TWI579002B - 皮膚之動力蛋白之調節 - Google Patents
皮膚之動力蛋白之調節 Download PDFInfo
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- TWI579002B TWI579002B TW100146323A TW100146323A TWI579002B TW I579002 B TWI579002 B TW I579002B TW 100146323 A TW100146323 A TW 100146323A TW 100146323 A TW100146323 A TW 100146323A TW I579002 B TWI579002 B TW I579002B
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- skin
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- keratinocytes
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Description
本發明一般係關於一種改善人類皮膚美學表觀及健康之方法，及亦關於一種識別可用於治療皮膚之化合物之方法。特定言之，本發明係關於一種調節皮膚中動力蛋白濃度之化合物。
動力蛋白係一種在微管中沿負端方向移動之蛋白家族。動力蛋白將來自ATP水解作用之化學能轉化為沿微管逐步移動時所需之機械能。僅出現在具有纖毛及鞭毛之細胞中之纖毛軸動力蛋白會造成彼等結構之軸絲發生微管滑動。另一方面，據信細胞質動力蛋白存在於所有動物細胞中。相較於纖毛軸動力蛋白，其在最近才經識別及分析特徵。參見Paschal B.M.、Shpetner H.S.、Vallee R.B.，「MAP 1C is a microtubule-acivated ATPase which translocates microtubules in vitro and has dynein-like properties」J. Cell Biol.,1987 Sep；105(3)：1273-82；及Shpetner H.S.、Paschal B.M.及Vallee R.B.，「Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein(MAP 1C)」J. Cell Biol.,1988 Sep；107(3)：1001-9，該等內容係以引用之方式併入本文。
細胞質動力蛋白係負責有絲分裂、細胞極化；小泡、內囊胞及溶酶體之細胞內傳輸及細胞內之其他移動之細胞骨架分子馬達。細胞質動力蛋白具有約1.5百萬道爾頓(MDa)之分子量及具有十二個或更多個多肽亞單位，該等亞單位包含每條鏈約530 kDa之兩條相同重鏈、約74 kDa之兩條中間鏈、約53至59 kDa之四條中間鏈及約10至約14 kDa之數條輕鏈。該等重鏈各含有四個ATP結合位點，包括ATP-水解位點，及一個微管結合位點。據信該等中間鏈可結合動力蛋白作為其承載體。
由於動力蛋白使細胞中各種不同材料移動，故其等係多種細胞及發育功能所必需，該等功能包括細胞器移動、發育決定子定位及神經元中之潛在長程訊號傳導。動力蛋白亦在單層細胞培養物之傷口癒合期間調節中心體重新定向、細胞骨架重塑、引導邊緣形成及細胞遷移。其等亦調控人類角質細胞中被吞噬之黑色素小體之核周聚集，導致核上黑色素形成。
動力蛋白之功能缺陷已涉及運動神經元中由軸突運輸受損所引起之運動神經元疾病之神經元退化。編碼細胞質動力蛋白鏈之基因之瓦解已與呼吸疾病，原發性纖毛運動異常(卡塔格氏症候群(Kartagener syndrome))有關且可能涉及運動神經元疾病(如肌肉萎縮性側索硬化)。至今，動力蛋白在維持健康皮膚及減緩皮膚老化中之作用仍未被確認。
本發明之一目的係提供一種用於治療、改善、抑制及/或防止皮膚老化跡象之組合物及方法。本發明之另一目的係提供一種藉由調節皮膚細胞中動力蛋白濃度來治療、改善、抑制及/或防止皮膚老化跡象之方法。本發明之又一目的係提供一種基於調節皮膚細胞中動力蛋白之濃度之能力來識別可用於治療、改善、抑制及/或防止皮膚老化跡象之化合物之方法。
以上論述僅用於更佳理解本技藝所面臨之問題的屬性且不應視為以任何方式承認先前技藝。
根據以上目的及其他內容，本發明提供動力蛋白(較佳為細胞質動力蛋白)之調節劑，以改良一或多種皮膚老化跡象。該等調節劑可依所需作用進行正調節或負調節皮膚之動力蛋白濃度。據信，在皮膚纖維母細胞及角質細胞中所產生之動力蛋白正調節劑會導致細胞內增加產生蛋白質，包括纖維母細胞特異性蛋白質：膠原蛋白、彈性蛋白及原纖維蛋白，及角質細胞蛋白質：角蛋白。由於纖維母細胞所產生之蛋白質對整體皮膚強度及健康甚為重要，故動力蛋白之正調節作用將對減少皮膚出現老化具有有益作用。
本發明亦涵蓋負調節皮膚中動力蛋白濃度之調節劑。由於動力蛋白亦調控人類角質細胞中被吞噬之黑色素小體之核周聚集(此聚集導致核上黑色素形成)，故動力蛋白(尤其是角質細胞及/或黑細胞中之動力蛋白)之負調節劑可用於治療老年斑及皮膚中其他過度色素沉積形式。動力蛋白之負調節劑亦可有效治療皮膚之增生性障礙，包括牛皮癬。
於本發明之一態樣中，提供一種篩選候選物質以識別可用於改善皮膚美學表觀之活性劑之方法。該方法包括分析候選物質調節皮膚纖維母細胞或角質細胞中動力蛋白表現之能力。該方法一般涉及以候選物質培養人類皮膚纖維母細胞，及隨後藉由諸如定量聚合酶鏈反應(qPCR)之定量技術測定編碼細胞質動力蛋白重鏈亞單位DYNC1H1之mRNA之濃度。
因此，提供一種改善人類皮膚美學表觀之方法，其包括對有此需求之皮膚局部施用有效量之可調節人類皮膚纖維母細胞及角質細胞中動力蛋白濃度之物質。動力蛋白調節劑在本文中稱為「活性劑」，且可為正調節劑或負調節劑，及一般係以美容上可接受載劑調配，及局部施用至人體表面，如：臉、頸、手、胸、腿等之皮膚，並持續充分長時間，以增強其健康或美學表觀。
