Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Orientadores: Marilda Mazzali, José Butori Lopes de Faria / Tese ( doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T18:16:39Z (GMT). No. of bitstreams: 1 Mazali_FernandaCristina_D.pdf: 2274720 bytes, checksum: ce2fe44ee92ab230ab3a6648462a850b (MD5) Previous issue date: 2011 / Resumo: A nefrotoxicidade por ciclosporina caracteriza-se, do ponto de vista histológico, por fibrose intersticial em faixa, atrofia tubular e hialinose de arteríolas aferentes glomerulares, ou seja, um quadro compatível com doença renal isquêmica. Esta isquemia provocada pela ciclosporina leva a redução da taxa de filtração glomerular, com consequente elevação dos níveis séricos de ácido úrico. Além disto, a ciclosporina altera o transporte tubular de urato, favorecendo o desenvolvimento de hiperuricemia. No modelo experimental de nefropatia pela ciclosporina, a elevação dos níveis de ácido úrico apresenta associação com lesão túbulo intersticial mais severa, além de maior frequência de hialinose de arteríola aferente. Em estudos anteriores demonstramos que a hiperuricemia agrava a nefrotoxicidade pela ciclosporina e também que, a administração concomitante de agentes hipouricemiantes previne a lesão renal pela CsA. Assim, consideramos a hipótese de que, em um modelo experimental de nefropatia crônica pela ciclosporina, instalada, a normalização dos níveis de ácido úrico com alopurinol ou benzbromarona poderia reverter a lesão renal estabelecida. Nefropatia pela ciclosporina foi induzida em ratos Sprague Dawley com injeções subcutâneas diárias de ciclosporina, em associação com dieta hipossódica, por 5 semanas. Ao final deste período, grupos experimentais foram divididos com interrupção da ciclosporina, tratamento com CsA isolada ou em associação com alopurinol ou benzbromarona por um período adicional de 4 semanas. Ao final de 9 semanas de estudo, foram realizadas avaliações funcionais e histológicas. Neste modelo, a co-administração de alopurinol ou benzbromarona cursou com redução dos níveis de ácido úrico e minimizou o quadro de nefrotoxicidade estabelecida por ciclosporina, através da redução de hialinose arteriolar, glomeruloesclerose e fibrose intersticial, além da melhora da função renal, do estresse oxidativo e da apoptose, porém sem efeito anti inflamatório, avaliado pelo infiltrado de macrófagos e pela expressão de osteopontina. Os resultados mais significativos no grupo tratado com alopurinol sugerem que, além do efeito hipouricemiante, o alopurinol pode também apresentar um mecanismo antioxidante, conforme demonstrado pela redução da peroxidação lipídica e da geração de radicais livres, resultando em menor intensidade de apoptose de células tubulares renais. Assim, a redução dos níveis de ácido úrico neste modelo atuou como protetor na progressão da lesão de microvasculatura e na redução da área de fibrose intersticial, mas não da lesão inflamatória. No modelo de nefropatia pela ciclosporina, assim como no de hiperuricemia, ocorre elevação da atividade de renina, sugerindo a participação do sistema renina angiotensina aldosterona na fibrose renal. Para determinar este efeito, um segundo estudo utilizou a associação de um inibidor de enzima conversora da angiotensina (enalapril), um bloqueador de receptor AT1 de angiotensina (losartan) ou um inibidor competitivo da aldosterona (espironolactona) ao tratamento com ciclosporina, após a instalação da lesão. Os animais experimentais receberam ciclosporina por 5 semanas, e após a instalação da nefropatia, foram divididos em um dos grupos experimentais e acompanhados por um período adicional de 4 semanas. A utilização de moduladores do SRAA também cursou com melhora funcional e histológica da nefropatia pela ciclosporina, sem alteração dos marcadores de inflamação intersticial. A melhora da vasculopatia pode ser atribuída à redução do remodelamento vascular com estas drogas, porém com efeito limitado sobre a geração de radicais livres de oxigênio e apoptose de células tubulares. Em resumo, os resultados do presente estudo indicam que em modelo experimental de nefrotoxicidade por CsA, o uso de hipouricemiantes ou de modeladores do sistema-renina-angiotensina-aldosterona apresentaram um importante efeito renoprotetor, comparável, do ponto de vista funcional, à interrupção do tratamento com ciclosporina. As duas abordagens terapêuticas foram eficientes na limitação da progressão da nefropatia, com reversão parcial da fibrose intersticial, provavelmente mediada por melhora de oxigenação tecidual secundária à redução da vasculopatia e do remodelamento vascular. A manutenção do estímulo tóxico da ciclosporina, com manutenção da inflamação, da geração de radicais livres de O2 e da apoptose de células tubulares, entretanto, não foi completamente neutralizado pela intervenção farmacológica / Abstract: Chronic allograft nephropathy is characterized by stripped tubular atrophy and interstitial