WO2017175457A1 - Therapeutic/prophylactic agent for alzheimer disease - Google Patents

Abstract

Provided is a therapeutic/prophylactic agent for Alzheimer disease, which uses a safe food-derived compound and which has an excellent therapeutic/preventive effect. This therapeutic/prophylactic agent for Alzheimer disease is formed by combining a first active ingredient, which is at least one compound selected from the group consisting of ferulic acid (FA) and pharmacologically acceptable salts thereof, and a second active ingredient, which is at least one compound selected from the group consisting of epigallocatechin gallate (EG) and pharmacologically acceptable salts thereof, and is characterized in that a mass ratio of the first active ingredient to the second active ingredient is within a range of 1:10 to 10:1 when the first active ingredient mass is converted to the ferulic acid mass, and the second active ingredient mass is converted to epigallocatechin gallate mass. The synergetic effect of the first active ingredient and the second active ingredient significantly reduces the abundance of Aβ oligomer for which association with behavioral/recognition functional disorders is strongly indicated.

Description

Therapeutic and preventive agent for Alzheimer's disease

The present invention relates to a therapeutic and preventive agent for Alzheimer's disease.

The number of patients with dementia who visit a medical institution is increasing rapidly as we are facing a super-aging society. According to the 2015 World Alzheimer report, 46.8 million people worldwide have been recognized in 2015. It is expected that patients with illness will survive and increase to 131.5 million by 2050. This figure doubles every 20 years and warns that dementia is a global crisis. For this reason, there is an urgent need to establish effective treatments and prevention methods for dementia.

Alzheimer's disease, the most common cause of dementia, is a neurodegenerative disease that causes a decline in cognitive function and neuropsychiatric function. As the severity of symptoms increases, eating, changing clothes, and communication become difficult. And eventually become bedridden. In the middle of the course of symptoms, peripheral symptoms such as paranoia, hallucinations, rants, and violence occur, which is a problem in nursing care. As a cause of the pathogenesis of Alzheimer's disease, aggregation of amyloid β protein in the brain accompanying the abnormal metabolism of amyloid precursor protein (hereinafter, amyloid precursor protein is expressed as “APP” and amyloid β protein is expressed as “Aβ”).・ Increased deposition (amyloid cascade hypothesis), hyperphosphorylation of tau protein (tau hypothesis), abnormal increase in glial cell / cytokine cycle (neuroinflammatory hypothesis), increased reactive oxygen species (free radical hypothesis), decreased acetylcholine synthesis (Choline hypothesis) is mentioned, but no conclusion has been reached.

FIG. 5 shows the production pathway of Aβ, which is one of the causative substances related to the onset and progression of Alzheimer's disease. Aβ is produced by stepwise enzymatic cleavage from APP, a type I transmembrane protein. As a first step, the extracellular region of APP is enzymatically cleaved by β-secretase. This cleavage produces a C-terminal fragment (β-CTF, C99: 12 kDa) and a soluble N-terminal fragment (sAPP-β: ˜100 kDa). Subsequently, Aβ of about 40 amino acids is produced as an N-terminal fragment by multiple enzymatic cleavages (ε, γ and ζ cleavage) of γ-secretase in the transmembrane region of β-CTF left behind in the cell membrane. The produced Aβ moves from a soluble monomer to an oligomer type. Oligomer type Aβ has synaptic toxicity and is strongly suggested to be associated with behavioral and cognitive impairment. Eventually, Aβ deposits in the tissue to form β-amyloid protein plaques (senile plaques). Aβ has a subtype composed of 40 amino acids or 42 amino acids by final enzymatic cleavage. Under physiological conditions, Aβ is produced at a constant low level mainly from APP existing mainly in the cell membrane of nerve cells. A part of Aβ deposited in the brain is taken up by glial cells. In addition, it is eliminated from the brain into the cerebrospinal fluid or serum through the blood cerebrospinal barrier clearance system. However, in the brains of Alzheimer's disease patients, Aβ production in the brain increases due to the increased production of Aβ or the failure of the elimination system, and deposition in tissues progresses.

Treatment methods for Alzheimer's disease focusing on Aβ include methods for inhibiting Aβ aggregation / deposition, methods for promoting degradation, methods for reducing neurotoxicity, methods for reducing production, and draining from the brain to peripheral blood A method for promoting the above has been tried (see Non-Patent Document 1 (Mol. Psychiatry, 14, 469 (2009))). However, despite vigorous development efforts, no new drugs that can be prescribed in medical institutions have been born since 2003. This is due to the fact that the toxicity of the test drug that was not confirmed in the preclinical study was expressed in Phase I to III clinical trials and that the same efficacy as the results of the preclinical study was not obtained. large. Under such circumstances, it is considered important to search for substances that affect the production and deposition of Aβ in the brain from among food-derived compounds whose safety is ensured. The use of these compounds has been proposed.

