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Anm20-6(06)河村

Annals of Nuclear Medicine Vol. 20, No. 6, 417–424, 2006 Synthesis and evaluation of vesamicol analog (−)-o-[11C]methylvesamicol
as a PET ligand for vesicular acetylcholine transporter
Kazunori KAWAMURA,*,** Kazuhiro SHIBA,*** Hideo TSUKADA,**** Shingo NISHIYAMA,**** Hirofumi MORI*** and Kiichi ISHIWATA* *Positron Medical Center, Tokyo Metropolitan Institute of Gerontology **SHI Accelerator Service Ltd. ***Advanced Science Research Center, Kanazawa University ****Central Research Laboratory, Hamamatsu Photonics K.K. (−)-o-Methylvesamicol ((−)-OMV) exhibited in vitro a high affinity for vesicular acetylcholinetransporter (VAChT) (Ki, 6.7 nM) and a relatively low affinity for sigma1 receptor (Ki, 33.7 nM).
We prepared (−)-[11C]OMV by a palladium-promoted cross-coupling reaction using [11C]methyliodide, in a radiochemical yield of 38 ± 6.9% (n = 3), a radiochemical purity of 98 ± 2.3% (n = 5),and a specific activity of 11 ± 7.0 TBq/mmol at 30 minutes after EOB (n = 5). Then, we evaluatedin vivo whether (−)-[11C]OMV has properties as a PET radioligand for mapping VAChT. In rats,the brain uptake of (−)-[11C]OMV was 1.1%ID/g at 5 minutes postinjection, and was retained of ahigh level for 60 minutes. The brain uptake was significantly inhibited by the co-injection (500nmol/kg) of cold (−)-OMV (58–66%), (−)-vesamicol (57–65%), and two sigma receptor ligandswith modulate affinities for VAChTs: SA4503 (56–71%) and haloperidol (39–64%) in all of thebrain regions, including the cerebellum with a low density of VAChTs, but not of sigma1-selectiveligand (+)-pentazocine. However, the pretreatment with a large excess amount of (±)-pentazocine(50 µmol/kg) reduced the uptake in a different manner in the brain regions: 25% reduction in thestriatum with a high density of VAChTs, and a 50–55% reduction in the other regions with a lowerdensity of VAChTs. Ex vivo autoradiography using (−)-[11C]OMV showed a similar regional braindistribution of [3H](−)-vesamicol. In the PET study of the monkey brain, the regional braindistribution pattern of (–)-[11C]OMV was different from that of [11C]SA4503. The uptake of (−)-[11C]OMV was relatively higher in the striatum, was reversible, and an apparent equilibrium statewas found at 20–40 minutes. In conclusion, (−)-[11C]OMV exhibited appropriate brain kineticsduring the time frame of 11C-labeled tracers and bound mainly to VAChTs; however, the bindingto sigma1 receptors was not disregarded. Therefore, (−)-[11C]OMV-PET together with help of[11C]SA4503-PET may evaluate VAChTs.
Key words: (−)-o-[11C]methylvesamicol, VAChT, PET, vesamicol
has been established as a reliable marker for presynapticcholinergic terminals. Consequently, a selective radio- THE VESICULAR ACETYLCHOLINE TRANSPORTER (VAChT) is ligand for VAChT may be used as a tool for studying the localized exclusively in cholinergic neurons1–4 and thus function of cholinergic neurons with positron emissiontomography (PET) and single photon emission computedtomography (SPECT).
