A participação da COX-1 na inflamação sistêmica tem sido questionada. Nós investigamos os mecanismos pelos quais a COX-1 participa da inflamação sistêmica mais grave. O inibidor da COX-1 (SC-560) atenuou a hipotermia, o hipometabolismo e a hipotensão induzidos por LPS. Este efeito atenuante teve duas fases: 30-60 min e 60-100 min. Em animais esplenectomizados, o efeito do SC-560 foi observado apenas na primeira fase. O SC-560 não alterou o nível plasmático das citocinas TNF-α, IL-1β e IL-10 em ambas as fases. No entanto, reduziu a expressão de IL-10 no baço, com tendência a aumentar IL-1β (80 min). Eicosanoides (PGE2, PGD2, PGF2, TXB2 e LTC4) foram detectados no baço e na circulação (80 min). Nossos resultados indicam que o mecanismo da COX-1 no choque endotóxico tem duas fases: (i) na fase inicial a COX-1 não é proveniente do baço e age independentemente de citocinas; (ii) na fase tardia da resposta a COX-1 parece agir de forma dependente do baço e através da produção de eicosanoides, independentemente de TNF-α, mas modulando a síntese de IL-10 e IL-1β. / The participation of COX-1 in systemic inflammation has been questioned. We investigated the mechanisms by which COX-1 participates in the most severe systemic inflammation. The COX-1 inhibitor (SC-560) attenuated the hypothermia, hypometabolism and hypotension induced by LPS. This attenuating effect had two phases: 30-60 min and 60-100 min. In splenectomized animals, the effect of SC-560 was observed only in the first phase. SC-560 did not alter the plasma levels of cytokines TNF-α, IL-1β and IL-10 in both phases. However, it reduced IL-10 expression in the spleen, with tendency to increase IL-1β (80 min). Eicosanoids (PGE2, PGD2, PGF2, TXB2 and LTC4) were detected in spleen and circulation (80 min). Our results indicate that the mechanism of COX-1 in endotoxic shock has two phases: (i) COX-1 does not originate from the spleen and acts independently of cytokines; (ii) in the late phase the COX-1 it seems to act in a spleen-dependent manner and through the production of eicosanoids, independently of TNF-α, but modulating the synthesis of IL-10 and IL-1β.

Metastasis is the major cause of cancer related mortality, due to a poor understanding of the metastatic process and a subsequent lack of effective anti-metastatic therapies. Evidence from experimental studies and clinical trials has shown that aspirin reduces the incidence of distant metastases. It is well established that aspirin inhibits cyclooxygenase (COX)-1 and COX-2, triggering anti-thrombotic and anti-inflammatory effects, respectively. However, the mechanisms underlying the anti-metastatic effect of aspirin are still largely unknown. By using an experimental model of pulmonary metastasis, we have found that the anti-metastatic effect of aspirin is associated with the inhibition of COX-1. In support of this, metastasis establishment was impaired in COX-1 deficient mice, suggesting a pivotal role of this isoform in the metastatic process. Looking in more detail into the metastatic cascade, we found that COX-1 contributes to the intravascular phase of metastasis and promotes the early persistence of tumour cells in the lung vasculature. In particular, COX-1 inhibition decreased the interaction of platelets with tumour cells and was associated with the reduction of endothelial activation, of tumour cell adhesion to the endothelium, of recruitment of metastasis-promoting monocytes/macrophages and of transendothelial migration. We have identified platelet-derived thromboxane A₂ (TXA₂) as the main product of COX-1 responsible for its permissive effect on metastasis. Indeed, TXA₂ delivered to mice in combination with aspirin was able to abrogate the anti-metastatic effect of aspirin. Taken together, our data suggest that the inhibition of COX-1:TXA₂ axis by aspirin is sufficient to exert an anti-metastatic effect. In particular, the inhibition of platelet-derived TXA₂ seems to affect multiple early steps of the haematogenous transit of tumour cells. In this perspective, TXA₂ might represent a more selective therapeutic target for the prevention of metastasis.

