(2)-N-[11C]Propyl-Norapomorphine: A Positron-LabeledDopamine Agonist for PET Imaging of D2 Receptors

Dah-Ren Hwang,1,* Lawrence S. Kegeles1 and Marc Laruelle1,2

DEPARTMENTS OF 1PSYCHIATRY AND 2RADIOLOGY, COLUMBIA UNIVERSITY COLLEGE OF PHYSICIANS AND SURGEONS,

NEW YORK STATE PSYCHIATRIC INSTITUTE, NEW YORK, NEW YORK, USA

ABSTRACT. Imaging neuroreceptors with radiolabeled agonists might provide valuable information on thein vivo agonist affinity states of receptors of interest. We report here the radiosynthesis, biodistribution inrodents, and imaging studies in baboons of [11C]-labeled (2)-N-propyl-norapomorphine [(2)-NPA].(2)-[11C]NPA was prepared by reacting norapomorphine with [11C]propionyl chloride and a lithiumaluminum hydride reduction. [11C]Propionyl chloride was prepared by reacting [11C]CO2 with ethylmagne-sium bromide, followed by reacting with phthaloyl chloride. The radiochemical yield of (2)-[11C]NPA was2.5% at end of synthesis (EOS), and the synthesis time was 60 min. The specific activity was 1700 6 1900mCi/mmol (N 5 7; ranged 110–5200 mCi/mmol at EOS). Rodent biodistribution studies showed high uptakeof [11C](2)-NPA in D2 receptor-rich areas, and the striatum/cerebellum ratios were 1.7, 3.4, and 4.4 at 5min, 30 min, and 60 min postinjection, respectively. Pretreating the animals with haloperidol (1 mg/kg)decreased the striatum/cerebellum ratio at 30 min postinjection to 1.3. (2)-[11C]NPA was also evaluated viababoon positron emission tomography (PET) studies. Under control conditions (N 5 4), rapid uptake of thetracer was observed and the striatum/cerebellum ratio reached 2.86 6 0.15 at 45 min postinjection. Followinghaloperidol pretreatment (0.2 mg/kg IV), the striatum/cerebellum ratio was 1.29 at 45 min postinjection. Theresult demonstrated the existence of specific binding of this new tracer to the D2 receptor. To our knowledge,the current finding of a striatum/cerebellum ratio of 2.8 in baboon was the highest reported with aradiolabeled D2 agonist. (2)-[11C]NPA is a promising new D2 agonist PET tracer for probing D2 receptorsin vivo using PET. NUCL MED BIOL 27;6:533–539, 2000. © 2000 Elsevier Science Inc. All rights reserved.

KEY WORDS. D2 receptor, agonist, carbon-11, (2)-NPA, PET

INTRODUCTION

The majority of radiotracers used to visualize G-protein-linkedneuroreceptor with positron emission tomography (PET) imagingare antagonists. These tracers display equal affinity for receptorsconfigured in states of high and low affinity for the agonists. Yet, theability to preferentially label receptors configured in the state ofhigh affinity for the agonist might provide important informationregarding neurotransmission. For example, several groups showedthat the acute increase in dopamine synaptic concentration follow-ing administration of the potent dopamine releaser amphetamine isassociated with a reduction in the binding potential of D2 receptorradiotracers, such as ([123I]IBZM) and [11C]raclopride (3, 5, 11, 12,15). Using this technique, three studies reported that the reductionin [123I]IBZM or [11C]raclopride binding induced by amphetaminewas elevated in schizophrenia (1, 3, 14). The mechanism of thisexaggerated stimulation of dopamine transmission remains to beelucidated. In patients with schizophrenia, increased displacementof [123I]IBZM or [11C]raclopride by dopamine might result from alarger increase in synaptic dopamine concentration following am-phetamine (i.e., presynaptic factor), from an increased affinity of D2

receptors for dopamine (i.e., postsynaptic factor), or from somecombination of both factors.

