A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter.

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition.

Steroid profiles of professional soccer players: an international comparative study.

BACKGROUND AND OBJECTIVES: Urinary steroid profiling is used in doping controls to detect testosterone abuse. A testosterone over epitestosterone (T/E) ratio exceeding 4.0 is considered as suspicious of testosterone administration, irrespectively of individual heterogeneous factors such as the athlete's ethnicity. A deletion polymorphism in the UGT2B17 gene was demonstrated to account for a significant part of the interindividual variability in the T/E between Caucasians and Asians. Here, the variability of urinary steroid profiles was examined in a widely heterogeneous cohort of professional soccer players. Method: The steroid profile of 57 Africans, 32 Asians, 50 Caucasians and 32 Hispanics was determined by gas chromatography-mass spectrometry. RESULTS: Significant differences have been observed between all ethnic groups. After estimation of the prevalence of the UGT2B17 deletion/deletion genotype (African: 22%; Asian: 81%; Caucasian: 10%; Hispanic: 7%), ethnic-specific thresholds were developed for a specificity of 99% for the T/E (African: 5.6; Asian: 3.8; Caucasian: 5.7; Hispanic: 5.8). Finally, another polymorphism could be hypothesised in Asians based on specific concentration ratio of 5alpha-/5beta-androstane-3alpha,17beta-diol in urine. CONCLUSION: These results demonstrate that a unique and non-specific threshold to evidence testosterone misuse is not fit for purpose. An athlete's endocrinological passport consisting of a longitudinal follow-up together with the ethnicity and/or the genotype would strongly enhance the detection of testosterone abuse. Finally, additional genotyping studies should be undertaken to determine whether the remaining unexplained disparities have an environmental or a genetic origin.

Biochemical markers of fatal hypothermia.

The aim of this study was to investigate the usefulness of postmortem biochemical investigations in the diagnosis of fatal hypothermia. 10 cases of fatal hypothermia and 30 control cases were selected. A series of biochemical parameters, such as glucose, acetone, 3-beta-hydroxybutyrate, isopropyl alcohol, free fatty acids, adrenaline, growth hormone, adrenocorticotropic hormone, thyroid-stimulating hormone, cortisol, calcium, magnesium, C-reactive protein, procalcitonin as well as markers of renal and cardiac functions were measured in blood, postmortem serum from femoral blood, urine, vitreous and pericardial fluid. The results suggested that deaths due to hypothermia, especially in free-ethanol cases, are characterized by increased ketone levels in blood and other biological fluids, increased adrenaline concentrations in urine, increased cortisol levels in postmortem serum from femoral blood and increased free cortisol values in urine. Increased or decreased levels of other biological parameters are either the result of terminal metabolic changes or the expression of preexisting diseases and may provide information to elucidate the death process on a case-by-case basis.

Urinary analysis of four testosterone metabolites and pregnanediol by gas chromatography-combustion-isotope ratio mass spectrometry after oral administrations of testosterone.

The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5 alpha-androstanediol, and 5 beta-androstanediol) together with an endogenous reference (5 beta-pregnanediol) were extracted from the urines and the delta(13)C/(12)C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum delta(13)C-value variations (parts per thousand difference of delta(13)C/(12)C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5 alpha-androstanediol delta(13)C-values returned to the baseline 15 h after the second T administration, a decrease of the 5 beta-androstanediol delta-values could be detected for over 40 h. This suggests that measurements of 5 beta-androstanediol delta-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites delta(13)C-values or than the usual T/E ratio approach.

