TWI844795B - Autophagy activators - Google Patents
Autophagy activators Download PDFInfo
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- TWI844795B TWI844795B TW110134884A TW110134884A TWI844795B TW I844795 B TWI844795 B TW I844795B TW 110134884 A TW110134884 A TW 110134884A TW 110134884 A TW110134884 A TW 110134884A TW I844795 B TWI844795 B TW I844795B
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- gene
- autophagy
- tocopherol
- tocopherol phosphate
- disease
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Abstract
含有生育酚磷酸酯或其鹽作為有效成分之自噬活化劑。含有前述自噬活化劑及藥學上可容許之載體之自噬活化用組成物。An autophagy activator containing tocopherol phosphate or its salt as an active ingredient. An autophagy activating composition containing the aforementioned autophagy activator and a pharmaceutically acceptable carrier.
Description
本發明係關於自噬活化劑及自噬活化用組成物。 本申請案係基於在2020年9月17日於日本申請之日本專利特願2020-156458號主張優先權,並將其內容援用於此。 The present invention relates to an autophagy activator and an autophagy activating composition. This application claims priority based on Japanese Patent Application No. 2020-156458 filed in Japan on September 17, 2020, and the contents thereof are hereby incorporated by reference.
自噬(autophagy)為對飢餓、生長因子缺乏及病原體感染等細胞外或細胞內之壓力及訊息進行應答,並藉由將已衰老化或損傷之細胞內物質及胞器進行分解而進行能量的再生產及損傷物質的去除之機制,對維持正常的細胞的恆定性而言實屬重要。從過去的研究中,已報導老化越進展,細胞內之自噬活性越急遽地減少(非專利文獻1)。此外,在抑制自噬之情況,衰老粒線體或經錯誤折疊之蛋白質等會過量地蓄積於細胞內,由於細胞內之氧化壓力增加而誘導細胞死亡,就結果而言,導致細胞老化。Autophagy is a mechanism that responds to extracellular or intracellular stress and messages such as hunger, growth factor deficiency, and pathogen infection, and regenerates energy and removes damaged substances by decomposing aging or damaged intracellular substances and organelles. It is important for maintaining normal cell homeostasis. Previous studies have reported that the more advanced aging is, the more rapidly the autophagic activity in cells decreases (non-patent document 1). In addition, when autophagy is inhibited, aging mitochondria or misfolded proteins will accumulate excessively in cells, inducing cell death due to increased oxidative stress in cells, ultimately leading to cell aging.
從而,藉由活化將細胞內之已老化之物質及胞器進行分解並回收再利用其分解產物之自噬,便可藉由迅速地去除細胞內之不需要物而提高細胞的恆定性。Therefore, by activating autophagy, which decomposes aged substances and organelles in cells and recycles the decomposition products, it is possible to improve cell homeostasis by quickly removing unnecessary substances in cells.
另一方面,作為老化對自噬所帶來之影響,已知在人類的腦中,ATG5基因、ATG7基因及Beclin 1基因的表現量會隨著年齡增長而降低。此外,一般認為在阿茲海默症(Alzheimer’s disease)等神經退化性疾患中,可看出自噬的活性降低。因此,已知自噬的活化係有助於治療及預防以阿茲海默症為首之亨丁頓氏症(Huntington’s disease)、帕金森氏症(Parkinson’s disease)等神經退化性疾患(非專利文獻2、3)。特定而言,在阿茲海默症中,自噬的機能受到阻礙,因而被稱為類澱粉β之凝集蛋白質蓄積於生體內,一般認為此與發病有關(非專利文獻4)。此外,針對屬於伴隨腦的黑質及蒼白球中之鐵的沉積及大腦的萎縮之神經退化性疾患之SENDA病(SENDA:static encephalopathy of childhood with neurodegeneration in adulthood)、屬於在消化道引起嚴重的炎症或潰瘍之炎症性腸疾患之克隆氏症(Crohn's disease)以及癌症,一般認為自噬相關基因或涉及選擇性自噬之基因的變異亦產生影響(非專利文獻6)。On the other hand, as an effect of aging on autophagy, it is known that the expression levels of ATG5, ATG7, and Beclin 1 genes in the human brain decrease with age. In addition, it is generally believed that the activity of autophagy decreases in neurodegenerative diseases such as Alzheimer’s disease. Therefore, it is known that the activation of autophagy contributes to the treatment and prevention of neurodegenerative diseases such as Huntington’s disease and Parkinson’s disease, including Alzheimer’s disease (Non-patent Documents 2, 3). Specifically, in Alzheimer’s disease, the function of autophagy is blocked, and aggregated proteins called starch-like β accumulate in the body, which is generally considered to be related to the onset of the disease (Non-patent Document 4). In addition, it is generally believed that mutations in autophagy-related genes or genes involved in selective autophagy also have an impact on SENDA disease (static encephalopathy of childhood with neurodegeneration in adulthood), a neurodegenerative disease accompanied by iron deposition in the substantia nigra and globus alba and brain atrophy, Crohn's disease, an inflammatory bowel disease causing severe inflammation or ulcers in the digestive tract, and cancer (Non-patent document 6).
自噬的過程已在酵母及哺乳類兩者中進行研究,最多已利用36種蛋白質。該等之中,從自噬體的形成至其內容物的分化係由自噬相關基因(ATG)所編碼出之Atg蛋白質所控制,可分類成包含Atg12-ATG5結合系統、LC3-Phosphatidyl Ethanolamine(PE)結合系統之6個組,各自在各過程中階段性地產生作用。The process of autophagy has been studied in both yeast and mammals, and up to 36 proteins have been used. Among them, the formation of autophagosomes and the differentiation of their contents are controlled by Atg proteins encoded by autophagy-related genes (ATG), which can be classified into 6 groups including Atg12-ATG5 binding system and LC3-Phosphatidyl Ethanolamine (PE) binding system, each of which plays a role in each stage of the process.
在另一方面,自噬在穩定狀態下被壓抑至較低的水平,在飢餓等壓力下則被活化。已知mTOR(mammalian target of rapamycin)係作為從酵母至哺乳類之自噬的主要抑制因子而發揮作用,但在飢餓條件下,mTOR被去活化,誘導自噬(非專利文獻5)。On the other hand, autophagy is suppressed to a low level under stable conditions and activated under stress such as starvation. mTOR (mammalian target of rapamycin) is known to function as a major inhibitor of autophagy from yeast to mammals, but under starvation conditions, mTOR is inactivated, inducing autophagy (Non-patent document 5).
作為自噬活化劑,已報導增加屬於自噬的活性狀態的標記之LC3相關的因子並活化自噬之化合物,以及促進自噬通量(包含自噬體向溶酶體之融合)之化合物(專利文獻1~4)。 [先前技術文獻] [專利文獻] As autophagy activators, compounds that increase LC3-related factors that are markers of the active state of autophagy and activate autophagy, as well as compounds that promote autophagic flux (including fusion of autophagosomes to lysosomes) have been reported (patent documents 1 to 4). [Prior art document] [Patent document]
[專利文獻1]國際公開第2018/173653號 [專利文獻2]日本專利特開2018-80204號公報 [專利文獻3]日本專利特表2018-510902號公報 [專利文獻4]日本專利特表2019-529514號公報 [非專利文獻] [Patent Document 1] International Publication No. 2018/173653 [Patent Document 2] Japanese Patent Publication No. 2018-80204 [Patent Document 3] Japanese Patent Publication No. 2018-510902 [Patent Document 4] Japanese Patent Publication No. 2019-529514 [Non-patent Document]
[非專利文獻1]Yogendra S. Rajawat et al., Aging: Central role for autophagy and the lysosomal degradative system. Ageing Research Reviews 8 (2009) 199-213. [非專利文獻2]Aaron Barnett et al., Autophagy in Aging and Alzheimer's Disease: Pathologic or Protective?. J Alzheimers Dis. 2011; 25(3): 385-394. [非專利文獻3]Marta M. Lipinski et al., Genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in Alzheimer's disease. Proc Natl Acad Sci USA. 2010, 107, 14164-14169. [非專利文獻4]Xiangqing Li et al., Cubeben induces autophagy via PI3K-AKT-mTOR pathway to protect primary neurons against amyloid beta in Alzheimer's disease. Cytotechnology (2019) 71: 679-686. [非專利文獻5]豬俁等人,「自噬與老化之相關性」,岐齒學誌,2018年,第45卷,第1號,1-7 [非專利文獻6]蔭山等人,「自噬及疾患」,領域融合評論,2014年,3,e006 [Non-patent document 1]Yogendra S. Rajawat et al., Aging: Central role for autophagy and the lysosomal degradative system. Ageing Research Reviews 8 (2009) 199-213. [Non-patent document 2]Aaron Barnett et al., Autophagy in Aging and Alzheimer's Disease: Pathologic or Protective?. J Alzheimers Dis. 2011; 25(3): 385-394. [Non-patent document 3]Marta M. Lipinski et al., Genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in Alzheimer's disease. Proc Natl Acad Sci USA. 2010, 107, 14164-14169. [Non-patent document 4]Xiangqing Li et al., Cubeben induces autophagy via PI3K-AKT-mTOR pathway to protect primary neurons against amyloid beta in Alzheimer's disease. Cytotechnology (2019) 71: 679-686. [Non-patent document 5] Zhu Yu et al., "The correlation between autophagy and aging", Journal of Qiya, 2018, Vol. 45, No. 1, 1-7 [Non-patent document 6] Yingshan et al., "Autophagy and disease", Convergence Review, 2014, 3, e006
[發明所欲解決之課題][The problem that the invention wants to solve]
如上述,已知在阿茲海默症等各式各樣的疾患中,自噬機能降低,遂謀求可有效地活化自噬之藥劑。然而,就以往所知之藥劑而言,其效果可謂仍不充分。As mentioned above, it is known that in various diseases such as Alzheimer's disease, the autophagy function is reduced, and drugs that can effectively activate autophagy are sought. However, the effects of the drugs known in the past are still insufficient.
於是,本發明之目的為提供可有效地活化自噬之自噬活化劑及含有前述自噬活化劑之自噬活化用組成物。 [解決課題之手段] Therefore, the purpose of the present invention is to provide an autophagy activator that can effectively activate autophagy and an autophagy activating composition containing the aforementioned autophagy activator. [Means for solving the problem]
本發明包含以下態樣。 [1]一種自噬活化劑,其含有生育酚磷酸酯或其鹽作為有效成分。 [2]如[1]所記載之自噬活化劑,其中,前述生育酚磷酸酯或其鹽為選自由α-生育酚磷酸酯及γ-生育酚磷酸酯所組成之群組之至少一種生育酚磷酸酯或其鹽。 The present invention includes the following aspects. [1] An autophagy activator, which contains tocopherol phosphate or a salt thereof as an active ingredient. [2] The autophagy activator as described in [1], wherein the tocopherol phosphate or a salt thereof is at least one tocopherol phosphate or a salt thereof selected from the group consisting of α-tocopherol phosphate and γ-tocopherol phosphate.
[3]如[1]或[2]所記載之自噬活化劑,其含有前述生育酚磷酸酯的鹽作為有效成分,前述生育酚磷酸酯的鹽為生育酚磷酸酯的鈉鹽。 [4]如[1]~[3]中任一項所記載之自噬活化劑,其促進LC3基因的表現。 [5]如[1]~[4]中任一項所記載之自噬活化劑,其促進ATG5基因的表現。 [3] The autophagy activator described in [1] or [2], which contains the salt of tocopherol phosphate as an active ingredient, wherein the salt of tocopherol phosphate is sodium salt of tocopherol phosphate. [4] The autophagy activator described in any one of [1] to [3], which promotes the expression of LC3 gene. [5] The autophagy activator described in any one of [1] to [4], which promotes the expression of ATG5 gene.
[6]如[1]~[5]中任一項所記載之自噬活化劑,其促進ATG7基因的表現。 [7]如[1]~[6]中任一項所記載之自噬活化劑,其促進Beclin 1基因的表現。 [8]如[1]~[7]中任一項所記載之自噬活化劑,其抑制mTOR基因的表現。 [6] An autophagy activator as described in any one of [1] to [5], which promotes the expression of the ATG7 gene. [7] An autophagy activator as described in any one of [1] to [6], which promotes the expression of the Beclin 1 gene. [8] An autophagy activator as described in any one of [1] to [7], which inhibits the expression of the mTOR gene.
