Dissertations / Theses on the topic 'Nitroindazole'

Orientador: Elenice Aparecida de Moraes Ferrari
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O óxido nítrico (NO), um neurotransmissor não convencional, tem papel importante em processos neurobiológicos de comportamento e de memória. Sua síntese é mediada por três isoformas de sintase do óxido nítrico (NOS): a neuronal (nNOS), a endotelial (eNOS) e induzível (iNOS). Este trabalho analisou o efeito do 7-nitroindazole (7-NI), um inibidor seletivo da nNOS, no condicionamento clássico aversivo em pombos. Foram usados 4 grupos: tratados com 7-NI (grupo 7-nitroindazole; G7-NI, n=5), tratados com óleo de amendoim (grupo veículo; GV, n=5), controle/sem tratamento (grupo controle; GC, n=5) e grupo não tratado/não condicionado (grupo manipulação; GM, n=5). A administração i.p. de 7-NI (25 mg/kg), ou do óleo de amendoim foi feita imediatamente após o treinamento. O G7-NI, o GV e o GC receberam três associações som-choque (5°, 10° e 15º minutos) numa sessão de 20 min. O teste a o contexto foi realizado 24 horas depois. As sessões foram gravadas para posterior transcrição e análise comportamental. A ocorrência da resposta de congelamento durante o treino não diferiu entre os grupos (p>0,05), mas durante o teste foi menor para o G7-NI em comparação ao treino (p<0.01) e aos demais grupos no teste (p<0.001). A atividade da NOS dependente de Ca++ no hipocampo foi menor no G7-NI do que nos outros grupos (p<0,01). Análise por Western blot indicou aumento na expressão de nNOS no G7-NI (p<0,05). A administração sistêmica de 7-NI teve um efeito amnésico sobre a memória contextual aversiva, indicando que a atividade da NOS dependente de Ca++ é importante para os processos de condicionamento clássico aversivo em pombos.
Abstract: Nitric oxide (NO) is an unsual neurotransmitter that plays an important role in neurobiological functions underlying behavior and memory. NO synthesis and release can be mediated by three isoforms of NO synthases (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). This study examined the effect of 7-nitroindazole (7-NI), a selective nNOS inhibitor, on contextual fear conditioning in pigeons. Four groups of pigeons were used: treated with 7-NI (7-NI; n=5), treated with peanut oil (Vehicle; n=5), non treated controls (Control; n=5) and non treated and no-trained controls (Non-trained; n=5). Treatment consisted in 7-NI (25 mg/kg; i.p.) or vehicle (peanut oil) administration, immediately after training. All the animals were trained in one 20 min session during which three tone-shock pairings (5th, 10th and 15th minutes) were presented. The test to the context was conducted 24h later. Behavioral categories were analyzed through the transcription of video-tapes of the sessions. The groups 7-NI, Vehicle and Control showed no significant differences in freezing during the conditioning session (p>0.05). During the test to the context the group 7-NI expressed significantly lower freezing as compared to Vehicle and Control (p<0.05). The 7-NI pigeons showed lower hippocampal activity of Ca++ dependent-NOS than Vehicle and Control groups (p<0.01). Western blot analysis indicated significant increase in nNOS expression (p<0.05). The systemic administration of 7-NI induced amnestic effects on contextual fear memory that evidence that Ca++-dependent NOS activity is required for fear conditioning in pigeons.
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular

Parkinson's disease (PD) is a progressive neurodegenerative disorder of unknown cause that afflicts about 1.5 million Americans. The characteristic feature of PD is a deficiency of dopamine in the terminals of nigrostriatal neurons. Two enzyme systems, the neuronal form of nitric oxide synthase (nNOS) and monoamine oxidase B (MAO-B), have been linked to neurodegenerative pathways leading to PD. Several MAO-B and nNOS inhibitors have been evaluated for their neuroprotective properties in the mouse model of neurodegeneration which employs the parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). One such compound is 7-nitroindazole (7-NI), a compound which is reported to inhibit both enzymes.

This thesis focuses on the synthesis and biological evaluation of a potential prodrug form of 7-NI and related indazolyl containing compounds which are designed to release the active drugs following a metabolic bioactivation process. These studies have led to a detailed description of the nucleophilic aromatic substitution reactions between 4-chloro-1-methylpyridinium iodide and the indazolyl reactants that were employed as the initial step in the synthesis of the target compounds. The MAO-B substrate and inhibition properties of these "prodrugs" as well as the parent indazolyl compounds were examined. The results are discussed in relation to a previously developed active site model of MAO-B.
