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In Vitro and In Vivo Evaluation of [‘231]IBZM: A Potential CNS D-2 Receptor Imaging Agent

Hank F. Kung, Sangren Pan, Mei-Ping Kung, Jeffrey Billings, Ravindra Kasliwal, John Reilley, and Abass Alavi Division ofNuclear Medicine, Department ofRadiology, University of Pennsylvania, Philadelphia, Pennsylvania

In vitro binding characteristics of a CNS dopamine D-2 receptor imaging agent, (S)-N-[(1-ethyl 2-pyrrolidinyl)] methyl-2-hydroxy-3-iodo-6-methoxybenzamide ([125I]IBZM),was carried out in rats. Also brain images, as well as organ biodistribution were determined in a monkey following the administration of 123l-Iabeledcompound. The S-(-)-l[125l]IBZMshowed high specific dopamine D-2 receptor binding in rat striatum (K@,= 0.426 ±0.082 nM, Bmax = 480 ±22 fmol/mg of protein). Competition of various ligands for the IBZM binding displayed the followingrankorderof potency:> S(-)IBZM>>R(+)IBZM S(-)BZM> dopamine > > SCH-23390 >> , norepinephrine, . In vivo planar images of a monkey injected with [1201]IBZMdemonstrated a high concentration in basal ganglia of brain. The ratios of activity in the basal ganglia to cerebellum and the cortex to cerebellum in monkey brain were 4.93 and 1.44, respectively, at 120 mm postinjection. These preliminary results indicate that [123l]IBZMis a potentially promising imaging agent for the investigation of dopamine D-2 receptors in humans.

J NucIMed30:88—92,1989

ecently, positron emission tomography (PET) has , IBZM, (S)-N-[[(1-ethyl-2-pyrrolidinyl)] attracted attention as a useful tool for studying the methyl-2-hydroxy-3-iodo-6-methoxybenzamide, has biochemical properties of the living human brain (1— been reported (11—13). 3). Several agents for the imaging of CNS dopamine IBZM (Fig. 1) belongs to a group of structurally receptors by PET have been reported: [‘‘C]-23390for related which display significant antidopa D-l receptors (4), [‘‘C]- (5—7), N-[' ‘C] minergic activity (14—19). These benzamides show a methylspiperone (NMSP)(8,9) and 4'-['8F]-N-methyl high specificity for the CNS i>2 and spiperone (10) for r-2 receptors. They have been dem selectively block -induced hyperactivity onstrated to be useful in the evaluation of CNS dopa in vivo. The in vitro affinity constant (Kd) for these mine receptors, which are essential for normal brain agents in rat striatum tissue preparation was found to functions. be: 13, 1.1, and 0. 17 nM for , raclopride and Because of the complexity and the cost associated , respectively (17). with radiopharmaceuticals used for PET imaging there To further investigate the potential of this agent as a is a need for comparable imaging agents labeled with SPECT imaging agent for i-2 dopamine receptors in single photon emitting radionuclides, which may be man, in vitro binding characteristics of [‘25I]IBZMin more suitable for a widespread clinical application with rats and the biodistribution of [‘23I]IBZMin a monkey single photo emission computed tomography (SPECT) were investigated. instrumentation. In developing the new CNS i-2 dopamine receptor imaging agents, an iodinated MATERIALS AND METHODS ReceivedApr. 25, 1988;revisionaccepted Aug. 18, 1988. For reprints contact: Hank F. Kung, PhD, Div. of Nuclear General Medicine, Dept. Radiology, Hospital of the University of Penn Male Sprague-Dawleyrats weighing200—250g were used. sylvania,Philadelphia,PA 19104. The rats were housed in an animal facility with 12 hr light

88 Kung,Pan,Kungetal The Journal of Nuclear Medicine obtained in the presence of 10 mM spiperone. Radioactivity bound on the filters was counted in a gamma counter (Beck man 5500) at an efficiency of 70%.