Glucocorticoids (GCs) are endogenous steroid hormones that regulate a number of critical physiological processes. Psychological stress increases the level of GCs in the circulating system. The biological effect of elevated GCs on the heart is not well understood. We found that GCs induced Cyclooxygenase-1 (COX-1) and COX-2 gene expression in cardiomyocytes. COX-1 or COX-2 encodes the rate-limiting enzyme in the biosynthesis of prostanoids, which modulate crucial physiological and pathophysiological responses. The present studies aim to elucidate the signaling transduction pathway and the mechanism underlying GC induced COX expression.Our data demonstrate that GCs activate COX-1 gene expression through transcriptional regulation. COX-1 gene promoter studies support a role of Sp binding site in CT induced COX-1 gene expression. The nuclear protein binding to this site appears to be Sp3 transcription factor. Co-immunoprecipitation assays indicated a physical interaction between GR and Sp3 protein. Silencing of Sp3 transcription factor with small interfering RNA suppressed CT-induced COX-1 promoter activation. These data suggest that the activated GR interacts with Sp3 transcription factor that binds to COX-1 promoter to up-regulate COX-1 gene expression in cardiomyocytes.We also found that administration of GC in adult mice increased the level of COX-2 in the ventricles. With isolated neonatal cardiomyocytes, corticosterone (CT) induces the transcription of COX-2 gene. This response appears to be cardiomyocyte cell type specific and GC receptor (GR)-dependent. CT causes activation of p38 MAPK and subsequently CREB phosphorylation that mediates COX-2 gene expression. Mifepristone, a GR antagonist, failed to inhibit p38 and CREB activation and p38 inhibition failed to prevent activation of GR. These data suggest that two parallel signaling pathways, GR and p38 MAPK, act in concert to regulate the expression of COX-2 gene in cardiomyocytes.In addition to the investigation of mechanism and signaling transduction pathway, I have explored pharmacological agents that modulate COX expression. LY294002, a commonly used PI3K inhibitor, inhibited COX-2 gene expression via a PI3K-independent mechanism. Whereas GSK-3 inhibitors, such as lithium chloride, upregulated COX-2 gene expression, but suppressed GC-induced COX-1 expression. These data have paved the foundation for pharmacological manipulation of COX-1 and COX-2 gene expression in the heart.

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography. / <p>De två (2) första författarna delar förstaförfattarskapet.</p>

Made available in DSpace on 2016-06-02T20:21:29Z (GMT). No. of bitstreams: 1 4439.pdf: 2006623 bytes, checksum: 21bda32fc532bf7d57753bbd03222990 (MD5) Previous issue date: 2012-04-25 / Universidade Federal de Sao Carlos / Roads generate a large ecological impact to wildlife by raising barriers among ranges of species originally continuous and for causing road-kills. After road-killing, a morphological identification becomes impossible for the loss of diagnostic features, especially in the case of small vertebrates. Use of molecular tools for identification is an alternative that has shown successful results, especially when looking towards the species level. We identified samples from animals road-killed, collected on Guilherme Scatena road, through the use of DNA barcode markers, contributing to the local biodiversity knowledge. Another objective was to know the places that have a higher incidence of road kill and the type of landscape coverage in them. During 20 weeks we collected 123 vertebrates: six mammals (5% of total), 30 birds (24%), 38 amphibians (30%) and 49 reptiles (40%), which meant an average of 0.051 animals/Km road-killed. Through morphological characteristics and molecular (mitochondrial genes cox1 and 16S) we identified 111 samples until species status. Analyses among taxonomic classes showed significant differences, confirming that the effects of roads on biodiversity