JP7637435B2 - Method for producing long-acting adrenomedullin derivative - Google Patents

Description

The present invention relates to a method for producing a long-acting adrenomedullin derivative.

Adrenomedullin (hereinafter, also referred to as "AM") is a physiologically active peptide that was isolated and identified from brown cell tissue in 1993 (Non-Patent Document 1). When first discovered, AM was found to exert a strong vasodilatory antihypertensive effect. For example, Patent Document 1 describes a peptide with antihypertensive effect that contains the amino acid sequence of human AM.

Subsequent research has revealed that AM exerts a variety of pharmacological actions, including cardiovascular protection, anti-inflammatory, angiogenic, and tissue repair promoting effects. Furthermore, with the aim of applying AM's pharmacological actions to disease treatment, research has been conducted into the administration of AM to patients with various diseases. In particular, AM is expected to be useful as a treatment for inflammatory bowel disease, pulmonary hypertension, peripheral vascular disease, and acute myocardial infarction.

For example, Patent Document 2 describes a preventive or therapeutic agent for non-bacterial inflammatory bowel disease that contains as an active ingredient adrenomedullin or a derivative thereof that has activity in suppressing non-bacterial inflammation, or a salt thereof that has activity in suppressing non-bacterial inflammation.

Patent Document 3 describes a method for preventing or treating inflammatory bowel disease in a patient in need of such prevention or treatment for whom the use of steroid preparations, immunosuppressants, or biological preparations is difficult or insufficiently effective, the method comprising administering to the patient an effective amount of adrenomedullin, a modified form thereof having inflammation-suppressing activity, or a salt of the adrenomedullin or the modified form thereof having inflammation-suppressing activity.

In addition, structure-activity relationship studies of AM have led to the identification of essential sequences that may contribute to the biological activity of AM (Non-patent literature 2-9).

In general, peptides are known to have a short half-life in the body due to metabolic reactions in the body (e.g., in blood). For this reason, when a peptide is used as an active ingredient in a pharmaceutical, it may be possible to extend the half-life in the body and improve the pharmacokinetics by making the peptide into a peptide derivative in which another group is linked to the peptide.

For example, US Pat. No. 5,399,633 describes biologically active intermedin or adrenomedullin peptides that are characterized by a serum half-life of more than 1.5 hours. The document describes linking an alkyl group to a peptide moiety via an amide bond.

Patent Document 5 describes an AM derivative linked to a polyethylene glycol (hereinafter also referred to as "PEG") group via the phenolic hydroxyl group of Tyr ¹ of the AM.

Patent Document 6 describes a method for producing a peptide derivative in which a PEG group is linked to a free amino group of a peptide by reacting PEG-aldehyde with the free amino group of the peptide. The document describes AM as the peptide.

Non-Patent Document 10 describes an AM derivative in which a PEG group is linked to the α-amino group at the N-terminus of AM via an amide bond. The document describes that the AM derivative linked to a PEG group has an extended half-life in blood.

Patent Document 7 describes a fusion protein comprising a first segment located at the amino terminus of the fusion protein and containing the sequence of a first biologically active peptide or protein; and a second segment located at the carboxyl terminus of the fusion protein and containing the sequence of a second biologically active protein or peptide, the first and second segments being functionally covalently linked. The document describes that the fusion protein may further comprise a linker segment, such as an Fc fragment of an immunoglobulin or a functional equivalent thereof, which links the first and second segments. The document does not mention adrenomedullin.

Patent Document 8 describes an engineered polypeptide comprising an albumin binding domain polypeptide (ABD) and a first peptide hormone domain (HD1) selected from leptin, a leptin analog, or an active fragment thereof. The document describes an Fc protein as a water-soluble polymer moiety contained in HD1. The document describes that the engineered polypeptide has a good duration of action. The document exemplifies amylin, such as adrenomedullin, or an analog thereof as a drug that may be administered in combination with the engineered polypeptide.

Patent Document 9 describes an Fc-interferon-β fusion protein with improved folding and reduced aggregation, comprising (i) an immunoglobulin Fc region; and (ii) an interferon-β protein linked to the carboxy terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence. The document describes that the fusion protein can improve the half-life of interferon-β in the blood. The document does not mention adrenomedullin.

Patent Document 10 describes a compound represented by the formula (I): A-L-B (I) [wherein A is an Fc region of an immunoglobulin, B is a peptide moiety derived from adrenomedullin or a modified form thereof having adrenomedullin activity, and L is a linking group consisting of a peptide having any amino acid sequence], or a salt thereof, or a hydrate thereof.

Patent No. 2774769 Patent No. 4830093 International Publication No. 2012/096411 International Publication No. 2012/138867 International Publication No. 2013/064508 US Patent Application Publication No. 2009/0252703 Patent Publication No. 2009-510999 Patent Publication No. 2014-528917 Patent No. 4808709 International Publication No. 2018/181638

Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun, April 30, 1993, Volume 192(2), pp. 553-560 Belloni, A.S. et al., Structure-activity relationships of adrenomedullin in the adrenal gland. Endocr Res, 1998, 24(3-4), 729-30. Champion, H.C. et al., Catecholamine release mediates pressor effects of adrenomedullin-(15-22) in the rat. Hypertension, 1996, Vol. 28(6), p. 1041-6. Champion, H.C., G.G. Nussdorfer, and P.J. Kadowitz, Structure-activity relationships of adrenomedullin in the circulation and adrenal gland. Regul Pept, 1999, vol. 85(1), p. 1-8. Eguchi, S. et al., Structure-activity relationship of adrenomedullin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle cells. Endocrinology, 1994, Vol. 135(6), p. 2454-8. Garcia, M.A. et al., Synthesis, biological evaluation, and three-dimensional quantitative structure-activity relationship study of small-molecule positive modulators of adrenomedullin. J Med Chem, 2005, Vol. 48(12), p. 4068-75. Mitsuda, Y. et al., Large-scale production of functional human adrenomedullin: expression, cleavage, amidation, and purification. Protein Expr Purif, 2002, Vol. 25(3), p. 448-55. Roldos, V. et al., Small-molecule negative modulators of adrenomedullin: design, synthesis, and 3D-QSAR study. ChemMedChem, 2008, Vol. 3(9), p. 1345-55. Watanabe, T.X. et al., Vasopressor activities of N-terminal fragments of adrenomedullin in anesthetized rats. Biochem Biophys Res Commun, 1996, vol. 219(1), pp. 59-63. Kubo, K. et al., Biological properties of adrenomedullin conjugated with polyethylene glycol. Peptides, 2014, Vol. 57, p. 118-21 Kato, J., Kitamura, K.. Bench-to-bedside pharmacology of adrenomedullin. European Journal of Pharmacology, 2015, Volume 764, p. 140-148.

As mentioned above, in order to improve the pharmacokinetics of AM from the viewpoint of improving the sustained activity in vivo, AM derivatives in which other groups such as PEG groups are linked to AM are known. However, there is room for improvement in the known AM derivatives. For example, when a relatively large group such as a PEG group is linked to a relatively small peptide such as AM, various properties of the resulting AM derivative may vary greatly depending on the molecular weight of the PEG group. In addition, when other groups are linked to the side chain of the amino acid residue of AM, as in the AM derivative described in Patent Document 5, the three-dimensional structure of the AM portion may change, and the affinity with the AM receptor that recognizes AM may decrease. In such a case, the resulting AM derivative may have a decreased pharmacological action as an AM.

AM has a strong vasodilatory effect in addition to pharmacological effects such as cardiovascular protection, anti-inflammatory, angiogenic, and tissue repair promotion. For this reason, when AM or an AM derivative is administered to a subject, it may cause undesirable side effects such as an excessive drop in blood pressure due to its strong vasodilatory effect. The occurrence of such side effects can be problematic, particularly when AM or an AM derivative is used in the hope of exerting a pharmacological effect other than vasodilatory effect. In order to avoid the occurrence of the above-mentioned problems, pharmaceuticals containing AM or its derivatives as an active ingredient in the prior art had to be administered to a subject by continuous intravenous infusion at a dose that does not substantially cause undesirable side effects. Such an administration method may impose a burden on the subject.

An AM derivative that maintains the pharmacological action of AM and has improved durability in the body is expected to be able to exert the pharmacological effect of AM without causing any undesirable side reactions, even when administered to a subject in a single dose. Patent Document 10 describes a long-lasting AM derivative having a structure in which the α-amino group at the N-terminus of AM is linked to the Fc region of an immunoglobulin via a linking group of a peptide having a specific amino acid sequence. However, in the case of a method for producing an AM derivative by a culture method using E. coli as a host cell, as shown in the example of Patent Document 10, a process of refolding, C-terminal amidation, and purification of the recombinant protein produced from E. coli is usually required. Therefore, when a culture method using a prokaryote such as E. coli as a host cell is applied to a method for producing an AM derivative, time and/or economic costs may increase.

Therefore, the present invention aims to provide a means for producing long-lasting AM derivatives that can substantially suppress undesirable side reactions while maintaining the pharmacological activity of AM, at lower time and/or economic cost.

The present inventors have investigated various means for solving the above problems. The present inventors have found that by using mammalian cells as host cells for producing AM derivatives, it is possible to obtain AM derivatives with high adrenomedullin activity without refolding the recombinant protein and amidating the C-terminus. The present inventors have completed the present invention based on the above findings.

