RESUMO
Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.
Assuntos
Canal de Potássio Kv1.3 , Bloqueadores dos Canais de Potássio , Proteínas Recombinantes de Fusão , Animais , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/química , Ligantes , Biblioteca de Peptídeos , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Linhagem CelularRESUMO
Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.
Assuntos
Glioblastoma , Metaloproteinase 2 da Matriz , Neuropilina-1 , Venenos de Escorpião , Humanos , Glioblastoma/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Neuropilina-1/metabolismo , Venenos de Escorpião/metabolismo , Linhagem Celular Tumoral , Ligação ProteicaRESUMO
A new total synthesis of the natural δ-lactone cleistenolide (1) and its (6S)-stereoisomer 2 was achieved starting from d-glucose. Key steps in the synthesis of 1 involved: oxidative cleavage of the C1-C2 bond in partially protected d-glucose derivative (20), and chain extension of resulting aldehyde 20a with a single C2 fragment using (Z)-selective Wittig olefination. Synthesis of 2 involves the following key steps: periodate cleavage of the C5-C6 bond in the commercially available monoacetone d-glucose (24), followed by C2 chain elongation by using the (Z)-selective Wittig olefination. This new approach is also applied to prepare a few new 4-substituted cleistenolide analogues (3 - 18). Compounds 3 - 7 were designed using molecular hybridization, while the remaining eleven analogues were designed using the bioisosterism method. MTT assay showed that most analogues were more active than lead 1 against several malignant cells, but were completely inactive in the culture of normal foetal lung fibroblasts (MRC-5). The K562 cells appeared to be the most sensitive to the synthesized analogues. The strongest antiproliferative activity against this cell line was shown by 4-O-cinnamoyl derivative 3 and 4,6-di-O-benzyl derivative 17, with submicromolar IC50 values (0.76 and 0.67 µM, respectively). Structural features important for the activity of this class of compounds were identified by SAR analysis.
Assuntos
Antineoplásicos , Proliferação de Células , Humanos , Proliferação de Células/efeitos dos fármacos , Relação Estrutura-Atividade , Estereoisomerismo , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral , Estrutura Molecular , Relação Dose-Resposta a Droga , Furanos/química , Furanos/farmacologia , Furanos/síntese química , PironasRESUMO
Using the "chiral pool" approach, two modified total syntheses of the biologically active δ-lactone cleistenolide (1) have been achieved starting from d-glucose. These approaches also enabled the preparation of novel analogues and derivatives of natural product 1. The applied strategy for the synthesis of 1 involves: the initial degradation of the chiral precursor for a single C-atom, C2-fragment chain extension using Z-selective Wittig reaction, and the final δ-lactonization. All tested cleistenolide analogues displayed antimicrobial activity against a panel of nine microbial strains, most of them superseding the activity of cleistenolide itself, and, in some cases, coming close in value to the observed minimal inhibitory concentrations of chloramphenicol. Increased lipophilicity of the derivatives and the non-sterically congested conjugated lactone moiety were a prerequisite for analogues with high inhibitory activity against S. aureus and, in general, Gram-positive bacteria.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Pironas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pironas/síntese química , Pironas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
An HTS campaign of our corporate compound library, and hit-to lead development resulted in thieno[2,3-b]pyridine derivative leads with mGluR5 negative allosteric modulator effects. During the lead optimization process, our objective was to improve affinity and metabolic stability. Modification of the first two targeted regions resulted in compounds with nanomolar affinity, then optimal substitution of the third region improved metabolic stability. One of the most promising compounds showed excellent in vivo efficacy and is a potential development candidate.