are taxon-specific. We found 19 different types of landscape around the road, being the most common farm buildings and pasture, showing the strong anthropic pressure on environments of this region. The results shown in this study are intended to be an important basis for planning future actions that provide the best management of this area rich in endemic biodiversity of the Brazilian cerrado, as well as mitigation measures to lessen the impact caused by the road. / As estradas geram um grande impacto ecológico à fauna por criar barreiras que separam áreas originalmente contínuas da distribuição das espécies e por ocasionar atropelamentos. Após os atropelamentos, a identificação morfológica dos animais tornase impossível pela perda de caracteres diagnósticos, sobretudo, nos vertebrados pequenos. A utilização de ferramentas moleculares para a identificação é uma alternativa que tem apresentado resultados bem sucedidos, principalmente quando se pretende conhecer a espécie. Neste contexto, o presente trabalho identificou as amostras de animais mortos por atropelamentos, coletados na estrada Guilherme Scatena, mediante o uso de marcadores de DNA barcode, contribuindo assim para o conhecimento da biodiversidade local. Também foi objetivo do mesmo conhecer os locais de maior incidência de atropelamentos e o tipo de coberturas da paisagem desses. Durante 20 semanas de coleta foram encontrados 123 vertebrados: seis mamíferos (5% do total); 30 aves (24%); 38 anfíbios (30%) e 49 répteis (40%), para uma média de atropelamentos de 0,051 animais/Km. Por meio de caraterísticas morfológicas e moleculares (genes mitocondriais cox1 e 16S) foi possível identificar o status de espécie de 111 amostras do total coletado. As análises realizadas entre classes taxonômicas mostraram diferenças significativas, corroborando que os efeitos das estradas sobre a biodiversidade são táxon-específico. Foram encontrados 19 tipos diferentes de cobertura da paisagem em torno da estrada, sendo mais comuns as construções rurais e as pastagens, evidenciando a forte pressão antrópica sobre os ambientes naturais desta região. Os resultados mostrados neste trabalho pretendem ser uma base importante para o planejamento de ações futuras que prevejam o melhor manejo desta região rica em biodiversidade endêmica do cerrado brasileiro, assim como medidas mitigatórias para diminuir o impacto da estrada.

No / There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.

Die Bedeutung genetischer Polymorphismen im Enzym Cytochrom P450 2C9 für Pharmakokinetik und Wirkungen von Diclofenac und Ibuprofen Es wird angenommen, dass Cytochrom-P450 2C9 die 4’-Hydroxylierung des Nichtsteroidalen Antiphlogistikums Diclofenac und die Hydroxylierung von S-Ibuprofen beim Menschen katalysiert. Es existieren zwei Varianten von Cyp2C9. Deren Auswirkungen auf die Diclofenac- bzw. Ibuprofen-Pharmakokinetik und die Hemmung von Cox-1 und -2 wurde an 21 gesunden Probanden, die sämtliche Kombination der genetischen Varianten *2 und *3 aufwiesen, untersucht. Es zeigten sich keinerlei Hinweise auf eine Einschränkung des Metabolismus von Diclofenac bei heterozygoten und homozygoten Trägern der Cyp2C9-Allele *2 und *3. Darüber hinaus lagen auch die Serumkonzentrationen des Metaboliten 4’-OH-Diclofenac bei Trägern der Allele Cyp2C9 *2 und *3 nicht niedriger. Obwohl verschiedene in vitro-Untersuchungen Cyp2C9 als metabolisierendes Enzym identifizierten, ist die Pharmakokinetik von Diclofenac ist beim Menschen entweder überhaupt nicht oder nur in geringem Ausmaß von Cyp2C9-Aminosäurenpolymorphismen abhängig. Möglicherweise sind die Auswirkungen der Cyp2C9-Aminosäurenvarianten substratabhängig, oder es ist in vivo ein anderes Enzym als Cyp2C9 verantwortlich für die Bildung von 4’-OH-Diclofenac. Im Unterschied dazu hing die Pharmakokinetik von razemischem und von S-Ibuprofen vom Cyp2C9 *3-Polymorphismus ab. Die Bildung von Tx B2 (Cox-1) hing signifikant von Cyp2C9 *3-Polymorphismus ab, derselbe Trend ließ sich auch