Biogeochemical evolution of a marine shore tailings deposit during bioremediation

Summary : Mining activities produce enormous amounts of waste material known as tailings which are composed of fine to medium size particles. These tailings often contain sulfides, which oxidation can lead to acid and metal contamination of water; therefore they need to be remediated. In this work a tailings bioremediation approach was investigated by an interdisciplinary study including geochemistry, mineralogy and microbiology. The aim of the work was to study the effect of the implementation of wetland above oxidizing tailings on the hydrogeology and the biogeochemical element cycles, and to assess the system evolution over time. To reach these goals, biogeochemical processes occurring in a marine shore tailings deposit were investigated. The studied tailings deposit is located at the Bahìa de Ite, Pacific Ocean, southern Peru, where between 1940 and 1996 the tailings were discharged from the two porphyry copper mines Cuajone and Toquepala. After the end of deposition, a remediation approach was initiated in 1997 with a wetland implementation above the oxidizing tailings. Around 90% of the tailings deposits (total 16 km2) were thus remediated, except the central delta area and some areas close to the shoreline. The multi-stable isotope study showed that the tailings were saturated with fresh water in spite of the marine setting, due to the high hydraulic gradient resulting from the wetland implementation. Submarine groundwater discharge (SGD) was the major source of SO4 2-, C1-, Na+, Fe2+, and Mn2+ input into the tailings at the original shelf-seawater interface. The geochemical study (aquatic geochemistry and X-Ray diffraction (XRD) and sequential extractions from the solid fraction) showed that iron and sulfur oxidation were the main processes in the non-remediated tailings, which showed a top a low-pH oxidation zone with strong accumulation of efflorescent salts at the surface due to capillary upward transport of heavy metals (Fe, Cu, Zn, Mn, Cd, Co, and Ni) in the arid climate. The study showed also that the implementation of the wetland resulted in very low concentrations of heavy metals in solution (mainly under the detection limit) due to the near neutral pH and more reducing conditions (100-150 mV). The heavy metals, which were taken from solution, precipitated as hydroxides and sulfides or were bound to organic matter. The bacterial community composition analysis by Terminal Restriction Fragment Length Polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes combined with a detailed statistical analysis revealed a high correlation between the bacterial distribution and the geochemical variables. Acidophilic autotrophic oxidizing bacteria were dominating the oxidizing tailings, whereas neutrophilic and heterotrophic reducing bacteria were driving the biogeochemical processes in the remediated tailings below the wetland. At the subsurface of the remediated tailings, an iron cycling was highlighted with oxidation and reduction processes due to micro-aerophilic niches provided by the plant rhizosphere in this overall reducing environment. The in situ bioremediation experiment showed that the main parameter to take into account for the effectiveness was the water table and chemistry which controls the system. The constructed remediation cells were more efficient and rapid in metal removal when saturation conditions were available. This study showed that the bioremediation by wetland implementation could be an effective and rapid treatment for some sulfidic mine tailings deposits. However, the water saturation of the tailings has to be managed on a long-term basis in order to guarantee stability. Résumé : L'activité minière produit d'énormes quantités de déchets géologiques connus sous le