mine transporter and not to the norepinephrine or the Se rotonin transporters(1). In vitro studies have shown that cocaine binds to both high- and low-affinity sites on the dopamine transporter. The range of Kd values is between 16 nM and 210 nM for high-affinitysites and between 660 riM and 26,400 nM for low-affinity binding sites (2—6).By using PET and [â€oeC]cocaine we demonstrated cocaine bindingto dopamine transportersin vivo (7). Because the studies with [â€oeC]cocaine were done at subpharmacological levels of cocaine, [â€oeC]cocaine's binding to the dopamine transporter probably represented high-affinity sites. This may, however, not be the only pharmacologically relevant binding site when cocaine is administered in behaviorally active, pharmacological doses. Recent studies using in vitro human brain autoradiography showed significantly differentprofflesofdistribution oftritiated cocaine at 1 j.tM concentration (8), which is similar to that found in the brains of cocaine abusers, than at 10 nM concentration which is in the range of the subpharmacological [‘ ‘C]co caine studies done with PET (7). At the higher concentra tion, binding is more homogeneous and low-affinity binding sites are observed in the hippocampus and in the temporal cortex. Although these low-affinity sites have yet to be characterized pharmacologically, they may play a signifi cant role in the pharmacological and toxicological spec trum of cocaine. The ability of PET to measure moment-to-moment changes in the distributionof positron-labeled compounds makes it an ideal technique to investigate binding charac teristics of pychoactive drugs in vivo. Since pharmacolog ical responses are observed in living subjects, it is impor tant to validate the binding parameters of drugs under in vivo conditions. Because of the possible relevance of low affinitysites in the behavioral, addictive and toxic proper ties ofcocaine, we used PET to examine low-affinitybind ing sites in the living baboon brain. For this purpose, we compared in vivo [â€oeC]cocaine bindingin the baboon brain at subpharmacological (18 pg) and at pharmacological(8 mg) doses. Pharmacologicaldoses were used to assess the degree of [â€oeC]cocainebinding to low-affinity sites. Per centage occupancy of high- and low-affinity cocaine bind We have charactenzedcocainebindinginthe brainto a high affinitysite on the dopamine transporter using PET and tracer doses of[11C]cocaineinthe baboon Invivo.Thebindingpattern, however,of cocaine at tracer (subpharmacological)doses may differfromthat observed when the drug is taken in beha@oratly active doses particularlysince invftrostudies have shown that cocaine also binds to lowaffinitybindingsites. Methods: PET was used to compare and characterize[11C]cocainebindingin the baboon brainat low subpharmacologlcal (18 @.mg average dose) and at pharmacological(8000 @.tg)doses. Serialstudies on the same day in the same baboon were used to assess the reprOdUablllty of repeated measures andto assess the effects of drugswhichinhibithedopammne, norepmnephrlne andserotonin transporters. Time-activitycurves from brain and the artenal plasmainputfunctionwere used to calculatethe steady-state distributionvolume (DV). Results: At subpharmacological doses, [11C]cocainehad a higherbindingand slowerdearance instnatum than inother brainregions.Atpharmacologicaidoses, [11C]cocainehad a more homogeneous distribution.Bmax/Kd forsub-pharmacological[11C]cocainecorresponded to 0.5-0.6 and for pharmacological[11Cjcocaineit corresponded to 0.1- 0.2.Two-pointScatchardanalysisgave Bmax= 2300 pmde/g and Kd'= 3600 nM.Bmax/Kdforsub-pharmacologicaldoses of [1 1C]cocaine was decreased by cocaine and drugs that inhibft thedopamine transporter, to0.1—0.2, butnotbydrugsthatinhibit the serotoninor the norepinephrine transporter. None of these drugs changed BmaQXdfora pharmacologicaldose of[11C]co caine. ConclusIon: Atsubpharmacologlcaidoses, [11C]cocaine bindspredominantlyto a high-affinity siteonthe dopaminetrans porter. KeyWords:cocaine; positron emission tomography; pharma cokinetics;low-affinitybindingsites;dopaminetransporters J Nuci Med 1995; 36:1289-lW ocaine is considered one of the most reinforcing and addictive drugs of abuse. The reinforcing properties of cocaine have been associated with its binding to the dopa ReceivedFeb. 28, 1994;revIsionacceptedSept. 29, 1994. Forcorrespondenceor rep@nt requestscont@ NoraD.Volkow, MD,Medoal Department@ BrookhavenNationalLaboratory, Upton,NewYorkI1973. 1289 Carbon-i 1-CocaineBindingat Subpharmacokgical and PharmacologicalDoses • Volkowet al Carbon-11-Cocaine Binding Compared at Subpharmacological and Pharmacological Doses: A PET Study Nora D. Volkow, Joanna S. Fowler, Jean Logan, S. John Gatley, Stephen L. Dewey, Robert R. MacGregor, David J. Schlyer, Naomi Pappas, Payton King, Gene-Jack Wang and Alfred P. Wolf Medical and Chemistry Departments, Brookhaven National Laboratory, Upton, New York; and Department ofPsychiatry, State University ofNew York, Stony Brook, New York by on April 8, 2018. For personal use only. jnm.snmjournals.org Downloaded from
Transcript
mine transporter and not to the norepinephrine or the Serotonin transporters(1). In vitro studies have shown thatcocaine binds to both high- and low-affinity sites on thedopamine transporter. The range of Kd values is between16 nM and 210 nM for high-affinitysites and between 660riM and 26,400 nM for low-affinity binding sites (2—6).Byusing PET and [“C]cocainewe demonstrated cocainebindingto dopamine transportersin vivo (7). Because thestudies with [“C]cocainewere done at subpharmacologicallevels of cocaine, [“C]cocaine'sbinding to the dopaminetransporter probably represented high-affinity sites. Thismay, however, not be the only pharmacologicallyrelevantbinding site when cocaine is administered in behaviorallyactive, pharmacological doses. Recent studies using invitro human brain autoradiography showed significantlydifferentprofflesofdistribution oftritiated cocaine at 1 j.tMconcentration (8), which is similar to that found in thebrains of cocaine abusers, than at 10 nM concentrationwhich is in the range of the subpharmacological [‘‘C]cocaine studies done with PET (7). At the higher concentration, binding is more homogeneous and low-affinity bindingsites are observed in the hippocampus and in the temporalcortex. Although these low-affinity sites have yet to becharacterized pharmacologically, they may play a significant role in the pharmacological and toxicological spectrumof cocaine.