[9]如[1]~[8]中任一項所記載之自噬活化劑,其係用於預防或治療阿茲海默症。 [10]一種自噬活化用組成物,其含有如[1]~[9]中任一項所記載之自噬活化劑及藥學上可容許之載體。 [11]如[10]所記載之自噬活化用組成物,其中,前述生育酚磷酸酯或其鹽之合計含量係相對於自噬活化用組成物的全量而言為0.01~10質量%。 [發明效果] [9] An autophagy activator as described in any one of [1] to [8], which is used for preventing or treating Alzheimer's disease. [10] An autophagy activating composition, which contains an autophagy activator as described in any one of [1] to [9] and a pharmaceutically acceptable carrier. [11] An autophagy activating composition as described in [10], wherein the total content of the tocopherol phosphate or its salt is 0.01 to 10% by weight relative to the total amount of the autophagy activating composition. [Effect of the invention]
藉由本發明,係提供可有效地活化自噬之自噬活化劑及含有前述自噬活化劑之自噬活化用組成物。The present invention provides an autophagy activator that can effectively activate autophagy and an autophagy activating composition containing the autophagy activator.
(自噬活化劑)(Autophagy Activator)
在一實施形態中,本發明係提供一種自噬活化劑,其含有生育酚磷酸酯或其鹽作為有效成分。 在此處,所謂「自噬(autophagy)」,係藉由將已衰老或損傷之細胞內物質及胞器進行分解而進行能量的再生產及損傷物質的去除之機制。 本實施形態之自噬活化劑可促進屬於自噬標記之LC3基因以及自噬體中所包含之ATG5基因、ATG7基因及Beclin 1基因的表現,使自噬活化。此外,可藉由抑制作為自噬的抑制因子而發揮作用之mTOR基因的表現而使自噬活化。 In one embodiment, the present invention provides an autophagy activator containing tocopherol phosphate or a salt thereof as an active ingredient. Here, "autophagy" refers to a mechanism for regenerating energy and removing damaged substances by decomposing aged or damaged intracellular substances and organelles. The autophagy activator of this embodiment can promote the expression of the LC3 gene, which is an autophagy marker, and the ATG5 gene, ATG7 gene, and Beclin 1 gene contained in the autophagosome, thereby activating autophagy. In addition, autophagy can be activated by inhibiting the expression of the mTOR gene, which acts as an inhibitor of autophagy.
本實施形態之自噬活化劑只要包含生育酚磷酸酯或其鹽作為有效成分,即無特別限定。作為用於本實施形態之自噬活化劑之生育酚磷酸酯,可列舉下述一般式(1)所示之化合物。The autophagy activator of the present embodiment is not particularly limited as long as it contains tocopherol phosphate or a salt thereof as an effective ingredient. Examples of the tocopherol phosphate used in the autophagy activator of the present embodiment include compounds represented by the following general formula (1).
[式中,R 1、R 2及R 3互相獨立地表示氫原子或甲基。] [In the formula, R 1 , R 2 and R 3 independently represent a hydrogen atom or a methyl group.]
在生育酚磷酸酯中,依據上述一般式(1)中之R 1、R 2、R 3,存在α-生育酚磷酸酯(R 1、R 2、R 3=CH 3)、β-生育酚磷酸酯(R 1、R 3=CH 3,R 2=H)、γ-生育酚磷酸酯(R 1、R 2=CH 3,R 3=H)、δ-生育酚磷酸酯(R 1=CH 3,R 2、R 3=H)、ζ 2-生育酚磷酸酯(R 2、R 3=CH 3,R 1=H)、η-生育酚磷酸酯(R 2=CH 3,R 1、R 3=H)等。 Among the tocopherol phosphates, according to R 1 , R 2 , and R 3 in the above general formula (1), there are α-tocopherol phosphate (R 1 , R 2 , R 3 = CH 3 ), β-tocopherol phosphate (R 1 , R 3 = CH 3 , R 2 = H), γ-tocopherol phosphate (R 1 , R 2 = CH 3 , R 3 = H), δ-tocopherol phosphate (R 1 = CH 3 , R 2 , R 3 = H), ζ 2 -tocopherol phosphate (R 2 , R 3 = CH 3 , R 1 = H), η-tocopherol phosphate (R 2 = CH 3 , R 1 , R 3 = H), and the like.
生育酚磷酸酯並無特別限定,可為此等生育酚磷酸酯中之任何者。在此等之中,較佳為α-生育酚磷酸酯及γ-生育酚磷酸酯,更佳為α-生育酚磷酸酯。The tocopherol phosphate is not particularly limited and may be any of these tocopherol phosphates. Among these, α-tocopherol phosphate and γ-tocopherol phosphate are preferred, and α-tocopherol phosphate is more preferred.
上述一般式(1)所示之化合物在二氫苯并哌喃環的2位具有不對稱碳原子,因而存在d體及l體的立體異構物,以及dl體。生育酚磷酸酯可為此等立體異構物中之任何者,較佳為dl體。The compound represented by the general formula (1) has an asymmetric carbon atom at the 2-position of the dihydrobenzopyran ring, and thus has d-isomers, l-isomers, and dl-isomers. Tocopherol phosphate may be any of these stereoisomers, and is preferably dl-isomers.
在上述之中,作為生育酚磷酸酯,較佳為dl-α-生育酚磷酸酯及dl-γ-生育酚磷酸酯,更佳為dl-α-生育酚磷酸酯。Among the above, as the tocopherol phosphate, dl-α-tocopherol phosphate and dl-γ-tocopherol phosphate are preferred, and dl-α-tocopherol phosphate is more preferred.
生育酚磷酸酯的鹽並無特別限定,可列舉例如與無機鹼的鹽、與有機鹼的鹽等。The salt of tocopherol phosphate is not particularly limited, and examples thereof include salts with inorganic bases and salts with organic bases.
作為與無機鹼的鹽,可列舉例如鈉鹽、鉀鹽等鹼金屬鹽;鈣鹽、鎂鹽等鹼土族金屬鹽;鋁鹽;銨鹽;鋅鹽等。 作為與有機鹼的鹽,可列舉例如烷基銨鹽、與鹼性胺基酸的鹽等。 Examples of salts with inorganic bases include alkaline metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts; ammonium salts; zinc salts, etc. Examples of salts with organic bases include alkylammonium salts and salts with alkaline amino acids, etc.
在上述之中,作為生育酚磷酸酯的鹽,較佳為鹼金屬鹽,更佳為鈉鹽。生育酚磷酸酯的鹼金屬鹽,特定而言,鈉鹽,具有對水之溶解性較高,此外,由於性狀呈粉末,因而容易處理之優點。Among the above, the salt of tocopherol phosphate is preferably an alkali metal salt, more preferably a sodium salt. The alkali metal salt of tocopherol phosphate, in particular the sodium salt, has the advantages of being highly soluble in water and being easy to handle due to its powdery state.
作為生育酚磷酸酯之較佳態樣,可列舉上述一般式(1)所示之化合物的鹼金屬鹽(例如鈉鹽)、α-生育酚磷酸酯的鹼金屬鹽(例如鈉鹽)、γ-生育酚磷酸酯的鹼金屬鹽(例如鈉鹽)、dl-α-生育酚磷酸酯的鹼金屬鹽(例如鈉鹽)、dl-γ-生育酚磷酸酯的鹼金屬鹽(例如鈉鹽)等。Preferred embodiments of tocopherol phosphate include alkali metal salts (e.g., sodium salts) of the compound represented by the general formula (1), alkali metal salts (e.g., sodium salts) of α-tocopherol phosphate, alkali metal salts (e.g., sodium salts) of γ-tocopherol phosphate, alkali metal salts (e.g., sodium salts) of dl-α-tocopherol phosphate, alkali metal salts (e.g., sodium salts) of dl-γ-tocopherol phosphate, and the like.
在生育酚磷酸酯的鹼金屬鹽之中,較佳為α-生育酚磷酸酯的鈉鹽及γ-生育酚磷酸酯的鈉鹽,更佳為α-生育酚磷酸酯的鈉鹽。Among the alkaline metal salts of tocopherol phosphate, sodium salt of α-tocopherol phosphate and sodium salt of γ-tocopherol phosphate are preferred, and sodium salt of α-tocopherol phosphate is more preferred.
dl-α-生育酚磷酸酯的鈉鹽係以TPNa(註冊商標)(標示名稱:生育酚磷酸酯鈉)的製品名由昭和電工加以市售。前述TPNa係例示為生育酚磷酸酯之較佳例。Sodium salt of dl-α-tocopherol phosphate is commercially available from Showa Denko under the product name of TPNa (registered trademark) (label name: sodium tocopherol phosphate). The above-mentioned TPNa is exemplified as a preferred example of tocopherol phosphate.
本實施形態之自噬活化劑可單獨使用選自生育酚磷酸酯及其鹽中之1種,亦可併用2種以上。本實施形態之自噬活化劑較佳係包含生育酚磷酸酯的鹽,更佳係單獨使用生育酚磷酸酯的鹼金屬鹽(例如鈉鹽)。The autophagy activator of the present embodiment can be used alone or in combination of two or more selected from tocopherol phosphate and its salts. The autophagy activator of the present embodiment preferably comprises a salt of tocopherol phosphate, and more preferably, an alkali metal salt (e.g., sodium salt) of tocopherol phosphate is used alone.
生育酚磷酸酯或其鹽可藉由公知的製造方法來予以製造。生育酚磷酸酯或其鹽可藉由例如日本專利特開昭59-44375號公報、國際公開公報第97/14705號等所記載之方法來予以製造。 例如,藉由使氧氯化磷等磷酸化劑作用於已溶解於溶媒中之生育酚,在反應結束後適宜進行精製,便可獲得生育酚磷酸酯。再者,藉由將所獲得之生育酚磷酸酯以氧化鎂等金屬氧化物、氫氧化鈉等金屬氫氧化物或者氫氧化銨或氫氧化烷基銨等進行中和,便可獲得生育酚磷酸酯的鹽。生育酚磷酸酯或其鹽的製造方法並不限定於上述方法。 Tocopherol phosphate or its salt can be produced by a known production method. Tocopherol phosphate or its salt can be produced by a method described in, for example, Japanese Patent Publication No. 59-44375, International Publication No. 97/14705, etc. For example, by allowing a phosphorylating agent such as phosphorus oxychloride to act on tocopherol dissolved in a solvent, and then appropriately purifying it after the reaction is completed, tocopherol phosphate can be obtained. Furthermore, by neutralizing the obtained tocopherol phosphate with a metal oxide such as magnesium oxide, a metal hydroxide such as sodium hydroxide, or ammonium hydroxide or alkylammonium hydroxide, a salt of tocopherol phosphate can be obtained. The production method of tocopherol phosphate or its salt is not limited to the above method.
以下,有時將生育酚磷酸酯及其鹽總括記載為「生育酚磷酸酯等」。Hereinafter, tocopherol phosphate and its salts may be collectively described as "tocopherol phosphate, etc."
本實施形態之自噬活化劑在治療阿茲海默症、亨丁頓氏症、帕金森氏症等神經退化性疾患之目的下,可將其本身投予至患者而使用。此外,本實施形態之自噬活化劑在活化自噬之目的下,亦可摻合至醫藥品或化妝品中而使用。此外,亦可摻合至後述之自噬活化用組成物中而使用。The autophagy activator of this embodiment can be administered to a patient for the purpose of treating neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc. In addition, the autophagy activator of this embodiment can also be mixed into a pharmaceutical or cosmetic product for the purpose of activating autophagy. In addition, it can also be mixed into the autophagy activating composition described below for use.
本實施形態之自噬活化劑可藉由促進LC3基因的表現而有效地活化自噬。 本實施形態之自噬活化劑可藉由促進ATG5基因的表現而有效地活化自噬。 本實施形態之自噬活化劑可藉由促進ATG7基因的表現而有效地活化自噬。 本實施形態之自噬活化劑可藉由促進Beclin 1基因的表現而有效地活化自噬。 本實施形態之自噬活化劑可藉由抑制mTOR基因的表現而有效地活化自噬。 本實施形態之自噬活化劑可有效地活化自噬,故可用於預防或治療阿茲海默症。 The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the LC3 gene. The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the ATG5 gene. The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the ATG7 gene. The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the Beclin 1 gene. The autophagy activator of the present embodiment can effectively activate autophagy by inhibiting the expression of the mTOR gene. The autophagy activator of the present embodiment can effectively activate autophagy, and thus can be used to prevent or treat Alzheimer's disease.