The development of a radiolabeled agonist is needed to resolve

this issue. D2 receptors, like all G-protein-linked receptors, exist instates of high or low affinities for agonists. Antagonists, such as[11C]raclopride or [123I]IBZM, bind to both configurations withsimilar affinity. Thus, the in vivo binding of these antagonists doesnot provide any information about the affinity of D2 receptors foragonists. In contrast, PET studies performed with a radiolabeled D2

agonist would allow direct in vivo measurement of the affinity of D2

receptors for agonists.Previously, several groups have prepared and evaluated D2

agonists as candidate radiotracers. Halldin et al. (9) demonstratedthat 2-hydroxy-[11C]NPA (Ki 5 0.053 nM) did not cross theblood-brain barrier and 2-[11C]methoxy-NPA (Ki 5 0.17 nM) hada striatum/cerebellum ratio of 2 in Cynomolgus monkeys. [11C]A-pomorphine was shown by Zijlstra et al. (31) to have a striatum/cerebellum ratio of 1.5 in rats. [18F]Fluoropropyl derivatives ofnorapomorphine and tetralin exhibited low striatum uptake (30,32). More recently, Mukherjee et al. reported two new N-[11C-propyl]amino-5-hydroxytetralin derivatives, ZYY339 (Ki 5 0.01nM) and 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (Ki 50.65 nM), and the striatum/cerebellum ratios in rats were 1.9 and1.2, respectively (20, 26).

We have selected the specific D2 receptor agonist (2)-NPA,(2)-N-propyl-norapomorphine as our lead compound for tracerdevelopment. (2)-NPA is an apomorphine derivative synthesizedin 1973 (21). It is a potent dopamine agonist and has differentaffinities for the high and low affinity states of the D2 receptor. TheKhigh and Klow values are 0.4 and 23 nM, respectively (25). Studieswith (2)-[3H]NPA have established that (2)-NPA crosses theblood-brain barrier and accumulates in the striatum in mice (13).

Address correspondence to: Dr. Dah-Ren Hwang, New York State Psychi-atric Institute, 1051 Riverside Drive, Unit 31, New York, NY 10032, USA;e-mail: hwang@neuron.cpmc.columbia.edu.

Received 4 March 2000.Accepted 20 May 2000.

Nuclear Medicine & Biology, Vol. 27, pp. 533–539, 2000 ISSN 0969-8051/00/$–see front matterCopyright © 2000 Elsevier Science Inc. All rights reserved. PII S0969-8051(00)00144-X

The uptake is selective for the D2 receptor as demonstrated by theprevention of uptake by D2 agonists and antagonists and by the lackof effects of other receptor ligands, such as mianserin (5-hydroxy-tryptamine), phenoxybenzamine (a-adrenergic), or propranolol (b-adrenergic) (13). We report here the synthesis of (2)-[11C]NPA aswell as rodent biodistribution studies and initial PET baboonstudies.

MATERIAL AND METHODS

Chemicals and anhydrous ethyl ether were purchased from AldrichChemical Co. (Milwaukee, WI, USA) and were used withoutfurther purification. High performance liquid chromatography(HPLC) grade solvents were purchased from Fisher Scientific Inc.(Springfield, NJ, USA). Anhydrous tetrahydrofuran (THF) wasdistilled from sodium under nitrogen using benzophenone as theindicator. Norapomorphine, apomorphine, and (2)-NPA wereobtained from Research Biochemical Inc. (Natick, MA, USA). Adiluted ethylmagnesium bromide solution in ether was preparedinside a nitrogen-filled glove box by adding 0.4 mL of the ethyl-magnesium bromide in THF (1 M) to 2.8 mL of anhydrous ether.

The semipreparative HPLC system consisted of a DynamaxSD200 pump (Varian Instrument, Walnut Creek, CA, USA), aRheodyne 7010 injector (Phenomenex, Torrance, CA, USA), asemipreparative C-18 column (Alltech, Deerfield, IL, USA) (Sym-metry, 10 m, 10 3 250 mm), a Spectroflow 757 UV detector (KratosAnalytical, Chestnut Ridge, NY, USA), and a custom-made gammadetector. The system was connected to a PC running Peak Simpledata acquisition software. The semipreparative HPLC conditionswere as follows: solvent—20% acetonitrile and 80% 0.1 M ammo-nium formate/0.5% acetic acid (pH 5 4.2); flow rate—0–2 min, 2mL/min; 2–5 min, 5 mL/min; and . 8 min, 8 mL/min. [11C](2)-NPA had a retention time of 10 min. The analytical systemconsisted of a Dynamax SD200 pump, a Rheodyne 7125 injector, ananalytical C-18 column (Phenomenex Symmetry, 5 m, 4 3 250mm), a PDA detector (Waters, Milford, MA, USA), and a FlowCell gamma detector (Bioscan, Washington DC, USA). A PC withthe Millenium (Waters) data acquisition software was used for theanalytical system. The solvent used was a mixture of 30% acetoni-trile and 70% ammonium formate (0.1 M), and the flow rate was 2mL/min. (2)-[11C]NPA had a retention time of 4.8 min.

