JP6478222B2 - Apoptosis inducer for undifferentiated cells - Google Patents
Apoptosis inducer for undifferentiated cells Download PDFInfo
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- JP6478222B2 JP6478222B2 JP2015102175A JP2015102175A JP6478222B2 JP 6478222 B2 JP6478222 B2 JP 6478222B2 JP 2015102175 A JP2015102175 A JP 2015102175A JP 2015102175 A JP2015102175 A JP 2015102175A JP 6478222 B2 JP6478222 B2 JP 6478222B2
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Description
本発明は新規ネオ糖脂質及びその用途に関する。 The present invention relates to novel neoglycolipids and their uses.
神経幹細胞は高い自己複製能と分化能を併せ持つ神経系の未分化細胞である。近年、パーキンソン病などの難治性疾患の治療に神経幹細胞を利用すること（細胞医療）が試みられている。こうした治療を行うためには、移植に用いる神経幹細胞の分化過程を正確に制御する必要がある。特に、移植後に未分化細胞が残存することは、細胞の異常増殖に伴う細胞隗の形成等の問題を引き起こすおそれがあり、癌化のリスクも伴う。 Neural stem cells are undifferentiated cells of the nervous system having high self-replication ability and differentiation ability. In recent years, utilization of neural stem cells for the treatment of intractable diseases such as Parkinson's disease (cellular medicine) has been attempted. In order to perform such treatment, it is necessary to precisely control the differentiation process of neural stem cells used for transplantation. In particular, the survival of undifferentiated cells after transplantation may cause problems such as formation of cell mass associated with abnormal cell proliferation, and also has a risk of canceration.
一方、細胞医療の一つとして、iPS細胞や間葉系幹細胞（例えば骨髄由来、脂肪組織由来）等を利用した再生医療の実現に向けた研究・開発が世界的規模で進行している。このような再生医療では、利用する細胞の分化能が高く、そのまま移植すると癌化のリスクが高いことから、通常は予め生体外で特定の細胞系譜に沿って分化誘導して得られた細胞（即ち、分化細胞）又はそれを含有する組成物を移植に供する。十分に分化していない細胞（未分化細胞）が移植材料中に混在することは、移植に伴う塞栓の形成や意図しない細胞が移植後に出現すること、或いはがん化等の問題を引き起こす可能性があり、治療成績や安全性に多大な影響を及ぼす。
尚、本願で開示する化合物（ネオ糖脂質）の構造と一部において共通する化合物が報告されている（非特許文献１）。
On the other hand, research and development for the realization of regenerative medicine using iPS cells and mesenchymal stem cells (for example, bone marrow-derived and adipose tissue-derived) as one of cell medicine are progressing on a global scale. In such regenerative medicine, since the differentiation ability of the cells to be used is high and the risk of canceration is high if transplanted as it is, cells obtained by differentiation induction along a specific cell lineage in advance in advance (generally That is, the differentiated cells) or compositions containing them are subjected to transplantation. Inclusion of cells that are not sufficiently differentiated (undifferentiated cells) in the graft material may cause problems such as formation of an embolism during transplantation, appearance of unintended cells after transplantation, or canceration, etc. Impact on treatment outcome and safety.
In addition, a compound common to the structure of the compound (neoglycolipid) disclosed in the present application in part has been reported (Non-patent Document 1).
未分化細胞（例えば神経幹細胞）の分化誘導や幹細胞性の維持を行うための技術は数多く開発されている。しかしながら、未分化細胞の除去（死滅）に有効な技術の報告は少ない。尚、ガレクチン−１とインテグリンβ１との結合を阻害することが神経幹細胞の増殖抑制に有効であるとの報告（特許文献１）があるが、直接的に細胞死を誘導するものではなく、細胞医療におけるその有用性は限定的といえる。
そこで本発明は、未分化細胞に対して特異的ないし選択的に細胞死を誘導する技術を提供し、細胞医療の更なる発展、及び新たな細胞医療の実現に貢献することを課題とする。
A number of techniques have been developed to induce differentiation and maintain stemness of undifferentiated cells (eg, neural stem cells). However, there are few reports of techniques effective for removal (death) of undifferentiated cells. Although it has been reported that inhibiting the binding of galectin-1 to integrin β1 is effective for suppressing the proliferation of neural stem cells (Patent Document 1), it does not directly induce cell death, and cells Its usefulness in medicine can be said to be limited.
