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In Vitro Evaluation of [3H]CPPC as a Tool Radioligand for CSF-1R
Ashley C Knight1,2, Cassis Varlow1,2, Tong Zi3
1Azrieli Centre for Neuro-Radiochemistry, Brain Health Imaging Centre, Centre for Addiction and Mental Health (CAMH), Toronto, ON M5T 1R8, Canada.
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.
Insights
The novel radioligand [3H]CPPC shows promise for screening macrophage colony stimulating factor-1 receptor (CSF-1R) inhibitors in peripheral tissues, but exhibits limitations for central nervous system imaging due to nonspecific binding.
Area of Science:
- Neuroscience
- Radiochemistry
- Pharmacology
Background:
- Microglia are key players in central nervous system (CNS) diseases, making them a significant target for positron emission tomography (PET) imaging and therapies.
- Macrophage colony stimulating factor-1 receptor (CSF-1R) is a target for microglia-related CNS disorders.
- 5-Cyano-N-(4-(4-[11C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([11C]CPPC) is a radiopharmaceutical developed to target CSF-1R on microglia.
Purpose of the Study:
- To conduct the first preclinical evaluation of the radioligand [3H]CPPC.
- To assess its utility for evaluating potential CSF-1R inhibitors in rodent models of neuroinflammation.
- To investigate the binding characteristics and distribution of [3H]CPPC in preclinical models.
Main Methods:
- Radioligand binding assays were employed for the preclinical evaluation.
- Autoradiography was used to determine the distribution of [3H]CPPC in CNS tissues.
- Displacement assays with unlabeled CPPC and known CSF1R inhibitors (BLZ-945, PLX3397) were performed.
Main Results:
- Autoradiography showed [3H]CPPC distribution did not correlate with microglial markers (IBA-1) or TSPO.
- Significant nonspecific binding of [3H]CPPC was observed in the CNS, with low displacement by unlabeled CPPC and inhibitors.
- The spleen demonstrated a suitable signal-to-noise ratio for screening CSF-1R inhibitors using [3H]CPPC.
- Unlabeled CPPC exhibited off-target binding to numerous kinase targets (204/403) at relevant concentrations.
Conclusions:
- While [3H]CPPC shows potential as a radioligand tool for peripheral target evaluation and CSF-1R inhibitor screening, its utility for in vivo CNS imaging is limited.
- High nonspecific binding and lack of selectivity in the CNS hinder its application as a CNS imaging probe.
- Further research may be needed to optimize CPPC or develop alternative probes for CNS applications.
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