Composition for preventing or treating neurotrophic keratitis containing PACAP peptide or stabilized peptide of PACAP

Abstract

An object of the present invention is to provide a composition for preventing or treating neurotrophic keratitis. The present inventors found that PACAP peptide exhibits an inhibitory effect on corneal injury in a neurotrophic keratitis model and promotes neurite outgrowth in a cultured nerve cell model. Furthermore, there are provided a composition for preventing or treating neurotrophic keratitis containing a PACAP peptide or a stabilized PACAP peptide, in which the carboxyl group of aspartic acid at positions 3 and / or 8 in the sequence of PACAP is replaced with tetrazole, and the stability is significantly enhanced, and a neurite outgrowth promoter.

Description

Technical field

[0001] The present invention relates to a composition for preventing or treating neurotrophic keratitis containing a stabilized peptide having the physiological activity of PACAP. [Background Technology]

[0002] Corneal perception, through the blink and tear reflexes, is crucial for preventing damage and is controlled by the corneal nerves. These nerves not only mediate perception but also influence corneal epithelial cell proliferation through the secretion of neurotransmitters and nerve growth factors released from corneal nerve terminals. Damage to the corneal nerves alters corneal epithelial cell metabolism, impairing proliferation and leading to dystrophic ulcers. Neurotrophic keratitis, or neuroplegic keratitis, is a degenerative corneal disease caused by damage to the trigeminal nerve, resulting in damage to the corneal epithelium. In its most severe form, corneal ulceration, melting, and perforation can occur, impairing the patient's visual perception. Neurotrophic keratitis can be caused by a variety of clinical conditions, including viral infections such as corneal herpes, ocular trauma, corneal surgery, and systemic symptoms of diabetes. Herpes infection, in particular, has been reported to account for 27% of cases of neurotrophic keratitis. Although neurotrophic keratitis has been known for a long time, no effective treatment has been developed.

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

[0004] Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide discovered from sheep hypothalamic extracts. PACAP has the activity of stimulating cAMP formation in anterior pituitary cells. PACAP consists of 38 amino acid residues, PACAP38, and 27 amino acid residues, respectively, both of which have equivalent effects (Non-Patent Documents 1 and 2). PACAP belongs to the vasoactive intestinal polypeptide (VIP) / secretin / glucagon superfamily, and the sequence of human PACAP27 shares 68% identity with vasoactive intestinal polypeptide (VIP). PACAP and VIP bind to the PAC1 receptor (PAC1R), VPAC1 receptor (VPAC1R), and VPAC2 receptor (VPAC2R), differing in their affinity for these receptors. PAC1R binds to PACAP with high selectivity, with an affinity for PACAP that is over 1000 times higher than its affinity for VIP. On the other hand, VPAC1R and VPAC2R both have similar affinity for PACAP and VIP. PACAP is known to have various physiological effects, including as a neuroprotective substance, an immunosuppressive factor, a vasodilator factor, an exocrine mitochondrial secretion-promoting factor (Patent Document 3), and a neurite outgrowth-promoting factor (Patent Document 4).

[0005] Pharmaceuticals are being developed by utilizing the diverse physiological activities of PACAP. However, relatively short peptides like PACAP are often unstable in aqueous solutions and have a short half-life in vivo due to their lack of protease resistance.

[0006]

Prior art literature

[0007] [Patent Literature]

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

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2012-232952

[0010] Patent Document 3: Japanese Patent Application Laid-Open No. 2009-269818

[0011] Patent Document 4: International Publication No. 2005 / 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] Non-patent document 3: Alessandro Lambiase (2012) Investigative Ophthalmology & Visual Science, vol. 53, No. 13, pp. 8280-8287

[0016] [Summary of the Invention]

[0017] [Problems to be solved by the invention]

[0018] The present invention aims to provide a composition for preventing or treating neurotrophic keratitis.

[0019]

Methods for solving the problem

[0020] The present inventors have discovered that PACAP peptide or a stabilized peptide thereof inhibits corneal damage in a neurotrophic keratitis model and promotes axonal extension in a cultured neural cell model, thereby completing the present invention. Furthermore, the present inventors discovered that the stability of PACAP peptide in aqueous solution can be significantly improved by replacing the carboxyl group of aspartic acid in PACAP peptide with a tetrazole as a stabilizing peptide.

[0021] Therefore, the present invention relates to the following technical solutions:

[0022] [1-1] A pharmaceutical composition for preventing or treating neurotrophic keratitis, comprising a peptide consisting of the sequence represented by the following sequence or a stabilized sequence thereof:

[0023] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0024] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0025] [1-2] A peptide for use in the prevention or treatment of neurotrophic keratitis, comprising a sequence represented by the following sequence or a stabilized sequence thereof:

[0026] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0027] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0028] [1-3] A method for preventing or treating neurotrophic keratitis, comprising administering a peptide consisting of the sequence represented by the following sequence, or a stabilized sequence thereof, to a subject in need of treatment or prevention of neurotrophic keratitis:

[0029] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0030] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0031] [1-4] Use of a peptide consisting of the sequence represented by the following sequence or a stabilized sequence thereof for the manufacture of a drug for the treatment of neurotrophic keratitis:

[0032] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0033] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0034] [2] The peptide composed of the above-mentioned stabilizing sequence is a sequence in which the carboxyl group of the aspartic acid residue at position 3 and / or position 8 in the sequence of SEQ ID NO: 1 or 2 is substituted with a tetrazole, or a sequence in which a portion of the sequence is changed, wherein when the above-mentioned stabilizing sequence is a sequence in which a portion of the sequence is changed, the peptide composed of the stabilizing sequence has binding properties to PAC1R, VPAC1R and VPACR2, the preventive or therapeutic composition described in item 1-1, the peptide described in item 1-2, the method described in item 1-3, or the use described in item 1-4.

[0035] [3] The peptide consisting of the above-mentioned stabilizing sequence is a peptide consisting of a sequence represented by the following formula wherein the carboxyl group of the aspartic acid residue at position 3 and / or position 8 is substituted with a tetrazole or a modified sequence thereof:

[0036] H-X1-DGIFTD-X2-Y-X3-RYR-X4-X5-X6-A-X7-X8-X9-YLAAVX 10 (SEQ ID NO: 3)

[0037] (wherein, X1 is a neutral amino acid, X2 is a neutral amino acid, X3 is a neutral amino acid, X4 is a basic amino acid, X5 is a neutral amino acid or egTz, X6 is a non-polar amino acid, X7 is a non-polar amino acid, X8 is a basic amino acid, X9 is a basic amino acid, X 10 is a neutral amino acid),

[0038] The above-mentioned peptide has binding properties to PAC1R, VPAC1R and VPACR2, and the above-mentioned altered sequence is a sequence in which one or more amino acids are deleted or added to the sequence of SEQ ID NO: 3. The preventive or therapeutic composition described in Item 1-1 or Item 2, the peptide described in Item 1-2 or Item 2, the method described in Item 1-3 or Item 2, or the use described in Item 1-4 or Item 2 containing the above-mentioned peptide.

[0039] [4] The preventive or therapeutic composition according to item 1-1, the preventive or therapeutic composition, peptide, method, or use according to item 3, wherein the neutral amino acid in X1, X2, and X3 is alanine or serine.

[0040] [5] The preventive or therapeutic composition, peptide, method, or use according to item 3 or 4, wherein the basic amino acid in X4, X8, and X9 is lysine or arginine.

[0041] [6] The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 5, wherein X5 is glutamine, alanine, or egTz.

[0042] [7] Furthermore, X6 is methionine, norleucine, alanine or leucine, and the preventive or therapeutic composition, peptide, method or use according to any one of items 3 to 6.

[0043] [8] The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 7, wherein X7 is valine or alanine.

[0044] [9]X 10 The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 8, wherein the amino acid is leucine or alanine.

[0045]

[10] The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 9, wherein the carboxyl groups of the aspartic acid at positions 3 and 8 of SEQ ID NO: 3 are substituted with tetrazole.

[0046]

[11] The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 10, wherein the N-terminus of the peptide is acetylated or mesylated.

[0047]

[12] The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 11, wherein the N-terminus of the above-mentioned peptide is acetylated.

[0048]

[13] The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 12, wherein one or two amino acids are deleted.