Assuntos
Pirimidinas/química , Receptor de Glutamato Metabotrópico 5/química , Regulação Alostérica , Animais , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica , Pirimidinas/metabolismo , Ratos , Receptor de Glutamato Metabotrópico 5/metabolismo , Relação Estrutura-AtividadeRESUMO
Isolated hearts with reduced repolarization reserve would be suitable for assessing the proarrhythmic liability of drugs. However, it is not known which proarrhythmia biomarkers indicate the increased susceptibility to torsades de pointes arrhythmia (TdP) in such experimental setting. Thus, we estimated the efficacy of proarrhythmia biomarkers in isolated hearts with attenuated repolarization reserve. Langendorff-perfused rabbit hearts were used. Repolarization reserve was reduced by concomitant inhibition of the rapid (IKr) and slow (IKs) delayed rectifier potassium currents by dofetilide and HMR-1556, respectively. Rate corrected QT (QTc) interval and beat-to-beat variability of the QT interval measured in sinus rhythm or irrespective of rhythm even during arrhythmias (sinus and absolute QT variability, respectively) were tested. QTc failed to predict increased proarrhythmic risk. Sinus QT variability indicated proarrhythmic liability when low concentration of dofetilide was used. However, when arrhythmias compromised sinus variability measurement during coperfusion of catecholamines and elevated concentration of dofetilide, only absolute QT variability indicated increased proarrhythmic risk. Absolute QT variability parameters seem to be the most practical and sensitive biomarkers of proarrhythmic liability in rabbit hearts with reduced repolarization reserve. Absolute QT variability parameters could serve as surrogates for torsades de pointes in drug-safety investigations in isolated rabbit hearts with attenuated repolarization reserve.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Cromanos/toxicidade , Fenetilaminas/toxicidade , Sulfonamidas/toxicidade , Torsades de Pointes/induzido quimicamente , Animais , Arritmias Cardíacas/fisiopatologia , Biomarcadores/metabolismo , Cromanos/administração & dosagem , Cromanos/farmacologia , Relação Dose-Resposta a Droga , Eletrocardiografia , Feminino , Fenetilaminas/administração & dosagem , Fenetilaminas/farmacologia , Coelhos , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Torsades de Pointes/fisiopatologiaRESUMO
An HTS campaign of our corporate compound library resulted in thieno[2,3-b]pyridines derivative hits with mGluR5 negative allosteric modulator effects. During the hit-to-lead development our objective was to improve affinity, and to keep the ligand efficiency values at an acceptable level. After different modifications of the linker resulted in a 2-sulfonyl-thieno[2,3-b]pyridines derivative, which fulfilled the lead criteria.
Assuntos
Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Tienopiridinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tienopiridinas/síntese química , Tienopiridinas/químicaRESUMO
A new series of quinolinyl- and phenantridinyl-acetamides were synthesizer and evaluated against bradykinin B1 receptor. In vitro metabolic stability data were reported for the key compounds.The analgesic effect of compound 20 from the phenantridine series was proved in-vivo.
Assuntos
Acetamidas/síntese química , Antagonistas de Receptor B1 da Bradicinina , Fenantrenos/síntese química , Quinolinas/síntese química , Acetamidas/farmacologia , Analgésicos/síntese química , Analgésicos/farmacologia , Linhagem Celular , Humanos , Metabolismo , Fenantrenos/farmacologia , Quinolinas/farmacologiaRESUMO
It is known that non-steroidal anti-inflammatory drugs increase cardiovascular (CV) morbidity and mortality. In this study, we examined whether a novel anti-inflammatory drug, bradykinin B1 receptor antagonist (FGY-1153) treatment could influence the development of hypertensive organ damages in spontaneously hypertensive rats (SHR). SHRs were treated with low (FGY-120) or high dose FGY-1153 (FGY-400) and with placebo (Control) for 26 weeks. Wistar-Kyoto rats were used as aged-matched, normotensive controls (WKY). Body weight, food consumption and blood pressure were measured regularly. Echocardiography was performed at the beginning and at the end of the study. Light and electron microscopic analysis of heart and great vessels were performed, and the extent of fibrotic areas was measured. The phosphorylation state of prosurvival Akt-1/glycogen synthase kinase (GSK)-3ß pathway and the activation of signaling factors playing part in the fibrotic processes - mitogen activated protein kinases (MAPKs), and TGF-ß/Smad2 - were monitored using Western-blot. Body weight and food consumption as well as the elevated blood pressure in SHRs was not influenced by FGY-1153 treatment. However, both doses of FGY-1153 treatment decreased left ventricular (LV) hypertrophy and diastolic dysfunction in hypertensive animals. Moreover systolic LV function was also preserved in FGY-120 group. Increased intima-media thickness and interstitial fibrosis were not significantly diminished in great vessels. FGY-1153 treatment inhibited the expression of TGFß and the phosphorylation of SMAD2 in the heart. Our results suggest that the tested novel anti-inflammatory compound has no deleterious effect on CV system, moreover it exerts moderate protective effect against the development of hypertensive cardiopathy.
RESUMO
(4-Benzylpiperidine-1-yl)-(6-hydroxy-1H-indole-2-yl)-methanone (6a) derived from (E)-1-(4-benzylpiperidin-1-yl)-3-(4-hydroxy-phenyl)-propenone (5) was identified as a potent NR2B subunit-selective antagonist of the NMDA receptor. To establish the structure-activity relationship (SAR) and to attempt the improvement of the ADME properties of the lead, a series of compounds were prepared and tested. Several derivatives showed low nanomolar activity both in the binding and in the functional assay. In a formalin-induced hyperalgesia model in mice, 6a and (4-benzylpiperidine-1-yl)-[5(6)-hydroxy-1H-benzimidazol-2-yl]-methanone (60a) were as active as besonprodil (2) after oral administration. A CoMSIA model was developed based on binding data of a series of indole- and benzimidazole-2-carboxamides.