Preparation of (2)-N-[11C] Propylnorapomorphine ((2)-[11C]NPA)11CO2 was produced by the 14N(p, a)11C nuclear reaction on a gastarget, filled with 2% oxygen in nitrogen to 200 psi, using theColumbia University RDS-112 negative ion cyclotron. A carbo-sphere column was used to trap 11CO2 at room temperature (19).The trapped 11CO2 was released by heating the column under astream of argon into an ether solution of ethylmagnesium bromide(0.3 mL). After transfer, only a small amount of solvent remained.Phthaloyl chloride (0.6 mL) was then added, and the reaction vesselwas heated, with cooling water running, using a heat gun for 2 min.Then, 2,6-di-t-butylpyridine (0.3 mL) was introduced (16). Thereaction mixture was heated under a stream of argon (initial flow of25 mL/min and increased to 250 mL/min) to distill [11C]propionylchloride into a THF (1 mL) solution of norapomorphine (1 mg) andtriethylamine (5 mL). The solution was heated at 110°C for 5 min,followed by addition of lithium aluminum hydride (LAH, 1 M inTHF, 0.5 mL). After 5 min of reaction time at 110°C, excess LAHwas hydrolyzed by adding 1 mL of 3 N HCl and refluxing at 110°C

for 5 min. After brief cooling, the crude mixture was mixed with 0.1N HCl (30 mL) and passed through a C-18 Sep-Pak (Waters).Crude product was recovered from C-18 Sep-Pak using 1 mL ofacetonitrile. 3 N HCl (50 mL) was added to the acetonitrilesolution, and the solution was concentrated under a stream of argonto about 0.5 mL. An equal volume of HPLC solvent was added. Themixture was then purified by semipreparative HPLC. The fractioncontaining the product was diluted with 0.1 N HCl (100 mL) andpassed through a C-18 Sep-Pak. The Sep-Pak was washed with 10mL of 0.1 N HCl, and [11C](2)-NPA was recovered from theSep-Pak using 1 mL of ethanol. To stabilize the product, 50 mL of3 N HCl was added to the ethanol solution. The solution was thenfiltered through a 0.22-micron filter into a vented sterile vial.Specific activity of (2)-NPA (ethanol solution) was determined byanalytical HPLC.

It is well known that catecholamines decompose readily insolution. Efforts were made to examine the stability of the solutionof [11C](2)-NPA. During the purification of crude product andafter the removal of HPLC solvents, the Sep-Pak was washed with10 mL of 0.1 N HCl. To the final ethanol solution (1 mL) ofpurified [11C](2)-NPA, 50 mL of 3 N HCl was added. The solutionwas then diluted with 9 mL of saline and filtered. The stability ofthis solution was monitored by HPLC, and the [11C](2)-NPAsolution was found to be stable up to 2 h after end of synthesis(EOS).

Biodistribution Studies

Rodent biodistribution studies were performed according to proto-cols approved by the Columbia Presbyterian Medical Center Insti-tutional Animal Care and Use Committee. Male Sprague Dawleyrats (average weight, 300 g) were used for the biodistributionstudies. The tracers and blocking drugs were administered via tailvein. Each animal received 0.1 mCi of [11C](2)-NPA (specificactivity, 0.1–0.8 Ci/mmol) in 0.1 mL of saline. At the predeter-mined time points (5 min, 30 min, 60 min) animals were anesthe-tized with CO2 and sacrificed by decapitation. For blocking studies,animals received a dose of haloperidol (1 mg/kg) 5 min prior totracer administration and were sacrificed at 30 min postinjection.The selected organs were dissected. For the brain tissues, the wholebrain was removed, and frontal cortex, striatum, cerebellum, andrest of brain were dissected on an ice-cold glass plate, weighed, andcounted (Wallac 1480 Wizard 3M automatic gamma counter,Perkin Elmer, Gaithersburg, MD, USA). The percent injected doseper gram (%ID/g) of each tissue was calculated as the ratio of tissueactivity per gram of tissue to the net injected dose, measured as thedifference between injected radioactivity and the radioactivity leftin the tail.

