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A rabbit lateral meniscectomy model was used to induce osteoarthritis. Separate studies were conducted to evaluate the progression of osteoarthritis and to identify possible biological markers. First, 21 male, New Zealand White rabbits were divided into 3 groups (n = 7 / group). A randomly selected left or right stifle underwent a lateral meniscectomy. The 3 groups were: corticosteroid administration, forced exercise and surgical control. An open field maze was used to assess mobility weekly. The rabbits were euthanitized 47 days after surgery. Histopathologic examination found that the lateral meniscectomy induced more severe lesions than in the non-surgical contralateral stifle. It also showed a significant sparing effect on erosion of cartilage in the corticosteroid group. The corticosteroid group, but not the exercise group, caused a significant increase in mobility (p = 0.008) compared to the surgical control. Secondly, synovial fluid was harvested from the 12 rabbits on days 0, 6, 26, 40, and 57 with surgery occurring on day 12. Trypan blue was used in the lavage fluid to estimate the volume of harvested synovial fluid. There was a significant increase in the volume harvested on day 26 (p < 0.001). Superoxide dismutase concentration in synovial fluid increased after surgery, although not significantly. These studies verify that the lateral meniscectomy model produce histopathologic lesions consistent with osteoarthritis. Furthermore, use of trypan blue appears to be a reliable concentration marker in a lavage sample to measure harvested synovial fluid. / Master of Science

<p>The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease.</p><p>Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells.</p><p>S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. </p><p>Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity.</p><p>Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.</p>

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

In recent years, endocrine disruption of corticosteroid signaling pathways in wildlife and humans by environmental chemicals have attracted increasing attention. The integrated potential of chemicals in the aquatic environment that disrupt corticosteroid actions have been evaluated using in vitro glucocorticoid receptor (GR) mediated bioassays. Exogenous natural and synthetic corticosteroids (CSs), which are widely used in human and animal therapeutic applications, were demonstrated to be the most important GR agonists, that can potentially cause adverse effects, especially on aquatic organisms. To date, only a few studies have investigated the occurrence and behavior of GR agonists in the aquatic environment and their removal in conventional wastewater treatment plants. Furthermore, there are hardly any data reported on the removal of GR agonists by advanced water and wastewater treatment, especially those synthetic CSs with high potency. To further understand the fate of GR agonists in water and wastewater treatment processes, a sensitive and robust LC-MS/MS method was successfully developed for analyzing a wide range of GR agonists in various environmental waters. The occurrence of GR agonists in surface water and groundwater was monitored along the Lower Santa Cruz River (SCR). Several GR agonists were detected, and a trend of degradation was observed downstream the two WWTP outfalls for both surface water and groundwater. The fate of GR agonists in a local wastewater treatment plant (WWTP) was investigated, and up to 14 GR agonists were detected at different stages. Highly potent synthetic CSs, including clobetasol propionate (CBP), fluticasone propionate (FTP), fluocinolone acetonide (FCA), and triamcinolone acetonide (TCA), were poorly removed in WWTP. Negative removal of some CSs was observed in primary treatment, which may due to the deconjugation of CS conjugates. Removal of GR agonists in secondary effluent during various advanced water treatment processes, including UV, ozonation, MF, RO and chlorination, were studied. UV and RO appeared to be the most efficient treatment process for the attenuation of GR agonists, followed by ozone, while chlorination had little effects on GR agonists in water. Bench-scale experiments were then carried out to investigate the removal of GR agonists by ultraviolet based advanced oxidation processes (UV/AOPs), and powder activated carbon (PAC). UV/chlorine and UV/H2O2 were demonstrated to be effective in removal GR agonists in wastewater, and UV photolysis would be the predominant mechanism in UV/AOP processes. Four types of PACs were tested for removing GR agonists in wastewater effluent, and Cabot HDB carbon was suggested, while Calgon PWA carbon was not recommended due to its low removal efficiency.

