PACAP stabilizing peptide

Abstract

The present invention aims to provide a PACAP peptide with improved stability. The present inventors have arrived at the present invention based on the discovery that stability is significantly enhanced by substituting aspartic acid at positions 3 and 8 in the sequence of PACAP with at least one amino acid selected from tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), tetrazole-substituted homoglutamic acid (nvTz), sulfoxyl-substituted aspartic acid (cya) and oxadiazolone-substituted aspartic acid (5Oxa).

Description

[Technical field]

[0001] The present invention relates to a stabilized peptide having the physiological activity of PACAP, a neuroprotectant containing the peptide, an agent for improving vascular endothelial function, an agent for promoting corneal neurite formation, an agent for promoting tear secretion, an agent for treating dry eyes, an agent for treating corneal epithelial or corneal endothelial disorders, an agent for treating neurotrophic keratitis, an anti-inflammatory agent, or a pharmaceutical composition. [Background technology]

[0002] Neurons are cells that constitute the nervous system, which is largely divided into the central nervous system and the peripheral nervous system. Neurons are susceptible to damage due to external factors such as cerebrovascular disorders such as stroke and cerebral infarction, or internal factors such as accumulation of abnormal proteins, oxidative stress, and inflammation. At the same time, their regeneration ability is low, and once a disorder occurs, it becomes a factor that significantly reduces the QOL of the patient. Neurodegenerative diseases accompanied by neurodegeneration and loss of central nervous system nerves include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis, as well as optic nerve degeneration diseases such as glaucoma and optic neuropathy, and sensorineural deafness.

[0003] With the development of neuroscience, various neuroprotective factors have been found, and it is expected that they will be developed as preventive or therapeutic drugs for neurological disorders. It has been found that agents that reduce free radicals or excitatory amino acids that cause neurodegeneration or agents that can protect and / or repair nerve cells (e.g., immunophilin ligands of neurotrophic factors or immunosuppressants, etc.) have neuroprotective effects. On the one hand, it has been found that proteins in vivo such as pituitary adenylate cyclase activating polypeptide (PACAP), CD44, or human brain carboxypeptidase B (HBCPB) have neuroprotective effects (Patent Documents 1 and 2).

[0004] Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide found from sheep hypothalamus extract. PACAP can have the activity of stimulating cAMP formation in anterior pituitary cells. As PACAP, there are PACAP38 composed of 38 amino acid residues and PACAP27 composed of 27 amino acid residues, both of which have equivalent effects (non-patent literature 1 and 2). PACAP belongs to the vasoactive intestinal polypeptide (VIP) / secretin / glucagon superfamily, and the sequence of human PACAP27 has 68% identity with vasoactive intestinal polypeptide (VIP). PACAP and VIP bind to PAC1 receptor (PAC1R), VPAC1 receptor (VPAC1R) and VPAC2 receptor (VPAC2R), and differ in the affinity for these receptors. PAC1R binds to PACAP with high selectivity. The affinity of PACAP for PAC1R is more than 1000 times higher than the affinity of VIP for PAC1R. On the one hand, VPAC1R and VPAC2R have the same affinity for PACAP and VIP. PACAP has various physiological effects, and is known to have physiological effects as a neuroprotective substance, an immunosuppressive factor, a vasodilator factor, an exocrine segment secretion promoting factor (Patent Document 3), and a neurite formation promoting factor (Patent Document 4).

[0005] Pharmaceuticals are being developed using the various physiological activities of PACAP, but it is known that many peptides such as PACAP are unstable in aqueous solutions and have a short half-life due to lack of protease resistance.

[0006] [Prior art literature]

[0007] [Patent Literature]

[0008] [Patent Document 1] Japanese Patent Publication No. 2014-510101

[0009] [Patent Document 2] Japanese Patent Application Publication No. 2012-232952

[0010] [Patent Document 3] Japanese Patent Application Publication No. 2009-269818

[0011] [Patent Document 4] WO2005 / 102375

[0012]

Non-patent literature

[0013] [Non-patent document 1] S. Bourgault (2009) Current Medicinal Chemistry 16, 4462-4480

[0014] [Non-patent document 2] Louise Dickson (2009) Pharmacology & Therapeutics, 12, 294-316

[0015] [Summary of the Invention]

[0016] [Problems to be solved by the invention]

[0017] The present invention aims to provide a stabilized peptide of PACAP having high stability in aqueous solution, a neuroprotectant containing the peptide, a neurite formation promoter, a lacrimal gland secretion promoter, a pharmaceutical composition for treating or preventing dry eye, a pharmaceutical composition for treating or preventing corneal epithelial or corneal endothelial disorders, a pharmaceutical composition for treating or preventing neurological disorders, or a composition for treating or preventing neurotrophic keratitis.

[0018]

Methods for solving the problem

[0019] As a result of intensive studies conducted by the present inventors to improve the stability of PACAP in aqueous solutions, it was found that the stability in aqueous solutions can be greatly improved by independently replacing the aspartic acid at positions 3 and 8 in the PACAP peptide with residues selected from tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), tetrazole-substituted homoglutamic acid (nvTz), sulfooxy-substituted aspartic acid (cya), and oxadiazolone-substituted aspartic acid (5Oxa), thereby completing the present invention.

[0020] Therefore, the present invention relates to the following technical solutions:

[0021] [1] A peptide comprising a sequence in which the aspartic acid at positions 3 and 8 of the sequence represented by the following sequence is independently substituted with a residue selected from the group consisting of tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), tetrazole-substituted homoglutamic acid (nvTz), sulfooxy-substituted aspartic acid (cya), and oxadiazolone-substituted aspartic acid (5Oxa), or a modified sequence thereof:

[0022] H-X1-DG-X2-F-X3-D-X4-Y-X5-RYRK-X6-X7-AVKKYLAAVL(SEQ ID NO:3)

[0023] (Where,

[0024] X1 is a neutral amino acid,

[0025] X2 is a neutral amino acid,

[0026] X3 is a neutral amino acid.

[0027] X4 is alanine, serine, Ai or Cn,

[0028] X5 is alanine, serine, Ai or Cn,

[0029] X6 is glutamine or alanine

[0030] X7 is a non-polar amino acid)

[0031] The peptide has the ability to bind to PAC1R and VPAC1R, and the altered sequence is a sequence in which one or more amino acids are deleted or added to the sequence of SEQ ID NO: 3.

[0032] [2] The peptide according to item 1, wherein X1 is serine or alanine.

[0033] [3] The peptide according to item 1 or 2, wherein X2 is isoleucine or alanine.

[0034] [4] The peptide according to any one of items 1 to 3, wherein X3 is alanine or threonine.

[0035] [5] The peptide according to any one of items 1 to 4, wherein X7 is methionine, leucine, norleucine or alanine.

[0036] [6] A peptide according to any one of items 1 to 5, wherein the amino acids at positions 3 and 8 are tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), or tetrazole-substituted homoglutamic acid (nvTz), and these amino acids are L-isomer, D-isomer, or racemic isomer.

[0037] [7] The peptide according to any one of items 1 to 6, wherein the N-terminus of the above-mentioned peptide is modified with a protecting group.

[0038] [8] The peptide according to item 7, wherein the protecting group is an acyl group.

[0039] [9] The peptide according to any one of items 1 to 8, wherein the C-terminus of the above peptide is modified with a protecting group.

[0040]

[10] The peptide according to item 9, wherein the protecting group is an amide group or an ester group.

[0041]

[11] The altered sequence is a peptide according to any one of items 1 to 10, wherein 1 to 3 amino acids are deleted from the above sequence at the C-terminus.

[0042]

[12] The altered sequence is a peptide according to any one of items 1 to 10, wherein a sequence of 1 to 3 amino acids is added to the N-terminus of the above sequence.

[0043]

[13] The peptide according to any one of items 1 to 10 or 12, wherein the altered sequence is a sequence in which one sequence selected from the following sequences is added to the C-terminus of the sequence:

[0044] GKRYKQRVKNK (SEQ ID NO: 43);

[0045] GKRYKQRVKN (SEQ ID NO: 44);

[0046] GKRYKQRVK (SEQ ID NO: 45);

[0047] GKRYKQRV (SEQ ID NO: 46);

[0048] GKRYKQR (SEQ ID NO: 47);

[0049] GKRYKQ (SEQ ID NO: 48);

[0050] GKRYK (SEQ ID NO: 49);

[0051] GKRY (SEQ ID NO:50)

[0052] GKR;

[0053] GRR;

[0054] GK;

[0055] GR; and

[0056] G.

[0057]

[14] A neuroprotective agent comprising the peptide according to any one of items 1 to 13.

[0058]

[15] A tear secretion enhancer comprising the peptide according to any one of items 1 to 13.

[0059]

[16] An anti-inflammatory agent comprising the peptide according to any one of items 1 to 13.

[0060]

[17] A vascular endothelial function improving agent comprising the peptide according to any one of items 1 to 13.

[0061]

[18] A therapeutic agent for corneal epithelial or corneal endothelial disorders, comprising the peptide according to any one of items 1 to 13.

[0062]

[19] A therapeutic agent for dry eye comprising the peptide according to any one of items 1 to 13.

[0063]

[20] A pharmaceutical composition comprising the peptide according to any one of items 1 to 13.

[0064]

[21] A method for neuroprotection in a subject in need of neuroprotection, comprising administering to the subject an effective amount of the peptide according to any one of items 1 to 13.

[0065]

[22] A method for promoting tear secretion in a subject in need of tear secretion, comprising administering the peptide according to any one of items 1 to 13 to the subject.

[0066]

[23] A method for improving vascular endothelial function in a subject in need of improvement of vascular endothelial function, comprising administering the peptide according to any one of items 1 to 13.

[0067]

[24] A method for suppressing inflammation in a subject suffering from inflammation, comprising administering the peptide according to any one of items 1 to 13.

[0068]

[25] A method for improving corneal epithelial or corneal endothelial disorders in a subject suffering from such disorders, comprising administering the peptide according to any one of items 1 to 13.

[0069]

[26] A method for treating or preventing dry eye in a subject suffering from dry eye, comprising administering the peptide according to any one of items 1 to 13.

[0070]

[27] The peptide according to any one of items 1 to 13 for use as a medicine.

[0071]

[28] The peptide according to any one of items 1 to 13 for use in the treatment of a neurological disorder.

[0072]

[29] The peptide according to any one of items 1 to 13 for use in the treatment or prevention of a neurological disorder via neuroprotection.

[0073]

[30] The peptide according to any one of items 1 to 13 for use in the treatment or prevention of dry eye or a disease associated with dry eye.

[0074]

[31] The peptide according to any one of items 1 to 13 for use in the treatment or prevention of corneal epithelial or corneal endothelial disorders.

[0075]

[32] The peptide according to any one of items 1 to 13 for use in the treatment or prevention of inflammatory diseases

[0076]

[33] The peptide according to any one of items 1 to 13 for use in the treatment or prevention of vascular endothelial disorders.

[0077]

[34] Use of the peptide according to any one of items 1 to 13 for the manufacture of a medicament selected from a neuroprotective agent, a tear secretion promoter, an anti-inflammatory agent, an agent for improving vascular endothelial function, a therapeutic agent for corneal epithelial or corneal endothelial disorders, and a therapeutic agent for dry eye.

