Cyclosporine is used in veterinary medicine to treat a number of inflammatory and immune-mediated conditions, however firm oral dosing protocols have yet to be established in the dog. Traditionally a pharmacokinetic approach, through measurement of blood drug concentrations, has been the primary method of establishing if the given dose is effectively suppressing the immune system. However, there is some debate over how well blood drug concentrations correspond to immunosuppression, since individuals can vary in response to the same drug concentration. Our research group believes that a pharmacodynamic approach could alternatively be used to accurately determine cyclosporine dosages in individual patients since this will give a measurement of the immune system’s response to the drug, rather than simply how the body is processing it. This method will give a more accurate assessment of the patient’s immune system, and allow for better immunosuppressant therapy. The objective of this thesis was to develop a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that could reliably predict patient outcome during cyclosporine treatment. This assay would essentially work as a diagnostic tool that clinicians can use to help determine if they were using an appropriate cyclosporine dose for their patients. The assay measures cytokine expression of activated T cells, which are the target cell for the active metabolite of cyclosporine. Our objectives were achieved, firstly, through validation of the assay. Since this assay will be used by clinicians throughout the nation, we first established if shipping conditions affected the sample, and therefore assay results. Once the effect of sample storage time and temperature were determined, optimal sample collection timing was established. Finally, cytokine levels were measured in samples from clinical cases and healthy control dogs to examine the difference in cytokine expression between these two groups. An effective and reliable treatment method for cyclosporine has yet to be established in the dog; therefore the results of this thesis will lead to better therapeutic monitoring and more efficient use of cyclosporine therapy in canine patients.

Le muscle strié squelettique subit de graves lésions d’ischémie-reperfusion (IR) au cours de la progression de l’artériopathie oblitérante des membres inférieurs et lors d’interventions chirurgicales qui nécessitent l’interruption transitoire du flux sanguin dans les artères des membres. Dans ce contexte, nos objectifs étaient de mettre à profit deux modèles expérimentaux d’IR des membres inférieurs par clampage aortique et garrotage unilatéral pour : ° tester l’efficacité d’une alternative médicamenteuse au postconditionnement ischémique par l’utilisation de la cyclosporine A (CsA). En se liant à la cyclophiline D, la CsA empêche l’ouverture du pore de transition mitochondrial (mPTP) à un niveau très distal de la cascade d’évènements qui conduit à la nécrose après IR. ° déterminer de quelle façon deux comorbidités fréquemment retrouvées chez des patients souffrant d’atteinte artérielle (le diabète et l’âge) influencent l’effet de la cyclosporine. Avec les protocoles de conditionnement et aux doses que nous avons utilisées, la cyclosporine a des effets différents sur les conséquences musculaires de l’ischémie-reperfusion des membres inférieurs, dépendant de la pathologie sous-jacente des animaux étudiés. Il semble intéressant d’étudier l’effet dose-réponse de la cyclosporine A pour déterminer l’intervalle thérapeutique optimal, celui-ci pouvant être différent chez l’animal sain et pathologique. D’autre part, étant donné l’importance considérable du stress oxydant chez les animaux diabétiques et sénescents, la co-administration de cyclosporine et d’un antioxydant au moment de la reperfusion pourrait rétablir une protection. / Peripheral arterial disease and many surgical procedures (requiring vascular clamping or tourniquet application) induce severe skeletal muscle ischemia-reperfusion (IR) injuries. As a result, using experimental hind limb ischemia-reperfusion models, our goals were: ° To test a pharmacologic substitute to ischemic postconditioning by using cyclosporine A, that acts on a very downstream step of IR injury cascade by binding to cyclophilin D and inhibiting mitochondrial transition pore opening. We wondered if cyclosporine could alleviate mitochondrial dysfunction and reduce ROS production in skeletal muscles submitted to IR. ° To determine how diabetes and senescence would influence cyclosporine A protective effects. In conclusion, the protective effects of pharmacologic postconditioning with cyclosporine A seem to critically depend on the model under study. A variable and narrow dose-effect relationship is likely and makes it necessary to perform a dose finding study in every pathologic model. Considering the narrow relationships between mitochondrial protection and oxidative stress, combing cyclosporine A postconditioning with antioxidant therapy may restore a more robust protective effect but this hypothesis has to be validated.