Master of Science

Orientador: Elenice Aparecida de Moraes Ferrari
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O óxido nítrico (NO) é um neurotransmissor não convencional o qual tem papel importante em processos neurobiológicos de comportamento e de memória. Sua síntese é mediada por três isoformas de sintase do óxido nítrico (NOS): a neuronal (nNOS), a endotelial (eNOS) e a induzível (iNOS). Este trabalho investigou os efeitos da administração do 7- nitroindazol (7-NI), inibidor preferencial da nNOS, na consolidação e reconsolidação da memória do condicionamento clássico aversivo. Pombos adultos foram atribuídos a 5 grupos: Foram usados 5 grupos: grupo 7-nitroindazole (7-NI) (100nmol/0.5?/l; DMSO (20%), NaOH (50mM) e Tween-80 (16%) diluído em PBS; i.c.v.), grupo veículo (VEIC) (0,5?/l; DMSO (20%), NaOH (50mM) e Tween-80 (16%) diluído em PBS, i.c.v.), grupo condicionado/não tratado (COND), grupo contexto/não-tratado (CONT) e grupo não tratado/não condicionado (NÄIVE). Sete dias após implante de microcânula intracerebroventricular (i.c.v.), ocorreu o condicionamento com três associações contextochoque numa sessão de 20 min. O teste e o re-teste consistiram na re-exposição ao contexto do condicionamento por 5 min. O intervalo entre sessões foi de 24h. A administração de 7-NI ou do veículo ocorreu imediatamente após o treino (Experimento 1) ou após o re-teste (Experimento 2). A atividade enzimática da NOS dependente e independente de Ca2+ e da expressão protéica da nNOS foram realizadas no tecido hipocampal. No Experimento 1, a ocorrência de congelamento no teste do 7-NI foi menor do que no treino (p<0.01) e no teste do COND e VEIC (p < 0.001). A atividade da NOS dependente de Ca++ no 7-NI foi menor do que no COND e VEIC (p<0,01), mas não diferiu do CONT e do NÄIVE. A expressão protéica de nNOS não diferiu entre os grupos (p<0,05). No Experimento 2, houve diminuição dos comportamentos defensivos, incluindo o congelamento, no re-teste do 7-NI comparado com VEIC e COND (p<0.05), mas os grupos não diferiram quanto à atividade de NOS dependente de Ca2+ ou à expressão protéica da nNOS. Conclui-se que o 7-NI interferiu na consolidação e a reconsolidação da memória, indicando a ativação da via de sinalização do óxido nítrico no hipocampo em processos da memória de medo condicionado ao contexto em pombos
Abstract: Nitric oxide (NO) is an unconventional neurotransmitter which plays an important role in neurobiological processes of behavior and memory. Its synthesis is mediated by three isoforms of nitric oxide synthase (NOS): the neuronal (nNOS), the endothelial (eNOS) and the inducible (iNOS). This study investigated the effects of the administration of 7- nitroindazole (7-NI), a preferential nNOS inhibitor, in the consolidation and reconsolidation of aversive classical conditioning memory. Adult male pigeons were assigned to 5 groups: 7-nitroindazole, 7-NI (100nmol/0.5?/l; DMSO (20%), NaOH (50 mM) and Tween-80 (16%) diluted in PBS; i.c.v.) Vehicle group; VEH (0.5 ? / L; DMSO (20%), NaOH (50 mM) and Tween-80 (16%) diluted in PBS; i.c.v.), conditioning/non-treated group (COND), context/non-treated group (CONT) and non-conditioning/non-treated group (NÄIVE). Seven days after implantation of intracerebral ventricular (i.c.v.) microcannula the conditioning occurred with three context-shock associations in a session of 20 min. During the testing and retesting sessions pigeons were reexposed to the conditioning context for 5 min. The between sessions interval was 24h. Administration of 7-NI or vehicle occurred immediately after training (Experiment 1) or after testing (Experiment 2). The enzymatic activity of Ca2+ dependent and independent NOS and protein expression of nNOS in the hippocampus tissue were carried out following the behavioral test or retest. In Experiment 1, the occurrence of freezing in the testing session of 7-NI group was lower than in the training (p <0.01) and the testing sessions of COND and VEH groups (p <0.001). The activity of Ca2+ dependent NOS in the 7-NI group was lower than in COND and VEH groups (p <0.01) but did not differ from CONT and NÄIVE groups. The nNOS protein expression in the hippocampus did not differ among the different groups (p<0.05). In Experiment 2, there was a decrease of defensive behaviors, which include freezing, in the retest of the 7-NI compared with VEH and COND groups (p <0.05), but the groups did not differ in the activity of Ca2+ dependent NOS or the protein expression of nNOS. We conclude that 7-NI interfered on the consolidation and reconsolidation of memory, indicating activation of the nitric oxide signaling pathway in the hippocampus and in memory processes of conditioned fear context in pigeons
