Novel brain disease treatment agent and its use

Abstract

The present invention relates to a novel therapeutic agent for brain diseases and its use. The peptide or pharmaceutical composition of the present invention has a high blood-brain barrier penetration level, which can enhance delivery and reach to the site of action (particularly, inflammatory sites in the brain), and can increase the blood half-life. Furthermore, by improving inflammation levels, it is possible to reduce inflammation and treat or prevent brain diseases.

Description

[Technical Field]

[0001] The present invention relates to a novel therapeutic agent for brain diseases and its use. [Background technology]

[0002] Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease primarily affect elderly people, and the number of patients is increasing exponentially with the aging of society. Furthermore, a number of early-onset neurodegenerative diseases have been reported in younger people. This has led to considerable interest in developing various therapies to halt disease progression and restore damaged brain tissue.

[0003] To improve or treat neurodegenerative diseases, methods for delivering drugs to restore damaged brain tissue can be used, but for this purpose, the drugs must cross the blood-brain barrier (BBB). However, the BBB prevents most substances in the blood, such as most macromolecules (e.g., immunoglobulins, antibodies, complement, albumin, as well as drugs and small molecules), from entering the brain, limiting the treatment of these diseases.

[0004] There is a demand and need for the development of new formulations that can effectively deliver biological materials into the body without cytotoxicity. Recently, new alternatives have been proposed, and among them, cell permeable peptides have attracted attention because they can enhance the utility of macromolecules such as therapeutic proteins and genes, which have previously been difficult to use as drugs due to their low cell membrane permeability and short in vivo half-lives.

[0005] Therefore, the present inventors conducted research to develop a composition that can improve blood-brain barrier permeability and exhibit preventive or therapeutic effects against neurodegenerative brain diseases, and completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect of the present invention aims to provide a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.

[0007] Other aspects of the present invention aim to provide an anti-inflammatory pharmaceutical composition containing the above peptide, a method for treating an inflammatory disease comprising administering the above peptide to a patient with the inflammatory disease, and use of the above peptide for treating or preventing an inflammatory disease. [Means for solving the problem]

[0008] In order to achieve the above-mentioned object, one embodiment of the present invention provides a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.

[0009] In one embodiment of the present invention, the peptide may suppress the expression of at least one of IL-1b, IL-6, Iba1, and TNFα, and increase the expression of Arg-1.

[0010] Another aspect of the present invention provides an anti-inflammatory pharmaceutical composition containing the peptide.

[0011] In one embodiment of the present invention, the inflammation may occur in the brain.

[0012] In one embodiment of the present invention, the pharmaceutical composition may be used for treating or preventing brain diseases.

[0013] In one embodiment of the present invention, the brain disease may be at least one of Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD).

[0014] Other aspects of the invention provide a method for treating inflammatory diseases comprising administering the peptide to a patient suffering from the disease, as well as therapeutic or prophylactic uses of the peptide for anti-inflammatory purposes. [Effects of the Invention]

[0015] The peptide or pharmaceutical composition of the present invention can cross the blood-brain barrier (BBB) ​​with high efficiency, enhancing delivery to the site of action (particularly, an inflammatory site in the brain). Furthermore, it can contribute to anti-inflammation and the treatment or prevention of brain diseases by prolonging its half-life in the blood and improving inflammatory conditions. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1A shows the results of the BBB permeability enhancement and plasma pharmacokinetics (PK) of exendin-4 by ACP. Specifically, Figure 1A shows the results of analysis of ISF (interstitial fluid) samples collected at time intervals after administration of exendin-4 and dose-balanced ACP2-exendin-4 and ACP4-exendin-4. Figure 1B shows the results of measuring pharmacokinetic parameters in the plasma of white rats to investigate whether the time-dependent differences in exendin-4 profiles observed in the ISF were due to in vivo distribution. [Figure 2] The results confirmed the increased distribution of ACP-exendin-4 in each region of the rat brain. [Figure 3] FIG. 1 shows the in vitro anti-inflammatory effect of ACP-exendin-4. [Figure 4-6] The experimental process and results of the protective effect of ACP-exendin-4 on Parkinson's disease-like symptoms are shown below. Specifically, Figure 4A outlines the experimental design, and Figure 4B shows the results of the pole test to examine motor function improvement. Figures 5A and 6A show the reduction of TH in the striatum and Substantia Nigra pars compacta (SNpc) in the MPTP-treated group compared with the control group, in which no neuronal damage was induced. Figures 5B and 6B show the changes in the expression of inflammatory cytokines (IL-1b, IL-6, Iba1, TNFa), the inflammation-related factor COX-2, and the anti-inflammatory marker Arg-1 in each region. [Figure 7] FIG. 7 shows the results regarding the ability of ACP-exendin-4 to produce cAMP and the recruitment of β-arrestin-2. Specifically, FIG. 7A shows cAMP production, and FIG. 7B shows β-arrestin-2 recruitment. DETAILED DESCRIPTION OF THE INVENTION