於本發明之一態樣中，提供一種改善人類皮膚美學表觀之方法，該方法包括對有此需求之皮膚區域局部施用有效量之可調節(例如，正調節或負調節)細胞質動力蛋白之細胞濃度之活性劑，其中藉由分析法測定該活性劑調節細胞質動力蛋白之能力，該分析法測量已與該活性劑接觸之細胞中之細胞質動力蛋白之表現程度。用於該分析法之細胞可係表現動力蛋白之任何細胞，但較佳係哺乳動物細胞，及更佳係人類或小鼠細胞。該細胞可係皮膚細胞，例如，纖維母細胞或角質細胞。該分析法可測量動力蛋白之任何片段或標記物(包括完整複合物)之濃度，但一般而言，其係測量細胞質動力蛋白重鏈亞單位DYNC1H1之表現程度，該亞單位係由人類基因DYNC1H1或小鼠基因Dync1h1表現。人類基因DYNC1H1或小鼠基因Dync1h1之表現程度可例如，藉由任何適宜技術(如定量聚合酶鏈反應(qPCR))測量相應mRNA之表現程度來確定。
提供一種治療皺紋及/或細紋之方法，該方法包括對有此需求之皮膚區域局部施用有效量之正調節動力蛋白之活性劑，其中已藉由分析法確定該活性劑調節細胞質動力蛋白之能力，該分析法測量已與該活性劑接觸之細胞中之細胞質動力蛋白之表現程度。該分析步驟最一般而言包括以候選物質培養人類皮膚纖維母細胞，及隨後測量編碼細胞質動力蛋白重鏈亞單位DYNC1H1之mRNA之濃度，其中部份基於其正調節動力蛋白表現之能力來選擇該候選物質用作活性劑。
亦提供一種治療過度色素沉積之方法，其包括對有此需求之皮膚區域局部施用有效量之負調節動力蛋白之活性劑，其中已藉由分析法確定該活性劑調節細胞質動力蛋白之能力，該分析法測量已與該活性劑接觸之細胞中之細胞質動力蛋白之表現程度。於此實施方案中，該分析步驟一般包括以候選物質培養人類皮膚角質細胞，及隨後測量編碼細胞質動力蛋白重鏈亞單位DYNC1H1之mRNA之濃度，其中部份基於其負調節動力蛋白表現之能力來選擇該候選物質用作活性劑。
本發明之其他態樣、特徵及優點將基於閱讀本發明實施方式而得以更加瞭解。
除非另外說明，否則本文中所使用之所有術語係具有其等一般含義。「美容上可接受」意指其特定組分在所採用之濃度下基本上視為安全及無毒性。如本文中所使用之術語「防止」包括延遲特定皮膚老化跡象之發生或進程。術語「薄皮膚」包括隨著年齡增長而變薄之皮膚及過早薄化之皮膚，其等可係，例如，由光老化引起。用語「有此需求之個體」係指可因改善皮膚表觀或健康而受益之人類，包括男性或女性。術語「皮膚」包括，但不限於：脣；臉、手、手臂、頸及胸之皮膚。如本文中所使用，術語「實質上由...組成」意欲將本發明限制於指定材料或步驟，但不會實質上影響本發明之基礎及新穎特性之彼等內容，該等內容將經由閱讀本說明書來理解。
如本文中所使用，術語動力蛋白係指細胞質動力蛋白及纖毛軸動力蛋白，而本發明之較佳調節劑將調節細胞質動力蛋白之濃度。細胞質動力蛋白包括細胞質動力蛋白1複合物，及細胞質動力蛋白2複合物，而本發明較佳調節劑將調節細胞質動力蛋白1複合物之濃度。動力蛋白可來自任何動物，但一般係人類或小鼠動力蛋白，及較佳係人類動力蛋白。本文中用於描述動力蛋白之命名法係Pfister等人「Cytoplasmic dynein nomenclature」J. Cell Biol.,2005 November 7；171(3)：411-413之命名法，該案係以引用之方式併入本文，該命名法示於表1中。
術語「調節劑」涵蓋任何物質，包括，但不限於：有機分子；生物分子(例如，肽、蛋白質、抗體、核酸寡聚物，等)及該等物質之組合，如：植物抽出物。調節劑調節動力蛋白之細胞濃度，其意指藉由活性劑增加或減少動力蛋白之細胞濃度。術語「調節」係指正調節、誘導、刺激、增強及/或減輕抑制，及抑制、減弱及/或負調節或壓制。調節劑可係，但不限於：抑制劑或拮抗劑，其等可係例如，會結合、部份或完全阻斷刺激、減小、防止、延遲活化、滅活、減敏或負調節基因或動力蛋白蛋白質或肽之表現程度之化合物。調節劑亦可係，但不限於：活化劑或激動劑，其等係例如，會結合、刺激、增加、打開、活化、促進、增強活化、敏化或正調節基因或動力蛋白蛋白質或肽之表現程度之化合物。調節蛋白質濃度之機轉並不重要。
如本文中所使用，術語「表現程度」係指基因及/或蛋白質在細胞中之表現量。如本文中所使用，「基因」包括含有至少一個可編碼特定多肽之開放讀碼框之聚核苷酸。如本文中所使用，術語「聚核苷酸」係與「寡核苷酸」同義且包括任何長度核苷酸之聚合形式，其可為脫氧核糖核苷酸或核糖核苷酸，或其等類似物，包括，但不限於：mRNA、DNA、cDNA、引子、探針及類似者。
動力蛋白之表現程度與皮膚老化之間之關係的發現提供一種識別有用動力蛋白調節劑之篩選方法。於一實施例中，提供一種用於在以候選物質處理、培養或在其他情況中，接觸細胞之後確定動力蛋白之表現程度之分析法。術語「候選物質」係指用於測試作為動力蛋白之調節劑之活性之任何物質，不論該物質是否疑似擁有此活性。該細胞可係表現動力蛋白(較佳指細胞質動力蛋白)之任何細胞。於一實施例中，該細胞係哺乳動物纖維母細胞。於另一實施例中，該細胞係哺乳動物角質細胞。較佳，該細胞係人類或小鼠細胞。於藉由候選物質培養細胞充分長時間以提供可測量之表現程度變化之後(一般為至少一小時，及更一般而言為約12小時至約72小時之後)，溶解該細胞，以釋放細胞組分，如動力蛋白及編碼動力蛋白之mRNA。然後藉由用於偵測及定量肽與蛋白質及/或聚核苷酸(例如，mRNA)之任何適宜技術，測量動力蛋白或其任何亞單位之含量。
用於測量動力蛋白之表現程度之較佳方法涉及mRNA表現之定量法。用於測定mRNA表現之適宜方法包括如本技藝中熟知之定量PCR(QPCR)、實時QPCR、反轉錄PCR(RT-PCR)及定量反轉錄PCR(QRT-PCR)。如以引用方式併入本文之美國專利案7,101,663及7,662,561中所詳細描述，用於偵測mRNA之定量反轉錄聚合酶鏈反應(QRT-PCR)可包括步驟：(a)由來自溶胞物之RNA樣本使用反轉錄酶及高濃度之標的序列專一性反轉錄酶引子，在適宜產生cDNA之條件下培養；(b)隨後添加適宜聚合酶鏈反應(PCR)試劑至反轉錄酶反應，包括添加高濃度之針對cDNA之特異性PCR引子群及熱穩定性DNA聚合酶至反轉錄酶反應中；及(c)在適宜條件下，使該PCR反應循環進行所需偱環次數，以產生針對cDNA之特異性PCR產物(「擴增子」)。經過固定之PCR偱環次數之後，比對QRT-PCR製程產物，藉由(例如)南方墨點法，與指定之報導基因比對，來決定RNA物質之相對量。更一般而言，藉由例如，以下兩種物質來分析各PCR引子群之相對擴增子產生速率來追蹤PCR反應進程：(1)可嵌入任何雙股DNA中之非專一性螢光染料及/或(2)由僅容許在探針與其互補DNA標的物雜交之後才可偵測到之螢光報導子標記之寡核苷酸所組成之序列專一性DNA探針。
