JP2015067579A - Cell permeable peptide and pharmaceutical composition comprising peptide concerned - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide which improves an expression of an antioxidation stress enzyme group by Nrf2 transcriptional activity, and to provide medicaments for diseases which can be caused by the expression of the above antioxidation stress enzyme group.SOLUTION: The invention provides a peptide consisting of an amino acid sequence in which one or some amino acids are deleted, substituted, or added and a pharmaceutical composition comprising the peptide concerned, in a peptide consisting of an amino acid sequence represented by a formula (1): (R)LQLDEETGEFLPIQ (1) [where, n represents a natural number of 6-12], or the amino acid sequence represented by the above formula (1).

Description

  The present invention relates to a cell-penetrating peptide and a pharmaceutical composition containing the peptide.

  Oxidative stress is considered to be one of the factors of bone destruction diseases such as periodontitis. Oxidative stress caused by reactive oxygen (ROS) plays a role in osteoclast formation as an intracellular signal molecule following, for example, RANK ligand (RANKL) signaling. It is known that ROS (oxidative stress) works as an intracellular signaling substance in the activation of this osteoclast. In addition, since oxidative stress can be a hindrance factor for cells, there is a mechanism for preventing it, and a mechanism for controlling the expression of antioxidant stress enzymes through the transcription factor Nrf2 is known. Nrf2 normally binds to the protein Keap1 and is ubiquitinated and degraded. The binding site between Nrf2 and Keap1 is the ETGE sequence.

  However, when the ETGE sequence itself is simply added to the cell, a significant amount of ETGE sequence does not reach the cell, resulting in binding of Nrf2 to Keap l and subsequent degradation of Nrf2, resulting in antioxidant stress. Enzyme group expression control could not be performed sufficiently.

Free Radie Biol Med. 2012 Jan 15; 52 (2) 444-5l ACS Med Chem Lett. 2012 May 10; 3 (5): 407-410. Kanzaki H, Shinohara F, Kajiya M, Kodama T. The keap1 / Nrf2 Axis Plays A Role In Osteoclast Diffefentiation By Regulating Intracellular ROS Signaling, Journal of Biological Chemistry 2013.

  The problem to be solved by the present invention is to provide a peptide that improves the expression of the antioxidant stress enzyme group by the transcriptional activity of Nrf2, and to provide a pharmaceutical that can be affected by the expression of the antioxidant stress enzyme group. .

  Under the circumstances as described above, as a result of intensive studies, the present inventor has found that when a fusion peptide in which a certain number of arginines are bound to the N-terminus of the ETGE sequence peptide is used, the peptide penetrates the cell membrane, Was found to improve the transcriptional activity of Nrf2. Furthermore, the present inventors have found that the above fusion peptide is useful for the prevention and treatment of diseases that can be treated by improving the transcriptional activity of Nrf2. The present invention is based on these new findings.

  The peptide of the present invention can be delivered into a cell to prevent the binding of Nrf2 and Keapl, and increase the expression of the antioxidant stress enzyme group by preventing the degradation of Nrf2. Therefore, the peptide of the present invention can be used for prevention and treatment of diseases caused by oxidative stress.

