RESUMEN
Evidence suggests that 30-50% of patients suffering from major depressive disorder (MDD) are classified as suffering from treatment resistant depression (TRD) as they have an inadequate response to standard antidepressants. A key feature of this patient population is the increased incidence of co-morbid symptoms like anxiety and pain. Recognizing that current standards of care are largely focused on monoaminergic mechanisms of action (MOAs), innovative approaches to drug discovery for TRD are targeting glutamate hyperfunction. Here we describe the in vitro and in vivo profile of GRN-529, a novel negative allosteric modulator (NAM) of metabotropic glutamate receptor 5 (mGluR5). In cell based pharmacology assays, GRN-529 is a high affinity (Ki 5.4 nM), potent (IC50 3.1 nM) and selective (>1000-fold selective vs mGluR1) mGluR5 NAM. Acute administration of GRN-529 (0.1-30 mg/kg p.o.) had dose-dependent efficacy across a therapeutically relevant battery of animal models, comprising depression (decreased immobility time in tail suspension and forced swim tests) and 2 of the co-morbid symptoms overrepresented in TRD, namely anxiety (attenuation of stress-induced hyperthermia, and increased punished crossings in the four plate test) and pain (reversal of hyperalgesia due to sciatic nerve ligation or inflammation). The potential side effect liability of GRN-529 was also assessed using preclinical models: GRN-529 had no effect on rat sexual behavior or motor co-ordination (rotarod), however it impaired cognition in mice (social odor recognition). Efficacy and side effects of GRN-529 were compared to standard of care agents (antidepressant, anxiolytic or analgesics) and the tool mGluR5 NAM, MTEP. To assess the relationship between target occupancy and efficacy, ex vivo receptor occupancy was measured in parallel with efficacy testing. This revealed a strong correlation between target engagement, exposure and efficacy across behavioral endpoints, which supports the potential translational value of PET imaging to dose selection in patients. Collectively this broad spectrum profile of efficacy of GRN-529 supports our hypothesis that negative allosteric modulation of mGluR5 could represent an innovative therapeutic approach to the treatment of TRD. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Trastorno Depresivo Resistente al Tratamiento/tratamiento farmacológico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Regulación Alostérica/fisiología , Analgésicos/farmacología , Animales , Ansiolíticos/farmacología , Antidepresivos/efectos adversos , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Conducta Animal/efectos de los fármacos , Benzamidas/efectos adversos , Benzamidas/farmacología , Benzamidas/uso terapéutico , Calcio/metabolismo , Trastorno Depresivo Resistente al Tratamiento/metabolismo , Trastorno Depresivo Resistente al Tratamiento/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/psicología , Antagonistas de Aminoácidos Excitadores/efectos adversos , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Células HEK293 , Humanos , Ratones , Piridinas/efectos adversos , Piridinas/farmacología , Piridinas/uso terapéutico , Ensayo de Unión Radioligante/métodos , Ratas , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/fisiologíaRESUMEN
The neuropeptide S receptor (NPSR) is a G-protein coupled receptor that is potently activated by the linear 20 amino acid peptide, neuropeptide S (NPS). Central administration of NPS promotes arousal and anxiolytic-like effects in rodents, and fails to promote such effects in NPSR knockout animals or in the presence of NPSR-selective antagonists. In situ hybridization (ISH) studies in rat brain have revealed that the mRNAs encoding the NPS precursor and the NPS receptor are expressed at high levels in discrete regions of the rat CNS. The distribution of the NPSR protein in brain has not been reported due to a lack of available antibodies. We have generated and validated a NPSR-specific antibody and used it to determine the distribution of the NPSR in male Sprague-Dawley (SD) rat brain. The anti-NPSR antibody identified a single protein by Western blot with an estimated molecular weight of 65 kD, which was prevented by pre-incubation of the antibody with the immunizing peptide. The protein distribution identified with this antibody in rat brain was consistent both with the mRNA distribution identified by in situ hybridization, and to the localization pattern identified by a second NPSR-specific antibody against a distinct NPSR epitope. NPSR protein was identified in the medial amygdala (MeA), substantia nigra pars compacta, subiculum, dorsal raphe, and several hypothalamic and thalamic regions. Additionally, NPSR protein was localized in the pyramidal cell layer of the ventral hippocampus, the medial habenula (MHb), and was widely distributed in the cortex. The distribution of NPSR protein provides further insight into the organization of the NPS system and may guide future studies on the role of the NPSR in brain.
