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PET Quantification of 5-HT2AReceptors in the Human : A Constant Infusion Paradigm with [1 8F]

Christopher H. van Dyck, Ping-Zhong Tan, Ronald M. Baldwin, Louis A. Amici, Pradeep K. Garg, Chin K. Ng, Robert Soufer, Dennis S. Charney, and Robert B. Innis

Departments ofPsychiatry and Diagnostic Radiology, Yale University School ofMedicine, New Haven; and West Haven Veterans' Affairs Medical Center@West Haven, Connecticut

(6,7). Recently,theuseof PEThasallowedin vivoimaging [18F]altansennhas been usedto labelserotonin5-HT@recep of the regional brain distribution of 5-HT2A receptors (8). tors, whichare believedto be importantin the pathophysiologyof However, PET for 5-HT2A receptor imaging, schizophreniaanddepression.The purposeofthisstudywasto such as “C-3-N-methyl-(NMSP) (9) and [18F]- testthefeasibilityofa constantinfusionparadigmforequilibrium (10), lack specificity, binding with moderate to modeling of [18F]altanserin with PET. Kinetic modeling with [18F]altansennmay be hamperedby the presenceof lipophilic high affinity to D2 receptors. As a result, consider radiometabolitesobservedin plasmaafterintravenousadminis able effort has been directed to the development of more tration. Methods: Eight healthy volunteers were injected with selective 5HT2A ligands for PET (11,12). [18F]altanserinas a bolus(208 ±9 MBq[5.62 ±0.25 mCi])plus [‘8F}altanserin(11, 13), a 4-fluorobenzoyl-piperidine de constantinfusion(65 ±3 MBq/h[1.76 ±0.08 mCi/h])ranging rivative structurally related to and setoperone, has from 555 to 626 mm (615 ±24 mm) after injection. PET emerged as a promising PET ligand for 5-HT2A receptors. acquisitions (10—20mm) and venous blood sampling were performedevery 30—60mm throughoutthe infusionperiod. Compared with previous 5-HT2A radiotracers, [‘8F]altan Results:Linearregressionanalysisrevealedthattime—activityserin yields higher ratios of specific-to-nonspecific binding curves for both brain activity and plasma [18l9altansennand and greater selectivity for 5-HT2A receptors compared with metaboliteconcentrationsstabilizedafter about 6 h. This permit other monoamine receptors (14). Altanserin has more than a ted equilibriummodelingand estimationof V3' (ratioof specific 20-fold greater selectivity for 5-HT2A than any other 5-HT uptake[cortical—cerebellar]tototalplasmaparentconcentration receptor subtype, including the 5-HT2C, which has signifi after 6 h). Valuesof V3' rangedfrom 1.57 ±0.38 for anterior cingulate cortex to 1.02 ±0.39 for frontal cortex. The binding cant homologies to 5-HT2A (15). Kinetic modeling with potentialV3 (ratio of specificuptake to free plasma parent [‘8F]altanserinhas previously been used in initial human concentrationafter6 h,usinggroupmeanf1)wasalsocalculated studies (16—19),but may be hampered by the presence of andrangedfrom169 ±41 foranteriorcingulatecortexto 110 ± nonspecifically bound lipophilic radiometabolites observed 42 forfrontalcortex. From6 h onward,the rate of change forV3' in plasma after intravenous administration (15,20,21), result and V3was only1.11 ±I .69 %Th.Conclusion:These results ing in the need to identify a large number of rate constants. demonstratethefeasibilityofequilibriumimagingwith[18F]altan senn over more than 5 radioactivehalf-livesand suggesta The purpose of this study was to test the feasibility of a methodto overcomedifficultiesassociatedwith lipophilicradiola constant infusion paradigm for equilibrium modeling of beledmetaboiites.The stabilityinV3andV3'onceequilibriumis [‘8F]altanserinwith PET. Because of problems associated achievedsuggeststhata singlePET acquisitionobtainedat 6 h with kinetic analysis of single bolus experiments, we mayprovidea reasonablemeasureof5-HT@receptordensity. implemented an alternative paradigm based on constant KeyWords:[18F]altanserin;5-HT@receptor;;PET infusion of the radiotracer. This paradigm creates and J NucIMed2000;41:234—241 maintains a prolonged state of equilibrium at the level of the receptors, allowing direct measurement of the distribution volumes of the tracer (22,23). The presence of lipophilic radiometabolites poses a challenge for an equilibrium ap n vitro studies on human brain autopsy material have proach as well, but the modeling is considerably simpler. revealed alterations in serotonin 5-HT2A receptors in a That is, if the metabolites are inactive (15,21) and distribute variety of conditions, including depression/suicide (1,2), uniformly in brain, then subtraction of background from a (3,4), aging (5), and Alzheimer's disease target region at the time of equilibrium provides specific receptor binding, corrected for the presence of metabolites. Received Jul. 27, 1998; revision accepted Jul. 30, 1999. Forcorrespondenceor reprintscontact:christopherH.vanDyck,MD,Yale Other advantages of an equilibrium approach include the University SchoolotMedicine, CB 2041, 333 Cedar St., New Haven, CT 06510. absence of arterial plasma sampling and the relatively short

