JP2024033770A - COMPOSITION FOR PREVENTION, TREATMENT, OR MITIGATION OF BRAIN FUNCTION DECLINE, AND COMPOSITION FOR INHIBITION OF AMYLOID β AGGREGATE ACCUMULATION - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for use in prevention, treatment, or mitigation of brain function decline, and a composition for inhibition of amyloid β aggregate accumulation.

SOLUTION: The present invention provides a composition for use in prevention, treatment, or mitigation of brain function decline, comprising one or more components selected from a group consisting of dipeptide comprising an aspartic acid, an aspartic acid itself, and methionine as an active ingredient.

SELECTED DRAWING: None

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention relates to a composition for preventing, treating, or alleviating a decline in brain function, and a composition for suppressing accumulation of amyloid β aggregates.

As humans age, brain functions such as learning ability and memory gradually decline, making them more susceptible to forgetfulness, anxiety disorders, loss of motivation, and poor sleep quality. In addition to this natural law, when a person suffers from dementia, it can lead to a serious situation that threatens the dignity of the person. Research into the prevention and treatment of dementia is currently being actively pursued.

Dementia is caused by various disorders caused by the death or deterioration of brain cells due to various causes, resulting in an inability to make decisions and a rapid decline in memory. Refers to a condition that continues to interfere with daily life. The most common cause of dementia is a neurodegenerative disease in which nerve cells in the brain slowly die, followed by cerebrovascular dementia. Examples of this neurodegenerative disease include Alzheimer's disease, frontotemporal dementia, and Lewy body dementia. In addition, cerebrovascular dementia is caused by cerebral infarction, cerebral hemorrhage, cerebral arteriosclerosis, etc., which causes nerve cells in the brain to die or nerve networks to break down.

Alzheimer's disease is a neurodegenerative disease that accounts for 50% to 60% of dementia cases. A major pathological change in the early stages of Alzheimer's disease is the formation in the brain of senile plaques mainly composed of amyloid beta protein (hereinafter also referred to as "amyloid β" or "Aβ"). Therefore, the amyloid hypothesis that Aβ in the brain causes the onset and progression of Alzheimer's disease is powerful. Based on this amyloid hypothesis, it is considered possible to suppress the progression of Alzheimer's disease by discharging Aβ from the brain. Furthermore, there is support for the oligomer hypothesis that relatively small Aβ oligomers, in which about 12 Aβ molecules are aggregated, have high toxicity. Since Aβ oligomers are soluble, they easily diffuse, and they also bind to various functional proteins and exhibit neurotoxicity. An anti-Aβ monoclonal antibody therapy against Aβ oligomers has been developed, and an application has been filed for marketing and manufacturing of the drug.
Aβ having such high hydrophobicity is highly susceptible to aggregation, forming soluble and highly toxic oligomers, but also forming larger, insoluble, amorphous Aβ aggregates. Since these large Aβ aggregates are hardly soluble in water, they are difficult to excrete from the brain.

Here, Patent Document 1 describes that chlorogenic acid (5-CQA) has an activity of decomposing aggregated amyloid in a dose-dependent manner.

Patent No. 6529634

In contrast to the above-mentioned prior art, an object of the present invention is to provide a composition for use in preventing, treating, or alleviating decline in brain function.
Another object of the present invention is to provide a composition for use in suppressing the accumulation of amyloid β aggregates.

As a result of intensive research to solve the above problems, the present inventors found that specific amino acids, dipeptides, etc. contribute to suppressing the aggregation of amyloid β monomers and/or fragmenting amyloid β aggregates. The present invention was completed based on the discovery that this is effective in preventing, treating, or alleviating the decline in brain function.

The present invention to solve the above problems is directed to a method for preventing or treating a decline in brain function, which contains as an active ingredient one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine. A composition for use in relief.

The present invention also provides a composition for inhibiting the accumulation of amyloid β aggregates, which contains as an active ingredient one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine. be.

In a preferred form of the invention, the dipeptide is Gly-Asp or Thr-Asp.

In a preferred form of the invention, the invention further comprises straight chain saturated fatty acids.

In a preferred form of the invention, the straight chain saturated fatty acid is palmitic acid.

In a preferred form of the present invention, the decline in brain function is due to dementia.

In a preferred form of the invention, the dementia is Alzheimer's dementia.

In a preferred embodiment of the present invention, the composition for use in treating Alzheimer's type dementia is used for inhibiting aggregation of amyloid β monomers and/or fragmenting amyloid β aggregates.

In a preferred form of the invention, the invention is a composition for administration to a subject who has not developed dementia.

In a preferred form of the invention, the invention is a pharmaceutical composition.
Moreover, in a preferable form of the present invention, the present invention is a composition for food or drink.

According to the present invention, a composition effective for preventing, treating, or alleviating a decline in brain function can be provided.
Further, according to the present invention, it is possible to provide a composition that is effective in suppressing accumulation of amyloid β by suppressing aggregation of amyloid β monomers and/or fragmenting amyloid β aggregates.
Note that the effects described here are not necessarily limited, and may be any effects described in this specification.

1 is a graph showing the effect of inhibiting amyloid β (Aβ) aggregation by amino acids in the presence of palmitic acid in Test Example 1.

Embodiments for implementing the present invention will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be interpreted narrowly thereby.

