Abstract
Over the past two decades, our understanding of the endocannabinoid system has greatly improved due to the wealth of results obtained from exploratory studies. Currently, two cannabinoid receptor subtypes have been well-characterized. The cannabinoid receptor type 1 (CB1) is widely expressed in the central nervous system, while the levels of the cannabinoid receptor type 2 (CB2) in the brain and spinal cord of healthy individuals are relatively low. However, recent studies demonstrated a CB2 upregulation on activated microglia upon neuroinflammation, an indicator of neurodegeneration. Our research group aims to develop a suitable positron emission tomography (PET) tracer to visualize the CB2 receptor in patients suffering from neurodegenerative diseases. Herein we report two novel thiophene-based 11C-labeled PET ligands designated [11C]AAT-015 and [11C]AAT-778. The reference compounds were synthesized using Gewald reaction conditions to obtain the aminothiophene intermediates, followed by amide formation. Saponification of the esters provided their corresponding precursors. Binding affinity studies revealed Ki-values of 3.3 ± 0.5 nM (CB2) and 1.0 ± 0.2 μM (CB1) for AAT-015. AAT-778 showed similar Ki-values of 4.3 ± 0.7 nM (CB2) and 1.1 ± 0.1 μM (CB1). Radiosynthesis was carried out under basic conditions using [11C]iodomethane as methylating agent. After semi-preparative HPLC purification both radiolabeled compounds were obtained in 99% radiochemical purity and the radiochemical yields ranged from 12 to 37%. Specific activity was between 96 and 449 GBq/μmol for both tracers. In order to demonstrate CB2 specificity of [11C]AAT-015 and [11C]AAT-778, we carried out autoradiography studies using CB2-positive mouse/rat spleen tissues. The obtained results revealed unspecific binding in spleen tissue that was not blocked by an excess of CB2-specific ligand GW402833. For in vivo analysis, [11C]AAT-015 was administered to healthy rats via tail-vein injection. Evaluation of the CB2-positive spleen, however, showed no accumulation of the radiotracer. Despite the promising in vitro binding affinities, specific binding of [11C]AAT-015, and [11C]AAT-778 could not be demonstrated.
Highlights
Cannabinoid receptors belong to the large family of G protein-coupled receptors (GPCRs) exhibiting the characteristics of a glycosylated extracellular N-terminus, seven transmembrane α-helixes, and an intracellular C-terminus (Mackie, 2008; Rom and Persidsky, 2013)
Solvents for thin layer chromatography (TLC), column chromatography and extractions were purchased as commercial grade
Organic chemistry reactions were monitored by TLC using SigmaAldrich silica gel 60 plates under UV light at 254 nm
Summary
Cannabinoid receptors belong to the large family of G protein-coupled receptors (GPCRs) exhibiting the characteristics of a glycosylated extracellular N-terminus, seven transmembrane α-helixes, and an intracellular C-terminus (Mackie, 2008; Rom and Persidsky, 2013) They are key players in a series of physiological processes resulting in an overall auto-protective effect in mammalians (Pertwee, 2005). The development of a successful CB2 PET ligand might improve our understanding of the mechanisms underlying disease pathogenesis as well as provide a tool for therapy monitoring and development of novel therapeutic drug candidates. This would be beneficial for patients suffering from rare diseases with no current effective drug treatment like ALS (Zinman and Cudkowicz, 2011). We report the development of two novel thiophene-based PET radioligands designated [11C]AAT-015 and [11C]AAT-778 for a diagnostic approach toward CB2 receptor imaging
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