Therefore, the present invention has an object to provide a technology for inducing cell death specifically or selectively to undifferentiated cells, and to contribute to the further development of cell medicine and the realization of new cell medicine.
本発明者らは、ネオ糖脂質の構造解析や生理機能を研究する中で、特徴的な３糖構造からなるLewis X型糖鎖に着目し、Lewis X型糖鎖を含有するネオ糖脂質を各種合成し、その機能を調べることにした。検討の結果、Lewis X型糖鎖にスペーサーを介してアシル鎖が連結された構造のネオ糖脂質が神経幹細胞特異的な細胞死（アポトーシス）を誘導することが明らかとなった。即ち、神経幹細胞特異的な細胞死を誘導できる新規化合物を見出すことに成功した。また、ネオ糖脂質の構造とアポトーシス誘導効果との関係について重要且つ興味深い知見も得られた。 The present inventors focused on the Lewis X-type sugar chain consisting of a characteristic trisaccharide structure in studying the structural analysis and physiological function of neoglycolipid, and used neoglycolipid containing Lewis X-type sugar chain. I made various synths and decided to investigate its function. As a result of examination, it was revealed that neoglycolipid having a structure in which an acyl chain is linked to Lewis X-type sugar chain via a spacer induces neural stem cell specific cell death (apoptosis). That is, they succeeded in finding a novel compound capable of inducing neural stem cell-specific cell death. Also, important and interesting findings were obtained regarding the relationship between the structure of neoglycolipid and the apoptosis-inducing effect.
ここで、神経幹細胞の表面にはLewis X型糖鎖が発現している（例えばJ Biol Chem. 2012 Jul 13;287(29):24356-64.を参照）。後述の実験結果（実施例）を踏まえると、ネオ糖脂質のLewis X型糖鎖と、神経幹細胞表面のLewis X型糖鎖が相互作用することがトリガーとなって細胞死が誘導されたと考察できる。一方、過去の報告（例えばNeurochem Res. 2011 Sep;36(9):1623-35.）から、未分化細胞のいくつかは、神経幹細胞と同様、Lewis X型糖鎖を発現していることが知られている。Lewis X型糖鎖を発現している未分化細胞については、神経幹細胞で観察されたものと同様の現象が起きると予測できる。従って、Lewis X型糖鎖にスペーサーを介してアシル鎖が連結されたという、特徴的な構造を備えるネオ糖脂質は、神経幹細胞に限らず、Lewis X型糖鎖を発現している他の未分化細胞に対しても、選択的な細胞死を誘導できるといえる。即ち、当該ネオ糖脂質は、未分化細胞に対する選択的なアポトーシス誘導剤として汎用性が高いと評価できる。
本発明は以上の知見及び考察に基づくものである。
［１］Lewis X型糖鎖がスペーサーを介してアシル鎖に連結した構造のネオ糖脂質からなる、未分化細胞のアポトーシス誘導剤。
［２］アシル鎖の炭素数が１６〜２４である、［１］に記載のアポトーシス誘導剤。
［３］アシル鎖の炭素数が１６〜２０である、［１］に記載のアポトーシス誘導剤。
［４］未分化細胞が神経幹細胞であり、アシル鎖の炭素数が１８である、［１］に記載のアポトーシス誘導剤。
［５］神経幹細胞の移植を受けた個体に投与されることになる、［４］に記載のアポトーシス誘導剤。
［６］脳腫瘍を標的として個体に投与されることになる、［４］に記載のアポトーシス誘導剤。
［７］スペーサーが、１〜１０個の構成単位が連結した構造を有し、各構成単位が、独立して、糖、アミノ酸、核酸、メチレン鎖及びエチレングリコール鎖からなる群より選択されるいずれかの分子からなる、［１］〜［６］のいずれか一項に記載のアポトーシス誘導剤。
［８］スペーサーが、２〜６個の構成単位が連結した構造からなる、［７］に記載のアポトーシス誘導剤。
［９］スペーサーが、Lewis X構造側に配置される単糖又はオリゴ糖と、アシル鎖側に配置されるアルキル鎖が連結した構造からなる、［１］〜［６］のいずれか一項に記載のアポトーシス誘導剤。
［１０］オリゴ糖がラクトースである、［９］に記載のアポトーシス誘導剤。
［１１］アルキル鎖がアミノエチル基又はヒドロキシエチル基である、［９］又は［１０］に記載のアポトーシス誘導剤。
［１２］スペーサーが、Lewis X構造側に配置されるラクトースと、アシル鎖側に配置されるアミノエチル基が連結した構造からなる、［１］〜［６］のいずれか一項に記載のアポトーシス誘導剤。
［１３］ネオ糖脂質の構造が下記の化学式で表される、［１］に記載のアポトーシス誘導剤。
(1)特定の細胞系譜に沿った分化を誘導する条件下で未分化細胞を培養するステップ；
(2)［１］〜［１３］のいずれか一項に記載のアポトーシス誘導剤の存在下で培養を継続するステップ。
