WO2024005133A1 - Therapeutic agent for meniscus - Google Patents

Therapeutic agent for meniscus Download PDF

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Publication number
WO2024005133A1
WO2024005133A1 PCT/JP2023/024150 JP2023024150W WO2024005133A1 WO 2024005133 A1 WO2024005133 A1 WO 2024005133A1 JP 2023024150 W JP2023024150 W JP 2023024150W WO 2024005133 A1 WO2024005133 A1 WO 2024005133A1
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Prior art keywords
amino acid
acid sequence
seq
peptide
meniscus
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PCT/JP2023/024150
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French (fr)
Japanese (ja)
Inventor
克人 玉井
敬史 新保
恭太 石橋
尊彦 山崎
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国立大学法人大阪大学
株式会社ステムリム
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Publication of WO2024005133A1 publication Critical patent/WO2024005133A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to a pharmaceutical composition for the treatment of meniscus, comprising a fragment peptide of high mobility group box 1 (HMGB1) protein.
  • HMGB1 high mobility group box 1
  • the pharmaceutical composition of the present invention exerts its therapeutic effect by regenerating the meniscus.
  • the meniscus is located within the knee joint and has the function of distributing the weight of the joint and maintaining the joint's compatibility. Damage to the meniscus includes deformation, tearing, loss, etc. caused by some force being applied to the meniscus. Causes of meniscal damage include cases resulting from injuries such as sports, and cases caused by external force being applied to the meniscus, which is more susceptible to damage due to aging.
  • meniscus damage If the meniscus is damaged, you will experience pain and be unable to walk or exercise normally. Meniscal damage also causes osteoarthritis. Treatments for meniscus injuries include conservative treatments such as intra-articular injection of hyaluronic acid, rest, and rehabilitation, and surgical treatments such as meniscectomy and suturing.
  • Non-Patent Document 1 Non-Patent Document 2
  • HMGB1 fragment peptide having a specific amino acid sequence was found to be effective in the red-white zone and white-white zone of the meniscus in meniscal defect model animals.
  • the present invention was completed based on the discovery that it is possible to reproduce .
  • a pharmaceutical composition for regenerating the red-white zone or white-white zone of the meniscus comprising the substance according to any one of the following (a) to (c): (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with. [2] The pharmaceutical composition according to [1], which is administered systemically.
  • a pharmaceutical composition for treating the resection site of a meniscus comprising the substance according to any one of the following (a) to (c): (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with. [4] The pharmaceutical composition according to [3], wherein the resection site is a red-white zone or a white-white zone. [5] The pharmaceutical composition according to [3] or [4], which is administered systemically.
  • the invention further provides: [6] A method for regenerating the red-white zone or white-white zone of the meniscus in a subject, comprising administering to the subject a substance according to any one of the following (a) to (c): (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with. [7] The method according to [6], wherein the administration is systemic administration.
  • a method for treating the resection site of a meniscus in a subject comprising administering to the subject a substance according to any one of the following (a) to (c): (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with. [9] The method according to [8], wherein the resection site is a red-white zone or a white-white zone. [10] The method according to [8] or [9], wherein the administration is systemic administration.
  • the invention further provides: [11] The substance described in any one of the following (a) to (c) for use in regenerating the red-white zone or white-white zone of the meniscus: (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with. [12] The substance according to [11] which is administered systemically.
  • the invention further provides: [16] Use of the substance according to any of the following (a) to (c) in the manufacture of a drug for regenerating the red-white zone or white-white zone of the meniscus: (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with. [17] The use according to [16], wherein the drug is administered systemically.
  • a pharmaceutical composition containing the peptide of the present application can regenerate and treat the red-white zone and white-white zone of the meniscus, which cannot be regenerated naturally.
  • FIG. 1 is a scheme showing the procedure for animal experiments.
  • FIG. 2 is a tissue specimen showing the results of examining meniscal regeneration using Safranin-O staining.
  • Figure 3 is a photograph of the regenerated meniscus.
  • FIG. 4 is a graph showing changes over time in the regenerated area of the meniscus. The circles indicate the HMGB1 administration group, and the squares indicate the physiological saline administration group.
  • FIG. 5 shows the results of a circulating cell colony-forming fibroblast (CFU-F) assay of partially meniscectomized mice administered saline (left dish) or HMGB1(1-44) (right dish).
  • CFU-F circulating cell colony-forming fibroblast
  • the bar graph shows a comparison of the number of colonies between the physiological saline administration group and the HMGB1(1-44) administration group.
  • Figure 6 shows an overview of experiments using the parabiosis model.
  • FIG. 7 shows Texas Red stained and bright field images of the regenerated meniscus.
  • FIG. 8 is a microscopic image showing the results of examining regeneration in the outer and inner regions of the meniscus using red fluorescence from tdTomato.
  • HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
  • a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or
  • the amino acid sequence set forth in SEQ ID NO: 1 Provided is a pharmaceutical composition for regenerating the red-white zone or white-white zone of the meniscus, comprising a peptide comprising an amino acid sequence having about 80% or more sequence identity with.
  • the vascular area on the outside of the meniscus is classified as the red-red zone, the avascular area on the inside of the meniscus (on the intercondylar side) as the white-white zone, and the area between them as the red-white zone.
  • the red-red zone has blood flow, so even if it is damaged, it is expected to heal. However, since the red-white zone has poor blood flow and the white-white zone has no blood flow, these areas cannot be expected to heal if damaged. Therefore, if these parts of the meniscus remain defective, inconveniences such as persistent pain and difficulty walking and exercise occur, leading to a decline in the patient's QOL.
  • Regenerating the red-white zone or white-white zone of the meniscus includes not only regenerating these damaged areas but also partially regenerating these areas.
  • regeneration means that normal meniscal cells including fibroblasts and cartilage-like cells (safranin-O staining positive cells) are induced, and meniscal tissue (also called fibrocartilage tissue) is formed. Say something. Such regeneration can eliminate or reduce symptoms associated with meniscal damage, such as pain and difficulty walking and exercising.
  • the pharmaceutical composition of the invention described above may be used to treat injuries in the red-white zone or white-white zone of the meniscus.
  • Meniscal damage includes meniscus tear, loss, and deformation.
  • the regeneration of the red-white zone or white-white zone of the meniscus in the present invention is based on the action of the HMGB1 fragment peptide.
  • the HMGB1 fragment peptide includes a peptide consisting of a portion of the HMGB1 protein containing the amino acid sequence set forth in SEQ ID NO: 1, and variants thereof.
  • the peptide containing the amino acid sequence set forth in SEQ ID NO: 1 will be referred to as "HMGB1 peptide”
  • the HMGB1 fragment peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 will be abbreviated as "HMGB1 (1-44).” There is.
  • HMGB1 peptide and its variants, or HMGB1(1-44) and its variants can be obtained using known methods.
  • a polynucleotide encoding HMGB1 peptide and its variants or HMGB1(1-44) and its variants may be obtained as a genetic recombinant by incorporating it into an appropriate expression system, or it may be obtained by artificially synthesizing it. (for example, chemical synthesis).
  • HMGB1 peptide or HMGB1 (1-44) variant is a peptide containing an amino acid sequence in which one or more amino acid residues have been modified (substituted, deleted, inserted, or added) in the amino acid sequence set forth in SEQ ID NO: 1.
  • This peptide is functionally equivalent to the HMGB1 fragment peptide containing the amino acid sequence set forth in SEQ ID NO:1.
  • HMGB1 peptides or variants of HMGB1(1-44) include, but are not limited to: i) One or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 (for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1) A peptide comprising an amino acid sequence in which ⁇ 5, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, deleted, inserted, or added; ii) One or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 (for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1) A peptide consisting of an amino acid sequence in which ⁇ 5, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, deleted, inserted, or added; iii) About 80% or more, for example about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more of the amino acid sequence set forth
  • HMGB1 peptide or HMGB1(1-44) may be used, a variant of HMGB1 peptide or HMGB1(1-44) may be used, or a combination of these may be used.
