PET study using [11C]FTIMD with ultra-high specific activity to evaluate I2-imidazoline receptors binding in rat brains

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Nuclear Medicine and Biology 39 (2012) 199 – 206 www.elsevier.com/locate/nucmedbio

PET study using [ 11 C]FTIMD with ultra-high specific activity to evaluate I2-imidazoline receptors binding in rat brains☆ Kazunori Kawamura a,⁎, Yuichi Kimura b , Joji Yui a , Hidekatsu Wakizaka c , Tomoteru Yamasaki a , Akiko Hatori a , Katsushi Kumata a , Masayuki Fujinaga a , Yuichiro Yoshida a, d , Masanao Ogawa a, d , Nobuki Nengaki a, d , Toshimitsu Fukumura a , Ming-Rong Zhang a

a

Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555, Japan b Planning and Promotion Unit, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555, Japan c Department of Biophysics, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555, Japan d SHI Accelerator Service Ltd., Tokyo 141-0032, Japan Received 2 June 2011; received in revised form 29 June 2011; accepted 26 July 2011

Abstract Introduction: We recently developed a selective 11C-labeled I2-imidazoline receptor (I2R) ligand, 2-(3-fluoro-4-[ 11C]tolyl)-4,5-dihydro1H-imidazole ([ 11C]FTIMD). [ 11C]FTIMD showed specific binding to I2Rs in rat brains having a high density of I2R, as well as to I2Rs those in monkey brains, as illustrated by positron emission tomography (PET) and autoradiography. However, [ 11C]FTIMD also showed moderate non-specific binding in rat brains. In order to increase the specificity for I2R in rat brains, we synthesized [ 11C]FTIMD with ultra-high specific activity and evaluated its binding. Methods: [ 11C]FTIMD with ultra-high specific activity was prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [ 11C]methyl iodide, which was produced by iodination of [ 11C]methane using the single-pass method. Dynamic PET scans were conducted in rats, and the kinetic parameters were estimated. Results: [ 11C]FTIMD with ultra-high specific activity was successfully synthesized with an appropriate level of radioactivity and ultra-high specific activity (4470±1660 GBq/μmol at end of synthesis, n=11) for injection. In the PET study, distribution volume (VT) values in all the brain regions investigated whether I2R expression was greatly reduced in BU224-pretreatead rats compared with control rats (29–45% decrease). Differences in VT values between control and BU224-pretreated rats using [ 11C]FTIMD with ultra-high specific activity were greater than those using [ 11C]FTIMD with normal specific activity (17–34% decrease) in all brain regions investigated. Conclusion: Quantitative PET using [ 11C]FTIMD with ultra-high specific activity can contribute to the detection of small changes in I2R expression in the brain. © 2012 Elsevier Inc. All rights reserved. Keywords: Ultra-high specific activity; Imidazoline receptors; I2; FTIMD;

1. Introduction The dose of positron emission tomography (PET) probe may modify both binding and pharmacokinetics in small ☆

This work was supported by a Grant-in-Aid for Young Scientists (B) (21791231; 23791466) from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. ⁎ Corresponding author. Tel.: +81 43 206 3192; fax: +81 43 206 3261. E-mail address: [email protected] (K. Kawamura). 0969-8051/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2011.07.008

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C; PET

animal imaging studies [1–4]. Dynamic interplays between specific and nonspecific binding in blood circulation, transient lung retention, kidney excretion, liver–gallbladder flow, soft tissue retention and metabolism could play a significant role in determining the probe concentration in the target regions [3]. We recently reported that the PET probe [ 11C]DAC for translocator protein (TSPO, 18 kDa) with ultra-high specific activity (∼3700 GBq/μmol) is a useful and sensitive biomarker for the visualization of early infarction and characterization of TSPO expression which

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(n-C4 H9)3Sn

N F

a

H3 C N F

N

N

Fig. 1. Synthesis of [ 11C]FTIMD with ultra-high specific activity. a Reagents, conditions and yields: tris(dibenzylideneacetone)dipalladium(0), tri(o-tolyl)phosphine, copper (I) chloride, cesium fluoride, N-methyl-2pyrolidine, [ 11C]methyl iodide, 80°C, 5 min, radiochemical yield 4.2±0.7% from [ 11C]methane (n=6), specific activity 2100–6900 GBq/μmol (at the end of synthesis, n=11).

