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ArticleInVitroandInVivoCharacterizationofDibenzothiopheneDerivatives[125I]Iodo-ASEMand[18F]ASEMasRadiotracersofHomo-andHeteromericα7NicotinicAcetylcholineReceptorsCorneliusK.
Donat1,2,*,HenrikH.
Hansen1,HanneD.
Hansen1,RonnieC.
Mease3,AndrewG.
Horti3,MartinG.
Pomper3,ElinaT.
L'Estrade1,4,5,MatthiasM.
Herth4,5,DanPeters6,GitteM.
Knudsen1andJensD.
Mikkelsen1,*1NeurobiologyResearchUnit,CopenhagenUniversityHospital,Rigshospitalet,DK-2100Copenhagen,Denmark;hbh@gubra.
dk(H.
H.
H.
);Hanne.
D.
Hansen@nru.
dk(H.
D.
H.
);elina.
nyberg@sund.
ku.
dk(E.
T.
L.
);Gitte.
Knudsen@nru.
dk(G.
M.
K.
)2DepartmentofBrainSciences,ImperialCollegeLondon,LondonW120LS,UK3RussellH.
MorganDepartmentofRadiologyandRadiologicalScience,TheJohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD21287,USA;rmease1@jhmi.
edu(R.
C.
M.
);ahorti1@jhmi.
edu(A.
G.
H.
);mpomper@jhmi.
edu(M.
G.
P.
)4DepartmentofDrugDesignandPharmacology,UniversityofCopenhagen,Jagtvej162,2100Copenhagen,Denmark;matthias.
herth@nru.
dk5DepartmentofClinicalPhysiology,NuclearMedicine&PET,Rigshospitalet,Blegdamsvej9,2100Copenhagen,Denmark6DanPETAB,21619Malm,Sweden;info@danpet.
eu*Correspondence:cdonat@imperial.
ac.
uk(C.
K.
D.
);jens_mikkelsen@dadlnet.
dk(J.
D.
M.
);Tel.
:+45-40205378(J.
D.
M)AcademicEditor:PeterBrustReceived:24January2020;Accepted:27February2020;Published:20March2020Abstract:Theα7nicotinicacetylcholinereceptor(α7nAChR)isinvolvedinseveralcognitiveandphysiologicprocesses;itsexpressionlevelsandpatternschangeinneurologicandpsychiatricdiseases,suchasschizophreniaandAlzheimer'sdisease,whichmakesitarelevantdrugtarget.
Developmentofselectiveradioligandsisimportantfordeningbindingpropertiesandoccupancyofnovelmoleculestargetingthereceptor.
Wetestedtheinvitrobindingpropertiesof[125I]Iodo-ASEM[(3-(1,4-diazabycyclo[3.
2.
2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene5,5-dioxide)]inthemouse,ratandpigbrainusingautoradiography.
Theinvivobindingpropertiesof[18F]ASEMwereinvestigatedusingpositronemissiontomography(PET)inthepigbrain.
[125I]Iodo-ASEMshowedspecicanddisplaceablehighanity(~1nM)bindinginmouse,rat,andpigbrain.
Bindingpatternoverlappedwith[125I]α-bungarotoxin,specicbindingwasabsentinα7nAChRgene-decientmiceandbindingwasblockedbyarangeofα7nAChRorthostericmodulatorsinananity-dependentorderinthepigbrain.
Interestingly,relativetothewild-type,bindinginβ2nAChRgene-decientmicewaslowerfor[125I]Iodo-ASEM(58%±2.
7%)than[125I]α-bungarotoxin(23%±0.
2%),potentiallyindicatingdierentbindingpropertiestoheteromericα7β2nAChR.
[18F]ASEMPETinthepigshowedhighbrainuptakeandreversibletracerkineticswithasimilarspatialdistributionaspreviouslyreportedforα7nAChR.
BlockingwithSSR-180,711resultedinasignicantdecreasein[18F]ASEMbinding.
Ourndingsindicatethat[125I]Iodo-ASEMallowssensitiveandselectiveimagingofα7nAChRinvitro,withbettersignal-to-noiseratiothanprevioustracers.
Preliminarydataof[18F]ASEMinthepigbraindemonstratedprincipalsuitablekineticpropertiesforinvivoquanticationofα7nAChR,comparabletopreviouslypublisheddata.
Keywords:alpha7;nicotinicacetylcholinereceptors;PET;nAChR;autoradiographyMolecules2020,25,1425;doi:10.
3390/molecules25061425www.
mdpi.
com/journal/moleculesMolecules2020,25,14252of191.
IntroductionTheα7nicotinicacetylcholinereceptor(α7nAChR)belongstothesuperfamilyofligand-gatedionchannelsandisexpressedacrossallmammalianspecies[1–4].
Thereceptorplaysanimportantroleincognition[5],mood[6]andconsistentwiththis,α7nAChRareparticularlyabundantinhippocampusandprefrontalcortex[7,8].
Furthermore,α7nAChRareimpliedinneuro-immune[9]andimmunefunctions[10]underhomeostaticconditions.
ChangesinproteinandmRNAlevelsofα7nAChRhavebeenreportedinanumberofneuropsychiatricandneurodegenerativediseases[1,11–15].
Notably,certainpolymorphismsinthepromoterregionoftheα7nAChRgene(CHRNA7)[16]areprobableriskfactorsforneuropsychiatricdiseases,suchasmajordepression[17]andschizophrenia[18]andareassociatedwithdevelopmentaldisordersandcognitiveimpairments[19].
Additionally,α7nAChRsareexpressedbyseveralcentralandperipheralimmunecellsandactivationviaagonistsandpositiveallostericmodulatorsshowedneuroprotectiveandimmunomodulatoryecacyindierentpreclinicaldiseasemodels[20–24].
Changesofα7nAChRinthehealthyanddiseasedbraincanonlybedetectedinvivobymolecularimaging,suchaspositronemissiontomography(PET)usingspecicradiotracers.
Aclinicallyusableradiotracerrequiressucientselectivity,specicityandsuitableanity,dependingonthetarget[25].
Mostofthepreviouslydescribedα7nAChRPETtracers,amongthose[18F]NS14490,[11C]NS14492,[11C]CHIBA-1001and[11C]A-582941(Table1),studiedinmice,pigsandnon-humanprimatesexhibitedsomeshortcomings,suchaspoorspecicand/orhighnonspecicbindingorradiometabolitescrossingtheblood–brainbarrier[26–32].
Furthermore,thespecicityofnovelα7nAChRtracershasnotalwaysbeentestedinrespectivegene-decientmice,e.
g.
,usinginvitroautoradiography.
Whiledevelopmentofnoveltracersfromdierentleadstructuresisstillongoing[33–37],tilorone[38]providedalead-structureforanumberofderivativessubsequentlydevelopedintoα7nAChRPETtracers.
Fromthose,[18F]ASEM(JHU82132)[39]andthestructurallyrelated[18F]DBT-10(JHU82108)[40]havebeenmostwidelyinvestigated.
InitialstudieshaveshownthatASEMisapotentantagonist[39]withsubnanomolaranityandhighselectivity[39,41],furthersubstantiatedbytheradiolabelledcompounds[18F]ASEM[26]and[125I]Iodo-ASEM[42]astestedinhumanandratrecombinantα7nAChR.
[18F]ASEMand[125I]Iodo-ASEMreadilyenterthemousebrain,aredisplaceable,andaccumulateinregionswithhighestα7nAChRdensity[26,39,42,43].
Morerecentstudiesusing[18F]ASEMand[18F]DBT-10furthersupportedthesuitabilityofthetracers,showinghighandreversiblebrainuptakewitharegionalbindingpatternconsistentwiththedistributionofα7nAChRreceptorsinthenon-humanprimatebrain[39,44,45].
Favourablebrainpharmacokinetics,excellenttest-retestreproducibilityandregionaluptake[18F]ASEMpatternconsistentwithpost-mortemα7nAChRdistributionhavebeenreportedinhumanPETstudies[43,46].
Severalrecentstudiesextendedtheavailabledatainhumansubjects,showinggoodagreementwithpreviousdistributionvolumes(VT)andtest-retestvaluesinnonhumanprimatesandhealthyvolunteers[44].
Astudyinageingsubjectsshowedasignicantpositivecorrelationbetweenageand[18F]ASEMVTinstriatumandseveralcorticalregions[47],howeverwithoutanycorrelationbetweenVTandcognitivemeasures.
Asmallsampleofindividualswithschizophreniaonstableantipsychoticmedicationshowedlower[18F]ASEMVTincingulatecortexandhippocampus[46]andindividualswithrecentonsetofpsychosiswerealsoreportedtoshowlower[18F]ASEMVTinhippocampus,aftercontrollingforage[48].
Interestingly,patientswithmildcognitiveimpairmentshowedhigher[18F]ASEMVTwhenadjustedforageascomparedtothecontrolgroup[49],consistentwithpost-mortemndingsfrompatientsandanimalmodels.
Additionally,[18F]ASEMhasbeenemployedinarat6-OHDAlesionmodelofParkinson'sdisease,showinganinitialincreaseof[18F]ASEMSUVrintheipsilateralstriatumandsubstantianigrabetween3and7days,whichcoincidedwithseveralhistologymarkersofgliaactivation[50].
Molecules2020,25,14253of19Whilethisdatashowsthegeneralapplicabilityof[18F]ASEM,bindingpropertiesandinterpretationofnovelα7nAChRtracersmightbecomplicatedbythefactthatα7subunitscanformheteromericreceptorstogetherwithothersubunits,specicallyβ2[51].
Thesereceptorscanbeheterologouslyexpressedinoocytesandarefoundintherodentandhumanbasalforebrainandcortex[52,53].
Whiletheseheteromericreceptorsdisplaydierentpharmacologicalproperties[52,54],itisnotclearhowthistranslatestoradiotracerbinding.
Invitrobindingstudiesof[18F]ASEMor[125I]ASEMingene-decientmicecouldanswerthequestion,andwouldalsorevealthesuitabilityof[125I]ASEMforinvitroautoradiographicstudies.
ThelatterwouldoerabettercomparabilitytoinvivoPETdataoverthecurrentgold-standardtracer[125I]α-bungarotoxin.
Wethereforeinvestigatedthepotentialof[125I]ASEMforinvitrostudiesoftheα7nAChR,bycomparingbindingof[125I]Iodo-ASEMinrat,mouseandpigbrainsections.
Furthermore,[18F]ASEMwascharacterizedforinvivobrainuptakeandtargetselectivityinaPETstudyconductedinthepig.
Table1.
Commonα7nAChRligandsandtheirstructure,previouslyevaluatedasradiotracers.
TracerStructure[11C]CHIBA-1001[11C]A-582941[18F]NS14490[11C]NS14492[18F]ASEM[18F]DBT-10[125I]ASEM2.
Results2.
1.
InVitroAutoradiographyCerebralbindingof[125I]Iodo-ASEMwasinvestigatedacrossseveralmammalianspecies,i.
e.
,rat(Figure1A,upperrow),mouse(Figure2A)andpigbrain(Figure1A,lowerrow)andcomparedto[125I]α-bungarotoxin(Figure1B/2B).
