FundaÃÃo de Amparo à Pesquisa do Estado do Amazonas / Os efeitos dos triterpenos pentaciclicos -amirina e -amirina, isolados a partir da resina de Protium heptaphyllum March. (Burseraceae), foram testados preliminarmente em modelos de nocicepÃÃo oral, sendo que -amirina apresentou significantes efeitos antinociceptivos, norteando a pesquisa com este isolado na investigaÃÃo de seus efeitos em modelos de dor orofacial induzida por capsaicina ou formalina e na dor induzida por capsaicina na cÃrnea de camundongos; na dor tÃrmica (testes de imersÃo de cauda em Ãgua quente e placa quente); e na nocicepÃÃo visceral induzida por Ãcido acÃtico 0,6%. Camundongos Swiss machos (n = 8 / grupo) foram prÃ-tratados com β-amirina (10, 30 e 100 mg / kg, v.o.), morfina (5 mg / kg, s.c.) ou controle (Ãgua destilada + 0,05% de Tween 80, v.o.), uma hora antes de capsaicina (20 L, 1,5 g) ou formalina (20L/animal) serem administradas na vibrissa direita. β-amirina tambÃm foi avaliada em teste comportamental relacionado à dor, desta vez por aplicaÃÃo tÃpica de capsaicina na conjuntiva do camundongo (âeye wiping testâ). Neste teste foi medido o tempo, em segundos, que o animal passou âlimpandoâ o olho durante um perÃodo de 10 minutos. O triterpenÃide demonstrou principalmente um efeito antinociceptivo dose-independente em todos os modelos de nocicepÃÃo testados. Na dor orofacial induzida por capsaicina, -amirina (30 e 100 mg/kg) e morfina foram mais eficazes na reduÃÃo da resposta nociceptiva. Nestas doses, as reduÃÃes foram de 81 e 90% para -amirina e 97% para morfina, respectivamente. No modelo de dor orofacial, a nocicepÃÃo produzida pela capsaicina à acompanhada por um aumento na resposta tÃrmica localizada (que foi mensurada por termometria), e reduzida significantemente pelo prÃ-tratamento dos animais com -amirina ou L-NAME, um inibidor da NOS. Em animais diabÃticos, a capsaicina injetada na vibrissa promoveu um menor grau de nocicepÃÃo orofacial comparada com os nÃo-diabÃticos. No teste da formalina, morfina e β-amirina apresentaram antinocicepÃÃo significativa reversÃvel nas duas fases por naloxona. No entanto, β-amirina (30 mg/kg) inibiu a segunda fase com maior eficiÃncia. Os valores de DE50 para β-amirina e morfina foram 16,44 mg/kg (LC 10,0-38,41) e 3 mg/kg (LC 2,5-5,0) na primeira fase e 43,37 mg/kg (LC 30,52-39,30) e 3 mg/kg (LC 2,5-5,0) na segunda fase, respectivamente. A co-administraÃÃo de β-amirina e morfina, em seus respectivos nÃveis de dose de DE50, nÃo apresentou qualquer efeito aditivo ou potencializador antinociceptivo. No entanto, as combinaÃÃes das doses DE25 e DE12,5 apresentaram uma antinocicepÃÃo comparÃvel ao efeito combinado da DE50, sugerindo que atravÃs da utilizaÃÃo de β-amirina, a dose analgÃsica de morfina poderia ser minimizada para evitar a sua alta dose e os efeitos colaterais associados. β-amirina tambÃm foi eficaz em aumentar o limiar de dor tÃrmica no teste da imersÃo da cauda (mais nÃo no teste placa quente) e, na reduÃÃo das contorÃÃes induzidas por Ãcido acÃtico. A antinocicepÃÃo produzida por β-amirina, foi significativamente bloqueada em animais prÃ-tratados com os respectivos antagonistas vermelho de rutÃnio (2 mg/kg, s.c.) e naloxona (1 mg/kg, i.p.), indicando o envolvimento de receptores da capsaicina (TRPV1) e opiÃides em seu mecanismo. No teste da formalina, de forma similar à morfina, β-amirina bloqueou significativamente a inibiÃÃo da ingestÃo alimentar associada a dor. Assim como morfina, β-amirina apresentou aÃÃo inibitÃria sobre o trÃnsito intestinal, efeito esse revertido pelo prÃ-tratamento com antagonista opiÃide nÃo seletivo, naloxona. Estes dados sugerem que β-amirina apresenta um potencial antinociceptivo comparÃvel à analgesia perifÃrica produzida pela morfina, evidencia a exploraÃÃo