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The P2X7 receptor (P2X7R) is an imaging biomarker of glioblastoma-associated microglia/macrophages (GAMMs), yet no SPECT tracer is currently available for GAMM imaging. Guided by the high-affinity P2X7R scaffold JNJ-64413739 and molecular docking, we designed two radioiodination-ready analogues, FJR01 and FJR02. Compounds were screened in vitro using homogenates from cell lines stably expressing mouse or human P2X7R; FJR01 was prioritised (hP2X7R, IC₅₀ = 8.8 nM). Radioiodination afforded [¹³¹I]FJR01 in high radiochemical yield (95%) with excellent stability. In normal mice, biodistribution showed high brain uptake and rapid clearance. In a rat C6 glioma model, in vivo SPECT demonstrated focal tumour accumulation, which was corroborated by ex vivo autoradiography; immunofluorescence confirmed P2X7R expression in GAMMs. Histopathology (H&E) and mouse-to-human dosimetry supported a favourable safety profile and the clinical translation potential of [¹³¹I]FJR01. In addition, [¹³¹I]FJR01 is well suited as a probe for competitive binding assays to screen next-generation P2X7R-targeted ligands.