JP2002173445A - Wound healing drug - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a wound healing drug free from side effects even by continuous use, having excellent effect and high safety and promoting the regeneration of the skin by the treatment of injury such as wound. SOLUTION: The wound healing drug contains a polypeptide consisting of a decomposed product of collagen and/or gelatin and having a molecular weight of >=500 and <2,500.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a wound healing agent for oral or external use on the skin.

[0002]

2. Description of the Related Art Collagen is about one-third of in vivo protein.
It is abundant in blood vessels, skin, and bones, and plays an important role as a scaffold for cells in the formation and construction of these tissues. Collagen is considered to be a protein with low nutritional value because it is hardly degraded by digestive enzymes. However, metabolism is promoted by ingesting collagen (JP-A-7-27801).
2) The hair diameter increases (Nutrition Reports Inter
national, 13, 579, 1976) and its use as a drug for the treatment of arthropathy (JP-A-63-39821), etc., and its efficacy has been reviewed. Collagen and gelatin have attracted attention as medical materials utilizing their cell support function. For example, a hemostatic agent, an artificial blood vessel, an anti-adhesion film, a contact lens worn continuously, and the like are being developed for many uses. In particular, use as artificial skin for wounds such as cut wounds, stab wounds, split wounds, bruises, lacerations, and wounds has been attracting attention (Japanese Patent Laid-Open No. 5-18466).
2).

However, its use is to protect the injured skin from drying and to serve as a scaffold for invasion of self-tissues, which is different from the function of promoting active cell migration. Also,
Collagen and gelatin, and their degradation products, bactericidal action,
It has been proposed to combine with urea which promotes skin regeneration, such as a wound healing action and a cell activating action (Japanese Patent Application Laid-Open No. 61-1986).
33105), which is intended to stabilize urea in an aqueous solution and is not a wound healing agent having a function of promoting active cell migration.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and has no fear of side effects even when used continuously.
It is an object of the present invention to provide an effective and highly safe wound healing agent that promotes skin regeneration against injury such as a wound.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned objects, and as a result, have found that a decomposition product of collagen or gelatin having a specific molecular weight has a wound healing effect. Based on this, the present invention has been completed.

That is, the present invention provides: 1. A wound healing agent comprising a polypeptide having an average molecular weight of 500 or more and less than 2500, which is a degradation product of collagen and / or gelatin. 2. The wound healing agent according to the above 1, which comprises one or more ascorbic acid and its derivatives. 3. the wound healing agent according to the above 1 or 2, which is for oral use; 4. The wound healing agent according to the above 1 or 2, which is for external use on the skin; The polypeptide content is 0.01% with respect to the total amount of the composition.
1 to 50% by weight
5. The wound healing agent as described above; The polypeptide content is 0.001 relative to the total amount of the composition.
1, 2 or 4 above, wherein
The wound healing agent according to the above. 7. The polypeptide content is 0.01% with respect to the total amount of the composition.
7. The wound healing agent according to the above 2 or 3, wherein the content of ascorbic acid and a derivative thereof is 0.001 to 80% by weight, and The polypeptide content is 0.00% relative to the total amount of the composition.
1 to 20% by weight and 0.001 to 60% by weight of ascorbic acid and its derivatives.
Or the wound healing agent according to 4.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Collagen used as a raw material to obtain the collagen or gelatin degradation product of the present invention is, for example, skin extracted from animals such as cows, pigs and fish, extracted from connective tissues such as bones and tendons, or heat-denatured collagen. Everything such as gelatin can be used. The material is heated and dissolved in about 2 to 40 times, preferably about 5 to 10 times the weight of the raw material to prepare a collagen or gelatin solution. The water used may be tap water, pure water, distilled water, or the like.

[0008] Hydrolysis of collagen or gelatin to obtain a degradation product thereof can be performed using an enzyme such as a proteolytic enzyme. The enzyme used for preparing the hydrolyzate is not particularly limited,
Trypsin, chymotrypsin, subtilisin, elastase, proline-specific protease, proteases produced by Streptococcus microorganisms, papain, pepsin, thermolysin and the like can be used. Carboxypeptidase Y as an exopeptidase, a protease produced by Aspergillus, a protease produced by a microorganism of Streptomyces, a protease produced by a microorganism of the genus Rhizopus, a protease produced by a lactic acid bacterium, and the like can be used. And collagenases derived from bacteria such as Clostridium and Streptomyces, actinomycetes and fungi. In addition, other microorganisms are produced by genetic recombination techniques, and enzymes having similar substrate specificities can be fermented with these microorganisms without any problem. Further, a plurality of enzymes may be mixed and used.