Mestrado
Fisiologia
Mestra em Biologia Funcional e Molecular

Post-traumatic stress disorder (PTSD), an anxiety disorder, may develop after experiencing or witnessing a severe traumatic event. Characteristic symptoms include hyper arousal and amnesic symptoms, while volume reductions in the hippocampus of these patients appear correlated with illness severity and the degree of cognitive deficit. Stress-induced increases in plasma cortisol have been implicated in this apparent atrophy of the hippocampus, although, clinical studies have described a marked suppression of plasma cortisol in PTSD. Given this hypocortisolemia, the basis for hippocampal neuro degeneration and cognitive decline remains unclear. While stress-related hippocampal structural changes have been linked to the neurotoxic effects of glucocorticoids and glutamate. NMDA-NO pathways have been found to play a causal role in anxiety-related behaviours. Prior exposure to trauma is an important risk factor for PTSD. In most instances the disorder becomes progressively worse over time, possibly with a delayed onset, suggesting a role for sensitization. In this study a time-dependent sensitization (TDS) model was used to induce PTSD-like sequelae in male Spraque-Dawley rats. The TDS-model is based on exposure to acute stressors, with a reminder of the trauma, in the form of re-exposure to one of the acute stressor, seven days later. NOS-activity, NMDA receptor parameters (Bmax and Kd) and GABA levels in the hippocampus of rats, as well as plasma corticosterone levels were determined 21 days after exposure to the TDS-model. Increased levels of corticosterone were measured after exposure to acute stress, but these levels were found to decrease below basal levels 21 days after the re-exposure, thus mimicking glucocorticoid levels in patients with PTSD. These findings may also imply that the increase in glucocorticoid levels after stress exposure is only the initial step in a cascade of events leading to neuronal damage in the hippocampus. This study also found that stress-restress evoked a long-lasting increase in hippocampal NOS activity that was accompanied by a reactive down-regulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. Subsequently, animals were chronically treated with certain pharmacological agents prior to exposure to the TDS-model to determine possible approaches for inhibiting the induction of PTSD. Pre-treatment with fluoxetine, currently indicated in the treatment of PTSD. and the nNOS inhibitor, 7-nitroindazole, had no effect on the increased NOS activity measured 21 days afler exposure to the TDS-model. Pre-treatment with the iNOS inhibitor, aminoguanidine, however, resulted in inhibition of the observed increase in hippocampal NOS-activity, implicating a possible role for the iNOS isoform in the etiology of PTSD. Treatment with ketoconazole, an inhibitor of glucoccfticoid synthesis, resulted in inhibition of the increase in NOS-activity observed after exposure to TDS-stress, thus indicating a possible link between stress glucocorticoid-release and NO synthesis. These perturbations may have importance in explaining the increasing evidence for stress-related hippocampal degenerative pathology and cognitive deficits seen in patients with PTSD. Uncovering and understanding the role of NO in PTSD will hopefully lead to the development of selective therapeutic agents in disorders like PTSD. as well as providing a better understanding of basic processes underlying normal and pathological neuronal functions in PTSD.
Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Posttraumatic stress disorder (PTSD) is a severe anxiety disorder characterised by hypothalamic-pituitary-adrenal (HPA)-axis abnormalities, hyperarousal, anxiety, flashbacks of trauma memories and avoidance. Increasing evidence is now accumulating that the disorder is also associated with shrinkage of the hippocampus and cognitive dysfunction that may have its origin in stress-induced excitotoxicity. Animal studies have indeed highlighted a potential role of the excitotoxic glutamatenitric oxide (NO) pathway in the stress response. Since PTSD appears to be an illness that progresses and worsens over time after an initial severe traumatic event, this study has used an animal model that emphasises repeated trauma to investigate the effect of stress on hippocampal NO synthase (NOS) activity, the release of the nitrogen oxide metabolites of NO (NOx), and also the evoked release of cGMP. Furthermore, the modulation and dependency of these responses on glutamate, NO and cGMP activity using drugs selective for these targets, will also be investigated. Rats (n=10/group) were exposed to repeated stress together with saline or drug administration immediately after the stress procedure and continuing for one week post-stress. The animals were then sacrificed for assay of hippocampal NOS activity, NO, and cGMP accumulation. Animals received either the glutamate-NMDA receptor antagonist, memantine (MEM;5mg/kg ip/d), the neuronal NOS selective inhibitor, 7- nitroindazole monosodium salt (7-NINA;20mg/kg ip/d), the cGMP-specific PDE inhibitor, sildenafil (SIL;10mg/kg ip/d) or the NFkb antagonist, pyrollidine dithiocarbamate (PDTC;70mg/kg ip/d). The latter inhibits the nuclear transcription factor, NFkb, responsible for inducing the expression of iNOS, while it also appears to mediate the glutamatergic actions on NOS expression, Stress significantly increased hippocampal NOS activity, as well as significantly increased hippocampal cGMP and NO, levels. These increases were blocked by pretreatment with either PDTC or 7-NINA, while memantine was without effect. Sildenafil significantly augmented stress induced NO, accumulation, as well as cGMP. although the latter failed to reach significance. 7-NINA and memantine significantly blocked the increase in cGMP evoked by time-dependent sensitisation (TDS)-stress, with PDTC attenuating this response, but not significantly. Additionally, administration of each drug separately for seven days without exposure to stress, did not evoke significant changes in NOx levels, compared to the control group. However, significant increases in cGMP levels, compared to the control group, were found with all four drugs. Repeated trauma therefore activates the NO-cGMP pathway, possibly involving actions on both nNOS and iNOS. The NMDA receptor appears less involved after chronic repeated stress, and may have limited therapeutic implications. Sub-cellular NO-modulation, however, may represent an important therapeutic strategy in preventing the effects of severe stress and in treating PTSD.
Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

L'existence d'un stress oxydatif (SO) a été rapportée dans diverses pathologies cérébrales. Dans ce contexte, notre travail a consisté à rechercher les mécanismes déclenchés par le SO ainsi que leur contribution à la mort neuronale. Le SO a été induit par l'injection de malonate (3 æmol), une toxine mitochondriale, dans le striatum de rats. Dans ce modèle, nous avons montré une chute précoce du glutathion total, le principal antioxydant endogène, associée à une lésion massive et à un œdème cérébral. L'a-phényl tert-butyl nitrone, un composé antioxydant, a exercé dans ce modèle un effet antiœdémateux. L'injection de malonate entraîne également une production de NO, par la NO-synthase neuronale. Ce NO module l'activation de la caspase 3, une des enzymes mises en jeu lors de la mort par apoptose. Cependant, l'inhibition de la caspase-3 n'a pas exercé d'effet neuroprotecteur. Le SO induit par le malonate déclenche donc des mécanismes conduisant principalement à une mort de type nécrotique
Oxidative stress (OS) has been reported in various cerebral pathogenesis. In this context, our work aimed to elucidate the mechanisms triggered by OS and their contribution to neuronal death. Our model of OS consisted in an injection of malonate (3 æmol), a mitochondrial toxin, in the striatum of rats. In this model, we showed an early drop of total glutathione, the major endogenous antioxidant, associated with a massive lesion and a cerebral edema. A-phenyl tert-butyl nitrone, an antioxidant compound, exerts in this model an antiedematous effect. Malonate injection was followed by NO production by the neuronal NO-synthase. This NO modulates caspase-3 activation, one of the enzymes implicated in apoptotic cell death. Nevertheless, inhibition of caspase-3 did not exert a neuroprotective effect. Thus the OS induced by malonate leads to neuronal death mainly through necrotic mechanisms