已知類澱粉β會引起神經細胞中之自噬的降低。此外,已知類澱粉β會誘使自噬的降低及其所引發之神經細胞的被稱為細胞凋亡之細胞死亡。 本實施形態之自噬活化劑可促進在類澱粉β存在下之LC3基因表現。 本實施形態之自噬活化劑可促進在類澱粉β存在下之ATG5基因表現。 本實施形態之自噬活化劑可促進在類澱粉β存在下之ATG7基因表現。 本實施形態之自噬活化劑可抑制在類澱粉β存在下之細胞凋亡。 本實施形態之自噬活化劑,特定而言,在神經細胞中,可促進在類澱粉β存在下之選自由LC3基因、ATG5基因及ATG7基因所組成之群組之至少1種基因的表現。此外,本實施形態之自噬活化劑,特定而言,在神經細胞中,可抑制在類澱粉β存在下之細胞凋亡。 It is known that amyloid β causes a decrease in autophagy in nerve cells. In addition, amyloid β is known to induce a decrease in autophagy and the resulting cell death of nerve cells called apoptosis. The autophagy activator of the present embodiment can promote the expression of the LC3 gene in the presence of amyloid β. The autophagy activator of the present embodiment can promote the expression of the ATG5 gene in the presence of amyloid β. The autophagy activator of the present embodiment can promote the expression of the ATG7 gene in the presence of amyloid β. The autophagy activator of the present embodiment can inhibit apoptosis in the presence of amyloid β. The autophagy activator of this embodiment, in particular, can promote the expression of at least one gene selected from the group consisting of LC3 gene, ATG5 gene and ATG7 gene in the presence of starch beta in nerve cells. In addition, the autophagy activator of this embodiment, in particular, can inhibit apoptosis in the presence of starch beta in nerve cells.
所謂在類澱粉β存在下促進LC3基因表現,係意味藉由在類澱粉β存在下投予本實施形態之自噬活化劑,而使LC3基因的表現量相較於未投予前述自噬活化劑之情況而言上升。針對ATG5基因及ATG7基因亦相同。 所謂在類澱粉β存在下抑制細胞凋亡,係意味藉由在類澱粉β存在下投予本實施形態之自噬活化劑,而使細胞凋亡相較於未投予前述自噬活化劑之情況而言受到抑制。 The so-called promotion of LC3 gene expression in the presence of amyloid β means that by administering the autophagy activator of the present embodiment in the presence of amyloid β, the expression amount of LC3 gene is increased compared to the case where the aforementioned autophagy activator is not administered. The same is true for the ATG5 gene and the ATG7 gene. The so-called inhibition of cell apoptosis in the presence of amyloid β means that by administering the autophagy activator of the present embodiment in the presence of amyloid β, cell apoptosis is inhibited compared to the case where the aforementioned autophagy activator is not administered.
LC3(microtubule associated protein 1 light chain 3 alpha:NCBI Gene ID:84557)係在自噬的訊息傳遞上游被附加磷脂醯乙醇胺,轉換成會被自噬體膜所吸引之LC3-II,結合至自噬體膜。LC3係用作自噬體的標記。作為人類LC3基因的鹼基序列,可列舉例如登錄於NCBI Reference Sequence資料庫之NM_032514.4及NM_181509.3等。LC3 (microtubule associated protein 1 light chain 3 alpha: NCBI Gene ID: 84557) is attached with phosphatidylethanolamine upstream of the autophagy signaling pathway and converted into LC3-II, which is attracted to the autophagosome membrane and binds to the autophagosome membrane. LC3 is used as a marker for autophagosomes. Examples of the base sequence of the human LC3 gene include NM_032514.4 and NM_181509.3 registered in the NCBI Reference Sequence database.
ATG5(autophagy related 5:NCBI Gene ID:9474)係與ATG12進行結合,在泛素樣共軛系統中作為E1樣活化酵素而發揮機能。作為人類ATG5基因的鹼基序列,可列舉例如登錄於NCBI Reference Sequence資料庫之NM_001286106.1、NM_001286107.1、NM_001286108.1、NM_001286111.1及NM_004849.4等。ATG5 (autophagy related 5: NCBI Gene ID: 9474) binds to ATG12 and functions as an E1-like activating enzyme in the ubiquitin-like conjugate system. The base sequences of the human ATG5 gene include NM_001286106.1, NM_001286107.1, NM_001286108.1, NM_001286111.1, and NM_004849.4 registered in the NCBI Reference Sequence database.
ATG7(autophagy related 7:NCBI Gene ID:10533)係作為ATP依存性地活化LC3及ATG12之E1樣活化酵素而發揮機能。作為人類ATG7基因的鹼基序列,可列舉例如登錄於NCBI Reference Sequence資料庫之NM_001136031.3、NM_001144912.2、NM_001349232.2、NM_001349233.2、NM_001349234.2等。ATG7 (autophagy related 7: NCBI Gene ID: 10533) functions as an E1-like activating enzyme that activates LC3 and ATG12 in an ATP-dependent manner. Examples of the base sequence of the human ATG7 gene include NM_001136031.3, NM_001144912.2, NM_001349232.2, NM_001349233.2, and NM_001349234.2 registered in the NCBI Reference Sequence database.
Beclin 1(NCBI Gene ID:8678)係與ATG14L、Vps34mp150共同地形成第III類PI3K複合體,作為自噬體形成的正控制因子而發揮機能。作為人類Beclin 1基因的鹼基序列,可列舉例如登錄於NCBI Reference Sequence資料庫之NM_001313998.2、NM_001313999.1、NM_001314000.1及NM_003766.4等。Beclin 1 (NCBI Gene ID: 8678) forms a class III PI3K complex together with ATG14L and Vps34mp150, and functions as a positive control factor for autophagosome formation. The base sequences of the human Beclin 1 gene include, for example, NM_001313998.2, NM_001313999.1, NM_001314000.1, and NM_003766.4 registered in the NCBI Reference Sequence database.
mTOR(mechanistic target of rapamycin kinase:NCBI Gene ID:2475)為磷脂醯肌醇激酶相關激酶中之1種,其介導針對於DNA損傷及營養缺乏等壓力之細胞應答。mTOR係作為自噬的抑制因子而發揮機能。作為人類mTOR基因的鹼基序列,可列舉例如登錄於NCBI Reference Sequence資料庫之NM_004958.4等。mTOR (mechanistic target of rapamycin kinase: NCBI Gene ID: 2475) is a phosphatidylinositol kinase-related kinase that mediates cellular responses to stresses such as DNA damage and nutrient deficiency. mTOR functions as an inhibitor of autophagy. The base sequence of the human mTOR gene can be listed, for example, as NM_004958.4 registered in the NCBI Reference Sequence database.
本實施形態之自噬活化劑可投予至阿茲海默症、亨丁頓氏症、帕金森氏症等神經退化性疾患的發病風險較高的患者,用於預防阿茲海默症、亨丁頓氏症、帕金森氏症等神經退化性疾患。此外,本實施形態之自噬活化劑亦可投予至阿茲海默症、亨丁頓氏症、帕金森氏症等神經退化性疾患已發病之患者,用於抑制神經退化性疾患的進展或惡化。The autophagy activator of this embodiment can be administered to patients with a higher risk of developing neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc., to prevent neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc. In addition, the autophagy activator of this embodiment can also be administered to patients who have already developed neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc., to inhibit the progression or deterioration of neurodegenerative diseases.
本實施形態之自噬活化劑可藉由與後述之自噬活化用組成物同樣的方法來投予至患者,可經口投予,亦可非經口投予,可進行靜脈內、動脈內、肌肉內、皮內、皮下、腹腔內等投予,亦可以栓劑之形式進行直腸內投予,亦可以皮膚外用劑之形式投予至皮膚。The autophagy activator of this embodiment can be administered to a patient by the same method as the autophagy activating composition described below, and can be administered orally or parenterally, and can be administered intravenously, intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally. It can also be administered intrarectally in the form of a suppository, or can be administered to the skin in the form of a topical preparation.
(自噬活化用組成物) 在一實施形態中,本發明係提供一種自噬活化用組成物,其含有上述之自噬活化劑及藥學上可容許之載體。 (Autophagy Activating Composition) In one embodiment, the present invention provides an autophagy activating composition, which contains the above-mentioned autophagy activator and a pharmaceutically acceptable carrier.
本實施形態之自噬活化用組成物可依照常法(例如日本藥典所記載之方法),藉由將上述之自噬活化劑、藥學上可容許之載體及視情況其他成分進行混合並加以製劑化而予以製造。The autophagy activating composition of the present embodiment can be produced by mixing the above-mentioned autophagy activator, a pharmaceutically acceptable carrier and other components as appropriate and preparing the mixture according to conventional methods (for example, the method described in the Japanese Pharmacopoeia).
在本說明書中,所謂「藥學上可容許之載體」,係意味不會阻礙有效成分的生理活性,且對其投予對象不會顯示出實質的毒性之載體。另外,所謂「不會顯示出實質的毒性」,係意味該成分在通常所使用之投予量下,對投予對象不會顯示出毒性。作為藥學上可容許之載體,並無特別限制,可列舉賦形劑、黏合劑、崩解劑、潤滑劑、乳化劑、安定劑、稀釋劑、注射劑用溶劑、油性基劑、保濕劑、觸感提升劑、界面活性劑、高分子/增黏/凝膠化劑、溶劑、噴射劑、抗氧化劑、還原劑、氧化劑、螯合劑、酸、鹼、粉體、無機鹽、水、含有金屬之化合物、不飽和單體、多元醇、高分子添加劑、輔助劑、潤濕劑、增黏劑、黏著賦予物質、油性原料、液狀基質、脂溶性物質、高分子羧酸鹽等。In this manual, the so-called "pharmaceutically acceptable carrier" means a carrier that does not hinder the physiological activity of the active ingredient and does not show substantial toxicity to the subject to which it is administered. In addition, the so-called "does not show substantial toxicity" means that the ingredient does not show toxicity to the subject to which it is administered at the dosage normally used. There are no special restrictions on pharmaceutically acceptable carriers, and examples thereof include excipients, adhesives, disintegrants, lubricants, emulsifiers, stabilizers, diluents, solvents for injections, oily bases, moisturizers, touch enhancers, surfactants, polymers/thickening/gelling agents, solvents, sprays, and anti- Oxidants, reducing agents, oxidants, chelating agents, acids, bases, powders, inorganic salts, water, metal-containing compounds, unsaturated monomers, polyols, polymer additives, auxiliary agents, wetting agents, thickeners, adhesion-imparting substances, oily raw materials, liquid bases, fat-soluble substances, polymer carboxylates, etc.
作為此等成分之具體例,可列舉例如國際公開公報第2016/076310號所記載者等。再者,作為高分子/增黏/凝膠化劑之具體例,可列舉甲基丙烯醯基氧基乙基磷醯膽鹼、甲基丙烯酸丁酯或此等的聚合物等。藥學上可容許之載體可單獨使用1種,亦可併用2種以上。Specific examples of such ingredients include those described in International Publication No. 2016/076310. Specific examples of polymers/thickening/gelling agents include methacryloyloxyethylphosphatidylcholine, butyl methacrylate, or polymers thereof. Pharmaceutically acceptable carriers may be used alone or in combination of two or more.
