A 211At-labelled mGluR1 inhibitor induces cancer senescence to elicit long-lasting anti-tumor efficacy.

Metabotropic glutamate receptor 1 (mGluR1), a key mediator of glutamatergic signaling, is frequently overexpressed in tumor cells and is an attractive drug target for most cancers. Here, we present a targeted radiopharmaceutical therapy strategy that antagonistically recognizes mGluR1 and eradicates mGluR1+ human tumors by harnessing a small-molecule alpha (α)-emitting radiopharmaceutical, 211At-AITM. A single dose of 211At-AITM (2.96 MBq) in mGluR1+ cancers exhibits long-lasting in vivo antitumor efficacy across seven subtypes of four of the most common tumors, namely, breast cancer, pancreatic cancer, melanoma, and colon cancers, with little toxicity. Moreover, complete regression of mGluR1+ breast cancer and pancreatic cancer is observed in approximate 50% of tumor-bearing mice. Mechanistically, the functions of 211At-AITM are uncovered in downregulating mGluR1 oncoprotein and inducing senescence of tumor cells with a reprogrammed senescence-associated secretory phenotype. Our findings suggest α-radiopharmaceutical therapy with 211At-AITM can be a useful strategy for mGluR1+ pan-cancers, regardless of their tissue of origin.

Scandium Triflate-Catalyzed N-[18F]Fluoroalkylation of Aryl- Or Heteroaryl-Amines with [18F]Epifluorohydrin under Mild Conditions.

The scandium triflate-catalyzed N-[18F]fluoroalkylation of aryl- or heteroaryl-amines with [18F]epifluorohydrin ([18F]2) was investigated. This reaction is mild and provides one-step access to N-[18F]fluoroalkylated aryl- or heteroaryl-amines, which are used for positron emission tomography imaging. The use of 2,2,2-trifluoroethanol as a cosolvent improved the reaction efficiency. The use of (S)- and (R)-[18F]2 produced the corresponding enantiomeric N-[18F]fluoroalkylated anilines.

Automated radiosynthesis of [11 C]MTP38-a phosphodiesterase 7 imaging tracer-using [11 C]hydrogen cyanide for clinical applications.

We have developed 8-amino-3-(2S,5R-dimethyl-1-piperidyl)-[1,2,4]triazolo[4,3-a]pyrazine-5-[11 C]carbonitrile ([11 C]MTP38) as a positron emission tomography (PET) tracer for the imaging of phosphodiesterase 7. For the fully automated production of [11 C]MTP38 routinely and efficiently for clinical applications, we determined the radiosynthesis procedure of [11 C]MTP38 using [11 C]hydrogen cyanide ([11 C]HCN) as a PET radiopharmaceutical. Radiosynthesis of [11 C]MTP38 was performed using an automated 11 C-labeling synthesizer developed in-house within 40 min after the end of irradiation. [11 C]MTP38 was obtained with a relatively high radiochemical yield (33 ± 5.5% based on [11 C]CO2 at the end of irradiation, decay-corrected, n = 15), radiochemical purity (>97%, n = 15), and molar activity (47 ± 12 GBq/µmol at the end of synthesis, n = 15). All the results of the quality control (QC) testing for the [11 C]MTP38 injection complied with our in-house QC and quality assurance specifications. We successfully automated the radiosynthesis of [11 C]MTP38 for clinical applications using an 11 C-labeling synthesizer and sterile isolator. Taken together, this protocol provides a new radiopharmaceutical [11 C]MTP38 suitable for clinical applications.

Automated radiosynthesis of two 18F-labeled tracers containing 3-fluoro-2-hydroxypropyl moiety, [18F]FMISO and [18F]PM-PBB3, via [18F]epifluorohydrin.

