Kindling epileptogenesis has been associated with a number of different forms of neuroplasticity in the hippocampus, including mossy fibre sprouting and an increase in both intracellular calcium and zinc. The purpose of this thesis was to determine whether interfering with the influx of calcium via the voltage gated calcium channels would interfere with kindling- induced plasticity. Both kindled and control rats were injected with either 5 or 25mg/kg of the L-type voltage gated calcium channel blocker, nifedipine, or a control vehicle, DMSO (dimethylsulfoxide). The kindled groups received a kindling stimulation twice a day for 11 days. It was revealed that both doses of nifedipine significantly increased afterdischarge duration (p<0.001) and furthermore, both doses of nifedipine were capable of significantly interfering with the rate of kindling (p<0.001). Three weeks following the last kindling stimulation, rats were perfused and brain tissue was processed according to the Timm method. The density of Timm granules, an indication of the level of intracellular zinc in the mossy fibre pathway, was quantified. The results of this analysis revealed that 25mg/kg of nifedipine is capable of significantly reducing the amount of intracellular zinc in both the IML (p<0.04) and the CA3 (p<0.01) region of the mossy fibre pathway, regardless of whether the rats had received kindling stimulations or not. These results provide support for the notion that nifedipine (5 or 25mg/kg) is an effective anticonvulsant agent. These results also suggest that, at a sufficient dose (25mg/kg), nifedipine can reduce the amount of intracellular zinc in the mossy fibre pathway in both kindled and non-kindled animals, suggesting that nifedipine may be a useful therapeutic agent for pathologies that have been associated with zinc-induced neurotoxicity. / Thesis / Master of Science (MSc)

A nifedipina atua como bloqueador de canais de cálcio inibindo o fluxo transmembrânico de ions Ca2+ no interior das células do músculo cardíaco e células do músculo liso vascular, o qual induz ao relaxamento do músculo liso e diminuição da resistência vascular periférica. É utilizado no Brasil no tratamento de hipertensão arterial sistêmica (HAS), faz parte da Relação Nacional de Medicamentos Essenciais do Ministério da Saúde e vem sendo distribuída pela Secretaria da Saúde do Estado de São Paulo. Uma vez que a determinação plasmática do fármaco contribui para maior segurança de seu uso, o objetivo deste trabalho se prendeu em padronizar e validar um método analítico em cromatografia em fase gasosa com detecção por captura de elétrons, sensível, específico e reprodutível para a quantificação das concentrações plasmáticas com a finalidade de avaliar a relação entre a dose diária de 60mg e a concentração plasmática versus a resposta da pressão arterial sistêmica, observando a ocorrência de possíveis reações adversas, alterações bioquímicas e hematológicas, em pacientes portadores de HAS, submetidos à farmacoterapia. O método apresentou linearidade na faixa de 10 a 200 ng.mL-1 , com coeficiente de correlação (r) igual a 0,9977. Coeficientes de variação de precisão intra e interensaio menor que 10% e recuperação absoluta da nifedipina de 74,47 a 75,97%. Os limites de detecção e quantificação do método foram de 1,0 e 2,0 ng.mL-1, respectivamente e o fármaco demonstrou ser estável por 90 dias quando armazenado a -70°C ao abrigo da luz. Os dados observados no presente estudo permitiram evidenciar que os pacientes apresentaram concentrações plasmáticas no intervalo terapêutico preconizado, as quais foram efetivas na redução da pressão arterial sistólica e diastólica. Estas concentrações não acarretaram efeitos adversos em nível do sistema hematológico e bioquímico estatisticamente significativos e em relação às reações adversas relacionadas ao medicamento, tais como: cefaléia, edema periférico vascular, tontura, hipotensão arterial, rubor, tosse e cãibras, apesar de relatadas de forma significativa pelos pacientes no inicio do tratamento, foram ao longo do mesmo minimizadas e pouco relatadas. / Nifedipine, a compound of dihydropyridine class, is a calcium-channel antagonists