FIG. 1 shows the inhibition of osteoclast formation by the 7R-ETGE peptide in the examples. (A) RAW 264.7 cells were stimulated with RANKL (100 ng / ml). (B) RANKL and 7R-ETGE were added, (C) RANKL and ETGE-7R were added, (D) RANKL and ETGE peptide were added, and TRAP staining was performed. The part indicated by the arrow is a TRAP + multinucleated cell. Bar = 100 μm. (E) The number of TRAP + multinucleated cells per well was counted. Independent tests were performed in triplicate and representative data are shown in the figure. ^* Indicates p <0.05. NS indicates no significant difference. (F) Control mouse primary peritoneal macrophages, (G) RANKL (100 ng / ml) -stimulated primary macrophages, (H) RANKL (100 ng / ml) -stimulated primary macrophages with 7R-ETGE added, TRAP staining went. The part indicated by the arrow is a TRAP + multinucleated cell. Bar = 100 μm. A representative photograph is shown. (I) The number of TRAP + multinucleated cells per well was counted. Independent tests were performed in triplicate and representative data are shown in the figure. ^* Indicates p <0.05. FIG. 2 shows the decrease in osteoclast marker gene expression by 7R-ETGE in Examples of the present application. The expression of osteoclast marker genes ATP6v0d2, DC-STAMP and OSCAR was tested in RAW 264.7 cells by real-time PCR. Indicates the relative value to the control. Independent tests were performed in triplicate and representative data are shown in the figure. ^{* Indicates} p <0.05 compared to the RANKL single stimulation group. NS: No significant difference compared to RANKL single stimulation group. FIG. 3 shows the reduction of osteoclast resorption activity by 7R-ETGE peptide in Examples of the present application. RAW 264.7 cells stimulated with (A) RANKL (100 ng / ml), (B) 7R-ETGE peptide added, (C) ETGE-7R peptide added, and (D) ETGE peptide added The cultures were cultured on bone resorption assay plates. The part indicated by the arrow is the part where absorption is lost. Bar = 100 μm. A representative photograph is shown. (E) The percentage of the area of the absorbed region was calculated. Independent tests were performed in triplicate and representative data are shown in the figure. ^{* Indicates} p <0.05 compared to the RANKL single stimulation group. NS: No significant difference compared to RANKL single stimulation group. FIG. 4 shows the results of flow cytometry analysis of intracellular ROS in the examples of the present application. Intracellular ROS levels in RAW 264.7 cells were tested using flow cytometry. (A) RANKL induced intracellular ROS. The solid line indicates the control, and the dotted line indicates the RANKL stimulation group. Independent tests were performed in triplicate and representative data are shown in the figure. (B) 7R-ETGE peptide decreased intracellular ROS in RAW264.7 induced by RANKL, but not ETGE-7R peptide or ETGE 7R-ETGE peptide. The dotted line indicates addition of RANKL and 7R-ETGE peptide, and the solid line indicates addition of RANKL alone, addition of RANKL and ETGE-7R peptide, or addition of RANKL and ETGE. Independent tests were performed in triplicate and representative data are shown in the figure. FIG. 5 shows the induction by the 7R-ETGE peptide of Nrf2-mediated upregulation of cytoprotective enzyme expression at the mRNA and protein levels in the Examples of the present application. (A) Western blot analysis of nuclear Nrf2. The extracted equal amount of nucleoprotein was electrophoresed and transferred to a PDVF membrane. In order to confirm the equivalence of the loaded nucleoprotein, the chopped membrane was subjected to Western blot analysis using anti-Histone H3 antibody. Concentration analysis is used to measure the ratio to the control and the average of three tests is shown on each panel. (B) HO1, NQO1 and GCS mRNA expression was examined by real-time PCR. The relative value of each gene relative to the control is shown. White: control, black: 7R-ETGE added cells. ^{* Indicates} p <0.05 relative to control. Independent tests were performed in triplicate and representative data are shown in the figure. (C) Western blot analysis of HO1, NQO1 and GCS. The extracted equal amount of nucleoprotein was electrophoresed and transferred to a PDVF membrane. Independent tests were performed in triplicate and representative data are shown in the figure. (D) In order to confirm the equivalence of the loaded nuclear protein, TGX Stain-Free Precast Gels (BioRad Laboratories) was used, and UV treatment was performed to visualize the total protein. The visualized protein on the membrane was imaged with ImageQuant LAS-4000 under UV light transmission. (E) The relative expression levels of HO1, NQO1 and GCS with respect to the control were calculated by concentration analysis of each band shown in FIG. 5C. These values are normalized by the amount of total protein visualized in FIG. 5D. The average of three tests is shown. FIG. 6 shows inhibition of bone destruction by ETGE-containing peptides in experimental animals in Examples of the present application. Each mouse was injected 5 times every other day with LPS (10 μg / site) or PBS with or without 7R-ETGE peptide added. (A) Real-time PCR analysis was performed using 3 RNA samples per group. ^{* Indicates} p <0.05. (B) MicroCT images are obtained with 5 samples per group and representative photographs are shown. The arrow indicates a defect or hole in the skull due to absorption. Bar = 1 mm. (C) The area of the resorption area in the skull was calculated using Image-J software. Shown is the average percentage of resorbed parts (calculated from the ratio of the number of pixels in the skull to the absorbed area to the number of pixels in the skull in the analyzed image). * Indicates p <0.05 relative to the control. NS shows no significant difference from the control. †: p <0.05.

Cell penetrating peptide The present invention relates to (R) _n LQLDEETGEFLPIQ (1) (SEQ ID NO: 1).
[Wherein n represents a natural number of 6 to 12]
Or a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (1).

