Imaging the Receptor for Advanced Glycation Endproducts with [¹⁸F]RAGER

Objectives: The receptor for advanced glycation endproducts (RAGE) is an inflammatory membrane bound receptor implicated in diseases of inflammation such as diabetic neuropathy. Increased levels of RAGE have also been identified in post-mortem brain sections of patients with neurodegenerative disorders. With the goal understanding RAGE and inflammation’s, role in neurodegenerative disease, we have previously reported synthesis and evaluation of [¹⁸F]RAGER, the first small molecule PET tracer for RAGE.¹ We have undertaken secondary evaluation of [¹⁸F]RAGER, including off-target screening and competitive blocking studies and report results herein.

Methods: [¹⁸F]RAGER was synthesized as reported,¹ and baseline PET imaging was conducted in Sprague-Dawley rat and rhesus monkey. Blocking experiments with known RAGE inhibitor FPS-ZM1 (1 mg/kg) to evaluate non-specific binding were done in rat. Autoradiography and immunohistochemistry (IHC) were performed as reported.¹ The RAGER profiling screen was performed by Cerep Eurofin Panlabs under the LeadProfileScreen, which consists of 68 primary molecular targets, including several CNS targets recommended to evaluate during drug development. Targets were screened against 10 mM of RAGER and % inhibition reported. For hits with >50% inhibition, secondary dose response assays were performed and K_i values reported. Additionally IHC was performed with the Melatonin Receptor 1 antibody, a possible off target of RAGER. Blocking experiments with melatonin (10ug/kg) to evaluate any off-target binding in vivo were performed in rat and rhesus macaque.

Results: [¹⁸F]RAGER was synthesized in a TRACERLab (44 ± 10 mCi (2.9% non-decay-corrected radiochemical yield), excellent radiochemical purity (>99%), and high specific activity (3740 ± 495 Ci/mmol); n = 6). The tracer has high affinity for RAGE (Kd = 15 nM), shows specific binding in Alzheimer's brain samples that colocalizes with RAGE identified by IHC, has good CNS penetration in rat and monkey, and increased uptake in areas of the brain known to express RAGE (e.g. hippocampus). Blocking studies also confirmed specific displacable binding in vivo. One target from the LeadProfileScreen returned >90% inhibition at 10 µM (melatonin 1; K_i = 93 nM). When compared to the K_d for RAGE (15 nM), [¹⁸F]RAGER maintains ~6-fold preference for RAGE, and off target binding is not expected to complicate clinical imaging studies. In comparison to the anti-Melatonin Receptor 1 antibody, the binding of [¹⁸F]RAGER does not match in tissue. Blocking experiments with melatonin (10ug/kg) showed no displacement in [¹⁸F]RAGER binding.

Conclusion: [¹⁸F]RAGER is a high affinity PET radioligand for the receptor for advanced glycation endproducts. It is selective for RAGE, and demonstrates good CNS penetration in rat and monkey. Given the role of RAGE in multiple diseases, rapid translation of a PET tracer for the target into clinical imaging studies is warranted. References [1] B.P. Cary et al. ACS Chem Neurosci, 2016, 7, 391-98. Research Support: Work was funded by National Institutes of Health (T32-GM007767; T32-EB005172; P50-AG08671), Alzheimer’s Association (NIRP-14-305669) and the University of Michigan (College of Pharmacy and Undergraduate Research Opportunity Program). The authors also thank Brian Cary for preliminary synthesis and labeling experiments.