Development of TASP0410457 (TASP457), a novel dihydroquinolinone derivative as a PET radioligand for central histamine H3 receptors
© Koga et al. 2016
Received: 19 October 2015
Accepted: 30 January 2016
Published: 9 February 2016
Histamine H3 receptor (H3R) is a potential therapeutic target of sleep- and cognition-related disorders. The purpose of the present study is to develop a novel positron emission tomography (PET) ligand for H3Rs from dihydroquinolinone derivatives, which we previously found to have high affinity with these receptors.
Six compounds were selected from a dihydroquinolinone compound library based on structural capability for 11C labeling and binding affinity for H3Rs. Their in vivo kinetics in the rat brain were examined in a comparative manner by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Chemicals with appropriate kinetic properties were then labeled with 11C and evaluated in rats and monkeys using PET.
Of the six compounds, TASP0410457 (also diminutively called TASP457) and TASP0434988 exhibited fast kinetics and relatively high brain uptakes in ex vivo LC-MS/MS and were selected as candidate PET imaging agents. PET data in rat brains were mostly consistent with LC-MS/MS findings, and rat and monkey PET scans demonstrated that [11C]TASP0410457 was superior to [11C]TASP0434988 for high-contrast H3R PET imaging. In the monkey brain PET, distribution volume for [11C]TASP0410457 could be quantified, and receptor occupancy by a nonradioactive compound was measurable using this radioligand. The specific binding of [11C]TASP0410457 to H3Rs was confirmed by autoradiography using rat and monkey brain sections.
We developed [11C]TASP0410457 as a radioligand enabling a robust quantification of H3Rs in all brain regions and demonstrated the utility of ex vivo LC-MS/MS and in vivo PET assays for selecting appropriate imaging tracers. [11C]TASP0410457 will help to examine the implication of H3Rs in neuropsychiatric disorders and to characterize emerging therapeutic agents targeting H3Rs.
Histaminergic neurotransmission in the brain appears to play roles in drinking and feeding, learning and memory, and wake and sleep [1, 2]. Histamine H3 receptor (H3R) acts as either presynaptic autoreceptor or heteroreceptor, producing negative feedback regulation of histamine release or regulating release of non-histamine neurotransmitters including glutamate, norepinephrine, dopamine, and serotonin [3–6]. Therefore, H3R can be a potential therapeutic target for various diseases such as sleep-wake disorders including narcolepsy, attention-deficit hyperactivity disorder, Alzheimer’s disease, and schizophrenia [7–9].
Positron emission tomography (PET) imaging techniques offer analytical approaches for examining neuroreceptor systems. Neuroreceptor ligands applicable to PET imaging could provide tools to investigate alterations in specific neurotransmissions under pathological conditions and to quantify the receptor occupancy by therapeutic agents. However, developing PET ligands for new targets has often been challenging, as an appropriate PET ligand needs to satisfy several criteria including the following properties: sufficient binding affinity and selectivity for the target receptor, adequate lipophilicity and molecular size for blood–brain barrier penetration, minimal interference of radiometabolites with imaging assays, low nonspecific binding, presence of a radiolabeling sites, and sufficiently fast kinetics in the brain .
Two compounds have been used to image central H3Rs in clinical PET studies. GSK189254, a benazepin derivative, is an inverse H3R agonist developed to improve cognitive functions in Alzheimer’s disease  and was labeled with 11C for PET imaging of H3Rs. [11C]GSK189254 could visualize H3Rs in the human brains, but its slow kinetics impeded accurate quantification of the available binding sites in brain regions with high-level H3R expression . MK-8278, a spiro-isobenzofuranone derivative, and an inverse agonist with high potency to H3Rs as well , was also labeled with 11C. [11C]MK-8278 showed more favorable kinetics than [11C]GSK189254 , although it still remains to be established whether the specific binding of this radioligand reaches an equilibrium state within the PET image acquisition time. Although these ligands were used for clinical trials to quantitatively estimate occupancy of H3Rs [11, 13–15], an additional new PET ligand with appropriate pharmacokinetic, metabolic, and binding characteristics can be developed along with a suitable method for quantitative determinations of kinetic parameters.