  As described above, the peptide of the present invention has a structure in which 6 to 12 arginines are bonded to the N-terminal of the sequence LQLDEETGEFLPIQ (SEQ ID NO: 2) corresponding to the binding site with Keap1 in the amino acid sequence of Nrf2. In the formula (1), the number of N-terminal arginines is 6 to 12, preferably 7 to 10, and more preferably 7 to 9.

  Moreover, the peptide of the present invention may be a peptide consisting of the amino acid sequence represented by the above formula (1), and the amino acid represented by the above formula (1) as long as the effects of the present invention are brought about. It may be a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence. In the embodiment in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (1), the deletion, substitution or addition is represented by the above formula (1). Although it may occur at any position in the amino acid sequence, one in which one or several amino acids are deleted, substituted or added in the portion of the sequence LQLDEETGEFLPIQ is preferable.

  The peptide of the present invention can be produced by a method known per se, for example, organic synthetic chemical peptide synthesis such as solid layer synthesis method, genetic engineering method and the like.

Pharmaceutical composition This invention provides the pharmaceutical composition containing the said peptide. The pharmaceutical composition of the present invention can be used for treating and / or preventing a disease caused by oxidative stress of cells.

  The pharmaceutical composition of the present invention may be a preparation blended with various pharmaceutically acceptable carriers (including excipients, binders, disintegrants, lubricants, etc.) in addition to the above peptides. The formulation may contain conventional additives.

  The form of the preparation is not particularly limited. For example, tablets, pills, capsules, powders, granules, syrups and the like orally administered; injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, drops And various preparation forms such as external preparations and parenteral administration agents such as suppositories. Preferable pharmaceutical forms include, for example, topical injections, coating agents for gingival mucosa, gargles and the like.

  The content of the peptide of the present invention in the preparation varies depending on the administration route, patient age, body weight, symptoms, etc., and cannot be specified unconditionally, but the daily dose of the peptide is usually about 10 to 1000 mg, more preferably 100 to 500 mg. What is necessary is just to be the amount which becomes about. When it is administered once a day, it is sufficient that this amount is contained in one preparation, and when it is administered three times a day, it is sufficient that this one-third amount is contained in one preparation.

  The pharmaceutical composition of the present invention is administered to a patient such as a mammal. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep and the like.

  The peptide of the present invention penetrates the cell membrane and binds to the transcription factor Nrf2 in the cell to prevent the binding of Nrf2 and Keap1, and prevents the degradation of Nrf2, thereby increasing the expression of the antioxidant stress enzyme group. Works. Therefore, the pharmaceutical composition comprising the peptide of the present invention as an active ingredient is effective for the prevention and treatment of diseases caused by oxidative stress, particularly diseases that can be treated by the antioxidant stress enzyme group expressed via the transcription factor Nrf2. . Examples of diseases caused by cellular oxidative stress can include a wide range of diseases that can be prevented and treated by an antioxidant stress enzyme group expressed via the transcription factor Nrf2, such as periodontal disease, rheumatoid arthritis, Examples include osteoporosis and bone metastasis of cancer.

  Periodontal disease is one of lifestyle-related diseases whose morbidity increases with age, and it develops when an excessive immune reaction is induced in periodontal tissues by infection with periodontal pathogens. In severe periodontal disease, the alveolar bone fracture causes loss of tooth support tissue, resulting in masticatory disorders and markedly lower quality of life. This bone destruction is known to be caused by the promotion of excessive bone resorption by osteoclasts, and RANKL (Yasuda H, ShimaN, Nakagawa N, Yamaguchi K, Kinosaki M) is an essential factor for osteoclast formation and activation. , Mochizuki S, TomoyasuA, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, UdagawaN, Takahashi N, Suda T. 1998 0steoclast differentiation factor is a ligand for osteoprotegerin / osteoclastogenesis-inhibitory factor and is identical to TRANCE / RANKL. Proc Natl Acad Sci US A. 95 (7): 3597-602.) Has also been reported to have increased expression at the mRNA and protein levels in periodontal tissues with periodontal disease (Kawai T, Matsuyama T, Hosokawa Y, Makihira S, Seki M, Karimbux NY, GoncalvesRB, Valverde P, Dibart S, Li YP, Miranda LA, Ernst CW, Izumi Y, Taubman MA. 2006 B and T Lymphocytes Are the Primary Sources of RANKL in the Bone Resorptive Lesion of PeriodontaI Disease. Am J Pathol 169: 987-998; and Teng YT, Nguy en H, Gao X, Kong YY, Gorczynski RM, Singh B, Ellen RP, Pennlnger JM. 2000 Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection.J Clin Invest. 106 (6): R59-67 ). In addition, regarding the type of cells that express RANKL in periodontal disease, it has been reported that lymphocytes (B cells and T cells) infiltrating the gingival tissue are the main source of this increased RANKL. (Kawai T et al., Above).