<Composition>
The composition of the present invention is a composition used for preventing, treating, or alleviating a decline in brain function (hereinafter also referred to as "prevention of a decline in brain function, etc."), and is a dipeptide containing aspartic acid (hereinafter, " This is a composition containing one or more components selected from the group consisting of a peptide according to the present invention), aspartic acid, and methionine.
Furthermore, the composition of the present invention contains as an active ingredient one or more components selected from the group consisting of aspartic acid-containing dipeptide, aspartic acid, and methionine. It is also a composition.

In the present invention, the term "prevention" refers to preventing or delaying the onset of a symptom or disease in a subject, or reducing the risk of developing a symptom or disease in a subject.
In the present invention, the term "treatment" means (i) completely or partially inhibiting a disease, disorder or condition, e.g. halting its onset; (ii) completely or partially inhibiting a disease, disorder or condition; (iii) alleviate the disease, disorder and/or condition in patients who may be predisposed to, but have not yet been diagnosed with, the disease, disorder and/or condition; Refers to completely or partially preventing a condition from occurring. Similarly, "treatment" refers to both therapeutic treatment and prophylactic measures.
In the present invention, the term "alleviation" means improvement of a symptom or disease, prevention or delay of worsening of a symptom or disease, reversal, prevention or delay of progression of a symptom or disease, treatment of a symptom or disease, and the like.

Furthermore, in the present invention, "suppression of accumulation of amyloid β aggregates" specifically means inhibition of aggregation of amyloid β monomers and/or fragmentation of amyloid β aggregates.

In the following, aspartic acid residues are Asp, methionine residues are Met, glycine residues are Gly, alanine residues are Ala, serine residues are Ser, threonine residues are Thr, asparagine residues are Asn, and glutamine residues are Gln, Glu for glutamic acid residue, Lys for lysine residue, Arg for arginine residue, His for histidine residue, Val for valine residue, Leu for leucine residue, Ile for isoleucine residue, Tyr for tyrosine residue, A phenylalanine residue is also written as Phe, a tryptophan residue as Trp, a proline residue as Pro, and a cysteine residue as Cys. Further, in this specification, any amino acid may be either D-type or L-type.

The peptide according to the present invention is a dipeptide containing Asp, specifically Asp-Gly, Asp-Ala, Asp-Ser, Asp-Thr, Asp-Asn, Asp-Gln, Asp-Asp, Asp-Glu, Asp-Lys, Asp-Arg, Asp-His, Asp-Val, Asp-Leu, Asp-Ile, Asp-Tyr, Asp-Phe, Asp-Trp, Asp-Pro, Asp-Met, Asp-Cys, Gly- Asp, Ala-Asp, Ser-Asp, Thr-Asp, Asn-Asp, Gln-Asp, Asp-Asp, Glu-Asp, Lys-Asp, Arg-Asp, His-Asp, Val-Asp, Leu-Asp, Examples include He-Asp, Tyr-Asp, Phe-Asp, Trp-Asp, Pro-Asp, Met-Asp, and Cys-Asp.

In the peptide according to the present invention, the C-terminus of the peptide is preferably Asp, and the peptide is more preferably Gly-Asp or Thr-Asp.

In the present invention, the amino acids and peptides may be in the form of their salts. Examples of acid addition salts include inorganic salts such as hydrochlorides, sulfates, nitrates, phosphates, hydrobromides, and perchlorates, as well as citric, succinic, maleic, fumaric, and apple acids. acid, salts of organic acids such as tartaric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. Examples of basic salts include salts of alkali metals such as sodium, potassium, and lithium; salts of alkaline earth metals such as calcium and magnesium. Exemplary bases that can be used to make base addition salts include sodium hydroxide, potassium hydroxide, alkali metal bases such as lithium hydroxide, calcium hydroxide.

In the present invention, the peptide according to the present invention preferably contains an effective amount for preventing decline in brain function, etc., and/or inhibiting accumulation of amyloid β aggregates.
Further, the concentration of the peptide according to the present invention in the composition is not particularly limited, but is preferably 10 μM or more, more preferably 100 μM or more, even more preferably 1 mM or more, and 1M or less. It is preferably 100mM or less, more preferably 50mM or less. In terms of efficacy and ease of oral ingestion, it is preferably 100 μM to 100 mM, more preferably 1 mM to 50 mM, even more preferably 5 mM to 20 mM.

In the present invention, aspartic acid is preferably contained in an amount effective for preventing decline in brain function, etc., and/or inhibiting accumulation of amyloid β aggregates.
The concentration of aspartic acid in the composition is not particularly limited, but is preferably 10 μM or more, more preferably 100 μM or more, even more preferably 1 mM or more, and preferably 1 M or less. , more preferably 100mM or less, even more preferably 50mM or less. In terms of efficacy and ease of oral ingestion, it is preferably 100 μM to 100 mM, more preferably 1 mM to 50 mM, even more preferably 5 mM to 20 mM.

In the present invention, it is preferable that methionine be contained in an amount effective for preventing decline in brain function, etc., and/or inhibiting accumulation of amyloid β aggregates.
The concentration of methionine in the composition is not particularly limited, but is preferably 10 μM or more, more preferably 100 μM or more, even more preferably 1 mM or more, and preferably 1 M or less, It is more preferably 100mM or less, even more preferably 50mM or less. In terms of efficacy and ease of oral ingestion, it is preferably 100 μM to 100 mM, more preferably 1 mM to 50 mM, even more preferably 5 mM to 20 mM.