Assuntos
Analgésicos/síntese química , Benzimidazóis/síntese química , Indóis/síntese química , Piperazinas/síntese química , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Analgésicos/química , Analgésicos/farmacologia , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Cálcio/metabolismo , Células Cultivadas , Técnicas In Vitro , Indóis/química , Indóis/farmacologia , Espaço Intracelular/metabolismo , Masculino , Camundongos , Modelos Moleculares , Medição da Dor , Técnicas de Patch-Clamp , Piperazinas/química , Piperazinas/farmacologia , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Relação Quantitativa Estrutura-Atividade , Ensaio Radioligante , Ratos , Ratos WistarRESUMO
INTRODUCTION: Majority of the work for establishing nitroglycerin (NTG)-induced migraine models in animals was done in rats, though recently some studies in mice were also reported. Different special formulations of NTG were investigated in various studies; however, NTG treated groups were often compared to simple saline treated control groups. The aim of the present studies was to critically assess the utility of a panel of potential outcome measures in mice by revisiting previous findings and investigating endpoints that have not been tested in mice yet. METHODS: We investigated two NTG formulations, Nitrolingual and Nitro Pohl, at an intraperitoneal dose of 10mg/kg, in comparison with relevant vehicle controls, and evaluated the following outcome measures: light aversive behaviour, cranial blood perfusion by laser Doppler imaging, number of c-Fos- and neuronal nitrogen monoxide synthase (nNOS)-immunoreactive neurons in the trigeminal nucleus caudalis (TNC) and trigeminal ganglia, thermal hyperalgesia and tactile allodynia of the hind paw and orofacial pain hypersensitivity. RESULTS: We could not confirm previous reports of significant NTG-induced changes in light aversion and cranial blood perfusion of mice but we observed considerable effects elicited by the vehicle of Nitrolingual. In contrast, the vehicle of Nitro Pohl was apparently inert. Increased c-Fos expression in the TNC, thermal hyperalgesia, tactile allodynia and orofacial hypersensitivity were apparently good endpoints in mice that were increased by NTG-administration. The NTG-induced increase in c-Fos expression was prevented by topiramate but not by sumatriptan treatment. However, the NTG-induced orofacial hypersensitivity was dose dependently attenuated by sumatriptan. DISCUSSION: Our results pointed to utilisable NTG formulations and outcome measures for NTG-induced migraine models in mice. Pending further cross-validation with positive and negative control drugs in these mouse models and in the human NTG models of migraine, these tests might be valuable translational research tools for development of new anti-migraine drugs.
Assuntos
Transtornos de Enxaqueca/tratamento farmacológico , Nitroglicerina/farmacologia , Animais , Modelos Animais de Doenças , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Masculino , Camundongos , Transtornos de Enxaqueca/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Avaliação de Resultados em Cuidados de Saúde , Proteínas Proto-Oncogênicas c-fos/metabolismo , Núcleos do Trigêmeo/efeitos dos fármacos , Núcleos do Trigêmeo/metabolismo , Vasodilatadores/farmacologiaRESUMO
INTRODUCTION: Accurate preclinical modeling of diabetic complications such as retinopathy, nephropathy and neuropathy is crucial to enable the development of novel preventative therapies. The aims of this study were to establish a model of long-term diabetes with sustained medium scale hyperglycemia and characterize the pathological changes detectable after 4months, with particular respect to dependence on the degree of hyperglycemia. METHODS: Streptozotocin-induced diabetic CFY rats were subjected to four different insulin substitution protocols to achieve different levels of glycemic control (Diabetic 1-4 groups). Eyes were investigated by ophthalmoscopy, kidney function by urine analysis, and neuropathy by functional tests. Retinal and renal morphological evaluations were performed by histology, immuno-histochemistry and electron microscopy. RESULTS: Rats of the Diabetic 3 group showed massive hyperglycemia-dependent anterior segment neovascularization, enhanced total retinal score and retinal apoptotic cell number, degeneration of dopaminergic amacrine cells, increased glomerular PAS-positivity, altered excreted total protein/creatinine ratio and cold allodynia, parallel with medium scale hyperglycemia (blood glucose level between 22 and 25mmol/L) and satisfying state of health. DISCUSSION: We established a treatment protocol in rats enabling complex investigation of diabetic retinopathy, nephropathy and neuropathy on a long-term period. Clearly hyperglycemic dependent parameters of these complications serve as good outcome measures for preclinical trials. Our results provide a useful basis for designing studies for testing preventative treatments as well as other translational medical research in this field.
