JP7479623B2 - Agents promoting polarity conversion to anti-inflammatory M2 phenotype microglia - Google Patents

Description

The present invention relates to a polarity conversion promoter that can promote polarity conversion of microglia to the anti-inflammatory M2 phenotype.

Microglia, a type of glial cell, are thought to be cells differentiated from the mesoderm-derived yolk sac. It has been found that microglia act as immune cells that regulate the environment inside and outside the brain, such as removing damaged cells and maintaining synapses. For example, when protecting the brain from traumatic brain injury, microglia become activated and release inflammatory cytokines, which transform astrocytes into reactive astrocytes and act to protect the brain. Activated microglia are also known to induce brain inflammation and promote neurodegenerative diseases.

Meanwhile, methods for treating neurodegenerative diseases that target microglia have been disclosed. For example, a method using a gap junction inhibitor, which is a type of glycyrrhetinic acid derivative (Non-Patent Document 1, Patent Document 1) and a method using iso-α acid (Non-Patent Document 2) have been proposed.

It is known that there are two activation types of microglia (Non-Patent Document 3). That is, as shown in Figure 1, 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 carbon monoxide synthase (iNOS), etc. IL-1β is an effector, and in the presence of IL-1β, microglia cause intracerebral inflammation and release free radicals such as radical active oxygen. In addition, in microglia that produce IL-1β, the ability to phagocytose waste products such as aged (broken) cells is reduced. Therefore, pro-inflammatory M1 phenotype microglia act in a neuronal cell-damaging manner. On the other hand, 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 that protects nerve cells by releasing it. Anti-inflammatory M2 phenotype microglia suppress inflammation in the brain and are highly capable of phagocytosing waste products such as aged cells, and therefore act as an anti-inflammatory, i.e., neuroprotective, agent.

Incidentally, the present inventors have provided a novel cyclic peptide derivative in Patent Document 2. This cyclic peptide derivative is extracted from Cordyceps sinensis, a type of Cordyceps sinensis, and has been shown to have a proliferation activity on astrocytes, but it is not known that it acts on microglia to promote polarity conversion to anti-inflammatory M2 phenotype microglia.

International Publication No. 2007/088712 International Publication No. 2016/047638

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 amyotrophic lateral sclerosis and Alzheimer’s disease. ProsOne, 6(6), e21108. 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. Ni J, Wu Z, Peters C, Yamamoto K, Qing H,Nakanishi H. (2015): The Critical Role of Proteolytic Relay throughCathepsins Band E in the Phenotypic Change of Microglia/Macrophage. J Neurosci. 35(36):12488-501. Wu Z, Sun L, Hashioka S, Yu S, Schwab C,Okada R, Hayashi Y,McGeerPL, Nakanishi H. (2013) Differential pathways for interleukin-1β production activated by chromogranin A and amyloid β in microglia. NeurobiologyAging.34(12):2715-25. 1582087454977_0.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8864135&query_hl=131&itool=pubmed_docsum (1996) Regulation of microglial activation by TGF-β, IL-10, and CSF-1. J. Leukoc Biol. 60, 502-508.

The present invention aims to provide a novel agent for promoting polarity conversion to anti-inflammatory M2 phenotype microglia that acts to protect neuronal cells.

式（１）中、ｍは０～３、ｎ≧１であり、Ｒ_１～Ｒ_６はそれぞれ独立に水素原子または炭化水素基であり、Ｒ_７およびＲ_８はそれぞれ独立にカルボキシ基もしくはその塩、またはアルコキシカルボニル基であり、Ｒ_９は炭化水素基、ヒドロキシ基、アルコキシ基またはアルキルカルボニルオキシ基であり、Ｒ_１０およびＲ_１１はそれぞれ独立に水素原子、炭化水素基またはアルキルカルボニルオキシ基であり、Ｒ_１２～Ｒ_１６はそれぞれ独立に水素原子または炭化水素基である。 The agent for promoting polarity conversion to anti-inflammatory M2 phenotype microglia according to an embodiment of the present invention comprises a cyclic peptide derivative represented by the following general formula (1).

In formula (1), m is 0 to 3 and n is 1 or greater; R ₁ to R ₆ are each independently a hydrogen atom or a hydrocarbon group; R ₇ and R ₈ are each independently a carboxy group or a salt thereof, or an alkoxycarbonyl group; R ₉ is a hydrocarbon group, a hydroxy group, an alkoxy group, or an alkylcarbonyloxy group; R ₁₀ and R ₁₁ are each independently a hydrogen atom, a hydrocarbon group, or an alkylcarbonyloxy group; and R ₁₂ to R ₁₆ are each independently a hydrogen atom or a hydrocarbon group.

