WO2011040564A1 - Ophthalmic agent comprising specific peptide compound as active ingredient - Google Patents

Abstract

The purpose of the present invention is to produce a novel substance capable of promoting tear secretion and provide an ophthalmic agent comprising the same as the active ingredient. Disclosed are an ophthalmic agent, which comprises at least one member selected from the group consisting of peptide compounds (a) to (c) and salts thereof, and so on: (a) R1-Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R2; (b) R1-Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R2; and (c) R1-Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R2. In formulae (a) to (c), R1's are the same or different and represent a hydrogen atom, a straight-chain or branched C10-22 alkanoyl group or a cell membrane-permeable peptide; and R2's are the same or different and represent an -OH group or an -NH2 group. The aforesaid ophthalmic agent can promote the secretion of a large amount of tear within a short period of time. Therefore, said ophthalmic agent can exhibit excellent effects of preventing and/or treating dry eye syndrome, treating corneal epithelial disorder caused by tear reduction, and so on.

Description

Ophthalmic agent containing a specific peptide compound as an active ingredient

The present invention relates to an ophthalmic agent, and more particularly to an ophthalmic agent containing a specific peptide compound as an active ingredient.

Tear fluid is a thin liquid layer having a thickness of about 7 μm that exists at the boundary between the eyeball and the atmosphere and covers the outermost layer of the eyeball. Tear fluid has a three-layer structure consisting of an oil layer, an aqueous layer, and a mucin layer from the outside, and each layer plays an important role in preventing dryness of the eyeball. The aqueous layer occupying most of the thickness of the tears is present between the oil layer and the mucin layer, thereby preventing the aqueous layer from decreasing and maintaining the wettability of the eyeball. Tear fluid has various functions as well as prevention of dry eye. Other functions of tears include, for example, protection of the cornea and conjunctiva, bacteriostatic action, defense against infection from bacteria, fungi and viruses, supply of oxygen and various nutrients to the cornea, and carbon dioxide and metabolites. Removal, dilution and removal of damaging stimuli in the event of damage to the cornea and conjunctiva, transport of humoral components such as epidermal growth factor and blood components such as fibronectin involved in wound healing to damaged sites, cornea and conjunctiva Such as epithelial cell retention and wound healing regulation.

A state in which an abnormality has occurred on the keratoconjunctival surface due to a lacrimal secretion disorder is generally called dry eye. When corneal and conjunctival disorders occur due to dry eye, supplementation of artificial tears, instillation of highly moisturizing viscoelastic substances such as hyaluronic acid, use of eyeglasses for dry eye to keep the eye surface moist and reduce dry symptoms Etc. have been treated. However, these therapies are coping therapy and can relieve symptoms, but they are not causal therapies for radical treatment. As mentioned earlier, tears are thought to have an effect of healing keratoconjunctival damage caused by dry eye due to its original function. Therefore, creating a substance that acts directly on the lacrimal gland and promotes lacrimal secretion. Is expected to be a useful prophylactic and therapeutic drug for dry eye and diseases associated with dry eye. For this reason, research and development of substances that promote lacrimation are performed and various findings have been obtained.

For example, carbachol is known as a substance that promotes lacrimation. However, carbachol also causes miosis and is difficult to use frequently. Further, phenylephrine is known as a substance that promotes the secretion of peroxidase, which is a tear fluid protein, by tear fluid asinner cells. However, phenylephrine has an anesthetic action and causes mydriasis when instilled. In fact, phenylephrine is used as a mydriatic. For this reason, it is difficult to use frequently.

Also, a method of promoting tear secretion using a peptide has been studied. For example, Patent Documents 1 and 2 describe that a specific peptide is applied to the eye to promote tear secretion.

JP 2000-169387 A JP 2005-272445 A

An object of the present invention is to create a novel lacrimal secretion promoting substance and to provide an ophthalmic agent containing this as an active ingredient.

The present inventors have surprisingly found that a specific peptide can promote lacrimation. In addition, the peptide secretion in which an alkanoyl group is bonded to the N-terminus of the specific peptide, a liposome containing the specific peptide, or the like can exhibit the lacrimal secretion effect of the specific peptide even more effectively. The headline and further improvements were repeated to complete the present invention.

That is, the present invention includes, for example, the subject matters described in the following sections.
Item 1-1. An ophthalmic agent comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
In the peptide compounds (a), (b), and (c), the amino acid sequences of the portions excluding R ¹ are the sequences described in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. is there.
Item 1-2. The ophthalmic agent according to Item 1-1, which is used for promoting lacrimal secretion.
Item 1-3. The ophthalmic agent according to Item 1-1, which is used for improving at least one symptom selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis.
Item 1-4. The ophthalmic preparation according to any one of Items 1-1 to 1-3, which is an eye drop, an eye ointment, an eye wash, or a contact lens composition.
Item 1-5. Item 5. The ophthalmic agent according to any one of Items 1-1 to 1-4, which is a liposome preparation. (In other words, the ophthalmic agent according to Item 1-1 to 1-4, comprising a liposome comprising at least one selected from the group consisting of the peptide compounds (a) to (c) and salts thereof) The liposome preparation can be rephrased as in the following items 2-1 to 2-4.
Item 2-1. An ophthalmic agent comprising a liposome comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 2-2. Item 2. The ophthalmic agent according to Item 2-1, which is used for promoting lacrimal secretion.
Item 2-3. Item 2. The ophthalmic agent according to Item 2-1, which is used for improving at least one symptom selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis.
Item 2-4. The ophthalmic agent according to any one of Items 2-1 to 2-3, which is an eye drop, an eye ointment, an eye wash, or a contact lens composition.
Item 3-1. A method for promoting lacrimal secretion, comprising a step of applying an ophthalmic agent comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof to the eyes of a mammal.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 3-2. Item 3. The method according to Item 3-1, comprising a step of applying an effective amount of an ophthalmic agent comprising at least one selected from the group consisting of the peptide compounds of (a) to (c) and salts thereof to the eyes of a mammal. For promoting lacrimal secretion.
Item 3-3. Item 3. The method for promoting lacrimal secretion according to Item 3-1 or 3-2, wherein the mammal has a lacrimal secretion disorder.
Item 3-4. Any of paragraphs 3-1 to 3-3, wherein the mammal has at least one symptom selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis A method for promoting lacrimal secretion according to claim 1.
Item 3-5. Item 5. The method for promoting lacrimal secretion according to any one of Items 3-1 to 3-4, wherein the ophthalmic agent is an eye drop, an eye ointment, an eye wash, or a composition for contact lenses.
Item 3-6. Item 6. The method for promoting lacrimal secretion according to any one of Items 3-1 to 3-5, wherein the ophthalmic agent is a liposome preparation. (In other words, the ophthalmic agent contains a liposome containing at least one selected from the group consisting of the peptide compounds (a) to (c) and salts thereof) The method for promoting lacrimal secretion according to the description.)
Item 4-1. A method for preventing or treating dry eye, comprising a step of applying an ophthalmic agent comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof to the eyes of a mammal.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 4-2. Item 4-1 includes a step of applying an effective amount of an ophthalmic agent comprising at least one selected from the group consisting of the peptide compounds of (a) to (c) and salts thereof to the eyes of a mammal. Dry eye prevention or treatment method.
Item 4-3. Item 3. The dry eye prevention or treatment method according to Item 4-1, wherein the mammal has a tear secretion disorder.
Item 4-4. Any of paragraphs 4-1 to 4-3, wherein the mammal has at least one symptom selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis The dry eye prevention or treatment method according to 1.
Item 4-5. Item 5. The dry eye prevention or treatment method according to any one of Items 4-1 to 4-4, wherein the ophthalmic agent is an eye drop, an eye ointment, an eye wash, or a composition for contact lenses.
Item 4-6. Item 6. The dry eye prevention or treatment method according to any one of Items 4-1 to 4-5, wherein the ophthalmic agent is a liposome preparation. (In other words, the ophthalmic agent comprises a liposome containing at least one selected from the group consisting of the peptide compounds (a) to (c) and salts thereof) The dry eye prevention or treatment method as described.)
Item 5-1. Use of at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof for the production of an ophthalmic agent.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 5-2. Item 5. The use according to Item 5-1, wherein the ophthalmic agent is an eye drop, an eye ointment, an eye wash, or a contact lens composition.
Item 5-3. Item 5. The use according to Item 5-1 or 5-2, wherein the ophthalmic agent is used for treatment or amelioration of lacrimal secretion disorder.
Item 5-4. Item 5. The use according to any one of Items 5-1 to 5-3, wherein the ophthalmic agent is a liposome preparation.
Item 6-1. At least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof for use in treating or improving tear secretion disorders.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 6-2. Item 6. The item according to Item 6-1, wherein the tear secretion disorder is a disorder caused by at least one selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis. at least one selected from the group consisting of peptide compounds of a) to (c) and salts thereof.
Item 6-3. A liposome comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof for use in treating or improving tear secretion disorders.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 6-4. Item 6. The liposome according to item 6-3, wherein the tear secretion disorder is a disorder caused by at least one selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis. .
Item 7-1. At least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof for use in the treatment or amelioration of dry eye.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 7-2. (A) The dry eye is dry eye caused by at least one selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis (a ) To (c) at least one selected from the group consisting of peptide compounds and salts thereof.
Item 7-3. A liposome comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof for use in the treatment or improvement of dry eye.
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]
Item 7-4. Item 4. The liposome according to Item 7-3, wherein the dry eye is dry eye caused by at least one selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis.

