Absenceofentourage:TerpenoidscommonlyfoundinCannabis

sativadonotmodulatethefunctionalactivityof∆9-THCat

humanCB1andCB2receptors

MarinaSantiago1,ShivaniSachdev1,JonathonCArnold2,3,IainSMcGregor2,4,andMarkConnor1

1DepartmentofBiomedicalSciences,MacquarieUniversity,NSW,Australia

2TheLambertInitiativeforCannabinoidTherapeutics,TheUniversityofSydney,NSW,Australia

3DisciplineofPharmacology,TheUniversityofSydney,NSW,Australia

4SchoolofPsychology,TheUniversityofSydney,NSW,Australia

Briefrunningtitle:THCandterpenoidactionsonCB1/CB2

Correspondingauthor:MarinaSantiago

Emailaddress:marina.junqueirasantiago@mq.edu.au

Keywords:phytocannabinoid,cannabinoidreceptor,terpenoid,entourageeffect,THC,signalling

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Footnotesandabbreviations:

SomeofthesedatawerepresentedatTheAustralasianSocietyofClinicalandExperimental

PharmacologistsandToxicologistsConferenceinMelbourne,Australia(2016).

β-Car β-caryophyllene

CBD cannabidiol

HBSS Hank'sbalancedsaltsolution

RFU relativefluorescenceunits

SEM standarderrorofthemean

SST somatostatin

∆9-THC ∆9-tetrahydrocannabinol

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Abstract

Introduction:CompoundspresentinCannabissativasuchasphytocannabinoidsandterpenoids,mayact

inconcerttoelicittherapeuticeffects.Cannabinoidssuchas∆9-tetrahydrocannabinol(∆9-THC)directly

activatecannabinoidreceptor1(CB1)andcannabinoidreceptor2(CB2),however,itisnotknownif

terpenoidspresentinCannabisalsoaffectcannabinoidreceptorsignalling.Therefore,weexamined6

commonterpenoidsalone,andincombinationwithcannabinoidreceptoragonists,onCB1andCB2

signallinginvitro.

MaterialsandMethods:PotassiumchannelactivityinAtT20FlpIncellstransfectedwithhumanCB1orCB2

receptorswasmeasuredinreal-timeusingFLIPR®membranepotentialdyeinaFlexStation3platereader.

Terpenoidsweretestedindividuallyandincombinationforperiodsupto30minutes.Endogenous

somatostatinreceptorsservedasacontrolfordirecteffectsofdrugsonpotassiumchannels.

Results:α-Pinene,β-pinene,β-caryophyllene,linalool,limoneneandβ-myrcene(upto30-100µM)didnot

changemembranepotentialinAtT20cellsexpressingCB1orCB2,oraffecttheresponsetoamaximally

effectiveconcentrationofthesyntheticcannabinoidCP55,940.Thepresenceofindividualora

combinationofterpenoidsdidnotaffectthehyperpolarizationproducedbyΔ9-THC(10µM):(CB1:control,

59±7%;withterpenoids(10µMeach)55±4%;CB2:Δ9-THC16±5%,withterpenoids(10µMeach)17±4%).

ToinvestigatepossibleeffectondesensitizationofCB1responses,allsixterpenoidswereaddedtogether

with∆9-THCandsignallingmeasuredcontinuouslyover30min.Terpenoidsdidnotaffectdesensitization,

after30minutesthecontrolhyperpolarizationrecoveredby63±6%,inthepresenceoftheterpenoids

recoverywas61±5%.

Discussion:NoneofthesixofthemostcommonterpenoidsinCannabisdirectlyactivatedCB1orCB2,or

modulatedthesignallingofthephytocannabinoidagonist∆9-THC.Theseresultssuggestthatifa

phytocannabinoid-terpenoidentourageeffectexists,itisnotattheCB1orCB2receptorlevel.Itremains

possiblethatterpenoidsactivateCB1andCB2signallingpathwaysthatdonotinvolvepotassiumchannels,

however,itseemsmorelikelythattheymayactatdifferentmoleculartarget(s)intheneuronalcircuits

importantforthebehaviouraleffectofCannabis.