nom de « tailings » composées de particules de taille fine à moyenne. Ces déchets contiennent souvent des sulfures dont l'oxydation conduit à la formation d'effluents acides contaminés en métaux, d'où la nécessité d'effectuer une remédiation des sites de stockage concernés. Le but de ce travail est dans un premier temps d'étudier l'effet de la bio-remédiation d'un dépôt de tailings oxydés sur l'hydrogéologie du système et les cycles biogéochimiques des éléments et en second lieu, d'évaluer l'évolution du processus de remédiation dans le temps. Le site étudié dans ce travail est situé dans la Bahía de Ite, au sud du Pérou, au bord de l'Océan Pacifique. Les déchets miniers en question sont déposés dans un environnement marin. De 1940 à 1996, les déchets de deux mines de porphyre cuprifère - Cuajone et Toquepala - ont été acheminés sur le site via la rivière Locumba. En 1997, une première remédiation a été initiée avec la construction d'une zone humide sur les tailings. Depuis, environ 90% de la surface du dépôt (16 km2) a été traité, les parties restantes étant la zone centrale du delta du Locumba et certaines zones proches de la plage. Malgré la proximité de l'océan, les études isotopiques menées dans le cadre de ce travail ont montré que les tailings étaient saturés en eau douce. Cette saturation est due à la pression hydraulique résultant de la mise en place des zones humides. Un écoulement d'eau souterrain sous-marin a été à détecté à l'interface entre les résidus et l'ancien fond marin. En raison de la géologie locale, il constitue une source d'entrée de SO4 2-, Cl-, Na+, FeZ+, et Mn2+ dans le système. L'analyse de la géochimie aquatique, la Diffraction aux Rayons X (XRD) et l'extraction séquentielle ont montré que l'oxydation du fer et .des sulfures est le principal processus se produisant dans les déchets non remédiés. Ceci a entraîné le développement d'une zone d'oxydation à pH bas induisant une forte accumulation des sels efflorescents, conséquence de la migration capillaire des métaux lourds (Fe, Cu, Zn, Mn, Cd, Co et Ni) de la solution vers la surface dans ce climat aride. Cette étude a montré également que la construction de la zone humide a eu comme résultats une précipitation des métaux dans des phases minérales en raison du pH neutre et des conditions réductrices (100-150mV). Les métaux lourds ont précipité sous la forme d'hydroxydes et de sulfures ou sont adsorbés à la matière organique. L'analyse de la composition de la communauté bactérienne à l'aide la technique T-RFLP (Terminal Restriction Fragment Length Polymorphism) et par le clonage/séquençage des gènes de l'ARNr 16S a été combinée à une statistique détaillée. Cette dernière a révélé une forte corrélation entre la distribution de bactéries spécifiques et la géochimie : Les bactéries autotrophes acidophiles dominent dans les déchets oxydés non remédiés, tandis que des bactéries hétérotrophes neutrophiles ont mené les processus microbiens dans les déchets remédiés sous la zone humide. Sous la surface de la zone humide, nos analyses ont également mis en évidence un cycle du fer par des processus d'oxydoréduction rendus possibles par la présence de niches micro-aérées par la rhizosphère dans cet environnement réducteur. L'expérience de bio-remédiation in situ a montré que les paramètres clés qui contrôlent l'efficacité du traitement sont le niveau de la nappe aquifère et la chimie de l'eau. Les cellules de remédiation se sont montrées plus efficaces et plus rapides lorsque le système a pu être saturé en eau. Finalement, cette étude a montré que la bio-remédiation de déchets miniers par la construction de zones humides est un moyen de traitement efficace, rapide et peu coûteux. Cependant, la saturation en eau du système doit être gérée sur le long terme afin de garantir la stabilité de l'ensemble du système.

