Home » Drugs » Pharmacokineticandbehaviouralprofile
of THC,CBD,andTHC+CBD combination
after pulmonary,oral,andsubcutaneous
administrationinratsandconfirmation
of conversion in vivo of CBDtoTHC

Pharmacokineticandbehaviouralprofile
of THC,CBD,andTHC+CBD combination
after pulmonary,oral,andsubcutaneous
administrationinratsandconfirmation
of conversion in vivo of CBDtoTHC

Introduction
Given theon-goingresearchanduseofcannabisand
cannabis-derived productsformedicalpurposes,itisparti-
cularly crucialtounderstandthenatureofcannabinoid
interactions withintheorganism.Eventhoughmorethan
100 phytocannabinoidshavebeenidentified incannabis
plants (Bhattacharyya etal.,2010; Englund etal.,2016;
Hanus etal.,2016; Mechoulam etal.,2014), thetwomajor
phytocannabinoids are Δ9-tetrahydrocannabinol (THC)and
cannabidiol (CBD),bothofwhichhavebeenshowntohave
distinct therapeuticandadverseeffects(Alexander,2016;
Schubart etal.,2014; Whiting etal.,2015; Zhornitsky and
Potvin,2012). WhileTHCistheprimarypsychoactive
constituent ofcannabis,CBDisprimarilynon-psychoactive
and hasbeenshowntoattenuatethebehaviouraland
metabolic effectsofTHC(Bhattacharyya etal.,2010;
Englund etal.,2013). Inrelationtothis,recentresearch
on medicalcannabisfocusesonvariousTHC:CBDratiosin
order totargetparticulartherapeuticrequirements.Owing
to thefactthatthereisgreatcomplexityinthenatureof
THC -CBDinteractions(McPartlandetal.,2015; Reidand
Bornheim, 2001; Toddetal.,2016; Zuardi etal.,2012a), the
pharmacokinetics andbehaviouraleffectsofTHCandCBD,
and especiallytheircombination,acrossvariousformsof
administration, arenotyetfullydescribed.Furthermore,
the translationbetweenpre-clinicalandhumandatahas
frequently beenproblematicbecausetheroutesofadmin-
istration typicallyusedbyhumansforcannabinoids,i.e.,
pulmonary (e.g.,smoking,vaporisation);dermal(e.g.,
application ofointmentorlotion)andoral(e.g.,consump-
tion infood)arenotoftenemployedinanimalstudies.
In humans,pulmonaryadministrationofcannabispro-
duces thegreatestbioavailabilityofTHC,withserum
concentrations peakingwithinminutes,andsubjective
effects apparentalmostimmediately(Huestis etal.,
1992), similartointravenous(iv.)administrationofTHC
(Bhattacharyya etal.,2010; Englund etal.,2016). By
contrast, afteroraladministrationofcannabinoids/cannabis
preparations, theonsetofsubjectiveeffectsistypically
delayed by30to130minandpeakserumTHCconcentra-
tions areloweranddelayedforabout1 – 6 hoursafter
ingestion, sometimesshowingtwopeaksdueto
enterohepatic circulation.Dueto first-pass livermetabo-
lism, higherlevelsofthepsychoactivemetabolite11-
hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC)arealso
typical (forreviewsee Huestis, 2007). Comparedtopul-
monary,iv.andoraladministrationofcannabinoids,very
little isknownaboutthekineticswhenadministeredviaskin
compartment (transdermalandsubcutaneousadministra-
tion; Paudeletal.,2010; Stinchcomb etal.,2004).
Whilst THCbindstoanumberofnon-cannabinoidrecep-
tors inthebrain,theprimarymechanismofactionofTHC
responsible foritspsychoactiveeffectsismediatedvia
partial agonismatcannabinoidCB1 receptors (Campos
et al.,2012; Mechoulam etal.,2014). Itisalsothemajor
mechanism, viawhichTHCmediatesitspro-psychotic
adverse effects(Bhattacharyya etal.,2010; Englund
et al.,2013). Inrodents,CB1 receptor agonists,likeTHC,
induce whathasbeentermedthe “cannabinoid tetrad”
which ischaracterisedbyanti-nociception,catalepsy,
hypothermia andsuppressionofmotoractivity(El-Alfy
et al.,2010; Katsidoni etal.,2013). Thepsychotomi-
metic-like effectsofTHCandotherCB1 receptor agonists
in preclinicalexperimentsfocusingonsensorimotorgating
produced inconsistent findings, withsomestudiesshowing
no effects,someadisruptionofPPIandothersafacilitation
(Gomes etal.,2014; Gururajan etal.,2011; Levin etal.,
2014; Long etal.,2010a; Long etal.,2010b, 2013; Malone
and Taylor,2006; Nagai etal.,2006; Peresetal.,2016).
