Fusion peptide

Abstract

The present invention addresses the problem of providing a novel method for treating cerebral infarction that does not depend on cell transplantation.　Provided is a fusion peptide obtained by linking adrenomedullin or an active fragment thereof with a self-assembling peptide.

Description

Fusion peptide

[0001] The present invention relates to a fusion peptide, a gelling composition, a sustained-release gel, a pharmaceutical composition, a method for producing a gel, a method for promoting angiogenesis, a method for suppressing neurodegeneration, a method for treating nerve tissue damage and / or ischemia, and a method for treating cerebral infarction, etc.

[0002] In cerebral infarction, cerebral blood vessels become blocked, reducing blood flow and causing dysfunction of brain nerve cells. Patients with cerebral infarction exhibit severe symptoms such as hemiplegia, sensory impairment, aphasia, and impaired consciousness.

[0003] In Japan, approximately 200,000 people suffer from cerebral infarction each year. Many patients who suffer from cerebral infarction have difficulty recovering their neurological functions, and approximately 25% of them require nursing care level 4 or 5. As a result, medical expenses currently amount to approximately 1 trillion yen per year, and there are concerns that medical expenses will increase further as the aging population continues.

[0004] In recent years, cell transplantation therapy has attracted attention as a new treatment for cerebral infarction (Non-Patent Document 1). Cell transplantation therapy is a treatment method that restores cranial nerve function by transplanting cells such as neurons into the site of cerebral infarction. Studies using animal models have shown that stem cell transplantation can improve brain function by replacing neurons, promoting angiogenesis, and providing neuroprotection.

[0005] The feasibility of cell transplantation therapy has increased with the development of technologies for producing induced pluripotent stem cells and inducing their differentiation. However, major problems remain, such as the need for complex procedures and the difficulty of maintaining quality due to the handling of cells. Another problem is that it is difficult to reduce costs due to the inability to mass-produce cells.

[0006] Therefore, in order to more easily treat cerebral infarction, a new therapeutic method that does not rely on cell transplantation is desired.

[0007] Olle Lindvall and Zaal Kokaia, Stroke (2011), 42(8):2369-75.

[0008] An object of the present invention is to provide a new method for treating cerebral infarction without relying on cell transplantation.

[0009] Gelling agents such as collagen and amphiphilic peptides composed of alternating hydrophilic and hydrophobic amino acids are known to be able to solidify from a sol state in water or an aqueous solution under specific temperature and pressure conditions to form a gel, and these peptides are collectively known as self-assembling peptides. Self-assembling peptides may be used as regenerative medical materials by fusing them with proteins or other substances that have specific biological functions, thereby conjugating them with biofunctional molecules.

[0010] In order to solve the above problems, the present inventors have created a new fusion peptide, AM-JigSAP, by linking jigsaw-shaped self-assembling peptide (JigSAP) with adrenomedullin (hereinafter also referred to as "AM").

[0011] Adrenomedullin is a physiologically active peptide discovered to possess potent vasodilatory antihypertensive effects. It is known to have diverse pharmacological activities, including cardiovascular protection, anti-inflammatory effects, angiogenesis, and tissue repair promotion. Because adrenomedullin has a relatively short amino acid sequence, the AM-JigSAP fusion peptide fused with JigSAP also has a short sequence. Therefore, it can be synthesized chemically at low production costs, rather than produced in cells, which requires enormous production costs. Adrenomedullin is also advantageous in that it is highly safe and has already been used clinically.

[0012] On the other hand, adrenomedullin is known to have low stability in the blood, and therefore it is difficult to achieve effects such as angiogenesis, which require sustained effects over a long period of time, using adrenomedullin alone.

[0013] The inventors complexed the above-mentioned fusion peptide with a gel composed of jigsaw self-assembling peptide (JigSAP) that was not complexed with adrenomedullin, and administered the complex to a mouse cerebral infarction model. As a result, they found that the angiogenesis-promoting function based on adrenomedullin contained in the fusion peptide is provided over a long period of time due to the sustained release effect from the gel, and that this significantly promotes recovery from gait dysfunction caused by cerebral infarction. The present invention is based on the above-mentioned research results and provides the following.

[0014] (1) A fusion peptide comprising adrenomedullin or an active fragment thereof linked to a self-assembling peptide. (2) The fusion peptide according to (1), wherein the adrenomedullin consists of: (i) the amino acid sequence set forth in any one of SEQ ID NOs: 36 to 41; (ii) an amino acid sequence in which 1 to 15 amino acid residues are deleted, substituted, or added in the amino acid sequence set forth in any one of SEQ ID NOs: 36 to 41; or (iii) an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 36 to 41. (3) The fusion peptide according to (1), wherein, in the adrenomedullin, cysteine ​​residues corresponding to positions 16 and 21 of the amino acid sequence set forth in SEQ ID NO: 36 form a disulfide bond, or the disulfide bond is substituted with an ethylene group. (4) The fusion peptide according to (1), wherein the adrenomedullin or an active fragment thereof contains a glycine residue added to its C-terminus. (5) The self-assembling peptide is: (a) an amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly); (b) an amino acid sequence represented by any of the following formulas II to IV: Arg-Gly-Asp-Ala-(Arg-Ala-Asp-Ala) (formula II), (Arg-Ala-Asp-Ala)-Arg-Gly-Asp-Ala (formula III), or (Arg-Ala-Asp-Ala)-Arg-Ala-Asp-Gly (formula IV); or (c) an amino acid sequence represented by the following formula V: (Arg-Ala-Asp-Ala) p(Formula V) (wherein p is an integer of 1 or greater). (6) The fusion peptide according to any one of (1) to (4), comprising an amino acid sequence represented by formula I, formulas II to IV, or formula V, wherein Arg is added to the N-terminus and / or C-terminus of the amino acid sequence represented by formula I, formulas II to IV, or formula V, or wherein two Arg amino acids are added to the N-terminus and / or C-terminus of the amino acid sequence represented by formula I, formulas II to IV, or formula V. (7) The fusion peptide according to any one of (1) to (4), wherein the adrenomedullin or an active fragment thereof is linked to the C-terminus and / or N-terminus of the self-assembling peptide. (8) The fusion peptide according to any one of (1) to (4), wherein the adrenomedullin or an active fragment thereof and the self-assembling peptide are linked via a linker. (9) The fusion peptide according to any one of (1) to (4), wherein the C-terminus is amidated. (10) A gelling composition comprising the fusion peptide according to any one of (1) to (4) as an active ingredient. (11) A sustained-release gel comprising the gelling composition according to (10). (12) The gelling composition according to (10), further comprising the self-assembling peptide not linked to the adrenomedullin or an active fragment thereof. (13) The gelling composition according to (10), further comprising one or more anions selected from the group consisting of bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions. (14) A pharmaceutical composition comprising the gelling composition according to (10). (15) The pharmaceutical composition according to (14), for promoting angiogenesis and / or suppressing neurodegeneration. (16) The pharmaceutical composition according to (14), for use in treating and / or preventing nervous tissue damage and / or ischemia. (17) The pharmaceutical composition according to (16), wherein the nervous tissue damage is cerebral infarction, stroke, or traumatic brain injury. (18) A method for producing a gel, comprising: a mixing step of mixing the gelling composition according to (10) with water or an aqueous solution; and a gelling step of gelling the mixture obtained after the mixing step by maintaining the mixture at a temperature equal to or lower than the gelling temperature.(19) The method according to (18), wherein the mixing step further comprises mixing one or more anions selected from the group consisting of bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions. (20) The method according to (18), wherein the concentration of the fusion peptide in the gel is 0.4% by weight to 10% by weight. (21) The method according to (18), wherein the gel is for implantation and / or sustained release. This specification incorporates the disclosure of Japanese Patent Application No. 2023-192464, from which the present application claims priority.

