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.

Impact of duration of untreated psychosis on pre-treatment, baseline, and outcome characteristics in an epidemiological first-episode psychosis cohort.

INTRODUCTION: To assess the impact of duration of untreated psychosis (DUP) on baseline and 18-month follow-up characteristics controlling for relevant confounders in an epidemiological first-episode psychosis (FEP) cohort. METHOD: The Early Psychosis Prevention and Intervention Centre (EPPIC) in Australia admitted 786 FEP patients from January 1998 to December 2000. Data were collected from medical files using a standardized questionnaire. Data from 636 patients were analyzed. RESULTS: Median DUP was 8.7 weeks. Longer DUP was associated with worse premorbid functioning (p<0.001), higher rate of schizophrenia-spectrum disorders (p<0.001), and younger age at onset of psychosis (p=0.004). Longer DUP was not associated with baseline variables but with a lower rate of remission of positive symptoms (p<0.001) and employment/occupation (p<0.001), a higher rate of persistent substance use (p=0.015), worse illness severity (p<0.001) and global functioning (p<0.001) at follow-up after controlling for relevant confounders, explaining approximately 5% of variance of remission of positive symptoms (p<0.001) in the total sample and 3% in schizophrenia-spectrum disorders excluding bipolar I disorder (p=0.002). Outcome was significantly worse when DUP exceeded 1-3 months. CONCLUSION: Avoiding pitfalls of non-epidemiological studies, DUP appears to be a modest independent predictor of prognosis in the medium-term. Results support the need for assertive early detection strategies.

Long-term treatment of eosinophilic esophagitis esophagitis with budesonide

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic-inflammatory disease of the esophagus, characterized by esophagus-related symptoms and a dense tissue eosinophilia, both refractory to proton pump inhibitors. Topical corticosteroids have proven effective in inducing clinical and histologic remission. However, a long-term strategy for the management of this chronic disease is not yet defined. METHODS: In a randomized, double-blind, placebocontrolled, long-term trial, we evaluated the efficacy of twice-daily 0.25 mg swallowed budesonide in maintaining a remission in adult EoE with prior response to induction therapy. Pre- and post-treatment disease activity was assessed clinically, endoscopically, histologically, by immunofluorescence and by high-resolution endosonography. The primary end point was the ability to maintain histologic remission (<5 eos/hpf) of EoE in. Secondary end points were the efficacy on symptom control and on tissue remodeling as well as the determination of the safety of long-term esophageal administration of topical corticosteroids. RESULTS: During a 50-week therapy of quiescent EoE with low-dose budesonide the esophageal eosinophil load (ECP staining) increased from 1.1 to 29.9 eos/hpf, but under placebo the increase was significantly larger (0.5 to 51.1 eos/hpf; p=0.01). At the end of the studyperiod, 35.7% (5/14) of the budesonide patients were in complete and 14.3% (2/14) in partial histologic remission; with placebo no patient was in complete and 28.6% (4/14) were in partial remission (p=0.0647). The increase of the symptom score was markedly lower in budesonide- (0.79 to 2.29 points) than in placebo-patients (0.71 to 4.00 points; p=0.0875). The median time to relapse of symptoms was >125 days in the budesonide and 95 days in the placebo group (p = 0.14). Measured by high-resolution endosonography, all EoE patients had pre-treatment a highly thickened esophageal wall compared with healthy controls (3.05±1.08 mm vs. 2.18±0.35 mm; p<0.0001). Long-term topical budesonide reduced mainly the thickness of the superficial wall layers (mucosa, 0.75 mm to 0.45 mm; p=0.025) whereas the response of the deeper layers was less pronounced (submucosa 1.31 to 1.08 mm; p=0.19 and muscularis 0.82 to 0.76 mm; p=0.72). Budesonide did not evoke any mucosal atrophy. CONCLUSIONS: This study clearly demonstrates that 1) Untreated eosinophil inflammation results in an impressive remodeling of the esophagus; 2) A therapy is therefore needed; 3) The high relapse rate after short-term therapy requires a long-term management and 4) Maintenance treatment with budesonide is well tolerated and keeps half of the patients in remission.

IL28B single nucleotide polymorphisms in the treatment of hepatitis C.

Recent genome-wide association studies (GWAS) have identified genetic variations near the IL28B gene which are strongly associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infection. Protective IL28B variations are strongly associated with on-treatment viral kinetics and approximately 2-fold increased sustained virologic response (SVR) rates in HCV genotype 1 and 4 patients. In HCV genotype 1 patients, IL28B variations were shown to be the strongest pre-treatment predictor of virologic response. In the treatment of HCV genotype 2 and 3 infected patients, IL28B variations play only a minor role. Preliminary data indicate that IL28B variations are also associated with treatment outcome of regimens, including directly acting antiviral (DAA) agents, though their impact seems to be attenuated compared to standard treatment. Here, we review these important findings and discuss possible implications for clinical decision making in the treatment of HCV infection.