According to an embodiment of the present invention, it is possible to provide a polarity conversion promoter for anti-inflammatory M2 phenotype microglia that acts in a neuroprotective manner.

FIG. 1 is a diagram for explaining the activation mechanism of microglia. 1 is a graph showing the relationship between the concentration of a cyclic peptide derivative and the cell viability of MG6 microglia. This is a graph showing the results of a polarization test of MG6 microglia in an example, 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, respectively. 1 is a graph showing the results of a safety test of a cyclic peptide derivative. 1 is a graph showing the relationship between the concentration of cyclic peptide derivatives and cell viability in primary microglia. 1 is a graph showing the results of a polarization test on primary microglia. The graph shows the gene expression levels of cytokines produced by primary microglia in a non-stimulated control, treatment with a cyclic peptide derivative alone, treatment with CGA alone, and treatment with a combination of CGA and a cyclic peptide derivative, where (a) shows the gene expression level of TGF-1β after 24 hours of treatment, (b) shows the gene expression level of TGF-1β after 72 hours of treatment, (c) shows the gene expression level of IL-1β after 24 hours of treatment, and (d) shows the gene expression level of IL-1β after 72 hours of treatment. IL-1β is a neuronal disorder, and TGF-β1 is a neuronal protection.

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

In the formula, m is an integer of 0 to 3, and n is an integer of 1 or more. R ₁ to R ₆ are each independently a hydrogen atom or a hydrocarbon group. R ₇ and R ₈ are each independently a carboxy group or a salt thereof, or an alkoxycarbonyl group. R ₉ is a hydrocarbon group, a hydroxy group, an alkoxy group, or an alkylcarbonyloxy group. R ₁₀ and R ₁₁ are each independently a hydrogen atom, a hydrocarbon group, or an alkylcarbonyloxy group. R ₁₂ to R ₁₆ are each independently a hydrogen atom or a hydrocarbon group.

Here, examples of the hydrocarbon group include aliphatic hydrocarbon groups, that is, linear or branched, saturated or unsaturated hydrocarbon groups, or alicyclic hydrocarbon groups. The number of carbon atoms in the hydrocarbon group is not particularly limited, but is preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. The same applies to the hydrocarbon moieties in alkoxy groups, alkoxycarbonyl groups, and alkylcarbonyloxy groups. As a preferred example, the hydrocarbon group and the hydrocarbon moiety are each an alkyl group having 1 to 4 carbon atoms.

With respect to _R7 and _R8 , examples of the salt of the carboxy 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.

In formula (1), for example, R ₁ , R ₂ , R ₃ and R ₄ each independently represent an alkyl group, n is 2 to 4, R ₅ and R ₆ each independently represent a hydrogen atom, and R ₇ and R ₈ each independently represent a carboxy group or a salt thereof.

In formula (1), for example, R ₁ , R ₂ , R ₃ and R ₄ are each independently an alkyl group, particularly preferably a methyl group or an ethyl group, n=2 to 4, R ₅ and R ₆ are each independently a hydrogen atom, R ₇ and R ₈ are each independently a carboxy group or a salt thereof, m=0, R ₁₀ and R ₁₁ are each independently a hydrogen atom, R ₁₂ and R ₁₃ are each independently a hydrogen atom, R ₁₄ and R ₁₅ are each independently a hydrogen atom or an alkyl group (particularly preferably a methyl group), and R ₁₆ is a hydrogen atom.

この式（２）で表される化合物は、Ｎ－メチル－β－ヒドロキシドーパ、バリン、β－ヒドロキシロイシン、グルタミン酸の４種のアミノ酸からなるペプチドが環状構造をとったものである。 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.

The compound represented by formula (2) is a peptide having a cyclic structure, which is composed of four amino acids, N-methyl-β-hydroxydopa, valine, β-hydroxyleucine, and glutamic acid.

The method for producing the cyclic peptide derivative represented by formula (1) is not particularly limited, and for example, as described in Patent Document 2 (WO2016/047638), it may be extracted from Polyporus arbutus using various extraction and separation methods, or it may be produced by combining various known chemical synthesis methods such as peptide synthesis.

