TRI, as significant correlations were measured between the dextromethorphan/dextrorphan ratios and the (L)-TRI/(L)-TRIOH (r = 0.45, p = 0.019), the (L)-DTRI/(L)-DTRIOH (r = 0.47, p = 0.014), and the (D)-DTRI/(D)-DTRIOH (r = 0.51, p = 0.006), but not with the (D)-TRI/(D)-TRIOH ratios (r = 0.29, NS). CYP2C19, but not CYP2D6, appears to be involved in the demethylation pathway, with a stereoselectivity toward the (D)-enantiomer of TRI, as a significant positive correlation was calculated between the mephenytoin (S)/(R) ratios and the concentrations to dose-to-weight ratios of (D)-TRI (r = 0.69, p = 0.00006). CYP3A4/5 appears to be involved in the metabolism of (L)-TRI to a presently not determined metabolite. The CYP2D6 poor metabolizer had the highest (L)-DTRI and (D)-DTRI concentrations to dose-to-weight ratios, and the CYP2C19 poor metabolizer had the highest (L)-TRI and (D)-TRI concentrations to dose-to-weight ratios of the group.

Vitamin E but not 17B-estradiol protect against vascular toxicity induced by B-amyloid wild type and the Dutch amyploid variant

Amyloid β-peptide (Aβ) fibril deposition on cerebral vessels produces cerebral amyloid angiopathy that appears in the majority of Alzheimer's disease patients. An early onset of a cerebral amyloid angiopathy variant called hereditary cerebral hemorrhage with amyloidosis of the Dutch type is caused by a point mutation in Aβ yielding AβGlu22→Gln. The present study addresses the effect of amyloid fibrils from both wild-type and mutated Aβ on vascular cells, as well as the putative protective role of antioxidants on amyloid angiopathy. For this purpose, we studied the cytotoxicity induced by Aβ1–40 Glu22→Gln and Aβ1–40 wild-type fibrils on human venule endothelial cells and rat aorta smooth muscle cells. We observed that AβGlu22→Gln fibrils are more toxic for vascular cells than the wild-type fibrils. We also evaluated the cytotoxicity of Aβ fibrils bound with acetylcholinesterase (AChE), a common component of amyloid deposits. Aβ1–40 wild-type–AChE fibrillar complexes, similar to neuronal cells, resulted in an increased toxicity on vascular cells. Previous reports showing that antioxidants are able to reduce the toxicity of Aβ fibrils on neuronal cells prompted us to test the effect of vitamin E, vitamin C, and 17β-estradiol on vascular damage induced by Aβwild-type and AβGlu22→Gln. Our data indicate that vitamin E attenuated significantly the Aβ-mediated cytotoxicity on vascular cells, although 17β-estradiol and vitamin C failed to inhibit the cytotoxicity induced by Aβ fibrils.

Infarctus cérébraux artériels d'origine hématologique : expérience lausannoise et revue de la littérature

