JPH0881387A - Wound healing agent - Google Patents

Abstract

(57) [Summary] [Structure] The peptides of SEQ ID NOs: 1 to 31, the pharmacologically acceptable derivatives of these peptides, the pharmacologically acceptable salts of these peptides, or two or more thereof. A wound healing agent containing a mixture as an active ingredient. [Effect] Since it has few side effects, is heat resistant, and is stable in an aqueous solution, it is stable as a drug and has an antibacterial action. Therefore, it is not necessary to use an antiseptic agent for formulation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a preparation for use in healing wounds, pressure sores, etc., which are accompanied by skin damage such as burns and lack of damage. Specifically, the present invention describes the peptides represented by SEQ ID NOs: 1 to 31, the pharmacologically acceptable derivatives of these peptides, and the pharmacologically acceptable salts of these peptides (hereinafter referred to as the peptides). Or a mixture of two or more thereof as an active ingredient.

As used herein, percentages are by weight unless otherwise noted, and wounds are surgical operations,
Ulcers due to trauma or burns, open pressure sores, incisions and the like caused by bed sores.

[0003]

2. Description of the Related Art At present, wound healing is performed by excision of the ulcer surface, surgical treatment such as skin grafting, or conservative treatment with an external preparation. For local therapy in conservative treatment, an external preparation having the action of promoting granulation, improving local circulation, promoting epidermal formation, etc. is used after wound surface purification such as protection of wound surface, removal of necrotic tissue, prevention of secondary infection, etc. There is. Among them, the promotion of granulation and epidermis formation is directly related to the mechanism of wound healing, and solcoceryl ointment (manufactured by Taiho Pharmaceutical Co., Ltd., Applied Pharmacology, Vol. 22, No. 4, 565-565) is used as an external preparation for these. 5
79 pages, 1981), Reflap ointment (manufactured by Nippon Kayaku Co., Ltd., Basic and Clinical, Vol. 18, No. 12, No. 194-2.
00 page, 1984), Orsenone ointment (manufactured by Nippon Redery Co., Ltd. Applied Pharmacology, Vol. 43, No. 2, 87-95).
Page, 1992 and Applied Pharmacology, Vol. 43, No. 2,
121-127, 1992) and the like are used.

However, the effects of these drugs are not remarkable, and a more effective wound healing agent has been desired.
Furthermore, with the arrival of an aging society expected, development of therapeutic agents for pressure ulcers is also emphasized, and an external preparation Eupasta (produced by Kowa Shinyaku Co., Pharmacology and Treatment, Vol. 17, Special issue No. 1, page 7, 1989 and West Japan Dermatology, Vol. 47, page 915, 1985) were marketed, but they contained bovidone iodine, so that the range of use was limited. there were.

On the other hand, lactoferrin is an iron-binding protein contained in tears, saliva, peripheral blood, milk and the like, and is known to exhibit an antibacterial action against harmful microorganisms such as Escherichia coli, Candida and Clostridia. [Journal of Pediatrics]
, Vol. 94, page 1, 1979].

The present inventors isolated peptides having a strong antibacterial activity from lactoferrin degradation products, or synthesized peptides having the same amino acid sequence as those peptides or derivatives of those peptides to obtain 20 amino acids. Antibacterial peptide consisting of residues (JP-A-5-92994)
No.), an antibacterial peptide consisting of 11 amino acid residues (JP-A-5-78392), an antibacterial peptide consisting of 6 amino acid residues (JP-A-5-148297), and five amino acids. Antibacterial peptide consisting of residues (JP-A-5-1498296), antibacterial peptide consisting of 3 to 6 amino acid residues (JP-A-5-14882)
No. 95) have already been applied for patents.

Furthermore, the present inventors have found that a peptide derived from lactoferrin having a specific amino acid sequence has a brain-protecting action (JP-A-6-172200) and a cell growth activating action (Japanese Patent Application No. 5-352422). , Antifungal action (Japanese Patent Application No. 6-126882), antioxidant (JP-A-6-1996)
No. 87) and the like, and already applied for a patent.

[0008] As a method for treating a wound using a peptide having a biological activity, Magainin et al.
No. 4566) is known. However, since these peptides are derived from the skin of frogs, insect body fluids and the like, it is difficult to obtain a natural product, an isolated purified product becomes expensive, and production on an industrial scale is difficult.