Among them, ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid) is one of the most abundant phenolic compounds present in foods consumed by humans, such as rice, wheat, barley and other grains, tomatoes It is a compound often found in fruits such as vegetables such as carrots, pineapples and oranges. It is known that this compound has various physiological activities including an active oxygen removing action, an antioxidant action, and an anti-inflammatory action, and also has an action of suppressing Aβ aggregation / deposition. Non-Patent Document 2 (PLoS ONE, 8, e55774 (2013)) shows that the administration of ferulic acid to Alzheimer's disease model mice significantly improves behavioral disorders such as mouse overactivity and object recognition disorders. In addition, the presence of various Aβ species including Aβ deposits and oligomers in the brain parenchyma and cerebral blood vessels are significantly reduced, and the pathogenesis of Alzheimer's disease is due to the effect of inhibiting the amyloid production system by targeting β-secretase of ferulic acid. It has been reported that the effectiveness and safety of improving Patent Document 1 (Japanese Patent Publication No. 2002-541194) reports that memory reduction by administration of Aβ into the ventricle was effectively prevented in mice pre-administered with ferulic acid. Furthermore, a health food containing ferulic acid as a kind of active ingredient is sold under the name of Felgard (registered trademark) (see Non-Patent Document 3 (http://www.glovia.co.jp/info/)). ).

Epigallocatechin gallate ([(2R, 3R) -5,7-dihydroxy-2- (3,4,5-trihydroxyphenyl) chroman-3-yl] 3,4,5-trihydroxybenzoate) Is one of the catechins most abundantly contained in green tea among plants. This compound has a variety of physiologically active actions, and it has been reported that it exhibits therapeutic effects on many diseases including cancer due to its powerful antioxidant and anti-inflammatory actions. It is known to have an action of suppressing the above. Non-Patent Document 4 (J. Neurosci., 25, 8807 (2005)) shows that administration of epigallocatechin gallate to Alzheimer's disease model mice significantly reduced the abundance of Aβ species and senile plaques. It has been reported that the efficacy and safety of improving the pathology of Alzheimer's disease have been confirmed by the effect of enhancing the non-amyloid production system by targeting α-secretase of epigallocatechin acid. Patent Document 2 (Japanese Patent Application Laid-Open No. 10-245342) discloses that rat embryonic brain hippocampal neurons cultured in a medium containing epigallocatechin gallate are cultured by adding Aβ to increase the survival rate of hippocampal neurons. As a result of the measurement, it has been reported that epigallocatechin gallate effectively reduced the neurotoxicity caused by Aβ.

Special Table 2002-541194 Japanese Patent Laid-Open No. 10-245342

Mol. Psychiatry, 14, 469 (2009) PLoS ONE, 8, e55774 (2013) http: // www. glovia. co. jp / info / J. et al. Neurosci. , 25, 8807 (2005)

As mentioned above, the use of food-derived compounds is safe in that the safety is ensured, but the therapeutic and preventive effects of Alzheimer's disease of the compounds proposed so far are not always satisfactory, treatment There is a constant demand for improved preventive effects.

Therefore, an object of the present invention is to provide a therapeutic and preventive agent for Alzheimer's disease, which is excellent in therapeutic and preventive effects, using a food-derived compound that ensures safety and can satisfy the above-mentioned requirements.

The inventors of the present invention provide ferulic acid having an effect of suppressing amyloid production system by targeting β-secretase, and epigallocatechin gallate having an effect of enhancing α-secretase as a target and non-amyloid production system by a predetermined method. When combined in proportion, we discovered that the synergistic action of both would significantly improve the therapeutic and preventive effects of Alzheimer's disease and expect therapeutic effects until Alzheimer's disease has not occurred. Completed. The inhibitory effect on the production and accumulation of Aβ by the combined administration of ferulic acid and epigallocatechin gallate is predicted by adding the effect of administration of ferulic acid alone and the effect of administration of epigallocatechin gallate alone. It is also remarkable compared with the effect.

Accordingly, the present invention provides at least one compound selected from the group consisting of ferulic acid as a first active ingredient and pharmacologically acceptable salts thereof, epigallocatechin gallate as a second active ingredient, and A therapeutic and prophylactic agent for Alzheimer's disease comprising a combination of at least one compound selected from the group consisting of pharmacologically acceptable salts thereof, and the mass of the first active ingredient and the second active ingredient The ratio ranges from 1:10 to 10: 1 by converting the mass of the first active ingredient into the mass of ferulic acid and the mass of the second active ingredient into the mass of epigallocatechin gallate. The present invention relates to a therapeutic and prophylactic agent for Alzheimer's disease. In the present invention, when determining the mass ratio of the first active ingredient and the second active ingredient, if ferulic acid or epigallocatechin gallate constitutes a salt, the mass of these salts Is converted to the mass of the same mole of ferulic acid or epigallocatechin gallate to calculate the mass ratio.

Knowledge about the combined effect of a compound with an action mechanism that suppresses the amyloid production system with β-secretase as a target and a compound with an effect to enhance the non-amyloid production system with α-secretase as a target However, the synergistic effect of the first active ingredient and the second active ingredient could not be predicted. In addition, Fergard (registered trademark) contains a green tea extract containing epigallocatechin gallate as an antioxidant, but the ratio of epigallocatechin gallate to ferulic acid is several percent or less. It is not an amount that can confirm the combined effect. In the present invention, the mass ratio of the first active ingredient to the second active ingredient is calculated by converting the mass of the first active ingredient into the mass of ferulic acid, and the mass of the second active ingredient is gallic acid. In terms of the mass of epigallocatechin, it is in the range of 1:10 to 10: 1, preferably in the range of 2:10 to 10: 2, more preferably in the range of 5:10 to 10: 5. Particularly preferably in the range of 8:10 to 10: 8.

