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Sigma1 and dopamine D2 receptor occupancy in the mouse brain after a single administration of haloperidol and two dopamine D2-like receptor ligands
Nuclear Medicine and Biology 30 (2003) 429 – 434
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Sigma1 and dopamine D2 receptor occupancy in the mouse brain after a single administration of haloperidol and two dopamine D2-like receptor ligands Kiichi Ishiwataa,*, Kazunori Kawamuraa,b, Tadayuki Kobayashic, Kiyoshi Matsunod a
Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1 Naka-cho, Itabashi-ku, Tokyo 173-0022, Japan b SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan c M’s Sciences Co., Ltd., 5-5-2 Minatoshima-Minami-machi, Chuo-ku, Kobe 650-0047, Japan d Nara Research and Development Center, Santen Pharmaceutical Co. Ltd., 8916-16 Takayama-machi, Ikoma 630-0101, Japan Received 28 August 2002; received in revised form 21 October 2002; accepted 21 November 2002
Abstract We investigated sigma1 and dopamine D2 receptor occupancy in mouse brain after a single injection of haloperidol, nemonapride, or spiperone using [11C]SA4503 and [11C]raclopride, respectively. Co-injection of the three compounds significantly blocked the uptake of each radioligand. Six hours later, only haloperidol blocked [11C]SA4503 uptake, while all three reduced [11C]raclopride uptake. Sigma1 receptor occupancy by haloperidol was reduced to 19% at day 2 when D2 receptor occupancy disappeared. [11C]SA4503 would be applicable to the investigation of sigma1 receptor occupancy of antispychotic drugs using PET. © 2003 Elsevier Inc. All rights reserved. Keywords: Haloperidol; Sigma1 receptor; Dopamine D2 receptor; [11C]SA4503; [11C]Raclopride; Positron emission tomography
1. Introduction Receptor occupancy by antipsychotic drugs in the human brain has been investigated extensively by positron emission tomography (PET) and single-photon emission computed tomography with appropriate radioligands [12,21]. Typical antipsychotic drugs such as haloperidol occupied predominantly dopamine D2 receptors in the striatum, and there is no association between striatal dopamine D2 receptor occupancy rates and antipsychotic efficacy. On the other hand, atypical antipsychotic drugs such as clozapine and risperidone occupied mainly serotonin (5-HT)2 receptors in the frontal cortex, and the 5-HT2A occupancy rates are associated with favorable treatment for depressive symptoms within schizophrenia and improvements of cognitive function [12]. It is also well known that a number of neuroleptics possess moderate to high affinity for sigma binding sites, suggesting the possibility that sigma receptors mediate some of the antipsychotic effects of neuroleptics [3,26]. A * Corresponding author. Tel.: ⫹81-3-3964-3241; fax: ⫹81-3-39642188. E-mail address: [email protected] (K. Ishiwata). 0969-8051/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 3 ) 0 0 0 0 3 - 9