Unlike THC,CBDlackssignificant psychotomimetic
effects; rather,itseemstocounteractthepsychotomimetic
and behaviouraleffectsofTHC,aswellasshowinganxio-
lytic andantipsychoticpropertiesinandofitself
(Bhattacharyya etal.,2010; Englund etal.,2013; Long
et al.,2010b; Morgan etal.,2010; Pertwee,2008; Schubart
et al.,2011; Varveletal.,2006). Inanimalstudies,againin
contrast toTHC,CBDdoesnotelicittheclassicCB1-
mediated cannabinoidtetrad(inmice)anditproduces
minimal disruptionofbehaviouraltasksinhumans,monkeys
and rodents(Lichtman etal.,1995; Winsauer etal.,1999).
This islikelybecauseCBDhasalowaffinity for,andonly
weakly antagonisesCB1 and CB2 receptors (Pertwee,2008;
Thomas etal.,1998; Zuardi etal.,2012a, 2012b); more
specifically atCB1 receptors itactsasanegativeallosteric
modulator (Laprairie etal.,2015). Instead,themajor
T.Hložek etal. 1224

effects ofCBDarevianegativemodulationofendocanna-
binoid tonethroughtheinhibitionoffattyacid-binding
proteins (FABPs)whichtransportendocannabinoidsintra-
cellularly toametabolisingenzymefattyacidamidohydro-
lase (FAAH)(Elmes etal.,2015) andviaitsinhibitoryaction
at anandamidetransporters(Campos etal.,2013; Rakhshan
et al.,2000; Watanabeetal.,1996). CBDhas,however,
further complexpharmacologicalactionsinvolvingother
neurotransmitter systemsandreceptors(Campos and
Guimaraes, 2009; Campos etal.,2012; Kathmann etal.,
2006; McPartlandetal.,2015; Ryberg etal.,2007). At
sufficiently highdoses(120mg/kgintraperitoneallyin
mice), CBDmayalsoinhibithepaticmicrosomaldrug
metabolism, whichmayleadtoincreasedTHClevelsin
blood owingtodelayedhydroxylationofTHCto11-OH-THC.
In consequence,athighdoses,CBDcanpotentiate(rather
than ameliorate)theaforementionedeffectsofTHC
(Bornheim etal.,1995).
The mainaimofourstudywastoevaluatepharmacoki-
netic andbehaviouraleffectsofTHCandCBDalone,andin
combination, inratsacrossthreeroutesofadministration:
sc., pulmonaryandoral.Specifically,weaimedtocompare
24 hourpharmacokineticprofiles ofthetwonaturalcanna-
binoids andtheirco-administrationata1:1ratioinratsera
and brainsandeffectsonlocomotorbehaviourintheopen
field andsensorimotorgatinginthetestofprepulse
inhibition ofacousticstartlereaction(PPIASR).Theprofiles
of thepsychoactivemetabolite11-OH-THC,andnon-psy-
choactive metabolite11-nor-9-carboxy-THC(THC-COOH)
were alsoevaluated.Fromthebehaviouralperspectivewe
hypothesised thatTHCwouldhaveinhibitoryeffectson
locomotion, thatitwoulddisruptsensorimotorgating,and
that CBDwouldcounteracttheseeffects.Wealsoexpected
that theTHCinducedchangesmightbemorepronounced
after oraladministrationbecauseoftheexpectedpresence
of thepotentpsychoactivemetabolite11-OH-THC(Huestis,
2007).
Finally,sinceTHCandCBDarechemicallyrelatedcom-
pounds, ithasbeenreportedthatundercertain(acidic)
conditions, CBDcanbecyclisedtoTHC in vitro: apartial
cyclisation ofCBDtoTHCwasreportedwhenCBDwas
machine smokedwithtobacco,mostlikelyduetoacidic
conditions producedbytheburnedtobaccoandbythe
acidity ofsimulatedgastric fluids (Merrick etal.,2016;
Quarles etal.,1973; Watanabeetal.,2007). Morerecently,
the importantquestionhasbeenraisedastowhetherCBD
can alsobeconvertedtoTHC in vivo (Merrick etal.,2016).
Therefore ourlastaimwastoascertainwhethertreatment
with CBD(orally,orbyanyotherroute)canresultinthe
presence ofTHCinserum,andifso,whetherthisis
accompanied byTHC-mediatedbehaviouraleffects.

