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 neuroinflammed 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 affects their binding. Here, we describe the ability of a new TSPO ligand, [18F]BS224, to identify abnormal TSPO expression in neuroinflammation 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-affinity binder (HAB) and low-affinity 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 aromatic 18F-fluorination. 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-specific uptake of [18F]BS224, lipopolysaccharide (LPS)-induced inflammatory and ischemic stroke rat models were used. Results: BS224 exhibited a high affinity (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 affinity 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 that the binding mode of BS224 is not affected 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 inflammatory 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 inflammatory and ischemic stroke rat models, respectively) without skull uptake. Conclusion: Our results suggest that [18F]BS224 may be a promising TSPO ligand to gauge neuroinflammatory disease-related areas in a broad range of patients irrespective of the common rs6971 polymorphism.