Longitudinal [18F]GE-180 PET Imaging Facilitates In Vivo Monitoring of TSPO Expression in the GL261 Glioblastoma Mouse Model.

The 18 kDa translocator protein (TSPO) is increasingly recognized as an interesting target for the imaging of glioblastoma (GBM). Here, we investigated TSPO PET imaging and autoradiography in the frequently used GL261 glioblastoma mouse model and aimed to generate insights into the temporal evolution of TSPO radioligand uptake in glioblastoma in a preclinical setting. We performed a longitudinal [18F]GE-180 PET imaging study from day 4 to 14 post inoculation in the orthotopic syngeneic GL261 GBM mouse model (n = 21 GBM mice, n = 3 sham mice). Contrast-enhanced computed tomography (CT) was performed at the day of the final PET scan (±1 day). [18F]GE-180 autoradiography was performed on day 7, 11 and 14 (ex vivo: n = 13 GBM mice, n = 1 sham mouse; in vitro: n = 21 GBM mice; n = 2 sham mice). Brain sections were also used for hematoxylin and eosin (H&E) staining and TSPO immunohistochemistry. [18F]GE-180 uptake in PET was elevated at the site of inoculation in GBM mice as compared to sham mice at day 11 and later (at day 14, TBRmax +27% compared to sham mice, p = 0.001). In GBM mice, [18F]GE-180 uptake continuously increased over time, e.g., at day 11, mean TBRmax +16% compared to day 4, p = 0.011. [18F]GE-180 uptake as depicted by PET was in all mice co-localized with contrast-enhancement in CT and tissue-based findings. [18F]GE-180 ex vivo and in vitro autoradiography showed highly congruent tracer distribution (r = 0.99, n = 13, p < 0.001). In conclusion, [18F]GE-180 PET imaging facilitates non-invasive in vivo monitoring of TSPO expression in the GL261 GBM mouse model. [18F]GE-180 in vitro autoradiography is a convenient surrogate for ex vivo autoradiography, allowing for straightforward identification of suitable models and scan time-points on previously generated tissue sections.

Radiosynthesis and Biological Investigation of a Novel Fluorine-18 Labeled Benzoimidazotriazine- Based Radioligand for the Imaging of Phosphodiesterase 2A with Positron Emission Tomography.

A specific radioligand for the imaging of cyclic nucleotide phosphodiesterase 2A (PDE2A) via positron emission tomography (PET) would be helpful for research on the physiology and disease-related changes in the expression of this enzyme in the brain. In this report, the radiosynthesis of a novel PDE2A radioligand and the subsequent biological evaluation were described. Our prospective compound 1-(2-chloro-5-methoxy phenyl)-8-(2-fluoropyridin-4-yl)-3- methylbenzo[e]imidazo[5,1-c][1,2,4]triazine, benzoimidazotriazine (BIT1) (IC50 PDE2A = 3.33 nM; 16-fold selectivity over PDE10A) was fluorine-18 labeled via aromatic nucleophilic substitution of the corresponding nitro precursor using the K[18F]F-K2.2.2-carbonate complex system. The new radioligand [18F]BIT1 was obtained with a high radiochemical yield (54 ± 2%, n = 3), a high radiochemical purity (≥99%), and high molar activities (155-175 GBq/µmol, n = 3). In vitro autoradiography on pig brain cryosections exhibited a heterogeneous spatial distribution of [18F]BIT1 corresponding to the known pattern of expression of PDE2A. The investigation of in vivo metabolism of [18F]BIT1 in a mouse revealed sufficient metabolic stability. PET studies in mouse exhibited a moderate brain uptake of [18F]BIT1 with a maximum standardized uptake value of ~0.7 at 5 minutes p.i. However, in vivo blocking studies revealed a non-target specific binding of [18F]BIT1. Therefore, further structural modifications are needed to improve target selectivity.

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain.

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([11C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC50: 26 nM) for mGluR2 overexpressed in human cells. [11C]1 was synthesized by O-[11C]methylation of the phenol precursor 2 with [11C]methyl iodide. After the reaction, HPLC purification and formulation, [11C]1 of 7.4 ±â€¯2.8 GBq (n = 8) was obtained from [11C]carbon dioxide of 22.5 ±â€¯4.8 GBq (n = 8) with >99% radiochemical purity and 70 ±â€¯32 GBq/µmol (n = 8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [11C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [11C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [11C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.