Totalcortical[125I]Iodo-ASEMbindingwashighestinthepig,andlowerintheratandmouse(Figures1Cand2C),ascomparedto[125I]α-bungarotoxin.
[125I]Iodo-ASEMbindingwasdisplaceablewith(-)-nicotine(1mMol/L,datanotshown)andSSR-180,711(10Mol/L,Figures1Aand2A,nonspecicbinding)inallspecies.
Molecules2020,25,14254of19Figure1.
(A,B)Representativeautoradiographsshowing[125I]Iodo-ASEMand[125I]α-bungarotoxintotalandnon-specicbinding(determinedwith1mMol/L(-)-nicotinefor[125I]α-bungarotoxinand10Mol/LSSR-180,711for[125I]Iodo-ASEM)in12msectionsoftherat(upperrow)andpigbrain(lowerrow).
Arrowheadsindicateresidualwhitematterbinding.
(C)Comparativequantitativeanalysisofspecicbinding(±S.
E.
M.
)of[125I]Iodo-ASEMand[125I]α-bungarotoxinfromautoradiographyintherat(n=1)andpigcortex(n=2).
Allautoradiographicexperimentsandquanticationsarecarriedoutin3-4sectionsperanimal.
Anon-specicbindingcomponentremaineddetectableunderthedescribedexperimentalconditionsatlowlevelsinwhitematterstructures(arrowheads,Figures1Aand2A).
Thedistributionpatternof[125I]Iodo-ASEMbindingintherat,mouseandpigbrainwascomparabletothatof[125I]α-bungarotoxin(Figures1Band2B).
Inthepig,[125I]Iodo-ASEMshowedalaminarbindingpatterninthefrontalcortex,withhighestdensityincorticallayers(1–3)(Figure1A,lowerrow),whileintherat,bindinginmotor,cingulateandsomatosensorycortexwasmoreprominentinlayers5–6.
However,thisspeciesdierencewasalsoobservedfor[125I]α-bungarotoxin(Figure1B).
Figure2.
(A,B)Representativeautoradiographsshowingtotal[125I]Iodo-ASEM(A)and[125I]a-bungarotoxin(B)andnon-specic(determinedwith1mMol/L(-)-nicotinefor[125I]α-bungarotoxinand10Mol/LSSR-180,711for[125I]Iodo-ASEM)bindingin12mbrainsectionsofα7andβ2nAChRwild-typevs.
correspondinggene-decient(def)mice(n=1each).
Arrowheadsindicateresidualwhitematterbinding.
(C)Comparativequantitativeanalysisofspecicbinding(±S.
E.
M.
)of[125I]Iodo-ASEMand[125I]a-bungarotoxininα7andβ2nAChRwild-typevs.
correspondinggene-decientmice(n=1).
Allautoradiographicexperimentsandquanticationsarecarriedoutin3-6sectionsperanimal.
Molecules2020,25,14255of19Thespecicityof[125I]Iodo-ASEMtoα7nAChRisfurthersubstantiatedbytracerbindingexperimentsinα7nAChRgene-decientmice.
Specic[125I]Iodo-ASEMbindingwaslackinginα7nAChRgene-decientmice(Figure2A),asindicatedbytheoverallreductionintotalbindingby93%±1.
7%,comparedtowild-typeanimals(Figure2C).
Similarly,[125I]α-bungarotoxintotalbinding(Figure2B)was96%±0.
4%lowerinα7nAChRgene-decientmice(Figure2C).
Inwild-typemice,nodierencein[125I]Iodo-ASEMand[125I]α-bungarotoxinbindingwasobserved(Figure2A,B).
However,tracesofnonspecicbindingwereagainnotedinwhitematterstructures(arrowheadsinFigure2A).
[125I]Iodo-ASEMbindinginβ2nAChRgene-decientmicewasdierentcomparedto[125I]α-bungarotoxin.
Anoverall58%±2.
7%lowerspecic[125I]Iodo-ASEMbindingwasobserved,ascomparedtocorrespondingwild-typecontrols(Figure2C).
Incontrast,[125I]α-bungarotoxinbindingwasreducedby23%±0.
2%,beinglessaectedbyβ2nAChRgene-deciencyascomparedtothereductionin[125I]Iodo-ASEMbinding.
Saturationbindinginratandpigbrainsectionsindicatedthat[125I]Iodo-ASEMbindingwassaturable.
Intherat,non-linearregressionanalysisrevealedanequilibriumdissociationconstant(Kd)of1.
14nM(cortex,Figure3B)and1.
17nM(hippocampus,Figure3A)withcorrespondingreceptordensity(Bmax)of0.
70fmol/mgprotein(cortex)and1.
44fmol/mgprotein(hippocampus),respectively(Figure3A,B).
Incomparison,thepigcortexshowedaKdof1.
21nMwithaBmaxof5.
47fmol/mgprotein(Figure3C).
Thenon-specicbindingof[125I]Iodo-ASEMatconcentrationsneartheKdwaslow(rathippocampus,20%;ratcortex,30%;pigcortex,10%).
Figure3.
Saturationbindingof[125I]Iodo-ASEM(0.
02-10nMol/L)to12msectionsfromtherathippocampusandcortex(A,B,n=1)andpigfrontalcortex(C,n=1)brain.
Non-specicbindingwasdeterminedinthepresenceof10Mol/LSSR-180,711.
Opticaldensityoftheautoradiogramswasconvertedintoligandbinding(fmol/mgprotein±S.
E.
M.
)fromarepresentativeexperiment.
Datafromsaturationbindingexperimentswereanalysedbynon-linearregression.
IndividualKdandBmaxvaluesareindicatedinSection2.
1.
Allautoradiographicexperimentsandquanticationsarecarriedoutin2–4sectionsperanimalusing10radioligandconcentrations.
Arangeofselectiveα7nAChRligands(10Mol/Leach),includingtheα7nAChRpreferringantagonistmethyllycaconitine(MLA),wereusedtotestwhetherinvitro[125I]Iodo-ASEMbinding(0.
5nMol/L)couldbeblockedinthepigcortex(Table2).
Thepartialagonists,NS14492,TC-5619,EVP-6124,A-582941,andSSR-180,711,showedalmostcomplete(>90%)blockingof[125I]Iodo-ASEMbindinginreceptordenseareasofthecortex,e.
g.
layers1–3.
Incontrast,GTS-21(weakα7nAChRagonist,~70%reduction)andMLA(α7nAChRpreferringantagonist,~80%reduction)exhibitedlessecaciousblockadeof[125I]Iodo-ASEMbindinginthepigcortex.
Molecules2020,25,14256of19Table2.
Blockingof[125I]Iodo-ASEMbindinginthepigcortexbyaseriesofα7nAChRligands.
Ligand(10Mol/L)[125I]Iodo-ASEMBindinginthePigCortex,Layers1–3(%,mean±S.
E.
M.
)[125I]Iodo-ASEMBindinginthePigCortex,Layers4–6(%,mean±S.
E.
M.
)NS144924.
04±0.
559.
25±0.
83TC-56197.
88±1.
657.
60±0.
30EVP-61242.
50±0.
205.
50±0.
07A-5829413.
22±0.
284.
44±0.
93SSR-180,7112.
92±0.
353.
53±0.
50GTS-2130.
92±2.
5531.
39±2.
15MLA20.
46±2.
1818.
16±2.
41Resultsaregivenin%remainingbindingoftotalbinding(mean±S.
E.
M.
).
2.
2.
InVivoPETImaginginthePigUsing[18F]ASEM[18F]ASEMreadilyenteredthepigbrainandhighesttraceraccumulationwasfoundinthethalamusfollowedbycortex,striatumandcerebellum(Figure4A,C).
[18F]ASEMuptakeinthewhitematterwasinitiallylowerthaninthegreymatterregions,howeverthetracerkineticswerealsoslower,resultinginlowergreytowhitematterratioattheendofthescans.
Themetabolismof[18F]ASEMinpigswasrelativelyslow,with60%oftheradioactivityat120minstillbeingparentradioligand(datanotshown).
Kineticmodellingwasperformedtoquantifythetraceruptake.
BaselineVTvaluesvariedbetweenanimalsbutaftercorrectingforfreefractioninplasma(fP),therewasonlya5%dierenceinVT/fPvaluesbetweenthetwobaselineanimals(Table3).
ThisalsosuggestthatVT/fPvaluesareunaectedbyrelativelylargedierencesininjectedmass(0.
35gand1.
78g).
Table3.
Kineticmodellingof[18F]ASEMwiththeLoganGraphicalAnalysismodelindierentpigbrainregions.
ComparisonofBaselineVTValues.
KineticModellingAnimal1Animal20–90minVTVT/fPVTVT/fPFrontalcortex7.
8743.
703.
7541.
66Somatosensorycortex8.
3346.
274.
1546.
14Occipitalcortex8.
0344.
633.
7741.
86Remainingcortex7.
6342.
373.
7841.
94Thalamus8.
8349.
064.
1245.
73Striatum7.
4141.
173.
7741.
94Hippocampus7.
5341.
833.
5939.
93Cerebellum6.
6636.
993.
1635.
07ComparisonofVTValuesatBaselineandAfterPre-treatmentwithSSR-180,711KineticModellingAnimal1Animal30–150minVTVT/fPVTVT/fPFrontalcortex6.
7337.
383.
6122.
57Somatosensorycortex7.
2640.
353.
9424.
60Occipitalcortex6.
9638.
653.
9024.
40Remainingcortex6.
6837.
083.
6022.
50Thalamus7.
4241.
244.
3427.
15Striatum6.
5136.
193.
8323.
92Hippocampus6.
5536.
413.
3721.
09Cerebellum5.
5530.
823.
3921.
17Molecules2020,25,14257of19Inathirdanimal,weevaluatedthespecicityof[18F]ASEMbindinginvivo,byadministeringSSR-180,711(1mg/kg)priortoinjectionof[18F]ASEM.
Comparedtothebaselinestudies,wefoundanincreasein[18F]ASEMuptakeinallbrainareasinvestigated(Figure4A).
QuanticationofuptakeandsubsequentcorrectionforfPrevealedthatSSR-180,711administrationdecreasedtheVT/fPcomparedtobaseline(Table3).
OccupancywascomputedwiththeLassenplotusingVT/fPvaluescomparingbaselinedatafromanimal1andblockingdatafromanimal3(0-150minscandata).
Wefoundthatthe1mg/kgSSR-180,711doseresultedina49%occupancy(Figure4B).
FromtheLassenplot,thevolumeofnon-displaceablebinding(VND/fP)wasfoundtobe9.
2mL/cm3.
WhencomparingtheVND/fPtotheVT/fPinthethalamus,wefoundthat78%ofthesignalobservedinthethalamusisspecicbinding,leaving22%asnon-displaceablebinding.
Inoneanimal,[18F]ASEMacquisitiontimewas240min,whichallowedsubsequentanalysisofthetime-stabilityoftheparametersestimatedwithkineticmodelling.
Again,theLGAmodelwasusedtodetermineVTwithdierentscanlengthandVTvalueswerefoundtodecreasewithmoretimeincludedinthekineticmodelling.
Usingalldata(0–240min),VTvalueswere5.
4mL/cm3(thalamus),5.
0mL/cm3(frontalcortex)and4.
0mL/cm3(cerebellum).