Making SENSE - Sustained Effort Network for treatment of Status Epilepticus as a multicenter prospective registry.

BACKGROUND: Evidence regarding the different treatment options of status epilepticus (SE) in adults is scarce. Large randomized trials cover only one treatment at early stage and suggest the superiority of benzodiazepines over placebo, of intravenous lorazepam over intravenous diazepam or over intravenous phenytoin alone, and of intramuscular midazolam over intravenous lorazepam. However, many patients will not be treated successfully with the first treatment step. A large randomized trial covering the treatment of established status (ESETT) has just been funded recently by the NIH and will not start before 2015, with expected results in 2018; a trial on the treatment of refractory status with general anesthetics was terminated early due to insufficient recruitment. Therefore, a prospective multicenter observational registry was set up; this may help in clinical decision-making until results from randomized trials are available. METHODS/DESIGN: SENSE is a prospective, multicenter registry for patients treated for SE. The primary objective is to document patient characteristics, treatment modalities and in-house outcome of consecutive adults admitted for SE treatment in each of the participating centres and to identify predictors of outcome. Pre-treatment, treatment-related and outcome variables are documented systematically. To allow for meaningful multivariate analysis in the patient subgroups with refractory SE, a cohort size of 1000 patients is targeted. DISCUSSION: The results of the study will provide information about risks and benefits of specific treatment steps in different patient groups with SE at different points of time. Thus, it will support clinical decision-making and, furthermore, it will be helpful in the planning of treatment trials. TRIAL REGISTRATION: DRKS00000725.

Photodynamic therapy : a pathway for enhanced delivery of liposomal doxorubicin to tumors