  It has been reported that Reactive Oxygen Species (ROS) works as a signal transduction system in RANKL osteoclasts (Exp. Cell Res. (2004) 301: 119-127). ROS not only acts as an intracellular signaling substance, but generally acts as a harmful substance for cells such as oxidation of nucleic acids, proteins, and lipids (Free Radic. Biol. Med. (1991) 11: 81-128; Toxicol Sci. (2009) 108: 4-18). Therefore, cells have a defense mechanism that regulates the expression of enzymes that eliminate oxidative stress via the transcription factor Nrf2 (Annu Rev Pharmacol Toxicol (2007) 47: 89-116). Nrf2 is ubiquitinated and degraded by binding to Keap1, and its transcription activity is kept low (Trends Mol Med (2004) 10: 549-557).

  The peptide of the present invention is a peptide imparted with cell membrane permeability by adding a cell membrane permeation sequence RRRRRRR to the main sequence LQLDEETGEFLPIQ mimicking the site where Nrf2 binds to Keap1, and even when administered outside the target cell, the cell membrane It has the effect of enhancing the expression of antioxidant stress enzymes via Nrf2 by shifting from permeability to cytoplasm, inhibiting the binding of Nrf2 and Keap1 and preventing Nrf2 from being degraded. When osteoclasts are targeted cells, ROS, which is an intracellular signaling substance of RANKL, is eliminated by enhancing the expression of antioxidant stress enzymes via Nrf2, and osteoclasts are inhibited by inhibiting RANKL signaling. It is possible to inhibit the differentiation and activation of. Therefore, it has a function of suppressing RANKL-dependent osteoclast formation and bone destruction, and is therefore very effective for the treatment and prevention of periodontal disease.

  In addition, similarities between bone destruction and periodontal disease in rheumatoid arthritis and osteoarthritis have been pointed out (Inflamm Res. 2004 Nov; 53 (11): 596-600. Bone lysis and inflammation. Haynes DR.), Activated lymphocytes express RANKL and RANKL and TNF-alpha play an important role in joint destruction in rheumatoid arthritis (Bone. 2002 Feb; 30 (2): 340-6. Involvement of receptor activator of NFkappaB ligand and tumor necrosis factor-alpha in bone destruction in rheumatoid arthritis. Romas E, Gillespie MT, Martin TJ.

  Regarding inhibition of bone metastasis of tumors, it is known that tumor cells express RANKL and promote osteoclast differentiation and activation, thereby causing bone metastasis.

  EXAMPLES Hereinafter, specific embodiments of the present invention will be described in detail using examples, but the present invention is not limited to these examples.

1.Cell
1.1. Mouse cells derived from RAW 264.7 monocyte cell lineage
RAW 264.7 cells were obtained from RIKEN BioResource Center.

1.2. Three primary mouse peritoneal macrophages C57 / B6 mice (Japan SLC Inc., Hamamatsu, Japan) were killed and cold phosphate buffered saline (PBS) was injected into the peritoneal cavity. The peritoneal lavage fluid was collected and centrifuged to collect cells. The cells recovered from the pelleted pellet are pre-cultured with 50 ng / ml of recombinant macrophage colony stimulating factor (M-CSF; Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 4 days, and the attached cells are peritoneally injected. Used as macrophages (Hata K, Kukita T, Akamine A, Kukita A, Kurisu K. Trypsinized osteoclast-like multinucleated cells formed in rat bone marrow cultures efficiently form resorption lacunae on dentine. Bone 1992; 13: 139-46). Recombinant M-CSF (20 ng / ml) was added to the culture medium.

2. Cell culture All cells contained 10% (V / V) fetal calf serum (HyClone; Thermo Scientific, South Logan, UT) and added antibiotics (100 U / ml penicillin and 100 mg / ml streptomycin) The cells were cultured in an alpha-modified Eagle medium (a-MEM; Wako Pure Chemical Industries, Ltd.) at 37 ° C, 5% CO2 incubator.