  1. Experimentalprocedures
    2.1. Subjects
    For alloftheexperiments,maleWistarrats(Velaz,CzechRepublic)
    weighing 200–250 gwereused.Animalswerehousedinstandard
    laboratory cagesintheanimalfacilitywithcontrolledtemperature
    (22 7 2 1C), humidity(30–70%), light/darkcycle(6a.m.lightson/
    6 p.m.lightsoff)and ad libitum access towaterandstandarddiet.
    Before testing,theanimalswereacclimatisedtotheanimalfacility
    for 7–10 days,andallexperimentswereperformedbetween8:00h
    and 13:00h,duringthelightphaseunderstandardtemperatureand
    humidity conditionsalreadydescribed.Unlessotherwisestated,
    experimentalgroups(drug  route) forthebehaviouralexperi-
    ments consistedof10animalsandeachsubjectwastestedonly
    once. Brainsandseraoftheseratswereusedalsoforkinetic
    analyses, with6animalsperexperimentalgroup(see Section 2.4).
    All oftheexperimentsadheredtotheGuidelinesoftheEuropean
    Union (86/609/EU)andthedirectivesoftheCzechNational
    Committee fortheCareandUseofLaboratoryAnimals.Ethical
    approval forthestudieswasobtainedfromtheCzechMinistryof
    Health.
    2.2. Drugpreparationandadministration
    THC andCBDwereobtainedfromTHC-PharmGmbHinapowder
    form. Forthepulmonaryadministrations,20mgofTHC,CBD,or
    their combination(THC:CBDat1:1doseratio)weredissolvedin
    98% ethanolinavolumeof200 μl anddroppedonthemetal-wired
    liquid padpurchasedwiththevaporiseranddriedfor1min.New
    liquid padswereusedforeachadministration.Cannabinoidswere
    then deliveredviaanin-houseset-upconsistingofaVolcanos
    vaporiser andahermeticallyclosedplasticinhalationbox(volume
    9.5 L).Vaporisationwasheldat226 1C for45sec;4animalswere
    kept togetherintheboxandinhaledthevaporisedairfor5min
    (including45secofvaporization).Intactanimalsplacedforthe
    same periodoftimeinthebox,withoutvaporizing,servedas
    controls. Forsc.,andoraladministration,THC,CBDandTHC+CBD
    were dissolvedinpharmaceuticalgradesunflower oil(oleum
    helanti) andadministeredatadoseof10mg/kgofeachdrugora
    mixture of10mg/kgTHC+10 mg/kgCBDinavolumeof0.5ml/kg,
    which wasthendeliveredbysc.injection,ordirectlytothe
    stomach byoralgavage.Controlanimalswereadministeredcorre-
    sponding amountsofsunflower oilasvehicle.Fororaladministra-
    tion, inordertocontrolforeffectsofstomachcontentson
    absorption, theratsweredeniedfoodfor12hpriortodrug
    administration.Thedosesusedinthecurrentstudywereselected
    accordingtoanimalandclinicalstudiesinwhich:1)THCshows
    behavioural locomotoreffectsandinducespsychotic-likesymptoms
    in animals(El-Alfy etal.,2010; Katsidoni etal.,2013; Nagai etal.,
    2006; WileyandBurston,2014); 2)dosesofCBDthatwereeffective
    in humanstotreatschizophrenia,haveshownsomeantagonising
    effects onTHCandhavealsoshownsomeantipsychotic-like
    properties inrodents(Bhattacharyyaetal.,2010; Englund etal.,
    2013; Gomes etal.,2014; Leweke etal.,2012; Pedrazzietal.,
    2015).
    2.3. Bloodandtissuecollection
    In ordertominimisethenumberofanimalsused,theanimalsfrom
    behavioural experimentsweresubsequentlyusedforthekinetic
    study.Theratswerehumanelykilledat0.5,1,2,4,8and24h(in
    batches ofsix)afteroralandsc.administration.Afterpulmonary
    administration,samplesattwoadditionaltimingswerealsocol-
    lected at0and15minafterremovalfromtheinhalationbox.All
    experimentalgroupsforkineticscomprisedsixanimalspertime
    point foreachseparatedrugxroutetreatment.Additionalanimals
    had tobeusedfortimepointsshorterthan1hour,someofwhich
    came fromthecontrolgroup.Toallowanintervalbetween
    behavioural testingandkineticsdatacollection,theseanimals
    remainedundisturbedinahomecageforatleasttwomoredays
    and subsequentlytheywereexposedtoinhalationofcannabinoids.
