NUCLEAR NEUROLOGY LABORATORY STUDIES Congeners as PET Imaging Probes for Terminals

Anna-Liisa Brownell, David R. Elmaleh, Peter C. Meltzer, Timothy M. Shoup, Gordon L. Brownell, Alan J. Fischman and Bertha K. Madras Department ofRadiology, Massachusetts General Hospital, Boston, Massachusetts; Organix Inc., Woburn, Massachusetts; and Harvard Medical School and New England Regional Primate Research Center, Southborough, Massachusetts

Several brain imaging agents targeted to the dopamine The PET imaging properties of three drugs with neurons and associated nerve terminals have been developed to differing affintt@sand selectMties for the dopamirie over serotonin image Parkinson's disease. These positron emission tomo transporter, were compared. Methods Carbon-i 1-CFT (WIN 35,428, 2p-carbomethoxy-3p-(4-fluorophenyl)),11C-CCT graphic agents are aimed at monitoring dopamine neurons (RT1-131, 2@3-carbomethoxy-3f3-(4-monochlorophenyI)tropane), and (8—26).The most commonly used ligand is 18F-fluoro-L-dopa, 11C-CDCT (, 2@3-carbomethoxy-3@3-(3,4-dichlorophe an analog of the naturally occuring compound, L-DOPA (8— nyl)tropane) were evaluated as imaging probes for dopamine neu 10). However, this compound is metabolizable and striatal rons in five normal and in two MPTP-treated cynomolgus monkeys accumulation of radioactivity is a reflection, in part, of the (macaca fascicularis) using a high-resolution PET imaging system activity of an amino acid transporter and of metabolism to (PCR-I).Results For “C-CFT,the specific binding ratk (as defined 18F-fluoro-dopamine and other 1 F-labeled metabolites. Other by the ratioof radioactivitylevels instriatumversus cerebellum)was ligands have been developed, particularly those targeted to the 4.2 ±0.8 in caudateand 4.9 ±1.2 in putamen at6O mm and 4.9 ± including I‘C-S- (11,12), 12 and 5.5 ±1.1 at 90 mm in controlanimals.In MPTP-treated 18F-GBR 13119 (13—15)and I C-cocaine (16—17). However, monkeys the corresponding ratios were 1.4 ±0.1 in caudate and 1.5 ±0.1 inputamenat 60 mmand 1.3 ±0.1 incaudateand 1.4 ± nonselective binding of these ligands to sites other than the 0.3 in putamenat 90 mm.Forthe monochloroanalogof CFT, dopamine transporter has been a major limiting factor for use of 11C-CCT, the ratios in control caudate and putamen were 2.7 ± 0.4 these compounds for in vivo imaging. and 3.4 ±0.3, respectively, at 60 mm and 3.7 ±0.5 and 4.4 ±0.6, In search of in vivo probes with high affinity and selectivity respect@ve@', at 90 mm. In MPTP-treated animals, corresponding for dopamine transporter/neurons, Madras et al. proposed (18— ratios were 1.4 ±0.4 and 1.5 ±0.3 at 60 mm and 1.4 ±0.4 and 1.6 23) the use of phenyltropane analogs of cocaine based on ±0.4 at 90 mm. The dichloro analog of CFT, CDCT, which has the promising data obtained with 3H-2j3-carbomethoxy-3@-(4-flu highest affinityfor the dopamine transporter, generated the lowest orophenyl) tropane (CFT or WIN 35,428) (18—20). In particu ratios in control brains, 2.3 ±0.4 in caudate and 2.4 ±0.5 in lar, studies conducted with 3H-CFT in postmortem human putamen at 60 mm. In one MPTP-treated monkey, the correspond Parkinson's diseased caudate and putamen showed that the ing ratios were 1.6 ±0.4 and 1.8 ±0.3. In comparison with 11C-CFT,both 11C-CCTand 11C-CDCTwerelessselectuveand had depletion of the transporter on dopamine neurons corresponded high uptake inthe thalamus. Conclusion: The present results clearly to the reported loss of dopamine (21). indicate that 11C-CFT is a useful ligand for monitoring dopamine Tritiated-CFT binding sites in primate striatum displayed a neuronal degeneration. pharmacological specificity consistent with that of the dopa Key Words CFT (WIN 35,428); CCT (R11-131); CDCT (dichloro mine transporter (18,19) and distributed primarily to brain pane); positron emission tomography; Parkinson's disease dopamine systems (20—22). Although selective for the dopa J NuciMed1996 37:1186-1192 mine transporter, the affinity of CFT for the transporter is relatively moderate, 11.0 ± 1.0 nM (18,20,24,25). Mono or dichloro substitution of the fluoro group on the phenyltropane Parkinson'sdiseaseisaneurologicaldisorderwhichischarring yields compounds with approximately 10-fold higher acterized by the degeneration ofdopamine neurons in substantia affinity for the dopamine transporter than CFT (WIN 35,428) nigra and depletion of dopamine in striatal terminal fields of but with reduced selectivity for the dopamine transporter (24). these neurons (1 ). A primary neurochemical consequence of In this study, the imaging characteristics of I1C-CFT and the Parkinson's disease is a marked decrease in the concentration of higher affinity chloro analogs of CFT, ‘‘C-CCTor RTI-l3 1 dopamine in the striatum (2). The original observation led to (2f3-carbomethoxy-3j3-(4-monochlorophenyl)tropane and 1 experiments aimed at developing animal models for Parkin CDCT or dichloropane (2@3-carbomethoxy-3f3-(3,4-dichloro son's disease using surgical techniques or neurotoxins selective phenyl)tropane) are described and compared in control and in for dopamine neurons (3 ). Since cases of human Parkinsonism MPTP-treated monkeys. were discovered after intravenous self-administration of a mependine analogue, l-methyl-4-phenyl-1,2,5,6 tetrahydropy MATERIALS AND MEASUREMENTS ridine (MPTP) (4), primate models of Parkinsonism have been MPTP Model of Parkinson's Disease developed using different MPTP administration protocols To induce symptomatic parkinsonism, adult (>3.3 kg) male and (5—7). female cynomolgus monkeys (macaca fascicularis) were anaesthe tized (15 mg/kg ) and three sequential doses of MPTP ReceivedMarch20, 1995;revisionaccepted Oct.8, 1995. (dose 0.7 mg/kg) were administered into the femoral vein within 10 Forcorrespondenceorreprintscontact Mna-UisaBrownell,PhD,PhysicsResearch Laboratory,Departmentof Radk@ogy,Masseshusetts Genetal Hospital,Edwards days (6). The primates developed Parkinsonian symptoms shortly Building, 5th floor, Boston, MA02114. after the last injection of MPTP and symptoms persisted as long as