When formulating the Alzheimer's disease treatment / prevention agent of the present invention, it may be in the form of a combination containing both the first active ingredient and the second active ingredient, and the first effective ingredient It may be in the form of a kit comprising a therapeutic and prophylactic agent containing a component and a therapeutic and prophylactic agent containing the second active ingredient. Each treatment preventive agent constituting the combination and kit may contain components other than the first active ingredient and the second active ingredient as long as the effects of the present invention are not adversely affected. When it is in the form of a kit, there is no restriction on the order of administration, and any therapeutic / prophylactic agent may be administered first.

Summing up the components of each therapeutic and prophylactic agent constituting the kit, at least one compound selected from the group consisting of ferulic acid and its pharmacologically acceptable salt, epigallocatechin gallate and its pharmacologically acceptable The mass ratio with the at least one compound selected from the group consisting of the salts is from 1:10 to 10: 1, preferably from 2:10 to 1 in terms of ferulic acid and epigallocatechin gallate as described above. The range is 10: 2, more preferably 5:10 to 10: 5, and particularly preferably 8:10 to 10: 8. The present invention also provides an agent for the treatment and prevention of Alzheimer's disease comprising, as an active ingredient, at least one compound selected from the group consisting of ferulic acid and pharmacologically acceptable salts thereof for constituting such a kit, and And an agent for treating and preventing Alzheimer's disease comprising, as an active ingredient, at least one compound selected from the group consisting of epigallocatechin gallate and pharmacologically acceptable salts thereof.

The therapeutic / prophylactic agent for Alzheimer's disease of the present invention can be administered in both oral and parenteral forms, and can be administered in the form of pharmaceuticals, quasi drugs, health foods (including supplements), and the like. . Since a food-derived compound with guaranteed safety is used, a large amount can be continuously administered, and it is preferably administered orally as a health food from the viewpoint of simplicity.

According to the therapeutic and preventive agent for Alzheimer's disease of the present invention in which the first active ingredient and the second active ingredient are combined in a predetermined amount, the synergistic effect of the first active ingredient and the second active ingredient In addition, the effect of suppressing the production / accumulation of Aβ is remarkably improved, and a therapeutic effect is expected until Alzheimer's disease is not caused.

It is a figure showing the results of evaluating changes in recognition frequency as an index of episodic memory when ferulic acid, epigallocatechin gallate, and both were administered to a pathological model mouse and wild type mouse of Alzheimer's disease, (A) shows the recognition frequency in the training phase test, and (B) shows the recognition frequency in the retention phase test. It is the figure which showed the result of the Y-maze test when ferulic acid, epigallocatechin gallate, and both were administered to a disease state model mouse and wild type mouse of Alzheimer's disease, and (A) shows each maze. (B) shows the voluntary alternation action rate, respectively. It is the figure which showed the result of having evaluated the abundance of A (beta) in the cerebral tissue piece after administering ferulic acid, epigallocatechin gallate, and both to the pathological model mouse of Alzheimer's disease, (A), (B) And (C) are Aβ _1-40 and Aβ in the Tris-buffered saline (TBS) solution soluble fraction, NP-40 lysis buffer (TNE) solution soluble fraction, and guanidine hydrochloride solution soluble fraction, respectively. _The result of measuring the amount of _1-42 is shown. The result of having measured the quantity of Aβ oligomer in the TNE solution soluble fraction of a cerebral tissue piece after administering ferulic acid, epigallocatechin gallate, and both to a pathological model mouse of Alzheimer's disease is shown. It is a figure explaining the production pathway of amyloid beta protein.

The agent for the treatment and prevention of Alzheimer's disease of the present invention is selected from the group consisting of ferulic acid as a first active ingredient having the effect of inhibiting the amyloid production system by targeting β-secretase and a pharmaceutically acceptable salt thereof. Selected from the group consisting of at least one compound, epigallocatechin gallate as a second active ingredient having the effect of enhancing the non-amyloid production system with α-secretase as a target, and pharmacologically acceptable salts thereof. And at least one compound in combination at a predetermined mass ratio. By this combined administration, the effect of suppressing the production / accumulation of Aβ is remarkably improved, and a therapeutic effect is expected until a state in which Alzheimer's disease has not occurred is reached.

Ferulic acid can be extracted from plants containing the same, for example, grains such as rice, wheat and barley, vegetables such as tomatoes and carrots, fruits such as pineapples and oranges, preferably extracted from rice. In addition, ferulic acid can be obtained by chemical synthesis or can be obtained commercially. In the present invention, a known extraction method can be used without particular limitation as a method for extraction from plants. For example, rice bran pitch produced in the process of refining rice bran oil obtained from rice bran is subjected to alkali hydrolysis, and solvent extraction. A method for obtaining a crude crystal of ferulic acid and purifying the crude crystal can be mentioned. Examples of a method for obtaining ferulic acid by chemical synthesis include a method in which vanillin (4-hydroxy-3-methoxybenzaldehyde) and malonic acid are condensed and decarboxylated in pyridine in the presence of piperidine.

