JPH09157183A - Preparation for external use for skin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a promoter for the synthesis of collagen and a glycosaminoglycan, comprising a midkeine as an active ingredient, having promoting actions on the synthesis of the collagen and the glycosaminoglycan in a human dermal fibroblast and useful as a therapeutic agent, etc., for dermatopathy such as a wound or burn. SOLUTION: This preparation for external use for skin comprises a midkeine(MK) as an active ingredient. When the medicine is preferably used for treating dermatopathy, it is administered in the dosage form such as an ointment, a cream, a lotion, a milky lotion or a gel for external use. A pharmacodynamically effective ingredient such as a tumor growth factor-β, interleukin-1, an epithelial growth factor, γ-interferon or an antimicrobial agent and further a mineral oil, an animal or a vegetable oil, an emulsifying agent, a gelling agent, water polyethylene glycols, a synthetic oil, etc., can be blended in addition to the MK. The amount of the MK blended is, e.g. 0.0001-50wt.%, especially preferably 0.0001-10wt.%.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an external preparation for skin,
More specifically, it relates to a skin external preparation having a collagen and glycosaminoglycan synthesis promoting action and useful for treating skin diseases such as wounds and for skin makeup.

[0002]

2. Description of the Related Art The dermis of the skin has many extracellular spaces, and that part is filled with a macromolecular network called extracellular matrix. The main constituents of this extracellular matrix are fibrous proteins typified by collagen and glycosaminoglycans (also called acidic mucopolysaccharides, including hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate). Glycosaminoglycans are usually present in the form of proteoglycans bound to proteins, retain a large amount of water and have a gel form, and have a structure in which fibrous proteins are embedded. On the other hand, collagen is a major protein in the extracellular matrix and functions to maintain the normal morphology of tissues. As described above, collagen and glycosaminoglycan maintain the skin morphology and retain water in the dermis and are said to be deeply involved in the recovery of scleroderma, keloid and injury. Further, collagen and glycosaminoglycan are also contained in a large amount in connective tissues such as joints, and are deeply involved in the progression of symptoms such as arthritis and rheumatism.

The biosynthesis of these collagens and glycosaminoglycans is carried out by TGF-β, IL-1, EGF and γ-I.
It is said that it is controlled by factors such as FN, but its action is not yet satisfactory. Also, some substances that promote the synthesis of collagen and glycosaminoglycan are known, but these are also not sufficiently satisfactory.

[0004]

Accordingly, an object of the present invention is to provide a new collagen and glycosaminoglycan synthesis promoter useful for treating skin diseases such as wounds.

[0005]

Therefore, the present inventor has focused on midkine (Midkine, hereinafter referred to as “MK”), which is a retinoic acid-induced growth / differentiation control factor during embryogenesis, and has various actions. After considering,
It was found that this MK has an action of promoting collagen and glycosaminoglycan synthesis in human skin fibroblasts and is useful as a therapeutic drug for skin diseases, and completed the present invention.

That is, the present invention provides a collagen synthesis promoter containing MK as an active ingredient. The present invention also provides a glycosaminoglycan synthesis promoter containing MK as an active ingredient. Furthermore, the present invention provides a therapeutic agent for skin diseases containing MK as an active ingredient. Furthermore, the present invention provides an external preparation for skin containing MK.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The MK used in the present invention is known as a retinoic acid-induced growth / differentiation factor during embryogenesis as described above (Biochem. Biop.
hys. Res. Commun. , 151: 1312-
1318 (1988), J. Biol. Chem. , 2
65, 10765-10770 (1990), Bioc.
hem. Biophys. Res. Commun. , 1
77: 652-658 (1991)). This MK is widely expressed in the middle stage of mouse embryogenesis, and then its expression is restricted to several organs, and finally it is found only in the kidney after 15 days of the fetal period. MK is a secretory protein with a molecular weight of 13 kilodaltons, which is composed of amino acids rich in basic amino acids and cysteine. This polypeptide has a heparin-binding ability in vitro and is
It is known to have an action of promoting the proliferation of cell lines, rat chromaffin cell lines and NIH3T3 cells as well as neurite outgrowth of rat fetal brain cells (J. Neurosci. Res). ., 35:53
0-539 (1993), Dev. Brain Re
s. , 75: 201-205 (1993), Neuro.
sci. Lett. , 160: 9-12 (199
3)). However, nothing is known about the action of MK on collagen and glycosaminoglycan.

