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The cannabinoid receptor type 2 (CB2R) is an attractive target for the diagnosis and therapy of neurodegenerative diseases and cancer. In this study, we aimed at the development of a novel

Purposes We present the first in-human brain PET imaging data of the new α4β2 nicotinic acetylcholine receptor (nAChR)–targeting radioligand (+)-[18F]Flubatine. Aims were to develop a kinetic modeling-based approach to quantify (+)-[18F]Flubatine and compare the data of healthy controls (HCs) and patients with Alzheimer’s disease (AD); to investigate the partial volume effect (PVE) on regional (+)-[18F]Flubatine binding; and whether (+)-[18F]Flubatine binding and cognitive test data respective β-amyloid radiotracer accumulation were correlated. Methods We examined 11 HCs and 9 mild AD patients. All subjects underwent neuropsychological testing and [11C]PiB PET/MRI examination. (+)-[18F]Flubatine PET data were evaluated using full kinetic modeling and regional as well as voxel-based analyses. Results With 270-min p.i., the unchanged parent compound amounted to 97 ± 2%. Adequate fits of the time-activity curves were obtained with the 1 tissue compartment model (1TCM). (+)-[18F]Flubatine distribution volume (binding) was significantly reduced in bilateral mesial temporal cortex in AD patients compared with HCs (right 10.6 ± 1.1 vs 11.6 ± 1.4, p = 0.049; left 11.0 ± 1.1 vs 12.2 ± 1.8, p = 0.046; one-sided t tests each). PVE correction increased not only (+)-[18F]Flubatine binding of approximately 15% but also standard deviation of 0.4–70%. Cognitive test data and (+)-[18F]Flubatine binding were significantly correlated in the left anterior cingulate, right posterior cingulate, and right parietal cortex (r > 0.5, p 18F]Flubatine binding and [11C]PiB standardized uptake value ratios were negatively correlated in several regions; whereas in HCs, a positive correlation between cortical (+)-[18F]Flubatine binding and [11C]PiB accumulation in the white matter was found. No adverse event related to (+)-[18F]Flubatine occurred. Conclusion (+)-[18F]Flubatine is a safe and stable PET ligand. Full kinetic modeling can be realized by 1TCM without metabolite correction. (+)-[18F]Flubatine binding affinity was high enough to detect group differences. Of interest, correlation between white matter β-amyloid PET uptake and (+)-[18F]Flubatine binding indicated an association between white matter integrity and availability of α4β2 nAChRs. Overall, (+)-[18F]Flubatine showed favorable characteristics and has therefore the potential to serve as α4β2 nAChR–targeting PET ligand in further clinical trials.

Cannabinoid receptors type 2 (CB2R) represent an attractive therapeutic target for neurodegenerative diseases and cancer. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor receptor density and/or occupancy during a CB2R-tailored therapy, we herein describe the radiosynthesis of cis-[18F]1-(4-fluorobutyl-N-((1s,4s)-4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide ([18F]LU14) starting from the corresponding mesylate precursor. The first biological evaluation revealed that [18F]LU14 is a highly affine CB2R radioligand with >80% intact tracer in the brain at 30 min p.i. Its further evaluation by PET in a well-established rat model of CB2R overexpression demonstrated its ability to selectively image the CB2R in the brain and its potential as a tracer to further investigate disease-related changes in CB2R expression.

The σ2 receptor (transmembrane protein 97), which is involved in cholesterol homeostasis, is of high relevance for neoplastic processes. The upregulated expression of σ2 receptors in cancer cells and tissue in combination with the antiproliferative potency of σ2 receptor ligands motivates the research in the field of σ2 receptors for the diagnosis and therapy of different types of cancer. Starting from the well described 2-(4-(1H-indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline class of compounds, we synthesized a novel series of fluorinated derivatives bearing the F-atom at the aromatic indole/azaindole subunit. RM273 (2-[4-(6-fluoro-1H-pyrrolo[2,3-b]pyridin-1-yl)butyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline) was selected for labelling with 18F and evaluation regarding detection of σ2 receptors in the brain by positron emission tomography. Initial metabolism and biodistribution studies of [18F]RM273 in healthy mice revealed promising penetration of the radioligand into the brain. Preliminary in vitro autoradiography on brain cryosections of an orthotopic rat glioblastoma model proved the potential of the radioligand to detect the upregulation of σ2 receptors in glioblastoma cells compared to healthy brain tissue. The results indicate that the herein developed σ2 receptor ligand [18F]RM273 has potential to assess by non-invasive molecular imaging the correlation between the availability of σ2 receptors and properties of brain tumors such as tumor proliferation or resistance towards particular therapies.

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

Monoamine oxidases (MAOs) play a key role in the metabolism of major monoamine neurotransmitters. In particular, the upregulation of MAO-B in Parkinson’s disease, Alzheimer’s disease and cancer augmented the development of selective MAO-B inhibitors for diagnostic and therapeutic purposes, such as the anti-parkinsonian MAO-B irreversible binder l-deprenyl (Selegiline®). Herein we report on the synthesis of novel fluorinated indanone derivatives for PET imaging of MAO-B in the brain. Out of our series, the derivatives 6, 8, 9 and 13 are amongst the most affine and selective ligands for MAO-B reported so far. For the derivative 6-((3-fluorobenzyl)oxy)-2,3-dihydro-1H-inden-1-one (6) exhibiting an outstanding affinity (Ki MAO-B = 6 nM), an automated copper-mediated radiofluorination starting from the pinacol boronic ester 17 is described. An in vitro screening in different species revealed a MAO-B region-specific accumulation of [18F]6 in rats and piglets in comparison to L-[3H]deprenyl. The pre-clinical in vivo assessment of [18F]6 in mice demonstrated the potential of indanones to readily cross the blood–brain barrier. Nonetheless, parallel in vivo metabolism studies indicated the presence of blood–brain barrier metabolites, thus arguing for further structural modifications. With the matching analytical profiles of the radiometabolite analysis from the in vitro liver microsome studies and the in vivo evaluation, the structure’s elucidation of the blood–brain barrier penetrant radiometabolites is possible and will serve as basis for the development of new indanone derivatives suitable for the PET imaging of MAO-B. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4592]

Purpose The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. Methods [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A2A receptor–specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. Conclusions The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application. Supplementary Information The online version contains supplementary material available at 10.1007/s00259-020-05164-4.

