The plant cannabinoid Δ9-tetrahydrocannabivarin can decrease signs of inflammation and inflammatory pain in mice

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<ul><li><p>THEMED ISSUE: CANNABINOIDS</p><p>RESEARCH PAPER</p><p>The plant cannabinoid D9-tetrahydrocannabivarincan decrease signs of inflammation andinflammatory pain in micebph_756 677..687</p><p>Daniele Bolognini1,2, Barbara Costa3, Sabatino Maione4, Francesca Comelli3, Pietro Marini1,Vincenzo Di Marzo5, Daniela Parolaro2, Ruth A Ross1, Lisa A Gauson1, Maria G Cascio1 andRoger G Pertwee1</p><p>1Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK, 2DBSF, Pharmacology Section and Neuroscience Centre,University of Insubria, Varese, Italy, 3Department of Biotechnology &amp; Bioscience, University of Milano-Bicocca, Milano, Italy,4Endocannabinoid Research Group, Division of Pharmacology L Donatelli, Department of Experimental Medicine, SecondUniversity of Naples, Naples, Italy and 5Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR,Pozzuoli, Naples, Italy</p><p>Background and purpose: The phytocannabinoid, D9-tetrahydrocannabivarin (THCV), can block cannabinoid CB1 receptors.This investigation explored its ability to activate CB2 receptors, there being evidence that combined CB2 activation/CB1blockade would ameliorate certain disorders.Experimental approach: We tested the ability of THCV to activate CB2 receptors by determining whether: (i) it inhibitedforskolin-stimulated cyclic AMP production by Chinese hamster ovary (CHO) cells transfected with human CB2 (hCB2)receptors; (ii) it stimulated [35S]GTPgS binding to hCB2 CHO cell and mouse spleen membranes; (iii) it attenuated signs ofinflammation/hyperalgesia induced in mouse hind paws by intraplantar injection of carrageenan or formalin; and (iv) any suchanti-inflammatory or anti-hyperalgesic effects were blocked by a CB1 or CB2 receptor antagonist.Key results: THCV inhibited cyclic AMP production by hCB2 CHO cells (EC50 = 38 nM), but not by hCB1 or untransfected CHOcells or by hCB2 CHO cells pre-incubated with pertussis toxin (100 ngmL-1) and stimulated [35S]GTPgS binding to hCB2 CHOand mouse spleen membranes. THCV (0.3 or 1 mgkg-1 i.p.) decreased carrageenan-induced oedema in a manner that seemedto be CB2 receptor-mediated and suppressed carrageenan-induced hyperalgesia. THCV (i.p.) also decreased pain behaviour inphase 2 of the formalin test at 1 mgkg-1, and in both phases of this test at 5 mgkg-1; these effects of THCV appeared to beCB1 and CB2 receptor mediated.Conclusions and implications: THCV can activate CB2 receptors in vitro and decrease signs of inflammation and inflammatorypain in mice partly via CB1 and/or CB2 receptor activation.British Journal of Pharmacology (2010) 160, 677687; doi:10.1111/j.1476-5381.2010.00756.xThis article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit</p><p>Keywords: D9-Tetrahydrocannabivarin; CP55940; CB2 receptor; CB1 receptor; pertussis toxin; pain; inflammation; carrageenan;formalin</p><p>Abbreviations: AM630, 6-iodopravadoline; CHO, Chinese hamster ovary; CP55940 ()-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; DMSO, dimethyl sulphoxide; PMSF, phenyl-methylsulphonyl fluoride; rimonabant, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; SR144528, N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; THCV,D9-tetrahydrocannabivarin</p><p>Introduction</p><p>We have reported previously that the plant cannabinoid,D9-tetrahydrocannabivarin (THCV; Figure 1), can behave as aCB1 receptor antagonist, both in vitro and in vivo (Pertwee</p><p>Correspondence: Professor RG Pertwee, School of Medical Sciences, Institute ofMedical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD,UK. E-mail: 29 October 2009; revised 19 January 