JP7613667B2 - Composition for inhibiting amyloid beta toxicity - Google Patents

Description

The present disclosure relates to compositions for suppressing amyloid beta toxicity, etc.

Amyloid beta protein (sometimes referred to as "Aβ" in this specification) is one of the candidates for the causative agent of Alzheimer's disease. A well-known hypothesis is that Aβ aggregates to form amyloid fibrils, which induces neuronal cell death, thereby causing Alzheimer's disease. It is also known that Aβ has the ability to self-aggregate, and that zinc ions promote this.

Singhuber J, Baburin L, Khom S et al.: GABAA receptor modulators from the Chinese herbal drug junci medulla-the pith of juncus effuses. Planta med 78: 455-458 (2012) Wang YG, Wang YL, Zhai HF et al. : Phenanthrens from juncus effusus with anxiolytic and sedative activities. Nat Prod Res. 26: 1234-1239 (2012) Liao YJ, Zhai HF, Zhang B et al. : Anxiolytic and sedative effects of dehydroeffusol from juncus effusus in mice. Planta med. 77: 416-420 (2011)

The main body of Aβ toxicity is thought to be zinc ions themselves. Even without suppressing the self-aggregation ability of Aβ, by suppressing the toxicity of zinc ions, which is thought to be the main body of toxicity, it is thought that the neurotoxicity of Aβ (particularly Aβ _1-42 ) can be suppressed as a result. More specifically, although Aβ (particularly Aβ _1-42 ) is considered to be one of the causes of Alzheimer's disease, its toxicity is due to zinc toxicity, and it is thought that zinc promotes the entry of Aβ into nerve cells, and finally zinc toxicity occurs in nerve cells. Therefore, it is thought that neurotoxicity can be suppressed by reducing the amount of zinc ions in nerve cells.

Therefore, the inventors considered the possibility that the neurotoxicity of Aβ could be suppressed by reducing the amount of zinc ions in nerve cells in the presence of Aβ, and continued their investigation.

The inventors discovered that dehydroefsol may be able to reduce the amount of zinc ions in nerve cells, and have made further improvements.

The present disclosure includes, for example, the subject matter described in the following sections:
Item 1.
A composition for suppressing amyloid beta toxicity containing dehydroefsol.
Item 2.
A composition for inhibiting hippocampal neuronal cell death, comprising dehydroefsol.
Item 3.
A composition for inducing metallothionein in nerve cells, comprising dehydroefsol.
Item 4.
The composition according to claim 3, for a subject having an intracellular zinc ion concentration at which zinc ions can be released from amyloid beta aggregates.
Item 5.
The composition according to any one of claims 1 to 4, for a subject having amyloid beta aggregates in the hippocampus.

The present disclosure provides a method that is useful for reducing the amount of zinc ions in neurons and suppressing neuronal toxicity in the presence of Aβ.

The figure shows the results of investigating neuronal cell death in the dentate gyrus of the hippocampus when Aβ _1-42 was administered into the lateral ventricle of mice. It can be seen that dehydroefsol suppressed the cell death marker PI in the dentate gyrus of the hippocampus, which was increased by human Aβ _1-42 , to the control level. The figure shows the results of investigating neuronal cell death in the dentate gyrus of the hippocampus when Aβ _1-42 was administered into the lateral ventricle of mice. It can be seen that dehydroefsol suppressed the neurodegeneration marker FJB in the dentate gyrus of the hippocampus, which was increased by human Aβ _1-42 , to the control level. This shows the results of immunostaining with anti-Aβ antibody in the hippocampal dentate gyrus after Aβ _1-42 was administered into the lateral ventricle of mice. It is clear that dehydroefsol does not inhibit Aβ uptake into neurons. This shows the results of detecting zinc ions in the dentate gyrus of the hippocampus when Aβ _1-42 was administered into the lateral ventricle of mice, using a fluorescent probe for detecting zinc ions, ZnAF-2DA (Goryo Chemical Co., Ltd.). It shows that dehydroefsol inhibits the increase in zinc ions induced by Aβ _1-42 in neurons. This shows the results of immunostaining with anti-metallothionein antibody in the hippocampal dentate gyrus of mice administered Aβ _1-42 into the lateral ventricle, demonstrating that dehydroefsol increases metallothionein expression in neurons. FIG. 1 is a model diagram showing that dehydroefsol increases metallothionein (MT) expression in nerve cells and suppresses Aβ _1-42- induced increase in zinc ions (Zn ²⁺ ). The chromatograms obtained by HPLC analysis of an extract of the whole rush plant with a 50% aqueous ethanol solution or an extract of the rush core with a 50% aqueous ethanol solution are shown.

Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes compositions containing dehydroefsole, as well as uses and intended users thereof, but is not limited thereto, and the present disclosure includes everything disclosed in the present specification and recognizable by a person skilled in the art.

The composition included in the present disclosure (hereinafter sometimes referred to as the "composition of the present disclosure") contains dehydroefsole. Dehydroefsole is a compound represented by the following structural formula:

Dehydroefsol may be a synthetic product or may be obtained by extracting and purifying a product that exists in nature. It can be obtained by synthesis using a known method or a method that can be easily conceived of from a known method. When obtained from nature, for example, dehydroefsol is known to exist in rush (wick grass), and can be obtained by extracting (and purifying as necessary) from rush. More specifically, it can be obtained by extracting, for example, rush with water, ethanol, or a mixture thereof. As the extract, a 40 to 60% ethanol aqueous solution is particularly preferred, and a 50% ethanol aqueous solution is particularly preferred. If necessary, the obtained extract may be further fractionated and/or purified. Fractionation can be performed, for example, by liquid-liquid partition extraction. The partition can be performed, for example, using water and ethyl acetate. Purification can be performed, for example, by high performance liquid chromatography (HPLC). There is also no particular limitation on the part of the rush used for extraction, and for example, the whole rush plant may be used for extraction, or the core of the rush may be used for extraction. It is particularly preferable to use the core of rush grass, as it contains a large amount of dehydroefsol.

It is believed that the aggregation of Aβ and the formation of amyloid fibrils causes neuronal death, and it is also known that the aggregation of Aβ is promoted by zinc ions (Zn ²⁺ ). The inventors have discovered that dehydroefsol induces metallothionein in neurons (preferably hippocampal neurons), thereby reducing the zinc ion concentration within neurons and suppressing zinc toxicity (and thus suppressing Aβ toxicity).

For this reason, the composition of the present disclosure is particularly useful, for example, for suppressing amyloid beta toxicity, for suppressing neuronal cell (particularly hippocampal neuronal cell) death (particularly for suppressing neuronal cell death in the presence of Aβ), or for inducing metallothionein (particularly for inducing metallothionein in neuronal cells, etc.). Although not particularly limited, when used for inducing metallothionein in neuronal cells, it is preferable to use the composition for a subject whose intraneuronal zinc ion concentration is equal to or higher than the concentration that induces amyloid beta-induced zinc ion toxicity. In addition, zinc ions are thought to promote the formation of Aβ aggregates, but zinc ions are released from Aβ aggregates taken up into cells and neurons, and it is thought that the zinc ions cause neuronal toxicity (see FIG. 6). For this reason, when used for inducing metallothionein in neuronal cells, it is preferable that the zinc ion concentration in the neuronal cells is a concentration at which zinc ions can be released from the taken-up Aβ aggregates (i.e., the zinc ion concentration in the neuronal cells is relatively low). It is preferable that the Aβ in the Aβ aggregates is Aβ _1-42 . The Aβ aggregates preferably contain Aβ _1-42 and zinc ions. The nerve cells are more preferably hippocampal nerve cells.

Although not wishing to be bound by theory, the extracellular zinc ion concentration is estimated to be about 10 nM, and in the presence of about 100 to 500 pM Aβ _1-42 , Zn-Aβ _1-42 aggregates are formed and can be rapidly taken up into hippocampal neurons. Since the zinc ion concentration inside the cells is usually low at about 100 nM, Zn ²⁺ is released from Aβ _1-42 and becomes toxic.

Metallothionein is a protein that is rich in cysteine thiol groups and binds to metals such as zinc via the thiol groups. It is thought to function in maintaining zinc homeostasis, removing heavy metal toxicity, and eliminating radicals derived from reactive oxygen species.

In addition, the subject for which the composition of the present disclosure is used is not particularly limited, but is preferably a subject in which amyloid beta aggregates (and thus amyloid fibers) are present in the hippocampus. Examples of such subjects include Alzheimer's disease patients.

The composition of the present disclosure can be used not only in humans but also in non-human mammals. In particular, mammals kept as pets and livestock are preferred. Specific examples include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc.

The composition of the present disclosure is preferably used, for example, in the pharmaceutical and food fields. As described in detail below, the composition may consist of only dehydroefsole, or may contain dehydroefsole and other components (various bases, carriers, additives, etc.). In addition, the biological extract (preferably rush extract) itself is also included in the composition. In other words, the biological extract containing dehydroefsole (and further containing other components as necessary) is also included in the composition containing dehydroefsole of the present disclosure. Note that a composition is usually composed of multiple components, so if the composition of the present disclosure consists only of dehydroefsole, it may not be appropriate to refer to it as a composition, but in this specification, even if it consists only of dehydroefsole, it will be referred to as a composition. (In other words, the term "composition" is synonymous with the term "agent.")

