Efeito gastroprotetor e cicatrizante da bosentana e sildenafila isolados ou associados em úlceras pépticas em ratos

Orientadora: Profa. Maria Fernanda de Paula WernerTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa : Curitiba, 19/12/2020Inclui referênciasResumo: A úlcera gástrica é um problema de saúde pública mundial, caracterizada por extensas lesões na mucosa do estômago e/ou esôfago que podem se estender por todas as camadas do órgão, causando sérias complicações como sangramentos e perfurações que requerem hospitalização do paciente e intervenção cirúrgica. Como principais causas da úlcera gástrica temos o uso de anti-inflamatórios não esteroidais (AINEs), que diminuem a produção de muco-bicarbonato gastroprotetor, e a infecção pela bactéria Helicobacter pylori. Além da erradicação da H. pylori, o tratamento convencional visa a inibição da produção de ácido clorídrico pelo uso dos inibidores da bomba de prótons (e.g. omeprazol), ou de antagonistas de receptores H2 da histamina (e.g. ranitidina). Contudo, estudos demonstram que a inibição da secreção ácida gástrica está associada ao aumento do risco de desenvolvimento de diversas doenças como osteoporose, infecções intestinais e até doença renal crônica. Além da secreção ácida gástrica, a ulcerogênese é um processo complexo que também envolve o fluxo sanguíneo. Os sistemas endotelinérgico e da via do óxido nítrico modulam a microcirculação bem como a inflamação e o estresse oxidativo na mucosa gástrica. Assim, o objetivo deste trabalho foi avaliar o efeito gastroprotetor e cicatrizante de drogas vasoativas, mais especificamente da bosentana e da sildenafila, em modelos de úlcera gástrica aguda e crônica. A administração oral de bosentana (antagonista dual de receptores ETA e ETB) e da sildenafila (inibidor da fosfodiesterase 5), isoladas ou em associação foram investigadas no modelo de úlcera aguda induzida por isquemia-reperfusão. O efeito cicatrizante da monoterapia com a bosentana foi investigada no modelo de úlcera crônica induzida por ácido acético. Por fim, o modelo de ligadura do piloro foi utilizado para investigar o efeito da bosentana na secreção ácida gástrica. Na úlcera aguda por IR, o pré-tratamento com bosentana, sildenafila ou a associação de baixas doses de ambos reduziram a área ulcerada, impediram a depleção de muco e das defesas antioxidantes no tecido gástrico. Complementarmente, esse pré-tratamento reduziu a infiltração de neutrófilos, importante marcador de inflamação tecidual. No modelo de úlcera crônica induzida por ácido acético foi constatado um aumento na expressão de receptores ETA e ETB na área ulcerada dos animais do grupo veículo comparado com animais naïve. O tratamento com a bosentana por cinco dias acelerou a cicatrização das úlceras crônicas, aumentou o conteúdo de mucina e a proliferação de células epiteliais da mucosa gástrica, reduziu a infiltração de neutrófilos e os níveis de citocinas inflamatórias, como fator de necrose tumoral-alfa e interleucina 1 beta. Os parâmetros plasmáticos da função renal e hepática não foram alterados pelo tratamento subcrônico com a bosentana. A partir dos resultados do modelo agudo, conclui-se que o pré-tratamento com bosentana e sildenafila ou a associação de baixas doses dos fármacos exibe efeito gastroprotetor, e no modelo de úlcera crônica, o pós-tratamento com bosentana induziu a cicatrização de úlceras crônicas, sem modificar a secreção ácida gástrica. Nossos resultados reforçam a tendência mundial do reposicionamento de fármacos (uso off label), e de maneira especial este estudo demonstra que outras vias podem ser exploradas como adjuvantes no tratamento de úlceras gástricas, contornar os possíveis efeitos adversos do tratamento convencional.Abstract: Gastric ulcer is a worldwide public health problem, characterized by extensive lesions in the mucosa of the stomach and / or esophagus that can extend through all layers of the organ, causing serious complications such as bleeding and perforations that require hospitalization and surgical intervention. The main causes of gastric ulcer are the use of non-steroidal anti-inflammatory drugs (NSAIDs), which decrease the production of gastroprotective mucus-bicarbonate, and infection by the bacterium Helicobacter pylori. In addition to the eradication of H. pylori, conventional treatment aims to inhibit the production of hydrochloric acid by the use of proton pump inhibitors (e.g. omeprazole), or histamine H2 receptor antagonists (e.g. ranitidine). However, studies show that inhibition of gastric acid secretion is associated with an increased risk of developing several diseases such as osteoporosis, intestinal infections and even chronic kidney disease. In addition to gastric acid secretion, ulcerogenesis is a complex process that also involves blood flow. The endothelinergic and nitric oxide pathway systems modulate microcirculation as well as inflammation and oxidative stress in the gastric mucosa. Thus, the objective of this work was to evaluate the gastroprotective and healing effect of vasoactive drugs, more specifically bosentan and sildenafil, in models of acute and chronic gastric ulcers. Oral administration of bosentan (dual antagonist of ETA and ETB receptors) and sildenafil (phosphodiesterase 5 inhibitor), either alone or in combination, were investigated in the ischemia-reperfusion-induced acute ulcer model. The healing effect of bosentan monotherapy was investigated in the acetic acid-induced chronic ulcer model. Finally, the pyloric ligation model was used to investigate the effect of bosentan on gastric acid secretion. In acute IR ulcers, pretreatment with bosentan, sildenafil or the combination of low doses of both reduced the ulcerated area, prevented mucus depletion and antioxidant defenses in gastric tissue. In addition, this pre-treatment reduced the infiltration of neutrophils, an important marker of tissue inflammation. In the acetic acidinduced chronic ulcer model, an increase in the expression of ETA and ETB receptors was found in the ulcerated area of animals in the vehicle group compared with naïve animals. Treatment with bosentan for five days accelerated the healing of chronic ulcers, increased mucin content and proliferation of gastric mucosal epithelial cells, reduced neutrophil infiltration and inflammatory cytokine levels, such as alpha-tumor necrosis factor and interleukin 1 beta. Plasma parameters of renal and liver function were not altered by sub-chronic treatment with bosentan. From the results of the acute model, it is concluded that pretreatment with bosentan and sildenafil or the association of low doses of the drugs exhibits a gastroprotective effect, and in the chronic ulcer model, post-treatment with bosentan induced the healing of ulcers, without changing gastric acid secretion. Our results reinforce the worldwide trend of drug repositioning (off label use), and in a special way this study demonstrates that other routes can be explored as adjuvants in the treatment of gastric ulcers, to circumvent the possible adverse effects of conventional treatment