The cyclic peptide derivative represented by formula (1) has the effect of promoting the polarity conversion of microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype, and can therefore be used as a polarity conversion promoter for anti-inflammatory M2 phenotype microglia. That is, by acting the cyclic peptide derivative represented by formula (1) on microglia, which is a type of glial cell, the polarity of the microglia can be converted (polarized) to an anti-inflammatory M2 phenotype, and the M2 phenotype allows the microglia to act in a neuroprotective manner. Therefore, the polarity conversion promoter according to this embodiment can be used in medicines and foods for the treatment and prevention of neuronal damage associated with brain diseases and aging.

In one embodiment, the polarity conversion promoter may be incorporated into a pharmaceutical composition or food composition as an active ingredient thereof, thereby providing a pharmaceutical composition or food composition for promoting polarity conversion to anti-inflammatory M2 phenotype microglia. These pharmaceutical compositions or food compositions can be used, for example, for humans or non-human mammals (laboratory animals, pets, etc.).

The pharmaceutical composition can be produced by using known techniques, such as by mixing the polarity conversion promoter with various medicament-acceptable additives and other ingredients. The pharmaceutical composition can be administered orally or parenterally. For oral administration, the pharmaceutical composition may be in the form of, for example, tablets, pills, powders, granules, capsules, liquid preparations (including elixirs, syrups, suspensions, and solutions), and for parenteral administration, the pharmaceutical composition may be in the form of, for example, injections, infusions, or may be administered directly into the brain.

The food composition can be produced using known techniques, such as by mixing the polarity conversion promoter with various additives and other ingredients that are acceptable for use in food. Examples of food compositions include health functional foods (nutritional functional foods, foods for specified health uses, and foods with functional claims), supplements, and the like. Examples of the form of the food composition include forms similar to those of pharmaceutical compositions for oral administration, and may also be in the form of beverages, confectioneries, etc.

Additives that can be added to pharmaceutical or food compositions include, for example, excipients, antioxidants, flavorings, seasonings, sweeteners, coloring agents, thickening and stabilizing agents, colorants, bleaching agents, gum bases, emulsifiers, binders, diluents, preservatives, stabilizers, and coagulants.

The amount of active ingredient per dose or day of the pharmaceutical composition, and the amount of active ingredient per meal or day of the food composition can be appropriately set according to the age, weight, sex, and disease or condition of the subject to be administered or ingested, and based on non-clinical or clinical test results. Although not particularly limited, the oral intake amount of the polarity conversion promoter may be, for example, 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 for mammals including humans, as the amount of the cyclic peptide derivative of formula (1).

The polarity transfer promoter is effective even at a low concentration in an in vitro experimental system. The concentration of the polarity transfer promoter in an in vitro experimental system is not particularly limited, and may be, for example, 0.01 μM or more and 5 μM or less (i.e., 1×10 ⁻⁸ to 5×10 ⁻⁶ mol/L) or 0.01 μM or more and 1 μM or less, or 0.03 μM or more and 0.3 μM or less, as the concentration of the cyclic peptide derivative of formula (1).

1. MG6 microglial cell culture In the following tests using the MG6 microglial cell line (Riken Cell Bank), MG6 microglial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) containing 100 μM β-mercaptoethanol, 10 μg/ml insulin, 10% fetal bovine serum (Gibco), penicillin-streptomycin (Gibco), and 450 mg/ml glucose (Gibco).

2. Measurement of MG6 microglial cell viability MG6 microglial cells were cultured in a 96-well plate at 37°C for 24 hours (5 x ¹⁰³ cells/well), and then incubated with various concentrations of cyclic peptide derivatives at 37°C for 48 hours. As the cyclic peptide derivative, a diethylamine salt of the compound represented by the above formula (2) obtained by the method described in the Examples of Patent Document 2 was used (the same applies to each of the following tests). The concentrations of the cyclic peptide derivative in the culture medium were adjusted to 0 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, and 1 μM.

The evaluation of cell viability was performed using Cell-Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer's instructions as follows: after treatment with the cyclic peptide derivatives, 10 μL of CCK-8 was transferred to a 96-well plate and then incubated at 37 °C for 2 h. Following the instructions, the optical density was read 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 treatment group/optical density of the control group) × 100%

48 hours after administration of the cyclic peptide derivative, the survival rate of microglia was examined. As shown in Figure 2, no microglial proliferation was observed within the range of 0.01-1 μM of the cyclic peptide derivative concentration, and survival was not inhibited. Therefore, in subsequent experiments, the optimal concentration of the cyclic peptide derivative was set at 1 μM and used for functionality analysis. In Figure 2, "ns" means non-significant.

