Asparagine endopeptidase (AEP) inhibitor formulation via zein-based nanoparticle improves the therapeutic efficacy toward Alzheimer's disease.

Asparagine endopeptidase (AEP) plays a critical role in Alzheimer's disease (AD) by cleaving amyloid precursor protein (APP) at N585 and tau protein at N368. Genetic deletion or pharmacological inhibition of AEP using compound 11a ameliorates AD pathology in murine models. To improve the therapeutic potential of 11a, we synthesized structural analogs and developed a zein-based nanoparticle delivery system to enhance pharmacokinetics. Structural modification, specifically isopropyl substitution of the N-methyl group in 11a, markedly improved blood-brain barrier permeability. The lead compound, 11a-isopropyl, formulated in zein nanoparticles, exhibited superior oral bioavailability and brain exposure. In vivo pharmacodynamic/pharmacokinetic (PK/PD) analyses confirmed dose-dependent AEP inhibition and enhanced substrate stabilization, with the nanoparticle formulation further increasing efficacy. One-month oral administration in 3xTg AD mice demonstrated that 11a-isopropyl, particularly in nanoparticle form, significantly reduced Aβ and tau pathology and improved cognitive performance. These findings indicate that zein-based nanoparticles enhance AEP inhibitor delivery and therapeutic efficacy in AD.