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Supplemental material, sj-pdf-1-jcb-10.1177_0271678X231152025 for Small-animal PET study for noninvasive quantification of transmembrane AMPA receptor regulatory protein γ-8 (TARP γ-8) in the brain by Tomoteru Yamasaki, Hideki Ishii, Atsuto Hiraishi, Katsushi Kumata, Hidekatsu Wakizaka, Yiding Zhang, Yusuke Kurihara, Masanao Ogawa, Nobuki Nengaki, Jiahui Chen, Yinlong Li, Steven Liang, Ming-Rong Zhang in Journal of Cerebral Blood Flow & Metabolism

Recently, we produced 11C‐labeled 2‐((1E,3E)‐4‐(6‐(methylamino)pyridin‐3‐yl)buta‐1,3‐dienyl)benzo[d]thiazol‐6‐ol ([11C]PBB3) as a clinically useful positron emission tomography (PET) tracer for in vivo imaging of tau pathologies in the human brain. To overcome the limitations (i.e., rapid in vivo metabolism and short half‐life) of [11C]PBB3, we further synthesized 18F‐labeled 1‐fluoro‐3‐((2‐((1E,3E)‐4‐(6‐(methylamino)pyridine‐3‐yl)buta‐1,3‐dien‐1‐yl)benzo[d]thiazol‐6‐yl)oxy)propan‐2‐ol ([18F]PM‐PBB3). [18F]PM‐PBB3 is also a useful tau PET tracer for imaging tau pathologies. In this study, we developed a routine radiosynthesis and quality control testing of [18F]PM‐PBB3 for clinical applications. [18F]PM‐PBB3 was synthesized by direct 18F‐fluorination of the tosylated derivative, followed by removal of the protecting group. [18F]PM‐PBB3 was obtained with sufficient radioactivity (25 ± 6.0% of the nondecay‐corrected radiochemical yield at the end of synthesis, EOS), radiochemical purity (98 ± 0.6%), and molar activity (350 ± 94 GBq/μmol at EOS; n = 53). Moreover, [18F]PM‐PBB3 consistently retained >95% of radiochemical purity for 60 min without undergoing photoisomerization using a new UV‐cutoff light (yellow light) fixed in the hot cell to monitor the synthesis. All the results of the quality control testing for the [18F]PM‐PBB3 injection complied with our in‐house quality control and quality assurance specifications. We have accomplished >200 production runs of [18F]PM‐PBB3 in our facility for various research purposes.

先進核医学基盤研究部では、臨床診断・治療や生体機能の計測に有用な放射性核種標識薬剤の開発、標識薬剤合成に必要な放射性核種の製造及び標識・分析技術の開発研究を行っている。また、新規放射性薬剤を開発し、多種多様な動物モデルを含めた動物実験による薬剤の有効性と前臨床評価、臨床共同研究を実施している。さらに、安全で高品位な標識核種及び診断・治療用の放射性核種を製造し、脳機能イメージング研究部、分子イメージング診断治療研究部のみならずQST病院や外部の大学・研究機関・企業の研究者に広く提供している。 当研究部によって製造される臨床研究用の放射性薬剤は、1) HIMACや量子メスを用いた腫瘍の治療効果の評価や転移の有無などの判定、2) がん患者への診断・治療研究、3) 認知症をはじめとする各種の脳疾患の診断、治療効果の評価及び病態発生メカニズムの解明研究などに利用されている。本報告書では、新規標識薬剤の開発状況及び放射性核種・薬剤の製造状況を報告する。

Metabotropic glutamate receptor 2 (mGlu2) is a known target for treating several central nervous system (CNS) disorders. To develop a viable positron emission tomography (PET) ligand for mGlu2, we identified new candidates 5a–i that are potent negative allosteric modulators (NAMs) of mGlu2. Among these candidates, 4-(2-fluoro-4-methoxyphenyl)-5-((1-methyl-1H-pyrazol-3-yl)methoxy)picolinamide (5i, also named as [11C]MG2-1812) exhibited high potency, high subtype selectivity, and favorable lipophilicity. Compound 5i was labeled with positron-emitting carbon-11 (11C) to obtain [11C]5i in high radiochemical yield and high molar activity by O-[11C]methylation of the phenol precursor 12 with [11C]CH3I. In vitro autoradiography with [11C]5i showed heterogeneous radioactive accumulation in the brain tissue sections, ranked in the order: cortex > striatum > hippocampus > cerebellum ≫ thalamus > pons. PET study of [11C]5i indicated in vivo specific binding of mGlu2 in the rat brain. Based on the [11C]5i scaffold, further optimization for new candidates is underway to identify a more suitable ligand for imaging mGlu2.