Synthesis and in vitro evaluation of three novel radiotracers for imaging of metabotropic glutamate receptor subtype 2 in rat brain.

The purpose of this study was to develop three new radiotracers, 1-(cyclopropylmethyl)-4-([11C/18F]substituted-phenyl)piperidin-1-yl-2-oxo-1,2-dihydropyridine-3-carbonitrile ([11C]1, [11C]2, and [18F]4), and to examine their specific bindings with metabotropic glutamate receptor subtype 2 (mGluR2) in rat brain sections by using in vitro autoradiography. These compounds were found to possess potent in vitro binding affinities (Ki: 8.0-34.1nM) for mGluR2 in rat brain homogenate. [11C]1, [11C]2, and [18F]4 were synthesized by [11C/18F]alkylation of the corresponding phenol precursors with [11C]methyl iodide or [18F]fluoroethyl bromide with >98% radiochemical purity and 80-130GBq/µmol specific activity at the end of synthesis. In vitro autoradiography indicated that these radiotracers showed heterogeneous specific bindings in mGluR2-rich brain regions, such as the cerebral cortex, striatum, hippocampus, and granular layer of the cerebellum.

Radiosynthesis and Evaluation of [11C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites.

γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report the 11C-labeling and subsequent evaluation of [11C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative autoradiography on sections of pig brain was performed using [3H]HOCPCA. In vivo evaluation of [11C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [11C]HOCPCA.

Synthesis and evaluation of 1-(cyclopropylmethyl)-4-(4-[11C]methoxyphenyl)-piperidin-1-yl-2-oxo-1,2-dihydropyridine-3-carbonitrile ([11C]CMDC) for PET imaging of metabotropic glutamate receptor 2 in the rat brain.

Brain metabotropic glutamate receptor 2 (mGluR2) has been proposed as a therapeutic target for the treatment of schizophrenia-like symptoms arising from increased glutamate transmission in the forebrain. However, there does not exist a reliable tool for the study of mGluR2 in human neuroimaging. The purpose of this study was to radiosynthesize 1-(cyclopropylmethyl)-4-(4-[11C]methoxyphenyl)piperidin-1-yl-2-oxo-1,2-dihydropyridine-3-carbonitrile ([11C]CMDC) and evaluate its potential as a positron emission tomography (PET) radiotracer for imaging mGluR2 in the rat brain. CMDC, a positive allosteric modulator of mGluR2, showed potent functional activity (EC50: 98nM) for human mGluR2 in vitro. [11C]CMDC was synthesized by O-[11C]methylation of 1-(cyclopropylmethyl)-4-(4-hydroxyphenyl)piperidin-1-yl-2-oxo-1,2-dihydropyridine-3-carbonitrile (1) with [11C]methyl iodide. [11C]CMDC (2.2±0.9GBq; n=20) was obtained from [11C]CO2 of 14.0-17.8GBq with >98% radiochemical purity and 86-150GBq/µmol specific activity at the end of synthesis. In vitro autoradiography indicated that [11C]CMDC binding was expressed (>50% of total binding) in mGluR2-rich brain regions including the cerebral cortex, striatum and hippocampus. However, small-animal PET showed low in vivo specific binding of [11C]CMDC in the rat brain. While [11C]CMDC has limited potential as a PET tracer for brain mGluR2, it can be used to develop new radiotracers with improved behaviors.

Discovery of a fluorinated 4-oxo-quinoline derivative as a potential positron emission tomography radiotracer for imaging cannabinoid receptor type 2.

