In Vivo Labeling of the Dopamine D2 Receptor with N-' ‘C-Methyl-Benperidol

Makiko Suehiro, Robert F. Dannals, Ursula Scheffel, Mango Stathis, Alan A. Wilson, Hayden T. Ravert, Victor L. Villemagne, Patricia M. Sanchez-Roa, and Henry N. Wagner, Jr.

Division ofNuclear Medicine and Radiation Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland

been reported to be close to 50% ofthe amount bound A new dopamine D2 receptor radiotracer, N-11C-methyl to the dopamine receptor in striatum (11). With the benperidol(11C-NMB), was prepared and its in vivo biologic methylated derivative, N-methyl-spiperone, @—‘2O%of behavior in mice and a baboon was studied. Carbon-i 1- the binding of this compound in the striatum and 90% NMB was determined to bind to specific sites character in the frontal cortex was characterized as binding to the ized as dopamine D2 receptors. The binding was satura serotonin S2 receptor (15). As a result, in PET brain ble, reversible, and stereospecific. Kineticstudies in the images with spiperone or its derivatives, the tissues with dopamine D2 receptor-rich stnatum showed that 11C-NMB low densities of dopamine receptors but an abundance was retained five times longer than in receptor-devoid of serotonin S2 receptors, such as frontal cortex and regions, resulting in a high maximum stnatal-to-cerebellar ratio of 11:1 at 60 mm after injection.From frontal cortex cortex, are visualized as well as dopamine receptor-rich and cortex, on the other hand, the tracer washed out as tissues. rapidlyas it did fromcerebellum,resultingin tissue-to This poor selectivity is a disadvantage ofthese ligands cerebellar ratios dose to one in these regions at any time as dopamine D2 receptor radiotracers. Among the series after injection. Blockingstudies confirmed the specificity of the butyrophenone neuroleptic compounds devel and selectivityof the ‘1C-NMBbindingto the dopamine D2 oped by Janssen et al. (16), haloperidol and benperidol receptor. A PET study with 11C-NMBof the baboon brain have better selectivity (12,16,1 7) than spiperone. Halo revealed highlyselective labelingof dopamine D2 receptor peridol, however, revealed high uptake in tissues (e.g., siteswhichwasblockedbypreinjectionofraclopnde. the cerebellum), which are devoid of dopamine D2 J NucIMed 1990;31:2015—2021 receptors. As a result, 3H or ‘8F-haloperidollabels the brain almost uniformly in vivo (1,18). Benperidol has high affinity for the dopamine D2 receptor (1) and was classified by Janssen et al. as a neuroleptic drug of high he dopamine D2 receptor in the living brain has apomorphine antagonistic effect and low anti-trypta been visualized by the positron emission tomographic mine and anti-norepinephrine potencies (16). Fluorine (PET) technique using carbon-ll- (11C),fluorine-18-, 18-benperidol has been synthesized and evaluated in and bromine-76- (76Br) labeled neuroleptic drugs as the baboon brain and has shown high target-to-non radiotracers. Among these drugs, spiperone, a butyro target ratios (19). A brominated derivative 77Br-brom phenone dopamine antagonist of high affinity for the benperidol has also been reported (20). However, this receptor with a Kd value of 0.14 nM (1), its alkylated compound exhibited relatively low in vivo target-to (2—5)and brominated (6, 7) derivatives have been eval non-target ratios. uated as in vivo ligands and have found widespread use This report describes the in vivo kinetics, distribu for studying the dopamine D2 receptor (8—10).With tion, and pharmacology of N-' ‘C-methyl-benperidol these compounds, the receptor-rich regions in the brain (I ‘C-NMB), a derivative of benperidol, as a potentially can be clearly visualized with PET. However, it is well more selective radiotracer ofthe dopamine D2 receptor. recognized that spiperone and its derivatives have mul tiple specific binding sites other than the dopamine D2 MATERIALSAND METHODS receptor (1,11—15).In particular, the binding of spipe rone to the serotonin S2 receptor in frontal cortex has Benperidol was prepared according to literature procedures (21). N-methyl-benpendol (NMB) was synthesized by a pro cedure similar to the one described below. (+)-Butaclamol, (—)-butaclamol, ketanserin, SCH-23390, spiperone, and halo ReceivedFeb.20,1990;revisionacceptedMay15,1990. peridol were purchased from Research Biochemicals Inc., For reprintscontact: Robert F. Dannals,MD,Div.of NuclearMedicine andRadiationHealthSciences,JohnsHopkinsMedicalInstitutions,615N. Natick, MA. Raclopride was gratefully obtained as a gift from WolfeSt., Baltimore,MD21205-2179. Astra Alab AB, SOdertälje,Sweden.