[0017] One aspect of the present invention provides a peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.

[0018] The amino acid sequence of SEQ ID NO: 1 or 2 has a structure in which the cell-penetrating peptide ACP2 or ACP4 is bound to exendin-4.

[0019] [Table 1]

[0020] As used herein, the term "cell penetrating peptides (CPPs)" refers to short peptides consisting of approximately 10 to 60 amino acids that translocate into cells without damaging the cell membrane, enabling the intracellular delivery of DNA and proteins that are difficult to penetrate membranes.

[0021] Exendin-4 is a peptide consisting of 39 amino acids derived from the salivary glands of the gila monster (Heloderma suspectum). Exendin-4 activates the GLP-1 (glucagon-like peptide-1) receptor but does not significantly activate the glucagon receptor. In 2005, Eli Lilly and Amylin developed a synthetic exendin-4 called Byetta (registered trademark), which was approved as a therapeutic agent for diabetes.

[0022] Previously, active ingredients such as Exendin-4 had difficulty penetrating the blood-brain barrier (BBB), making it difficult for them to reach their site of action. However, this peptide improves permeability by binding the cell-penetrating peptides ACP2 or ACP4 to Exendin-4, thereby increasing delivery and reach to inflammatory sites in the brain and increasing its half-life in the blood.

[0023] The peptide can suppress the expression of any one or more of IL-1b, IL-6, Iba1, and TNFα, and increase the expression of Arg-1.

[0024] The above-mentioned IL-1b, IL-6, Iba1, and TNFa are inflammatory indicator factors, and the peptides of the present invention can reduce the expression of these inflammatory indicator factors, specifically, the expression of intracerebral inflammatory indicator factors, compared to when Exendin-4 is administered. Arg-1 is a marker of M2 polarization of microglia, and the peptides of the present invention can increase the expression of this marker compared to when Exendin-4 is administered (Figures 3, 5, and 6).

[0025] The term "binding" as used in the present invention means that the cell-penetrating peptide and a substance having biological or pharmacological activity are linked together by chemical, physical, covalent or non-covalent bonds.

[0026] Another aspect of the present invention provides an anti-inflammatory pharmaceutical composition containing the peptide.

[0027] The pharmaceutical composition may contain the peptide alone or in combination with known ingredients as an active ingredient.

[0028] In another aspect of the present invention, the inflammation may occur in the brain, and specifically, the pharmaceutical composition is used to treat or prevent a brain disease, which may be any one or more of Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD).

[0029] The pharmaceutical composition of the present invention may contain, in addition to the present fusion protein, a pharmacologically acceptable carrier, if necessary.

[0030] Such pharmacologically acceptable carriers are those commonly used in pharmaceutical manufacturing, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition of the present invention may further contain additives such as lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0031] The carrier may be contained in the pharmaceutical composition of the present invention in an amount of about 1% to about 99.99% by weight, preferably about 90% to about 99.99% by weight, based on the total weight of the composition, and the additive may be contained in an amount of about 0.1% to about 20% by weight.

[0032] On the other hand, the pharmaceutical composition of the present invention can be administered orally or parenterally, but can also be administered directly to the skin by topical administration.

[0033] The pharmaceutical compositions of the present invention may be formulated using pharmacologically acceptable carriers and / or excipients to be prepared in unit dose form or filled in multi-dose containers, and the formulation may include solutions, suspensions, emulsions, elixirs, extracts, powders, granules, tablets, plasters, liniments, lotions, ointments, etc.

