RESUMEN
It has recently been demonstrated that pharmacological blockade of the glycine transporter 1 (GlyT1) reduced alcohol intake and relapse in rats. The aim of the present study was to further explore the role of GlyT1 in alcohol relapse-like behavior. For this purpose we used three different GlyT1 blockers-SSR504734, A-1246399, and RO4993850-and tested their effect on alcohol-seeking and relapse-like consumption. Two behavioral models, the alcohol deprivation effect model and the cue-induced reinstatement model, were used. Our data show that all three GlyT1 blockers reduce relapse-like alcohol consumption and cause either minimal or no side effects, measured as changes in home-cage activity, water intake, and body weight. In the reinstatement test, GlyT1 blockers completely abolished alcohol-seeking responses. Furthermore, we tested other drug/cue associations and found that cocaine-seeking responses were also abolished by GlyT1 blockade. Our data confirm that GlyT1 can be used as a target to develop novel anticraving and antirelapse drugs.
Asunto(s)
Consumo de Bebidas Alcohólicas/tratamiento farmacológico , Consumo de Bebidas Alcohólicas/metabolismo , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Etanol/farmacología , Glicina/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Cocaína/farmacología , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Masculino , Ratas , Ratas Wistar , RecurrenciaRESUMEN
Development of disease-modifying therapies requires an innovative approach to drug development where novel drugs are designed to target mechanisms of interest rather than to produce preclinical effects similar to those of currently used antipsychotics. Application of such novel strategy will undoubtedly require a very deep understanding of the disease biology that is just starting to emerge. Alternatively, one may let environmental experiences of the diseased individual guide the repair process and use drugs only to facilitate the effects of experience. Such an approach would bring together functional experience that is age-, environment- and disease-dependent with the plasticity resources that may otherwise not be available. There are currently no preclinical drug-environment interaction models that can be claimed to have significant degrees of validity. Therefore, from a drug development perspective, principles that combine acute symptomatic and disease-modifying properties are clearly preferred. The question arises then how such treatments can be differentiated from those that have only symptomatic effects (i.e., most currently used antipsychotic medications). One expectation is that the former will show superior and broader efficacy (especially with longer treatment duration). Another possibility is that disease-modifying drugs will be particularly useful at the very earliest stages of the disease. Society and medical communities may not be ready yet to initiate the treatment as early as during the prodromal phase, but the situation may change by the time the science advances enough to bring a convincing case of a drug with disease-modification potential.
Asunto(s)
Antipsicóticos/uso terapéutico , Esquizofrenia/tratamiento farmacológico , Animales , Epigénesis Genética , Humanos , Neurogénesis , Plasticidad Neuronal , Esquizofrenia/fisiopatología , Sinapsis/fisiologíaRESUMEN
Dopamine D2 receptor blockade has been an obligate mechanism of action present in all medications that effectively treat positive symptoms of schizophrenia (e.g., delusions and hallucinations) and have been approved by regulatory agencies since the 1950s. Blockade of 5-hydroxytryptamine(2A) receptors plays a contributory role in the actions of the second generation of antipsychotic drugs, the so-called atypical antipsychotics. Nevertheless, substantial unmet medical needs remain for the treatment of negative symptoms and cognitive dysfunction. Recognition that dissociative anesthetics block the N-methyl-D-aspartate (NMDA) receptor channel has inspired a search for glutamatergic therapeutic mechanisms because ketamine and phencyclidine are known to induce psychotic-like symptoms in healthy volunteers and exacerbate the symptoms of patients with schizophrenia. Current pathophysiological theories of schizophrenia emphasize that hypofunction of NMDA receptors at critical sites in local circuits modulate the function of a given brain region or control projections from one region to another (e.g., hippocampal-cortical or thalamocortical projections). The demonstration that a metabotropic glutamate 2/3 (mGlu2/3) receptor agonist prodrug decreased both positive and negative symptoms of schizophrenia raised hopes that glutamatergic mechanisms may provide therapeutic advantages. In addition to discussing the activation of mGlu2 receptors with mGlu2/3 receptor agonists or mGlu2 receptor positive allosteric modulators (PAMs), we discuss other methods that may potentially modulate circuits with hypofunctional NMDA receptors such as glycine transporter inhibitors and mGlu5 receptor PAMs. The hope is that by modulating glutamatergic neurotransmission, the dysfunctional circuitry of the schizophrenic brain (both local circuits and long-loop pathways) will be improved.
