JP7498882B2 - Treatment for neurodegenerative diseases caused by abnormal protein accumulation - Google Patents

Description

The present invention relates to a therapeutic and/or preventive agent for an abnormal protein accumulation neurodegenerative disease, which comprises adrenomedullin or a modified form thereof having adrenomedullin activity, or a derivative or a salt thereof. The present invention also relates to a therapeutic and/or preventive method for the above disease, which comprises administering adrenomedullin or a modified form thereof having adrenomedullin activity, or a derivative or a salt thereof to a subject suffering from an abnormal protein accumulation neurodegenerative disease.

Against the backdrop of the accelerating aging of society in recent years, the number of patients suffering from neurodegenerative diseases caused by the accumulation of abnormal proteins (hereinafter referred to as "abnormal protein accumulation neurodegenerative diseases") (e.g. Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS), Huntington's disease, age-related macular degeneration, frontotemporal dementia (FTD), multiple sclerosis (MS), etc.) is rapidly increasing. Among these, the number of patients suffering from Alzheimer's disease and age-related macular degeneration, which are neurodegenerative diseases that impose a heavy social burden, currently amounts to tens of millions worldwide.

Alzheimer's disease (hereinafter referred to as "AD") is a progressive dementia that begins with a decline in short-term memory and mild learning disabilities, and is accompanied by higher brain dysfunction, particularly visuospatial agnosia, ideational apraxia, and constructional apraxia, and ultimately leads to motor disorders and personality destruction. Currently, although there are drugs such as cholinesterase inhibitors and N-Methyl-D-Aspartate (NMDA) receptor antagonists, no definitive treatment has been found. Age-related macular degeneration (hereinafter referred to as "AMD") is largely divided into two types, exudative and atrophic. Vascular endothelial growth factor (hereinafter referred to as "VGEF") inhibitors are available for the exudative type, but for the atrophic type, no effective drugs exist, although new drug candidates have been developed.

In this context, the relationship between Alzheimer's disease and the complement system has recently been attracting attention. Specifically, when soluble oligoamyloid beta begins to accumulate in synapses through polymerization, it directly activates C1q, and C3, a complement component activated by C1q, is first deposited in synapses. Next, microglial cells gather around the deposited C3, causing pruning and loss of synapses, resulting in the progression of Alzheimer's disease (Non-Patent Document 1). It is also becoming clear that the complement system, centered on C3, may be involved in AMD, which may cause retinal degeneration, but the use of complement system inhibitors has not yet achieved clinical effectiveness (Non-Patent Document 2).

Although mechanisms related to the complement system as described above have been proposed, no compounds that could be effective therapeutic agents for Alzheimer's disease or age-related macular degeneration have yet been discovered.

Soyon Hong et al., Science 2016 May 6; 352(6286):712-716 Heping Xu et al., European Journal of Pharmacology 787 (2016) 94-104

An object of the present invention is to provide an agent for treating and/or preventing an abnormal protein accumulation neurodegenerative disease, comprising adrenomedullin or a modified form thereof having adrenomedullin activity, or a derivative or a salt thereof. Another object of the present invention is to provide a fundamental method for treating and/or preventing the disease, comprising administering adrenomedullin or a modified form thereof having adrenomedullin activity, or a derivative or a salt thereof to a subject suffering from an abnormal protein accumulation neurodegenerative disease.

It has been pointed out that adrenomedullin (hereinafter referred to as "AM") may mediate neurodegeneration in Alzheimer's disease and thus worsen the disease (Molecular Neurobiology (2018) 55:8799-8814). This was based on the fact that the concentration of AM in the brain and the number of microglial cells increase in correlation with each other. Regarding the conclusion of the paper, the inventors first considered that although it is true that microglial cells proliferate in the disease, the concentration of AM, which is thought to control homeostasis in the body, in the brain may be high in order to improve the above-mentioned condition. In other words, considering the ability of AM to promote the degradation of C3b, they came up with the idea that in the body, it may actually have the effect of calming down the activity of microglia via complement and suppressing the progression of AD. As a result of intensive research, it was surprisingly discovered that AM exerts a therapeutic effect against AD caused by neurodegeneration resulting from the accumulation of amyloid beta in the body that is equal to or greater than that of an existing cholinesterase inhibitor (donepezil), leading to the completion of the present invention.

That is, the present invention relates to the following.
[1] A therapeutic and/or preventive agent for an abnormal protein accumulation neurodegenerative disease, comprising adrenomedullin or a modified form thereof having adrenomedullin activity, or a salt thereof.
[2] The adrenomedullin or a modified form thereof having adrenomedullin activity is a derivative of adrenomedullin, and the derivative has the following formula (I):

[Wherein,
A is a modifying group comprising one or more polyethylene glycol groups;
B is a peptide moiety derived from adrenomedullin or a modification thereof having adrenomedullin activity,
However, peptide portion B is linked to the remainder by a covalent bond between the nitrogen atom of its N-terminal α-amino group and the carbon atom of a methylene group.
The agent according to [1], which is a compound represented by the following formula (I):
[3] A is represented by the following formula (II):

[Wherein,
a is an integer equal to or greater than 1;
m is an integer equal to or greater than 1;
^L1 is an (m+1)-valent linear or branched linking group, provided that when there are multiple ^L1s , the multiple ^L1s may be the same or different from each other;
^L2 and L2 ^' each independently represent a bond or a divalent linking group, provided that when there are multiple L2 ^' , the multiple L2 ^' may be the same or different from each other;
_M1 is represented by the following formula (III):

[Wherein,
n is an integer equal to or greater than 1,
** indicates the bonding position with ^L1 ,
# is the bonding position to O or L2 ^' .
In the case where M ¹ is a plurality of M 1, the plurality of M ¹ may be the same or different from each other,
^M2 is a bond or a polyethylene glycol group represented by formula (III), provided that when there are multiple ^M2 , the multiple ^M2 may be the same or different from each other;
R ¹ is hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C 2 -C ₂₀ alkenyl, substituted or unsubstituted C ₂ -C ₂₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₄ -C 20 cycloalkenyl, substituted or unsubstituted C ₄ -C ₂₀ cycloalkynyl, substituted or unsubstituted _3-6 membered heterocycloalkyl, substituted or unsubstituted C ₇ -C ₂₀ cycloalkylalkyl, substituted or unsubstituted 3-6 membered heterocycloalkyl-C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₄ -C ₂₀ aryl, substituted _or unsubstituted C ₅ -C ₂₀ arylalkyl, substituted or unsubstituted 5-15 membered heteroaryl, substituted or unsubstituted 5-15 membered heteroaryl-C ₁ -C ₂₀ alkyl, or substituted or unsubstituted acyl;
* indicates the position of attachment to the remainder.
The agent according to [2], wherein the modifying group is represented by the following formula:
[4] A is represented by the following formula (V), (VI), (VII) or (VIII):

[Wherein,
a is an integer equal to or greater than 1;
M ³ , M ^{3 '} , M ^{3 ''} , M ^{3 '''} and M ^{3 ''''} each independently represent a bond or a group represented by the following formula (III):

