8 PhadmaCOdynamics，ShowaUniversity，Tbkyo，Japan 9 2DepartmeI止OfPharmacology，FacultyofPharmaCy，MahidoIUniversity，Ban或ok，Thailand

(1)

1

♪卯gJ如椚g〝fげα胸作J肋〟〃劇〝〟ぁわ〝鞠〃〝〟加わ〝わ血甲州γg

2 血血如αJd鮎Ⅵ伊上わ〟q′α〝〝αふ肋J

3

4 YhkakoNakan01，MasatakaT勾imal，ErikaSugiyamal，VilasineeHirunPamichSato2，

5 HitoshiSato1

6

7 LDepartmentofPharmaCOlogy，ToxicologyandTherapeutics，DivisionofPharmaCOkineticsand 8 PhadmaCOdynamics，ShowaUniversity，Tbkyo，Japan

9 2DepartmeI止OfPharmacology，FacultyofPharmaCy，MahidoIUniversity，Ban或ok，Thailand

lO

ll Shorttitle：bhanCedabsorptionofcamabidiolbynano−eⅡmlsincadon 12

13 14 ☆CorrespondingAtLthor 15 ShowaUhiversity

16 1−5−8HatanOdai，Shinagawa−ku 17 Tbkyo142−8555，Japan

18 Tel．：＋81−3−3784−8612 19 Fax：＋8ト3−5498−1148

20 E−mail：yutanPO．ny＠cmed・Showaiu・aCjp 21

22 Keywords：Carmabidiol・CBD・Nanoemulsion・PharmaCOkinetics・Absorption

23

24

1

(2)

25 Abstract

26 BacAgTOundtCannabidiol（CBD）ishigh1ylipophilicanditsoralbioamilabilityiskn0wntObe 27 verylowinhumanS・Inthisstudy，WedevelopedanovelnanoemulsionpreparationofCBD（CBD−

28 NE）toirnprovethepoorsolubilityandabsorptionofCBD・ThepharmacokineticprofilesofCBD 29 hlratSWereeValuatedafteroraladministrationsofCBDoilandCBD−NE，andthee飴ctofbile

30 secretiononCBDabsorptionwasalsoevaluated・肋tho血TheCBDJNEformulationdeveloped 31inthis study consisted ofvitamin E acetate，ethanOl，T粥en−20，and distilled water 32（1．7／3．8／70／24．5，V／v％）．ACBDoilformlation（CBDoil，COntrOl）100mgn唱OrCBD⊥NE50 33 m釘kgwasorallyadministeredtoratsandthebloodsampleswerecollectedovertime・Moreover，

34 theCBDoilorCBD朋Ewasorallyadministeredtobile−fistulatedratsandthepharmaCOkinetic 35 profilesofCBDwerealsoevaluated・CBDconcentrationsinplasmaweremeasuredusingLC−

36 MS／MS．Res〟Lts：TheparticlesizeofCBD馴Ewas35・3士11・8nm・ThemeanTmaxofCBD−NE 37 wasshortenedsigni丘cantlyby3−fo1d（丘om8・00hto2・40h，P＜0・001）andtheAUCo−JDose

38increasedby65％（fromO．272土0．045toO・448主0・087hUk由COmParedwithCBDoil・The 39 AUC。＿JDoseandCmax／DoseafteroraladministrationofCBDoilweresignificantlyreducedby 40 27−and23−fold（P＜0．05andP＜0．01），reSPeCtively，inbile−fistulatedratscomparedwiththe 41 untreatedrats．Incontrast，allpharmacokineticparameterSafteroraladmimistrationsofCBD−NE 42 werenotsignificantlydi飴rentbetweentheuntreatedandbile−fistulatedrats・Therefore，these 43 resultsdem）nStratedthatconventionalCBDoilformulationbutnotCBD−NErequlreSbile−

44 mediatedmicelleformation．CbncL〟Sions：ThenovelNEfonmlationdevelopedinthisstudy 45 successfu11yimprovedtheabsorptionofCBDregardlessofbilesecretion・Thenewlydeveloped

46 CBD−NEoralpreparationcouldbeusefu1toachievemorestableandquickeronsetofactionby

47 CBD．

48

(3)

49 IntrodⅦedon

50 Cannabidiol（CBD）isoneofthemqjorcamabinoidconstituentsofmarihuanaObtainedfrom 51 CbnnabLssativaL．AlthoughCBDhasasimilarChemicalstructuretothatoftetrahydrocannabino1

52 （THC），ithasnopsydhotropicactivity［1−5］．CBDisknowntomodulatetheactivityofmany 53 ce11ulare飴ctorsincludingCBlandCB2receptors［2，6］，5HTIAreceptors［7］，GPR55［8］，Ll−

54 and8−OPioidreceptors［9］，TRPVIcationchannels［10］，PPARY［11］，andFAAH［10］．

55 CBD has been reported to have severale飴cts such as prolongation ofsleep，anti−

56 inflammation，anticonvulsant，anXiolytic，andreliefofneuropathicpain［1，3，12，13］．Manykinds 57 0fformulationscontainingCBDhavebeenwidebFdisbibutedforthesee飴cts［14］．Moreover，in

58 June2018，highlypurifiedCBD Epidiolex⑧ ，aCBDoilpreparation，WaSaPPrOVedbytheUS 59 FDAforthetreatmentofseiz∬eSaSSOCiatedwithLennox−GastautsyndromeorDravetsymdrome 60 ［15，16】．

61 CBDishighlylipophilic，anditisusua11ysuppliedasanOilpreparation．Sincetheoral

62 bioavaihbilityofCBDiskn0wntObeapproximately6％inlmmanS［1】，CBDiscommonly

63 administeredviathesdblingualroute．CBDismetabolizedafteradministrationand severa1

64 previousstudieshaveidenti丘edCYP3A4andCYP2C19asthem句Orisoformsmediatingthe 65 metabolismofCBD［17，18］．Therefore，theoralbioavailabilityofCBDisa飴ctedbyboththe 66 poorsolubility，i．e．，lowal）SOrption，andthelarge負rst−paSSe飴ct．