Received November 30, 2005, revision accepted May 16, For reprint contact: Kiichi Ishiwata, Ph.D., Positron Medical been reported to bind to VAChTs on presynaptic acetyl- Center, Tokyo Metropolitan Institute of Gerontology, Naka-cho choline storage vesicles.5,6 Therefore, many radioligands 1–1, Itabashi-ku, Tokyo 173–0022, JAPAN.
based on vesamicol have been developed for mapping VAChTs with PET7–13 and SPECT.14–18 Substituted Vol. 20, No. 6, 2006 positions and optical isomerization of the vesamicol de- Table 1 In vitro affinities of (–)-OMV, (–)-vesamicol and three
rivatives altered their affinities for VAChTs and sigma sigma receptor ligands for vesicular acetylcholine transporter receptors.11,19 Shiba et al. synthesized (−)-o-methyl- (VAChT) and sigma receptors vesamicol ((−)-OMV), wihch exhibited a high affinity for VAChTs (Ki, 6.7 nM) and a relatively low affinity for 1 receptor in vitro (Ki for sigma1, 33.7 nM; Ki for sigma2, 266 nM) (Table 1).20 The VAChT/sigma1 recep- tor selectivity of (−)-OMV (5.0) would not be sufficient for VAChTs; however, in general, a Ki = 33.7 nM is not optimal if one is seeking PET and SPECT radioligands.
Recently, Efange et al. reported [18F](+)-4-fluoro- benzyltrozamicol ([18F](+)-FBT) as a potential PET ligand for mapping VAChTs.10 (+)-FBT was found to Data from Shiba et al.20 have a high affinity (Ki, 0.22 nM) for VAChTs and a lower *Values are the average of three experiments.
affinity for sigma1 receptors (Ki, 21.6 nM) and sigma2 #Rat cerebral membranes were incubated with [3H](−)-vesamicol receptors (Ki, 35.9 nM).21 In the in vivo study, [18F](+)- in 50 mmol/l Tris-HCl (pH 7.8) for 60 minutes at 37°C in the FBT showed a high uptake and a slow rate of washout presence of 200 nmol/l DTG to mask the sigma receptors. †Rat from the striatum of rats10 and monkeys.21 Compared with cerebral membranes were incubated with [3H](+)-pentazocine (+)-FBT, (−)-OMV has a lower affinity for VAChTs, a in 50 mmol/l Tris-HCl (pH 7.8) for 90 minutes at 37°C. $Rat liver slightly lower affinity for sigma membranes were incubated with [3H]DTG in 50 mmol/l Tris- 1 receptors and much lower affinity for sigma HCl (pH 7.8) for 90 minutes at 37°C in the presence of 0.001 2 receptors. Therefore, it is consid- ered that (−)-o-[11C]methylvesamicol ((−)-[11C]OMV) mmol/l (+)-pentazocine to mask the sigma1 receptors.
shows a faster rate of washout from the striatum than[18F](+)-FBT without affinity for sigma receptors. Thebrain kinetics that the receptor-ligand binding reaches the Institute of Gerontology.
apparent equilibrium state during the time-scale for PET Two male rhesus monkeys (Macaca mulatta, 7 years measurement is preferable for quantitative analysis. Neg- old, 6.4 and 6.6 kg) were used for the PET measurements.
ligible binding of (−)-[11C]OMV to sigma receptors is They were trained to sit on a chair twice a week for more also expected. Here, we prepared (−)-[11C]OMV by sub- than three months. The study was performed in accor- stitution of the trimethylstannyl group with [11C]methyl dance with recommendations of the US National Institute iodide in a palladium-promoted cross-coupling reaction of Health and the guidelines of the Central Research (Fig. 1)22 and evaluated in vivo in rats whether (−)- Laboratory, Hamamatsu Photonics K.K.
[11C]OMV has properties as a PET radioligand for map-ping VAChT, or whether it shows affinity for both VAChTs Radiolabeling of (−)-[11C]OMV and sigma receptors. We also performed PET imaging of A solution of tris(dibenzylideneacetone)dipalladium(0) the monkey brain with (−)-[11C]OMV, compared with the (2.8–3.4 mg, 3.0–3.6 µmol), tri(o-tolyl)phosphine (3.7– PET imaging with [11C]SA4503 that has been developed 4.0 mg, 12–13 µmol) in N,N-dimethylformamide (DMF) as a selective PET ligand for mapping sigma1 recep- (0.2 ml) was prepared in a dry septum equipped vial and heated for a few minutes (until the color in the solutionchanged to yellow), and then added to the mixture of MATERIALS AND METHODS
copper chloride (1.2–3.0 mg, 12–30 µmol), potassiumcarbonate (1.7–2.5 mg, 12–18 µmol) and (−)-o- trimethylstannyl-vesamicol (0.6 mg) in DMF (0.2 ml).
