JP6143270B2 - Myostatin inhibitory peptide - Google Patents

Description

  The present invention relates to a novel peptide that selectively inhibits the action of myostatin (GDF8), a skeletal muscle growth inhibitory factor, and a pharmaceutical composition containing the peptide.

  Muscular dystrophy is a hereditary disease in which muscular dystrophy progresses mainly due to skeletal muscle degeneration / necrosis. The expression of muscle force requires a mechanism that transmits tension generated in intracellular myofibrils to the extracellular basement membrane via a plurality of proteins. A malfunction of a gene encoding a protein group involved in this impairs the function of the protein, resulting in muscular dystrophy. For example, in the most severe Duchenne type, the dystrophin gene is mutated, and the deletion or dysfunction of the protein is the main cause. Therefore, as a means to combat degenerative necrosis of skeletal muscle in muscular dystrophy, the development of a treatment method that increases muscle mass by inhibiting the function of “myostatin”, a factor that negatively regulates skeletal muscle mass. It is considered particularly useful.

Myostatin is secreted as a precursor protein in the same manner as TGF-β, and the prodomain part remains as late associated protein (LAP) and interacts with the myostatin body to inactivate myostatin. And it is latent in an extracellular matrix through the coupling | bonding with latent TGF-beta binding protein (LTBP), and acts as a myostatin reservoir in the body. That is, when necessary, LAP is enzymatically removed to become an active form. From this, it was predicted that a peptide structure capable of binding to myostatin and suppressing its activity exists in the LAP part.
In 2004, Jiang et al. Reported that this recognition sequence exists at positions 42 to 115 of the precursor sequence (Non-patent Document 1). More recently, Walton et al. Pointed out the importance of the α-helix present at positions 42-57 of the TGF-β1 precursor sequence (corresponding to positions 50-65 of mouse myostatin) as this recognition sequence ( Non-patent document 2).

In addition, several studies have been reported that show that inhibition of myostatin function leads to an increase in skeletal muscle mass. For example, myostatin-related myopathy has been reported due to a myostatin gene abnormality in humans and a plurality of animals (Non-patent Document 3). Furthermore, it has been reported that in mice in which the gene is deficient, muscle mass is increased 2-3 times (Non-patent Document 4). These results not only position myostatin as an effective molecular target in drug development for the purpose of enhancing skeletal muscle mass, but also increase muscle mass and strengthen muscle strength by inhibiting myostatin activity. Proof of concept (POC) This is an established drug discovery issue.
Therefore, if a highly active compound with excellent target molecule selectivity and high safety can be created, it is extremely useful for the development of a therapeutic drug that overcomes muscular dystrophy. However, specific examples of drug discovery at present are mainly examples of using high molecular weight proteins. That is, in a clinical trial (Phase II) using a specific neutralizing antibody against myostatin (MYO-029), a muscle hypertrophy effect in patients with Becker and limb girdle muscular dystrophy has been confirmed (Non-patent Document 5). In studies using myostatin soluble receptor (ActRIIB-Fc), cancer cachexia has been reported to be improved by increasing muscle mass in cancer-bearing mice and reducing muscle destruction by cancer cells (non-patented). Reference 6). On the other hand, a kinase inhibitor SB431542 that inhibits phosphorylation of Smad2 responsible for intracellular signal transmission of the TGF-beta family is known. Although this inhibitor is known to suppress intracellular signal transduction of myostatin (Non-patent Document 7), the selectivity between TGF-beta families is low. Therefore, it can be said that a molecule that inhibits the function of myostatin itself is useful as an inhibitor of myostatin signal having high selectivity.

  Thus, a compound useful as a pharmaceutical having excellent target molecule selectivity and high safety and high inhibitory activity against human and animal myostatin has not yet been found.

BBRC, 315, 525 (2004) JBC, 285, 17029 (2010) Schuelke, M., et al., N Engl J Med, 350, 2682 (2004) McPherron, A. C., et al., Nature, 387, 83 (1997) Wagner, K. R., et al., Ann Neurol, 63, 561 (2008) Zhou, X., et al., Cell, 142, 531 (2010) Watt, K.I., et al., Muscle Nerve, 41, 624 2010).

  An object of the present invention is to provide a therapeutic agent that is selective against myostatin and has a high inhibitory activity, and enables muscle hyperplasia and muscle weight increase.

  The inventors of the present invention synthesized a partial peptide in a mouse-derived myostatin prodomain sequence and intensively searched for a peptide that inhibits the activity of human / mouse myostatin. It was demonstrated for the first time in the world that it has inhibitory activity and strongly inhibits the signal through the human myostatin receptor in a concentration-dependent manner.

That is, the present invention
[1] Any peptide selected from the following (a) to (d) or a pharmaceutically acceptable salt thereof:
(A) any one of SEQ ID NOs: 1 to 9, or a peptide comprising the amino acid sequence represented by 40
Sequence number 1: AWRQNTRYSRIEAIKIQILSKLRLETA
Sequence number 2: AWRQNTRYSRIEAIKIQILSKLRL
Sequence number 3: NTRYSRIEAIKIQILSKLRLETA
Sequence number 4: SRIEAIKIQILSKLRLETA
Sequence number 5: NTRYSRIEAIKIQILSKLRL
Sequence number 6: WRQNTRYSRIEAIKIQILSKLRL
Sequence number 7: AWRQNTRYSRIEAIKIEILSKLRL
Sequence number 8: AWRQNTRYSRIEAIKIQILSKLR
Sequence number 9: AWRQNTRYSRIEAIKIQILSKL
SEQ ID NO: 40: WRTRYSRIEAIKIQILSKLRL
(B) any one of SEQ ID NOs: 1 to 9, or a peptide comprising the amino acid sequence represented by 40 and having a length of 30 amino acids or less;
(C) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 or 40, wherein the amino acid sequence is substituted, deleted or added with 1 to 6 amino acids, and selectively inhibits the activity of myostatin A peptide to inhibit; or (d) a derivative of the peptide of any one of (a) to (c) above.
[2] A peptide in which the derivative is chemically modified by covalent bonding of any type of molecule to any of the peptides (a) to (c), or any of the above (a) to (c) The peptide according to [1] or a pharmaceutically acceptable salt thereof, which is an Fc fusion protein comprising the peptide.
[3] The derivative is represented by the following formula (I):
(Chemical formula 1)
R ¹ —X—R ² (I)
(Wherein R ¹ represents a hydrogen atom, an acyl group, a sulfonyl group or a hydrocarbon group:
R ² represents a hydrogen atom, an alkoxy group, a polyethylene glycol-like group, a hydrocarbon group, or an amine group:
X is a peptide residue of the peptide according to any one of (a) to (c) of claim 1 or a peptide residue from which one amino acid residue at both ends or one end of the peptide residue is deleted. Represent. ) Or a pharmaceutically acceptable salt thereof according to [1] or [2].
[4] In the peptide of (c), in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 or 40, at least one of the following substitutions:
(1) The alanine at position 32 from the N-terminus of the mouse-derived myostatin prodomain is substituted with phenylalanine, homophenylalanine, histidine or tyrosine;
(2) Glutamic acid number 31 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(3) The lysine having the number 40 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(4) Tyrosine number 27 from the N-terminus of the myostatin prodomain derived from mouse is replaced with phenylalanine, tryptophan, histidine or arginine;
(5) One or more amino acids of numbers 22, 23, 24, 28, 31, 34, 36, 39, 40, 42 from the N-terminus of the mouse-derived myostatin prodomain are replaced with another amino acid:
(6) Carboxylic acid wherein the tryptophan number 21 from the N-terminus of the mouse-derived myostatin prodomain has a C1-C10 fatty chain or at least one of an aromatic ring and / or a heterocyclic ring Or substitution with amino acids:
(7) one or more of isoleucines of numbers 30, 33, 35 and 37 from the N-terminus of the mouse-derived myostatin prodomain are each replaced by leucine or norleucine;
(8) The leucine numbered 43 from the N-terminus of the mouse-derived myostatin prodomain is replaced by isoleucine or norleucine;
The peptide according to any one of [1] to [3], or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence obtained.
[5] In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, the peptide of (c) is an alanine at position 32 from the N-terminus of the mouse-derived myostatin prodomain is phenylalanine, The peptide according to any one of [1] to [3] or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence substituted with homophenylalanine, histidine or tyrosine.
[6] The peptide of (c) is an amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, and glutamic acid number 31 from the N-terminus of the mouse-derived myostatin prodomain is α-aminoisobutane The peptide according to any one of [1] to [3], which consists of an amino acid sequence substituted with an acid, or a pharmaceutically acceptable salt thereof.
[7] In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, the peptide of (c) is α-aminoisobutane whose lysine is number 40 from the N-terminal of the mouse-derived myostatin prodomain The peptide according to any one of [1] to [3], which consists of an amino acid sequence substituted with an acid, or a pharmaceutically acceptable salt thereof.
[8] The peptide of (c) is an amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, wherein tyrosine having a number 27 from the N-terminus of a mouse-derived myostatin prodomain is phenylalanine, tryptophan, The peptide according to any one of [1] to [3], or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence substituted with histidine or arginine.
[9] In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, the peptide of (c) has a number from the N-terminus of the mouse-derived myostatin prodomain of 22, 23, 24, 28, The peptide according to any one of [1] to [3], or a pharmacy thereof, comprising an amino acid sequence in which any one or more of 31, 34, 36, 39, 40, 42 is substituted with another amino acid. Acceptable salt.
[10] In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, the peptide of (c) has a number from the N-terminal of the mouse-derived myostatin prodomain of 22, 23, 24, 28, The peptide according to [9] or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence in which any one or more of 31, 34, 36, 39, 40, 42 is substituted with alanine.
[11] The peptide of (c) is the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, and the tryptophan number 21 from the N-terminus of the mouse-derived myostatin prodomain is carbon number [1] to [3], comprising an amino acid sequence substituted with a carboxylic acid or an amino acid having at least one of a C1-C10 fatty chain or an aromatic ring and / or a heterocyclic ring. Or a pharmaceutically acceptable salt thereof.
[12] The carboxylic acid is 1-adamantaneacetic acid, tetrahydroisoquinoline-3-carboxylic acid, 3- (2-fluorophenyl) -propionic acid, 3- (3-fluorophenyl) -propionic acid or 3- (4- The peptide according to [11] or a pharmaceutically acceptable salt thereof selected from fluorophenyl) -propionic acid.
[13] The peptide of [11] or a pharmaceutically acceptable salt thereof, wherein the amino acid is acetyl-L-tryptophan.
[14] The peptide of (c) is an amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, and the numbers from the N-terminus of the mouse-derived myostatin prodomain are 30, 33, 35 and 37 The peptide or a pharmaceutically acceptable salt thereof according to any one of [1] to [3], wherein any one or more isoleucines are each composed of an amino acid sequence substituted with leucine or norleucine.
[15] In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, the peptide of (c) is a leucine having a number 43 from the N-terminus of a mouse-derived myostatin prodomain is isoleucine or norleucine The peptide according to any one of [1] to [3], which consists of a substituted amino acid sequence, or a pharmaceutically acceptable salt thereof.
[16] The peptide of any one of [1] to [3] or a pharmaceutically acceptable salt thereof, wherein the peptide of (c) consists of the amino acid sequence represented by SEQ ID NOs: 10 to 48 .
[17] In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 or 40, at least one of the following substitutions:
(1) The alanine at position 32 from the N-terminus of the mouse-derived myostatin prodomain is substituted with phenylalanine, homophenylalanine, histidine or tyrosine;
(2) Glutamic acid number 31 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(3) The lysine with number 40 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(4) Tyrosine number 27 from the N-terminus of the myostatin prodomain derived from mouse is replaced with phenylalanine, tryptophan, histidine or arginine;
(5) One or more amino acids of numbers 22, 23, 24, 28, 31, 34, 36, 39, 40, 42 from the N-terminus of the mouse-derived myostatin prodomain are replaced with another amino acid:
(6) The tryptophan number 21 from the N-terminus of the mouse-derived myostatin prodomain is a carboxylic acid having at least one of a C1-C10 aliphatic chain, or an aromatic ring and / or a heterocyclic ring Replace with amino acid:
(7) one or more of isoleucines of numbers 30, 33, 35 and 37 from the N-terminus of the mouse-derived myostatin prodomain are each replaced by leucine or norleucine;
(8) The leucine numbered 43 from the N-terminus of the mouse-derived myostatin prodomain is replaced by isoleucine or norleucine;
Or a derivative thereof, or a pharmaceutically acceptable salt thereof.
[18] A pharmaceutical composition for treating a myostatin-related disorder, comprising the peptide according to any one of [1] to [17] or a pharmaceutically acceptable salt thereof.
[19] The pharmaceutical composition according to [18], wherein the myostatin-related disorder is a muscular atrophy disorder.
[20] The pharmaceutical composition according to [19], wherein the muscular atrophy disorder is at least one selected from Duchenne muscular dystrophy, progressive muscular dystrophy, Becker muscular dystrophy, Dejerin Landusy muscular dystrophy, elve muscular dystrophy, or pediatric neuroaxon muscular dystrophy object.
Is provided.

  The peptide of the present invention has selective and high inhibitory activity against myostatin. In addition, since the peptide of the present invention realizes the principle of myostatin inhibition in the living body with a small peptide molecule as it is, it is possible to create a highly selective pharmaceutical compound with few side effects.