3. Construction of ETGE-containing peptides
The ETGE peptide (LQLDEETGEFLPIQ) sequence is reported to be identical to the binding domain of Nrf2 for the ubiquitination-promoting protein Keap1, and is reported to inhibit the binding of Keap1 to Nrf2. ETGE peptide and ETGE peptide-containing cell-permeable sequence (7R-ETGE:
RRRRRRRRLQLDEETGEFLPIQ (SEQ ID NO: 3) and ETGE-7R: LQLDEETGEFLPIQRRRRRRRR (SEQ ID NO: 4)) were synthesized by requesting the Institute of Medical Biology. The synthesis of these peptides was confirmed by HPLC analysis . About 7R-ETGE peptide, ETGE-7R peptide and ETGE peptide,
The actual purity measured by each peak ratio of signals at UV 210 nm of the HPLC fraction was 87.5%, 95%, and 98.1%, respectively. Moreover, the measured molecular weights measured by TOF-Mass for the 7R-ETGE peptide, ETGE-7R peptide, and ETGE peptide were 2882.5, 2882.4, and 1633.0, respectively. These peptides were dissolved in N, N-dimethylformamide (DMF) and stored in a (50 mM) freezer. In this example, 7R-ETGE peptide, ETGE-7R peptide, and ETGE peptide are collectively referred to as ETGE-containing peptides.

4. Osteoclast formation assay
RAW 264.7 cells in the presence or absence of recombinant sRANKL (100 ng / ml final concentration; Wako Pure Chemical Industries) and with or without the addition of ETGE-containing peptides (final concentration 50 micromolar) Then, the cells were seeded at 10 ³ cells / well in a 96-well plate. The cultured cells were stained with tartrate-resistant acid phosphatase (TRAP) using an acid phosphatase kit (Sigma Chemical Co., St Louis, MO) according to the manufacturer's instructions. Dark red diversified cells ( > 3 nuclei) were counted as TRAP positive diversified cells.

5. Real-time PCR analysis For RAW 264.7 cells treated in the same way as in “4. Osteoclastogenesis assay” above, use the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich Co., St Louis, MO) according to the manufacturer's instructions. RNA was extracted. After measuring the RNA concentration, isolated RNA (100 ng each) is reverse transcribed using iScript cDNA Supermix (Bio-Rad Laboratories, Hercules, CA) and the cDNA stock diluted in Tris-EDTA (TE) buffer (× 5) Real-time RT-PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad Laboratories) and CFX96 instrument (Bio-Rad Laboratories). PCR primers used were those used in the experiments of Kanzaki H, Shinohara F, Kajiya M. Kodama T. The Keap1 / Nrf2 Axis Plays A Role In Osteoclast Differentiation By Regulating Intracellular ROS Signaling. Journal of Biological Chemistry 2013.

6. Absorption assay
RAW 264.7 cells were seeded on a synthetic calcium phosphate substrate (bone resorption assay plate; PG Research, Tokyo, Japan) and stimulated with RANKL (100 ng / ml). For the ETGE-containing peptide addition group, the ETGE-containing peptide was added to a final concentration of 50 microM at the start of RANKL stimulation. After 7 days of culture, the cells were removed by bleach, the calcium phosphate substrate was rinsed with distilled water and dried. Photographs were taken and the average of the percentage of absorption area in the area was calculated from 12 images for each sample using Image-Jsoftware (National Institutes of Health, Bethesda, MD).

7. Intracellular ROS detection Intracellular ROS levels were detected using Total ROS / Superoxide Detection Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) according to the manufacturer's instructions. Cells were precultured for 24 hours with or without ETGE-containing peptides and then washed with PBS. Cells were stimulated with sRANKL (100 ng / ml) for 6 hours, and for the ETGE-containing peptide addition group, ETGE-containing peptide was added to a final concentration of 50 μM at the start of RANKL stimulation, washed, and 2% fetal bovine serum (FBS) It collect | recovered with PBS which added. The cell suspension was incubated with oxidative stress detection reagent for 30 minutes on ice. After washing with PBS, intracellular ROS was detected with an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ). Live monocyte / macrophage fractions were gated on a monocyte / macrophage fraction was gated on an FSC / SSC plot and ROS levels were monitored with the FL-1 channel.

