The Repurposing of R-Etodolac for Cerebrovascular Dysfunction in ADRD: In Vitro and In Vivo Studies

Abstract

Alzheimer’s disease (AD) is the most common type of dementia, defined as a progressive neurodegenerative disorder. AD is pathologically characterized by amyloid-β (Aβ) accumulation, tau hyperphosphorylation, blood–brain barrier (BBB) dysfunction, and neuroinflammation. Growing evidence indicates that BBB disruption is an early pathological event in AD. Currently, there is no curative therapy for AD, and all existing treatments mainly provide symptomatic relief while often being associated with adverse effects. Therefore, therapeutic strategies aimed at restoring BBB integrity and improving Aβ clearance may represent promising disease-modifying approaches. In this study, we investigated the potential of repurposing R-etodolac as a modulator of BBB function and Aβ transport. In vitro experiments with bEnd3 mouse brain endothelial cells demonstrated that R-etodolac significantly improved endothelial barrier integrity under amyloid-toxic conditions. Moreover, treatment reduced paracellular permeability and restored junctional organization by increasing the expression of β-catenin, VE-cadherin, claudin-5, and occludin. Importantly, R-etodolac enhanced regulated Aβ transport across endothelial monolayers and, at high concentrations, increased the expression of key Aβ transporters, LRP1 and P-gp. Mechanistic investigations indicated that these effects were associated with modulation of the canonical Wnt/β-catenin signaling pathway, including increased expression of upstream receptors and stabilization of β-catenin signaling, which was further supported by Transcriptomic analysis. Additionally, for pharmacokinetic studies, we established a sensitive and validated HPLC method for the analysis of etodolac and R-etodolac in mouse plasma and brain. R-etodolac exhibited higher systemic exposure and lower clearance compared with racemic etodolac, while both compounds showed high oral bioavailability. Brain distribution was low. Next, the in vivo efficacy of R-etodolac was tested in the TgSwDI mouse model of AD and cerebral amyloid angiopathy (CAA). Preventive administration of R-etodolac (5 mg/kg) restored endothelial β-catenin signaling and increased the levels of key BBB junctional proteins, including VE-cadherin, claudin-5, and occludin. Additionally, Gadolinium-enhanced MRI further demonstrated that R-etodolac reduced cortical BBB permeability in TgSwDI mice, supporting functional restoration of vascular integrity in vivo. These effects were associated with a significant reduction in Aβ accumulation. Behavioral testing showed no significant differences in spatial memory among groups, likely reflecting the early stage of pathology in young TgSwDI mice. Together, these findings demonstrate that R-etodolac enhances BBB integrity and promotes Aβ clearance by modulating endothelial Wnt/β-catenin signaling. By stabilizing the neurovascular unit and facilitating physiological mechanisms of Aβ transport, R-etodolac reduces Aβ burden in a mouse model of AD and CAA. These results highlight the therapeutic potential of targeting BBB endothelial signaling pathways and support the repurposing of R-etodolac as a vascular-protective strategy for ADRD.