desta para o desenvolvimento de um analgÃsico nÃo-opiÃide Ãtil na farmacoterapia de patologias do trigÃmeo e visceral. / The effects of pentacyclic triterpene β-amiryn and β-amyrin, isolated from resin of Protium heptaphyllum March. (Burseraceae), were preliminarily showed significant tested in models of nociception oral, and antinociceptives effects, guiding the search with this isolate in the investigation of their effects in models of orofacial pain induced by capsaicin or formalin and against capsaicin-induced corneal pain; thermal pain (tail immersion test in hot water and hot-plate) and in acetic acid 0,6%-induced visceral nociception in mice. Male Swiss mice (n = 8 per group) were pre-treated with β-Amyrin (10, 30, and 100 mg/kg, p.o.), morphine (5 mg/kg, s.c.) or vehicle (distlled water + 0,05% Tween 80), one hour before the capsaicin (20 μl, 1.5 μg) or formalin (20 μl, 1.5%) injection into the right vibrissa. β-Amyrin was also assessed on pain-related behavioral test (Eye-wiping) by topical application of capsaicin (20 μl, 1.5 μg) on to the mouse conjuctiva and the time (sec) that the animal spent in eye wiping was determined during a 10 min period. The triterpenoid demonstrated mostly a dose-unrelated antinociception in all the test models of nociception. Against the orofacial pain induced by capsaicin, β-Amyrin (30 e 100 mg/kg, p.o.) and morphine showed greater potency in reducing the nociceptive response. At the doses employed, the reductions were 81 and 90% to β-Amyrin and 97% for the morphine, respectively. Capsaicin nociception in orofacial test is accompanied by a localized thermal flare (measured by thermometry), which was significantly diminished by pretreatment of animals with β-Amyrin or L-NAME, an NOS inhibitor. In four weeks diabetic mice, capsaicin injected into vibrissa pad demonstrated a lesser degree of orofacial nociception compared to non-diabetics. In formalin test, both morphine and β-Amyrin showed significant naloxone reversible antinociception in both phases. However, β-Amyrin inhibited the second phase response, more prominently, at 30 mg/kg. The caliculated ED50 values for β-Amyrin and morphine were 16,44 mg/kg (CL 10,0 - 38,41) and 3 mg/kg (CL 2,5 - 5,0) in the first phase and 43,37 mg/kg (CL 30,52 - 39,30) and 3 mg/kg (CL 2,5 - 5,0) in the second phase, respectively. Co-administration of β-Amyrin and morphine at their respective ED50 dose levels failed to demonstrate any additive or potentiating effect on anti-nociception. However, at ED25 and ED12.5 dose-combinations exhibited an antinociception that equalled their ED50 combination effect, suggesting that by the use of β-Amyrin, the analgesic dose of morphine could be minimised to avoid its high-dose-associated side-effects. Similar to morphine, β-Amyrin significantly blocked the pain-related suppression of food intake in formalin test. β-Amyrin (30 and 100 mg/kg was also effective in increasing the thermal pain threshold in hot-water tail immersion test (but not in hot-plate test), and in reducing the acetic acid-induced writhes. The antinociception produced by 30 mg/kg β-Amyrin was significantly blocked in animals pre-treated with the respective antagonists capsazepine (5 mg/kg, s.c.), and naloxone (1 mg kg/kg, i.p.), indicating the involvement of capsaicin (TRPV1) and opioid receptors in its mechanism. Like morphine, β-Amyrin showed an inhibitory effect on intestinal transit, an effect reversed by pretreatment with nonseletive opiÃide antagonist, naloxona. These data indicate that β-Amyrin has the antinociceptive potential comparable to peripheral analgesia produced by morphine that could be explored further on its suitability in developing a non-opioid analgesic useful in pharmacotherapy of trigeminal and visceral pathologies.

