JP2003231695A - New peptide and its medicinal use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To search for the minimum activity-developing unit of an insulin-like growth factor to find out its medicinal uses in ophthalmological and dermatological fields. <P>SOLUTION: When a peptide containing an amino acid sequence represented by Ser-Ser-Ser-Arg which is the minimum activity-developing unit or the insulin- like growth factor and a peptide containing an amino acid sequence represented by Phe-Gly-Leu-Met-NH<SB>2</SB>are used, the cures of corneal damages and skin wounds are remarkably promoted. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Ser-Ser-Ser-A.
Novel peptide containing the amino acid sequence represented by rg, a pharmaceutical composition containing the peptide as an active ingredient, and Se
r-Ser-Ser-Arg peptide and Phe-Gly-Leu-Met- NH corneal disorder treatment agent or skin wound healing containing a peptide as an active ingredient comprising the amino acid sequence represented by ₂ comprising the amino acid sequence represented by Regarding accelerators.

[0002]

BACKGROUND OF THE INVENTION The cornea is a transparent avascular tissue having a diameter of about 1 cm and a thickness of about 1 mm. Corneal transparency has an important effect on visual function, and various physiological and biochemical phenomena in the cornea function mainly for the purpose of maintaining the transparency of the cornea.

Corneal epithelial defects caused by various diseases such as corneal ulcer, corneal epithelial detachment, keratitis or dry eye are naturally repaired unless there is a concurrent infection. However, if the repair is delayed for some reason or the epithelial defect is prolonged without repair, not only the normal construction of the epithelium is adversely affected, but also the structure and function of the parenchyma and the endothelium are damaged. The conventional therapeutic principle is passive in that by protecting the corneal surface from external stimuli, the epithelium naturally expands to recover the defect.
In recent years, along with the development of cell biology, factors involved in cell division, migration, adhesion, spreading, etc. have been elucidated, and compounds that promote the spreading of corneal epithelium play an important role in repairing corneal epithelial defects. It is reported to be responsible for this (see Non-Patent Document 1).

By the way, insulin-like growth factor is one of the growth factors that regulate the growth of normal human cells, such as epidermal growth factor, fibroblast growth factor, platelet-derived growth factor and transforming growth factor. Then, there are insulin-like growth factor-I (hereinafter referred to as "IGF-I") and insulin-like growth factor-II (hereinafter referred to as "IGF-II"). Recently, it has been reported that IGF-I stimulates thyroid cell proliferation (see Non-Patent Document 2), IGF-II regulates muscle growth and differentiation (see Non-Patent Document 3), and the like. In the field of ophthalmology, IGF-I, IG
F-II and functional derivatives thereof promote survival of retinal neurons (see Patent Document 1), IGF-II
Is effective for treatment of a wide range of wounds including damage at the time of corneal transplantation (see Patent Document 2), and by using the solution containing the above growth factor, the eye tissue such as cornea to be transplanted It is disclosed that it can be preserved in a fresh tissue state even in a low temperature state (see Patent Documents 3 and 4). Further, it is also disclosed that a gel composition containing a growth factor is generally effective for healing a wound including the anterior segment of the eye (see Patent Document 5).

On the other hand, substance P is a polypeptide consisting of 11 amino acids which exhibits vasodilation, smooth muscle contraction, salivary gland secretion promotion, diuretic action and the like. Regarding substance P, improvement of abnormal secretion of conjunctival goblet cells in ocular disorders has been disclosed (see Patent Document 6), and kinetics of substance P during inflammation such as keratitis has been reported in the field of ophthalmology (non- Various references have been made. Further, in Patent Document 7, Phe-G which is a tetrapeptide on the C-terminal side of substance P
_{ly-Leu-Met-NH 2} ( hereinafter referred to as "FGLM") and I
It is described that when used in combination with GF-I, FGLM promotes wound healing of corneal epithelium, and FGLM is the minimum unit of partial peptide of substance P that exerts this action. However, IGF-I is a polypeptide in which as many as 70 amino acids are bound, and which amino acid sequence site of IGF-I contributes to the effect expression has not been clarified at all.

