Promoter of polar transformation toward anti-inflammatory M2 phenotype microglia

Abstract

The present invention provides a polarity conversion promoter to anti-inflammatory M2 phenotype microglia. The polarity conversion promoter to anti-inflammatory M2 phenotype microglia comprises a cyclic peptide derivative represented by formula (1). In the formula, m=0-3, n≥1, R1-R6 are hydrogen atoms or hydrocarbon groups, R7 and R8 are carboxyl groups or salts thereof or alkoxycarbonyl groups, R9 is a hydrocarbon group, a hydroxyl group, an alkoxy group or an alkylcarbonyloxy group, and R 10 and R 11 is a hydrogen atom, a hydrocarbon group or an alkylcarbonyloxy group, R 12 ~R 16 It is a hydrogen atom or a hydrocarbon group. [Chemical formula 1] #imgabs0#

Description

Technical Field

[0001] The present invention relates to a polarity conversion promoter capable of promoting polarity conversion of microglia to anti-inflammatory M2 phenotype. Background Art

[0002] Microglia, a type of glial cell, can be considered to be cells derived from the mesoderm and differentiated from the yolk sac. Microglia are known to have the role of immune cells that regulate the environment inside and outside the brain, such as removing damaged cells and maintaining and inspecting synapses. For example, in the case of protecting the brain from traumatic brain injury, microglia are activated and release inflammatory cytokines, thereby changing astrocytes into reactive astrocytes and playing a role in protecting the brain. In addition, activated microglia are known to induce encephalitis and promote neurodegenerative diseases.

[0003] On the other hand, methods for treating neurodegenerative diseases targeting microglia have been disclosed, for example, methods using a gap junction blocker as a glycyrrhetinic acid derivative (Non-Patent Document 1, Patent Document 1) and methods using iso-α acids (Non-Patent Document 2) have been proposed.

[0004] It is known that there are two types of microglial activation (Non-patent Document 3). Figure 1 As shown, there are pro-inflammatory M1 phenotype microglia and anti-inflammatory M2 phenotype microglia. Pro-inflammatory M1 phenotype microglia produce interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS), etc. IL-1β is an effector, and in the presence of IL-1β, microglia cause inflammation in the brain and release free radicals such as free radicals such as reactive oxygen species. In addition, in microglia that produce IL-1β, the ability to phagocytize metabolic waste such as aging (destroyed) cells is reduced. Therefore, pro-inflammatory M1 phenotype microglia damage nerve cells. Anti-inflammatory M2 phenotype microglia produce arginase-1 (ARG1), interleukin-10 (IL-10), interleukin-4 (IL-4), transforming growth factor (TGF)-β1, etc. ARG1 is an effector, and acts to protect nerve cells by releasing ARG1. Anti-inflammatory M2 phenotype microglia have a high ability to phagocytize metabolic waste such as aging cells while inhibiting inflammation in the brain. Therefore, they have an anti-inflammatory effect, that is, a protective effect on nerve cells.

[0005] However, the present inventors provided a novel cyclic peptide derivative in Patent Document 2. Although it is known that the cyclic peptide derivative is extracted from Paecilomyces tenuipes, a wintergrass, and exhibits proliferative activity on astrocytes, it is not known that it can act on microglia and promote polar transformation to anti-inflammatory M2 phenotype microglia.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2007 / 088712

[0009] Patent Document 2: International Publication No. 2016 / 047638

[0010] Non-patent literature

[0011] Non-patent document 1: Takeuchi, H., Mizoguchi, H., Doi, Y., Jin, S., Noda, M., Liang, J., Li, H., Zhou, Y., Mori, R., Yasuka, S., Li, E., Parajuli, B., Kawanokuchi, J., Sonobe, Y., Sato, J., Yamanaka, K. and Sobue G. (2011) Blockade of gap junction hemichannel suppresses disease progression in mouse models of sclerosis and Alzheimer's disease. Pros One, 6 (6), e21108.

[0012] Non-patent document 2: Ano, Y., Dohata, A., Taniguchi, Y., Hoshi, A., Uchida, K., Takashima, A. and Nakayama, H. (2017) Iso-α-acids, bitter components of beer, prevent inflammation and cognitive decline induced in a mouse model of Alzheimer's disease. Journal of Biological Chemistry, 292, 3720-3728.