Data Analysis Both Scatchard and competition experiments were ana lyzed using the iterative nonlinear least squares curve-fitting program LIGAND (21). Each saturation analysis used 11—12 concentrations of['25I]IBZM between 0.05 and 1.5 nM. Scat OCH3 chard analysis ofthe binding data using tissues from striatum, HO hippocampus and frontal cortex always resulted in linear plots with saturability.The Hill coefficientswere nearlyone in all cases. Re-extractionofI'@IJIBZM fromRat Striatum IBZM Thirty minutesafter the i.v. injectionof [‘25I]IBZMinto a rat, the striatal tissues were removed, homogenized in 1 ml I Tris buffer (50 mM, pH 7.4) and extracted with ethyl acetate FIGURE 1 (3 x 1 ml) in the presenceof cold IBZM carrier (400 mg). Chemicalstructureof IBZM(S)-N-[(1-ethyl-2-pyrrciidinyl)]The combined organic extracts were condensed and the resi methyl-2-hydroxy-3-iodo-6-methoxybenzamide. due injected into the high performance liquid chromatography (HPLC)with the same solvent system as describedabove. The retention time of the major peak (>98%) was comparable to and dark cycle with access to food and water ad lib. All the original [‘251]IBZMunder the same conditions. A total of chemicalsusedwereof chemicalgrade.Spiperone,(+)-buta three rats was used for the experiment. clamol, (—)-butaclamol, (±)-6,6-ADTN, serotonin, ketan scm, dopamine, eticlopride .HCL, clonidine, apomorphine, Biodistribution Study in Monkey and papaverine were obtained from Research Biochemical A monkey(Cynomologous, 5 lb)was sedated with mc, Wayland, MA; , norepinephrine and pro (50 mg) andan intravenousinfusion ofpentobarbital (0.2 ml, pranolol were purchased from Aldrich Chemical Company. 25 mg/mI) and followed 30 mm later by an intravenous injection of the [‘231]IBZM(1.7 mCi/l.2 ml, radiochemical Radiolabeling purity 94.4%). Immediately after the i.v. injection, lateral The iodine-l25 (l251)and iodine-l23 (1231)labeled IBZM images of the head (1 mm per frame for 120 mm) were were prepared by a procedure reported previously (radiochem obtained using a Picker Digital Dyna Camera equipped with @ ical purity 94%, overall yield @@-60%,no uv detectable an all purpose collimator on line to a GE Star II computer impurities) (11). The theoretical specific activities for [‘2@I] system with a 20% window set at an energy peak of 159 keY. IBZM and [‘231]IBZMare 2.2 x i0@and 2.4 x l0@Ci/mmol, For brain washoutanalysisthe dynamic planar imageswere respectively. After dilution with saline, these agents were used summed (5 mm x 24). Regions of interest for whole brain in the studies described below. were defined. The net counts were obtained by subtracting TissuePreparation the counts in the brain from that ofthe same number of pixels Male Sprague-Dawley rats (200—250g) were killed and the in the soft tissuenearthe neck.The net counts of the whole brains removed and placed on ice. Striatal tissues were excised, brain versus time were plotted. pooled, and homogenized (glass and teflon) in 100 volumes At the end of 120 mm the monkey was killed. The brain (w/v) of ice-cold Tris-HCI buffer (50 mM), pH 7.4. The and other tissues of interest were removed and weighed. The homogenates were centrifuged at 20,000 g for 20 mm. The brain was sliced into four sections along the cantho meatal resultant pellets were rehomogenized in the same buffer and line. Planarimagesof the brain sectionswereobtained using centrifuged again. The final pellets were resuspended in assay the same camera. The brain slices were then cut into smaller buffer containing: 50 mM Tris-HC1 buffer pH 7.4, 120 mM samples and counted along with standards (a diluted initial NaC1,5 mM KCI, 2 mMCaCl2 and 1mM MgCl2.The protein dose) and samples of other tissues: liver, lung, kidney, spleen, concentration of homogenate was determined by the method thyroid, and muscle. The % dose/g and % dose/organ were of Lowry (20). calculatedby comparingthe net counts of the tissue sample to that of the standard and using 40% and 7% of the body BindingAssays weight for total muscle and blood, respectively. Binding assays were carried out by incubating 50 ml of tissue preparations containing a variable amount of protein (striatum, 40—60mg; hippocampus, 300 mg; cortex, 400 mg) RESULTS AND DISCUSSION with appropriate amounts of['25I]IBZM and competitors in a total volume of0.2 ml ofthe assay buffer. After an incubation period of 15 mm at 37°C,samples were rapidly filtered with a Saturation Analysis cell harvester(BrandelM-24R) under vacuumthrough What Results of the binding studies show specific binding man GF/B glass fiber filters pretreated with 0.2% poly-L of [‘25I]IBZMin the striatum membrane preparation lysine, and the filter paper washed with 3 x 5 ml ofcold (4°C) with a Kd value of 0.426 nM (Table 1). The B,,,@of 50 mM Tris-HC1 buffer, pH 7.4. Nonspecific binding was [‘251]IBZMin the striatum of rats was 480 fmol/mg of

