JP2015531881A - 相乗作用する美容成分の組み合わせを特定する方法 - Google Patents
相乗作用する美容成分の組み合わせを特定する方法 Download PDFInfo
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Abstract
Description
「相加作用」は、有効成分の組み合わせにより提供される効果が、それらの個々の効果の合計と等しい又は実質的に等しいことを意味する。例えば、相加作用は、第1の有効成分が、単独で使用した場合に、酸素消費速度において10%の改善をもたらし、かつ第2の有効成分が、単独で使用した場合に、酸素消費において20%の改善をもたらし、組み合わせて使用した場合にの酸素消費速度に対し30%の改善が提供される場合に、実証される。本例において、「相加作用を超える」は、第1及び第2の有効成分の組み合わせが酸素消費速度を30%超改善する場合に記載される。
角化細胞は、一般に表皮において顕著な細胞型として認識されており、典型的には表皮で見られる細胞の約95%を構成する。角化細胞は、表皮の基底層の下部に存在する表皮幹細胞から分化により形成される。角化細胞は分裂し、表皮層(例えば、有棘細胞層及び顆粒層)内を上方に移動するにつれて分化し、最終的には角質層中で角質細胞となる。分化プロセス中、角化細胞はますますケラチンを産生するようになり(「角質化」)、最終的には、細胞周期から永久に離脱して、角質層の硬性の外層を構成する角質細胞を形成するようになる。角質細胞は、新しい細胞が加わるにつれ、落屑を介し最終的には脱離する。酸化的ストレスが角化細胞の代謝を低下させる場合に、角化細胞の倍加及び分化速度は低下し、又は更には停止する場合がある。これは同様に、損失分の角質細胞を角質層に補充する速度も低下させ、最終的には皮膚のバリア特性の望ましくない低下を招き得る。したがって、角化細胞に対するある種のストレッサー(例えば、紫外線A波、紫外線B波、巻き煙草/煙草の煙、煙霧、オゾン、エンジン排気、揮発性有機化合物)からの酸化的ストレスに関する代謝への望ましくない影響を軽減、予防及び/又は逆転する、スキンケア有効成分の相乗作用する組み合わせを特定することは望ましいものであり得る。
場合によっては、線維芽細胞、並びに場合により、皮膚において通常見られる他の種類の細胞に対する、特定のストレッサー由来の酸化的ストレスに関する望ましくない影響を、軽減、予防及び/又は逆転させるスキンケア有効成分の相乗作用する組み合わせを特定することが望ましいものであり得る。線維芽細胞皮膚及び皮下(すなわち、皮膚下)層の真皮層に見られ、一般に、細胞外マトリックス(「ECM」)及びコラーゲンを合成して哺乳類組織の構造骨格を提供する細胞として認識されている。ECM及びコラーゲンは、皮膚に引張り強さ及び弾性を提供することにより、身体に対する応力及びひずみを和らげるよう機能する。線維芽細胞は、創傷治癒において重要な役割も果たす。酸化的ストレスが線維芽細胞の代謝を低下させると、身体がコラーゲン及びECMを合成する能力が低下して、結果として皮膚の外見は垂れ下がり、薄くなる場合がある。及び、身体が創傷を治癒する能力が妨害され得る。
皮膚の健康及び/又は外観上の効果は健康な皮膚細胞により提供されることから、本願における方法により特定された、2つ以上のスキンケア有効成分の相乗作用する組み合わせを化粧品組成物に組み込むことは望ましいものであり得る。すなわち、化粧品組成物中にスキンケア有効成分を原料として含ませることは望ましいものであり得る。このような化粧品組成物には、皮膚科学的に許容可能な担体、並びに角化細胞、線維芽細胞及び/又は皮膚において通常見られるその他の種類の細胞(例えば、メラニン産生細胞、筋細胞、幹細胞、脂腺細胞、神経細胞、及び脂肪細胞)に対する酸化的ストレスに関する望ましくない代謝効果を軽減、予防及び/又は逆転するスキンケア有効成分の相乗作用する組み合わせを含有させることができる。特に好適な例では、組成物は、安全かつ有効な量のナイアシンアミド及びトコキノンを含有し得る。この実施例では、ナイアシンアミドは、0.001%〜20%(例えば、0.1〜10%又は0.5〜5%)で加えることができ、トコキノンは、0.0001%〜20%(例えば、0.01〜10%、又は0.1%〜5%、又は0.5%〜3%)で加えることができる。ナイアシンアミド及びトコキノン単独の使用と比較して、過酸化水素に曝露された線維芽細胞の酸化的リン酸化及び解糖代謝経路を改善させるのに十分なナイアシンアミド及びトコキノンの組み合わせを添加することが望ましい場合がある。
この方法は、調節された環境下での酸化的リン酸化及び解糖代謝経路に関連する代謝指標を、非致死的に同時に検出することを可能にする。この方法は、動態データの取得も可能にする。ストレッサー、被検物質、又は被検物質の組み合わせに対する代謝応答を評価する場合に、2つの代謝経路がどのようにしてストレッサー又は被検物質と相互作用するのか及び/又は互いに相互作用するのかを理解するためには、代謝経路の両方を同時に評価することが重要である。加えて、代謝経路をリアルタイムで(すなわち、周期的な測定を繰り返して)モニターして、静的なデータのみを提供する方法を使用したときには損なわれ得る傾向及び/又は一過性の応答を観察することが重要である。したがって、破壊試験は、典型的にはそれらの試験は単回測定のみを可能にすることから、本願における使用に好適ではない。