Construction of prodomain-Fc fusion protein expression plasmid Analysis of activity inhibition center of prodomain by in vitro myostatin transcription activity measurement system Identification result of myostatin activity inhibition center by co-expression of prodomain-Fc fusion protein Identification result of myostatin activity inhibition center by co-expression of prodomain-Fc fusion protein Results of study on inhibition of myostatin activity by addition of synthetic peptides Comparison of inhibition of myostatin activity by various synthetic peptides Results of evaluation of myostatin inhibitory activity of peptides 1, 2, 3 and 5 by in vitro reporter assay Concentration dependence of peptide 1 myostatin inhibitory activity Concentration dependence of peptide 2 myostatin inhibitory activity Concentration dependence of myostatin inhibitory activity of peptide 3 Evaluation result of myostatin inhibitory activity of peptides 1, 4, 6 and 7 by in vitro reporter assay Evaluation results of myostatin signal inhibitory activity of peptides 1-3 and 8 by Western blot Results of evaluation of TGF-β3 inhibitory activity of peptide 1 by in vitro reporter assay Effects of peptide 1 on wild-type mouse thigh muscles (in vivo evaluation) Results of evaluation of TGF-β3 inhibitory activity of peptide 10 by in vitro reporter assay Evaluation of myostatin inhibitory activity of peptides 13 and 14 by in vitro reporter assay Evaluation results of myostatin inhibitory activity of peptides 15 to 22 by in vitro reporter assay Evaluation results of myostatin inhibitory activity of peptides 23-29 by in vitro reporter assay Evaluation results of myostatin inhibitory activity of peptide 30 and peptide 31 by in vitro reporter assay Evaluation result of myostatin inhibitory activity of peptides 32-36 by in vitro reporter assay Evaluation results of myostatin inhibitory activity of peptide 37 and peptide 38 by in vitro reporter assay Results of evaluation of myostatin inhibitory activity of peptides 39-44 by in vitro reporter assay Results of evaluation of myostatin inhibitory activity of peptide 45 by in vitro reporter assay Evaluation result of myostatin inhibitory activity of peptide 46 by in vitro reporter assay Results of evaluation of myostatin inhibitory activity of peptides 47-53 by in vitro reporter assay Effect of peptide 1 on C2C12 myoblasts (in vitro evaluation) Effect of peptide 1 on wild-type mouse thigh muscle (in vivo evaluation) (HE staining) Effect of peptide 1 on wild-type mouse thigh muscle (in vivo evaluation) (statistical analysis of cell number) Effect of peptide 1 on thigh muscle of wild type mouse (in vivo evaluation) (immunostaining)

One embodiment of the present invention is any peptide selected from the following (a) to (d) or a pharmaceutically acceptable salt thereof:
(A) any one of SEQ ID NOs: 1 to 9, or a peptide comprising the amino acid sequence represented by 40
(B) any one of SEQ ID NOs: 1 to 9, or a peptide comprising the amino acid sequence represented by 40 and having a length of 30 amino acids or less;
(C) the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 or 40, wherein the amino acid sequence is substituted, deleted or added with 1 to 6 amino acids, and selectively activates myostatin activity A peptide to inhibit; or (d) a derivative of any of the peptides (a) to (c). The peptide of the present invention or a pharmaceutically acceptable salt thereof can selectively inhibit the action of the skeletal muscle growth inhibitory factor myostatin (GDF8).

  As used herein, “inhibiting myostatin activity” is demonstrated by in vitro assays such as, for example, myostatin activity assays based on CAGA12-luc reporter-introduced cells, or by in vivo animal tests as described below. As such, it refers to the ability of a peptide or pharmaceutical composition comprising it to reduce or block myostatin activity or signaling.

The amino acid sequence of the peptide (a) is shown in the following table.

Peptide As used herein, the term “peptide” refers to a molecule of amino acids linked by peptide bonds. The peptides of the present invention are preferably between about 10-30 amino acids in length, more preferably between 15-25 amino acids in length, and are capable of binding to myostatin protein.

The peptides of the present invention comprise an active sequence present in the prodomain of native mouse myostatin.
The peptides consisting of the amino acid sequences represented by SEQ ID NOs: 1-6 and 8-9 of the present invention are partial peptides of the prodomain of mouse-derived myostatin. For example, taking SEQ ID NO: 1 as an example, mMPS (20-46) described in Table 1 indicates that it has an amino acid sequence from the 20th to the 46th amino acid sequence from the N-terminus in the mouse-derived myostatin prodomain sequence. .
In addition, mMPS (20-43) Q36E of SEQ ID NO: 7 is an amino acid sequence from the 20th to the 43rd amino acid sequence from the N-terminus in the mouse-derived myostatin prodomain sequence, and the 36th Q (glutamine) from the N-terminus is E (glutamic acid). ) To have the amino acid sequence substituted. In addition, mMPS (21-43) ΔQ23N24 of SEQ ID NO: 40 lacks the 23rd Q and the 24th N from the N-terminal in the 21st to 43rd amino acid sequences from the N-terminal in the mouse-derived myostatin prodomain sequence. Indicates having a missing amino acid sequence.

  As the peptide of the above (a), any one of SEQ ID NOs: 1 to 9 or a peptide consisting of the amino acid sequence represented by 40 is preferred, The peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 6 to 7, or 40 is more preferable.

  The peptide of the present invention includes any one of SEQ ID NOS: 1 to 9, or an amino acid sequence represented by 40, and a peptide having a length of 30 amino acids or less (hereinafter also referred to as “(b) peptide”). . The length of the amino acid is preferably 10-30, more preferably 15-25.

The peptide of the present invention consists of an amino acid sequence in which any one of SEQ ID NOs: 1 to 9, or 40 is substituted, deleted or added in the amino acid sequence represented by 40, and has the activity of myostatin. Peptides that selectively inhibit (hereinafter also referred to as “peptide of (c)”).
Here, in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 or 40, 1 to 6 amino acids are substituted, deleted or added means any and one or more amino acids in the same sequence Meaning that there is a substitution, deletion or addition of 1 to 6 amino acid residues (preferably 1 or more, 5 or less, more preferably 1 or more and 3 or less, more preferably 1 or 2) at positions in the sequence. , Substitutions, deletions or additions may occur simultaneously. In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9, or 40, examples of the amino acid sequence in which 1 to 6 amino acid residues are substituted, deleted or added include (1) SEQ ID NOs: 1 to 1 1 or 6 or less (preferably 1 or more and 5 or less, more preferably 1 or more and 3 or less, more preferably 1 or 2) of the amino acid sequence represented by any one of 9 or 40 , (2) any one of SEQ ID NOs: 1 to 9, or 40 to the amino acid sequence represented by 40 (preferably 1 or more and 5 or less, more preferably 1 or more and 3 or less, more preferably 1 or 2) An amino acid sequence to which an amino acid is added, (3) any one of SEQ ID NOS: 1 to 9, or 1 to 6 in the amino acid sequence represented by 40 (preferably 1 to 5 and preferably 1 to 3; Further Preferably, the amino acid sequence in which the amino acid of 1 or 2) is substituted with another amino acid, or (4) an amino acid sequence in which the mutations in (1) to (3) above are combined (in this case, the sum of the mutated amino acids is 1 Or more and 6 or less (preferably 1 or more and 5 or less, more preferably 1 or more and 3 or less, and still more preferably 1 or 2)).

  Examples of amino acids to be substituted or added include normal amino acids (for example, 20 kinds of L-amino acids known as amino acids constituting proteins, namely L-alanine, L-asparagine, L-aspartic acid, L-arginine, L- Glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L- Examples include but are not limited to tyrosine, L-valine and L-cysteine, such as tert-leucine, norleucine, norvaline, 2-aminobutanoic acid, O-methylserine, t-butylglycine, t-butyl. Alanine, cyclohexylalanine, isoaspartic acid, a Other amino acids such as glutamic acid, 2-aminoadipic acid, 2-aminosuberic acid, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, 3-hydroxyproline, 4-hydroxyproline, homoserine, and In addition to naturally occurring amino acids, abnormal amino acids (for example, natural amino acids may be obtained, amino acids that can be created by chemical synthesis, protecting groups and fluorescent groups used in peptide synthesis, amines and nitrogen in the side chain. If there is an acylated, sulfonated, phosphorylated amino acid, if there is a carboxyl group in the side chain, it is esterified, amidated, reduced amino acid, if there is a hydroxyl group in the side chain, etherified, phosphorylated, sulfated Or an amino acid substituted with a halogen or nitro group, an amino acid having another functional group, Aminoalkyl carboxylic acids, α-alkyl amino acids such as aminoisobutanoic acid, amino acids having a heterocyclic ring in the side chain such as pyridine, mono- and polysubstituted β-amino acids, heterocyclic compounds having an amino group and a carboxyl group as substituents , Cyclic compounds having amine and carboxylic acid as substituents, such as aminocyclohexane and aminofurylacetic acid, amino acids such as aminophenylacetic acid, tryptophan derivatives with modified indole ring, histidine derivatives with modified imidazole ring, β-haloalanine , A haloalkylalanine, an amino acid having a carboxyl group or a carboxylic acid in the carbon part of the cyclic amine, tranexamic acid, levodoba, methyldopa, melphalan, baclofen), or both.

  In the peptide of (c) above, the addition can be located at either or both ends of the peptide, or can be located within an internal region of the amino acid sequence of the peptide.

  In the peptide (b) above, the deletion can also be achieved by removal of one or more residues at one or both ends of the peptide or within the amino acid sequence of the peptide.

In addition, the peptide of (c) above includes substitution mutants of peptides consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 or 40. These mutants may be anything that can improve activity or stability to biological enzymes, but substitution mutants have one or more amino acid residues removed and one or more other amino acids. And the amino acid to be exchanged can be naturally occurring or non-naturally occurring. A substitution variant produces a peptide that is “similar” to the original peptide in that the two molecules have the same amino acid in a certain proportion. The number of substitutions can be up to 20 percent of the amino acids of the peptide.
Furthermore, the variants include sequences due to conservative structural changes of each amino acid residue. For example, leucine to isoleucine, serine to threonine, glutamine to glutamic acid, asparagine to aspartic acid, lysine to arginine, and the like.

  The identity and similarity of related peptides can be easily calculated by known methods. Such methods include Computational Molecular Biology, Lesk, A. et al. M.M. Supervision, Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D. W. Supervised, Academic Press, New York (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A .; M.M. And Griffin, H .; G. Supervised, Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G. , Academic Press (1987); Sequence Analysis Primer, Gribskov, M .; And Devereux, J. et al. Supervised by M.M. Stockton Press, New York (1991); and Carillo et al., SIAM J. et al. Applied Math. 48: 1073 (1988), but is not limited thereto.

  In one aspect of the present invention, the peptide of (c) is any one of SEQ ID NOS: 1 to 9, or the amino acid sequence represented by 40, wherein the number from the N-terminal of the mouse-derived myostatin prodomain is 32nd position. Is a peptide consisting of an amino acid sequence substituted with alanine. Here, the amino acid substituting alanine at position 32 is preferably selected from phenylalanine, homophenylalanine, histidine or tyrosine.

  In one aspect of the present invention, the peptide of the above (c) is any one of SEQ ID NOS: 1 to 9, or the amino acid sequence represented by 40, wherein the number from the N-terminal of the mouse-derived myostatin prodomain is position 31. It is a peptide consisting of an amino acid sequence substituted with glutamic acid. Here, the amino acid that replaces glutamic acid at position 31 is preferably α-aminoisobutanoic acid.

  In one aspect of the present invention, in the peptide of (c), the amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 or 40, the number from the N-terminal of the mouse-derived myostatin prodomain is position 40. It is a peptide consisting of an amino acid sequence substituted with lysine. Here, the amino acid that substitutes lysine at position 40 is preferably α-aminoisobutanoic acid.

  In one aspect of the present invention, the peptide of (c) above is any one of SEQ ID NOs: 1 to 9, or the amino acid sequence represented by 40, wherein the number from the N-terminal of the mouse-derived myostatin prodomain is at position 27. It is a peptide consisting of an amino acid sequence in which the tyrosine is substituted. Here, the amino acid replacing tyrosine at position 27 is preferably selected from phenylalanine, tryptophan, histidine or arginine.

  In one aspect of the present invention, the peptide of (c) is any one of SEQ ID NOs: 1 to 9, or the amino acid sequence represented by 40, wherein the number from the N-terminal of the mouse-derived myostatin prodomain is 22, It is a peptide comprising an amino acid sequence in which any one or more amino acids at positions 23, 24, 28, 31, 34, 36, 39, 40, and 42 are substituted. Moreover, in a preferred aspect, the peptide of (c) above is any one of SEQ ID NOS: 1 to 9, or the amino acid sequence represented by 40, wherein the numbers from the N-terminus of the mouse-derived myostatin prodomain are 22, 23, It is a peptide consisting of an amino acid sequence in which any one or more amino acids of 24, 28, 31, 34, 36, 39, 40, 42 are substituted with alanine.

In one aspect of the present invention, the peptide of (c) is any one of SEQ ID NOS: 1 to 9, or the amino acid sequence represented by 40, wherein the number from the N-terminal of the mouse-derived myostatin prodomain is 21st position. It is a peptide consisting of an amino acid sequence in which tryptophan is substituted. Here, tryptophan at the 21-position is preferably substituted with a carboxylic acid or amino acid having a C1-C10 aliphatic chain, or an aromatic ring and / or a heterocyclic ring.
The fatty chain may be a saturated chain, an unsaturated chain, a chain or a cyclic chain, and may have a branched chain structure. In addition, the fatty chain is a C5 or lower alkoxy chain, amino group, substituted amino group, carboxyl group, ester group, carbamoyl group, substituted carbamoyl group, amide group, substituted amide group, guazino group, nitro group, sulfonic acid group, sulfone. It may have a substituent such as amide or halogen (fluorine, chlorine, bromine, iodine).
In the carboxylic acid having an aromatic ring and / or a heterocyclic ring, the aromatic ring and the heterocyclic ring having a C1-C4 aliphatic chain carboxylic acid, the aromatic ring having a carbon number of C12 or less, and the heterocyclic ring is , Oxygen, nitrogen and sulfur atoms in the ring, the number of heteroatoms being 4 or less and 8 or less carbon atoms. These rings may have a substituent having 0 to 3 substituents. Examples of the substituent include a C6 or less fatty chain, a C5 or less alkoxy chain, an amino group, a substituted amino group, a carboxyl group, an ester group, A carbamoyl group, a substituted carbamoyl group, an amide group, a substituted amide group, a guazino group, a nitro group, a sulfonic acid group, a sulfonamide, a halogen and the like. The substituent is composed of 6 atoms or less.
As the carboxylic acid having a C1-C10 aliphatic chain or an aromatic ring and / or a heterocyclic ring, 1-adamantaneacetic acid, tetrahydroisoquinoline-3-carboxylic acid, 3- (2-fluorophenyl) is preferable. ) -Propionic acid, 3- (3-fluorophenyl) -propionic acid or 3- (4-fluorophenyl) -propionic acid. Moreover, as an amino acid which has a C1-C10 aliphatic chain or an aromatic ring and / or a heterocyclic ring, Preferably, it is acetyl-L-tryptophan.