    Separated seraandbrainswerecollectedandkeptat 20 1C until
    the toxicologicalanalysis.
    P 1225 harmacokineticandbehaviouralprofile ofTHC,CBD,andTHC+CBD

2.4. Quantification ofTHC,11-OH-THC,THC-COOHand
CBD
Cannabinoidsweredeterminedbyanin-housevalidatedand
certified GC–MS method(certified byPolicePresidiumoftheCR,
ref. no.:PPR-31123-7/CJ-2015–990530 /evidenceno.:16/2015).A
total of10 μl ofdeuteratedCBD-d3/THC-d3/11-OH-THC-d3(5ng/
μl) internalstandardsolutionwasaddedtoeach1.0mlsampleof
serum orbrainmethanolhomogenate(5ml).Forbrainanalysis,1g
of braintissuewashomogenisedin5mlofmethanol.Homogenised
brain sampleswerefrozenat 20 1C inanethanolbathfor10min
and thencentrifugedat4200rpmfor2min.Supernatant(4ml)was
placed inaglasstubeandevaporatedto200–300 μl. Serumand
brain extractsweredilutedwitha4mlsodiumacetatebufferwith
a pHof4.0(0.01mol/l).Serumandbraincannabinoidswere
extractedwithSPEcolumns(Bond-ELUT,130mg,AgilentTechnol-
ogies) andelutedwithhexan/ethylacetate(1:4v/v)anddried
under anitrogengasstreamina400 μl glassinsertplacedina
1.5 glassvial.Thesampleswerederivatisedwith100 μl of
N-Methyl-N-(trimethylsilyl)trifluoroacetamide(MSTFA)for20min
at 80 1C. Quantification ofextractedcannabinoidswasperformed
by gaschromatography-massspectrometry(GC–MS) (GC7860/5742C
MSD, AgilentTechnologies)usingelectronimpactionizationinthe
selective ionmode(CBD:m/z391;CBD-d3:m/z394;THC:m/z386;
THC-d3: m/z389;11-OH-THC:m/z371;11-OH-THC-d3:m/z374;
THC-COOH: m/z371;THC-COOH:-d3:m/z374).Calibrationcurve
ranges werepreparedbyspikingdrug-freebovineserumordrug-
free ratbrainhomogenateforserumandbrainanalysis,respec-
tively,atconcentrationsof(i)2–100 ng/mlCBD,THC,11-OH-THC
and THC-COOH;(ii)100–1 000ng/mlCBD,THC,11-OH-THCand
THC-COOH. Limitsofdetection(LOD)andquantification (LOQ)were
1 ng/mland2ng/ml,respectively.Thespikedsampleswere
vortexed andtreatedidenticallytotheexperimentalsamples.
2.5. Behaviouraltesting
Behaviouraltestingwasperformed5minafterinhalation,60min
after sc.administrationand120minafteroraladministrationofthe
cannabinoidsandvehicleaccordingtotheestimatedmaximalpeak
of cannabinoidserumlevels(Huestis etal.,1992), theselectionof
which wassubsequentlyconfirmed byourkinetic findings. The
general behaviouraltestingprocedureswereidenticaltoour
previousstudies(Horsley etal.,2016; Paleniceketal.,2013;
Paleniceketal.,2016), briefly:
Open field test:asquareblackplasticopen field arena(68  68
 30 cm)wasplacedinasoundproofanddiffuselylitroom.
Animals wereplacedintothecentreofthearenaandthelength
and spatialcharacteristicsoftheirtrajectorywereregisteredfor
30 minandpre-processedbyanautomaticvideotrackingsystemfor
recordingbehaviouralactivity(EthoVisionColorProv.3.1.1,
Noldus, Netherlands).Locomotoractivitywasanalysedin5min
time intervals.Thespatialcharacteristicsoflocomotoractivity
were recordedin5  5 gridofvirtualzoneswith16located
peripherallyand9centrally.Frequency(f) oflinecrossingsinto
differentzonesofthearenawasusedtocalculatethigmotaxis( =
Sfperipheral zones/Sfall zones) whichindicatestheprobabilityofappear-
ance inperipheralzones.Timespentinthecentreofthearena
(Tcentre) wascalculatedasasummationofthetimespentinall
9 centralzones(Σcentre zones).
