JP2022544701A - 発毛促進活性を有するペプチド、及びその用途 - Google Patents
発毛促進活性を有するペプチド、及びその用途 Download PDFInfo
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- JP2022544701A JP2022544701A JP2022511060A JP2022511060A JP2022544701A JP 2022544701 A JP2022544701 A JP 2022544701A JP 2022511060 A JP2022511060 A JP 2022511060A JP 2022511060 A JP2022511060 A JP 2022511060A JP 2022544701 A JP2022544701 A JP 2022544701A
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Abstract
Description
自動ペプチド合成器(Milligen 9050、Millipore、米国)を利用し、下記表1に記載された配列番号1のアミノ酸配列を有するペプチドを合成し、C18逆相高性能液体クロマトグラフィ(HPLC)(Waters Associates、米国)を利用し、それら合成されたペプチドを純水分離した。カラムは、ACQUITY UPLC BEH300 C18(2.1mmX100mm、1.7μm、Waters Co.、米国)を利用した。
1.毛乳頭細胞(dermal papilla cell)の増殖促進効果確認
配列番号1のアミノ酸配列からなるペプチドの毛嚢細胞の増殖促進効果を確認する。具体的には、ヒト毛嚢毛乳頭細胞(human hair follicle dermal papilla cells)を、5x103細胞/ウェルの密度で、96ウェルプレートにシーディングした後、16時間培養した。無血清培地に替えた後、前記ペプチドを、0.5μM、5μMまたは50μMそれぞれの濃度で処理して72時間培養した。陽性対照群として、脱毛治療剤として使用されるミノキシジル(minoxidil)5μMを処理した。その後、細胞増殖を測定するために、MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide))法を遂行した。具体的には、3-(4,5-ジメチルチアゾール-2-イル)-2,5-ジフェニルテトラゾリウムプロミド(MTT)4μg/mlを処理し、4時間後、DMSOを処理してポルマザンを溶解させ、それを、分光光度計を利用し、560nmにおける吸光度を測定した。
配列番号1のアミノ酸配列からなるペプチドの毛嚢細胞の増殖と係わる因子の活性化効果を確認する。具体的には、ヒト毛嚢毛乳頭細胞を、4x105細胞/ウェルの密度で、6ウェルプレートにシーディングした後、16時間培養した。無血清培地に替えた後、前記ペプチドを、0.5μM、5μMまたは50μMそれぞれの濃度で処理し、30分間培養した。陽性対照群として、ミノキシジル5μMを処理した。細胞を収穫し、細胞溶解物を準備した後、p-ERK抗体及びp-AKT抗体(Santacruz Biotechnology、米国)を利用し、リン酸化されたERK、及びリン酸化されたAKTに対するウェスタンブロッティングを進めた。
配列番号1のアミノ酸配列からなるペプチドの毛嚢細胞の増殖メカニズム活性化効果を確認する。具体的には、ヒト毛嚢毛乳頭細胞を、4x105細胞/ウェルの密度で、6ウェルプレートにシーディングした後、16時間培養した。無血清培地に替えた後、前記ペプチドを、0.5μM、5μMまたは50μMそれぞれの濃度で処理して24時間培養した。陽性対照群として、ミノキシジル5μMを処理した。細胞を収穫し、核タンパク質抽出キット(Merck、米国)を使用し、核タンパク質を分離した。β-カテニン抗体(Santacruz Biotechnology、米国)を利用し、毛乳頭細胞の増殖と係わる因子であるβ-カテニンに対するウェスタンブロッティングを進めた。
1.毛乳頭細胞でDKK-1発現抑制効果の確認
配列番号1のアミノ酸配列からなるペプチドの毛嚢細胞においけ、脱毛関連因子であるDKK-1の発現が抑制されるか否かということを確認する。具体的には、ヒト毛嚢毛乳頭細胞を、4x105細胞/ウェルの密度で、6ウェルプレートにシーディングした後、16時間培養した。無血清培地に替えた後、刺激剤(stimulator)として、ジヒドロテストステロン(DHT:dihydrotestosterone)10μg/ml、及び前記ペプチドの、0.5μM、5μMまたは50μMそれぞれの濃度で処理して24時間培養した。細胞を収穫し、核タンパク質抽出キットを使用し、核タンパク質を分離した。DKK-1抗体(Santacruz Biotechnology、米国)を利用し、DKK-1に対するウェスタンブロッティングを進めた。前記DHTは、アンドロゲン受容体を活性化させるものであり、脱毛誘発因子であるDKK-1(Dickkopf-related protein1)の発現を増大させる脱毛ホルモンであると知られている。
その結果、図4に示されているように、配列番号1のアミノ酸配列からなるペプチドは、DHT処理による脱毛誘発因子であるDKK-1の発現を抑制することができるということを確認した。
配列番号1のアミノ酸配列からなるペプチドの毛嚢細胞において、脱毛関連因子であるTGFβ-1の発現が抑制されるか否かということを確認する。具体的には、ヒト毛嚢毛乳頭細胞を、4x105細胞/ウェルの密度で、6ウェルプレートにシーディングした後、16時間培養した。無血清培地に替えた後、前記ペプチドを、0.5μM、5μMまたは50μMそれぞれの濃度で処理して24時間培養した。陽性対照群として、ミノキシジル5μMを処理した。細胞収獲及びRNA分離後、cDNA合成キット及びPCR pre-mixを利用してcDNA合成し、下記表2に記載されたTGFβ-1プライマー及びGAPDHプライマーを利用してPCR(polymerase chain reaction)を進めた。前記TGFβ-1は、毛嚢細胞において、アンドロゲン誘導(androgen-inducible)因子であり、脱毛と関連していると知られている。
Claims (6)
- 配列番号1のアミノ酸配列からなる、ペプチド。
- 前記ペプチドのN-末端は、アセチル基、フルオレニルメトキシカルボニル基、ホルミル基、パルミトイル基、ミリスチル基、ステアリル基、ブトキシカルボニル基(Boc)、アリルオキシカルボニル基(Alloc)及びポリエチレングリコール(PEG)からなる群のうちから選択されるいずれか1つの保護基と結合されたものである、請求項1に記載のペプチド。
- 前記ペプチドのC-末端は、アミノ基(-NH2)、三次アルキル基及びアザイド(-NHNH2)からなる群のうちから選択されるいずれか1つの保護基と結合されたものである、請求項1に記載のペプチド。
- 前記ペプチドは、下記のところのような特性から選択されるいずれか1以上を示すものである、請求項1に記載のペプチド:
(a)毛嚢細胞の活性促進、
(b)毛嚢細胞の増殖促進、
(c)毛嚢細胞の死滅抑制、及び
(d)DKK-1(Dickkopf-related protein 1)またはTGF-β1(transforming growth factor-beta 1)の発現抑制。 - 請求項1ないし4のうちいずれか1項に記載のペプチドを有効成分として含む、脱毛改善用または発毛促進用の化粧料組成物。
- 請求項1ないし4のうちいずれか1項に記載のペプチドを有効成分として含む、脱毛予防用または脱毛治療用の薬剤学的組成物。
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JP7323705B2 (ja) | 2023-08-08 |
CN114174314B (zh) | 2023-09-12 |
EP4019531A4 (en) | 2023-04-05 |
KR20210022408A (ko) | 2021-03-03 |
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US20220265538A1 (en) | 2022-08-25 |
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