[0015] According to the present invention, a new method for treating cerebral infarction without relying on cell transplantation is provided.

[0016] 1A shows the structure and activity of the AM-JigSAP fusion peptide. FIG. 1A is a schematic diagram of the structure of the AM-JigSAP fusion peptide. In the AM-JigSAP fusion peptide, adrenomedullin (AM) is fused to the C-terminus of JigSAP. FIG. 1B shows the results of measuring the activity of adrenomedullin (AM) not fused to JigSAP and the AM-JigSAP fusion peptide. This figure shows a method for analyzing gait function in a mouse cerebral infarction model. FIG. 2A shows a mouse slipping its hind paws off a net. FIG. 2B shows normal walking. This figure shows the results of gait function analysis in a mouse cerebral infarction model. FIG. 3A shows a method for analyzing gait function in a mouse cerebral infarction model. FIG. 3B shows the results of gait function analysis in a mouse cerebral infarction model administered intracerebrally with an AM-JigSAP / JigSAP composition or a JigSAP composition. This figure shows gene expression in the mouse brain after administration of various compositions. Figure 4A shows the results of comparing gene expression in the brains of mice administered with the VEGF-JigSAP / JigSAP composition with that in the brains of mice administered with PBS (VEGF-JigSAP vs. PBS), and Figure 4B shows the results of comparing gene expression in the brains of mice administered with the AM-JigSAP / JigSAP composition with that in the brains of mice administered with the JigSAP composition (AM-JigSAP vs. JigSAP).

[0017] 1. Fusion Peptides 1-1. Overview A first aspect of the present invention is a fusion peptide. The fusion peptide of this aspect is a peptide formed by linking adrenomedullin or an active fragment thereof to a self-assembling peptide. The fusion peptide of this aspect can provide angiogenesis-promoting function based on the adrenomedullin in the fusion peptide. Furthermore, in the fusion peptide of this aspect, adrenomedullin is stabilized by being linked to the self-assembling peptide, and the angiogenesis-promoting function is maintained for a long period of time.

[0018] 1-2. Definition of Terms The following terms frequently used in this specification are defined below. As used herein, "self-assembly" refers to the spontaneous assembly of small molecules in a dispersion medium through intermolecular interactions or the like to form a three-dimensional structure. In this specification, a substance that gels through self-assembly is often referred to as a "gelator." As used herein, self-assembling peptides are a type of gelator.

[0019] As used herein, the term "self-assembling peptide" refers to a peptide that can solidify from a sol state dissolved in water or an aqueous solution to a gel state under specific temperature and pressure conditions. Examples of such peptides include collagen (including glue, gelatin, and jelly), (RADA)4 peptide (SEQ ID NO: 35), and JigSAP, which will be described in the Examples below.

[0020] In this specification, the term "gel" refers to a substance in which colloidal particles self-assemble in a dispersion medium, lose fluidity, solidify, and become solid.

[0021] In this specification, the term "gel state" refers to a state in which colloidal particles self-assemble in a dispersion medium, lose fluidity, and solidify. Generally, this refers to a state in which a sol is solidified by lowering its temperature. "Gellation" refers to a phase transition phenomenon from a sol state to a gel state.

[0022] As used herein, the term "sol" refers to a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity, such as a gel in which a colloid made of a gelling agent is fluidized in the dispersion medium by heating the gel.

[0023] The "sol state" refers to a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity. For example, this refers to a state in which a gelling agent is dispersed in a dispersion medium such as water or an aqueous solution, or a state in which a gel is fluidized by heating. "Solation" is a phase transition phenomenon from a gel state to a sol state.

[0024] As used herein, the term "gelation temperature" refers to the temperature at which a gelling agent undergoes a phase transition from a sol state to a gel state. The term "solation temperature" refers to the temperature at which a gelling agent undergoes a phase transition from a gel state to a sol state.

[0025] As used herein, the term "peptide" refers to an amino acid polymer having one or more peptide bonds. The term "peptide" is not limited by the number of amino acid residues contained in the peptide. Therefore, "peptide" encompasses everything from oligopeptides containing a few amino acid residues, such as dipeptides and tripeptides, to polypeptides containing many amino acid residues. Therefore, it encompasses not only so-called proteins, but also fragments and peptides linked to other peptides by peptide bonds.

[0026] As used herein, the term "fusion peptide" refers to a self-assembling peptide to which a functional peptide such as adrenomedullin is linked. The link between the self-assembling peptide and the functional peptide may be a covalent bond or a supramolecular interaction. The covalent bond is not limited, and examples include a peptide bond and a disulfide bond. Although not limited thereto, the functional peptide is preferably linked to the N-terminus and / or C-terminus of the self-assembling peptide, and the bond is preferably a covalent bond, with a preferred covalent bond being a peptide bond.

[0027] As used herein, the term "functional peptide" refers to a peptide that has a specific biological function in vivo or ex vivo, or intracellularly or extracellularly. As used herein, the term "specific biological function" is not limited to a function that can have any effect on biomolecules such as proteins and nucleic acids, cells, tissues, or individuals. Specific biological functions may be natural or non-natural, and examples include cell adhesion function, signal transduction function, binding function, linking function, labeling function, metabolic function, etc. An example of a functional peptide is adrenomedullin.

[0028] As used herein, "biocompatibility" refers to the property of being capable of being introduced into a living organism. In particular, it refers to the property of a material having no toxicity or side effects to the living organism, or having only very slight toxicity or side effects, and / or the property of not being recognized as a foreign body in the living organism and not being eliminated. As used herein, "a peptide having biocompatibility" refers, for example, to a peptide that is free from biological contamination and therefore has no or very little risk of causing allergies or unknown infectious diseases in the human body. Examples of biocompatible peptides include chemically synthesized peptides.

[0029] As used herein, the term "living body" refers to cells (including cultured cells), tissues, organs, or individuals. Examples include, but are not limited to, cells, tissues, or organs derived from humans or non-human individuals, or cells derived from humans or non-human individuals, such as cells or tissues obtained by differentiation from ES cells or iPS cells. Preferably, the organism is a human-derived cell, a tissue or organ composed of human-derived cells, or a human individual.

[0030] As used herein, "physiological conditions" refers to conditions such as temperature and pH that do not substantially impair the structure or activity of biomolecules, the structure or function of cells or tissues, or the activity or survival of individuals. More specifically, these conditions refer to conditions that may exist in a living organism or within a cell. As used herein, physiological conditions refer to conditions under which biomolecules, particularly proteins, do not denature or are unlikely to denature. As used herein, physiological pH is not limited to any pH at which biomolecules, such as proteins, do not denature or are unlikely to denature. For example, it is within the range of pH 4.0 to 10.0, pH 5.0 to 9.0, pH 6.0 to 8.0, or pH 6.5 to 7.5, e.g., pH 7.4. As used herein, physiological temperature is not limited to any temperature at which biomolecules, such as proteins, do not denature or are unlikely to denature. For example, it is within the range of 0 to 65°C, 4 to 60°C, 20 to 50°C, or 30 to 40°C, e.g., 37°C.

[0031] As used herein, the term "sustained release" refers to the gradual release of a substance into space. In this specification, it particularly refers to the gradual dissipation of a substance contained in a gel from the gel. For example, it refers to the dissipation of the substance at a rate slower than the rate at which the substance would dissipate if it were not encapsulated in the gel. When a functional molecule is slowly released from a gel implanted in a living body, the functional molecule remains in the surrounding space (e.g., the diseased site, damaged site, tissue, or organ into which the gel is implanted) for a long period of time, thereby providing its function.