BACKGROUND: [18F]Fluoromisonidazole ([18F]FMISO) and 1-[18F]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 or [18F]APN-1607) are clinically used radiotracers for imaging hypoxia and tau pathology, respectively. Both radiotracers were produced by direct 18F-fluorination using the corresponding tosylate precursors 1 or 2 and [18F]F-, followed by the removal of protecting groups. In this study, we synthesized [18F]FMISO and [18F]PM-PBB3 by 18F-fluoroalkylation using [18F]epifluorohydrin ([18F]5) for clinical applications. RESULTS: First, [18F]5 was synthesized by the reaction of 1,2-epoxypropyl tosylate (8) with [18F]F- and was purified by distillation. Subsequently, [18F]5 was reacted with 2-nitroimidazole (6) or PBB3 (7) as a precursor for 18F-labeling, and each reaction mixture was purified by preparative high-performance liquid chromatography and formulated to obtain the [18F]FMISO or [18F]PM-PBB3 injection. All synthetic sequences were performed using an automated 18F-labeling synthesizer. The obtained [18F]FMISO showed sufficient radioactivity (0.83 ± 0.20 GBq at the end of synthesis (EOS); n = 8) with appropriate radiochemical yield based on [18F]F- (26 ± 7.5 % at EOS, decay-corrected; n = 8). The obtained [18F]PM-PBB3 also showed sufficient radioactivity (0.79 ± 0.10 GBq at EOS; n = 11) with appropriate radiochemical yield based on [18F]F- (16 ± 3.2 % at EOS, decay-corrected; n = 11). CONCLUSIONS: Both [18F]FMISO and [18F]PM-PBB3 injections were successfully synthesized with sufficient radioactivity by 18F-fluoroalkylation using [18F]5.

Radiosynthesis of 18F-fluoroethylated tracers via a simplified one-pot 18F-fluoroethylation method using [18F]fluoroethyl tosylate.

Recently, a straightforward one-pot method for 18F-fluoroethylation without azeotropic drying of cyclotron-produced [18F]F- was developed. In this study, we have attempted to simplify the automated radiosynthesis of two [18F]fluoroethylated tracers, [18F]FEDAC and [18F]FET, using a desmethyl labeling precursor and [18F]fluoroethyl tosylate, based on the above-mentioned method. The radiochemical yields of [18F]FEDAC and [18F]FET were 26 ± 3.7% (n = 5) and 14 ± 2.2% (n = 4), respectively, based on total [18F]F- at the end of irradiation.

Radiosynthesis and quality control testing of the tau imaging positron emission tomography tracer [18 F]PM-PBB3 for clinical applications.

Recently, we produced 11 C-labeled 2-((1E,3E)-4-(6-(methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol ([11 C]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 [11 C]PBB3, we further synthesized 18 F-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 ([18 F]PM-PBB3). [18 F]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 [18 F]PM-PBB3 for clinical applications. [18 F]PM-PBB3 was synthesized by direct 18 F-fluorination of the tosylated derivative, followed by removal of the protecting group. [18 F]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, [18 F]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 [18 F]PM-PBB3 injection complied with our in-house quality control and quality assurance specifications. We have accomplished >200 production runs of [18 F]PM-PBB3 in our facility for various research purposes.

Simultaneous measurements of the molar radioactivity, radiochemical purity and chemical impurity in the [11C]choline injection using radio-HPLC with a corona-charged aerosol detector.

[11C]choline has a weak UV absorption chromophore, and it is challenging to detect less than 1 µg/mL choline using radio-HPLC-UV. In this study, we established an analytical procedure of [11C]choline using the radio-HPLC coupled with the corona-charged aerosol detector. As a result, we achieved more than 100 GBq/µmol (over 0.1 µg/mL of choline) of molar radioactivity at the end of synthesis, over 95% of radiochemical purity, and more than 0.1 µg/mL of 2-dimethyoaminoethanol as a chemical impurity.

Radiosynthesis and in vivo evaluation of 11C-labeled BMS-193885 and its desmethyl analog as PET tracers for neuropeptide Y1 receptors.