Theupperhalfofthetableshowsthebaselinedistributionvolumes(VT)valueswithandwithoutcorrectionforfreefractioninplasma(fP)intwodierentanimals.
BottompartofthetabledescribesVTvalueswithandwithoutcorrectionfor(fP)atbaseline(animal1)andafterpre-treatmentwithSSR-180,711(animal3).
SeeTable4(MaterialandMethods)forfPvaluesintheindividualanimals.
Becauseanimal2wasonlyscannedfor90min,theacquisitiontimeofanimal1wastruncatedto90mintoallowforcomparison.
Figure4.
[18F]ASEMbindinginthepigbrain.
(A)Time-activitycurvesof[18F]ASEMinthreedierentanimals:Animal1,bsl,blue;Animal2,bsl,black;Animal3,SSR-180,711pre-treated,red.
Theregionsshownare:Thalamus(tha,triangles)andcerebellum(cb,circles).
(B)Lassenplotwithtotaldistributionvolumes(VT)correctedforfreefractioninplasma(fP)usingvaluesfromanimal1andanimal3.
Eachpointrepresentsoneregionofinterest(ROI),pleaserefertothemethodsectionforthecompletelistofROIs.
(C)SummedPETimage(0–240min)fromanimal1showingthedistributionof[18F]ASEMinthepigbrain.
SUV:standarduptakevalue.
Bsl:baseline.
SSR:SSR-180,711(1mg/kg).
Molecules2020,25,14258of193.
DiscussionInthisstudy,weinvestigatedbindingpropertiesofradiolabelledASEMinvitro([125I]Iodo-ASEM]andinvivo([18F]ASEM).
Autoradiographywasusedtodeterminetheapplicabilityof[125I]Iodo-ASEMforinvitroassessmentofα7nAChRreceptordistributionandoccupancyinthemammalianbrain.
[125I]Iodo-ASEMshowedhigh-anityandspecicbindingtoα7nAChRintherat,mouseandpigbrain.
Specicbindingwasabsentinα7gene-decientmice,indicatinghighspecicityandselectivity.
SaturationbindingexperimentsinratandpigbrainsectionsrevealedlownanomolarKdvalues(approximately1nM)inbothspecies.
Bmaxinthepigcortexwasconsiderablyhigherascomparedtothebindingfoundinthemouseandratbraincortex.
Suchspeciesdierencesarewelldocumentedintheliterature,e.
g.
,formetabotropicglutamate5receptorsandthe18kDatranslocatorproteinbetweenmonkeyandhumans[55,56].
Asanityandselectivityaremajorcriteriaforradiotracers,ourdatafurthersubstantiatesthesuitabilityofASEMderivativesasfavourableα7nAChRtracers[57,58]Wefoundthattheanityof[125I]Iodo-ASEMinthepigbrainasdeterminedwithautoradiographywasinasimilarrangeas[3H]NS14492[59].
Incontrast,higheranitiesandreceptordensitiesarereportedforradioligandbindingassayinbrainhomogenatesforanumberofdierentα7nAChRligands,suchas[3H]NS14492and[3H]A-585539[60],includingotherdibenzothiophenes[26,38].
Acrossthebrainandspecicallyregionswithhighα7nAChRexpression,suchashippocampusandsupercialcorticallayers,non-specicbindingof[125I]Iodo-ASEMatconcentrationsneartheKdwaslow(10%–30%oftotalbinding)andproducedarobustspecicsignal.
However,aconsistentnonspecicbindingcomponentinwhitematterwasobservedinallinvestigatedspecies,inparticularthecorpuscallosumandsubcorticaltracts.
Thisissupportedbypreviouslyreportedinvivondingsinhumanandnon-humanprimatesubjects[39,43],wheretraceruptakewaslowestinwhitematterstructures,suchasthecorpuscallosum.
Furthermore,wealsoobservedslowerinvivokineticsinwhitematterstructuresinthepigbrain.
Thiscouldbecausedbylowerperfusionorkineticsmaybedierentwhenthetracerinteractswithlipidmembranes,comparedtointeractionwiththereceptor.
Asinvitrobindingconditionsaredistinctlydierentduetoabsentmetabolismandbloodow,itispossiblethattheseeectsmaylimitpronouncednon-specicwhitematterbindinginvivoorthatthenonspecicbindingexhibitsmuchslowerkinetics.
Undertheemployedincubationconditions,[125I]α-bungarotoxinshowsnowhitematterresidualbinding.
However,underthesameconditions,theoverallnon-specicbindingingreymatterfor[125I]α-bungarotoxinisapproximately45%inhuman(datanotshown)and55%inpigbraintissue,whereitismuchlowerfor[125I]Iodo-ASEM(~10%–30%).
[125I]Iodo-ASEMbindingenablesanimportantdistinctionbetweengreyandwhitematterstructures,e.
g.
,thedistinctcorticallaminarbindingpatternobservableinthepig.
While[125I]Iodo-ASEMbindingwasprominentinthedeepercorticallayersinthemouseandrat,supercialcorticallayerswereintenselylabelledinthepig.
Usinginvitroautoradiography,similarlaminarcorticalbindingpatterninthepigbrainhasalsorecentlybeenreportedforastructurallydierentα7nAChRradioligand,[3H]NS14492[61].
Bindingofbothtracerswasmatchingthepatternof[125I]α-bungarotoxin,theinvitrogold-standardradioligandforα7nAChR.
However,thespatialbindingpatternintherodentbrainwasonlysimilarbetween[125I]Iodo-ASEMand[125I]α-bungarotoxin,butnotfor[3H]NS14492,suggestingdierentbindingprolesofantagonistsandagonists,orspeciesdierencesinanity.
[125I]Iodo-ASEMprovedspecictotheα7nAChR,asevidencedbythelackofspecicbindinginα7nAChRgene-decientmiceandavirtuallycompleteblockofcortical[125I]Iodo-ASEMbindingbyawiderangeofstructurallydierentα7nAChRselectiveligandsandMLA,withtherankorderbeingNS14492=TC-5619=EVP-6124=A-582941=SSR-180,711>MLA>GTS-21.
Whilethiscorrespondswellwiththeindividualhighanitiesinthenanomolarrange(NS14492,TC5619,EVP-6124,A-582941,SSR-180,711)[31,62–65],ascomparedtotheloweranityofapartialagonist(GTS-21)[66],itcouldalsoreectthegeneraldierencesbetweenantagonistsandagonistintermsofbindingsitesandkinetics.
Molecules2020,25,14259of19Whencomparingto[125I]α-bungarotoxin,thespecicbindingof[125I]Iodo-ASEMwaslowerintheratandmousebrain,buthigherinthepigcortex.
Whilespeciesdierencesinreceptorstructuremayaccountforthediscrepancies,itshouldalsobeconsideredthatdierentincubationprotocolswereusedforthedeterminationofoptimal[125I]Iodo-ASEMand[125I]α-bungarotoxinbinding.
Hence,arelativelyhighdetergentconcentrationwasrequiredintheexperimentstoobtainoptimaltotaltissuebindingof[125I]Iodo-ASEM,whichmaypotentiallyaectbindingoftheradioligandinthemammalianspeciestested,e.
g.
,throughdierencesinlipidcontentandmyelination.
Forexample,[125I]Iodo-ASEMshowedsomedegreeofnon-displaceablebindingtowhitematterstructures,whichcouldbecausedbytheligands'lipophilicityand/ordierentkineticsinwhitematterstructures.
Interestingly,radioligandbindinginβ2nAChRgene-decientmicewasmorestronglyreducedfor[125I]Iodo-ASEMthan[125I]α-bungarotoxin.
Thisobservationsuggestsdierentbindingpropertiesandsubtypeselectivitytoheteromericα7β2nAChR,comparedtothehomomericreceptors.
IntheCNS,heteromericα7β2nAChRareidentiedinthemouseforebrainandhippocampalneurons,ratbasalforebraincholinergicneurons,aswellasinthehumanbasalforebrainandcerebralcortex.
Importantly,α7β2nAChRdisplaydistinctfunctionalpropertiesascomparedtohomomericα7nAChR[51,67],owingtotheirslowerwholecelldecaykineticsandcurrentamplitudesinbothtransfectedcellsystemsandnativerodentneurons[53,54,68–70].
Accordingly,co-expressionofα7andβ2nAChRsubunitsinXenopusoocytesalsoresultsinlowermaximalresponses(evokedcurrentamplitudes)ofselectiveα7nAChRagonistsbutdoesnotshiftpharmacologytoamoreβ2-likeprole[52–54,71].
Theseinvitrostudiesintransfectedcellsystemsthereforesuggestthatα7nAChRagonistsbindtotheα7-α7subunitinterface,andβ2subunitslikelydonotcontributetotheligandbindingsiteonheteromericα7β2nAChR[67,71].
Whenusingselectiveα7nAChRantagonists,includingMLAandα-bungarotoxin,toaltertheresponsetosomenicotinicagonistsineitherhomomericα7andheteromericα7β2nAChR,resultshavebeenlessconsistent,astheyshowunaltered[54]orreducedpotency[52]andecacy[53]incomparisontohomomericα7nAChRexpressedinXenopusoocytes.
Thefunctionalsignicanceofheteromericα7nAChRexpressionisnotwellunderstood,withrecentworksuggestingthatthissubtypecombinationmightbemoresensitivetoinhibitionbyoligomericamyloidβ1–42[68,69]andisourane[72],ascomparedtohomomericα7nAChR.
Ourndingthatbindingof[125I]Iodo-ASEM,anantagonist,wasmarkedlyreducedintheforebrainofβ2gene-decientmicethereforemaysuggestthat[125I]Iodo-ASEMbindstoheteromericα7β2nAChRinthebrain,asopposedto[125I]α-bungarotoxin.
Whetherthisisduetodierentanityforhomomericα7andheteromericα7β2nAChRrequiresfurtherinvitrostudies.
Althoughspeculative,thismayoerachancetoprobethebindingofamyloidβ1–42toheteromericα7β2nAChRinvivousingPET.
Invivouptakeof[18F]ASEMintothepigbrainoccurredrapidlywithintherst10–20minandareversiblebutslowerwashoutwasfound,asobservedinhumanandnon-humanprimatesubjects[39,43].
Theinvivodistributionof[18F]ASEMfoundhereisverysimilartothatof[11C]NS14492andimportantly,alsoinaccordancewiththedistributionofα7nAChRinthepigbrain[31,73].
Furthermore,ourdatawith[18F]ASEMmatchespreviousreportswiththestructurallysimilaranalogue[18F]DBT-10inpiglets[40].
WefoundvariationsinbrainuptakeandfPinthetwobaselineanimals,andthisresultisconsistentwiththeinterpretationthatlowerfPwillleadtolowerbrainuptake[74].
Duetothelimitednumberofanimalsinthisstudy,thisobservationmeritsfurtherinvestigations.
OurndingishoweversupportedbyPETstudiesinnon-humanprimateswith[18F]ASEMand[18F]DBT-10,whereVT/fPwasshowntobeamorestableoutcomemeasurethanVT[44,45].
Thishasalsobeenshownforradiotracersbindingtootherneurotransmitterreceptors[75].
VTwasfoundtoincreaseslightlywhenthescantimewasprolonged.