RÉSUMÉ Introduction. Les hémopathies représentent une cause rare d'accident vasculaire cérébral (AVC), faisant l'objet de peu de publications, mais sont très fréquemment recherchées après un AVC par de coûteux bilans dont la rentabilité reste à définir. Matériel et Méthodes. Dans le registre lausannois des AVC, nous nous sommes intéressés de façon rétrospective aux dossiers des patients hospitalisés entre 1979 et 2001 pour un premier AVC ischémique artériel, dont la cause a été attribuée à une pathologie hématologique. Sur 4 697 patients, 22 (0,47 p. 100) ont vu leur AVC imputé à l'une des causes hématologiques suivantes : maladie de Vaquez , polyglobulie secondaire , thrombocytémie essentielle , thrombocytose secondaire , myélome multiple , MD , déficit en protéine S , syndrome anticorps antiphospholipides , hyperhomocystéinémie . Chaque hémopathie retrouvée a donné lieu à une revue de la littérature. Conclusion. À la lumière de ces données, nous concluons qu'une formule sanguine représente un bilan hématologique de dépistage suffisant pour l'immense majorité des patients hospitalisés pour un premier AVC artériel ischémique. Les recherches d'anticorps antiphospholipides, de thrombophilies héréditaires et d'hyperhomocystéinémie peuvent être limitées à des cas sélectionnés. SUMMARY Cerebral infarction of arterial origin and haematological causation: the Lausanne experience and a review of the literature. Introduction. Hematological diseases are seldom found as the etiology of ischemic strokes, but are frequently investigated by expensive laboratory tests after a first cerebral vascular event. Methods. In the Lausanne Stroke Registry, we retrospectively reviewed the cases of patients hospitalized between 1979 and 2001 for a first ischemic arterial stroke which was attributed to a hematological etiology. Of 4697 patients, 22 (0.47 per cent) had a stroke due to one of the following hematological pathology: polycythemia vera, secondary polycythemia, essential thrombocytemia, secondary thrombocytosis, multiple myeloma, CIVD, protein S deficiency, antiphospholipid antibody syndrome, moderate homocysteinemia. A literature review was undertaken for each hemopathy. Conclusion. In light of the results of these data, we concluded that a complete blood count provides sufficient hematological screening for the majority of patients hospitalized for an arterial stroke. The antiphospholipid antibody syndrome is a rare cause of cerebral infarction, which needs to be investigated in young patients, in cases of multiple or recurring stroke or in the presence of a typical history. Inherited thrombophilias are not a significant risk factor for arterial cerebral infarction and their investigation is only warranted for a sub-group of young patients with a cryptogenic stroke, in which group the prevalence is slightly increased. Moderate homocysteinemia must be considered as a cerebrovascular risk factor of minor importance, but potentially treatable by a substitution of vitamin B12, B 6 and folates. The efficacy of this substitution in the prevention of cardiovascular events needs yet to be demonstrated.

Vitamin C requirements in parenteral nutrition.

Some biochemical functions of vitamin C make it an essential component of parenteral nutrition (PN) and an important therapeutic supplement in other acute conditions. Ascorbic acid is a strong aqueous antioxidant and is a cofactor for several enzymes. The average body pool of vitamin C is 1.5 g, of which 3%-4% (40-60 mg) is used daily. Steady state is maintained with 60 mg/d in nonsmokers and 140 mg/d in smokers. Shocked surgical, trauma, and septic patients have a drastic reduction of circulating plasma ascorbate concentrations. These low concentrations require 3-g doses/d to restore normal plasma ascorbate concentrations, questioning the recommended PN dose of 100 mg/d. Determination of intravenous requirements is usually based on plasma concentrations, which are altered during the inflammatory response. There is no clear indicator of deficiency: serum or plasma ascorbate concentrations <0.3 mg/dL (20 micromol/L) indicates inadequate vitamin C status. On the basis of available pharmacokinetic data the 100 mg/d dose for patients receiving home PN and 200 mg/d for stable adult patients receiving PN are adequate, but requirements have been shown to be higher in perioperative, trauma, burn, and critically ill patients, paralleling oxidative stress. One recommendation cannot fit all categories of patients. Large vitamin C supplements may be considered in severe critical illness, major trauma, and burns because of increased requirements resulting from oxidative stress and wound healing. Future research should distinguish therapeutic use of high-dose ascorbic acid antioxidant therapy from nutritional PN requirements.

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Effects of carbamazepine coadministration on plasma concentrations of the enantiomers of mianserin and of its metabolites.

