Avaliação pré-clínica da segurança e do mecanismo de ação analgésico do polissacarídeo glucomanana obtido do líquen Heterodermia obscurata

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2016.Os produtos naturais são uma grande fonte de moléculas bioativas, entre elas destacam-se os polissacarídeos que apresentam efeitos imunomoduladores, anti-inflamatórios e analgésicos. O polissacarídeo glucomanana (GM) obtido do líquen Heterodermia obscurata já demonstrou ter efeito antinociceptivo (analgésico), indicando ser uma molécula interessante. Assim, o presente trabalho pretende avançar na caraterização do efeito antinociceptivo da GM, bem como na compreensão do seu mecanismo de ação neurobiológico e segurança em camundongos. Neste trabalho, foi confirmado que a administração sistêmica da GM (100 mg/kg, ip.) é capaz de reduzir a nocicepção espontânea de origem inflamatória induzida por formalina ou PGE2. Ademais, a redução da nocicepção induzida por PGE2 causada pela GM (92+15%) foi semelhante à produzida pela indometacina (93+12%), inibidora não seletiva da COX. Além disso, a administração periférica (local, intraplantar) da GM (10 µg/pata) também reduziu a nocicepção causada pela PGE2 de maneira similar à indometacina e ao KT5720 (inibidor não seletivo da PKA). A GM (100 mg/kg) também reduziu significativamente a hiperalgesia térmica ao calor, mas não a alodinia mecânica causada pela injeção intraplantar de PGE2 e CFA (Adjuvante Completo de Freund), bem como aquela causada pela incisão plantar (modelo de dor pós-operatória). Por outro lado, a GM (10 µg/pata) promoveu redução da nocicepção espontânea e hiperalgesia térmica ao calor induzida pela forscolina (FSK, ativadora da adenililciclase). Quando administrado perifericamente (intraplantar), a GM (10 µg/pata) também reduziu a nocicepção espontânea induzida por FSK, assim como o KT5720 (10 nmol/pata). Além disso, a GM (100 mg/kg) foi capaz de reduzir o aumento da fosforilação da PKA (p-PKA) induzida por FSK, na pata de camundongos. A nocicepção espontânea induzida pelo ativador de PKC, o 13- acetato12-miristato de forbol (PMA), foi completamente inibida pela GM (100 mg/kg). Quando avaliado frente à ativação de termo-receptores, a GM (30 e 100 mg/kg, ip.) reduziu a nocicepção espontânea induzida pela capsacina (agonista TRPV1), cinamaldeído (agonista TRPA1), mentol (agonista TRPM8) e pela salina acidificada (agonista ASICs). No entanto, a administração periférica (local, intraplantar) da GM (30 µg/pata) reduziu (70±4%) somente a nocicepção causada pelo cinamaldeído. A nocicepção observada no modelo de PGE2 foi completamente dependente das fibras aferentes TRPV1+. Ainda, a GM, na dose que promove importante efeito analgésico, não causou efeitos sedativos e tampouco alterou o peso corpóreo dos animais. Contudo, a administração prolongada (11 dias) promoveu aumento do peso do baço, mas não alterou o peso do coração, pulmões, fígado e rins. Ademais, a GM não promoveu alterações hematológicas e bioquímicas importantes. Em conjunto, os dados deste trabalho demonstram que a GM é capaz de reduzir a nocicepção (dor) aguda e persistente, principalmente a de origem inflamatória, por um mecanismo dependente da inibição da via de sinalização ativada por PGE2 (AMPc/PKA) e dos canais TRPs, principalmente o TRPV1 e o TRPA1. Assim, a GM é uma molécula com potencialidade para o desenvolvimento de fármacos de interesse terapêutico no controle da dor.Abstract : Natural products are a wide source of bioactive molecules, which include the polysaccharides with analgesic, anti-inflammatory and immunomodulatory properties. It was already demonstrated that the polysaccharide Glucomannan (GM) obtained from the lichen Heterodermia obscurata has antinociceptive (analgesic) effect, making it an interesting molecule for further studies. Thus, the present work intends to advance in the characterization of GM antinociceptive effect, as well as in the comprehension of its neurobiological mechanism of action in mice. In this study, it was demonstrated that systemic administration of GM (100 mg/kg, ip.) is able to reduce spontaneous nociception of inflammatory origin induced by formalin or PGE2. Besides, the reduction of PGE2-induced nociception caused by GM (93±15%) was similar to that produced by indometacin (93±12%), a non-selective COX inhibitor. Peripheral (intraplantar) administration of GM (10 µg/paw) also reduced PGE2-induced nociception similarly to indometacin and KT5720 (non-selective PKA inhibitor). GM (100 mg/kg, ip.) also reduced significantly the thermal hyperalgesia to heat, but not the mechanical allodynia induced by intraplantar injection of PGE2 and CFA (Complete Freund s Adjuvant), as well as that caused by plantar incision (post operatory model of pain). On the other hand, GM (10 µg/paw) reduced both spontaneous nociception and thermal hyperalgesia to heat induced by forskolin (FSK, adenylyl cyclase activator). When peripherically administrated (intraplantar), GM (10 µg/paw) also reduced spontaneous nociception induced by FSK, as well as KT5720 (10 nmol/paw). Moreover, GM (100 mg/kg, ip) was able to reduce the increase on PKA phosphorylation (p-PKA) induced by FSK in mice paw. Spontaneous nociception induced by PKC activator phorbol-12-myristate-13-acetate (PMA) was completely inhibited by GM (100 mg/kg, ip). When evaluated against thermal receptor activators, intraperitoneal administration of GM (30 and 100 mg/kg) reduced the spontaneous nociception induced by capsaicin (TRPV1 agonist), cinnamaldehyde (TRPA1 agonist), menthol (TRPM8 agonist) and acidified saline (ASICs agonist). However, peripheral (intraplantar) administration of GM (30 µg/paw) reduced only the cinnamaldehyde-induced nociception (70±4%). The nociception observed in PGE2 model was completely dependent on TRPV1+ afferent fibers. Furthermore, at the doses that promote antinociceptive effect, GM caused no sedative effect and did not alter mice body weight. However, daily administration (11 days) of GM promoted an increase in spleen weight, with no effect on the weight of heart, lungs, liver and kidneys. Also, GM did not promote any significant hematologic or biochemical alteration. Together, the data presented in this study demonstrate that GM is able to reduce acute and persistent nociception (pain), mainly of inflammatory origin, by a mechanism that is dependent on inhibition of signaling pathways activated by PGE2 (cAMP/PKA) and TRP channels, mainly TRPV1 and TRPA1. Thus, GM is a molecule with great potential for further development of drugs with therapeutic interest for pain control

