RESUMO
N-methyl-d-aspartate receptors (NMDARs) mediate both physiological and pathophysiological processes, although selective ligands lack broad clinical utility. NMDARs are composed of multiple subunits, but N-methyl-d-aspartate receptor subunit 2 (GluN2) is predominately responsible for functional heterogeneity. Specifically, the GluN2A- and GluN2B-containing subtypes are enriched in adult hippocampus and cortex and impact neuronal communication via dynamic trafficking into and out of the synapse. We sought to understand if ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl) butanamide (NYX-2925), a novel NMDAR modulator, alters synaptic levels of GluN2A- or GluN2B-containing NMDARs. Low-picomolar NYX-2925 increased GluN2B colocalization with the excitatory post-synaptic marker post-synaptic density protein 95 (PSD-95) in rat primary hippocampal neurons within 30 min. Twenty-four hours following oral administration, 1 mg/kg NYX-2925 increased GluN2B in PSD-95-associated complexes ex vivo, and low-picomolar NYX-2925 regulated numerous trafficking pathways in vitro. Because the NYX-2925 concentration that increases synaptic GluN2B was markedly below that which enhances long-term potentiation (mid-nanomolar), we sought to elucidate the basis of this effect. Although NMDAR-dependent, NYX-2925-mediated colocalization of GluN2B with PSD-95 occurred independent of ion flux, as colocalization increased in the presence of either the NMDAR channel blocker (5R,10S)-(-)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate or glycine site antagonist 7-chlorokynurenic acid. Moreover, while mid-nanomolar NYX-2925 concentrations, which do not increase synaptic GluN2B, enhanced calcium transients, functional plasticity was only enhanced by picomolar NYX-2925. Thus, NYX-2925 concentrations that increase synaptic GluN2B facilitated the chemical long-term potentiation induced insertion of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA1 subunit levels. Basal (unstimulated by chemical long-term potentiation) levels of synaptic GluA1 were only increased by mid-nanomolar NYX-2925. These data suggest that NYX-2925 facilitates homeostatic plasticity by initially increasing synaptic GluN2B via metabotropic-like NMDAR signaling. Cover Image for this issue: doi: 10.1111/jnc.14735.
Assuntos
Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Compostos de Espiro/farmacologia , Sinapses/metabolismo , Animais , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacosRESUMO
STUDY OBJECTIVES: The present studies examine the effects of NMDAR activation by NYX-2925 diurnal rhythmicity of both sleep and wake as well as emotion. METHODS: Twenty-four-hour sleep EEG recordings were obtained in sleep-deprived and non-sleep-deprived rats. In addition, the day-night cycle of both activity and mood was measured using home cage ultrasonic-vocalization recordings. RESULTS: NYX-2925 significantly facilitated non-REM (NREM) sleep during the lights-on (sleep) period, and this effect persisted for 3 days following a single dose in sleep-deprived rats. Sleep-bout duration and REM latencies were increased without affecting total REM sleep, suggesting better sleep quality. In addition, delta power during wake was decreased, suggesting less drowsiness. NYX-2925 also rescued learning and memory deficits induced by sleep deprivation, measured using an NMDAR-dependent learning task. Additionally, NYX-2925 increased positive affect and decreased negative affect, primarily by facilitating the transitions from sleep to rough-and-tumble play and back to sleep. In contrast to NYX-2925, the NMDAR antagonist ketamine acutely (1-4 hours post-dosing) suppressed REM and non-REM sleep, increased delta power during wake, and blunted the amplitude of the sleep-wake activity rhythm. DISCUSSION: These data suggest that NYX-2925 could enhance behavioral plasticity via improved sleep quality as well as vigilance during wake. As such, the facilitation of sleep by NYX-2925 has the potential to both reduce symptom burden on neurological and psychiatric disorders as well as serve as a biomarker for drug effects through restoration of sleep architecture.