1186 [email protected]'@i.OFNUCLEARMEDICINE•Vol. 37 •No. 7 •July 1996 animals and in five studies with two MPTP-treated monkeys. ,CH3 ‘‘C-CCT was used in two studies in control animals and in one N study in an MPTP-treated primate. All MPTP-treated animals had control studies before MPTP treatment. Carbon-i l-CDCT was used in one primate before and after MPTP treatment. For PET imaging, the animals were anaesthetized with ketamine/ FiGURE1. Schematic structure of Y CFT (WIN 35,428, 2@3-carbome xylazine (15/1.5 mg/kg, intramuscular, initial dose) and anaesthesia thoxy-3j3-(4-fluorophenyl)tropane), was maintained with half doses hourly as needed. Catheters were 1.CFT(W1N35,248); X—F,Y—H CCT (R11-131,2@-carbomethoxy placed into the femoral artery and vein for collection of blood 2. CCT ; x—Cl,Y—H 3fr.(4-monochlorophenyl)tropane) samples and injection of labeled ligand. The animal was placed and CDCT (dkhloropane, 2f3-car ventrally on imaging table. The head was secured in a customized 3. CDCT ; X—Cl,Y—Cl bomethoxy-3f3-(3,4-dichlorophe nyl)tropane). acrylic head holder equipped with ear and eye bars specially designed to ensure reproducible stereotactic head positioning from for three months, until euthanasia. Animals were maintained with study to study. Dynamic imaging data were collected at seven daily intramuscular injections (0.1—0.5mg/kg) ofquinelorane, aD2 different coronal planes: 30, 25, 20, 15 and 10 mm anterior from dopamine receptor agonist. Animals used in this study were the origin, 5 mm and 10 mm posterior from the origin. After maintained according to the guidelines of the Committee on injection 1.5 ±0.3 nmole/kg ofradiolabeled ‘‘C-CFT,or ‘‘C-CCT Animals of the Harvard Medical School and Massachusetts Gen or 1‘C-CDCT(3.8—7.9mCi, specific activity 600—1000 mCi! eral Hospital and of the “Guidefor Care and Use of Laboratory @mole)into the femoral vein, imaging data were collected stepwise Animals―of the Institute of Laboratory Animal Resources, Na at each level using an initial collection time of 15 sec. The tional Research Council, Department of Health, Education and collection time was increased up to 60 sec and the total imaging Welfare, Publication No. (NIH)85-23, revised 1985. time was 90 mm. Calibration of the positron tomograph was performed prior to each study using a cylindrical plastic phantom Radiopharmaceutical Production Carbon-11-CFT, CCT (diameter 6 cm) containing a solution of ‘8F.Six arterial blood and CDCT samples (600 pi) were collected at 5, 10, 20, 30, 60 and 90 mm Carbon-l l-CFT, ‘1C-CCT, and 11C-CDCT (Fig. 1) were pre after injection of labeled compound for determination of metabo pared by direct 1‘C-methyliodide methylation of their nor N- lites. Blood analysis was done by thin-layer chromatography demethylated precursors. The nor precursors were synthesized by (TLC). A plasma aliquot was developed on a silica gel TLC plate Organix, Inc. ‘‘C-methyliodide was produced in an automated after drying. The eluant system was methanol:ethylacetate:trieth system following proton bombardment of N2 containing 0.5% 02 ylamine (10:90:0.01). At 5 mm more than 90% of the activity was to yield ‘‘CO2.The 1‘CO2was trapped in 0.4 ml ofLAH (0.25 M) associated with ‘‘C-CFT,Rf 0.45. The rest of the activity was in THF and dried under a stream of N2 gas by heating at 120°C. associated with more polar metabolic product Rf = 0.1—0.2 The A1LiO-' ‘CH3was treated with 0.8 ml of a 57%-solution of (possibly hydrolysis ofthe 2B-carbomethoxy ester). The metabolic hydroiodic acid. The resulting I1C methyl iodide was