Hiroki Hashimoto

Radiosynthesis, Photoisomerization, Biodistribution, and Metabolite Analysis of 11C-PBB3 as a Clinically Useful PET Probe for Imaging of Tau Pathology

2-((1E,3E)-4-(6-(11C-methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol (11C-PBB3) is a clinically useful PET probe that we developed for in vivo imaging of tau pathology in the human brain. To ensure the availability of this probe among multiple PET facilities, in the present study we established protocols for the radiosynthesis and quality control of 11C-PBB3 and for the characterization of its photoisomerization, biodistribution, and metabolism. Methods: 11C-PBB3 was synthesized by reaction of the tert-butyldimethylsilyl desmethyl precursor (1) with 11C-methyl iodide using potassium hydroxide as a base, followed by deprotection. Photoisomerization of 11C-PBB3 under fluorescent light was determined. The biodistribution and metabolite analysis of 11C-PBB3 was determined in mice using the dissection method. Results: 11C-PBB3 was synthesized with 15.4% ± 2.8% radiochemical yield (decay-corrected, n = 50) based on the cyclotron-produced 11C-CO2 and showed an averaged synthesis time of 35 min from the end of bombardment. The radiochemical purity and specific activity of 11C-PBB3 were 98.0% ± 2.3% and 180.2 ± 44.3 GBq/μmol, respectively, at the end of synthesis (n = 50). 11C-PBB3 showed rapid photoisomerization, and its radiochemical purity decreased to approximately 50% at 10 min after exposure to fluorescent light. After the fluorescent light was switched off, 11C-PBB3 retained more than 95% radiochemical purity over 60 min. A suitable brain uptake (1.92% injected dose/g tissue) of radioactivity was observed at 1 min after the probe injection, which was followed by rapid washout from the brain tissue. More than 70% of total radioactivity in the mouse brain homogenate at 5 min after injection represented the unchanged 11C-PBB3, despite its rapid metabolism in the plasma. Conclusion: 11C-PBB3 was produced with sufficient radioactivity and high quality, demonstrating its clinical utility. The present results of radiosynthesis, photoisomerization, biodistribution, and metabolite analysis could be helpful for the reliable production and application of 11C-PBB3 in diverse PET facilities.

Identification of a major radiometabolite of [11C]PBB3.

INTRODUCTION [(11)C]PBB3 is a clinically used positron emission tomography (PET) probe for in vivo imaging of tau pathology in the brain. Our previous study showed that [(11)C]PBB3 was rapidly decomposed to a polar radiometabolite in the plasma of mice. For the pharmacokinetic evaluation of [(11)C]PBB3 it is important to elucidate the characteristics of radiometabolites. In this study, we identified the chemical structure of a major radiometabolite of [(11)C]PBB3 and proposed the metabolic pathway of [(11)C]PBB3. METHODS Carrier-added [(11)C]PBB3 was injected into a mouse for in vivo metabolite analysis. The chemical structure of a major radiometabolite was identified using LC-MS. Mouse and human liver microsomes and liver S9 samples were incubated with [(11)C]PBB3 in vitro. In silico prediction software was used to assist in the determination of the metabolite and metabolic pathway of [(11)C]PBB3. RESULTS In vivo analysis showed that the molecular weight of a major radiometabolite of [(11)C]PBB3, which was called as [(11)C]M2, was m/z 390 [M+H(+)]. In vitro analysis assisted by in silico prediction showed that [(11)C]M2, which was not generated by cytochrome P450 enzymes (CYPs), was generated by sulfated conjugation mediated by a sulfotransferase. CONCLUSION The major radiometabolite, [(11)C]M2, was identified as a sulfated conjugate of [(11)C]PBB3. [(11)C]PBB3 was metabolized mainly by a sulfotransferase and subsidiarily by CYPs.