Asunto(s)
Lóbulo Frontal/fisiología , Red Nerviosa/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatología , Animales , Lóbulo Frontal/efectos de los fármacos , Humanos , Interneuronas/metabolismo , Interneuronas/patología , Red Nerviosa/efectos de los fármacos , Receptores AMPA/agonistas , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Esquizofrenia/tratamiento farmacológico , Potenciales Sinápticos/fisiologíaRESUMEN
In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Because glycine is a coagonist of the N-methyl-D-aspartate (NMDA) receptor (NMDAR), which has been implicated in schizophrenia, inhibition of GlyT1 is thought to provide an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N-methyl-glycine (sarcosine) derivatives (e.g., (R)-N[3-(4'fluorophenyl)-3-(4'phenyl-phenoxy)propyl]-sarcosine [NFPS], (R)-N[3-phenyl-3-(4'-(4-toluoyl)phenoxy)-propyl]sarcosine [(R)-NPTS], and (R,S)-(+/-)N-methyl-N-[(4-trifluoromethyl)phenoxy]-3-phenyl-propylglycine [Org24589]), and non-sarcosine-containing inhibitors, such as 2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride (SSR504734), have been described. In the present study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus laevis oocytes, and with two binding assays, using [(3)H](R)-NPTS or 2-chloro-N-[(S)-phenyl[(2S)-N-methylpiperidin-2-yl]-methyl]-3-trifluoromethyl benzamide monohydrochloride ([(3)H]N-methyl-SSR504734) as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently irreversible and independent of glycine concentration. The latter indicates a noncompetitive mode of action. In contrast, both SSR504734 and N-methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the binding of the novel radioligand [(3)H]N-methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734 but noncompetitively by sarcosine-based compounds. Inversely, [(3)H](R)-NPTS binding was competitively inhibited by sarcosine-based compounds, whereas glycine, SSR504734, and N-methyl-SSR504734 noncompetitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a noncompetitive or competitive mode of inhibition. The divergent modes of inhibition may significantly affect the efficacy and tolerability of these drugs.
Asunto(s)
Benzamidas/farmacología , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Glicina/metabolismo , Piperidinas/farmacología , Sarcosina/análogos & derivados , Sarcosina/farmacología , Animales , Astrocitos/metabolismo , Benzamidas/química , Sitios de Unión , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Línea Celular , Cricetinae , Cricetulus , Femenino , Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Piperidinas/química , Ensayo de Unión Radioligante , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Sarcosina/química , Relación Estructura-Actividad , Xenopus laevisRESUMEN
The glycine transporter 1 (GlyT1) has emerged as a key novel target for the treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of aminotetralines and aminochromanes as novel classes of competitive GlyT1 inhibitors. Starting from a high-throughput screening hit, structure-activity relationship studies led first to the discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable pharmacokinetic properties. Systematic investigations of various parameters (e.g., topological polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy evaluation resulted in lead compounds possessing favorable brain penetration properties as for (7 S,8 R)-27a. Further optimization revealed compounds with reduced efflux liabilities as for aminochromane 51b. In an in vivo efficacy model (7 S,8 R)-27a, dose-dependently reversed L-687,414 induced hyperlocomotion in mice with an ED50 of 0.8 mg/kg. All these results suggest (7 S,8 R)-27a and 51b as new GlyT1 inhibitors worthy of further profiling.
Asunto(s)
Encéfalo/efectos de los fármacos , Cromanos/química , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Tetrahidronaftalenos/química , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Unión Competitiva , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Masculino , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Pirrolidinonas/efectos adversos , Ratas Sprague-Dawley , Relación Estructura-Actividad , XenopusRESUMEN
The development of glycine transporter 1 (GlyT1) inhibitors may offer putative treatments for schizophrenia and other disorders associated with hypofunction of the glutaminergic N-methyl-d-aspartate (NMDA) receptor. Herein, we describe the synthesis and biological evaluation of a series of 3,4-disubstituted pyrrolidine sulfonamides as competitive GlyT1 inhibitors that arose from de novo scaffold design. Relationship of chemical structure to drug-drug interaction (DDI) and bioactivation was mechanistically investigated. Murine studies were strategically incorporated into the screening funnel to provide early assessments of in vivo target occupancy (TO) by ex vivo binding studies. Advanced compounds derived from iterative structure-activity relationship (SAR) studies possessed high potency in ex vivo binding studies and good brain penetration, promising preliminary in vivo efficacy, acceptable preclinical pharmacokinetics, and manageable DDI and bioactivation liabilities.