Assuntos
Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/patologia , Retinopatia Diabética/complicações , Retinopatia Diabética/patologia , Animais , Glicemia/metabolismo , Peso Corporal/fisiologia , Diabetes Mellitus Experimental/sangue , Nefropatias Diabéticas/sangue , Retinopatia Diabética/sangue , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
INTRODUCTION: Centrally muscle relaxants (CMRs) are used mainly for treating muscle spasticities of neurological origin, and painful muscle spasms due to rheumatologic conditions. Their use is frequently associated with dose-limiting adverse effects. New drugs with improved side-effect characteristics are badly needed. However, there is no general agreement in the pharmacological literature on what methods are adequate to assess CMR effect and side effects in behaving rodents, which may hinder the development of new drugs. Here we report on the establishment of a simple pharmacological test battery, which was used to compare efficacies and side effect profiles of 11 compounds with central muscle relaxant action, in mice (intraperitoneal application). METHODS: For measuring muscle relaxant activity, (1) a new tremor model (GYKI 20039-induced tremor) and (2) the morphine-induced Straub-tail assay were used. The former, newly developed method has advantages over harmaline- or LON-954-induced tremor. For detecting side effect liability (ataxia, sedation, impairment of voluntary motor functions), (1) the rota-rod test, (2) measurement of spontaneous motility, (3) the weight-lifting test and (4) the thiopental sleep test were used. RESULTS: Among the 11 muscle relaxant compounds tested (tolperisone, eperisone, silperisone, diazepam, baclofen, tizanidine, afloqualon, mephenesin, zoxazolamine, memantine and carisoprodol), the calculated safety ratios (i.e. ID50 for side effect/ID50 for muscle relaxant effect) varied in a wide range. Silperisone seems to have the most advantageous profile (safety ratios range between 1.7 and 3.3 in the different pairs of assays) compared to the other tested drugs with lower (one or more ratios below 1.5, and often far below 1) and more varying ratios. DISCUSSION: Therapeutic indices calculated from the results of these in vivo experiments for the clinically used muscle relaxants are in agreement with their adverse effect profiles in humans. Thus the present test battery seems to be suitable for predicting the possible clinical utility of newly synthesized compounds.
Assuntos
Relaxantes Musculares Centrais/farmacologia , Tremor/prevenção & controle , Animais , Baclofeno/farmacologia , Derivados de Benzeno/farmacologia , Citalopram/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Harmalina/toxicidade , Ketanserina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Morfina/farmacologia , Atividade Motora/efeitos dos fármacos , Relaxantes Musculares Centrais/efeitos adversos , Relaxamento Muscular/efeitos dos fármacos , Piperidinas/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Sertralina/farmacologia , Sono/efeitos dos fármacos , Especificidade da Espécie , Comportamento Estereotipado/efeitos dos fármacos , Tiazóis/toxicidade , Tiopental/farmacologia , Tolperisona/farmacologia , Resultado do Tratamento , Tremor/induzido quimicamente , Ureia/análogos & derivados , Ureia/toxicidadeRESUMO
The trigeminovascular system has a pivotal role in the pathomechanism of migraine. The aim of the present study was to further develop existing models of migraine making them more suitable for testing the effects of compounds with presumed antimigraine activity in anaesthetised rats. Simultaneous recording of ongoing activity of spontaneously active neurons in the trigeminocervical complex as well as their discharges evoked by electrical stimulation of the dura mater via activation of A- and C-sensory fibres were carried out. Effects of sumatriptan, propranolol and topiramate were evaluated prior to and after application of a mixture containing inflammatory mediators on the dura. Propranolol (10 mg/kg s.c) and topiramate (30 mg/kg s.c.) resulted in a tendency to decrease the level of both spontaneous and evoked activity, while sumatriptan (1 mg/kg s.c.) did not exhibit any effect on recorded parameters. Application of an inflammatory soup to the dura mater boosted up spontaneous activity, which could be significantly attenuated by propranolol and topiramate but not by sumatriptan. In addition, all compounds prevented the delayed increase of spontaneous firing. In contrast to the ongoing activity, evoked responses were not augmented by inflammatory mediators. Nevertheless, inhibitory effect of propranolol and topiramate was evident when considering A- or C-fibre responses. Findings do not support the view that electrically evoked responses are useful for the measurement of trigeminal sensitization. It is proposed however, that inhibition of enhanced firing (immediate and/or delayed) evoked by inflammatory mediators as an endpoint have higher predictive validity regarding the clinical effectiveness of compounds.
