WO2017150371A1 - Agent thérapeutique pour lésion tissulaire - Google Patents

Agent thérapeutique pour lésion tissulaire Download PDF

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WO2017150371A1
WO2017150371A1 PCT/JP2017/007069 JP2017007069W WO2017150371A1 WO 2017150371 A1 WO2017150371 A1 WO 2017150371A1 JP 2017007069 W JP2017007069 W JP 2017007069W WO 2017150371 A1 WO2017150371 A1 WO 2017150371A1
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myd88
amino acid
inhibitor
cells
acid sequence
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PCT/JP2017/007069
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English (en)
Japanese (ja)
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健太 丸山
ミカエル エム． マルティーノ
審良　静男
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国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention relates to a therapeutic agent for tissue damage and the like.
  • Living tissue can be damaged in various diseases and the course of treatment thereof.
  • skin tissue, muscle tissue, bone tissue, or the like is damaged due to trauma, bed slip, or the like, or the tissue is artificially damaged during surgery.
  • Treatment of these tissue damages is usually performed by protecting the damaged site and promoting natural healing.
  • regeneration of this tissue damage can be promoted by locally administering mesenchymal stem cells to the damaged site (Non-patent Documents 1 and 2).
  • QOL patient quality of life
  • an inflammatory reaction usually occurs at the damaged site, followed by cell migration to the damaged site, cell division, production of extracellular matrix, etc., and finally the tissue is reconstructed.
  • the first inflammatory reaction is important, and if this is inhibited by any factor, it is said that the period until healing is prolonged.
  • Interleukin-1 receptor type 1 is a type of receptor with interleukin-1 (IL-1) as a ligand, and signals downstream via intracellular adapter molecules such as MyD88. It is known to control the expression of genes related to inflammatory reaction, immune reaction, cell proliferation and the like. These signal pathways are called Toll-like receptor pathways and play an important role in inducing inflammation.
  • An object of the present invention is to provide a therapeutic agent for tissue damage that can further promote regeneration of tissue damage.
  • the present inventor surprisingly reduced the expression level or function of IL-1R1 or MyD88, thereby migrating mesenchymal stem cells to the damaged site and It has been found that proliferation can be promoted and further regeneration of tissue damage can be promoted. Furthermore, it has also been found that these effects are enhanced by using mesenchymal stem cells in combination. As a result of further research based on these findings, the present inventor has completed the present invention.
  • the present invention includes the following embodiments: Item 1. A therapeutic agent for tissue damage comprising at least one selected from the group consisting of an IL-1R1 function inhibitor, an IL-1R1 expression inhibitor, a MyD88 function inhibitor, and a MyD88 expression inhibitor.
  • Item 2. Item 2. The tissue damage therapeutic agent according to Item 1, further comprising a scaffold.
  • Item 3. Item 3. The tissue damage therapeutic agent according to Item 2, wherein the scaffold is a gel or porous material of a biocompatible material.
  • Item 4. Item 4. The tissue damage therapeutic agent according to Item 2 or 3, wherein the scaffold is fibrin gel.
  • Item 6. Item 6.
  • the tissue damage therapeutic agent according to Item 5 wherein the mesenchymal stem cells are mesenchymal stem cells derived from a tissue to be treated.
  • Item 7. Item 7. The tissue damage therapeutic agent according to any one of Items 1 to 6, further comprising a growth factor.
  • the tissue damage therapeutic agent according to any one of Items 1 to 7, comprising at least one selected from the group consisting of an IL-1R1 function inhibitor and a MyD88 function inhibitor.
  • Item 9. Item 9. The tissue damage therapeutic agent according to any one of Items 1 to 8, wherein the tissue is bone tissue.
  • An oligopeptide comprising the following amino acid sequence (i) or (j) and having MyD88 dimerization inhibitory activity: (I) In order from the N-terminal side, a scaffold binding sequence, an enzyme cleavage sequence, a membrane permeation sequence, an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 18 or 19 is arranged, or (j) the amino acid sequence of (i) above An amino acid sequence in which one or several amino acids are substituted, deleted, added or inserted.
  • Item 13 A method for treating tissue damage, comprising administering at least one selected from the group consisting of an IL-1R1 function inhibitor, an IL-1R1 expression inhibitor, a MyD88 function inhibitor, and a MyD88 expression inhibitor.
  • Item 14 At least one drug selected from the group consisting of an IL-1R1 function inhibitor, an IL-1R1 expression inhibitor, a MyD88 function inhibitor, and a MyD88 expression inhibitor for the treatment of tissue damage.
  • Item 15. Use of at least one drug selected from the group consisting of an IL-1R1 function inhibitor, an IL-1R1 expression inhibitor, a MyD88 function inhibitor, and a MyD88 expression inhibitor for the manufacture of a tissue damage therapeutic agent.
  • Item 16 Use of at least one drug selected from the group consisting of IL-1R1 function inhibitor, IL-1R1 expression inhibitor, MyD88 function inhibitor, and MyD88 expression inhibitor for the treatment of tissue damage.
  • the present invention includes the following embodiments: Item 12.
  • a method for treating tissue damage comprising administering at least one selected from the group consisting of an IL-1R1 function inhibitor, an IL-1R1 expression inhibitor, a MyD88 function inhibitor, and a MyD88 expression inhibitor.
  • Item 13 At least one agent selected from the group consisting of an IL-1R1 function inhibitor, an IL-1R1 expression inhibitor, a MyD88 function inhibitor, and a MyD88 expression inhibitor for use as a tissue damage treatment agent.
  • a therapeutic agent for tissue damage can be provided. Furthermore, at least one promoter selected from the group consisting of proliferation and accumulation of mesenchymal stem cells can also be provided. By using these, tissue damage can be efficiently treated in a shorter period of time.
  • FIG. 1 It is a graph which shows the micro CT analysis result at the time of using the MyD88 knockout mouse of Example 1.
  • the vertical axis (Coverage (%)) is the average value of the ratio of the area of the damaged part in the circle (damage recovery rate) to the area of the damaged part range (circle with a diameter of 5 mm) at the time of damage (n ⁇ ) 6) is shown.
  • “wt” indicates a case where a wild type mouse is used
  • “Myd88 ⁇ / ⁇ ” indicates a case where a MyD88 knockout mouse is used.
  • the bar at the top of each column indicates the standard deviation, and “***” indicates that the P value is less than 0.001.
  • FIG. 3 is a photograph showing the results of micro CT analysis in Example 1 using MyD88 knockout mice.
  • “wt” indicates a case where a wild-type mouse is used
  • “Myd88 ⁇ / ⁇ ” indicates a case where a MyD88 knockout mouse is used.
  • the circle drawn with a dotted line shows the range of the damaged part at the time of damage (circle with a diameter of 5 mm).
  • white areas indicate areas where bone tissue exists, and black areas indicate areas where bone tissue does not exist.
  • the vertical axis (Coverage (%)) is the average value of the ratio of the area of the damaged part in the circle (damage recovery rate) to the area of the damaged part range (circle with a diameter of 5 mm) at the time of damage (n ⁇ ) 6) is shown.
  • “wt” indicates the case where a wild type mouse is used
  • “Il1r1 ⁇ / ⁇ ” indicates the case where an IL-1R1 knockout mouse is used.
