Agent for suppressing or repairing DNA damage

Abstract

The present invention addresses the problem of repairing or suppressing DNA damage in cells caused by various types of in vivo and ex vivo factors.　The present invention pertains to an agent that is for repairing or suppressing DNA damage in cells, and that contains adrenomedullin or a modified product thereof having an adrenomedullin activity, or a salt thereof, or an adrenomedullin receptor agonist.

Description

Technical Field

[0001] The present invention relates to an agent for suppressing or repairing DNA damage for suppressing or repairing DNA damage of cells caused by various factors in vivo or in vitro.

Background Art

[0002] Adrenomedullin (hereinafter also referred to as "AMD") is a bioactive peptide isolated and identified from pheochromocytoma tissue in 1993 (Non-Patent Document 1). At the time of discovery, AMD was found to exhibit a potent vasodilatory hypotensive effect. For example, Patent Document 1 describes a peptide having a blood pressure lowering effect containing the amino acid sequence of human AM. Subsequent studies have revealed that AMD exhibits pharmacological effects such as an anti-inflammatory effect, an antioxidant stress effect, and an anti-apoptotic effect.

[0003] In the living body, DNA damage also occurs during nuclear DNA replication during normal cell division, and a repair mechanism is activated. On the other hand, many anticancer agents target nuclear DNA in cells and act harmfully, and cell death is caused when damage exceeding the DNA repair ability of the cells is caused. Since the action of anticancer agents has low cell specificity, not only cancer cells but also normal cells cause cell death. In addition to DNA damage caused by anticancer agents, ultraviolet rays, radiation, chemical substances, etc., oxidative DNA damage in various pathological conditions, such as tissue ischemia-reperfusion, hyperoxic environment, lifestyle diseases such as obesity and diabetes, and chronic inflammation, is also known to cause cell death, apoptosis, or cell senescence and carcinogenesis.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] DNA damage in cells caused by various factors not only leads to changes and losses of genetic information in biological cells, but also has a significant and long - term impact on transcriptional regulation and intracellular functions. If DNA damage can be suppressed or repaired, it is considered beneficial in medicine. However, conventionally, no effective components for suppressing or repairing DNA damage in cells have been found.

[0007] Therefore, the present invention aims to solve the problem of suppressing or repairing DNA damage caused by various factors in cells in vivo or in vitro.

Means for Solving the Problems

[0008] The present inventors surprisingly found that an analog of cAMP, an intracellular signaling substance generated by the activation of adrenomedullin or the adrenomedullin receptor, has the effect of suppressing DNA damage in normal cells caused by anticancer agents or oxidative stress or promoting its repair, and thus completed the following present invention.

[0009] (1) An agent for suppressing or repairing DNA damage in cells, containing adrenomedullin or a modified form thereof having adrenomedullin activity or a salt thereof, or an agonist of the adrenomedullin receptor ​

[0010] (2) Adrenomedullin or a modified form thereof having adrenomedullin activity is (I) a peptide consisting of the amino acid sequence of adrenomedullin, (II) a peptide consisting of the amino acid sequence of adrenomedullin and having two cysteine residues in the amino acid sequence forming a disulfide bond; (III) in the peptide of (II) above, the disulfide bond is replaced by an ethylene group and the peptide has adrenomedullin activity; (IV) in any of the peptides of (I) to (III) above, 1 to 15 amino acid residues are deleted, substituted or added and the peptide has adrenomedullin activity; (V) in any of the peptides of (I) to (IV) above, a peptide in which the C-terminus is amidated, and (VI) in any of the peptides of (I) to (IV) above, a peptide in which a glycine residue is added to the C-terminus and is a peptide selected from the group consisting of the DNA damage inhibitor or repair agent in the cell according to (1).

[0011] (3) Adrenomedullin or a modified form thereof having adrenomedullin activity is (a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (d) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (e) A peptide consisting of the amino acid sequence of SEQ ID NO: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (f) A peptide consisting of the amino acid sequence of SEQ ID NO: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (g) A peptide having adreno-medullin activity, wherein in any of the peptides of (a) to (f) above, the disulfide bond is substituted by an ethylene group; (h) A peptide having adreno-medullin activity, wherein in any of the peptides of (a) to (g) above, 1 to 15 amino acid residues are deleted, substituted or added; (i) A peptide having an amidated C-terminus in any of the peptides of (a) to (h) above; and (j) A peptide having a glycine residue added to the C-terminus in any of the peptides of (a) to (h) above; A peptide selected from the group consisting of, a DNA damage inhibitor or repair agent in the cell according to (1) or (2).

[0012] (4) For use in suppressing or repairing DNA damage in cells in vivo or in vitro, adrenomedullin or a modified form thereof having adrenomedullin activity or salts thereof, or an agonist of an adrenomedullin receptor. (5) For use in preventing or treating a disease or condition caused by DNA damage, adrenomedullin or a modified form thereof having adrenomedullin activity or salts thereof, or an agonist of an adrenomedullin receptor. (6) Adrenomedullin, a modified form thereof having adrenomedullin activity, or a salt thereof, or an agonist of an adrenomedullin receptor for use in the manufacture of a medicament for suppressing or repairing DNA damage in cells in vivo or in vitro. (7) Adrenomedullin, a modified form thereof having adrenomedullin activity, or a salt thereof, or an agonist of an adrenomedullin receptor for use in the manufacture of a medicament for preventing or treating a disease or condition caused by DNA damage. (8) Administering to a cell in vivo or in vitro an effective amount of adrenomedullin, a modified form thereof having adrenomedullin activity, or a salt thereof, or an agonist of an adrenomedullin receptor and suppressing or repairing DNA damage in said cell, a method for suppressing or repairing DNA damage in cells in vivo or in vitro, comprising. (9) Administering to a subject in need of prevention or treatment of a disease or condition caused by DNA damage an effective amount of adrenomedullin, a modified form thereof having adrenomedullin activity, or a salt thereof, or an agonist of an adrenomedullin receptor and suppressing or repairing DNA damage in said subject, a method for preventing or treating a disease or condition caused by DNA damage, comprising. (10) In any one of (4) to (9) above, the adrenomedullin or the modified form thereof having adrenomedullin activity is preferably a peptide selected from the group consisting of (I) to (VI) described in (2) above. (11) (12)

[0013] (13) (14) In any one of the above (4) to (9), the adrenomedullin or the modified form thereof having adrenomedullin activity is preferably a peptide selected from the group consisting of the above (I) to (VI) described in (2). In any one of (4) to (9) above, adrenomedullin or a modified form thereof having adrenomedullin activity is preferably a peptide selected from the group consisting of (a) to (j) described in (3) above.

[0014] In (4), (6) or (8) above, the cell is preferably a mammalian cell, and particularly preferably a human cell. In (5), (7) or (9) above, the disease or condition caused by the DNA damage is preferably a disease or condition in a mammal, and particularly preferably a disease or condition in a human. In (5), (7) or (9) above, the subject is preferably a mammal, and particularly preferably a human. In (4), (6) or (8) above, the cell is preferably a cell derived from a blood vessel, such as a vascular endothelial cell. In (4), (6) or (8) above, the cell is preferably a cell derived from the kidney, such as a renal tubular cell. In (5), (7) or (9) above, the disease or condition caused by the DNA damage is preferably a disease or condition in a blood vessel or an organ containing a blood vessel, such as a vascular dysfunction. In (5), (7) or (9) above, the disease or condition caused by the DNA damage is preferably a disease or condition in the kidney, such as a renal tubular dysfunction.

[0015] This specification incorporates the disclosure of Japanese Patent Application No. 2019-222050, which is the basis of the priority of this application.

Effects of the Invention

[0016] According to the present invention, it is possible to suppress or repair DNA damage caused by various factors in cells in vivo or in vitro.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] Hereinafter, the mode for carrying out the present invention will be specifically described.