ThisphenomenonwasmostpronouncedinthethalamusandleastpronouncedinthewhitematterandisevidentfromtheTACs(Figure4A),wheretheratiobetweene.
g.
,thalamusandcerebellumwaslowerat240minthanat90min.
Thisndingisincontrastwiththenon-humanprimateandhumandata,whereVTwasunderestimatedwhenreducingthePETdatafrom180to60min[44].
GiventhatASEMisanantagonist,Molecules2020,25,142510of19itisunlikelythatinternalizationofthereceptor-ligandcomplexisanexplanationforthedecreaseinVT.
Wecannotexcludethatotherreceptoradaptations,suchas(de)sensitization,couldberesponsibleforthisobservation.
Desensitizationcouldoccurifexperimentswerenotconductedattracerdose,i.
e.
,butwedidnotattempttoidentifythemassdoselimitofunlabelledASEM.
AlthoughtheinjecteddosesofASEMvariedinthetwobaselineanimals,weonlyfounda5%dierenceinthecalculatedVT/fP,whichsuggestthatthestudieswereconductedattracerdoses.
Theinjecteddosesinthisstudy(0.
007–0.
085ug/kg)arehigherthanthedosesusedinthenon-humanprimateevaluationof[18F]ASEM,whereinjecteddosesrangedfrom0.
009to0.
056g/kg[44].
FurtherstudiesareneededtoidentifythemassdoselimitofunlabelledASEM.
Whilepre-treatmentwith1mg/kgSSR-180,711resultedinanincreaseduptakeof[18F]ASEM,kineticmodellingforquanticationoftraceruptakeshowedthatSSR-180,711atthisdoseresultedin49%occupancy.
Asimilarphenomenonhasalsobeenreportedinpigletswhen[18F]DBT-10wasblockedbytheweakagonistNS6740,whichwasascribedtoapotentialbloodow-driveneectofNS6740leadingtogreatercentraluptakeof[18F]DBT-10[40].
Theincreasedtraceruptakecouldalsobearesultofperipheralα7nAChRbindingsiteshavingbeenblockedbySSR-180,771.
TheoccupancyfoundbyusisinlinewithpreviousworkofHortietal.
,reporting39%and81%occupancyfordosesof0.
5and5mg/kgSSR-180,711,respectively[39].
Theoccupancycomputedinthisstudyshouldbeinterpretedwithcare,asthebaselineandblockingstudyisconductedintwodierentpigs.
Duetothehalf-lifeof[18F]ASEM,itwasnotpossibletoconductthestudyinthesameanimalonthesameday.
AfurtherlimitationtothisinvivostudyisthelownumberofPETscansandanimalsandthuswecanonlyprovideadescriptivepresentationofthedata,withoutstatisticalevaluations.
Fromourresults,[125I]Iodo-ASEMthereforeoersseveraladvantagesover[125I]α-bungarotoxin:1)lownonspecicbinding,2)similarhighanityandselectivityand3)invivoapplicabilityanddirectcomparisonofPETdatawithautoradiographicdata.
Thelowernonspecicbindingof[125I]Iodo-ASEMisadvantageousprimarilyinvitro,asitallowsforabettersignal-to-noiseratioover[125I]α-bungarotoxinatverysimilaranities.
Whilealownonspecicbindingwouldalsobefavourableunderinvivoconditions(e.
g.
,PET),havingtwonearlyidenticalmoleculesastracersoersinterestingavenues,especiallyforpreclinicalstudies.
InvivoPET/SPECTimagingdatacanbeacquiredthrough[18F]ASEM,[18F]DBT10or[123I]Iodo-ASEMandresultscanbevalidatedorextendedbyusingtheadvantagesofinvitroautoradiography(e.
g.
resolution)with[125I]Iodo-ASEM.
Inconclusion,[125I]-Iodo-ASEMisapplicableforvisualizingα7nAChRbindinginvitro,itsbindingisdierentbetweenspecies,andmaypotentiallybindtoheteromericα7β2nAChR.
Inaddition,[18F]ASEMisdemonstratedtohavesuitablekineticpropertiesforinvivoquanticationofα7nAChRinthepig.
4.
MaterialsandMethods4.
1.
CompoundsandRadioligands[125I]Iodo-ASEM[(3-(1,4-diazabycyclo[3.
2.
2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene5,5-dioxide)]waslabelledaccordingtopreviouslypublishedprocedures[42].
Meanmolaractivitywas59.
94±6.
25TBq/mmol.
[125I]Tyr-54-mono-Iodo-α-bungarotoxin(81.
4TBq/mmol)waspurchasedfromPerkin-Elmer(Skovlunde,Denmark).
(-)-nicotinetartratewaspurchasedfromSigma-Aldrich(St.
Louis,MO).
UnlabelledASEMandprecursorforradiosynthesiswasprovidedbyDanPET(Malmoe,Sweden).
Theα7-selectiveligandswerepurchasedfromSigma-Aldrich(MLA)orprovidedbyDanPET(NS11492)orNeuroSearchA/S(Copenhagen,Denmark)(SSR-180,711,TC-5619,EVP-6124,A-58294,andGTS-21.
Molecules2020,25,142511of194.
2.
TissueOriginandSectioningforInVitroAutoradiographyAllanimalprocedureswereapprovedbytheDanishAnimalExperimentationInspectorate(J.
No.
2012-15-2034-00156)andtreatedinconcordancewiththeEuropeanCommunitiesCouncilDirectiveof24thNovember1986(86/609ECC).
OnefemaleSprague-Dawleyrat(250g,obtainedfromCharlesRiver,Sulzfeld,Germany)waseuthanizedwithanintraperitonealoverdoseofpentobarbital,thebrainwasquicklyremovedandsnap-frozenin50C2-methylbutane,thenstoredat80Cuntilfurtherprocessing.
Micedecientfortheα7subunits(TheJacksonLaboratory)andβ2(InstitutPasteur,Paris,France)andtheircorrespondingwild-typelittermateswerebred(C57BL/6Jbackground)inananimalcarefacilityatVirginiaCommonwealthUniversity.
Brainsfromα7andβ2gene-decientmiceandcorrespondingwild-typelittermateswerekindlyprovidedbyDr.
M.
ImadDamaj(Dept.
ofPharmacologyandToxicology,VirginiaCommonwealthUniversity,Richmond,VA,USA).
Onetwo-montholdfemaleDanishdomesticpig(LandracexYorkshirexDuroc,22kg)waseuthanizedwithanintravenousinjectionofpentobarbital,thebrainwasquicklyexcised,separatedintwohemispheresandfrozenondryice,beforebeingstoredat80C.
Allbrainspecimenswerecutin12mserialsectionsonacryostat(MicromHM500OM,Walldorf,Germany),thaw-mountedontoSuperFrostslides(ThermoScientic,Hvidovre,Denmark),brieyairdriedandstoredat80Cuntilfurtherprocessing.
ProteinconcentrationwasdeterminedfromsingleorthreesectionswiththeBio-RadProteinAssay(Bio-Rad,Hercules,CA,USA)basedonthemethodofBradford[76].
4.
3.
InVitroAutoradiographywith[125I]Iodo-ASEMInitialoptimizationofassayconditionswasperformedtomaximizetotalbindingwhilekeepingnon-specicbindinglow.
AdjustmentsincludedbuercompositionandpH,detergentconcentration,washandincubationtimeandtemperature.
Anassaybuerwith50mMol/LTris-HClpH7.
4,21C(termedTris-HClbuer)providedbestpreservedtissueintegrityandlowestnon-specicbinding,ascomparedtophysiologicTris,Tris-EDTA-EGTAorHEPES-KRHbuer(datanotshown).
Forallfurtherexperiments,tissuefrom1–2animalswasused,withexperimentsandquanticationscarriedoutusing3–4sectionsforpigandrattissueand3–6sectionsformousetissue.
Adjacentsectionswereusedforautoradiographyforallsimilarexperiments(e.
g.
saturationbinding).
Sectionswerebroughttoroomtemperatureandpre-incubatedfor20mininTris-HClbuer(pH7.
4,21C),thenincubatedfor60mininthesamebuer(21C)containing1.
5%TritonX-100(v/v)and0.
5-1.
0nMol/L[125I]Iodo-ASEMinahumidiedchamber.
Non-specicbindingof[125I]Iodo-ASEMwasassessedinthepresenceof10Mol/LSSR-180,711,addedtothebuer.
Blockingof[125I]Iodo-ASEMbindingwasinvestigatedwithaseriesofindividualα7nAChRselectivecompounds(10Mol/L)addedtotheincubationbuer,i.
e.
,methyllycaconitine(MLA)[77],SSR-180,711[63],NS14492[31],TC-5619[64],EVP-6124[65],A-582941[62],andGTS-21[66].
Followingincubation,slideswererinsedinTris-HClbuer(pH7.
4,21C),washed2*5mininTris-HClbuer(pH7.
4,4C)andrinsed(5sec)inice-colddistilledwater.
Slidesweregentlydriedunderanairstreamandexposedto4%paraformaldehydevapourovernightat4C,followedbyanotherdryingstepinadesiccatorfor1h.
Adjacentsectionswereusedforautoradiography.
4.
4.
InVitroAutoradiographywith[125I]α-bungarotoxinSlideswerethawedatroomtemperature(21C)for30min,followedby30minofrehydrationin50mMol/LTrisbuerwith0.
1%BSA(w/v),pH7.
3(bindingbuer).
Forassessmentofα-bungarotoxinbinding,thebindingbuercontained0.
5mMol/L[125I]α-bungarotoxinand4.
5nMol/Lunlabelledα-bungarotoxin(Tocris,Denmark)yieldingatotalof5nMol/Lα-bungarotoxin(incubationbuer).
Totalbindingwasdeterminedusingonesetofslidesincubatedwiththeradioligandfor2hatroomtemperatureinahumidiedchamber.
Non-specicbindingwasdeterminedinthepresenceMolecules2020,25,142512of19of1mMol/L(-)-nicotineaddedtotheincubationbuer.
Afterwards,slideswerebrieyrinsedinbindingbuer,followedby2*30minofwashinginice-coldbindingbuer(4C).
Finally,slideswerebrieyrinsed(5sec)inice-colddistilledwater,driedunderagentleairstreamandexposedto4%paraformaldehydevapourovernightat4C.
Onthenextday,theslidesweredriedfor1hinadesiccator.
4.
5.
SaturationBindingandKineticAnalysisUsingInVitroAutoradiographySaturationbindingwascarriedoutinratandpigbrainsectionsasdescribedabove(Section4.
3).
Sectionswereincubatedwithtenserialdilutionsof[125I]Iodo-ASEMrangingfrom0.
02to10nMol/L,withconcentrationsmeasuredbygamma-counting.
Non-specicbindingwasdeterminedinadjacentsectionsinthepresenceof10Mol/LSSR-180,711foreachradioligandconcentration.
Bindingwasterminatedbywashingthesectionsinice-coldbindingbuer.
Theequilibriumdissociationconstant(Kd)andmaximumnumberofbindingsites(Bmax)weredeterminedbynon-linearregressionanalysisofaone-sitesaturationbindingmodelusingGraphPadPrism6.
0(GraphPadSoftware,Inc.