［１５］以下のステップ(i)を含む、分化細胞の調製方法：
(i)未分化細胞と分化細胞が混在する試料を［１］〜［１３］のいずれか一項に記載のアポトーシス誘導剤の存在下で培養するステップ。
［１６］以下のステップ(3)を更に含む、［１４］又は［１５］に記載の調製方法：
(3)分化細胞を回収するステップ。
Here, Lewis X-type sugar chain is expressed on the surface of neural stem cells (see, for example, J Biol Chem. 2012 Jul 13; 287 (29): 24356-64.). Based on the experimental results described below (Examples), it can be considered that the interaction between the Lewis glycosyl sugar of neoglycolipid and the Lewis glycosyl on the surface of neural stem cells is triggered to induce cell death. . On the other hand, according to previous reports (for example, Neurochem Res. 2011 Sep; 36 (9): 1623-35.), Some undifferentiated cells express Lewis X-type sugar chains like neural stem cells. Are known. For undifferentiated cells expressing Lewis X-type sugar chains, it can be predicted that the same phenomenon as that observed in neural stem cells will occur. Therefore, neoglycolipids having a characteristic structure in which an acyl chain is linked to a Lewis X-type sugar chain via a spacer are not limited to neural stem cells, but other non-neurogenic stem cells expressing Lewis X-type sugar chains. It can be said that selective cell death can also be induced to differentiated cells. That is, the neoglycolipid can be evaluated to be highly versatile as a selective apoptosis inducer for undifferentiated cells.
The present invention is based on the above findings and considerations.
[1] An apoptosis inducer for undifferentiated cells, which comprises neoglycolipid having a structure in which a Lewis X-type sugar chain is linked to an acyl chain via a spacer.
[2] The apoptosis inducer according to [1], wherein an acyl chain has 16 to 24 carbon atoms.
[3] The apoptosis inducer according to [1], wherein the carbon number of the acyl chain is 16 to 20.
[4] The apoptosis inducer according to [1], wherein the undifferentiated cell is a neural stem cell, and the carbon number of the acyl chain is 18.
[5] The apoptosis inducer according to [4], which is to be administered to an individual who has received a transplant of neural stem cells.
[6] The apoptosis inducer according to [4], which will be administered to an individual targeting a brain tumor.
[7] The spacer has a structure in which 1 to 10 constituent units are linked, and each constituent unit is independently selected from the group consisting of a sugar, an amino acid, a nucleic acid, a methylene chain and an ethylene glycol chain The apoptosis inducer according to any one of [1] to [6], which comprises one or more molecules.
[8] The apoptosis inducer according to [7], wherein the spacer has a structure in which 2 to 6 structural units are linked.