Baboon PET Studies

Baboon PET studies (N 5 5) were performed according to protocolsapproved by the Columbia Presbyterian Medical Center Institu-tional Animal Care and Use Committee. All experiments wereperformed on the same 12-kg male baboon. The baboon wasimmobilized with ketamine (10 mg/kg IM) and anesthetized with1.8% isoflurane via endotracheal tube. Vital signs were monitoredevery 10 min and the temperature was kept constant at 37°C withheated water blankets. An IV perfusion line was used for hydrationand injection of radiotracers and nonradioactive drugs. A catheterwas inserted in a femoral artery for arterial sampling. The head waspositioned at the center of the field of view as defined by imbedded

534 D.-R. Hwang et al.

laser lines. The PET scans were performed with the ECAT EXACTHR1 (Siemens/CTI, Knoxville, TN, USA). A 15-min transmissionscan was obtained prior to radiotracer injection. A bolus of 6.25 60.82 mCi of [11C](2)-NPA (specific activity, 1.7 6 1.5 Ci/mmol)was injected IV over 30 s. Emission data were collected in the 3-Dmode for 121 min as 24 successive frames of increasing duration(6 3 10 s, 2 3 1 min, 4 3 2 min, 2 3 5 min, 10 3 10 min). Fourexperiments were acquired under control conditions. One experi-ment was performed 5 min following administration of haloperidol(0.2 mg/kg IV). PET emission data were attenuation-correctedusing the transmission scan, and frames were reconstructed using aShepp filter (cutoff 0.5 cycles/projection rays). Reconstructed imagefiles were then processed by the MEDX image analysis software(Sensor Systems, Sterling, VA, USA). PET images were coregis-tered to a T1-weighted magnetic resonance image (MRI) of thebaboon’s brain using an automated registration algorithm (28).Regions of interest (ROI) were drawn on the MRI and transferredto the registered PET frames. Time activity curves for each ROIwere measured and decay-corrected. Right and left regions wereaveraged.

RESULTSChemistry

[11C]Propionyl chloride was prepared according to a modifiedliterature procedure (16). Starting from 1000–1300 mCi of 11CO2,200–340 mCi of [11C]propionyl chloride were distilled into theTHF solution of norapomorphine. [11C](2)-NPA was successfullyprepared by a one-pot, two-step procedure: the reaction of norapo-morphine with [11C]propionyl chloride and an LAH reduction(Scheme 1):(2)-[11C]NPA was then purified using semipreparative HPLC (Fig.1). The average synthesis time was 60 min. Starting with 1300 mCi

of 11CO2, about 30–50 mCi of (2)-[11C]NPA were obtained atEOS. The radiochemical yield was 2.5% at EOS (or 16% at the endof bombardment). The specific activity was 1700 6 1900 mCi/mmol(N 5 7, ranged from 110–5200 mCi/mmol at EOS).

Biodistribution Studies

The results of the rodent brain uptake studies are presented in Table1. At all time points, the uptake of (2)-[11C]NPA in the striatum(D2 receptor-rich area) was higher than in receptor-poor areas(cerebellum and frontal cortex). The striatal to cerebellar activitiesratios were 1.73 6 0.18, 3.47 6 0.35, and 4.40 6 0.48, at 5, 30, and60 min postinjection, respectively. The striatum uptake was blockedby haloperidol pretreatment, and the striatum to cerebellum ratio at30 min postinjection was 1.29 6 0.13 as compared to 3.47 6 0.35in control conditions. In the peripheral tissues, the highest uptakewas observed in adrenal gland (Table 2). Other high uptake organsincluded kidney, spleen, liver, and lung.