Ce travail est consacré au rôle pléotropique des microparticules endothéliales dans la réponse et l'homéostasie vasculaire. Un modèle de sénescence réplicative a été caractérisé en utilisant des cellules endothéliales de coronaires de porc en culture primaire. Ce modèle a mis en évidence des changements drastiques du phénotype endothélial avec la production de ROS, la dépolarisation de la membrane mitochondriale et la surexpression de régulateurs clés du cycle cellulaire incluant p53, p21 et p16. La sénescence a transformé le phénotype endothélial vers un statut procoagulant indiqué par la libération de microparticules (MP). L'induction d'activité Facteur Tissulaire (FT) et une réduction drastique de l'inhibition de l'agrégation plaquettaire due à la sécrétion réduite de NO endothélial. Simultanément, une augmentation importante des protéines du système de l'angiotensine à la surface des cellules sénescentes et des MPs qu'elles émettent a été mesurée. D'autres résultats obtenus avec les MPs circulantes de patients transplantés ou atteints de syndrome coronarien suggèrent l'existence dune boucle d'amplification des effets délétères des MPs au travers de la signalisation Redox et l'altération des fonctions vasculaires résultant d'une sénescence exacerbée. En plus de ses qualités reconnues d'immunosuppresseur, la cyclosporine A (CsA) est un inhibiteur puissant de l'ouverture des pores mitochondriaux (mPTP). Certaines études ont présenté le traitement bref et contrôlé par CsA comme un moyen de limiter les dommages vasculaires de l'ischémie reperfusion.Nos données suggèrent une possible modulation de la sénescence endothéliale induite par les MPs grâce au préconditionnement avec des concentrations faibles de CsA. nos résultats suggèrent aussi que de faibles doses de Cs A peuvent avoir un effet bénéfique dans les pathologies cardiovasculaires lorsque la sénescence est exacerbée et contribue les fonctions vasculaires de l’endothélium. / This scientific work has tackled the issue of the pleitropic role mediated by endothelial microparticles function and homeostasis. A replicative model of senescence using coronary endothelial cells was set showing drastic phenotype changes characterized by ROS production, mitochondrial membrane depolarization and the up-regulation of key regulators of cell cycle arrest including p53, p21 and p16.Replicative senescence shifted the coronary endothelial phenotype toward a procoagulant status as evidenced by (i) procoagulant MP shedding (ii) enhanced tissue factor (TF) expression and (iii) a marked decrease in the endothelial NO-mediated inhibition of platelet aggregation. In parallel, a drastic up regulation of the angiotensin system could be evidenced at the surface of senescent cells or derived MP. Results obtained with MPs from patients with acute coronary artery syndrome and from grafted patients,suggested a feedback loop disseminating the deleterious effect of circulating MPs redox signaling and alteration of vascular function owing to exaggerated senescence. In addition to its well-known immunosuppressive properties, cyclosporine A (Cs A) is a potent inhibitor of the opening of the mitochondrial permeability transition pore (mPTP), and several reports have indicated that a brief and timely administration of Cs A can limit ischemia-reperfusion injuries. Our data evidenced the possible pharmacological modulation of endothelial MP-mediated senescence by cell preconditioning with low concentrations of Cs A. our data are thus suggestive of a beneficial effect of CsA in cardiovascular disorders where senescence is altering the endothelial vascular functions.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