[9] In any one of [1] to [6], wherein the spacer comprises a structure in which a monosaccharide or oligosaccharide placed on the Lewis X structure side and an alkyl chain placed on the acyl chain side are linked The apoptosis inducer as described.
[10] The apoptosis inducer according to [9], wherein the oligosaccharide is lactose.
[11] The apoptosis inducer according to [9] or [10], wherein the alkyl chain is an aminoethyl group or a hydroxyethyl group.
[12] The apoptosis according to any one of [1] to [6], wherein the spacer comprises a structure in which lactose disposed on the Lewis X structure side is linked to an aminoethyl group disposed on the acyl chain side. Inducer.
[13] The apoptosis inducer according to [1], wherein the structure of neoglycolipid is represented by the following chemical formula.
(1) culturing undifferentiated cells under conditions that induce differentiation along a specific cell lineage;
(2) A step of continuing the culture in the presence of the apoptosis inducer according to any one of [1] to [13].
[15] A method of preparing differentiated cells, comprising the following step (i):
(i) culturing a sample in which undifferentiated cells and differentiated cells coexist in the presence of the apoptosis-inducing agent according to any one of [1] to [13].
[16] The preparation method according to [14] or [15], further comprising the following step (3):
(3) Recovering differentiated cells.
１．未分化細胞のアポトーシス誘導剤
本発明の第１の局面は未分化細胞のアポトーシス誘導剤に関する。本発明のアポトーシス誘導剤は、標的の未分化細胞に対して特異的／選択的にアポトーシスを誘導する。従って、本発明のアポトーシス誘導剤を用いれば、標的の未分化細胞を選択的に死滅させることができる。本発明のアポトーシス誘導剤は特定の構造のネオ糖脂質からなる。天然に存在するのではなく、人工的に得られる糖脂質（即ち人工糖脂質）であることを表現するため、慣例に倣い、本発明の糖脂質を「ネオ糖脂質」と呼称する。
1. Apoptosis-Inducing Agent for Undifferentiated Cells The first aspect of the present invention relates to an apoptosis-inducing agent for undifferentiated cells. The apoptosis inducer of the present invention specifically / selectively induces apoptosis in target undifferentiated cells. Thus, the apoptosis inducer of the present invention can selectively kill target undifferentiated cells. The apoptosis inducer of the present invention consists of neoglycolipid of a specific structure. The glycolipid of the present invention is referred to as "neoglycolipid" in accordance with the conventional manner in order to express that it is an artificially obtained glycolipid (ie, an artificial glycolipid) rather than occurring naturally.
本発明を構成するネオ糖脂質は、Lewis X型糖鎖がスペーサーを介してアシル鎖に連結した構造を備える。本発明を構成するネオ糖脂質では、Lewis X型糖鎖、スペーサー、及びアシル鎖が直鎖状に連なり、片方の末端にLews X型糖鎖が、他方の末端にアシル鎖が配置されることになる。 The neoglycolipid constituting the present invention has a structure in which a Lewis X-type sugar chain is linked to an acyl chain via a spacer. In the neoglycolipid constituting the present invention, a Lewis X-type sugar chain, a spacer, and an acyl chain are linearly linked, a Lews X-type sugar chain is disposed at one end, and an acyl chain is disposed at the other end. become.
（１）Lewis X型糖鎖
Lewis X型糖鎖は、ガラクトース、フコース及びN-アセチルグルコサミンからなる３糖構造（Galβ1,4(Fucα1,3)GlcNAc）の糖鎖である。生体中に見出されるLewis X構造と同様のLewis X構造が形成されるように、GlcNacにスペーサーが結合している。Lewis X型糖鎖はstage-specific embryonic antigen-1 (SSEA-1)とも呼ばれ、ヒト神経幹細胞、ヒト間葉系幹細胞、ヒト脳腫瘍細胞、マウスiPS細胞、マウスES細胞、マウス羊膜由来幹細胞等に発現していることが知られている。
(1) Lewis X-type sugar chain
The Lewis X-type sugar chain is a sugar chain of a trisaccharide structure (Galβ1,4 (Fucα1,3) GlcNAc) composed of galactose, fucose and N-acetylglucosamine. A spacer is attached to GlcNac such that a Lewis X structure similar to that found in living organisms is formed. Lewis type X sugar chain is also called stage-specific embryonic antigen-1 (SSEA-1), and it is used for human neural stem cells, human mesenchymal stem cells, human brain tumor cells, mouse iPS cells, mouse ES cells, mouse amniotic membrane-derived stem cells, etc. It is known to be expressed.