8. Preparation of nucleoprotein lysate Using the DUALXtractcytoplasmic and nuclear protein extraction kit (Dualsystems Biotech AG, Schlieren, Switzerland) according to the manufacturer's instructions, Prepared from similarly processed RAW 264.7. Cultured cells were washed with PBS and treated with cell lysis buffer. After centrifugation, the nuclear pellet was washed twice and lysed using a nucleating reagent. After centrifugation, the clear supernatant was used as the nucleoprotein extract. The protein concentration of each nuclear lysate was measured using Quick Start Protein Assay Kit (Bio-Rad Laboratories) and adjusted so that the concentrations were the same. After mixing x4 sample buffer containing mercaptoethanol, the sample was heat denatured at 70 ° C. for 10 minutes.

9. Western blot analysis The nuclear lysate containing the same amount of protein prepared above was electrophoresed on TGX Precast gel (BioRad Laboratories), and iBlot blotting system (Life Technologies Japan Ltd.,
The protein was transferred to a polyvinylidene difluoride (PVDF) membrane using Tokyo, Japan). The membrane showing the protein was treated with Qentix Western Blot Signal Enhancer (Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer's instructions. After washing with deionized water, the membrane was blocked with BlockAce (DS Pharma Biomedical Inc., Osaka, Japan) for 30 minutes, and then the rabbit IgG anti-Nrf2 step (Santa Cruz Biotechnology Inc.) in Can Get Signal Solution-1 ( And Toyobo Co. Ltd, Tokyo, Japan) for 1 hour. After washing with PBS containing 0.5% Tween-20 (PBS-T), the membrane was subjected to horseradish peroxidase (HRP) -conjugated protein A / G (Thermo Fisher Scientific Inc.) Can Get Signal Solution-2 (Toyobo Co. Ltd. ) Incubated with solution for 1 hour and washed with PBS-T. Chemiluminescence was generated using Luminata Forte (EMD Millipore Corporation, Billerica, MA) and detected with the ImageQuant LAS-4000 digital imaging system (GE Healthcare Japan, Tokyo, Japan). To confirm equivalence of the loaded nucleoprotein, the membrane was re-probed with Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific Inc.) for 15 minutes, washed, blocked and anti-histone H3 Blotting was performed with an antibody (Cell Signaling Technology Japan, Tokyo, Japan) and then with HRP-conjugated protein A / G. Cytoplasmic protein samples were used for HO-1, NQO1 and GCS measurements. The antibodies used in these experiments are anti-HO-1 antibody (StressMarq Biosciences Inc, Victoria, BC), anti-NQO1 antibody (Abcam plc, Cambridge, MA) and anti-GCS antibody (Thermo Fisher Scientific Inc.). TGX Stain-Free Precast Gels (BioRad Laboratories) were used to confirm equivalence of loaded cytoplasmic proteins. Proteins in the electrophoresis gel were visualized by ultraviolet (UV) treatment before being transferred to PVDF. The visualized protein on the membrane was imaged with ImageQuant LAS-4000 under UV transillumination, and the value of total protein band density was used for calibration.

10. All animal experiment protocols for in vivo bone destruction models have been reviewed and approved by the Tohoku University Environmental and Safety Committee Animal Experiment Committee. Animal experiments were conducted in accordance with the rules for animal experiments and related activities at Tohoku University. In the in vivo bone destruction model, LPS (10 μg / site, 5 times) was repeatedly injected [Ayon Haro ER, Ukai T, Yokoyama M, Kishimoto T, Yoshinaga Y, Hara Y. Locally administered interferon-gamma accelerates lipopolysaccharide -induced osteoclastogenesis independent of immunohistological RANKL upregulation. J Periodontal Res 2011: 46: 361-73.]. This bone destruction model caused RANKL-dependent osteoclast formation and extensive bone destruction. Thirty-two 8-week-old C57B / 6 female mice (Japan SLC Inc., Hamamatsu, Japan) were used in the experiment. These mice were randomly divided into the following four groups: PBS injection group (control group); 7R-ETGE peptide injection group (7R-ETGE group); LPS-induced bone resorption group (LPS group); and LPS Induced bone resorption and 7R-ETGE peptide injection group (LPS + 7R-ETGE group). Purified LPS derived from Escherichia coli O111: B4 (Sigma Chemical Co.) was dissolved in PBS to a concentration of 1 μg / μL. 10 μL of LPS solution containing 2 μL of DMF or 7R-ETGE peptide (50 mM) was injected into LPS group and LPS + 7R-ETGE group, respectively. 10 μL of PBS containing 2 μL of DMF or 7R-ETGE peptide (50 mM) was injected into the control group and 7R-ETGE group animals, respectively. Injections were made on days 1, 3, 5, 7, and 9 with a 30 gauge needle at the point on the midline between both eyes of the skull under anesthesia. On day 11, mice were killed by cervical dislocation. RNA from the cranial tissue near the excised injection site (3 RNA samples per group) was extracted and reverse transcribed as detailed above. Gene expression was tested by real-time PCR. Other skull tissue samples (5 samples per group) were fixed overnight in 4% paraformaldehyde in PBS and the sections were X-ray microtomography (microCT) system (ScanXmate-E090; Comscan Tecno Co., Ltd., Scanned by Kanagawa, Japan). The scanned microCT image was reconstructed using ConeCTexpress software (Comscan Tecno Co., Ltd.). After reconstruction and obtaining DICOM files, 3D-volume rendering was performed using Pluto software (http://pluto.newves.org/trac).