Volume 30 •Number I •January1989 89 TABLE I TABLE 2 Binding Constants of [125l]IBZMin Different Regions Inhibition Constants of Compounds on [1@l]lBZMBinding of Rat Brain to Rat Striatal Membranes° BmaxRegionKd Compounds Ki(nM) protein)Striatum0.426 (nM)(fmol/mg S(—)IBZM 0.633 ± 22Frontal ±0.082480 ± R(+)IBZM 30.3 ±0.84 6.2Hippocampus0.914cortex0.984 ±0.19147.0 ± S(-)BZM 31.1 ±5.78 ±0.15487.1 ±7.7 (+)Butaclamol 0.851±0.174 Chloropromazine 4.01±0.80 (±)ADTN 52.6±6.60 protein, which greatly exceeded the values for hippo Dopamine 296±59 SCH23390 600±15 campus (87 fmol/mg) and frontal cortex (47 fmol/mg) Ketansetin 359 ±79 (Table 1). The B,,,@value is comparable to that reported Apomorphine 262±38 in the literature using a similar procedure and [3H] spiperone as the ligand (22). The nonspecific binding . Each value represents the mean ± s.e.m. of four to six in striatum was 5% of the total bound at the K@con determinations.Kd= 0.426±0.082nM,Bmax= 480±22tmol/ centration, and lower than 50% found in the hippocam mgofprotein.Norepinephrine,propranolol,serotonan,(-)-butacla pus and frontal cortex. The result is consistent with the mcidisplayedKi> I ,000nM. fact that the striatum has more i-2 dopamine receptors than that in any other region of the brain. glia. The total brain washout curve (Fig. 4) shows a Competition Studies single component with a T½of 100 mm. t-2 Dopamine antagonists, e.g., spiperone, (+)- The ex vivo image of the sectioned brain shows a butaclamol and chlorpromazine inhibit [‘25IJIBZMclear cut concentration ofthe agent in the basal ganglia, binding more potently than the D-2 agonist (±)-ADTN where the i-2 dopamine receptors are located (Fig. 4). and the i-l antagonist SCH-23390 (Table 2). Agonists This observation is confirmed by the in vitro experi or antagonists of other receptors including serotonin, ments. The ratios between the basal ganglia and cere ketanserin, norepinephrine, propranolol, prazosin and bellum, and the cortex and cerebellum were 4.93 and clonidine, do not compete with the binding of IBZM. 1.44, respectively at 120 mm postinjection (Table 3). The data demonstrate that the ligand is a very specific These findings are consistent with those observed in ligand with which to study r-2 dopamine receptors. rats (11). Using the same tissue counting technique the The fact that the displacement of the S(—)-isomerof total brain uptake was 3.71 % dose/organ (total brain = IBZM was more effective than its R(+)-isomer (by a 55 g), which suggests that there is high residual uptake factor of―-300),and that (—)-butaclamoldid not inhibit in the brain at 2 hr postinjection. Based on the fact that the binding of IBZM proves that the binding of [12511 IBZM is highly stereoselective. 1000 Re-Extraction of IIThIIIBZM from Rat Striatum 1-123IBZMin MonkeyBrain At 30 mm after an i.v. injection, the radioactivity in rat striatal tissue can be re-extracted (> 95%) by eth ,@ ylacetate. The extractable material displaced the same retention time on the HPLC as that of authentic [1251] IBZM. The data strongly suggests that the radioactivity in striatum is the original compound and in vivo me > U tabolism is minimum (if any) in the target tissue con 0 taming a high density of i-2 dopamine receptors. @0