[0013] Non-patent document 3: Ni J, Wu Z, Peters C, Yamamoto K, Qing H, Na kanishi H. (2015): The Critical Role of Proteolytic Relay through Cat hepsins Band E in the Phenotypic Change of Microglia / Macrophage. J Neurosci. 35(36): 12488-501.

[0014] Non-patent document 4: Wu Z, Sun L, Hashioka S, Yu S, Schwab C, Okada R, Hayashi Y, McGeer PL, Nakanishi H. (2013) Differential pathways for interleukin-1β production activated by chromogranin A and amyloid β in microglia. Neurobiology Aging. 34(12): 2715-25.

[0015] Non-patent literature 5: 1582087454977_0.fcgi? cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8864135&query_hl=131&itool=pubmed_docsu m(1996) Regulation of microglialactivation by TGF-β, IL-10, and C SF-1. J. Leukoc Biol. 60, 502-508. Summary of the invention

[0016] Technical problem that the invention aims to solve

[0017] The object of the present invention is to provide a novel polarity conversion promoter to anti-inflammatory M2 phenotype microglia that has a protective effect on nerve cells.

[0018] Technical solutions to the problem

[0019] The polarity conversion promoter for the transformation into anti-inflammatory M2 phenotype microglia according to an embodiment of the present invention comprises a peptide derivative represented by the following general formula (1).

[0020] [Chemical formula 1]

[0021]

[0022] In the general formula (1), m is 0 to 3, n ≥ 1, R1 to R6 are each independently a hydrogen atom or a hydrocarbon group, R7 and R8 are each independently a carboxyl group or a salt thereof or an alkoxycarbonyl group, R9 is a hydrocarbon group, a hydroxyl group, an alkoxy group or an alkylcarbonyloxy group, and R 10 and R 11 are independently a hydrogen atom, a hydrocarbon group or an alkylcarbonyloxy group, R 12 ~R 16 Each independently represents a hydrogen atom or a hydrocarbon group.

[0023] Effects of the Invention

[0024] According to an embodiment of the present invention, a polarity conversion promoter for converting microglia into anti-inflammatory M2 phenotype microglia that has a protective effect on nerve cells can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram for explaining the activation mechanism of microglia.

[0026] Figure 2 This is a graph showing the relationship between the concentration of the cyclic peptide derivative and the cell survival rate in MG6 microglial cells.

[0027] Figure 3 It is a graph showing the results of the polarization test of MG6 microglia in the Examples, and is a graph showing the gene expression levels of IL-1β and ARG1 produced by MG6 microglia treated with CGA alone and MG6 microglia treated with a cyclic peptide derivative in the presence of CGA.

[0028] Figure 4 This is a graph showing the results of safety tests on cyclic peptide derivatives.

[0029] Figure 5 This is a graph showing the relationship between the concentration of the cyclic peptide derivative and the cell survival rate in primary microglial cells.

[0030] Figure 6 : is a graph showing the results of the polarization test of primary microglia. : is a graph showing the gene expression levels of cytokines produced by primary microglia obtained by the unstimulated control group, the cyclic peptide derivative alone treatment, the CGA alone treatment, and the combination of CGA and the cyclic peptide derivative, respectively. (a) represents the gene expression level of TGF-1β after 24 hours of treatment, (b) represents the gene expression level of TGF-1β after 72 hours of treatment, (c) represents the gene expression level of IL-1β after 24 hours of treatment, and (d) represents the gene expression level of IL-1β after 72 hours of treatment. In addition, IL-1β is a neuronal cell injury (Japanese: nervus necrosis), and TGF-β1 is a neuronal cell protection. DETAILED DESCRIPTION

[0031] The polarity conversion promoter for conversion to anti-inflammatory M2 phenotype microglia (hereinafter, also simply referred to as "polarity conversion promoter") according to the present embodiment contains a cyclic peptide derivative represented by the following general formula (1) as an active ingredient.