11. Statistical analysis All data are shown as mean ± standard deviation (SD). All in vitro data are the average of three independent tests. Comparison of multiple data was performed by Tukey's test (MEPHAS; http://www.gen-info.osaka-u.ac.jp/testdocs/tomocom/tukey-e.html). P <0.05 was considered statistically significant.

result
1. Inhibition of osteoclast formation and resorption activity by ETGE-containing peptides First, it was tested whether ETGE-containing peptides have an inhibitory activity on osteoclast formation via RANKL. By TRAP staining, 7R-ETGE peptide (Figure 1B) inhibited osteoclast formation (Figure 1A), but ETGE-7R (Figure 1C) and ETGE peptide (Figure 1D) did not inhibit osteoclast formation. It was. TRAP-positive multinucleated cells were statistically different between RANKL alone and RANKL and 7R-ETGE peptides. There was no statistical difference between RANKL alone, RANKL and ETGE-7R peptides, and RANKL and ETGE peptides. Similar results were obtained with mouse primary peritoneal macrophages (FIG. 1F-I). The 7R-ETGE peptide (FIG. 1H) inhibited osteoclast formation (FIG. 1G) via RANKL. The number of TRAP-positive multinucleated cells was statistically different between RANKL alone and RANKL and 7R-ETGE peptides (FIG. 1I).

  Next, the effect of ETGE-containing peptides on RANKL-mediated osteoclast marker gene induction was examined. 7R-ETGE peptide inhibited the induction of ATP6v0d2, DC-STAMP and OSCAR via RANKL, respectively, but ETGE-7R peptide and ETGE peptide did not inhibit the induction of these genes (FIG. 2). The effect of ETGE-containing peptides on RANKL-mediated absorption activity was tested (FIG. 3). Compared to RANKL alone (FIG. 3A), the 7R-ETGE peptide inhibited RANKL-mediated absorption activity (FIG. 3B). ETGE-7R and ETGE peptides did not show an inhibitory effect on the absorption activity via RANKL (FIGS. 3C and D). FIG. 3E shows the percentage of assay plate area absorbed. Only RANKL alone and RANKL and 7R-ETGE showed statistically significant differences. These results indicate that cell-permeable 7R-ETGE peptide inhibits RANKL-mediated osteoclast formation and osteoclast activation, but ETGE-7R and ETGE peptide do not inhibit them.

2. Reduction of intracellular ROS via RANKL by 7R-ETGE peptide Next, whether or not the 7R-ETGE peptide interferes with ROS signaling via RANKL was examined using flow cytometry. 4A and B, the more the cell number peak goes to the right, the more ROS is produced in the cell. Addition of RANKL induced the formation of intracellular ROS (FIG. 4A). This ROS induction via RANKL was reduced by the addition of 7R-ETGE peptide, but no reduction was observed with ETGE-7R and ETGE peptide (FIG. 4B). These results indicate that the cell-permeable 7R-ETGE peptide decreases RANKL-mediated intracellular ROS signaling.

3. Dose-dependent nuclear Nrf2 induction by 7R-ETGE peptide
To further investigate the mechanism of RANKL-mediated osteoclastogenesis inhibition by the 7R-ETGE peptide, nuclear Nrf2 was examined by Western blot analysis (FIG. 5A). The 7R-ETGE peptide induced nuclear Nrf2 in a dose-dependent manner. These data suggest that the 7R-ETGE peptide is suitable for Nrf2-dependent transcription of the cytoprotective enzyme.