The ophthalmic preparation of the present invention can provide a very excellent tear secretion promoting effect. Specifically, the ophthalmic agent of the present invention has a stronger lacrimal secretion action than a known lacrimal secretion promoter, and can promote lacrimation in a short time. For this reason, the prophylactic and / or therapeutic effect of dry eye syndrome, the therapeutic effect of corneal epithelial injury caused by tear reduction, etc. can also be obtained.

It is the figure which showed the SDS-PAGE image which detected the tear protein (60 minutes after addition) secreted from the rat lacrimal gland assiner cell by mSIRK. From the leftmost side of the lane, a group to which a molecular weight marker, 0 μM mSIRK (control), 0.1 μM mSIRK, 1 μM mSIRK and 10 μM mSIRK was added is shown. It is the figure which showed the lacrimal protein secretion promotion effect (10 minutes after addition) from a rat lacrimal gland asiner cell by mSIRK and mSCAR by the peroxidase activity value. FIG. 4 is a diagram (a) showing the effect of mSIRK and mSIRK (N7A) on lacrimal protein secretion from rat lacrimal gland asiner cells (10 minutes after addition) by peroxidase activity value and a diagram (b) showing albumin secretion. The test results for the control are also shown. It is the figure which compared the tear protein secretion promotion effect of mSIRK and mSCAR, and the tear protein secretion promotion effect (after 90 minutes of addition) of phenylephrine by the peroxidase activity value. The test results for the control are also shown. It is the figure which showed the lacrimal protein secretion promotion effect from rat lacrimal gland asiner cell by mSIRK, mSIRK (N7A) and phenylephrine (30 minutes, 60 minutes and 90 minutes after addition) by peroxidase activity value. It is the figure which compared the tear protein secretion promotion effect of mSIRK, mSIRK (N7A) and mSCAR, and the tear protein secretion promotion effect (30 minutes after addition) of carbachol by the peroxidase activity value. It is the figure which showed the SDS-PAGE image which detected tears protein (20 minutes after addition) secreted from the rabbit lacrimal gland asiner cell by mSIRK, mSIRK (L9A), or carbachol. In the figure, “L9A” indicates mSIRK (L9A). The compounds added to the cells when preparing the sample applied to each lane are as follows (where M indicates that a molecular weight marker was migrated). M: Molecular weight marker (bands from the top 250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa, 25 kDa, 20 kDa, 15 kDa, 10 kDa) 1: DMSO, 2: mSIRK 0.1 μM, 3: mSIRK 1 μM, 4: mSIRK 10 μM, 5: mSIRK (L9A) 0.1 μM, 6: mSIRK (L9A1) μM, 7: mSIRK (L9A) 10 μM, 8: Carbachol 1 μM, 9: Carbachol 10 μM, 10: Carbachol 100 μM The upper figure is the figure which detected the lipocalin secreted from the rabbit lacrimal gland assiner cell by the immunoblot. The figure below compares the amount of lipocalin secreted from rabbit lacrimal gland asinator cells under each condition from the density of the band detected by immunoblotting. The meanings of sample numbers 1 to 7 in the figure are the same as those described in the explanation of FIG. 7a. It is the figure which detected the various tear fluid proteins secreted from the monkey lacrimal gland asinator cell by the immunoblot. The chromatogram at the time of measuring a known amount of mSIRK by a high performance liquid chromatography (HPLC) is shown. The arrow indicates a peak derived from mSIRK. “a” to “d” show the results of 50 μL injection of mSIRK having the following concentrations, respectively. a: mSIRK 0.05 mg / mL, b: mSIRK 0.025 mg / mL, c: mSIRK 0.005 mg / mL, d: mSIRK 0.0025 mg / mL. A calibration curve created from the chromatogram results shown in FIG. 9 and Table 1 is shown. When the liposome suspension containing the liposome containing mSIRK is centrifuged to separate the liposome fraction and the supernatant fraction, and mSIRK contained in each fraction is detected by high performance liquid chromatography. A chromatogram is shown. a shows the result when 50 μL of the liposome fraction (diluted 10 times) was injected, and b shows the result when 50 μL of the supernatant fraction (diluted 5 times) was injected. The arrow indicates a peak derived from mSIRK.

Hereinafter, the present invention will be described in more detail. In the present specification, amino acids may be represented by one letter or three letters. The three-letter code and the one-character code corresponding to each amino acid are described below in the order of “one-character code: three-character code: amino acid name”.
A: Ala: Alanine V: Val: Valine L: Leu: Leucine I: Ile: Isoleucine P: Pro: Proline F: Phe: Phenylalanine W: Trp: Tryptophan M: Met: Methionine G: Gly: Glycine S: Ser : Serine T: Thr: Threonine C: Cys: Cysteine Q: Gln: Glutamine N: Asn: Asparagine Y: Tyr: Tyrosine K: Lys: Lysine R: Arg: Arginine H: His: Histidine D: Asp: Aspartic Acid E: Glu: Glutamic acid The ophthalmic agent of the present invention contains at least one of the following peptide compounds (a) to (c) or a salt thereof (that is, the following peptide compounds (a) to (c) and At least one selected from the group consisting of the salts).
(a) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(b) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(c) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²

In the peptide compounds (a) to (c) above, R ¹ is the same or different and represents a hydrogen atom, an alkanoyl group, or a cell penetrating peptide (CPP). In general, peptides are often hydrophilic and difficult to permeate cell membranes. However, a technique for improving the cell permeability of many peptides (peptide cell membrane permeability improving technique) is already known. As such a technique, for example, a technique for containing a peptide in a liposome, which will be described later, is mentioned. The peptide contained in the liposome can be efficiently transported into the cell. Even if the peptide membrane permeability of the peptide compound is not good, the effect of the present invention can be exhibited more efficiently by using a known peptide cell membrane permeability improving technique. In particular, when R ¹ is an alkanoyl group or a cell membrane-penetrating peptide, the peptide compounds (a) to (c) can easily penetrate the cell membrane. That is, the peptide compounds (a) to (c) can be efficiently permeated through the cell membrane by using a known peptide cell membrane permeability improving technique, or when R ¹ is an alkanoyl group or a cell membrane permeable peptide. It becomes possible to enter inside. Although not particularly limited, R ¹ is more preferably an alkanoyl group. Further, even when R ¹ is an alkanoyl group or a cell membrane-permeable peptide, a known peptide cell membrane permeability improving technique may be used in combination.

When R ¹ is an alkanoyl group, since the alkanoyl group is hydrophobic, the peptide compounds (a) to (c) have an affinity for the cell membrane at the R ¹ part. Therefore, the peptide compounds (a) to (c) can efficiently permeate the cell membrane. (A) As long as the peptide compounds of ~ (c) has a cell membrane permeability, but alkanoyl group represented by R ¹ is particularly limited, usually R ¹ are the same or different, straight-chain or branched having 10 to 22 carbon atoms A chain alkanoyl group is shown. The number of carbon atoms of the alkanoyl group is preferably 10 to 20, more preferably 10 to 18, and still more preferably 12 to 18. The alkanoyl group preferably has 0 to 3 double bonds, more preferably 0, 1 or 2. The alkanoyl group is preferably linear. In particular, the alkanoyl group is preferably a saturated or unsaturated fatty acid residue having the above carbon number. Further, in the group represented by —CO— (CH ₂ ) _n —CH ₃ (n represents a natural number), a group of n = 8 to 18 is preferable, a group of n = 8 to 16 is more preferable, and n = More preferred are 10 to 16 groups.