[0032] [Chemical formula 2]

[0033]

[0034] In the formula, m is an integer of 0 to 3, and n is an integer greater than 1. R1 to R6 are each independently a hydrogen atom or a hydrocarbon group. R7 and R8 are each independently a carboxyl group or a salt thereof or an alkoxycarbonyl group. R9 is a hydrocarbon group, a hydroxyl group, an alkoxy group or an alkylcarbonyloxy group. 10 and R 11 R are independently a hydrogen atom, a hydrocarbon group or an alkylcarbonyloxy group. 12 ~R 16 Each independently represents a hydrogen atom or a hydrocarbon group.

[0035] Here, as the hydrocarbon group, for example, there can be mentioned: an aliphatic hydrocarbon group, i.e., a linear or branched saturated or unsaturated hydrocarbon group, or an alicyclic hydrocarbon group. The number of carbon atoms in the hydrocarbon group is not particularly limited, but preferably the number of carbon atoms is 1 to 6, and more preferably the number of carbon atoms is 1 to 4. The same applies to the hydrocarbon part in the alkoxy group, alkoxycarbonyl group, and alkylcarbonyloxy group. As a preferred example, the hydrocarbon group and the hydrocarbon part are alkyl groups having 1 to 4 carbon atoms, respectively.

[0036] As for R7 and R8, examples of the salt of the carboxyl group include metal salts such as alkali metal salts and alkaline earth metal salts, ammonium salts such as ammonium acetate salts, and amine salts such as diethylamine salts.

[0037] In formula (1), for example, R1, R2, R3 and R4 may be independently an alkyl group, n=2 to 4, R5 and R6 may be a hydrogen atom, and R7 and R8 may be independently a carboxyl group or a salt thereof.

[0038] In formula (1), for example, the following may be mentioned: R1, R2, R3 and R4 are each independently an alkyl group, particularly preferably a methyl group or an ethyl group, n=2 to 4, R5 and R6 are each a hydrogen atom, R7 and R8 are each independently a carboxyl group or a salt thereof, m=0, R 10 and R 11 are hydrogen atoms, R 12 and R 13 are hydrogen atoms, R 14 and R 15 are each independently a hydrogen atom or an alkyl group (particularly preferably a methyl group), R 16 A hydrogen atom.

[0039] In one embodiment, the cyclic peptide derivative represented by formula (1) may be a compound represented by the following formula (2) or a salt thereof.

[0040] [Chemical formula 3]

[0041]

[0042] The compound represented by formula (2) is a compound in which a peptide containing four amino acids, namely, N-methyl-β-hydroxydopa, valine, β-hydroxyleucine, and glutamic acid, is in a cyclic structure.

[0043] The method for producing the cyclic peptide derivative represented by formula (1) is not particularly limited. For example, as described in Patent Document 2 (WO2016 / 047638), it can be collected from Hanasana Kitake using various extraction and separation methods, or it can be produced by combining various known chemical synthesis methods such as peptide synthesis.

[0044] The cyclic peptide derivative shown in formula (1) has the effect of promoting the polarity conversion of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and can therefore be used as a polarity conversion promoter to the anti-inflammatory M2 phenotype microglia. That is, by causing the cyclic peptide derivative shown in formula (1) to act on microglia, which is a type of glial cell, the polarity conversion (polarization) of microglia can be made to be anti-inflammatory M2 phenotype, and by becoming M2 phenotype, microglia can be made to protect nerve cells. Therefore, the polarity conversion promoter involved in the present embodiment can be used in medicines or foods for treating and preventing nerve cell damage caused by brain diseases or aging.

[0045] In one embodiment, the polarity conversion promoter can be incorporated as an active ingredient in a pharmaceutical composition or a food composition, and a pharmaceutical composition or a food composition for promoting polarity conversion to anti-inflammatory M2 phenotype microglia can also be provided. These pharmaceutical compositions or food compositions can be used, for example, for humans or mammals other than humans (experimental animals, pets, etc.).

[0046] The pharmaceutical composition can be prepared by mixing the above-mentioned polarity conversion promoter with various additives or other ingredients permitted as pharmaceuticals. The pharmaceutical composition can be administered orally or parenterally. As its form, if it is for oral administration, for example, tablets, pills, powders, granules, capsules, liquid preparations (including elixirs, syrups, suspensions, solutions), etc., if it is for parenteral administration, for example, injections, drips, etc., can also be directly administered into the brain.

