SPECT Measurement of Benzodiazepine Receptors in Human Brain with Iodine-123-Iomazenil: Kinetic and Equilibrium Paradigms

SPECT Measurement of Benzodiazepine Receptors in Human Brain with Iodine-123-Iomazenil: Kinetic and Equilibrium Paradigms

Anissa Abi-Dargham, Marc Laruelle, John Seibyl, Zachary Rattner, Ronald M. BaldWin, Sami S. Zoghbi, Yolanda Zea-Ponce, J. Douglas Bremner, Thomas M. Hyde, Dennis S. Charney, Paul B. Hoffer and RobertB. !nnis

DepartmenL@ ofPsychiatiy and Diagnostic RadiOlOgy, Yale UniVeTSÃœySchool ofMedicine, New Haven, Connecticut and West Haven VA Medical Center, West Haven@Connecticut; Clinical Brain Disorders Branch, IRP, NIMH Neuroscience Center at St. Elizabeths, Washington, DC

been used as a PETradiotracer forvisualization and quan Iodine-123-iomazenilbindingto benzoduazepinereceptorsin hu tification ofbenzodiazepine receptors in humans (Z12-16). manbrainwasmeasuredwithSPECTusingkineticandequilib Recently, an iodinated analog of fluinazenil, iomazenil (Ro nummethods.Mthods: In the kineticexperiments(n = 6), 16-0154), has been introduced as a SPECT radiotracer (17). regionalthne-activitycurvesaftera singlebolusinjec@onof the SPECT studies in nonhuman primates (18) and healthy tracerwerefit to a three-compartmentmodelto provideesti humansubjects (19) have shown that [‘@IJiomazenilhad a matesofthe rateconstantsK1tok. Thebindingpotential(equal high brain uptake (iO%—i2%of the injected dose). About to the productof the receptordensityandaffinity)wasderived 90% of this brain activity is displaceable and therefore, fromthe rateconstants.Inthe equilibriummethod(n = 8),the tracerbolusinjectionwasfollowedbya constanttracerinfusion associated with specific binding to benzodiazepine recep toinduceasustalnedequilibriumstate.Theregionalequilibriumtors. volumeof distributionwascalculatedastheratiooftheregional Analysis of SPECT neuroreceptor imaging studies have brainCOnCentrabOn-ID-thefreeparenttracersteady-stateplasma been typically restricted to empirical, semiquantitative concentration.Inthreeexperiments,a receptor-saturatingdose methods such as the ratio of activities in regions of interest offlumazenil was injectedfor direct measurementofthe nondis (RO!s) or the RO! washout rates. Since these empirical placeablecompartmentdistributionvolume.Results: The Id measures do not control for factors such as peripheral neticandequilibriummethodresuftswereingoodagreementin clearance, binding to plasma proteins and cerebral blood all regions investigated. Iodine-125-iomazenilbinding potential flow, the ability of these measures to provide quantitative measuredin vitroin 12 postmortemsampleswasfoundto be information about the receptors under study is question consistent wfth SPECT in @vomeasurements. Conclusion: able (20). The goal of the studies reported here was to Thesestudiesdemonstratedthe fea@brIftyof quantificationof receptorbindingwithSPECT. develop model-based methods for SPECT measurementof [‘@!]iomazenilbinding to benzodiazepine receptors in the KeyWords:SPECT;brain;benzodlazeplnereceptors;10- humanbrain. dlno-123-Iomazsnll Model-based methods for in vivo quantification of neu J Nuci Md 1994; 35:228-238 roreceptors can be broadly divided into kinetic and equi librium methods. Kinetic methods yield quantitative inior mationaboutthe receptorsby estimatingthe rateconstants which characterizethe transferbetween plasma, brainand terations of central benzodiazepine receptors have receptor compartments (21). Equilibrium methods derive been described in several neuropsychiatricconditions, in this information by analyzing the activity distribution at cluding epilepsy (1—3),Alzheimer's disease (4,5), Hunting equilibrium, i.e., when the receptor-ligand association and ton's chorea (6—8)and schizophrenia (9—11).Carbon-li dissociation rates are equal (22). With both approaches, flumazenil (Ro 15-1788),a benzodiazepine antagonist, has the outcome measure of experiments performed at tracer doses is a unitless number, the binding potential (BP), which equals the product of the receptor density (Bm@, ReceivedJul.6, 1993;revisionaxepled Nov.4, 1993. nM) and affinity (i/Ks, @J@1)(21). The BP is also the Forcorrespondenceand reprirdscon@ As@ssaAbi-Dar@wn,MD,De@of Psychiatry,Y@eUniversityand West Haven VA MedicalCerter/116A2,950 equilibrium distribution volume of the receptor compart CaIT;@bstAve.,West Haven,CT06516. ment, i.e., the ratio of specific binding in the brain-to