then trans product increased >20% at 30 mm. At later times low levels of ferred as a gas using N2 as carrier to a reaction vial containing 400 radioactivity in blood did not allow for accurate quantification of I_Lgof N-demethylated precursor (norCFT, norCCT or norCDCT) the peaks. in 0.3 ml of DMSO and the mixture was heated at 110°Cfor 5 mm. Data Analysis. Imaging data were corrected for attenuation, HPLC purification of ‘‘C-CFT(or ‘‘C-CCTor ‘‘C-CDCT)was decay and collection time. PET images were reconstructed using performed with a Waters C ,8 reverse-phase column (1 X 10 cm) Hanning weighted convolution backprojection (28). Regions of using a solvent system of 60% methanol and 40% pH 7.3 buffer interest (ROIs), including left and right caudate and putamen, (2.75 g KH2PO4, 2 ml TEA in 1000 ml water) and flow rate of 2 frontal, parietal and temporal cortex, thalamus and cerebellum, ml/min. Retention times were 6.5, 7.7 and 9.0 mm for ‘‘C-CFT, were drawn on different planes and activity per unit volume, I ‘C-CCT and ‘‘C-CDCT, respectively. Radiochemical yield was percent activity of injected dose and ligand concentration were 15—20%and radiochemical purity >98%. After solvent evapora calculated. The values corresponding to time points of 60 and 90 tion, the residue was dissolved in 6-ml saline and the solution was mm were interpolated for each ROl. Putamen data were averages filtered through a 0.22-p@mMillipore filter. The specific activity of of data from A-P levels 20 and 15 mm from the left and right sides the final product was between 600—1000Ci/mmole. The prepara using the area of ROI as a weight. Caudate and cortical data were tion time of the labeled compound was 23 mm. averaged similarly from A-P levels 25 and 20 mm from the left and PET Studies right sides. Thalamus data were obtained from A-P level 10 mm Imaging System. PET was performed using a high-resolution and cerebellum data were averaged from levels —5and —10 mm. PET scanning system equipped with one ring of 360 BGO In vivo specific binding for each subject was defined by the ratio detectors and a computer-controlled imaging table (27). The of average radioactivity concentration in caudate, putamen, cortex resolution of PCR-I for a point source at the center of the imaging and thalamus to the corresponding cerebellum value based on the field is 4.5 mm and the sensitivity is 46,000 counts per sec for a assumption that the cerebellum does not have specific binding sites source 20 cm in diameter with a concentration of 1 @Ci/ml,when and the measured radioactivity represents nonspecific binding. the images are reconstructed using Hanning weighted convolution In studies involving MPTP-treated animals, the ROIs obtained backprojection with cutoffvalue of 1.0 (28). The overall efficiency during the pre-MPTP phase (control study) were used as guides, is 64% of the theoretical maximum for a plane thickness corre because the striatum is not clearly identifiable in brains of sponding to the 2-cm high detectors. A plane thickness of 5 mm, to MPTP-treated monkeys. be used in this study, is obtained by limiting the effective height of the detectors with cylindrical collimators. The coincidence resolv ing time of PCR-I is 6 nsec (FWHM). RESULTS Study Protocol. PET studies were performed with the three Carbon-I 1-CFT PET Imaging different ligands in normal and MPTP-treated cynomolgus mon Time-activity curves were generated from various coronal keys (weight 3.3—4.6kg). Carbon-ll-CFT was used in five control images, which included planes of frontal cortex, caudate nu