Memoria para optar al título de Químico Farmacéutico
En esta tesis se estudió el efecto que tendrían la βCD y la 2,6-DMβCD sobre la solubilidad de un nuevo nitroderivado antichagásico, el 1-(2-(dimetilamino)etil)-3-metoxi-5- nitro-1H-indazol. Además se propuso la estructura tridimensional que adopta el 5-nitroindazol al interior de la cavidad de las diferentes CDs. Por medio de la metodología de variación continua y de solubilidad de fase, se encontró que ambas CDs generan compuestos de inclusión con una estequiometría de 1:1. Las constantes de asociación, Ka, encontradas fueron 186,5 M-1 y 3687,8 M-1 para la βCD y la DMβCD respectivamente. Se observó que 10 mM de βCD aumenta al doble la solubilidad del 5- NI; mientras que la DMβCD, a la misma concentración, aumenta seis veces la solubilidad del compuesto. El espectro ROESY del 5-NI libre permitió determinar la geometría que adopta el compuesto en solución acuosa. Se observó que el protón 7 del anillo aromático establece una interacción con los protones de la cadena alifática lateral, 1’, 2’ y 4’. Esto significaría que en solución, la cadena se encuentra plegada orientándose hacia la región aromática. Estos espectros también permiten establecer cómo es la inclusión del 5-NI al interior de las CDs. En el caso del complejo entre el 5-NI y βCD, el espectro ROESY indica que existen interacciones entre los protones aromáticos, 4, 6 y 7 del 5-NI y los protones del interior de la cavidad de la CD. Junto con esto se encontró que los protones que forman parte de la cadena alifática, 2’ y 4’, se desplazan cuando se encuentran formando el complejo de inclusión, lo que significaría que también interactúan con el interior de la cavidad. Lo anterior sugiere que el compuesto se incluye en la cavidad de la CD casi completamente. Por otro lado, también se observan interacciones entre el protón 7 del anillo aromático del 5-NI y los protones de la cadena alifática, indicando que ésta se encuentra plegada hacia la región aromática, de la misma forma que el 5-NI libre en solución. El complejo entre el 5-NI y la DMβCD, presenta una geometría de inclusión distinta, ya que las interacciones de la región aromática del 5-NI y los protones de la cavidad de la CD, indican que la orientación del 5-NI deja el núcleo bencénico hacia la abertura más ancha, mientras que el núcleo pirazólico, del indazol, hacia la abertura de diámetro menor. Además los desplazamientos de las señales de los protones de la cadena alifática, indican que se encuentra completamente incluida en la CD; sin embargo, se puede decir que la cadena se encuentra menos plegada en este complejo, pues no se observan interacciones con el protón 7 del 5-NI
In this thesis we studied the impact of the βCD and 2,6-DMβCD on the solubility of a new antichagasic nitroderivative, 1-(2-(dimethylamino) ethyl) -3-metoxi-5-nitro-1H-indazol . In addition, we proposed the three-dimensional structure that adopts 5-nitroindazol inside the cavity of different CDs. With the continuous variation and phase solubility methodologies, we found that both generate inclusion complexes with the different CDs. The stoichiometry was 1:1, and the asosiation constants, Ka, was 186.5 M-1 and 3687.8 M-1, for both βCD and DMβCD respectively. The geometry of the 5-NI derivative in aqueous solution, was determined by 2DROESY spectra. The aliphatic amino chain is folded and interacts with the aromatic H-7. The 2D-ROESY spectrum of the complex of 5-NI inside the CD, indicates that there are interactions between the aromatic protons, 4, 6 and 7 of 5-NI and protons inside the cavity of the CD. Besides the protons that are part of the aliphatic chain, 2' and 4’, they shift to a high filed when they are forming the inclusion complex. This suggests, that the compound is included in the cavity of the CD almost completely. On the other hand, there are certain interactions between the proton 7 of the aromatic ring of 5-NI and protons in the aliphatic chain, indicating that it is folded toward the aromatic region in the same way that 5-NI free solution . Whereas the complex between 5-NI and DMβCD, shows a different geometry of inclusion. Interactions of aromatic protons of the 5-NI with the CD, show that the orientation of 5-NI includes benzenic core towards the secondary rim, while the pirazolic core, is near the primary rim.

