RESUMO
Mouse models of Alzheimer's disease (AD) are valuable but do not fully recapitulate human AD pathology, such as spontaneous Tau fibril accumulation and neuronal loss, necessitating the development of new AD models. The transgenic (TG) TgF344-AD rat has been reported to develop age-dependent AD features including neuronal loss and neurofibrillary tangles, despite only expressing APP and PSEN1 mutations, suggesting an improved modelling of AD hallmarks. Alterations in neuronal networks as well as learning performance and cognition tasks have been reported in this model, but none have combined a longitudinal, multimodal approach across multiple centres, which mimics the approaches commonly taken in clinical studies. We therefore aimed to further characterise the progression of AD-like pathology and cognition in the TgF344-AD rat from young-adults (6 months (m)) to mid- (12 m) and advanced-stage (18 m, 25 m) of the disease. Methods: TgF344-AD rats and wild-type (WT) littermates were imaged at 6 m, 12 m and 18 m with [18F]DPA-714 (TSPO, neuroinflammation), [18F]Florbetaben (Aß) and [18F]ASEM (α7-nicotinic acetylcholine receptor) and with magnetic resonance spectroscopy (MRS) and with (S)-[18F]THK5117 (Tau) at 15 and 25 m. Behaviour tests were also performed at 6 m, 12 m and 18 m. Immunohistochemistry (CD11b, GFAP, Aß, NeuN, NeuroChrom) and Tau (S)-[18F]THK5117 autoradiography, immunohistochemistry and Western blot were also performed. Results: [18F]DPA-714 positron emission tomography (PET) showed an increase in neuroinflammation in TG vs wildtype animals from 12 m in the hippocampus (+11%), and at the advanced-stage AD in the hippocampus (+12%), the thalamus (+11%) and frontal cortex (+14%). This finding coincided with strong increases in brain microgliosis (CD11b) and astrogliosis (GFAP) at these time-points as assessed by immunohistochemistry. In vivo [18F]ASEM PET revealed an age-dependent increase uptake in the striatum and pallidum/nucleus basalis of Meynert in WT only, similar to that observed with this tracer in humans, resulting in TG being significantly lower than WT by 18 m. In vivo [18F]Florbetaben PET scanning detected Aß accumulation at 18 m, and (S)-[18F]THK5117 PET revealed subsequent Tau accumulation at 25m in hippocampal and cortical regions. Aß plaques were low but detectable by immunohistochemistry from 6 m, increasing further at 12 and 18 m with Tau-positive neurons adjacent to Aß plaques at 18 m. NeuroChrom (a pan neuronal marker) immunohistochemistry revealed a loss of neuronal staining at the Aß plaques locations, while NeuN labelling revealed an age-dependent decrease in hippocampal neuron number in both genotypes. Behavioural assessment using the novel object recognition task revealed that both WT & TgF344-AD animals discriminated the novel from familiar object at 3 m and 6 m of age. However, low levels of exploration observed in both genotypes at later time-points resulted in neither genotype successfully completing the task. Deficits in social interaction were only observed at 3 m in the TgF344-AD animals. By in vivo MRS, we showed a decrease in neuronal marker N-acetyl-aspartate in the hippocampus at 18 m (-18% vs age-matched WT, and -31% vs 6 m TG) and increased Taurine in the cortex of TG (+35% vs age-matched WT, and +55% vs 6 m TG). Conclusions: This multi-centre multi-modal study demonstrates, for the first time, alterations in brain metabolites, cholinergic receptors and neuroinflammation in vivo in this model, validated by robust ex vivo approaches. Our data confirm that, unlike mouse models, the TgF344-AD express Tau pathology that can be detected via PET, albeit later than by ex vivo techniques, and is a useful model to assess and longitudinally monitor early neurotransmission dysfunction and neuroinflammation in AD.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Espectroscopia de Ressonância Magnética , Placa Amiloide/metabolismo , Tomografia por Emissão de Pósitrons , Proteínas tau/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Doença de Alzheimer/patologia , Animais , Escala de Avaliação Comportamental , Disfunção Cognitiva/genética , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Feminino , Radioisótopos de Flúor , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Gliose/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Inflamação/metabolismo , Locomoção/genética , Locomoção/fisiologia , Masculino , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Transgênicos , Receptores Colinérgicos/metabolismo , Tálamo/metabolismo , Tálamo/patologiaRESUMO
Adamantanyl benzamide 1 was identified as a potent P2X7R antagonist but failed to progress further due to poor metabolic stability. We describe the synthesis and SAR of a series of bioisosteres of benzamide 1 to explore improvements in the pharmacological properties of this lead. Initial efforts investigated a series of heteroaromatic bioisosteres, which demonstrated improved physicochemical properties but reduced P2X7R antagonism. Installation of bioisosteric fluorine on the adamantane bridgeheads was well tolerated and led to a series of bioisosteres with improved physicochemical properties and metabolic stability. Trifluorinated benzamide 34 demonstrated optimal physicochemical parameters, superior metabolic stability (ten times longer than lead benzamide 1), and an improved physicokinetic profile and proved effective in the presence of several known P2X7R polymorphisms.
