Drug abuse, brain calcification and glutamate-induced neurodegeneration

Positive and negative reinforcing systems are part of the mechanism of drug dependence. Drugs with abuse potential may change the manner of response to negative emotional stimuli, activate positive emotional reactions and possess primary reinforcing properties. Catecholaminergic and peptidergic processes are of importance in these mechanisms. Current research needs to understand the types of adaptations that underlie the particularly long-lived aspects of addiction. Presently, glutamate is candidate to play a role in the enduring effects of drugs of abuse. For example, it participates in the chronic pathological changes of corticostriatal terminals produced by methamphetamine. At the synaptic level, a link between over-activation of glutamate receptors, [C(a2+)](i) increase and neuronal damage has been clearly established leading to neurodegeneration. Thus, neurodegeneration can start after an acute over-stimulation whose immediate effects depend on a diversity of calcium-activated mechanisms. If sufficient, the initial insult results in calcification and activation of a chronic on-going process with a progressive loss of neurons. At present, long-term effects of drug dependence underlie an excitotoxicity process linked to a polysynaptic pathway that dynamically regulates synaptic glutamate. Retaliatory mechanisms include energy capability of the neurons, inhibitory systems and cytoplasmic calcium precipitation as part of the neuron-glia interactions. This paper presents an integrated view of these molecular and cellular mechanisms to help understand their relationship and interdependence in a chronic pathological process that suggest new targets for therapeutic intervention.

Drug-induced mitochondrial dysfunction and cardiotoxicity.

Mitochondria has an essential role in myocardial tissue homeostasis; thus deterioration in mitochondrial function eventually leads to cardiomyocyte and endothelial cell death and consequent cardiovascular dysfunction. Several chemical compounds and drugs have been known to directly or indirectly modulate cardiac mitochondrial function, which can account both for the toxicological and pharmacological properties of these substances. In many cases, toxicity problems appear only in the presence of additional cardiovascular disease conditions or develop months/years following the exposure, making the diagnosis difficult. Cardiotoxic agents affecting mitochondria include several widely used anticancer drugs [anthracyclines (Doxorubicin/Adriamycin), cisplatin, trastuzumab (Herceptin), arsenic trioxide (Trisenox), mitoxantrone (Novantrone), imatinib (Gleevec), bevacizumab (Avastin), sunitinib (Sutent), and sorafenib (Nevaxar)], antiviral compound azidothymidine (AZT, Zidovudine) and several oral antidiabetics [e.g., rosiglitazone (Avandia)]. Illicit drugs such as alcohol, cocaine, methamphetamine, ecstasy, and synthetic cannabinoids (spice, K2) may also induce mitochondria-related cardiotoxicity. Mitochondrial toxicity develops due to various mechanisms involving interference with the mitochondrial respiratory chain (e.g., uncoupling) or inhibition of the important mitochondrial enzymes (oxidative phosphorylation, Szent-Györgyi-Krebs cycle, mitochondrial DNA replication, ADP/ATP translocator). The final phase of mitochondrial dysfunction induces loss of mitochondrial membrane potential and an increase in mitochondrial oxidative/nitrative stress, eventually culminating into cell death. This review aims to discuss the mechanisms of mitochondrion-mediated cardiotoxicity of commonly used drugs and some potential cardioprotective strategies to prevent these toxicities.

Multigenerational effects of the anticancer drug tamoxifen and its metabolite 4-hydroxy-tamoxifen on Daphnia pulex.

