In Vivo Characterization of ARN14140, a Memantine/Galantamine-Based Multi-Target Compound for Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is a chronic pathological condition that leads to neurodegeneration, loss of intellectual abilities, including cognition and memory, and ultimately to death. It is widely recognized that AD is a multifactorial disease, where different pathological cascades (mainly amyloid and tau) contribute to neural death and to the clinical outcome related to the disease. The currently available drugs for AD were developed according to the one-target, one-drug paradigm. In recent times, multi-target strategies have begun to play an increasingly central role in the discovery of more efficacious candidates for complex neurological conditions, including AD. In this study, we report on the in vivo pharmacological characterization of ARN14140, a new chemical entity, which was obtained through a multi-target structure-activity relationship campaign, and which showed a balanced inhibiting profile against the acetylcholinesterase enzyme and the NMDA receptor. Based on the initial promising biochemical data, ARN14140 is here studied in mice treated with the amyloidogenic fragment 25-35 of the amyloid-β peptide, a consolidated non-transgenic AD model. Sub-chronically treating animals with ARN14140 leads to a prevention of the cognitive impairment and of biomarker levels connected to neurodegeneration, demonstrating its neuroprotective potential as new AD agent.

Figure 1. 2D structure of ARN14140 (MW = 506.73) along with biological activities values against AChE and NMDAR as reported in Simoni et al..

Figure 2. Effects of ARN14140 infusion for 7 days on Aβ_25-35-induced spontaneous alternation deficits in mice.

V, vehicle solution. N = 11–12 per group. ANOVA: F_(3,45) = 20.1, p < 0.0001 in (a), F_(3,45) = 1.22, p > 0.05 in (b). ***p < 0.001 vs. the Sc.Aβ/V-treated group; ^###p < 0.001 vs. the Aβ_25-35/V-treated group; Dunnett’s test in (a). Effects of ARN14140 infusion for 7 days on Aβ_25-35-induced passive avoidance deficits in mice. (c) Step-through latency and (d) escape latency measured during the retention session. V, vehicle solution. Data show median and interquartile range (25–75%). N = 11–12 per group. Kruskal-Wallis ANOVA: H = 22.8, p < 0.001 in (c), H = 20.3, P < 0.001 in (d), *p < 0.05, **p < 0.01, ***p < 0.001 vs. the Sc.Aβ/V-treated group; ^##p < 0.01 vs. the Aβ_25-35/V-treated group; Dunn’s test in (c,d).

Figure 3. Effects of ARN14140 infusion for 7 days on Aβ_25-35-induced toxicity.

(a) Increase in lipid peroxidation in the mouse hippocampus. ANOVA: F_(3,22) = 52.2, p < 0.0001, ***p < 0.001 vs. the Sc.Aβ/V-treated group; ^###p < 0.001 vs. the Aβ_25-35/V-treated group; Dunnett’s test (b) increase in TNFα levels in the mouse hippocampus. ANOVA: F_(3,22) = 2.47, p > 0.05. Note that a Dunnett’s test between the Sc.Aβ- and Aβ_25-35-treated groups led to a significant (p < 0.05) difference (c) decrease of synaptophysin level in the mouse hippocampus. ANOVA: F_(3,22) = 9.15, p < 0.001, **p < 0.01, ***p < 0.001 vs Sc.Aβ/V, ^##p < 0.01 vs Aβ_25-35/V; Dunnett’s test. V, vehicle solution. N = 5–7 per group.

Figure 4. Effect of ARN14140 infusion for 7 days on Aβ_25-35-induced toxicity.

(a) Bax level in the mouse hippocampus; (b) Bax/Bcl-2 ratio in the mouse hippocampus. V, vehicle solution. N = 5–7 per group in (a,b), ANOVA: F_(3,22) = 12.9, p < 0.0001 in (a), F_(3,22) = 9.13, p < 0.0001 in (b), ***p < 0.001 vs. Sc.Aβ/V, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Aβ_25–35/V; Dunnett’s test. Note that Bcl-2 levels did not differ among groups (F_(3,22) = 0.04, p > 0.05).

Figure 5. Effects of ARN14140 infusion for 7 days on Aβ_25-35-induced decrease in viable pyramidal cells in the CA1 layer of the mouse hippocampus.

(a) Typical CA1 field in zone c; (b) typical CA1 field in zone b; (c) quantification including zones a–c. V, vehicle solution. N = 10–11 animals per group, with 3 areas counted per animal. ANOVA: F_(3,40) = 3.01, p < 0.05, *p < 0.05 vs. Sc.Aβ/V, ^#p < 0.05 vs. Aβ_25-35/V; Dunnett’s test. Scale bar = 50 μm in (a,b).

Figure 6. Immunohistochemical staining of VAChT in Aβ_25-35-treated mouse.

Hippocampus (a,c) and cortex (b,d) CA3 field in: (a,b) Sc.Aβ/V-treated mouse and (c,d) Aβ_25-35/V-treated mouse. Cholinergic nerve terminals were pointed out on the figures by arrowheads and cholinergic punctae labeling by arrows. Hippocampus (e,g) and cortex (f,h) CA3 field in: (e,f) Aβ_25-35/ARN14140 (2.5)-treated mouse and (g,h) Aβ_25-35/ARN14140 (7.5)-treated mouse. Scale bar = 200 μm.