Monocarboxylate transporters 1-4 (MCT1-4) are involved in several metabolism-related diseases, especially cancer, providing the chance to be considered as relevant targets for diagnosis and therapy. [18F]FACH was recently developed and showed very promising preclinical results as a potential positron emission tomography (PET) radiotracer for imaging of MCTs. Given that [18F]FACH did not show high blood-brain barrier permeability, the current work is aimed to investigate whether more lipophilic analogs of FACH could improve brain uptake for imaging of gliomas, while retaining binding to MCTs. The 2-fluoropyridinyl-substituted analogs 1 and 2 were synthesized and their MCT1 inhibition was estimated by [14C]lactate uptake assay on rat brain endothelial-4 (RBE4) cells. While compounds 1 and 2 showed lower MCT1 inhibitory potencies than FACH (IC50 = 11 nM) by factors of 11 and 25, respectively, 1 (IC50 = 118 nM) could still be a suitable PET candidate. Therefore, 1 was selected for radiosynthesis of [18F]1 and subsequent biological evaluation for imaging of the MCT expression in mouse brain. Regarding lipophilicity, the experimental log D7.4 result for [18F]1 agrees pretty well with its predicted value. In vivo and in vitro studies revealed high uptake of the new radiotracer in kidney and other peripheral MCT-expressing organs together with significant reduction by using specific MCT1 inhibitor &alpha, cyano-4-hydroxycinnamic acid. Despite a higher lipophilicity of [18F]1 compared to [18F]FACH, the in vivo brain uptake of [18F]1 was in a similar range, which is reflected by calculated BBB permeabilities as well through similar transport rates by MCTs on RBE4 cells. Further investigation is needed to clarify the MCT-mediated transport mechanism of these radiotracers in brain.

Whether α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) expression is reduced in early Alzheimer’s disease is controversial. Using (-)-[18F]Flubatine PET, Sabri, Meyer et al. report α4β2-nAChR deficiency in mild Alzheimer’s dementia, especially within the basal forebrain-cortical and septohippocampal cholinergic projections. Reduced α4β2-nAChR availability correlates with impaired episodic memory and executive function/working memory., In early Alzheimer’s dementia, there is a need for PET biomarkers of disease progression with close associations to cognitive dysfunction that may aid to predict further cognitive decline and neurodegeneration. Amyloid biomarkers are not suitable for that purpose. The α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) are widely abundant in the human brain. As neuromodulators they play an important role in cognitive functions such as attention, learning and memory. Post-mortem studies reported lower expression of α4β2-nAChRs in more advanced Alzheimer’s dementia. However, there is ongoing controversy whether α4β2-nAChRs are reduced in early Alzheimer’s dementia. Therefore, using the recently developed α4β2-nAChR-specific radioligand (−)-18F-flubatine and PET, we aimed to quantify the α4β2-nAChR availability and its relationship to specific cognitive dysfunction in mild Alzheimer’s dementia. Fourteen non-smoking patients with mild Alzheimer’s dementia, drug-naïve for cholinesterase therapy, were compared with 15 non-smoking healthy controls matched for age, sex and education by applying (−)-18F-flubatine PET together with a neuropsychological test battery. The one-tissue compartment model and Logan plot method with arterial input function were used for kinetic analysis to obtain the total distribution volume (VT) as the primary, and the specific binding part of the distribution volume (VS) as the secondary quantitative outcome measure of α4β2-nAChR availability. VS was determined by using a pseudo-reference region. Correlations between VT within relevant brain regions and Z-scores of five cognitive functions (episodic memory, executive function/working memory, attention, language, visuospatial function) were calculated. VT (and VS) were applied for between-group comparisons. Volume of interest and statistical parametric mapping analyses were carried out. Analyses revealed that in patients with mild Alzheimer’s dementia compared to healthy controls, there was significantly lower VT, especially within the hippocampus, fronto-temporal cortices, and basal forebrain, which was similar to comparisons of VS. VT decline in Alzheimer’s dementia was associated with distinct domains of impaired cognitive functioning, especially episodic memory and executive function/working memory. Using (−)-18F-flubatine PET in patients with mild Alzheimer’s dementia, we show for the first time a cholinergic α4β2-nAChR deficiency mainly present within the basal forebrain-cortical and septohippocampal cholinergic projections and a relationship between lower α4β2-nAChR availability and impairment of distinct cognitive domains, notably episodic memory and executive function/working memory. This shows the potential of (−)-18F-flubatine as PET biomarker of cholinergic α4β2-nAChR dysfunction and specific cognitive decline. Thus, if validated by longitudinal PET studies, (−)-18F-flubatine might become a PET biomarker of progression of neurodegeneration in Alzheimer’s dementia.

International audience; With the aim to develop a specific radioligand for imaging the cyclic nucleotide phosphodiesterase 5 (PDE5) in brain by positron emission tomography (PET), seven new fluorinated inhibitors (3-9) were synthesized on the basis of a quinoline core. The inhibitory activity for PDE5 together with a panel of other PDEs was determined in vitro and two derivatives were selected for IC50 value determination. The most promising compound 7 (IC50 = 5.92 nM for PDE5A), containing a 3-fluoroazetidine moiety, was further radiolabeled by aliphatic nucleophilic substitution of two different leaving groups (nosylate and tosylate) using [18F]fluoride. The use of the nosylate precursor and tetra-n-butyl ammonium [18F]fluoride ([18F]TBAF) in 3-methyl-3-pentanol combined with the addition of a small amount of water proved to be the best radiolabeling conditions achieving a RCY of 4.9 ± 1.5% in an automated procedure. Preliminary biological investigations in vitro and in vivo were performed to characterize this new PDE5 radioligand. Metabolism studies of [18F]7 in mice revealed a fast metabolic degradation with the formation of radiometabolites which have been detected in the brain.