Binding potential of (E)-[11C]ABP688 to metabotropic glutamate receptor subtype 5 is decreased by the inclusion of its 11C-labelled Z-isomer

INTRODUCTION [(11)C]ABP688 is a promising positron emission tomography (PET) ligand for imaging of metabotropic glutamate receptor subtype 5 (mGlu5 receptor). Of the two geometric isomers of ABP688, (E)-ABP688 has a greater affinity towards mGlu5 receptors than (Z)-ABP688. Therefore, a high ratio of E-isomer is required when using [(11)C]ABP688 as a PET probe for imaging and quantification of mGlu5 receptors. The aim of this study was to evaluate the effect (Z)-[(11)C]ABP688 on the synthesis of [(11)C]ABP688 to be used for binding (E)-[(11)C]ABP688 in the brain. METHODS We synthesized and separated (E)- and (Z)-[(11)C]ABP688 by purification using an improved preparative high-performance liquid chromatography (HPLC) method equipped with a COSMOSIL Cholester column. We performed an in vitro binding assay in rat brain homogenates and PET studies of the rat brains using (E)- and (Z)-[(11)C]ABP688. RESULTS (E)- and (Z)-[(11)C]ABP688 were successfully obtained with suitable radioactivity for application. In the in vitro assay, the Kd value of (E)-[(11)C]ABP688 (5.7 nmol/L) was higher than that of (Z)-[(11)C]ABP688 (140 nmol/L). In the PET study of the rat brain, high radioactivity after injection of (E)-[(11)C]ABP688 was observed in regions rich in mGlu5 receptors such as the striatum and hippocampus. In contrast, after injection of (Z)-[(11)C]ABP688, radioactivity did not accumulate in the brain. Furthermore, BPND in the striatum and hippocampus was highly correlated (R(2) = 0.99) with the percentage of (E)-[(11)C]ABP688 of the total radioactivity of (E)- and (Z)-[(11)C]ABP688 in the injection. CONCLUSION We demonstrated that including (Z)-[(11)C]ABP688 in the [(11)C]ABP688 injection can decrease BPND in regions rich in mGlu5 receptors. Routine production of (E)-[(11)C]ABP688 will be helpful for imaging and quantification of mGlu5 receptors in clinical studies.

Synthesis, metabolite analysis, and in vivo evaluation of [11C]irinotecan as a novel positron emission tomography (PET) probe

INTRODUCTION Irinotecan is a semisynthetic derivative of camptothecin that exerts potent antitumor activity by inhibiting topoisomerase I. Despite much research into the complex pharmacokinetic profile and pharmacodynamic effects of irinotecan, unpredictable and severe side effects are still commonly observed. In this study, we synthesized [(11)C]irinotecan as a positron emission tomography (PET) probe, performed the metabolite analysis, and evaluated the biodistribution and kinetics of [(11)C]irinotecan using small animal PET. METHODS [(11)C]Irinotecan was synthesized by two routes using [(11)C]phosgene and [(11)C]carbon dioxide fixation. Metabolites in the plasma of mice following injection of [(11)C] irinotecan were investigated using a combination of column-switching high-performance liquid chromatography (HPLC) and on-line solid-phase extraction (SPE). Whole-body PET studies were conducted in wild-type mice and P-glycoprotein and breast cancer resistance protein (Pgp/Bcrp) knockout mice. RESULTS [(11)C]Irinotecan was successfully synthesized by the two abovementioned routes. Decay-corrected radiochemical yields based on [(11)C]carbon dioxide using [(11)C]phosgene and [(11)C]carbon dioxide fixation were 8.8 ± 2.0% (n=8) and 16.9 ± 2.9 % (n=5), respectively. Metabolite analysis of the plasma of mice following injection of [(11)C]irinotecan was successfully performed using the column-switching HPLC and on-line SPE combination resulting in greater than 87 % recovery of radioactivity from HPLC. In the PET study in mice, the radioactivity levels in the brain, liver, and small intestine were slightly increased by inhibition of the Pgp/Bcrp function for more than 30 min after [(11)C]irinotecan injection. This result demonstrated that in vivo behavior of [(11)C] irinotecan and radioactive metabolites are influenced by the Pgp/Bcrp function. CONCLUSION PET studies using [(11)C]irinotecan combined with metabolite analysis may be a useful tool for evaluating irinotecan pharmacokinetics and toxicity.

Dynamic Changes in Striatal mGluR1 But Not mGluR5 during Pathological Progression of Parkinson's Disease in Human Alpha-Synuclein A53T Transgenic Rats: A Multi-PET Imaging Study

