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.

中文翻译：

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[18F]BS224 的放射合成和表征：对 rs6971 多态性不敏感的下一代 TSPO PET 配体。

目的 转运蛋白 18-kDa (TSPO) 正电子发射断层扫描 (PET) 是检测广泛神经退行性疾病中的神经炎症区域的宝贵工具。然而，由于存在影响其结合的人类 rs6971 多态性，第二代 TSPO 配体作为生物标志物的临床应用受到限制。在这里，我们描述了一种新的 TSPO 配体，[18F]BS224，在独立于 rs6971 多态性的情况下识别神经炎症中异常 TSPO 表达的能力。方法 BS224 的体外竞争性抑制试验是用 [3H]PK 11195 使用从表达 TSPO-野生型 (WT) 或 TSPO-突变体 A147T (Mut) 的 293FT 细胞中分离的膜蛋白进行的，对应于高亲和力结合剂 (HAB ) 和低亲和力粘合剂 (LAB)。进行分子对接以研究BS224与大鼠TSPO-WT和TSPO-Mut结合位点的相互作用。我们分别使用硼酸频哪醇酯 6 或碘甲苯甲苯磺酸盐前体 7，通过芳香族 18F-氟化合成了一种新的 18F 标记的咪唑并吡啶乙酰胺 ([18F]BS224)。向健康小鼠注射[18F]BS224后90分钟内进行动态PET扫描，并获得PET成像数据以估计其在器官中的吸收剂量。为了评估 [ 18 F] BS224 的体内 TSPO 特异性摄取，使用脂多糖 (LPS) 诱导的炎症和缺血性中风大鼠模型。结果 BS224 对 TSPO 表现出高亲和力 (Ki = 0.51 nM) 和选择性。BS224对LAB与HAB的IC50比值表明BS224的TSPO结合亲和力对rs6971多态性的结合敏感性较低，与对多态性不敏感的PK 11195相当。对接模拟表明 BS224 的结合模式不受 A147T 突变的影响，因此支持观察到的 [18F] BS224 的体外选择性，无论多态性如何。[18F]BS224 具有最佳放射化学产量（39 ± 6.8%，衰减校正）和纯度（> 99%），在两种动物模型（BPND = 1.43 ± 0.17 和 1.57 ± 0.37 在 LPS 诱导的炎症和缺血性中风大鼠模型中，分别）没有颅骨摄取。