Tamoxifen and its metabolite 4-hydroxy-tamoxifen (4OHTam) are two potent molecules that have anticancer properties on breast cancers. Their medical use is expected to increase with the increasing global cancer rate. After consumption, patients excrete tamoxifen and the 4OHTam metabolite into wastewaters, and tamoxifen has been already detected in wastewaters and natural waters. The concentrations of 4OHTam in waters have never been reported. A single study reported 4OHTam effects on the microcrustacean Daphnia pulex. The effects of tamoxifen and 4OHTam over more than two generations are unknown in aquatic invertebrates. The main goal of this study was to assess the long-term sensitivity of the microcrustacean D. pulex over four generations, based on size, reproduction, viability and the intrinsic rate of natural increase (r). Additional experiments were carried out to observe whether the effects of tamoxifen and 4OHTam were reversible in the next generation after descendants were withdrawn from chemical stress (i.e., recovery experiment), and whether the lowest test concentration of each chemical induced toxic effects when both concentrations were combined (i.e., mixture experiments). Our results showed that tamoxifen and 4OHTam induced the adverse effects at environmentally relevant concentrations. Tamoxifen and 4OHTam impaired size, viability, reproduction and the r in four generations of treated D. pulex, but these effects were not clearly magnified over generations. Tamoxifen was more potent than 4OHTam on D. pulex. When used in a mixture, the combination of tamoxifen and 4OHTam induced effects in offspring, whereas no effects were observed when these chemicals were tested individually. In the recovery experiment, the reproduction and size were reduced in offspring withdrawn from chemical exposures. Our results suggested that tamoxifen and its metabolite may be a relevant pharmaceutical to consider in risk assessment.

Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.

Assessing the added value of wastewater-based epidemiology to monitor illicit drug use