Specifically, for example, a decanoyl group, an undecanoyl group, a lauroyl group, a tridecanoyl group, a myristoyl group, a palmitoyl group, a stearoyl group, an oleoyl group, an icosanoyl group, an eicosanoyl group, a docosanoyl group, and the like are preferable.

Also when R ¹ is a cell penetrating peptide, as long as the cell membrane penetrating peptides represented by R ¹ is not particularly limited, R ¹ are the same or different with a peptide compound cell membrane permeability of (a) ~ (c), known Cell membrane permeation peptides are shown. Although not particularly limited, examples of suitable known cell membrane-penetrating peptides include HIV-1 Tat peptide or (Arg) _n . The HIV-1 Tat peptide is a basic peptide derived from HIV-1 Tat, in particular HIV-1 Tat _49-57 peptide (RKKRRQRRR; SEQ ID NO: 8) or HIV-1 Tat _48-60 peptide (GRKKRRQRRRRPPQ; SEQ ID NO: 9 ) Is preferred. In addition, (Arg) _n represents a peptide in which n arginines are continuously bonded, and n is preferably 6, 7, 8, 9, or 10, and more preferably 8 or 9.
In addition, for example, peptides described in Table 2 of Trends in Pharmaceutical Sciences, Volume 21, Issue 3, 1 March 2000, Pages 99-103 can also be used as suitable cell membrane-penetrating peptides.

Furthermore, the three types of peptides (I) to (III) described below can be used as particularly preferred cell membrane-penetrating peptides.
(I) Gly-Gly-Gly-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly (SEQ ID NO: 4)
(II) Ac-Arg-Lys-Lys-Arg-Arg-Xaa-Arg-Arg-Arg (SEQ ID NO: 5)
(III) Ac-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 6)
In the peptides (II) and (III), Ac represents an acetyl group. In peptide (II), Xaa represents ornithine.

In the cell membrane-penetrating peptide, each C-terminal amino acid is preferably peptide-bonded with a serine residue to become peptide compounds (a) to (c). That is, when R ¹ is a cell membrane-penetrating peptide, the C-terminal carboxyl group of the cell membrane-penetrating peptide and the amino group of the serine residue form the “R ¹ -Ser” bond of the peptide compounds (a) to (c) above. It is preferable.

In the peptide compounds (a) to (c), R ² is the same or different and represents an —OH group or —NH ₂ group.

R ¹ is bonded to the amino group at the N-terminal of the peptide (ie, 1 hydrogen atom of the amino group at the N-terminal of the peptide is substituted with R ¹ ), and the N-terminal of the peptide is —NH—R ¹ can be labeled. When R ¹ is a hydrogen atom, the N-terminus of the peptide is an amino group (—NH ₂ ). R ² is bonded to the C-terminal carboxyl group of the peptide (ie, —OH of the C-terminal carboxyl group of the peptide is substituted with R ² ), and the C-terminal of the peptide is —CO—R ² Can be labeled. When R ² is an —OH group, the C-terminus of the peptide is a carboxyl group (—COOH).

When R ¹ is a hydrogen atom and R ² is an —OH group, the above peptide compounds (a) to (c) correspond to the following peptides (A) to (C).
(A) Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (SEQ ID NO: 1)
(B) Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (SEQ ID NO: 2)
(C) Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro (SEQ ID NO: 3)

In the present invention, particularly preferred peptide compounds include the following peptide compounds (i) to (iii). In the following, amino acids constituting these peptide compounds are represented by one letter.
(I) mSIRK: myr-SIRKALNILGYPDYD-OH
(Ii) mSCAR: myr-SCARFFGTPCP-NH ₂
(Iii) mSIRK (N7A): myr-SIRKALAILGYPDYD-OH
mSIRK, mSCAR, and mSIRK (N7A) represent the names of these peptide compounds, respectively. “Myr-” indicates that the N-terminus of the peptide is myristoylated. That is, “myr-” represents “—CO— (CH ₂ ) ₁₂ —CH ₃ ” (myristoyl group).

mSIRK is a peptide compound in which the amino group of serine (S) is myristoylated in the peptide represented by SIRKALNILYPYPDY (SEQ ID NO: 1) in terms of one amino acid. That is, it has a structure (—NH-myr) in which the hydrogen atom of the amino group of serine of the peptide is substituted with a myristoyl group. mSIRK corresponds to a compound in which R ¹ is a myristoyl group and R ² is an —OH group in the peptide compound of (a).

mSCAR is a peptide represented by SCARFFGTCPCP (SEQ ID NO: 3) in amino acid one letter code, wherein the amino group of serine (S) is myristoylated and the carboxyl group of proline (P) at the C-terminal is amidated. It is a peptide compound. That is, it has a structure (—CO—NH ₂ ) in which the hydrogen atom of the amino group of serine of the peptide is substituted with a myristoyl group (—NH-myr) and the carboxyl group of proline at the C-terminal is amidated. mSCAR corresponds to a compound in which R ¹ is a myristoyl group and R ² is a —NH ₂ group in the peptide compound of (c).

mSIRK (N7A) is a peptide compound in which asparagine, the seventh amino acid of mSIRK, is substituted with alanine. mSIRK (N7A) corresponds to the compound (b) in which R ¹ is a myristoyl group and R ² is an —OH group.

In the peptide compounds (a) to (c) above, the various amino acids constituting these peptide compounds (when R ¹ is a cell membrane permeation peptide, including all amino acids constituting the cell membrane permeation peptide) are usually all L-forms. However, as long as the effect of the present invention is not impaired, D-form may be included. When D-form is included, in each peptide compound of (a) to (c), preferably all amino acids are D-form, more preferably 1 to 3 amino acids are D-form, more preferably 1 ~ 2 amino acids are in D form. The D-form amino acid is preferably contained in the protease recognition sequence. This is because resistance to cleavage by protease can be imparted.

Although not particularly limited, when R ¹ is a peptide represented by (II) or (III) above, all of the amino acids constituting the peptide represented by (II) or (III) are D-forms. Is preferred. It is within the technical scope of those skilled in the art to appropriately select and use such cell membrane permeation peptides for transferring peptide compounds into cells.

In addition, in the amino acid sequences of the peptide compounds (a) to (c) above (in the case where R ¹ is a cell membrane permeable peptide, including the amino acid sequence constituting the cell membrane permeable peptide), unless the effects of the present invention are impaired, 1 to 5 (preferably 1 to 3, more preferably 1 to 2) amino acids may be substituted, deleted or added.

The peptide compounds (a) to (c) are known compounds or can be produced by appropriately selecting an appropriate production method from known methods. For example, it can be synthesized by a solid phase synthesis method or a liquid phase synthesis method. For example, in the solid-phase synthesis method, the carboxyl group of an N-protected amino acid obtained by protecting the amino group of the amino acid most corresponding to the C-terminus with a protecting group such as a 9-fluorenylmethyloxycarbonyl (Fmoc) group is used as the amino group. After binding to the insoluble resin, the amino protecting group is removed, and the protected amino acid is condensed sequentially in the N-terminal direction, and then the insoluble resin and the amino acid protecting group are deprotected. ) Peptide compounds can be obtained. When R ¹ is an alkanoyl group, it can be obtained by condensing a carboxylic acid having a R ¹ —OH structure or a salt thereof with a peptide by a known method. The carboxylic acid having a structure of R ¹ —OH or a salt thereof is known or can be easily produced by a known method.