The ability of PET to measure moment-to-momentchanges in the distributionof positron-labeledcompoundsmakes it an ideal technique to investigate binding characteristics of pychoactive drugs in vivo. Since pharmacological responses are observed in living subjects, it is important to validate the binding parameters of drugs under invivo conditions. Because of the possible relevance of lowaffinitysites in the behavioral, addictive and toxic properties ofcocaine, we used PET to examine low-affinitybinding sites in the living baboon brain. For this purpose, wecomparedin vivo [“C]cocainebindingin the baboon brainat subpharmacological(18 pg) and at pharmacological(8mg) doses. Pharmacologicaldoses were used to assess thedegree of [“C]cocainebinding to low-affinity sites. Percentage occupancy of high- and low-affinity cocaine bind
We have charactenzedcocaine bindingin the brainto a highaffinitysite on the dopamine transporter using PET and tracerdoses of [11C]cocaineinthe baboon Invivo.The bindingpattern,however,of cocaine at tracer (subpharmacological)doses maydifferfromthat observed when the drug is taken in beha@oratlyactive doses particularlysince in vftrostudies have shown thatcocaine also binds to low affinitybindingsites. Methods: PETwas used to compare and characterize [11C]cocainebindinginthe baboon brainat low subpharmacologlcal(18 @.mgaveragedose) and at pharmacological(8000 @.tg)doses.Serialstudiesonthe same day in the same baboonwere used to assess thereprOdUablllty of repeated measures andto assess the effects ofdrugswhichinhibitthe dopammne,norepmnephrlneandserotonintransporters. Time-activitycurves from brain and the artenalplasma inputfunctionwere used to calculatethe steady-statedistributionvolume (DV). Results: At subpharmacologicaldoses, [11C]cocainehad a higher bindingand slowerdearanceinstnatumthan inother brainregions.Atpharmacologicaidoses,[11C]cocainehad a more homogeneous distribution.Bmax/Kdfor sub-pharmacological[11C]cocainecorresponded to 0.5-0.6and for pharmacological[11Cjcocaineit corresponded to 0.1-0.2. Two-pointScatchardanalysisgave Bmax= 2300 pmde/gand Kd'= 3600 nM.Bmax/Kdforsub-pharmacologicaldoses of[1 1C]cocaine was decreased by cocaine and drugs that inhibft
thedopaminetransporter,to0.1—0.2,butnotbydrugsthatinhibitthe serotoninor the norepinephrinetransporter.None of thesedrugs changed BmaQXdfora pharmacologicaldose of [11C]cocaine. ConclusIon: Atsubpharmacologlcaidoses, [11C]cocainebindspredominantlytoa high-affinitysiteonthe dopaminetransporter.
ocaine is considered one of the most reinforcing andaddictive drugs of abuse. The reinforcing properties ofcocaine have been associated with its binding to the dopa
1289Carbon-i 1-CocaineBindingat Subpharmacokgical and PharmacologicalDoses •Volkowet al
Carbon-11-Cocaine Binding Compared atSubpharmacological and PharmacologicalDoses: A PET StudyNora D. Volkow, Joanna S. Fowler, Jean Logan, S. John Gatley, Stephen L. Dewey, Robert R. MacGregor,David J. Schlyer, Naomi Pappas, Payton King, Gene-Jack Wang and Alfred P. Wolf
Medical and Chemistry Departments, Brookhaven National Laboratory, Upton, New York;and Department ofPsychiatry, State University ofNew York, Stony Brook, New York
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S@es Region Kd(nM) (pmol>issue) BmaxFXd Ref (18 @g)(7500 ig)(15,000 @g)
*ReporI@ as I .47 pmol&mg protein (2).tReportadas42.2 @mgprotein(2)convertedtopmole/gtissuebymuWp@ngby100conaldedngprot&nCOntentOfthebraintobe 10%(36).