,SanDiego,CA,USA).
4.
6.
AutoradiographicImageAcquisitionandAnalysisBASSR2040phosphorimagingplates(Fujilm,Toyko,Japan)wereexposedtothesamplesalongwith[125I]standards(ARI0133A;AmericanRadiolabeledChemicals,St.
Louis,USA)for24-72hours.
ImagingplateswerescannedusingaPhosphorImagerBAS-2500(FujilmEuropeGmbH,Düsseldorf,Germany).
ImageswereconvertedtoTIF-lesusingthemanufacturer'ssoftwareandanalysedinQuantityOne(BioRad,Waltham,MA,USA).
Regionsofinterest(ROIs)weredrawnovergreyandwhitematterstructures,dependingontheinvestigatedspecies.
Highintensitycircularspotswereoccasionallyobservedin[125I]Iodo-ASEMautoradiographsandwereexcludedfromtheanalysis.
Forα7andβ2gene-decientmice,onlyoneROIwasdrawnoverthewholebrain,againexcludingspotsandirregularwhitematterbinding.
Intheratbrain,theROIsweredrawnovercortexandhippocampus.
Fromthepigbrain,onlyfrontalcortexsectionswerecut,theROIsthereforecontainedthefrontalcortexandwhitemattertracts.
Themeanvaluesofopticaldensitypermm2(averagedfromthereplicates)wereconvertedtoradioactiveconcentrationusingalinearregressionderivedfromthe[125I]radioactivestandards.
Aglobalbackgroundoftheimagingplateandindividualnon-specicbindingweresubtracted.
Finalvalueswereexpressedasfmol/mgprotein,basedontheproteinmeasurementsfromindividualsections.
4.
7.
Radiosynthesisof[18F]ASEMTheradiosynthesisof[18F]ASEMwasperformedaspreviouslypublished[26].
No-carrier-addedaqueous18F-uoridefromthetargetwascollectedatanon-conditionedactivated(10mLethanol,20mLwateranddriedwithair)anion-exchangecartridge(QMA).
Asolutionof20mgof1,10-diaza-4,7,13,16,21,24-hexaoxabicyclo[8.
8.
8]hexacosane(Kryptox-222)and3.
3mgofK2CO3dissolvedina0.
65mLmethanol-watermixture(97/3v/v)wasusedtoelutethe18F-uorideothecartridge.
Theelutewasthereafterdriedbyevaporationat90Cundernitrogenandthenfurtherdriedtwicewith1mLdryacetonitrile.
TothedriedKryptox222/[18F]uoridecomplex,2.
4mg(0.
006mmol)/Lof3-(1,4-Diazabicyclo[3.
2.
2]nonan-4-yl)-6-nitrodibenzo[b,d]-thiophene5,5-Dioxidedissolvedin0.
8mLDMSOwasadded.
Thereactionwasperformedat160Cfor15minandafterwardsthecrudewasquenchedwith3.
5mLH2O.
Reactantsandby-productswereseparatedfrom[18F]ASEMbysemi-preparativeHPLC[Lunacolumn,PhenomenexLtd.
Aschaenburg,Germany;10mC18(2)10*250mmcolumn,owrate6mL/min,eluent:Ethanol/0.
1%H3PO4inwater(25:75)with6mMascorbicacidtopreventradiolysis].
Theretentiontimefor[18F]ASEMwas400-450sandtheproductwascollectedintoavialcontaining9mLofPBS(phosphate-bueredsaline).
Theproductwasvisuallyinspectedforclarity,absenceofcolourandvisibleparticles.
ChemicalandradiochemicalpuritieswereassessedbyanalyticalHPLC[Kinetexcolumn,PhenomenexLtd.
Aschaenburg,Germany;2.
6C18Molecules2020,25,142513of194.
60*50mm,eluent:ACN/0.
1%H3PO4inwater(25:75)RT:[18F]ASEM=1.
3min;nitroprecursor=1min;owrate1.
5mL/min].
Molaractivity(Am)oftheradiotracerwasdeterminedasfollows:theareaoftheUVabsorbancepeakcorrespondingtotheradiolabelledproductwasmeasured(integrated)ontheHPLCchromatogram.
Thisvaluewasthenconvertedintoamolarmassbycomparisonwithanaverageofintegratedareas(triplet)ofaknownstandardofthereferencecompound.
4.
8.
InVivoImaginginthePigThreefemalepigs(21,22and23kg)wereusedforinvivoPETimagingonaHRRTPETscanner(SiemensHealthcare,Erlangen,Germany).
AllanimalprocedureswereapprovedbytheDanishCouncilforAnimalEthics(journalno.
2012-15-2934-00156).
4.
8.
1.
AnimalProceduresBeforescanning,anaesthesiawasinducedwithi.
m.
injectionof0.
13mL/kgZoletilveterinarymixture(Virbac,Kolding,Denmark;10.
87mg/kgxylazine+10.
87mg/kgketamine+1.
74mg/kgmethadone+1.
74mg/kgbutorphanol+10.
87mg/kgtiletamine+10.
87mg/kgzolezepam).
Hereafter,anaesthesiawasmaintainedwithconstantpropofolinfusion(1.
5mg/kg/hintravenous(i.
v.
);B.
Braun,Melsungen,Germany).
Anarteriali.
v.
catheterwasemployedforbloodsamplingfromtherightfemoralarteryandtwovenousi.
v.
cathetersforinjectionswereplacedintheleftandrightmammaryveins.
Duringanaesthesia,animalswereendotracheallyintubatedandventilated.
Vitalparameters(heartrate,bodytemperature,bloodpressure,oxygensaturationandendtidalCO2)werecontinuouslymonitoredduringthescan.
4.
8.
2.
PETScanning[18F]ASEMwasgivenasintravenousi.
v.
bolus,withexperimentaldetailsdescribedinTable4.
Table4.
Experimentaldetailsof[18F]ASEMPETscansinpigs.
DetailsAnimal1Animal2Animal3TypeofexperimentBaselineBaselineSSR-180,711;1mg/kgScanlength240min90min150minMolaractivity20GBq/mol345GBq/mol388GBq/molInjectedactivity99MBq335MBq189MBqInjectedmass1.
78g0.
35g0.
18gFreeplasmafraction18%16%9%4.
8.
3.
BloodSamplingDuringtherst30minofthescans,radioactivityinthewholebloodwascontinuouslymeasuredusinganABSSautosampler(AlloggTechnology,Mariefred,Sweden)countingcoincidencesinalead-shieldeddetector.
Concurrently,arterialwholebloodwassampledmanuallyattimes2.
5,5,10,20,30,40,50,70,89,91,120and150minafterinjectionof[18F]ASEM.
Totalradioactivityinplasma(500L)andwholeblood(500L)wasmeasuredinawellcounter(Cobra5003;PackardInstruments,Meriden,CT,USA),whichwascross-calibratedtotheHRRTscannerandautosampler.
Allmeasurementsofradioactivityweredecaycorrectedtothetimeofradioligandinjection.
4.
8.
4.
MetaboliteAnalysisRadiolabelledparentcompoundandmetabolitesweredeterminedbydirectinjectionofplasmaintoaradio-HPLCsystem(DionexUltimate3000;ThermoFisherScientic,Hvidovre,Denmark)conguredforcolumnswitching.
Manuallydrawnarterialwholebloodsampleswerecentrifuged(1500g,7min,4C),andplasmawaslteredthroughasyringelter(WhatmanGD/X13mmor25mm,PVDFmembrane,0.
45mporesize;FrisenetteApS,Knebel,Denmark)priortotheanalysisbyHPLCMolecules2020,25,142514of19aspreviouslydescribed[78].
Toincreasesensitivityongammacountsfromsampleswithlowlevelsofradioactivity,eluentfromtheHPLCwascollectedintofractions(10mL)usingafractioncollector(FoxyJrFC144;Teledyne,Lincoln,NE,USA)andcountedoineinawellcounter(2480Wizard2AutomaticGammaCounter,WallacOy,Turku,Finland).
4.
8.
5.
DeterminationofFreeFractionThefree,non-proteinboundfractionof[18F]ASEMinpigplasma,fp,wasestimatedusinganequilibriumdialysischambermethodaspreviouslydescribed[79].
4.
8.
6.
ReconstructionandPre-ProcessingofPETData150-minutelist-modePETdatawerereconstructedin58dynamicframes(6*10,6*20,6*30,6*60,4*120,14*300,8*150,8*300s).
Oneanimalwasscannedfor240minusingthementionedframingprotocolbutadding9framesof600s).
Imagesconsistedof207planesof256*256voxelsof1.
22*1.
22*1.
22mm.
Asummedpictureofallcountsinthe150-minscanwasreconstructedforeachpigandusedforco-registrationtoastandardizedMRI-basedatlasofthedomesticpigbrain,similartothatpreviouslypublished[80].
Thetimeactivitycurveds(TACs)werecalculatedforthefollowingvolumesofinterest(VOIs):thalamus,striatum,hippocampus,cerebellum,whitematter,frontalcortex,somatosensorycortex,occipitalcortex,restofthecortex.
RadioactivityinallVOIswascalculatedastheaverageofradioactiveconcentration(Bq/mL)intheleftandrightsides.
OutcomemeasureintheTACswascalculatedasradioactiveconcentrationinVOI(inkBq/mL)normalizedtotheinjecteddosecorrectedforanimalweight(inkBq/kg),yieldingstandardizeduptakevalues(g/mL).
4.
8.
7.
KineticModellingofPETDataThePETimagingdatawereanalysedwiththeLogangraphicalanalysis(LGA)model,usingthemetabolitecorrectedarterialplasmaconcentrationtocalculatetheprimaryoutcomemeasure:totaldistributionvolume(VT).
ThesecondaryoutcomemeasurewasVTvaluescorrectedforfreefractioninplasma(VT/fP).
Theparentfractioncurvefor[18F]ASEMwasttedwithaWatabet.
Bothcurveswereconstrainedto1.
0attime=0.
KineticmodelingwasperformedinPMODversion3.
0(PMODTechnologies).
AuthorContributions:Conceptualization:C.
K.
D.
,J.
D.
M.
,A.
G.
H.
,M.
G.
P.
,D.
P;Methodology:C.
K.
D.
,H.
D.
H.
,G.
M.
K.
,M.
M.
H.
;Formalanalysis:C.
K.
D.
;H.
D.
H.
,H.
H.
H.
;Investigation:C.
K.
D.
;H.
D.
H.
,E.
T.
L.
;Resources:D.
P.
,R.
C.
M.
,M.
M.
H.
,A.
G.
H.
;Writing—originaldraftpreparation:C.
K.
D.
;H.
H.
H.
,H.
D.
H.
;Writing—reviewandediting:C.
K.
D.
,H.
D.
H.
,J.
D.
M.
,A.
G.
H.
,M.
G.
P.
,G.
M.
K.
,M.
M.
H.
,;Visualization:C.
K.
D.
,H.
D.
H.
;Supervision:J.
D.
M.
,G.
M.
K.
;Fundingacquisition:J.
D.
M.
,G.
M.
K.
,M.
G.
P.
;Allauthorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:ThisstudywassupportedbytheDanishStrategicResearchCouncil(projectCOGNITO),EB024495,theAugustinus,ElsassandTheLundbeckFoundations.