PET Studies

Figure 2 presents the time activity curves in striatum and cerebel-lum following injection of (2)-[11C]NPA in the same baboon,under control conditions and following haloperidol blockade. Rep-resentative images of control and blocked experiments are shown inFigure 3. Under control conditions, activity accumulated rapidly inthe striatum. Striatal activity peaked at 306 6 30 nCi/mCi ID pergram of tissue (0.031 6 0.003 %ID per gram of tissue) at 7 6 1 min.This peak uptake was followed by a rapid washout. Cerebellaractivity peaked earlier (2.8 6 0.7 min) at 224 6 14 nCi/mCi ID pergram of tissue (0.022 6 0.001 %ID per gram of tissue). At 45 minpostinjection, the striatum to cerebellum ratio was 2.86 6 0.15.Specific binding, calculated as striatal minus cerebellar activities,peaked at 15 6 3 min. At the peak of specific binding, the striatalto cerebellar activity ratio was 2.33 6 0.15. Thus, as a firstapproximation, the specific to nonspecific activity ratio at equilib-rium would be in the range of 1 to 1.5.

In the blocking study with haloperidol, the striatum uptake was

FIG. 1. Chromatogram from preparative separation of (2)-[11C]NPA by reverse-phase HPLC. The major radioactivepeak is that of (2)-[11C]NPA.

TABLE 1. Brain Uptake of (2)-[11C]NPA in Male Sprague Dawley Rats

Time (min) Conditoin

%ID/g Tissue/cerebellum ratios

Cerebellum Frontal cortex Striatum Striatum Frontal cortex

5 Control 0.56 6 0.18 0.71 6 0.17 0.96 6 0.31 1.73 6 0.18 1.31 6 0.1830 Control 0.25 6 0.01 0.36 6 0.09 0.88 6 0.14 3.47 6 0.35 1.44 6 0.2730 Posthaloperidol 0.22 6 0.02 0.21 6 0.08 0.28 6 0.03 1.29 6 0.13 1.00 6 0.3760 Control 0.13 6 0.03 0.24 6 0.15 0.56 6 0.13 4.40 6 0.48 1.79 6 0.78

Data are mean 6 SD (N 5 3).

SCHEME 1. Reaction scheme for preparation (2)-[11C]NPA.

D2 Receptor Agonist (2)-[11C]NPA 535

decreased to the level of the cerebellar uptake. At 45 min, thestriatum/cerebellum ratio was 1.29 (compared to 2.8 6 0.7 incontrol experiments.

DISCUSSION

(2)-[11C]NPA was successfully prepared by the formation of[11C]propionamide of norapomorphine followed by a reduction(Scheme 1). Recently, a similar procedure was used for thepreparation of N-[11C]propyl-5-hydroxy-tetralin derivatives (20).This two-step procedure was chosen based on our previous experi-ence with the preparation of another dopamine agonist,[11C]pramipexole (11). In that study, a direct N-[11C]propylationprocedure using [11C]propyl iodide was studied. However, in ourhands, the yield of [11C]propyl iodide was consistently low, and thecoproduction of [11C]methyl iodide was always observed. These twofactors led to a low overall radiochemical yield of the directN-alkylation approach. On the contrary, the two-step procedure(amide formation followed by a reduction) resulted in a higher yield

of [11C]pramipexole. The same procedure with slight modificationwas used for the preparation of (2)-[11C]NPA. The major variationof the reaction condition was the use of lithium aluminum hydrideas the reducing agent.

The two-step procedure requires [11C]propionyl chloride as theradioactive precursor. To produce [11C]propionyl chloride, a mod-ified literature procedure was used (16). The procedure usedphthaloyl chloride as the chlorinating agent, and the acid chlorideformation and distillation was done in the presence of 2,6-di-t-butyl-pyridine. When the reaction was carried out with phthaloylchloride only, the yield of distilled [11C]propionyl chloride was low(, 100 mCi). In the presence of 2,6-di-t-butylpyridine, the distil-lation of [11C]propionyl chloride was more efficient (. 250 mCi).We also observed that the more Grignard reagent used, the lowerwas the distillation yield of [11C]propionyl chloride. A detailedstudy of this effect may lead to an even higher yield of the desiredacid chloride.

The preparation of (2)-[11C]NPA was successfully achieved byan one-pot, two-step procedure starting from [11C]propionyl chlo-

TABLE 2. Organ biodistribution of (2)-[11C]NPA in Sprague Dawley Rats

Time(min)

%ID/g

Blood Adrenal GI tracks Heart Kidney Liver Lungs Spleen Testes

5 0.18 6 0.08 1.60 6 0.72 0.26 6 0.07 0.31 6 0.14 1.09 6 0.27 0.52 6 0.20 1.15 6 0.37 0.72 6 0.21 0.07 6 0.0530 0.11 6 0.04 0.66 6 0.19 0.14 6 0.10 0.13 6 0.07 1.00 6 0.17 0.72 6 0.13 0.31 6 0.25 0.25 6 0.11 0.15 6 0.0260 0.07 6 0.01 0.58 6 0.18 0.13 6 0.07 0.08 6 0.02 0.45 6 0.29 0.34 6 0.10 0.26 6 0.08 0.35 6 0.42 0.18 6 0.20

Data are mean 6 SD (N 5 3).