In addition, if the amino acids contained are all L-form peptide compounds, the peptide portion of the peptide compounds can be obtained by genetic engineering techniques. Thereafter, the alkanoyl group corresponding to R ¹ and the —NH ₂ group corresponding to R ² can be bonded by a known organic chemical method (for example, dehydration condensation reaction). Furthermore, synthesis can be ordered from a peptide synthesis company, and a peptide compound corresponding to (a) in which R ¹ is a myristoyl group and R ² is an —OH group is commercially available from Calbiochem. It can also be purchased. In addition, you may refine | purify the various peptide compounds obtained in this way by a well-known method (for example, high performance liquid chromatography (HPLC)).
The peptide compounds (a) to (c) can also be produced by placing an order with a company that has received an order for peptide compound synthesis. For example, you can place an order with Biologica Co., Ltd.

The ophthalmic preparation of the present invention may contain salts of the peptide compounds (a) to (c) as active ingredients, and the salts are preferably pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include acid addition salts (organic acid salts / inorganic acid salts), metal salts, ammonium salts, organic acid amine addition salts, and the like. Examples of the organic acid salt include acetate, maleate, fumarate, tartrate, and citrate; examples of the inorganic acid salt include hydrochloride, sulfate, nitrate, phosphate, and the like; As the metal salt, for example, lithium salt, sodium salt, potassium salt, magnesium salt, calcium salt, aluminum salt, zinc salt and the like can be preferably used, but not limited thereto. In addition, the salt of the said peptide compound can be used 1 type or in combination of 2 or more types.

That is, the ophthalmic agent of the present invention contains at least one of the peptide compounds (a) to (c) or a salt thereof as an active ingredient. In other words, the ophthalmic agent of the present invention contains at least one compound selected from the group consisting of the peptide compounds (a) to (c) and salts thereof as an active ingredient.

The ophthalmic preparation of the present invention may be in the form of a liquid, gel, ointment, etc., and is preferably liquid. The ophthalmic agent of the present invention is an ophthalmic pharmaceutical composition comprising the active ingredient and a pharmaceutically acceptable base. As the substrate, for example, pharmaceutically acceptable (preferably ophthalmically acceptable) water (for example, purified water, ultrapure water, etc.) can be preferably used.

In addition to the above active ingredients, other optional ingredients may be added to the ophthalmic preparation of the present invention as necessary. For example, pharmaceutically acceptable additives such as cooling agents, tonicity agents, pH regulators, buffers, thickeners, antioxidants, emulsifiers, and preservatives can be added as necessary. . Specific examples of such optional components include, but are not limited to, the following.

Examples of the refreshing agent include l-menthol, camphor, borneol and the like.

Examples of the isotonic agent include sodium chloride, boric acid, sodium nitrate, potassium nitrate, D-mannitol, glucose and the like.

Examples of the pH regulator include boric acid, anhydrous sodium sulfite, hydrochloric acid, citric acid, sodium citrate, acetic acid, potassium acetate, sodium carbonate, borax, sodium hydroxide and the like.

Examples of the buffer include ascorbic acid, benzoic acid, ammonium chloride, potassium chloride, sodium chloride, citric acid, acetic acid, tartaric acid, maleic acid, sodium hydrogen phosphate, boric acid and the like.

Examples of the thickener include methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, and the like.

Examples of the antioxidant include ascorbic acid, cysteine hydrochloride, tocopherol, sodium edetate and the like.

Examples of the emulsifier include polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyoxy hydrogenated castor oil, and the like.

Examples of the preservative include benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylethyl alcohol, dehydroacetic acid, paraoxybenzoic acid esters, and the like.

Furthermore, in addition to the above active ingredients, other active ingredients (pharmacologically active substances) usually used for ophthalmic medicines may be added to the ophthalmic preparations of the present invention. As such an active ingredient, for example,
Bactericides such as acrinol, cetylpyridinium, benzalkonium, benzethonium, chlorhexidine, sulfamethoxazole,
Anti-inflammatory agents such as epsilon aminocaproic acid, glycyrrhizic acid, glycyrrhetinic acid, allantoin, berberine, methyl salicylate, glycol salicylate, azulene, azulene sulfonic acid, guaiazulene, tranexamic acid, lysozyme, licorice, epinephrine, ephedrine, naphazoline, tetrahydrozoline, oxymetalin Vasoconstrictors such as zolin, phenylephrine, methylephedrine,
Examples include vitamins such as pyridoxine hydrochloride, riboflavin phosphate, cyanocobalamin, panthenol, flavin adenine dinucleotide sodium, d-α tocopherol acetate, pin regulators such as neostigmine methyl sulfate, antihistamines such as iproheptin, diphenhydramine, chlorpheniramine, etc. be able to.

In addition, such arbitrary components and other active ingredients can be used alone or in combination of two or more. Such optional ingredients and other active ingredients can be appropriately selected by those skilled in the art according to the purpose, and conditions such as the amount added can also be appropriately set.

The usage form of the ophthalmic preparation of the present invention is not particularly limited. For example, eye drops, eye ointments, eye washes, contact lens compositions (contact lens mounting solutions, cleaning solutions, preservatives; eye drops when wearing contact lenses) or other agents that are applied directly or indirectly to the eye Can be preferably used.

The ophthalmic preparation of the present invention can be prepared according to a known method to the above-mentioned usage form. For example, in an aqueous solvent such as purified water and physiological saline, at least one compound selected from the group consisting of the peptide compounds (a) to (c) or salts thereof, and optional components and An aqueous ophthalmic preparation can be prepared by adding other active ingredients to a desired concentration. As a preferred embodiment, an aqueous ophthalmic agent containing 20% by mass or more, preferably 50% by mass or more, more preferably 80% by mass or more, and further preferably 90% by mass or more is desirable. In addition, for example, it can be produced according to the method described in the section “General eye drops” of the 15th revised Japanese Pharmacopoeia.

In addition, the ophthalmic agent of the present invention can be in the form of a liposome preparation. For example, at least one of the peptide compounds (a) to (c) above or a salt thereof can be encapsulated in a liposome to form a liposome preparation. In addition, at least one of the peptide compounds (a) to (c) or a salt thereof may be contained in the lipid membrane of the liposome. In particular, in the peptide compounds (a) to (c) or salts thereof, when R ¹ is an alkanoyl group, the alkanoyl group is hydrophobic and has an affinity for the cell membrane, so that the form contained in the liposome membrane A liposomal formulation can be prepared. In this case, the alkanoyl group is embedded in the lipid constituting the liposome membrane, and the peptide portion has a form protruding from the liposome membrane to the outside or inside of the liposome. It is thought that.

In this specification, “included in liposome” includes the case of being included in the liposome and the case of being included in the liposome membrane. The liposome preparation of the present invention can be said to be an agent comprising at least one peptide compound of any of the above (a) to (c) or a salt thereof contained in the liposome.

As described above, the peptide compounds (a) to (c) or salts thereof have an alkanoyl group or a cell membrane-permeable peptide as R ¹ to obtain efficient cell membrane permeability. For this, the peptide compounds (a) to (c) or salts thereof may not necessarily have cell membrane permeability. This is because the substance contained in the liposome can be transported into the cell by fusing the liposome with the cell membrane. Therefore, when the ophthalmic agent of the present invention is a liposome preparation, R ¹ of the peptide compounds (a) to (c) is not particularly limited as long as the effects of the present invention are not impaired. For example, R ¹ may be a hydrogen atom. In addition, R ¹ may be an alkanoyl group (for example, an acetyl group) that does not have cell membrane permeability. Of course, the alkanoyl group which has the above-mentioned cell membrane permeability may be sufficient. It may be a linear or branched alkanoyl group having 1 to 30 carbon atoms. Accordingly, the ophthalmic agent of the present invention is a liposome preparation containing at least one peptide compound of any one of (a) to (c) or a salt thereof, for example, wherein R ¹ is a hydrogen atom, an alkanoyl group or a cell membrane permeable peptide. Is also included.

The liposome preparation as described above can be easily fused with the cell membrane and can transport the peptide compounds (a) to (c) or salts thereof into cells.

Liposomes usually have a lipid bilayer structure, but the structure of the liposome in the liposome preparation of the present invention is not particularly limited, and includes bilayer liposomes, as well as multilamellar liposomes (MLV), small monolayer liposomes (SUV), Large single membrane liposomes (LUV) may be used. The particle size of the liposome is not particularly limited, and an appropriate particle size can be selected and used. For example, a particle size of 100 to 5000 nm can be used. The particle diameter can be determined by a dynamic light scattering method (Dynamic Light Scattering = DLS).