Assuntos
Adamantano/análogos & derivados , Benzamidas/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/efeitos dos fármacos , Adamantano/farmacologia , Animais , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacocinética , Biotransformação , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Humanos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredução , Polimorfismo de Nucleotídeo Único , Antagonistas do Receptor Purinérgico P2X/síntese química , Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Ratos , Receptores Purinérgicos P2X7/genética , Relação Estrutura-AtividadeRESUMO
The sigma-1 receptor (S1R) has attracted a great deal of attention as a prospective drug target due to its involvement in numerous neurological disorders and, more recently, for its therapeutic potential in neuropathic pain. As there was no crystal structure of this membrane-bound protein reported until 2016, ligand generation was driven by pharmacophore refinements to the general model suggested by Glennon and co-workers. The generalised S1R pharmacophore comprises a central region where a basic amino group is preferred, flanked by two hydrophobic groups. Guided by this pharmacophore, S1R ligands containing piperazines, piperazinones, and ethylenediamines have been developed. In the current work, we systematically deconstructed the piperazine core of a prototypic piperazine S1R ligand (vide infra) developed in our laboratories. Although we did not improve the affinity at the S1R compared to the lead, we identified several features important for affinity and selectivity. These included at least one basic nitrogen atom, conformational flexibility and, for S1R, a secondary or tertiary amine group proximal to the anisole. Furthermore, S2R selectivity can be tailored with functional group modifications of the N-atom proximal to the anisole.
Assuntos
Diaminas/química , Diaminas/metabolismo , Receptores sigma/metabolismo , Animais , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Etilenodiaminas/química , Cobaias , Ligantes , Piperazina , Piperazinas/química , Ensaio Radioligante , Ratos , Receptores sigma/química , Relação Estrutura-Atividade , Receptor Sigma-1RESUMO
Synthetic cannabinoid (SC) designer drugs based on indole and indazole scaffolds and featuring l-valinamide or l-tert-leucinamide side chains are encountered with increasing frequency by forensic researchers and law enforcement agencies and are associated with serious adverse health effects. However, many of these novel SCs are unprecedented in the scientific literature at the time of their discovery, and little is known of their pharmacology. Here, we report the synthesis and pharmacological characterization of AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, 5F-ADBICA, and several analogues. All synthesized SCs acted as high potency agonists of CB1 (EC50 = 0.24-21 nM) and CB2 (EC50 = 0.88-15 nM) receptors in a fluorometric assay of membrane potential, with 5F-ADB-PINACA showing the greatest potency at CB1 receptors. The cannabimimetic activities of AB-FUBINACA and AB-PINACA in vivo were evaluated in rats using biotelemetry. AB-FUBINACA and AB-PINACA dose-dependently induced hypothermia and bradycardia at doses of 0.3-3 mg/kg, and hypothermia was reversed by pretreatment with a CB1 (but not CB2) antagonist, indicating that these SCs are cannabimimetic in vivo, consistent with anecdotal reports of psychoactivity in humans.
Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Drogas Desenhadas/farmacologia , Indazóis/farmacologia , Indóis/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/síntese química , Agonistas de Receptores de Canabinoides/química , Antagonistas de Receptores de Canabinoides/farmacologia , Linhagem Celular Tumoral , Estudos de Coortes , Drogas Desenhadas/síntese química , Drogas Desenhadas/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Frequência Cardíaca/efeitos dos fármacos , Humanos , Indazóis/síntese química , Indazóis/química , Indóis/síntese química , Indóis/química , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Estrutura Molecular , Ratos Wistar , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismoRESUMO
Synthetic cannabinoid (SC) designer drugs featuring bioisosteric fluorine substitution are identified by forensic chemists and toxicologists with increasing frequency. Although terminal fluorination of N-pentyl indole SCs is sometimes known to improve cannabinoid type 1 (CB1) receptor binding affinity, little is known of the effects of fluorination on functional activity of SCs. This study explores the in vitro functional activities of SC designer drugs JWH-018, UR-144, PB-22, and APICA, and their respective terminally fluorinated analogues AM-2201, XLR-11, 5F-PB-22, and STS-135 at human CB1 and CB2 receptors using a FLIPR membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 2.8-1959 nM) and CB2 (EC50 = 6.5-206 nM) receptors, with the fluorinated analogues generally showing increased CB1 receptor potency (â¼2-5 times). Additionally, the cannabimimetic activities and relative potencies of JWH-018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135 in vivo were evaluated in rats using biotelemetry. All SCs dose-dependently induced hypothermia and reduced heart rate at doses of 0.3-10 mg/kg. There was no consistent trend for increased potency of fluorinated SCs over the corresponding des-fluoro SCs in vivo. Based on magnitude and duration of hypothermia, the SCs were ranked for potency (PB-22 > 5F-PB-22 = JWH-018 > AM-2201 > APICA = STS-135 = XLR-11 > UR-144).
Assuntos
Canabinoides/química , Canabinoides/farmacologia , Drogas Desenhadas/química , Drogas Desenhadas/farmacologia , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipotermia/induzido quimicamente , Indóis/química , Indóis/farmacologia , Masculino , Camundongos , Estrutura Molecular , Naftalenos/química , Naftalenos/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Ratos Wistar , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , TelemetriaRESUMO
In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo-synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo-synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1-2 d for bio-orthogonal labeling; and, with regard to analysis, 3-4 d for western blotting, 5-6 d for immunofluorescence or ~3 weeks for mass spectrometry.
Assuntos
Aminoácidos/metabolismo , Proteínas de Caenorhabditis elegans/análise , Técnicas de Química Analítica/métodos , Química Click/métodos , Coloração e Rotulagem/métodos , Alanina/análogos & derivados , Animais , Western Blotting , Proteínas de Caenorhabditis elegans/metabolismo , Imunofluorescência , Espectrometria de MassasRESUMO
Much recent work is investigating the role of oxidative stress and inflammatory mechanisms in the aetiology of neurodegeneration in Parkinson's disease. The present study evaluated whether the green tea constituent epigallocatechin gallate (EGCG) which has both anti-oxidant and anti-inflammatory properties, exerts neuroprotection and symptomatic effects when administered orally as a pre-treatment prior to 6-hydroxydopamine (6-OHDA) lesions. Groups of rats were given either 1mg/kg, 2mg/kg EGCG or vehicle solution for 14 days. Sham or 6-OHDA surgery was performed on day 11 of the drug administration protocol. Behavioural analysis was conducted before drugs/vehicle solution, again during the treatment period and then repeated at fortnightly intervals for 2 months post-operatively. Whilst some subtle behavioural improvements in postural abnormalities and ability to cross a narrow beam were observed in lesioned rats after EGCG (vs. vehicle) there was no evidence of neuroprotection on post-mortem quantification of degree of nigral dopaminergic neuronal loss when comparing the lesioned groups given the various treatments.
Assuntos
Adrenérgicos/toxicidade , Antioxidantes/farmacologia , Catequina/análogos & derivados , Discinesia Induzida por Medicamentos/tratamento farmacológico , Neurônios/efeitos dos fármacos , Oxidopamina/toxicidade , Administração Oral , Animais , Antioxidantes/administração & dosagem , Catequina/administração & dosagem , Catequina/farmacologia , Contagem de Células , Dopamina/metabolismo , Feminino , Imuno-Histoquímica , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Testes Neuropsicológicos , Postura , Ratos , Ratos Sprague-Dawley , Rotação , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismoRESUMO
The purinergic P2X(7) receptor is a unique member of the ATP-gated P2X family. This receptor has been implicated in numerous diseases and many structurally diverse ligands have been discovered via high throughput screening. This perspective will attempt to highlight some of the most recent key findings in both the biology and chemistry.