major role for sigma receptors might be to regulate the activity of the glutamatergic system via the modulation of the NMDA receptor subtype. This modulation of the glutamatergic system could in turn interfere with the dopaminergic neurotransmission with which, however, sigma ligands could also interact directly [3]. However, the sigma receptor occupancy by the neuroleptics has not been evaluated in humans and animal models, because no in vivo selective radioligand was available up to now. Recently, we have developed 1-([3-O-methyl-11C]3,4-dimethoxyphenethyl)-4(3-phenylpropyl)piperazine ([11C]SA4503) as a selective PET ligand for sigma1 receptors [8,9,13,14]. Preliminarily, we have successfully performed imaging of the sigma1 receptors of the human brain by PET with [11C]SA4503 [7, 22]. The aim of the present study is to investigate preliminarily in mice whether [11C]SA4503 is available as an in vivo probe for evaluating the sigma1 receptor occupancy by the neuroleptics using PET. By measuring in vivo the binding of [11C]SA4503 , we evaluated the blockade of sigma1 receptors in the brain after a single administration of haloperidol or two other dopamine D2–like receptor ligands having high and moderate affinity for sigma receptors: nemonapride and spiperone. We also used [11C]raclopride
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K. Ishiwata et al. / Nuclear Medicine and Biology 30 (2003) 429 – 434
Table 1 The in vitro affinity of haloperidol, three dopamine D2-like receptor ligands and SA4503 for dopamine D2 and sigma receptors. Dopamine D2 Ki(nM)a IC50(nM) Haloperidol* Nemonapride Spiperone Raclopride SA4503
19e 470e
0.5 0.06 0.05 1.8
Sigmai IC50(nM) 3b 5.6* 2269d 11800e 17.4b,f
Sigma2 IC50(nM) 120b 38.8* ⬎10,000d 4950e 1780b,f
Data from a van Tol et al., 1991 [25]; b Matsuno et al., 1996 [16]; present study; d Akunne et al., 1997 (Ki value) [1]; e Ishiwata et al., 2001 [9]; and f Matsuno et al., 1997 [19]. A higher affinity of haloperidol for sigma2 receptors was also reported (Ki values of 3.3 nM and 18.9 nM for sigma1 and sigma2 receptors, respectively, [26]), but the data obtained by the same assay conditions in our group were represented, except for spiperone. c
for evaluating dopamine D2 receptor occupancy rates of the three compounds. The typical antipsychotic haloperidol is considered to be a reference compound in sigma receptor binding assay [26]. The benzamide derivative nemonapride is now used clinically as a neuroleptic [15,16], but it has been shown to have a high affinity for sigma receptors both in vitro [6,24] and in vivo [11]. Finally, Coccini et al., [2] reported that spiperone, which is also used clinically, binds in vivo to sigma receptors in rat and human lymphocytes. Therefore, the occupancy rates of haloperidol, nemonapride, and spiperone to sigma1 and dopamine D2 receptor s were measured over a seven day period following a single dose of each drug. The goal of this study is to elucidate which receptors are involved in mediating the CNS effects of these drugs. 2. Materials and methods 2.1. General Haloperidol and spiperone were purchased from Research Biochemicals International (Natick, MA, USA). Nemonapride was supplied by Santen Pharmaceutical Co. Ltd. (Osaka, Japan). [11C]SA4503 [13] and [11C]raclopride [10] were prepared by [11C ]methylation of the respective demethyl compounds. The in vitro affinity of nemonapride for the sigma1 and sigma2 receptors was determined using [3H](⫹)-pentazocine (5 nM) and [3H]1,3-di-O-tolylguanidine (5 nM) in the presence of (⫹)-pentazocine, respectively, as radioligands, and the guinea pig cortex membrane as described [18]. The affinities of the three ligands investigated, SA4503 and raclopride for sigma and dopamine D2 receptors are summarized in Table 1. Male ddY mice (7-week-old) were obtained from Tokyo Laboratory Animals Co., Ltd. (Tokyo, Japan). The animal studies were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology.