für Pg E2 (Cox-2) beobachten. Die eingeschränkte Clearance von S-Ibuprofen, die mit einer erhöhten pharmakodynamischen Aktivität einhergeht, legt nahe, dass Träger des Allels Cyp2C9*3 ein höheres Risiko tragen, nach Einnahme einer oralen Standarddosis unerwünschte Nebenwirkungen zu erleiden. / Consequences of genetic polymorphisms in Cytochrome P450 2C9 for pharmacokinetics and effects of Diclofenac and Ibuprofen. Cytochrome-P450 2C9 is considered to catalyse the 4’-hydroxylation of the nonsteroidal analgesic drug diclofenac and the hydroxylation of S-ibuprofen in humans. There are two variants of Cyp2C9. Their impact on diclofenac/ ibuprofen pharmacokinetics and on the inhibition of cyclooxygenases 1 and 2 was studied in 21 healthy volunteers with all combinations of the Cyp2C9 variants *2 and *3. Blood concentrations of diclofenac/ racemic ibuprofen (and of S-ibuprofen and R-ibuprofen) were measured by HPLC. Thromboxane B2 and prostaglandin E2 were measured with use of an enzyme immunoassay. There was no evidence of impaired metabolism of diclofenac in heterozygous and homozygous carriers of the Cyp2C9 alleles *2 and *3 compared to the wildtype. Furthermore, plasma concentrations of the metabolite 4’-OH-diclofenac were not lower in carriers of Cyp2C9*2 and *3. Marked diclofenac mediated inhibition of Cox-1- and Cox-2 activity was detected in all individuals without any Cyp2C9 genotype dependent differences. Even though several in vitro studies identified Cyp2C9 as the metabolising enzyme, Diclofenac pharmacokinetics in humans is either not or only to a minor extend dependent on the Cyp2C9 amino acid polymorphisms. It may be that the Cyp2C9 amino acid variants have differential effects depending on the substrates. Alternatively, an enzyme other than Cyp2C9 may be responsible for 4’-OH-Diclofenac formation in vivo.In contrast, the pharmacokinetics of racemic and of S-ibuprofen depended on the Cyp2C9 *3-polymorphism. The Cyp2C9 variant *2 exhibited no significant effect. Formation of Tx B2 (cox-1) depended significantly on the Cyp2C9 polymorphism, the same trend was observed for Pg E2 (cox-2). The reduced S-ibuprofen total clearance accompanied by increased pharmacodynamic activity indicates an increased risk for carriers of Cyp2C9*3 to suffer from adverse effects after intake of a standard oral dose.

Connaître le sexe d’un oiseau est important pour divers domaines notamment pour les vétérinaires, les écologistes ainsi que pour les éleveurs d’oiseaux qui veulent former des couples qui serviront à la reproduction. Plusieurs espèces d’oiseaux, juvéniles et adultes, n’ont pas de dimorphisme sexuel. L’utilisation de l’ADN est une façon rapide de déterminer le sexe à partir d’un échantillon de sang, de muscle, de plumes ou de fèces. Par contre, la méthode devrait être validée pour chaque espèce et idéalement, standardisée. Le premier objectif de cette étude est de développer une méthode de sexage par séquençage des oiseaux à partir des séquences du gène CHD, en utilisant les oiseaux de proie et les perroquets vus en clinique au Québec. Un deuxième objectif est de faire l’identification de l’espèce à sexer, à partir du gène mitochondrial COX-1 et aussi à partir des séquences CHD-Z et CHD-W, utilisés pour le sexage. Un troisième objectif est d’évaluer les séquences sorties (CHD-Z, CHD-W et COX-1) en vue d’une étude phylogénique. Une extraction d’ADN a été effectuée chez 27 espèces de perroquets, 34 espèces d’oiseaux de proie, une corneille (Corvus brachyrhynchos) et un poulet (Gallus gallus). Une amplification par PCR a été exécutée pour les exons partiels 23 et 24 du gène CHD. Le séquençage de cet amplicon permettait de savoir s’il s’agissait d’un mâle (séquence simple CHD-Z) ou d’une femelle (séquences CHD-Z et CHD-W qui se chevauchent). Afin d’avoir des séquences CHD-W distinctes, un sous-clonage a été fait chez les femelles de chaque espèce. De cette manière, les séquences partielles du gène CHD, Z et W, ont été trouvées pour les espèces