Dalton L. Ferreira-Alves

Selective inhibitors of cyclo-oxygenase-2 (COX-2) induce hypoalgesia in a rat paw model of inflammation

It is well‐established that inhibitors of cyclo‐oxygenase (COX) and hence of prostaglandin (PG) biosynthesis reverse inflammatory hyperalgesia and oedema in both human and animal models of inflammatory pain. Paw oedema and hyperalgesia in rats were induced by injecting carrageenan (250 μg paw−1) into a hindpaw. Both inflammatory responses were followed for 24 h after the injection, measuring hyperalgesia by decreased pain threshold in the paws and oedema by plethysmography. Three selective inhibitors of cyclo‐oxygenase‐2 (COX‐2), celecoxib, rofecoxib and SC 236, given systemically in a range of doses, before the inflammatory stimulus, abolished carrageenan‐induced hyperalgesia with little reduction of oedema. These inhibitors also induced hypoalgesia, increasing nociceptive thresholds in the inflamed paw above normal, non‐inflamed levels. This hypoalgesia was lost at the higher doses of the selective inhibitors, although hyperalgesia was still prevented. In paws injected with saline only, celecoxib, given at the dose inducing the maximum hypoalgesia after carrageenan, did not alter the nociceptive thresholds. Two non‐selective inhibitors of COX‐2, indomethacin and piroxicam, abolished hyperalgesia and reduced oedema but did not induce hypoalgesia. Celecoxib given locally into the paw also abolished inflammatory hyperalgesia and induced hypoalgesia without reducing oedema. We conclude that hypoalgesia is expressed only over a critical range of COX‐2 inhibition and that concomitant inhibition of COX‐1 prevents expression of hypoalgesia, although hyperalgesia is still prevented. Our results suggest a novel anti‐nociceptive pathway mediating hypoalgesia, involving COX‐2 selectively and having a clear peripheral component. This peripheral component can be further explored for therapeutic purposes.

Evidence of the involvement of biogenic amines in the antinociceptive effect of a vouacapan extracted from Pterodon polygalaeflorus Benth

In view of the extensive use of Pterodon species in Brazilian folk medicine, the present investigation was performed to examine the involvement of biogenic amines in antinociceptive by a vouacapan (6 alpha-7 beta-dihydroxy vouacapan-17 beta-oate), extracted from seeds of Pterodon polygalaeflorus Benth), using acetic acid writhing test in mice. The alpha 2-adrenergic (yohimbine) and D2-dopaminergic (domperidone) antagonists and the pretreatment with the peripheral noradrenergic depletor, guanethidine partially inhibited the antinociceptive effect of vouacapan. Dopamine and D2 dopaminergic agonist (Ly 17155) caused antinociceptive that was not antagonized by naloxone but by domperidone, whereas noradrenaline induce pain. A synergistic analgesic effect was obtained when vouacapan was associated with clonidine or dopamine. These results indicate that vouacapan acts, at least in part, through activation of the catecholaminergic system.

Possible participation of endogenous opioid peptides on the mechanism involved in analgesia induced by vouacapan

The involvement of opioid peptides in the mechanism of action of vouacapan, a new experimental compound extracted from seeds of Pterodon poligalaeflorus Benth, was investigated both in mice utilizing acetic acid writhing response and in rats utilizing inflammatory hyperalgesia induced by carrageenan and modified Randall-Selitto method. Vouacapan, in both models, caused a dose-dependent analgesia when injected p.o., s.c. and i.p. The analgesic effect was partially blocked by naloxone, nalorphine and n-methyl-nalorphine. Significant tolerance to analgesic effect was observed following repeated administration of vouacapan or morphine. On the last day of treatment, cross administration revealed symmetrical and asymmetrical cross-tolerance between vouacapan and morphine, in rats and mice, respectively. We conclude that a release of endorphins could be involved in the analgesic mechanism of vouacapan in both models tudied.

Endogenous opioids mediate the hypoalgesia induced by selective inhibitors of cyclo-oxygenase 2 in rat paws treated with carrageenan.

Mechanical hyperalgesia induced in rat paws by carrageenan (250microg) was modified by pre-treatment with three selective inhibitors of cyclo-oxygenase-2 (COX-2); celecoxib, rofecoxib and SC236. These inhibitors raised the nociceptive threshold above the normal, non-inflamed, level, inducing a state of hypoalgesia. Such hypoalgesia was observed in different strains of rat (Holtzman, Wistar and Sprague-Dawley) and after different modes of administration of the COX-2 inhibitor (locally, in the paw, or systemically). A selective inhibitor of COX-1 (SC 560; 1-10mg kg(-1)) decreased hyperalgesia but did not induce hypoalgesia. Pre-treatment with naltrexone (3mg kg(-1)), an opioid receptor antagonist, did not affect carrageenan-induced hyperalgesia but abolished the hypoalgesic effects of COX-2 inhibitors, without diminishing the anti-hyperalgesic effect of indomethacin. In rats made tolerant to the anti-nociceptive effects of morphine, all anti-nociceptive effects of SC236 were abolished but the anti-hyperalgesic effects of indomethacin or SC 560 were unaffected. We conclude that, in our model of inflammatory hyperalgesia, the anti-nociceptive effect of selective COX-2 inhibitors involved the participation of endogenous opioids.

Tension generation and increase in voltage-activated Na+ current by crotamine

We performed the present experiments to study the action of crotamine, a toxin isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus, on macroscopic Na+ currents in frog skeletal muscle by using the loose patch clamp technique. Crotamine at 50 microM increased the peak Na+ current by 50% (P < 0.05). In addition, the voltage dependence of inactivation was shifted by +8 mV. Other parameters of Na+ currents (reversal potential, voltage-dependence of activation and time courses of inactivation, of activation and of removal of inactivation) were not significantly affected. We suggest that crotamine inhibits the direct transition of channels from closed to inactivated states, thereby forcing their transition through the open states.