[0078] [Effects of the invention]

[0079] According to the present invention, PACAP which is unstable in an aqueous solution can be significantly stabilized. [Specific implementation method]

[0080] The present invention relates to a peptide composed of a sequence in which the aspartic acid residues at positions 3 and 8 of PACAP are independently substituted with residues selected from tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), tetrazole-substituted homoglutamic acid (nvTz), sulfooxy-substituted aspartic acid (cya), and oxadiazolone-substituted aspartic acid (5Oxa), or a sequence in which a part of the sequence is changed, and the peptide of the present invention has the same degree of binding to PAC1R, VPAC1R, and / or VPAC2R as PACAP. The same degree of binding is sufficient as long as the EC50 value of the peptide to each receptor is within 100 times that of PACAP. PACAP may also be either PACAP38 composed of 38 residues or PACAP27 composed of 27 residues. PACAP38 and PACAP27 have the following sequences:

[0081] PACAP38:HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK(SEQ ID NO:1)

[0082] PACAP27:HSDGIFTDSY SRYRKQMAVK KYLAAVL(SEQ ID NO:2)

[0083] In the present invention, tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), and tetrazole-substituted homoglutamic acid (nvTz) refer to non-natural amino acid residues having a structure in which the carboxyl group of the side chain of aspartic acid, glutamic acid, and homoglutamic acid is substituted with tetrazole, which is an isostere of the carboxyl group. Specifically, they are represented by the following formula:

[0084]

Chemistry 1

[0085]

[0086] Tetrazolyl substituted aspartic acid (Tz) Tetrazolyl substituted glutamic acid (egTz) Tetrazolyl substituted homoglutamic acid (nvTz)

[0087] In the present invention, sulfo-substituted aspartic acid (cya) and oxadiazolone-substituted aspartic acid (5Oxa) refer to residues having a structure in which the carboxyl group of the side chain of aspartic acid is substituted with a sulfo-oxy group or 1,2,4-oxadiazol-5-one (oxadiazolone), which is an isostere of the carboxyl group. Specifically, they are represented by the following formula:

[0088]

Chemistry 2

[0089]

[0090] The non-natural amino acid residues in which the aspartic acid at positions 3 and 8 are substituted may be L-form, D-form, or racemic as long as they have the same degree of physiological activity as PACAP. In particular, the peptides in which the tetrazole-substituted aspartic acid is substituted with the L-form and the peptides in which the tetrazole-substituted aspartic acid is substituted with the D-form have the same degree of binding to PAC1R, VPAC1R, and / or VPAC2R. Without intending to be bound by theory, the amino acid containing the D-form may become difficult to be decomposed by enzymes and improve stability. For non-natural amino acids other than protein-constituting amino acids, the protective group may be introduced into the peptide by the Fmoc method or the like in the same manner as the protein-constituting amino acids.

[0091] In the present invention, a sequence in which a part of the sequence is changed refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence. More preferably, a sequence in which a part of the sequence is changed refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence.

[0092] Amino acid substitution may occur at any position as long as it does not change the binding of the peptide composed of a sequence in which a part of the sequence is changed to PAC1R, VPAC1R, and / or VPAC2R. In the peptide composed of a sequence in which a part of the sequence is changed, amino acid substitution may occur at positions 2, 5, 7, 9, 11, 16, and 17 of PACAP27 from the viewpoint of maintaining binding. In a particularly preferred embodiment, in the following sequence:

[0093] H-X1-DG-X2-F-X3-D-X4-Y-X5-RYRK-X6-X7-AVKKYLAAVL(SEQ ID NO:3)

[0094] The amino acids at the positions shown in X1 to X7 may be substituted.

[0095] Thus, one embodiment of the present invention relates to a peptide consisting of a sequence in which the aspartic acid at the 3-position and the 8-position in the sequence represented by the following formula is independently substituted with a residue selected from tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), tetrazole-substituted homoglutamic acid (nvTz), sulfooxy-substituted aspartic acid (cya), and oxadiazolone-substituted aspartic acid (5Oxa), or a modified sequence thereof:

[0096] H-X1-DG-X2-F-X3-D-X4-Y-X5-RYRK-X6-X7-AVKKYLAAVL(SEQ ID NO:3)

[0097] (Where,

[0098] X1 to X6 are neutral amino acids.

[0099] X7 is a non-polar amino acid),

[0100] A peptide having the ability to bind to PAC1R, VPAC1R, and / or VPAC2R.

[0101] It can be conservatively substituted with amino acids having the same properties. As an example, conservative substitutions refer to substitutions of amino acids within the following groups:

[0102] (i) Non-polar amino acids: Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, Ala

[0103] (ii) Neutral amino acids: Ala, Ser, Thr, Tyr, Cys, Asn, Gln, Gly

[0104] (iii) Basic amino acids: Lys, Arg, His

[0105] (iv) Acidic amino acids: Asp, Glu

[0106] Among the neutral amino acids, α-aminoisobutyric acid (Ai) in which a methyl group is substituted at the α position of alanine, or β-cyanoalanine (Cn) in which a hydrogen at the β position of alanine is substituted with a cyano group can be used instead of alanine. In particular, α-aminoisobutyric acid (Ai) and β-cyanoalanine (Cn) can be used as neutral amino acids at positions 9 and 11. α-aminoisobutyric acid (Ai) and β-cyanoalanine (Cn) have the following structures:

[0107]

Chemistry 3

[0108]

[0109] In the sequence represented by SEQ ID NO: 3 of the present invention, the amino acids represented by X1 to X7 are preferably as follows:

[0110] The neutral amino acid in X1 is particularly alanine or serine.

[0111] The neutral amino acid in X2 is specifically isoleucine or alanine.

[0112] The neutral amino acid in X3 is specifically alanine or threonine.

[0113] The neutral amino acid in X4 and X5 is particularly alanine, serine, α-aminoisobutyric acid, or β-cyanoalanine.

[0114] The neutral amino acid in X6 is specifically glutamine or alanine.

[0115] The non-polar amino acid in X7 is in particular methionine, leucine, norleucine, or alanine.

[0116] In the present invention, an altered sequence refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence. More preferably, a sequence in which a portion of the sequence is altered refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence.

[0117] Amino acid substitution may be 1 or more amino acids substituted, and from the viewpoint of maintaining activity, any number of 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted. Among them, substitution of 1 to 4 amino acids is particularly preferred, substitution of 3 amino acids is more preferred, substitution of 2 amino acids is more preferred, and substitution of 1 amino acid is particularly preferred.

[0118] The deletion of amino acids can occur at any location as long as it does not change the binding of the peptide composed of a sequence in which a part of the sequence is changed to PAC1R, VPAC1R, and / or VPAC2R. The number of deleted amino acids is selected from any number of 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. From the viewpoint of not changing the binding property, the deletion of 1 or 2 amino acids, or the deletion of 1 amino acid is particularly preferred. The deletion of amino acids can be deleted from the N-terminal side or the C-terminal side of the original sequence, or can be deleted from the inside of the sequence. Since PACAP27 and PACAP38 each have the same binding to PAC1R, VPAC1R, and / or VPAC2R, even if the amino acid present on the C-terminal side of PACAP38 is deleted, it is believed that the binding property has little effect.

[0119] The addition of amino acids can occur at any location as long as it does not change the binding of the peptide composed of a sequence in which a part of the sequence is changed to PAC1R, VPAC1R, and / or VPAC2R. The number of amino acids added is selected from any number from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acids can be added to the N-terminal side or C-terminal side of the original sequence, or can be added inside the sequence. Since PACAP27 and PACAP38 each have the same binding to PAC1R, VPAC1R, and / or VPAC2R, it is believed that the addition of any amino acid to the C-terminal of PACAP27 has little effect on the binding.

[0120] As a modified sequence in which the aspartic acid residues at positions 3 and 8 in the sequence of SEQ ID NO: 3 are independently substituted with a residue selected from tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), tetrazole-substituted homoglutamic acid (nvTz), sulfooxy-substituted aspartic acid (cya), and oxadiazolone-substituted aspartic acid (5Oxa), preferably, a sequence in which one or more amino acids are deleted or added to the amino acid sequence represented by SEQ ID NO: 3. More preferably, one or two amino acids may be deleted from the amino acid sequence represented by SEQ ID NO: 3, and the following C-terminal additional sequence may be added to the amino acid sequence represented by SEQ ID NO: 3. In addition, 1 to 3 amino acids (e.g., methionine) may be added to the N-terminal of the amino acid sequence represented by SEQ ID NO: 3.

[0121] Without intending to be bound by theory, since PACAP27 and PACAP38 each have equivalent binding properties to PAC1R, VPAC1R, and / or VPAC2R, the addition of amino acids to the C-terminal side of PACAP27 does not affect the binding property to PAC1R. Therefore, as a peptide with a modified sequence of the present invention, it can contain a C-terminal additional sequence at the C-terminus of the amino acid sequence of SEQ ID NO: 3, similarly to PACAP27. The C-terminal additional sequence refers to a sequence consisting of 1 to 11 arbitrary amino acids. The C-terminal additional sequence preferably corresponds to the amino acids at positions 28 to 38 of PACAP38. Therefore, as C-terminal sequences, the following sequences can be cited:

[0122] GKRYKQRVKNK (SEQ ID NO: 43);

[0123] GKRYKQRVKN (SEQ ID NO: 44);

[0124] GKRYKQRVK (SEQ ID NO: 45);

[0125] GKRYKQRV (SEQ ID NO: 46);

[0126] GKRYKQR (SEQ ID NO: 47);

[0127] GKRYKQ (SEQ ID NO: 48);

[0128] GKRYK (SEQ ID NO: 49);

[0129] GKRY (SEQ ID NO:50)

[0130] GKR;

[0131] GRR;

[0132] GK;

[0133] GR; and

[0134] G.

[0135] As long as the peptides of the present invention do not lose their binding properties to PAC1R, VPAC1R, and / or VPAC2R, D- or L-form amino acids can be used, or racemic amino acids can also be used. In addition, similarly, the peptides of the present invention can be composed of non-natural amino acids such as 2-aminoisobutyric acid, and contain derivatives in which the amino group at the N-terminus, the carboxyl group at the C-terminus, or the functional groups of the amino acid side chains are arbitrarily modified. As examples of modifications, there can be cited the addition of protective groups to the amino groups (for example, acylation (formylation, acetylation, propionylation, butyrylation, isobutyrylation, etc.), mesylation, ureidation, aminomethoxylation, Bocization, Fmocization), esterification of carboxyl groups (ethylation, etc.), etc. In addition, modifications that usually occur in vivo, such as phosphorylation, amidation, methylation, esterification, acetylation, etc., can also be included, which occur during the synthesis process or make purification easier, such as biotinylation. In addition, modifications such as PEGylation can also be performed for the purpose of extending the half-life of the peptide in vivo. In particular, from the viewpoint of improving stability, the free amino group of the amino acid at the N-terminus can be protected by a protecting group (e.g., acyl group). For example, the free amino group of the amino acid at the N-terminus can be acylated (e.g., acetylated, propionylated, butyrylated, isobutyrylated) or mesylated. Similarly, the free carboxyl group of the amino acid at the C-terminus can be protected by a protecting group (e.g., amide group, ester group). For example, the free carboxyl group of the amino acid at the C-terminus can be amidated or esterified. In the peptides to which the present invention relates, stability is further improved by acetylation or mesylation of the N-terminus. The C-terminus can also be any of a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH2) or an ester (-COOR). The peptides to which the present invention relates can also be further attached with sugar chains (see, for example, WO2017 / 027848).

[0136] In a specific embodiment of the present invention, the present invention relates to peptides having the sequences shown in the following Table 1, or their altered sequences:

[0137]

Table 1

[0138]

[0139]

[0140] In the table, Tz refers to tetrazole-substituted aspartic acid, egTz refers to tetrazole-substituted glutamic acid, nvTz refers to tetrazole-substituted homoglutamic acid, cya refers to sulfo-substituted aspartic acid, (5Oxa) refers to oxadiazolone-substituted aspartic acid, Ai refers to α-aminoisobutyric acid, or Cn refers to β-cyanoalanine. Tz(D) indicates that the tetrazole-substituted aspartic acid is the D-isomer.

[0141] The peptides of the present invention can be produced by any production method. For example, they can be produced by solid phase synthesis or liquid phase synthesis using the Boc method or the Fmoc method. In addition, in another method, the nucleic acid encoding the peptide of the present invention can be introduced into a host cell by a gene introduction method and synthesized by the host cell. In this case, by designing a tag peptide such as a polyhistidine tag at the end of the peptide, purification after expression can be facilitated.

[0142] The peptides of the present invention contain pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts with inorganic acids (e.g., hydrochlorides, hydrobromides, sulfates, phosphates, etc.), salts with organic acids (e.g., methanesulfonates, benzenesulfonates, p-toluenesulfonates, formates, acetates, trifluoroacetates, oxalates, citrates, malonates, fumarates, maleates, tartrates, succinates, malates, etc.), or salts with bases (e.g., ammonium salts, methylpyridinium salts, acetylpyridinium salts, etc.). The peptides of the present invention also contain hydrates or solvents.

