Large-Scale Oral Treatment Study with the Four Most Promising D3-Derivatives for the Treatment of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is associated with the aggregation of the amyloid β protein (Aβ). Aβ oligomers are currently thought to be the major neurotoxic agent responsible for disease development and progression. Thus, their elimination is highly desirable for therapy development. Our therapeutic approach aims at specific and direct elimination of toxic Aβ oligomers by stabilizing Aβ monomers in an aggregation-incompetent conformation. We have proven that our lead compound "D3", an all d-enantiomeric-peptide, specifically eliminates Aβ oligomers in vitro. In vivo, D3 enhances cognition and reduces plaque load in several transgenic AD mouse models. Here, we performed a large-scale oral proof of concept efficacy study, in which we directly compared four of the most promising D3-derivatives in transgenic mice expressing human amyloid precursor protein with Swedish and London mutations (APP_SL), transgenic mice, to identify the most effective compound. RD2 and D3D3, both derived from D3 by rational design, were discovered to be the most effective derivatives in improving cognition in the Morris water maze. The performance of RD2- and D3D3-treated mice within the Morris water maze was significantly better than placebo-treated mice and, importantly, nearly as good as those of non-transgenic littermates, suggesting a complete reversal of the cognitive deficit of APP_SL mice.

Keywords: ">d-enantiomeric peptides; Alzheimer’s disease; Aβ oligomers; amyloid β; cognition; large-scale; oral treatment; transgenic mice.

Figure 1

RD2RD2 and RD2D3 eliminated toxic amyloid β protein (Aβ) oligomers and reduced Aβ induced cell toxicity. Potency of RD2RD2 and RD2D3 to reduce toxic Aβ oligomers was demonstrated in vitro by the QIAD assay (A). Both compounds were able to reduce the toxic Aβ oligomers present in fractions 4–6. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) after incubation of PC-12 cells (B) with 1 µM Aβ(1–42) co-incubated with 1 µM RD2RD2 (orange), 1 µM RD2D3 (red), or 1 µM D3D3 (blue). All compounds significantly increase the cell viability after co-incubation with Aβ(1–42), without having negative impact on cell viability when incubated alone (patterned bars). Data is represented as mean ± SD (standard deviation), one-way ANOVA (analysis of variance) with Fisher post hoc analysis, *** p ≤ 0.001.

Figure 2

Treatment with RD2 and D3D3 improved cognitive performance as shown in the Morris water maze (MWM). During the acquisition phase of the MWM, RD2- (green, (A,E)) and D3D3- (blue, (D,E)) treated mice showed significantly improved learning behaviors compared to those of placebo- (black) treated mice (two-way repeated measurements (RM) ANOVA, Fisher post hoc analysis, * p < 0.05, ** p < 0.001) (A–C,E). The improved cognitive performance is indistinguishable to that of non-transgenic littermates (ntg, pink) ((A–C)). Neither RD2RD2- (orange (B,E)) nor RD2D3- (red, (C,E)) treated mice showed improved cognitive performance compared to the behavior of placebo-treated mice. (E) is a combination of (A) to (D) for direct comparison of all four tested compounds. The probe trial did not reveal significant differences between the groups, but showed the tendency of RD2- and D3D3-treated mice to spend more time in the target quadrant F.

Figure 3

Immunofluorescent labeling of amyloid and glia cells. The images show exemplary immunofluorescent labeling of amyloid (antibody 6E10, red), astrocytes (GFAP (glial fibrillary acidic protein), blue), and activated microglia (CD11b, green) in mice of all groups (A–E). Nuclei are labeled with DAPI (white) to provide the requested comparison between the different groups. Abbreviations: CX, Cortex; CA1, cornu Ammonis area 1; HC, hippocampus; DG, dentate gyrus.