  In one aspect of the present invention, the peptide of (c) above has an amino acid sequence represented by any one of SEQ ID NOs: 1 to 9 and 40, and the number from the N-terminus of the mouse-derived myostatin prodomain is 30,33. , 35, and 37. A peptide comprising an amino acid sequence substituted with at least one isoleucine. Here, the amino acid substituting for isoleucine is preferably leucine or norleucine. When two or more isoleucines are substituted, all may be substituted with leucine, all may be substituted with norleucine, or may be substituted with leucine and norleucine.

  In one aspect of the present invention, the peptide of (c) above is a leucine having a number 43 from the N-terminus of a mouse-derived myostatin prodomain in the amino acid sequence represented by any of SEQ ID NOs: 1 to 9 and 40 Is a peptide consisting of a substituted amino acid sequence. Here, the amino acid substituting leucine is preferably isoleucine or norleucine.

In another aspect of the present invention, the peptide of (c) above is any one of SEQ ID NOS: 1 to 9, or 40 in the amino acid sequence represented by 40:
(1) The alanine at position 32 from the N-terminus of the mouse-derived myostatin prodomain is substituted with phenylalanine, homophenylalanine, histidine or tyrosine;
(2) Glutamic acid number 31 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(3) The lysine with number 40 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(4) Tyrosine number 27 from the N-terminus of the myostatin prodomain derived from mouse is replaced with phenylalanine, tryptophan, histidine or arginine;
(5) One or more amino acids of numbers 22, 23, 24, 28, 31, 34, 36, 39, 40, 42 from the N-terminus of the mouse-derived myostatin prodomain are replaced with another amino acid:
(6) Tryptophan number 21 from the N-terminus of the mouse-derived myostatin prodomain is replaced with a carboxylic acid or amino acid substituted with a bulky fatty chain structure or aromatic ring:
(7) one or more of isoleucines of numbers 30, 33, 35 and 37 from the N-terminus of the mouse-derived myostatin prodomain are each replaced by leucine or norleucine;
(8) The leucine numbered 43 from the N-terminus of the mouse-derived myostatin prodomain is replaced by isoleucine or norleucine;
It is a peptide consisting of an amino acid sequence.

  In still another aspect of the present invention, the peptide (c) is a peptide consisting of the amino acid sequence represented by SEQ ID NOs: 10 to 48.

  The peptide of the present invention includes derivatives of any of the peptides (a) to (c) (hereinafter also referred to as “(d) peptide”). As used herein, the term “derivative” refers to modifications other than or in addition to insertions into amino acid residues, deletions or substitutions of amino acid residues that retain the ability to bind to myostatin. Specifically, a peptide chemically modified by covalent bonding of any type of molecule to any one of the peptides (a) to (c) above, or any one of the peptides (a) to (c) above An Fc fusion protein comprising

Preferably, the modifications made to the peptides of the invention to yield a derivative are covalent and include, for example, chemical linkage with polymers, lipids, other organic moieties, and inorganic moieties.
On the other hand, the peptide of the present invention is a peptide having an α-helix as a secondary structure, and for enhancing the inhibitory activity of myostatin, a derivative or in vivo enzyme that further enhances the α-helix property of the peptide of the present invention. Derivatives converted into a structure that is difficult to be decomposed by the above are also encompassed in the present invention. The former may include a derivative in which a salt bridge is formed at the i, i + 4 position, etc., and a derivative whose outer side is cross-linked by a disulfide bond, a carbon-carbon bond, or the like as the amino acid arrangement. For example, a derivative that forms a salt bridge is SEQ ID NO: 7. Furthermore, the derivative which improved the structure which is easy to be decomposed | disassembled by the in-vivo enzyme is also contained.

The typical derivative may be anything as long as it can improve activity or improve stability to biological enzymes, and particularly includes the following compounds:
1. The N-terminus is derivatized. Typically, the N-terminus can be modified with (1) a C1-C20 fatty acid, which includes an alkyl group, alkenyl group, alkynyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group Furthermore, benzoic acid derivatives such as p-fluorobenzoyl, aryl and heteroaryl carboxylic acids, etc., and (2) modified with nucleic acid or vitamin derivatives containing carboxylic acid, such as orotic acid and 5-fluoroorotic (3) including those that are PEGylated or saccharified with polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, etc., and (4) protecting groups commonly used in peptide synthesis (for example, Boc group, Z group, etc.) and those modified with carbamate derivatives (5) including those modified with carboxylic acids that are easily derived from amino acids (eg, pyroglutamic acid, morotanoic acid, etc.), and (7) sulfonic acid derivatives such as benzenesulfonic acid, phosphoric acid derivatives, Furthermore, it can be modified with a C1-C20 hydrocarbon group. Furthermore, they may contain an N-terminal amino group to form a cyclic structure.
Typical N-terminal derived groups include acetyl, propionyl, caproyl, palmitoyl, benzoyl, substituted benzoyl, oxazole carboxyl, imidazole carboxyl, orotynyl, 5-fluoroorotynyl, lactic acid, pyruvate, gluconic acid, tertiary butyloxycarbonyl , Benzyloxycarbonyl, pyroglutamyl, ethyl, dimethyl, acetamidomethyl, benzenesulfonyl, piperidino, morpholino, piperazino, imidazolino, tisazolidino and pyrrolidino groups in which the amino group itself is converted.

  2. The C-terminus is derivatized. Typically, the C-terminus is amidated (eg, an alkylamide such as ethylamide) or esterified. The amine part and the alcohol part of the amide and ester may be anything as long as they can improve the activity or improve the stability to the biological enzyme, but generally may contain carbon atoms of C0 to C10. , Amines and alcohols containing nitrogen, sulfur, halogen, and the like. The alcohol includes polyhydric alcohols, sugars, alcohols having an ether structure, and the like. The amine can also be modified with biogenic amines or amines that are easily derived from amino acids. Typical C-terminal derivative groups include ethylamide, isopropylamide, benzylamide, cyanoethylamide, ethyl ester, isopropyl ester, benzyl ester, glucose and the like. In addition, a polyethylene glycol analog having a hydroxyl group or an amino group at the terminal that contributes to the improvement of pharmacokinetics may be bonded by an ester bond or an amide bond, and the polyethylene glycol has 4 to 20 carbon atoms, and the polyethylene glycol group The terminal on the opposite side may be modified with a general protecting group used for protecting a hydroxyl group such as an alkyl group. Polyethylene glycol analogs include polyethylene glycol, polyoxyethylene glycol, polypropylene glycol, and the like.

式中、Ｒ_３は、水素原子、アルキル基又はＯ−アルキル基であり、好ましくは、水素原子、低級アルキル基又は低級Ｏ−アルキル基であり；Ｒ_４は、水素原子、アルキル基、脂肪鎖スルホキシル基又は芳香族スルホキシル基であり、好ましくは、水素原子又は低級アルキル基であり；Ｒ_５は、水素原子、アルキル基又はＯ−アルキル基であり、好ましくは、水素原子、低級アルキル基又は低級Ｏ−アルキル基である。式（１）において、各々のＲ_５は同じであっても異なっていてもよい。また、ｑは０〜３の整数である。
Ｒ^１のアシル基として用いられるビタミンおよびビタミン誘導体はとくに限定されない。例えば、ニコチン酸、ｐ−パントテン酸、ビオチン、プテロイルグルタミン酸、オロチン酸、フルオロオロチン酸、リポ酸、５−ピリドキシン酸、ビオチンｐ−，Ｌ−スルホキシド、ビオチンスルホン、ビオシチン、プテロイン酸、１０−ホルミルプテロイン酸、７，８−ジヒドロ葉酸、（−）Ｌ−Ｈ，葉酸、ホモプロテイン酸、６−カルボキシプテリン、ジヒドロリポ酸、ハイドロオロチン酸などが挙げられる。
さらに、式（Ｉ）における核酸の塩基またはその誘導体とは、一般にヌクレオチドを構成する塩基成分およびその誘導体を指すが、好ましくはピリミジン誘導体等、例えば５−カルボキシメチルウラシル、５−カルボキシチオウラシル等を例示することができる。
Ｒ^１がスルホニル基である場合は、上記アシル基で説明したのと同等であり、スルホニル基には、アシル基のカルボニル構造をスルホン構造に変換した構造を有するものが含まれる。
Ｒ^１が炭化水素基である場合は、その炭素数がＣ１〜Ｃ１０であって、炭化水素鎖は飽和鎖、不飽和鎖、鎖状、環状のものであって良く、さらに分岐鎖構造を取ってもよい。芳香族環および複素環を有する炭化水素基においては、炭素数Ｃ０〜Ｃ３の炭化水素鎖を有する芳香族環および複素環で、芳香族環はＣ１２以下の炭素数であり、複素環は、酸素、窒素、イオウ原子を環内に含むもので、ヘテロ原子の数が４個以下で炭素数８以下のものを指す。これらの環には置換基数０〜３の置換基があっても良く、置換基としては、それぞれＣ６以下の脂肪鎖、Ｃ５以下のアルコキシ鎖、アミノ基、置換アミノ基、カルボキシル基、エステル基、カルバモイル基、置換カルバモイル基、アミド基、置換アミド基、グアジノ基、ニトロ基、スルホン酸基、スルホンアミド、ハロゲンなどである。置換基は、原子６以下からなる。
Ｒ^１として、例えば、アセチル、プロピオニル、カプロイル、パルミトイル、ベンゾイル、置換ベンゾイル、オキサゾールカルボキシル、イミダゾールカルボキシル、オロチニル、５−フルオロオロチニル、ラクチル、ピルビル、グルコリル又はピログルタミル、ターシャリーブチルオキシカルボニル、ベンジルオキシカルボニル、エチル、ジメチル、アセトアミドメチル、ベンゼンスルホニル、アミノ基自体が変換されたピペリジノ、モルホリノ、ピペラジノ、イミダゾリノ、チソゾリジノ又はピロジニノが挙げられるが、これらに限定されない。In one aspect of the present invention, the peptide of (d) (a derivative of any peptide of (a) to (c)) is represented by the following formula (I):
Is a peptide represented by ^{R 1 -X-R 2 (I} ).
In formula (I), R ¹ represents a hydrogen atom, an acyl group, a sulfonyl group, or a hydrocarbon group.
When R ¹ is an acyl group, acyl groups derived from various carboxylic acids are included. That is, an acyl group derived from a carboxylic acid having an aliphatic chain, a carboxylic acid having an aromatic ring and a heterocyclic ring. The acyl group also includes an acyl group selected from the group consisting of amino acid derivatives, vitamins having a free acyl group and vitamin derivatives, and nucleobase derivatives having a free acyl group.
In the carboxylic acid having an aliphatic chain, the carbon number thereof is C1 to C10, and the aliphatic chain may be a saturated chain, an unsaturated chain, a chain, or a cyclic chain, and may have a branched chain structure. good. In addition, the fatty chain is a C5 or lower alkoxy chain, amino group, substituted amino group, carboxyl group, ester group, carbamoyl group, substituted carbamoyl group, amide group, substituted amide group, guazino group, nitro group, sulfonic acid group, sulfone. It may have a substituent such as amide or halogen (fluorine, chlorine, bromine, iodine). The fatty chain may be a C4 to C15 polyether chain having a plurality of oxygen atoms.
The carboxylic acid having an aromatic ring and a heterocyclic ring is an aromatic ring and a heterocyclic ring having a C1-C4 aliphatic chain carboxylic acid, the aromatic ring having a carbon number of C12 or less, and the heterocyclic ring is an oxygen , Nitrogen and sulfur atoms are included in the ring, and the number of heteroatoms is 4 or less and 8 or less carbon atoms. These rings may have a substituent having 0 to 3 substituents. Examples of the substituent include a C6 or less fatty chain, a C5 or less alkoxy chain, an amino group, a substituted amino group, a carboxyl group, an ester group, A carbamoyl group, a substituted carbamoyl group, an amide group, a substituted amide group, a guazino group, a nitro group, a sulfonic acid group, a sulfonamide, a halogen and the like. The substituent is composed of 6 atoms or less.
Examples of the acyl group derived from a carboxylic acid having an aromatic ring and a heterocyclic ring when the hetero ring is an indole derivative include various indole rings, and the acyl group represented by the following formula (1) is Can be mentioned.