234 THE JOURNALOF NUCLEARMEDICIr@ir@•Vol. 41 •No. 2 •February 2000 imaging time (20—40mm), once the methodology has been along the canthomeatal line and glabella. The Posicam 6.5 is a validated for clinical studies. 21-slice camera with 5.125-mm interslice distance. The resolution of the camera is 5.8 mm in-plane and 11.9 mm in the z axis. MATERIALSAND METHODS In-planeresolutionafterfilteringwith the Butterworthfilteris 7.5 mm. The sensitivity of the camerameasuredin a 20-cm-diameter RadiopharmaceuticalPreparationandPurity cylinder phantom is 165 kcts/s/pCi/mL (27). Five fiducial markers [‘tF]altanserinwas synthesized in a complete remote control filled with 0.5—1.0pCi ‘8Fwere attached to the skin along the system (24) modified from a method previously described (11).The canthomeatalplane to identify this plane during image analysis. precursor nitro-altanserin, 3-(2-(4-(4-nitrobenzoyl)-l-piperidinyl)- PETwas performedthroughoutthe [‘8F]altanserininfusion period ethyl)-l,2-dihydro-2-thioxo-4-quinazolinone, was prepared from accordingto the following protocol: 10-mm frame mode acquisi commercial reagents with >99.0% purity (25). For these 8 PET tions starting at approximately 45, 90, 120, 180, 240, and 300 mm studies, 2057 ±322 MBq (55.6 ±8.7 mCi; with these and after injection; then 20-mm frame mode acquisitions starting at subsequent data expressed as mean ±SD, unless otherwise approximately360, 390, 450, 480, 510, 540, 570, and600 mmafter specified; SEM is used in Figs. 1, 2, and 4 for legibility) injection. PET and plasma datawere more densely sampled from [‘8Flaltanserinwere produced, with an overall radiochemical yield 6 h onward to evaluate the assumptions of the equilibrium model of 10.3% ±2.5% at the end ofsynthesis. Radiochemical purity was more reliably late in the study.To minimize repositioningerrors, 99.0% ±I.3% and specific activity 46,546 ±18,981 MBq/pmol volunteersremainedin the scannerfor up to 3 h at a time, taking2 (I 258 ±513 mCi4imol) (range, 22,533—75,591MBq/pmol [609— breaks out of the scanner during the experiment. 2043 mCi/pmol]) at the time of injection and start of infusion, as determined by high-performance liquid chromatography (HPLC) PETimageAnalysis (EMRPSelect-B [EMScience, Gibbstown,NJ];250 X 4.6 mm, 10 PETimageswerereconstructedusinga deconvolutiontechnique @.im,tetrahydrofuran-to-sodiumacetatebuffer,35:65 [pH5.0] at 2.5 for scatter correction and a Butterworthfilter (cutoff, 0.09/mm; mL/min). The synthesis time was 112 ±9 mm (range, 97—124 power factor, 10) anddisplayedas a 256 X 256 matrix(pixel size, mm). The total mass dose of [‘8F]altanserinadministeredduring 1.7 X 1.7 mm; slice thickness, 5.125 mm; voxel volume, 14.81 the bolus plus constant infusion study was 0.14 ±0.05 pg/kg mm3). Attenuationcorrection was performedassuming uniform (range, 0.10—0.24pg/kg). Sterility was confirmed by lack of growth attenuation equal to that of water (attenuation coefficient, p = in 2 media: fluid thioglycollate and soybean-casein digest (26). 