Ex vivo assays of an exogenously administered compound in tissues by liquid chromatography and tandem mass spectrometry (LC-MS/MS) have been used to evaluate kinetics of compounds in the brain without radiolabeling and to predict the capability of compounds as PET radioligands for several target molecules, such as opioid κ receptor and nociception/orphanin FQ receptor [16–20]. With this method, it is possible to select candidate compounds with preferable in vivo kinetics with a relatively high throughput. The recent advances of the LC-MS/MS device have also offered highly sensitive measurements of compound retaining in the brain following administration at a dose nearly equivalent to PET studies (approximately 2 to 10 μg/kg).
Here, we developed a novel PET ligand for H3Rs from dihydroquinolinone derivatives, which were shown to exert high affinity for H3Rs in our previous work . Six compounds were initially selected from a library of dihydroquinolinone analogs in consideration of the presence of 11C labeling site and binding reactivity with H3Rs. Then, the pharmacokinetics of these candidate compounds in the rat brain were compared by ex vivo LC-MS/MS assays. Based on these kinetic data, two compounds were chosen for labeling with 11C and were evaluated in rats and monkeys using PET. A compound that exhibited more favorable kinetics and higher imaging contrast for H3Rs in the brain was considered as the best radioligand, and its utility in receptor occupancy studies was further demonstrated by PET in monkeys.
In vitro binding of compounds to H3Rs
Rat cerebral cortex and monkey frontal cortex were dissected, homogenized with 50 mM Tris buffer (pH 7.4) containing 5 mM EDTA and Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Indianapolis, IN) and centrifuged at 48,000×g for 15 min at 4 °C. The pellets were then suspended in binding buffer (50 mM Tris containing 5 mM EDTA, pH 7.4) and stored at −80 °C as membrane preparations.
The membrane preparations were thawed and incubated in the binding buffer containing 0.1 % bovine serum albumin, 1 nM [3H]N-α-methylhistamine (PerkinElmer, Waltham, MA) with various concentrations of a dihydroquinolinone derivative (i.e., test compound) at 25 °C for 1 h. The reaction was terminated by rapid filtration through 96-well GF/C filter plates (PerkinElmer) presoaked with 0.5 % polyethyleneimine (Wako, Osaka, Japan). After washes with ice-cold binding buffer, the plates were dried and filled with Microscint-o (PerkinElmer). The radioactivity retained on the filters was counted using a TopCount NXT (PerkinElmer). Total and nonspecific radioligand binding was determined by reaction without and with 10 μM thioperamide and clobenpropit, respectively. The specific radioligand binding was then calculated by subtracting the nonspecific binding from the total binding. The IC50 value for inhibition of the radioligand binding by the test compound was determined using a nonlinear fit of the concentration-response curve for each test compound using SAS 9.2 (SAS Institute Inc., Cary, NC). Data were expressed as the geometric means of IC50 values from three independent experiments run in duplicate.
Other in vitro profiling of compounds
Lipophilicity, off-target binding, and P-glycoprotein-mediated efflux were determined as described in Additional file 1: Supplemental methods.
Ex vivo pharmacokinetic assessments of compounds in the rat brain using LC-MS/MS
The procedures of this animal experiment were reviewed and approved by the Animal Care Committee of Taisho Pharmaceutical Co., Ltd. Six-week-old male Sprague-Dawley rats were purchased from Charles River (Yokohama, Kanagawa, Japan).
Summary of radiosynthesis
Radiochemical yieldab (%)
Average synthesis time (min)
Specific activityac (GBq/mol)
Radiochemical purityc (%)
10.1 ± 3.1
95 ± 23
1.7 ± 0.3
104 ± 7
15.5 ± 4.6
111 ± 16
PET imaging of rat brains
All PET experiments using animals were approved by the Committee for the Care and Use of Laboratory Animals of the National Institute of Radiological Sciences (NIRS).
Six-week-old male Sprague-Dawley rats were purchased from Japan SLC (Shizuoka, Japan). PET scans were performed using a microPET Focus 220 small-animal scanner (Siemens Medical Solutions USA, Knoxville, TN) . The rats were anesthetized with 1.5–2 % isoflurane during the imaging session, while their heart rate and blood oxygen saturation were monitored by pulse oximeter (CANL-425SVA; Med Associates, St. Albans, VT). Body temperature was kept at 37 °C with a heating pad (BWT-100; Bio Research Center, Aichi, Japan). Following the transmission scans for attenuation correction using a 68Ge-68Ga point source, emission scans were performed for 90 min in 3D list-mode with an energy window of 350–750 keV, immediately after the intravenous injection of each radioligand. Mean ± S.D. of the injected radioactivity of [11C]TASP0410457, [11C]TASP0434988, and [11C]TASP0390136 was 119 ± 26 MBq, 132 ± 34 MBq, and 124 ± 17 MBq, respectively (n = 3–5). Mean ± S.D. of the mass doses of [11C]TASP0410457, [11C]TASP0434988, and [11C]TASP0390136 was 2.4 ± 1.2 μg/kg, 1.9 ± 0.4 μg/kg, and 4.2 ± 1.6 μg/kg, respectively (n = 3–5).