  • the bar at the top of each column indicates the standard deviation
  • “***” indicates that the P value is less than 0.001.
  • 2 is a photograph showing the results of micro CT analysis in Example 1 using IL-1R1 knockout mice.
  • the vertical axis (Coverage (%) is the average value of the ratio of the area of the damaged part in the circle (damage recovery rate) to the area of the damaged part range (circle with a diameter of 5 mm) at the time of damage (n ⁇ ) 6) is shown.
  • FIG. 6 is a graph showing the results of flow cytometry in Example 3.
  • wt indicates that a mesenchymal stem cell derived from a wild type mouse is used
  • Il1r1 ⁇ / ⁇ indicates that a mesenchymal stem cell derived from an IL-1R1 knockout mouse indicates “Myd88 ⁇ / ⁇ ”.
  • FIG. 6 is a graph showing the flow cytometry result of Example 4.
  • “wt” indicates a case where a wild type mouse is used
  • “Il1r1 ⁇ / ⁇ ” indicates a case where an IL-1R1 knockout mouse is used
  • Myd88 ⁇ / ⁇ ” indicates a case where a MyD88 knockout mouse is used.
  • the bar at the top of each left column indicates the standard deviation, and “**” indicates that the P value is less than 0.01.
  • 10 is a graph showing the micro CT analysis results of Example 5.
  • “Fibrin only” indicates that when nothing is added to the polymerized fibrin matrix
  • “IL-1Ra” indicates that when an IL-1R1 inhibitor is added
  • “ ⁇ 2 -PI 1-8 -MyD88-I” indicates When a MyD88 inhibitor is added
  • “MSCs” is added when a mesenchymal stem cell is added
  • “MSCs + IL-1Ra” is added when a mesenchymal stem cell and an IL-1R1 inhibitor are added
  • “MSCs + ⁇ 2 -PI 1-8 -MyD88- “I” shows the case where mesenchymal stem cells and MyD88 inhibitor are added.
  • Example 5 is a photograph showing the results of micro CT analysis of Example 5.
  • “Fibrin only” indicates that when nothing is added to the polymerized fibrin matrix
  • “IL-1Ra” indicates that when an IL-1R1 inhibitor is added
  • “ ⁇ 2 -PI 1-8 -MyD88-I” indicates When a MyD88 inhibitor is added
  • “MSCs” is added when a mesenchymal stem cell is added
  • “MSCs + IL-1Ra” is added when a mesenchymal stem cell and an IL-1R1 inhibitor are added
  • “MSCs + ⁇ 2 -PI 1-8 -MyD88- "I” shows the case where mesenchymal stem cells and MyD88 inhibitor are added.
  • the circle drawn with a dotted line shows the range of the damaged part at the time of damage (circle with a diameter of 5 mm).
  • white areas indicate areas where bone tissue exists, and black areas indicate areas where bone tissue does not exist.
  • a is a graph showing the micro CT analysis results of Example 6
  • b is a photograph showing the micro CT analysis results of Example 6.
  • “Fibrin” is added to the polymerized fibrin matrix
  • “FGF-2”, “PDGF-BB” and “BMP-2” are “P” when these growth factors are added.
  • “” Indicates a case where a MyD88 inhibitor is added
  • “PDGF-BB + P” and “BMP-2 + P” indicate a case where a MyD88 inhibitor and a growth factor are added.
  • the bar at the top of each column indicates the standard deviation, and “***” indicates that the P value is less than 0.001.
  • identity of amino acid sequences means the degree of amino acid sequences of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the identity of two amino acid sequences, the higher the identity or similarity of those sequences.
  • the level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters.
  • FASTA a sequence analysis tool
  • the algorithm BLAST by Karlin and Altschul Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessing the statisticalsignificance of molecular sequence features by using general scoringschemes” Proc. Natl Acad Sci USA. 87: 2264-2268 (SF), Karlin, Sul.
  • conservative substitution means that an amino acid residue is substituted with an amino acid residue having a similar side chain.
  • substitution with amino acid residues having basic side chains such as lysine, arginine, histidine and the like is a conservative substitution technique.
  • 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 non-polar side chains such as isoleucine, proline, phenylalanine, methionine, tryptophan
  • amino acid residues having ⁇ -branched side chains such as threonine, valine, isoleucine
  • aromas such as tyrosine, phenylalanine, tryptophan, histidine
  • substitutions between amino acid residues having a family side chain are conservative substitutions.
  • Tissue damage therapeutic agent mesenchymal stem cell proliferation promoter, mesenchymal stem cell accumulation promoter
  • This invention relates to IL-1R1 function inhibitor, IL-1R1 expression inhibitor, MyD88 function inhibitor, and MyD88 expression inhibitor Proliferation of mesenchymal stem cells and mesenchymal stem cells containing at least one selected from the group consisting of these (in the present specification, these may be collectively referred to as “active ingredients of the present invention”).
  • An accelerator and an agent for promoting mesenchymal stem cell accumulation are provided. Hereinafter, these will be described.
  • IL-1R1 interleukin-1 receptor type 1
  • IL-1R1 interleukin-1 receptor type 1
  • IL-1R1 is an IL expressed by cells (especially mesenchymal stem cells) present in or around the damaged site to be treated There is no particular limitation as long as the function of -1R1 can be suppressed.
  • the “cells present at the damaged site” include not only cells originally present at the damaged site of the living body but also cells added from outside the living body (for example, cells contained in the drug of the present invention (especially mesenchymal system).
  • the concept also includes stem cells)).
  • “Suppressing IL-1R1 function” means, for example, directly acting on IL-1R1 to inhibit signal transduction via IL-1R1, more specifically, acting directly on IL-1R1, It means to suppress the production of cytokines (for example, IL-6, CCL2, CXCL1, CXCL2, etc.) by (for example, IL-1 ⁇ ).
  • cytokines for example, IL-6, CCL2, CXCL1, CXCL2, etc.
  • the IL-1R1 that is the target of function suppression is IL-1R1 that is expressed by cells present in or around the damaged site that is the target of treatment. Therefore, depending on the species from which the cells are derived, the species from which IL-1R1 is derived also changes. Examples of the biological species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.
  • human IL-1R1 includes a protein (NCBI Reference Reference Sequence: NP_000868.1, NP_001275635.1) having the amino acid sequence shown in SEQ ID NOS: 1-2, and mouse IL-1R1 has a sequence.
  • proteins NCBI Reference Sequence: NP_001116854.1, NP_032388.1 having the amino acid sequences shown in Nos. 3-4.
  • IL-1R1 may be partially deleted due to physiological processing as long as its function is not impaired.
  • IL-1R1 accepts ligands (eg IL-1 ⁇ ) and induces downstream signal transduction to promote cytokine production (eg IL-6, CCL2, CXCL1, CXCL2 etc.) As long as it has (activity), it may have mutations such as amino acid substitution, deletion, addition, and insertion.
  • the mutation preferably includes substitution, more preferably conservative substitution, from the viewpoint that the cytokine production promotion mediating activity is less likely to be impaired.
  • IL-1R1 include proteins described in (a) below and proteins described in (b) below: (A) a protein comprising the amino acid sequence shown in any of SEQ ID NOs: 1 to 4, and (b) consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 4. And at least one selected from the group consisting of proteins having cytokine production promotion mediating activity.