The compositions of the invention can further include straight chain saturated fatty acids.
In the present invention, it is preferable that the linear saturated fatty acid is contained in an amount effective for preventing a decline in brain function, etc., and/or inhibiting the accumulation of amyloid β aggregates.
Further, the concentration of the straight chain saturated fatty acid in the composition is not particularly limited, but is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and 0.1% by mass or more. It is more preferably 90% by mass or less, more preferably 60% by mass or less, even more preferably 30% by mass or less, and particularly preferably 10% by mass or less. In terms of efficacy and ease of oral ingestion, the amount is preferably 0.01 to 2% by weight, more preferably 0.1 to 1.5% by weight, and even more preferably 0.5 to 1% by weight.

The straight chain saturated fatty acid is preferably a straight chain saturated fatty acid having 10 to 20 carbon atoms, more preferably 13 to 20 carbon atoms, even more preferably 16 to 20 carbon atoms, and Most preferably the number is 16, ie palmitic acid.

Here, as shown in the Examples described later, a mixture of methionine and palmitic acid, and a mixture of aspartic acid and palmitic acid can further fragment amyloid β aggregates.
That is, in a preferred form of the present invention, the present invention can be used as a composition for fragmenting amyloid β aggregates, which contains methionine and palmitic acid as active ingredients.

In addition, methionine, palmitic acid, a mixture thereof, and the dipeptides Gly-Asp and Thr-Asp have the effect of suppressing the aggregation of amyloid β monomers, as described in the Examples.
That is, in a preferred form of the present invention, the present invention can be used as a composition for inhibiting aggregation of amyloid β monomer, which contains methionine and/or palmitic acid as an active ingredient, and more preferably, methionine and palmitic acid. It can be used as a composition for inhibiting aggregation of amyloid β monomer, which contains as an active ingredient.
Furthermore, in a preferred form of the present invention, the present invention can be used as a composition for inhibiting aggregation of amyloid β monomer, which contains Gly-Asp and/or Thr-Asp as an active ingredient.

The product form of the composition of the present invention is not particularly limited, but forms such as pharmaceutical compositions, food and drink products, etc. described below are preferred.

<Administration target>
Subjects to whom the composition of the present invention is administered are those in need of prevention, treatment, or alleviation of decline in brain function. More specifically, it is a subject in which amyloid β aggregation or accumulation has occurred or is likely to occur.

The subject is usually a human, but it can also be administered to mammals other than humans, such as pet animals such as dogs and cats, and livestock such as cows, sheep, and pigs. Below, a case where the subject is a human will be explained.

Specifically, the composition of the present invention can be used to prevent symptoms such as dementia, depression, schizophrenia, delirium, amnesia, decreased judgment/thinking ability, decreased cognitive ability, and intellectual disability. It can be used for treatment, treatment, or relief.

Among these, the composition of the present invention is preferably used for preventing, treating, or alleviating symptoms or diseases caused by dementia.

In addition, dementia of the Alzheimer's type, dementia with Lewy bodies, frontotemporal dementia, cerebrovascular dementia, etc. are generally known. The composition of the present invention can be used to treat symptoms caused by a decline in one or more brain functions selected from the group consisting of Alzheimer's dementia, Lewy body dementia, frontotemporal dementia, and cerebrovascular dementia. More preferably, it is used for prevention, treatment, or alleviation of diseases.

Generally, patients with Alzheimer's disease have brain atrophy (atrophy of the cerebral cortex and hippocampus, and enlargement of the ventricles), and pathological findings called senile plaques in the cerebral cortex. can be seen. It is known that the main component of this senile plaque is Aβ.

The cause of Alzheimer's dementia is not completely clear, but Aβ aggregates and accumulates in the brain, causing neurotoxicity in the process of forming senile plaques, degenerating neurofibrillary tangles, and eventually It is assumed that the main cause of this is death of nerve cells.

Based on the results of the Examples described below, the composition of the present invention is considered to be particularly useful for Alzheimer's dementia among the above-mentioned dementias.

Subjects to whom the composition of the present invention is administered include subjects who have not developed dementia.
The subject who has not developed dementia is preferably one of the following (1) to (4).
(1) Subjects who have not developed dementia but are concerned about the decline in brain cognitive function associated with aging.
(2) Subjects who have not developed dementia but are concerned about the complex decline in cognitive function associated with aging.
(3) A subject selected from the group consisting of healthy individuals, the group consisting of mildly cognitively impaired individuals, and the group consisting of healthy individuals and mildly cognitively impaired individuals.
(4) Subjects with an MMSE score of 22 or higher and a total RBANS score of 65 or lower or a head health check score of 75 or lower. Note that the RBANS total score is preferably 41 or less. The score of the head health check is preferably 50.2 or less.

Here, the MMSE score is a score calculated by MMSE (Mini Mental State Examination). MMSE is a screening method for cognitive dysfunction that is widely used internationally in clinical practice and research. He received a perfect score of 30 on tests related to orientation, verbal memory, general attention/calculation, language, and figure imitation. The lower the MMSE score, the more advanced the cognitive decline.
The RBANS score is a score calculated by RBANS (Repeatable Battery for the Assessment of Neuropsychological Status). RBANS is a nationally standardized neuropsychological test developed by Randolph in 1998 that evaluates five cognitive areas: immediate memory, delayed memory, visuospatial/organizational, language, and attention. It consists of 12 tests, and the evaluation score for each cognitive area is calculated from the rough score of each test using a conversion table that takes into account factors such as age (provided by Regilio Co., Ltd.). The lower the RBANS score, the more advanced the decline in cognitive function is.

