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The antimycobacterial activity of thioridazine derivatives against drug resistant Mycobacterium tuberculosis: in vitro, ex vivo and in vivo studies
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O objectivo principal desta tese foi o de avaliar a acção da tioridazina (TZ), bem como de compostos derivados desta, obtidos por manipulação química, como agentes com actividade contra Mycobacterium tuberculosis, em particular contra M. tuberculosis
multi-resistente (MDR-TB). Desta forma foram obtidos vinte e dois derivados e efectuados testes de toxicidade e mutagenicidade pelo método de exclusão com azul de trypan (realizado em linfócitos humanos) e o teste de Ames, respectivamente. Todos os derivados não tóxicos e não mutagénicos foram testados in vitro contra estirpes de
Staphylococcus aureus resistente à meticilina (MRSA), que foi o microrganismo modelo utilizado durante todo o trabalho, e estirpes de MDR-TB. Visto que a tuberculose é uma infecção do macrófago alveolar, estes estudos foram subsequentemente aplicados a macrófagos infectados. Desta forma, é importante analisar a actividade que estes compostos apresentam dentro do macrófago, local onde
normalmente a micobactéria se encontra. Iniciaram-se estudos animais com a TZ, de forma a verificar a eficácia deste composto em curar murganhos Balb/C infectados com M. tuberculosis H37Rv ATCC27294. Desta forma foi possível optimizar parâmetros, tais como, a via de infecção, a concentração de composto a administrar, entre outros. Os
resultados obtidos demonstraram que dos vinte e dois derivados nenhum apresentava toxicidade ou efeitos mutagénicos, nas condições testadas. Desta forma, os vinte e dois derivados foram seleccionados para estudos in vitro contra estirpes de S. aureus e de M. tuberculosis. Dos estudos in vitro foi possível verificar que seis derivados apresentaram uma maior actividade do que a TZ e desta forma foram seleccionados para os estudos ex vivo. Quando testados em macrófagos infectados três derivados demonstraram um efeito
marcado na activação das células fagocitárias (“enhancement of the killing activity”), sendo um dos derivados ainda mais activo do que a TZ. Dos estudos em animais, foi possível seleccionar as condições a serem implementadas em estudos futuros com os derivados mais activos. De todos os resultados obtidos durante esta tese foi possível desenvolver um modelo baseado na interacção do macrófago com a bactéria e a
subsequente acção destes compostos. O modelo desenvolvido (“macrophage model”) pode assim contribuir para clarificar o que ocorre a nível intracelular aquando da adição dos compostos ao meio de cultura.
The main objective of this Thesis was to evaluate thioridazine (TZ) and chemically derived derivatives for anti-Mycobacterium tuberculosis activity in particular against multi-drug resistant (MDR) M. tuberculosis. Twenty-two TZ derivatives were obtained and screened for toxicity and mutagenicity by the trypan blue exclusion assay in human lymphocytes and the Ames test, respectively. The derivatives that were devoid of any toxicity and mutagenicity were then tested against Methicillin-resistant Staphylococcus aureus (MRSA) used as a model during the entire work, and against antibiotic resistant M. tuberculosis (MDR-TB) strains in vitro and subsequently in the macrophage that has phagocytosed the organism. Since tuberculosis is an infection of the alveolar macrophage it is important to evaluate the activity of these compounds inside the phagocytic cell where the mycobacteria are to be found. Thioridazine was also tested for its ability to cure the mouse of infection by M. tuberculosis. Therefore, animal studies using Balb/C mice infected with M. tuberculosis H37Rv ATCC27294 strain were initiated to parameterize the route of infection, dose of compound to be administered, among others. The results obtained showed that from the twenty-two TZ derivates none was toxic or mutagenic under the conditions tested. Therefore, all the twenty-two derivatives were selected for in vitro evaluation against S. aureus and M. tuberculosis strains. From the in vitro results six derivatives showed greater activity than TZ and were selected for ex vivo studies. Three of these derivatives were shown to enhance the macrophage killing activity and one of the derivatives was even more active than TZ. From the animal studies it was possible to select the conditions to apply in further studies with the most active derivatives. From all the data obtained during this Thesis it was possible to develop a model based on the macrophage interaction with the bacteria and the subsequent action of the compounds. The macrophage model can elucidate what takes place inside the human macrophage when these compounds are added to the medium. Due to the uniqueness of the approaches developed during the Thesis research, other potential sources of anti-tubercular compounds were explored: plants and organosilicon compounds (SILA). These studies demonstrated that extracts from the nuisance plant Carpobrotus edulis could enhance the killing of intracellular bacteria such as MDR-TB and MRSA. In addition, the extract was shown to modulate the immune system thereby indicating its potential use for cellular immune deficient disorder as well as render MDR mouse lymphoma cells carrying the human mdr1 gene completely susceptible to cytotoxic drugs to which they were initially resistant. The results of this component of my research have resulted in the design of new experiments which are now being carried out by another Ph.D. student in our Unit and at the Medical University of Szeged, Hungary. In conclusion, the results obtrained from my Thesis research has paved the way for clinical trial consideration of the many compounds studied shown to have significant intracellular activity against XDR-TB/MDR-TB at concentrations that are non-toxic and that can be readily achieved in the infected human.
