Composition for prevention or treatment of alzheimer's disease

Abstract

Provided is a novel means capable of efficiently inhibiting beta-secretase (BACE1). Specifically, a BACE1-inhibiting composition containing a specific glycerophospholipid is provided.

Description

Composition for preventing or treating Alzheimer's disease

[0001] The present disclosure relates to a composition for preventing or treating Alzheimer's disease.

[0002] Amyloid β peptide (Aβ), especially Aβ aggregation, is widely known as the most important factor in the development of Alzheimer's disease (AD). Aβ is known to be generated by cleavage of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase.

[0003] J. Neuropathol. Exp. Neurol. (1999) 50:740J. Neuroinflammation (2012) 13:97Neurobiol. Dis. (2011) 42:404

[0004] If β-secretase (BACE1) can be efficiently inhibited, it is thought that the production of Aβ and, in turn, Aβ aggregation can be suppressed, potentially leading to the prevention and treatment of Alzheimer's disease.

[0005] Therefore, the present inventors attempted to develop a novel means capable of efficiently inhibiting β-secretase (BACE1).

[0006] The present inventors have found that glycerophospholipids having a specific structure can efficiently inhibit BACE1, and have made further improvements.

[0007] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. Formula (1):

[0008]

[0009] (In the formula, R １ represents an alkyl or alkenyl group having 14 to 20 carbon atoms, which may be substituted with an OH group; R ２ is R ２ -COOH represents a group representing a fatty acid having 18 carbon atoms, and R ３ is -CH ２ CH ２ N ＋ (CH ３ ) ３Item 2. A composition for preventing or treating Alzheimer's disease, comprising a compound represented by formula (1):

[0010]

[0011] (In the formula, R １ represents an alkyl or alkenyl group having 14 to 20 carbon atoms, which may be substituted with an OH group; R ２ is R ２ -COOH represents a group representing a fatty acid having 18 carbon atoms, and R ３ is -CH ２ CH ２ N ＋ (CH ３ ) ３ Item 3. A composition for inhibiting BACE1, comprising a compound represented by the formula (1): １ represents a linear alkyl group or linear alkenyl group having 16 or 18 carbon atoms, which may be substituted with one OH group; R ２ But -(CH ２ ) ７ CH=CH(CH ２ ) ７ CH ３ Item 4. The composition according to any one of Items 1 to 3, which is a pharmaceutical composition or a food composition.

[0012] This provides a novel means for efficiently inhibiting BACE1, which is expected to enable the effective prevention and treatment of Alzheimer's disease.

[0013] The left panel shows the ratio of the expression levels of C83 and C99 (C83 / C99) produced from APP when SH-SY5Y cells were treated with PC-PLS-18 or PC-DPL-18. The right panel shows the results of Western blotting to examine the expression levels of α-secretase (ADAM9, ADAM10, TACE) when the same treatment was performed. An overview of the mechanism by which Aβ is produced from APP is shown. The results of examining the expression level of BACE1 when SH-SY5Y cells were treated with PC-PLS-18 or PC-DPL-18 are shown. The results of examining the expression level of BACE1 when SH-SY5Y cells were treated with PE-PLS-18 or PE-DPL-18 are shown. The results of examining the expression levels of CLU and Tau proteins when SH-SY5Y cells were treated with PC-PLS-18 are shown.

[0014] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes a composition for inhibiting BACE1 containing a glycerophospholipid having a specific structure, and further includes a composition for preventing or treating Alzheimer's disease, but is not limited thereto. The present disclosure includes all of the compositions disclosed herein and recognizable by a person skilled in the art.

[0015] A composition containing a glycerophospholipid having a specific structure encompassed by the present disclosure may be referred to as a composition of the present disclosure. As described above, the composition of the present disclosure is useful as a composition for inhibiting BACE1, and thus as a composition for preventing or treating Alzheimer's disease. Furthermore, a glycerophospholipid having a specific structure contained in a composition of the present disclosure may be referred to as a glycerophospholipid of the present disclosure.