Asunto(s)
Sistema Nervioso Central/química , Sistema Nervioso Central/metabolismo , Receptores de Neuropéptido/química , Receptores de Neuropéptido/metabolismo , Secuencia de Aminoácidos , Animales , Inmunohistoquímica , Hibridación in Situ , Masculino , Mesencéfalo/metabolismo , Datos de Secuencia Molecular , Prosencéfalo/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido/genéticaRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons. We studied HCN1-4 channel mRNA and protein expression and correlated these findings with I(h) functional properties in rat DRG neurons of different size. Quantitative RT-PCR (TaqMan) analysis demonstrated that HCN2 and HCN1 mRNAs were more abundantly expressed in large diameter (55-80 microm) neurons, while HCN3 mRNA was preferentially expressed in small diameter (20-30 microm) neurons. HCN4 mRNA expression was very low in neurons of all sizes. At the protein level, subunit-selective polyclonal antibodies and immunofluorescence indicated that HCN1 and HCN3 are present in large diameter neurons and small diameter neurons. Staining in small diameter neurons was in IB4-positive (non-peptidergic) and IB4-negative (peptidergic) cells. HCN2 immunofluorescent staining was heterogeneous and predominantly in large diameter neurons and in small diameter IB4-negative neurons. HCN4 was poorly expressed in all neurons. Functionally, I(h) amplitude and density were significantly larger, and activation kinetics faster, in large diameter neurons when compared with small neurons. I(h) activation rates in small and large diameter DRG neurons were consistent with the relative abundance of HCN subunits in the respective cell type, considering the reported HCN channel activation rates in heterologous systems (HCN1>HCN2 approximately HCN3>HCN4), suggesting exclusivity of roles of different HCN subunits contributing to the excitability of DRG neurons of different size. Additionally, a functional role of I(h) in small DRG neuron excitability was evaluated using a computational model.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Ganglios Espinales/citología , Neuronas/clasificación , Neuronas/fisiología , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Simulación por Computador , Canales Catiónicos Regulados por Nucleótidos Cíclicos/clasificación , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Lectinas/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Modelos Neurológicos , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Epidemiological studies have yet to identify a single cause for the most common late-onset form of Alzheimer's disease. The common respiratory pathogen Chlamydia pneumoniae recently has been implicated as a risk factor for this form of Alzheimer's disease. Were this true, there would be a dramatic shift in current paradigms of Alzheimer's disease research and treatment. In the absence of published confirmation, we obtained postmortem brain tissue from late-onset Alzheimer's disease patients (n = 15) and representative controls (n = 5) and extracted DNA from up to six separate brain regions in each instance, including those areas particularly relevant to Alzheimer's disease neuropathology. Each sample of DNA (n = 101) was assayed five times or more for the presence of C. pneumoniae DNA using a nested-PCR protocol targeting a species-specific gene sequence coding for the major outer membrane protein of this organism. We were unable unequivocally to detect C. pneumoniae in any of the 101 samples tested by PCR and failed to culture the organism from tissue samples. We conclude that C. pneumoniae is neither strongly nor uniquely associated with the neuropathology seen in late-onset Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/microbiología , Encéfalo/microbiología , Chlamydophila pneumoniae/aislamiento & purificación , Anciano , Anciano de 80 o más Años , Autopsia , Chlamydophila pneumoniae/genética , Medios de Cultivo , ADN Bacteriano/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Polymorphisms of three different dopaminergic genes, dopamine D2 receptor (DRD2), dopamine beta-hydroxylase (D beta H), and dopamine transporter (DAT1), were examined in Tourette syndrome (TS) probands, their relatives, and controls. Each gene individually showed a significant correlation with various behavioral variables in these subjects. The additive and substractive effects of the three genes were examined by genotyping all three genes in the same set of subjects. For 9 of 20 TS associated comorbid behaviors there was a significant linear association between the degree of loading for markers of three genes and the mean behavior scores. The behavior variables showing the significant associations were, in order attention deficit hyperactivity disorder (ADHD), stuttering oppositional defiant, tics, conduct, obsessive-compulsive, mania, alcohol abuse and general anxiety-behaviors that constitute the most overt clinical aspects of TS. For 16 of the 20 behavior scores there was a linear progressive decrease in the mean score with progressively lesser loading for the three gene markers. These results suggest that TS, ADHD, stuttering oppositional defiant and conduct disorder, and other behaviors associated with TS, are polygenic, due in part to these three dopaminergic genes, and that the genetics of other polygenic psychiatric disorders may be deciphered using this technique.