[0019] <Adrenomedullin or a modified form thereof having adrenomedullin activity or a salt thereof> In one or more embodiments of the DNA damage suppression or repair agent in cells according to the present invention, as an active ingredient, it contains adrenomedullin or a modified form thereof having adrenomedullin activity or a salt thereof.

[0020] In the present invention, adrenomedullin (AMD) is not only a human-derived peptide (SEQ ID NO: 1) isolated and identified from human pheochromocytoma tissue, but also a peptide (ortholog) derived from other non-human mammals (e.g., warm-blooded animals) such as pig (SEQ ID NO: 3), dog (SEQ ID NO: 5), cow (SEQ ID NO: 7), rat (SEQ ID NO: 9) or mouse (SEQ ID NO: 11). In vivo, in these peptides, two cysteine residues in their amino acid sequences form a disulfide bond, and the C-terminus is amidated. In this specification, the peptide having a disulfide bond and a C-terminal amide group may sometimes be simply described as "adrenomedullin". The present invention can be applied to any of the above peptides.

[0021] As used herein, "C-terminal amidation" refers to a form of post-translational modification of a peptide in vivo, and specifically refers to a reaction in which the main-chain carboxyl group of the C-terminal amino acid residue of a peptide is converted into an amide group. Also, as used herein, "formation of a disulfide bond of a cysteine residue" or "disulfidization of a cysteine residue" refers to a form of post-translational modification of a peptide in vivo, and specifically refers to a reaction in which two cysteine residues in the amino acid sequence of a peptide form a disulfide bond (-S-S-). Many bioactive peptides produced in vivo are initially biosynthesized as precursor proteins with a larger molecular weight, and during the process of intracellular translocation, they undergo post-translational modification reactions such as C-terminal amidation and / or disulfidization of cysteine residues to become mature bioactive peptides. C-terminal amidation usually proceeds by the action of a C-terminal amidating enzyme on the precursor protein. In the case of a bioactive peptide having a C-terminal amide group, in its precursor protein, a Gly residue is bound to the C-terminal carboxyl group to be amidated, and the Gly residue is converted into a C-terminal amide group by a C-terminal amidating enzyme. Also, in the C-terminal propeptide of the precursor protein, there is a repeating sequence of a combination of basic amino acid residues such as Lys-Arg or Arg-Arg (Mizuno, Biochemistry, Vol. 61, No. 12, pp. 1435-1461 (1989)). Disulfidization of cysteine residues can proceed under oxidative conditions. In vivo, disulfidization of cysteine residues usually proceeds by the action of a protein disulfide isomerase on the precursor protein.

[0022] In the present invention, the modified adrenomedullin contained as an active ingredient is a modified adrenomedullin having adrenomedullin activity, specifically, an activity of suppressing or repairing DNA damage in cells. In the present invention, "adrenomedullin or a modified form thereof having adrenomedullin activity" means adrenomedullin or a modified form of adrenomedullin that retains at least a part of adrenomedullin activity, and "modified form of adrenomedullin" means a peptide in which one to several amino acids contained in the above-described adrenomedullin are modified by a chemical reaction with another chemical substance.

[0023] The adrenomedullin or a modified form thereof having adrenomedullin activity is (I) a peptide consisting of the amino acid sequence of adrenomedullin, (II) a peptide consisting of the amino acid sequence of adrenomedullin and having two cysteine residues in the amino acid sequence forming a disulfide bond, (III) a peptide in which the disulfide bond in the peptide of (II) is substituted by an ethylene group and has adrenomedullin activity, (IV) a peptide in which 1 to 15 amino acid residues are deleted, substituted or added in any of the peptides of (I) to (III) and has adrenomedullin activity, (V) a peptide in which the C-terminus is amidated in any of the peptides of (I) to (IV), and (VI) a peptide in which a glycine residue is added to the C-terminus in any of the peptides of (I) to (IV) and is preferably a peptide selected from the group consisting of.

[0024] As used herein, "adrenomedullin activity" specifically refers to an activity of suppressing or repairing DNA damage in cells.

[0025] Among the peptides (I) to (VI), the peptide consisting of the amino acid sequence of adrenomedullin included in (V), having an amidated C-terminus, and in which two cysteine residues in the amino acid sequence form a disulfide bond corresponds to mature native adrenomedullin. The peptide consisting of the amino acid sequence of adrenomedullin of (I) corresponds to the native form of adrenomedullin in an immature form (i.e., before undergoing post-translational modifications of C-terminal amidation and disulfidization of cysteine residues). Among the peptides (I) to (VI), other peptides excluding the peptides described above correspond to modified forms of adrenomedullin.

[0026] The peptide of (II) can be formed by air-oxidizing the thiol groups of the two cysteine residues of the peptide of (I) or by oxidizing them using an appropriate oxidizing agent to convert them into a disulfide bond. The three-dimensional structure of the peptide of (II) is substantially equivalent to the three-dimensional structure of native adrenomedullin. Thereby, the adrenomedullin activity of the modified form of adrenomedullin can be made substantially equivalent to that of native adrenomedullin.

[0027] The peptide of (III) can be formed by converting the disulfide bond of the peptide of (II) into an ethylene group. The substitution of a disulfide bond with an ethylene group can be carried out by a method well-known in the art (O. Keller et al., Helv. Chim. Acta, 1974, Vol. 57, p. 1253). By using the peptide of (III), the three-dimensional structure of the peptide can be stabilized. Therefore, the modified form of adrenomedullin consisting of the peptide of (III) can continuously exhibit adrenomedullin activity in vivo.

[0028] In the peptide of (IV) above, the amino acid residues that are deleted, substituted or added are preferably in the range of 1 to 12, more preferably in the range of 1 to 10, still more preferably in the range of 1 to 8, particularly preferably in the range of 1 to 5, particularly preferably in the range of 1 to 3, particularly preferably 1 or 2, and most preferably 1. The preferred peptide of (IV) above is a peptide in which the amino acids at positions 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5 or 1 to 3 from the N-terminal side are deleted in any of the peptides of (I) to (III) above and which has adrenomedullin activity. In the above preferred peptide, one or more (for example, 1 to 5, 1 to 3, or 1 or 2) amino acids may be further deleted, substituted or added. By using the peptide of (IV) above, the adrenomedullin activity of the peptide can be made substantially equivalent to that of natural adrenomedullin.

[0029] The peptide of (VI) above can be converted into the peptide of (V) above by the action of a C-terminal amidating enzyme, in which the glycine residue at the C-terminal is converted into a C-terminal amide group. Therefore, by administering the peptide of (VI) above to a subject, a C-terminal amidated peptide can be formed in the subject's body after a certain period of time. Thereby, a modified form of adrenomedullin consisting of the peptide of (VI) above can continuously exhibit adrenomedullin activity in the body.

[0030] More specifically, the adrenomedullin or its modified form is (a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (c) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (d) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (e) A peptide consisting of the amino acid sequence of SEQ ID NO: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (f) A peptide consisting of the amino acid sequence of SEQ ID NO: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (g) A peptide in any one of the peptides (a) to (f) above, wherein the disulfide bond is substituted by an ethylene group and has adrenomedullin activity; (h) A peptide in any one of the peptides (a) to (g) above, wherein 1 to 15 amino acid residues are deleted, substituted or added and has adrenomedullin activity; (i) A peptide in any one of the peptides (a) to (h) above, wherein the C-terminus is amidated; and (j) A peptide in any one of the peptides (a) to (h) above, wherein a glycine residue is added to the C-terminus; It can be a peptide selected from the group consisting of.

[0031] In the peptide of (h) above, the amino acid residues that are deleted, substituted or added are preferably in the range of 1 to 12, more preferably in the range of 1 to 10, still more preferably in the range of 1 to 8, particularly preferably in the range of 1 to 5, particularly preferably in the range of 1 to 3, particularly preferably 1 or 2, and most preferably 1. The preferred peptide of (h) above is a peptide in which the amino acids at positions 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5 or 1 to 3 from the N-terminal side are deleted in any of the peptides of (a) to (g) above and has adrenomedullin activity. In the above preferred peptide, one or more (for example, 1 to 5, 1 to 3, or 1 or 2) amino acids may be further deleted, substituted or added. By using the peptide of (h) above, the adrenomedullin activity of the peptide can be made substantially equivalent to that of natural adrenomedullin.