2010; accepted 28 January 2010</p><p>British Journal of Pharmacology (2010), 160, 677687 2010 The AuthorsJournal compilation 2010 The British Pharmacological Society All rights reserved 0007-1188/</p></li><li><p>et al., 2007; receptor nomenclature follows Alexander et al.,2009), and also that this phytocannabinoid opposes theability of the cannabinoid receptor agonist, CP55940, tostimulate [35S]GTPgS binding to human CB2 receptors inChinese hamster ovary (CHO) cell membranes (Thomas et al.,2005). In this paper, we present evidence that THCV canbehave in vitro as a CB2 receptor partial agonist whenthe measured response is inhibition of forskolin-inducedstimulation of cyclic AMP production by CHO cells express-ing very high densities of human CB2 receptors or stimulationof [35S]GTPgS binding to membranes obtained either fromthese cells or from mouse spleen. No such effects wereinduced by THCV in human CB1 CHO cells, in mouse wholebrain membranes or in mouse spleen membranes obtainedfrom CB2-/- mice, findings that are in line with previousreports that THCV can behave as a CB1 receptor antagonist(Thomas et al., 2005; Pertwee et al., 2007; Dennis et al., 2008;Ma et al., 2008). Because there is convincing pre-clinical evi-dence that combined activation of CB2 receptors and block-ade of CB1 receptors would ameliorate disorders such aschronic liver diseases and stroke (Mallat et al., 2007; Zhanget al., 2009), our discovery that THCV can behave as a CB2receptor agonist in vitro prompted us to investigate whetherthis compound can also produce signs of CB2 receptor activa-tion in vivo. This we did by determining whether THCV sharesthe ability of established selective CB2 receptor agonists toreduce signs of inflammation and inflammatory pain in amanner that can be antagonized by a selective CB2 receptorantagonist (Guindon and Hohmann, 2008). These experi-ments were performed with mice in which paw oedema andsigns of hyperalgesia were induced by intraplantar injectionsof either carrageenan or formalin.</p><p>Methods</p><p>AnimalsAll animal care and experimental procedures complied withItalian (D.L. 116/92) and EEC (O.J. of EC L358/1 18/12/1986)regulations on the protection of laboratory animals, and withthe UK Animals (Scientific Procedures) Act 1986 and associ-ated guidelines for the use of experimental animals. Guide-lines of the International Association for the Study of Painwere also followed. Brain and spleen tissue was obtained fromadult male C57BL/6J mice weighing 2540 g and maintainedon a 12/12 h light/dark cycle with free access to food andwater. These animals were either wild-type mice (Harlan UKLtd, Blackthorn, UK) or mice from which the CB2 receptor hadbeen genetically deleted as described by Buckley et al., 2000.All in vivo experiments were also performed with male</p><p>C57BL/6J mice (Harlan, Milan, Italy). These mice were 9weeks old and housed three per cage under controlled illumi-nation (12:12 h light : dark cycle; light on at 0600 h) andstandard environmental conditions (room temperature 22 1C; humidity 60 10%) for at least 1 week before experi-mental use. Mouse chow and tap water were available adlibitum. All the in vivo experiments were performed in a ran-domized manner by an experimenter, unaware of the phar-macological treatments.</p><p>CHO cellsCHO cells either untransfected or transfected with cDNAencoding human cannabinoid CB2 or CB1 receptors weremaintained in Dulbeccos modified Eagles medium nutrientmixture F-12 HAM, supplemented with 1 mM L-glutamine,10% fetal bovine serum and 0.6% penicillinstreptomycin forall cells together with G418 (400 mgmL-1) for the CHOhCB2cells or with hygromycin B (300 mgmL-1) and G418(600 mgmL-1) for the CHOhCB1 cells. All cells were main-tained at 37C and 5% CO2 in their respective media, andwere passaged twice a week using non-enzymatic cell disso-ciation solution.