L’oxaliplatine, anticancéreux utilisé en première intention dans le traitement du cancercolorectal, engendre des neuropathies qui se caractérisent par des dommages du systèmenerveux périphériques responsables de l’apparition de troubles sensitifs douloureux dès lapremière cure ainsi que de troubles moteurs mais également des comorbidités qui s’installentavec la répétition des cures. Les traitements actuellement disponibles sont peu efficaces pourtraiter ces neuropathies qui sont à l’origine d’une altération de la qualité de vie des patients etpeuvent conduire à une diminution des doses de chimiothérapie utilisées voire même à l’arrêtdu traitement, compromettant ainsi les chances de guérison. Des données du laboratoire ont misen avant plusieurs canaux ioniques comme étant des cibles moléculaires potentielles pour letraitement de la neuropathie aigue. Le but de ce travail de thèse a été de poursuivre les étudespermettant de comprendre le mécanisme de survenue de ces effets indésirables et de rechercherles canaux dont la modulation pharmacologique permettrait un traitement efficace et bien tolérépour ces neuropathies.Pour se faire nous avons décidé de mieux caractériser un modèle murin de neuropathiedouloureuse aigue et de mettre au point et caractériser un modèle de neuropathie chroniqueinduites respectivement, par des injections unique ou répétées d’oxaliplatine. Un travailéthologique a été entrepris chez ces modèles afin d’évaluer la survenue et le suivi longitudinaldes symptômes retrouvés en clinique : symptômes douloureux céphaliques et extracéphaliques,déficits moteurs, dépression, anxiété. Chez ces mêmes animaux, des analyses moléculaires nousont permis de sélectionner des gènes codant pour des canaux ioniques impliqués dans laphysiologie et la pharmacologie de la douleur (canaux potassiques TREK et TRAAK, canauxHCN1 & HCN2). Nous avons parallèlement entrepris de valider pharmacologiquement ces cibleschez l’animal. Les résultats de nos travaux sont présentés séquentiellement et concernent :l’étude de l’implication des canaux HCN dans l’hypersensibilité douloureuse céphalique etextracéphalique induite par l’oxaliplatine ; l’étude de l’implication des canaux TREK et TRAAKdans la neuropathie chronique induite par l’oxaliplatine et la validation pharmacologique de cescibles chez un modèle murin de cancer colorectal.Dans un premier temps, nous avons utilisé un modèle de neuropathie aigue induit parune unique injection d’oxaliplatine (6mg/kg), et avons caractérisé les symptômes décrits enclinique au niveau du territoire céphalique. Nous avons observé l’existence de symptômesdouloureux au niveau orofacial et nous avons pu les corréler à une surexpression des canauxHCN1& HCN2. Il avait précédemment été démontré une surexpression de ses mêmes canaux auniveau extracéphalique, ce qui en fait des cibles particulièrement pertinentes compte tenu desdifférences anatomiques et pharmacologiques connues entre ces deux territoires somatiques. Deplus, l’utilisation d’ivabradine (Procoralan®), antagoniste non sélectif de ces canaux, a permis deréverser l’hypersensibilité douloureuse chez ce modèle animal de neuropathie douloureuseaigue.En conclusion, l’ensemble de ces résultats expérimentaux a permis l’identification denouvelles pistes pour la compréhension et le traitement des douleurs chimio-induites àl’oxaliplatine. Dans une perspective de recherche translationnelle, cette approche préclinique esten cours de transposition dans un protocole de recherche clinique. Un essai clinique de phase II(RILUZOX) devrait débuter prochainement afin de confirmer l’intérêt du riluzole chez despatients recevant une chimiothérapie à base d’oxaliplatine. / Oxaliplatin causes neuropathies which are characterized by damage of peripheralnervous system responsible for the onset of disorders such as neuropathic pain, sensory andmotor symptoms and comorbidities. Currently, available treatments are not very effective fortreating these symptoms in acute or chronic form. However, these symptoms are theconsequence of an impaired quality of life for patients and can lead to a decrease ofchemotherapy doses used or even stopping treatment, compromising the chances of surviving.Therefore, it appears essential for further studies to understand the mechanism of occurrence ofthese pains and identify potential targets for the development of new analgesics.We decided to further characterize a mouse model of acute painful neuropathy and todevelop and characterize a chronic neuropathy model induced respectively by single or repeatedinjections of oxaliplatin. An ethological work was realized in these models to assess theoccurrence of clinical symptoms: painful symptoms at cephalic and extracephalic levels, motordeficits, depression, anxiety. In these animals, molecular analyzes allowed us to select the genesencoding ion channels involved in the physiology and pharmacology of pain (potassium channelTREK and TRAAK, HCN1 & HCN2 channels). We undertook to pharmacologically validate thesetargets in animal. The results of our work are presented sequentially and concern: the study ofthe involvement of HCN channels in the cephalic and extracephalic painful hypersensitivityinduced by oxaliplatin; the study of the involvement of TREK and TRAAK channels in chronicoxaliplatin-induced neuropathy and pharmacological validation of these targets in a mousemodel of colorectal cancer.Initially, we used a model of acute neuropathy induced by single injection of oxaliplatin(6mg / kg) to study the clinical symptoms described at the orofacial sphere. Indeed, peripheralsymptoms had already been studied and the hypothesis of the ion channels expressionremodelling was confirmed. So, we verified the existence of orofacial pain symptoms with newbehavioral tests and we have correlated these results with an over expression of HCN1 & HCN2channels. In addition, use of ivabradine (Procoralan®), a non-selective antagonist of thesechannels, allowed reversing hypersensitivity to cold at low doses and mechanicalhypersensitivity when we used higher doses.In conclusion, all of these experimental results allowed the identification of newmechanisms for understanding and treating oxaliplatin-induced neuropathy. First, involvementsof HCN1 & HCN2 channels have showed in the acute neuropathy induced by oxaliplatin for bothperipheral and oral symptoms. In particular, blocking of these channels by ivabradine has alsohelped to reverse the cold hypersensitivity. Also, the TREK-1 channel showed great interest ininvolvement in chronic neuropathy induced by oxaliplatin and riluzole showed many interestingproperties to overcome the symptoms described in this neuropathy. From the perspective oftranslational research, this preclinical approach is being transposed in a clinical researchprotocol. A phase II clinical trial (RILUZOX) should begin shortly to confirm the interest ofriluzole in patients receiving chemotherapy with oxaliplatin.