Wastewater-based epidemiology consists in acquiring relevant information about the lifestyle and health status of the population through the analysis of wastewater samples collected at the influent of a wastewater treatment plant. Whilst being a very young discipline, it has experienced an astonishing development since its firs application in 2005. The possibility to gather community-wide information about drug use has been among the major field of application. The wide resonance of the first results sparked the interest of scientists from various disciplines. Since then, research has broadened in innumerable directions. Although being praised as a revolutionary approach, there was a need to critically assess its added value, with regard to the existing indicators used to monitor illicit drug use. The main, and explicit, objective of this research was to evaluate the added value of wastewater-based epidemiology with regards to two particular, although interconnected, dimensions of illicit drug use. The first is related to trying to understand the added value of the discipline from an epidemiological, or societal, perspective. In other terms, to evaluate if and how it completes our current vision about the extent of illicit drug use at the population level, and if it can guide the planning of future prevention measures and drug policies. The second dimension is the criminal one, with a particular focus on the networks which develop around the large demand in illicit drugs. The goal here was to assess if wastewater-based epidemiology, combined to indicators stemming from the epidemiological dimension, could provide additional clues about the structure of drug distribution networks and the size of their market. This research had also an implicit objective, which focused on initiating the path of wastewater- based epidemiology at the Ecole des Sciences Criminelles of the University of Lausanne. This consisted in gathering the necessary knowledge about the collection, preparation, and analysis of wastewater samples and, most importantly, to understand how to interpret the acquired data and produce useful information. In the first phase of this research, it was possible to determine that ammonium loads, measured directly in the wastewater stream, could be used to monitor the dynamics of the population served by the wastewater treatment plant. Furthermore, it was shown that on the long term, the population did not have a substantial impact on consumption patterns measured through wastewater analysis. Focussing on methadone, for which precise prescription data was available, it was possible to show that reliable consumption estimates could be obtained via wastewater analysis. This allowed to validate the selected sampling strategy, which was then used to monitor the consumption of heroin, through the measurement of morphine. The latter, in combination to prescription and sales data, provided estimates of heroin consumption in line with other indicators. These results, combined to epidemiological data, highlighted the good correspondence between measurements and expectations and, furthermore, suggested that the dark figure of heroin users evading harm-reduction programs, which would thus not be measured by conventional indicators, is likely limited. In the third part, which consisted in a collaborative study aiming at extensively investigating geographical differences in drug use, wastewater analysis was shown to be a useful complement to existing indicators. In particular for stigmatised drugs, such as cocaine and heroin, it allowed to decipher the complex picture derived from surveys and crime statistics. Globally, it provided relevant information to better understand the drug market, both from an epidemiological and repressive perspective. The fourth part focused on cannabis and on the potential of combining wastewater and survey data to overcome some of their respective limitations. Using a hierarchical inference model, it was possible to refine current estimates of cannabis prevalence in the metropolitan area of Lausanne. Wastewater results suggested that the actual prevalence is substantially higher compared to existing figures, thus supporting the common belief that surveys tend to underestimate cannabis use. Whilst being affected by several biases, the information collected through surveys allowed to overcome some of the limitations linked to the analysis of cannabis markers in wastewater (i.e., stability and limited excretion data). These findings highlighted the importance and utility of combining wastewater-based epidemiology to existing indicators about drug use. Similarly, the fifth part of the research was centred on assessing the potential uses of wastewater-based epidemiology from a law enforcement perspective. Through three concrete examples, it was shown that results from wastewater analysis can be used to produce highly relevant intelligence, allowing drug enforcement to assess the structure and operations of drug distribution networks and, ultimately, guide their decisions at the tactical and/or operational level. Finally, the potential to implement wastewater-based epidemiology to monitor the use of harmful, prohibited and counterfeit pharmaceuticals was illustrated through the analysis of sibutramine, and its urinary metabolite, in wastewater samples. The results of this research have highlighted that wastewater-based epidemiology is a useful and powerful approach with numerous scopes. Faced with the complexity of measuring a hidden phenomenon like illicit drug use, it is a major addition to the panoply of existing indicators. -- L'épidémiologie basée sur l'analyse des eaux usées (ou, selon sa définition anglaise, « wastewater-based epidemiology ») consiste en l'acquisition d'informations portant sur le mode de vie et l'état de santé d'une population via l'analyse d'échantillons d'eaux usées récoltés à l'entrée des stations d'épuration. Bien qu'il s'agisse d'une discipline récente, elle a vécu des développements