[0032] As used herein, "long-term" or "prolonged period" refers to a period longer than the period during which a substance continues to be released under normal conditions. Specifically, it refers to a period longer than the period during which a substance not encapsulated in a gel continues to be released under the same conditions. The specific period varies depending on the type of substance, but examples include periods of 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, half a day or more, 1 day or more, 2 days or more, 3 days or more, 1 week or more, 2 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 6 months or more, or 1 year or more.

[0033] As used herein, the term "amino acid" encompasses both natural and unnatural amino acids. Unnatural amino acids are, for example, amino acids having any chemically modified group or substituent. As used herein, amino acids include any optical isomers, and may be either D- or L-isomers.

[0034] As used herein, the term "hydrophobic amino acid" refers to an amino acid having hydrophobicity or a highly hydrophobic amino acid. Examples include alanine (Ala / A), glycine (Gly / G), proline (Pro / P), valine (Val / V), leucine (Leu / L), isoleucine (Ile / I), methionine (Met / M), cysteine ​​(Cys / C), phenylalanine (Phe / F), tyrosine (Tyr / Y), and tryptophan (Trp / W). Among these amino acids, cysteine ​​has low hydrophobicity and is therefore sometimes classified as a hydrophilic amino acid.

[0035] As used herein, the term "hydrophilic amino acid" refers to an amino acid that has hydrophilicity or has high hydrophilicity, including, for example, aspartic acid (Asp / D), glutamic acid (Glu / E), lysine (Lys / K), histidine (His / H), and arginine (Arg / R).

[0036] As used herein, "plurality" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 3, or 2. Additionally, as used herein, "several" refers to 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2.

[0037] As used herein, "amino acid identity" refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two peptides being compared are aligned by inserting appropriate gaps into one or both of them as needed to maximize the number of identical amino acid residues. Alignment of two amino acid sequences to calculate amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW.

[0038] As used herein, unless otherwise specified, "(amino acid) substitution" refers to a substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to alter the properties of peptides.

[0039] 1-3. Structure The structure of the fusion peptide of this embodiment is specifically described below. The fusion peptide of this embodiment is a peptide formed by linking adrenomedullin or an active fragment thereof with a self-assembling peptide.

[0040] As used herein, "adrenomedullin (hereinafter also referred to as "AM")" is a physiologically active peptide discovered to have potent vasodilatory antihypertensive effects. Adrenomedullin is known to have a variety of pharmacological effects, including cardiovascular protection, anti-inflammatory effects, angiogenesis, and tissue repair promotion.

[0041] A specific example of adrenomedullin is human-derived adrenomedullin (human AM) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 36. Further specific examples include porcine-derived adrenomedullin consisting of the amino acid sequence shown in SEQ ID NO: 37, dog-derived adrenomedullin consisting of the amino acid sequence shown in SEQ ID NO: 38, bovine-derived adrenomedullin consisting of the amino acid sequence shown in SEQ ID NO: 39, rat-derived adrenomedullin consisting of the amino acid sequence shown in SEQ ID NO: 40, and mouse-derived adrenomedullin consisting of the amino acid sequence shown in SEQ ID NO: 41.

[0042] Adrenomedullin also encompasses adrenomedullin variants, adrenomedullin mutants, and adrenomedullin orthologs of other organisms that have activity functionally equivalent to that of the adrenomedullin shown in SEQ ID NOs: 36 to 41. Specific examples include amino acid sequences in which one or several amino acids have been deleted, substituted, or added in the amino acid sequences shown in SEQ ID NOs: 36 to 41, or adrenomedullin variants, adrenomedullin mutants, or adrenomedullin orthologs that have 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity to the amino acid sequences shown in SEQ ID NOs: 36 to 41.

[0043] As used herein, the term "active fragment" of adrenomedullin refers to a fragment of any of the above adrenomedullins that has physiological activity such as angiogenic activity, for example, a fragment having 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of any of the above adrenomedullins, or an activity equivalent to or greater than that. The amino acid length of the peptide constituting the active fragment is not particularly limited, and may be, for example, a region of any of the above adrenomedullins that has 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acids.

[0044] In one embodiment, in the fusion peptide of this aspect, the cysteine ​​residues corresponding to positions 16 and 21 of the amino acid sequence shown in SEQ ID NO: 36 in adrenomedullin form a disulfide bond, or the disulfide bond is substituted with a linking group such as an ethylene group.

[0045] In one embodiment, adrenomedullin or an active fragment thereof comprises a glycine (Gly) residue added to its C-terminus.

[0046] The self-assembling peptide contained in the fusion peptide of this embodiment is not limited to a particular type, as long as it is a peptide that can be solidified from a sol state dissolved in water or an aqueous solution under specific temperature and pressure conditions to form a gel state.

[0047] In one embodiment, the self-assembling peptide contained in the fusion peptide of this embodiment is an amphipathic peptide in which hydrophilic and hydrophobic amino acid residues are alternately linked, such as a peptide comprising or consisting of any of the following amino acid sequences (a) to (c): (a) an amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (Formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly); (b) an amino acid sequence represented by any of the following formulas II to IV: Arg-Gly-Asp-Ala-(Arg-Ala-Asp-Ala) (Formula II), (Arg-Ala-Asp-Ala)-Arg-Gly-Asp-Ala (Formula III), or (Arg-Ala-Asp-Ala)-Arg-Ala-Asp-Gly (Formula IV); (c) an amino acid sequence represented by the following formula V: (Arg-Ala-Asp-Ala) p An amino acid sequence represented by formula V (wherein p is an integer of 1 or more)

[0048] WO 2022 / 025209 discloses that self-assembling peptides consisting of the amino acid sequence shown in Formula I are capable of gelation. Specific examples of the amino acid sequence shown in Formula I include IRARMDADI (SEQ ID NO: 1), IRADMRADI (SEQ ID NO: 2), IRADMDARI (SEQ ID NO: 3), IDARMRADI (SEQ ID NO: 4), IDARMDARI (SEQ ID NO: 5), IDADMRARI (SEQ ID NO: 6), IRGDIRGDI (SEQ ID NO: 7), IRGDMRGDI (SEQ ID NO: 8), IRADIRADM (SEQ ID NO: 9), IDARMRADM (SEQ ID NO: 10), MDARIDARI (SEQ ID NO: 11), MDADMRARI (SEQ ID NO: 12), IRGDMRADI (SEQ ID NO: 13), IRADMRGDI (SEQ ID NO: 14), IRGDIRGDI (SEQ ID NO: 15), IRGDIRADI (SEQ ID NO: 16), and IRADIRGDI (SEQ ID NO: 17).

[0049] In one embodiment, Arg is linked to the N-terminus and / or C-terminus of a self-assembling peptide comprising the amino acid sequence shown in Formula I. In a further embodiment, Arg is linked to both of the two amino acids at the N-terminus and / or C-terminus of a self-assembling peptide comprising the amino acid sequence shown in Formula I. Examples of self-assembling peptides comprising the amino acid sequence shown in Formula I above in which the N-terminus and C-terminus are Arg include RIRARMDADIR (SEQ ID NO: 18), RIRADMRADIR (SEQ ID NO: 19), RIRADMDARIR (SEQ ID NO: 20), RIDARMRADIR (SEQ ID NO: 21), RIDARMDARIR (SEQ ID NO: 22), RIDADMRARIR (SEQ ID NO: 23), RIRGDIRGDIR (SEQ ID NO: 24), RIRGDMRGDIR (SEQ ID NO: 25), RIRADIRADMR (SEQ ID NO: 26), RIDARMRADMR (SEQ ID NO: 27), RMDARIDARIR (SEQ ID NO: 28), RMDADMRARIR (SEQ ID NO: 29), RIRGDMRADIR (SEQ ID NO: 30), RIRADMRGDIR (SEQ ID NO: 31), RIRGDIRGDIR (SEQ ID NO: 32), RIRGDIRADIR (SEQ ID NO: 33), and RIRADIRGDIR (SEQ ID NO: 34).