COMPARISON OF 1‘C-CFT, ‘1C-CCT AND 11C-CDCT •Brownell et al. 1187 Control C-CFT 18 18

E 15 15 0 0

0 12 12

9 a U 0 @0 6 6

3 3 0 0 0 15 30 45 60 75 90 0 15 30 45 60 75 90 $t-cCT 18 18

E 15 15 0 0

0 12 12

a 9 U 0 6 6

3 0 3 0 0 15 30 45 60 75 90 0 15 30 45 60 75 90 It-.cDCT 20 20

E 17 0 17 0 0 14 14

11 11 U a a 0 6 6

3 0 3 0 0 15 30 45 60 75 90 0 15 30 45 60 75 90 Tims (mini Tim. (mini

FIGURE 2@lime-acthifty distilbution of 11C-CFT,uC@CCT,and @C-CDCTin a control and MPTP-freated ptimate (macace fasciculails) in five brain areas. Putamendata are the average of level15 mm on the leftand tightaldes, caudate data of level20 mm, and frontalcortexdata of level25 mm, respecth,ely. Thalamicdata are obtained fromlevel10 mm and cerebellumdata of level-5 mm. cleus, putamen, thalamus and cerebellum. In Figure 2, putamen left and right sides at A-P level 25 mm. Thalamic data were data are averaged from the left and right sides at A-P level 15 obtained from A-P level 10 mm and cerebellar data were from mm. Caudate data are averaged from the left and right sides at level —5mm. Time activity curves for “C-CFTin normal A-P level 20 mm. Data for frontal cortex are averaged from the caudate and putamen showed a rapid increase in radioactivity

1188 THEJOURNALOFNUCLEARMEDICINE•Vol. 37 •No. 7 •July 1996 TABLE I Specific binding of [11C]CFF, @1cJccT and r1cicoc@ in Normal and MPW-treated Primates at 60 and 90 Min*