本明細書に記載の方法により試験される細胞は、当該技術分野において既知の任意の好適な手法により得ることができる。例えば、細胞は、天然環境から単離することができ(例えば、ヒトの皮膚)、又は好適な市販元から購入することもできる。細胞は、初代細胞株であっても(すなわち、生体組織源から単離し、通常の組織培養条件下で増殖させた細胞株)、不死化細胞株であってもよい(すなわち、増殖及び倍加指数が、初代細胞株より得られるものよりも大幅に増加するよう、化学的又は遺伝的改変により改変を受けている細胞株)。いくつかの実施態様では、好適な市販元から凍結初代又は不死化細胞株を得て、組織培養フラスコ(例えば、BD Biosciencesから入手可能なコラーゲンコートT−75フラスコ)内で適切な増殖培地に希釈しインキュベートする(例えば、37℃)ことができる。試験用の細胞が用意できたならば、好適な試験容器(例えば、マルチウェル容器、1ウェル容器、1本以上のチューブ、及び12ウェルプレート、24ウェルプレート又は96ウェルプレートなどの従来の試験プレート)内に入れることができる。試験培地は、試験容器内で調製して、細胞外環境を提供することができる。試験培地は、試験中(例えば、少なくとも4時間、8時間、12時間、24時間、48時間、72時間)、細胞を生存させかつ健康に維持する必要がある。各ウェル中の細胞数は、好適な測定値を取得するのに十分なものとすべきである(例えば、角化細胞については4×104個、及び線維芽細胞については1×105個)。試験中、細胞は、試験培地に懸濁してよく、試験容器内に配置された好適な基質に接着させてよく、及び/又は試験容器に接着させてもよいことは理解される。
細胞は、当該技術分野において既知の任意の好適な手法によりストレッサー又は被検物質に曝露することができる。いくつかの実施態様では、細胞は、代謝パラメータを検出するのに使用される機器に配置する前にストレッサーに曝露することができる。例えば、細胞は、XF Extracellular Flux Analyzerに細胞を配置する前に、BIO−SUN製太陽シミュレーター(Vilber Lourmat(France)から入手可能)を使用して紫外線に曝露することもできる。この実施例では、試験培地をプレートウェルから除去し、100uL PBSにより置き換えて、試験培地が紫外線により受ける望ましくない影響(例えば、吸光度又は反射率)を低減させることもできる。代謝指標が曝露後の好適な時間内に検出されない実施形態では(例えば、24時間で測定値を取得した場合に)、ストレッサーへの曝露後すぐに(但しインキュベータ内に細胞を移動させる前に)、PBSを適切な培地(例えば、上記の角化細胞又は線維芽細胞用培地)で置き換えてもよい。非照射細胞からデータを取得するため、非照射細胞を入れるプレートの一部分は、アルミホイルなどのUV不透過性材料により覆うこともできる。場合によっては、ストレッサー及び/又は被検物質は、細胞を試験装置に配置する前及び/又は後に試験容器内に導入され得る。例えば、試験装置は、試験前、中及び/又は後に、剤を直接試験容器に導入することのできる、1つ以上の注入口を含み得る。
酸素消費速度及び細胞外酸性化速度は、XF Extracellular Flux Analyzer又は等価物を使用して検出することができる。装置は、制御環境下で、酸化的リン酸化及び解糖経路の代謝指標を非致死的かつ同時に検出でき、並びに動態データを提供できるものであるべきである。細胞は、機器に使用するのに好適なマルチウェルプレート(例えば、24ウェル又は96ウェルプレート)中に提供し、試験前に洗浄してもよい。細胞は、当該技術分野において既知の任意の好適な手法により(例えば、Seahorse Biosciences XF prep stationを使用して)洗浄することもできる。Seahorse Biosciencesから入手可能なXF prep stationなどの好適な調製ステーションを使用することは望ましいものであり得る。細胞を洗浄する場合に、ウェルからは培地を除去し、その後、細胞は好適な量の試験培地で3回洗浄することが望ましいものであり得る(例えば、96ウェルプレートでは180μL、又は24ウェルプレートでは600μL)。細胞の洗浄後、好適な量(例えば、180μL)の適切な試験培地を各ウェル中に入れ、細胞は、試験のためプレートを機器内に配置する前に、CO2−インキュベータ内、37℃下で、1〜1.5時間平衡化する。平衡期間後、細胞を含有しているプレートを機器に装填し、製造元の指示に従い平衡化する。試験全体は37℃で実施する。いくつかの実施態様では、機器は、角質細胞については、3分の混合サイクル、2分の待機サイクル、及び3分の測定サイクルを、並びに線維芽細胞については、2分の混合サイクル、2分の待機サイクル、及び3分の測定サイクルを提供するものであり得る。このサイクルは少なくとも88分間反復すべきである。サイクル及び時間は、所望される細胞型及び実験デザインに従い変更することができることは認識されるであろう。