Memoria para optar al título de Químico
Este estudio fue desarrollado para investigar los mecanismos de reducción y reactividad de compuestos derivados de 5-nitroindazol, los cuales podrían tener actividad antitripanocida a través de un análisis de capacidad antioxidante por medio de la técnica de fluorescencia ORAC-FL y un estudio cinético UV-Vis, además de un estudio electroquímico. La formación del anión radical nitro fue estudiada por medio de voltametría cíclica, a través del cual se propuso un mecanismo de reducción común para todos los compuestos en estudio; una reducción monoelectrónica, seguido de una reacción ácido base y finalmente una segunda reducción monoelectrónica. Este mecanismo es debido a que estos compuestos poseen un hidrógeno lábil en sus estructuras. Para determinar la reactividad del hidrógeno lábil presente, se realizó un estudio de la capacidad antioxidante, a través del estudio ORAC-FL el cual determinó que el hidrogeno lábil presente en las estructuras de los compuestos estudiados es donado al radical libre AAPH en razón 1:1, por lo tanto se comprueba la labilidad de este hidrógeno. Por otra parte, se realizó el estudio de reactividad a través de la determinación de las constantes cinéticas de reacción por medio de la espectroscopia UV-Vis. En este estudio se pudo ver que la reacción de donación del hidrogeno lábil de los compuestos estudiados al radical DPPH es lenta, ya que se mostraron valores bajos de constantes de velocidad.
This study was undertaken to investigate the reduction mechanism and the reactivity of nitroindazole derivates, wich have antitrypanosida activity through a free radical-scavenging analysis by ORAC-FL assay and an UV-Vis kinetic study, moreover an electrochemical study. The nitro radical´s species formation was studied by cyclic voltametric, through which a common reduction mechanism was proposed; a monoelectronic reduction, followed by an acid-base equilibrium and finally a second monoelectronic reduction. The mechanism by which these compounds possess labile hydrogen in it structures. The antioxidant capacity of these compounds was determinate by the oxygen radical absorbing capacity assay using fluorescein (ORAC-FL), it showed that the labile proton present in this structures is given to the free radical AAPH in rate 1:1 therefore it is found the lability of this hydrogen. On the other hand, the reactivity of the nitroindazole derivates was studied through a kinetic study, by this assay were obtained the rate constants of the reduction reaction of DPPH radical for the antioxidant, it were obtained under pseudo-first-order conditions, lo which is studied by UV-Vis. The experimental results showed that the labile donation reaction of the hydrogen of the compounds studied the DPPH radical are slow, as it showed low rate constants.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia, Florianópolis, 2016
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A participação do óxido nítrico (NO) proveniente da isoforma neuronal da óxido nítrico sintase (NOS-1) tem sido reconhecida como um fator importante em diversas doenças, como a sepse. A inibição de alta seletividade in vivo dessa isoforma pelo 7-nitroindazol (7-NI) melhora parâmetros como mortalidade e reatividade vascular na sepse. Porém este composto possui curto tempo de meia vida plasmático (~2 h), limitando seu uso. Com o objetivo de melhor evidenciar o papel da NOS-1 em condições patológicas, desenvolvemos uma formulação de nanoemulsão de óleo de rícino contendo 7-NI e, então, a caracterizamos pelos aspectos de tamanho, índice de polidispersão, teor e eficiência de encapsulação. Uma metodologia analítica para a detecção do composto na nanoemulsão foi desenvolvida e validada. Também, investigamos a estabilidade térmica e à luz no armazenamento durante 30 dias. O estudo de estabilidade mostrou que sistema de nanoemulsões de 7-NI é fotossensível, sendo mais estáveis quando mantidas a 25oC, na ausência de luz. Avaliamos a produção de NO de maneira indireta pela mensuração de nitrito em cultura de células, 48 horas após a inibição com 7-NI livre ou nanoemulsionado. A nanoemulsão de 7-NI reduziu a produção de NO pela NOS-1 de maneira prolongada em células de músculo liso A7r5. Não houve inibição pelo 7-NI livre. Nem o composto livre e nem a nanoemulsão de 7-NI apresentaram citotoxicidade. A nanoemulsão e o composto livre mostraram o mesmo perfil dose-resposta de inibição da produção de NO proveniente da NOS-1 de maneira direta, através da intensidade de fluorescência da sonda DAF-FM DA, seletiva para o NO. Por fim, testamos os efeitos do pré-tratamento com a nanoemulsão de 7-NI em ratos sépticos pelo modelo de ligadura e perfuração do ceco (CLP) e em camundongos com sepse por pneumonia. A nanoemulsão de 7-NI preveniu em 40% a mortalidade nos dois modelos, enquanto que o 7-NI livre preveniu na mesma proporção apenas no modelo de sepse por CLP. Nossos resultados mostram que nanoemulsões melhoram o tempo de ação do 7-NI, podendo ser ferramenta interessante para o estudo de doenças que envolvam o desequilíbrio da produção de NO pela NOS-1.