drug that inhibits the transmembrane influx of Ca+2 into cardiac muscle cells and vasculas smooth muscle cells throught specific ion channels. It induces smooth muscle relaxation and decreases peripheral vascular resistance. It is widely used for the treatment of high blood pressure, and is considered as a essencial medicine by the Brazilian government. In Sao Paulo state, this drug has been distributed to hypertensive patients in treatment. Since the drug quantification in plasma contributes for a drug safety use, the objective of the present study was to develop and validate a accurate, specific and reproducible method for the determination of nifedipine in plasma by gas chromatography with eletron capture detection. The validated method was applied in samples of hipertensive patients on 60 mg daily dose of nifedipine with the purpose to evaluate the relation between drug plasma concentration and it\'s daily dose versus the hemodymamic effects, possible side effects and biochemical and hematologic alterations. The method was linear over a concentration range of 10 -200 ng.mL-1 (r2>0,99). The coefficient of variation of intra- and inter-assay precision less than 10% and the recovery was higher than 74%. The limit of detection and quantification were 1,0 and 2,0 ng.mL-1, respectively. Nifedipine was found to be stable in samples stored at -70ºC for 90 days and protect from light. The result showed that patients with drug plasma concentration within therapeutics levels also showed systolic and diastolic blood pressure succesfully controled. Therefore, these patients do not manifested any adverse effects specially in biochemical and hematologic systems. Other adverse efects of nifedipine such as headache, peripheral edema, hypotension, redness, cramp and cough reported by the patients at the beggining of thetreatment, were gradually diminishing and rarely related.

A nifedipina atua como bloqueador de canais de cálcio inibindo o fluxo transmembrânico de ions Ca2+ no interior das células do músculo cardíaco e células do músculo liso vascular, o qual induz ao relaxamento do músculo liso e diminuição da resistência vascular periférica. É utilizado no Brasil no tratamento de hipertensão arterial sistêmica (HAS), faz parte da Relação Nacional de Medicamentos Essenciais do Ministério da Saúde e vem sendo distribuída pela Secretaria da Saúde do Estado de São Paulo. Uma vez que a determinação plasmática do fármaco contribui para maior segurança de seu uso, o objetivo deste trabalho se prendeu em padronizar e validar um método analítico em cromatografia em fase gasosa com detecção por captura de elétrons, sensível, específico e reprodutível para a quantificação das concentrações plasmáticas com a finalidade de avaliar a relação entre a dose diária de 60mg e a concentração plasmática versus a resposta da pressão arterial sistêmica, observando a ocorrência de possíveis reações adversas, alterações bioquímicas e hematológicas, em pacientes portadores de HAS, submetidos à farmacoterapia. O método apresentou linearidade na faixa de 10 a 200 ng.mL-1 , com coeficiente de correlação (r) igual a 0,9977. Coeficientes de variação de precisão intra e interensaio menor que 10% e recuperação absoluta da nifedipina de 74,47 a 75,97%. Os limites de detecção e quantificação do método foram de 1,0 e 2,0 ng.mL-1, respectivamente e o fármaco demonstrou ser estável por 90 dias quando armazenado a -70°C ao abrigo da luz. Os dados observados no presente estudo permitiram evidenciar que os pacientes apresentaram concentrações plasmáticas no intervalo terapêutico preconizado, as quais foram efetivas na redução da pressão arterial sistólica e diastólica. Estas concentrações não acarretaram efeitos adversos em nível do sistema hematológico e bioquímico estatisticamente significativos e em relação às reações adversas relacionadas ao medicamento, tais como: cefaléia, edema periférico vascular, tontura, hipotensão arterial, rubor, tosse e cãibras, apesar de relatadas de forma significativa pelos pacientes no inicio do tratamento, foram ao longo do mesmo minimizadas e pouco relatadas. / Nifedipine, a compound of dihydropyridine class, is a calcium-channel