Agent for preventing or treating osteoarthritis

Abstract

The present invention addresses the problem of providing an agent which is for preventing or treating osteoarthritis and which is capable of suppressing the M1 polarization of macrophages. The problem is solved by an agent for preventing or treating osteoarthritis, the agent comprising an oligopeptide composed of an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β strand D sequence disposed adjacent to an N-terminal side of the DE loop sequence.

Description

Preventive or therapeutic agent for osteoarthritis

[0001] The present invention relates to a preventive or therapeutic agent for osteoarthritis.

[0002] In a super-aging society, the most common cause of needing nursing care or support is disorders of the musculoskeletal system, with knee osteoarthritis accounting for the largest number of patients at approximately 25.3 million. Existing non-steroidal anti-inflammatory drugs (NSAIDs) and intra-articular injections of hyaluronic acid have a certain analgesic effect, but are insufficient in inhibiting the progression of the disease, and intra-articular injections of steroids raise concerns about cartilage necrosis.

[0003] NF-κB is a transcription factor that controls inflammation, immunity, and cell differentiation. In osteoarthritis, RANKL-RANK-NF-κB signaling promotes cartilage degeneration, while LPS-CD14 (expressed in macrophages and functions as a TLR)-NF-κB signaling induces arthritis by macrophages and promotes cartilage degeneration. Activation of Wnt signaling in cartilage also promotes cartilage degeneration. Steroids have been used as drugs that directly inhibit NF-κB. Steroids bind to the glucocorticoid receptor (GR) in cells and inhibit NF-κB transcriptional activity by directly binding to it. However, GR itself acts as a transcription factor, causing various side effects (chondronecrosis, osteoporosis, elevated blood sugar levels, gastrointestinal ulcers, etc.).

[0004] Patent Document 1 reports that RANKL peptides have an inhibitory effect on inflammatory cytokines mediated by TLRs, but there have been no reports on the preventive or therapeutic effects of RANKL peptides on osteoarthritis.

[0005] International Publication No. 2016 / 186071

[0006] The Journal of Clinical Investigation, No.7, Vol.108, 2001, pages 971-979.Mol Endocrinol, January 2009, 23(1):35-46.

[0007] One of the causes of osteoarthritis is M1 polarization of macrophages, which is thought to be one of the causes of synovitis, cartilage degeneration, bone sclerosis, osteoclast activation, etc., leading to the onset and worsening of osteoarthritis.

[0008] An objective of the present invention is to provide a preventive or therapeutic agent for osteoarthritis that can suppress M1 polarization of macrophages. Another objective that is more desirable, though not essential, is to effectively prevent or treat osteoarthritis by suppressing multiple phenomena such as worsening synovitis, cartilage degeneration, osteosclerosis, and osteoclast activation.

[0009]

[0006] In view of the above problems, the present inventors have conducted extensive research and found that an oligopeptide consisting of an amino acid sequence containing the DE loop sequence of RANKL protein and the β-strand D sequence of RANKL protein located adjacent to the N-terminus of the DE loop sequence can be used as an active ingredient in a preventive or therapeutic agent for osteoarthritis that can suppress M1 polarization of macrophages. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects:

[0010] Item 1. A preventive or therapeutic agent for osteoarthritis, comprising an oligopeptide consisting of an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is either (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is either (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0011] Item 2. The preventive or therapeutic agent according to Item 1, wherein the DE loop sequence is the amino acid sequence of (a) above.

[0012] Item 3. The preventive or therapeutic agent according to Item 1 or 2, wherein the 1 to 3 amino acids are 1 or 2 amino acids.

[0013] Item 4. The preventive or therapeutic agent according to any one of Items 1 to 3, wherein, in the amino acid sequences of (d), the N-terminal leucine residue of SEQ ID NOs: 2 and 4 and the leucine residue that is the fourth amino acid residue from the N-terminus of SEQ ID NOs: 3 and 5 are not substituted or deleted.

[0014] Item 5. The preventive or therapeutic agent according to any one of Items 1 to 4, wherein the amino acid sequence of the oligopeptide comprises a RANKL protein β-strand E sequence located adjacent to the C-terminus of the DE loop sequence.

[0015] Item 6. The preventive or therapeutic agent according to Item 5, wherein the β strand E sequence is the following (e) or (f): (e) an amino acid sequence set forth in any one of SEQ ID NOs: 6 to 9, or (f) an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in any one of SEQ ID NOs: 6 to 9.

[0016] Item 7. The preventive or therapeutic agent according to any one of Items 1 to 6, wherein the amino acid sequence of the oligopeptide does not contain the CD loop sequence of RANKL protein.

[0017] Item 8. The preventive or therapeutic agent according to any one of Items 1 to 7, wherein the amino acid sequence of the oligopeptide is either (i) or (j) below: (i) an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 21, or (j) an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in any one of SEQ ID NOs: 12 to 21.

[0018] Item 9. The preventive or therapeutic agent according to any one of Items 1 to 7, wherein the amino acid sequence of the oligopeptide is the following (i') or (j'): (i') the amino acid sequence shown in SEQ ID NO: 12, 17, 20, or 21, or (j') the amino acid sequence shown in SEQ ID NO: 12, 17, 20, or 21 in which 1 to 3 amino acids have been substituted, deleted, added, or inserted.