[0050] WO2020 / 171161 discloses that self-assembling peptides consisting of the amino acid sequences shown in Formulas II to IV above can gel.

[0051] A self-assembling peptide consisting of the amino acid sequence shown in Formula V above, where p=4, is known as a RADA16 peptide and is known to be capable of gelation. p in Formula V above is an integer of 1 or greater, such as, but not limited to, 2 or greater, 3 or greater, or 4 or greater, and / or 8 or less, 7 or less, 6 or less, or 5 or less.

[0052] The self-assembling peptide contained in the fusion peptide of this embodiment comprises at least one amino acid sequence shown in any one of Formulas I to V, or consists of one or more amino acid sequences shown in any one of Formulas I to V. For example, the self-assembling peptide contained in the fusion peptide of this embodiment may comprise one or two amino acid sequences shown in any one of Formulas I to V.

[0053] When the self-assembling peptide contained in the fusion peptide of this embodiment contains two amino acid sequences represented by any one of Formulas I to V, the two or more amino acid sequences represented by any one of Formulas I to V may consist of the same amino acid sequence or different amino acid sequences.

[0054] In one embodiment, the self-assembling peptide contained in the fusion peptide of this embodiment may or may not contain additional amino acid residues on the N-terminal and / or C-terminal sides of the amino acid sequence shown in any of Formulas I to V.

[0055] The amino acids other than glycine that make up the self-assembling peptide contained in the fusion peptide of this embodiment can be used regardless of their optical isomers. That is, either the D- or L-form may be used. For example, all of the amino acids other than glycine that make up the self-assembling peptide may be D- or L-forms.

[0056] The total length of the amino acid sequence constituting the self-assembling peptide contained in the fusion peptide of this embodiment is, but is not limited to, for example, 50 or fewer amino acids or 25 or fewer amino acids. Specific examples of amino acid lengths include 20 or fewer amino acids, 15 or fewer amino acids, or 10 or fewer amino acids, and / or 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, or 8 or more amino acids, for example, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, or 9 amino acids.

[0057] In one embodiment, the fusion peptide of this aspect may comprise a peptide consisting of one amino acid residue or multiple amino acid residues at the N-terminus and / or C-terminus of the self-assembling peptide. The amino acid residues at the N-terminus and / or C-terminus of the self-assembling peptide may be hydrophilic amino acids.

[0058] In the fusion peptide of this embodiment, the link between adrenomedullin or an active fragment thereof and the self-assembling peptide is a covalent bond or a supramolecular interaction. The covalent bond is not limited to, and examples thereof include a peptide bond or a disulfide bond.

[0059] In one embodiment, in the fusion peptide of this aspect, adrenomedullin or an active fragment thereof and the self-assembling peptide are linked via a linker. The specific structure of the linker is not limited, but it may be, for example, a peptide linker. The length of the peptide linker is not limited, but is typically 1 to 50 amino acids long, preferably 5 to 20 amino acids long. Peptides containing many amino acids with relatively small side chains, such as serine and glycine, are often used.

[0060] The amino group of the N-terminal amino acid residue and the carboxyl group of the C-terminal amino acid residue of the fusion peptide of this embodiment may optionally have a modifying group added. For example, an acetyl group may be added to the N-terminus of the fusion peptide of this embodiment. Furthermore, an NH2 amide may be added to the C-terminus of the modified peptide of this embodiment.

[0061] In one embodiment, the fusion peptide of this embodiment is linked to adrenomedullin or an active fragment thereof, as well as other functional peptides. The type of such functional peptide is not limited, and may be, for example, a peptide that supports adrenomedullin activity, VEGF, which has an angiogenesis-promoting effect similar to adrenomedullin, a peptide with labeling function, or a peptide tag for purification. Labeling peptides may include, but are not limited to, fluorescent proteins such as GFP, photoproteins such as luciferin or aequorin, or enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Furthermore, peptide tags are artificially synthesized oligopeptides consisting of several to a dozen amino acids, and examples include epitope tags such as FLAG tags, histidine tags, HA tags, and DAP tags, as well as His tags, GST tags, and Halo tags. Furthermore, the fusion peptide of this embodiment may optionally be linked to one or more chemical modification groups other than by peptide bonds. The structure of the chemical modification group is not particularly limited, but it is a moiety that confers a desired function to the fusion peptide to which it is attached. Examples of desired functions include labeling, linker, linking, and binding functions. Examples of chemical modification groups that impart labeling functions include chromophores and fluorescent groups (e.g., fluorescein). Examples of chemical modification groups that impart linker functions include any polymer (e.g., alkylene). Examples of chemical modification groups that impart binding functions include compounds such as biotin. Other chemical modification groups that can be attached to the fusion peptide of this embodiment include lipids, sugars, aptamers, receptor ligands, and the like. Examples of lipids include cholesterol, fatty acids, and other lipids (e.g., vitamin E, vitamin A, vitamin D), fat-soluble vitamins such as vitamin K, intermediate metabolites such as acyl-CoA, glycolipids, glycerides, and derivatives thereof. Examples of sugars include glucose and sucrose.

[0062] 1-4. Effects The fusion peptide of this embodiment can provide angiogenesis-promoting function based on adrenomedullin in vivo. For example, by combining the fusion peptide of this embodiment with a self-assembling peptide that is not fused to adrenomedullin and administering it to a target site such as a cerebral infarction site, a gel containing the fusion peptide is formed, and the fusion peptide is gradually released from the gel. This sustained release effect can promote angiogenesis over a long period of time around the target site where the gel is implanted.

[0063] The fusion peptide of this aspect provides a method for treating and / or preventing a disease, comprising administering any of the fusion peptides described above to a subject such as a human. The fusion peptide of this aspect also provides any of the fusion peptides described above for use in treating and / or preventing a disease in a subject such as a human. The present invention also provides any of the fusion peptides described above for use in the manufacture of a medicament for treating and / or preventing a disease.

[0064] 2. Gelling Composition 2-1. Overview The second aspect of the present invention is a gelling composition. The gelling composition of this aspect contains the fusion peptide described in the first aspect as an essential active ingredient, and also contains a gelling agent, a gelation-promoting component, and / or a carrier. The gelling composition of this aspect can be gelled by maintaining it in water or an aqueous solution at a temperature below its gelling temperature.

[0065] 2-2. Composition 2-2-1. Constituent Components The gelling composition of the present invention is composed of an active ingredient and other ingredients. The components other than the active ingredient are not particularly limited, but examples include components that can promote gelation of the gelling composition and carriers. Each of the constituent components will be described in detail below.

[0066] (1) Active Ingredient The gelling composition of this embodiment contains the fusion peptide described in the first embodiment as an essential active ingredient.

