Abstract
PurposeTranslocator 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.MethodsAn 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.ResultsBS224 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.ConclusionOur 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.
Highlights
The translocator protein 18-kDa (TSPO) is expressed predominantly in steroidogenic tissues [1]
TSPO rs6971 polymorphism appeared to have a little influence on the binding sites for [18F]FEBMP, but this radioligand showed a metabolic stability not eligible in rats as its radiolabeled metabolite rapidly increased in the plasma and its was found in the brain tissue [29]. (R,S)-[18F]GE387 overcame the binding sensitivity of the tricyclic ligand [18F]GE180; further investigation of each enantiomer for brain imaging remained [30]
Results of docking simulations offered a molecular rationale for the low sensitivity to the rs6971 polymorphism exhibited by this new TSPO positron emission tomography (PET) ligand
Summary
The translocator protein 18-kDa (TSPO) is expressed predominantly in steroidogenic tissues [1]. Interpretation of PET results of second-generation TSPO ligands is not straightforward, as clinical trial participants may have a high-affinity binder (HAB) or low-affinity binder (LAB) phenotype depending on their particular TSPO gene polymorphism and TSPO binding features. Most second-generation TSPO ligands display a desirable high-affinity for TSPO visualization in the HAB phenotype, but exhibit poor TSPO affinity in the LAB phenotype [10] This variation in ligand-binding affinity complicates the quantitative assessment of PET data. Many studies have focused on developing new TSPO ligands to overcome the clinical issues of second-generation TSPO ligands caused by TSPO polymorphism In this respect, previously, we reported that the imidazo[1,2-a]pyridine scaffold shows a high affinity and selectivity for TSPO, and we prepared its 18F-labeled form [18F]CB251 as a new TSPO ligand [14, 15]. To the best of our knowledge, we present the first biological characterization of [18F]BS224 demonstrating that it is a new promising TSPO ligand and that its binding is not dependent on rs6971 polymorphism
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