Le manque de thérapies innovantes en chimiothérapie humaine incite la communauté scientifique à s'intéresser à de nouvelles sources de composés bioactifs. Nous pouvons citer les métabolites secondaires de plantes, auxquels appartiennent les acides hydroxy pentacycliques triterpénoiques (AHPTs) et plus particulièrement les Acides Ursolique (AU), Oléanolique (AO) et Bétulinique (AB). Ces molécules font l'objet de nombreuses études qui tendent à démontrer leurs propriétés : anti-infectieuses, anticancéreuses, antiprolifératives, anti-inflammatoires, hépatoprotectrices. Le principal obstacle à leur utilisation à des fins thérapeutiques, reste l'insolubilité de ces AHPTs dans l'eau. L'objectif de ce travail a donc été d'augmenter leur hydrosolubilité. Dans un premier temps, en accord avec les recommandations et/ou normes existantes, nous avons démontré que le spectre d'activité antibactérienne de l'AU et de l'AO se limitait aux bactéries à Gram positif. Aucun AHPT n'a montré d'activité antifongique. Seul l'AB a montré une activité intéressante sur le Cytomégalovirus humain (hCMV) ; aucune activité antivirale n'ayant été retrouvée sur le Poliovirus. Enfin, l'AB, mais encore plus l'AU ont montré une activité anticancéreuse à l'encontre de cellules modèles de leucémie myéloïde chronique (LMC). Dans un deuxième temps, nous avons procédé à la fabrication et à l'étude de complexes entre les AHPTs et des cyclodextrines. Nous avons retenu la gamma-cyclodextrine (gamma-CD), qui présentait l'avantage de complexer les 3 AHPTs avec une constante de formation « moyenne » à « élevée ». Ces complexes AHPTs :gamma-CD ont été caractérisés en utilisant diverses techniques : chromatographiques, thermiques et spectrométriques. Nous avons conclu à l'obtention de complexes d'inclusion qui ont permis d'augmenter la solubilité des AHPTs. Dans une dernière partie, nous avons évalué les activités biologiques des complexes AHPTs : gamma-CD. Les résultats montrent que les complexes AU : gamma-CD et AO : gamma-CD restent actifs à l'encontre des bactéries à Gram positif (mais avec une efficacité plus faible) ; tandis que le complexe AB : gamma-CD se révèle être actif sur certaines bactéries. Le complexe AB : gamma-CD, et de façon surprenante le complexe AU : gamma-CD présentent une activité antivirale à l'encontre du hCMV. Enfin, la diminution de la cytotoxicité liée à la complexation des AHPTs accroit l'intérêt des molécules d'AU et d'AB sur les cellules de LMC / The lack of innovative therapies in human chemotherapy incites the scientific community to be interested in new sources of bioactive compounds. We can quote the secondary metabolites of plants, to which belong hydroxy pentacyclic triterpene acids (HPTAs) and more particularly Ursolic (UA), Oleanolic (OA) and Betulinic Acids(BA). These molecules are the subjetc of numerous studies which tend to demonstrate their properties: anti-infective, anticancer, antiproliferatives, anti-inflammatory and hepatoprotectrive. The main obstacle to their use in therapeutic purposes stays the insolubility of these AHPTs in the water. Thus the objective of this work was to increase their hydrosolubility. At first, in agreement with the recommendations and/or the existing standards, we demonstrated that the antibacterial spectra of UA and OA are limited to Gram-positive bacteria. No HPTA showed antifungal activity. Only the BA showed an interesting antiviral activity on human Cytomegalovirus (hCMV); no antiviral activity was on Poliovirus. Finally, the BA, but even more UA showed an anticancer activity against cellular model of chronic myeloid leukemia (CML). Secondly, we proceeded to the manufacturing and to the study of complexes between HPTAs and cyclodextrines. We retained gamma-cyclodextrine (gamma-CD), which presented the advantage to complex the 3 AHPTs with a constant of "average" to "raised". These HPTAs:gamma-CD complexes were characterized by using diverse techniques: chromatographic, thermal and spectrometric. We concluded in the obtaining of inclusion complexes which allowed increasing the solubility of the HPTAs. In a last part, we estimated the biological activities of the HPTAs:gamma-CD complexes. The results show that the UA:gamma-CD and OA:gamma-CD complexes remain active against Gram-positive bacteria (but with a weaker efficiency); whereas the BA:gamma-CD complex shows to be active on certain bacteria. The BA:gamma-CD complex, and in a surprising way the UA:gamma-CD complex presents an antiviral activity against the hCMV. Finally, the decrease of the cytotoxicity linked to the complexation of the HPTAs believes the interest of UA and of BA on CML cells