Thesis (Ph.D.)--University of Western Australia, 2006.

Orientador: Stephen Hyslop / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T04:49:26Z (GMT). No. of bitstreams: 1 Smaal_Adriana_M.pdf: 6478282 bytes, checksum: 2107d35eeaa596500136ca0dd4e07119 (MD5) Previous issue date: 2010 / Resumo: O envenenamento por serpentes do gênero Bothrops resulta em alterações sistêmicas que incluem coagulopatia, hemorragia sistêmica, choque hipovolêmico e insuficiência renal aguda. Neste estudo investigamos a ação de da peçonha de B. jararacussu e suas frações cromatográficas sobre alguns parâmetros cardiovasculares (pressão arterial, freqüências cardíaca e respiratória, e eletrocardiograma) em ratos anestesiados com uretana. Ratos Wistar (300-400 g) foram canulados na arteria carótida esquerda para monitoramento contínuo da pressão arterial sistêmica e na veia femoral para administração das peçonhas e frações cromatográficas. A freqüência cardíaca e o ECG foram monitorados por um eletrocardiógrafo. No final do experimento, coração, rins, fígado e pulmão foram processados para avaliação histológica. Os ratos foram monitorados durante 120 min. Para análise histológica, foram coletados amostras de coração, pulmão, rim e fígado. A peçonha (0.25, 0.5 and 0.75 mg/kg, i.v.) casou queda acentuada da pressão arterial nos até os 5 minutos após administração das peçonhas, tendo recuperação para valores de níveis basais nas doses menores, e a dose maior não houve recuperação. As doses menores não causaram alterações nas freqüências cardíacas e respiratórias, porem a dose maior causou queda irreversivel destes parâmetros. Alterações transitórias vistas no ECG foram extra sístole, taquicardia ventricular e alteração na amplitude do complexo QRS. Ratos injetados com a menor dose sobreviveram durante os 120 minutos monitorados no experimento, já os injetados com a maior dose morreram com aproximadamente 15 minutos após a administração da peçonha. A gel filtração resultou em 5 picos (PI-PV), sendo que a administração dos 2 primeiros picos mostraram alterações semelhantes a peçonha bruta. O fracionamento do pico I por troca iônica, não resultou em alterações cardiovasculares,pois provavelmente eles agem em sinergismo. Similarmente, a Bothropstoxina isolada do pico III não causou alterações. Analise histológica mostraram alterações em órgãos, principalmente em pulmões. Adicionalmente,foi visualizado mionecrose, edema e infiltrado inflamatório no coração, perda da arquitetura tecidual no fígado, e microaneurismas capilares, presença de proteína na luz tubular, e infiltrado inflamatório nos rins. Os picos I, II e III mostraram alterações similares a peçonha bruta. Estes resultados mostram que as peçonhas botrópicas produzem alterações cardiovasculares importantes, alem de alterações morfológicas em diversos órgãos. A predominância de hemorragia e presença de atividade hemorrágica nos picos I, II e III indicam a presença de diversas proteínas hemorrágicas nesta peçonha. / Abstract: Envenoming by Bothrops snakes results in systemic alterations that include coagulopathy, internal hemorrhage, hypovolemic shock and acute renal failure. In this work, we investigated the action of Bothrops jararacussu snake venom and its chromatographic fractions on arterial blood pressure, heart and respiratory rates, and electrocardiogram in urethane-anesthetized male Wistar rats. The rats were cannulated for blood pressure measurements (carotid artery) and the injection of venom or fraction (femoral vein). The heart rate and electrocardiogram (ECG) were measured with an ECG recorder. At the end of the experiments, samples of heart, liver, lung and kidney were processed for histological analysis. Venom (0.25, 0.5 and 0.75 mg/kg, i.v.) caused immediate hypotension that lasted for 5 min followed by a gradual recovery to pre-venom levels with the two lowest doses, whereas there was no recovery with the highest dose. The two lowest doses did not significantly affect the heart rate or respiratory rate, but these parameters were depressed with the highest dose. Transient changes in the ECG seen during the first 5 min included ventricular extrasystoles, ventricular tachycardia and alterations in the QRS complex amplitude. Rats injected with the two lowest doses survived for 120 min post-venom, whereas with the highest dose the survival time was 15 min at the most. Gel filtration (Sephacryl 75) of venom resulted in five peaks (PI - PIV), the first two of which produced cardiovascular alterations similar to those of the venom. Fractionation of PI resulted in two major peaks, neither of which produced significant cardiovascular alterations, perhaps because they normally act in synergy. Similarly, a PLA2 (bothropstoxin) isolated from peak PIII did not alter the parameters investigated. Histological analysis revealed hemorrhage in the organs examined, particularly the lungs. In addition, there were focal lesions in hepatic and renal tissue, as well as renal epithelial desquamation, glomerular microaneurysms, an increase in the capsular space and the presence of protein-like material in the lumen of proximal and distal tubules. Peaks PI, PII and PIII produced histological alterations similar to those of the venom. These results show that B. jararacussu venom causes important cardiovascular changes in anesthetized rats, in addition to morphological alterations in a variety of organs. The predominance of hemorrhage and the presence of hemorrhagic activity in peaks PI - PIII indicate the presence of a variety of hemorrhagic proteins in this venom. / Mestrado / Farmacologia / Mestre em Farmacologia