60 mm 90 mm PET Numberof imaging animala/ Frontal Frontal agent studlas Caudate Putarnen cortex Thalamus Caudate Putamen cortex Thalamus C1CICFT Control[5/5] 4.2 ±0.8 4.9 ±12 1.4 ±0.2 1.4 ±02 4.9 ±1.2 5.5 ±1.1 1.2 ±0.2 1.1 ±0.2 MPTP[2/5] 1.4±0.1 1.5±0.1 1.2±0.2 1.2±0.2 1.3±0.1 1.4±0.3 1.2±02 1.1±0.1 rTC]CCT Control[2/2] 2.7±0.4 3.4±0.3 1.2±0.2 1.5±0.3 3.7±0.5 4.4±0.6 1.3±0.3 1.6±0.3 MPTP[1/1] 1.4±0.4 1.5±0.3 1.2±0.2 1.5±0.2 1.4±0.4 1.6±0.4 1.2±0.2 1.5±0.3 r1C]CDCT Control[1/1] 2.3 ±0.4 2.4 ±0.5 1.2 ±0.2 1.4 ±0.2 — — — — MPTP[1/1J 1.6±0.4 1.8±0.3 1.5±02 1.8±0.4 1.8±0.2 1.9±0.4 1.5±0.2 1.8±0.4

*Specfficbindingwas determined as a ra@oof uptake (mean ±s.d.) of each region[caudate = average fromAP levela25 and 20 mm on leftand nght sides; putamen = averagefromAPlevels20 and 15mmon leftand rightsides; frontalcortex = averagefromAPlevels25 and 20 mmon leftand rightsides; thalamus from AP level 10 mm] to cerebellum (average from AP levels -5 and -10 mm).

over the first 10 min after injection and a slower increase up to Carbon-II-CCT PE@ Imaging 90 mm (Fig. 2, top). In the cortex, thalamus and cerebellum The regional accumulation of radioactivity following intra @ radioactivity increased sharply within minutes but declined venous injection of monochioro analog of CFT, ‘C-CCTin after 6—7mm. In MPTP-treated primates (Fig. 2, top right), normal and MPTP-treated monkeys was similar to that of radioactivity increased sharply in all brain regions although the 1‘C-CFT (Fig. 2, middle panel). In the control caudate and plateau for putamen and caudate occured later than for other putamen, the radioactivity increased through the imaging period regions. A rapid decline in all regions was observed. (90 mm) while the radioactivity washed out from other areas of For control studies interpolated values for specific binding brain. In MPTP-treated monkey brain radioactivity washed out ratios in caudate, putamen, frontal cortex and thalamus at 60 of all brain areas. A relatively high striatum-to-cerebellum ratio and 90 mm are given in Table 1. The striatum-to-cerebellum was achieved (2.7 and 3.4) at 60 mm, which increased to 3.7 ratio increased sharply to >4 within 60 min and was approxi and 4.4 by 90 mm (Table 1). The thalamus-to-cerebellum ratio mately 5 within 90 mm. In the other brain areas, frontal cortex (1.5) was higher than the corresponding ratio for the frontal and thalamus, the ratios were equivalent and did not exceed 1.4 cortex. for the duration of the experiment. This distribution cone sponds closely to known distribution of 3H-CFT (WIN 35,428) Carbon-II-CDCT PET Imaging on the dopamine transporter, dopamine and dopamine receptors In vitro, CDCT or dichloropane, the dichloro analog of CFT (19—23).In MPTP-treated primates, the striatum-to-cerebellum has one of the highest affinities for the dopamine transporter ratio was lightly higher at 60 mm than at 90 min (1.4 and 1.5 (25). The time-activity curves for 1‘C-CDCTrevealed rapid versus 1.3 and 1.4) indicating washout of radioactivity. In accumulation in putamen and caudate, and significant levels in addition, binding ratio in striatum was lightly higher than in thalamus, frontal cortex and cerebellum in normal monkeys cortex and thalamus, 1.2 at 60 min and 1.2 and 1.1 at 90 miii, (Fig. 2, lower left panel). Radioactivity increased in caudate and @ respectively. The distribution of 1 a normal and an putamen through the imaging period (70 mm) and accumulated MPTP-treated primate are compared at 4 different levels (20, in the thalamus for at least 45 mm while it decreased in frontal 15, 10 and —5mm) at 60—64mm after injection ofthe labeled cortex and cerebellum after an initial rise. In an MPTP-treated ligand (Fig. 3). animal, radioactivity increased and thereafter remained constant

•18600 @ Control ‘@,t@ @,, 0 1.