DopamineD2ReceptorsandN-11C-Methyl-Benpendol•Suehiroetal 2015 Radiotracer Synthesis according to the following procedures. For both receptor Carbon-l l-NMB was synthesized by N-methylation of the assays, rats were killed by decapitation and the brains were precursorbenpendol with ‘‘C-iodomethane(22).Briefly,ben removed quickly. peridol and ‘C-iodomethane were reactedat 80°Cfor 1 mm For the dopamine D2 receptorassay, striatawere dissected in dimethylformamide. Carbon-l 1l-NMB was purified by and homogenized in ice-cold 50 mM Tris buffer (pH 7.4 at reversed-phase high-performance liquid chromatography 37°C).The homogenate was centrifuged at 48,000 g for 15 (HPLC). After evaporation of the HPLC solvent, the labeled mm. The pellets were suspended in ice-cold buffer and reho compound was dissolved in normal saline and sodium bicar mogenized. After recentrifugation, a tissue suspension of 6 bonate and filtered for injection. Radiochemical yields were mg/ml of buffer was made. To glass test tubes, 0.1 ml of 6 20%-50% with a radiochemical purity of 100%. The specific nM 3H-spiperone (32.4 Ci/mmol; NEN, N. Billerica, MA) in activities were determined to be @@-2200mCi/@imole at the end buffer and 0.1 ml of a l0@ to l0@ M NMB solution were of synthesis. The full synthetic details will be reported else placed; then, 0.8 ml of the tissue suspension was added. For where (unpublished results). N) ‘C-methyl-spiperone(‘‘C- comparison, samples with l0@ to l0@ M benperidol were NMSP) was synthesizedas previouslyreported(23). also prepared. For determination of nonspecific binding, 1 @tM(+)butaclamolwas used. The sampleswereincubatedat Brain Biodistribution Study 37°Cfor 20 mm. At the end of incubation, 3 ml of the ice Mice weighing between 25 to 30 g were injected intrave coldbufferwereadded.Boundandfreeradioligandswere nously via tail vein with 110—160@iCiof ‘‘C-NMB(this separated by pouring the samples onto OF/B glass filter discs corresponds to a dose of 1.6 ±0.4 @igNMB/kg body weight). (Whatman) under vacuum. The discs were washed three times The mice were killed by cervical dislocation at 5, 15, 30, 60, with 3 ml of bufferand placedin glassvials,to which 10ml and 90 mm after injection and blood (100 @tl)was collected scintillation cocktail (NEN NEF-963) was added. The radio from the carotid artery. The brains were removed, placed on activity concentrations on the filter discs were determined ice, and dissected into the following five regions: cerebellum, using liquid scintillation counting. striatum, frontal cortex, remaining cortex except the area To evaluate the affinity for the serotonin 52 receptor, superior to the corpus striatum, and the rest ofthe brain. After receptor assays were performed in ice-cold 50 mM Iris buffer each tissuewas weighed,the radioactivity concentration in containing 120 mM NaCI, 5 mM KC1, 2 mM CaC12, 10 mM these tissues and in the blood was determined using an auto MgCl2, and 0.5 mM EDTA (pH 7.7) using rat frontal cortex mated gamma counter. as the tissue and 3H-ketansenn(64.9 Ci/mmol) as the radioli BlockingStudy gand. Nonspecific binding was determined with 5 @Mcinan The selectivity of' ‘C-NMBbinding to dopamine D2 recep serin. Assay procedures were similar to the dopamine D2 tor sites was examined as follows. Mice were injected intra receptorassay. venously with 1 mg/kg of (+)-butaclamol, (—)-butaclamol, Data analysisto determineIC@and K@(inhibitionconstant) ketanserin, SCH-23390, prazosin, propranolol, spiperone, ra values was performed using microcomputer programs by clopnde (1-5 mg/kg), haloperidol, or unlabeled NMB. The McPherson (25,26). drugs were dissolved in saline and administered at 15 mm priorto the ‘‘C-NMBinjection. The animals were once again killedby cervicaldislocation15mm after the injectionof the RESULTS radiotracer. After injection of ‘‘C-NMBinto mice, the time Comparative blocking experiments were performed with course of ‘‘Cradioactivity in cerebellum, striatum, another dopamine D2 radiotracer, ‘‘C-NMSP.The mice in frontal cortex, and remaining cortex was determined these experiments were killed at 30 mm after injection of “C- NMSP. This time point (instead of 15 mm for ‘‘C-NMB)was (Fig. 1). As early as 5 mm after injection, a high uptake chosenbecauseofthe slowerin vivo kineticsof ‘‘C-NMSP. by the striatum was observed. The radioactivity reached Statistical evaluation of the data was performed by the a peak of 12% dose per gram of tissue at 15 mm and Student's t-test. declined exponentially with a slope ofO.Ol min', which corresponded to a washout rate of -‘-0.1%dose/g/min Baboon PET Study or 2—6pg/g/min. The biologic half-life of' ‘C-NMBin A 30-kg male baboon (Papio Anubis) was anesthetized with the striatum was 62 mm. In contrast, the radioactivity alfadolone and alfaxolone acetate. Anesthesia was maintained throughout the study by continuous i.v. infusion drip of 6—9 in cerebellum,frontal cortex,and remainingcortex mg/kg/hr (20% solution). Twenty millicuries of high-specific washed out rapidly. The biologic half-lives in these activity ‘‘C-NMB(2250 mCi/@tmole)wereadministeredintra tissues were 12, 12, and 13 mm, respectively, which venously. In a competition study, raclopride (2 mg/kg) was were ..@5times shorter than in the striatum. As a result, injected i.v. 5 mm prior to the ‘‘C-NMBinjection. Sequential at 30 mm after injection, the radioactivity concentra tomographic slices passing through the caudates, cerebellum, tions in these three tissues (cerebellum, frontal cortex, and temporal lobes were obtained over a period of 110 mm. and remaining cortex) were as low as 1.4% injected Regions ofinterest (3 x 3 pixels) were placed over the different dose per gram of tissue while there still remained 11% areas ofthe baboon brain based on a baboon PET atlas (24). dose per gram in the striatum. The time course of the In VitroExperiments tissue to cerebellar ratios is shown in Figure 2. The To evaluatethe affinityof NMBfor the dopamine D2 and ratios of frontal cortex-to-cerebellum and of remaining serotonin 52 receptors, in vitro receptor assays were performed cortex-to-cerebellum were 1. 15 ±0. 14 (n = 15) and