Assuntos
Transtornos de Enxaqueca/patologia , Neurônios/fisiologia , Sumatriptana/farmacologia , Nervo Trigêmeo/fisiopatologia , Núcleos do Trigêmeo/patologia , Potenciais de Ação/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Estimulação Elétrica , Frutose/análogos & derivados , Frutose/farmacologia , Frutose/uso terapêutico , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Masculino , Transtornos de Enxaqueca/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Valor Preditivo dos Testes , Propranolol/farmacologia , Propranolol/uso terapêutico , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacos , Sumatriptana/uso terapêutico , Topiramato , Vasodilatadores/farmacologia , Vasodilatadores/uso terapêuticoRESUMO
Windup, the frequency dependent build-up of spinal neuronal responses, is implicated in the development of central sensitization of nociceptive pathways. N-methyl-D-aspartate (NMDA) receptors have been shown to be involved in these processes but the role of various receptor subtypes at the spinal level is not fully understood. In our experiments, we compared the inhibitory effect of MK-801 (a nonselective NMDA receptor antagonist, 0.01-3 mg/kg i.v.) and CI-1041 (an NR2B subunit specific NMDA receptor antagonist, 0.3-10 mg/kg i.v.) on the formation of dorsal horn neuronal windup in spinalized rats, in vivo. Both types of antagonist blocked windup considerably at doses not affecting the normal synaptic transmission. These results are in agreement with the well-documented effectivity of NR2B subtype selective NMDA receptor antagonists in chronic pain models and give the first direct evidence that spinal mechanisms are involved in this effect.
Assuntos
Células do Corno Posterior/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Medula Espinal/citologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/efeitos da radiação , Análise de Variância , Animais , Benzoxazóis/farmacologia , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Masculino , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/efeitos da radiação , Piperidinas/farmacologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
N-Methyl-D-aspartate (NMDA) receptor-mediated glutamatergic neurotransmission is thought to play a central role in the development of alcohol dependence and this alteration is supposed to be due to a differential up-regulation of the NR2B type of subunits. In this work, we examined the effect of some known (CP-101,606; CI-1041 and Co-101,244) and novel indole-2-carboxamide derivative NR2B subunit selective NMDA receptor antagonists (SSNAs) (RG-13579 and RG-1103) on the neurotoxic effect of withdrawal in ethanol pre-treated cultures of rat cortical neurones. The extent of neurotoxicity was estimated by measuring the activity of lactate dehydrogenase (LDH) that was released into the culture medium during the 24h withdrawal period. Here, we demonstrate that NR2B SSNAs given in the course of the withdrawal potently reduced the LDH release in ethanol pre-treated cultures. One of our novel compound, RG-1103, proved to be more potent than the reference NR2B SSNAs tested in this work having similar potency as the most potent but non-subunit selective NMDA receptor antagonist dizocilpine (MK-801). Acamprosate, a currently used therapeutic drug for the treatment of alcoholism was also effective although only in high micromolar concentrations. According to these observations, NR2B SSNAs are potent inhibitors of ethanol-withdrawal-induced neurotoxicity and considering that these agents have acceptable side effect profiles, they could be promising therapeutic candidates in the pharmacotherapy for physical signs of acute alcohol-withdrawal and associated neuronal damage.
Assuntos
Córtex Cerebral/citologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Síndrome de Abstinência a Substâncias/patologia , Acamprosato , Dissuasores de Álcool/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Células Clonais , Citosol/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/biossíntese , Proteínas Recombinantes/biossíntese , Taurina/análogos & derivados , Taurina/farmacologiaRESUMO
Eleven analogues of nifedipine (NP) showed synergistic interactions with ampicillin (Ap) and erythromycin (Er) on Escherichia coli K12LE140/F'lac. The antibacterial effect of Ap was enhanced by most analogues but compound (G9) and (+/-)-verapamil (VP) were antagonistic. Two of the 11 compounds (G7, G8) were synergistic with Er and four were additive. With a sensitive clinical isolate of E. coli Gy-1/Ap(sens)Er(res), compound G1 antagonized the antibacterial effect of Ap and a synergistic effect was found in the combination of Er with G4, G5, G6 or G7. None of the drugs had any effect on a multidrug resistant (MDR) clinical isolate of E. coli Gy-2/Ap(res)Er(res).