Evaluation of Tetrazine Tracers for Pretargeted Imaging within the Central Nervous System

The pretargeting approach separates the biological half-life of an antibody from the physical half-life of the radioisotope label, providing a strategy for reducing the radiation burden. A widely explored pretargeting approach makes use of the bioorthogonal click reaction between tetrazines (Tzs) and trans-cyclooctenes (TCOs), combining the targeting specificity of monoclonal antibodies (mAbs) with the rapid clearance and precise reaction of Tzs and TCOs. Such a strategy can allow for the targeting and imaging (e.g., by positron emission tomography (PET)) of molecular markers, which cannot be addressed by solely relying on small molecules. Tz derivatives that undergo inverse electron-demand Diels-Alder (IEDDA) reactions with an antibody bearing TCO moieties have been investigated. This study describes the synthesis and characterization of 11 cold Tz imaging agent candidates. These molecules have the potential to be radiolabeled with ¹⁸F or ³H, and with the former label, they could be of use as imaging tracers for positron emission tomography studies. Selection was made using a multiparameter optimization score for the central nervous system (CNS) PET tracers. Novel tetrazines were tested for their pH-dependent chemical stability. Those which turned out to be stable in a pH range of 6.5-8 were further characterized in in vitro assays with regard to their passive permeability, microsomal stability, and P-glycoprotein transport. Furthermore, selected Tzs were examined for their systemic clearance and CNS penetration in a single-dose pharmacokinetic study in rats. Two tetrazines were successfully labeled with ¹⁸F, one of which showed brain penetration in a biodistribution study in mice. Another Tz was successfully tritium-labeled and used to demonstrate a bioorthogonal click reaction on a TCO-modified antibody. As a result, we identified one Tz as a potential fluorine-18-labeled CNS-PET agent and a second as a ³H-radioligand for an IEDDA-based reaction with a modified brain-penetrating antibody.