échantillonnées. Une étude phylogénique a été effectuée avec les séquences de COX-1, CHD-Z et CHD-W grâce au site « Clustal-Omega ». La méthode de sexage des oiseaux par séquençage du gène CHD est standard et efficace. Le gène COX-1 permet une meilleure identification des espèces parentes et le gène CHD-Z est le plus utile pour étudier la phylogénie profonde. / Knowing the sex of a bird is important for many disciplines, notably for veterinary, ecological and evolutionary studies, not to mention for bird breeders who need to form pairs for reproduction. For many species of birds, juveniles and adults do not display a sexual dimorphism. The use of DNA, derived from a sample of blood, muscle, feathers or feces, is a rapid method to determine a bird’s sex. However, this method must be validated for each species, and ideally, standardised. The first objective of this work was to develop a method of sexing birds by sequencing portions of their CHD gene, for birds of prey and parrots seen in veterinary clinics in Quebec. A second objective was to identify the species being sexed, first of all using the mitochondrial gene COX-1, and second of all using the CHD-Z and CHD-W sequences used for sexing. The third objective of these studies was to evaluate the sequences obtained (CHD-Z, CHD-W and COX-1) for performing phylogenetic studies. DNA was extracted from 27 species of parrots, 34 species of birds of prey, from one species of crow (Corvus brachyrhynchos) and from the chicken (Gallus gallus). A PCR amplification was performed for partial exons 23 and 24 of the CHD gene. Sequencing this amplicon resulted in simple CHD-Z sequences for a male and overlapping CHD-Z and CHD-W sequences for a female, allowing sexing of the bird. In order to obtain distinct CHD-W sequences, a sub-cloning was performed for females of each species. In this fashion, partial sequences of the CHD gene, both -Z and –W, were generated for the species studied. A phylogenetic study was performed using COX-1, CHD-Z and CHD-W sequences and the site “Clustal-Omega”. The method of sexing birds by sequencing was found to be standard and efficient. The COX-1 gene permitted a better resolution of closely related species, while the CHD-Z gene was the most useful for estimating deep phylogenetic relationships.

We are unceasingly exposed to potentially harmful microorganisms. The battle against threatening infectious agents includes activation of both the innate and of the adaptive immune systems. Illness responses are elicited and include inflammation, fever, decreased appetite, lethargy and increased sensitivity to painful stimuli in order to defeat invaders. While many of these signs of disease are controlled by the central nervous system, it has remained an enigma how signals from the peripheral immune system reach the brain through its blood-brain barrier, which precludes macromolecules, including cytokines, from diffusing into the brain parenchyma. Previous findings indicate the existence of a pathway across the blood-brain barrier, which includes binding of the cytokine interleukin-1 (IL-1) to its receptor in the brain vessels, thereby inducing the production of the prostaglandin E2 (PGE2) synthesizing enzymes cyclooxygenase-2 (Cox-2) and microsomal prostaglandin E synthase-1 (mPGES-1), which ultimately synthesize PGE2. PGE2 subsequently binds to any of the four prostaglandin E2 (EP) -receptors. Previous results from our laboratory have suggested that this pathway plays a critical role in the febrile response to infectious stimuli. The present thesis aims at further investigating the molecular events underlying immune-to-brain signalling, with special emphasis on fever, hypothalamic-pituitary-adrenal (HPA) -axis activation and anorexia and their connection to signalling molecules of the cytokine and prostaglandin families, respectively. In paper I, the molecular processes linking the proinflammatory cytokine interleukin-6 (IL-6) and PGE2 in the febrile response were investigated. Both IL-6 