[0034] The daily dose of the pharmaceutical composition of the present invention is usually in the range of 0.001 to 150 mg / kg body weight, and can be administered once or in several divided doses. However, since the dose of the pharmaceutical composition of the present invention is determined based on multiple relevant factors such as the administration route, the patient's age, sex, and weight, and the severity of the patient's condition, the above dose should not be construed as limiting the technical scope of the present invention in any aspect.

[0035] As mentioned above, conventional active ingredients such as Exendin-4 have difficulty penetrating the blood-brain barrier (BBB), making it difficult for them to reach their site of action. However, the present peptides conjugate Exendin-4 with the cell-penetrating peptides ACP2 or ACP4, thereby improving the blood-brain barrier permeability of the peptides of the present invention, thereby increasing their delivery and reach to the site of action (especially inflammatory sites in the brain) and increasing their blood half-life. Furthermore, the peptides of the present invention suppress the expression of one or more of IL-1b, IL-6, Iba1, and TNFα and increase the expression of Arg-1 compared to administration of Exendin-4. Through these effects, the peptides or pharmaceutical compositions of the present invention can be used to suppress inflammation in brain tissue and to treat or prevent brain diseases such as Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD).

[0036] The present invention provides a method for treating an inflammatory disease, which comprises administering the peptide to a patient suffering from the disease, and a use of the peptide for treating or preventing an inflammatory disease. [Example]

[0037] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0038] Example 1: Study Method Pharmacokinetic study using SD rats after intravenous administration Test substances (exendin-4, ACP2-exendin-4, and ACP4-exendin-4) dissolved in PBS were intravenously administered to rats. Whole blood samples were collected before administration (0 min) and 1, 5, 15, 30, and 45 min and 1, 2, 4, and 6 h after administration. Plasma was separated and stored in a deep freezer at approximately -70°C until analysis. Plasma concentrations were measured using LC-MS / MS analysis. Exendin-4 (SEQ ID NO: 3), ACP2-exendin-4 (SEQ ID NO: 1), and ACP4-exendin-4 (SEQ ID NO: 2) used in this method were synthesized by Chempeptide Limited. The purity of the synthesized peptides was 98% or higher.

[0039] Cell lines and cell culture All cells were cultured at 37°C under 5% CO2 humidified air conditions. BV2 cells were cultured in DMEM containing 10% FBS, and Pathhunter® CHO-K1 GLP1R bioassay cells were cultured in the medium provided by the kit.

[0040] Real-time PCR BV2 cells were seeded in 12-well plates and cultured overnight. They were then treated with the test substances at various concentrations. After 1 hour of culture, lipopolysaccharide (LPS) was added at 1 μg / mL and the cells were cultured for 6 hours. Total RNA was extracted using TRI Reagent, and cDNA was synthesized. The expression of inflammation-related genes was then examined using real-time PCR analysis.

[0041] β-arrestin-2 recruitment assay (Pathhunter® CHO-K1 GLP1R bioassay) A β-arrestin-2 recruitment assay was performed using Pathhunter® CHO-K1 GLP1R bioassay cells. After seeding, cells were treated with test substances at concentrations ranging from 25.6 pM to 10 μM. After 30 minutes of incubation, each well was treated with Detection Reagent 1 and incubated at room temperature for 15 minutes in the dark. Detection Reagent 2 was then added per well, and the incubation was continued for 1 hour at room temperature in the dark, after which luminescence was measured.

[0042] cAMP assay Intracellular cAMP production was measured using the cAMP Parameter Assay Kit (R&D Systems). BV2 cells were seeded in 6-well plates and cultured overnight. After incubation, the cells were treated with test substances at a final concentration of 100 nM for 15 minutes. After incubation, the cells were washed three times with cold PBS and harvested using a scraper. After centrifugation, the supernatant was removed and the cell pellet was resuspended in 300 μL of Cell Lysis Buffer 5. The cells were subjected to two freeze-thaw cycles and centrifuged to prepare cell lysates. The assay was performed according to the kit's instructions. 200 μL of Substrate Solution was added, and the reaction was incubated at room temperature for 30 minutes in the dark. The reaction was then terminated by adding 100 μL of Stop Solution. After the reaction, the absorbance of the sample was measured at 450 nm using a microplate reader, and the cAMP concentration was calculated according to the kit's instructions.