[Wherein,
n is an integer equal to or greater than 1,
** indicates the position of binding to R ³ , R ^3′ or CH;
# is the bond position to O.
wherein when there are a plurality of M3, ^M3 ', M3 ^'' , M3 ^{'' '} and M3 ^'''' , the plurality of ^M3 , M3 ^' , M3 ^'' , M3 ^''' and M3 ^'''' may be the same or different, and at least one of ^M3 , M3 ^' , M3 ^'' , M3 ^{''' and M3'' ''} is a polyethylene glycol group represented by formula (III),
R ¹ , R ^1' , R ^1'' and R ^1''' are each independently selected from hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkenyl, substituted or unsubstituted C ₂ -C ₂₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₄ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ⁴ -C ₂₀ cycloalkynyl, substituted or unsubstituted 3- to 6-membered heterocycloalkyl, substituted or unsubstituted C ₇ -C ₂₀ cycloalkylalkyl, substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₄ -C ₂₀ aryl, substituted or unsubstituted C ₅ -C ₂₀ arylalkyl, substituted or unsubstituted 5- to 15-membered heteroaryl, substituted or unsubstituted 5- to 15-membered heteroaryl-C ₁ -C ₂₀ alkyl, or substituted or unsubstituted acyl;
R ² is a bond, a substituted or unsubstituted C ₁ -C ₂₀ alkylene, a substituted or unsubstituted C ₂ -C ₂₀ alkenylene, a substituted or unsubstituted C ₂ -C ₂₀ alkynylene, a substituted or unsubstituted C 3 -C 20 cycloalkylene, a substituted or unsubstituted C ₄ -C ₂₀ cycloalkenylene, a substituted or unsubstituted C ₄ -C ₂₀ cycloalkynylene, a substituted or unsubstituted _3- to 6-membered heterocycloalkylene, a substituted or unsubstituted C ₇ -C ₂₀ cycloalkylalkylene, a substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C ₁ -C ₂₀ alkylene, a substituted or unsubstituted C ₄ -C ₂₀ arylene, a substituted or unsubstituted C 5 -C ₂₀ arylalkylene, a substituted or unsubstituted _5- to 15 _- membered heteroarylene, or a substituted or unsubstituted 5- to 15-membered heteroaryl-C ₁ -C ₂₀ alkylene (wherein the said groups may contain one or more heteroatoms, amide groups (-CO-NH-), ester groups (-CO-O-), or urethane groups (-O-CO-NH-), amide groups (-CO-NH-), ester groups (-CO-O-), or urethane groups (-O-CO-NH-);
R ³ , R ^3′ , and R ^3″ are each independently a bond, a substituted or unsubstituted C ₁ -C ₂₀ alkylene, a substituted or unsubstituted C ₂ -C ₂₀ alkenylene, a substituted or unsubstituted C ₂ -C ₂₀ alkynylene, a substituted or unsubstituted C ₃ -C ₂₀ cycloalkylene, a substituted or unsubstituted C ₄ -C ₂₀ cycloalkenylene, a substituted or unsubstituted C ₄ -C ₂₀ cycloalkynylene, a substituted or unsubstituted 3- to 6-membered heterocycloalkylene, a substituted or unsubstituted C ₇ -C ₂₀ cycloalkylalkylene, a substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C ₁ -C ₂₀ alkylene, a substituted or unsubstituted C ₄ -C ₂₀ arylene, a substituted or unsubstituted C ₅ -C _arylalkylene , substituted or unsubstituted 5-15 membered heteroarylene, or substituted or unsubstituted 5-15 membered heteroaryl-C ₁ -C ₂₀ alkylene (which may contain one or more heteroatoms, amide groups (-CO-NH-), ester groups (-CO-O-), or urethane groups (-O-CO-NH-)), amide groups (-CO-NH-), ester groups (-CO-O-), or urethane groups (-O-CO-NH-), provided that when R ³ , R ^3' , and R ^3'' are multiple, the multiple R ³ , R ^3' , and R ^3'' may be the same or different from each other;
* indicates the position of attachment to the remainder.
The agent according to [2] or [3], wherein the modifying group is represented by the following formula:
[5] The agent according to [3] or [4], wherein the polyethylene glycol groups represented by formula (III) have a weight-average molecular weight in total ranging from 1 to 100 kDa.
[6] The adrenomedullin or a modified form thereof having adrenomedullin activity is any one 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) A peptide according to (ii), in which the disulfide bond is replaced by an ethylene group, and which has adrenomedullin activity;
(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), and which has adrenomedullin activity.
The agent according to any one of [1] to [5], which is a peptide 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.
[7] The adrenomedullin or a modified form thereof having adrenomedullin activity is any one 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;
The agent according to [6], which is a peptide selected from the group consisting of (v) peptides of (i) or (ii) in which the C-terminus is amidated, and (vi) peptides of (i) or (ii) in which a glycine residue has been added to the C-terminus.
[8] The adrenomedullin or a modified form thereof having adrenomedullin activity is any one of the following:
(iv') a peptide in which the amino acid residues at positions 1 to 15, 1 to 10, or 1 to 5 from the N-terminus are deleted in any one of the peptides (i) to (iii), and which has adrenomedullin activity;
The agent according to [6], which is a peptide selected from the group consisting of peptides (v) and (iv') having an amidated C-terminus, and peptides (vi) and (iv') having a glycine residue added to the C-terminus.
[9] The agent according to any one of [1] to [8], wherein the abnormal protein is amyloid beta.
[10] The agent according to any one of [1] to [9], wherein the abnormal protein accumulation neurodegenerative disease is Alzheimer's disease.
[11] The agent according to any one of [1] to [10], which is an agent for treating and/or preventing neurodegeneration caused by accumulation of amyloid beta in Alzheimer's disease.
[12] The agent according to any one of [1] to [11], which is used in combination with one or more agents selected from the group consisting of a cholinesterase inhibitor, an NMDA receptor antagonist, and a tau protein remover and production inhibitor.
[13] The agent according to any one of [1] to [12], which is for nasal administration.
[14] The method according to any one of [1] to [13], wherein 0.06 to 600 μg/kg of adrenomedullin is administered per subject.
[15] A method for treating and/or preventing an abnormal protein accumulation neurodegenerative disease, comprising administering to a subject an effective amount of adrenomedullin or a modified form thereof having adrenomedullin activity, or a salt thereof.

According to the present invention, a therapeutic and/or prophylactic agent for abnormal protein accumulation neurodegenerative diseases can be provided, which contains adrenomedullin or a modified form thereof having adrenomedullin activity, or a derivative or a salt thereof. The agent of the present invention contains adrenomedullin as an active ingredient, and acts in particular on Alzheimer's disease or age-related macular degeneration based on neurodegeneration caused by accumulation of amyloid β, by a mechanism of action different from that of cholinesterase inhibitors and NMDA receptor antagonists, which are existing therapeutic agents for Alzheimer's disease, or VEGF inhibitors, which are existing therapeutic agents for age-related macular degeneration, and is considered to be a fundamental therapy, and therefore can be a new drug for patients who could not expect effects from the above-mentioned drugs, and can be used in combination with existing drugs.
Furthermore, according to the present invention, a method for treating and/or preventing an abnormal protein accumulation neurodegenerative disease can be provided, which comprises administering adrenomedullin or a modified form thereof having adrenomedullin activity, or a derivative or a salt thereof, to a subject suffering from the disease. The method for treating and/or preventing the disease of the present invention comprises administering the adrenomedullin or the like to a subject suffering from the disease, and in particular, in Alzheimer's disease or age-related macular degeneration based on neurodegeneration caused by accumulation of amyloid β, the adrenomedullin acts by a different mechanism of action from existing Alzheimer's disease treatment agents such as cholinesterase inhibitors and NMDA receptor antagonists, or existing age-related macular degeneration treatment agents such as VEGF inhibitors, and is considered to be a fundamental therapy, so that it can be a new method for treating and/or preventing the disease for patients who could not expect any effect from the above-mentioned drugs, and can be used in combination with existing drugs.

Figure 1 shows the results of the Y-maze test. In the figure, **p<0.01 indicates a significant difference compared to the sham-operated group (Student's t-test), ††p<0.01 indicates a significant difference compared to the vehicle control group (Dunnett's test), and ##p<0.01 indicates a significant difference compared to the vehicle control group (Student's t-test).

1. Agent for treating and/or preventing abnormal protein accumulation neurodegenerative disease The agent for treating and/or preventing abnormal protein accumulation neurodegenerative disease of the present invention (hereinafter also referred to as "agent of the present invention") contains adrenomedullin as an active ingredient. As used herein, "abnormal protein accumulation neurodegenerative disease" refers to a progressive disease in which neurons in the central nervous system gradually degenerate due to the accumulation of aggregates of proteins misfolded into abnormal structures (i.e., abnormal proteins), resulting in dysfunction and cell death.

Examples of proteins that are misfolded include amyloid β protein (hereinafter also referred to as "Aβ"), tau protein, α-synuclein, polyglutamine, TDP-43, etc. Examples of abnormal protein accumulation neurodegenerative diseases include, but are not limited to, Alzheimer's disease, mild cognitive impairment (MCI), amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar degeneration, multiple sclerosis, Creutzfeldt-Jakob disease, progressive multifocal leukoencephalopathy, dementia with Lewy bodies, corticobasal degeneration, Parkinson's disease, Parkinson's syndrome, frontotemporal dementia (FTD), spastic paraplegia, non-herpetic acute limbic encephalitis, and age-related macular degeneration (including precursor lesions, atrophic type, and exudative type). The abnormal protein accumulation neurodegenerative diseases that are treated and/or prevented by the agent of the present invention are preferably Alzheimer's disease, age-related macular degeneration, etc.

The adrenomedullin contained in the agent of the present invention may be not only a peptide of human origin (SEQ ID NO: 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: 3), dogs (SEQ ID NO: 5), cows (SEQ ID NO: 7), rats (SEQ ID NO: 9) or mice (SEQ ID NO: 11). 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".

In this specification, "amidation of the C-terminus" refers to one aspect of post-translational modification of a peptide in a living body, specifically, a 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 a peptide in a living body, specifically, a reaction in which two cysteine residues in the amino acid sequence of a peptide form a disulfide bond (-S-S-).

The adrenomedullin contained in the agent of the present invention may be the adrenomedullin as described above, or may be a modified form of the adrenomedullin, or a salt, hydrate, solvate, or the like thereof. In the present specification, adrenomedullin or modified form of adrenomedullin includes derivatives thereof, or precursors that are converted to adrenomedullin or its modified form by metabolism in the body, or prodrug compounds thereof. Specific examples of modified adrenomedullin include those described in International Publication No. 2012/096411, International Publication No. 2015/141819, International Publication No. 2017/047788, or International Publication No. 2018/181638. Specific examples of derivatives of adrenomedullin or modified form of adrenomedullin include those described in International Publication No. 2017/047788. Adrenomedullin and modified adrenomedullin, as well as their derivatives, etc., contained in the agent of the present invention will be described in detail below.

The adrenomedullin derivative contained in the agent of the present invention has the following formula (I):

, or a salt thereof, or a hydrate thereof.

In formula (I), B must be a peptide moiety derived from adrenomedullin or a modified form thereof having adrenomedullin activity (hereinafter also referred to as a "modified form of adrenomedullin"). In the present invention, a "peptide moiety derived from adrenomedullin or a modified form thereof having adrenomedullin activity" refers to 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 α-amino group at the N-terminus) has been removed from adrenomedullin or a modified form thereof having adrenomedullin activity.

In the present invention, "modified adrenomedullin" refers to a peptide in which the native adrenomedullin described above is chemically modified. In addition, in the present invention, "adrenomedullin activity" refers to the effect of improving neurodegeneration caused by the accumulation of abnormal proteins contained in adrenomedullin. The compound represented by formula (I) of the present invention contains peptide portion B derived from adrenomedullin or a modified form thereof contained in the agent of the present invention, and thereby can exhibit adrenomedullin activity substantially equivalent to that of native adrenomedullin.

The above-mentioned adrenomedullin or a modified form thereof having adrenomedullin activity 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) A peptide according to (ii), in which the disulfide bond is replaced by an ethylene group, and which has adrenomedullin activity;
(iv) A peptide in which 1 to 15 amino acids are deleted, substituted or added in any one of the peptides (i) to (iii) and which has adrenomedullin activity.
(v) any of the peptides (i) to (iv) above, the C-terminus of which is amidated, and (vi) any of the peptides (i) to (iv) above, the C-terminus of which is added with a glycine residue. In the present specification, the addition of an amino acid includes the insertion of an amino acid.

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

In another embodiment, the adrenomedullin or a modification thereof having adrenomedullin activity is selected from the group consisting of:
(iv') a peptide in which the amino acid residues at positions 1 to 15, 1 to 10, or 1 to 5 from the N-terminus are deleted in any one of the peptides (i) to (iii), and which has adrenomedullin activity;
It is more preferable that the peptide is selected from the group consisting of peptides (v)(iv') having an amidated C-terminus and peptides (vi)(iv') having a glycine residue added to the C-terminus.