  • a cell that secretes HMGB1 peptide or a variant thereof or HMGB1 (1-44) or a variant thereof, a polynucleotide encoding the peptide or a variant thereof, a vector into which the polynucleotide is inserted , cells containing the vector may be used.
  • the pharmaceutical composition of the present invention contains an effective amount of HMGB1 fragment peptide or variant thereof.
  • An effective amount is an amount that can regenerate the red-white zone or white-white zone of the meniscus, or can partially regenerate these damaged areas. Refers to the amount that can be done.
  • the effective amount is an amount that results in regeneration of 30% or more, preferably 50% or more, more preferably 90% or more, where 100% regeneration is defined as regeneration to the original meniscus. Good too.
  • the effective amount may be an amount that eliminates or alleviates symptoms associated with meniscal damage, such as pain and difficulty walking and exercising. Regeneration of the meniscus can be confirmed using known methods such as MRI imaging and ultrasound echo.
  • Subjects to which the pharmaceutical composition of the present invention is administered are not particularly limited, and include mammals, birds, fish, and the like. Mammals include humans and non-human animals, including, but not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales. isn't it. As used herein, the term “subject” is used interchangeably with “patient,” “individual,” and "animal.”
  • composition is used interchangeably with “medicine,” “drug,” or “pharmaceutical composition.”
  • the administration site of the pharmaceutical composition of the present invention may be the knee joint (for example, the meniscus or its vicinity) or a site different from the knee joint.
  • the pharmaceutical composition of the present invention can exert its effects no matter where it is administered.
  • Methods for administering the pharmaceutical composition of the present invention include oral administration and parenteral administration.
  • parenteral administration methods include intravascular administration (intraarterial administration, intravenous administration, etc.), intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, nasal administration, pulmonary administration, and transdermal administration. but not limited to.
  • the present invention also provides that the pharmaceutical compositions of the invention can be administered systemically or locally (e.g., subcutaneously, intradermally, on the skin surface, in the nasal cavity, etc.) by injection, e.g., intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. It may also be administered to mucous membranes, oral and gastrointestinal mucosa, vaginal/uterine mucosa, or injured areas).
  • the administration method and dosage can be appropriately selected depending on the patient's age and symptoms.
  • the HMGB1 fragment peptide or variant thereof may be administered in a range of about 0.0000001 mg to about 1000 mg per kg of patient body weight per administration.
  • it may be administered in a range of about 0.00001 to about 100,000 mg/kg of patient body weight.
  • the amount of the peptide can be administered within the above range. can.
  • the dosage of the pharmaceutical composition of the present invention is not limited to these amounts.
  • compositions of the present application can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, United States). .S.A), pharmaceutically acceptable carriers and additives It may also include things. For example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity promoters, and flavoring agents. These include, but are not limited to, and other commonly used carriers and additives can be used as appropriate.
  • Examples include polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, purified water, physiological saline, and glycerin.
  • the pharmaceutical composition of the second aspect of the present invention may regenerate the red-white zone or white-white zone excised by resection. Such regeneration cures the ablation site.
  • Regeneration of the resection site of the meniscus by the pharmaceutical composition of the second aspect of the present invention may be performed to restore the site to its original state or to partially regenerate it. Regeneration may be in an amount that results in regeneration of 30% or more, preferably 50% or more, more preferably 90% or more, where 100% regeneration is regeneration to the original meniscus.
  • Treatment of the resection site of the meniscus with the pharmaceutical composition of the second aspect of the present invention may eliminate or alleviate symptoms associated with meniscal damage, such as pain and difficulty walking and exercising.
  • the invention provides the methods, materials and uses described above.
  • the statements regarding the pharmaceutical composition of the first aspect and the pharmaceutical composition of the second aspect also apply to the methods, materials and uses.
  • mice All experimental mice were housed in cages with a 12 hour light/dark cycle. Feed and water were available ad libitum. C57BL/6J mice were purchased from Claire Japan (Tokyo, Japan). PDGFR ⁇ /H2B-GFP knockin (P ⁇ -GFPKI) mice in which all PDGFR ⁇ -positive (P ⁇ ) cells emit GFP fluorescence in the nucleus (Hamilton TG, Mol Cell Biol. 2003), P ⁇ -Cre (JAX, 013148) and ROSAtdTomato ( JAX, 007909) mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA).
  • HMGB1(1-44) A peptide consisting of amino acid residues 1-44 (SEQ ID NO: 1) of human-derived HMGB1 protein (SEQ ID NO: 2) was chemically synthesized by PolyPeptide Laboratories (San Diego).
  • parabiosis mouse model A parabiosis mouse model was generated as previously described (Duyverman MA. et al., Nat. Protc., 7(4):763-770, 2012). Wild type mice (C57/BL6) and Pa-Cre; ROSAtdTomato mice were parabiotically combined. The torso was sutured from the olecranon to the knee joint on the corresponding sides. Prior to the start of the experiment, pairs were kept individually in cages for over 4 weeks under normal husbandry conditions as described above.
  • mice were euthanized 1, 2, 4, or 6 weeks after partial meniscectomy (6 mice per group). Tissues were fixed with 4% paraformaldehyde for 1 day, decalcified with 0.5 mol/L ethylenediaminetetraacetic acid (EDTA) solution for 14 days, and then embedded in paraffin wax. Samples were sectioned at 6 ⁇ m in the sagittal plane and stained with Safranin O and Fast Green (ref.). Microscopic evaluation of histological slices was performed under an Olympus BX 53 microscope (Olympus, Tokyo, Japan).
  • EDTA ethylenediaminetetraacetic acid
  • HMGB1 (1-44) The therapeutic potential of HMGB1 (1-44) administration for meniscal damage in mice was investigated.
  • Physiological saline (100 ⁇ l) or HMGB1 (1-44) (5 mg/kg in 100 ⁇ l of physiological saline) was administered intravenously for 5 consecutive days starting from the day of partial meniscectomy.
  • the experimental procedure is shown in Figure 1.
  • Figure 2 shows the histological observation results.
  • the saline-administered group only fibrous scar tissue was observed at the meniscal defect site one week after surgery, and there was almost no change at 2, 4, and 6 weeks after surgery. There were almost no cells positive for Safranin-O staining. Fibrillation or division was observed on the surface of the regenerated meniscus.
  • the HMGB1(1-44) administration group regeneration of chondroid cells (ie, cells positive for Safranin-O staining) was significantly induced two weeks after surgery. Furthermore, the area of Safranin-O positive meniscal cells gradually increased, and the surface of the regenerated meniscus was smoother.
  • the cell morphology of the regenerated meniscus at 4 and 6 weeks after surgery was similar to normal meniscal cells and surrounded by cells positive for safranin-O staining.
  • a parabiosis model was established using wild type (WT) mice and P ⁇ -Cre; RosatdTomato mice, and the presence or absence of recruitment of P ⁇ Lin+ cells to the injured site was investigated.
  • a partial meniscectomy was performed on the knee of the WT mouse, and physiological saline or HMGB1 (1-44) was administered through the tail vein of the P ⁇ -Cre; RosatdTomato mouse (FIG. 6).
  • HMGB1(1-44) In the saline-administered group, there was almost no contribution from systemically recruited P ⁇ Lin+ cells at the defect site.
  • HMGB1(1-44) administration group recruitment of P ⁇ Lin+ cells was significantly induced to both the outer and inner regions of the deletion site (FIGS. 7 and 8). From these results, it was considered that systemic administration of HMGB1(1-44) mobilizes P ⁇ Lin+ cells to the damaged meniscus and regenerates the meniscus.
  • the present invention can be used in fields such as pharmaceuticals, medical research, and pharmaceutical research.
  • SEQ ID NO: 1 shows the amino acid sequence of a peptide consisting of amino acid residues 1-44 of human HMGB1 protein.
  • SEQ ID NO: 2 shows the amino acid sequence of human HMGB1 protein.
