Composition for preventing or treating neurotrophic keratitis which contains pacap peptide or stabilizing peptide of pacap

Abstract

To provide a composition for preventing or treating neurotrophic keratitis.SOLUTION: It is found that a PACAP peptide exerts an inhibitory effect of cornea disorder in a neurotrophic keratitis model, and model axon extension is promoted in a cultured nerve cell. Furthermore, provided are a composition for preventing or treating neurotrophic keratitis which contains a PACAP peptide or a stabilized PACAP peptide in which stability significantly enhances by substituting a carboxyl group of aspartic acid at the 3- and / or 8-position in a sequence of PACAP with tetrazole, and an axon extension promoter.SELECTED DRAWING: Figure 4

Description

Technical Field

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

Background Art

[0002] Corneal sensation is important for preventing damage through the blink reflex and tearing reflex and is innervated by the corneal nerves. On the other hand, corneal nerves not only mediate sensation but also affect corneal epithelial cell proliferation through the release of neurotransmitters and the secretion of nerve growth factors from corneal nerve endings. When corneal nerves are damaged, the metabolism of corneal epithelial cells changes, their proliferative ability is impaired, and trophic ulcers are known to occur. Neurotrophic keratitis and neuroparalytic keratopathy are degenerative corneal diseases caused by damage to the trigeminal nerve and cause damage to the corneal epithelium. In the most severe cases, corneal ulceration, melting, and perforation may occur, impairing the patient's visual recognition ability. Various clinical conditions, such as viral infections like herpes of the cornea, ocular trauma, corneal surgery, and systemic conditions such as diabetes, can cause neurotrophic keratitis. In particular, it has been reported that herpes infection accounts for 27% of neurotrophic keratitis. Although neurotrophic keratitis has been known to exist for a long time, an effective treatment method has not yet been developed.

[0003] With the development of neuroscience, various neuroprotective factors have been discovered and are expected to be developed as preventive or therapeutic agents for nerve disorders. Agents that reduce free radicals and excitatory amino acids that cause neurodegeneration, and agents that can protect and / or repair nerve cells (such as immunophilins 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 ovine hypothalamus extracts. PACAP may have the activity to stimulate cAMP formation in anterior pituitary cells. There are PACAP38 consisting of 38 amino acid residues and PACAP27 consisting of 27 amino acid residues as PACAP, and both have equivalent actions (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 differ in terms of affinity for these receptors. PAC1R binds to PACAP with high selectivity, and the affinity for PACAP is more than 1000 times higher compared to 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 actions and is known to have physiological actions as a neuroprotective substance, an immunosuppressive factor, a vasodilator, an exocrine gland secretion promoting factor (Patent Document 3), and a neurite formation promoting factor (Patent Document 4).

[0005] Utilizing the various physiological activities of PACAP, the development of pharmaceuticals has been carried out. However, relatively short peptides such as PACAP are often unstable in aqueous solutions and are known to have 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

[0007] [Non-Patent Document 1] S. Bourgault (2009) Current Medicinal Chemistry 16, 4462-4480 [Non-Patent Document 2] Louise Dickson (2009) Pharmacology & Therapeutics, 12, 294-316 [Non-Patent Document 3] Alessandro Lambiase (2012) Investigative Ophthalmology & Visual Science, vol. 53, No.13, p.8280-8287 [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] An object of the present invention is to provide a composition for preventing or treating neurotrophic keratitis. [Means for Solving the Problems]

[0009] The present inventors have found that a PACAP peptide or a stabilized peptide thereof exerts an inhibitory effect on corneal injury in a neurotrophic keratitis model and promotes neurite outgrowth in a cultured nerve cell model, leading to the present invention. Furthermore, as a stabilized peptide, a peptide in which the carboxy group of aspartic acid present in the PACAP peptide is replaced with tetrazole has been found to significantly enhance the stability in an aqueous solution.