Head Health Check (JMCIS) (registered trademark) is a test that can easily and accurately quantitatively express minute changes in cognitive function in healthy individuals, borderline areas, and MCI areas with an interactive check that takes about 10 minutes. It is. A cognitive function index is calculated using a proprietary algorithm and database based on information such as the subject's gender, age, years of study, and race (provided by Millenia Co., Ltd.). MPI (Memory Performance Index) score (hereinafter referred to as "MPI score" or "head health check score"), which is an index that objectively indicates the cognitive function of an individual, evaluated and calculated by head health check. ) indicates that cognitive function decline is progressing.

In the present invention, prevention, treatment, or alleviation of decline in brain function is achieved by significantly increasing one or more of the MMSE score, RBANS total score, and head health check score before and after administration of the composition of the present invention. This can be determined by not seeing a decrease in score, or by seeing an increase in one or more of the MMSE score, RBANS total score, and head health check score.

The effectiveness of the composition of the present invention in preventing, treating, or alleviating decline in brain function can also be evaluated based on animal models such as mouse, rabbit, or monkey models.

<Intake (administration) timing, amount, frequency, route>
The timing of ingestion (administration) of the composition of the present invention is not particularly limited, and can be appropriately selected depending on the condition of the subject to be administered.

The amount of the composition of the present invention to be ingested (administered) is appropriately selected depending on the age, sex, condition, other conditions, etc. of the subject to be ingested (administered).
The intake (administration) amount of the composition of the present invention is preferably 0.1 mg/day or more for adults, more preferably 1 mg/day or more, as the intake amount of the peptide according to the present invention. Moreover, it is preferably 1000 mg/day or less, and more preferably 100 mg/day or less. Specifically, it is preferably 0.1 to 1000 mg/day.
The intake (administration) amount of the composition of the present invention is preferably 0.1 mg/day or more, more preferably 1 mg/day or more, and 1000 mg/day as an intake amount of aspartic acid for adults. The amount is preferably at most 100 mg/day, more preferably at most 100 mg/day. Specifically, it is preferably 0.1 to 1000 mg/day.
The intake (administration) amount of the composition of the present invention is preferably 0.1 mg/day or more, more preferably 1 mg/day or more, and 1000 mg/day in terms of methionine intake for adults. It is preferably at most 100 mg/day, more preferably at most 100 mg/day. Specifically, it is preferably 0.1 to 1000 mg/day.
The intake (administration) amount of the composition of the present invention is preferably 0.1 mg/day or more for adults, more preferably 1 mg/day or more, and , preferably 1000 mg/day or less, more preferably 100 mg/day or less. Specifically, it is preferably 0.1 to 1000 mg/day.
Incidentally, regardless of the amount and period of intake (administration), the composition of the present invention can be administered once a day or divided into multiple doses.

The frequency and period of intake (administration) of the composition of the present invention are not particularly limited. Although it may be administered in a single dose, it is preferably administered once or more a week, more preferably once or more every 3 days, even more preferably once or more every 2 days, and continued every day. Most preferably, it is administered as The effect is more likely to be obtained if the treatment period is preferably 4 weeks or more, more preferably 12 weeks or more, even more preferably 24 weeks or more. Further, there is no particular upper limit to the intake (administration) period, and continuous long-term intake (administration) is possible.

The ingestion (administration) routes for the composition of the present invention include, for example, oral administration, tube administration (nasally, gastrostomy, intestinal fistula, etc.), intraperitoneal administration, intramuscular administration, transmucosal administration, intranasal administration, and rectal administration. Examples include internal administration, but oral administration is particularly preferred.

<How to obtain peptides>
The peptide according to the present invention can be obtained, for example, by (1) a method of decomposing a protein or peptide containing the amino acid sequence with a hydrolase or the like and separating and purifying the resulting decomposed product, (2) a method of synthesizing the peptide. After synthesizing the peptide according to the present invention, the desired peptide according to the present invention is obtained by separating and purifying the obtained crude synthetic product, (3) from plants, animals, or microorganisms that produce the peptide according to the present invention. It can be obtained by a method of extraction and separation and purification from the obtained extract.
Methods (1), (2), and (3) will be specifically explained below.

(1) Method for obtaining the peptide by hydrolysis The peptide according to the present invention can be obtained from the hydrolyzate obtained by hydrolyzing a protein or peptide containing the amino acid sequence with a protein hydrolase, an acid, an alkali, etc. It can be obtained by separating and purifying the peptide. The protein or peptide used as a raw material is not particularly limited as long as it contains the amino acid sequence. Specific proteins include proteins derived from milk, soybeans, eggs, wheat, barley, oats, rice, potatoes, sweet potatoes, peas, corn, livestock meat, fish meat, seafood, and the like.

Examples of hydrolysis treatments for proteins and peptides include enzyme treatment, acid treatment, alkali treatment, heat treatment, etc., and two or more of these treatments may be combined as appropriate.
For the enzyme treatment, proteolytic enzymes derived from plants, animals, microorganisms, etc. can be used, and one type or a combination of two or more of these can be used. Examples of the protease include endoprotease.
Examples of the endoprotease include serine protease, metalloprotease, cysteine protease, and aspartic protease, and one or more of these can be selected and used.