0.096/cm) within an ellipse drawn around the skull as identified by Apyrogenicity was confirmed by the LAL test (Endosafe, Charles the markers.A cylindrical (20-cm diameter) fluid-filled phantom ton, SC). was scanned on the day of each study to obtain a calibration factor foreachof the 21 slices forconversionof radioactivityintounitsof StudyPopulation nCi/mL. The study population included 8 healthy volunteers (3 men, 5 Region of interest (ROl) analysis was performed by an investiga women; age range, 25—42y; mean age, 32.1 ±5.6 y) and ranged in tor using the AnalyzelC image analysis program(CNSoftware, weight from 43 to 82 kg (63.8 ±12.5 kg). Volunteers underwent Wilmington, DE) running on a Sun SPARC station 20 (Sun clinical examinations by a research psychiatrist to exclude any Microsystems, Inc.; Mountain View, CA). ROt analysis was neurological or psychiatric disease or or substance abuse. conducted for 6 cortical structures (frontal, anterior cingulate, Screening procedures included a physical and neurological exami orbitofrontal,temporal,parietal,andoccipitalcortices)andcerebel nation, electrocardiography, serum chemistries, thyroid function lum.These regionswere identifiedby comparisonwitha neuroana studies, complete blood cell count, urinalysis, and urine toxicology tomical atlas (28). ROIs were drawn on the PET image using a screen.In addition,eachvolunteerunderwentbrain MRI that was mouse-driven cursor. The ROl map was drawn individually for read as normalby a neuroradiologist.No volunteer,accordingto each volunteer with respect to the first PET acquisition and was self-report, had taken centrally active medication within I mo of applied uniformly to all subsequent acquisitions for that person. PETscanning.All volunteersgave writteninformedconsent to the RO!analysiswas performedin 17of the 21 slices, withthe inferior research protocol approved by the local human investigation slice chosen to correspond best to the level of the fiducial markers committee. (canthomeatal plane). The superior slice was thus centered approxi mately8.7 cm above thecanthomeatalplane. PETDataAcquisition A mean count density (cpm/pixel) was obtained for each Based on preliminary studies in 5 healthy volunteers (data not anatomic structure in each acquisition by averaging values across shown), a bolus/infusion (hourly infusion rate) ratio of 3.2 was all slices in which the structuresappeared.Count densities were selected for the present analysis. [‘8F]altansenn(208 ±9 MBq then decay-corrected for the time of injection and expressed in [5.62 ± 0.25 mCi]) was injected intravenously (through the nCi/mL to generate brain time—activitycurves. constant infusion line) as a single bolus over 30 s at approximately 12 noon, and a constant infusion of 65 ±3 MBq/h (1.76 ±0.08 PlasmaMetaboliteAnalysis mCi/h)was startedandranin durationfrom555 to 626 mm(615 ± An intravenous line was inserted in the arm opposite the 24 mm). Thus, the total administered dose (incorporating decay of constant infusion line for obtaining venous blood samples. Venous the isotope) was 377 ±19 MBq (10.2 ±0.5 mCi). The actual blood samples were obtained throughout the [‘8F]altanserininfu bolus/infusion ratio for the 8 volunteers was 3.20 ±0.02. sion period at approximately 30, 60, 120, 180, 300, 360, 390, 420, Volunteers were placed in the Posicam 6.5 camera (Positron 450, 480, 510, 540, 570, 600, and630 mmafterinjection. Corp., Houston, TX) at the Yale/Veterans'Affairs PET Center, with Metabolite analysis was based on a published procedure (16). head positioned by laser along the canthomeatal line and immobi Plasma(2 mL) was dilutedwith 20 mL50 mmol/Lacetic acid and lized withstraps.Repositioningafterperiodsoutofthe scannerwas trapped on a C18solid-phase extraction cartridge (Sep-Pak Lite; accomplished by realigning the laser lights with marks placed WatersAssociates, Milford, MA) preconditionedwith methanol