Assuntos
Afeto/fisiologia , Ritmo Circadiano/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Privação do Sono/fisiopatologia , Sono/fisiologia , Compostos de Espiro/farmacologia , Afeto/efeitos dos fármacos , Animais , Ritmo Circadiano/efeitos dos fármacos , Eletroencefalografia/métodos , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistas , Sono/efeitos dos fármacos , Privação do Sono/tratamento farmacológico , Compostos de Espiro/uso terapêutico , Vigília/efeitos dos fármacos , Vigília/fisiologiaRESUMO
NYX-2925 [(2S,3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide] is a novel N-methyl-d-aspartate (NMDA) receptor modulator that is currently being investigated in phase 2 clinical studies for the treatment of painful diabetic peripheral neuropathy and fibromyalgia. Previous studies demonstrated that NYX-2925 is a member of a novel class of NMDA receptor-specific modulators that affect synaptic plasticity processes associated with learning and memory. Studies here examined NYX-2925 administration in rat peripheral chronic constriction nerve injury (CCI) and streptozotocin-induced diabetic mechanical hypersensitivity. Additionally, NYX-2925 was examined in formalin-induced persistent pain model and the tail flick test of acute nociception. Oral administration of NYX-2925 resulted in rapid and long-lasting analgesia in both of the neuropathic pain models and formalin-induced persistent pain, but was ineffective in the tail flick model. The analgesic effects of NYX-2925 were blocked by the systemic administration of NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid. Microinjection of NYX-2925 into the medial prefrontal cortex of CCI rats resulted in analgesic effects similar to those observed following systemic administration, whereas intrathecal administration of NYX-2925 was ineffective. In CCI animals, NYX-2925 administration reversed deficits seen in a rat model of rough-and-tumble play. Thus, it appears that NYX-2925 may have therapeutic potential for the treatment of neuropathic pain, and the data presented here support the idea that NYX-2925 may act centrally to ameliorate pain and modulate negative affective states associated with chronic neuropathic pain.
Assuntos
Analgésicos/farmacologia , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Compostos de Espiro/farmacologia , Analgésicos/uso terapêutico , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Compostos de Espiro/uso terapêutico , Vocalização Animal/efeitos dos fármacosRESUMO
Background: N-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-ß-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil. Methods: The in vitro and in vivo pharmacological properties of NYX-2925 were examined. Results: NYX-2925 has a low potential for "off-target" activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100-500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01-1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour. Conclusions: NYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders.
Assuntos
Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Memória/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Oligopeptídeos/farmacologia , Animais , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Emoções/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/líquido cefalorraquidiano , Fármacos Atuantes sobre Aminoácidos Excitatórios/química , Células HEK293 , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Aprendizagem/efeitos dos fármacos , Aprendizagem/fisiologia , Masculino , Memória/fisiologia , Estrutura Molecular , Plasticidade Neuronal/fisiologia , Oligopeptídeos/líquido cefalorraquidiano , Oligopeptídeos/química , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Pirazinas/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
BACKGROUND: Rapastinel (GLYX-13) is a NMDA receptor modulator with glycine-site partial agonist properties. It is a robust cognitive enhancer and shows rapid and long-lasting antidepressant properties in both animal models and in humans. METHODS: Rapastinel was derived from a monoclonal antibody, B6B21, is a tetrapeptide (threonine-proline-proline-threonine-amide) obtained from amino acid sequence information obtained from sequencing one of the hypervariable regions of the light chain of B6B21. The in-vivo and in-vitro pharmacology of rapastinel was examined. RESULTS: Rapastinel was found to be a robust cognitive enhancer in a variety of learning and memory paradigms and shows marked antidepressant-like properties in multiple models including the forced swim (Porsolt), learned helplessness and chronic unpredictable stress. Rapastinel's rapid-acting antidepressant properties appear to be mediated by its ability to activate NMDA receptors leading to enhancement in synaptic plasticity processes associated with learning and memory. This is further substantiated by the increase in mature dendritic spines found 24 hrs after rapastinel treatment in both the rat dentate gyrus and layer five of the medial prefrontal cortex. Moreover, ex vivo LTP studies showed that the effects of rapastinel persisted at least two weeks post-dosing. CONCLUSION: These data suggest that rapastinel has significant effects on metaplasticity processes that may help explain the long lasting antidepressant effects of rapastinel seen in the human clinical trial results.