[0019] Item 10. The preventive or therapeutic agent according to any one of Items 1 to 9, wherein the oligopeptide is 50 amino acid residues or less in length.

[0020] Item 11. The preventive or therapeutic agent according to any one of Items 1 to 10, wherein the oligopeptide is 40 amino acid residues or less in length.

[0021] Item 12. The preventive or therapeutic agent according to any one of Items 1 to 11, wherein the osteoarthritis is osteoarthritis of at least one joint selected from the group consisting of knee joints, shoulder joints, elbow joints, hip joints, ankle joints, and finger joints.

[0022] Item 13. A bone sclerosis inhibitor comprising an oligopeptide consisting of an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is either (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is either (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0023] Item 14. A method for preventing or treating osteoarthritis, comprising administering to a patient with osteoarthritis an oligopeptide consisting of an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0024] Item 15. A method for inhibiting bone sclerosis, comprising administering to a patient with osteoarthritis an oligopeptide consisting of an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0025] Item 16. An oligopeptide for use as an agent for preventing or treating osteoarthritis, comprising an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0026] Item 17. An oligopeptide for use as a bone sclerosis inhibitor, comprising an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is either (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is either (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0027] Item 18. Use of an oligopeptide for the manufacture of an agent for the prevention or treatment of osteoarthritis, comprising an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is either (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is either (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0028] Item 19. Use of an oligopeptide for the manufacture of a bone sclerosis inhibitor, comprising an amino acid sequence including a RANKL protein DE loop sequence and a RANKL protein β-strand D sequence located adjacent to the N-terminus of the DE loop sequence, wherein the DE loop sequence is either (a) or (b) below: (a) the amino acid sequence set forth in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and the β-strand D sequence is either (c) or (d) below: (c) the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which one to three amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 2 to 5.

[0029] According to the present invention, it is possible to provide a preventive or therapeutic agent for osteoarthritis that can suppress M1 polarization of macrophages.

[0030] 1 shows the results of OARSI score measurement in Test Example 1. The vertical axis indicates the OARSI score. On the horizontal axis, "control" indicates the sham surgery group, "DMM" indicates the untreated osteoarthritis model group, and "DMM+MHP1" indicates the MHP1-administered osteoarthritis model group. The legend indicates the number of weeks since the sham surgery or the surgery to create the osteoarthritis model.

[0044] Figure 1 shows the results of immunostaining for type X collagen in Test Example 1. The vertical axis indicates the proportion of COL-X-positive cells on the articular surface. The legend for the horizontal axis is the same as in Figure 1.

[0045] Figure 1 shows the results of immunostaining for IL-1β in Test Example 1. The vertical axis indicates the proportion of IL-1β-positive cells in the synovium. The legend for the horizontal axis is the same as in Figure 1.

[0046] Figure 1 shows the results of synovitis score measurement in Test Example 1. The vertical axis indicates the synovitis score. The legend for the horizontal axis is the same as in Figure 1.

[0047] Figure 1 shows the results of immunostaining for CD86 in Test Example 1. The vertical axis indicates the proportion of CD86-positive cells in the synovium. The legend for the horizontal axis is the same as in Figure 1. 1 shows the measurement results of BV / TV of subchondral bone in Test Example 1. The vertical axis shows BV / TV (unit: %). The horizontal axis is the same as in Figure 1. 1 shows the results of immunostaining for TRAP in Test Example 1. The vertical axis shows the number of TRAP-positive cells (osteoclasts) per mm of the tibial subchondral bone section. The horizontal axis is the same as in Figure 1. 1 shows the results of immunostaining for phosphorylated Smad2 / 3 in Test Example 1. The vertical axis shows the number of TRAP-positive cells (osteoclasts) per mm of the tibial subchondral bone section. The horizontal axis is the same as in Figure 1.2 The graph shows the number of phosphorylated Smad2 / 3-positive (TGF-β signal-positive) cells per 1 mm of the tibial subchondral bone section. The legend on the horizontal axis is the same as in Figure 1. The graph shows the results of immunostaining for CD31 in Test Example 1. The vertical axis shows the number of phosphorylated Smad2 / 3-positive (TGF-β signal-positive) cells per 1 mm of the tibial subchondral bone section. 21 shows the number of CD31-positive cells (degree of angiogenesis) per 1000 cells. The legend on the horizontal axis is the same as in Figure 1. This shows the results of real-time PCR when mouse bone marrow-derived macrophages from Test Example 2 were stimulated with LPS. The measured mRNA is shown above each graph. The vertical axis shows the relative amount of mRNA. The horizontal axis shows the administered drug and its dose. This shows the results of Western blot when mouse bone marrow-derived macrophages from Test Example 2 were stimulated with LPS. The measured protein is shown on the left side of the photograph. The administered drug and its dose are shown below the photograph. This shows the results of FACS analysis when mouse bone marrow-derived macrophages from Test Example 2 were stimulated with LPS. The administered drug is shown on the left side of the graph. The vertical axis of each graph shows the expression level of the M2 marker, and the horizontal axis of each graph shows the expression level of the M1 marker. The M1 / M2 ratio is shown at the bottom left of each graph. This shows the results of TRAP staining when mouse bone marrow-derived macrophages from Test Example 2 were induced to differentiate into osteoclasts. In the graphs, the vertical axis indicates the number of TRAP-positive cells, and the horizontal axis indicates the administered drug and its dose. Figure 1 shows the results of real-time PCR in Test Example 2, where mouse bone marrow-derived macrophages were induced to differentiate into osteoclasts. Measured mRNA is shown above each graph. The vertical axis indicates the relative amount of mRNA. The horizontal axis indicates the administered drug and its dose. Figure 1 shows the results of Western blot in Test Example 2, where mouse bone marrow-derived macrophages were induced to differentiate into osteoclasts. Detected proteins are shown on the left side of the photograph. Administered drug and its dose are shown above the photograph. Figure 1 shows the results of Western blot in Test Example 2, where bone marrow-derived mesenchymal stem cells were stimulated with TGF-β. Detected proteins are shown on the left side of the photograph. Administered drug and its dose are shown above the photograph. Figure 1 shows the results of real-time PCR in Test Example 2, where bone marrow-derived mesenchymal stem cells were stimulated with TGF-β. Measured mRNA is shown above each graph. The vertical axis indicates the relative amount of mRNA. Administered drug and its dose are shown above the photograph.