A commercially available product can be used as the protease. Examples of the proteolytic enzyme include Bioprase (manufactured by Nagase ChemteX), Proleather (manufactured by Amano Enzymes), Protease S (manufactured by Amano Enzymes), PTN6.0S (manufactured by Novozymes), and Sabinase (manufactured by Novozymes). , GODO B. A. P (manufactured by Godo Shusei Co., Ltd.), Protease N (manufactured by Amano Enzyme Co., Ltd.), GODO B. N. P (manufactured by Godo Shuusei Co., Ltd.), Neutrase (manufactured by Novozymes Co., Ltd.), Alcalase (manufactured by Novozymes Co., Ltd.), Trypsin (manufactured by Novozymes Co., Ltd.), Chymotrypsin (manufactured by Novozymes Co., Ltd.), Subtilisin (manufactured by Novozymes Co., Ltd.), Papain (manufactured by Amano Enzyme Co., Ltd.) ), bromelain (manufactured by Amano Enzymes), etc., and one or more enzymes may be selected from these and used.

In addition, in the present invention, the target peptide content can be measured by the following method.
(a) The sample powder was diluted and dissolved in a 0.2% formic acid aqueous solution to a concentration of 1.0 mg/mL, crushed by ultrasonic waves for 10 minutes, and then filtered with a PVDF filter (manufactured by Millipore) with a diameter of 0.22 μm. A powder solution is prepared, and LC/MS analysis is performed under the following measurement conditions. On the other hand, several solutions of a chemically synthesized standard peptide (manufactured by Peptide Research Institute) of the peptide to be measured are prepared at different concentrations, and LC/MS analysis is performed under the following measurement conditions to create a calibration curve.
Among the peaks in the analysis of the powder solution, those whose molecular weight and retention time match those of the standard peptide are identified as having the same sequence as the standard peptide. By comparing the peak area of the standard peptide and the peak area of the sample powder, the content of the target peptide in the powder solution is determined.

(b) Target peptide content (mg/1 g of hydrolyzate)
Target peptide content (mg/1 g of hydrolyzate) = [measured value of target peptide in the obtained hydrolyzate (mg)]/[mass of the obtained hydrolyzate (g)]
[Measurement value of the target peptide in the obtained hydrolyzate (mg)] is the measurement value of the target peptide in the sample by "LC/MS" described below.

(c) Equipment used for LC/MS Mass spectrometer: TSQ Quantum Discovery MAX (manufactured by Thermo Fisher Scientific).
High performance liquid chromatograph: Prominence (manufactured by Shimadzu Corporation), column: XBridge BEH300 C18 φ2.1 mm×250 mm, 3.5 μm (manufactured by Waters Corporation).

(d) LC/MS measurement conditions Mobile phase A: 0.2% by weight formic acid-aqueous solution Mobile phase B: 0.2% by weight formic acid-acetonitrile solution Time program: 2%B (0.0 min) - 25%B ( 5.0 minutes) - 65% B (5.1 minutes) - 65% B (10 minutes) - 85% B (10.1 minutes) - 85% B (13.0%) - 2% B (13. 1 minute) - STOP (30.0 minutes).
Sample injection amount: 10 μL, column temperature: 40°C, liquid flow rate: 200 μL/min
Analysis mode: SRM measurement.
Product Mass: m/z = 260.10 (Parent m/z = 375.21)

The obtained hydrolyzate can exhibit its efficacy even when used in an unpurified state. That is, the hydrolyzate may be ingested or administered in the form of a pharmaceutical composition or a food/beverage composition described below.

Further, the obtained protein hydrolyzate may be subjected to appropriate separation and purification using known methods. For example, the obtained protein hydrolyzate can be subjected to molecular weight fractionation to obtain a protein hydrolyzate containing a fraction corresponding to the molecular weight of the peptide according to the present invention.
As the molecular weight fractionation, methods such as ultrafiltration and gel filtration can be employed, thereby increasing the removal rate of peptides and free amino acids with unnecessary molecular weights.
In the case of ultrafiltration, a desired ultrafiltration membrane may be used, and in the case of gel filtration, a desired gel filtration agent used for size exclusion chromatography may be used.
Furthermore, in order to desalt, remove impurities, and increase purity, various chromatography methods such as ion exchange chromatography, adsorption chromatography, reversed phase chromatography, and partition chromatography, as well as solvent Methods such as precipitation, salting out, partitioning between two liquid phases, etc. may also be used.

For the purpose of confirming whether the separated and purified peptide fraction contains the peptide according to the present invention, the peptide can be identified by the above-mentioned mass spectrometry method.

The peptide according to the present invention thus obtained can be used as a peptide solution, or, if necessary, the solution can be concentrated by a known method and used as a concentrate. Alternatively, the concentrate can be dried by a known method and used as a powder.

(2) Method for obtaining by synthesis The peptide according to the present invention can also be produced by chemical synthesis or biosynthesis.
Chemical synthesis of peptides can be carried out by a liquid phase method or a solid phase method commonly used for peptide synthesis. The synthesized peptide can be deprotected as necessary to remove unreacted reagents, by-products, etc., and the peptide according to the present invention can be isolated. Such peptide synthesis can be performed using a commercially available peptide synthesizer.
Peptide biosynthesis can be carried out by a conventional method such as introducing a peptide expression vector into a host organism and causing production and secretion.
Obtaining the desired peptide can be confirmed in the same manner as described in "(1) Method for obtaining by hydrolysis."

(3) Method for extracting, separating and purifying from plants, animals, or microorganisms The peptide according to the present invention is extracted from the culture supernatant of the plant, animal, microorganism, or microorganism that produces the peptide according to the present invention. It can be obtained by a method of separating and purifying from.
Examples of the microorganisms include Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Escherichia coli, and yeast.
After collecting the above-mentioned separated and purified product, it can be confirmed that the desired peptide has been obtained using the same method as described in "(1) Method for obtaining by hydrolysis."