Asunto(s)
Encéfalo/efectos de los fármacos , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Pirrolidinas/química , Sulfonamidas/química , Animales , Encéfalo/metabolismo , Técnicas de Química Sintética , Perros , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones Endogámicos , Microsomas Hepáticos/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Pirrolidinonas/efectos adversos , Ratas Sprague-Dawley , Relación Estructura-Actividad , XenopusRESUMEN
Amphetamine and methylphenidate are known to have stimulatory effect in healthy subjects but not in humans with attention deficit hyperactivity disorder and in rodents with impaired dopamine transporter (DAT) function. This phenomenon is called the paradoxical calming effect of psychostimulants. It has been previously demonstrated that psychostimulants may regulate the enzymatic activity of tyrosine hydroxylase (TH). Hence, the objective of the present study was to determine whether the lack of activity-stimulating effects of amphetamine in hyperactive rats is associated with changes in TH activity. To model hyperactivity in rats, acute administration of DAT inhibitor GBR12909 was used. Changes in TH activity, assessed as L-DOPA accumulation and TH phosphorylation levels, were measured in amphetamine treated rats with or without pretreatment with GBR12909. Our results showed that amphetamine treatment alone increased locomotor activity in rats, whereas pretreatment of rats with GBR12909 counteracted this effect, a finding consistent with the paradoxical calming effect. GBR12909, while having no effect on its own, blocked amphetamine-induced elevation of TH activity in dorsal striatum and nucleus accumbens, measured as increased tissue L-DOPA concentration. However, the phosphorylation levels of TH were not affected by treatment with amphetamine, GBR12909 or the combination of both. Our findings indicate that other mechanisms than phosphorylation-regulated TH activity changes are responsible for the paradoxical calming effect of amphetamine under conditions of impaired DAT activity.
Asunto(s)
Anfetamina/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Inhibidores de Captación de Dopamina/farmacología , Piperazinas/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Levodopa/metabolismo , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Ratas Sprague-DawleyRESUMEN
The research domain criteria (RDoC) matrix has been developed to reorient psychiatric research towards measurable behavioral dimensions and underlying mechanisms. Here, we used a new genetic rat model with a loss-of-function point mutation in the dopamine transporter (DAT) gene (Slc6a3_N157K) to systematically study the RDoC matrix. First, we examined the impact of the Slc6a3_N157K mutation on monoaminergic signaling. We then performed behavioral tests representing each of the five RDoC domains: negative and positive valence systems, cognitive, social and arousal/regulatory systems. The use of RDoC may be particularly helpful for drug development. We studied the effects of a novel pharmacological approach metabotropic glutamate receptor mGluR2/3 antagonism, in DAT mutants in a comparative way with standard medications. Loss of DAT functionality in mutant rats not only elevated subcortical extracellular dopamine concentration but also altered the balance of monoaminergic transmission. DAT mutant rats showed deficits in all five RDoC domains. Thus, mutant rats failed to show conditioned fear responses, were anhedonic, were unable to learn stimulus-reward associations, showed impaired cognition and social behavior, and were hyperactive. Hyperactivity in mutant rats was reduced by amphetamine and atomoxetine, which are well-established medications to reduce hyperactivity in humans. The mGluR2/3 antagonist LY341495 also normalized hyperactivity in DAT mutant rats without affecting extracellular dopamine levels. We systematically characterized an altered dopamine system within the context of the RDoC matrix and studied mGluR2/3 antagonism as a new pharmacological strategy to treat mental disorders with underlying subcortical dopaminergic hyperactivity.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Trastornos Mentales/diagnóstico , Trastornos Mentales/genética , Animales , Conducta Animal , Modelos Animales de Enfermedad , Dopamina , Células HEK293 , Humanos , Mutación con Pérdida de Función , Masculino , Proteínas Mutantes/metabolismo , Fenotipo , Mutación Puntual , Psiquiatría , Ratas Endogámicas F344RESUMEN
In this study, we describe a new rapid and versatile method to determine the BrdU content of DNA in brain tissues dissected from BrdU-treated rats. Different to already existing BrdU ELISAs the method is suitable for the assessment of BrdU incorporation in ex vivo experiments as it is based on the analysis of tissue extracts instead of immobilized cells. The method comprises the preparation of DNA extracts from dissected tissues, the immobilization of BrdU-containing DNA with an anti-BrdU antibody and quantification of the incorporated BrdU by a peroxidase-conjugated anti-BrdU antibody. Validating the new assay in vitro, we found a clear-cut dependency of the ELISA signal from the time SKNSH neuroblastoma cells had been exposed to BrdU. Parallel studies with existing ELISAs and a parallel immunocytochemical determination of BrdU positive cells revealed comparable results. In vivo experiments showed a virtually linear relationship between the BrdU immunoreactivity in the hippocampus and the time rats have been exposed to BrdU. Repeating the determination of the BrdU content of the same set of tissue samples revealed reproducible relative differences of the ELISA signals. This was true for protocols using purified DNA as well as crude DNA extracts. For the sensitivity and reproducibility of the method heat denaturation of the DNA prior to the analysis in the ELISA was crucial. In rats treated with electroconvulsion the BrdU content of the hippocampus, determined by the new ELISA, was increased to 225% of controls. In a parallel immunohistochemical study, the number of BrdU positive cells was comparably increased to 251% of controls. The assay thus provides a rapid method to detect changes of cell proliferation in dissected brain tissues and other proliferative tissues. With appropriate protocols, the assay may also be used to assess the generation of particular cell types like neurons in neurogenic areas.
