Initial evaluation of C-preladenant 1
(Journal of Nuclear Medicine)
Title: Initial Evaluation of a Novel Adenosine A2A Receptor Ligand,
11C-Preladenant, in Healthy Human Subjects
Authors: Muneyuki Sakata 1, Kenji Ishibashi 1, Masamichi Imai 2, Kei
Wagatsuma 1, Kenji Ishii 1, Xiaoyun Zhou 3, Erik F.J. de Vries 3, Philip H. Elsinga
3, Kiichi Ishiwata 1, 4, 5, Jun Toyohara 1
Affiliations: 1 Research Team for Neuroimaging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; 2 Department of Radiology, Toranomon Hospital, Tokyo Japan; 3 Department of Nuclear Medicine and Molecular Imaging, University Medical Center of Groningen, University of Groningen, Groningen, The Netherlands; 4 Institute of Cyclotron and Drug Discovery Research, Southern Tohoku Research Institute for Neuroscience, Koriyama, Japan; 5 Department of Biofunctional Imaging, Fukushima Medical University, Fukushima, Japan
First author: Muneyuki Sakata, Ph.D.
Research Team for Neuroimaging, Tokyo Metropolitan Institute of Gerontology 35-2 Sakae-cho, Itabashi-ku, 173-0015 Tokyo, Japan
Phone: +81-3-3964-3241; Fax: +81-3-3964-1148 E-mail: sakata@pet.tmig.or.jp
Address correspondence to: Jun Toyohara, Ph.D.
Research Team for Neuroimaging, Tokyo Metropolitan Institute of Gerontology 35-2 Sakae-cho, Itabashi-ku, 173-0015 Tokyo, Japan
Phone: +81-3-3964-3241; Fax: +81-3-3964-1148 E-mail: toyohara@pet.tmig.or.jp
ORIGINAL ARTICLE: 5642 words (all data).
Financial disclosure
No potential conflict of interest relevant to this article was reported.
Clinical trial registration number: UMIN000019999
Abbreviated title: Initial evaluation of 11C-preladenant
ABSTRACT
11C-Preladenant is a novel selective antagonist for mapping of cerebral adenosine A2A receptors (A2ARs) by positron emission tomography (PET). This is a
first-in-human study to examine the safety, radiation dosimetry, and brain imaging of 11C-preladenant in healthy human subjects. Methods: Dynamic
11C-preladenant PET scans (90 min) were performed in 5 healthy male subjects.
During the scan, arterial blood was sampled at various time intervals, and the fraction of the parent compound in plasma was determined. For anatomic coregistration, T1-weighted magnetic resonance imaging was performed. The total distribution volume (VT) was estimated using one- and two-tissue
compartment models (1T and 2T, respectively). Distribution volume ratio (DVR) was calculated from VT of target and reference region, and obtained with a non-invasive Logan graphical reference tissue method (LGRM) (t* = 30 min).
The applicability of a shortened protocol as an alternative to the 90 min PET scan was investigated. Tracer biodistribution and dosimetry were determined in 3 healthy male subjects, using serial whole-body PET scan acquired over 2 h post
11C-preladenant injection. Results: There were no serious adverse events in any of the subjects throughout the study period. 11C-Preladenat readily entered the brain, with a peak uptake in the putamen and head of the caudate nucleus 30−40 min after tracer injection. Other brain regions showed rapid clearance of radioactivity.
The regional distribution of 11C-preladenant was consistent with known A2AR
densities in the brain. At pseudoequilibrium (reached at 40 min after injection), stable target-to-cerebellar cortex ratios of around 3.8−10.0 were obtained. The 2T fit better than the 1T in the low-density A2AR regions. In contrast, there were no significant differences between 1T and 2T in the high A2AR density regions.
DVRs in putamen and head of the caudate nucleus were around 3.8−10.3 when estimated using a LGRM with cerebellum as the reference region. Fifty or 70 min PET scan is capable of providing the stable DVR estimates within 10 % or 5 % differences at most, respectively. The radioactivity was mainly excreted through the hepatobiliary system after 11C-preladenant injection. As a result, the absorbed dose (μGy/MBq) was highest in the gallbladder wall (mean ± standard deviation (SD), 17.0 ± 2.5) and liver (11.7 ± 2.1). The estimated effective dose for
11C-preladenant was 3.7 ± 0.4 μSv/MBq. Conclusion: This initial evaluation indicated that 11C-preladenat is suitable for imaging of A2ARs in the brain.
Word count: 390 words
Key Words: Adenosine A2A receptor; 11C-Preladenant; Radiation dosimetry;
Human brain; Positron emission tomography
INTRODUCTION
Adenosine acts as an endogenous modulator of synaptic function through adenosine receptors (ARs) in the central nervous system (1,2). ARs are members of the G-protein-coupled superfamily (3) and are divided into at least four subtypes: A1, A2A, A2B, and A3 (4–6). Postmortem studies showed that A2ARs are abundant in the putamen, caudate nucleus, nucleus accumbens, and globus pallidus pars lateralis (7). A2ARs are known to stimulate adenylate cyclase and interact negatively with dopamine D2 receptors at the level of second messengers and beyond (8). A2ARs also have a key role in adenosine-mediated sleep-promoting effects (9). In addition, A2ARs are involved in many neuropsychiatric disorders such as Parkinson's disease, Huntington's disease, Alzheimer's disease, drug
addiction, alcohol abuse, epilepsy seizures, sleep disorders, and schizophrenia (10). Notably, selective A2AR antagonists have been tested for non-dopaminergic treatment of Parkinson's disease (11), resulting in the approval of istradefylline as an anti-parkinsonian agent in Japan in 2013 (12).