In the peptides (i) to (vi) and (iv') above, the peptide included in (v) consisting of the amino acid sequence of adrenomedullin, in which the C-terminus is amidated and in which two cysteine residues in the amino acid sequence form a disulfide bond, corresponds to mature native adrenomedullin. The peptide consisting of the amino acid sequence of adrenomedullin in (i) corresponds to native adrenomedullin in its (i.e. immature) form prior to post-translational modification of C-terminus amidation and disulfidation of cysteine residues. In the peptides (i) to (vi) and (iv') above, the other peptides except for those described above correspond to modified forms of adrenomedullin.

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

The peptide (iii) can be formed by replacing the disulfide bond of the peptide (ii) with an ethylene group. The replacement 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 exert a sustained 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 preferred 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 which has adrenomedullin activity, and a more preferred 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) and which has adrenomedullin activity (peptide (iv')). In the suitable peptide, one or more (e.g., 1 to 5, 1 to 3, or 1 or 2) amino acid residues may be further deleted, substituted, or added. By using the peptide (iv) or (iv'), the adrenomedullin activity of the compound represented by formula (I) can be made substantially equivalent to that of natural adrenomedullin. Furthermore, by using the peptide (iv) or (iv'), the compound represented by formula (I) can exert sustained adrenomedullin activity in vivo.

The peptide (vi) or (iv') 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) or (iv') 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 exert adrenomedullin activity sustainably in the body.

The adrenomedullin or a modified form thereof is
(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and 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: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 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: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 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: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 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: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 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: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(g) any one of the peptides (a) to (f), in which the disulfide bond is replaced by an ethylene group, and the peptide has adrenomedullin activity;
(h) a peptide in which 1 to 15 amino acids are deleted, substituted or added in any one of the peptides (a) to (g) and which has adrenomedullin activity;
(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 has been added to the C-terminus;
More preferably, the peptide is selected from the group consisting of:

In one embodiment, the adrenomedullin or a modified form thereof is
(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and 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: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 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: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 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: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 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: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 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: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(i) any one of peptides (a) to (f) in which the C-terminus is amidated; and (j) any one of peptides (a) to (f) in which a glycine residue has been added to the C-terminus;
It is even more preferable that the peptide is selected from the group consisting of:

In another embodiment, the adrenomedullin or a modified form thereof is selected from the group consisting of:
(h') a peptide in which the 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 (a) to (d) and which has adrenomedullin activity, or a peptide in which the amino acid residues at positions 1 to 13, 1 to 8, or 1 to 5 from the N-terminus are deleted in the peptides (e) or (f) and which has adrenomedullin activity;
(i) a peptide having an amidated C-terminus in the peptide (h'); and (j) a peptide having a glycine residue added to the C-terminus in the peptide (h');
It is even more preferable that the peptide is selected from the group consisting of:

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 suitable 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 are deleted in any of the peptides (a) to (g), and the peptide has adrenomedullin activity. A more suitable 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 are deleted in any of the peptides (a) to (d), and the peptide has adrenomedullin activity, or a peptide in which amino acid residues at positions 1 to 13, 1 to 8, or 1 to 5 from the N-terminus are deleted in the peptides (e) or (f), and the peptide has adrenomedullin activity (peptide (h')). In the suitable peptides, one or more amino acids (e.g., 1 to 5, 1 to 3, or 1 or 2) may be further deleted, substituted, or added. By using the peptide (h) or (h'), the adrenomedullin activity of the compound represented by formula (I) can be made substantially equivalent to that of natural adrenomedullin. In addition, by using the peptide (h) or (h'), the compound represented by formula (I) can exert the adrenomedullin activity sustainably in vivo.

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 an N-terminal α-amino group) has been removed from the above-mentioned adrenomedullin or a modified form thereof having adrenomedullin activity corresponds to B in formula (I). In addition, in formula (I), A must be a modifying group containing one or more PEG groups. The embodiment in which the modifying group A contains one or more PEG groups is not particularly limited. For example, one or more PEG groups may be located at the terminal end of the modifying group A, or may be located inside the modifying group A. In addition, the modifying group A may be any of various groups known in the art as a linear or branched group containing a PEG group. Known groups that can be used as the modifying group A include, but are not limited to, groups disclosed in, for example, WO 1995/11924, WO 2006/084089, WO 98/41562, WO 2005/079838, WO 2002/060978, WO 2001/048052, WO 1998/055500, WO 1996/021469, WO 2003/040211, and JP-A-04-108827. By using a group containing one or more PEG groups as the modifying group A, the compound represented by formula (I) can exert adrenomedullin activity sustainably in vivo.

A is represented by the following formula (II):

It is preferable that the modifying group is represented by:

In formula (II),
a is an integer equal to or greater than 1;
m is an integer equal to or greater than 1;
^L1 is an (m+1)-valent linear or branched linking group, provided that when there are multiple ^L1s , the multiple ^L1s may be the same or different from each other;
^L2 and L2 ^' each independently represent a bond or a divalent linking group, provided that when there are multiple L2 ^' , the multiple L2 ^' may be the same or different;
M ¹ is a PEG group, provided that when there are multiple M ¹ s, the multiple M ¹ s may be the same or different from each other;
^M2 is a bond or a PEG group, provided that when there is a plurality of ^M2 , the plurality of ^M2 may be the same or different from each other;
R ¹ is hydrogen or a monovalent group;
* indicates the position of attachment to the remainder.

m is the number of branches of the linking group ^L1 . For example, when m is 1, ^L1 is a divalent linking group that is unbranched toward the terminal, i.e., a linear group. When m is 2 or more, ^L1 is a trivalent or more linking group that is two or more branches toward the terminal. m is usually an integer of 1 or more and 5 or less, preferably in the range of 1 to 5, more preferably in the range of 1 to 4, and more preferably in the range of 1 to 3. When the number of branches m of the linking group ^L1 is within the above range, the modifying group A containing a PEG group can have a linear or branched structure.

a is the number of repeats of the units of the PEG groups ^M1 and ^M2 and the linking groups ^L1 and L2 ^' . For example, when a is 1, the units do not have a repeat structure. When a is 2 or more and m is 1, the units have a linear repeat structure. When a is 2 or more and m is 2 or more, the units have a dendritic branched repeat structure. a is usually an integer of 1 or more and 5 or less, preferably in the range of 1 to 5, and more preferably in the range of 1 to 2. When the number of repeats a of the units of the PEG groups ^M1 and ^M2 and the linking groups ^L1 and L2 ^' is within the above range, the modifying group A containing a PEG group can have a linear or branched structure.

In ^M1 and ^M2 , the PEG group is usually represented by the following formula (III):

In formula (III), ** is the bonding position with ^L1 , and # is the bonding position with O or L2 ^' . The weight average molecular weight of the PEG group represented by formula (III), in total in the modifying group A, is usually 1 kDa or more, preferably 5 kDa or more, more preferably 10 kDa or more, and even more preferably 20 kDa or more, and is usually 2000 kDa or less, preferably 1000 kDa or less, more preferably 100 kDa or less, even more preferably 80 kDa or less, and particularly preferably 60 kDa or less. The PEG group represented by formula (III) has a weight average molecular weight in the range of 1 to 2000 kDa, for example, 1 to 1000 kDa in total in the modifying group A, preferably 1 to 100 kDa, more preferably 5 to 80 kDa, even more preferably 10 to 60 kDa, and particularly preferably 20 to 60 kDa. When the total weight average molecular weight of the PEG group represented by formula (III) in the modifying group A is within the above range, the adrenomedullin activity of the compound represented by formula (I) can be substantially equivalent to that of natural adrenomedullin. In addition, the compound represented by formula (I) can exert adrenomedullin activity sustainably in vivo while substantially suppressing undesirable side reactions.

In formula (III), n is the number of repeats of ethylene oxide units defined based on the weight average molecular weight. When defined based on the preferred range of the weight average molecular weight, n is usually an integer of about 20 or more, preferably about 110 or more, more preferably about 230 or more, and even more preferably about 460 or more, and is usually an integer of about 45,000 or less, preferably about 22,000 or less, more preferably about 2,200 or less, even more preferably about 1,820 or less, and especially preferably about 1,360 or less. When defined based on the preferred range of the weight average molecular weight, n is usually in the range of about 20 to 45,000, for example, in the range of about 20 to 22,000, preferably in the range of about 1 to 2,200, more preferably in the range of about 110 to 1,820, even more preferably in the range of about 230 to 1,360, and especially preferably in the range of about 460 to 1,360. When the number of repeats n is in the above range, the total weight average molecular weight of the PEG groups contained in the modifying group represented by formula (II) is in the above range. Therefore, when the repeat number n is within the above range, the adrenomedullin activity of the compound represented by formula (I) can be substantially equivalent to that of natural adrenomedullin. In addition, the compound represented by formula (I) can exert the adrenomedullin activity sustainably in vivo while substantially suppressing undesirable side reactions.

R ¹ is preferably hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C 2 -C ₂₀ alkenyl, substituted or unsubstituted C ₂ -C ₂₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₄ -C 20 cycloalkenyl, substituted or unsubstituted C ₄ -C ₂₀ cycloalkynyl, substituted or unsubstituted _3-6 membered heterocycloalkyl, substituted or unsubstituted C ₇ -C ₂₀ cycloalkylalkyl, substituted or unsubstituted 3-6 membered heterocycloalkyl-C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₄ -C ₂₀ aryl, substituted or unsubstituted C _{5 -C 20 arylalkyl, substituted or unsubstituted 5-15 membered heteroaryl, substituted or unsubstituted 5-15 membered heteroaryl-C 1 -C 20 alkyl , or} substituted or unsubstituted acyl, and is preferably hydrogen, substituted or unsubstituted C More preferably, it is a ₁ - _C20 alkyl, a substituted or unsubstituted _C2 - _C20 alkenyl, or a substituted or unsubstituted _C2 - _C20 alkynyl, even more preferably, it is hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl, and especially preferably, it is methyl. When the group is substituted, the substituents are each independently preferably a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, substituted or unsubstituted C ₁ -C ₅ alkyl, substituted or unsubstituted C ₂ -C ₅ alkenyl, substituted or unsubstituted C ₂ -C ₅ alkynyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, substituted or unsubstituted C ₃ -C ₆ cycloalkenyl, substituted or unsubstituted C ₃ -C ₆ cycloalkynyl, substituted or unsubstituted amino, and substituted or unsubstituted C ₁ -C ₅ alkoxy, and more preferably a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, unsubstituted C ₁ -C ₅ alkyl, unsubstituted C ₂ -C ₅ alkenyl, unsubstituted C ₂ -C ₅ alkynyl, unsubstituted C ₃ -C ₆ cycloalkyl, unsubstituted C ₃ -C ₆ cycloalkenyl, unsubstituted C ₃ It is more preferable that R 1 is a monovalent group selected from the group consisting of C 1-C ₆ cycloalkynyl, unsubstituted amino, and unsubstituted C ₁ -C ₅ alkoxy. When R ¹ is the above group, the adrenomedullin activity of the compound represented by formula (I) can be substantially equivalent to that of natural adrenomedullin. In addition, the compound represented by formula (I) can continuously exert adrenomedullin activity in vivo while substantially suppressing undesirable side reactions.