[0049]

[14] The preventive or therapeutic composition, peptide, method, or use according to any one of items 3 to 13, wherein one sequence selected from the following sequences is added to the C-terminus of the peptide:

[0050] GKRYKQRVKNK (SEQ ID NO: 37);

[0051] GKRYKQRVKN (SEQ ID NO: 38);

[0052] GKRYKQRVK (SEQ ID NO: 39);

[0053] GKRYKQRV (SEQ ID NO: 40);

[0054] GKRYKQR (SEQ ID NO: 41);

[0055] GKRYKQ (SEQ ID NO: 42);

[0056] GKRYK (SEQ ID NO: 43);

[0057] GKRY (SEQ ID NO: 44);

[0058] GKR;

[0059] GRR;

[0060] GK;

[0061] GR; and

[0062] G.

[0063] [15-1] A neuroaxon extension promoter comprising a peptide consisting of the sequence represented by the following sequence or a stabilized sequence thereof:

[0064] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0065] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0066] [15-2] A method for promoting neurite outgrowth, comprising administering a peptide consisting of a sequence represented by the following sequence, or a stabilized sequence thereof, to a subject in need of promotion of neurite outgrowth:

[0067] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0068] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0069] [15-3] Use of a peptide consisting of the sequence represented by the following sequence or a stabilized sequence thereof for the manufacture of a neuroaxon extension promoter:

[0070] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0071] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0072] [15-4] A peptide consisting of the following sequence or a stabilized sequence thereof for use in treating neurotrophic keratitis, nerve damage, or nerve regeneration by promoting axonal elongation:

[0073] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0074] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0075]

[16] The axon extension promoter according to Item 15-1, the method according to Item 15-2, the use according to Item 15-3, or the peptide according to Item 15-4, wherein the peptide causes the axon of the trigeminal nerve to extend.

[0076] [17-1] A composition for treating nerve damage or for nerve regeneration, comprising the neurite outgrowth promoter according to item 15-1 or 16.

[0077] [17-2] A method for treating nerve damage or regenerating nerves using the method described in item 15-2 or 16.

[0078] [17-3] The use described in item [15-3], wherein the above-mentioned axon extension promoter is used for treating nerve damage or nerve regeneration.

[0079]

[18] A method for promoting axonal extension in vitro or in vivo, comprising applying a peptide consisting of a sequence represented by the following sequence, or a stabilized sequence thereof:

[0080] HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 1), or

[0081] HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2).

[0082]

[19] The method for promoting axonal extension in vitro according to item

[18] , comprising culturing neural cells in a culture medium supplemented with the above-mentioned peptide.

[0083] Effects of the Invention

[0084] According to the present invention, PACAP or stabilized PACAP can be used for the prevention or treatment of neurotrophic keratitis, or for the promotion of neurite outgrowth.

[0085] [Brief description of the accompanying drawings]

[0086] Figure 1 The cAMP-inducing effects of each peptide (peptides 1, 2, 6 to 9) in a cell line highly expressing PAC1R were shown.

[0087] Figure 2 The cAMP-inducing effects of each peptide (peptides 1, 2, 6 to 9) in a cell line highly expressing VPAC1R were shown.

[0088] Figure 3 The tear secretion promoting effect of each peptide (peptide 1 and peptide 9) in neurotrophic keratitis model rats was shown.

[0089] Figure 4 Shown are changes in the epidermal punctate cornea (SPK) score in rats with neurotrophic keratitis models using each peptide (peptide 1 and peptide 9).

[0090] Figure 5 The peptides showed a promoting effect on axonal extension in cultured trigeminal nerves. [Specific implementation method]

[0091] The present invention relates to a pharmaceutical composition for the treatment or prevention of neurotrophic keratitis containing a PACAP peptide or a stabilized PACAP peptide. In another embodiment of the present invention, the present invention relates to a neurite extension promoter containing a PACAP peptide or a stabilized PACAP peptide. Furthermore, in another embodiment, the present invention relates to a method for promoting neurite extension in vitro or in vivo, including the application of a PACAP peptide or a stabilized PACAP peptide. A stabilized PACAP peptide refers to a peptide in which amino acids are changed in a manner that maintains the physiological activity of PACAP while improving stability. Specifically, the stabilized PACAP peptide relates to a peptide composed of a sequence in which the carboxyl group of the aspartic acid residue at position 3 and / or 8 in the amino acid sequence of the PACAP peptide is replaced with tetrazole or a sequence in which a part of the sequence is changed. The stabilized PACAP peptide according to the present invention has the same degree of binding to PAC1R, VPAC1R, and / or VPAC2R as PACAP. The same degree of binding means that the EC50 value of the peptide to each receptor is within 10-fold compared to PACAP, preferably within 5-fold, more preferably within 3-fold. PACAP can also be either PACAP38 composed of 38 residues or PACAP27 composed of 27 residues. PACAP38 and PACAP27 have the following sequences:

[0092]

Chemical 1

[0093] PACAP38: HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO:1)

[0094] PACAP27: HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO:2)

[0095] In the present invention, a sequence in which a part of the sequence is changed refers to a sequence in which 1 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 1 or several amino acids are substituted, deleted, or added to the original sequence.

[0096] Amino acid substitution can occur at any position as long as it does not change the binding of the peptide composed of the sequence in which a part of the sequence is changed to PAC1R, VPAC1R, and / or VPAC2R. In the peptide composed of the sequence in which a part of the sequence is changed, from the viewpoint of maintaining binding, amino acid substitution can occur at positions 2, 9, 11, 15-17, 19-21, and 27 of PACAP27. In a particularly preferred embodiment, the amino acids at the positions shown by X1-X 10 can be substituted:

[0097] H-X1-DGIFTD-X2-Y-X3-RYR-X4-X5-X6-A-X7-X8-X9-YLAAVX 10 (SEQ ID NO: 3).

[0098] Amino acid substitutions may be 1 or more amino acids. From the perspective 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 these, substitution of 1 to 4 amino acids is particularly preferred, substitution of 3 amino acids is more preferred, substitution of 2 amino acids is even more preferred, and substitution of 1 amino acid is particularly preferred.

[0099] Amino acid deletions may occur at any location as long as they do not alter the binding of a peptide composed of a partially altered sequence to PAC1R, VPAC1R, and / or VPAC2R. The number of amino acids deleted can be selected from any number between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Deletion of one or two amino acids is particularly preferred because it does not alter binding. Amino acid deletions can occur from the N-terminal or C-terminal side of the original sequence, or from within the sequence. Since PACAP27 and PACAP38 each have comparable binding to PAC1R, VPAC1R, and / or VPAC2R, deletion of amino acids located at the C-terminal end of PACAP38 is expected to have minimal effect on binding.

[0100] Amino acids may be added at any location as long as they do not alter the binding of a peptide composed of a partially altered sequence to PAC1R, VPAC1R, and / or VPAC2R. The number of added amino acids can be selected from any number between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Amino acids may be added to the N-terminus or C-terminus of the original sequence, or within the sequence. Since PACAP27 and PACAP38 each have comparable binding to PAC1R, VPAC1R, and / or VPAC2R, the addition of any amino acid to the C-terminus of PACAP27 is expected to have minimal effect on binding.

[0101] One embodiment of the present invention relates to a peptide consisting of a sequence of the following formula wherein the carboxyl groups of the aspartic acid residues at positions 3 and / or 8 are substituted with tetrazole, or a modified sequence thereof:

[0102] H-X1-DGIFTD-X2-Y-X3-RYR-X4-X5-X6-A-X7-X8-X9-YLAAVX 10 (SEQ ID NO: 3)

[0103] (wherein, X1 is a neutral amino acid, X2 is a neutral amino acid, X3 is a neutral amino acid, X4 is a basic amino acid, X5 is a neutral amino acid or egTz, X6 is a non-polar amino acid, X7 is a non-polar amino acid, X8 is a basic amino acid, X9 is a basic amino acid, X 10 are non-polar amino acids),

[0104] A peptide having the ability to bind to PAC1R, VPAC1R, and / or VPAC2R.