[0010] Therefore, the present invention relates to the following: [1-1]The following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) A pharmaceutical composition for preventing or treating neurotrophic keratitis, comprising a peptide consisting of the sequence represented thereby, or a stabilized sequence thereof. [1-2] A peptide for use in preventing or treating neurotrophic keratitis, comprising the following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) A peptide consisting of the sequence represented thereby, or a stabilized sequence thereof. [1-3] A method for preventing or treating neurotrophic keratitis, comprising the following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) The method, comprising administering a peptide consisting of the sequence represented thereby, or a stabilized sequence thereof, to a subject in need of treatment or prevention of neurotrophic keratitis. [1-4] For use in manufacturing a medicament for treating neurotrophic keratitis, comprising the following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) Use of a peptide consisting of the sequence represented thereby, or a stabilized sequence thereof. [2] The peptide consisting of the stabilizing sequence is a sequence in which the carboxyl group of the aspartic acid residue at the 3rd and / or 8th positions in the sequence of SEQ ID NO: 1 or 2 is substituted with tetrazole, or a sequence in which a part of the sequence is modified. Here, when the stabilizing sequence is a sequence in which a part of the sequence is modified, the peptide consisting of the stabilizing sequence has binding properties to PAC1R, VPAC1R, and VPACR2. The prophylactic or therapeutic composition according to item 1-1, the peptide according to item 1-2, the method according to item 1-3, or the use according to item 1-4. [3] The peptide consisting of the stabilizing sequence is represented by 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) {In the formula, 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 nonpolar amino acid 、 X7 is a nonpolar amino acid, X8 is a basic amino acid, X9 is a basic amino acid, X 10 is a neutral amino acid} A peptide consisting of a sequence in which the carboxyl group of the aspartic acid residue at the 3rd and / or 8th positions in the sequence represented by the formula 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. The prophylactic or therapeutic composition according to item 1-1 or item 2, the peptide according to item 1-2 or item 2, the method according to item 1-3 or item 2, or the use according to item 1-4 or item 2, comprising the peptide. [4] The neutral amino acids in X1, X2, and X3 are alanine or serine. The prophylactic or therapeutic composition according to item 1-1, the prophylactic or therapeutic composition according to item 3, the peptide, the method, or the use. [5] The basic amino acids in X4, X8, and X9 are lysine or arginine. The prophylactic or therapeutic composition, the peptide, the method, or the use according to item 3 or 4. [6] The prophylactic or therapeutic composition, peptide, method, or use according to any one of items 3 to 5, wherein X5 is glutamine, alanine, or egTz. [7] The prophylactic or therapeutic composition, peptide, method, or use according to any one of items 3 to 6, wherein X6 is further methionine, norleucine, alanine, or leucine. [8] The prophylactic or therapeutic composition, peptide, method, or use according to any one of items 3 to 7, wherein X7 is valine or alanine. [9] X 10 is leucine or alanine, and the prophylactic or therapeutic composition, peptide, method, or use according to any one of items 3 to 8.

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

[11] The prophylactic 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.

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

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

[14] 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 prophylactic or therapeutic composition, peptide, method, or use according to any one of items 3 to 13, wherein one sequence selected from the group consisting of is added to the C-terminus of the peptide. [15-1] The following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) A neurite outgrowth promoter comprising a peptide consisting of the sequence represented by, or a stabilized sequence thereof. [15-2] The following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) A method for promoting neurite outgrowth, comprising administering to a subject in need of promoting neurite outgrowth a peptide consisting of the sequence represented by, or a stabilized sequence thereof. [15-3] For the manufacture of a neurite outgrowth promoter, the following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) Use of a peptide consisting of the sequence represented by, or a stabilized sequence thereof. [15-4] For use in neurotrophic keratitis, the treatment of nerve injury or nerve regeneration through the promotion of neurite outgrowth, the following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) A peptide consisting of the sequence represented by, or a stabilized sequence thereof.