Concentrations of the enantiomers of unconjugated and of total (unconjugated plus conjugated) mianserin, desmethylmianserin and 8-hydroxymianserin were measured in 12 patients before and after the introduction of carbamazepine. The dose of mianserin was 60 mg/d, carbamazepine was coadministered at 400 mg/d for 4 weeks, and blood samples were taken at weekly intervals after the introduction of carbamazepine. Each week, carbamazepine significantly decreased plasma concentrations of unconjugated and total (S)-mianserin (the more potent enantiomer) and of unconjugated and total (R)-mianserin. On average, plasma concentrations of unconjugated and total (S)-mianserin and of unconjugated and total (R)-mianserin were 55%, 56%, 66%, and 55%, respectively, of the corresponding values before introduction of carbamazepine. These results strongly suggest the involvement of CYP3A4, the major CYP enzyme induced by carbamazepine, in the metabolism of both enantiomers of mianserin. A strong decrease in the concentrations of (S)-8-hydroxymianserin was also measured (on average, the concentrations were 69% of the corresponding values before carbamazepine introduction). Conversely, plasma concentrations of unconjugated and of total (S)-desmethylmianserin, (R)-desmethylmianserin, and (R)-8-hydroxymianserin were only slightly modified by carbamazepine. From a clinical point of view, as a therapeutic window for (S)-mianserin has been recently suggested, the dose of racemic mianserin for a patient whose (S)-mianserin concentrations have been stabilized within this therapeutic window would need to be approximately doubled if carbamazepine, at 400 mg/d, is introduced as a comedication.