That is, the present invention includes the following aspects and embodiments.
(1) Formula (I):
ALB (I)
[Wherein,
A is an immunoglobulin Fc region,
B is a peptide moiety derived from adrenomedullin or a modified form thereof,
L is a linking group consisting of a peptide having any amino acid sequence.
A method for producing a compound represented by the following formula (I), a salt thereof, or a hydrate thereof:
an expression step of mass-expressing the compound in a host mammalian cell capable of producing the compound;
The method comprising:
(2) The method according to the above embodiment (1), which does not include a refolding step of refolding the compound expressed in large amounts in the expression step.
(3) The method according to the above embodiment (1) or (2), which does not include a step of amidating the C-terminus of the compound expressed in large amounts in the expression step.
(4) The method according to any one of the above embodiments (1) to (3), which does not include a purification step of purifying the compound represented by formula (I).
(5) The method according to any one of the above embodiments (1) to (4), which comprises only an expression step.
(6) L is the following:
GGGGSGGGGSGGGGS (SEQ ID NO: 18);
is a linking group consisting of a peptide having an amino acid sequence of
The method according to any one of the above embodiments (1) to (5), wherein the Fc region A is linked to the remainder by forming a peptide bond between the C-terminal carboxyl group and the N-terminal α-amino group of the linking group L, and the peptide portion B is linked to the remainder by forming a peptide bond between the N-terminal α-amino group and the C-terminal carboxyl group of the linking group L.
(7) The method according to any one of the preceding embodiments (1) to (6), wherein A is an Fc region of immunoglobulin G1 (IgG1) or an Fc region of immunoglobulin G4 (IgG4).
(8) The adrenomedullin or a modified form thereof is one selected from the group consisting of the following:
(i) a peptide consisting of the amino acid sequence of adrenomedullin;
(ii) a peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond;
(iii) The peptide of (ii), wherein the disulfide bond is replaced by an ethylene group;
(iv) A peptide in which 1 to 15 amino acid residues are deleted, substituted or added in any one of the peptides (i) to (iii).
The method according to any one of the preceding embodiments (1) to (7), wherein the peptide is selected from the group consisting of: (v) any one of peptides (i) to (iv) having an amidated C-terminus; and (vi) any one of peptides (i) to (iv) having a glycine residue added to the C-terminus.
(9) The method according to embodiment (8), wherein the adrenomedullin or a modified form thereof is a peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond, and the C-terminus is amidated.
(10) The adrenomedullin or a modified form thereof is one of the following:
(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 residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(g) any one of the peptides (a) to (f), wherein the disulfide bond is substituted with an ethylene group;
(h) any one of the peptides (a) to (g) in which 1 to 15 amino acid residues have been deleted, substituted or added;
(i) any one of peptides (a) to (h) in which the C-terminus is amidated; and (j) any one of peptides (a) to (h) in which a glycine residue is added to the C-terminus;
The method according to any one of embodiments (1) to (7), wherein the peptide is selected from the group consisting of:
(11) The adrenomedullin or a modified form thereof is one selected from the group consisting of the following:
(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 residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(i) any one of the peptides (a) to (f) above, wherein the C-terminus is amidated; and (j) any one of the peptides (a) to (f) above, wherein a glycine residue is added to the C-terminus;
The method of embodiment (10), wherein the peptide is selected from the group consisting of:
(12) The adrenomedullin or a modified form thereof is selected from the group consisting of the following:
(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 residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond; and (f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
The method according to embodiment (11), wherein the peptide is amidated at the C-terminus, and is selected from the group consisting of:
(13) The compound represented by formula (I) is
(Ea-1) a peptide consisting of the amino acid sequence of SEQ ID NO: 15, or a peptide consisting of the amino acid sequence of SEQ ID NO: 15 and in which the cysteine residues at positions 259 and 264 form a disulfide bond;
(Ea-2) a peptide consisting of the amino acid sequence of SEQ ID NO: 17, or a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and in which the cysteine residues at positions 256 and 261 form a disulfide bond;
(Ea-3) a peptide consisting of the amino acid sequence of SEQ ID NO: 23, or a peptide consisting of the amino acid sequence of SEQ ID NO: 23 and in which the cysteine residues at positions 254 and 259 form a disulfide bond;
(Ea-4) a peptide consisting of the amino acid sequence of SEQ ID NO: 25, or a peptide consisting of the amino acid sequence of SEQ ID NO: 25 in which the cysteine residues at positions 251 and 256 form a disulfide bond;
(Eg) A peptide according to any one of (Ea-1) to (Ea-4), in which the disulfide bond is substituted with an ethylene group;
(Eh) A peptide in which 1 to 15 amino acid residues are deleted, substituted or added in any of peptides (Ea-1) to (Eg);
(Ei) any one of peptides (Ea-1) to (Eh) in which the C-terminus is amidated; and (Ej) any one of peptides (Ea-1) to (Eh) in which a glycine residue has been added to the C-terminus;
The method according to any one of embodiments (1) to (7), wherein the peptide is selected from the group consisting of:
(14) The compound represented by formula (I) is
(Ea-1) a peptide consisting of the amino acid sequence of SEQ ID NO: 15, or a peptide consisting of the amino acid sequence of SEQ ID NO: 15 and in which the cysteine residues at positions 259 and 264 form a disulfide bond;
(Ea-2) a peptide consisting of the amino acid sequence of SEQ ID NO: 17, or a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and in which the cysteine residues at positions 256 and 261 form a disulfide bond;
(Ea-3) a peptide consisting of the amino acid sequence of SEQ ID NO: 23, or a peptide consisting of the amino acid sequence of SEQ ID NO: 23 and in which the cysteine residues at positions 254 and 259 form a disulfide bond;
(Ea-4) a peptide consisting of the amino acid sequence of SEQ ID NO: 25, or a peptide consisting of the amino acid sequence of SEQ ID NO: 25 in which the cysteine residues at positions 251 and 256 form a disulfide bond; and (Ei) a peptide in which the C-terminus is amidated in any one of the peptides (Ea-1) to (Ea-4);
The method of embodiment (13), wherein the peptide is selected from the group consisting of:
(15) The compound represented by formula (I) is
(Ea-1) a peptide consisting of the amino acid sequence of SEQ ID NO: 15, or a peptide consisting of the amino acid sequence of SEQ ID NO: 15 and in which the cysteine residues at positions 259 and 264 form a disulfide bond;
(Ea-2) a peptide consisting of the amino acid sequence of SEQ ID NO: 17, or a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and in which the cysteine residues at positions 256 and 261 form a disulfide bond;
(Ea-3) a peptide consisting of the amino acid sequence of SEQ ID NO: 23, or a peptide consisting of the amino acid sequence of SEQ ID NO: 23 and in which the cysteine residues at positions 254 and 259 form a disulfide bond; and (Ea-4) a peptide consisting of the amino acid sequence of SEQ ID NO: 25, or a peptide consisting of the amino acid sequence of SEQ ID NO: 25 and in which the cysteine residues at positions 251 and 256 form a disulfide bond;
The method according to embodiment (14), wherein the peptide is amidated at the C-terminus, and is selected from the group consisting of:

The present invention makes it possible to provide a means for producing long-lasting AM derivatives that can substantially suppress undesirable side reactions while maintaining the pharmacological activity of AM, at lower time and/or economic cost.

This specification includes the contents described in the specification and/or drawings of Japanese Patent Application No. 2021-025877, which is the basis of the priority of this application.

1 is a graph showing a dose-response curve of the concentration of added AM or AM derivatives and the intracellular cAMP concentration in Experiment III-1, in which the horizontal axis represents the concentration of added AM or AM derivatives (M) and the vertical axis represents the intracellular cAMP concentration (fmol/well (plate)). 2 is a graph showing the time course of the blood concentration of the AM derivative in the subcutaneous administration of the AM derivative of Example 2 or 4 in Experiment III-2. A is a graph showing the time course of the blood concentration of the AM derivative corresponding to mAM, and B is a graph showing the time course of the blood concentration of the AM derivative corresponding to tAM. In the figure, the horizontal axis is the period (days) after administration, and the vertical axis is the blood concentration (pM) of the AM derivative. FIG. 3 is a graph showing the effect of subcutaneous administration of the AM derivative of Example 4 on the suppression of blood pressure rise in spontaneously hypertensive rats (SHR) in Experiment III-3. A is a graph showing the change in systolic blood pressure (SBP) over time, and B is a graph showing the change in diastolic blood pressure (DBP) over time. In the figure, the horizontal axis shows the period (days) after administration, and the vertical axis shows blood pressure (mmHg). * indicates that the p value against the saline-administered control group, calculated by Student's t-test (n=5), is less than 0.05. 4 is a graph showing the blood concentration of the AM derivative in SHR 12 days after subcutaneous administration of the control or the AM derivative of Example 4 in Experiment III-3. Graph A shows the blood concentration of the AM derivative corresponding to mAM, and graph B shows the blood concentration of the AM derivative corresponding to tAM. In the figure, the vertical axis shows the blood concentration (pM) of the AM derivative corresponding to mAM or tAM.

1. Method for producing adrenomedullin derivative
One aspect of the present invention is a compound of formula (I):
ALB (I)
The present invention relates to a method for producing a compound represented by the formula (I) or a salt thereof, or a hydrate thereof. In formula (I), A is an immunoglobulin Fc region, B is a peptide moiety derived from adrenomedullin (AM) or a modified form thereof, and L is a linking group consisting of a peptide having any amino acid sequence. In this specification, the compound represented by formula (I) may be referred to as an "immunoglobulin Fc region-linked adrenomedullin derivative" or an "immunoglobulin Fc region-linked AM derivative", or simply as an "adrenomedullin derivative" or "AM derivative".

AM, a known physiologically active substance, is a peptide. For this reason, a medicine containing AM as an active ingredient may have an extremely short period of time during which it can effectively act in the body of a subject (e.g., a human patient). Therefore, attempts have been made to improve the pharmacokinetics by extending the half-life in the body by forming an AM derivative in which other groups such as polyethylene glycol (PEG) are linked to AM (Patent Documents 4 to 6 and 10, and Non-Patent Document 10). However, when a relatively large group such as a PEG group is linked to a relatively small peptide such as AM, various properties of the resulting AM derivative may vary greatly depending on the molecular weight of the PEG group. In addition, when other groups are linked to the side chain of the amino acid residue of AM, the three-dimensional structure of the AM portion may change, decreasing the affinity with the AM receptor that recognizes AM. In such a case, the resulting AM derivative may have a decreased pharmacological action as AM.