(−)-Vesamicol and haloperidol were purchased from Sigma [11C]Methyl iodide was produced from [11C]CO2 with an Chemical (St. Louis, MO). SA4503 and (+)-pentazocine automated system (Sumitomo Heavy Industries, Tokyo, were provided from M's Science (Kobe, Japan). (±)- Japan) and was trapped in the mixture of DMF (0.4 ml) Pentazocine (PENTAGIN® injection) was purchased with air cooling. The reaction mixture was heated at 80°C from Sankyo (Tokyo, Japan). (−)-OMV and (−)-2-(4-(2- for 3 minutes. After adding 1.2 ml of high-performance liquid chromatography (HPLC) eluent [acetonitrile/ 50 trimethylstannyl-vesamicol) were synthesized as described mmol/l ammonium acetate, (30/70, v/v)], the reaction previously.20 All chemicals were obtained from commer- mixture was passed through the glass filter (20 µm) and cial sources.
followed by injection onto the preparative HPLC: YMC- Male Wistar rats were obtained from Tokyo Labora- Pack ODS-A column (10 mm inner diameter (i.d.) × 250 tory Animals (Tokyo, Japan). Animal experiments were mm length, YMC, Kyoto, Japan) with a mobile phase of carried out in compliance with the Guidelines for Animal acetonitrile/ 50 mmol/l ammonium acetate (30/70) at a Care and Use Committee of the Tokyo Metropolitan flow rate of 5.0 ml/minute (UV detector at 260 nm). The Kazunori Kawamura, Kazuhiro Shiba, Hideo Tsukada, et al Annals of Nuclear Medicine Fig. 1 Synthesis of (−)-[11C]OMV.
retention time of (−)-[11C]OMV was 17 minutes. The 1 minute at 4°C to obtain the plasma, which was denatured fraction of (−)-[11C]OMV was collected and evaporated with a 1/3 equivalent volume of 20% trichloroacetic acid to dryness. The residue was dissolved in physiological (TCA) in acetonitrile. The mixture was centrifuged in the saline. The final product was analyzed by HPLC using a same condition, and the precipitate was re-suspended in TSKgel super-ODS column (4.6 mm i.d. × 100 mm 0.5 ml of 10% TCA in acetonitrile followed by centrifu- length, Toso, Tokyo, Japan) with a mobile phase of gation. This procedure was repeated three times. The acetonitrile/ 50 mmol/l acetic acid/ 50 mmol/l ammonium brain was homogenized in 1 ml of 20% TCA in acetoni- acetate (25/37.5/37.5, v/v/v) at a flow rate of 1.0 ml/ trile/water (1/1, v/v). The homogenate was treated as minute (UV detector at 260 nm). The retention time of described above. The combined supernatant was ana- (−)-[11C]OMV was 4.4 minutes.
lyzed by HPLC with a radioactivity detector (Radiomatic150TR, Packard, Meriden, CT). A Radial-Pak C18 col- Tissue distribution in rats umn equipped in an RCM 8 × 10 module (8 mm × 100 mm, (−)-[11C]OMV (9.7 MBq/2.2 nmol) was intravenously Waters, Milford, MA) was used with a mixture of 35% injected into Wistar rats (7 weeks old, 210–250 g). Rats acetonitrile and 65% 50 mmol/l acetic acid/sodium ac- were sacrificed by cervical dislocation 5, 15, 30 and 60 etate (1/1) at a flow rate of 2 ml/minute.
minutes after injection (n = 4). The blood was collected byheart puncture, and tissues were harvested and weighed.