and PGE2 have been shown to be critical players in the febrile response, although the molecular connections are not known, i.e. if IL-6 exerts its effects up- or downstream of PGE2. Mice deficient in IL-6 were unable to respond to bacterial lipopolysaccharide (LPS) with a febrile response, but displayed similar induction of Cox-2 and mPGES-1, and similar concentrations of PGE2 in the cerebrospinal fluid as wild-type mice. Paradoxically, the IL-6 deficient mice responded with a dose-dependent elevation of body temperature in response to intracerebroventricularly injected PGE2. Furthermore, IL-6 per se was not pyrogenic when injected peripherally in mice, and did not cause increased levels of PGE2 in cerebrospinal fluid. IL-6 deficient mice were not refractory to the action of PGE2 because of excess production of some hypothermia-producing factor, since administration of a Cox-2 inhibitor in LPS-challenged IL-6 deficient mice did not unmask any hypothermic response, and neutralization of tumor necrosis factor α (TNFα), associated with hypothermia, did not produce fever in LPS-challenged IL-6 deficient mice. These data indicate that IL-6 rather than exerting its effects up- or down-stream of PGE2 affects some process in parallel to PGE2, perhaps by influencing the diffusion and binding of PGE2 onto its target neurons. In papers II and III, we injected the proinflammatory cytokine IL-1β in free-fed wild-type mice, in mice with a deletion of the gene encoding mPGES-1, or in mice deficient in the EP1, EP2 and EP3. Food intake was continuously measured during their active period, revealing that mPGES-1 deficient mice were almost completely resistant to anorexia induced by IL-1β. However, all of the investigated EP receptor deficient mice exhibited a normal profound anorexic response to IL-1β challenge, suggesting that the EP4 is the critical receptor that mediates IL-1β-induced anorexia. We also investigated the role of mPGES-1 in anorexia induced by lipopolysaccharide (LPS) in mPGES-1 deficient mice. The profound anorexic response after LPS-challenge was similar in mPGES-1 deficient and wild-type mice. To further investigate the anorectic behaviour after LPS injection, we pre-starved the animals for 22 hours before injecting them with LPS. In this paradigm, the anorexia was less profound in mPGES-1 knock-out mice. Our results suggest that while the inflammatory anorexia elicited by peripheral IL-1β seems largely to be dependent on mPGES-1-mediated PGE2 synthesis, similar to the febrile response, the LPS-induced anorexia is independent of this mechanism in free-fed mice but not in pre-starved animals. In papers IV and V, the role of prostanoids for the immune-induced HPA-axis response was investigated in mice after genetic deletion or pharmacological inhibition of prostanoid-synthesizing enzymes, including Cox-1, Cox-2, and mPGES-1. The immediate LPS-induced release of ACTH (adrenocorticotropic hormone and corticosteroids was critically dependent on Cox-1 derived prostanoids and occurred independently of Cox-2 and mPGES-1 derived PGE2. In contrast, the delayed HPA-axis response was critically dependent on immune-induced PGE2, synthesized by Cox-2 and mPGES-1, and occurred independently of Cox-1 derived enzymes. In addition, in the mPGES-1 deficient mice, the synthesis of CRH hnRNA and mRNA was decreased in the paraventricular nucleus of the hypothalamus after LPS-challenge, indicating that the delayed hormone secretion was mediated by PGE2-induced gene-transcription of CRH in the hypothalamus. The expression of the c-fos gene and Fos protein, an index of synaptic activation, was maintained in the paraventricular nucleus and its brainstem afferents both after unselective and Cox-2 selective inhibition as well as in Cox-1, Cox-2, and mPGES-1 knock-out mice. This suggests that the immune-induced neuronal activation of autonomic relay nuclei occurs independently of prostanoid synthesis and that it is insufficient for eliciting stress hormone release.