Parkinsons disease (PD) is a prevalent degenerative disorder affecting the CNS that is primarily characterized by resting tremor and movement deficits. Group I metabotropic glutamate receptor subtypes 1 and 5 (mGluR1 and mGluR5, respectively) are important targets for investigation in several CNS disorders. In the present study, we investigated the in vivo roles of mGluR1 and mGluR5 in chronic PD pathology by performing longitudinal positron emission tomography (PET) imaging in A53T transgenic (A53T-Tg) rats expressing an abnormal human α-synuclein (ASN) gene. A53T-Tg rats showed a dramatic decline in general motor activities with age, along with abnormal ASN aggregation and striatal neuron degeneration. In longitudinal PET imaging, striatal nondisplaceable binding potential (BPND) values for [11C]ITDM (N-[4-[6-(isopropylamino) pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methyl-4-[11C]methylbenzamide), a selective PET ligand for mGluR1, temporarily increased before PD symptom onset and dramatically decreased afterward with age. However, striatal BPND values for (E)-[11C]ABP688 [3-(6-methylpyridin-2-ylethynyl)-cyclohex-2-enone-(E)-O-[11C]methyloxime], a specific PET ligand for mGluR5, remained constant during experimental terms. The dynamic changes in striatal mGluR1 BPND values also showed a high correlation in pathological decreases in general motor activities. Furthermore, declines in mGluR1 BPND values were correlated with decreases in BPND values for [18F]FE-PE2I [(E)-N-(3-iodoprop-2E-enyl)-2β-carbo-[18F]fluoroethoxy-3β-(4-methylphenyl) nortropane], a specific PET ligand for the dopamine transporter, a biomarker for dopaminergic neurons. In conclusion, our results have demonstrated for the first time that dynamic changes occur in mGluR1, but not mGluR5, that accompany pathological progression in a PD animal model. SIGNIFICANCE STATEMENT Synaptic signaling by glutamate, the principal excitatory neurotransmitter in the brain, is modulated by group I metabotropic glutamate receptors, including the mGluR1 and mGluR5 subtypes. In the brain, mGluR1 and mGluR5 have distinct functional roles and regional distributions. Their roles in brain pathology, however, are not well characterized. Using longitudinal PET imaging in a chronic rat model of PD, we demonstrated that expression of mGluR1, but not mGluR5, dynamically changed in the striatum accompanying pathological PD progression. These findings imply that monitoring mGluR1 in vivo may provide beneficial information to further understand central nervous system disorders.

Carbon-11 radiolabeling of an oligopeptide containing tryptophan hydrochloride via a Pictet-Spengler reaction using carbon-11 formaldehyde

A procedure for the synthesis of a11C‐labeled oligopeptide containing [1‐11C]1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ([1‐11C]Tpi) from the corresponding Trp•HCl‐containing peptides has been developed involving a Pictet‐Spengler reaction with [11C]formaldehyde. The synthesis of [1‐11C]Tpi from Trp and [11C]formaldehyde was examined as a model reaction with the aim of developing a facile and effective method for the labeling of peptides with carbon‐11. The Pictet‐Spengler reaction of Trp and [11C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1‐11C]Tpi in a moderate radiochemical yield. Herein, the application of a Pictet‐Spengler reaction to an aqueous solution of Trp•HCl gave the desired product with a radiochemical yield of 45.2%. The RGD peptide cyclo[Arg‐Gly‐Asp‐D‐Tyr‐Lys] was then selected as a substrate for the labeling reaction with [11C]formaldehyde. The radiolabeling of a Trp•HCl‐containing RGD peptide using the Pictet‐Spengler reaction was successful. Furthermore, the remote‐controlled synthesis of a [1‐11C]Tpi‐containing RGD peptide was attempted by using an automatic production system to generate [11C]CH3I. The radiochemical yield of the [1‐11C]Tpi‐containing RGD at the end of synthesis (EOS) was 5.9 ± 1.9% (n = 4), for a total synthesis time of about 35 min. The specific activity was 85.7 ± 9.4 GBq/µmol at the EOS. Copyright

The use of tetrabutylammonium fluoride to promote N‐ and O‐11C‐methylation reactions with iodo[11C]methane in dimethyl sulfoxide

The N- or O-methylation reactions of compounds bearing amide, aniline, or phenol moieties using iodo[(11) C]methane (1) with the aid of a base are frequently applied to the preparation of (11) C-labeled radiopharmaceuticals. Although sodium hydride and alkaline metal hydroxides are commonly employed as bases in these reactions, their poor solubility properties in organic solvents and hydrolytic activities have sometimes limited their application and made the associated (11) C-methylation reactions difficult. In contrast to these bases, tetrabutylammonium fluoride (TBAF) is moderately basic, highly soluble in organic solvents, and weakly nucleophilic. Although it was envisaged that TBAF could be used as the preferred base for (11) C-methylation reactions using 1, studies concerning the use of TBAF to promote (11) C-methylation reactions are scarce. Herein, we have evaluated the efficiency of the (11) C-methylation reactions of 13 model compounds using TBAF and 1. In most cases, the N-(11) C-methylations were efficiently promoted by TBAF in dimethyl sulfoxide at ambient temperature, whereas the O-(11) C-methylations required heating in some cases. Comparison studies revealed that the efficiencies of the (11) C-methylation reactions with TBAF were comparable or sometimes greater than those conducted with sodium hydride. Based on these results, TBAF should be considered as the preferred base for (11) C-methylation reactions using 1.

