Discovery of a novel PET tracer for the Excitatory Amino Acid Transporter 2 (EAAT2) in the CNS

Objectives Our objective is to realize a PET imaging tracer for the CNS L-glutamate (L-Glu) transporter EAAT2 (Excitatory Amino Acid Transporter 2) found on astrocytes. Transport by EAAT2 is the primary mechanism through which extracellular levels of L-Glu are regulated. Excitotoxic injury promoted by elevated L-Glu can be a result from altered regional astrocyte EAAT2 activity and/or cell surface expression. Thus, an EAAT2 PET tracer might afford quantitative assessments of these in vivo CNS changes.

Methods A series of aspartylamide ligands underwent EAAT2 screening adjunct to other CNS targets. Screening identified ligand 1 as an EAAT2 candidate agent. CNS penetration studies of non-radiolabeled (cold) ligand 1 and cognate pro-drug forms, including pro-drug 2, used wild type (WT) mice (i.v. dosing 1-20 mg/Kg, brain and plasma concentrations of 1 by LC/MS-MS). An electrophilic fluorine-18 ([18F]) radiosynthesis was developed for pro-drug tracer [18F]2 with rat microPET scans using an Inveon DPET scanner, 0-90 min.

Results Ligand 1 has high EAAT2 potency (IC50 < 3 nM) and non-EAAT selectivity (>500x). Injection of cold pro-drug 2 afforded 1 in brain and plasma, where the PK curves for 1 were PET tracer suitable. Ligand 2 penetrates brain 4x better than parent 1. Radioligand [18F]2 was prepared in > 95% radiochemical purity and mean specific activity ~2.3 Ci/mmol (EOS, 3 h, n=5). Tracer [18F]2 results in high uptake of radioactivity in thalamus, hippocampus, frontal cortex, which is consistent with regional EAAT2 density determinations from ex vivo tissue immunostaining.

Conclusions Pro-drug 2 affords 1 in rat brain and plasma. In rats PET pro-drug tracer [18F]2 penetrates brain and its hydrolyzed form, [18F]1, provides good specific binding signals in EAAT2 rich regions. Ligand [18F]2 shows promise as a first-in-class CNS [18F]-tracer for EAAT2.

Research Support The Robert Packard Center for ALS Research, Johns Hopkins University; P2ALS; ALS Association; NIH P30-NS055022; and the Center for Molecular & Genomic Imaging, University of California, Davis