</p><p>Membrane preparationBinding assays with [3H]CP55940 or [35S]GTPgS were per-formed with CHOhCB2 cell membranes (Ross et al., 1999a),with mouse whole brain membranes (Vigan et al., 2003) orwith mouse spleen membranes, the preparation of which wasbased on a method described by Hillard et al. (1999) for pre-paring rat spleen membranes. The hCB2 receptor-transfectedcells were removed from flasks by scraping and then frozen asa pellet at -20C until required. Before use in a radioligandbinding assay, cells were defrosted, diluted in Tris buffer(50 mM TrisHCl and 50 mM Trisbase) and homogenizedwith a 1 mL hand-held homogenizer. Protein assays were per-formed using a Bio-Rad DC Kit (Hercules, CA, USA). Spleenswere cut into several pieces and placed in a Choi lysis buffer(TrisHCl 20 mM, sucrose 0.32 M, EDTA 0.2 mM, EGTA0.5 mM, pH 7.5) containing Roche protease inhibitor cocktail(1:40 v/v; Roche Diagnostics, Mannheim, Germany) and phe-nylmethylsulphonyl fluoride (PMSF; 150 mM), and thenhomogenized. The homogenate was centrifuged at 500 g for2 min, and the resulting supernatant was re-centrifuged at16 000 g for 20 min. The harvested membranes were resus-pended in TME buffer (50 mM TrisHCl; EDTA 1.0 mM; MgCl23.0 mM; pH 7.4) and stored at -80C for no more than 1month. Mouse brains were homogenized in ice-cold Choilysis buffer containing Roche protease inhibitor cocktail (1:40v/v) and PMSF (1 mM). The homogenate was centrifuged at13 500 g for 15 min, and the resulting pellet was kept at-80C for at least 2 h. The pellet was then resuspended in TMEbuffer, homogenized and stored at -80C.</p><p>Cyclic AMP assayAdherent CHOhCB1 or CHOhCB2 cells were washed oncewith Dulbeccos phosphate-buffered saline (PBS) anddetached using non-enzymatic cell dissociation solution.</p><p>O</p><p>OH</p><p>Figure 1 The structure of THCV.</p><p>D9-Tetrahydrocannabivarin and inflammation678 D Bolognini et al</p><p>British Journal of Pharmacology (2010) 160 677687</p></li><li><p>After centrifugation, cells were resuspended (2 106 cellsmL-1) in buffer containing PBS (calcium and magne-sium free), 1% BSA and 10 mM rolipram. Cells were incubatedfor 30 min at 37C with the cannabinoid under investigation.A further 30 min incubation was carried out with 10 mM offorskolin in a total volume of 500 mL. The reaction was ter-minated by the addition of 0.1 M HCl, followed by centrifu-gation to remove cell debris. The pH was then adjusted tobetween 8 and 9 by the addition of 1 M of NaOH, and cyclic-AMP content was measured using a radioimmunoassay kit(GE Healthcare Amersham Ltd, Little Chalfont, Buckingham-shire, UK). Forskolin and rolipram were dissolved in dimethylsulphoxide (DMSO) and stored at -20C as 10 mM stock solu-tions. Some CHO cells were pretreated overnight with pertus-sis toxin (100 ngmL-1; Coutts et al., 2001). The pertussis toxinused in these experiments was dissolved in distilled water andstored at 4C.</p><p>Radioligand displacement assayThe assays were carried out with [3H]CP55940 and Tris-binding buffer (50 mM TrisHCl, 50 mM Trisbase, 0.1% BSA,pH 7.4), total assay volume 500 mL, using the filtration pro-cedure described previously by Ross et al. (1999b). Bindingwas initiated by the addition of transfected hCB2 cells (50 mgprotein per well). All assays were performed at 37C for60 min before termination by the addition of ice-cold Tris-binding buffer and vacuum filtration using a 24-well samplingmanifold (Brandel Cell Harvester; Brandel Inc, Gaithersburg,MD, USA) and Brandel GF/B filters that had been soaked inwash buffer at 4C for at least 24 h. Each reaction well waswashed six times with a 1.2 mL aliquot of