[0143] The Fmoc-N protected amino acid in which the side chain carboxyl group of the amino acid (aspartic acid) is substituted with tetrazole which is an isostere of the carboxyl group can be produced by the method shown in Reaction Scheme 1 or a method based thereon, using Fmoc-N protected asparagine as a starting material.

[0144]

Chemistry 4

[0145]

[0146] In the formula,

[0147] Trt represents a trityl group,

[0148] Additionally, the H or Trt group is substituted on N1 or N2 on the tetrazole.

[0149] In the method of reaction formula 1, in step A, the compound represented by (I) (for example, asparagine with amino group protected by Fmoc) is reacted with EDCI in the presence of a base to generate compound (II). In step A, the base is used in an amount of 1 to 50 equivalents, preferably 10 to 25 equivalents, relative to compound (I). Examples of the base used include pyridine, 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, etc., preferably pyridine. EDCI is used in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents. The solvent is not particularly limited as long as it does not affect the reaction. For example, N,N-dimethylformamide, N,N-dimethylacetamide, toluene, dichloromethane, tetrahydrofuran, acetonitrile, etc., preferably acetonitrile. The reaction temperature is usually -10 to 60°C, preferably 0 to 30°C, and the reaction time is 0.5 to 12 hours, preferably 1 to 5 hours.

[0150] In step B, compound (II) is reacted with trimethylsilyl azide and dibutyltin oxide in toluene to generate compound (III). Trimethylsilyl azide is used in an amount of 1 to 5 equivalents, preferably 2 to 4 equivalents, relative to compound (II). Dibutyltin oxide is used in an amount of 0.3 to 1 equivalent, preferably 0.7 to 0.95 equivalents, relative to compound (II). The solvent is not particularly limited as long as it does not affect the reaction, and examples thereof include benzene, toluene, xylene, etc., preferably toluene. The reaction time is generally 0.5 to 24 hours, preferably 0.5 to 2 hours. The reaction time is generally 80 to 150°C, preferably 80 to 100°C. The heating method may include oil bath, microwave, etc., preferably microwave.

[0151] Next, in step C, compound (IV) can be produced by reacting trityl chloride in the presence of a base. Examples of the base used in step C include pyridine, 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, and preferably N-methylmorpholine. The amount of the base used is 1 to 3 equivalents, preferably 1 to 1.5 equivalents, relative to compound (IX). Trityl chloride is used in an amount of 1 to 3 equivalents, preferably 1.05 to 1.1 equivalents. The solvent is not particularly limited as long as it does not affect the reaction. For example, examples include N,N-dimethylformamide, N,N-dimethylacetamide, toluene, dichloromethane, tetrahydrofuran, acetonitrile, and preferably tetrahydrofuran. The reaction temperature is generally -10 to 60°C, preferably 0 to 30°C. The reaction time is 0.5 to 12 hours, preferably 1 to 2 hours.

[0152] Fmoc-nvTz(Trt)-OH in which tetrazole is bonded to the α-position of the amino acid via an alkyl group having 3 carbon atoms can be produced by the method shown in Reaction Scheme 2 or a method based thereon.

[0153]

Chemistry 5

[0154]

[0155] In step D of the method of reaction formula 2, a compound represented by the general formula compound (V) is reacted with Boc2O in the presence of a base to generate compound (VI). The base is used in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents, relative to compound (V). Examples of the base used include triethylamine, N,N-diisopropylamine, N-methylmorpholine, sodium hydroxide, potassium carbonate, sodium bicarbonate, etc., preferably sodium hydroxide. Boc2O is used in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents, relative to compound (V). The solvent is not particularly limited as long as it does not affect the reaction, and examples include THF, DMF, 1,4-dioxane, etc., and 1,4-dioxane is preferably used. The reaction temperature is generally 0 to 25°C, preferably 10 to 25°C. The reaction time is 1 to 24 hours, preferably 18 to 24 hours. In addition, N-methylpiperazine may be added to treat excess Boc2O as long as it does not affect the reaction.

[0156] Next, in step E, compound (VI) is reacted with nickel sulfate hexahydrate, sodium peroxodisulfate and sodium hydroxide to generate compound (VII). In step E, sodium peroxodisulfate is used in an amount of 1 to 4 equivalents, preferably 1 to 2 equivalents, relative to compound (VI). Nickel sulfate hexahydrate is used in an amount of 0.01 to 0.1 equivalents, preferably 0.08 to 0.1 equivalents, relative to compound (VI). Sodium hydroxide is used as an example of the base used, and 1 to 10 equivalents, preferably 6 to 8 equivalents, relative to compound (VI) are used as a whole. Water is used as a solvent. The reaction temperature is 10 to 40° C., preferably 10 to 25° C. The reaction time is usually 1 to 24 hours, preferably 12 to 24 hours.

[0157] Next, in step F, compound (VII) is reacted with TFA, the pH is adjusted to alkaline, and the compound (VIII) is reacted with Fmoc-OSu to generate compound (VIII). Examples of the acid used in step F include hydrochloric acid, sulfuric acid, trifluoroacetic acid, etc., and trifluoroacetic acid is preferably used. The solvent is not particularly limited as long as it does not affect the reaction, and examples include THF, dichloromethane, 1,4-dioxane, etc., and dichloromethane is preferably used. The reaction temperature is 0 to 25°C, preferably 10 to 25°C. The reaction time is usually 0.5 to 5 hours, preferably 0.5 to 2 hours. Examples of inorganic bases used as the base and the pH adjustment after the reaction include sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium carbonate, etc., and potassium carbonate is preferably used. Fmoc-OSu is used in 1 to 3 equivalents, preferably 1 to 1.5 equivalents, relative to compound (VII). The solvent is not particularly limited as long as it does not affect the reaction, and examples thereof include THF, N,N-dimethylformamide, dichloromethane, 1,4-dioxane, etc., and 1,4-dioxane is preferably used. The reaction temperature is 0 to 25° C., preferably 10 to 25° C. The reaction time is usually 1 to 36 hours, preferably 1 to 24 hours.

[0158] Next, in step G, compound (VIII) is reacted with trimethylsilyl azide and dibutyltin oxide in toluene to generate compound (IX). In step G, trimethylsilyl azide is used in 1 to 10 equivalents, preferably 2 to 4 equivalents, relative to compound (II). Dibutyltin oxide is used in 0.3 to 1 equivalents, preferably 0.7 to 1.0 equivalents, relative to compound (II). The solvent is not particularly limited as long as it does not affect the reaction, and examples thereof include benzene, toluene, xylene, etc., and preferably toluene. The reaction time is generally 0.5 to 48 hours, and preferably 0.5 to 2 hours. The reaction time is generally 80 to 150°C, and preferably 80 to 100°C. The heating method may include oil bath, microwave, etc., and preferably microwave.

[0159] Next, in step H, compound (X) can be produced by reacting compound (IX) with trityl chloride in the presence of a base. In the production of compound (X), examples of the base include pyridine, 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, and preferably N-methylmorpholine. The amount of the base used is 1 to 3 equivalents, preferably 1 to 1.5 equivalents, relative to compound (IX). 1 to 3 equivalents, preferably 1.05 to 1.1 equivalents, relative to trityl chloride compound (IX). The solvent is not particularly limited as long as it does not affect the reaction. For example, examples include N,N-dimethylformamide, N,N-dimethylacetamide, toluene, dichloromethane, tetrahydrofuran, acetonitrile, and preferably tetrahydrofuran. The reaction temperature is usually -10 to 60°C, preferably 20 to 30°C, and the reaction time is 0.5 to 12 hours, preferably 1 to 2 hours.

[0160] Fmoc-(5Oxa)-OH in which the carboxyl group of aspartic acid is substituted with 1,2,4-oxadiazol-5-one can be produced by the method shown in Reaction Scheme 3 or a method based thereon.

[0161]

Chemistry 6

[0162]

[0163] In the method of reaction formula 3, in step I, a compound represented by the general formula compound (XI) is reacted with 2-bromo-2-methylpropane in the presence of benzyltriethylammonium chloride and a base to generate compound (XII). In step I, the base is used in an amount of 10 to 30 equivalents, preferably 10 to 25 equivalents, relative to compound (XI). The base used can be exemplified by potassium carbonate. Benzyltriethylammonium chloride is used in an amount of 1 to 3 equivalents, preferably 1 to 1.2 equivalents, relative to compound (XI). 2-bromo-2-methylpropane is used in an amount of 10 to 50 equivalents, preferably 10 to 40 equivalents, relative to compound (XI). N,N-dimethylacetamide is used as a solvent. The reaction temperature is generally 1 to 24 hours, preferably 18 to 24 hours. The reaction time is generally 25-70°C, preferably 25 to 55°C.

[0164] Next, in step J, compound (XIII) is produced by reacting compound (XII) with hydroxylamine hydrochloride in the presence of a base. In step J, the base used may include triethylamine, N,N-diisopropylethylamine, etc., and triethylamine is preferably used. The base added to compound (XII) is used in an amount of 1 to 3 equivalents, preferably 1 to 2 equivalents. Hydroxylamine hydrochloride is used in an amount of 1 to 2 equivalents, preferably 1 to 1.5 equivalents, relative to compound (XII). The solvent is not particularly limited as long as it does not affect the reaction, and examples thereof include methanol, ethanol, propanol, and isopropanol, and ethanol is preferably used. The reaction temperature is generally 80 to 100° C., preferably 80 to 95° C. The reaction time is generally 1 to 6 hours, preferably 1 to 4 hours.

[0165] Next, in step K, compound (XIV) is generated by reacting compound (XIII) with carbonyldiimidazole. In step K, carbonyldiimidazole is used in an amount of 1 to 2 equivalents, preferably 1 to 1.2 equivalents, relative to compound (XIII). The solvent is not particularly limited as long as it does not affect the reaction, and examples include THF, 1,4-dioxane, etc., preferably THF. The reaction temperature is 40 to 60°C, preferably 40 to 55°C. The reaction time is usually 1 to 6 hours, preferably 1 to 3 hours. In the continuous cyclization reaction, the solvent is not particularly limited as long as it does not affect the reaction, and examples include toluene, xylene, benzene, etc., preferably toluene. The reaction temperature is usually 100 to 130°C, preferably 100 to 120°C.

[0166] Next, in step L, compound (XV) can be produced by reacting compound (XIV) with TFA in the presence of a scavenger, adjusting the pH to alkaline, and reacting with Fmoc-OSu. In step L, the acid used may include hydrochloric acid, sulfuric acid, trifluoroacetic acid, etc., and trifluoroacetic acid is preferably used. The scavenger used may include triethylsilane and triisopropylsilane, and triethylsilane is preferably used. The solvent is not particularly limited as long as it does not affect the reaction, and examples include THF, dichloromethane, 1,4-dioxane, etc., and dichloromethane is preferably used. The reaction temperature is 0 to 25°C, preferably 10 to 25°C. The reaction time is usually 0.5 to 5 hours, preferably 0.5 to 3 hours. The inorganic base used for adjusting the pH after the reaction and the base may include sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium carbonate, and preferably sodium bicarbonate. Fmoc-OSu is used in 1 to 3 equivalents, preferably 1 to 1.5 equivalents, relative to compound (VII). The solvent is not particularly limited as long as it does not affect the reaction, and examples thereof include THF, N,N-dimethylformamide, dichloromethane, and 1,4-dioxane. Preferably, when 1,4-dioxane is used, the reaction temperature is 0 to 25° C., preferably 10 to 25° C. The reaction time is usually 1 to 36 hours, preferably 1 to 24 hours.

[0167] The peptide according to the present invention can exert the same physiological activity as PACAP by binding to PAC1R, VPAC1R, and / or VPAC2R. That is, the peptide according to the present invention, as an example, can exert physiological effects as a neuroprotective substance, a nerve regeneration factor, a wound healing promoting factor, an inflammation suppressing factor, and an exocrine line secretion promoting factor. Therefore, in another embodiment of the present invention, it may also involve a neuroprotective agent, a nerve regeneration agent, a wound healing agent, an inflammation suppressing agent, a corneal epithelial or corneal endothelial disorder therapeutic agent, a dry eye therapeutic agent or an exocrine line secretion promoting agent, or a neurotrophic keratitis therapeutic agent containing the peptide according to the present invention. Furthermore, in another embodiment, it also involves a pharmaceutical composition containing the peptide according to the present invention.

