RESUMEN
Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.
Asunto(s)
Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Colinérgicos/farmacología , Inhibidores de la Colinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Modelos Animales de Enfermedad , Diseño de Fármacos , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , FosforilaciónRESUMEN
DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.
Asunto(s)
Enfermedades del Sistema Nervioso/metabolismo , Trastornos Psicóticos/metabolismo , Receptores de Dopamina D1/metabolismo , Animales , Antipsicóticos/uso terapéutico , Parpadeo/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Dopaminérgicos/uso terapéutico , Isoquinolinas/uso terapéutico , Levodopa/uso terapéutico , Macaca mulatta , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Trastornos Psicóticos/tratamiento farmacológico , Receptores de Dopamina D1/genética , Reserpina/uso terapéutico , Sueño/efectos de los fármacos , Vigilia/efectos de los fármacosRESUMEN
BACKGROUND: Human cerebrospinal fluid (CSF) is often acquired in Phase I clinical trials to assess the CNS penetration of new pharmacological agents and to search for biomarkers associated with PD effects. Robust methods for neurotransmitter metabolites in CSF have proven elusive, in part due to inadequate reversed phase LC retention. RESULTS: Benzoyl chloride derivatization was used to promote retention for LC-MS/MS for a panel of neurotransmitter metabolites while delivering a concise method for sample preparation. CONCLUSION: A validated assay in human CSF was obtained for 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3,4-dihydroxyphenylglycol and 5-hydroxyindoleacetic acid. This method is differentiated from other LC-MS/MS methods by delivering results in line with full regulatory expectations.
Asunto(s)
Benzoatos/química , Pruebas de Química Clínica/métodos , Neurotransmisores/líquido cefalorraquídeo , Espectrometría de Masas en Tándem , Ácido 3,4-Dihidroxifenilacético/líquido cefalorraquídeo , Ácido 3,4-Dihidroxifenilacético/normas , Animales , Cromatografía Líquida de Alta Presión/normas , Semivida , Ácido Homovanílico/líquido cefalorraquídeo , Ácido Homovanílico/normas , Humanos , Ácido Hidroxiindolacético/líquido cefalorraquídeo , Ácido Hidroxiindolacético/normas , Masculino , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/líquido cefalorraquídeo , Metoxihidroxifenilglicol/normas , Neurotransmisores/química , Neurotransmisores/metabolismo , Control de Calidad , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem/normasRESUMEN
BACKGROUND: (18)F-Fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging of atherosclerosis in the clinic is based on preferential accumulation of radioactive glucose analog in atherosclerotic plaques. FDG-PET is challenging in mouse models due to limited resolution and high cost. We aimed to quantify accumulation of nonradioactive glucose metabolite, FDG-6-phosphate, in the mouse atherosclerotic plaques as a simple alternative to PET imaging. METHODOLOGY/PRINCIPAL FINDINGS: Nonradioactive FDG was injected 30 minutes before euthanasia. Arteries were dissected, and lipids were extracted. The arteries were re-extracted with 50% acetonitrile-50% methanol-0.1% formic acid. A daughter ion of FDG-6-phosphate was quantified using liquid chromatography and mass spectrometry (LC/MS/MS). Thus, both traditional (cholesterol) and novel (FDG-6-phosphate) markers were assayed in the same tissue. FDG-6-phosphate was accumulated in atherosclerotic lesions associated with carotid ligation of the Western diet fed ApoE knockout mice (5.9 times increase compare to unligated carotids, p<0.001). Treatment with the liver X receptor agonist T0901317 significantly (2.1 times, p<0.01) reduced FDG-6-phosphate accumulation 2 weeks after surgery. Anti-atherosclerotic effects were independently confirmed by reduction in lesion size, macrophage number, cholesterol ester accumulation, and macrophage proteolytic activity. CONCLUSIONS/SIGNIFICANCE: Mass spectrometry of FDG-6-phosphate in experimental atherosclerosis is consistent with plaque inflammation and provides potential translational link to the clinical studies utilizing FDG-PET imaging.