[0009]

DISCLOSURE OF THE INVENTION The present inventors have been earnestly researching a more effective drug in view of the prior art of the above wound healing agent. The present invention has been completed by finding that the proliferation of blast cells and the promotion of wound healing in a wound model animal, that is, that there is a wound healing effect.

It is an object of the present invention to provide a safer, more effective and cheaper wound healing agent.

[0011]

Means for Solving the Problems The present invention for solving the above-mentioned problems comprises the peptides of SEQ ID NO: 1 to SEQ ID NO: 31,
A wound healing agent containing, as an active ingredient, a pharmacologically acceptable derivative of these peptides, a pharmacologically acceptable salt of these peptides, or a mixture of two or more thereof. That the active ingredient is contained at least 0.2 mg per 1 g of the external preparation, the wound healing agent is an oral preparation, and the active ingredient is orally administered at a rate of at least 100 mg per 1 kg of body weight. It is also a desirable embodiment that the wound healing agent is a subcutaneous administration agent and that the active ingredient is subcutaneously administered at a rate of at least 20 mg per wound surface.

Next, the constitution and preferred embodiments of the present invention will be described in detail.

When the peptides, which are the active ingredients of the wound healing agent of the present invention, are produced from lactoferrins, the lactoferrins used as starting materials are commercially available lactoferrin, mammals (eg, human, bovine, sheep, goat,
Horse) colostrum, transitional milk, normal milk, end-stage milk, etc., or skim milk, whey, etc., which are processed products of these milks, in a conventional manner (for example,
Lactoferrin separated by ion exchange chromatography), apolactoferrin deironed with hydrochloric acid, citric acid, etc., metal saturated or partially saturated lactoferrin obtained by chelating apolactoferrin with a metal such as iron, copper, zinc, manganese, etc. It is also possible to use a product or a preparation produced by a known method.

The peptides used in the present invention are peptides obtained by a separation means from lactoferrin degradation products, peptides having the same amino acid sequence as this peptide, homologous amino acid sequences, derivatives of these peptides, and these peptides. The pharmaceutically acceptable salts thereof or any mixture thereof can be chemically synthesized by a known method. These peptides are described in, for example, JP-A-5-92994 and JP-A-5-78.
392, JP-A-5-148297, JP-A-5-1498296, and JP-A-5-148295.
It can be obtained by the method described in each invention of the publication.

As the peptide obtained by the above method, a peptide having the following amino acid sequence, its derivative or salt can be exemplified as a desirable embodiment. For example, peptides having the amino acid sequences of SEQ ID NOs: 1, 2 and 27, salts thereof or derivatives thereof (JP-A-5-78392),
Peptides having the amino acid sequences of SEQ ID NOs: 3, 4, 5 and 6, salts thereof or derivatives thereof (JP-A-5-14829).
7), peptides having the amino acid sequences of SEQ ID NOS: 7, 8, 9 and 31, salts thereof or derivatives thereof (JP-A-5-1498296), peptides having the amino acid sequences of SEQ ID NOS: 10 to 21, salts thereof. Or a derivative thereof (JP-A-5-148295), peptides having the amino acid sequences of SEQ ID NOs: 22 to 26, 28, 29 and 30, salts thereof or derivatives thereof (JP-A-5-9299).
No. 4).

Examples of the pharmaceutically acceptable salts of the above peptides include acid addition salts such as hydrochloride, phosphate, sulfate, citrate, lactate, tartrate, and the like. The derivative is a carboxyl group. Examples thereof include amidated or acylated derivatives.

The obtained peptides have extremely low toxicity as shown in Test Example 5, and can be appropriately used as a drug such as ointment, liquid coating agent, lotion agent, aerosol (spray) agent and suppository. Yes, by known methods, tablets,
Capsules, troches, syrups, granules, powders,
It is also possible to process it into an injection or the like.

The compounding amount of the peptide which is the active ingredient of the wound healing agent of the present invention can be appropriately selected according to the symptoms and the like.
In the case of subcutaneous administration, it is at least 20 mg per wound surface, and in the case of oral preparation, it is at least 100 mg per kg body weight.

Next, the present invention will be described in detail by showing test examples.

Test Example 1 This test was carried out using mouse Balb / c 3T3 to examine the effect of the peptides on cell proliferation.