fibrosis, in presence of arteriolar hyalinosis, resembling an ischemic pattern of chronic kidney disease. Chronic ischemia is associated with reduced glomerular filtration rate, and increase in serum uric acid levels. Cyclosporine per se also has a direct effect on tubular urate handling that facilitates the development of hyperuricemia. Hyperuricemia exacerbates chronic cyclosporine nephropathy, with a more severe tubulointerstitial fibrosis and atrophy, as well as worsening of arteriolar hyalinosis. In a previous study we have shown that concomitant treatment with uric acid lowering agents limits the development of experimental CsA nephropathy. The hypothesis of the present study was that treatment with uric acid lowering agents, after the development of CsA nephropathy could reverse or reduce the severity of tubulointerstitial disease. Male Sprague Dawley rats received daily SC injections of cyclosporine in presence of low salt diet, during 5 weeks. At the end of this period, experimental groups were assigned for CsA withdrawal, maintenance of daily CsA alone or associated with allopurinol or benzbromarone in drinking water for an additional period of 4 weeks. At the end of 9 weeks of study, rats were sacrificed for functional and morphological analysis of kidneys. In this model, concomitant treatment with allopurinol or benzbromaroes was associated with reduction of serum uric acid levels, improvement in renal function and renal disease, characterized by lower arteriolar hyalinosis index, less glomerulosclerosis and significant reduction in interstitial fibrosis area. Other findings included reduction in oxidative stress markers and apoptotic cells, despite of maintenance of inflammatory status, quantified by macrophage infiltration and osteopontin expression. Allopurinol treatment was associated with more significant changes, with reduction of free radical generation, and lower grade of apoptotic cells in renal cortex, suggesting a participation of antioxidant effects in association with uric acid reduction. Taken together, these datsa suggests that reduction of serum uric acid in the stablished model of CsA nephropathy has a protective effect in microvascular lesions and progression of interstitial disease, despite the maintenance of interstitial inflammation. In cyclosporine nephropathy, as well as in the experimental hyperuricemia model, renal disease is associated with increased renin activity, suggesting the participation of renin angiotensin aldosterone system (RAS) in the mechanism of disease. In order to analyze the effect of RAS in CsA nephropathy model, a second study tested the treatment with angiotensin converting enzyme inhibitor (enalapril), a angiotensin II AT1 receptor blocker (losartan) or an aldosterone inhibitor (espironolactone) in association with cyclosporine after the development of chronic nephropathy. Experimental animals were treated with cyclosporine and low salt diet for 5 weeks, and then assigned for one treatment group, including cyclosporine withdrawal, cyclosporine alone, CsA and enalapril, CsA and losartan or CsA and espironolactone for an additional period of 4 weeks. RAS blockade in the established model of CsA nephropathy was associated with improvement in renal function and interstitial fibrosis, despite the maintenance of interstitial inflammation. The most striking finding was the improvement of arteriolar hyalinosis and glomerulosclerosis, suggesting that the most important effect was protecting against vascular remodeling. The improvement in vasculopathy was associated with reduction in tissue hypoxia, with a partial reduction in oxidative stress and tubular cell apoptosis. Both therapeutic interventions proved to be efficient in limiting progression of renal disease, with a partial reversion of interstitial fibrosis. The main mechanism is associated with improvement in renal tissue O2 delivery, as a consequence of recovery of arteriolar hyalinosis and control of vascular remodeling. However, maintenance of CsA therapy was associated with a persistent toxic effect, with maintained interstitial inflammation, free radical generation and tubular cell apoptosis that was not neutralized by intervention / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Inflammatory ocular disease is an important cause of blindness and uveitis accounts for 1.0% of blind patients in Canada.¹ This disease can be particularly troublesome to treat, because the nature of the causal factor or factors and mechanisms of progressi n are usually unknown. Non-specific anti - inflammatory agents have been used orally and systemically with some success to treat uveitis, ²⁻⁸ but they may produce serious side effects both locally and elsewhere in the body.⁹̛¹²̛¹⁴ With prolonged use tolerance to these drugs may develop , making them ineffective. Recently a powerful immuno suppressive agent, Cyclosporine (Cy), used orally and systemically in the treatment of uveitis has shown promising results.