Abstract : The role of nitric oxide (NO) derived fom the neuronal isoform of NO synthase (NOS-1) has been recognized as an important factor in several conditions such as sepsis. The inhibition of this isoform by a highly selective in vivo compound 7-nitroindazol (7-NI) improves parameters such as mortality and vascular reactivity in sepsis. However, 7-NI has a very short plasma half-life (~ 2 h), limiting its use. In order to provide a tool for better characterization of the role of NOS-1 in pathological conditions, we developed a formulation of castor oil nanoemulsion containing 7-NI and, then, characterized it by size, polydispersion index, content and encapsulation efficiency. An analytical method for detecting the compound in the nanoemulsion has been developed and validated. Also, we investigated the thermal and light stability for 30 days. The stability study showed that the system with the nanoemulsions of 7-NI is photosensitive, being more stable when maintained at 25°C in the absence of light. NO production was assessed indirectly by measuring nitrite in cell culture 48 hours after inhibition with free or nanoemulsion 7-NI. The nanoemulsion reduced NO production by NOS-1 in a long-lasting manner in A7r5 smooth muscle cells. NO inhibitory effect was induced by free 7-NI. Neither the free compound or the nanoemulsion and 7-NI showed cytotoxicity. The nanoemulsion and free compound showed the same profile of dose-response inhibition of NO production measured by the fluorescence intensity of DAF-FM probe, selective to NO. Finally, we tested the effects of pretreatment with the nanoemulsion 7-NI in septic rats by cecal ligation and puncture model (CLP) and in mice rendered septic by pneumonia. The nanoemulsion reduced the mortality by 40% mortality in both models, whereas the free 7-NI reduced it by the same amount only in the CLP model. Our results show that nanoemulsions extend the effective time of 7-NI, which may be an interesting tool to study diseases involving the imbalance of NO production by NOS-1.

Tesis para optar al grado académico de Doctor en Química
La presente tesis doctoral exhibe un estudio químico y biológico de dos familias de nitrocompuestos: derivados de Nitrofurano y derivados de Nitroindazol, con el propósito principal de obtener nuevos aspectos acerca de los mecanismos de acción tripanocida de estos potenciales nuevos fármacos, capaces de superar las limitancias que presentan las actuales drogas empleadas en el tratamiento de la enfermedad de Chagas. Nifurtimox, Benznidazol y otros fármacos empleados en contra el mal de Chagas y algunos tipos de cánceres son capaces de producir radicales libres, los cuales en presencia de oxígeno, generan a través de un ciclo redox, especies reactivas de oxígeno, las cuales son sustancias altamente reactivas. El exceso de radicales libres puede generar daño celular (estrés oxidativo), este daño ocurre sólo si la acción de estos radicales no es remediada por los mecanismos de defensa presentes en cada organismo, como son los antioxidantes. En el mismo orden de ideas, se utilizó la Resonancia de Espín Electrónico para obtener información directa de las especies radicalarias. Además el uso de la técnica de spin trapping permitió evidenciar especies reactivas de oxígeno en medio biológico, durante la metabolización de los compuestos testeados. Voltamperometría cíclica se empleó como una herramienta valiosa para evaluar el mecanismo de reducción de ambas familias, donde fue posible identificar un mecanismo de autoprotonación. Además resultó útil para la determinación parámetros de significancia biológica. Por otra parte, también se realizó la exploración de los confórmeros de mínima energía y de las propiedades electrónicas empleando cálculos teóricos, específicamente teoría de funcionales de la densidad (DFT). Estos indicaron que los derivados de nitrofurano neutros tendían a adoptar una conformación plana estabilizada, mientras que los radicalarios lo hacían a través del plegado de su cadena lateral respecto del anillo. La familia de nitroindazoles también mostró que las conformaciones más estables dependían de la influencia de los sustituyentes. La correlación de las propiedades electrónicas con el potencial de pico catódico fue posible empleando el funcional híbrido B3LYP/6-31G*+(d,p). Finalmente, los ensayos biológicos mostraron que los derivados de nitrofurano resultan ser activos contra T. cruzi cepa Dm28C, frente a sus formas morfológicas epimastigotes y tripomastigote, encontrando valores de IC50 comparables y mejores a los reportados para las drogas parentales