Skin wounds include lacerations, abrasions, surgical incisions,
Skin tissue ulcers, burns and other surface tissue damage. In the treatment of such a skin wound, it is general to give a first aid treatment to the injured site and then wait for it to heal naturally by the resilience of the living body itself, but such natural healing requires a long period of time for recovery. In addition, since pain continues, it is desirable to actively promote wound healing by administering a wound healing agent to the injured site.

During the wound healing process, new epithelial tissues and connective tissues are formed due to the migration and proliferation of cells.
A drug for promoting or stimulating the migration, differentiation, and proliferation of cells involved in wound healing can be a wound therapeutic agent. Lysozyme chloride, sorboserine and the like are known as such wound healing agents.

[0008] However, the existing wound healing agents have a problem in that they do not have a sufficient effect of promoting wound healing and cannot completely heal a wound in a short period of time. It is considered that these wound healing agents have a small contribution to recoating of epidermis, synthesis of collagen, improvement of peripheral circulation, granulation, and angiogenesis, which are important factors in the healing process of wounds. ing.

By the way, there is no report on the minimum activity expression unit of IGF-I, and no report on the peptide itself consisting of the amino acid sequence represented by Ser-Ser-Ser-Arg. Further, the action on the corneal disorder and the action on the skin disease in which the peptide containing the amino acid sequence represented by Ser-Ser-Ser-Arg and the peptide containing the amino acid sequence represented by Phe-Gly-Leu-Met-NH ₂ are used in combination. Has not been reported.

[0010]

[Refer to Non-Patent Document 1] Rikio, 46 , 738-743 (1992), Ophthalmic surgery, 5 , 719-727 (1992)

[0011]

[Non-Patent Document 2] J. Biol. Chem., 264 , 18485-18488.
(1989)

[0012]

[Non-Patent Document 3] Hum. Mol. Genet., 3 , 1117-1121 (19
94)

[0013]

[Patent Document 1] Japanese Patent Publication No. 7-500839

[0014]

[Patent Document 2] Japanese Patent Laid-Open No. 63-233925

[0015]

[Patent Document 3] Japanese Unexamined Patent Publication No. 5-2501

[0016]

[Patent Document 4] JP-A-6-48901

[0017]

[Patent Document 5] Japanese Patent Application Laid-Open No. 2-112

[0018]

[Patent Document 6] International Publication No. WO95 / 13087 Pamphlet

[0019]

[Non-Patent Document 4] Journal of Japan Ophthalmological Society, 91 , 982-987 (198)
7)

[0020]

[Non-patent document 5] See non-patent document Japanese Society of Ophthalmology, 9
2 , 448-452 (1988)

[0021]

[Patent Document 7] Japanese Patent Laid-Open No. 10-17489.

[0022]

Generally, when a peptide consisting of a large number of amino acids is administered in vivo, the peptide bond is easily cleaved by metabolism or the like, and it is easily decomposed at the stage of formulation for use as a medicine. Therefore, it is desirable to make the peptide chain as short as possible, but since the pharmacological activity must be maintained, finding the minimum active expression unit of the long-chain peptide is an important subject in drug development. IGF-I is a long-chain peptide consisting of as many as 70 amino acids.
Finding the minimum active expression unit of is a very important issue in creating a more useful drug. In addition, it is a very interesting subject to study the specific pharmacological action, that is, the action on corneal disorders and the action on skin wounds, using the minimum activity expression unit.

[0023]

Means for Solving the Problems The present inventors have found that IGF-
IGF-I was synthesized by synthesizing various partial peptides of I and carrying out a pharmacological test for corneal epithelial extension using the partial peptides in combination with substance P or FGLM.
It was found that the minimum activity expression unit of is an amino acid sequence represented by Ser-Ser-Ser-Arg (hereinafter referred to as "SSSR"). In addition, a peptide containing the amino acid sequence represented by SSSR and substance P or FGLM
Was found to promote corneal lesion healing and skin wound healing. That is, (1) the peptide whose amino acid sequence is represented by Ser-Ser-Ser-Arg or its analogs or their pharmaceutically acceptable salts, and (2) the amino acid sequence is Phe-Gly-Leu-Met-NH ₂ The composition containing the peptide represented by or a pharmaceutically acceptable salt thereof is a corneal ulcer, corneal epithelial detachment, keratitis or dry eye in which the cornea is damaged due to various factors. It has been found to be useful as a therapeutic agent for corneal disorders such as and a healing agent for diseases such as lacerations, abrasions, surgical incisions, skin ulcers, burns and other skin wounds and gangrene resulting therefrom. The corneal disorder therapeutic agent and the skin wound healing promoter of the present invention contain ascorbic acid, ascorbic acid ester, a salt of ascorbic acid, a salt of ascorbic acid, pantothenic acid, a salt of pantothenic acid, etc., which have already been recognized for the wound healing effect. Can be used.