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Introduction

AnenduringnotioninthemedicinalCannabisandcannabinoidfieldisthatofentourage:theideathatuse

ofthewholeplantmayexertsubstantiallygreatereffectsthanthesumofitsindividualparts.1Entourage

isusuallyconstruedasapositiveattribute,withtheassumptionthatsuperiortherapeuticactions,ora

morefavourable“high”,willbeobtainedfromconsumingthewholeCannabisplantratherthanindividual

componentssuchas∆9-tetrahydrocannabinol(THC).Somewhatsurprisingly,theevidenceforthiswidely

citednotionisrelativelysparse.

Cannabiscontainsalmost150phytocannabinoids,themostcommonofwhichareTHCandcannabidiol

(CBD),togetherwiththeiracidprecursorsTHCAandCBDA2.Cannabisalsocontainsalargenumberof

monoterpeneandsesquiterpenecompounds(togethercalledterpenoids),themostcommonofwhich

includeα-pinene,β-pinene,linalool,limoneneandβ-myrcene(monoterpenes)andβ-caryophylleneand

caryophylleneoxide(sesquiterpenes).3Terpenoidsarevolatilecompoundsthataresynthesisedalongside

phytocannabinoidsmainlyinthetrichomesofthecannabisplant,andprovidecannabiswithitsdistinctive

aromaandflavour.4Terpenoidsareoftenlostiftheextractionprocessinvolvesheating.5

Theentourageconceptappliedtocannabiscanencompassthepotentialforbothcannabinoid-

cannabinoidandcannabinoid-terpenoidinteractions.Withregardtotheformer,∆9-THC-CBDsynergyin

producinganalgesiawasreportedinananimalmodelofneuropathicpain6whileinhumans,CBDhasbeen

proposedtoamelioratesomeoftheadversepsychotomimeticandanxiogeniceffectsof∆9-THC.7,8This

claimiscontroversial,however,withanumberofcontraryfindings9,10CBDmaymodulate∆9-THCeffects

atthereceptorlevelactingasaCB1negativeallostericmodulator11,providingsomebiologicalplausibility

toamodulatoryinteraction.

Scientificevidenceforcannabinoid-terpenoidinteractionsisessentiallyabsentandmostlycomesfrom

websitesanddispensariesextollingthevirtuesofproprietaryCannabischemicalvarieties,or

chemovars.12,13However,someterpenoidsdohaveintrinsicpsychoactiveandphysiologicaleffects,and

modulatoryeffectson∆9-THCactionsisnotfarfetched.1,14Forexample,instudieswithlaboratory

animals,limonenedisplayedanxiolyticeffects,pineneincreasedgastrointestinalmotility,linaloolwas

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sedative,anticonvulsantandanxiolytic,whilemyrceneproducedsedation,analgesiaandmusclerelaxant

effects(summarisedinRussoandMarcu14).However,compellingevidenceforcannabinoid-terpenoid

interactionsorsynergydoesnotyetexist,anditisalsoworthnotingtherelativelylowconcentrationsof

terpenoidspresentinherbalcannabis.5,13,15,16

Withsomanybioactivecomponentspresentincannabis,thesystematic,granularelucidationofpossible

entourageeffectsposesasubstantialcombinatorialpuzzleandscientificchallenge.Asapreliminary

approachtoaddressingthischallenge,thepresentstudyexaminedwhethertheeffectsof∆9-THConits

cognatecannabinoidreceptors(CB1andCB2)wouldbemodifiedinthepresenceofterpenoidsthatare

commonlyfoundincannabis,eitheraloneorincombination.Thedemonstrationofsuchareceptor-level

entourageeffectmightleadtopredictionsregardingfunctionalcannabinoid-terpenoidinteractioneffects

thatcouldbetestedinvivo.

MaterialsandMethods

Cellculture

Experimentsusedmousewild-typeAtT20FlpIncells(AtT20-WT),orthesecellsstablytransfectedwith

humanCB1orCB2receptorswith3xN-terminushaemagglutinintags(AtT20-CB1andAtT20-CB2

respectively).17CellswerecultivatedinDulbecco’sModifiedEagle’sMedium(DMEM;Sigma-Aldrich)

supplementedwith10%foetalbovineserum(FBS;SIGMA/SAFC)and100Upenicillin/100µgstreptomycin

mL-1(Gibco).Selectionantibioticswere80µgmL-1Zeocin(Invivogen)forAtT20-WTor80µgmL-1

hygromycinBGold(Invivogen)fortransfectedcells.

Cellsweregrownin75mm2flasksat37°C/5%CO2andpassagedwhen80-90%confluent.Assayswere

carriedoutoncellsupto20passagesinculture.