Asunto(s)
Arterias/metabolismo , Química Farmacéutica/métodos , Glucosa-6-Fosfato/análogos & derivados , Glucosa/metabolismo , Placa Aterosclerótica/metabolismo , Animales , Apolipoproteínas E/genética , Aterosclerosis/terapia , Arterias Carótidas/metabolismo , Línea Celular , Colesterol/metabolismo , Cromatografía Liquida/métodos , Diagnóstico por Imagen/métodos , Modelos Animales de Enfermedad , Diseño de Fármacos , Glucosa/análogos & derivados , Glucosa-6-Fosfato/metabolismo , Humanos , Hidrocarburos Fluorados/farmacología , Iones , Receptores X del Hígado , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Tomografía de Emisión de Positrones/métodos , Sulfonamidas/farmacología , Factores de TiempoRESUMEN
We have reported that [methyl- (11)C] (3 R,5 R)-5-(3-methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one ([(11)C] 8, [(11)C]MePPEP) binds with high selectivity to cannabinoid type-1 (CB 1) receptors in monkey brain in vivo. We now describe the synthesis of 8 and four analogues, namely, the 4-fluorophenyl (16, FMePPEP), 3-fluoromethoxy (20, FMPEP), 3-fluoromethoxy- d 2 (21, FMPEP- d 2), and 3-fluoroethoxy analogues (22, FEPEP), and report their activity in an ex vivo model designed to identify compounds suitable for use as positron emission tomography (PET) ligands. These ligands exhibited high, selective potency at CB 1 receptors in vitro (K b < 1 nM). Each ligand (30 microg/kg, iv) was injected into rats under baseline and pretreatment conditions (3, rimonabant, 10 mg/kg, iv) and quantified at later times in frontal cortex ex vivo with liquid chromatography-mass spectrometry (LC-MS) detection. Maximal ligand uptakes were high (22.6-48.0 ng/g). Under pretreatment, maximal brain uptakes were greatly reduced (6.5-17.3 ng/g). Since each ligand readily entered brain and bound with high selectivity to CB 1 receptors, we then established and here describe methods for producing [(11)C] 8, [(11)C] 16, and [(18)F] 20- 22 in adequate activities for evaluation as candidate PET radioligands in vivo.
Asunto(s)
Pirrolidinonas/síntesis química , Pirrolidinonas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Cromatografía Liquida , Ligandos , Espectroscopía de Resonancia Magnética , Tomografía de Emisión de Positrones , Pirrolidinonas/farmacología , Ratas , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
[11C]MePPEP is an inverse agonist and a radioligand developed to image cannabinoid CB1 receptors with positron emission tomography (PET). It provides reversible, high specific signal in monkey brain. We assessed [11C]MePPEP in rodent brain with regard to receptor selectivity, susceptibility to transport by P-glycoprotein (P-gp), sensitivity to displacement by agonists, and accumulation of radiometabolites. We used CB1 receptor knockout mice and P-gp knockout mice to assess receptor selectivity and sensitivity to efflux transport, respectively. Using serial measurements of PET brain activity and plasma concentrations of [11C]MePPEP, we estimated CB1 receptor density in rat brain as distribution volume. CB1 knockout mice showed only nonspecific brain uptake, and [11C]MePPEP was not a substrate for P-gp. Direct acting agonists anandamide (10 mg/kg), methanandamide (10 mg/kg), CP 55,940 (1 mg/kg), and indirect agonist URB597 (0.3 and 0.6 mg/kg) failed to displace [11C]MePPEP, while the inverse agonist rimonabant (3 and 10 mg/kg) displaced >65% of [11C]MePPEP. Radiometabolites represented ~13% of total radioactivity in brain between 30 and 120 min. [11C]MePPEP was selective for the CB1 receptor, was not a substrate for P-gp, and was more potently displaced by inverse agonists than agonists. The low potency of agonists suggests either a large receptor reserve or non-overlapping binding sites for agonists and inverse agonists. Radiometabolites of [11C]MePPEP in brain caused distribution volume to be overestimated by approximately 13%.