TABLE IPercent Oceupancy of High-and Low-AffinityBindingSites for Cocaine at Subpharmacological(18 @g)and PharmaCOkgical
Doses (0.5 mg/kgand 1.0 mg/kg)UsingEquation4 and StriatalUptakeof 0.05%Dose/cc as MeasuredwithPET
Highaffinity
Highaffk@hy
Lowaffinhy
Lowaffinity
Human
Nonhuman
Human
14r
28.3
431
0.7 2 7
1.47 3 41
0.16 2 0.06
0.38 3 1
98
99
30
91
Putamen 210
Caudate- 192@men
Putamen 26,400
99
100
46
96Nonhuman Caudate- 1120primate putamen
ing sites by the 18 @zgand the 8 mg doses were calculatedusing literaturevalues ofbinding parametersdeterminedinvitro. To compare the degree of specific versus nonspecfficbinding, we assessed the effects ofcocaine pretreatment onsubpharmacological and pharmacological doses of[―C]cocaine. To compare the degree of binding to serotonin,dopamine and norepinephrine transporters, we assessedthe effects of pretreatmentwith citalopram(9) and fluoxetine (10), nomifensine (11) and methylphenidate(12), anddesipramine (13) and tomoxetine (14), respectively, on[1 ‘C]cocaine binding at subpharrnacological and at pharma
cological doses.
METhODS
[N-―C-methyl]cocainewas preparedby the methylationofnorcocaine (7) using [“C]methyliodide prepared by the methodof Langstrom(15).Cocaineand norcocainewere suppliedby theNationalInstituteon DrugAbuse.
ScanningProtocolSevenadultfemalebaboons(Papioanubis)wereusedin this
investigation.A total of 16 pairedstudieswere done in theseanimals2 hr apart. The first scan was obtainedas a baselineandwas used to comparechangesindUcedby the pharmacologicalinterventionon the second scan. Paired studiesare requiredbecause of the largeintrasubjectvariabilitywhenscanningbaboonson separatedays(unpublisheddata).Baboonswereanesthetized,catheterized and prepared for the PET study as previously described (16). Imaging was performed on a CTI 931 tomograph(ComputerTechnologies,Inc., Knoxville,‘FN)(spatialresolutionof 6.5 mm x 5.9 mmFWHMat the centerof the fieldof view).Dynamic scans were started immediately after injection of a fastbolus of [“C]cocaine(5—8mCi; SA 100mCi4zmoleat time ofinjection) and were continued for a total of 54 rain. Sequence fordynamicscans correspondedto four 30-sec, four 60-nec,four120-sec and four 600-sec scans. For the subpharmacological doseof [“C]cocaine,an averageof 17.8 ±12.2pg of cocainewasinjected and for the pharmacological dose [“C]cocaine,8 mg ofunlabeledcocaine was coadministeredwith [“Cicocaine.A calculation ofthe percent occupancy ofhigh- and low-affinity sites at
these doses usingbindingparametersreportedin the literature(2,3) is presentedin Table 1. Vital signs(heartrate andbloodpressure) were monitored during the entire scanning period and
during drug administration.Pharmacological Challenge. To assess binding of [“Cjcocaine
at subpharmacologicalandatpharmacologicaldosesto thedopamine, serotonin and norepinephrine transporters, various pharmacologicalchallengeexperimentswere conducted.The drugs, theirdoses and the timingof their administrationfor these studiesaredescribedin Table2. In addition,a pairedstudywas also done tocompare subpharmacological and pharmacological dose of[“Cjcocainein the same baboon and two paired studies weredoneto assessthetest-retestreproducibilityforsubpharmacological and pharmacologicaldose of [“C]cocainewhen repeatedstudieswere done with no intervention.Test and re-test studieswere performedto serve as comparisonswith which to monitorthe magnitudeof the changesinducedby pharmacologicalinterventions.
Analyses. Procedures regarding blood sampling and quantification of total radioactivityand unchangedlabeledtracer in plasmawerepreviouslydescribed(7).
Regions for the corpus striatum,cerebellum, thalamus, cortexandmesencephalonwereobtainedinthesliceor sliceswheretheregions were identified(16). The radioactivityconcentration inthese regions of interest (ROIs) was used to obtain the timeactivity curve for regional tissue concentration. An approximatevalueforwholebrainuptakewas obtainedby averagingthe radioactivityin the fivecentral slices.
Time-activity curves for tissue concentration and for Unchangedtracerin plasmawereusedto calculatedthe plasmatotissue transport constant (K!) and the distribution volume (DV)for the variousregionsusing graphicalanalysisfor reversiblesystems(17). Forthecerebellum,theDV is givenby (18):
DV(CB)= K1/k2(1+ NS), Eq.!
whereNS representsthe ratioof nonspecificbindingconstants(17,19). For the striatum:
DV(ST)= K1/k2(1+ NS +@ (Bmax@'/Kd@, Eq. 2
wherei designatesthe typeof receptorandBmax'refersto thefree receptor concentration. The ratio of DVs for striatum tocerebellum is given by:
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TABLE 2Tabulation of Paired Baboon Stud@s with Subpharmaco1og@al and Pharmacological Doses of Carbon-I 1-Cocaine
DV(ST)IDV(CB)= 1+ @(Bmaxi'fKd1'),
Kd' = Kd(l + NS).
