ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol. 14, No. 4, 285-292, 2000 
Preclinical evaluation of [11C]SA4503: radiation dosimetry, in vivo selectivity and PET imaging of sigma1 receptors in the cat brain 
Kazunori KAWAMURA,*,** Kiichi ISHIWATA,* Yuhei SHIMADA,* Yuichi KIMURA,* Tadayuki KOBAYASHI,*** Kiyoshi MATSUNO,*** Yoshio HOMMA** and Michio SENDA* 
*Positron Medical Center, Tokyo Metropolitan Institute of Gerontology
**Kyoritsu College of Pharmacy 
***Central Research Laboratories, Sanren Pharmaceutical Co., Ltd. 
Our previous in vivo study with rats has demonstrated that 11C-labeled 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine ([11C]SA4503) is a potential radioligand for mapping CNS sigma1 receptors by positron emission tomography (PET). In the present study, we further characterized this ligand. The radiation absorbed-dose of [11C]SA4503 in humans estimated with the tissue distribution in mice, was higher in the liver, kidney and pancreas than in other organs studied, but was low enough for clinical use. The brain uptake of [11C]SA4503 in mice was reduced to approximately 60-70% by co-injection of carrier SA4503 and haloperidol, but not by co-injection of any of six ligands for sigma2 or other receptors, for which SA4503 showed in vitro >100 times weaker affinity than for sigma1 receptor. In the cat brain, the uptake in the cortex was higher than that in the cerebellum. The radioactivity in the cortex and cerebellum accumulated for the first 10 min and then gradually decreased until 81.5 min in the baseline measurement, but rapidly decreased in the carrier-loading condition. The receptor-mediated uptake was estimated to be approximately 60-65% of the total radioactivity in the cortex and cerebellum at 76 min after tracer injection. We have concluded that [11C]SA4503 has the potential for mapping sigma1 receptor by PET. 
Key words: sigma1 receptor, [11C]SA4503, central nervous system, positron emission tomography 
INTRODUCTION 
THE SIGMA1 RECEPTOR is considered to be involved in some diseases of the central nervous system (CNS), such as schizophrenia,1,27 depression,18 dementia1,18,23 and ischemia.23 Furthermore, the sigma receptors have been found in the endocrine, immune and other peripheral organ systems and peripheral nervous systems,7,22,31,36,40 and are also expressed in a variety of human tumors2,37 such as neuroblastoma, glioma and melanoma. 
Several radioligands have been synthesized and evaluated for imaging the sigma receptors by positron emission tomography (PET)3-5,9,16,17,26,33,34,38 and by single photon 

Received February 9, 2000, revision accepted June 19, 2000. 
For reprint contact: Kazunori Kawamura, M.Sc., Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1 Naka-cho, Itabashi, Tokyo 173-0022, JAPAN. 
E-mail: kawamura@pet.tmig.or.jp 

emission tomography.10-14,25,39 In a previous study, we have found 11C-labeled [4-O-11C-methyl]-1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride ([11C]SA4503, shown in Fig. 1) to be a promising PET ligand for mapping sigma1 receptors.15 In the in vivo study with rats, the brain uptake of [11C]SA4503 was high, and approximately 75-85% of the total radioactivity in the brain reflected the receptor-specific uptake.15 No 11C-labeled metabolite was detected in the rat brain tissue, and metabolic alteration seemed to be slow: approximately 80% of the radioactivity was found as an unchanged form in plasma 30 min postinjection.15 The regional brain distribution of [11C]SA4503 was clearly demonstrated by ex vivo ARG. In previous in vitro studies, SA4503 had a selective affinity for sigma1 receptors (IC50, 17.4 nM; selectivity sigma1/sigma2, 103).6,19,20 The SA4503 has weak affinity for alpha1-adrenargic, dopamine D2, serotonin (5-HT)1A, 5-HT2, histamine H1, muscarinic M1, and muscarinic M2 receptors.19,20 These affinities were over 100 times weaker than that for the sigma1 receptor. The affinity may be low for the in vivo binding of [11C]SA4503 to the receptors. Moreover, SA4503 had no affinity for 29 other receptors (such as N-methyl-D-aspartate, phencyclidine and opiate), ion channels or second messenger systems.19,20 
In the present study, we estimated the radiation dosimetry of [11C]SA4503 for humans from mice data and further characterized the in vivo selectivity of the compound as a sigma1 receptor ligand. We evaluated whether [11C]SA4503 binds in vivo to lower affinity sites such as a1-adrenargic, dopamine D2, 5-HT2/5-HT1C histamine H1 and cholinergic receptors. The study was performed in mice, because slight species difference in the density of sigma receptors was reported,30 and because the receptor-specific binding of [11C]SA6298, an analog of [11C]SA4503, was slightly different in rats and mice.16 We also performed imaging of sigma receptors in the cat brain by PET with [11C]SA4503. 