Diagnostic performance of urinary metanephrines for the postmortem diagnosis of hypothermia.

The purpose of this study was to assess the diagnostic potential of urinary metanephrines and 3-methoxytyramine compared to urinary catecholamine determination in diagnosing antemortem cold exposure and fatal hypothermia. 83 cases of fatal hypothermia and 144 control cases were included in this study. Catecholamines (adrenaline, noradrenaline and dopamine), metanephrines (metanephrine, normetanephrine) and 3-methoxytyramine were measured in urine collected during autopsy. All tested analytes were significantly higher in hypothermia cases compared to control subjects and displayed a generally satisfying discriminative value, thus indicating urinary catecholamines and their metabolites as reliable markers of cold-related stress and hypothermia related-deaths. Metanephrine and adrenaline had the best discriminative value between hypothermia and control cases compared to other tested analytes, though with different sensitivity and specificity. These can therefore be considered the most suitable markers of cold-related stress.

Smoked cannabis and doping control: looking for the wrong target analyte?

Introduction: Since 2004, cannabis is prohibited by the World Anti-Doping Agency (WADA) for all sports in competition. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In most cases, the athletes plausibly claim to have consumed cannabis several days or even weeks before competition and only for recreational purposes not related to competition. In doping analysis, the target analyte in urine samples is 11-nor-delta-9-tetrahydrocannabinol- 9-carboxylic acid (THC-COOH), the reporting threshold for laboratories is 15 ng/mL. However, the wide detection window of this long-term THC metabolite in urine does not allow a conclusion concerning the time of consumption or the impact on the physical performance. Aim: The purpose of the present pharmacokinetic study on volunteers was to evaluate target analytes with shorter urinary excretion time. Subsequently, urines from athletes tested positive for cannabis should be reanalyzed including these analytes. Methods: In an one-session clinical trial (approved by IRB, Swissmedic, and Federal Office of Public Health), 12 healthy, male volunteers (age 26 ± 3 yrs, BMI 24 ± 2 kg/m2) with cannabis experience (> once/month) smoked a Cannabis cigarette standardized to 70 mg THC/cigarette (Bedrobinol® 7%, Dutch Office for Medicinal Cannabis) following a paced-puffing procedure. Plasma and urine was collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-THC (THC-OH), and THC-COOH were determined after enzymatic hydrolyzation followed by SPE and GC/MS-SIM. The limit of quantitation (LOQ) for all analytes was 0.1 ng/mL. Visual analog scales (VAS) and vital functions were used for monitoring psychological and somatic side-effects at every timepoint of specimen collection (up to 480 min). Results: Eight puffs delivered a mean THC dose of 45 mg. Mean plasma levels of total THC, THC-OH and THC-COOH were measured in the range of 0.1-20.9, 0.1-1.8, and 1.8-7.5 ng/mL, respectively. Peak concentrations were observed at 5, 10, and 90 min. Mean urine levels were measured in the range of 0.1-0.7, 0.10-6.2, and 0.1-13.4 ng/mL, respectively. The detection windows were 2-8, 2-96, and 2-120 h. No or only mild effects were observed, such as dry mouth, sedation, and tachycardia. Besides high to very high THC-COOH levels (0-978 ng/mL), THC (0.1-24 ng/mL) and THC-OH (1-234 ng/mL) were found in 90 and 96% of the cannabis-positive urines from athletes. Conclusion: Instead of or in addition to THC-COOH, the pharmacologically active THC and THC-OH should be the target analytes for doping urine analysis. This would allow the estimation of more recent Cannabis consumption, probably influencing performance during competition. Keywords: cannabis, doping, clinical trial, plasma and urine levels, athlete's samples

Rapid affinity purification of erythropoietin from biological samples using disposable monoliths.

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 ?L volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 ?L eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.

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

Testosterone and doping control.

BACKGROUND AND OBJECTIVES: Anabolic steroids are synthetic derivatives of testosterone, modified to enhance its anabolic actions (promotion of protein synthesis and muscle growth). They have numerous side effects, and are on the International Olympic Committee's list of banned substances. Gas chromatography-mass spectrometry allows identification and characterisation of steroids and their metabolites in the urine but may not distinguish between pharmaceutical and natural testosterone. Indirect methods to detect doping include determination of the testosterone/epitestosterone glucuronide ratio with suitable cut-off values. Direct evidence may be obtained with a method based on the determination of the carbon isotope ratio of the urinary steroids. This paper aims to give an overview of the use of anabolic-androgenic steroids in sport and methods used in anti-doping laboratories for their detection in urine, with special emphasis on doping with testosterone. METHODS: Review of the recent literature of anabolic steroid testing, athletic use, and adverse effects of anabolic-androgenic steroids. RESULTS: Procedures used for detection of doping with endogenous steroids are outlined. The World Anti-Doping Agency provided a guide in August 2004 to ensure that laboratories can report, in a uniform way, the presence of abnormal profiles of urinary steroids resulting from the administration of testosterone or its precursors, androstenediol, androstenedione, dehydroepiandrosterone or a testosterone metabolite, dihydrotestosterone, or a masking agent, epitestosterone. CONCLUSIONS: Technology developed for detection of testosterone in urine samples appears suitable when the substance has been administered intramuscularly. Oral administration leads to rapid pharmacokinetics, so urine samples need to be collected in the initial hours after intake. Thus there is a need to find specific biomarkers in urine or plasma to enable detection of long term oral administration of testosterone.