2.2. Treatment with haloperidol and two dopamine D2like receptor ligands Mice weighing 35– 40 g (8- to 9-week-old) were used. In a group of mice, haloperidol, nemonapride, or spiperone was co-injected intravenously with each radiotracer as described below. In another group of mice, each of the three compounds was injected intraperitoneally into mice 6 hours before the tracer injection. In the other group of mice, haloperidol was injected intraperitoneally, and after various time intervals up until seven days, the regional brain uptake of each of the radiotracers was measured. The administered dose of each of the three ligands was 100 nmol/animal (0.94 –1.03 mg/kg). As a control for each group, physiological saline was injected. 2.3.Regional brain uptake of [11C]SA4503 and [11C]raclopride Regional uptake of [11C]SA4503 in the mouse brain was measured 30 min after intravenousinjection, because the uptake of [11C]SA4503 was highest at 5 min, then decreased slightly and remained in an equilibrium state from 30 to 60 min [14]. Regional uptake of [11C]raclopride was measured 15 min after injection, because the ratio of specific to nonspecific binding determined as (striatal uptake – cerebellar uptake)/(cerebellar uptake) was maximum 15 min after the tracer injection [9]. [11C]SA4503 (2.0 MBq/21– 46 pmol) or [11C]raclopride (2.0 MBq/18 –36 pmol) was injected intravenously into mice through the tail vein, and 30 min or 15 min later the mice were killed by cervical dislocation. The brain was removed and divided into the striatum, cerebral cortex, cerebellum and rest. The tissues were measured for radioactivity with an auto-gamma counter and weighed. The uptake was expressed as a percentage of the injected dose per gram of the tissue (%ID/g). Student’s t test was performed to compare the control and each group, and P ⬍ 0.05 was considered significant. The receptor-specific binding of the tracer was determined as the difference between the uptake in each group and the uptake in the group co-injected with haloperidol, while the receptor occupancy (%) was defined as 100 ⫻ {1 ⫺ (the receptor-specific binding in each group)/(the receptor-specific binding in the control). 3. Results The regional brain uptake of [11C]SA4503 or [11C]raclopride in the control and other groups is represented in Fig. 1. In the control, [11C]SA4503 was taken up in the three regions, whereas [11C]raclopride was highly concentrated in the striatum. By co-injection of each of the three ligands, the uptake of [11C]SA4503 was significantly reduced to 39 – 82% of the control in all three regions. The highest reduction was found for haloperidol followed by spiperone and
K. Ishiwata et al. / Nuclear Medicine and Biology 30 (2003) 429 – 434
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Fig. 1. Blockade of the regional uptake of [11C]SA4503 (A) and [11C]raclopride (B) in the mouse brain by intravenous co-injection of haloperidol, nemonapride or spiperone or by intraperitoneal injection of each of the three compounds 6 hours before the tracer injection. Mean ⫾ SD (N ⫽ 5–7). *Any significant reduction (P ⬍ 0.05, Student’s t test, compared with the control) is presented as a percentage of the control.
nemonapride. On the other hand, the uptake of [11C]raclopride in the striatum was decreased to 14 –18% of the control by each of the three compounds. Six hours after the treatment with haloperidol but not with each of the two others, the uptake of [11C]SA4503 was significantly reduced to 45– 47% of the control, whereas the striatal uptake of [11C]raclopride was reduced to 28 – 42% of the control by all the three ligands. The time course of blocking effects of haloperidol on the tracer uptake and the receptor occupancy over seven days are summarized in Figs. 2 and 3, respectively. The largest reduction was found 0.5 h after the haloperidol treatment for two tracers: 38 – 41% of the control in all tissues investigated for [11C]SA4503 and 22% of the control in the striatum for [11C]raclopride. The blocking effects for [11C]SA4503 decreased with time in the three regions of the
brain. At day 2 the uptake of [11C]SA4503 by the cerebral cortex was 84% of the control, which corresponded to 24% receptor occupancy (Fig. 3). The whole brain uptake was also significantly reduced to 90% (21% occupancy) at day 2 (Fig. 3). The reduction was not significant in the cerebellum (93%) and striatum (92%). On the other hand, the striatal uptake of [11C]raclopride returned to the control level at day 2.
4. Discussion A number of neuroleptics possess moderate to high affinity for sigma binding sites, suggesting the possibility that sigma receptors mediate some of the antipsychotic effects of neuroleptics [3]. Furthermore, the number of sigma binding
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Fig. 2. Time course of the blockade of the regional uptake of [11C]SA4503 (A) and [11C]raclopride (B) in the mouse brain after intraperitoneal injection of haloperidol. The uptake of the tracer was measured 0.5 and 6 hours, and 1, 2, 3, and 7 days after the haloperidol treatment. Mean ⫾ SD (N ⫽ 5– 6). *Significant reduction (P ⬍ 0.05, Student’s t test, compared with the control) is presented as a percentage of the control.