228 TheJournalofNudearMedicine•Vol.35•No.2 •February1994 plasma level of free parenttracer at equilibrium.Benzodi yieldof the [@!Jiomazenilpreparationsaveraged66.4%±8.2% azepine receptors have been measured with [11C]flumazenil (with these and subsequent data expressed as mean ±s.d., n = 14) and PET with kinetic (1423,24) and various “equilibrium―and the radiochemical purity averaged 97.6% ±1.7%(n = 14).The methods (2,25-28). specific activity was determined by comparh@gthe UV absorbance Among equilibrium methods used to measure [“C]flu ofthelabeledpmductwithastandardcurvegeneratedfromknown concentrations of nonradioactive iomazenil. The specific activity of mazenil BP, it is important to distinguish the “peakup E'23!liomazenilin these preparations was found to be greater than take―equilibriummethod (2) and the “transient―equiib 5000 Ci/mmole and may be estimated to be on the order of 180,000 rium method (25,27,29). “Peakequilibrium― methods CL'mmole(37).Sterilitywas assuredby lackof bacterialgrowthin measure the BP at peak uptake of the specific binding, twomediaandapyrogenicitywasconfirmedusingthelimulusame usually obtained by subtracting the activity in the white bocyte lysate (LAL)test. The specificactivity of [1@I]iomazenilwas matter(2) or the pons (2428) from the activity in the RO!. estimated by UV detection to be 1090 Ci/mmole on the day of At peak uptake, the BP is calculated as the ratio of activity labelin@ @ in the ROl-to-activity in a reference region. The difficulty He&thy of this method is the proper identificationof peak uptake Fourteen healthy subjects (age 26 ±6 yr. weight 75 ±7 kg, 11 for ligands such as [1@I]iomazenilwhich exhibit a pro males and 2 females) were recruited for these studies. Inclusion tracted plateau phase (19). The “transient―equilibrium criteriawere: (1) absence of current medicalconditionsand (2) method, also called quasi-equilibrium(27) or pseudo-equi absence of neumpsychiatric illness, alcohol or substance use. A librium (25), refers to the measure of the BP when the physical examination, EKG and routine blood and urine tests specific-to-nonspecific ratio or the specific binding-to were performed in the screening procedure. All subjects gave plasma tracer concentration ratio becomes constant over written informed consent. Protocols were approved by the insti time (i.e., when both are decreasing at the same rate). tutional human investigation committee. Subjects received potas Carson et a!. (30) proposed the term “transient―equilib sium iodide (SSK! solution 0.6 g in the 24 hr period prior to rium to describe these conditions. As opposed to the peak imaging). uptakeequilibriummethod, the transientequilibriummeth DataAcquisition ods do not satisfy the Michaelis Menten equilibriumequa SPECF datawere acquiredwith the multislicebrain-dedicated tion and can result in an overestimation of the BP (30). To CERASPECF camera (Digital Scintigraphics,Waltham, MA) overcome the technical and theoretical difficulties associ (38). The transaxial and axial resolution in air are 7.7 and 5.9 mm ated with the use of equilibrium methods after single bolus FWHM,respectively(39).Inwater,theresolutionis 10—12mmin thethreeplanes. injection of the tracer, we implemented in humans the Four fiducialmarkersfilledwith 10 j@Ciof [@Tc]sodium constant infusion/sustained equilibrium method (30—32) pertechnetatewere attachedon both sidesof the subjects's head that we previously described in baboons (33—35). at the level of the cantho-meatalline to control positioningof the In the study reportedhere, benzodiazepine receptor BP head in the gantrybeforetracerinjectionand to identifythe was measured in 14 healthy subjects with [‘@!Jiomazenilcantho-meatal plane during image analysis. and SPECT using both a kinetic (i.e., bolus only) and a Singlebolus experimentswere performedin six subjects(age sustained equilibrium (i.e., bolus plus constant infusion) 23 ±0.7yr,weight78 ±11kg).Iodine-123-iomazenil(12.8±4.2 paradigm. Plasma clearance of the tracer estimated from mCi)was injectedas a singlebolus over 30 sec. Scans were the single bolus experiments was used to design the tracer acquired in continuous mode every 2 mm for 145 ±5 mm. Arterial administration protocol of the bolus plus constant infusion blood samples (1—2ml) were collected every 20 sec for the first 2 experiments. A direct measure of the nonspecific compart mis with a peristalticpump(Harvard2501-001,SouthNatick, ment was obtained in three of the eight constant infusion MA)andthenmanuallyat3, 4, 6, 8, 12,16,20,30,45and60mm. After the first hour, samples were drawn every 30 mis until the experiments by injecting a receptor saturating dose of flu endof theexperiment. mazenil (0.2 mg/kg) at the end of the study. In addition, the Constant infusion experiments without flumazenil displace accuracy of SPECT measurements was confirmed with mentwere performedin five subjects (age 26 ±9 yr, weight 75 ± [‘@!]iomazenilbinding studies to postmortem human brain 4kg).Thedosewasdividedinabolusof3.89 ±0.39mCifollowed samples. byaconstantinfusionof 1.08±0.12mCi/hr(IMEDpump,Jemini PC-i, San Diego, CA) given over 450 miii, resultingin a bolus-to infusionratioof3.61 ±0.27hr.Scanswereacquiredincontinuous METhODS modeevery5 ruinfrom250 to 450 ruinpostinjection.In three Radlolab.llng subjects, data were also acquired from 0 to 150mis postinjection. Iodine-123-iomazenil and [‘@!Jiomazenilwere prepared by io Arterialsampleswere collectedin a pattern similarto the single dodestsnnylation of ethyl 7-(thbuty@tannyl)-5,6-dihydro-5-meth bolus experiments in the first three experiments, and every 15 mm yI-6-oxo-4H-hnidazo[1,5.a][i,4jbenzodiazepthe-3-carboxylate with starting 2 to 4 hr postradiotracer injection in the last five experi chloramine-Tin methanolicaceticacidat 120°C(36).Radiolabeled ments. productswere purifiedby reversed-phaseHPLC(C-18column, Humazenildisplacementduring[‘@I]iomazenilconstantlain 3.9x 300mm,55%CH3OH/H20,0.7mI/rain;[email protected]),formu sion was performedin threesubjects(age27 ±2.6 yr. weight latedin 5%ethanolin normalsalinecontaining0.1mML-ascorbic 75 ±12 kg). The injection and acquisition protocols were similar acid, pH 5-6, and filtered through a O.2-@membrane filter into a to the previous constant infusion studies (bolus 4.1 ±0.54 mCi, sterile 10-mI serum vial or sterile 10-mi syringe. The radiochemical infusion 1.12 ±0.16 mCi/hr resulting in a bolus-to-iniusion ratio of

