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The cure from poison

Toxin of the urutu pit viper acts as a cicatrizant and in the formation of the blood vessels

MIGUEL BOYAYANA protein found in the venom of the urutu (Bothrops alternatus) has shown in tests potential for acting as a cicatrizant and regenerator of damaged tissues, as in the cases of myocardial infarction. Depending on the concentration employed, the alternagin-C, or ALT-C, the name given to the toxin isolated from the venom, can either promote or inhibit the formation of new blood vessels. “They are two opposite effects”, says Professor Heloísa Sobreiro Selistre de Araújo, from the Physiological Sciences Department of the Federal University of São Carlos (UFSCar), the coordinator of a group that is researching snake venoms to isolate natural compounds and pharmaceutical applications.

In low concentrations, the isolated proteins fosters the formation of new vessels, which makes the molecule a candidate for the development of medicines for the treatment of pathologies that result in an inadequate vascularization, like heart attacks, wounds that are hard to cicatrize in the lower members, particularly in diabetics, and even in erectile dysfunction. In high concentrations, it inhibits the formation of new vessels, an interesting activity for treating cancers and metastases. Although the two effects have been observed in tests in vitro and in vivo with mice, the researchers are concentrating their studies on the tissue regeneration activities shown by the toxin, which proved to be more promising.

Two companies from the pharmaceutical area have shown interest in entering into a partnership with the university to work on the development of the new molecule. The UFSCar laboratory will be in charge of all the tests up to the preclinical tests, which consists of tests with larger animals. In the stage of clinical tests, which will be done by the company, the researchers are going to produce and supply the necessary quantity of the protein. Before formalizing the commitment with the university, the companies are awaiting the results of further, more specific tests, to assess what the safe concentration range of the protein is. “These tests are necessary, because the line that separates the desired effect from the toxic effect is very tenuous”, Heloísa says.

Alternated horseshoes
The urutu was chosen because the researchers wanted to work with a typically South American snake. Besides Brazil, it is found in Argentina, Uruguay and Paraguay. Of the same genus as the (Bothrops), it is given the alternatus denomination because of the patterns distributed over its body, in the form of horseshoes. “At the beginning, we only knew of the effects arising from poisoning”, says researcher Oscar Henrique Pereira Ramos, who began to take part in the research during his studies for a master’s and doctor’s degrees, at the Physiological Sciences Department of UFSCar. Today, he is doing postdoctoral studies at the Biophysics and Biochemistry Laboratory of the Butantan Institute. The effects after the bite of an urutu are mainly hemorrhagic, local or systemic. When they are systemic, they may produce toxicity in the kidneys, leading to kidney failure, and hemorrhage in the brain or lungs.

“We noticed that the venom had some very active components”, Ramos says. Amongst these components are the disintegrins, proteins that interact with the integrins, a class of attaching molecules located on the cell surface. The attachment processes occur when a cell makes contact with another or with the extracellular matrix. These interactions are fundamental for various biological processes, such as cell differentiation, immune response, maintenance of the cell structure, cicatrization of wounds and formation of metastases.

The search for proteins of interest led to alternagin-C toxin, a disintegrin isolated for the first time by researcher Dulce Helena Ferreira de Souza, who was doing postdoctoral studies at the laboratory and is today a professor of UFSCar’s Chemistry Department. The toxin alters the cell behavior, because it binds itself to the surface receptors, in this case the integrins, and sets off a cascade of signals inside the cell, that culminates with an alteration in the expression of certain genes. Some proteins that are activated inside the cell and some genes that start being more or less expressed inside the cells have now been tested by researcher Márcia Regina Cominetti, from UFSCar, and identified in relation to the cicatrizing effects. The biological tests of the activity of the toxin in endothelial cells of the umbilical cord were led by Professor Verônica Maria Morandi da Silva, from the Biology Institute of the Rio de Janeiro State University (UERJ). The endothelial cells form the blood capillaries, and it is they that need to divide to make the growth of new vessels possible.

Effect reproduced
Studies carried out with mice have proved the effect of ALT-C on the induction and inhibition of angiogenesis, a process for forming new vessels that occurs naturally in the organism during the cicatrization of wounds and tissue regeneration, to restore the flow of blood in the damaged tissues. The studies were done by researcher Cristina Helena Bruno Terruggi, currently at the Health Sciences Department of the Municipal University of São Caetano do Sul, an industrial suburb of São Paulo, during part of her doctorate, done at Université Paris 13, in France.

For the tests, a commercially available gel containing proteins from the extracellular matrix called Matrigel was used, which was injected into the subcutaneous abdominal tissue of the animals, with two different combinations. In the control group of animals, the growth factor of fibroblasts was incorporated into the gel: these are cells that are involved in the production of several kinds of fibers and cause angiogenesis. The group of treated animals was given, besides this combination, the alternagin-C toxin in several concentrations. After 14 days, a histological study was done to check the formation of new vessels inside the Matrigel. “The effect observed in vivo reproduced what had been observed in vitro”, Ramos says. Accordingly, revascularization in vivo was proven.

The obtainment of the protein in the laboratory occurs by passing the untreated venom through chromographic columns, a relatively simple process used to separate the chemical substances into well defined ranges, and with a satisfactory yield for small scales. For large scale production, the protein can be obtained by the recombinant DNA technology, a study that is currently being done at the UFSCar laboratory. With this technology, the gene of interest is put inside a host cell, such as bacteria, yeasts, or cells of insects or mammals, to produce the toxins with the original biological properties, in a large quantity.

The best formulations for alternagin-C are being studied in UFSCar’s laboratories. Amongst them, are the microcapsulation of the protein in liposomes, which has the objective of protecting the medicine, for it to be delivered at the place where it has to act, application in a free form by means of a catheter, in cases of heart attacks, and in the form of an ointment or cream for superficial wounds.

The market for products based on alternagin-C is very promising. Until now, no other protein extracted from snake venom and with the same structural characteristics as ALT-C has been described as a molecule capable of inducing the formation of new blood cells. Not to mention that there are few options in the pharmaceutical market for this purpose.

The project
Use of alternagin-C as coadjuvant molecule in the process of forming and inhibiting the formation of new blood vessels (nº 04/07052-7); Modality Intellectual Property Support Program (Papi); Coordinator Heloísa Sobreiro Selistre de Araújo – UFSCar; Investment R$ 6,000.00 (FAPESP)