BACKGROUND: Neuropeptide Y (NPY) has been implicated in a wide variety of physiological processes, including feeding, learning, memory, emotion, cardiovascular homeostasis, hormone secretion, and circadian rhythms. NPY Yl receptor (NPY Y1-R) is the most widely studied NPY receptor, and is involved in many of these processes. BMS-193885 (1) was previously developed as a potent and selective NPY Y1-R antagonist, which has good systemic bioavailability and brain penetration. To evaluate the characteristics of 1 in vivo, we developed 11C-labeled BMS-193885 ([11C]1) and its desmethyl analog ([11C]2) for potential use as two new positron emission tomography (PET) tracers. RESULTS: [11C]1 was synthesized from [11C]methyl iodide using 2. [11C]2 was synthesized from [11C]phosgene using its aniline and amine derivatives. The mean ± S.D. decay-corrected radiochemical yields of [11C]1 and [11C]2 from 11CO2 at the end of radionuclide production were 23 ± 3.2% (n = 6) and 24 ± 1.5% (n = 4), respectively. In biodistribution on mice, radioactivity levels for both tracers were relatively high in the kidney, small intestine, and liver at 60 min post-injection. The radioactivity levels in the kidney, lung, and spleen of mice at 30 min post-injection with [11C]1 were significantly reduced by pretreatment with 1 (10 mg/kg), and levels of [11C]1 in the brain of mice were significantly increased by pretreatment with the P-glycoprotein and breast cancer resistance protein inhibitor elacridar (10 mg/kg). In metabolite analysis using mouse plasma, [11C]1 and [11C]2 were rapidly metabolized within 30 min post-injection, and [11C]1 was mainly metabolized into unlabeled 2 and radiolabeled components. CONCLUSION: [11C]1 and [11C]2 were successfully synthesized with sufficient amount of radioactivity and high quality for use in vivo. Our study of [11C]1 and its desmethyl analog [11C]2 was useful in that it helped to elucidate the in vivo characteristics of 1.

Development of 2-(2-(3-(4-([18F]Fluoromethoxy- d2)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione for Positron-Emission-Tomography Imaging of Phosphodiesterase 10A in the Brain.

Phosphodiesterase 10A (PDE10A) is a newly identified therapeutic target for central-nervous-system disorders. 2-(2-(3-(4-([18F]Fluoroethoxy)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([18F]MNI-659, [18F]5) is a useful positron-emission-tomography (PET) ligand for imaging of PDE10A in the human brain. However, the radiolabeled metabolite of [18F]5 can accumulate in the brain. In this study, using [18F]5 as a lead compound, we designed four new 18F-labeled ligands ([18F]6-9) to find one more suitable than [18F]5. Of these, 2-(2-(3-(4-([18F]fluoromethoxy- d2)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([18F]9) exhibited high in vitro binding affinity ( Ki = 2.9 nM) to PDE10A and suitable lipophilicity (log D = 2.2). In PET studies, the binding potential (BPND) of [18F]9 (5.8) to PDE10A in the striatum of rat brains was significantly higher than that of [18F]5 (4.6). Furthermore, metabolite analysis showed much lower levels of contamination with radiolabeled metabolites in the brains of rats given [18F]9 than in those given [18F]5. In conclusion, [18F]9 is a useful PET ligand for PDE10A imaging in brain.

[18F]DAA1106: Automated radiosynthesis using spirocyclic iodonium ylide and preclinical evaluation for positron emission tomography imaging of translocator protein (18 kDa).

DAA1106 (N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide), is a potent and selective ligand for the translocator protein (18 kDa, TSPO) in brain mitochondrial fractions of rats and monkey (Ki = 0.043 and 0.188 nM, respectively). In this study, to translate [18F]DAA1106 for clinical studies, we performed automated syntheses of [18F]DAA1106 using the spirocyclic iodonium ylide (1) as a radiolabelling precursor and conducted preclinical studies including positron emission tomography (PET) imaging of TSPO in ischemic rat brains. Radiofluorination of the ylide precursor 1 with [18F]F-, followed by HPLC separation and formulation, produced the [18F]DAA1106 solution for injection in 6% average (n = 10) radiochemical yield (based on [18F]F-) with >98% radiochemical purity and molar activity of 60-100 GBq/µmol at the end of synthesis. The synthesis time was 87 min from the end of bombardment. The automated synthesis achieved [18F]DAA1106 with sufficient radioactivity available for preclinical and clinical use. Biodistribution study of [18F]DAA1106 showed a low uptake of radioactivity in the mouse bones. Metabolite analysis showed that >96% of total radioactivity in the mouse brain at 60 min after the radiotracer injection was unmetabolized [18F]DAA1106. PET study of ischemic rat brains visualized ischemic areas with a high uptake ratio (1.9 ±â€¯0.3) compared with the contralateral side. We have provided evidence that [18F]DAA1106 could be routinely produced for clinical studies.

