Stabilizing peptide of PACAP

Abstract

The purpose of this study is to provide a PACAP peptide with improved stability. The problem is solved by finding that stability is significantly improved by substituting the carboxyl groups of the aspartic acids at positions 3 and / or 8 of the PACAP sequence with tetrazole.

Description

Technical Field

[0001] The present invention relates to a stabilized peptide having the physiological activity of PACAP, a neuroprotective agent containing the peptide, a corneal neurite formation promoter, a tear secretion promoter, a dry eye therapeutic agent, a corneal epithelial disorder therapeutic agent, a corneal endothelial disorder therapeutic agent, a vascular endothelial function improver, an anti-inflammatory agent, or a pharmaceutical composition.

Background Art

[0002] Neurons are cells that make up the nervous system, which is roughly divided into the central nervous system and the peripheral nervous system. Neurons are easily damaged by external factors such as cerebrovascular disorders such as stroke and cerebral infarction, and internal factors such as the accumulation of abnormal proteins, oxidative stress, and inflammation. On the other hand, since their regenerative ability is low, once a disorder occurs, it becomes a factor that significantly reduces the QOL of the patient. Neurodegenerative diseases accompanied by neurodegeneration and dropout of the central nervous system include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis, as well as degenerative diseases of the optic nerve such as glaucoma and sensory neurodegenerative diseases such as neural deafness.

[0003] With the development of neuroscience, various neuroprotective factors have been discovered and are expected to be developed as preventive or therapeutic drugs for nerve disorders. Drugs that reduce free radicals and excitatory amino acids that cause neurodegeneration, and drugs that can protect and / or repair neurons (such as immunophilin ligands such as neurotrophic factors and immunosuppressants) have been found to have a neuroprotective effect. On the other hand, pituitary adenylate cyclase-activating polypeptide (PACAP), in vivo proteins such as CD44 and human brain carboxypeptidase B (HBCPB) have been found to have a neuroprotective effect (Patent Documents 1 and 2).

[0004] Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide discovered from sheep hypothalamus extract. PACAP can have the activity to stimulate cAMP formation in anterior pituitary cells. As PACAP, there exist PACAP38 consisting of 38 amino acid residues and PACAP27 consisting of 27 amino acid residues, and both 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 has 68% identity with vasoactive intestinal polypeptide (VIP). PACAP and VIP bind to PAC1 receptor (PAC1R), VPAC1 receptor (VPAC1R) and VPAC2 receptor (VPAC2R), but their affinities are different. PAC1R binds to PACAP with high selectivity, and the affinity for PACAP is more than 1000 times that for VIP compared with the affinity for VIP. On the other hand, both VPAC1R and VPAC2R have the same degree of affinity for PACAP and VIP. PACAP has various physiological effects, and its physiological effects as a neuroprotective substance, immunosuppressive factor, vasodilator, exocrine gland secretion promoting factor (Patent Document 3), neurite formation promoting factor (Patent Document 4) are known.

[0005] Utilizing the various physiological activities possessed by PACAP, pharmaceutical development has been carried out. However, relatively short peptides such as PACAP are often unstable in aqueous solution, and there are known problems such as a short half-life in vivo due to lack of protease resistance.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention aims to provide a stabilizing peptide of PACAP with high stability in an aqueous solution, a neuroprotective agent containing the peptide, a neurite outgrowth promoter, a lacrimal gland secretion promoter, a pharmaceutical composition for treating or preventing dry eye, a pharmaceutical composition for treating or preventing corneal epithelial disorders, a pharmaceutical composition for treating or preventing neuropathy, a pharmaceutical composition for treating or preventing corneal endothelial disorders, and an agent for improving vascular endothelial function.

Means for Solving the Problems

[0009] As a result of intensive studies by the present inventors to enhance the stability of PACAP in an aqueous solution, it has been found that by substituting the carboxy group of aspartic acid present in the PACAP peptide with tetrazole, the stability in an aqueous solution can be significantly enhanced, leading to the present invention.

[0010] Therefore, the present invention relates to the following: [1] The following formula: H-X1-D-G-I-F-T-D-X2-Y-X3-R-Y-R-X4-X5-X6-A-X7-X8-X9-Y-L-A-A-V-X 10 (SEQ ID NO: 3) {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, and 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 non-polar amino acid} In the sequence represented by , a peptide consisting of a sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions is substituted with tetrazole or a modified sequence thereof, wherein the peptide has binding properties to PAC1R, VPAC1R, and VPACR2, and the modified sequence is a sequence in which one or more amino acids are deleted or added in the sequence of SEQ ID NO: 3, said peptide. [2] The peptide according to item 1, wherein the neutral amino acids in X1, X2, and X3 are alanine or serine. [3] The peptide according to item 1 or 2, wherein the basic amino acids in X4, X8, and X9 are lysine or arginine. [4] The peptide according to any one of items 1 to 3, wherein X5 is glutamine, alanine, or egTz. [5] The peptide according to any one of items 1 to 4, wherein further X6 is methionine, norleucine, leucine, or alanine. [6] The peptide according to any one of items 1 to 5, wherein X7 is valine or alanine. [7] X 10 is leucine or alanine, the peptide according to any one of items 1 to 6. [8] The peptide according to any one of items 1 to 7, wherein the carboxy groups of the aspartic acids at the 3rd and 8th positions of SEQ ID NO: 3 are substituted with tetrazole. [9] The peptide according to any one of items 1 to 8, wherein the N-terminus of the peptide is acetylated or mesylated.

[10] The peptide according to any one of items 1 to 9, wherein the N-terminus of the peptide is acetylated.

[11] The peptide according to any one of items 1 to 10, wherein one or two amino acids are deleted.