Therapeutic drug monitoring and metabolites profil analysis of antiviral drugs

The widespread use of combination antiretroviral therapy (ARVs) has considerably improved the prognosis of patients infected with HIV. Conversely, considerable advances have been recently realized for the therapy of hepatitis C infection with the recent advent of potent new anti-HCV drugs that allow an increasing rate HCV infection cure. Despite their overall efficacy, a significant number of patients do not achieve or maintain adequate clinical response, defined as an undetectable viral load for HIV, and a sustained virological response (or cure) in HCV infection. Treatment failure therefore still remains an important issue besides drugs toxicities and viral resistance which is not uncommon in a significant percentage of patients who do not reach adequate virological suppression. The reasons of variability in drug response are multifactorial and apart from viral genetics, other factors such as environmental factors, drug- drug interactions, and imperfect compliance may have profound impact on antiviral drugs' clinical response. The possibility of measuring plasma concentration of antiviral drugs enables to guide antiviral drug therapy and ensure optimal drug exposure. The overall objective of this research was to widen up the current knowledge on pharmacokinetic and pharmacogenetic factors that influence the clinical response and toxicity of current and newly approved antiretroviral and anti-HCV drugs. To that endeavour, analytical methods using liquid chromatography coupled with tandem mass spectrometry have been developed and validated for the precise and accurate measurement of new antiretroviral and anti-HCV drugs . These assays have been applied for the TDM of ARVs and anti-HCV in patients infected with either HIV or HCV respectively, and co-infected with HIV- HCV. A pharmacokinetic population model was developed to characterize inter and intra-patient variability of rilpivirine, the latest marketed Non Nucleoside Reverse transcriptase (NNRTI) Inhibitor of HIVand to identify genetic and non genetic covariates influencing rilpivirine exposure. None of the factors investigated so far showed however any influence of RPV clearance. Importantly, we have found that the standard daily dosage regimen (25 mg QD) proposed for rilpivirine results in concentrations below the proposed therapeutic target in about 40% of patients. In these conditions, virologie escape is a potential risk that remains to be further investigated, notably via the TDM approach that can be a useful tool to identify patients who are at risk for being exposed to less than optimal levels of rilpivirine in plasma. Besides the last generation NNRTI rilpivirine, we have studied efavirenz, the major NNRTI clinically used so far. Namely for efavirenz, we aimed at identifying a potential new marker of toxicity that may be incriminated for the neuropsychological sides effects and hence discontinuation of efavirenz therapy. To that endeavour, a comprehensive analysis of phase I and phase II metabolites profiles has been performed in plasma, CSF and in urine from patients under efavirenz therapy. We have found that phase II metabolites of EFV constitute the major species circulating in blood, sometimes exceeding the levels of the parent drug efavirenz. Moreover we have identified a new metabolite of efavirenz in humans, namely the 8-OH-EFV- sulfate which is present at high concentrations in all body compartments from patients under efavirenz therapy. These investigations may open the way to possible alternate phenotypic markers of efavirenz toxicity. Finally, the specific influence of P-glycoprotein on the cellular disposition of a series ARVs (NNRTIs and Pis] has been studies in in vitro cell systems using the siRNA silencing approach. -- Depuis l'introduction de la thérapie antirétrovirale (ARVs) la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Une réponse imparfaite ou la toxicité du traitement est certainement multifactorielle. Le suivi thérapeutique des médicaments [Therapeutic Drug Monitoring TDM) à travers la mesure des concentrations plasmatiques constitue une approche importante pour guider le traitement médicamenteux et de s'assurer que les patients sont exposés à des concentrations optimales des médicaments dans le sang, et puissent tirer tout le bénéfice potentiel du traitement. L'objectif global de cette thèse était d'étudier les facteurs pharmacocinétiques et pharmacogénétiques qui influencent l'exposition des médicaments antiviraux (ARVs et anti- VHC) récemment approuvés. A cet effet, des méthodes de quantification des concentrations plasmatiques des médicaments antirétroviraux, anti-VHC ainsi que pour certains métabolites ont été développées et validées en utilisant la Chromatographie liquide couplée à la spectrométrie de masse tandem. Ces méthodes ont été utilisées pour le TDM des ARVs et pour les agents anti-VHC chez les patients infectés par le VIH, et le VHC, respectivement, mais aussi chez les patients co-infectés par le VIH-VHC. Un modèle de pharmacocinétique de population a été développé pour caractériser la variabilité inter-et intra-patient du médicament rilpivirine, un inhibiteur non nucléosidique de la transcriptase de VIH et d'identifier les variables génétiques et non génétiques influençant l'exposition au médicament. Aucun des facteurs étudiés n'a montré d'influence notable sur la clairance de la rilpivirine. Toutefois, la concentration résiduelle extrapolée selon le modèle de pharmacocinétique de population qui a été développé, a montré qu'une grande proportion des patients présente des concentrations minimales inférieures à la cible thérapeutique proposée. Dans ce contexte, la relation entre les concentrations minimales et l'échappement virologique nécessite une surveillance étroite des taux sanguins des patients recevant de la rilpivirine. A cet effet, le suivi thérapeutique est un outil important pour l'identification des patients à risque soient sous-exposés à lai rilpivirine. Pour identifier de nouveaux marqueurs de la toxicité qui pourraient induire l'arrêt du traitement, le profil des métabolites de phase I et de phase II a été étudié dans différentes matrices [plasma, LCR et urine) provenant de patients recevant de l'efavirenz. Les métabolites de phase II, qui n'avaient à ce jour jamais été investigués, constituent les principales espèces présentes dans les matrices étudiées. Au cours de ces investigations, un nouveau métabolite 8- OH-EFV-sulfate a été identifié chez l'homme, et ce dernier est. présent à des concentrations importantes. L'influence de certains facteurs pharmacogénétique des patients sur le profil des métabolites a été étudiée et ouvre la voie à de possibles nouveaux marqueurs phénotypiques alternatifs qui pourraient possiblement mieux prédire la toxicité associée au traitement par l'efavirenz. Finalement, nous nous sommes intéressés à étudier dans un modèle in vitro certains facteurs, comme la P-glycoprotéine, qui influencent la disposition cellulaire de certains médicaments antirétroviraux, en utilisant l'approche par la technologie du siRNA permettant de bloquer sélectivement l'expression du gène de cette protéine d'efflux des médicaments. -- Depuis l'introduction de la thérapie antiretrovirale (ARVs] la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Il a pu être démontré que la concentration de médicament présente dans l'organisme est corrélée avec l'efficacité clinique pour la plupart des médicaments agissant contre le VIH et contre le VHC. Les médicaments antiviraux sont généralement donnés à une posologie fixe et standardisée, à tous les patients, il existe cependant une importante variabilité entre les concentrations sanguines mesurées chez les individus. Cette variabilité peut être expliquée par plusieurs facteurs démographiques, environnementaux ou génétiques. Dans ce contexte, le suivi des concentrations sanguines (ou Therapeutic Drug Monitoring, TDM) permet de contrôler que les patients soient exposés à des concentrations suffisantes (pour bloquer la réplication du virus dans l'organisme) et éviter des concentrations excessives, ce qui peut entraîner l'apparition d'intolérence au traitement. Le but de ce travail de thèse est d'améliorer la compréhension des facteurs pharmacologiques et génétiques qui peuvent influencer l'efficacité et/ou la toxicité des médicaments antiviraux, dans le but d'améliorer le suivi des patients. A cet effet, des méthodes de dosage très sensibles et ont été mises au point pour permettre de quantifier les médicaments antiviraux dans le sang et dans d'autres liquides biologiques. Ces méthodes de dosage sont maintenant utilisées d'une part dans le cadre de la prise en charge des patients en routine et d'autre part pour diverses études cliniques chez les patients infectés soit par le HIV, le HCV ou bien coinfectés par les deux virus. Une partie de ce travail a été consacrée à l'investigation des différents facteurs démographiques, génétiques et environnementaux qui pourraient l'influencer la réponse clinique à la rilpivirine, un nouveau médicament contre le VIH. Toutefois, parmi tous les facteurs étudiés à ce jour, aucun n'a permis d'expliquer la variabilité de l'exposition à la rilpivirine chez les patients. On a pu cependant observer qu'à la posologie standard recommandée, un pourcentage relativement élevé de patients pourrait présenter des concentrations inférieures à la concentration sanguine minimale actuellement proposée. Il est donc utile de surveiller étroitement les concentrations de rilpivirine chez les patients pour identifier sans délai ceux qui risquent d'être sous-exposés. Dans l'organisme, le médicament subit diverses transformations (métabolisme) par des enzymes, notamment dans le foie, il est transporté dans les cellules et tissus par des protéines qui modulent sa concentration au site de son action pharmacologique. A cet effet, différents composés (métabolites) produits dans l'organisme après l'administration d'efavirenz, un autre médicament anti-VIH, ont été étudiés. En conclusion, nous nous sommes intéressés à la fois aux facteurs pharmacologiques et génétiques des traitements antiviraux, une approche qui s'inscrit dans l'optique d'une stratégie globale de prise en charge du patient. Dans ce contexte, le suivi des concentrations sanguines de médicaments constitue une des facettes du domaine émergent de la Médecine Personnalisée qui vise à maximiser le bénéfice thérapeutique et le profil de tolérance des médicaments antiviraux