Assuntos
Depressão/tratamento farmacológico , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Fatores Etários , Animais , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Depressão/patologia , Depressão/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Comportamento Exploratório/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Oligopeptídeos/química , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Natação , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Vocalização Animal/efeitos dos fármacosRESUMO
INTRODUCTION: Docosahexaenoic acid (DHA) and DHA-containing ethanolamine plasmalogens (PlsEtn) are decreased in the brain, liver and the circulation in Alzheimer's disease. Decreased supply of plasmalogen precursors to the brain by the liver, as a result of peroxisomal deficits is a process that probably starts early in the AD disease process. To overcome this metabolic compromise, we have designed an orally bioavailable DHA-containing ether lipid precursor of plasmalogens. PPI-1011 is an alkyl-diacyl plasmalogen precursor with palmitic acid at sn-1, DHA at sn-2 and lipoic acid at sn-3. This study outlines the oral pharmacokinetics of this precursor and its conversion to PlsEtn and phosphatidylethanolamines (PtdEtn). METHODS: Rabbits were dosed orally with PPI-1011 in hard gelatin capsules for time-course and dose response studies. Incorporation into PlsEtn and PtdEtn was monitored by LC-MS/MS. Metabolism of released lipoic acid was monitored by GC-MS. To monitor the metabolic fate of different components of PPI-1011, we labeled the sn-1 palmitic acid, sn-2 DHA and glycerol backbone with (13)C and monitored their metabolic fates by LC-MS/MS. RESULTS: PPI-1011 was not detected in plasma suggesting rapid release of sn-3 lipoic acid via gut lipases. This conclusion was supported by peak levels of lipoic acid metabolites in the plasma 3 hours after dosing. While PPI-1011 did not gain access to the plasma, it increased circulating levels of DHA-containing PlsEtn and PtdEtn. Labeling experiments demonstrated that the PtdEtn increases resulted from increased availability of DHA released via remodeling at sn-2 of phospholipids derived from PPI-1011. This release of DHA peaked at 6 hrs while increases in phospholipids peaked at 12 hr. Increases in circulating PlsEtn were more complex. Labeling experiments demonstrated that increases in the target PlsEtn, 16:0/22:6, consisted of 2 pools. In one pool, the intact precursor received a sn-3 phosphoethanolamine group and desaturation at sn-1 to generate the target plasmalogen. The second pool, like the PtdEtn, resulted from increased availability of DHA released during remodeling of sn-2. In the case of sn-1 18:0 and 18:1 plasmalogens with [(13)C(3)]DHA at sn-2, labeling was the result of increased availability of [(13)C(3)]DHA from lipid remodeling. Isotope and repeated dosing (2 weeks) experiments also demonstrated that plasmalogens and/or plasmalogen precursors derived from PPI-1011 are able to cross both the blood-retinal and blood-brain barriers. CONCLUSIONS: Our data demonstrate that PPI-1011, an ether lipid precursor of plasmalogens is orally bioavailable in the rabbit, augmenting the circulating levels of unesterified DHA and DHA-containing PlsEtn and PtdEtn. Other ethanolamine plasmalogens were generated from the precursor via lipid remodeling (de-acylation/re-acylation reactions at sn-2) and phosphatidylethanolamines were generated via de-alkylation/re-acylation reactions at sn-1. Repeated oral dosing for 2 weeks with PPI-1011 resulted in dose-dependent increases in circulating DHA and DHA-containing plasmalogens. These products and/or precursors were also able to cross the blood-retinal and blood-brain barriers.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Diglicerídeos/administração & dosagem , Administração Oral , Animais , Disponibilidade Biológica , Caproatos/sangue , Diglicerídeos/farmacocinética , Ácidos Docosa-Hexaenoicos/sangue , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Fosfatidiletanolaminas/sangue , Plasmalogênios/sangue , Coelhos , Distribuição TecidualRESUMO