[0032] As a further form of adrenomedullin or its modified form, Formula (AI): A-L n -B (AI) [In the formula, A is a modifying group, L is a divalent linking group, n is an integer of 0 or 1, B is a peptide, and peptide B is adrenomedullin or a modified form of adrenomedullin that retains at least a part of the adrenomedullin activity, provided that peptide B is bound to the modifying group A or the linking group L via its N-terminal amino group.] Examples include the peptide complex represented by the formula or a salt thereof, or a hydrate thereof.

[0033] In formula (AI), the modifying group A is a palmitoyl group, a polyethylene glycol group, a myristoyl group, a peptide group, a sugar group, an organic group containing at least one polyethylene glycol group (hereinafter referred to as "organic group A" PEGIt may be referred to as "」. ), and may be selected from the group consisting of the Fc region of an immunoglobulin. The sugar group may be a monovalent group (e.g., a glycosyl group) derived from a monosaccharide, disaccharide, oligosaccharide or polysaccharide. The peptide group may be a monovalent group (e.g., a monovalent group that forms a bond via an N-terminal amino group, a C-terminal carboxyl group or a side chain group) derived from polyglycine, polyglutamic acid, polylysine or polyasparagine, etc.

[0034] In formula (AI), the divalent linking group L may be a linking group consisting of a hydrocarbon group or a peptide having an arbitrary amino acid sequence.

[0035] Specific examples of the peptide B in formula (AI) include peptides consisting of any of the peptides (I) to (VI) or any of the peptides (a) to (h).

[0036] In formula (AI), the peptide B is bonded to the modifying group A or the linking group L via its N-terminal α-amino group. Due to this bonding form, the peptide complex represented by formula (AI) exhibits an activity equivalent to that of natural-type adrenomedullin in vivo and can continuously express such activity.

[0037] In one embodiment, in formula (AI), the modifying group A is selected from the group consisting of a palmitoyl group, a polyethylene glycol group, a polyethylene glycol group, and an organic group containing at least one polyethylene glycol group, and L is a hydrocarbon group. The hydrocarbon group may be selected from a substituted or unsubstituted divalent aliphatic hydrocarbon group, a substituted or unsubstituted divalent alicyclic group, or a substituted or unsubstituted divalent aromatic group. The above aliphatic hydrocarbon group is a C1-C 10 alkylene group, a C2-C 10 alkenylene group or a C2-C 10It may be an alkynylene group. When the hydrocarbon group is an aliphatic hydrocarbon group having a substituent, an alicyclic group having a substituent, or an aromatic group having a substituent, the substituent may be a divalent group containing a heteroatom such as an oxo (carbonyl) group, a thiocarbonyl group, a carbamate group, or a carboximideoyl group. The divalent group containing a heteroatom may be disposed at one or both ends of the divalent linking group L. Specific examples of the hydrocarbon group that can be used as L include a methylene group (-CH2-), an oxo (carbonyl) group (-C(=O)-), and a 1-oxo-1,6-hexanediyl group.

[0038] In one embodiment, in formula (AI), A is a polyethylene glycol group, and L may be a hydrocarbon group. The average molecular weight of the polyethylene glycol group can be, for example, in the range of about 200 to 40,000, about 500 to 20,000, or about 500 to 10,000. When the average molecular weight of the polyethylene glycol group is 200 or more, the peptide complex represented by formula (AI) can continuously exhibit adrenomedullin activity in vivo over a long period of time. The polyethylene glycol group is usually a polyethylene glycol group protected by a protecting group such as a methoxypolyethylene glycol group or an ethoxypolyethylene glycol group, but may also be a hydroxypolyethylene glycol group. The hydrocarbon group is the same as above and can be selected from a substituted or unsubstituted divalent aliphatic hydrocarbon group, a substituted or unsubstituted divalent alicyclic group, or a substituted or unsubstituted divalent aromatic group.

[0039] [Alkylamine-linked adrenomedullin derivative] In one embodiment, in formula (AI), A is an organic group containing one or more polyethylene glycol groups (organic group A PEG ), L is a methylene group, and n is the integer 1. In this embodiment, the nitrogen atom of the α-amino group at the N-terminus of peptide B is bonded to the carbon atom of the methylene group by a covalent bond. Organic group A PEGThe peptide complex represented by formula (AI) in which peptide A and peptide B are linked in the above-described linking manner via a methylene group may be referred to as an "alkylamine-linked adrenomedullin derivative". The alkylamine-linked adrenomedullin derivative has high adrenomedullin activity.

[0040] Organic group A PEG In this case, the embodiment containing one or more PEG groups is not particularly limited. For example, one or more PEG groups may be arranged at the terminal portion of the organic group A PEG or may be arranged inside the organic group A PEG The organic group A PEG may be various groups known in the art as a linear or branched group containing a PEG group. Known groups that can be used as the modifying group A include, but are not limited to, for example, the groups disclosed in WO1995 / 11924, WO2006 / 084089, WO98 / 41562, WO2005 / 079838, WO2002 / 060978, WO2001 / 048052, WO1998 / 055500, WO1996 / 021469, WO2003 / 040211, and JP-A-04-108827.

[0041] Organic group A PEG may be a modifying group represented by the following formula (II):

Chemical formula

[0042] a is an integer of 1 or more, m is an integer of 1 or more, m is an integer of 1 or more, L 1 is a linear or branched linking group having a valence of m + 1, provided that when there are a plurality of L 1 the plurality of L 1 may be the same as or different from each other, L 2 and L 2’ are, independently of each other, a bond or a divalent linking group, provided that L 2’When there are a plurality of them, the plurality of L 2’ may be the same as or different from each other, M 1 is a PEG group, provided that M 1 When there are a plurality of them, the plurality of M 1 may be the same as or different from each other, M 2 is a bonding or PEG group, provided that M 2 When there are a plurality of them, the plurality of M 2 may be the same as or different from each other, R 1 is hydrogen or a monovalent group, * is the bonding position with L in formula (AI) n and thereof.

[0043] m is the number of branches of the linking group L 1 For example, when m is 1, L 1 is a divalent linking group and is unbranched, i.e., a linear group, in the terminal direction. When m is 2 or more, L 1 is a trivalent or higher-valent linking group and is a group with two or more branches in the terminal direction. m is usually an integer of 1 or more and 5 or less. m can be, for example, in the range of 1 to 5, 1 to 4, or 1 to 3. When the number of branches m of the linking group L 1 is in the above range, the organic group A PEG can have a linear or branched-chain structure.

[0044] a is the number of repetitions of the units of the PEG groups M 1 and M 2 , and the linking groups L 1 and L 2’ For example, when a is 1, the above units do not have a repeating structure. When a is 2 or more and m is 1, the above units have a linear repeating structure. When a is 2 or more and m is 2 or more, the above units have a dendritic branched-chain repeating structure. a is usually an integer of 1 or more and 5 or less and can be, for example, in the range of 1 to 5 or 1 to 2. The PEG groups M 1 and M 2 , and the linking groups L 1 and L2’ When the repeat number a of the unit is within the above range, the organic group A PEG can have a linear or branched structure.

[0045] M 1 and M 2 In the formula (III), the PEG group is typically represented by the formula (III): # -(CH2CH2O) n - ** (III) In formula (III), ** represents L 1 and # is O or L. 2’ The weight average molecular weight of the PEG group represented by formula (III), in total with respect to the modifying group A, can usually be 1 kDa or more, for example, 5 kDa or more, 10 kDa or more, or 20 kDa or more. It can usually be 2000 kDa or less, for example, 1000 kDa or less, 100 kDa or less, 80 kDa or less, or 60 kDa or less. The PEG group represented by formula (III) is a PEG group having an organic group A PEG The total weight average molecular weight of the copolymer usually ranges from 1 to 2000 kDa, for example, from 1 to 1000 kDa, and may range from 1 to 100 kDa, 5 to 80 kDa, 10 to 60 kDa, or 20 to 60 kDa.