[0047] The food composition can be manufactured by mixing the above-mentioned polarity conversion promoter with various additives or other ingredients permitted as food. As food compositions, health functional foods (nutritional functional foods, specific health foods and functional foods), dietary supplements, etc. can be mentioned. As examples of the form of food compositions, the same form as the pharmaceutical composition for oral administration can be mentioned, and it can also be in the form of beverages, snacks, etc.

[0048] Examples of additives that can be incorporated into pharmaceutical compositions or food compositions include excipients, antioxidants, spices, seasonings, sweeteners, colorants, thickeners and stabilizers, coloring agents, bleaching agents, gum bases, emulsifiers, binders, diluents, preservatives, stabilizers, coagulants, and the like.

[0049] The amount of the active ingredient per administration or per day of the pharmaceutical composition and the amount of the active ingredient per meal or per day of the food composition can be appropriately set according to the age, weight, sex, disease or condition of the subject of administration or ingestion, or based on non-clinical or clinical test results, etc. Although not particularly limited, the oral intake of the above-mentioned polar conversion promoter, in terms of the amount of the cyclic peptide derivative of formula (1), for example, for mammals including humans, can be 0.1 μg / kg or more and 50 μg / kg or less, or 1 μg / kg or more and 25 μg / kg or less per day.

[0050] In addition, the polarity conversion promoter can obtain an effect even at a low concentration in an in vitro experimental system. The concentration of the polarity conversion promoter in the in vitro experimental system is not particularly limited, and can be, for example, 0.01 μM or more and 5 μM or less (i.e., 1×10 -8 ~5×10 -6 mol / L), may be 0.01 μM or more and 1 μM or less, or may be 0.03 μM or more and 0.3 μM or less.

[0051] Example

[0052] 1. Cell Culture of MG6 Microglia

[0053] In the following experiments using the cell line of MG6 microglia (Riken Cell Bank), MG6 microglia were cultured using Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific). DMEM contains 100 μM β-mercaptoethanol, 10 μg / ml insulin, 10% fetal bovine serum (Gibco), penicillin-streptomycin (Gibco), and 450 mg / ml glucose (Gibco).

[0054] 2. Determination of cell viability of MG6 microglia

[0055] MG6 microglia were cultured in 96-well plates at 37°C for 24 h (5 × 10 3Cells / well), and then cultured at 37°C for 48 hours together with cyclic peptide derivatives at various concentrations. As the cyclic peptide derivative, the diethylamine salt of the compound represented by the above formula (2) obtained by the method described in the examples of Patent Document 2 (the same in the following respective tests) was used. As the concentration of the cyclic peptide derivative, the concentration in the culture solution was adjusted to 0 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, and 1 μM.

[0056] The evaluation of cell viability was carried out as follows according to the instructions of the manufacturer using Cell-Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). That is, after treatment with the cyclic peptide derivative, 10 μL of CCK-8 was transferred to a 96-well plate, and then cultured at 37°C for 2 hours. According to this instruction, the optical density was measured at a wavelength of 450 nm using a microplate reader. The cell viability was calculated using the following formula. Cell viability = (optical density of the experimental group / optical density of the control group) × 100%

[0057] After administration of the cyclic peptide derivative for 48 hours, the viability of microglial cells was investigated. The results were as Figure 2 shown. No proliferation of microglial cells was confirmed in the range of 0.01 to 1 μM of the concentration of the cyclic peptide derivative, and their survival was not inhibited. Therefore, in subsequent experiments, the optimal concentration of the cyclic peptide derivative was set to 1 μM and used for functional analysis. In addition, Figure 2 "ns" in

[0058] 3. Polarization test of MG6 microglial cells

[0059] The neurotoxicity of chromogranin A (CGA), which is a component of senile plaques in the brain of Alzheimer's dementia, is stronger than that of amyloid-β. In addition, a large number of microglial cells were confirmed around the senile plaques accumulating CGA, and it is considered that the reason is the activation of microglial cells (Non-Patent Document 4). Therefore, as conditions close to the brain environment, a polarization test of microglial cells based on cyclic peptide derivatives was carried out under pathological conditions in the presence of CGA.