BACKGROUND: To develop effective strategies in cancer chemoprevention, an increased understanding of endogenous biochemical mediators that block metastatic processes is critically needed. Dietary lipids and non-steroidal anti-inflammatory drugs (NSAIDs) have a published track record of providing protection against gastrointestinal malignancies. In this regard, we examined the effects of membrane plasmalogens and ibuprofen on regulation of cellular levels of diamines, polyamine mediators that are augmented in cancer cells. For these studies we utilized Chinese hamster ovary (CHO) cells and NRel-4 cells, a CHO cell line with defective plasmalogen synthesis. RESULTS: NRel-4 cells, which possess cellular plasmalogen levels that are 10% of control CHO cells, demonstrated 2- to 3-fold increases in cellular diamine levels. These diamine levels were normalized by plasmalogen replacement and significantly reduced by ibuprofen. In both cases the mechanism of action appears to mainly involve increased diamine efflux via the diamine exporter. The actions of ibuprofen were not stereospecific, supporting previous studies that cyclooxygenase (COX) inhibition is unlikely to be involved in the ability of NSAIDs to reduce intracellular diamine levels. CONCLUSIONS: Our data demonstrate that ibuprofen, a drug known to reduce the risk of colorectal cancer, reduces cellular diamine levels via augmentation of diamine efflux. Similarly, augmentation of membrane plasmalogens can increase diamine export from control and plasmalogen-deficient cells. These data support the concept that membrane transporter function may be a therapeutic point of intervention for dietary and pharmacological approaches to cancer chemoprevention.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Cadaverina/metabolismo , Membrana Celular/metabolismo , Ibuprofeno/farmacologia , Neoplasias/prevenção & controle , Plasmalogênios/farmacologia , Putrescina/metabolismo , Aminoácidos/metabolismo , Animais , Células CHO , Cricetinae , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Plasmalogênios/fisiologiaRESUMO
BACKGROUND: Childhood peroxisomal disorders and leukodystrophies are devastating diseases characterized by dysfunctional lipid metabolism. Plasmalogens (ether glycerophosphoethanolamine lipids) are decreased in these genetic disorders. The biosynthesis of plasmalogens is initiated in peroxisomes but completed in the endoplasmic reticulum. We therefore undertook a study to evaluate the ability of a 3-substituted, 1-alkyl, 2-acyl glyceryl ether lipid (PPI-1011) to replace plasmalogens in rhizomelic chrondrodysplasia punctata type 1 (RCDP1) and rhizomelic chrondrodysplasia punctata type 2 (RCDP2) lymphocytes which possess peroxisomal mutations culminating in deficient plasmalogen synthesis. We also examined plasmalogen synthesis in Pelizaeus-Merzbacher disease (PMD) lymphocytes which possess a proteolipid protein-1 (PLP1) missense mutation that results in abnormal PLP1 folding and it's accumulation in the endoplasmic reticulum (ER), the cellular site of the last steps in plasmalogen synthesis. In vivo incorporation of plasmalogen precursor into tissue plasmalogens was also evaluated in the Pex7 mouse model of plasmalogen deficiency. RESULTS: In both RCDP1 and RCDP2 lymphocytes, PPI-1011 repleted the target ethanolamine plasmalogen (PlsEtn16:0/22:6) in a concentration dependent manner. In addition, deacylation/reacylation reactions resulted in repletion of PlsEtn 16:0/20:4 in both RCDP1 and RCDP2 lymphocytes, repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP2 lymphocytes, and partial repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP1 lymphocytes. In the Pex7 mouse, oral dosing of labeled PPI-1011 demonstrated repletion of tissue levels of the target plasmalogen PlsEtn 16:0/22:6 with phospholipid remodeling also resulting in significant repletion of PlsEtn 16:0/20:4 and PlsEtn 16:0/18:1. Metabolic conversion of PPI-1011 to the target plasmalogen was most active in the liver. CONCLUSIONS: Our data demonstrate that PPI-1011 is activated (removal of 3-substitution) and converted to PlsEtn in vitro in both RCDP1 and RCDP2 lymphocytes and in vivo in the Pex7 mouse model of RCPD1 effectively bypassing the peroxisomal dysfunction present in these disorders. While PPI-1011 was shown to replete PlsEtns 16:0/x, ether lipid precursors of PlsEtn 18:0/x and PlsEtn 18:1/x may also be needed to achieve optimal clinical benefits of plasmalogen replacement in these complex patient populations. In contrast, only limited plasmalogen replacement was observed in PMD lymphocytes suggesting that the effects of protein misfolding and accumulation in the ER negatively affect processing of plasmalogen precursors in this cellular compartment.