We report the design, synthesis, and evaluation of a series of 1-oxa-8-azaspiro[4.5]decane and 1,5-dioxa-9-azaspiro[5.5]undecane derivatives as selective σ1 receptor ligands. All seven ligands exhibited nanomolar affinity for σ1 receptors (Ki(σ1) = 0.47 – 12.1 nM) and moderate selectivity over σ2 receptors (Ki(σ2)/ Ki(σ1) = 2 – 44). Compound 8, with the best selectivity among these ligands, was selected for radiolabeling and further evaluation. Radioligand [18F]8 was prepared via nucleophilic 18F-substitution of the corresponding tosylate precursor, with an overall isolated radiochemical yield of 12–35%, a radiochemical purity of greater than 99%, and molar activity of 94 – 121 GBq/μmol. Biodistribution studies of [18F]8 in mice demonstrated high initial brain uptake at 2 min. Pretreatment with SA4503 resulted in significantly reduced brain-to-blood ratio (70% − 75% at 30 min). Ex vivo autoradiography in ICR mice demonstrated high accumulation of the radiotracer in σ1 receptor-rich brain areas. These findings suggest that [18F]8 could be a lead compound for further structural modifications to develop potential brain imaging agents for σ1 receptors.

The adenosine A2A receptor (A2AR) is regarded as a particularly appropriate target for non-dopaminergic treatment of Parkinson&rsquo, s disease (PD). An increased A2AR availability has been found in the human striatum at early stages of PD and in patients with PD and dyskinesias. The aim of this small animal positron emission tomography/magnetic resonance (PET/MR) imaging study was to investigate whether rotenone-treated mice reflect the aspect of striatal A2AR upregulation in PD. For that purpose, we selected the known A2AR-specific radiotracer [18F]FESCH and developed a simplified two-step one-pot radiosynthesis. PET images showed a high uptake of [18F]FESCH in the mouse striatum. Concomitantly, metabolism studies with [18F]FESCH revealed the presence of a brain-penetrant radiometabolite. In rotenone-treated mice, a slightly higher striatal A2AR binding of [18F]FESCH was found. Nonetheless, the correlation between the increased A2AR levels within the proposed PD animal model remains to be further investigated.

Purine nucleotides such as ATP and ADP are important extracellular signaling molecules in almost all tissues activating various subtypes of purinoreceptors. In the brain, the P2Y

On the basis of a pyrazine core structure, three new adenosine A

Sigma-1 receptors (Sig1R) are highly expressed in various human cancer cells and hence imaging of this target with positron emission tomography (PET) can contribute to a better understanding of tumor pathophysiology and support the development of antineoplastic drugs. Two Sig1R-specific radiolabeled enantiomers (S)-(−)- and (R)-(+)-[18F]fluspidine were investigated in several tumor cell lines including melanoma, squamous cell/epidermoid carcinoma, prostate carcinoma, and glioblastoma. Dynamic PET scans were performed in mice to investigate the suitability of both radiotracers for tumor imaging. The Sig1R expression in the respective tumors was confirmed by Western blot. Rather low radiotracer uptake was found in heterotopically (subcutaneously) implanted tumors. Therefore, a brain tumor model (U87-MG) with orthotopic implantation was chosen to investigate the suitability of the two Sig1R radiotracers for brain tumor imaging. High tumor uptake as well as a favorable tumor-to-background ratio was found. These results suggest that Sig1R PET imaging of brain tumors with [18F]fluspidine could be possible. Further studies with this tumor model will be performed to confirm specific binding and the integrity of the blood-brain barrier (BBB).

Specific radioligands for in vivo visualization and quantification of cyclic nucleotide phosphodiesterase 2A (PDE2A) by positron emission tomography (PET) are increasingly gaining interest in brain research. Herein we describe the synthesis, the 18F-labelling as well as the biological evaluation of our latest PDE2A (radio-)ligand 9-(5-Butoxy-2-fluorophenyl)-2-(2-([18F])fluoroethoxy)-7-methylimidazo[5,1-c]pyrido[2,3-e][1,2,4]triazine (([18F])TA5). It is the most potent PDE2A ligand out of our series of imidazopyridotriazine-based derivatives so far (IC50 hPDE2A = 3.0 nM; IC50 hPDE10A > 1000 nM). Radiolabelling was performed in a one-step procedure starting from the corresponding tosylate precursor. In vitro autoradiography on rat and pig brain slices displayed a homogenous and non-specific binding of the radioligand. Investigation of stability in vivo by reversed-phase HPLC (RP-HPLC) and micellar liquid chromatography (MLC) analyses of plasma and brain samples obtained from mice revealed a high fraction of one main radiometabolite. Hence, we concluded that [18F]TA5 is not appropriate for molecular imaging of PDE2A neither in vitro nor in vivo. Our ongoing work is focusing on further structurally modified compounds with enhanced metabolic stability.

We report the syntheses and evaluation of series of novel piperidine compounds with low lipophilicity as σ1 receptor ligands. 8-(4-(2-Fluoroethoxy)benzyl)-1,4-dioxa-8-azaspiro[4.5]decane (5a) possessed high affinity (K(i) = 5.4 ± 0.4 nM) for σ1 receptors and selectivity for σ2 receptors (30-fold) and the vesicular acetylcholine transporter (1404-fold). [(18)F]5a was prepared using a one-pot, two-step labeling procedure in an automated synthesis module, with a radiochemical purity of >95%, and a specific activity of 25-45 GBq/μmol. Cellular association, biodistribution, and autoradiography with blocking experiments indicated specific binding of [(18)F]5a to σ1 receptors in vitro and in vivo. Small animal positron emission tomography (PET) imaging using mouse tumor xenograft models demonstrated a high accumulation in human carcinoma and melanoma. Treatment with haloperidol significantly reduced the accumulation of the radiotracer in tumors. These findings suggest that radiotracer with suitable lipophilicity and appropriate affinity for σ1 receptors could be used for tumor imaging.