antagonists drug that inhibits the transmembrane influx of Ca+2 into cardiac muscle cells and vasculas smooth muscle cells throught specific ion channels. It induces smooth muscle relaxation and decreases peripheral vascular resistance. It is widely used for the treatment of high blood pressure, and is considered as a essencial medicine by the Brazilian government. In Sao Paulo state, this drug has been distributed to hypertensive patients in treatment. Since the drug quantification in plasma contributes for a drug safety use, the objective of the present study was to develop and validate a accurate, specific and reproducible method for the determination of nifedipine in plasma by gas chromatography with eletron capture detection. The validated method was applied in samples of hipertensive patients on 60 mg daily dose of nifedipine with the purpose to evaluate the relation between drug plasma concentration and it\'s daily dose versus the hemodymamic effects, possible side effects and biochemical and hematologic alterations. The method was linear over a concentration range of 10 -200 ng.mL-1 (r2>0,99). The coefficient of variation of intra- and inter-assay precision less than 10% and the recovery was higher than 74%. The limit of detection and quantification were 1,0 and 2,0 ng.mL-1, respectively. Nifedipine was found to be stable in samples stored at -70ºC for 90 days and protect from light. The result showed that patients with drug plasma concentration within therapeutics levels also showed systolic and diastolic blood pressure succesfully controled. Therefore, these patients do not manifested any adverse effects specially in biochemical and hematologic systems. Other adverse efects of nifedipine such as headache, peripheral edema, hypotension, redness, cramp and cough reported by the patients at the beggining of thetreatment, were gradually diminishing and rarely related.

Microemulsions (MEs) are dispersed systems, thermodynamically stable, isotropic, transparent, and stabilized by an interfacial film of surfactant compounds. In this study were obtained MEs from differents co - surfactant (ethanol - ET, isopropanol-ISO and propylene glycol - PG), Citrus sinensis (L.) Osbeck essential oil (CSEO), Tween 80 ® and phosphate buffer (pH 5.0). The co - surfactants were selected because they are tolerated by the skin. Therefore, the aim of the study was to evaluate the influence of co - surfactants in the formation of MEs containing Tween 80, CSEO and evaluate the influence of nifedipine (NFD) in the structure of systems formed. The MEs were obtained by constructing diagrams of pseudo-ternary phase in order to evaluate the influence of co-surfactant in the formation of MEs. The structural characterization of these systems was obtained by Polarized Light Microscopy (MLP), test electrical conductivity and Small angle X-ray scattering (SAXS). In each diagram, two formulations were selected, which was the criterion sense phase, in the O / A and bicontinuous, in which the NFD is incorporated. The formulations were evaluated by the techniques mentioned, to observe the influence of NFD and characterized by pH, droplet size, polydispersity index (PDI), surface tension and rheology. Interaction studies with model stratum corneum (SC) were performed using the techniques of differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). In the diagrams showed the greatest formation region of the MEs to the ISO (above 40 % T / Cot) instead of the co- surfactants ET and PG. When assessing the electrical conductivity has been observed that increasing the aqueous fraction resulted in an increase in conductivity. The SAXS curves demonstrated that all systems are characteristic of micellar structures and as the water content was added, it was observed the formation of larger structures. Therefore, from the results obtained, the NFD has been incorporated in the formulations selected. The MEs presented as stable systems, isotropic, translucent droplet size between 16 and 34 nm, PDI below 0.5 and pH in the range of 5.64 to 6.7, compatible with the skin and pH stability of the NFD. In conductivity tests for each co - surfactant, the MEs were