1) Preparation of sample Using a commercially available mouse submandibular gland-derived EGF (manufactured by Takara Shuzo) and the peptide of SEQ ID NO: 26 produced by the same method as in Reference Example 1, a sample without addition (Sample 1), EGF 10 ng
/ Ml spiked sample (Sample 2), peptide 2 of SEQ ID NO: 26
0 μg / ml spiked sample (Sample 3) and EGF 10 ng /
A sample (Sample 4) to which 20 ml of the peptide of SEQ ID NO: 26 and 20 μg / ml of the peptide was added was prepared.

2) Test method Mouse fibroblast Balb / c 3T3 (American Typ)
(Purchased from the e Culture Collection) is plated on a 24-well culture plate, 10,000 pieces per well, and a Dulbecco's modified Eagle culture medium containing 10% fetal bovine serum (hereinafter referred to as FBS-D).
The cells were cultured to a confluent state (described as MEM) (the culture solution was 1 ml per well). Culture medium
The cells were replaced with 1% FBS-DMEM, and the cells were further cultured for 3 days to introduce the cells in the stationary phase. 1% FBS containing each sample in culture solution
-The medium was replaced with DMEM (0.5 ml), and the cells were cultured for 18 hours. Culture medium containing 3H-thymidine (1 μC / ml) D
The medium was replaced with MEM (0.35 ml), and the cells were further cultured for 2 hours. 2 cells with phosphate buffer (PBS (-), 2 ml)
And then washed with ice-cold 5% trifluoroacetic acid (TFA, 2,
ml) was added and left in the refrigerator for 1 hour. Cells with 5% T
The cells were lysed by washing twice with FA (2 ml), adding 1 N sodium hydroxide (0.4 ml), and allowing to stand at 37 ° C. for 1 hour. 6N hydrochloric acid (0.08 ml) was added to neutralize, and then the amount of ³ H-thymidine incorporated into DNA was measured by a liquid scintillation counter (manufactured by LKB) to test cell proliferation activity.

3) Test Results The results of this test are shown in FIG. Figure 1
The ³ H-thymidine uptake of each sample in Balb / c 3T3 is shown, and the vertical axis and the horizontal axis show the ³ H-thymidine uptake and the sample, respectively.

As is apparent from FIG. 1, 10 ng of EGF
/ Ml and the peptide of SEQ ID NO: 26 (20 μg / ml), the amount of 3H-thymidine incorporated in Sample 4 was 23,4.
34 cpm, that of the non-added sample (1,176 cp
m), about 20 times, and about 2 times that of the EGF 10 ng / ml-added sample (12,300 cpm).

Therefore, it was revealed that the incorporation of ³ H-thymidine in the presence of EGF was dramatically increased by the peptide of SEQ ID NO: 26. In addition, the type of the peptides was changed and tested, but almost the same results were obtained.

Test Example 2 This test was carried out to examine the granulation-promoting activity of the peptide using the cotton ball method.

1) Test Material Physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) and the same peptide of SEQ ID NO: 26 as in Test Example 1 were used.

2) Test method 7-week-old male Wistar rats (purchased from Charles River)
Fifteen animals were randomly divided into 3 groups (5 animals per group).

The method of Sakyo et al. (Applied Pharmacology, Vol. 43, No. 2)
No. 87-95, 1992).

One cotton ball impregnated with each physiological saline solution of the peptide of SEQ ID NO: 26 in an amount of 3.3 mg / ml, similarly 33 mg / ml and physiological saline, 300 μl each, was used.
Group (control), 2 groups (corresponding to 2 mg per animal) and 3 groups (corresponding to 20 mg per animal) were inserted by the following method.

The rat dorsal skin under Nembutal anesthesia was incised about 2 cm along the midline, and 1 cotton ball (2 per animal) was inserted subcutaneously in each of the left and right shoulders, and the incision was immediately made. Sutured. On the 4th day after inserting the cotton ball, the rat
The animal was sacrificed under anesthesia, and the granulation tissue formed into a capsule around the cotton ball was removed together with the cotton ball. Exclude fat mass, mucous membrane, subcutaneous tissue, etc. other than granulation tissue, peel the granulation tissue from the cotton ball under ice cooling, wash the obtained granulation tissue with ice-cold physiological saline, and remove blood components and exudates. After that, the water was sufficiently removed with a filter paper, and then degreased and dried, and the dry weight was measured and tested.

3) Test Results The results of this test are shown in Table 1. As is clear from Table 1, in the 3 group, the granulation increased about 2 times as compared with the 1 group (control), but in the 2 group, the remarkable increase of the granulation was not recognized as compared with the 1 group. . From this result, it was confirmed that the administration of 20 mg of the peptide of SEQ ID NO: 26 per animal has a granulation formation promoting action. still,
When the type of the peptides was changed and tested, almost the same results were obtained.