67 Selfrmicro or selflnanO emulsifying drug delivery systems（SMEDDS or SNEDDS，

68 respectively）canPrOducenanOemulsionPE）andimprovetheoralbioavailabilityofhigh1y 69 1ipophilic compounds［19−21】．NEformulation comprises temary phases；Oil，Water，and 70 surfactant．NEisathermodynamica11ystablesystemusedasavehicletodeliverhighlylipophilic

71 dmgs［22，23］．

72 Inthisstudy，WedevelopedanovelNEformulationcontainingCBD（CBD−NE）toimprovethe

3

1 芯1

(4)

73 intestinalabsorptionofCBDandevaluateditspharmaCOkineticpro丘1esinrats．Moreover，We 74 evaluatedthee飴ctofbile secretionontheintestinalabsorptionofCBD丘omoilandNE 75 fommlationsusingbile−Lstulatedrats．

76 77 Matedal＄andmet血ods 78

肋ねrねね

79 CBD powder（purity，99％）was provided bythe ENDOCA（Hoofddorp，Netherlands）．

80 ClobetasoIpropionateastheintemalstandard（IS）andaceticacidwereobtainedfromSigma−

81 Aldrich（St．Lo扇s，MO，USA）．Polyoxyethylenesorbitanmonolaurate（Tween−20）waspurchased 82 formMPBiomedicals，Inc．（OH，USA）．Acetomitri1ewasobtained丘omHoneywellInternationa1 83 Inc．（Seelze，Gemany）．Otherreagentssuchasammoniumformate，01iveoil，（⇒−α−tOCOPhero1 84 acetate（vitaminEacetate），andsodiumaCetateWerePurChasedfromFUJWILMⅥねkoPure 85 ChemicalCorporation（Osaka，Japan）．

86 87 乃甲αrα∫わ乃げC毘Dr〃百

88 EmulsionforrmlationsaregenerallycomposedofternaryPhases：Oil，Water，andsurfactant．

89 WeusedvitaminEacetate，TⅣeen−20，andethanolastheoilphase，Sur払ctant，andco−Surfactant，

90 respectively．hapreliminarystudy，WeOPtimisedtheratiosofvitaminEacetate，Tween−20，and 91 water．WhenthepreparedmiⅩturebecametranSparentandclear，WedeteminedthattheCBD−

92 NEhadbeensuccessfu11yformulated．TheparticlesizeofCBD−NEwasalsomeasuredusinga 93 lasernanoparticleanalyser（SALD−7100，Shimadzu，Kyoto，Japan）．

94 TheCBD−NEanalysedinthesubsequentpharmaCOkineticstudieswasfinallypreparedas

95 fo1lows．CBDpowder（30mg）wasdissoIvedwithvitaminEacetateandgentlystirredusinga

96 magneticstirrerinethan01forlOmin．Then，themixturewasstirredafteraddingTween−20for

(5)

97 10minandagitatedatroomtemperature（23土5OC）forlOmin，fo1lowedbytheadditionof 98 disti11edwateIlhgredientsandeachphasevolume（V／v％）forpreparingtheCBD−NEformulation

99 aresumisedThblel（totalvolumewaslmLforCBD30mg）．TheCBD−NEformulations lOO preparedwerekeptat40Canddiluted3−foldwithdistilledwaterjustbeforeadministrationtothe

lOl rats．

102 A扉〝は血

MaleWistarrats（SankyoLaboServiceCorporation，Tbkyo，Japan）weighingapproximately 105 240gwereallowedfoodandwaterunderstandardconditions（temperature−COntrO11edfacility）

106 0na12hlight／d訂kcycle．Allanimdstudieswerecarriedoutaccordingtotheguidelinesfor lO7 animalexperimentationofShowatjhiversity．

108 ．1〃か〃J／丘v）ぐ′J川棚J・ゞ

Theratswerefastedovemight（approximately12h）．UnderisofluraneinhalationanaeSthesia，

111 theleftftmoralarteryofeachratwascannulatedwithsilicontubing（SP−31，NatsumeSeisakusho

l12 Co．，Ltd．，Tbkyo，Japan）tofacilitatebloodsamPling．Thecommonbileductwasalsocannulated

l13 usingsiliconthbing（SP−10，Natsume、SeisaknshoCo・，Ltd・）topreparethebile−fistulatedmodel l14 asdescribedpreviously［24］．Aftertheoperations，eaChratwaskeptinaBolmanCageandinvitv l15 e叩erimentswerecamiedoutafterrecoveryfromanaesthesia・

116 d鮎0甲まわ刀瑚J由q／CaDノ払椚OfJα〝d〃百凡r椚〟加わ〝ざ

CBDoil（01iveoilsolution，100mgn（g／2．5mL）orCBD−NE（50mgn g／5mL）wasorally 119 admimisteredtorats，tOCOmParetheabsorptionpronlesofCBDoilandCBDJNE・Approximately 120 200llLbloodsampleswerecollectedfromthefemoralarteryintoheparimizedtubesatO・5，1，2，

5

(6)

121 4，8，12，and24h（CBDoil）orO．25，0．5，l，2，4，8，10，and24h（CBDJNE）afteradmimitration・

122 Pl訟皿aSamPleswereobtainedbycentrifugingtheblood（3，000g，15min）at40Candk甲tat−

123 230Cuntiltheanalysis．

124 砂蕗げ戯ね0〃d鮎0甲′わ〝げCaD

CBDoil（01iveoilsohtion，50mg／kg／2．5mL）orCBDJNE（50mg／kg／5mL）wasora11y 127 administeredtobile一航stulatedrats，tOeXaminethee飴ctsofbilesecretionontheintestinal 128 absorptionofCBD・Approximately200pLbloodsampleswereco11ectedO・5，1，2，4，8，andlOh 129 a鮎radministration・Bilesampleswerealsocollectedinplastictubes丘omOto24haftertheoral 130 administration．Plasmasampleswereobtainedbycentrifugation（3，000g，15min）ofbloodat40C

131 andkeptat−230Cuntiltheanalysis・

132 ．㌦J〃p／ビハ叩（JⅢJわ〝、ルr眈 …JⅣJ肝JJJ

TheIS solution（ClobetasoIpropionatelO nghnLin acetonitri1e）was dispensedinto 135 microtubesandevaporatedunderanitrogenstreamat50T700C，andthen50pLoftheplasma