Plant derived aporphinic alkaloid S-(+)-dicentrine induces antinociceptive effect in both acute and chronic inflammatory pain models: evidence for a role of TRPA1 channels.

S-(+)-dicentrine is an aporphinic alkaloid found in several plant species, mainly from Lauraceae family, which showed significant antinociceptive activity in an acute model of visceral pain in mice. In this work, we extended the knowledge on the antinociceptive properties of S-(+)-dicentrine and showed that this alkaloid also attenuates mechanical and cold hypersensitivity associated with cutaneous inflammation induced by Complete Freund's Adjuvant in mice. Given orally, S-(+)-dicentrine (100 mg/kg) reversed CFA-induced mechanical hypersensitivity, evaluated as the paw withdrawal threshold to von Frey hairs, and this effect lasted up to 2 hours. S-(+)-dicentrine also reversed CFA-induced cold hypersensitivity, assessed as the responses to a drop of acetone in the injured paw, but did not reverse the heat hypersensitivity, evaluated as the latency time to paw withdrawal in the hot plate (50°C). Moreover, S-(+)-dicentrine (100 mg/kg, p.o.) was effective in inhibit nociceptive responses to intraplantar injections of cinnamaldehyde, a TRPA1 activator, but not the responses induced by capsaicin, a TRPV1 activator. When administered either by oral or intraplantar routes, S-(+)-dicentrine reduced the licking time (spontaneous nociception) and increased the latency time to paw withdrawal in the cold plate (cold hypersensitivity), both induced by the intraplantar injection of cinnamaldehyde. Taken together, our data adds information about antinociceptive properties of S-(+)-dicentrine in inflammatory conditions, reducing spontaneous nociception and attenuating mechanical and cold hypersensitivity, probably via a TRPA1-dependent mechanism. It also indicates that S-(+)-dicentrine might be potentially interesting in the development of new clinically relevant drugs for the management of persistent pain, especially under inflammatory conditions

Effect of S-(+)-dicentrine (DCTN, 100 mg/kg, p.o.) on mechanical hypersensitivity induced by CFA 50%.