mRNA可係與動力蛋白相關之任何mRNA，包括編碼表1中所識別之亞單位之mRNA。於一實施例中，該mRNA編碼重鏈亞單位DYNC1H1，該亞單位在文獻上之同義字亦(尤其)稱為「細胞質動力蛋白1重鏈」或「習知細胞質動力蛋白重鏈」。於較佳實施例中，該mRNA編碼人類DYNC1H1。
可與未經候選物質處理之對照組比較mRNA表現程度，以確定相對調節程度。於一些實施例中，該候選物質會正調節mRNA表現至少約10%，更適宜為至少約20%，至少約30%，至少約40%或至少約50%。較佳，候選物質會正調節mRNA表現至少約60%，至少約70%，至少約80%，至少約90%或至少約100%。於其他實施例中，候選物質會負調節mRNA表現至少約10%，更適宜為至少約20%，至少約30%，至少約40%，或至少約50%。可選擇符合此等準則之候選物質用於進一步評估之用途。
藉由以上篩選計劃所識別之動力蛋白調節劑可用作美容用製劑中之活性劑，且可與美容上可接受組分(如載劑)一起調配成用於皮膚局部施用之組合物。將有效量組合物局部施用至皮膚意指足以達到可測量到之皮膚健康改善或一或多種皮膚老化跡象減輕時之用量。改善此等皮膚老化跡象包括，但不限於以下情況：
(a)治療、減少及/或防止細紋或皺紋；
(b)縮小皮膚孔徑；
(c) 改善皮膚厚度、飽滿度及/或緊實度；
(d) 改善皮膚柔軟性及/或柔軟度；
(e) 改善膚色、光澤及/或潔淨度；
(f) 改善膠原蛋白原及/或膠原蛋白產生；
(g) 改善彈性蛋白之維持及重塑；
(h) 改善皮膚紋理及/或促進再紋理化；
(i) 改善皮膚屏障修復及/或功能；
(j) 改善皮膚輪廓表觀；
(k) 修復皮膚光澤及/或亮度；
(l) 補充皮膚中之必需營養物及/或成份；
(m) 改善因老化及/或停經而下降之皮膚表觀；
(n) 改善皮膚保濕力；
(o) 增大皮膚彈性及/或回彈性；
(p) 治療、減少及/或防止皮膚下垂，及/或
(q) 減少色素斑。
於實際中，本發明之組合物，包括調節動力蛋白表現程度之製劑，係單獨或以溶於美容上可接受載劑之方式施用至需要治療之皮膚。亦即，缺乏或喪失以上情況中任一者或可因改善以上任一種皮膚狀況而受益之皮膚。該皮膚一般每日接受一次或兩次治療。該治療可持續達一週、兩週、四週、八週、六個月或更長時間。
於一實施例中，該等活性劑係以溶於美容上可接受載劑之方式局部施用至出現細紋及/或皺紋之皮膚，以防止、治療及/或改善皮膚中之細紋及/或皺紋之出現。於此情況中，將組合物施用至需要治療之皮膚意指已出現皺紋及/或細紋之皮膚或存在形成細紋及/或皺紋之風險之皮膚。較佳，將組合物直接施用至臉、頸、胸及/或手之皮膚上細紋及/或皺紋。根據此實施例之較佳製劑係動力蛋白表現之正調節劑。於較佳實施例中，動力蛋白表現之正調節劑可促進皮膚纖維母細胞中產生膠原蛋白、彈性蛋白及/或原纖維蛋白。
於一實施例中，本發明係關於一種藉由促進皮膚中產生膠原蛋白、彈性蛋白及/或原纖維蛋白來改善皮膚美學表觀之方法，該方法包括對有此需求之皮膚區域局部施用有效量之正調節動力蛋白表現之製劑，其中該化合物已藉由分析法識別可用，該分析法確定物質調節動力蛋白之表現程度之能力，包括本文中所描述之分析法。較佳，該分析法測量皮膚纖維母細胞中動力蛋白mRNA表現程度。
於一實施例中，動力蛋白調節劑包含諸如植物抽出物之天然植物材料。正調節動力蛋白之天然植物材料及植物抽出物中，可提及由來自以下物種之抽出物所製成者：大花雞腳蔘(Orthosiphon grandiflorus)、大花鰐嘴花(Clinacanthus nutans)、管花肉蓯蓉(Cistanche tubulosa)、刺桐(Erythrina indica)、盒果藤(Operculina turpethum)、白花菜(Gynandropsis gynandra)、風車草(Erthrina Flabelliformis)、香蠟菊(Helichrysum Odoratissimum)、伊利諾草合歡(Desmathus illinoensis)、倒心類蠟菊(Ozothamnus Obcordatus)、萬壽菊(Tagetes erecta Linn)、蘇木(Caesalpinia sappan Linn)、俾斯麥棕櫚(Medemia nobilis)、皇冠花(Calatropis gigantean)、檵木(Loropetalum chinense)、狗尾草(Heliotropium indicum)、山菅蘭(Dianella ensifolia)、鬼畫符(Breynia fruticosa)、野菊(Dendranthema indicum)、接骨草(Sambucus chinensis)、野甘草(Scoparis dulcis)、黑木相思(Acacia melanoxylon)、多花大青(Clerodendrum floribundum)、山麻樹(Commersonia bartramia)、車桑子(Dodonaea viscose)、車前草(Duboisa myoporoides)、厚殼樹(Ehretia acuminate)、花球枇杷螺(Ficus coronata)、卵形山羊草(Goodenia ovata)、黃海桐花(Hymenosporum flavum)、花葵(Lavatera plebeian)、白千層(Melaleuca quinquernervia)、血桐(Omolanthes populifolius)及其組合。
正調節動力蛋白之其他物質包括以下：
亦適宜正調節動力蛋白者係查米林(chalmicrin)，其係連接至d-甘露醇之10-甲基-反式-單環金合歡醇，係自微孢子橫節黴菌(Chalara microspora)單離之烯丙醚；亦即，1-O-[(E)-3-甲基-5-(2,5,6,6-四甲基-1-環己烯-1-基)-2-戊烯基]-D-甘露醇。