L'aspect expérimental de la pharmacocinétique nécessite le développement de techniques analytiques dans les milieux biologiques pour la quantification des médicaments dans les échantillons prélevés à des temps choisis. L'interprétation des données pharmacocinétiques implique ensuite le développement de modèles et de méthodes statistiques permettant l'évaluation des paramètres pharmacocinétiques caractéristiques des molécules considérées. Dans la première partie de ce travail, une méthode LC/MS/MS sensible et spécifique, a été développée et validée pour le dosage simultané de pralidoxime, diazépam et atropine dans le plasma. Une étude pharmacocinétique de chacun des trois composés chez le sujet sain recevant par voie intramusculaire l'association avizafone-atropine-pralidoxime, traitement recommandé pour l'intoxication par les agents organophosphorés, a ensuite été conduite. Les données issues du dosage plasmatique ont été analysées afin de caractériser la cinétique d'hydrolyse de l'avizafone et la pharmacocinétique de la pralidoxime. La modélisation individuelle, sujet par sujet, a permis de déterminer le meilleur modèle pharmacocinétique de la transformation d'avizafone en diazépam après injection intramusculaire d'avizafone seule ou en association avec atropine et pralidoxime. Une approche de pharmacocinétique de population a enfin été appliquée pour caractériser la pharmacocinétique de la pralidoxime après injection intramusculaire seule ou en association avec atropine et avizafone. Dans la deuxième partie de cette thèse, l'approche non compartimentale et la modélisation individuelle ont été appliquées à des données obtenues au cours d'une étude de pharmacocinétique évaluant l'administration orale de 50 mg de riluzole chez des enfants atteints d'amyotrophie spinale. Par simulations, il a été possible de montrer que l'administration de riluzole, à la dose de 50 mg deux fois par jour se traduirait par des concentrations résiduelles et maximales comparables à celles obtenues chez l'adulte après une dose de 100 mg deux fois par jour. La dose de 50 mg une fois par jour a donc été retenue pour l'étude clinique évaluant l'efficacité du riluzole chez les patients atteints d'amyotrophie spinale infantile. Mots-clés : Pharmacocinétique, Organophosphorés, Amyotrophie spinale infantile, Riluzole.

The biophysical properties and distribution of voltage gated ion channels shape the spatio-temporal pattern of synaptic inputs and determine the input-output properties of the neuron. Of the various voltage-gated ion channels, persistent Na⁺ current (INaP) is of interest because of its activation near rest, slow inactivation kinetics, and consequent effects on excitability. Overshadowed by transient Na⁺ current (INaT) of large amplitude and fast inactivation, various quantitative characterizations of INaP have yet to provide a clear understanding of their role in neuronal excitability. We addressed this question using quantitative electrophysiology to compare somatic INaP and INaT in 4–7 week old Sprague-Dawley rat hippocampal CA1 pyramidal neurons. INaP was evoked with 0.4 mV/ms ramp voltage commands and INaT with step commands in hippocampal neurons from in vitro brain slices utilizing nucleated patch-clamp recording. INaP was found to have a density of 1.4 ± 0.7 pA/pF in the soma. Compared to INaT, it has a much smaller amplitude (2.38% of INaT) and distinct voltage dependence of activation (16.7 mV lower half maximal activation voltage and 41.3% smaller slope factor than those of INaT). The quantitative measurement of INaT gave the activation time constant ([tau]m) of 22.2 ± 2.3 [mu]s at 40 mV. Hexanol, which has anesthetic effects, was shown to preferentially block INaP compared to INaT with a significant voltage threshold elevation (4.6 ± 0.7 mV) and delayed 1st spike latency (221 ± 54.6 ms) suggesting reduced neuronal excitability. The number of spikes evoked by either given step current injections or [alpha]-EPSP integration was also significantly decreased. The differential blocking of INaP by halothane, a popularly used volatile anesthetic, further supports the critical role of INaP in setting voltage threshold. Taken together, the presence of INaP in the soma demonstrates an intrinsic mechanism utilized by hippocampal CA1 pyramidal neurons to regulate axonal spike initiation through different biophysical properties of the Na⁺ channel. Furthermore, INaP becomes an interesting target of intrinsic plasticity because of its profound effect on the input-output function of the neuron. / text