The present investigation show a chemical and biological study about two families of nitro compounds: Nitrofurane and Nitroindazole derivatives, whith the target to obtain the main characteristic about tripanocide action mechanism of its new potential drugs, able to overcome the limitation the current treatment of Chagas disease. Nifirtimox, benznidazol and other drugs used against Chagas disease and some of the other types of cáncer disease are able to produce free radicals in which the presence of the oxygen, create through the redox cicle a reactive oxygen species (ROS) they are higly reactive subtances . The excess of the free radical can produce a celular damage (oxidative stress) this damage ocurs only if the action of these free radicals is not avoided by the action of antioxidant mechanism present in the organism. In the same order we used the Electronic Spin Resonance to get direct information about the free radicals. Besides the technique use of Spin Trapping allowed to show the ractive oxygen species of in a biological enviroment, during the methabolism of the testing compound. We used a very important tool called Cyclic Voltametry in order to valued the reduction mechanism of these compund. It was posible to identify an autoprotonation mechanism, besides CV was used to obtain parameters of biologycal significance. In the other hand we also realized a conformational study and the electronic properties using theorical calculations, specifically Density Functional Theory (DFT). This study indicates that the neutral nitrofurane derivatives adopt one plane stabilized conformation, meanwhile the nitro radical structures adopted the stabilised conformation trough the plication of the lateral chains respect to the nitrofurane ring. The Nitroindazoles family showed that stabilised conformation depends on the influence of the subtituents. The correlation of the electronic properties with the cathodic peak potential was possbile using the Hybrid functional B3LYP/6-31G*+(d,p). Finally the biologycal essays showed that the nitrofuranes derivates are actives against T. cruzi strain Dm28C, obtained values of IC50, better and comparables with the parental drugs
Conicyt

Title: Neurological deficit after focal cerebral ischemia in rats - pharmacological intervention Objectives: The goal of the thesis was to determine the effect of 7-nitroindazole, a selective inhibitor of neuronal nitric oxide synthase, after focal ischemic stroke in rats. Methods: Twenty adult male Wistar rats were used in this experiment. The rats were randomly divided into four groups: ischemic stroke was given to half of them, the rest were sham operated. 10 animals were given 7-nitroindazole (25mg/kg) to protect neuronal ischemic brain damage. After a few weeks the rats were tested with a set of behavioral tests: Ladder rung walking test, Bar holding test, Rotarod test and Open field test. To evaluate the volume of brain damage the stereotactic method was used. The brain sections were cut and compared with atlas. This study was supported by Institute of Physiology, Academy of Sciences ČR in Prague. Results: The present results show that the 7-nitroindazole has no side effects on healthy rats. The long-term effect on rats after ischemic stroke was not proved. There were a few positive trends observed such as an increase of locomotor speed, increased explorative behaviour and better coordination outcome on RotaRod. On the other hand the brain tissue damage was bigger and the time of hanging in...

Name of the thesis Effect of nitric oxide on cerebral blood flow during neuronal activity Aim of the thesis The aim of this thesis is to determine whether the application of 7-nitroindazole, relatively specific inhibitor of neuronal nitric oxide synthase, affects the baseline blood pressure. Furthermore, to determine whether the application of the substance affects the baseline cerebral blood flow and whether it influences blood flow in brain during transcallosal stimulation with increasing frequency. Research method The research took place at the premises of the Institute of Physiology, Academy of Sciences of the Czech Republic. Experiments were carried out on laboratory albino Wistar rats. The group contained both experimental and control sample. General anesthesia was performed to rats, stimulating and sensing electrodes were implanted in epidural area of sensorimotor cortex and Laser Doppler flow probe was implanted into the contralateral hemisphere. A plastic catheter was applied in the carotid artery for measuring systemic blood pressure. In the first part of the experiment, we tested the effects of 7-nitroindazole on the systemic blood pressure. In the second part of the experiment, we investigated the effects of 7-nitroindazole on baseline cerebral blood flow. The third part of the...