[0033]

[Table 1] Test Example 3 This test was performed to examine the healing effect of peptides on burns in vivo using rats.

1) Preparation of sample 66 g of Veterinary Pharmacopoeia (manufactured by Yoshida Pharmaceutical Co., Ltd.) per 1 g
g, cetanol (manufactured by Wako Pure Chemical Industries, Ltd.) 3.5 g,
Cholesterol (Nacalai Tesque, Inc.) 0.5
g, distilled water 25 g, methyl p-hydroxybenzoate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.16 g, and butyl p-hydroxybenzoate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.02 g. Pharmacology, Vol. 22, No. 4, 56
5 to 579, 1981), the same peptide of SEQ ID NO: 26 as in Test Example 1 was added to 0.1% (Sample 2), 1.0
% (Sample 3) and 5.0% (Sample 4),
An ointment was prepared. An ointment containing no peptide of SEQ ID NO: 26 (Sample 1) was similarly prepared.

2) Test method 7-week-old Wister rat (purchased from Charles River)
The 32 animals were randomly divided into 4 groups (8 animals per group). Method of Sakyo et al. (Applied Pharmacology, Vol. 43, No. 2, 121-127
Page, 1992) and tested as follows.

Under Nembutal anesthesia, the back of the rat was shaved and depilated, and a brass cylinder (diameter 11 mm × height 45 mm) heated to 100 to 105 ° C. was self-weighted at a position 2 cm from the armpit on the midline on the caudal side. (Approximately 33 g) presses for 10 seconds to inflict a burn, and the size of the burn site (major axis x minor axis)
Was measured with a caliper. The burned site was covered with a gauze coated with 0.2 g of each sample and fixed with an elastic bandage. Test start 1
Do the administration of the sample and the measurement of the wound area every day until 2 days later,
The wound healing situation was tested.

3) Test Results A part of the results of this test (from 8 days after the start of the test) is shown in Table 2.
As shown in. As is clear from Table 2, sample 2
The wound area of burns decreased quickly in the administration group, and a significant difference was observed from the sample 1 administration group 8, 10, 11 and 12 days after the start of the test. Also, in the groups administered with Sample 3 and Sample 4, no significant difference was observed with respect to Sample 1, but it was confirmed that the reduction of the wound area was promoted.

Therefore, an ointment containing at least 1 mg of peptide per gram was found to be effective as a wound healing agent. In addition, the type of the peptides was changed and tested, but almost the same results were obtained.

[0039]

[Table 2] Test Example 4 This test was conducted to examine the healing effect of peptide-deficient wounds in vivo using rats.

1) Preparation of sample Based on the pharmacopoeia macrogol ointment (manufactured by Yoshida Pharmaceutical Co., Ltd.),
The peptide of SEQ ID NO: 26, which is the same as in Test Example 1, was added to 0.02
An ointment containing% (Sample 2) and 0.1% (Sample 3) was prepared. As a control, a pharmacopoeia macrogol ointment base (Sample 1) containing no peptide of SEQ ID NO: 26 and a commercially available defect-creating agent solcoceryl ointment (manufactured by Taiho Pharmaceutical Co., Ltd., Sample 4) were used.

2) Test Method [Method] 7-week-old Wister rat (purchased from Charles River)
Twenty animals were randomly divided into 4 groups (5 animals per group). Method of Sakyo et al. (Applied Pharmacology, Vol. 43, No. 2, 121-127
The test was conducted as follows according to the method of page, 1992).

After shaving and removing the back of the rat under Nembutal anesthesia, a skin defect wound reaching a muscle layer having a diameter of about 14 mm was made by using a cork polar at the midline of the back, 2 cm caudal from the armpit, and the wound surface. Was measured with a caliper. The skin defect site was covered with a gauze coated with 0.2 g of each sample and fixed with an elastic bandage. The state of wound healing was examined by administering the sample and measuring the wound area every day until 12 days after the start of the test.

3) Test Results The results of this test are shown in Table 3. As is clear from Table 3, the defect wound area decreased quickly in the sample 2 administration group, and a significant difference was observed from the sample 1 administration group 7 and 8 days after the start of the test, and the sample 4 (commercially available drug) administration group The effect was equal to or higher than that. Even in the sample 3 administration group, no significant difference was observed with respect to sample 1,
It was observed that it promotes the reduction of the defect wound area.