To verify vesamicol as lead structure in the development of radioligands for imaging of VAChT in the brain by PET, we systematically modified this molecule and investigated five different groups of derivatives. Structural changes were conducted in all three ring systems A, B, and C resulting in a library of 67 different vesamicol analogs. Based on their in vitro binding affinity toward VAChT as well as σ1 and σ2 receptors, we performed an extensive structure-affinity relationship (SAR) study regarding both affinity and selectivity. The compounds possessed VAChT affinities in the range of 1.32 nM (benzovesamicol) to > 10 µM and selectivity factors from 0.1 to 73 regarding σ1 and σ2 receptors, respectively. We could confirm the exceptional position of benzovesamicols as most affine VAChT ligands. However, we also observed that most of the compounds with high VAChT affinity demonstrated considerable affinity in particular to the σ1 receptor. Finally, none of the various vesamicol analogs in all five groups showed an in vitro binding profile suitable for specific VAChT imaging in the brain.

The ghrelin receptor (GhrR) is a widely investigated target in several diseases. However, the current knowledge of its role and distribution in the brain is limited. Recently, the small and non-peptidic compound (S)-6-(4-bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4(3H)-one ((S)-9) has been described as a GhrR ligand with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical properties. These results identified compounds (S)-9, (R)-9, and (S)-16 as suitable parent molecules for 18F-labeled positron emission tomography (PET) radiotracers to enable future investigation of GhrR in the brain.

Recent pharmacologic data revealed the implication of α3β4 nicotinic acetylcholine receptors (nAChRs) in nicotine and drug addiction. To image α3β4 nAChRs in vivo, we aimed to establish the synthesis of a [

The ghrelin receptor (GhrR) is a widely investigated target in several diseases. However, the current knowledge of its role and distribution in the brain is limited. Recently, the small and non-peptidic compound (S)-6-(4-bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4(3H)-one ((S)-9) has been described as a GhrR ligand with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical properties. These results identified compounds (S)-9, (R)-9, and (S)-16 as suitable parent molecules for 18F-labeled positron emission tomography (PET) radiotracers to enable future investigation of GhrR in the brain.

Several spirocyclic piperidine derivatives were designed and synthesized as σ 1 receptor ligands. In vitro competition binding assays showed that the fluoroalkoxy analogues with small substituents possessed high affinity towards σ 1 receptors and subtype selectivity. Particularly for ligand 1′-((6-(2-fluoroethoxy)pyridin-3-yl)methyl)-3 H -spiro[2-benzofuran-1,4′-piperidine] ( 2 ), high σ 1 receptor affinity ( K i = 2.30 nM) and high σ 1 /σ 2 subtype selectivity (142-fold) as well as high σ 1 /VAChT selectivity (234-fold) were observed. [ 18 F] 2 was synthesized using an efficient one-pot, two-step reaction method in a home-made automated synthesis module, with an overall isolated radiochemical yield of 8–10%, a radiochemical purity of higher than 99%, and specific activity of 56–78 GBq/μmol. Biodistribution studies of [ 18 F] 2 in ICR mice indicated high initial brain uptake and a relatively fast washout. Administration of haloperidol, compound 1 and different concentrations of SA4503 (3, 5, or 10 μmol/kg) 5 min prior to injection of [ 18 F] 2 significantly decreased the accumulation of radiotracer in organs known to contain σ 1 receptors. Ex vivo autoradiography in Sprague–Dawley rats demonstrated high accumulation of radiotracer in brain areas with high expression of σ 1 receptors. These encouraging results prove that [ 18 F] 2 is a suitable candidate for σ 1 receptor imaging with PET in humans.

We report the design, synthesis, and evaluation of a series of novel cyclopentadienyl tricarbonyl 99mTc complexes as potent σ1 receptor radioligands. Rhenium compounds 3-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)-propylcarbonylcyclopentadienyl tricarbonyl rhenium (10a) and 4-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)-butylcarbonylcyclopentadienyl tricarbonyl rhenium (10b) possessed high in vitro affinity for σ1 receptors and moderate to high selectivity for σ2 receptors and the vesicular acetylcholine transporter. Biodistribution studies in mice demonstrated high initial brain uptake for corresponding 99mTc derivatives [99mTc] 23 and [99mTc]24 of 2.94 and 2.13% injected dose (ID)/g, respectively, at 2 min postinjection. Pretreatment of haloperidol significantly reduced the radiotracer accumulation of [99mTc]23 or [99mTc]24 in the brain. Studies of the cellular uptake of [99mTc]23 in C6 and DU145 tumor cells demonstrated a reduction of accumulation by incubation with haloperidol, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine (SA4503), or 1,3-di-otolyl-guanidine (DTG). Furthermore, blocking studies in C6 glioma-bearing mice confirmed the specific binding of [99mTc]23 to σ1 receptors in the tumor.

Both enantiomers of the epibatidine analogue flubatine display high affinity towards the α4β2 nicotinic acetylcholine receptor (nAChR) in vitro, accompanied by negligible interactions with diverse off-target proteins. Extended single dose toxicity studies in rodent indicated a NOEL (No Observed Effect Level) of 6.2μg/kg for (-)-flubatine and 1.55μg/kg for (+)-flubatine. We developed syntheses for both flubatine enantiomers and their corresponding precursors for radiolabeling. The newly synthesized trimethylammonium precursors allowed for highly efficient (18)F-radiolabelling in radiochemical yields >60% and specific activities >750GBq/μmol, thus making the radioligands practical for clinical investigation.

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 &plusmn, 2%, n = 3), a high radiochemical purity (&ge, 99%), and high molar activities (155&ndash, 175 GBq/&mu, mol, 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.

The σ

Cyclic nucleotide phosphodiesterase 10A (PDE10A) regulates the level of the second messengers cAMP and cGMP in particular in brain regions assumed to be associated with neurodegenerative and psychiatric diseases. A better understanding of the pathophysiological role of the expression of PDE10A could be obtained by quantitative imaging of the enzyme by positron emission tomography (PET). Thus, in this study we developed, radiolabeled, and evaluated a new PDE10A radioligand, 8-bromo-1-(6-[

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.