[0033] Figure 1 shows the results of ALP staining when bone marrow-derived mesenchymal stem cells of Test Example 2 were induced to differentiate into bone in the presence of TGF-β. Also shown are the results of real-time PCR performed during this process. Measured mRNA is shown above the graph. The vertical axis indicates the relative amount of mRNA. The horizontal axis indicates the administered drug and its dose.

[0034] Figure 1 shows the results of Alizarin red staining when bone marrow-derived mesenchymal stem cells of Test Example 2 were induced to differentiate into bone in the presence of TGF-β.The absorbance at 450 nm at that time is also shown. The vertical axis represents absorbance. The horizontal axis represents the administered drug and its dose. The results of the cell migration assay using human umbilical vein endothelial cells in Test Example 2 are shown. Quantitative evaluation thereof is also shown. The vertical axis represents cell migration rate. The horizontal axis represents the administered drug and its dose. The results of Western blot when human umbilical vein endothelial cells in Test Example 2 were stimulated with TGF-β are shown. The detected proteins are shown on the left side of the photograph. The administered drug and its dose are shown at the top of the photograph. Quantitative evaluation is also shown. The vertical axis represents p-Smad2 / Smad2. The horizontal axis represents the administered drug and its dose.

[0031] 1. Definitions In this specification, all amino acid sequences are represented by single letter codes.

[0032] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

[0033] "Identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF, "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin S, Altschul SF, "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0034] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0035] 2. Oligopeptides

[0049] Below, oligopeptides (sometimes referred to herein as "oligopeptides of the present invention") consisting of an amino acid sequence containing the DE loop sequence of RANKL protein and the β-strand D sequence of RANKL protein located adjacent to the N-terminus of the DE loop sequence are described.

[0036] The DE loop sequence of the RANKL protein includes the following amino acid sequence (a) or (b): (a) the amino acid sequence shown in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid has been substituted, deleted, added or inserted relative to the amino acid sequence shown in SEQ ID NO: 1.

[0037] Preferably, the substitution in the amino acid sequence of (b) is a conservative substitution. Also, preferably, the addition in the amino acid sequence of (b) is an addition at the N-terminus or C-terminus.

[0038] The β-strand D sequence of the RANKL protein is not particularly limited, as long as it is an amino acid sequence that forms β-strand D in the amino acid sequence of the RANKL protein. The biological species from which the β-strand D sequence is derived is not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits. Among these, preferred are humans, monkeys, mice, and rats, more preferred are humans and mice, and even more preferred are humans. The β-strand D sequences of various biological species are known, and for example, the cases of mice and rats are described in Non-Patent Documents 1 and 2. Furthermore, even if the β-strand D sequence from a certain biological species is not known, its sequence can be easily determined based on known information (e.g., Non-Patent Documents 1 and 2). Specific examples of the β-strand D sequence of RANKL protein include the amino acid sequence shown in SEQ ID NO: 2 (a part of the β-strand D sequence of mouse-derived RANKL protein), the amino acid sequence shown in SEQ ID NO: 3 (the β-strand D sequence of mouse-derived RANKL protein), the amino acid sequence shown in SEQ ID NO: 4 (a part of the β-strand D sequence of human-derived RANKL protein), and the amino acid sequence shown in SEQ ID NO: 5 (the β-strand D sequence of human-derived RANKL protein). The β-strand D sequence may be mutated, as long as the oligopeptide of the present invention can exert an inhibitory effect on M1 polarization of macrophages, etc.

[0039] Among SEQ ID NOS: 2 to 5, the leucine residue at the N-terminus of SEQ ID NOS: 2 and 4 and the leucine residue that is the fourth amino acid from the N-terminus of SEQ ID NOS: 3 and 5 are important for suppressing macrophage M1 polarization, etc. In the β-strand D sequence, it is desirable that this leucine residue is not mutated.

[0040] The β-strand D sequence of the RANKL protein preferably includes the following amino acid sequence (c) or (d): (c) an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5.

[0041] In the above (d), the number of mutated (substituted, deleted, added or inserted) amino acids is preferably one or two, more preferably one.

[0042] In addition, in the above (d), preferred mutations include conservative substitutions. Furthermore, preferred examples of the amino acid sequence of (d) above include the following amino acid sequence (d'): (d') an amino acid sequence in which 1 to 3 amino acids have been added to the N-terminus or C-terminus (preferably the C-terminus) of the amino acid sequence shown in any of SEQ ID NOs: 2 to 5.