The amount of the enzyme used for the hydrolysis is preferably about 0.01% to 10%, preferably about 1% by weight based on the raw material.
The temperature is from room temperature to 55 ° C., preferably 37 ° C. to 40 ° C., and the reaction time is 1 to 24 hours, preferably 1 to 4 hours. Also, p
Adjust the H condition to the optimum pH before adding the enzyme. For collagenase, a pH of 6-8 is appropriate. After completion of the hydrolysis, the enzyme may be deactivated by heating, and after cooling, filtration, desalting, concentration and drying may be performed as necessary.

[0010] By separating according to molecular weight, collagen, which is a polypeptide having an average molecular weight used in the present invention,
A decomposed product of gelatin can be obtained. Separation by molecular weight can be preferably performed by a gel filtration method. The detection wavelength of the molecular weight is 210-290 nm, preferably 210-220 nm.
It can be determined using the UV wavelength.

As a degradation product of collagen or gelatin, a polypeptide having an average molecular weight of 500 or more and less than 2500 is used. Such a polypeptide has an average molecular weight of 500 or more2
Less than 500 polypeptides are its major constituent. More preferably, it is a polypeptide having an average molecular weight of 1000-1500. Particularly, a polypeptide having an average molecular weight of 1100 to 1300 is preferable.

Further, ascorbic acid used in the present invention includes L-ascorbic acid, and derivatives thereof include esters and salts thereof. As more specific examples, the esters include L-ascorbic acid monostearate, L-ascorbic acid monopalmitate, L-ascorbic acid monooleate and other ascorbic acid monoalkyl esters, L-ascorbic acid monophosphate ester, L-ascorbic acid monophosphate, -Ascorbic acid monoester derivatives such as ascorbic acid-2-sulfuric acid, ascorbic acid dialkyl esters such as L-ascorbic acid distearate, L-ascorbic acid dipalmitate and L-ascorbic acid dioleate, and ascorbic acids such as L-ascorbic acid diphosphate ester. Acid diester derivatives, L-ascorbic acid tristearate, L-ascorbic acid tripalmitate, L-ascorbic acid triolate, and other ascorbic acid trialkyl esters; L-ascorbic acid triphosphate, such as ascorbic acid It can be mentioned acid triester derivatives such as, but not limited thereto. Examples of the salt include salts of ascorbic acid and various bases. Examples thereof include, but are not limited to, alkali metal salts (eg, sodium salts) and alkaline earth metal salts (eg, calcium salts, magnesium salts, etc.).

The polypeptide of the present invention has an excellent function of promoting cell migration, and can be used as an oral or external skin wound healing agent. In addition, the use of the above-described ascorbic acid and / or a derivative thereof exhibits more excellent effects.

The collagen and gelatin decomposition product of the present invention
It is useful for wounds such as cuts, punctures, split wounds, bruises, lacerations, and wounds. It promotes cell migration of 3 ', 5'-cyclic adenosine monophosphate (cAMP), a mediator of wound healing, to rapidly condense cells at the wound site. Also, collagen production,
Enhances cell activation such as cell proliferation and differentiation, and promotes skin regeneration. Therefore, the present invention provides an excellent wound healing agent.

[0015] The "oral" wound healing agent of the present invention can be used as food, medicine and the like. It may be used as a dietary supplement, a functional food, a health food, a food for specified health use, and the like. In the form of powder, powder, granules,
It can be in the form of tablets, capsules, liquids and the like. In addition, as a food, it can be blended with a drink such as juice. For oral use, the amount of polypeptide added is preferably 0.01 to 50% by weight, particularly preferably 15% by weight or more and 50% by weight or less in the whole preparation. When ascorbic acid or a derivative thereof is contained, the content is preferably 0.001 to 80% by weight, particularly preferably,
It is not less than 0.1% by weight and not more than 80% by weight. There is no particular limitation on the amount of food or medicine taken, but it is preferably about 0.03 to 3 g / day.

Further, the wound healing agent of "external use for skin" of the present invention can be a product contained in cosmetics, quasi-drugs, pharmaceuticals and the like as defined in the Pharmaceutical Affairs Law. Used for skin care products, hair care products, ointments and the like. Although not particularly limited, the dosage form is an aqueous solution type, a solubilizing type, an emulsifying type,
Powder, oil-liquid, gel, ointment, aerosol, water
It can take a wide range of dosage forms, such as a two-layer oil system and a three-layer water-oil-powder system. The polypeptide content in the external composition for skin of the present invention is about 0.001 to 20% by weight of the total weight, preferably 0.1 to 20% by weight.
10% by weight. When ascorbic acid or a derivative thereof is contained, the content is preferably 0.001 to 60% by weight, particularly preferably 0.001 to 10% by weight. The amount to be applied for external use on the skin is appropriately selected depending on the difference in symptoms, but is usually determined as the amount of polypeptide per day.
It is in the range of about 0.00001 to 0.5 g. Also, it is of course possible to administer the drug in 1 to 4 times / day. Oral preparations and skin external preparations are usually used for food, medicine,
It can be produced by a formulation method used in the field of cosmetics. It can be said that the collagen and gelatin hydrolyzate of the present invention have no toxicity problem due to the properties of collagen and gelatin.

