Design, Synthesis and Preclinical Evaluation of a Novel PET Tracer for P2Y12R Imaging in the Brain

Microglia, the innate immune cells of the central nervous system (CNS), act as first responders in the context of brain injury or neurodegeneration. Their ability to switch between different neuroprotective and neurotoxic phenotypes, plays a central role in maintaining brain homeostasis. Recently, the P2Y12 receptor (P2Y12R) has been identified as a promising molecular biomarker for microglia, as its expression level is directly dependent on microglia phenotype and function. A decline in P2Y12R expression is indicative of damage, microglial malfunction or chronic neuroinflammation, as observed in neurodegenerative diseases such as Alzheimer's disease (AD). Novel therapeutic strategies aiming to modulate microglia phenotypes directly depend on matching diagnostic tools for success control. Accordingly, a suitable P2Y12R positron emission tomography (PET) tracer is hypothesised to provide valuable in vivo information regarding microglia activation within the CNS. However, P2Y12R PET tracers with sufficient brain retention for neuroimaging have not been reported so far. Herein, we report the first brain-permeable P2Y12R PET tracer for in vivo imaging of P2Y12R-positive microglia. Nicotinate [18F]12 exhibited nanomolar affinity for the target receptor and favourable in silico parameters. Tracer specificity was proven by in vitro autoradiography (ARG) and brain uptake was confirmed by PET imaging in wild-type (WT) mice and ex vivo biodistribution. Ex vivo metabolite analysis indicated the exclusive presence of intact tracer in the mouse brain, with no evidence of any radio-metabolites. The tracer showed a reduced uptake in microglia-depleted mice after PLX-5622 diet, in comparison to WT and Trem2 knock-out (Trem2-/-) mice. Ex vivo immunohistochemistry (IHC) results and PET data revealed a strong correlation between microglia, P2Y12R expression levels and tracer uptake giving strong evidence that P2Y12 PET signal reflects P2Y12R binding. This novel tracer represents an important step forward in P2Y12 PET imaging in the context of neuroinflammation.