[16] The nerve axon elongation 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 extends the axons of the trigeminal nerve. [17-1] A composition for treating nerve injury or promoting nerve regeneration, comprising the nerve axon elongation promoter according to item 15-1 or 16. [17-2] A method for treating nerve injury or promoting nerve regeneration by the method according to item 15-2 or 16. [17-3] The use according to item [15-3], wherein the nerve axon elongation promoter is used for treating nerve injury or promoting nerve regeneration.

[18] A method for promoting nerve axon elongation in vitro or in vivo, comprising the following: Applying a peptide consisting of the sequence represented by HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) or a stabilized sequence thereof.

[19] The method for promoting nerve axon elongation in vitro according to item

[18] , comprising culturing nerve cells in a culture medium supplemented with the peptide.

Effect of the Invention

[0011] According to the present invention, PACAP or stabilized PACAP can be used for the prevention or treatment of neurotrophic keratitis or for promoting nerve axon elongation.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0013] 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 aspect of the present invention, the present invention relates to a nerve axon elongation promoter containing a PACAP peptide or a stabilized PACAP peptide. In yet another aspect, the present invention relates to a method for promoting nerve axon elongation in vitro or in vivo, which includes applying a PACAP peptide or a stabilized PACAP peptide. The stabilized PACAP peptide refers to a peptide in which amino acids are modified to enhance stability while maintaining the physiological activity of PACAP. Specifically, the stabilized PACAP peptide relates to a peptide consisting of a sequence in which the carboxy group of the aspartic acid residue at the 3rd and / or 8th positions in the amino acid sequence of the PACAP peptide is substituted with tetrazole or a sequence in which a part of the sequence is modified. The stabilized PACAP peptide according to the present invention has a comparable binding affinity to PAC1R, VPAC1R, and / or VPAC2R as compared to PACAP. The comparable binding affinity means that the EC50 value of each peptide to its respective receptor is within 10-fold, preferably within 5-fold, more preferably within 3-fold as compared to 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 sequence in which a part of the sequence is modified refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence. More preferably, a sequence in which a part of the sequence is modified 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 ability of the peptide consisting of the sequence in which a part of the sequence is modified to PAC1R, VPAC1R, and / or VPAC2R. In the peptide consisting of the sequence in which a part of the sequence is modified, from the viewpoint of maintaining the binding ability, amino acid substitution can occur at the 2nd, 9th, 11th, 15th to 17th, 19th to 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 to X 10 can be substituted.

[0016] One or more amino acids may be substituted for amino acid substitution, but 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 to 4 amino acids is particularly preferred, more preferably 3 amino acids are substituted, still 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. The amino acid deletion may be 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 have equivalent binding to PAC1R, VPAC1R, and / or VPAC2R, respectively, it is considered that the amino acids present on the C-terminal side of PACAP38 can be deleted with 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. The amino acid 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 have equivalent binding to PAC1R, VPAC1R, and / or VPAC2R, respectively, 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) {In the formula, 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 substituted by 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 non-polar amino acids in X7 are Val, Leu, Ile, Met, Phe, Trp, Pro, Nle, or Ala, and more preferably Val or Ala.

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

[0027] In the present invention, the modified sequence refers to a sequence in which one or more amino acids are substituted, deleted, or added to the original sequence. More preferably, the modified sequence refers to a sequence in which one or several amino acids are substituted, deleted, or added to the original sequence.

[0028] 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 position is substituted with tetrazole is preferably a sequence in which one or more amino acids are deleted or added to the amino acid sequence represented by SEQ ID NO: 3. More preferably, one or two amino acids can be deleted from the amino acid sequence represented by SEQ ID NO: 3, and the following C-terminal addition sequence can be added to the amino acid sequence represented by SEQ ID NO: 3.

[0029] Although not intended to be limited by theory, since PACAP27 and PACAP38 each have equivalent binding properties to PAC1R, VPAC1R, and / or VPAC2R, the addition of amino acids to the C-terminal side in PACAP27 does not affect the binding property to PAC1R. Therefore, when the present invention relates to PACAP27 or its stabilized peptide, it can further contain a C-terminal addition sequence, or the C-terminal addition sequence may not be present. 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 (SEQ ID NO: 44) GKR; GRR; GK; and GR; G.