  • the identity is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more.
  • the presence or absence of cytokine production promotion mediating activity can be determined according to or according to a known method. For example, cytokines produced by contacting a ligand (eg IL-1 ⁇ ) of IL-1R1 with a cell (especially a mesenchymal stem cell) in which the test protein is expressed on the cell membrane (eg IL-6, If the amount of CCL2, CXCL1, CXCL2, etc.) is greater than the amount of cytokine similarly measured using cells that do not express the test protein, it can be determined that the test protein has a cytokine production promotion mediating activity it can.
  • a ligand eg IL-1 ⁇
  • a cell especially a mesenchymal stem cell
  • the amount of CCL2, CXCL1, CXCL2, etc. is greater than the amount of cytokine similarly measured using cells that do not express the test protein, it can be determined that the test protein has a cytokine production promotion mediating activity it can.
  • (b ′) one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4. And a protein having an activity of promoting cytokine production.
  • the plurality is, for example, 2 to 85, preferably 2 to 30, more preferably 2 to 10, even more preferably 2 to 5, More preferably, it is 2 or 3.
  • IL-1R1 function inhibitor examples include IL-1R1 antagonists, neutralizing antibodies, and the like, and when these are proteins, their expression vectors.
  • IL-1R1 antagonists include endogenous IL-1R antagonists (IL-1 receptor antagonist (IL-1Ra)); anakinra, rilonacept and the like that are commercially available as IL-1 receptor antagonists.
  • endogenous IL-1R antagonists are preferable from the viewpoint of more reliably exerting a tissue damage treatment effect.
  • endogenous IL-1R antagonists for example, endogenous IL-1R antagonists derived from various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits and the like can be employed. Among them, an endogenous IL-1R antagonist of a species to be treated for tissue damage is preferable.
  • endogenous IL-1R antagonists derived from various species are known.
  • a human endogenous IL-1R antagonist a protein comprising the amino acid sequence shown in SEQ ID NOs: 5 to 8 (NCBI Reference Reference Sequence: NP_000568.1, NP_776214.1, NP_776215.1, NP_776213.1)
  • mouse endogenous IL-1R antagonists include proteins having the amino acid sequences shown in SEQ ID NOs: 9 to 11 (NCBI Reference Sequence: NP_001034790.1, NP_001153034.1, NP_112444.1) and the like.
  • the endogenous IL-1R antagonist may have mutations such as amino acid substitution, deletion, addition and insertion as long as it has IL-1R1 antagonist activity.
  • the mutation preferably includes substitution, more preferably conservative substitution, from the viewpoint that the IL-1R1 antagonist activity is more difficult to be impaired.
  • Preferred specific examples of the endogenous IL-1R antagonist include the protein described in the following (c) and the protein described in the following (d): (C) a protein comprising the amino acid sequence shown in any of SEQ ID NOs: 5 to 11, and (d) an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 5 to 11 And at least one selected from the group consisting of proteins having IL-1R1 antagonist activity.
  • the identity is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more.
  • the presence or absence of IL-1R1 antagonist activity can be determined according to or according to a known method.
  • cytokines for example, IL-6, CCL2, CXCL1, CXCL2, etc.
  • a test substance for example, mesenchymal stem cells
  • a ligand for example, IL-1 ⁇
  • (d ′) one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in any of SEQ ID NOs: 5 to 11. And a protein having a T-cadherin binding ability.
  • the number is, for example, 2 to 25, preferably 2 to 10, more preferably 2 to 5, and further preferably 2 or 3.
  • the endogenous IL-1R antagonist may be chemically modified as long as it has IL-1R1 antagonist activity.
  • the endogenous IL-1R antagonist may be any one of a carboxyl group (—COOH), a carboxylate (—COO ⁇ ), an amide (—CONH 2 ) and an ester (—COOR) at the C-terminus.
  • R in the ester for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C 6-12 aryl groups such as ⁇ -naphthyl; C 7- such as phenyl-C 1-2 alkyl groups such as benzyl and phenethyl; ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl; 14 aralkyl group; pivaloyloxymethyl group is used.
  • a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl
  • a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl
  • a carboxyl group (or carboxylate) other than the C-terminus may be amidated or esterified.
  • ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
  • the amino group of the N-terminal amino acid residue is protected with a protecting group (for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as formyl group, acetyl group, etc.).
  • a protecting group for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as formyl group, acetyl group, etc.
  • N-terminal glutamine residues that can be cleaved in vivo, pyroglutamine oxidized, substituents on the side chain of amino acids in the molecule (eg —OH, —SH, amino group, imidazole) Group, indole group, guanidino group, etc.) protected with an appropriate protecting group (for example, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group, acetyl group, etc.), or sugar chain Also included are complex proteins such as so-called glycoproteins to which is bound.
  • substituents on the side chain of amino acids in the molecule eg —OH, —SH, amino group, imidazole) Group, indole group, guanidino group, etc.
  • an appropriate protecting group for example, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group, acetyl group, etc.
  • complex proteins such as so-called glycoprotein
  • the endogenous IL-1R antagonist may have a known protein tag added as long as it has IL-1R1 antagonist activity.
  • protein tags include histidine tags, FLAG tags, GST tags, and the like.
  • the endogenous IL-1R antagonist 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 a pharmaceutically acceptable salt, and both an acidic salt and a basic salt can be adopted.
  • acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, apple Organic acid salts such as acid salts, citrate salts, methanesulfonate salts, paratoluenesulfonate salts; amino acid salts such as aspartate salts and glutamate salts.
  • basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts.
  • the endogenous IL-1R antagonist 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, acetic acid and the like.
  • An endogenous IL-1R antagonist can be obtained from a transformant containing a polynucleotide encoding an endogenous IL-1R antagonist according to a known method, for example, chemical synthesis, purification from mammalian cells or tissues (eg, serum). It can be obtained by purification of When obtained by purification from a transformant, the transformant is not particularly limited as long as it is a cell capable of expressing an endogenous IL-1R antagonist from a polynucleotide encoding the endogenous IL-1R antagonist. Various cells such as bacteria, insect cells, mammalian cells, etc. can be used.
  • insect cells for example, Sf cells, MG1 cells, High Five TM cells, BmN cells and the like are used.
  • Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells and the like are used.
  • animal cells examples include monkey COS-7 cells, monkey Vero cells, Chinese hamster cells CHO, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells.
  • IL-1R1 neutralizing antibody refers to an antibody having the property of inhibiting IL-1R1's cytokine production promotion mediated activity by binding to IL-1R1.
  • the antibody includes a part of the above antibody having antigen binding properties such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a fragment generated by a Fab fragment, a Fab expression library, or the like.
  • the antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of at least continuous amino acid sequence of IL-1R1, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.
  • the antibody is preferably an antibody having an antigen-binding property with respect to the amino acid sequence of the IL-1R1 ligand (preferably IL- ⁇ ) binding site, from the viewpoint that neutralizing activity can be more reliably exhibited.
  • the ligand (preferably IL- ⁇ ) binding site of IL-1R1 is known, and based on this known information, such an antibody can be produced.
  • the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)).
  • an oligopeptide having a partial amino acid sequence of IL-1R1 using IL-1R1 expressed and purified in Escherichia coli according to a conventional method or according to a conventional method Can be synthesized to immunize non-human animals such as rabbits, and obtained from the sera of the immunized animals according to a conventional method.
