ORIGINAL 

Annals of Nuclear Medicine Vol. 7, No. 3, 153-156, 1993 

Myocardial accumulation of a dopamine D2 receptor-binding radioligand, 2'-iodospiperone 

Hideo SAJI,* Yoshiharu YONEKURA,** Kiyoko TANAHASHI,* Yasuhiko IIDA,* Yasushi IWASAKI,** Yasuhiro MAGATA,** Junji KONISHI,** and Akira YOKOYAMA* 

*Faculty of Pharmaceutical Sciences and * *School of Medicine, Kyoto University, Kyoto 606-01, Japan 

125I-2'-iodospiperone (2'-ISP), which has a high and selective affinity for dopamine D2 receptors, produced a high myocardial accumulation of radioactivity in the early phase after intravenous injection into mice. A human scintigraphic study also showed that the myocardium was clearly visualized soon after intravenous injection of the tracer. Analysis of the myocardial homogenate obtained from mice showed that 125I-2'-ISP was metaboli-cally stable and was taken up the myocardium in its intact form. Administration of spiperone significantly reduced the myocardial uptake of 125I-2'-ISP in mice. Treatment with halo-peridol and (+). butaclamol, which have a high affinity for dopamine D2 receptors, also tended to reduce the myocardial uptake of radioactivity, while ( -)-butaclamol, which has no affinity for dopamine D2 receptors, caused no change in uptake. These findings suggest that the myocardial accumulation of 2'-ISP occurred in association with dopamine D2 (DA2) receptors. 
Key words : radioiodinated 2'-iodospiperone, myocardium, dopamine receptor, SPECT. 

INTRODUCTION 

We recently developed 2'-iodospiperone (2'-ISP), a spiperone derivative iodinated at the ortho position of the p-fluorobutyrophenone moiety, as a radio-ligand for single photon emission computed tomo-graphy (SPECT) studies of the dopamine D2 receptor1-3 (Fig. 1). In vitro and in vivo studies in mice and rats have shown that this compound binds to dopamine D2 receptors in the central nervous system with a high affinity and stereospecificity.1,2 Furthermore, a preliminary human imaging study with 123I-2'-ISP showed its specific uptake by the basal ganglia, a region of the brain known to have a high density of dopamine D2 receptors.3 
In the myocardium, dopamine D2 (DA2) receptors are located on postsynaptic sympathetic nerves and inhibit the release of norepinephrine from sym-pathetic nerve storage sites.4,5 In this study, the myocardial accumulation of radioiodinated 2'-ISP was investigated in mice and humans, and its useful-ness for imaging of the myocardium was assessed. 

MATERIALS AND METHODS 

123I-sodium iodide produced by the indirect method using the 1271 (p, 5n) 123Xe reaction was obtained from Nihon Medi-Physics Co. Ltd. (specific activity : 8.88 TBq/pmol), and 1251-sodium iodide (specific activity: 81.4 GBq/umol) was purchased from Amersham International, Plc. All other chemicals used were of reagent grade. Male ddY mice were supplied by Japan SLC Co. Ltd. (Hamamatsu, Ja pan) . 

Synthesis of 123I- and 125I-2'-ISP 
123I- and 125I-2'-ISP were synthesized by a bromine-radioiodine exchange reaction. A solution of 123I-or 125I-sodium iodide was evaporated to dryness. To the residue was added 37.5 ul of an 80 % aqueous solution of dimethylformamide (DMF) containing 200 ug of 2'-bromospiperone, 13 peg of sodium iodine, 200 ug of copper sulfate pentahydrate, and 1 80 peg of 1-naphthalenesulfonic acid dihydrate. After heating at 95'C for 1 hr, the product was purified by high-performance liquid chromato-graphy (HPLC) (Lichrosorb RP-18, H2O/CH3OH/ (C2H5)3N=50/75/1 ; flow rate : 2 ml/min). 
The radiochemical purity of the products was more than 98 % as determined by thin-layer chro-atography (TLC) (CH2Cl2/C2H2OH=6/1, Rf= 0.6-0.7) and HPLC (Lichrosorb RP-18, H2O/ CH3OH/(C2H5)3N=50/75/1; flow rate : 2 ml/min; Rt =39 min). The specific radioactivity of 123I- and 125I-2'-ISP was 9.25 GBq/umol and 1.1 GBq//umol respectively, as estimated by the ultraviolet absorb-ance at 249 nm. 