[0030] The peptides according to the present invention can use D-form or L-form amino acids, or racemic amino acids, as long as the binding affinity for PAC1R, VPAC1R, and / or VPAC2R is not lost. Similarly, the peptides according to the present invention may be composed of non-natural amino acids such as 2-aminoisobutyric acid or L-2-aminoisobutyric acid, and include derivatives in which the functional groups of the amino group at the N-terminus, the carboxy group at the C-terminus, or the amino acid side chain are arbitrarily modified. Examples of the modification include addition of a protecting group to the amino group (e.g., acylation (formylation, acetylation, etc.), mesylation, ureation, carbamation, Boc protection, Fmoc protection), esterification of the carboxy group (ethylation, etc.). 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 N-terminal amino acid can be protected by a protecting group (e.g., acyl group). For example, the free amino group of the N-terminal amino acid 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 acetylating or mesylating 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 may further have a sugar chain added (see, for example, WO2017 / 027848).

[0031] 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 modified sequences thereof:

Table 1

[0032] The amino acid in which the carboxy group of aspartic acid is substituted with tetrazole has the following structure: [Chemical formula] In addition, in this specification, "Tz" shall be used as the notation in the sequence.

[0033] Furthermore, the amino acid in which the amide of glutamine is replaced by tetrazole has the following structure: [Chemical formula] In addition, in this specification, "egTz" shall be used as the notation in the sequence.

[0034] 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 can be facilitated after expression.

[0035] 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.

[0036] Histidine with 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.}

[0037] 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.

[0038] 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, 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 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.

[0039] 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, 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 reagents or solvents used, but is usually 0.5 hour to 12 hours, preferably 0.5 hour to 3 hours. The reaction temperature varies depending on the reagents or solvents used, but is usually 0°C to 100°C, preferably 60°C to 100°C.

[0040] The peptide according to the present invention can exert the same physiological activity as PACAP by binding to PAC1R, VPAC1R, and / or VPAC2R. Although not intended to be limited by theory, the prophylactic or therapeutic use for neurotrophic keratitis may be an action mediated through binding to these receptors. More specifically, the peptide according to the present invention can suppress the decrease in tear volume caused by neurotrophic keratitis and also suppress superficial punctate keratitis. Furthermore, the peptide according to the present invention promotes axonal elongation of nerves. By the axonal elongation promoting action of the peptide according to the present invention, damage to the trigeminal nerve can be treated or the trigeminal nerve can be regenerated. The peptide according to the present invention can treat or prevent neurotrophic keratitis through these actions. In addition, the peptide according to the present invention can promote axonal elongation of nerves in vivo or in vitro. When promoting axonal elongation in vitro, nerve cells are cultured in a medium containing the peptide according to the present invention. The method for culturing nerve cells can be performed by any method well-known in the art, and a nerve cell culture medium widely known in the art can be used.

[0041] The present invention relates to a pharmaceutical composition for treating or preventing neurotrophic keratitis containing a therapeutically effective amount of the above peptide. By administering the pharmaceutical composition of the present invention to a patient, neurotrophic keratitis can be treated by the physiological action of PACAP, or by administering it to a patient who may be suffering from such a disease, neurotrophic keratitis can be prevented. 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. Patients who may be suffering from neurotrophic keratitis include, for example, patients suffering from viral infections such as herpes of the cornea, ocular trauma, corneal surgery, and furthermore diabetes.

