In Vitro Evaluation of [³H]CPPC as a Tool Radioligand for CSF-1R

Microglia play a role in several central nervous system (CNS) diseases and are a highly sought target for positron emission tomography (PET) imaging and therapeutic intervention. 5-Cyano-N-(4-(4-[¹¹C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([¹¹C]CPPC) is a radiopharmaceutical designed to selectively target microglia via macrophage colony stimulating factor-1 receptor (CSF-1R) in the CNS. Herein, we report the first preclinical evaluation of [³H]CPPC using radioligand binding methods for the evaluation of putative CSF-1R inhibitors in rodent models of neuroinflammation. The distribution of [³H]CPPC by autoradiography did not align with 18 kDa translocator protein (TSPO) distribution using [³H]PBR28 and IBA-1 staining for microglia. In the CNS, [³H]CPPC had considerable nonspecific binding, as indicated by a low displacement of the tritiated ligand by unlabeled CPPC and the known CSF1R inhibitors BLZ-945 and PLX3397. Spleen was identified as a tissue that provided an adequate signal-to-noise ratio to enable screening with [³H]CPPC and a library of 20 novel PLX3397 derivatives. However, unlabeled CPPC lacked selectivity and showed off-target binding to a substantial number of kinase targets (204 out of 403 tested) at a concentration relevant to in vitro radioligand binding assays (10 μM). These findings suggest that, while [³H]CPPC may have utility as a radioligand tool for the evaluation of peripheral targets and screening of CSF-1R inhibitors, it may have limited utility as an in vivo CNS imaging probe on the basis of the current evaluation.