SPECTMeasurementofBenzodiazepineReceptors•Abr-Darghametal. 229 3.64 ±0.13 hr), with the exception that a receptor-saturating dose (f1= freeparent/Ca(t))(21)was assumedto be constantforall of flumazenil(0.2 mg/kgwas given at 384 miii, as an intravenous blood samples in a single experiment. infusionover 10mmn(40). Forsinglebolusexperiments,the measuredplasmafreecon A brainMR image was acquiredin one subject on a 1.5 Tesla centrationdatawerefitto a sumof threeexponentials Signasystem(GeneralElectricCo., Milwaukee,WI). Transaxial Ti-weighted,3-mmcontiguousimageswereobtainedusinga spin @ echo sequence (TE 26, TR 500, FOV 24, 2 nex with a 256 x 256 f1C@(t)= ± C@@eAl Eq. 1 matrix). MIII was performed on the same day as the SPECT i= 1 experiment. Coreglstrationof MR and SPECT images was done relative to a set of 10common fiducialmarkersfilledwith 20 mM whereC01is thezero-timeintercept(@CV@)of eachexponential CUSO4 and approximately 10 @Ci[@Tc]sodium pertechnetate and A@the elimination rate constant (min@1)associated with each (41). The complete datasets (32 SPEC.!' and 45 MR images) were exponential. Plasma concentration at time zero, C@,was related to reslicedparallelto thecantho-meatalplane. theinjecteddose,D (pCi),andtotheinitialvolumeofdistribution of thetracer,V@1(L),by Image Analysis Imageswere reconstructedfromcounts set on the iodinepho D topeak (159 keV) with a 10% symmetric window using a Butter C0=@:C01=—. Eq.2 worth filter (cutoff = 1 cm, power factor = 10) and displayed on 1=1 Vbc@ a 64 x 64 x 32 matrix(pixelsize = 3.3 mm x 3.3 mm, slice thickness = 3.3 mm, voxel volume = 36.7 mm3). Attenuation The fractionofzero-time interceptassociatedwith each exponen correction was performed assuming uniform attenuation equal to tial, f@,was calculated as @ that of water (attenuationcoefficient = 0.150 cm2/g)within an fth=Co,/Co, Eq.3 ellipse drawn aroundthe skull as identifiedby the markers. !m @ ageswerereorientedso thatthecantho-meatalplane,asidentified and the clearance liter/hr) was calculated as by thefourfiducialmarkers,correspondedtothetransaxialplane of the dataset. D CL= Eq.4 The level of highest occipital concentrationwas identifiedby 3 inspection of the 32 images of an acquisitionobtained after peak brain uptake, usually 30 mis postinjection. This level was invari @: 1=1 ably located 9—10slices above the cantho-meatal plane. A set of threeslices,includingoneimagebelowandoneabovethelevelof The clearance parameterswere used to derive the interpolated maximal occipital activity, were then summed (upper slice). Three values of the input function to the compartment model and to sliceswerealsosummedatthelevelofthecantho-meatalplaneas estimate the bolus-to-constant infusion ratio for the constant in defined by the markers (lower slice). Seven ROIs were drawn on fusion experiments. the coregistered MRI slices and applied to the corresponding Forbolusplusconstantinfusionexperiments,the concentra SPEC!' slices fromthe coregistrationstudy. The same RO!s were lionof freeparentcompoundis givenby used, without shape or size modification, to analyze all experl ments. Pontiac (1 cm@)and cerebellarROIs (4.3 cm2)were posi @ tioned on the lower slice. Occipital (4.0 cm2), frontal (3.3 cm2), f1C@(t)= [email protected] A@ f@(i—e Alt), Eq. 5 temporal(2.8cm2),striatal(1.5cm2)andthalamic(0.8cm2)ROIs were positioned on the upper slice. Averagecpm/pixelROl activitieswere measured,decay-cor whereC5@(@CW@)is thefreeconcentrationatsteady-state.C@ rected for the time of injection and expressed in MCi/ccusing a is relatedto CLandtherateof infusionR@,(@Cl/hr),by calibration factor of 0.0069 @CWcpm.This calibration factor was C@=R@/CL. Eq.6 measuredwith an ‘@!distributedsource of 13.5 cm diameter(0.8 @Ci/@)acquiredandreconstructedusingthe sameprotocol.The FromEquation5, in theabsenceof a bolus(C@= 0), thetimeto sensitivity of the camera was monitored weekly between these reachsteady-stateis determinedby the smallestexponent(A3), studies and varied byless than 5%. A RO! was also placed on one i.e., theterminalhalf-life(ln(2)1A3).Approximately90%of Css 15 marker to assess the uniformity of the sensitivity during the cx reachedafter 3.3half-lives.The timeto reach 90%of C@can be periments. No attempts were made to correct for the scatter reduced if a bolus injection is given just priorto the initiationof fraction within the photopeak window nor for partial volume the constant Infusion. The optimal bolus-to-hourly infusion ratio effects. (B/!)wascalculatedusingthekineticparametersderivedfromthe single bolus experiments. Arterial Plasma Analysis Blood samples were analyzed as previously described (42). Kinetic and EquIlIbrium Analysis Extraction(ethylacetate)was followedby reverse-phase@HPLC Compamnental MOdeL Equilibrium and kinetic analyses were to measure the metabolite-correctedtotal plasma activity (Ca(t), performed using the same three-compartment model (Fig. 1): (1) @CWmm).Plasma protein bindingwas measured by ultracentrifu the arterialcompartment(C11);(2) the intracerebralnondisplace gation through Centrifree membrane ifiters (Amicon Division, ablecompartment(Ca)inwhichthetracercanbe freeornonspe W.R. Grace& Co., Danvers,MA).Thebindingto plasmapro cificallybound;and(3)thespecificallyboundcompartment(Ci). teins was assumed to be rapid as compared to the other processes Equilibration of the nonspecifically bound tracer is assumed to measured in these experiments. As a consequence, the free frac be rapidincomparisonto theotherprocessesso thattheratioof tionof plasmaparentcompoundmeasuredby ultracentrifugationthe free-to-the total tracer concentration in the nondisplaceable

230 TheJournalofNuclearMedicine•Vol.35•No.2 •February1994 @ K1 = FE = F(1—e (ml . g ‘. miii Eq.14

15k3k2 K1/V2f1(min@ 1),Eq.

16k4=k0ff(miii@'),Eq.17kcn@mJ2(mm ‘),Eq.

FiGURE1. Thethree-comparb@nentmodelusedforbothkinetic where F is the regionalblood flow (ml •g' . min1); E the and equilibriumanalyses.C, = arterialplasmaconcentration,me unidirectional extraction fraction; PS the permeability surface taboiftecorre@ed;C2= tra@erconcentrationinIntr@erebralnondis @abIecompartment(freeandnonspecificbinding);C, = specif areaproductof the tracer(ml •.g'. @i@@');and k@,and k.@the Icallyboundconcentration;K, to k, = fractionalrateconstants association and dissociation rate constants for ligand-receptor describingthekineticsoftracertransferbetweencompartments. binding. Equation 15 can be derived from Equation 12 by setting k3, k4and the derivates to zero and multiplyingboth sides by f1. Attracerdoses,Equations12and13haveconstantcoefficients and can be solved analytically (43,44), and BP can be derived compartment (f') is constant over time. The vascular activity from the rate constants using Equations 15, 16 and 17: present within an RO! was estimated assuming a blood volume equal to 5% of the ROI volume (21) and subtracted from the total BP Bmax koaBmax k3 K1k3 i8 RO! activityprior to analysis.The activityconcentrationin the RO! at time t, C@01(t),is the sum of the concentrations in the — KD 1(01! k42k2k41 Eq. secondand third compartments: Equilibnum [email protected] equilibrium, the rate of association and dissociation of the tracer-receptor complex are equal: CRo@(t)= C,Jt)+ C@(t). Eq. 7 @ @f2C@B@= Eq. 19 The equilibriumvolume of distributionof compartmenti is the ratio of the tracer concentration in compartmenti to the free Thus, tracer concentration in the arterial plasma at equilibrium, i.e., when no net transferbetweenthe plasmaand compartmenti Bmax C3 C3 exists: BP-@=@=@-@, Eq.20