3. MG6 Microglia Polarization Test Chromogranin A (CGA), a component of senile plaques in the brain of Atzheimer's dementia, is more neurotoxic than amyloid beta. In addition, many microglia are found around senile plaques with CGA accumulation, which is thought to be caused by microglial activation (Non-Patent Document 4). Therefore, a microglia polarization test using cyclic peptide derivatives was performed under pathological conditions in which CGA is present, which is a condition similar to the brain environment.

MG6 microglial cells were cultured in a 96-well plate at 37°C for 24 hours (5 x ¹⁰³ cells/well), and then 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, followed by incubation at 37°C. The cells were incubated in the same manner as above with the addition of CGA without the addition of the cyclic peptide derivative, and with neither the cyclic peptide derivative nor CGA (control).

24 and 72 hours after the 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 are polarized into M1 phenotype, which acts on neuronal cells, and M2 phenotype, which acts on neuronal cells, depending on the factors they produce, and that M1 phenotype microglia produce IL-1β and M2 phenotype microglia produce ARG1 (Non-Patent Document 3). Therefore, the M1 phenotype was identified by the gene expression of IL-1β, and the M2 phenotype was identified by the gene expression of ARG1. The methods of real-time quantitative PCR (RT-PCR) analysis and statistical analysis are as follows.

Real-time quantitative PCR (RT-PCR) analysis
mRNA was isolated from MG6 microglial cell line after treatment with cyclic peptide derivatives, and total RNA was extracted using RNAiso Plus (Takara, Japan) according to the manufacturer's instructions. A total of 800 ng of extracted RNA was reverse transcribed into cDNA using QuantiTect ReverseTranscription Kit (Qiagen, Japan). After an initial denaturation step at 95°C for 5 min, temperature cycling was started. Each cycle consisted of denaturation at 95°C for 5 s, annealing at 60°C for 10 s, and extension for 30 s. A total of 40 cycles were performed. cDNA was amplified in duplicate using Rotor-Gene SYBR Green RT-PCR kit (Qiagen, Japan) using Corbett Rotor-Gene RG-3000A Real-Time PCR System. Data were evaluated using the RG-3000A software program (version Rotor-Gene 6.1.93, Corbett). The sequences of primer pairs (Invitrogen, custom primers) are listed below.
IL-1β: 5′-CAACCAACAAGTGATATTCTCCATG-3′ (SEQ ID NO: 1);
and 5'-GATCCACACTCTCCAGCTGCA-3' (SEQ ID NO: 2)
ARG: 5'-CGCCTTTCTCAAAAGGACAG-3' (SEQ ID NO: 3),
and 5'-CCAGCTCTTCATTGGCTTTC-3' (SEQ ID NO: 4)
For data normalization, an endogenous control (actin) was evaluated to control for cDNA input, and relative units were calculated by the comparative Ct method. All real-time RT-PCR experiments were repeated three times, and results were presented as the mean ± SEM.

[Statistical analysis]
Data are expressed as mean ± SEM. Statistical analysis was performed by one-way or two-way ANOVA with post-hoc Tukey's test using the GraphPadPrism software package. A value of p<0.05 was considered to indicate statistical significance (GraphPadSoftware).

The results are shown in Figure 3 as the relative expression level of mRNA for the sample containing CGA and a cyclic peptide derivative (Example) and the sample containing only CGA (Comparative Example) compared to the control (no cyclic peptide derivative or CGA added).

When treated with the cyclic peptide derivative together with CGA, 24 hours after addition, IL-1β gene expression was about 5 times higher than ARG1 gene expression, and microglia were polarized to the pro-inflammatory M1 phenotype, but after 72 hours, the situation was reversed, with ARG1 gene expression being about 9 times higher than IL-1β gene expression. This result indicates that long-term treatment with the cyclic peptide derivative polarizes microglia to the anti-inflammatory M2 phenotype, which acts to protect neuronal cells. It is also believed that by first polarizing microglia to the pro-inflammatory M1 phenotype, the vitality of microglia can be increased in a short period of time, allowing efficient and rapid polarization to the anti-inflammatory M2 phenotype.