AM has a strong vasodilatory effect. For this reason, when a therapeutically effective amount of AM or its derivatives is administered once, the strong vasodilatory effect may cause undesirable side effects (e.g., excessive drop in blood pressure, tachycardia associated with reflex increased sympathetic activity, and/or increased renin activity). The occurrence of such side effects can be problematic, particularly when AM or its derivatives are used in the hope of exerting a pharmacological effect other than vasodilatory effect. In order to avoid the occurrence of such problems, medicines containing AM or its derivatives as an active ingredient have had to be administered to subjects by continuous intravenous infusion. Such an administration method may impose a burden on the subject.

It is expected that an AM derivative that maintains the pharmacological action of AM and has improved durability in the body can exert the pharmacological effect of AM without causing any undesirable side reactions even when administered to a subject in a single dose. From this perspective, the present inventors have developed a long-lasting AM derivative having a structure in which the α-amino group at the N-terminus of AM and the Fc region of an immunoglobulin are linked via a linking group of a peptide having a specific amino acid sequence (Patent Document 10). 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 a subject when administered to a subject, compared to the parent compound protein or peptide (e.g., Patent Documents 8 and 9). By applying the AM derivative disclosed in Patent Document 10 to symptoms, diseases and/or disorders that can be prevented or treated by AM, the symptoms, diseases and/or disorders can be prevented or treated sustainably while substantially suppressing undesirable side reactions.

However, in the case of the method for producing AM derivatives by a culture method using E. coli as a host cell, as shown in the examples of Patent Document 10, steps of refolding, C-terminal amidation, and purification of the recombinant protein produced from E. coli are usually required. Therefore, when a culture method using a prokaryote such as E. coli as a host cell is applied to a method for producing AM derivatives, there is a possibility that the time and/or economic costs will increase.

The present inventors have found that by using mammalian cells as host cells for producing AM derivatives, it is possible to obtain AM derivatives having high adrenomedullin activity without refolding and C-terminal amidation of recombinant proteins. Therefore, the method of this embodiment includes an expression step of mass-expressing the compound represented by formula (I) in a host mammalian cell capable of producing the compound. By using the method of this embodiment using a host mammalian cell capable of producing the compound represented by formula (I), it is possible to produce a long-lasting AM derivative that can substantially suppress undesirable side reactions while maintaining the pharmacological action of AM, at a lower time and/or economic cost.

[1-1. Host mammalian cell production process]
The method of this embodiment may, if desired, include a host mammalian cell preparation step of preparing a host mammalian cell capable of producing a compound represented by formula (I).

This process can be carried out by linking an isolated nucleic acid having a base sequence encoding the compound represented by formula (I) to a vector, introducing it into a mammalian cell, and transforming the cell.

The isolated nucleic acid used in this step preferably has a base sequence corresponding to various embodiments of the compound represented by formula (I) described below, and more preferably has a base sequence selected from the group consisting of SEQ ID NOs: 14, 16, 22 and 24. Alternatively, as described below, when a plasmid vector for preparing an immunoglobulin Fc region fusion protein is used as a protein expression vector, the isolated nucleic acid preferably has a base sequence selected from the group consisting of SEQ ID NOs: 2, 5, 7, 9, 11 and 13 corresponding to mammalian AM. Such isolated nucleic acids are disclosed, for example, in Patent Document 10. Those skilled in the art can prepare the isolated nucleic acid by purchasing the isolated nucleic acid, applying an appropriate conversion reaction to the isolated nucleic acid purchased, or preparing it themselves, based on the above literature.

Examples of vectors used in this process include plasmid vectors such as pUC119, pUC118, and pGEM T-Easy vectors for gene expression, and plasmid vectors such as pET-3, pET-11, pET-32, and pCMV-TNT for protein expression, as well as plasmid vectors for preparing immunoglobulin Fc region fusion proteins such as pFUSEN-hG1Fc, pFUSE-hIgG4-Fc2, and pCAG-NeO-IgGFc. It is preferable to use a plasmid vector for preparing an immunoglobulin Fc region fusion protein. By using a plasmid vector for preparing an immunoglobulin Fc region fusion protein in combination with an isolated nucleic acid corresponding to mammalian AM, a vector for expressing a large amount of the compound represented by formula (I) can be easily prepared. By using the vectors exemplified above in this process, transformation can be performed with high efficiency.

Mammalian cells used in this process include, for example, HEK293 and CHO cells. It is preferable to use HEK293.

In this process, the ligation of the isolated nucleic acid to a vector and the transformation of mammalian cells with the ligated vector can be carried out under conditions commonly used in the art.

By carrying out this process, a host mammalian cell capable of producing the compound represented by formula (I) can be obtained.

[1-2. Expression process]
The method of this embodiment includes an expression step of expressing a large amount of the compound represented by formula (I) in a host mammalian cell capable of producing the compound.

In this process, the mass expression of the compound represented by formula (I) can be carried out based on conditions commonly used in the technical field, taking into consideration the vector and cell type of the host mammalian cell used in this process.

In this process, the compound represented by formula (I) that is expressed in large quantities can be obtained from the cells or culture supernatant of the host mammalian cells, preferably from the culture supernatant.

By carrying out this process, a compound represented by formula (I) having adrenomedullin activity can be obtained.

[1-3. Purification process]
The method of this embodiment may, if desired, include a purification step of purifying the compound represented by formula (I) obtained in the expression step.

In this step, examples of the means for purifying the compound represented by formula (I) include extraction, filtration, centrifugation, adsorption, recrystallization, and various types of chromatography. Examples of chromatography include adsorption, normal phase or reverse phase partitioning, ion exchange, and gel filtration. Each of the above means can be carried out under conditions commonly used in the technical field. Each of the above means may be repeated multiple times under the same or different conditions, as desired.

By carrying out this process, a compound represented by formula (I) having adrenomedullin activity can be obtained in high purity.

In the method of this embodiment, it has been found that the compound represented by formula (I) obtained in the expression step can express adrenomedullin activity in its original form without refolding and amidation of the C-terminus. It is known that when a physiologically active protein derived from a mammal is expressed in large quantities using a prokaryote such as Escherichia coli as a host cell, the protein may accumulate as an inclusion body in the cell without undergoing post-translational modifications such as formation of a higher-order structure and/or amidation of the C-terminus, which are necessary for the expression of physiological activity. In such a case, in order to obtain a protein in a form having a higher-order structure capable of expressing physiological activity and/or a form in which the C-terminus is amidated, a step of refolding the protein expressed in large quantities and/or amidating the C-terminus is required. In contrast, it is known that the mammalian cells used as host cells in the method of this embodiment can produce a physiologically active protein derived from a mammal in a form having a higher-order structure and/or a C-terminal amide structure capable of expressing physiological activity, and can secrete it outside the cell. Therefore, it is preferable that the method of this embodiment does not include a refolding step of refolding the compound expressed in large quantities in the expression step. In addition, the method of this embodiment preferably does not include a C-terminal amidation step of amidating the C-terminus of the compound expressed in large quantities in the expression step. By carrying out the method of this embodiment without including either a refolding step and/or a C-terminal amidation step, particularly without a refolding step and a C-terminal amidation step, it is possible to obtain a compound represented by formula (I) having a higher-order structure required for the expression of adrenomedullin activity and/or having an amidated C-terminus. This allows the compound represented by formula (I) to be produced at a lower time and/or economic cost than the conventional method using a prokaryote such as Escherichia coli as a host cell.

In a particular embodiment, it has been found that the compound represented by formula (I) obtained in the expression step is of high purity without carrying out a purification step. As described above, it is known that the mammalian cells used as host cells in the method of this aspect can produce physiologically active proteins derived from mammals in a form having a higher-order structure and/or a C-terminal amide structure capable of expressing physiological activity, and can be secreted outside the cell. For this reason, the compound represented by formula (I) obtained in the expression step can preferably be obtained from the culture supernatant of the host mammalian cells. Since the culture supernatant does not substantially contain any components derived from the host mammalian cells other than the compound represented by formula (I) expressed in large quantities, the culture supernatant obtained in the expression step contains the compound represented by formula (I) in high purity. Therefore, it is preferable that the method of this aspect does not include a purification step of purifying the compound represented by formula (I). By carrying out the method of this aspect without including a purification step, it is possible to obtain a compound represented by formula (I) in high purity, which has a higher-order structure required for the expression of adrenomedullin activity and/or has an amidated C-terminus. This allows the compound of formula (I) to be produced at lower time and/or economic cost compared to prior art methods that use prokaryotes such as E. coli as host cells.

<2. Adrenomedullin Derivatives>
Another aspect of the present invention relates to a compound represented by formula (I) or a salt thereof, or a hydrate of the compound or the salt, which can be obtained by the method according to one aspect of the present invention, preferably obtained by said method.

It has been found that the Fc domain-linked AM derivative, which is a compound represented by formula (I) obtained by the method of one embodiment of the present invention, has a higher-order structure required for the expression of adrenomedullin activity and/or has an amidated C-terminus, and thus has significantly higher adrenomedullin activity and persistence in vivo compared to Fc domain-linked AM derivatives obtained by conventional methods using prokaryotes such as Escherichia coli as host cells. Therefore, the compound represented by formula (I) that can be obtained by the method of one embodiment of the present invention, preferably obtained by said method, can express sustained high adrenomedullin activity in vivo.