Abstract Part I : Background : Isolated lung perfusion (ILP) was designed for the treatment of loco-regional malignancies of the lung. In contrast to intravenous (IV) drug application, ILP allows for a selective administration of cytostatic agents such as doxorubicin to the lung while sparing non-affected tissues. However, the clinical results with ILP were disappointing. Doxorubicinbased ILP on sarcoma rodent lungs suggested high overall doxorubicin concentrations within the perfused lung but a poor penetration of the cytostatic agent into tumors. The same holds true for liposomal-encapsulated macromolecular doxorubicin (LiporubicinTM) In specific conditions, low-dose photodynamic therapy (PDT) can enhance the distribution of macromolecules across the endothelial bamer in solid tumors. It was recently postulated that tumor neovessels were more responsive to PDT than the normal vasculature. We therefore hypothesized that Visudyne®-mediated PDT could selectively increase liposomal doxorubicin (LiporubicinTM) uptake in sarcoma tumors to rodent lungs during intravenous (IV) drug administration and isolated lung perfusion (ILP). Material and Methods : A sarcoma tumor was generated in the left lung of Fisher rats by subpleural injection of a sarcoma cell ,suspension via thoracotomy. Ten days later, LiporubicinTM is administered IV or by single pass antegrade ILP, with or without Visudyne® -mediated low-dose PDT pre-treatment of the sarcoma bearing lung. The drug concentration and distribution were assessed separately in tumors and lung tissues by high pressure liquid chromatography (HPLC) and fluorescence microscopy (FNI~, respectively. Results : PDT pretreatment before IV LiporubicinTM administration resulted in a significantly higher tumor drug uptake and tumor to lung drug ratio compared to IV drug injection alone without affecting the blood flow and drug distribution in the lung. PDT pre-treatment before LiporubicinTM-based ILP also resulted in a higher tumor drug uptake and a higher tumor to lung drug ratio compared to ILP alone, however, these differences were not significant due to a heterogeneous blood flow drug distribution during ILP which was further accentuated by PDT. Conclusions : Low-dose Visudyne®-mediated PDT pre-treatment has the potential to selectively enhance liposomal encapsulated doxorubicin uptake in tumors but not in normal lung tissue after IV drug application in a rat model of sarcoma tumors to the lung which opens new perspectives for the treatment of superficially spreading chemoresistant tumors of the chest cavity such as mesothelioma or malignant effusion. However, the impact of PDT on macromolecular drug uptake during ILP is limited since its therapeutic advantage is circumvented by ILP-induced heterogeneicity of blood flow and drug distribution Abstract Part II Background : Photodynamic therapy (PDT) with Visudyne® acts by direct cellular phototoxicity and/or by an indirect vascular-mediated effect. Here, we demonstrate that the vessel integrity interruption by PDT can promote the extravasation of a macromolecular agent in normal tissue. To obtain extravasation in normal tissue PDT conditions were one order of magnitude more intensive than the ones in tissue containing neovessels reported in the literature. Material and Methods : Fluorescein isothiocyanate dextran (FITC-D, 2000kDa), a macromolecular agent, was intravenously injected 10 minutes before (LKO group, n=14) or 2 hours (LK2 group, n=16) after Visudyne® mediated PDT in nude mice bearing a dorsal skin fold chamber. Control animals had no PDT (CTRL group, n=8). The extravasation of FITC-D from blood vessels in striated muscle tissue was observed in both groups in real-time for up to 2500 seconds after injection. We also monitored PDT-induced leukocyte rolling in-vivo and assessed, by histology, the corresponding inflammatory reaction score in the dorsal skin fold chambers. Results : In all animals, at the applied PDT conditions, FITC-D extravasation was significantly enhanced in the PDT treated areas as compared to the surrounding non-treated areas (p<0.0001). There was no FITC-D leakage in the control animals. Animals from the LKO group had significantly less FITC-D extravasation than those from the LK2 group (p = 0.0002). In the LKO group FITC-D leakage correlated significantly with the inflammation (p < 0.001). Conclusions: At the selected conditions, Visudyne-mediated PDT promotes vascular leakage and FITC-D extravasation into the interstitial space of normal tissue. The intensity of vascular leakage depends on the time interval between PDT and FITC-D injection. This concept could be used to locally modulate the delivery of macromolecules in vivo. Résumé : La perfusion cytostatique isolée du poumon permet une administration sélective des agents cytostatiques sans implication de la circulation systémique avec une forte accumulation au niveau du poumon mais une faible pénétration dans les tumeurs. La thérapie photodynamique (PDT) qui consiste en l'application d'un sensibilisateur activé par lumière laser non- thermique d'une longueur d'onde définie permet dans certaines conditions, une augmentation de la pénétration des agents cytostatiques macromoléculaires à travers la barrière endothéliale tumorale. Nous avons exploré cet avantage thérapeutique de la PDT dans un modèle expérimental afin d'augmenter d'une manière sélective la pénétration tumorale de la doxorubicin pegylée, liposomal- encapsulée macromoléculaire (Liporubicin). Une tumeur sarcomateuse a été générée au niveau du poumon de rongeur suivie d'administration de Liporubicin, soit par voie intraveineuse soit par perfusion isolée du poumon (ILP). Une partie des animaux ont reçus un prétraitement de la tumeur et du poumon sous jacent par PDT avec Visudyne comme photosensibilisateur. Les résultats ont démontrés que la PDT permet, sous certaines conditions, une augmentation sélective de Liporubicin dans les tumeurs mais pas dans le parenchyme pulmonaire sous jacent. Après administration intraveineuse de Liporubicin et prétraitement par PDT, l'accumulation dans les tumeurs était significative par rapport au poumon, et aux tumeurs sans PDT. Le même phénomène est observé après ILP du poumon. Cependant, les différences avec ou sans PDT n'étaient pas significatives lié à und distribution hétérogène de Liporubicin dans le poumon perfusé après ILP. Dans une deuxième partie de l'expérimentation, nous avons exploré la microscopie intra-vitale pour déterminer l'extravasion des substances macromoléculaires (FITS) à travers la barrière endothéliale avec ou sans Visudyne-PDT au niveau des chambres dorsales des souris nues. Les résultats montrent qu'après PDT, l'extravasion de FITS a été augmentée de manière significative par rapport au tissu non traité. L'intensité de l'extravasion de FITS dépendait également de l'intervalle entre PDT et injection de FITS. En conclusion, les expérimentations montrent que la PDT est capable, sous certaines conditions, d'augmenter de manière significative l'extravasion des macromolécules à travers la barrière endothéliale et leur accumulation dans des tumeurs mais pas dans le parenchyme pulmonaire. Ces résultats permettent une nouvelle perspective de traitement pour des tumeurs superficielles intrathoraciques chimio-résistent comme l'épanchement pleural malin ou le mésothéliome pleural.

Effect of vitamin D on Epstein-Barr (EBV)-specific CD8+T cells in patients with early multiple sclerosis (MS)