importants depuis sa première mise en oeuvre en 2005, notamment dans le domaine de l'analyse des résidus de stupéfiants. Suite aux retombées médiatiques des premiers résultats de ces analyses de métabolites dans les eaux usées, de nombreux scientifiques provenant de différentes disciplines ont rejoint les rangs de cette nouvelle discipline en développant plusieurs axes de recherche distincts. Bien que reconnu pour son coté objectif et révolutionnaire, il était nécessaire d'évaluer sa valeur ajoutée en regard des indicateurs couramment utilisés pour mesurer la consommation de stupéfiants. En se focalisant sur deux dimensions spécifiques de la consommation de stupéfiants, l'objectif principal de cette recherche était focalisé sur l'évaluation de la valeur ajoutée de l'épidémiologie basée sur l'analyse des eaux usées. La première dimension abordée était celle épidémiologique ou sociétale. En d'autres termes, il s'agissait de comprendre si et comment l'analyse des eaux usées permettait de compléter la vision actuelle sur la problématique, ainsi que déterminer son utilité dans la planification des mesures préventives et des politiques en matière de stupéfiants actuelles et futures. La seconde dimension abordée était celle criminelle, en particulier, l'étude des réseaux qui se développent autour du trafic de produits stupéfiants. L'objectif était de déterminer si cette nouvelle approche combinée aux indicateurs conventionnels, fournissait de nouveaux indices quant à la structure et l'organisation des réseaux de distribution ainsi que sur les dimensions du marché. Cette recherche avait aussi un objectif implicite, développer et d'évaluer la mise en place de l'épidémiologie basée sur l'analyse des eaux usées. En particulier, il s'agissait d'acquérir les connaissances nécessaires quant à la manière de collecter, traiter et analyser des échantillons d'eaux usées, mais surtout, de comprendre comment interpréter les données afin d'en extraire les informations les plus pertinentes. Dans la première phase de cette recherche, il y pu être mis en évidence que les charges en ammonium, mesurées directement dans les eaux usées permettait de suivre la dynamique des mouvements de la population contributrice aux eaux usées de la station d'épuration de la zone étudiée. De plus, il a pu être démontré que, sur le long terme, les mouvements de la population n'avaient pas d'influence substantielle sur le pattern de consommation mesuré dans les eaux usées. En se focalisant sur la méthadone, une substance pour laquelle des données précises sur le nombre de prescriptions étaient disponibles, il a pu être démontré que des estimations exactes sur la consommation pouvaient être tirées de l'analyse des eaux usées. Ceci a permis de valider la stratégie d'échantillonnage adoptée, qui, par le bais de la morphine, a ensuite été utilisée pour suivre la consommation d'héroïne. Combinée aux données de vente et de prescription, l'analyse de la morphine a permis d'obtenir des estimations sur la consommation d'héroïne en accord avec des indicateurs conventionnels. Ces résultats, combinés aux données épidémiologiques ont permis de montrer une bonne adéquation entre les projections des deux approches et ainsi démontrer que le chiffre noir des consommateurs qui échappent aux mesures de réduction de risque, et qui ne seraient donc pas mesurés par ces indicateurs, est vraisemblablement limité. La troisième partie du travail a été réalisée dans le cadre d'une étude collaborative qui avait pour but d'investiguer la valeur ajoutée de l'analyse des eaux usées à mettre en évidence des différences géographiques dans la consommation de stupéfiants. En particulier pour des substances stigmatisées, telles la cocaïne et l'héroïne, l'approche a permis d'objectiver et de préciser la vision obtenue avec les indicateurs traditionnels du type sondages ou les statistiques policières. Globalement, l'analyse des eaux usées s'est montrée être un outil très utile pour mieux comprendre le marché des stupéfiants, à la fois sous l'angle épidémiologique et répressif. La quatrième partie du travail était focalisée sur la problématique du cannabis ainsi que sur le potentiel de combiner l'analyse des eaux usées aux données de sondage afin de surmonter, en partie, leurs limitations. En utilisant un modèle d'inférence hiérarchique, il a été possible d'affiner les actuelles estimations sur la prévalence de l'utilisation de cannabis dans la zone métropolitaine de la ville de Lausanne. Les résultats ont démontré que celle-ci est plus haute que ce que l'on s'attendait, confirmant ainsi l'hypothèse que les sondages ont tendance à sous-estimer la consommation de cannabis. Bien que biaisés, les données récoltées par les sondages ont permis de surmonter certaines des limitations liées à l'analyse des marqueurs du cannabis dans les eaux usées (i.e., stabilité et manque de données sur l'excrétion). Ces résultats mettent en évidence l'importance et l'utilité de combiner les résultats de l'analyse des eaux usées aux indicateurs existants. De la même façon, la cinquième partie du travail était centrée sur l'apport de l'analyse des eaux usées du point de vue de la police. Au travers de trois exemples, l'utilisation de l'indicateur pour produire du renseignement concernant la structure et les activités des réseaux de distribution de stupéfiants, ainsi que pour guider les choix stratégiques et opérationnels de la police, a été mise en évidence. Dans la dernière partie, la possibilité d'utiliser cette approche pour suivre la consommation de produits pharmaceutiques dangereux, interdits ou contrefaits, a été démontrée par l'analyse dans les eaux usées de la sibutramine et ses métabolites. Les résultats de cette recherche ont mis en évidence que l'épidémiologie par l'analyse des eaux usées est une approche pertinente et puissante, ayant de nombreux domaines d'application. Face à la complexité de mesurer un phénomène caché comme la consommation de stupéfiants, la valeur ajoutée de cette approche a ainsi pu être démontrée.