In each aspect of the present invention, the AM may be not only a human-derived peptide (SEQ ID NO: 1, Non-Patent Document 1) isolated and identified from human brown cell tissue, but also a peptide (orthologue) derived from other non-human mammals (e.g., warm-blooded animals), such as pigs (SEQ ID NO: 4), dogs (SEQ ID NO: 6), cows (SEQ ID NO: 8), rats (SEQ ID NO: 10) or mice (SEQ ID NO: 12). In vivo, these peptides have two cysteine residues in their amino acid sequences that 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 be referred to as "natural adrenomedullin" or simply "adrenomedullin". Each aspect of the present invention can be applied to any of the above peptides.

In this specification, "C-terminal amidation" refers to one aspect of post-translational modification of peptides in vivo, specifically, the reaction in which the main chain carboxyl group of the C-terminal amino acid residue of a peptide is converted to the form of an amide group. In addition, in this specification, "formation of a disulfide bond of a cysteine residue" or "disulfidation of a cysteine residue" refers to one aspect of post-translational modification of peptides in vivo, specifically, the 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 first biosynthesized as precursor proteins with larger molecular weights, which undergo post-translational modification reactions such as C-terminal amidation and/or disulfidation of cysteine residues during intracellular translocation 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 physiologically active peptide having a C-terminal amide group, a Gly residue is bound to the C-terminal carboxyl group to be amidated in the precursor protein, and the Gly residue is converted to a C-terminal amide group by a C-terminal amidating enzyme. In addition, the C-terminal propeptide of the precursor protein has a repeat sequence of a combination of basic amino acid residues such as Lys-Arg or Arg-Arg (Mizuno, Seikagaku vol. 61, no. 12, pp. 1435-1461 (1989)). Disulfidation of cysteine residues can proceed under oxidative conditions. In vivo, disulfidation of cysteine residues usually proceeds by the action of protein disulfide isomerase on precursor proteins.

In formula (I), B is a peptide moiety derived from adrenomedullin or a modified form thereof. In each embodiment of the present invention, the "peptide moiety derived from adrenomedullin or a modified form thereof" means a monovalent free group having a structure in which one hydrogen atom (usually one hydrogen atom of an amino group, typically one hydrogen atom of the N-terminal α-amino group) has been removed from AM or a modified form thereof. In the present invention, the "modified form of adrenomedullin" means a peptide in which the natural AM described above has been chemically modified. The modified form of adrenomedullin preferably has adrenomedullin activity. In addition, in the present invention, the "adrenomedullin activity" means the biological activity of AM. Examples of adrenomedullin activity include the following.

(1) Cardiovascular system: vasodilatory effect, blood pressure lowering effect, blood pressure elevation suppression effect, increased cardiac output/improvement of heart failure, improvement of pulmonary hypertension, angiogenic effect, lymphangiogenic effect, improvement of vascular endothelial function, vascular permeability control, endothelial cell-cell adhesion control, vascular endothelial barrier protection effect, anti-atherosclerosis effect, myocardial protective effect (e.g., myocardial protective effect in ischemia-reperfusion injury or inflammation), suppression of remodeling after myocardial infarction, suppression of cardiac hypertrophy, and angiotensin-converting enzyme suppression effect.
(2) Kidneys and water-electrolyte system: diuretic effect, natriuretic effect, antidiuretic hormone suppression effect, aldosterone lowering effect, renal protective effect (e.g., myocardial protective effect in hypertension or ischemia-reperfusion injury), inhibitory effect on diabetic nephropathy, inhibitory effect on C3 nephropathy, inhibitory effect on drinking behavior, and inhibitory effect on salt requirement.
(3) Brain and nervous system: neuroprotective effect/brain damage inhibition effect, anti-inflammatory effect, apoptosis inhibition effect (e.g., apoptosis inhibition effect in ischemia-reperfusion injury or inflammation), autoregulation function maintenance effect, oxidative stress inhibition effect, dementia improvement effect, and sympathetic nervous system suppression effect.
(4) Genitourinary organs: erection improvement effect, blood flow improvement effect, and implantation promotion effect.
(5) Digestive system: Anti-ulcer effect, tissue repair effect, mucosal regeneration effect, intestinal barrier protection effect, blood flow improvement effect, anti-inflammatory effect, and liver function improvement effect.
(6) Orthopedics: Stimulating effect on osteoblasts and improving arthritis.
(7) Endocrine metabolism: adipocyte differentiation effect, lipolysis control effect, insulin sensitivity improvement effect, insulin secretion control effect, antidiuretic hormone secretion inhibition effect, and aldosterone secretion inhibition effect.
(8) Respiratory system: bronchodilator effect, lung protection effect, improvement of emphysema, inhibition of pulmonary fibrosis, inhibition of pneumonia, inhibition of bronchitis, and improvement of breathing.
(9) Immune system: Promoting the degradation of C3b.
(10) Others: Circulation improving effect, anti-inflammatory effect, cytokine control effect, organ protection effect, oxidative stress suppression effect, tissue repair effect (e.g., anti-bedsore effect), sepsis improvement effect, septic shock improvement effect, multiple organ failure suppression effect, autoimmune disease suppression effect, diabetic retinopathy suppression effect, antibacterial effect, hair growth effect, and hair nourishing effect.

The blood pressure lowering effect is preferably a vasodilatory antihypertensive effect. The anti-inflammatory effect in the digestive system is preferably a preventive or therapeutic effect on inflammatory bowel disease such as steroid-resistant or steroid-dependent inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, or intestinal Behçet's disease).

The adrenomedullin activity exemplified above, which is expressed by AM, is usually expressed via an increase in the intracellular cAMP concentration. Therefore, an increase in the intracellular cAMP concentration can be used as an index of adrenomedullin activity. In each aspect of the present invention, the intracellular cAMP concentration increasing effect can be evaluated, for example, by adding the target compound to a cultured cell line (HEK293 cell line) in which the AM type 1 receptor (AM1 receptor) is stably expressed, and measuring the amount of intracellular cAMP produced. By containing the peptide portion B derived from the AM or its modified form having the above-mentioned biological activity, the compound represented by formula (I) has an intracellular cAMP concentration increasing effect substantially equivalent to that of natural AM. Therefore, the compound represented by formula (I) can express a biological activity (i.e., adrenomedullin activity) substantially equivalent to that of natural AM through an increase in the intracellular cAMP concentration.

The AM or a modified form thereof is as follows:
(i) a peptide consisting of the amino acid sequence of adrenomedullin;
(ii) a peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond;
(iii) The peptide of (ii), wherein the disulfide bond is replaced by an ethylene group;
(iv) A peptide in which 1 to 15 amino acid residues are deleted, substituted or added in any one of the peptides (i) to (iii).
(v) any one of the peptides (i) to (iv) having an amidated C-terminus; and (vi) any one of the peptides (i) to (iv) having a glycine residue added to the C-terminus.

In one embodiment, the AM or a modification thereof is
(i) a peptide consisting of the amino acid sequence of adrenomedullin;
(ii) a peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond;
It is more preferable that the peptide is selected from the group consisting of (v) a peptide having an amidated C-terminus in the peptide (i) or (ii), and (vi) a peptide having a glycine residue added to the C-terminus in the peptide (i) or (ii).

In another embodiment, the AM or a modification thereof is
(i) a peptide consisting of the amino acid sequence of adrenomedullin;
It is further preferable that the peptide is selected from the group consisting of (ii) a peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond, and (v) a peptide of (i) or (ii) in which the C-terminus is amidated.

In another embodiment, the AM or its modified form is particularly preferably a peptide having an amidated C-terminus, the peptide consisting of the amino acid sequence of adrenomedullin and in which two cysteine residues in the amino acid sequence form a disulfide bond.

Among the peptides (i) to (vi) above, the peptide included in (v) consisting of the amino acid sequence of AM, in which the C-terminus is amidated and in which two cysteine residues in the amino acid sequence form a disulfide bond, corresponds to a mature native AM having a higher-order structure required for the expression of adrenomedullin activity. The peptide consisting of the amino acid sequence of AM in (i) corresponds to a native AM in its (immature) form prior to the post-translational modifications of C-terminus amidation and disulfidation of cysteine residues. Among the peptides (i) to (vi) above, the other peptides, except for those described above, correspond to modified forms of AM.

The peptide (ii) can be formed by converting the thiol groups of the two cysteine residues of the peptide (i) into disulfide bonds by air oxidation or by oxidation with an appropriate oxidizing agent. By using the peptide (ii), the three-dimensional structure of the peptide portion B can be made similar to that of natural AM. This makes it possible to make the adrenomedullin activity of the compound represented by formula (I) substantially equivalent to that of natural AM.

The peptide (iii) can be formed by converting the disulfide bond of the peptide (ii) to an ethylene group. The substitution of the disulfide bond with an ethylene group can be performed by a method well known in the art (O. Keller et al., Helv. Chim. Acta, 1974, Vol. 57, p. 1253). The peptide (iii) can be used to stabilize the three-dimensional structure of the peptide portion B. This allows the compound represented by formula (I) to continuously express adrenomedullin activity in vivo.

In the peptide (iv), the number of deleted, substituted or added amino acid residues is preferably in the range of 1 to 15, more preferably in the range of 1 to 10, even more preferably in the range of 1 to 8, particularly preferably in the range of 1 to 5, and most preferably in the range of 1 to 3. A suitable peptide (iv) is a peptide in which amino acid residues at positions 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5 or 1 to 3 from the N-terminus are deleted in any of the peptides (i) to (iii), and a more suitable peptide (iv) is a peptide in which amino acid residues at positions 1 to 15, 1 to 10 or 1 to 5 from the N-terminus are deleted in any of the peptides (i) to (iii). In the suitable peptide, one or more amino acid residues (e.g., 1 to 5, 1 to 3, or 1 or 2) may be further deleted, substituted or added. By using the peptide (iv), the adrenomedullin activity of the compound represented by formula (I) can be substantially equivalent to that of natural AM. In addition, by using the peptide (iv), the compound represented by formula (I) can continuously express the adrenomedullin activity in vivo.