Background: Infection with EBV and a lack in vitamin D may be important environmental triggers of MS. 1,25-(OH)2D3 mediates a shift of antigen presenting cells (APC) and CD4+ T cells to a less inflammatory profile. Although CD8+ T cells do express the vitamin D receptor, a direct effect of 1,25(OH)2D3 on these cells has not been demonstrated until now. Since CD8+ T cells are important immune mediators of the inflammatory response in MS, we examined whether vitamin D directly affects the CD8+ T cell response, and more specifically if it modulates the EBV-specific CD8+ T cell response. Material and Methods: To explore whether the vitamin D status may influence the pattern of the EBV-specific CD8+ T cell response, PBMC of 10 patients with early MS and 10 healthy controls (HC) were stimulated with a pool of immunodominant 8-10 mer peptide epitopes known to elicit CD8+ T cell responses. PBMC were stimulated with this EBV CD8 peptide pool, medium (negative control) or anti- CD3/anti-CD28 beads (positive control). The following assays were performed: ELISPOT to assess the secretion of IFN-gamma by T cells in general; cytometric beads array (CBA) and ELISA to determine whichcytokines were released by EBV-specific CD8+ T cells after six days of culture; and intracellular cytokine staining assay to determine by which subtype of T cells secreted given cytokines. To examine whether vitamin D could directly modulate CD8+ T cell immune responses, we depleted CD4+ T cells using negative selection. Results: We found that pre-treatment of vitamin D had an antiinflammatory action on both EBV-specific CD8+ T cells and on CD3/ CD28-stimulated T cells: secretion of pro-inflammatory cytokines (IFNgamma and TNF-alpha) was decreased, whereas secretion of antiinflammatory cytokines (IL-5 and TGF-beta) was increased. At baseline, CD8+ T cells of early MS patients showed a higher secretion of TNFalpha and lower secretion of IL-5. Addition of vitamin D did not restore the same levels of both cytokines as compared to HC. Vitamin D-pretreated CD8+T cells exhibited a decreased secretion of IFN-gamma and TNF-alpha, even after depletion of CD4+ T cells from culture. Conclusion: Vitamin D has a direct anti-inflammatory effect on CD8+ T cells independently from CD4+ T cells. CD8+ T cells of patients with earlyMS are less responsive to the inflammatory effect of vitamin D than HC, pointing toward an intrinsic dysregulation of CD8+ T cells. The modulation of EBV-specific CD8+T cells by vitaminDsuggests that there may be interplay between these twomajor environmental factors of MS. This study was supported by a grant from the Swiss National Foundation (PP00P3-124893), and by an unrestricted research grant from Bayer to RDP.

Illicit drug profiling, reflection on statistical comparisons

This paper presents reflexions about statistical considerations on illicit drug profiling and more specifically about the calculation of threshold for determining of the seizure are linked or not. The specific case of heroin and cocaine profiling is presented with the necessary details on the target profiling variables (major alkaloids) selected and the analytical method used. Statistical approach to compare illicit drug seizures is also presented with the introduction of different scenarios dealing with different data pre-treatment or transformation of variables.The main aim consists to demonstrate the influence of data pre-treatment on the statistical outputs. A thorough study of the evolution of the true positive rate (TP) and the false positive rate (FP) in heroin and cocaine comparison is then proposed to investigate this specific topic and to demonstrate that there is no universal approach available and that the calculations have to be revaluate for each new specific application.

Photodynamic drug delivery enhancement in tumours does not depend on leukocyte-endothelial interaction in a human mesothelioma xenograft model.

OBJECTIVES: The pre-treatment of tumour neovessels by low-level photodynamic therapy (PDT) improves the distribution of concomitantly administered systemic chemotherapy. The mechanism by which PDT permeabilizes the tumour vessel wall is only partially known. We have recently shown that leukocyte-endothelial cell interaction is essential for photodynamic drug delivery to normal tissue. The present study investigates whether PDT enhances drug delivery in malignant mesothelioma and whether it involves comparable mechanisms of actions. METHODS: Human mesothelioma xenografts (H-meso-1) were grown in the dorsal skinfold chambers of 28 nude mice. By intravital microscopy, the rolling and recruitment of leukocytes were assessed in tumour vessels following PDT (Visudyne(®) 400 μg/kg, fluence rate 200 mW/cm(2) and fluence 60 J/cm(2)) using intravital microscopy. Likewise, the distribution of fluorescently labelled macromolecular dextran (FITC-dextran, MW 2000 kDa) was determined after PDT. Study groups included no PDT, PDT, PDT plus a functionally blocking anti-pan-selectin antibody cocktail and PDT plus isotype control antibody. RESULTS: PDT significantly enhanced the extravascular accumulation of FITC-dextran in mesothelioma xenografts, but not in normal tissue. PDT significantly increased leukocyte-endothelial cell interaction in tumour. While PDT-induced leukocyte recruitment was significantly blunted by the anti-pan-selectin antibodies in the tumour xenograft, this manipulation did not affect the PDT-induced extravasation of FITC-dextran. CONCLUSIONS: Low-level PDT pre-treatment selectively enhances the uptake of systemically circulating macromolecular drugs in malignant mesothelioma, but not in normal tissue. Leukocyte-endothelial cell interaction is not required for PDT-induced drug delivery to malignant mesothelioma.