2016 The Journal of Nudear Medicine•Vol.31 •No. 12 •December1990 5 for dopamine Dl, serotonin S2, and a-adrenergic sites (Fig. 3). Two dopamine D2 receptor ligands, spiperone and haloperidol, blocked the accumulation of' ‘C-NMB

0) in the striatum by 99% and 97%, respectively, indicat @ ing high specificity of ‘‘C-NMBbinding to dopamine @E: D2 receptor sites in striatum. (+)-Butaclamol blocked g 10 100%ofthespecificbindingwhile(—)-butaclamoldid @ not (p > 0.5); this suggests that the binding of ‘‘C-NMB

@. to the dopamine D2 binding site in striatum is stereo @ specific (27). Unlabeled NMB (1000 times the amount @ injected during the control studies) blocked 99% of the 5 @ 1‘C-NMB specific binding; this demonstrates the sat @ urability of the NMB binding site in the striatum.

@• Raclopride, a dopamine D2 receptor antagonist of a different type (14,28,29), inhibited 87.7% ±3.9% of the ‘‘C-NMBbinding at a dose of 1 mg/kg (2.9 @imole/ @ r . . kg). 0 30 60 90 SCH-23390, a dopamine Dl receptor-specific ligand Time (minutes) (3032), showed a consistent blockade of 20% (p < FIGURE1 0.0001).Ketanserin,a ligandfortheserotoninS2recep Time course of 11Cradioactivity in mouse stnatum (str), frontal tor (1 7), did not cause significant inhibition either in cortex ¶r@eixb(clt@ an@cerebeOurn(cb)after i.v.injec- striatum (p > 0.1) (Fig. 3), in frontal cortex ( p > 0.1), %dose/g ±s.d.(n =3—5). zg). a are mean or the remaining cortex (p > 0. 1) (data not shown). The al and a2 adrenergic blockers, prazosin and propran

1.24 ±0.11 (n = 15), respectively,at any time after @ injection (5—90mm). In contrast, striatal-to-cerebellar ratios increased with time until a plateau value of 11:1 was reached at ‘@@60mm after injection. In vivo characterization ofthe ‘‘C-NMBbinding was carried out by studying the effect of several dopamine 4.0 D2 receptor blockers as well as drugs with high affinity 0

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-;J C; 30 60 Time (minutes) FIGURE3 Effect of various drugs on 11C-NMBbinding in the mouse FIGURE2 striatum. Drugs (1—5mg/kg) were injected intravenously 15 Time course of tissue to cerebellar ratios of 11C radioactivity mm before administrationof 11C-NMB.Stnatal to cerebellar after i.v. injection of 11C-NMB.Data are mean ratios ±s.d. ratiosweredetermined15 mmafterradiotracerapplication. (n = 3—5). Dataare meanratios ±s.d. (n = 4—12).

Dopamine D2 Receptors and N-11C-Methyl-Benperidol •Suehiro et al 2017 olol (33), respectively, also did not block the accumu striatum by 75%. In frontal cortex and remaining cor lation of' ‘C-NMB(p > 0. 1 for both drugs). tex, no significant effects were observed with this com In the frontal cortex and the rest of the cortex, none pound (p > 0.1). of the drugs tested had any effect on ‘‘C-NMBbinding After evaluation of' ‘C-NMBin mice, the compound (p > 0.05). Mean tissue-to-cerebellarratios after block was further tested in a baboon. The corpus striatum ing were 1.13 ±0.10 (n = 30) for the frontal cortex and was the area of highest accumulation of' ‘C-NMB(Fig. 1.10 ±0.06 (n = 30) for the cortex. 