Fabrice G. Siméon

Single-Step High-Yield Radiosynthesis and Evaluation of a Sensitive 18F-Labeled Ligand for Imaging Brain Peripheral Benzodiazepine Receptors with PET

Elevated levels of peripheral benzodiazepine receptors (PBR) are associated with activated microglia in their response to inflammation. Hence, PBR imaging in vivo is valuable for investigating brain inflammatory conditions. Sensitive, easily prepared, and readily available radioligands for imaging with positron emission tomography (PET) are desirable for this purpose. We describe a new 18F-labeled PBR radioligand, namely [18F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([18F]9). [18F]9 was produced easily through a single and highly efficient step, the reaction of [18F]fluoride ion with the corresponding bromo precursor, 8. Ligand 9 exhibited high affinity for PBR in vitro. PET showed that [18F]9 was avidly taken into monkey brain and gave a high ratio of PBR-specific to nonspecific binding. [18F]9 was devoid of defluorination in rat and monkey and gave predominantly polar radiometabolite(s). In rat, a low level radiometabolite of intermediate lipophilicity was identified as [18F]2-fluoro-N-(2-phenoxyphenyl)acetamide ([18F]11). [18F]9 is a promising radioligand for future imaging of PBR in living human brain.

Quantification of Translocator Protein (18 kDa) in the Human Brain with PET and a Novel Radioligand, 18F-PBR06

Translocator protein (TSPO) (18 kDa), formerly called the peripheral benzodiazepine receptor, is upregulated on activated microglia and macrophages and is, thus, a biomarker of inflammation. We previously reported that an 11C-labeled aryloxyanilide (half-life, 20 min) was able to quantify TSPOs in the healthy human brain. Because many PET centers would benefit from a longer-lived 18F-labeled radioligand (half-life, 110 min), the objective of this study was to evaluate the ability of a closely related aryloxyanilide (18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline [18F-PBR06]) to quantify TSPOs in the healthy human brain. Methods: A total of 9 human subjects were injected with 18F-PBR06 (∼185 MBq) and scanned for 5 h, with rest periods outside the camera. The concentrations of 18F-PBR06, separated from radiometabolites, were measured in arterial plasma. Results: Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model. Even if data for all time points were used for the fitting, concentrations of brain activity measured with PET were consistently greater than the modeled values at late (280–300 min) but not at early time points. The greater values may have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume (VT), which is the summation of receptor binding and nondisplaceable activity, we investigated which scan duration would be associated with maximal or near-maximal identifiability. We found that a scan of 120 min provided the best identifiability of VT (∼2%). The images showed no significant defluorination. Conclusion: 18F-PBR06 can quantify TSPOs in the healthy human brain using 120 min of image acquisition and concurrent measurements of radioligand in plasma. Although brain activity is likely contaminated with radiometabolites, the percentage contamination is thought to be small (<10%), because values of distribution volume are stable during 60–120 min and vary by less than 10%. 18F-PBR06 is a longer-lived and promising alternative to 11C-labeled radioligands to measure TSPOs as a biomarker of inflammation in the brain.

Metabotropic Glutamate Subtype 5 Receptors Are Quantified in the Human Brain with a Novel Radioligand for PET