136 samplewas addedtoeachmicrotube・SolidiPhaseextractionwasperformedasdescribed 137 previously［25］withsomemodifications・BondElutPlexacartridges（60mg，3mL，Agi1ent 138 Tbclm0logies，CA，USA）weresetonvacuum，OnWhich2miJeaChofmethanolandwaterwas 139 sequentiallyadded（COnditioning）．Then，50pLofthebloodsamplewasappliedtothecartridge，

140 whichwassubsequentlywashedwith2mLwashingsolution（Water：aCetOnitrile：1％glacialacetic

141 acidinacetonitrile，79：20：1，V／v％）．Aftervacmingfor50s，0．75mLl％glacialaceticacidin

142 acetonitrilewasaddedtwice，theextractwascollectedintoasampletube，eVaPOratedundera

143 nitrogenstream，andlefttostandatroomtemperatureovemight・Theresiduewasreconstituted

144 in200pLacetmitorile：1mMammoniumformate（80：20，ⅥV％），VOrteXed，andcentrifugedat

(7)

145 10，000gforlOminat40C・S止bsequently，thesupematantwas航1teredusingasymge丘Iterunit

146（Ekicrodisc⑧3CR，diameter3mm，POreSizeO・45pm，NihonPallManufacturingLtd・，Ibaraki，

147 Japan）．Theextractsolutionwasstoredandkeptat130CⅦ血theanalysis・

148 149 e〟α乃J豆粒α血〝げC及Dw〜gゐエCA棚

150 TheconcentrationsofCBDinplasmaweremeasuredusingLC−MS／MSasdescribed 151 previously［26−29］withsomemodi丘cations・TheMSsystem（QTRAP5500，ABSicex，

152 Framingham，MA，USA）wasusedintheelectrosprayionisationmode・Thescantypewasinthe 153 multiplereactionmonitoringmode（MRM）runinthepositivemodefortheIS（Cldbetasol）atO−

154 9min，andinthenegativemodeforCBD・

155 Areversed十phasecohmn，XBridge⑳c18（5p叫2・1×50mm，Waters，MA，USA）witha

156 guardcamidgeXBridge⑳c18（5pm，2・lxlOmm，Waters，MA，USA），WaSusedfortheLC・

157 ThemobilephasesAandB（1mManmoniumformateinwaterandacetonitri1e，reSPeCtively）

158 wereusedinagradientmodeforthechromatographicelution・Then，Eachsarnpleswere 159i両ectedlOLLLandmeasuredatatotalflowrateofO・3miJminusingthefo1lowinggradient：the 160 ratioofmobilephaseB（B−COnC）wasinitia11ysetat25％，afterthemeasurementcormncedit 161increasedto50％over2min，andthenitwasheldfor6min・TheB−COnCWaSincreasedagainto 162 65％overlminandheldfor5min・Finally，B−COnCWaSincreasedtolOO％overlminandheld 163 for5min，thenitwasrettmedto25％over2minandheldfor8min・Theautosamplerandthe 164 colurrmovenweremaintainedat14OCand40OC，reSPeCtively．

165 TheMS／MSparameterSettingsareshowninThbles2（A）and（B）・Theretentiontimesofthe

166 1SandCBDwere5．71andll．77min，reSPeCtively・Dataacquisitionandanalysiswereperformed

167 uslngaSOftwareimplementedintheLC−MS／MSsystem・

168

7

(8)

169 αJ仏和gわ〝C〟Ⅳ甜♪rCβ∂臆∬〟柁椚e〝J

170 CalibrationcuⅣeSforCBDplasmaconcentrationswereobtainedattherangeSOflO−500and 171200T2000ng／mL．StandardsolutionsofCBD（10andlOOng／mLinacetomitri1e）wereusedfor 172 calibration・Anq）PrOPriateamountoftheCBDstandardsolutionwasdispensedintoeadh 173 micro血be，andlOOpLISsolution（10nghnLclobetasoIpropionateinacetomitri1e）wasaddedto 174 thetube．A鮎revaporationunderanitrogenstreamat50−700C，100pLoftheblankplasmawas 175 addedandvortexed．Aportion（50pL）ofthepreparedplasmasamplewasextractedusingthe 176 samemethoddescribedabove．Moreover，WeeValuatedtheintra−dayandinterdayvariationsat 177 CBDconce血ationsoflOandlOOng／mLinplasma・

178 179 上場α′澗αCO彪乃efわα乃d肋おおcαJA乃α桓甜

180 Areasundertheconcentrationversustimecurve（AUC）fromzerotoaparticulartime（AUCo−

181．），AUCfromzerotoinfinity（AUCo棚），teminalhalfllifb（Tl佗），tOtalclearance（CLtoJF），VOlume 182 0fdistribution（VdsJF），themaximmbloodconcentration（Cmax），timetoreachCmax（Tmax）and 183 meanreSidencetime（MRT）werecalculatedusingtheMicrosoftExcelso氏wareprogram［30］

184 usmgamomentanalysis・Theobtainedphamacokineticparameterswereexpressedasthemean 185 j＝Standarderrorofthemean（SEM）．Tbcomparethetwofomulationsortwogroupsoftreatments，

186 parameterValueswithequalvarianceweretestedusingtheStudent，stLteStandtheothersuslng

187 theWelchtest．Di飴renceswereconsideredstatistica11ysigni丘cantatP＜0・05・

(9)

188 Results 189

190 e〟α〝J節cαfわ乃q／C以h扇喝■エC朗

191 LinearcalibrationcurveSWereObtainedatrangesoflO−500and200一つ000ng／mLofCBD，and 192 theircorrelationcoe伍cients（R2）were＞0．985andO．998，re甲eCtively．Thelowerlimitof 193 detectionwas10ng／mL．htheintra−dayanalysis，PreCisionandaccuraCyWere15．8％and3．82％

194 atlOng／mLand3．58％and3．62％atlOOnghnL，reSPeCtively．Intheinter−dayanalysis，PreCision

195 andaccuracywere8．20％and16．2％at10ng／mLand2．88％andl．83％atlOO nghmi，，

196 respectively．TheseresultssatisfiedtheacceptanCeCriteria（＜20％and15％forthelowerand 197 higherconcentrations，reSPeCtively）．