Assuntos
Condrodisplasia Punctata Rizomélica/metabolismo , Diglicerídeos/farmacologia , Linfócitos/efeitos dos fármacos , Doença de Pelizaeus-Merzbacher/metabolismo , Plasmalogênios/metabolismo , Animais , Células Cultivadas , Olho/metabolismo , Humanos , Rim/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Neocórtex/metabolismo , Doença de Pelizaeus-Merzbacher/genética , Receptor 2 de Sinal de Orientação para Peroxissomos , Plasmalogênios/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Distribuição TecidualRESUMO
While new anti-cytokine disease-modifying anti-arthritic drugs for rheumatoid arthritis have been designed via mechanistic approaches, the mechanism of action of a number of more established disease-modifying anti-arthritic drugs has not been elucidated. In the case of d-penicillamine and sodium aurothiomalate, the key structural feature appears to be a free thiol group. However, the role thiol groups play in the therapeutic efficacy of these drugs has not been defined. A number of lines of evidence have demonstrated increased generation of reactive aldehydes and the associated depletion of free thiol pools in rheumatoid arthritis. These observations have led to the suggestion that reactive aldehydes may be the ultimate mediators of cell destruction in rheumatoid arthritis joints. Our data clearly demonstrate that thiol-containing disease-modifying anti-arthritic agents both directly sequester reactive aldehydes and augment intracellular thiol pools, which also can buffer increased aldehyde load and oxidative stress. These data are consistent with clinical data that penicillamine lowers synovial aldehyde levels and augments plasma thiols. We suggest that these actions are the pivotal mechanism of action of thiol-containing disease-modifying anti-arthritic drugs. Understanding the mechanism of action of these drugs provides the opportunity for the design of more potent and safer thiol drug candidates.
Assuntos
Aldeídos/toxicidade , Antirreumáticos/farmacologia , Tiomalato Sódico de Ouro/farmacologia , Penicilamina/farmacologia , Compostos de Sulfidrila/metabolismo , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/fisiopatologia , Linhagem Celular , Camundongos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Cellular thiol pools have been shown to be important in the regulation of the redox status of cells, providing a large antioxidant pool consisting of free thiols, thiols bound in the disulfide form and thiols bound to proteins. However, experimental studies with the thiol cysteamine and its disulfide cystamine have demonstrated dramatic cytoprotection in experimental models where antioxidants provide only minor protection. These data suggest that an alternate action of thiols is important in their cytoprotective actions. A common feature of the in vitro and in vivo models, where these thiol agents demonstrate cytoprotection, is the generation of cytotoxic aldehydes. We therefore studied the actions of cystamine, cysteamine and several reference thiol agents as cytoprotectants against cell death induced by increased "aldehyde load". We found that all the thiol agents examined provided dramatic protection against aldehyde-induced cell death in SN56 cholinergic neurons, under conditions in which acrolein induced 100% cell death. With regard to mechanism of action, the reference thiols cysteine, N-acetylcysteine, 2-mercaptoethanesulfonic acid, mercapto-propionyglycine, and cysteamine can directly sequester aldehydes. In addition, these thiols were all found to augment intracellular cysteine levels via disulfide interchange reactions. Cysteamine and cystamine also augmented basal intracellular cysteamine levels. Our data, for the first time, demonstrate the importance of intracellular thiols in sequestering toxic reactive aldehyde products of lipid peroxidation and polyamine metabolism. In addition it appears that pharmacological manipulation of intracellular thiol pools might offer a new approach in the design of neuroprotective drug candidates.