[0043] In the above (d'), the number of added amino acids is preferably one or two, more preferably one.

[0044] The phrase "located adjacent to the N-terminus" means that the N-terminal amino acid of the DE loop sequence and the C-terminal amino acid of the β-strand D sequence are linked by a peptide bond.

[0045] The amino acid sequence of the oligopeptide of the present invention may contain a sequence other than the above-mentioned sequences, so long as the oligopeptide of the present invention can exert an inhibitory effect on M1 polarization of macrophages, etc. The other sequence is not particularly limited, but it is desirable to determine it from the viewpoint of a longer intracellular half-life. Hydrophilicity and intracellular half-life can be predicted on various websites (e.g., EXPATHY (http: / / web.expasy.org / protparam / )). When using EXPATHY, it is preferable to design the sequence so that the "Grand average of hydropathicity" shows a negative value for hydrophilicity.

[0046] As another sequence, the β-strand E sequence of the RANKL protein is particularly preferred from the viewpoint of the effect of suppressing M1 polarization of macrophages. The β-strand E sequence is preferably located adjacent to the C-terminus of the DE loop sequence. The phrase "adjacent to the C-terminus" refers to the linkage between the C-terminal amino acid of the DE loop sequence and the N-terminal amino acid of the β-strand E sequence via a peptide bond.

[0047] The β-strand E sequence of the RANKL protein is not particularly limited, as long as it is an amino acid sequence that forms β-strand E in the amino acid sequence of the RANKL protein. The biological species from which the β-strand E sequence is derived is not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits. Among these, preferred are humans, monkeys, mice, and rats, more preferred are humans and mice, and even more preferred are humans. The β-strand E sequences of various biological species are known, and for example, the cases of mice and rats are described in Non-Patent Documents 1 and 2. Furthermore, even if the β-strand E sequence from a certain biological species is not known, its sequence can be easily determined based on known information (e.g., Non-Patent Documents 1 and 2). Specific examples of the β-strand E sequence of RANKL protein include the amino acid sequence shown in SEQ ID NO: 6 (the β-strand E sequence of mouse-derived RANKL protein), the amino acid sequence shown in SEQ ID NO: 7 (a part of the β-strand E sequence of mouse-derived RANKL protein), the amino acid sequence shown in SEQ ID NO: 8 (the β-strand E sequence of human-derived RANKL protein), and the amino acid sequence shown in SEQ ID NO: 9 (a part of the β-strand E sequence of human-derived RANKL protein). The β-strand E sequence may be mutated as long as the oligopeptide of the present invention can exert an inhibitory effect on M1 polarization of macrophages, etc.

[0048] The β-strand E sequence of the RANKL protein preferably includes the following amino acid sequence (e) or (f): (e) an amino acid sequence set forth in any of SEQ ID NOs: 6 to 9, or (f) an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 6 to 9.

[0049] In the above (f), the number of mutated (substituted, deleted, added or inserted) amino acids is preferably one or two, more preferably one.

[0050] In addition, in the above (f), preferred mutations include conservative substitutions. Furthermore, preferred examples of the amino acid sequence of (f) above include the following amino acid sequence (f'): (f') an amino acid sequence in which 1 to 3 amino acids have been added to the N-terminus or C-terminus (preferably the N-terminus) of the amino acid sequence set forth in any of SEQ ID NOs: 6 to 9.

[0051] In the above (f'), the number of added amino acids is preferably 1 or 2, more preferably 1.

[0052] The amino acid sequence of the oligopeptide of the present invention preferably does not contain the CD loop sequence of the RANKL protein.

[0053] The CD loop sequence of a RANKL protein is an amino acid sequence that forms the CD loop in the amino acid sequence of the RANKL protein. The CD loop sequences of various biological species are known, and for example, those of mice and rats are described in Non-Patent Documents 1 and 2. Specific examples of the CD loop sequence of a RANKL protein include the amino acid sequence shown in SEQ ID NO: 10 (the CD loop sequence of mouse-derived RANKL protein) and the amino acid sequence shown in SEQ ID NO: 11 (the CD loop sequence of human-derived RANKL protein).

[0054] The length of the oligopeptides of the present invention is not particularly limited, as long as it is a typical length for an oligopeptide. The length is, for example, 50 amino acid residues or less, preferably 40 amino acid residues or less, more preferably 35 amino acid residues or less, more preferably 30 amino acid residues or less, and even more preferably 25 amino acid residues or less. The length is, for example, 11 amino acid residues or more, preferably 13 amino acid residues or more, more preferably 15 amino acid residues or more, even more preferably 20 amino acid residues or more, and even more preferably 25 amino acid residues or more. The length ranges, for example, from 11 to 50 amino acid residues, preferably from 15 to 40 amino acid residues, more preferably from 20 to 35 amino acid residues, and even more preferably from 25 to 30 amino acid residues.

[0055] Preferred amino acid sequences of the oligopeptides of the present invention include oligopeptides consisting of the amino acid sequence (i) or (j) below: (i) an amino acid sequence set forth in any of SEQ ID NOs: 12 to 21, or (j) an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 12 to 21.