[0105] X1~X 10 The amino acids indicated may be conservatively substituted with amino acids having the same properties. As an example, conservative substitutions are substitutions of amino acids within the following groups:

[0106] 1. Non-polar amino acids: Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, Ala

[0107] 2. Neutral amino acids: Ala, Ser, Thr, Tyr, Cys, Asn, Gln, Gly

[0108] 3. Basic amino acids: Lys, Arg, His

[0109] 4. Acidic amino acids: Asp, Glu

[0110] The neutral amino acid in X1, X2, and X3 is Ala, Ser, Thr, Tyr, Cys, Asn, Gln, or Gly, more preferably Ala or Ser.

[0111] The basic amino acid in X4, X8, and X9 is Lys, Arg, or His, more preferably Lys or Arg.

[0112] The neutral amino acid in X5 is Ala, Ser, Thr, Tyr, Cys, Asn, Gln, or Gly, more preferably Ala or Gln. X5 may also be an amino acid in which the amide of glutamine is further substituted with a tetrazole (egTz).

[0113] The non-polar amino acid in X6 is Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, or Ala, more preferably Met, Nle, Leu, or Ala.

[0114] The non-polar amino acid in X7 is Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, or Ala, more preferably Val or Ala.

[0115] X 10 The non-polar amino acid is Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, or Ala, more preferably Leu or Ala.

[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, an altered sequence refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence.

[0117] In the sequence of SEQ ID NO: 3, the altered sequence in which the carboxyl group of the aspartic acid residue at position 3 and / or position 8 is substituted with a tetrazole preferably refers to 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 addition sequence may be added to the amino acid sequence represented by SEQ ID NO: 3.

[0118] 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-terminus of PACAP27 does not affect its binding to PAC1R. Therefore, when the present invention relates to PACAP27 or a stabilized peptide thereof, the C-terminal addition sequence may or may not be present. The C-terminal addition sequence refers to a sequence consisting of 1 to 11 arbitrary amino acids. The C-terminal addition sequence preferably corresponds to amino acids 28 to 38 of PACAP38. Thus, the following sequences can be cited as C-terminal sequences:

[0119] GKRYKQRVKNK (SEQ ID NO: 37);

[0120] GKRYKQRVKN (SEQ ID NO: 38);

[0121] GKRYKQRVK (SEQ ID NO: 39);

[0122] GKRYKQRV (SEQ ID NO: 40);

[0123] GKRYKQR (SEQ ID NO: 41);

[0124] GKRYKQ (SEQ ID NO: 42);

[0125] GKRYK (SEQ ID NO: 43);

[0126] GKRY (SEQ ID NO:44)

[0127] GKR;

[0128] GRR;

[0129] GK;

[0130] GR; and

[0131] G.

[0132] The peptides of the present invention may be composed of D- or L-amino acids, or racemic amino acids, as long as they retain their binding properties to PAC1R, VPAC1R, and / or VPAC2R. Similarly, the peptides of the present invention may be composed of unnatural amino acids such as 2-aminoisobutyric acid or L-2-aminobutyric acid, and may contain derivatives with arbitrarily modified functional groups, such as an N-terminal amino group, a C-terminal carboxyl group, or amino acid side chains. Examples of modifications include the addition of protecting groups to amino groups (e.g., acylation (formylation, acetylation, etc.), mesylation, ureidation, carbamidomethylation, Boc-ylation, Fmoc-ylation), and esterification (ethylation, etc.) of carboxyl groups. In addition to modifications that typically occur in vivo, such as phosphorylation, amidation, methylation, esterification, and acetylation, modifications that occur during synthesis or facilitate purification, such as biotinylation, may also be included. Furthermore, modifications such as PEGylation may be performed to extend the in vivo half-life of the peptide. In particular, from the perspective of improving stability, the free amino group of the amino acid at the N-terminus can be protected by a protecting group (e.g., an acyl group). For example, the free amino group of the amino acid at the N-terminus can be acetylated or mesylated. In a peptide composed of a sequence in which the carboxyl group of the aspartic acid residue at position 3 and / or position 8 of PACAP is substituted with a tetrazole, stability is further improved by acetylation or mesylation of the N-terminus. The C-terminus can be any of a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH2), or an ester (-COOR), and a sugar chain can also be attached (see, for example, WO2017 / 027848).

[0133] In a specific embodiment of the present invention, the present invention relates to peptides 3 to 34 having the sequences shown in Table 1 below, or altered sequences thereof:

[0134]

Table 1

[0135] Peptide name N-terminus sequence C-terminus Peptide 1 H- HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 2) <![CDATA[NH2]]> Peptide 2 Ac- HSDGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 4) <![CDATA[NH2]]> Peptide 3 H- HSTzGIFTDSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 5) <![CDATA[NH2 <!-- 7 -->]]> Peptide 4 H- HSDGITTzSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 6) <![CDATA[NH2]]> Peptide 5 H- HSTzGIFTTzSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 7) <![CDATA[NH2]]> Peptide 6 H- HSTzGIFTTzSY SRYRKQNIAVK KYLAAVL (SEQ ID NO: 8) <![CDATA[NH2]]> Peptide 7 H- HSTzGIFTTzSY SRYRKQLAVK KYLAAVL (SEQ ID NO: 9) <![CDATA[NH2]]> Peptide 8 Ac- HSTzGIFTTzSY SRYRKQNIAVK KYLAAVL (SEQ ID NO: 10) <![CDATA[NH2]]> Peptide 9 Ac- HSTzGIFTTzSY SRYRKQLAVK KYLAAVL (SEQ ID NO: 11) <![CDATA[NH2]]> Peptide 10 Ac- HSTzGIFTTzSY SRYRKQMAVK KYLAAVL (SEQ ID NO: 12) <![CDATA[NH2]]> Peptide 11 Ms- HSTzGIFTTzSY SRYRKQNIAVK KYLAAVL (SEQ ID NO: 13) <![CDATA[NH2]]> Peptide 12 Ms- HSTzGIFTTzSY SRYRKQLAVK KYLAAVL (SEQ ID NO: 14) <![CDATA[NH2]]> Peptide 13 Ac- HSTzGIFTTzSY SRYRKegTzLAVK KYLAAVL (SEQ ID NO: 15) <![CDATA[NH2]]> Peptide 14 Ac- HSTzGIFTTzSY SRYRKQLAVK KYLAAVL (SEQ ID NO: 16) <![CDATA[NH2]]> Peptide 15 Ac- HSTzGIFTTzAY SRYRKQLAVK KYLAAVL (SEQ ID NO: 17) <![CDATA[NH2]]> Peptide 16 Ac- HSTzGIFTTzSY ARYRKQLAVK KYLAAVL (SEQ ID NO: 18) <![CDATA[NH2]]> Peptide 17 Ac- HSTzGIFTTzSY SRYRKALAVK KYLAAVL (SEQ ID NO: 19) <![CDATA[NH2]]> Peptide 18 Ac- HSTzGIFTTzSY SRYRKQAAVK KYLAAVL (SEQ ID NO: 20) <![CDATA[NH2]]> Peptide 19 Ac- HSTzGIFTTzSY SRYRKQLAAK KYLAAVL (SEQ ID NO: 21) <![CDATA[NH2]]> Peptide 20 Ac- HSTzGIFTTzSY SRYRKQLAVK KYLAAVA (SEQ ID NO: 22) <![CDATA[NH2]]> Peptide 21 Ac- HSTzGIFTTzSY SRYRRQLAVR RYLAAVL (SEQ ID NO: 23) <![CDATA[NH2]]> Peptide 22 Ac- HSTzGIFTTzSY SRYRRQLAVR RYLAAVLGRR (SEQ ID NO: 24) <![CDATA[NH2]]> Peptide 23 Ac- HSTzAIFTTzSY SRYRKQLAVK KYLAAVL (SEQ ID NO: 25) <![CDATA[NH2]]> Peptide 24 Ac- HSTzGIFTTzAY SRYRKQLAVK KYLAAVL (SEQ ID NO: 26) <![CDATA[NH2]]> Peptide 25 Ac- HSTzGIFTTzSY SRYRKQAAVK KYLAAVL (SEQ ID NO: 27) <![CDATA[NH2]]> Peptide 26 Ac- HSTzGIFTTzAY SRYRKALAVK KYLAAVL (SEQ ID NO: 28) <![CDATA[NH2]]> Peptide 27 Ac- HSTzGIFTTzAY SRYRKQAAVK KYLAAVL (SEQ ID NO: 29) <![CDATA[NH2]]> Peptide 28 Ac- HSTzGIFTTzSY SRYRKAAAVK KYLAAVL (SEQ ID NO: 30) <![CDATA[NH2]]> Peptide 29 Ms- HSTzGIFTTzAY SRYRKQLAVK KYLAAVL (SEQ ID NO: 31) <![CDATA[NH2]]> Peptide 30 Ms- HSTzAIFTTzSY SRYRKQLAVK KYLAAVL (SEQ ID NO: 32) <![CDATA[NH2]]> Peptide 31 Ms- HSTzAIFTTzSY SRYRKQAAVK KYLAAVL (SEQ ID NO: 33) <![CDATA[NH2]]> Peptide 32 Ac- HSTzAIFTTzSY SRYRKQAAVK KYLAAVL (SEQ ID NO: 34) <![CDATA[NH2]]> Peptide 33 Ac- HATzAIFTTzSY SRYRKQAAVK KYLAAVL (SEQ ID NO: 35) <![CDATA[NH2]]> Peptide 34 Ac- HSTzGIFTTzSY SRYRKAAAVK KYLAAVL (SEQ ID NO: 36) <![CDATA[NH2]]>