Non-invasive in vivo imaging of A2ARs with PET represents a potentially useful method for monitoring changes in A2AR density during the disease course, and the assessment of receptor occupancy of investigational drugs. Several compounds have been tested as PET tracers for A2AR imaging in human subjects (13−16). Among them,
7-(2-(4-(4-(2-18F-fluoroethoxy)phenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-pyrazolo[4,3 -e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (18F-MNI-444) seems to have the best properties as an A2AR imaging agent so far, with highest target-to-nontarget ratios
(putamen-to-cerebellum ratio at 60−90 min: 5.0) (16). 18F-MNI-444 is the 18F-fluoroethoxy derivative of
2-(furan-2-yl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin-1-yl)ethyl)-7H-pyrazolo[4,3- e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (preladenant), which has already been tested in
phase III clinical trials as a potential treatment for Parkinson's disease.
Recently, Zhou et al. developed 11C-preladenant for use as a PET tracer and showed a high striatum-to-cerebral ratio in rats by PET imaging (17). In vivo blocking and A2AR quantification in rat PET studies showed that 11C-preladenant is a suitable tracer to quantify striatal A2AR density and assess A2AR occupancy by istradefylline (18). The toxicological profile of preladenant in humans is already known from clinical phase I/II/III studies, in which preladenant was investigated as a drug for the treatment of Parkinson's disease (19). We conducted an additional preclinical safety and radiation dosimetry study of
11C-preladenant (Supplemental Materials, Supplemental Tables 1-3) (supplemental materials are available online only at http://jnm.snmjournals.org). The radiation-absorbed dose estimated from mouse distribution data was highest in the small intestine, but the effective dose was somewhat lower than that obtained from rat data (20). Nevertheless, the effective dose was similar in magnitude to most other 11C-labelled PET tracers (21). The absence of any abnormalities in rats in the acute toxicity test of 11C-preladenant injections demonstrated the clinical suitability of 11C-preladenant for use in PET studies in humans.
These findings prompted us to undertake an initial evaluation of 11C-preladenant in human subjects as a phase 1 study. Here, we report the first-in human study to examine the safety, radiation dosimetry, and brain imaging of 11C-preladenant in healthy human subjects.
MATERIALS AND METHODS Subjects
All experiments were approved by the Tokyo Metropolitan Institute of
Gerontology institutional review board and were performed in accordance with the institutional review board rules and policies. All subjects gave study-specific informed consent to participate in the study and all experiments were carried out in accordance with the relevant guidelines. The study was registered in UMIN-CTR (UMIN000019999) on November 30th, 2015. Eight healthy male volunteers, aged 21−25 y (mean age ± SD, 23 ± 1 y), were enrolled in this study (Supplemental Table 4). Although there were no
restrictions of diet, all subjects were required to refrain from drinking caffeinated beverages up to 12 h before PET imaging. Five of the 8 subjects were recruited into the safety
monitoring and dynamic brain PET study. The subjects weighed 61.0−72.2 kg (mean weight ± SD, 63.7 ± 5.9 kg). For anatomical co-registration, magnetic resonance imaging was performed using a GE Discovery MR750w 3.0T scanner (GE Healthcare, Wauwatosa, WI). A 3-dimensional (3D) fast spoiled gradient-echo (repetition time = 7.6 ms, echo time = 3.1 ms, inversion time = 400 ms, matrix = 256 × 256 × 196 voxels) T1-weighted
whole-brain image was acquired for each subject. The other 3 subjects participated in the whole-body PET distribution study. The subjects weighed 62.5−86.1 kg (mean weight ± SD, 71.5 ± 12.8 kg). All subjects were free of somatic and neuropsychiatric illnesses, according to their medical history and the findings of physical examination, and had no brain
abnormalities on magnetic resonance imaging.
Preparation of 11C-Preladenant
11C-Preladenant was prepared by O-methylation of the corresponding desmethyl precursor using 11C-methyl iodide in the presence of potassium hydroxide according to a previously described procedure (17) with slight modification. Details on the method, results, and quality control data of 11C-preladenant preparations are provided in the Supplemental
Materials.
Safety Monitoring
Safety data were collected after administration of 11C-preladenant and throughout the follow-up period of 1 week in 5 subjects. Safety monitoring included the recording of adverse events, changes in vital signs, physical examination, electrocardiogram, and laboratory parameters (serum biochemistry and hematology analyses). The detailed protocol for investigating safety monitoring was the same as that reported previously (22).
Brain PET Scanning
PET scanning was performed using a Discovery PET/computed tomography (CT) 710 scanner (GE Healthcare) in 3D mode. This scanner has an axial field of view of 15.7 cm, a spatial resolution of 4.5 mm full width at half maximum, and a Z-axis resolution of 4.8 mm full width at half maximum. We acquired 47 slices for each 3D reconstruction.
After low-dose CT scanning to correct for attenuation, 11C-preladenant (508 − 691 MBq/9.3
− 24.3 nmol) was injected in the antecubital vein of each subject as a bolus for 1 min, and a 90-min dynamic scan in 3D mode (20 s × 3 frames, 30 s × 3 frames, 60 s × 5 frames, 150 s
× 5 frames, and 300 s × 14 frames) was performed. Arterial blood (0.5 mL each) was sampled from the catheter that has been placed in the radial artery at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 135, 150, and 180 s, as well as at 5, 7, 10, 15, 20, 30, 40, 50, 60, 75, and 90 min. The whole blood and separated plasma were weighed, and radioactivity was measured with a NaI (Tl) well scintillation counter (BeWell Model-QS03 F/B;
Molecular Imaging Labo, Suita, Japan). To analyze the labeled metabolites, an additional 1.5 mL of blood was obtained at 3, 10, 20, 30, 40, and 60 min. Plasma protein binding free fraction could not be measured due to the undesirable adsorption of 11C-preladenant to the
ultrafiltration membrane. After the PET scan, urine was obtained from each subject and radioactivity was measured. Unaltered 11C-preladenant and radioactive metabolites in the plasma and urine were determined with high-performance liquid chromatography (HPLC).