The discovery of new immunosuppressive drugs has resulted in an improvement of short-term graft survival. Despite this achievement, long-term cardiac allograft survival has not been correspondingly improved. Cyclosporine A (CsA), an effective immunosuppressive drug, has been shown to increase the risk of hyperlipidemia, hypertension, kidney injuries and chronic rejection despite its extensive use in the clinical setting. Therefore, these side-effects of CsA, may further contribute to graft failure over long-term. Early studies have shown that fish oil may reduce side-effects of CsA. These beneficial effects of fish oil may be related to n-3 fatty acids (n-3 FAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Flaxseed oil is another major source of an n-3 FA, namely α-linolenic acid (ALA). However, its impact on heart transplantation has not been fully explored. The current study aimed to investigate whether dietary flaxseed oil and fish oil reduce post-transplant complications and prolong graft function in a rat cardiac allograft model. Male Fischer and Lewis rats were used as donors and recipients, respectively, to generate a heterotopic cardiac allograft model. After transplant, animals were randomly assigned into 3 groups and fed a diet supplemented with: a) 5% w/w safflower oil (control n=7), b) 5% w/w flaxseed oil (n=8) or c) 2% w/w fish oil (n=7) and an intraperitoneal injection of cyclosporine A (CsA) (1.5 mg/kg/d) over 12 weeks. Body weight, blood pressure (BP), plasma levels of lipids, CsA, and select cytokines, fatty acid profile of hearts (native and graft) and liver tissues as well as graft function and chronic rejection features were assessed. Body weight and blood CsA levels were similar among the groups. As compared to controls, both diet treated groups demonstrated a significantly lower systolic blood pressure (SBP) (p<0.001), diastolic blood pressure (pressure (DBP) (p<0.001), mean arterial pressure (MAP) (p<0.001), heart rate (p<0.05), abdominal fat (p<0.05) and plasma levels of macrophage chemoattractant protein-1 (MCP-1) (p<0.05). Moreover, the fish oil group had significantly (p<0.05) lower plasma levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL), as compared to the control group. High-density lipoprotein cholesterol (HDL) concentrations were significantly higher (P<0.05) in the flaxseed oil-treated group as compared to the other two groups. Data of this study suggest that both flaxseed oil and fish oil may provide similar biochemical, hemodynamic and inflammatory improvements after heart transplantation; however, these apparent beneficial changes were not accompanied with significant reductions in chronic rejection states or apparent histological evidence of cyclosporine-induced nephrotoxicity in this model.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

The discovery of new immunosuppressive drugs has resulted in an improvement of short-term graft survival. Despite this achievement, long-term cardiac allograft survival has not been correspondingly improved. Cyclosporine A (CsA), an effective immunosuppressive drug, has been shown to increase the risk of hyperlipidemia, hypertension, kidney injuries and chronic rejection despite its extensive use in the clinical setting. Therefore, these side-effects of CsA, may further contribute to graft failure over long-term. Early studies have shown that fish oil may reduce side-effects of CsA. These beneficial effects of fish oil may be related to n-3 fatty acids (n-3 FAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Flaxseed oil is another major source of an n-3 FA, namely α-linolenic acid (ALA). However, its impact on heart transplantation has not been fully explored. The current study aimed to investigate whether dietary flaxseed oil and fish oil reduce post-transplant complications and prolong graft function in a rat cardiac allograft model. Male Fischer and Lewis rats were used as donors and recipients, respectively, to generate a heterotopic cardiac allograft model. After transplant, animals were randomly assigned into 3 groups and fed a diet supplemented with: a) 5% w/w safflower oil (control n=7), b) 5% w/w flaxseed oil (n=8) or c) 2% w/w fish oil (n=7) and an intraperitoneal injection of cyclosporine A (CsA) (1.5 mg/kg/d) over 12 weeks. Body weight, blood pressure (BP), plasma levels of lipids, CsA, and select cytokines, fatty acid profile of hearts (native and graft) and liver tissues as well as graft function and chronic rejection features were assessed. Body weight and blood CsA levels were similar among the groups. As compared to controls, both diet treated groups demonstrated a significantly lower systolic blood pressure (SBP) (p<0.001), diastolic blood pressure (pressure (DBP) (p<0.001), mean arterial pressure (MAP) (p<0.001), heart rate (p<0.05), abdominal fat (p<0.05) and plasma levels of macrophage chemoattractant protein-1 (MCP-1) (p<0.05). Moreover, the fish oil group had significantly (p<0.05) lower plasma levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL), as compared to the control group. High-density lipoprotein cholesterol (HDL) concentrations were significantly higher (P<0.05) in the flaxseed oil-treated group as compared to the other two groups. Data of this study suggest that both flaxseed oil and fish oil may provide similar biochemical, hemodynamic and inflammatory improvements after heart transplantation; however, these apparent beneficial changes were not accompanied with significant reductions in chronic rejection states or apparent histological evidence of cyclosporine-induced nephrotoxicity in this model.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 200-242). Also available on the World Wide Web.

Thesis (M.A.)--Ball State University, 2009. / Title from PDF t.p. (viewed on Mar. 25, 2010). Research paper (M.A.), 3 hrs. Includes bibliographical references (p. [49]-53).