Assuntos
Aldeídos/toxicidade , Cisteamina/metabolismo , Cisteína/metabolismo , Neurônios/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Cisteamina/farmacologia , Cisteína/farmacologia , Interações Medicamentosas , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Camundongos , Modelos Biológicos , Neurônios/metabolismo , Septo do Cérebro/citologia , Fatores de TempoRESUMO
In neurodegenerative diseases augmented polyamine metabolism results in the generation of hydrogen peroxide and a number of reactive aldehydes that participate in the death of compromised tissue. The major aldehydes produced by polyamine oxidase and amine oxidases include the 2-alkenal acrolein, the acetoamidoaldehyde 3-acetamidopropanal (3-AAP) and the aminoaldehydes 3-aminopropanal (3-AP) and 4-aminobutanal (4-AB). Using retinal ganglion cell (E1A-NR.3) cultures, we confirmed the cytotoxicity of acrolein and 3-AP. For the first time we also demonstrated the cytotoxicity of 4-AB and the lack of toxicity of 3-AAP. Our data with 3-AAP, a product of N-acetylspermine and N-acetylspermidine metabolism, indicate that the aldehyde function of aminoaldehydes is insufficient to express toxicity since the free amino group of aminoaldehydes is also required to gain access to lysosomes where their cytotoxic actions are expressed via leakage of cathepsins that compromise mitochondrial integrity. Metabolism of 3-AP to beta-alanine by aldehyde dehydrogenase was also evaluated in retinal ganglion cell cultures and found to proceed at a linear rate of 24.3+/-1 nmol/mg protein/h. These are the first data demonstrating the dynamic cellular detoxification of 3-AP by neural cells and support the concept that decrements in aldehyde elimination leading to an increase in "aldehyde load" may play pivotal roles in the development and progression of neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis and Parkinson's disease.
Assuntos
Aldeídos/metabolismo , Poliaminas Biogênicas/metabolismo , Degeneração Neural/metabolismo , Neurotoxinas/metabolismo , Células Ganglionares da Retina/metabolismo , Acroleína/metabolismo , Acroleína/toxicidade , Aldeído Desidrogenase/metabolismo , Aldeídos/toxicidade , Animais , Poliaminas Biogênicas/toxicidade , Química Encefálica/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Taxa de Depuração Metabólica/fisiologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/fisiopatologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Neurotoxinas/toxicidade , Propilaminas/metabolismo , Propilaminas/toxicidade , Ratos , Células Ganglionares da Retina/efeitos dos fármacos , beta-Alanina/metabolismoRESUMO
In ongoing studies of the neuroprotective properties of monoamine oxidase inhibitors, we found that phenelzine provided robust neuroprotection in the gerbil model of transient forebrain ischemia, with drug administration delayed up to 3 h post reperfusion. Since ischemia-reperfusion brain injury is associated with large increases in the concentrations of reactive aldehydes in the penumbra area, we investigated if the hydrazine function of phenelzine was capable of sequestering reactive aldehydes. Both aminoaldehydes and acrolein are generated from the metabolism of polyamines to putrescine by polyamine oxidase. These toxic aldehydes in turn compromise mitochondrial and lysosomal integrity and initiate apoptosis and necrosis. Previous studies have demonstrated that pharmacological neutralization of reactive aldehydes via the formation of thioacetal derivatives results in significant neuroprotection in ischemia-reperfusion injury, in both focal and global ischemia models. In our studies of acrolein and 3-aminopropanal toxicity, using an immortalized retinal cell line, we found that aldehyde sequestration with phenelzine was neuroprotective. The neuroprotection observed with phenelzine is in agreement with previous studies of aldehyde sequestering agents in the treatment of ischemia-reperfusion brain injury and supports the concept that "aldehyde load" is a major factor in the delayed cell losses of the ischemic penumbra.