PotassiumChannelActivityMeasurements

ChangesinmembranepotentialweremeasuredusingtheFLIPR®bluemembranepotentialdye

(MolecularDevices)inaFlexStation3,asoutlinedinKnapman2013.18Cellsfroma90-100%confluent

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75mm2flaskwereresuspendedinLeibovitz'sL-15Medium(Gibco)supplementedwith1%FBS,100U

penicillin/100µgstreptomycinmL-1andglucose(15mM)andplatedin96wellblackwalledclearbottom

microplates(Costar)inavolumeof90µLperwell.Cellswereincubatedovernightinhumidifiedambient

airat37°Cincubator.Membranepotentialdye,usedat50%ofthemanufacturerrecommended

concentration,wasresuspendedinHank'sBalancedSaltsolution(HBSS)ofcomposition(inmM):NaCl

145,HEPES22,Na2HPO40.338,NaHCO34.17,KH2PO40.441,MgSO40.407,MgCl20.493,CaCl21.26,

glucose5.55(pH7.4,osmolarity315±15).Dyewasloadedintoeachwell(90µLperwell)andequilibrated

at37°Cforatleast1hourpriortoassay.Fluorescencewasmeasuredevery2seconds(λexcitation=

530nm,λemission=565nm,λemissioncut-off=550nm).Assayswerecarriedoutat37°Canddrugs

wereautomaticallyaddedinvolumesof20µL.

DeterminingtheEffectsofTerpenoidsonAcuteHyperpolarization

Terpenoidswereaddedafteratleast60secondsofbaselinerecordingandincubatedfor5minutes

beforecannabinoid(CP55,940orΔ9-THC)addition.InAtT20-WTcells,somatostatin(SST)wasadded

insteadofcannabinoid.

DeterminingtheEffectsofTerpenoidsonSignallingDesensitization

HomologousdesensitizationwasmeasuredbysimultaneouslyaddingΔ9-THCwithterpenoidsafter120

secondsofbaselinerecording.Signallingdesensitizationwascalculatedaspercentagedecreasefrom

peakΔ9-THCresponseafter30minutesindrugs.SST(100nM)wasadded30minutesafterΔ9-THC

additiontoexaminethepotentialeffectsofprolongedcannabinoidreceptoractivationonnative

somatostatinreceptors(heterologousdesensitization).TheSSTresponsewascomparedbetweengroups

(withorwithoutterpenoids).

DrugDilution

Alldrugs(exceptSST)weremadeupinDMSOandstoredasfrozenstocksataconcentrationof10mM–

100mM.Terpenoidstocksolutionconcentrationswere100mM,withexceptionofβ-myrcene(30mM)

whichwasinsolubleat100mM.SSTwasdissolvedinwater.Freshaliquotswereusedeachday,withthe

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drugsdilutedinHBSScontaining0.1%bovineserumalbumin(Sigma-Aldrich)immediatelybeforethe

assay.ThefinalconcentrationofDMSOineachwellwas0.1to0.11%;thislimitedthemaximum

concentrationofterpenoidsabletobetested.Avehicle(HBSSplussolventalone)wellwasincludedin

eachcolumnofthe96wellplateandthechangesinfluorescenceproducedbyvehiclealonewere

subtractedbeforedeterminingthemaximumhyperpolarizationaftereachdrugexposure.

DrugsandReagents

Δ9-THCwasfromTHCPharm(Frankfurt,Germany).TerpenoidswerefromSigma-Aldrich;(+)-α-pinene,

(+)-β-pinene,(-)-β-caryophyllene,(+/-)-linalool,(R)-(+)-limoneneandβ-myrcene.Somatostatinwasfrom

AuspepandCP55,940fromCayman.Unlessotherwiseindicated,theotherchemicalsandreagentswere

fromSigma-Aldrich.

DataAnalysis

Eachexperimentwasindependentlyrepeatedatleast5times,with2technicalreplicatesineach

determination.Dataareexpressedasapercentagechangeinthefluorescencecomparedwiththepre-

drugbaseline(30sbeforedrugaddition),oraspercentageof1µMCP55,940response.Graphswere

plottedusingGraphpadPrism7.02,andscatterdotplotsshowmeanswithstandarderrorofthemean

(SEM).MeanswerecomparedusingunpairedStudent'st-testornomatchingone-wayANOVAfollowed

bycorrectionformultiplecomparisons(Dunnett);andnullhypothesiswasrejectedifp-valuewaslower

than0.05(p>0.05=notsignificant).