Ferulic acid can improve water solubility and increase physiological effectiveness by making it into a salt. The salt of ferulic acid is not particularly limited as long as it is a pharmacologically acceptable salt. For example, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, ammonium salt And basic amino acid salts such as arginine salt and lysine salt, and amine salts such as monoethanolamine salt and diethanolamine salt. In particular, an alkali metal salt or an alkaline earth metal salt is preferable.

In addition, ferulic acid and its salt may exist in the form of a pharmacologically acceptable solvate, for example, a solvate of water or alcohols. Ferulic acid may be present in the form of an inclusion compound with α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, preferably γ-cyclodextrin.

Epigallocatechin gallate can be isolated from green tea leaf extract by techniques such as high performance liquid chromatography, synthetic adsorbent chromatography, ion exchange chromatography and gel filtration, and is commercially available. You can also. In the present invention, a known extraction method can be used without particular limitation as an extraction method from green tea leaves. For example, green tea leaves were extracted with hot water, aqueous methanol solution, aqueous ethanol solution or aqueous acetone solution, and obtained. A method may be mentioned in which the extract is dissolved in an organic solvent and then concentrated.

Epigallocatechin gallate may be in the form of a pharmacologically acceptable salt. Examples of the salt include alkali metal salts such as sodium salt and potassium salt, and alkaline earth metal salts such as magnesium salt and calcium salt. Moreover, epigallocatechin gallate or a salt thereof may exist in the form of a pharmacologically acceptable solvate, for example, a solvate of water or alcohols.

In the therapeutic and preventive agent for Alzheimer's disease of the present invention, the first active ingredient and the second active ingredient are 1:10 to 10: 1, preferably 2:10 to 10: 2, more preferably 5: They are combined in a mass ratio of 10 to 10: 5, particularly preferably 8:10 to 10: 8. The combined administration of the first active ingredient and the second active ingredient, as shown in the following examples, markedly improves the effect of suppressing the production and accumulation of Aβ, and Alzheimer's disease has not occurred Expected to have therapeutic effects up to

The therapeutic / prophylactic agent for Alzheimer's disease of the present invention can be administered in both oral and parenteral forms, and can be administered in the form of pharmaceuticals, quasi drugs, health foods (including supplements), and the like. . A large amount can be administered continuously because of the use of food-derived compounds with guaranteed safety. From the viewpoint of simplicity, it is preferably administered orally as a supplement.

In the case of formulating, it may be in the form of a mixture containing both the first active ingredient and the second active ingredient, and the therapeutic and prophylactic agent containing the first active ingredient, It may be in the form of a kit comprising a therapeutic and prophylactic agent containing two active ingredients. When in the form of a kit, the order of administration is not limited, and any of the treatment / prevention agents may be administered first, or each treatment / prevention agent may be administered sequentially or administered after a time interval. May be.

In the case of a combination or in the case of each therapeutic / preventive agent constituting the kit, depending on the intended use, preparations for oral administration such as capsules, chewables, tablets, powders, granules, syrups, etc., or injections It can be formulated in the form of preparations for parenteral administration such as pills, drops, and suppositories. In the case of kit form, the dosage form of each therapeutic / preventive agent may be the same or different.

In the production of these preparations, the first active ingredient and / or the second active ingredient is usually a pharmacologically acceptable excipient selected according to the purpose of use, ie, dextrin, lactose, etc. These are mixed with liquid excipients such as solid excipient, water, salt water, glycerin, etc. and formulated into a predetermined shape. In these preparations, functions of other components such as vitamins such as vitamin C and vitamin E, minerals such as iron and zinc, ginkgo biloba extract, docosahexaenoic acid, and the like, as long as the effects of the present invention are not adversely affected In addition to the sex component, conventional additives such as a binder, a thickener, a lubricant, a fragrance, a sweetener, a buffer, a preservative, and an antibacterial agent may be contained.

Further, the Alzheimer's disease treatment and prevention agent of the present invention is blended in various foods and beverages such as mineral waiters, beverages such as soft drinks, dairy products such as cheese and yogurt, confectionery such as jelly, biscuits, and candy. You can also. *

The dose of the therapeutic and preventive agent for Alzheimer's disease of the present invention can be appropriately determined according to the symptoms, age, body weight, etc. of the patient with Alzheimer's disease, the dosage form, the number of administrations, combined use with other agents, etc. In this case, the total amount of the first active ingredient and the second active ingredient per day for an adult is generally converted into ferulic acid and epigallocatechin gallate as described above. The range is 20 to 1000 mg, preferably 100 to 700 mg, particularly preferably 200 to 500 mg. However, the dose of the first active ingredient and the dose of the second active ingredient are 1:10 to 10: 1 in terms of ferulic acid and epigallocatechin gallate, preferably 2: The mass ratio should be selected from 10 to 10: 2, more preferably from 5:10 to 10: 5, particularly preferably from 8:10 to 10: 8.

Each therapeutic / preventive agent constituting the kit described above is also provided as a therapeutic / preventive agent for Alzheimer's disease in the present invention. However, these therapeutic / prophylactic agents are converted into ferulic acid and epigallocatechin gallate in a ratio of 1:10 to 10: 1, preferably 2:10 to 10: 2, more preferably 5:10 to 10: 5. Particularly preferably, it must be used in combination with another therapeutic / preventive agent so as to ensure a mass ratio in the range of 8:10 to 10: 8.