The peptide (vi) can be converted to the peptide (v) by the action of a C-terminal amidating enzyme, which converts the C-terminal glycine residue into a C-terminal amide group. Therefore, by administering the peptide (vi) to a subject, a C-terminally amidated peptide can be formed in the subject's body after a certain period of time. This allows the compound represented by formula (I) to continuously express adrenomedullin activity in the body.

The AM or a modified form thereof is as follows:
(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 residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(g) any one of the peptides (a) to (f), wherein the disulfide bond is substituted with an ethylene group;
(h) any one of the peptides (a) to (g) in which 1 to 15 amino acid residues have been deleted, substituted or added;
(i) any one of the peptides (a) to (h) above, wherein the C-terminus is amidated; and (j) any one of the peptides (a) to (h) above, wherein a glycine residue is added to the C-terminus;
More preferably, the peptide is selected from the group consisting of:

In one embodiment, the AM or a modification 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 in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(i) any one of the peptides (a) to (f) above, wherein the C-terminus is amidated; and (j) any one of the peptides (a) to (f) above, wherein a glycine residue is added to the C-terminus;
It is even more preferable that the peptide is selected from the group consisting of:

In another embodiment, the AM or a modification 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 in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond; and (i) any of the peptides (a) to (f) whose C-terminus is amidated;
It is even more preferable that the peptide is selected from the group consisting of:

In another embodiment, the AM or a modification 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 in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond; and (f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
Among the peptides selected from the group consisting of the above, those having an amidated C-terminus are particularly preferred.

In the peptide (h), the number of deleted, substituted or added amino acid residues is preferably in the range of 1 to 12, more preferably in the range of 1 to 10, even more preferably in the range of 1 to 8, particularly preferably in the range of 1 to 5, and most preferably in the range of 1 to 3. A preferred peptide (h) is a peptide in which 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-terminus in any of the peptides (a) to (g), and a more preferred peptide (h) is a peptide in which amino acid residues at positions 1 to 15, 1 to 10 or 1 to 5 from the N-terminus in any of the peptides (a) to (d), or a peptide in which amino acid residues at positions 1 to 13, 1 to 8 or 1 to 5 from the N-terminus in any of the peptides (e) or (f). In the suitable peptide, one or more (e.g., 1 to 5, 1 to 3, or 1 or 2) amino acids may be further deleted, substituted, or added. By using the peptide (h), the adrenomedullin activity of the compound represented by formula (I) can be made substantially equivalent to that of natural AM. Furthermore, by using the peptide (h), the compound represented by formula (I) can continuously express adrenomedullin activity in vivo.

In formula (I), A is the Fc region of an immunoglobulin. A is preferably the Fc region of immunoglobulin G1 (IgG1) or the Fc region of immunoglobulin G4 (IgG4). It is known in the art that a fusion protein in which an immunoglobulin Fc region is linked to a specific protein or peptide can extend the half-life in the subject's body when administered to a subject, compared to the parent protein or peptide (e.g., Patent Documents 8 and 9). Therefore, the compound represented by formula (I) of this embodiment having the immunoglobulin Fc region A can continuously express adrenomedullin activity in the body.

In formula (I), the mammal from which the Fc region of the immunoglobulin used as A originates can be appropriately selected based on the subject to which the pharmaceutical containing the compound represented by formula (I) of one embodiment of the present invention as an active ingredient is applied, as described below. A is preferably an 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, Mandrill or chimpanzee), and more preferably an Fc region of an immunoglobulin derived from the same human or non-human mammal as the subject to which the pharmaceutical of one embodiment of the present invention is applied. By having the Fc region of an immunoglobulin derived from the human or non-human mammal, the compound represented by formula (I) of this embodiment can sustain adrenomedullin activity in vivo while maintaining the pharmacological action of natural AM.

In formula (I), L is a linking group consisting of a peptide having any amino acid sequence. L is not limited, but can be a linking group consisting of a peptide having an amino acid sequence of (GGGS)n (SEQ ID NO: 20) (n is an integer ranging from 2 to 10, preferably an integer ranging from 4 to 6) or (GGGGS)n (SEQ ID NO: 21) (n is an integer ranging from 2 to 6, preferably 3), where n is the number of repeats. In the amino acid sequence, the number of Gs in the repeat unit and the number of repeats n can be appropriately changed. L can be any of the following:
GGGGSGGGGSGGGGS (SEQ ID NO: 18);
It is particularly preferable that the linking group is a peptide having the amino acid sequence of the following formula (I): By linking the Fc region A of an immunoglobulin to the peptide moiety B derived from an AM or a modified form thereof via the linking group L having the above amino acid sequence, the compound represented by formula (I) of this embodiment can sustainably express adrenomedullin activity in vivo while maintaining the pharmacological action of natural AM.

In formula (I), it is preferable that the Fc region A is linked to the remainder by forming a peptide bond between its C-terminal carboxyl group and the N-terminal α-amino group of the linking group L, and that the peptide portion B is linked to the remainder by forming a peptide bond between its N-terminal α-amino group and the C-terminal carboxyl group of the linking group L. That is, the compound represented by formula (I) of this embodiment has a protein or polypeptide structure as a whole. By having such a structure, the compound represented by formula (I) of this embodiment can have high biocompatibility. Therefore, the compound represented by formula (I) of this embodiment can continuously express adrenomedullin activity in the body while suppressing undesirable side reactions.

A preferred compound of formula (I) is
A is an Fc region of immunoglobulin G1 (IgG1) or an Fc region of immunoglobulin G4 (IgG4),
B is the following:
(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 residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(i) any one of the peptides (a) to (f) above, wherein the C-terminus is amidated; and (j) any one of the peptides (a) to (f) above, wherein a glycine residue is added to the C-terminus;
A peptide portion derived from adrenomedullin or a modified form thereof, which is a peptide selected from the group consisting of:
L is the following:
GGGGSGGGGSGGGGS (SEQ ID NO: 18);
is a linking group consisting of a peptide having an amino acid sequence of
The Fc region A is linked to the remainder by forming a peptide bond between its C-terminal carboxyl group and the N-terminal α-amino group of the linking group L, and the peptide portion B is linked to the remainder by forming a peptide bond between its N-terminal α-amino group and the C-terminal carboxyl group of the linking group L.

More preferred compounds of formula (I) are
A is an Fc region of immunoglobulin G1 (IgG1) or an Fc region of immunoglobulin G4 (IgG4),
B is the following:
(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 residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond; and (i) any of the peptides (a) to (f) whose C-terminus is amidated;
A peptide portion derived from adrenomedullin or a modified form thereof, which is a peptide selected from the group consisting of:
L is the following:
GGGGSGGGGSGGGGS (SEQ ID NO: 18);
is a linking group consisting of a peptide having an amino acid sequence of
The Fc region A is linked to the remainder by forming a peptide bond between its C-terminal carboxyl group and the N-terminal α-amino group of the linking group L, and the peptide portion B is linked to the remainder by forming a peptide bond between its N-terminal α-amino group and the C-terminal carboxyl group of the linking group L.

Further preferred compounds of formula (I) are
A is an Fc region of immunoglobulin G1 (IgG1) or an Fc region of immunoglobulin G4 (IgG4),
B is the following:
(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 residues at positions 16 and 21 form a disulfide bond;
(b) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(c) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(d) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8 and in which the cysteine residues at positions 16 and 21 form a disulfide bond;
(e) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and in which the cysteine residues at positions 14 and 19 form a disulfide bond; and (f) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
A peptide portion derived from adrenomedullin or a modified form thereof, which is a peptide having an amidated C-terminus, selected from the group consisting of:
L is the following:
GGGGSGGGGSGGGGS (SEQ ID NO: 18);
is a linking group consisting of a peptide having an amino acid sequence of
The Fc region A is linked to the remainder by forming a peptide bond between its C-terminal carboxyl group and the N-terminal α-amino group of the linking group L, and the peptide portion B is linked to the remainder by forming a peptide bond between its N-terminal α-amino group and the C-terminal carboxyl group of the linking group L.

Particularly preferred compounds of formula (I) are
(Ea-1) a peptide consisting of the amino acid sequence of SEQ ID NO: 15, or a peptide consisting of the amino acid sequence of SEQ ID NO: 15 and in which the cysteine residues at positions 259 and 264 form a disulfide bond;
(Ea-2) a peptide consisting of the amino acid sequence of SEQ ID NO: 17, or a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and in which the cysteine residues at positions 256 and 261 form a disulfide bond;
(Ea-3) a peptide consisting of the amino acid sequence of SEQ ID NO: 23, or a peptide consisting of the amino acid sequence of SEQ ID NO: 23 and in which the cysteine residues at positions 254 and 259 form a disulfide bond;
(Ea-4) a peptide consisting of the amino acid sequence of SEQ ID NO: 25, or a peptide consisting of the amino acid sequence of SEQ ID NO: 25 in which the cysteine residues at positions 251 and 256 form a disulfide bond;
(Eg) A peptide according to any one of (Ea-1) to (Ea-4), in which the disulfide bond is substituted with an ethylene group;
(Eh) A peptide in which 1 to 15 amino acid residues are deleted, substituted or added in any of peptides (Ea-1) to (Eg);
(Ei) any one of peptides (Ea-1) to (Eh) in which the C-terminus is amidated; and (Ej) any one of peptides (Ea) to (Eh) in which a glycine residue has been added to the C-terminus;
The peptide is selected from the group consisting of:

