Formulação farmacêutica à base de epiisopiloturina, alcaloide de Pilocarpus microphyllus, reduz a hipernocicepção inflamatória: papel da via NO/GMPc/PKG/K+ATP e modulação da resposta imune

Abstract

Epiisopiloturine (EPI) is an imidazole alkaloid obtained from industrial waste generated by extracting pilocarpine from the leaves of Pilocarpus microphyllus, popularly known as jaborandi. Given the above, the aim of the present study was to investigate the effect of epiisopiloturine (EPI) and pharmaceutical formulation (EPI drops) in experimental models of acute and persistent nociception with description of the possible mechanism of action. For this purpose, the method of increasing pressure on the animals' paws through the electronic Von Frey was used to evaluate mechanical hypernociception induced by carrageenan (Cg), epinephrine or complete Freund's adjuvant (CFA). Swiss mice (n = 5-8 / group, 25-30g) were used. Initially, the effect of EPI in the salt and base forms (0.5 to 50 mg/kg, po) on inflammatory hypernociception induced by Cg (300 μg/paw) was evaluated and the evaluation occurred at 1, 3 and 5h after administration of Cg. Assessing the rational use of this molecule, the form and dose chosen to follow the study was the EPI based on a dose of 1 mg/kg which reduced 50.13% mechanical hypernociception and neutrophil migration measured by the activity of the enzyme myeloperoxidase (MPO) in approximately 45% in the subplantar tissue. This effect was related to the modulation of the production of pro- and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6, KC, and IL-10). To assess the participation of the NO/cGMP/PKG/K+ATP pathway, NOS inhibitors (L-NAME, 30 mg/kg, ip), and cGMP (ODQ, 10 mg/ kg, ip), PKG (KT5823, 0.5 µg/paw) or K+ATP channel blocker (glibenclamide, 5 mg/kg, ip), which significantly reversed mechanical hypernociception, thus showing the participation of this pathway in the effect antinociceptive of EPI. In mechanical hypernociception induced by epinephrine (100 ng/paw), treatment with EPI (1, 5 and 10 mg/kg, p.o.) reduced hypernociception by up to 52.2%. In the CFA-induced persistent pain model, mechanical hypernociception and paw edema were evaluated for 7 days. Animals were treated with saline (control, p.o); EPI (1, 5 and 10 mg/kg, p.o.) and dexamethasone (5 mg/kg, p.o.) daily before and after administration of CFA (20 μL/paw, i.pl, single administration, day 0). On the 7th day, subplantar tissue was collected to quantify cytokines, MPO activity, iNOS enzyme expression (Western blotting), as well as tissue/stomach collection, to assess the effect of EPI on the gastric mucosa. Daily administration of EPI reduced mechanical hypernociception, paw edema, neutrophil migration, production of pro-inflammatory cytokines (IL-1β and IL-6) and expression of iNOS not associated with gastric mucosal damage, as well as to the development of tolerance. The oral liquid formulation based on epiisopiloturine (EPI Drops) showed an increase of 40% in the antinociceptive effect when compared to the treatment of EPI (Active Pharmaceutical Ingredient). Based on the results obtained, the antinociceptive effect of EPI seems to be related to a block in the course of sensitization and activation of nociceptors by activating the NO/GMPc/PKG/K+ATP pathway and also indirectly preventing nociceptor sensitization through modulation of the immune/inflammatory response. The liquid formulation based on EPI (API), showed promise for the treatment of acute and persistent inflammatory pain.