[0168] The neuroprotective agent according to the present invention refers to a drug having a neuroprotective effect. Thus, the neuroprotective agent can protect nerves from obstacles accompanied by damage, degeneration and / or cell death of nerve cells, and can also be called a nerve cell death (apoptosis and / or necrosis) inhibitor, a nerve cell degeneration inhibitor, a nerve cell stress reducer, an improver of nerve cell toxicity resistance, an improver of nerve cell viability, and an inhibitor of abnormal protein accumulation.

[0169] In this specification, neuroprotection refers to the effect of protecting nerve cells from damage, degeneration, and / or cell death, preferably refers to the effect of protecting nerve cells from death. More specifically, in neuroprotection, it may also contain the inhibition of nerve cell death (apoptosis and / or necrosis), the inhibition of nerve cell degeneration, the reduction of nerve cell stress, the improvement of nerve cell toxicity resistance, the improvement of nerve cell survival, the inhibition of abnormal protein accumulation, etc. In addition to physical damage to nerve cells, nerve cells are exposed to neurotoxic substances, oxygen or lack of nutrients, and damage exceeds a certain level, causing cell death. In addition, nerve cells are denatured by accumulating neurotoxic substances, and eventually cause cell death. Neurotoxic substances are largely divided into exogenous toxic substances and endogenous toxic substances. As exogenous toxic substances, chemical substances such as heavy metals or alcohols, botulinum toxins, etc. can be cited. As endogenous toxic substances, reactive oxygen species, neurotransmitters such as glutamate, abnormal proteins, etc. are known. Neuroprotection can be easily measured as long as it is a person skilled in the art. As an example, under conditions of exposure to various stresses, such as hypoxia load, neurotoxic substances, nutrient depletion, ultraviolet irradiation, etc., nerve cells can be cultured with a medium containing a test substance (drug group) or a medium without a test substance (control group), the number of surviving cells and the number of dead cells in the culture medium are measured, and the ratio of the number of surviving cells relative to the number of whole cells is calculated. When the ratio of the number of living cells in the drug group is higher than the ratio of the number of living cells in the control group, it is judged that the test substance has a neuroprotective effect. In a more preferred embodiment, it can be compared with a positive control group to which a substance known to have a neuroprotective effect, such as IGF-1 or NGF, is added, and whether it has a protective effect equal to or higher than that of the positive control group is measured. As another example, the neuroprotective effect can also be measured by performing an in vivo animal experiment.

[0170] The peptide of the present invention has the exocrine gland secretion promoting effect possessed by PACAP. As the exocrine gland, lacrimal gland, salivary gland, etc. can be cited, it can be used as an exocrine gland secretion promoter, such as a tear secretion promoter or a saliva secretion promoter. Without intending to be bound by theory, PACAP and the peptide of the present invention promote the secretion of saliva or tears by binding to receptors (PAC1R, VPAC1R, VPAC2R) expressed in the acinar cells of the exocrine glands. Tears cover the surface of the cornea and keep its wetness. At the same time, tears fill the depressions of the microvilli on the surface of the cornea to make the surface smooth, so that it becomes possible to obtain a clear image. In addition, the epithelial cells of the cornea actively metabolize, and unnecessary cells or metabolites are shed and discharged from the outermost surface. While washing them with tears, the necessary oxygen or nutrients are supplied. Furthermore, tears wash foreign matter mixed into the surface of the cornea, and the antibacterial effect of tears plays a role in infection defense against viruses, bacteria, fungi, etc. entering from the outside. In addition, as the synovial fluid between the eyelids and the cornea, it moves in a way that blinking or eye movement proceeds smoothly. Thus, tears are a trace amount of liquid that forms a tiny film on the surface of the cornea and conjunctiva, and the transparency or stability of the cornea cannot be omitted due to various delicate structures. The state in which an abnormality occurs on the surface of the cornea and conjunctiva due to a secretion disorder of tears is generally called dry eye. When a cornea and conjunctival disorder caused by dry eye occurs, a compound that promotes tear secretion becomes a useful preventive and therapeutic drug for dry eye and diseases associated with dry eye. The peptide according to the present invention can be used as a tear secretion promoter in eye drops such as eye drops.

[0171] The peptide of the present invention can be used as an inflammatory inhibitor because it has the inflammatory inhibitory effect of PACAP. Without intending to be bound by theory, PACAP and the peptide of the present invention can inhibit the production of inflammatory cytokines (TNF-α, IL-6, IL-12, etc.). Thus, the peptide of the present invention can be used as an anti-inflammatory agent. In particular, VPAC1R and VPAC2R, which are receptors of PACAP, can also show anti-inflammatory effects because they are widely distributed in the digestive tract, and are particularly used for the treatment of inflammatory bowel diseases, etc.

[0172] In another aspect, the present invention also relates to a pharmaceutical composition containing a therapeutically effective amount of the above-mentioned peptide. The pharmaceutical composition of the present invention can be used in the treatment or prevention of diseases that are improved by the physiological effects of PACAP. As diseases that are improved by the physiological effects of PACAP, neurological disorders, diseases associated with reduced tears, inflammatory diseases, neurotrophic keratitis, etc. can be cited. The pharmaceutical composition of the present invention can be administered to patients to treat diseases that are improved by the physiological effects of PACAP, or can be administered to patients who are likely to suffer from such diseases to prevent diseases. In addition, "treatment" refers to preventing the deterioration of their state when suffering from a disorder or disease, maintaining their current state, alleviating or disappearing, and "prevention" refers to preventing the onset of a disorder or disease before it occurs.

[0173] Neurological disorders refer to pathological conditions caused by the degeneration of nerve cells - cell death, which damages their functions, and include cerebrovascular disorders and neurodegenerative diseases.

[0174] Examples of cerebrovascular disorders include hemorrhagic disorders, such as cerebral hemorrhage, subarachnoid hemorrhage, and disorders caused by occlusion of cerebral blood vessels, such as cerebral thrombosis, cerebral infarction, cerebral circulatory insufficiency, etc. Even in the case of any of hemorrhagic disorders and occlusive disorders, the nerve cells in the brain are placed in a hypoxic state and cell death occurs. Therefore, for such cerebrovascular disorders, the peptide, neuroprotectant or pharmaceutical composition of the present invention can be administered for the purpose of treatment or prevention.

[0175] As neurodegenerative diseases, there can be mentioned, without limitation, degenerative diseases of the brain-central nerves such as dementia, Parkinson's disease, spinocerebellar degeneration, Creutzfeldt-Jakob disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, mad cow disease, epilepsy, motor neurodegenerative diseases such as spinal muscular atrophy, amyotrophic lateral sclerosis, bulbar muscular atrophy, and sensory neurodegenerative diseases. As sensory neurodegenerative diseases, there can be mentioned, without limitation, degenerative diseases of the visual, auditory, tactile, taste, and olfactory nerves, visual degenerative diseases such as glaucoma, retinal pigment degeneration, age-related macular degeneration, diabetic retinopathy, and auditory neurodegenerative diseases such as deafness.

[0176] Examples of diseases associated with tear reduction include, but are not limited to, dry eyes, keratoconjunctivitis sicca, and tear reduction syndrome.

[0177] Neurotrophic keratitis is a degenerative corneal disease caused by damage to the trigeminal nerve, which causes damage to the corneal epithelium. In the most severe cases, it can cause ulceration, melting, and perforation of the cornea, impairing the patient's visual recognition ability.

[0178] Endothelial function improvers refer to drugs that improve the function of endothelial cells. Endothelial cells are cells located in the innermost layer of blood vessels. They start with the contraction and relaxation of the vascular wall (the hardness or flexibility of the blood vessel), and regulate the adhesion of inflammatory cells to the vascular wall, vascular permeability, and coagulation-fibrinolysis system. The peptide of the present invention has the effect of improving endothelial disorders such as arteriosclerosis due to its vasodilatory effect as a physiological effect of PACAP.

[0179] Corneal epithelial or endothelial disorder refers to a disease in which corneal epithelial cells and corneal endothelial cells are disordered. Corneal epithelial cells are proliferative cells. In corneal epithelial cell disorders, the proliferation ability is inhibited, or epithelial shedding is hyperactive, then the balance of epithelial homeostasis collapses and the disease occurs. Corneal endothelial cells do not proliferate in vivo and decrease with age. In addition, corneal epithelial or corneal endothelial disorder refers to endogenous diseases such as corneal ulcers, corneal epithelial detachment, diabetic keratitis, keratoconjunctivitis sicca, chronic epidermal keratitis, punctate epidermal keratitis, corneal erosion, persistent corneal epithelial defects, exogenous diseases caused by drugs, trauma, contact lens installation, etc., or physical or chemical disorders that damage the corneal epithelium or endothelium.

[0180] Dry eyes refer to chronic diseases of tears and corneal and conjunctival epithelium caused by various factors, and are accompanied by eye discomfort or abnormal visual function. Abnormalities of tears include quantitative abnormalities of reduced tear volume and qualitative abnormalities of changes in tear properties or the ability to retain tears. In addition, dry eyes include, for example, tear hypotriasis, hyperevaporative dry eyes, dry eyes accompanied by Sjogren's syndrome, Stevens-Johnson syndrome, corneal epithelial erosion, blepharitis, ocular pemphigoid, spring mucositis, allergic conjunctivitis, vitamin A deficiency, etc.

[0181] Examples of inflammatory diseases include, but are not limited to, asthma, atopic dermatitis, urticaria, hay fever, anaphylactic shock, paranasal inflammation (including eosinophilic paranasal inflammation), rheumatoid arthritis, multiple sclerosis, arthritis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, gluten-sensitive bowel disease, etc.), Sjogren's syndrome, chronic graft-versus-host disease (GVHD), corneal infection, allergic conjunctivitis, corneal trauma, polymyositis, dermatomyositis, myasthenia gravis, chronic obstructive pulmonary disease (COPD), scleroderma, and the like.

[0182] The peptide according to the present invention, or the neuroprotectant, nerve regeneration agent, inflammation inhibitor, wound healing promoter or exocrine line secretion promoter containing the peptide, or the pharmaceutical composition can be administered orally in accordance with the treatment target disease instead of oral administration. As oral administration, sublingual, intraoral, and oral administration can be cited. As parenteral administration, it can be administered, for example, by intravenous, intraarterial, subcutaneous, topical, intraperitoneal, intramuscular, nasal, transdermal, transmucosal, intramedullary, rectal, intramuscular, intracerebral, intramedullary, subarachnoid, intradural, epidural, eye drops, ear drops, nose drops, and intraocular pathways. As intraocular pathways, more specifically, pathways under the conjunctiva, under the eyeball fascia capsule, and in the vitreous body can be cited. The pharmaceutical composition containing the peptide according to the present invention can be in a suitable dosage form according to its administration route, such as eye drops, injections, powders, infusion preparations, granules, tablets, suppositories, etc., preferably eye drops, injections, infusion preparations, powders for preparation at the time of use, etc. from the perspective of parenteral administration. As preparations for intraocular administration, for example, intravitreal injections, subconjunctival injections, and subocular muscular sac injections can be cited. In addition, these preparations may also contain various pharmaceutically acceptable auxiliary agents, that is, carriers or other auxiliary agents, such as additives such as stabilizers, preservatives, pain-relieving agents, emulsifiers, etc. In addition, it can also be used in combination with other agents having a neuroprotective effect, an inflammatory inhibitory effect, or an exocrine secretion effect.

[0183] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0184] The embodiments of the present invention described below are intended to be illustrative only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the patent claims. The present invention may be modified, for example, by adding, deleting and replacing the constituent elements of the present invention, without departing from the purpose of the present invention.

[0185] [Example]

[0186] [Example 1A: Synthesis of Fmoc-(D)-Tz(Trt)-OH (Compound IV)]

[0187] Fmoc-D-Tz(Trt)-OH (Compound IV) was synthesized according to the following reaction formula.