classified as bicontinuous and O / A. The MEs exhibited reduced surface tension and the formulations containing ISO submitted a surface tension value (24,764 mN / m) lower than the other co - surfactants (ET - 29.512 mN/m PG - 31.784 mN/m), confirmed by the evaluation of the droplet diameter and obtaining the phase diagram. The MEs exhibited Newtonian behavior, with consistency indices (k) higher in formulations with higher proportions of T / Cot, result corroborated the structures observed by SAXS. The DSC and FTIR techniques have demonstrated the use of the MEs as permeation promoters, provided from interactions with SC model. Therefore, the diagrams have elucidated the effect of co- surfactant in the formation of MEs and the same interference suffered no NFD, suggesting their use as permeation enhancers on the skin on application. / As microemulsões (MEs) são sistemas dispersos, termodinamicamente estáveis, isotrópicos, transparentes, estabilizados por um filme interfacial de compostos tensoativos. Neste trabalho foram obtidas MEs a partir de diferentes co-tensoativos (etanol-ET, isopropanol-ISO e propilenoglicol-PG), óleo essencial de Citrus sinensis (L.) Osbeck (OECS), Tween 80® e tampão fosfato (pH 5.0). Os co-tensoativos foram selecionados por serem toleráveis pela pele. Assim, o objetivo deste trabalho foi avaliar a influência dos co-tensoativos na formação de MEs contendo Tween 80, OECS bem como avaliar a influência da nifedipina (NFD) na estrutura dos sistemas formados. As MEs foram obtidas pela construção de diagramas de fase pseudoternário com o propósito de avaliar a influência do co-tensoativo na zona de formação das MEs. A caracterização estrutural desses sistemas foi realizada por Microscopia de Luz Polarizada (MLP), ensaios de condutividade elétrica e espalhamento de raios-X a baixo ângulo (SAXS). De cada diagrama, foram selecionadas duas formulações, cujo critério foi o sentido de fases, na região de óleo-água (O/A) e bicontínuas, nas quais a NFD foi incorporada. As formulações foram reavaliadas pelas técnicas citadas, a fim de observar a influência da NFD, além de caracterizadas por pH, tamanho de gotícula, índice de polidispersividade (IPD), tensão superficial e reologia. Estudos de interação com modelo de estrato córneo (EC) foram realizados através das técnicas de Calorimetria exploratória diferencial (DSC) e Espectroscopia na região de infravermelho com transformada de Fourier (FTIR). Nos diagramas foi observada uma maior região de formação das MEs para o ISO (acima de 40% de T/Cot) ao contrário dos co-tensoativos ET e PG. Ao avaliar a condutividade elétrica foi observado que o aumento da fração aquosa proporcionou um aumento na condutividade. As curvas de SAXS demonstraram que todos os sistemas foram característicos de estruturas micelares e à medida que o conteúdo aquoso foi adicionado, observou-se a formação de estruturas com maiores dimensões. Logo, a partir dos resultados obtidos, a NFD foi incorporada nas formulações selecionadas. As MEs apresentaram-se como sistemas estáveis, isotrópicos, translúcidos, tamanho de gotículas entre 16 e 34 nm, IPD abaixo de 0,5 e pH na faixa de 5,64 a 6,07, compatíveis com a pele e pH de estabilidade da NFD. Nos ensaios de condutividade para cada co-tensoativo, as MEs foram classificadas como bicontínuas e O/A. As MEs apresentaram redução da tensão superficial, sendo que as formulações contendo ISO apresentaram um valor de tensão (24,764 mN/m) inferior aos demais co-tensoativos (ET- 29,512 mN/m e PG- 31,784 mN/m), confirmado pela avaliação do diâmetro de gotas e obtenção do diagrama de fases. As MEs apresentaram comportamento newtoniano, com os índices de consistência (k) maiores nas formulações com proporções mais elevadas de T/Cot, resultado que corroborou com as estruturas observadas por SAXS. As técnicas de DSC e FTIR demonstraram a utilização das MEs como promotores de permeação, a partir das interações proporcionadas com modelo de EC. Logo, os diagramas elucidaram a influência do co-tensoativo na área de formação de MEs e os mesmos não sofreram interferência da NFD, sugerindo a sua utilização como promotores de permeação em aplicação sobre a pele.