In Vivo Biodistribution of t@I1IBZM in a Monkey Immediately after the i.v. injection of [‘23I]IBZM significant uptake was observed in the monkey brain. The uptake appeared to reach a maximum by -@‘l0mm 0 20 40 60 80 100 120 140 postinjection (Fig. 2). Since the agent is a lipid-soluble Time (mm) material (partition coefficient: 1-octanol/buffer = 111 and 21 1 at pH 7.0 and 7.4, respectively), it is likely that FIGURE 2 lime activity curve for the whole brain of a monkey after IBZM penetrates the blood-brain barrier by simple an i.v. injection of [1@l]lBZM.The total brain curve appears diffusion (23). The summed images (Fig. 3) demon to show a single component wash out with a T½of ‘@-100 strates that the agent is concentrated in the basal gan mm.

90 Kung,Pan,Kungetal The Journal of Nudear Medicine TABLE 3 Uptake Ratios of [1@l]lBZMin Different Regions of Monkey Brain ratio4.93Cortex/cerebellumBasalganglia/cerebellum .44@. ratio1

Ratio of % dose/g for each region.

j@&@ IBZM, for imaging of one human brain has recently been reported, however, no data on the chemical char @@1-9CiMINUTES 91-120 MINUTES acterization were described (24). This agent showed lower brain uptake than that for [77Br]bromospiperone. In conclusion, the results of the in vitro binding studies suggest that the binding is highly selective for c-2 dopamine receptors. The in vivo images and tissue counting data described in this paper clearly demon strate that [‘23I]IBZMis localized in basal ganglia of a FIGURE 3 monkey. The planar images (lateral view of the head) Summed images (lateral view) of a monkey head at 1—30 with this agent clearly demonstrate that D-2 dopamine mm, 31—60mm, 61—90mm and 91—120mm after an i.v. receptors can be visualized in a species close to human injection of [123I]IBZM.The early images demonstrate that being. Based on this data it is our beliefthat with single the agent is initially localized in the whole brain as well as photon emission computed tomography (SPECT), in the basal ganglia. However, delayed images show wash out ofthis agent from the cortex while basai gangliaappear which improves the image quality and provides quan to retain high concentrations of ligand. titative information, the use of this agent will allow the generation of images of these receptors in the human brain. the T½in the brain is ‘@@@1®mm, the maximum total brain uptake is estimated to be 7% dose/organ. Other TABLE 4 tissue and organ uptake is summarized in Table 4. Liver Biodistnbution of [123I]IBZMin a MonkeyS and kidney were the organs with higher uptake. The Organ % Dose/organ % Dose/g use of [‘23IJ@@@981, chemical equivalent of [12311 B. 0.212 Cerebellum 0.043 B. Stem 0.062 Hip. 0.160 Gray 0.062 White 0.054 Brain(total) 3.71 0.0675 Liver 4.19 0.0873 Musde 12.64• 0.0126 Lungs 1.885 0.134 Thyroid 0.146 Spleen 0.281 0.0385 Kidney 1.198 0.0974

I . T@ne monkey was killed at 1 20 mm after the i.v. injection of . ..,@..,-...I.... V.. [@lJIBZM. .: •‘@.

ACKNOWLEDGMENT The authors thank Dr. Thomas Goodwin ofthe University Laboratory Animal Resources, University ofPennsylvania for assistingwith the animal preparation and Mr. Daniel Beer FIGURE 4 bohm for assisting with the data analysis. The authors also Ex vivo image of brain slice at the level of the basal ganglia along the cantho meatal line after an i.v. injection of [1231] thank Dr. P. Molinoffofthe Department of Pharmacology at IBZM. This image showed a clear cut concentration of the the University of Pennsylvania for his useful suggestions on agent in the basal ganglia (BG), where D-2 dopamine this paper. This project is supported by grants from NIH (NS receptors are located, and low uptake in the cerebellum 24538) and Institute fur Diagonstikforschung, Schering-Freie (CB) with a low concentration of these receptors. Universitat Berlin.

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92 Kung,Pan,Kungetal The Journal of Nudear Medicine