The main objective of this Thesis was to evaluate thioridazine (TZ) and chemically derived derivatives for anti-Mycobacterium tuberculosis activity in particular against multi-drug resistant (MDR) M. tuberculosis. Twenty-two TZ derivatives were obtained and screened for toxicity and mutagenicity by the trypan blue exclusion assay in human lymphocytes and the Ames test, respectively. The derivatives that were devoid of any toxicity and mutagenicity were then tested against Methicillin-resistant Staphylococcus aureus (MRSA) used as a model during the entire work, and against antibiotic resistant M. tuberculosis (MDR-TB) strains in vitro and subsequently in the macrophage that has phagocytosed the organism. Since tuberculosis is an infection of the alveolar macrophage it is important to evaluate the activity of these compounds inside the phagocytic cell where the mycobacteria are to be found. Thioridazine was also tested for its ability to cure the mouse of infection by M. tuberculosis. Therefore, animal studies using Balb/C mice infected with M. tuberculosis H37Rv ATCC27294 strain were initiated to parameterize the route of infection, dose of compound to be administered, among others. The results obtained showed that from the twenty-two TZ derivates none was toxic or mutagenic under the conditions tested. Therefore, all the twenty-two derivatives were selected for in vitro evaluation against S. aureus and M. tuberculosis strains. From the in vitro results six derivatives showed greater activity than TZ and were selected for ex vivo studies. Three of these derivatives were shown to enhance the macrophage killing activity and one of the derivatives was even more active than TZ. From the animal studies it was possible to select the conditions to apply in further studies with the most active derivatives. From all the data obtained during this Thesis it was possible to develop a model based on the macrophage interaction with the bacteria and the subsequent action of the compounds. The macrophage model can elucidate what takes place inside the human macrophage when these compounds are added to the medium. Due to the uniqueness of the approaches developed during the Thesis research, other potential sources of anti-tubercular compounds were explored: plants and organosilicon compounds (SILA). These studies demonstrated that extracts from the nuisance plant Carpobrotus edulis could enhance the killing of intracellular bacteria such as MDR-TB and MRSA. In addition, the extract was shown to modulate the immune system thereby indicating its potential use for cellular immune deficient disorder as well as render MDR mouse lymphoma cells carrying the human mdr1 gene completely susceptible to cytotoxic drugs to which they were initially resistant. The results of this component of my research have resulted in the design of new experiments which are now being carried out by another Ph.D. student in our Unit and at the Medical University of Szeged, Hungary. In conclusion, the results obtrained from my Thesis research has paved the way for clinical trial consideration of the many compounds studied shown to have significant intracellular activity against XDR-TB/MDR-TB at concentrations that are non-toxic and that can be readily achieved in the infected human.
Descrição
Palavras-chave
Microbiologia médica Biologia molecular Tioridazina Mycobacterium tuberculosis Infecção Terapêutica Bactérias Macrófagos
Contexto Educativo
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Instituto de Higiene e Medicina Tropical