IGF-I is composed of A, B, C and D domains. Since the A domain and B domain have a structure similar to that of insulin or IGF-II, the present inventors have Focusing on the C domain and D domain of I, the corneal epithelial spreading action was examined. Then, a corneal epithelial extension test was carried out by using a peptide forming the C domain or a peptide forming the D domain in combination with substance P, and it was found that the peptide forming the C domain was Gly-Tyr-Gly-Ser-Ser-Ser. −Arg−Arg−Ala−Pro
-Gln-Thr (hereinafter referred to as "GYGSSSRRAPQT") was found to have activity. Next, GYGSSSRRA
Since the activity was maintained even if 2 amino acids were removed from both ends of PQT, Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro (hereinafter,
"GSSSRRAP") amino acids are sequentially converted to alanine to synthesize substance P or F
When a corneal epithelial extension test was carried out in the presence of GLM, all peptides containing the amino acid sequence represented by SSSR expressed activity. Therefore, SSSR is essential and minimal for expressing corneal epithelial extension. It was found to be a partial peptide of IGF-I.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION
Completed two inventions.

The first invention is that the amino acid sequence is Ser-Ser-
The present invention relates to a novel peptide represented by Ser-Arg, a derivative thereof, or a pharmaceutically acceptable salt thereof.

The feature of the first invention is to find out a novel peptide which is a minimum activity expression unit of IGF-I, that is, a novel peptide whose amino acid sequence is represented by Ser-Ser-Ser-Arg. Therefore, the amino acid sequence is Ser-S
peptide represented by er-Ser-Arg or its derivative (hereinafter, the peptide and its derivative are referred to as "SSSR derivative").
Are collectively referred to as), and are not particularly limited as long as they are novel peptides containing an amino acid sequence represented by Ser-Ser-Ser-Arg. The derivative of the peptide means Ser-Ser-Se.
One in which one or more amino acids that do not affect the activity expression are bound to the peptide represented by r-Arg, one in which the N-terminal is protected by a protecting group commonly used in peptides such as an acyl group, one in which the C-terminal is an ester , Amide, etc., which are protected by a protecting group commonly used for peptides, etc.
It also includes those in which the hydroxy group of the r residue is protected by a commonly used protecting group, and those in which the amino group of the Arg residue is protected by a commonly used protecting group. More specifically, examples of the SSSR derivative include SSSR, GSSSRRAP, Ser-Ser-Ser-Arg-Arg (hereinafter, referred to as "SSSRR").
Abbreviated), Gly-Ser-Ser-Ser-Arg (hereinafter, "G
"SSSR"), Gly-Ser-Ser-Ser-Arg-A
rg (hereinafter abbreviated as "GSSRR"), Ala-Ser-
Ser-Ser-Arg-Arg-Ala-Pro (hereinafter referred to as "ASSSRR"
Abbreviated as "AP"), Gly-Ser-Ser-Ser-Arg-Ala-
Ala-Pro (hereinafter abbreviated as "GSSSRAAP"),
Gly-Ser-Ser-Ser-Arg-Ala-Ala-Ala-Pro (hereinafter abbreviated as "GSSSRAAAP") and the like can be mentioned. The amino acids that make up these peptides include L-
There are body, D-body, and DL-body, all of which are included in the present invention. As specifically shown in the pharmacological test section, all SSSR derivatives containing the amino acid sequence represented by SSSR in the peptide chain were identified as substance P.
Alternatively, in combination with FGLM, a corneal epithelial spreading effect and a skin wound healing promoting effect are exhibited.

The SSSR derivative of the present invention can be produced by a known method using an automatic peptide synthesizer, the details of which will be described in Examples.

The second invention is that the amino acid sequence is Ser-Ser-
The present invention relates to a pharmaceutical composition comprising a novel peptide represented by Ser-Arg or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable additive compounded therein.