Asunto(s)
Bromodesoxiuridina/metabolismo , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática/métodos , Pesos y Medidas , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Bromodesoxiuridina/farmacocinética , Recuento de Células , Línea Celular Tumoral , ADN/análisis , ADN/efectos de los fármacos , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Endopeptidasa K/farmacología , Fijadores/farmacología , Formaldehído/farmacología , Calor , Humanos , Neuroblastoma , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Accumulating evidence suggests that cognitive processes may be regulated by glycine concentration in the local environment of glutamate N-methyl-d-aspartate receptor (NMDAR). The concentration of glycine is controlled, among other factors, by the glycine transporter 1 (GlyT1). While GlyT1 inhibitors are developed for a number of indications including cognitive improvement, little is known about their effects in tasks depending on prefrontal cortical function. We examined the effect of GlyT1 inhibitor SSR-504734 on cognitive flexibility assessed in the attentional set-shifting task in rats (ASST). The second goal was to elucidate whether SSR-504734 effect has been due to the compound's action at glycine/NMDAR site. Rats treated with SSR-504734 (3 and 10 mg/kg, IP) required significantly less trials to criteria during extra-dimensional shift (EDs) phase of the ASST. The effect of SSR-504734 (3 mg/kg) was completely prevented by the glycine/NMDAR site antagonist, L-687,414 (30 mg/kg, IP) that by itself exerted no effect on cognitive flexibility. Present study demonstrates that the elevation of glycine concentration through the blockade of its reuptake facilitates cognitive flexibility. As this effect was fully blocked by glycine/NMDAR antagonist, SSR-504734-induced cognitive improvement is likely mediated through glycine action at NMDAR. It is suggested that GlyT1 inhibitors like SSR-504734 may represent a useful pharmacological approach for cognitive enhancement, especially in domains critically affected in schizophrenia.
Asunto(s)
Benzamidas/farmacología , Cognición/efectos de los fármacos , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Piperidinas/farmacología , Receptores de Glicina/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Cognición/fisiología , Aprendizaje Discriminativo/efectos de los fármacos , Aprendizaje Discriminativo/fisiología , Proteínas de Transporte de Glicina en la Membrana Plasmática/fisiología , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Neural progenitor cells reside in the hippocampus of adult rodents and humans and generate granule neurons throughout life. Knowledge about the molecular processes regulating these neurogenic cells is fragmentary. In order to identify genes with a role in the proliferation of adult neural progenitor cells, a protocol was elaborated to enable the staining and isolation of such cells under RNA-preserving conditions with a combination of immunohistochemistry and laser capture microdissection. We increased proliferation of neural progenitor cells by electroconvulsive treatment, one of the most effective antidepressant treatments, and isolated Ki-67-positive cells using this new protocol. RNA amplification via in vitro transcription and subsequent microarray analysis revealed over 100 genes that were differentially expressed in neural progenitor cells due to electroconvulsive treatment compared to untreated control animals. Some of these genes have already been implicated in the functioning of neural progenitor cells or have been induced by electroconvulsive treatment; these include brain-derived neurotrophic factor (Bdnf), PDZ-binding kinase (Pbk) and abnormal spindle-like microcephaly-associated (Aspm). In addition, genes were identified for which no role in the proliferation of neurogenic progenitors has been described so far, such as enhancer of zeste homolog 2 (Ezh2).