198 199 CみααCねrねgfαげCaD−〃E

200 ThetemaryphasediagramoftheCBDJNEsystemcontainingTⅣeen−20，vitamhEacetate，

201 anddistilledwaterisshowninFig．1．Intheshadedareaofthediagram，theappearanCeOfthe 202 mixturesbecameclearandtransparent，indicatingtheformationofamonophasicliquid．Themean 203 土SDparticlesizeofthesetranSParentforrmlationsimmediatelyafterthepreparation，Onthe 204 fonowingday，andlweeka銑er，Weredeteminedtobe35．3土Il．8nm（n＝12），43．8士22．Onm 205 （n＝7），and44．5土14．Onm（n＝4），reSPeCtively．Oneweeklater，theliquidsti11appearedclear 206 and仕組Spa托nt．

207 ThemeanTmaxofCBD−NEwasshortenedby3−foldsigni丘cantly（from8．00hto2．40h）（P 208 ＜0．001）andtheAUCoJDoseincreasedbyl．65−fold（fromO．272j＝0．045toO．448±0．087h 209 L／kg）ascomparedwithCBDoil．

210 d∂∫0甲′わ〝P′℃βJ甜」毎∽C毘DOgJα〝♂CaD−〃E

9

(10)

211 Figure2showstheabsorptionprofi1esofCBDoil（100mg／kg）andCBDJNE（50mgkg）a

212 0raladministrationstorats．Thble3presentsthepharmacokineticparameterSOfCBDoiland 213 CBDヰ寸E．The Tmaxwith CBD−NEindicated a statistically signi丘cant（P＜0・001）3・3−fold

214 decreasefrom8．00hto2．40hascomparedwithCBDoil（COntrOl）．TheAUCoJDoseandAUCo−

215 24rDoseincreasedby65％（丘omO272土0．0445toO．448土0．0866hL／短）andby28％（from 216 0．253j＝0．0440toO．324士0．532hL此g），reSPeCtively，withtheself・emulsifyingNEformulation 217 0fCBDcomparedwiththeCBDoil．Therefore，therelativebioaNailabilityoftheCBD−NE 218 formulationcomparedwiththatoftheCBDoilwascalculatedtobel．65．Ontheotherhand，the 219 valueoftheAUC／DosedbtainedafteranintravenousbolusadministrationofCBDwasrqported

220 tobeO．345kgnl［31］，Usingthisliteraturedataandtheequation（AUCp。／Dosep。）／（AUCi，／Doseiv），

221 theabsol血ebioavailabilityofCBDoilandCBDJNEwasestimatedtobe73．3％and93・9％，

222 respectively．

223 224 聯cねq／朗わ0乃d∂∫0甲gわ乃・オC昆D

225 Figure3showstheabsorptionpro丘1esofCBDfromoilandNEforrmlationsinthebile−

226 fistulatedrats．ThepharmaCOkineticparametersofCBDoilandCBD−NEarepresentedinThble 227 4．IntheCBD−NEgroup，theAUCo4WaSSignificantlyhigherU｝＜0．001）thanthatintheCBD

228 0ilgroupwas，WhereastheVdss仔wassigni丘cantlylowerげ＜0．001）．Ontheotherhand，the 229 CLt。JF，Tl／2，Cmax，MRT，andAUCo＿10WerenOtSignificantlydi飴rentbetweentheCBDoiland 230 CBD−NEgroups．ThemeanAUCo−のandAUCo−100fCBD−NEweremarkedlyhigherby21・5−fold 231 and19．7−fold，reSPeCtivelythanthoseoftheCBDoilwere．TheTmaxvalueswerecompletelythe 232 sameamongallrats（4h）．

233 In the CBD oilgroup，AUCo−JDoseandAUCo−24／Dose ofthe bile−nStulatedrats were

234 signi負cantlydecreasedbyO．058−andO．020−foldcomparedwiththoseoftheuntreatedrats（P＜

(11)

235 0．05，Thbles3and4）．TheCmaxofthebile−nStulatedratswasalsosigni丘cantlydecreasedO．022−

236 fold（P＜0．01）．Incontrast，a11pharmaCOkineticparametersafteroraladministrationsofCBDJNE 237 werenotsignifica血1ydi脆rentbetweentheuntreatedandbile一員stulatedrats・

238 239 Ⅰ）iscⅦSSion

240 241 Tbimprove the absorption andbioavailabilityofCBD，We developedanew CBD−NE 242 formulation and examined the difftrencesinthe absorptionpromes betweenthe CBD−NE

243 preparationandconventionalCBDoilsolution．FortheCBD−NEpreparation，WeuSedtheshaded

244 areaoftheternaryPhasediagramsystem（Fig．1），Whchissimi1artothatusedforcyclosporine 245 Amicroemulsion（ME）［20］．TheappearanCeandparticlediameterofCBD−NEwerethesame

246 duringstorageat40Cforatleast6months．TheappearanCeOftheCBD−NEpreparationdeveloped 247 inthisstudywastransparentanditsparticlediameterwassmallenoughtobeevaluatedasanNE

248 （＜45nmasthemeanvalue）．TheNEformulationsolubilizedCBDandremainedtransparent 249 evenafterextensivedilution（e．g．morethan100timesdilution）withwater，indicatingthatthis 250 wasaself−emulsifyingsystem．Tbthebestofourknowledge，thisisthe丘rststudytodevelopa 251 self−errmlsifyingNEforCBDtodate・

252 Inthisstudy，WeuSedfourlngredients，Water，ethanOl，Tween−20，andvitaminEacetateto 253 emulsifyCBDforCBD−NE．EthanoIwasusedasaco−SOlventtodissoIveCBDpowderinthe 2541ipidphase，WhichwasalsousedforNeoral⑧【32］，thecommerciallyavailableMEformulation

255 0fcyclosporineA．Tween−20，Whichwasusedasasurfactantisgenerallyknowntobeanon−tOXic