[12] The following: GKRYKQRVKNK (SEQ ID NO: 37); GKRYKQRVKN (SEQ ID NO: 38); GKRYKQRVK (SEQ ID NO: 39); GKRYKQRV (SEQ ID NO: 40); GKRYKQR (SEQ ID NO: 41); GKRYKQ (SEQ ID NO: 42); GKRYK (SEQ ID NO: 43); GKRY (SEQ ID NO: 44) GKR; GRR; GK; and GR G; The peptide according to any one of items 1 to 11, wherein one sequence selected from the group consisting of is added to the C-terminus of the peptide.

[13] A neuroprotective agent comprising the peptide according to any one of items 1 to 12.

[14] A lacrimal secretion promoter comprising the peptide according to any one of items 1 to 12.

[15] An anti-inflammatory agent comprising the peptide according to any one of items 1 to 12.

[16] An agent for improving vascular endothelial function comprising the peptide according to any one of items 1 to 12.

[17] A therapeutic agent for corneal epithelial disorder or corneal endothelial disorder comprising the peptide according to any one of items 1 to 12.

[18] A therapeutic agent for dry eye comprising the peptide according to any one of items 1 to 12.

[19] A pharmaceutical composition comprising the peptide according to any one of items 1 to 12.

[20] A method for neuroprotection, lacrimal secretion promotion, improvement of vascular endothelial function or inflammation suppression, comprising administering the peptide according to any one of items 1 to 12.

[21] The peptide according to any one of items 1 to 12 for use in the treatment or prevention of neuropathy, lacrimal hyposecretion disorder, corneal epithelial disorder or corneal endothelial disorder, inflammatory disease or dry eye.

[22] Use of the peptide according to any one of items 1 to 12 in the manufacture of a neuroprotective agent, a lacrimal secretion promoter, a corneal epithelial disorder therapeutic agent, a corneal endothelial disorder therapeutic agent, an anti-inflammatory agent or a dry eye therapeutic agent.

Advantages of the Invention

[0011] According to the present invention, PACAP, which was unstable in an aqueous solution, can be significantly stabilized.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0013] The present invention relates to a peptide comprising a sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th position of PACAP is substituted with tetrazole or a modified sequence thereof, and such a peptide has a comparable binding affinity to PAC1R, VPAC1R, and / or VPAC2R as compared with PACAP. The EC50 for each receptor may be within 10 times, preferably within 5 times, more preferably within 3 times as compared with PACAP. PACAP may be either PACAP38 consisting of 38 residues or PACAP27 consisting of 27 residues. PACAP38 and PACAP27 have the following sequences:

Chemical Formula

[0014] In the present invention, a modified sequence refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence. More preferably, a modified sequence refers to a sequence in which one or several amino acids are substituted, deleted, or added to the original sequence.

[0015] Amino acid substitution can occur at any position as long as it does not change the binding affinity of the peptide consisting of the modified sequence to PAC1R, VPAC1R, and / or VPAC2R. In the peptide consisting of the modified sequence, from the viewpoint of maintaining the binding affinity, amino acid substitution can occur at the 2nd, 9th, 11th, 15th - 17th, 19th - 21st, and 27th positions of PACAP27. In a particularly preferred embodiment, the following sequence: H-X1-D-G-I-F-T-D-X2-Y-X3-R-Y-R-X4-X5-X6-A-X7-X8-X9-Y-L-A-A-V-X 10 (SEQ ID NO: 3) in which the amino acids at the positions represented by X1 - X 10 can be substituted.

[0016] One or more amino acids may be substituted for amino acid substitution. From the viewpoint of maintaining the activity, any number from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be substituted. Among them, substitution of 1 - 4 amino acids is particularly preferred, more preferably 3 amino acids are substituted, even more preferably 2 amino acids are substituted, and particularly preferably 1 amino acid is substituted.

[0017] Amino acid deletions can occur anywhere, as long as they do not change the binding of the peptide consisting of the modified sequence to PAC1R, VPAC1R, and / or VPAC2R. The number of amino acids deleted can be any number from 1 to 10, for example, selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. From the perspective of not changing the binding, deletions of 1 or 2 amino acids are particularly preferred. Amino acid deletions may be deleted from the N-terminal side or C-terminal side of the original sequence, or may be deleted from within the sequence. Since PACAP27 and PACAP38 each have equivalent binding to PAC1R, VPAC1R, and / or VPAC2R, it is considered that deleting the amino acids present on the C-terminal side of PACAP38 has little effect on the binding.

[0018] Amino acid additions can occur anywhere, as long as they do not change the binding of the peptide consisting of the modified sequence to PAC1R, VPAC1R, and / or VPAC2R. The number of amino acids added can be any number from 1 to 10, for example, selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Amino acids may be added to the N-terminal side or C-terminal side of the original sequence, or may be added within the sequence. Since PACAP27 and PACAP38 each have equivalent binding to PAC1R, VPAC1R, and / or VPAC2R, it is considered that adding any amino acid to the C-terminal of PACAP27 has little effect on the binding.

[0019] One aspect of the present invention is the following formula: H-X1-D-G-I-F-T-D-X2-Y-X3-R-Y-R-X4-X5-X6-A-X7-X8-X9-Y-L-A-A-V-X 10 (SEQ ID NO: 3) {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 non-polar amino acid} In the sequence, a peptide consisting of a sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions is substituted with tetrazole, or a modified sequence thereof, and having binding affinity to PAC1R, VPAC1R, and / or VPAC2R.

[0020] X1~X 10 The amino acids represented by are conservatively substitutable with amino acids having the same attributes. As an example, conservative substitution refers to the substitution of amino acids within the following groups: 1 Non-polar amino acids: Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, Ala 2 Neutral amino acids: Ala, Ser, Thr, Tyr, Cys, Asn, Gln, Gly 3 Basic amino acids: Lys, Arg, His 4 Acidic amino acids: Asp, Glu

[0021] The neutral amino acids at X1, X2, and X3 are Ala, Ser, Thr, Tyr, Cys, Asn, Gln, or Gly, more preferably Ala or Ser.