The present invention will be described using the following examples, but the present invention is not limited to the following examples. In the following Examples, genetically modified mice introduced with human APP and presenelin mutant genes as Alzheimer's disease state model mice are referred to as “PSAPP mice”, wild-type mice are referred to as “WT mice”, and Ferla The acid is expressed as “FA”, epigallocatechin gallate is expressed as “EG”, and the combined administration of ferulic acid and epigallocatechin gallate is expressed as “FAEG”. In addition, PSAPP mice administered with FAEG are represented as “PSAPP-FAEG mice”, PSAPP mice administered with EG are represented as “PSAPP-EG mice”, and PSAPP mice administered with FA are represented as “PSAPP-FA mice”. A PSAPP mouse fed with distilled water containing no EG and FA is referred to as a “PSAPP-V mouse”. Furthermore, a WT mouse administered with FAEG is represented as “WT-FAEG mouse”, a WT mouse administered with EG is represented as “WT-EG mouse”, and a WT mouse administered with FA is represented as “WT-FA mouse”. And WT mice fed with distilled water containing no EG and FA are referred to as “WT-V mice”.

(1) Experimental procedure (a) Animal, administration group, breeding environment Male PSAPP mouse [B6. _{Cg-Tg (APP swe, PSEN1dE9} ) 85Dbo / Mmjax mice [bearing APP "Swedish" APP K595N / M596L (APP swe) and presenilin 1 (PS1) exon 9 deleted (PS1 △ E9) mutant human transgenes] is, The Jackson Laboratory (Bar Harbor, ME, USA). Male PSAPP mice were crossed with female C57BL / 6J mice, and female and male F1 PSAPP mice and WT mice were obtained by this crossing. The genetic background of all mice used in the experiment was of the same strain.

FA and EG were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in distilled water. For the following behavioral analysis and Sandwich ELISA analysis, 12 months old WT mice and PSAPP mice with behavioral / cognitive dysfunction and cerebral amyloidosis were treated with FA (30 mg / day). kg (coefficient of conversion of species difference between human and mouse “10” (refer to “Regul. Toxicol. Pharmacol. 24, 108-120”), human 60 kg adult dose is 180 mg / day)) EG (30 mg / kg), FAEG (FA 30 mg / kg + EG 30 mg / kg) and distilled water were orally administered once a day for 3 months. A total of 32 mice (4 males and 4 females) were used as PSAPP-FAEG mice, PSAPP-EG mice, PSAPP-FA mice, and PSAPP-V mice. A total of 32 mice (4 males and 4 females) were used as WT-FAEG mice, WT-EG mice, WT-FA mice, and WT-V mice. In addition to these administration groups, for the purpose of investigating the effects of the doses of FA and EG by Sandwich ELISA analysis, 8 mice each of 12-month-old WT mice and PSAPP mice (4 males and 4 females) In addition, FAEG (FA 15 mg / kg + EG 15 mg / kg) was orally administered once a day for 3 months using a stomach tube. Each mouse was housed in a specific pathogen-free (SPF) area with free access to food and water in a 12/12 hour light / dark lighting cycle environment.

(B) Behavioral analysis In order to evaluate new object recognition and memory maintenance, a mouse administered with each drug or distilled water is placed in an individual cage for 4 hours, and two differently shaped objects A and B are simultaneously added. Placed in cage for 10 minutes. The first 5 minutes, the mouse nose the object A, the 2cm or number which it approached within relative B, as the number of times of searching the objects A, B (the number of times that evaluates to interested), respectively N _A and counted as _{N B,} the recognition frequency as an indicator of episodic memory _R AB (%),
R _AB (%) = N _B / (N _A + N _B ) × 100
(Training phase). After 24 hours, in order to see the storage maintenance, instead of the object B to the object C of different shapes, count the number of times in the same manner for 5 minutes and exploring each object as N _A, and N _C respectively, as an indicator of episodic memory Recognition frequency R _AC (%)
R _AC (%) = N _C / (N _A + N _C ) × 100
(Retention phase).

Subsequently, a Y-maze composed of a lustrous gray acrylic radial / symmetric maze (in order to evaluate exploratory activity and spatial working memory by the total number of entries into the Y-maze and voluntary exchange behavior ( Place the mouse in one maze with a maze length of 40 cm, a width of 4 cm, and a maze wall height of 40 cm, and record the entry trajectory to each maze (number each maze as A, B, C and enter the maze) Recorded as ABCCABBCAACABAC ...) and the total number of times of entering each maze was recorded for 8 minutes. Calculation of spontaneous alternation behavior index A (%) is, A using the total entry number A ₂ of spontaneous alternation behavior number to (3 times different times when the maze route selected in consecutive) A ₁ and the labyrinth (%) = A ₁ / (A ₂ −2) × 100
Calculated according to

