Inhibitor for diseases associated with microglial activation

Abstract

To provide an inhibitor that can effectively inhibit diseases associated with microglial activation and that, owing to its comparative safety, allows regular oral administration.SOLUTION: The inhibitor for diseases associated with microglial activation according to the present invention is characterized by containing, as active ingredients, two or more aromatic amino acids selected from phenylglycine, phenylalanine, tyrosine, and dihydroxyphenylalanine, or salts thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a suppressant that can effectively suppress diseases associated with microglial activation and that can be administered regularly because it is relatively safe. [Background technology]

[0002] Glial cells, also known as neuroglial cells, are a collective term for cells that make up the nervous system but are not neurons. Microglia are a type of glial cell present in the cerebrospinal cord and have been suggested to potentially play a role as immune cells in the central nervous system. Specifically, microglia possess macrophage-like neurophagocytic behavior, and when neural tissue is damaged by inflammation or degeneration, microglia are activated and participate in the repair of the lesion. For example, in Alzheimer's disease, microglia have been observed surrounding amyloid plaques, spatially suppressing plaque growth and initiating their removal by phagocytosis.

[0003] On the other hand, activated microglia secrete inflammatory cytokines such as IL-1β, IL6, and TNFα, which damage neurons. Activated microglia have been reported to be involved in various pathologies, such as depression, bipolar disorder, autism, myocardial infarction, malocclusion, alcohol use, sleep disorders, obesity, hyperglycemia, hypertension, and stroke. Microglia-induced neuroinflammation is also known to be involved in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis.

[0004] In addition, activated microglia are known to produce excessive amounts of nitric oxide. Nitric oxide is known to function as a neurotransmitter and intracellular second messenger in the central nervous system, but it is also known to be involved in glutamate-induced neuronal damage and various neurodegenerative diseases. For example, it has been reported that nitric oxide is involved in glutamate-mediated neuronal cell death, and that the large amounts of nitric oxide derived from activated microglia observed in pathological conditions are involved in cell damage.

[0005] Known drugs for treating neurodegenerative diseases include donepezil, memantine, galantamine, and rivastigmine. While these drugs may slow the progression of symptoms, they do not provide a fundamental cure and there are safety concerns.

[0006] In response to this, drugs have been developed that contain relatively safe active ingredients, can be taken regularly, and are also applicable to the prevention of neurodegenerative diseases. For example, Patent Document 1 discloses an antioxidant-promoting composition that contains an extract of Bacopa monniera, a moist perennial plant distributed in Europe, etc., and that increases the enzymatic activity level of antioxidant enzymes and reduces the plasma concentration level of thiobarbituric acid-reactive chemical species. Patent Document 2 discloses an inhibitor of the expression of nuclear transcription factor AP-1, containing chamameloside, which is contained in Roman chamomile extract. Patent Document 3 discloses a nitric oxide production inhibitor that contains an extract of the leaves of a plant belonging to the genus Erythronium. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2007-530574 [Patent Document 2] International Publication No. 2007 / 148697 Brochure [Patent Document 3] Japanese Patent Application Laid-Open No. 2018-135294 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, various drugs containing active ingredients that are considered to be relatively safe and that suppress neurodegenerative diseases and nitric oxide production are being investigated. However, the reality is that there are no or very few safe compounds that have been put to practical use as inhibitors of neurodegenerative diseases, etc., and effective candidate compounds are still eagerly awaited. Therefore, an object of the present invention is to provide an inhibitor that can effectively suppress diseases associated with microglial activation and that can be taken regularly because it is relatively safe. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and as a result have found that a combination of two or more specific aromatic amino acids can significantly reduce the amount of nitric oxide produced by microglia, thereby completing the present invention. The present invention will now be described.

[0010] [1] An agent for suppressing diseases associated with microglial activation, characterized by containing as active ingredients two or more aromatic amino acids selected from phenylglycine, phenylalanine, tyrosine, and dihydroxyphenylalanine or salts thereof. [2] The inhibitor according to [1], wherein the phenylglycine is 2-phenylglycine or N-phenylglycine. [3] The inhibitor according to [1] or [2], wherein the dihydroxyphenylalanine is 3,4-dihydroxyphenylalanine. [4] The inhibitor according to [1], which contains the dihydroxyphenylalanine and the phenylglycine as active ingredients. [5] The inhibitor according to [1], which contains two or more aromatic amino acids selected from the group consisting of phenylalanine, tyrosine, and dihydroxyphenylalanine as active ingredients. [6] The inhibitor according to [1], which contains the phenylalanine, the tyrosine, and the dihydroxyphenylalanine as active ingredients. [7] The inhibitor according to any one of [1] to [6] above, which inhibits the production of nitric oxide by the microglia. [Effects of the Invention]