Rôle et mécanisme d'action de la metformine et du telmisartan sur la régulation de la prise alimentaire

Dans ce travail de thèse, nous avons étudié les mécanismes d'action de deux médicaments connus pour diminuer la prise alimentaire et pondérale : la metformine et le telmisartan. Nous avons dans un premier temps étudié les effets de la metformine, un antidiabétique oral connu pour avoir des effets anorexigènes. Les mécanismes hypothalamiques potentiellement impliqués dans la modulation de la prise alimentaire par la metformine ont été étudiés dans trois groupes de rats : un groupe de rats obèses (DIO), un groupe de rats résistants à l'obésité (DR) ainsi qu'un groupe contrôle. A la fin de la période de prise pondérale de six mois, les rats DIO avaient des taux d'ARNm de NPY hypothalamique plus élevés que leurs congénères résistants et contrôles. Chez les DIO ainsi que chez les DR un traitement par metformine induit une baisse significative de la prise alimentaire accompagnée par une baisse du poids. Nous avons pu d'autre part constater que la perte de poids obtenue par un traitement de metformine était corrélée aux taux circulants de leptine avant le traitement. Cet effet s'accompagne d'une augmentation de l'expression du récepteur ObRb au niveau hypothalamique. Dans un second temps, nous avons étudié les effets du telmisartan, un inhibiteur du récepteur à l'angiotensine II ayant une activité agoniste partielle PPARγ. L'influence du telmisartan associé à la pioglitazone sur la prise alimentaire et pondérale a été examinée en étudiant leur effet sur les neuropeptides hypothalamiques responsables du contrôle de la prise alimentaire. Quatre groupes de souris soumises à un régime riche en graisse ont été formés : un groupe placebo, un groupe pioglitazone, un groupe telmisartan et un groupe pioglitazone-telmisartan. Le telmisartan a aboli la prise pondérale induite par une diète riche en graisse ou par un traitement de pioglitazone. Cette diminution était corrélée à une baisse de la prise alimentaire et de l'expression hypothalamique d'AgRP. Cette étude confirme donc les effets anorexigènes du telmisartan et démontre pour la première fois le rôle fonctionnel du telmisartan sur l'expression hypothalamique d'AgRP. English Abstract : In this work, we investigated the effect of two drugs known to have interessants effects on food intake and body weight. First we investigated the hypothalamic mechanisms potentially implicated in the modulation of feeding by the glucose-lowering drug metformin in three different groups of animals: diet-induced obese (DIO) and diet-resistant (DR) male rats as well as lean controls (CT). At the end of the high fat diet period, despite higher leptin levels, DIO rats had higher levels of hypothalamic NPY expression than DR or CT, suggesting a central leptin resistance. In DIO but also in DR rats, metformin treatment induced significant reductions of food intake accompanied by decreases in body weight. Interestingly, the weight loss achieved by metformin was correlated with pre-treatment plasma leptin levels. This effect was paralleled by a stimulation of the expression of the leptin receptor gene (ObRb) in the arcuate nucleus. Next we investigated the antihypertensive drug Telmisartan, an angiotensin II receptor blocker with PPARγ agonistic properties. The influence of telmisartan, of pioglitazone and of their association on weight gain and food intake was assessed by studying their effects on neuro-endocrine mediators involved in food intake. Mice were fed a high fat diet, weightmatched and randomized in four treatment groups: vehicle, pioglitazone, telmisartan and pioglitazone-telmisartan. Telmisartan treatment was found to abolish weight and fat gain in either vehicle or pioglitazone treated mice. This effect was accompanied by a decrease in food intake. The hypothalamic expression of the agouti-related protein and plasma leptin levels show also a decrease under metformin treatment. This study confirms the anorexigenic effects of telmisartan in mice fed a high fat diet, and suggests for the first time a functional role of telmisartan on hypothalamic orexigenic agouti-related protein regulation.

Biomedical nanoparticles modulate specific CD4(+) T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4(+) T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4(+) T cells, and induce cytokines. The decreased antigen processing and CD4(+) T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

Rate and predictors of service disengagement in an epidemiological first-episode psychosis cohort.

OBJECTIVES: To assess the prevalence and predictors of service disengagement in a treated epidemiological cohort of first-episode psychosis (FEP) patients. METHODS: The Early Psychosis Prevention and Intervention Centre (EPPIC) in Australia admitted 786 FEP patients from January 1998 to December 2000. Treatment at EPPIC is scheduled for 18 months. Data were collected from patients' files using a standardized questionnaire. Seven hundred four files were available; 44 were excluded, because of a non-psychotic diagnosis at endpoint (n=43) or missing data on service disengagement (n=1). Rate of service disengagement was the outcome of interest, as well as pre-treatment, baseline, and treatment predictors of service disengagement, which were examined via Cox proportional hazards models. RESULTS: 154 patients (23.3%) disengaged from service. A past forensic history (Hazard ratio [HR]=1.69; 95%CI 1.17-2.45), lower severity of illness at baseline (HR=0.59; 95%CI 0.48-0.72), living without family at discharge (HR=1.75; 95%CI 1.22-2.50) and persistence of substance use disorder during treatment (HR=2.30; 95%CI 1.45-3.66) were significant predictors of disengagement from service. CONCLUSIONS: While engagement strategies are a core element in the treatment of first-episode psychosis, particular attention should be paid to these factors associated with disengagement. Involvement of the family in the treatment process, and focusing on reduction of substance use, need to be pursued in early intervention services.