The cannabinoid receptor type 2 (CB2) is part of the endocannabinoid system and has gained growing attention in recent years because of its important role in neuroinflammatory/neurodegenerative diseases. Recently, we reported on a carbon-11 labeled 4-oxo-quinoline derivative, designated RS-016, as a promising radiotracer for imaging CB2 using PET. In this study, three novel fluorinated analogs of RS-016 were designed, synthesized, and pharmacologically evaluated. The results of our efforts led to the identification of N-(1-adamantyl)-1-(2-(2-fluoroethoxy)ethyl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-126) as the most potent candidate for evaluation as a CB2 PET ligand. [(18) F]RS-126 was obtained in ≥ 99% radiochemical purity with an average specific radioactivity of 98 GBq/µmol at the end of the radiosynthesis. [(18) F]RS-126 showed a logD7.4 value of 1.99 and is stable in vitro in rat and human plasma over 120 min, whereas 55% intact parent compound was found in vivo in rat blood plasma at 10 min post injection. In vitro autoradiographic studies with CB2-positive rat spleen tissue revealed high and blockable binding which was confirmed in in vivo displacement experiments with rats by dynamic PET imaging. Ex vivo biodistribution studies confirmed accumulation of [(18) F]RS-126 in rat spleen with a specificity of 79% under blocking conditions. The moderate elevated CB2 levels in LPS-treated mice brain did not permit the detection of CB2 by [(18) F]RS-126 using PET imaging. In summary, [(18) F]RS-126 demonstrated high specificity toward CB2 receptor in vitro and in vivo and is a promising radioligand for imaging CB2 receptor expression. Cannabinoid receptor type 2 (CB2) is an interesting target for PET imaging. Specific binding of [(18) F]RS-126 in CB2-positive spleen tissue (white arrow head) was confirmed in in vivo displacement experiments with rats. Time activity curve of [(18) F]RS-126 in the spleen after the addition of GW405833 (CB2 specific ligand, green) demonstrates faster radiotracer elimination (blue) compared to the tracer only (red).

[¹²5I]AT-1012, a new high affinity radioligand for the α3ß4 nicotinic acetylcholine receptors.

Recent genetic and pharmacological studies have implicated the α3, ß4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3ß4* nAChR subtype has been shown to co-assemble with the α5 or ß3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3ß4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [(125)I]-radiolabeled analog of a high affinity, selective small-molecule α3ß4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3ß4* nAChR. Binding of [(125)I]AT-1012 was characterized at the rat α3ß4 and α4ß2 nAChR transfected into HEK cells, as well as at the human α3ß4α5 nAChR in HEK cells. Binding affinity of [(125)I]AT-1012 at the rat α3ß4 nAChR was 1.4 nM, with a B(max) of 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [(3)H]epibatidine. Saturation isotherms suggested that [(125)I]AT-1012 binds to a single site on the α3ß4 nAChR. Similar high binding affinity was also observed for [(125)I]AT-1012 at the human α3ß4α5 nAChR transfected into HEK cells. [(125)I]AT-1012 did not bind with high affinity to membranes from α4ß2 nAChR-transfected HEK cells. Binding studies with [(3)H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3ß4 over α4ß2 nAChR. K(i) values determined for known nAChR compounds using [(125)I]AT-1012 as radioligand were comparable to those obtained with [(3)H]epibatidine. [(125)I]AT-1012 was also used to label α3ß4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3ß4 nAChR. We demonstrate that [(125)I]AT-1012 is an excellent tool for labeling the α3ß4 nAChR in the presence of other nAChR subtypes.

Quantitative Autoradiography on Ontogenic Development of alpha-Adrenoceptor in the Rat Cerebral Cortex / 대한해부학회지

Adrenoceptors mediate response to catecholamines throughout the body. To investigate postnatal ontogenic development of alpha1- and alpha2- adrenoceptors in the rat cerebral cortex, in vitro autoradiography was done on frontal, parietal and temporal cortex in P0, P5, P10, P15, P20, P30 and adult animals. Binding sites for the alpha1-adrenergic receptor ligand, [3H]-prazosin, and the alpha2-adrenergic receptor ligand, [3H]-rauwolscine, were visualized by in vitro autoradiography, and anatomically localized by comparing the autoradiogram to Nissl-stained sections. Nonspecific binding was detected with unlabeled phentolamine (alpha1) and yohimbine (alpha2). There is uniform increase in alpha1- and alpha2- adrenoceptors from birth through first three or four postnatal weeks, followed by a decrease to adult level. Two alpha-adrenoceptors have very different ontogenic patterns of distribution during postnatal development. alpha1- adrenoceptors were expressed differentially in different cortical (frontal, temporal, parietal) regions and in different cortical layers (layers V, II-IV, VI) at same age. alpha2- adrenoceptor was expressed homogenously in throughout regions and layers. These findings may provide evidence that alpha1- adrenoceptors are involved in regulating cortical development or function more specifically than alpha2- adrenoceptors during postnatal development.