[0022] The basic amino acids at X4, X8, and X9 are Lys, Arg, or His, more preferably Lys or Arg.

[0023] The neutral amino acid at X5 is Ala, Ser, Thr, Tyr, Cys, Asn, Gln, or Gly, more preferably Ala or Gln. X5 may further be an amino acid (egTz) in which the amide of glutamine is substituted with tetrazole.

[0024] The non-polar amino acids at X6 are Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, or Ala, more preferably Met, Nle, Leu, or Ala.

[0025] The nonpolar amino acids at X7 are Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, or Ala, more preferably Val or Ala.

[0026] X 10 The nonpolar amino acids are Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, or Ala, more preferably Leu or Ala.

[0027] In the sequence of SEQ ID NO: 3, the modified sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions is substituted with tetrazole is 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 can be deleted from the amino acid sequence represented by SEQ ID NO: 3, and the following C-terminal addition sequences can be added to the amino acid sequence represented by SEQ ID NO: 3.

[0028] Although not intended to be limited by theory, since PACAP27 and PACAP38 have equivalent binding properties to PAC1R, VPAC1R, and / or VPAC2R, respectively, the addition of amino acids to the C-terminal side in PACAP27 does not affect the binding property to PAC1R. Therefore, when the present invention relates to PACAP27, it can further contain a C-terminal addition sequence, or the C-terminal addition sequence may not exist. The C-terminal addition sequence refers to a sequence consisting of any amino acids from 1 to 11. The C-terminal addition sequence preferably corresponds to the amino acids at positions 28 to 38 of PACAP38. Therefore, the following sequences can be mentioned as the C-terminal sequence: GKRYKQRVKNK (SEQ ID NO: 37); GKRYKQRVKN (SEQ ID NO: 38); GKRYKQRVK (SEQ ID NO: 39); GKRYKQRV (SEQ ID NO: 40); GKRYKQR (SEQ ID NO: 41); GKRYKQ (SEQ ID NO: 42); GKRYK (SEQ ID NO: 43); GKRY (Accession No. 44) GKR; GRR; GK; and GR; G.

[0029] The peptide according to the present invention may be composed of D-form or L-form amino acids, unnatural amino acids such as 2-aminoisobutyric acid and L-2-aminoisobutyric acid, as long as the binding property to PAC1R, VPAC1R, and / or VPAC2R is not lost, and derivatives in which the amino group at the N-terminus, the carboxy group at the C-terminus, or the functional group of the amino acid side chain is arbitrarily modified are included. Examples of the modification include addition of a protecting group to the amino group (for example, acetylation, mesylation, ureation, carbamation, formylation, Boc protection, Fmoc protection), esterification of the carboxy group (such as ethyl esterification), and the like. In addition, modifications that can occur in vivo, such as phosphorylation, amidation, methylation, esterification, acetylation, etc., as well as modifications that occur during the synthesis process or facilitate purification, such as biotinylation, may be included. Further, for the purpose of extending the in vivo half-life of the peptide, modifications such as PEGylation may be performed. In particular, from the viewpoint of enhancing stability, the free amino group of the amino acid at the N-terminus can be acetylated or mesylated. In a peptide consisting of a sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions of PACAP is substituted with tetrazole, the stability is further improved by acetylation or mesylation of the N-terminus. The C-terminus may be any of a carboxy group (-COOH), a carboxylate (-COO-), an amide (-CONH2), or an ester (-COOR), and furthermore, a sugar chain may be added (for example, see WO2017 / 027848).

[0030] 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:

Table 1

[0031] The amino acid in which the carboxy group of aspartic acid is substituted with tetrazole has the following structure: [Chemical Formula] Also, in this specification, "Tz" shall be used as the notation in the sequence.

[0032] Furthermore, the amino acid in which the amide of glutamine is substituted with tetrazole has the following structure: [Chemical Formula] Also, in this specification, "egTz" shall be used as the notation in the sequence.

[0033] The peptide according to the present invention can be produced by any production method. For example, it can be produced by performing solid-phase synthesis or liquid-phase synthesis using the Boc method, the Fmoc method, or the like. Alternatively, in another method, the nucleic acid encoding the peptide of the present invention can be introduced into a host cell by a gene transfer method and synthesized by the host cell. In this case, by designing to add a tag peptide such as a polyhistidine tag to the end of the peptide, purification after expression can be facilitated.

[0034] The peptide according to the present invention includes pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts with inorganic acids (e.g., hydrochloride, hydrobromide, sulfate, phosphate, etc.), salts with organic acids (e.g., methanesulfonate, benzenesulfonate, p-toluenesulfonate, formate, acetate, trifluoroacetate, oxalate, citrate, malonate, fumarate, maleate, tartrate, succinate, malate, etc.), or salts with bases (e.g., ammonium salt, methylpyridinium salt, acetylpyridinium salt, etc.). The peptide according to the present invention also includes hydrates or solvates.

[0035] Histidine with an N-terminal mesylation can be produced by the method shown in Reaction Scheme 1 or a method analogous thereto. [Chemical formula] {In the formula, Trt represents a trityl group and Me represents a methyl group.}

[0036] In the method of Reaction Scheme 1, Compound (II) can be produced by reacting a compound represented by General Formula Compound (I) with methanesulfonyl chloride in the presence of a base, and then Compound (III) can be produced by hydrolyzing Compound (II) with a base in methanol.

[0037] 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 that can be used include triethylamine, N,N-diisopropylethylamine, pyridine, and 4-dimethylaminopyridine, and preferably triethylamine can be mentioned. 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, and examples thereof include tetrahydrofuran, dichloromethane, toluene, etc., and preferably dichloromethane can be mentioned. The reaction temperature is usually 1°C to 30°C, preferably 15°C to 25°C, and the reaction time is usually 0.5 hour to 12 hours, preferably 0.5 hour to 2 hours.

