“Atividade inibitória de substâncias isoladas a partir de plantas da floresta amazônica frente aos agentes da criptococose”

Abstract

ABSTRACT The search for new drugs is one of the main challenges facing cryptococcosis. This severe disease has limited therapy with the use of amphotericin B and fluconazole, substances described as having limitations on its toxicity, microbial resistance and clinical failure. In this sense, the screening of new substances is necessary. The present thesis has 3 chapters: To compose the chapter cryptococcosis in Amazonas (Chapter 1) a bibliographical survey was carried out comparing the current literature and the works produced in our region. For the screening of new substances (Chapter 2), the antifungal activity of 15 substances (plant and semi-synthetic isolates) was determined using the methodology described by the Clinical and Laboratory Standards Institute - CLSI (document M-27 A3) the Cryptococcus neoformans VNI and C. gattii VGII. Finally, the mechanism of antifungal action and cytotoxicity of the substances desdro-iso-α-lapachone (NAF2) and 8 nitroharmano (AHON1) were investigated in Chapter 3, and the mechanism of action on the cell wall (sorbitol tests) was investigated in membrane ergosterol (exogenous ergosterol assays) and in cell extravasation (dosing of substances that absorb at 260 nm). The cytotoxicity of these substances was assessed against the MRC-5 lineage of human fibroblasts in a dose-response assay. As for Chapter 1 results, it was observed that cryptococcosis in the state of Amazonas is an illness that affects an average of 25 people annually. These are mainly men (73%), young (39.2 ± 12.3, 19-68%), with HIV (70-85%). The region also presents around 3 cases / year of cryptococcosis in a patient without apparent immunocompromising, including 1-2 cases of children. The main clinical forms of the disease in the region are neurocryptococcosis (96%) and fungemia (42%). As for laboratory diagnosis, the Tropical Medicine Foundation Heitor Vieira Dourado, a reference center for infectology in the northern region, centralizes most of the diagnoses. Direct examinations and culture of cerebrospinal fluid are the most common form of diagnosis. More recently, the research with Cryptococcal Antigen Lateral Flow Assay (CrAg LFA) in blood samples has been used and is notable for its sensitivity and speed of execution, especially useful when the patient has low fungal load (early stages of the disease). As for the agents, Cryptococcus neoformans complex genotypes are the main causative agents (86%), and the main genotypes in the region are VNI (86%) and VGII (14%) and MLST is ST93 (86%). As for the screening results (Chapter 2), of the 15 substances tested (04 isolates of plants and 11 obtained from semi-synthesis), two substances showed prominent antifungal activity NAF2 and AHON1. NAF2 had a minimum inhibitory concentration (MIC) of 80 μg / mL against C. neoformans VNI and 160 μg / mL against C. gattii. AHON1 had MIC of 40 μg / mL against C. neoformans VNI and C. gattii VGII. Regarding the results of mechanism of action and cytotoxicity (Chapter 3), it was observed that the presence of sorbitol (cell wall assay) did not interfere in the MIC of substances NAF2 and AHON1. Similarly, it was observed that the presence of ergosterol (membrane ergosterol assay), did not interfere with their MIC. However, both caused extravasation of substances absorbing at 260 nm (extravasation test). In the normal human fibroblast (MRC-5) cytotoxicity assay, AHON1 and NAF2 were found to have IC 50> 50 μg / mL. Keywords - Cryptococcosis, Drugs, Mechanism of Action, Antifungal Sensitivity