More particularly preferred compounds of formula (I) are
(Ea-1) a peptide consisting of the amino acid sequence of SEQ ID NO: 15, or a peptide consisting of the amino acid sequence of SEQ ID NO: 15 and in which the cysteine residues at positions 259 and 264 form a disulfide bond;
(Ea-2) a peptide consisting of the amino acid sequence of SEQ ID NO: 17, or a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and in which the cysteine residues at positions 256 and 261 form a disulfide bond;
(Ea-3) a peptide consisting of the amino acid sequence of SEQ ID NO: 23, or a peptide consisting of the amino acid sequence of SEQ ID NO: 23 and in which the cysteine residues at positions 254 and 259 form a disulfide bond;
(Ea-4) a peptide consisting of the amino acid sequence of SEQ ID NO: 25, or a peptide consisting of the amino acid sequence of SEQ ID NO: 25 in which the cysteine residues at positions 251 and 256 form a disulfide bond; and (Ei) a peptide in which the C-terminus is amidated in any one of the peptides (Ea-1) to (Ea-4);
The peptide is selected from the group consisting of:

Particularly preferred compounds of formula (I) are
(Ea-1) a peptide consisting of the amino acid sequence of SEQ ID NO: 15, or a peptide consisting of the amino acid sequence of SEQ ID NO: 15 and in which the cysteine residues at positions 259 and 264 form a disulfide bond;
(Ea-2) a peptide consisting of the amino acid sequence of SEQ ID NO: 17, or a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and in which the cysteine residues at positions 256 and 261 form a disulfide bond;
(Ea-3) a peptide consisting of the amino acid sequence of SEQ ID NO: 23, or a peptide consisting of the amino acid sequence of SEQ ID NO: 23 and in which the cysteine residues at positions 254 and 259 form a disulfide bond; and (Ea-4) a peptide consisting of the amino acid sequence of SEQ ID NO: 25, or a peptide consisting of the amino acid sequence of SEQ ID NO: 25 and in which the cysteine residues at positions 251 and 256 form a disulfide bond;
The peptide is amidated at the C-terminus.

The compound represented by formula (I) of this embodiment having the above characteristics can sustainably express adrenomedullin activity in the body while maintaining the pharmacological action of natural AM and substantially suppressing undesirable side reactions.

In each aspect of the present invention, the compound represented by formula (I) includes not only the compound itself but also its salt. When the compound represented by formula (I) is in the form of a salt, it is preferably a pharma- ceutically acceptable salt. Counterions of the salt of the compound represented by formula (I) include, but are not limited to, cations such as sodium ion, potassium ion, calcium ion, magnesium ion, or substituted or unsubstituted ammonium ion, or chloride ion, bromide ion, iodide ion, phosphate ion, nitrate ion, sulfate ion, carbonate ion, bicarbonate 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, benzoic acid ... Anions such as acid ion, nicotinic acid ion, cinnamic acid ion, ascorbic acid ion, pamoic acid ion, succinic acid ion, salicylic acid ion, bismethylene salicylic acid ion, oxalic acid ion, tartrate ion, malic acid ion, citrate ion, gluconate ion, aspartic acid ion, stearate ion, palmitic acid ion, itaconate ion, glycolic acid ion, glutamic acid ion, benzenesulfonic acid ion, cyclohexylsulfamic acid ion, methanesulfonic acid ion, ethanesulfonic acid ion, isethionate ion, benzenesulfonic acid ion, p-toluenesulfonic acid ion, or naphthalenesulfonic acid ion are preferred. When the compound represented by formula (I) is in the form of a salt with the counter ion, the adrenomedullin activity of the compound can be substantially equivalent to that of natural AM.

In each aspect of the present invention, the compound represented by formula (I) includes not only the compound itself but also a solvate of the compound or a salt thereof. When the compound represented by formula (I) or a salt thereof is in the form of a solvate, it is preferably a pharma- ceutically acceptable solvate. The solvent capable of forming a solvate with the compound or a salt thereof is, but is not limited to, water or an organic solvent such as methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine, acetonitrile, or ethyl acetate. When the compound represented by formula (I) or a salt thereof is in the form of a solvate with the solvent, the adrenomedullin activity of the compound can be substantially equivalent to that of natural AM.

In each embodiment of the present invention, the compounds represented by formula (I) also include mixtures of stereoisomers of the compounds, such as individual enantiomers and diastereomers of the compounds, as well as racemates.

3. Medicinal uses of adrenomedullin derivatives
The compound represented by formula (I) obtainable by the method of one aspect of the present invention, preferably obtained by the method, can continuously express in vivo a biological activity (i.e., adrenomedullin activity) substantially equivalent to that of the parent molecule AM. Therefore, another aspect of the present invention relates to a pharmaceutical comprising the compound represented by formula (I) obtainable by the method of one aspect of the present invention, preferably obtained by the method, as an active ingredient.

When the compound represented by formula (I) of one aspect of the present invention is applied to pharmaceutical use, the compound may be used alone or in combination with one or more pharma- ceutically acceptable ingredients. The pharmaceutical of this aspect can be formulated into various dosage forms commonly used in the art, depending on the desired administration method. Therefore, the pharmaceutical of this aspect can also be provided in the form of a pharmaceutical composition containing the compound represented by formula (I) of one aspect of the present invention and one or more pharma- ceutically acceptable carriers. The pharmaceutical composition of one aspect of the present invention may contain, in addition to the above-mentioned ingredients, one or more pharma- ceutically acceptable carriers, excipients, binders, vehicles, solubilizers, preservatives, stabilizers, swelling agents, lubricants, surfactants, oily liquids, buffers, soothing agents, antioxidants, sweeteners, flavoring agents, and the like.

A pharmaceutical containing the compound represented by formula (I) of one aspect of the present invention as an active ingredient can also be used in combination with one or more other pharmaceutical drugs. In this case, the pharmaceutical of this aspect may be provided in the form of a single pharmaceutical containing the compound represented by formula (I) of one aspect of the present invention and one or more other pharmaceutical drugs, or may be provided in the form of a pharmaceutical combination or kit containing multiple preparations in which the compound represented by formula (I) of one aspect of the present invention and one or more other pharmaceutical drugs are separately formulated. In the case of a pharmaceutical combination or kit form, each preparation can be administered simultaneously or separately (e.g., consecutively).

When the compound represented by formula (I) of one embodiment of the present invention is applied to a medicinal use, the compound represented by formula (I) includes not only the compound itself but also pharma- ceutically acceptable salts of the compound and pharma-ceutically acceptable solvates thereof. The pharma-ceutically acceptable salts of the compound represented by formula (I) of one embodiment of the present invention and pharma-ceutically acceptable solvates thereof are, but are not limited to, preferably the salts or solvates exemplified above. When the compound represented by formula (I) is in the form of the above-mentioned salt or solvate, the compound can be applied to a desired medicinal use.

A medicine containing the compound represented by formula (I) of one embodiment of the present invention as an active ingredient can similarly prevent or treat various symptoms, diseases and/or disorders that are prevented or treated by AM. The symptoms, diseases and/or disorders include, but are not limited to, the following:

(1) Cardiovascular diseases: heart failure, pulmonary hypertension, arteriosclerosis obliterans, Buerger's disease, myocardial infarction, lymphedema, Kawasaki disease, myocarditis, arrhythmia (e.g., arrhythmia after catheter ablation surgery), atrial fibrillation, aortitis, pulmonary hypertension, hypertension, organ damage due to hypertension, peripheral vascular disease, and arteriosclerosis.
(2) Kidney/hydrolyte system diseases: renal failure and nephritis.
(3) Brain and neurological diseases: cerebral infarction, dementia, vascular dementia, Alzheimer's disease, and encephalitis.
(4) Genitourinary diseases: erectile dysfunction (ED).
(5) Gastrointestinal diseases: inflammatory diseases (e.g., inflammatory bowel disease or Crohn's disease), ulcerative diseases (e.g., ulcerative colitis), intestinal Behcet's disease, hepatitis, liver fibrosis, cirrhosis, and liver failure.
(6) Orthopedic diseases: arthritis.
(7) Endocrine metabolic diseases: diabetes and diabetic organ damage (e.g., diabetic nephropathy or diabetic retinopathy), and primary aldosteronism.
(8) Respiratory diseases: bronchial asthma, emphysema, pulmonary fibrosis, pneumonia, acute bronchitis, chronic bronchitis, and acute respiratory distress syndrome (ARDS).
(9) Immunologic diseases: diseases related to the complement system (e.g., C3 nephropathy).
(10) Other diseases: sepsis, septic shock, autoimmune diseases, multiple organ failure, bedsores, wound healing, and alopecia.

The compound represented by formula (I) of one embodiment of the present invention has a structure in which AM, a natural physiologically active peptide, and the Fc region of immunoglobulin are linked via a peptide linking group. Therefore, the compound represented by formula (I) of one embodiment of the present invention is safe and low toxic. Therefore, a medicine containing the compound represented by formula (I) of one embodiment of the present invention as an active ingredient can be applied to various subjects requiring prevention or treatment of the above-mentioned symptoms, diseases and/or disorders. The subject is preferably a human or non-human mammalian subject or patient (e.g., a warm-blooded animal such as a pig, a dog, a cow, a rat, a mouse, a guinea pig, a rabbit, a chicken, a sheep, a cat, a monkey, a Mandrill or a chimpanzee). By administering the medicine of this embodiment to the subject, various symptoms, diseases and/or disorders that are prevented or treated by AM can be prevented or treated.

As used herein, "prevention" means substantially preventing the occurrence (onset or manifestation) of a symptom, disease, and/or disorder. Also, as used herein, "treatment" means inhibiting (e.g. inhibiting progression), alleviating, repairing, and/or curing a symptom, disease, and/or disorder that has occurred (onset or manifestation).