[0038] 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 that can be mentioned include lithium hydroxide, sodium hydroxide, and potassium hydroxide, and preferably potassium hydroxide can be mentioned. The solvent is a mixed solvent of an organic solvent (for example, methanol, ethanol, isopropanol, acetonitrile, 1,4-dioxane, and tetrahydrofuran) and water, and preferably a mixed solvent of methanol and water can be mentioned. The reaction time varies depending on the reagent or solvent used, but is usually 0.5 hour to 12 hours, preferably 0.5 hour to 3 hours. The reaction temperature varies depending on the reagent or solvent used, but is usually 0°C to 100°C, preferably 60°C to 100°C.

[0039] The peptide according to the present invention can exhibit physiological activities similar to PACAP by binding to PAC1R, VPAC1R, and / or VPAC2R. That is, the peptide according to the present invention can, for example, exhibit physiological actions as a neuroprotective substance, a nerve regeneration factor, a wound healing promoter, an inflammation inhibitor, and an exocrine gland secretion promoter. Therefore, in another aspect of the present invention, it may also relate to a neuroprotective agent, a nerve regeneration agent, a wound healing agent, an anti-inflammatory agent, a corneal epithelial disorder therapeutic agent, a corneal endothelial disorder therapeutic agent, a vascular endothelial function improver, a dry eye therapeutic agent, or an exocrine gland secretion promoter containing the peptide according to the present invention. In yet another aspect, it also relates to a pharmaceutical composition containing the peptide according to the present invention.

[0040] The neuroprotective agent according to the present invention refers to an agent having a neuroprotective effect. Therefore, the neuroprotective agent can protect nerves from disorders accompanied by nerve cell damage, degeneration, and / or cell death, and can also be referred to as a nerve cell death (apoptosis and / or necrosis) inhibitor, a nerve cell degeneration inhibitor, a nerve cell stress reducer, an improver of nerve cell cytotoxic resistance, an improver of nerve cell viability, and an abnormal protein accumulation inhibitor.

[0041] As used herein, the term "neuroprotective effect" refers to the effect of protecting nerve cells from damage, degeneration, and / or cell death, preferably the effect of protecting nerve cells from cell death. More specifically, the neuroprotective effect may include suppression of nerve cell death (apoptosis and / or necrosis), suppression of nerve cell degeneration, reduction of nerve cell stress, improvement of resistance to nerve cell toxicity, improvement of nerve cell viability, suppression of abnormal protein accumulation, and the like. Nerve cells are damaged not only by physical injury but also by exposure to neurotoxic substances and lack of oxygen and nutrients, and cell death is caused when the damage exceeds a certain level. In addition, nerve cells are degenerated by accumulating neurotoxic substances, eventually leading to cell death. Neurotoxic substances are broadly classified into exogenous toxic substances and endogenous toxic substances. Examples of exogenous toxic substances include heavy metals, chemicals such as alcohol and botulinum toxin. Endogenous toxic substances are known to include reactive oxygen species, neurotransmitters such as glutamate, and abnormal proteins. The neuroprotective effect can be easily measured by those skilled in the art. As an example, under various stress conditions, such as hypoxia, exposure to neurotoxic substances, nutrient depletion, ultraviolet irradiation, etc., nerve cells are cultured in a medium containing the test substance (drug group) or a medium not containing the test substance (control group), the number of viable cells and dead cells in the medium are measured, the ratio of the number of viable cells to the total number of cells is calculated, and when the ratio of the number of viable cells in the drug group is higher than the ratio of the number of viable cells in the control group, it can be determined that the test substance has a neuroprotective effect. In a more preferred embodiment, it can be measured by comparing with a positive control group added with a substance known to have a neuroprotective effect, such as IGF-1 or NGF, to determine whether it has a protective effect equal to or greater than that of the positive control group. As another example, the neuroprotective effect may be measured by performing in vivo animal experiments.

[0042] The peptide of the present invention has an action of promoting the secretion of exocrine glands possessed by PACAP. Since exocrine glands include lacrimal glands, salivary glands, etc., it can be used as an exocrine gland secretion promoter, for example, a tear secretion promoter or a saliva secretion promoter. Although not intended to be limited by theory, PACAP and the peptide of the present invention promote the secretion of saliva and tears by binding to receptors (PAC1R, VPAC1R, VPAC2R) expressed in the acinar cells of exocrine glands. Tears cover the surface of the cornea and conjunctiva, maintain its wetness, and fill the depressions formed by the microvilli on the corneal surface with tears to smooth the surface, enabling a clear image to be obtained. In addition, the epithelial cells of the cornea and conjunctiva actively metabolize, and cells and metabolites that have become unnecessary are shed and excreted from their outermost surface. Tears wash them away while supplying necessary oxygen and nutrients. Furthermore, tears wash away foreign substances mixed on the corneal surface and play a role in infection prevention against viruses, bacteria, fungi, etc. that enter from the outside by the bacteriostatic action of tears. Also, it acts as a lubricating fluid between the eyelid and the cornea and conjunctiva to enable smooth blinking and eye movement. Thus, although tears are a small amount of liquid that forms a thin film on the corneal surface, they are indispensable for maintaining the transparency and constancy of the cornea through various sophisticated mechanisms. A state in which abnormalities occur on the corneal surface due to a tear secretion disorder is generally called dry eye. When corneal damage occurs due to dry eye, creating a compound that promotes tear secretion will be a useful prophylactic and therapeutic agent for dry eye and diseases associated with dry eye. The peptide according to the present invention can be formulated into eye drops such as eye medications as a tear secretion promoter.

[0043] Since the peptide of the present invention has an anti-inflammatory action possessed by PACAP, it can be used as an anti-inflammatory agent. Although not intended to be limited by theory, PACAP and the peptide of the present invention can suppress the production of VEGF and inflammatory cytokines (TNF-α, IL-6, IL-12, etc.). In particular, VPAC1R and VPAC2R, which are receptors for PACAP, are widely distributed in the gastrointestinal tract and further exhibit an anti-inflammatory action, so they can be particularly used for the treatment of inflammatory bowel diseases and the like.