（２）スペーサー
スペーサーは、Lewis X型糖鎖とアシル鎖の連結という基本的な役割の他、Lewis X型糖鎖とアシル鎖の間の空間的距離の形成、及び柔軟性の付与という役割も担う。これらの役割を果たすため、本発明に使用するスペーサーは、１〜１０個の構成単位が連結した構造を有する。好ましくは、協働性の観点から、２〜６個の構成単位が連結した構造のスペーサーを用いる。構成単位毎にそれを構成する分子を選択できる。従って、スペーサーは一種又は二種以上の分子から構成されることになる。スペーサーを構成する分子（即ち、構成単位として採用可能な分子）の例は、糖、アミノ酸、核酸、メチレン鎖（CH2）及びエチレングリコール鎖（HOCH2CH2OH）である。
(2) Spacer In addition to the basic role of linking a Lewis X-type sugar chain and an acyl chain, the spacer also plays a role of forming a spatial distance between the Lewis X-type sugar chain and an acyl chain and imparting flexibility. Bear. In order to fulfill these roles, the spacer used in the present invention has a structure in which 1 to 10 structural units are linked. Preferably, from the viewpoint of cooperativity, a spacer having a structure in which 2 to 6 structural units are linked is used. For each constituent unit, it is possible to select the molecules that constitute it. Thus, the spacer will be composed of one or more molecules. Examples of molecules that constitute the spacer (that is, molecules that can be adopted as a constituent unit) are sugars, amino acids, nucleic acids, methylene chains (CH 2 ) and ethylene glycol chains (HOCH 2 CH 2 OH).
スペーサーの構造の好ましい一例として、Lewis X構造側に配置される単糖又はオリゴ糖と、アシル鎖側に配置されるアルキル鎖が連結した構造を挙げることができる。単糖はアルドース又はケトースであり、具体例はグルコース、ガラクトース、マンノース、キシロース、フルクトース、N-アセチルグルコサミン、N-アセチルガラクトサミンである。オリゴ糖の重合度は特に限定されない。例えば重合度２〜６のオリゴ糖を用いることができる。特に好ましいオリゴ糖の一例としてラクトースを挙げることができる。ラクトースを採用した場合、本発明のネオ糖脂質は、ラクトースにLewis X構造が連結した５糖構造という、生体中に存在する構造を備えることになり、非免疫原性であるという利点ないし効果を期待できる。 As a preferable example of the structure of a spacer, the structure which the monosaccharide or oligosaccharide arrange | positioned at the Lewis X structure side and the alkyl chain arrange | positioned at the acyl chain side can be mentioned. The monosaccharide is aldose or ketose, and specific examples are glucose, galactose, mannose, xylose, fructose, N-acetylglucosamine, N-acetylgalactosamine. The degree of polymerization of the oligosaccharide is not particularly limited. For example, oligosaccharides having a polymerization degree of 2 to 6 can be used. Lactose can be mentioned as an example of a particularly preferred oligosaccharide. When lactose is employed, the neoglycolipid of the present invention has a structure existing in the living body, that is, a pentasaccharide structure in which Lewis X structure is linked to lactose, and the advantage or effect of being non-immunogenic I can expect it.
アルキル鎖としては、例えば、アミノエチル基又はヒドロキシエチル基を用いる。アミノエチル基を採用した場合、スペーサーとアシル鎖がアミド結合を介して連結されることになる。一方、ヒドロキシエチル基を採用した場合の結合様式はエステル結合となる（エチレンオキシ基が形成されることになる）。 As the alkyl chain, for example, an aminoethyl group or a hydroxyethyl group is used. When an aminoethyl group is employed, the spacer and the acyl chain will be linked via an amide bond. On the other hand, in the case of employing a hydroxyethyl group, the bonding mode is an ester bond (an ethyleneoxy group will be formed).