Tomographic images were reconstructed using a 3D ordered subset expectation maximization algorithm (subset, 16; iteration, 4) with incorporated time of flight
information. The dynamic images were post-smoothed with a 4-mm full width at half maximum Gaussian filter. The data were reconstructed in a 128 128 47 matrix, and the voxel size was 2 2 3.27 mm. Partially overlapping circular regions of interest (ROIs) that were 10 mm in diameter were placed on the frontal and cerebellar cortices, thalamus, putamen, and head of the caudate nucleus with reference to the co-registered magnetic resonance imaging (Fig. 1C). Time-activity curves (TACs) for these ROIs were calculated as Bq/mL or as standardized uptake value (SUV): (activity/mL tissue)/(injected
activity/body weight). Using the TACs of tissues and the metabolite-corrected TAC of plasma, the VT for 11C-preladenant was evaluated using the 1T (K1/k2) and 2T (K1/k2 × (1 k3/k4)). The goodness of fit by the 2 model analyses was evaluated using Akaike's
information criterion. DVR was calculated as the ratio of VT in the target and reference regions and estimated with LGRM (t* = 30 min) (23). Cerebellum was used as a candidate of the reference region. Finally, using the DVRs from LGRM, the applicability of shortened protocols (45, 50, 55, 60, 65, 70, 75, 80, and 85 min) as alternatives to the standard 90 min PET scan for stable DVR quantification was investigated.
Metabolite Analysis
The parent and metabolites of 11C-preladenant in the plasma sampled at 3, 10, 20,
30, 40, and 60 min and urine recovered at 97−101 min were analyzed by HPLC. The blood was centrifuged at 8700 × g for 40 s at 2 °C to obtain the plasma, which was denatured with an equivalent volume of 100% acetonitrile (final concentration: 50% acetonitrile) in an ice-water bath. The suspension was centrifuged using the same conditions and divided into soluble and precipitated fractions. The precipitate was resuspended in the same volume of 100% acetonitrile followed by centrifugation. This procedure was repeated twice.
Radioactivity in the three soluble fractions and precipitates was measured with an
auto-gamma counter (LKB Wallac, Turku, Finland). In this treatment of plasma, < 4% of the total radioactivity was left in the final precipitates. The soluble fractions were combined and analyzed by HPLC with a radioactivity detector (FLO-ONE 150TR; Packard
Instrument, Meriden, CT). A YMC-Pack ODS-A column (10-mm i.d. × 250-mm length;
YMC, Kyoto, Japan) was used with acetonitrile/50 mM aqueous acetic acid/50 mM
aqueous sodium acetate, pH 4.5 (80/10/10, v/v/v) at a flow rate of 4 mL/min. The retention time of 11C-preladenant was 8.0 min. The urine sample was directly applied to HPLC.
Whole-Body Imaging
The protocol for investigating radiation dosimetry in human subjects using
whole-body imaging was essentially the same as that reported previously (24). Whole-body PET/CT scans were obtained using a Discovery 710 PET/CT scanner in 3D mode.
Low-dose CT was used for attenuation correction of the PET emission scan. The first PET acquisition was started 1 min after the intravenous bolus injection of 751 ± 36 MBq (7.1−11.3 nmol) of 11C-preladenant. After the initial whole-body scan, 18 bed position scans (overlap of 23 of 47 slices per bed position, 15 s/bed position × 4 frames, 30 s/bed position × 12 frames, and 60 s/bed position × 2 frames) from the top of the head to
mid-thigh were performed. Images were reconstructed using 3D ordered subset expectation maximization algorithm (subset, 24; iteration, 2) with a 6.4 mm Gaussian filter.
ROIs were manually placed over 15 organs that could be identified from PET or Low-dose CT: adrenals, brain, gallbladder, small intestine, stomach, heart wall, kidneys, liver, lungs, pancreas, bone marrow (thoracic and lumbar vertebrae), spleen, thymus, thyroid, and urinary bladder. The decay-uncorrected and decay-corrected TACs of organs were calculated as the percentage injected dose per organ. Then, the normalized number of disintegrations (MBq-h/MBq administered) for each source organ was calculated, which is equal to the area under the time course curve multiplied by the volume of the organ ROI.
The volume of bone marrow, in which only part of the organ could be measured, was substituted by the volume, which was calculated from the mass of red marrow in the adult male phantom (1.12 kg for 73.7 kg of body weight) adjusted by the subject’s body weight and 1 g/mL as the specific gravity (25). The area under the time course curve was
calculated by summing the area from time 0 to the endpoint and the area from the endpoint to infinity of the uncorrected TACs. The former area was calculated by trapezoidal
integration. The latter area was calculated by integration of radioactive decay from the endpoint.
The absorbed doses in 25 target organs of the adult male phantom was estimated from the normalized number of disintegrations of source organs by implementing the Medical Internal Radiation Dose method using OLINDA/EXM (Vanderbilt University, Nashville, TN) (26). The effective dose was also calculated by OLINDA/EXM using the methodology described in International Commission on Radiological Protection
Publication 60 (27).
RESULTS
Safety Monitoring
The mean ± SD of the administered mass of 11C-preladenant was 6.5 ± 3.3 μg (range, 3.6–12.2 μg). The mean ± SD of administered activity was 677 ± 86 MBq (range, 508−786 MBq). The administration of 11C-preladenant was well tolerated by all subjects.
There were no adverse or clinically detectable pharmacologic effects in any of the 5 subjects. No clinically important trends indicative of a safety signal were noted for laboratory parameters, vital signs, or electrocardiogram parameters.
Brain PET Scanning
Figure 1 shows representative static 11C-preladenant images (Fig. 1A), parametric DVR images (Fig. 1B) and magnetic resonance images (Fig. 1C) of the corresponding slices. Figure 2A shows the mean TACs in 5 brain regions of healthy subjects (n = 5) after intravenous injection of 11C-preladenant. The radioactivity levels were high in the putamen and head of the caudate nucleus, regions known to contain high densities of A2AR, with a peak uptake in the putamen of 3.2−6.6 SUV at around 40–60 min after injection. The caudate showed a lower peak uptake of 2.3−4.9 SUV at around 20–50 min after injection.
Radioactivity levels in the other 3 brain regions known to contain low densities of A2AR peaked within 2 min and rapidly washed out, followed by steady baseline levels until the end of the scan.