[0044] In another aspect, the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of the above peptide. The pharmaceutical composition of the present invention can be used for the treatment or prevention of diseases improved by the physiological action of PACAP. Diseases improved by the physiological action of PACAP include neuropathy, diseases related to decreased tear production, inflammatory diseases, and the like. The pharmaceutical composition of the present invention can treat diseases improved by the physiological action of PACAP by administering to a patient, or can prevent diseases by administering to a patient who may be suffering from such diseases. Also, "treatment" means preventing the deterioration of those conditions, delaying the progression, maintaining, reducing or regressing those conditions when a disorder or disease has developed, and "prevention" means preventing the onset of a disorder or disease before its onset.

[0045] Neuropathy refers to a pathological condition in which the function of nerve cells is impaired due to degeneration and cell death of nerve cells, and includes cerebral and retinal vascular disorders and neurodegenerative diseases.

[0046] Examples of vascular disorders include hemorrhagic disorders such as cerebral hemorrhage and subarachnoid hemorrhage, and disorders due to occlusion of cerebral blood vessels such as cerebral thrombosis, cerebral infarction, and cerebral circulatory insufficiency. In any of the hemorrhagic and obstructive disorders, nerve cells in the brain are placed in a hypoxic state and cell death occurs. Also, examples of vascular disorders in the retina include hypertensive retinopathy, diabetic retinopathy, central retinal artery occlusion, central retinal vein occlusion, etc., and nerve cells in the retina are placed in a hypoxic state and cell death occurs. Therefore, the peptide, neuroprotective agent, vascular endothelial function improving agent, or pharmaceutical composition according to the present invention can be administered for the purpose of treatment or prevention against such vascular disorders.

[0047] Neurodegenerative diseases include, but are not limited to, brain and central nervous system degenerative diseases such as dementia, Parkinson's disease, spinocerebellar degeneration, Creutzfeldt-Jakob disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, mad cow disease, epilepsy, etc., motor neuron degenerative diseases such as spinal progressive muscular atrophy, amyotrophic lateral sclerosis, bulbospinal muscular atrophy, etc., and sensory nerve degenerative diseases. Sensory nerve degenerative diseases include, but are not limited to, degenerative diseases of the visual, auditory, tactile, gustatory, and olfactory nerves. Examples of visual degenerative diseases include glaucoma, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, etc. Examples of auditory nerve degenerative diseases include hearing loss, etc.

[0048] Diseases associated with decreased tear production include, but are not limited to, dry eye, dry keratoconjunctivitis, hyposecretion of tears, etc.

[0049] Corneal epithelial disorder is a disease that occurs when the balance of epithelial homeostasis is disrupted due to suppression of corneal epithelial cell proliferation ability or enhanced epithelial exfoliation. Also, corneal epithelial disorder means that the corneal epithelium is damaged by endogenous diseases such as corneal ulcer, corneal epithelial detachment, diabetic keratopathy, dry keratoconjunctivitis, chronic superficial keratitis, punctate superficial keratosis, corneal erosion, and persistent corneal epithelial defect, exogenous diseases caused by drugs, trauma, contact lens wear, etc., or physical or chemical damage.

[0050] Dry eye is a chronic disease of tears and corneal and conjunctival epithelium caused by various factors, and is a disease accompanied by eye discomfort and visual function abnormalities. Abnormalities of tears include quantitative abnormalities in which the amount of tears decreases and qualitative abnormalities in which the nature of tears or the ability to retain tears changes. Also, dry eye includes, for example, hyposecretion of tears, evaporative dry eye, and dry eye associated with Sjogren's syndrome, Stevens-Johnson syndrome, corneal epithelial erosion, blepharitis, ocular pemphigoid, vernal catarrh, allergic conjunctivitis, vitamin A deficiency, etc.

[0051] Examples of inflammatory diseases include, but are not limited to, asthma, atopic dermatitis, urticaria, allergic rhinitis, anaphylactic shock, rhinosinusitis (including eosinophilic rhinosinusitis), rheumatism, multiple sclerosis, arthritis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy, etc.), Sjögren'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.

[0052] Examples of corneal endothelial disorders include Fuchs corneal endothelial dystrophy, persistent reduction in corneal endothelial density after corneal transplantation, trauma, ophthalmic surgery, aging, and disorders associated with corneal endotheliitis.

[0053] Examples of diseases in which vascular endothelial cell disorders are observed include, but are not limited to, diabetes, hypertension, arteriosclerosis, and the like.

[0054] The peptide according to the present invention, or a neuroprotective agent, a nerve regenerating agent, an anti-inflammatory agent, a wound healing promoting agent, an exocrine gland secretion promoting agent, or a pharmaceutical composition containing the peptide can be administered parenterally or orally depending on the disease to be treated. Examples of oral administration include sublingual, intraoral, and oral administration. Examples of parenteral administration include intravenous, intraarterial, subcutaneous, topical, intraperitoneal, intramuscular, intranasal, transdermal, transmucosal, intrathecal, rectal, intramuscular, intracerebral, intrathecal, subarachnoid, intradural, epidural, ophthalmic, otic, nasal, and intraocular routes. More specifically, examples of the intraocular route include subconjunctival, sub-Tenon's capsule, and intravitreal routes. The pharmaceutical composition containing the peptide according to the present invention can be formulated into an appropriate dosage form according to the administration route, and can be, for example, an eye drop, an injection, a powder, an infusion preparation, a granule, a tablet, a suppository, or the like. From the viewpoint of parenteral administration, eye drops, injections, infusion preparations, powders for preparation at the time of use, etc. are preferable. Examples of preparations for intraocular administration include intravitreal injection agents, subconjunctival injection agents, and sub-Tenon's capsule injection agents. These preparations may also contain various pharmaceutically acceptable adjuvants, that is, carriers and other aids, for example, additives such as stabilizers, preservatives, soothing agents, and emulsifying agents. It can also be used in combination with another drug having a neuroprotective effect, an anti-inflammatory effect, or an exocrine gland secretion effect.

