Investigação da leucurolisina-a após administração intravítrea em ratos como potencial alternativa para o tratamento das oclusões de veia da retina.

Abstract

Retinal vein occlusion is characterized by a thrombotic vascular obstruction and is an important cause of blindness worldwide. The treatment of this condition is still very challenging due to restricted drug access to the posterior segment of the eye, caused by anatomical and physiological barriers designed to keep the ocular tissues isolated from the systemic circulation. Thus, invasive interventions, such as intravitreal injection, are necessary for drugs to reach therapeutic concentrations in the retina. Several substances with thrombolytic potential have been studied and employed in the dissolution of thrombi, so that, in this work, we sought to evaluate leucurolysin-a (leuc-a), a non-hemorrhagic metalloproteinase, purified from the Bothrops leucurus snake venom, which has a fibrinolytic activity already described. In this context, the objective of this work was to investigate the toxicity and safety of leuc-a in vitro and in vivo, employing different clinical tests and animal models. In addition, the clearance of leuc-a after intravitreal injection into the eyes of healthy rats and the potential degradation of Matrigel components by leuc-a were evaluated. The cell viability study indicated that none of the tested concentrations showed cell viability above 80% against ARPE-19 cells, and for the IC50 a concentration of 26.64 µg/mL was obtained. In the in vivo studies, doses of 12.5, 25, 50, and 100 µg/mL were evaluated. The first step was performed in healthy Wistar rats and evaluated by electroretinography exam; in it, only the highest dose caused alteration in the functional activity of the retina. The scintigraphic images obtained after the radiolabeling of leuc-a with technetium-99m showed that the clearance of this protein in the vitreous cavity occurred slowly, remaining constant for up to 6 h. However, in the second step, also in healthy Wistar rats, optical coherence tomography and angiography revealed that all doses tested showed some degree of toxicity, including hemorrhagic events. This result was corroborated by the third step, which evaluated the effects of the highest dose of leuc-a in healthy Long Evans rats and gave evidence of vascular changes and recruitment of inflammatory cells by immunohistochemistry analyses. The degradation of laminin and nidogen, components of Matrigel and basement membranes, by leuc-a may explain the hemorrhagic events observed earlier. This study showed controversial results regarding the safety of leuc-a for intravitreal use and its hemorrhagic potential, so it was not directly tested in a retinal vein occlusion model. However, this work provides clinical results of great relevance for ocular toxicity studies as well as for further elucidation of the behavior of leuc-a in vivo and in further applications.