[0016] The glycerophospholipids of the present disclosure have the formula (1):

[0017]

[0018] (In the formula, R １ represents an alkyl or alkenyl group having 14 to 20 carbon atoms, which may be substituted with an OH group; R ２ is R ２ -COOH represents a group representing a fatty acid having 18 carbon atoms, and R ３ is -CH ２ CH２ N ＋ (CH ３ ) ３ The compound is represented by the formula:

[0019] R １ As described above, represents an alkyl group or alkenyl group having 14 to 20 carbon atoms (14, 15, 16, 17, 18, 19, or 20), which may be substituted with an OH group. The alkyl group or alkenyl group may be linear or branched, and linear groups are preferred. In the alkenyl group, the carbon-carbon double bond is preferably R １ Counting from the carbon atom to which R １ is bonded to), it is preferably present between the 6th and 7th carbon atoms or between the 8th and 9th carbon atoms. In addition, when substituted with an OH group, it is preferable that one or two OH groups are substituted, and it is particularly preferable that one hydrogen atom of the terminal methyl group of the alkyl group or alkenyl group is substituted with an OH group.

[0020] R １ As R, a linear alkyl group or linear alkenyl group having 16 or 18 carbon atoms, which may be substituted with an OH group, is more preferred. １ As for R １ A linear alkyl group or linear alkenyl group in which —COOH represents palmitic acid, stearic acid, or oleic acid and which may have a structure in which one hydrogen atom of the terminal methyl group is substituted with an OH group is most preferred.

[0021] R ２ As mentioned above, R ２ —COOH represents a group representing a fatty acid having 18 carbon atoms. The fatty acid having 18 carbon atoms may be a saturated fatty acid or an unsaturated fatty acid, and more specific examples include stearic acid, oleic acid, vaccenic acid, linoleic acid, and linolenic acid, with oleic acid being particularly preferred.

[0022] The glycerophospholipid of the present disclosure can be said to be a type of plasmalogen. Plasmalogens are generally formed by attaching an aliphatic hydrocarbon group or an aliphatic alcohol group (in formula (1), -CH=CH-R bonded to the glycerol skeleton) via a vinyl ether bond to the 1-position (sn-1 position) of the glycerol skeleton. １ It refers to a glycerophospholipid having a group represented by the formula:

[0023] Plasmalogen is a compound represented by the formula (1): ２ is usually an aliphatic hydrocarbon group derived from a fatty acid residue, and the number of carbon atoms therein is not particularly limited. However, in the glycerophospholipids of the present disclosure, R ２ is R ２ -COOH is a group representing a fatty acid having 18 carbon atoms. ２ When the group is such, a particularly favorable BACE1 inhibitory effect, and therefore an effect of preventing or treating Alzheimer's disease, is exhibited.

[0024] In addition, plasmalogen is a compound represented by the formula (1), ３ represents various polar groups, and may be, for example, ethanolamine, serine, inositol, or glycerol, but in the glycerophospholipids of the present disclosure, -CH ２ CH ２ N ＋ (CH ３ ) ３ (i.e. choline).

[0025] The glycerophospholipid of the present disclosure can be, for example, a synthetic product or an extract. Among extracts, those extracted from biological tissues are preferred. When using an extract, the crude extract may be further purified by a known method (e.g., HPLC, etc.) and used as the glycerophospholipid of the present disclosure.

[0026] The content of the glycerophospholipid of the present disclosure in the composition of the present disclosure is not particularly limited as long as the effect is obtained. For example, it may be 0.1 to 100% by mass, or 0.5 to 99% by mass. In particular, when the composition of the present disclosure is a solid composition (particularly a dry composition), the glycerophospholipid of the present disclosure is preferably contained in an amount of, for example, 0.1 to 10% by mass. The lower limit of this range may be, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% by mass. The upper limit of this range may be, for example, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3% by mass.

[0027] The administration form of the composition of the present disclosure is not particularly limited as long as the effect can be obtained. For example, oral administration, intravascular administration (intravenous administration, intraarterial administration), subcutaneous administration, etc. are included, but oral administration is particularly preferred. In other words, it is particularly preferred that the composition of the present disclosure is an oral composition.

[0028] The composition of the present disclosure can be preferably used as, for example, a pharmaceutical composition or a food composition.

[0029] When used as a pharmaceutical composition, the glycerophospholipid of the present disclosure, which is the active ingredient, can be blended with pharmaceutically acceptable bases, carriers, additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavorings, odorants, surfactants, humectants, preservatives, pH adjusters, thickeners, etc.) as needed. The formulation form is also not particularly limited, and the active ingredient and other ingredients can be mixed using conventional methods to prepare formulations such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, emulsions, jellies, chewable tablets, and soft tablets.