Prepulseinhibitionofacousticstartleresponse:PPIASRwas
tested inastartlechamber(SR-LAB,SanDiegoInstruments,
California,USA).Twoventilatedstartlechambers(SR-LAB,San
Diego Instruments,California,USA)werecalibratedtoensure
equivalent stabilimetersensitivitybetweenthechambers.Therats
were acclimatisedtothestartlechambertwodaysbeforetreat-
ment withashortparadigmconsistingof5minofbackgroundnoise
(75 dBwhitenoise)andasubsequentpresentationof6pulsealone
stimuli (125dB/20ms).Startledatawerenotrecordedforaccli-
matisation. Onthedayofthetest,atotalof72trialswereheld
with aninter-trialinterval(ITI)of4–20 s(meanITI:12.27s).Rats
were acclimatisedtothestartlechamberfor5min,duringwhich
time a75dBbackgroundwhitenoisewascontinuouslypresented.
Six 125dB/40msdurationpulsealonetrialswerethendeliveredto
establish thebaselineacousticstartleresponse(ASR)forthe
subsequent calculationofhabituation.Subsequently,60trialswere
presented inapseudorandomorderasfollows:(A)pulsealone:
40 ms125dB;(B)prepulse-pulse:20ms83dBor91dBprepulse,a
variable (30,60or120ms)inter-stimulusinterval(ISI:mean70ms),
then 40ms125dBpulse;(C)60msnostimulus.Finally,sixpulse
alone (40ms125dB)trialsweredelivered.Habituationwascalcu-
lated bythepercentagereductioninstartlevaluesfromtheinitial
six baselinetrialstothe final sixtrials.PPIwascalculatedas:
[100(mean prepulsepulse trials/meanpulsealonetrials) ∗
100]. MeanASRwasderivedfromthepulsealonetrials.The
average startleresponse(theareaunderthecurveinarbitrary
units, AVG)wasusedforthecalculationofthedependentvariable.
Animals withanASRAVGresponselowerthan10wereexcluded
from furtheranalysisasnon-responders.
2.6. Statisticalanalysisofbehaviouraldata
Analyses ofbehaviouraldatausedIBMSPSSversion22orMSOffice
Excel. Inallcases,thecriterionforrejectionofthenullhypothesis
for po0.05, andalltestsweretwotailed.Factorialandone-way
ANOVAs(AnalysisofVariance)wereusedtoanalysethedata,
according tothespecific experimentaldesigninuse.Wheretime
blocks wasincludedintheANOVA(locomotortrajectorydata),
pairwise comparisonsoftheoverallbehaviouralcurveswere
planned usingcontrastanalysesaccordingtothemethoddescribed
by (Abelson andPrentice,1997), otherwise(forPPI,totallocomo-
tion, thigmotaxisandTcentre), independentt-testswereplannedto
follow upsignificant maineffectsorinteractions.WhereMauchly’s
test ofsphericity(repeatedmeasuresANOVA)orwhereLevene’s
test forunequalvariances(independentt-tests)weresignificant,
adjusted statisticsarereported.ForAbelson’scontrasts,sincea
mixed designwasused,thepoolederrortermandpooleddegrees
of freedomwerecalculatedandarereported.Recalculatedand
adjusted degreesoffreedomareroundedtowholenumbersfor
presentationalpurposes.
2.6.1. Open field
An overalltotallocomotionwasanalysedbya4(drug)  3 (route)
independent ANOVAwithdrug(THC,CBD,THC+CBD orvehicle)and
route (sc.,pulmonary,oral)asindependentfactors.Locomotor
trajectory datain5mintimeblockswereanalysedusinga4  3 
6 mixedfactorialANOVAwithdrug(THC,CBD,THC+CBD orvehicle)
and route(sc.,pulmonary,oral)independentfactors,andtime
blocks (6  5 min)asarepeatedmeasuresfactor,followedby
Abelson’s contrastanalysestocompare(withineachroute)the
characteristic patternsoflocomotoreffects(acrosstimeblocks)of
the differentcannabinoidswithvehicle,andwithoneanother.
Tcentre and thigmotaxisdatawereeachanalysedusinga4 
3 factorialANOVAwithdrug(THC,CBD,THC+CBD orvehicle)and
route (sc.,pulmonary,oral)asindependentfactors.
2.6.2. Prepulseinhibition
ASR datawerescreenedfornon-responders(startleamplitude
o10); 14oralTHCandTHC+CBD ratswereexcludedonthisbasis.
As aresult,orallyadministeredgroupswereexcludedentirelyfrom
subsequent habituationandPPIanalyses,andtheoralroutewas
dropped asaleveloftheroutefactor(leavingsc.andpulmonary
administrationgroupsforanalysis).HabituationandPPIwere
therefore analysedusinga4  2 factorialANOVA,withdrug
T.Hložek etal. 1226

independentfactors.