was slightly elevated in the infarcted brain using PET [5,6]. Therefore, ultra-high specific activity may be useful for the development and application of PET probes for imaging and assessment of receptors present at extremely low densities or new binding sites in the brain. I2-imidazoline receptors (I2Rs) are widely distributed throughout the tissues of various species, including humans [7], and are mostly located on the outer mitochondrial membrane [8]. There is evidence of their involvement in various disorders of the central nervous system; the density of I2Rs in the brain was decreased in suicide victims and depressed patients [9,10], increased in patients with Alzheimer's disease [11] and decreased in patients with Huntington's disease [12]. Probably, changes in the density of I2Rs are directly or indirectly related to particular diseases. In addition, high-affinity I2R ligands promote food intake [13], which is presumed to be an effect mediated by the arcuate nucleus of the hypothalamus which has a markedly high density of I2Rs [14]. I2R ligands may be useful probes for investigating these diseases in PET studies. Therefore, a number of PET probes for imaging I2Rs have been synthesized and evaluated [15–17]. However, not a single I2R ligand suitable for PET has not been reported. We recently developed a selective 11C-labeled I2R ligand, 2-(3fluoro-4-[ 11 C]tolyl)-4,5-dihydro-1H-imidazole ([ 11 C] FTIMD; Fig. 1) [18]. FTIMD has a high and selective affinity for I2R (Ki for I2Rs, 8.0 nM; I1Rs/I2Rs N3000; Ki for α1-ARs and α2-ARs, N10 μM) [19]. [ 11C]FTIMD specifically bound to I2Rs in rat brains having a high density of I2R [18], as well as to I2Rs in monkey brains [20], as illustrated by PET and autoradiography. However, it also showed moderate nonspecific binding (approximately 70%) in the rat brain [18]. In order to increase its I2R binding specificity, we synthesized [ 11C]FTIMD with ultra-high specific activity and evaluated its binding to I2R in the rat brain. 2. Materials and methods 2.1. Materials FTIMD was prepared in our laboratory as described previously [19] with certain modifications. BU224 hydrochloride was purchased from Tocris Bioscience (Bristol, UK). High-performance liquid chromatography (HPLC) was performed using a Jasco HPLC system (Jasco, Tokyo,

Japan). Effluent radioactivity was monitored using a NaI (Tl) scintillation detector system. Reagents and organic solvents were commercially available and were used without further purification. Unless otherwise stated, radioactivity was determined using an IGC-3R Curiemeter (Aloka, Tokyo, Japan). 2.2. Animals Male Sprague–Dawley rats (aged 7 weeks) were purchased from Japan SLC (Shizuoka, Japan). Animals were maintained and handled in accordance with recommendations of the US National Institutes of Health and the guidelines of the National Institute of Radiological Sciences (Chiba, Japan). Animal studies were approved by the Animal Ethics Committee of the National Institute of Radiological Sciences. 2.3. Radiosynthesis of [ 11C]FTIMD with ultra-high specific activity [ 11C]FTIMD was synthesized as described previously [20] using an in-house automated synthesis system (Fig. 1) [2,21]. A mixture of tris(dibenzylideneacetone)dipalladium (0) (1.3 mg, 1.4 μmol), cesium fluoride (1.1 mg, 7 μmol) and tri(o-tolyl)phosphine (6.5 mg, 22 μmol) in anhydrous N-methyl-2-pyrolidine (NMP; 0.1 ml) and copper (I) chloride (0.28 mg, 2.7 μmol) in NMP (0.1 ml) was added to the NMP solution (0.2 ml) of the tributylstannyl precursor (1.7 mg, 4.1 μmol) [18] in a dry septumequipped vial immediately before radiosynthesis. [ 11C]Methyl iodide was produced by iodination of [ 11C]methane, which was produced by a cyclotron (CYPRIS HM-18; Sumitomo Heavy Industries, Tokyo, Japan), using the single-pass method [2]. [ 11C]Methyl iodide was trapped in the mixture of the tributylstannyl precursor (0.4 ml) at room temperature. The reaction mixture was heated to 80°C for 5 min. After cooling, 1.0 ml of the preparative HPLC eluent was added to the mixture. The solution was filtered through a pre-filter glass fiber (GF53, 30 mm; Agilent Technologies, Santa Clara, CA, USA) and applied to the preparative HPLC column. Preparative HPLC purification was performed on a Hydrosphere C18 column [5 μm, 10 mm internal diameter (i.d.)×250 mm length; YMC, Kyoto, Japan] using a mixture of acetonitrile and 50 mmol/L ammonium acetate (20:80, v/v) as the mobile phase, at a flow rate of 4 ml/min with an ultraviolet detector at 260 nm and a radioactivity detector. The retention time of [ 11C]FTIMD was approximately 7 min. The HPLC fraction of [ 11C]FTIMD was collected in a flask to which 25% ascorbic acid (100 μl) had been added before radiosynthesis and subsequently evaporated to dryness. The residue was dissolved in physiological saline. The final product (100 μl) was analyzed with HPLC on Zorbax Bonus-RP (5 μm, 4.6 mm i.d.×150 mm length; Agilent Technologies) using a mixture of acetonitrile and 0.1 mol/L ammonium formate (10:90, v/v) as the mobile phase at a