Lipids used for liposome production are not particularly limited. For example, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, glycerophospholipid, sphingomyelin Such as sphingophospholipid, cholesterol, egg yolk lecithin, soybean lecithin and the like. These lipids may be used alone or in combination of two or more. By mixing these lipid compounds (a) to (c) or their salts with these lipids and using them as a liposome production material, liposomes that can be used in the ophthalmic preparation of the present invention can be produced. The mixing ratio of the lipid and the peptide compound is not particularly limited as long as the liposome can be produced, and can be appropriately set.

The method for producing the liposome is not particularly limited, and can be appropriately produced by a known method. For example, a thin film method, reverse layer evaporation method, ethanol injection method, dehydration-rehydration method and the like can be mentioned. Moreover, it is also possible to adjust the particle diameter of the liposome using an ultrasonic irradiation method, an extrusion method, a French press method, a homogenization method, or the like. In addition, the said other active ingredient etc. may be included in the liposome manufacturing material, for example, and the said active ingredient etc. may be included in a liposome.

As an example of a specific production method, a liposome production lipid (for example, soybean lecithin and cholesterol) is dissolved in chloroform, and the above peptide compound (for example, mSIRK) dissolved in an organic solvent (for example, methanol) is added and mixed. An example is a method for producing liposomes by removing a solvent with an evaporator or the like to form a lipid thin film and permeating the thin film with a buffer solution injection.

Using the thus-obtained liposome containing at least one compound selected from the group consisting of the peptide compounds (a) to (c) or salts thereof, and other components as necessary, An ophthalmic agent can be prepared. For example, it can be prepared as described above. For example, it is added to an aqueous solvent such as purified water or physiological saline so as to obtain a desired concentration, and an ophthalmic agent (for example, an eye drop, an eye ointment, an eye wash, etc.) according to a known method (preferably conventional method). Agents, contact lens compositions, etc.) can be prepared.

The amount of the active ingredient ((a) to (c) peptide compound or salt thereof) contained in the ophthalmic preparation of the present invention is not particularly limited. For example, the active ingredient concentration may be about 0.0001 to 100 mg / mL, preferably about 0.0005 to 100 mg / mL. That is, about 0.00001 to 10 w / v%, preferably about 0.00005 to 10 w / v% can be exemplified.

In particular, for example, when the ophthalmic agent is an eye drop, an eye wash, a contact lens composition, or the like, the active ingredient concentration is preferably 0.0001 to 100 mg / mL, more preferably 0.0005 to 100 mg / mL, More preferably, 0.001 to 100 mg / mL, even more preferably 0.005 to 100 mg / mL, still more preferably about 0.01 to 100 mg / mL, and particularly preferably about 0.01 to 10 mg / mL. That is, preferably about 0.00001 to 10 w / v%, more preferably about 0.00005 to 10 w / v%, still more preferably about 0.0001 to 10 w / v%, and still more preferably about 0.0005 to 10 w%. / V%, preferably about 0.001 to 10 w / v%, particularly preferably about 0.001 to 1 w / v%. It may be about 0.1-10 mg / mL (0.01-1 w / v%).

The number of times of use is not particularly limited. For example, about 20 to 50 μL can be instilled once or several times a day (preferably 2 to 8 times, more preferably 2 to 6 times). Also, for example, when the ophthalmic agent is an eye wash or a contact lens composition (particularly a contact lens cleaning solution), the active ingredient concentration is, for example, about 0.01 to 1 times the concentration of the above eye drops. It can be used once or several times a day (preferably 2 to 8 times, more preferably 2 to 6 times) to wash eyes or contact lenses.

In addition, the application amount of the active ingredient (peptide compound of (a) to (c) or a salt thereof) is, for example, 0.00005 mg to 50 mg, preferably 0.0001 mg to 20 mg, more preferably 0.001 mg / day for one adult eye. Examples include 001 mg to 10 mg.

When the active ingredient (the peptide compound of (a) to (c) or a salt thereof) is contained in the liposome (that is, when the ophthalmic agent of the present invention is a liposome preparation), a surfactant (for example, By adding Triton X-100) and destroying the liposome membrane, the amount of the peptide compound contained in the liposome can be quantified by detecting the peptide compound by liquid chromatography. More specifically, for example, the amount of peptide compound can be quantified by the method described in the Examples.

The subject of administration of the ophthalmic preparation of the present invention is not particularly limited, but a subject with reduced lacrimation is preferable. In particular, subjects with tear secretion disorders are preferred. Tear fluid protects the cornea and conjunctiva, bacteriostatic, protects against bacteria, fungi and viruses, supplies oxygen and various nutrients to the cornea, removes carbon dioxide and metabolites, and damages the cornea and conjunctiva Dilution and removal of damaging stimuli when applied, transport of humoral components such as epidermal growth factor and blood components such as fibronectin involved in wound healing to the site of injury, retention of corneal and conjunctival epithelial cells and wound healing It plays various roles such as regulation. Therefore, the ophthalmic agent of the present invention that promotes the secretion of tears can be used for the prevention and treatment of dry eye syndrome. Furthermore, it is particularly effective for treating corneal epithelial disorders caused by tear reduction. Furthermore, the ophthalmic preparation of the present invention is used for subjects suffering from lacrimation, dry eye syndrome (dry eye syndrome), corneal epithelial disorder, Sjogren's syndrome, keratitis and / or conjunctivitis (especially dry keratoconjunctivitis). On the other hand, it can be preferably used as a preventive and / or therapeutic agent or an improving agent. Furthermore, it can be used for replenishing tears when wearing a contact lens. In addition, even those who do not have such symptoms can be administered prophylactically in order to prevent a decrease in tear secretion. Thus, the ophthalmic agent of the present invention is selected, for example, from the group consisting of lacrimal secretion promotion, dry tear syndrome, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis It can be preferably used for the prevention and / or treatment or improvement of at least one symptom. Furthermore, the administration target of the ophthalmic agent of the present invention can be applied not only to humans but also to animals such as domestic animals and pets (for example, dogs, cats, cows, horses, monkeys, sheep, pigs, etc.). .

In producing an ophthalmic agent, it is common to adjust the osmotic pressure with an inorganic salt (for example, sodium chloride), a polyhydric alcohol (for example, glycerin), a saccharide, etc. Ophthalmic agents can also be used to adjust the osmotic pressure using conventional methods. The osmotic pressure ratio is usually about 0.8 to 1.2. The osmotic pressure can be measured by, for example, an osmotic pressure measuring method described in the 15th revised Japanese Pharmacopoeia (a method of measuring the osmolality of a sample using a freezing point depression method).

The pH is not particularly limited as long as it is within the range acceptable for ophthalmic preparations, but is usually about pH 6 to 9, and preferably about 6 to 8. The pH can be adjusted with an appropriate pH adjuster known in the art.

The method for applying the ophthalmic preparation of the present invention to the eye is not particularly limited, and can be appropriately selected according to the form and dosage form of the ophthalmic preparation. For example, the ophthalmic preparation of the present invention can be applied to the eye by instilling, washing, applying to the eye, etc., or by attaching the contact lens to the eye after being attached to the contact lens. Can do. Further, the eye may be instilled with respect to the eye wearing the contact lens.

By applying the ophthalmic preparation of the present invention obtained as described above to the eye, secretion of a large amount of tears can be promoted in a relatively short time. For this reason, it is possible to obtain excellent effects such as a prophylactic and / or therapeutic effect on dry eye syndrome and a therapeutic effect on corneal epithelial injury caused by a decrease in tears.

The present invention also relates to a method for promoting lacrimal secretion including the step of applying the above-mentioned ophthalmic agent to a mammal's eye, and dry eye prevention or including the step of applying the above-mentioned ophthalmic agent to a mammal's eye. Treatment methods are also encompassed. The mammal that is the subject in these methods is as described above. In addition, the above-described contents are also applied to various conditions such as application methods and application amounts in these methods.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the following examples. In the following examination, for basic operations and the like, textbooks well-known in the field (for example, Molecular Cloning: A Laboratory Manual (3 Vol. Set); Cold Spring Harbor Laboratory) may be referred to as appropriate.