MATERIALS AND METHODS 
1-(4-Hydroxy-3-methoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (4-O-demethyl SA4503) was synthesized in Tokyo Metropolitan Institute of Gerontology as previously described.6 1-(3,4-Dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (SA4503) was prepared by Santen Pharmaceutical Co. (Osaka, Japan). Raclopride tartrate was supplied by Astra Arcus AB (Sodertalje, Sweden). Benoxathian hydrochloride, 1,3-di(2-tolyl)guanidine (DTG), haloperidol, pyrilamine malate and ritanserine were purchased from Research Biochemicals International (Natick, MA, USA). Atropine sulfate was obtained from Wako Chemical Industry Ltd. (Tokyo, Japan). 
Male ddY mice were obtained from Tokyo Laboratory Animals Co., Ltd. (Tokyo, Japan). An adult male cat was supplied by IEFA CREDO (IOC cats, Lyon, France). The animal studies were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology. 
Radiosynthesis 
[11C]SA4503 (Fig. 1) was prepared by methylation of 4-O-demethyl SA4503 with [11C]CH3I in a solution of N,N-dimethylformamide containing NaH, followed by high performance liquid chromatography (HPLC) purification, according to the previously described method.15 The specific activity was 24-76 TBq/mmol. 
Tissue distribution study 
[11C]SA4503 (1.1-2.0 MBq/16-34 pmol) was intravenously injected into mice (8 weeks old). They were killed by cervical dislocation at 1, 5, 15, 30, 60 and 90 min after injection (n = 4). The blood was collected by heart puncture, and the tissues were harvested and weighed. The 11C in the samples was measured with an auto-gamma counter. The tissue uptake of 11C was expressed as the percent injected dose per organ (%ID/organ), the percent injected dose per gram tissue (%ID/g) or the standardized uptake value (SUV, [tissue activity/gram tissue]/injected activity/gram body weight). Based on the mice data, radiation dosimetry for human adults was estimated by the MIRD method as described previously.8,24 
Blocking study 
The effect of carrier doses on the brain uptake of [11C]SA4503 was examined. [11C]SA4503 was co-injected with different amounts of carrier SA4503 into mice. The co-injected doses of SA4503 were 7, 70, 700, 2000 or 5000 nmol/kg. The mice were killed at 30 min after injection, and the brain uptake was measured. 
In another group of mice, [11C]SA4503 was co-injected with one of the following receptor ligands: haloperidol for sigma1 receptor, DTG for sigma1/sigma2 receptor, benoxathian for a1 adrenoreceptor, raclopride for dopamine D2 receptor, ritanserin for serotonin 5-HT2/5-HT1c receptor, pyrilamine for histamine H1 receptor and atropine for cholinergic receptors. The co-injected dose of the ligands was 2000 nmol/kg except for DTG (1600 nmol/kg). The brain uptake of [11C]SA4503 was measured at 30 min postinjection. 
PET study on a cat 
A cat weighing 4.4 kg was anesthetized with atropine (0.1 mL/kg) and thiopental sodium (15 mg/kg), and was placed in the prone position on a holder. Catheters were inserted into the femoral vein and femoral artery. [11C]SA4503 (200 MBq/57 nmol) was intravenously injected through the catheter, and an 85 min PET scan was done (one 30 sec frame, one 1 min frame, two 2 min frames, three 4 min frames, three 6 min frames, one 10 min frame and three 12 min frames). After the radioactivity decayed out, the tracer (190 MBq/19 nmol) was injected together with cold SA4503 (1.0 mg/kg), and the 85-min PET scan was done. The PET camera was a model SHR 2000 (Hamamatsu Photonics, Hamamatsu, Japan). The camera consists of four ring detectors and acquires seven slices at a center-to-center interval of 6.5 mm with a resolution of 4.0 mm full width at half maximum in the transaxial plane. The PET images were contrasted to the standard magnetic resonance imaging (MRI) of the cat brain prepared by our laboratory,32 which had been acquired at the positions in a cat brain atlas and had been reoriented to match the PET images. Major intracranial structures were identified on the PET images by referring to the MRI and to the atlas,29,35 and regions of interest (ROIs) were placed over the cortex and cerebellum, and regional time-activity curves were obtained for each scan as described.28 Decay-corrected radioactivity was expressed as a percentage of the injected dose per mL tissue volume (%ID/mL). 