[0056] More preferred amino acid sequences of the oligopeptides of the present invention include oligopeptides consisting of the amino acid sequence (i') or (j') below: (i') the amino acid sequence shown in SEQ ID NO: 12, 17, 20 or 21, or (j') the amino acid sequence shown in SEQ ID NO: 12, 17, 20 or 21 in which 1 to 3 amino acids have been substituted, deleted, added or inserted.

[0057] In the above (j) and (j'), the number of mutated (substituted, deleted, added or inserted) amino acids is preferably one or two, more preferably one.

[0058] Preferably, the substitutions in the amino acid sequences (j) and (j') are conservative substitutions.

[0059] The oligopeptides of the present invention may be chemically modified, as long as they can exert the effect of suppressing M1 polarization of macrophages.

[0060] The oligopeptide of the present invention has a C-terminal carboxyl group (-COOH) or a carboxylate group (-COO ‐ ), amide (-CONH2) or ester (-COOR).

[0061] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1－2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1－2C such as alkyl group 7－14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0062] In addition, the oligopeptide of the present invention may contain a protecting group (e.g., a C group such as a formyl group or an acetyl group) in which the amino group of the N-terminal amino acid residue is protected. 1－6 C such as alkanoyl 1－6 Also included are oligopeptides protected with an acyl group or the like.

[0063] Among the above chemical modifications, preferred are amidation of the C-terminus and protection of the N-terminus with an acetyl group. Oligopeptides with an acetylated N-terminus are particularly preferred because they have excellent activity. From the viewpoint of stability, oligopeptides with an amidated C-terminus are superior. More preferred oligopeptides include those with an amidated C-terminus and an acetylated N-terminus. Particularly preferred oligopeptides include MHP1-7, which is the oligopeptide of the present invention shown in SEQ ID NO: 12, in which the C-terminus of MHP1 is amidated and the N-terminus is acetylated; MHP6-AcN, which is the oligopeptide of MHP6 with an amidated C-terminus and an acetylated N-terminus; MHP24-AcN, which is the oligopeptide of MHP24 with an amidated C-terminus and an acetylated N-terminus; and MHP24h-AcN, which is the oligopeptide of MHP24h with an amidated C-terminus and an acetylated N-terminus.

[0064] Furthermore, the oligopeptides of the present invention may be modified with known modifications used to improve drug stability, pharmacokinetics, bioavailability, etc. Examples of such modifications include polyethylene glycol chains.

[0065] The oligopeptides of the present invention may be in various forms, such as linear, branched, or cyclic, but are preferably linear. Furthermore, the oligopeptides of the present invention may be crosslinked according to or in accordance with known methods, as long as they are capable of exerting M1 polarization of macrophages or the like.

[0066] The oligopeptides of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is pharmaceutically acceptable, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0067] The oligopeptide of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0068] The oligopeptide of the present invention can be produced according to the amino acid sequence thereof by known peptide synthesis methods.

[0069] 3. Uses The oligopeptides of the present invention can inhibit M1 polarization of macrophages and can be used as an active ingredient in a preventive or therapeutic agent for osteoarthritis. Furthermore, in addition to their inhibitory effect on M1 polarization of macrophages, the oligopeptides of the present invention can also inhibit cartilage degeneration, changes in subchondral bone, inflammation of the articular surface, synovitis, macrophage infiltration, osteosclerosis of subchondral bone, osteoclast differentiation, TGF-β signaling, angiogenesis in subchondral bone, and vascular endothelial cell migration, and can therefore be used as an active ingredient in pharmaceuticals for these purposes. Furthermore, the oligopeptides of the present invention can also be used as an active ingredient in a bone sclerosis inhibitor.

[0070] In this specification, the above-mentioned agents may be collectively referred to as "agents of the present invention." The agents of the present invention can be used in various fields, such as medicine. The agents of the present invention can be applied (e.g., administered, ingested, inoculated, etc.) to animals and humans either as they are or in the form of various compositions together with conventional components.

[0071] In the agent of the present invention, the oligopeptide of the present invention may be used alone or in combination of two or more.

[0072] Examples of osteoarthritis include osteoarthritis of at least one joint selected from the group consisting of knee joints, shoulder joints, elbow joints, hip joints, ankle joints, and finger joints. Osteoarthritis can be, for example, primary osteoarthritis or secondary osteoarthritis.

[0073] As used herein, "treatment" can include concepts such as cure, remission, alleviation, mitigation, and suppression of progression of symptoms. Furthermore, "prevention" can include concepts such as not only preventing the onset of a disease, but also delaying the onset of the disease and suppressing symptoms once the disease has occurred.

[0074] The content of the active ingredient in the agent of the present invention can be appropriately determined taking into consideration the type of target disease, the desired therapeutic effect, the administration method, the treatment period, the age and body weight of the patient, etc. For example, the content of the active ingredient in the agent of the present invention can be about 0.0001 to 100 parts by weight, assuming that the total amount of the agent of the present invention is 100 parts by weight.

[0075] The administration form of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and it can be administered to mammals, including humans, by either oral administration or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration). Parenteral administration is preferred. Dosage forms for oral and parenteral administration and their preparation methods are well known to those skilled in the art, and can be prepared by standard methods, such as by mixing the active ingredient with a pharmaceutically acceptable carrier.