Examination Example 1: Examination of tear protein secretion promoting effect of peptide compound <Preparation of peptide compound>
The following four peptide compounds (i) to (iv) were obtained by purchasing commercially available products or consigning synthesis for use in the study. In the following, amino acids constituting these peptide compounds are represented by one letter. “Myr-” indicates that the N-terminus of the peptide is myristoylated. That is, “myr-” represents a myristoyl group.
(I) mSIRK: myr-SIRKALNILGYPDYD-OH
(Ii) mSCAR: myr-SCARFFGTPCP-NH ₂
(Iii) mSIRK (N7A): myr-SIRKALAILGYPDYD-OH
(Iv) mSIRK (L9A): myr-SIRKALNIAGYPDYD-OH

MSIRK is a peptide compound in which the amino group of serine (S) is myristoylated in a peptide represented by SIRKALNILGYPDYD (SEQ ID NO: 1) in terms of one amino acid. That is, it has a structure (—NH-myr) in which the hydrogen atom of the amino group of serine of the peptide is substituted with a myristoyl group.

In mSCAR, the amino group of serine (S) of the peptide represented by SCARFFGTCPCP (SEQ ID NO: 3) in amino acid one letter code is myristoylated, and the carboxyl group of C-terminal proline (P) is amidated, It is a peptide compound. That is, it has a structure (—CO—NH ₂ ) in which the hydrogen atom of the amino group of serine of the peptide is substituted with a myristoyl group (—NH-myr) and the carboxyl group of proline at the C-terminal is amidated.

mSIRK (N7A) is a peptide compound in which asparagine which is the seventh amino acid of mSIRK is substituted with alanine, and mSIRK (L9A) is a peptide compound in which leucine which is the ninth amino acid of mSIRK is substituted with alanine.
mSIRK and mSCAR were purchased from Calbiochem or commissioned to Biologica. In addition, mSIRK (N7A) and mSIRK (L9A) were outsourced to Biologica Corporation for synthesis. The peptide compounds commissioned for synthesis were synthesized by a solid phase synthesis method using Fmoc as a protecting group and further purified by HPLC before delivery.

[Study using rat asinar cells]
<Preparation of rat lacrimal gland asinator cells>
Rat lacrimal glands are minced in DMEM / F12 (Invitrogen, 11330) and gently shaken in an incubator at 37 ° C. for 20 minutes in HBSS (Invitrogen, 14175) containing 0.76 mg / mL EDTA (WAKO, 345-01865). After centrifugation, the supernatant was removed. To this, in a DMEM / F12 medium containing 200 U / mL collagenase A (Roche, 10103586001), 698 U / mL Hyralonidase (Worthington, 2592), 10 U / mL DNase (Roche, 4536282) (CHD) for 20 minutes at 37 ° C. in an incubator. The process of gently shaking was performed twice.

However, the second treatment was performed at 37 ° C. for 30 minutes. Thereafter, the tissue derived from the lacrimal gland was suspended by pipetting, and 20% FBS (Invitrogen, 10082-147) was added to stop the enzymatic reaction, whereby isolated cells were obtained. After removing the residue with 100 μm (FALCON, 352360) and 40 μm (FALCON, 352340) cell strainers, the cells were separated with 10%, 30%, 60% Percoll (Sigma, P1644-100ML), 30% and 60% Cells collected during the period were defined as asiner cells.

The thus prepared asiner cells were cultured in Keratinocyte SFM medium (Invitrogen, 10724) and coated with 0.01 mg / cm ² Collagen I (BD, 354236) (IWAKI, 3810-006). Alternatively, each well of a 96-well plate (IWAKI, 3860-096) is seeded with cells at a culture density of 1.03 × 10 ⁵ cells / cm ² and cultured overnight in a CO ₂ incubator (SANYO, MCO-19AIC) at 37 ° C. After that, it was used for the following examination.

<Examination of the effect of various peptide compounds on promoting secretion of tear protein>
After the overnight culture, the medium of rat asiner cells was changed to normal DMEM / F12 (Invitrogen) without supplement, and preincubated at 37 ° C. for 30 minutes.

In addition, each of the above peptide compounds (i) to (iv), phenylephrine (WAKO, 163-11791) or carbachol (Sigma Aldrich, C4382-10G) at each concentration was added to the DMEM / F12 medium to obtain a medium for examination. . After the pre-incubation, the medium was replaced with the examination medium. After incubation at 37 ° C. for 10 minutes, 30 minutes, 60 minutes, or 90 minutes, the medium was collected. The tear protein secretion into this medium was examined. Phenylephrine is a kind of adrenergic agonist and is a compound known to have a lacrimal secretion promoting action. Carbachol is one of cholinergic drugs and is known to have a lacrimal secretion promoting action.

These were each used as a positive control.

When cultured for 60 minutes in a study medium supplemented with various concentrations (0 μM, 0.1 μM, 1 μM, 10 μM) of mSIRK, the tear proteins secreted into each medium were analyzed by SDS-PAGE (in the following manner). (CBB staining).

400 μL of the collected medium after drying was dried with a freeze dryer (TAITEC, Freeze dryer), dissolved in 80 μL of MilliQ water (Millipore, MQ synthesis,), and ReadyPrep 2D cleanup kit (Bio-Rad, 163-2130). ) Was used to obtain a protein pellet. This was dissolved in 50 μL of LDS sample buffer (Invitrogen, NP0007), heat-denatured at 70 ° C. for 10 minutes, and 20 μL of the sample together with Precision Plus Standard (Bio-Rad, 161-0373) was added to a 4-12% NuPAGE gel ( Applied to Invitrogen, NP0322BOX), and subjected to SDS-PAGE at room temperature for 35 minutes in MES buffer (Invitrogen, NP0002) to separate proteins. The gel was washed 3 times for 5 minutes in MilliQ water and then stained with Simply Blue Safe Stain (Invitrogen, LC6065) to detect the protein in the medium.

The results are shown in FIG. From the results, it was confirmed that 1 μM or 10 μM mSIRK promotes tear protein secretion from lacrimal gland asinar cells, that is, promotes tear secretion.

Furthermore, tear protein secretion was examined by measuring peroxidase activity using Amplex Red Hydroxide Peroxide / Peroxidase Assay Kit (Invitrogen, A22188). Specifically, 50 μL of reaction buffer containing Amplex red, H ₂ O ₂ was added to 50 μL of the collected medium, incubated at room temperature for 30 minutes, and then a fluorescent plate reader (Corona, MTP set to EX / EM = 530 nm / 610 nm). Fluorescence was measured at −800 AFC). The measured value was quantified using a standard prepared with peroxidase. The peroxidase activity 1 U (unit) here represents the amount of enzyme that produces 1.0 mg purpurogallin from Pyrogallol in 2 seconds at 20 ° C. and pH 6.0.

In addition, using an albumin measurement kit Rat Albumin ELISA Quantitation Set (Bethyl Laboratories), the amount of albumin in 100 μL of the collected culture broth was measured according to the protocol of the kit. Specifically, it was performed as follows. That is, 100 μL of anti-albumin antibody diluted 1: 100 with a coating buffer was added to each 96-well plate and reacted at room temperature for 1 hour to coat the antibody. Wash 3 times with Wash buffer and add 200 μl of blocking buffer. After incubating at room temperature for 30 minutes, the plate was washed 3 times with a wash buffer, and 100 μL of standards and samples (each culture solution) were added. The mixture was reacted for 60 minutes while slowly shaking at room temperature, and then washed 3 times with a wash buffer. 100 μL of HRP detection antibody diluted 1: 10000 with Sample-Conjugate Diluent solution was added, and the mixture was allowed to react for 60 minutes while slowly shaking at room temperature. After washing 3 times with a wash buffer, 100 μL of TMB Substance Solution was added and allowed to react in the dark for 3 minutes. 100 μL of 0.2 MH ₂ SO ₄ was added, and the absorbance was measured at 450 nm. A calibration curve was prepared from the standard measurement results, and the amount of albumin in each culture solution was determined using the calibration curve.

It should be noted that peroxidase and albumin are contained in tear fluid as tear proteins, and methods using these as indices of tear secretion are well known. A method of using a peroxidase activity value as an index of lacrimal secretion is also well known (see, for example, Am J Physiol Cell Physiol 275: 278-284, 1998).