Ischemia-reperfusión coronaria: papel del óxido nitro y endothelina-1. Una Revisión

This Review focus on the myocardial ischemia-reperfusion, paying particular attention to the role of nitric oxide (NO) and endothelin-1 (ET-1) in the regulation of the coronary circulation under normal conditions and after ischemia-reperfusion. Coronary atheromatosis is usually induced by endothelium dysfunction/damage, and is the main cause of acute coronary syndromes (e. g., acute myocardial infarction, AMI). The assessment of endothelial function of patients with coronary artery disease may provide useful information. The most effective treatment of AMI is timely reperfusion for restoring the blood flow to the ischemic myocardial territory, but this procedure may also damage myocardium (reperfusion injury), of which the pathophysiology and treatment remain uncertain. The interaction between NO and ET-1 may be relevant for regulating the coronary circulation, with predominance of NO over ET-1 under normal conditions. The coronary circulation plays a crucial role in ischemia-reperfusion since it is the cause and victim of consequences of this condition. Ischemia-reperfusion damages not only the myocardium but also coronary vasculature, including the endothelium, increases plasma levels of ET-1, induces functional predominace of ET-1 over NO, augments the coronary response to ET-1, and alters the role of endothelin receptors in this response. All these alterations may lead to dysregulation of coronary vasculature and the non-reflow phenomenon, which may underly reperfusion injury. Thus, ET-1 could be of significance in pathophsysiology of ischemia-reperfusion and reperfusion injury, and the use of antagonists for endothelin ETA/ETB receptors could protect the heart against reperfusion injury.Esta Revisión se centra en la isquemia-reperfusión del miocardio, prestando particular atención al papel del óxido nítrico (NO) y endothelina-1 (ET-1) en la regulación de la circulación coronaria en condiciones normales y tras la isquemia-reperfusión. La ateromatosis coronaria suele estar causada por disfunción/daño endotelial, y lidera las causas de los síndromes coronarios agudos (p. e., infarto agudo de miocardio, IAM). El tratamiento más eficaz del IAM es la reperfusión del territorio de miocardio isquémico, pero este procedimiento también causa lesión del miocardio (lesión por reperfusión), cuya fisiopatología y tratamiento siguen siendo inciertos. La interacción entre el NO y ET-1 es relevante en la regulación de la circulación coronaria, y en condiciones normales predomina el NO sobre la ET-1. La circulación coronaria desempeña un papel crucial en la isquemia-reperfusión puesto que ella es la causa y víctima de las consecuencias de esta situación. La isquemia-reperfusión daña no solo el miocardio, sino también el lecho vascular coronario, aumenta los niveles plasmáticos de ET-1, induce el predominio de la ET-1 sobre el NO, aumenta la respuesta coronaria alaET-1 y altera el papel de sus receptores en esta respuesta. Todo ello podría contribuir a la disfunción de la circulación coronaria y el fenómeno de no-reflujo y, como consecuencia, a la lesión por reperfusión. Así, la ET-1 podría ocupar un papel destacado en la fisiopatología de la lesión por reperfusión, y el uso de antagonistas de los receptores ETA/ETB podría proteger al corazón frente a la lesiones por reperfusión