This thesis discussed the cardiovascular effects of the aqueous leaf extract and a fraction of the methanol extract of Leonotis leonurus, a plant commonly used in traditional medicine in South Africa for the treatment of hypertension and other cardiac problems. The cardiovascular effects was tested on anaesthetized normotensive male Wistar rats and isolated perfused rat hearts.

The mechanical contraction of the heart is tightly coupled to rapid and concerted electrical excitation of the cardiac muscle. This electrical activity is facilitated by a highly synchronized conduction system consisting of channels, pumps, and transporters that facilitate the flow of charged ions between cellular compartments, the cytoplasm, and the interstitial fluid between cells. The biophysical properties of these membrane proteins have been studied for many years, but their role in the generation of potentially lethal cardiac arrhythmias and their interactions with drugs remains an important field of research. The cardiac isoform of the voltage-gated Na+-channel, Nav1.5, has garnered widespread interest because of its role in the generation of electrical impulses in the cardiac myocyte, its association with congenital conduction disorders and acquired cardiac arrhythmias, and its unique pharmacological properties. The Congenital Long QT Syndrome Type 3 (LQT3) arises from heritable mutations in SCN5A - the gene encoding Nav1.5 - that disrupt the inactivation process responsible for imparting a refractory period and that often cause a sustained depolarizing late current (INaL). The gain of function depolarizing currents arising from LQT3 mutant channels cause a prolongation of the ventricular action potential and leave patients susceptible to asynchronous electrical activity, ventricular arrhythmias, and sudden cardiac death. The disruption of channel inactivation can arise through a wide range of modalities, including changes in inactivation voltage-dependence and kinetics, and has been shown to occur with varying degrees of severity. Because of this range of phenotypes there is heterogeneity in the risk factors for arrhythmia and sudden cardiac death and in the utility of Na+-channel blocking antiarrhythmic drugs. Moreover, INaL has been implicated as a proarrhythmic and potentiating factor in several acquired cardiac ailments including heart failure, ischemia, and hypertrophy. There is therefore a large unmet need for improved understanding of INaL and mechanisms of its selective inhibition, and LQT3 mutant channels provide a reliable experimental model for this class of cardiac arrhythmias. This study will employ a combination of electrophysiological and computational methods to unravel mechanisms by which mutant Nav1.5 produces pro-arrhythmic currents and the interactions of different disease-causing mutant channels with a set of clinically relevant antiarrhythmic drugs. Chapter 1 of this study presents a functional characterization of one LQT3 mutation, F1473C, that was discovered in a patient with severe QT prolongation, frequent ventricular arrhythmias, and a poor response to pharmacological intervention. This mutation gives rise to INaL by a mechanism that is functionally distinct from the mechanism discovered previously in the canonical LQT3 mutation, ΔKPQ (1505-1507del), and causes a unique response to channel inhibitors. In order to better understand the mechanisms of this divergent pharmacology, Chapter 2 presents the development of a series of computational models which explore the gating dysfunctions that cause INaL and how these pathological changes can influence the predicted safety and efficacy of pharmacological intervention. These models predict that the majority of mutation- specific drug effects can be attributed to differential mutant channel gating, but raise the possibility that mutations may directly alter the physical chemical interaction between drugs and channels. Finally, Chapter 3 presents an attempt to explore this possibility using an innovative chemical biology technique - the site-specific incorporation of unnatural amino acids - that allows for the measurement of precise chemical interactions hypothesized to vary in a mutation-dependent manner. The findings presented in this work promote the need for patient-specific screening of antiarrhythmic agents and lay the groundwork for the use of in silico systems analysis of cardiovascular pharmacology.

