Alzheimer's disease-like symptom alleviation

Abstract

To provide a material that can be safely ingested, and has an effect of alleviating Alzheimer disease-like symptoms.SOLUTION: An agent for alleviating Alzheimer disease-like symptoms has one kind selected from β-nicotinamide mononucleotide, a pharmacologically acceptable salt thereof, and solvate thereof as an active ingredient. A health supplement contains the symptom alleviating agent, and is ingested in order to alleviate Alzheimer disease-like symptoms. A feeding stuff contains the symptom alleviating agent, and is ingested in order to alleviate Alzheimer disease-like symptoms.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a material that can be safely ingested and that can alleviate the symptoms of Alzheimer's disease, and to an agent for alleviating Alzheimer's disease-like symptoms that contains the material as an active ingredient. [Background technology]

[0002] Alzheimer's disease is a disease that gradually deteriorates cognitive function and is the most common type of dementia among the elderly. It is a global problem, with an estimated 46.8 million patients worldwide in 2015, and is expected to increase to 74.7 million by 2030. Alzheimer's disease is believed to be caused by the accumulation of amyloid beta proteins and tau proteins in the brain, which causes nerve cell death and brain atrophy, resulting in cognitive impairment.

[0003] β-Nicotinamide mononucleotide (hereinafter referred to as "β-NMN") is a coenzyme NAD + It is a biosynthetic intermediate metabolic product of β-NMN. In recent years, it has been reported that β-NMN has the effect of improving insulin secretion in aging mice, dramatically improving insulin sensitivity and secretion in mouse models of type 2 diabetes caused by a high-fat diet and aging (see, for example, Patent Document 1), that it is involved in the control of circadian rhythms (see, for example, Patent Document 2), and that it has the effect of significantly enhancing mitochondrial function in aging muscles. Furthermore, it has also been reported that administration of β-NMN is useful for improving and preventing the symptoms of various age-related diseases such as obesity, elevated blood lipid levels, decreased insulin sensitivity, memory loss, and deterioration of ocular function such as macular degeneration (see, for example, Patent Document 3). Furthermore, administration of β-NMN increases NAD levels in the body. + By increasing the amount of NMN and activating the sirtuin gene, it is expected that the decline in physical functions associated with aging will be suppressed and delayed, resulting in anti-aging effects (see, for example, Patent Document 4).Recently, clinical trials have been conducted in which NMN was orally administered to humans, and it has been confirmed that there are no safety issues (see, for example, Non-Patent Document 1).

[0004] Research into administering β-NMN to Alzheimer's disease animal models has been conducted for some time, and it has been reported that β-NMN improves cognitive function (see, for example, Non-Patent Documents 2 and 3). However, in Non-Patent Document 2, β-NMN is administered to the model animals subcutaneously, while in Non-Patent Document 3, it is administered intraperitoneally. Both administration methods are parenteral, and the "Guidelines for Administration of Test Substances to Experimental Animals (Administration Route, Administration Volume) and Blood Collection" states that it is necessary to consider the administration volume, stability of the formulation before and after administration, pH, viscosity, osmotic pressure, buffer capacity, sterility of the formulation, and biocompatibility. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 7,737,158 [Patent Document 2] US Patent Application Publication No. 2011 / 123510 [Patent Document 3] International Publication No. 2014 / 146044 [Patent Document 4] International Publication No. 2017 / 200050 [Non-patent literature]

[0006] [Non-Patent Document 1] Irie J et al., “Effect of oral administration of nicotinamide mononucleotide on clinical parameters and nicotinamide metabolite levels in healthy Japanese men.”, Endocr J, Vol. 67, No. 2, pp. 153-160, 2020. [Non-patent document 2] Long AN et al., “Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model.”, BMC Neurol, Vol. 15, No. 19, 2015. [Non-patent document 3] Wang X et al., “Nicotinamide mononucleotide protects against β-amyloid oligomer-induced cognitive impairment and neuronal death.”, Brain Res, Vol. 1643, pp. 1-9, 2016. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a material that can be safely ingested and has the effect of alleviating Alzheimer's disease-like symptoms. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above problems, the present inventors discovered that β-NMN has the effect of alleviating Alzheimer's disease-like symptoms, and thus completed the present invention.