(C) Sandwich ELISA analysis At the age of 15 months after oral administration for 3 months, euthanasia was performed under deep anesthesia by intraperitoneal administration of pentobarbital sodium salt (50 mg / kg). For the ELISA analysis, the right hemisphere was weighed and stored frozen at -80 ° C. Cerebral tissue pieces were treated with TissueLyser LT (Qiagen, Valencia, CA, USA) in a Tris-buffered saline (TBS: 25 mM Tris-HCl, pH 7.4, 150 mM NaCl) solution containing a protease inhibitor cocktail (Sigma-Aldrich). The mixture was homogenized and centrifuged at 4 ° C. and 18,800 G for 60 minutes, and the supernatant was collected as a TBS solution-soluble fraction. The remaining pellet was added to a NP-40 lysis buffer (TNE: 10 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 150 mM NaCl) solution containing the same protease inhibitor cocktail using TissueLyser LT. The mixture was homogenized and centrifuged at 4 ° C. and 18,800 G for 60 minutes, and the supernatant was collected as a TNE solution-soluble fraction. Further, the remaining pellet was dissolved in 5M guanidine hydrochloride solution by stirring on ice for 30 minutes, centrifuged at 4 ° C and 18,800G for 60 minutes, and the supernatant was dissolved in a fraction soluble in guanidine hydrochloride solution. As collected. The abundances of Aβ _1-40 and Aβ _1-42 were individually measured using ELISA kits (Aβ _1-40 : catalog number 27718, Aβ _1-42 : catalog number 27712, IBL, Gunma, Japan). The amount of Aβ oligomer present in the TNE solution-soluble fraction was measured using an Aβ oligomer ELISA kit (Catalog No. 27725, IBL). All sample measurements were within the linear range of the calibration curve.

(D) Statistical method All data were expressed as mean ± standard error. When multiple comparisons were made between groups, one-way analysis of variance (one-way ANOVA) was used, and Bonferroni method or Dunnett's T3 method was used as a post hoc test for comparison between groups. Which analysis result is selected depends on the result of the equivariance test, the result of Bonferroni method is selected when p> 0.05, and the result of Dunnett's T3 method is selected when p <0.05. did. All statistical analysis results were significant at p <0.05. Statistical analysis was performed using SPSS, release 19.0 (IBM, Armonk, NY, USA).

(2) Results (a) Improvement effect of behavior / cognitive dysfunction In Alzheimer's disease, various behavioral / cognitive dysfunctions occur, making it difficult to conduct home and social life due to the progression of the disease state. In evaluating drugs, it is important to evaluate their effectiveness for behavioral and cognitive impairment. The test for evaluating novel object recognition and memory maintenance is a test using the curiosity of mice. When two different objects are placed in the cage, the mouse usually shows the behavior of both sides touching the rostral area. Remember the object and observe the reaction when changing one of the objects after 24 hours. The increase / decrease in the search behavior to a new object is measured and used as an index of memory learning (new object recognition ability).

In the training phase test, the results of which are shown in FIG. 1 (A), all eight groups showed similar recognition frequencies (R _AB : 50.3-52.5%). However, in the retention phase test, the results of which are shown in Fig. 1 (B), PSAPP-V mice lack the memory of the previous day's object and do not notice the new object. Showed a decrease in the ability to recognize new objects, PSAPP-FAEG mice administered with 30 mg / kg of FA and 30 mg / kg per dose, PSAPP-EG mice administered with 30 mg / kg of EG, and PSAPP-FA mice administered with FA30mg / kg per time recognized the replaced object as a new object, and took many actions to contact the new object with the rostral part, showing improved new object recognition ability .

That is, the one-way ANOVA and post hoc analysis results, the recognition frequency _{R AC} statistically significant difference with respect to the recognition frequency _{R AC} other seven groups of PSAPP-V groups of mice ^(* p <0.05) PSAPP-FAEG mice, PSAPP-EG mice and PSAPP-FA mice (R _AC : 60.8-71.6%) are significantly more significant than PSAPP-V mice (R _AC : 50.0%). The new object recognition frequency increased. Furthermore, the PSAPP-FAEG mouse group showed a novel object recognition frequency that was significantly increased compared to the PSAPP-EG mouse group and the PSAPP-FA mouse group ( ⁺ p <0.05), and the combined administration of FA and EG A synergistic improvement effect was observed. The recognition frequency (R _AC : 71.6%) of the PSAPP-FAEG mouse group was found to be statistically different from the recognition frequency (R _AC : 71.0-71.5%) of the other four WT mouse groups. Not (p> 0.05). This indicates that combined administration of FAEG completely improved the novel object recognition impairment in PSAPP mice expressing the APP mutation.

Next, the results of the Y-maze test are shown. Originally, when selecting the route of the Y-maze, the mouse has a property of selecting a route different from the route selected immediately before. If the mouse is placed in a Y-maze with three different routes A, B, and C, if A is selected first, the next action will take another B or C route. The Y-maze test is a test for evaluating exploratory activity and short-term memory (spatial working memory) using this property. In this test, the case where different routes were selected three times in succession was regarded as “voluntary alternation behavior”. When memory impairment develops, the tendency to choose the same route over and over increases, resulting in reduced voluntary alternation behavior.

In PSAPP-V mice, the total number of entries into the Y-maze, which reflects overactivity, increased, and spontaneous alternation behavior decreased, reflecting short-term memory (spatial working memory) impairment. On the other hand, in PSAPP-FAEG mice administered with both 30 mg / kg FA and 30 mg / kg per administration, PSAPP-EG mice administered with 30 mg / kg EG and PSAPP-FA mice administered with 30 mg / kg per administration Overactivity has subsided (Y—reduced total number of mazes) and short-term memory (spatial working memory) impairment has improved.

