Identification and Preclinical Evaluation of a Radiofluorinated Benzazepine Derivative for Imaging the GluN2B Subunit of the Ionotropic NMDA Receptor

Abstract

The previously reported ¹¹C-labeled GluN2B PET radioligand ¹¹C-Me-NB1 served as a starting point for derivatization and led to the successful development of a radiofluorinated analog designated (<i>R</i>)<i>-</i>¹⁸F-OF-Me-NB1. Given the short physical half-life of 20.3 min for ¹¹C, (<i>R</i>)-¹⁸F-OF-Me-NB1 with a physical half-life of 109.8 min would allow satellite distribution to nuclear medicine facilities without an on-site cyclotron. <b>Methods:</b> Two fluorinated Me-NB1 derivatives, OF-Me-NB1 and PF-Me-NB1, were synthesized. On chiral resolution, the respective enantiomers were radiolabeled with ¹¹C and assessed in a proof-of-concept study by applying in vitro autoradiography on rodent brain sections. On the basis of the autoradiograms, (<i>R</i>)<i>-</i>OF-Me-NB1 was selected for radiofluorination and preclinical evaluation by ex vivo autoradiography, PET imaging, biodistribution, and metabolite studies on Wistar rats. To rule out off-target binding to the σ₁ receptor (σ₁R), brain uptake of (<i>R</i>)<i>-</i>¹⁸F-OF-Me-NB1 in wild-type mice was compared with that in σ₁R knock-out mice. <b>Results:</b> Autoradiographic assessment revealed that both enantiomers of ¹¹C-PF-Me-NB1 distributed homogeneously across all brain regions on rodent brain sections. In contrast, the 2 enantiomers of ¹¹C-OF-Me-NB1 exhibited an entirely different behavior. Although (<i>S</i>)<i>-</i>¹¹C-OF-Me-NB1 bound to virtually all brain regions, with considerable σ₁R binding, (<i>R</i>)<i>-</i>¹¹C-OF-Me-NB1 exhibited high selectivity and specificity for the GluN2B-rich rat forebrain. These findings were confirmed for the radiofluorinated analog (<i>R</i>)<i>-</i>¹⁸F-OF-Me-NB1, which was obtained via copper-mediated radiofluorination in radiochemical yields of 13%–25% and molar activities ranging from 61 to 168 GBq/μmol. PET imaging and biodistribution studies on Wistar rats indicated an appropriate pharmacokinetic profile and high in vivo specific binding of (<i>R</i>)<i>-</i>¹⁸F-OF-Me-NB1 as revealed by blocking studies with the GluN2B antagonist CP101,606. Off-target binding to the σ₁R was excluded by PET imaging of σ₁R knock-out mice. Half-maximal receptor occupancy by CP101,606 occurred at 8.3 μmol/kg (intravenous). <b>Conclusion:</b> (<i>R</i>)<i>-</i>¹⁸F-OF-Me-NB1 is a promising radiofluorinated probe that exhibits specificity and selectivity for the GluN2B-containing <i>N</i>-methyl-d-aspartate complex and enables in vivo target occupancy studies on rodents.