[0009] That is, the present invention provides the following agents for alleviating Alzheimer's disease-like symptoms, dietary supplements, and feeds. [1] An agent for alleviating Alzheimer's disease-like symptoms, characterized by containing as an active ingredient one selected from β-NMN, its pharmacologically acceptable salts, and solvates thereof. [2] The symptom-relieving agent according to [1] above, which is orally administered. [3] The symptom-relieving agent according to [1] or [2] above, which is used for the prevention or treatment of spatial cognitive impairment caused by the accumulation of amyloid beta in the brain. [4] The symptom-relieving agent according to [1] or [2] above, which is used to prevent learning disability or memory disability caused by the accumulation of amyloid beta in the brain. [5] A dietary supplement containing any of the symptom-relieving agents [1] to [4], taken to alleviate Alzheimer's disease-like symptoms. [6] A feed containing any of the symptom-relieving agents [1] to [4], which is ingested to alleviate Alzheimer's disease-like symptoms. [Effects of the Invention]

[0010] The agent for alleviating Alzheimer's disease-like symptoms according to the present invention contains β-NMN, which is naturally present in the body, as an active ingredient and can alleviate Alzheimer's disease-like cognitive impairment. Therefore, the agent for alleviating Alzheimer's disease-like symptoms according to the present invention can be taken safely without causing side effects and is effective in preventing and alleviating diseases caused by the accumulation of amyloid β in the brain. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing the cell viability (%) of SH-SY5Y cells evaluated by luciferase assay for toxicity to amyloid β42 (Aβ42) in each group of cells in Example 1, where the medium was replaced daily with a medium containing 0.2 mM β-NMN for one week, and the medium was replaced daily with a β-NMN-free medium. [Figure 2] In Example 2, spatial cognitive ability was evaluated based on the spontaneous alternation rate in a behavioral test in a Y-maze for mice from a group administered β-NMN one week before (protective effect group), a group administered β-NMN after Aβ administration (therapy effect group), a sham operation group, and a group administered only water (vehicle group). This graph shows the spontaneous alternation rate (%). [Figure 3]In Example 2, learning and memory abilities were evaluated based on response latency in a passive avoidance test for mice from the group administered β-NMN one week before (protective effect group), the group administered β-NMN after Aβ administration (therapy effect group), the sham operation group, and the group administered only water (vehicle group). This graph shows the response latency (sec). DETAILED DESCRIPTION OF THE INVENTION

[0012] The agent for alleviating Alzheimer's disease-like symptoms according to the present invention (hereinafter sometimes referred to as the "agent for alleviating symptoms according to the present invention") contains NMN (chemical formula: C 11 H 15 NMN contains N2O8P) as an active ingredient and has the effect of preventing or alleviating Alzheimer's disease-like cognitive impairment. Therefore, the symptom-relieving agent of the present invention is suitable as an active ingredient of oral compositions or topical compositions for preventing or treating symptoms caused by Alzheimer's disease. Examples of Alzheimer's disease-like cognitive impairment include memory impairment, disorientation, judgment impairment (executive dysfunction), and higher-order functional disorders (aphasia, agnosia, apraxia). As shown in the examples below, NMN can improve cognitive impairment caused by amyloid-β accumulation, particularly impaired spatial cognition. Furthermore, NMN can prevent amyloid-β toxicity in neurons and suppress cell death, making it effective as a preventative agent for Alzheimer's disease-like symptoms. Specifically, ingesting NMN before amyloid-β accumulation can prevent cognitive impairment caused by amyloid-β accumulation, specifically impairments in spatial cognition and learning and memory abilities. Thus, the symptom-relieving agent according to the present invention is particularly useful as a preventive or therapeutic agent for spatial cognitive impairment caused by the accumulation of amyloid beta in the brain, a preventive agent for learning ability impairment caused by the accumulation of amyloid beta in the brain, and a preventive agent for memory impairment caused by the accumulation of amyloid beta in the brain.Furthermore, the symptom-relieving agent according to the present invention is also useful as an agent for suppressing neuronal cell death caused by amyloid beta.

[0013] NMN exists as two optical isomers, α and β, but the NMN that serves as the active ingredient in the symptom-relieving agent of the present invention is β-NMN (CAS number: 1094-61-7). The structure of β-NMN is shown below.

[0014] [ka]

[0015] The β-NMN used as the active ingredient may be prepared by any method. For example, β-NMN artificially synthesized by chemical synthesis, enzymatic methods, fermentation, etc., and then purified can be used as the active ingredient. Furthermore, since β-NMN is widely found in living organisms, β-NMN obtained by extraction and purification from natural sources such as animals, plants, and microorganisms can also be used as the active ingredient. Commercially available purified β-NMN may also be used.

[0016] Chemical synthesis of β-NMN involves, for example, reacting NAM with L-ribose tetraacetate and phosphorylating the resulting nicotinamide mononucleoside to produce β-NMN. Enzymatic synthesis involves, for example, producing β-NMN from NAM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) using nicotinamide phosphoribosyltransferase (NAMPT). Fermentation can also involve producing β-NMN from NAM using the metabolic system of a microorganism expressing NAMPT.