To assess the specificity of radioligand binding, thioperamide was injected intravenously at a dose of 10 mg/kg at 30 s before the injection of each radioligand.
Analyses of rat PET data
All list-mode data were sorted into 3D sinograms, which were then Fourier rebinned into 2D sinograms (frames: 1 min × 4, 2 min × 8 and 5 min × 14). Images were reconstructed using 2D-filtered back-projection with a 0.5-mm Hanning filter.
The binding potentials relative to the non-displaceable uptake (BPND) in the striatum and hippocampus were estimated using the simplified reference tissue model (SRTM)  with PMOD software using the cerebellum as a reference region, as the rat cerebellum is devoid of H3R .
PET study in monkeys
A male rhesus monkey (Macaca mulatta, weighing 6–7 kg) was used for a comparative PET analysis of [11C]TASP0410457 and [11C]TASP0434988. PET scans were performed using a high-resolution SHR-7700 animal PET camera (Hamamatsu Photonics, Hamamatsu, Japan). Following transmission scans for attenuation correction using a 68Ge-68Ga point source, emission scans were performed on a conscious monkey as described previously , in 3D acquisition mode (frames: 10 s × 12, 30 s × 6, 1 min × 5, 2 min × 5 and 5 min × 14) for 90 min after the intravenous injection of [11C]TASP0410457 (169–176 MBq) or [11C]TASP0434988 (173–188 MBq). Briefly, the monkey was immobilized by joining the acrylic cap on the monkey head with the fixation device during PET scans. To assess a homologous competition for the target binding sites, unlabeled TASP0410457 or TASP0434988 was injected intravenously at a dose of 1 mg/kg at 30 s before injection of the corresponding radioligand.
For a detailed pharmacokinetic PET study using [11C]TASP0410457 with arterial blood sampling, three male rhesus monkeys weighing 5–7 kg were scanned with the SHR-7700 PET camera. The monkeys were initially anesthetized with an intramuscular injection of ketamine (10 mg/kg), intubated, and subsequently kept anesthetized with 1–2 % isoflurane. Electrocardiogram and blood oxygen saturation levels were monitored, and body temperature was maintained by a heating pad throughout the experiment. Arterial blood (1–2.5 mL) was collected from the saphenous artery at 10, 20, 30, 40, 50, 60, and 90 s and at 2, 3, 4, 5, 10, 15, 30, 60, and 90 min after the intravenous injection of [11C]TASP0410457 (injected radioactivity, 159 ± 30 MBq; mass dose, 1.2 ± 0.3 μg; mean ± S.D., n = 3).
The arterial blood was centrifuged at 20,000×g for 3 min at 4 °C to separate the plasma. Radioactivity in total blood and plasma was determined using a 1480 WIZARD gamma counter (PerkinElmer), and was decay-corrected to the injection time. Arterial plasma collected at 1, 5, 15, 30, 60, and 90 min was used to analyze radioactive metabolites of [11C]TASP0410457. Two hundred microliters of plasma was mixed with 200 μL of ice-cold acetonitrile, and the mixtures were centrifuged at 20,000×g for 3 min at 4 °C. Fifty to five hundred microliters of the resultant supernatants were separated using HPLC (JASCO, Tokyo, Japan) on an Atlantis T3 column (4.6 × 50 mm, Waters, Milford, MA) with a mobile phase consisting of 10 mM of ammonium acetate/acetonitrile (68:32) at a flow rate of 1 mL/min. The fraction of unmetabolized [11C]TASP0410457 was determined by measuring the areas of the HPLC peaks corresponding to [11C]TASP0410457 and its radiometabolites.
Analyses of monkey PET data
Emission scan images were reconstructed using a 4.0-mm Colsher filter. ROIs were placed on the anterior cingulate cortex, striatum, thalamus, hippocampus, cerebellum, frontal cortex, temporal cortex, occipital cortex, and pons-medulla using PMOD software, with reference to an MRI template as described previously . Tissue TACs were generated as in the rat PET image analysis.