The results are shown in FIGS. 2 to 6, only FIG. 3 b shows the albumin amount measurement results, and the other figures show the peroxidase activity measurement results. 2 and 3 show the results of measurement using a medium cultured for 10 minutes after the addition of the peptide compound, FIG. 4 shows the results of measurement using a medium cultured for 90 minutes after the addition of the peptide compound, and FIG. 6 shows the peptide compound. The result measured using the culture medium cultured for 30 minutes after the addition is shown. FIGS. 2 to 4 and 6 are results of using a culture solution obtained by culturing cells in wells of a 6-well plate and FIG. 5 in wells of a 96-well plate. 2 to 6, “Phe” indicates phenylephrine, “Cch” indicates carbachol, and “control” indicates a control in which nothing is added to the medium. In FIGS. 2 to 6, negative numbers or mathematical expressions are described after each peptide compound name, Phe or Cch. The negative number represents the concentration (M) of each peptide compound, phenylephrine, or carbachol added to the medium as an index. That is, when the negative number is “α”, the concentration of the peptide compound, phenylephrine, or carbachol added to the medium is 10 ^α (M). For example, “mSIRK-6” indicates that the experiment was performed by adding the peptide compound mSIRK to the medium to a final concentration of 10 ⁻⁶ (M). In the case of mathematical expressions, for example, “−0.3 × 5” or “−0.3 × 4”, “0.3 × 10 ⁻⁵ (M)” and “0.3 × 10 ⁻⁴ ”, respectively. (M) ”. Specifically, for example, “mSIRK-0.3 × 5” indicates that the experiment was performed by adding the peptide compound mSIRK to the medium to a final concentration of 0.3 × 10 ⁻⁵ (M). .

FIG. 2 shows that mSIRK and mSCAR have an action of promoting tear protein secretion in a concentration-dependent manner. It was also found that tear protein secretion was promoted in a short time of 10 minutes after addition to the asiner cell culture medium.

FIG. 3 shows that mSIRK and mSIRK (N7A) have an action of promoting tear protein secretion in a concentration-dependent manner, whereas mSIRK (L9A) does not show an action of promoting tear secretion. Both mSIRK (N7A) and mSIRK (L9A) are peptide compounds in which only one amino acid is substituted among the amino acids constituting mSIRK. It was thought that the effect of promoting lacrimal secretion could be greatly changed by substitution.

From FIG. 4, it was found that when compared at the same concentration, mSIRK and mSCAR have a lacrimal secretion promoting action higher than phenylephrine. In particular, when mSIRK was added with 10 ⁻⁵ M, the lacrimal secretion was promoted more than when 10 ⁻³ M phenylephrine was added. From this, it was found that mSIRK has a particularly strong tear secretion promoting action.

FIG. 5 shows the results of examining the lacrimal secretion promoting action over time by using 96-well plates and incubating asiner cells by adding mSIRK, mSIRK (N7A) and phenylephrine on the same plate. It was reconfirmed that mSIRK and mSIRK (N7A) have a stronger tear secretion promoting action than phenylephrine. Further, phenylephrine promotes tear secretion by adding a relatively high concentration of about 10 ⁻³ M, but it can be seen that it takes about 90 minutes to obtain the effect (FIG. 5: Phe-3). However, mSIRK and mSIRK (N7A) are at lower concentrations than phenylephrine and show a strong lacrimal secretion promoting effect after 30 minutes (FIG. 5: mSIRK-5 and mSIRK (N7A) -5). Further, FIG. 2 and FIG. 3 show that mSIRK, mSCAR and mSIRK (N7A) show a lacrimal secretion promoting effect in about 10 minutes. Therefore, it was confirmed that these peptide compounds promote tear secretion in a short time (at least about 10 to 30 minutes) after the action.

From FIG. 6, it was found that mSIRK, mSIRK (N7A), and mSCAR have a lacrimal secretion promoting action more than carbachol, especially when compared at the same concentration (10 ⁻⁵ M). Furthermore, these three types of peptide compounds exhibited a lacrimal secretion promoting effect when carbachol was added at 10 ⁻³ M when 10 ⁻⁵ M was added. In particular, mSIRK protruded and showed a strong lacrimal secretion promoting action. From this, it was found that, among these three types of peptide compounds, mSIRK has a particularly strong tear secretion promoting action.

[Study using rabbit asinar cells]
<Preparation of Rabbit Lacrimal Gland Asiner Cells>
Rabbit lacrimal gland asiner cells were prepared from rabbit lacrimal gland in the same manner as in the method for preparing rat lacrimal gland asinator cells.

<Examination of the effect of various peptide compounds on promoting lacrimal protein secretion>
Rabbit lacrimal gland asinner cells were seeded at a rate of 1 × 10 ⁻⁶ cells in ^6- well plates (IWAKI 3810-006) coated with collagen I (BECTON DICKINSON 354236), and in keratinocyte-SFM (Invitrogen 17005-042) in CO _The cells were cultured in _two incubators (SANYO). After overnight culture, the medium was replaced with DMEM / F12 (Invitrogen 11330-057), mSIRK, mSIRK (L9A), or carbachol (Sigma C4382) was added and cultured for 20 minutes, and then the medium was collected. Proteins secreted into the medium were purified by the same protocol as in the above rat, separated by electrophoresis on a 4-12% NuPGAE gel in MES buffer (Invitrogen NP0002) at 200V for 35 minutes at room temperature. Bands were detected by staining the gel with blue safe stain (Invitrogen LC6065). The result is shown in FIG.

In addition, in order to confirm the secretion of lipocalin (a type of tear protein) as an index of tear protein, it is electrically charged at room temperature for 35 minutes in a MES buffer (Invitrogen NP0002) at 4-12% NuPGAE gel (Invitrogen NP0322) at room temperature for 35 minutes. After separation by electrophoresis, the protein was transferred to a PVDF membrane (Millipore). Block the membrane for 30 minutes at room temperature using TBS (Bio-Rad 170-6435) containing 0.05% Tween20 (Bio-Rad 170-6653) in which 0.5% skim milk (Wako Pure Chemical 198-10605) is dissolved. Lipocalin antibody (consigned to Takara bio) diluted 1: 10000 with TBS (Bio-Rad 170-6435) containing 0.05% Tween20 (Bio-Rad 170-6653) in which 1% BSA (Sigma A3059-100G) is dissolved Using a partial sequence of rabbit lipocalin as an immunizing antigen, the reaction was carried out overnight at 4 ° C. in a solution of an antibody prepared in a rabbit (detailed preparation method is described below). Then, it was washed with TBS (Bio-Rad 170-6435) containing 0.05% Tween 20 (Bio-Rad 170-6651), and 0.05% Tween 20 (0.5% skim milk (Wako Pure Chemicals 198-10605) was dissolved. Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (GE Healthcare Bioscience NA 934-100UL) diluted 1: 10000 with TBS (Bio-Rad 170-6431) with Bio-Rad 170-6431) as a secondary antibody And allowed to react for 1 hour at room temperature. After the reaction, the membrane was washed with 0.05% Tween 20 (Bio-Rad 170-6431) in TBS (Bio-Rad 170-6435) and TBS, and with chemiluminescence reagent ECL Plus Western Blotting Detection (GE Healthcare Bioscience RPN2132). After treatment, the band was detected by Chemi doc (Bio-Rad). In addition, 5 μL of all the culture media migrated in lanes 2 to 10 in FIG. 7a were mixed to obtain a standard sample. The standard sample was applied to each well 1.0, 2.5, 5.0, 10, 15 μL, and electrophoresis and band detection were performed in the same manner as described above. A calibration curve was created from the density of the detected standard sample band, and the amount of lipocalin secreted from rabbit lacrimal gland asinator cells under each condition was compared. The result is shown in FIG.

[Method for producing anti-rabbit lipocalin antibody]
The antibodies that react with the rabbit lipocalin used in this study are described in Azzarolo et al. (Presence of tear lipocalin and other major promoters in laminar of O reb, K., z. Comp Biochem Physiol B Biochem Mol Biol.2004 Jun; 138 (2): 111-7.). Specifically, a cysteine residue is added to the C-terminus of a peptide consisting of 20 amino acids from the N-terminus of the rabbit tear lipocalin sequence (VDPAQVSGSWRTAAIADSPSP; SEQ ID NO: 10) for binding to a carrier protein, and KLH (keyhole limpet) is added thereto. Hemocyanin) was used as an immunizing antigen. The antigen was subcutaneously administered to the back of the rabbit, immunized for 45 days, and then serum was collected to obtain an antibody.