[0055] All documents mentioned in this specification are hereby incorporated by reference in their entirety.

[0056] The examples of the present invention described below are for illustrative purposes 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 in the claims. Changes to the present invention, for example, addition, deletion, and substitution of the constituent elements of the present invention, can be made on the condition that the gist of the present invention is not deviated from.

Examples

[0057] Example 1: Peptide Synthesis 1 Synthesis of Ms-His(Trt)-OMe

Chemical formula

[0058] Synthesis of Ms-His(Trt)-OH

Chemical formula

[0059] Example 2: Peptide Synthesis 2 Using a peptide synthesizer (Model: PSSM-8, manufactured by Shimadzu Corporation), the peptides used in the test were synthesized by solid-phase synthesis method according to the Fmoc method. The unnatural amino acids Fmoc-TZ-OH, Fmoc-TZ(trt)-OH, and Fmoc-egTZ(trt)-OH used in solid-phase synthesis were purchased from Astatech. Peptides 1 to 34 with the following sequences were synthesized, and the molecular weights of the synthesized peptides were determined by mass spectrometry (MALDI TOF). As shown in Table 2 below, all measured values were in good agreement with the theoretical values.

Table 2

[0060] Example 3: Peptide Stability Test 1 [Preparation of Measurement Samples] 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. Further, the 1.0 mM peptide solution was diluted to 100 μM with phosphate buffer. The solution was filtered through a chromatodisk (Millex-GV, 0.22 μm, manufactured by Merck millipore), and the filtrate was dispensed into an LC vial (Waters Deactivated Qsert Vial). The prepared peptide solution was incubated in a thermostat at 40 °C for 1 month or 2 months to obtain post-storage samples. At the same time, a sample of the peptide solution prepared but not used for storage was used as a standard sample (initial sample). The standard sample and the post-storage samples were stored at -30 °C until sample analysis.

[0061] [Water permeability] The total weight of the aqueous peptide solution and the storage container was regarded as the sample weight. The sample weight before storage was weighed, and the sample weight after storage was also weighed in the same manner after storage under each condition. Also, the empty weight of the storage container was weighed, and the water permeability was determined based on the following formula. [Equation]

[0062] After stirring the standard sample and the sample after storage 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 shown in Table 3 below to analyze the peptide solution and obtain a chromatogram. [HPLC analysis conditions 1] Column: XSelect CSH C18, 5 μm, 4.6×250 mm manufactured by Waters Guard column: XSelect CSH C18, 5 μm, 4.6×20 mm Guard Cartridge manufactured by Waters Detection wavelength: 220 nm Mobile phase A: 0.1% aqueous formic acid solution Mobile phase B: 0.1% formic acid acetonitrile solution Measurement time: 30 minutes Injection volume of measurement sample: 50 μL Flow rate: 1.0 mL / min Sample cooler: 4°C Column temperature: 40°C Delivery of mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 3 below to perform linear concentration gradient control. [Table 3]

[0063] In the chromatogram, the peak area value of the peptide was determined, and the residual ratio (residual ratio before moisture correction) was calculated using Equation (2). Further, with respect to the residual ratio before moisture correction, the residual ratio after moisture correction was calculated by taking into account the moisture permeability of the container according to Equation (3).

Number

Number

[0064] The residual ratio after moisture correction of the peptide in the sample after storage is shown in Table 4.

Table 4

[0065] Example 4: Stability Test 2 of Peptide Peptides 1, 2, and 6 - 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 through a chromatodisk (Millex - GV, 0.22 μm, manufactured by Merckmillipore). 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 thermostat at 60°C for 1 week or 2 weeks to obtain post - storage samples. At the same time, samples of the peptide solution that were not used for storage were used as standard samples (initial samples). The standard samples and post - storage samples were stored at - 30°C until sample analysis.

[0066] The post - storage samples were measured under the following HPLC analysis conditions 2, and the residual ratio of the peptide in the post - storage samples after moisture correction was calculated in the same manner as in Example 3. The results are shown in Table 6.

[0067] [HPLC Analysis Conditions 2] Column: XSelect CSH C18, 5 μm, 4.6×250 mm, manufactured by Waters Guard Column: XSelect CSH C18, 5 μm, 4.6×20 mm Guard Cartridge, manufactured by Waters Detection Wavelength: 220 nm Mobile Phase A: 0.1% Aqueous Formic Acid Solution Mobile Phase B: 0.1% Acetonitrile Solution with Formic Acid Measurement Time: 20 minutes Injection Volume of Measurement Sample: 50 μL Flow Rate: 1.0 mL / min Column Temperature: 40°C Sample Cooler: 25°C 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.

Table 5

Table 6

[0068] Example 5: Stability Test 3 of Peptides Peptides 12 - 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 through a chromatodisk (Millex-GV, 0.22 μm, manufactured by Merckmillipore). 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 constant temperature bath at 60°C for 1 week or 2 weeks to obtain post-storage samples. At the same time, a sample of the peptide solution that was not used for storage was used as a standard sample (initial sample). The standard sample and the post-storage samples were stored at -30°C until sample analysis.

[0069] The residual rate of the peptide in the post-storage sample after moisture correction was calculated in the same manner as in Example 4, and the results are shown in Table 7.

Table 7

[0070] Example 5 - 2: Stability Test 3 of Peptides Peptides 29 - 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 through a chromatodisk (Millex - GV, 0.22 μm, manufactured by Merckmillipore). 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 constant temperature bath at 60°C for 2 weeks to obtain post - storage samples. At the same time, a sample of the peptide solution that was not used for storage was used as a standard sample (initial sample). The standard sample and the post - storage samples were stored at - 30°C until sample analysis.