The Pathophysiologic Significance of Endothelins in the Cerebral Circulation

This thesis examined the pathophysiologic role of the endothelins in the cerebral circulation. Endothelin receptors were characterised in the rabbit basilar artery in vitro and in feline cerebral resistance arterioles in situ. Investigations in the feline cerebral resistance arterioles were designed to examine the cerebrovascular effects and blood-brain barrier penetration of non-peptide and peptide endothelin receptor antagonists. The pathophysiologic role of the endothelins were examined, using validated doses of endothelin receptor antagonists (primarily Bosentan), in experimental models of focal cerebral ischaemia in the cat, transient global cerebral ischaemia in the rat and subdural haematoma in the rat. In the rabbit basilar artery in vitro, pre-incubation with either the combined ETA/ETB endothelin receptor antagonist Bosentan (4-tert-Butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzene sulphonamide) or the endothelin ETA receptor antagonist BQ-123 (cyclo D-Aspartate-D-Tryptophan-L-Leucine-D-Valine-L-Proline) had minimal effect on the resting tone of arterial segments. The ETB receptor agonist BQ-3020 (N-Acetyl [11 Ala,15 Ala] ET-1 (6-21)) elicited a small constriction of the arterial segments. Bosentan (10 muM) elicited a rightward shift of the ET-1 concentration response curve (pA2 = 5.1). BQ-123 (0.1 - 10 muM) elicited a concentration dependent rightward shift of the ET-1 concentration response curve (pA2 = 5.3). BQ-123 (1 muM) elicited a substantial rightward shift of the ET-3 concentration response curve (pA2 = 7.2). The observations suggested the presence of an 'atypical' endothelin ETA receptor mediating vasoconstriction in the rabbit basilar artery. The receptors mediating the cerebrovascular actions of endothelins were examined in feline cerebral resistance arterioles in vivo. The adventitial microapplication of the endothelin ETA receptor antagonist BQ-123 (0.1 - 10 muM) per se had minimal effect on cerebral resistance arterioles examined. The adventitial microapplication of endothelin-1 (10 nM) elicited a marked vasoconstriction of cerebral resistance arterioles (-29.1 +/- 1.9 % from pre-injection baseline). The endothelin-1 induced vasoconstriction was attenuated, in a dose dependent manner, by the adventitial co-application of BQ-123 and endothelin-1 (estimated IC50 0.7 muM). The adventitial microapplication of the endothelin ETB receptor agonist BQ-3020 (0.001 - 1 muM) effected a dose dependent vasodilatation (EC50 30 nM, maximum response 25 +/- 5 % from pre-injection baseline). The magnitude of the vasodilatation elicited by BQ-3020 (100 nM and 1 muM) was dependent on the pre-injection calibre of the arterioles examined. The intracarotid infusion (via the lingual artery) of BQ- 3020 (0.5-500 pmol/min) had no significant effect on the calibre of cerebral resistance arterioles. These results suggest that the peptide endothelin ETB receptor agonist fails to gain access to the cerebrovascular endothelin ETB receptors following its intraluminal administration. These investigations indicate that endothelin ETA receptors mediate vasoconstriction and endothelin ETB receptors mediate vasodilatation in feline cerebral resistance arterioles in vivo. The cerebrovascular actions of Bosentan, a novel endothelin antagonist with effects at ET

Influencing the macro- and microcirculatory complications of nonocclusive mesenteric ischemia by complement C5a inhibitor treatments