Micelles (MIs) and microemulsions (MEs) are classified as stabilized systems by surfactants and are very similar with respect to their structure and physico-chemical properties. As for the applicability, they have been deployed by being able to carry drugs which have a limited systemic bioavailability by oral route. The formation of these systems is mainly dependent on the types of components used, making it important to study its influence on the structure. The goal of this study was to obtain and characterize micellar and microemulsion systems (containing essential oil of Citrus sinensis (L.) Osbeck as oil phase) stabilized by a nonionic surfactant (Tween 80) and short-chain cosurfactant (alcohol ethyl) which may be used as a delivery system for nifedipine (NFD a model drug) seeking to verify the influence of the phases and the interaction of the drug in the structure of these systems. Ternary and pseudoternary phase diagrams for MIs and MEs, respectively, and were obtained from the formation regions, formulations were selected for physico-chemical characterization and incorporation of NFD. The macro and microscopic aspects were evaluated using polarized light microscopy (MLP), measures pH, electrical conductivity and surface tension. The average droplet size was measured by dynamic light scattering (DLS) and small angle x-ray scattering (SAXS). The results demonstrated that such systems are stable, optically isotropic and transparent in the absence and presence of drug. The droplet size decreases with increasing amount of surfactant to MIs and the mixture surfactant/cosurfactant to the MEs. The influence of the cosurfactant in MIs was negligible. The increase of the amount of oily phase in MEs caused an increase in the droplet size. The presence of NFD no influence on the structure of MIs, but for the MEs increased droplet size, suggesting that the NFD is the internal phase of the MEs. The modeling by SAXS curves for MIs and MEs most diluted were made and showed the interaction between the NFD and systems, confirming the previous results. Studies by Fourier Transformed Infra Red (FTIR) confirmed the interaction between MIs and MEs with the stratum corneum (EC) , which allows the use of these systems as permeation enhancers of NFD. / As micelas (MIs) e as microemulsões (MEs) são classificadas como sistemas estabilizados por tensoativos e se assemelham bastante com relação a sua estrutura e propriedades físico-químicas. Quanto a sua aplicabilidade, elas têm sido destacadas por serem capazes de veicular fármacos que possuem uma biodisponibilidade sistêmica limitada por via oral. A formação destes sistemas é dependente principalmente dos tipos de componentes utilizados, tornando-se relevante o estudo da sua influência na estrutura. O objetivo do presente trabalho foi a obtenção e caracterização de sistemas micelares e microemulsionados (contendo óleo essencial de Citrus sinensis (L.) Osbeck como fase oleosa), estabilizados por um tensoativo não iônico (Tween 80) e um cotensoativo de cadeia curta (álcool etílico), que possam ser utilizados como sistema de liberação para a nifedipina (NFD, um fármaco modelo), buscando verificar a influência das fases e a interação do fármaco na estrutura desses sistemas. Diagramas de fase ternário e pseudoternário para MIs e MEs, respectivamente, foram obtidos e a partir das regiões de formação, formulações foram selecionadas para caracterização físico-química e incorporação da NFD. Os aspectos macro e microscópicos foram avaliados utilizando microscopia de luz polarizada (MLP), medidas de pH, condutividade elétrica e tensão superficial. O tamanho médio de gotículas foi avaliado por espalhamento dinâmico de luz (DLS) e espalhamento de raios-x a baixos ângulos (SAXS). Os resultados demonstraram que esses sistemas são estáveis, isotrópicos e opticamente transparentes na ausência e presença de fármaco. O tamanho das gotículas diminuiu com o aumento da quantidade de tensoativo para as MIs e de mistura de tensoativo/cotensoativo para as MEs. A influência do cotensoativo nas MIs foi praticamente desprezível. O aumento da quantidade de fase oleosa nas MEs ocasionou um aumento do tamanho das gotículas. A presença da NFD não exerceu influência na estrutura das MIs, entretanto para as MEs aumentou o tamanho de gotículas, sugerindo que a NFD se encontra na fase interna das MEs. Os modelamentos das curvas de SAXS para as MIs e MEs mais diluídas foram realizados e mostraram a interação existente entre a NFD e os sistemas, corroborando com os resultados anteriores. Estudos por espectrofotometria de absorção na região do infravermelho com transformada de Fourier (FTIR) comprovaram a interação das MIs e das MEs com o estrato córneo (EC), o que possibilita o uso destes sistemas como promotores de permeação da NFD.