Nous avons mis au point par atomisation-séchage un système vecteur innovant de corticostéroïdes destiné à être administré par voie pulmonaire pour le traitement des maladies inflammatoires des voies respiratoires dont la plus fréquente est l’asthme. Cette forme pharmaceutique appelée "grosse particule poreuse" consiste en une matrice de deux excipients biocompatibles et biodégradables, la 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) et l'acide hyaluronique (AH) encapsulant le palmitate de dexaméthasone (DXP). La première partie de ce travail a consisté à optimiser l’encapsulation d’un glucocorticoïde modèle, la DXP, une prodrogue lipophile de la dexaméthasone (DXM) au sein de microparticules poreuses par atomisation séchage. Les résultats des tests physicochimiques ont permis d’isoler la poudre à 5% de DXP dont les propriétés morphologiques et aérodynamiques répondent bien aux critères des grosses particules poreuses. La cinétique de libération in vitro a montré une libération relativement lente avec moins de 5% de DXP dans le surnageant après 21 jours. Nous avons ensuite mis au point une méthode d’extraction et de de dosage de la DXP et de son métabolite actif, la DXM dans le plasma et le liquide de lavage bronchoalveolaire (BALF). La dernière partie de notre travail a été consacrée à l’étude pharmacocinétique après administration pulmonaire de la formulation optimisée chez le rat sain. Les résultats in vivo montrent une libération relativement lente de la DXP dans le BALF tandis que la concentration en DXM diminue rapidement en 4 heures. En revanche les concentrations dans le plasma restent faibles. Ces résultats indiquent une bonne distribution pulmonaire de la DXP et la DXM avec une faible absorption sanguine de ces molécules ce qui est prometteur pour le traitement local de l’asthme avec la possibilité de réduire le nombre d’administrations et d’améliorer l’observance thérapeutique des patients. / We have developed by spray drying an innovative carrier system of corticosteroids to be administered by pulmonary route for the treatment of airway inflammatory diseases, among which the most common is asthma. This pharmaceutical form known as "large porous particles" consists of a matrix composed of two biocompatible and biodegradable excipients, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hyaluronic acid (HA) encapsulating dexamethasone palmitate (DXP). The first part of this work was to optimize the encapsulation of a model glucocorticoid, DXP, a lipophilic prodrug of dexamethasone (DXM) within porous microparticles by spray drying. The results of the physicochemical characterization allowed to isolate microparticles loaded with 5% DXP for which morphological and aerodynamic properties meet the criteria of large porous particles. In vitro release kinetics showed a relatively slow release with less than 5% of DXP in the supernatant after 21 days. Then we developed an extraction method and a HPLC method for the determination of DXP and its active metabolite, DXM in plasma and bronchoalveolar lavage fluid (BALF). In vivo results show a relatively slow release of DXP in BALF while the DXM concentration decreases rapidly in 4 hours. By contrast, the plasma concentrations remained low. These results indicate good pulmonary delivery of DXP and DXM with low blood absorption of these molecules which is promising for the local treatment of asthma with the possibility of reducing the number of administrations and improve patient compliance.

Objetivos: Comparar os efeitos dos tratamentos com montelucaste e dexametasona no recrutamento eosinofílico e na avaliação de células positivas para eotaxina, RANTES, fibronectina, IGF-I e NF-B tanto no parênquima pulmonar distal, quanto nas vias aéreas de cobaias com inflamação alérgica crônica. Métodos: As cobaias receberam inalação com ovoalbumina (grupo OVA- 2 vezes semanais, durante 4 semanas, totalizando 7 inalações). Após a quarta inalação, as cobaias foram tratadas com montelucaste (grupo OVA-M: 10mg/Kg/VO/dia) ou dexametasona ( grupo OVA-D: 5mg/Kg/IP/dia). Após 72 horas da sétima inalação, as cobaias foram anestesiadas e os pulmões foram removidos e submetidos a avaliação histopatológica. Resultados: Os tratamentos com montelucaste e dexametasona reduziram o número de eosinófilos tanto no parênquima pulmonar distal quanto nas vias aéreas, quando comparados ao grupo OVA (p<0.05). No parênquima pulmonary distal, ambos os tratamentos foram efetivos na redução de células positivas para RANTES, NF-B e fibronectina, quando comparados ao grupo OVA (p<0.001). O tratamento com montelucaste mostrou melhor eficácia na redução de células positivas para eotaxina, quando comparado ao tratamento com dexametasona (p<0.001), por outro lado, o tratamento com dexametasona mostrou-se mais significativo na redução de células positivas para IGF-I, quando comparado ao tratamento com montelucaste (p<0.001). Nas vias aéreas, ambos os tratamentos foram efetivos na redução de células positivas para IGF-I, RANTES e fibronectina, quando comparados ao grupo OVA (p<0.05). O tratamento com dexametasona foi mais efetivo na redução de células positivas para eotaxina e NF-B, quando comparado ao tratamento com montelucaste (p<0.05). Conclusões: Neste modelo animal, ambos os tratamentos foram efetivos no controle da resposta inflamatória, tanto no parênquima pulmonar distal, quanto nas vias aéreas / Aims: Compare the effects of montelukast or dexamethasone treatments on eosinophilic recruitment, eotaxin, RANTES, fibronectin, IGF-I and NF-B positive cells of distal lung parenchyma and also in airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods: GP were inhaled with ovalbumin (OVA group-2x/week/4weeks). After 4th inhalation, GP were treated with montelukast (M group: 10mg/Kg/PO/day) or dexamethasone (D group: 5mg/Kg/IP/day). After 72 hrs of 7th inhalation, GP were anesthetised, lungs were removed and submitted to histopathological evaluation. Results: Montelukast and dexamethasone treatments reduced the number of eosinophils both in airway wall as well as in distal lung parenchyma compared to OVA group (p<0.05). On distal parenchyma both montelukast and dexamethasone were effective in reducing RANTES, NF-B and fibronectin positive cells compared to OVA group (p<0.001). Montelukast was more effective in reducing the eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (p<0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (p<0.001). On airway walls, both montelukast and dexamethasone were effective in reducing IGF-I, RANTES and fibronectin positive cells compared to OVA group (p<0.05). Dexamethasone was more effective reducing the number of eotaxin and NF-kB positive cells than Montelukast (p<0.05). Conclusions: In this animal model, both treatments were effective in modulating the eosinophilic response in distal lung parenchyma and in airway wall, contributing to a better control of the inflammatory response in distal lung parenchyma as well as in airway walls. Dexamethasone treatment induced a greater reduction of NF-B expression in airway walls which suggests one of the mechanisms that explains the higher efficacy of this therapeutic approach