The third invention is (1) the amino acid sequence is Ser
-Ser-Ser-Arg represented by a peptide or an analogue thereof or a pharmaceutically acceptable salt thereof and (2) a peptide represented by Phe-Gly-Leu-Met-NH ₂ in amino acid sequence or an analogue thereof Alternatively, the present invention relates to a therapeutic agent for corneal disorders, which contains a pharmaceutically acceptable salt thereof as an active ingredient.

The fourth invention is the above component (1) and the above component (2).
Is an invention relating to a skin wound healing promoter containing as an active ingredient.

The features of the third and fourth inventions are a peptide whose minimum activity expression unit is represented by Ser-Ser-Ser-Arg,
By minimum unit for the exhibition of the activity is used together with peptide represented by Phe-Gly-Leu-Met- NH 2, are in was found that superior corneal epithelial migration effect and promoting the skin wound healing effect appears.

In the third and fourth inventions, a peptide whose amino acid sequence is represented by Ser-Ser-Ser-Arg or an analogue thereof (hereinafter, the peptide and its analogue are referred to as "SSSR").
The term “analog” is collectively not limited as long as it is a peptide containing an amino acid sequence represented by Ser-Ser-Ser-Arg. The analog of the peptide means Ser-Ser-
A peptide represented by Ser-Arg to which one or more amino acids that do not affect its activity expression are bound, N
It means that the terminal is protected by a protecting group commonly used for peptides such as acyl group, or the C-terminal is protected by a protecting group commonly used for peptides such as ester or amide. It also includes those in which the hydroxy group of the group is protected by a commonly used protecting group, and those in which the amino group of the Arg residue is protected by a commonly used protecting group. More specifically,
Examples of the SSSR analogs include GYGSSSSRRAPQT and the like in addition to the above-mentioned SSSR derivative. S
The amino acids that make up the peptide of the SSR analog include L-
There are body, D-body, and DL-body, all of which are included in the present invention. A more preferable form is a peptide consisting of all L-amino acids.

The amino acid sequence is Phe-Gly-Leu-Met.
-NH ₂ peptide or its analogue (hereinafter,
The peptide and its analogs are collectively referred to as “FGLM analog”), and are not particularly limited as long as they are peptides containing the amino acid sequence represented by Phe-Gly-Leu-Met-NH ₂ . The analog of the peptide means Phe-Gly-Leu-Met-
Means a peptide represented by NH _{2 to} which one or more amino acids that do not affect its activity expression are bound, or a peptide whose N-terminus is protected by a protecting group commonly used in peptides such as an acyl group. To do. More specifically, FGL
Examples of the M analog include Tyr-Arg-Pro-Lys-Pro-Gln, which is a polypeptide composed of 4 to 12 amino acids specifically disclosed in U.S. Pat. No. 3,862,114, including substance P and FGLM.
-Gln-Phe-Phe-Gly-Leu-Met-
_{NH 2, Pro-Gln-Gln} -Phe-Phe-G
_{ly-Leu-Met-NH 2} , Gln-Gln-Ph
e-Phe-Gly-Leu- Met-NH 2, Gln
-Phe-Phe-Gly-Leu- Met-NH 2,
Phe-Phe-Gly-Leu- Met-NH 2, T
yr-Phe-Gly-Leu- Met-NH 2, Gl
such as y-Phe-Gly-Leu- Met-NH 2 and the like. As a more preferable example, the substance P,
There is FGLM. The amino acids that make up these are also L-
There are body, D-body, and DL-body, all of which are included in the present invention. All of the preferred forms of the present invention are
It is a peptide consisting of L-form amino acids.

In the present invention, the pharmaceutically acceptable salts include, for example, hydrochloride, sulfate, phosphate, lactate, maleate, fumarate, oxalate, methanesulfonate, paratoluenesulfone. Examples thereof include acid salts.

In the present invention, SSSR analogs and FGL
The combination of M analogues exerts an effect of promoting corneal epithelial spreading and cutaneous wound healing. The types of SSSR analogs and FGLM analogs that express these actions are not particularly limited, but in a more preferred embodiment, the minimum activity expression unit of SSGF which is the minimum activity expression unit of IGF-1 and the minimum activity expression unit of substance P are And the use of FGLM which is

The therapeutic agent for corneal disorders and the agent for promoting skin wound healing of the present invention can be prepared by using a generally used technique, and the SSSR analogue or a pharmaceutically acceptable salt thereof and the FGLM analogue or The pharmaceutically acceptable salts thereof can be administered either parenterally or orally as a single preparation or a combined preparation, but parenteral administration is more preferable.