Results

TerpenoidsinAtT20-WTcells

Wefirstexaminedterpenoidactioninnon-transfectedAtT20cells.Weusedsomatostatin(100nM)asa

positivecontrolbecauseithyperpolarizesAtT20-WTcellsviaactivationofendogenousSSTreceptors(Fig.

1AandB).18,19Additionofα-pinene,β-pinene,β-caryophyllene,linalool,limonene(100μM)orβ-myrcene

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(30μM)didnotaffectthemembranepotentialofAtT20-WTcells(Fig.1C,opencircles).Thepresenceof

terpenoids(100μM/30μM)hadnoeffectonthesubsequentsomatostatinresponse(Fig.1C).

TerpenoidsinAtT20-CB1and-CB2cells

TheabsenceofaterpenoidresponseinAtT20-WTcellsenabledthestudyoftheireffectonmembrane

potentialinAtT20cellsexpressinghumanCB1orCB2.Weexaminedwhetherterpenoids(1nM–100μM,β-

myrcene300pM-30μM)hyperpolarisedcellsviathesereceptorsand,inparallel,whethertheyaffecteda

subsequentresponsetoamaximallyeffectiveconcentrationofCP55,940(1µM,Fig.2).17Asummaryof

thefluorescencechangeafterterpenoidadditiontoAtT20-CB1cellsisshowninFigure3(closedcircles).

Nodifferencebetweenvehicleandterpenoidswasobserved.Further,noneoftheterpenoidschangedthe

membranepotentialofcellsexpressingCB2(SupplementaryFig.S1).Thechangeinfluorescenceproduced

bythesubsequentadditionofthenon-selectivecannabinoidagonistCP55,940(1μM)wasalsounaffected

inbothAtT20-CB1and-CB2(Fig.3andSupplementaryFig.S1–opencircles).

CP55,940isahighefficacyagonistofbothCB1andCB2receptors.20However,inCannabis,∆9-THCisthe

principlecannabinoidagonistandithasalowerefficacythanCP55,940,whichisapparentinthe

hyperpolarizationassayasalowermaximalresponse.20Wenexttestedtheeffectofalowandhigh

concentrationofterpenoids(100nMand10μM)onthehyperpolarizationproducedbythree

concentrationsof∆9-THC(100nM,1μMand10μM).Afterfiveminutesofindividualterpenoidapplication,

applicationof∆9-THCproducedfluorescencechangesthatwerenotsignificantlydifferentfromthose

producedby∆9-THCaloneinbothAtT20-CB1and-CB2cells(10μM∆9-THCFigs.5and6,100nM∆9-THC

SupplementaryFigs.S2andS3).Toexplorethepossibilityofanemergententourageeffect,wecombined

allsixterpenoids(10μMeach)andtestedtheeffectofthemixtureonthe∆9-THC-induced

hyperpolarization.Similartoindividuallytestedterpenoids,theeffectsof∆9-THCwerenotchangedbythe

mixture(Fig.7).

TerpenoidsanddesensitizationinAtT20-CB1

Wehavepreviouslyreporteddesensitizationcannabinoid-mediatedcellularhyperpolarizationinAtT20

cellsexpressingratorhumanCB1receptors21,22,andwefoundthisreversalofCP55,940-induced

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hyperpolarizationwasacceleratedbynegativeallostericmodulatorssuchasORG27569andPSNCBAM-1.

Therefore,wetestedwhetherterpenoidsmayactinasimilarwaytoORG27569andothernegative

allostericmodulators,alteringdesensitizationtime-course.Weused∆9-THCinsteadofCP55,940,as∆9-

THCisthemainphytocannabinoidagonist.Prolongedapplicationof∆9-THC(10μM)produceda

hyperpolarizationthatreversedsubstantiallyover30minutes.Representativetracesforthisexperiment

areillustratedinFigure8A.Wemeasuredthepeakresponseto∆9-THCandthesignalremaining30

minutesafteragonistexposureandquantifieddesensitizationasapercentdeclineinthepeakresponse.