We report the design, synthesis and biological evaluation of a novel (99m)Tc 4-(4-cyclohexylpiperazine-1-yl)-butan-1-one-1-cyclopentadienyltricarbonyl technetium ([(99m)Tc]5) as a potential SPECT tracer for imaging of σ(2) receptors in tumors. [(99m)Tc]5 was prepared in 25±5% isolated radiochemical yield with radiochemical purity of99% via double-ligand transfer (DLT) reaction from the ferrocene precursor 2b (4-(4-cyclohexylpiperazine-1-yl)-1-ferrocenylbutan-1-one). The corresponding Re-complex 4 and the ferrocenyl complex 2b showed relatively high affinity towards σ(2) receptors in in vitro competition binding assay (K(i) values of 4 and 2b were 64.4±18.5 nM and 43.6±21.3 nM, respectively) and moderate to high selectivity versus σ(1) receptors (K(i)σ(1)/K(i)σ(2) ratios were 12.5 and 95.5, respectively). The logD value of [(99m)Tc]5 was determined to be 2.52±0.33. Biodistribution studies in mice revealed comparably high initial brain uptake of [(99m)Tc]5 and slow washout. Administration of haloperidol 5 min prior to injection of [(99m)Tc]5 significantly reduced the radiotracer uptake in brain, heart, lung, and spleen by 40-50% at 2h p.i.. Moreover, [(99m)Tc]5 showed high uptake in C6 glioma cell lines (8.6%) after incubation for 1h. Blocking with haloperidol to compete with [(99m)Tc]5 significantly reduced the cell uptake. Preliminary blocking study in C6-brain-tumor bearing rats showed that [(99m)Tc]5 binds to σ receptors in the brain-tumor specifically. These results are encouraging for further exploration of (99m)Tc-labeled probes for σ(2) receptor tumor imaging in vivo.

Molecular imaging of brain structures by highly sensitive non-invasive techniques offers unique possibilities in the understanding of physiological and pathological processes in the central nervous system. In particular, the quantitative analysis by positron emission tomography (PET) of α7 nicotinic acetylcholine receptors (α7 nAChR), which are involved in different signalling pathways in the brain, is assumed to provide important information on the relation between receptor dysfunction and the pathogeneses of neuropsychiatric brain diseases, but the applicability of this imaging approach is still hampered due to insufficient imaging agents. This paper presents the recent efforts made to develop PET radiotracers targeting α7 nAChR as well as the current state of the evaluation of the most promising radiolabelled compounds in animal models and humans.

Traumatic brain injury is a leading cause of death and disability in children. Studies using adult animal models showed alterations of the central cholinergic neurotransmission as a result of trauma. However, there is a lack of knowledge about consequences of brain trauma on cholinergic function in the immature brain. It is hypothesized that trauma affects the relative acetylcholine esterase activity and causes a loss of cholinergic neurons in the immature brain. Severe fluid percussion trauma (FP-TBI, 3.8 0.3 atm) was induced in 15 female newborn piglets, monitored for 6 h and compared with 12 control animals. The hemispheres ipsilateral to FP-TBI obtained from seven piglets were used for acetylcholine esterase istochemistry on frozen sagittal slices, while regional cerebral blood flow and oxygen availability was determined in the remaining eight FP-TBI animals. Post-fixed slices were immunohistochemically labelled for choline acetyltransferase as well as for lowaffinity neurotrophin receptor in order to characterize cholinergic neurons in the basal forebrain. Regional cerebral blood flow and brain oxygen availability were reduced during the first 2 h after FPTBI (P < 0.05). In addition, acetylcholine esterase activity was significantly increased in the neocortex, basal forebrain, hypothalamus and medulla after trauma (P < 0.05), whereas the number of choline acetyltransferase and low-affinity neurotrophin receptor positive cells in the basal forebrain were unaffected by the injury. Thus, traumatic brain injury evoked an increased relative activity of the acetylcholine esterase in the immature brain early after injury, without loss of cholinergic neurons in the basal forebrain. These changes may contribute to developmental impairments after immature traumatic brain injury.

A series of various N-substituted 3-(3-fluoropropyl)-3H-spiro[[2]benzofuran-1,4'-piperidines] (7) has been synthesized. In receptor binding studies, the N-benzyl derivative 7a (WMS-1813) revealed extraordinarily high sigma(1) receptor affinity (K(i) = 1.4 nM) and excellent sigma(1)/sigma(2) selectivity (600 fold). In vitro biotransformation of 7a with rat liver microsomes led to three main metabolites. N-Debenzylation was inhibited by introduction of an N-phenylethyl residue (7 g). The PET tracer [(18)F]7a was synthesized by nucleophilic substitution of the tosylate 13 with K[(18)F]F-K222-carbonate complex. The decay corrected radiochemical yield of [(18)F]7a was 35-48% with a radiochemical purity of99.5% and a specific activity of 150-238 GBq/micromol. The radiotracer properties were evaluated in female CD-1 mice by organ distribution and ex vivo brain autoradiography. The radiotracer uptake in the brain was fast and sufficient, with values of approximately 4% injected dose per gram. Target specificity of [(18)F]7a was validated in blocking studies by preapplication of haloperidol, and significant reduction in the uptake of radioactivity was observed in the brain and peripheral organs expressing sigma(1) receptors.

Functional changes of nicotinic acetylcholine receptors (nAChR) are important during age-related neuronal degeneration. Recent studies demonstrate the applicability of the nAChR ligand 2-[(18)F]F-A-85380 for neuroimaging of patients with dementias. However, its binding kinetics demands a 7-h acquisition time limiting its practicality for clinical PET studies. Thus, the authors developed [(18)F]norchloro-fluoro-homoepibatidine ([(18)F]NCFHEB) for nAChR imaging. The kinetics of the two enantiomers of [(18)F]NCFHEB were compared with 2-[(18)F]F-A85380 in porcine brain to evaluate their potential for human neuroimaging. Twenty-four juvenile female pigs were studied with PET using [(18)F]NCFHEB. Nine animals received an additional i.v. injection (1 mg/kg) of the nAChR agonist A81418 before radiotracer administration followed by infusion (2 mg/kg/7h) thereafter. Several compartment models were applied for quantification. (-)- and (+)-[(18)F]NCFHEB showed a twofold to threefold higher brain uptake than 2-[(18)F]F-A-85380. All three radiotracers displayed spatially heterogeneous binding kinetics in regions with high, moderate, or low specific binding. The equilibrium of specific binding of (-)-[(18)F]NCFHEB was reached earlier than that of (+)-[(18)F]NCFHEB or 2-[(18)F]F-A85380. Continuous administration of the nAChR agonist A81418 inhibited the specific binding of (-)- and (+)-[(18)F]NCFHEB but not of 2-[(18)F]F-A85380. The peripheral metabolism of (+)-[(18)F]NCFHEB proceeded somewhat slower than that of the other radiotracers. Both enantiomers of [(18)F]NCFHEB are appropriate radiotracers for neuroimaging of nAChR in pigs. Their binding profile in vivo appears to be more selective than that of 2-[(18)F]F-A85380. (-)-[(18)F]NCFHEB offers a faster equilibrium of specific binding than 2-[(18)F]F-A85380.

