ALZ-801 prevents amyloid β-protein assembly and reduces cytotoxicity: A preclinical experimental study.

Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder, mainly characterized by amyloid β (Aβ) accumulation in the brain. Numerous new agents are currently undergoing clinical trials as disease-modifying therapies (DMTs) targeting Aβ. ALZ-801 is a promising candidate DMT for AD, with a phase 3 trial of ALZ-801 ongoing specifically for apolipoprotein E (APOE) ε4 homozygous patients with early-stage AD. This study aimed to examine the effects of ALZ-801 on Aβ assembly and explore its toxicological profile. Thioflavin T (ThT) assays and two imaging modalities-transmission electron microscopy (TEM) and high-speed atomic force microscopy (HS-AFM)-were used to evaluate ALZ-801's effects on Aβ assembly. To assess the effect of ALZ-801 on Aβ42-induced cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and lactate dehydrogenase (LDH) assays were performed. ThT assays revealed increased lag time and decreased fluorescence in the presence of ALZ-801, confirming inhibition of Aβ42 fibril formation, as confirmed by TEM. Real-time observation using HS-AFM revealed that ALZ-801 inhibited the formation of Aβ42 fibril from low-molecular-weight (LMW)-Aβ42 in the presence of Aβ42 seeds. HS-AFM also revealed that globular aggregates from LMW-Aβ42 were significantly larger with ALZ-801, with few fibrils noted. MTT and LDH assays indicated that ALZ-801 prevented LMW-Aβ42-induced cytotoxicity but did not reduce cytotoxicity induced by high-molecular-weight-Aβ42. ALZ-801 can inhibit Aβ42 aggregation by preventing both nucleus formation and fibril elongation, while promoting large globular oligomer formation, and can significantly reduce LMW-Aβ42-induced cytotoxicity. These findings underscore the potential of ALZ-801 as an effective DMT for APOE ε4 homozygous patients with AD.