  • IL-1R1 which is used as an immunizing antigen for the production of antibodies, is based on known gene sequence information. DNA cloning, construction of each plasmid, transfection into a host, culture of transformants, and the transfer of proteins from the culture. It can be obtained by a recovery operation. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
  • a recombinant DNA capable of expressing a gene encoding IL-1R1 in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured.
  • a protein as an immunizing antigen for producing the antibody of the present invention can be obtained.
  • the partial peptide of IL-1R1 can also be produced by a general chemical synthesis method (peptide synthesis) in accordance with known gene sequence information.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of IL-1R1.
  • the oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has the same immunogenic properties as IL-1R1.
  • An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of IL-1R1 can be exemplified.
  • Such an antibody against an oligo (poly) peptide can also be produced by enhancing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.
  • the expression vector comprises a polynucleotide comprising a promoter sequence and a coding sequence (and optionally a transcription termination signal sequence) such as an IL-1R1 antagonist, neutralizing antibody.
  • the expression vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids; virus vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses.
  • plasmid vectors such as animal cell expression plasmids
  • virus vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses.
  • the promoter is not particularly limited, and examples thereof include CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, ⁇ actin promoter, CAG promoter and the like.
  • the expression vector may contain other elements that the expression vector can contain.
  • other elements include a replication origin and a drug resistance gene.
  • the drug resistance gene is not particularly limited. For example, chloramphenicol resistance gene, tetracycline resistance gene, neomycin resistance gene, erythromycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, puromycin resistance gene, etc. Is mentioned.
  • the expression vector can be easily obtained according to a known genetic engineering technique. For example, it can be prepared using PCR, restriction enzyme cleavage, DNA ligation technology, and the like.
  • IL-1R1 expression inhibitor can reduce the amount of IL-1R1 expressed in cells (especially mesenchymal stem cells) present at or around the damaged site to be treated As long as it is not particularly limited.
  • the definitions of “cells present at the site of injury” and “inhibit IL-1R1 function” are the same as those described above in “2-1-1. IL-1R1 function inhibitor”.
  • IL-1R1 that is subject to expression suppression is the same as IL-1R1 in “2-1-1. IL-1R1 function inhibitor” above.
  • IL-1R1 expression inhibitor examples include IL-1R1-specific siRNA, IL-1R1-specific miRNA, IL-1R1-specific antisense nucleic acid, and expression vectors thereof.
  • IL-1R1-specific siRNA is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of a gene encoding IL-1R1.
  • the siRNA is preferably 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length, for example.
  • the siRNA preferably has a length of 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. It is assumed that the upper limit value and the lower limit value of the siRNA length described here are arbitrarily combined.
  • the lower limit is 18 bases
  • the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases
  • the lower limit is 19 bases
  • the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases
  • Length lower limit is 20 bases
  • upper limit is 25 bases, 24 bases, 23 bases, or 22 bases
  • lower limit is 21 bases
  • upper limit is 25 bases, 24 bases, 23 bases, or 22
  • a length that is a base is assumed.
  • the siRNA may be shRNA (small hairpin RNA).
  • shRNA can be designed so that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is a sequence a and the complementary strand to the sequence a is a sequence b, these sequences are present in a single RNA strand in the order of sequence a, spacer, and sequence b. And can be designed to be 45-60 bases in total.
  • the sequence a is a sequence of a partial region of the base sequence encoding IL-1R1 to be a target, and the target region is not particularly limited, and any region can be a candidate.
  • the length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.
  • the IL-1R1-specific siRNA may have an additional base at the 5 'or 3' end.
  • the length of the additional base is usually about 2 to 4 bases.
  • the additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples include, but are not limited to, ', uuuuuu-3'.
  • SiRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specifically includes those to which dTdT (dT represents deoxyribonucleic acid) is added. Further, it may be a blunt end (blunt end) without end addition.
  • the siRNA may have a different number of bases in the sense strand and the antisense strand, for example, “aiRNA” in which the antisense strand has a protruding sequence (overhang) at the 3 ′ end and the 5 ′ end. be able to.
  • a typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and has an overhang structure of 3 bases at each end of the antisense strand.
  • the target sequence of the IL-1R1-specific siRNA is not particularly limited, in one embodiment, the target sequence is from the 5′-UTR and the start codon to about 50 bases, and from a region other than the 3′-UTR. It is desirable to select.
  • a sense strand having a 3 'terminal overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a 3 ′ end overhang of UU may be designed as an siRNA.
  • siRNA short hairpin RNA
  • an arbitrary linker sequence for example, about 5-25 bases
  • the sense strand and the antisense strand are combined with each other. It can be designed by linking via a linker sequence.
  • siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include the following. SiRNA Target Finder provided by Ambion (Http://www.ambion.com/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer (R) Expression Vector (http://www.ambion.com/techlib/misc/psilencer_converter .html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).
  • the siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing a short hairpin RNA (shRNA) serving as a precursor of siRNA and cleaving it with a dicer.
  • shRNA short hairpin RNA
  • IL-1R1-specific miRNA is optional as long as it inhibits translation of the gene encoding IL-1R1.
  • miRNA miRNA
  • the miRNA may pair with the 3 'untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA like siRNA.
  • the miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA, and mature miRNA.
  • the length of miRNA is not particularly limited, the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 ⁇ 30 bases.
  • the IL-1R1-specific miRNA is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA.
  • Such IL-1R1-specific miRNA may be synthesized by a known method or purchased from a company that provides synthetic RNA.
  • IL-1R1-specific antisense nucleic acid is a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of mRNA of a gene encoding IL-1R1, or a part thereof, and is specific to the mRNA. It is a nucleic acid having a function of suppressing IL-1R1 protein synthesis by forming and binding an ideal and stable duplex.
  • the antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera.
  • the antisense nucleic acid is DNA
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA is recognized by the endogenous RNase H and causes selective degradation of the target RNA.
  • the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of IL-1R1 gene.
  • the intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the IL-1R1 gene using a homology search program such as BLAST or FASTA.
  • the target region of the IL-1R1-specific antisense nucleic acid is not limited in length as long as the antisense nucleic acid hybridizes and as a result, translation into the IL-1R1 protein is inhibited.
  • the IL-1R1-specific antisense nucleic acid may be the entire sequence or partial sequence of mRNA encoding IL-1R1.
  • an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto.
  • 5 ′ end hairpin loop of IL-1R1 gene 5 ′ end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3 ′ end untranslated region, 3 ′ end palindromic region or A 3 ′ end hairpin loop or the like can be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
  • IL-1R1-specific antisense nucleic acids not only hybridize with the mRNA and initial transcripts of the IL-1R1 gene to inhibit translation into proteins, but also bind to these genes, which are double-stranded DNA, to form a triplex. It may be one that forms a strand (triplex) and can inhibit transcription to RNA (antigene).
  • nucleotide molecules constituting the IL-1R1-specific siRNA, IL-1R1-specific miRNA, and IL-1R1-specific antisense nucleic acid are stable (chemical and / or anti-enzyme) and specific activity (with RNA).
  • Various chemical modifications may be included to improve (affinity).
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate etc. It can be substituted with a phosphate residue.