In the formula, R ₃ is a hydrogen atom, an alkyl group or an O-alkyl group, preferably a hydrogen atom, a lower alkyl group or a lower O-alkyl group; R ₄ is a hydrogen atom, an alkyl group or an aliphatic chain. A sulfoxyl group or an aromatic sulfoxyl group, preferably a hydrogen atom or a lower alkyl group; R ₅ is a hydrogen atom, an alkyl group or an O-alkyl group, preferably a hydrogen atom, a lower alkyl group or a lower group; O-alkyl group. In the formula (1), each R ₅ may be the same or different. Moreover, q is an integer of 0-3.
The vitamin and vitamin derivative used as the acyl group for R ¹ are not particularly limited. For example, nicotinic acid, p-pantothenic acid, biotin, pteroylglutamic acid, orotic acid, fluoroorotic acid, lipoic acid, 5-pyridoxic acid, biotin p-, L-sulfoxide, biotinsulfone, biocytin, pteroic acid, 10-formyl Examples include pteroic acid, 7,8-dihydrofolic acid, (−) LH, folic acid, homoprotein acid, 6-carboxypterin, dihydrolipoic acid, hydroorotic acid and the like.
Furthermore, the nucleic acid base or derivative thereof in the formula (I) generally refers to a base component constituting a nucleotide and a derivative thereof, and preferably a pyrimidine derivative such as 5-carboxymethyluracil, 5-carboxythiouracil, etc. It can be illustrated.
When R ¹ is a sulfonyl group, it is the same as described for the acyl group, and the sulfonyl group includes those having a structure obtained by converting the carbonyl structure of the acyl group into a sulfone structure.
When R ¹ is a hydrocarbon group, the carbon number thereof is C1 to C10, the hydrocarbon chain may be a saturated chain, an unsaturated chain, a chain or a ring, and further has a branched chain structure. May be. In the hydrocarbon group having an aromatic ring and a heterocyclic ring, the aromatic ring and the heterocyclic ring having a hydrocarbon chain having a carbon number of C0 to C3, the aromatic ring having a carbon number of C12 or less, and the heterocyclic ring is oxygen , Nitrogen and sulfur atoms are included in the ring, and the number of heteroatoms is 4 or less and 8 or less carbon atoms. These rings may have a substituent having 0 to 3 substituents. Examples of the substituent include a C6 or less fatty chain, a C5 or less alkoxy chain, an amino group, a substituted amino group, a carboxyl group, an ester group, A carbamoyl group, a substituted carbamoyl group, an amide group, a substituted amide group, a guazino group, a nitro group, a sulfonic acid group, a sulfonamide, a halogen and the like. The substituent is composed of 6 atoms or less.
R ¹ may be, for example, acetyl, propionyl, caproyl, palmitoyl, benzoyl, substituted benzoyl, oxazole carboxyl, imidazole carboxyl, orotynyl, 5-fluoroorotynyl, lactyl, pyruvir, glucolyl or pyroglutamyl, tertiary butyloxycarbonyl, benzyloxy Examples include, but are not limited to, carbonyl, ethyl, dimethyl, acetamidomethyl, benzenesulfonyl, piperidino, morpholino, piperazino, imidazolino, tisazolidino, or pirodinino in which the amino group itself is converted.

R ² represents a hydrogen atom, an alkoxy group, a polyethylene glycol-like group, a hydrocarbon group, or an amine group.
In the ester structure in which R ² has an alkoxy group, the carbon number thereof is C1 to C18, and the hydrocarbon portion may be a saturated chain, an unsaturated chain, a chain, or a ring, and further has a branched chain structure. May be. In the hydrocarbon portion having an aromatic ring and a heterocyclic ring, the aromatic ring and the heterocyclic ring having a C0-C4 hydrocarbon chain, the aromatic ring has a carbon number of C12 or less, and the heterocyclic ring is oxygen , Nitrogen and sulfur atoms are included in the ring, and the number of heteroatoms is 4 or less and 8 or less carbon atoms. These rings may have a substituent of 0 to 3 substituents, and the substituents are C6 or less hydrocarbon chain, C5 or less alkoxy chain, amino group, substituted amino group, carboxyl group, ester group, respectively. Carbamoyl group, substituted carbamoyl group, amide group, substituted amide group, guazino group, nitro group, sulfonic acid group, sulfonamide, halogen and the like. The substituent is composed of 6 atoms or less.
In the structure of ester or amide in which R ² has a polyethylene glycol-like group, polyethylene glycol having a hydroxyl group or an amino group at the terminal, which contributes to the improvement of pharmacokinetics, may be bound by an ester bond or an amide bond. The number of carbon atoms of the glycol is 4 to 20, and the terminal on the opposite side of the polyethylene glycol group may be modified with a general protecting group used for protecting a hydroxyl group such as an alkyl group.
The ketone structure in which R ² has a hydrocarbon group is also defined in the same manner as the alkoxy group.
When R ² is an amine group, the substituent on the nitrogen may be hydrogen, a hydrocarbon group or an amine group, and either may be on one or both on the nitrogen atom.
The hydrocarbon group has C1 to C18 carbon atoms, and the hydrocarbon portion may be a saturated chain, unsaturated chain, chain, or cyclic group, and may have a branched chain structure. In the hydrocarbon portion having an aromatic ring and a heterocyclic ring, the aromatic ring and the heterocyclic ring having a C0-C4 hydrocarbon chain, the aromatic ring has a carbon number of C12 or less, and the heterocyclic ring is oxygen , Nitrogen and sulfur atoms are included in the ring, and the number of heteroatoms is 4 or less and 8 or less carbon atoms. These rings may have a substituent of 0 to 3 substituents, and the substituents are C6 or less hydrocarbon chain, C5 or less alkoxy chain, amino group, substituted amino group, carboxyl group, ester group, respectively. Carbamoyl group, substituted carbamoyl group, amide group, substituted amide group, guazino group, nitro group, sulfonic acid group, sulfonamide, halogen and the like. The substituent is composed of 6 atoms or less.
The amine group is an amino group (which becomes hydrazine) or a substituted amino group, and the substituent is hydrogen or a hydrocarbon group, both of which may be on one or both on the nitrogen atom. The hydrocarbon group has C1 to C18 carbon atoms, and the hydrocarbon portion may be a saturated chain, unsaturated chain, chain, or cyclic group, and may have a branched chain structure. In the hydrocarbon portion having an aromatic ring and a heterocyclic ring, the aromatic ring and the heterocyclic ring having a C0-C4 hydrocarbon chain, the aromatic ring has a carbon number of C12 or less, and the heterocyclic ring is oxygen , Nitrogen and sulfur atoms are included in the ring, and the number of heteroatoms is 4 or less and 8 or less carbon atoms. These rings may have a substituent of 0 to 3 substituents, and the substituents are C6 or less hydrocarbon chain, C5 or less alkoxy chain, amino group, substituted amino group, carboxyl group, ester group, respectively. Carbamoyl group, substituted carbamoyl group, amide group, substituted amide group, guazino group, nitro group, sulfonic acid group, sulfonamide, halogen and the like. The substituent is composed of 6 atoms or less.
R ² is, for example, a hydrogen atom, an ethylamino group, an isopropylamino group, a benzylamino group, a cyanoethylamino group, an ethoxy group, an isopropoxy group, a benzyloxy group, or polyethylene glycol, polyoxyethylene glycol, polypropylene glycol, or glucosyl group. However, it is not limited to these.

  X represents a peptide residue of the peptide according to any one of the above (a) to (c) or a peptide residue in which one amino acid residue at both ends or one end of the peptide residue is deleted. ).

  The Fc fusion protein containing any peptide of (a) to (c) above is obtained by adding Fc to the N-terminal or / and C-terminal of any peptide of (a) to (c). Here, Fc is a non-antigen-binding fragment of an antibody, specifically, human complete Fc derived from human immunoglobulin or the like. Other proteins or peptides include antibodies, fragments of polypeptides that are part of antibodies. These antibodies or polypeptide fragments can be, for example, glycosylated, acetylated, PEGylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, Others may be chemically modified.

  The peptides of the present invention can be prepared by any method known in the art, including chemical synthesis, protein digestion, or recombinant techniques. Phage display and RNA-peptide screening, and other affinity screening techniques are particularly useful for generating peptides that can bind to myostatin.

  In order to prepare the peptide of the present invention by chemical synthesis, each amino acid is prepared by a method commonly used in peptide chemistry, for example, “The Peptides”, Volume 1 [by Schroder and Luhke, Academic Press, New York, USA. (1966)], “Basics and Experiments of Peptide Synthesis” (Nobuo Izumiya et al., Maruzen Co., Ltd. (1985)) and the like. It can be produced by any of the methods. Furthermore, any of a column method and a batch method can be used.

  Condensation methods for forming peptide bonds include azide method, acid halide method, acid anhydride method, carbodiimide method, carbodiimide-additive method, active ester method, carbonylimidazole method, redox method, enzyme method, Woodward reagent K , Methods using HATU reagent, Bop reagent, and the like. In addition, the condensation reaction in the solid phase method includes the acid anhydride method, the carbodiimide method, and the active ester method as the main methods among the methods described above.

  Further, when the peptide chain is extended by the solid phase method, it is bound to a support such as a resin insoluble in an organic solvent using a C-terminal amino acid. Here, a resin with a functional group introduced for the purpose of binding an amino acid to the resin, a resin with a spacer inserted between the resin and the functional group, and a chain called a handle that can be cut at various points depending on conditions are introduced. The resin can also be used depending on the purpose. Examples of such resins include halomethyl resins such as chloromethyl resins, oxymethyl resins, 4- (oxymethyl) -phenylacetamidomethyl resins, 4- (oxymethyl) -phenoxymethyl resins, and C-terminal amidation resins. And so on. Before carrying out these condensation reactions, protective means such as a carboxyl group, amino group, hydroxyl group and amidino group which are not involved in the condensation reaction can be applied by a generally known means. Conversely, carboxyl groups and amino groups that are directly involved in the condensation reaction can also be activated.

  Protective groups used for protecting functional groups that do not participate in the condensation reaction of each unit are those commonly used in organic chemistry, such as `` Protective Groups in Organic Synthesis (by Greene, It can be protected by a protecting group described in John Wiley & Sons, Inc. (1981)].

  Examples of the protecting group for the carboxyl group include conventionally known protecting groups such as various methyl esters, ethyl esters, benzyl esters, p-nitrobenzyl esters, t-butyl esters, and cyclohexyl esters. Examples of amino-protecting groups include benzyloxycarbonyl group, t-butoxycarbonyl group, isobornyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group and the like.

  Examples of activated carboxyl groups include acid anhydrides corresponding to the carboxyl groups; azides; pentafluorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, and N-hydroxysuccinic acid. Examples thereof include active esters with imide, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxyphthalimide, 1-hydroxybenzotriazole and the like.

  Examples of activated amino groups include phosphoric acid amides corresponding to the amino groups. The condensation reaction for peptide synthesis is usually performed in a solvent. Examples of the solvent include chloroform, dichloromethane, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, N-methylpyrrolidone, water, methanol, and the like, or a mixture thereof. . Moreover, the reaction temperature of the said condensation reaction can be performed in the range of -30 degreeC-50 degreeC similarly to the normal case.

  Furthermore, the type of the protecting group elimination reaction in the production process of the peptide of the present invention should be selected according to the type of the protecting group used as long as the protecting group can be eliminated without affecting the peptide bond. Can do. For example, acid treatment with hydrogen chloride, hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof, sodium hydroxide, potassium hydroxide, hydrazine, diethylamine, piperidine, etc. And alkali treatment with liquid ammonia, sodium treatment in liquid ammonia, reduction with palladium carbon, and silylation treatment of trimethylsilyl triflate, trimethylsilyl bromide, and the like. In the above deprotection group reaction by treatment with an acid or silylating agent, the addition of a cation scavenger such as anisole, phenol, cresol, thioanisole, ethanedithiol effectively performs the deprotection group reaction. It is preferable in that

  The method for cleaving the peptide of the present invention synthesized from the solid phase method from the solid phase also follows a generally known method. For example, the treatment with the above acid or silylating agent can be mentioned as the cleavage method. For the peptide of the present invention thus produced, generally known separation and purification means can be used after completion of the above series of reactions. For example, the peptide of the present invention can be obtained in a more pure form by extraction, distribution, reprecipitation, recrystallization, column chromatography and the like.

  Phage display technology is also described, for example, in Scott et al., Science 249: 386 (1990); Devlin et al., Science 249: 404 (1990); US Pat. No. 5,223,409, each incorporated herein by reference. , Issued June 29, 1993; US Pat. No. 5,733,731, issued March 31, 1998; US Pat. No. 5,498,530, issued March 12, 1996; No. 432,018, issued July 11, 1995; US Pat. No. 5,338,665, issued August 16, 1994; US Pat. No. 5,922,545, issued July 13, 1999; WO 96/40987, published 19 December 1996; and WO 98/15833, published 16 April 1998. A phage library is used to display random peptide sequences by fusion with the coat protein of filamentous phage.

  Other methods of preparing the peptides of the present invention include additional affinity selection techniques known in the art. It is also possible to fuse the peptide library at the carboxyl terminus of the lac repressor and express it in E. coli. Another method based on E. coli allows display on the outer membrane of cells by fusion with peptidoglycan-associated lipoprotein (PAL). In the present specification, these methods and related methods are hereinafter collectively referred to as “E. coli display”. In another method, translation of random RNA is stopped prior to ribosome release, resulting in a library of polypeptides with associated RNA still attached.

Examples of the pharmaceutically acceptable salt of the peptide of the present invention include acid addition salts, metal salts, organic base addition salts and the like. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate and phosphate, and organic acid salts such as acetate, maleate, fumarate, tartrate and citrate. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like. Examples of organic base addition salts include primary amines such as methylamine, ethylamine and aniline, secondary amines such as dimethylamine, diethylamine, pyrrolidine, piperidine, morpholine and piperazine, trimethylamine, triethylamine, N, N-dimethylaniline and pyridine. And salts formed with tertiary amines such as ammonium salts and the like.

  The peptide of the present invention or a pharmaceutically acceptable salt thereof is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed. Purity of the peptide of the present invention or a pharmaceutically acceptable salt thereof isolated or purified (the peptide of the present invention or a pharmaceutically acceptable salt thereof relative to the total weight of the peptide or the pharmaceutically acceptable salt thereof) The weight ratio is usually 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more (for example, 100%).

Peptide Activity A peptide of the invention or a pharmaceutically acceptable salt thereof binds to myostatin and blocks or inhibits myostatin signaling within the targeted cell.
The peptides of the invention or pharmaceutically acceptable salts thereof preferably bind to human myostatin and block or inhibit myostatin signaling within the targeted cell.

Any type of assay or animal test can be used to determine the ability of the peptides of the invention or pharmaceutically acceptable salts thereof to bind to myostatin and inhibit or block myostatin activity. It is. Several assays used to characterize the peptides of the invention are described in the examples below.
One assay is a reporter assay, in which a reporter (luciferase) gene that responds to a myostatin signal is introduced into a cell, cultured in a medium containing 0.1% serum, and then a reporter upon addition of myostatin. The second assay described below is a myostatin signal activity evaluation by Western blot, and the evaluation method is a myostatin after culturing in a medium containing 0.1% serum. , And the cell lysate separated by polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular weight is electrically transferred to a PVDF (polyvinylidene fluoride) membrane to recognize phosphorylated Smad-2 Phosphorylation of Smad-2 (myostatin signal activation) by adding primary antibody and primary antibody recognition antibody (secondary antibody) It is intended to detect and measure the amount as a band.