^L1 is a linear or branched linking group having a valence of m+1. ^L1 is preferably a substituted or unsubstituted linear or branched hydrocarbon group having a valence of m+1. The group may contain one or more heteroatoms, alicyclic groups, aromatic groups, amide groups (-CO-NH-), ester groups (-CO-O-), or urethane groups (-O-CO-NH-). When the group is substituted, the substituents are preferably each independently a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine, or iodine), cyano, nitro, and substituted or unsubstituted linear or branched hydrocarbon groups.

^L2 and L2 ^' are each independently a bond or a divalent linking group. When ^L2 and L2 ^' are divalent linking groups, ^L2 and L2 ^' are preferably each independently a substituted or unsubstituted divalent hydrocarbon group, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-), and are preferably a substituted or unsubstituted _C1 - _C20 alkylene, a substituted or unsubstituted _C2 - _C20 alkenylene, a substituted or unsubstituted _C2 - _C20 alkynylene, a substituted or unsubstituted _C3 - _C20 cycloalkylene, a substituted or unsubstituted _C4 - _C20 cycloalkenylene, a substituted or unsubstituted _C4 - _C20 cycloalkynylene, a substituted or unsubstituted 3- to 6-membered heterocycloalkylene, a substituted or unsubstituted _C7 - _C20 cycloalkylalkylene, a substituted or unsubstituted 3- to 6-membered heterocycloalkyl- _C1 - _C20 alkylene, a substituted or unsubstituted C More preferably, it is a _C4 - _C20 arylene, a substituted or unsubstituted _C5 - _C20 aryl alkylene, a substituted or unsubstituted 5-15 membered heteroarylene, or a substituted or unsubstituted 5-15 membered heteroaryl- _C1 - _C20 alkylene, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-), which may contain one or more heteroatoms, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). When the group is substituted, the substituents are preferably each independently a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, and substituted or unsubstituted straight-chain or branched-chain hydrocarbon groups, and more preferably a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, unsubstituted _C1 - _C5 alkyl, unsubstituted _C2 - _C5 alkenyl, unsubstituted _C2 - _C5 alkynyl, unsubstituted _C3 - _C6 cycloalkyl, unsubstituted _C3 - _C6 cycloalkenyl, unsubstituted _C3 - _C6 cycloalkynyl, unsubstituted amino, and unsubstituted _C1 - _C5 alkoxy.

When ^L1 , ^L2 , and L2 ^' are the above groups, the adrenomedullin activity of the compound represented by formula (I) can be substantially equivalent to that of natural adrenomedullin. In addition, the compound represented by formula (I) can sustain the adrenomedullin activity in vivo while substantially suppressing undesirable side reactions.

Suitable modifying groups A are of the following formula (V), (VI), (VII) or (VIII):

is a modifying group represented by.

In formulas (V), (VI), (VII) and (VIII),
a is an integer equal to or greater than 1;
M ³ , M ^{3 '} , M ^{3 ''} , M ^{3 '''} and M ^{3 ''''} are each independently a bond or a PEG group;
provided that when there are a plurality of M ³ , M ^3' , M ^3'' , M ^3''' , and M ^3'''' , the plurality of M ³ , M ^3' , M ^3'' , M ^3''' , and M ^3'''' may be the same or different from each other, and at least one of M ³ , M ^3' , M ^3'' , M ^3'''' , and M ^3'''' is a PEG group;
R ¹ , R ^{1 '} , R ^{1 ''} and R ^{1 '''} are each independently hydrogen or a monovalent group;
^R2 is a bond or a divalent group;
R ³ , R ^3′ and R ^3″ are each independently a bond or a divalent group, provided that when there are a plurality of R ³ , R ^3′ and R ^3″ , the plurality of R ³ , R ^3′ and R ^3″ may be the same or different from each other;
* indicates the position of attachment to the remainder.

a is the number of repeats of the units containing PEG groups M ³ , M ^3' , M ^3'' , M ^3''' and M ^3'''' . For example, when a is 1, the units do not have a repeat structure. When a is 2 or more in formula (V), the units have a linear repeat structure. When a is 2 or more in formulas (VI), (VII) and (VIII), the units have a dendritic branched repeat structure. a is usually an integer of 1 or more and 5 or less, preferably in the range of 1 to 5, and more preferably in the range of 1 to 2. When the number of repeats a of the units containing PEG groups M ³ , M ^3' , M ^3'' , M ^3''' and M ^3'''' is within the above range, the modifying group A containing a PEG group can have a linear or branched structure.

When ^M3 , M3 ^' , M3 ^'' , M3 ^''' , and M3 ^'''' are PEG groups, the PEG group is usually a group represented by formula (III). The PEG group represented by formula (III) has the same meaning as above. In this case, the adrenomedullin activity of the compound represented by formula (I) can be made substantially equivalent to that of natural adrenomedullin. In addition, the compound represented by formula (I) can exert adrenomedullin activity sustainably in vivo while substantially suppressing undesirable side reactions.

^R1 has the same meaning as above. R1 ^' , R1 ^'' , and R1 ^''' have the same meaning as above ^R1 . In this case, the adrenomedullin activity of the compound represented by formula (I) can be substantially equivalent to that of natural adrenomedullin. Furthermore, the compound represented by formula (I) can sustainably exert adrenomedullin activity in vivo while substantially suppressing undesirable side reactions.

R ² is preferably a bond, a substituted or unsubstituted divalent hydrocarbon group, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-), and is preferably a bond, a substituted or unsubstituted C ₁ -C ₂₀ alkylene, a substituted or unsubstituted C ₂ -C ₂₀ alkenylene, a substituted or unsubstituted C ₂ -C ₂₀ alkynylene, a substituted or unsubstituted C ₃ -C ₂₀ cycloalkylene, a substituted or unsubstituted C ₄ -C ₂₀ cycloalkenylene, a substituted or unsubstituted C ₄ -C ₂₀ cycloalkynylene, a substituted or unsubstituted 3- to 6-membered heterocycloalkylene, a substituted or unsubstituted C ₇ -C ₂₀ cycloalkylalkylene, a substituted or unsubstituted 3- to 6-membered heterocycloalkyl-C ₁ -C ₂₀ alkylene, a substituted or unsubstituted C ₄ -C ₂₀ arylene, a substituted or unsubstituted C ₅ -C More preferably, the divalent hydrocarbon group is a C ₁ -C 20 _aryl alkylene, a substituted or unsubstituted 5-15 membered heteroarylene, or a substituted or unsubstituted 5-15 membered heteroaryl-C 1 -C ₂₀ alkylene, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). The divalent hydrocarbon group may contain one or more heteroatoms, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). When the group is substituted, the substituents are each independently preferably a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, substituted or unsubstituted C ₁ -C ₅ alkyl, substituted or unsubstituted C ₂ -C ₅ alkenyl, substituted or unsubstituted C ₂ -C ₅ alkynyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, substituted or unsubstituted C ₃ -C ₆ cycloalkenyl, substituted or unsubstituted C ₃ -C ₆ cycloalkynyl, substituted or unsubstituted amino, and substituted or unsubstituted C ₁ -C ₅ alkoxy, and more preferably a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, unsubstituted C ₁ -C ₅ alkyl, unsubstituted C ₂ -C ₅ alkenyl, unsubstituted C ₂ -C ₅ alkynyl, unsubstituted C ₃ -C ₆ cycloalkyl, unsubstituted C ₃ -C ₆ cycloalkenyl, unsubstituted C ₃ ^R2 is preferably a bond or _a substituted or unsubstituted _{C1 - C10} alkylene group, more preferably a bond, methylene _, ethylene, propylene or butylene, and even more preferably a bond or ethylene.

R ³ , R ^3′ and R ^3″ are each independently preferably a bond, a substituted or unsubstituted divalent hydrocarbon group, an amide group (—CO—NH—), an ester group (—CO—O—), or a urethane group (—O—CO—NH—), and are preferably a bond, a substituted or unsubstituted C ₁ to C ₂₀ alkylene, a substituted or unsubstituted C ₂ to C ₂₀ alkenylene, a substituted or unsubstituted C ₂ to C ₂₀ alkynylene, a substituted or unsubstituted C ₃ to C ₂₀ cycloalkylene, a substituted or unsubstituted C ₄ to C ₂₀ cycloalkenylene, a substituted or unsubstituted C ₄ to C ₂₀ cycloalkynylene, a substituted or unsubstituted 3 to 6 membered heterocycloalkylene, a substituted or unsubstituted C ₇ to C ₂₀ cycloalkylalkylene, a substituted or unsubstituted 3 to 6 membered heterocycloalkyl-C ₁ to C ₂₀ alkylene, a substituted or unsubstituted C ₄ to C More preferably, the divalent hydrocarbon group is a C ₁ -C 20 arylene, a substituted or unsubstituted C ₅ -C ₂₀ aryl alkylene, a substituted or unsubstituted 5-15 membered heteroarylene, or a substituted or unsubstituted 5-15 membered heteroaryl-C ₁ -C ₂₀ alkylene, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). The divalent hydrocarbon group may contain one or more heteroatoms, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO-NH-). When the group is substituted, the substituents are each independently preferably a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, substituted or unsubstituted C ₁ -C ₅ alkyl, substituted or unsubstituted C ₂ -C ₅ alkenyl, substituted or unsubstituted C ₂ -C ₅ alkynyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, substituted or unsubstituted C ₃ -C ₆ cycloalkenyl, substituted or unsubstituted C ₃ -C ₆ cycloalkynyl, substituted or unsubstituted amino, and substituted or unsubstituted C ₁ -C ₅ alkoxy, and more preferably a monovalent group selected from the group consisting of halogen (fluorine, chlorine, bromine or iodine), cyano, nitro, unsubstituted C ₁ -C ₅ alkyl, unsubstituted C ₂ -C ₅ alkenyl, unsubstituted C ₂ -C ₅ alkynyl, unsubstituted C ₃ -C ₆ cycloalkyl, unsubstituted C ₃ -C ₆ cycloalkenyl, unsubstituted C ₃ More preferably, R ³ , R ^3' , and R ^3'' are each independently a bond, a substituted _or unsubstituted C ₁ -C ₁₀ alkylene group, a substituted or unsubstituted C _{1 -C 10} alkylene group including an amido group, or an amido group (-CO-NH-), and more preferably each independently a bond, methylene, ethylene, -CO-NH-(CH ₂ ) ₄ -, -CH ₂ -O-CO-NH-(CH _{2 ) 3} -, or -CO-NH-.