vi= @a. 1@ 8 which demonstrates that BP is equal to the equilibrium volume of f1C5 £.d@1. distribution of the bound compartment (V3, Equation 8). At equilibrium,VTwas calculatedwithEquation9. Forcon V2istheequilibriumvolumeofdistributionofthenondisplaceable stant infusion experiments followed by an injection of a saturating compartment and V3 is the equilibrium distribution volume of the dose of flumazenil,V2was calculatedas the ratio of the nondis specifically bound compartment. The total tissue equilibrium vol. placeableactivity-to-theplasma-freeparentcompound(from15to ume of distribution,V.@.,is the sum of both compartments: 45miiipostflumazenilinjection)andBP(V@)wascalculatedasthe differencebetweenVTandV2(Equation9). CR0! VT j@ = V2+ V3. Eq. 9 Cwve-Fitting Plvcedwe. Rate constants for arterialclearance and brain uptake ofthe tracerwere estimated by nonlinear regres sion usinga Levenberg-Marquartleast-squaresminimizationpro Passive diffusion is assumed to be the mechanism of transfer of cedure (45) implementedin MATLAB (The Math Works, !nc., the tracer across the blood-brainbarrier(BBB). When the non South Natick, MA) on a Macintosh Quadra 950. The number of displaceable compartment is at equilibrium with the plasma, the exponential terms used to describe the plasma clearance was free concentration is assumed to be equal on both sides of the determinedby the F-test (46) and the AIC criteria (47). The BBB, standard error of the parameters was given by the diagonal of the f1C5= f2C@, Eq.10 covanance matrix (48) and expressed as percent of the parame ters (coefficientof variation,%CV).Thus, %CVindicatesthe which yields the inverse relationship between f2 and V2: identifiabilityofthe parameterby the least-squaresprocedureand should not be confused with the Standard deviation (s.d.) of the f2= i/V2. Eq. ii distributionof the parameteramong subjects. Kinetic Anc4ySiS. The tracer concentration over time in each inVitror@'iiom.@[email protected] compartment is given by: Twelve brains were collected from the District of Columbia Medical Examiner's Office. The brains were examined by a fo dC@(t) .—@;.—=K1C@(t)—k2C@(t)—k3C@(t)+k@C@(t),Eq.12 rensic pathologistor a neuropathologistfor the detection of gross abnormalities and were cut into i-cm thick coronal slices. Sam pleawere dissectedon ice and immediatelyfrozenat -70°Cuntil dC@(t) assayed. Inclusion criteria were absence of gross pathological —@i--=k3C@(t)—k@C@(t), Eq.13 abnormalitieson examinationof the brain and absenceof neuro psychiatricdisordersas noted by reviewof the medicalrecords. wherethekineticparametersK1to k, aredefinedas follows: Ages varied from 22 to 87 yr (50 ±22 yr). Brains from four

SPECTMeasurementofBenzodlazeplneReceptors•Abr-Darghamatal. 231 females and eight males were used. Time between death and freezingof tissue was 24 ±10hr. Preservationtime at —80°Cwas A 1000 76 ±12 mo. Onthedayof theassay,brainsampleswereweighed,thawed iii C.) @ and homogenizedwith a Polytron(setting6 for 105cc) in 1/40wet £100 weight in mg/vol in ml (wAr)of buffer(25 mM KH2PO4'150 mM z NaCl, pH 7.4). After homogenization, tissues were centrifuged @ (20,000g, 4°C,10min). The supernatantwas discardedandpellets 10 were resuspended and recentrifuged. This procedure was re Ef 2 peated twice. Incubation(22°C,45mm)was initiatedby the suc cessive additionof 100 @tlof [‘@!]iomazenil,100 @dof bufferor unlabeled iomazenil and 800 @dof tissue solution. A final tissue dilution of 1/2,400 w/v was selected so that total binding was 40 80 120 160 between 5% and 10%of total ligand concentration. Incubation was terminated by rapid filtration though OF/B ifiters on a 48- channelCell Harvester(Brandel,Gaithersburg,MD). Filterswere B 1500 rapidly washed three times with 5 ml of ice-cold buffer and counted in a COBRA 5010 gamma counter (Packard, Menden, i200 CT) with an efficiency of 80%. Saturationexperiments (n = 12) C.) were performedby the isotopic dilutionmethod (“cold―satura C 900 lion) on the occipital cortex from each brain using one concentra 0 tion of [‘@!]iomazethl(0.02 nM) and 15 concentrations of unla > 600 beled iomazenil ranging from 10_14 to 106 M. Saturation F experiments were analyzed by weighted nonlinear regression @ 300 analysis using the programLIGAND (NIH, Bethesda, MD) (49).

RESULTS 80 160 SingleBolusEXperimentS TiME(mln) After single bolus injection of [‘@!]iomazenil,the six FiGURE2. (A@Arterialtlme-actMtycurveof free parent subjects exhibited similar kinetics of plasma clearance of [1@lJiomazenilfollowinga bolusInjectionof 12.5mCIIna 22-yr-old the parentcompound (Table 1). In all cases, a sum of three male.Valueswerefittoatriexponentlalmodel(solIdlIne)toderive exponentials provided a statistically significant improve the peripheralclearance(Cl = 8.5lIter/hr/kg).(B)Regionalbrain time-actMtycurvesfromthesamesubjectasInpanelA Measured ment in the fit as compared to a two-exponential fit. A values (drcles,trianglesand squares)werefitto a three-comport four-exponential fit provided a statistically significant im mentmodel(solIdlines).BPvalueswere272,174and58 inthe provement of the fit in only two of six subjects. Therefore, occipitalcortex,frontalcortexandcerebellum,respectivaly. a three-exponential model was chosen (Fig. 2A). !mtial volume of distributionwas 0.44 ±0.14 liter/kg and clear ance was 7.4 ±1.1 liter/hr/kg.The average A3(0.013 ±0.01 RO!s peaked earlier and at lower values (in decreasing min') corresponded to a terminalhalf-lifeof 97 mm. The order temporal > frontal > striatum, thalamus, cerebellum free fraction ofthe parent compound was between 20% and > pons). RO! time-activity curves were fit to the three 30% of the total parent compound (f1 = 0.23 ±0.02). compartment model (Fig. 2B). The iteration procedure The occipital region showed the highest and latest up converged for all regions, providing estimates of the re take, peaking at about 30 mm (Fig. 2B). Other investigated gional rate constants (K1to k@)and values for the outcome measures (‘@2@f2, BP and VT, Table 2). K1rangedfrom 0.385 nil . g@ •@Ji@@1(pons) to 0.546 ml