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flow rate of 1.5 ml/min with an ultraviolet detector at 260 nm and a radioactivity detector. The retention time was 5.0 min. 2.4. PET study in rats PET scans were acquired with an Inveon Dedicated PET Scanner (Siemens Medical Solutions, Knoxville, TN, USA); the total duration was for 60 min as described previously [18]. Group of four rats (aged 7–8 weeks, weighing 246–292 g) received no treatment (control condition) or were pretreated with BU224 (1.0 mg/kg, 0.1 ml) (pretreatment condition) 5 min before intravenous injection of [ 11C]FTIMD (58–91 MBq/17–47 pmol/0.6 ml). Decay-corrected radioactivity was expressed as standard uptake value (SUV). Arterial blood samples were manually collected every 5 s from 0 to 30 s, followed by collection at 0.5, 1, 2, 3, 5, 15, 30 and 60 min after the injection of [ 11 C]FTIMD. For metabolite analysis, plasma samples were obtained 0.5, 1, 5, 15, 30 and 60 min after the injection. Plasma treatment and metabolite analysis were carried out as described previously [18]. The percentages of unchanged forms were then determined. The unchanged fraction data fitted with the sum of exponentials. 2.5. Measurement of plasma protein binding in rats Plasma protein binding was determined by an ultrafiltration method using a Microcon YM-30 filter (Millipore) according to a previously described method [18]. Blood samples (approximately 1.0 ml) were obtained from rats. In rats pretreated with 1.0 mg/kg BU224, blood samples (approximately 1.0 ml) were obtained 5 min after the injection. The plasma free fraction (fp) was calculated as the ratio of the ultrafiltrate activity to the total plasma activity corrected for the stick factor. The stick factor is the radioactivity in a known volume of ultrafiltered saline divided by the radioactivity in the same volume of saline. 2.6. Kinetic modeling of [ 11C]FTIMD using PET in rats Regions of interest were manually placed on the summed PET images with reference to a typical magnetic resonance imaging (MRI) of rat brains using ASIPro VM (Analysis Tools and System Setup/Diagnostics Tool; Siemens Medical Solutions) to generate six regional time–activity curves (TACs) in the following regions: arcuate nucleus, interpeduncular nucleus, ependymal cell layer, cerebellum, hippocampus and cortex. TACs were analyzed using a multilinear analysis 1 (MA1) with a metabolite-corrected input function as previously described [18]. 2.7. Statistical analysis Quantitative data are expressed as the mean±S.D. Differences between the kinetic parameters of control rats and those of pretreated rats were calculated using Student's t tests with Welch's correction; differences between TACs of control rats and those of pretreated rats

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were calculated using two-way analysis of variance and were considered significant if Pb.05. The data were analyzed GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).