Targeting anticancer therapy : clinical and preclinical approaches

AbstractCancer treatment has shifted from cytotoxic and nonspecific chemotherapy to chronic treatment with targeted molecular therapies. These new classes of drugs directed against cancer-specific molecules and signaling pathways, act at a particular level of the tumor cell development. However, in both types of therapeutic approaches (standard cytotoxic chemotherapy and targeted signal transduction inhibitions), toxicity and side effects can occur. The aim of this thesis was to investigate various approaches to improve the activity and tolerability of cancer treatment, in a clinical setting, a) by molecular targeting through the use of tyrosine kinase inhibitors (TKIs), whose dosage can be adapted to each patient according to plasma levels, and, b) in a preclinical model, by tissue targeting with locoregional administration of cytotoxic chemotherapy to increase drug exposure in the target tissue while reducing systemic toxicity of the treatment.A comprehensive program for the Therapeutic Drug Monitoring (TDM) of the new class of targeted anticancer drugs of TKIs in patient's blood has been therefore initiated comprising the setting up, validation and clinical application of a multiplex assay by liquid chromatography coupled to tandem mass spectrometry of TKIs in plasma from cancer patients. Information on drugs exposure may be clinically useful for an optimal follow-up of patients' anticancer treatment, especially in case of less than optimal clinical response, occurrence of adverse drug reaction effects and the numerous risks of drug-drug interactions. In this context, better knowledge of the potential drug interactions between TKIs and widely prescribed co- medications is of critical importance for clinicians, to improve their daily care of cancer patients. For one of the first TKI imatinib, TDM interpretation is nowadays based on total plasma concentrations but, only the unbound (free) form is likely to enter cell to exert its pharmacological action. Pharmacokinetic analysis of the total and free plasma level of imatinib measured simultaneously in patients have allowed to refine and validate a population pharmacokinetic model integrating factors influencing in patients the exposure of pharmacological active species. The equation developed from this model may be used for extrapolating free imatinib plasma concentration based on the total plasma levels that are currently measured in TDM from patients. Finally, the specific influence of Pglycoprotein on the intracellular disposition of TKIs has been studies in cell systems using the siRNA silencing approach.Another approach to enhance the selectivity of anticancer treatment may be achieved by the loco-regional administration of a cytostatic agent to the target organ while sparing non- affected tissues. Isolated lung perfusion (ILP) was designed for the treatment of loco-regional malignancies of the lung but clinical results have been so far disappointing. It has been shown in a preclinical model in rats that ILP with the cytotoxic agent doxorubicin alone allows a high drug uptake in lung tissue, and a low systemic toxicity, but was characterized by a high spatial tissular heterogeneity in drug exposure and doxorubicin uptake in tumor was comparatively smaller than in normal lung tissue. Photodynamic therapy (PDT) is a new approach for the treatment of superficial tumors, and implies the application of a sensitizer activated by a laser light at a specific wavelength, that disrupts endothelial barrier of tumor vessels to increase locally the distribution of cytostatics into the tumor tissue. PDT pre-treatment before intravenous administration of liposomal doxorubicin was indeed shown to selectively increase drug uptake in tumors in a rat model of sarcoma tumors to the lung.RésuméLe traitement de certains cancers s'est progressivement transformé et est passé de la chimiothérapie, cytotoxique et non spécifique, au traitement chronique des patients avec des thérapies moléculaires ciblées. Ces médicaments ont une action ciblée en interférant à un niveau spécifique du développement de la cellule tumorale. Dans les deux types d'approches thérapeutiques (chimiothérapie cytotoxique et traitements ciblés), on est confronté à la présence de toxicité et aux effets secondaires du traitement anticancéreux. Le but de cette thèse a donc été d'étudier diverses approches visant à améliorer l'efficacité et la tolérabilité du traitement anticancéreux, a) dans le cadre d'une recherche clinique, par le ciblage moléculaire grâce aux inhibiteurs de tyrosines kinases (TKIs) dont la posologie est adaptée à chaque patient, et b) dans un modèle préclinique, par le ciblage tissulaire grâce à l'administration locorégionale de chimiothérapie cytotoxique, afin d'augmenter l'exposition dans le tissu cible et de réduire la toxicité systémique du traitement.Un programme de recherche sur le suivi thérapeutique (Therapeutic Drug Monitoring, TDM) des inhibiteurs de tyrosine kinases a été ainsi mis en place et a impliqué le développement, la validation et l'application clinique d'une méthode multiplex par chromatographie liquide couplée à la spectrométrie de masse en tandem des TKIs chez les patients souffrant de cancer. L'information fournie par le TDM sur l'exposition des patients aux traitements ciblés est cliniquement utile et est susceptible d'optimiser la dose administrée, notamment dans les cas où la réponse clinique au traitement des patients est sous-optimale, en présence d'effets secondaires du traitement ciblé, ou lorsque des risques d'interactions médicamenteuses sont suspectés. Dans ce contexte, l'étude des interactions entre les TKIs et les co-médications couramment associées est utile pour les cliniciens en charge d'améliorer au jour le jour la prise en charge du traitement