TABLE 1 . g' . min' (occipital), with an average value for all PeripheralClearance Parametersin Singie-BolusExperiments regions of 0.467 ml . g@ . piJ1@1.‘flij@parameter was reasonably identified in the frontal, occipital, temporal and [email protected]/kg)(lfter/hr/kg) cerebellarregions (%CVbetween 5%and 6%)butwas less well identified in the striatum(%CV ±s.d.; 16% ±9%), 10.296.80.2220.528.20.2330.487.30.2340.418.50.2350.295.60.2760.648.40.21Mean±s.d.0.44thalamus (%CV ±s.d.; 18% ±15%) and pons (%CV ± s.d.; 19% ±7%). The rate constant k2 showed a large variation across regions, from 0.094 ±0.118 min1 (tem poral) to 0.300 ±0.214 (pons)with a mean regional value of 0.151 ±0.150 min', and was poorly identified in all re ±0.147.4 ±1.10.23 ±0.02 gions, with %CV ranging from 26.6% ±8.9% in the thal amus to 127% ±159%in the striatum. The value of k3 VboI initial volume of distribution In plasma CL = P@riP'@' dEW varied from 0.175 ±0.095 min' (occipital) to 0.104 ± ance;andf1= freefractionInplasma. 0.037 min@' (cerebellum) but was as poorly identifiable as

232 TheJournalofNuclearMedicine•Vol.35•No.2 •February1994 Estimation of the Optimal Bolus-to-Infusion Ratio for 800 Con@antIn@&on @m@tS Simulationswere performedto estimate the optimal bo 600 lus-to-hourly infusion ratio needed to reach equilibrium 0. rapidly in all ROIs. The system was considered at equiib num if both plasma and brain activity were within 5% of @400 I the asymptotic equilibrium values (C@@in plasma and VT in @ :i: .•...... - .•. brain). Simulations were based on the average clearance parameters and brain rate constants derived from the bolus 200 only experiments (n = 6, Tables 1 and 2). The first simu lation (mean clearance) was performed using the mean ,@ I I I values of the group(7.4 liter/hr/kgfor a 70-kgsubject, A3= 0 40 80 120 160 TIME(mln) 0.013 min'). Additional simulationswere performedwith clearancevalues correspondingto 2 s.d. below (slow clear ance) and 2 s.d. above (fast clearance) the average clear FiGURE3. Bindingpotentialversuslengthofdataanalysis.Fol lowingbolusinjectionof12.5mCiof[1@l]Iomazenil,theplasmaand ance of the sampled group (398 liter/hr and 708 liter/hr. braindatafromSubject3 wereanalyzedusingthecompletedataset respectively). These scenarios were implemented with the acquiredover155mmandforvasiabtyshorterintervalsfromtime0 corresponding A3values of 0.007 and 0.022 min' for slow tothatindicatedonthex-axls.ValuesrepresentthekinetiCallyde and fast clearance simulations, respectively. Simulations uivedBP ±s.e.e.TheseresultsshowthatBPwouldhavebeen were performedfor two ROIs with high (occipital, BP = overestimatedif thescanningses&onhadbeenlessthan60mm andreached100%ofthe valuedeterminedfromthecompletestudy 215, VT 233) and low (cerebellum, BP = 91, VT = 108) by80mm. densities of receptors. For each of the three clearance conditions, time-activity curves were projected in the plasma, occipital and cerebel larROIsforvarious bolus-to-hourlyinfusionratios ranging k2,with %CVrangingfrom23.9 ±7.3 in the cerebellumto from 1 to 5. Bolus-to-hourly infusion ratios between 3.2 142% ±84% in the thalamus. The rank order of k3 was: and 4.0 fulfilledthe equilibriumcriteriain both ROIs in all occipital > frontal = striatum > thalamus > pons > tem three situations. For example, with a ratio of 3.4, the value poral = cerebellum. Values of k@were low in all regions of plasma-freeparent activity integratedbetween 300 and (regionalaverage = 0.022 ±0.009 min', correspondingto 420 mm was 101%, 103% and 100% of C@5for the control, a dissociation half-lifeof 31 min). Identifiabilitywas better slow clearance and fast clearance simulation, respectively than k2and k3but worse than K1(with %CVrangingfrom (Fig. 4A). The occipital total volume of distributioninte 9.5% ±2.9% in the cerebellum to 55% ±30% in the grated during the same period was 102%, 98% and 103% of thalamus). VTforcontrol, slow andfast clearance, respectively. Inthe BPvalues were significantlydifferentamongregions(oc cerebellum, the corresponding values were 105%, 102% cipital, 240 ±41; temporal, 189 ±3; frontal, 161 ±15; and 104% (Fig. 4B). Thus, based on these simulations, cerebellum, 101 ±23; striatum, 81 ±12; thalamus, 78 ± equilibriumshould be attainedby 300—420miii in all ROIs, 27; pons, 30 ±ii; ANOVA: p < 0.001), and %CV of with bolus-to-infusion ratios of 3.4:4. The average bolus regional means varied from 36%in the pons to 2% in the to-infusion ratio used in the experiments reported here was temporal. VTalso exhibited significantregionaldifferences 3.63 ±0.21 (n = 8). (ANOVA: p < 0.001), with %CV ranging from 8% in frontalto 30%in pons. No statistically significantregional Bolus Pius Constant Infusion Experiments differences in V2 were observed (ANOVA, p = 0.17). V2 !n the experiment depicted in Figure 5A, arterial sam regional average was 31 ±15. The kinetic analysis esti ples were collected from 0 to 450 min and plasma values were fit according to Equation 5. Parameters estimated mated the average regional nonspecific binding at 20% ± from the regression were: A1 = 1.17; A2 = 0.137; A3 = 7% of the total activity. The extravascular free fraction 0.0246; f01 = 1.5; f@ = 0.0824; f1J3 0.0560; the clearance represented 7% ±4% of the nondisplaceable compart @ was estimated as 6.7 liter/hr/kgand the was estimated ment. as 2.1 nCi/ml.The Css obtained by averagingthe last four The minimaldurationof scanning needed to derive sta values (over the last hour of the experiment), 2.2 nCi/ml ble BP values was evaluated by fittingthe occipital ROIs was fairly close to the C@ estimated by the regression. forvarious periodsoftime rangingfrom0—50to 0—150miii, Measuredactivities in threerepresentativeROIs(occipital, with 20-miii incrementalsteps (Fig. 3). In the majorityof temporal and cerebellum) from this experiment are shown cases, the program did not converge for less than 80 miii in Figure5B. SPEC!' datawere acquiredfrom0 to 150min and when it did, BP values were markedly overestimated. and from 250 to 420 min. Plasma and RO! activity were After 80 min, BP values were within 5% of that derived by stable from 180mm untilthe end of the infusion (420min). fitting the entire experiment (0—150min). Similarobservations were made in two additionalexpen