When R ² , R ³ , R ^3' and R ^3'' are the above groups, the adrenomedullin activity of the compound represented by formula (I) can be made substantially equivalent to that of natural adrenomedullin. Furthermore, the compound represented by formula (I) can exert sustained adrenomedullin activity in the body while substantially suppressing undesirable side reactions.

Particularly preferred modifying groups A are of the following formula (V-1-1), (VI-1-1), (VII-1-1), (VII-1-2), (VII-2-1), or (VIII-1-1):

[Wherein,
n has the same meaning as defined above,
n' has the same meaning as defined above for n,
* indicates the position of attachment to the remainder.
It is a modifying group represented by the formula:

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

In formula (VI-1-1), the PEG groups preferably have a combined weight average molecular weight of 40 kDa.

In formula (VII-1-1), the PEG groups preferably have a combined weight average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa or 80 kDa.

In formula (VII-1-2), the PEG groups preferably have a total weight average molecular weight of 50 kDa, where the ethylene _oxide units of ( _CH2CH2O ) _n typically have a total weight average molecular weight of 40 kDa, and the ethylene _oxide units of ( _CH2CH2O ) _n' typically have a total weight average molecular weight of 10 kDa.

In formula (VII-2-1), the PEG group preferably has a total weight average molecular weight of 40 kDa. In this case, the ethylene oxide units of (CH ₂ CH ₂ O) _n usually have a total weight average molecular weight of 30 kDa, and the ethylene oxide units of (CH ₂ CH ₂ O) _{n '} usually have a total weight average molecular weight of 10 kDa. Alternatively, the PEG group preferably has a total weight average molecular weight of 60 kDa. In this case, the ethylene oxide units of (CH ₂ CH ₂ O) _n usually have a total weight average molecular weight of 50 kDa, and the ethylene oxide units of (CH ₂ CH ₂ O) _{n '} usually have a total weight average molecular weight of 10 kDa. Alternatively, the PEG group preferably has a total weight average molecular weight of 80 kDa. In this case, typically, the ethylene oxide units of (CH ₂ CH ₂ O) _n have a total weight average molecular weight of 70 kDa and the ethylene oxide units of (CH ₂ CH ₂ O) _{n '} have a total weight average molecular weight of 10 kDa.

In formula (VIII-1-1), the PEG groups preferably have a combined weight average molecular weight of 40 kDa.

By using the above group as the modifying group A, the compound represented by formula (I) can maintain the pharmacological action of natural adrenomedullin while substantially suppressing undesirable side reactions, and can exert sustained adrenomedullin activity in the body.

In formula (I), the peptide portion B must be linked to the remainder by covalently bonding the nitrogen atom of the α-amino group at its N-terminus to the carbon atom of the methylene group. In the present invention, when the modifying group A containing one or more PEG groups and the peptide portion B are linked in the above-mentioned manner, they may be described as "alkylamine-linked adrenomedullin derivatives". The alkylamine-linked adrenomedullin derivatives have higher adrenomedullin activity than the adrenomedullin derivatives in which the nitrogen atom of the α-amino group at the N-terminus of adrenomedullin is linked to the remainder by forming an amide bond, such as the adrenomedullin derivatives described in Kubo, K et al., Biological properties of adrenomedullin conjugated with polyethylene glycol. Peptides, 2014, Vol. 57, p. 118-21 (hereinafter also referred to as "amide-linked adrenomedullin derivatives"). Furthermore, the alkylamine-linked adrenomedullin derivatives of the present invention represented by formula (I) are more suppressed in undesirable side reactions (e.g., excessive blood pressure reduction, tachycardia associated with reflex increased sympathetic nerve activity, and/or increased renin activity, etc.) than amide-linked adrenomedullin derivatives. Therefore, the compounds of the present invention represented by formula (I) are more capable of exerting adrenomedullin activity in vivo while more suppressing undesirable side reactions than known adrenomedullin derivatives.

Particularly preferred compounds of formula (I) are
A is a modifying group containing a PEG group represented by formula (V-1-1), (VI-1-1), (VII-1-1), (VII-1-2), (VII-2-1), or (VIII-1-1),
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: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 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: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 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: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 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: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 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: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(i) any one of peptides (a) to (f) in which the C-terminus is amidated; and (j) any one of peptides (a) to (f) in which a glycine residue has been added to the C-terminus; or
below:
(h') a peptide in which the 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 (a) to (d) and which has adrenomedullin activity, or a peptide in which the amino acid residues at positions 1 to 13, 1 to 8, or 1 to 5 from the N-terminus are deleted in the peptides (e) or (f) and which has adrenomedullin activity;
(i) a peptide having an amidated C-terminus in the peptide (h'); and (j) a peptide having a glycine residue added to the C-terminus in the peptide (h');
The compound represented by formula (I) having the above characteristics is capable of exerting the adrenomedullin activity sustainably in vivo while maintaining the pharmacological action of natural adrenomedullin and substantially suppressing undesirable side reactions.

Another embodiment of the adrenomedullin derivative contained in the agent of the present invention is the following (X):

or a salt thereof, or a hydrate thereof. In this specification, the compound represented by formula (X) may be referred to as a "urethane-linked adrenomedullin derivative."

In formula (X), B must be a peptide moiety derived from adrenomedullin or a modified form thereof having adrenomedullin activity contained in the agent of the present invention. The peptide moiety B has the same meaning as defined above for the compound represented by formula (I).

A' must be a modifying group containing one or more PEG groups. However, A' must be linked to the remainder of the moiety by a covalent bond between the oxygen atom of the modifying group containing a PEG group and the carbon atom of the carbonyl group. When the modifying group A' has such a structure, the compound represented by formula (X) can have a structure in which the modifying group A' and the peptide portion B are linked via a urethane bond.

A' is represented by the following formula (XI), (XI') or (XII):

It is preferable that the modifying group is represented by the formula:

In formulas (XI), (XI') and (XII), * indicates the bonding position to the remainder.

In formulas (XI), (XI') and (XII), a, ^R1 , R1 ^' , ^R2 , ^R3 , R3 ^' , R3 ^'' , ^M1 , ^M3 , M3 ^' and ^M3'' have the same meanings as defined above for compounds represented by formula (I).

Particularly preferred modifying groups A' are represented by the following formulae (XI-1-1), (XII-1-1) or (XII-2-1):

[Wherein,
n has the same meaning as defined above,
n' has the same meaning as defined above for n,
* indicates the position of attachment to the remainder.
It is a modifying group represented by the formula:

In formula (XI-1-1), the PEG groups preferably have a combined weight average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa or 80 kDa.

In formula (XII-1-1), the PEG groups preferably have a combined weight average molecular weight of 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 60 kDa or 80 kDa.

In formula (XII-2-1), the PEG group preferably has a total weight average molecular weight of 40 kDa. In this case, the ethylene oxide units of (CH ₂ CH ₂ O) _n usually have a total weight average molecular weight of 30 kDa, and the ethylene oxide units of (CH ₂ CH ₂ O) _{n '} usually have a total weight average molecular weight of 10 kDa. Alternatively, the PEG group preferably has a total weight average molecular weight of 60 kDa. In this case, the ethylene oxide units of (CH ₂ CH ₂ O) _n usually have a total weight average molecular weight of 50 kDa, and the ethylene oxide units of (CH ₂ CH ₂ O) _{n '} usually have a total weight average molecular weight of 10 kDa. Alternatively, the PEG group preferably has a total weight average molecular weight of 80 kDa. In this case, typically, the ethylene oxide units of (CH ₂ CH ₂ O) _n have a total weight average molecular weight of 70 kDa and the ethylene oxide units of (CH ₂ CH ₂ O) _{n '} have a total weight average molecular weight of 10 kDa.

By using the above group as the modifying group A', the compound represented by formula (X) can exert sustained adrenomedullin activity in the body while maintaining the pharmacological action of natural adrenomedullin.

In formula (X), the peptide portion B must be linked to the remainder by covalent bonding of the nitrogen atom of the α-amino group at its N-terminus to the carbon atom of the carbonyl group. The urethane-linked adrenomedullin derivative has higher adrenomedullin activity than the amide-linked adrenomedullin derivative described in Kubo, K et al., Biological properties of adrenomedullin conjugated with polyethylene glycol. Peptides, 2014, Vol. 57, p. 118-21. Therefore, the compound represented by formula (X) of the present invention can sustainably exert higher adrenomedullin activity in vivo than known adrenomedullin derivatives.