The∆9-THC(10μM)signaldesensitizedby63.3±6.3%,inthepresenceoftheterpenoidmixdesensitization

was60.8±4.9%(Fig.8B).Thus,terpenoidsdidnotinterferewithdesensitizationofCB1signallingproduced

by∆9-THC.Wealsoassessedthecapacityof∆9-THCalone,terpenoidsalone(10μMeach)orterpenoids

combinedwith∆9-THCtoaffectsomatostatinreceptorsignallinginAtT20-CB1cells(heterologous

desensitization).Somatostatin(100nM)wasapplied30minutesafterfirstdrugapplication(Figs.8Aand

9Aandthehyperpolarizationproducedbysomatostatinafter∆9-THC,terpenoidsaloneor∆9-THCwith

terpenoidswerenotsignificantlydifferenttosomatostatinalone(p>0.05,Fig.8B).

Discussion

TheprincipalfindingofthisstudyisthatagonistactivationofCB1andCB2receptorsisnotobviously

alteredbyanyorallofthe6majorterpenoidsfromCannabissativa.Theterpenoidstesteddidnot

activateCB1orCB2bythemselves,nordidtheymodifythesignallingofthehighefficacyagonistCP55,940

orthelowerefficacyagonistΔ9-THC.Inparticular,Δ9-THCeffectswouldbeexpectedtobeverysensitive

tothepresenceofdrugswhichinhibited(orenhanced)signallingatthereceptor.Therearenospare

receptorsforΔ9-THCinthisassay,andchangesinligandbindingwouldbedirectlyreflectedasachange

inthemaximumresponse.Thelackofeffectofterpenoidsontheresponsetothesyntheticcannabinoid

CP55,940indicatesthatterpenoidsdonotinterferewithmaximalcannabinoidreceptor-mediated

hyperpolarization,suggestingnodirectmodulationofthepotassiumchannelresponse.Thiswas

confirmedbythelackofeffectofterpenoidsontheresponsetosomatostatin.

Apreviousstudyprovidedevidencethatβ-caryophylleneisaCB2agonist23.However,wewereunableto

detectanyeffectofβ-caryophylleneonCB2signallinginthepresentstudy.Thereasonsforthisare

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unclear,buttheefficacyofβ-caryophyllenehasnotbeendefinedincellularassaysandmaybelower

thanΔ9-THC.TheCB2responsetoevenhighconcentrationsofΔ9-THCinourassayissmall,suggesting

thatproductivecouplingofCB2toendogenouspotassiumchannelsinAtT-20cellsrequireshighefficacy

agonists.Theaffinityofβ-caryophylleneforCB2(155nM)hasbeendeterminedinmembranesfrom

HEK293cellsheterologouslyexpressingCB223,butisnotknowninintactcells.ItsEC50forinhibitionof

forskolin-inducedadenylylcyclaseinCHO-K1expressingCB2wasaround2µM,23suggestingalow

functionalaffinity,whichmaynotbesufficienttosignificantlyaffecttherapidresponsetothehigher

affinityagonistΔ9-THC.

PositiveandnegativeallostericmodulatorshavebeenreportedforCB124,25,andtheeffectsofseveral

negativeallostericmodulatorshavebeendefinedinthehyperpolarizationassayusedhere.21Both

PSNCBAM-1andORG27569enhancedCP55,940signaldesensitization,whilePSNCBAM-1alsoinhibited

theinitialCP55,940hyperpolarization.Co-applicationoftheterpenoidswithΔ9-THCfailedtoaffectthe

peakresponse,orthedegreeoftachyphylaxisobservedovera30-minuteexposuretodrug,suggesting

thattheyarenotactingasallostericmodulatorsofthisCB1signallingpathway.

AlimitationofthepresentstudyisthatweonlyexaminedCB1andCB2signallingthroughonepathway,

involvingGi/o.ThehyperpolarizationoftheAtT20cellslikelyrepresentsG-proteinmediatedactivationof

inwardlyrectifyingpotassiumchannels(GIRK),aspreviouslydescribedforCB1andotherGPCRinthese

cellsaswellasinseveraldifferentneurons.26,27,28CannabinoidreceptorscoupletomultipleGproteinsas

wellassignallingthroughotherpathwayssuchasthosedependentonarrestinsandItispossiblethat

entourageeffectsofterpenoidsaremediatedthroughmodulationofasubsetofthecannabinoid

receptorsignallingrepertoire26.CB1andCB2receptorscanbeactivatedinaligandbiasedmanner–the

phenomenonwhereadrugpreferentiallyactivatesasubsetofthesignallingpathwaysthatthereceptor

canaccess.29Ingeneral,thisbiashasbeenbestdefinedforGproteincouplingversusactivationof

arrestin-mediatedsignalling,buttoourknowledgetherearenoexamplesofcannabinoidligandsonly

affectingarrestin-mediatedsignalling.20,30Itremainspossiblethatterpenoidshavesuchanabsolutebias,

butthiswouldbeunprecedented,andinanycaserecruitmentofarrestinwouldbeexpectedtoproduce

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enhanceddesensitizationoftheCB1responsestoprolongedagonistexposure21,29.Anysubtlechangeto

receptorsignallingshouldbeclearwithuseofthelowefficacyagonistΔ9-THC.