That is, as shown in FIG. 2 (A), as a result of one-way ANOVA and post hoc analysis, the PSAPP-V mouse group showed a significant increase in the total number of entries into the Y-maze as compared to the other seven groups. ( ^* P <0.05. The total number of entries was 48.3 times in the PSAPP-V mouse group, 29.0 times in the PSAPP-FAEG mouse group, 39.0 times in the PSAPP-EG mouse group, PSAPP- The FA mouse group was 38.4 times, the WT-V mouse group was 28.3 times, the WT-FAEG mouse group was 29.8 times, the WT-EG mouse group was 28.4 times, and the WT-FA mouse group was 28. 5 times). Interestingly, in the PSAPP-FAEG mouse group, compared to the PSAPP-EG mouse group and the PSAPP-FA mouse group, the hyperactivity was significantly reduced and stabilized ( ⁺ p <0.05), and FA and EG Synergistic improvement effect was recognized by combined administration. The data of this PSAPP-FAEG mouse group had been reduced to no significant difference compared to the data of the other 4 WT mouse groups. This indicates that the combined administration of FAEG completely stabilized the exploratory activity that was increased in PSAPP mice.

PSAPP-V mice showed a low spontaneous alternation behavior rate of 50% or less and had short-term memory (spatial working memory) impairment. As a result of the one-way ANOVA and post hoc analysis, the PSAPP-V mouse group showed a significant decrease in spontaneous alternation behavior rate compared to the other seven groups ( ^* p <0.05. Spontaneous alternation behavior rate. Are 41.5% in the PSAPP-V mouse group, 67.5% in the PSAPP-FAEG mouse group, 54.1% in the PSAPP-EG mouse group, 52.5% in the PSAPP-FA mouse group, and WT-V mice. 67.3% in the group, 69.9% in the WT-FAEG mouse group, 68.9% in the WT-EG mouse group, and 66.2% in the WT-FA mouse group). Importantly, in the PSAPP-FAEG mouse group, the spontaneous alternation behavior rate was significantly increased ( ⁺⁺ p <0.01) compared with the PSAPP-EG mouse group and the PSAPP-FA mouse group, and FA and EG. Synergistic improvement effect was recognized by combined administration. The data of this PSAPP-FAEG mouse group increased to the extent that there was no significant difference compared to the data of the other 4 WT mouse groups. This indicates that the combined administration of FAEG completely improved the short-term memory (spatial working memory) decline that had been impaired in PSAPP mice.

Interestingly, the combined administration of FAEG and single administration of FA or EG did not affect the enhancement of behavior / cognitive function of WT mice in any of the test results.

(B) Inhibitory effect on production / accumulation of Aβ Aβ is considered to be one of causative substances involved in the onset / pathological progression of Alzheimer's disease. Therefore, in evaluating drugs, it is important to see the effect of suppressing Aβ production / accumulation.

In the analysis of the soluble fraction of the TBS solution shown in FIG. 3 (A), it is expressed as a PSAPP-FAEG mouse (hereinafter referred to as “PSAPP-FAEG (30) mouse”) in which FA 30 mg / kg and EG 30 mg / kg are administered in combination. .) PSAPP-FAEG mice administered together with 15 mg / kg FA and 15 mg / kg per dose (hereinafter referred to as “PSAPP-FAEG (15) mice”) PSAPP- administered 30 mg / kg EG per dose EG mice and PSAPP-FA mice dosed with 30 mg / kg of FA per Aβ _1-40 level (reduction rate was 58.5, 49.4, 34.0, 28.5% in each treatment group) and Aβ _{1 -42} levels (rate of decrease each treatment group 55.8,50.7,42.2,44.0%) showed a significant reduction in ^(** p <0 01). Furthermore, PSAPP-FAEG (30) mice have a synergistically significant decrease in Aβ _1-40 levels compared to PSAPP-EG mice ( ⁺ p <0.05). In the analysis of the TNE solution soluble fraction shown in FIG. 3 (B), PSAPP-FAEG (30) mice, PSAPP-FAEG (15) mice, PSAPP-EG mice, and PSAPP-FA mice showed Aβ _1-40 levels ( The rate of decrease was 61.1, 58.5, 32.4, 36.2%) and the Aβ _1-42 level (the rate of decrease was 37.6, 36.8, 35.5, 30.30 for each dose group). 3%) ( ^** P <0.01). Furthermore, PSAPP-FAEG (30) mice have a synergistically significant decrease in Aβ _1-40 levels ( ⁺ p <0.05) compared to PSAPP-EG mice and PSAPP-FA mice, and PSAPP-FAEG (15) in mice, as compared to PSAPP-EG mice, a [beta] _1-40 levels were synergistically decreased significantly ^(# p <0.05).