¹⁶⁻¹⁹̛²⁸ However, its routine use is limited because of a narrow therapeutic index and renal toxicity. Several studies have shown that subconjunctival injection of a number of antineoplastic agents enhanced ocular absorption ²⁰⁻²⁴ in a traditional pharmacological sanctuary,¹³̛¹⁴ and circumvented the associated systemic side effects. Therefore, if Cy were administered subconjunctivally it might be possible to avoid the side effects associated with the oral and systemic routes, and at the same time provide higher levels of Cy to the eye. A protocol for the administration of Cy subconjunctivally was developed in New Zealand white rabbits, to study toxicity, ocular pharmacokinetics following equidose administration subconjunctivally and systemically and the effects of Cy on an animal model of uveitis. Subconjuntival administration of 5mg of Cy in O.lcc (Sandimmune I.V.(R) 50 mg/ml) weekly was found to be the maximum tolerated dose by the rabbitsˈ eye, and was superior to intravenous injection for ocular penetration while minimizing systemic exposure. The uveitis model showed that Cy was effective in reducing the inflammatory response and the earlier the application of Cy the milder the uveitis. The results from our study support the contention that local administration of Cy would lead to higher levels of Cy absorption and circumvent the side effects of systemic administration. This may facilitate the routine use of Cy in ocular inflammatory disease. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Cyclosporine is a potent immunosuppressive agent used to treat a wide range of canine inflammatory diseases. Unfortunately, optimal dosing protocols for achieving immunosuppression with cyclosporine in dogs remain unclear, and standard methods that objectively monitor effectiveness of immunosuppression have not been established. We evaluated an already established panel of biomarkers of immunosuppression in vivo with two oral dosages of cyclosporine in seven normal dogs, a high dosage known to induce immunosuppression and a lower dosage used to treat atopy, with a washout period between the two dosages. The biomarker panel included the flow cytometric evaluation of T-lymphocyte cytokine expression (IL-2, IL-4, and IFN-gamma). High dosage cyclosporine resulted in significant decreases in IL-2 and INF-gamma expression, but not IL-4 expression. Low dosage cyclosporine was associated with a significant decrease in INF-gamma expression, while IL-2 expression was not affected. The results demonstrated suppression of biomarkers in a dose-dependent manner.

no / Cyclosporine A (CsA) has long been recognized as a potent hair growth stimulator in both humans and rodent. The induction of a dose-dependent hypertrichosis is one of the most frequent adverse effects of long-term CsA therapy (Lutz, 1994). However, it is unclear how this immunosuppressant induces hypertrichosis in patients or stimulates hair growth in human scalp skin transplanted on nude mice (Gilhar et al., 1988; Gilhar et al., 1991).

No description available.

L'infarctus du myocarde est la première cause de mortalité dans le monde. La reperfusion précoce est le traitement central de la prise en charge thérapeutique. Mais bien que salvatrice, la reperfusion s'accompagne elle-même de lésion dite de reperfusion. La mitochondrie et l'ouverture du pore de transition de perméabilité mitochondriale (mPTP) sont au centre de ces lésions de reperfusion. Le dysfonctionnement mitochondrial et la nécrose entraine par ailleurs une intense réponse inflammatoire locale et systémique. Le post-conditionnement ischémique et pharmacologique (par la cyclosporine A, CsA) constitue une voie de recherche importante afin de limiter les lésions de reperfusion. Mes travaux de thèse se sont attachés à étudier le rôle de la Sirtuine 3 dans la cardioprotection ainsi que d'étudier la réponse inflammatoire locale et systémique induite par l'ischémie/reperfusion (I/R) myocardique.Nous avons précisé le mécanisme d'action du post-conditionnement, qui semble médié par l'activation de la sirtuine 3 et la désacétylation de la cyclophiline D qui contribue à inhiber l'ouverture du mPTP. Nous avons ensuite montré que l'I/R myocardique induisait une intense réponse inflammatoire chez l'homme avec un rôle particulier d'IL-17A, IL-6, IL-8 et IL-10. Cependant, cette réponse inflammatoire n'était pas modifiée par l'utilisation de CsA. Enfin, nous avons pu montrer que le facteur induit par l'hypoxie (HIF-1a), qui est surexprimé lors de l'I/R est un important activateur la réponse inflammatoire, notamment sur l'inflammasome, et que le Nicotinamide Mononucléotide possède un rôle anti-inflammatoire en empêchant la stabilisation de HIF-1a. Tous ces éléments sont autant de cibles thérapeutiques potentielles à développer avec de nouvelles études / Myocardial infarction (MI) is the first cause of death in the world. Reperfusion is the key treatment