SPECTMeasurementofBenzodlazepneReceptors•AbI-Darghamatal. 233 TABLE 2 KineticAnalyaisof SingleBolusExperiments Parameters Frontal Occiplal Temporal Striatum Thalamus Cerebellum Pore mc@@jn@1)0.4630.5460.4950.4460.5340.3980.385±(ml. g1 s.d.0.2190.1670.2220.2000.3550.1710.172k2 (m1n1)0.1560.1360.0940.1230.1430.1060.300± s.d.0.1940.0980.1180.1340.2120.0800.214k3(mln-1)0.1450.1750.1080.1340.1180.1040.114±

s.d.0.1540.0950.0610.0800.1330.0370.064k4 (m1n1)0.0170.0140.0170.0340.0290.0180.024± s.d.0.0050.0040.0060.0270.0100.0040.008BP161.00240.00189.0081.0078.00101.0030.00±

s.d.15.0041.003.0012.0027.0023.0011.00f20.070.050.040.050.050.060.17±

s.d.0.090.030.030.030.040.030.09V237.0031.0054.0027.0041.0020.008.00±

.004.00VT196.00271.00242.00108.00119.00121.0037.00±s.d.25.0030.0054.0019.0030.001 1

.0011.00%s.d.16.0029.0055.00180034.0031 .0019.0024.0033.0016.0022.00±NSB18.001 1 s.d.12.0011.0015.0013.0020.006.0013.00

Valuesaremean±s.d.of sixexperiments. mctoi@,= transferrateconstants;BP= bindingpotentialasdertvedfromkineticanalysis;@2= freefr@IonInthenondispleceablecompartment V2- nondlspIaceai@Ieequilbiumvolumeofdl@rIbutIon;VT= t@aItissueequiblumvolumeofdistribution;and% NSB= percentnonspecific t@ndIng

ments. Therefore, in subsequent experiments, arterial InVitroraswom@eniIBindinginPostmortem plasmasamples and scan datawere acquiredonly from250 HumanBrain to 450 min. In the occipital cortex, [‘@!JiomazemlBm@was 162±52 To quantifythe stability of the plasma andbrainuptake, nM and KD was 0.59 ±0.14 n.M (n = 12). These values a linear regression was performedon plasmavalues (last 4 corresponded to a BP of 287 ±95. No significantcorrela hr, mean number of points = 12) and on RO! values (last 2 tion was observed between age and [‘@!]iomazenilB@,, or hr of the experiment, mean numberof points = 22). !n the KD. plasma, the mean change was 2.5% ±2.1%/hr.With the exception of the pons, all RO!s showed changes of less DISCUSSION than 5%/hr. RegionalVTwas measuredby calculatingthe ratioof the These studies demonstratedthe feasibility of measuring averaged RO! activity and the free unmetabolized plasma benzodiazepine receptors regional binding potential with activity over the last 2 hr (Table 3). SPECF. Two methods were tested: kinetic analysis of a single bolus injection and equilibrium analysis ofbolus plus Bolus Plus Infusion Plus Dlsplacsmsnt Experimsnts constant infusion. With the exception of the pons (where Flumazenil injection was well tolerated by all three sub the low level of counts may have contributed to a higher jects with no side effects reported at the dose of 0.2 mg/kg. level of noise), both methods gave similar regional results. Flumazenil induced 88% displacement of the tracer in the For example, the occipital BP was 240 ±41 (n = 5) and248 occipital, 87% in the temporal, 86% in the frontal, 84% in ±53 (n = 3) for kinetic and equilibrium experiments, the thalamic and cerebellar, 81%in the striataland 60%in respectively, and veiy close to the in vitro homogenate the pontine RO!s (n = 3). Plasma tracer activity exhibited binding value of 287 ±95. a slight (11%±3%)and transient(5—15mm) increase after The three-compartment kinetic analysis applied here flumazenilinjection. At the time ofthe measurementof V2, was comparable to the kinetic analysis developed for re the plasma tracer concentration was back to baseline. Re versible PET tracers such as [‘1Cjflumazenil(16,24). As gional V2 varied from 34 (occipital) to 23 (pons, Striatum previously observed with PET, the rate constants K1 to k@ and thalamus; Table 3 and Fig. 6). The value of f2, calcu were poorly identified (high standard errors due to covari lated as 1/V2,varied from 0.043 (pons) to 0.030 (occipital ance) when unconstrained kinetic analysis was applied and temporal).An importantobservation was the presence (30,44). Furthermore, the values of the kinetic parameters of 60% displaceable binding in the pons, indicating that the wereinconsistentwiththeirphysiologicalinterpretation. pons cannot be used as an area devoid of benzodiazepine Regional differences in K1 and k2 should reflect regional receptors sites. differences in blood flow. Their ratio, V2 should reflect the

234 TheJournalof NucisarMedicine•Vol.35•No.2 •February1994 @ a

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t4 0.1 0 90 180 270 360 450 B OCCIPITAL w B 600 •IdL!s!@• 500 ••%.•-w•? -J • • • * 0 8 ••••••S••. •• .• ••.• •• > •? •••• z o@400 @s 0 @.E. TEMPORAL • • 4 @- 300 mg200 4 CEREBELLAR I 00

