Protective effect of a metalloprotease (marimastat) on haemostatic and neuromuscular disfunctions induced by Bothrops alternatus (Viperidae: Crotalinae) venom in rodents

Abstract

Accidents caused by snakes of the genus Bothrops induce severe clinical manifestations such as local and systemic myonecrosis, renal dysfunction, hemorrhage, coagulopathy, and hypotension. The treatment for envenomation by Bothrops spp. is restricted to polyvalent serotherapy. B. alternatus (Urutu-Cruzeiro) venom exhibits peculiar neurotoxic action in rodent nerve-muscle preparation in vitro, accompanied by muscle injury. Recent studies have shown that marimastat, a metalloprotease synthetic peptide inhibitor, can be used as a potential coadjutant agent to serotherapy in envenomation by Viperidae and Elapidae snakes, although there are no studies with venoms of South American snakes. In this project, we aim to assess the protective action of marimastat, associated or not to a commercial polyvalent anti-Bothrops serum, on the haemostatic and neuromuscular alterations by Bothrops alternatus venom in animal experimentation models in vitro and in vivo. Initially, we will carry out an enzymatic kinetic protocol for caseinolytic and esterase activities to determine the minimum inhibitory concentration of marimastat on the major proteases of this venom; it will be applied colorimetric methods, with the reactions being read in a spectrophotometer. For in vitro protocols, the inhibitory action of marimastat (alone or associated with antivenom) on the haemostatic activity of the B. alternatus venom will be determined from the minimal coagulant dose, activation of prothrombin and activated partial thromboplastin, and thrombin-like activity in rat citrated plasma using commercial kits for coagulometry. In addition, the inhibitory action of the drug alone or associated with antivenom will be assessed on the neurotoxicity of the B. alternatus venom in mouse isolated nerve phrenic diaphragm preparation mounted in a multifunction myographic system. In vivo, the inhibitory action of both agents on the haemorrhagic effect of B. alternatus venom will be assess through formation of subcutaneous hemorrhagic halo in rats. The results of this study will contribute to understand the therapeutic potential of marimastat on the effects induced by Bothrops venoms.