[0042] The peptide according to the present invention, or a pharmaceutical composition containing the peptide, can be administered parenterally or orally depending on the disease to be treated. Oral administration includes sublingual, intraoral, and oral administration. Parenteral administration includes, for example, 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, the intraocular route includes subconjunctival, sub-Tenon's capsule, and intravitreal routes. The pharmaceutical composition containing the peptide according to the present invention can be appropriately formulated according to the administration route, and can be, for example, eye drops, injections, powders, infusion preparations, granules, tablets, suppositories, etc., but from the perspective of parenteral administration, eye drops, injections, infusion preparations, powders for preparation at the time of use, etc. are preferred. Examples of preparations for intraocular administration include intravitreal injections, subconjunctival injections, and sub-Tenon's capsule injections. In addition, these preparations may contain various pharmaceutically acceptable adjuvants, that is, carriers and other auxiliaries, for example, additives such as stabilizers, preservatives, soothing agents, emulsifiers, etc. Further, it can also be used in combination with another drug having a neuroprotective effect, anti-inflammatory effect, or exocrine gland secretion effect.

[0043] All documents mentioned in this specification are hereby incorporated by reference in their entirety into this specification.

[0044] 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

[0045] Example 1: Peptide Synthesis 1 Synthesis of Ms-His(Trt)-OMe

Chemical formula

[0046] Synthesis of Ms-His(Trt)-OH

Chemical Structure

[0047] Example 2: Peptide Synthesis 2 The peptides used in the test were synthesized by solid-phase synthesis using the Fmoc method with 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 solid-phase synthesis were purchased from Astatech. Peptides 1 - 34 having 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]

[0048] 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 a 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 the 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 or 2 months to obtain samples after storage. 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 the samples after storage were stored at -30 °C until sample analysis.

[0049] [Water permeability] The total weight of the peptide aqueous solution and the storage container was taken 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]

[0050] After stirring the standard samples and the samples 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 the measurement sample: 50 μL Flow rate: 1.0 mL / min Sample cooler: 4°C Column temperature: 40°C Liquid delivery of the mobile phase: The mixing ratio of mobile phase A and mobile phase B was changed as shown in Table 3 below, and linear concentration gradient control was performed. [Table 3]

[0051] In the chromatogram, the peak area value of the peptide was determined, and the residual rate (residual rate before moisture correction) was calculated by Equation (2). Also, for the residual rate before moisture correction, the residual rate after moisture correction was calculated by taking into account the moisture permeability of the container according to Equation (3). [Equation] [Equation]

[0052] The residual rate after moisture correction of the peptide in the sample after storage is shown in Table 4. [Table 4] Peptide 1 (PACAP) had low stability, with 37.4% remaining after storage at 40°C for 1 month and 21.3% remaining after storage at 40°C for 2 months. For Peptide 1, Peptide 3, in which only the carboxy group of the aspartic acid side chain at the 3rd residue of Peptide 1 was replaced with tetrazole, showed higher stability than Peptide 1. Also, Peptide 4, in which only the carboxy group of the aspartic acid side chain at the 8th residue was replaced with tetrazole, had improved stability compared to Peptide 1. However, in Peptide 5, in which only the carboxy groups of the aspartic acid side chains at the 3rd and 8th residues of Peptide 1 were replaced with tetrazole, the stability was further improved compared to Peptide 3 and Peptide 4, indicating that two tetrazole substitutions are more effective than one tetrazole substitution for improving the stability of PACAP.

[0053] Example 4: Stability Test 2 of Peptides 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 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.

[0054] The post - storage samples were measured under the following HPLC analysis conditions 2, and the residual rate 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.

[0055] [HPLC Analysis Conditions 2] Column: XSelect CSH C18, 5 μm, 4.6×250 mm, manufactured by Waters Guard Column: Waters XSelect CSH C18, 5 μm, 4.6×20 mm Guard Cartridge 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 Liquid Delivery of Mobile Phase: The mixing ratio of Mobile Phase A and Mobile Phase B was changed as shown in Table 5 below, and linear concentration gradient control was performed.

Table 5

Table 6

[0056] Example 5: Stability Test 3 of Peptides 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 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, 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.

[0057] 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.

Table 7

[0058] Example 5-2: Stability Test 3 of Peptide The 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 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 a post-storage sample. At the same time, a sample of the peptide solution prepared that was not used for storage was used as a standard sample (initial sample). The standard sample and the post-storage sample were stored at -30°C until sample analysis.