Blood was collected from the femoral artery at 1, 5, 10, 15, 20, 30, 45, 60 and 90 min after the tracer injection. The radioactivity level of the plasma was assessed as the %ID/mL and the labeled metabolites were analyzed by HPLC as previously described.15 
RESULTS 
The tissue distribution of the radioactivity after injection of [11C]SA4503 into mice is summarized in Tables 1 and 2. The lung showed the highest initial uptake followed by a gradual decrease. The mean radioactivity level in the blood, heart and muscle gradually decreased. The uptake by the pancreas and testis gradually increased over 90 min. In the spleen and bone, uptake increased for the first 15 min and then decreased until 90 min. In the liver, uptake increased for the first 60 min and then decreased until 90 min. The brain uptake was increased for the first 5 min and was gradually decreased until 90 min. 
The estimated radiation-absorbed doses are summarized in Table 3. In the liver, pancreas and kidney, the radiation-absorbed doses were higher than in the other organs studied. 
Figure 2 shows the time radioactivity curves expressed as the SUV of the mouse brain in the present study and of the rat brain in a previous study.15 The brain uptake gradually decreased in mice, but gradually increased in rats. 
The effects of the carrier dose on the brain uptake of [11C]SA4503 at 30 min after tracer injection are summarized in Figure 3. The brain uptake was significantly decreased at a dose of 70 nmol/kg and over. At a dose of 2000 nmol/kg, the brain uptake was reduced to approximately 30% of the control. The radioactivity level in the blood slightly increased with the dose. 
Figure 4 shows the effects of the co-injection of one of the eight receptor ligands or cold SA4503 on the brain uptake of [11C]SA4503. The brain uptake was reduced to approximately 30-40% of the control by SA4503 or haloperidol, but not by any of the other six ligands. 
Two PET scans were performed on the same individual: baseline measurement and carrier-loading condition. In the baseline measurement, a high uptake of radioactivity was observed in the cortex (Fig. 5A). The time-radioactivity curves for the cortex and cerebellum are shown in Figure 6. In the baseline measurement, the radioactivity increased for the first 10 min and then gradually decreased until 90 min. In the carrier-loading condition, the brain radioactivity disappeared (Fig. 5B). The radioactivity in the cortex and the cerebellum rapidly decreased after the initial uptake (Fig. 6). The radioactivity levels in the cortex were 67%, 47% and 41% of the baseline at 30 min, 60 min and 90 min, respectively, after the tracer injection. In the cerebellum, the radioactivity levels were 53%, 39% and 35% of the baseline at 30 min, 60 min and 90 min, respectively, after the tracer injection. 
Figure 7 shows time-activity curves of blood and plasma. The radioactivity rapidly decreased and was higher in blood than in plasma for 90 min after tracer injection. The time course of unchanged [11C]SA4503 in plasma is also shown in Figure 7. In the baseline measurement and carrier-loading condition, percentages of unchanged [11C]SA4503 rapidly decreased: 26.3% at 15 min, 21.9% at 30 min, 12.5% at 60 min and 11.0% at 90 min in the baseline measurement; and 24.0% at 15 min, 19.4% at 30 min, 13.1% at 60 min and 7.3% at 90 min in the carrier-loading condition. 