3. Results 3.1. Radiosynthesis of [ 11C]FTIMD with ultra-high specific activity [ 11C]FTIMD with ultra-high specific activity was successfully synthesized with an appropriate level of radioactivity for injection by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [ 11C]methyl iodide, which was produced by iodination of [ 11C]methane using the single-pass method [2]. The decay-corrected radiochemical yield of [ 11C]FTIMD from [ 11C]methane was 4.2±0.7 % at the end of irradiation (n=6), and the specific activity was 4470±1660 GBq/μmol at the end of synthesis (30 min after the end of irradiation; n=11). The present value of specific activity was obtained by comparison of the assayed radioactivity with the mass in 100 μl product solution from the carrier ultraviolet peak at 260 nm. Radiochemical purity was N99%. 3.2. PET study in rats Typical coronal brain PET images acquired 15–60 min after the injection of [ 11 C]FTIMD with ultra-high specific activity are shown in Fig. 2. In control rats, high radioactivity was observed in the arcuate nucleus, interpeduncular nucleus, hippocampus and ependymal cell layer (Fig. 2A). The regional distribution of radioactivity using [ 11 C]FTIMD with ultra-high specific activity was similar to that in a previous PET study using [ 11 C]FTIMD with normal specific activity [18]. Pretreatment with BU224 (1.0 mg/kg) reduced the radioactivity across the whole brain (Fig. 2B). TACs in the arcuate nucleus showing high levels of I2R and in the cerebellum showing low levels of I2R after the injection of [ 11C]FTIMD with ultra-high or normal specific activity are shown in Fig. 3. In control rats, the radioactivity level in the arcuate nucleus after the injection of [ 11C]FTIMD gradually decreased from initial uptake until 60 min (Fig. 3A), and no statistical difference was observed between the two TACs after the injection of [ 11C]FTIMD with ultra-high and normal specific activities (Fig. 3A). In BU224-pretreated rats, the radioactivity level in the arcuate nucleus decreased as compared with that in control rats over 5 min after the injection, and the radioactivity level in the cerebellum slightly decreased as compared with that in the control rats (Fig. 3A). The decreased levels in the arcuate nucleus and cerebellum obtained using ultra-high specific activity were slightly higher than those obtained using normal specific activity (Fig. 3). TACs after the injection of [ 11C]FTIMD with ultrahigh specific activity in the other regions of the brain that

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SUV 0.8

A

0

B Ep

Cor

Cer

Hip

C Arc

Ipn

Fig. 2. Typical coronal PET images using [ 11C]FTIMD with ultra-high specific activity in the rat brain of control rats (injected radioactivity, 84 MBq/46 pmol) (A) and BU224-pretreated (1.0 mg/kg) rats (injected radioactivity, 64 MBq/35 pmol) (B) and typical coronal MRI images of rat brains on which six regions of interest were placed (C). PET images were acquired for 45 min, starting 15 min after the injection. Rats were anesthetized with isoflurane (1.0–1.5%) and placed in the prone position on the bed of the scanner. The scale of radioactivity was expressed as SUV. Ep, ependymal cell layer; Cor, cortex; Hip, hippocampus; Arc, arcuate nucleus; Ipn, interpeduncular nucleus; Cer, cerebellum.

express I2R are shown in Fig. 4. The radioactivity level in these other brain regions gradually decreased from initial uptake until 60 min in control rats while it slightly decreased in BU224-pretreated rats (Fig. 4). The metabolite corrected TACs and percentage of unchanged form in plasma after the injection of [ 11C] FTIMD with ultra-high specific activity are shown in Fig. 5. The radioactivity level in the metabolite-corrected plasma of control rats (Fig. 5A) was slightly lower than that of BU224pretreated rats (Fig. 5B). The percentage of unchanged [ 11C]

A

3.3. Measurement of plasma protein binding The values of fp of [ 11C]FTIMD with ultra-high specific activity were 84.3±4.3% (n=4) and 85.6±2.9% (n=4) in control and BU224-pretreated rats, respectively. The plasma free fraction of [ 11C]FTIMD in rats did not change in control or BU224-pretreated rats.

B

Arcuate nucleus 4

Cerebellum 4

Ultra-high SA (control) Ultra-high SA (pretreatment) Normal SA (control)* Normal SA (pretreatment)*

Radioactivity level (SUV)

Radioactivity level (SUV)

FTIMD in the plasma of control rats (Fig. 5A) was similar to that of BU224-pretreated rats (Fig. 5B).

2

Ultra-high SA (control) Ultra-high SA (pretreatment) Normal SA (control)* Normal SA (pretreatment)*

2

0

0 0

30

Time after injection (min)

60

0

30

60

Time after injection (min)

Fig. 3. Time–activity curves of the arcuate nucleus (A) and cerebellum (B) in the control and BU224-pretreated (1.0 mg/kg) rats after injection of [ 11C]FTIMD with ultra-high SA (58–91 MBq/17–47 pmol) and normal SA (46–117 MBq/0.26–1.38 nmol) [18]. Radioactivity level was expressed as mean SUV. ⁎Data quoted from Ref. [18]. SA, specific activity.