（３）アシル鎖
例えば、炭素数が１６〜２４のアシル鎖が用いられる。好ましくは、炭素数が１６〜２０のアシル鎖を用いる。アシル鎖は、典型的には、非修飾のアシル鎖である。但し、本発明の効果、即ち、未分化細胞に対してアポトーシスを誘導すること、に実質的な影響のない限り、修飾されたアシル鎖であってもよい。ここでの修飾の例として、ニトロベンゾオキサジアゾールをはじめとする蛍光色素の付加を挙げることができる。
(3) Acyl chain For example, an acyl chain having 16 to 24 carbon atoms is used. Preferably, an acyl chain having 16 to 20 carbon atoms is used. Acyl chains are typically unmodified acyl chains. However, as long as the effects of the present invention, ie, induction of apoptosis in undifferentiated cells, are not substantially affected, it may be a modified acyl chain. As an example of the modification here, mention may be made of the addition of fluorescent dyes including nitrobenzoxadiazole.
後述の実施例に示す通り、炭素数１８のアシル鎖を用いて構成したネオ糖脂質が神経幹細胞に対して優れた効果を発揮した事実に鑑み、本発明のアポトーシス誘導剤の標的となる細胞（即ち、アポトーシスを誘導することになる細胞）が神経幹細胞の場合には、好ましくは炭素数が１８のアシル鎖を採用する。 In view of the fact that the neoglycolipid composed of an acyl chain having 18 carbon atoms exhibited an excellent effect on neural stem cells as shown in the examples described later, cells targeted by the apoptosis inducer of the present invention ( That is, when the cell to be induced apoptosis is a neural stem cell, preferably, an acyl chain having 18 carbon atoms is employed.
（４）ネオ糖脂質の具体例
本発明のアポトーシス誘導剤を構成するネオ糖脂質の構造の具体例を示す。
（５）合成方法
本発明のアポトーシス誘導剤を構成するネオ糖脂質は化学合成で調製することができる。例えば、グルコサミン、ガラクトース、フコースの誘導体を用いて、チオグリコシドを用いた反応やシュミットのグリコシル化反応でLewis X型糖鎖誘導体を合成する。また、ラクトースをアセテート体とし、これを用いたグリコシル化反応でスペーサーを合成する。このようにして得たLewis X型糖鎖誘導体とスペーサーを、糖供与体、受容体としたグリコシル化反応で5糖構造とし、次いで各保護基を塩基または還元剤等によって脱保護した後、脱水縮合反応で脂肪酸と連結することにより、目的のネオ糖脂質を得る。
(5) Synthetic Method The neoglycolipid constituting the apoptosis inducer of the present invention can be prepared by chemical synthesis. For example, derivatives of glucosamine, galactose and fucose are used to synthesize Lewis X-type sugar chain derivatives by a reaction using thioglycoside or a glycosylation reaction of Schmidt. In addition, lactose is made into an acetate form, and a spacer is synthesized by a glycosylation reaction using this. The Lewis X-type sugar chain derivative thus obtained and the spacer are converted to a pentasaccharide structure by a glycosylation reaction using a sugar donor and acceptor, and then each protective group is deprotected by a base or a reducing agent or the like and then dehydrated. The desired neoglycolipid is obtained by linking with a fatty acid by condensation reaction.