[0011] The inhibitor of a disease associated with microglial activation according to the present invention contains a proteinogenic amino acid or a derivative thereof as an active ingredient, and is therefore considered safe, allowing for long-term, consistent administration. Therefore, it may be used not only for treatment after the onset of the disease, but also for prevention. Therefore, the inhibitor of the present invention is industrially very advantageous as an inhibitor of a disease associated with microglial activation. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1(1) is a graph showing the synergistic inhibitory effect of a combination of L-dopa (L-3,4-dihydroxyphenylalanine) and D-2-phenylglycine on the amount of nitric oxide produced by microglia. Figure 1(2) is a graph showing the synergistic inhibitory effect of a combination of L-dopa and L-2-phenylglycine on the amount of nitric oxide produced by microglia. Figure 1(3) is a graph showing the synergistic inhibitory effect of a combination of L-dopa and N-phenylglycine on the amount of nitric oxide produced by microglia. [Figure 2] Figure 2(1) is a graph showing the synergistic inhibitory effect of the combination of L-phenylalanine and L-dopa on the amount of nitric oxide produced by microglia. Figure 2(2) is a graph showing the synergistic inhibitory effect of the combination of L-phenylalanine and L-tyrosine on the amount of nitric oxide produced by microglia. Figure 2(3) is a graph showing the synergistic inhibitory effect of the combination of L-dopa and L-tyrosine on the amount of nitric oxide produced by microglia. [Figure 3]FIG. 3 is a graph showing the synergistic inhibitory effect of a combination of L-dopa, L-tyrosine, and L-phenylalanine on the amount of nitric oxide produced by microglia. DETAILED DESCRIPTION OF THE INVENTION

[0013] The inhibitor of a disease associated with microglial activation according to the present invention contains two or more aromatic amino acids selected from phenylglycine, phenylalanine, tyrosine, and dihydroxyphenylalanine as active ingredients.

[0014] In the present invention, "suppression" includes not only "treatment" after the onset of the disease, but also the concept of "prevention" which inhibits the onset of the disease itself. In other words, the inhibitor according to the present invention can be said to be a therapeutic agent and / or a prophylactic agent.

[0015] The main role of microglia is to remove dead cells and repair brain tissue as immune cells, but excessive nitric oxide produced by activated microglia is thought to be an important mediator of neuronal damage seen in neurodegenerative processes, leading not only to apoptosis of microglia themselves but also to neuronal cell death. Therefore, if we can suppress the production of nitric oxide by activated microglia, we may be able to suppress diseases caused by nitric oxide and neurodegenerative diseases.

[0016] Phenylglycine is a glycine substituted with a phenyl group, and mainly includes 2-phenylglycine and N-phenylglycine as shown below. 2-phenylglycine has two optical isomers, L-2-phenylglycine and D-2-phenylglycine.

[0017] [ka]

[0018] Phenylalanine and tyrosine are proteinogenic amino acids, each of which has optical isomers called L- and D-isomers.

[0019] Dihydroxyphenylalanine is a phenylalanine with two hydroxyl groups substituted on the phenyl group. Depending on the position of the hydroxyl groups, there are structural isomers such as 3,4-dihydroxyphenylalanine and 3,5-dihydroxyphenylalanine, as well as optical isomers called L- and D-forms.

[0020] Since aromatic amino acids have at least an amino group and a carboxy group as functional groups, they may be in the form of a salt. The salt is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, perchlorate, and phosphate; organic acid salts such as oxalate, malonate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, and trifluoromethanesulfonate; amino acid salts such as glutamate and aspartate; alkali metal salts such as sodium salt and potassium salt; Group 2 metal salts such as calcium salt and magnesium salt; and ammonium salt.

[0021] The inhibitor of the present invention contains two or more aromatic amino acids or salts thereof as active ingredients. The number of aromatic amino acids as active ingredients is not particularly limited as long as it is two or more, but can be, for example, five or less. The number is preferably four or less, and more preferably two or three.