[0076] Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, lotions, creams, and gels), suppositories, inhalants, ophthalmic preparations, eye ointments, nasal drops, ear drops, and liposomes. For example, injectable preparations are prepared by dissolving the oligopeptide of the present invention in distilled water for injection, to which solubilizing agents, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as needed. The agents of the present invention can also be made into lyophilized preparations for preparation immediately before use.

[0077] The agent of the present invention may further contain other drugs that are effective in treating or preventing diseases. The agent of the present invention may also contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids, as needed.

[0078] Carriers used in formulating the agent of the present invention include excipients, binders, disintegrants, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, and the like that are commonly used in the art.

[0079] The dosage of the agent of the present invention can be determined based on various factors, such as the route of administration, the type of disease, the severity of symptoms, the patient's age, sex, and body weight, the severity of the disease, pharmacological findings such as pharmacokinetic and toxicological characteristics, whether a drug delivery system is used, and whether the agent is administered as part of a combination of other drugs. The dosage of the agent of the present invention can be, for example, approximately 1 μg / kg (body weight) to 10 g / kg (body weight) per day. The administration schedule of the agent of the present invention can also be determined taking into account factors similar to those for the dosage. For example, the above daily dosage can be administered 1 to 5 times per day to 1 month.

[0080] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0081] Production Example 1. Synthesis of Oligopeptides The synthesis of oligopeptides with the amino acid sequences shown in Table 1 was outsourced to ILS Inc. Except for MHP24-AcN and MHP24h-AcN, the N- and C-termini are unmodified, with the N-terminus being an amino group and the C-terminus being a carboxy group. MHP24-AcN and MHP24h-AcN are oligopeptides in which the N-terminus is acetylated and the C-terminus is amidated. All amino acid residues in these peptides are in the L-configuration.

[0082] Furthermore, we commissioned ILS Inc. to synthesize the following oligopeptides: an oligopeptide in which the methionine residue of MHP1 is replaced with the D-form (MHP1-3); an oligopeptide in which the N-terminus of MHP1 is acetylated (MHP1-6); an oligopeptide in which the N-terminus of MHP1 is acetylated and the C-terminus is amidated (MHP1-7 or MHP1-AcN); and an oligopeptide in which the C-terminus of MHP1 is amidated (MHP1-8).

[0083] Furthermore, an oligopeptide (biotinylated MHP1) in which the N-terminus of MHP1 was acetylated and a biocytin (biotinylated lysine) residue was added to the C-terminus was commissioned to be synthesized by BACHEM.

[0084] HPLC and MS confirmed that oligopeptides of the desired sequences were synthesized with high purity. Hereinafter, these peptides may be collectively referred to as "RANKL peptides."

[0085]

[0086] Test Example 1. In vivo test <1-1. Preparation of model animals and administration of RANKL peptide> A medial meniscus destabilization (DMM) osteoarthritis model was prepared using C57BJ / 6 mice (male, 8-10 weeks old). Specifically, the procedure is as follows.

[0087] After intraperitoneal administration of a triple-anesthesia mixture (medetomycin, betolfar, and midazolam) to relieve pain and provide sedation, a 1.5-cm longitudinal incision was made on the medial side of the knee joint with a scalpel. The vastus medialis muscle was then longitudinally incised proximally from the medial side of the patellar tendon, and the patella was laterally dislocated and entered the joint. The meniscotibial ligament, which connects the medial meniscus to the tibia, was then transected anteriorly with a scalpel. Finally, the patella was repositioned, and the joint capsule and skin were sutured with 5-0 nylon suture. This surgery destabilizes the medial meniscus, allowing osteoarthritis to develop through a more physiological mechanism. Cartilage degeneration was observed two weeks after surgery, and osteoarthritis was complete within approximately eight weeks. The joint pain and cartilage degeneration seen in osteoarthritis were reproduced. Starting on the day of surgery, 200 μl (600 μg) of MHP1 dissolved in 0.45% NaCl was administered intraperitoneally five times a week, and the results were compared with those of a 0.45% NaCl-treated group (untreated group). A sham-operated group also received 0.45% NaCl. The following evaluations were performed 2, 4, and 8 weeks after surgery.

[0088] <1-2. Histological Evaluation> Mice were euthanized, and the knee joints were removed by separating the mid-femur and mid-tibia. After fixation in formalin, coronal sections (5 μm) of paraffin blocks were prepared and stained with hematoxylin-eosin, safranin O, and immunostained (type X collagen, IL-1β, CD86, TRAP, phosphorylated Smad2 / 3) to evaluate the cartilage matrix, subchondral bone, and osteophyte formation. Synovial tissue was evaluated using the synovitis score proposed by Krenn et al., and cartilage tissue was evaluated using the O'Driscoll score and OARSI score.

[0089] 1-3. Results The results are shown in Figures 1 to 9. Figure 1 shows that RANKL peptide inhibits cartilage degeneration in osteoarthritis. Figure 2 shows that RANKL peptide inhibits changes in subchondral bone in osteoarthritis. Figure 3 shows that RANKL peptide inhibits inflammation of the articular surface in osteoarthritis. Figure 4 shows that RANKL peptide inhibits synovitis in osteoarthritis. Figure 5 shows that RANKL peptide inhibits macrophage infiltration in osteoarthritis. Figure 6 shows that RANKL peptide inhibits bone sclerosis (increased bone mineral density) in the subchondral bone in osteoarthritis. Figure 7 shows that RANKL peptide inhibits osteoclast counts in the tibial subchondral bone in osteoarthritis. Figure 8 shows that RANKL peptide inhibits TGF-β signaling in the tibial subchondral bone in osteoarthritis. Figure 9 shows that RANKL peptide inhibits angiogenesis in the tibial subchondral bone in osteoarthritis.