Synthesis of two novel [18F]fluorobenzene-containing radiotracers via spirocyclic iodonium ylides and positron emission tomography imaging of translocator protein (18 kDa) in ischemic brain.

Two novel radiotracers, namely, N-(4-[18F]fluorobenzyl)-N-methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide ([18F]5) and 2-(5-(4-[18F]fluorophenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide ([18F]6), were developed for positron emission tomography (PET) imaging of translocator protein (18 kDa) (TSPO) in ischemic brain in this study. The two radiotracers with a [18F]fluorobenzene ring were derived from the corresponding [18F]fluoroethyl tracers [18F]7 and [18F]8 which underwent [18F]defluoroethylation in vivo easily. [18F]5 or [18F]6 was synthesized by the radiofluorination of the spirocyclic iodonium ylide precursor 10 or 17 with [18F]F- in 23 ± 10% (n = 7) or 56 ± 9% (n = 7) radiochemical yields (decay-corrected, based on [18F]F-). [18F]5 and [18F]6 showed high in vitro binding affinities (Ki = 0.70 nM and 5.9 nM) for TSPO and moderate lipophilicities (log D = 2.9 and 3.4). Low uptake of radioactivity for both radiotracers was observed in mouse bones. Metabolite analysis showed that the in vivo stability of [18F]5 and [18F]6 was improved in comparison to the parent radiotracers [18F]7 and [18F]8. In particular, no radiolabelled metabolite of [18F]5 was found in the mouse brains at 60 min after the radiotracer injection. PET studies with [18F]5 on ischemic rat brains revealed a higher binding potential (BPND = 3.42) and maximum uptake ratio (4.49) between the ipsilateral and contralateral sides. Thus, [18F]5 was shown to be a useful PET radiotracer for visualizing TSPO in neuroinflammation models.

Automated Synthesis of (rac)-, (R)-, and (S)-[18 F]Epifluorohydrin and Their Application for Developing PET Radiotracers Containing a 3-[18 F]Fluoro-2-hydroxypropyl Moiety.

To introduce the 3-[18 F]fluoro-2-hydroxypropyl moiety into positron emission tomography (PET) radiotracers, we performed automated synthesis of (rac)-, (R)-, and (S)-[18 F]epifluorohydrin ([18 F]1) by nucleophilic displacement of (rac)-, (R)-, or (S)-glycidyl tosylate with 18 F- and purification by distillation. The ring-opening reaction of (R)- or (S)-[18 F]1 with phenol precursors gave enantioenriched [18 F]fluoroalkylated products without racemisation. We then synthesised (rac)-, (R)-, and (S)- 2-{5-[4-(3-[18 F]fluoro-2-hydroxypropoxy)phenyl]-2-oxobenzo[d]oxazol-3(2H)-yl}-N-methyl-N-phenylacetamide ([18 F]6) as novel radiotracers for the PET imaging of translocator protein (18 kDa) and showed that (R)- and (S)-[18 F]6 had different radioactivity uptake in mouse bone and liver. Thus, (rac)-, (R)-, and (S)-[18 F]1 are effective radiolabelling reagents and can be used to develop PET radiotracers by examining the effects of chirality on their in vitro binding affinities and in vivo behaviour.

Comparison between [18F]fluorination and [18F]fluoroethylation reactions for the synthesis of the PDE10A PET radiotracer [18F]MNI-659.