Overall,ourdatasuggestthatitisunlikelythattheterpenoidsstudiedhereaffect∆9-THCinteractions

withcannabinoidreceptors.However,thisisnotadefinitiverebuttaloftheentourageeffect.Thereare

manyotherwaysthatthesemoleculescouldinteractwithcannabinoidstoinfluencetheoverall

therapeuticandsubjectiveoutcomesofcannabisadministrationanditshouldbeacknowledgedthat∆9-

THCinfluencessignallingatawidevarietyofothernon-cannabinoidreceptortargets(seeBanisteretal31

forreview).Theseincludeinteractionwithmetabolicpathways,otherG-proteincoupledreceptors,

ligand-gatedionchannels,signallingcascadespresentonthesamecellsthatexpresscannabinoid

receptors,oronothercellsupordownstreamofthecannabinoid-receptorexpressingcells.Terpenoids

mayevenhaveprimaryeffectsondistinctfunctionalmodulesthattogetherwithcannabinoidreceptor-

modulatedpathwaysareultimatelyintegratedintoabehaviouralorphysiologicaloutput.Sothequest

forentouragedoesnotendhere;inmanywaysithasonlyjustbegun.

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12. RussoEB.TheCasefortheEntourageEffectandConventionalBreedingofClinicalCannabis:No"Strain,"NoGain.Frontiersinplantscience.2018;9:1969.13. LewisMA,RussoEB,SmithKM.PharmacologicalFoundationsofCannabisChemovars.Plantamedica.2018;84(04):225-233.14. RussoEB,MarcuJ.CannabisPharmacology:TheUsualSuspectsandaFewPromisingLeads.AdvPharmacol.2017;80:67-134.15. PotterD.Thepropagation,characterisationandoptimisationofCannabissativaL.asaphytopharmaceutical[DoctoralThesis].London:KingʼsCollege;2009.16. SuraevA,LintzerisN,StuartJ,etal.CompositionandUseofCannabisExtractsforChildhoodEpilepsyintheAustralianCommunity.Scientificreports.2018;8(1):10154.17. BanisterSD,LongworthM,KevinR,etal.PharmacologyofValinateandtert-LeucinateSyntheticCannabinoids5F-AMBICA,5F-AMB,5F-ADB,AMB-FUBINACA,MDMB-FUBINACA,MDMB-CHMICA,andTheirAnalogues.ACSChemNeurosci.2016;7(9):1241-1254.18. KnapmanA,SantiagoM,DuYP,etal.Acontinuous,fluorescence-basedassayofmu-opioidreceptoractivationinAtT-20cells.JBiomolScreen.2013;18(3):269-276.19. GuntherT,CullerM,SchulzS.ResearchResource:Real-TimeAnalysisofSomatostatinandDopamineReceptorSignalinginPituitaryCellsUsingaFluorescence-BasedMembranePotentialAssay.MolEndocrinol.2016;30(4):479-490.20. SoethoudtM,GretherU,FingerleJ,etal.CannabinoidCB2receptorligandprofilingrevealsbiasedsignallingandoff-targetactivity.NatCommun.2017;8:13958.21. CawstonEE,RedmondWJ,BreenCM,etal.Real-timecharacterizationofcannabinoidreceptor1(CB1)allostericmodulatorsrevealsnovelmechanismofaction.BrJPharmacol.2013;170(4):893-907.22. CawstonEE,ConnorM,DiMarzoV,etal.DistinctTemporalFingerprintforCyclicAdenosineMonophosphate(cAMP)SignalingofIndole-2-carboxamidesasAllostericModulatorsoftheCannabinoidReceptors.Journalofmedicinalchemistry.2015;58(15):5979-5988.23. GertschJ,LeontiM,RadunerS,etal.Beta-caryophylleneisadietarycannabinoid.ProcNatlAcadSciUSA.2008;105(26):9099-9104.24. PriceMR,BaillieGL,ThomasA,etal.AllostericmodulationofthecannabinoidCB1receptor.MolPharmacol.2005;68(5):1484-1495.25. Ignatowska-JankowskaBM,BaillieGL,KinseyS,etal.ACannabinoidCB1Receptor-PositiveAllostericModulatorReducesNeuropathicPainintheMousewithNoPsychoactiveEffects.Neuropsychopharmacology.2015;40(13):2948-2959.26. BacciA,HuguenardJR,PrinceDA.Long-lastingself-inhibitionofneocorticalinterneuronsmediatedbyendocannabinoids.Nature.2004;431(7006):312-316.27. MarinelliS,PacioniS,CannichA,etal.Self-modulationofneocorticalpyramidalneuronsbyendocannabinoids.Natureneuroscience.2009;12(12):1488-1490.28. FelderCC,JoyceKE,BrileyEM,etal.ComparisonofthepharmacologyandsignaltransductionofthehumancannabinoidCB1andCB2receptors.MolPharmacol.1995;48(3):443-450.29. IbsenMS,ConnorM,GlassM.CannabinoidCB1andCB2ReceptorSignalingandBias.CannabisCannabinoidRes.2017;2(1):48-60.30. AtwoodBK,Wager-MillerJ,HaskinsC,etal.FunctionalselectivityinCB(2)cannabinoidreceptorsignalingandregulation:implicationsforthetherapeuticpotentialofCB(2)ligands.MolPharmacol.2012;81(2):250-263.31. BanisterSD,ArnoldJC,ConnorM,etal.DarkClassicsinChemicalNeuroscience:Δ9-Tetrahydrocannabinol.ACSChemicalNeuroscience.2019.