5). The activity increased gradually and striatal-to-cer Carbon-l l-NMSP on the other hand showed quite ebellar ratios of 2.0:1 were observed between 60 and 90 different binding characteristics in striatum, frontal cor mm after injection (Fig. 6a). There was no apparent tex, and cortex (Fig. 4) than ‘‘C-NMB.Whereas stria accumulation of ‘‘C-NMBin the frontal cortex or other tum-to-cerebellar ratios of ‘‘C-NMSPat 30 mm after cortical regions, which suggests that this tracer retained injection were comparable to those observed with ‘‘C- its specificity of binding in this species (Fig. 6a). After NMB, the ratios of frontal cortex-to-cerebellum and of pretreatment with 2 mg/kg of raclopride, the striatal cortex-to-cerebellum were six and three times higher uptake of ‘‘C-NMBwas significantly inhibited (stria (7.3 ±0.7 and 3.9 ±0.3), respectively. The specific tum-to-cerebellar ratio = 1:1; Fig. 6b). binding of' ‘C-NMSPin the striatum was not blocked In vitro receptor assays revealed that K@values against significantly by ketanserin (p > 0.5) while 79% of the 3H-spiperone binding to rat striatum were 5.2 nM for binding in the frontal cortex and 76% in the cortex NMB and 1. 1 nM for benperidol. They also revealed were blocked by this drug, which suggests that NMSP that NMB had a significantly lower affinity for the binding in these tissues was mostly to serotonin S2 serotonin S2 receptor (K@= 50 nM) than benperidol receptor sites. SCH-23390, the dopamine Dl receptor (K@= 4.0 nM) and NMSP (K@= 0.45 nM). ligand which has been reported to interact with the serotonin S2 receptor (31,32), also showed 66% and DISCUSSION 55% blockade in frontal cortex and cortex, respectively, while no significant blockade was seen in the striatum This investigation demonstrates specific and selective (p > 0. 1). Raclopride at a dose of 1 mg/kg (2.9 @imole/ in vivo labeling of the dopamine D2 receptor by “C- kg) blocked only 55% ofthe “C-NMSPbinding sites in NMB. The binding was found to be saturable, reversi the striatum. With a higher dose (10 mg/kg), 83% of ble, and stereospecific. Because of its high selectivity, these sites were blocked. However, at this dosage, sig specificity and reversibility, ‘‘C-NMBmay be a more nificant blockade was observed in frontal cortex and appropriate dopamine D2 receptor radiotracer than the remaining cortex as well as in the striatum, while at I ‘C-NMSP. a lower dose of 1-5 mg/kg, no significant blockade was At all times after i.v. injection, ‘‘C-NMBconcentra observed in cortical areas. Unlabeled NMB (1 mg/kg tions were highest in the dopamine D2 receptor-rich or 2.5 @mole/kg)inhibited ‘‘C-NMSPbinding in the striatal region (Fig. 1). Unlike ‘‘C-NMSP, ‘‘C-NMB