1000

I FIGURE3. Dlatnbutlonof @C-CFTat fourcoronalbrainlevels(20, 15, lOand —5mm) in a control (upper row)and MPTP •.•‘ MPTP-treated Qowerrow) pilmate at J5cm 60-64 mm after injection.Aftercorrec tions for decay, collectiontime and in meted act@Atythe highest pixelvalueof the eight images is nOrmalIZedto 10,000 and the lowest to 0. ,6J@the images are nOrmalIZedaccording this 20mm 15mm 10mm -5mm scale.

@ COMPARISON OF 1C-CFT, 1‘C-CCT AND 1‘C-CDCT •Brownell et al. 1189 [email protected] largest loss was detected using CFT, indicating that 50 1 C-CFT is the most sensitive of the three ligands for detecting loss of dopamine transporters. In a comparative distribution of the three ligands in MPTP-treated primates at levels 20, 15, 10 25 C and —5mm binding can clearly be seen in extrastriatal regions E (e.g., thalamus) for the chloronated ligands (Fig. 5). . 0 0. DISCUSSION I- 0. The development of selective high-affinity ligands for imag —25 S ing the dopamine transporter on dopamine neurons is desirable S for a number of reasons. The higher the affinity of the drug, the S lower the dose needed for detecting its targets by in vivo C a —50 .C imaging, and the more likely the ligand will remain associated C.) with its target for the duration of the study. Furthermore, dopamine may compete with the probe for binding to the —75 dopamine transporter ifthe affinity is low. Finally, selectivity is needed to minimize binding to the or other FIGURE 4. Average change in spedflc binding after MPTP treatment in four sites, as nonspecific binding may reduce the level ofaccuracy of bralnareas (putamen,caudate, frontalcortexand thalamus)using11C-CFT, 11C-CCTand 11C-CDCTat 60-64 mm after injectionof labeled ligand. imaging data. Specific binding was defined as ratios of radkactMty concentrations to The affinity and selectivity of ‘‘C-CFT,‘1C-CCT and ‘‘C- cerebellum basing on the assumption that cerebellar radioactivity represents CDCT were previously determined in vitro (24). The IC50- nonspecificbinding. values for the dopamine transporter were 11.0 ±1.0 nM for CFT, 1.40 ±0.O4nMforCCT(RTI-l3l)and 1.09 ±0.O2nM in the caudate, putamen and thalamus and declined in the cortex for CDCT. Corresponding values for the serotonin transporter and cerebellum after an initial increase (Fig. 2, bottom right were 160 ±2OnM, 5.87 ±2.8 nMand2.47 ±0.14 nM. panel). In a control study the binding ratios were 2.3 and 2.4 for Transporter selectivities (dopamine/serotonin) calculated from caudate and putamen and 1.2 and 1.4 for cortex and thalamus at the in vitro data were 15-fold for CFT, 4-fold for CCT and 60 miii (Table 1). In an MPTP-treated monkey the thalamus/ 2-fold for CDCT. Thus, CFT is the most selective of the three cerebellum ratio (1 .8) was the same as the putamen/cerebellum phenyltropane analogs, although it has lower affinity than the ratio at 60 min. However, at 90 mm the binding ratio of other halogenated phenyltropane analogs. The imaging data putamen (1.9) was lightly higher than that of thalamus. The supports these observations. The specific binding expressed as thalamus to cerebellum ratio was higher than frontal cortex to striatum/cerebellum ratio was highest using ‘‘C-CFT;4.9 ±1.2 cerebellum ratio in both the control and MPTP-treated primate at 60 mm and 5.5 ±1.1 at 90 mm for putamen and lowest with brain. I ‘C-CDCT, 2.4 ± 0.5 in putamen at 60 mm. Further, the loss in Comparison of Carbon-11-CFT, CCT and CDCT in MPTP specific binding after MPTP treatment was most prominent @ Treated Monkeys when 1 was used to image the striatum (Figs. 4, 5). The loss ofspeciflc binding in striatum after MPTP treatment Interestingly, using ‘‘C-CDCT,the calculated specific binding differed for 1‘C-CFT,‘1C-CCT and 11C-CDCT (Table 1, Fig. ratio in cortex area and thalamus increased after MPTP treat

_A(@ . 10000 CFT&@ IT'@:

1@ CCT “I

J5cm CDCT RGURE 5@Comparathiedistributionof 0 . ., the uganda11C-CFT,11C-CCTand “C CDCT in MPTP-treated p.imates at four coronal brain levels @20,15, 10 and —5 1@ mm) at 60-64 mm after mjection.The normalizationofimage set lathe same es 20mm 15mm 10mm -5mm in Figure3.