Eq. 3 where Bmax1 —RL1represents the concentration of free bindingsitesforreceptortypei andL representstheconcentrationof freecocaine in striatum. The total concentration of [cocaine] is given
The derivationof the equationsrelatingDV andBmax'/Kd'as- Underequilibriumconditions:sumeda constantfree receptorconcentration.Althoughthis doesnotapplyto mostof theexperimentsreportedhereinwhichdrugpretreatment and low specificactivity tracer result in changingfreebindingsiteconcentrationsover the courseofthe experiment,data analysiswith this methodproducedlinearplots. Thisallowsthe interpretationof changesin DVs fromthese experimentsinterms of changes in an effective free bindingsite concentration.
Resultswere also analyzedusingthe equilibriummethodofFarde et al. (20). By subtracting cerebeflar radioactivity from thatin striatum,the timeat which the rateofchange ofspecific bindingis zero can be determined, and at this time:
Estimation of Dopamine Transporters OccupancyBinding parameters from two different in vitro studies,
one with human putamen tissue (2) and one with monkeycaudate-putamen (3), report Kds of2IO tiM and 19 tiM forthe high-affinitysites and 26.4 @Mand I . 12 p@Mfor thelow-affinity sites for the human and monkey tissues, respectively. Using these values along with the masses ofcocaine administered, and the striatal uptake as measuredby PET, we calculate that a maximum of 7%-41% of thehigh-affinitysites are occupied when the subpharmacological dose was given while a maximum of 0. l%—l%of thelow-affinity sites would be occupied at this dose. At pharmacological doses (7.5—15.0mg), a maximum of99% of thehigh-affinity sites and a maximum of 30%-96% of the lowaffinity sites would be occupied. These occupancy estimates represent a “maximum―since these are not equilibrium conditions. Binding parameters and percentoccupancy are tabulated in Table 1. The large range ofoccupancies reflect a very large difference in in vitro bind
Eq. 5 ing parameters reported in these two studies (2,3).
where by:
Eq. 4 [cocaine]= L + RL@+ RL@. Eq. 6
Eq. 7
Eq.8
(Bmax1-RL1)ffcocaine]—RL, —RL,J = Kd1RL@,
(Bmax2-RL@J@[cocaine]—RL, —RL@)= Kd2RL@,
and % occupancy for each species is given by:
l0@RLj
Bmax1
RESULTS
RL (Bmax - RL)
@1@Kdwhere RL is the concentrationofbound cocaine andF thatof freecocaine. This allows a Scatchard-type analysis, assuming the cereheliumcan be used to represent the free concentration.
Inaddition,time-activitycurvesfortheratioof theactivityinstriatum over that in cerebellum were plotted. Only pharmacological interventionswhich inducedchangesthree times the size ofthe test-retestvariabilitywere consideredsignificant.Studentt-tests (unpaired) were used to compare peak uptake and K1values for subpharmacologicaland pharmacologicaldoses of[1 ‘C]cocaine.
Calculation of Receptor OccupancyA maximumexpectedoccupancyof cocainereceptorsin ba
boon striatum can be calculated for the high- and low-affinitybinding sites using values reported in the literature for the bindingparameters (Bmax and Kd) (2,3) for both sites and assumingequilibriumconditionsfor both sites. Under these conditions:
(Bmax1 - RL@)L= Kd@RL@,
1291Carbon-i 1-Cocaine Bindingat Subpharmacokgical and PharmacologicalDoses •Volkowet al.
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peakPeakactivityTime to peakdearancePeak activity%Time topeakdearanceRegions
%dos&ccactivity (Mn)(mm)Dose/ceactivity (mm)(mm)
•Valuesrepresent an average from 11 studies at an average injected mass of 18 pg and from 6 studIes with an injected mass of 8 mg of[11C)cocaine.Slgnrncantdifferencesinpeakuptakebetweensubpharmacclogiciandpharmacological[11Cjcocainearedenotedwith @=p < 0.05;*p < 0.01 ; p < 0.0005.
TABLE 3BrainUptake and Clearance Rates for Subpharmacologicaland PharmacologicalDoses of Carbon-i I @Cocaine*
Repeated measurements on the same day with no intervention were reproducible. Table 4 provides the values forthe DV in STh and in CB as well as for the ratioofthe DVin STR to CB for these repeated measures, as well as forthe results of the pharmacological challenges. Test andretest percent change for the subpharmacological dose of[“C]cocainecorresponded to 2% and to 4% for the pharmacological dose. The time-course of the STR/CB ratioswere also very stable (Fig. 4).
We used the technique ofFarde et al. (20) andfound thatthe maximum amount of specifically bound cocaine was 9
FIGURE1. Brainimagesof the baboonobtainedwithsubpharmacologicaldose of[11Cjcocaine(upperimages)and withpharmaooIog@ai dose of [11C)cocaine (kwier images) 15 mm after injection.Images correspond to r@anes where striatum and cerebellum arelocated. Each study was nOrmalized to the ma,dmal activity.