[0060] MG6 microglial cells were cultured at 37°C for 24 hours in a 96-well plate (5×10 3Then, CGA (Peptide Institute, Osaka) was added to a concentration of 10 nM and the cyclic peptide derivative was added to a concentration of 1 μM, and cultured at 37° C. The cells to which CGA was added without adding the cyclic peptide derivative and the cells to which neither the cyclic peptide derivative nor CGA was added (control group) were cultured in the same manner.

[0061] After 24 hours and 72 hours of the added treatment, real-time quantitative PCR (RT-PCR) analysis and statistical analysis were performed to determine the gene expression levels of interleukin-1β (IL-1β) and arginase-1 (ARG1). It is known that stimulated microglia polarize into an M1 phenotype that damages nerve cells and an M2 phenotype that protects nerve cells according to the factors produced. M1 phenotype microglia produce IL-1β, and M2 phenotype microglia produce ARG1 (non-patent literature 3). Therefore, the M1 phenotype is determined based on the gene expression of IL-1β, and the M2 phenotype is determined based on the gene expression of ARG1. The methods of real-time quantitative PCR (RT-PCR) analysis and statistical analysis are described as follows.

[0062] [Real-time quantitative PCR (RT-PCR) analysis]

[0063] mRNA was isolated from the cell line of MG6 microglia treated with cyclic peptide derivatives, and total RNA was extracted using RNAisoPlus (Takara, Japan) according to the manufacturer's instructions. A total of 800 ng of the extracted RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Japan). After the initial denaturation step at 95°C for 5 minutes, the temperature cycle was started. Each cycle included: denaturation at 95°C for 5 seconds, annealing at 60°C for 10 seconds, and extension for 30 seconds. A total of 40 cycles were performed. cDNA was amplified for 2 rounds using the Rotor-Gene SYBR Green RT-PCR Kit (Qiagen, Japan) using the Corbett Rotor-Gene RG-3000AReal-Time PCR System. The data was evaluated using the RG-3000A software program (version Rotor-Gene 6.1.93, Corbett). The sequence of the primer pair (Invitrogen, Custom primer) is recorded below.

[0064] IL-1β: 5′-CAACCAACAAGTGATATTCTCCATG-3′ (SEQ ID NO: 1), and 5′-GATCCACACTCTCCAGCTGCA-3′ (SEQ ID NO: 2);

[0065] ARG: 5′-CGCCTTTCTCAAAAGGACAG-3′ (SEQ ID NO: 3), and 5′-CCAGCTCTTCATTGGCTTTC-3′ (SEQ ID NO: 4)

[0066] For normalization of data, an endogenous control (actin) was evaluated to control for cDNA input and relative units were calculated according to the comparative Ct method. All real-time RT-PCR experiments were repeated three times and the results are expressed as mean ± SEM.

[0067] [Statistical Analysis]

[0068] Data are expressed as mean ± SEM. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) using post hoc Tukey's test with GraphPad Prism software package. Values ​​of p < 0.05 were considered statistically significant (GraphPad software).

[0069] The results are shown in the form of relative expression levels of mRNA relative to the control group (without addition of cyclic peptide derivative and CGA) for the case where CGA and cyclic peptide derivative were added (Example) and the case where only CGA was added (Comparative Example). Figure 3 .

[0070] When treated with CGA and cyclic peptide derivatives together, although the gene expression of IL-1β was about 5 times higher than that of ARG1 24 hours after the addition treatment, and the microglia were polarized to the pro-inflammatory M1 phenotype, it was reversed after 72 hours, and the gene expression of ARG1 was about 9 times higher than that of IL-1β. This result shows that as the polarization of microglia, polarization to the anti-inflammatory M2 phenotype through long-term treatment with cyclic peptide derivatives has a protective effect on nerve cells. In addition, it can be considered that by temporarily polarizing to the pro-inflammatory M1 phenotype, the activity of microglia can be increased in a short time, and polarization to the anti-inflammatory M2 phenotype can be efficiently and quickly achieved.