PET QUANTIFICATIONOF 5-HT2A RECEPTORS•van Dyck et al. 235 andwater,washed with 10 mL0.1% Et3Nfollowed by 5 mL H20, equilibrium: then eluted with 1 mL MeOH. The methanol eluate was diluted withanequalvolume H20, andtheentiresolutionwas analyzedby vi, = C./C@. HPLC on a Waters Novapak C18,3.9 X 300 mm column; mobile At equilibrium,2 differentoutcome measures can be derived. phase CH3CN/0.353 mol/L HOAc/0.17 mollL NH.@OAc(pH 4.9) V3,theequilibriumvolume of distributionof the specificallybound (40:50:10), at a flow rate of 1.0 mLlmin, with in-line ultraviolet compartment, is an estimate of the BP and is calculated as the ratio detector(291 nm) and flow-throughNa! scintillationdetector/rate of the specific to the plasmafree parentconcentration: meter.Recoveryofradioactivity usingthisprocedurewas 88.5% ± 6.0%, measured by adding tracer to blood and carrying out the V3 = BP = C3/f1C@. same procedure. For identification of radiometabolites, the radioac At tracer concentration, BP equals Bmss/KD, @fl tive methanol/water solution was mixed with 3 authentic corn which B,,,,,@,is the density of sites and KD is the equilibrium pounds (altanserin, altanserinol, and 4-(,p-fluorobenzoyl)piperidine dissociation constant (34). V3' is a similar ratio to the total parent [FBP]) and injected on the HPLC. (which therefore does not depend on f1—onlyabout 1% for Plasma protein binding was measured by ultrafiltrationas altanserin): previously described (29). The binding to plasma proteins was assumed to be rapid compared with the other processes measured in V3' = C3/C@. these experiments.As a consequence, the free fractionof plasma parent compound (f1 = free parent/C@,,(t))(30) was assumed to be StatisticalAnalysis constant for all blood samples in a single experiment. The proposedequilibriummodel relies on several assumptions, the most importantof which is the stability over time of specific EquilibriumModel tracer uptake and free plasma parent concentrations. Average time—activitycurves for both PET and plasma measurements were Equilibrium analysis was performed using a 3-compartment generated and visually examined to determine when a state of model, comprising the plasma compartment (Cr); the intracerebral sustained equilibrium appeared to be achieved. To measure the nondisplaceablecompartment(C2),in which the tracercan be free stability of PET and plasma parameters from this time onward, the or nonspecificallybound;andthe specifically boundcompartment hourlyrateofchange foreachparameterwas computedby dividing (C3).At equilibrium,the venous concentrationof traceris assumed the linearregressionslope by the meanvalue of the parameter. to approximatethe arterialconcentrationandis takenas a measure The equilibriummodel was thenappliedto the entiresample to of [email protected] equilibriumdistributionvolume of a compartmenti (V1) calculate the distribution volumes V3 (C@/f1C@)and V3' (C@/C@). is defined as the equilibrium ratio of the tracer concentration in that Theeffect of age on V3andV3'was examinedby Pearson'sproduct compartment to the free plasma concentration: V = moment correlation coefficient (r). Finally, the stability of the where f1 = free fraction of parent tracer in plasma. V3 is equal to distribution volumes V3 and V3' at equilibrium was assessed by the bindingpotential(BP), which is theratioofthe receptordensity computing the hourly rate of change of the distribution volumes for @ to thedissociationconstant(KD)(30). each volunteer and for the entire sample. The activityconcentrationin a receptor-richcorticalROl attime t, C@@(t),is the sum of the concentrations of the nondisplaceable [‘8F]altanserin,the specifically bound [‘8F]altanserin,and the RESULTS radioactive metabolites (which are also assumed to be nondisplace None of the volunteers experienced any subjective or able): objective reactions. All vital signs (pulse, blood pressure, C@@(t)= C2(t)+ C3(t)+ C,,,..@(t). and respirations) were recorded before and 15—60mm after bolus injection and start of constant infusion, and serum has a relatively low concentration of 5-HT2A chemistry profile, complete blood count, and urinalysis were receptors (31—33).Ifcerebellum is assumed to be devoid of 5-HT@ repeated the day after the [‘8flaltanserin PET study. No receptors,then the activity concentrationthereat time t, CCbI(t),is clinically significant effects on vital signs or laboratory thesumofthe concentrationsofthe nondisplaceable[‘@F}altanserin studies were observed. and the radioactive metabolites: Seven of 8 volunteers completed planned [‘8Fjaltanserin CCbI(t)= C2(t)+ C,ret(t). infusions of at least 620 mm. Because of a technical problem, 1 volunteer completed an infusion of only 555 mm; If C2andC@1arefurtherassumedto be uniformthroughoutthe her data were not used to determine stability of regional brain,thenthe specific bindingin any receptor-richcorticalregion may be estimatedas: brain uptake and plasma parent activity but were included in the final computations of V3 and V31. Inspection of average C3(t)= C@@(t)—CCb,(t). plasma metabolite concentrations (Fig. 1A) and average The equilibriumvolume of distributionof compartmenti is the PET time—activitycurves (Fig. 2) suggested that equilibrium ratioof the tracerconcentrationin compartmenti to the free tracer was attained after approximately 6 h of [18F]altanserin concentration in the plasma at equilibrium, i.e., when no net infusion. To examine the stability of plasma and brain transfer between the plasma and compartment i exists: parameters after 6 h, the hourly rate of change for each parameter was computed by dividing the linear regression Vi = C1/FIC@. slope by the average value of the parameter from 6 to 10.5 h Vi, is defined as the ratio of the tracer concentration in for the 7 volunteers who completed the full infusion compartment i to the total tracer concentration in the plasma at experiment.