For PET study in anesthetized monkeys, metabolite-corrected plasma TACs were used as arterial input functions. The regional total volume of distribution (V T) was estimated by Logan’s graphical plot and one-tissue compartment model using PMOD software.
Receptor occupancy assays
Autoradiography of [11C]TASP0410457 using rat and monkey brain sections
Twenty-micrometer-thick sagittal rat and monkey brain sections were incubated in an autoradiography buffer containing 5 nM [11C]TASP0410457 as described in Additional file 1: Supplemental methods. The binding was also performed in the presence of 10 μM of unlabeled TASP0410457, thioperamide, or ciproxifan for the rat sections and 10 μM of TASP0410457 or ciproxifan for the monkey sections to assess the specific radioligand binding.
Selection of candidate compounds for PET imaging
TASP0410457 and TASP0434988 were selected as candidates for a PET radioligand, because they exhibited rapid entry into the brain followed by fast washout (Fig. 2a, b) and the highest and second highest uptakes, respectively, in the forebrain versus the cerebellum. TASP0390136 was also chosen for radiolabeling as a control compound with insufficient brain uptake in order to assess the consistency between ex vivo LC-MS/MS and in vivo PET data.
In vitro properties of TASP0390136, TASP0410457, and TASP0434988
Affinity for H3Rs
Efflux ratio (MDR1)
Sigma 1, IC50
Adrenergic α2c, IC50
nM (n = 3)
nM (n = 3)
PET imaging of the rat brain with [11C]TASP0410457 and [11C]TASP0434988 versus [11C]TASP0390136
Regional BPND values for radioligands in rat PET
1.26 ± 0.12
0.53 ± 0.06
0.92 ± 0.21
0.36 ± 0.11
The regional brain kinetics of the three compounds exhibited by PET was mostly consistent with the ex vivo LC-MS/MS results. The ranked order of peak radioligand uptakes in the striatum agreed with the order in the forebrain determined by LC-MS/MS (TASP0410457 > TASP0434988 > TASP0390136; Fig. 2a–c versus Fig. 4a–c). Meanwhile, the cerebellar peak radioligand uptakes assayed by PET but not ex vivo LC-MS/MS were inversely correlated with the ER values of the compounds. As a consequence, the LC-MS/MS analysis seemed to overestimate the specific binding (difference in SUV between the target reference regions) of TASP0434988 as compared with the quantification by PET.
Comparative PET imaging of monkey brains with [11C]TASP0410457 and [11C]TASP0434988
Quantitative analyses of [11C]TASP0410457-PET data in monkeys
Time-stability of regional V T values for [11C]TASP0410457 in the monkey brain
Logan graphical analysis
One-tissue compartment model
Scan duration (min)
Scan duration (min)
21.2 ± 3.0
21.5 ± 3.0
21.9 ± 3.3
22.6 ± 3.8
23.2 ± 3.6
Anterior cingulate cortex
18.4 ± 2.7
18.6 ± 2.8
19.1 ± 3.2
19.5 ± 3.4
20.3 ± 3.3
15.1 ± 1.7
15.4 ± 1.9
15.6 ± 1.9
16.0 ± 2.1
16.1 ± 1.8
13.0 ± 1.0
13.2 ± 1.0
13.6 ± 1.2
13.9 ± 1.4
13.9 ± 1.3
12.3 ± 1.6
12.5 ± 1.7
12.8 ± 1.8
13.0 ± 2.0
13.4 ± 1.8
12.1 ± 1.2
12.4 ± 1.3
12.7 ± 1.5
13.0 ± 1.7
12.8 ± 1.7
12.3 ± 1.6
12.4 ± 1.7
12.5 ± 1.8
12.9 ± 2.1
13.3 ± 2.1
11.7 ± 1.6
11.8 ± 1.6
12.0 ± 1.8
12.1 ± 1.9
12.7 ± 2.0
8.5 ± 0.6
8.6 ± 0.7
8.8 ± 0.7
9.1 ± 0.9
8.8 ± 0.9
Pretreatment by intravenous administration of an H3R antagonist, ciproxifan, at a dose of 3 mg/kg substantially reduced the radioactivity in all ROIs including the cerebellum and pons-medulla. Lassen’s plot displayed robust linearity (Fig. 6e), demonstrating uniform receptor occupancy among brain regions, and the receptor occupancy of ciproxifan at this dose was stably estimated as ~75 %. Non-displaceable distribution volume was estimated as 4.0 mL/cm3 (Fig. 6e), and thus, the specific radioligand binding accounted for more than 80 % of the radioactivity in the striatum, taking this result together with the finding that the striatal V T exceeded 20 mL/cm3 (Table 4). We also treated a monkey with 10 mg/kg of ciproxifan but could not complete PET imaging due to its profound effects on heart rates and body motions even in an anesthetized condition with isoflurane.