of MI. However, reperfusion can cause reperfusion injuries. Mitochondria and mitochondrial permeablility transition pore are the target of reperfusion injuries. Mitochondrial dysfonction and necrosis lead to an intense local and systemic inflammation. Ischemic post-conditioning (PC) and pharmacologic PC (with cyclosporine A, CsA) are used to limit reperfusion injuries. During my thesis, I worked on cardioprotective effet of sirtuin 3 and I studied inflammation induced by myocardial ischemia/reperfusion (I/R). I have shown that ischemic PC involve sirtuin 3 and deacetylation of cyclophilin D. I demonstrated that myocardial I/R induce an intense inflammatory response in Human with a key role of IL-17A, IL-6, IL-8 and IL-10. However, this inflammatory response is not modulated by the administration of CsA. A least, we studied the role of HIF-1a that is over expressed during I/R. We showed that HIF-1a activate inflammasome and the secretion of IL-1beta and IL-18. Furthermore, Nicotinamied Mononucleotide has anti-inflammatory effets with an action of HIF-1a. Taken together, these data contribute to develop new target for cardioprotection

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Dans cette étude, la stabilité de préparations intraveineuses de cyclosporine (0.2 et 2.5 mg/mL dans NaCl 0.9% ou dextrose 5%) entreposées dans des seringues de polypropylène, des sacs de polypropylène-polyoléfine et des sacs de vinyle acétate d’éthylène a été évaluée. Une méthode HPLC indicatrice de la stabilité à base de méthanol a été développée et validée suite a des études de dégradation forcée. Les solutions évaluées ont été préparées de façon aseptique, puis entreposées à 25°C. La stabilité chimique a été évaluée par HPLC et la stabilité physique a été évaluée par inspection visuelle et aussi par diffusion dynamique de la lumière (DLS). Tous les échantillons sont demeurés stables chimiquement et physiquement dans des sacs de polypropylène-polyoléfine (>98% de cyclosporine récupérée après 14 jours). Lorsqu’entreposés dans des seringues de polypropylène, des contaminants ont été extraits des composantes de la seringue. Toutefois, aucune contamination n’a été observée après 10 min de contact entre la préparation de cyclosporine non-diluée et ces mêmes seringues. Les préparations de 2.5 mg/mL entreposées dans des sacs de vinyle acétate d’éthylène sont demeurés stables chimiquement et physiquement (>98% de cyclosporine récupérée après 14 jours). Toutefois, une adsorption significative a été observée avec les échantillons 0.2 mg/mL entreposés dans des sacs de vinyle acétate d’éthylène (<90% de cyclosporine récupéré après 14 jours). Une étude cinétique a démontré une bonne corrélation linéaire entre la quantité adsorbée et la racine carrée du temps de contact (r2 > 0.97). Un nouveou modèle de diffusion a été établi. En conclusion, les sacs de polypropylène-polyoléfine sont le meilleur choix; les seringues de polypropylène présentent un risque de contamination, mais sont acceptables pour un transfert rapide. Les sacs de vinyle acétate d’éthylène ne peuvent être recommandés à cause d’un problème d’adsorption. / In the present study, the stability of intravenous cyclosporine preparations (0.2 and 2.5 mg/mL in 0.9% sodium chloride injection or 5% dextrose injection) stored in polypropylene (PP) syringes, polypropylene–polyolefin (PP-PO) bags and ethylene vinyl acetate (EVA) bags was evaluated. A methanol-based high-performance liquid chromatography (HPLC) method was developed and validated to be stability-indicating by stress degradation tests. The test solutions were aseptically prepared and stored at 25 °C. Chemical stability was evaluated by HPLC assay. Physical stability was assessed by visual inspection and a dynamic light scattering (DLS) method. All samples were chemically stable (> 98% of recovered cyclosporine) and physically stable when stored in polypropylene–polyolefin bags for 14 days. When stored in polypropylene syringes, some impurities were leached. However, no leaching was detected when the syringes were exposed to undiluted intravenous cyclosporine for 10 minutes. The preparations of 2.5 mg/mL were chemically and physically stable as stored in ethylene vinyl acetate bags for a period of 14 days (> 98% of recovered cyclosporine), while significant cyclosporine adsorption occurred on the samples of 0.2 mg/mL (< 90 % of recovered cyclosporine) after 14 days. Kinetic study showed that good linear correlations were achieved by plotting the adsorption amount versus square root of contact time (r2 > 0.97). A novel diffusion model was established and successfully predicted long-term drug stability. In conclusion, polypropylene–polyolefin bags were the best choice; syringes were inferior because of leachables. However, they were safe for preparation and transferring undiluted intravenous cyclosporine. Ethylene vinyl acetate bags cannot be recommended due to cyclosporine adsorption.