Validating P2x7 PET tracer [18F]JNJ-64413739 on postmortem brain tissue

Introduction: Background: The P2x7 receptor is an adenosine triphosphate-gated ion channel, which is abundantly expressed in microglial cells within the central nervous system (CNS), such as cortex, hippocampus, and striatum, regions involved in neurodegenerative diseases. Previous work demonstrated that the activation and expression level of P2x7 mediate several neuroinflammatory and neurodegenerative processes. These results suggest that antagonism of P2x7 pathway could be a novel therapeutic strategy and molecular imaging tool for neurodegenerative processes. In particular, PET imaging targeting P2x7 could provide a non-invasive method for evaluating the expression and function of P2x7 in the CNS, as a proxy marker for neuroinflammation. Herein we describe autoradiography and in vitro binding assays with [¹⁸F]JNJ-64413739 and validate the binding affinity and region selectivity for P2x7 in postmortem rat, nonhuman primates and human brain tissues.

Methods: Methods: We applied [¹⁸F]JNJ-64413739 autoradiographic imaging and in vitro binding assays to study the distribution and expression of P2x7 with the brain tissue from rats, nonhuman primates and human subjects. The synthesis of [¹⁸F]JNJ-64413739 was carried out by heating precursor (3 mg) and [¹⁸F]fluoride/Kryptofix222 in DMSO at 180 ℃ for 4 min. The radio-synthesized tracers were used to evaluate the P2x7 signal and region selectivity with brain slices. In addition, in vitro binding assays were performed to assess the binding selectivity of [¹⁸F]JNJ-64413739 with validated inhibitors of P2x7, including JNJ-64413739, A-740003, JNJ-47965567 and JNJ-54175446.

Results: Results: The labeling of [¹⁸F]JNJ-64413739 was achieved in 6% radiochemical yield, and the molar activity was higher than 37 GBq/μmol (Fig. 1A). The in vitro autoradiography indicated heterogeneous distribution for the bound radioactivity in rat and nonhuman primate brain tissue. The uptake of [¹⁸F]JNJ-64413739 in the cortex, putamen, corpus callosum (white matter), midbrain, hindbrain, and cerebellum were observed in line with the P2x7 gene expression level in rodents (Fig. 1B). Similar labeling trends were detected in the brain of nonhuman primate brain tissues containing cortex, white matter, putamen, globus pallidus, and hippocampus (Fig. 1C). The in vitro binding assays were performed with inhibitors of P2x7 and resulted in 45-81% reduction of bound activity in rat brain and 53-86% reduction in nonhuman primate brain. In blocking studies with JNJ-54175446, high-level blockade in binding characteristics has been observed in postmortem human cortex (65−85% reduction) and cerebellum tissue (50−87% reduction) (Fig 1D). These results suggested that [¹⁸F]JNJ-64413739 had excellent in vitro binding specificity toward P2x7.

Conclusions: Conclusions: We evaluated [¹⁸F]JNJ-64413739 by autoradiography and in vitro binding assays in rats, nonhuman primates, and human subject postmortem brain tissues. [¹⁸F]JNJ-64413739 was shown excellent specific binding in rat and higher species. These results provide important insights for supporting and translating [¹⁸F]JNJ-64413739 in human use, particularly for neurodegenerative diseases.

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