[0017] The active ingredient of the symptom-relieving agent of the present invention may be a pharmacologically acceptable salt of β-NMN. Pharmacologically acceptable salts of β-NMN may be inorganic acid salts or organic acid salts containing a basic moiety such as an amine. Examples of acids that constitute such acid salts include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Furthermore, pharmacologically acceptable salts of β-NMN may be alkali salts or organic salts containing an acidic moiety such as a carboxylic acid. Examples of bases that constitute such acid salts include alkali metal salts or alkaline earth metal salts, such as those derived from bases such as sodium hydride, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, trimethylammonia, triethylammonia, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, and tetramethylammonium hydroxide.

[0018] The active ingredient of the symptom-relieving agent of the present invention may be a solvate of free β-NMN or a solvate of a pharmacologically acceptable salt of β-NMN. Solvents that form such solvates include water, ethanol, etc.

[0019] The symptom-relieving agent of the present invention may contain other active ingredients in addition to β-NMN. The other active ingredients are not particularly limited as long as they do not impair the effect of β-NMN in alleviating Alzheimer's disease-like symptoms. Examples of such other active ingredients include functional materials other than β-NMN that are known to have an effect of alleviating Alzheimer's disease-like symptoms. Examples of known functional materials that are known to have an effect of alleviating Alzheimer's disease-like symptoms include ginkgo, omega-3 fatty acids (fish oil), vitamins B and E, ginseng, grape seed extract, and curcumin.

[0020] The symptom-relieving agent of the present invention may consist of only the active ingredient, or may contain other ingredients. For example, the symptom-relieving agent of the present invention can be formulated into various dosage forms by combining the active ingredient with a non-toxic pharmaceutical carrier using conventional pharmaceutical methods. Among the dosage forms of the symptom-relieving agent of the present invention, oral administration forms include solid forms such as tablets, granules, powders, capsules, and soft capsules; liquid forms such as solutions, suspensions, and emulsions; and lyophilized preparations. Parenteral administration forms include injections, suppositories, sprays, transdermal absorption agents, and the like.

[0021] Examples of non-toxic pharmaceutical carriers used in formulation include sugars such as glucose, lactose, sucrose, fructose, reduced maltose, etc.; carbohydrates such as starch, hydroxyethyl starch, dextrin, β-cyclodextrin, crystalline cellulose, hydroxypropyl cellulose, etc.; sugar alcohols such as mannitol, erythritol, sorbitol, xylitol, etc.; esters such as fatty acid glycerides and polyoxyethylene sorbitan fatty acid esters; polyethylene glycol, ethylene glycol, amino acids, albumin, casein, silicon dioxide, water, physiological saline, etc. In formulating the symptom-relieving agent of the present invention, conventional additives such as stabilizers, lubricants, humectants, emulsifiers, suspending agents, binders, disintegrants, solvents, solubilizers, buffers, isotonicity agents, preservatives, flavorings, coloring agents, etc. may be added as needed for the formulation.

[0022] The symptom-relieving agent according to the present invention is preferably administered to humans or non-human animals. Examples of non-human animals include mammals such as cows, pigs, horses, sheep, goats, donkeys, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs. The symptom-relieving agent according to the present invention is preferably administered to or ingested by humans, livestock, laboratory animals, or pets, and more preferably administered to or ingested by humans.

[0023] The dosage or intake amount of the symptom-relieving agent according to the present invention is appropriately selected and determined depending on the species, age (months), body weight, symptoms, severity of disease, administration schedule, formulation, etc. For example, the daily dose per adult is 0.1 mg to 10 g, preferably 0.5 mg to 7 g, more preferably 10 mg to 5 g, and even more preferably 100 mg to 2 g, of β-NMN, which can be administered once or in divided doses.

[0024] β-NMN is a biological component and is also found in foods, and is therefore considered to be highly safe. Therefore, the symptom-relieving agent of the present invention can be used as an active ingredient in dietary supplements taken to alleviate Alzheimer's disease-like symptoms. Dietary supplements are foods and beverages that provide nutritional supplements for the purpose of maintaining or improving health, and include foods for specified health uses, foods with nutrient functions, and health foods. Because the symptom-relieving agent of the present invention is highly safe and suitable for long-term continuous ingestion, dietary supplements containing the symptom-relieving agent are expected to have excellent effects on preventing and alleviating Alzheimer's disease.