anticancéreux. Pour le premier TKI imatinib, l'interprétation TDM est actuellement basée sur la mesure des concentrations plasmatiques totales alors que seule la fraction libre (médicament non lié aux protéines plasmatiques circulantes) est susceptible de pénétrer dans la cellule pour exercer son action pharmacologique. L'analyse pharmacocinétique des taux plasmatiques totaux et libres d'imatinib mesurés simultanément chez les patients a permis d'affiner et de valider un modèle de pharmacocinétique de population qui intègre les facteurs influençant l'exposition à la fraction de médicament pharmacologiquement active. L'équation développée à partir de ce modèle permet d'extrapoler les concentrations libres d'imatinib à partir des concentrations plasmatiques totales qui sont actuellement mesurées lors du TDM des patients. Finalement, l'influence de la P-glycoprotéine sur la disposition cellulaire des TKIs a été étudiée dans un modèle cellulaire utilisant l'approche par la technologie du siRNA permettant de bloquer sélectivement l'expression du gène de cette protéine d'efflux des médicaments.Une autre approche pour augmenter la sélectivité du traitement anticancéreux consiste en une administration loco-régionale d'un agent cytostatique directement au sein de l'organe cible tout en préservant les tissus sains. La perfusion isolée du poumon (ILP) a été conçue pour le traitement loco-régional des cancers affectant les tissus pulmonaires mais les résultats cliniques ont été jusqu'à ce jour décevants. Dans des modèles précliniques chez le rat, il a pu être démontré que l'ILP avec la doxorubicine, un agent cytotoxique, administré seul, permet une exposition élevée au niveau du tissu pulmonaire, et une faible toxicité systémique. Toutefois, cette technique est caractérisée par une importante variabilité de la distribution dans les tissus pulmonaires et une pénétration du médicament au sein de la tumeur comparativement plus faible que dans les tissus sains.La thérapie photodynamique (PDT) est une nouvelle approche pour le traitement des tumeurs superficielles, qui consiste en l'application d'un agent sensibilisateur activé par une lumière laser de longueur d'onde spécifique, qui perturbe l'intégrité physiologique de la barrière endothéliale des vaisseaux alimentant la tumeur et permet d'augmenter localement la pénétration des agents cytostatiques.Nos études ont montré qu'un pré-traitement par PDT permet d'augmenter sélectivement l'absorption de doxorubicine dans les tumeurs lors d'administration i.v. de doxorubicine liposomale dans un modèle de sarcome de poumons de rongeurs.Résumé large publicDepuis une dizaine d'année, le traitement de certains cancers s'est progressivement transformé et les patients qui devaient jusqu'alors subir des chimiothérapies, toxiques et non spécifiques, peuvent maintenant bénéficier de traitements chroniques avec des thérapies ciblées. Avec les deux types d'approches thérapeutiques, on reste cependant confronté à la toxicité et aux effets secondaires du traitement.Le but de cette thèse a été d'étudier chez les patients et dans des modèles précliniques les diverses approches visant à améliorer l'activité et la tolérance des traitements à travers un meilleur ciblage de la thérapie anticancéreuse. Cet effort de recherche nous a conduits à nous intéresser à l'optimisation du traitement par les inhibiteurs de tyrosines kinases (TKIs), une nouvelle génération d'agents anticancéreux ciblés agissant sélectivement sur les cellules tumorales, en particulier chez les patients souffrant de leucémie myéloïde chronique et de tumeurs stromales gastro-intestinales. L'activité clinique ainsi que la toxicité de ces TKIs paraissent dépendre non pas de la dose de médicament administrée, mais de la quantité de médicaments circulant dans le sang auxquelles les tumeurs cancéreuses sont exposées et qui varient beaucoup d'un patient à l'autre. A cet effet, nous avons développé une méthode par chromatographie couplée à la spectrométrie de masse pour mesurer chez les patients les taux de médicaments de la classe des TKIs dans la perspective de piloter le traitement par une approche de suivi thérapeutique (Therapeutic Drug Monitoring, TDM). Le TDM repose sur la mesure de la quantité de médicament dans le sang d'un patient dans le but d'adapter individuellement la posologie la plus appropriée: des quantités insuffisantes de médicament dans le sang peuvent conduire à un échec thérapeutique alors qu'un taux sanguin excessif peut entraîner des manifestations toxiques.Dans une seconde partie préclinique, nous nous sommes concentrés sur l'optimisation de la chimiothérapie loco-régionale dans un modèle de sarcome du poumon chez le rat, afin d'augmenter l'exposition dans la tumeur tout en réduisant la toxicité dans les tissus non affectés.La perfusion isolée du poumon (ILP) permet d'administrer un médicament anticancéreux cytotoxique comme la doxorubicine, sélectivement au niveau le tissu pulmonaire où sont généralement localisées les métastases de sarcome. L'administration par ILP de doxorubicine, toxique pour le coeur, a permis une forte accumulation des médicaments dans le poumon, tout en épargnant le coeur. Il a été malheureusement constaté que la doxorubicine ne pénètre que faiblement dans la tumeur sarcomateuse, témoignant des réponses cliniques décevantes observées avec cette approche en clinique. Nous avons ainsi étudié l'impact sur la pénétration tumorale de l'association d'une chimiothérapie cytotoxique avec la thérapie photodynamique (PDT) qui consiste en l'irradiation spécifique du tissu-cible cancéreux, après l'administration d'un agent photosensibilisateur. Dans ce modèle animal, nous avons observé qu'un traitement par PDT permet effectivement d'augmenter de façon sélective l'accumulation de doxorubicine dans les tumeurs lors d'administration intraveineuse de médicament.

