JP2012062266A - 皮膚線維芽細胞のタンパク質産生促進剤および角化細胞遊走・増殖促進剤 - Google Patents
皮膚線維芽細胞のタンパク質産生促進剤および角化細胞遊走・増殖促進剤 Download PDFInfo
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Abstract
【解決手段】本発明に係る皮膚線維芽細胞のタンパク質産生促進剤および角化細胞遊走・増殖促進剤は、γ−グルタミルトランスペプチダーゼを阻害する化合物を有効成分とする。
【選択図】なし
Description
(式中、R1およびR2の少なくともいずれか一方が一般式(2)〜一般式(6)
(式中、R1がOR10であり、R2がOR11であり、R10およびR11が水素原子を除く。)で示されるホスホン酸ジエステル誘導体。
GGT阻害剤のエラスチン産生能向上作用、α−SMA産生能向上作用、HSP47産生能向上作用、および成熟コラーゲン産生能向上作用を調べるために、以下の実験を行なった。具体的な実験方法は以下のとおり。
ヒト皮膚由来線維芽細胞(CCD-1059SK、DSファーマメディカル株式会社)を、10%FBS(fetal bovine serum)を含むDMEM培地で継代培養し、10%FBSを含むDMEM培地で細胞数を1.0×105個/mlに調整して、直径60mmのプラスチックシャーレ(株式会社グライナー・ジャパン)に6.0×105個ずつ播種し、本培養時に10μMのGGT阻害剤を添加して24時間培養し、10μM GGT阻害剤添加群の細胞を調製した。GGT阻害剤としては、2−アミノ−4−{[3−(カルボキシメチル)フェニル](メチル)ホスホノ}ブタン酸(GGT5a)を用いた。
1)コントロール群
2)10μM GGT阻害剤添加群。
(1)で調製した1)〜2)のサンプル群について、本培養終了後に、細胞を回収し、エラスチンの産生量を、エラスチンを認識する抗体(Monoclonal Antibody to Elastin-Ascites、Acris Antibodies GmbH)を用いたウエスタンブロット法によって解析した。
(1)で調製した1)〜2)のサンプル群について、本培養終了後に、細胞を回収し、α−SMAアクチンの産生量を、α−SMAを認識する抗体(Mouse Anti-Human Smooth Muscle Actin(1A4)、Dako A/S)を用いたウエスタンブロット法によって解析した。
(1)で調製した1)〜2)のサンプル群について、本培養終了後に、細胞を回収し、HSP47の産生量を、HSP47を認識する抗体(Anti-HSP47 Mouse Monoclonal、Stressgen BIOREAGENTS.CORP)を用いたウエスタンブロット法によって解析した。
(1)で調製した1)〜2)のサンプル群について、本培養終了後に、細胞を回収し、I型コラーゲンの産生量を、I型コラーゲンを認識する抗体(COL1A1(C-18), SANTA CRUZ BIOTECHNOLOGY, INC)を用いたウエスタンブロット法によって解析した。上記抗体は成熟コラーゲンおよび前駆体コラーゲンの両方を認識する抗体であり、成熟コラーゲンおよび前駆体コラーゲンは異なる分子量のタンパク質として検出される。成熟コラーゲンを表す分子量70〜90kDaのバンドの濃さを定量的に解析することによって、成熟コラーゲン量を測定することができる。
皮膚の真皮に存在する皮膚線維芽細胞は、コラーゲンやエラスチンなどの細胞外マトリックスを産生する。エラスチンはコラーゲンにからみつくように存在し、皮膚に弾力性を与えることが知られている。このため、皮膚線維芽細胞のコラーゲンおよびエラスチン産生能が亢進することが、皮膚の張りを維持し、皺の予防に重要であると考えられる。
(1)試料の作製
GGT阻害剤の角化細胞の遊走・増殖能向上作用を調べるために、以下の実験を行なった。具体的な実験方法は以下のとおり。
1)コントロール群
2)1μM GGT阻害剤添加群
3)10μM GGT阻害剤添加群。
ケラチノサイトの遊走・増殖能を評価するための指標として、(1)で調製した1)〜3)のサンプル群について、スクラッチ箇所の修復程度を測定した。具体的には、スクラッチ直後(0時間後)のスクラッチ幅をモニター画面上で測定し、a(cm)とし、24時間後に同一箇所のスクラッチ幅を測定し、b(cm)とした。
修復幅=(0時間後のスクラッチ幅a)−(24時間後のスクラッチ幅b)…(1)
修復率は、コントロール群における修復幅を100%としたときの、相対値として求めた。
皮膚の表皮を形成する角化細胞は、基底層で分裂・増殖した後、角層へと移行し、最終的には垢となってはがれ落ちる。このため、角化細胞の遊走・増殖能が亢進することは、皮膚の新陳代謝の促進、皮膚のバリア機能の維持に重要な働きを示す。実験例2によって、GGT阻害剤が角化細胞の遊走・増殖能を亢進させることが確認され、GGT阻害剤が皮膚の新陳代謝の促進効果、皮膚のバリア機能の維持効果をもたらすことが示唆された。
以下に示す処方の美容液を常法により製造した。
(組成) (重量%)
ソルビット 4.0
グリセリン 3.0
ブチレングリコール 3.0
ポリエチレングリコール 1500 5.0
POE(20)オレイルアルコールエーテル 0.5
ショ糖脂肪酸エステル 0.2
メチルセルロース 0.2
GGT阻害剤 0.005
精製水 全体で100となる量
〔処方例:乳液〕
以下に示す処方の乳液を常法により製造した。
(処方) (重量%)
グリセリン 1.5
ショ糖脂肪酸エステル 1.5
モノステアリン酸ソルビタン 1.0
スクワラン 7.5
ジプロピレングリコール 5.0
GGT阻害剤 0.005
精製水 全体で100となる量
〔処方例:クリーム〕
以下に示す処方のクリームを常法により製造した。
(処方) (重量%)
グリセリン 3.0
ブチレングリコール 3.0
スクワラン 19.0
ステアリン酸 5.0
モノステアリン酸グリセリン 5.0
モノステアリン酸ソルビタン 12.0
モノステアリン酸ポリエチレンソルビタン 38.0
エデト酸ナトリウム 0.03
GGT阻害剤 0.005
精製水 全体で100となる量。
Claims (5)
- γ−グルタミルトランスペプチダーゼを阻害する化合物を有効成分とすることを特徴とする、皮膚線維芽細胞のタンパク質産生促進剤。
- 上記タンパク質は、エラスチン、ヒートショックプロテイン47、α−平滑筋アクチンおよび成熟コラーゲンからなる群から選択される1以上のタンパク質であることを特徴とする、請求項1に記載の皮膚線維芽細胞のタンパク質産生促進剤。
- 上記化合物は、脱離基を有するホスホン酸ジエステル誘導体を含む、請求項1または2に記載の皮膚線維芽細胞のタンパク質産生促進剤。
- γ−グルタミルトランスペプチダーゼを阻害する化合物を有効成分とすることを特徴とする、角化細胞遊走・増殖促進剤。
- 上記化合物は、脱離基を有するホスホン酸ジエステル誘導体を含む、請求項4に記載の角化細胞遊走・増殖促進剤。
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