[0071] In addition, compared with the case of treatment with CGA alone, when CGA and cyclic peptide derivatives were used together, although the gene expression level of IL-1β was as high as about 4 times after 24 hours of addition treatment, and the activation effect of the pro-inflammatory M1 phenotype was high, after 72 hours of addition treatment, the activity of the pro-inflammatory M1 phenotype was reduced to a level that was not significantly different from the case of treatment with CGA alone. On the other hand, for the gene expression level of ARG1, when CGA and cyclic peptide derivatives were used together, compared with the case of treatment with CGA alone, the activation effect was as high as about 4 times after 24 hours of addition treatment, and the activation effect was more than 2 times after 72 hours of addition treatment. It can be seen from this that in an environment close to the brain environment where CGA exists (i.e., under pathological conditions where microglia are activated), the polar transformation to the anti-inflammatory M2 phenotype microglia that has a protective effect on nerve cells is promoted by long-term treatment with cyclic peptide derivatives.

[0072] Microglia are cells in the first line of brain immunity that control the brain environment and encephalitis. M2 phenotype microglia have the ability to collect dead neurons in the brain or metabolic wastes such as amyloid proteins accumulated in the brain and discharge them outside the brain. Furthermore, during encephalitis, M2 phenotype microglia quickly end encephalitis and protect neurons. Therefore, it can be considered that cyclic peptide derivatives promote the polar transformation of microglia to anti-inflammatory M2 phenotypes, thereby delaying brain aging, and contributing to the prevention and morbid recovery of acute / chronic encephalitis-related diseases such as traumatic brain injury, cerebral stroke, and dementia. Furthermore, it can be considered to contribute to the prevention and morbid recovery of mental illnesses including autism and depression.

[0073] 4. Safety testing of cyclic peptide derivatives

[0074] MG6 microglial cells were cultured in 96-well plates at 37°C for 24 h (5 × 10 3 cells / well), then the cyclic peptide derivative was added to a concentration of 1 μM and cultured at 37° C. 24 hours and 72 hours after the addition treatment, the same real-time quantitative PCR (RT-PCR) analysis and statistical analysis as above were performed to determine the gene expression levels of interleukin-1β (IL-1β) and arginase-1 (ARG1).

[0075] The results are as follows Figure 4 As shown in the results, when the cyclic peptide derivative was administered alone, microglia were not activated after 24 hours and 72 hours, and were neither polarized to the pro-inflammatory M1 phenotype nor to the anti-inflammatory M2 phenotype. This indicates that the cyclic peptide derivative is not recognized as a foreign substance by microglia and does not activate microglia under non-pathological conditions, thus confirming the safety of the cyclic peptide derivative for microglia.

[0076] 5. Primary Microglial Cell Culture

[0077] CD11b primary microglia were isolated from the brain of adult mice (8 weeks old, male, Japan SLC, Inc. Hamamatsu, Japan) using the magnetic cell sorting (MACS) method. + Cells. In detail, the brain was cut into small pieces, and the obtained cell suspension was mechanically dissociated using an enzyme digestion method using a neural tissue separation kit (Miltenyi Biotec) and a Gentle MACS Dissociator (fully automatic tissue single cell separator, Miltenyi Biotec), and further transferred to a 30 mm cell sieve to obtain a single cell suspension. After magnetic labeling with CD11b microbeads (Miltenyi Biotec), the cell suspension was injected into a magnetic (MACS) column provided in a magnetic separator (Miltenyi Biotec). After rinsing the MACS column with phosphate-buffered saline (PBS), CD11b positive components were collected according to the method described in non-patent document 4. In the following experiments using primary microglia, primary microglia were cultured using Eagle MEM (Nissui Pharmaceutical Co., Ltd.) culture medium.

[0078] 6. Determination of the survival rate of primary microglia

[0079] Primary microglial cells were seeded in 96-well plates (10 4 Cells / well) were cultured at 37°C for 24 hours and 72 hours with various concentrations of cyclic peptide derivatives. The concentration of the cyclic peptide derivative was adjusted to 0 μM, 0.01 μM, 0.1 μM, 1 μM, and 5 μM in the culture medium. The evaluation of cell survival rate (24 hours and 72 hours) was as described in the determination of MG6 microglial cell survival rate in "2." above.