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FiGURE4. (A)Simulatedplasmafree[1@l)momazenilconcenta FIGURE5. (A)Arterialtime-activitycurveoffreeunmetabolized tionaftera bolusinjectionof3.4 mCIfollowedbya constantInfusion (1@qioru@i after a bolus of 3.68 ma followedby a constant of 1 mCI/hrfor420mm.Thesoftdlinerepresentstheprojectedmnfuslonof 1.12 m@libr(B/I ratio of 3.68 hr) in a 21-yr-oldmale. time-ectivitycurvein a 70-kgsubjectwith [1@IJiomazenilclearance Valueswere fit to a tilexponentlalmodel (solidlIne)to derivethe of553lfter/hr(= averageclearancemeasuredInsixsubjects).The peripheralclearance(CI = 6.7 liter/hr/kg).(B) RegionalAOl time dottedline( . . . . ) andthebrokenline(———)areprojectedactMtles activitycurves.TotalAOl activityat equllibdum(averageof the last insubjectsw@,slow(398Ilter/hr)andhigh(708llter/hr)clearance,180 mW@)was 0.61 @CWrnlin the occipital [email protected] @CWmlin the respectivaly.In all cases,Steady-Stateis achlevedat 300 mm.Sim temporalcortexand 0.19 @&CIfmlIn the cerebellum.The average ulatlonsweregeneratedusingthe followingparameters:V@ =34 leveloffree parentradlotrecerInplasma(2.2nCl/mI)duringthe last @ lfterf01 = 1.0;f@= 0.11;f@= 0.026;A1= 1.18m1n1; 0.127 2hrwasassumedtobeequaltothefreeredictrecerinthebrain.The min1; A3= 0.013 mlrr1, 0.007 min@ and 0.022 mWr1 In mean, ratiooftotal brainactMtytothefreegaveVTvaluesof 276,242and sk@wand fast clearancescenarios,respectively.(B) Simulatedvol 89 Inoccipital, temporal and cerebellar ROIs, respectively. umes of distribution(regionalactivity/freeplasma activity)in the occlpftalcortex and cerebellum.In both regionsand In all three clearancescenarios,the volumeof distributionreechedan equ@Ib numvalue(‘IT)at 360mm.Parametersusedforthissimulationdance with the known distribution of BDZ receptors in were:f1 = 0.231; K, = 0.546ml. mmn@. g1; k@= 0.136m1n@;k@ human brain (50—52). = 0.175 min1 (ooclpltaJ) and 0.104 min1 (cerebellum), k, = A similarsituation is observed in plasma. Whereas it is 0.0141 m1n1; V2 = 17; BR = 215 (occipital),91 (cerebellum). unclear ha defined physiological process is associated with each exponential, the overall outcome measure, the clear nonspecific distribution volume and is not expected to vaiy ance of the compound from the plasma, is adequately de much between regions. In fact, V2. as derived fromkinetic scribed by a three-exponential model. Similarly, the total analysis (Table 2), showed more regional variation than V2 tissue volume of distribution is a well defined outcome as measured directly by flumazenil displacement (Table 3). measure. According to the model, k3 is the parameter that includes Theidentifiabilityandphysiologicalmeaningoftherate the receptor density (B@, Equation 16), leading theoret constants can be improved by constraining the regression ically to a correlation between VT and k3. No such corre process to values obtained in a region devoid of receptors lation was observed (Table 2). The molecular dissociation (16,44,53).K1,k2ortheirratiocanbederivedfromthese constant, k4 (k@) is expected to be similar across regions, regions andused in fittingreceptor-richregions. This strat which was not the case (Table 2). Thus, the rate constants egy could not be implemented with [‘@I]iomazemldue to did not reflectthe physiological processes ascribedto them the absence of a region devoid of receptors. Flumazenil by the model. However, the regional distributionof the displacement (n = 3) clearly showed that 60% of the activ outcome measure VT was better identified and in accor ity measured in the pons was displaceable. This displace

SPECTMeasurementof Benzod,azepneReceptors•Ab@Darghamatal. 235 TABLE 3 EquilibriumMalysis of BolusPlusConstantInfusionExperimentS Frontal Ocdpital Temporal Striatum Thalamus Cerebellum Pore 5)VTBolusplusconstantInfusions(n = 23512513814360±s.d.209.00 267.00 3910283419Bolus28.00 32.00 3)VT plusconstantinfusionplusflumazenildisplacement(n = 26612715217063±s.d.234.00 281.00 5523253527V235.00 53 3323232723±s.d.31.00 34 13414f2 5.00 5 0.0300.0430.0440.0370.043±0.032 0.030 0.0010.0060.0080.0020.007BPs.d. 0.005 0.004 23210312914339±s.d.20300 248 5620293624%NSB38.00 53 1319161640±s.d.14.00 12 4.00 3 316510

@ VT tissueequllblumvolumeofdistributIon;V2= nondisplaceableequdibilumvolumeofd@trIbutIon;f2= treefractioninthenondisplace ablecompartmentBP = bindingpotentialasderivedfromequllbriumanalysisand% NSB= percentnonspecificbinding.