INTRODUCTION: 2-(2-(3-(4-(2-[18F]Fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione ([18F]MNI-659, [18F]1) is a useful PET radiotracer for imaging phosphodiesterase 10A (PDE10A) in human brain. [18F]1 has been previously prepared by direct [18F]fluorination of a tosylate precursor 2 with [18F]F-. The aim of this study was to determine the conditions for the [18F]fluorination reaction to obtain [18F]1 of high quality and with sufficient radioactivity for clinical use in our institute. Moreover, we synthesized [18F]1 by [18F]fluoroethylation of a phenol precursor 3 with [18F]fluoroethyl bromide ([18F]FEtBr), and the outcomes of [18F]fluorination and [18F]fluoroethylation were compared. METHODS: We performed the automated synthesis of [18F]1 by [18F]fluorination and [18F]fluoroethylation using a multi-purpose synthesizer. We determined the amounts of tosylate precursor 2 and potassium carbonate as well as the reaction temperature for direct [18F]fluorination. RESULTS: The efficiency of the [18F]fluorination reaction was strongly affected by the amount of 2 and potassium carbonate. Under the determined reaction conditions, [18F]1 with 0.82±0.2GBq was obtained in 13.6%±3.3% radiochemical yield (n=8, decay-corrected to EOB and based on [18F]F-) at EOS, starting from 11.5±0.4GBq of cyclotron-produced [18F]F-. On the other hand, the [18F]fluoroethylation of 3 with [18F]FEtBr produced [18F]1 with 1.0±0.2GBq and in 22.5±2.5 % radiochemical yields (n=7, decay-corrected to EOB and based on [18F]F-) at EOS, starting from 7.4GBq of cyclotron-produced [18F]F-. Clearly, [18F]fluoroethylation resulted in a higher radiochemical yield of [18F]1 than [18F]fluorination. CONCLUSION: [18F]1 of high quality and with sufficient radioactivity was successfully radiosynthesized by two methods. [18F]1 synthesized by direct [18F]fluorination has been approved and will be provided for clinical use in our institute.

Developing new PET tracers to image the growth hormone secretagogue receptor 1a (GHS-R1a).

INTRODUCTION: `The growth hormone secretagogue receptor 1a (GHS-R1a) is the orphan G-protein-coupled receptor, and its endogenous ligand is ghrelin. GHS-R1a contributes to regulation of glucose homeostasis, memory and learning, food addiction, and neuroprotection. Several PET tracers for GHS-R1a have been developed, but none have been reported to be clinically applicable to GHS-R1a imaging. In this study, we developed three new PET tracers for GHS-R1a: 18F-labeled 6-(4-chlorophenyl)-3-((1-(2-fluoroethyl)piperidin-3-yl)methyl)-2-(o-tolyl)quinazolin-4(3H)-one (1), 11C-labeled 6-(4-chlorophenyl)-3-((1-(2-methoxyethyl)piperidin-3-yl)methyl)-2-(o-tolyl)quinazolin-4(3H)-one (2), and 11C-labeled (S)-(4-(1H-indole-6-carbonyl)-3-methylpiperazin-1-yl)(4'-methoxy-[1,1'-biphenyl]-4-yl)methanone (3). METHODS: [18F]1 was synthesized by the 18F-fluoroethylation; [11C]2 or [11C]3 was synthesized by the 11C-methylation. Biodistribution studies and PET studies were conducted in mice. RESULTS: We successfully radiosynthesized [18F]1, [11C]2, and [11C]3 with appropriate radioactivity for the animal study. In the ex vivo biodistribution study, 60min following injection, the radioactivity level of [18F]1 was relatively high in the small intestine, that of [11C]2 was high in the liver, and that of [11C]3 was high in the pancreas. The radioactivity levels of the three PET tracers were relatively low in the brain. Under pretreatment with YIL781 (a selective and high affinity antagonist for GHS-R1a), the pancreas radioactivity level at 30min following [11C]3 injection was significantly reduced to 55% of control, but the radioactivity in the brain was not changed. In the PET study under control conditions, high radioactivity levels in the liver and pancreas were observed following [11C]3 injection. With YIL781 pretreatment, the accumulated radioactivity in the pancreas 15-60min after [11C]3 injection was significantly decreased to 78% of control. CONCLUSION: [11C]3 exhibited relatively high uptake and in vivo specific binding to GHS-R1a in the mouse pancreas. [11C]3 may be a useful PET tracers for in vivo imaging of GHS-R1a in the pancreas.

Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein.

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.