[0080] The results are as follows Figure 5 As shown, in the range of 0.01 to 5 μM concentration of the cyclic peptide derivative, no proliferation of primary microglial cells was observed, and there was almost no inhibition of survival. In addition, “*” indicates P<0.05.

[0081] 7. Polarization assay of primary microglia

[0082] Microglia, the resident immune cells of the brain, regulate pro-inflammatory cytokines and anti-inflammatory cytokines, thereby playing an important role in neuroinflammation of neurodegenerative diseases including Alzheimer's disease. Interleukin-1β (IL-1β) is known to be the main inflammatory cytokine, and transforming growth factor (TGF)-β1 is an important anti-inflammatory cytokine (non-patent literature 5). As mentioned above, chromogranin A (CGA), which is a neurosecretory acidic glycoprotein, is found in senile plaques of Alzheimer's disease as a candidate factor for the activation of microglia. Therefore, IL-1β and TGF-β1 are used as cells and markers of primary microglia to study the effects of cyclic peptide derivatives and CGA on inflammation or anti-inflammatory.

[0083] Primary microglia were cultured in 96-well plates at 37°C for 24 h (10 4 Cells / well), then CGA (American Peptide Company, Anaspec) was added to a concentration of 10 nM and the cyclic peptide derivative was added to a concentration of 1 μM, and cultured at 37° C. The cells without adding the cyclic peptide derivative but adding CGA, the cells without adding CGA but adding the cyclic peptide derivative, and the cells without adding the cyclic peptide derivative and CGA (control group) were cultured in the same manner.

[0084] Real-time quantitative PCR (RT-PCR) analysis and statistical analysis were performed 24 hours and 72 hours after the addition treatment to determine the gene expression levels of TGF-β1 and IL-1β. The methods of real-time quantitative PCR (RT-PCR) analysis and statistical analysis are as described in the polarization test of MG6 microglia in "3." above. Among them, the primer pair of TGF-β1 (Invitrogen, Customprimer) is the following sequence.

[0085] TGF-β1: 5′-TCAGACATTCGGGAAGCAGTG-3′ (SEQ ID NO: 5), and 5′-ATTCCGTCTCCTTGGTTCAGC-3′ (SEQ ID NO: 6)

[0086] The results are as follows Figure 6 In addition, Figure 6 "*" in the table indicates P<0.05, "##" indicates P<0.01, and "###" indicates P<0.001. In addition, "con" indicates a control group, "peptide" indicates treatment with a cyclic peptide derivative alone, "CGA" indicates treatment with CGA alone, and "peptide+CGA" indicates treatment with a cyclic peptide derivative and CGA in combination.

[0087] like Figure 6As shown in (a) and (b), in primary microglial cells, when treated with CGA alone, the expression of TGF-β1 increased somewhat at 24 hours and 72 hours compared with the control group. When treated with CGA in combination with the cyclic peptide derivative, the expression of TGF-β1 increased significantly (about 3 times on average) at 24 hours and 72 hours. It should be noted that compared with the control group, the cyclic peptide derivative alone did not affect the expression of TGF-β1.

[0088] On the other hand, for the expression of IL-1β, e.g. Figure 6 As shown in (c) and (d), the expression of IL-1β increased significantly when treated with CGA alone, and decreased to the same level or lower than that of the control when treated with CGA and the cyclic peptide derivative in combination, which had an opposite tendency to the expression of TGF-β1. It should be noted that the cyclic peptide derivative alone did not affect the expression of IL-1β compared with the control group.

[0089] This result shows that the cyclic peptide derivative increases TGF-β1 and inhibits IL-1β, thereby converting microglia activated by CGA (pro-inflammatory phenotype M1) to anti-inflammatory phenotype M2, that is, polarity conversion. Considering that the cyclic peptide derivative itself does not affect the expression of TGF-β1 or IL-1β in primary microglia, the safety of cyclic peptide derivatives for microglia can also be confirmed based on this experiment using primary microglia, and it is understood that cyclic peptide derivatives can be effectively used to improve neurodegenerative diseases.

Claims

1. Use of a compound represented by the following formula (2) or a salt thereof in the preparation of a drug for treating Alzheimer's disease, characterized in that:

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