256 andnon−irritantmaterial［33，34］．However，theJointFAO／WHOExpertComitteeonFood 257 Additives（JECFA）recommendedtheacceptabledailyintake（ADI）ofTween−20withoutany 258 sidee脆ctstobe25mgn（gbodyweight．SincetheefEbctiveCBDdoseusedtotreatchronicpain

ll

(12)

259 inht山鳩皿WaSlOO抑釘bodyweight，［35］，thedoseofTwe孤−20containedintheprepared 260 CBDJNE（70％ratioforTween−20）canbecalculatedtobe20・2mgWdayfora70kghuman，

261 血ichislessthantheabove−mentionedADIvalue（25mgTⅣeen−20此g），aSSumingthatthe 262 bioavai1abilityofCBDwasincreasedbytheNEformulationby65％・

263 TbevolumeoforaladmitrationforCBD−NEwaschosentobe5mL此g，Sincethisisthe 264 optimalvolume［36］．Ontheotherhand，anOraladministrationofCBDoilat5mg耽ginduced 2（i5 diarrheilinrn。St。ftllもratstls占d：thcrcfbre．thevoIumcふasrauccdto2．5mLkglbrthcCBD■oi1

266 鮎mml如iom

267 hthisstudy，theAUCoaandCEnaXfo1lowingoraladministrationoftheCBDoilweresimilar

268 totheseofpublishedinpreviousstudies［27，28，31，37］・Thepresentresultsdemonstratedthatthe

269 novelCBD朋EfomdationimpfOVedthe bioavailabuityof CBD（AUGJDose）by

270 approximately65％，a他oughthischangewasnotsignificant・Incontrast，theTmxwithCBDヰ肥 271 wassigmi航cantlylowerthanthatwithCBDoilwas（2・4hvs・8h，P＜0・001），aSShowninFig・2 272 andThble3，indicdinganeXtenSively（33−fold）enhancedintestinalabsorptionofCBDbythe 273 NEfomlation．Across−SeCtionalstudyofcannabidioluserS［38］reportedthatmorethan60％

274 血endedtotreatpain，followedbyarthritispain，anXiety，anddq）reSSion・Therefore，therapid 275 absorptionofCBDfromNEformulationswouldbepreferableforthemqorpurposesofCBD

276 intake，eSPeCiallypaln−reliefanddqpression・

277 血止estinalabsorptionofahighlylipophiricdrugisgenera11ya脆ctedbybileproductionand

278 high−fatmeals［39］．Infact，theCmaxandAUCofEpidiolex⑧withahigh−fa仙ighーCal0riemeal 279 wererqportedtoincrease5−and4−fold，reSPeCtively，COmParedtonormalmeals［40］・This

280 suggeststhataRerplacingtheoilformulationofFpidiolex㊨insidethecheek，itislargely

281 transferredintotheintestineandabsofbedviathebile−mediatedmicelleformationwithlipids

282 ￠eleased丘omfatbylipase），butnotviaoralmucosalabsorption・

(13)

283 hthisstudy，anenOrmOuSreductioninCBDal）SOq）tion鉦omtheoilpreparationwasobserved 284 inbile一丘stulatedrats，indicatingthattheabsorptionfromtheCBDoildependedonthebile−

285 mediatedmicelleformation（i．e．solubilization），difhsionofthedrug−COntainingmicellesbeyond 286 theunstirredwaterlayerNWL）attheproximityofintestinalepithelialcells，andsubsequent 287 absorption（i．e．，tranSlocation）ofCBDmoleculesthroughtheepithelialcellmembraneS・This 288 notionisthebasisofthelipid−basedformulationsofwater−insolubledrugs［4l，42］．hcontrast，

289 ther6werenosignificantchangesinthepharmacokineticparametersbetweenthebile一丘stulated 290 anduntreatedrats admhisteredthe CBD−NEformulation，SuggeStingthatCBDJNErapidly 291 diffusedintheintestinal1iquidsandtranSVerSedtheUWL，SOthatthelipophilicCBDmolecules

292 couldbee瓜cientlyabsofbedintotheintestinalepithelialcellswithouttheneedforbile−mediated

293 micelleformation．Tlms，itisreasonablythattheT．mxfollowlngOraladmimistrationofCBD−NE 294 wasmuchlowerthatoftheCBD oil，Whichwasprobal）1yduetobypasslngOfthemicelle 295 formationprocessintheintestinebyformulatingCBDintoanNE．

296 ThisstudyestimatedthattheabsolutebioavailabilityofCBDoilinratsisapproximately70％，

297 whichismuchhigherthanthatinlmanS（approximately6％）．Severalstudiesshowedthatthe 298 metabolismofCBDinratswasdi飴rent丘omthatinlmmanS［37，43，44］，SuggeStingthatthe

299 human−ratdi飴renceintheabsolutebioavailability（i．e．verylowinhumanbutmoderateinrats）

300 maybeduetothelower血st−PaSSmetabolismofCBDinrats．Consideringafutureclimicalstudy 301 wherethepresentCBDJNEisora11yadmimisteredanditspharmaCOkineticprofileiscompared

302 witha conventionaloilsolutionin humanS，We COuld expect theincrement ofthe CBD

303 bioavailabilitybynano−ermlsi丘cationtobehigherthanthat（65％）observedinthepresentstudy．

304 ThisisbecausethebasalbioavailabilityofCBDinanOilformulationinhumanissolow（dueto

305 poorabsorptionandextensivefirst−PaSSmetabolism）thatthebioavailal）ility−enhancinge飴ctof 306 theNEcouldbemoregreatlypronouncedthanthatinrats．

1：）

(14)

307 Sincebilesecretionwouldbea飴ctedbyvarious払ctorsincludingfoodintake，biliarytract 308 infbction，andliverfunction，theanticipatede飴ctsofCBDmaybemorestableandconsistent 309 withtheNEforrmlationsthanCOnVentionaloilformulations．hanycase，thenovelCBDJNE 310 formulation，WhichdemonstratedamuchhigherabsorptionprofileofCBDinthisstudy，Should 311 betestedinclimicaltrialsanditshmanbioavailabilityneedstobeclarifiedinfuture・