  • SEQ ID NO: 3 shows the base sequence of a nucleic acid encoding human HMGB1 protein.

Abstract

Provided are: a pharmaceutical composition for treating damage to the white-white zone or red-white zone of the meniscus, said composition containing an HMGB1 fragment peptide containing an amino acid sequence delimited by SEQ ID NO 1, or a variant thereof; and a pharmaceutical composition for treating a meniscus excision site, said composition containing an HMGB1 fragment peptide containing an amino acid sequence delimited by SEQ ID NO 1, or a variant thereof.

Description

半月板の治療薬Medications for treating meniscus
　本発明は、Ｈｉｇｈ　ｍｏｂｉｌｉｔｙ　ｇｒｏｕｐ　ｂｏｘ　１（ＨＭＧＢ１）タンパク質の断片ペプチドを含む、半月板の治療のための医薬組成物に関する。詳細には、本発明の医薬組成物は、半月板を再生することにより治療効果を発揮する。 The present invention relates to a pharmaceutical composition for the treatment of meniscus, comprising a fragment peptide of high mobility group box 1 (HMGB1) protein. Specifically, the pharmaceutical composition of the present invention exerts its therapeutic effect by regenerating the meniscus.
　半月板は膝関節内にあり、関節に加わる体重の負荷を分散させる機能と、関節の適合性を保つ機能を有している。半月板の損傷は、半月板に何らかの力が加わることにより生じる変形、断裂および欠損などを包含する。半月板損傷の原因としては、スポーツなどのケガから生じる場合と、加齢により傷みやすくなっている半月板に外力が加わって生じる場合などが挙げられる。 The meniscus is located within the knee joint and has the function of distributing the weight of the joint and maintaining the joint's compatibility. Damage to the meniscus includes deformation, tearing, loss, etc. caused by some force being applied to the meniscus. Causes of meniscal damage include cases resulting from injuries such as sports, and cases caused by external force being applied to the meniscus, which is more susceptible to damage due to aging.
　半月板が損傷すると、痛みを感じ、正常な歩行や運動も妨げられる。半月板の損傷は、変形性関節症も引き起こす。半月板損傷の治療は、ヒアルロン酸の関節内注射、安静、リハビリテーションなどの保存療法、および半月板切除術や縫合術などの手術療法がある。 If the meniscus is damaged, you will experience pain and be unable to walk or exercise normally. Meniscal damage also causes osteoarthritis. Treatments for meniscus injuries include conservative treatments such as intra-articular injection of hyaluronic acid, rest, and rehabilitation, and surgical treatments such as meniscectomy and suturing.
　半月板の治癒を促進するために、滑膜液間葉系幹細胞（ＭＳＣ）や多血小板血漿などのいくつかの補完療法が用いられている（非特許文献１、非特許文献２）。しかし、これらの補完的な治療法はいずれも半月板の再生に十分ではない。 Several complementary therapies, such as synovial fluid mesenchymal stem cells (MSCs) and platelet-rich plasma, have been used to promote meniscal healing (Non-Patent Document 1, Non-Patent Document 2). However, none of these complementary treatments are sufficient for meniscal regeneration.
　半月板のなかでも血流が乏しいｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅ または血流がないｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅにおける損傷の場合は、保存療法や縫合術によっては治癒が望めないことがある。そのような場合は切除術が適用となるが、切除によって失った部位を再生することができない。この切除術によって失った部位を再生することができれば、非常に有益である。また、保存療法や縫合術を行う場合であっても、損傷した半月板を再生することができれば、治癒を促進することができる。
　以上より、損傷、欠損または切除した半月板を治療する薬剤、特にｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅおよびｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するための新たな薬剤が必要とされている。 In the case of an injury in the red-white zone of the meniscus where blood flow is poor or the white-white zone where there is no blood flow, healing may not be expected with conservative treatment or suturing. In such cases, excision is applicable, but the area lost through excision cannot be regenerated. It would be extremely beneficial if the area lost through this resection could be regenerated. Furthermore, even when conservative treatment or suturing is performed, healing can be promoted if the damaged meniscus can be regenerated.
Accordingly, there is a need for new agents to treat damaged, missing, or excised menisci, particularly for regenerating the red-white zone and white-white zone.
　本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、特定のアミノ酸配列を有するＨＭＧＢ１断片ペプチドが、半月板欠損モデル動物において、半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅやｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生できることを見出し、本発明を完成するに至った。 As a result of intensive research to solve the above problems, the present inventors found that a HMGB1 fragment peptide having a specific amino acid sequence was found to be effective in the red-white zone and white-white zone of the meniscus in meniscal defect model animals. The present invention was completed based on the discovery that it is possible to reproduce .
　すなわち、本発明は、以下を提供する：
　［１］以下の（ａ）から（ｃ）のいずれかに記載の物質を含む、半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するための医薬組成物：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［２］全身投与される［１］記載の医薬組成物。
　［３］以下の（ａ）から（ｃ）のいずれかに記載の物質を含む、半月板の切除部位を治療するための医薬組成物：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［４］切除部位がｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅである、［３］記載の医薬組成物。
　［５］全身投与される［３］または［４］記載の医薬組成物。 That is, the present invention provides:
[1] A pharmaceutical composition for regenerating the red-white zone or white-white zone of the meniscus, comprising the substance according to any one of the following (a) to (c):
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[2] The pharmaceutical composition according to [1], which is administered systemically.
[3] A pharmaceutical composition for treating the resection site of a meniscus, comprising the substance according to any one of the following (a) to (c):
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[4] The pharmaceutical composition according to [3], wherein the resection site is a red-white zone or a white-white zone.
[5] The pharmaceutical composition according to [3] or [4], which is administered systemically.
　本発明は、さらに以下を提供する：
　［６］以下の（ａ）から（ｃ）のいずれかに記載の物質を対象に投与することを含む、該対象における半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するための方法：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［７］該投与が全身投与である［６］記載の方法。
　［８］以下の（ａ）から（ｃ）のいずれかに記載の物質を対象に投与することを含む、該対象における半月板の切除部位を治療するための方法：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［９］切除部位がｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅである、［８］記載の方法。
　［１０］該投与が全身投与である［８］または［９］記載の方法。 The invention further provides:
[6] A method for regenerating the red-white zone or white-white zone of the meniscus in a subject, comprising administering to the subject a substance according to any one of the following (a) to (c):
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[7] The method according to [6], wherein the administration is systemic administration.
[8] A method for treating the resection site of a meniscus in a subject, the method comprising administering to the subject a substance according to any one of the following (a) to (c):
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[9] The method according to [8], wherein the resection site is a red-white zone or a white-white zone.
[10] The method according to [8] or [9], wherein the administration is systemic administration.
　本発明は、さらに以下を提供する：
　［１１］半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅの再生に用いるための、以下の（ａ）から（ｃ）のいずれかに記載の物質：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［１２］全身投与される［１１］記載の物質。
　［１３］半月板の切除部位の治療に用いるための、以下の（ａ）から（ｃ）のいずれかに記載の物質：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［１４］切除部位がｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅである、［１３］記載の物質。
　［１５］全身投与される［１３］または［１４］記載の物質。 The invention further provides:
[11] The substance described in any one of the following (a) to (c) for use in regenerating the red-white zone or white-white zone of the meniscus:
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[12] The substance according to [11] which is administered systemically.
[13] The substance according to any one of the following (a) to (c) for use in treating the resection site of the meniscus:
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[14] The substance according to [13], wherein the excision site is a red-white zone or a white-white zone.
[15] The substance according to [13] or [14] which is administered systemically.