此外,作為其他成分,並無特別限制,可列舉防腐劑、抗菌劑、紫外線吸收劑、美白劑、消炎劑、抗炎症劑、生髮用藥劑、血液循環促進劑、刺激劑、激素類、抗皺劑、抗老化劑、緊緻劑、冷感劑、溫感劑、創傷治癒促進劑、刺激緩和劑、鎮痛劑、細胞賦活劑、植物/動物/微生物萃取物、種子油、鎮癢劑、角質剝離/溶解劑、制汗劑、清涼劑、收斂劑、酵素、核酸、香料、色素、著色劑、染料、顏料、消炎鎮痛劑、抗真菌劑、抗組織胺劑、催眠鎮靜劑、精神安定劑、抗高血壓劑、降壓利尿劑、抗生物質、麻醉劑、抗菌性物質、抗癲癇劑、冠狀血管擴張劑、生藥、止癢劑、角質軟化剝離劑、紫外線遮斷劑、防腐殺菌劑、抗氧化物質、pH調整劑、添加劑、金屬皂等。作為此等成分之具體例,可列舉例如國際公開公報第2016/076310號所記載者等。再者,作為植物/動物/微生物萃取物之具體例,可列舉歐洲稻槎菜(Lapsana communis)花/葉/莖、茶葉等。作為種子油之具體例,可列舉辣木種子油。作為香料之具體例,可列舉紫蘇醛。其他成分可單獨使用1種,亦可併用2種以上。In addition, other ingredients are not particularly limited, and include preservatives, antibacterial agents, ultraviolet absorbers, whitening agents, anti-inflammatory agents, anti-inflammatory agents, hair growth agents, blood circulation promoters, stimulants, hormones, anti-wrinkle agents, anti-aging agents, firming agents, cooling agents, warming agents, wound healing promoters, irritation relievers, analgesics, cell activators, plant/animal/microorganism extracts, seed oils, anti-itching agents, keratin peeling/dissolving agents, and antiperspirants. , coolants, astringents, enzymes, nucleic acids, fragrances, pigments, colorants, dyes, pigments, anti-inflammatory analgesics, antifungal agents, antihistamines, hypnotic sedatives, tranquilizers, antihypertensives, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptics, coronary vasodilators, herbal medicines, antipruritics, keratin softening and exfoliating agents, ultraviolet light blocking agents, preservatives and bactericidal agents, antioxidant substances, pH adjusters, additives, metal soaps, etc. As specific examples of such ingredients, for example, those described in International Publication No. 2016/076310 can be cited. Furthermore, as specific examples of plant/animal/microorganism extracts, Lapsana communis flowers/leaves/stems, tea leaves, etc. can be cited. As specific examples of seed oils, Moringa seed oil can be cited. As specific examples of spices, perilla aldehyde can be cited. Other ingredients can be used alone or in combination of two or more.
本實施形態之自噬活化用組成物可含有治療有效量的前述自噬活化劑。所謂「治療有效量」,係意味有效於治療或預防患者的疾患之藥劑的量。治療有效量可依投予對象的疾患的狀態、年齡、性別及體重等而變動。 在本實施形態之自噬活化用組成物中,上述自噬活化劑的治療有效量可為生育酚磷酸酯等可活化自噬之量。此外,上述自噬活化劑的治療有效量可為生育酚磷酸酯等可促進選自由LC3基因、ATG5基因、ATG7基因及Beclin 1基因所組成之群組之至少1種基因的表現之量。或者,可為生育酚磷酸酯等可抑制屬於自噬抑制因子之mTOR基因的表現之量。此外,上述自噬活化劑的治療有效量可為生育酚磷酸酯等可在類澱粉β的存在下抑制細胞凋亡之量。本實施形態之自噬活化用組成物中之前述自噬活化劑的治療有效量係生育酚磷酸酯或其鹽之合計含量相對於自噬活化用組成物的全量而言,可例如為0.01~10質量%,亦可例如為0.1~10質量%,亦可例如為0.1~5質量%,亦可例如為0.1~3質量%,亦可例如為0.1~2質量%,亦可例如為0.3~2質量%,亦可例如為0.6~1.5質量%。 The composition for activating autophagy of the present embodiment may contain a therapeutically effective amount of the aforementioned autophagy activator. The so-called "therapeutically effective amount" means the amount of the agent that is effective for treating or preventing the patient's disease. The therapeutically effective amount may vary depending on the state of the disease, age, gender, and weight of the subject to be administered. In the composition for activating autophagy of the present embodiment, the therapeutically effective amount of the aforementioned autophagy activator may be an amount of tocopherol phosphate, etc. that can activate autophagy. In addition, the therapeutically effective amount of the aforementioned autophagy activator may be an amount of tocopherol phosphate, etc. that can promote the expression of at least one gene selected from the group consisting of LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene. Alternatively, it may be an amount of tocopherol phosphate, etc. that can inhibit the expression of mTOR gene, which is an autophagy inhibitor. In addition, the therapeutically effective amount of the above-mentioned autophagy activator can be an amount of tocopherol phosphate, etc. that can inhibit cell apoptosis in the presence of starch beta. The therapeutically effective amount of the above-mentioned autophagy activator in the autophagy activating composition of this embodiment is the total content of tocopherol phosphate or its salt relative to the total amount of the autophagy activating composition, which can be, for example, 0.01-10 mass%, or 0.1-10 mass%, or 0.1-5 mass%, or 0.1-3 mass%, or 0.1-2 mass%, or 0.3-2 mass%, or 0.6-1.5 mass%.
上述生育酚磷酸酯等在自噬活化用組成物中之含量,在單獨使用1種生育酚磷酸酯等之情況,係意味該化合物的含量,在組合使用2種以上生育酚磷酸酯等之情況,係意味此等化合物之合計的含量。The content of the above-mentioned tocopherol phosphate ester in the composition for activating autophagy means the content of the compound when one tocopherol phosphate ester is used alone, and means the total content of these compounds when two or more tocopherol phosphate esters are used in combination.
本實施形態之自噬活化用組成物可為醫藥組成物,亦可為化妝料。The composition for activating autophagy of this embodiment may be a pharmaceutical composition or a cosmetic.
(醫藥組成物) 在一實施形態中,本發明係提供一種自噬活化用醫藥組成物,其含有上述之自噬活化劑及藥學上可容許之載體。 (Pharmaceutical composition) In one embodiment, the present invention provides a pharmaceutical composition for activating autophagy, which contains the above-mentioned autophagy activator and a pharmaceutically acceptable carrier.
在本實施形態之醫藥組成物中,作為藥學上可容許之載體,並無特別限制,除了上述所列舉者以外,尚可使用一般使用於醫藥品之載體。例如,可使用記載於日本藥典、日本藥典外醫藥品規格、醫藥品添加物規格2013(藥事日報社,2013年)、醫藥品添加物辭典2016(日本醫藥品添加劑協會編,藥事日報社,2016年)、Handbook of Pharmaceutical Excipients, 7th edition(Pharmaceutical Press,2012年)等之一般的原料。藥學上可容許之載體可單獨使用1種,亦可併用2種以上。In the pharmaceutical composition of the present embodiment, there is no particular limitation on the pharmaceutically acceptable carrier, and in addition to the above-mentioned carriers, carriers generally used in pharmaceuticals can be used. For example, general raw materials described in the Japanese Pharmacopoeia, Japanese Pharmacopoeia Non-Pharmacopoeia Pharmaceutical Specifications, Pharmaceutical Additives Specifications 2013 (Yakuji Nichipōsha, 2013), Pharmaceutical Additives Dictionary 2016 (Japan Pharmaceutical Additives Association, Yakuji Nichipōsha, 2016), Handbook of Pharmaceutical Excipients, 7th edition (Pharmaceutical Press, 2012), etc. can be used. The pharmaceutically acceptable carrier may be used alone or in combination of two or more.
本實施形態之醫藥組成物係除了前述自噬活化劑及藥學上可容許之載體以外,亦可含有其他成分。作為其他成分,並無特別限制,可使用一般的醫藥品添加物。此外,作為其他成分,亦可使用上述之自噬活化劑以外之活性成分。作為當作其他成分之醫藥品添加物及活性成分,除了上述所列舉者以外,尚可使用例如記載於日本藥典、日本藥典外醫藥品規格、醫藥品添加物規格2013(藥事日報社,2013年)、醫藥品添加物辭典2016(日本醫藥品添加劑協會編,藥事日報社,2016年)、Handbook of Pharmaceutical Excipients, 7th edition(Pharmaceutical Press,2012年)等之一般的原料。其他成分可單獨使用1種,亦可併用2種以上。The pharmaceutical composition of the present embodiment may contain other ingredients in addition to the aforementioned autophagy activator and pharmaceutically acceptable carrier. As other ingredients, there are no particular restrictions, and general pharmaceutical additives can be used. In addition, as other ingredients, active ingredients other than the aforementioned autophagy activator can also be used. As pharmaceutical additives and active ingredients used as other ingredients, in addition to those listed above, general raw materials such as those recorded in the Japanese Pharmacopoeia, Japanese Pharmacopoeia Non-Pharmacopoeia Pharmaceutical Specifications, Pharmaceutical Additives Specifications 2013 (Yakuji Nichipōsha, 2013), Pharmaceutical Additives Dictionary 2016 (Compiled by the Japanese Pharmaceutical Additives Association, Yakuji Nichipōsha, 2016), Handbook of Pharmaceutical Excipients, 7th edition (Pharmaceutical Press, 2012), etc. can also be used. Other ingredients can be used alone or in combination of two or more.
作為本實施形態之醫藥組成物的劑型,並無特別限制,可製成一般用作醫藥品製劑之劑型。可列舉例如錠劑、被覆錠劑、丸劑、散劑、顆粒劑、膠囊劑、液劑、懸浮劑、乳劑等經口投予之劑型;以及注射劑、栓劑、皮膚外用劑等非經口投予之劑型等。此等劑型的醫藥組成物可依照常規法(例如日本藥典所記載之方法)來予以製劑化。The dosage form of the pharmaceutical composition of this embodiment is not particularly limited, and can be prepared into dosage forms generally used as pharmaceutical preparations. Examples include oral dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, and parenteral dosage forms such as injections, suppositories, and skin external preparations. Pharmaceutical compositions of these dosage forms can be prepared according to conventional methods (e.g., methods described in the Japanese Pharmacopoeia).
本實施形態之醫藥組成物的投予方法並無特別限制,可藉由一般用作醫藥品的投予方法之方法來進行投予。例如,可以錠劑、被覆錠劑、丸劑、散劑、顆粒劑、膠囊劑、液劑、懸浮劑、乳劑等之形式進行經口投予,亦可以注射劑、輸液製劑等之形式單獨地或與葡萄糖液、林格氏液等一般的輸液進行混合,進行靜脈內、動脈內、肌肉內、皮內、皮下、腹腔內等投予,亦可以栓劑之形式進行直腸內投予,亦可以皮膚外用劑之形式投予至皮膚。The administration method of the pharmaceutical composition of the present embodiment is not particularly limited, and can be administered by methods generally used for the administration of pharmaceuticals. For example, it can be administered orally in the form of tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., or it can be administered intravenously, intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally in the form of injections, infusion preparations, etc., alone or mixed with common infusions such as glucose solution and Ringer's solution, or it can be administered intrarectally in the form of suppositories, or it can be administered to the skin in the form of topical preparations for skin application.
本實施形態之醫藥組成物的投予量可設為治療有效量。治療有效量只要依患者的症狀、體重、年齡及性別等以及醫藥組成物的劑型及投予方法等而適宜決定即可。例如,本實施形態之醫藥組成物的投予量可例示如下:在經口投予之情況,就生育酚磷酸酯等而言每投予單位形態0.01~500mg,在注射劑之情況,就生育酚磷酸酯等而言每投予單位形態0.02~250mg,在栓劑之情況,就生育酚磷酸酯等而言每投予單位形態0.01~500mg等。此外,在皮膚外用劑之情況,可例示例如就生育酚磷酸酯等而言每投予單位形態0.15~500mg,亦可例如為0.15~300mg,亦可例如為0.15~200mg,亦可例如為0.2~100mg。The dosage of the pharmaceutical composition of this embodiment can be set to a therapeutically effective amount. The therapeutically effective amount can be appropriately determined according to the patient's symptoms, weight, age, and gender, as well as the dosage form and administration method of the pharmaceutical composition. For example, the dosage of the pharmaceutical composition of this embodiment can be exemplified as follows: in the case of oral administration, 0.01 to 500 mg per administration unit form of tocopherol phosphate, etc., in the case of injections, 0.02 to 250 mg per administration unit form of tocopherol phosphate, etc., in the case of suppositories, 0.01 to 500 mg per administration unit form of tocopherol phosphate, etc. In the case of a skin external preparation, for example, the dosage per administration unit of tocopherol phosphate is 0.15 to 500 mg, 0.15 to 300 mg, 0.15 to 200 mg, or 0.2 to 100 mg.
