Radiosynthesis and characterization of [18F]BS224: a next-generation TSPO PET ligand insensitive to the rs6971 polymorphism

Purpose Translocator protein 18-kDa (TSPO) positron emission tomography (PET) is a valuable tool to detect neuroinfammed areas in a broad spectrum of neurodegenerative diseases. However, the clinical application of second-generation TSPO ligands as biomarkers is limited because of the presence of human rs6971 polymorphism that afects their binding.Here, we describe the ability of a new TSPO ligand, [ 18F]BS224, to identify abnormal TSPO expression in neuroinfammation independent of the rs6971 polymorphism.Methods An in vitro competitive inhibition assay of BS224 was conducted with [3H]PK 11195 using membrane proteins isolated from 293FT cells expressing TSPO-wild type (WT) or TSPO-mutant A147T (Mut), corresponding to a high-afnity binder (HAB) and low-afnity binder (LAB), respectively. Molecular docking was performed to investigate the interaction of BS224 with the binding sites of rat TSPO-WT and TSPO-Mut. We synthesized a new 18F-labeled imidazopyridine acetamide ([18F]BS224) using boronic acid pinacol ester 6 or iodotoluene tosylate precursor 7, respectively, via aromatic18F-fuorination. Dynamic PET scanning was performed up to 90 min after the injection of [18F]BS224 to healthy mice, and PET imaging data were obtained to estimate its absorbed doses in organs. To evaluate in vivo TSPO-specifc uptake of [18F] BS224, lipopolysaccharide (LPS)-induced infammatory and ischemic stroke rat models were used.Results BS224 exhibited a high afnity (Ki=0.51 nM) and selectivity for TSPO. The ratio of IC50 values of BS224 for LAB to that for HAB indicated that the TSPO binding afnity of BS224 has low binding sensitivity to the rs6971 polymorphism and it was comparable to that of PK 11195, which is not sensitive to the polymorphism. Docking simulations showed thatthe binding mode of BS224 is not afected by the A147T mutation and consequently supported the observed in vitro selectivity of [ 18F]BS224 regardless of polymorphisms. With optimal radiochemical yield (39±6.8%, decay-corrected) and purity (>99%), [18F]BS224 provided a clear visible image of the infammatory lesion with a high signal-to-background ratio in both animal models (BPND =1.43±0.17 and 1.57±0.37 in the LPS-induced infammatory and ischemic stroke rat models, respectively) without skull uptake.Conclusion Our results suggest that [ 18F]BS224 may be a promising TSPO ligand to gauge neuroinfammatory disease-relatedareas in a broad range of patients irrespective of the common rs6971 polymorphism.