As a result of confirming the protein secreted in the medium, secretion promotion was confirmed by carbachol as a positive control, and secretion promotion was also confirmed by stimulation with mSIRK (FIG. 7a). In particular, mSIRK secreted more tear proteins than carbachol in the 10 μM addition group. When lipocalin was detected with an antibody, secretion was confirmed in mSIRK and carbachol as in the case of SDS-PAGE (upper part of FIG. 7b). Furthermore, comparing the density of this band, it was confirmed that mSIRK and carbachol had a concentration-dependent secretion promoting effect (bottom of FIG. 7b).

[Study using monkey asinar cells]
<Preparation of monkey lacrimal gland asiner cells>
Monkey lacrimal gland assiner cells were prepared from monkey lacrimal gland in the same manner as in the method for preparing rat lacrimal gland asinator cells.
<Examination of the effect of various peptide compounds on promoting lacrimal protein secretion>
Monkey lacrimal gland assiner cells were seeded at a rate of 1 × 10 ⁻⁶ cells in a ^6- well plate (IWAKI 3810-006) coated with collagen I (BECTON DICKINSON 354236), and 10 ng / ml dexamethazone (Sigma-Aldrich Corp., Sigma-Aldrich Corp., Japan). .Louis, MO), 1 mM putrescine (Sigma), 50 ng / ml EGF (Invitrogen), 25 μg / ml L-ascorbic acid (Sigma), 1 × ITS (Invitrogen), and 25 μg / ml gentamicin (Invitrogen) among F12 (Invitrogen), and cultured in a CO ₂ incubator (Fisher scientific) After overnight culture, the medium was replaced with DMEM / F12, and mSIRK or carbachol (Sigma C4382) was added and cultured for 10 minutes, and the medium was collected. The protein secreted into the medium was purified by the same protocol as that for rats and rabbits, and transferred to SDS-PAGE and PVDF membranes by the same protocol as that for rabbit asinar experiments. After blocking, lipocalin antibody diluted 1: 100 with TBS (Bio-Rad 170-6435) containing 0.05% Tween20 (Bio-Rad 170-6653) in which 1% BSA (Sigma A3059-100G) was dissolved (Santa cruz sc-34680), lacritin antibody diluted 1: 1000 (made by the method described in Nakajima et al, (2007) Exp Eye Res 85: 651-658), or lactoferrin antibody diluted 1: 1000 ( Sigma L3262-1VL) at 4 ° C. overnight, washed with TBS (Bio-Rad 170-6435) containing 0.05% Tween 20 (Bio-Rad 170-6653), and 0.5% skim milk (Bio-Rad 170). -6404 TU) and 0.05% Tween20 (Bio-Rad 170-6651) in TBS (Bio-Rad 170-6435) diluted 1: 10000 Anti-Rabbit IgG, HRP-Linked Hole Ab Donkey (GE Healthcare Bioscience) NA934-100UL), or donkey anti-goat IgG conjugated with HRP (Santa cruz sc-2020) for 1 hour at room temperature. After the reaction, the membrane was washed with TBS (Bio-Rad 170-6435) containing 0.05% Tween20 (Bio-Rad 170-6431) and TBS, and then with a chemiluminescence reagent ECL Plus Western Blotting Detection (GE Healthcare Bioscience RPN2132). Treatment was followed by detection of bands with FluorChem FC2 imager (Alpha Innotech). The results are shown in FIG.

As shown in FIG. 8, as a result of stimulation with 10 μM mSIRK or carbachol, secretion of lactoferrin, lacritin and lipocalin was promoted after 10 minutes of stimulation. Note that lactoferrin, lacritin, and lipocalin are all well known proteins for tear secretion. This also confirmed that mSIRK promoted lacrimation.

Study Example 2: Investigation of liposome containing peptide compound [Preparation of liposome containing peptide compound]
Liposome lipid composition soybean lecithin (Tokyo Kasei Kogyo) 20 mg and cholesterol (Nacalai Tesque) 5.76 mg were weighed into an eggplant flask, and 4.75 mL of chloroform (Wako) was added and dissolved. To this solution, 0.5 mg of mSIRK dissolved in 0.25 mL of methanol (Wako) was added and mixed with stirring. The solvent was distilled off with a rotary evaporator to form a lipid thin film. The lipid thin film was shaken in 3 mL of Dulbecco-phosphate buffered saline (D-PBS, Nacalai Tesque) to obtain a liposome suspension.

[Measurement of liposome content of peptide compound]
<Quantitative determination of mSIRK>
To measure the liposome content of mSIRK, a known amount of mSIRK was measured by high performance liquid chromatography (HPLC) to create a calibration curve. Specifically, 0.05, 0.025, 0.005, and 0.0025 (mg / mL) mSIRK solutions were chromatographed under the following liquid chromatographic conditions to detect mSIRK-derived peaks, and peak areas Was recorded.

[Liquid chromatography (HPLC) conditions]

The results are shown in FIG. FIG. 9 shows a chromatogram, and Table 1 shows a peak area (Area) and a retention time (RT).

The analytical results were plotted with the detected peak area as the horizontal axis and the mSIRK concentration as the vertical axis, and a calibration curve was created (FIG. 10).

Next, the liposome suspension obtained as described above was centrifuged (4 ° C., 15000 rpm, 60 min) to separate the liposome fraction and the supernatant fraction. 10% Triton X-100 was added to each fraction to disrupt the liposome membrane. Thereafter, each fraction was appropriately diluted, and the mSIRK concentration was measured by liquid chromatography. The conditions for liquid chromatography were the same as above.

The results are shown in FIG. “Liposome” in Table 2 represents the liposome fraction, and “Supernatant” represents the supernatant fraction. Further, Encapsulation in Table 2 indicates the amount of mSIRK contained in each fraction determined from the calibration curve based on the area of the mSIRK-derived peak in the chromatogram.

From Table 2, the liposome content of mSIRK is
(0.139 / 0.536) × 100≈26 (%)
It was possible to calculate.

Sequence number 1: Synthetic peptide Sequence number 2: Synthetic peptide sequence number 3: Synthetic peptide sequence number 4: Synthetic peptide sequence number 5: Synthetic peptide sequence number 6: Synthetic peptide sequence number 7: Synthetic peptide

Claims (10)

An ophthalmic agent comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof.
(A) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(B) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(C) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide,
R ² is the same or different and represents an —OH group or —NH ₂ group. ] The ophthalmic agent according to claim 1, which is used for promoting lacrimal secretion. The ophthalmic preparation according to claim 1, which is used for improving at least one symptom selected from the group consisting of tear reduction, dry eye syndrome, corneal epithelial disorder, Sjogren's syndrome, keratitis, and conjunctivitis. The ophthalmic preparation according to any one of claims 1 to 3, which is an eye drop, an eye ointment, an eye wash, or a contact lens composition. The ophthalmic preparation according to any one of claims 1 to 4, comprising a liposome comprising at least one selected from the group consisting of the peptide compounds (a) to (c) and salts thereof. A method for promoting lacrimal secretion, comprising a step of applying an ophthalmic agent comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof to the eyes of a mammal.
(A) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(B) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(C) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ] The method for promoting lacrimal secretion according to claim 6, wherein the mammal has a disorder of lacrimal secretion. A method for preventing or treating dry eye, comprising a step of applying an ophthalmic agent comprising at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof to the eyes of a mammal.
(A) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(B) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(C) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ] Use of at least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof for the production of an ophthalmic agent.
(A) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(B) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(C) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ] At least one selected from the group consisting of the following peptide compounds (a) to (c) and salts thereof for use in treating or improving tear secretion disorders.
(A) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(B) R ¹ -Ser-Ile-Arg-Lys-Ala-Leu-Ala-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp-R ²
(C) R ¹ -Ser-Cys-Ala-Arg-Phe-Phe-Gly-Thr-Pro-Cys-Pro-R ²
[In the above (a) to (c)
R ¹ is the same or different and represents a hydrogen atom, a linear or branched alkanoyl group having 10 to 22 carbon atoms, or a cell membrane permeation peptide, and R ² is the same or different and represents an —OH group or —NH _Two groups are shown. ]

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