  • the base moiety pyrimidine, purine
  • a part of nucleotide molecules constituting siRNA or miRNA may be replaced with natural DNA.
  • IL-1R1-specific siRNA, IL-1R1-specific miRNA, IL-1R1-specific antisense nucleic acid, etc. determine the target sequence of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of IL-1R1 gene However, it can be prepared by synthesizing a complementary sequence using a commercially available DNA / RNA automatic synthesizer. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a known method.
  • the expression vector of IL-1R1-specific siRNA, IL-1R1-specific miRNA, or IL-1R1-specific antisense nucleic acid is the same as the expression vector in “2-1-1. IL-1R1 function inhibitor” above. is there.
  • a promoter of the expression vector a polII promoter can be used as in the above “2-1-1. IL-1R1 function inhibitor”, but in order to perform transcription of a short RNA accurately, It is preferable to use a polIII promoter.
  • the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter and the like.
  • IL-1R1-specific ribozyme in a narrow sense, means RNA having an enzymatic activity to cleave nucleic acid, but in this specification, DNA is also included as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has an advantage that it does not attack genomic DNA because it uses only RNA as a substrate.
  • the target sequence is single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
  • ribozymes when used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • MyD88 function inhibitor is not particularly limited as long as it can suppress the function of MyD88 expressed by cells (particularly mesenchymal stem cells) present in or around the damaged site to be treated.
  • the “cells present at the damaged site” include not only cells originally present at the damaged site of the living body but also cells added from outside the living body (for example, cells contained in the agent of the present invention (especially mesenchymal system).
  • the concept also includes stem cells)).
  • “Suppressing the function of MyD88” means, for example, directly acting on MyD88 to inhibit signal transduction via MyD88, more specifically acting directly on MyD88 (for example, by binding to MyD88. It means inhibiting the production of cytokines (eg IL-6, CCL2, CXCL1, CXCL2, etc.) by a ligand (eg IL-1 ⁇ ) by inhibiting dimerization).
  • cytokines eg IL-6, CCL2, CXCL1, CXCL2, etc.
  • a ligand eg IL-1 ⁇
  • MyD88 which is the target of function suppression, is MyD88 in which cells present in or around the damaged site to be treated are expressed. Therefore, depending on the species from which the cells are derived, the species from which MyD88 is derived also changes. Examples of the biological species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.
  • the amino acid sequences of MyD88 derived from various species are known. Specifically, for example, a protein consisting of the amino acid sequence shown in SEQ ID NOs: 12 to 16 as human MyD88 (NCBI Reference Reference Sequence: NP_001166037.1, NP_001166040.1, NP_001166039.1, NP_001166038.1, NP_002459.2) Examples of the mouse MyD88 include a protein having the amino acid sequence shown in SEQ ID NO: 17 (NCBI Reference Sequence: NP_034981.1). In addition, MyD88 may have been partially deleted through physiological processing as long as its function is not impaired.
  • MyD88 ultimately promotes production of cytokines (eg, IL-6, CCL2, CXCL1, CXCL2, etc.) by mediating signal transduction caused by binding of ligands (eg, IL-1 ⁇ ) to IL-1R1
  • cytokines eg, IL-6, CCL2, CXCL1, CXCL2, etc.
  • ligands eg, IL-1 ⁇
  • the mutation preferably includes substitution, more preferably conservative substitution, from the viewpoint that the cytokine production promotion mediating activity is less likely to be impaired.
  • MyD88 include proteins described in (e) below and proteins described in (f) below: (E) a protein comprising the amino acid sequence shown in any of SEQ ID NOs: 12 to 17, and (f) an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 12 to 17 And at least one selected from the group consisting of proteins having cytokine production promotion mediating activity.
  • the identity is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more.
  • the presence or absence of cytokine production promotion mediating activity can be determined according to or according to a known method. For example, cytokines produced by contacting IL-1R1 ligands (eg IL-1 ⁇ ) with cells (especially mesenchymal stem cells) in which the test protein is expressed in the cells (eg IL-6, If the amount of CCL2, CXCL1, CXCL2, etc.) is greater than the amount of cytokine similarly measured using cells that do not express the test protein, it can be determined that the test protein has a cytokine production promotion mediating activity it can.
  • IL-1R1 ligands eg IL-1 ⁇
  • cells especially mesenchymal stem cells
  • the amount of CCL2, CXCL1, CXCL2, etc. is greater than the amount of cytokine similarly measured using cells that do not express the test protein, it can be determined that the test protein has a cytokine production promotion mediating activity it can.
  • (f ′) one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in any of SEQ ID NOS: 12 to 17. And a protein having an activity of promoting cytokine production.
  • the plurality is, for example, 2 to 25, preferably 2 to 10, more preferably 2 to 5, and further preferably 2 or 3.
  • MyD88 function inhibitor examples include MyD88 inhibitor, neutralizing antibody and the like, and further, when these are proteins, their expression vectors.
  • the MyD88 inhibitor is not particularly limited, and a known inhibitor can be used.
  • Examples of MyD88 inhibitors include MyD88 dimerization inhibitors.
  • MyD88 dimerization inhibitors are commercially available as peptides, and these can be widely used in the present invention.
  • Preferable specific examples of the MyD88 dimerization inhibitor include the following oligopeptide (oligopeptide 1).
  • An oligopeptide comprising the following amino acid sequence (g) or (h) and having MyD88 dimerization inhibitory activity: (G) an amino acid sequence shown in SEQ ID NO: 18 or 19, or (h) an amino acid sequence in which one or several amino acids are substituted, deleted, added or inserted into the amino acid sequence shown in SEQ ID NO: 18 or 19 .
  • SEQ ID Nos: 18 and 19 are known sequences as MyD88 dimerization inhibitory sequences.
  • the oligopeptide 1 may have other amino acid sequences in addition to the amino acid sequence (g) or (h).
  • Other amino acid sequences are not particularly limited, and examples include membrane permeation sequences, enzyme cleavage sequences, and scaffold binding sequences. Among these, a membrane permeation arrangement is preferable.
  • oligopeptide 1 As a more preferred embodiment of the oligopeptide 1, the following oligopeptide (oligopeptide 2) can be mentioned.
  • An oligopeptide comprising the following amino acid sequence (i) or (j) and having MyD88 dimerization inhibitory activity: (I) In order from the N-terminal side, a scaffold binding sequence, an enzyme cleavage sequence, a membrane permeation sequence, an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 18 or 19 is arranged, or (j) the amino acid sequence of (i) above An amino acid sequence in which one or several amino acids are substituted, deleted, added or inserted.
  • Oligopeptide 2 can exhibit the effect of the present invention more efficiently by combining with the scaffold described in “2-2.
  • the scaffold binding sequence is not particularly limited as long as it is an amino acid sequence that can bind to the scaffold, and can be appropriately selected from known sequences according to the type of scaffold.