Mutations that increase leucine-rich repeat kinase 2 (LRRK2) activity in the brain are associated with Parkinson's disease. Here, we synthesized a novel compound 4-(6-fluoro-4-(5-isopropoxy-1H-indazol-3-yl)pyridin-2-yl)morpholine (FIPM) and labeled it with fluorine-18 (18F), to develop a positron emission tomography (PET) tracer for in vivo visualization of LRRK2 in the brain. FIPM showed high in vitro binding affinity for LRRK2 (IC50 = 8.0 nM). [18F]FIPM was prepared in 5% radiochemical yield (n = 5), by inserting 18F into a pyridine ring, followed by removal of the protecting group. After HPLC separation and formulation, [18F]FIPM was acquired with >97% radiochemical purity and 103–300 GBq μmol−1 of molar activity at the end of radiosynthesis. Biodistribution and small-animal PET studies in mice indicated a low in vivo specific binding of [18F]FIPM. While [18F]FIPM presented limited potential as an in vivo PET tracer for LRRK2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.

Fatty acid amide hydrolase (FAAH) is a key enzyme in the endocannabinoid system. N-(3,4-Dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide ([11C]DFMC) was developed as an irreversible-type positron emission tomography (PET) ligand for FAAH. Here, we attempted to noninvasively estimate rate constant k3 as a direct index for FAAH in the rat brain. First, the two-tissue compartment model analysis including three parameters (K1–k3, 2TCMi) in PET study with [11C]DFMC was conducted, which provided 0.21 ± 0.04 mL·cm−3·min−1 of the net uptake value (Ki), an indirect index for FAAH, in the FAAH-richest region (the cingulate cortex). Subsequently, to noninvasively estimate Ki value, the reference model analysis (Patlak Reference, PGAREF) was tried using a time-activity curve of the spinal cord. In that result, the noninvasive Ki value (KREF) was concisely estimated with high correlation (r > 0.95) to Ki values based on 2TCMi. Using estimated KREF value, we tried to obtain calculated-k3 based on previously defined equations. The caluculated-k3 was successfully estimated with high correlation (r = 0.95) to direct k3 in 2TCMi. Finally, the dose relationship study using calculated-k3 demonstrated that in vivo ED50 value of URB597, a major inhibitor of FAAH, was 66.4 µg/kg in rat brain. In conclusion, we proposed the calculated-k3 as an alternative index corresponding to regional FAAH concentrations and suggested that PET with [11C]DFMC enables occupancy study for new pharmaceuticals targeting FAAH.

The 18 kDa translocator protein (TSPO) is a viable target for imaging of inflammation in brain. In the recent past we have explored a pharmacophore skeleton acetamidobenzoxazolone for positron emission tomography (PET) imaging of TSPO expression in brain. Here we evaluated a new radioligand for visualization of TSPO, namely [11C]N-(2-methoxyoxyphenyl)-N-methyl-2-(5-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)acetamide ([11C]N′-MPB). This PET ligand exhibited high binding affinity towards TSPO (Ki = 4.9 nM) and a suitable lipophilicity (log D) of 2.08 for brain imaging. A biodistribution study on mice showed high accumulation of radioactivity in TSPO-rich organs, such as the lungs, heart, kidneys, and adrenal glands. Metabolite analysis of rat brain homogenate showed 98% intact [11C]N′-MPB at 30 min after injection. To determine the specific binding of the radioligand with TSPO on neuroinflammation of the brain, in vitro autoradiography and PET studies were performed in an ischemic rat model. In vitro autoradiography indicated significantly increased binding on the ipsilateral side compared with that on the contralateral side of ischemic rat brains. This result was supported firmly by the contrast of radioactivity in PET images. Displacement experiments with PK11195 minimized the difference in radioactivity uptake between the two sides. In summary, [11C]N′-MPB is a potential PET imaging radioligand for TSPO and, consequently, for gaining more insight for up-regulation of microglia during neuroinflammation.