Second-Generation of Deuterium-Substituted Glutamate Uptake Enhancers Exhibit Superior Drug-Like Properties in Preclinical Evaluation

Strategic deuterium–hydrogen exchange applied to the first-in-class positive allosteric modulators (PAMs) of the glutamate transporter EAAT2/GLT-1, (R)-AS-1 and (R)-AS-7, yielded novel analogues with improved drug-like properties. Specifically, incorporation of deuterium into the pyrrolidine-2,5-dione ring significantly prolonged the elimination half-life and increased both plasma and brain exposure in mice. These enhancements translated into more sustained antiseizure activity and a more favorable pharmacokinetic/pharmacodynamic (PK/PD) relationship. Similar to their nondeuterated counterparts, the new deuterated analogues displayed broad-spectrum antiseizure efficacy across multiple in vivo mouse seizure models, including maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazole (PTZ), and PTZ-induced kindling. Among these compounds, d₆-(R)-AS-7 demonstrated the most robust antiseizure effects and the most advantageous overall pharmacokinetic profile following both intraperitoneal and oral administration. Mechanistic studies revealed that d₆-(R)-AS-7 markedly enhanced glutamate uptake in COS-7 cells expressing EAAT2 as well as in primary astrocyte cultures. Furthermore, electrophysiological recordings in acute mouse hippocampal slices, together with two-electrode voltage-clamp recordings in Xenopus laevis oocytes expressing EAAT2, confirmed increased transporter-mediated currents. Collectively, these findings identify d₆-(R)-AS-7 as a potent EAAT2 PAM with improved pharmacokinetic properties and strong antiseizure efficacy, supporting its further development as a therapeutic candidate for epilepsy and other disorders associated with glutamate excitotoxicity.