No description available.

yes / Background: Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson’s disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle. Results: Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion. Conclusions: The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson’s disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.

yes / Despite intense attention from biomedical and chemical researchers, there are few approved treatments for amyotrophic lateral sclerosis (ALS), with only riluzole (Rilutek) and edaravone (Radicava) currently available to patients. Moreover, the mechanistic basis of the activity of these drugs is currently not well-defined, limiting the ability to design new medicines for ALS. This Letter describes the synthesis of triazole-containing riluzole analogues, and their testing in a novel neuroprotective assay. Seven compounds were identified as having neuroprotective activity, with two compounds having similar activity to riluzole.

No description available.

Obsessive-compulsive disorder is a chronic psychiatric disease. It seriously limits the quality of life of patients. Treatment of OCD is not yet fully successful and still many patients are left with debilitating symptoms without functioning medication. Animal models of genetic, behavioral, pharmacological, and optogenetic origins are beneficial in the achievement of new understandings of the disease. Chronic sensitization of serotonin 1A and 7-receptors with an agonist 8-OH-DPAT ((8- hydroxy-2-(di-propylamino)-tetralin hydrobromide) induces perseverative and compulsive behaviors, which is considered to constitute an animal model of OCD. In this thesis, the 8-OH- DPAT model has been tested in the active place avoidance task on Carousel maze to provide information about the model on learning. Second, this model is used to determine, whether co- administration of memantine or riluzole alleviates the cognitive and learning deficits of this model. To uncover these effects, an active place avoidance task on a Carousel maze was used. Measured criteria were total distance, entrances to the shock sector, total number of shocks, and median speed after the shock. During habituation, the animals were sensitized to 8-OH-DPAT (with a control group that did not receive 8-OH-DPAT but only saline). In an...

Amyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)-mediated inhibition of the PI3K/AKT pathway leading to Fas receptor activation and caspase dependent death. The relevance of Hsp90 for motor neuron survival was investigated in mutant Cu/Zn superoxide dismutase (SOD) transgenic animal models for ALS. Nitrated Hsp90, a posttranslational modification known to induce cell death (Franco, Ye et al. 2013), was present in motor neurons after intracellular release of zinc deficient (Zn, D83S) and the SOD in which copper binding site was genetically ablated (Q) but not after copper deficient (Cu) wild type SOD. Zn deficient and Q mutant SOD induced motor neuron death in a peroxynitrite mediated and copper dependent mechanism. Nitrated Hsp90 was not detected in the spinal cord of transgenic animals for ALS-mutant SOD animal models until disease onset. Increased nitrated Hsp90 concentrations correlated with disease progression. Addition of Zn or Q SOD to nontransgenic brain homogenate treated with peroxynitrite led to an increase level of nitrotyrosine in comparison to wild type controls. However, in the same samples there was a 2 to 10 time increase in Hsp90 nitration as compared to nitrotyrosine. The selective increase is likely due to the binding of Hsp90 to Zn deficient and Q SOD as oppose to wild type SOD. These results suggest that Hsp90 nitration facilitated by mutant SOD may cause motor neuron degeneration in ALS. Targeted inhibition of nitrated Hsp90 may be a novel therapeutic approach for ALS. An alternative therapeutic strategy is to target the production of survival factors by glial cells. Riluzole is the only FDA approved drug for the treatment of ALS and it shows a small but significant increase in patient lifespan. Our results show that acute riluzole treatment stimulated trophic factor production by astrocytes and Schwann cells. However long-term exposure reversed and even inhibited the production of trophic factors, an observation that may explain the modest increase in patient survival in clinical trials. Discontinuous riluzole treatment can maintain elevated trophic factor levels and prevent trophic factor reduction in spinal cords of nontransgenic animals. These results suggest that discontinuous riluzole administration may improve ALS patient survival. In summary, we demonstrated that Hsp90 has an essential function in the regulation of motor neuron survival. We have also shown that Hsp90 was nitrated in the presence of mutant SOD and was present during symptom onset and increases as disease progresses, which may explain the toxic gain of function of mutant SOD. Finally we demonstrate a biphasic effect of riluzole on trophic factor production and propose changes in administration to improve effects in ALS patients.

Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and (3)H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 muM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 muM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 muM-1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.