[0188]

Chemistry 7

[0189]

[0190] Step A: Synthesis of Fmoc-D-Ala(β-CN)-OH:

[0191]

Chemistry 8

[0192]

[0193] Compound (II): Fmoc-D-Ala(β-CN)-OH

[0194] To a solution of Fmoc-D-Asn-OH (5.04 g, 14 mmol) in acetonitrile (35 mL) was added a solution of pyridine (30 mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.96 g, 21 mmol) suspended in acetonitrile (35 mL) at 0°C, and the mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed with 3M hydrochloric acid and saturated saline, dried over sodium sulfate, and the solvent was removed by reduced pressure distillation. The obtained crude product was dissolved in acetone, and hexane / diethyl ether (1 / 1) was added to precipitate, and the mixture was concentrated under reduced pressure and filtered to obtain the target compound (4.76 g, 99%) as a white solid.

[0195] 1 H NMR (400MHz, DMSO-d6) δ13.20(1H,brs),8.08(1H,d,J=8.4Hz),7.90(2H,d,J=7.6Hz),7.72(2H,d,J=7.2Hz),7.42(2H,t,J=7.2H z), 7.33 (2H, t, J = 7.6Hz), 4.39-4.32 (3H, m), 4.25 (1H, t, J = 7.2Hz), 2.98 (1H, dd, J = 16.8, 4.8Hz), 2.88 (1H, dd, J = 16.8, 9.2Hz).

[0196] Step B: Synthesis of Fmoc-D-Tz-OH:

[0197]

Chemistry 9

[0198]

[0199] Compound (III): Fmoc-D-Tz-OH

[0200] In the above structural formula, it means that H is bonded to either N1 or N2 on the tetrazole.

[0201] Dibutyltin oxide (0.710 g, 2.9 mmol) and trimethylsilyl azide (1.8 mL, 14 mmol) were added to a toluene (18 mL) solution of Fmoc-D-Ala(β-CN)-OH (1.01 g, 3.0 mmol), and the mixture was heated at 100°C for 1.5 hours using a microwave. The reaction solution was returned to room temperature and filtered. The filtrate was washed with toluene and hexane, dissolved in ethanol / dichloromethane (3 / 2.25 mL), cooled to 4°C, and then 1M hydrochloric acid (5 mL) was added and stirred at room temperature for 2 hours for 15 minutes. The reaction solution was washed with 1M hydrochloric acid and saturated saline, the solvent was removed by vacuum distillation, hexane was added to suspend, and filtered to obtain the target compound (1.03 g, 90%) as a thin yellow solid.

[0202] 1 H NMR (400MHz, DMSO-d6) δ16.00(1H,brs),13.07(1H,brs),7.89(2H,d,H=7.6Hz),7.83(1H,d,J=8.4Hz),7.65(2H,t,J=8.0Hz),7.41(2H ,t,J=7.6Hz),7.31(2H,t,J=7.6Hz),4.53-4.48(1H,m),4.27-4.18(3H,m),3.40(1H,dd,J=15.2,6.0Hz),3.28(1H,dd,J=15.2,8.8Hz).

[0203] Step C: Synthesis of Fmoc-D-Tz(Trt)-OH:

[0204]

Chemistry 10

[0205]

[0206] Compound (IV): Fmoc-D-Tz(Trt)-OH

[0207] In the above structural formula, it means that a Trt group is bonded to N1 or N2 on the tetrazole.

[0208] To a solution of Fmoc-D-Tz-OH (1.99 g, 5.3 mmol) in THF (10 mL) were added N-methylmorpholine (0.72 mL, 7.2 mmol) and trityl chloride (1.63 g, 5.9 mmol), and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was filtered, the filtrate was washed with THF, combined with the filtrate, and the solvent was removed by vacuum distillation. The residue was purified by column chromatography (ODS, H2O / CH3CN=80 / 20→30 / 70→20 / 80), and the fraction containing the target compound was extracted with ethyl acetate, washed with saturated saline, dried over sodium sulfate, and the solvent was removed by vacuum distillation. The target compound (1.29 g, 40%) was obtained as a white solid.

[0209] 1 H NMR(400MHz,DMSO-d6)δ13.03(1H,brs),7.91(2H,d,J=7.6Hz),7.79(1H,d,J=8.8Hz),7.67(2H,brd,J=6.8Hz),7.44-7.23(1 3H,m),7.01-6.99(6H,m),4.52-4.46(1H,m),4.28-4.15(3H,m),3.40(1H,dd,J=15.2,5.2Hz),3.27(1H,dd,J=15.2,9.2Hz).

[0210] [Example 1B: Synthesis of Fmoc-nvTz(Trt)-OH (Synthesis of Compound X)]

[0211] Fmoc-nvTz(Trt)-OH (compound X) was synthesized according to the following reaction formula.

[0212]

Chemistry 11

[0213]

[0214] Process D: Boc-N ε -Synthesis of trifluoroacetyl-L-lysine:

[0215]

Chemistry 12

[0216]

[0217] Compound (VI): Boc-N ε -Trifluoroacetyl-L-lysine

[0218] To N εA solution of trifluoroacetyl-L-lysine (10.0 g, 41 mmol) in 1,4-dioxane (70 mL) was added with 1 M aqueous sodium hydroxide solution (42 mL, 42 mmol) and Boc2O (30% THF solution, 49.8 g, 68 mmol), and stirred at room temperature for 1 day. N-methylpiperazine (3.5 mL) was added to the reaction solution, and after stirring at room temperature for 2 hours, 1 M hydrochloric acid was added to the reaction solution to adjust the pH to 2, and the solution was extracted with ethyl acetate and washed with saturated physiological saline. The organic layer was dried over sodium sulfate, and the solvent was removed by vacuum distillation to obtain the title compound (15.9 g, >100%) as a white solid.

[0219] 1 H NMR (400MHz, CDCl3) δ6.69 (1H, brs), 5.12 (1H, d, J = 6.8Hz), 4.30 (1H, brs), 3.38 (2H ,q,J=6.8Hz),1.94-1.86(1H,m),1.74-1.57(3H,m),1.52-1.41(3H,m),1.45(9H,s).

[0220] Step E: Synthesis of (S)-2-[(tert-butoxycarbonyl)amino]-5-cyanopentanoic acid

[0221]

Chemistry 13

[0222]

[0223] Compound (VII): (S)-2-[(tert-butoxycarbonyl)amino]-5-cyanopentanic acid

[0224] Add Boc-N to the bottle e -trifluoroacetyl-L-lysine (1.00 g, 2.9 mmol), sodium hydroxide (276.4 mg, 6.9 mmol) and water (15 mL), further added with an aqueous solution (1.0 mL) of nickel sulfate (II) hexahydrate (77.0 mg, 0.29 mmol), sodium peroxodisulfate (1.43 g, 6.0 mmol) and sodium hydroxide (529.5 mg, 13 mmol), and stirred at room temperature for 19.5 hours. The reaction solution was adjusted to pH 2 with 1 M hydrochloric acid, extracted with ethyl acetate, and the obtained organic layer was washed with saturated physiological saline, dried over sodium sulfate, and the solvent was removed by reduced pressure distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 → 95 / 5) to obtain the title compound (661.5 mg, 86%) as a white solid.

[0225] 1H NMR(400Mz,DMSO-d6)δ12.53(1H,brs),7.14(1H,d,J=8.4Hz),3.93-3.88(1 H,m),2.53-2.48(2H,m),1.81-1.74(1H,m),1.69-1.56(3H,m),1.39(9H,m)

[0226] Step G: Synthesis of (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-cyanopentanoic acid:

[0227]

Chemistry 14

[0228]

[0229] Compound (VIII): (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-cyanopentanoic acid A solution of (S)-2-[(tert-butoxycarbonyl)amino]-5-cyanopentanoic acid (586.3 g, 2.4 mmol) in dichloromethane (2.5 mL) was cooled to 0°C, trifluoroacetic acid (2.5 mL) was added, and the mixture was stirred at room temperature for 1 hour. The solvent of the reaction solution was removed by distillation under reduced pressure. 1,4-dioxane (3.0 mL) was added to the obtained residue, and a 10% aqueous potassium carbonate solution and solid potassium carbonate were added until the pH reached 9, and then Fmoc-OSu (1.26 g, 3.7 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate / hexane (1 / 1) solution, washed with 0.5 M hydrochloric acid and saturated saline, and the obtained organic layer was dried over sodium sulfate, and the solvent was removed by distillation under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate / hexane=10 / 90→31 / 69) to obtain the title compound (731.1 mg, 83%) as a white solid.

[0230] 1 H NMR (4000MHz, CDCl3) δ7.76 (2H, d, J = 7.2Hz), 7.58 (2H, brd, J = 6.8Hz), 7.40 (2H, t, J = 7.2Hz), 7.31 (2H, t, J = 6.8Hz), 5.3 8(1H,d,J=7.6Hz),4.64-4.36(3H,m),4.20(1H,J=6.4Hz),2.38(1H,brt,J=6.4Hz),2.28-2.07(2H,m),1.88-1.41(3H,m)

[0231] Step H: Synthesis of Fmoc-nvTz-OH

[0232]

Chemistry 15

[0233]

[0234] Compound (IX): Fmoc-nvTz-OH

[0235] In the above structural formula, it means that H is bonded to either N1 or N2 on the tetrazole.

[0236] Trimethylsilyl azide (1.2 mL, 9.2 mmol) was added to a toluene (17 mL) solution of (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-cyanopentanoic acid (851 mg, 2.3 mmol) and dibutyltin oxide (581 mg, 2.3 mmol), and the mixture was heated at 100°C for 1.5 hours using a microwave. The reaction solution was returned to room temperature and filtered, and the obtained solid was washed with toluene and hexane to obtain a thin orange solid. Ethanol / dichloromethane (3 / 2.50 mL) was added to the solid, and after cooling to 0°C, 1M hydrochloric acid (5.0 mL) was added, and the mixture was stirred at room temperature for 2 hours. Ethyl acetate, 1M hydrochloric acid, and saturated saline were added to the reaction solution, and the mixture was dried over sodium sulfate, and the solvent was removed by reduced pressure distillation. The obtained residue was purified by silica gel column chromatography (dichloromethane / methanol=100 / 0→95 / 5) to obtain the title compound (367 mg, 39%).

[0237] 1 H NMR (400MHz, DMSO-d6) δ7.88(2H,d,J=7.6Hz),7.72(2H,d,J=7.2Hz),7.63(1H,d,J=7.6Hz),7.40(2H,t, J=7.6Hz),7.31(2H,t,J=7.2Hz),4.29-4.20(3H,m),4.01-3.96(1H,m),2.89(2H,m),1.78-1.46(5H,m).

[0238] Step I: Synthesis of Fmoc-nvTz(Trt)-OH

[0239]

Chemistry 16

[0240]

[0241] Compound (X): Fmoc-nvTz(Trt)-OH

[0242] In the above structural formula, it means that a Trt group is bonded to either N1 or N2 on the tetrazole.

[0243] To a solution of Fmoc-nvTz-OH (365 mg, 0.89 mmol) in THF (4.0 mL) were added N-methylmorpholine (110 μL, 1.0 mmol) and trityl chloride (261 mg, 0.94 mmol), and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the mixture was filtered, and the solvent of the obtained filtrate was removed by reduced pressure distillation to obtain the title compound (540 mg, 93%) as a white solid.

[0244] 1 H NMR(400MHz,DMSO-d6)δ12.67(1H,s),7.89(2H,d,J=7.2Hz),7.72(2H,m),7.66(1H,brs),7.39-7.19( 13H,m),7.02-6.98(6H,m),4.34-4.20(3H,m),3.98-3.94(1H,m),2.88-2.83(2H,m),1.77-1.62(4H,m)

[0245] [Example 1C: Synthesis of (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanoic acid (Compound XV)]

[0246] (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanoic acid (Compound XV) was synthesized according to the following reaction formula.

