Development of novel ¹⁸F-labeled PET tracers for imaging TSPO with insensitivity to rs6971 polymorphism

Objectives: The translocator protein 18 kDa (TSPO), whose expression is upregulated by the activation of glia cells, is a continuously attractive biomarker for exploring neuroinflammation and neurologic disorders such as AD, PD and multiple sclerosis by PET. However, most TSPO PET tracers are sensitive to rs6971 polymorphism in human. In this study, we aim to identify some potential ¹⁸F-labeled PET tracers with high affinities, good brain uptake as well as reasonable insensitivity to rs6971 polymorphism.

Methods: Through the structure-activity relationship study, two novel candidates with aryl fluorine moiety, namely LW-1-F and LW-2-F, were screened with high TSPO binding affinities. The corresponding 18F-isotopologues, ¹⁸F-LW-1 and ¹⁸F-LW-2, were synthesized automatically via spirocyclic iodonium ylide (SCIDY) strategy by CFN-MPS200 module. Dynamic neuroPET imaging were performed on wild-type Sprague-Dawley (SD) rats. The specificities and sensitivities to rs6971 polymorphism were evaluated by autoradiography (ARG) with LAB and HAB postmortem human brain slices. The pharmacokinetics were explored on mice by ex vivo biodistribution (bioD).

Results: Both LW-1-F and LW-2-F showed excellent binding affinity with K_i < 0.1 nM. For radiofluorination, the corresponding SCIDY precursors were successfully obtained as colorless solids. The radiochemical yields were 16 ± 5% (n.d.c, n = 4, ¹⁸F-LW-1) and 15 ± 7% (n.d.c., n = 4, ¹⁸F-LW-2), respectively. The total radiosynthesis time was less than 90 min with good molar activities (> 1 Ci/μmol) and radiochemical purities (> 99%). MicroPET imaging showed that ¹⁸F-LW-1 penetrated the blood-brain-barrier (BBB) of SD rat, with the highest whole brain uptake as 1.38 SUV at 2 min post-injection, and gradually washed out to 0.7 SUV after 20 min and remained at this level. The ARG results from human brain slices showed a certain level of sensitivity to rs6971 (ratio_HAB/LAB = 1.7) and nonspecific binding. ¹⁸F-LW-2 showed reasonable insensitivity (ratio_HAB/LAB = 1.1) and excellent in vitro specific binding by ARG study. Ex vivo biodistribution of ¹⁸F-LW-2 showed high radioactivity accumulation in the lungs, heart, kidney and adrenal glands, as well as urinary and hepatobiliary excretion.

Conclusions: We have efficiently synthesized a series of novel TSPO ligands with aryl fluorine skeleton. Specifically, LW-1-F and LW-2-F showed good binding affinity to TSPO, which were successfully radiolabeled for imaging study. The microPET, in vitro ARG and ex vivo bioD study revealed that ¹⁸F-LW-2 showed good specificity, pharmacokinetics as well as reasonable insensitivity to rs6971 polymorphism, which is a potential PET tracer for visualizing TSPO in human.