236 ThE JOURNALOFNUCLEARMEDICINE•Vol. 41 •No. 2 •February 2000 A 100@ . . .- - *altansenn -J .. •0@-- “FBP― E 80 J ..A- C.)

C 60. 0

L. 40 C G) U C 0 20- C.) 4'@4&:-@ A w 0 100 200 300 400 500 600 Minutes B 70 . . .. . *aft@nsenn .. . 0••- “FBP― FiGURE 1. Average plasma metabolite

. . A- . altansennoi concentrations (A) and percentage compo ..@..+.. sition (B) (mean ±SEM used for legibility) after[18F]altanserininfusionin7 volunteers studiedforat least620 mm.(Onevolunteer whoseinfusiondurationwas555mmisnot included.)Two missingplasma samples (out of 105 total) were handled by using linearinterpolationofprecedingandsubse quentsamplesfor that subject.From6 h onward, rates of change were low for altan C@ serin (—0.69±5.21 %/@)and major lipo philic metabolite fraction (“FBP―;3.64 ± 1.90 %/@),whereas some increase was still I' observedinminorlipophilicmetabolitealtan serinol(5.78 ±9.02 %ft@)Percentageof @ 0 1ÔÔ@2Ô@@ à óóióó‘êóóunchanged[18F]altanserinfellfrom63% at Minutes 37 mm after injection and stabilized at approximately45%from6 honward.

PlasmaMetabolfteConcentrationsAfter[‘8F]Altanserinare displayed in Figure lÀ.The average time—activitycurve Infusion for [18F]altanserinappeared to level offby approximately 6 h Three major plasma components were observed in these after injection. From 6 h onward, mean rates of change were experiments. [‘8F]altanserinparent (HPLC retention time tR, low for altanserin (0.69 ±5.21 %/h) and the major lipophilic 10.7 ±0.3 mm) and the ketone reduction product [‘8F]altan metabolite fraction (“FBP―; 3.64 ± 1.90 %/h), whereas serinol(tR,8.1 ±0.2 mm) were identifiedby comparisonto some increase was still observed in the minor lipophilic authentic standards co-injected with the radioactive plasma metabolite altanserinol (5.78 ±9.02 %/h). samples. The third metabolite fraction had retention time (tR, The kinetics ofunmetabolized [‘8F]altanserinand metabo 6.2 ±0.1 mm) close but not identical to that of authentic lites as a fraction of the plasma activity are shown in Figure FBP (tR, 5.6 ±0.1 mm), the product of N-dealkylation on lB. The percentage of unchanged [18F]altanserin fell from the piperidine ring. Results from other studies (15,20,21) 63% at 37 mm after injection and stabilized at approxi suggest that this fraction actually consists of a mixture of mately 45% from 6 h onward. metabolites, some of which may be secondary metabolites of FBP. In the present analysis, these additional metabolites are presumed to be contained in the FBP peak on HPLC and PETTime—ActivityCurvesAfter(@9AItanserIninfusion are included in the “FBP―time—activitycurves. Representative [‘8FjaltanserinPET images from early and Average plasma metabolite concentrations after [‘8F]altan late time points are displayed in Figure 3. [‘8F]altanserin serin infusion in the 7 volunteers studied for at least 620 mm uptake is high and relatively uniform in all cerebral cortical

PET QUANTIFICATIONOF 5@'T2A RECEPTORS•van Dyck et al. 237 @ 0

regions and low in basal ganglia, thalamus, and cerebel 200- .•..o.@. .. . CortexCerebellum lum. ::@ ••A•Specific Average PET time—activity curves after [18flaltanserin infusion in the 7 volunteers studied for at least 620 mm are @ 150 displayed in Figure 2. From 6 h onward, rates of change were low for receptor-rich cortical regions (2.46 ± 1.03 %Ih), receptor-poor cerebellum (3.45 ± 2.10 %/h), and t 100@ specifically bound (cortex—cerebellum) tracer uptake (1 .16 ± 2.00 %/h) (Table 1). @ 50 EquilibriumOutcomeMeasures The outcome measures derived at equilibrium (averaging each volunteer's data from 6 to 10.5 h after injection) for 0 100 200 300 400 500 600 cortical brain regions in all 8 volunteersare displayed in Minutes Table 2. Ratios of total-to-nonspecific (cerebellar) binding FIGURE2. AveragePETtime-activitycurves(mean±SEM between 6 and 10.5 h after injection were 1.97 ±0.16 for the usedfor legibility)after [18F]altanserininfusionin 7 volunteers structure of highest uptake (anterior cingulate) and 1.75 ± studiedfor at least 620 mm. (One volunteerwhose infusion 0.15 for wholecortex.Valuesof V3' (C@/C@)rangedfroma durationwas555 mmisnotincluded.)ThreemissingPETscans high of 1.57 ±0.38 for anterior cingulate cortex to 1.02 ± (out of 98 total) were handled using linear interpolationof 0.39 for frontal cortex. Measurements of f1 were used to precedingand subsequentsamplesfor that subject.Cortex representscompositeofall6 corticalregionsanalyzed.From6 h calculate V3 (C@/f1C@)as an estimate of the BP. Values of f1 onward, rates of change were low for receptor-richcortical ranged from 0.36% to 2.00% (0.93% ±0.63%). V3 was regions(2.46 ±I .03 %/@),receptor-poorcerebellum(3.45 ± computed using both group mean and individual values of f1. 2.10 0k/h),and specificallybound(cortex—cerebellum)tracer Values of V3' (and therefore V3. using a mean f1) were uptake(1.16±2.00 %Ih). significantly correlated with age in this small sample for whole cortex (r = —0.73;P < 0.05) as well as orbitofrontal cortex (r = —0.79;P < 0.05), frontal cortex (r = —0.79; P < 0.05), anterior cingulate cortex (r = —0.70;P < 0.05),

