JP2017515829A - 抗微生物ペプチドの生成を誘導するためのナイアシンアミド - Google Patents
抗微生物ペプチドの生成を誘導するためのナイアシンアミド Download PDFInfo
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- niacinamide
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/67—Vitamins
- A61K8/673—Vitamin B group
- A61K8/675—Vitamin B3 or vitamin B3 active, e.g. nicotinamide, nicotinic acid, nicotinyl aldehyde
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
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- A—HUMAN NECESSITIES
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
[実施例1から6]
ナイアシンアミドはLL−37遺伝子発現を誘導するがプソリアシン発現を変化させないことを証明するための実施例
表1に示されている通りの下記の試料を、プソリアシン発現に対するそれらの効果および発現されたプソリアシンAMP(LL−37)を測定するために調製した。
正常ヒト新生児表皮初代ケラチノサイト(NHEK)、ケラチノサイト成長培地(KGM)および成長サプリメント、抗生物質(ペニシリンおよびストレプトマイシン)、大腸菌(E.coli)株(ATCC株10536)、大腸菌055:B5株由来のリポ多糖(LPS)、1×リン酸緩衝食塩液、10mMリン酸ナトリウム緩衝液、ナイアシンアミド、無菌カップ(両端はオープンバレル形状であり、開口端面積は2.27cm2であり、ロッドは、ハードテフロン(付録1)、ヴァセリン原料(付録2)、プソリアシン(S100A7)(サイクレックス)およびLL−37ELISAキット(ハイカルト)からできている丸型先端である、マッコンキー寒天培地、TSA培地(ユニリーバ研究報告書番号:BL110086)。
新生児包皮から調製したヒト初代ケラチノサイトを(ロンザインディア)から入手し、定義された成長サプリメント(インビトロジェン)を加えた無血清ケラチノサイト成長培地中で培養した。ケラチノサイトを、12および24ウェル組織培養プレート(BDファルコン)中で培養し、60〜70%の集密で細胞を処理した。ケラチノサイト刺激は、成長サプリメントを加えたKGM中で実施し、すべての実験を第3から5代継代の間に行った。
NHEKを、12ウェルプレート(BD、ファルコン)中、ウェル当たり6×104細胞で培養した。24時間のインキュベーション後、50%の培地を新鮮培地で置きかえた。3日目に、細胞を、異なる濃度のナイアシンアミドで新鮮培地とともに16〜18時間処理した。細胞をRLT緩衝液(キアゲン)中に採取し、試料を、RNA単離に使用するまで−70℃で貯蔵した。
リアルタイムPCRは、製造業者(バイオ・ラッド、米国)の説明書のように、熱制御されたクロモ4マシン(バイオ・ラッド、米国)を使用するSYBRグリーン法を使用して行った。5μlのcDNAの1:10希釈試料および1.5pmoleの特定のプライマーを、各反応において、標準化されたPCRプログラム(すなわち、94℃で5分間、続いて、50サイクルの「94℃で15秒間、60℃で30秒間、72℃で30秒間)を使用することによって使用し、2−ΔΔCT法を使用することによって結果を分析した。
初代ケラチノサイトを、12ウェルプレート(BDファルコン)に播種した。60から70%の密集度において、細胞を異なる濃度のナイアシンアミドで72時間処理した。72時間のインキュベーション後、細胞培養上清を、ELISAに使用するまで−70℃で貯蔵した。すべてのELISA試薬および試料を、アッセイ前に室温にした。細胞培養上清試料を、使用前に5000rpmで10分間遠心分離した。100μlの細胞培養上清の未希釈試料を、サイクレックス製のELISAキットを使用することにより、プソリアシンレベルを確認するために使用した。臨床試料を希釈緩衝液中で1:25に希釈し、ELISAによってプソリアシンレベルを確認するために希釈試料から100μlを使用した。LL−37の事例においては、ELISA(ハイカルトバイオテック)によってLL−37レベルを確認するために、インビトロから100μlの未希釈試料および臨床試料を使用した。
皮膚におけるプソリアシンおよびLL−37の分泌誘導を証明するための、ナイアシンアミドを含有するスキンローションを用いるインビボ実験
インビトロ実験からの所見を裏付けるために、健康なヒトボランティアに対するナイアシンアミドの効果をインビボで試験した。ボランティアに、ナイアシンアミド(3%)を加えたおよび加えていないローションを、1日2回、前腕に塗布するように求めた。7日間の塗布後、その箇所をPBS中で抽出し、プソリアシンおよびLL−37AMPの検出のためにELISAを実施した。
大腸菌培養調製物
大腸菌(10536)のグリセロールストックを、30mlのTSB培地中に接種し、培養物を、シェーカーインキュベーター内、37℃で終夜インキュベートした。終夜、培養物をTSAスラント上で二次培養し、37℃で終夜インキュベートし、次いで、スラントを4℃で貯蔵した(15日に一度、スラントを新たに調製した)。
この技術は、ASTM法、E2752−10(抗菌性の残効性の評価のための標準ガイド)に含まれる。カップおよびロッドはテフロンで構成されており、カップの面積は2.84cm2である。カップは皮膚上に緩衝液を保持するために使用し、ロッドは、スクラビングのため、皮膚AMPおよび/または皮膚からの残留細菌のより良好な回収のために使用した。スクラビングの持続時間は1分であった。抽出された試料を、分析(付録1)のために1.5mlのエッペンドルフ管中に収集した。
10μlの大腸菌の1OD620培養物を、前腕の印を付けた領域に2.27cm2面積で塗布した(クリーム、基剤クリームおよびナイアシンアミドクリームが塗布されていない箇所)。培養物を各箇所に均一に広げた。15分間の接触時間後、1mlのPBS中、カップスクラブ法によって試料を抽出した。試料を無菌微量遠心管中に収集した。
1. ボランティアに、毎朝入浴後に研究施設に来るように求めた。
ナイアシンアミドによる誘導によって分泌された抗微生物ペプチド(AMP)の種類および量を決定するための実験
ケラチノクテ(keratinocte)細胞においてナイアシンアミドの結果として誘導されたAMPを決定するために、下記の手順を使用した。
感染に対して自身を防御する皮膚の能力についてのヒトボランティア研究
下記のプロトコールを使用する実験を行った:
1. ボランティアに、使用するためのラックス(商標)石鹸を与え、研究開始前の7日間、前腕にいかなる他の石鹸も保湿剤も使用しないように求めた。
Claims (7)
- 人体の外部表面に塗布された場合に、抗微生物ペプチド(AMP)の分泌を誘導するためのナイアシンアミドの使用。
- ナイアシンアミドが、ケラチノサイトからのAMPの分泌を誘導する、請求項1に記載の使用。
- 前記外部表面の免疫を改善させるための、請求項1に記載の使用。
- 前記外部表面が、皮膚、頭皮または口腔を含む、請求項1または2に記載の使用。
- 前記使用が非治療的使用である、請求項1から4のいずれか一項に記載の使用。
- 人体の外部表面に塗布された場合に、抗微生物ペプチド(AMP)の分泌を予防的に誘導するための、ナイアシンアミド。
- 抗微生物ペプチド(AMP)の分泌を誘導することにより、頭皮に保護を提供するための方法であって、ナイアシンアミドを、人物の毛髪/頭皮に塗布するステップを含む、方法。
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US11793741B2 (en) | 2017-12-18 | 2023-10-24 | Conopco, Inc. | Topical composition |
CN111683649A (zh) * | 2017-12-18 | 2020-09-18 | 荷兰联合利华有限公司 | 基于烟酰胺衍生物的局部组合物 |
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