Ex vivo autoradiography in rats The 11C radioactivity in the samples was measured with Ex vivo autoradiography of the brain was carried out in an auto-gamma scintillation counter. The tissue uptake of rats. (−)-[11C]OMV (111 MBq/7.2 nmol) was intrave- 11C was expressed as the percentage of the injected dose nously injected into the rat (8 weeks old, 270 g). The rat per gram of tissue (%ID/g).
was sacrificed by cervical dislocation at 30 minutes after To determine the specific binding, we performed the injection. The brain was rapidly dissected, frozen, and blocking experiment by co-injection with cold (−)-OMV, coronally cut into 20 µm thick sections using a cryotome (−)-vesamicol, SA4503 (sigma1 receptor ligand), halo- (Bright Instrument, Huntingdon, UK). The brain sections peridol (non-selective sigma receptor ligand) and (+)- were dried on a hot plate at 60°C and were apposed to a pentazocine (sigma1 receptor ligand), and by pretreat- storage phosphor screen (PhosphorImager SI system, ment with (±)-pentazocine (PENTAGIN® injection) Molecular Dynamics, Sunnyvale, CA) for 2 hours.
(non-selective sigma receptor ligand). A mixture of (−)-[11C]OMV (9.9 MBq/0.68 nmol) and one of blockers, PET measurement in the monkey brain except for (±)-pentazocine, at a dose of 500 nmol/kg in 0.2 A monkey was fixed on a PET camera, a model SHR-7700 ml physiological saline/dimethyl sulfoxide (1/1, v/v) was (Hamamatsu Photonics K.K., Hamamatsu, Japan), which injected into the rats. In the other group of rats, 50 µmol/ acquires 31 slices at a center-to-center interval of 3.6 mm kg of (±)-pentazocine was intravenously administrated 10 with a resolution of 2.6 mm full width at half maximum in minutes prior to the tracer injection (14 MBq/ 0.87 nmol).
the transaxial plane. (−)-[11C]OMV (745 MBq/ 8.8 nmol) The rats were sacrificed by cervical dislocation 30 min- was injected into the monkey through a posterior tibial utes after injection (n = 4–6, 7–8 weeks old, 230–270 g).
vein cannula. The PET scanning was performed for 61 The sample collection and measurement were performed minutes with 6 time frames at 10 second intervals, 6 time as described above.
frames at 30 seconds, 12 time frames at 1 minute, fol-lowed by 15 time frames at 3 minutes. In the other Metabolite study in rats monkey, the PET study with [11C]SA4503 (537 MBq/8.4 (−)-[11C]OMV (111–118 MBq/7.2–25 nmol) was intra- nmol) was performed in the same way. Regions of interest venously injected into rats (7–8 weeks old, 240–270 g), were placed on the striatum, occipital cortex, frontal and 15 (n = 3) or 30 (n = 1) minutes later they were cortex, temporal cortex and cerebellum. The decay-cor- sacrificed by cervical dislocation. Blood was removed by rected radioactivity was expressed as the percentage of heart puncture using a heparinized syringe, and the brain the injected dose per ml tissue volume (%ID/ml).
was removed. The blood was centrifuged at 7,000 × g for Vol. 20, No. 6, 2006

Table 2 Tissue distribution of radioactivity after intravenous injection of (–)-[11C]OMV in rats
Radioactivity level (%ID/g)* *Radioactivity levels are represented as the mean % injection dose per gram of tissue ± S.D. (n = 4).
Fig. 2 The blocking effects of (−)-OMV, (−)-vesamicol, SA4503, haloperidol, (+)-pentazocine and (±)-
pentazocine on the regional brain distribution of (−)-[11C]OMV in rats at 30 minutes after intravenous
injection. *Co-injected dose with (−)-OMV, (−)-vesamicol and (+)-pentazocine was 500 nmol/kg.
**Pretreatment dose with (±)-pentazocine was 50 µmol/kg. Radioactivity levels are represented as the
mean % injection dose per gram of tissue (%ID/g) ± S.D. (n = 4–6). #p < 0.005, Student's t-test compared
with the control.
(3.4%ID/g) and kidney (2.3%ID/g), while the uptake at60 minutes was high in the pancreas (2.2%ID/g) and Radiolabeling of (−)-[11C]OMV small intestine (1.8%ID/g). The uptake in the brain (−)-[11C]OMV was synthesized by methylation of the showed a tendency to decrease over 60 minutes, and the radioactivity levels of (−)-[11C]OMV decreased in the iodide in a palladium-promoted cross-coupling reaction blood, heart, lung, kidney and muscle over 60 minutes.