Preferred dosage forms of the therapeutic agent for corneal disorders include eye drops and eye ointments. These can be prepared using a commonly used technique. For example, eye drops can be prepared using a tonicity agent such as sodium chloride, a buffering agent such as sodium phosphate, a preservative such as benzalkonium chloride, and the like. The pH may be in the range acceptable for ophthalmic preparations, but is preferably in the range of 4-8.

The dose of the therapeutic agent for corneal disorders can be appropriately selected depending on the symptoms, age, dosage form and the like. In the case of eye drops, the amount of the SSSR analog or a pharmaceutically acceptable salt thereof is 0. 001 to 10% (w / v), preferably 0.01 to 1% (w / v) may be instilled 1 to several times a day. The amount of the FGLM analog or a pharmaceutically acceptable salt thereof is from 0.00001 to
0.1% (w / v), preferably 0.0001 to 0.0
A 1% (w / v) solution may be applied once to several times a day.

Of course, the above-mentioned both components can be mixed at the same time to prepare a preparation such as an eye drop.

Further, as a preferable formulation form of the skin wound healing promoter, an ointment, a jelly, a poultice, a patch,
Examples include lotions, creams, sprays, aerosols, plasters, suspensions, emulsions, and the like, and liquids can be prepared by selecting an appropriate solvent. Also,
A filler, an excipient, a base, a bulking agent, a pH adjusting agent, a solubilizing agent, a suspending agent, a buffering agent, a stabilizing agent, depending on the dosage form, for preparing a skin wound healing promoter. Preservatives, surfactants, antioxidants, dispersants, emulsifiers, solubilizers, solubilizers and the like can be added.

Examples of the carrier for the above-mentioned preparation include white petrolatum, liquid paraffin, gelled hydrocarbon, cetyl alcohol, polyethylene glycol, gelatin, corn starch, sodium alginate, methyl cellulose,
Polymers containing hydroxyethyl cellulose, carboxymethyl cellulose, plastibase hydrophilic, gelatin, dextrin, cetyl alcohol, stearyl alcohol, polyethylene glycol, polyvinyl alcohol, methoxyethylene-maleic anhydride copolymer, polyvinyl ether, vinylpyrrolidone. -Copolymer, sodium stearate, magnesium stearate, benzalkonium chloride, olive oil,
Examples include oils and fats such as camellia oil and soybean oil, lactose, and water.

The skin wound healing promoter of the present invention can be administered in various forms depending on the wound site and the degree of the wound. For example, when used as an external preparation, it is desirable to directly apply, spray or stick this agent on a required site (affected area) such as skin.

The dose of the skin wound healing promoter of the present invention is
It can be appropriately selected in consideration of symptoms, age, dosage form and the like. SSS
The dose of the R analogue or a pharmaceutically acceptable salt thereof is usually 0.001 to 1000 mg, preferably 0.01 to 500 mg per day, which is administered once or in several divided doses. The dose of the FGLM analog or a pharmaceutically acceptable salt thereof is usually 0.01 to 5000 mg, preferably 0.1 to 1 mg per day.
The dose is 000 mg, which is administered once or in several divided doses.

Of course, both of the above components can be blended simultaneously to give a preparation such as an ointment.

The results of production examples, formulation examples and pharmacological tests are shown below, but these examples are for the purpose of better understanding of the present invention, and do not limit the scope of the present invention.

[0047]

EXAMPLES [Production Examples] Representative production examples of the SSSR analog used in the present invention are shown below.