Claims (14)
- 化粧品組成物に使用するための有効成分の相乗作用する組み合わせを特定又は評価する方法であって、
a.それぞれ、培地中に入れた細胞を含む、第1、第2及び第3の試験容器を提供することと、
b.前記第1の試験容器内で、前記細胞を第1の被検物質と接触させることと、
c.解糖及び酸化的リン酸化のそれぞれと関連付けられる代謝指標を非致死的に検出して、前記第1の被検物質に対するそれぞれの代謝経路に関する応答を提供すること、
d.前記第2の試験容器内で、前記細胞を第2の被検物質と接触させることと、
e.解糖及び酸化的リン酸化のそれぞれと関連付けられる代謝指標を非致死的に検出して、前記第2の被検物質に対するそれぞれの代謝経路に関する応答を提供することと、
f.前記第3の試験容器内で、前記細胞を前記第1及び前記第2の被検物質の組み合わせと接触させることと、
g.解糖及び酸化的リン酸化のそれぞれと関連付けられる代謝指標を非致死的に検出して、前記第1及び前記第2の被検物質の組み合わせに対するそれぞれの代謝経路の応答を提供することと、
h.前記(g)における応答のうち少なくとも1つが、前記(c)又は(e)における相当する応答に対し相乗的な改善を示す場合に、前記第1及び前記第2の組成物の前記組み合わせを有効成分の相乗作用する組み合わせとして特定することと、を含む、方法。 - 前記改善が、相加作用に相当する、あるいは相加作用を上回る、請求項1に記載の方法。
- 前記細胞を前記第1又は前記第2の被検物質と接触させる前に、前記第1、前記第2及び前記第3の試験容器の少なくとも1つ内で前記細胞を酸化的ストレッサー又はROSに曝露させることを更に含む、請求項1又は2のいずれか一項に記載の方法。
- 前記代謝指標が、前記酸化的ストレッサー又は前記ROSに対する曝露から少なくとも1時間後に検出される、請求項3に記載の方法。
- 前記酸化的ストレッサーが、紫外線照射、煙草の煙、オゾン、エンジン排気、ディーゼル排気、煙霧、界面活性剤、及びコンピューターのモニター又はテレビからの放射線からなる群から選択される、請求項3に記載の方法。
- 前記細胞が、ケラチノサイト及び線維芽細胞からなる群から選択され、前記ストレッサーが、紫外線B波及び紫外線A波照射からなる群から選択される、請求項5に記載の方法。
- 前記代謝指標の前記検出が、制御環境下で実施される、請求項1〜6のいずれか一項に記載の方法。
- 前記酸化的リン酸化経路に関連付けられる前記代謝指標が、酸素消費速度及び二酸化炭素生成速度のうち少なくとも1つで測定される、請求項1〜7のいずれか一項に記載の方法。
- 前記解糖経路に関連付けられる前記代謝指標が、細胞外酸性化速度、細胞外乳酸生成速度及び乳酸濃縮速度のうち少なくとも1つを測定することにより検出される、請求項1〜8のいずれか一項に記載の方法。
- 前記皮膚細胞が、線維芽細胞、角化細胞、メラニン産生細胞、脂腺細胞、幹細胞、脂肪細胞、及び神経細胞からなる群から選択される、請求項1〜9のいずれか一項に記載の組成物。
- 前記相乗作用する組み合わせにおける少なくとも1つの有効成分が、非代謝性効果を提供する、請求項1〜10のいずれか一項に記載の組成物。
- 前記皮膚細胞が、線維芽細胞、角化細胞、メラニン産生細胞、脂腺細胞、幹細胞、脂肪細胞、及び神経細胞からなる群から選択される、請求項1〜11のいずれか一項に記載の組成物。
- 皮膚健康効果を提供し、皮膚への局所塗布に好適である化粧品組成物の作製方法であって、前記方法が、
a.請求項1〜12のいずれか一項に記載の方法による有効成分の相乗作用する組み合わせを特定することと、
b.安全かつ有効な量の前記有効成分を製薬上許容され得る担体に組み込むことと、を含む、方法。 - 皮膚の健康を改善する方法であって、
a.スキンケア効果を必要としている皮膚の対象領域を特定すること、
b.請求項13に記載の方法により作製された美容上安全かつ有効な量の化粧品組成物を前記対象領域に塗布することと、を含む、方法。
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US11137387B2 (en) | 2021-10-05 |
US20150316537A1 (en) | 2015-11-05 |
WO2014059009A1 (en) | 2014-04-17 |
CN110151591A (zh) | 2019-08-23 |
US20170299577A1 (en) | 2017-10-19 |
CN104703585A (zh) | 2015-06-10 |
US9726663B2 (en) | 2017-08-08 |
EP2906197A1 (en) | 2015-08-19 |
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