(Fig. 1).22 The total synthesis time was about 30 minutes.
The uptake levels in the pancreas, spleen and small in- The decay corrected radiochemical yield was 38 ± 6.9% testine increased until 15 minutes and then decreased (n = 3) calculated from [11C]methyl iodide, and the for 60 minutes. In the liver, the uptake slightly increased radiochemical purity determined by analytical HPLC was for 60 minutes.
98 ± 2.3% (n = 5). The specific activity was 11 ± 7.0 TBq/ The regional distribution of (−)-[11C]OMV in the brain mmol (n = 5) at 30 minutes after the end of bombardment.
and the blocking effects of (−)-OMV, (−)-vesamicol, andthree sigma receptor ligands on the uptake were investi- Tissue distribution in rats gated at 30 minutes after the tracer injection. In the control The tissue distribution of the radioactivity after injection group, the uptake values of the striatum, hippocampus, of (−)-[11C]OMV into rats is summarized in Table 2. The cerebral cortex, medulla oblongata, cerebellum and the initial uptake of (−)-[11C]OMV was high in the lung rest of brain were 1.07 ± 0.14, 0.97 ± 0.12, 1.16 ± 0.12, Kazunori Kawamura, Kazuhiro Shiba, Hideo Tsukada, et al Annals of Nuclear Medicine

Fig. 3 Ex vivo autoradiography of the coronal sections of the rat brain at 15 minutes after injection of
(−)-[11C]OMV.
Fig. 4 The brain images of (−)-[11C]OMV of a conscious monkey. The PET images of (−)-[11C]OMV
were acquired for 45 minutes starting at 60 minutes after injection.
1.21 ± 0.12, 0.97 ± 0.10 and 1.08 ± 0.12, respectively. As Metabolite study in rats shown in Figure 2, the uptake of (−)-[11C]OMV was Metabolite analysis was carried out in the brain and significantly decreased by the co-injection of (−)-OMV plasma at 15 minutes and 30 minutes after injection. The (34–42% of control), (−)-vesamicol (35–43% of control), recovery of the radioactivity in the HPLC analysis was SA4503 (29–44% of control), and haloperidol (36–61% essentially quantitative. At 15 minutes after injection of of control) at a dose of 500 nmol/kg in all of the brain (−)-[11C]OMV, the percentages of the unchanged form in regions. The blocking effects of (−)-OMV and (−)- the brain and in the plasma were 96 ± 1.3% and 58 ± 0.5% vesamicol were equivalent in all of the regions (57–66% (n = 3), respectively, and at 30 minutes after injection, that blockade of control), whereas those of each sigma recep- in the brain and in the plasma were 98% and 58% (n = 1), tor ligand were smaller in the striatum (56% by SA4503 and 39% by haloperidol) than in the other regions (65–71% by SA4503 and 66–74% by haloperidol). On the Ex vivo autoradiography in rats other hand, co-injection of (+)-pentazocine at the dose of Figure 3 shows the coronal images of the rat brain visual- 500 nmol/kg did not reduce the uptake of (−)-[11C]OMV ized by ex vivo autoradiography with (−)-[11C]OMV at 15 in any of the brain regions, although the uptake in the minutes after tracer injection. A slightly higher 11C den- cerebellum seemed to be reduced. However, pretreatment sity was observed in the striatum, pyramidal cell layer of with a large excess of (±)-pentazocine (50 µmol/kg) the hippocampus, hypothalamus, thalamus and nuclei of significantly decreased the uptake of (−)-[11C]OMV in all the cranial motor nerves. A moderate 11C density was of the brain regions, and the effect was relatively small in observed in the cortex and amygdaloid.
the striatum (25% reduction) as compared to that in theother regions (50–55%).