　本発明は、さらに以下を提供する：
　［１６］半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するための薬剤の製造における、以下の（ａ）から（ｃ）のいずれかに記載の物質の使用：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［１７］該薬剤が全身投与されるものである［１６］記載の使用。
　［１８］半月板の切除部位を治療するための薬剤の製造における、以下の（ａ）から（ｃ）のいずれかに記載の物質の使用：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。
　［１９］切除部位がｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅである、［１８］記載の使用。
　［２０］該薬剤が全身投与されるものである［１８］または［１９］記載の使用。 The invention further provides:
[16] Use of the substance according to any of the following (a) to (c) in the manufacture of a drug for regenerating the red-white zone or white-white zone of the meniscus:
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[17] The use according to [16], wherein the drug is administered systemically.
[18] Use of the substance according to any of the following (a) to (c) in the manufacture of a medicament for treating a meniscal resection site:
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
[19] The use according to [18], wherein the resection site is a red-white zone or a white-white zone.
[20] The use according to [18] or [19], wherein the drug is administered systemically.
　本願のペプチドを含む医薬組成物は、自然には再生し得ない半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅおよびｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生・治療することができる。 A pharmaceutical composition containing the peptide of the present application can regenerate and treat the red-white zone and white-white zone of the meniscus, which cannot be regenerated naturally.
図１は、動物実験の手順を示すスキームである。FIG. 1 is a scheme showing the procedure for animal experiments. 図２は、サフラニン－Ｏ染色にて半月板の再生を調べた結果を示す組織標本である。FIG. 2 is a tissue specimen showing the results of examining meniscal regeneration using Safranin-O staining. 図３は、再生された半月板の写真である。Figure 3 is a photograph of the regenerated meniscus. 図４は、半月板再生面積の経時的変化を示すグラフである。丸がＨＭＧＢ１投与群、四角が生理食塩水投与群を示す。FIG. 4 is a graph showing changes over time in the regenerated area of the meniscus. The circles indicate the HMGB1 administration group, and the squares indicate the physiological saline administration group. 図５は、生理食塩水（左シャーレ）またはＨＭＧＢ１（１－４４）（右シャーレ）を投与した半月板部分切除マウスの循環細胞のコロニー形成線維芽細胞（ＣＦＵ－Ｆ）アッセイの結果を示す。棒グラフは、生理食塩水投与群とＨＭＧＢ１（１－４４）投与群のコロニー数の比較を示す。FIG. 5 shows the results of a circulating cell colony-forming fibroblast (CFU-F) assay of partially meniscectomized mice administered saline (left dish) or HMGB1(1-44) (right dish). The bar graph shows a comparison of the number of colonies between the physiological saline administration group and the HMGB1(1-44) administration group. 図６は、パラバイオーシスモデルを用いた実験の概要を示す。Figure 6 shows an overview of experiments using the parabiosis model. 図７は、再生された半月板のテキサスレッド染色像および明視野像を示す。FIG. 7 shows Texas Red stained and bright field images of the regenerated meniscus. 図８は、半月板の外側領域と内側領域での再生を、ｔｄTｏｍａｔｏの赤色蛍光を用いて調べた結果を示す顕微鏡像である。FIG. 8 is a microscopic image showing the results of examining regeneration in the outer and inner regions of the meniscus using red fluorescence from tdTomato.
　本発明は、第１の態様において、
　以下の（ａ）から（ｃ）のいずれかに記載の物質：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド
を含む、半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するための医薬組成物を提供する。 In a first aspect of the present invention,
Substances described in any of the following (a) to (c):
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 Provided is a pharmaceutical composition for regenerating the red-white zone or white-white zone of the meniscus, comprising a peptide comprising an amino acid sequence having about 80% or more sequence identity with.
　半月板の外側（関節包側）の血管領域はｒｅｄ－ｒｅｄ　ｚｏｎｅ、半月板の内側（顆間側）の無血行野はｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅ、その間はｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅと分類されている。ｒｅｄ－ｒｅｄ　ｚｏｎｅには血流があるため損傷を受けても治癒が期待される。しかしｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅは血流が乏しく、ｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅには血流がないため、これらの領域は、損傷を受けた場合に治癒が期待できない。したがって、半月板のこれらの部分が欠損したままだと、痛みが持続する、歩行や運動に支障が出る等の不都合を生じ、患者のＱＯＬが低下する。 The vascular area on the outside of the meniscus (on the joint capsule side) is classified as the red-red zone, the avascular area on the inside of the meniscus (on the intercondylar side) as the white-white zone, and the area between them as the red-white zone. The red-red zone has blood flow, so even if it is damaged, it is expected to heal. However, since the red-white zone has poor blood flow and the white-white zone has no blood flow, these areas cannot be expected to heal if damaged. Therefore, if these parts of the meniscus remain defective, inconveniences such as persistent pain and difficulty walking and exercise occur, leading to a decline in the patient's QOL.
　半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するとは、損傷したこれらの領域を元通りに再生することだけでなく、これらの領域を部分的に再生することも包含する。また、再生するとは、線維芽細胞、軟骨様細胞（すばわち、サフラニン－О染色陽性細胞）を含む正常半月板細胞が誘導され、半月板組織（線維軟骨組織ともいう）が形成されることをいう。このような再生によって、痛み、歩行・運動困難などの半月板損傷に伴う症状をなくす、あるいは軽減することができる。したがって、上記の本発明の医薬組成物を、半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅにおける損傷を治療するために用いてもよい。 Regenerating the red-white zone or white-white zone of the meniscus includes not only regenerating these damaged areas but also partially regenerating these areas. In addition, regeneration means that normal meniscal cells including fibroblasts and cartilage-like cells (safranin-O staining positive cells) are induced, and meniscal tissue (also called fibrocartilage tissue) is formed. Say something. Such regeneration can eliminate or reduce symptoms associated with meniscal damage, such as pain and difficulty walking and exercising. Accordingly, the pharmaceutical composition of the invention described above may be used to treat injuries in the red-white zone or white-white zone of the meniscus.
　半月板損傷とは、半月板の断裂、欠損および変形を包含する。 Meniscal damage includes meniscus tear, loss, and deformation.
　本発明における半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅの再生は、ＨＭＧＢ１断片ペプチドの作用に基づく。本発明において、ＨＭＧＢ１断片ペプチドは、配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１タンパク質の一部からなるペプチドおよびその変異体を包含する。以下において、配列番号：１に記載のアミノ酸配列を含むペプチドを「ＨＭＧＢ１ペプチド」といい、配列番号：１に記載のアミノ酸配列からなるＨＭＧＢ１断片ペプチドを「ＨＭＧＢ１（１－４４）」と略称することがある。 The regeneration of the red-white zone or white-white zone of the meniscus in the present invention is based on the action of the HMGB1 fragment peptide. In the present invention, the HMGB1 fragment peptide includes a peptide consisting of a portion of the HMGB1 protein containing the amino acid sequence set forth in SEQ ID NO: 1, and variants thereof. In the following, the peptide containing the amino acid sequence set forth in SEQ ID NO: 1 will be referred to as "HMGB1 peptide," and the HMGB1 fragment peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 will be abbreviated as "HMGB1 (1-44)." There is.
ＨＭＧＢ１ペプチドおよびその変異体、又はＨＭＧＢ１（１－４４）およびその変異体を公知の方法を用いて得ることができる。例えば、ＨＭＧＢ１ペプチドおよびその変異体又はＨＭＧＢ１（１－４４）およびその変異体をコードするポリヌクレオチドを適当な発現系に組み込んで遺伝子組換え体（ｒｅｃｏｍｂｉｎａｎｔ）として得てもよく、あるいは人工的に合成（例えば化学合成）してもよい。 HMGB1 peptide and its variants, or HMGB1(1-44) and its variants can be obtained using known methods. For example, a polynucleotide encoding HMGB1 peptide and its variants or HMGB1(1-44) and its variants may be obtained as a genetic recombinant by incorporating it into an appropriate expression system, or it may be obtained by artificially synthesizing it. (for example, chemical synthesis).