  1. Results
    3.1. Cannabinoidlevelsinbloodandbraintissue
    In thecaseofsc.administration,theconcentrationofTHCwas
    approximatelyfourtimeshigherandCBDtwotimeslowerduring
    THC+CBD co-administrationinboththeserumandbraintissue
    compared tosinglecannabinoidadministration.Administrationby
    the sc.routealsorevealedtwopeaks,possiblyindicativeofatwo-
    compartment model.CBDsc.resultedinameasurableTHCserum
    concentrationbutonly4hand8hafterdosing(Figure 1).
    After pulmonaryadministrationofcannabinoids,theirserum
    levels peakedjustafterremovalfromtheinhalationboxwhilethe
    brain levelspeakedat15minafterdosing,andthengradually
    decreased. Thepharmacokineticprofile afterco-administrationof
    THC+CBD wasnotdifferenttotheprofiles ofCBDorTHCalone.The
    maximum braincannabinoidconcentrationswereapproximately
    three timeslowerincomparisonwithserumconcentrations
    (Figure 2).
    Followingoraladministrationeachofthecannabinoidsortheir
    combination, bothserumandbrainlevelspeakedat2hpost
    administrationandwithTHCalonethebrainconcentrations
    remained highforanother2h.Serumandbrainconcentrationsof
    CBD wereapproximatelytwotothreefoldlowerduringTHC+CBD
    co-administrationthaninthecaseofadministrationofCBDalone.
    On thecontrary,concentrationsofTHCwereapproximatelytwo
    times higherduringTHC+CBD co-administrationincomparisonwith
    THC alone.Importantly,oraladministrationofCBDagainresultedin
    measurable THCserumconcentrationsalongwithCBDconcentra-
    tions, butherewerenodetectablelevelsinthebrain(Figure 3). To
    find outwhetherthiswasrelatedtothefactthatthelevelsinthe
    brain werebelowthelimitofdetection(LOD)ofthemethod,we
    administeredanadditionaltwoanimalswith60mg/kgoforalCBD,
    and theseraandbrainswereanalysed2hafteradministration.The
    concentrationsofcannabinoidsdetectedwereasfollows:(A)forrat
    1 serumCBDwas990.9ng/mlandTHC19.1ng/ml,brainlevels
    were 1075.9ng/gforCBDand33.3ng/gforTHC,(B)forrat2serum
    CBD was723.1ng/mlandTHCbelowthelimitofdetection,brain
    levels were871.9ng/gforCBDand6.8ng/gforTHC.Theoral
    administrationalsoresultedinasignificant accumulationofcanna-
    binoids inbraintissue,whichinthecaseofTHCwasmorethan
    double thelevelsintheserum.
    The pharmacokineticprofile of11-OH-THCaftereachrouteof
    administrationreachedthehighestconcentrationsafteroraladmin-
    istration; itaccumulatedinthebrainatconcentrationsofapproxi-
    mately 200ng/g,whichiscomparabletoTHCbrainlevelsafter
    vaporisation(Figs. 1–3). Itwasnotdetectedatmeasurablelevels
    after administrationofCBDalone.
    Thenon-psychoactivemetaboliteTHC-COOHhadadelayedpeak
    between4–8 hafteradministration,remaineddetectable24hlater
    and hadverylowlevelscomparedto11-OH-THC;afterpulmonaryand
    sc.administrationitwasnotdetectedinthebraintissue(Table1).
    3.2. Behaviouraltests
    3.2.1. Open field
    Totallocomotionsummedacrossthetimeblocks(Figure 4) showed
    a significant maineffectofdrug,routeandasignificant interaction,
    minimum F(2,18) = 13.49, p = 0.001. Independent t-tests (within
    route) showedaftersc.treatment,therewasnosignificant
    differencebetweenvehicleandeachofthecannabinoids,butafter
    pulmonary treatment,thereweresignificant reductionoflocomo-
    tion aftereachofthecannabinoidscomparedtovehicle,minimum t
    (18) = 2.96, p = 0.01. SimilarlyafteroraltreatmentTHCandTHC
    +CBD significantly reducedtotallocomotion,butonthecontrary
    Figure 1 Pharmacokineticprofiles ofCBD,THCandCBD+THC inserumandbraintissueaftersubcutaneousadministration10mg/
    kg (sixratspertimepoint). n.d. = not detected.