Submitted by ALEXANDRE BORGES null (alex.brgs@hotmail.com) on 2016-06-28T19:11:21Z No. of bitstreams: 1 ESTUDOS DE MODELAGEM MOLECULAR DE LIGNANAS EM COMPLEXOS COM CICLOOXIGENASES-1 E 2.pdf: 4325586 bytes, checksum: 2f6ab56677aea7746bd28dad4b24ea23 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-06-29T17:41:12Z (GMT) No. of bitstreams: 1 borges_a_dr_ilha.pdf: 4325586 bytes, checksum: 2f6ab56677aea7746bd28dad4b24ea23 (MD5) / Made available in DSpace on 2016-06-29T17:41:12Z (GMT). No. of bitstreams: 1 borges_a_dr_ilha.pdf: 4325586 bytes, checksum: 2f6ab56677aea7746bd28dad4b24ea23 (MD5) Previous issue date: 2016-05-11 / Os inibidores seletivos da ciclooxigenase-2 (COX-2), como o rofecoxibe (2) e o celecoxibe (1), formam uma importante classe de medicamentos anti-inflamatórios desenvolvidos a partir da descoberta das duas isoformas das ciclooxigenases (COX-1 e COX-2) na década de 1979. A isoforma 1 esta relacionada com a citoproteção gástrica, agregação plaquetária e função renal e a isoforma 2 relacionada a processos inflamatórios. Estes inibidores seletivos apesar de não apresentarem os efeitos colaterais (ulceras e gastrites) dos anti-inflamatórios não esteroidais (AINEs) clássicos por inibirem apenas a COX-2, apresentam grave risco cardiovascular, o que motivou à retirada do rofecoxibe do mercado. Porém, por ser um eficiente inibidor seletivo da COX-2 a estrutura do rofecoxibe tornou-se referência no estudo de novas substâncias capazes de inibir seletivamente a COX-2. Dentre as ferramentas utilizadas na busca destas novas estruturas está a modelagem molecular através de programas como o GOLD 5.1, que foi utilizado neste trabalho. O uso do GOLD 5.1 possibilitou o estudo do comportamento das estruturas avaliadas em ligação com as ciclooxigenases. O objetivo foi obtenção de estruturas com comportamento semelhante ao rofecoxibe (em relação às COXs) como potenciais candidatos ao desenvolvimento de novos inibidores seletivos para a COX-2. O estudo foi realizado com 480 estruturas modeladas a partir de lignanas naturais como a hinoquinina, cubebina, deoxipodofilotoxina e podofilotoxina, que apresentam atividade anti-inflamatória in vivo ou in vitro, além de semelhanças estruturais com o rofecoxibe. A deoxipodofilotoxina por apresentar seletividade para a COX-2 em ensaio in vitro também foi utilizada como estrutura de referência além do rofecoxibe. Os resultados observados a partir da simulação molecular permitiram concluir que embora tanto o rofecoxibe como a deoxipodofilotoxina (3) inibam seletivamente a COX-2 in vitro, o fazem de modo diferente. Em relação a COX-2 as duas estruturas ocupam a mesma região do sítio ativo, mas o rofecoxibe apresenta interações mais fortes com o bolso hidrofílico desta isoforma (condição necessária para a inibição seletiva para os coxibes). Já para a COX-1 enquanto o rofecoxibe ocupa a porção superior do canal hidrofóbico (sítio ativo) como os demais AINEs, a deoxipodofilotoxina ocupa uma região vizinha. Pelos resultados obtidos é possível sugerir que tanto a maior flexibilidade das estruturas como a presença do anel lactônico, são importantes para um comportamento análogo ao rofecoxibe ou à deoxipodofilotoxina. Com relação à interação com o bolso hidrofílico da COX-2, os resultados sugerem que a presença de grupos aceptores de prótons menos volumosos nas posições C3 e C4, C3’ e C4’ ou C4 levam a resultados melhores que grupos aceptores de maior volume. A presença de grupos doadores de prótons apesar de permitirem forte interação com o bolso hidrofílico da COX-2 leva a resultados globais insatisfatórios, pois formam interações fortes com o resíduo Arg120 do sítio ativo da COX-1, interação considerada importante para a inibição não seletiva. Resultado