[0090] Test Example 2. In vitro test <2-1. Evaluation of apoptosis, inflammatory cytokines, and matrix-degrading enzyme production in chondrocytes and synovial cells> Primary mouse chondrocytes were collected from the knee joint surface of 5-day-old C57B / 6J mice and used in the experiment. Human synovial cells were collected from the synovium during total knee replacement surgery and used as synovial membrane-derived mesenchymal stem cells in the experiment.

[0091] The above cells were plated in a 96-well plate at 4 × 10 3 Cells were seeded at a concentration of 1000 cells / well and cultured for 24 hours. Then, 25 μl / well of MHP1 peptide was administered at a concentration of 4 mg / ml, followed by further culture for 2 hours. IL-1β (10 ng / ml) was then administered at 100 μl / well. 24, 48, and 72 hours after administration, the viable cell count reagent SF was administered, and absorbance was measured 2 hours later to assess apoptosis.

[0092] Also, 8x10 cells were cultured in a 24-well plate. 4The above cells were seeded and cultured for 24 hours. Then, 25 μl / well of MHP1 peptide was added at a concentration of 4 mg / ml and cultured for another 2 hours. IL-1β (10 ng / ml) was added at 100 μl / well and the cells were harvested 24 hours later. Real-time PCR and Western blotting were used to quantitatively evaluate inflammatory cytokines such as IL-6 and TNF-α, and extracellular matrix degrading enzymes such as MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5.

[0093] <2-2. Evaluation of macrophage polarization and anti-inflammatory activity> Macrophages are known to polarize into M1 and M2 subtypes that induce inflammatory responses depending on the surrounding conditions, and are thought to play an important role in maintaining articular cartilage homeostasis.

[0094] Murine bone marrow-derived macrophages (BMDMs) were generated by harvesting bone marrow contents from the femoral and tibial shafts of 8-week-old C57BL / 6J mice, removing blood cells, and stimulating them with M-CSF.

[0095] BMDM were plated in a 24-well plate at 8.0 × 10 4 The cells were seeded at a concentration of 1000 cells / well and cultured for 24 hours. Then, 25 μl / well of MHP1 peptide was added at a concentration of 4 mg / ml and cultured for 2 hours. The cells were then stimulated with inflammatory cytokines such as LPS and IL-1β, and harvested 24 hours later. Real-time PCR and Western blotting were used to evaluate the expression of markers characteristic of the M1 subtype, such as CD86 and iNOS, and markers characteristic of the M2 subtype, such as CD206 and IL-10. The anti-inflammatory effect was also evaluated by changes in the expression of inflammatory cytokines such as IL-6 and TNF-α. Furthermore, FACS analysis was performed using M1 and M2 markers.

[0096] <2-3. Evaluation of inhibition of osteoclast differentiation induction from macrophages> BMDMs were prepared in the same manner as above.

[0097] <2-3-1> BMDM, 2.0x10 5 The cells were seeded onto a 96-well plate at a concentration of 1 / well. M-CSF 10 ng / ml, RANKL, and MHP1 were added at the respective concentrations to induce differentiation into osteoclasts. Osteoclast formation was confirmed four days after seeding onto the 96-well plate, and TRAP staining was performed.

[0098] <2-3-2> BMDM 8.0x10 4 The cells were seeded onto a 24-well plate at a concentration of 1 / well. M-CSF 10 ng / ml, RANKL 50 ng / ml, and MHP1 were added at the respective concentrations to induce osteoclast differentiation. Cells were harvested on day 4 of treatment, and the mRNA expression levels of TRAP, MMP9, DCSTAMP, Cathepsin-K, and TRAF6 were evaluated by real-time PCR.

[0099] <2-3-3> BMDM 1.0x10 6 M-CSF (10 ng / ml), RANKL (50 ng / ml), and MHP1 (50 ng / ml) were added to induce osteoclast differentiation. On day 4, the cells were harvested and analyzed by Western blotting for p-p65, p65, p-ERK, ERK, p-p38, p38, p-IKKα, and p52.

[0100] <2-3-4> Bone marrow contents were collected from the femoral and tibial shafts of C57BL / 6J mice (8 weeks old), and blood cell and macrophage components were removed to obtain bone marrow-derived mesenchymal stem cells. 8.0 × 10 cells were placed in a 24-well plate. 4 Cells were seeded at a concentration of 1000 cells / well, and 25 μl / well of MHP1 peptide was added at a concentration of 4 mg / ml and cultured for 24 hours. TGF-β was then added at a concentration of 2 ng / ml, and cells were harvested 1 hour later. p-Smad2 and Smad2 were evaluated by Western blotting. Furthermore, MHP1 was added at various concentrations, and cells were harvested 24 hours later and TGF-β receptor 1 and 2 were evaluated by real-time PCR.