於一實施例中，本發明係關於一種減少皮膚中老年斑出現及過度色素沉積之其他症狀之方法，該方法包含對有此需要之皮膚區域局部施用有效量之可負調節動力蛋白表現之製劑，其中該化合物已藉由分析法識別可用，該分析法測定該物質調節動力蛋白之表現程度之能力，包括本文中所描述之分析法。較佳，該分析法測量皮膚角質細胞中動力蛋白mRNA表現程度。動力蛋白之負調節劑亦可有效治療皮膚增生性疾病，包括牛皮廯。
其中，負調節動力蛋白之天然植物材料及植物抽出物係疣柄魔芋(Amorphophallus campanulatus)、圓仔花(Gomphrena globosa Linn)、根黃藤(Fibraretinum resica Pierre(黃藤素(Fibrauretine)))、小花斑鳩菊(Vernonia cinerea Linn. Less)、烏蘞莓(Cayratia japonica)、半邊羽裂鳳尾蕨(Pteris semipinnata)及其組合。
負調節動力蛋白之其他物質包括5-氯-水楊酸、3,6-壬二烯醇、順式-6-壬烯醇及以下：
於實施例中，提供一種改善人類皮膚美學表觀及/或改善老化及/或被光破壞之皮膚外觀之方法，該方法包括局部施用有效量之組合物，該組合物包含選自由泰國黃果木(Mammea siamensis)(花抽出物)、俾斯麥棕櫚(Medemia nobilis)(種子抽出物)、羅非亞椰子(Raphia farinifera)(種子抽出物)、花葉刺桐(Erythrina indica)(葉抽出物)、臭茉莉(Clerodendron fragrans)(全植物抽出物)、盒果藤(Operculina turpethum)(全植物抽出物，不包括根)或其組合組成之群之天然植物抽出物，及美容上可接受載劑。
該等植物材料可呈任何形式，包括，但不限於：全植物、乾燥植物、經研磨植物或其部分，包括，但不限於：種子、針葉、葉部、根、樹皮、毬果、莖、根莖、癒傷組織細胞、原生質體、花及分生組織，或在天然植物材料中發現或單離之組分及/或成份，及/或植物之部分，或其任何組合。於一實施例中，天然植物材料係呈自全植物或自植物中之選定部分(如植物之葉子)所獲得之抽出物之形式。應理解，「天然植物材料」亦包括自天然植物材料獲得之成份、組分、組成分或抽出物。於另一實施例中，如本文中所使用之植物抽出物亦包括「合成」抽出物，亦即已知植物組分及/或組成份之各種組合，該等組分及/或成份係經過組合，以便實質上擬似天然來源植物抽出物之組成及/或活性。此等合成抽出物包括在術語「植物抽出物」中。合成抽出物具有在一種植物中常見之兩種或更多種、三種或更多種、或四種或更多種活性成份。更佳，該等合成抽出物具有與天然抽出物實質上相同數量之活性成份。合成抽出物與植物或天然抽出物之間之活性成份發生率數值對應程度亦以術語「一致性百分比」描述。較佳，合成抽出物與植物或天然抽出物之化學組成具有50%或更高之一致性。換言之，該合成抽出物具有50%或更高之在植物或天然抽出物中所發現之活性成份。更佳，合成抽出物之化學組成與植物或天然抽出物之化學組成具有約70%或更高之一致性。最佳，合成抽出物與植物或天然抽出物之化學組成具有約90%或更高之一致性。
就本發明組合物之用途而言，該植物抽出物或組分及/或活性組成份較佳係直接自植物獲得。該等組分可呈純物質形式、半純物質形式或未純化形式。於一實施例中，該等組分係呈藉由水性或有機溶劑萃取所獲得抽出物之形式。有機溶劑之非限制性實例包括乙酸、乙酸乙酯、低碳數醇(例如，甲醇、乙醇、異丙醇、丁醇)、二氯甲烷、己烷、甲苯、二甲苯、石油醚及其組合。該溶劑可係極性或非極性、質子性或非質子性、水可混溶或水不可混溶。pH可係酸性、中性或鹼性。可採用本技藝中熟知方法進行水性或有機溶劑萃取。萃取時間一般宜在約30℃至約90℃下持續約1至8小時。所收集之抽出物隨後可經過精細過濾，以移除碎片，並可直接使用，或藉由(例如)蒸餾溶劑或藉由習知加工方法進行濃縮，且抽出物亦可呈粉末形式提供。
本發明之美容用組合物可調配成用於局部施用之各種不同形式，且包含約0.00001%至約90重量%之一或多種調節動力蛋白之活性劑，及較佳包含含量約0.001%至約25重量%，及更佳約0.001%至約1重量%之此等活性劑。
該等組合物可包括美容上可接受之載劑。此等載劑可呈相關技藝中任何已知適宜施用至皮膚之形式，且可包括，但不限於：水；植物油；諸如棕櫚酸辛酯、肉豆蔻酸異丙酯及棕櫚酸異丙酯之酯類；諸如二辛醚及異山梨醇二甲基醚之醚類；諸如乙醇及異丙醇之醇類；諸如鯨蠟醇、鯨蠟硬脂醇、硬脂醇及聯苯醇之脂肪醇；諸如異辛烷、異十二烷及異十六烷之異鏈烷烴；諸如環甲聚矽氧烷之聚矽氧油；諸如礦物油、石蠟脂、異二十碳烷及聚異丁烯之烴油；諸如丙二醇、甘油、丁二醇、戊二醇及己二醇之多元醇；脂質體；蠟類；或以上物質之任何組合或混合物。
該載劑可包含水相、油相、醇、聚矽氧相或其混合物，且可呈乳液形式。適宜乳液之非限制性實例包括油包水乳液、水包油乳液、水包聚矽氧乳液、聚矽氧包水乳液、水包甘油乳液、水包蠟乳液、水-油-水三重乳液或類似者。該乳液可包括乳化劑，如：非離子性、陰離子性或兩親性表面活性劑，或膠凝劑。
局部組合物一般具有約2至約8之pH範圍，以3至7範圍內之pH為較佳。於一些實施例中，該組合物具有3.5至5.5範圍內之pH。可添加適宜pH調整劑(如檸檬酸及三乙醇胺)，將pH調整至所需範圍內。
於本發明之一實施例中，該等組合物可包括其他皮膚活性劑，包括，但不限於：植物製劑、角質層分解劑、剝落劑、角質細胞增生增強劑、膠原蛋白酶抑制劑、彈性蛋白酶抑制劑、脫色素劑、消炎劑、類固醇、消除粉刺劑、抗氧化劑及後期糖基化終產物(AGE)抑制劑。