When the composition of the present disclosure is used in the pharmaceutical field (including drugs and quasi-drugs), the composition (hereinafter sometimes referred to as the "pharmaceutical composition of the present disclosure") may consist of dehydroefsole alone, or may be a composition containing other ingredients. For example, in the pharmaceutical composition of the present disclosure, the active ingredient dehydroefsole may be mixed with pharma- ceutical acceptable bases, carriers, additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavorings, odorants, surfactants, moisturizers, preservatives, pH adjusters, thickeners, etc.) as necessary. Such bases, carriers, additives, etc. are specifically described, for example, in the Pharmaceutical Additives Dictionary 2016 (Yakuji Nippo Co., Ltd.), and those described therein may be used. The formulation form is not particularly limited, and the active ingredient and other ingredients can be mixed in a conventional manner to prepare formulations such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, emulsions, jellies, chewable tablets, and soft tablets. For example, tablets can be produced by tableting. Either a direct tableting method in which the mixed raw materials are directly tableted, or a granule tableting method in which the mixed raw materials are granulated and then tableted, can be used. For example, in the case of capsules, either a soft capsule or a hard capsule can be used.

The amount of dehydroefsole in the pharmaceutical composition according to the present disclosure is not particularly limited as long as the effect is exhibited, and can be appropriately set according to the subject. It is preferably 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.05 to 80% by mass. The lower limit may be about 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, 60% by mass, 70% by mass, or 80% by mass.

The timing of administration of the pharmaceutical composition according to the present disclosure is not particularly limited, and can be appropriately selected taking into consideration, for example, the formulation form, the age of the subject, and the severity of the symptoms of the subject. The form of administration is not particularly limited, but oral administration and intravascular administration (intravenous administration, intraarterial administration) are preferred, and oral administration is more preferred.

The dosage of the pharmaceutical composition according to the present disclosure may be appropriately selected depending on the age of the subject, the severity of symptoms of the subject, and other conditions. In particular, the amount of dehydroefsole contained may be set appropriately within a range that does not impair the effects of the present disclosure. Although not particularly limited, for example, the amount of dehydroefsole administered to an adult per day is preferably about 1 to 50 mg, more preferably about 5 to 30 mg, about 6 to 24 mg, about 10 to 24 mg, or about 12 to 24 mg. The amount may be administered once or multiple times (preferably 2 to 3 times) per day. In the case of a non-human mammal, the dosage may be set appropriately by referring to the case of the person.

When the composition of the present disclosure is used as a food composition (e.g., food and beverage products or food additives), the composition (hereinafter sometimes referred to as the "food composition of the present disclosure") is a composition in which dehydroefsol and a base, carrier, additive, or other ingredient or material that can be used as a food or beverage product that is acceptable from the viewpoint of food hygiene are appropriately blended. Examples of such food compositions that contain dehydroefsol include processed foods, beverages, health foods, functional foods, nutritional supplements, supplements, health functional foods, foods for specified health uses, nutritional functional foods, foods with functional claims, or foods for the sick (hospital foods, sick foods, or nursing care foods, etc.) for enhancing learning and memory ability. Although not particularly limited, when the dehydroefsol blended in the food composition is a biological extract (preferably a rush grass extract), the food composition may be, for example, a processed food, health food, nutritional functional foods, foods for specified health uses, supplements, foods for the sick, etc., in which the extract is blended. Dehydroefsol may also be powdered and added to various foods and beverages such as beverages (juices, etc.), confectioneries, breads, soups (including powdered soups, etc.), and processed foods. The food composition according to the present disclosure can also be used preventively when Aβ aggregation (particularly Aβ aggregation in the hippocampus) is predicted to occur in the near future. Hospital food is food provided when hospitalized, sick food is food for sick people, and nursing food is food for people receiving care. The food composition according to the present disclosure can be preferably used as hospital food, sick food, or nursing food for patients who are hospitalized or recuperating at home, or patients receiving care, and who need to prevent and/or inhibit Aβ aggregation.

When preparing the food composition according to the present disclosure as a health food (nutritional functional food, food for specified health uses, etc.) or supplement, it is preferable to prepare it in the form of, for example, granules, capsules, tablets (including chewable tablets, etc.), beverages (drinks), etc., so that it can be easily taken continuously, and among these, capsules, tablets, and tablets are preferable in terms of ease of intake. However, this is not particularly limited. The food composition according to the present disclosure in the form of granules, capsules, tablets, etc. can be appropriately prepared according to conventional methods using medicaments and/or food hygienically acceptable carriers, etc. Furthermore, even when preparing it in other forms, conventional methods may be followed.

The amount of dehydroefsol in the food composition according to the present disclosure is not particularly limited as long as the effect can be exhibited. It is preferably 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.05 to 80% by mass. The lower limit may be about 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, 60% by mass, 70% by mass, or 80% by mass.