[0030] When used as a food composition, the glycerophospholipid of the present disclosure may be appropriately blended with hygienically acceptable bases, carriers, additives, and other ingredients and materials that can be used in foods and beverages. Examples of food compositions that contain the glycerophospholipid of the present disclosure include processed foods, beverages, health foods (nutrient-functional foods, foods for specified health uses, etc.), supplements, and foods for the sick (hospital meals, sick meals, or nursing care meals, etc.). While not particularly limited, examples include processed meat foods such as burgers, meatballs, sausages, minced chicken, and chicken skin chips, as well as health foods (nutrient-functional foods, foods for specified health uses, etc.), supplements, and foods for the sick that contain processed meat foods. Furthermore, plasmalogens may be powdered and incorporated into various foods and beverages, such as beverages (juices, etc.), confectioneries (e.g., gum, chocolate, candy, biscuits, cookies, rice crackers, rice crackers, puddings, almond tofu, etc.), breads, soups (including powdered soups, etc.), and processed foods.

[0031] When preparing the food and beverage products of the present invention as health foods (nutritional functional foods, foods for specified health uses, etc.) or supplements, it is preferable to prepare them in the form of, for example, granules, capsules, tablets (including chewable tablets), beverages (drinkable supplements), etc., so that they can be easily taken continuously. Of these, capsule, tablet, and tablet forms are preferred in terms of ease of intake, but the present invention is not limited to these.

[0032] The glycerophospholipids of the present disclosure can efficiently inhibit β-secretase (BACE1), thereby suppressing Aβ production, and can also suppress the production of clusterin α (CLU) and tau protein, which are factors that affect Aβ levels.

[0033] For these reasons, the composition of the present disclosure can be preferably used as a composition for inhibiting BACE1, and therefore as a composition for preventing or treating Alzheimer's disease.

[0034] It should be noted that in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0035] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0036] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0037] Human neuroblastoma SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium / Ham's F-12 (DMEM / Ham's F-12) containing 10% fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 IU / mL penicillin under conditions of 95% air, 5% CO2, and 37°C.

[0038] Glycerophospholipids used: Four types of glycerophospholipids, PC-PLS-18, PC-DPL-18, PE-PLS-18, and PE-DPL-18, were purchased from Avanti. These four types of glycerophospholipids are shown below.

[0039] PC-PLS-18 and PE-PLS-18 have the formula (1):

[0040]

[0041] In this case, R １ ~R ３ represents the following group. ２ ) ７ CH=CH(CH２ ) ７ CH ３ is oleic acid.

[0042] PC-PLS-18R １ :-(CH ２ ) １５ CH ３ R ２ :-(CH ２ ) ７ CH=CH(CH ２ ) ７ CH ３ R ３ :-CH ２ CH ２ N ＋ (CH ３ ) ３

[0043] PE-PLS-18R １ :-(CH ２ ) １５ CH ３ R ２ :-(CH ２ ) ７ CH=CH(CH ２ ) ７ CH ３ R ３ :-CH ２ CH ２ NH ２

[0044] PC-DPL-18 and PE-DPL-18 are represented by the formula (2):

[0045]

[0046] In this case, R １ ~R ３ represents the group described below.