　ＨＭＧＢ１ペプチド又はＨＭＧＢ１（１－４４）の変異体は、配列番号：１に記載のアミノ酸配列において１つ以上のアミノ酸残基が改変（置換、欠失、挿入若しくは付加）されたアミノ酸配列を含むペプチドであって、配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチドと機能的に同等なペプチドである。 HMGB1 peptide or HMGB1 (1-44) variant is a peptide containing an amino acid sequence in which one or more amino acid residues have been modified (substituted, deleted, inserted, or added) in the amino acid sequence set forth in SEQ ID NO: 1. This peptide is functionally equivalent to the HMGB1 fragment peptide containing the amino acid sequence set forth in SEQ ID NO:1.
　ＨＭＧＢ１ペプチド又はＨＭＧＢ１（１－４４）の変異体の例としては、以下のものが挙げられるが、これらに限定されるものではない：
　ｉ） 配列番号：１に記載のアミノ酸配列において１若しくは複数個（例えば１個～１０個、１個～９個、１個～８個、１個～７個、１個～６個、１個～５個、１個～４個、１個～３個、または１個若しくは２個）のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含むペプチド；
　ｉｉ） 配列番号：１に記載のアミノ酸配列において１若しくは複数個（例えば１個～１０個、１個～９個、１個～８個、１個～７個、１個～６個、１個～５個、１個～４個、１個～３個、または１個若しくは２個）のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列からなるペプチド；
　ｉｉｉ） 配列番号：１に記載のアミノ酸配列と約８０％以上、例えば約８５％以上、約９０％以上、約９１％以上、約９２％以上、約９３％以上、約９４％以上、約９５％以上、約９６％以上、約９７％以上または約９８％以上の配列同一性を有するアミノ酸配列を含むペプチド；
　ｉｖ） 配列番号：１に記載のアミノ酸配列と約８０％以上、例えば約８５％以上、約９０％以上、約９１％以上、約９２％以上、約９３％以上、約９４％以上、約９５％以上、約９６％以上、約９７％以上または約９８％以上の配列同一性を有するアミノ酸配列からなるペプチド。 Examples of HMGB1 peptides or variants of HMGB1(1-44) include, but are not limited to:
i) One or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 (for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1) A peptide comprising an amino acid sequence in which ~5, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, deleted, inserted, or added;
ii) One or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 (for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1) A peptide consisting of an amino acid sequence in which ~5, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, deleted, inserted, or added;
iii) About 80% or more, for example about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more of the amino acid sequence set forth in SEQ ID NO: 1 % or more, about 96% or more, about 97% or more, or about 98% or more sequence identity;
iv) About 80% or more, for example about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more of the amino acid sequence set forth in SEQ ID NO: 1 % or more, about 96% or more, about 97% or more, or about 98% or more.
　本発明の医薬組成物において、ＨＭＧＢ１ペプチド又はＨＭＧＢ１（１－４４）を用いてもよく、ＨＭＧＢ１ペプチド又はＨＭＧＢ１（１－４４）の変異体を用いてもよく、あるいはこれらを組み合わせて用いてもよい。本発明の医薬組成物において、ＨＭＧＢ１ペプチド若しくはその変異体又はＨＭＧＢ１（１－４４）若しくはその変異体を分泌する細胞、該ペプチド若しくはその変異体をコードするポリヌクレオチド、該ポリヌクレオチドが挿入されたベクター、該ベクターを含む細胞を用いてもよい。 In the pharmaceutical composition of the present invention, HMGB1 peptide or HMGB1(1-44) may be used, a variant of HMGB1 peptide or HMGB1(1-44) may be used, or a combination of these may be used. . In the pharmaceutical composition of the present invention, a cell that secretes HMGB1 peptide or a variant thereof or HMGB1 (1-44) or a variant thereof, a polynucleotide encoding the peptide or a variant thereof, a vector into which the polynucleotide is inserted , cells containing the vector may be used.
　通常は、本発明の医薬組成物は、有効量のＨＭＧＢ１断片ペプチドまたはその変異体を含有する。有効量とは、半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するとは、損傷したこれらの領域を元通りに再生することができる量、あるいはこれらの領域を部分的に再生することができる量をいう。例えば、有効量は、元の半月板にまで再生することを１００％の再生とした場合に、３０％以上の、好ましくは５０％以上、より好ましくは９０％以上の再生をもたらす量であってもよい。また例えば、有効量は、痛み、歩行・運動困難などの半月板損傷に伴う症状をなくす、あるいは軽減する量であってもよい。半月板の再生については、公知の方法、例えばＭＲＩ撮影や超音波エコーなどを用いて確認することができる。 Usually, the pharmaceutical composition of the present invention contains an effective amount of HMGB1 fragment peptide or variant thereof. An effective amount is an amount that can regenerate the red-white zone or white-white zone of the meniscus, or can partially regenerate these damaged areas. Refers to the amount that can be done. For example, the effective amount is an amount that results in regeneration of 30% or more, preferably 50% or more, more preferably 90% or more, where 100% regeneration is defined as regeneration to the original meniscus. Good too. Also, for example, the effective amount may be an amount that eliminates or alleviates symptoms associated with meniscal damage, such as pain and difficulty walking and exercising. Regeneration of the meniscus can be confirmed using known methods such as MRI imaging and ultrasound echo.
　本発明の医薬組成物を投与する対象としては、特に制限はなく、哺乳類、鳥類、魚類等が挙げられる。哺乳類としては、ヒト又は非ヒト動物が挙げられ、例えば、ヒト、マウス、ラット、サル、ブタ、イヌ、ウサギ、ハムスター、モルモット、ウマ、ヒツジ、クジラなどが例示できるが、これらに限定されるものではない。本明細書において、「対象」という用語は、「患者」、「個体」および「動物」と互換的に用いられる。 Subjects to which the pharmaceutical composition of the present invention is administered are not particularly limited, and include mammals, birds, fish, and the like. Mammals include humans and non-human animals, including, but not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales. isn't it. As used herein, the term "subject" is used interchangeably with "patient," "individual," and "animal."
　本明細書において、「医薬組成物」という用語は、「医薬」、「薬剤」または「薬学的組成物」と互換的に用いられる。 As used herein, the term "pharmaceutical composition" is used interchangeably with "medicine," "drug," or "pharmaceutical composition."
　本発明の医薬組成物の投与部位は膝関節（例えば半月板もしくはその近傍）であってもよく、膝関節とは異なる部位であってもよい。いかなる部位に投与されても、本発明の医薬組成物は、その効果を発揮することができる。 The administration site of the pharmaceutical composition of the present invention may be the knee joint (for example, the meniscus or its vicinity) or a site different from the knee joint. The pharmaceutical composition of the present invention can exert its effects no matter where it is administered.
　本発明の医薬組成物の投与方法としては、経口投与または非経口投与が挙げられる。非経口投与方法としては、血管内投与（動脈内投与、静脈内投与等）、筋肉内投与、皮下投与、皮内投与、腹腔内投与、経鼻投与、経肺投与、経皮投与などが挙げられるが、これらに限定されない。また、本は発明の医薬組成物を、注射により、例えば、静脈内注射、筋肉内注射、腹腔内注射、皮下注射などにより、全身的または局部的（例えば、皮下、皮内、皮膚表面、鼻腔粘膜、口腔内および消化管粘膜、膣・子宮内粘膜、または損傷部位など）に投与してもよい。 Methods for administering the pharmaceutical composition of the present invention include oral administration and parenteral administration. Examples of parenteral administration methods include intravascular administration (intraarterial administration, intravenous administration, etc.), intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, nasal administration, pulmonary administration, and transdermal administration. but not limited to. The present invention also provides that the pharmaceutical compositions of the invention can be administered systemically or locally (e.g., subcutaneously, intradermally, on the skin surface, in the nasal cavity, etc.) by injection, e.g., intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. It may also be administered to mucous membranes, oral and gastrointestinal mucosa, vaginal/uterine mucosa, or injured areas).