[0071] The residual ratio of the peptide in the post - storage sample after moisture correction was calculated in the same manner as in Example 4, and the results are shown in Table 7 - 2.

Table 7 - 2

[0072] Example 6: Stability Test 4 of Peptides Peptides 10 and 11 synthesized in Example 2 were weighed and dissolved in phosphate buffer (pH 7.0) to prepare a 1.0 mM peptide solution. This solution was filtered through a chromatodisk (Millex - GV, 0.22 μm, manufactured by Merckmillipore). The filtrate was diluted to 100 μM with phosphate buffer (pH 7.0) and dispensed into tubes (Protein Lobind Tube manufactured by eppendorf). The prepared peptides were incubated in a thermostat at 40°C for 1 month or 2 months to obtain samples after storage. At the same time, a sample of the peptide solution that was not used for storage was used as a standard sample (initial sample). The standard sample and the samples after storage were stored at - 30°C until sample analysis. The residual rate of the peptide after moisture correction in the samples after storage was measured in the same manner as in Example 4, and the results are shown in Table 8.

Table 8

[0073] Example 7: Stability Test 5 of Peptides 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 through a chromatodisk (Millex-GV, 0.22 μm, manufactured by Merckmillipore). 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 constant temperature bath at 60 °C for 1 week or 2 weeks to obtain post-storage samples. At the same time, a sample of the peptide solution that was not used for storage was used as a standard sample (initial sample). The standard sample and the post-storage samples were stored at -30 °C until sample analysis.

[0074] The post-storage samples were measured under the following HPLC analysis conditions 3, and the residual ratio of the peptide in the post-storage samples after moisture correction was calculated in the same manner as in Example 3. The results are shown in Table 10.

[0075] [HPLC Analysis Conditions 3] Column: XSelect CSH C18, 5 μm, 4.6×250 mm, manufactured by Waters Guard Column: XSelect CSH C18, 5 μm, 4.6×20 mm Guard Cartridge, manufactured by Waters Detection Wavelength: 220 nm Mobile Phase A: 0.1% Aqueous Formic Acid Solution Mobile Phase B: 0.1% Acetonitrile Solution of Formic Acid Measurement Time: 20 minutes Injection Volume of Measurement Sample: 50 μL Flow Rate: 1.0 mL / min Column Temperature: 40 °C Sample Cooler: 25 °C 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.

Table 9

Table 10

[0076] Example 7: cAMP assay of PACAP27 and its modified peptides [Cell culture] Frozen CHO-K1 cells treated with mitomycin (highly expressing PAC1 or VPAC1 receptor cell line: purchased from DiscoveRx) were prepared with Cell plating reagent (manufactured by DiscoveRx) to a concentration of 1.35×10 4 cells / 100 μl / well and then seeded into a 96-well culture plate. The cells were cultured at 37 °C in a 5% CO2 incubator for 18 - 24 hours to allow the cells to adhere to the plate.

[0077] [Reagent preparation] The 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 with Cell assay buffer (manufactured by DiscoveRx) (containing 0.5 mM IBMX and 0.001% BSA) to a concentration of 20 μM. A 5-fold dilution series was then prepared with the same Cell assay buffer and used for the assay.

[0078] [cAMP assay] 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 was mixed with the peptide 1, 2, 6-9 solutions at each diluted concentration to prepare a peptide-cAMP antibody mixture. Subsequently, the medium was removed from the culture plate of CHO-K1 cells, washed with PBS, and then the peptide-cAMP antibody mixture was added to the cells, which were incubated for 30 minutes in a 5% CO2 atmosphere at 37°C. Next, the Working detection solution was added, and after covering the culture plate with aluminum foil to block light, it was incubated at 25°C for 1 hour. After incubation, Solution A was added, and after covering the culture plate with aluminum foil to block light, it was incubated at 25°C for 3 hours. Finally, the chemiluminescence signal was detected using a GloMax detector (manufactured by Promega) under the conditions of Luminescence, Integration time (1 sec). The obtained Relative luminescence unit (RLU) values were analyzed using GraphPad Prism Ver 6.05 (manufactured by Graph Pad), and the EC 50 values for each peptide were calculated. The results of the EC 50 values of the cAMP induction effect in the PAC1R or VPAC1R overexpressing cell lines of each peptide are shown in Figure 1, Figure 2, and Table 11.

Table 11

[0079] Synthesized peptides 1, 2, and 6-9 showed cAMP induction ability against the respective overexpressing cells of PAC1R and VPAC1R, and their EC 50 values were comparable to those of the native peptide (peptide 1: PACAP27).

[0080] Summarizing the results in Table 6 and Table 11, the peptides according to the present invention (Peptides 6-9) have extremely improved stability in aqueous solution compared to PACAP and maintain the same physiological activity as PACAP. In particular, having a shelf life exceeding two years at room temperature enables development as liquid formulations, such as products like vials, ampoules, eye drops, etc.

[0081] Example 8: cAMP assay 2 of PACAP27 modified peptide [Reagent preparation] The powders of Peptides 3-5 and 10-28 synthesized in Example 2 were each dissolved in water to a concentration of 0.1 mM, and then diluted with Cell assay buffer (manufactured by DiscoveRx) (containing 0.5 mM IBMX and 0.001% BSA) to a concentration of 20 μM. From this, a 5-fold dilution series was prepared with the same Cell assay buffer and used for the assay. In the same manner as in Example 7, the EC 50 values for Peptides 3-5 and 10-28 were calculated. The results of the EC 50 values of the cAMP induction effect in the PAC1 or VPAC1 highly expressing cell lines of each peptide are shown in Table 12.