sites has been reported to be reduced in the brain of schizophrenic patients [6,27]. Therefore, sigma receptors are believed to play an important role in the pathogenesis of schizophrenia. Frieboes et al. [4] reported that the specific sigma ligand panamesine has similar antipsychotic properties regarding immunomodulation and sleep-electroencephalographic changes as haloperidol, in contrast to an atypical antipsychotic clozapine. These findings suggest indirectly the binding of haloperidol to sigma receptors. However, the sigma receptor occupancy by haloperidol and other neuroleptics has not been evaluated in vivo, because no selective radioligand was available. In the present study we investigated preliminarily whether [11C]SA4503, a new PET probe, is available for evaluating the sigma1 receptor occupancy by neuroleptics. The present pilot study demonstrates directly that haloperidol and two other dopamine
D2-like receptor ligands bind in vivo to sigma1 receptors to a different extent after a single administration. Because no binding of [11C]SA4503 to dopamine D2 receptors was detected in vivo [9], the reduction in [11C]SA4503 uptake by co-injection of each of the three ligands investigated, demonstrates the in vivo binding of these ligands to sigma1 receptors. The highest reduction was found for haloperidol, followed by spiperone and nemonapride. The blocking effects of spiperone and nemonapride disappeared 6 hours later. Among the three ligands, the blocking effect of nemonapride did not parallel the affinity in vitro (spiperone ⬍ nemonapride ⬍ haloperidol). Although the reason for the weaker blocking effect of nemonapride remains unknown, these findings suggest that spiperone and nemonapride bind much weaker to sigma1 receptors than does haloperidol, and also bind
K. Ishiwata et al. / Nuclear Medicine and Biology 30 (2003) 429 – 434
433
Fig. 3. Time course of the sigma1 and dopamine D2 receptor occupancy in mice after intraperitoneal injection of haloperidol. Mean ⫾ SD (N ⫽ 5– 6).
much weaker to sigma1 receptors than to striatal dopamine D2 receptors. By co-injection of haloperidol, the highest blockade rates were found for both [11C]SA4503 and [11C]raclopride: 62% of the total uptake in the whole brain of [11C]SA4503 and 84% of the striatal uptake of [11C]raclopride, suggesting that the specific binding rate of [11C]raclopride was larger than that of [11C]SA4503. Therefore, in principle, the dopamine D2 receptor occupancy can be evaluated more sensitively than the sigma1 receptor occupancy using [11C]SA4503 and [11C]raclopride as in vivo probes. Approximately 40% of the brain uptake of [11C]SA4503 that was not blocked is considered to involve non-specific binding, because of very low in vitro affinity of SA4503 for 36 other receptors, ion channels and second messenger systems [16]and because of the in vivo selectivity of [11C]SA4503 [8,14]. A notable finding is that after the single administration of haloperidol, the sigma1 receptor blockade continued in all regions of the brain slightly longer than the dopamine D2 receptor blockade in the striatum (Figs. 2 and 3). Although the dopamine D2 receptor occupancy was negligible at day 2, a slight blockade of the uptake of [11C]SA4503 at day 2 reflects the sigma1 receptor occupancy by haloperidol in the cerebral cortex (24%) and in whole brain (21%). Thus, the present data clearly demonstrate the potential of [11C]SA4503 for evaluating the sigma1 receptor occupancy rates of antipsychotic drugs, but shows no association between sigma1 receptor occupancy rates and antipsychotic efficacy. It is also pointed out that one administration as a single dose does not reflect the regimen of drug treatment, although the dose of 100 nmol/animal (0.94 –1.03 mg/kg) is usually used in pharmacological studies and is enough to achieve the highest blockade in the striatal uptake of [11C]raclopride. Therefore, PET evaluation of the sigma1
receptor occupancy by antipsychotic drugs in relation to behavioral potency in humans using [11C]SA4503 is of great interest. In conclusion, all three compounds investigated blocked sigma1 receptors to a different extent immediately after the injection. The sigma1 receptor blockade with haloperidol continued slightly longer than the dopamine D2 receptor blockade after a single administration. [11C]SA4503 is applicable to investigation of sigma1 receptor occupancy by antispychotic drugs using PET.
Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (B) No. 13557077 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Sigma1 and dopamine D2 receptor occupancy in the mouse brain after a single administration of haloperidol and two dopamine D2-like receptor ligands Kiichi Ishiwataa,*, Kazunori Kawamuraa,b, Tadayuki Kobayashic, Kiyoshi Matsunod a
Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1 Naka-cho, Itabashi-ku, Tokyo 173-0022, Japan b SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan c M’s Sciences Co., Ltd., 5-5-2 Minatoshima-Minami-machi, Chuo-ku, Kobe 650-0047, Japan d Nara Research and Development Center, Santen Pharmaceutical Co. Ltd., 8916-16 Takayama-machi, Ikoma 630-0101, Japan Received 28 August 2002; received in revised form 21 October 2002; accepted 21 November 2002
Abstract We investigated sigma1 and dopamine D2 receptor occupancy in mouse brain after a single injection of haloperidol, nemonapride, or spiperone using [11C]SA4503 and [11C]raclopride, respectively. Co-injection of the three compounds significantly blocked the uptake of each radioligand. Six hours later, only haloperidol blocked [11C]SA4503 uptake, while all three reduced [11C]raclopride uptake. Sigma1 receptor occupancy by haloperidol was reduced to 19% at day 2 when D2 receptor occupancy disappeared. [11C]SA4503 would be applicable to the investigation of sigma1 receptor occupancy of antispychotic drugs using PET. © 2003 Elsevier Inc. All rights reserved. Keywords: Haloperidol; Sigma1 receptor; Dopamine D2 receptor; [11C]SA4503; [11C]Raclopride; Positron emission tomography
1. Introduction Receptor occupancy by antipsychotic drugs in the human brain has been investigated extensively by positron emission tomography (PET) and single-photon emission computed tomography with appropriate radioligands [12,21]. Typical antipsychotic drugs such as haloperidol occupied predominantly dopamine D2 receptors in the striatum, and there is no association between striatal dopamine D2 receptor occupancy rates and antipsychotic efficacy. On the other hand, atypical antipsychotic drugs such as clozapine and risperidone occupied mainly serotonin (5-HT)2 receptors in the frontal cortex, and the 5-HT2A occupancy rates are associated with favorable treatment for depressive symptoms within schizophrenia and improvements of cognitive function [12]. It is also well known that a number of neuroleptics possess moderate to high affinity for sigma binding sites, suggesting the possibility that sigma receptors mediate some of the antipsychotic effects of neuroleptics [3,26]. A * Corresponding author. Tel.: ⫹81-3-3964-3241; fax: ⫹81-3-39642188. E-mail address: [email protected] (K. Ishiwata). 0969-8051/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 3 ) 0 0 0 0 3 - 9
major role for sigma receptors might be to regulate the activity of the glutamatergic system via the modulation of the NMDA receptor subtype. This modulation of the glutamatergic system could in turn interfere with the dopaminergic neurotransmission with which, however, sigma ligands could also interact directly [3]. However, the sigma receptor occupancy by the neuroleptics has not been evaluated in humans and animal models, because no in vivo selective radioligand was available up to now. Recently, we have developed 1-([3-O-methyl-11C]3,4-dimethoxyphenethyl)-4(3-phenylpropyl)piperazine ([11C]SA4503) as a selective PET ligand for sigma1 receptors [8,9,13,14]. Preliminarily, we have successfully performed imaging of the sigma1 receptors of the human brain by PET with [11C]SA4503 [7, 22]. The aim of the present study is to investigate preliminarily in mice whether [11C]SA4503 is available as an in vivo probe for evaluating the sigma1 receptor occupancy by the neuroleptics using PET. By measuring in vivo the binding of [11C]SA4503 , we evaluated the blockade of sigma1 receptors in the brain after a single administration of haloperidol or two other dopamine D2–like receptor ligands having high and moderate affinity for sigma receptors: nemonapride and spiperone. We also used [11C]raclopride
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K. Ishiwata et al. / Nuclear Medicine and Biology 30 (2003) 429 – 434
Table 1 The in vitro affinity of haloperidol, three dopamine D2-like receptor ligands and SA4503 for dopamine D2 and sigma receptors. Dopamine D2 Ki(nM)a IC50(nM) Haloperidol* Nemonapride Spiperone Raclopride SA4503
19e 470e
0.5 0.06 0.05 1.8
Sigmai IC50(nM) 3b 5.6* 2269d 11800e 17.4b,f
Sigma2 IC50(nM) 120b 38.8* ⬎10,000d 4950e 1780b,f
Data from a van Tol et al., 1991 [25]; b Matsuno et al., 1996 [16]; present study; d Akunne et al., 1997 (Ki value) [1]; e Ishiwata et al., 2001 [9]; and f Matsuno et al., 1997 [19]. A higher affinity of haloperidol for sigma2 receptors was also reported (Ki values of 3.3 nM and 18.9 nM for sigma1 and sigma2 receptors, respectively, [26]), but the data obtained by the same assay conditions in our group were represented, except for spiperone. c
for evaluating dopamine D2 receptor occupancy rates of the three compounds. The typical antipsychotic haloperidol is considered to be a reference compound in sigma receptor binding assay [26]. The benzamide derivative nemonapride is now used clinically as a neuroleptic [15,16], but it has been shown to have a high affinity for sigma receptors both in vitro [6,24] and in vivo [11]. Finally, Coccini et al., [2] reported that spiperone, which is also used clinically, binds in vivo to sigma receptors in rat and human lymphocytes. Therefore, the occupancy rates of haloperidol, nemonapride, and spiperone to sigma1 and dopamine D2 receptor s were measured over a seven day period following a single dose of each drug. The goal of this study is to elucidate which receptors are involved in mediating the CNS effects of these drugs. 2. Materials and methods 2.1. General Haloperidol and spiperone were purchased from Research Biochemicals International (Natick, MA, USA). Nemonapride was supplied by Santen Pharmaceutical Co. Ltd. (Osaka, Japan). [11C]SA4503 [13] and [11C]raclopride [10] were prepared by [11C ]methylation of the respective demethyl compounds. The in vitro affinity of nemonapride for the sigma1 and sigma2 receptors was determined using [3H](⫹)-pentazocine (5 nM) and [3H]1,3-di-O-tolylguanidine (5 nM) in the presence of (⫹)-pentazocine, respectively, as radioligands, and the guinea pig cortex membrane as described [18]. The affinities of the three ligands investigated, SA4503 and raclopride for sigma and dopamine D2 receptors are summarized in Table 1. Male ddY mice (7-week-old) were obtained from Tokyo Laboratory Animals Co., Ltd. (Tokyo, Japan). The animal studies were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology.