    P 1227 harmacokineticandbehaviouralprofile ofTHC,CBD,andTHC+CBD

CBD increasedit,minimum t (18) = 3.92, p = 0.001. Comparisons
of routesofadministrationwithineachdrugtreatmentshowedthat
THC andTHC+CBD orallyhadsignificantly lowerlocomotion
compared tosc.andpulmonaryadministrationofthesecompounds,
by contrastCBDincreasedlocomotion,minimum t (18) = 4.36, p =
0.0004.
The analysisoflocomotordatain5mintimeblocks(Figure 5)
showed significant maineffectsforblocks(shownasatypical
progressive declineinlocomotoractivityover30min),fordrugand
for route,minimumF(2,108) = 13.49, p = 0.001. Allpossibletwo-
way interactionsweresignificant (drug  blocks, route  blocks,
drug  route), minimumF(10,371) = 2.66, p = 0.003. Thedrug
 route  blocks interactionwasalsosignificant: F(21,371) =
3.46, p = 0.001. Figure 6 shows characteristicstrajectories.
Subsequent contrastanalysisbetweencannabinoidsandvehicle
for thesc.routeshowedmarginallysignificant differencesbetween
the behaviouralcurvesofvehicle versus THC andCBD,F(1,256) =
3.01, p = 0.08, andF(1,256) = 3.23, p = 0.07, respectively;the
THC+CBD combinationdidnotdifferfromvehicleandnodiffer-
ences betweenthecannabinoidswhendeliveredviathesc.route
were observed.
Withinthepulmonaryroute,THCwasdifferenttovehicle,
contrast F(1,256) = 6.00, p = 0.015, butCBDandTHC+CBD did
not differfromvehicle.Therewerenosignificant differences
between CBD versus THC, orCBD versus THC+CBD, althoughthe
differencebetweenTHC versus THC+CBD wasmarginallysignificant
(contrast F(1,256) = 3.03, p = 0.08). Thiswasmanifestedas
slightly higheractivityforTHCinearlyblocks,andsomeminor
fluctuationsinrespondingfromtheTHC+CBD group,whilstrequir-
ing comment,doesnotmeritfurtherdiscussion.
When administeredorally,THCshowedsignificantly reduced
locomotor responding,whichpersistedovertimecomparedto
vehicle, contrastF(1,256) = 24.63, p = 0.001, andasimilar
pattern wasshownafterTHC+CBD, F(1,256) = 22.87, p = 0.001.
Even thoughthetotaldistancetravelledwaslongerinCBDthanin
vehicle, thecontrastsforbehaviouralcurveswerenotsignificantly
different. Locomotoractivitywasalsosignificantly reducedunder
THC andtheTHC+CBD combinationcomparedtoCBDalone,
minimum contrastF(1,256) = 15.51, p = 0.001, andTHCand
THC+CBD didnotdiffersignificantly fromoneanother.
Withindrugcontrastsofthecurvesovertimeshowedthatfor
CBD, thereweresignificant differencesbetweenroutes,whereoral
administrationinducedhigherlocomotoractivitythansc.or
pulmonary administration,minimumcontrastF(1,256) = 4.10, p
= 0.05. OralTHCandTHC+CBD significantly reducedlocomotion
compared tosc.andpulmonaryadministration,minimumcontrastF
(1, 256) = 10.23, p = 0.001, andtherewerenosignificant
differencesbetweenthepulmonaryandsc.routes.
For Tcentre (Figure 7), therewerenosignificant maineffectsof
drug orroute,maximumF(2,108) = 1.02, p = 0.37, buttherewas
Table1 Pharmacokineticprofiles ofTHC-COOHinserumandbraintissueaftersubcutaneous,pulmonaryandoral
administration ofTHC(20mgvaporisedper4rats,inhalationfor5minand10mg/kgfororalandsubcutaneous
administration, sixratspertimepoint). n.d. = not detected.
TimeofSampling
Route0min15min0.5h1h2h4h8h24h
Subcutaneous serum(ng/ml) – – 1.0 6.34.411.95.42.4
brain (ng/g) – – n.d. n.d.n.d.n.d.n.d.n.d.