Nonocclusive mesenteric ischemia (NOMI) can develop in the absence of apparent anatomical obstruction of the mesenteric circulation in a variety of low flow states. The pathophysiology of NOMI is unexplored, the early diagnosis is challenging and the available treatments are of questionable effectiveness. In this respect, new experimental models are sought to clarify the exact pathomechanisms and new, effective therapeutic ways are needed to reduce the increasingly high mortality. Our first aim was to develop clinically relevant in vivo models to investigate the macro- and microcirculatory effects of NOMI. Also, we hypothesized the role of complement activation in the acute and subacute consequences of NOMI and our objectives were to characterize the effects of the inhibition of complement protein known as C5a during this condition. Acetyl-peptide-A (AcPepA) is an antisense-homology box-derived peptide, which is capable to inhibit the C5a effects by binding directly to the anaphylatoxin. We hypothesized that the inhibition of C5a can decrease the intensity of inflammatory reactions and in parallel, to normalize the impaired mesenteric circulation. Acute experimental pericardial tamponade (PT) was established in anesthetized minipigs, while partial aorta occlusion (PAO) was induced in rats to investigate the circulatory and inflammatory changes of NOMI in clinically relevant time frames. After the relief of PT, elevated levels of oxidative stress markers and inflammatory mediators were detected in association with the signs of diminished splanchnic microcirculation. 24 hours after PAO the macrocirculatory parameters improved significantly, while the intramural microcirculation was significantly impaired and accompanied by increased leukocyte infiltration. The in vivo histology confirmed the structural and microvascular damage of the mucosa. In both animal models of NOMI, the administration of AcPepA moderated the hemodynamic changes, improved the intramural microcirculation, reduced the inflammatory activations and the histological signs of mucosal damage. In conclusion we can say that our newly developed animal models provide a cross section for events in the short and long time frames and proved to be suitable for the investigations of the pathophysiology of NOMI. The hemodynamic changes in the acute PT together with those observed after PAO suggest that complement activation plays central role in the early and late macro- and microcirculatory disturbances during NOMI. The results suggest that C5a inhibitor treatment influences favourably the hemodynamic effects and reduces the potentially harmful inflammatory activation after experimental NOMI as well

Role of endothelin in the pathogenesis of acute laminitis in horses

Acute laminitis is a severely debilitating disease of the laminae of the equine digit; however, the mechanism(s) of pathogenesis have yet to be fully elucidated. In physiologic states, the endothelium synthesizes substances, such as nitric oxide (NO; vasodilator) and endothelin-1 (ET-1; profound vasoconstrictor), which play a crucial role in vasomotor regulation. The overall hypothesis is that the initiating factor in the onset of acute laminitis is a disruption in the balance between NO and ET-1, which leads to digital vasoconstriction and subsequent laminar ischemic necrosis. In vitro studies with digital vessels from healthy horses and horses with naturally-acquired laminitis determined that ET-1 caused concentration-dependent, sustained contraction of arteries and more profound contraction of veins, and incubation with the nonselective ET receptor antagonist (PD145065) at a 10-5 M concentration abolished these contractile effects. ET-1 was then administered into the digit of healthy conscious horses, which resulted in reduced blood flow and the ET antagonist, especially in combination with a NO donor, reversed these reductions. Naturally-acquired laminitic horses had a trend for increased jugular and cephalic venous plasma ET-like immunoreactivity, and horses during the development of black walnut extract (BWE)-induced laminitis developed increased digital venous plasma ET-like immunoreactivity. After validation for equine tissues, ET-1 immunohistochemical staining was conducted on digital vascular and laminar tissues, but no notable differences were found between healthy and naturally-acquired or experimentally-induced laminitic horses. During the developmental stages of BWE-induced laminitis, digital blood flow initially decreased followed by hyperemia, corresponding with demonstration of clinical signs of laminitis. Administration of the ET antagonist, and the antagonist combined with a NO donor, improved Starling force alterations by improving digital vascular resistances and blood flow. Utilizing digital vessel rings from BWE-treated horses, ET-1 caused a concentration-dependent contraction in vitro that was abolished by the ET antagonist. Endothelium-dependent vasodilation was decreased in these vessels, demonstrating possible altered endothelial function due to BWE administration. Based on the results of these studies, ET-1 appears to play a role in the pathophysiology of acute laminitis in horses and continued investigations evaluating ET antagonists as preventative and therapeutic agents for this devastating disease are warranted