1. Production of SSSR Using an automatic peptide synthesizer 430A (manufactured by Applied Biosystems), a protected peptide resin was synthesized by the tertiary butyloxycarbonyl (BOC) method according to the existing software.
4- (Oxymethyl) phenylacetamidomethyl [Boc-Arg (Tos) PAM] resin carrier as starting material (0.5 mmol
Scale) was used. In this synthetic method, 30% trifluoroacetic acid (T
FA) / dichloromethane and 70% TFA / dichloromethane were used, and N-methyl-2-pyrrolidone (NMP) / dichloromethane was used for the washing. The condensing agents N, N '-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) and the N-protected amino acid Boc-Ser (OB
The zl) derivative was used in an amount of 4 equivalents based on each amino group, and dimethyl sulfoxide (DMSO) -NMP (8:
2) was used. After completion of the condensation reaction, acetic anhydride / N, N-diisopropylethylamine (DIEA) was used as an operation to completely block the remaining amino groups to prevent the production of defective peptides. The removal of the Boc group and the condensation of Boc-Ser (OBzl) were repeated to construct the final protected peptide. Cleavage of the peptide from the resulting protected peptide resin and elimination of all protecting groups were treated with anhydrous hydrogen fluoride (HF) (HF: p-cresol,
8: 2 (V / V), -2 to -5 ° C, 60 minutes). After the reaction, HF was distilled off, the peptide was extracted with 0.1% trifluoroacetic acid water, and a crude product was obtained as a lyophilized powder, which was then subjected to preparative purification. Preparative purification is HPLC LC8A manufactured by Shimadzu (column: ODS30 X 24 manufactured by YMC).
0mm), acetonitrile / water system (0.1% TFA
Content) with a gradient of 0.5-2% (80 min). The high-purity fractions of the obtained target substance were collected, acetonitrile was distilled off, and the residue was lyophilized to give TFA salt of the target compound 70 mg.
(Yield 32%) was obtained.

Amino acid analysis (hydrolysis conditions: 6N HCl, 11
0 ° C, 22 hours) Ser (3) 2.74, Arg (1) 1.00 HPLC analysis [Column: YMC Pak ODS-A (4.6mml.D. × 150m
m), Eluent: 1-60% CH ₃ CN / 5mM CF ₃ CF ₂ COOH (25min), T
emp .: 25 ℃, Flow rate: 1.0ml / min, Detector: 220nm] Purity (HPLC): 98.5% Mass Spectrometry (ESI-MS) MH ⁺ = 436.2 (Theor. = 436.2, mono isotopic)

2. Manufacture of SSSR analogs Perform the same operation as SSSR, GSSSR, SSSR
R, GSSSRR, GSSSRRAP, ASSSRRA
P, GSSSRAAP and GSSSRAAAP were manufactured. The physical properties of typical peptides are shown below.

(1) GSSSR amino acid analysis (hydrolysis condition: 6N HCl, 110 ° C., 22 hours) Ser (3) 2.76, Gly (1) 1.00, Arg (1) 1.00 HPLC analysis [Column: YMC Pak ODS-A (4.6mm l.D. × 150m
m), Eluent: 1-60% CH ₃ CN / 5mM CF ₃ CF ₂ COOH (25min), T
emp .: 25 ℃, Flow rate: 1.0ml / min, Detector: 220nm] Purity (HPLC): 98.5% Mass spectrometry (ESI-MS) MW = 492.3 (Theor. = 492.5)

(2) SSSRR amino acid analysis (hydrolysis condition: 6N HCl, 110 ° C., 22 hours) Ser (3) 2.76, Arg (2) 2.00 HPLC analysis [Column: YMC Pak ODS-A (4.6 mm l.D. × 150m
m), Eluent: 1-60% CH ₃ CN / 0.1% CF ₃ COOH (25min), Tem
p .: 25 ℃, Flow rate: 1.0ml / min, Detector: 220nm] Purity (HPLC): 99.7% Mass spectrometry (ESI-MS) MW = 591.5 (Theor. = 591.6)

(3) GSSSRR Amino acid analysis (hydrolysis condition: 6N HCl, 110 ° C., 22 hours) Ser (3) 2.73, Gly (1) 0.98, Arg (2) 2.00 HPLC analysis [Column: YMC Pak ODS-A (4.6mm l.D. × 150m
m), Eluent: 1-60% CH ₃ CN / 0.1% CF ₃ COOH (25min), Tem
p .: 25 ℃, Flow rate: 1.0ml / min, Detector: 220nm] Purity (HPLC): 99.3% Mass spectrometry (ESI-MS) MW = 648.5 (Theor. = 648.7)

(4) GSSSRRAP amino acid analysis (hydrolysis condition: 6N HCl, 110 ° C., 22 hours) Ser (3) 2.68, Gly (1) 0.99, Ala (1) 1.01, Arg (2) 2.0
0 HPLC analysis [Column: YMC Pak ODS-A (4.6 mm l.D. × 150 m
m), Eluent: 1-60% CH ₃ CN / 0.1% CF ₃ COOH (25min), Tem
p .: 25 ℃, Flow rate: 1.0 ml / min, Detector: 220 nm] Purity (HPLC): 98.6% Mass spectrometry (ESI-MS) MW = 816.7 (Theor. = 816.9)

[Formulation Example] A typical formulation example used in the present invention is shown below.