As described above, the present invention relates to collagen and / or collagen.
Or a decomposed product of gelatin with an average molecular weight of 500 or more2
It is an oral or external skin wound healing agent characterized by containing less than 500 polypeptides, and has already reported the cell activating effect of ascorbic acid and its derivatives (Ayumi of Medicine, June Supplement, 39-44, 1996), a more excellent wound healing effect is exhibited. In other words, by promoting cell migration against injuries such as cuts, stab wounds, split wounds, bruises, lacerations, and wounds, cells can be quickly condensed at the wound site, and collagen production, cell proliferation, differentiation, etc. It is an excellent wound healing agent that promotes skin regeneration by enhancing cell activation.

[0018]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Production Example 1 [Collagenase digestion degradation product] As a collagen protein, 30 g of gelatin prepared from bovine dermis was heated and dissolved in 300 ml of distilled water. Collagenase Type I (Worthington Biochemical
Corp.) (300 mg), adjusted to pH 7.5 with aqueous ammonia, and allowed to stand at 37 ° C for 1 hour. After the reaction is completed, heat the reaction solution to 100 ° C.
For 3 minutes to inactivate the enzyme and then sterile filtered through a 0.45 μm filter.

The filtrate was subjected to gel filtration using Sephadex LH-20 (manufactured by Pharmacia) equilibrated with distilled water, fractionated into 6 fractions, and lyophilized. The results of the gel filtration chromatography are shown in FIG. Each peak was measured using Superdex Peptide HR10 / 30 (Pharmacia) in 0.1M sodium phosphate buffer (p.
H7.2) When the average molecular weight was determined using the eluate, the test samples were 10,000, 2500, 1300, 1030, 490, and 320, respectively.
Met. Each fraction was used in the test of Example 1.
The fractions 1300 and 320 were also used in the test of Example 2.

Production Example 2 [Pepsin digestion degradation product] As a collagen protein, 30 g of gelatin prepared from bovine dermis was dissolved by heating in 300 ml of distilled water.
300 mg of pepsin (manufactured by Biozyme Lab., LTD) was added, the pH was adjusted to 2.0 with dilute hydrochloric acid, and the mixture was allowed to stand at 37 ° C for 1 hour. After the completion of the reaction, the pH was re-adjusted to 7 with an aqueous sodium hydroxide solution.
Heat reaction at 100 ° C for 3 minutes to inactivate pepsin, then
The solution was sterile-filtered with a 0.45 μm filter.

The filtrate was subjected to gel filtration using Sephadex LH-20 (Pharmacia) equilibrated with distilled water. When the average molecular weight of this product was measured, it was found to be 20,000
~ 60 widely distributed. 12,000 of these fractions were lyophilized and used in the test of Example 1. FIG. 2 shows the results of the gel filtration chromatography.

Example 1 [Cell migration test 1] CCD45SK normal human dermal fibroblasts (Dainippon Pharmaceutical Co., Ltd.) were immobilized in 10% immobilized fetal bovine serum (hereinafter abbreviated as FBS) and 0.1% non-immobilized. The cells were cultured in a MEM medium containing essential amino acids (hereinafter abbreviated as NEAA) at 37 ° C. in the presence of 5% carbon dioxide. Confluent cells are 0.
The medium was replaced with a medium containing 5% FBS and 0.1% NEAA. After acclimation for 24 hours, the medium was removed by suction, and a cell layer was partially peeled off with a cell scraper to create a wound. The cells that created the wound
After washing with PBS, 1 μM N ⁶ , 2 ′ was used as a cell migration initiator.
-O-dibutyryl adenosine 3 ': 5'-cyclic monophosphat
e (hereinafter abbreviated as dbcAMP) was pre-incubated for 24 hours, and then a test sample was added at 10 μg / ml (or ascorbic acid; 200 μM) to examine the effect of promoting cell migration. 48
After an hour, the cells were fixed in 10% formalin-containing PBS, stained with hematoxylin, and the number of migrated cells was determined by photographing. The results are shown in Table 1. From Table 1, it can be seen that the cell migration-promoting effect is higher than that obtained by adding a degraded product having an average molecular weight of 1030 or 1300 (Table 1; Nos. 4 and 5). In addition, ascorbic acid administration exhibited a more excellent effect (Table 1; No. 9).