Table 12

[0082] Example 8-2: cAMP assay 2 of PACAP27 modified peptide [Reagent preparation] The powders of Peptides 29-34 synthesized in Example 2 were each dissolved in water to a concentration of 0.1 mM, and then diluted with Cell assay buffer (manufactured by DiscoveRx) (containing 0.5 mM IBMX and 0.001% BSA) to a concentration of 20 μM. From this, a 5-fold dilution series was prepared with the same Cell assay buffer and used for the assay. The EC in peptides 27 - 34 was calculated in the same manner as in Example 7. 50 The EC values of the cAMP induction effect in PAC1 or VPAC1 highly expressed cell lines of each peptide are shown in Table 12 - 2. 50 The results of the EC values are shown in Table 12 - 2.

Table 12 - 2

[0083] Formulation Example The medicament containing the peptide of the present invention as an active ingredient can be produced, for example, by the following formulations. Although the medicament of the present invention will be described in more detail with formulation examples, the present invention is not limited only to these formulation examples.

[0084] 1. Capsules (1) Peptide 5 40 mg (2) Lactose 70 mg (3) Microcrystalline cellulose 9 mg (4) Magnesium stearate 1 mg 1 capsule 120 mg After mixing the total amounts of (1), (2), (3) and 1 / 2 of (4), granulation is carried out. The remaining (4) is added thereto and the whole is encapsulated in a gelatin capsule.

[0085] 2. Tablets (1) Peptide 6 40 mg (2) Lactose 58 mg (3) Corn starch 18 mg (4) Microcrystalline cellulose 3.5 mg (5) Magnesium stearate 0.5 mg 1 tablet 120 mg After mixing the total amounts of (1), (2), (3), 2 / 3 of (4) and 1 / 2 of (5), granulation is carried out. The remaining (4) and (5) are added to this granule and compression molded into tablets.

[0086] 3. Vitreous Body Injection In 1 ml (1) Peptide 5 40 mg (2) Refined Sucrose 50 mg (3) Sodium Chloride 2.34 mg (4) Polysorbate 80 appropriate amount (5) Disodium Hydrogen Phosphate appropriate amount (6) Sodium Dihydrogen Phosphate appropriate amount (7) Sterile Purified Water appropriate amount Dissolve (1) - (6) in (7) Sterile Purified Water to prepare the vitreous body injection.

[0087] 4. Eye Drops In 100 mL (1) Peptide 6 100 mg (2) Trometamol 300 mg (3) Sodium Chloride 900 mg (4) Benzalkonium Chloride appropriate amount (5) Sterile Purified Water appropriate amount Dissolve (1) - (4) in (5) Sterile Purified Water, adjust the pH, and prepare the eye drops.

Claims

1. The following formula: H-X 1 -D-G-I-F-T-D-X 2 -Y-X 3 -R-Y-R-X 4 -X 5 -X 6 -A-X 7 -X 8 -X 9 -Y-L-A-A-V-X 10 (SEQ ID NO: 3) {In the formula, X 1 is a neutral amino acid, X 2 is a neutral amino acid, X 3 is a neutral amino acid, X 4 is a basic amino acid, X 5 is a neutral amino acid or egTz, X 6 is a non-polar amino acid 、 X 7 is a non-polar amino acid, X 8 is a basic amino acid, X 9 is a basic amino acid, X 10 is a neutral amino acid} In the sequence represented by, a peptide consisting of a sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions is substituted with tetrazole or a modified sequence thereof, wherein the peptide has binding affinity to PAC1R, VPAC1R and VPACR2, and the modified sequence is in the sequence represented by SEQ ID NO: 3, in the sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions is substituted with tetrazole, The following: GKRYKQRVKNK (SEQ ID NO: 37); GKRYKQRVKN (SEQ ID NO: 38); GKRYKQRVK (SEQ ID NO: 39); GKRYKQRV (SEQ ID NO: 40); GKRYKQR (SEQ ID NO: 41); GKRYKQ (SEQ ID NO: 42); GKRYK (SEQ ID NO: 43); GKRY (SEQ ID NO: 44); GKR; GRR; GK; GR; and G; One sequence selected from the group consisting of is added to the C-terminus of the sequence represented by SEQ ID NO: 3 where the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions is substituted with tetrazole, or In the sequence represented by SEQ ID NO: 3, the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions is substituted with tetrazole, one amino acid at the N-terminus of the sequence is deleted, or 1 to 3 amino acids at the C-terminus are deleted, the said peptide.

2. X 1 X 2 and X 3 The neutral amino acids in are alanine or serine, the peptide according to claim 1.

3. X 4 X 8 and X 9 The basic amino acids in are lysine or arginine, the peptide according to claim 1 or 2.

4. X 5 is glutamine, alanine, or egTz, the peptide according to any one of claims 1 to 3.

5. Further X 6 is methionine, norleucine, alanine or leucine, the peptide according to any one of claims 1 to 4.

6. X 7 is valine or alanine, the peptide according to any one of claims 1 to 5.

7. X 10 is leucine or alanine, the peptide according to any one of claims 1 to 6.

8. The carboxy groups of the aspartic acids at the 3rd and 8th positions of SEQ ID NO: 3 are substituted with tetrazole, the peptide according to any one of claims 1 to 7.

9. The peptide according to any one of claims 1 to 8, wherein the N-terminus is acetylated or mesylated.

10. The peptide according to any one of claims 1 to 9, wherein the N-terminus is acetylated.

11. A neuroprotective agent comprising the peptide according to any one of claims 1 to 10.

12. A lacrimal secretion promoter comprising the peptide according to any one of claims 1 to 10.

13. An anti-inflammatory agent comprising the peptide according to any one of claims 1 to 10.

14. An agent for improving vascular endothelial function comprising the peptide according to any one of claims 1 to 10.

15. A therapeutic agent for corneal epithelial disorder or corneal endothelial disorder comprising the peptide according to any one of claims 1 to 10.

16. A therapeutic agent for dry eye comprising the peptide according to any one of claims 1 to 10.

17. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 10.

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