Acknowledgments:TheauthorswouldliketothanktheanimalfacilitiesattheDepartmentofExperimentalMedicine,UniversityofCopenhagen.
ConictsofInterest:Theauthorsdeclarenoconictofinterest.
References1.
Marutle,A.
;Zhang,X.
;Court,J.
;Piggott,M.
;Johnson,M.
;Perry,R.
;Perry,E.
;Nordberg,A.
Laminardistributionofnicotinicreceptorsubtypesincorticalregionsinschizophrenia.
J.
Chem.
Neuroanat.
2001,22,115–126.
[CrossRef]2.
Kulak,J.
M.
;Carroll,F.
I.
;Schneider,J.
S.
[125I]Iodomethyllycaconitinebindstoalpha7nicotinicacetylcholinereceptorsinmonkeybrain.
Eur.
J.
Neurosci.
2006,23,2604–2610.
[CrossRef][PubMed]Molecules2020,25,142515of193.
Whiteaker,P.
;Davies,A.
R.
;Marks,M.
J.
;Blagbrough,I.
S.
;Potter,B.
V.
;Wolstenholme,A.
J.
;Collins,A.
C.
;Wonnacott,S.
Anautoradiographicstudyofthedistributionofbindingsitesforthenovelalpha7-selectivenicotinicradioligand[3H]-methyllycaconitineinthemousebrain.
Eur.
J.
Neurosci.
1999,11,2689–2696.
[CrossRef][PubMed]4.
Quik,M.
;Choremis,J.
;Komourian,J.
;Lukas,R.
J.
;Puchacz,E.
Similaritybetweenratbrainnicotinicalpha-bungarotoxinreceptorsandstablyexpressedalpha-bungarotoxinbindingsites.
J.
Neurochem.
1996,67,145–154.
[CrossRef][PubMed]5.
Lendvai,B.
;Kassai,F.
;Szajli,A.
;Nemethy,Z.
alpha7nicotinicacetylcholinereceptorsandtheirroleincognition.
BrainRes.
Bull.
2013,93,86–96.
[CrossRef][PubMed]6.
Picciotto,M.
R.
;Lewis,A.
S.
;vanSchalkwyk,G.
I.
;Mineur,Y.
S.
Moodandanxietyregulationbynicotinicacetylcholinereceptors:Apotentialpathwaytomodulateaggressionandrelatedbehavioralstates.
Neuropharmacology2015,96,235–243.
[CrossRef][PubMed]7.
Albuquerque,E.
X.
;Pereira,E.
F.
;Alkondon,M.
;Rogers,S.
W.
Mammaliannicotinicacetylcholinereceptors:Fromstructuretofunction.
Physiol.
Rev.
2009,89,73–120.
[CrossRef]8.
Dani,J.
A.
;Bertrand,D.
Nicotinicacetylcholinereceptorsandnicotiniccholinergicmechanismsofthecentralnervoussystem.
AnnuRevPharm.
Toxicol2007,47,699–729.
[CrossRef]9.
Maurer,S.
V.
;Williams,C.
L.
TheCholinergicSystemModulatesMemoryandHippocampalPlasticityviaItsInteractionswithNon-NeuronalCells.
FrontImmunol.
2017,8,1489.
[CrossRef]10.
Bosmans,G.
;ShimizuBassi,G.
;Florens,M.
;Gonzalez-Dominguez,E.
;Matteoli,G.
;Boeckxstaens,G.
E.
CholinergicModulationofType2ImmuneResponses.
FrontImmunol.
2017,8,1873.
[CrossRef]11.
Freedman,R.
;Hall,M.
;Adler,L.
E.
;Leonard,S.
Evidenceinpostmortembraintissuefordecreasednumbersofhippocampalnicotinicreceptorsinschizophrenia.
Biol.
Psychiatry1995,38,22–33.
[CrossRef]12.
Guillozet-Bongaarts,A.
L.
;Hyde,T.
M.
;Dalley,R.
A.
;Hawrylycz,M.
J.
;Henry,A.
;Hof,P.
R.
;Hohmann,J.
;Jones,A.
R.
;Kuan,C.
L.
;Royall,J.
;etal.
Alteredgeneexpressioninthedorsolateralprefrontalcortexofindividualswithschizophrenia.
Mol.
Psychiatry2014,19,478–485.
[CrossRef]13.
Kunii,Y.
;Zhang,W.
;Xu,Q.
;Hyde,T.
M.
;McFadden,W.
;Shin,J.
H.
;Deep-Soboslay,A.
;Ye,T.
;Li,C.
;Kleinman,J.
E.
;etal.
CHRNA7andCHRFAM7AmRNAs:Co-LocalizedandTheirExpressionLevelsAlteredinthePostmortemDorsolateralPrefrontalCortexinMajorPsychiatricDisorders.
Am.
J.
Psychiatry2015.
[CrossRef][PubMed]14.
Thomsen,M.
S.
;Weyn,A.
;Mikkelsen,J.
D.
Hippocampalalpha7nicotinicacetylcholinereceptorlevelsinpatientswithschizophrenia,bipolardisorder,ormajordepressivedisorder.
BipolarDisord.
2011,13,701–707.
[CrossRef][PubMed]15.
Sugaya,K.
;Giacobini,E.
;Chiappinelli,V.
A.
Nicotinicacetylcholinereceptorsubtypesinhumanfrontalcortex:ChangesinAlzheimer'sdisease.
J.
Neurosci.
Res.
1990,27,349–359.
[CrossRef]16.
Araud,T.
;Graw,S.
;Berger,R.
;Lee,M.
;Neveu,E.
;Bertrand,D.
;Leonard,S.
ThechimericgeneCHRFAM7A,apartialduplicationoftheCHRNA7gene,isadominantnegativeregulatorofalpha7*nAChRfunction.
Biochem.
Pharmacol.
2011,82,904–914.
[CrossRef]17.
Gillentine,M.
A.
;Lozoya,R.
;Yin,J.
;Grochowski,C.
M.
;White,J.
J.
;Schaaf,C.
P.
;Calarge,C.
A.
CHRNA7copynumbergainsareenrichedinadolescentswithmajordepressiveandanxietydisorders.
J.
Aect.
Disord.
2018,239,247–252.
[CrossRef]18.
Sinkus,M.
L.
;Graw,S.
;Freedman,R.
;Ross,R.
G.
;Lester,H.
A.
;Leonard,S.
ThehumanCHRNA7andCHRFAM7Agenes:Areviewofthegenetics,regulation,andfunction.
Neuropharmacology2015.
[CrossRef]19.
Gillentine,M.
A.
;Berry,L.
N.
;Goin-Kochel,R.
P.
;Ali,M.
A.
;Ge,J.
;Guey,D.
;Rosenfeld,J.
A.
;Hannig,V.
;Bader,P.
;Proud,M.
;etal.
TheCognitiveandBehavioralPhenotypesofIndividualswithCHRNA7Duplications.
J.
AutismDev.
Disord.
2017,47,549–562.
[CrossRef]20.
Hua,S.
;Ek,C.
J.
;Mallard,C.
;Johansson,M.
E.
Perinatalhypoxia-ischemiareducesalpha7nicotinicreceptorexpressionandselectivealpha7nicotinicreceptorstimulationsuppressesinammationandpromotesmicroglialMoxphenotype.
BiomedRes.
Int.
2014,2014,718769.
[CrossRef]21.
Han,Z.
;Li,L.
;Wang,L.
;Degos,V.
;Maze,M.
;Su,H.
Alpha-7nicotinicacetylcholinereceptoragonisttreatmentreducesneuroinammation,oxidativestress,andbraininjuryinmicewithischemicstrokeandbonefracture.
J.
Neurochem.
2014,131,498–508.
[CrossRef][PubMed]Molecules2020,25,142516of1922.
Dash,P.
K.
;Zhao,J.
;Kobori,N.
;Redell,J.
B.
;Hylin,M.
J.
;Hood,K.
N.
;Moore,A.
N.
ActivationofAlpha7CholinergicNicotinicReceptorsReduceBlood-BrainBarrierPermeabilityfollowingExperimentalTraumaticBrainInjury.
J.
Neurosci.
2016,36,2809–2818.
[CrossRef][PubMed]23.
Mavropoulos,S.
A.
;Khan,N.
S.
;Levy,A.
C.
J.
;Faliks,B.
T.
;Sison,C.
P.
;Pavlov,V.
A.
;Zhang,Y.
;Ojamaa,K.
Nicotinicacetylcholinereceptor-mediatedprotectionoftheratheartexposedtoischemiareperfusion.
Mol.
Med.
2017,23.
[CrossRef][PubMed]24.
Gatson,J.
W.
;Simpkins,J.
W.
;Uteshev,V.
V.
Hightherapeuticpotentialofpositiveallostericmodulationofalpha7nAChRsinaratmodeloftraumaticbraininjury:Proof-of-concept.
BrainRes.
Bull.
2015,112,35–41.
[CrossRef][PubMed]25.
Pike,V.
W.
ConsiderationsintheDevelopmentofReversiblyBindingPETRadioligandsforBrainImaging.
Nicotin42016,23,1818–1869.
26.
Gao,Y.
;Kellar,K.
J.
;Yasuda,R.
P.
;Tran,T.
;Xiao,Y.
;Dannals,R.
F.
;Horti,A.
G.
Derivativesofdibenzothiopheneforpositronemissiontomographyimagingofalpha7-nicotinicacetylcholinereceptors.
J.
Med.
Chem.
2013,56,7574–7589.
[CrossRef]27.
Toyohara,J.
;Hashimoto,K.
α7NicotinicReceptorAgonists:PotentialTherapeuticDrugsforTreatmentofCognitiveImpairmentsinSchizophreniaandAlzheimer'sDisease.
OpenMed.
Chem.
J.
2010,4,37–56.
[CrossRef]28.
Hashimoto,K.
;Nishiyama,S.
;Ohba,H.
;Matsuo,M.
;Kobashi,T.
;Takahagi,M.
;Iyo,M.
;Kitashoji,T.
;Tsukada,H.
[11C]CHIBA-1001asanovelPETligandforα7nicotinicreceptorsinthebrain:APETstudyinconsciousmonkeys.
PLoSONE2008,3,e3231.
[CrossRef]29.
Rtering,S.
;Scheunemann,M.
;Fischer,S.
;Hiller,A.
;Peters,D.
;Deuther-Conrad,W.
;Brust,P.
Radiosynthesisandrstevaluationinmiceof[(18)F]NS14490formolecularimagingofalpha7nicotinicacetylcholinereceptors.
BioorganicMed.
Chem.
2013,21,2635–2642.
30.
Kim,S.
W.
;Ding,Y.
S.
;Alexo,D.
;Patel,V.
;Logan,J.
;Lin,K.
S.
;Shea,C.
;Muench,L.
;Xu,Y.
;Carter,P.
;etal.
SynthesisandpositronemissiontomographystudiesofC-11-labeledisotopomersandmetabolitesofGTS-21,apartialalpha7nicotiniccholinergicagonistdrug.
Nucl.
Med.
Biol.
2007,34,541–551.
[CrossRef]31.
Ettrup,A.
;Mikkelsen,J.
D.
;Lehel,S.