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Fig.1.TerpenoidandsomatostatinmediatedfluorescencechangeinAtT20-WT.Representativetraces

showingchangeinfluorescencesignalafterterpenoidandsomatostatin(SST,100nM)application.Adecrease

insignalcorrespondstomembranehyperpolarization.Additionofterpenoids(A.β-pinene,β-caryophyllene

andβ-myrcene;B.α-pinene,linaloolandlimonene)didnotchangebaselinefluorescence,whilesomatostatin

mediatedaclearhyperpolarization.C.Percentagechangeoffluorescencefrombaselineaftereachterpenoid

(opencircle)andsomatostatin(closedcircles)application.Terpenoidswereaddedattwominutes;five

minutesbeforesomatostatin.Whencomparedtopositive(SST)ornegative(vehicle)controls,noneofthe

terpenoidstestedaffectedbaselinemembranepotentialorpeaksomatostatinresponse.β-Car=β-

caryophyllene.n=5,SEM,one-wayANOVAp>0.05.Drugswereaddedforthedurationofthebar.

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Fig.2.Representativetracesofβ-caryophylleneandCP55,940inAtT20-CB1and-CB2.Fluorescencewas

recordedfor10minuteswhereβ-caryophyllene(100nMand100µM)wasaddedat2minutesfollowedby

incubationfor5minutes,before1µMCP55,940application.β-caryophyllenedidnothyperpolarizeA.AtT20-

CB1andB.AtT20-CB2cells,oraffecttheresponsetoCP55,940(1µM).Drugswereaddedforthedurationof

thebar.

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Fig.3.EffectofterpenoidsatvaryingconcentrationsonAtT20-CB1membranepotentialandon1μM

CP55,940inducedhyperpolarization.Terpenoids(A.α-pinene,B.β-pinene,C.β-caryophyllene,D.β-myrcene,

E.linaloolandF.limonene)wereaddedtoAtT20-CB1cellsandincubatedfor5minutes.Maximum

fluorescencechangeswerenotdifferentfromnegativecontrol(closedcircles,n=5,SEM,one-wayANOVAp>

0.05).CP55,940(1µM)additiontoAtT20-CB1cellsinducedfluorescencechangesfrom33.1±1.7%to34.6

±0.7%.PeakCP55,940responseswerenotaffectedbythepresenceofterpenoids(opencircles,n=5,SEM,

one-wayANOVAp>0.05).V=Vehicle.