<p>On 1^st and 14^th days, evaluations were done 1, 2, 3, 4 and 24 hours post-DCTN treatment; all other evaluations were done 1 hour post-treatment. Each point represents the mean ± S.E.M. of 8 animals and significance levels are indicated by *p<0.05 and **p<0.01 when compared to the CFA i.pl. group (two-way anova and Bonferroni post hoc test).</p

Effect of S-(+)-dicentrine (DCTN) administered by oral (100 mg/kg) or intraplantar (100 µg/paw) routes, or the TRPA1 antagonist camphor by subcutaneous (7.6 mg/kg) or intraplantar (3.8 µg/paw) routes on cinnamaldehyde-induced nociception.

<p>Each bar represents the mean ± S.E.M. of 6 - 8 animals, being column C indicative of control values. Significance levels are indicated by ***p<0.001 when compared to control group (one-way anova and Student-Newman-Keuls post hoc test).</p

Effect of S-(+)-dicentrine (DCTN, 100 mg/kg, p.o.) on mechanical hypersensitivity induced by CFA 80%.

<p>Panel A: time-course effect of DCTN evaluated at 1, 2, 3, 4 and 24 hours post-DCTN administration; each point represents the mean ± S.E.M. of 10 animals and significance levels are indicated by *p<0.05 and ***p<0.001 when compared to the CFA i.pl. group (two-way anova and Bonferroni post hoc test). Panel B: effect of DCTN on 7^th and 10^th days post-CFA injection, evaluated before DCTN administration (basal) and 1 hour post-DCTN administration; each bar represents the mean ± S.E.M. of 10 animals and significance levels are indicated by ***p<0.001 when compared to control group and ^##p<0.01 when compared to the respective basal of CFA i.pl. group (one-way anova and Student-Newman-Keuls post hoc test).</p

Effect of S-(+)-dicentrine (DCTN) administered by oral route (10 - 100 mg/kg, panels A and B) or by intraplantar route (10 - 100 µg/paw, panels C and D), or the TRPA1 antagonist camphor by subcutaneous (7.6 mg/kg) or intraplantar (3.8 µg/paw) routes on cinnamaldehyde-induced nociception.

<p>Panels A and C represents the spontaneous nociception (licking time) and panels B and D represents the hypersensitivity to cold (latency time to paw withdrawal). Each bar represents the mean ± S.E.M. of 6 - 8 animals, being column C indicative of control values (cinnamaldehyde i.pl. injection) and column V indicative of group receiving only vehicle i.pl. injection. Significance levels are indicated by ***p<0.001 when compared to vehicle (V) group and ^##p<0.01 and ^###p<0.001 when compared to respective control (C) groups, and the values above the symbols represent the percent of inhibition (one-way anova and Student-Newman-Keuls post hoc test).</p

Effect of S-(+)-dicentrine (DCTN) administered by oral (100 mg/kg) or intraplantar (100 µg/paw) routes, or the TRPV1 antagonist AMG9810 by intraperitonial (30 mg/kg) or intraplantar (30 µg/paw) routes on capsaicin-induced nociception.

<p>Each bar represents the mean ± S.E.M. of 6 - 8 animals, being column C indicative of control values. Significance levels are indicated by **p<0.01 when compared to control group (one-way anova and Student-Newman-Keuls post hoc test).</p

Effect of S-(+)-dicentrine (DCTN, 100 mg/kg, p.o.) on thermal hypersensitivity to cold (panel A) and heat (panel B), induced by CFA 80%.

<p>Each bar represents the mean ± S.E.M. of 10 animals. Significance levels are indicated by *p<0.05, **p<0.01 and ***p<0.001 when compared to control group and ^#p<0.05 and ^##p<0.01 when compared to the CFA i.pl. group (one-way anova and Student-Newman-Keuls post hoc test).</p