[0047] PC-DPL-18R １ :-(CH ２ ) １５ CH ３ R ２ :-(CH ２ ) ７ CH=CH(CH ２ ) ７ CH ３ R ３ :-CH２ CH ２ N ＋ (CH ３ ) ３

[0048] PE-DPL-18 R １ :-(CH ２ ) １５ CH ３ R ２ :-(CH ２ ) ７ CH=CH(CH ２ ) ７ CH ３ R ３ :-CH ２ CH ２ NH ２

[0049] Western blotting 1.6×10 6SH-SY5Y cells were seeded at 100 cells / well in a 6-well plate and incubated in DMEM / Ham's F-12 containing 10% FBS, 100 μg / mL streptomycin, and 100 IU / mL penicillin under conditions of 95% air, 5% CO2, and 37°C for 24 hours. The medium was then changed to Opti-MEM, and the reagents were added. After 2 hours, the cells were treated with 1 μg / mL of choline-type plasmalogen with oleic acid (18:1) attached at the sn-2 position (PC-PLS-18), phosphatidylcholine-type diacylphospholipid with oleic acid (18:1) attached at the sn-2 position (PC-DPL-18), ethanolamine-type plasmalogen with oleic acid (18:1) attached at the sn-2 position (PE-PLS-18), and ethanolamine-type diacylphospholipid with oleic acid (18:1) attached at the sn-2 position (PE-DPL-18), respectively. Control (Ctrl) cells were treated with a solution of chloroform:ethanol (1:9) dissolved in Opti-MEM. After 16–20 hours, the cells were harvested using a cell scraper with 100 μL / well of RIPA buffer. After incubation in RIPA buffer at 4°C for 15 minutes, the samples were centrifuged at 14,800 rpm at 4°C for 20 minutes, and the supernatant was collected and used as the sample. Protein concentration was measured by Bradford assay, adjusted to 20–60 μg / sample, and then 5× Laemmli buffer was added. The sample was heated at 95°C for 5 minutes to desaturate it with SDS. The resulting gel was then applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4% stacking gel, 10–14% running gel). After electrophoresis at 80 V for 150 minutes or 120 V for 120 minutes, the gel was transferred to a PVDF membrane at 0.1 A for 60 minutes. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated with primary antibody for 1 hour at room temperature or 6 hours at 4°C, followed by overnight incubation at 4°C.The membrane was then washed three times for 10 minutes with TBS-T (0.1% Tween 20 in TBS) and incubated with the secondary antibody for 1 hour at room temperature. After washing three times for 10 minutes, the membrane was incubated with Super Signal West Pico PLUS Chemiluminescent Substrate for 1.5 minutes and detected using an ImageQuant LAS-4000 (GE Healthcare).

[0050] Antibody dilution conditions used ・APP, C-Terminal Primary antibody: anti-APP C-Terminal (1:1000) in Solution1 in TBS-T Secondary antibody: Immun-Star Goat Anti-Rabbit (GAM) - HRP (1:10000) in 5% BSA in TBS-T・APP-C99 Primary antibody: anti-APP-C99 (1:1000) in Solution1 in TBS-T secondary antibody: Immun-Star Goat Anti-Mouse (GAM) - HRP (1:10000) in 5% BSA in TBS-T・BACE1 Primary antibody: anti-BACE (1:1000) in Solution1 in TBS-T Secondary antibody: Immun-Star Goat Anti-Mouse (GAM) - HRP (1:5000) in 5% BSA in TBS-T・α-secretase Primary antibody: ADAM10 antibody (A-3); sc-48400, TACE / ADAM17 antibody (B-6); sc-390859, and ADAM9 antibody (G-1); sc-377233 in Solution1 in TBS-T Secondary antibody: Immun-Star Goat Anti-Mouse (GAM) - HRP (1:5000) in 5% BSA in TBS-T

[0051] ・CLU primary antibody: anti-Clusterin-α (1:1000) in Solution1 in TBS-T Secondary antibody: Immun-Star Goat Anti-Mouse (GAM) - HRP (1:5000) in 5% BSA in TBS-T ・Tau primary antibody: anti-Tau (1:1000) in Solution1 in TBS-T Secondary antibody: Immun-Star Goat Anti-Mouse (GAM) - HRP (1:10000) in 5% BSA in TBS-T ・β-tubulin Primary antibody: anti-β-tubulin (1:2000) in 5%BSA in TBS-T Secondary antibody: Immun-Star Goat Anti-Mouse (GAM) - HRP (1:10000) in 5% BSA in TBS-T

[0052] ELISA 1.6 x 10 6 SH-SY5Y cells were seeded in 6-well plates at 100 cells / well and incubated in DMEM / Ham's F-12 medium containing 100% FBS, 100 μg / mL streptomycin, and 100 IU / mL penicillin under 95% air, 5% CO2, and 37°C for 24 hours. The medium was then changed to Opti-MEM, and the cells were treated with PC-PLS-18 and PC-DPL-18 at 1 μg / mL each. After 16–20 hours, the cells were treated with 1 μg / mL arachidonic acid for 4 hours. The supernatant was collected and centrifuged for 5 minutes. Amyloid-β (Aβ) levels were measured using 100 μL / well of the supernatant using the Human β-Amyloid (1-42) ELISA Kit (Wako, #298-62401, Fujifilm Wako Pure Chemical Corporation).

[0053] The effect of PC-PLS-18 treatment on the C83 / C99 ratio was investigated. SH-SY5Y cells were treated with 1 μg / mL PC-PLS-18 or 1 μg / mL PC-DPL-18 (control) for 18–20 hours, and the C83 and C99 expression ratios were examined by Western blotting. PC-PLS-18 treatment significantly increased the C83 / C99 ratio. The results are shown in Figure 1A. The C83 and C99 bands in Figure 1A (detected by Western blotting) were detected using the antibody described above under "APP, C-Terminal" in the "Antibody Dilution Conditions" section, and the C99 band was detected using the antibody described above under "APP-C99" in the "Antibody Dilution Conditions" section.