[0136] The amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole has the following structure:

[0137]

Chemistry 2

[0138]

[0139] In addition, in this specification, "Tz" is used as an expression in a sequence.

[0140] Furthermore, the amino acid in which the amide of glutamine is replaced by a tetrazole has the following structure:

[0141]

Chemistry 3

[0142]

[0143] In addition, in this specification, "egTz" is used as an expression in a sequence.

[0144] 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. Alternatively, they can be produced by introducing a nucleic acid encoding the peptide of the present invention into a host cell by gene transfer, and synthesizing the peptide in the host cell. In this case, by designing a peptide with a tag such as a polyhistidine tag attached to the end of the peptide, purification after expression can be facilitated.

[0145] The peptides of the present invention include 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, and malates, etc.), or salts with bases (e.g., ammonium salts, methylpyridinium salts, acetylpyridinium salts, etc.). The peptides of the present invention also include hydrates or solvates.

[0146] Histidine with mesylated N-terminus can be produced by the method shown in Reaction Scheme 1 or a method based thereon.

[0147]

Chemistry 4

[0148]

[0149] In the formula, Trt represents a trityl group, and Me represents a methyl group.

[0150] In the method of Reaction Scheme 1, a compound represented by the general formula (I) is reacted with methanesulfonyl chloride in the presence of a base to produce Compound (II). Subsequently, Compound (II) can be hydrolyzed with a base in methanol to produce Compound (III).

[0151] In the production of compound (II), the base is used in an amount of 0.2 to 5 equivalents, preferably 1 to 3 equivalents, relative to compound (I). Examples of the base used include triethylamine, N,N-diisopropylethylamine, pyridine, and 4-dimethylaminopyridine, preferably triethylamine. Methanesulfonyl chloride is used in an amount of 0.1 to 5 equivalents, preferably 1 to 2 equivalents. The solvent is not particularly limited as long as it does not affect the reaction. For example, tetrahydrofuran, dichloromethane, toluene, etc. can be mentioned, and dichloromethane is preferably mentioned. The reaction temperature is generally 1°C to 30°C, preferably 15°C to 25°C, and the reaction time is generally 0.5 to 12 hours, preferably 0.5 to 2 hours.

[0152] In the production of compound (III), the base is used in an amount of 0.1 to 10 equivalents, preferably 1 to 3 equivalents, relative to compound (II). Examples of the base include lithium hydroxide, sodium hydroxide, and potassium hydroxide, preferably potassium hydroxide. Examples of the solvent include a mixed solvent of an organic solvent (for example, methanol, ethanol, isopropanol, acetonitrile, 1,4-dioxane, and tetrahydrofuran) and water, preferably a mixed solvent with methanol and water. The reaction time varies depending on the reagent or solvent used, and is generally 0.5 to 12 hours, preferably 0.5 to 3 hours. The reaction temperature varies depending on the reagent or solvent used, and is generally 0°C to 100°C, preferably 60°C to 100°C.

[0153] The peptides according to the present invention can exert the same physiological activity as PACAP by binding to PAC1R, VPAC1R, and / or VPAC2R. Without intending to be bound by theory, the prevention or treatment of neurotrophic keratitis may be achieved through binding to these receptors. More specifically, the peptides according to the present invention can inhibit the reduction in tear volume caused by neurotrophic keratitis and inhibit corneal punctate epidermatitis. Furthermore, the peptides according to the present invention can promote axonal extension. The axonal extension-promoting effect of the peptides according to the present invention can be used to treat trigeminal nerve damage or regenerate the trigeminal nerve. The peptides according to the present invention can treat or prevent neurotrophic keratitis through these effects. In addition, the peptides according to the present invention can promote axonal extension in vivo or in vitro. To promote axonal extension in vitro, neural cells are cultured in a culture medium containing the peptides according to the present invention. The neural cell culture method can be carried out by any method known in the art, and widely known neural cell culture media can be used.

[0154] The present invention relates to a pharmaceutical composition for treating or preventing neurotrophic keratitis containing a therapeutically effective amount of the above-mentioned peptide. The pharmaceutical composition of the present invention can be administered to a patient to treat neurotrophic keratitis through the physiological effects of PACAP, or can be administered to a patient who is likely to suffer from such a disease to prevent neurotrophic keratitis. In addition, "treatment" refers to preventing the deterioration of the condition of a disorder or disease, retarding the progression, maintaining the current condition, alleviating or eliminating the disorder or disease, and "prevention" refers to preventing the onset of a disorder or disease before the onset of the disorder or disease. Examples of patients who are likely to suffer from neurotrophic keratitis include patients suffering from corneal viral infections such as herpes, eye injuries, corneal surgery, and diabetes.

[0155] The peptide according to the present invention, or the pharmaceutical composition containing the peptide, can be administered orally or non-orally, corresponding to the disease to be treated. As oral administration, sublingual, oral, and oral administration can be mentioned. As parenteral administration, it can be administered, for example, by intravenous, intraarterial, subcutaneous, topical, intraperitoneal, intramuscular, nasal, transdermal, transmucosal, intrathecal, rectal, intramuscular, intracerebral, intrathecal, subarachnoid, intradural, epidural, eye drops, ear drops, nose drops, and intraocular routes. As intraocular routes, more specifically, subconjunctival, subocular, and intravitreal routes can be mentioned. The pharmaceutical composition containing the peptide according to the present invention can be in any suitable dosage form, such as eye drops, injections, powders, infusion preparations, granules, tablets, suppositories, etc., corresponding to its administration route. From the perspective of parenteral administration, eye drops, injections, infusion preparations, and powders for preparation at the time of use are preferred. Examples of preparations for intraocular administration include intravitreal injections, subconjunctival injections, and subtenon injections. These preparations may also contain various pharmaceutically acceptable adjuvants, i.e., carriers or other adjuvants, such as stabilizers, preservatives, analgesics, emulsifiers, and the like. Furthermore, they may be used in combination with other agents having neuroprotective, anti-inflammatory, or exocrine effects.

[0156] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0157] 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 defined solely by the claims. Modifications to the present invention, such as additions, deletions, and substitutions of constituent elements, may be made without departing from the spirit of the present invention.

[0158] [Example]

[0159] [Example 1: Peptide Synthesis 1]

[0160] Synthesis of Ms-His(Trt)-OMe

[0161]

Chemistry 5

[0162]

[0163] To a dichloromethane solution (40 mL) of N-im-trityl-L-histidine methyl ester hydrochloride (2.02 g, 4.5 mmol) was added methanesulfonyl chloride (314.7 mg, 2.7 mmol) dissolved in N,N-diisopropylethylamine (1.0 mL, 5.7 mmol) and dichloromethane (2.0 mL). The mixture was reacted at 25°C for 2 hours (40 min). Furthermore, methanesulfonyl chloride (174.7 mg, 1.5 mmol) dissolved in dichloromethane (1.0 mL) was added and the mixture was reacted at 25°C for 1 hour. The reaction was then terminated by the addition of saturated aqueous sodium bicarbonate solution, followed by extraction with ethyl acetate. The resulting organic layer was washed with 10% aqueous citric acid solution, saturated aqueous sodium bicarbonate solution, and saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Since the reaction was not complete, dichloromethane (15 mL) was added to the resulting residue, followed by triethylamine (1.0 mL, 7.2 mmol) and a dichloromethane solution (1.0 mL) of methanesulfonyl chloride (326.8 mg, 2.9 mmol), and the mixture was stirred at room temperature for 1 hour. The reaction was then quenched by the addition of saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The resulting organic layer was washed with 10% aqueous citric acid solution, saturated aqueous sodium bicarbonate solution, and saturated saline, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a pale yellow solid (2.35 g, >100%). The resulting solid was used in the following reaction without further purification.