[0043] Blood-brain barrier permeation assay of ACP-Exendin-4 using microdialysis Microdialysis was performed in brain interstitial fluid (ISF) to examine ACP-induced enhancement of BBB permeability to Exendin-4. After fixing the rat's head in a stereotaxic frame, the rat was moved to the coordinates of the striatum (basal ganglia) using bregma as the reference point. A microdialysis guide cannula was inserted into the exposed rat skull using stereotaxic surgery, drilling a hole with a microdrill. After the surgery and recovery period, the rat was placed in a microdialysis cage, a microdialysis probe was inserted into the guide cannula, and the microdialysis tubing was connected to a syringe pump and a fraction collector. The microdialysis solution was then allowed to flow through the tubing outlet, allowing it to stabilize. After intravenous administration of 10 mg / kg of exendin-4 and equilibrated doses of ACP2-exendin-4 (16 mg / kg) and ACP4-exendin-4 (14 mg / kg) into rat tail veins, microdialysis sampling (ISF sampling) was performed over a time course. The concentrations of exendin-4, ACP2-exendin-4, and ACP4-exendin-4 in the microdialysis samples were analyzed using LC-MS / MS.

[0044] Example 2: Experimental Results Enhancement of BBB penetration of Exendin-4 by ACP and plasma PK results (Figure 1) After intravenous administration of 10 mg / kg exendin-4, ACP2-exendin-4, and ACP4-exendin-4 equilibrated with the exendin-4 dose, ISF samples were collected over a time course. At 10 min post-administration, ACP2- and ACP4-exendin-4 complexes (ACP2-exendin-4, 12.63 ± 1.35 ng / mL; ACP4-exendin-4, 5.56 ± 0.13 ng / mL) were detected at 6- to 12-fold higher concentrations than exendin-4 (0.99 ± 1.38 ng / mL) (Figure 1A).

[0045] Exendin-4 was detected in the ISF at low concentrations only 10 min after administration, whereas ACP2-Exendin-4 and ACP4-Exendin-4 were detected in the ISF even 1 h after administration, and ACP4-Exendin-4 in particular was confirmed to maintain its level even after 1 h (Fig. 1A).

[0046] To investigate whether the differences in the time-course profiles of exendin-4 observed in ISF were due to the distribution of exendin-4 in vivo, we investigated the pharmacokinetic (PK) parameters of each exendin-4 in rat plasma. After administering 1 mg / kg of exendin-4 and the dose-balanced ACP2-exendin-4 and ACP4-exendin-4 formulations via the tail vein of rats, blood samples were collected over time, and the amount of test substance was measured using LC-MS / MS. The concentrations of all three test substances were highest at 1 min after administration and decreased over time (Figure 1B).

[0047] The area under the blood concentration-time curve (AUClast) and plasma half-life of ACP4-Exendin-4 were significantly increased compared to Exendin-4 and ACP2-Exendin-4, and the ACP4-Exendin-4 profile in brain ISF correlated with that in plasma. These results suggest that the binding of ACP2 and ACP4 to Exendin-4 not only improves blood-brain barrier penetration but also the in vivo stability of the active ingredient, potentially leading to the development of effective drugs for the treatment of brain diseases.

[0048] Increased distribution of ACP-Exendin-4 in rat brain tissue regions was confirmed (Figure 2). To develop effective brain disease therapeutics, the ultimate goal of CNS drug development is to ensure that drugs penetrate (deliver) into brain tissue for sufficient time to exert biological efficacy at the site of action. Therefore, we investigated the distribution of exendin-4 and ACP-exendin-4 in different brain regions. Exendin-4 and ACP2-exendin-4 and ACP4-exendin-4, which were equilibrated with exendin-4, were administered intravenously to rats at 10 mg / kg. After 10 minutes, the rats were sacrificed and brain tissue was extracted. The distribution of the administered compounds in different brain regions was analyzed using LC-MS / MS. The four regions analyzed were the hippocampus, prefrontal cortex, striatum, and substantia nigra pars compacta, which are known to be severely damaged by typical neurodegenerative brain diseases such as Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD). Analysis showed that no Exendin-4 was detected in the prefrontal cortex, while ACP2-Exendin-4 and ACP4-Exendin-4 were detected at 307 ng / mL and 161 ng / mL, respectively.