The apelin system is an evolving transmitter system consisting of the G protein coupled apelin receptor and two endogenous peptide ligands, apelin and elabela. It is implicated as a potential therapeutic for a number of diseases; however, the endogenous peptides are limited by half-life and bioavailability. This study aims to identify and pharmacologically characterise apelin agonists in vitro and in vivo and to evaluate their therapeutic potential in pulmonary arterial hypertension as a model disease. CMF-019 was identified as the first G protein biased apelin agonist. To date, suitable small molecule apelin agonists as experimental tool compounds have been limited and CMF-019 represents an important advance. CMF-019 was active in vivo, producing an increase in cardiac contractility and vasodilatation, similar to apelin. These effects were achieved without receptor desensitisation, supporting the remarkable G protein bias observed in vitro. Furthermore, it was disease-modifying in vitro in an endothelial cell apoptosis assay but despite this, did not prevent pulmonary arterial hypertension in a monocrotaline rat model of the disease. An apelin mimetic peptide possessing an unnatural amino acid, MM202, conjugated chemically via a polyethylene glycol linker to an anti-serum domain antibody (AlbudAb) was also characterised. The product MM202-AlbudAb represents the first time an AlbudAb has been conjugated chemically to an unnatural peptide mimetic, providing protection from proteolysis and glomerular filtration. Importantly, it retained binding to albumin and demonstrated in vitro and in vivo activity at the apelin receptor. In conclusion, this thesis has identified and pharmacologically characterised two novel apelin agonists that possess significant advantages over the endogenous peptides. CMF-019 is suitable as an experimental tool compound and, as the first G protein biased small molecule, provides a starting point for more suitable therapeutics. In addition, MM202-AlbudAb proves that unnatural peptides can be conjugated to AlbudAb, supporting use of this technology in other small-peptide ligand transmitter systems.

This thesis discussed the cardiovascular effects of the aqueous leaf extract and a fraction of the methanol extract of Leonotis leonurus, a plant commonly used in traditional medicine in South Africa for the treatment of hypertension and other cardiac problems. The cardiovascular effects was tested on anaesthetized normotensive male Wistar rats and isolated perfused rat hearts.