[0067] The amount (content) of the fusion peptide incorporated into the gelling composition is not particularly limited. When the gelling composition of the present invention is administered to a living body, the amount may be determined appropriately depending on the fusion peptide and the type and / or effective amount of gelling agent contained in the gelling composition, information about the subject, the formulation of the gelling composition, and the type of carrier or additive described below. Specifically, the concentration of the fusion peptide in the gelling composition is not limited, but may be, for example, 1 / 100,000 or more, 1 / 10,000 or more, 1 / 1000 or more, 1 / 100 or more, or 1 / 10 or more, and / or 2 times or less, 1.5 times or less, 1 times or less, 0.8 times or less, 0.5 times or less, or 0.2 times or less of the concentration of the gelling agent (self-assembling peptide) described below. Specific concentrations of the fusion peptide include, but are not limited to, 1 x 10 -6 Weight% or more, 1×10 -5 Weight% or more, 1×10 -4 Weight% or more, 1×10 -3 % by weight or more, or 1 x 10 -2The concentration may be 10% by weight or more and / or 10% by weight or less, 5% by weight or less, 2% by weight or less, 1% by weight or less, 0.5% by weight or less, 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.05% by weight or less, or 0.02% by weight or less. The ratio of the concentration of the fusion peptide to the concentration of the gelling agent (self-assembling peptide) in the gelling composition can be adjusted appropriately depending on the treatment target, administration site, etc. As used herein, the term "effective amount" refers to the amount of the fusion peptide in the gelling composition necessary to function as an active ingredient and which causes little or no harmful side effects in the living body to which it is applied. This effective amount may vary depending on various conditions, such as information about the subject, the route of administration, and the number of administrations. Here, the term "subject" refers to the living body to which the gelling composition or pharmaceutical composition is applied. Examples of such animals include humans, livestock (cattle, horses, sheep, goats, pigs, chickens, ostriches, etc.), racehorses, pets (dogs, cats, rabbits, etc.), and laboratory animals (mice, rats, guinea pigs, monkeys, marmosets, etc.). Humans are preferred. Furthermore, "subject information" refers to various individual information about the living body to which the gelling composition is applied. For example, in the case of a subject, this includes the overall health condition, the progression and severity of any disease or injury, age, weight, sex, diet, drug sensitivity, the presence or absence of concomitant medications, and tolerance to treatment. The final effective dose of the gelling agent and the dosage calculated based thereon are ultimately determined by the judgment of a physician, dentist, veterinarian, or the like, depending on the individual subject's information, etc.

[0068] The gelling composition of this embodiment can also contain drugs and the like as other active ingredients. As used herein, the term "drug" refers to a concept that includes low molecular weight compounds, peptides (including enzymes and antibodies), or nucleic acids (including RNAi molecules such as miRNA, siRNA, and shRNA, antisense nucleic acids, aptamers, and the like). Drugs include, but are not limited to, various types of pharmaceuticals, such as therapeutic drugs intended to treat diseases or alleviate symptoms. The gelling composition of this embodiment may contain not only one type of drug, but also two or more types.

[0069] (2) Gelling Agent The gelling composition of this embodiment contains a gelling agent as an optional component.

[0070] The type of gelling agent contained in the gelling composition of this embodiment is not limited, but is preferably one that can contain the fusion peptide (active ingredient) in the gel formed by the gelling agent and can sustainedly release the fusion peptide. The gelling agent may be a self-assembling peptide or a non-self-assembling peptide. Specific examples of gelling agents include the self-assembling peptide described in the first embodiment that is not linked to adrenomedullin or an active fragment thereof. The amino acid sequence of the self-assembling peptide described in the first embodiment that is not linked to adrenomedullin or an active fragment thereof may be the same as or different from the amino acid sequence of the self-assembling peptide contained in the fusion peptide (active ingredient), but is preferably the same amino acid sequence. Note that the self-assembling peptide described in the first embodiment that is not linked to adrenomedullin or an active fragment thereof may be bound to a peptide such as the above-mentioned peptide with a labeling function or a peptide tag for purification.

[0071] The amount (content) of gelling agent to be incorporated into the gelling composition is not particularly limited. It may be determined appropriately taking into consideration the gelling conditions. Furthermore, when the gelling composition of the present invention is administered to a living body, it may be determined appropriately depending on the type and / or effective amount of gelling agent contained in the gelling composition, information on the subject, the dosage form of the gelling composition, and the type of carrier or additive described below. Specifically, the concentration of the gelling agent (self-assembling peptide) in the gelling composition is not limited, but may be, for example, 1 x 10 -4 Weight% or more, 1×10 -3 % by weight or more, or 1 x 10 -2 The effective amount of the gelling agent may be 0.1% by weight or more, 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight or more, or 0.4% by weight or more, and / or 10% by weight or less, 5% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less, for example, 0.1% by weight or more and 2.0% by weight or less. The final effective amount of the gelling agent and the dosage calculated thereon will ultimately be determined by the judgment of a physician, dentist, veterinarian, or the like, depending on information about the individual subject, etc.

[0072] (3) Gelling-Promoting Component The gelling composition of this embodiment may contain a component capable of promoting its gelation, if necessary. Examples of gelling-promoting components include, but are not limited to, components that have the effect of reducing the solubility of proteins. This is because components that have the effect of reducing the solubility of proteins generally promote the gelation of peptide gelators.

[0073] Components that have the effect of reducing protein solubility are not limited, and examples include cations and anions that have the effect of reducing protein solubility. Cations and anions that have this effect are well known to those skilled in the art as the Hofmeister series. Examples of anions include bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions. Examples of cations include lithium ions, sodium ions, potassium ions, magnesium ions, and calcium ions.

[0074] The concentrations of cations and anions that have the effect of reducing protein solubility are not particularly limited. For example, they may be 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, or 40 mM or more. When bicarbonate ions or carbonate ions are used, taking into consideration that bicarbonate ions and carbonate ions are usually in equilibrium in an aqueous solution, the total concentration of bicarbonate ions and carbonate ions may be 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, or 40 mM or more, for example, 44 mM.

[0075] (4) Carrier The gelling composition of this embodiment may contain a pharmaceutically acceptable carrier as needed. As used herein, the term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include solvents, excipients, fillers, emulsifiers, flow control agents, lubricants, and human serum albumin.

[0076] The solvent may be, for example, water or another pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent, preferably water or another pharmaceutically acceptable aqueous solution. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other supplements, phosphate buffer, sodium acetate buffer, and any medium used in cell culture or tissue culture. Examples of supplements include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like. Commercially available media may be used, including, for example, DMEM medium, Ham's F12 medium, DMEM / F12 medium, McCoy's 5A medium, Eagle's MEM medium, αMEM medium, MEM medium, RPMI1640 medium, Iscove's modified Dulbecco's medium, MCDB131 medium, William's medium E, IPL41 medium, and Fischer's medium.

[0077] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metallic salts, citric acid, tartaric acid, glycine, polyethylene glycol, Pluronic®, kaolin, silicic acid, or combinations thereof.

[0078] Examples of fillers include petrolatum, the aforementioned sugars and / or calcium phosphate.

[0079] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0080] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0081] In addition to the above, if necessary, the composition may appropriately contain solubilizers, suspending agents, diluents, dispersing agents, surfactants, soothing agents, stabilizers, pH adjusters, absorption enhancers, bulking agents, moisturizing agents, humectants, wetting agents, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, antioxidants, fragrances, flavoring agents, sweeteners, buffers, isotonic agents, and the like that are commonly used in pharmaceuticals.

[0082] Such carriers are primarily used to facilitate the formation of dosage forms, maintain the dosage form and drug efficacy, and also to make the active ingredient, the gelling agent, less susceptible to degradation by enzymes in the body, etc., and may be used appropriately as needed.

[0083] 2-2-2. Properties of the Gelling Composition The pH of the gelling composition of this embodiment is not limited. For example, it may be a physiological pH, such as a pH in the range of 4.0 to 10.0, 5.0 to 9.0, 6.0 to 8.0, or 6.5 to 7.5, e.g., pH 7.4.

[0084] The gelling composition of this embodiment can be gelled by maintaining it in water or an aqueous solution at a temperature below its gelling temperature. The gelling temperature is essentially based on that of the self-assembling peptide or fusion peptide that constitutes the gelling agent, which is the active ingredient. Therefore, the gelling composition of this embodiment can gel at a temperature within the range of 4 to 80°C, 10 to 70°C, 15 to 60°C, 20 to 50°C, or 30 to 40°C, such as 37°C, under 1 atmosphere.