We developed a radioligand, 3-fluoro-5-(2-(2-18F-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile (18F-SP203), for metabotropic glutamate subtype 5 (mGluR5) receptors that showed both promising (high specific binding) and problematic (defluorination) imaging characteristics in animals. The purposes of this initial evaluation in human subjects were to determine whether 18F-SP203 is defluorinated in vivo (as measured by uptake of radioactivity in the skull) and to determine whether the uptake in the brain can be quantified as distribution volume relative to concentrations of 18F-SP203 in plasma. Methods: Seven healthy subjects were injected with 18F-SP203 (323 ± 87 MBq) and scanned over 5 h, with rest periods outside the camera. The concentrations of 18F-SP203, separated from radiometabolites, were measured in arterial plasma. Results: The skull was difficult to visualize on PET images in the initial 2 h, because of high radioactivity in the brain. Although radioactivity in the skull and adjacent cortex showed some cross-contamination, the concentration of radioactivity in the skull was less than half of that in the adjacent cortex during the initial 2 h. Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model, consistent with measurable amounts of both receptor-specific and nonspecific binding. The concentrations of activity in the brain measured with PET were consistently greater than the modeled values at late but not early time points and may well have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume, we selected a scan duration (i.e., 2 h) associated with maximal or near-maximal identifiability. Distribution volume was well identified (∼2%) by only 2 h (and even just 1) of image acquisition. Conclusion: This initial evaluation of 18F-SP203 in healthy human subjects showed that defluorination is relatively small and that brain uptake can be robustly calculated as distribution volume. The values of distribution volume were well identified and had relatively small variation in this group of 7 subjects. These results suggest that 18F-SP203 will have good sensitivity to measure mGluR5 receptors for both within-subject studies (e.g., receptor occupancy) and between-subject studies (e.g., patients vs. healthy subjects).

Radiodefluorination of 3-Fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a Radioligand for Imaging Brain Metabotropic Glutamate Subtype-5 Receptors with Positron Emission Tomography, Occurs by Glutathionylation in Rat Brain

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[18F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [18F]-fluoride ion from [18F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [18F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [18F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [18F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [18F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M + H]+ of S-glutathione (SP203Glu) and N-acetyl-S-l-cysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M + H]+ of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [18F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.

Biodistribution and Radiation Dosimetry in Humans of a New PET Ligand, 18F-PBR06, to Image Translocator Protein (18 kDa)

As a PET biomarker for inflammation, translocator protein (18 kDa) (TSPO) can be measured with an 18F-labeled aryloxyanilide, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), in the human brain. The objective of this study was to estimate the radiation absorbed doses of 18F-PBR06 based on biodistribution data in humans. Methods: After the injection of 18F-PBR06, images were acquired from head to thigh in 7 healthy humans. Urine was collected at various time points. Radiation absorbed doses were estimated by the MIRD scheme. Results: Moderate to high levels of radioactivity were observed in organs with high densities of TSPO and in organs of metabolism and excretion. Bone had low levels of radioactivity. The effective dose was 18.5 μSv/MBq. Conclusion: The effective dose of 18F-PBR06, compared with other 18F radioligands, was moderate. This radioligand had negligible defluorination, as indirectly assessed by bone radioactivity. Doses to the gallbladder wall and spleen may limit the amount of permissible injected radioactivity.

Quantification of metabotropic glutamate subtype 5 receptors in the brain by an equilibrium method using 18F-SP203

A new PET ligand, 3-fluoro-5-(2-(2-(18)F-(fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile (18F-SP203) can quantify metabotropic glutamate subtype 5 receptors (mGluR5) in human brain by a bolus injection and kinetic modeling. As an alternative approach to a bolus injection, binding can simply be measured as a ratio of tissue to metabolite-corrected plasma at a single time point under equilibrium conditions achieved by administering the radioligand with a bolus injection followed by a constant infusion. The purpose of this study was to validate the equilibrium method as an alternative to the standard kinetic method for measuring 18F-SP203 binding in the brain. Nine healthy subjects were injected with 18F-SP203 using a bolus plus constant infusion for 300 min. A single ratio of bolus-to-constant infusion (the activity of bolus equaled to that of infusion over 219 min) was applied to all subjects to achieve equilibrium in approximately 120 min. As a measure of ligand binding, we compared total distribution volume (VT) calculated by the equilibrium and kinetic methods in each scan. The equilibrium method calculated VT by the ratio of radioactivity in the brain to the concentration of 18F-SP203 in arterial plasma at 120 min, and the kinetic method calculated VT by a two-tissue compartment model using brain and plasma dynamic data from 0 to 120 min. VT obtained via the equilibrium method was highly correlated with VT obtained via kinetic modeling. Inter-subject variability of VT obtained via the equilibrium method was slightly smaller than VT obtained via the kinetic method. VT obtained via the equilibrium method was ~10% higher than VT obtained via the kinetic method, indicating a small difference between the measurements. Taken together, the results of this study show that using the equilibrium method is an acceptable alternative to the standard kinetic method when using 18F-SP203 to measure mGluR5. Although small differences in the measurements obtained via the equilibrium and kinetic methods exist, both methods consistently measured mGluR5 as indicated by the highly correlated VT values; the equilibrium method was slightly more precise, as indirectly measured by the smaller coefficient of variability across subjects. In addition, when using 18F-SP203, the equilibrium method is more efficient because it requires much less data.

