A radioligand for in vitro autoradiography of CSF1R in post-mortem CNS tissues

Reactive microglia and recruited peripheral macrophages contribute to the pathogenesis of Alzheimer's dementia (AD). Monocytes, macrophages and microglia all express the marker colony-stimulating factor 1 receptor (CSF1R). 4-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)-1H-pyrrole-2-carboxamide (1) is a high-affinity antagonist for CSF1R. We report the radiosynthesis of both [³H]1 and [¹¹C]1. The PET imaging properties of [¹¹C]1 in mice and baboon were investigated. [³H]1 was studied in B_max measurement in post-mortem autoradiography in the frontal cortex, inferior parietal cortex and hippocampus from donors diagnosed with AD and age-matched controls. In vitro binding affinity of 1 was measured commercially. Nor-methyl-1 precursor was radiolabeled with [¹¹C]iodomethane or [³H]iodomethane to produce [¹¹C]1 and [³H]1, respectively. Ex vivo brain biodistribution of [¹¹C]1 was compared in normal mice versus lipopolysaccharide-administered (LPS) murine model of neuroinflammation. Dynamic PET imaging was performed in a healthy male Papio anubis baboon. Post-mortem autoradiography with [³H]1 was performed in frozen sections using a standard saturation binding technique.

Compound 1 exhibits a high in vitro CSF1R binding affinity (0.59 nM). [¹¹C]1 was synthesized with high yield. [³H]1 was synthesized similarly (commercially). Biodistribution of [¹¹C]1 in healthy mice demonstrated moderate brain uptake. In LPS-treated mice the brain uptake of [¹¹C]1 was ~ 50% specific for CSF1R. PET/CT [¹¹C]1 study in baboon revealed low brain uptake (0.36 SUV) of [¹¹C]1. Autoradiography with [³H]1 gave significantly elevated B_max values in AD frontal cortex versus control (47.78 ± 26.80 fmol/mg vs. 12.80 ± 5.30 fmol/mg, respectively, P = 0.023) and elevated, but not significantly different binding in AD hippocampus grey matter and inferior parietal cortex (IPC) white matter.

Compound 1 exhibits a high in vitro CSF1R binding affinity. [¹¹C]1 specifically labels CSF1R in the mouse neuroinflammation, but lacks the ability to efficiently cross the blood-brain barrier in baboon PET. [³H]1 specifically labels CSF1R in post-mortem human brain. The binding of [³H]1 is significantly higher in the post-mortem frontal cortex of AD versus control subjects.