;Madsen,J.
;Nielsen,E.
O.
;Palner,M.
;Timmermann,D.
B.
;Peters,D.
;Knudsen,G.
M.
11C-NS14492asanovelPETradioligandforimagingcerebralalpha7nicotinicacetylcholinereceptors:Invivoevaluationanddrugoccupancymeasurements.
J.
Nucl.
Med.
2011,52,1449–1456.
[CrossRef][PubMed]32.
Deuther-Conrad,W.
;Fischer,S.
;Hiller,A.
;Becker,G.
;Cumming,P.
;Xiong,G.
;Funke,U.
;Sabri,O.
;Peters,D.
;Brust,P.
Assessmentofalpha7nicotinicacetylcholinereceptoravailabilityinjuvenilepigbrainwith[(18)F]NS10743.
Eur.
J.
Nucl.
Med.
Mol.
Imaging2011.
[CrossRef][PubMed]33.
Ouach,A.
;Vercouillie,J.
;Bertrand,E.
;Rodrigues,N.
;Pin,F.
;Serriere,S.
;Boiaryna,L.
;Chartier,A.
;Percina,N.
;Tangpong,P.
;etal.
Bis(het)aryl-1,2,3-triazolequinuclidinesasalpha7nicotinicacetylcholinereceptorligands:Synthesis,structureanityrelationships,agonismactivity,[(18)F]-radiolabelingandPETstudyinrats.
Eur.
J.
Med.
Chem.
2019,179,449–469.
[CrossRef][PubMed]34.
Huan,W.
;Aiqin,W.
;Jianping,L.
;Qianqian,X.
;Xia,L.
;Lei,Y.
;Yu,F.
;Huabei,Z.
Radiosynthesisandin-vivoevaluationof[125I]IBT:Asingle-photonemissioncomputedtomographyradiotracerforalpha7-nicotinicacetylcholinereceptorimaging.
Nucl.
Med.
Commun.
2017,38,683–693.
[CrossRef][PubMed]35.
Wang,S.
;Fang,Y.
;Wang,H.
;Gao,H.
;Jiang,G.
;Liu,J.
;Xue,Q.
;Qi,Y.
;Cao,M.
;Qiang,B.
;etal.
Design,synthesisandbiologicalevaluationof1,4-Diazobicylco[3.
2.
2]nonanederivativesasalpha7-NicotinicacetylcholinereceptorPET/CTimagingagentsandagonistsforAlzheimer'sdisease.
Eur.
J.
Med.
Chem.
2018,159,255–266.
[CrossRef][PubMed]36.
Teodoro,R.
;Scheunemann,M.
;Wenzel,B.
;Peters,D.
;Deuther-Conrad,W.
;Brust,P.
Synthesisandradiouorinationofnoveluoren-9-onebasedderivativesfortheimagingofalpha7nicotinicacetylcholinereceptorwithPET.
Bioorg.
Med.
Chem.
Lett.
2018,28,1471–1475.
[CrossRef]37.
Sarasamkan,J.
;Scheunemann,M.
;Apaijai,N.
;Palee,S.
;Parichatikanond,W.
;Arunrungvichian,K.
;Fischer,S.
;Chattipakorn,S.
;Deuther-Conrad,W.
;Schuurmann,G.
;etal.
VaryingChiralityAcrossNicotinicAcetylcholineReceptorSubtypes:SelectiveBindingofQuinuclidineTriazoleCompounds.
AcsMed.
Chem.
Lett.
2016,7,890–895.
[CrossRef]Molecules2020,25,142517of1938.
Schrimpf,M.
R.
;Sippy,K.
B.
;Briggs,C.
A.
;Anderson,D.
J.
;Li,T.
;Ji,J.
;Frost,J.
M.
;Surowy,C.
S.
;Bunnelle,W.
H.
;Gopalakrishnan,M.
;etal.
SARofalpha7nicotinicreceptoragonistsderivedfromtilorone:Explorationofanovelnicotinicpharmacophore.
Bioorg.
Med.
Chem.
Lett.
2012,22,1633–1638.
[CrossRef]39.
Horti,A.
G.
;Gao,Y.
;Kuwabara,H.
;Wang,Y.
;Abazyan,S.
;Yasuda,R.
P.
;Tran,T.
;Xiao,Y.
;Sahibzada,N.
;Holt,D.
P.
;etal.
18F-ASEM,aradiolabeledantagonistforimagingthealpha7-nicotinicacetylcholinereceptorwithPET.
J.
Nucl.
Med.
2014,55,672–677.
[CrossRef]40.
Teodoro,R.
;Scheunemann,M.
;Deuther-Conrad,W.
;Wenzel,B.
;Fasoli,F.
M.
;Gotti,C.
;Kranz,M.
;Donat,C.
K.
;Patt,M.
;Hillmer,A.
;etal.
APromisingPETTracerforImagingofalpha(7)NicotinicAcetylcholineReceptorsintheBrain:Design,Synthesis,andInVivoEvaluationofaDibenzothiophene-BasedRadioligand.
Molecules2015,20,18387–18421.
[CrossRef]41.
Horti,A.
G.
Developmentof[(18)F]ASEM,aspecicradiotracerforquanticationofthealpha7-nAChRwithpositron-emissiontomography.
Biochem.
Pharmacol.
2015,97,566–575.
[CrossRef][PubMed]42.
Gao,Y.
;Mease,R.
C.
;Olson,T.
T.
;Kellar,K.
J.
;Dannals,R.
F.
;Pomper,M.
G.
;Horti,A.
G.
[(125)I]Iodo-ASEM,aspecicinvivoradioligandforalpha7-nAChR.
Nucl.
Med.
Biol.
2015,42,488–493.
[CrossRef][PubMed]43.
Wong,D.
F.
;Kuwabara,H.
;Pomper,M.
;Holt,D.
P.
;Brasic,J.
R.
;George,N.
;Frolov,B.
;Willis,W.
;Gao,Y.
;Valentine,H.
;etal.
Humanbrainimagingofalpha7nAChRwith[(18)F]ASEM:AnewPETradiotracerforneuropsychiatryanddeterminationofdrugoccupancy.
WinnieBbb132014,16,730–738.
44.
Hillmer,A.
T.
;Li,S.
;Zheng,M.
Q.
;Scheunemann,M.
;Lin,S.
F.
;Nabulsi,N.
;Holden,D.
;Pracitto,R.
;Labaree,D.
;Ropchan,J.
;etal.
PETimagingofalpha7nicotinicacetylcholinereceptors:Acomparativestudyof[18F]ASEMand[18F]DBT-10innonhumanprimates,andfurtherevaluationof[18F]ASEMinhumans.
Eur.
J.
Nucl.
Med.
Mol.
Imaging2017,44,1042–1050.
[CrossRef]45.
Hillmer,A.
T.
;Zheng,M.
Q.
;Li,S.
;Scheunemann,M.
;Lin,S.
F.
;Holden,D.
;Labaree,D.
;Ropchan,J.
;Teodoro,R.
;Deuther-Conrad,W.
;etal.
PETimagingevaluationof[(18)F]DBT-10,anovelradioligandspecictoalpha7nicotinicacetylcholinereceptors,innonhumanprimates.
Eur.
J.
Nucl.
Med.
Mol.
Imaging2016,43,537–547.
[CrossRef]46.
Wong,D.
F.
;Kuwabara,H.
;Horti,A.
G.
;Roberts,J.
M.
;Nandi,A.
;Cascella,N.
;Brasic,J.
;Weerts,E.
M.
;Kitzmiller,K.
;Phan,J.
A.
;etal.
BrainPETImagingofalpha7-nAChRwith[18F]ASEM:Reproducibility,Occupancy,ReceptorDensity,andChangesinSchizophrenia.
Int.
J.
Neuropsychopharmacol.
/O.
Sci.
J.
Coll.
Int.
Neuropsychopharmacol.
2018,21,656–667.
47.
Coughlin,J.
M.
;Du,Y.
;Rosenthal,H.
B.
;Slania,S.
;MinKoo,S.
;Park,A.
;Solomon,G.
;Vranesic,M.
;Antonsdottir,I.
;Speck,C.
L.
;etal.
Thedistributionofthealpha7nicotinicacetylcholinereceptorinhealthyaging:Aninvivopositronemissiontomographystudywith[(18)F]ASEM.
NeuroImage2017,165,118–124.
[CrossRef]48.
Coughlin,J.
;Du,Y.
;Crawford,J.
L.
;Rubin,L.
H.
;BehnamAzad,B.
;Lesniak,W.
G.
;Horti,A.
G.
;Schretlen,D.
J.
;Sawa,A.
;Pomper,M.
G.
Theavailabilityofthealpha7nicotinicacetylcholinereceptorinrecent-onsetpsychosis:Astudyusing(18)F-ASEMPET.
J.
Nucl.
Med.
2018.
[CrossRef]49.
Coughlin,J.
M.
;Rubin,L.
H.
;Du,Y.
;Rowe,S.
P.
;Crawford,J.
L.
;Rosenthal,H.
B.
;Frey,S.
M.
;Marshall,E.
S.
;Shinehouse,L.
K.
;Chen,A.
;etal.
Highavailabilityofthealpha7nicotinicacetylcholinereceptorinbrainsofindividualswithmildcognitiveimpairment:Apilotstudyusing(18)F-ASEMPET.
J.
Nucl.
Med.
2019.
[CrossRef]50.
Vetel,S.
;Vercouillie,J.
;Buron,F.
;Vergote,J.
;Tauber,C.
;Busson,J.
;Chicheri,G.
;Routier,S.
;Serriere,S.
;Chalon,S.
LongitudinalPETImagingofalpha7NicotinicAcetylcholineReceptorswith[(18)F]ASEMinaRatModelofParkinson'sDisease.
WinnieBbb132019.
[CrossRef]51.
Wu,J.
;Liu,Q.
;Tang,P.
;Mikkelsen,J.
D.
;Shen,J.
;Whiteaker,P.
;Yakel,J.
L.
Heteromericalpha7beta2NicotinicAcetylcholineReceptorsintheBrain.
TrendsPharm.
Sci.
2016,37,562–574.
[CrossRef][PubMed]52.
Thomsen,M.
S.
;Zwart,R.
;Ursu,D.
;Jensen,M.
M.
;Pinborg,L.
H.
;Gilmour,G.
;Wu,J.
;Sher,E.
;Mikkelsen,J.
D.
alpha7andbeta2NicotinicAcetylcholineReceptorSubunitsFormHeteromericReceptorComplexesthatAreExpressedintheHumanCortexandDisplayDistinctPharmacologicalProperties.
PLoSONE2015,10,e0130572.
[CrossRef][PubMed]53.
Moretti,M.
;Zoli,M.
;George,A.
A.
;Lukas,R.
J.
;Pistillo,F.
;Maskos,U.
;Whiteaker,P.
;Gotti,C.
Thenovelalpha7beta2-nicotinicacetylcholinereceptorsubtypeisexpressedinmouseandhumanbasalforebrain:Biochemicalandpharmacologicalcharacterization.
Mol.
Pharmacol.
2014,86,306–317.
[CrossRef][PubMed]Molecules2020,25,142518of1954.
Zwart,R.
;Strotton,M.
;Ching,J.
;Astles,P.
