Pharmacokinetic Studies of Amyloid-Targeting Bis(styryl)benzene Agents for Alzheimer's Disease.

The pharmacokinetics (PK) and biodistribution of radiolabeled amyloid-β (Aβ)-targeting probes have been extensively characterized in the context of Alzheimer's disease (AD), whereas the corresponding nonradiolabeled scaffolds remain largely unexplored in vivo. Herein, we report in vivo PK studies of the widely used amyloid probe methoxy-X04 (MeX04) in transgenic 5xFAD and wild-type (WT) mice. In plasma, MeX04 shows comparable exposure between 5xFAD and WT mice, indicating no major genotype-dependent differences in systemic clearance. In contrast, brain exposure was markedly increased in 5xFAD mice, with higher brain Cmax and AUC parameters and slower washout, consistent with Aβ-dependent sequestration and retention in the AD brain tissue. Notably, fluorescence microscopy revealed that the fluorescence intensity of amyloid plaque-bound MeX04 closely mirrored the total brain concentration, and we employed ex vivo fluorescence intensity quantification to evaluate the PK of a related bis(styryl)benzene compound, LS-4, which is proposed to exhibit increased affinity for soluble Aβ aggregates. We then performed age-dependent ex vivo staining of compound-bound Aβ aggregates in 5xFAD brains, and LS-4 exhibits appreciable amyloid-bound fluorescence intensity in younger 5xFAD mice, consistent with its higher affinity for Aβ oligomers, whereas MeX04 exhibited stronger fluorescence intensity in older mice. Consequently, we propose an efficient approach to track temporal changes in the PK of Aβ-binding compounds by ex vivo evaluation of their amyloid-bound fluorescence intensity, providing a rapid assessment of the general PK trends of newly developed amyloid-binding probes.