本實施形態之醫藥組成物的投予間隔只要依患者的症狀、體重、年齡及性別等以及醫藥組成物的劑型及投予方法等而適宜決定即可。例如,可設為1日1次或2~3次左右等。The administration interval of the pharmaceutical composition of this embodiment can be appropriately determined according to the patient's symptoms, weight, age, gender, etc., as well as the dosage form and administration method of the pharmaceutical composition, etc. For example, it can be set to once a day or about 2 to 3 times a day.
本實施形態之醫藥組成物可用於治療或預防起因於自噬活性的降低之疾患。作為該種疾患,可列舉例如阿茲海默症、亨丁頓氏症、帕金森氏症、SENDA病等神經退化性疾患;克隆氏症等炎症性腸疾患;及癌症等。The pharmaceutical composition of this embodiment can be used to treat or prevent diseases caused by decreased autophagy activity. Examples of such diseases include neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and SENDA disease; inflammatory bowel diseases such as Crohn's disease; and cancer.
本實施形態之醫藥組成物可投予至例如阿茲海默症、亨丁頓氏症、帕金森氏症、SENDA病等神經退化性疾患;克隆氏症等炎症性腸疾患;或癌症的患者,用於抑制神經退化性疾患、炎症性腸疾患或癌症的進展。此外,本實施形態之醫藥組成物可投予至阿茲海默症、亨丁頓氏症、帕金森氏症、SENDA病等神經退化性疾患;克隆氏症等炎症性腸疾患;或癌症的患者,用於治療神經退化性疾患、炎症性腸疾患或癌症。此外,本實施形態之醫藥組成物可用於治療起因於類澱粉β之疾患。此外,本實施形態之醫藥組成物可用於治療起因於LC3基因、ATG5基因、ATG7基因或Beclin 1基因的表現量降低之疾患。此外,本實施形態之醫藥組成物可用於治療起因於mTOR的表現量上升之疾患。 在上述之中,本實施形態之醫藥組成物可特別適合地用於治療阿茲海默症。 The pharmaceutical composition of this embodiment can be administered to patients suffering from neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, SENDA disease, inflammatory bowel diseases such as Crohn's disease, or cancer, to inhibit the progression of neurodegenerative diseases, inflammatory bowel diseases, or cancer. In addition, the pharmaceutical composition of this embodiment can be administered to patients suffering from neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, SENDA disease, inflammatory bowel diseases such as Crohn's disease, or cancer, to treat neurodegenerative diseases, inflammatory bowel diseases, or cancer. In addition, the pharmaceutical composition of this embodiment can be used to treat diseases caused by starch beta. In addition, the pharmaceutical composition of this embodiment can be used to treat diseases caused by reduced expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene. In addition, the pharmaceutical composition of this embodiment can be used to treat diseases caused by increased expression of mTOR. Among the above, the pharmaceutical composition of this embodiment can be particularly suitable for treating Alzheimer's disease.
本實施形態之醫藥組成物亦可投予至以阿茲海默症為首之亨丁頓氏症、帕金森氏症、SENDA病等神經退化性疾患的發病風險較高的患者,用於預防神經退化性疾患。此外,本實施形態之醫藥組成物亦可投予至克隆氏症等炎症性腸疾患的發病風險較高的患者,用於預防炎症性腸疾患。此外,本實施形態之醫藥組成物亦可投予至癌症的發病風險較高的患者,用於預防癌症。The pharmaceutical composition of this embodiment can also be administered to patients at high risk of developing neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and SENDA disease, for the prevention of neurodegenerative diseases. In addition, the pharmaceutical composition of this embodiment can also be administered to patients at high risk of developing inflammatory bowel diseases such as Crohn's disease, for the prevention of inflammatory bowel diseases. In addition, the pharmaceutical composition of this embodiment can also be administered to patients at high risk of developing cancer, for the prevention of cancer.
(化妝料) 在一實施形態中,本發明係提供一種用於活化自噬之化妝料,其含有上述之自噬活化劑及藥學上可容許之載體。 (Cosmetics) In one embodiment, the present invention provides a cosmetic for activating autophagy, which contains the above-mentioned autophagy activator and a pharmaceutically acceptable carrier.
在本實施形態之化妝料中,作為藥學上可容許之載體,並無特別限制,除了上述所列舉者以外,尚可使用一般使用於化妝料之載體。例如,可使用記載於化妝品原料基準第二版註解(日本公定書協會編,藥事日報社,1984年)、化妝品原料基準外成分規格(日本厚生省藥務局審查課監修,藥事日報社,1993年)、化妝品原料基準外成分規格追補(日本厚生省藥務局審查課監修,藥事日報社,1993年)、化妝品種別許可基準(日本厚生省藥務局審查課監修,藥事日報社,1993年)、化妝品原料辭典(日光Chemicals公司,日本平成3年)、International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol.1~4, by CTFA等之一般的原料。藥學上可容許之載體可單獨使用1種,亦可併用2種以上。In the cosmetic of this embodiment, there is no particular limitation on the pharmaceutically acceptable carrier, and in addition to the above-mentioned carriers, carriers generally used in cosmetics can also be used. For example, general raw materials described in the Notes to the Second Edition of the Cosmetic Raw Materials Standard (edited by the Japan Kondo Association, Yakuji Nichipō, 1984), Specifications for Ingredients Outside the Cosmetic Raw Materials Standard (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Supplement to the Specifications for Ingredients Outside the Cosmetic Raw Materials Standard (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Category Permit Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Raw Materials Dictionary (Nikko Chemicals, Japan, 1993), International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol.1~4, by CTFA, etc. can be used. The pharmaceutically acceptable carriers may be used alone or in combination of two or more.
本實施形態之化妝料係除了自噬活化劑及藥學上可容許之載體以外,亦可含有其他成分。作為其他成分,並無特別限制,可使用一般的化妝品添加物。此外,作為其他成分,亦可使用上述之自噬活化劑以外之活性成分。作為當作其他成分之化妝品添加物及活性成分,除了上述所列舉者以外,尚可使用例如記載於化妝品原料基準第二版註解(日本公定書協會編,藥事日報社,1984年)、化妝品原料基準外成分規格(日本厚生省藥務局審查課監修,藥事日報社,1993年)、化妝品原料基準外成分規格追補(日本厚生省藥務局審查課監修,藥事日報社,1993年)、化妝品種別許可基準(日本厚生省藥務局審查課監修,藥事日報社,1993年)、化妝品原料辭典(日光Chemicals公司,日本平成3年)、International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol.1~4, by CTFA等之一般的原料。其他成分可單獨使用1種,亦可併用2種以上。The cosmetic of this embodiment may contain other ingredients in addition to the autophagy activator and the pharmaceutically acceptable carrier. There is no particular limitation on the other ingredients, and general cosmetic additives may be used. In addition, active ingredients other than the above-mentioned autophagy activator may also be used as other ingredients. In addition to the above, cosmetic additives and active ingredients that are deemed to be other ingredients may include those listed in the Notes to the Second Edition of the Cosmetic Ingredient Standards (edited by the Japan KOKU Association, Yakuji Nichipō, 1984), Specifications for Ingredients Outside the Cosmetic Ingredient Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Supplement to the Specifications for Ingredients Outside the Cosmetic Ingredient Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Category Permit Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Ingredient Dictionary (Nikko Chemicals, Japan, 1993), International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol.1~4, by CTFA and other general raw materials. Other ingredients may be used alone or in combination of two or more.
作為本實施形態之化妝料的形態,並無特別限制,可製成一般用作化妝料之形態。可列舉例如洗髮精、潤絲精、整髮劑等毛髮用化妝料;洗顏料、清潔劑、化妝水、乳液、護膚液、乳霜、凝膠、防曬劑、面膜、臉膜、美容液等基礎化妝料;粉底類、妝前修飾品、口紅類、唇蜜、腮紅類等彩妝化妝料;身體洗淨料、身體粉、防臭化妝料等身體化妝料等。此等化妝料可依照常規法來予以製造。The form of the cosmetics of this embodiment is not particularly limited, and can be made into forms commonly used as cosmetics. Examples include hair cosmetics such as shampoo, conditioner, and hair spray; basic cosmetics such as facial cleansers, cleaners, toners, emulsions, skin lotions, creams, gels, sunscreens, facial masks, face packs, and beauty serums; makeup cosmetics such as foundations, primers, lipsticks, lip glosses, and blushers; and body cosmetics such as body washes, body powders, and deodorant cosmetics. These cosmetics can be manufactured according to conventional methods.
此外,作為本實施形態之化妝料的劑型,並無特別限制,可列舉例如水中油(O/W)型、油中水(W/O)型、W/O/W型、O/W/O型等乳化型、乳化高分子型、油性、固形、液狀、糊狀、棒狀、揮發性油型、粉狀、膠凍狀、凝膠狀、膏狀、乳霜狀、片狀、膜狀、霧狀、噴霧型、氣溶膠狀、多層狀、泡狀、碎片狀等。In addition, the dosage form of the cosmetic of this embodiment is not particularly limited, and examples include emulsion types such as oil in water (O/W), water in oil (W/O), W/O/W, and O/W/O, emulsified polymer types, oily, solid, liquid, paste, stick, volatile oil type, powder, jelly, gel, ointment, cream, sheet, film, mist, spray, aerosol, multilayer, bubble, and fragments.
本實施形態之化妝料的使用量並無特別限制,可設為有效於活化自噬之量。例如,本實施形態之化妝料的使用量可例示就生育酚磷酸酯等的量而言每1次使用0.15~500mg,亦可例如為0.15~300mg,亦可例如為0.15~200mg,亦可例如為0.2~100mg。The amount of the cosmetic used in this embodiment is not particularly limited and can be set to an amount effective for activating autophagy. For example, the amount of the cosmetic used in this embodiment can be 0.15 to 500 mg, 0.15 to 300 mg, 0.15 to 200 mg, or 0.2 to 100 mg per use, for example, in terms of the amount of tocopherol phosphate, etc.
本實施形態之化妝料的使用間隔並無特別限制,例如,可設為1日1次或2~3次左右等。The usage interval of the cosmetic of this embodiment is not particularly limited, and can be set to, for example, once a day or 2 to 3 times a day.
本實施形態之化妝料可用於緩和起因於自噬活性的降低之症狀。或者,亦可為了預防起因於自噬活性的降低之症狀的發病,而由此等的發病風險較高的被驗者使用於日常的肌膚保養或彩妝。The cosmetic of this embodiment can be used to alleviate symptoms caused by reduced autophagic activity. Alternatively, it can also be used by subjects with a high risk of developing symptoms caused by reduced autophagic activity for daily skin care or makeup in order to prevent the onset of such symptoms.
[其他實施形態] 在一實施形態中,本發明係提供一種活化自噬之方法,其包含將生育酚磷酸酯或其鹽投予至對象之步驟。 [Other embodiments] In one embodiment, the present invention provides a method for activating autophagy, which comprises the step of administering tocopherol phosphate or a salt thereof to a subject.
在一實施形態中,本發明係提供一種促進LC3基因、ATG5基因、ATG7基因或Beclin 1基因的表現之方法,其包含將生育酚磷酸酯或其鹽投予至對象之步驟。In one embodiment, the present invention provides a method for promoting the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene, comprising the step of administering tocopherol phosphate or a salt thereof to a subject.
在一實施形態中,本發明係提供一種抑制mTOR基因的表現之方法,其包含將生育酚磷酸酯或其鹽投予至對象之步驟。In one embodiment, the present invention provides a method for inhibiting the expression of mTOR gene, comprising the step of administering tocopherol phosphate or a salt thereof to a subject.
