0022-3565/02/3013-1020–1024$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 0/986104 JPET 301:1020–1024, 2002 Printed in U.S.A. Characterization of a Novel Endocannabinoid, Virodhamine, with Antagonist Activity at the CB1 Receptor AMY C. PORTER, JOHN-MICHAEL SAUER, MICHAEL D. KNIERMAN, GERALD W. BECKER, MICHAEL J. BERNA, JINGQI BAO, GEORGE G. NOMIKOS, PETRA CARTER, FRANK P. BYMASTER, ANDREA BAKER LEESE, and CHRISTIAN C. FELDER Lilly Research Laboratories, Neuroscience Division (A.C.P., G.G.N., P.C., F.P.B., A.B.L., C.C.F.), Drug Disposition (J.-M.S., M.J.B., J.B.), and Research Technologies and Proteins (M.D.K., G.W.B.), Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana Received December 19, 2001; accepted February 18, 2002 This article is available online at http://jpet.aspetjournals.org Downloaded from ABSTRACT The first endocannabinoid, anandamide, was discovered in rodhamine concentrations were 2- to 9-fold higher than anan- 1992. Since then, two other endocannabinoid agonists have damide. In contrast to previously described endocannabinoids, been identified, 2-arachidonyl glycerol and, more recently, no- virodhamine was a partial agonist with in vivo antagonist activ- ladin ether. Here, we report the identification and pharmaco- ity at the CB1 receptor. However, at the CB2 receptor, vi- 14 logical characterization of a novel endocannabinoid, vi- rodhamine acted as a full agonist. Transport of [ C]anandam- jpet.aspetjournals.org rodhamine, with antagonist properties at the CB1 cannabinoid ide by RBL-2H3 cells was inhibited by virodhamine. receptor. Virodhamine is arachidonic acid and ethanolamine Virodhamine produced hypothermia in the mouse and acted as joined by an ester linkage. Concentrations of virodhamine mea- an antagonist in the presence of anandamide both in vivo and sured by liquid chromatography atmospheric pressure chemi- in vitro. As a potential endogenous antagonist at the CB1 cal ionization-tandem mass spectrometry in rat brain and hu- receptor, virodhamine adds a new form of regulation to the man hippocampus were similar to anandamide. In peripheral endocannabinoid system. tissues that express the CB2 cannabinoid receptor, vi- at ASPET Journals on January 24, 2020 Following the discovery of two G protein-coupled receptors, brain microdialysate, a second analyte was seen that had the CB1 and CB2, which respond to ⌬9-tetrahydrocannabinol, same molecular weight as anandamide but a shorter reten- the active principal in marijuana, a search was initiated to tion time, and therefore, could not be anandamide (Fig. 1). identify endogenous ligands for these receptors. Anandamide The peak was hypothesized to be O-arachidonoyl ethanol- (N-arachidonyl ethanolamide) was the first endocannabinoid amine, and an authentic standard was subsequently synthe- discovered in 1992 by screening porcine brain extracts for sized (BIOMOL Research Laboratories, Plymouth Meeting, compounds that bound to the cannabinoid receptor (Devane PA). Based on its chromatographic and mass spectrometric et al., 1992). It was later shown that anandamide could properties compared with the synthesized standard, the un- stimulate cannabinoid receptor-mediated signal transduc- known analyte was confirmed to be O-arachidonoyl ethanol- tion (Felder et al., 1993). The second endocannabinoid iden- amine. O-Arachidonoyl ethanolamine is arachidonic acid and tified was 2-arachidonoyl glycerol (2-AG), which was isolated ethanolamine joined by an ester linkage, the opposite of the from canine gut and shown to have in vivo effects similar to amide linkage found in anandamide. Based on this opposite ⌬9-tetrahydrocannabinol (Mechoulam et al., 1995). Very re- orientation, the molecule was named virodhamine from the cently, a third endocannabinoid, noladin ether, was also iso- Sanskrit word virodha, which means opposition. lated from porcine brain (Hanus et al., 2001). Both anand- Synthetic virodhamine was used to measure and characterize amide and 2-AG are agonists at both the CB1 and CB2 its in vitro and in vivo function. Tissue concentrations were receptors. Noladin ether has been shown to bind to the CB1 measured in rat and human brain as well as rat peripheral receptor with nanomolar affinity and to the CB2 receptor tissues and rat striatal microdialysate and compared with with low micromolar affinity, but functional activity has not anandamide. The in vitro functional activity of virodhamine at yet been determined (Hanus et al., 2001). CB1 and CB2 receptors was measured by antibody capture In the course of development of a bioanalytical method to GTP binding. The ability of virodhamine to decrease body tem- measure anandamide in brain and peripheral tissues and perature in mouse, a property of CB1 cannabinoid receptor ABBREVIATIONS: Anandamide, N-arachidonyl ethanolamide; 2-AG, 2-arachidonoyl glycerol; LC, liquid chromatography; APCI-MS-MS, atmo- spheric pressure chemical ionization-tandem mass spectrometry; DMSO, dimethyl