It is preferable that the subjects of intake of the food composition according to the present disclosure are the same as those of the pharmaceutical composition according to the present disclosure described above.

The present disclosure also includes a method for producing the composition of the present disclosure, which includes a step of compounding dehydroefsole, as well as a method for suppressing amyloid beta toxicity, a method for suppressing hippocampal neuronal cell death, a method for inducing metallothionein in the hippocampus, and the like, by administering dehydroefsole (preferably the composition of the present disclosure containing the above-mentioned dehydroefsole). The various conditions in these methods are as described above.

In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

Furthermore, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described in this specification.

The present invention will be described in more detail below, but the present invention is not limited to the following examples.

In the following study, human amyloid beta protein (1-42) was purchased and used as the amyloid beta protein (Synthetic human Aβ1-42, ChinaPeptides, Shanghai, China). The amino acid sequence of the human amyloid beta protein is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. The human amyloid beta protein may also be referred to as Aβ _1-42 .

Aβ _1-42 (25 μM, 20 μl) or saline as a control was administered into the lateral ventricle of mice. Two weeks later, frozen brain sections or whole brain slices were prepared, and hippocampal dentate gyrus granule cell death was evaluated using Fluoro-Jade-B (FJB), a neurodegeneration marker, and propidium iodide (PI), a cell death marker, under a confocal laser microscope (GCL: dentate gyrus granule cell layer). Mice were orally administered dehydroefsol (15 mg/kg, 10 mL/kg/day) once a day for a total of six times, and then Aβ _1-42 or saline was administered into the lateral ventricle for evaluation. Neurodegeneration and cell death in the hippocampal dentate gyrus due to Aβ _1-42 were suppressed to the control level (Figures 1 and 2; in these figures, vehicle means 0.5% carboxymethyl cellulose sodium).

This pre-administration of dehydroefsol did not inhibit Aβ uptake into neurons as assessed by anti-Aβ antibody (Figure 3). However, it suppressed the increase in intracellular Zn ²⁺ levels induced by Aβ _1-42 to the control level (Figure 4). The photographs in Figure 4 were taken using ZnAF-2DA (Goryo Chemical Co., Ltd.), a fluorescent probe for detecting zinc ions.

Next, we investigated the induction of metallothionein (MT), an intracellular zinc-binding protein, by dehydroefsol. Immunostaining of MT revealed that MT levels were significantly increased in the hippocampal dentate gyrus after six doses of dehydroefsol (Fig. 5). It was revealed that MT induced by dehydroefsol suppressed the increase in intracellular Zn2 ⁺ levels induced by _Aβ1-42 and prevented hippocampal neuronal cell death induced by _Aβ1-42 (Fig. 6).

Dehydroefsol was obtained by further purifying the 50% ethanol aqueous solution extract of rush grass (whole plant or core). Figure 7 shows the chromatogram obtained by HPLC analysis of the 50% ethanol aqueous solution extract of the whole plant or the 50% ethanol aqueous solution extract of the rush grass core. The extraction, fractionation and purification of the rush grass core are explained in detail below. 5 kg of commercially available wick grass (dried pith of the stem of rush grass of the Juncaceae family) was extracted twice by refluxing at 80°C for 2 hours in 20 times the amount of 50% aqueous ethanol and filtered. The filtrate was concentrated under reduced pressure, 1.3 liters of distilled water was added to the concentrated liquid to suspend it, and 1.3 liters of ethyl acetate was added and liquid-liquid partition extraction was performed using a separatory funnel. This was repeated three times, and the water layer and ethyl acetate layer were separated. The ethyl acetate was distilled off under reduced pressure and dried to obtain 26.2 g of ethyl acetate extract. This ethyl acetate extract was fractionated into 13 fractions, A to M, using silica gel column chromatography (510 milliliters) (eluent: chloroform containing 2% methanol to chloroform containing 40% methanol). Fractions F and G were purified using preparative HPLC to obtain compound 1 (0.218 grams) and compound 2 (1.725 grams). Compounds 1 and 2 were identified as efsole and dehydroefsole by NMR spectroscopy, respectively.

Claims (4)

A composition containing dehydroefsol for use in a subject having an intracellular zinc ion concentration of 100 nM or less , the concentration at which zinc ions can be released from amyloid beta aggregates, for inducing metallothionein in the neurons of the subject. The composition of claim 1 for a subject having amyloid beta aggregates in the hippocampus. The composition according to claim 1 or 2 for suppressing hippocampal neurodegeneration and/or hippocampal neuronal cell death. The composition according to claim 1 or 2 for suppressing the toxicity of zinc ions released from amyloid beta aggregates in neuronal cells.

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