在一實施形態中,本發明係提供一種抑制在類澱粉β存在下之細胞凋亡之方法,其包含將生育酚磷酸酯或其鹽投予至對象之步驟。In one embodiment, the present invention provides a method for inhibiting apoptosis in the presence of amyloid β, comprising the step of administering tocopherol phosphate or a salt thereof to a subject.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽,其係用於活化自噬。In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof for activating autophagy.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽,其係用於促進LC3基因、ATG5基因、ATG7基因或Beclin 1基因的表現。In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof, which is used to promote the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽,其係用於抑制mTOR基因的表現。In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof, which is used to inhibit the expression of the mTOR gene.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽,其係用於抑制在類澱粉β存在下之細胞凋亡。In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof for inhibiting apoptosis in the presence of starch beta.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽,其係用於預防或治療阿茲海默症、亨丁頓氏症、帕金森氏症、SENDA病、克隆氏症或癌症。In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof for preventing or treating Alzheimer's disease, Huntington's disease, Parkinson's disease, SENDA disease, Crohn's disease or cancer.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造自噬活化劑。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing an autophagy activator.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造LC3基因、ATG5基因、ATG7基因或Beclin 1基因表現的促進劑。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for producing an agent for promoting the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造mTOR基因表現的抑制劑。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for producing an inhibitor of mTOR gene expression.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造在類澱粉β存在下之LC3基因、ATG5基因或ATG7基因表現的促進劑。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for producing an agent for promoting the expression of LC3 gene, ATG5 gene or ATG7 gene in the presence of starch beta.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造在類澱粉β存在下之細胞凋亡的抑制劑。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing an inhibitor of cell apoptosis in the presence of starch beta.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造自噬活化用組成物。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for activating autophagy.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造LC3基因、ATG5基因、ATG7基因或Beclin 1基因表現的促進用組成物。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for promoting the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造mTOR基因表現的抑制用組成物。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for inhibiting mTOR gene expression.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造在類澱粉β存在下之LC3基因、ATG5基因或ATG7基因表現的促進用組成物。In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for promoting the expression of LC3 gene, ATG5 gene or ATG7 gene in the presence of starch beta.
在一實施形態中,本發明係提供一種生育酚磷酸酯或其鹽的使用,其係用於製造在類澱粉β存在下之細胞凋亡的抑制用組成物。 [實施例] In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for inhibiting cell apoptosis in the presence of starch beta. [Example]
以下,基於實施例來更具體地說明本發明,但本發明並不限定於此等實施例。The present invention is described in more detail below based on embodiments, but the present invention is not limited to these embodiments.
[生育酚類] 在以下實施例及配方例中,係使用下述生育酚磷酸酯的鈉鹽。 α-TPNa:dl-α-生育酚磷酸酯鈉(標示名稱:生育酚磷酸酯鈉,製品名;TPNa(註冊商標),昭和電工公司製) γ-TPNa:dl-γ-生育酚磷酸酯鈉(昭和電工公司製) α-TPNa溶液:將α-TPNa溶解於0.05%(V/V)乙醇水溶液中而得。 γ-TPNa溶液:將γ-TPNa溶解於0.05%(V/V)乙醇水溶液中而得。 在以下比較例中,係使用下述生育酚類。 生育酚醋酸酯:Eisai股份有限公司製 α-生育酚:Sigma-Aldrich公司製 γ-生育酚:Sigma-Aldrich公司製 生育酚醋酸酯溶液:將生育酚醋酸酯溶解於0.05%(V/V)乙醇水溶液中而得。 α-生育酚溶液:將α-生育酚溶解於0.05%(V/V)乙醇水溶液中而得。 γ-生育酚溶液:將γ-生育酚溶解於0.05%(V/V)乙醇水溶液中而得。 [Tocopherols] In the following examples and formulations, the following sodium salts of tocopherol phosphates were used. α-TPNa: dl-α-tocopherol sodium phosphate (label name: tocopherol sodium phosphate, product name; TPNa (registered trademark), manufactured by Showa Denko Co., Ltd.) γ-TPNa: dl-γ-tocopherol sodium phosphate (manufactured by Showa Denko Co., Ltd.) α-TPNa solution: obtained by dissolving α-TPNa in a 0.05% (V/V) ethanol aqueous solution. γ-TPNa solution: obtained by dissolving γ-TPNa in a 0.05% (V/V) ethanol aqueous solution. In the following comparative examples, the following tocopherols were used. Tocopherol acetate: manufactured by Eisai Co., Ltd. α-Tocopherol: manufactured by Sigma-Aldrich γ-Tocopherol: manufactured by Sigma-Aldrich Tocopherol acetate solution: obtained by dissolving tocopherol acetate in a 0.05% (V/V) ethanol aqueous solution. α-Tocopherol solution: obtained by dissolving α-tocopherol in a 0.05% (V/V) ethanol aqueous solution. γ-Tocopherol solution: obtained by dissolving γ-tocopherol in a 0.05% (V/V) ethanol aqueous solution.
<人類老化纖維母細胞中之LC3基因、ATG5基因、ATG7基因、Beclin 1基因的表現促進效果的評估> 為了製作經人為誘發老化之細胞,係使用纖維母細胞來施行試驗。老化纖維母細胞係藉由以下順序來予以製作。 <Evaluation of the effect of promoting the expression of LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene in human aged fibroblasts> In order to produce artificially aged cells, fibroblasts were used for the experiment. Aged fibroblasts were produced by the following procedure.
(老化纖維母細胞的製作) 在經添加10%胎牛血清(MP Biomedicals公司製)之D-MEM培養基(Sigma-Aldrich公司製)中,將人類正常纖維母細胞(NB1RGB;RIKEN BRC Cell Bank)進行培養,直至呈匯合。然後,以250μM過氧化氫水處理2小時,以新的經添加10%胎牛血清之D-MEM培養基培養24小時。將此經由過氧化氫水之處理及細胞培養的操作重複進行3次,使所獲得之纖維母細胞成為老化纖維母細胞。 (Preparation of aged fibroblasts) Human normal fibroblasts (NB1RGB; RIKEN BRC Cell Bank) were cultured in D-MEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (MP Biomedicals) until they became confluent. Then, they were treated with 250 μM hydrogen peroxide for 2 hours and cultured in new D-MEM medium supplemented with 10% fetal bovine serum for 24 hours. This process of hydrogen peroxide treatment and cell culture was repeated three times to obtain aged fibroblasts.
(基因的表現促進效果的評估) 以10000個/cm 2的接種密度準備所製作而得之老化纖維母細胞,以經添加10%胎牛血清(MP Biomedicals公司製)之D-MEM培養基(Sigma-Aldrich公司製)培養24小時。接著,在實施例1中,以α-TPNa的終濃度成為1μM之方式,將α-TPNa溶液添加至培養基中。在實施例2中,以α-TPNa的終濃度成為10μM之方式,將α-TPNa溶液添加至培養基中。在實施例3中,以γ-TPNa的終濃度成為10μM之方式,將γ-TPNa溶液添加至培養基中。在比較例2中,以生育酚醋酸酯的終濃度成為10μM之方式,將生育酚醋酸酯溶液添加至培養基中。在比較例3中,以α-生育酚的終濃度成為10μM之方式,將α-生育酚溶液添加至培養基中。在比較例4中,以γ-生育酚的終濃度成為10μM之方式,將γ-生育酚溶液添加至培養基中。 此外,在比較例1中,僅在培養基中添加0.05%(V/V)乙醇水溶液。 接著,將各培養基在37℃、5%CO 2下培養24小時。 (Evaluation of the effect of promoting gene expression) The prepared aged fibroblasts were prepared at an inoculation density of 10,000 cells/ cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 1, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa became 1 μM. In Example 2, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa became 10 μM. In Example 3, a γ-TPNa solution was added to the medium in such a manner that the final concentration of γ-TPNa became 10 μM. In Comparative Example 2, a tocopherol acetate solution was added to the culture medium in such a manner that the final concentration of tocopherol acetate became 10 μM. In Comparative Example 3, an α-tocopherol solution was added to the culture medium in such a manner that the final concentration of α-tocopherol became 10 μM. In Comparative Example 4, a γ-tocopherol solution was added to the culture medium in such a manner that the final concentration of γ-tocopherol became 10 μM. In addition, in Comparative Example 1, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium. Then, each culture medium was cultured at 37°C and 5% CO2 for 24 hours.
作為參考例1,係以10000個/cm 2的接種密度準備上述人類正常纖維母細胞,以經添加10%胎牛血清(MP Biomedicals公司製)之D-MEM培養基(Sigma-Aldrich公司製)培養24小時,僅在該培養基中添加0.05%(V/V)乙醇水溶液。接著,在37℃、5%CO 2下培養24小時。 As Reference Example 1, the human normal fibroblasts were prepared at a seeding density of 10,000 cells/ cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Only a 0.05% (V/V) ethanol aqueous solution was added to the medium. Then, the cells were cultured for 24 hours at 37°C and 5% CO2 .
然後,使用Nucleospin(註冊商標)RNA套組(Takara Bio公司製),從各例的老化纖維母細胞或人類正常纖維母細胞中萃取出RNA,從所獲得之RNA合成cDNA。接著,以此cDNA當作模板,藉由定量即時PCR,各自使用LC3基因、ATG5基因、ATG7基因及Beclin 1基因特異性引子(Takara Bio公司製)對各基因的表現量進行定量。Then, RNA was extracted from aged fibroblasts or normal human fibroblasts of each case using Nucleospin (registered trademark) RNA Kit (manufactured by Takara Bio), and cDNA was synthesized from the obtained RNA. Then, using this cDNA as a template, quantitative real-time PCR was performed to quantify the expression level of each gene using primers specific to the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene (manufactured by Takara Bio).
對作為內部標準基因且不會因添加化合物而在表現上出現變動之管家基因GAPDH(引子;Takara Bio公司製)的表現量進行定量,藉由其值來對各基因的表現量進行標準化。針對前述各例中之基因表現量,求出將比較例1中之各基因的表現量各自設為1.00時之相對基因表現量。將其結果示於表1。The expression level of the housekeeping gene GAPDH (primer; manufactured by Takara Bio) which is an internal standard gene and does not change in expression due to the addition of compounds was quantified, and the expression level of each gene was standardized by using its value. For the gene expression levels in the above examples, the relative gene expression level was calculated when the expression level of each gene in Comparative Example 1 was set to 1.00. The results are shown in Table 1.
[表1]
如表1所示,在參考例1及比較例1中,就各自僅添加0.05%(V/V)乙醇水溶液所培養而得之人類正常纖維母細胞及老化纖維母細胞而言,比較例1的老化纖維母細胞係LC3基因、ATG5基因、ATG7基因及Beclin 1基因的表現量降低或幾乎未改變。 此外,就實施例1及2的添加α-TPNa所培養而得之老化纖維母細胞以及實施例3的添加γ-TPNa所培養而得之老化纖維母細胞而言,與比較例1相比,可確認到LC3基因、ATG5基因、ATG7基因及Beclin 1基因的表現促進效果。再者,與以往所利用之比較例2的添加生育酚醋酸酯所培養而得之老化纖維母細胞相比,亦可確認到較高的表現促進效果。 As shown in Table 1, in Reference Example 1 and Comparative Example 1, for human normal fibroblasts and aged fibroblasts cultured with only 0.05% (V/V) ethanol aqueous solution, the expression levels of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene in the aged fibroblasts of Comparative Example 1 were reduced or almost unchanged. In addition, for the aged fibroblasts cultured with α-TPNa in Examples 1 and 2 and the aged fibroblasts cultured with γ-TPNa in Example 3, the expression-promoting effect of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene was confirmed compared with Comparative Example 1. Furthermore, compared with the aged fibroblasts cultured with tocopherol acetate added in Comparative Example 2 used in the past, a higher performance-enhancing effect was also confirmed.
與參考例1的人類正常纖維母細胞相比,就比較例1的老化纖維母細胞而言,可見到LC3基因的表現降低,而實施例1~3的添加生育酚磷酸酯鹽所培養而得之老化纖維母細胞相較於參考例1的正常纖維母細胞而言,LC3基因的表現量增加,可確認到優異的LC3基因的表現促進效果。另一方面,就比較例2~3的添加生育酚醋酸酯或α-生育酚所培養而得之老化纖維母細胞而言,並未見到LC3基因的表現促進效果,就比較例4的添加γ-生育酚所培養而得之老化纖維母細胞而言,LC3基因的表現促進效果微弱。Compared with the human normal fibroblasts of Reference Example 1, the expression of LC3 gene was reduced in the aged fibroblasts of Comparative Example 1, while the expression of LC3 gene was increased in the aged fibroblasts cultured with the addition of tocopherol phosphate in Examples 1 to 3 compared with the normal fibroblasts of Reference Example 1, and an excellent LC3 gene expression promoting effect was confirmed. On the other hand, no LC3 gene expression promoting effect was observed in the aged fibroblasts cultured with the addition of tocopherol acetate or α-tocopherol in Comparative Examples 2 to 3, and the LC3 gene expression promoting effect was weak in the aged fibroblasts cultured with the addition of γ-tocopherol in Comparative Example 4.