312 313 COnClusio叫

314 Thisstudyisthe血sttodevelppaCBD−NEformulation，Whichextensivelyenhancedthe 315 absorptionofCBDandimproveditsbioavailabilitytosomeextent・Wedemonstratedthatthe 316 CBDabsorption丘omtheformlationwasnota飴ctedbybilesecretion，Whereasthatfromthe 317 conventionaloilformulationwasgrea瓜yreducedbybile丘stulation，SuggeStingamorerdbust 318 absorptionofCBDfromNEwithoutfoode飴cts・Consideringthesaf如yoftheTween−20used 319 asasur払ctantinthisstudy，thenewNEpreparationofCBDmightbeusefulforclinicalusein

320 丘ItWe．

321 322 DiselosⅦreStatement

323 TheauthorsdeclarenOCOnflictsofinterestassociatedwiththisrmnuscript．

324 325 Acknowledgement

326 TheauthorswishtothankENDOCAforkindlysupplyingthepureCBDcrystalsfbrour

327 researcb．

328 329 Refbrences

330 1WorldHealthOrganizationExpertComitteeonDrugDependence：Cannal）idiol（CBD）Pre−

(15)

331 ReviewReportAgendaItem5．2andPeerReview，2017・

332 ∠ McPartlandJM，Duncan M，DiMarzo V Pertwee RG：Are cannabidioland A（9）−

333 tetrahydrocannabivarinnegativemodulatorsoftheendocannabinoidsystem？Asystematic 334 review．BrJPharmaCO12015；172（3）：737−53．

335 3 RongC，LeeY，CarmonaNE，ChaDS，RagguettM，RosenblatJD，etal：Cannabidiolin 336 medicalmart）uana：Research vistas and potentialopportunities・PharmaCOIRes

・337・・2017；121：213−8．

^−▲

338 4IbeasBihC，ChenT，NunnAⅥBazelotM，Da11asM，WhalleyBJ：Moleculartargetsof 339 cannabidiolinneurologicaldisorders．Netm）therapeutics2015；12（4）：699−730・

340 5IfnandK，GrotenhermenF：Anupdateonsafetyandsidee鮎ctsofcannabidiol：areViewof 341 clinicaldataandrelevantanimalstudies．CannabisCannabinoidRes2017；2（1）：139r54．

342 6 HayakawaK，MishimaK，HazekawaM，SanOK，IrieK，0ritoK，etal：Cannabidio1 343 potentiatespharmaCOlogicale飴ctsofDelta（9）−tetrahydrocannabinoIviaCB（1）receptor−

344 dependentmechanism・BrainRes2008；1188：157−64・

345 7 RussoEB，BurnettA，HallB，ParkerKK：Agonisticpropertiesofcannabidiolat5−HTla 346 receptors・NeurochemRes2005；30‥1037−43・

347 8 RybergE，LarssonN，句6grenS，鴫orthS，HermanSSOnNO，LeonovaJ，etal：Theorphan 348 receptorGPR55isanovelcannabinoidreceptor・BrJPharmaCO12007；152：1092LlOl・

349 9 KathmannM，FlauK，RedmerA，Trankle C，SchlickerE：Cannabidiolis anallosteric 350 modulator at mu−and delta−OPioid receptors・Naunyn SclmiedebergsArch Pharmaco1

351 2006；372：354Jl．

352 10BisognoT，HanusL，DePetroce11isL，TchilibonS，PondeDE，BrandiI，etal・Molecular 353 targetsforcannabidiolanditssyntheticanalogues：e飴ctonvamilloidVRlreceptorsandon 354 thecellularuptakeandenzymatichydrolysisofanandamide・BrJPharmaCO12001；134：845−

1F

(16)

355 52．

356 11CamposAC，MoreiraFA，GomesFV；DelBelEA，Guimar互esFS：Multiplemechanisms 357 invoIvedinthelarge−SPeCtrumtherapeuticpotentialofcannabidiolinpsychiatricdisorders・

358 PhilosTransRSocLondBBioISci2012；367：3364−78．

359 12PisantiS，Mal丘tanoAM，CiagliaE，LambertiA，RanieriR，CuomoG，etal：Cannabidiol‥

360 State of the art and new cha11enges for therapeutic applications．PharmaCOITher 361 2017；175：133−150．

362 13BruniN，DellaPepaC，Oliar0−Bosso S，PessioneE，GastaldiD，Dosio F：Cannabinoid 363 deliverysystemsforpainandinfladiontreatment．Molecules2018；27：23（10）・

364 14PavlovicR，NennaG，CalviL，PanSeriS，BorgonovoG，GiupponiL，etal：QualityTraitsof 365 ‖Cannabidio10ils‖：Cannabinoids content，terPene航ngerprintandoxidationstabilityof

366 Europeancommercia11yavailablepreparations．Molecules2018；23（5）・

367 15 ThieleEA，MarshED，FrenchJA，MazurkiewiczLBeldzinskaM，BenbadisSR，JoshiC，etal：

368 Cannabidiolin patients with seizures associated with Lennox−Gastaut syndrome

369 （GWPCARE4）：a randomised，double−blind，Placebo−COntrOlled phase3 trial・LanCet 370 2018；391（10125）：1085−96．

37116DevinskyO，CrossJH，LauxL，MarShE，Mi11erI，NabboutR，etal：Trialofcannabidiolfor 372 drug−reSistantseizuresinthedravetsyndrome．NEnglJMed2017；377（7）：699−700・

373 17JiangR，YamaoriS，ThkedaS，YamamotoI，WatanabeK：Identi丘cationofcytochromeP450

374 enzymesresponsibleformetabolismofcannabidiolbyhumanlivermicrosomes・LifeSci

375 2011；89（5−6）：165−70．

376 18Cannabidiol（CBD）Pre−ReviewReportAgendaItem5．2WorldHealthOrganization2017

377 19YenCC，ChenYC，WuMT，WangCC，WuYT：Nanoemulsionasastrategyforimprovlng

378 the oralbioavailability and anti−inflarrmatory activity of andrographolide．IntJ

(17)

379 NanOmedicine2018；13：669−80．

380 20HirunPanichV；SatoH：lmprovementofcyclosporineAbioavailabilitybyincorporatingethy1 381 docosahexaenoatein themicroemulsion as an oilexcIPlent．EurJPharmBiopharm