sulfoxide; [35S]GTP␥S, guanosine 5Ј-O-(3-[35S]thio)triphos- phate; methanandamide, R(ϩ)-arachidonyl-1Ј-hydroxy-2Ј-propylamide; AM404, N-(4-hydroxyphenyl)arachidonylamide; WIN 55,212-2, R-(ϩ)- [2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone. 1020 Characterization of a Novel Endogenous CB1 Antagonist 1021 ligands, was characterized in vivo. Together, these data indi- cate that virodhamine acts as a unique endogenous cannabinoid ligand with antagonist activity at the CB1 receptor. Materials and Methods Drugs. WIN 55,212-2 [R-[ϩ]-[2,3-dihydro-5-methyl-3-(4-morpholi- nylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmeth- anone] was purchased from Tocris Cookson (St. Louis, MO). AM404 [N-(4-hydroxyphenyl)arachidonylamide], anandamide, and methanan- damide [R(ϩ)-arachidonyl-1Ј-hydroxy-2Ј-propylamide] were purchased from Sigma/RBI (Natick, MA). 2-AG was purchased from BIOMOL Research Laboratories. Virodhamine (O-arachidonoyl ethanolamine) 2 and [ H8]anandamide were custom synthesized by BIOMOL Research Laboratories. Liquid Chromatography (LC) Atmospheric Pressure Chem- ical Ionization-Tandem Mass Spectrometry (APCI-MS-MS) Fig. 1. LC APCI-MS-MS quantification of anandamide and virodhamine. ϩ ϩ Analysis. A quantitative bioanalytical method utilizing LC APCI- Using m/z 348 ([M H] ) as a precursor ion and m/z 62 as a product ion in a selected reaction monitoring mode, both anandamide and vi- Downloaded from MS-MS was used to measure anandamide and virodhamine. Follow- rodhamine concentrations were quantified. Anandamide and vi- ing liquid extraction with organic solvent and further purification by rodhamine were easily separated chromatographically using a C-18 col- solid phase extraction, samples were separated using a Zorbax umn under isocratic conditions. Eclipse XDB C-18 column (75 ϫ 4.6 mm, 3.5 ␮m, 80 A; Phenomenex, Torrance, CA) under isocratic conditions with a 90:10, methanol/5 (coordinates relative to bregma: anterioposterior ϭϩ0.7, mediolat- mM ammonium acetate, 0.25% glacial acetic acid mobile phase at a flow eral ϭϩ3.5, and dorsoventral ϭϪ3.6) according to the atlas of Paxinos rate of 1 ml/min. Quantification of anandamide and virodhamine was and Watson (1986). While the animal was still anesthetized, a commer- jpet.aspetjournals.org ϩ accomplished using m/z 348 ([M ϩ H] ) as a precursor ion and m/z 62 cially available microdialysis probe (Bioanalytical Systems, Inc.) with as a product ion in a selected reaction monitoring mode using an active membrane surface of 4.0 mm was inserted through the guide 2 [ H8]anandamide as an internal standard. Anandamide and cannula and secured in place. After surgery, rats were housed individ- 2 [ H8]anandamide eluted at approximately 6 min, whereas virodhamine ually in Plexiglas cages. Principles of laboratory animal care (Guide for eluted at approximately 3.5 min. The lower limit of quantification for the Care and Use of Laboratory Animals, National Academy Press, both anandamide and virodhamine was 25 pg/ml (8.7 fmol/ml). 1996) were followed, and the Animal Care and Use Committee of Eli Antibody Capture Scintillation Proximity GTP-Binding As- Lilly & Co. approved all protocols. 35 say. [ S]GTP␥S binding was measured in a 96-well format, using a Microdialysis Experiments. The day of the microdialysis exper- at ASPET Journals on January 24, 2020 modified antibody capture technique previously described (DeLapp iments, rats were transported from their home cage to the microdi- et al., 1999). Briefly, Sf9 cell membranes expressing CB1 or CB2 and alysis testing room. All experiments were performed approximately G␣i3␤1␥2 (Packard Biosciences, Meriden, CT), drug, and 500 pM 24 h postsurgery in awake, freely moving animals during the light [35S]GTP␥S (PerkinElmer Life Sciences, Boston, MA) were incu- period. Microdialysis probes were perfused with a modified Ringer’s bated in a total volume of 200 ␮l at room temperature for 60 min in solution (1.3 mM CaCl2, 1.0 mM MgCl2, 3.0 mM KCl, 147.0 mM GTP-binding assay buffer (20 mM Hepes, 100 mM NaCl, and 5 mM ϫ ϫ NaCl, 1.0 mM Na2HPO4 7H2O, 0.2 mM NaH2PO4 2H2O, and 1% MgCl2, pH 7.4). The labeled membranes were then solubilized with bovine serum albumin, pH 7.2) at a rate of 5.0 ␮l/min set by a 0.27% Nonidet P-40 detergent (Roche Applied Science, Indianapolis, microinfusion pump (SciPro, North Tonawanda, NY). Sample collec- IN) for 30 min followed by the addition of a rabbit anti-Gi antibody at tion started 1 h after the beginning of perfusion. Microdialysis sam- a final dilution of 1:400. Following a 60-min incubation at room ples were collected every 20 min in polypropylene tubes containing temperature, anti-rabbit antibody scintillation proximity assay ␮ 2 150 lof[H8]anandamide standard in methanol. The microdialysis beads (Amersham Biosciences, Piscataway, NJ) were added, and the samples were kept at 8°C in a microsampler until the end of the day and plates were incubated at room temperature for an additional 3 h.