Team:USP-EEL-Brazil/Implementation


According to the World Health Organization (WHO), it is estimated that, annually, the number of people bitten by snakes can reach 5.4 million. Of this amount, the number of deaths varies between 81,000 and 138,000. In addition, up to 400,000 victims are incapacitated due to the action of the toxin in the bloodstream.

The WHO includes, in its list of neglected tropical diseases, accidents with venomous animals, including snakes that produce venom. This is a worrying factor, since the poison weakens the victim, being an obstacle for them to be able to seek care in a timely manner, considering affected people who are from regions that do not have adequate infrastructure.

Snake venom, considered by some authors as the most complex mixture in nature, is composed of 90 to 95% of proteins. Metalloproteases and phospholipases A2 (PLA2) are the main component enzymes of the venom, being also the most harmful to the affected cells. In bothropic venom, there are also toxins called hemorrhages, which are metalloproteases capable of disrupting the integrity of the vascular endothelium and inhibiting platelet aggregation, causing hemorrhagic activity (PICELLI, 2013).

Snakes have their own inhibitors for PLA2, and it can be found in their plasma. They are called Phospholipase Inhibitors (PLI). Based on amino acid sequence homology, PLIs are divided into 3 classes, ⍺-PLI, β-PLI and 𝛾-PLI, which will be better characterized throughout the project.

Despite the existence of inhibitor variations, the project is focused on the production of 𝛾PLI via synthetic biology, as it has a broad spectrum of action, being able to inhibit most classes of PLA2 found in venoms, while the other classes of inhibitors cannot.

In the current process, the antivenom serum is produced through the wide use of large animals, such as horses. In this process, the snake's venom is injected into the animal in successive doses and, after days with the venom in its body, the horse's blood is collected with the presence of antibodies capable of fighting the venom. (LOPES, 2011).

Since the beginning of our project, we have thought about who is the target audience of the Honorato project, so we have always been concerned with thinking about a product that serves the victims of snakebites and health professionals as well. To this end, we got in touch, via telephone, with institutions and professionals in the area so that it was possible to mold a product that best served these people. For this, we contacted “Instituto Vital Brazil”, a laboratory that serves the entire Brazilian public sector with the production of serums and medicines, and also conducts studies and research in the field of different areas. We were referred to a nurse named Oséias, who gladly talked to our team and answered some questions. He told us that the main causes of aggravation in snakebites are the delay in the care, as it leads to severe necrosis, use of a tourniquet at the site of the bite, as it concentrates poison at the site, and infection due to bacteria present in the body of the snake’s preys. It was also commented that rural workers are the most vulnerable to this type of accident, and children are also at great risk, as the effects can be more serious.

We also spoke with Samira Rodrigo dos Santos Silva, a nurse in the city of Cruzeiro, in the countryside of the state of São Paulo. In our conversation, she reinforced what we talked about with Oséias, and added that identifying the snakes that caused the accident could help in a more agile treatment.

Based on these conversations, and others that are better cited in Human Practices, we concluded that the best way to make Honorato available would be through ampoules, both in health centers and in ambulances, so the service time problem can be solved, and these ampoules can also be applied in order to prevent necrosis even without the identification of snakes, as this inhibitor is specific for PLA2. We also performed bioinformatics analysis and found that PLA2 from different snakes have high similarity, which indicates that there is a high chance that our project has high efficacy in reducing the myotoxic activity of several snake species, and not only snakes of the Bothrops genus. Due to the impact of the COVID-19 pandemic caused around the world and more specifically in Brazil we did not have the opportunity to prove it experimentally.

In order to make it possible to make the inhibitor available in ambulances and emergency rooms, for helping victims in emergency situations, it is necessary to obtain a license from the Brazilian Health Regulatory Agency (Anvisa). This is the organization responsible for defining the criteria that make a drug suitable for use by the population. The medicine approval process is long and indispensable, and requires an authorized company interested in granting the documentation, which we do not have and are not authorized to request.

The approval process involves the steps:

  • Non-clinical: Research phase of safety and action of the molecule in cells and animals, understanding the safe dosage.
  • Development: Phase related to product quality, as well as investigation of better form of application and pharmaceutical formulation.
  • Clinical: Involves clinical trials in volunteers, following ethical and technical instances. The results make up the medicine insert.
  • Record: It is the phase in which the documents that prove the quality of the drug, as well as its safety and efficacy, are presented. As well as certifications of principles of good manufacturing practices and control and sanitation of the production environment.
  • Aftermarket: Monitoring the use of the drug, checking if there are any adverse effects.

Brasil. Ministério da Saúde. Ofidismo: Análise Epidemiológica. Brasília, 1991.

PICELLI, Carina Godoy. Caracterização de fosfolipases A2 da peçonha de Bothrops bilineata e identificação do gene de inibidores de fosfolipases A2 de classe gama em Bothrops atrox e Micrurus lemniscatus: um estudo com ênfase no potencial biotecnológico. 2013, Porto Velho - RO.

Boeno, Charles Nunes, et al. "Inflammasome activation induced by a snake venom Lys49-phospholipase A2 homologue." Toxins 12.1 (2020): 22.

Picadas de cobras matam até 138 mil pessoas por ano. OMS, 2019. Available at: https://news.un.org/pt/story/2019/05/1671281. Access on: September 23, 2021

BRAZILIAN GOVERNMENT (Brazil). Brazilian Health Regulatory. Registro de novos medicamentos: saiba o que é preciso. Brazil, 1 nov. 2018. Available in: http://antigo.anvisa.gov.br/registro-de-novos-medicamentos-saiba-o-que-e-preciso. Access: 23 aug. 2021.