Os corticosteróides são substância largamente empregadas em dertatologia devido ao seu potente efeito anti-inflamatório na pela. Entretanto, associado a este efeito benéfico tem-se o risco da ocorrência de efeitos colaterais, decorrentes principalmente da absorção sistêmica dos corticosteróides pela via cutânea. A penetração transcutânea e retenção cutânea dos corticosteróides é influenciada pelo veículo no qual estes princípios ativos são incorporados. Assim sendo, os objetivos desta pesquisa foram investigar, in vitro, formulações que proporcionassem uma alta retenção cutânea dos corticosteróides na pele e também mínima penetração transcutânea. O estudo foi realizado com géis de Poloxamer 407 contendo diferentes concentrações dos promotores de absorção cutânea, uréia ou lecitina. Os parâmetros liberação, penetração transcutânea e retençâo cutânea dos acetatos de hidrocortisona e dexametasona, desonida e triamcinolona acetonida foram avaliados por metodologia in vitro, utilizando-se de célula de difusão e membranas. A quantificação dos corticosteróides nas diferentes fases do experimento foi realizada por cromatografia líquida de alta eficiência. Os esultados obtidos mostraram a influência do veículo nos parâAmetros avaliados. As formulações obedeceram cinética de 1ª. ordem para a liberação e penetração transcutânea. As preparações contendo lecitina promoveram uma maior retenção cutânea dos corticosteróides, sugerindo, assim, que géis de Poloxamer 407, associados com lecitina, formam veículos adequados para a incorporação de corticosteróides. / Corticoids are drugs often used in dermatology due to its anti-inflamatory effect in the skin. Meanwhile, together with this beneficial effect it can have side effects, due to the systemic absorption of the corticoids by cutaneous way. Transcutaneous penetration and cutaneous retention of the corticoids is influenciated by the vehicle in which this drogs are incorporated. In this way, the objetive of this research was to investigate, in vitro, formulations that would provide both, high cutaneous retention of the corticoids in the Skin and minimmn transcutaneous penetration. For this work was used Poloxamer 407 gels containing different concentrations of the absorptions enhancers, urea and lecithin. The release transcutaneous penetration and cutaneous retention of hydrocortisone and dexamethasone acetate, desonide and triamcinolone acetonide was evaluated by in vitro methodology using difiusion cells and membranes. The corticoids was analysed by HPLC. The results obtained showed the influence of the vehicle in the evaluated parameters. The corticoid relesse and transcutaneous penetration appeared to fit first order kinetic. The formulations containing lecithin promoved higher cutaneous retetion of the corticoids than the containing urea, sugesting, in this way, that Poloxamer 407 gels in the presence of lecithin are adequated preparations to the corticoids.