[0247]

Chemistry 17

[0248]

[0249] Step I: Synthesis of tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-3-cyanopropionate

[0250]

Chemistry 18

[0251]

[0252] Compound (XII): tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-3-cyanopropionate

[0253] 2-Bromo-2-methylpropane (46 ml, 409 mmol) was added to a suspension of Boc-Ala(CN)-OH (2.23 g, 10.4 mmol), benzyltriethylammonium chloride (2.38 g, 10.4 mmol) and potassium carbonate (37.0 g, 268 mmol) in DMA (60 mL), and the mixture was stirred at 55°C for 1 day. The reaction solution was returned to room temperature, water was added, and the mixture was extracted with ethyl acetate. The obtained organic layer was washed with water and saturated saline, dried over sodium sulfate, and the solvent was removed by reduced pressure distillation. The obtained residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 1 / 0 → 1 / 4) to obtain the title compound (2.79 g, 99%) as a transparent oily substance.

[0254] 1 H NMR(400MHz, CDCl3)δ5.45(1H,brd,J=4.8Hz),4.37(1H,brq,J=5.2Hz),2.94(1 H,dd,J=16.8,5.2Hz), 2.88(1H,dd,J=16.8,4.8Hz), 1.51(9H,s), 1.45(9H,s).

[0255] Step J: Synthesis of tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-4-(hydroxyamino)-4-imidobutyrate

[0256]

Chemistry 19

[0257]

[0258] Compound (XIII): tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-4-(hydroxyamino)-4-imidobutyrate

[0259] Triethylamine (2.8 mL, 20 mmol) and hydroxylamine hydrochloride (1.08 g, 16 mmol) were added to a solution of tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-3-cyanopropionate (2.79 g, 10 mmol) in ethanol (30 mL), and the mixture was stirred at 95° C. for 4 hours. The reaction solution was returned to room temperature, water was added, extracted with ethyl acetate, dried over sodium sulfate, the solvent was removed by reduced pressure distillation, and vacuum drying was performed to obtain the title compound (2.85 g, 91%) as white amorphous.

[0260] 1 H NMR (400MHz, CDCl3) δ6.47(1H,brs),5.47(1H,brs),4.63(2H,brs),4.37(1H,brs),2.62-2.54(2H,m),1.46(9H,s),1.44(9H,s).

[0261] Step K: Synthesis of tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanoate

[0262]

Chemistry 20

[0263]

[0264] Compound (XIV): tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanoate

[0265] 1,1-Carbonyldiimidazole (627 mg, 3.9 mmol) was added to a solution of tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-4-(hydroxyamino)-4-iminobutanoate (1.04 g, 3.4 mmol) in THF (20 mL), and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated, toluene (20 mL) was added, and the mixture was stirred at 100°C for 2 hours. The reaction solution was returned to room temperature, the solvent was removed by vacuum distillation, and the obtained residue was purified by flash silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to obtain the title compound (658.9 mg, 58%) as a white amorphous substance.

[0266] 1 H NMR (400MHz, DMSO-d6) δ12.23 (1H, brs), 7.36 (1H, d, J = 8.0Hz), 4.24 (1H, q, J = 8.0Hz), 2.90(1H,dd,J=15.2,7.2Hz), 2.81(1H,dd,J=15.2,8.4Hz), 1.38(9H,s), 1.37(9H,s).

[0267] Step L: Synthesis of (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanoic acid

[0268]

Chemistry 21

[0269]

[0270] Compound (XV): (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanoic acid

[0271] To a solution of tert-butyl (S)-2-[(tert-butoxycarbonyl)amino]-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)propanoate (658.9 mg, 2.0 mmol) in dichloromethane (4.0 mL) were added triethylsilane (1.0 mL) and trifluoroacetic acid (5.0 mL) at 0°C, followed by stirring at room temperature for 3 hours. The solvent was removed by vacuum distillation, diethyl ether and hexane were added, the solvent was removed by vacuum distillation again, 1,4-dioxane (10 mL) was added, and a saturated sodium bicarbonate aqueous solution was added until the pH became 9. Thereafter, Fmoc-OSu (813 mg, 2.4 mmmol) was added at 0°C, and the mixture was stirred at room temperature overnight. The reaction solution was adjusted to pH 3 by adding 10% aqueous citric acid solution, extracted with ethyl acetate and dried over sodium sulfate. The solvent was removed by reduced pressure distillation and the resulting residue was purified by silica gel column chromatography (dichloromethane / methanol=100 / 0→93 / 7) to obtain the title compound (465.3 mg, 59%) as white amorphous.

[0272] 1 H NMR (400MHz, DMSO-d6) δ7.89 (2H, d, J = 7.2Hz), 7.70-7.63 (3H, m), 7.42 (2H, t, J = 7.2Hz), 7.32 (2H,t,J=7.2Hz),4.33-4.20(4H,m),2.96(1H,dd,J=15.2,5.6Hz),2.80(1H,dd,15.2,8.8Hz),

[0273] [Example 2: Peptide Synthesis]

[0274] The peptides used in the experiment were synthesized by solid phase synthesis using the Fmoc method using a peptide synthesizer (model: PSSM-8 manufactured by Shimadzu Corporation). Fmoc-Tz-OH, Fmoc-Tz(trt)-OH, and Fmoc-egTz(trt)-OH, which are non-natural amino acids used in the solid phase synthesis, were purchased from Astatech, and Fmoc-cya-OH was purchased from Anaspec. Furthermore, compounds (IV), (X), and (XV) synthesized in Example 1 were used. In addition, F-moc-Ai-OH and Fmoc-Cn-OH were purchased from Watanabe Chemical Industries, Ltd., and peptides 1 to 28 having the following sequences were synthesized. The molecular weight of the synthesized peptides was analyzed by mass spectrometry (MALDI TOF). As shown in Table 2 below, all measured values ​​were in good agreement with the theoretical values. Modification of the N-terminus and the C-terminus was performed by methods known to those skilled in the art.

[0275]

Table 2

[0276]

[0277]

[0278]

[0279] In the table, Tz refers to tetrazole-substituted aspartic acid, egTz refers to tetrazole-substituted glutamic acid, nvTz refers to tetrazole-substituted homoglutamic acid, cya refers to sulfo-substituted aspartic acid, (5Oxa) refers to oxadiazolone-substituted aspartic acid, Ai refers to α-aminoisobutyric acid, or Cn refers to β-cyanoalanine. Tz(D) indicates that the tetrazole-substituted aspartic acid is the D-isomer.

[0280] Tz introduced into peptides 1 to 9 was a racemic form. Amino acids used in other peptides were L-forms unless otherwise specified.

[0281] [Example 3: Stability Test 1]

[0282] [Preparation of measurement sample 1]

[0283] The peptides synthesized in Example 2 (peptides 1 to 9) were weighed and dissolved in 0.1% phosphate buffer (pH 7.0) to prepare a 1.0 mM peptide solution. The 1.0 mM peptide solution was then diluted to 100 μM with phosphate buffer. Filtered using a chromatographic disk (Millex-GV, 0.22 μm, manufactured by Merck Millipore), the filtrate was dispensed into an LC bottle (Waters Deactivated Qsert Vial). The prepared peptide solution was incubated in a thermostatic bath at 40°C for 1 month or 2 months to obtain a stored sample. In addition, a sample that was not stored in the peptide solution prepared at the same time was used as a standard sample (initial sample). The standard sample and the stored sample were stored at -30°C until the sample was analyzed.

[0284] [Water permeability]

[0285] The total weight of the peptide aqueous solution and the storage container was taken as the subject weight, and the subject weight before storage was weighed. After storage under each condition, the subject weight after storage was also weighed. In addition, the empty weight of the storage container was weighed, and the water permeability was calculated based on the following formula.

[0286]

Number 1

[0287]

[0288] After stirring the standard sample and the stored sample with a vortex mixer, the peptide solution was transferred to an HPLC vial (Deactivated Qsert vial manufactured by Waters). Reverse phase HPLC (HPLC system: Prominence manufactured by Shimadzu Corporation) was operated under the conditions of Table 3 below to analyze the peptide solution and obtain a chromatogram.

[0289] [HPLC analysis conditions]

[0290] Column: XSelect CSH C18 manufactured by Waters, 5 μm, 4.6×250 mm,

[0291] Guard column: Waters XSelect CSH C18, 5 μm, 4.6×20 mm Guard Cartridge Detection wavelength: 220 nm

[0292] Mobile phase A: 0.1% formic acid aqueous solution,

[0293] Mobile phase B: 0.1% formic acid in acetonitrile

[0294] Measurement time: 30 minutes

[0295] Measurement sample injection volume: 50μL

[0296] Flow rate: 1.0mL / min

[0297] Column temperature: 40°C

[0298] Liquid delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 3 below, and linear concentration gradient control was performed.

[0299]

Table 3

[0300] Time after sample injection (min) Mobile phase A (vol%) Mobile phase B (vol%) 0～30.0 90→75 10→25 30.0～30.5 75→0 25→100 30.5～36.5 0 100 36.5～37.0 0→90 100→10 37.0～49.5 90 10

[0301] In the chromatogram, the peak area value of the peptide was determined and the residual rate (residual rate before water correction) was calculated by formula (2). In addition, for the residual rate before water correction, the residual rate after water correction was calculated by formula (3) by taking into account the permeability of the container.

[0302]

Number 2

[0303]

[0304]

Number 3

[0305]

[0306] Table 4 shows the moisture-corrected residual ratios of peptides in the samples after storage.

[0307]

Table 4

[0308]

[0309]

[0310] All peptides showed higher aqueous solution stability at 40°C for 4 weeks and 8 weeks than PACAP27.

[0311] [Preparation of measurement sample 2]

[0312] For the peptides synthesized in Example 2 (peptides 13 to 16), the peptide solutions prepared by the same method as [Preparation of measurement samples 1] were incubated in a thermostatic chamber at 40°C for 2 weeks and 8 weeks to obtain stored samples. In addition, the sample that was not used for storage among the peptide solutions prepared at the same time was used as a standard sample (initial sample). The standard sample and the stored sample were stored at -30°C until the sample analysis.

[0313] [HPLC analysis conditions]

[0314] Only the measurement time and the liquid feeding conditions of the mobile phase were changed as follows, and the measurement was performed under the same conditions as those in the column of [Preparation 1 of measurement sample]:

[0315] Measurement time: 20 minutes

[0316] Liquid delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 5 below, and linear concentration gradient control was performed.

[0317]

Table 5

[0318] Time after sample injection (min) Mobile phase A (vol%) Mobile phase B (vol%) 0～20.0 85→75 15→25 20.0～20.1 75→0 25→100 20.1～24.5 0 100 24.5～25.0 0→85 100→15 25.0～40.5 85 15

[0319] Table 6 shows the moisture-corrected residual ratios of peptides in the samples after storage.

[0320]

Table 6

[0321] Peptide name 40℃, 2 weeks 40℃, 8 weeks Peptide 13 96.2％ 84.6％ Peptide 14 96.3％ 84.5％ Peptide 15 95.6％ 81.3％ Peptide 16 94.4％ 80.3％ PACAP27 64.3％ 23.7％

[0322] Peptides with differences in tetrazole steric structure and length between tetrazole and peptide main chain also showed higher stability than PACAP27.

[0323] [Preparation of measurement sample 3]

[0324] For the peptides synthesized in Example 2 (peptides 17 to 21), the peptide solution prepared by the same method as [Preparation of the measurement sample 1] was incubated in a thermostatic chamber at 40°C for 4 weeks to obtain a stored sample. In addition, a sample that was not used for storage among the peptide solutions prepared at the same time was used as a standard sample (initial sample). The standard sample and the stored sample were stored at -30°C until the sample analysis.

[0325] [HPLC analysis conditions]

[0326] Only the measurement time and the liquid feeding conditions of the mobile phase were changed as follows, and the measurement was performed under the same conditions as those in the column of [Preparation 1 of measurement sample]:

[0327] Measurement time: 20 minutes

[0328] Liquid delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 7 below, and linear concentration gradient control was performed.

[0329]

Table 7

[0330] Time after sample injection (min) Mobile phase A (vol%) Mobile phase B (vol%) 0～20.0 85→75 15→25 20.0～20.1 75→0 25→100 20.1～24.5 0 100 24.5～25.0 0→85 100→15 25.0～40.5 85 15

[0331] Table 8 shows the moisture-corrected residual ratios of peptides in the samples after storage.