該組合物可包含具有抗衰老效益之其他活性成份，因為據信可利用此等組合來獲得協同改善效果。示例性抗衰老組分包括，但不限於：植物製劑(例如，甄叔迦樹(Butea Frondosa)抽出物)；植醇；硫代二丙酸(TDPA)及其酯；類視黃素(例如，9-順式視黃酸、13-順式視黃酸、全反式視黃酸及其等衍生物、植烷酸、視黃醇(維生素A)及其酯類，如棕櫚酸視黃醇酯、乙酸視黃醇酯及丙酸視黃醇酯，及其等鹽，及其他)；羥酸(包括，α-羥酸及β-羥酸)、水楊酸及水楊酸烷酯；去角質劑(例如，乙醇酸、3,6,9-三氧雜十一碳烷二酸，等)、雌激素合成酶刺激化合物(例如，咖啡因及衍生物)；可抑制5α-還原酶活性之化合物(例如，亞麻酸、亞油酸、柔沛(finasteride)及其混合物)；及屏障功能增強劑(例如，神經醯胺、甘油酯、膽固醇及其酯類、α-羥基及ω-羥基脂肪酸及其酯類，等)，僅舉數例。示例性類視黃素包括，但不限於：視黃酸(例如，全反式或13-順式)及其衍生物、視黃醇(維生素A)及其酯類，如棕櫚酸視黃醇酯、乙酸視黃醇酯及丙酸視黃醇酯，及其鹽類。
於另一實施例中，本發明之局部組合物亦可包括以下物質中之一或多者：皮膚滲透增強劑、潤膚劑，如肉豆蔻酸異丙酯、石蠟脂、聚矽氧(例如，甲聚矽氧烷、二甲聚矽氧烷)、油類、礦物油及脂肪酸酯類；保濕劑(如：甘油或辛醯二醇)、皮膚豐滿劑(如：棕櫚醯基寡肽、膠原蛋白，或膠原蛋白及/或葡糖胺聚醣(GAG)增強劑)、防曬霜(如：亞佛苯酮(avobenzone))、去角質劑及抗氧化劑。
適宜去角質劑包括，例如，α-羥酸、β-羥酸、氧雜酸、氧雜二酸及其衍生物，如：其酯類、酸酐類及鹽類。適宜羥酸包括，例如，乙醇酸、乳酸、蘋果酸、酒石酸、檸檬酸、2-羥基烷酸、扁桃酸、水楊酸及其衍生物。較佳去角質劑係乙醇酸。當存在時，去角質劑可佔該組合物之約0.1重量%至約80重量%。
可用於本發明中之抗氧化劑實例包括具有酚系羥基官能機之化合物，如：抗壞血酸及其衍生物/酯類；β-胡蘿蔔素；兒茶素；薑黃素；阿魏酸衍生物(例如，阿魏酸乙酯、阿魏酸鈉)；沒食子酸衍生物(例如，沒食子酸丙酯)；番茄紅素；還原酸；迷迭香酸；單寧酸；四氫薑黃素；生育酚及其衍生物；尿酸；或其任何混合物。其他適宜抗氧化劑係具有一或多個巰基官能基(-SH)，呈還原或未還原形式之彼等物，如榖胱甘肽、硫辛酸、硫代乙醇酸及其他氫硫基化合物。抗氧化劑可係無機物，如亞硫酸氫鹽、偏亞硫酸氫鹽、亞硫酸鹽或含有硫之其他無機鹽及酸。本發明組合物可包含佔組合物總重量之較佳約0.001重量%至約10重量%，及更佳約0.01重量%至約5重量%之抗氧化劑。
其他習知添加劑包括：維生素，如：生育酚及抗壞血酸；維生素衍生物，如：單棕櫚酸抗壞血酸酯；增稠劑，如：羥烷基纖維素；膠凝劑；結構劑，金屬螯合劑，如EDTA；色素；著色劑及pH調整劑。該組合物可視需要包含熟習本項技術者已知之其他組分，包括，但不限於：成膜劑、潤濕劑、礦物質、黏度及/或流變改質劑、消除粉刺劑、驅蟲劑、爽膚化合物、護膚劑、潤滑劑、芳香劑、防腐劑、穩定劑及其混合物。除以上物質外，本發明之美容用組合物可含有用於治療皮膚疾病之任何其他化合物。
該組合物可調配成各種不同產物形式，如，例如，乳液、洗劑、乳霜、精華液、噴液、氣溶膠、餅狀物、軟膏、香精、凝膠、糊膏、貼片、畫筆、小毛巾、敷膜、棒、泡沫劑、酏劑、濃縮物及類似者，尤其是用於局部投與之形式。較佳，將該組合物調配成乳液、洗劑、乳霜、軟膏、精華液或凝膠。
本發明提供一種治療老化皮膚之方法，該方法將包含較佳溶於美容上可接受載劑中之調節動力蛋白之活性劑之組合物局部施用於受影響區域並持續充分長時間，以減少、改善、逆轉或防止皮膚老化跡象。
一般而言，病況及/或美學表觀之改善係選自由以下情況組成之群：減少皮膚隨年齡增長、光老化、內分泌老化及/或光化老化之跡象；防止及/或減少細紋及/或皺紋出現；減少臉部細紋及皺紋之顯著性、臉頰、前額之臉部皺紋、眼間之垂紋、眼睛上方及嘴部周圍之橫紋、木偶線，及尤其是深皺紋或折紋；改善眼眶下線及/或眼眶周線之外觀；減少爪形條紋之出現；重建及/或修護皮膚，尤指老化皮膚；減輕皮膚脆性；防止及/或逆轉葡糖胺聚糖及/或膠原蛋白之流失；改善雌激素失衡之作用；防止皮膚萎縮；防止、減少及/或治療過度色素沉積或色素不足；盡可能減小皮膚褪色；改善膚色、光澤、潔淨度及/或緊實度；防止、減少及/或改善皮膚下垂；改善皮膚堅實度、飽滿度、柔軟性及/或柔軟度；改善膠原蛋白原及/或膠原蛋白產生；改善皮膚紋理及/或促進再紋理化；改善皮膚屏障修補及/或功能；改善皮膚輪廓表觀；修復皮膚光澤及/或亮度；盡可能減少皮膚疲勞及/或緊張跡象；抵抗環境應力；補充皮膚中因老化及/或停經而減少之成份；改善皮膚細胞間之聯通；增強細胞增殖及/或複製；增強因老化及/或停經而減弱之皮膚細胞代謝；阻止細胞老化；改善皮膚保濕力；增強皮膚厚度；減慢或中止皮膚薄化；增加皮膚彈性及/或回彈性；增強去角質化；改善微循環；減少及/或防止皮下脂肪團形成；及其任何組合。
於一實施例中，該組合物包含約0.00001%至約20%，更一般而言，約0.001%至約10%，包括約0.01至約1重量%之動力蛋白調節劑。較佳，該調節劑可係纖維母細胞中之動力蛋白之正調節劑。亦較佳，該調節劑可係角質細胞中之動力蛋白之正調節劑。該調節劑可係角質細胞中之動力蛋白之負調節劑。次佳，但仍在本發明之範圍內，該調節劑可係纖維母細胞中之動力蛋白之負調節劑。於一實施例中，該調節劑係纖維母細胞及角質細胞中之動力蛋白之正調節劑。亦涵蓋調節劑之組合。於一實施例中，該調節劑係作為纖維母細胞及/或角質細胞中之動力蛋白之正調節劑之兩或更多種物質之組合。
該組合物一般每日一次、兩次或三次施用至皮膚，依需要直到獲得所需結果為止。治療方案可包括每日施用並持續至少一週、至少兩週、至少四週、至少八週或至少十二週或更長時間。亦涵蓋慢性治療方案。組合物對細紋及皺紋之形成或出現之效用可藉由例如，視覺檢查定性評估，或藉由例如，顯微鏡或電腦輔助測量皺紋形態(例如，每單位面積皮膚之皺紋數量、深度、長度、面積、體積及/或寬度)來定量評估。於一實施例中，本發明之組合物將以約0.001至約100 mg/cm2，更一般而言，約0.01至約20 mg/cm2，及較佳約0.1至約10 mg/cm2之量施用至皮膚。
本發明組合物亦可藉由將該組合物局部施用至有此需要之個體之薄皮膚上，來治療薄皮膚。「薄皮膚」包括因年齡增長、停經或光破壞而薄化之皮膚及過早薄化之皮膚。於一些實施例中，該治療係針對男性薄皮膚，而其他實施例則治療女性停經前或停經後之薄皮膚，因為據信皮膚會隨著男性及女性年齡不同而不同地變薄，且尤其對於在不同生命階段之女性。