A

FIGURE3. TransaxiaiPET imagesof [18F]altanserinuptake in healthy volunteer at early and late time points:(A) 10-mm acquisitionstarting52 mm after injection and(B)40-mmacquisitionstarting376 mm B. after injection. Uptake of [18F]altanserinis consistentwithknowndistributionof5-HT@ receptorsasseeninautoradiographicstud ies,withhighand relativelyuniformuptake in all cerebralcortical regions,low uptakein basal ganglia and thalamus, and much I,, j loweruptakeincerebellum.Slicesare situ ated approximately80 (high cortex), 45 (thalamusand basalganglia),and 10 mm / (cerebellum)above canthomeatalplane. Laterimagesare fromsinglelongacquisi tion obtained at equilibrium, as could be usedinclinicalstudy.

238 THE JOURNALOF NUCLEARMEDICINE•Vol. 41 •No. 2 •February 2000 TABLE1 Rates of Change in PlasmaMetabolitesand PET BrainROls 2- at Equilibrium

@ % Change/h* -C,) 1.5- +-+ @ MetabolitesandROls Mean SD i 1-•@... Plasmametabolite Altanserint 0.69 ±5.21 “FBP― 3.64 ±1.90 Altanserinol 5.78±9.02 C.) BrainAOl Anteriorcingulatecortex 3.33 ±1.13 Frontalcortex 1.38±1.84 2.70 ±3.11 Parietalcortex 2.78 ±1.22 0 100 200 300 400 500 600 Temporalcortex 2.50±1.30 Occipitalcortex 2.67 ±1.61 Minutes Combinedcortex 2.46 ±1.03 Cerebellum 3.45±2.10 FIGURE4. Stabilityof outcomemeasureV3' forcombined Corticalspecificbinding 1.16±2.00 cortex (mean ±SEM used for legibility)after [18F]altanserin infusion in 7 volunteers studied for at least 620 mm. (One volunteerwhoseinfusiondurationwas555 mmis notincluded.) “FBP―= majorlipophilicmetabolitefraction,probablyderived ForeachPETacquisitionofeachvolunteer,V3'(ratioofspecific from 4-(p-fluoro-benzoyl)piperidine, as explained in text. uptake[cortical—cerebeilar]to total plasmaparentconcentration) *Valuesare obtainedby dividingthe linearregressionslopeby wasderivedusingplasmasamplenearestintimetomidpointof meanvaluefrom6 to 10.5h afterinjectionin7 volunteersstudiedfor PETacquisition.From6 honward,meanrateofchangewaslow at least 620 mm. forcorticalV3'(1.11±1.69%/h). tParentcompound.

%Ih for the 7 volunteers studied for at least 620 mn. and parietal cortex (r —0.69;P < 0.05). However, values Moreover, this rate of change did not exceed ±5%/h for any for V3 using individual f1 values were uncorrelated with age. volunteer (Table 3). This age-related decline in 5-HT2A receptors has been previously reported in both postmortem (5) and in vivo PET DISCUSSION (35—37)studies. The major finding of this study is that [‘8F]altanserinis a Once equilibrium was achieved, values of V3' (and suitable radioligand for bolus plus constant infusion studies therefore V3, using mean or individual values of f1) demon for equilibrium modeling with PET. Stable time—activity strated excellent stability as shown in Figure 4 and Table 3. curves for both plasma and PET measurements suggest that From 6 h onward, the rate of change for V3' was 1.11 ±1.69 a state of sustained equilibrium is achieved by approxi mately 6 h, permitting direct measurement of distribution TABLE2 volumes V3 and V3'. These results afford an alternative to OutcomeMeasuresat Equilibriumwith[18F]Altansermn kinetic modeling for quantification of 5-HT2A receptors in in 8 Volunteers* the human brain using [‘8F]altanserinand PET. The proposed equilibrium model for [‘8F]altanserinsug Totat/V3V3Cortex cerebellar@ V31t (meanfi)t(mdiv. f1)@ TABLE3 Antenorcingulate1.97149Frontal1.63±0241.02±0.39110±42179±130Orbitofrontal1±0.161.57 ±0.38169 ±41248 ± Stabilityof V3' at Equilibriumin IndividualVolunteers