382 2009；73（2）：247−52．

383 21GuptaS，KesarlaR，OmriA：Formulationstrategiestoimprovethebioavailabilityofpoorly 384 absotbed drugS With specialemphasis on self−emulsifying systems・ISRN Pharm 385 2013；2013：848043．

386 22SnezanaA，DavidWM，RaginiS：HybridneuralnetworksastooIsforpredictingthephase 387 behaviorofco1loidalsystems．ColloidsandSurfacesA：PhysicEngAsp2012；415：59−67

388 23TbshiyukiS：BasicsonemulsionteclmologleSWerereViewedfromthefo1lowlngVleWPOints・

389 JSocCosmetChemJpn2010；44（2）：103−17・

390 24T8nSbergH，HolmR，均erregaardTG，BollJB，JacobsenJ，MnllertzA：Anupdatedand 391 simpli丘edmethodforbileductcannulationofrats・LabAnim2010；44（4）：373−j・

392 25Prego−MeleiroIILendoiroE，ConcheiroM，CruZA，L6pez−RivadullaM，deCastroA：

393 Developmentandvalidationofaliquidchromatogr叩hytandemmassspectrometrymethod

394 for the detemination ofcannabinoidsandphaseIandIImetabolitesin meconium・J 395 ChromatogrA2017；1497：118，−26・

396 26NamYS，KwonIK，Lee KB：Monitoring ofclobetasoIproplOnate and betamethasone 397 diprop10nateaSundeclaredsteroidsincosmeticproductsmanufacturedinKorea・Forensic 398 SciInt2011；210（1−3）：144−8．

399 27DeianaS，WatanabeA，ⅥlmaSakiY，AmadaN，ArthurM，FlemingS，etal：Plasmaandbrain 400 pharmaCOkinetic profile of cannabidiol（CBD），Cannabidivarine（CBDV），A9−

401 tetrahydrocannabivarin（THCV）andcannabigerol（CBG）inratsandmicefo1lowingoraland 402 intraperitonealadmimistration and CBD action on obsessive−COmpulsive behaviour・

17

(18)

403 Psychophawlogy（Berl）2012；219（3）：859−73．

404 28HloZekT；U旭L，Kade臨bekL，BalikovaM，LhotkovaE，HorsleyRR，etal：Pharmacokinetic 405 andbehaviouralprofileofTHC，CBD，andTHC＋CBDcombinationa食erpulmonary，Oral，

406 andsdbcutaneOuSadministrationinratsandconfirmationofconversioninvivoofCBDto 407 THC．EurNewopsychophawlio17；7（12）‥1223−37．

408 29ScheidweilerKB，NewmeyerMN，BamesAJ，HuestisMA：Quant漬cationofcannabinoids 409 andtheirBTeeandglucuronidem血olitesinw血01ebloodbydisposablepipetteeXtraCtion 410 andliquidchromatogrq）hy−tandemmassspectrometry．JChromatogrA2016；1453：34A2・

411 30Ke桓iT：KyotoUmiversitygraduateschodpharmacygraduatecourseclinicalcondition

412 information pharmacy［1ast update 2016 0ct 22］．如tp：〟wwpharm．kvoto−

413 u．ac．il）／bvovakuhtics／downloadhtm1

414 31ZgairA，WongJC，LeeJB，MistryJ，SivakO，WasanKM：Dietaryfatsandphameutical 415 1ipidexcipientsincreasesystemicexpo甜retOOranya血血isbredcannal）isandcannabisL

416 basedmedicines．AmJTtanSIR飴2016；8（8）：3448−59．

417 32ASNarang，DDelmarre，GaoD：StabledrugenCaPSulationinmicellesandmicroem山sion．

418 血tJPbam2007；345：9−25．

419 33 SeebanuckF，Lawless E，AshfordMB，ODriscollCM：Stinmitionoftriglyceride−rich 420 1ipoproteinsecretionbypotysotbate80：invitroandinvivocorrelationusiqgCaco−2cells 421 andacannulatedratintestinal1ymPhaticmode．pharmRes2004；21（12）‥2320J．

422 34KawG，MehtaSK：Developmentsofpolysofbate（Tween）basedmicroemulsions：PreClimical 423 drugdelivery，tOXicityandantimicrobialapplications．IntJPlm2017；529（1「2）：134−60．

424 35Cu色ettiL，MamL，PeyraubeR，AmakJ，CuriL，OrihuelaS：ChronicPainTr飽tmentWith 425 CannabidiolinKidneyTransplantPatientsinUruguay．TransplantProc2018；50（2）：461−464・

426 36TurnerPV，BrabbT，PekowC，V加わinderMA：Admimistrationofsubstancestolaboratoryanimals：

(19)

427 routesofadmimistrationa皿dぬ．ctorstoconsider．JAmAssocLabAnimSci．2011；50（5）：600−13．

428 37StottC，WhiteL，WdghtS，WilbrahamD，GuyG：APhaseI，OPen−1abel，randomized，

429 crossoverstudyinthreeparallelgroupstoevaluatethee飴ctofrifampicin，ketoconazole，and 430 0mePraZOleonthepharmaCOkineticsofTHC／CBDoromucosalsprayinhealthyvolunteers・

431 Springerplus2013；2（1）：236・

432 38CorroonJ，Phi11ipsJA：Across−SeCtionalstudyofcannabidioluserS・CamabisCannabinoid 433 Res2018；3（1）：152Jl．

434 39ZimmermanJJ，FerronGM，LimHK，P訂kerV：Thee飴ctofahigh−fhtmealontheoral 435 bioavailability of theirLmOSuPPreSSant Sirolimus（叩amyCin）JClin PharmaCO1 436 1999；39（11）：1155−61．

437 40Epidiolex（Cannabidiol）oralsolution［prescribinginformation］・Carlsbad，USA：Greenwich 438 Biosciences，Lic．；June2018．

439 41Wi11iamstID，取evaskisNL，Ye叩YY；AnbyMU，PoutonCW，PorterCJ：LipidJ）aSed 440 forndationsand drug SuPerSaturation：hameSSlng theun1que benefits of thelipid

441 digestionhbsorptionpadlWay・PhamRes2013；30（12）：2976−92・

442 42HolmR，M揖1ertzA，MuH：BilesaltsandtheirimportanCefordrugabsorption・IntJPharm 443 2013；453（1）：中ト55．