In addition, when treated with CGA and the cyclic peptide derivative, the gene expression level of IL-1β was about four times higher at 24 hours after addition treatment, indicating a high activation effect of the pro-inflammatory M1 phenotype, but after 72 hours after addition treatment, the activity of the pro-inflammatory M1 phenotype had decreased to a level that was not significantly different from that of treatment with CGA alone. On the other hand, when treated with CGA and the cyclic peptide derivative, the gene expression level of ARG1 was about four times higher at 24 hours after addition treatment, and showed a high activation effect of more than twice as much at 72 hours after addition treatment, compared to treatment with CGA alone. From this, it was found that long-term treatment with the cyclic peptide derivative in an environment where CGA is present, which is similar to the brain environment (i.e., under pathological conditions in which microglia are activated), promotes polarity conversion to anti-inflammatory M2 phenotype microglia that acts in a neuroprotective manner.

Microglia are cells located at the first line of immunity in the brain that control the brain environment and brain inflammation. M2 phenotype microglia have the ability to take in waste products such as dead nerve cells and amyloid proteins accumulated in the brain and excrete them from the brain. Furthermore, in the event of brain inflammation, M2 phenotype microglia quickly resolve brain inflammation and protect nerve cells. Therefore, by promoting the polarity conversion of microglia to the anti-inflammatory M2 phenotype, cyclic peptide derivatives are thought to delay brain aging and to be useful in preventing and recovering from acute and chronic brain inflammation-related diseases such as traumatic brain injury, stroke, and dementia. They are also thought to be useful in preventing and recovering from mental disorders including autism and depression.

4. Safety test of cyclic peptide derivatives MG6 microglial cells were cultured in a 96-well plate at 37°C for 24 hours (5 x ¹⁰³ cells/well), and then the cyclic peptide derivatives were added to a concentration of 1 μM and incubated at 37°C. 24 and 72 hours after the addition treatment, real-time quantitative PCR (RT-PCR) analysis and statistical analysis were performed as described above to determine the gene expression levels of interleukin-1β (IL-1β) and arginase-1 (ARG1).

The results are shown in Figure 4. Administration of the cyclic peptide derivative alone did not activate microglia, either 24 or 72 hours later, and did not polarize them to either the pro-inflammatory M1 phenotype or the anti-inflammatory M2 phenotype. This confirmed the safety of the cyclic peptide derivative for microglia, as it is not recognized as a foreign substance by microglia and does not activate microglia under non-pathological conditions.

5. Primary Microglial Culture Primary microglial cells, CD11b ⁺ cells, were isolated from adult mouse brains (8 weeks old, male, Japan SLC, Hamamatsu, Japan) by magnetic cell sorting (MACS) method. In detail, the brains were cut into small pieces, and the resulting cell suspension was mechanically dissociated using a gentle MACS Dissociator (Milteny Biotec) along with enzymatic digestion using a Neural Tissue Dissociation Kit (MiltenyiBiotec), and further transferred to a 30 mm cell strainer to obtain a single cell suspension. After magnetic labeling with CD11b MicroBeads (Miltenyi Biotec), the cell suspension was injected into a magnetic (MACS) column installed in a magnetic separator (Milteny Biotec). After rinsing the MACS column with phosphate-buffered saline (PBS), the CD11b positive fraction was collected according to the method described in Non-Patent Document 4. In the following tests using primary microglial cells, the primary microglial cells were cultured in Eagle's MEM (Nissui Pharmaceutical Co., Ltd.) medium.

6. Measurement of primary microglial cell viability Primary microglial cells were seeded on a 96-well plate ( ¹⁰⁴ cells/well) and cultured with various concentrations of cyclic peptide derivatives at 37°C for 24 and 72 hours. The concentrations of the cyclic peptide derivatives in the culture medium were adjusted to 0 μM, 0.01 μM, 0.1 μM, 1 μM, and 5 μM. The cell viability was evaluated (24 hours and 72 hours) as described in the measurement of MG6 microglial cell viability in 2 above.

The results are shown in Figure 5. Within the range of 0.01 to 5 μM of the cyclic peptide derivative concentration, no proliferation of primary microglial cells was observed, and their survival was hardly inhibited. Note that "*" indicates P<0.05.