　また、患者の年齢、症状により適宜投与方法および投与量を選択することができる。本発明の医薬組成物を投与する場合、例えば、一回の投与につき、ＨＭＧＢ１断片ペプチドまたはその変異体を、患者体重１ｋｇあたり約０．００００００１ｍｇから約１０００ｍｇの範囲で投与してもよい。例えば、患者体重１ｋｇあたり約０．００００１から約１０００００ｍｇの範囲で投与してもよい。本願のペプチドを分泌する細胞や該ペプチドをコードするポリヌクレオチドが挿入された遺伝子治療用ベクターを含む医薬組成物を投与する場合も、該ペプチドの量が上記範囲内となるように投与することができる。しかしながら、本発明の医薬組成物の投与量はこれらの量に限定されるものではない。 Furthermore, the administration method and dosage can be appropriately selected depending on the patient's age and symptoms. When administering the pharmaceutical composition of the present invention, for example, the HMGB1 fragment peptide or variant thereof may be administered in a range of about 0.0000001 mg to about 1000 mg per kg of patient body weight per administration. For example, it may be administered in a range of about 0.00001 to about 100,000 mg/kg of patient body weight. When administering a pharmaceutical composition containing cells that secrete the peptide of the present application or a gene therapy vector into which a polynucleotide encoding the peptide has been inserted, the amount of the peptide can be administered within the above range. can. However, the dosage of the pharmaceutical composition of the present invention is not limited to these amounts.
　本願の医薬組成物は、常法に従って製剤化することができ（例えば、Ｒｅｍｉｎｇｔｏｎ’ｓ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｓｃｉｅｎｃｅ，　ｌａｔｅｓｔ　ｅｄｉｔｉｏｎ，　Ｍａｒｋ　Ｐｕｂｌｉｓｈｉｎｇ　Ｃｏｍｐａｎｙ，　Ｅａｓｔｏｎ，　Ｕ．Ｓ．Ａ）、医薬的に許容される担体や添加物を共に含むものであってもよい。例えば界面活性剤、賦形剤、着色料、着香料、保存料、安定剤、緩衝剤、懸濁剤、等張化剤、結合剤、崩壊剤、滑沢剤、流動性促進剤、矯味剤等が挙げられるが、これらに制限されず、その他常用の担体や添加剤が適宜使用できる。具体的には、軽質無水ケイ酸、乳糖、結晶セルロース、マンニトール、デンプン、カルメロースカルシウム、カルメロースナトリウム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルアセタールジエチルアミノアセテート、ポリビニルピロリドン、ゼラチン、中鎖脂肪酸トリグリセライド、ポリオキシエチレン硬化ヒマシ油６０、白糖、カルボキシメチルセルロース、コーンスターチ、無機塩類、精製水、生理食塩水、グリセリン等を挙げることができる。 The pharmaceutical compositions of the present application can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, United States). .S.A), pharmaceutically acceptable carriers and additives It may also include things. For example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity promoters, and flavoring agents. These include, but are not limited to, and other commonly used carriers and additives can be used as appropriate. Specifically, light silicic anhydride, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, Examples include polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, purified water, physiological saline, and glycerin.
　本発明は、第２の態様において、
　以下の（ａ）から（ｃ）のいずれかに記載の物質：
　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド
を含む、半月板の切除部位を治療するための医薬組成物を提供する。 In a second aspect of the present invention,
Substances described in any of the following (a) to (c):
(a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 Provided is a pharmaceutical composition for treating a meniscal resection site comprising a peptide comprising an amino acid sequence having about 80% or more sequence identity with.
　上述のごとく、ｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅにおける損傷であって、保存療法や縫合術によっては治癒が望めない場合は切除術が適用となる。切除術を行った後に切除部位を再生することができれば、患者にとって非常に有益である。本発明の第２の態様の医薬組成物は、切除術によって切除されたｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅの部位を再生するものであってもよい。このような再生によって、当該切除部位が治療される。 As mentioned above, if the injury is in the red-white zone or white-white zone and cannot be cured by conservative treatment or suturing, excision is applied. It would be of great benefit to patients if the resection site could be regenerated after a surgical excision. The pharmaceutical composition of the second aspect of the present invention may regenerate the red-white zone or white-white zone excised by resection. Such regeneration cures the ablation site.
　本発明の第２の態様の医薬組成物による半月板の切除部位の再生は、元通りに再生するものであってもよく、部分的に再生するものであってもよい。再生は、元の半月板にまで再生することを１００％の再生とした場合に、３０％以上の、好ましくは５０％以上、より好ましくは９０％以上の再生をもたらす量であってもよい。 Regeneration of the resection site of the meniscus by the pharmaceutical composition of the second aspect of the present invention may be performed to restore the site to its original state or to partially regenerate it. Regeneration may be in an amount that results in regeneration of 30% or more, preferably 50% or more, more preferably 90% or more, where 100% regeneration is regeneration to the original meniscus.
　本発明の第２の態様の医薬組成物による半月板の切除部位の治療は、痛み、歩行・運動困難などの半月板損傷に伴う症状をなくす、あるいは軽減するものであってもよい。 Treatment of the resection site of the meniscus with the pharmaceutical composition of the second aspect of the present invention may eliminate or alleviate symptoms associated with meniscal damage, such as pain and difficulty walking and exercising.
　第１の態様の医薬組成物に関する他の説明は、第２の態様の医薬組成物についてもあてはまる。 Other explanations regarding the pharmaceutical composition of the first aspect also apply to the pharmaceutical composition of the second aspect.
　さらなる態様において、本発明は、上記の方法、物質および使用を提供する。第１の態様の医薬組成物および第２の態様の医薬組成物に関する説明は、該方法、物質および使用についてもあてはまる。 In further aspects, the invention provides the methods, materials and uses described above. The statements regarding the pharmaceutical composition of the first aspect and the pharmaceutical composition of the second aspect also apply to the methods, materials and uses.
　なお、本明細書において引用されたすべての先行技術文献は、参照として本明細書に組み入れられる。本願は、２０２２年６月３０日出願の日本国特許出願第２０２２－１０５６９６号に対して優先権を主張するものであり、該日本国特許出願の全内容を、参照により本明細書に組み入れられる。本明細書中の用語は、医学、薬学、生物学、生化学、化学等の分野において通常に理解されている意味に解される。 Furthermore, all prior art documents cited in this specification are incorporated herein by reference. This application claims priority to Japanese Patent Application No. 2022-105696 filed on June 30, 2022, and the entire contents of the Japanese Patent Application No. 2022-105696 are incorporated herein by reference. . The terms used herein are understood to have meanings commonly understood in the fields of medicine, pharmacy, biology, biochemistry, chemistry, and the like.
　本発明は、下記の実施例によってさらに例示されるが、それらに限定されるものではない。 The present invention is further illustrated by, but not limited to, the following examples.
　実験で使用した材料および方法は以下のとおりであった。 The materials and methods used in the experiment were as follows.
　倫理声明 
　すべての動物は、大阪大学大学院医学研究科動物委員会の承認されたガイドラインに従って取り扱われた。プロトコルは、大阪大学大学院医学研究科の動物委員会によって承認された。 ethics statement
All animals were handled in accordance with the approved guidelines of the Osaka University Graduate School of Medicine Animal Committee. The protocol was approved by the Animal Committee of Osaka University Graduate School of Medicine.
