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Effect of crotalphine on the cannabinoid system: release of endocannabinoids or binding to the CB2 receptors

Grant number: 12/01423-0
Support type:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): September 01, 2012
Effective date (End): December 31, 2012
Field of knowledge:Biological Sciences - Pharmacology
Principal Investigator:Gisele Picolo
Grantee:Franciele Corrêa Machado
Supervisor abroad: Teresa Iuvone
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Local de pesquisa : Università degli Studi di Napoli Frederico II (UNINA), Italy  
Associated to the scholarship:10/12917-8 - Contribution of cannabinoid system to the antinociceptive effect of crotalphine and its interation with opioid system, BP.MS

Abstract

Studies develop in the last years in our lab, using compounds obtained from animal venoms or toxins, resulted in the discovery of crotalfina, a peptide of 14 amino acids that had its sequence based on natural painkiller factor obtained from the venom of Crotalus durissus terrificus. Studies demonstrated that crotalfina induces long-lasting antinociception (2-3 days in the chronic pain models and 5 days in acute pain models) mediated by the activation of kappa or kappa and delta opioid receptors, depending on the pain model evaluated. Prolonged treatment with the synthetic peptide does not cause the development of tolerance to antinociceptive effect or delayed hyperalgesia, subsequently the end of the analgesic effect.Although the blocked of crotalphine analgesia-induced by opioid antagonistas, the sequence of crotalfina shows no similarity with any other known opioid. Still, preliminary studies indicate that the crotalfina not directly activates opioid receptors since the peptide is not able to displace naloxone marked ([3H] naloxone) in studies of "binding" (Correa, F., personal communication). Then, based on data from the literature showing that there is a strong relationship between the cannabinoid and opioid systems, and in order to better understand the mechanisms involved in antinociception induced crotalfina, it was investigated the participation of cannabinoid receptors in this effect. Our results indicated that the effect of crotalfina involves the participation of opioid receptors, as well as peripheral cannabinoid receptors, besides endogenous opioids release, particularly dynorphin A, the endogenous agonist of kappa receptors. The same assays also suggest that this release of dynorphin A-induced by crotalfina is dependent of the activation of peripheral cannabinoid receptors, particularly CB2. Data of Literature demonstrate, in relation to the connection between opioids and cannabinoids systems, that the analgesic effect of cannabinoids may be mediated by release of endogenous opioids, in the same manner that opioid drugs may cause the release of endocannabinoids. So, in order to investigate the involvement of the endocannabinoid system in the antinociception induced by the crotalfina, MAFP (methyl araquidonil fluorofosfonado), an inhibitor of fatty acid amide hydrolase (an enzyme that hydrolyzes endocannabinoids), and Orlistat, an inhibitor of diacylglycerol (precursor of endocannabinoids, produced from the hydrolysis of phosphoinositides membrane) were used. The results demonstrated that MAFP was able to potentiate the antinociception while the treatment with Orlistat caused no change in animals treated with crotalphine. Literature describes that MAFP is a poor selective inhibitor of the enzyme that hydrolyzes endocannabinoids, been able to hydrolyze other fatty acids. Then, the involvement of endocannabinoids in the effect of crotalfina remains unclear. For this reason, the purpose of this project for application to the "Training Scholarship for Foreign Research" is to evaluate if the activation of cannabinoid receptors induced by crotalfina occurs directly or indirectly, due to liberation of endocannabinoids. (AU)