1190 [email protected]•Vol. 37 •No. 7 ‘July 1996 ment in a single study. These areas contain moderately high PhD, John Zaknun, MD, Department of Radiology, and Steven B. level of the serotonin transporter and as ‘‘C-CDCThas high Tatter, MD, Department of Neurosurgery, Massachusetts General affinity (2.47 nM) for serotonin transporters, it may bind to Hospital for helping with the image production. This work was these sites when the dopamine transporter is depleted after supported in part by National Institutes of Health grant MPTP treatment. Taken together, the studies support the use of CA32873-i 1 and Department of Energy grant DE-FGO2- more selective agents for monitoring dopamine terminals in 87ER60519 to Massachusetts General Hospital and grants neurodegenerative diseases. DA06303, NS30556, RROO168 and National Institute on Drug It is shown that tracer/receptor complex may be affected by Abuse GrantDA09462 to the Primate Center. A preliminary report radioactive metabolites and changes in blood flow (29). How of this work appeared in the 1994 proceedings of the annual ever, metabolism of CFT is minimal (23) and the observed meeting of the Society Nuclear Medicine. metabolite structure is different than that of the ‘‘C-CFT.The main metabolite is a less lipophilic compound, the hydrolyzed REFERENCES methylester and its affinity is one order ofmagnitude lower than I. Bernheimer H, Birkmayer W, Hornykiewicz 0. Brain dopamine and the syndromes of the labeled parent compound. This strongly suggests that Parkinson and Huntington. Clinical, morphological and neurochemical correlations. ‘‘C-labeled metabolites do not significantly affect PET imaging J Neurol Sd 1973;20:415—455. 2. Kish Si, Shannak K, Hornykiewicz 0. Uneven paftern ofdopamine loss in the striatum studies. In addition, ketamine anesthesia may decrease blood of patients with idiopathic Parkinson's disease. 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Cocaine receptors labeled by [3H12@- demonstrate a loss of dopamine neurons equivalent to the loss carbomethoxy-3@-(4-fluorophenyl)tropane. Mol Pharmacol 1989;36:5 I8—524. @ of dopamine in the human postmortem Parkinson's diseased 19. Madras BK. ‘C-WIN35,428 for detecting dopamine depletion in mild Parkinson's brain (21 ). Subsequently, ‘‘C-CFTbinding was found to disease. Ann Neurol 1994;35:376—377. 20. Canfleld DR. Kaufman Mi, Madras BK. Autoradiographic localization of cocaine correlate with behavioral symptoms in a primate model of receptors by [3H] CFT in monkey brain. Synapse 1990;5:189—195. Parkinson's disease (37,38) and in early Parkinson's disease in 21. Kaufman Mi, Madras BK. Severe depletion of cocaine recognition sites associated with the dopamine transporter in Parkinson's diseased striatum. Synapse l991;9:43— humans (30,39). The present results clearly indicate that ‘‘C- 49. CFT has the highest selectivity of the three ligands studied and 22. Kaufman Mi, Madras BK. 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Age-Related Diminution of Dopamine Antagonist Stimulated Vesamicol Receptor Binding