‘5'

El1 S c-I I NMSP(control) @ Ketanserin @ SCH-23390 0 Raclopride C 10' 0 NMB

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FiGURE4 Effect of various drugs on 11C-NMSP bindingin differentareasof the mouse brain.Drugs(1—5mg/kg)wereinjected intravenously15 mmbeforeadministra tionof 11C-NMSP.Tissue-to-cerebellar 0@ ratios were determined 30 mm after 2 3 1: striatum/cerebellum radiotracer application. Data are mean 2: frontal cortex/cerebellum ratios ±s.d. (n = 3—4). 3: cortex/cerebellum

2018 The Journal of Nudear Medicine•Vol.31 •No. I2 •December1990 rapidly as from the cerebellum, a region devoid of dopamine D2 receptors (Fig. 1). Carbon-l l-NMB c-Il Labeled NMethylbenperldol cleared twelve times faster from the frontal cortex and PETStudy Ina Baboonat 985 Mlnutss four times faster from the cerebellum than NMSP (15). Carbon-l l-NMB cleared significantly slower from the striatum than from all other regions (Fig. 1). As a result, a high ratio between striatum and the other regions of 11:1 was reached as early as 60 mm after injection (Fig. 2). The peak striatal-to-cerebellar ratio for ‘‘C-NMBwas six times higher than 77Br-bromben peridol (20) and twice as high as 3H- and ‘8F-spiperone (15,18). mockIngStudy The high in vivo selectivity of' ‘C-NMBto the do withRaclopridi pamine D2 receptor, which was strongly indicated by the in vivo kinetics in different areas of the CNS (Fig. 1), was confirmed by the blocking experiments. Neither FIGURE 5 serotonin S2 or a-adrenergic receptor antagonists had PET studiesin baboon98.5 mmafterinjectionof “C-NMB.any significant effect on the ‘‘C-NMBbinding in the Control study on right and blocking study by preinjection on regions abundant in dopamine D2 receptors (Fig. 3). In racloprideon left. vitro data suggest that N-methylation of benperidol resulted in decreased affinity for both the dopamine D2 showed no specific binding in the frontal cortex or and serotonin S2 receptors. However, larger effects were remaining cortex, which are the tissues abundant in observed on the affinity for the serotonin S2 receptor serotonin S2 or a-adrenergic receptors (Figs. 1 and 6a). than for the dopamine D2 receptor. The affinity of From these brain regions, ‘‘C-NMBwashed out as NMB for the dopamine D2 receptor was 4-5 times lower than that of benperidol and 20 times lower than that ofNMSP (34), whereas its affinity for the serotonin S2 was one order of magnitudelower than that of benperidol and two orders of magnitude lower than that of NMSP. Therefore, the high selectivity of “C- NMB shown in vivo was most likely due to a weak interaction with the serotonin S2 receptor and/or a low incidenceof rebindingof this compoundto receptor sites, and consequently, a rapid washout from the S2 receptor-rich tissues (35). Blocking experiments confirmed that the long reten tion of ‘‘C-NMBin the striatum was the result of 40 60 80 100 interaction with the dopamine D2 receptor (Figs. 3, 5, Time (minutes) and 6b). With halopendol and raclopride, both highly specific dopamine D2 receptor ligands, at concentra b.@ tions of 1000 times the mass dose of NMB, 88%—99% 5000 of the ‘‘C-NMBbinding sites in the striatum were blocked. In the baboon brain, raclopride almost com @ 4000 a. pletely blocked the binding sites for ‘‘C-NMB(Figs. 5 c)3000 and 6b) at a concentration 2000 times the mass dose of E NMB. Therefore, it can be concluded that ‘‘C-NMB ‘I) e@ labels the dopamine D2 receptor site. In contrast, the —0@——Caudate blocked I ‘C-NMSP binding was not inhibited completely by a 1000 - --0-- Cerebellum blocked fr... TemporalLobeblocked 1000—2000times excess amount of unlabeled NMB, a butyrophenone ligand similar to spiperone, or raclo 20 40 60 80 100 pride, an example of a different class of dopamine D2 Time (minutes) receptor ligands (Fig. 4). After blockade by these li FIGURE6 gands, 30%-45% of the binding sites for ‘‘C-NMSP Thne-activity curves in baboon caudates, temporal cortex, remained unblocked. A very high dose of raclopride and cerebellumafterinjectionof ‘1C-NMBwithout(a)and with (b)preinjectionof raclopride(2 mg/kg)at 5 mmbefore radio (e.g., 10 mg/kg or 29 @mole/kg)blocked 83% of the tracer administration. I ‘C-NMSP binding sites. However, with this dose of