[0024]

[Table 1]

Example 2 [Cell migration test 2] In this experiment, cell migration was performed by a method using a transwell chamber different from the above method.
Motility was evaluated. CCD45SK normal human dermal fibroblasts (Dainippon Pharmaceutical Co., Ltd.) contain 10% FBS and 0.1% NEAA
The cells were cultured in a MEM medium at 37 ° C. in the presence of 5% carbon dioxide until they became confluent. Thereafter, cells were collected and 1 × 10 ⁵ cel
150 μl of the ls / ml suspension was added to an 8 μm pore size transwell chamber (manufactured by NUNC), and dbcAMP and each test sample (100 μg / ml) were added. Transwell chamber is 10% FBS, 500 μl MEM or ascorbic acid 2
After incubating for 48 hours on a 24-well plate (IWAKI) in which 00 μM was dispensed, the cells in the transwell chamber were removed with a cotton swab, and the number of cells that had migrated to the back was determined by Di.
After staining with an ff-quik (manufactured by Kokusai Reagent Co., Ltd.) reagent, measurement was performed under an optical microscope. Table 2 shows the results. Average molecular weight 1300
It can be seen that the degradation product of (Table-2; No. 2) has a high cell migration promoting effect. In addition, ascorbic acid administration showed a more excellent effect (Table 2; No. 4).

[0026]

[Table 2]

Formulation Example 1 [Preparation of tablet] A tablet having the following composition was prepared by a conventional method using a fraction having an average molecular weight of 1300 in the gelatin decomposition product obtained in Preparation Example 1. (Composition) (Blend;% by weight) Decomposed gelatin (average molecular weight: 1300) 15 Lactose 63 Corn starch 20 Guar gum 1.0 L-Ascorbic acid 1.0

Formulation Example 2 [Manufacture of Juice] A juice having the following composition was manufactured according to a conventional method using a fraction having an average molecular weight of 1300 in the gelatin decomposition product obtained in Production Example 1. (Composition) (Blend;% by weight) Fructose sugar solution sugar 5.00 Citric acid 10.4 L-Ascorbic acid 0.20 Flavor 0.02 Pigment 0.10 Gelatin decomposed product (average molecular weight 1300) 1.00 Water 83.28

Formulation Example 3 [Production of skin care product (hand cream)] Production Example 1
Using the fraction having an average molecular weight of 1300 in the gelatin decomposition product obtained in the above, a hand cream having the following composition was produced according to a conventional method. (Composition) (Blend; wt%) Isopropyl isostearate 8.0 Jojoba oil 6.0 Cetanol 8.0 Stearyl alcohol 2.0 Polyoxyethylene lauryl ether 1.5 Propylene glycol 6.0 Sorbitol 1.0 Paraben 0.4 Gelatin degradation product (average molecular weight 1300) 0.8 Vitamin E 0.5 L-ascorbic acid 1.0 Fragrance 0.1 Purified water 64.7

[0030]

As apparent from the above, the average molecular weight is 500
Collagen and / or gelatin hydrolyzate consisting of a region of not less than 2,500 has a cell migration promoting effect, and further shows a more excellent effect by combining ascorbic acid and / or a derivative thereof. It is effective as a wound healing agent for skin and external use.

[Brief description of the drawings]

FIG. 1 shows LH- of gelatin degraded by collagenase.
1 shows each fraction prepared in Production Example 1 on 20 chromatographs.

FIG. 2 is a chromatogram of LH-20 of gelatin decomposed with pepsin, showing the fraction prepared in Production Example 2.

Continued on front page F term (reference) 4B064 AG01 CA21 CB01 CC30 DA01 4C084 AA02 BA05 BA23 BA44 CA20 CA25 CA34 DB52 MA28 MA35 MA52 MA63 NA14 ZA891 ZB212 ZC282 4C086 AA01 AA02 BA18 MA02 MA04 MA52 MA63 NA14 ZA89

Claims (8)

[Claims] 1. A wound healing agent comprising a polypeptide having an average molecular weight of 500 or more and less than 2500, which is a degradation product of collagen and / or gelatin. 2. The wound healing agent according to claim 1, comprising one or more of ascorbic acid and a derivative thereof. 3. The wound healing agent according to claim 1, which is for oral use. 4. The wound healing agent according to claim 1, which is for external use on the skin. 5. The composition according to claim 1, wherein the polypeptide content is 0.01 to 50% by weight based on the total amount of the composition.
4. The wound healing agent according to any one of 3. 6. The wound healing agent according to claim 1, wherein the polypeptide content is 0.001 to 20% by weight based on the total amount of the composition. 7. The composition according to claim 2, wherein the content of the polypeptide is 0.01 to 50% by weight and the content of ascorbic acid and its derivatives is 0.001 to 80% by weight based on the total amount of the composition. Wound healing agent. 8. The composition according to claim 2, wherein the content of the polypeptide is 0.001 to 20% by weight and the content of ascorbic acid and its derivatives is 0.001 to 60% by weight based on the total amount of the composition. Wound healing agent.