PET measurement in the monkey brainFigure 4 shows PET images of (−)-[11C]OMV in the Vol. 20, No. 6, 2006 Fig. 5 Time-activity curves of the regional brain tissues and the uptake ratio of tissue-to-cerebellum
after intravenous injection of (−)-[11C]OMV (A) and [11C]SA4503 (B) in a conscious monkey using
PET. The radioactivity levels are expressed as the percent of injected dose per ml tissue volume.
monkey brain (images of (−)-[11C]SA4503 not shown24).
the present study is whether (−)-[11C]OMV specifically The uptake of (−)-[11C]OMV was relatively higher in the binds to VAChTs (Ki, 6.7 nM),20 but not to sigma1 striatum. In Figure 5, the time-activity curves of (−)- receptors (Ki for sigma1, 33.7 nM; Ki for sigma2, 266 [11C]OMV and [11C]SA4503 showed different regional nM)20 in vivo.
distribution patterns. (−)-[11C]OMV showed the highest The density of VAChTs was the highest in the striatum uptake in the striatum and lowest uptake in cerebellum, and low in the cerebellum,11,28,29 whereas sigma1 recep- while the regional differences in the uptake of (−)- tors are distributed more uniformly in the brain.30 In a [11C]SA4503 were small. The binding of the two tracers blocking study, (−)-OMV, (−)-vesamicol and two sigma1 was reversible, and an apparent equilibrium state was receptor ligands (SA4503 and haloperidol) having a mod- found at 20–40 minutes for (−)-[11C]OMV and at 5–15 erate affinity (Ki = 50.2 and 41.4 nM, respectively)20 for minutes for [11C]SA4503.
VAChT at the dose of 500 nmol/kg significantly reducedthe brain uptake of (−)-[11C]OMV (60–70% of specific binding), but the same dose of sigma1 receptor ligand, (+)-pentazocine, having a low affinity (Ki = 315 nM)20 for Synthesis of (−)-[11C]OMV was achieved by a palladium- VAChT, did not. Haloperidol was sometimes used to promoted cross-coupling reaction with [11C]methyl evaluate whether the VAChT radioligands bind to sigma iodide.22 (−)-[11C]OMV was prepared in a sufficient receptor.10,12,31 The moderate affinity of haloperidol for radiochemical yield (38 ± 6.9%) for the in vivo studies.
VAChTs (Ki = 41.4 nM)20; however, reasonably blocked The specific radioactivity was relatively low (11 ± 7.0 the binding of the radioligands to VAChTs. Previously, TBq/mmol at 30 minutes after the end of bombardment), we reported that [3H](+)-pentazocine showed a lower compared with the other 11C-labeled tracers prepared in brain uptake and lower specific binding by the modulation our laboratory, such as [11C]SA4503.23 However, this of P-glycoproteins.32 Then, we performed a further block- specific radioactivity is sufficient for the in vivo studies.
ing study by pretreatment with a large excess dose of (±)- The reason for the low specific radioactivity might be pentazocine (50 µmol/kg), where the uptake of (−)- explained by the fact that a cross-coupling between one of [11C]OMV significantly decreased in all of the brain the methyl groups on the tin and the nucleoside part can regions (44–75% of control). A low affinity of (+)- occur yielding (−)-OMV.26 pentazocine (Ki = 315 nM)20 at the dose of 50 µmol/kg Vesamicol and its analogs have been reported to bind to may also block the binding of (−)-[11C]OMV to VAChTs.
VAChTs and sigma receptors.27 The issue addressed in When comparing the blocking effects among the tissues Kazunori Kawamura, Kazuhiro Shiba, Hideo Tsukada, et al Annals of Nuclear Medicine investigated, the uptake of (−)-[11C]OMV in the striatum Aid for Creative Scientific Research of the Japan Society for the was decreased to a lesser extent by the co-injection of Promotion of Science.
SA4503 (44% of control) and haloperidol (61% of con-trol), as well as by the pretreatment with a large excess of (±)-pentazocine (75% of control). Although selectiveligands for VAChT or sigma receptors were not applied to 1. Schafer MK, Weihe E, Varoqui H, Eiden LE, Erickson JD.
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