[0046] In formula (III), n is the number of repetitions of ethylene oxide units defined based on the weight-average molecular weight. When n in formula (III) is defined based on the preferred range of the weight-average molecular weight, the lower limit can usually be about 20, about 110, about 230, about 460. The upper limit can usually be about 45000, about 22000, about 2200 or less, about 1820, about 1360 or less. When n in formula (III) is defined based on the preferred range of the weight-average molecular weight, it is usually in the range of about 20 to 45000, for example, in the range of about 20 to 22000, about 20 to 2200, about 110 to 1820, about 230 to 1360, about 460 to 1360. When the number of repetitions n in formula (III) is within the above range, the total weight-average molecular weight of the PEG groups contained in the modifying group represented by formula (II) is within the above range.

[0047] R 1 is hydrogen, substituted or unsubstituted C1-C 20 alkyl, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 20 alkynyl, substituted or unsubstituted C3-C 20 cycloalkyl, substituted or unsubstituted C4-C 20 cycloalkenyl, substituted or unsubstituted C4-C 20 cycloalkynyl, substituted or unsubstituted 3- to 6-membered heterocycloalkyl, substituted or unsubstituted C7-C 20 cycloalkylalkyl, substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C1-C 20 alkyl, substituted or unsubstituted C4-C 20 aryl, substituted or unsubstituted C5-C 20 arylalkyl, substituted or unsubstituted 5- to 15-membered heteroaryl, substituted or unsubstituted 5- to 15-membered heteroaryl-C1-C 20It may be alkyl, or substituted or unsubstituted acyl. When the group is substituted, the substituents may each independently be a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C2-C5 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C3-C6 cycloalkynyl, substituted or unsubstituted amino, and substituted or unsubstituted C1-C5 alkoxy.

[0048] L 1 is an m+1-valent linear or branched linking group. L 1 may be a substituted or unsubstituted m+1-valent linear or branched hydrocarbon group. The above group may contain one or more heteroatoms, alicyclic groups, aromatic groups, amide groups (-CO-NH-), ester groups (-CO-O-), or urethane groups (-O-CO-NH-). When the group is substituted, the substituents may each independently be a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, and substituted or unsubstituted linear or branched hydrocarbon groups.

[0049] L 2 and L 2’ are, independently of each other, a bond or a divalent linking group. L 2 and L 2’ When L and L are divalent linking groups, 2 and L 2’ may, independently of each other, be a substituted or unsubstituted divalent hydrocarbon group, amide group (-CO-NH-), ester group (-CO-O-), or urethane group (-O-CO-NH-). The substituted or unsubstituted divalent hydrocarbon group includes substituted or unsubstituted C1-C 20 alkylene, substituted or unsubstituted C2-C 20 alkenylene, substituted or unsubstituted C2-C 20 alkynylene, substituted or unsubstituted C3-C 20Cycloalkylene, substituted or unsubstituted C4 - C 20 Cycloalkenylene, substituted or unsubstituted C4 - C 20 Cycloalkynylene, substituted or unsubstituted 3 - to 6 - membered heterocycloalkylene, substituted or unsubstituted C7 - C 20 Cycloalkylalkylene, substituted or unsubstituted 3 - to 6 - membered heterocycloalkyl - C1 - C 20 Alkylene, substituted or unsubstituted C4 - C 20 Arylene, substituted or unsubstituted C5 - C 20 Arylalkylene, substituted or unsubstituted 5 - to 15 - membered heteroarylene, or substituted or unsubstituted 5 - to 15 - membered heteroaryl - C1 - C 20 Alkylene may be included. The substituted or unsubstituted divalent hydrocarbon group may contain one or more heteroatoms, an amide group (-CO - NH-), an ester group (-CO - O-), or a urethane group (-O - CO - NH-). When the group is substituted, the substituents may each independently be a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine, or iodine), cyano, nitro, and a substituted or unsubstituted linear or branched hydrocarbon group.

[0050] In the alkylamine - linked adrenomedullin derivative, the organic group A PEG is a modifying group represented by the following formula (V), (VI), (VII), or (VIII):

Chemical formula

[0051] In formulas (V), (VI), (VII), and (VIII), a is an integer of 1 or more, M 3 、M 3’ 、M 3’’ 、M 3’’’ and M 3’’’’ are each independently a bond or a PEG group, provided that M 3 、M 3’ 、M 3’’ 、M 3’’’and M 3’’’’ When there are a plurality of M, the plurality of M 3 M 3’ M 3’’ M 3’’’ and M 3’’’’ may be the same as or different from each other, and M 3 M 3’ M 3’’ M 3’’’ and M 3’’’’ at least one of them is a PEG group, R 1 R 1’ R 1’’ and R 1’’’ are, independently of each other, hydrogen or a monovalent group, R 2 is a bond or a divalent group, R 3 R 3’ and R 3’’ are, independently of each other, a bond or a divalent group, provided that R 3 R 3’ and R 3’’ When there are a plurality of them, the plurality of R 3 R 3’ and R 3’’ may be the same as or different from each other, * is the bonding position with L in formula (AI). n

[0052] a is the repeating number of units containing the PEG groups M 3 M 3’ M 3’’ M 3’’’ and M 3’’’’ For example, when a is 1, the said unit has no repeating structure. In formula (V), when a is 2 or more, the said unit has a linear repeating structure. In formulas (VI), (VII) and (VIII), when a is 2 or more, the said unit has a dendritic branched-chain repeating structure. a is usually an integer of 1 or more and 5 or less, and can be, for example, in the range of 1 to 5, or in the range of 1 to 2. The PEG groups M 3 M 3’ M 3’’ M 3’’’ and M 3’’’’ ​When the number of repetitions a of the unit containing it is within the above range, the modifying group A containing a PEG group can have a linear or branched structure.

[0053] M 3 , M 3’ , M 3’’ , M 3’’’ and M 3’’’’ When M is a PEG group, the PEG group is usually a group represented by the formula (III). The PEG group represented by the formula (III) has the same meaning as described above.

[0054] R 1 has the same meaning as R in the formula (II). Also, R 1 , R 1’ , R 1’’ and R 1’’’ have the same meaning as the above R 1 .

[0055] R 2 can be a bonding, substituted or unsubstituted divalent hydrocarbon group, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). The substituted or unsubstituted divalent hydrocarbon group includes substituted or unsubstituted C1-C 20 alkylene, substituted or unsubstituted C2-C 20 alkenylene, substituted or unsubstituted C2-C 20 alkynylene, substituted or unsubstituted C3-C 20 cycloalkylene, substituted or unsubstituted C4-C 20 cycloalkenylene, substituted or unsubstituted C4-C 20 cycloalkynylene, substituted or unsubstituted 3- to 6-membered heterocycloalkylene, substituted or unsubstituted C7-C 20 cycloalkylalkylene, substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C1-C 20 alkylene, substituted or unsubstituted C4-C 20 arylene, substituted or unsubstituted C5-C 20Arylalkylene, substituted or unsubstituted 5- to 15-membered heteroarylene, or substituted or unsubstituted 5- to 15-membered heteroaryl-C1-C 20 alkylene may be included. The divalent hydrocarbon group may contain one or more heteroatoms, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). When the group is substituted, the substituents may each independently be a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C2-C5 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C3-C6 cycloalkynyl, substituted or unsubstituted amino, and substituted or unsubstituted C1-C5 alkoxy.