[0025] Furthermore, the symptom-relieving agent according to the present invention can be used as an active ingredient in feed to be ingested by animals to alleviate Alzheimer's disease-like symptoms. By feeding the feed to livestock, pets, laboratory animals, etc., toxicity caused by the accumulation of amyloid beta in the brains of the animals that ingested the feed is prevented, and neuronal cell death is suppressed, which is expected to prevent Alzheimer's disease and improve the pathology.

[0026] The dietary supplement and feed of the present invention can be produced by adding appropriate auxiliary agents to β-NMN or the like, and then forming it into an edible form, such as a powder, granules, tablets, capsules, soft capsules, or paste, using conventional means. The dietary supplement of the present invention may be consumed as is, or may be consumed after being mixed with various foods or beverages. For example, a powdered dietary supplement can be ingested by dissolving or dispersing it in beverages such as water, alcoholic beverages, fruit juice, milk, or soft drinks. The feed of the present invention may also be ingested by animals as is, or may be ingested by animals after being mixed with other solid feed or drinking water.

[0027] The dietary supplement and feed according to the present invention may contain other food ingredients and various additives, such as vitamins, carbohydrates, proteins, lipids, dietary fiber, and fruit juice. Specific examples include B vitamins such as vitamin B1 derivatives, vitamin B2, vitamin B6, vitamin B12, vitamin B13, biotin, pantothenic acid, nicotinic acid, and folic acid; fat-soluble vitamins such as vitamin E, vitamin D or its derivatives, vitamin K1, vitamin K2, and β-carotene; minerals such as calcium, potassium, iron, and zinc; yeast, L-carnitine, creatine, α-lipoic acid, glutathione, glucuronic acid, taurine, collagen, soy isoflavones, lecithin, peptides, amino acids, γ-aminobutyric acid, diacylglycerol, DHA, EPA, capsaicin, chondroitin sulfate, agaricus mushroom extract, carrot extract, garlic extract, green juice, lecithin, royal jelly, propolis, octacosanol, flavangenol, pycnogenol, maca, chitosan, Garcinia extract, chondroitin, and glucosamine. Examples of additives include sweeteners, acidulants such as organic acids, stabilizers, flavorings, and coloring agents. [Example]

[0028] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0029] [mouse] The Slc:ddY mice used in the following experiments were kept in an SPF environment throughout the entire experimental period. Solid feed (MF, Oriental Yeast Co., Ltd.) and drinking water were available ad libitum throughout the entire experimental period.

[0030] [Oral administration] Oral administration was performed by forcibly administering a solution of β-NMN (manufactured by Oriental Yeast Co., Ltd.) dissolved in water for injection (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) using a disposable polypropylene syringe (Terumo Corporation) attached with a disposable oral probe for rats (Fuchigami Instruments, Ltd.).

[0031] [Creation of Alzheimer's disease model mice by intracerebroventricular injection of Aβ] The animals were anesthetized with 40 mg / kg of pentobarbital sodium (Tokyo Chemical Industry Co., Ltd.) administered intraperitoneally (10 mL / kg). After anesthesia, levobupivacaine hydrochloride (Popscaine® 0.25% injection, Maruishi Pharmaceutical Co., Ltd.) was administered subcutaneously (0.1 mL) to the scalp. The hair on the parietal area of ​​the animal was shaved, and the head was fixed in a stereotaxic apparatus. The parietal area was disinfected with isodine and then incised to expose the skull. The connective tissue on the skull was removed with a cotton swab and then dried with a blower to make the location of the bregma easier to visualize. A hole for inserting a stainless steel pipe was drilled into the skull 1 mm lateral (right side) and 0.2 mm posterior to the bregma using a dental drill. A stainless steel pipe connected to a 0.5 mm outer diameter silicone tube and a microsyringe was inserted vertically to a depth of 2.5 mm from the bone surface. 3 μL of amyloid β solution (6 nmol / 3 μL) was injected into the ventricle over 3 minutes using a microsyringe pump (3 μL of water for injection was injected in the sham-operated group). After injection, the stainless steel pipe was left inserted for 3 minutes and then slowly removed. The skull hole was sealed with a non-absorbable bone marrow hemostatic agent (Nestop (registered trademark), Alfresa Pharma Corporation), and the scalp was sutured. The animal was removed from the stereotaxic apparatus and returned to its cage. The stainless steel pipe and silicone tubing used were sterilized.