本發明之方法可在老化前作為預防手段使用，包括用於未出現皮膚老化跡象之個體，最常見用於年齡小於25歲之個體。該方法亦可逆轉或治療已出現之老化跡象，常見用於超過25歲之個體，或用於減慢此等個體中皮膚老化之進程。
以下實例描述本發明之具體態樣以說明本發明，但不應視為對本發明之限制，係因該等實例僅提供可用於理解及實施本發明及其各態樣之具體方法。
皮膚纖維母細胞中與年齡相關之動力蛋白之表現
將來自新生兒(n=3)及年齡在31至39歲之間之女性供體(n=3)之正常皮膚纖維母細胞塗佈於含DMEM(10% FBS，4.5 g/L葡萄糖)之10 cm培養皿上並生長至半滿程度。以冷PBS清洗細胞及利用RNAqueous套組，依照製造商說明來單離RNA。藉由Agilent Bioanalyzer 2100，利用RNA Nano Chip驗證RNA完整度及濃度。利用經單離之RNA及ABI 7900 HT RT-PCR儀器進行基因表現分析。所使用之動力蛋白探針係智人(Homo sapiens)細胞質重鏈DYNCIH1，其具有ABI碼DYNCIH1-Hs00300243_ml。引入內部對照物，並相對於GAPDH校正動力蛋白基因表現。發現30歲以上之供體之動力蛋白表現比新生兒細胞顯著(~14%)下降。內因性(校正後)基因表現程度提供於下表2中。
於另一實驗中，在曝露於UV輻射後進行人類活體組織檢查，以分析DYINC1H1之表現。自年輕(18至25歲，n=15)及年長(45-65歲，n=15)健康女性高加索人供體之前臂背側(光曝露)及臂上部(光保護)收集探索用活體皮膚組織，以福爾馬林固定及以石蠟包埋。切成五微米切片，及置於載玻片上。利用針對動力蛋白之兔多株抗體進行IHC，及利用與載體藍共軛之羊抗兔二級抗體，以便目視檢測。在較年輕群組中，62%受試者展現動力蛋白表現提高，而僅有33%較年長受試者之表現提高。此等數據證實，當有需要時，較年長皮膚較無法有效產生動力蛋白。受到UV曝露時之基因表現程度之增加亦示於下表2中。
表2中之數據說明，細胞質動力蛋白表現會隨年齡增長而下降，且皮膚受到諸如UV曝露之刺激反應時正調節動力蛋白表現之能力亦隨年齡增長而下降。因此，據信動力蛋白之調節係克服年齡及環境相關之皮膚老化之合理方法。
動力蛋白調節分析法
在經組織培養物處理且含有適當培養基之96-孔分析板中培養正常人類皮膚纖維母細胞或角質細胞。於適當載劑(水/乙醇/丙二醇)中製造活性劑母液。於含10% CO2之含濕氣37℃培養箱中，以於生長介質中稀釋之測試材料或各載劑對照物處理細胞24小時。培養之後，自各分析板中移除生長介質，及添加100 μL溶胞緩衝液至各孔，並置於含10% CO2之37℃培養箱中30分鐘。培養結束時，將細胞收集於冷凍分析板中，置於-80℃冷凍庫中，直至分析。利用採用分支DNA技術之Panomics Quantigene多重分析法，依照製造商說明(Affymetrix,CA)來分析經處理後之動力蛋白之mRNA變化。比對測試結果與對照組結果，計算細胞質動力蛋白之重鏈DYNC1H1之mRNA之增加(正調節)或減少(負調節)百分比。將正調節或負調節百分比換算為以下表3中所示之量級得分。
動力蛋白之正調節
依照實例2之方法測試各種不同植物抽出物正調節動力蛋白之能力。結果提供於以下表4中。各抽出物之濃度係基於指定植物抽出物之乾重表示，其意指已排除揮發性萃取溶劑後之抽出物重量。所測試之細胞係角質細胞(K)或纖維母細胞(F)。
亦依照實例2之方法測試化合物1至5及查米林(chalmicrin)正調節動力蛋白之能力。結果出示於表5中。
動力蛋白之負調節
依照實例2之方法測試各種不同化合物及植物抽出物負調節動力蛋白之能力。結果示於下表6中，其中植物抽出物之濃度係基於指定植物抽出物之乾重表示。於各例中，於p<0.05或更佳基準下達到統計學顯著性。
水楊酸衍生物對細胞質動力蛋白之重鏈DYNC1H1中mRNA產生最高之負調節程度，於角質細胞中之結果為下降45.03%(p=0.02)，其次為泰國亞南葉(Tliliacora Triandra)，其在纖維母細胞中之結果為下降36.7%(p=0.01)。
動力蛋白之體內正調節
測試植物抽出物在體內正調節動力蛋白之能力。以成份處理具有II或III型皮膚之33個30至65歲健康女性高加索人的前臂背側達3週(在半封閉下連續進行3輪5×24個貼片)。測試物及載劑係以隨機分配方式施用於各前臂之五個位置上。施用劑量係2 mg/cm2。經處理後，自各處理位置鑽取一片2 mm活體組織切片，並固定於4%多聚甲醛中，及針對IHC研究進行處理。利用針對動力蛋白之兔多株抗體進行IHC，及利用與載體藍共軛之羊抗兔二級抗體(如實例1中所描述)以便目視檢測。示於下表7中之結果係以動力蛋白濃度出現正調節、無變化或負調節之病患之數量(N)表示。
如表7中所示，當局部施用至皮膚時，幾乎所有經測試之植物抽出物均在活體內正調節動力蛋白。基於其活體外效能，雖然盒果藤(Operculina turpethum)在此研究中未展現顯著正調節或負調節，然而，據信該等結果可藉由使用滲透增強劑改善向皮膚之傳遞性及/或提高劑量或延長處理時間而得以改良。
本文中所引述之所有參考文獻，包括專利申請案及公開案均係以引用其等全文之方式及針對相同內容之所有目的併入本文，正如各單獨公開案或專利案或專利申請案係專門及獨立地以引用其全文之方式針對所有目的併入本文。在不脫離本發明之精神及範圍下，可對本發明進行許多修改及變化，如熟習本項技術者所瞭解。本文中所描述之具體實施例僅以實例方式提供，且本發明僅限制於附錄申請專利範圍及此等技術方案所主張之等效內容之完整範圍。
Claims (12)
- 一種調節細胞質動力蛋白之細胞濃度之活性劑之用途，其係用於製造供改善人類皮膚美學表觀之調配物，其中該化合物係選自以下物質組成之群：
- 如請求項1之用途，其中該活性劑會增加細胞質動力蛋白之表現程度。
- 如請求項1之用途，其中該皮膚美學之改善係選自由以下情況組成之群：(a)治療、減少及/或防止細紋或皺紋；(b)縮小皮膚毛孔大小；(c)改善皮膚厚度、飽滿度及/或緊實度；(d)改善皮膚柔韌性及/或柔軟度；(e)改善皮膚色澤、光澤及/或透明度；(f)改善彈性蛋白之維持及重塑；(g)改善皮膚紋理及/或促進再紋理化；(h)改善皮膚屏障修復及/或功能；(i)改善皮膚輪廓之外觀；(j)恢復皮膚光澤及/或明亮；(k)改善因停經而下降之皮膚表觀；(l)改善皮膚保濕力；(m)增加皮膚彈性及/或回彈性；(n)處理、減少及/或防止皮膚鬆弛；或(o)減少色素斑點。