[0164] 1 H-NMR (400MHz, CDCl3) δ7.38(1H,d,J=1.2Hz),7.34-7.31(9H,m),7.12-7.08(6H,m),6.57(1H,d,J=2.4H z), 6.30 (1H, d, J = 8.4Hz), 4.40 (1H, dt, J = 8.4, 5.2Hz), 3.65 (3H, s), 3.05 (2H, d, J = 5.2Hz), 2.97 (3H, s).

[0165] Synthesis of Ms-His(Trt)-OH

[0166]

Chemistry 6

[0167]

[0168] To a methanol solution (15 mL) of Ms-His-OMe (2.21 g, 4.5 mmol) was added a 1 M aqueous potassium hydroxide solution (9.0 mL, 9.0 mmol) and refluxed for 1.5 hours. The reaction solution was cooled to room temperature, acidified to pH 5 with a 10% aqueous citric acid solution, and extracted with dichloromethane. The resulting organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. Dichloromethane and hexane were added to the residue to precipitate a white solid, which was collected by filtration (2.32 g, >100%).

[0169] 1 H-NMR(400MHz, CDCl3)δ7.84(1H,s),7.31-7.33(9H,m),7.12-7.09(6H,m),6.82(1H,s) ,4.13(1H,m),3.34(1H,dd,J=14.8,3.6Hz),3.17(1H,dd,J=14.8,6.8Hz),2.90(3H,s).

[0170] [Example 2: Peptide Synthesis 2]

[0171] 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). The unnatural amino acids Fmoc-TZ-OH, Fmoc-TZ(trt)-OH, and Fmoc-egTZ(trt)-OH used in the solid-phase synthesis were purchased from Astatech. Peptides 1 to 34 having the following sequences were synthesized, and the molecular weights of the synthesized peptides were analyzed by mass spectrometry (MALDI TOF). As shown in Table 2 below, all measured values ​​were in good agreement with the theoretical values.

[0172]

Table 2

[0173]

[0174]

[0175]

[0176] [Example 3: Peptide Stability Test 1]

[0177] [Preparation of measurement samples]

[0178] Peptides 1 and 3 to 5 synthesized in Example 2 were weighed and dissolved in phosphate buffer (pH 7.0) to prepare a 1.0 mM peptide solution. Furthermore, the 1.0 mM peptide solution was 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 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 was analyzed.

[0179] [Water permeability]

[0180] The total weight of the aqueous peptide solution and the storage container was used as the subject weight. The subject weight before storage was weighed. After each storage 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.

[0181]

Number 1

[0182]

[0183] 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). The peptide solution was analyzed by reversed-phase HPLC (HPLC system: Prominence manufactured by Shimadzu Corporation) under the conditions shown in Table 3 below, and a chromatogram was obtained.

[0184] [HPLC analysis conditions 1]

[0185] Column: Waters XSelect CSH C18, 5 μm, 4.6 × 250 mm

[0186] Guard column: Waters XSelect CSH C18, 5 μm, 4.6 × 20 mm Guard Cartridge

[0187] Detection wavelength: 220nm

[0188] Mobile phase A: 0.1% formic acid aqueous solution

[0189] Mobile phase B: 0.1% formic acid in acetonitrile

[0190] Measurement time: 30 minutes

[0191] Measurement sample injection volume: 50 μL

[0192] Flow rate: 1.0 mL / min

[0193] Sample cooler: 4°C

[0194] Column temperature: 40°C

[0195] Mobile phase delivery: 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.

[0196]

Table 3

[0197] 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

[0198] In the chromatogram, the peak area of ​​the peptide was determined, and the residual rate (pre-moisture correction residual rate) was calculated using formula (2). Furthermore, the post-moisture correction residual rate was calculated using formula (3) by taking into account the water permeability of the container for the pre-moisture correction residual rate.

[0199]

Number 2

[0200]

[0201]

Number 3

[0202]

[0203] Table 4 shows the moisture-corrected residual rates of peptides in the samples after storage.

[0204]

Table 4

[0205] PeptideNo. 40℃, 1 month storage 40℃, 2 months storage Peptide 1 37.4％ 21.3％ Peptide 3 76.5％ 60.7％ Peptide 4 42.8％ 28.2％ Peptide 5 94.7％ 85.6％

[0206] The stability of peptide 1 (PACAP) was 37.4% after storage at 40°C for one month, and dropped to 21.3% after storage at 40°C for two months. Peptide 3, in which only the carboxyl group of the aspartic acid side chain at the third residue of peptide 1 was substituted with a tetrazole, showed higher stability than peptide 1. Furthermore, peptide 4, in which only the carboxyl group of the aspartic acid side chain at the eighth residue was substituted with a tetrazole, showed improved stability compared to peptide 1. However, peptide 5, in which only the carboxyl groups of the aspartic acid side chains at the third and eighth residues of peptide 1 were substituted with tetrazole, showed further improved stability compared to peptides 3 and 4. This suggests that two tetrazole substitutions are more effective than a single tetrazole substitution in improving PACAP stability.

[0207] [Example 4: Peptide Stability Test 2]

[0208] Peptides 1, 2, and 6 to 9 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 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 sample analysis.

[0209] The stored samples were measured under the following HPLC analysis conditions 2, and the moisture-corrected residual rate of the peptide in the stored samples was calculated in the same manner as in Example 3. The results are shown in Table 6.

[0210] [HPLC analysis conditions 2]

[0211] Column: Waters XSelect CSH C18, 5 μm, 4.6 × 250 mm

[0212] Guard column: Waters XSelect CSH C18, 5 μm, 4.6 × 20 mm Guard Cartridge

[0213] Detection wavelength: 220nm

[0214] Mobile phase A: 0.1% formic acid aqueous solution

[0215] Mobile phase B: 0.1% formic acid in acetonitrile

[0216] Measurement time: 20 minutes

[0217] Measurement sample injection volume: 50 μL

[0218] Flow rate: 1.0 mL / min

[0219] Column temperature: 40°C

[0220] Sample cooler: 25°C

[0221] Mobile phase delivery: 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.

[0222]

Table 5

[0223] 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

[0224]

Table 6

[0225] PeptideNo. 60℃, store for 1 week 60℃, store for 2 weeks Peptide 1 26.6％ 15.8％ Peptide 2 28.7％ 17.2％ Peptide 6 91.3％ 82.4％ Peptide 7 91.7％ 84.8％ Peptide 8 98.1％ 92.9％ Peptide 9 98.4％ 92.8％

[0226] Peptide 1 (PACAP) showed 26.6% preservation after one week at 60°C, but after two weeks, its stability deteriorated to 15.8%. Furthermore, peptide 2, in which the N-terminus of PACAP was acetylated, had only comparable stability. Meanwhile, peptides 6 and 7, in which the carboxyl groups of aspartic acid at positions 3 and 8 were substituted with tetrazole (in peptide 6, methionine at position 17 was further substituted with norleucine (denoted as "N1" in the sequence), and in peptide 7, methionine at position 17 was further substituted with leucine), showed 91.3% and 91.7% preservation after one week at 60°C, and their stability significantly improved to 82.4% and 84.8% after two weeks. Furthermore, peptides 8 and 9, in which the N-terminus of peptides 6 and 7 were acetylated, showed 98.1% and 98.4% preservation after one week at 60°C, and their stability further improved to 92.9% and 92.8% after two weeks. The accelerated tests of 1 week and 2 weeks at 60°C are equivalent to about 1 year and about 2 years at room temperature (25°C). Therefore, peptides 8 and 9 are expected to be stable at room temperature for 2 years (with a residual rate of 90% or more).