此外,與參考例1的人類正常纖維母細胞相比,就比較例1的老化纖維母細胞而言,ATG5基因及ATG7基因的表現量降低,而就實施例1~3的添加生育酚磷酸酯鹽所培養而得之老化纖維母細胞而言,相較於比較例1的老化纖維母細胞而言ATG5基因及ATG7基因的表現受到促進。再者,就實施例1~3的添加生育酚磷酸酯鹽所培養而得之老化纖維母細胞而言,亦可確認到Beclin 1基因的表現促進效果。 即,就實施例1~3的添加屬於自噬活化劑之生育酚磷酸酯鹽所培養而得之老化纖維母細胞而言,可確認到LC3基因、ATG5基因、ATG7基因及Beclin 1基因的表現促進效果。 In addition, compared with the human normal fibroblasts of Reference Example 1, the expression levels of ATG5 and ATG7 genes in the aged fibroblasts of Comparative Example 1 were reduced, while the expressions of ATG5 and ATG7 genes in the aged fibroblasts cultured with the addition of tocopherol phosphate in Examples 1 to 3 were promoted compared with the aged fibroblasts of Comparative Example 1. Furthermore, the expression promotion effect of Beclin 1 gene was also confirmed in the aged fibroblasts cultured with the addition of tocopherol phosphate in Examples 1 to 3. That is, in the aged fibroblasts cultured with the addition of tocopherol phosphate, which is an autophagy activator in Examples 1 to 3, the expression-promoting effect of the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene was confirmed.
<人類老化纖維母細胞中之mTOR基因的表現抑制效果的評估> 以10000個/cm 2的接種密度準備上述所製作而得之老化纖維母細胞,以經添加10%胎牛血清(MP Biomedicals公司製)之D-MEM培養基(Sigma-Aldrich公司製)培養24小時。接著,在實施例4中,以α-TPNa的終濃度成為1μM之方式,將α-TPNa溶液添加至培養基中。在實施例5中,以α-TPNa的終濃度成為10μM之方式,將α-TPNa溶液添加至培養基中。在實施例6中,以γ-TPNa的終濃度成為10μM之方式,將γ-TPNa溶液添加至培養基中。在比較例6中,以生育酚醋酸酯的終濃度成為10μM之方式,將生育酚醋酸酯溶液添加至培養基中。在比較例7中,以α-生育酚的終濃度成為10μM之方式,將α-生育酚溶液添加至培養基中。在比較例8中,以γ-生育酚的終濃度成為10μM之方式,將γ-生育酚溶液添加至培養基中。 此外,在比較例5中,僅在培養基中添加0.05%(V/V)乙醇水溶液。 接著,將各培養基在37℃、5%CO 2下培養24小時。 <Evaluation of the inhibitory effect on the expression of the mTOR gene in human aged fibroblasts> The aged fibroblasts prepared as described above were prepared at a seeding density of 10,000 cells/ cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 4, an α-TPNa solution was added to the culture medium in such a manner that the final concentration of α-TPNa became 1 μM. In Example 5, an α-TPNa solution was added to the culture medium in such a manner that the final concentration of α-TPNa became 10 μM. In Example 6, a γ-TPNa solution was added to the culture medium in such a manner that the final concentration of γ-TPNa became 10 μM. In Comparative Example 6, a tocopherol acetate solution was added to the culture medium in such a manner that the final concentration of tocopherol acetate was 10 μM. In Comparative Example 7, an α-tocopherol solution was added to the culture medium in such a manner that the final concentration of α-tocopherol was 10 μM. In Comparative Example 8, a γ-tocopherol solution was added to the culture medium in such a manner that the final concentration of γ-tocopherol was 10 μM. In addition, in Comparative Example 5, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium. Then, each culture medium was cultured at 37°C and 5% CO 2 for 24 hours.
作為參考例2,以10000個/cm 2的接種密度準備上述人類正常纖維母細胞,以經添加10%胎牛血清(MP Biomedicals公司製)之D-MEM培養基(Sigma-Aldrich公司製)培養24小時,僅在該培養基中添加0.05%(V/V)乙醇水溶液。接著,在37℃、5%CO 2下培養24小時。 As Reference Example 2, the human normal fibroblasts were prepared at a seeding density of 10,000 cells/ cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Only a 0.05% (V/V) ethanol aqueous solution was added to the medium. Then, the cells were cultured at 37°C and 5% CO2 for 24 hours.
然後,使用Nucleospin(註冊商標)RNA套組(Takara Bio公司製),從各例的老化纖維母細胞或人類正常纖維母細胞中萃取出RNA,從所獲得之RNA合成cDNA。接著,以此cDNA當作模板,藉由定量即時PCR,使用mTOR基因特異性引子(Takara Bio公司製)對mTOR基因的表現量進行定量。Then, RNA was extracted from aged fibroblasts or normal human fibroblasts of each case using Nucleospin (registered trademark) RNA Kit (manufactured by Takara Bio), and cDNA was synthesized from the obtained RNA. Then, the expression level of the mTOR gene was quantified by quantitative real-time PCR using mTOR gene-specific primers (manufactured by Takara Bio) using this cDNA as a template.
對作為內部標準基因且不會因添加化合物而在表現上出現變動之管家基因GAPDH(引子;Takara Bio公司製)的表現量進行定量,藉由其值來對各基因的表現量進行標準化。針對前述各例中之基因表現量,求出將比較例5中之mTOR基因的表現量設為1.00時之相對基因表現量。將其結果示於表2。The expression level of the housekeeping gene GAPDH (primer; manufactured by Takara Bio) which is an internal standard gene and does not change in expression due to the addition of compounds was quantified, and the expression level of each gene was standardized by using its value. For the gene expression levels in the above examples, the relative gene expression level was calculated when the expression level of the mTOR gene in Comparative Example 5 was set to 1.00. The results are shown in Table 2.
[表2]
如表2所示,在參考例2及比較例5中,就各自僅添加0.05%(V/V)乙醇水溶液所培養而得之人類正常纖維母細胞及老化纖維母細胞而言,與參考例2的人類正常纖維母細胞相比,就比較例5的老化纖維母細胞而言可見到mTOR基因的表現亢進。就實施例4及5的添加α-TPNa所培養而得之老化纖維母細胞以及實施例6的添加γ-TPNa所培養而得之老化纖維母細胞而言,相較於參考例2的正常纖維母細胞而言mTOR基因的表現降低,可確認到mTOR基因的表現抑制效果。As shown in Table 2, in Reference Example 2 and Comparative Example 5, for human normal fibroblasts and aged fibroblasts cultured with only 0.05% (V/V) ethanol aqueous solution, the expression of mTOR gene was increased in the aged fibroblasts of Comparative Example 5 compared with the human normal fibroblasts of Reference Example 2. In the aged fibroblasts cultured with the addition of α-TPNa in Examples 4 and 5 and the aged fibroblasts cultured with the addition of γ-TPNa in Example 6, the expression of mTOR gene was decreased compared with the normal fibroblasts of Reference Example 2, and the mTOR gene expression inhibitory effect was confirmed.
另一方面,就比較例6的添加生育酚醋酸酯所培養而得之老化纖維母細胞、比較例7的添加α-生育酚所培養而得之老化纖維母細胞及比較例8的添加γ-生育酚所培養而得之老化纖維母細胞而言,mTOR基因的表現量皆與比較例5為同等以上,無法確認到mTOR基因的表現抑制效果。On the other hand, in the aged fibroblasts cultured with the addition of tocopherol acetate in Comparative Example 6, the aged fibroblasts cultured with the addition of α-tocopherol in Comparative Example 7, and the aged fibroblasts cultured with the addition of γ-tocopherol in Comparative Example 8, the expression levels of the mTOR gene were all equal to or higher than those in Comparative Example 5, and no mTOR gene expression inhibitory effect was confirmed.
<人類神經母細胞瘤中之LC3基因、ATG5基因及ATG7基因的表現促進效果的評估> 藉由以下試驗方法來測定人類神經母細胞瘤(SH-SY5Y;從ATCC取得)中之LC3基因、ATG5基因及ATG7基因的表現量,對生育酚磷酸酯鹽所引發之LC3基因、ATG5基因及ATG7基因的表現促進效果進行評估。 在以下實施例及比較例中,係將已知會引起神經細胞中之自噬的降低之類澱粉β添加至各培養基中而施行試驗。 <Evaluation of the effect of promoting the expression of LC3 gene, ATG5 gene and ATG7 gene in human neuroblastoma> The expression levels of LC3 gene, ATG5 gene and ATG7 gene in human neuroblastoma (SH-SY5Y; obtained from ATCC) were measured by the following test method, and the effect of promoting the expression of LC3 gene, ATG5 gene and ATG7 gene induced by tocopherol phosphate was evaluated. In the following examples and comparative examples, starch β, which is known to cause a decrease in autophagy in neurons, was added to each culture medium to perform the test.
以10000個/cm 2的接種密度準備SH-SY5Y細胞,以經添加10%胎牛血清(MP Biomedicals公司製)之D-MEM/Ham’s F-12培養基(Sigma-Aldrich公司製)培養24小時。接著,在實施例7中,以α-TPNa的終濃度成為1μM之方式,將α-TPNa溶液添加至培養基中。在實施例8中,以α-TPNa的終濃度成為10μM之方式,將α-TPNa溶液添加至培養基中。在實施例9中,以γ-TPNa的終濃度成為10μM之方式,將γ-TPNa溶液添加至培養基中。 在比較例10中,以生育酚醋酸酯的終濃度成為10μM之方式,將生育酚醋酸酯溶液添加至培養基中。在比較例11中,以α-生育酚的終濃度成為10μM之方式,將α-生育酚溶液添加至培養基中。在比較例12中,以γ-生育酚的終濃度成為10μM之方式,將γ-生育酚溶液添加至培養基中。 此外,在比較例9中,僅將0.05%(V/V)乙醇水溶液添加至培養基中。 SH-SY5Y cells were prepared at a seeding density of 10,000 cells/ cm2 and cultured for 24 hours in D-MEM/Ham's F-12 medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 7, α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 1 μM. In Example 8, α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 10 μM. In Example 9, γ-TPNa solution was added to the medium in such a manner that the final concentration of γ-TPNa was 10 μM. In Comparative Example 10, a tocopherol acetate solution was added to the culture medium so that the final concentration of tocopherol acetate was 10 μM. In Comparative Example 11, an α-tocopherol solution was added to the culture medium so that the final concentration of α-tocopherol was 10 μM. In Comparative Example 12, a γ-tocopherol solution was added to the culture medium so that the final concentration of γ-tocopherol was 10 μM. In Comparative Example 9, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium.
接著,調製將類澱粉β(Sigma-Aldrich公司製)溶於0.01%(V/V)DMSO水溶液中而得之類澱粉β溶液,以各培養基中之類澱粉β的終濃度成為20μM之方式,將該類澱粉β溶液添加至各培養基中。 另外,在參考例3中,僅添加0.05%(V/V)乙醇水溶液,並未添加類澱粉β溶液。 接著,將各培養基在37℃、5%CO 2下培養48小時。 Next, a starch β-like solution was prepared by dissolving a starch β-like solution (manufactured by Sigma-Aldrich) in a 0.01% (V/V) DMSO aqueous solution, and the starch β-like solution was added to each culture medium so that the final concentration of the starch β-like solution in each culture medium was 20 μM. In addition, in Reference Example 3, only a 0.05% (V/V) ethanol aqueous solution was added, and the starch β-like solution was not added. Then, each culture medium was cultured at 37°C and 5% CO 2 for 48 hours.
然後,使用Nucleospin(註冊商標)RNA套組(Takara Bio公司製),從各例的SH-SY5Y細胞中萃取出RNA,從所獲得之RNA合成cDNA。接著,以此cDNA當作模板,藉由定量即時PCR,各自使用LC3基因、ATG5基因及ATG7基因特異性引子(Takara Bio公司製)對各基因的表現量進行定量。Then, RNA was extracted from SH-SY5Y cells of each case using Nucleospin (registered trademark) RNA Kit (manufactured by Takara Bio), and cDNA was synthesized from the obtained RNA. Then, using this cDNA as a template, quantitative real-time PCR was performed to quantify the expression level of each gene using primers specific to the LC3 gene, ATG5 gene, and ATG7 gene (manufactured by Takara Bio).