The preliminary kinetic analysis revealed that the 2T provided significantly lower Akaike's information criterion scores than the 1T (paired t test, P < 0.05) in brain regions with low A2AR densities (i.e., frontal and cerebellar cortices and thalamus), and that Akaike's information criterion scores did not show any significant differences in regions
with high A2AR density. The average SUVr (SUV ratio to the reference region) graph showed pseudoequilibrium from about 40–60 min after injection onward, with target-to-reference region ratios of 3.8–10 (Figure 2B).
The VT obtained with 1T and 2T, and DVR calculated from VT with 2T, and obtained with LGRM are summarized in Table 1. The highest VT values were measured in the putamen and head of the caudate nucleus. The VT values of the frontal cortex and thalamus were close to those in the reference candidate region (cerebellar cortex). The SD of DVR calculated from VT with 2T was larger than that of DVR obtained with LGRM. If excluding an outlier (DVR = 13.1) of 2T estimates occurred in the head of the caudate nucleus, DVR calculated from VT with 2T, and obtained with LGRM were matched with each other well (DVR(LGRM) = 0.95 × DVR(2T) + 0.05, R2 = 0.96). Figure 3 shows the
impact of the shortened scan protocol in the estimates of DVR with LGRM. Fifty or 70 min PET scan of a shortened protocol as an alternative to the standard 90 min scan is capable of providing the stable DVR estimates within 10 % or 5 % differences at most, respectively.
Metabolite Analysis
Plasma radioactivity rapidly decreased after bolus injection (Fig. 4A). The radioactivity concentration in plasma was higher than in whole blood (Fig. 4B). The results of the HPLC analysis of plasma are summarized in Table 2.
Three hydrophilic metabolites (HM1; 3.2 min, HM2; 4.8 min, and HM3; 6.2 min) and a lipophilic metabolite (LM1; 9.9 min) were detected in plasma. At 60 min after injection, intact 11C-preladenant remained dominant (77.8% ± 7.2%, n = 5). The mean radioactivity voided into urine at 111 ± 17 min (range, 97−132; n = 8) was 1.2% ± 0.2% of
injected activity (range, 1.0−1.5%, n = 8). In urine, hydrophilic metabolite HM2 was dominant (75.4% ± 2.7%, n = 3) and a small amount of the parent radioligand was observed (3.0% ± 3.4%, n = 3).
Whole-body Imaging
A representative whole-body distribution of 11C-preladenant is shown in Figure 5, and decay-corrected TACs of source organs for the same subject are shown in Figure 6. In the first frame (1−5 min) of Figure 5, the liver showed the highest uptake of radioactivity, which gradually decreased up to 90 min and remained stable thereafter. The radioactivity level of liver remained the highest in the last frame. The gallbladder was clearly visible and its radioactivity was secreted into the small intestine, illustrating the hepatobiliary excretion of 11C radioactivity. The urinary bladder showed a gradual increase in radioactivity, and was visible after the middle frame, indicating some urinary excretion of 11C radioactivity.
The mean and SD of radioactivity voided into urine at 132 ± 1 min (n = 3) was 1.3% ± 0.2% (n = 3) of injected activity.
The normalized number of disintegrations is shown in Supplemental Table 5, and the organ absorbed and effective doses are shown in Table 3. The highest absorbed dose was observed in the gallbladder wall, followed by the liver, heart wall, small intestine, stomach wall, and pancreas. The mean and SD of the estimated effective dose are 3.7 ± 0.4 μSv/MBq.
DISCUSSION
This is the first clinical study to assess the safety, radiation dosimetry, and initial brain imaging of 11C-preladenant in a small population of healthy human subjects.
We determined 11C-preladenant to be safe and well tolerated, with no adverse
effects in the 5 subjects included in this study. The radiation-absorbed dose was higher in the gallbladder wall, liver, heart wall, small intestine, stomach wall, pancreas, and kidneys than in the other organs studied, but was nonetheless sufficiently low for clinical use. The individual organ and total-body doses of 11C-preladenant were comparable with other
11C-labeled tracers (28,29).
The regional distribution of 11C-preladenant is consistent with the A2AR density in the healthy human brain. The localization of A2AR density in the normal human brain has been demonstrated by in vitro autoradiographic studies with 3H-CGS21680 and
3H-SCH58261 (7). The highest signal level of A2ARs was observed in the basal ganglia, followed by cerebral cortex and thalamus. The estimated Bmax of A2AR in the putamen and caudate were 7 and 6 nM, respectively, which is sufficiently high for visualization by radioligands with affinity of preladenant (Ki = 1.1 nM). In contrast, A2AR expression levels in the extrastriatal regions were low (Bmax < 1 nM) and theoretically cannot be visualized using 11C-preladenant.
In this preliminary study, we observed large individual differences in
11C-preladenant VT (around 30% coefficient of variation) in the target regions. There were no individual differences in plasma input function and area under the time course curve of metabolite-corrected plasma radioactivity. This variance may be attributable to individual differences in A2AR density, affinity, or competition by endogenous (i.e., adenosine) and exogenous A2AR binding ligands. One limitation of this study is the lack of analysis of plasma caffeine concentration in the subjects. Caffeine is a well-known antagonist of central nervous system A2AR (30). Although all subjects were required to refrain from drinking caffeinated beverages up to 12 h before PET imaging, residual caffeine may have
affected the distribution of 11C-preladenat in the target regions.
Significant amounts of the radiometabolite of 11C-preladenant were found in the rat brain (17% of radioactivity in the brain 60 min after injection) (18). In the human plasma, 11C-preladenant was also metabolized, and radiometabolites profile was identical to that observed in the rat plasma. The ratio of cerebellum/plasma TACs was stable over time.
However, the ratio of cerebellum/metabolite-corrected plasma TACs was slightly increased over time (Supplemental Fig. 1). Therefore, we estimated a certain amount of radiometabolites would enter the human brain.