Binding ParametersThere were no measureable changes in heart rate or
blood pressure when 18 @gof cocaine was administered.Heart rate and blood pressure, however, increased transiently during the administrationof 7.5—iSmg of cocaineand during methyiphenidate administration. Whole brainand regional brain peak uptake (except for striatum)wassignificantly higher with pharmacological than with subpharmacological doses of [‘‘C]cocaine(Table 3). The largest differences were in cortex (t = 6.7, p < 0.0003) and inthalamus (t = 5, p < 0.0005). The values for K1 (transportfrom plasma to tissue) were also higher for pharmacological than subpharmacologicaldoses of[―C]cocaine:1.07 ±0.21 versus 0.68 ±0.26 (t = 3.0, p < 0.01). The bindingpattern also differed markedly between pharmacologicaland subpharmacological doses [“C]cocaine.Carbon-ilcocaine bound predominantly to the striatum at subpharmacological doses, but bound more homogeneously withequivalent uptake in striatum,thalamusand cortex (Fig. 1)at pharmacological doses.
The uptake and clearance of [“Cicocaineat pharmacological doses was similarfor the various brain regions; peakuptake was achieved 1.5-3 mm after injection and half peakclearance 10—15mm after injection. In contrast, the uptakeand clearance for subpharmacological doses of [“C]cocaine differed among brain regions. Peak uptake occurredat 3—5mm in striatumand at 1—2mm in cerebellum, andclearance was slower in striatum (half peak clearance = 20miii) than in thalamus (half-peak clearance = 15 ruin) andcerebellum (half@ clearance = 11miii). The time-activitycurves for subpharmacological and pharmacological doses of[“C]cocaineare shown in Figure 2 and the uptake and clearance rates for the various brain regions are shown in Table 3.The slower clearanceofa subpharmacologicaldose [“C]cocaine from striatum than that with the pharmacological doseled to higher STR/CB values (Fig. 3). Graphical analysisplots ofthe ratio ofthe distribution volume (DV) in striatum(STR)to that in cerebellum (CB) for the subpharmacological
1292 TheJournal of NudearMedicine•Vol.36•No. 7•July1995
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Effects of MonoamIne Transporter BlockersPretreatmentwith nomifensine or with methylphenidate
decreased the uptake of a subpharmacological dose of[“C]cocainein striatum but did not affect other brain regions. Nomifensine did not affect binding of a pharmacological dose of [“C]cocaine(Table 4, Fig. 4).
Citalopram significantly increased the uptake (Fig. 4) ofa subpharmacological dose [“C]cocaineand it increasedboth the STR/CB ratio and the ratio of the DV in STR tothat in CB (Table 4). Fluoxetine pretreatment also increased (though to a lesser extent than citalopram) striatalbinding of the subpharmacological dose of [“C]cocaine.Neither citalopram nor fluoxetine affected binding of apharmacological dose of [“C]cocaine(Fig. 4).
Though it was not possible to calculate DVs for thestudies with desipramine and tomoxetine, the STR/CBtime-activity curves were not changed by either of these
drugs (Fig. 4).
DISCUSSION
A numberof studies have reported the characterizationof high- and low-affinitybinding sites for cocaine in vitro(2—6)and the range of values is quite large probably reflecting differences in methodology. We selected bindingparameters from two different studies: one with humanputamen tissue (2) and one with nonhuman primate caudate-putamen tissue (3) to estimate the percentage occupancy which might be expected with the doses of cocaineused in this study. Binding parameters, as well as Bmaxvalues for these two studies vary dramatically (Table 1)with Kds differingby factors of 10and 25 for the high- andlow-affinity sites respectively. Because of this, the calculated percent occupancy for the high-affinitysites when thesubpharmacological dose of 18 j@gis administered rangedfrom 7% to 40%, whereas percentage occupancies for thelow-affinity sites ranged from 26% to 95% for the pharmacological doses depending on the binding parameters used(Table 1). The large range of values reported in vitro emphasizes the need for measuring binding parameters invivo. It is interesting that the ratio of Bmax to Kd for[“C]cocainedetermined by graphical analysis (17) is0.62 ±0.21 for human brain which is in close agreementwith the in vitro binding parameters for a Kd of2lO nM anda Bmax of 147pmole/g tissue (0.7) (2). It is also importantto note that the relationshipbetween the affinityof cocaineand cocaine-related compounds for the dopamine transporter and their reinforcing properties was done using a Kivalue for dopamine uptake of 640 nM (1), which is similarto that reported in the in vitro human study (2) and is inaccordance with cocaine doses required to observe pharmacological effects (21).