A series of fluorinated diphenylchalcogen derivatives, possessing a sulfur or an oxygen bridge, has been prepared with the aim to get a suitable radiotracer to image the SERT in vivo using positron emission tomography (PET). The compounds were synthesized and assayed toward the serotonin (SERT), dopamine (DAT), and norepinephrine (NET) transporters. Among the developed series, five compounds display a high SERT affinity (Ki: 0.27–2.91 nM range) and can be labeled either with carbon-11 or fluorine-18.

Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs) in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET). We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR) of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a) for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10) has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

Positron emission tomography (PET) radioligands specific to α7 nicotinic acetylcholine receptors (nAChRs) afford in vivo imaging of this receptor for neuropathologies such as Alzheimer’s disease, schizophrenia, and substance abuse. This work aims to characterize the kinetic properties of an α7-nAChR-specific radioligand, 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-[18F]-fluorodibenzo[b,d]thiophene 5,5-dioxide ([18F]DBT-10), in nonhuman primates. [18F]DBT-10 was produced via nucleophilic substitution of the nitro-precursor. Four Macaca mulatta subjects were imaged with [18F]DBT-10 PET, with measurement of [18F]DBT-10 parent concentrations and metabolism in arterial plasma. Baseline PET scans were acquired for all subjects. Following one scan, ex vivo analysis of brain tissue was performed to inspect for radiolabeled metabolites in brain. Three blocking scans with 0.69 and 1.24 mg/kg of the α7-nAChR-specific ligand ASEM were also acquired to assess dose-dependent blockade of [18F]DBT-10 binding. Kinetic analysis of PET data was performed using the metabolite-corrected input function to calculate the parent fraction corrected total distribution volume (V T/f P). [18F]DBT-10 was produced within 90 min at high specific activities of 428 ± 436 GBq/μmol at end of synthesis. Metabolism of [18F]DBT-10 varied across subjects, stabilizing by 120 min post-injection at parent fractions of 15–55 %. Uptake of [18F]DBT-10 in brain occurred rapidly, reaching peak standardized uptake values (SUVs) of 2.9–3.7 within 30 min. The plasma-free fraction was 18.8 ± 3.4 %. No evidence for radiolabeled [18F]DBT-10 metabolites was found in ex vivo brain tissue samples. Kinetic analysis of PET data was best described by the two-tissue compartment model. Estimated V T/f P values were 193–376 ml/cm3 across regions, with regional rank order of thalamus > frontal cortex > striatum > hippocampus > occipital cortex > cerebellum > pons. Dose-dependent blockade of [18F]DBT-10 binding by structural analog ASEM was observed throughout the brain, and occupancy plots yielded a V ND/f P estimate of 20 ± 16 ml/cm3. These results demonstrate suitable kinetic properties of [18F]DBT-10 for in vivo quantification of α7-nAChR binding in nonhuman primates.

Paediatric traumatic brain injury (TBI) is a leading cause of death and disability. Previous studies showed neuroprotection after TBI by (endo)cannabinoid mechanisms, suggesting involvement of cannabinoid receptors (CBR). We therefore determined CBR densities and expression of the translocator protein 18 kDA (TSPO) in newborn piglets after experimental TBI. Newborn female piglets were subjected to sham operation (n=6) or fluid-percussion (FP) injury (n=7) under controlled physiological conditions. After six hours, brains were frozen, sagittally cut and incubated with radioligands for CBR ([3HCP-55,940, [3H]SR141716A) and TSPO ([3H]PK11195), an indicator of gliosis/brain injury. Early after injury, FP-TBI elicited a significant ICP increase at a temporary reduced cerebral perfusion pressure; however, CBF and CMRO2 remained within physiological range. At 6 hours post injury, we found a statistically significant increase in binding of the non-selective agonist [3H]CP-55,940 in 15 of the 24 investigated brain regions of injured animals. By contrast, no significant changes in binding of the CB1R-selective antagonist [3H]SR141716A were observed. A non-significant trend towards increased binding of [3H]PK11195 was observed, suggesting an incipient microglial activation. We therefore conclude that in this model and time span after injury, the increase in [3H]CP-55,940 binding reflects changes in CB2R density, while CB1R density is not affected. The results may provide explanation for the neuroprotective properties of cannabinoid ligands and future therapeutic strategies of TBI.

A series of spirocyclic piperidine derivatives were designed and synthesized as σ1 receptor ligands. In vitro competition binding assays showed that 1'-(4-(2-fluoroethoxy)benzyl)-3H-spiro[2-benzofuran-1,4'-piperidine] (19) possessed high σ1 receptor affinity (Ki= 0.79 nM) and excellent σ1/σ2 subtype selectivity (350-fold) as well as high σ1/VAChT selectivity (799-fold). The radiolabeled compound [18F]19 was synthesized by substitution of the tosylate precursor 24 with [18F]fluoride, with isolated radiochemical yield of 35–60%, radiochemical purity of >99%, and specific activity of 30–55 GBq/Qmol. Biodistribution studies in ICR mice indicated that [18F]19 displayed excellent initial brain uptake and slow washout. Ex vivo autoradiography in Sprague-Dawley rat demonstrated high accumulation of the radiotracer in brain areas known to express high levels of σ1 receptors. MicroPET imaging and blocking studies confirmed the specific binding of [18F]19 to σ1 receptors in vivo.