[0008] Such MK can be extracted from mouse embryos as described above, but can also be synthesized using gene recombination technology (Biochem. Biophy.
s. Res. Commun. , 177: 652-658.
(1991)).

MK has an action of promoting the synthesis of collagen and glycosaminoglycan in human fibroblasts, as shown in the Examples below, and causes damage to the fibrous form of skin such as wounds, burns, scleroderma, and keloids. It is useful as a therapeutic agent for skin diseases accompanied by abnormalities. Further, as described above, since collagen and glycosaminoglycan are deeply involved in the fiber morphology maintenance and water retention in the skin, MK is also useful as a moisturizing component of a skin external preparation including skin cosmetics.

The dosage form of the therapeutic agent for skin diseases of the present invention may be oral, injection or external use, but external use is particularly preferable. Examples of the form of the external preparation include ointments, creams, lotions, emulsions and gels. In addition to MK, these external preparations include TGF-β, IL-1, EGF, γ-
In addition to other medicinal components such as IFN and antibacterial agent, mineral oil, animal and vegetable oil, emulsifier, gelling agent, water, polyethylene glycols, synthetic oil and the like can be added. Further, when MK is used as an external preparation for skin, such a form can be adopted.

The amount of MK to be added to these external preparations is not particularly limited, but 0.0001 to 50% by weight, particularly 0.0
001-10 weight% is preferable.

[0012]

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

Example 1 (1) Normal fibroblasts were transplanted from a skin biopsy sample. 10% using a 35 mm Petri dish
The cells were grown in Dulbecco's modified Eagle medium (DMEM medium) containing fetal bovine serum (FBS), and subcultured every 6 days. Cells that had been subcultured 8 to 10 times were used in the following experiments.

(2) Recombinant mouse MK (Biochem. Biophys. Re) at various concentrations in DMEM containing 0.5% dialyzed FBS of the sufficiently cultured cells.
s. Commun. , 177: 652-658 (199
The cells were further cultured with 1)) for 48 hours. The last 18 of the culture
During the time, the cells were treated with [2,3-tritium] proline (20 uCi / ml) in the presence of ascorbic acid (30 μg / ml).
Labeled with. Then, the culture solution was collected and mixed with a solution of an inhibitor of proteolytic enzyme such that each of N-ethylmaleimide, EDTA (pH 7.0), and phenylmethylsulfonylfluoride had a concentration of 1 mM. Cells were harvested with 0.05% trypsin. And the cell number was measured by the Coulter counter. After centrifugation, the cells are recombined with the culture medium until ready for analysis-2.
Stored at 0 ° C. The amount of radioactivity incorporated into collagen and non-collagen proteins depends on the collagenase (Arch. Biochem. Bi) of purified bacteria (E. coli).
ophys. , 295: 397-403 (1992) and Biochemistry 10: 988-994.
(1971)). Collagenase-sensitive cpm synthesis of collagen and non-collagen proteins
/ Cell or collagenase resistant cpm / cell.

As a result, as shown in Table 1, 100 ng /
Treatment with ml of MK for 48 hours increased collagen synthesis in human fibroblasts up to 1.6-fold. Under these conditions, MK had no effect on cell proliferation.

[0016]

[Table 1]

Example 2 The MK concentration was 100 ng / ml, and the culture time was 48 hours.
The collagen synthesis promoting action of MK was examined in the same manner as in Example 1 except that the action was changed to 4 to 72 hours. As a result, as shown in Table 2, it was revealed that the action of MK to promote collagen synthesis increased with the treatment time.