[0227] [Example 5: Peptide Stability Test 3]

[0228] Peptides 12 to 25, 27, and 28 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 60°C for 1 or 2 weeks to obtain a stored sample. In addition, a sample that was not stored 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 sample analysis.

[0229] The moisture-corrected residual rate of peptides in the samples after storage was calculated in the same manner as in Example 4. The results are shown in Table 7.

[0230]

Table 7

[0231]

[0232]

[0233] Peptide 12 (a peptide in which the N-terminal acetyl group of peptide 9 was substituted with a methylsulfonyl group) exhibited stability comparable to that of peptide 9, demonstrating high stability even with the N-terminal methylsulfonyl substitution. Peptide 13 (a peptide in which the glutamine side chain at residue 16 of peptide 9 was substituted with a tetrazole) exhibited stability comparable to that of peptide 9, demonstrating that peptides substituted with tetrazole for the glutamine side chain also maintained high stability. Peptides 14-25, 27, and 28, in which any residue of peptide 9 was substituted or added with alanine or arginine, also maintained high stability.

[0234] [Example 5-2: Peptide Stability Test 3]

[0235] Peptides 29 to 34 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 60°C for 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 sample analysis.

[0236] The moisture-corrected residual rate of the peptide in the sample after storage was calculated in the same manner as in Example 4. The results are shown in Table 7-2.

[0237]

Table 7-2

[0238] PeptideNo. 60℃, store for 2 weeks Peptide 29 91.9％ Peptide 30 92.2％ Peptide 31 90.1％ Peptide 32 88.7％ Peptide 33 85.3％ Peptide 34 96.9％

[0239] Peptide 29 (a peptide in which the N-terminal acetyl group of peptide 15 was substituted with a methylsulfonyl group) exhibited stability comparable to that of peptide 15, and even with the N-terminal methylsulfonyl group substituted, it exhibited high stability. Peptide 30 (a peptide in which the N-terminal acetyl group of peptide 23 was substituted with a methylsulfonyl group) exhibited stability comparable to that of peptide 23, and even with the N-terminal methylsulfonyl group substituted, it exhibited high stability. Peptides 32 to 34, in which any residue of peptide 9 was substituted with alanine, maintained high stability. Peptide 31 (a peptide in which the N-terminal acetyl group of peptide 32 was substituted with a methylsulfonyl group) exhibited stability comparable to that of peptide 32.

[0240] [Example 6: Peptide Stability Test 4]

[0241] The peptides 10 and 11 synthesized in Example 2 were weighed and dissolved in a phosphate buffer solution (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 a phosphate buffer solution (pH 7.0) and dispensed into a tube (Protein LobindTube manufactured by Eppendorf). The prepared peptides were 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.

[0242] The moisture-corrected residual rate of the peptide in the sample after storage was measured in the same manner as in Example 4. The results are shown in Table 8.

[0243]

Table 8

[0244] PeptideNo. 40℃, 1 month storage 40℃, 2 months storage Peptide 10 94.0％ 87.8％ Peptide 11 94.2％ 88.8％

[0245] Compared with the results of peptide 10 having an acetyl group at the N-terminus and peptide 11 having an N-methylsulfonyl group, it was shown that the peptide was stabilized to the same extent regardless of whether the N-terminal substituent was an acetyl group or an N-methylsulfonyl group.

[0246] [Example 7: Peptide Stability Test 5]

[0247] The peptide 26 synthesized in Example 2 was 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 a tube (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 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 sample analysis.

[0248] The stored samples were measured under the following HPLC analysis conditions 3, and the moisture-corrected residual rate of the peptide in the stored samples was calculated in the same manner as in Example 3. The results are shown in Table 10.

[0249] [HPLC analysis conditions 3]

[0250] Column: Waters XSelect CSH C18, 5 μm, 4.6 × 250 mm

[0251] Guard column: Waters XSelect CSH C18, 5 μm, 4.6 × 20 mm Guard Cartridge

[0252] Detection wavelength: 220nm

[0253] Mobile phase A: 0.1% formic acid aqueous solution

[0254] Mobile phase B: 0.1% formic acid in acetonitrile

[0255] Measurement time: 20 minutes

[0256] Measurement sample injection volume: 50 μL

[0257] Flow rate: 1.0 mL / min

[0258] Column temperature: 40°C

[0259] Sample cooler: 25°C

[0260] Mobile phase delivery: 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.

[0261]

Table 9

[0262] 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

[0263]

Table 10

[0264] PeptideNo. 60℃, store for 1 week 60℃, store for 2 weeks Peptide 26 98.0％ 94.8％

[0265] Peptide 26 also showed high stability.

[0266] [Example 7: cAMP Assay of PACAP27 and Its Stabilized Peptides]

[0267] [Cell culture]

[0268] 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 Cells were prepared using a cell plating reagent (DiscoveRx) 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.

[0269] [Reagent preparation]

[0270] Powders of peptides 1, 2, and 6-9 synthesized in Example 2 were dissolved in water to a concentration of 0.1 mM and then diluted to 20 μM using Cell assay buffer (DiscoveRx) (containing 0.5 mM IBMX and 0.001% BSA). A series of 5-fold dilutions was then prepared using the same Cell assay buffer and used in the assay.

[0271] [cAMP measurement]

[0272] 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 solutions of peptides 1, 2, 6 to 9 at 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 at 37°C in a 5% CO2 atmosphere. Next, the Working detection solution was added, the culture plate was shielded from light with 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 EC values ​​for cAMP induction of each peptide in cell lines overexpressing PAC1R or VPAC1R 50 The results of the values ​​are shown in Figure 1 、 Figure 2 and Table 11.

[0273]

Table 11

[0274] PeptideNo. <![CDATA[EC 50 (PAC1R)]]> <![CDATA[EC 50 (VPAC1R)]]> Peptide 1 0.052nM 0.097nM Peptide 2 0.032nM 0.062nM Peptide 6 0.079nM 0.093nM Peptide 7 0.112nM 0.114nM Peptide 8 0.072nM 0.046nM Peptide 9 0.063nM 0.043nM

[0275] The synthesized peptides 1, 2, and 6 to 9 showed cAMP induction ability in cells with high expression of PAC1R and VPAC1R, respectively. 50 The value was comparable to that of the natural peptide (peptide 1: PACAP27).

[0276] Taking the results in Tables 6 and 11 into consideration, the peptides according to the present invention (peptides 6-9) exhibit significantly improved stability in aqueous solutions compared to PACAP while maintaining comparable physiological activity. In particular, their shelf life exceeding two years at room temperature enables development of liquid formulations such as bottles, ampoules, and eye drops.

[0277] [Example 8: cAMP Assay 2 of PACAP27 Stabilized Peptide]

[0278] [Reagent preparation]

[0279] Powders of peptides 3 to 5 and 10 to 28 synthesized in Example 2 were dissolved in water to 0.1 mM each and then diluted to 20 μM using Cell assay buffer (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.

[0280] The EC values ​​of peptides 3 to 5 and 10 to 28 were calculated by the same method as in Example 7. 50 EC values ​​for cAMP induction of each peptide in cell lines overexpressing PAC1 or VPAC1 50 The results of the values ​​are shown in Table 12.

[0281]

Table 12

[0282] PeptideNo. <![CDATA[EC 50 (PAC1R)]]> <![CDATA[EC 50 (VPAC1R)]]> Peptide 3 0.060nM 0.101nM Peptide 4 0.040nM 0.053nM Peptide 5 0.033nM 0.064nM Peptide 10 0.076nM 0.101nM Peptide 11 0.038nM 0.190nM Peptide 12 0.023nM 0.087nM Peptide 13 0.132nM 0.072nM Peptide 14 0.074nM 0.099nM Peptide 15 0.061nM 0.149nM Peptide 16 0.330nM 0.065nM Peptide 17 0.099nM 0.132nM Peptide 18 0.061nM 0.033nM Peptide 19 0.094nM 0.044nM Peptide 20 0.080nM 0.040nM Peptide 21 0.102nM 0.268nM Peptide 22 0.130nM 0.457nM Peptide 23 0.079nM 0.072nM Peptide 24 0.083nM 0.088nM Peptide 25 0.088nM 0.028nM Peptide 26 0.066nM 0.075nM Peptide 27 0.060nM 0.044nM Peptide 28 0.084nM 0.029nM

[0283] The peptides according to the present invention (peptides 3 to 5, 10 to 28) have significantly improved stability in aqueous solutions compared to PACAP, while maintaining the same physiological activity as PACAP.