DISCUSSION 
In the previous in vivo study on rats, we demonstrated that [11C]SA4503 has the potential for mapping sigma1 receptors in the CNS as a PET ligand.15 The high receptor-specific uptake of [11C]SA4503 by the brain was confirmed by competition with cold SA4503 and haloperidol, a representative sigma receptor ligand. The previous in vitro study has shown that SA4503 has a selective affinity for sigma1 receptors, and has over 100 times weaker affinities for sigma2, a1-adrenargic, dopamine D2, 5-HT1A, 5-HT2, histamine H1, muscarinic M1 and muscarinic M2 receptors.19,20 In the present in vivo study on mice, we confirmed that [11C]SA4503 did not bind to these receptors (Fig. 4). On the other hand, as shown in Figure 4, the brain uptake of [11C]SA4503 was reduced by co-injection of haloperidol, suggesting that approximately 70% of the radioactivity in the mouse brain reflects the sigma1 receptor-mediated uptake. The brain uptake of [11C]SA4503 was not saturated up to 7 nmol/kg body weight of the injected dose (Fig. 3). In the present study, it is not clear whether [11C]SA4503 is bound to sigma2 receptors. Because no selective sigma2 ligand with a high affinity is commercially available. DTG was used as a blocker. DTG which has relatively low affinity for sigma1 (IC50 = 246 nM) and sigma2 (IC50 = 362 nM) receptors compared with SA4503,20 did not reduce but rather enhanced the brain uptake of [11C]SA4503, possibly due to low penetration across the blood-brain barrier and/or the pharmacological effect. The co-injected dose of DTG (1600 nmol/kg) was close to the lethal dose. 
The present study evaluated the radiation absorbed dose of [11C]SA4503 in humans. The radiation absorbed-dose was higher in the liver, kidney and pancreas than in other organs studied, but was low enough for clinical use. The PET study of the cat brain demonstrated the saturable binding of [11C]SA4503. A high density of radioactivity was observed in the cortex. Approximately 50-60% of the radioactivity reflects the receptor-specific binding in the cortex and cerebellum at 60-80 min after injection. In a previous ex vivo ARG study of the rat brain, we found a high uptake of [11C]SA4503 in the vestibular nucleus, temporal cortex, cingulate cortex, inferior colliculus, thalamus and frontal cortex, and confirmed 75-85% of the total radioactivity to be the receptor-specific at 30 min after the tracer injection.15 The brain uptake of [11C]SA4503 in both rats15 and mice was blocked to a similar degree by a sigma1 receptor ligand haloperidol (IC50 = 3 nM for sigma1 and IC50 = 120 nM for sigma2)20 and non-radioactive SA4503. Therefore, the saturable binding sites of [11C]SA4503 measured in the cat brain by PET may reflect sigma1 receptors. 
An in vitro study suggested that there was a species difference for sigma receptors: the concentration of sigma1 receptors was slightly higher in the guinea pig brain than in the rat brain.30 In the present in vivo study, a slight species difference was found in the time-radioactivity curves of the brain (Figs. 2 and 6). The brain uptake measured by tissue dissection gradually decreased for 90 min in mice, but gradually increased in rats. In the cat measured by PET, the brain uptake increased for the first 10 min and then gradually decreased. These results may reflect the different affinity of the tracer for sigma1 receptors on the brain of the three species, but, regarding specific binding, no species difference was found. The specific binding of [11C]SA4503 in the cat brain measured by PET was estimated to be approximately 60% of the total uptake at 60-80 min after injection, which was slightly lower than that in the rat brain (approximately 70% at 30 min after injection with tissue dissection and 75-85% at 30 min with ex vivo ARG) or that in the mouse brain (approximately 70% at 30 min with tissue dissection).15 The slight discrepancy in these results may be due to a technical problem, not to the species difference. In general, the activity on small ROIs on measured by PET appears to be lower due to the partial volume effect, whereas the activity on the ROIs placed on autoradiograms is determined exactly. With tissue dissection, the uptake of [11C]SA4503 by the whole brain, where sigma1 receptors are present heterogeneously, was measured. Recently we preliminary performed imaging of the conscious monkey brain by PET with [11C]SA4503, and found that the specific binding was approximately 50-60% of the total uptake at 60-80 min (unpublished data). A clear species difference was observed in the peripheral metabolism of [11C]SA4503. In the rat, metabolic change seemed to be slow: approximately 80% of the radioactivity was found in the unchanged form in plasma 30 min postinjection15 but the unchanged form of [11C]SA4503 in plasma of the cat rapidly decreased: 21.9% of total radioactivity at 30 min, 12.5% at 60 min and 11.0% at 90 min in the baseline measurement. 
In conclusion, we have demonstrated that [11C]SA4503 has the potential for mapping sigma1 receptors by PET. 
ACKNOWLEDGMENTS 
The authors thank the staff of the Positron Medical Center, TMIG, for their cooperation. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan. 
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