Notwithstanding the range of occupancies calculatedfrom in vitro binding parameters and from tissue cocaineconcentration as determined with PET for the two doses,we were able to document specffic bindingof [“C]cocaineonly when given at subpharmacological doses. The inabil
FiGURE 2. I@neticsof subpharmacobgical(left)and pharmacological (right) doses of [11C]cocaine in striatum (STh), thalamus(THL) and cerebellum (CBL). Notice the relatively slower clearancein striatumfor subpharmacobgicaldoses but not pharmacologicaldoses of [11C]cocaine.Noticethe similarkineticsforpharmacological dose of [11C]cocaine in various brain regions.
pmole/cc in the subpharmacological[“C]cocainestudyand1450 pmole/cc in the pharmacological [“C]cocainestudy(Table 5). If the 9 pmole value is bound to the high-affinitysite, the occupancy would be 6% (assuming Bmax = 147pmole/g, Table 1). The bound-to-free fractions were 0.6and 0.23 for the subpharmacologicaiand the pharmacological [“C]cocainestudies, respectively. A Scatchardanalysis with two points gives Bmax = 2300 pmole/g and Kd =3600 nM. For comparison, the calculation of specfficallybound cocaine was applied to the test/retest study withpharmacological [“C]cocaine,giving 1900 to 2000pmole/cc of specifically bound cocaine at the maximumforboth studies. The maximumin the specificallyboundcurve(STR-CB) was found to occur between 7 and 11 mm afterinjection.
Cocaine pretreatment decreased the uptake and distribution volume of the subpharmacologicaldose of [1‘C]cocaine in striatum but not in cerebellum. In contrast, theuptake and distribution volume of a pharmacological doseof [“C}cocainein striatum was not affected by cocainepretreatment(Table 4). Cocaine pretreatmentwith a subpharmacological dose of [“C]cocainedecreased the peakin the STR/CB ratiofrom 2.0 to 1.35for the 0.5-mgfkgdoseand from 1.75 to 1.1for the 2-mg/kgdose (Fig. 4). It had noeffect on a pharmacologicaldose of [“C]cocaine.
FIGURE3. (A)RatiosforSTR/CBforsubpharmacologicalandpharmacologicaldoses of [11C]cocaine.Closeddrcles are the subpharmacologicaldose and open squares are the pharmacologicaldose. Peak STR/CBratioforsubpharmacologicaldose was 1.8andI .2 forthe pharmacologicaldose. (B)Graphicalanalysleplots(17)forsubpharmacologicaiand pharmacologicaldose [11C]cocaineforstilatum and cerebellum.
*Bbod samples for this second study were lost so no DVvalues are available.@Thepaired baseline scan was lost due to technical error.
Percent change is expressed with respect to the baseline of the paired study. Drug pretreatrnents prior to the second PET scan (P1)included:cocaine1:0.5mg,kgi.v.,2 mmP1;cocaine@:2.0mg/kgi.v.,2 mmP1;nomifensine:2.0mg/kgi.v.,10mmP1;methylphenidate:0.5mg/kgi.v.,20mmP1;citalopram1:2.0 mg/kg i.v., 180 mm P1;citalopram@:2.0 mgflcgi.v., 30 mm P1;fluoxetine: 0.5 mg/kg iv., 20 mm P1;desiprammne:0.5 mg/kg iv.,30 mm P1;tomoxetine: 2.0 mg/kg i.v., 20 mm P1.
ity to observe specffic binding ofa pharmacological dose of[“C]cocaineto low-affinity sites with PET could be interpreted as indicating that in vivo cocaine binding occurspredominantly to high-affinity sites. Alternatively, it mayreflectpoor sensitivity ofPET. Althoughit has been arguedthat low-affinitybindingsites could be an artifactofin vitroexperiments, since their detection requires multiplewashings (with a single rinse, only one binding site is evident)(22), the proportion of high- to low-affinity sites is highlyvariable and is dependent on assay and tissue conditions(23), most studies have consistently demonstrated thepresence of a high- and a low-affinity binding site. It ismore likely that the poor sensitivity of PET did not allowus to demonstrate specific binding of [“C]cocaineto lowaffinity binding sites.
From Table 4, the values for the total Bmax-to-Kd observed are on the order of 0.5—0.6which are similar tovalues determined from in vitro data (Table 1). It is some
what surprisingthat the Bmax-to-Kd ratios are as close tothe in vitro values as they are, because the Kd valuedetermined by PET contains a contribution from nonspecific binding (Equation 2). The apparent Kd should begreaterthanthe in vitro Kd according to Equation 4. Sincethe DV in CB can be related to the free fractionof tracerinplasma, fp (19):
DV(CB) = fp(1 + NS), Eq. 9
iffp is on the order of0.1 and DV(CB) is 4, then Kd' = 40Kd. Therefore BmaxlKd should be 0.002 instead of 0.7 or0.04 instead of 1.5. Since DV(STR)/DV(CB)-1 is on theorder of 0.5, either the Kd observed in vivo with PET issmaller than the in vitro value or binding is not restricted toonly the free fraction of tracer but can occur directly fromthe nonspecific compartment so that no Kd correction isrequired. Assuming that the in vitro values for Bmax/Kd
1294 The Journal of Nudear Medicine•Vol.36 •No. 7 •July 1995
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SpecificactivityBoundCocaineBound/free(mCV@zmole)(pmole/cc)(pmole/cc)DV (ST) DV (GB) [DV(ST)/DV(CB)J-1
l@76@ 9
SUB PHRRMACOLGGICNI@tRtt
::1J@@-18 B ie 28 38 48 5i!oc@lne
•@1oa io 28 38 Nornirenslni
@ @::::@:::49.,1o- a@ io 20 30 40 @aloprarn
*ThemaamumintheSpecificallyboundcurve(ST- GB)occurredbetween7and11mmafterinjection.TheSpeCifiCallyboundandfreecocainewere determined as the average of the values in this time range.