Smoking, Alcohol, Drug Use, Abuse and Dependence in Narcolepsy and Idiopathic Hypersomnia: A Case-Control Study.

STUDY OBJECTIVES: Basic experiments support the impact of hypocretin on hyperarousal and motivated state required for increasing drug craving. Our aim was to assess the frequencies of smoking, alcohol and drug use, abuse and dependence in narcolepsy type 1 (NT1, hypocretin-deficient), narcolepsy type 2 (NT2), idiopathic hypersomnia (IH) (non-hypocretin-deficient conditions), in comparison to controls. We hypothesized that NT1 patients would be less vulnerable to drug abuse and addiction compared to other hypersomniac patients and controls from general population. METHODS: We performed a cross-sectional study in French reference centres for rare hypersomnia diseases and included 450 adult patients (median age 35 years; 41.3% men) with NT1 (n = 243), NT2 (n = 116), IH (n = 91), and 710 adult controls. All participants were evaluated for alcohol consumption, smoking habits, and substance (alcohol and illicit drug) abuse and dependence diagnosis during the past year using the Mini International Neuropsychiatric Interview. RESULTS: An increased proportion of both tobacco and heavy tobacco smokers was found in NT1 compared to controls and other hypersomniacs, despite adjustments for potential confounders. We reported an increased regular and frequent alcohol drinking habit in NT1 versus controls but not compared to other hypersomniacs in adjusted models. In contrast, heavy drinkers were significantly reduced in NT1 versus controls but not compared to other hypersomniacs. The proportion of patients with excessive drug use (codeine, cocaine, and cannabis), substance dependence, or abuse was low in all subgroups, without significant differences between either hypersomnia disorder categories or compared with controls. CONCLUSIONS: We first described a low frequency of illicit drug use, dependence, or abuse in patients with central hypersomnia, whether Hcrt-deficient or not, and whether drug-free or medicated, in the same range as in controls. Conversely, heavy drinkers were rare in NT1 compared to controls but not to other hypersomniacs, without any change in alcohol dependence or abuse frequency. Although disruption of hypocretin signaling in rodents reduces drug-seeking behaviors, our results do not support that hypocretin deficiency constitutes a protective factor against the development of drug addiction in humans.

An Experimental Model of Prostatic Inflammation for Drug Discovery

There is increasing evidence to support a significant role for chronic non-bacterial, prostatic inflammation in the development of human voiding dysfunction and prostate cancer. Their increased prevalence with age suggests that the decrease of testosterone concentration and/or the ratio of testosterone-to-estradiol in serum may have a role in their development. The main objective of this study was to explore prostatic inflammation and its relationship with voiding dysfunction and prostate carcinogenesis by developing an experimental model. A novel selective estrogen receptor modulator (SERM), fispemifene, was tested for the prevention and treatment of prostatic inflammation in this model. Combined treatment of adult Noble rats with testosterone and estradiol for 3 to 6 weeks induced gradually developing prostatic inflammation in the dorsolateral prostatic lobes. Inflammatory cells, mainly T-lymphocytes, were first seen around capillaries. Thereafter, the lymphocytes migrated into the stroma and into periglandular space. When the treatment time was extended to 13 weeks, the number of inflamed acini increased. Urodynamical recordings indicated voiding dysfunction. When the animals had an above normal testosterone and estradiol concentrations but still had a decreased testosterone-to-estradiol ratio in serum, they developed obstructive voiding. Furthermore, they developed precancerous lesions and prostate cancers in the ducts of the dorsolateral prostatic lobes. Interestingly, inflammatory infiltrates were observed adjacent to precancerous lesions but not in the adjacency of adenocarcinomas suggesting that inflammation has a role in the early stages of prostate carcinogenesis. Fispemifene, a novel SERM tested in this experimental model, showed anti-inflammatory action by attenuating the number of inflamed acini in the dorsolateral prostate. Fispemifene exhibited also antiestrogenic properties by decreasing expression of estrogen-induced biomarkers in the acinar epithelium. These findings suggest that SERMs could be considered as a new therapeutic possibility in the prevention and in the treatment of chronic prostatic inflammation

Drug loading of mesoporous silicon particles

Porous silicon (PSi) is a promising material to be utilized in drug delivery formulations. The release rate of the drug compound can be controlled by changing the pore properties and surface chemistry of PSi. The loading of a poorly soluble drug into mesoporous silicon particles enhances its dissolution in the body. The drug loading is based on adsorption. The attainable maximum loaded amount depends on the properties of the drug compound and the PSi material, and on the process conditions. The loading solvent also essentially affects the adsorption process. The loading of indomethacin into PSi particles with varying surface modification was studied. Solvent mixtures were applied in the loading, and the loaded samples were analyzed with thermal analysis methods. The best degree of loading was obtained using a mixture of dichloromethane and methanol. The drug loads varied from 7.7 w-% to 26.8 w-%. A disturbing factor in the loading experiments was the tendency of indomethacin to form solvates with the solvents applied. In addition, the physical form and stability of indomethacin loaded in PSi and silica particles were studied using Raman spectroscopy. In the case of silica, the presence of crystalline drug as well as the polymorph form can be detected, but the method proved to be not applicable for PSi particles.