<Pharmaceutical composition>
The composition of the invention may be a pharmaceutical composition. The pharmaceutical composition can be formulated into a desired dosage form as appropriate. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; liquid preparations such as solutions, syrups, suspensions, and emulsions.
In formulation, in addition to the peptide/amino acid that is the active ingredient of the present invention, components such as excipients, pH adjusters, coloring agents, and flavoring agents that are commonly used in formulation can be used. In addition, other medicinal ingredients, known or future discovered ingredients that are effective in suppressing the accumulation of amyloid β aggregates, and other ingredients that can prevent, treat, or alleviate the decline in brain function, etc. It is also possible to use medicines in combination.
In addition, other amino acids and peptides may be present together as long as they do not impede the effects of the present invention.

In addition, formulation can be carried out by appropriately known methods depending on the dosage form. During formulation, carriers commonly used in formulation may be added as appropriate. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, and the like.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Cellulose derivatives such as hydroxypropyl methylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium metasilicate aluminate; phosphate derivatives such as calcium phosphate; carbonic acid Examples include carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate.

Examples of the binder include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

Examples of the disintegrant include, in addition to the above excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.

Examples of lubricants include talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gay wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid. ; carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives, etc. It will be done.

Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

Examples of flavoring agents include sweeteners, acidulants, fragrances, and the like.
In addition, examples of carriers used in the case of liquid preparations for oral administration include solvents such as water.

The timing of ingesting the pharmaceutical composition of the present invention is not particularly limited, such as before a meal, after a meal, between meals, or before going to bed, but preferably before a meal.

Furthermore, the peptide, aspartic acid, methionine, and straight chain saturated fatty acid according to the present invention can be used for producing these pharmaceutical compositions.

<Composition for food and drink>
The composition of the present invention may be a composition for food or drink. The composition for food and drink products is not particularly limited in form or properties as long as it does not impair the effects of the present invention and can be ingested orally, and can be produced by conventional methods using raw materials commonly used for food and drink products. can.

Food and drink compositions may be in the form of liquids, pastes, gel-like solids, powders, etc., such as tablets, liquid foods (nutritive foods for tube administration), bread, macaroni, spaghetti, noodles, and cakes. Flour products such as mixes, fried flour, and bread crumbs; instant noodles, cup noodles, retort/cooked foods, cooked canned foods, microwave foods, instant soups/stews, instant miso soup/suimono, canned soups, freeze/dried foods, and other instant foods. Instant foods such as food; Agricultural processed products such as canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (processed grain products); Canned marine products, fish hams and sausages, marine paste products, Processed seafood products such as seafood delicacies and boiled fish; Processed livestock products such as canned livestock products and pastes, livestock hams and sausages; Processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams, and powdered milk products Milk and dairy products such as , cream, and other dairy products; Fats and oils such as butter, margarine, and vegetable oil; Basic seasonings such as soy sauce, miso, sauces, tomato processed seasonings, mirin, and vinegar; Cooking mixes , complex seasonings and foods such as curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as raw frozen foods, semi-cooked frozen foods, and cooked frozen foods; Confectionery such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice sweets, bean sweets, dessert sweets, jellies, and other sweets; carbonated drinks, natural fruit juices, fruit juice drinks, Soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other recreational drinks, etc. Examples include other commercially available foods such as recreational drinks, baby food, furikake, and ochazuke nori; infant formula; enteral nutrition; and functional foods (foods for specified health uses, foods with nutritional function functions), and the like.

Moreover, it can also be used as feed as one aspect of the composition for food and drink. Examples of the feed include pet food, livestock feed, and fish feed.
The form of the feed is not particularly limited, and in addition to the peptides and amino acids according to the present invention, for example, grains such as corn, wheat, barley, rye, and milo; plants such as soybean oil meal, rapeseed oil meal, coconut oil meal, and linseed oil meal. Rice bran such as wheat bran, wheat bran, rice bran, and defatted rice bran; Production meal such as corn gluten meal and corn jam meal; Animal feed such as fish meal, skim milk powder, whey, yellow grease, and tallow; Torla It may contain yeasts such as yeast and brewer's yeast; mineral feeds such as tricalcium phosphate and calcium carbonate; oils and fats; simple amino acids; saccharides and the like.

When the composition of the present invention is in the form of a composition for food and drink (including feed), it can be provided and sold as a composition for food and drink labeled with uses such as prevention of brain function decline and prevention of memory decline. It is possible. Furthermore, the peptide, aspartic acid, methionine, and straight chain saturated fatty acid according to the present invention can be used for producing these food and drink compositions.

Such acts of "display" include all acts to inform the consumer of the above-mentioned use, and if the expression is such that it may remind or infer the above-mentioned use, the purpose of the display, the content of the display, Regardless of the object or medium to be displayed, all of them fall under the act of "display" in the present invention.
Moreover, it is preferable that the "display" be performed using expressions that allow the consumer to directly recognize the above-mentioned use. Specifically, the act of transferring, handing over, exhibiting for transfer or delivery, or importing food and drink products or products with the above-mentioned usage written on their packaging, advertisements related to products, price lists, or transaction documents. Examples include actions such as displaying or distributing information with the above-mentioned uses written thereon, or writing the above-mentioned uses on information containing these items and providing it by electromagnetic (Internet, etc.) method.