[0284] [Example 8-2: cAMP Assay 2 of PACAP27 Stabilized Peptide]

[0285] [Reagent preparation]

[0286] Powders of peptides 29 to 34 synthesized in Example 2 were dissolved in water to 0.1 mM each and then diluted to 20 μM using Cell assay buffer (DiscoveRx) (containing 0.5 mM IBMX and 0.001% BSA). A 5-fold dilution series was prepared using the same Cell assay buffer and used in the assay.

[0287] The EC values ​​of peptides 29 to 34 were calculated in the same manner as in Example 7. 50EC values ​​for cAMP induction of each peptide in cell lines overexpressing PAC1 or VPAC1 50 The results of the values ​​are shown in Table 12-2.

[0288]

Table 12-2

[0289]

[0290]

[0291] The peptides according to the present invention (peptides 29 to 34) have significantly improved stability in aqueous solutions compared to PACAP, while maintaining the same physiological activity as PACAP.

[0292] Example 9: Evaluation of the neurotrophic keratitis model using PACAP27 and its stabilized peptides

[0293] Capsaicin was administered subcutaneously to the back of rats to create a rat model that induces corneal neurological impairment and decreased corneal sensation, accompanied by a decrease in tear volume and epidermal punctate cornea (SPK) (Non-Patent Document 3: Investigative Ophthalmology & Visual Science (2012), vol. 53, No. 13, pp. 8280-8287). PACAP27 and its stabilized peptide were then administered to the eyes of these rats to investigate their inhibitory effects on corneal damage. The specific experimental method was as follows.

[0294] [Capsaicin administration]

[0295] Capsaicin (manufactured by SIGMA) was dissolved in PBS containing 10% EtOH and 10% Tween 80, and the solution was subcutaneously administered to the back of 4-day-old male Wistar / ST rats at a dose of 50 mg / kg·bw.

[0296] [Preparation of eye drops]

[0297] Base: Dissolve 0.3 g of trishydroxymethylaminomethane (manufactured by Nacalai Tesque) and 4.4 g of D(-)-mannitol (manufactured by Nacalai Tesque) in 100 mL of sterile water, and add 1N HCl to adjust the pH to 7. Peptide 1 and Peptide 9 are dissolved in the base to a 0.1% concentration. Each eye drop should be stored at room temperature. Apply 10 μL of each solution three times a day to both eyes starting three weeks after capsaicin administration.

[0298] [Measurement of tear volume]

[0299] Measurement of tear volume ZONe QUICK (Bu Pharmaceutical) was inserted into the lower outer corner of the rat's eye. After 20 seconds, the length of the red-stained portion was measured using the attached scale. Tear volume was measured 10 minutes before and 5 minutes before the peptide was applied to the eye, and the average value was used as the tear volume before the application. After the peptide was applied to the eye, the tear volume was measured 5 minutes later, and the difference from the tear volume before the application was calculated. The results are shown in Figure 3 .

[0300] In rats with induced neurotrophic keratitis, eye application of peptide 1 and peptide 9 showed comparable tear secretion effects.

[0301] [SPK's Observation]

[0302] SPK was evaluated 2 weeks after the start of eye drops. Under isoflurane inhalation anesthesia, 1 μL of 1% sodium fluorescein (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution dissolved in PBS was dropped onto the ocular surface. After forcibly closing the eyes for 1 minute, the excess fluorescein staining solution was rinsed with physiological saline. Thereafter, the ocular surface was observed with a slit lamp microscope, and the degree of corneal disorder was evaluated based on the method of Murakami et al. in Ophthalmology 21(1): 87-90 (2004). Specifically, the cornea was divided into three areas: upper, central, and lower. Each area was scored according to the following criteria, and the total value of each score of the three areas was used as the SPK score. <Criteria> 0: No punctate staining, 1: Sparse (punctate fluorescein staining is separated), 2: Intermediate (intermediate between 1 and 3), 3: Dense (punctate fluorescein staining is almost adjacent). The results are shown in Figure 4 .

[0303] Eye application of peptide 1 and peptide 9 improved the increase in SPK score caused by capsaicin administration.

[0304] [Measurement of corneal sensation]

[0305] Corneal perception was measured using a Cochet-Bonnet corneal sensory meter (Handaya Shoten). The filament length of the corneal sensory meter was set to 60 mm, and the filament was brought into vertical contact with the center of the cornea to stimulate. The eyelid reaction was observed, and the filament length was recorded when a reaction was observed. When no reaction was observed, the filament length was shortened by 5 mm and measured again. The above operation was repeated 3 times, and the average value of the obtained filament lengths was used as the individual corneal perception threshold (CST).

[0306] Eye application of peptide 1 and peptide 9 improved the reduction in corneal sensation caused by capsaicin administration.

[0307] [Corneal nerve staining]

[0308] After euthanizing the rat, the eyeball was removed, immersed in Zamboni solution (manufactured by Wako Pure Chemical Industries, Ltd.), and allowed to stand at room temperature for 15 minutes. The cornea was collected from the eyeball, immersed in Zamboni solution again, and allowed to stand at room temperature for 45 minutes. Thereafter, it was replaced with a 0.1M phosphate buffer containing 30% sucrose. After confirming the sedimentation of the corneal tissue, a clamp was inserted into the incision, and the eyeball was immersed in a 0.1M phosphate buffer (pH 5.3) containing 0.1% EDTA and 0.01% type IV-S hyaluronidase (manufactured by SIGMA), and allowed to stand at 37°C overnight. After washing with PBS-T containing 0.3% TritonX-100, the eyeball was immersed in PBS-T containing 1% BSA and blocked at room temperature for 2 hours. Alexa Fluor 488 was combined with mouse anti-β-tubulin and Class III antibody (manufactured by BD) to react at room temperature overnight. After washing with PBS-T, the corneal tissue was attached to a glass slide and mounted with Vectashield hard set mounting medium. The mounted sample was observed using a confocal microscope to obtain images of the subepithelial nerve plexus. The nerve density of the whorl structure of the nerve layer was quantified using Sholl analysis using Image J.

[0309] Eye application of peptide 1 and peptide 9 improved the decrease in nerve density induced by capsaicin administration.

[0310] [Example 10: Evaluation of the Axonal Extension Effect of PACAP27 and Its Stabilized Peptides on Rat Trigeminal Neurons]

[0311] [Isolation and Culture of Trigeminal Nerve]

[0312] Rats purchased from SLC Co., Ltd. of Japan (Slc: Sprague-Dawley 17 days old, both males and females) were euthanized by carbon dioxide inhalation, and the trigeminal ganglia were collected. The collected trigeminal ganglia were washed with Hanks' balanced salt solution (HBSS) and treated with collagenase A (Roche) at 37°C for 30 minutes. Thereafter, the cells were dispersed using a nerve cell dispersion solution (Wako) according to the procedure. Furthermore, Debris and myelin were removed using a Debris removal solution (Miltenyi biotec) and Myelin removal beads II (Miltenyi biotec) according to the procedure. The isolated trigeminal neurons were suspended in culture medium and seeded on 8-well chamber slides (Corning) coated with polylysine / laminin to a density of 2.5×10 3cells / well. The culture medium used was Neurobasal-A containing B27 adjuvant (final concentration 2%), GlutaMAX (final concentration 2mM), and penicillin / streptomycin (final concentration 1%). The culture conditions were set to a carbon dioxide concentration of 5%, an air concentration of 95%, a humidity of 100%, and a temperature of 37°C. After sowing, the cells were cultured for 24 hours and then replaced with a culture medium containing peptide 1, peptide 9, peptide 12, peptide 18, and peptide 31 to a final concentration of 1μM. The control was replaced with a culture medium without peptide. After the culture medium was replaced, the cells were cultured for another 24 hours.