[0059] 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

[0060] 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. Also, among the simultaneously prepared peptide solutions, a sample 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 ratio 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

[0061] Example 7: Stability Test 5 of Peptides 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 thermostat at 60 °C for 1 week or 2 weeks to obtain samples after storage. Also, among the simultaneously prepared peptide solutions, a sample 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.

[0062] The stored samples were measured under the following HPLC analysis conditions 3, and the residual ratio of the peptide in the stored samples after moisture correction was calculated in the same manner as in Example 3. The results are shown in Table 10.

[0063] [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 Liquid Delivery of Mobile Phase: The mixing ratio of Mobile Phase A and Mobile Phase B was changed as shown in Table 9 below, and linear concentration gradient control was performed.

Table 9

Table 10

[0064] Example 7: cAMP Assay of PACAP27 and Its Stabilized Peptide [Cell Culture] Frozen CHO-K1 cells treated with mitomycin (a high-expressing cell line of PAC1 or VPAC1 receptor: 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 adhere the cells to the plate.

[0065] [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.

[0066] [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 diluted peptide solutions of each concentration of Peptides 1, 2, 6 - 9 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 and incubated at 37°C in a 5% CO2 atmosphere for 30 minutes. Next, the Working detection solution was added, the culture plate was covered with aluminum foil to block light, and incubated at 25°C for 1 hour. After incubation, Solution A was added, the culture plate was covered with aluminum foil to block light, and 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 EC 50 values of the cAMP induction effects in the PAC1R- or VPAC1R-high expressing cell lines of each peptide are shown in Figure 1, Figure 2, and Table 11. [Table 11]

[0067] The synthesized peptides 1, 2, and 6 - 9 showed cAMP induction ability against the high - expressing cells of PAC1R and VPAC1R respectively, and their EC 50 values were comparable to those of the native peptides (peptide 1: PACAP27).

[0068] 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 physiological activities equivalent to those of PACAP. In particular, having a shelf - life exceeding 2 years at room temperature enables development as products such as liquid formulations, for example, vials, ampoules, eye drops, etc.

[0069] Example 8: cAMP assay 2 of PACAP27 - stabilized 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 in peptides 3 - 5 and 10 - 28 were calculated. The EC 50 values of the cAMP induction effect of each peptide in the PAC1 - or VPAC1 - high - expressing cell line are shown in Table 12.

Table 12

[0070] Example 8 - 2: cAMP assay 2 of PACAP27 - stabilized peptide [Reagent preparation] After dissolving the powders of Peptides 29 to 34 synthesized in Example 2 in water to a concentration of 0.1 mM each, they were 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. EC in Peptides 29 to 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 expressing 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

[0071] Example 9: Evaluation of PACAP27 and its stabilized peptide using a neurotrophic keratitis model By administering capsaicin subcutaneously to the back of rats, a model rat was created in which corneal neuropathy and attenuation of corneal sensation were induced, and along with that, a decrease in tear volume and superficial punctate keratitis (hereinafter, SPK) were induced (Non-Patent Document 3: Investigative Ophthalmology & Visual Science (2012), vol. 53, No. 13, p. 8280-8287). PACAP27 and its stabilized peptide were instilled into the eyes of the rats, and the inhibitory effect on corneal injury was examined. The specific experimental method was carried out as follows.

[0072] [Capsaicin administration] Capsaicin (manufactured by SIGMA) was dissolved in PBS containing 10% EtOH and 10% Tween 80 and administered subcutaneously to the back of 4-day-old male Wistar / ST rats at a dose of 50 mg / kg·b.w.