[0049] Parkinson's disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain, resulting in decreased dopamine levels in the striatum. These two regions are known to be the primary lesions in Parkinson's disease. While Exendin-4 itself reached these two regions at low concentrations of 22-24 ng / mL, ACP-bound Exendin-4 reached these two regions at levels ranging from 2.3-fold to over 40-fold higher than Exendin-4. Furthermore, ACP-Exendin-4 was also observed to efficiently reach the hippocampus, a major site of Alzheimer's disease pathology. These results suggest that ACP-Exendin-4 may have superior therapeutic effects to Exendin-4 in the treatment of neurodegenerative brain diseases such as Parkinson's disease and Alzheimer's disease due to its differential brain delivery.

[0050] In vitro anti-inflammatory effects of ACP-Exendin-4 (Figure 3) The anti-inflammatory effects of test substances were confirmed using BV2 cells, microglia involved in the brain's inflammatory response (neuroinflammation). Neuroinflammation is an important and unavoidable pathological process associated with all types of central nervous system injury and disease and is an environmental indicator of neurodegenerative diseases. In response to various microenvironmental disturbances, microglia can polarize to either an M1 pro-inflammatory phenotype, which exacerbates neurotoxicity, or an M2 anti-inflammatory phenotype, which has neuroprotective functions. Microglia have been reported to be activated to the M1 phenotype by LPS. In BV2 cells, LPS-induced inflammation was confirmed to upregulate the inflammatory cytokines IL-6, IL-1β, and TNF-α, as well as Iba1, a marker of microglial activation. Pretreatment with test substances reduced the expression of IL-6, IL-1β, TNF-α, and Iba1 compared to the LPS-treated group (Figure 3). Pretreatment with Exendin-4 and ACP-Exendin-4 significantly reduced the expression of IL-6 and Iba1, with ACP-Exendin-4 showing a greater inhibitory effect than Exendin-4. The anti-inflammatory effects of AVI-6110 on IL-1β and TNF-α were confirmed, but the reductions were not significant (Figure 3).

[0051] Arg-1, a known marker of M2 polarization in microglia, is an anti-inflammatory factor whose expression has been reported to be reduced by LPS treatment, a finding confirmed in this study in which BV2 cells were stimulated with LPS. Arg-1 mRNA expression, which was reduced by LPS treatment, was restored by pretreatment with Exendin-4 and ACP-Exendin-4, and ACP-Exendin-4 was found to be more effective than Exendin-4 in restoring Arg-1 expression, indicating that the anti-inflammatory effect of ACP-Exendin-4 is superior to that of Exendin-4.

[0052] Confirmation of the protective effect of ACP-Exendin-4 against Parkinson's disease-like symptoms (Figures 4-6) (1) Pole test To investigate the effects of ACP-conjugated Exendin-4 on Parkinson's disease (PD)-like symptoms, we performed a study using an MPTP-treated PD mouse model (Fig. 4A). To assess the improvement in motor ability, we performed a pole test and measured the time it took for the mice to turn down the pole (time to turn) and the time it took to climb down (time to climb down). The MPTP-treated group (G2) significantly increased the time to turn, climb down, and total time to reach, whereas the ACP2-Exendin-4 (G4) and ACP4-Exendin-4 (G5) groups significantly decreased the total time and turn time (Fig. 4B).

[0053] (2) TH (tyrosine hydroxylase) staining To confirm the effect of ACP-Exendin-4 on MPTP-induced dopamine cell death, the mouse brain was removed, and the striatum and substantia nigra were isolated and immunohistochemically stained to observe the expression of TH (tyrosine hydroxylase).

[0054] TH is known to regulate the rate of dopamine and epinephrine synthesis from tyrosine in dopamine biosynthetic neurons. TH is highly tissue-specific, being expressed only in specific regions of the mammalian central nervous system, including the hypothalamus, midbrain, brainstem, and olfactory bulb. In particular, Parkinson's disease pathology is associated with inappropriate TH gene expression regulation following the death of midbrain dopaminergic neurons due to various causes or due to inappropriate differentiation of dopaminergic neurons. A significant decrease in TH was observed in the striatum and substantia nigra of the MPTP-treated group compared with the control group, where no neuronal damage was induced. No significant differences were observed in the striatum and substantia nigra of the test substance-treated group compared with the MPTP-induced group (Figures 5A and 6A).