[0056] R 3 、R 3’ and R 3’’ may independently of one another be a bond, a substituted or unsubstituted divalent hydrocarbon group, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). The substituted or unsubstituted divalent hydrocarbon group includes substituted or unsubstituted C1-C 20 alkylene, substituted or unsubstituted C2-C 20 alkenylene, substituted or unsubstituted C2-C 20 alkynylene, substituted or unsubstituted C3-C 20 cycloalkylene, substituted or unsubstituted C4-C 20 cycloalkenylene, substituted or unsubstituted C4-C 20 cycloalkynylene, substituted or unsubstituted 3- to 6-membered heterocycloalkylene, substituted or unsubstituted C7-C 20 cycloalkylalkylene, substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C1-C 20 alkylene, substituted or unsubstituted C4-C 20 arylene, substituted or unsubstituted C5-C 20Arylalkylene, substituted or unsubstituted 5- to 15-membered heteroarylene, or substituted or unsubstituted 5- to 15-membered heteroaryl-C1-C 20 Alkylene may be included. The divalent hydrocarbon group may contain one or more heteroatoms, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). When the group is substituted, the substituent may each independently be a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C2-C5 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C3-C6 cycloalkynyl, substituted or unsubstituted amino, and substituted or unsubstituted C1-C5 alkoxy.

[0057] In one embodiment, the organic group A PEG is of the following formula (V-1-1), (VI-1-1), (VII-1-1), (VII-1-2), (VII-2-1), or (VIII-1-1):

Chemical formula

[0058] In formula (V-1-1), the PEG group may have a weight average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa or 80 kDa in total.

[0059] In formula (VI-1-1), the PEG group may have a weight average molecular weight of 40 kDa in total.

[0060] In formula (VII-1-1), the PEG group may have a weight average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa, or 80 kDa in total.

[0061] In formula (VII-1-2), the PEG group has a weight average molecular weight of 50 kDa in total. In this case, usually, the ethylene oxide units of (CH2CH2O) n have a weight average molecular weight of 40 kDa in total, and the ethylene oxide units of (CH2CH2O) n’ have a weight average molecular weight of 10 kDa in total.

[0062] In formula (VII-2-1), the PEG group may have a weight average molecular weight of 40 kDa in total. In this case, usually, the ethylene oxide units of (CH2CH2O) n have a weight average molecular weight of 30 kDa in total, and the ethylene oxide units of (CH2CH2O) n’ may have a weight average molecular weight of 10 kDa in total. Alternatively, the PEG group may have a weight average molecular weight of 60 kDa in total. In this case, usually, the ethylene oxide units of (CH2CH2O) n have a weight average molecular weight of 50 kDa in total, and the ethylene oxide units of (CH2CH2O) n’ may have a weight average molecular weight of 10 kDa in total. Alternatively, the PEG group may have a weight average molecular weight of 80 kDa in total. In this case, usually, the ethylene oxide units of (CH2CH2O) n have a weight average molecular weight of 70 kDa in total, and the ethylene oxide units of (CH2CH2O) n’ may have a weight average molecular weight of 10 kDa in total.

[0063] In formula (VIII-1-1), the PEG group may have a weight average molecular weight of 40 kDa in total.

[0064] [Amide-linked adrenomedullin derivative] In certain embodiments, in formula (AI), A is an organic group containing one or more polyethylene glycol groups (organic group APEG ) and L is a carbonyl (oxo) group (-C(=O)-) and n is the integer 1. In this embodiment, the nitrogen atom of the α-amino group of peptide B forms a covalent bond with the carbon atom of the carbonyl group. Organic group A PEG is one of the following formulae (XI), (XI') or (XII): R 1 -O-M 1 -* (XI)

Chemical formula

[0065] [Urethane-linked adrenomedullin derivative] In one embodiment, in formula (AI), A contains one or more polyethylene glycols and has an ether group at the terminal on the bonding side with the linking group L (hereinafter sometimes referred to as "organic group A" PEGO "), L is a carbonyl (oxo) group (-C(=O)-), and n is an integer 1. The nitrogen atom of the α-amino group of peptide B is bonded to the carbon atom of the carbonyl group by a covalent bond. The oxygen atom derived from the ether group of organic group A PEGO forms a urethane bond with the carbonyl group and the α-amino group at the N-terminus of peptide B. In formula (AI), the peptide complex having the aforementioned urethane bond may sometimes be referred to as a "urethane-linked adrenomedullin derivative".

[0066] Organic group A PEGO may be a group represented by the following formula (XI-1-1), (XII-1-1) or (XII-2-1). CH3O-(CH2CH2O) n -* (XI-1-1) [Chemical formula] [In the formula, n has the same meaning as the definition of n in formula (III), n' has the same meaning as the aforementioned definition of n, * is L in formula (AI) n and is the bonding position with.] is a modifying group represented by.

[0067] In formula (XI-1-1), the PEG groups may have a weight-average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa, or 80 kDa in total.

[0068] In formula (XII-1-1), the PEG groups may have a weight-average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa, or 80 kDa in total.

[0069] In formula (XII-2-1), the PEG group may have a weight-average molecular weight of 40 kDa in total. In this case, usually, the ethylene oxide units of (CH2CH2O) n have a weight-average molecular weight of 30 kDa in total, and the ethylene oxide units of (CH2CH2O) n’ may have a weight-average molecular weight of 10 kDa in total. Alternatively, the PEG group may have a weight-average molecular weight of 60 kDa in total. In this case, usually, the ethylene oxide units of (CH2CH2O) n have a weight-average molecular weight of 50 kDa in total, and the ethylene oxide units of (CH2CH2O) n’ may have a weight-average molecular weight of 10 kDa in total. Alternatively, the PEG group may have a weight-average molecular weight of 80 kDa in total. In this case, usually, the ethylene oxide units of (CH2CH2O) n have a weight-average molecular weight of 70 kDa in total, and the ethylene oxide units of (CH2CH2O) n’ may have a weight-average molecular weight of 10 kDa in total.

[0070] [Releasing-type adrenomedullin derivative] In one aspect, in formula (AI), A is selected from the group consisting of a palmitoyl group, a polyethylene glycol group, a polyethylene glycol group, and an organic group containing at least one polyethylene glycol group; L is a divalent hydrocarbon group containing a heteroatom; and n is the integer 1. The divalent hydrocarbon group containing a heteroatom includes a hydrocarbon group having a group containing a nitrogen atom or an oxygen atom such as an oxo (carbonyl) group, a thiocarbonyl group, a carbamate group, or a carboximido group in the structure. When a group containing a nitrogen atom or an oxygen atom is disposed at the terminal forming a bond with peptide B, the peptide complex represented by formula (AI) can be cleaved at the portion of the divalent linking group L by an in vivo metabolic reaction such as hydrolysis, to release adrenomedullin or a modified form thereof having adrenomedullin activity. An example of the linking group L that can be used for the releasable adrenomedullin derivative is 1-oxo-1,6-hexanediyl.

[0071] [Fc-linked adrenomedullin derivative] In one aspect, the modifying group A is the Fc region of an immunoglobulin (hereinafter may be referred to as "modifying group A" Fc .), L is a linking group consisting of a peptide having an arbitrary amino acid sequence, and n is the integer 1.

[0072] The modifying group A Fc The Fc region of the immunoglobulin used as may be the Fc region of immunoglobulin G1 (IgG1) or the Fc region of immunoglobulin G4 (IgG4). In the art, it is known that a fusion protein in which the Fc region of an immunoglobulin is linked to a specific protein or peptide can extend the half-life in the body of the subject as compared with the protein or peptide that is the parent compound when administered to the subject.

[0073] The Fc region of the immunoglobulin can be the Fc region of an immunoglobulin derived from a human or non-human mammal (e.g., a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, baboon or chimpanzee, etc.). The Fc region of an immunoglobulin derived from the same human or non-human mammal to which the inhibitor or repair agent of the present invention is applied can be used.

[0074] L is, but not limited to, taking p as the number of repetitions, (GGGS) p (p is an integer in the range of 2 to 10, or an integer in the range of 4 to 6), (GGGGS) p (p is an integer in the range of 2 to 6, or 3), (GGGS) p +GGGK (p is an integer in the range of 1 to 9, or an integer in the range of 3 to 5), or (GGGGS) p +GGGGK (p is an integer in the range of 1 to 5, or 2), and a linking group composed of a peptide having such an amino acid sequence can be used. In the said amino acid sequence, the number of Gs in the repeating unit and the number of repetitions p can be appropriately changed. L can be a linking group composed of a peptide having an amino acid sequence of GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGK.