t18/.Lg 10.4 mCi.*7500 p9 in 10.9 mGi.
low-affinity site is still difficult to detect because it is expected to be small compared to 1 (Eq. 2). A small residualcontributionof low-affinitybinding sites is consistent withthe results reported in Table 4 for which the values ofDV(STR)/DV(CB)-1 after pretreatment and for pharmacological [“C]cocaineare on the order ofO.1—0.2.Failuretodetect changes in the uptake of pharmacologicaldoses of[“C]cocaineafter cocaine pretreatment (15 mg), or to reduce the DV(STR)IDV(CB)-1 below 0. 1 to 0.2 may alsoindicate that pretreatment doses were insufficient. Usingthe values for binding parameters for the low-affinity sitereported for human putamen allows us to estimate that a200-mg dose of cocaine would be required to occupy 92%of the low-affinitysites (2). Since such a high dose wouldjeopardize the health of the baboon, it could not be exammed.
The relatively large amount of specifically bound cocaine (1400 pmole) found in the pharmacological dose[1 ‘C]cocame study reflects the difference between striatum
and cerebellum which at the relevant time points is 0.006%dose/cc (Table 5). This is also consistent with a highcapacity, low-affinity site. The validity of this conclusiondepends, however, on the appropriateness of the key assumption used for its determination, namely that the cerebellumat these time points represents the appropriatecorrection for the free and nonspecifically bound cocaine instriatum.From the two-point Scatchardanalysis, only oneset of parameters can be determined, Bmax = 2300pmole/cc and Kd 3600tiM with Bmax/Kd = 0.63. WhereasBmax-to-Kd reflects the effects of both types of sites, thelarge Bmax and Kd appear to be more characteristic of alow-affinity site.
Although these data are not conclusive, they are consistent with other experimental data suggesting the existenceof a low-affinitybindingsite. In general, it is more difficultto determine receptor binding parameters for low-affinitysites with imaging techniques because of the restrictionsimposed by Equation2 (thatis that (1 + Bmax/Kd) must besignificantly greater than 1.0 to be detected) even thoughsuch binding is easily determined in vitro.
The pharmacologicalchallenge experiments confirmthatat subpharmacological doses, [“Cicocainebinds predominantly to the dopamine transporterin striatum.Failure to
FiGURE4. Time-actMtycurvesforthestiiatum-to-cerebellumratb (STR/CB)forsubpharmacologicaland pharmacologicaldoses of[11C]cocainefor the baseline studies (test-retest),as wellas thepharmacologicalinterventions.Datawereobtainedinthe same daywith injections performed 2 hr apart, except for tomoxetine studywhere the time-actMty curves for the baseline correspond to theaveragefromthe baselinesforthe otherstudies(indMdualbaselinewas lost). Circles represent baseline scan (first study) and squaresrepresent pharmacological intervention (second study).
are also appropriatefor the low-affinitysites, the contribution from the BmaxlKd term in the DV is small comparedto 1 + Bmax/Kd (Eq. 2) for the high-affinitysite. If thehigh-affinity site is effectively saturated, the effect of the
TABLE5Scatthard Analysis of Subpharmacological and Pharmacological Comparison of Carbon-I I -Cocaine in the Same Animal Using
the Methodof Farde at al. (20)*
0.630.23
6.405.0
4.264.26
0.500.150.44* 1450
1295Carbon-il-Cocaine Bindingat Subpharmacological and Pharmacological Doses •Volkow et al.
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corroborate previous in vitro studies documenting cocainebinding to serotonin and norepinephrine transporters probably reflects differences between in vitro and PET studies.The relatively poor counting statistics of dynamic PETstudies preclude measurements of sites with relatively lowtarget-to-nontargetratios. Because there is a relatively lowconcentration of serotonin transportersites in the striatum(24), particularlywhen compared with the concentration ofdopamine transporters,the signalgeneratedfroma relativedecrease in binding from striatal serotonin transporterswould be undetected. This limitationwas recently demonstrated in a SPEC!' study in which citalopram failed todisplace [‘@I]Cff,a ligand that binds to dopamine andserotonin transporters, from striatum, an area with a dopamine-to-serotonin concentration ratio of at least 4,whereas it displaced it from a mesencephalic region, anareawith a dopamine-to-serotoninratioof 0.3 (25). Also, aPET study that compared different cocaine analogs reported almost identical stnatal kinetics for [‘1C]cocaineand 4'['8F}fluorococaine despite a 100-fold higher affinityof 4'-fluorococaine for the serotonin transporter (26). Inthe current study, neither fluoxetine nor citalopram pretreatment changed the uptake of [‘1C]cocainein mesencephalon (data not shown). The limitations from the partialvolume effect in an area as small as the mesencephalon aswell as the short half-lifeof 11Candconsequent poor counting statistics reduce confidence of our measurements inthis region.