In the analysis of the soluble fraction of the guanidine hydrochloride solution shown in FIG. 3 (C), PSAPP-FAEG (30) mice, PSAPP-FAEG (15) mice, PSAPP-EG mice and PSAPP-FA mice have Aβ _1-40 levels. (Reduction rate is 55.9, 54.0, 34.6, 32.5% in each administration group) and Aβ _1-42 level (reduction rate is 58.0, 53.2, 33.5, 30 in each administration group) 0.0%) ( ^*** p <0.001). Furthermore, PSAPP-FAEG (30) mice have a synergistically significant decrease in Aβ _1-40 levels ( ⁺ p <0.05) compared to PSAPP-EG mice and PSAPP-FA mice, and Aβ _{1- 1 42} levels synergistically significantly reduced ⁽⁺⁺ p <0.01), even PSAPP-FAEG (15) mice, compared to PSAPP-EG mice and PSAPP-FA mice, a [beta] _1-40 levels synergistically significantly decreased ^{(# p <0.05), Aβ} 1-42 levels were synergistically decreased significantly ^(# p <0.05).

The total amount of FA and EG administered to PSAPP-FAEG (15) mice (15 mg / kg + 15 mg / kg = 30 mg / kg per dose) is the amount of EG administered to PSAPP-EG mice (30 mg per dose) / Kg) and the amount of FA administered to PSAPP-FA mice (30 mg / kg per dose), but from FIG. 3, the Aβ abundance in PSAPP-FAEG (15) mice is the same as that in PSAPP-EG mice and Aβ abundance in PSAPP-FAEG (30) mice administered with a dose of FA and EG (30 mg / kg + 30 mg / kg = 60 mg / kg per dose) significantly smaller than the amount of Aβ in PSAPP-FA mice You can see that they are approaching. This is significant even when the effect of suppressing Aβ production / accumulation by the combined administration of FA and EG is compared with the effect predicted by adding the effect of administering FA alone and the effect of administering EG alone. It shows that a synergistic effect of FA and EG is recognized. In addition, the combined effect of FA and EG includes an Aβ aggregate that is insoluble in TBS solution and TNE solution, and is considered to most reflect the presence of β-amyloid protein plaque (senile plaque). It is also noticeable in the soluble fraction. From this, effective suppression of the production | generation of (beta) -amyloid protein plaque (senile plaque) is anticipated with the treatment-prevention agent of Alzheimer's disease of this invention which used FA and EG together.

Next, FIG. 4 shows the results of analysis of Aβ oligomers on the TNE solution soluble fraction. Interestingly, PSAPP-FAEG (30) mice, PSAPP-FAEG (15) mice, PSAPP-EG mice and PSAPP-FA mice have Aβ oligomer levels (decrease rates are 59.7, 55.8, 32 for each treatment group). 0.7, 29.2%) ( ^* p <0.05). Furthermore, PSAPP-FAEG (30) mice have a synergistically significant decrease in Aβ oligomer levels ( ⁺ p <0.05) compared to PSAPP-EG mice and PSAPP-FA mice, and PSAPP-FAEG (15 ) in mice, as compared to PSAPP-FA mice, a [beta] oligomers levels were synergistically decreased significantly ^(# p <0.05).

The total amount of FA and EG administered to PSAPP-FAEG (15) mice (15 mg / kg + 15 mg / kg = 30 mg / kg per dose) is the amount of EG administered to PSAPP-EG mice (30 mg per dose) / Kg) and the amount of FA administered to PSAPP-FA mice (30 mg / kg per dose), but from FIG. 4, the amount of Aβ oligomers in PSAPP-FAEG (15) mice is And Aβ oligomers in PSAPP-FAEG (30) mice that were significantly smaller than the amount of Aβ oligomers present in PSAPP-FA mice and were administered twice as much FA and EG (30 mg / kg + 30 mg / kg = 60 mg / kg per dose) It turns out that it is almost the same as the abundance. This is significant even when the inhibitory effect on the production of Aβ oligomers by the combined administration of FA and EG is compared with the effect expected by adding the effect of administering FA alone and the effect of administering EG alone. It shows that a synergistic effect of FA and EG is recognized. Therefore, the therapeutic and preventive agent for Alzheimer's disease of the present invention using both FA and EG effectively produces Aβ oligomers that have synaptic toxicity and are strongly suggested to be associated with behavioral and cognitive impairment. Suppression is expected.

The present invention provides a therapeutic and preventive agent for Alzheimer's disease that is safe and has excellent therapeutic and preventive effects.

Claims (4)

At least one compound selected from the group consisting of ferulic acid as a first active ingredient and pharmacologically acceptable salts thereof;
A therapeutic and prophylactic agent for Alzheimer's disease comprising a combination of at least one compound selected from the group consisting of epigallocatechin gallate and a pharmacologically acceptable salt thereof as a second active ingredient,
The mass ratio of the first active ingredient and the second active ingredient is such that the mass of the first active ingredient is converted to the mass of ferulic acid, and the mass of the second active ingredient is converted to epigallocatechin gallate. An agent for the treatment and prevention of Alzheimer's disease, characterized by being in the range of 1:10 to 10: 1 in terms of the mass of The therapeutic / preventive agent for Alzheimer's disease according to claim 1, which is a combination comprising both the first active ingredient and the second active ingredient. The therapeutic and preventive agent for Alzheimer's disease according to claim 1, which is a kit comprising a therapeutic and prophylactic agent comprising the first active ingredient and a therapeutic and prophylactic agent comprising the second active ingredient. The therapeutic / preventive agent for Alzheimer's disease according to any one of claims 1 to 3, which is orally administered as a health food.

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