Assuntos
Aldeídos/metabolismo , Fármacos Neuroprotetores/farmacologia , Fenelzina/farmacologia , Traumatismo por Reperfusão/enzimologia , Células Ganglionares da Retina/efeitos dos fármacos , Acroleína/metabolismo , Acroleína/toxicidade , Aldeídos/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/enzimologia , Isquemia Encefálica/patologia , Células Cultivadas , Modelos Animais de Doenças , Gerbillinae , Masculino , Inibidores da Monoaminoxidase/farmacologia , Propilaminas/metabolismo , Propilaminas/toxicidade , Ratos , Traumatismo por Reperfusão/prevenção & controle , Retina/citologia , Células Ganglionares da Retina/enzimologia , Fatores de TempoRESUMO
The pharmacokinetics (PK) and hepatic extraction (E(H)) of human PTH (1-34), hPTH (1-34), were characterized in rat, dog, and monkey, following intraportal (IPO) and intravenous (IV) bolus administration. hPTH (1-34) was administered to Sprague-Dawley rats (2, 10, 100 microg/kg), beagle dogs (3, 6 microg/kg), and rhesus monkeys (6, 30 microg/kg). Serum concentrations of immunoreactive hPTH (1-34) were used to derive PK parameters. IPO bioavailability (F(IPO)) was determined by comparing dose-normalized serum exposure (i.e., AUC(IPO)/AUC(IV)). E(H) was estimated as 1-F(IPO). In all species, greater than dose-proportional increases in exposure (i.e., C(max) and AUC) were observed for both routes. Dose-dependent disposition (i.e., time-average clearance (CL) and half-life (t(1/2)) were observed in all three species. In rats, E(H) values of 71% (2 microg/kg), 35% (10 microg/kg), and <1% (100 microg/kg) were obtained. In dogs, E(H) values of 90% (3 microg/kg) and 66% (6 microg/kg) were obtained. In monkeys, E(H) values of 25% (6 microg/kg) and <1% (30 microg/kg) were observed. In conclusion, hPTH (1-34) is subject to hepatic first pass extraction in rat, dog, and monkey with evidence of saturation in the rat. Saturable hepatic extraction in dog and monkey is inconclusive due to the limited dose range investigated.
Assuntos
Fígado/metabolismo , Hormônio Paratireóideo/farmacocinética , Fragmentos de Peptídeos/farmacocinética , Animais , Área Sob a Curva , Disponibilidade Biológica , Interpretação Estatística de Dados , Cães , Indicadores e Reagentes , Injeções Intraperitoneais , Injeções Intravenosas , Macaca mulatta , Masculino , Hormônio Paratireóideo/administração & dosagem , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/metabolismo , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinéticaRESUMO
The concept of "oxidative stress" has become a mainstay in the field of neurodegeneration but has failed to differentiate critical events from epiphenomena and sequalae. Furthermore, the translation of current concepts of neurodegenerative mechanisms into effective therapeutics for neurodegenerative diseases has been meager and disappointing. A corollary of current concepts of "oxidative stress" is that of "aldehyde load". This relates to the production of reactive aldehydes that covalently modify proteins, nucleic acids, lipids and carbohydrates and activate apoptotic pathways. However, reactive aldehydes can also be generated by mechanisms other than "oxidative stress". We therefore hypothesized that agents that can chemically neutralize reactive aldehydes should demonstrate superior neuroprotective actions to those of free radical scavengers. To this end, we evaluated hydroxylamines as aldehyde-trapping agents in an in vitro model of neurodegeneration induced by the reactive aldehyde, 3-aminopropanal (3-AP), a product of polyamine oxidase metabolism of spermine and spermidine. In this model, the hydroxylamines N-benzylhydroxylamine, cyclohexylhydroxylamine and t-butylhydroxylamine were shown to protect, in a concentration-dependent manner, against 3-AP neurotoxicity. Additionally, a therapeutic window of 3 h was demonstrated for delayed administration of the hydroxylamines. In contrast, the free radical scavengers TEMPO and TEMPONE and the anti-oxidant ascorbic acid were ineffective in this model. Extending these tissue culture findings in vivo, we examined the actions of N-benzylhydroxylamine in the trimethyltin (TMT) rat model of hippocampal CA3 neurodegeneration. This model involves augmented polyamine metabolism resulting in the generation of reactive aldehydes that compromise mitochondrial integrity. In the rat TMT model, NBHA (50 mg/kg, sc, daily) provided 100% protection against neurodegeneration, as reflected by measurements of KCl-evoked glutamate release from hippocampal brain slices and septal high affinity glutamate uptake. In contrast, ascorbic acid (100 mg/kg, sc, daily) failed to protect CA3 neurons from TMT toxicity. In summary, our data support further evaluation of the concept of "aldehyde load" in neurodegeneration and the potential clinical investigation of agents that are effective traps for reactive aldehydes.