Influences of β-HCG administration on carbon isotope ratios of endogenous urinary steroids.

Several factors influencing the carbon isotope ratios (CIR) of endogenous urinary steroids have been identified in recent years. One of these should be the metabolism of steroids inside the body involving numerous different enzymes. A detailed look at this metabolism taking into account differences found between steroids excreted as glucuronides or as sulphates and hydrogen isotope ratios of different steroids pointed out possibility of unequal CIR at the main production sites inside the male body - the testes and the adrenal glands. By administration of β-HCG it is possible to strongly stimulate the steroid production within the testes without influencing the production at the adrenal glands. Therefore, this treatment should result in changed CIR of urinary androgens in contrast to the undisturbed pre-treatment values. Four male volunteers received three injections of β-HCG over a time course of 5 days and collected their urine samples at defined intervals after the last administration. Those samples showing the largest response in contrast to the pre-administration urines were identified by steroid profile measurements and subsequent analysed by GC/C/IRMS. CIR of androsterone, etiocholanolone, testosterone, 5α- and 5β-androstanediol and pregnanediol were compared. While pregnanediol was not influenced, most of the investigated androgens showed depleted values after treatment. The majority of differences were found to be statistically significant and nearly all showed the expected trend towards more depleted δ(13)C-values. These results support the hypothesis of different CIR at different production sites inside the human body. The impact of these findings on doping control analysis will be discussed.

Malignant pleural mesothelioma: leukocyte recruitment is not required for drug delivery induced by photodynamic therapy in a human xenograft model

Objective: The pre-treatment of tumor neo-vessels by photodynamic therapy (PDT) was shown to improve the distribution of chemotherapy administered subsequently. However, the precise mechanism by which PDT modifies the tumor vasculature is unknown. We have recently shown that leukocyteendothelial cell interaction was essential for PDT induced drug delivery to normal tissue. Our purpose was to determine if PDT could enhance drug distribution in malignant mesothelioma and if a comparable role for leucocytes existed.Methods: We grew human mesothelioma xenografts (H-meso-1) in the dorsal skinfold chambers of nude mice (n = 28). The rolling, sticking and recruitment of leucocytes was assessed in tumor and normal vessels following PDT (Visudyne 0?4 mg/kg, fluence rate 200 mW/cm2, fluence 60 J/cm2) using intravital microscopy. In parallel, the distribution of a macromolecule (FITC dextran, 2000 kDa) administered after PDT was determined. We compared these variables in control (no PDT), PDT + IgG (non specific antibody) and PDT + pan-selectin antibody (monoclonal P-E-L selectin antibody).Results: PDT significantly enhanced the distribution of FITC dextran in mesothelioma xenografts compared to controls. Interestingly, PDT enhanced the leukocyte-endothelial interaction significantly (rolling and recruitment)in tumor and surrounding normal vessels compared to controls. Leukocyte recruitment was significantly down-regulated by pan-selectin antibodies in tumor tissues. However, the suppression of leucocyte recruitement did not affect the extravasation of FITC-dextran in tumor tissue.Conclusion:PDTpre-treatment of the mesothelioma vasculature can enhance the distribution of macromolecular drugs administered subsequently. However, unlike normal vessels, leukocyte-endothelial cell interaction is not required for PDT induced leakage.