　マウス　
　すべての実験用マウスは、１２時間の明暗サイクルでケージに収容された。飼料と水は自由に摂取可能とした。Ｃ５７ＢＬ／６Jマウスは、クレア　Ｊａｐａｎ（東京、日本）から購入した。すべてのＰＤＧＦＲα陽性（Ｐα）細胞が核内でＧＦＰ蛍光を発するＰＤＧＦＲα／Ｈ２Ｂ－ＧＦＰノックイン（Ｐα－ＧＦＰＫＩ）マウス（Ｈａｍｉｌｔｏｎ　ＴＧ，　Ｍｏｌ　Ｃｅｌｌ　Ｂｉｏｌ．２００３）、Ｐα－Ｃｒｅ（ＪＡＸ、０１３１４８）およびＲＯＳＡｔｄＴｏｍａｔｏ（ＪＡＸ、００７９０９）マウスは、ＪａｃｋｓｏｎＬａｂｏｒａｔｏｒｙ（Ｂａｒ　Ｈａｒｂｏｕｒ、ＭＥ、ＵＳＡ）から入手した。 mouse
All experimental mice were housed in cages with a 12 hour light/dark cycle. Feed and water were available ad libitum. C57BL/6J mice were purchased from Claire Japan (Tokyo, Japan). PDGFRα/H2B-GFP knockin (Pα-GFPKI) mice in which all PDGFRα-positive (Pα) cells emit GFP fluorescence in the nucleus (Hamilton TG, Mol Cell Biol. 2003), Pα-Cre (JAX, 013148) and ROSAtdTomato ( JAX, 007909) mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA).
　半月板部分切除モデルマウスの作製　
　１．５－２．０％（ｖ／ｖ）のイソフルラン吸入麻酔下で、半月板部分切除する側（右膝前側）の下肢の剃毛を行い、右膝前側の皮膚に縦切開を行った。次に、関節包の前内側を切離し、膝蓋骨を外側に脱臼させた後、顕微手術技術を用いて内側半月板の前方半分（前節、中節）を内側側副靱帯レベルで切除した。その後、関節包を８－０バイクリルで縫合し、皮膚を５－０バイクリルで縫合した。術後、マウスはケージの中を自由に歩行させた。手術の精度と再現性を確保するために、すべての手順は実体顕微鏡（Ｚｅｉｓｓ　Ｓｔｅｍｉ　２０００Ｃ、ドイツ）下で実施された。 Preparation of partial meniscectomy model mouse
Under 1.5-2.0% (v/v) isoflurane inhalation anesthesia, the lower leg on the side where the partial meniscus was to be removed (the front side of the right knee) was shaved, and a vertical incision was made in the skin on the front side of the right knee. . Next, after dissecting the anteromedial joint capsule and dislocating the patella laterally, the anterior half of the medial meniscus (anterior segment, middle segment) was resected at the level of the medial collateral ligament using microsurgical techniques. Thereafter, the joint capsule was sutured with 8-0 Vicryl, and the skin was sutured with 5-0 Vicryl. Postoperatively, the mice were allowed to walk freely in the cage. All procedures were performed under a stereomicroscope (Zeiss Stemi 2000C, Germany) to ensure precision and reproducibility of the surgery.
　ＨＭＧＢ１（１－４４）　
　ヒト由来のＨＭＧＢ１タンパク質（配列番号：２）のアミノ酸残基１－４４（配列番号：１）からなるペプチドを、ＰｏｌｙＰｅｐｔｉｄｅ　Ｌａｂｏｒａｔｏｒｉｅｓ（サンディエゴ）に委託して化学合成した。 HMGB1(1-44)
A peptide consisting of amino acid residues 1-44 (SEQ ID NO: 1) of human-derived HMGB1 protein (SEQ ID NO: 2) was chemically synthesized by PolyPeptide Laboratories (San Diego).
　パラバイオーシスマウスモデル　
　パラバイオーシスマウスモデルは、以前に説明されたように生成された（Duyverman MA. et al., Nat. Protc., 7(4):763-770, 2012）。野生型マウス（Ｃ５７／ＢＬ６）とＰａ－Ｃｒｅ；ＲＯＳＡｔｄＴｏｍａｔｏマウスを並体結合した。胴体を、対応する側面で肘頭から膝関節まで縫合した。実験開始前に、ペアを上記の通常の飼育条件下で４週間以上ケージに個別に保管した。 parabiosis mouse model
A parabiosis mouse model was generated as previously described (Duyverman MA. et al., Nat. Protc., 7(4):763-770, 2012). Wild type mice (C57/BL6) and Pa-Cre; ROSAtdTomato mice were parabiotically combined. The torso was sutured from the olecranon to the knee joint on the corresponding sides. Prior to the start of the experiment, pairs were kept individually in cages for over 4 weeks under normal husbandry conditions as described above.
　組織学的検査　
　半月板部分切除の１、２、４、または６週間後にマウスを安楽死させた（１群あたりマウス６匹）。組織を４％パラホルムアルデヒドで１日間固定し、０．５ｍｏｌ／Ｌ　エチレンジアミン四酢酸（ＥＤＴＡ）溶液で１４日間脱灰した後、パラフィンワックスに包埋した。サンプルを矢状面で６μｍに切断し、サフラニンＯとファストグリーンで染色した（参考文献）。組織学的スライスの顕微鏡評価は、オリンパスＢＸ　５３顕微鏡（オリンパス、東京、日本）下で実施した。 histological examination
Mice were euthanized 1, 2, 4, or 6 weeks after partial meniscectomy (6 mice per group). Tissues were fixed with 4% paraformaldehyde for 1 day, decalcified with 0.5 mol/L ethylenediaminetetraacetic acid (EDTA) solution for 14 days, and then embedded in paraffin wax. Samples were sectioned at 6 μm in the sagittal plane and stained with Safranin O and Fast Green (ref.). Microscopic evaluation of histological slices was performed under an Olympus BX 53 microscope (Olympus, Tokyo, Japan).
　肉眼観察 
　内側半月板を、脛骨プラトーから注意深く分離した。巨視的画像は、ライカＭ　２０５Ｆ顕微鏡（ライカ社、ハイデルベルク、ドイツ）を使用して撮影した後、Ｆｉｊｉを使用して内側半月板の面積の定量化を実施した（Ozeki N. et al., Arthritis Rheum., 65(11):2876-2886, 2013 Nov.）。 naked eye observation
The medial meniscus was carefully separated from the tibial plateau. Macroscopic images were taken using a Leica M 205F microscope (Leica GmbH, Heidelberg, Germany), after which quantification of the area of the medial meniscus was performed using Fiji (Ozeki N. et al., Arthritis Rheum., 65(11):2876-2886, 2013 Nov.).
　統計分析 
　値は平均±標準偏差として表される。２つのグループ間のデータは、ウィルコクソンの符号付き順位検定を使用して比較した。多重比較分析では、分散分析（ＡＮＯＶＡ）と事後分析としてのＴｕｋｅｙ検定を実行した。データの入力と計算は、ＪＭＰ　Ｐｒｏソフトウェアバージョン１３（ＳＡＳ　Ｉｎｓｔｉｔｕｔｅ　Ｉｎｃ．，米国ノースカロライナ州ケアリー）で実行した。０．０５未満のｐ値は統計的に有意であると見なした。 statistical analysis
Values are expressed as mean ± standard deviation. Data between two groups were compared using the Wilcoxon signed rank test. For multiple comparison analysis, analysis of variance (ANOVA) and Tukey test as post hoc analysis were performed. Data entry and calculations were performed with JMP Pro software version 13 (SAS Institute Inc., Cary, NC, USA). P values less than 0.05 were considered statistically significant.
　マウスの半月板損傷に対するＨＭＧＢ１（１－４４）投与の治療可能性を調べた。生理食塩水（１００μｌ）またはＨＭＧＢ１（１－４４）（生理食塩水１００μｌ中５ｍｇ／ｋｇ）を、半月板部分切除後当日から５日間連続して静脈内投与した。実験手順を図１に示す。 The therapeutic potential of HMGB1 (1-44) administration for meniscal damage in mice was investigated. Physiological saline (100 μl) or HMGB1 (1-44) (5 mg/kg in 100 μl of physiological saline) was administered intravenously for 5 consecutive days starting from the day of partial meniscectomy. The experimental procedure is shown in Figure 1.