able activity could be due to contamination by activity with tissue distributionvolume (includingboth specific and originating in surrounding regions, due to the limited res nonspecific binding) as outcome measure. The kinetic olution of the SPECF device. However, the presence of method proposed here is equivalent to this solution in the specific binding in the ponshas previously been reported sense that the total volume of distributionis the outcome with in vitro studies in the humanbrain (50,51). Our data measure. are consistent with these reports and clearly suggest that A constant infusion plus flumazenil displacement para the use of the pons as reference region is questionable. digm was developed to provide direct measurements of In cortical regions, the nondisplaceable binding repre regional V2. VTvalues as measured duringconstant infu sented only 13%of the total binding.The total equilibrium sion were close to VTvalues derived fromkinetic analysis. volume of distributionis thus primarilyaffected by recep This method presents several advantages over the kinetic tor density. Koeppe et al. (16) proposed a two-compart method: ment model for the analysis of [“CiflumazenilPET data, 1. Direct measurement of V2 by flumazenil injection. This displacement method was developed because of the difficulties associated with the proper derivation 350 Flumazenll of V2 by the kinetic method. 2. Direct measure of the free tracer. The prolonged ‘z;@ 300 :@ OCCIPITAL •• plasma steady-state allows the free tracer to equili @ C.) 250 •••oO@ brate on both sides of the BBB. The validity of this @ £ FRONTAL o@@oo@000000 @ 200 method has been demonstrated in rodents and ba boons with CSF measurements (3Z35) and microdi @ 150 CEREBELLAR alysis (54). 4 ioo 3. Computational simplicity. The outcome measure is o 0 0 •o00 obtained by a simple ratio. This would allow an easy @ 50 PONTINE ••@@°@o.@‘“@P@! implementationof a pixel-by-pixel representation of 0 •0@ 0 . . , S. • BP. 150 200 250 300 350 400 450 4. Prolongedacquisitiontime. At equilibrium,the activ TIME (mm) ity level being stable, the image acquisition time can be increased. Thus, this method appears to be well FiGURE 6. RegionalAOl time-activitycurvesfrom a constant infusionexperimentfollowedbyflumazenildisplacementina 24-yr suited to SPECT, which has a lower sensitivity than old female. Scanningstartedat 280 mm and endedat 450 mm. PET. Injectionof flumazenil(0.2mg/kg)resuftedIn 84% dIsplacementIn 5. Reduced scanning time. For proper derivation of VT theoccipital,82%Inthefrontal,78%in cerebellarand50%in with the kinetic method, the subject has to stay in the pontineROls.Specificblndmngwasmeasureddirectlybysubtracting camera for 100-120 mm. With the equilibrium para thenonspeclflcfromthetotalactMty.Theratioofthe specificbinding tothefree,measuredInplasmaoverthelast2 hr,gaveBRvalues digm, this time can be reducedto 60 minbecause only of 199,163,102and 22 Inthe occipitalcortex@frontalcortex@cere one acquisitionbefore and one after flumazenilinjec bellum and pons, respectively. tion is needed.

236 The Journal of Nuclear Methane •Vol. 35 •No. 2 •February 1994 6. No arterial sampling. At steady-state, the arterial and benzodiazepinereceptors:oppositechangesin humanepileptogenictissue. venous plasma concentrations equilibrate, allowing Neurology 199242:811—815. 4. OwenF, PoulterM,WaddingtonJL,MaShaIRD,CrowTJ.[3H]Ro05.4864 the measurement of tracer C@ concentration from and[3Hjflunitrazepambindinginkainate-lesionedratstriatwnandtemporal one venous blood sample, which further facilitates cortex of brainsfrompatientswith seniledementiaof the Alzheimertype. the experimental procedure. &ainRes 1983;278:373—375. 5. ShimohamaS, TaniguchiT, FujiwaraM, KameyamaM. Changeinbenzo Several disadvantagesofthe constant infusiontechnique diazepinereceptorsin Alzheimertype dementia.Ann Newvl 1988;23:404- 4@. must be noted. Although scanning time is reduced, the 6. PenneyJB, YoungAB. Quantitativeautoradiographyof neurotransmitter total experimental time is increased from 120 miii to 500 receptorsin Huntingtondisease.NeuroIogj@198232:1391-1395. min. There is a potential for pump failure and finally, if 7. WalkerFO, YoungAB, PenneyJB, Dovorini-ZisK, ShoulsonI. Benzodi. subjectperipheralclearance is extremely fast or slow, equi azepine and GABA receptors in early Huntington's disease. Neurology 198434:1237—1240. librium may not be reached by the time of scan acquisition. 8. Whitehouse PJ, Trifiletti RR, Jones BE, et at. Neurotransmitter receptor Low specific activity [‘@I}iomazenilconstant infusion alterationsinHuntington'sdisease:autoradiographicandhomogenatestud experiments were used in primates to derive in vivo icswithspecialreferenceto benwdiazcpinereceptorcomplexes.AnnNeu ivi 1985;18:202—210. [‘@I]iomazenilK@and Bm@(33,35). Derivation of these 9. KiuchiY, Koba@ T, TakeuchiJ, ShimizuH, OgataH, Toni M. Ben. parameters with kinetic analysis is complex since the zodiazepinereceptorsincreaseinpostmortembrainofchronic schizophren model becomes nonlinear at significantlevels of receptor ice.EwA,th P3ychNewvl Sd 1989;239:71-78. 10. BenesFM,VincentSL, Alsterberg0, BirdED, SanGiovanniJP. Increased occupancy. In contrast, Scatchard analysis can easily be GABASreceptorbindinginsuperficiallayersofcingulatecortexinschizo performed on equilibriumdata. We are in the process of phrenics.JNeWV,Sci1992;12:924-929. extending these low specific activity experiments to hu 11. Squires RF, Lajtha A, SaesderUp E, Palkovits M. Reduced mans. [3Hjflunitrazepambindingincingulatecortexandhippocampusof postmor tern schizophrenic brains: is selective loss ofglutamatergic neurons associ Assuming an in vivo K@of 0.59 nM (value measured in ated with majorpsychoses?NeurochemRes 1993;18:219-223. primates with low specific activity SPECT experiments) 12. HantrayeP, KaijimaM; PrenantC, et al. Centraltype benzodiazepine (33), the occipital BP value of 244 reported here would bindingsites: a positionemissiontomographystudy in the baboon's brain. Neurosci Let: 1984;48:115-120. correspond to a Bm,, of 141nM. This value is close to the 13. SamsonY, HantrayeP, BaronJC, SoussalineF, ComarD, MaziereM. Bm@ measured in vitro in 12 subjects (Bm@ of 162 ± 52 Kineticsanddisplacementof [11CJRo 15.1788,a benzodiazepineantago nM) . In vitro and in vivo values were thus consistent. tiist, studiedin humanbrain in vivo by positrontomography.EurJ Phar macol 1985;110:247—251. In conclusion, both kinetic and equilibrium methods pro 14. PerssonA, EhlingE, EriksonI, Ctat.Imagingof[@q-laheledRo 15-1788 vide adequate measures of regional total equilibriumdis to benzodiazepinereceptors in the humanbrain by positronemissionto tribution volume of [@I]iomazenil. Following establish mography.IPsychiat Res 1985;19:609-622. 15. ShinotohH, YamasakiT, Inouc0, Ctal. 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238 The Journal of Nuclear Medicrne•Vol. 35 •No. 2 •February 1994