Pharmaceutical Composition Another aspect of the present invention is a medicament for treating a myostatin-related disorder, comprising any peptide selected from the above (a) to (d) or a pharmaceutically acceptable salt thereof. It is a composition.
The pharmaceutical composition of the present invention can reduce the amount or activity of myostatin in a subject by administering an effective dose to the subject. In one aspect, the present invention provides a method and pharmaceutical composition for treating a myostatin-related disorder in a subject, wherein the subject comprises an effective pharmaceutical composition comprising one or more peptides or pharmaceutically acceptable salts thereof. What includes administering a dose is provided.
The subject to which the pharmaceutical composition of the present invention is administered includes animals including fish, but preferably humans and industrially useful mammals such as mice, cows, pigs, horses (including racehorses), poultry, etc. Includes animals such as birds.

  These myostatin-related disorders include, but are not limited to, various types of muscle atrophy, as well as metabolic disorders such as diabetes and related disorders, and bone degenerative diseases such as osteoporosis. Muscular atrophy disorders include dystrophies such as Duchenne muscular dystrophy, progressive muscular dystrophy, Becker muscular dystrophy, Degelin Landusy muscular dystrophy, elb muscular dystrophy, and pediatric neuroaxonous muscular dystrophy.

In addition, the pharmaceutical composition of the present invention is also effective for strengthening the anterior tibial muscle by local administration for elderly people who are usually impaired in walking. Simply strengthening the anterior tibial muscle makes it easy to do dorsiflexion of the ankle joint and prevents falls.
The pharmaceutical composition of the present invention can also be used for disuse muscle atrophy. For example, muscular strength reduction by astronauts is particularly significant in muscles that hold posture and walk. If continuous local administration at the time of staying in space can be carried out, it can contribute to shortening the rehabilitation period after returning.
The pharmaceutical composition of the present invention can also be used for inclusion body myositis (IBM).
Furthermore, examples of the systemic administration of the pharmaceutical composition of the present invention include disuse muscle atrophy of bedridden patients and sarcopenia with senile syndrome.

  Additional amyotrophic disorders arise from chronic diseases such as amyotrophic lateral sclerosis, chronic obstructive pulmonary disease (COPD), cancer, AIDS, renal failure, and rheumatoid arthritis. For example, systemic administration of myostatin induced cachexia or muscle atrophy and weight loss in athymic nude mice (Zimmers et al.). Therefore, the pharmaceutical composition of the present invention can be used to improve cachexia accompanied by muscle atrophy.

  In addition, increasing muscle mass by reducing myostatin levels can improve bone strength and reduce osteoporosis and other degenerative bone diseases. For example, it has been found that myostatin-deficient mice show an increase in muscle mass with an increase in the mineral content and density of the mouse humerus and an increase in both the trabecular and cortical bone mineral content of the area where the muscles are attached. (Hamrick et al., Calc Tissue Int 71 (1): 63-8 (2002)).

The present invention also provides an edible animal by administering to the animal an effective dosage of a myostatin binding agent comprising any peptide selected from (a) to (d) above or a pharmaceutically acceptable salt thereof. Methods and reagents for increasing muscle mass in are also provided.
Because mature C-terminal myostatin polypeptides are highly conserved in all species tested, myostatin binding agents are found in any of the agriculturally important species, including cattle, chickens, turkeys, and pigs. Are also expected to be effective in increasing muscle mass and reducing fat.

  The pharmaceutical composition of the invention comprises a therapeutically or prophylactically effective amount of a peptide or a pharmaceutically acceptable salt thereof, as described herein, in combination with a pharmaceutically acceptable agent. . In a preferred embodiment, the pharmaceutical composition comprises an antagonist binding agent that partially or completely inhibits myostatin mixed with a pharmaceutically acceptable agent. Typically, the myostatin binding agent will be sufficiently purified for administration to a subject.

  The pharmaceutical composition of the present invention modifies or maintains, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption or penetration of the composition. It is also possible to contain formulation substances that are to be preserved. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); (Borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids, etc.); bulking agents (such as mannitol or glycine), chelating agents (ethylenediaminetetraacetic acid (EDTA)) Complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (glucose, mannose, or dextrin) Etc.); protein (blood Colorants; flavoring agents and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (benzalkco chloride) Nitrogen, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvent (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohol (such as mannitol or sorbitol); suspension Agent: Surfactant or wetting agent (pluronic, PEG, sorbitan ester, polysorbate 20, polysorbate 80 and other polysorbates, triton, tromethamine, lecithin, cholesterol Stability enhancer (sucrose or sorbitol); tonicity enhancing agent (alkali metal halide (preferably sodium chloride or potassium chloride), mannitol sorbitol); carrier vehicle; Diluents; include excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18th edition, supervised by AR Gennaro, Mack Publishing Company, 1990).

  The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binder.

  The primary vehicle or carrier in the pharmaceutical composition can be either aqueous or non-aqueous. For example, a suitable vehicle or carrier is water for injection, physiological saline solution or artificial cerebrospinal fluid, optionally supplemented with other substances commonly used for parenteral administration. It is also possible. Neutral buffered saline or saline mixed with serum albumin is a further typical vehicle. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or even a suitable substitute. It can also be included. In one embodiment of the invention, the binder composition comprises an optional formulation (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution and a selected composition having the desired degree of purity. It is also possible to prepare a binder composition for storage by mixing. In addition, the binder product can be formulated as a lyophilizate using appropriate excipients such as sucrose.

  It is also possible to select the pharmaceutical composition for parenteral delivery. Alternatively, the composition can be selected for inhalation or enteral delivery, for example oral, ear, eye, rectal or vaginal delivery. Such pharmaceutically acceptable compositions are within the skill of the art.

  Moreover, the pharmaceutical composition of the present invention can be administered in a sustained release system. Suitable examples of sustained release compositions include semipermeable polymer materials in the form of shaped articles, such as films or microcapsules. Sustained release materials include polylactide (US Pat. No. 3,773,919, EP58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamic acid ester (Sidman, U. et al., Biopolymers 22: 547 -556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12 : 98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) Or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). The sustained release pharmaceutical composition may contain a compound encapsulated in liposomes. The liposome-containing pharmaceutical composition is prepared by a method known per se: E 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad Sci. (USA) 77: 4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; Japanese Patent Application No. 83-118008; US Patent No. 4,485 , 045 and 4,544,545; and EP 102,324. Typically, liposomes are of a small (about 200-800 angstrom) monolayer type with a lipid content of about 30 mole percent or more of cholesterol and the selected proportion is adjusted for optimal treatment. . The pharmaceutical composition can also be introduced transdermally by iontophoresis for topical administration. Furthermore, it can be introduced by inhalation into the oral cavity, nasal cavity, pharynx, trachea and lung. On the other hand, in site-selective delivery by systemic and local administration, introduction by a combination of bubble liposomes and ultrasonic waves is also possible.

  In the pharmaceutical composition of the present invention, the peptide or a pharmaceutically acceptable salt thereof is present in an acceptable concentration at the administration site. For example, a buffer is used to maintain the composition within a physiological pH or slightly lower pH, typically within a pH range of about 5 to about 8.

  The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[Example 1]
(1) Identification of Myostatin Prodomain Activity Inhibition Center To identify the activity inhibition center of the prodomain corresponding to the N-terminus of the myostatin precursor on the C-terminal mature myostatin, As shown in Fig. 1, each prodomain region and an immunoglobulin Fc fusion protein expression plasmid were constructed.
Next, as shown in FIG. 2, a reporter plasmid incorporating a luciferase gene downstream of the Smad2 / 3 recognition sequence [(CAGA _{) 12} ], which is an intracellular effector of myostatin, and a prodomain-Fc fusion protein expression plasmid are human. Co-expression in nephroblasts. Transcriptional inhibitory activity was assayed by recombinant myostatin stimulation of the medium.

  The results of searching for the myostatin activity inhibition center by co-expression of the prodomain-Fc fusion protein are shown in FIGS. 3A and 3B. Of the prodomain regions (1 to 8), Prod-2 consisting of 110 amino acid residues showed higher inhibitory activity than the full length prodomain (Prod-1) consisting of 262 amino acid residues. Prod-7 (29 amino acid residues) showing about 70% inhibitory activity was narrowed down from this Prod-2.

(2) Examination of Myostatin Activity Inhibition by Addition of Synthetic Peptide Next, a peptide corresponding to Prod-7 (Prod-7 peptide) and a peptide having a reverse amino acid sequence of Prod-7 (Rev. Prod-7 peptide) as a control ) Were chemically synthesized to compare myostatin activity. As shown in FIG. 4, the addition of synthetic peptide to the medium inhibited myostatin activity by 80% at a final concentration of 1 μM.

(3) Comparison of Myostatin Activity Inhibition of Various Synthetic Peptides Results of measuring myostatin inhibitory activity of Prod-7 peptide and various synthetic peptides (the synthesis methods are described in Examples described later) are shown in FIG. Shown in Each final 1 μM synthetic peptide was added to the medium. Here, peptide 0 is a peptide represented by SEQ ID NO: 1, and has a sequence obtained by removing one residue at the N-terminus and one residue at the C-terminus relative to the Prod-7 peptide. As shown in FIG. 5, Peptide 1 showed almost the same myostatin inhibitory activity as Prod-7 peptide.

mMPS：マウス由来マイオスタチンプロドメインの部分ペプチド
（ ）内は、プロドメインのＮ末端からの番号
hMPS：ヒト由来マイオスタチンプロドメインの部分ペプチド
mMPS(26-41)mut：α−へリックスを破壊した部分ペプチド
Human/mouse TGFβ3 precursor (30-53)：mMPS(20-43)に相当するヒト／マウス由来TGFβ3部分ペプチドPeptides having the following sequence numbers were synthesized in the examples and reference examples.

mMPS: Partial peptide of the myostatin prodomain derived from mouse () is the number from the N-terminal of the prodomain
hMPS: Partial peptide of human myostatin prodomain
mMPS (26-41) mut: Partial peptide with disrupted α-helix
Human / mouse TGFβ3 precursor (30-53): Human / mouse-derived TGFβ3 partial peptide equivalent to mMPS (20-43)

[Example 2]
Synthesis of peptide 1
AWRQNTRYSRIEAIKIQILSKLRL-NH ₂ (Peptide 1)
Peptide 1 having SEQ ID NO: 2 was synthesized by the Fmoc solid phase peptide synthesis method shown below.
Rink amide resin (0.47 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.047 mmol) is weighed in a reaction vessel for solid phase synthesis and left in a dimethylformamide (DMF) solution at room temperature for 2 hours to swell the resin. Then, the protecting group Fmoc (9-fluorenylmethyoxycarbonyl) group on the resin was removed by reacting in a 20% piperidine / DMF solution (3 mL) at room temperature for 20 minutes. The resin was washed 10 times with DMF (3 mL), 1-hydroxybenzotriazole (HOBt) 22 mg (0.141 mmol, 3 eq.), N, N-diisopropylcarbodiimide (DIPCD) 0.023 mL (0.141 mmol, 3 eq.) In the presence, 48 mg (0.141 mmol, 3 eq.) Of Fmoc-Leu-OH was reacted in DMF at room temperature for 2 hours to introduce an amino acid on the resin. In order to condense the next amino acid, the Fmoc group on the resin was removed by reacting in a 20% piperidine / DMF solution (3 mL) for 20 minutes. Thereafter, in the same manner as in the case of Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH (91 mg, 0.141 mmol, 3 eq.), Fmoc-Leu-OH (48 mg, 0.141 mmol, 3 eq. .), Fmoc-Lys (Boc) -OH (66 mg, 0.141 mmol, 3 eq.), Fmoc-Ser (tBu) -OH (91 mg, 0.141 mmol, 3 eq.), Fmoc-Leu-OH (48 mg, 0 141 mmol, 3 eq.), Fmoc-Ile-OH (48 mg, 0.141 mmol, 3 eq.), Fmoc-Gln (Trt) -OH (86 mg, 0.141 mmol, 3 eq.), Fmoc-Ile-OH (48 mg, 0.141 mmol, 3 eq.), Fmoc-Lys (Boc) -OH (6 mg, 0.141 mmol, 3 eq.), Fmoc-Ile-OH (48 mg, 0.141 mmol, 3 eq.), Fmoc-Ala-OH (44 mg, 0.141 mmol, 3 eq.), Fmoc-Glu (OtBu) -OH (60 mg, 0.141 mmol, 3 eq.), Fmoc-Ile-OH (48 mg, 0.141 mmol, 3 eq.), Fmoc-Arg (Pbf) -OH (91 mg, 0.141 mmol, 3 eq.), Fmoc-Ser ( tBu) -OH (91 mg, 0.141 mmol, 3 eq.), Fmoc-Tyr (tBu) -OH (64 mg, 0.141 mmol, 3 eq.), Fmoc-Asn (Trt) -OH (84 mg, 0.141 mmol, 3 eq.) .), Fmoc-Gln (Trt) -OH (86 mg, 0 141 mmol, 3 eq.), Fmoc-Arg (Pbf) -OH (91 mg, 0.141 mmol, 3 eq.), Fmoc-Trp (Boc) -OH (74 mg, 0.141 mmol, 3 eq.), Fmoc-Ala-OH ( 44 mg, 0.141 mmol, 3 eq.) Were introduced to extend the peptide chain. The N-terminal Fmoc group was removed by reacting in a 20% piperidine / DMF solution (3 mL) for 20 minutes, and DMF (3 mL, 10 times), methanol (3 mL, 5 times), chloroform (3 mL, 5 times) in this order. The resin was washed and dried. Trifluoroacetic acid (TFA) in the presence of m-cresol (0.125 mL), thioanisole (0.125 mL), 1,2-ethanedithiol (0.050 mL) for removal of side chain protecting groups and deresin 5. Reacted in 0 mL for 2 hours. After removing the resin by filtration using a funnel with a glass filter, TFA was distilled off under reduced pressure using a rotary evaporator, and 100 mL of diethyl ether was added to precipitate a crude purified peptide. The crude purified peptide was dissolved in 1M acetic acid and purified using high performance liquid chromatography to give a white solid (15.3 mg, 0.0038 mmol, 8.1%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 986.2442, found 986.2116.

