Desenvolvimento, avaliação da atividade leishmanicida e toxicológica de nanosistema de cumarina (1,2-benzopirona)

Abstract

Coumarin (CM - 1,2-benzopyranone), found in Caatinga plant species such as Amburana cearensis AC Smith and Justicia pectoralis Jacq, is used in the clinic to treat varicose veins, hemorrhoids and pre and postoperative thrombosis prophylaxis and in pregnancy. Studies prove its anti-inflammatory, immunomodulatory and anti-parasitic activity. However, it is a volatile molecule, with low solubility in water and its lactone is easily hydrolyzed, which makes it difficult to take advantage of its pharmacological properties. On the other hand, nanosystems have been successfully used to overcome or reduce challenges in the development of drugs with biopharmaceutical problems. Given the above, the objective of this work was the development of a nanosystem of CM, with evaluation of toxicity and leishmanicidal activity (Leishmania braziliensis) in vitro and in vivo. For this purpose, an analytical method was developed and validated for the identification and quantification of CM by High Performance Liquid Chromatography (HPLC), according to Resolution No. 166 (BRASIL, 2017). Two different nanocarriers were produced to encapsulate CM: CM-loaded Nanostructured Lipid Carriers (CM-NLC) and CM-loaded polymeric Nanocapsules (CM- NC). The CM-NLCs were produced (homogenization in high pressure at heat) with different concentrations of lipid phase (5% and 10%) using Precirol ATO5® and Miglyol 812®. CM- NC were prepared (interfacial deposition of the preformed polymer) with the aid of Central Rotational Composite Design (CRCD) 23 using the polymer Eudragit® RS100. The characterization and evaluation of the nanosystems stability for nine months was carried out in relation to the size (S), polydispersion index (PDI), zeta potential (ZP), active content (AC) and encapsulation efficiency (EE). Toxicity tests were performed in vitro (MTT test - Bromide 3 [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium in macrophages/J774) and in vivo (acute toxicity in Swiss mice). The parasitic load (intracellular amastigotes) in J774 macrophages infected with L. braziliensis (24 or 48 h) was determined and the leishmanicidal activity was evaluated in Mesocricetus auratus hamsters. For the purposes of characterizing the CM-NC, the analytical method (277 nm) was validated, showing to be specific, linear, precise, accurate and robust under certain conditions. CM-NLC with 10% lipid phase and 5 homogenization cycles (CM-NLC 10%5C) showed better physical-chemical characteristics compared to other formulations of lipid carriers, but it was cytotoxic in macrophages (5 - 100 μg/ml), therefore was passed over from the study. The CM-NC formulation from the selected CRCD showed higher EE (58.01 ± 0.2%) in relation to the other nanocapsule suspensions, with S, PDI and ZP of 210.0 ± 0.24 nm; 0.13 ± 7.27; 75.9 ± 0.73 mV, respectively and a AC of 102.5 ± 8.8%, showing stability during the investigated period (nine months). CM-NC (100 μg/ml) did not show cytotoxicity in macrophages and in acute toxicity studies (10 - 40 mg/kg, v.o.), it did not cause behavioral, biochemical or hematological changes in rodents. The CM- NC (50 μg/ml) reduced the parasitic load of macrophages infected by L. braziliensis by 31.1% and 5.4% after 24 and 48 h of incubation, respectively. The treatment of animals with CM-NC (10 mg/kg, v.o.) reduced the parasitic load at the lesion site and protected the spread of the parasites to the lymph node. The study provided the development and characterization of CM- NC, which showed technological characteristics of interest and stability, not associated with toxicity and with leishmanicidal activity in vitro and in vivo.