On the other hand, the display content is preferably a display approved by the government (for example, a display that is approved based on various systems established by the government and performed in a manner based on such approval). Further, it is preferable to attach such display contents to packaging, containers, catalogs, pamphlets, promotional materials at sales sites such as POP, and other documents.

In addition, "labeling" includes health foods, functional foods, enteral nutritional foods, special purpose foods, foods with health claims, foods for specified health uses, foods with nutritional function claims, foods with functional claims, quasi-drugs, etc. Display can also be mentioned. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, labeling approved under the system related to food for specified health uses, food with nutritional function claims, or food with functional claims, or a system similar to these, etc. can be mentioned. Specifically, labeling as a food for specified health uses, labeling as a food for specified health uses with conditions, labeling that it affects the structure or function of the body, labeling to reduce disease risk, and labeling as a food for specified health uses based on scientific evidence. More specifically, the Cabinet Office Ordinance on Permission for Special Purpose Labeling as stipulated in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) Typical examples include labeling as food for specified health uses (in particular, labeling for health uses) and similar labeling as stipulated in the Act.

Examples of such displays include, ``For those who are concerned about memory decline,'' ``For those who want to improve their cognitive function,'' ``For those who are concerned about their cognitive function,'' and ``I want to prevent Alzheimer's type dementia in the future.'' ``For those who want to improve their cognitive accuracy'' ``For those who want to support information such as numbers, words, shapes, situations, etc.'' ``For those who want to improve the accuracy of memory and judgment, which are part of cognitive functions.'' ``For those who want to maintain their memory'' ``For healthy middle-aged and elderly people who want to support the maintenance of cognitive function'' ``For those who are concerned about forgetfulness or mild cognitive impairment (MCI)'' ``For those who are concerned about forgetfulness For middle-aged and elderly people who are concerned about mild cognitive impairment (MCI),'' and so on.

Another aspect of the present invention is one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine for use in preventing, treating, or alleviating decline in brain function. be.
It is also one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine for use in suppressing the accumulation of amyloid β aggregates.

Another aspect of the present invention is the use of one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine for preventing, treating, or alleviating decline in brain function. It is.
It is also the use of one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine to suppress the accumulation of amyloid β aggregates.

Another aspect of the present invention is one or more selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine as an active ingredient for preventing, treating, or alleviating decline in brain function. This is the use of ingredients.
It is also the use of one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine as active ingredients for inhibiting the accumulation of amyloid β aggregates.

Another aspect of the present invention is to apply one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine to subjects in need of prevention, treatment, or alleviation of brain function decline. This is a method for preventing, treating, or alleviating a decline in brain function, including administering to a patient.
In addition, amyloid β aggregation, which involves administering one or more components selected from the group consisting of aspartic acid-containing dipeptide, aspartic acid, and methionine to a subject who needs to suppress the accumulation of amyloid β aggregates. It is also a method of suppressing the accumulation of aggregates.

Another aspect of the present invention is one or more selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine, in the production of a composition for preventing, treating, or alleviating decline in brain function. This is the use of ingredients.
It is also the use of one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine in the production of a composition for suppressing the accumulation of amyloid β aggregates.

The present invention will be explained in more detail below using examples. Note that the embodiment described below is merely a representative example of the present invention, and the present invention is not limited thereto.

<Test Example 1: Subdivision effect of amino acids and palmitic acid on solubilized Aβ aggregates>
(1) Preparation of solubilized Aβ aggregates 500 μg of Aβ ₄₂ (Peptide Institute Co., Ltd.) was placed in a microtube and dissolved in 200 μL of 1,1,1,3,3,3-hexafluoro-2-propanol. It was left standing at 4°C for 16 hours. Thereafter, the mixture was allowed to stand at 37° C. for 3 hours, and then freeze-dried. Solubilized Aβ ₄₂ aggregates were prepared by performing this dissolution and freeze-drying twice, and phosphate buffered saline (PBS) was added and stored at -80°C.

(2) Evaluation of Aβ ₄₂ aggregate fragmentation effect The solubilized Aβ ₄₂ aggregates prepared in (1) were treated with 0.033 w/v% palmitic acid, amino acids (isoleucine, phenylalanine, valine, leucine, tryptophan, methionine, Alanine, glycine, cysteine, tyrosine, proline, threonine, serine, histidine, glutamic acid, asparagine, glutamine, aspartic acid, lysine, or arginine) were added at 10 mM or 20 mM. In addition, since tyrosine (Y* in FIG. 1) has low solubility, a 2 mM solution was used.

The fragmentation effect of _Aβ42 aggregates was then evaluated by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against _Aβ42 aggregates (antibody 83-3).
Here, Fig. from the literature (Shimizu et al., Global J. Res. Anal., 4: 117-119 (2015)). As shown in Figure 3, the antibody 83-3 mainly reacts with amorphous Aβ ₄₂ aggregates with a size of 0.22 μm or less and 300 kDa or more. That is, by using the above antibody 83-3, subdivided Aβ can be detected. It can be said that the larger the amount of Aβ detected with antibody 83-3, the more fragmented the Aβ aggregate is.