126Parietal1.88±0.131.43±0.34153±37224±[email protected]±0.151 .28±0.35138 ±37204 ± Volunteer no. % Change/h*

127Occipital1.70±0.131.17±0.41126±44175±102Combined1.74 ±0.181 .20±0.38129 ±41187 ± 1 3.08 2 2.79 .75 ±0.151 23 ±0.32132 ±34198 ±128 3 —1.68 4 —0.21 5 1.83 *All valuesare obtainedas the averageof data between6 and 6 1.41 10.5hafterinjection. 7 0.59 tV3' @5calculatedas ratioof specific(cortex—cerebellum)tothe Mean 1.11 plasmatotalparentconcentration(C@/C@).V3iscalculatedas ratioof SD 1.69 specificto plasmafree parentconcentration(C@/f1C@;equivalentto BP). tV3 (meanf1)usesgroupmeanvalueoff1(0.93%). *Valuesareobtainedbydividinglinearregressionslopebymean §V3(mdiv.f1)usesindividualvaluesoff1. valuefrom6to 10.5h afterinjection.

PET QUANTIFICATIONOF 5-HT2A RECEPTORS•van Dyck et al. 239 gestsa novel methodto overcomedifficultiesassociated approximately 10% protein in wet brain and 50% white with lipophilic radiolabeled metabolites. Whereas kinetic matter in our PET ROIs, we would expect a B,@ on the studies with [‘8F]altanserin(16—19)have required complex order of 10—13.5nmol/L. The previously published K1value modeling to identify a large number of rate constants, (0.13 nmol/L) of altanserin for 5-HT2 receptors (13) would equilibrium modeling with [‘8F}altanserin(assuming urn thus predict BP values in the range of 77—104for our cortical form uptake of nondisplaceable [‘8F]altanserinand metabo ROIs. lites) permits the estimation of specific binding in a receptor The outcome measure of choice for clinical studies rich cortical region by simply subtracting cerebellar activity. remains to be determined. The stability in V3 and V3' once equilibrium is achieved suggests that a single long PET PlasmaMeasurements acquisition (e.g., 40 mm) obtained at 6 h may provide a The plasma analysis used in these experiments allowed reasonable measure of 5-HT2A receptor density. V3 (using quantitating parent [‘8F]altanserinseparated from metabo individual measurements of f1) has the theoretical advantage lites, but only clearly resolved the reduced alcohol [‘8F]altan ofdirectly estimating the BP but may lack reliability because serinol. What we have called the “FBP―fraction actually of the difficulty of measuring f1. The wide intersubject consists of a mxture of metabolites that probably result variability in f1points to the need to examine the intrasubject from secondary metabolism of FBP. Detailed studies by the variability in f1 as well. V3' (or the proportionate measure of PET group at the University of Pittsburgh have shown that V3 using a group mean f1) rests on the questionable [‘8F]FBPis converted to at least 1 other metabolite in assumption of negligible intersubject variability in f1, but and baboons (20). They reported that after administration of may constitute a more reliable measure. Further test—retest [‘8FJFBPto baboons, radioactivity was uniformly distrib studies are needed to establish the within-subjectreliability uted and was not displaced by the 5-HT2A antagonist SR of both outcome measures obtained from a single PET scan 46349B (21). In this study, the fact that about 60% of 6 h postinjection. [‘8F]altanserinremained unmetabolized in plasma at early Assumptionsof @8F]AItanserInEquilibriumModeling time points (37 and 67 mn after injection) is consistent with The assumptions underlying the present model include: previous kinetic studies (1 7). This value later stabilized at that cerebellum is relatively devoid of 5-HT@ receptors and about 45% during the period of sustained equilibrium. thus lacks displaceable uptake; and that the nondisplaceable PETMeasurements C2 and C,@ are uniformly distributed throughout the brain. The overall regional variability of uptake of [‘8F]altan Support for the second assumption is furnished by both serin in this equilibrium study agreed with that of previous autoradiographic studies (39) as well as human PET kinetic kinetic studies (16—19)as well as the known distribution of studies in which ketanserin pretreatment produced nonspe S@T2A receptors as seen in autoradiographic studies (31— cific binding of [‘8F]altanserin and metabolites that was 33). After intravenous injection of a bolus plus constant uniform in all brain regions (17). infusion of [‘8F]altanserin,radioactivity was high in all With regard to the first assumption, the cerebellum has cerebral cortical regions and low in basal ganglia, thalamus, previously been used as a reference region for determination and cerebellum. of S@T2A receptors in PET studies using [“C]NMSP(9,35), Ratios of total-to-nonspecific (cerebellar) binding be [‘8F]setoperone(10,40), and [‘8F]altanserin(16). The choice tween 6 and 10.5 h after injection were 1.97 ±0.16 for the of cerebellum as a reference region is based on the low structure of highest uptake (anterior cingulate) and 1.75 ± levels of S@T2A receptors from postmortem studies (31— 0.15 for whole cortex. The fact that these ratios were 33). The aforementioned ketanserin pretreatment studies somewhat lower than previously reported for [‘@F]altanserinwith [‘8F]altanserin(17) supported the validity of estimating kinetic studies (2.6) (17) is likely the result of: the greater the nonspecific binding in cerebellum, revealing similar age of our volunteers (mean, 32 versus 25 y); and the lower [‘8F]altanserinkinetics in cerebellum during blocked and ratio of unmetabolized [‘8F]altanserininherent in a pro unblocked studies in the same individuals. Further competi longed equilibrium compared with a kinetic paradigm (45% tion studies should be conducted in nonhuman primates with after 6 h of constant infusion versus 60% at 1 h after bolus competitors of well-defined specificity. The presence of a injection). Our group is currently exploring the small component of specific binding in cerebellum may substitution ofthe 2'-hydrogen ofaltanserin ([‘8F]deuteroalt result in the underestimation of specific uptake in receptor anserin), yielding a metabolically more stable radiotracer rich cortical regions using the proposed equilibrium model. with higher ratio of parent tracer to radiometabolites (38). CONCLUSION DistributionVolumes These results demonstrate the feasibility of equilibrium The values of V3 (as an estimate of the BP) calculated in imaging with [‘8F]altanserinand suggest a method to this study (Table 2) are on the order of magnitude predicted overcome difficulties associated with lipophilic radiolabeled from in vitro data. Pazos et al. (32) reported 5-HT2 receptor metabolites. Further studies are needed to verify assump densities of approximately 200—270fmol/mg protein for tions of the proposed model and to establish the reliability of those cortical regions analyzed in this study. Assuming outcome measures obtained from a single PET scan.