- 一種識別可用於改善皮膚美學表觀之活性劑之方法，其包括分析候選物質增加皮膚纖維母細胞或角質細胞中動 力蛋白表現之能力。
- 如請求項4之方法，其中該分析步驟包括由人類皮膚纖維母細胞或角質細胞與該候選物質培養，及隨後測量編碼細胞質動力蛋白重鏈亞單位DYNC1H1之mRNA之濃度。
- 如請求項5之方法，其中該測量步驟係藉由定量聚合酶鏈反應(qPCR)進行。
- 如請求項5之方法，其中該細胞係皮膚纖維母細胞。
- 如請求項5之方法，其中該細胞係皮膚角質細胞。
- 一種正調節動力蛋白之活性劑之用途，其係用於製造供治療皺紋及/或細紋之調配物，其中該化合物係選自以下物質組成之群：
- 一種組合物，其包含正調節皮膚纖維母細胞或角質細胞中動力蛋白之有效量植物抽出物，及局部施用上可接受之載劑，其中該植物抽出物係選自以下物質組成之群：大花雞腳蔘(Orthosiphon grandiflorus)、大花鰐嘴花(Clinacanthus nutans)、刺桐(Erythrina indica)、盒果藤(Operculina turpethum)、白花菜(Gynandropsis gynandra)、風車草(Erthrina Flabelliformis)、香蠟菊(Helichrysum Odoratissimum)、伊利諾草合歡(Desmanthus illinoensis)、萬壽菊(Tagetes erecta Linn)、蘇木(Caesalpinia sappan Linn)、俾斯麥棕櫚(Medemia nobilis)、皇冠花(Calatropis gigantean)、檵木(Loropetalum chinense)、狗尾草(Heliotropium indicum)、鬼畫符(Breynia fruticosa)、野菊(Dendranthema indicum)、接骨草(Sambucus chinensis)、野甘草(Scoparis dulcis)、黑木相思(Acacia melanoxylon)、多花大青(Clerodendrum floribundum)、山麻樹(Commersonia bartramia)、車桑子(Dodonaea viscose)、車前草(Duboisa myoporoides)、厚殼樹(Ehretia acuminate)、花球枇杷螺(Ficus coronata)、卵形山羊草(Goodenia ovata)、黃海桐花(Hymenosporum flavum)、花葵(Lavatera plebeian)、白千層(Melaleuca quinquernervia)、血桐(Omolanthes populifolius)、泰國黃果木(Mammea siamensis)及其等組合。
- 一種組合物，其包含來自蜜茱萸(Melicope hayesii)之抽出物與另一種可有效正調節皮膚纖維母細胞或角質細胞中動力蛋白之植物抽出物，及局部施用上可接受之載劑，其中該另一種植物抽出物係選自以下物質組成之群：大花雞腳蔘(Orthosiphon grandiflorus)、大花鰐嘴花(Clinacanthus nutans)、刺桐(Erythrina indica)、盒果藤(Operculina turpethum)、白花菜(Gynandropsis gynandra)、風車草(Erthrina Flabelliformis)、香蠟菊(Helichrysum Odoratissimum)、伊利諾草合歡(Desmanthus illinoensis)、萬壽菊(Tagetes erecta Linn)、 蘇木(Caesalpinia sappan Linn)、俾斯麥棕櫚(Medemia nobilis)、皇冠花(Calatropis gigantean)、檵木(Loropetalum chinense)、狗尾草(Heliotropium indicum)、鬼畫符(Breynia fruticosa)、野菊(Dendranthema indicum)、接骨草(Sambucus chinensis)、野甘草(Scoparis dulcis)、黑木相思(Acacia melanoxylon)、多花大青(Clerodendrum floribundum)、山麻樹(Commersonia bartramia)、車桑子(Dodonaea viscose)、車前草(Duboisa myoporoides)、厚殼樹(Ehretia acuminate)、花球枇杷螺(Ficus coronata)、卵形山羊草(Goodenia ovata)、黃海桐花(Hymenosporum flavum)、花葵(Lavatera plebeian)、白千層(Melaleuca quinquernervia)、血桐(Omolanthes populifolius)、泰國黃果木(Mammea siamensis)及其等組合。
- 一種組合物，其包含正調節皮膚纖維母細胞或角質細胞中動力蛋白之有效量化合物及局部施用上可接受之載劑，其中該化合物係選自以下物質組成之群：
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| ES (1) | ES2645011T3 (zh) |
| MX (1) | MX354734B (zh) |
| PL (2) | PL2659004T3 (zh) |
| TW (1) | TWI579002B (zh) |
| WO (1) | WO2012091832A2 (zh) |
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| KR102499738B1 (ko) * | 2009-08-26 | 2023-02-13 | 마리 케이 인코포레이티드 | 식물 추출물을 포함하는 국소 피부 케어 제형 |
