Preclinical evaluation of radiolabeled negative allosteric modulators for PET imaging of metabotropic glutamate receptor 2

Objectives: Negative allosteric modulation of metabotropic glutamate receptor 2 (mGlu2) represents a novel specific and selective approach for treating brain disorders. Positron emission tomography (PET) imaging of mGlu2 would facilitate disease diagnosis and negative allosteric modulators (NAMs) discovery. However, few efforts have been disclosed to visualize mGlu2 in vivo, indicating an unmet and urgent clinical demand. Herein we report the proof-of-concept development of PET tracers derived from NAMs for successfully imaging of mGlu2. Methods: A library seven mGlu2 NAMs 1a-1g were synthesized and subjected to the investigation of pharmacological and physicochemical properties. As a result, 1a and 1b showed excellent potency and selectivity, and then radiolabeled with 11C. The specificities of both PET tracers [11C]1a and [11C]1b were validated by in vitro autoradiography (ARG) with rat brain slices. Dynamic PET imaging were performed on normal Sprague-Dawley (SD) rats to assess the capability for brain penetration and in vivo specificity by the simplified reference tissue model (SRTM) with the pons as the reference region. Whole body biodistribution and metabolite studies were conducted in rodents to evaluate the pharmacokinetic properties of [11C]1b. Results: All of the novel compounds were synthesized in five steps with 14-20% overall yields.1a and 1b showed good binding potency (IC50 to mGlu2 30 μM), together with reasonable lipophilicity and tPSA. 11C radiolabeling were conducted within 40 min on GE TracerLab FXC, and radiochemical yields were 36.6 ± 7.3% (n = 7, [11C]1a) and 6.53 ± 1.5% (n = 10, [11C]1b) respectively, with high radiochemical purities and molar activities. ARG results showed that [11C]1b was much more specific to mGlu2 with highest radioactivity accumulated in the striatum and cerebral cortex, followed by hippocampus and cerebellum. The heterogeneous distribution was diminished by pre-incubation with the corresponding standard compounds or QCA. The non-displaceable binding potential (BPND) of [11C]1b were calculated by PET, and the highest values were observed in striatum and cingulate cortex (0.72), followed by cerebral cortex (0.42) and hippocampus (0.48), and the lowest BPND was identified in the cerebellum (0.12) under baseline conditions. The BPND values were decreased substantially by pretreatment with 1b (41-88% reduction, 1 mg/kg) or 1a (48-90% reduction, 3 mg/kg). Biodistribution studies of [11C]1b demonstrated that high brain uptake (ca. 3.6 %ID/g) was detected after 5 min post injection, which was consistent with PET imaging data. Several peripheral organs with high mGlu2 expression were also observed with high radioactivity accumulation. Notably, metabolite analysis suggested that [11C]1b showed excellent in vivo stability in brain with more than 99% unchanged fraction at 20 min post injection. Conclusion: We have developed [11C]1b as a potent and promising PET tracer for neuroimaging of mGlu2. Translational PET imaging in nonhuman primates are on the way and will be reported in due course.SNMMI 2020 Annual Meetin