Cloning of the guinea pig 5-lipoxygenase gene and nucleotide sequence of its promoter

The guinea pig 5-lipoxygenase (5-LO) gene and its promoter were cloned from a guinea pig genomic DNA library. Sequencing analysis of the guinea pig promoter revealed that expression of the 5-LO gene in this rodent is probably governed by cis acting nucleotide sequences quite similar to the human gene. Nucleotide sequences that bind factors like Sp-1, AP-2, NF-kB and c-Ha-ras were identified in the guinea pig 5-LO promoter region.

Synthesis of 6a,7b-dihydroxyvouacapan-17b-oic acid derivatives. Part IV: mannich base derivatives and its activities on the electrically stimulated Guinea-pig ileum preparation

O acido 6a,7b-di-hidroxivouacapan-17b-oico (ADV, 1), furanoditerpeno isolado dos frutos de Pterodon polygalaeflorus Benth, apresenta atividade analgesica em modelos laboratoriais utilizando roedores. Visando reunir dados para estudar a acao biologica de vouacapanoides, foram obtidas seis novas amidas derivadas (4-9), contendo grupos alquilaminas no anel furânico, preparadas pela reacao de Mannich com sais de iminios pre-formados. Esses derivados tiveram suas estruturas determinadas pela analise de dados espectroscopicos no IV, RMN de 1H e de 13C e foram submetidos a triagem farmacologica in vitro, destinada a detectar substâncias com acao depressora sobre celulas neuronais.

Peripheral μ-, κ- and δ-opioid receptors mediate the hypoalgesic effect of celecoxib in a rat model of thermal hyperalgesia

AIMS The endogenous opioids mediate the analgesic effects of celecoxib in a model of mechanical hyperalgesia in rats. As responses to thermal stimuli may differ from those to mechanical stimuli, we have here assessed celecoxib in a rat model of thermal hyperalgesia and the possible involvement of endogenous opioids and their corresponding receptors in these effects. MAIN METHODS Injection of carrageenan (CG) into one hind paw induced a dose-related hyperalgesia (decreased time for paw withdrawal) to thermal stimuli (infra-red light beam), over 6h. KEY FINDINGS Celecoxib (sc) 30 min before CG (250 microg per paw) induced a dose-dependent reversal of hyperalgesia, with withdrawal times well above basal levels, characterizing development of hypoalgesia. Indomethacin (sc) reversed CG-induced hyperalgesia only to basal levels (an anti-hyperalgesic effect). Naltrexone (sc) prevented hypoalgesia after celecoxib but did not change the response to indomethacin. Local (intraplantar) injection of either a selective antagonist of mu-(beta-funaltrexamine), kappa-(nor-binaltorphimine) or of delta-(naltrindole) opioid receptors also reversed the hypoalgesic effects of celecoxib, without modifying the hyperalgesia due to CG or affecting the nociceptive thresholds in the non-injected paw. SIGNIFICANCE Our data show that celecoxib, unlike indomethacin, was hypoalgesic in this model of thermal hyperalgesia, and that this effect was mediated by peripheral mu-, kappa- and delta-opioid receptors.

Effect of 6α,7β-dihydroxyvouacapan-17β-oic acid and its lactone derivatives on the growth of human cancer cells

The furanditerpene 6 alpha,7 beta-dihydroxyvouacapan-17 beta-oic acid (1) is a natural product biosynthesized by some species from the genus Pterodon (Leguminosae). This secondary metabolite has multiple biological activities that include anti-inflammatory, analgesic, plant growth regulatory, anti-edematogenic, photosystem II inhibitory and photosynthesis uncoupler, and antifungal properties. However, few studies on the antiproliferative profile of compound 1 and/or its derivatives have been reported up to date. Here, we describe the isolation of compound 1 from hexane extract of P. polygalaeflorus fruits as well as the semisynthesis of three lactone derivatives: 6 alpha-hydroxyvouacapan-7 beta,17 beta-lactone (2), 6 alpha-acetoxyvouacapan-7 beta,17 beta-lactone (3), and 6-oxovouacapan-7 beta,17 beta-lactone (4). Additionally, antiproliferative activity of these compounds against nine human cancer cell lines was investigated. Our results revealed that 6 alpha-hydroxyvouacapan-7 beta,17 beta-lactone (2) was the most potent furanditerpene against all cancer cell lines studied. The presence of non-substituted hydroxyl group at C-6 and the presence of 7 beta,17 beta-lactone ring are important for the antiproliferative activity of these compounds.

Synthesis, antiproliferative activity in cancer cells and theoretical studies of novel 6α,7β-dihydroxyvouacapan-17β-oic acid Mannich base derivatives

Natural products are great prototypes for the design of new anticancer agents. The plant-derived natural product 6α,7β-dihydroxyvouacapan-17β-oic acid (1) is promising for the development of more potent antiproliferative agents against human cancer cells. Indeed, its lactone derivative 6α-hydroxyvouacapan-7β,17β-lactone (2), a non-natural furanoditerpene, exhibited higher anticancer activity than compound 1. Herein, we describe the synthesis and antiproliferative activity of six new Mannich derivatives of compound 2 against nine cancer cell lines. Overall, our results revealed that Mannich derivatives 3-8 were more potent than compound 2 in inhibiting the proliferation of cancer cells. Theoretical studies also supported our findings, revealing the nucleophilic character of furan ring as an important feature for antiproliferative activity of the studied Mannich derivatives.