Chronic kidney disease (CKD) is defined as glomerular filtration rate (GFR) less than 60 mL/min/1.73 m2 for 3 months and is characterized by progressive loss of renal function. The second leading cause of CKD is hypertension. More than half of CKD patients also suffer from hypertension. Arteries and arterioles adjust to the fluctuations in the systematic blood pressure through a mechanism called autoregulation. In the kidneys, autoregulation protects the delicate glomeruli capillaries from high blood pressure and occurs through myogenic constriction (MC). MC refers to contraction of arterioles in response to an increase in the blood pressure. Chronically hypertensive individuals and animal models have an enhanced MC, leading to minimal renal injury despite their elevated blood pressure. Experimental and clinical evidence point to a role for the MC in the pathogenesis of the CKD, however, the mechanism through which preglomerular arterial MC contributes to CKD has not been fully elucidated. This thesis showed that augmented MC in chronically hypertensive animal models was due to increased thromboxane A2 prostaglandin that was not released from the endothelium (Chapter 2). Nevertheless, inhibiting MC while also reducing the blood pressure prevented salt-induced renal injury even though the blood pressure was still not normalized compared to the normotensive controls (Chapter 3). The resulting improvement in renal structure and function could be attributed to the reduction in the blood pressure, albumin, and uromodulin (UMOD) excretion (Chapter 3). UMOD is a kidney-specific glycoprotein that, based on a genome-wide association study have the strongest association to CKD (Chapter 3). Comparing two CKD hypertensive animal models further revealed that CKD progression was independent of the blood pressure and strongly associated with UMOD excretion levels (Chapter 4). Collectively, the data discussed in this thesis demonstrates potential therapeutic targets in CKD hypertensive animal models. / Dissertation / Doctor of Philosophy (PhD)

The kinetics of drugs photodegradation have traditionally been treated using thermal kinetic analysis methods consisting most commonly in zero and first order kinetics. These treatment strategies were shown to lack specificity and present a number of limitations when applied to photoreactions kinetics. Nevertheless, these methods have widely been used due to a lack of integrated rate-laws for the majority of photoreactions types, in turn, due to the presence of a variable time-dependent factor in most photoreactions rate-laws that prevents their mathematical integration. To address these limitations, a new methodology for the development and validation of semi-empirical integrated rate-laws that faithfully describe photoreactions kinetics and photoreactions simulated cases generated by numerical integration methods (NIMs), is hereby presented. Using this methodology, a new kinetic order was ascribed to photoreactions namely the Φ-order kinetics. Semi-empirical integrated rate-laws were, thus, developed for three photoreaction types namely, unimolecular, AB(1Φ), photoreversible ,AB(2Φ), and consecutive, AB4(4Φ), photoreactions. The proposed models were further tested experimentally on drugs following these photodegradation mechanisms using; nifedipine and nisoldipine for unimolecular photoreactions; montelukast and fluvoxamine for photoreversible reactions; and riboflavin for consecutive photoreactions. The developed models not only accurately described the photoreaction kinetics of these drugs but also allowed the determination of all the kinetic parameters that characterise them. Furthermore, the above studied drugs were shown to act as precise and simple actinometers when analytically treated with the Φ-order kinetic methods, hereby presented. A universal standard method for the precise and worldwide reproducible study of drugs stability and compounds photoreactions, based on monochromatic irradiation and Φ-kinetics data analysis, is also detailed and adopted throughout the thesis. Finally, two new kinetic parameters namely, the pseudo-rate-constant and pseudo-initial velocity have been identified and shown to be more reliable and accurate in the description and universal comparison of photoreactions kinetics.