[0018]

[Table 2]

Example 3 Well-cultured cells treated for 48 hours in the absence or presence of MK (60 ng / ml) were labeled as in Example 1. The culture solution was collected, the cell layer was peeled off, and the mixture was homogenized with 0.5 M acetic acid. Then 13,000 at 4 ° C
It was centrifuged at × g. The supernatant after centrifugation is combined with the culture solution and
It was dialyzed at 0 ° C. against 0.1% acetic acid and lyophilized. The lyophilized product was pepsin (50 μg for 18 hours at 4 ° C).
/ Ml). Ammonium sulfate (ammonium sulfate) (176
After precipitation with (mg / ml), pepsin-resistant protein was separated on 4-15% SDS-PAGE in a non-reducing state and subjected to autoradiography. The intensity of bands corresponding to α1 (type III collagen), α1 (type I collagen), and α2 (type I collagen) was determined by a scanning densitometer (Cliniscan Helena).
Laboratories). As a result, the relative amount of type III collagen before and after treatment with MK did not change. Therefore, it was revealed that MK stimulates both type I and type III collagen to the same degree.

Example 4 A sufficient number of fibroblasts were dialyzed with 0.5 ml of dialyzed FBS.
48% with a given concentration of MK in DMEM containing a concentration of 48%
Time processed. Among them, in the latter half 24 hours 6-tritium glucosamine (1.2 TBq / mmol, Amersham) (2
5 μCi / ml). The culture solution and the trypsin-treated (0.25%) cell layer precipitate were combined together as a carrier glycosaminoglycan mixture consisting of hyaluronic acid, dermatan sulfate, chondroitin 4- or 6-sulfate, and heparan sulfate, and freeze-dried. Sample is 0.5M
It was dissolved in NaOH and stored overnight at 4 ° C. pH 7.0
And then added an equal volume of 0.1 containing 5 mM CaCl _2.
M Tris-HCl buffer (pH 7.8) was added. The sample was boiled for 30 minutes and then subjected to actinase (Kaken Scientific) degradation at 50 ° C. for 48 hours. It was subsequently precipitated with 10% trichloroacetic acid. The supernatant after precipitation was dialyzed against distilled water overnight at 4 ° C. and then freeze-dried. A portion was taken to measure total glycosaminoglycan synthesis and measured in a liquid scintillation spectrometer (Beckman 9800). The separated glycosaminoglycan was subjected to two-dimensional electrophoresis on a cellulose acetate membrane. The first dimension is 0.1M pyridine / 0.45M
1.5 mA for 1 hour at 1 mA / cm using formic acid (pH 3), 1 mA / cm for 2nd dimension using 0.1 M barium acetate (pH 8)
I went there for 5 hours. Membranes were stained with 0.1% Alcian blue and bleached with 0.1% acetic acid. A part of the membrane containing glycosaminoglycan was scraped off and redissolved with 1 ml of dioxane. Then, their radioactivity was measured by a liquid scintillation spectrometer. The isolated material was subjected to chemical and enzymatic treatments to identify glycosaminoglycans. For example, a sample of glycosaminoglycan having a total radioactivity of up to 10 ⁵ cpm was digested with hyaluronidase from Streptomyces actinomycetes, hyaluronidase from testis and chondroitinase ABC, AC. In addition, it was treated with sodium nitrite for the identification of N-sulfated glycosaminoglycan (Anal. Bio.
chem. , 45: 462-468 (1972), ibid.5.
2: 652-656 (1973)).

As a result, as shown in Table 3, MK is 10
Treatment with a concentration of 0 ng / ml for 48 hours increased total glycosaminoglycan synthesis 1.9-fold. The major constituent of glycosaminoglycans stimulated by MK treatment was hyaluronic acid.

[0022]

[Table 3]

[0023]

The present invention is useful as a therapeutic agent for skin diseases such as wounds, burns, scleroderma and keloids, and as a skin cosmetic.

Front page continuation (72) Inventor Takashi Muramatsu 2845 Kuroishi, Hira Needle, Tenpaku-ku, Nagoya-shi, Aichi Prefecture 6 block, Hira-needle house 35

Claims (4)

[Claims] 1. A collagen synthesis promoter containing midkine as an active ingredient. 2. A glycosaminoglycan synthesis promoter containing midkine as an active ingredient. 3. A therapeutic agent for skin diseases, which comprises midkine as an active ingredient. 4. A skin external preparation containing midkine.