[0085] The gelling composition of this embodiment can be gelled under physiological conditions without substantially losing the activity of the adrenomedullin that constitutes the fusion peptide, or while retaining at least a portion of that activity. Therefore, after gelling, the gelling composition of this embodiment can exert the physiological activity of the adrenomedullin that constitutes the fusion peptide, such as the ability to promote angiogenesis. For example, angiogenesis can be induced by implanting the gelling composition into a living body by surgical or other methods.

[0086] After the gelling composition of this embodiment has been introduced into a subject, and has sufficiently promoted angiogenesis and formed or regenerated tissues or organs at the administration site, the gelling composition can be removed from the transplantation site, for example, by incising the target site by surgery or the like.

[0087] 2-2-3. Dosage Form The dosage form of the gelling composition of this embodiment is not particularly limited. For example, it may be a liquid or solid formulation that can be introduced into the target site. In the case of a solid formulation, its shape is not important. It may be in the form of a general solid dosage form such as a powder, granules, tablet, or a shape suitable for a transplantation member.

[0088] 2-2-4. Application Method The method of application of the gelling composition of this embodiment is not particularly limited, but is preferably parenteral administration, and more preferably local administration. Local administration includes, for example, intramuscular administration, subcutaneous administration, tissue administration, and organ administration. When the gelling composition of this embodiment is administered locally, the gelling composition of this embodiment may be introduced into the target site in a gel state. For example, the target site can be incised by surgical operation and the gel state can be transplanted. Note that the gel state is preferably achieved when the composition is still soft and gel formation is not complete. The dosage may be an amount effective for the active ingredient to be effective. The effective amount is selected appropriately depending on the subject information.

[0089] The gelling composition of this embodiment can be removed from the administration site as needed. For example, the administration site can be incised by surgical operation and the composition can be surgically removed in its gel state.

[0090] 3. Pharmaceutical Composition 3-1. Configuration A third aspect of the present invention is a pharmaceutical composition. The pharmaceutical composition of this aspect comprises the gelling composition of the second aspect. Therefore, the configuration of the pharmaceutical composition of this aspect is similar to that of the second aspect, except for the following components.

[0091] In one embodiment, the pharmaceutical composition of this aspect can be used to promote angiogenesis and / or inhibit neurodegeneration.

[0092] In one embodiment, the pharmaceutical composition of this aspect is for implantation. The pharmaceutical composition of the present invention can be implanted, for example, by surgically incising the target site.

[0093] The target diseases for the pharmaceutical composition of this embodiment include, but are not limited to, nerve tissue damage and / or ischemia.

[0094] As used herein, "nervous tissue damage" includes both central nervous tissue damage and peripheral nervous tissue damage. Central nervous tissue damage includes brain damage and spinal cord damage. Brain damage includes, for example, traumatic brain injury, cerebrovascular disease, etc. Cerebrovascular disease includes both cerebral infarction (ischemic cerebrovascular disease) and cerebral hemorrhage. Spinal cord injury includes, for example, cervical spinal cord injury, thoracic spinal cord injury, lumbar spinal cord injury, sacral spinal cord injury, etc. Peripheral nervous tissue damage includes damage to any peripheral nervous tissue. Examples include damage to motor nerves, sensory nerves, and autonomic nerves.

[0095] As used herein, "ischemia" refers to ischemia in any organ or tissue, including, for example, lower limb ischemia (e.g., arteriosclerosis obliterans and its severe form, critical lower limb ischemia), ischemic heart disease (e.g., myocardial infarction), and the above-mentioned cerebrovascular disorders.

[0096] As used herein, treatment includes, but is not limited to, curative treatment and preventative treatment. Prevention also includes prevention of onset, prevention of progression, and prevention of recurrence.

[0097] 3-2. Effects By transplanting the pharmaceutical composition of the present invention into a diseased, injured, or ischemic area of ​​a subject, or into a nearby area, it becomes possible to continuously release the fusion peptide containing adrenomedullin over a long period of time, thereby promoting angiogenesis and suppressing neurodegeneration. Alternatively, by transplanting the pharmaceutical composition of the present invention into a specific area within the body of a subject, it is possible to continuously release the fusion peptide containing adrenomedullin throughout the body over a long period of time.

[0098] The risk of cerebrovascular disorders can be detected by imaging tests such as CT scans and MRI scans, and the onset of such disorders can be prevented by the pharmaceutical composition of the present invention. Furthermore, cerebrovascular disorders such as cerebral infarction have a high recurrence rate, and the pharmaceutical composition of the present invention can also prevent such recurrence.

[0099] 4. Gel Preparation Method 4-1. Overview A fourth aspect of the present invention is a gel preparation method. According to this gel preparation method, it is possible to prepare a gel (e.g., a sustained-release gel) containing a fusion peptide containing adrenomedullin or an active fragment thereof that maintains at least a portion of its activity.

[0100] 4-2. Method The method for producing a gel of the present invention includes a mixing step and a gelling step as essential steps.

[0101] (Mixing Step) The mixing step is a step of mixing the gelling composition according to the second embodiment with water or an aqueous solution. The water or aqueous solution may be, for example, a cell culture medium or other medium. The medium can be appropriately selected from known media. For example, the medium can be prepared by using any liquid medium for animal cell culture as a basal medium and appropriately adding other components (serum, serum replacement reagents, growth factors, etc.; N2 supplement, B27(R) supplement, insulin, bFGF, activin A, heparin, ROCK inhibitor, and / or GSK-3 inhibitor, etc.) as needed. Specific examples of media include DMEM medium, Ham's F12 medium, DMEM / F12 medium, McCoy's 5A medium, Eagle's MEM medium, αMEM medium, MEM medium, RPMI1640 medium, Iscove's modified Dulbecco's medium, MCDB131 medium, William's medium E, IPL41 medium, Fischer's medium, etc. The mixing method in this step is not particularly limited; sufficient mixing can be achieved by stirring or the like.

[0102] In this step, a component that can promote gelation in the gelation step described below may be further mixed. The gelation-promoting component is not limited to, but may be, for example, a component that has the effect of reducing the solubility of proteins. Examples of anions that have this effect are as described above in the second embodiment.

[0103] The concentration of the fusion peptide contained in the gelling composition used in this step is not limited, but may be, for example, 1 / 100,000 or more, 1 / 10,000 or more, 1 / 1000 or more, 1 / 100 or more, or 1 / 10 or more of the concentration of the gelling agent, and / or 2 times or less, 1.5 times or less, 1 times or less, 0.8 times or less, 0.5 times or less, or 0.2 times or less. Specific concentrations of the fusion peptide are not limited to the following, but may be, for example, 1 x 10 -6 Weight% or more, 1×10 -5 Weight% or more, 1×10 -4 Weight% or more, 1×10 -3 % by weight or more, or 1 x 10 -2 The concentration may be 10% by weight or more and / or 10% by weight or less, 5% by weight or less, 2% by weight or less, 1% by weight or less, 0.5% by weight or less, 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.05% by weight or less, or 0.02% by weight or less, for example, 0.4% by weight to 10% by weight. The ratio of the concentration of the fusion peptide to the concentration of the gelling agent (self-assembling peptide) in the gelling composition can be adjusted as appropriate depending on the treatment subject, administration site, etc.

[0104] (Gelling Step) The gelling step is a step in which the mixture obtained after the mixing step is gelled by maintaining the mixture at a temperature equal to or lower than the gelling temperature.