Autoradiography of [11C]TASP0410457
The present work provides a clear demonstration that a new PET radioligand with adequate binding and kinetic properties suitable for high-contrast and quantitative PET assays of central H3Rs can be generated from dihydroquinolinone derivatives. Screening of non-radiolabeled chemicals was carried out by the conjunctive use of information from in vitro assays and ex vivo LC-MS/MS measurements, resulting in the identification of two candidate compounds. Subsequent in vivo PET analyses of rat and monkey brains indicated the utility of these two ligands as imaging agents and revealed that the pyridinated compound [11C]TASP0410457 yielded a higher signal-to-background ratio, reflecting the abundance H3Rs, and good linearity of Lassen’s graphical plot across all examined brain regions, offering accurate quantitative measures of the receptor density at baseline and under occupancy by a drug with a sufficient dynamic range. Supported by these promising non-clinical items of evidence, [11C]TASP0410457 (a diminutive name, [11C]TASP457 is used in clinical works) has been applied to a human PET study and has been demonstrated to permit robust quantification of H3Rs in diverse brain regions (Kimura et al., manuscript submitted to Eur J Nucl Med Mol Imaging). These clinical data accordingly indicate the validity of our translational workflow for the identification of an appropriate H3R radioligand.
We employed ex vivo LC-MS/MS to select candidate compounds for PET imaging, because this technique has been successfully applied to label-free evaluations of potential PET ligands for opioid κ receptor and nociception/orphanin FQ receptor [16, 18]. Our present data also verified the capability of LC-MS/MS measurements in predicting in vivo performances of the H3R ligands when employed for PET examinations. Indeed, the relative order of ligand uptake in the rat forebrain determined by LC-MS/MS was overall in good agreement with the observations in rat PET. In light of the present and previous [16–20] demonstrations, LC-MS/MS quantifications of ex vivo samples can serve for capturing kinetic profiles of probable microdose imaging compounds with a reasonably high throughput. Meanwhile, a noticeable discrepancy between ex vivo and in vivo measures was also found in a small subset of data, including the peak cerebellar uptake of TASP0434988. In addition, ex vivo LC-MS/MS failed to determine concentrations of the compounds at 60 and 120 min after administration, as these values did not exceed the low limit of quantification of MS (0.15 ng/g tissue). These issues may be attributed to the sensitivity of the detection device and sample preparation for LC-MS/MS, which will need further improvements and optimizations.
It should also be considered that a radiometabolite of [11C]TASP0410457 in plasma gradually entered the CNS and raised PET signals in the brain while not affecting LC-MS/MS detection of the parent compound. We performed a preliminary analysis of metabolites in the rat brain after injection of non-radiolabeled TASP0410457 and TASP0434988 using an HPLC and ion trapping MS system and found that the sole detectable metabolite was generated by desmethylation of each parent compound at the 11C labeling site. Accordingly, it is likely that the major metabolic pathway leads to the production of a small, polar radiometabolite, such as [11C]formaldehyde, which is in accord with our HPLC analysis of plasma radiometabolites in monkeys. Moreover, our pilot assays in mice have suggested that this metabolite accounts for approximately 50 % of radioactivity in the brain at 30 min after intravenous injection of [11C]TASP0410457 (K. Kawamura, personal communication, 2015). In view of these factors potentially causing differences between LC-MS/MS and PET data, a practical workflow for the identification of the best PET ligand from an array of chemicals may be to select a few compounds exhibiting qualified kinetic properties in label-free ex vivo assays, followed by the determination of a radiolabeled ligand with the highest capability in rodent and nonhuman primate PET imaging, as conducted in the present work.