[0055] (3) Suppression of inflammatory cytokine expression The anti-inflammatory effects of ACP-Exendin-4 were confirmed by qPCR using brain tissue regions of the striatum and substantia nigra pars compacta (SNpc) from an MPTP-induced PD mouse model. In the striatum, the MPTP-induced group (G2) showed the highest expression of inflammatory cytokines (IL-1b, IL-6, Iba1, and TNFα) and the inflammatory mediator COX-2. The Exendin-4 and ACP-Exendin-4-treated groups showed lower expression of inflammatory cytokines and COX-2 compared to the MPTP-induced group. The order of potency was ACP4-Exendin-4 > ACP2-Exendin-4 > Exendin-4. Furthermore, ACP4-Exendin-4 showed significant differences compared to Exendin-4 in IL-1b and COX-2, indicating that ACP4-Exendin-4 has a stronger anti-inflammatory effect than Exendin-4 (Figure 5B). Experimental results using SNpc brain tissue sections also showed the highest inflammatory response in the MPTP-induced group, confirming that administration of the test substance suppressed these inflammatory responses, similar to the results in the striatum (Figure 6B). Although the SNpc experimental results did not consistently demonstrate the order of anti-inflammatory potency, ACP4-Exendin-4 > ACP2-Exendin-4 > Exendin-4, the ACP-bound Exendin-4 still demonstrated superior anti-inflammatory effects to Exendin-4. The anti-inflammatory factor Arg-1 was confirmed to be decreased by MPTP induction in both the striatum and SNpc, and increased by the administration of the test substances. Although there was no significant difference, the ACP2-Exendin-4 (G4) and ACP4-Exendin-4 (G5) groups were found to be more effective in restoring Arg-1 expression than Exendin-4 (G3), indicating that the anti-inflammatory effect of ACP-Exendin-4 is superior to that of Exendin-4 (Figures 5B and 6B).

[0056] (4) cAMP production by ACP-Exendin-4 and β-arrestin-2 recruitment (Figure 7) To elucidate the mechanism of action of ACP-Exendin-4, we first assessed whether ACP-bound Exendin-4 mediates signal transduction via the Exendin-4 receptor, GLP-1R, using a cAMP production assay. Treatment of BV2 with the test substance significantly increased cAMP production compared to vehicle. Furthermore, cAMP production by ACP2-Exendin-4 was significantly increased compared to Exendin-4 (Figure 7A). Exendin-4 is internalized into cells via its receptor, GLP-1R, and cAMP is produced during this process. Therefore, these results suggest that ACP-Exendin-4 is internalized into cells via GLP-1R.

[0057] To elucidate the mechanism of ACP-Exendin-4's GLP-1R activity, we used the PathHunter® Bioassay Detection Kit, which can detect activation of β-arrestin signaling, one of the downstream mechanisms of GLP-1R. Exendin-4 induced β-arrestin-2 recruitment in a concentration-dependent manner. In contrast, ACP-bound Exendin-4 induced almost no β-arrestin-2 recruitment (Figure 7B). These results suggest that ACP-bound Exendin-4 is a G protein-biased agonist that is internalized via the GLP-1R but does not activate β-arrestin signaling.

[0058] The present invention has been discussed above with a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be embodied in various modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the foregoing description, and all variations within the scope of equivalents thereto should be construed as being within the scope of the present invention.

Claims

1. A peptide represented by the amino acid sequence of SEQ ID NO: 1 or 2.

2. 2. The peptide of claim 1, The peptide suppresses the expression of any one or more of IL-1b, IL-6, Iba1 and TNFa, and increases the expression of Arg-1.

3. A pharmaceutical composition for anti-inflammatory use, comprising the peptide of claim 1.

4. 4. The pharmaceutical composition of claim 3, The pharmaceutical composition, wherein the inflammation occurs in the brain.

5. 4. The pharmaceutical composition of claim 3, The pharmaceutical composition is for treating or preventing a brain disease.

6. 6. The pharmaceutical composition of claim 5, The pharmaceutical composition, wherein the brain disease is any one or more of Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD).

7. A method for treating an inflammatory disease, comprising administering the peptide of claim 1 to a patient suffering from the inflammatory disease.

8. 10. Use of the peptide of claim 1 for treating or preventing an anti-inflammatory disease.

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