[0075] In a certain aspect, the carboxyl group at the C-terminus of the modifying group A Fc forms a peptide bond with the α-amino group at the N-terminus of the linking group L, and the α-amino group at the N-terminus of the peptide B and the carboxyl group at the C-terminus of the linking group L form a peptide bond. The Fc-linked adrenomedullin derivative as a whole has the structure of a protein or polypeptide.

[0076] Examples of the counter ion of the salt of the peptide complex represented by formula (AI) include, but are not limited to, cations such as sodium ion, potassium ion, calcium ion, magnesium ion, or substituted or unsubstituted ammonium ion, or anions such as chloride ion, bromide ion, iodide ion, phosphate ion, nitrate ion, sulfate ion, carbonate ion, hydrogen carbonate ion, perchlorate ion, formate ion, acetate ion, trifluoroacetate ion, propionate ion, lactate ion, maleate ion, hydroxymaleate ion, methylmaleate ion, fumarate ion, adipate ion, benzoate ion, 2-acetoxybenzoate ion, p-aminobenzoate ion, nicotinate ion, cinnamate ion, ascorbate ion, pamoate ion, succinate ion, salicylate ion, bismethylenesalicylate ion, oxalate ion, tartrate ion, malate ion, citrate ion, gluconate ion, aspartate ion, stearate ion, palmitate ion, itaconate ion, glycolate ion, glutamate ion, benzenesulfonate ion, cyclohexylsulfamate ion, methanesulfonate ion, ethanesulfonate ion, isethionate ion, benzenesulfonate ion, p-toluenesulfonate ion, or naphthalenesulfonate ion.

[0077] Examples of the solvent capable of forming a solvate with the peptide complex represented by formula (AI) or a salt thereof include, but are not limited to, water, or organic solvents such as methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine, acetonitrile, or ethyl acetate.

[0078] The peptide complex represented by formula (AI) includes not only the peptide complex itself described above or below, but also its protected forms. As used herein, "protected form" means a form in which a protecting group is introduced into one or more functional groups (e.g., the side-chain amino group of a lysine residue). Also, as used herein, "protecting group" is a group that is introduced into a specific functional group to prevent the progress of unwanted reactions, is quantitatively removed under specific reaction conditions, and is substantially stable, i.e., reaction-inactive, under other reaction conditions. Examples of protecting groups that can form the protected form of the peptide complex include, but are not limited to, t-butoxycarbonyl (Boc), 2-bromobenzyloxycarbonyl (BrZ), 9-fluorenylmethoxycarbonyl (Fmoc), p-toluenesulfonyl (Tos), benzyl (Bzl), 4-methylbenzyl (4-MeBzl), 2-chlorobenzyloxycarbonyl (ClZ), cyclohexyl (cHex), and phenacyl (Pac); other protecting groups for amino groups include benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, benzhydryloxycarbonyl, 2-(p-biphenyl)isopropyloxycarbonyl, 2-(3,5-dimethoxyphenyl)isopropyloxycarbonyl, p-phenylazobenzyloxycarbonyl, triphenylphosphonoethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, t-amyloxyoxycarbonyl, diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethyloxycarbonyl, allyloxycarbonyl, 2-methylsulfonylethyloxycarbonyl, 2,2,2-trichloroethyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, benzenesulfonyl, mesitylenesulfonyl, methoxytrimethylphenylsulfonyl, 2-nitrobenzenesulfonyl, 2-nitrobenzenesulfenyl, 4-nitrobenzenesulfonyl, and 4-nitrobenzenesulfenyl;As other protecting groups for the carboxyl group, methyl ester, ethyl ester, t-butyl ester, p-methoxybenzyl ester, and p-nitrobenzyl ester; as other side chain protecting groups for Arg, 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, and 2-methoxybenzenesulfonyl; as other protecting groups for Tyr, 2,6-dichlorobenzyl, t-butyl, and cyclohexyl; as other protecting groups for Cys, 4-methoxybenzyl, t-butyl, trityl, acetamidomethyl, and 3-nitro-2-pyridinesulfenyl; as other protecting groups for His, benzyloxymethyl, p-methoxybenzyloxymethyl, t-butoxymethyl, trityl, and 2,4-dinitrophenyl; and as other protecting groups for Ser and Thr, t-butyl, etc. can be mentioned. When the peptide complex represented by the formula (AI) is in a protected form by the above protecting groups, the adrenomedullin activity of the peptide complex can be made substantially equivalent to that of natural-type adrenomedullin.;

[0079] Further, the peptide complex represented by the formula (AI) also includes individual enantiomers and diastereomers of the peptide complex, and mixtures of stereoisomers of the peptide complex such as a racemate.

[0080] In the present invention, adrenomedullin or a modified form thereof can be used not only in the form of adrenomedullin or a modified form thereof itself, but also in the form of a salt. When adrenomedullin or a modified form thereof is in the form of a salt, it can be a pharmaceutically acceptable salt. Examples of counter ions of salts of adrenomedullin or a modified form thereof include, but are not limited to, cations such as sodium ion, potassium ion, calcium ion, magnesium ion, or substituted or unsubstituted ammonium ion, or anions such as chloride ion, bromide ion, iodide ion, phosphate ion, nitrate ion, sulfate ion, carbonate ion, hydrogen carbonate ion, perchlorate ion, formate ion, acetate ion, trifluoroacetate ion, propionate ion, lactate ion, maleate ion, hydroxymaleate ion, methylmaleate ion, fumarate ion, adipate ion, benzoate ion, 2-acetoxybenzoate ion, p-aminobenzoate ion, nicotinate ion, cinnamate ion, ascorbate ion, pamoate ion, succinate ion, salicylate ion, bismethylenesalicylate ion, oxalate ion, tartrate ion, malate ion, citrate ion, gluconate ion, aspartate ion, stearate ion, palmitate ion, itaconate ion, glycolate ion, glutamate ion, benzenesulfonate ion, cyclohexylsulfamate ion, methanesulfonate ion, ethanesulfonate ion, isethionate ion, benzenesulfonate ion, p-toluenesulfonate ion, or naphthalenesulfonate ion.

[0081] In the present invention, adrenomedullin, a modified form thereof or a salt thereof may be in the form of a solvate. Examples of solvents capable of forming a solvate include, but are not limited to, water, or organic solvents such as methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine, acetonitrile or ethyl acetate.

[0082] The adrenomedullin, its modified form or its salt used in the present invention can be produced by means commonly used in the art. The production of adrenomedullin, its modified form or its salt may, for example, use a solid-phase or liquid-phase peptide synthesis method, or a method of purifying a natural peptide from tissues or cells of a human or non-human mammal capable of producing adrenomedullin. Alternatively, a method of mass-expressing a recombinant protein in a transformation system such as Escherichia coli or budding yeast may be used using DNA encoding adrenomedullin (for example, SEQ ID NO: 2, 4, 6, 8, 10 or 12) in a human or non-human mammal capable of producing adrenomedullin. Alternatively, a pre-produced peptide may be purchased and used.

[0083] In the peptide produced by the above means, by disulfidizing the thiol groups of two cysteine residues in the amino acid sequence, a peptide in which the two cysteine residues in the amino acid sequence form a disulfide bond can be obtained. Further, in the peptide produced by the above means, by substituting the disulfide bond formed between two cysteine residues in the amino acid sequence with an ethylene group, a peptide in which the disulfide bond is substituted with an ethylene group can be obtained. The disulfidization reaction and the substitution reaction with an ethylene group can be carried out based on conditions commonly used in the art.

[0084] <Agonist of adrenomedullin receptor> In one or more embodiments of the DNA damage suppression or repair agent in cells according to the present invention, an agonist of an adrenomedullin receptor may be included as an active ingredient.