Orientador: Stephen Hyslop / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T23:44:30Z (GMT). No. of bitstreams: 1 Dias_Lourdes_D.pdf: 2781638 bytes, checksum: 00a6161d21fa428051e65ae4993b92c1 (MD5) Previous issue date: 2009 / Resumo: As peçonhas do gênero bothrops causam hipotensão, no entanto as alterações cardíacas e hemodinâmicas, associadas com este fenômeno não têm sido bem investigadas. Neste estudo, analisamos as alterações cardiovasculares causadas pela peçonha de Bothrops alternatus (urutu) em cães e, avaliamos a cardiotoxicidade desta peçonha em átrio direito isolado de rato. Em cães anestesiados com isoflurano, a peçonha (0,3 mg/kg, i.iv.) causou hipotensão imediata atingindo o ápice aos 5 minutos, seguida por uma lenta recuperação para níveis não significativamente diferentes do basal após 2h. Não foi observada recuperação no grupo tratado com a dose de 1,0 mg/kg. A hipotensão foi acompanhada por uma queda abrupta do débito cardíaco, dos trabalhos tanto do ventrículo direito quanto do esquerdo, bem como do volume e índice sistólico, os quais permaneceram reduzidos até o final do experimento. Não ocorreu alteração significativa na freqüência cardíaca, no ECG, nos valores pressóricos pulmonares, nos níveis dos gases sanguíneos (pO2, pCO2, HCO3, SBCe e SBEc), e nem nos parâmetros metabólicos (pH, lactato, glicose e creatinoquinase), porém, foi observado um aumento significativo nos níveis de lactato desidrogenase logo no início do envenenamento. Nenhuma alteração histológica no tecido cardíaco foi observada, no entanto, observou-se microaneurismas e alguma descamação epitelial nos túbulos renais. Houve uma diminuição rápida da peçonha circulante, após a administração i.v., que ainda foi detectada aos 240 minutos. A peçonha de B. alternatus (0,5; 1,0 e 2,0 mg/ml) não alterou a frequência atrial, mas reduziu significativamente a força contrátil (redução máxima de ~76%) e aumentou acentuadamente a liberação de creatinoquinase (CK) e creatinoqunase - MB (CK-MB). A análise histopatológica mostrou extensa mionecrose dos cardiomiócitos. A peçonha dialisada (1,0 mg/ml, membrana de 2000 Da) contra NaCl 0,9 % (24h a 4 °C) não alterou a redução progressiva na força contrátil, enquanto que o aquecimento (100 °C, 20 min) aboliu esta redução; A liberação de CK e CK-MB não foi alterada pela diálise e pelo aquecimento. A atropina, o atenolol e propranol, a cimetidina ou a indometacina não alteraram a força contrátil atrial, no entanto o L-NAME - inibidor da NOS, atenuou a redução na contratilidade, sugerindo um possível envolvimento do NO na resposta induzida pela peçonha. Estes resultados mostram que em cães, a peçonha de B. alternatus produziu acentuadas alterações cardiovasculares, envolvendo uma ação cardíaca direta, com poucas alterações metabólicas. A peçonha é também tóxica para átrio isolado de rato, provavelmente em função dos efeitos proteolíticos e/ou de PLA2. / Abstract: Bothrops snake venoms cause hypotension, but the hemodynamic and cardiac alterations associated with this phenomenon have not been extensively investigated. In this study, we examined the cardiovascular changes caused by Bothrops alternatus (urutu) venom in anesthetized dogs and examined the cardiotoxicity of the venom in rat isolated rat atria. In isofluorane-anesthetized dogs, venom (0.3 mg/kg, i.v.) caused immediate hypotension that was maximal within 5 min followed by a slow recovery to levels not significantly different from pre-venom values after 2 h; no recovery was seen with a venom dose of 1 mg/kg. The hypotension was accompanied by an abrupt decrease in cardiac output, left and right ventricular systolic work, and systolic indices and volume that persisted without recovery until the end of the experiment. There were no significant changes in heart rate, ECG, pulmonary hemodynamics, blood gas levels (pO2, pCO2, HCO3, SBCe and SBEc) and metabolic parameters (blood pH, lactate, glucose and creatine kinase); however, a slight, significant increase in lactate dehydrogenase was seen soon after venom. There were no histological alterations in cardiac tissue, but microaneurysms and epithelial desquamation were seen in renal tubules. Circulating venom decreased rapidly after i.v. administration, but was still detectable after 240 min. Venom (0.5, 1.0 and 2.0 mg/ml) did not affect the beating rate of rat isolated right atria but significantly reduced the contractile force (maximal reduction of ~76%) and markedly increased the release of creatine kinase (CK) and CK-MB. Histological analysis revealed extensive myonecrosis. Venom dialysis (1.0 mg/ml; membrane nominal MW cut-off = 2000 Da) against 0.9% NaCl (24 h, 4°C) did not affect the decrease in contractile force whereas heating (100°C, 20 min) abolished the venom-induced reduction; CK and CK-MB release was also unaltered by dialysis but attenuated by heating. The decrease in atrial contractility was unaffected by atropine, atenolol, propranolol, cimetidine or indomethacin, but was attenuated by L-NAME, an inhibitor of nitric oxide synthase, indicating a possible role for nitric oxide in the venom-induced response. These results show that in dogs B. alternatus produces marked cardiovascular alterations involving a direct cardiac action, with little role for metabolic changes. The venom is also toxic to rat atria, probably as a result of venom proteolytic and/or phospholipase A2 activity. / Doutorado / Farmacologia / Doutor em Farmacologia