240 THE JOURNALOF NUCLEARMEDICINE•Vol. 41 •No. 2 •February 2000 ACKNOWLEDGMENTS 19. Smith GS, Price JC, Mathis CA, et al. Test-retest variability ofserotonin 5-HT@ receptorbinding measured with positron emission tomography and [‘@F]a1tanserin The authors thank Dayton Rich and Quinn Ramsby for in the human brain. Synapse. 1998:30:380—392. excellent technical assistance and Dr. Yolanda Zea-Ponce for 20. Mason NS, Huang Y, Holt DP,Perevuznik JJ, Lopresti Bi, Mathis CA. Synthesis helping with initial metabolite analysis method develop and characterization of [F-18]4-(4-fluorobenzoyl)piperidine, an [F-18]altanserin metabolite [abstract]. JNucl Med@1997;38:56P. ment. This research was supported by funds from the 21. Mason NS, Huang Y, Holt DP. Perevuznik JJ, Lopresti BJ. Mathis CA. Synthesis Department of Veterans Affairs (Schizophrenia Research of two radiolabeled metabolites of [“F]altanserin:[@F]3-[2-[4-(4-fluorophenyl Center and the VA Mental Illness Research, Education, and methanol)-1-piperidinyl]ethyl]-2.3-dihydro-2.thioxo-4-(IH)-quinawlinone and Clinical Center) and the Public Health Service (MH 30929). [‘8F]4-(4-fluorobenzoyl)piperidine.J Labelled Compds Radiopharm. 1997:40:5! 161—162. 22. Frey MA, Ehrenkaufer LE, Beaucage 5, Agranoff BW. Quantitative in vivo REFERENCES receptor binding I. Theory and application of the muscarinic cholinergic receptor. I. Arango V. Ernsberg P. Mazuk PM, et al. Autoradiographic demonstration of JNeurosci. 1985:5:421—428. increased 5-HT2 and 8-adrenergic receptors in the of suicide victims. Arch 23. Carson RE, Channing MA, BIaSbCrg RG, et al. Comparison ofbolus and infusion GenPsvchiatrj.1990;47:1038—1047. methods for receptor quantitation: applications to [‘8F]cyclofoxyand positron 2. Stanley M, Mann ii. 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