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Abstract(s)
O Acidente Vascular Cerebral Isquémico (AVCI) ocorre quando o fluxo sanguíneo
cerebral é interrompido ou diminuído devido à obstrução de uma artéria, levando
consequentemente a danos no tecido cerebral. Quando não revertido rapidamente, o
AVCI leva a sequelas duradouras que podem comprometer as funções motoras,
cognitivas e emocionais do doente. Em lesões graves, os tratamentos existentes não são
eficazes na reversão dos danos causados pelo evento isquémico, sendo crucial o
desenvolvimento de novas estratégias terapêuticas.
Com o presente trabalho, pretendemos avaliar o potencial efeito neuroprotetor dos
fármacos acetato de segesterona, um potente agonista do recetor da progesterona, e a
sinvastatina, uma estatina com ação antioxidante, num modelo in vitro de AVCI, e testar
a segurança de duas novas formulações (nanoemulsão e microemulsão) para o acetato
de segesterona. Para o efeito, foram usadas como modelo de AVCI culturas de córtex de
rato submetidas a privação de oxigénio e de glucose. Numa primeira fase, otimizámos o
modelo celular de forma a obter uma percentagem de morte celular que permitisse
detetar possíveis efeitos protetores dos fármacos em estudo. A citotoxicidade foi avaliada
pelo ensaio de redução do sal de tetrazólio. Posteriormente, otimizámos as condições de
cultura de forma a eliminar possíveis ações protetoras endógenas, que pudessem
camuflar a ação protetora dos compostos em estudo. Com este objetivo, procedemos à
remoção do soro da cultura e testámos a lesão isquémica em culturas enriquecidas em
neurónios, para eliminar a ação protetora dos astrócitos. Apesar destas estratégias
conduzirem ao aumento da morte celular, a análise da viabilidade celular com o ensaio
MTT (do inglês, Thiazolyl Blue Tetrazolium Bromide) não demonstrou efeito protetor do
acetato de segesterona ou da sinvastatina em nenhuma das concentrações testadas
(0,001 mM a 10000 mM para o acetato de segesterona e 0,1 mM a 10000 mM para a
sinvastatina), sugerindo que o efeito protetor destes compostos, observado em estudos
in vivo, pode depender da ação destes em outros alvos.
A segunda parte do trabalho consistiu na análise da segurança de uma nanoemulsão e de
uma microemulsão para o acetato de segesterona, tendo-se verificado que nenhuma das
formulações apresentou citoxicidade nas concentrações estudadas.
Concluindo, as formulações de acetato de segesterona são promissoras para uso in vivo,
de modo a aumentar a biodisponibilidade do fármaco na administração intranasal.
Embora não tenhamos conseguido obter os efeitos neuroprotetores pretendidos, ainda existe necessidade de efetuar mais estudos, para compreender os efeitos protetores
descritos pela literatura tanto para o acetato de segesterona como da sinvastatina em
AVCI, bem como o impacto da interação com outros alvos na neuroprotecção exercida
por esses fármacos.
Ischemic stroke occurs when cerebral blood flow is interrupted or reduced due to the obstruction of an artery, consequently leading to damage to brain tissue. In Portugal, ischemic stroke is the leading cause of death and permanent disability. When not reversed quickly, it leads to lasting sequelae that can compromise motor, cognitive and emotional functions. In severe injuries, existing treatments are not effective in reversing the damage caused by the ischemic event, hence the strong need for new therapeutic strategies. The aim of this study was to evaluate the potential neuroprotective effect of the drugs segesterone acetate, a potent progesterone receptor agonist, and simvastatin, a statin with antioxidant action, in an in vitro model of ischemic stroke, and to test the safety of two new formulations (nanoemulsion and microemulsion) for segesterone acetate. For this purpose, rat cortex cultures subjected to oxygen and glucose deprivation were used as a stroke model. Initially, we optimized the cell model to obtain a percentage of cell death that would allow us to detect possible protective effects of the drugs under study. Cytotoxicity was assessed using the MTT reduction assay. Subsequently, we adjusted the culture conditions to eliminate possible endogenous protective actions that could mask the protective action of the compounds under study. With this aim, we removed the serum from the culture and tested the ischemic injury in cultures enriched in neurons, to eliminate the protective action of astrocytes. Although these strategies led to increased cell death, analysis of cell viability using the MTT assay did not show a protective effect of segesterone acetate or simvastatin at any of the concentrations tested (0.001 mM - 10000 mM for segesterone acetate and 0.1 mM - 10000 mM for simvastatin), suggesting that the protective effect observed in the in vivo studies of these compounds may depend on other targets. A second part of the work consisted of analyzing the safety of a nanoemulsion and a microemulsion for segesterone acetate. None of the formulations showed toxicity at the concentrations studied (up to 10000 mM of segesterone acetate). In conclusion, segesterone acetate formulations are promising for in vivo use, to increase the bioavailability of the drug in intranasal administration. Although we were unable to achieve the desired neuroprotective effects, there is still a great need for further studies to understand the protective effects described in the literature for both segesterone acetate and simvastatin in ischemic strokes, as well as the impact of interaction with other targets on the neuroprotection exerted by these drugs.
Ischemic stroke occurs when cerebral blood flow is interrupted or reduced due to the obstruction of an artery, consequently leading to damage to brain tissue. In Portugal, ischemic stroke is the leading cause of death and permanent disability. When not reversed quickly, it leads to lasting sequelae that can compromise motor, cognitive and emotional functions. In severe injuries, existing treatments are not effective in reversing the damage caused by the ischemic event, hence the strong need for new therapeutic strategies. The aim of this study was to evaluate the potential neuroprotective effect of the drugs segesterone acetate, a potent progesterone receptor agonist, and simvastatin, a statin with antioxidant action, in an in vitro model of ischemic stroke, and to test the safety of two new formulations (nanoemulsion and microemulsion) for segesterone acetate. For this purpose, rat cortex cultures subjected to oxygen and glucose deprivation were used as a stroke model. Initially, we optimized the cell model to obtain a percentage of cell death that would allow us to detect possible protective effects of the drugs under study. Cytotoxicity was assessed using the MTT reduction assay. Subsequently, we adjusted the culture conditions to eliminate possible endogenous protective actions that could mask the protective action of the compounds under study. With this aim, we removed the serum from the culture and tested the ischemic injury in cultures enriched in neurons, to eliminate the protective action of astrocytes. Although these strategies led to increased cell death, analysis of cell viability using the MTT assay did not show a protective effect of segesterone acetate or simvastatin at any of the concentrations tested (0.001 mM - 10000 mM for segesterone acetate and 0.1 mM - 10000 mM for simvastatin), suggesting that the protective effect observed in the in vivo studies of these compounds may depend on other targets. A second part of the work consisted of analyzing the safety of a nanoemulsion and a microemulsion for segesterone acetate. None of the formulations showed toxicity at the concentrations studied (up to 10000 mM of segesterone acetate). In conclusion, segesterone acetate formulations are promising for in vivo use, to increase the bioavailability of the drug in intranasal administration. Although we were unable to achieve the desired neuroprotective effects, there is still a great need for further studies to understand the protective effects described in the literature for both segesterone acetate and simvastatin in ischemic strokes, as well as the impact of interaction with other targets on the neuroprotection exerted by these drugs.
Description
Keywords
Acetato de Segesterona Acidente Vascular Cerebral Isquémico Isquemia Cerebral Microemulsão Nanoemulsão Sinvastatina