Our current results imply the applicability of [11C]TASP0410457 to investigations of H3Rs and evaluations of drugs acting on these receptors in the living human brain, which has been evidenced in clinical PET analyses (Kimura et al., manuscript submitted to Eur J Nucl Med Mol Imaging). Two radioligands, [11C]GSK189254 and [11C]MK-8278, have been non-clinically and clinically applied to quantify central H3Rs. Although [11C]GSK189254 exhibited sufficient brain uptakes, the kinetics of this radioligand exhibited a continuous increase over 90 min, hampering the estimation of regional V T in the baboon and human striatum [11, 14, 29]. [11C]MK-8278 displayed more preferable kinetics (a rapid increase followed by a gradual decline in PET imaging time) than [11C]GSK189254, and but its time-stability data of V T in the human brain showed a gradual increase in V T over time . The present study indicated that V T of [11C]TASP0410457 in the monkey brain was relatively stable over time but increased by 7 % at maximum from 60 to 90 min, and this is presumably due to gradually accumulating radiometabolites in the brain but not stemming from slow kinetics of the radioligand binding. Hence, dynamic PET scans acquired for 60 min provide adequate quantitative measures of the radioligand kinetics in the monkey brain. As mentioned above, the major metabolite accounted for approximately 50 % of total radioactivity in the mouse brain at 30 min after radioligand injection (K. Kawamura, personal communication, 2015). In light of the finding that 86 and 53 % of total plasma activity in mice and monkeys, respectively, were derived from the radiometabolite at 30 min, the entry of the radiometabolite in the monkey brain should be less prominent than that in the mouse brain. Influences of the radiometabolite on V T values in the human brain would be even less profound, because only 13 % of total plasma radioactivity at 30 min in humans arose from a metabolite identical to the molecule detected in animals (Kimura et al., manuscript submitted to Eur J Nucl Med Mol Imaging). Correspondingly, our clinical PET results showed smaller increase of V T values along with extension of dynamic scan time from 60 to 90 min than the changes in a monkey (Kimura et al., manuscript submitted to Eur J Nucl Med Mol Imaging).
It is also noteworthy that pharmacokinetic evaluations of H3R ligands were quantitatively performable in rats using the cerebellum as a reference region lacking the target receptor. By contrast, the use of the cerebellum as a reference region to quantify H3R in the human and nonhuman primate brains remains to be revisited. A clinical PET study [11C]GSK189254 indicated that V T values for this radioligand were markedly reduced in all ROIs including the cerebellum and pons by treatment with an H3R ligand, AZD5213, and it is accordingly likely that no reference region can be defined in the human brain . However, PET imaging of the monkey and human brains with [11C]MK-8278 employed the pons as a reference region to estimate the occupancy of H3Rs by an antagonist/inverse agonist, MK-0249 [13, 30]. Our present data demonstrated the presence of displaceable binding sites for [11C]TASP0410457 and [11C]TASP0434988 in the monkey cerebellum and brainstem including the pons, similar to the previous reports on [11C]GSK189254 [11, 14, 29]. This finding was also consistent with the presence of specific binding components for H3R ligands in the monkey cerebellum demonstrated previously [30, 31]. Likewise, reference tissue models may not be available for describing the kinetics of [11C]TASP0410457 in the human brain, and thereby Lassen’s plot of V T values estimated with an arterial input function will be required for clinical assays of the H3R occupancy by a drug.
We developed a radioligand, [11C]TASP0410457, for a robust quantification of H3Rs in all brain regions and demonstrated the utility of ex vivo LC-MS/MS and in vivo PET assays for selecting appropriate imaging tracers from candidate compounds. In light of the current data, [11C]TASP0410457 will facilitate clarification of roles played by H3Rs in neuropsychiatric disorders and characterizations of emerging therapeutic agents acting on these receptors.
The animal experiments for ex vivo LC-MS/MS in rats were approved by the Animal Care Committee of Taisho Pharmaceutical Co., Ltd. All PET experiments using rats and monkeys were approved by the Committee for the Care and Use of Laboratory Animals of NIRS. All procedures performed in the studies were in accordance with the ethical standards of both institutions where the studies were conducted.
The authors thank Mr. T. Minamihisamatsu for his technical assistance and the staff of the Molecular Probe Group, NIRS, for support with the radiosynthesis. This study was supported in part by Grants-in-Aid for Japan Advanced Molecular Imaging Program, and Scientific Research on Innovative Areas (“Brain Environment”) (23111009) (to M.H.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
This study was supported in part by Grants-in-Aid for Japan Advanced Molecular Imaging Program, and Scientific Research on Innovative Areas (“Brain Environment”) (23111009) (to M.H.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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