[Example 3]
Synthesis of peptide 2
NTRYSRIEAIKIQILSKLRLETA-NH ₂ (Peptide 2)
Peptide 2 having SEQ ID NO: 3 was synthesized and purified in the same manner as in Example 2 using 50 mg (0.024 mmol) of Rink amide resin (0.47 mmol / g, Watanabe Chemical Co., Ltd.) (16.1 mg, 0.0045 mmol, 19%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 543.9262, found 543.9313.

[Example 4]
Synthesis of peptide 3
SRIEAIKIQILSKLRLETA-NH ₂ (Peptide 3)
Peptide 3 having SEQ ID NO: 4 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.47 mmol / g, Watanabe Chemical Co., Ltd.) 50 mg (0.024 mmol) (12.6 mg, 0.0044 mmol, 19%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 437.2657, found 437.2602.

[Example 5]
Synthesis of peptide 4
NTRYSRIEAIKIQILSKLRL-NH ₂ (Peptide 4)
Peptide 4 having SEQ ID NO: 5 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.055 mmol) (8.8 mg, 0.0029 mmol, 5.4%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 805.4960, found 805.4984.

[Example 6]
Synthesis of peptide 11
WRQNTRYSRIEAIKIQILSKLRL-NH ₂ (peptide 11)
Peptide 11 having SEQ ID NO: 6 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.055 mmol) (14.1 mg, 0.0040 mmol, 7.2%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7549.

[Example 7]
Synthesis of peptide 12
AWRQNTRYSRIEAIKIEILSKLRL-NH ₂ (peptide 12)
Peptide 12 having SEQ ID NO: 7 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.055 mmol) (10.8 mg, 0.00264 mmol, 9.6%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 986.2494, found 986.2457.

[Example 8]
Synthesis of peptide 13
AWRQNTRYSRIEAIKIQILSKLR-NH ₂ (Peptide 13)
Peptide 13 having SEQ ID NO: 8 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.055 mmol) (6.7 mg, 0.0018 mmol, 3.3%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 711.422, found 711.4211.

[Example 9]
Synthesis of peptide 14
AWRQNTRYSRIEAIKIQILSKL-NH ₂ (peptide 14)
Peptide 14 having SEQ ID NO: 9 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.055 mmol) (9.5 mg, 0.0026 mmol, 4.7%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 896.1930, found 896.1958.

[Example 10]
Synthesis of peptide 15
WAQNTRYSRIEAIKIQILSKLRL-NH ₂ (peptide 15)
Peptide 15 having SEQ ID NO: 10 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (4.7 mg). 0.0016 mmol, 5.5%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 700.6677, found 700.6661.

[Example 11]
Synthesis of peptide 16
WRANTRYSRIEAIKIQILSKLRL-NH ₂ (Peptide 16)
Peptide 16 having SEQ ID NO: 11 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (4.7 mg). 0.0013 mmol, 5.3%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 566.3442, found 566.3473.

[Example 12]
Synthesis of peptide 17
WRQATRYSRIEAIKIQILSKLRL-NH ₂ (peptide 17)
Peptide 17 having SEQ ID NO: 12 was synthesized and purified in the same manner as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (4.8 mg). , 0.0014 mmol, 6.2%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 711.1823, found 711.1854.

[Example 13]
Synthesis of peptide 18
WRQNARYSRIEAIKIQILSKLRL-NH ₂ (peptide 18)
Peptide 18 having SEQ ID NO: 13 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (2.2 mg 0.00062 mmol, 2.5%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 714.4311, found 714.4288.

[Example 14]
Synthesis of peptide 19
WRQNTAYSRIEAIKIQILSKLRL-NH ₂ (peptide 19)
Peptide 19 having SEQ ID NO: 14 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 80 mg (0.044 mmol) (4.6 mg, 0.0014 mmol, 3.1%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 702.9259, found 702.9250.

[Example 15]
Synthesis of peptide 20
WRQNTRASRIEAIKIQILSKLRL-NH ₂ (Peptide 20)
Peptide 20 having SEQ ID NO: 15 was synthesized and purified by the same method as in Example 2 using 80 mg (0.044 mmol) of Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) (16.6 mg, 0.0048 mmol, 10.8%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 698.9272, found 698.9250.

[Example 16]
Synthesis of peptide 21
WRQNTRYARIEAIKIQILSKLRL-NH ₂ (peptide 21)
Peptide 21 having SEQ ID NO: 16 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 80 mg (0.044 mmol) (35.1 mg, 0.0099 mmol, 21.4%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 574.55496, found 574.5444.

[Example 17]
Synthesis of peptide 22
WRQNTRYSRIAAIKIQILSKLRL-NH ₂ (Peptide 22)
Peptide 22 having SEQ ID NO: 17 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 80 mg (0.044 mmol) (27.7 mg, 0.0079 mmol, 17.0%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 566.1475, found 566.1432.

[Example 18]
Synthesis of peptide 23
WRQNTRYSRIEAIAIQILSKLRL-NH ₂ (peptide 23)
Peptide 23 having SEQ ID NO: 18 was synthesized and purified in the same manner as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (14.0 mg). , 0.0041 mmol, 16.9%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 707.66693, found 707.6689.

[Example 19]
Synthesis of peptide 24
WRQNTRYSRIEAIKIAILSKLRL-NH ₂ (Peptide 24)
Peptide 24 having SEQ ID NO: 19 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (19.2 mg). 0.0055 mmol, 22.7%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 566.3442, found 566.3425.

[Example 20]
Synthesis of peptide 25
WRQNTRYSRIEAIKIQILAKLRL-NH ₂ (Peptide 25)
Peptide 25 having SEQ ID NO: 20 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (7.9 mg). 0.0022 mmol, 11.1%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 574.5496, found 574.5496.

[Example 21]
Synthesis of peptide 26
WRQNTRYSRIEAIKIQILSALRL-NH ₂ (Peptide 26)
Peptide 26 having SEQ ID NO: 21 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.025 mmol) (3.2 mg). 0.00094 mmol, 3.8%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 943.2331, found 943.2232.

[Example 22]
Synthesis of peptide 27
WRQNTRYSRIEAIKIQILSKARL-NH ₂ (peptide 27)
Peptide 27 having SEQ ID NO: 22 was synthesized and purified in the same manner as in Example 2 using 81.5 mg (0.045 mmol) of Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) (18. 1 mg, 0.0051 mmol, 11.4%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 711.4220, found 711.4183.

[Example 23]
Synthesis of peptide 28
WRQNTRYSRIEAIKIQILSKLAL-NH ₂ (peptide 28)
Peptide 28 having SEQ ID NO: 23 was synthesized and purified by the same method as in Example 2 using 81.5 mg (0.045 mmol) of Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) (28. 8 mg, 0.0085 mmol, 19.1%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 700.6677, found 700.6633.

[Example 24]
Synthesis of peptide 29
WRQNTRYSRIEAIKIQILSKLRA-NH ₂ (Peptide 29)
Peptide 29 having SEQ ID NO: 24 was synthesized and purified by the same method as in Example 2 using 81.5 mg (0.045 mmol) of Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) (31. 2 mg, 0.0088 mmol, 19.7%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 569.3391, found 569.3355.

[Example 25]
Synthesis of peptide 30
WRQNTRYSRIEAIKIQILSKLRA-NH ₂ (Peptide 30)
Peptide 30 having SEQ ID NO: 25 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 55 mg (0.014 mmol) (6.0 mg). 0.0017 mmol, 12.3%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 717.9350, found 717.9410.

[Example 26]
Synthesis of peptide 31
WRQNTRWSRIEAIKIQILSKLRL-NH ₂ (peptide 31)
Peptide 31 having SEQ ID NO: 26 was synthesized and purified by the same method as in Example 2 using TentaGel S RAM resin (0.25 mmol / g, Watanabe Chemical Co., Ltd.) 55 mg (0.014 mmol) (5.6 mg). 0.0016 mmol, 11.3%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 727.6877, found 727.6931.

[Example 27]
Synthesis of peptide 32
WRQNTRYSRIEFIKIQILSKLRL-NH ₂ (peptide 32)
Peptide 32 having SEQ ID NO: 27 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (18.2 mg, 0.005 mmol, 24.6%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 592.9548, found 592.9555.

[Example 28]
Synthesis of peptide 33
WRQNTRYSRIEYIKIQILSKLRL-NH ₂ (peptide 33)
Peptide 33 having SEQ ID NO: 28 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (16.0 mg, 0.0044 mmol, 21.5%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 596.1537, found 596.1620.

[Example 29]
Synthesis of peptide 34
WRQNTRYSRIEWIKIQILSKLRL-NH ₂ (peptide 34)
Peptide 34 having SEQ ID NO: 29 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (22.5 mg, 0.0061 mmol, 30.1%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 600.7570, found 600.7562.

[Example 30]
Synthesis of peptide 35
WRQNTRYSRIEHIKIQILSKLRL-NH ₂ (peptide 35)
Peptide 35 having SEQ ID NO: 30 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (13.8 mg, 0.0038 mmol, 18.7%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 590.9529, found 590.9570.

[Example 31]
Synthesis of peptide 36
WRQNTRYSRIEXIKIQILSKLRL-NH ₂ , X = homophenylalanine (peptide 36)
Peptide 36 having SEQ ID NO: 31 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (19.3 mg, 0.0053 mmol, 26.0%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 595.7579, found 595.7608.

[Example 32]
Synthesis of peptide 37
WRQNTRYSRIXAIKIQILSKLRL-NH ₂ , X = α-aminoisobutanoic acid (peptide 37)
Peptide 37 having SEQ ID NO: 32 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (9.7 mg, 0.0028 mmol, 14.0%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 568.9506, found 568.9470.

[Example 33]
Synthesis of peptide 38
WRQNTRYSRIEAIKIQILSXLRL-NH ₂ , X = α-aminoisobutanoic acid (peptide 38)
Peptide 38 having SEQ ID NO: 33 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (27.5 mg, 0.0081 mmol, 39.7%).
HRMS (ES +) calcd for (M ⁴⁺ + 4H) 711.1732, found 711.1719.

[Example 34]
Synthesis of peptide 39
XRQNTRYSRIEAIKIQILSKLRL-NH ₂ , X = 1-adamantaneacetic acid (peptide 39)
Peptide 39 having SEQ ID NO: 34 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (4.2 mg, 0.0012 mmol, 5.8%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 575.7567, found 575.7684.

[Example 35]
Synthesis of peptide 40
XRQNTRYSRIEAIKIQILSKLRL-NH ₂ , X = tetrahydroisoquinoline-3-carboxylic acid (peptide 40)
Peptide 40 having SEQ ID NO: 35 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (3.1 mg, 0.00088 mmol, 4.3%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 572.3464, found 572.3454.

[Example 36]
Synthesis of peptide 41
_{XRQNTRYSRIEAIKIQILSKLRL-NH 2, X = 3-} (2- fluorophenyl) - propionic acid (peptide 41)
Peptide 41 having SEQ ID NO: 36 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (6.5 mg, 0.0018 mmol, 9.1%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 570.5423, found 570.5367.

[Example 37]
Synthesis of peptide 42
XRQNTRYSRIEAIKIQILSKLRL-NH ₂ , X = 3- (3-fluorophenyl) -propionic acid (peptide 42)
Peptide 42 having SEQ ID NO: 37 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (4.4 mg, 0.0012 mmol, 6.1%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 570.5423, found 570.5423.

[Example 38]
Synthesis of peptide 43
XRQNTRYSRIEAIKIQILSKLRL-NH ₂ , X = 3- (4-fluorophenyl) -propionic acid (peptide 43)
Peptide 43 having SEQ ID NO: 38 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (6.5 mg, 0.0018 mmol, 9.1%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 570.5423, found 570.5455.

[Example 39]
Synthesis of peptide 44
XRQNTRYSRIEAIKIQILSKLRL-NH ₂ , X = acetyl-L-tryptophan (peptide 44)
Peptide 44 having SEQ ID NO: 39 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (12.0 mg, 0.0033 mmol, 16.4%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 586.1506, found 586.1483.

[Example 40]
Synthesis of peptide 45
WRTRYSRIEAIKIQILSKLRL-NH ₂ (peptide 45)
Peptide 45 having SEQ ID NO: 40 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (15.6 mg, 0.0047 mmol, 23.1%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 529.3282, found 529.3278.

[Example 41]
Synthesis of peptide 46
WRTRYSRIEAIKIQILSKLRL-OH (Peptide 46)
Peptide 46 having SEQ ID NO: 41 was synthesized and purified in the same manner as in Example 2 using Wang resin (0.73 mmol / g, Watanabe Chemical Co., Ltd.) 30 mg (0.0219 mmol) (10.4 mg, 0 .0029 mmol, 13.2%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.9453, found 577.9456.

[Example 42]
Synthesis of peptide 47
WRQNTRYSRIEAIKIQIISKIRI-NH ₂ (peptide 47)
Peptide 47 having SEQ ID NO: 42 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (10.4 mg, 0.0020 mmol, 10.0%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7452.

[Example 43]
WRQNTRYSRIEAIKIQIXSKXRX-NH ₂ , X = norleucine (peptide 48)
Peptide 48 having SEQ ID NO: 43 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (15.5 mg, 0.0043 mmol, 21.7%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7516.

[Example 44]
WRQNTRYSRLEALKIQILSKLRL-NH ₂ (Peptide 49)
Peptide 49 having SEQ ID NO: 44 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (15.0 mg, 0.0042 mmol, 21.0%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7452.

[Example 45]
WRQNTRYSRIEAIKLQLLSKLRL-NH ₂ (Peptide 50)
Peptide 50 having SEQ ID NO: 45 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (19.3 mg, 0.0054 mmol, 27.0%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7500.