O corticóide é usado nas várias especialidades médicas por ser um fármaco de potente efeito anti-inflamatório e imunossupressor, no entanto, é capaz de produzir algumas alterações metabólicas quando administrado por uso prolongado. Desta forma o presente estudo teve como objetivo avaliar as possíveis alterações nas propriedades mecânicas de osso cortical e trabecular de coelhas albinas, quando submetidas à administração de corticoesteróide em altas doses e por tempo prolongado, através de ensaio mecânico de flexão em três pontos em tíbia e fêmures avaliados como estrutura, além de ensaios mecânicos em corpos de prova por flexão em três pontos em fêmures e compressão em vértebras (L5). Para o estudo foram utilizados 39 animais, divididos aleatoriamente em dois grupos, experimental (GE) e controle (GC). Estes grupos foram divididos em quatro subgrupos, sendo 2 experimentais e 2 controles. Na investigação estrutural foram avaliadas as tíbias e fêmures direito por ensaios mecânicos de flexão em 3 pontos, e na investigação como material foram realizados ensaios mecânicos de flexão em 3 pontos para fêmures e compressão para vértebras (L5) através de corpos de prova. A metodologia utilizada para esta pesquisa foi igual para todos os animais, no entanto, o grupo experimental foi administrado durante 21 dias com o medicamento metilprednisolona (Solumedron) diluída em solução salina numa proporção de 2 mg/Kg/dia, e o grupo controle foi somente administrada solução salina na mesma dosagem. Todos os ensaios mecânicos foram realizados na Máquina Universal de Ensaio do Laboratório de Bioengenharia - FMRP/USP. Durante os ensaios foram registrados os valores das deflexões e as respectivas cargas aplicadas, sendo posteriormente confeccionadas curvas carga versus deflexão para cada osso ensaiado. As propriedades mecânicas determinadas para os fêmures e tíbias analisadas de forma estrutural foram: a carga aplicada no limite de proporcionalidade, deflexão no limite de proporcionalidade, carga máxima, rigidez e a resiliência de cada osso. Já para os fêmures e vértebras analisados como material ósseo, foram avaliados suas tensões máximas através da curva carga versus deflexão. Comparações estatísticas foram realizadas entre os resultados encontrados, através do teste T-Student, com nível de significância estabelecido em 5% para todos os parâmetros analisados. Nas análises estatísticas entre os grupos investigados não foram observadas diferenças significativas em nenhuma das comparações, porém observou-se ao final deste estudo uma perda de peso corporal significativa do grupo experimental. Desta forma foi evidenciado através dos resultados encontrados nesta pesquisa que as propriedades mecânicas investigadas não apresentaram alterações significativas com o protocolo de administração de medicamento, sugerindo-se que novos experimentos sejam realizados, com modificações de dosagem e tempo de aplicação. / Corticoteroids are used in many clinical conditions because they present strong anti-inflammatory and imunessupressor activities. But, at the same time, they can cause many metabolic alterations and side effects mainly when there is prolonged. In the present research we studied the possible alterations caused by steroids on the mechanical properties of lamellar and trabecular bone of rabbits. The mechanical properties were assessed by bending tests performed on intact femurs and tibial as well as in samples of cortical bone. Compression tests were performed in L5 vertebral. Thirty-seven female rabbits were randomly distributes in one experimental group (EG-animals) and control (CG animals). Such groups were divided into four subgroups: two experimentals and two controls. The experimental animals received 2mg/kg/day of methylprednisolone (Solumedron ® ) during three weeks. The control animals received the same volume of intramuscular injections of saline, once a day, during three weeks. From the load x deformation curves the load and deflexion were obtained at the yielding point. The ultimate load as well as resilience were also obtained for the intact bones. When the specimens were analysed the ultimate tension was determined. The statisitical analyses did not show any difference between treated and untreated animals for the mechanical properties. But the treated animals showed a significant loose of body weight. We ful that such results require a deepening in the investigation.

In this thesis, two potential applications of transdermal iontophoresis in the field of sports medicine were studied: (1) the local delivery of dexamethasone phosphate (Dex-Phos), a corticosteroid used to treat musculoskeletal inflammation, and (2) the extraction of systemic amino acids (AAs), potential biological markers of fatigue in athletes. The iontophoretic delivery of Dex-Phos was studied, in vitro, in order to evaluate the effects of competing ions and electroosmosis, and identify the optimal conditions for its delivery. The iontophoretic extraction of AAs from the skin was first studied in vitro, before evaluating the method in a group of human volunteers. Dex-Phos was best delivered by iontophoresis from the cathode in absence of background electrolyte in the drug solution. In this situation, the delivery of Dex-Phos is limited principally by the competition with counter-ions (mainly Na+) present subdermally and the small mobility of the drug inside the membrane. The accumulation of Cl-, released by the Ag/AgCl cathode in the drug solution during current passage, can also reduce Dex-Phos delivery. The extraction of zwitterionic AAs from the skin during iontophoresis was highly influenced by their presence in the outermost layer of the skin, the stratum corneum (SC). In the pig skin model, the amount of the AAs extracted during a short extraction period (1 hour) correlated with their abundance in the SC. Once this ‘reservoir’ was emptied (after ~3 hours of iontophoresis), the subdermal compartment could be sampled, suggesting that the method could be used to monitor systemic levels of AAs. The experiments in human volunteers revealed, however, that a 4-hour iontophoretic extraction period was insufficient to deplete the AAs SC ‘reservoir’. It follows that the method can be used to evaluate the abundance of AAs in the SC, but is unpractical for the clinical monitoring of their systemic levels.