[0313] [2. Dyeing]

[0314] The culture medium containing the peptide was replaced, and rat trigeminal neurons, cultured for 24 hours, were fixed by immersing them in 2% paraformaldehyde at room temperature for 20 minutes. The cells were washed with PBS and 2% bovine serum albumin containing 0.1% Triton X-100 was added and allowed to react for 30 minutes. After washing with PBS, the cells were reacted with a phospho-neurofilament H antibody (Merk, MAB1592-C), which specifically recognizes the neurofilaments that constitute the cell bodies and neurites, at room temperature for one hour. After the one-hour reaction, the specimen was fluorescently stained with a fluorescently labeled secondary antibody (Invitrogen, #A-11031) at room temperature for one hour, and the stained cells were observed under a fluorescence microscope. Images of the stained cells were captured as images from the fluorescence microscope and uploaded to a computer.

[0315] [3. Image Analysis]

[0316] In order to evaluate the degree of neurite formation in rat trigeminal neurons, cells with neurites that were at least twice the cell body diameter in the images of stained cells collected on a computer were defined as neurite-forming cells, and the ratio (%) of the number of such cells to the total number of cells was calculated (Otori Y, Wei JY, Barnstable CJ. Invest. Ophthalmol Vis Sci (1998) 39, 972-981). The results are shown in Figure 5 (N=8) Significant differences relative to the control were determined by Dunnett's test (P<0.001).

[0317] Peptide 1, peptide 9, peptide 12, peptide 18, and peptide 31 showed an effect of promoting axonal extension of trigeminal neurons.

[0318] [Preparation Example]

[0319] The medicine containing the peptide of the present invention as an active ingredient can be prepared, for example, into the following formulation: The pharmaceutical agent of the present invention will be described in more detail with reference to formulation examples, but the present invention is not limited to these formulation examples.

[0320] 1. Capsules

[0321] (1) Peptide 5 40mg (2) Lactose 70mg (3) Microcrystalline cellulose 9mg (4) Magnesium stearate 1mg 1 capsule 120mg

[0322] After mixing all of (1), (2), and (3) and 1 / 2 of (4), the mixture is granulated. The remaining (4) is added to the mixture and the whole is sealed in a gelatin capsule.

[0323] 2. Tablets

[0324] (1) Peptide 6 40mg (2) Lactose 58mg (3) Corn starch 18mg (4) Microcrystalline cellulose 3.5mg (5) Magnesium stearate 0.5mg 1 ingot 120mg

[0325] After mixing all of (1), (2), and (3), 2 / 3 of (4), and 1 / 2 of (5), the mixture is granulated. The remaining (4) and (5) are added to the granulated mixture, and the mixture is compressed to form tablets.

[0326] 3. Vitreous injection

[0327] 1ml

[0328] (1) Peptide 5 40mg (2) Purified white sugar 50mg (3) Sodium chloride 2.34mg (4) Polysorbate 80 appropriate amount (5) Disodium hydrogen phosphate appropriate amount (6) Sodium dihydrogen phosphate appropriate amount (7) Sterile purified water appropriate amount

[0329] (1) to (6) are dissolved in sterile purified water (7) to prepare a vitreous injection solution.

[0330] 4. Eye drops

[0331] 100mL

[0332] (1) Peptide 6 100mg (2) Tromethamine 300mg (3) Sodium chloride 900mg (4) Benzalkonium chloride appropriate amount (5) Sterile purified water appropriate amount

[0333] (1) to (4) were dissolved in sterile purified water (5) to adjust the pH and prepare an eye drop. Sequence Listing <110> Senju Pharmaceutical Co., Ltd. <120> Composition for preventing or treating neurotrophic keratitis containing PACAP peptide or stabilized PACAP peptide <130> P200319WO <150> JP2019-091700 <151> 2019-05-14 <160> 44 <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> synthetic peptides <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is a neutral amino acid <220> <221> MUTAGEN <222> (9)..(9) <223> Xaa is a neutral amino acid <220> <221> MUTAGEN <222> (11)..(11) <223> Xaa is a neutral amino acid <220> <221> MUTAGEN <222> (15)..(15) <223> Xaa is a basic amino acid <220> <221> MUTAGEN <222> (16) <223> Xaa is a neutral amino acid or an amino acid in which the amide of glutamic acid is replaced by a tetrazole <220> <221> MUTAGEN <222> (17) <223> Xaa is a nonpolar amino acid <220> <221> MUTAGEN <222> (19)..(19) <223> Xaa is a nonpolar amino acid <220> <221> MUTAGEN <222> (20)..(20) <223> Xaa is a basic amino acid <220> <221> MUTAGEN <222> (21)..(21) <223> Xaa is a basic amino acid <220> <221> MUTAGEN <222> (27) <223> Xaa is a nonpolar amino acid <400> 3 His Xaa Asp Gly Ile Phe Thr Asp Xaa Tyr Xaa Arg Tyr Arg Xaa Xaa 1 5 10 15 Xaa Ala Xaa Xaa Xaa Tyr Leu Ala Ala Val Xaa 20 25 <210> 4 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(1) <223> Acetylation <400> 4 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> 5 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MUTAGEN <222> (3) <223> Xaa is an amino acid in which the carboxyl group of aspartic acid is replaced by a tetrazole <400> 5 His Ser Xaa 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> 6 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <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> 6 His Ser Asp 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 Leu 20 25 <210> 7 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <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> 7 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 Leu 20 25 <210> 8 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <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> 8 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> 9 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <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> 9 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> 10 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 10 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> 11 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 11 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> 12 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 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 Leu 20 25 <210> 13 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(1) <223> Mesylation <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> 13 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> 14 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(1) <223> Mesylation <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> 14 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> 15 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> (16) <223> Xaa is an amino acid in which the amido group of glutamic acid is replaced by a tetrazole <400> 15 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Xaa 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 16 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 16 His Ala 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> 17 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 17 His Ser Xaa Gly Ile Phe Thr 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> 18 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 18 His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ala Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 19 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 19 His Ser 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> 20 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 20 His Ser Xaa Gly Ile Phe Thr 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> twenty one <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> twenty one His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Leu Ala Ala Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> twenty two <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> twenty two 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 Ala 20 25 <210> twenty three <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> twenty three His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Ala Ala Val Leu 20 25 <210> twenty four <211> 30 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> twenty four His Ser Xaa Gly Ile Phe Thr Xaa Ser Tyr Ser Arg Tyr Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Ala Ala Val Leu Gly Arg Arg 20 25 30 <210> 25 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 25 His Ser Xaa Ala 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> 26 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 26 His Ala Xaa Gly Ile Phe Thr 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> 27 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 27 His Ala Xaa Gly Ile Phe Thr 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> 28 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 28 His Ser Xaa Gly Ile Phe Thr Xaa Ala 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> 29 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 29 His Ser Xaa Gly Ile Phe Thr Xaa Ala 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> 30 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 30 His Ser Xaa Gly Ile Phe Thr Xaa Ser 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> 31 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(1) <223> Mesylation <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> 31 His Ser Xaa Gly Ile Phe Thr 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> 32 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(1) <223> Mesylation <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> 32 His Ser Xaa Ala 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> 33 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(1) <223> Mesylation <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> 33 His Ser Xaa Ala Ile Phe Thr 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> 34 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 34 His Ser Xaa Ala Ile Phe Thr 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> 35 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 35 His Ala Xaa Ala Ile Phe Thr 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> 36 <211> 27 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <220> <221> MOD_RES <222> (1)..(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> 36 His Ser Xaa Ala Ile Phe Thr Xaa Ser 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> 37 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 37 Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn Lys 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 38 Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn 1 5 10 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 39 Gly Lys Arg Tyr Lys Gln Arg Val Lys 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 40 Gly Lys Arg Tyr Lys Gln Arg Val 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 41 Gly Lys Arg Tyr Lys Gln Arg 1 5 <210> 42 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 42 Gly Lys Arg Tyr Lys Gln 1 5 <210> 43 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 43 Gly Lys Arg Tyr Lys 1 5 <210> 44 <211> 4 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 44 Gly Lys Arg Tyr 1

Claims

1. Use of a peptide consisting of the amino acid sequence of SEQ ID NO: 11 in the manufacture of a pharmaceutical composition for the prevention or treatment of neurotrophic keratitis.

2. Method for promoting axonal outgrowth in vitro, which comprises culturing nerve cells in a culture medium supplemented with a peptide selected from the peptides consisting of the amino acid sequences of SEQ ID NO: 11, 14, 20 and 33.

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