7. Polarization study of primary microglial cells Microglia, the resident immune cells of the brain, play an important role in neuroinflammation in neurodegenerative diseases, including Alzheimer's disease, by regulating pro-inflammatory and anti-inflammatory cytokines. It is known that interleukin-1β (IL-1β) is the main inflammatory cytokine, and transforming growth factor (TGF)-β1 is an important anti-inflammatory cytokine (Non-Patent Document 5). As mentioned above, chromogranin A (CGA), a neurosecretory acidic glycoprotein, has been found in senile plaques of Alzheimer's disease as a candidate factor for microglial activation. Therefore, the effects of cyclic peptide derivatives and CGA on inflammation or anti-inflammation were investigated using primary microglial cells and IL-1β and TGF-β1 as markers.

Primary microglial cells were cultured in a 96-well plate at 37° C. for 24 hours ( ¹⁰⁴ cells/well), and 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, followed by incubation at 37° C. The cells were incubated in the same manner as above with the addition of CGA without the addition of the cyclic peptide derivative, the addition of the cyclic peptide derivative without the addition of CGA, and the addition of neither the cyclic peptide derivative nor CGA (control).

24 and 72 hours after the treatment, real-time quantitative PCR (RT-PCR) analysis and statistical analysis were performed to determine the gene expression levels of TGF-β1 and IL-1β. The methods for real-time quantitative PCR (RT-PCR) analysis and statistical analysis were as described in the polarization test of MG6 microglia in 3 above. However, the primer pair for TGF-β1 (Invitrogen, custom primers) had the following sequences:
TGF-β1: 5′-TCAGACATTCGGGAAGCAGTG-3′ (SEQ ID NO: 5),
and 5'-ATTCCGTCTCCTTGGTTCAGC-3' (SEQ ID NO: 6)

The results are shown in Figure 6. In Figure 6, "*" indicates P<0.05, "##" indicates P<0.01, and "###" indicates P<0.001. "Con" indicates the control, "peptide" indicates treatment with the cyclic peptide derivative alone, "CGA" indicates treatment with CGA alone, and "peptide + CGA" indicates treatment with a combination of the cyclic peptide derivative and CGA.

As shown in Figures 6(a) and (b), in primary microglia, treatment with CGA alone slightly increased the expression of TGF-β1 at 24 and 72 hours compared to the control. Treatment with CGA in combination with the cyclic peptide derivative significantly increased the expression of TGF-β1 at 24 and 72 hours (approximately 3-fold on average). Furthermore, treatment with the cyclic peptide derivative alone did not affect the expression of TGF-β1 compared to the control.

On the other hand, as shown in Figures 6(c) and (d), the expression of IL-1β was significantly increased by treatment with CGA alone, and by treatment with a combination of CGA and the cyclic peptide derivative, the expression of IL-1β was equal to or less than that of the control, showing the opposite tendency to the expression of TGF-β1. Furthermore, compared to the control, treatment with the cyclic peptide derivative alone did not affect the expression of IL-1β.

This result means that the cyclic peptide derivatives increase TGF-β1 and suppress IL-1β, thereby shifting microglia activated by CGA (pro-inflammatory phenotype M1) to the anti-inflammatory phenotype M2, i.e., reversing polarity. Considering that the cyclic peptide derivatives themselves do not affect the expression of TGF-β1 or IL-1β in primary microglial cells, this test using primary microglia also confirms the safety of the cyclic peptide derivatives for microglia, and it can be understood that the cyclic peptide derivatives will be effective in improving neurodegenerative diseases.

Claims (2)

A promoter for promoting polarity conversion of microglia to an anti-inflammatory M2 phenotype , comprising a cyclic peptide derivative represented by the following general formula (1):
In formula (1), m is 0 to 3 and n is 1 or greater; R ₁ to R ₆ are each independently a hydrogen atom or a hydrocarbon group; R ₇ and R ₈ are each independently a carboxy group or a salt thereof, or an alkoxycarbonyl group; R ₉ is a hydrocarbon group, a hydroxy group, an alkoxy group, or an alkylcarbonyloxy group; R ₁₀ and R ₁₁ are each independently a hydrogen atom, a hydrocarbon group, or an alkylcarbonyloxy group; and R ₁₂ to R ₁₆ are each independently a hydrogen atom or a hydrocarbon group. The agent for promoting polarity conversion of microglia to an anti-inflammatory M2 phenotype according to claim 1, wherein, in the general formula (1), R ₁ , R ₂ , R ₃ and R ₄ are each independently an alkyl group, n=2 to 4, R ₅ and R ₆ are each a hydrogen atom, and R ₇ and R ₈ are each independently a carboxy group or a salt thereof.