Therefore, at least 0.2 mg per gram
It has been found that an ointment containing the peptide of 1. is effective as a wound healing agent. In addition, the type of the peptides was changed and tested, but almost the same results were obtained.

[0045]

[Table 3] Test Example 5 This test was conducted to examine the acute toxicity of the peptides.

1) Preparation of sample The same sample as in Test Example 1 was used.

2) Test Method Male and female were randomly divided into 2 groups (5 animals per group) using 6-week-old CD (SD) strain rats (purchased from Japan SLC).

1000 and 2000 mg / kg body weight
The sample was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a ratio of 4 and a single forced oral administration was carried out at a ratio of 4 ml per 100 g of body weight using a needle with a metal ball, and acute toxicity was tested.

3) Test Results As a result of this test, no deaths were observed in the groups to which this sample was administered at the rates of 1000 mg / kg body weight and 2000 mg / kg body weight. Therefore, LD of this peptide
₅₀ was 2000 mg / kg body weight or more, and the toxicity was found to be extremely low. In addition, the type of the peptides was changed and tested, but almost the same results were obtained.

Reference Example 1 50 mg of commercially available bovine lactoferrin (manufactured by Sigma) was dissolved in 0.9 ml of purified water, pH was adjusted to 2.5 with 0.1N hydrochloric acid, and then commercially available porcine pepsin (manufactured by Sigma). (Manufactured by Mitsui Chemicals Co., Ltd.) and hydrolyzed at 37 ° C for 6 hours. Then, adjust the pH to 7.0 with 0.1 N sodium hydroxide, heat at 80 ° C. for 10 minutes to inactivate the enzyme, cool to room temperature, and centrifuge at 15,000 rpm for 30 minutes,
A clear supernatant was obtained. 100 μl of this supernatant was added to TSK gel O
It was subjected to high performance liquid chromatography using DS-120T (manufactured by Tosoh Corporation) and eluted with 20% acetonitrile containing 0.05% TFA (trifluoroacetic acid) for 10 minutes after injecting the sample at a flow rate of 0.8 ml / min. , Then 30 minutes 0.0
Elution was carried out with a gradient of 20 to 60% acetonitrile containing 5% TFA, and the fractions eluted during 24 to 25 minutes were collected and dried under vacuum. This dried product was dissolved in purified water at a concentration of 2% (W / V), and the TSK gel ODS-120T was dissolved again.
(Manufactured by Tosoh Corporation), and subjected to high performance liquid chromatography at a flow rate of 0.8 ml / min for 10 minutes after injection of the sample.
Elute with 24% acetonitrile containing 05% TFA, then elute with a gradient of 24-32% acetonitrile containing 0.05% TFA for 30 minutes, 33.5-35.
Fractions eluting during 5 minutes were collected. The above operation was repeated 25 times, and vacuum drying was performed to obtain about 1.5 mg of the peptide.

The above peptide was hydrolyzed with 6N hydrochloric acid,
The amino acid composition was analyzed by an ordinary method using an amino acid analyzer. Identical sample is vapor phase sequencer (Applied
Edman degradation was performed 25 times using Biosystems) to determine the sequence of 25 amino acid residues. Further, a disulfide bond analysis method using DTNB [5,5-dithio-bis (2-nitrobenzoic acid)] [Analytical B chemistry (Analytical B
iochemistry), vol. 67, p. 493, 1975]
Confirmed that a disulfide bond was present.

As a result, this peptide was composed of 25 amino acid residues, the 3rd and 20th cysteine residues were disulfide-bonded, and the 3rd cysteine residue resulted in N
It was confirmed to have the amino acid sequence of SEQ ID NO: 26 in which two amino acid residues were bound to the -terminal side and 5 amino acids were bound to the C-terminal side from the 20th cysteine residue, respectively. .

Reference Example 2 Using a peptide automatic synthesizer (Pharmacia LKB Biotechnology, LKB Biolynx 4170), a solid phase peptide synthesis method using a shepherd or the like [Journal of Chemical Society Part] Kin I (Journal of Chemi
cal Society Perkin I), p. 538, 1981]
Based on the above, the antibacterial peptide was synthesized as follows.