[0101] <2-3-5> Bone marrow contents were collected from the femoral and tibial shafts of C57BL / 6J mice (8 weeks old), and blood cell and macrophage components were removed to obtain bone marrow-derived mesenchymal stem cells. 8.0 × 10 cells were placed in a 24-well plate. 4 MHP1 peptide was administered at 25 μl / well at a concentration of 4 mg / ml and cultured for 24 hours. 2 ng / ml TGF-β, 10 mM β-glycerophosphate, and 50 μg / ml ascorbic acid were added to induce osteogenic differentiation. On day 4, ALP staining and real-time PCR were performed to evaluate osteogenic differentiation.

[0102] <2-3-6> Bone marrow contents were collected from the femoral and tibial shafts of C57BL / 6J mice (8 weeks old), and blood cell and macrophage components were removed to obtain bone marrow-derived mesenchymal stem cells. 8.0 × 10 cells were placed in a 24-well plate. 4 MHP1 peptide was administered at 25 μl / well at a concentration of 4 mg / ml and cultured for 24 hours. 2 ng / ml TGF-β, 10 mM β-glycerophosphate, and 50 μg / ml ascorbic acid were added to induce osteogenic differentiation. On day 20, cells were stained with Alizarin red and quantified by absorbance measurement.

[0103] <2-3-7> Human umbilical vein endothelial cells (HUVEC) were plated on a 24-well plate at 8.0 × 10 4 Cells were seeded at a concentration of 1000 cells / well and cultured until confluent. A line was drawn using the tip of a pipette, and 25 μl / well of MHP1 peptide was administered at a concentration of 4 mg / ml. After 24 hours of culture, the extent of cell migration was quantitatively assessed.

[0104] 2-4. Results The results are shown in Figures 10 to 21. Figures 10 to 12 show that RANKL peptide reduces the response of bone marrow-derived macrophages to LPS stimulation, inhibiting M1 polarization and the increase in inflammatory cytokines. Figures 13 to 15 show that RANKL peptide inhibits osteoclast differentiation of bone marrow-derived macrophages. Figures 16 and 17 show that RANKL peptide reduces TGF-β stimulation in bone marrow-derived mesenchymal stem cells. Figures 18 and 19 show that RANKL peptide inhibits bone formation in bone marrow-derived mesenchymal stem cells. Figures 20 to 21 show that RANKL peptide reduces HUVEC cell migration.

Claims

1. An amino acid sequence comprising an RANKL protein DE loop sequence and an RANKL protein β-strand D sequence arranged adjacent to the N-terminal side of the DE loop sequence, wherein the DE loop sequence is the following (a) or (b): (a) the amino acid sequence shown in SEQ ID NO: 1, or (b) an amino acid sequence in which one amino acid is substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and the β-strand D sequence is the following (c) or (d): (c) the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5, or (d) an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. A preventive or therapeutic agent for osteoarthritis containing an oligopeptide which is.

2. The preventive or therapeutic agent according to claim 1, wherein the DE loop sequence is the amino acid sequence of (a).

3. The preventive or therapeutic agent according to claim 2, wherein the 1 to 3 amino acids are 1 or 2 amino acids.

4. The preventive or therapeutic agent according to claim 1, wherein in the amino acid sequence of (d), the leucine residues at the N-terminals of SEQ ID NOs: 2 and 4 and the leucine residues which are the 4th amino acid residues from the N-terminals of SEQ ID NOs: 3 and 5 are not substituted or deleted.

5. The preventive or therapeutic agent according to claim 1, wherein the amino acid sequence of the oligopeptide contains an RANKL protein β-strand E sequence arranged adjacent to the C-terminal side of the DE loop sequence.

6. The preventive or therapeutic agent according to claim 5, wherein the β-strand E sequence is the following (e) or (f): (e) the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9, or (f) an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9.

7. The preventive or therapeutic agent according to claim 1, wherein the amino acid sequence of the oligopeptide does not contain an RANKL protein CD loop sequence.

8. The preventive or therapeutic agent according to claim 1, wherein the amino acid sequence of the oligopeptide is the following (i) or (j): (i) the amino acid sequence shown in any one of SEQ ID NOs: 12 to 21, or (j) an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in any one of SEQ ID NOs: 12 to 21.

9. The amino acid sequence of the oligopeptide is the following (i') or (j'): (i') the amino acid sequence shown in SEQ ID NO: 12, 17, 20 or 21, or (j') the amino acid sequence in which 1 to 3 amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 12, 17, 20 or 21. The prophylactic or therapeutic agent according to claim 1.

10. The prophylactic or therapeutic agent according to any one of claims 1 to 9, wherein the length of the oligopeptide is 50 amino acid residues or less.

11. The prophylactic or therapeutic agent according to claim 10, wherein the length of the oligopeptide is 40 amino acid residues or less.

12. The prophylactic or therapeutic agent according to any one of claims 1 to 9, wherein the osteoarthritis is osteoarthritis in at least one joint selected from the group consisting of the knee joint, shoulder joint, elbow joint, hip joint, ankle joint, and finger joint.

13. An osteosclerosis inhibitor containing an oligopeptide consisting of an amino acid sequence including an RANKL protein DE loop sequence and an RANKL protein β-strand D sequence arranged adjacent to the N-terminal side of the DE loop sequence, wherein the DE loop sequence is the following (a) or (b): (a) the amino acid sequence shown in SEQ ID NO: 1, or (b) the amino acid sequence in which 1 amino acid is substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and the β-strand D sequence is the following (c) or (d): (c) the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5, or (d) the amino acid sequence in which 1 to 3 amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5.

Images