2.2. Treatment with haloperidol and two dopamine D2like receptor ligands Mice weighing 35– 40 g (8- to 9-week-old) were used. In a group of mice, haloperidol, nemonapride, or spiperone was co-injected intravenously with each radiotracer as described below. In another group of mice, each of the three compounds was injected intraperitoneally into mice 6 hours before the tracer injection. In the other group of mice, haloperidol was injected intraperitoneally, and after various time intervals up until seven days, the regional brain uptake of each of the radiotracers was measured. The administered dose of each of the three ligands was 100 nmol/animal (0.94 –1.03 mg/kg). As a control for each group, physiological saline was injected. 2.3.Regional brain uptake of [11C]SA4503 and [11C]raclopride Regional uptake of [11C]SA4503 in the mouse brain was measured 30 min after intravenousinjection, because the uptake of [11C]SA4503 was highest at 5 min, then decreased slightly and remained in an equilibrium state from 30 to 60 min [14]. Regional uptake of [11C]raclopride was measured 15 min after injection, because the ratio of specific to nonspecific binding determined as (striatal uptake – cerebellar uptake)/(cerebellar uptake) was maximum 15 min after the tracer injection [9]. [11C]SA4503 (2.0 MBq/21– 46 pmol) or [11C]raclopride (2.0 MBq/18 –36 pmol) was injected intravenously into mice through the tail vein, and 30 min or 15 min later the mice were killed by cervical dislocation. The brain was removed and divided into the striatum, cerebral cortex, cerebellum and rest. The tissues were measured for radioactivity with an auto-gamma counter and weighed. The uptake was expressed as a percentage of the injected dose per gram of the tissue (%ID/g). Student’s t test was performed to compare the control and each group, and P ⬍ 0.05 was considered significant. The receptor-specific binding of the tracer was determined as the difference between the uptake in each group and the uptake in the group co-injected with haloperidol, while the receptor occupancy (%) was defined as 100 ⫻ {1 ⫺ (the receptor-specific binding in each group)/(the receptor-specific binding in the control). 3. Results The regional brain uptake of [11C]SA4503 or [11C]raclopride in the control and other groups is represented in Fig. 1. In the control, [11C]SA4503 was taken up in the three regions, whereas [11C]raclopride was highly concentrated in the striatum. By co-injection of each of the three ligands, the uptake of [11C]SA4503 was significantly reduced to 39 – 82% of the control in all three regions. The highest reduction was found for haloperidol followed by spiperone and
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Fig. 1. Blockade of the regional uptake of [11C]SA4503 (A) and [11C]raclopride (B) in the mouse brain by intravenous co-injection of haloperidol, nemonapride or spiperone or by intraperitoneal injection of each of the three compounds 6 hours before the tracer injection. Mean ⫾ SD (N ⫽ 5–7). *Any significant reduction (P ⬍ 0.05, Student’s t test, compared with the control) is presented as a percentage of the control.
nemonapride. On the other hand, the uptake of [11C]raclopride in the striatum was decreased to 14 –18% of the control by each of the three compounds. Six hours after the treatment with haloperidol but not with each of the two others, the uptake of [11C]SA4503 was significantly reduced to 45– 47% of the control, whereas the striatal uptake of [11C]raclopride was reduced to 28 – 42% of the control by all the three ligands. The time course of blocking effects of haloperidol on the tracer uptake and the receptor occupancy over seven days are summarized in Figs. 2 and 3, respectively. The largest reduction was found 0.5 h after the haloperidol treatment for two tracers: 38 – 41% of the control in all tissues investigated for [11C]SA4503 and 22% of the control in the striatum for [11C]raclopride. The blocking effects for [11C]SA4503 decreased with time in the three regions of the
brain. At day 2 the uptake of [11C]SA4503 by the cerebral cortex was 84% of the control, which corresponded to 24% receptor occupancy (Fig. 3). The whole brain uptake was also significantly reduced to 90% (21% occupancy) at day 2 (Fig. 3). The reduction was not significant in the cerebellum (93%) and striatum (92%). On the other hand, the striatal uptake of [11C]raclopride returned to the control level at day 2.
4. Discussion A number of neuroleptics possess moderate to high affinity for sigma binding sites, suggesting the possibility that sigma receptors mediate some of the antipsychotic effects of neuroleptics [3]. Furthermore, the number of sigma binding
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Fig. 2. Time course of the blockade of the regional uptake of [11C]SA4503 (A) and [11C]raclopride (B) in the mouse brain after intraperitoneal injection of haloperidol. The uptake of the tracer was measured 0.5 and 6 hours, and 1, 2, 3, and 7 days after the haloperidol treatment. Mean ⫾ SD (N ⫽ 5– 6). *Significant reduction (P ⬍ 0.05, Student’s t test, compared with the control) is presented as a percentage of the control.