semelhante à deoxipodofilotoxina foi observado apenas para a estrutura 17. As estruturas 37, 188, 266, 267, 348 e a hinoquinina (4) apresentam resultados semelhantes ao rofecoxibe, para as duas isoformas. Deste modo permite-se sugerir a partir dos resultados obtidos neste estudo que a hinoquinina (4) e as estruturas 17, 37, 188, 266, 267 e 348 apresentam-se como possíveis protótipos de fármacos que atuem como inibidores seletivos para a COX-2. / The selective inhibitors of the cyclooxygenase-2 (COX-2) as rofecoxib (2) and celecoxib (1), form an important class of anti-inflammatory drugs developed from the discovery of two isoforms of cyclooxygenases (COX-1 and COX-2) in the late 1979. Isoform 1 is related to the gastric cytoprotection, platelet and renal function and isoform 2 related to inflammatory processes. These selective inhibitors although they did not side effects (ulcers and gastritis) of the classic NSAIDs to inhibit only COX-2, have severe cardiovascular risk, which led to the withdrawal of rofecoxib from the market. However, to be an effective selective COX-2 to rofecoxib structure has a reference in the study of new substances capable of selectively inhibiting COX-2. Among the tools used in the search of these new structures is by molecular modeling program such as GOLD 5.1, which was used in this work. Using GOLD 5.1 made it possible to study the behavior of structures evaluated in binding with the cyclooxygenases. With the objective of obtaining structures with similar behavior to rofecoxib (regarding behavior with COX) as potential candidates for the development of new selective inhibitors for COX-2. The study was conducted with 480 structures modeled from natural lignans as hinokinin, cubebin, deoxypodophyllotoxin and podophyllotoxin, which have anti-inflammatory activity in vivo or in vitro as well as structural similarities with rofecoxib. The deoxypodophyllotoxin for presenting selectivity for COX-2 in the in vitro assay was also used as a reference structure beyond rofecoxib. The results observed from the molecular simulation showed that although both rofecoxib (2) as deoxypodophyllotoxin (3) selectively inhibit COX-2 in vitro, they do differently. In relation to COX-2 the two structures occupy the same region of the active site, but rofecoxib has stronger interactions with the hydrophilic pocket of this isoform (a necessary condition for the selective inhibition for coxibs). As for the COX-1 while rofecoxib occupies the upper portion of the hydrophobic channel (active site) like other NSAIDs, the deoxypodophyllotoxin occupies a neighboring region. From the results it is possible to suggest that the greater flexibility of the structures such as the presence of the lactone ring, are important for a similar behavior to rofecoxib or deoxipodofilotoxina. With respect to the interaction with the hydrophilic pocket COX-2, the results suggest that the presence of acceptors groups less bulky protons in posítions C3 and C4, C3 ' and C4' and C4 lead to better results than acceptors groups of larger volume. The presence of proton donors groups despite allowing strong interaction with the hydrophilic pocket COX-2 lead to poor overall results, since they form strong interactions with Arg120 residue of COX-1 active site, considered important interaction for inhibiting non-selective. Results similar to deoxipodofilotoxina was only observed for structure 17. Structures 37, 188, 266, 267, 348 and hinokinin (4) show results similar to rofecoxib for the two isoforSA. Thus it allows suggest from the results obtained in this study hinokinin (4) and structures 17, 37, 188, 266, 267 and 348 are shown as possible prototype drugs that act as selective inhibitors for COX-2.