[0022] The ratio of aromatic amino acids as active ingredients is not particularly limited, but for example, the mass ratio of one aromatic amino acid to the other aromatic amino acid may be 0.5 or more and 1.5 or less. This mass ratio may be adjusted to 0.8 or more, 0.9 or more, 1.2 or less, or 1.1 or less. The mass ratio of 2-phenylglycine to dihydroxyphenylalanine may be adjusted to 0.5 or more, 0.8 or more, 0.9 or more, 1.5 or less, 1.2 or less, or 1.1 or less. The mass ratio of N-phenylglycine or phenylalanine to dihydroxyphenylalanine may be adjusted to 0.1 or more, 0.2 or more, 0.4 or more, 1.5 or less, 1 or less, or 0.8 or less. The mass ratio of tyrosine to dihydroxyphenylalanine may be adjusted to 1 or more, 1.2 or more, 1.5 or more, 1.8 or more, 5 or less, 4 or less, or 2.5 or less. The mass ratio of tyrosine to phenylalanine may be adjusted to 1 or more, 2 or more, 2.5 or more, 5 or less, 4 or less, or 3.5 or less.

[0023] The dosage form of the inhibitor of the present invention is not particularly limited, and may be, for example, oral preparations such as tablets, capsules, liquids, granules, powders, and syrups; inhalants such as aerosols; injections; and suppositories. Oral preparations such as oral preparations are preferred. The inhibitor of the present invention may be formulated with common additives depending on the dosage form. For example, excipients, disintegrants, bases, colorants, lubricants, flavoring agents, emulsifiers, thickeners, humectants, stabilizers, preservatives, solvents, solubilizers, suspending agents, surfactants, antioxidants, adjuvants, buffers, pH adjusters, sweeteners, and flavors may be added. The amounts of these additives may be appropriately determined as needed, as long as they do not interfere with the effects of the present invention. Furthermore, other medicinal ingredients may also be added.

[0024] As the raw material for the inhibitor according to the present invention, for example, commercially available aromatic amino acids may be used, or an extract containing aromatic amino acids may be used.

[0025] Microglia, a type of glial cell present in the brain and spinal cord, secrete inflammatory cytokines and may produce excessive amounts of nitric oxide upon activation. These inflammatory cytokines are known to cause neuroinflammation, and nitric oxide induces neuronal cell death. Therefore, diseases associated with microglial activation include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis. Other diseases associated with microglial activation include depression, bipolar disorder, autism, myocardial infarction, malocclusion, alcohol use, sleep disorders, obesity, hyperglycemia, hypertension, and stroke.

[0026] The inhibitor of the present invention is considered to be safe because the active ingredient is an amino acid or a derivative thereof, and may be administered regularly, for example, every day, every other day, or once every three days, or may be administered multiple times a day. Furthermore, it may be administered prophylactically before symptoms become apparent.

[0027] The aromatic amino acid or a salt thereof according to the present invention can suppress microglial activation, particularly reducing the amount of nitric oxide produced by activated microglia. Therefore, the inhibitor according to the present invention can reduce the possibility of developing diseases associated with microglial activation, particularly diseases caused by neuroinflammation or excessive nitric oxide, and can also alleviate the symptoms.

[0028] The method for suppressing a disease associated with microglial activation according to the present invention includes administering to a patient two or more aromatic amino acids selected from phenylglycine, phenylalanine, tyrosine, and dihydroxyphenylalanine or salts thereof as active ingredients. The amount of the aromatic amino acid or salt thereof used should be adjusted appropriately depending on the patient's condition, age, sex, etc., and is not particularly limited. For example, the dosage of the aromatic amino acid or salt thereof may be adjusted appropriately within a range in which the aforementioned effects are observed. For example, the daily dosage of the amino acid or salt thereof may be adjusted to approximately 0.5 mg to 5000 mg. The daily dosage of the amino acid or salt thereof per kg of patient body weight may be adjusted to approximately 0.005 mg to 100 mg. Furthermore, the number of daily administrations may be adjusted to approximately one to five times. Note that the aforementioned patients are not limited to those exhibiting symptoms or characteristics of a disease associated with microglial activation, but also include those in whom the disease should be prevented, even though they do not exhibit the symptoms or characteristics of the disease.

[0029] The inhibitor of the present invention can be administered not only to humans but also to animals other than humans, including, for example, livestock such as horses, cows, pigs, sheep, goats, camels, and llamas; sports animals such as racehorses; pets such as dogs and cats; laboratory animals such as mice, rats, guinea pigs, and rabbits; and poultry such as chickens, ducks, turkeys, and ostriches. [Example]

[0030] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the invention by making appropriate modifications within the scope of the above and below-described aims, and all such modifications are included in the technical scope of the present invention.