Vitamin E content in fish oil emulsion does not prevent lipoperoxidative effects on human colorectal tumors.

OBJECTIVE: The anticancer action exerted by polyunsaturated fatty acid peroxidation may not be reproduced by commercially available lipid emulsions rich in vitamin E. Therefore, we evaluated the effects of fish oil (FO) emulsion containing α-tocopherol 0.19 g/L on human colorectal adenocarcinoma cells and tumors. METHODS: HT-29 cell growth, survival, apoptosis, and lipid peroxidation were analyzed after a 24-h incubation with FO 18 to 80 mg/L. Soybean oil (SO) emulsion was used as an isocaloric and isolipidic control. In vivo, nude mice bearing HT-29 tumors were sacrificed 7 d after an 11-d treatment with intravenous injections of FO or SO 0.2 g ∙ kg(-1) ∙ d(-1) FO or SO to evaluate tumor growth, necrosis, and lipid peroxidation. RESULTS: The FO inhibited cell viability and clonogenicity in a dose-dependent manner, whereas SO showed no significant effect compared with untreated controls. Lipid peroxidation and cell apoptosis after treatment with FO 45 mg/L were increased 2.0-fold (P < 0.01) and 1.6-fold (P = 0.04), respectively. In vivo, FO treatment did not significantly affect tumor growth. However, immunohistochemical analyses of tumor tissue sections showed a decrease of 0.6-fold (P < 0.01) in the cell proliferation marker Ki-67 and an increase of 2.3-fold (P = 0.03) in the necrotic area, whereas malondialdehyde and total peroxides were increased by 1.9-fold (P = 0.09) and 7.0-fold (P < 0.01), respectively, in tumors of FO-treated compared with untreated mice. CONCLUSION: These results suggest that FO but not SO has an antitumor effect that can be correlated with lipid peroxidation, despite its vitamin E content.