Novel Thienopyrimidine-Based PET Tracers for P2Y₁₂ Receptor Imaging in the Brain

The P2Y₁₂ receptor (P2Y₁₂R) is uniquely expressed on microglia in the brain, and its expression level directly depends on the microglial activation state. Therefore, P2Y₁₂R provides a promising imaging marker for distinguishing the pro- and anti-inflammatory microglial phenotypes, both of which play crucial roles in neuroinflammatory diseases. In this study, three P2Y₁₂R antagonists were selected from the literature, radiolabeled with carbon-11 or fluorine-18, and evaluated in healthy Wistar rats. Brain imaging was performed with and without blocking of efflux transporters P-glycoprotein and breast cancer resistance protein using tariquidar. Low brain uptake in healthy rats was observed for all tracers at baseline conditions, whereas blocking of efflux transporters resulted in a strong (6-7 fold) increase in brain uptake for both of them. Binding of the most promising tracer, [¹⁸F]3, was further evaluated by in vitro autoradiography on rat brain sections, ex vivo metabolite studies, and in vivo P2Y₁₂R blocking studies. In vitro binding of [¹⁸F]3 on rat brain sections indicated high P2Y₁₂R targeting with approximately 70% selective and specific binding. At 60 min post-injection, over 95% of radioactivity in the brain accounted for an intact tracer. In blood plasma, still 40% intact tracer was found, and formed metabolites did not enter the brain. A moderate P2Y₁₂R blocking effect was observed in vivo by positron emission tomography (PET) imaging with [¹⁸F]3 (p = 0.04). To conclude, three potential P2Y₁₂R PET tracers were obtained and analyzed for P2Y₁₂R targeting in the brain. Unfortunately, the brain uptake appeared low. Future work will focus on the design of P2Y₁₂R inhibitors with improved physicochemical characteristics to reduce efflux transport and increase brain penetration.