sites has been reported to be reduced in the brain of schizophrenic patients [6,27]. Therefore, sigma receptors are believed to play an important role in the pathogenesis of schizophrenia. Frieboes et al. [4] reported that the specific sigma ligand panamesine has similar antipsychotic properties regarding immunomodulation and sleep-electroencephalographic changes as haloperidol, in contrast to an atypical antipsychotic clozapine. These findings suggest indirectly the binding of haloperidol to sigma receptors. However, the sigma receptor occupancy by haloperidol and other neuroleptics has not been evaluated in vivo, because no selective radioligand was available. In the present study we investigated preliminarily whether [11C]SA4503, a new PET probe, is available for evaluating the sigma1 receptor occupancy by neuroleptics. The present pilot study demonstrates directly that haloperidol and two other dopamine
D2-like receptor ligands bind in vivo to sigma1 receptors to a different extent after a single administration. Because no binding of [11C]SA4503 to dopamine D2 receptors was detected in vivo [9], the reduction in [11C]SA4503 uptake by co-injection of each of the three ligands investigated, demonstrates the in vivo binding of these ligands to sigma1 receptors. The highest reduction was found for haloperidol, followed by spiperone and nemonapride. The blocking effects of spiperone and nemonapride disappeared 6 hours later. Among the three ligands, the blocking effect of nemonapride did not parallel the affinity in vitro (spiperone ⬍ nemonapride ⬍ haloperidol). Although the reason for the weaker blocking effect of nemonapride remains unknown, these findings suggest that spiperone and nemonapride bind much weaker to sigma1 receptors than does haloperidol, and also bind
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Fig. 3. Time course of the sigma1 and dopamine D2 receptor occupancy in mice after intraperitoneal injection of haloperidol. Mean ⫾ SD (N ⫽ 5– 6).
much weaker to sigma1 receptors than to striatal dopamine D2 receptors. By co-injection of haloperidol, the highest blockade rates were found for both [11C]SA4503 and [11C]raclopride: 62% of the total uptake in the whole brain of [11C]SA4503 and 84% of the striatal uptake of [11C]raclopride, suggesting that the specific binding rate of [11C]raclopride was larger than that of [11C]SA4503. Therefore, in principle, the dopamine D2 receptor occupancy can be evaluated more sensitively than the sigma1 receptor occupancy using [11C]SA4503 and [11C]raclopride as in vivo probes. Approximately 40% of the brain uptake of [11C]SA4503 that was not blocked is considered to involve non-specific binding, because of very low in vitro affinity of SA4503 for 36 other receptors, ion channels and second messenger systems [16]and because of the in vivo selectivity of [11C]SA4503 [8,14]. A notable finding is that after the single administration of haloperidol, the sigma1 receptor blockade continued in all regions of the brain slightly longer than the dopamine D2 receptor blockade in the striatum (Figs. 2 and 3). Although the dopamine D2 receptor occupancy was negligible at day 2, a slight blockade of the uptake of [11C]SA4503 at day 2 reflects the sigma1 receptor occupancy by haloperidol in the cerebral cortex (24%) and in whole brain (21%). Thus, the present data clearly demonstrate the potential of [11C]SA4503 for evaluating the sigma1 receptor occupancy rates of antipsychotic drugs, but shows no association between sigma1 receptor occupancy rates and antipsychotic efficacy. It is also pointed out that one administration as a single dose does not reflect the regimen of drug treatment, although the dose of 100 nmol/animal (0.94 –1.03 mg/kg) is usually used in pharmacological studies and is enough to achieve the highest blockade in the striatal uptake of [11C]raclopride. Therefore, PET evaluation of the sigma1
receptor occupancy by antipsychotic drugs in relation to behavioral potency in humans using [11C]SA4503 is of great interest. In conclusion, all three compounds investigated blocked sigma1 receptors to a different extent immediately after the injection. The sigma1 receptor blockade with haloperidol continued slightly longer than the dopamine D2 receptor blockade after a single administration. [11C]SA4503 is applicable to investigation of sigma1 receptor occupancy by antispychotic drugs using PET.
Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (B) No. 13557077 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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