DopamineD2 Receptors and N-'1C-Methyl-Benperidol•Suehiro et al 2019 raclopride not only D2-specific binding sites but also In conclusion, ‘‘C-NMBhas excellent properties as nonspecific sites (36) would possibly be blocked. These an in vivo dopamine D2 receptor radiotracer. It gives data are in agreement with results by other investigators high specific-to-nonspecific binding ratios and the in who performed in vivo and in vitro inhibition studies vivo binding is highly specific and selective for the with 3H-spiperone or 3H-NMSP (1,29,37,38). Together, dopamine D2 receptor. the findings ofthis study and that of others suggest that NMSP and spiperone have multiple binding sites in ACKNOWLEDGMENTS dopamine D2 receptor-rich regions; some of which are The authors would like to thank Mr. Robert C. Smoot for not shared with NMB, haloperidol, or raclopride. his skillful assistance with the ‘‘C-NMBand ‘‘C-NMSP The 20% blockade of' ‘C-NMBbinding in the stria syntheses and Mr. David J. Clough for the PET acquisition tum by 1mg/kg ofSCH-23390, a dopamine Dl-specific and computer analysis of the PET data. They also thank Dr. antagonist, might suggest an interaction of this com Dean F. Wong for valuable discussion and suggestions. This pound with dopamine Dl receptors sites. Similarly, a work is supported in part by the U.S.P.H.S.grant numbers previous study from this laboratory indicated that “C- NS-15080 and CA-32845. SCH-23390 binding in the striatum is blocked to the same degree by 1 mg/kg of haloperidol (39). These REFERENCES findings considered together might indicate that dopa mine Dl and dopamine D2 receptor sites are linked. A 1. Leysen JE, Gommeren W, Laduron PM. Spiperone. A ligand of choice for neuroleptic receptors. 1. Kineticsand character pharmacologic linkage between these two different istics of in vitro binding. Biochem Pharm 1978; 27:307—316. types of dopaminergic receptors has been suggested by 2. Wagner HN Jr, Bums HD, Dannals RF, et al. 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DopamineD2 Receptors and N-11C-Methyl-Benpehdci•Suehiro et al 2021