[0031] Example 1 BV-2 cells, a mouse microglia-derived cell line, were cultured in Dulbecco's modified Eagle's medium (DMEM, Fujifilm Wako Pure Chemical Industries, Ltd.) at a density of 1.0 × 10 4 BV-2 cells were seeded at a ratio of 0.175 cells / mL and pre-cultured at 37°C for 24 hours. Subsequently, L-DOPA, D-2-phenylglycine, L-2-phenylglycine, or N-phenylglycine, or L-DOPA plus D-2-phenylglycine, L-2-phenylglycine, or N-phenylglycine, was added to the medium and incubated at 37°C for 3 hours. The concentration of each test compound in the medium was 200 μg / mL, but the concentration of N-phenylglycine was reduced to 100 μg / mL to more reliably assess the synergistic effect of L-DOPA and N-phenylglycine alone. Furthermore, BV-2 cells were stimulated with 0.5 μg / mL lipopolysaccharide (LPS) for 24 hours. Separately, an aqueous solution containing 1% sulfanilamide by mass, 0.1% N-1-naphthylethylenediamine by mass, and 2.5% phosphoric acid by mass was prepared as Griess reagent. After 24 hours, the culture supernatant was collected, and the amount of nitric oxide (NO), an indicator of neuroinflammation, was measured by the Griess method using the Griess reagent. For comparison, the amount of NO produced was measured in the same manner except that the test compound was not added. Measurements were performed four times for each group, and the average and standard deviation were calculated. The results are shown in Figure 1. In Figure 1, "Phg" represents phenylglycine, and "**" indicates a significant difference of p<0.01 based on one-way analysis of variance followed by Tukey's test.

[0032] As shown in Figure 1, when L-dopa was used in combination with D-2-phenylglycine, L-2-phenylglycine, or N-phenylglycine, NO production was significantly suppressed compared to when L-dopa, D-2-phenylglycine, L-2-phenylglycine, or N-phenylglycine was used alone. Since nitric oxide produced by activated microglia is thought to damage nerve cells, it has been demonstrated that the combined use of L-dopa and each phenylglycine can synergistically suppress diseases associated with microglial activation.

[0033] Example 2 The NO production was measured in the same manner as in Example 1, except that the test compound was changed to L-phenylalanine, L-dopa, L-tyrosine, or a combination of these two. The concentrations of each aromatic amino acid were determined in preliminary experiments to be concentrations at which NO production was suppressed by approximately 30% when used alone: ​​L-phenylalanine at 125 μg / mL, L-dopa at 200 μg / mL, and L-tyrosine at 400 μg / mL. The results are shown in Figure 2. In Figure 2, "Phe" represents phenylalanine, "Tyr" represents tyrosine, and "**" indicates a significant difference of p<0.01 by one-way analysis of variance followed by Tukey's test.

[0034] As shown in Figure 2, even when two of L-phenylalanine, L-dopa, and L-tyrosine were selected and used in combination, NO production was significantly suppressed, demonstrating that diseases associated with microglial activation can be synergistically suppressed.

[0035] Example 3 The NO production was measured in the same manner as in Example 1, except that the test compound was changed to L-dopa, L-tyrosine, L-phenylalanine, or a combination of these three compounds. The L-dopa concentration was 200 μg / mL, the L-tyrosine concentration was 400 μg, and the L-phenylalanine concentration was 125 μg / mL. The results are shown in Figure 3. In Figure 3, "Phe" represents phenylalanine, "Tyr" represents tyrosine, and "**" indicates a significant difference of p<0.01 by one-way analysis of variance followed by Tukey's test.

[0036] As shown in Figure 3, when L-dopa, L-tyrosine, and L-phenylalanine were used in combination, NO production was significantly suppressed, demonstrating that diseases associated with microglial activation can be synergistically suppressed.

Claims

1. An agent for suppressing diseases associated with microglial activation, characterized by containing, as active ingredients, two or more aromatic amino acids selected from phenylglycine, phenylalanine, tyrosine, and dihydroxyphenylalanine, or salts thereof.

2. 2. The inhibitor of claim 1, wherein the phenylglycine is 2-phenylglycine or N-phenylglycine.

3. 2. The inhibitor of claim 1, wherein the dihydroxyphenylalanine is 3,4-dihydroxyphenylalanine.

4. 2. The inhibitor according to claim 1, comprising the dihydroxyphenylalanine and the phenylglycine as active ingredients.

5. 2. The inhibitor according to claim 1, comprising two or more aromatic amino acids selected from the group consisting of phenylalanine, tyrosine, and dihydroxyphenylalanine as active ingredients.

6. 2. The inhibitor according to claim 1, comprising the phenylalanine, the tyrosine, and the dihydroxyphenylalanine as active ingredients.

7. The inhibitor according to claim 1, which inhibits the production of nitric oxide by microglia.

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