In this study, we synthesized and evaluated a new spirocyclic piperidine derivative 3, containing a 4-fluorobutyl side chain, as a PET radioligand for neuroimaging of σ₁ receptors. In vitro, compound 3 displayed high affinity for σ₁ receptors (K(i) =1.2 nM) as well as high selectivity. [¹⁸F]3 radiosynthesis was performed from the corresponding tosylate precursor, with high radiochemical yield (45-51 %), purity (98 %), and specific activity (201 GBq μmol⁻¹). Metabolic stability of [¹⁸F]3 in the brain of CD-1 mice was verified, and no penetration of peripheral radiometabolites into the cerebral tissue was observed. Results of ex vivo autoradiography revealed that the distribution of [¹⁸F]3 in the brain corresponded to regions with high σ₁ receptor density. The highest region-specific total-to-nonspecific ratio was determined in the facial nucleus (4.00). Biodistribution studies indicated rapid and high levels in brain uptake of [¹⁸F]3 (2.2 % ID per gram at 5 min p.i.). Pre-administration of haloperidol significantly inhibited [¹⁸F]3 uptake into the brain and σ₁ receptor-expressing organs, further confirming in vivo target specificity.

To conduct a quantitative PET assessment of the specific binding sites in the brain of juvenile pigs for [(18)F]NS10743, a novel diazabicyclononane derivative targeting α7 nicotinic acetylcholine receptors (α7 nAChRs).Dynamic PET recordings were made in isoflurane-anaesthetized juvenile pigs during 120 min after administration of [(18)F]NS10743 under baseline conditions (n = 3) and after blocking of the α7 nAChR with NS6740 (3 mg·kg(-1) bolus + 1 mg·kg(-1)·h(-1) continuous infusion; n = 3). Arterial plasma samples were collected for determining the input function of the unmetabolized tracer. Kinetic analysis of regional brain time-radioactivity curves was performed, and parametric maps were calculated relative to arterial input.Plasma [(18)F]NS10743 passed readily into the brain, with peak uptake occurring in α7 nAChR-expressing brain regions such as the colliculi, thalamus, temporal lobe and hippocampus. The highest SUV(max) was approximately 2.3, whereas the lowest uptake was in the olfactory bulb (SUV(max) 1.53 ± 0.32). Administration of NS6740 significantly decreased [(18)F]NS10743 binding late in the emission recording throughout the brain, except in the olfactory bulb, which was therefore chosen as reference region for calculation of BP(ND). The baseline BP(ND) ranged from 0.39 ± 0.08 in the cerebellum to 0.76 ± 0.07 in the temporal lobe. Pretreatment and constant infusion with NS6740 significantly reduced the BP(ND) in regions with high [(18)F]NS10743 binding (temporal lobe -29%, p = 0.01; midbrain: -35%, p = 0.02), without significantly altering the BP(ND) in low binding regions (cerebellum: -16%, p = 0.2).This study confirms the potential of [(18)F]NS10743 as a target-specific radiotracer for the molecular imaging of central α7 nAChRs by PET.

C. K. Donat, B. Walter, W. Deuther-Conrad, K. Nieber, R. Bauer and P. Brust (2010) Neuropathology and Applied Neurobiology36, 225–236 Alterations of cholinergic receptors and the vesicular acetylcholine transporter after lateral fluid percussion injury in newborn piglets Aims: Traumatic brain injury (TBI) is one of the leading causes of death and disability in children. Adult animal models of TBI showed cholinergic alterations. However, there is no comparable data on immature animals. Therefore, this study investigates cholinergic markers in a large animal model of juvenile TBI. Methods: Twenty-seven female newborn piglets were subjected to lateral fluid percussion (FP) injury and compared with 12 untreated animals. After 6 h, animals were sacrificed and the brains removed. The hemispheres ipsilateral to FP-TBI from seven piglets and corresponding hemispheres from six control animals were used for autoradiography. Receptor density was determined with [3H]epibatidine (nicotinic acetylcholine receptors) or [3H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was assessed with (−)-[3H]vesamicol. Cerebral blood flow was measured by coloured microsphere method. Results: Cerebral blood flow and brain oxygen delivery were transiently reduced early after FP-TBI (P

Traumatic brain injury (TBI) is one of the leading causes of death and disability. Cognitive deficits are believed to be connected with impairments of the cholinergic system. The present study was conducted to evaluate the cholinergic system in a model of focal brain injury with special attention to the time course of posttraumatic events in critical brain regions. Three groups of male Sprague–Dawley rats (post-TBI survival time: 2 h, 24 h and 72 h) were subjected to sham-operation (control) or controlled cortical impact injury. Receptor densities were determined on frozen ipsilateral sagittal brain sections with [3H]epibatidine (nicotinic acetylcholine receptors) and [3H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was evaluated with (−)[3H]vesamicol. Compared to control, vAChT was lowered (up to 50%) at each time point after trauma, with reductions in olfactory tubercle, basal forebrain, motor cortex, putamen, thalamic and hypothalamic areas and the gigantocellular reticular nucleus. Time-dependent reductions of about 20% of nAChR-density in the thalamus, hypothalamus, olfactory tubercle, gigantocellular reticular nucleus and motor cortex were observed post-TBI at 24 and 72 h. The same brain regions showed reductions of mAChR at 24 and 72 h after trauma with additional decreases in the corpus callosum, basal forebrain and anterior olfactory nucleus. In conclusion, cholinergic markers showed significant time-dependent impairments after TBI. Considering the role of the cholinergic system for cognitive processes in the brain, it seems likely that these impairments contribute to clinically relevant cognitive deficits.