[0105] The duration and temperature used in this step vary depending on the types of fusion peptide and self-assembling peptide in the gelling composition mixed in the mixing step, and can be determined appropriately depending on the types. For example, the gelling composition after the mixing step may be maintained for at least 1 minute, at least 10 minutes, or at least 1 hour at a temperature within the range of 4-80°C, 10-70°C, 15-60°C, 20-50°C, or 30-40°C, such as 37°C. Temperature conditions that maintain the activity of the adrenomedullin or active fragment that constitutes the fusion peptide, or at least partially retain its activity, are particularly preferred. For example, temperatures within the range of 4-80°C, 10-70°C, 15-60°C, 20-50°C, or 30-40°C, such as 37°C, can be used. Gelation may also be achieved under 5% CO2 conditions at any of the above temperature conditions (e.g., 37°C). The temperature control method used in this step is not particularly limited, and examples include placing the composition in a thermostatic bath.

[0106] The pH conditions used for gelation in this step are not limited, and may be, for example, within the range of pH 4.0 to 10.0, pH 5.0 to 9.0, pH 6.0 to 8.0, or pH 6.5 to 7.5, e.g., pH 7.4.

[0107] 4-3. Effects The gel production method of the present invention allows the production of transplantable and / or sustained-release gels. Furthermore, the gel production method of the present invention allows gelation under physiological conditions that do not impair the activity of adrenomedullin or an active fragment thereof contained in the fusion peptide, and further allows the fusion peptide containing adrenomedullin or an active fragment thereof to be sustained-released from the gel. The gel produced by the gel production method of the present invention has high uptake efficiency of the fusion peptide containing adrenomedullin or an active fragment thereof and excellent sustained-release properties.

[0108] The present invention will be specifically described below with reference to examples, but these examples are merely illustrative and the present invention is not limited to the scope described in the examples.

[0109] Example 1: Preparation of AM-JigSAP fusion peptide and activity measurement (Objective) To prepare an AM-JigSAP fusion peptide by fusing a jigsaw-shaped self-assembling peptide (JigSAP) to the N-terminus of adrenomedullin (hereinafter referred to as "AM"), and to measure its activity.

[0110] (Methods and Results) (1) Preparation of AM-JigSAP fusion peptide The AM-JigSAP fusion peptide (Figure 1A; hereinafter, also referred to as "fusion peptide" or "AM-JigSAP") was prepared by commissioning Peptide Institute, Inc., consisting of the amino acid sequence (SEQ ID NO: 42) in which adrenomedullin (AM), consisting of the amino acid sequence shown in SEQ ID NO: 36, was fused to the C-terminus of JigSAP, consisting of the amino acid sequence (RIDARMRADIR) shown in SEQ ID NO: 21.

[0111] (2) Activity Measurement of AM-JigSAP Fusion Peptide. To measure the activity of the AM-JigSAP fusion peptide, HEK-293 cells stably expressing the type 1 adrenomedullin receptor (AM1) were suspended in DMEM medium containing 10% FCS and seeded onto a 24-well plate coated with human fibronectin (Thermo Fisher Scientific) and cultured at 37°C under 5% CO2. The medium was replaced with Hank's balanced salt solution containing 0.035% NaHCO3 and 0.2% BSA. The AM-JigSAP fusion peptide prepared in (1) above was added in the presence of 0.5 mM isobutylmethylxanthine and incubated at 37°C for 15 minutes. Cell lysates were added, and the cAMP concentration in the supernatant was measured by enzyme immunoassay (Cayman Chemical Company). Adrenomedullin not fused to JigSAP (SEQ ID NO: 36; hereinafter, also referred to as "non-fused AM" or simply "AM") was produced by Peptide Institute, Inc. in the same manner as in (1) above, and the cAMP production ability relative to the non-fused AM was measured.

[0112] The results are shown in Figure 1B. It was revealed that the AM-JigSAP fusion peptide induced cAMP production to the same extent as the unfused AM, and had almost the same activity.

[0113] Example 2: Verification of the therapeutic effect of AM-JigSAP on cerebral infarction (Objective) The AM-JigSAP fusion peptide was administered intracerebrally to a mouse cerebral infarction model to verify its therapeutic effect on gait disturbance.

[0114] (Methods) (1) Preparation of Non-Fused JigSAP JigSAP not fused to adrenomedullin (amino acid sequence RIDARMRADIR, SEQ ID NO: 21; hereinafter, also referred to as "non-fused JigSAP" or simply "JigSAP") was synthesized on a 0.10 mmol scale by the Fmoc peptide solid-phase synthesis method described in WO 2022 / 025209. The synthesized peptide had an acetyl group bound to the N-terminus and an NH2 amide at the C-terminus. The synthesized JigSAP was dried, dispersed in ion-exchanged water, and lyophilized.

[0115] One mg of the lyophilized peptide powder and 90 μL of D-MEM (containing 4.0 mM HEPES and 44 mM NaHCO3; pH 7.4) were added to a 1.5 mL microtube, and JigSAP was dispersed at a concentration of 1.0 wt% by ultrasonication in a water bath ultrasonicator (AS12GTU, 35 kHz, 60 W).

[0116] (2) Preparation of AM-JigSAP / JigSAP Composition A composition containing the AM-JigSAP fusion peptide and JigSAP (hereinafter referred to as the "AM-JigSAP / JigSAP composition") was prepared by adding 10 μL of the aqueous solution containing 6 μg / μL of the AM-JigSAP fusion peptide prepared in Example 1 to 10 μL of the solution containing 1.0 wt% JigSAP prepared in (1) above. The AM-JigSAP / JigSAP composition contains the AM-JigSAP fusion peptide at a molar ratio of 1 / 2 to JigSAP.

[0117] In the following experiments, a composition (hereinafter referred to as the "JigSAP composition") was prepared by adding 10 μL of phosphate buffer to 10 μL of a solution containing 1.0 wt% JigSAP prepared in (1) above, but not containing the AM-JigSAP fusion peptide, and this composition served as a comparison control for the AM-JigSAP / JigSAP composition.

[0118] (3) Evaluation of the therapeutic effect on cerebral infarction The mouse cerebral infarction model used was the distal middle cerebral artery occlusion (dMCAO) model previously reported by the present inventors (Oshikawa, M. et al., Adv Healthc Mater. 2017;6(11):10.1002 / adhm.201700183, PMID: 28488337). In the dMCAO model, infarcts form in brain regions where blood supply from the middle cerebral artery is cut off.

[0119] On day 7 after the cerebral infarction model was established in the subacute phase, a foot-fault test was performed as a pre-administration gait function analysis (first gait function analysis; Figure 3A, "Gait Function Analysis 1"). The foot-fault test was performed according to a method previously described by the present inventors (Jinnou, H., et al., Cell Stem Cell., 2018, 22(1), 128-137.e9., PMID: 29276142). Specifically, mice trained to walk on a wire mesh for 10 minutes on day 6 after the onset of cerebral infarction were then subjected to a 10-minute gait test in which they walked on the wire mesh. During the test, the mouse feet were videotaped from below. After videotaping, the number of slipping steps (Figure 2A) and normal steps (Figure 2B) were counted. The percentage of slipping steps out of the total number of steps (number of slipping steps / total number of steps (%)) was calculated.

[0120] After the first gait function analysis, a glass needle (Drummond, Wiretrol II) filled with the AM-JigSAP / JigSAP composition or JigSAP composition prepared in (2) above was inserted into the 2 mm-diameter hole drilled in the skull during dMCAO model creation. The needle depth was adjusted using a micromanipulator (Narishige). 2 μL of the AM-JigSAP / JigSAP composition prepared in (2) above was administered in small increments over 5 min into the injured area near the middle cerebral artery (Figure 3A, "Injection"). The gel was kept at approximately 0°C on ice before administration, room temperature (25°C) in the needle immediately before administration, and near the mouse's internal temperature (37°C) during administration. The dose was 2 μL per mouse. A second gait function analysis was performed 7 days after administration (14 days after cerebral infarction model creation) (Figure 3A, "Gait Function Analysis 2"). The "recovery rate" was calculated as the percentage of steps in which the foot slipped out of the total number of steps in walking function analysis 1 compared to the percentage of steps in which the foot slipped out of the total number of steps in walking function analysis 2.

