Compositions and methods for the treatment of "plaques and tangles" in humans and animals
一种组合物、斑块的技术,应用在药物组合、医药配方、植物原材料等方向
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Embodiment 1
[0175] Example 1: Destruction / inhibition of Alzheimer's disease Aβ fibrils or aggregates
[0176] The compositions described herein were found to be potent disruptors / inhibitors of A[beta] protein fibrils or aggregates. This example investigates the efficacy of the composition in causing the breakdown / disruption / depolymerization of pre-formed amyloid fibrils (ie, consisting of Aβ1-42) of Alzheimer's disease.
[0177] Part A: Thioflavin T fluorescence method
[0178] In this study, the effect of the composition was determined using Thioflavin T fluorescence. Thioflavin T specifically binds to fibril amyloid, and this binding produces a fluorescence increase at 485 nm that is proportional to the number of amyloid fibrils formed. The higher the fluorescence, the more the number of amyloid fibrils formed (Nakai et al., Lab.Invest.65:104-110, 1991; Levine III, Protein Sci.2:404-410, 1993; Amyloid: Int. J. Exp. Clin. Invest. 2:1-6, 1995).
[0179] In this study, 40 μl of a 1 mg / ...
Embodiment 2
[0209] Example 2: Inhibition / Disruption of In Vitro Conversion of Aβ to β-Sheets Containing Fibrillar Structures Part A: Thioflavin T Fluorometric Assay
[0210] To test whether the compositions described herein can inhibit β-sheet formation of Aβ, the same Thioflavin T assay as described in Example 1 was utilized, but using Aβ1-40 as substrate instead. Similar to Aβ1-42, Aβ1-40 forms Thioflavin T-positive aggregates but requires >24 hours of incubation at 37°C with shaking to fully fibrillate. Since A[beta]1-40 is in a non-fibrillar state at the start of the assay, this protein can be aggregated in the presence of the composition to measure aggregation inhibition. Lyophilized human Aβ1-40 (rPeptide) was dissolved in dH20 to 1 mg / mL (220 μΜ). In separate tubes, different concentrations of test composition stock solutions were prepared in PBS such that a final reaction containing equal volumes of test composition stock solutions and Aβ solution would yield a final Aβ concentra...
Embodiment 3
[0218] Example 3: In vitro screening of tau aggregation inhibitors using recombinant Tau repeat domains
[0219] In previous studies on in vitro screens for the identification of tau aggregation inhibitors, the present inventors found that, under the same experimental conditions, paired helical filaments were formed from commercially available full-length tau protein (Tau441; from rPeptide). Filament (PHF) (>11 days; data not shown) is much slower than formation of paired helical filaments (>24 hours) by tau repeat domains (TauRD; Q244-E372 containing Tau441) (S. Barghorn et al. , Methods Mol Biol, 299:35-51, 2005).
[0220] Due to its apparently short turnaround time and common aggregation properties, TauRD was used to screen tau aggregation inhibitors in vitro. Since TauRD protein is not commercially available, TauRD was prepared according to the following procedure, which is also described in US 2016 / 0250273 (where the disclosure for this procedure is incorporated by refer...
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