The compound represented by formula (I) of one aspect of the present invention can be used to prevent or treat the symptoms, diseases, and/or disorders (e.g., cardiovascular disease, brain/nerve disease, or digestive disease) in a subject having the symptoms, diseases, and/or disorders described above. Therefore, the pharmaceutical of this aspect is preferably a pharmaceutical for use in the prevention or treatment of the symptoms, diseases, and/or disorders described above, and more preferably a pharmaceutical for use in the prevention or treatment of heart failure, acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular disease, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, sepsis, or septic shock. The present invention also relates to a preventive or therapeutic agent for cardiovascular disease, brain/nerve disease, or digestive disease, which contains the compound represented by formula (I) of one aspect of the present invention as an active ingredient. By using the compound represented by formula (I) according to one aspect of the present invention for the prevention or treatment of the symptoms, diseases and/or disorders described above, the symptoms, diseases and/or disorders can be prevented or treated continuously.

The compound represented by formula (I) of one aspect of the present invention can be used in a subject having the above-described symptoms, diseases, and/or disorders (e.g., cardiovascular disease, inflammatory disease, vascular disease, or renal disease) for the prevention or treatment of the symptoms, diseases, and/or disorders. Therefore, another aspect of the present invention is a method for preventing or treating the above-described symptoms, diseases, and/or disorders, comprising administering to a subject in need of prevention or treatment of the above-described symptoms, diseases, and/or disorders an effective amount of a compound represented by formula (I) of one aspect of the present invention or a pharma- ceutical acceptable salt thereof, or a pharma-ceutical acceptable hydrate thereof. The symptoms, diseases and/or disorders are preferably cardiovascular diseases, brain and nerve diseases, digestive diseases, endocrine and metabolic diseases, respiratory diseases or other diseases, and more preferably heart failure, acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular diseases, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, sepsis or septic shock. The symptoms, diseases and/or disorders can be prevented or treated by administering a compound represented by formula (I) according to one aspect of the present invention to a subject in need of prevention or treatment of the symptoms, diseases and/or disorders.

Another aspect of the present invention is a compound represented by formula (I) of one aspect of the present invention or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable hydrate thereof, for use in the prevention or treatment of the symptoms, diseases, and/or disorders described above. Yet another aspect of the present invention is the use of a compound represented by formula (I) of one aspect of the present invention or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable hydrate thereof, in the manufacture of a medicament for use in the prevention or treatment of the symptoms, diseases, and/or disorders described above. Yet another aspect of the present invention is the use of a compound represented by formula (I) of one aspect of the present invention or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable hydrate thereof, for use in the prevention or treatment of the symptoms, diseases, and/or disorders described above. The symptoms, diseases and/or disorders are preferably cardiovascular diseases, brain and nerve diseases, digestive diseases, endocrine and metabolic diseases, respiratory diseases or other diseases, and more preferably heart failure, acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular diseases, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, sepsis or septic shock. By using the compound or medicine represented by formula (I) according to one aspect of the present invention for the prevention or treatment of the symptoms, diseases and/or disorders described above, the symptoms, diseases and/or disorders can be prevented or treated continuously.

The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

<Experiment I: Construction of a recombinant gene encoding an adrenomedullin derivative>
[Experiment I-1: Design and analysis of recombinant genes]
A recombinant gene for an adrenomedullin derivative having the following structure was designed based on the Fc region of immunoglobulin G1 (IgG1), the Fc region of immunoglobulin G4 (IgG4), human adrenomedullin (h.AM(1-52) or h.AM(6-52)), and the following linking group: For the genes used, identification of restriction enzyme sites in the base sequence, confirmation of the base sequence, design of primers, and analysis of the amino acid sequence, molecular weight, isoelectric point, etc. of the corresponding protein were performed using genetic information processing software GENETIX Ver.13 (Genetex).
Example 1: (IgG1 Fc region) + (Linker S) + (h.AM(1-52)-Gly); (SEQ ID NOs: 14 and 15)
Example 2: (IgG1 Fc region) + (Linker S) + (h.AM(6-52)-Gly); (SEQ ID NOs: 22 and 23)
Example 3: (IgG4 Fc region) + (Linker S) + (h.AM(1-52)-Gly); (SEQ ID NOs: 16 and 17)
Example 4: (IgG4 Fc region) + (Linker S) + (h.AM(6-52)-Gly); (SEQ ID NOs: 24 and 25)
Linker S:
Amino acid sequence: GGGSGGGGSGGGGGS; (SEQ ID NO: 18)
Base sequence: GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA GGA GGA GGA GGA TCA
(SEQ ID NO: 19)

[Experiment I-2: Preparation of DNA fragments]
DNA fragments encoding the Fc region of IgG1, the Fc region of IgG4, and human adrenomedullin (h.AM(1-52) or h.AM(6-52)) were cloned. The Fc region of IgG1 was derived from Ellison et al. (Ellison JW, Nucleic Acids Res. 1982;10(10);4071-9) and GenBank: JN222933. The Fc region of IgG4 was derived from Labrijn et al. (Labrijn AF, J Immunol. 2011;187(6):3238-46.). Human adrenomedullin was derived from Kitamura et al. (Kitamura K et.al.BBRC.1993;194(2);720-5.). The vector and DNA fragment to be inserted were amplified by PCR using the In-Fusion HD Cloning Kit (Takara). The PCR reaction for the vector was carried out at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 25 seconds for 35 cycles. The PCR reaction for the fragment to be inserted was carried out at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 10 seconds for 35 cycles. The cloning enhancer from the kit was then added, and the reaction was carried out at 37°C for 15 minutes and 80°C for 15 minutes to decompose the template and primers.

[Experiment I-3: Ligation]
Using the In-Fusion HD Cloning Kit (Takara), the amplified plasmid vector DNA, the DNA fragment of the target protein, and the enzyme premix were added and reacted at 50°C for 15 minutes to produce a plasmid containing the sequence of the target protein.

[Experiment I-4: Mass production of plasmids]
The ligated plasmid was transformed into Stellar Competent cells (Takara) according to the protocol of the In-Fusion HD Cloning Kit (Takara). The cells were cultured with shaking at 37°C. The plasmid was then purified using the Plasmid Maxi Kit (Qiagen).

<Experiment II: Preparation of adrenomedullin derivative protein>
[Experiment II-1: Transformation and induction of target protein expression]
1.0 mg of plasmid was transfected into 1 L of HEK293 (Expi293F) cells at 2.5 x ¹⁰⁶ cells/mL. Enhancer was added the next day, and the cells and culture supernatant were collected. Gene transfection and protein expression induction were performed using a gene transfer reagent and enhancer set (NeoFectionEN-1) (Astec Co., Ltd.). The amount of AM derivatives contained in the cells or culture supernatant was measured using a specific fluorescent immunoassay (Tosoh Corporation) using two types of antibodies with different recognition sites. The first antibody binds to a cyclic structure in which the cysteine residues at positions 16 and 21 of AM form a disulfide bond, and the second antibody binds to the C-terminal portion of AM. By using these two antibodies, it is possible to quantitatively analyze proteins corresponding to both inactive and active AM (tAM) and only active AM (mAM) that can express activity (Ohta H et al., One-step direct assay for mature-type adrenomedullin with monoclonal antibodies. Clin Chem., February 1999, Vol. 45(2), p. 244-51; Kubo K et al., Biological properties of adrenomedullin conjugated with polyethylene glycol. Peptides, July 2014, Vol. 57, p. 118-21. doi: 10.1016/j.peptides.2014.05.005. Epub 2014 May 27.). The amount of AM derivative contained in the cells or culture supernatant is shown in Table 1. In the table, the amount of AM derivative is the value per 1 L of cell culture medium.

As shown in Table 1, the AM derivatives of Examples 1 to 4 accumulated in greater amounts in the culture supernatant than in the cells. In addition, from the results of quantitative analysis of the AM derivatives using two antibodies, it is presumed that the glycine residue added to the C-terminus of each of the AM derivatives of Examples 1 to 4 was converted to a C-terminal amide group by the action of a C-terminal amidating enzyme. Comparing Examples 2 and 4, the protein expression level of Example 4 having IgG4 was lower than that of Example 2 having IgG1. In addition, the cell viability of the host cells in which Examples 3 and 4 having IgG4 were induced to express was also lower than that of the host cells in which Examples 1 and 2 were induced to express. From the above results, it was revealed that, unlike the case in which the AM derivatives were prepared using E. coli as the host cell (Patent Document 10), the AM derivatives obtained by the method of this experiment were secreted into the culture medium of the host mammalian cells in the form of a peptide with an amidated C-terminus.

[Experiment II-2: Purification of recombinant proteins using ion exchange columns]
The culture supernatants of Examples 1 to 4 obtained in Experiment II-1 were purified by a stepwise method using an ion exchange column (SP). The culture supernatants of Examples 1 and 2 were eluted with 50 mM acetate (sodium) buffer + 300 mM NaCl (pH 5.5), and the culture supernatants of Examples 3 and 4 were eluted with 50 mM acetate (sodium) buffer + 300 mM NaCl (pH 6.0).

[Experiment II-3: Purification of recombinant proteins using affinity columns]
The recombinant proteins of Examples 1 to 4 obtained in Experiment II-2 were purified using a HiTrap Protein A HP column having specific binding ability to the Fc region of IgG and an Ab Buffer Kit (GE Healthcare) according to the manufacturer's protocol. The recombinant proteins purified with Protein A were diluted with 20 mM citrate buffer (pH 7.2) and then concentrated and solvent-exchanged using an Amicon Ultra-15 Ultracel-10K (Merck Millipore).