Particularly preferred compounds of formula (X) are:
A' is a modifying group containing a PEG group represented by formula (XI-1-1), (XII-1-1) or (XII-2-1),
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: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 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: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 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: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 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: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 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: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(i) any one of peptides (a) to (f) in which the C-terminus is amidated; and (j) any one of peptides (a) to (f) in which a glycine residue has been added to the C-terminus;
or a peptide selected from the group consisting of:
below:
(h') a peptide in which the 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 (a) to (d) and which has adrenomedullin activity, or a peptide in which the amino acid residues at positions 1 to 13, 1 to 8, or 1 to 5 from the N-terminus are deleted in the peptides (e) or (f) and which has adrenomedullin activity;
(i) a peptide having an amidated C-terminus in the peptide (h'); and (j) a peptide having a glycine residue added to the C-terminus in the peptide (h');
The compound represented by formula (X) having the above-mentioned characteristics is capable of exerting higher adrenomedullin activity in vivo for a sustained period compared to known adrenomedullin derivatives.

In the present invention, adrenomedullin and its modifications, and the compounds represented by formulae (I) and (X) include not only the compounds themselves but also their salts. When the compounds are in the form of salts, they are preferably pharma- ceutically acceptable salts. Counterions of the salts of the compounds include, but are not limited to, cations such as sodium ions, potassium ions, calcium ions, magnesium ions, or substituted or unsubstituted ammonium ions, or chloride ions, bromide ions, iodide ions, phosphate ions, nitrate ions, sulfate ions, carbonate ions, bicarbonate ions, perchlorate ions, formate ions, acetate ions, trifluoroacetate ions, propionate ions, lactate ions, maleate ions, hydroxymaleate ions, methylmaleate ions, fumarate ions, adipic acid ions, benzoate ions, 2-acetoxybenzoate ions, p-aminobenzoate ions, and the like. Anions such as nicotinate, cinnamate, ascorbate, pamoate, succinate, salicylate, bismethylenesalicylate, oxalate, tartrate, malate, citrate, gluconate, aspartate, stearate, palmitate, itaconate, glycolate, glutamate, benzenesulfonate, cyclohexylsulfamate, methanesulfonate, ethanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate, or naphthalenesulfonate are preferred. When the compound 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 adrenomedullin.

Adrenomedullin and its modifications, and compounds represented by formulas (I) and (X) include not only the compounds themselves, but also solvates of the compounds or their salts. When the compounds or their salts are in the form of solvates, the solvates are preferably pharma- ceutically acceptable. Solvents capable of forming solvates with the compounds or their salts are, but are not limited to, water, or organic solvents such as methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine, acetonitrile, or ethyl acetate. When the compounds or their salts are in the form of solvates with the solvents, the adrenomedullin activity of the compounds can be substantially equivalent to that of natural adrenomedullin.

Adrenomedullin and its modifications, and compounds represented by formulae (I) and (X) include not only the compounds themselves described above or below, but also their protected forms. In this specification, "protected form" refers to a form in which a protecting group is introduced into one or more functional groups (e.g., the side chain amino group of a lysine residue). In addition, in this specification, "protecting group" refers to a group that is introduced into a specific functional group to prevent the progression of an undesired reaction, is quantitatively removed under specific reaction conditions, and is substantially stable, i.e., inactive, under other reaction conditions. Protecting groups that may form the protected form of the compound include, but are not limited to, t-butoxycarbonyl (Boc), 2-bromobenzyloxycarbonyl (BrZ), 9-fluorenylmethoxycarbonyl (Fmoc), p-toluenesulfonyl (Tos), benzyl (Bzl), 4-methylbenzyl (4-MeBzl), 2-chlorobenzyloxycarbonyl (ClZ), cyclohexyl (cHex), and phenacyl (Pac); other protecting groups for amino groups include benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxycarbonyl, and p-methoxycarbonyl. dibenzyloxycarbonyl, benzhydryloxycarbonyl, 2-(p-biphenyl)isopropyloxycarbonyl, 2-(3,5-dimethoxyphenyl)isopropyloxycarbonyl, p-phenylazobenzyloxycarbonyl, triphenylphosphonoethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, t-amyloxycarbonyl, diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethyloxycarbonyl, allyloxycarbonyl, 2-methylsulfonylethyloxycarbonyl, 2,2,2-trichloroethyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, benzenesulfonyl, mesitylenesulfonyl, methoxytrimethylphenylsulfonyl, 2-nitrobenzenesulfonyl, 2-nitrobenzenesulfenyl, 4-nitrobenzenesulfenyl, and 4-nitrobenzenesulfenyl; other protecting groups for the carboxyl group include methyl ester, ethyl ester, t-butyl ester, p-methoxybenzyl ester, and p-nitrobenzyl ester; other side chain protecting groups for Arg include 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl, 4-methoxy -2,3,6-trimethylbenzenesulfonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, and 2-methoxybenzenesulfonyl; other protecting groups for Tyr include 2,6-dichlorobenzyl, t-butyl, and cyclohexyl; other protecting groups for Cys include 4-methoxybenzyl, t-butyl, trityl, acetamidomethyl, and 3-nitro-2-pyridinesulfenyl; other protecting groups for His include benzyloxymethyl, p-methoxybenzyloxymethyl, t-butoxymethyl, trityl, and 2,4-dinitrophenyl; and other protecting groups for Ser and Thr include t-butyl, etc. When the compound is in a form protected by the above-mentioned protecting groups, the adrenomedullin activity of the compound can be substantially equivalent to that of natural adrenomedullin.

Adrenomedullin and its modifications, and the compounds represented by formulas (I) and (X) also include mixtures of stereoisomers of the compounds, such as individual enantiomers and diastereomers of the compounds, and racemates.

By possessing the above characteristics, the compounds represented by formulas (I) and (X) can exert sustained adrenomedullin activity in the body while maintaining the pharmacological action of natural adrenomedullin and substantially suppressing undesirable side reactions.

The compounds represented by formulas (I) and (X) of the present invention can sustainably express biological activity (i.e., adrenomedullin activity) in vivo that is substantially equivalent to that of the parent molecule, adrenomedullin.

As can be understood from the examples in the above applications, the adrenomedullin derivatives described above and the adrenomedullin derivatives described in WO 2012/096411, WO 2015/141819, WO 2017/047788, and WO 2018/181638 are capable of inducing adrenomedullin in the body and are therefore suitable as components to be contained in the therapeutic agents of the present invention.

The agent of the present invention can be administered systemically or locally, orally or parenterally, such as intravenously, intraarterially, intramuscularly, subcutaneously, intradermally, intraperitoneally, nasally, rectally, orally, intraocularly, intraauricularly, or sublingually. In this specification, peripheral administration means an administration route other than intracerebral administration. The agent of the present invention may also be administered directly to the brain by an intraventricular pump. When administered orally, it may be administered before, after, or between meals. The preferred administration route of the agent of the present invention is intranasal administration.

The dosage form of the agent of the present invention may be, for example, a liquid preparation such as an injection, cream, ointment, beverage, aerosol, skin gel, eye drop, or nasal drop, in which an effective amount of adrenomedullin or its modified form, or a derivative thereof, or a salt thereof, is dissolved, dispersed, or emulsified in a diluent or dispersion medium such as water or physiological saline; or a solid preparation such as a tablet, capsule, powder, powder preparation, granule, sustained release agent, or suppository, which may be in the form of an inclusion body in which the active ingredient is encapsulated in a liposome or sustained release material, or a carrier in which the active ingredient is supported by a carrier. There is no particular restriction on the dosage form of the agent of the present invention, and it may be either an oral preparation or a parenteral preparation, but preferably it is a parenteral preparation, and more preferably it is a parenteral preparation for nasal administration. The dosage form of the agent of the present invention may be a unit dose preparation or a multiple dose preparation.

Oral preparations include tablets, powders, powder preparations, granules, fine granules, pills, capsules, troches, chewable preparations, liquids, emulsions, microcapsules, suspensions, elixirs, syrups, sustained-release preparations, etc., and these may be in the form of an inclusion body in which the active ingredient is encapsulated in a liposome or sustained-release material, etc., or a support body in which the active ingredient is supported by a carrier.

Parenteral preparations include liquid preparations such as injections, infusions, creams, ointments, aerosols, skin gels, eye drops, and nasal drops, in which an effective amount of adrenomedullin or its modified form, or a derivative or salt thereof is dissolved, dispersed, or emulsified in a diluent or dispersion medium such as water or physiological saline. Alternatively, the preparation may be in the form of a powder preparation, transdermal patch, lotion, ointment, cataplasm, or suppository.

The agent of the present invention may contain carriers, binders, excipients, bulking agents, lubricants, flow-improving agents, viscosity agents, sweeteners, flavoring agents, preservatives, emulsifiers, coating agents, diluents, etc., as necessary.

Specific components that may be contained in the agent of the present invention include, for example, binding agents such as tragacanth, gum arabic, corn starch, and gelatin; excipients such as D-mannitol, erythritol, maltitol, xylitol, palatinose, trehalose, sorbitol, starch, microcrystalline cellulose, crystalline cellulose, and dextran; leavening agents such as corn starch, pregelatinized starch, alginic acid, and dextrin; lubricants such as magnesium stearate and calcium stearate; flow agents such as fine silicon dioxide and methylcellulose. Examples of suitable additives include mobility improving agents; sweetening agents such as sucrose, lactose, aspartame, saccharin, and saccharin sodium; preservatives such as paraoxybenzoic acid esters, chlorobutanol, and benzalkonium chloride; flavoring agents such as peppermint, menthol, levomenthol, vanilla flavoring, and cherry or orange flavor; emulsifiers such as monoglycerides, polyglycerin fatty acid esters, sucrose fatty acid esters, and lecithin; coating agents such as glycerin fatty acid esters; and diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate.

Various other materials may also be included to change the physical form of the formulation. Examples of coating agents for tablets include shellac, sugar, or both. Syrups or elixirs may contain, for example, sucrose as a sweetener, methylparaben and propylparaben as preservatives, coloring, and cherry or orange flavoring. In addition, various vitamins and various amino acids may be included. When preparing an enteric formulation, for example, an aqueous solution of hydroxymethylcellulose may be used as a coating pretreatment agent, and an aqueous solution of hydroxypropylmethylcellulose phthalate and an aqueous solution of polyacetin may be used as coating agents to prepare an enteric formulation by a conventional method.