Efficient and regioselective halogenations of 2-amino-1,3-thiazoles with copper salts.

Monohalo and dihalo 1,3-thiazole derivatives can be efficiently and selectively prepared under mild conditions from 2-amino-1,3-thiazoles. Halogenations proceed easily in the presence of copper(I) or copper(II) chlorides, bromides, or iodides directly in solution or with supported copper halides.

Comparison of two PET radioligands, [11C]FPEB and [11C]SP203, for quantification of metabotropic glutamate receptor 5 in human brain

Of the two 18F-labeled PET ligands currently available to image metabotropic glutamate receptor 5 (mGluR5), [18F]FPEB is reportedly superior because [18F]SP203 undergoes glutathionlyation, generating [18F]-fluoride ion that accumulates in brain and skull. To allow multiple PET studies on the same day with lower radiation exposure, we prepared [11C]FPEB and [11C]SP203 from [11C]hydrogen cyanide and compared their abilities to accurately quantify mGluR5 in human brain, especially as regards radiometabolite accumulation. Genomic plot was used to estimate the ratio of specific-to-nondisplaceable uptake (BPND) without using a receptor blocking drug. Both tracers quantified mGluR5; however [11C]SP203, like [18F]SP203, had radiometabolite accumulation in brain, as evidenced by increased distribution volume (VT) over the scan period. Absolute VT values were ∼30% lower for 11C-labeled compared with 18F-labeled radioligands, likely caused by the lower specific activities (and high receptor occupancies) of the 11C radioligands. The genomic plot indicated ∼60% specific binding in cerebellum, which makes it inappropriate as a reference region. Whole-body scans performed in healthy subjects demonstrated a low radiation burden typical for 11C-ligands. Thus, the evidence suggests that [11C]FPEB is superior to [11C]SP203. If prepared in higher specific activity, [11C]FPEB would presumably be as effective as [18F]FPEB for quantifying mGluR5 in human brain.

11C-Labeling of Aryl Ketones as Candidate Histamine Subtype-3 Receptor PET Radioligands through Pd(0)-Mediated 11C-Carbonylative Coupling

Pd(0)-mediated coupling between iodoarenes, [11C]carbon monoxide and aryltributylstannanes has been used to prepare simple model [11C]aryl ketones. Here, we aimed to label four 2-aminoethylbenzofuran chemotype based molecules ([11C]1–4) in the carbonyl position, as prospective positron emission tomography (PET) radioligands for the histamine subtype 3 receptor (H3R) by adapting this methodology with use of aryltrimethylstannanes. Radiosynthesis was successfully performed on a platform equipped with a mini-autoclave and a liquid handling robotic arm, within a lead-shielded hot-cell. Candidate radioligands were readily formulated in saline containing ethanol (10%, v/v) and ascorbic acid (0.5 mg/10 mL). Yields for preclinical use were in the range of 5–9%, decay-corrected from cyclotron-produced [11C]CO2 and molar activities were >115 GBq/µmol at end of synthesis. Radiochemical purities exceeded >97%.