The assessment of cocaine's binding to serotonin transporters in vivo would require the use of a specffic serotonintransporter ligand to evaluate the effects of cocaine pretreatment on its binding. Since we have no specific PETligand for serotonin transporters, we have been unable toevaluate the degree of cocaine binding to serotonin transporters in vivo in the baboon brain. We have applied,however, the strategy of using specific radiotracerligandsto investigate cocaine's interactions with the norepinephrine transporter in myocardial tissue. Carbon-li-cocaineshows significantbinding in the human and baboon heartwhich is not inhibited by desipramine pretreatment (27,28).Cocaine, however, inhibited 6-['8Flfluoronorepinephrineuptake in heartto the same degree as did desipramine(28).Since uptake of 6-['8F@fiuoronorepinephrinein the heart isa function of its uptake by the norepinephrinetransporter(29), its inhibition by cocaine corroborates in vivo a significant interaction of cocaine with this transporter. Becauseof the similaritiesbetween peripheraland centralmonoaminc transporters(30), it is likely that cocaine would haveinduced a similar inhibitionof norepinephrinetransportinbrain. Though the different relative regional concentrationsof the transporters,as well as the differentrelative affinitiesof cocaine for the transporterscould account for the discrepancies in the binding studies, it is also possible thatthey could reflect different binding sites within the transporters as reviewed by Carroll et al. (6).
Another variable that needs to be taken into accountwhen comparing in vitro and in vivo studies is that phar
macological challenges in vivo will have secondary effectsdue to neurotransmitterinteractions (31). These interactions could account for the relative increases in [11C}cocaine bindingafterpretreatmentwith drugs that inhibit theserotonin transporter. Most studies investigating the interactions between serotonin and dopamine have concludedthat serotonin inhibits dopamine neurotransmission in thestriatum (32). PET can be used to evaluate these interactions, and a recent study confirmeddecreased striatal dopamine concentration following pretreatmentwith citalopram, a serotonin transporter inhibitor (33). Decreasedstnatal dopamine concentration after citalopram or fluoxetine would lead to a larger fraction of free dopaminetransporters in striatum with a consequent increase in[“C]cocainebinding. Increases in striatal binding after fluoxetine pretreatment have also been observed with thecocaine analogs [1@I]R11-55 (34) and 4l.[1@I] iodococaine(Gatley SJ, personal communication), ligands which bindto serotonin and dopamine transporters.The increase in asubpharmacologicaldose of [11C]cocainebinding, after citalopram and fluoxetine, could, however, reflect drug-induced changes in ligandbioavailabiity (35). Similarly, thehigher brain uptake and K1 for the pharmacological thanfor the subpharmacological dose of [11C]cocaine, couldresult from a largerfree fractionof ligand, due to displacement by cold cocaine of binding sites in plasma proteinsand cells.
Although the use of [11C]cocaineas a ligand for thedopamine transporter has been criticized on the basis ofthe nonselectivity of cocaine for monoamine transporters,this study shows that in vivo it is highly selective for thedopamine transporter. Since cocaine has a lower affinityfor the dopamine transporterand a lower specific-to-nonspecific binding ratio compared to other dopamine transporter PET ligands, it has rapid kinetics which facilitate itsmodeling and quantitation (17). Also, its relatively lowaffinity for the dopamine transporter may make it sensitiveto synaptic dopaminewhich may enable its use to monitorsynaptic changes in dopamine concentration in an analgous way to the use of [“C]raclopride(26).
CONCLUSION
Binding of a subpharmacological dose of [11C]cocainein brain is predominantly associated with a high-affinitysite in the dopamine transporter. Although we wereunable to definitively demonstrate low-affinity bindingsites for cocaine in vivo, the data is consistent withwhat has previously been reported for low-affinity binding sites in vitro.
ACKNOWLEDGMENTS
TheauthorsthankRobertCarcielloandBabeBarrettforCyclotronoperations;Alex Levy, David Alexoff andDonaldWarnerfor PET operations;ColleenShea, ThomasMartinand DarrinJenkins for radiotracerpreparationand analysis; Carol RedvanlyforschedulingandorganizationandCharlesAshby for manuscriptdiscussion and evaluation. This research was supported in part by
the U.S. Department of Energy under Contract DE-ACO2-76CH00016and National Institute on Drug Abuse grant 5R01-DA06278and NationalInstituteof NeurologicalandCommunicative Disorders and Stroke grants 5P01 NS 15638 and NS 15380.
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1297FunctionalStudies in Substance Abuse •Levinand Kaufman
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1995;36:1289-1297.J Nucl Med. Schlyer, Naomi Pappas, Payton King, Gene-Jack Wang and Alfred P. WolfNora D. Volkow, Joanna S. Fowler, Jean Logan, S. John Gatley, Stephen L. Dewey, Robert R. MacGregor, David J. Doses: A PET StudyCarbon-11-Cocaine Binding Compared at Subpharmacological and Pharmacological
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