Efeito relaxante de derivados N-sulfonilidrazônicos do rolipram em traqueia de cobaia : investigação do mecanismo de ação do LASSBio-1846

Abstract

To contribute in development of new drugs to treat the symptoms of airway diseases were synthesized new PDE4 inhibitors from rolipram. The new N - sulfonilhidrazone derivatives had been only tested on in vitro enzymatic assa ys. To investigate their effects on physiological conditions, the guinea - pig trachea model was selected. Thus, the present study aimed to assess a possible relaxant action of a new series from rolipram, a PDE4 inhibitor, as well as elucidate the mechanism of action of the more potent derivative. Therefore, from guinea - pigs was isolated the trachea, sectioned into rings and connected to a digital acquisition system to obtain the data. Initially, a pharmacological screening was carried out with 5 derivatives ( LASSBio - 1846, 1847, 1848, 1849 and 1851 ). All compounds relaxed the guinea - pig trachea pre - contracted with carbachol in the presence (pD 2 = 5.34 ± 0.04; 4 .83 ± 0.02; 4.51 ± 0.03; 4.20 ± 0.08 e 4.70 ± 0.06, respectively ) or absence of functional epithelium (pD 2 = 5.15 ± 0.08; 4.78 ± 0.04; 4.44 ± 0.03; 4.10 ± 0.07 e 4.85 ± 0.08, respectively ). According to the pD 2 values, LASSBio - 1846 was the most potent derivative and, so, selected to the investigation of its mechanism of action, at functional level. Since, the β 2 - adrenergic receptor on airway smooth muscle, we used propranolol, a non - selective β - adrenergic blocker, to assess the participation of these receptors. The relaxation curve of LASSBio - 1846 was shifted to the right (pD 2 = 5.13 ± 0. 07) with reduction of 1 .7 folds on blocker presence, suggesting a participation of β 2 - adrenergic receptor on derivative induced relaxation. The β 2 - adrenergic receptor leads to an adenylyl cyclase (AC) activation, so we decided to evaluate its participation on LASSBio - 1846 relaxa tion. Therefore, we used foskolin, an AC direct activator, and observed that the relaxation curve of forskolin (pD 2 = 6.46 ± 0.05) was shifted to the left (pD 2 = 7.56 ± 0.12) in the derivative presence, around 11 folds, indicating a potentiation of this ef fect and suggesting that LASSBio - 1846 could favor the activation of AC. The AC activation leads to intracellular increase of cAMP, an important nucleotide involved on airway relaxation and cAMP levels are regulated to PDEs. To evaluate its participation, a minophylline, a non - selective PDEs inhibitor, was used. The concentration - curve response to aminophylline (pD 2 = 4.17 ± 0.07) was shifted to the left in the presence of LASSBio - 1846 ( pD 2 = 6.61 ± 0.07 ) with potentiation of this effect, around 255 folds, sh owing a PDEs inhibition. An elevation of cAMP leads to a PKA activation, the kinase protein effector of this relaxation pathway on smooth muscle. The LASSBio - 1846 relaxation curve was shifted to the right, 3 folds, in the presence of H - 89 ( pD 2 = 4.83 ± 0. 0 4 ) , a PKA inhibitor, suggesting the participation of this protein. Thus, LASSBio - 1846 could be activating AC/cAMP/PKA pathway to promote airway smooth muscle relaxation. According to the data, the evaluated N - sulfonilhidrazone derivatives presented relaxan t activity on guinea - pig trachea in an epithelium independent manner, and LASSBio - 1846 was the most potent compound in induce relaxation due to PDEs inhibition and posi tive modulate of β - receptor - AC - PKA pathway .