[0085] The present inventors surprisingly found that not only adrenomedullin but also an analog of cAMP, an intracellular signaling substance generated by activation of the adrenomedullin receptor, has an effect of suppressing DNA damage of normal cells caused by anticancer agents or oxidative stress or promoting its repair. From this, it was shown that suppression or repair of DNA damage in cells by adrenomedullin is caused by activation of the receptor when adrenomedullin binds to the adrenomedullin receptor of the cell and subsequent intracellular signal transduction. This supports that not only adrenomedullin but also an agonist of the adrenomedullin receptor also has an activity of promoting suppression or repair of DNA damage in cells.

[0086] As the adrenomedullin receptor, CRLR (calcitonin receptor-like receptor), RAMP-2 (receptor activity-modifying protein-2), etc. are known.

[0087] The agonist of the adrenomedullin receptor that can be used in the present invention is not particularly limited as long as it binds to the adrenomedullin receptor and generates an intracellular signaling substance such as cAMP in the cell.

[0088] <Agent for suppressing or repairing DNA damage> In the present invention, the agent for suppressing or repairing DNA damage can suppress or repair DNA damage in cells existing in vivo or cells existing in vitro.

[0089] The cells are not particularly limited, and may be, for example, cells existing in the body of animals such as humans and non-human mammals (e.g., mice, rats, guinea pigs, rabbits, chickens, sheep, pigs, cows, cats, dogs, monkeys, baboons, chimpanzees, etc.), or cultured cells derived from these animals. The cells may be cells derived from blood vessels or cells derived from the kidney. Examples of cells derived from blood vessels include vascular endothelial cells. Examples of cells derived from the kidney include tubular cells.

[0090] The cause of DNA damage in the cells targeted by the present invention is not particularly limited. Examples of DNA damage include DNA damage caused by endogenous factors such as DNA damage during nuclear DNA replication during cell division or damage caused by reactive oxygen species produced as a byproduct of cell metabolism, and DNA damage caused by environmental factors. Examples of DNA damage caused by environmental factors include DNA damage caused by irradiation with ultraviolet rays or electromagnetic waves, biological toxins, tobacco, DNA mutagenic substances such as chemical substances, cancer chemotherapy and radiation therapy, ischemic hypoxia or hyperoxic environment in cells and tissues, lifestyle-related diseases, excessive exercise or lack of exercise, DNA damage caused by chronic inflammation, and the like. According to the present invention, it is possible to suppress or repair intracellular DNA damage caused by these various factors. DNA damage affects cell functions such as genetic information changes and transcriptional control disorders in addition to cell death, apoptosis, cell division arrest, cell aging, and carcinogenesis. According to the present invention, it is expected to maintain cell functions and homeostasis by preventing, suppressing, or promoting the repair system of DNA damage.

[0091] The agent for suppressing or repairing DNA damage in cells according to the present invention contains at least the above-mentioned active ingredient and may further contain other components as appropriate. Examples of other components other than the active ingredient include pharmaceutically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like.

[0092] For example, in an embodiment where the agent for suppressing or repairing DNA damage in cells according to the present invention is used for the use of suppressing or repairing DNA damage in cells in a living body after being administered to the living body, the active ingredient can be formulated into an orally administrable form such as tablets, capsules, elixirs, microcapsules, etc. with a sugar coating or a soluble film, or the active ingredient can be formulated into a parenterally administrable form (for example, an injection) such as a sterile solution or suspension agent combined with water or other pharmaceutically acceptable liquids. Such a preparation can be produced by mixing the active ingredient with other ingredients in a unit dosage form required for generally recognized pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained. It may further contain other ingredients useful as pharmaceuticals. When the active ingredient is administered to humans, the dosage varies depending on symptoms and the like, but typically it is 0.1 to 100 mg / 60 Kg / day in the case of intravenous administration.

Example

[0093] The adrenomedullin used in the following experiments is derived from humans, has the amino acid primary sequence shown in SEQ ID NO: 1, and in the amino acid sequence of SEQ ID NO: 1, the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond, and the C-terminus is amidated.

[0094] Example 1: Effect of adrenomedullin on reducing cisplatin-induced renal tubular injury (in vivo) Cisplatin is a DNA cross-linking agent. The administered cisplatin is reabsorbed in the renal tubules after glomerular filtration and directly damages the renal tubular cells.

[0095] C57BL / 6, 8-week-old male mice were arbitrarily divided into four groups. Osmotic pumps filled with human adrenomedullin (1 μg / kg / min (=ADM 1γ) or 5 μg / kg / min (=ADM 5γ)) or physiological saline (Cont) were implanted subcutaneously in three of the groups. On the following day, cisplatin at 16 mg / kg body weight was intraperitoneally administered to the three groups of mice to establish a model of tubulointerstitial injury. The remaining one group was intraperitoneally administered physiological saline as a negative control group (NC). Three days after cisplatin administration, urine and kidney tissues of the four groups of mice were collected to examine the degree of tubulointerstitial injury (Figure 1).

[0096] A part of the kidney was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned thinly to prepare tissue specimens. These tissue specimens were immunohistochemically stained with an anti-Phospho-Histone H2A.X (Ser139) antibody (pH2A.X, Cell Signaling Technology) to examine DNA damage due to double-strand breaks in tubular cells. Urinary albumin / creatinine was measured to examine the dysfunction of the renal tubules. Total RNA was extracted from a part of the kidney, and gene expression changes of representative inflammatory cytokines, Mcp1 / Ccl2 (monocyte chemotactic protein-1 / C-C motif chemokine ligand 2), Tnfa (Tumor necrosis factor-α), and Il1b (Interleukin-1β), were examined using quantitative PCR analysis.

[0097] Figure 2 shows the results of immunohistological analysis (immunohistochemical staining of pH2A.X). Cisplatin administration resulted in numerous pH2A.X-positive DNA damage cells in tubular cells, but the pH2A.X-positive cells decreased and suppression of DNA damage cells was observed in an adrenomedullin (ADM) dose-dependent manner.

[0098] Figure 3 shows the measurement results of urinary albumin / creatinine. In the cisplatin-administered group, the amount of urinary albumin / creatinine increased significantly, indicating a decline in tubular function. Administration of adrenomedullin decreased the amount of urinary albumin / creatinine, and a significant decrease in the amount of urinary albumin / creatinine was observed in the group administered with 5 μg / kg / min of adrenomedullin.

[0099] Figure 4 shows the results of the analysis of the expression of inflammatory cytokine genes. In the renal tissue of the cisplatin-administered group, the gene expressions of Mcp1 / Ccl2, Tnfa, and Il1b increased, indicating the induction of tissue inflammation. In contrast, in the adrenomedullin-administered group, a significant decrease in these gene expressions was observed at the time of administration of 5 μg / kg / min or in a dose-dependent manner.

[0100] From the above, although cisplatin causes DNA damage, reduced cell function, and tissue inflammation in mouse renal tubular cells, it was revealed that administration of adrenomedullin suppresses DNA damage in tubular cells and reduces subsequent functional decline and inflammatory expression.

[0101] Example 2: Effect of adrenomedullin on reducing DNA damage in tubular cells (in vitro) (Experiment 1) Effect of reducing DNA damage by cisplatin Using mouse proximal tubule (MCT) immortalized cells, the effect of ADM on DNA damage response and its mechanism of action were investigated. Since adrenomedullin is easily degradable and adsorptive to containers in cell culture medium, which is a limitation for long-term experiments, an analog of cAMP, 8-Bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cAMP), an intracellular signaling molecule (second messenger) of adrenomedullin, was used for the investigation.

[0102] As shown in the outline in Figure 5, MCT cells were seeded in a cell culture dish, cultured in a CO2 incubator under normal culture conditions for 24 hours, and then cisplatin was added at a final concentration of 3 μM. 30 minutes before the addition of cisplatin, pretreatment was performed with 8-Br-cAMP (final concentration 0.3 mM) or phosphate-buffered saline (PBS). After the addition of cisplatin, further culture was carried out in a CO2 incubator for 24 hours, and then analysis was performed.