  • ⁇ 2 -plasmin inhibitor N-terminal sequence for example, the 1st to 8th amino acid sequence on the N-terminal side (SEQ ID NO: 20: NQEQVSPL)
  • the amino acid sequence represented by SEQ ID NO: 28 YRGDTIGEGQQHHLGG
  • amino acid sequence represented by SEQ ID NO: 29 LRGDGAKDV
  • amino acid sequence represented by SEQ ID NO: 30 LRGKKKKG
  • amino acid sequence represented by SEQ ID NO: 31 LNQEQVSPLRGD
  • amino acid sequence represented by SEQ ID NO: 32 GAKDV
  • An amino acid sequence represented by SEQ ID NO: 33 (KKKK), an amino acid sequence represented by SEQ ID NO: 34 LNQEQVSPLGYIGSR
  • an amino acid sequence represented by SEQ ID NO: 35 LNQEQ
  • the scaffold is collagen, the amino acid sequence represented by SEQ ID NO: 39 (GVMGF), the amino acid sequence represented by SEQ ID NO: 40 (GAOGLRGGAGPOGPEGGKGAAGPOGPO), the amino acid sequence represented by SEQ ID NO: 41 (KGHRGF), represented by SEQ ID NO: 42 And an amino acid sequence represented by SEQ ID NO: 43 (GXXGER), an amino acid sequence represented by SEQ ID NO: 44 (GTGXR), an amino acid sequence represented by SEQ ID NO: 45 (GPGXR), and the like.
  • GVMGF amino acid sequence represented by SEQ ID NO: 39
  • SEQ ID NO: 40 GOGLRGGAGPOGPEGGKGAAGPOGPO
  • the amino acid sequence represented by SEQ ID NO: 41 KGHRGF
  • SEQ ID NO: 42 And an amino acid sequence represented by SEQ ID NO: 43 (GXXGER), an amino acid sequence represented by SEQ ID NO: 44 (GTGXR), an amino acid sequence represented by SEQ ID NO: 45 (GPGXR), and
  • the enzyme cleavage sequence is not particularly limited as long as it is an amino acid sequence that is recognized and cleaved by a protease present at the site of tissue damage, and known sequences can be widely used.
  • a protease that is activated in association with an inflammatory reaction due to tissue damage is used.
  • examples of such protease include matrix metalloprotease, plasmin, stromelysin, stromelysin elastase, collagenase, tPA, uPA and the like.
  • Examples of the enzyme cleavage sequence include the amino acid sequence represented by SEQ ID NO: 21 (VPMSMRGG) which is a plasmin / matrix metalloprotease cleavage sequence, and the amino acid sequence (LIKMKP) represented by SEQ ID NO: 46 which is a plasmin cleavage sequence.
  • SEQ ID NO: 49 PFELRA
  • SEQ ID NO: 50 which is a collagenase cleavage sequence
  • PHYGRSGG tPA cleavage sequence
  • oligopeptide 2 is cleaved at the damaged site, and a peptide (oligopeptide 3) containing a transmembrane sequence and a MyD88 dimerization inhibitory sequence (SEQ ID NO: 18, 19, etc.) Release from the scaffold.
  • the enzyme cleavage sequence is a sequence that is cleaved by a protease that is activated in association with an inflammatory reaction, the protease is activated at the site of tissue damage (usually accompanied by an inflammatory reaction). It becomes possible to release peptide 3 from the scaffold more efficiently.
  • the membrane permeation sequence is not particularly limited as long as it is a sequence capable of imparting cell membrane permeability to oligopeptide 3 released from the scaffold, and known sequences can be widely used.
  • Examples of the membrane permeation sequence include the amino acid sequence represented by SEQ ID NO: 22 derived from Antennapedia (RQIKIWFQNRRMKWKK), the amino acid sequence represented by SEQ ID NO: 25 derived from HIV-1 Tat (GRKKRRQRRRPPQ), and the sequence number 26 derived from transportan Amino acid sequence (GWTLNSAGYLLGKINLKALAALAKKIL), amino acid sequence shown by SEQ ID NO: 27 (AAVALLPAVLLALLP) derived from membrane transport sequence (MTS), amino acid sequence shown by SEQ ID NO: 53 (KRPAAIKKAGAQAKKK), amino acid sequence shown by SEQ ID NO: 54 (AGYLLGKINLKALAALAKKIL) , Amino acid sequence shown by SEQ ID NO: 55 (RRRRRR), amino acid sequence shown by SEQ
  • oligopeptide 3 By adding a membrane permeation sequence, oligopeptide 3 can be introduced into the cell, and can exhibit MyD88 dimerization inhibitory activity.
  • the plurality is, for example, 2 to 4, preferably 2 to 3, more preferably 2.
  • the mutation preferably includes substitution, more preferably conservative substitution, from the viewpoint that the MyD88 dimerization inhibitory activity is less likely to be impaired.
  • the length of the oligopeptide is not particularly limited as long as it is a general length as an oligopeptide.
  • the length is, for example, 5 to 50 amino acid residues, preferably 7 to 45 amino acid residues.
  • the presence or absence of MyD88 dimerization inhibitory activity can be determined according to or according to a known method.
  • cytokines for example, IL-6, CCL2, CXCL1, CXCL2, etc.
  • a test substance for example, mesenchymal stem cells
  • a ligand for example, IL-1 ⁇
  • the MyD88 dimerization inhibitor may be chemically modified as long as it has MyD88 dimerization inhibitory activity.
  • the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁇ ), an amide (—CONH 2 ), and an ester (—COOR).
  • R in the ester for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C 6-12 aryl groups such as ⁇ -naphthyl; C 7- such as phenyl-C 1-2 alkyl groups such as benzyl and phenethyl; ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl; 14 aralkyl group; pivaloyloxymethyl group is used.
  • a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl
  • a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl
  • the carboxyl group (or carboxylate) other than the C terminus may be amidated or esterified.
  • ester for example, the above-mentioned C-terminal ester or the like is used.
  • the amino group of the N-terminal amino acid residue is a protecting group (for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as formyl group, acetyl group, etc.).
  • substituents on the side chain of amino acids in the molecule for example, —OH, —SH, amino group, An imidazole group, an indole group, a guanidino group, etc.
  • an appropriate protecting group for example, a C 1-6 acyl group such as a C 1-6 alkanoyl group such as a formyl group or an acetyl group
  • complex proteins such as so-called glycoproteins with chains attached.
  • the MyD88 dimerization inhibitor may have a known protein tag added as long as it has MyD88 dimerization inhibitory activity.
  • protein tags include histidine tags, FLAG tags, GST tags, and the like.
  • the MyD88 dimerization inhibitor 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 a pharmaceutically acceptable salt, and both an acidic salt and a basic salt can be adopted.
  • acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, apple Organic acid salts such as acid salts, citrate salts, methanesulfonate salts, paratoluenesulfonate salts; amino acid salts such as aspartate salts and glutamate salts.
  • basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts.
  • the MyD88 dimerization inhibitor 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, acetic acid and the like.
  • the MyD88 dimerization inhibitor is transformed according to known methods, for example, chemical synthesis, purification from mammalian cells or tissues (eg, serum, etc.), and a polynucleotide containing a MyD88 dimerization inhibitor-encoding polynucleotide. It can be obtained by purification from the body.
  • the transformant is not particularly limited as long as it is a cell capable of expressing a MyD88 dimerization inhibitor from a polynucleotide encoding a MyD88 dimerization inhibitor.
  • Various cells such as bacteria such as Escherichia coli, insect cells, and mammalian cells can be used.
  • insect cells for example, Sf cells, MG1 cells, High Five TM cells, BmN cells and the like are used.
  • Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells and the like are used.
  • animal cells examples include monkey COS-7 cells, monkey Vero cells, Chinese hamster cells CHO, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells.
  • MyD88 neutralizing antibody refers to an antibody having the property of inhibiting the cytokine production promotion mediated activity of MyD88 by binding to MyD88.
  • the mode of the antibody is the same as the mode of the antibody in the above “2-1-1. IL-1R1 function inhibitor”.
  • the antibody is preferably an antibody having an antigen-binding property with respect to an amino acid sequence necessary for dimerization of MyD88 from the viewpoint that neutralization activity can be more reliably exhibited.
  • the amino acid sequence necessary for MyD88 dimerization is known, and such antibodies can be produced based on this known information.
  • these expression vectors are the same as the expression vectors in “2-1-1. IL-1R1 function inhibitor” above.
  • MyD88 expression inhibitor is not particularly limited as long as it can reduce the amount of MyD88 expressed by cells (especially mesenchymal stem cells) present in or around the damaged site to be treated.
  • the definitions of “cells present at the injury site” and “suppressing MyD88 function” are the same as those in the above “2-1-3. MyD88 function inhibitor”.
  • MyD88 which is the target of expression suppression, is the same as defined in “2-1-3. MyD88 function inhibitor” above.
  • MyD88 expression inhibitor examples include MyD88-specific siRNA, MyD88-specific miRNA, MyD88-specific antisense nucleic acid, and expression vectors thereof. These aspects are the same as those described above in “2-1-2. IL-1R1 expression inhibitor”.
  • the active ingredient of the present invention can promote the regeneration of tissue damage.
  • the active ingredient of the present invention can promote the migration of mesenchymal stem cells to the damaged site and the proliferation of the cells. Therefore, the active ingredient of the present invention can be used as a tissue damage therapeutic agent, a mesenchymal stem cell proliferation promoter, or a mesenchymal stem cell accumulation promoter.
  • target tissues of the drug of the present invention are not particularly limited. Since the active ingredient of the present invention can promote the accumulation of mesenchymal stem cells at the site of injury and further promote the proliferation of mesenchymal stem cells, it can promote the repair of these various tissues.
  • Specific examples of the target tissue include bone tissue, muscle tissue, adipose tissue, endothelial tissue, epithelial tissue and the like, preferably bone tissue.
  • the target organism of the drug of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats and rabbits.
  • the drug of the present invention preferably further contains a scaffold from the viewpoint that the effect of the active ingredient of the present invention can be exhibited more efficiently.
  • the scaffold is not particularly limited as long as it can slow down the diffusion rate of the active ingredient of the present invention.
  • the scaffold is preferably a gel or porous body of a biocompatible material. Examples of biocompatible materials include fibrin, hydroxyapatite, chitosan, various extracellular matrix components (for example, collagen, proteoglycan, hyaluronic acid, fibronectin, laminin, elastin, etc.), synthetic polymers, and the like.
  • fibrin gel is particularly preferable. The production of fibrin gel can be performed according to or according to a known method.
  • the content can be appropriately adjusted according to the size of the damaged site.
  • the content is, for example, about 5 to 500 ⁇ L, preferably about 10 to 200 ⁇ L, more preferably about 20 to 100 ⁇ L when the range of the damaged site is a circle having a diameter of 5 mm.
  • the drug of the present invention preferably further contains mesenchymal stem cells from the viewpoint that the effect of the active ingredient of the present invention can be exhibited more efficiently.
  • the tissue derived from mesenchymal stem cells is not particularly limited, and may be various tissues such as bone tissue (particularly bone marrow), adipose tissue, placental tissue, human body tissue, and dental pulp. From the viewpoint that the effect of the present invention can be exhibited more efficiently, it is desirable that the derived tissue is the same tissue as the tissue to be treated. For example, if the tissue to be treated is bone tissue, it is desirable that the mesenchymal stem cells contained in the agent of the present invention are bone tissue-derived mesenchymal stem cells.
  • the number of cells can be appropriately adjusted according to the size of the damaged site.
  • the number of cells is, for example, 1 ⁇ 10 4 to 1 ⁇ 10 8 cells, preferably 1 ⁇ 10 5 to 3 ⁇ 10 7 cells, more preferably when the damaged site is a circle having a diameter of 5 mm. It is about 5 ⁇ 10 5 to 8 ⁇ 10 6 cells, more preferably about 1 ⁇ 10 6 to 4 ⁇ 10 6 cells.
  • the drug of the present invention preferably further contains a growth factor from the viewpoint that the effect of the active ingredient of the present invention can be exhibited more efficiently.
  • the growth factor is not particularly limited as long as it is a protein that can promote cell growth and / or differentiation.
  • growth factors include FGF (preferably FGF2), PDGF (preferably PDGF-BB), BMP (preferably BMP2), EGF, IGF, TGF, NGF, BDNF, VEGF, G-CSF, GM-CSF, EPO , TPO, HGF, NT3 and the like, preferably FGF (preferably FGF2), PDGF (preferably PDGF-BB), BMP (preferably BMP2) and the like.
  • the content can be appropriately adjusted according to the size of the damaged site.
  • the content is, for example, about 0.01 to 100 ⁇ g, preferably about 0.1 to 10 ⁇ g, and more preferably about 0.3 to 5 ⁇ g when the range of the damaged site is a circle having a diameter of 5 mm.
  • the drug of the present invention can contain any additive in addition to the above components.
  • the additive is not particularly limited as long as it is a pharmaceutically acceptable ingredient.
  • a base a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder
  • examples include disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, and chelating agents.
  • the drug of the present invention can be used in appropriate dosage forms such as tablets, pills, powders, solutions, injections, suspensions, emulsions, powders, granules, in usage (in vitro or in vivo) and administration forms.
  • dosage form such as a capsule can be employed.
  • the content of the active ingredient of the present invention in the drug of the present invention can be appropriately adjusted according to the type of active ingredient, the size of the damaged site, and the like.
  • the content can be, for example, 0.0001 to 95% by weight, preferably 0.001 to 50% by weight.
  • Examples of the route of administration of the drug of the present invention include local administration, enteral administration, parenteral administration and the like. More specific administration routes include dermal administration, enema administration, oral administration, intravenous administration, intramuscular administration, subcutaneous administration, intraosseous administration, transmucosal administration, intraperitoneal administration and the like. From the viewpoint that the effects of the present invention can be exhibited more efficiently, the administration route of the drug of the present invention is preferably local administration, and the drug of the present invention is preferably a preparation for local administration suitable for local administration.
  • Example 1 Promotion of regeneration of bone damage by IL-1R1 or MyD88 knockout Test mouse (wild type mouse C57BL / 6, IL-1R1 knockout mouse (Immunity 11, 115-122 (1999)), and MyD88 knockout mouse ( Immunity 11, 115-122 (1999))) was physically damaged, and the degree of subsequent regeneration was evaluated. Specifically, it was performed as follows.
  • ⁇ 1-1 Creation of a calvarial injury model> Test mice 10-12 weeks old were anesthetized with isoflurane. The parietal hair was removed and a longitudinal incision was made to expose the skull bone tissue. Two injuries (5 mm in diameter) were created in the parietal bone on each side of the sagittal suture using a drill.