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Ependymal cell layer

Interpeduncular nucleus

4

4

Control

Radioactivity level (SUV)

Radioactivity level (SUV)

Control Pretreament

2

0

Pretreament

2

0 0

30

60

0

Time after injection (min)

30

60

Time after injection (min) Cortex

Hippocampus 4

4

Control

Control Pretreament

Pretreament Radioactivity level (SUV)

Radioactivity level (SUV)

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2

0

2

0 0

30

60

Time after injection (min)

0

30

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Time after injection (min)

Fig. 4. Time–activity curves of brain regions expressing I2R in control and BU224-pretreated (1.0 mg/kg) rats after injection of [ 11C]FTIMD with ultra-high specific activity (58–91 MBq/17–47 pmol). Radioactivity level was expressed as mean SUV.

3.4. Kinetic modeling of [ 11C]FTIMD using PET in rats Distribution volume (VT) values were derived using a MA1 model which provided a better fit than one-tissue compartment or two-tissue compartment models as described previously [18]. Results of VT values in the brain regions expressing I2R are presented in Table 1. No significant differences were found in within-subject standard deviations between outcome measures of VT and VT/fp. VT values were prominently affected by pretreatment with BU224. VT values in BU224-pretreated rats were significantly reduced by 29–45% of that control rats. The percentage of decrease in VT value compared with control rats was higher with the ultra-high specific activity probe than with the normal specific activity probe as described previously [18]. The VT values of the normal specific activity probe in the arcuate nucleus showing high level of I2R and in the cerebellum showing low level of I2R [18] are compared with those of the ultra-high specific activity probe in the

corresponding regions in Fig. 6. In control rats, VT values of the ultra-high specific activity probe in the arcuate nucleus (Fig. 6A) and cerebellum (Fig. 6B) were significantly higher than those of the normal specific activity probe. In BU224pretreated rats, VT values of both the ultra-high and normal specific-activity probes were significantly reduced compared with those of control rats (Fig. 6). 4. Discussion To determine the increase in specific binding to I2R, we compared the results of a PET study using [ 11C]FTIMD with ultra-high specific activity (N2000 GBq/μmol at the end of synthesis) in the rat brain with those of a PET study using [ 11C]FTIMD with normal specific activity (approximately 100 GBq/μmol at the end of synthesis) as described previously [18]. In the quantitative PET study, VT values of [ 11C]FTIMD with ultra-high specific activity in the arcuate nucleus expressing high levels of I2Rs (Fig. 6A) and

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A

B 4

Radioactivity level (SUV)

Radioactivity level (SUV)

4 Control

2

Pretreatment

2

0

0 0

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30

60

Time after injection (min)

100

100

Unchanged form (%)

Unchanged form (%)

Control

50

0

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50

0 0

30

60

Time after injection (min)

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30

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Fig. 5. Time-activity curves of plasma and the time courses of unchanged [ 11C]FTIMD in plasma in control rats (A) and BU224-pretreated (1.0 mg/kg) rats (B). The injected dose of [ 11C]FTIMD with ultra-high specific activity was 58–91 MBq/17–47 pmol. Radioactivity level was expressed as mean SUV. Unchanged [ 11C]FTIMD in plasma was expressed as mean percentage of the unchanged form.

the cerebellum expressing low levels of I2Rs (Fig. 6B) were higher than those of [ 11C]FTIMD with normal specific activity (Fig. 6A and B). Furthermore, VT values in all the investigated regions expressing I2Rs were greatly reduced in BU224-pretreated rats compared with control rats (29–45% decrease) (Table 1). Difference in VT values between control and BU224-pretreated rats using [ 11C]FTIMD with ultra-high specific activity (29–45% decrease) were approximately 1.3–1.7-fold higher than that using [ 11C]FTIMD with normal specific activity (17–34% decrease) (Table 1). These results may be explained by the lower dissociation constant of [ 11C]FTIMD with ultra-high specific activity compared with that of probe with normal specific activity as described previously [22]. These results indicate that specific binding to I2Rs is increased by the use of [ 11C] FTIMD with ultra-high specific activity. On the other hand, TACs of the arcuate nucleus and cerebellum in control rats after injection of [ 11C]FTIMD with ultra-high specific activity were not statistically different from those with normal specific activity (Fig. 3). This result indicated that such a relatively high concentration of ligand in the [ 11C] FTIMD with ultra-high specific activity did not occupy all