[Example 46]
WRQNTRYSRXEAXKIQILSKLRL-NH ₂ , X = norleucine (peptide 51)
Peptide 51 having SEQ ID NO: 46 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (17.6 mg, 0.0049 mmol, 24.7%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7500.

[Example 47]
WRQNTRYSRIEAXKXQILSKLRL-NH ₂ , X = norleucine (peptide 52)
Peptide 52 having SEQ ID NO: 47 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (20.9 mg, 0.0059 mmol, 29.3%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7520.

[Example 48]
WRQNTRYSRIEAIKXQXLSKLRL-NH ₂ , X = norleucine (peptide 53)
Peptide 53 having SEQ ID NO: 48 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.58 mmol / g, Watanabe Chemical Co., Ltd.) 35 mg (0.020 mmol) (13.0 mg, 0.0036 mmol, 18.2%).
HRMS (ES +) calcd for (M ⁵⁺ + 5H) 577.7485, found 577.7421.

[Example 49]
Synthesis of peptide 0
AWRQNTRYSRIEAIKIQILSKLRLETA-NH ₂ (peptide 0)
Peptide 0 having SEQ ID NO: 1 was synthesized and purified in the same manner as in Example 1 using Rink amide resin (0.47 mmol / g, Watanabe Chemical Co., Ltd.) 50 mg (0.024 mmol) (19.1 mg, 0.0048 mmol, 20.0%).
HRMS (ES +) calcd for (M ⁴⁺ + 5H) 652.1814, found 652.1826.

[Example 50]
Evaluation of Myostatin Inhibitory Activity of Peptide 1 by In Vitro Reporter Assay The evaluation of myostatin inhibitory activity of peptide 1 was performed by the following method. The result is shown in FIG. Furthermore, the result of having evaluated the concentration-dependent myostatin inhibitory ability of peptide 1 is shown in FIG. Peptide 1 had significant myostatin inhibitory activity at a concentration of 30 μM and an IC ₅₀ value of 4.1 μM.
(1) Cell culture Human hepatoma-derived HepG2 cells were prepared using a 10% FBS DMEM (Nakarai quest) medium supplemented with non-essential amino acids (WAKO) and Penicillin / Streptomycin (WAKO) at 37 ° C, 5% CO2. Cultured in ₂ incubators.
(2) In Vitro Reporter Assay One well or 7.5 × 10 ⁴ cells (500 μL DMEM + 10% FBS) of HepG2 cells were seeded on a 24-well white transparent plate (BD Falcon) and cultured for 24 hours. The next day, 0.05 μg (CAGA) 12-luc reporter plasmid per well and 0.01 μg β-galactosidase expression plasmid (pCH110, Pharmacia) as an internal control to a final concentration of PEI (Polyscience, Inc.) of 23 μg / mL And added to the cell culture medium. After culturing these cells at 37 ° C. for 24 hours, the cell culture medium was replaced with DMEM + 0.1% FBS and cultured for 16 hours. Peptide 1 was suspended in DMSO to a stock solution of 3 mM and stored at −30 ° C. One hour before adding to the cells, the cells were suspended in DMEM + 0.1% FBS medium to a final concentration of 30 μM (or 10 μM, 3 μM, 1 μM) and myostatin 10 ng / mL, and incubated at 37 ° C. for 1 hour. And stimulation was performed for 8 hours. The Smad-2 phosphorylation inhibitor SB-431542 (Wako) used as a positive control was suspended in DMSO to a stock solution of 5 mM and stored at -30 ° C. 1 hour before addition to cells, suspended in DMEM + 0.1% FBS medium to a final concentration of 5 μM and myostatin 10 ng / mL, incubated at 37 ° C. for 1 hour, then added to cells and stimulated for 8 hours. went. After stimulation, the culture solution was removed with an aspirator, and the cells were washed with 1 × PBS, and then 100 μL of Lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM CDTA, 10% glycerol, 1% triton x− 100) was added to lyse the cells. The lysate was centrifuged at 13000 rpm for 10 minutes, 30 μL of supernatant and 25 μL of luciferase assay reagent (Promega) were mixed in a white 96-well plate, and luminescence was detected with Centro XS ³ LB 960 (Berthold) for reporter assay. It was. Furthermore, 50 μL of the supernatant and 50 μL of β-gal substrate (200 mM phosphate buffer, ₂ mM MgCl ₂ , 100 mM 2-ME, 1.33 mg / mL ONPG) were mixed in a white transparent 96-well plate (BD Falcon) and incubated at 37 ° C. for 1 hour. After incubation, the absorbance at 420 nm was detected with Sunrise REMOTE (Wako) and used as an internal control.

[Example 51]
Evaluation of Myostatin Inhibitory Activity of Peptide 2 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 2 was evaluated by the same method as in Example 50. The result is shown in FIG. Furthermore, the results of evaluating the concentration-dependent myostatin inhibitory ability of peptide 2 are shown in FIG. Peptide 2 had a relatively high myostatin inhibitory activity at a concentration of 30 μM, and showed some inhibitory activity even at a concentration of 10 μM.

[Example 52]
Evaluation of Myostatin Inhibitory Activity of Peptide 3 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 3 was evaluated by the same method as in Example 50. The result is shown in FIG. Furthermore, the result of having evaluated the concentration-dependent myostatin inhibitory ability of the peptide 3 is shown in FIG. Peptide 3 showed about 50% myostatin inhibitory activity at a concentration of 30 μM.

[Example 53]
Evaluation of Myostatin Inhibitory Activity of Peptide 4 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 4 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptide 4 showed inhibitory activity at a concentration of 30 μM.

[Example 54]
Evaluation of Myostatin Inhibitory Activity of Peptides 13 and 14 by In Vitro Reporter Assay The myostatin inhibitory activity of peptides 13 and 14 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptides 13 and 14 showed inhibitory activity at a concentration of 30 μM.

[Example 55]
Evaluation of Myostatin Inhibitory Activity of Peptides 15-22 by In Vitro Reporter Assay The myostatin inhibitory activity of peptides 15-22 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptides 15-22 showed inhibitory activity at a concentration of 30 μM.

[Example 56]
Evaluation of Myostatin Inhibitory Activity of Peptides 23-29 by In Vitro Reporter Assay The myostatin inhibitory activity of peptides 23-29 was evaluated in the same manner as in Example 50. The result is shown in FIG. Peptides 23-29 showed inhibitory activity at a concentration of 30 μM.

[Example 57]
Evaluation of Myostatin Inhibitory Activity of Peptide 30 and Peptide 31 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 30 and peptide 31 was evaluated by the same method as in Example 50. The result is shown in FIG. Both peptides showed inhibitory activity at a concentration of 10 μM.

[Example 58]
Evaluation of Myostatin Inhibitory Activity of Peptides 32-36 by In Vitro Reporter Assay The myostatin inhibitory activity of peptides 32-36 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptides 32-36 showed inhibitory activity at a concentration of 10 μM.

[Example 59]
Evaluation of Myostatin Inhibitory Activity of Peptide 37 and Peptide 38 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 37 and peptide 38 was evaluated by the same method as in Example 50. The result is shown in FIG. Both peptides showed inhibitory activity at a concentration of 10 μM.

[Example 60]
Evaluation of Myostatin Inhibitory Activity of Peptides 39-44 by In Vitro Reporter Assay The myostatin inhibitory activity of peptides 39-44 was evaluated by the same method as in Example 50. The result is shown in FIG. Both peptides showed inhibitory activity at a concentration of 10 μM.

[Example 61]
Evaluation of Myostatin Inhibitory Activity of Peptide 45 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 45 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptide 45 showed inhibitory activity at a concentration of 10 μM.

[Example 62]
Evaluation of Myostatin Inhibitory Activity of Peptide 46 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 46 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptide 46 showed inhibitory activity at a concentration of 30 μM.

[Example 63]
Evaluation of myostatin inhibitory activity of peptides 47-53 by in vitro reporter assay The myostatin inhibitory activity of peptides 47-53 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptides 47-53 showed inhibitory activity at a concentration of 10 μM.

[Example 64]
Evaluation of Myostatin Signal Inhibitory Activity of Peptide 1 by Western Blot Evaluation of myostatin signal inhibitory activity of Peptide 1 was performed by the following method. The result is shown in FIG. Peptide 1 remarkably suppressed phosphorylation of Smad-2 at a concentration of 30 μM, which revealed that it has high myostatin signal inhibitory activity.
(1) Cell culture HepG2 cells were cultured in the same manner as in Example 50.
(2) Western blot HepG2 cells were seeded on a 12-well white transparent plate (BD Falcon) for 1 well or 1.5 × 10 ⁵ cells and cultured for 24 hours. On the next day, the cell culture medium was replaced with DMEM + 0.1% FBS and cultured for 16 hours. Peptide 1 is mixed with DMEM + 0.1% FBS medium to a final concentration of 30 μM and myostatin 10 ng / mL and incubated at 37 ° C. for 1 hour, and then added to the cells. Stimulated for hours. SB-431542 (Wako) was suspended in DMEM + 0.1% FBS medium to a final concentration of 5 μM and myostatin 10 ng / mL and incubated at 37 ° C. for 1 hour 1 hour before addition to the cells. Added and stimulated for 1 hour. After stimulation, the culture solution was removed with an aspirator, washed with 1 × PBS, and then 200 μL of Lysis buffer (10 mM Tris-phosphate (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 40 mM NaF, 20 mM pyrroline per well. (Sodium acid, 2 mM sodium orthovanadate, 1 mM PMSF, 5 μg / mL leupeptin, 20 units / mL aprotinin) were added to lyse the cells. The lysate was centrifuged at 13000 rpm, 4 ° C. for 10 minutes, and the supernatant and an equal volume of 2 × Sample Buffer (125 mM Tris, 4% SDS, 20% glycerol, 200 mg / mL bromophenol blue, 10% 2-ME). After mixing and heating at 98 ° C. for 5 minutes, 40 μL was applied to an 8% acrylamide gel and subjected to SDS-page. The gel separated by molecular weight was transferred to a PVDF membrane (BD bioscience) and subjected to Western blot analysis. The antibodies include homemade rabbit phosphorylated Smad2 antibody (x2000), mouse anti-Smooth muscle actin antibody (Shigma) (x4000), anti-Rabbit IgG-HRP (Amersham) (x10000), anti-mouse IgG-HRP (Amersham) (Amersham) The color development of HRP was detected with ImageQuant LAS4000 (GE Healthcare) using SuperSignal West Dura Extended Duration Substrate (Piece).

[Example 65]
Evaluation of Myostatin Signal Inhibitory Activity of Peptide 2 by Western Blot The myostatin signal inhibitory activity of Peptide 2 was evaluated by the same method as in Example 64. The result is shown in FIG. Peptide 2 clearly inhibited phosphorylation of Smad-2 at a concentration of 30 μM.

[Example 66]
Evaluation of Myostatin Signal Inhibitory Activity of Peptide 3 by Western Blot The myostatin signal inhibitory activity of Peptide 3 was evaluated by the same method as in Example 64. The result is shown in FIG. Peptide 3 showed Smad-2 phosphorylation inhibitory activity at a concentration of 30 μM.

[Example 67]
Evaluation of TGF-β3 Inhibitory Activity of Peptide 1 by In Vitro Reporter Assay TGF-β3 inhibitory activity of peptide 1 was evaluated by the following method. The result is shown in FIG.
Peptide 1 did not inhibit TGF-β3 at all at a concentration of 30 μM. Therefore, myostatin inhibition of peptide 1 is thought to be selective.
(1) Cell culture HepG2 cells were cultured in the same manner as in Example 50.
(2) In Vitro Reporter Assay One well or 7.5 × 10 ⁴ cells (500 μL DMEM + 10% FBS) of HepG2 cells were seeded on a 24-well white transparent plate (BD Falcon) and cultured for 24 hours. The next day, 0.05 μg (CAGA) 12-luc reporter plasmid per well and 0.01 μg β-galactosidase expression plasmid (pCH110, Pharmacia) as an internal control to a final concentration of PEI (Polyscience, Inc.) of 23 μg / mL And added to the cell culture medium. After culturing these cells at 37 ° C. for 24 hours, the cell culture medium was replaced with DMEM + 0.1% FBS and cultured for 16 hours. Peptide 1 was suspended in DMSO to a stock solution of 3 mM and stored at −30 ° C. 1 hour before addition to cells, suspended in DMEM + 0.1% FBS medium to a final concentration of 30 μM and TGF-β3 5 ng / mL, incubated at 37 ° C. for 1 hour, then added to cells and stimulated for 8 hours Went. The Smad-2 phosphorylation inhibitor SB-431542 (Wako) used as a positive control was suspended in DMSO to a stock solution of 5 mM and stored at -30 ° C. 1 hour before addition to cells, suspended in DMEM + 0.1% FBS medium to a final concentration of 5 μM and TGF-β3 5 ng / mL, incubated at 37 ° C. for 1 hour, then added to cells and stimulated for 8 hours Went. After stimulation, the culture solution was removed with an aspirator, and the cells were washed with 1 × PBS, and then 100 μL of Lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM CDTA, 10% glycerol, 1% triton x− 100) was added to lyse the cells. The lysate was centrifuged at 13000 rpm for 10 minutes, 30 μL of the supernatant and 25 μL of luciferase assay reagent (Promega) were mixed in a white 96-well plate, and luminescence was detected with Centro XS ³ LB960 (Berthold) for reporter assay. Furthermore, 50 μL of the supernatant and 50 μL of β-gal substrate (200 mM phosphate buffer, 2 mM MgCl ₂ , 100 mM 2-ME, 1.33 mg / mL ONPG) were mixed in a white transparent 96-well plate (BD Falcon), and 1 at 37 ° C. After incubation for a period of time, absorbance at 420 nm was detected with Sunrise REMOTE (Wako), and used as an internal control.

