Territo Paul R, Meyer Jill A, Peters Jonathan S, Riley Amanda A, McCarthy Brian P, Gao Mingzhang, Wang Min, Green Mark A, Zheng Qi-Huang, Hutchins Gary D
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
J Nucl Med. 2017 Mar;58(3):458-465. doi: 10.2967/jnumed.116.181354. Epub 2016 Oct 20.
The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood-brain barrier penetration, and the ability to be radiolabeled with C. We report the initial physical and biologic characterization of this novel ligand. C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association-disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity C-GSK1482160, receptor density and K were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant , dissociation constant , and binding potential (/) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min⋅nM, 0.2547 ± 0.0155 min, and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections showed a respective 1.8- and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30 min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160. The current study provides compelling data that support the suitability of C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.
嘌呤能受体亚型7（P2X7R）是通过正电子发射断层扫描（PET）成像神经炎症的新型分子靶点。强效P2X7R拮抗剂GSK1482160具有高受体亲和力、高血脑屏障通透性以及能用碳进行放射性标记的能力。我们报告了这种新型配体的初步物理和生物学特性。C-GSK1482160是根据已发表的方法合成的。对表达人P2X7R的人胚肾细胞系（HEK293-hP2X7R）进行细胞密度研究，并使用P2X7R多克隆抗体进行蛋白质印迹、免疫荧光测定和放射免疫组织化学分析。分别通过饱和及结合-解离动力学测定受体密度和结合潜能。在时间进程研究中测定小鼠对脂多糖治疗的峰值免疫反应，并通过Iba1和P2X7R蛋白质印迹以及Iba1免疫组织化学进行分析。在给予放射性示踪剂后15、30和60分钟，对用盐水或脂多糖处理的小鼠进行全动物生物分布研究。在给予盐水、脂多糖或脂多糖+阻断剂后72小时，对小鼠进行动态体内PET/CT检查，并对脑区进行二室、五参数示踪剂动力学建模。P2X7R随浓度或细胞数量呈线性变化。对于高比活度的C-GSK1482160，在HEK293-hP2X7R膜中，受体密度和Kd分别为1.15±0.12 nM和3.03±0.10 pmol/mg。HEK293-hP2X7R细胞中的结合常数、解离常数和结合潜能（BPND）分别为0.2312±0.01542 min·nM、0.2547±0.0155 min和1.0277±0.207。脂多糖处理小鼠的全脑Iba1免疫组织化学表达在72小时达到峰值，盐水和脂多糖处理脑切片的P2X7R蛋白质印迹分析显示，在72小时时信号增强分别增加了1.8倍和1.7倍。在所有研究组织中，72小时时C-GSK1482160在盐水和脂多糖处理小鼠中的生物分布具有统计学意义。给予盐水、脂多糖或脂多糖+阻断剂后72小时，小鼠体内动态C-GSK1482160 PET/CT显示，30分钟时增加了3.2倍且阻断率为97%。多个皮质区域和海马体的总分布容积显示有统计学意义的增加，并被过量的正品标准GSK1482160阻断。当前研究提供了令人信服的数据，支持C-GSK1482160作为靶向P2X7R（神经炎症生物标志物）的放射性配体的适用性。