The dosage form of the agent of the present invention is preferably an aerosol agent (particularly an aerosol agent for nasal inhalation), a nasal drop agent (particularly a nasal liquid agent), or a powder preparation (particularly a powdered nasal preparation).
In the case of a powder formulation, for example, a dry powder may be administered into the nasal cavity using an inhaler, which may contain the active ingredient alone, a powder mixture of the active ingredient and a diluent such as lactose, or a mixed component particle containing the active ingredient and a phospholipid such as phosphatidylcholine. For intranasal use, the powder may contain a bioadhesive such as chitosan or cyclodextrin. The powder formulation may be filled into capsules, blisters, and cartridges (e.g., made of gelatin or hydroxypropylmethylcellulose) for use in an inhaler or insufflator. When filled into the above capsules, the agent of the present invention may be formulated as a powder mixture of adrenomedullin or a modified form thereof, or a derivative thereof, or a salt thereof, with a suitable powder base such as lactose or starch, and a performance modifier such as L-leucine, mannitol, magnesium stearate, etc. Lactose may be anhydrous or in the form of a monohydrate. Other preferred bases include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, trehalose, and the like.

In the case of aerosols, aerosols may be generated from a solution or suspension of the active ingredient and one or more agents for dispersing, solubilizing, or extending the release of the active ingredient (e.g., aqueous or non-aqueous ethanol, one or more solvents acting as propellants (e.g., 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane), and an optional surfactant such as sorbitan trioleate, oleic acid, or oligolactic acid) using a pressurized container, pump, spray, atomizer, or nebulizer for administration intranasally. Aerosols may also be generated from a solution or suspension of the active ingredient and one or more agents for dispersing, solubilizing, or extending the release of the active ingredient (e.g., ethanol, with or without water, one or more solvents acting as propellants (e.g., 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane), and an optional surfactant such as sorbitan trioleate, oleic acid, or oligolactic acid, using a nebulizer that uses electrohydrodynamics for administration intranasally.

A suitable aerosol formulation for use in a nebulizer that uses electrohydrodynamics to generate a fine mist may contain about 1 μg to about 20 mg of active ingredient per actuation, and actuation volumes may vary from about 1 μl to about 100 μl. A typical aerosol formulation may include adrenomedullin or a modified form thereof, or a derivative thereof, or a salt thereof, propylene glycol, sterile water, ethanol, and NaCl. Alternative solvents that may be used in place of propylene glycol include glycerol and polyethylene glycol.

Formulations for administration by nasal inhalation, intranasal administration, or both, may be formulated to be immediate or modified release, for example, using PGLA. Suitable flavors, such as menthol, levomenthol, or sweeteners, such as saccharin or saccharin sodium, may also be added to formulations intended for nasal inhalation/intranasal administration.

The subjects to which the agent of the present invention is administered include humans and non-human mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.). When applied to mammals other than humans, the dosage of the agent of the present invention may be adjusted appropriately according to the body weight and size of the animal.

The daily dose of adrenomedullin, the active ingredient of the agent of the present invention, for an adult needs to be adjusted appropriately depending on the sex, age, body weight, symptoms, route of administration, dosage form, etc., but in the case of oral agents, it is usually in the range of about 0.1 μg/kg to about 100 mg/kg, preferably in the range of 0.6 μg/kg to 6 mg/kg. In the case of parenteral agents, it is usually in the range of about 0.01 μg/kg to about 100 mg/kg, preferably in the range of 0.06 μg/kg to 600 μg/kg. In particular, in the case of parenteral agents for nasal administration, for example, the concentration of adrenomedullin in the formulation is usually about 60 μg/ml to about 180 μg/ml, preferably about 80 μg/ml to about 160 μg/ml, more preferably about 120 μg/ml.

The agent of the present invention may be administered at the above-mentioned daily dose all at once or in multiple doses. The timing of administration may be before, after, or between meals. The administration interval is not particularly limited, but may be daily, every other day, or intermittent administration. The administration period is not particularly limited, but long-term administration is possible.

The agent of the present invention can be used in combination with other drugs. When used in combination, the agent of the present invention and the other drug may be provided in the form of a single pharmaceutical, or may be provided in the form of a pharmaceutical combination or kit containing multiple formulations that are separately formulated. The administration time of the drug used in combination with the agent of the present invention is not limited, and the agent of the present invention and the drug used in combination may be administered to the subject at the same time or separately (e.g., consecutively, at an interval, etc.). The dosage of the drug used in combination may be similar to the dosage used in clinical practice, and can be appropriately selected depending on the subject, administration route, disease, combination, etc.

Drugs that can be used in combination with the agent of the present invention are not particularly limited as long as they are used for the treatment and/or prevention of Alzheimer's disease, mild cognitive impairment (MCI), amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar degeneration, multiple sclerosis, Creutzfeldt-Jakob disease, progressive multifocal leukoencephalopathy, dementia with Lewy bodies, corticobasal degeneration, amyotrophic lateral sclerosis, Parkinson's disease, Parkinson's syndrome, frontotemporal dementia, spinocerebellar degeneration, spastic paraplegia, spinocerebellar degeneration, multiple sclerosis, progressive multifocal leukoencephalopathy, non-herpetic acute limbic encephalitis, and age-related macular degeneration (including precursor lesions, atrophic type, and exudative type).

Specifically, in the case of Alzheimer's disease, examples include cholinesterase inhibitors, NMDA receptor antagonists, amyloid β removers and production inhibitors, and tau protein removers and production inhibitors.
Cholinesterase inhibitors include, for example, donepezil, galantamine, rivastigmine, huperzine A, and tacrine.
NMDA receptor antagonists include, for example, memantine.
Examples of amyloid β removal agents and production inhibitors include amyloid β vaccines, amyloid β removal antibodies, amyloid β production enzyme inhibitors, amyloid β aggregation inhibitors, and amyloid β decomposition promoters. Specific examples include CNP-520, E-2609, aducanumab, solanezumab, gantenerumab, crenezumab, amilomotide, ASD-005, HSH-971, ELND-005, ALZT-OP1, nilvadipine, ACI-24, UB-311, and AFFITOPE. Examples include AD-02, LY-3002813, BAN-2401, Neurostem-AD, CT-1812, ID-1201, NIC5-15, BI-425809, Posiphen, PQ-912, bryostatin-1, Apabetalone, PBT-2, RIV-1061-IR, MEDI-1814, PF-05236812, SAR-228810, Lu-AF20513, PRI-002, IRX-4204, GC-021109, AAD-2004, CTS-21166, LY-3323795, benfotiamine, bisnorcymserine, MDR-1339, KHK-6640, and NPT-088.
Examples of tau protein removers and production inhibitors include tau protein vaccines, tau protein removal antibodies, tau protein modification inhibitors, tau protein aggregation inhibitors, and tau protein degradation promoters. Specific examples include TRx-237, TPI-287, ABBV-8E12, RG-6100, AADvac1, RO7105705, PTI-80, JNJ-63733657, UCB-0107, BIIB-076, MC-1, ACI-35, and AZP-2006.
The above drugs may be used in appropriate combination depending on the symptoms of the subject.

Specific examples of drugs for age-related macular degeneration include Aflibercept, Verteporfin, Pegaptanib sodium, Ranibizumab, etc. These drugs may be used in appropriate combinations depending on the symptoms of the subject.

In the present invention, the dosage of the above-mentioned concomitant drug can be set at any amount within the range in which side effects are not a problem. The daily dosage of the concomitant drug varies depending on the severity of symptoms, the age, sex, weight, and sensitivity difference of the subject, the timing and interval of administration, the properties, formulation, and type of pharmaceutical preparation, the type of active ingredient, etc., and is not particularly limited.

2. Method for treating and/or preventing abnormal protein accumulation neurodegenerative disease The method for treating and/or preventing abnormal protein accumulation neurodegenerative disease of the present invention (hereinafter also referred to as "method of the present invention") comprises the step of administering to a subject suffering from the disease an effective amount of adrenomedullin or a modified form thereof having adrenomedullin activity, or a derivative thereof, or a salt thereof, etc. All necessary content regarding the target disease to be treated, etc. in the method of the present invention, the administered adrenomedullin, its dosage, administration route, administration regimen, combined therapy, etc. may be incorporated in full in the above "1. Agent for treating and/or preventing abnormal protein accumulation neurodegenerative disease".

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these in any way.

Example 1: Investigation of the effect of AM on improving learning and memory impairment in mice receiving a single intracerebroventricular administration of Aβ The effect of adrenomedullin on improving learning and memory impairment in mice receiving a single intracerebroventricular administration of amyloid β, an Alzheimer's disease model animal, was evaluated using a Y-maze test.

(Test animals)
In the above test, 5-week-old Slc:ddY male mice (Japan SLC Co., Ltd.) were obtained and subjected to a 5-day quarantine period followed by a 3-day acclimation period. During this period, body weight was measured three times (electronic balance: PB3002-S/FACT, ME3002, Mettler Toledo Co., Ltd.) and general condition was observed once a day. Animals showing no abnormalities in body weight transition or general condition were used for group allocation. Group allocation was performed by stratifying the weight using a computer program (IBUKI, Japan Bio Research Center Co., Ltd.), and then random sampling was performed so that the average weight of each group was approximately equal.

(Group composition in the study)
Each group consisted of 8 animals, as follows:
(1) As a control, a sham-operated group was administered intraventricularly with water for injection, orally with a vehicle (0.5% methylcellulose (Metolose (registered trademark) SM-100 (Shin-Etsu Chemical Co., Ltd.) (hereinafter also referred to as "MC"), and intranasally with physiological saline),
(2) A vehicle control group (Vehicle) in which Aβ (PolyPeptide Laboratories) was administered intracerebroventricularly, vehicle (0.5% MC) was administered orally, and saline was administered intranasally.
(3) Aβ was administered intracerebroventricularly, AM (Peptide Institute, Inc.) was administered intranasally, and vehicle (0.5% MC) was administered orally, AM 1.2 μg/body group;
(4) an AM 12 μg/body group in which Aβ was administered intracerebroventricularly, AM was administered intranasally, and vehicle (0.5% MC) was administered orally; and (5) a donepezil 0.5 mg/kg group in which Aβ was administered intracerebroventricularly, donepezil hydrochloride was administered orally as a positive control, and saline was administered intranasally.