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Fig.4.Representativetracesofβ-myrceneandΔ9-THCinAtT20-CB1andAtT20-CB2.Fluorescencechange

mediatedbytwosub-maximalconcentrationsofΔ9-THC(100nMand1µM)inthepresenceofβ-myrcene

(10µM).Terpenoidwasaddedat1minutesandincubatedfor5minutesbeforeΔ9-THCapplication.CP55,940

addedaspositivecontrol.Drugswereaddedforthedurationofthebar.

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Fig.5.Effectof10μMterpenoidsonΔ9-THCinducedhyperpolarizationinAtT20-CB1.ResponsetoΔ9-THCat

twosub-maximalandonemaximalconcentration(n=6-7,SEM,unpairedt-testp>0.13).Datapresentedas%

ofmaximumCP55,940(1µM)response.

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Fig.6.Effectof10μMterpenoidsonΔ9-THCinducedhyperpolarizationinAtT20-CB2.ResponsetoΔ9-THCat

twosub-maximalandonemaximalconcentration(n=6-7,SEM,unpairedt-testp>0.26).Datapresentedas%

ofmaximumCP55,940(1µM)response.

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Fig.7.Testingthe“Entourageeffect”.Effectofcombinationofsixterpenoidsat10μMeachonΔ9-THC

inducedhyperpolarizationinA.AtT20-CB1andB.AtT20-CB2.ResponsetoΔ9-THCattwosub-maximalandone

maximalconcentration(n=5,SEM,unpairedt-testp>0.13).Datapresentedas%ofmaximumCP55,940(1µM)

response.

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Fig.8.TerpenoidsonΔ9-THCmediateddesensitizationinAtT20-CB1.A.Representativetracesof

hyperpolarizationandsignaldesensitizationmediatedbyΔ9-THCalone(10µM,black)orwithterpenoids

(10µMeach,red).Cellswerethenchallengedwithsomatostatin(100nM)after30minutestoexamine

heterologousdesensitization.B.Percentagedesensitizationafter30minutesexposuretoΔ9-THCalone(10µM)

orinthepresenceofterpenoids(10µMeach),comparedtopeakfluorescenceresponse.Terpenoidsdidnot

affectΔ9-THCmediateddesensitization(n=5,SEM,unpairedt-testp=0.76).SST=somatostatin.Drugswere

addedforthedurationofthebar.

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Fig.9.SomatostatinchallengeofAtT20-CB1cellstoinvestigateheterologousdesensitization.A.

Representativetracesofcellspreincubatedwith(black)orwithout(red)terpenoidsfor30minutesbefore

somatostatin(100nM)challenge.B.Comparisonofpeakhyperpolarization(%fluorescencechange)obtained

aftersomatostatin(100nM)challenge(n=5,one-wayANOVAp>0.05).SST=somatostatin.Drugswereadded

forthedurationofthebar.

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SupplementaryFig.S1.EffectofterpenoidsatvaryingconcentrationsonAtT20-CB2membranepotential

andon1µMCP55,940inducedhyperpolarization.Terpenoids(A.α-pinene,B.β-pinene,C.β-caryophyllene,

D.β-myrcene,E.linaloolandF.Limonene)wereaddedtoAtT20-CB2cellsandincubatedfor5minutes.

Maximumfluorescencechangesweredeterminedandcomparedtonegativecontrol(HBSS,closedcircles).No

significantfluorescencedifferencewasobservedwhencomparingmeansofterpenoidsandHBSS(n=5,SEM,

unpairedt-testp=0.72).CP55,940(1µM)additiontoAtT20-CB2cellsinducedfluorescencechangesfrom30.4

±2.4%to32.2±2.5%.PeakCP55,940responseswerenotaffectedbythepresenceofterpenoids(opencircles,

n=5,SEM,unpairedt-testp>0.09).V=Vehicle

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SupplementaryFig.S2.Effectof100nMTerpenoidsonpeakhyperpolarizationinducedbyΔ9-THCinAtT20-

CB1cells.ResponsetoΔ9-THCattwosub-maximalandonemaximalconcentration(n=5,SEM,unpairedt-test

p>0.24).Datapresentedas%ofmaximumCP55,940(1µM)response.

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SupplementaryFig.S3.Effectof100nMTerpenoidsonpeakhyperpolarizationinducedbyΔ9-THCinAtT20-

CB2cells.ResponsetoΔ9-THCattwosub-maximalandonemaximalconcentration(n=5,SEM,unpairedt-test

p>0.50).Datapresentedas%ofmaximumCP55,940(1µM)response.

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