[0121] (Results) Figure 3B shows the recovery rates of mice treated with the AM-JigSAP / JigSAP composition or the JigSAP composition. One week after administration, the AM-JigSAP / JigSAP composition (n = 7; Figure 3B, "AM-JigSAP") showed a more significant decrease in the percentage of slippages compared with the JigSAP composition (n = 7; Figure 3B, "JigSAP"), demonstrating a significant improvement in gait function (P = 0.041, Student's t-test). Furthermore, the AM-JigSAP-unfused AM model (n = 8; Figure 3B, "AM") showed no significant difference in gait function compared with the JigSAP composition (n = 7; Figure 3B, "JigSAP") model (P = 0.395).

[0122] These results suggest that administration of the AM-JigSAP fusion peptide together with JigSAP significantly promotes recovery from gait dysfunction caused by cerebral infarction. These results suggest that the AM-JigSAP fusion peptide is slowly released from the gel formed by JigSAP into the surrounding brain tissue, and the adrenomedullin-based angiogenesis-promoting function contained in the AM-JigSAP fusion peptide is supplied to the infarcted area and surrounding brain tissue over a long period of time, significantly promoting recovery of brain function.

[0123] Example 3: Analysis of gene expression changes based on AM-JigSAP (Objective) To analyze gene expression changes in a mouse cerebral infarction model administered with an AM-JigSAP fusion peptide, and to compare the changes with those in a mouse cerebral infarction model administered with a VEGF-JigSAP fusion peptide in which vascular endothelial growth factor (VEGF) is fused to the C-terminus of JigSAP.

[0124] (Methods) (1) Preparation of VEGF-JigSAP fusion polypeptide and VEGF-JigSAP / JigSAP composition A VEGF-JigSAP fusion peptide (SEQ ID NO: 43) consisting of mouse VEGF fused to the N-terminus of JigSAP, which consists of the amino acid sequence (RIDARMRADIR) shown in SEQ ID NO: 21, was prepared by the same method as in Example 1(1). Furthermore, a composition containing the VEGF-JigSAP fusion peptide and JigSAP (hereinafter referred to as the "VEGF-JigSAP / JigSAP composition") was prepared by the same method as in Example 2(2). The VEGF-JigSAP / JigSAP composition was prepared in a molar ratio of 10 to JigSAP. 5 Contains 1 / 2 of the VEGF-JigSAP fusion peptide.

[0125] (2) Analysis of gene expression changes The AM-JigSAP / JigSAP composition or JigSAP composition prepared in the same manner as in Example 2(2), or the VEGF-JigSAP / JigSAP composition or PBS prepared in (1) above was administered intracortically to a mouse cerebral infarction model. 14 days after administration, the damaged area, including the peri-injury area, was excised from the mouse brain and subjected to gene expression array analysis.

[0126] (Results) The results of gene expression analysis are shown in Figure 4. Figure 4 compares gene expression in the brains of mice administered the VEGF-JigSAP / JigSAP composition with that in the brains of mice administered PBS (Figure 4A, "VEGF-JigSAP vs. PBS") and in the brains of mice administered the AM-JigSAP / JigSAP composition with that in the brains of mice administered the JigSAP composition (Figure 4B, "AM-JigSAP vs. JigSAP"). The gene groups upregulated in the brains of mice administered the AM-JigSAP / JigSAP composition showed high similarity to the gene groups upregulated in the brains of mice administered the VEGF-JigSAP / JigSAP composition. This suggests that the AM-JigSAP / JigSAP composition restores brain function through a mechanism similar to that achieved by administration of the VEGF-JigSAP / JigSAP composition. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A fusion peptide comprising adrenomedullin or an active fragment thereof linked to a self-assembling peptide.

2. The fusion peptide according to claim 1, wherein the adrenomedullin consists of: (i) an amino acid sequence shown in any one of SEQ ID NOs: 36 to 41; (ii) an amino acid sequence in which 1 to 15 amino acid residues have been deleted, substituted or added in the amino acid sequence shown in any one of SEQ ID NOs: 36 to 41; or (iii) an amino acid sequence having 90% or more identity to the amino acid sequence shown in any one of SEQ ID NOs: 36 to 41.

3. The fusion peptide described in claim 1, wherein in the adrenomedullin, cysteine ​​residues corresponding to positions 16 and 21 of the amino acid sequence shown in SEQ ID NO: 36 form a disulfide bond or the disulfide bond is substituted by an ethylene group.

4. The fusion peptide of claim 1, wherein the adrenomedullin or an active fragment thereof comprises a glycine residue added to its C-terminus.

5. The self-assembling peptide is: (a) an amino acid sequence represented by the following formula I: Xaa-Yaa-Zaa-Yaa-Xaa-Yaa-Zaa-Yaa-Xaa (formula I) (wherein Xaa is independently Ile or Met, Yaa is independently Asp, Glu, Lys, or Arg, and Zaa is independently Ala or Gly); (b) an amino acid sequence represented by any of the following formulas II to IV: Arg-Gly-Asp-Ala-(Arg-Ala-Asp-Ala)3 (formula II), (Arg-Ala-Asp-Ala)3-Arg-Gly-Asp-Ala (formula III), or (Arg-Ala-Asp-Ala)3-Arg-Ala-Asp-Gly (formula IV); or (c) an amino acid sequence represented by the following formula V: (Arg-Ala-Asp-Ala) p The fusion peptide according to any one of claims 1 to 4, comprising an amino acid sequence represented by the formula V: (wherein p is an integer of 1 or more).

6. The fusion peptide according to claim 5, wherein Arg is added to the N-terminus and / or C-terminus of the amino acid sequence represented by formula I, formulas II to IV, or formula V, or wherein two amino acids of Arg are added to the N-terminus and / or C-terminus of the amino acid sequence represented by formula I, formulas II to IV, or formula V.

7. A fusion peptide according to any one of claims 1 to 4, wherein the adrenomedullin or an active fragment thereof is linked to the C-terminus and / or N-terminus of the self-assembling peptide.

8. The fusion peptide according to any one of claims 1 to 4, wherein the adrenomedullin or an active fragment thereof and the self-assembling peptide are linked via a linker.

9. A fusion peptide according to any one of claims 1 to 4, wherein the C-terminus is amidated.

10. A gelling composition comprising the fusion peptide according to any one of claims 1 to 4 as an active ingredient.

11. A sustained release gel comprising the gelling composition of claim 10.

12. The gelling composition of claim 10, further comprising the self-assembling peptide not linked to the adrenomedullin or an active fragment thereof.

13. The gelling composition according to claim 10, further comprising at least one anion selected from the group consisting of bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions.

14. A pharmaceutical composition comprising the gelling composition of claim 10.

15. The pharmaceutical composition according to claim 14, for promoting angiogenesis and / or suppressing neurodegeneration.

16. A pharmaceutical composition according to claim 14 for use in the treatment and / or prevention of nervous tissue injury and / or ischemia.

17. The pharmaceutical composition according to claim 16, wherein the nerve tissue damage is cerebral infarction, stroke, or traumatic brain injury.

18. A method for producing a gel, comprising: a mixing step of mixing the gelling composition according to claim 10 with water or an aqueous solution; and a gelling step of gelling the mixture obtained after the mixing step by maintaining the mixture at a temperature below the gelling temperature.

19. The method according to claim 18, wherein the mixing step further comprises mixing one or more anions selected from the group consisting of bicarbonate ions, carbonate ions, citrate ions, tartrate ions, and sulfate ions.

20. The method of claim 18, wherein the concentration of the fusion peptide in the gel is 0.4% to 10% by weight.

21. The method of claim 18, wherein the gel is implantable and / or sustained release.