I2Rs in brain regions, because I2Rs density (Bmax) in the rat brain is relatively high (144 fmol/mg protein) [14]. In control rats, VT values of the [ 11C]FTIMD with ultra-high specific activity in the arcuate nucleus and cerebellum were significantly higher than those with normal specific activity (Fig. 6). The density of I2Rs in human platelets (Bmax, 350 fmol/mg protein [23]) was similar level to that in the human brain (Bmax, 471 fmol/mg protein [24]), although the density of I2Rs in rat platelets has not been investigated. However, the radioactivity level in the metabolite-corrected plasma of control rats was slightly lower than that of BU224pretreated rats (Fig. 5). Therefore the binding to platelets may affected the specific binding to I2Rs of [ 11C]FTIMD with ultra-high specific activity in the rat brain to a very low extent. Therefore, quantitative PET measurements using [ 11C]FTIMD with ultra-high specific activity may sensitively detect small changes in I2R expression in the brain. In the PET study, the regional distribution (Fig. 2A) and VT values in rat brain regions (Table 1) showed a rank order of ependymal cell layerNhippocampusNarcuate nucleusNinterpeduncular nucleusNcortexNcerebellum. The rank order of VT values of [ 11C]FTIMD with ultra-high

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[14,25,26]. On the contrary, the difference in VT values of [ 11C]FTIMD with ultra-high specific activity between control and BU224-pretreated rats (Table 1) was almost similar to that of the specific binding of I2R ligands in rat brain tissues [14,25,26]. This discrepancy may be due to moderate nonspecific binding. Therefore, it is desirable to develop a PET probe that has low nonspecific binding in in vivo experiments in rats. Presently, it is recommended that [ 11C]FTIMD with ultra-high specific activity be used as a PET probe for elucidating I2Rs, because a PET probe with low nonspecific binding used for imaging I2Rs has not yet been reported.

Table 1 Values of total VT of [ 11C]FTIMD in rats obtained using multilinear analysis 1 VT (ml/cm 3)

Arcuate nucleus Interpeduncular nucleus Ependymal cell layer Cerebellum Hippocampus Cortex

Percentage of decrease compared with control rats

Control rats

BU224-pretreated rats a

17.10±0.74 16.14±0.83

9.36±0.39⁎ 10.63±0.58⁎

45 (34) b 34 (26) b

19.02±0.97

12.84±1.49⁎

32 (22) b

13.77±1.33 18.03±0.46 16.78±0.98

9.51±0.57⁎ 12.80±0.49⁎ 11.97±0.70⁎

31 (19) b 29 (18) b 29 (17) b

Data are expressed as mean±S.D. (n=4). The estimated kinetic parameter was expressed as the volume of VT. a Administered intravenously at 1.0 mg/kg BU224 5 min before PET scan. b Normal specific activity, quoted from Ref. [18]. ⁎ Pb.05 (Student's t tests with Welch's correction compared with control rats).

5. Conclusion [ 11C]FTIMD with ultra-high specific activity showed the specific-binding in rat brain tissues expressing I2Rs. Quantitative PET study using [ 11C]FTIMD with ultra-high specific activity would thus contribute to the detection of small changes in I2R expression and hence may be useful in elucidating I2R expression in the brain.

specific activity (Table 1) showed results similar to those of [ 11C]FTIMD with normal specific activity in all brain regions investigated as described previously [18]. However, the autoradiographical distribution of the typical high affinity I2R ligands [ 3H]2-BFI and [ 3H]BU224 in rat brain tissues showed high specific binding in the interpeduncular nucleus, arcuate nucleus and ependymal cell layer and moderate levels of binding in other brain regions such as the hippocampus, cortex and cerebellum [14,23,24]. The rank order of VT values differed slightly from that of the binding of I2R ligands in rat brain tissues

A

Acknowledgments We are grateful to Dr. Fujiko Konno (WDB) for her technical assistance with the synthesis and to the staff of the Cyclotron Operation Section [National Institute of Radiological Sciences (NIRS)] for their technical assistance in radioisotope production. We also thank the staff of the Department of Molecular Probes (NIRS) for general assistance.

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Fig. 6. Values of total VT by multilinear analysis in the arcuate nucleus (A) and cerebellum (B) using [ 11C]FTIMD with ultra-high SA (58–91 MBq/17–47 pmol) and normal SA (46–117 MBq/0.26–1.38 nmol) [18]. ⁎Data quoted from ref. [18]. #Pb.05 (Student's t tests with Welch's correction compared with control rats).

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