1. Eye drops The following eye drops were prepared using a commonly used method.

[0057]   Prescription example 1     In 100 ml       SSSR 1mg       Sodium chloride 900mg       Sodium hydroxide suitable amount       Hydrochloric acid       Sterile purified water

In the same manner as in Prescription Example 1, SSSR was set to 100.
0.01 mg, 0.05 mg, 0.1 mg in ml
0.5mg, 5mg, 10mg, 50mg, 100mg
The containing eye drop can be prepared.

[0059] Prescription example 2     In 100 ml       GSSSR 1mg       FGLM 100mg       Sodium chloride 900mg       Sodium hydroxide suitable amount       Hydrochloric acid       Sterile purified water

FGLM was added to 100 in the same manner as in Formulation Example 2.
1 mg, 5 mg, 10 mg, 50 mg, 500 in ml
It is possible to prepare eye drops containing 100 mg and 1000 mg.

[0061] Prescription example 3   In 100 ml       SSSR 1mg       FGLM 100mg       Sodium chloride 900mg       Sodium hydroxide suitable amount       Hydrochloric acid       Sterile purified water

SSSR was adjusted to 0.0 in the same manner as in Prescription Example 3.
1 mg, 0.05 mg, 0.1 mg, 0.5 mg, 10
mg, 50 mg, 100 mg, and FGLM for 1 m
g, 5 mg, 10 mg, 50 mg, 500 mg, 100
0 mg Any combination of eye drops can be prepared.

[0063] 2. Ointment Prescription example 4    In 100g      SSSR 10mg      FGLM 100mg      Liquid paraffin 10g      White vaseline

Ointments of various concentrations can be prepared by appropriately changing the addition amount of SSSR and the addition amount of FGLM.

[0065] Prescription example 5   In 100g       GSSSR 1mg       Substance P 100mg       Liquid paraffin 10g       White vaseline

By changing the addition amount of GSSSR and the addition amount of substance P in the same manner as in Formulation Example 5, ointments of various concentrations can be prepared.

[0067] Prescription example 6    In 100g      SSSRR 5mg   FGLM 100mg      Liquid paraffin 10g      White vaseline

Ointments of various concentrations can be prepared by appropriately changing the addition amount of SSSRR and the addition amount of FGLM.

[0069] Prescription example 7    In 100g      GSSSRR 50mg      Substance P 10mg      Ascorbic acid 3mg      Liquid paraffin 10g      Plastibase Hydrophilic Suitable amount

Ointments of various concentrations can be prepared by appropriately changing the amounts of GSSSRR and substance P.

[Pharmacological test] (1) Effect on corneal epithelial extension (in vitro) According to the method of Nishida et al. (J. Cell Biol., 97 , 1653-1657 (1983)) using cornea of male Japanese white rabbit. The effect of corneal epithelial extension on the extent of corneal epithelial extension in a tissue culture system was examined.

[0072] (Experimental Method) The corneas were excised from rabbit corneal piece block (6 per group), the culture medium containing the test compound (TC-199), 2 under the conditions of 37 ℃ · 5% CO ₂
Cultured for 4 hours. After culturing, the corneal block was
It was fixed in a mixed solution of glacial acetic acid (volume ratio 95: 5) and embedded in paraffin to prepare a section. After deparaffinizing the sections, they were stained with hematoxylin-eosin and the extension length of the epithelial cell layer was measured under a microscope.

As a control, one similarly cultured in a culture solution containing no test compound was used.

(Test compound) Table 1 shows typical examples of peptides used in the experiment.

(Results) Table 1 shows the experimental results. The extension rate in the table is based on the extension length of the control group (100
%) Is the average value of each of the 6 cases.

