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

Abstract

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.

Characterization of neuroinflammatory positron emission tomography biomarkers in chronic traumatic encephalopathy

“…[ 3 H]CPPC was similarly assayed at 10 nM utilizing previous conditions. 50 , 51 Although radiotracer binding was observed in post-mortem CTE tissues, it was not specific as defined by homologous blockade (15.34 ± 8.12%; n = 4). Because our preliminary ARG studies with [ 3 H]SMW-139 and [ 3 H]CPPC in CTE tissues showed little-to-no specific binding, these radiotracers were not evaluated further.…”

Synthesis and Evaluation of a 18F-Labeled Ligand for PET Imaging of Colony-Stimulating Factor 1 Receptor

“…However, most of the PET radioligands currently developed for CSF1R imaging have low uptake or high non-specific binding in the brains of rodents and non-human primates [ 25 , 27 , 28 ]. Moreover, a recent in vitro study of [ 3 H]CPPC showed off-target binding of CPPC to quite a number of kinases in the CNS; thus, [ 11 C]CPPC may have low selectivity for CSF1R [ 38 ]. In this context, further blocking studies of [ 18 F] 1 need to be conducted using the optimized injection time points of the blocking agents and using other potent CSF1R inhibitors as the blocking agents.…”

“…Although [ 11 C]CPPC was reported as a lead PET radioligand for CSF1R imaging [ 18 ], the recent study revealed that CPPC has off-target binding in the CNS [ 38 ]. Radioligand [ 18 F] 1 was designed based on CPPC, and therefore, further studies are warranted to confirm whether [ 18 F] 1 has selective binding to CSF1R over other targets, including kinases in the brain.…”

PET Imaging in Animal Models of Alzheimer’s Disease

“…Additionally, increased expression of CSF1R after LPS treatment and about 50% specific binding of [ 11 C]CPPC in LPS-treated mice were observed relative to sham controls. Autoradiography studies with [ 3 H]CPPC demonstrated the lack of specificity of [ 3 H]CPPC in brain tissues of LPS-treated Sprague-Dawley rats ( Knight et al, 2021 ). In another study, the imaging performance of [ 11 C]CPPC was compared with that of [ 11 C]GW2580 in mouse models of acute and chronic neuroinflammation and a rhesus monkey ( Zhou X. et al, 2021 ).…”