Assuntos
Aldeídos/toxicidade , Hidroxilaminas/uso terapêutico , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Neurotoxinas/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Linhagem Celular , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Cromatografia Gasosa-Espectrometria de Massas/métodos , Ácido Glutâmico/metabolismo , Hidroxilaminas/química , L-Lactato Desidrogenase/metabolismo , Masculino , Cloreto de Potássio/farmacologia , Putrescina/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Triacetonamina-N-Oxil/farmacologiaRESUMO
The GC-MS quantitation of a large number of neurochemicals utilizing a single derivatization step is not common but is provided by the reagent N-(tert-butyldimethylsilyl)-N-methyltrifluro-acetamide (MTBSTFA). Previous workers have utilized this derivative for GC-MS analyses of amino acids, carboxylic acids and urea with electron impact (EI) and with positive chemical ionization (PCI; methane as reagent gas). However, these conditions yield significant fragmentation, decreasing sensitivity and in some cases reducing specificity for quantitation with selected ion monitoring (SIM). Additionally, the majority of studies have used a single internal standard to quantitate many compounds. In this study we demonstrate that using isotopic dilution combined with ammonia as the reagent gas for PCI analyses, results in high precision and sensitivity in analyzing complex neurochemical mixes. We also demonstrate for the first time the utility of this derivative for the analysis of brain polyamines and the dipeptide cysteinyl glycine. In the case of ammonia as the reagent gas, all amino acids, polyamines and urea yielded strong [MH](+) ions with little or no fragmentation. In the case of carboxylic acids, [M+18](+) ions predominated but [MH](+) ions were also noted. This approach was used to analyze superfusates from hippocampal brain slices and brain tissue extracts from brain lesion studies. The advantages of this methodology include: (i) simple sample preparation; (ii) a single derivatization step; (iii) direct GC-MS analysis of the reaction mix; (iv) high precision as a result of isotopic dilution analyses; (v) high sensitivity and specificity as a result of strong [MH](+) ions with ammonia reagent gas; (vi) no hydrolysis of glutamine to glutamate or asparagine to aspartate; and (vii) applicability to a wide range of neurochemicals.
Assuntos
Aminoácidos/análise , Ácidos Carboxílicos/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hipocampo/química , Compostos de Organossilício/análise , Poliaminas/análise , Acetamidas , Amônia , Animais , Isótopos de Carbono , Deutério , Fluoracetatos , Hipocampo/efeitos dos fármacos , Masculino , Neurotransmissores/metabolismo , Isótopos de Nitrogênio , Compostos de Organossilício/química , Ratos , Ratos Sprague-Dawley , Ácido Trifluoracético/química , Compostos de Trimetilestanho/toxicidadeRESUMO
Conventional taste-masking strategies are used to overcome the bitter taste perception of pharmaceuticals by coating the drug particles and/or adding flavoring agents. However, for certain product categories such as rapid dissolve sublingual tablets, taste-masking is challenging. Programs exploring such formulation strategies in the LO-CS phase or post CS phase possess very little toxicological information available in order to conduct human taste panel studies. The potential of a bitter taste perception can present a significant business risk. The objective of the study was to validate a rat behavioral avoidance model that identifies bitter-tasting compounds. Most classic bitter substances elicit a response in the micromolar concentration range while most drugs elicit a response in the millimolar range, hence a validation exercise was conducted to examine if the existing biological model was sensitive enough to identify known bitter tasting drugs as such. Five compounds: ergotamine tartrate, fluoxetine, sucrose, sumatriptan and povidone were chosen to represent a spectrum of compounds. The bitter tasting compounds were identified as such in the model. Based on these results, the assay may serve as a useful surrogate test to identify compounds that may have bitter taste issues.
Assuntos
Aprendizagem da Esquiva , Avaliação Pré-Clínica de Medicamentos/métodos , Ergotamina/farmacologia , Fluoxetina/farmacologia , Sumatriptana/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Masculino , Modelos Animais , Ratos , Reprodutibilidade dos Testes , Limiar Gustativo/efeitos dos fármacos , Privação de ÁguaRESUMO
Extensive two-dimensional NMR analysis was employed to characterize the structural identity of the macrocyclic peptide lactam and the imide analog, a major side reaction product when allyl ester was used to protect the side chain of aspartic acid. A straightforward protocol modification was developed to minimize aspartimide formation during the synthesis of cyclic peptides.