Asunto(s)
Química Encefálica , Tomografía de Emisión de Positrones , Pirrolidinonas/farmacocinética , Radiofármacos/farmacocinética , Receptor Cannabinoide CB1/análisis , Animales , Ácidos Araquidónicos/farmacología , Unión Competitiva , Moduladores de Receptores de Cannabinoides/farmacología , Radioisótopos de Carbono/farmacocinética , Endocannabinoides , Masculino , Espectrometría de Masas , Ratones , Alcamidas Poliinsaturadas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The cannabinoid CB(1) receptor is one of the most abundant G protein-coupled receptors in the brain and is a promising target of therapeutic drug development. Success of drug development for neuropsychiatric indications is significantly enhanced with the ability to directly measure spatial and temporal binding of compounds to receptors in central compartments. We assessed the utility of a new positron emission tomography (PET) radioligand to image CB(1) receptors in monkey brain. [(11)C]MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) has high CB(1) affinity (K(b)=0.574+/-0.207 nM) but also moderately high lipophilicity (measured LogD(7.4)=4.8). After intravenous injection of [(11)C]MePPEP, brain activity reached high levels of almost 600% standardized uptake value (SUV) within 10-20 min. The regional uptake was consistent with the distribution of CB(1) receptors, with high radioactivity in striatum and cerebellum and low in thalamus and pons. Injection of pharmacological doses of CB(1)-selective agents confirmed that the tracer doses of [(11)C]MePPEP reversibly labeled CB(1) receptors. Preblockade or displacement with two CB(1) selective agents (ISPB; (4-(3-cyclopentyl-indole-1-sulfonyl)-N-(tetrahydro-pyran-4-ylmethyl)-benzamide) and rimonabant) showed that the majority (>89%) of brain uptake in regions with high receptor densities was specific and reversibly bound to CB(1) receptors in the high binding regions. [(11)C]MePPEP was rapidly removed from arterial plasma. Regional brain uptake could be quantified as distribution volume relative to the concentration of parent radiotracer in plasma. The P-glycoprotein (P-gp) inhibitor DCPQ ((R)-4-[(1a,6,10b)-1,1-dichloro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol) did not significantly increase brain uptake of [(11)C]MePPEP, suggesting it is not a substrate for this efflux transporter at the blood-brain barrier. [(11)C]MePPEP is a radioligand with high brain uptake, high specific signal to CB(1) receptors, and adequately fast washout from brain that allows quantification with (11)C (half-life=20 min). These promising results in monkey justify studying this radioligand in human subjects.
Asunto(s)
Encéfalo/diagnóstico por imagen , Pirrolidinonas/farmacocinética , Receptor Cannabinoide CB1/fisiología , Animales , Transporte Biológico , Encéfalo/metabolismo , Radioisótopos de Carbono , Procesamiento de Imagen Asistido por Computador , Cinética , Análisis de los Mínimos Cuadrados , Macaca mulatta , Masculino , Tomografía de Emisión de Positrones , Pirrolidinonas/sangre , Radiografía , Ensayo de Unión RadioliganteRESUMEN
Preclinical brain receptor occupancy measures have heretofore been conducted by quantifying the brain distribution of a radiolabeled tracer ligand using either scintillation spectroscopy or tomographic imaging. For smaller animals like rodents, the majority of studies employ tissue dissection and scintillation spectroscopy. These measurements can also be accomplished using liquid chromatography coupled to mass spectral detection to measure the brain distribution of tracer molecules, obviating the need for radioligands. In order to validate mass spectroscopy-based receptor occupancy methods, we examined dopamine D2 receptor dose-occupancy curves for a number of antipsychotic drugs in parallel experiments using either mass spectroscopy or radioligand-based approaches. Oral dose-occupancy curves were generated for 8 antipsychotic compounds in parallel experiments using either radiolabeled or unlabeled raclopride tracer. When curves generated by these two methods were compared and ED(50) values determined, remarkably similar data were obtained. Occupancy ED(50) values were (mg/kg): chlorpromazine, 5.1 and 2.7; clozapine, 41 and 40; haloperidol, 0.2 and 0.3; olanzapine, 2.1 and 2.2; risperidone, 0.1 and 0.4; spiperone, 0.5 and 0.4; thioridazine 9.2 and 9.5; and ziprasidone 1.4 and 2.1 (unlabeled and radiolabeled raclopride tracer, respectively). The observation that in vivo application of both techniques led to comparable data adds to the validation state of the mass spectroscopy-based approach to receptor occupancy assays.
Asunto(s)
Antipsicóticos/metabolismo , Antagonistas de Dopamina , Racloprida , Receptores de Dopamina D2/metabolismo , Animales , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Antagonistas de Dopamina/farmacocinética , Relación Dosis-Respuesta a Droga , Masculino , Espectrometría de Masas , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Racloprida/farmacocinética , Radiofármacos , Ratas , Ratas Sprague-DawleyRESUMEN
RATIONALE: Cannabinoid type 1 (CB(1)) receptor antagonists are reportedly effective in reducing food intake both preclinically and clinically. This may be due in part to their effects on monoamine release in the brain. The level of central CB(1) receptor occupancy underlying these neurobiological effects is unclear. OBJECTIVES: We explored the relationship between in vivo CB(1) receptor occupancy in the frontal cortex and changes in both monoamine release in the medial prefrontal cortex (mPFC) and feeding behavior in rats in response to two orally administered CB(1) receptor antagonists presently in clinical trials, SR141716A (rimonabant) and SLV319. METHODS: CB(1) receptor occupancy was measured using [(3)H] SR141716A, and these occupancies were related to potencies to mediate increases in dopamine (DA) and norepinephrine (NE) release measured with microdialysis and decreases in consumption of a highly palatable diet (HP). RESULTS: High receptor occupancy levels (>65%) were required to detect increases in monoamine release that were achieved with 3 and 10 mg/kg of SR141716A and 10 mg/kg of SLV319 for DA and 10 mg/kg of SR141716A for NE. Decreases in HP consumption were seen at occupancies higher than 65% for SR141716A that were achieved with 3 and 10 mg/kg. In contrast, decreases in HP consumption were seen at relatively low CB(1) receptor occupancies (11%) for SLV319. CONCLUSIONS: The occupancy method described here is an effective tool for interrelating central CB(1) receptor occupancy with neurobiological actions of CB(1) receptor antagonists and for furthering our understanding of the role of CB(1) receptors in central nervous system physiology and pathology.