[0103] After fixing MCT cells with 4% paraformaldehyde, fluorescence immunostaining was performed with an anti-pH2A.X antibody. The results of fluorescence immunostaining (images converted to black and white display) are shown in Figure 6. The lower part cAMP(+) in Figure 6 shows the results of immunostaining of cultured cells treated with 8-Br-cAMP 30 minutes before the addition of cisplatin, and the upper part 8-Br-cAMP(-) in Figure 6 shows the results of immunostaining of cultured cells treated with PBS 30 minutes before the addition of cisplatin. The results of 8-Br-cAMP(-) indicate that cisplatin addition induced pH2A.X-positive DNA damage in MCT cells. On the other hand, the results of 8-Br-cAMP(+) indicate that the number of cells with cisplatin-induced DNA damage decreased in the pretreatment with 8-Br-cAMP.

[0104] To clarify the mechanism of the adrenomedullin / cAMP effect on cisplatin-induced DNA damage, under the same culture conditions of MCT cells as above, the final concentration of cisplatin (CDDP) addition was set to 3 μM or 10 μM, and the effect of 8-Br-cAMP (final concentration 0.3 mM) on cisplatin-induced DNA damage was examined. As a negative control, MCT cells were cultured under the same conditions except that cisplatin and 8-Br-cAMP were not added.

[0105] Figure 7 shows the results of analyzing the gene expression of BRCA1 (breast cancer susceptibility gene 1) and TP53BP1 (tumor suppressor p53-binding protein 1), which are DNA repair factors, after culturing for 24 hours in a CO2 incubator after adding cisplatin. It was confirmed that the gene expression of BRCA1 and TP53BP1, which are DNA repair factors, was suppressed by cisplatin (10 μM). In contrast, it was confirmed that the gene expression was significantly improved by pretreatment with 8-Br-cAMP.

[0106] Figure 8 shows the measurement results of the cell viability of the cultured cells. A decrease in cell viability was observed with cisplatin (10 μM). In contrast, it became clear that the cell viability was significantly improved by pretreatment with 8-Br-cAMP.

[0107] From the above results, it became clear that adrenomedullin activates the DNA repair function at least via cAMP and reduces cell death against cisplatin-induced DNA damage and cytotoxicity.

[0108] (Experiment 2) Effect of reducing oxidative stress DNA damage In the culture of MCT cells in Experiment 1 of Example 2 above, MCT cells were cultured under the same conditions except that hydrogen peroxide solution (H2O2) was added at a final concentration of 0.3 mM instead of cisplatin (CDDP) (Figure 9). Specifically, 30 minutes before adding hydrogen peroxide solution (H2O2) (final concentration 0.3 mM), pretreatment was performed with 8-Br-cAMP (final concentration 0.3 mM) or phosphate-buffered saline (PBS). Then, after culturing in a CO2 incubator for 24 hours, the MCT cells were fixed with 4% paraformaldehyde and subjected to fluorescence immunostaining with an anti-pH2A.X antibody.

[0109] Figure 10 shows the results of fluorescence immunostaining (image converted to black and white). The lower part of Figure 10, 8-Br-cAMP(+), shows the results of immunostaining of cultured cells treated with 8-Br-cAMP 30 minutes before the addition of H2O2. The upper part of Figure 10, 8-Br-cAMP(-), shows the results of immunostaining of cultured cells treated with PBS 30 minutes before the addition of H2O2. The results of 8-Br-cAMP(-) indicate that DNA damage positive for pH2A.X was observed in MCT cells by the addition of H2O2. On the other hand, the results of 8-Br-cAMP(+) indicate that the number of cells with H2O2-induced DNA damage decreased in the pretreatment with 8-Br-cAMP.

[0110] Example 3: Effect of adrenomedullin on reducing DNA damage in human cells (in vitro) (Experiment 1) Effect of reducing DNA damage by H2O2 Instead of the MCT cells of Example 2 above, the effect of adrenomedullin on the DNA damage response in human cells and its mechanism of action were examined using human umbilical vein endothelial cells (HUVEC). 8-Br-cAMP was used to examine the effect of adrenomedullin as in Example 2. As shown in the outline in Figure 11, HUVEC were seeded in a cell culture dish and cultured in a CO2 incubator under normal culture conditions for 24 hours, and then H2O2 was added to a final concentration of 1.0 mM. Pretreatment was performed with 8-Br-cAMP (final concentration 0.3 mM) or PBS 30 minutes before the addition of H2O2. After the addition of H2O2, the cells were further cultured in a CO2 incubator for 6 hours.

[0111] After fixing HUVEC with 4% paraformaldehyde, fluorescence immunostaining was performed using an anti-pH2A.X antibody. The results of fluorescence immunostaining (image converted to black and white display) are shown in Fig. 12. The lower row 8-Br-cAMP(+) in Fig. 12 shows the results of immunostaining of cultured cells treated with 8-Br-cAMP 30 minutes before H2O2 addition, and the upper row 8-Br-cAMP(-) in Fig. 12 shows the results of immunostaining of cultured cells treated with PBS 30 minutes before H2O2 addition. The results of 8-Br-cAMP(-) indicate that pH2A.X-positive DNA damage was observed in HUVEC cells by H2O2 addition. On the other hand, the results of 8-Br-cAMP(+) indicate that the number of cells with H2O2-induced DNA damage decreased in the pretreatment with 8-Br-cAMP.

[0112] From the above results, it was clarified that the DNA damage suppression and repair function by the analog of cAMP generated by the activation of adrenomedullin and adrenomedullin receptor against H2O2-induced DNA damage and cytotoxicity also has an effect in human cells. In addition, it was clarified that this DNA damage suppression and repair function also has an effect in vascular endothelial cells. That is, the effect related to the suppression or repair of DNA damage by adrenomedullin in the blood vessels and all organs of the human body was suggested.

[0113] All publications, patents and patent applications cited in this specification are hereby incorporated by reference as they are.

Claims

1. containing adrenomedullin or a modified form thereof or a salt thereof, wherein the adrenomedullin or the modified form thereof is (a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (e) a peptide consisting of the amino acid sequence of SEQ ID NO: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (g) a peptide in any one of the peptides (a) to (f) above, wherein the disulfide bond is replaced by an ethylene group; (h) a peptide in any one of the peptides (a) to (g) above, wherein 1 to 5 amino acid residues are deleted, substituted or added, and having adrenomedullin activity; (i) a peptide in any one of the peptides (a) to (h) above, wherein the C-terminus is amidated; and (j) A peptide in which any one of the peptides of (a) to (h) above has a glycine residue added to the C-terminus; A suppressant or repair agent for DNA damage in normal cells by an anticancer agent, which is a peptide selected from the group consisting of.

2. Adrenomedullin or a modified form thereof is (a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (b) A peptide consisting of the amino acid sequence of SEQ ID NO: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (c) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (d) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 and having a disulfide bond formed between the cysteine residue at position 16 and the cysteine residue at position 21; (e) A peptide consisting of the amino acid sequence of SEQ ID NO: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (f) A peptide consisting of the amino acid sequence of SEQ ID NO: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and having a disulfide bond formed between the cysteine residue at position 14 and the cysteine residue at position 19; (i) A peptide in which any one of the peptides of (a) to (h) above has an amidated C-terminus; and (j) A peptide in which any one of the peptides of (a) to (h) above has a glycine residue added to the C-terminus; A suppressant or repair agent for DNA damage in normal cells by the anticancer agent according to claim 1, which is a peptide selected from the group consisting of.

3. The agent for suppressing or repairing DNA damage in normal cells by the anticancer agent according to claim 2, wherein adrenomedullin or a modified form thereof is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, in which a cysteine residue at position 16 and a cysteine residue at position 21 form a disulfide bond, and the C-terminus is amidated.

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