[Example 68]
Effect of peptide 1 on C2C12 myoblasts (in vitro evaluation)
In order to verify the myostatin inhibitory effect of peptide 1 on myoblasts, the following method was used for evaluation.
C2C12 cells were seeded at 2.0 × 10 ⁵ cells in a 60 mm dish (BD Falcon) and cultured. Peptide 1 was mixed with 5% bovine serum and 5% horse serum to a final concentration of 30 μM and myostatin 10 ng / mL, and cultured at 37 ° C. for 24 hours. The cells were collected, and total RNA was prepared to prepare cDNA. Produced. The prepared cDNA and muscle-specific genes (Myogenine and MylpF) primers were subjected to PCR to amplify the gene (95 ° C., 5 min; 95 ° C., 30 sec; 62 ° C., 45 sec; 72 ° C., 1 min; 72 ° C., 5 min, 30 cycles) .
The result of observing the target band by performing agarose electrophoresis is shown in FIG. Peptide 1 significantly inhibited myostatin in the same way as the positive control.

[Example 69]
Effects of peptide 1 on wild-type mouse thigh muscles (in vivo evaluation)
In order to verify the effect of peptide 1 in increasing muscle mass in vivo, evaluation was performed by the following method.
Peptide 1 was suspended in physiological saline to a concentration of 0.6 mM. 50 μL each was administered intramuscularly to the right buttock of 4-week-old C57 / BL6 male mice under anesthesia. Two weeks later, peptide 1 was administered again, and four more weeks later, the mice were safflicated and the muscles were necropsied. The excised muscle tissue was fixed overnight with formalin. After washing with PBS three times for 30 minutes, the mixture was infiltrated with 70% EtOH / PBS overnight, and a paraffin block was prepared. 3 μm slices were prepared and subjected to Hematoxylin, Eosin staining, or immunostaining. In Hematoxylin and Eosin staining, the number of nuclei was counted from three slice images, and the average number of cells in one field was statistically analyzed. Immunostaining of embryonic myosin heavy chain was performed by antigen activation in an autoclave at 121 ° C for 20 minutes, PBS washing 5 minutes 3 times, blocking (Blocking Reagent (perkinelmer)) room temperature 30 minutes, primary antibody (MF20, 200 times) Dilution, O / N), PBS washing 5 minutes 3 times, secondary antibody (HRP conjugated anti rabbit, 200-fold dilution), PBS washing 5 minutes 3 times, DAB color development procedure. Hematoxylin was used for counterstaining.
The results are shown in FIGS. 13 (appearance), 26 (HE staining), 27 (statistical analysis of the number of cells), and 28 (immunostaining). Peptide 1 statistically significantly increased the muscle mass of mouse thigh muscle based on its myostatin inhibitory activity, and a clear increase in embryonic myosin heavy chain immunostaining signal was observed.

[Reference Example 1]
Synthesis of peptide 5
AWRQNTRYSRIEAIKIQILSK-NH ₂ (Peptide 5)
Peptide 5 having SEQ ID NO: 49 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.47 mmol / g, Watanabe Chemical Co., Ltd.) 50 mg (0.024 mmol) (13.1 mg, 0.0037 mmol, 16%).
HRMS (ES +) calcd for (M2 ⁺ + 2H) 1287.2435, found 1287.2073.

[Reference Example 2]
Synthesis of peptide 6
SRIEAIKIQILSKLRL-NH ₂ (Peptide 6)
Peptide 6 having SEQ ID NO: 50 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.055 mmol) (26.1 mg, 0.011 mmol, 20%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 627.4110, found 627.4123.

[Reference Example 3]
Synthesis of peptide 7
TWRQNTKSSRIEAIKIQILSKLRL-NH ₂ (peptide 7)
Peptide 7 having SEQ ID NO: 51 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.55 mmol / g, Watanabe Chemical Co., Ltd.) 100 mg (0.055 mmol) (21.4 mg, 0.0060 mmol, 11%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 961.2457, found 961.2437.

[Reference Example 4]
Synthesis of peptide 8
RYSRIEAIKIQILSKL-NH ₂ (Peptide 8)
Peptide 8 having SEQ ID NO: 52 was synthesized and purified by the same method as in Example 2 using Rink amide resin (0.47 mmol / g, Watanabe Chemical Co., Ltd.) 50 mg (0.024 mmol) (12.9 mg, 0.0049 mmol, 21%).
HRMS (ES +) calcd for ( M 2+ + 2H) 966.0903, found 966.0923.

[Reference Example 5]
Synthesis of peptide 9
RYSRIEAIKAQAASKA-NH ₂ (peptide 9)
Peptide 9 having SEQ ID NO: 53 was synthesized and purified in the same manner as in Example 2 using Rink amide resin (0.47 mmol / g, Watanabe Chemical Co., Ltd.) 50 mg (0.024 mmol) (9.3 mg, 0.0038 mmol, 16%).
HRMS (ES +) calcd for (M ³⁺ + 3H) 588.0002, found 588.0001.

[Reference Example 6]
Synthesis of peptide 10
LDFGHIKKKRVEAIRGQILSKLRL-NH ₂ (Peptide 10)
Peptide 10 having SEQ ID NO: 54 was synthesized and purified in the same manner as in Example 2 (13.3 mg, 0.0037 mmol, 6.7%).
HRMS (ES +) calcd for (M2 ⁺ + 2H) 1409.3749, found 1409.3763.

[Reference Example 7]
Evaluation of Myostatin Inhibitory Activity of Peptide 5 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 5 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptide 5 showed no inhibitory activity at a concentration of 30 μM.

[Reference Example 8]
Evaluation of Myostatin Inhibitory Activity of Peptide 6 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 6 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptide 6 showed no inhibitory activity at a concentration of 30 μM.

[Reference Example 9]
Evaluation of Myostatin Inhibitory Activity of Peptide 7 by In Vitro Reporter Assay The myostatin inhibitory activity of peptide 7 was evaluated by the same method as in Example 50. The result is shown in FIG. Peptide 7 showed no inhibitory activity at a concentration of 30 μM.

[Reference Example 10]
Evaluation of Myostatin Signal Inhibitory Activity of Peptide 8 by Western Blot The myostatin signal inhibitory activity of peptide 8 was evaluated by the same method as in Example 64. The result is shown in FIG. Peptide 8 did not inhibit Smad-2 phosphorylation at all at a concentration of 30 μM.

[Reference Example 11]
Effect of peptide 9 on thigh muscle of wild type mouse (in vivo evaluation)
In order to verify the effect of peptide 9 in increasing muscle mass in vivo, evaluation was performed in the same manner as in Example 69. The result is shown in FIG. Peptide 9 used as a negative control has no effect on wild-type mouse thigh muscle mass.

[Reference Example 12]
Evaluation of TGF-β3 Inhibitory Activity of Peptide 10 by In Vitro Reporter Assay The TGF-β3 inhibitory activity of peptide 10 was evaluated by the same method as in Example 67. The result is shown in FIG. Peptide 10 showed no inhibitory activity at a concentration of 30 μM.

As described above, the peptides 1, 2, 3 and 4 of the examples strongly suppress the reporter activity by myostatin relative to the peptides 5 to 10 of the reference example, and the peptides 1 to 3 have a reporter activity in a dose-dependent manner. Inhibition was shown (FIGS. 6-10). In addition, the inhibition rates of peptides 11 and 12 at 30 μM were 89.2% and 94.7%, respectively, while peptide 1 was 92.4% under the same conditions, showing remarkable inhibitory activity.
Peptide 7 has the sequence of the partial peptide hMPS (20-43) of the human-derived myostatin prodomain, and the position of this sequence in the prodomain is the prodomain of mouse-derived myostatin in the sequence of peptide 1 Despite being the same as the position in the medium, it did not show myostatin inhibitory activity.
Moreover, as shown in FIG. 12, peptide 1 does not inhibit the reporter activity by TGF-β3. This suggests that the peptide of the present invention selectively inhibits the activity of myostatin.
Furthermore, the results of in vivo evaluation of peptide 1 in wild-type mouse thigh muscles showed that peptide 1 increases the muscle mass of mouse thigh muscles based on its myostatin inhibitory activity.

  Moreover, it was shown that the peptide 45 represented by sequence number 40 strongly suppresses the reporter activity by myostatin (FIG. 22). Furthermore, the substituted product of the peptide represented by SEQ ID NO: 40 exhibited a myostatin inhibitory activity equivalent to that of peptide 11 represented by SEQ ID NO: 6.

  Thus, the therapeutic agent containing the peptide of the present invention has selective and high inhibitory activity against myostatin, and enables muscle hyperplasia and muscle weight increase.

Claims (15)

The following peptide ( c ) or a pharmaceutically acceptable salt thereof:
(C) In the amino acid sequence represented by any one of SEQ ID NOs: 2, 5 to 9, or 40, an amino acid sequence in which 1 to 6 amino acids are substituted or deleted, or 1 or 2 amino acids are added A peptide comprising an amino acid sequence and selectively inhibiting the activity of myostatin ,
In the amino acid sequence represented by SEQ ID NO: 2, 5-9, or 40, the peptide of (c) is at least one of the following substitutions:
(1) The alanine at position 32 from the N-terminus of the mouse-derived myostatin prodomain is substituted with phenylalanine, homophenylalanine, histidine or tyrosine;
(2) Glutamic acid number 31 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(3) The lysine having the number 40 from the N-terminus of the mouse-derived myostatin prodomain is replaced with α-aminoisobutanoic acid;
(4) Tyrosine number 27 from the N-terminus of the myostatin prodomain derived from mouse is replaced with phenylalanine, tryptophan, histidine or arginine;
(6) Carboxylic acid wherein the tryptophan number 21 from the N-terminal of the mouse-derived myostatin prodomain has a C1-C10 fatty chain or at least one of an aromatic ring and / or a heterocyclic ring Or substitution with amino acids:
(7) one or more of isoleucines of numbers 30, 33, 35 and 37 from the N-terminus of the mouse-derived myostatin prodomain are each replaced by leucine or norleucine;
(8) The leucine numbered 43 from the N-terminus of the mouse-derived myostatin prodomain is replaced by isoleucine or norleucine;
It consists of the amino acid sequence made.
Sequence number 2: AWRQNTRYSRIEAIKIQILSKLRL
Sequence number 5: NTRYSRIEAIKIQILSKLRL
Sequence number 6: WRQNTRYSRIEAIKIQILSKLRL
Sequence number 7: AWRQNTRYSRIEAIKIEILSKLRL
Sequence number 8: AWRQNTRYSRIEAIKIQILSKLR
Sequence number 9: AWRQNTRYSRIEAIKIQILSKL
SEQ ID NO: 40: WRTRYSRIEAIKIQILSKLRL In the amino acid sequence represented by any one of SEQ ID NOs: 2, 5 to 9, and 40, the peptide of (c) is an alanine whose number is position 32 from the N-terminal of the mouse-derived myostatin prodomain is phenylalanine, homo The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence substituted with phenylalanine, histidine or tyrosine. Wherein the peptide of (c) is an amino acid sequence represented by any of SEQ ID NOs: 2, 5-9, and 40, and the glutamic acid number 31 from the N-terminus of the mouse-derived myostatin prodomain is α-aminoisobutanoic acid The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, which comprises the amino acid sequence substituted by. In the amino acid sequence represented by SEQ ID NO: 2, 5-9, or 40, wherein the peptide of (c) is α-aminoisobutanoic acid, the number 40 lysine from the N-terminal of the mouse-derived myostatin prodomain The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, which comprises the amino acid sequence substituted by. Wherein the peptide of (c) is an amino acid sequence represented by any one of SEQ ID NOs: 2, 5 to 9 and 40, and tyrosine having a number 27 from the N-terminal of a mouse-derived myostatin prodomain is phenylalanine, tryptophan, Or the peptide of Claim 1 which consists of an amino acid sequence substituted by arginine, or its pharmaceutically acceptable salt. In the amino acid sequence represented by any one of SEQ ID NOs: 2, 5-9, and 40, the peptide of (c) has numbers 22, 23, 24, 28, 31 from the N-terminus of the mouse-derived myostatin prodomain. The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, which consists of an amino acid sequence in which any one or more of amino acids 34, 36, 39, 40 and 42 are substituted with alanine. The peptide of (c) is an amino acid sequence represented by any one of SEQ ID NOs: 2, 5 to 9, and 40, and the tryptophan number 21 from the N-terminus of the mouse-derived myostatin prodomain is C1 The peptide according to claim 1 or a pharmaceutically acceptable peptide thereof, comprising an amino acid sequence substituted with a carboxylic acid or amino acid having at least one of a C10 fatty chain, or an aromatic ring and / or a heterocyclic ring salt. The carboxylic acid is 1-adamantaneacetic acid, tetrahydroisoquinoline-3-carboxylic acid, 3- (2-fluorophenyl) -propionic acid, 3- (3-fluorophenyl) -propionic acid or 3- (4-fluorophenyl) -The peptide of claim 7 or a pharmaceutically acceptable salt thereof, selected from propionic acid. The peptide according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the amino acid is acetyl-L-tryptophan. The peptide of (c) is an amino acid sequence represented by any one of SEQ ID NOs: 2, 5 to 9, and 40, and the number from the N-terminal of the mouse-derived myostatin prodomain is any of 30, 33, 35, and 37 The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the one or more isoleucines each have an amino acid sequence substituted with leucine or norleucine. In the amino acid sequence represented by SEQ ID NO: 2, 5-9, or 40, the peptide of (c) is substituted with isoleucine or norleucine in number 43 from the N-terminus of the mouse-derived myostatin prodomain The peptide according to claim 1 or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence obtained. A peptide consisting of an amino acid sequence represented by SEQ ID NOs: 10 to 48, or a pharmaceutically acceptable salt thereof, which selectively inhibits the activity of myostatin. A pharmaceutical composition for treating a myostatin-related disorder, comprising the peptide according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof. 14. The pharmaceutical composition according to claim 13 , wherein the myostatin-related disorder is a muscular atrophy disorder. 15. The pharmaceutical composition according to claim 14 , wherein the muscular atrophy disorder is at least one selected from Duchenne muscular dystrophy, progressive muscular dystrophy, Becker muscular dystrophy, Degelin Landusy muscular dystrophy, elb muscular dystrophy, or pediatric neuroaxon muscular dystrophy.

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