Using toxicokinetic modeling approach to determine biological reference values (BRVs) and to assess human exposure to pesticides

Exposure to various pesticides has been characterized in workers and the general population, but interpretation and assessment of biomonitoring data from a health risk perspective remains an issue. For workers, a Biological Exposure Index (BEI®) has been proposed for some substances, but most BEIs are based on urinary biomarker concentrations at Threshold Limit Value - Time Weighted Average (TLV-TWA) airborne exposure while occupational exposure can potentially occurs through multiple routes, particularly by skin contact (i.e.captan, chlorpyrifos, malathion). Similarly, several biomonitoring studies have been conducted to assess environmental exposure to pesticides in different populations, but dose estimates or health risks related to these environmental exposures (mainly through the diet), were rarely characterized. Recently, biological reference values (BRVs) in the form of urinary pesticide metabolites have been proposed for both occupationally exposed workers and children. These BRVs were established using toxicokinetic models developed for each substance, and correspond to safe levels of absorption in humans, regardless of the exposure scenario. The purpose of this chapter is to present a review of a toxicokinetic modeling approach used to determine biological reference values. These are then used to facilitate health risk assessments and decision-making on occupational and environmental pesticide exposures. Such models have the ability to link absorbed dose of the parent compound to exposure biomarkers and critical biological effects. To obtain the safest BRVs for the studied population, simulations of exposure scenarios were performed using a conservative reference dose such as a no-observed-effect level (NOEL). The various examples discussed in this chapter show the importance of knowledge on urine collections (i.e. spot samples and complete 8-h, 12-h or 24-h collections), sampling strategies, metabolism, relative proportions of the different metabolites in urine, absorption fraction, route of exposure and background contribution of prior exposures. They also show that relying on urinary measurements of specific metabolites appears more accurate when applying this approach to the case of occupational exposures. Conversely, relying on semi-specific metabolites (metabolites common to a category of pesticides) appears more accurate for the health risk assessment of environmental exposures given that the precise pesticides to which subjects are exposed are often unknown. In conclusion, the modeling approach to define BRVs for the relevant pesticides may be useful for public health authorities for managing issues related to health risks resulting from environmental and occupational exposures to pesticides.

Rapid sample pre-treatment prior to GC-MS and GC-MS/MS urinary toxicological screening.

Drug screening is an important issue in clinical and forensic toxicology. Gas chromatography coupled to mass spectrometry (GC-MS) remains the gold standard technique for the screening of unknown compounds in urine samples. However, this technique requires substantial sample preparation, which is time consuming. Moreover, some common drugs such as cannabis cannot be easily detected in urine using general procedures. In this work, a sample preparation protocol for treating 200 μL of urine in less than 30 min is described. The enzymatic hydrolysis of glucuro-conjugates was performed in 5 min thanks to the use of microwaves. The use of a deconvolution software allowed reducing the GC-MS run to 10 min, without impairing the quality of the compound identifications. Comparing the results from 139 authentic urine samples to those obtained using the current routine analysis indicated this method performed well. Moreover, additional 5-min GC-MS/MS programs are described, enabling a very sensitive target screening of 54 drugs, including THC-COOH or buprenorphine, without further sample preparation. These methods appeared as an interesting alternative to immuno-assays based screening. The analytical strategy presented in this article proved to be a promising approach for systematic toxicological analysis (STA) of drugs in urine.

alphaENaC-mediated lithium absorption promotes nephrogenic diabetes insipidus.

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.

L'éthylglucuronide: un marquer de la consommation d'alcool [Ethyl glucuronide: a biomarker of alcohol consumption]

Excessive alcohol consumption represents a major risk factor for morbidity and mortality. It is therefore indispensable to be able to detect at-risk drinking. Ethyl glucuronide (EtG) is a specific marker of alcohol consumption. The determination of ethyl glucuronide in urine or blood can be used to prove recent driving under the influence of alcohol, even if ethanol is no longer detectable. The commercialization of an EtG specific immunological assay now allows to obtain preliminary results rapidly and easily with satisfying sensitivity. Moreover, the detection of ethyl glucuronide in hair offers the opportunity to evaluate an alcohol consumption over a long period. The EtG concentration in hair is in correlation with the amount of ingested alcohol. Thus, the analysis of ethyl glucuronide can be used to monitor abstinence, to detect alcohol relapse and to identify at-risk drinkers. However, a cut off allowing to detect chronic alcohol abuser reliably still does not exist. Therefore, it is recommended to perform the analysis of ethyl glucuronide in complement to the existing blood markers. A study financed by the Swiss Foundation for Alcohol Research is actually conducted by the West Switzerland University Center of Legal Medicine in order to establish an objective cut-off.