| US8815265B2 (en) | 2010-06-30 | 2014-08-26 | Avon Products, Inc. | Cosmetic use of N-substituted sulfonyloxybenzylamines and related compounds |
| CN106727054A (zh) | 2010-06-30 | 2017-05-31 | 雅芳产品公司 | 用于刺激magp‑1以改善皮肤外观的组合物和方法 |
| US8455518B2 (en) * | 2010-12-28 | 2013-06-04 | Avon Products, Inc. | Method of treating skin with microRNA modulators |
| US8686013B2 (en) * | 2011-08-25 | 2014-04-01 | Avon Products, Inc. | Cosmetic use of substituted amino heterocylic carbamoyl analogs and related compounds |
| WO2014158877A1 (en) * | 2013-03-13 | 2014-10-02 | Avon Products, Inc | Vernonia cinerea extracts and methods of use |
| KR101821010B1 (ko) * | 2014-11-11 | 2018-01-23 | 건국대학교 산학협력단 | 클리나칸투스 누탄스 추출물을 유효성분으로 포함하는 피부 개선용 화장료 조성물 |
| CN106265211B (zh) * | 2015-06-03 | 2020-03-31 | 上海家化生物科技有限公司 | 一种肉苁蓉上清混合物、其制备方法及其皮肤屏障修复作用 |
| CN105147571A (zh) * | 2015-10-21 | 2015-12-16 | 广东药学院 | 黑面神提取物及其应用和含该黑面神提取物的美白抗皱霜 |
| CN110551828B (zh) * | 2019-09-19 | 2021-02-26 | 江苏省家禽科学研究所 | 一种与鸡背部毛孔密度相关的snp分子标记及其应用 |
| CN114957379B (zh) * | 2021-06-28 | 2024-03-26 | 河南省人民医院 | 一种二氢嘧啶硫酮类化合物及其制备方法与应用 |
| CN114149985A (zh) * | 2021-12-06 | 2022-03-08 | 杭州优玛达生物科技有限公司 | 一种分子马达的制备方法及其应用 |
| CN114478396B (zh) * | 2022-03-18 | 2023-12-01 | 河南省人民医院 | 二氢嘧啶(硫)酮类化合物及其制备方法与应用 |
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2011
- 2011-11-29 ES ES11853773.7T patent/ES2645011T3/es active Active
- 2011-11-29 WO PCT/US2011/062279 patent/WO2012091832A2/en active Application Filing
- 2011-11-29 MX MX2013007435A patent/MX354734B/es active IP Right Grant
- 2011-11-29 PL PL11853773T patent/PL2659004T3/pl unknown
- 2011-11-29 EP EP15152603.5A patent/EP2889384B1/en not_active Not-in-force
- 2011-11-29 CN CN201180063233.0A patent/CN103561718B/zh not_active Expired - Fee Related
- 2011-11-29 EP EP11853773.7A patent/EP2659004B1/en active Active
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2012091832A3 (en) | 2012-09-07 |
| BR112013016935A2 (pt) | 2016-08-30 |
| TW201304816A (zh) | 2013-02-01 |
| WO2012091832A2 (en) | 2012-07-05 |
| EP2659004A2 (en) | 2013-11-06 |
| CN103561718A (zh) | 2014-02-05 |
| EP2889384B1 (en) | 2016-09-28 |
| US20120171133A1 (en) | 2012-07-05 |
| ES2645011T3 (es) | 2017-12-01 |
| EP2889384A1 (en) | 2015-07-01 |
| JP2014507132A (ja) | 2014-03-27 |
| CA2822328A1 (en) | 2012-07-05 |
| PL2659004T3 (pl) | 2017-12-29 |
| EP2659004B1 (en) | 2017-08-30 |
| MX354734B (es) | 2018-03-16 |
| JP6249777B2 (ja) | 2017-12-20 |
| EP2659004A4 (en) | 2014-05-14 |
| MX2013007435A (es) | 2013-09-06 |
| CN103561718B (zh) | 2020-06-16 |
| PL2889384T3 (pl) | 2017-07-31 |
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