(Substances for producing animal models and test substances in tests, etc.)
Amyloid β protein (25-35) (PolyPeptide Laboratories) was dissolved in water for injection to a concentration of 2 mM to prepare an Aβ solution, which is a substance for producing an animal model.
AM (10 mg/vial (peptide content 83.6%) (Peptide Institute)) was completely dissolved in 1 ml of water for injection to obtain a homogeneous solution (8.36 mg/ml). It was then diluted (69.2-fold and 692-fold) with physiological saline to prepare the test substances AM (12 μg/ml) and AM (120 μg/ml).
As a positive control for the above test substances, donepezil hydrochloride (Eisai Co., Ltd.) was ground in an agate mortar, and 0.5% MC was added in small amounts to the specified concentration. The positive control substance was prepared in salt equivalent (conversion factor: 1.10).

(Trial Administration Protocol)
The volume of the 0.5% MC and positive control substance administered was set at 10 ml/kg based on the body weight of the mouse on the day of administration. The administration route was oral, and the number of administrations was once a day, a total of nine times. On the day of Aβ injection, the administration was performed after Aβ injection. On the day of Y-maze test, the administration was performed one hour before the test.
The amount and route of administration of physiological saline and test substance was 5 μl per nostril per mouse, and administered into both nasal cavities (10 μl/both nostrils). The number of administrations was once a day, a total of nine times. On the day of Aβ injection, administration was performed after Aβ injection. On the day of Y-maze test, administration was performed 1 hour before the test.

(Exam Schedule)
The administration start date was set as day 1, and the Aβ solution was injected on day 3. Then, the Y-maze test was performed on day 9.

(Observation of general condition and weight measurement)
The general condition of the mice was observed once a day before administration. The body weight was measured (electronic balance: ME3002, Mettler Toledo Co., Ltd.) on each day of administration. The body weight was measured before administration.

(Method of Aβ injection in the study, etc.)
Mice were anesthetized by intraperitoneal administration of 40 mg/kg of Somnopentyl (64.8 mg/mL as pentobarbital sodium, Kyoritsu Seiyaku Co., Ltd.) (administration volume: 10 mL/kg). After anesthesia, levobupivacaine hydrochloride (POPSCAINE (registered trademark) 0.25% injection, Maruishi Pharmaceutical Co., Ltd.) was administered subcutaneously (0.1 ml) to the scalp. The hair on the top of the mouse's head was shaved, and the head was fixed in a brain stereotaxic apparatus. The scalp was disinfected with isodine and then incised to expose the skull, and the connective tissue on the skull was removed with a cotton swab, and then dried with a blower to make the position of the bregma easier to see. A hole for inserting a stainless steel pipe was made in the skull 1 mm lateral (right side) and 0.2 mm posterior to the bregma using a dental drill. A stainless steel pipe connected to a silicone tube with an outer diameter of 0.5 mm and a microsyringe was inserted vertically to a depth of 2.5 mm from the bone surface. 3 μl (6 nmol/3 μL) of Aβ solution (3 μl of water for injection in the sham operation group) was injected into the ventricle over 3 minutes using a microsyringe pump. After injection, the stainless steel pipe was left inserted for 3 minutes and then slowly removed. The skull hole was blocked with a non-absorbable bone marrow hemostatic agent (Nestop (registered trademark) Alfresa Pharma Corporation), and the scalp was sutured. The mouse was removed from the stereotaxic apparatus and returned to the breeding cage. The stainless steel pipe and silicone tube used were sterilized.

(Y-maze test (spontaneous alternation behavior test) protocol)
For the test, a plastic Y-shaped maze (Unicom Co., Ltd.) was used, with each arm being 39.5 cm long, the floor being 4.5 cm wide, the walls being 12 cm high, and three arms each branching at 120 degrees.
Regarding the measurement method in the test, first, before the measurement, the illuminance of the maze floor was adjusted to 10-40 lx (actual measurement: 10.0-12.9 lx). The measurement was performed 1 hour after administration on the 9th day. The mouse was placed in one of the arms of the Y-shaped maze and allowed to freely explore the maze for 8 minutes. The order of the arms into which the mouse moved during the measurement time was recorded, and the number of times the mouse moved to an arm was counted and used as the total number of entries. Next, the combinations in which the mouse consecutively selected three different arms were examined, and this number was used as the number of spontaneous alternation behaviors. Furthermore, the following formula:

The rate of spontaneous alternation behavior was calculated using the formula: As an example of calculation, if the arms of a Y-maze are A, B, and C, and the animal moves in the order ACBABACBAB, the number of spontaneous alternation behaviors is ACB, CBA, BAC, ACB, CBA, which is 5. This is calculated by dividing the total number of entries (10) by 8 (subtracting 2 from the total number of entries), then multiplying the result by 100 to obtain 62.5, which is the rate of spontaneous alternation behavior (rate of spontaneous alternation behavior (%) = [5/(10-2)] x 100 = 62.5).

(Test result 1)
For the Y-maze test (total number of entries, number of spontaneous alternations, and rate of spontaneous alternations), the mean and standard error for each group were calculated.
Significant difference tests were performed between the sham surgery group and the vehicle control group, between the vehicle control group and the donepezil group, and between the vehicle control group and the AM-administered group. For the two-group comparison test, a test for equal variance was performed using the F test, and after confirming equal variance, a Student's t-test was performed. For the multiple comparison test, a test for equal variance was performed using the Bartlett test, and after confirming equal variance, a Dunnett test was performed.
The significance level was set at 5%, and the results were divided into less than 5% (p<0.05) and less than 1% (p<0.01). A commercially available statistical program (SAS System, SAS Institute Japan, Ltd.) was used to test for significance.

(Test result 2)
The results of the Y-maze test are shown in Table 1. The values in the table are the mean ± standard error (standard deviation).

As shown in Table 1, the spontaneous alternation behavior rate (%) was significantly lower in the vehicle control group compared to the sham-operated group, indicating short-term memory impairment. In addition, the spontaneous alternation behavior rate (%) was significantly higher in the positive control substance donepezil 0.5 mg/kg group compared to the vehicle control group, indicating improvement in short-term memory impairment. The above results first confirmed that this Y-maze test system was valid.

Next, the AM 1.2μg/body group showed a significant increase in the rate of spontaneous alternation behavior (%) compared to the vehicle control group, and the reproducibility of the improving effect on short-term memory impairment was confirmed. A significant increase in the rate of spontaneous alternation behavior (%) was also observed in the high-dose 12μg/body group, demonstrating the improving effect on short-term memory impairment. A significant increase in the rate of spontaneous alternation behavior (%) was observed in all AM groups compared to the vehicle control group, but the difference between the doses was small between the AM 1.2μg/body group and the AM 12μg/body group.
From the above results, the improvement effect of AM on short-term memory impairment in mice with a single intracerebroventricular administration of β-amyloid was observed at 1.2μg/body. Regarding dose-response, a plateau may have been reached in the AM 1.2μg/body group, suggesting a dose-response at a smaller dose. In addition, the clear effect of intranasal administration is considered to be a great advantage for the future spread of AM.

Furthermore, in this test system, Aβ was administered directly into the ventricles of the brain and accumulated therein to mimic the symptoms of Alzheimer's disease. Considering that short-term memory impairment in Alzheimer's disease was improved by administration of adrenomedullin, which is thought to have no cholinesterase inhibitory or NMDA receptor antagonistic effects, it was suggested that adrenomedullin inhibits the accumulation of Aβ in the ventricles of the brain or degrades Aβ itself. Therefore, it was suggested that the agent of the present invention is effective not only for Alzheimer's disease, but also for diseases based on neurodegeneration caused by the accumulation of Aβ, such as age-related macular degeneration.

Use of the agent of the present invention makes it possible to treat and/or prevent abnormal protein accumulation neurodegenerative diseases. In addition, since the agent of the present invention is thought to act through a different mechanism of action from cholinesterase inhibitors and NMDA receptor antagonists, which are existing treatments for Alzheimer's disease, or VEGF inhibitors, which are existing treatments for age-related macular degeneration, it may become a new drug for patients for whom the above drugs have not been effective.

This application is based on patent application No. 2019-220695 filed in Japan, the contents of which are incorporated in their entirety into this specification.

Claims (5)

A therapeutic and/or prophylactic agent for an amyloid β -accumulating neurodegenerative disease, comprising adrenomedullin or a modified form thereof having adrenomedullin activity, or a salt thereof,
The adrenomedullin or a modified form thereof having adrenomedullin activity is selected from the group consisting of: (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) A peptide according to (ii), in which the disulfide bond is replaced by an ethylene group, and which has adrenomedullin activity;
(iv) A peptide in which the 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 which has adrenomedullin activity.
(v) a peptide selected from the group consisting of any one of the peptides (i) to (iv) in which the C-terminus is amidated, and (vi) any one of the peptides (i) to (iv) in which a glycine residue has been added to the C-terminus. The adrenomedullin or a modified form thereof having adrenomedullin activity is
(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and 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: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 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: 5, or a peptide consisting of the amino acid sequence of SEQ ID NO: 5 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: 7, or a peptide consisting of the amino acid sequence of SEQ ID NO: 7 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: 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 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: 11, or a peptide consisting of the amino acid sequence of SEQ ID NO: 11 and in which the cysteine residues at positions 14 and 19 form a disulfide bond;
(g) A peptide according to any one of (a) to (f), in which the disulfide bond is replaced by an ethylene group, and which has adrenomedullin activity.
(h) a peptide in which the 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 (a) to (g), and which has adrenomedullin activity;
The agent according to claim 1, which is a peptide selected from the group consisting of: (i) any one of the peptides (a) to (h) in which the C-terminus is amidated; and (j) any one of the peptides (a) to (h) in which a glycine residue has been added to the C-terminus. The method according to claim 1 or 2 , wherein the amyloid β -accumulating neurodegenerative disease is Alzheimer's disease. The agent according to any one of claims 1 to 3 , which is for nasal administration. The agent according to any one of claims 1 to 4 , wherein 0.06 to 600 μg/kg of adrenomedullin is administered per subject.