Material for promoting skin basement formation

Abstract

A method for promoting skin basement membrane formation comprises administering matrix metalloproteinase inhibitor or matrix metalloproteinase inhibitor and matrix protein production promoting agent. The matrix metalloproteinase inhibitor is N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbutanamide hydrochloride.

Description

[0001] This application claims the priority of Japanese Patent Application No. 2000-87574, filed on Mar. 27, 2000, which is incorporated herein by reference. This application is a continuation of U.S. patent application Ser. No. 10 / 648,485, filed on Aug. 27, 2003, which is a continuation of U.S. patent application Ser. No. 09 / 979,712, filed on Nov. 26, 2001 and later abandoned (International Patent Application Number PCT / JP200102507 filed Mar. 27, 2001).FIELD OF THE INVENTION

[0002] The present invention relates to a skin basement membrane stabilizing agent. In addition, the present invention relates to artificial skin formation promoting agent and a production method for artificial skin. BACKGROUND ART

[0003] In the field of cosmetics and dermatology, a wide variety of measures have been proposed and attempted to alleviate or treat injuries caused by the effects of the external environment and aging including the exposure of the skin to sunlight. For example, examples of skin chang...

Examples

example 1

Production of Artificial Skin Using Matrix Metalloproteinase Inhibitor

[0078] Collagen gel was obtained by preparing 10 ml of a collagen solution of suspended fibroblasts (0.3 to 1×105 cells / ml) originating in human dermis (product I-AC of Koken Co., Ltd. was used for the collagen) on ice followed by gelling the collagen at 37° C. in a 60 mm Petri dish. Next, the gel was peeled from the walls of the Petri dish and the collagen gel was contracted in a CO2 incubator.

[0079] This contracted collagen gel equivalent to dermis was placed on a metal mesh and a glass ring (inner diameter: 12 mm) was placed over this gel. 0.4 ml of a suspension of epidermal keratinocytes (1×106 / ml) (mixed medium of KGM-DMEM containing 5% fetal calf serum (1:1)) were placed inside the glass ring to prevent leakage of liquid. After adhering keratinocytes in a CO2 incubator overnight, the ring was removed on the following day. The above medium was filled to the boundary of the epidermal layer and culturing was ...

example 2

Production of Artificial Skin Using Both Matrix Metalloproteinase Inhibitor and Matrix Protein Production Promoting

Agent

[0084] Although the same method as Example 1 was repeated, the following substances were used as test substances. [0085] (1) 10 μM matrix metalloproteinase inhibitor CGS27023A only; [0086] (2) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10 ng / ml of matrix metalloproteinase protein production promoting agent, Transforming Growth Factor α (TGFa); [0087] (3) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10 ng / ml of matrix protein production promoting agent, Transforming Growth Factor β1 (TGFb1); [0088] (4) 10 μM matrix metalloproteinase inhibitor CGS27023A, 10 ng / ml of matrix protein production promoting agent, Transforming Growth Factor α (TGFa) and 10 ng / ml of Transforming Growth Factor β1 (TGFb1); and [0089] (5) culturing of a control to which none of the above were added.

[0090] The formed artificial skin was stained by hematoxylin-eosin (H ...

example 3

Preparation of Artificial Skin Culture Medium

[0096] Matrix metalloproteinase inhibitor CGS27023A or MMP-inhibitor were added to a concentration of 10 μM to basal medium comprising a 1:1 mixture of KGM (keratinocyte growth medium), typically used to culture epidermic cells, and Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, typically used to culture fibroblasts. Furthermore, KGM was prepared by adding HEPES (6.7 g / L), NaHCO3 (1.2 g / L), insulin (5 mg / L), transferrin (10 mg / L), hydrocortisone (0.5 mg / L) and phosphorylethanolamine (14.1 mg / L) to MCDB153 medium (10.93 g / L) and adjusting the pH to 7.4 followed by the addition of 20 g of bovine pituitary extract (Far East Pharmaceutical).

[0001] This application claims the priority of Japanese Patent Application No. 2000-87574, filed on Mar. 27, 2000, which is incorporated herein by reference. This application is a continuation of U.S. patent application Ser. No. 10 / 648,485, filed on Aug. 27, 2003, which is a continuation of U.S. patent application Ser. No. 09 / 979,712, filed on Nov. 26, 2001 and later abandoned (International Patent Application Number PCT / JP200102507 filed Mar. 27, 2001).FIELD OF THE INVENTION

[0002] The present invention relates to a skin basement membrane stabilizing agent. In addition, the present invention relates to artificial skin formation promoting agent and a production method for artificial skin. BACKGROUND ART

[0003] In the field of cosmetics and dermatology, a wide variety of measures have been proposed and attempted to alleviate or treat injuries caused by the effects of the external environment and aging including the exposure of the skin to sunlight. For example, examples of skin chang...

Examples

example 1

Production of Artificial Skin Using Matrix Metalloproteinase Inhibitor

[0078] Collagen gel was obtained by preparing 10 ml of a collagen solution of suspended fibroblasts (0.3 to 1×105 cells / ml) originating in human dermis (product I-AC of Koken Co., Ltd. was used for the collagen) on ice followed by gelling the collagen at 37° C. in a 60 mm Petri dish. Next, the gel was peeled from the walls of the Petri dish and the collagen gel was contracted in a CO2 incubator.

[0079] This contracted collagen gel equivalent to dermis was placed on a metal mesh and a glass ring (inner diameter: 12 mm) was placed over this gel. 0.4 ml of a suspension of epidermal keratinocytes (1×106 / ml) (mixed medium of KGM-DMEM containing 5% fetal calf serum (1:1)) were placed inside the glass ring to prevent leakage of liquid. After adhering keratinocytes in a CO2 incubator overnight, the ring was removed on the following day. The above medium was filled to the boundary of the epidermal layer and culturing was ...

example 2

Production of Artificial Skin Using Both Matrix Metalloproteinase Inhibitor and Matrix Protein Production Promoting

Agent

[0084] Although the same method as Example 1 was repeated, the following substances were used as test substances. [0085] (1) 10 μM matrix metalloproteinase inhibitor CGS27023A only; [0086] (2) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10 ng / ml of matrix metalloproteinase protein production promoting agent, Transforming Growth Factor α (TGFa); [0087] (3) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10 ng / ml of matrix protein production promoting agent, Transforming Growth Factor β1 (TGFb1); [0088] (4) 10 μM matrix metalloproteinase inhibitor CGS27023A, 10 ng / ml of matrix protein production promoting agent, Transforming Growth Factor α (TGFa) and 10 ng / ml of Transforming Growth Factor β1 (TGFb1); and [0089] (5) culturing of a control to which none of the above were added.

[0090] The formed artificial skin was stained by hematoxylin-eosin (H ...

example 3

Preparation of Artificial Skin Culture Medium

[0096] Matrix metalloproteinase inhibitor CGS27023A or MMP-inhibitor were added to a concentration of 10 μM to basal medium comprising a 1:1 mixture of KGM (keratinocyte growth medium), typically used to culture epidermic cells, and Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, typically used to culture fibroblasts. Furthermore, KGM was prepared by adding HEPES (6.7 g / L), NaHCO3 (1.2 g / L), insulin (5 mg / L), transferrin (10 mg / L), hydrocortisone (0.5 mg / L) and phosphorylethanolamine (14.1 mg / L) to MCDB153 medium (10.93 g / L) and adjusting the pH to 7.4 followed by the addition of 20 g of bovine pituitary extract (Far East Pharmaceutical).

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