對作為內部標準基因且不會因添加化合物而在表現上出現變動之管家基因GAPDH(引子;Takara Bio公司製)的表現量進行定量,藉由其值來對各基因的表現量進行標準化。針對前述各例中之基因表現量,求出將參考例3中之各基因的表現量各自設為1.00時之相對基因表現量。將其結果示於表3。The expression level of the housekeeping gene GAPDH (primer; manufactured by Takara Bio) which is an internal standard gene and does not change in expression due to the addition of compounds was quantified, and the expression level of each gene was standardized by using its value. For the gene expression levels in the above examples, the relative gene expression level when the expression level of each gene in Reference Example 3 was set to 1.00 was calculated. The results are shown in Table 3.
[表3]
如表3所示,在參考例3及比較例9中,與僅添加0.05%(V/V)乙醇水溶液且並未添加類澱粉β之參考例3的SH-SY5Y細胞相比,就進一步添加類澱粉β所培養而得之比較例9的SH-SY5Y細胞而言,可見到LC3基因的表現降低。就實施例7~9的添加屬於自噬活化劑之生育酚磷酸酯鹽所培養而得之SH-SY5Y細胞而言,相較於比較例9的SH-SY5Y細胞而言LC3基因的表現量較高,可確認到生育酚磷酸酯鹽所引發之LC3基因的表現促進效果。另一方面,就比較例10~12的添加生育酚醋酸酯、α-生育酚或γ-生育酚所培養而得之SH-SY5Y細胞而言,與比較例9相比,無法確認到LC3基因表現的顯著增加。As shown in Table 3, in Reference Example 3 and Comparative Example 9, compared with the SH-SY5Y cells of Reference Example 3, which only added 0.05% (V/V) ethanol aqueous solution and did not add starch beta, the SH-SY5Y cells of Comparative Example 9, which were cultured by further adding starch beta, showed a decrease in the expression of the LC3 gene. In Examples 7 to 9, the SH-SY5Y cells cultured by adding tocopherol phosphate, which is an autophagy activator, showed a higher expression of the LC3 gene than the SH-SY5Y cells of Comparative Example 9, confirming the effect of promoting the expression of the LC3 gene caused by tocopherol phosphate. On the other hand, in the SH-SY5Y cells cultured in Comparative Examples 10 to 12 supplemented with tocopherol acetate, α-tocopherol, or γ-tocopherol, no significant increase in LC3 gene expression was observed compared to Comparative Example 9.
此外,與未添加類澱粉β之參考例3的SH-SY5Y細胞相比,就進一步添加類澱粉β所培養而得之比較例9的SH-SY5Y細胞而言,可見到ATG5基因及ATG7基因的表現降低。就實施例7~9的添加屬於自噬活化劑之生育酚磷酸酯鹽所培養而得之SH-SY5Y細胞而言,相較於比較例9的SH-SY5Y細胞而言ATG5基因及ATG7基因的表現量皆增加,可確認到生育酚磷酸酯鹽的ATG5基因及ATG7基因的表現促進效果。另一方面,就比較例10~12的添加生育酚醋酸酯、α-生育酚或γ-生育酚所培養而得之SH-SY5Y細胞而言,與比較例9相比,無法確認到ATG5基因及ATG7基因的表現量的顯著增加。In addition, compared with the SH-SY5Y cells of Reference Example 3 to which starch beta was not added, the SH-SY5Y cells of Comparative Example 9 obtained by further adding starch beta and cultured showed reduced expression of ATG5 gene and ATG7 gene. In the SH-SY5Y cells obtained by adding tocopherol phosphate, which is an autophagy activator, in Examples 7 to 9, the expression levels of ATG5 gene and ATG7 gene increased compared with the SH-SY5Y cells of Comparative Example 9, and the expression-promoting effect of tocopherol phosphate on ATG5 gene and ATG7 gene was confirmed. On the other hand, in the SH-SY5Y cells cultured in Comparative Examples 10 to 12 supplemented with tocopherol acetate, α-tocopherol, or γ-tocopherol, no significant increase in the expression levels of the ATG5 gene and the ATG7 gene was observed compared to Comparative Example 9.
<人類神經母細胞瘤中之起因於類澱粉β之細胞凋亡的抑制作用的評估> 藉由下述試驗方法來測定在生育酚磷酸酯鹽存在下之人類神經母細胞瘤(SH-SY5Y;從ATCC取得)中之凋亡細胞的比例,對生育酚磷酸酯鹽所引發之細胞凋亡的抑制作用進行評估。 在以下實施例及比較例中,係將已知會誘使自噬的降低及其所引發之神經細胞的被稱為細胞凋亡之細胞死亡之類澱粉β添加至各培養基中而施行試驗。 <Evaluation of the inhibitory effect of amyloid beta on apoptosis in human neuroblastoma> The ratio of apoptotic cells in human neuroblastoma (SH-SY5Y; obtained from ATCC) in the presence of tocopherol phosphate was measured by the following test method, and the inhibitory effect of tocopherol phosphate-induced apoptosis was evaluated. In the following examples and comparative examples, amyloid beta, which is known to induce a decrease in autophagy and the cell death of neurons called apoptosis, was added to each culture medium to perform the test.
以50000個/cm 2的接種密度準備SH-SY5Y細胞,以經添加10%胎牛血清(MP Biomedicals公司製)之D-MEM/Ham’s F-12培養基(Sigma-Aldrich公司製)培養24小時。接著,在實施例10中,以α-TPNa的終濃度成為1μM之方式,將α-TPNa溶液添加至培養基中。在實施例11中,以α-TPNa的終濃度成為10μM之方式,將α-TPNa溶液添加至培養基中。在實施例12中,以γ-TPNa的終濃度成為10μM之方式,將γ-TPNa溶液添加至培養基中。 在比較例14中,以生育酚醋酸酯的終濃度成為10μM之方式,將生育酚醋酸酯溶液添加至培養基中。在比較例15中,以α-生育酚的終濃度成為10μM之方式,將α-生育酚溶液添加至培養基中。在比較例16中,以γ-生育酚的終濃度成為10μM之方式,將γ-生育酚溶液添加至培養基中。 此外,在比較例13中,僅將0.05%(V/V)乙醇水溶液添加至培養基中。 SH-SY5Y cells were prepared at a seeding density of 50,000 cells/ cm2 and cultured for 24 hours in a D-MEM/Ham's F-12 medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 10, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 1 μM. In Example 11, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 10 μM. In Example 12, a γ-TPNa solution was added to the medium in such a manner that the final concentration of γ-TPNa was 10 μM. In Comparative Example 14, a tocopherol acetate solution was added to the culture medium so that the final concentration of tocopherol acetate was 10 μM. In Comparative Example 15, an α-tocopherol solution was added to the culture medium so that the final concentration of α-tocopherol was 10 μM. In Comparative Example 16, a γ-tocopherol solution was added to the culture medium so that the final concentration of γ-tocopherol was 10 μM. In Comparative Example 13, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium.
接著,調製將類澱粉β(Sigma-Aldrich公司製)溶於0.01%(V/V)DMSO水溶液中而得之類澱粉β溶液,以各培養基中之類澱粉β的終濃度成為30μM之方式,將該類澱粉β溶液添加至各培養基中。 另外,在參考例4中,僅添加0.05%(V/V)乙醇水溶液,並未添加類澱粉β溶液。 接著,將各培養基在37℃、5%CO 2下培養48小時。 Next, a starch β-like solution was prepared by dissolving starch β-like (manufactured by Sigma-Aldrich) in a 0.01% (V/V) DMSO aqueous solution, and the starch β-like solution was added to each culture medium so that the final concentration of starch β-like in each culture medium was 30 μM. In addition, in Reference Example 4, only a 0.05% (V/V) ethanol aqueous solution was added, and the starch β-like solution was not added. Then, each culture medium was cultured at 37°C and 5% CO 2 for 48 hours.
然後,在已移除培養基之各SH-SY5Y細胞中添加10μM Hoechst 33342(Sigma-Aldrich公司製)水溶液,於室溫(25℃)靜置10分鐘。去除溶液後,將各SH-SY5Y細胞以磷酸緩衝液(PBS,和光純藥公司製)洗淨,在螢光顯微鏡下計測可見到染色質凝集所引發之細胞凋亡樣強Hoechst螢光之細胞數(Hoechst(+)細胞)。在獨立試行的4個實驗中,各視野計測5000個細胞以上,將該4個實驗中之Hoechst(+)細胞的比例之平均值視為發生細胞凋亡之細胞的比例。針對各例,算出將參考例4中之凋亡細胞的比例設為1.00時之相對值,將其結果示於表4。Then, 10 μM Hoechst 33342 (Sigma-Aldrich) aqueous solution was added to each SH-SY5Y cell from which the culture medium had been removed, and the cells were left to stand at room temperature (25°C) for 10 minutes. After the solution was removed, each SH-SY5Y cell was washed with phosphate buffered saline (PBS, Wako Pure Chemical Industries, Ltd.), and the number of cells (Hoechst(+) cells) with strong Hoechst fluorescence, which indicated cell apoptosis caused by chromatin condensation, was counted under a fluorescent microscope. In four independent experiments, more than 5,000 cells were counted in each field of view, and the average of the proportion of Hoechst(+) cells in the four experiments was regarded as the proportion of cells undergoing cell apoptosis. For each example, the relative value was calculated when the ratio of apoptotic cells in Reference Example 4 was set to 1.00, and the results are shown in Table 4.
[表4]
如表4所示,在參考例4及比較例13中,與僅添加0.05%(V/V)乙醇水溶液且並未添加類澱粉β之參考例4的SH-SY5Y細胞相比,就進一步添加類澱粉β所培養而得之比較例13的SH-SY5Y細胞而言,可確認到凋亡細胞的比例增加。As shown in Table 4, in Reference Example 4 and Comparative Example 13, compared with the SH-SY5Y cells of Reference Example 4 to which only 0.05% (V/V) aqueous ethanol solution was added and no starch beta was added, it was confirmed that the proportion of apoptotic cells increased in the SH-SY5Y cells of Comparative Example 13 to which starch beta was further added.
就比較例14~16的添加生育酚醋酸酯、α-生育酚或γ-生育酚所培養而得之SH-SY5Y細胞而言,與比較例13的SH-SY5Y細胞相比,無法確認到凋亡細胞的比例降低。 另一方面,就實施例10~12的添加屬於自噬活化劑之生育酚磷酸酯鹽所培養而得之SH-SY5Y細胞而言,與比較例13的SH-SY5Y細胞相比,可確認到凋亡細胞的比例降低。 In the SH-SY5Y cells cultured with tocopherol acetate, α-tocopherol or γ-tocopherol in Comparative Examples 14 to 16, a decrease in the proportion of apoptotic cells was not observed compared with the SH-SY5Y cells in Comparative Example 13. On the other hand, in the SH-SY5Y cells cultured with tocopherol phosphate, which is an autophagy activator, in Examples 10 to 12, a decrease in the proportion of apoptotic cells was observed compared with the SH-SY5Y cells in Comparative Example 13.
[配方例] 作為自噬活化用組成物,係將外用劑的配方例1及2示於表5。 [Formulation Examples] As compositions for activating autophagy, the formulation examples 1 and 2 of external preparations are shown in Table 5.
[表5]
以上,對本發明之較佳實施例進行說明,但本發明並不限定於此等實施例。在不脫離本發明之主旨之範圍中,能夠進行構成的附加、省略、取代及其他變更。本發明並不受前述之說明所限定,僅受隨附之申請專利範圍所限定。 [產業上之可利用性] The above is a description of the preferred embodiments of the present invention, but the present invention is not limited to these embodiments. Additions, omissions, substitutions and other changes can be made to the structure without departing from the scope of the present invention. The present invention is not limited by the above description, but only by the scope of the attached patent application. [Industrial Applicability]
藉由本發明,係提供可有效地活化自噬之自噬活化劑及含有前述自噬活化劑之自噬活化用組成物。The present invention provides an autophagy activator that can effectively activate autophagy and an autophagy activating composition containing the autophagy activator.
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期刊 , A GUGLIANDOLO, et al., "α-Tocopherol modulates non-amyloidogenic pathway and autophagy in an in vitro model of Alzheimer's disease: A transcriptional study", Brain Sci., 9, MDPI, 2019: 196. |
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