[0073] [Preparation of eye drops] The base was prepared by dissolving 0.3 g of tris(hydroxymethyl)aminomethane (manufactured by Nacalai Tesque) and 4.4 g of D(-)-mannitol (manufactured by Nacalai Tesque) in 100 mL of sterilized water, and adding 1N·HCl to adjust the pH to 7. Peptide 1 and Peptide 9 were dissolved in the base to a concentration of 0.1%. Each eye drop solution was stored at room temperature. Eye drops were started 3 weeks after capsaicin administration, and 10 μL of each solution was instilled into both eyes 3 times a day, every day.

[0074] [Measurement of Tear Volume] The tear volume was measured by inserting Zone Quick (manufactured by Ayumi Pharmaceutical) into the lower outer canthus of the rat. After 20 seconds, the length of the reddish-stained area was measured using the attached scale. The tear volume was measured 10 minutes and 5 minutes before instilling the peptide, and the average value was taken as the tear volume before instillation. After instilling the peptide, the tear volume was measured 5 minutes later, and the difference in tear volume from before instillation was determined. The results are shown in Figure 3.

[0075] In rats induced with neurotrophic keratitis, instillation of Peptide 1 and Peptide 9 showed similar tear secretion effects.

[0076] [Observation of SPK] SPK was evaluated 2 weeks after the start of instillation. Under isoflurane inhalation anesthesia, a 1 μL aqueous solution of 1% sodium fluorescein (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in PBS was instilled onto the eye surface. After forced eye closure for 1 minute, the excess fluorescein staining solution was rinsed off with physiological saline. Then, the eye surface was observed with a slit lamp microscope, and the degree of corneal damage was evaluated according to the method of Murakami et al. in Ophthalmology 21(1):87 - 90 (2004). Specifically, the cornea was divided into three regions: upper, central, and lower. Each region was scored according to the following criteria, and the sum of the scores for the three regions was taken as the SPK score. <Criteria> 0: No punctate staining, 1: Sparse (punctate fluorescein staining is separated), 2: Intermediate (between 1 and 3), 3: Dense (almost all punctate fluorescein stainings are adjacent). The results are shown in Figure 4.

[0077] Instillation of Peptide 1 and Peptide 9 improved the increase in SPK score caused by capsaicin administration.

[0078] [Measurement of corneal sensation] Corneal sensation is measured using a Cochet - Bonnet corneal esthesiometer (Hatta - ya Shoten). The filament length of the corneal esthesiometer is set to 60 mm, and the filament is vertically contacted and stimulated at the center of the cornea. Observe the eyelid reaction. If there is a reaction, record the filament length. When no reaction is observed, measure by shortening the length by 5 mm each time. Repeat the above operation 3 times, and take the average value of the obtained filament lengths as the corneal sensation threshold (CST) of that individual.

[0079] Instillation of Peptide 1 and Peptide 9 improves the decrease in corneal sensation caused by capsaicin administration.

[0080] [Corneal nerve staining] After euthanizing the rats, enucleate the eyeballs, immerse them in Zamboni's solution (manufactured by Wako Pure Chemical Industries, Ltd.), and let them stand at room temperature for 15 minutes. Collect the corneas from the eyeballs, immerse them in Zamboni's solution again, and let them stand at room temperature for 45 minutes. Then, replace them with 0.1 M phosphate buffer containing 30% sucrose. After confirming the sedimentation of the corneal tissue, insert a cut with scissors and immerse it in 0.1 M phosphate buffer (pH 5.3) containing 0.1% EDTA and 0.01% type IV - S hyaluronidase (manufactured by Sigma), and let it stand at 37°C overnight. After washing with PBS - T containing 0.3% Triton X - 100, immerse it in PBS - T containing 1% BSA and block at room temperature for 2 hours. React with Alexa Fluor 488 - conjugated mouse anti - β - tubulin, ClassIII antibody (manufactured by BD) at room temperature overnight. After washing with PBS - T, attach the corneal tissue to a slide glass and mount it with Vectashield hard set mounting medium. Observe the enclosed samples with a confocal microscope and acquire images of the subepithelial nerve plexus of the cornea. The nerve density of the spiral structure of the nerve layer is quantified by performing Shohl analysis with image analysis software (Image J).