In the target regions, no significant differences in the goodness of fit, described by Akaike's information criterion, were observed between 1T and 2T. This may also indicate the difficulty in distinguishing between the specific and non-displaceable compartments in the 2T model. Therefore, estimated micro kinetic parameters in 2T (K1, k2, k3, k4) were unstable, though VT from the micro parameters were relatively stable. To estimate the specific binding of 11C-preladenant, a reference tissue model may be useful for
quantification of binding potential. Very recently, Zhou et al. assessed 11C-preladenat PET kinetics in the conscious monkey brain. They concluded cerebellum is favorable over other investigated reference regions in terms of low VT and robustness of binding potential estimation (31). Therefore, reference tissue model using cerebellum as the reference region may be applicable for quantification of A2AR density. Further blocking studies in the human brain may be needed to fully validate the presence of a reference region.
CONCLUSION
The initial findings of the present study of a small group of subjects indicated that imaging of A2AR in the brain with 11C-preladenant PET is feasible. The radiation dose is
acceptable and the tracer is pharmacologically safe at the dose required for adequate PET imaging. The regional distribution of 11C-preladenant was consistent with known A2AR densities in the brain. The brain uptake of 11C-preladenant can be calculated as VT and DVR, which is an index of A2AR density. This initial evaluation indicated that the use of
11C-preladenat represents a feasible strategy for imaging of adenosine A2AR in the brain.
Further blocking studies in the human brain may be needed to fully validate the presence of a reference region.
Financial disclosure
This work was supported in part by a Grant-in-Aid for Scientific Research (B) 16H05396 from the Japan Society for the Promotion of Science. No potential conflicts of interest relevant to this article were reported.
ACKNOWLEDGEMNTS
We thank Mr. Kunpei Hayashi and Mr. Masanari Sakai for technical support with the cyclotron operation and radiosynthesis, Mr. Shotaro Yamaguchi for assistance in data acquisition, Ms. Kiyomi Miura for care of subjects in the PET scanning, and Ms. Airin Onishi for coordination of the clinical study.
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Figure legends FIGURE 1
Representative 11C-preladenant PET and magnetic resonance images obtained from a 24-year-old male subject. (A) PET images (SUV summed 40−60 min). Radioactivity levels are expressed as standardized uptake values (SUVs). (B) Distribution volume ratio (DVR) images from Logan graphical reference tissue method (t* = 30 min). (C) Magnetic
resonance images. Regions of interest are indicated by the red circles: cerebellar cortex (Cb), head of the caudate nucleus (Cdt), frontal cortex (Frt), putamen (Put), and thalamus (Thm).
FIGURE 2
Mean decay-corrected time-activity curves of 5 brain regions after intravenous injection of
11C-preladenat into human subjects. (A) Radioactivity levels are expressed as standardized uptake values (SUVs): putamen (solid circles), head of the caudate nucleus (open circles), thalamus (solid squares), frontal cortex (open squares), and cerebellar cortex (solid
triangles). (B) SUV ratio (SUVr): putamen/cerebellar cortex (solid diamonds) and head of the caudate nucleus/cerebellar cortex (open diamonds). Data represent the mean ± SD for 5 subjects.
FIGURE 3
Differences between the distribution volume ratio (DVR) of 11C-preladenant in the standard 90 min scan protocol and those in the shortened protocol with Logan graphical reference tissue method. Box and whiskers shows the median, quartile, minimum and maximum of the differences.
FIGURE 4
Mean decay-corrected time-activity curves of whole blood (circle), plasma (triangle), metabolite-corrected plasma (inverted triangle), and fraction of intact 11C-preladenant (11C-PLC) (square) after intravenous injection of 11C-PLN into human subjects. (A) Radioactivity levels are expressed as standardized uptake value (SUV). (B) Values for the first 7 min after tracer injection were extracted from A. Data represent the mean ± SD for 5 subjects.
FIGURE 5
Representative whole-body decay-corrected maximum-intensity-projection images of
11C-preladenant. Images were obtained at 1−5, 38−45, and 95−102 min after intravenous injection of 11C-preladenant into a 23-year-old male subject.
FIGURE 6
Regional decay-corrected time-activity curves after intravenous injection of
11C-preladenant into the same subject shown in Figure 5. Time-activity curves of 15 source organs are expressed as % of injected dose (%ID) per organ. Activities in the bone marrow were estimated from thoracic and lumbar vertebrae.
TABLE 1
11C-Preladenant VT Estimated from 1- and 2-Tissue Compartment Models (1T, 2T) and Distribution Volume Ratio (DVR) from 2-Tissue Compartment Model, and Estimated from Logan Graphical Reference Tissue Method (LGRM).
Brain region VT(1T) VT(2T) DVR(2T) DVR(LGRM) Putamen 4.4 ± 1.2 4.5 ± 1.3 7.9 ± 2.3 7.7 ± 1.9 Head of the caudate nucleus 3.4 ± 1.0 4.1 ± 2.0 7.2 ± 3.6 6.1 ± 1.5 Frontal cortex 0.3 ± 0.0 0.5 ± 0.1 0.8 ± 0.1 0.9 ± 0.0 Thalamus 0.4 ± 0.0 0.8 ± 0.4 1.3 ± 0.8 1.0 ± 0.0
Cerebellar cortex 0.4 ± 0.0 0.6 ± 0.1 − −
Data are the mean ± SD for healthy male subjects (n =5).
TABLE 2
Percentages of Radiolabeled Metabolites in Plasma After Intravenous Injection of
11C-Preladenant
Time (min) HM1 HM2 HM3 11C-Preladenant LM1
3 1.1 ± 0.8 0.8 ± 0.6 0.3 ± 0.1 97.5 ± 2.7 0.3 ± 0.3 10 6.0 ± 2.4 1.3 ± 1.4 5.1 ± 1.9 87.3 ± 3.2 0.2 ± 0.2 20 8.2 ± 4.2 1.6 ± 2.1 7.4 ± 2.2 80.3 ± 6.0 0.5 ± 0.4 30 10.4 ± 3.0 0.2 ± 0.1 7.4 ± 2.7 81.7 ± 5.8 0.3 ± 0.3 40 10.2 ± 3.8 1.8 ± 3.1 7.9 ± 3.0 79.7 ± 5.9 0.4 ± 0.3 60 13.1 ± 3.5 0.5 ± 0.6 8.4 ± 3.3 77.8 ± 7.1 0.7 ± 0.6 HM = hydrophilic metabolite
LM = lipophilic metabolite
Data are the mean ± SD for healthy male subjects (n = 5).