An amino acid having an amine functional group protected by a 9-fluorenylmethoxycarbonyl group [hereinafter sometimes referred to as Fmoc-amino acid or Fmoc-specific amino acid (for example, Fmoc-asparagine)] has N, N- Dicyclohexylcarbodiimide was added to produce the anhydride of the desired amino acid and this Fmoc-amino acid anhydride was used in the synthesis. In order to produce a peptide chain, Fmoc-asparagine anhydride corresponding to the C-terminal asparagine residue is immobilized on Ultrosin A resin (Pharmacia LKB Biotechnology) via its carboxyl group using dimethylaminopyridine as a catalyst. To do. The resin is then washed with dimethylformamide containing piperidine to remove the amine functional group protecting group of the C-terminal amino acid. Thereafter, Fmoc-arginine anhydride, which corresponds to the second position from the C-terminal of the amino acid sequence, was coupled to the deprotected amine functional group of arginine fixed to the resin via the C-terminal amino acid residue. In the same manner, glutamine, tryptophan, glutamine and phenylalanine were fixed in the same manner. 94% TFA, 5% after completion of coupling of all amino acids and formation of a peptide chain having a desired amino acid sequence
The protective group other than acetamidomethyl was removed and the peptide was eliminated with a solvent consisting of phenol and 1% ethanol, the peptide was purified by high performance liquid chromatography, and the solution was concentrated and dried. , Peptide powder was obtained.

The amino acid composition of the above-mentioned peptide was analyzed by an ordinary method using an amino acid analyzer, and SEQ ID NO: 10 was used.
It was confirmed to have the amino acid sequence of

Next, the present invention will be described in more detail by showing examples, but the present invention is not limited to the following examples.

[0057]

【Example】

Example 1 Peptide of SEQ ID NO: 26 produced by the same method as Reference Example 0.1 (g) Squalane 10.0 White petrolatum 8.0 Cetostearyl alcohol 8.0 Glycerin monostearate 2.0 Polyoxyethylene monostearate 1.0 Methyl paraoxybenzoate 0.2 Propyl paraoxybenzoate 0.1 1,3-Butylene glycol 2.5 Sterile purified water 68.1 100 g of the above ointment ratio per 100 g It was produced by a conventional method. In addition, as raw materials other than peptides, commercially available products were used.

Example 2 A cream having the following blending ratio per 100 g was produced by a conventional method.

Peptide of SEQ ID NO: 26 produced by the same method as in Reference Example 1.0 (g) Polyoxyethylene stearyl ether 2.0 Polyoxyethylene cetyl ether 3.0 Mitsurou 4.0 cetano- Le 3.0 Lanolin 1.0 Isopropyl palmitate 2.0 Liquid paraffin 15.0 Polyethylene glycol monostearate 0.5 Methyl paraoxybenzoate 0.1 Purified water 68.4 Any raw material other than peptides Also, a commercially available product was used.

Example 3 A tablet having the following compounding ratio per tablet was produced.

Peptide of SEQ ID NO: 10 produced by the same method as Reference Example 10.0 (mg) Lactose monohydrate 30.0 Corn starch 19.8 Crystalline cellulose 28.0 Magnesium silicate pentahydrate Product 2.0 Magnesium stearate 0.2

The mixture of the peptide of Reference Example 1, lactose monohydrate, corn starch and crystalline cellulose was uniformly kneaded while appropriately adding sterilized water, and dried at 50 ° C. for 3 hours to obtain a dried product. Magnesium silicate pentahydrate and magnesium stearate were added to and mixed with each other, and the mixture was tableted by a tableting machine by a conventional method. In addition, as raw materials other than peptides, commercially available products were used.

Example 4 100 ml of the peptide powder of SEQ ID NO: 26 produced by the same method as in Reference Example 1 in 1 ml of water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.)
g and sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) at a ratio of 9 mg, adjusted to pH about 7 with sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.), and filtered. The ampoule was sterilized and filled in 1 ml portions by a conventional method to prepare a wound healing agent for injection.

[0064]

Industrial Applicability As described above in detail, the present invention is a wound healing agent containing, as an active ingredient, one or more peptides having a specific amino acid sequence, and the effects exhibited by the present invention are as follows. ,It is as follows.

1) There are few side effects. 2) It has heat resistance, is stable in an aqueous solution, and is stable as a drug. 3) Since it has an antibacterial action, it is not necessary to use an antiseptic agent for formulation.

[0066]

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 2 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 3 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 5 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 6 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 8 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 9 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys.

SEQ ID NO: 10 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

SEQ ID NO: 11 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment.