　組織学的観察結果を図２に示す。生理食塩水投与群では術後１週間で半月板の欠損部位に線維性瘢痕組織のみが観察され、術後２、４、６週間目でほとんど変化はなかった。サフラニン－Ｏ染色陽性細胞はほとんど存在しなかった。再生された半月板の表面は細線維化または分裂が認められた。対照的に、ＨＭＧＢ１（１－４４）投与群では、術後２週間目で軟骨様細胞（すなわち、サフラニン－Ｏ染色陽性細胞）の再生が有意に誘導された。さらに、サフラニン－Ｏ陽性半月板細胞の面積は徐々に大きくなり、再生された半月板の表面はより平滑であった。術後４週間目および６週間目での再生半月板の細胞の形態は、正常な半月板細胞と同様にサフラニン－Ｏ染色陽性細胞で囲まれていた。 Figure 2 shows the histological observation results. In the saline-administered group, only fibrous scar tissue was observed at the meniscal defect site one week after surgery, and there was almost no change at 2, 4, and 6 weeks after surgery. There were almost no cells positive for Safranin-O staining. Fibrillation or division was observed on the surface of the regenerated meniscus. In contrast, in the HMGB1(1-44) administration group, regeneration of chondroid cells (ie, cells positive for Safranin-O staining) was significantly induced two weeks after surgery. Furthermore, the area of Safranin-O positive meniscal cells gradually increased, and the surface of the regenerated meniscus was smoother. The cell morphology of the regenerated meniscus at 4 and 6 weeks after surgery was similar to normal meniscal cells and surrounded by cells positive for safranin-O staining.
　肉眼分析は、生理食塩水投与群では、再生領域が徐々に拡大することが示された。術後６週間目で、ほぼ再生された半月板は、両方の群で正常な半月板と同様のＣ字型の組織を示した（図３）。しかし、ＨＭＧＢ１（１－４４）投与群では、術後２週間目と４週間目で、再生面積は生理食塩水群よりも有意に大きかった（図３、図４）。これらの結果は、ＨＭＧＢ１（１－４４）の全身投与が、瘢痕の軽減および自然には発生しない機能的な半月板の治癒を促進することを明確に示している。 Macroscopic analysis showed that the regeneration area gradually expanded in the saline-administered group. At 6 weeks postoperatively, the nearly regenerated meniscus showed a C-shaped organization similar to normal menisci in both groups (Figure 3). However, in the HMGB1(1-44) administration group, the regenerated area was significantly larger than that in the saline group at 2 and 4 weeks after surgery (Figures 3 and 4). These results clearly demonstrate that systemic administration of HMGB1(1-44) promotes scarring reduction and non-naturally occurring functional meniscal healing.
　次に、ＨＭＧＢ１（１－４４）の全身投与により、骨髄由来ＰＤＧＦＲα陽性幹細胞（ＰαＬｉｎ＋細胞）が末梢循環を介して半月板切除部分に動員されることを確認するために、生理食塩水またはＨＭＧＢ１（１－４４）を投与した半月板部分切除マウスの循環細胞のコロニー形成線維芽細胞（ＣＦＵ－Ｆ）アッセイを評価した。半月板部分切除術とＨＭＧＢ１（１－４４）投与により、コロニー数が有意に増加した（図５）。 Next, to confirm that bone marrow-derived PDGFRα-positive stem cells (PαLin+ cells) are mobilized to the meniscectomy site via the peripheral circulation by systemic administration of HMGB1(1-44), we used saline or HMGB1( Colony-forming fibroblast (CFU-F) assay of circulating cells was evaluated in partially meniscectomized mice treated with C.1-44). Partial meniscectomy and administration of HMGB1(1-44) significantly increased the number of colonies (Figure 5).
　次に、野生型（ＷＴ）マウスとＰα－Ｃｒｅ；ＲｏｓａｔｄＴｏｍａｔｏマウス用いたパラバイオーシスモデルを確立して、損傷部位へのＰαＬｉｎ＋細胞の動員の有無を調べた。ＷＴマウス側の膝に半月板部分切除を行い、生理食塩水またはＨＭＧＢ１（１－４４）をＰα－Ｃｒｅ；ＲｏｓａｔｄＴｏｍａｔｏマウス側の尾静脈から投与した（図６）。生理食塩水投与群では、欠損部位で全身的に動員されたＰαＬｉｎ＋細胞の寄与はほとんどなかった。対照的に、ＨＭＧＢ１（１－４４）投与群では、欠損部位の外側領域と内側領域の両方にＰαＬｉｎ＋細胞の動員が有意に誘導された（図７、図８）。これらの結果から、ＨＭＧＢ１（１－４４）の全身投与により、ＰαＬｉｎ＋細胞が損傷した半月板に動員されて、半月板を再生させると考えられた。 Next, a parabiosis model was established using wild type (WT) mice and Pα-Cre; RosatdTomato mice, and the presence or absence of recruitment of PαLin+ cells to the injured site was investigated. A partial meniscectomy was performed on the knee of the WT mouse, and physiological saline or HMGB1 (1-44) was administered through the tail vein of the Pα-Cre; RosatdTomato mouse (FIG. 6). In the saline-administered group, there was almost no contribution from systemically recruited PαLin+ cells at the defect site. In contrast, in the HMGB1(1-44) administration group, recruitment of PαLin+ cells was significantly induced to both the outer and inner regions of the deletion site (FIGS. 7 and 8). From these results, it was considered that systemic administration of HMGB1(1-44) mobilizes PαLin+ cells to the damaged meniscus and regenerates the meniscus.
　本発明は、医薬品、医学研究、薬学研究などの分野において利用可能である。 The present invention can be used in fields such as pharmaceuticals, medical research, and pharmaceutical research.
　配列番号：１は、ヒトＨＭＧＢ１タンパク質のアミノ酸残基１－４４からなるペプチドのアミノ酸配列を示す。
　配列番号：２は、ヒトＨＭＧＢ１タンパク質のアミノ酸配列を示す。
　配列番号：３は、ヒトＨＭＧＢ１タンパク質をコードする核酸の塩基配列を示す。
　 SEQ ID NO: 1 shows the amino acid sequence of a peptide consisting of amino acid residues 1-44 of human HMGB1 protein.
SEQ ID NO: 2 shows the amino acid sequence of human HMGB1 protein.
SEQ ID NO: 3 shows the base sequence of a nucleic acid encoding human HMGB1 protein.

Claims (5)

  1. 　以下の（ａ）から（ｃ）のいずれかに記載の物質を含む、半月板のｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅを再生するための医薬組成物：
    　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
    　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
    　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。     A pharmaceutical composition for regenerating the red-white zone or white-white zone of the meniscus, comprising a substance according to any of the following (a) to (c):
    (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
    (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
  2. 　全身投与される請求項１記載の医薬組成物。 The pharmaceutical composition according to claim 1, which is administered systemically.
  3. 　以下の（ａ）から（ｃ）のいずれかに記載の物質を含む、半月板の切除部位を治療するための医薬組成物：
    　（ａ）配列番号：１に記載のアミノ酸配列を含むＨＭＧＢ１断片ペプチド；
    　（ｂ）配列番号：１に記載のアミノ酸配列において１個または複数個のアミノ酸が置換、欠失、挿入または付加されたアミノ酸配列を含むペプチド；または
    　（ｃ）配列番号：１に記載のアミノ酸配列と約８０％以上の配列同一性を有するアミノ酸配列を含むペプチド。     A pharmaceutical composition for treating a meniscal resection site, comprising a substance according to any of the following (a) to (c):
    (a) HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
    (b) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequence set forth in SEQ ID NO: 1; or (c) the amino acid sequence set forth in SEQ ID NO: 1 A peptide comprising an amino acid sequence having about 80% or more sequence identity with.
  4. 　切除部位がｒｅｄ－ｗｈｉｔｅ　ｚｏｎｅまたはｗｈｉｔｅ－ｗｈｉｔｅ　ｚｏｎｅである、請求項３記載の医薬組成物。 The pharmaceutical composition according to claim 3, wherein the resection site is a red-white zone or a white-white zone.
  5. 　全身投与される請求項３または４記載の医薬組成物。 The pharmaceutical composition according to claim 3 or 4, which is administered systemically.
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