Simon M.N. Efange, Rosemary B. Langason and Anul B. Khare Departments ofRadiology, Medicinal Chemistry and Neurosurgery, University ofMinnesota, Minneapolis, Minnesota

specific regions of the brain (4—7). Although qualitatively Previous studies of radiolabeled vesamicol receptor (VR) Iigands similar alterations in cognitive and motor function are associ suggest that the latter may be used in conjunclion with dopamine 02 ated with normal aging and age-related neuropathology, unves antagonists to measure changes in striatal tholinergic function. In this study, the effects of aging on vesicular acetyk@hoIinestorag& tigations into the neurochemistry of normal aging have largely release were investigated wfth the high-affinityVR Iigand (+)-meta yielded conflicting results. r25ok@dober@4tro@amicoI [(+)-@25@jMIBT].Methods Male Fischer With normal aging, alterations in presynaptic cholinergic 344 rats (aged 3 and 24 mo) were injected efther with a vehicle or a markers are either absent or of a much lower magnitude than 02 antagonist [ or S-(-)-eticlopridel. At prescribed inter in age-related neuropathology (3,8,9). In spite of these vals thereafter, allanimals were intravenoualy injected with 10 pCi of growing body of evidence suggests that the dynamic aspects (+)[email protected] hoursafter radktracerinjection,the animals of central cholinergic neurotransmission are impaired in were killed and their brains dissected. The concentration of radio aging (10). Specifically, studies of SDHAChT (an indicator tracer in the striatum, cortex and cerebellum were then determined. of ongoing neuronal activity and structural integrity) and Results: In control animals, comparable levels of (+)-r25@JMIBT vesicular ACh (1 ) release in rodents have were observed in corresponding brain regions of young adult and aged Fischer 344 rats. Moreover, in haloperklol- and S-(-)-eliclo consistently revealed significant age-related reductions (10— pride-treated young adult rats, striatal levels of (+)-r25I]MIBTwere 13). In the present study, we attempt to assess the effects of elevated by 35% and 66%, reSpeCtIVely,relative to controls. In aging on ACh storage/release mechanisms with the aid of the contrast, haloperkiol treatment failed to after the striatal levels of radiolabeled vesamicol receptor @VR) ligand (+)-meta (+)-r25@MIBTinaged ratswhileS-(—)-eticlopridedisplayeda two [‘251]iodobenzyltrozamicol ((+)@[12 I]MIBT) (14). fold reduction in potency in aged rats. Conclusion: Aging is asso The vesamicol receptor, a unique site on the cholinergic ciated wfth a reduction in striatal choIinergk@plasticity or striatal synaptic vesicle, is functionally linked to the vesicular acetyl cholir,er.gicieser@@eandthatthe02-stimulatedincreaseinyR Iigand choline (ACh) transporter (15). The prototypical ligand for the binding is a fUnCtiOnally relevant parameter. VR, (—)-2-(4-phenylpiperidinyl)cyclohexanol (AH5 183, vesa Key Words iodine-125-MIBT vesamicol receptor@cholinergic func micol; Fig. 1), is a potent noncompetitive inhibitor of ACh tion; aging storage (16). Binding of vesamicol to the VR results in the J NucIMedI99@37:1192-1197 blockade of cholinergic neurotransmission, attributable to the inhibition of both vesicular ACh storage and the subsequent r1;, e cholinergic system has been implicated in memory and quantal release of ACh. ACh synthesis is, however, unaffected. central motor functions (1—3).In age-related neurologic disor The location ofthis receptor presents opportunities for studying ders, such as Alzheimer's disease, a progressive neurodegen the mechanisms underlying the storage and release of ACh. As erative disorder associated with: loss of cognitive function and part ofour ongoing effort to validate the use ofradiolabeled VR changes in personality, marked reductions in the presynaptic ligands in the study of presynaptic cholinergic function, we cholinergic markers choline acetyltransferase (ChAT), acetyl demonstrate in this article, with the aid of the YR ligand cholinesterase (AChE) and sodium dependent high-affinity (+)-[‘25I]MIBT, that aging is accompanied by a diminution of choline transport (SDHAChT) have been observed in several striatal cholinergic ftmction. MATERIALS AND METHODS ReceivedJun. 2, 1995;revisionaccepted Oct. 18, 1995. [email protected],PhD,UniversityofMinnesota The radiotracers (+)- and (—)-[‘251]MIBTwere synthesized as Hospitaland @1mIC,Departmentof Radiology,Ma@,vBox292,420 DelawareSt S.E, described (14) at a specific activity of 1500 ±200 Ci/mmole. MinneapOliS, MN 55455.

1192 THEJou@tr'@u.OFNUCLEARMEDICINE•Vol. 37 •No. 7 . July 1996