TABLE 3
Organ Absorbed Doses
Organ Absorbed dose (μGy/MBq)
Adrenals 3.8 ± 0.2
Brain 2.0 ± 0.2
Breasts 2.2 ± 0.1
Gallbladder wall 17.0 ± 2.5
Heart wall 8.4 ± 1.1
Kidneys 5.1 ± 0.9
Liver 11.7 ± 2.1
Lower large intestine wall 2.8 ± 0.1
Lungs 4.3 ± 0.6
Muscle 2.4 ± 0.1
Osteogenic cells 3.9 ± 0.1
Ovaries 3.0 ± 0.1
Pancreas 5.7 ± 1.4
Red marrow 3.1 ± 0.1
Skin 1.9 ± 0.1
Small intestine 7.0 ± 0.3
Spleen 4.5 ± 0.8
Stomach wall 5.8 ± 2.0
Testes 2.2 ± 0.1
Thymus 1.7 ± 0.0
Thyroid 2.5 ± 0.4
Upper large intestine wall 3.4 ± 0.1 Urinary bladder wall 2.8 ± 0.3
Uterus 3.0 ± 0.1
Total body 2.8 ± 0.0
Effective dose (μSv/MBq) 3.7 ± 0.4
Data are the mean ± SD for healthy male subjects (n = 3).
Acute toxicity of 11C-preladenant injections in rats
Methods. The Institutional Animal Care and Use Committee of Tokyo
Metropolitan Institute of Gerontology approved the animal studies. Toxicity studies of 11C-preladenat injections were performed at the Cimic Bioresearch Center (Hokuto, Japan). Acute toxicity was assayed in 6-week-old Crl:CD(SD) rats (SPF) weighing 211−233 g (males, n = 3) and 155−168 g (females, n = 3).
The three lots of 11C-preladenant were assayed after decay-out of 11C. The first lot (7.50 μg/6.52 mL/kg body weight), which was equivalent to 200-fold of the postulated maximum administration dose of 740 MBq 11C-preladenat for humans, was injected intravenously into male and female rats (n = 3 each). The second and third lots of 11C-preladenant were individually injected
intravenously into male and female rats (n = 3 each) at doses of 8.64 μg/3.87 mL/kg body weight and 6.09 μg/4.74 mL/kg body weight, for each of the two lots, both of which are equivalent to 250-fold of the postulated maximum administration dose. Rats were observed on an ongoing basis until 30 min and 1, 2, 4, and 6 h after the injection on day 1 and thereafter once daily for clinical signs of toxicity until 14 days. Rats were weighed on days 1, 2, 4, 7, and 14. At the end of the 14-day observation period, the rats were euthanized by
exsanguination under isoflurane anesthesia, and a macroscopic analysis of autopsy samples was performed.
Results. Acute toxicity in rats was evaluated after a single intravenous injection
of one of the three lots of 11C-preladenant preparations at a dose range of 6.09–
8.64 µg/kg. No mortality was found in the rats during the 14-day observation period. All rat groups showed normal gains in body weight compared with the control animals, and no clinical signs of toxicity were observed over a 15-day period. No abnormalities were found with postmortem macroscopic
examination.
Radiation dosimetry estimated from mice bioditribution
Methods. Male ddY mice were obtained from Japan SLC (Hamamatsu, Japan).
The Institutional Animal Care and Use Committee approved the animal studies.
11C-Preladenant (10 MBq/106 pmol) was intravenously injected into male ddY mice (8 weeks old). Mice were killed by cervical dislocation 1, 5, 15, 30, 60, and 90 min after injection (n = 4 each). The blood was collected by heart puncture, and the tissues were harvested. The samples were measured for 11C radioactivity with an auto-gamma counter (1282 Commpugamma CS; LKB Wallac, Turku, Finland) and weighed. The tissue uptake of 11C was expressed as a percentage of the injected dose per organ (%ID/organ) or a percentage of the injected dose per gram of tissue (%ID/g). The tissue distribution data were extrapolated to an adult male phantom using the % kg/g method (1). The radiation absorbed dose and effective dose for human adults were estimated using the software OLINDA/EXM (Vanderbilt University, Nashville, TN) (2).
Results. The tissue distribution of the radioactivity after injection of 11C-
preladenant into mice is summarized in Supplemental Tables 1 and 2. The liver showed the highest initial uptake (%ID/organ) followed by the kidney and small intestine. The levels in other tissues investigated were low. The levels of radioactivity in the liver and stomach peaked at 5 and 15 min, respectively, and then gradually decreased. The radioactivity in small intestine peaked at 60 min and migrated to the large intestine. The radioactivity excreted in urine gradually increased, but remained at 3%ID at 90 min post-injection. From these data, we estimated the radiation absorbed doses for human adults (Table 3). The
effective dose according to the risk-weighting factors of ICRP60 (3) was estimated at 4.3 µSv/MBq.