Asunto(s)
Dopamina/metabolismo , Conducta Alimentaria/efectos de los fármacos , Lóbulo Frontal/efectos de los fármacos , Norepinefrina/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Lóbulo Frontal/metabolismo , Masculino , Microdiálisis , Piperidinas/farmacología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Pirazoles/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/metabolismo , Rimonabant , Sulfonamidas/farmacologíaRESUMEN
High performance liquid chromatography combined with either single quad or triple quad mass spectral detectors (LC/MS) was used to measure the brain distribution of receptor occupancy tracers targeting dopamine D2, serotonin 5-HT2A and neurokinin NK-1 receptors using the ligands raclopride, MDL-100907 and GR205171, respectively. All three non-radiolabeled tracer molecules were easily detectable in discrete rat brain areas after intravenous doses of 3, 3 and 30 microg/kg, respectively. These levels showed a differential brain distribution caused by differences in receptor density, as demonstrated by the observation that pretreatment with compounds that occupy these receptors reduced this differential distribution in a dose-dependent manner. Intravenous, subcutaneous and oral dose-occupancy curves were generated for haloperidol at the dopamine D2 receptor as were oral curves for the antipsychotic drugs olanzapine and clozapine. In vivo dose-occupancy curves were also generated for orally administered clozapine, olanzapine and haloperidol at the cortical 5-HT2A binding site. In vivo occupancy at the striatal neurokinin NK-1 binding site by various doses of orally administered MK-869 was also measured. Our results demonstrate the utility of LC/MS to quantify tracer distribution in preclinical brain receptor occupancy studies.
Asunto(s)
Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Neuroquinina-1/metabolismo , Animales , Antipsicóticos/farmacología , Aprepitant , Benzodiazepinas/farmacología , Clozapina/farmacología , Antagonistas de Dopamina/farmacocinética , Antagonistas de los Receptores de Dopamina D2 , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Evaluación Preclínica de Medicamentos , Fluorobencenos/farmacocinética , Gerbillinae , Haloperidol/farmacología , Masculino , Morfolinas/farmacología , Antagonistas del Receptor de Neuroquinina-1 , Olanzapina , Piperidinas/farmacocinética , Racloprida/farmacocinética , Ratas , Antagonistas del Receptor de Serotonina 5-HT2 , Antagonistas de la Serotonina/farmacocinética , Tetrazoles/farmacocinéticaRESUMEN
RATIONALE: The selective serotonin uptake inhibitor (SSRI) fluoxetine has been shown to not only increase the extracellular concentrations of serotonin, but also dopamine and norepinephrine extracellular concentrations in rat prefrontal cortex. The effect of other SSRIs on monoamine concentrations in prefrontal cortex has not been thoroughly studied. OBJECTIVE: The aim of this study was to compare the ability of five systemically administered selective serotonin uptake inhibitors to increase acutely the extracellular concentrations of serotonin, norepinephrine and dopamine in rat prefrontal cortex. METHODS: The extracellular concentrations of monoamines were determined in the prefrontal cortex of conscious rats using the microdialysis technique. RESULTS: Fluoxetine, citalopram, fluvoxamine, paroxetine and sertraline similarly increased the extracellular concentrations of serotonin from 2- to 4-fold above baseline. However, only fluoxetine produced robust and sustained increases in extracellular concentrations of norepinephrine and dopamine after acute systemic administration. Fluoxetine at the same dose blocked ex vivo binding to the serotonin transporter, but not the norepinephrine transporter, suggesting that the increase of catecholamines was not due to non-selective blockade of norepinephrine uptake. Prefrontal cortex extracellular concentrations of fluoxetine at the dose that increased extracellular monoamines were 242 nM, a concentration sufficient to block 5-HT(2C) receptors which is a potential mechanism for the fluoxetine-induced increase in catecholamines. CONCLUSION: Amongst the SSRIs examined, only fluoxetine acutely increases extracellular concentrations of norepinephrine and dopamine as well as serotonin in prefrontal cortex, suggesting that fluoxetine is an atypical SSRI.