<Experiment III: Example of use of adrenomedullin derivative>
[Experiment III-1: Effect of adrenomedullin derivatives on increasing intracellular cAMP concentration]
It is known that the physiological action of AM is expressed through an increase in the intracellular cAMP concentration (see Non-Patent Document 1). Therefore, the AM derivatives of Examples 1 to 4 were added to a cultured cell line (HEK293 cell line) expressing the AM receptor, and the amount of intracellular cAMP produced was measured. In the presence of 0.5 mM IBMX, 10 ^-8 to 10 ^-6 M of any of the AM derivatives of Examples 1 to 4, or adrenomedullin (h.AM(1-52)) as a positive control AM, were added to confluent HEK293 cells, and incubated for 15 minutes. Then, the intracellular cAMP concentration in the HEK293 cells of each test group was measured using a cAMP measurement ELISA kit (GE Healthcare, #RPN2251). The dose-response curve of the concentration of the added AM or AM derivative and the intracellular cAMP concentration is shown in FIG. 1. In the figure, the horizontal axis is the concentration of the added AM or AM derivative (M), and the vertical axis is the intracellular cAMP concentration (fmol/well (plate)).

As shown in Figure 1, for both IgG1- and IgG4-linked AM derivatives, Examples 2 and 4 having N-terminal deleted AM h.AM(6-52) tended to have relatively higher activity than Examples 1 and 3 having full-length AM, i.e., h.AM(1-52). In particular, at high concentrations, Examples 2 and 4 showed high activity almost equivalent to that of natural AM. Moreover, Example 4 showed high activity even at low concentrations.

As Comparative Examples 1 to 4, AM derivatives having the same amino acid sequences as those in Examples 1 to 4 were prepared using E. coli as the host cell based on Patent Document 10. Using the AM derivatives of Comparative Examples 1 to 4, the intracellular cAMP concentration was measured in HEK293 cells to which AM (h.AM(1-52)) or an AM derivative had been added, in the same manner as described above. The maximum activity when AM was added was taken as 100%, and the results when 10 ^-6 M or 10 ^-7 M of any of the AM derivatives of Comparative Examples 1 to 4 and Examples 1 to 4 were added were calculated as relative values. The results are shown in Table 2.

As shown in Table 2, the AM derivatives of Examples 1 to 4 exhibited substantially the same activity (Example 2) or significantly higher activity (Examples 1, 3, and 4) compared to the AM derivatives of Comparative Examples 1 to 4, all of which have the same amino acid sequence.

[Experiment III-2: Changes in blood concentration over time after subcutaneous administration of adrenomedullin derivative]
8-week-old Wistar rats (approximately 300 g) were subcutaneously administered 30 nmol/kg of a physiological saline solution of the AM derivative of Example 2 or 4. Before administration (day 0) and every day from 1 day after administration to 14 days after administration, the rats were anesthetized with isoflurane by inhalation. Under anesthesia, blood was collected from the tail vein using a simple tube (Eiken) with EDTA-2Na and aprotinin added. The blood obtained was centrifuged at 2,000×g for 1 minute using a simple centrifuge (Eiken) to obtain plasma. The concentration of the AM derivative in the plasma was measured by ELISA. The change over time of the blood concentration of the AM derivative of Example 2 or 4 when subcutaneously administered is shown in FIG. 2. A is a graph showing the change over time of the blood concentration of the AM derivative corresponding to mAM, and B is a graph showing the change over time of the blood concentration of the AM derivative corresponding to tAM. In the figure, the horizontal axis is the period (days) after administration, and the vertical axis is the blood concentration (pM) of the AM derivative. Table 3 shows the blood concentrations of the AM derivatives of Examples 2 and 4 14 days after subcutaneous administration.

As shown in Figure 2 and Table 3, when either the AM derivative of Example 2 or 4 was administered subcutaneously, sufficient amounts of the AM derivative were present in the blood to be in the therapeutic range even 14 days after administration.

[Experiment III-3: Antihypertensive effect of adrenomedullin derivatives in spontaneously hypertensive rats (SHR)]
The effect of subcutaneous administration of the AM derivative of Example 4 on blood pressure rise in SHR was examined by the following procedure. A single subcutaneous administration of 50 nmol/kg of the AM derivative of Example 4 in physiological saline was administered to 8-week-old SHR fed an 8% high-salt diet. As a control group, the same amount of physiological saline was administered subcutaneously once. Blood pressure was measured by tail cuff before administration (day 0) and 1, 3, 6, 9, and 12 days after administration. From the measured values, the blood pressure rise rate was calculated. At the end of the experiment (12 days after administration), blood was collected by decapitation into a tube containing EDTA-2Na. The blood obtained was centrifuged at 3,500 rpm and 4°C for 10 minutes to obtain plasma. The AM concentration of the derivative in the plasma was measured by ELISA. The effect of subcutaneous administration of the AM derivative of Example 4 on blood pressure rise in SHR is shown in Figure 3. A is a graph showing the change over time in systolic blood pressure (SBP), and B is a graph showing the change over time in diastolic blood pressure (DBP). In the figure, the horizontal axis indicates the period (days) after administration, and the vertical axis indicates blood pressure (mmHg). * indicates that the p-value calculated by Student's t-test (n=5) is less than 0.05 compared to the control group administered physiological saline. Table 4 shows the blood pressure of SHR 12 days after subcutaneous administration of the control or the AM derivative of Example 4. FIG. 4 also shows the blood concentration of the AM derivative in SHR 12 days after subcutaneous administration of the control or the AM derivative of Example 4. A is a graph showing the blood concentration of the AM derivative corresponding to mAM, and B is a graph showing the blood concentration of the AM derivative corresponding to tAM. In the figure, the vertical axis indicates the blood concentration (pM) of the AM derivative corresponding to mAM or tAM.

As shown in Figure 3 and Table 4, in the group administered the AM derivative of Example 4, a suppression of blood pressure rise of 10 mmHg or more was confirmed in both SBP and DBP, compared to the control group administered physiological saline. In addition, as shown in Figure 4, subcutaneous administration of the AM derivative of Example 4 resulted in sufficient AM derivative present in the blood to be in the therapeutic range even 12 days after administration.

[Experiment III-4: Tissue transfer after subcutaneous administration of adrenomedullin derivatives]
8-week-old Wistar rats (approximately 300 g) were subcutaneously administered 30 nmol/kg of the AM derivative of Example 2 or 4 in physiological saline solution, four rats each. After 7 days (two rats each) and 14 days (two rats each), tissues of the brain, lung, heart, kidney, adrenal gland, liver, pancreas, spleen, large intestine, small intestine and stomach were collected, flash frozen in liquid nitrogen and stored at -80°C until use. The stored tissues were crushed in the frozen state using a homogenizer in phosphate buffered saline (PBS) containing inhibitors. PBS containing inhibitors was prepared by diluting protease inhibitor cocktail (Nacalai Tesque) 50-fold with cooled PBS (Takara). 1 mL of PBS containing inhibitors was used for approximately 0.3 g of tissue. The crushed tissues were centrifuged at 20,000×g and 4°C for 20 minutes, and the supernatant was obtained as a tissue extract. The AM derivative concentration in the tissue extract was then measured by ELISA. After the measurement, the total protein amount in the tissue extract was measured using the Pierce BCA Protein Assay Kit (Thermo). The measured values of the AM derivative concentration were corrected by the total protein amount.

The concentration of the AM derivatives corresponding to tAM of Example 2 or 4 in each tissue extract is shown in Table 5, and the concentration of the AM derivatives corresponding to mAM of Example 2 or 4 in each tissue extract is shown in Table 6. The data in the tables are the average value of two rats each. As shown in Tables 5 and 6, the migration of the AM derivatives of Examples 2 and 4 was confirmed in all tissues measured, and it was revealed that they remained in the tissues even after 14 days. There was no significant difference in the results between the AM derivatives of Examples 2 and 4. In addition, the values in the kidney were the highest in both cases, followed by the small intestine, lung, large intestine, heart and stomach. Furthermore, there was no significant difference in the results between the concentration values of the AM derivatives corresponding to tAM and the concentration values of the AM derivatives corresponding to mAM. Since mAM reflects the active form of AM, it was thought that both Examples 2 and 4 existed in the active form in the tissues.

The present invention is not limited to the above-described embodiments, but includes various modified examples. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and are not necessarily limited to those having all of the configurations described. In addition, it is possible to add, delete, and/or replace part of the configuration of each embodiment with other configurations.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (1)

Formula (I):
ALB (I)
[Wherein,
A is an immunoglobulin Fc region,
B is a peptide moiety derived from adrenomedullin or a modified form thereof,
L is a linking group consisting of a peptide having any amino acid sequence.
A method for producing a compound represented by the following formula (I), a salt thereof, or a hydrate thereof:
The compound represented by formula (I) is
(Ea-1) a peptide consisting of the amino acid sequence of SEQ ID NO: 15, or a peptide consisting of the amino acid sequence of SEQ ID NO: 15 and in which the cysteine residues at positions 259 and 264 form a disulfide bond;
(Ea-2) a peptide consisting of the amino acid sequence of SEQ ID NO: 17, or a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and in which the cysteine residues at positions 256 and 261 form a disulfide bond;
(Ea-3) a peptide consisting of the amino acid sequence of SEQ ID NO: 23, or a peptide consisting of the amino acid sequence of SEQ ID NO: 23 and in which the cysteine residues at positions 254 and 259 form a disulfide bond; and
(Ea-4) a peptide consisting of the amino acid sequence of SEQ ID NO: 25, or a peptide consisting of the amino acid sequence of SEQ ID NO: 25 in which the cysteine residues at positions 251 and 256 form a disulfide bond;
The peptide is selected from the group consisting of:
an expression step of mass-expressing the compound in HEK293 cells, which are host mammalian cells capable of producing the compound;
Including,
a refolding step in which the compound expressed in large quantities in the expression step is refolded; and
A C-terminal amidation step in which the C-terminus of the compound mass-expressed in the expression step is amidated.
The method, which does not include

Images