[0081] Instillation of Peptide 1 and Peptide 9 improves the decrease in nerve density caused by capsaicin administration.

[0082] Example 10: Evaluation of the axonal outgrowth effect of PACAP27 and its stabilized peptides on rat trigeminal neurons [Isolation and culture of trigeminal nerve] Rats (Slc: Sprague-Dawley, 17 days old, including both males and females) purchased from Japan SLC, Inc. 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 then treated with collagenase A (Roche) at 37 °C for 30 minutes. Then, a neuronal cell dispersion solution (Wako) was used according to the protocol to disperse the cells. Furthermore, Debris removal solution (Miltenyi biotec) and Myelin removal beads II (Miltenyi biotec) were used according to the protocol to remove debris and myelin. The isolated trigeminal neurons were suspended in the culture medium and seeded onto polylysine / laminin-coated 8-well chamber slides (Corning) at a density of 2.5×103 cells / well. The culture medium used was Neurobasal-A containing B27 supplement (final concentration 2%), GlutaMAX (final concentration 2 mM), and penicillin / streptomycin (final concentration 1%). The culture conditions were a carbon dioxide concentration of 5%, an air concentration of 95%, a humidity of 100%, and a temperature of 37 °C. After seeding, the cells were cultured for 24 hours and then changed to a culture medium containing peptide 1, peptide 9, peptide 12, peptide 18, and peptide 31 to a final concentration of 1 μM. The control was changed to a culture medium without peptides. After the medium change, the cells were cultured for an additional 24 hours.

[0083] 2. Staining The culture solution containing the peptide was replaced with a medium, and rat trigeminal neurons after 24 hours of culture were immersed in 2% paraformaldehyde solution at room temperature for 20 minutes for fixation. The cells were washed with PBS, 2% bovine serum albumin containing 0.1% TritonX-100 was added, and the reaction was carried out for 30 minutes. After washing with PBS, the phospho-neurofilament H antibody (Merk, MAB1592-C) that specifically recognizes neurofilaments constituting the neuronal cell bodies and neurites was reacted at room temperature for 1 hour. After reacting for 1 hour, a fluorescently labeled secondary antibody (Invitrogen, #A-11031) was reacted at room temperature for 1 hour to fluorescently stain the specimen, and the stained cells were observed under a fluorescence microscope. The stained cell images were captured from the fluorescence microscope into the computer as images.

[0084] 3. Image analysis To evaluate the degree of neurite formation of rat trigeminal neurons, in the images of stained cells captured by the computer, cells having neurites with a length of more than twice the cell body diameter were defined as neurite-forming cells, and the ratio (%) of the number of these 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 Fig. 5 (N = 8). The significant difference from the control was determined by Dunnett's test (P < 0.001).

[0085] Peptide 1, Peptide 9, Peptide 12, Peptide 18, and Peptide 31 showed an effect of promoting axonal elongation of trigeminal neurons.

[0086] Formulation Example The medicament containing the peptide of the present invention as an active ingredient can be produced, for example, by the following formulations. The medicament of the present invention will be further specifically described by giving formulation examples, but the present invention is not limited only to these formulation examples.

[0087] 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 Mix the total amounts of (1), (2), (3), and half of (4), then granulate. Add the remaining (4) to this and encapsulate the whole in gelatin capsules.

[0088] 2. Tablet (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 Mix the total amounts of (1), (2), (3), two-thirds of (4), and half of (5), then granulate. Add the remaining (4) and (5) to this granule and press into tablets.

[0089] 3. Vitreous injection In 1 ml (1) Peptide 5 40 mg (2) Refined sugar 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 injection.

[0090] 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 (1) to (4) are dissolved in (5) sterilized purified water, the pH is adjusted, and an eye drop solution is prepared.

Claims

【Claim 1】 The following: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 1), or HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 2) A pharmaceutical composition for preventing or treating neurotrophic keratitis, comprising a peptide consisting of the represented sequence or a stabilized sequence thereof.

Images