Biodistribution in mice 
Male ddY mice weighing an average of 30 g were injected intravenously with 125I-2'-ISP (18.5 kBq in 0.1 ml of ethanolic saline solution, 0.29 ug/kg). At designated times afterward, the mice were killed by decapitation, blood samples were collected by cardiac puncture, and the organs of interest were removed. All samples were weighed, and the radioactivity was counted in a well-type NaI scintillation counter. 

Metabolic studies in mouse myocardium 
Mice were injected intravenously with 18.5 kBq of 125I-2'-ISP, and were killed at 10 min after injection. The hearts were removed immediately and homo-genized in a mixture of 340 pl of methanol, 120 Id of water, 30 ,d of dimethyl sulfoxide (DMSO) and 30 ul of 2N HCl. After centrifugation, the pre-cipitate was washed with a mixture of 340 pl of methanol, 120 pl of water, 30 pl of DMSO and 30 pl of 2N HC1, and the washings were combined with the supernatant. The solution was then analyzed by TLC (CH2Cl2/C2H5OH = 6/ 1). Effect of various drugs on myocardial uptake in mice Several dopaminergic drugs were injected intra-venously into mice along with 18.5 kBq of 125I-2'-ISP. Spiperone (1 mg/kg), haloperidol (1 mg/kg), or unlabeled 2'-ISP (1, 5, 10 mg/kg) in a mixture of ethanol and 2% acetic acid (2 : 100) was injected simultaneouly with the radioligand, and (+) or ( -)-butaclamol (5 mg/kg) in ethanolic saline solution was injected intraperitoneally 30 min before radioligand administration. The animals were killed 10 min after radioligand administration, their hearts were re-moved, and the radioactivity was counted as described above. 

Human scintigraphic study 
Imaging was performed with a scintillation camera with a low-energy collimator (Gamma View-E RC150E, Hitachi). 
An 123I-2'-ISP solution (59 MBq, 48 ng/kg) was injected into a healthy male volunteer and serial planar images of the chest and abdomen were obtained for 60 min afterwards. 

RESULTS AND DISCUSSION 

One strategy for imaging the myocardium and evaluating its function is to utilize a radiolabled ligand that is known to bind to specific myocardial receptors.6,7 Accordingly, this study examined the localization of 2'-ISP, a ligand for dopamine D2 receptors,1-3 which are located on postsynaptic sympathetic nerves in the myocardium.4,5 
Table 1 shows the biodistribution of 125I-2'-ISP in mice. Radioactivity was cleared rapidly from the blood. In the heart, a high uptake was observed during the early phase after injection, following which it declined with time. Therefore, a high heart-to-blood ratio of 4.4-10.8 was obtained during the first 10 min of the study. The lungs showed a high initial uptake, but they cleared rapidly. The liver and kidneys showed a steep increase in uptake until 5 min, and thereafter their radioactivity remained nearly constant. 
The scintigraphic study in the human volunteer was in agreement with the results of the biodistribu-tion study in mice, and a high myocardial accumu-lation of radioactivity was observed in the early phase after injection. Fig. 2 presents an image obtained at 15 min after injection of 123I-2'-ISP, a time when the myocardium was clearly visualized. The high accumulation in the myocardium and the success of myocardial imaging in the human volun-teer suggested that further in vivo evaluation of this tracer was justified. 
At 10 min after the intravenous injection of 125I-2'-ISP into mice, the radioactivity in the extract of myocardial homogenate was analyzed by TLC. Approximately 80% of the radioactivity in the homogenate was extractable by our organic solvent technique, and the extractable material displayed a single peak which co-migrated with authentic 2'-ISP. These results showed that most of the myo-cardial uptake and distribution of the tracer occurred in the intact form and indicated its metabolic stability in the myocardium. 
To assess the dopaminergic nature of the 125I-2'-ISP binding sites, the effects of several drugs on the myocardial uptake of this tracer were studied at 10 min after injection. As shown in Table 2, the administration of spiperone significantly reduced the myocardial uptake of radioactivity. Treatment with haloperidol and (+) butaclamol also tended to reduce the myocardial uptake. We also tested (-) butaclamol, which has no affinity for dopamine D2 receptors, and found that it caused no change in the myocardial uptake of radioactivity. Furthermore, the effect of the carrier itself on the myocardial uptake of 125I-2'-ISP at 10 min after injection was investigated with various doses of unlabeled 2'-lSP (1-10 mg/kg). As shown in Fig. 3, the myocardial uptake of radioactivity was decreased in a dose-dependent manner by unlabeled 2'-ISP injection. Since 2'-ISP is highly selective for dopamine D2 receptors,1,2 these displacement studies indicated that 125I-2'-ISP binds to dopamine D2 receptors (DA2 receptors) in the myocardium. However, a significant amount of radioactivity remained in the myocardium after displacement. Quantitative studies of neuroreceptors in vivo require the use of a radio-ligand that displays a fairly high specific to non-specific binding ratio (ideally>10).8,5 Thus, if the fraction displaced by dopaminergic drugs in this study represents the true level of specific binding, the specific binding of 2'-ISP may be considerably less than ideal for quantitatively evaluating myo-cardial dopamine DA2 receptors, although the imag-ing of such receptors would be possible. However, it is not clear at present that this displaced fraction represents the true specific binding, i.e., that the fraction remaining after displacement represents true nonspecific binding. As has been found for other receptor-binding radiotracers,10-13 the retention of radioactivity after displacement might have been due to the presence of high-capacity binding sites or the so-called "occultation" phenomenon in which the receptor becomes refractory to competing ligands following initial binding. However, this high reten-tion was not caused by metabolism of the compound, since the myocardial homogenate study showed that 2'-ISP remained intact in the myocardium. Further studies are required to evaluate the in vivo behavior of this ligand. 
In conclusion, the results obtained in this study indicate that 2'-ISP showed rapid and high myo-cardial uptake and that its distribution was partly associated with dopamine DA2 receptors. Although further investigations are still required, 2'-ISP seems to hold some promise for use in functional studies of the myocardium. 