Assuntos
Benzamidas/farmacologia , Produtos Biológicos/farmacologia , Guanidinas/farmacologia , Hidrazinas/farmacologia , Antagonistas do Receptor Purinérgico P2 , Triazóis/farmacologia , Animais , Benzamidas/química , Sítios de Ligação , Produtos Biológicos/química , Avaliação Pré-Clínica de Medicamentos , Guanidinas/química , Humanos , Hidrazinas/química , Estrutura Molecular , Ligação Proteica , Receptores Purinérgicos P2/análise , Receptores Purinérgicos P2/metabolismo , Triazóis/químicaAssuntos
Gânglios da Base/patologia , Transtornos dos Movimentos/patologia , Tálamo/patologia , Animais , Gânglios da Base/fisiopatologia , Humanos , Núcleos Intralaminares do Tálamo/patologia , Núcleos Intralaminares do Tálamo/fisiopatologia , Transtornos dos Movimentos/fisiopatologia , Rede Nervosa/patologia , Rede Nervosa/fisiopatologia , Tálamo/fisiopatologiaRESUMO
Recently, a novel series of amidines has been described, exhibiting high NR2B-subtype selective N-methyl-D-aspartate (NMDA) antagonist activity with nanomolar or subnanomolar affinity. Within the styrylamidine subclass, (E)-N-(2-methoxybenzyl)-3-phenyl-acrylamidine (1), displayed the highest affinity (Ki=0.7 nM versus [(3)H]ifenprodil) and was considered an appropriate candidate for isotopic labelling with carbon-11 (T(1/2): 20.38 min) at its methoxy group for imaging of NMDA receptors with PET. Derivative 1 has been labelled from the corresponding nor-analogue using [(11)C]methyl triflate and the following experimental conditions : (1) trapping at -10 degrees C of [(11)C]methyl triflate in 300 microL of acetone containing 0.6-0.8 mg of precursor 5 (2.4-3.2 micromol) and 5 microL of a 3M solution of NaOH in water (about 5 eq.); (2) concentration to dryness of the reaction mixture (at 110 degrees C, using a helium stream for 1-2 min); (3) taking up the residue with 0.5 mL of the HPLC mobile phase and (4) purification using semi-preparative HPLC (SymmetryPrep) C-18, Waters, 300 x 7.8 mm). Typically, starting from a 1.5 Ci (55.5 GBq) [(11)C]CO(2) production batch, 120-240 m Ci (4.44-8.88 GBq) of [(11)C]-1 (20-40% decay-corrected radiochemical yield, n=5) was obtained within a total synthesis time of 25-30 min. Specific radioactivities ranged from 0.8 to 1.2 Ci/micromol (29.6-44.4 GBq/micromol) at the end of radiosynthesis. No attempts were made to further optimise these reactions, as sufficient material was obtained to allow for preliminary pharmacological characterisation.
Assuntos
Amidinas/análise , Amidinas/síntese química , Marcação por Isótopo/métodos , Tomografia por Emissão de Pósitrons/métodos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Compostos Radiofarmacêuticos/análise , Compostos Radiofarmacêuticos/síntese químicaRESUMO
The cyclohexyl piperazine 1 (1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-propyl]-piperazine) has been shown to be a potent and selective sigma-2 receptor ligand. In the present study, we prepared [(11)C]1 by O-alkylation of the phenolic precursor 2 with [(11)C]CH(3)I. [(11)C]1 was obtained in a 29% non-decay corrected yield and specific activity of 9299 mCi/micromol calculated at end-of-synthesis. The biodistribution of [(11)C]1 in mouse brain demonstrated rapid and homogenous concentration in all brain structures, which included the cortex, thalamus, cerebellum and striatum. Co-administration of unlabelled 1 (1 mg/kg) or the sigma-2 selective ligand SM-21 (1 mg/kg) failed to show any significant inhibition of [(11)C]1 uptake in the mouse brain. The evaluation of this radioligand in vivo in the mouse clearly indicates that it does not possess the required properties for studying sigma-2 receptors in the brain using PET.