SEQ ID NO: 12 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment.

SEQ ID NO: 13 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment.

SEQ ID NO: 14 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment.

SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

SEQ ID NO: 16 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment.

SEQ ID NO: 17 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

SEQ ID NO: 18 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment.

SEQ ID NO: 19 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment.

SEQ ID NO: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

SEQ ID NO: 21 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

SEQ ID NO: 22 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the following sequence, Cys 2 and 19 are disulfide-bonded. Sequence: Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys Val 20

SEQ ID NO: 23 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. Cys * in the sequence below
Indicates cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond. Sequence: Lys Cys * Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys * Val 20

SEQ ID NO: 24 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the following sequence, Cys 2 and 19 are disulfide-bonded. Sequence: Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys Ile 20

SEQ ID NO: 25 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. Cys * in the sequence below
Indicates cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond. Sequence: Lys Cys * Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys * Ile 20

SEQ ID NO: 26 Sequence length: 25 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, Cys 3 and 20 are disulfide-bonded. Sequence: Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala 1 5 10 15 Pro Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25

SEQ ID NO: 27 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment.

SEQ ID NO: 28 Sequence length: 38 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. 16 in the sequence below
Cys No. 33 and Cys No. 33 have a disulfide bond. Sequence: Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro Glu Trp Phe Lys 1 5 10 15 Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser 20 25 30 Ile Thr Cys Val Arg Arg Ala Phe 35

SEQ ID NO: 29 Sequence length: 32 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. 10 in the sequence below
No. 27 Cys and No. 27 Cys are disulfide-bonded. Sequence: Thr Ile Ser Gln Pro Glu Trp Phe Lys Cys Arg Arg Trp Gln Trp 1 5 10 15 Arg Met Lys Lys Leu Gly Ala Pro Ser Ile Thr Cys Val Arg Arg 20 25 30 Ala Phe

SEQ ID NO: 30 Sequence length: 47 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment. In the following sequence, in the peptide having a sequence length of 36 and having Cys at positions 9, 26, and 35, the 9th Cys and the 26th Cys form a disulfide bond, and 35 of peptides
No. 10 Cys has a sequence length of 11 and has a disulfide bond with No. 10 Cys of a peptide having Cys at No. 10. Sequence: Val Ser Gln Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn 1 5 10 15 Met Arg Lys Val Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp 20 25 30 Ser Pro Ile Gln Cys Ile 35 Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala 1 5 10

SEQ ID NO: 31 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment. In the sequence below, R01
Indicates any amino acid residue except Cys

[0097]

[Brief description of drawings]

FIG. 1 ³ H-of each sample in Balb / c 3T3.
The amount of thymidine incorporated is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display location // C07K 14/79 ZNA 8318-4H A61K 37/18 ADS (72) Inventor Kazuzo Shinoda Kanagawa Prefecture 51-83 Higashihara, Zama-shi 51-83, Institute of Nutrition Science, Morinaga Dairy Co., Ltd. (72) Inventor, Tsuneharu Yamauchi 51-83 Higashihara, Zama-shi, Kanagawa, Japan 51-83, Institute of Nutrition Science, Morinaga Dairy Co., Ltd. (72) Hidefumi Kuwata 51, Higashihara, Zama-shi, Kanagawa −83 Morinaga Dairy Co., Ltd., Institute of Nutrition Science (72) Inventor Nantsuko Yamazaki 51-83 Morinaga Dairy Co., Ltd., Institute of Nutrition Science

Claims (7)

[Claims] 1. An active ingredient comprising the peptide of SEQ ID NO: 1 to SEQ ID NO: 31, a pharmacologically acceptable derivative of these peptides, a pharmacologically acceptable salt of these peptides, or a mixture of two or more thereof. As a wound healing agent. 2. The wound healing agent according to claim 1, wherein the wound healing agent is an external preparation. 3. The wound healing agent according to claim 2, wherein the active ingredient is contained in an amount of at least 0.2 mg per 1 g of the external preparation. 4. The wound healing agent according to claim 1, which is an oral agent. 5. The wound healing agent according to claim 4, wherein the active ingredient is orally administered at a rate of at least 100 mg / kg body weight. 6. The wound healing agent according to claim 1, which is a subcutaneous administration agent. 7. The active ingredient is at least 2 per wound surface.
The wound healing agent according to claim 6, which is subcutaneously administered at a rate of 0 mg.