Radiosynthesis and quality control of [11C]TASP457 as a clinically useful PET ligand for imaging of histamine H3 receptors in human brain

INTRODUCTION Recently, 6-[(1-cyclobutylpiperidin-4-yl)oxy]-1-(6-[11C]methoxypyridin-3-yl)-3,4-dihydroquinolin-2(1H)-one ([11C]TASP457, [11C]2) has been developed as a novel PET ligand for histamine H3 receptors in brain. [11C]2 is potentially suitable for imaging H3 receptors in rat and monkey brains, which has motivated us to perform first-in-human study of [11C]2 for qualifying H3 receptors in human brain. In this paper, we report an efficient radiosynthesis of [11C]2 to obtain sufficient radioactivity and high quality for clinical application. METHODS In manual synthesis, we optimized the reaction conditions of desmethyl precursor 1, which contains a 2-hydroxypyridine moiety, with [11C]MeI or [11C]MeOTf. After optimization, we performed automated synthesis and quality control of [11C]2. RESULTS Bubbling [11C]MeOTf into a heated mixture of precursor 1 and cesium carbonate in DMF at 100°C for 90s produced [11C]2 with decay-corrected radiochemical yields of (based on [11C]CO2) 7.9±1.8% (n=78). The specific activity of [11C]2 was 156±52GBq/μmol (n=78) at the end of synthesis. The total synthesis time was approximately 35min from the end of bombardment. All the quality control results of [11C]2 were in compliance with our in-house quality control/assurance specifications. CONCLUSION We radiosynthesized [11C]TASP457 ([11C]2) with sufficient amounts of radioactivity and high quality for clinical usefulness. This radioligand is being used for PET assessment of H3 receptors in human brain in our facility.

Efficient radiosynthesis and non-clinical safety tests of the TSPO radioprobe [18F]FEDAC: Prerequisites for clinical application

INTRODUCTION [(18)F]FEDAC ([(18)F]1) has potent binding affinity and selectivity for translocator protein (18kDa, TSPO), and has been used to noninvasively visualize neuroinflammation, lung inflammation, acute liver damage, nonalcoholic fatty liver disease, and liver fibrosis. We had previously synthesized [(18)F]1 in two steps: (i) preparation of [(18)F]fluoroethyl bromide and (ii) coupling of [(18)F]fluoroethyl bromide with the appropriate precursor (2) for labeling. In this study, to clinically utilize [(18)F]1 as a PET radiopharmaceutical and to transfer the production technique of [(18)F]1 to other PET centers, we simplified its preparation by using a direct, one-step, tosyloxy-for-fluorine substitution. We also performed an acute toxicity study as a major non-clinical safety test, and determined radiometabolites using human liver microsomes. METHODS [(18)F]1 was prepared via direct (18)F-fluorination by heating the corresponding tosylated derivative (3) with [(18)F]fluoride as its Kryptofix 222 complex in dimethyl sulfoxide at 110°C for 15min, following by HPLC purification. Non-clinical safety tests were performed for the extended single-dose toxicity study in rats, and for the in vitro metabolite analysis with human liver microsomal incubation. RESULTS High quality batches of [(18)F]1, compatible with clinical applications, were obtained. At the end of irradiation, the decay-corrected radiochemical yield of [(18)F]1 using 1 and 5mg of precursor based on [(18)F]fluoride was 18.5±7.9% (n=10) and 52.0±5.8% (n=3), respectively. A single-dose of [(18)F]1 did not show toxicological effects for 14 days after the injection in male and female rats. In human liver microsomal incubations, [(18)F]1 was easily metabolized to [(18)F]desbenzyl-FEDAC ([(18)F]10) by CYPs (4.2% of parent compound left 60min after incubation). CONCLUSION We successfully synthesized clinical grade batches of [(18)F]1 and verified the absence of innocuity of this radiotracer. [(18)F]1 will be used to first-in-human studies in our facility.

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.