Identification of the Plant Steroid -Spinasterol as a Novel Transient Receptor Potential Vanilloid 1 Antagonist with Antinociceptive Properties Gabriela Trevisan, Mateus Fortes Rossato, Cristiani Isabel Bandero ´ Walker, Jonatas Zeni Klafke, Fernanda Rosa, Sara Marchesan Oliveira, Raquel Tonello, Gustavo Petri Guerra, Aline Augusti Boligon, Ricardo Basso Zanon, Margareth Linde Athayde, and Juliano Ferreira Programa de Po ´ s-Graduac ¸a ˜o em Cie ˆ ncias Biolo ´ gicas:Bioquímica Toxicolo ´ gica (G.T., M.F.R., J.Z.K., F.R., S.M.O., R.T., G.P.G., J.F.), Programa de Po ´ s-Graduac ¸a ˜ o em Farmacologia (A.A.B., R.B.Z., M.L.A.), and Programa de Po ´ s-graduac ¸a ˜o em Cie ˆ ncias Farmace ˆ uticas (A.A.B., R.B.Z., M.L.A.), Universidade Federal de Santa Maria, Santa Maria, Brazil; and Departamento de Sau ´ de, Curso de Farma ´ cia, Universidade Regional Integrada do Alto Uruguai e Misso ˜ es, Santiago, Brazil (C.I.B.W.) Received April 30, 2012; accepted July 25, 2012 ABSTRACT The transient receptor potential vanilloid 1 (TRPV1) receptor is relevant to the perception of noxious information and has been studied as a therapeutic target for the development of new analgesics. The goal of this study was to perform in vivo and in vitro screens to identify novel, efficacious, and safe TRPV1 antagonists isolated from leaves of the medicinal plant Ver- nonia tweedieana Baker. All of the fractions and the hydroal- coholic extract produced antinociception in mice during the capsaicin test, but the dichloromethane fraction also had anti- oedematogenic effect. Among the compounds isolated from the dichloromethane fraction, only -spinasterol reduced the nociception and edema induced by capsaicin injection. More- over, -spinasterol demonstrated good oral absorption and high penetration into the brain and spinal cord of mice. -Spi- nasterol was able to displace [ 3 H]resiniferatoxin binding and diminish calcium influx mediated by capsaicin. Oral adminis- tration of the dichloromethane fraction and -spinasterol also produced antinociceptive effect in the noxious heat-induced nociception test; however, they did not change the mechanical threshold of naive mice. The treatment with -spinasterol did not produce antinociceptive effect in mice systemically pre- treated with resiniferatoxin. In addition, -spinasterol and the dichloromethane fraction reduced the edema, mechanical, and heat hyperalgesia elicited by complete Freund’s adjuvant paw injection. The dichloromethane fraction and -spinasterol did not affect body temperature or locomotor activity. In conclu- sion, -spinasterol is a novel efficacious and safe antagonist of the TRPV1 receptor with antinociceptive effect. Introduction The transient receptor potential vanilloid type 1 (TRPV1) receptor is a member of the TRP family (Caterina et al., 1997). It is composed of six transmembrane domains that form a nonselective cation channel (principally for calcium) (Schumacher, 2010). The TRPV1 receptor is expressed mainly in the nervous system and is particularly abundant in C and A nociceptive fibers, where it plays a key role in the detection of noxious painful stimuli (Szallasi et al., 2007). It is activated by exogenous substances, such as capsaicin (the active component of chili peppers), resiniferatoxin (RTX; iso- lated from Euphorbia resinifera), endogenous inflammatory agents, extracellular protons, and noxious heat (43°C) (Schumacher, 2010). Because the TRPV1 receptor plays an important role in the detection and integration of noxious stimuli and is sensitized by different mechanisms, including various inflammatory This study was supported by the Conselho Nacional de Desenvolvimento Científico, Financiadora de Estudos e Projetos, Programa de Apoio aos Nu ´ cleos de Excele ˆncia, Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado do Rio Grande do Sul, and Coordenac ¸a ˜ o de Aperfeic ¸oamento de Pessoal de Nível Superior. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. http://dx.doi.org/10.1124/jpet.112.195909. ABBREVIATIONS: TRPV1, transient receptor potential vanilloid 1; ANOVA, analysis of variance; BSA, bovine serum albumin; CNS, central nervous system; CFA, complete Freund’s adjuvant; Dcm, dichloromethane; DMSO, dimethyl sulfoxide, Act, ethyl acetate; HE, hydroalcoholic extract; But, n-butanol; PWT, paw withdrawal threshold; RTX, resiniferatoxin; TBS-T, Tween 20 in Tris-borate saline; AM, acetoxymethyl ester; HPLC, high-performance liquid chromatography; SB-366791, N-(3-methoxyphenyl)-4-chlorocinnamide; AMG-9810, [(E)-3-(4-t-butylphenyl)-N-(2,3-dihy- drobenzo[b][1,4] dioxin-6-yl)acrylamide]; AMG-517, N-(4-hydroxy-benzo[d]thiazol-2-yl)acetamide (N-acetyl benzothiazole). 1521-0103/12/3432-258–269$25.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 343, No. 2 Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics 195909/3797896 JPET 343:258–269, 2012 258 at ASPET Journals on August 23, 2016 jpet.aspetjournals.org Downloaded from