Orientador: Stephen Hyslop / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T05:11:43Z (GMT). No. of bitstreams: 1 Rodrigues_MarianaAcedoPitocco_M.pdf: 2686415 bytes, checksum: 93b2ceba582a84158f4ce0d23348a829 (MD5) Previous issue date: 2010 / Resumo: Serpentes do gênero Bothrops são a principal causa de acidentes ofídicos no Brasil. Acidentes envolvendo a Bothrops jararacussu (jararacuçu) resultam em envenenamento moderado e grave, devido principalmente à grande quantidade de peçonha injetada por esta espécie. Entre os efeitos observados, o quadro de hipotensão e de choque são fatores importantes a serem considerados. Neste trabalho, avaliamos as alterações cardíacas causadas pela peçonha de B. jararacussu em átrio direito isolado de rato. A incubação das preparações com peçonha (0,025, 0,050, 0,1 e 0,2 mg/ml) resultou em uma marcada contratura atrial em altas concentrações que levou a uma redução progressiva da força contrátil e a freqüência atrial, especialmente na maior concentração utilizada (0,2 mg/ml). A maior concentração da peçonha também causou uma marcada liberação de CK-BM e desorganização das fibras musculares atriais. A peçonha aquecida (100°C, 20 min) aboliu esta atividade enquanto a diálise não alterou significativamente essas respostas. O fracionamento da peçonha de B. jararacussu em cromatografia por gel filtração e troca iônica nos forneceu a fração (III3) que reproduziu todas as alterações funcionais e morfológicas vistas com a peçonha bruta. SDS-PAGE da fração III3, na presença e ausência de ditiotreitol e ß-mecaptoetanol revelaram uma massa molecular de ~28 KDa e 14 KDa, respectivamente, semelhantes às bothropstoxinas, as principais PLA2 miotóxicas desta peçonha. Os antivenenos comerciais utilizados (botrópico e botrópico/crotálico) se mostraram eficientes em neutralizar os efeitos produzidos pela peçonha bruta tanto na força e freqüência atriais como na liberação de CK-MB e nas alterações histológicas. A pré-incubação com antagonistas farmacológicos (bloqueadores de receptores adrenérgicos, histaminérgicos, muscarínicos, e inibidores da ciclooxigenase e óxido nítrico sintase) não afetou significativamente as alterações induzidas pela peçonha. Estes resultados indicam que as alterações induzidas pela peçonha de B. jararacussu estão envolvidas diretamente com o dano a fibra muscular atrial mediada provavelmente através da atividade das PLA2 miotóxicas. / Abstract: The genus Bothrops is the principal cause of snakebite in Brazil. Bites by Bothrops jararacussu (jararacuçu) result in moderate to severe envenoming partly because of the large amount of venom injected by this species. Hypotension and circulatory shock are important systemic manifestations associated with bites by this species. In this work, we examined the cardiac alterations caused by B. jararacussu venom in rat isolated right atria. Incubation of atria with venom (0.025, 0.050, 0.1 and 0.2 mg/ml) resulted in marked muscle contracture at high concentrations that led to a progressive decrease in contractile force and beating rate, especially at the highest concentration (0.2 mg/ml). The highest venom concentrations also caused a marked release of CK-MB and disorganization of atrial muscle fibers. Heating the venom (100ºC, 20 min) abolished this activity whereas dialysis did not significantly alter the responses. Fractionation of B. jararacussu venom by gel filtration and ion exchange chromatographies yielded a peak (III-3) that reproduced all of the morphological and functional changes seen with the venom. SDS-PAGE of the peak III-3 protein in the absence and presence - mercaptoethanol revealed a molecular mass of ~28 KDa and 14 KDa, respectively, similar to bothropstoxins, the main myotoxic PLA2 of this venom. Bothropic and bothropic/crotalic antivenoms prevented the venom (0.2 mg/ml)-induced decrease in contractile force and atrial rate, as well as the tissue damage (CK-MB release and histological alterations). Pre-incubation with adrenergic, histaminergic and muscarinic receptor antagonists, or with cyclooxygenase and nitric oxide synthase inhibitors, did not significantly affect the venom-induced changes. These results indicate that the B. jararacussu venom-induced alterations probably involve direct to atrial muscle fibers mediated by venom myotoxic PLA2. / Mestrado / Mestre em Farmacologia

Magister Pharmaceuticae - MPharm / This thesis discussed the cardiovascular effects of the aqueous leaf extract and a fraction of the methanol extract of Leonotis leonurus, a plant commonly used in traditional medicine in South Africa for the treatment of hypertension and other cardiac problems. The cardiovascular effects was tested on anaesthetized normotensive male Wistar rats and isolated perfused rat hearts. / South Africa

[Truncated abstract] Background: This thesis examines pharmacotherapy use in the secondary prevention of coronary heart disease. It includes antiplatelet agents, beta-blockers, statins and ACE inhibitors, all shown in landmark clinical trials and meta-analyses to reduce the risk of cardiac events in patients with known coronary disease. Underuse of effective preventive therapies represents a lost opportunity to reduce mortality and morbidity. Overseas studies have shown significant underuse of effective therapies at the time of hospital discharge following an acute event and later in ambulatory care. Australian data on prescribing practices following an acute coronary event and, ongoing use in ambulatory care are sparse. Aims: The aim of this thesis was to quantify the prescription of known effective therapies at the time of hospital discharge following an acute coronary event and ongoing use in ambulatory care. A secondary aim was to identify barriers to optimal secondary prevention thus providing an evidential basis to recommend change. Methods: This was an observational study of a cohort of post-MI patients admitted to a tertiary and affiliate hospital in Perth, Western Australia. The continuum of care from the treatment plan at discharge through to the treatment regimen and risk factor management 12 months post-MI was examined. The intermediate step, communication about the treatment plan with the patient and the primary health care provider was also examined. The study involved a review of hospital medical records and follow-up questionnaires to patients and their general practitioners at 3 and 12 months post-MI. All post-myocardial patients were included in the analysis of prescriptions at discharge. The follow-up study included patients 80 years and younger with no terminal conditions. Patient interviews at 3 months and interviews and focus groups with key hospital staff provided qualitative data to inform the quantitative data.