ACKNOWLEDGMENT 

The authors wish to thank Nihon Medi-Physics Co. Ltd, Japan, for providing 123I. 

REFERENCES 

1 . Nakatsuka I, Saji H, Shiba K, et al : In vitro evalu-ation of radioiodinated butyrophenones as radio-tracer for dopamine receptor study. Life Sci 41 : 1989-1997, 1987 
2. Saji H, Nakatsuka I, Shiba K, et al : Radioiodinated 
2'-iodospiperone : a new dopamine receptor study. 1987 
radioligand for in vivo Life Sci 41: 1999-2006, 
3. Saji H, Iida Y, Magata Y, et al : Preparation of 123I-labeled 2'-iodospiperone and imaging of D2 dop-amine receptors in the human brain using SPECT. Nucl Med Biol 19: 523-529, 1992 
4. Murphy MB, Bass AS, Goldberg LI : Modulation of the dopaminergic system in cardiovascular thera-peutics. In Enzymes as Targets for Drug Design, Palfreyman MG, McCann PP. Lovenberg W. Temple JG, Sjoerdsma A, (eds), San Diego, Academic Press, pp 17-29, 1990 
5. Terai M, Usuda S, Maeno H : Dopamine receptors. Protein Nucleic Acid Enzyme 29 1328-1337, 1984 
6. Eckelman WC, Grissom M. Conklin J, et al : In vivo competition studies with analogues of 3-quinuclidinyl benzilate. J Pharm Sci 73 : 529-534, 1984 
7. Syrota A, Comar D, Paillotin G, et al : Muscarinic cholinergic receptor in the human heart evidenced under physiological conditions by positron emission tomography. Proc Natl Acad Sci USA : 82: 584-588, 1985 
8. Eckelman WC, Reba RC, Gibson RE, et al : Re-ceptor-binding radiotracers : a class of potential radiopharmaceuticals. J Nucl Med 20: 350-357, 1979 
9. Kung HF : Radiopharmaceuticals for CNS receptor imaging with SPECT. Nucl Med Biol 17: 85-92, 1990 
10. Beer HF, Blauenstein PA, Hasler PH, et al : In vitro and in vivo evaluation of iodine-123-Ro 16-0154 : a new imaging agent for SPECT investigations of benzodiazepine receptors. J Nucl Med 31: 1007-1014, 1990 
11 . Pappata S, Cornu P, Sarnson Y, et al : PET study of carbon-11-PK 11195 binding to peripheral type benzodiazepine sites in glioblastoma : a case report. J Nucl Med 32: 1608-1610, 1991 
12. Rogers GA, Parsons SM: Very high-affinity analogs of vesamicol , and an acetylcholine storage inhibitor. Neuroreport 1 : 11-14, 1990 
13. Efange SMN, Dutta AK, Michelson RH, et al : Radioiodinated 2-hydroxy-3-4(-iodophenyl)-1-(4-phenylpiperidinyl)propane : poteitial radiotracer for mapping central cholinergic innervation in vivo. Nucl Med Biol 19: 337-348, 1992