FIG. 2. Regional activities following injection of (2)-[11C]NPA in a baboon under control conditions (circles) and followinghaloperidol pretreatment (0.2 mg/kg, triangles). Symbols are: control experiment—striatum (open circle); cerebellum (closedcircles); posthaloperidol experiment—striatum (open triangles); cerebellum (closed triangles). Under control conditions,activity concentrated in the striatum (striatum to cerebellum ratio of 2.8 at 45 min). Following haloperidol, striatal andcerebellar activities were similar (striatum to cerebellum ratio of 1.29 at 45 min). These experiments are consistent withspecific binding of (2)-[11C]NPA to D2 receptors in the striatum.

536 D.-R. Hwang et al.

ride and norapomorphine. After trapping [11C]propionyl chloride inthe THF solution of norapomorphine and triethylamine, a whiteprecipitate was observed in the vial. To ensure the formation of[11C]propionamide of norapomorphine, the reaction mixture washeated at 110°C for 5 min. A similar reaction condition has beenused successfully in the preparation of [11C]WAY100635 (10). LAHis the most effective reagent to reduce the [11C]propionamide ofnorapomorphine to (2)-[11C]NPA. A 5-min reaction time at110°C provided . 60% conversion of [11C]propionamide to (2)-[11C]NPA. Excess LAH was then destroyed using 3 N HCl. A C-18Sep-Pak was used to remove salt and radioactive by-product. Thecrude product was then recovered from C-18 Sep-Pak with aceto-nitrile and purified by semipreparative HPLC.

The purification of (2)-[11C]NPA is worth comment. Theamount of acetonitrile of the crude product solution affects thepurification. When the entire crude (2)-[11C]NPA acetonitrile

solution was injected directly, all radioactivity was eluted in lessthan 4 min, and no separation could be achieved. Therefore, thesolution was concentrated under a stream of argon to , 0.5 mL andthen diluted with HPLC solvent before injection. The initial flowrate was set to 2 mL/min for the first 2 min, and then increased to5 mL/min between 5 and 8 min, and 8 mL/min after 8 min. Usingthese conditions, a consistent retention time (10 min) of (2)-[11C]NPA was achieved. The specific activity of (2)-[11C]NPA hada wide range and is mainly due to the solvent used. For example, thetwo low specific activity runs (110 and 150 Ci/mmol) were theresult of using anhydrous ether with purity of only 99%. The specificactivity greatly improved (1100–5200 Ci/mmol) when higherpurity of anhydrous ether (. 99.8%) was used.

Biodistribution studies in rodents showed that (2)-[11C]NPAaccumulated in D2 receptor-rich brain regions, such as striatum. Astriatum to cerebellum ratio of 3.47 was achieved at 30 min

FIG. 3. Coregistered MRI and PET images acquired 30 to 40 min following injection of (2)-[11C]NPA in one baboon undercontrol condition (top row) and following pretreatment with the D2 antagonist haloperidol (0.2 mg/kg IV, 5 min prior to(2)-[11C]NPA injection).

D2 Receptor Agonist (2)-[11C]NPA 537

postinjection, and the ratio increased to 4.4 at 60 min postinjection.The striatal uptake was blocked by the administration of haloperi-dol. These findings agree with those of van der Werf et al. (27) whoused (2)-[3H]NPA in similar biodistribution studies and found astriatum/cerebellum ratio of 3.3 at 30 min post-tracer injection.Similar results were reported for (2)-[3H]NPA by Ross and Jackson(23).

The whole-body biodistribution studies also showed that manyperipheral tissues, such as kidney, liver, adrenal, and lung, had highuptake of radioactivity. The organ with the highest uptake was theadrenal gland. A similar uptake was observed with the [F-18]-labeled apomorphine derivative (32). The high uptake in thekidney and liver was also similar to that observed with [11C]apo-morphine (31). Zijlstra et al. suggested that the accumulation inthese organs was due to the conjugated metabolites of cat-echolamines (31).