Tris-binding buffer.The filters were oven-dried for 60 min and then placed in5 mL of scintillation fluid (Ultima Gold XR, PerkinElmer, SeerGreen, Buckinghamshire, UK). Radioactivity was quantifiedby liquid scintillation spectrometry. Specific binding wasdefined as the difference between the binding that occurred inthe presence and absence of 1 mM unlabelled CP55940. Theconcentration of [3H]CP55940 used in our displacementassays was 0.7 nM. The compounds under investigation werestored as stock solutions of 10 mM in DMSO, the vehicleconcentration in all assay wells being 0.1% DMSO. Thebinding parameters for [3H]CP55940 were 215 pmolmg-1</p><p>(Bmax) and 4.3 nM (Kd).</p><p>[35S]GTPgS binding assayThe method used for measuring agonist-stimulated bindingof [35S]GTPgS was based on previously described methods(Hillard et al., 1999; Thomas et al., 2005). The assays werecarried out with GTPgS binding buffer (50 mM TrisHCl,100 mM NaCl, 0.1% BSA) in the presence of [35S]GTPgS andGDP, in a final volume of 500 mL. The GTPgS binding bufferalso contained 50 mM Trisbase, 5 mM MgCl2, 1 mM dithio-threitol and 1 mM EDTA (CHO cell membrane experiments)or 3 mM MgCl2 and 0.2 mM EGTA (brain and spleen mem-brane experiments). Binding was initiated by the addition of[35S]GTPgS to the wells. Non-specific binding was measured inthe presence of 30 mMGTPgS. The drugs were incubated in theassay for 60 min at 30C. The reaction was terminated by a</p><p>rapid vacuum filtration method using Tris-binding buffer, andthe radioactivity was quantified by liquid scintillation spec-trometry. In all the [35S]GTPgS-binding assays, we used 0.1 nM[35S]GTPgS, 30 mM GDP and 10 mg (brain membranes), 40 mg(spleen membranes) or 50 mg (cell membranes) protein perwell. Additionally, mouse brain and spleen membranes werepre-incubated for 30 min at 30C with 0.5 UmL-1 adenosinedeaminase (200 UmL-1) to remove any endogenousadenosine.</p><p>Carrageenan-induced inflammationThe mice were anaesthetized with sodium pentobarbital(60 mgkg-1 i.p.). Acute inflammation was induced by intra-plantar injection of 20 mL of l-carrageenan (2% w/v in saline)into the right hind paw. The volume of the injected paw, aswell as of the contralateral paw, was measured with a plethys-mometer (Ugo Basile, Varese, Italy). Data are expressed asoedema (difference in volume between the right and leftpaws). Responses to thermal stimuli were measured in thesame animals used to monitor oedema. After recording base-line withdrawal latencies (s), withdrawal latencies of bothhind paws were estimated at different time-points after carra-geenan injection, starting 3 h after injection of this inflam-matory stimulus, when all mice had recovered fromanaesthesia. Heat hypersensitivity was tested according to theHargreaves procedure (Hargreaves et al., 1988) using theplantar test (Ugo Basile). Briefly, the animals were placed in aclear plexiglass box and allowed to acclimatize. A constantintensity radiant heat source was aimed at the midplantararea of the hind paw. The time, in seconds, from initial heatsource activation until paw withdrawal was recorded. Thecontrol animals received saline instead of carrageenan byintraplantar injection. All these experiments were performedby the same experimenter.</p><p>Formalin testFormalin injection induces biphasic stereotypical nocifensivebehaviour (Dubuisson and Dennis, 1977). Nociceptiveresponses are divided into an early, short lasting first phase(07 min) caused by a primary afferent discharge produced bythe stimulus and a subsequent second, prolonged phase (1560 min) of tonic pain (Sawynok and Liu, 2004). These twophases are separated by a transient quiescent period. The micereceived formalin (1.25% in saline, 30 mL) into the dorsalsurf...</p></li></ul>