[0076]

[Table 1]

As shown in Table 1, the SSSR analog alone,
Although substance P alone and FGLM alone had no effect on the extension of corneal epithelium, when cultured in a culture medium containing both SSSR analogue and substance P (or FGLM), significant extension of corneal epithelium was observed. It was

(2) Action on skin wound healing The skin wound healing effect can be tested by the following method.

The rat is anesthetized with diethyl ether inhalation, and the back hair is shaved with a hair clipper and the hair is removed with a hair removing cream. Twenty-four hours later, a 5 mm diameter trepan for skin biopsy was used to make 5 full-thickness epidermal / dermis wounds on the back skin at equal intervals.
Ointment containing SSSR after creating the site and confirming hemostasis,
The ointment containing FGLM and the ointment containing SSSR and FGLM are each applied once a day. Before applying each ointment, the rat's back wound is photographed and its area is measured.
The skin wound healing effect can be examined by comparing the respective wound areas after the application of the SSSR-containing ointment, the FGLM-containing ointment, and the SSSR-FGLM-containing ointment.

[0080]

EFFECT OF THE INVENTION From the results of the pharmacological test, an SSSR analog containing an amino acid sequence represented by Ser-Ser-Ser-Arg, which is the minimum activity expression unit of IGF-I, and Phe-Gly-Leu-Met-.
The combined use of an FGLM analog containing the amino acid sequence represented by NH ₂ significantly promotes corneal epithelial extension and cutaneous wound healing. Therefore, when the SSSR analog and the FGLM analog are administered in combination, these agents act synergistically to treat corneal ulcers, corneal epithelial detachment, keratitis, dry eye and other therapeutic agents for corneal disorders, lacerations, abrasions, Surgical incision,
It exerts its effect as a healing agent for skin wounds such as skin ulcers and burns, and diseases such as gangrene resulting therefrom.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 17/02 A61P 27/02 27/02 A61K 37/36 (72) Inventor Makoto Inui No. 6 Unoshima-cho, Ube City, Yamaguchi Prefecture 25-1001 (72) Inventor Masatoshi Nakamura 8916-16 Takayamacho, Ikoma-shi, Nara Santen Pharmaceutical Co., Ltd. Research Institute F-term (reference) 4C076 AA08 AA12 AA72 BB24 BB31 CC19 CC30 DD22Z DD23D DD30Z DD34A 4C084 AA01 AA02 AA03 AA07 BA01 BA08 BA16 BA23 CA59 DB58 MA17 MA28 MA32 MA58 MA63 NA05 ZA332 ZA892 4H045 AA10 AA30 BA13 DA38 EA20 FA40 HA02

Claims (8)

[Claims] 1. A peptide having an amino acid sequence represented by Ser-Ser-Ser-Arg, a derivative thereof, or a pharmaceutically acceptable salt thereof. 2. A peptide having an amino acid sequence represented by Ser-Ser-Ser-Arg or a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and containing a pharmaceutically acceptable additive. A pharmaceutical composition comprising: 3. The following components; 1) A peptide whose amino acid sequence is represented by Ser-Ser-Ser-Arg or an analogue thereof or a pharmaceutically acceptable salt thereof 2) An amino acid sequence is Phe-Gly-Leu-Met A therapeutic agent for corneal disorders, which comprises a peptide represented by NH ₂ or an analog thereof or a pharmaceutically acceptable salt thereof. 4. The therapeutic agent for corneal disorders according to claim 3, wherein the corneal disorder is corneal ulcer, corneal epithelial detachment, keratitis or dry eye. 5. The therapeutic agent for corneal disorders according to claim 3, wherein the dosage form is an eye drop. 6. The following components: 1) A peptide whose amino acid sequence is represented by Ser-Ser-Ser-Arg or an analogue thereof or a pharmaceutically acceptable salt thereof 2) An amino acid sequence is Phe-Gly-Leu-Met peptide or an analogue or skin wound healing promoting agent containing acceptable salts their pharmaceutically represented by -NH _2. 7. A skin wound is a laceration, abrasion, surgical incision,
The skin wound healing promoter according to claim 6, which is a skin ulcer or a burn. 8. The skin wound healing promoter according to claim 6, wherein the dosage form is an ointment or a patch.