[0332]

Table 8

[0333] Peptide name 40℃, 4 weeks Peptide 17 59.2％ Peptide 18 65.1％ Peptide 19 68.7％ Peptide 20 69.5％ Peptide 21 69.0％ PACAP27 23.9％

[0334] Among the peptides with different acyl groups substituted at the N-terminus, the peptides showed higher stability than PACAP27.

[0335] [Preparation of measurement sample 4]

[0336] For the peptide 22 synthesized in Example 2, the peptide solution prepared by the same method as [Preparation of the measurement sample 1] was incubated in a thermostatic chamber at 40°C for 1 week and 4 weeks to obtain a stored sample. In addition, a sample that was not used for storage among the peptide solutions prepared at the same time was used as a standard sample (initial sample). The standard sample and the stored sample were stored at -30°C until the sample analysis.

[0337] [HPLC analysis conditions]

[0338] Only the measurement time and the liquid feeding conditions of the mobile phase were changed as follows, and the measurement was performed under the same conditions as those in the column of [Preparation 1 of measurement sample]:

[0339] Measurement time: 20 minutes

[0340] Liquid delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 9 below, and linear concentration gradient control was performed.

[0341]

Table 9

[0342]

[0343]

[0344] Table 10 shows the moisture-corrected residual ratios of peptides in the samples after storage.

[0345]

Table 10

[0346] Peptide name 40℃, 1 week 40℃, 4 weeks Peptide 22 89.7％ 79.7％ PACAP27 74.9％ 33.0％

[0347] The peptides in which the tetrazole-substituted aspartic acid was substituted with oxadiazolone-substituted aspartic acid at positions 3 and 8 showed higher stability than PACAP27.

[0348] [Preparation of measurement sample 5]

[0349] Peptides 23 to 30 synthesized in Example 2 were weighed and dissolved in Tris buffer (pH 7.0) to prepare a 1.0 mM peptide solution. This solution was filtered using a chromatographic disk (Millex-GV, 0.22 μm, manufactured by Merck Millipore). The filtrate was diluted to 100 μM with Tris buffer (pH 7.0) and dispensed into tubes (Protein Lobind Tube manufactured by Eppendorf). The prepared peptide solution was incubated in a thermostatic bath at 40°C for 4 weeks or 8 weeks, and incubated in a thermostatic bath at 60°C for 1 week or 2 weeks to obtain a stored sample. In addition, a sample that was not stored in the peptide solution prepared at the same time was used as a standard sample (initial sample). The standard sample was stored at -30°C until the sample was analyzed, and the stored sample was stored at -30°C until the sample was analyzed.

[0350] [HPLC analysis conditions 1]

[0351] Only the measurement time and the liquid feeding conditions of the mobile phase were changed as follows, and the measurement was performed under the same conditions as those in the column of [Preparation 1 of measurement sample]:

[0352] Measurement time: 20 minutes

[0353] Liquid delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 11 below, and linear concentration gradient control was performed.

[0354]

Table 11

[0355] Time after sample injection (min) Mobile phase A (vol%) Mobile phase B (vol%) 0～20.0 85→75 15→25 20.0～20.1 75→0 25→100 20.1～24.5 0 100 24.5～25.0 0→85 100→15 25.0～40.5 85 15

[0356] For peptide 30 only, the following gradient times were used.

[0357]

Table 12

[0358] Time after sample injection (min) Mobile phase A (vol%) Mobile phase B (vol%) 0～20.0 82.5→72.5 17.5→27.5 20.0～20.1 72.5→0 27.5→100 20.1～24.5 0 100 24.5～25.0 0→82.5 100→17.5 25.0～40.5 82.5 17.5

[0359] Table 13 shows the moisture-corrected residual ratios of peptides in the samples after storage.

[0360]

Table 13

[0361]

[0362]

[0363] [Example 4: Stability Test 2]

[0364] For the peptides selected from peptides 1 to 16, peptides subjected to N-terminal acylation were prepared. For the peptides selected from peptides 17 to 30, peptides without N-terminal acylation were prepared. For the N-terminally modified peptides and the non-N-terminally modified peptides, each was dissolved in a Tris buffer (pH 7.0) to prepare a 1.0 mM peptide solution. This solution was filtered using a chromatographic disk (Millex-GV, 0.22 μm, manufactured by Merck Millipore). The filtrate was diluted to 100 μM with Tris buffer (pH 7.0) and dispensed into a tube (Protein Lobind Tube manufactured by Eppendorf). The prepared peptide solution was incubated in a thermostatic bath at 60°C for 1 week or 2 weeks to obtain a stored sample. In addition, a sample that was not stored in the peptide solution prepared at the same time was used as a standard sample (initial sample). The standard sample and the stored sample were stored at -30°C until the sample was analyzed.

[0365] The stored samples were analyzed under appropriate HPLC analysis conditions, and the moisture-corrected residual rate of the peptide in the stored samples was calculated in the same manner as in Example 3. Improved stability was observed in the N-terminally modified, particularly N-terminally acylated, and further acetylated peptides.

[0366] [Reference example: Stability test 3]

[0367] The peptides of reference examples 1 to 6 were weighed and dissolved in Tris buffer (pH 7.0) to prepare a 1.0 mM peptide solution. The solution was filtered using a chromatographic disk (Millex-GV, 0.22 μm, manufactured by Merck Millipore). The filtrate was diluted to 100 μM with Tris buffer (pH 7.0) and dispensed into tubes (Protein Lobind Tube manufactured by Eppendorf). The prepared peptide solution was incubated in a thermostatic bath at 60°C for 1 or 2 weeks to obtain a stored sample. In addition, a sample that was not stored in the peptide solution prepared at the same time was used as a standard sample (initial sample). The standard sample and the stored sample were stored at -30°C until the sample was analyzed.

[0368] The stored samples were measured under the following HPLC analysis conditions, and the moisture-corrected residual ratio of the peptide in the stored samples was calculated in the same manner as in Example 3. The results are shown in Table 15.

[0369] [HPLC analysis conditions]

[0370] Column: XSelect CSH C18, 5 μm, 4.6×250 mm manufactured by Waters

[0371] Guard column: Waters XSelect CSH C18, 5 μm, 4.6×20 mm Guard Cartridge Detection wavelength: 220 nm

[0372] Mobile phase A: 0.1% formic acid aqueous solution

[0373] Mobile phase B: 0.1% formic acid in acetonitrile

[0374] Measurement time: 20 minutes

[0375] Measurement sample injection volume: 50μL

[0376] Flow rate: 1.0mL / min

[0377] Column temperature: 40°C

[0378] Sample cooler: 25°C

[0379] Liquid delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 14 below, and linear concentration gradient control was performed.

[0380]

Table 14

[0381]

[0382]

[0383] Table 15 shows the moisture-corrected residual rates of peptides in the samples after storage.

[0384]

Table 15

[0385] Peptides 60℃, store for 1 week 60℃, 2 weeks Reference Example 1 26.6％ 15.8％ Reference Example 2 28.7％ 17.2％ Reference Example 3 91.3％ 82.4％ Reference Example 4 91.7％ 84.8％ Reference Example 5 98.1％ 92.9％ Reference Example 6 98.4％ 92.8％

[0386] Reference Example 1 (PACAP) was preserved at 60°C for 1 week at 26.6% and after 2 weeks, the stability was extremely poor at 15.8%. Reference Example 2 in which the N-terminus of PACAP was acetylated also had only the same stability. On the other hand, in Reference Examples 3 and 4 in which the carboxyl groups of aspartic acid at positions 3 and 8 were substituted with tetrazole (in Reference Example 3, methionine at position 17 was further substituted with norleucine, and in Reference Example 4, methionine at position 17 was further substituted with leucine), after 1 week at 60°C, 91.3% and 91.7% were preserved and after 2 weeks, the stability was greatly improved to 82.4% and 84.8%. Furthermore, in Reference Examples 5 and 6 in which the N-terminus of Reference Examples 3 and 4 were acetylated, after 1 week at 60°C, 98.1% and 98.4% were preserved and after 2 weeks, the stability was further improved to 92.9% and 92.8%. Among them, the accelerated test of 1 week and 2 weeks at 60° C. is equivalent to about 1 year and about 2 years at room temperature (25° C.). Therefore, Reference Examples 5 and 6 are expected to be stable at room temperature for 2 years (residual rate of 90% or more).

[0387] [Example 5: cAMP determination of PACAP27 and its altered peptides]

[0388] [Cell culture]

[0389] Frozen CHO-K1 cells (PAC1 or VPAC1 receptor-highly expressed cell lines purchased from DiscoveRx) treated with mitomycin were cultured at a concentration of 1.35×10 4 Cell plating reagent (DiscoveRx) was prepared at a concentration of cells / 100 μl / well and seeded onto a 96-well culture plate. The cells were cultured in a 5% CO 2 incubator at 37° C. for 18 to 24 hours to allow the cells to attach to the plate.

[0390] [Reagent preparation]

[0391] The powders of peptides 1 to 30 synthesized in Example 2 and Reference Example 1 (PACAP) were dissolved in water to 0.1 mM, and then diluted to 20 μM with Cell assay buffer (manufactured by DiscoveRx) (containing 0.5 mM IBMX and 0.001% BSA). A 5-fold dilution series was then prepared using the same Cell assay buffer and used in the assay.

[0392] [cAMP measurement]

[0393] The cAMP assay was performed using the Hit Hunter cAMP assay for Biologics kit (manufactured by DiscoveRx, Cat. No. 90-0075LM25) according to the instructions attached to the kit. The cAMP antibody solution and the diluted peptide 1 to 30 solutions of various concentrations were mixed to prepare a peptide-cAMP antibody mixture. Next, the culture medium was removed from the culture plate of CHO-K1 cells, washed with PBS, and the peptide-cAMP antibody mixture was added to the cells and incubated for 30 minutes in a 5% CO2 atmosphere at 37°C. Next, the Working detection solution was added, the culture plate was shielded from light in aluminum foil, and incubated at 25°C for 1 hour. After incubation, Solution A was added, the culture plate was shielded from light with aluminum foil, and incubated at 25°C for 3 hours. Finally, the chemiluminescent signal was detected using a GloMax detector (manufactured by Prω) under the conditions of Luminescence, Integration time (1 sec). The obtained relative luminescence unit (RLU) value was analyzed using GraphPad Prism Ver 6.05 (manufactured by GraphPad) to calculate the EC value of each peptide. 50 value.

[0394]

Table 16

[0395] Peptide name N-terminus C-terminus PAC1 VPAC1 Reference Example 1 (PACAP27) H <![CDATA[NH2]]> 0.052nM 0.097nM Peptide 1 H <![CDATA[NH2]]> 1.220nM 0.097nM Peptide 2 H <![CDATA[NH2]]> 0.610nM 0.085nM Peptide 3 H <![CDATA[NH2]]> 0.689nM 0.111nM Peptide 4 H <![CDATA[NH2]]> 2.360nM 0.121nM Peptide 5 H <![CDATA[NH2]]> 0.695nM 0.078nM Peptide 6 H <![CDATA[NH2]]> 1.170nM 0.110nM Peptide 7 H <![CDATA[NH2]]> 0.319nM 0.096nM Peptide 8 H <![CDATA[NH2]]> 0.679nM 0.045nM Peptide 9 H <![CDATA[NH2]]> 0.287nM 0.079nM Peptide 10 H <![CDATA[NH2]]> 0.447nM 0.057nM Peptide 11 H <![CDATA[NH2]]> 0.911nM 0.080nM Peptide 12 M <![CDATA[NH2]]> 4.280nM 0.651nM Peptide 13 H <![CDATA[NH2]]> 0.738nM 0.082nM Peptide 14 H <![CDATA[NH2]]> 0.857nM 0.107nM Peptide 15 H <![CDATA[NH2]]> 0.079nM 0.243nM Peptide 16 H <![CDATA[NH2]]> 1.620nM 0.142nM Peptide 17 <![CDATA[CH3CH2CO]]> <![CDATA[NH2]]> 0.398nM 0.071nM Peptide 18 <![CDATA[CH3CH2CH2CO]]> <![CDATA[NH2]]> 0.189nM 0.069nM Peptide 19 <![CDATA[(CH3)2CHCO]]> <![CDATA[NH2]]> 0.256nM 0.069nM Peptide 20 <![CDATA[CH3CH2CH2CH2CO]]> <![CDATA[NH2]]> 0.574nM 0.131nM Peptide 21 <![CDATA[(CH3)2CHCH2CO]]> <![CDATA[NH2]]> 1.130nM 0.141nM Peptide 22 <![CDATA[Ac(CH3CO)]]> <![CDATA[NH2]]> 0.376nM 0.077nM Peptide 23 Ac <![CDATA[NH2]]> 1.150nM 0.047nM Peptide 24 Ac <![CDATA[NH2]]> 1.260nM 0.042nM Peptide 25 Ac <![CDATA[NH2]]> 0.693nM 0.041nM Peptide 26 Ac <![CDATA[NH2]]> 0.209nM 0.068nM Peptide 27 Ac <![CDATA[NH2]]> 2.530nM 0.111nM Peptide 28 Ac <![CDATA[NH2]]> 2.120nM 0.072nM Peptide 29 Ac <![CDATA[NH2]]> 3.280nM 0.123nM Peptide 30 Ac <![CDATA[NH2]]> 2.290nM 0.113nM

[0396] Taking the above results into consideration, the peptides (peptides 1 to 30) according to the present invention have significantly improved stability in aqueous solutions compared to PACAP, while maintaining the same physiological activity as PACAP. In particular, since the peptides have a shelf life of more than 2 years at room temperature, they can be developed into liquid preparations such as bottles, ampoules, eye drops, etc.