The postnatal development of the binding of the GABA(A) receptor agonist [(3)H]gaboxadol in rat brain was investigated. Using brain tissue from rats obtained at postnatal days 1, 10, 25, and >25 (adult), the binding of [(3)H]gaboxadol and the benzodiazepine [(3)H]flunitrazepam to GABA(A) receptors was compared in homogenate binding assays and quantitative receptor autoradiography. Kinetic and equilibrium data obtained in homogenate binding studies revealed two different [(3)H]gaboxadol affinities. A kinetically derived K(D) of 3.7 nM in adult cerebellum, calculated from the association and dissociation rate constants k(on) (1.45 x 10(8) M(-1) min(-1)) and k(off) (0.54 min(-1)) was contrasted by an equilibrium K(D) of 38.6 nM, obtained by homologous competition experiments. Quantitative analysis of autoradiographic data revealed an increase in specific [(3)H]gaboxadol binding sites during brain development, which resembles the anatomical and temporal pattern of the postnatal expression of the extrasynaptic delta subunit of GABA(A) receptors. In conclusion, by the radioligand binding data obtained on native tissue, binding of gaboxadol to GABA(A) receptors located outside the synaptic junctions could be postulated.

Introduction Gamma amino butyric acid type A (GABA A ) receptors are involved in a variety of neurological and psychiatric diseases, which have promoted the development and use of radiotracers for positron emission tomography imaging. Radiolabeled benzodiazepine antagonists such as flumazenil have most extensively been used for this purpose so far. Recently, the non-benzodiazepine pyrazolopyrimidine derivative indiplon with higher specificity for the α 1 subtype of the GABA A receptor has been introduced for treatment of insomnia. The aim of this study was the development and biological evaluation of an 18 F-labeled derivative of indiplon. Methods Both [ 18 F]fluoro-indiplon and its labeling precursor were synthesized by two-step procedures starting from indiplon. The radiosynthesis of [ 18 F]fluoro-indiplon was performed using the bromoacetyl precursor followed by multiple-stage purification using semipreparative HPLC and solid phase extraction. Stability, partition coefficients, binding affinities and regional brain binding were determined in vitro. Biodistribution and radiotracer metabolism were studied in vivo. Results [ 18 F]Fluoro-indiplon was readily accessible in good yields (38–43%), with high purity and high specific radioactivity (>150 GBq/μmol). It displays high in vitro stability and moderate lipophilicity. [ 18 F]Fluoro-indiplon has an affinity to GABA A receptors comparable to indiplon ( K i =8.0 nM vs. 3.4 nM). In vitro autoradiography indicates high [ 18 F]fluoro-indiplon binding in regions with high densities of GABA A receptors. However, ex vivo autoradiography and organ distribution studies show no evidence of specific binding of [ 18 F]fluoro-indiplon. Furthermore, the radiotracer is rapidly metabolized with high accumulation of labeled metabolites in the brain. Conclusions Although [ 18 F]fluoro-indiplon shows good in vitro features, it is not suitable for in vivo imaging studies because of its metabolism. Structural modifications are needed to develop derivatives with higher in vivo stability.

Noninvasive molecular imaging of subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) will provide information on the role of these receptors in neurodegenerative diseases. The binding of the positron emission tomography ligand 2-[ 1 8 F]F-A-85380 to nAChRs was investigated in the porcine brain by quantitative autoradiography in vitro. The high-affinity binding of 2-[ 1 8 F]F-A-85380 to each of the investigated 12 brain areas was saturable and apparently monophasic (e.g., apparent K D value of 1.72 nM in the thalamus). The highest density of specific binding sites was observed in the thalamus (1,158 fmol/mg protein), and the lowest density was measured in the cerebellar gray matter (11 fmol/mg protein). An attempt to assess nAChR subtype specificity of 2-[ 1 8 F]F-A-85380 was made by competitive autoradiography. Binding of 2-[ 1 8 F]F-A-85380 coincubated with 2-F-A-85380, epibatidine, cytisine, or methyllycaconitine, respectively, indicated a specificity of 2-[ 1 8 F]F-A-85380 to β2-containing nAChRs in the porcine brain. The autoradiographic data confirmed the suitability of swine as a model for the evaluation of radioligands designed for imaging of nAChR subtypes in the living brain. Synapse 59:201-210, 2006. 02005 Wiley-Liss, Inc.

N,N-Dimethyl-2-(2-amino-4-methylthiophenylthio)benzylamine (SMe-ADAM, 1) is a highly potent and selective inhibitor of the serotonin transporter (SERT). This compound was labeled with carbon-11 by methylation of the S-desmethyl precursor 10 with [(11)C]methyl iodide to obtain the potential positron emission tomography (PET) radioligand [(11)C]SMe-ADAM. The radiochemical yield was 27 +/- 5%, and the specific radioactivity was 26-40 GBq/micromol at the end of synthesis. Ex vivo and in vivo biodistribution experiments in rats demonstrated a rapid accumulation of the radiotracer in brain regions known to be rich in SERT, such as the thalamus/hypothalamus region (3.59 +/- 0.41%ID/g at 5 min after injection). The specific uptake reached a thalamus to cerebellum ratio of 6.74 +/- 0.95 at 60 min postinjection. The [(11)C]SMe-ADAM uptake in the thalamus was significantly decreased by pretreatment with fluoxetine to 38 +/- 11% of the control value. Furthermore, no metabolites of [(11)C]SMe-ADAM could be detected in the SERT-rich regions of the rat brain. It is concluded that [(11)C]SMe-ADAM may be a suitable PET ligand for SERT imaging in the living brain.

The reaction of proteins with reducing sugars leads to the formation of 'advanced glycation endproducts' (AGEs). They accumulate in Alzheimer's disease brain in the vicinity of beta-amyloid plaques. AGEs are cytotoxic by a mechanism involving reactive oxygen species, which implies that they could compromise glutathione redox status. In this study, we show that AGEs (BSA-AGE and beta-amyloid-AGE) persistently increase the ratio of oxidized to reduced glutathione in a dose- and time-dependent manner in SH-SY5Y neuroblastoma cells. The level of oxidized glutathione accounted to 10-14% and persisted for up to 24 h in the presence of added AGEs. In contrast, the unmodified beta-amyloid peptides A beta (1-40) and A beta (25-35) had no significant effect on glutathione redox status. The AGE-induced increase in oxidized glutathione could be prevented by the radical scavengers N-acetylcysteine, alpha-lipoic acid and 17beta-estradiol or by application of catalase, indicating that superoxide and hydrogen peroxide production precedes the AGE-mediated depletion of reduced glutathione.