Discovery and Preclinical Evaluation of an mGluR2-NAM PET Radioligand.

290 Objectives It has been proposed that negative allosteric modulators of metabotropic glutamate receptor type 2 (mGluR2-NAM) have the potential to be utilized as therapeutic candidates for the treatment of neurological disorders. Measurement of central mGluR2 occupancy using positron emission tomography (PET) radioligands would offer several benefits, including: assessment of target engagement, establishing an occupancy-efficacy relationship, aid Go/NoGo decisions and guide dose selection for Phase II proof-of-concept studies. This presentation describes the discovery and characterization of an mGluR2-NAM PET tracer for use in the determination of central mGluR2 occupancy with mGluR2 NAM therapeutic candidates. Methods [11C]Compound 1 was prepared by methylation of the phenol precursor with [11C]MeI in good yield with high radiochemical purity and specific activity, with [3H]Compound 1 prepared in an analogous fashion. In vitro autoradiography and homogenate binding studies were performed in rat, monkey, and human brain tissue to assess specific binding and regional biodistribution. Potential off target binding was evaluated by screening Compound 1 against other mGluR subtypes, in an Pan Laboratories screen that included 119 potential off target binding sites, and finally with PET imaging using transgenic mGluR2 knock-out mice. Baseline PET scans in rhesus monkey were performed, with the regional brain distribution of [11C] Compound 1 compared to that predicted from the in vitro studies. In rhesus monkey, mGluR2 specific binding was established by conducting blocking studies and finally in vivo occupancy studies were carried with an mGluR2-NAM therapeutic candidate to examine the utility of [11C]Compound 1 to establish a drug plasma level / mGluR2 receptor occupancy relationship. Finally, in order to estimate the human radiation dosimetry of [11C]Compound 1, whole body biodistribution studies were obtained in three rhesus monkeys. Results In vitro autoradiography studies with [3H]Compound 1 in rat, monkey, and human brain slices demonstrated that the cerebellum gray matter, striatum and frontal cortex are regions with high binding density, consistent with known mGluR2 expression. Saturation binding studies with caudate-putamen homogenates indicate the concentration (Bmax) of mGluR2 is similar across species, suggesting Compound 1 will provide a signal of similar magnitude in rhesus monkey and human. Compound 1 shows weak binding (IC50 > 1 µM) to selected ion channels, mGluR subtypes, and the standard survey of receptors and enzymes and therefore off-target binding was not expected to be a concern. In addition, microPET studies in mGluR2 KO mice showed homogeneous tracer distribution throughout the brain, indicating a lack of specific binding to mGluR2 and consistent with the selectivity of [11C]Compound 1. Baseline PET scans in rhesus monkey demonstrated the expected regional distribution of [11C]Compound 1 in the brain, with the region of highest uptake being the cerebellum, with the thalamus identified as a reference region. mGluR2 specificity in vivo was confirmed by co-administering an mGluR2-NAM therapeutic candidate, which resulted in substantial reduction of uptake in the cerebellum. In vivo occupancy studies in rhesus monkey demonstrated the utility of [11C]Compound 1 to determine plasma concentration vs. receptor occupancy relationships with potential mGluR2-NAM therapeutic candidates. From the monkey whole body scans the average effective dose (ED) was estimated to be 5.5 µSv/MBq, which is in the typical range for a [11C] labelled PET tracer, and as such will not prohibit the administration of [11C]Compound 1 to human subjects. Conclusions [11C]Compound 1 has been qualified preclinically as a fit-for-purpose PET tracer for determining target engagement of central mGluR2 occupancy in monkey by mGluR2-NAM therapeutic candidates.