444 43SamaraE，BialerM，HarveyDJ：Metabolismofcamabidiolbytherat・EurJDrugMetab 445 Pbamacokhet1991；16（4）：305−13．

446 44StoutSM，CiminoNM：Exogenouscannabinoidsassubstrates，inhibitors，andinducersof 447 lmmandrugmetabolizingenzymeS：aSyStematicreviewDrugMetabRev2014；46（1）：86−95・

19

(20)

﹁車−．↓hete∃aryPhased訂gra∋fOrtheOPtぎ訂at6．⊃Of

theCBロコaコOe∃u一s6．コSySte∃︵↓きee⊃・NO＼≦ta∃ぎ﹇＼Wateユ

(21)

▲シ﹁車N．P一as∃anO⊃Ce⊃tratiO⊃・tぎecu﹁＜eSOfCBロ○ニa⊃dnB？Z﹇

ad∋iコ落eredOraニytOratSatthedOSeSOご00∃g＼kg︵nBロ○ニ︶

a⊃d岩∋g＼kg︵nB？ZE︶．

﹇achsy∋b〇一w彗barrepreseコtSthe∃eaコ什S﹇M︵⊃＝∽fOrnBロ

○ニa⊃d⊃＝のfOrCBロ・Z﹇︶．

CBDconcentration，LJg／ml

L

二 ∴⁚γ T

− ▼一

ー○

h﹁ ↓ぎ一e︑

ー∽ NO Nu

(22)

CBDconcentration，LJg／m［

﹇争ぃ．P一as∃aCO⊃Ce⊃trat5．⊃主∃2nu﹁＜2SOfnBロ○ニa⊃dnB？Z﹇

ad∃宣steredOraニytObニ2・f訂tu一at2drat〜atth2dO〜20f岩

∃g＼kg︵nBロ○ニ︶a⊃d岩∃g＼kg︵nBロ主﹇︶・

﹇anhsy∃b〇一w彗barreprese⊃tSthe∃2a⊃什s﹇M︵⊃‖半nBロ

was⊃Otdetented．at〇．いha⊃d−hafterthead∃宣stratiO⊃Of

CBロ○ニ．

h﹁ゴヨe︑

ー○

(23)

Thblel．Ingredientsandeachphasevolume（W／w％）forthe負nalCBDJNEfbrmulation

Ingrediemts Phasev01ume（w佃％）

1．70

3．80

70．0

24．5 VitaminE

EthanoI

Tween−20

Water

＿iT i ▲i− ■

「■■′−■巧■■

CBDformulationwaspreparedtocontain30mgCBD血血

ニこ÷恐赫J

虚言重職学生．

(24)

Thble2．LC−MS／MSconditionsforquantiBcationofCBD

（A）MassspeCtrOmetryParameterSforCBDandIS（clobetasoIpropionate）

CoⅡi＄iom

Ce皿Ex祉 Potential

（¶

Rete止血皿

Ti血e

（nlim）

Col敲sion

Emergy

（ヽ）

p。叫 QIMass甲Ma＄＄慧 pわ pa） Pg

M M

CBD Neg如ive 313・085 245・033 −30 −10 −28 −17

11・8

IS Positive 467．161 372．900 41 10 13 10 5．71

P）OptimizedsourceparameterS

SoⅦree Tbmper8伽Ⅳe

（℃）

IonSeoⅦree IonSeoⅦree Gas 1 Gas 2

Col旭sion IonSpray Gasbsi） Ⅵ此age（Ⅴ）

Curtain

Gas（psり

CBD 20

³

ヰ500 400 40 70

IS 40

⁵

5500 400 80 70

(25)

Table3．Pharmacokinetic parameterS Of CBD oil（100mg／kg）and CBD−NE（50mg丑g）

administeredorallytorats．

PKI）arameter CBD oil CBD−NE P

50 16．2 土 2．7

0．324 土 0．053 0．409 22．4 土 4．3

0．448 土 0．087 0．172 2．40 土 3．49 0．069 2．8l士 0．67 0．258 36．0 土 7．4 0．456 2．40 士 0．46

＜0．001＊＊＊

3．23 土 0．80 100

25．3 土 4．4 0．253 土 0．044

26．7 土 4．5 0．272 土 0．045

4．68 士 0．31 4．29 土1．67 46．7 士 9．6 8．00 士 0．00 2．72 土 0．37 Dose（mg／kg）

AUCo＿24（mgh／L）

AUC／Dose（hkg／L） 0＿24 AUCo＿叩（mgh／L）

AUC／Dose（hkg／L） 0

＿の

Tl′2匝）

CLt。t／F（L仙kg）

Ⅴ血s仔（Ukg）

Tmax（h）

Cmax（帽血⊥）

C／Dose（kg／L） maX MRT（h）

0．0272 土 0．0037 0．0646 土 0．0159 0．103

10．8 土 0．4 15．10 ± 3．7 0．356

＊＊＊P＜0．00lvs．CBDoil．Eachvalueisexpressedasthemean土SEM

n＝4forCBDoil，n＝5forCBD−ME

(26)

Table4．PharmaCOkineticpammetersofCBDoil（50mg／kg）andCBD−NE（50mg此g）administered Ora11ytobile一抗stulatedrats

PK parameter CBD oil CBD−NE P

50 50

0．300 士 0．019 5．9 土1．1 0．502 土 0．127 10．8 ± 0．1

5．46 土1．81 8．3 土 2．2 117 土 23 4．62 士 0．05 Dose（mg／kg）

AUCo

＿10

AUCo

＿の

T．′2（b）

CL．。t／F（L／M g）

Vdss／F（L此g）

Tm。X（h）

Cmax仙g血L）

MRT（h）

0．05（i

＜0．001＊＊＊

0．461 0．059

992 土 21 63．3 土16．0

＜0．001＊＊＊

4．00 土 0．00 4．00 土 0．00

0．0588 土 0．0015 0．90 土 0．20 0．077

10．0 士 2．6 13．7 土 3．4 0．520

＊＊＊P＜0．001vs．CBDoil．Eachvalueisexpressedasthemean土SEM（n＝3）