4. Up-regulation of cytoprotective enzyme expression by 7R-ETGE peptide at mRNA and protein level Next, the effect of 7R-ETGE peptide on the expression of cytoprotective enzymes such as HO1, NQO1 and GCS was tested (Fig. 5B). . Real-time PCR analysis revealed that all three genes were upregulated by 7R-ETGE peptide treatment. Similar to mRNA upregulation, protein level upregulation of HO1, NQO1 and GCS was also clearly detected by Western blot (FIG. 5C). In the relative band intensity normalized by total protein staining for the stain-free blot (Figure 5D), the expression of HO1, NQO1 and GCS increased by 2.1, 7.2, and 6.8 fold after 7R-ETGE treatment. (Figure 5E). These results suggest that the 7R-ETGE peptide upgrades cytoprotective enzyme expression through Nrf2-dependent transcription.

5. Inhibition of bone destruction by ETGE-containing peptides in experimental animals
Finally, it was tested in vivo whether 7R-ETGE peptide inhibits RANKL-mediated bone destruction. Repeated LPS injections upregulated osteoclast-forming cytokines (RANKL and TNF-α) mRNA (FIG. 6A). This increase in RANKL and TNF-α resulted in upregulation of osteoclast marker genes such as MMP9 and TRAP (FIG. 6A). The 7R-ETGE peptide significantly reduced both osteoclastogenic cytokine and osteoclast marker gene expression. MicroCT analysis revealed that repetitive injection of LPS resulted in resorption in the anti-range of the skull (FIG. 6B). The 7R-ETGE peptide was induced by this LPS and reduced bone destruction via RANKL (FIG. 6B). It was revealed that there was a statistically significant difference between the LPS alone group and the LPS and 7R-ETGE peptide added group in the area of the resorption area calculated in the skull (FIG. 6C). Furthermore, since there was no statistically significant difference between the control and the LPS and 7R-ETGE peptide addition group, the 7R-ETGE peptide was induced by LPS and almost completely suppressed bone destruction via RANKL. I understand. These data indicate that the 7R-ETGE peptide can suppress osteoclast formation even in vivo.

  As shown above, in the Examples of the present application, cell permeability is imparted without impairing the activity of the ETGE peptide by binding a certain number of arginines to the N-terminus of the ETGE peptide, resulting in osteoclast formation and resorption. Inhibition of activity, reduction of intracellular ROS, dose-dependent nuclear Nrf2 induction, up-regulation of cytoprotective enzyme expression, and suppression of bone destruction in experimental animals were revealed. In general, when an oligopeptide for imparting cell permeability is bound to a certain peptide, the action of the oligopeptide often affects the activity of the original peptide. Therefore, the effects shown in the examples of the present application are surprising.

  The peptide of the present invention can be delivered into a cell to prevent the binding of Nrf2 and Keapl, and increase the expression of the antioxidant stress enzyme group by preventing the degradation of Nrf2. Therefore, the peptide of the present invention can be used for prevention and treatment of diseases caused by oxidative stress. For example, the peptide of the present invention can prevent or suppress osteoclast activation by the mechanism described above. Therefore, the present invention leads to the development of a therapeutic agent that stops the progression of bone destruction by inhibiting osteoclast activation in bone destruction diseases, and is considered to be able to greatly contribute to the future super-aging society. .

SEQ ID NO: 1 Cell-permeable peptide SEQ ID NO: 2 ETGE peptide SEQ ID NO: 3 7R-ETGE peptide SEQ ID NO: 4 ETGE-7R peptide

Claims (5)

Following formula (1)
(R) _n LQLDEETGEFLPIQ (1)
[Wherein n represents a natural number of 6 to 12]
Or a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the above formula (1).   The peptide according to claim 1, wherein n represents a natural number of 7-9.   A pharmaceutical composition comprising the peptide according to claim 1 or 2.   The pharmaceutical composition according to claim 3, for treating and / or preventing a disease caused by cellular oxidative stress.   The pharmaceutical composition according to claim 4, wherein the disease caused by oxidative stress of the cell is at least one selected from the group consisting of periodontal disease, rheumatoid arthritis, osteoporosis, and bone metastasis of cancer.