[0397] [Preparation example]

[0398] The medicine containing the peptide of the present invention as an active ingredient can be prepared, for example, in the following formulation: Although the medicine of the present invention is described more specifically by giving formulation examples, the present invention is not limited to these formulation examples.

[0399] 【1. Capsules】

[0400] (1) Peptide 5 40mg (2) Lactose 70mg (3) Microcrystalline cellulose 9mg (4) Magnesium stearate 1mg 1 capsule 120mg

[0401] All of (1), (2) and (3) and 1 / 2 of (4) are mixed and granulated. The remaining (4) is added thereto and the whole is sealed in a gelatin capsule.

[0402] 【2. Tablets】

[0403] (1) Peptide 6 40mg (2) Lactose 58mg (3) Corn starch 18mg (4) Microcrystalline cellulose 3.5mg (5) Magnesium stearate 0.5mg 1 tablet 120mg

[0404] The whole amount of (1), (2) and (3), 2 / 3 of (4) and 1 / 2 of (5) are mixed and granulated. The remaining amount of (4) and (5) are added to the granulated mixture and pressed into tablets.

[0405] 【3. Vitreous injection】

[0406] 1ml

[0407] (1) Peptide 5 40mg (2) Purified white sugar 50mg (3) Sodium chloride 2.34mg (4) Polysorbate 80 Moderate (5) Disodium hydrogen phosphate Moderate (6) Sodium dihydrogen phosphate Moderate (7) Sterile purified water Moderate

[0408] (1) to (6) are dissolved in sterile purified water (7) to prepare a vitreous injection solution.

[0409] 【4. Eye drops】

[0410] 100mL

[0411] (1) Peptide 6 100mg (2) Tromethamine 300mg (3) Sodium chloride 900mg (4) Benzalkonium chloride Moderate (5) Sterile purified water Moderate

[0412] (1) to (4) are dissolved in sterile purified water (5) to adjust the pH to prepare an eye drop. Sequence Listing <110> Senju Pharmaceutical Co., Ltd. <120> PACAP stabilizing peptide <130> P200279WO <150> JP 2019-091696 <151> 2019-05-14 <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 38 <212> PRT <213> Homo sapiens <400> 1 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys 20 25 30 Gln Arg Val Lys Asn Lys 35 <210> 2 <211> 27 <212> PRT <213> Homo sapiens <400> 2 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 3 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (2) <223> Xaa is a neutral amino acid <220> <221> MUTAGEN <222> (5) <223> Xaa is a neutral amino acid <220> <221> MUTAGEN <222> (7) <223> Xaa is a neutral amino acid <220> <221> MUTAGEN <222> (9)..(9) <223> Xaa is Ala, Ser, Aib, or b-cyanoalanine <220> <221> MUTAGEN <222> (11)..(11) <223> Xaa is Ala, Ser, Aib, or b-cyanoalanine <220> <221> MUTAGEN <222> (16) <223> Xaa is Gln or Ala <220> <221> MUTAGEN <222> (17) <223> Xaa is a nonpolar amino acid <400> 3 His Xaa Asp Gly Xaa Phe Xaa Asp Xaa Tyr Xaa Arg Tyr Arg Lys Xaa 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 4 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 4 His Ser Xaa Gly Ile Phe Ala Xaa Ala Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 5 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 5 His Ser Xaa Gly Ile Phe Ala Xaa Ser Tyr Ala Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 6 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 6 His Ser Xaa Gly Ile Phe Ala Xaa Ala Tyr Ala Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 7 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (9)..(9) <223> Xaa is Aib <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 7 His Ser Xaa Gly Ile Phe Ala Xaa Xaa Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 8 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (11)..(11) <223> Xaa is Aib <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 8 His Ser Xaa Gly Ile Phe Ala Xaa Ser Tyr Xaa Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 9 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (9)..(9) <223> Xaa is Aib <220> <221> MUTAGEN <222> (11)..(11) <223> Xaa is Aib <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 9 His Ser Xaa Gly Ile Phe Ala Xaa Xaa Tyr Xaa Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 10 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (9)..(9) <223> Xaa is Aib <220> <221> MUTAGEN <222> (11)..(11) <223> Xaa is Aib <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 10 His Ser Xaa Gly Ile Phe Thr Xaa Xaa Tyr Xaa Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 11 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (24)..(24) <223> Amidation <400> 11 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala 20 <210> 12 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (26)..(26) <223> Amidation <400> 12 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val 20 25 <210> 13 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a sulfoxyl group <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a sulfoxyl group <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 13 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 14 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a sulfoxyl group <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a sulfoxyl group <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 14 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 15 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (4) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (9)..(9) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (28)..(28) <223> Amidation <400> 15 Met His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys 1 5 10 15 Gln Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 16 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is a D-amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is a D-amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 16 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 17 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is a D-amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 17 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 18 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is a D-amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 18 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 19 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of glutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 19 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 20 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> PROPYONYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 20 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> twenty one <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> BUTYRYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> twenty one His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> twenty two <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ISOBUTYRYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> twenty two His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> twenty three <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> PENTANOYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> twenty three His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> twenty four <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ISOPENTANOYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> twenty four His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 25 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by an oxadiazolone <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by an oxadiazolone <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 25 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 26 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 26 His Ser Xaa Gly Ala Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 27 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 27 His Ser Xaa Gly Ile Phe Ala Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 28 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 28 His Ala Xaa Gly Ile Phe Ala Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 29 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 29 His Ala Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Ala 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 30 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 30 His Ser Xaa Gly Ile Phe Ala Xaa Ala Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 31 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 31 His Ser Xaa Gly Ile Phe Ala Xaa Ser Tyr Ser Arg Tyr Arg Lys Ala 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 32 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 32 His Ser Xaa Gly Ile Phe Ala Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Ala Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 33 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 33 His Ala Xaa Gly Ile Phe Ala Xaa Ala Tyr Ser Arg Tyr Arg Lys Ala 1 5 10 15 Ala Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 34 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (11)..(11) <223> Xaa is b-cyanoalanine <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 34 His Ser Xaa Gly Ile Phe Ala Xaa Ser Tyr Xaa Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 35 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (9)..(9) <223> Xaa is b-cyanoalanine <220> <221> MUTAGEN <222> (11)..(11) <223> Xaa is b-cyanoalanine <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 35 His Ser Xaa Gly Ile Phe Thr Xaa Xaa Tyr Xaa Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 36 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of glutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 36 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 37 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of glutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of glutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 37 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 38 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of homoglutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 38 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 39 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of homoglutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 39 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 40 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of homoglutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of homoglutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 40 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 41 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of glutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of homoglutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 41 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 42 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of homoglutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of glutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <220> <221> MOD_RES <222> (27)..(27) <223> Amidation <400> 42 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 43 <211> 11 <212> PRT <213> Homo sapiens <400> 43 Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn Lys 1 5 10 <210> 44 <211> 10 <212> PRT <213> Homo sapiens <400> 44 Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn 1 5 10 <210> 45 <211> 9 <212> PRT <213> Homo sapiens <400> 45 Gly Lys Arg Tyr Lys Gln Arg Val Lys 1 5 <210> 46 <211> 8 <212> PRT <213> Homo sapiens <400> 46 Gly Lys Arg Tyr Lys Gln Arg Val 1 5 <210> 47 <211> 7 <212> PRT <213> Homo sapiens <400> 47 Gly Lys Arg Tyr Lys Gln Arg 1 5 <210> 48 <211> 6 <212> PRT <213> Homo sapiens <400> 48 Gly Lys Arg Tyr Lys Gln 1 5 <210> 49 <211> 5 <212> PRT <213> Homo sapiens <400> 49 Gly Lys Arg Tyr Lys 1 5 <210> 50 <211> 4 <212> PRT <213> Homo sapiens <400> 50 Gly Lys Arg Tyr 1 <210> 51 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <400> 51 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 52 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <400> 52 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 53 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <400> 53 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 54 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is Nle <400> 54 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Xaa Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 55 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Modified PACAP peptide <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (8) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <400> 55 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25

Claims

1. A peptide consisting of a sequence in which the aspartic acid at the 3-position and the 8-position of the sequence represented by the following formula is independently substituted with a residue selected from the group consisting of tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz), tetrazole-substituted homoglutamic acid (nvTz), sulfoxyl-substituted aspartic acid (cya) and oxadiazolone-substituted aspartic acid (5Oxa), or a modified sequence thereof: H-X1-DG-X2-F-X3-D-X4-Y-X5-RYRK-X6-X7-AVKKYLAAVL(SEQ ID NO:3) In the above formula, X1 is serine or alanine, X2 is isoleucine or alanine, X3 is alanine or threonine, X4 is alanine, serine or Ai, X5 is alanine, serine or Ai, X6 is glutamine or alanine, X7 is methionine, leucine, norleucine or alanine, The peptide has binding properties to PAC1R and VPAC1R, The sequence of changes is: A sequence selected from the following sequences is added to the C-terminus of the sequence of SEQ ID NO: 3: GKRYKQRVKNK (SEQ ID NO: 43), GKRYKQRVKN (SEQ ID NO: 44), GKRYKQRVK (SEQ ID NO: 45), GKRYKQRV (SEQ ID NO: 46), GKRYKQR (SEQ ID NO: 47), GKRYKQ (SEQ ID NO: 48), GKRYK (SEQ ID NO: 49), GKRY (SEQ ID NO: 50), GKR, GRR, GK, GR, and G; or A sequence having one amino acid added to the N-terminus of the sequence of SEQ ID NO: 3; or A sequence in which 1 to 3 amino acids are deleted from the C-terminus of the sequence of SEQ ID NO:

3.

2. The peptide of claim 1, wherein when the amino acids at positions 3 and 8 are respectively tetrazole-substituted aspartic acid (Tz), tetrazole-substituted glutamic acid (egTz) or tetrazole-substituted homoglutamic acid (nvTz), these amino acids are L-isomer, D-isomer or racemic isomer. The peptide according to claim 1 , wherein in the peptide, the N-terminus is modified with a protecting group.

4. The peptide of claim 3, wherein the protecting group is an acyl group. The peptide according to claim 1 , wherein in the peptide, the C-terminus is modified with a protecting group. The peptide of claim 5 , wherein the protecting group is an amide group or an ester group.

7. Use of the peptide according to any one of claims 1 to 6 in the manufacture of a neuroprotective agent.

8. Use of the peptide according to any one of claims 1 to 6 in the manufacture of a tear secretion promoting agent.

9. Use of the peptide according to any one of claims 1 to 6 in the manufacture of an anti-inflammatory agent.

10. Use of the peptide according to any one of claims 1 to 6 in the manufacture of an agent for improving vascular endothelial function.

11. Use of the peptide according to any one of claims 1 to 6 in the manufacture of a therapeutic agent for corneal epithelial or corneal endothelial disorders.

12. Use of the peptide according to any one of claims 1 to 6 in the manufacture of a therapeutic agent for dry eye.

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