Pulmonary serum(ng/ml)1.42.01.92.61.62.63.20.9
brain (ng/g)n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
Oral serum(ng/ml) – – 4.1 8.915.034.743.317.7
brain (ng/g) – – n.d. 1.55.315.712.84.6
F

significant drug  route interaction,F(6,108) = 6.28, p = 0.001
(Figure 7). Withinthesc.route,CBDaffectedthigmotaxis,reducing
the likelihoodofappearanceintheperipherycomparedtovehicle,
t (18) = 2.10, p = 0.05, butTHCandTHC+CBD didnot.After
pulmonary treatment,THCandCBDalonereducedthigmotaxis
compared tovehicle,minimum t (18) = 3.02, p = 0.007, butTHC
+CBD didnotsignificantly affectthismeasure.Withintheoral
route, THCandCBDalonedidnotsignificantly affectthigmotaxis,
however THC+CBD significantly reducedit,comparedtovehicle, t
(18) = 2.89, p = 0.010. Whencomparingcannabinoidswithone
another,therewerenodifferencesonthigmotaxisbetweenTHCor
CBD versus THC+CBD, whentreatedsc.andviapulmonaryadmin-
istration. Howeverafterpulmonaryadministration,THC+CBD
increased thigmotaxiscomparedtoCBDaloneandtoTHCalone,
minimum t (18) = 3.42, p = 0.003. Afteroraltreatment,THC+CBD
decreased thigmotaxiscomparedtoCBDandTHC, t (18) = 2.51, p
= 0.02, andtherewerenosignificant differencesbetweenTHCand
CBD.
3.2.2. Prepulseinhibition
ASR datawerescreenedpriortoanalysisfornon-responders(ASR o
10) andanumberofratswereexcludedonthisbasis,mostnotably
from thegroupsthatwereorallyadministeredTHC(beforeexclu-
sions meanASR = 20.58, SEM = 6.69; 5ratsneededexcluding)and
THC+CBD (beforeexclusionsmeanASR = 9.38 SEM = 3.13; 9rats
required excluding).Giventhe floor inresponding(whereany
further decreaseinstartlewouldnotbedetectable:(Palenicek
et al.,2013)) andthenumberofexclusions,theoralroutewas
dropped fromsubsequentfactorialanalysisofhabituationandPPI
data. Afterexcludingtheoralgroup,ASRanalysisshowedno
significant maineffectsorinteractions,maximumF(3,64) =
0.71, p = 0.55 (Table2). Likewise,habituationdatashowedno
significant maineffectsorinteractions,maximumF(3,63) = 0.32,
p = 0.81. ThereforeneitherASRnorhabituationshowedbaseline
differences thatmightconfoundinterpretationofPPIdata.
On PPItherewasasignificant maineffectofdrug,F(3,64) =
4.13, p = 0.01 (Figure 8), butnosignificant maineffectofroute,or
drug  route interaction.Independentt-testsbydrugonthemain
effect (irrespectiveofroute)showedthatneitherTHCnorTHC
+CBD weredifferentfromvehicle(THCwasmarginally,maximum t
(32) = 1.79, p = 0.08), buttherewasasignificant difference
between CBDandvehicle, t (33) = 2.30, p o 0.05. THCandCBDdid
not differfromoneanother,however,eachwassignificantly
different toTHC+CBD, minimum t (34) = 2.64, p o 0.01.
Table2 shows thedescriptivestatisticsforthenon-significant
interaction, andalsoincludesvalues(afterexclusions)forthe
orally administeredgroups.Independent t-tests wereusedto
compare useableoralPPIdata;theseshowedthatoralCBDdid
not differtooralvehicle,howeverthereweredifferencesbetween
the oralandthesc.andpulmonaryrouts,minimum t (18) = 2.09, p
= 0.05.

  1. Discussion
    The main findingsofthepharmacokineticexperimentswere:
    1) cannabinoidswerebestabsorbedafteroraladministra-
    tion,afterwhichbrainlevelswereseveraltimeshigher
    comparedtosc.andpulmonaryadministration;2)pulmonary
    administrationyieldedanalmostimmediatepeakincanna-
    binoidlevelsfollowedbytheirrapidelimination;incontrast,
    peaksappearedlateraftersc.andoraladministration,and
    cannabinoidswerestilldetectable24hourslater;3)sc.as
    wellasoral,butnotpulmonary,co-administrationofTHC
    withCBDyieldedanincreaseinserumandbrainlevelsof
    THC comparedtoTHCalone;4)thepsychoactivemetabolite
    11-OH-THCaccumulatedinthebraintissuecomparedtosera
    Figure 5 Mean trajectorylengthper5mintimeblockover30
    min aftersubcutaneous(A),pulmonary(B)andoral
    (C) administrationofvehicle(VEH),THC,CBD,THC+CBD. Error
    bars show 7 SEM. Asterisksindicatesignificant differencesfrom
    corresponding vehicle, po0.05.
    T.Hložek etal. 1230