FIG. 1 shows the ratio of the ELISA values when various amino acids were added, with the ELISA value for Aβ aggregates (containing only palmitic acid) to which no amino acids were added as 1 (reference). Furthermore, in FIG. 1, amino acids are indicated by one letter.
As a result of the evaluation, it was revealed that the ELISA value against the standard was high when a mixture of methionine and palmitic acid and a mixture of aspartic acid and palmitic acid were added. Furthermore, when methionine and aspartic acid were added as amino acids, the higher the amino acid concentration, the higher the ELISA value tended to be.
From the above, it has become clear that Aβ aggregates are further fragmented in the presence of a mixture of methionine and palmitic acid and a mixture of aspartic acid and palmitic acid.

From the above results, it has become clear that a composition containing methionine and palmitic acid, or aspartic acid and palmitic acid as active ingredients can be used for fragmenting Aβ aggregates. Furthermore, as mentioned above, it is possible to prevent, treat, or reduce Alzheimer's type dementia by fragmenting Aβ aggregates and suppressing the accumulation of Aβ aggregates. It can be said that a composition containing palmitic acid as an active ingredient can be used for prevention, treatment, or alleviation of decline in brain function.

<Test Example 2: Aggregation inhibitory effect of methionine and palmitic acid on Aβ monomer>
As the Aβ monomer, 26-O-acyl isoAβ1-42 (isoAβ42, Peptide Institute Co., Ltd.) was used. This isoAβ42 is in a monomer state in an organic solvent or under acidic conditions, but in a neutral aqueous solution it becomes _Aβ42 with a half-life of about 1 minute and immediately starts aggregating.

First, 1 mM methionine and/or 0.033 w/v% palmitic acid were added as evaluation substances to 10 μM isoAβ42 dissolved in dimethyl sulfoxide, and the mixture was left at 37° C. for 24 hours. As a control, only isoAβ42 was reacted.
Thereafter, the average diameter of the Aβ aggregates was measured using analysis software attached to an atomic force microscope (JASCO Corporation), and the degree of aggregate formation was evaluated. The results are shown in Table 1.

Compared to control conditions in which no evaluation substance was added, the average diameter of aggregates formed from Aβ monomers was smaller in the presence of methionine, palmitic acid, and a mixture thereof. That is, methionine, palmitic acid, and a mixture thereof were found to be effective in suppressing the formation of large Aβ aggregates.

From the above, it has become clear that a composition containing methionine, palmitic acid, or a mixture thereof as an active ingredient can be used for suppressing Aβ monomer aggregation. Furthermore, as mentioned above, it is possible to prevent, treat, or reduce Alzheimer's type dementia by suppressing the accumulation of Aβ aggregates, and therefore a composition containing methionine, palmitic acid, or a mixture thereof as an active ingredient. It can be said that it can be used for the prevention, treatment, or alleviation of decline in brain function.

<Test Example 3: Aggregation inhibitory effect of dipeptide on Aβ monomer>
The six types of dipeptides listed in Table 2 below (Glu-Glu, Gly-Asp, Gly-Gly, Ser-Glu, Thr-Asp, Tyr-Glu) and the Aβ aggregation inhibitory effect reported in the past 2 Synthetic peptides (manufactured by As One Corporation) of various dipeptides (Trp-Met, Trp-Tyr) were used. The effect of inhibiting Aβ monomer aggregation by adding 1 mM of these dipeptides was evaluated in the same manner as in Test Example 2. The results are shown in Table 2.

As a result, the Aβ aggregates formed in the presence of 1mM Gly-Asp or 1mM Thr-Asp were particularly small compared to the control condition in which no evaluation substance was added. It was shown that the effect of suppressing aggregation is high (Table 2). In addition, compared to the case of adding two types of dipeptides (Trp-Met, Trp-Tyr), which have been reported to have an Aβ aggregation inhibiting effect in the past, the addition of Gly-Asp or Thr-Asp was more effective on average. It is clear that the diameter becomes smaller.

Furthermore, the dipeptides Gly-Asp and Thr-Asp have in common that they contain Asp, and the other dipeptides evaluated in this test example do not contain Asp. That is, it was suggested that the dipeptide containing Asp is highly effective in suppressing the aggregation of Aβ monomers.

From the above, it has become clear that a composition containing a dipeptide containing Asp as an active ingredient can be used for suppressing Aβ monomer aggregation. Furthermore, as mentioned above, it is possible to prevent, treat, or reduce Alzheimer's type dementia by suppressing the accumulation of Aβ aggregates. It can be said that it can be used for the prevention, treatment, or alleviation of decline.

According to the present invention, it is possible to provide a new composition for preventing, treating, and improving brain function decline, particularly by preventing the accumulation of amyloid β aggregates.

Claims (11)

A composition for use in preventing, treating, or alleviating a decline in brain function, which contains as an active ingredient one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine. A composition for inhibiting the accumulation of amyloid β aggregates, which contains as an active ingredient one or more components selected from the group consisting of aspartic acid-containing dipeptides, aspartic acid, and methionine. The composition according to claim 1 or 2, wherein the dipeptide is Gly-Asp or Thr-Asp. The composition according to claim 1 or 2, further comprising a straight chain saturated fatty acid. 5. The composition according to claim 4, wherein the straight chain saturated fatty acid is palmitic acid. The composition according to claim 1, wherein the decline in brain function is due to dementia. 7. The composition according to claim 6, wherein the dementia is Alzheimer's dementia. The composition according to any one of claims 1, 2, 6, and 7, for inhibiting aggregation of amyloid β monomers and/or fragmenting amyloid β aggregates. The composition according to claim 1 or 2, for administration to a subject who has not developed dementia. The composition according to claim 1 or 2, which is a pharmaceutical composition. The composition according to claim 1 or 2, which is a composition for food or drink.