[0054] Furthermore, Western blotting analysis of the expression levels of α-secretase (ADAM9, ADAM10, TACE) revealed that PC-PLS-18 did not decrease the expression level of α-secretase (Fig. 1B), suggesting that PC-PLS-18 may suppress amyloid β production via a decrease in the expression level of β-secretase, rather than α-secretase.

[0055] The mechanism by which Aβ is produced from APP is outlined in Figure 2. As can be seen from Figure 2, the results shown in Figure 1, in which the expression level of C99 is reduced but the expression level of C83 is not suppressed (i.e., the C83 / C99 ratio is increased), are interpreted as indicating that Aβ production is suppressed.

[0056] Effect of PC-PLS-18 Treatment on BACE1 Expression: We investigated whether PC-PLS-18 treatment altered the expression of β-site APP cleaving enzyme (BACE) 1, a β-secretase that influences Aβ levels. SH-SY5Y cells were treated with 1 μg / mL PC-PLS-18 or 1 μg / mL PC-DPL-18 (control) for 18–20 hours, and BACE1 expression was examined using Western blotting. PC-PLS-18 treatment significantly decreased BACE1 expression. However, PC-DPL-18 treatment did not significantly alter BACE1 expression (Figure 3).

[0057] Effect of PE-PLS-18 treatment on BACE1 expression. SH-SY5Y cells were treated with 1 μg / mL PE-PLS-18 or 1 μg / mL PE-DPL-18 (control) for 18–20 hours, and BACE1 expression was examined by Western blotting. Unlike PC-PLS-18 treatment, PE-PLS-18 treatment did not significantly affect BACE1 expression (Fig. 4).

[0058] Effects of PC-PLS-18 Treatment on CLU and Tau Expression. It was possible that the reduction in intracellular Aβ levels by PC-PLS-18 treatment also affected downstream factors. Therefore, we focused on clusterin α (CLU) and tau protein, two factors that affect Aβ levels, and examined the effects of PC-PLS-18 treatment on these two factors. SH-SY5Y cells were treated with 1 μg / mL PC-PLS-18 or 1 μg / mL PC-DPL-18 (control) for 18–20 hours, and CLU and tau protein expression was examined by Western blotting. PC-PLS-18 treatment significantly reduced both CLU and tau protein expression (Fig. 5A and B).

[0059] CLU is known to co-localize with Aβ and enhance neurotoxicity.

[0060] These results indicate that treatment of SH-SY5Y cells with PC-PLS-18 reduces BACE1 expression and the production of C99, the precursor of Aβ(1-42), from APP. This may also result in a reduction in the expression of intracellular CLU and tau protein. Treatment with PC-PLS-18 may reduce the accumulation of Aβ and tau protein, which is believed to occur in the early stages of Alzheimer's disease, suggesting that it may delay the onset of subsequent neuronal death and cognitive impairment. These results suggest that PC-PLS-18 may have a preventive effect on Alzheimer's disease, potentially delaying its onset.

Claims

1. Formula (1): (In the formula, R １ represents an alkyl group or alkenyl group having 14 to 20 carbon atoms which may be substituted with an OH group, and R ２ represents a group in which R ２ -COOH represents a fatty acid having 18 carbon atoms, and R ３ represents -CH ２ CH ２ N ＋ (CH ３ ) ３ .) A composition for preventing or treating Alzheimer's disease containing the compound represented by the formula.) 2. Formula (1): (In the formula, R １ represents an alkyl group or alkenyl group having 14 to 20 carbon atoms which may be substituted with an OH group, and R ２ represents R ２ -COOH represents a group representing a fatty acid having 18 carbon atoms, and R ３ represents -CH ２ CH ２ N ＋ (CH ３ ) ３ .) A composition for inhibiting BACE1 containing the compound represented by the formula.

3. In formula (1), R １ represents a linear alkyl group or a linear alkenyl group having 16 or 18 carbon atoms, which may be substituted with one OH group, and R ２ is -(CH ２ ) ７ CH=CH(CH ２ ) ７ CH ３ The composition according to claim 1 or 2, which is shown as 4. The composition according to claim 1 or 2, which is a pharmaceutical composition or a food composition.

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