SUPPLEMENTAL TABLE 1 Tissue distribution of radioactivity in mice after intravenous injection of 11C-preladenant
% Injected dose/g tissue
1 min 5 min 15 min 30 min 60 min 90 min Blood 2.18 ±
0.18
1.66 ± 0.09
1.15 ± 0.09
0.85 ± 0.06
0.74 ± 0.05
0.66 ± 0.06 Heart 7.30 ±
1.27
3.39 ± 0.17
2.07 ± 0.17
1.36 ± 0.13
0.93 ± 0.07
0.78 ± 0.12 Lung 10.40 ±
1.31
4.48 ± 0.47
2.79 ± 0.15
1.80 ± 0.11
1.50 ± 0.17
1.43 ± 0.16 Liver 5.17 ±
2.68
11.92 ± 0.56
8.89 ± 0.78
6.79 ± 0.79
5.01 ± 0.09
4.05 ± 0.43 Pancreas 6.27 ±
0.72
5.63 ± 0.40
4.54 ± 0.39
4.95 ± 0.35
4.60 ± 0.30
4.07 ± 0.66 Spleen 3.84 ±
1.12
4.45 ± 0.48
2.91 ± 0.34
2.42 ± 0.22
2.19 ± 0.62
2.50 ± 0.65 Kidney 16.78 ±
1.71
8.69 ± 0.61
4.81 ± 0.26
3.40 ± 0.31
2.76 ± 0.34
2.50 ± 0.23 Stomach 3.64 ±
0.77
8.73 ± 1.85
13.33 ± 1.40
12.82 ± 1.93
7.73 ± 2.12
3.98 ± 2.01 Small
intestine
4.52 ± 0.48
5.59 ± 0.43
8.87 ± 1.25
12.23 ± 1.19
15.90 ± 0.95
13.14 ± 3.18 Large
intestine
2.39 ± 0.42
1.86 ± 0.30
2.37 ± 0.35
2.53 ± 0.09
3.00 ± 0.49
5.00 ± 4.61 Testes 1.39 ±
0.27
1.52 ± 0.13
1.39 ± 0.09
0.86 ± 0.10
0.54 ± 0.04
0.43 ± 0.07 Muscle 2.85 ±
0.08
1.87 ± 0.12
1.20 ± 0.10
0.79 ± 0.05
0.60 ± 0.07
0.46 ± 0.12 Bone 2.13 ±
0.54
1.72 ± 0.08
1.31 ± 0.07
1.22 ± 0.32
1.03 ± 0.06
0.93 ± 0.21 Brain 2.96 ±
0.47
1.99 ± 0.14
1.61 ± 0.19
1.39 ± 0.08
1.03 ± 0.15
0.77 ± 0.12 Data are the mean ± SD for male ddY mice (n = 4).
SUPPLEMENTAL TABLE 2 Organ distribution of radioactivity in mice after intravenous injection of 11C-preladenant
% Injected dose/organ
1 min 5 min 15 min 30 min 60 min 90 min
Heart 1.07 ±
0.09
0.58 ± 0.07
0.32 ± 0.04
0.20 ± 0.03
0.14 ± 0.01
0.11 ± 0.02
Lung 2.01 ±
0.21
1.00 ± 0.09
0.56 ± 0.01
0.37 ± 0.05
0.33 ± 0.04
0.31 ± 0.02
Liver 9.89 ±
5.01
25.32 ± 1.69
17.99 ± 0.70
13.78 ± 1.45
9.40 ± 0.59
7.84 ± 0.71 Pancreas 1.34 ±
0.20
1.21 ± 0.23
1.03 ± 0.25
0.97 ± 0.25
1.02 ± 0.09
0.87 ± 0.19 Spleen 0.51 ±
0.15
0.61 ± 0.13
0.38 ± 0.06
0.29 ± 0.07
0.30 ± 0.17
0.30 ± 0.12 Kidney 9.43 ±
0.64
5.24 ± 0.45
2.84 ± 0.49
2.13 ± 0.51
1.61 ± 0.37
1.50 ± 0.15 Stomach 1.78 ±
0.17
5.98 ± 1.31
8.03 ± 0.87
6.75 ± 1.38
3.75 ± 0.88
2.07 ± 1.09 Small intestine 8.11 ±
0.72
11.18 ± 1.00
17.75 ± 0.64
24.84 ± 2.61
29.14 ± 1.73
27.01 ± 7.26 Large intestine 2.65 ±
0.94
2.53 ± 0.39
3.14 ± 0.32
3.27 ± 0.53
3.78 ± 0.86
7.34 ± 7.96 Testes 0.36 ±
0.06
0.43 ± 0.09
0.38 ± 0.07
0.23 ± 0.06
0.13 ± 0.01
0.10 ± 0.02
Brain 1.31 ±
0.20
0.83 ± 0.10
0.70 ± 0.09
0.63 ± 0.07
0.46 ± 0.08
0.31 ± 0.06 Bladder 0.10 ±
0.07
0.10 ± 0.03
0.56 ± 0.66
0.72 ± 1.33
1.46 ± 1.74
1.22 ± 2.26
Urine 0.04 ±
0.01
0.32 ± 0.17
1.24 ± 0.35
2.37 ± 1.03
2.11 ± 2.07
1.68 ± 0.52 Bladder+Urine 0.11 ±
0.06
0.41 ± 0.20
1.69 ± 0.62
3.09 ± 1.46
3.57 ± 2.52
2.90 ± 2.28 Data are the mean ± SD for male ddY mice (n = 4).
SUPPLEMENTAL TABLE 3 Absorbed dose of 11C-preladenant for human adults estimated from mouse data
μGy/MBq μGy/MBq
Adrenals 2.6 Muscle 2.6
Brain 1.7 Ovaries 3.2
Breasts 1.7 Pancreas 5.0
Gallbladder wall 3.2 Red marrow 2.4
Lower large intestine wall 4.6 Osteogenic cells 2.9
Small intestine 10.2 Skin 1.6
Stomach wall 7.6 Spleen 3.4
Upper large intestine wall 5.0 Testes 1.8
Heart wall 3.1 Thymus 2.0
Kidneys 5.1 Thyroid 2.0
Liver 6.9 Urinary bladder wall 12.0
Lungs 3.0 Uterus 3.3
Total body 2.8
Effective dose 4.3 μSv/MBq
Preparation of 11C-preladenant
Methods. 2-(Furan-2-yl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin-1-
yl)ethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine
(preladenant) and its O-desmethyl precursor, 2-(4-(4-(2-(5-amino-2-(furan-2- yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-7-yl)ethyl)piperazin-1- yl)phenoxy)ethanol (O-desmethyl preladenant), were prepared according to a previously described method (4). All chemical reagents were obtained from commercial sources.