  • the damaged site was washed with physiological saline, and on the dura mater, a polymerized fibrin matrix (40 ⁇ L / damaged site, 10 mg / mL fibrinogen (manufactured by Enzyme Research Laboratories), 2 U / mL thrombin (manufactured by Sigma-Aldrich), It was covered with 5 mM calcium chloride, 25 ⁇ g / mL aprotinin (Roche), 2 ⁇ 10). The soft tissue was closed with sutures, and tramadol was administered subcutaneously as an analgesic (100 mg / kg).
  • Example 1-1 The skull injury model mouse prepared in Example 1-1 was bred for 4 weeks according to a conventional method, and then the skull was scanned with a micro CT scanner (manufactured by SCANCO Medical, microCT 40). In the scanned image, the periphery of the damaged site was surrounded by a circle having a diameter of 5 mm (the range of the damaged site at the time of damage), and the ratio of the area of the damaged playback site to the area of the circle (damage recovery rate) was obtained.
  • Example 2 Effect of IL-1R1 or MyD88 knockout on bone damage regeneration by mesenchymal stem cells
  • Example 1 except that mesenchymal stem cells are further added to the polymerized fibrin matrix (2 ⁇ 10 6 cells / injured site). Were tested in the same manner.
  • Example 3 Promotion of proliferation of mesenchymal stem cells by IL-1R1 or MyD88 knockout
  • IL-1R1 or MyD88 knockout mouse were administered to the damaged site.
  • the subsequent cell growth rate was evaluated. Specifically, it was performed as follows.
  • ⁇ 3-1 Administration of mesenchymal stem cells>
  • mesenchymal stem cells are collected from the bone marrow of wild-type mouse C57BL / 6, IL-1R1 knockout mouse, and MyD88 knockout mouse and labeled with carboxyfluorescein succinimidyl ester (CSFE, Life Technologies) did.
  • a calvarial injury model was prepared in the same manner as in Example 1-1, except that the obtained labeled mesenchymal stem cells were further added to the polymerized fibrin matrix (2 ⁇ 10 6 cells / injured site).
  • the obtained labeled mesenchymal stem cells were cultured using an ⁇ -MEM medium having an FBS concentration of 1%.
  • ⁇ 3-2 Evaluation of cell growth rate>
  • the skull injury model mouse prepared in Example 3-1 was bred for 7 days according to a conventional method, and then the matrix around the damaged site was collected. From the obtained matrix, cells were separated according to a conventional method and suspended in 10 mL of a flow cytometry buffer.
  • the non-proliferation control was cultured for 7 days according to a conventional method, and then the cells were detached from the plate and suspended in a flow cytometry buffer.
  • the CSFE fluorescence intensity of each cell in these cell suspensions was measured by flow cytometry (BD GACSCanto II, manufactured by BD Bioscience), and the median value was determined. Since CSFE fluorescence intensity is inversely proportional to cell proliferation, the lower the median CSFE fluorescence intensity, the faster the cell proliferation rate.
  • Example 4 Promotion of accumulation of mesenchymal stem cells at the injury site by IL-1R1 or MyD88 knockout Accumulation of mesenchymal stem cells at the injury site of wild-type mouse C57BL / 6, IL-1R1 knockout mouse, and MyD88 knockout mouse Evaluated. Specifically, it was performed as follows. A calvarial injury model was prepared in the same manner as in Example 1-1 except that mouse PDGF-BB was further added to the polymerized fibrin matrix (1 ⁇ g / injured site). The prepared skull injury model mice were bred for 7 days according to a conventional method, and then the matrix around the injury site was collected. Cells were separated from the resulting matrix according to conventional methods.
  • the isolated cells were treated with antibodies against various mesenchymal stem cell markers (1/200 dilution, manufactured by Biolegend) (anti-mouse CD29, anti-mouse Ly-6A / E (Sca-1), anti-mouse CD90.1, anti-mouse Immunostaining was performed by treatment with a flow cytometry buffer containing CD45.2, anti-mouse CD44) for 15 minutes. After washing the cells with a flow cytometry buffer solution, the mesenchymal stem cell-like cells (CD90 + , CD45 ⁇ , Sca-1 + , and so on) are analyzed by flow cytometry (BD GACSCanto II, manufactured by BD Bioscience). CD44 + and CD29 + cells) were measured. The higher the ratio, the more mesenchymal stem cells accumulated at the damaged site.
  • Example 5 Acceleration of bone damage regeneration by IL-1R1 or MyD88 inhibitor Additional IL-1R1 inhibitor or MyD88 inhibitor was added to the polymerized fibrin matrix (IL-1R1 inhibitor: 1 ⁇ g / injury site, MyD88 inhibitor: 4 ⁇ g / injury site) Or mesenchymal stem cells were further added (2 ⁇ 10 6 cells / injured site), and the breeding period after preparation of the injury model was set to 8 weeks. All the test mice are wild-type mice.
  • IL-1R1 inhibitor mouse IL-1Ra (manufactured by R & D Systems), which is an endogenous antagonist of mouse IL-1R, was used.
  • MyD88 inhibitors ⁇ 2 -plasmin inhibitor sequence (fibrin binding sequence) ( ⁇ 2 -PI 1-8 , NQEQVSPL (SEQ ID NO: 20)), plasmin / matrix metalloprotease sensitive sequence (VPMSMRGG (sequence) No.
  • IL-1R1 permeation sequence derived from Antennapedia / Homeobox
  • RQIKIWFQNRRMKWKK SEQ ID NO: 22
  • RQIKIWFQNRRMKWKK SEQ ID NO: 22
  • RQIKIWFQNRRMKWKK SEQ ID NO: 22
  • RDVLPGTCVNS MyD88 inhibitory peptide sequence
  • a peptide consisting of (SEQ ID NO: 24) was designed, synthesized by commissioning from Gene Script, and then dialyzed with HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.5). Both IL-1R1 inhibitor and MyD88 inhibitor can inhibit IL-1R1 / MyD88 pathway and suppress cytokine secretion by treating cells with IL-1 ⁇ .
  • the degree of increase in the damage regeneration rate due to the addition of the inhibitor was significantly higher when the mesenchymal stem cells were added than when the mesenchymal stem cells were not added.
  • Example 6 Promoting regeneration of bone damage by combination with a growth factor Further growth factor (FGF-2, PDGF-BB, or BMP-2: 1 ⁇ g / damage site) is added to the polymerized fibrin matrix, or to these. Further, a test was conducted in the same manner as in Example 1 except that a MyD88 inhibitor was added (MyD88 inhibitor: 4 ⁇ g / injured site), and the breeding period after preparation of the injury model was 8 weeks. All the test mice are wild-type mice.
  • FGF-2, PDGF-BB, or BMP-2 1 ⁇ g / damage site

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Abstract

La présente invention concerne un agent thérapeutique pour lésion tissulaire. Ledit agent thérapeutique pour lésion tissulaire contient au moins un élément choisi dans le groupe constitué par des inhibiteurs de la fonction de IL-1R1, des inhibiteurs de l'expression de IL-1R1, des inhibiteurs de la fonction de MyD88 et des inhibiteurs de l'expression de MyD88.
PCT/JP2017/007069 2016-02-29 2017-02-24 Agent thérapeutique pour lésion tissulaire WO2017150371A1 (fr)

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