Baboon PET studies with (2)-[11C]NPA showed that a striatumto cerebellum ratio of 2.8 was observed at 45 min postinjection. Theselective striatal uptake was decreased to 1.29 by the preadminis-tration of haloperidol, indicating the uptake is selective to the D2

receptor. To our knowledge, the ratio obtained with (2)-[11C]NPAis the highest reported for a D2 PET agonist. (2)-NPA is awell-established dopamine D2 receptor agonist. Administered inrodents, it induces a typical dopamine agonist response (i.e.,increased locomotion and stereotypies) (8, 18, 29). (2)-NPA is 35times more potent than apomorphine in producing stereotypedbehavior in rats. This effect of (2)-NPA is unaltered by alpha-methyl-para-tyrosine pretreatment, which is consistent with a directagonist effect (24). At low dose, (2)-NPA inhibits locomotorresponse, which is consistent with a preferential stimulation ofautoreceptors at low dose (2, 29). (2)-NPA is able to fullyeliminate firing of the dopamine cells in the nigra, an electrophys-iological response typical of dopamine agonist, which is inhibited byhaloperidol (17, 29). At the biochemical level, (2)-NPA induces aresponse typical of autoreceptor activation (antagonism of theg-butyrolactone-induced increase in dopa formation, antagonism ofreserpine and haloperidol-induced increase in tyrosine hydroxylaseactivity), of postsynaptic activation (elevation of acetylcholinelevel), and of diminution of both dopamine synthesis and ho-movanillic acid levels (4). (2)-NPA stimulates [35S]-GTPgS bind-ing in Chinese hamster ovary cells expressing D2 receptors, anagonist functional assay (6, 22), with an intrinsic activity of 114%relative to dopamine.

In vitro, the binding of (2)-NPA to D2 receptors is best fitted bya high and low affinity state model (6, 7, 25), and the high affinitystate is converted to a low affinity state in the presence of guaninenucleotides (6, 7, 25). In vitro, the affinity of (2)-NPA for the lowaffinity state (27 nM) is about 50-fold lower than the affinity for thehigh affinity state (0.4 nM) (25). Ross and Jackson (23) reportedthat [3H]NPA and [3H]raclopride had the same Bmax value in themouse striatum in vivo, indicating that (2)-NPA probably binds tothe low affinity state of the D2 receptors. Thus, the in vivo bindingpotential (BP 5 Bmax/KD) of [11C]NPA will reflect binding to bothhigh and low affinity sites of D2 receptors (BP 5 BPhigh 1 BPlow 5R1/Khigh 1 R2/Klow, with R1 and R2 being the number of sites inhigh and low affinity states, respectively). Yet, the contribution ofthe low affinity state to the binding measured at tracer dose shouldbe negligible. Assuming that the in vitro affinities predict the in vivoaffinities and that 30% of the available D2 receptors in vivo are inthe high affinity state, the contribution of the high affinity site to[11C]NPA binding at tracer dose is predicted to represent 96% of[11C]NPA binding [calculated as 100 z (0.3/0.4)/(0.3/0.4) 1 (0.7/

27)]. Therefore, (2)-[11C]NPA is a promising radiotracer andmight enable characterization of agonist affinity states of D2

receptors in vivo in diseased states. In addition, (2)-[11C]NPAmight provide a superior tool to study fluctuations of endogenousdopamine in vivo in humans.

CONCLUSIONS

(2)-[11C]NPA was successfully prepared with high specific activityvia a two-step procedure. Biodistribution studies and baboon PETstudies confirmed that (2)-[11C]NPA accumulates in D2 receptor-rich tissues. Additional studies, such as challenge studies withamphetamine and feasibility in determining the high affinity site ofthe D2 receptor, are warranted.

Supported by a Young Investigator Award from NARSAD (D.R.H.)and the U.S. Public Health Service (NIMH K02 MH01603-0 andNIMH MH59342-01). The authors would like to acknowledge theexpert assistance of Julie Montoya, Ning-Ning Guo, Ph.D., HenryHuang, Ph.D., Rikki Waterhouse, Ph.D., Kimchung Ngo, John Castil-lon, Rick Weiss, Norman Simpson, and Jennifer Bae.

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