C.
;Sher,E.
Uniquepharmacologyofheteromericalpha7beta2nicotinicacetylcholinereceptorsexpressedinXenopuslaevisoocytes.
Eur.
J.
Pharmacol.
2014,726C,77–86.
[CrossRef]55.
Patel,S.
;Hamill,T.
G.
;Connolly,B.
;Jagoda,E.
;Li,W.
;Gibson,R.
E.
SpeciesdierencesinmGluR5bindingsitesinmammaliancentralnervoussystemdeterminedusinginvitrobindingwith[18F]F-PEB.
Pet§Cholin22007,34,1009–1017.
[CrossRef]56.
Fujita,M.
;Imaizumi,M.
;Zoghbi,S.
S.
;Fujimura,Y.
;Farris,A.
G.
;Suhara,T.
;Hong,J.
;Pike,V.
W.
;Innis,R.
B.
Kineticanalysisinhealthyhumansofanovelpositronemissiontomographyradioligandtoimagetheperipheralbenzodiazepinereceptor,apotentialbiomarkerforinammation.
NeuroImage2008,40,43–52.
[CrossRef]57.
VandeBittner,G.
C.
;Ricq,E.
L.
;Hooker,J.
M.
AphilosophyforCNSradiotracerdesign.
AccChem.
Res.
2014,47,3127–3134.
[CrossRef]58.
Laruelle,M.
;Slifstein,M.
;Huang,Y.
Relationshipsbetweenradiotracerpropertiesandimagequalityinmolecularimagingofthebrainwithpositronemissiontomography.
Nicotin42003,5,363–375.
[CrossRef]59.
Magnussen,J.
H.
;Ettrup,A.
;Donat,C.
K.
;Peters,D.
;Pedersen,M.
H.
F.
;Knudsen,G.
M.
;Mikkelsen,J.
D.
Radiosynthesisandinvitrovalidationof3H-NS14492asanovelhighanityα7nicotinicacetylcholinereceptorradioligandMol.
Cell.
Neurosci.
2014.
[CrossRef]60.
Anderson,D.
J.
;Bunnelle,W.
;Surber,B.
;Du,J.
;Surowy,C.
;Tribollet,E.
;Marguerat,A.
;Bertrand,D.
;Gopalakrishnan,M.
[3H]A-585539[(1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.
2.
1]heptane],anovelhigh-anityalpha7neuronalnicotinicreceptoragonist:Radioligandbindingcharacterizationtoratandhumanbrain.
J.
Pharmacol.
Exp.
Ther.
2008,324,179–187.
[CrossRef]61.
Magnussen,J.
H.
;Ettrup,A.
;Donat,C.
K.
;Peters,D.
;Pedersen,M.
H.
;Knudsen,G.
M.
;Mikkelsen,J.
D.
Radiosynthesisandinvitrovalidationof(3)H-NS14492asanovelhighanityalpha7nicotinicreceptorradioligand.
Eur.
J.
Pharmacol.
2015,762,35–41.
[CrossRef][PubMed]62.
Bitner,R.
S.
;Bunnelle,W.
H.
;Anderson,D.
J.
;Briggs,C.
A.
;Buccafusco,J.
;Curzon,P.
;Decker,M.
W.
;Frost,J.
M.
;Gronlien,J.
H.
;Gubbins,E.
;etal.
Broad-spectrumecacyacrosscognitivedomainsbyalpha7nicotinicacetylcholinereceptoragonismcorrelateswithactivationofERK1/2andCREBphosphorylationpathways.
J.
Neurosci.
2007,27,10578–10587.
[CrossRef][PubMed]63.
Biton,B.
;Bergis,O.
E.
;Galli,F.
;Nedelec,A.
;Lochead,A.
W.
;Jegham,S.
;Godet,D.
;Lanneau,C.
;Santamaria,R.
;Chesney,F.
;etal.
SSR180711,anovelselectivealpha7nicotinicreceptorpartialagonist:(1)bindingandfunctionalprole.
Neuropsychopharmacology2007,32,1–16.
[CrossRef][PubMed]64.
Hauser,T.
A.
;Kucinski,A.
;Jordan,K.
G.
;Gatto,G.
J.
;Wersinger,S.
R.
;Hesse,R.
A.
;Stachowiak,E.
K.
;Stachowiak,M.
K.
;Papke,R.
L.
;Lippiello,P.
M.
;etal.
TC-5619:Analpha7neuronalnicotinicreceptor-selectiveagonistthatdemonstratesecacyinanimalmodelsofthepositiveandnegativesymptomsandcognitivedysfunctionofschizophrenia.
Biochem.
Pharmacol.
2009,78,803–812.
[CrossRef][PubMed]65.
Prickaerts,J.
;vanGoethem,N.
P.
;Chesworth,R.
;Shapiro,G.
;Boess,F.
G.
;Methfessel,C.
;Reneerkens,O.
A.
;Flood,D.
G.
;Hilt,D.
;Gawryl,M.
;etal.
EVP-6124,anovelandselectivealpha7nicotinicacetylcholinereceptorpartialagonist,improvesmemoryperformancebypotentiatingtheacetylcholineresponseofalpha7nicotinicacetylcholinereceptors.
Neuropharmacology2012,62,1099–1110.
[CrossRef][PubMed]66.
Meyer,E.
M.
;Tay,E.
T.
;Papke,R.
L.
;Meyers,C.
;Huang,G.
L.
;deFiebre,C.
M.
3-[2,4-Dimethoxybenzylidene]anabaseine(DMXB)selectivelyactivatesratalpha7receptorsandimprovesmemory-relatedbehaviorsinamecamylamine-sensitivemanner.
BrainRes.
1997,768,49–56.
[CrossRef]67.
Nielsen,B.
E.
;Minguez,T.
;Bermudez,I.
;Bouzat,C.
Molecularfunctionofthenovelα7β2nicotinicreceptor.
Cell.
Mol.
LifeSci.
Cmls2018,75,2457–2471.
[CrossRef]68.
Liu,Q.
;Huang,Y.
;Shen,J.
;Steensen,S.
;Wu,J.
Functionalalpha7beta2nicotinicacetylcholinereceptorsexpressedinhippocampalinterneuronsexhibithighsensitivitytopathologicallevelofamyloidbetapeptides.
BmcNeurosci.
2012,13,155.
[CrossRef]69.
Liu,Q.
;Huang,Y.
;Xue,F.
;Simard,A.
;DeChon,J.
;Li,G.
;Zhang,J.
;Lucero,L.
;Wang,M.
;Sierks,M.
;etal.
Anovelnicotinicacetylcholinereceptorsubtypeinbasalforebraincholinergicneuronswithhighsensitivitytoamyloidpeptides.
J.
Neurosci.
2009,29,918–929.
[CrossRef]Molecules2020,25,142519of1970.
Khiroug,S.
S.
;Harkness,P.
C.
;Lamb,P.
W.
;Sudweeks,S.
N.
;Khiroug,L.
;Millar,N.
S.
;Yakel,J.
L.
RatnicotinicAChreceptoralpha7andbeta2subunitsco-assembletoformfunctionalheteromericnicotinicreceptorchannels.
JPhysiol2002,540,425–434.
[CrossRef]71.
Murray,T.
A.
;Bertrand,D.
;Papke,R.
L.
;George,A.
A.
;Pantoja,R.
;Srinivasan,R.
;Liu,Q.
;Wu,J.
;Whiteaker,P.
;Lester,H.
A.
;etal.
alpha7beta2nicotinicacetylcholinereceptorsassemble,function,andareactivatedprimarilyviatheiralpha7-alpha7interfaces.
Mol.
Pharmacol.
2012,81,175–188.
[CrossRef][PubMed]72.
Mowrey,D.
D.
;Liu,Q.
;Bondarenko,V.
;Chen,Q.
;Seyoum,E.
;Xu,Y.
;Wu,J.
;Tang,P.
Insightsintodistinctmodulationofα7andα7β2nicotinicacetylcholinereceptorsbythevolatileanestheticisourane.
J.
Biol.
Chem.
2013,288,35793–35800.
[CrossRef][PubMed]73.
Teodoro,R.
;Moldovan,R.
P.
;Lueg,C.
;Gunther,R.
;Donat,C.
K.
;Ludwig,F.
A.
;Fischer,S.
;Deuther-Conrad,W.
;Wunsch,B.
;Brust,P.
RadiouorinationandbiologicalevaluationofN-aryl-oxadiazolyl-propionamidesaspotentialradioligandsforPETimagingofcannabinoidCB2receptors.
Org.
Med.
Chem.
Lett.
2013,3,11.
[CrossRef][PubMed]74.
Innis,R.
B.
;Cunningham,V.
J.
;Delforge,J.
;Fujita,M.
;Gjedde,A.
;Gunn,R.
N.
;Holden,J.
;Houle,S.
;Huang,S.
-C.
;Ichise,M.
;etal.
Consensusnomenclatureforinvivoimagingofreversiblybindingradioligands.
J.
Cereb.
BloodFlowMetab:O.
J.
Int.
Soc.
Cereb.
BloodFlowMetab.
2007,27,1533–1539.
[CrossRef][PubMed]75.
Gallezot,J.
-D.
;Weinzimmer,D.
;Nabulsi,N.
;Lin,S.
-F.
;Fowles,K.
;Sandiego,C.
;McCarthy,T.
J.
;Maguire,R.
P.
;Carson,R.
E.
;Ding,Y.
-S.
Evaluationof[(11)C]MRBforassessmentofoccupancyofnorepinephrinetransporters:Studieswithatomoxetineinnon-humanprimates.
NeuroImage2011,56,268–279.
[CrossRef]76.
Bradford,M.
M.
Arapidandsensitivemethodforthequantitationofmicrogramquantitiesofproteinutilizingtheprincipleofprotein-dyebinding.
Anal.
Biochem.
1976,72,248–254.
[CrossRef]77.
Ward,J.
M.
;Cockcroft,V.
B.
;Lunt,G.
G.
;Smillie,F.
S.
;Wonnacott,S.
Methyllycaconitine:Aselectiveprobeforneuronalalpha-bungarotoxinbindingsites.
FebsLett.
1990,270,45–48.
[CrossRef]78.
Gillings,N.
Arestrictedaccessmaterialforrapidanalysisof[(11)C]-labeledradiopharmaceuticalsandtheirmetabolitesinplasma.
Nucl.
Med.
Biol.
2009,36,961–965.
[CrossRef]79.
Kornum,B.
R.
;Lind,N.
M.
;Gillings,N.
;Marner,L.
;Andersen,F.
;Knudsen,G.
M.
Evaluationofthenovel5-HT4receptorPETligand[11C]SB207145intheGottingenminipig.
J.
Cereb.
BloodFlowMetab.
2009,29,186–196.
[CrossRef]80.
Villadsen,J.
;Hansen,H.
D.
;Jorgensen,L.
M.
;Keller,S.
H.
;Andersen,F.
L.
;Petersen,I.
N.
;Knudsen,G.
M.
;Svarer,C.
AutomaticdelineationofbrainregionsonMRIandPETimagesfromthepig.
J.
Neurosci.
Methods2018,294,51–58.
[CrossRef]SampleAvailability:Samplesofthecompoundsarenotavailable.
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