11C-CO2 was produced by proton irradiation of nitrogen gas at 50 µA for 15 min using the HM-20 cyclotron (Sumitomo Heavy Industries, Tokyo, Japan).
11C-Methyl iodide was produced from 11C-CO2 with an automated system (Sumitomo Heavy Industries). 11C-Methyl iodide was trapped in dry dimethylsulfoxide (0.4 mL) containing 0.5 mg (1.0 μmol) of O-desmethyl preladenant and 1 mg (17.8 μmol) of potassium hydroxide at room temperature.
The reaction mixture was heated at 40°C for 3 min. After adding 1.5 mL of acetonitrile/100 mM sodium dihydrogen phosphate (1/1, v/v), the reaction mixture was subjected to high-performance liquid chromatography (HPLC) using a reversed phase column [YMC-Pack Pro C18 RS 5 µm, 10 mm inner diameter (i.d.) × 250 mm length; YMC, Kyoto, Japan] and ultra violet (UV) absorbance (260 nm) with a semiconductor radiation detector. The mobile phase was a mixture of acetonitrile/50 mM aqueous ammonium acetate (45/55,
v/v) at a flow rate of 5 mL/min. The 11C-preladenant fraction [retention time (tR) = 7.7 min] was collected in a flask containing 0.1 mL of 250 mg/mL ascorbate injection (Nipro Pharma, Osaka, Japan) and evaporated to dryness.
The residue was dissolved in physiological saline containing 0.125% (v/v) polyoxyethylene (20) sorbitan monoolate (polysorbate 80) (Wako Pure Chemical Industries, Osaka, Japan), and the solution was filtered through a 0.22-µm membrane filter (Millex GV; Merck Millipore, Billerica, MA).
Radiochemical purity was analyzed by ultrafast liquid chromatography (Prominence UFLC; Shimadzu, Kyoto, Japan) using a TitanTM C18 UHPLC column, 1.9 μm (2.1 mm i.d. × 50 mm length; Sigma-Aldrich, St. Louis, MO) with a mobile phase of acetonitrile/50 mM aqueous acetic acid/50 mM aqueous ammonium acetate (27.5/36.25/36.25, v/v/v) at a flow rate of 0.4 mL/min (UV detector at 260 nm). The tR of 11C-preladenant was 2.5 min. To determine the specific activity, the mass (µmol) of the radioligand with known radioactivity (GBq) was determined by UFLC comparison of UV absorbance at 260 nm of the radioligand with those of known concentrations of the corresponding nonradioactive ligand.
Results. The total synthesis time was within 35 min from the end of
bombardment. The decay-corrected radiochemical yields of 11C-preladenant, based on 11C-methyl iodide, were 25.4% ± 7.2% (range, 15.8 – 32.1) (n = 8).
The radiochemical purity of 11C-preladenant was always greater than 98%. It
remained >98% for at least 90 min after preparation in the presence of
ascorbate. The specific activities of 11C-preladenant were 138 ± 28 GBq/μmol (range, 105 – 175), at 35 min after the end of irradiation (n = 8). The absence of any residual traces of the starting material was verified by UFLC analysis.
Finally, the sterility and apyrogenicity of the products were confirmed.
SUPPLEMENTAL TABLE 4 Subject characteristics and 11C-preladenent PET protocol Subject
No.
Age
(y) Sex Weight (kg)
Protocol
Urination (min) Region Dose
(MBq/nmol)
Duration (min)
1 24 M 66.8 Brain 624/9.3 90 97
2 24 M 56.7 Brain 508/24.3 90 101
3 24 M 61.9 Brain 690/16.6 90 98
4 23 M 72.2 Brain 649/5.7 90 101
5 31 M 61.0 Brain 590/12.6 90 99
6 25 M 86.1 Whole
body 786/8.5 120 131
7 21 M 62.5 Whole
body 754/11.3 120 132
8 23 M 65.9 Whole
body 714/7.1 120 131
SUPPLEMENTAL TABLE 5 Normalized number of disintegrations calculated from whole-body 11C-preladenant PET in human subjects Organ Normalized number of disintegrations (MBq-
h/MBq administered)
Adrenals 1.1E-04 ± 1.6E-05 (1.0E-04 – 1.3E-04) Brain 6.7E-03 ± 9.8E-04 (5.7E-03 – 7.7E-03) Gallbladder contents 5.7E-03 ± 1.1E-03 (4.4E-03 – 6.3E-03) Small intestine 1.4E-02 ± 8.7E-04 (1.3E-02 – 1.5E-02) Stomach 5.6E-03 ± 3.9E-03 (1.8E-03 – 9.6E-03) Heart wall 8.3E-03 ± 1.2E-03 (7.4E-03 – 9.7E-03) Kidneys 3.8E-03 ± 9.8E-04 (2.7E-03 – 4.7E-03) Liver 6.3E-02 ± 1.3E-02 (5.3E-02 – 7.7E-02) Lungs 1.2E-02 ± 2.4E-03 (9.1E-03 – 1.4E-02) Pancreas 1.3E-03 ± 4.4E-04 (9.1E-04 – 1.8E-03) Red marrow 8.2E-03 ± 9.7E-04 (7.1E-03 – 8.8E-03) Spleen 2.1E-03 ± 5.0E-04 (1.6E-03 – 2.6E-03) Thymus 2.4E-05 ± 2.3E-06 (2.2E-05 – 2.7E-05) Thyroid 1.2E-04 ± 3.9E-05 (8.7E-05 – 1.6E-04) Urinary bladder contents 3.8E-04 ± 2.9E-04 (1.2E-04 – 6.9E-04) Remainder 3.6E-01 ± 2.2E-02 (3.3E-01 – 3.7E-01)
Data are the mean ± SD (range) and the range for healthy male subjects (n = 3).
SUPPLEMENRAL FIGURE 1 Radioactivity ratios of cerebellum over plasma (solid circles) and metabolite-corrected plasma (open circles) after intravenous injection of 11C-praladenant into human subjects. Data represent the mean ± SD for 5 subjects.
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