[0032] [Y-maze test (spontaneous alternation test)] For the test, a plastic Y-shaped maze (Unicom Co., Ltd.) was used, with each arm 39.5 cm long, a floor 4.5 cm wide, walls 12 cm high, and three arms each branching at 120 degrees. Before measurement, the illuminance on the floor of the apparatus was adjusted to 10-40 lx. Measurements were performed approximately 1 hour (+10 min) after administration. The animals were placed in one of the arms of the Y-shaped maze and allowed to freely explore the maze for 8 min. The arm numbers (A-C) to which the animals moved during the measurement period were recorded, and the number of arm entries was counted and used as the total number of entries. Next, the combinations in which the animals consecutively selected three different arms were examined, and this number was used as the number of spontaneous alternations. The spontaneous alternation rate was calculated using the following formula.

[0033] Spontaneous alternation rate (%) = [Number of spontaneous alternations / (Total number of entries - 2)] × 100

[0034] [Contract Avoidance Testing] For the test, a step-through type passive avoidance response apparatus (light-dark box: manufactured in-house, SHOCK SCRAMBLER: Unicom Co., Ltd.) was used, which had a light chamber (W: 100 mm × D: 100 mm × H: 300 mm) separated by a central guillotine door and a dark chamber (W: 240 mm × D: 245 mm × H: 300 mm) in which electrical stimuli were applied via a grid on the floor. Measurements were taken approximately 1 hour (+10 minutes) after administration. The animals were placed in the lighted compartment, and 10 seconds later, the guillotine door was gently opened to measure the time it took for the animals to enter the dark compartment (response latency). In the acquisition trial (day 10 of administration), the animals entered the dark room, and the guillotine door was closed. An electric stimulus (0.2 mA, 2 seconds, scrambled) was administered. The animals were monitored for vocalizations during the electric stimulus. Response latencies were measured up to 300 seconds. The retention trial (day 11 of administration) ended when the animal entered the dark compartment or spent 300 seconds in the light compartment.

[0035] [Example 1] SH-SY5Y cells were cultured in medium containing 0.2 mM NMN for one week prior to the addition of Aβ42. During this period, the medium was changed daily. The day before the assay, the medium was changed to NMN-free medium and cells were seeded at 1 x 10 / well. The following day, different concentrations of Aβ42 were added to each well, and two days later, cell death was measured by luciferase assay. The measurement results are shown in Figure 1. Cell death was suppressed in the cell group to which NMN was added in advance compared to the group to which NMN was not added. These results suggest that administering NMN before Aβ administration is effective in preventing Aβ-induced toxicity.

[0036] [Example 2] The Alzheimer's disease model mice were divided into four groups, each consisting of 10 mice: one group administered β-NMN one week before Aβ administration (protective effect group), one group administered β-NMN after Aβ administration (therapy effect group), a sham operation group, and a vehicle control group administered only water (vehicle group) (Table 1). The protective effect group was administered β-NMN for 16 days. The therapy effect group was administered the vehicle (water for injection) from days 1 to 7 (before Aβ administration) and β-NMN from days 8 to 16 (after Aβ administration).

[0037] [Table 1]

[0038] (Y-maze test results) The first day of administration of the test substance was defined as day 1, and amyloid beta was injected on day 8. The Y-maze test was then performed on day 14. The measurement results are shown in Figure 1. A tendency for spontaneous alternation rate to improve was observed in both the protective effect group and the therapy effect group, which received beta-NMN. These results suggest that beta-NMN has both the effect of preventing and treating spatial cognitive impairment caused by Aβ.

[0039] (Measurement results of passive avoidance test) The first day of administration of the test substance was designated as day 1, and amyloid beta was injected on day 8. A passive avoidance test was then conducted on days 15-16. The measurement results are shown in Figure 2. Response latency did not improve in the therapy effect group, but a tendency for improvement was observed in the protective effect group, which had been administered β-NMN beforehand. These results suggest that prior intake of β-NMN may be able to prevent impairment of learning and memory ability caused by amyloid beta.

Claims

1. An inhibitor of neuronal cell death caused by amyloid beta, The present invention is characterized in that the active ingredient is one selected from the group consisting of β-nicotinamide mononucleotide, a pharmacologically acceptable salt thereof, and a solvate thereof, It is administered orally before amyloid beta accumulates in the subject's brain. Inhibitor of neuronal cell death caused by amyloid beta.

2. A composition for preventing symptoms caused by Alzheimer's disease, comprising the inhibitor of amyloid beta-induced neuronal cell death described in claim 1, and being orally administered before amyloid beta accumulates in the brain of the subject.

3. The composition according to claim 2, wherein the symptom caused by Alzheimer's disease is learning or memory impairment caused by amyloid beta accumulation in the brain.

4. The composition of claim 2 which is a dietary supplement.

5. The composition of claim 2 which is a feed.

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