（６）標的細胞
本発明のアポトーシス誘導剤は、Lewis X型糖鎖を発現している未分化細胞に適用される。即ち、Lewis X型糖鎖を発現していることを条件として様々な細胞に適用できる。未分化細胞を例示すると、成体幹細胞（例えば、神経幹細胞、上皮幹細胞、造血幹細胞）、間葉系幹細胞（骨髄由来、脂肪組織由来、歯髄由来、臍帯由来など）、ES細胞（胚性幹細胞）、iPS細胞（人工多能性幹細胞）、ntES細胞（体細胞由来ES細胞）、これらの細胞を分化誘導して得られた細胞（但し、更に分化する能力を有するもの）、脳腫瘍細胞である。未分化細胞の分化能や分化レベルは特に限定されない。従って、上記のごとき各種幹細胞はもとより、各種前駆細胞も未分化細胞に該当する。
(6) Target Cell The apoptosis inducer of the present invention is applied to undifferentiated cells expressing Lewis X-type sugar chain. That is, the present invention can be applied to various cells as long as they express Lewis X-type sugar chain. Examples of undifferentiated cells include adult stem cells (eg, neural stem cells, epithelial stem cells, hematopoietic stem cells), mesenchymal stem cells (bone marrow-derived, adipose tissue-derived, dental pulp-derived, umbilical cord-derived, etc.), ES cells (embryonic stem cells), iPS cells (artificial pluripotent stem cells), ntES cells (somatic cell-derived ES cells), cells obtained by inducing differentiation of these cells (with the ability to further differentiate), and brain tumor cells. There are no particular limitations on the differentiation ability or differentiation level of undifferentiated cells. Therefore, not only the above-described various stem cells but also various precursor cells correspond to undifferentiated cells.
未分化細胞の動物種はヒト、マウス、ラット等の哺乳動物である。好ましい標的細胞はヒト細胞である。即ち、好ましい一態様では、ヒト未分化細胞に対して本発明のアポトーシス誘導剤を適用する。 Animal species of undifferentiated cells are mammals such as humans, mice and rats. Preferred target cells are human cells. That is, in a preferred embodiment, the apoptosis inducer of the present invention is applied to human undifferentiated cells.
未分化細胞がLewis X型糖鎖を発現しているか否かは、Lewis X型糖鎖に特異的結合性を有する抗体を用いた免疫学的検出、クロマトグラフィーや質量分析等によって確認することができる。また、過去の報告を参考にしてLewis X型糖鎖の発現の有無を判断することにしてもよい。尚、ヒト神経幹細胞、ヒト間葉系幹細胞、ヒト脳腫瘍細胞、マウスiPS細胞、マウスES細胞、マウス羊膜由来幹細胞はLewis X型糖鎖を発現することが知られており、好適な標的細胞となる。 Whether or not undifferentiated cells express Lewis X-type sugar chain can be confirmed by immunological detection, chromatography, mass spectrometry, etc. using an antibody having specific binding property to Lewis X-type sugar chain it can. In addition, the presence or absence of expression of Lewis X-type sugar chain may be determined with reference to past reports. Human neural stem cells, human mesenchymal stem cells, human brain tumor cells, mouse iPS cells, mouse ES cells, and mouse amniotic membrane-derived stem cells are known to express Lewis X-type sugar chains and are suitable target cells. .
（７）適用方法
中枢神経の障害の治療・改善を目的として、中枢神経組織への神経細胞の移植（再生医療）が検討されている。これまでの報告によれば、分化した細胞（ニューロンなど）を直接移植すると死滅してしまうため、通常は神経幹細胞が移植される。移植された神経幹細胞は周囲の環境によって特定の機能を担う細胞（ニューロンなど）に分化し、治療効果を発揮する。この治療戦略では、神経幹細胞の増殖能が高いために移植部位において細胞塊を形成する可能性や、がん化等が懸念されている。本発明の一態様は、この課題に対する解決策を提供する。具体的には、神経幹細胞の移植を受けた個体に対して、移植後のある程度分化が進行した段階でアポトーシス誘導剤を投与し、移植部位に残存する未分化細胞（即ち神経幹細胞）の選択的除去を図る。即ち、残存神経幹細胞の除去剤として本発明のアポトーシス誘導剤を利用する。この態様では、移植部に送達されるようにアポトーシス誘導剤を投与する。例えば、移植部への直接投与（典型的には注射）、移植部近傍への投与（典型的には注射）等の投与形態で投与することができる。移植部に送達されることを条件にその他の投与形態を採用することにしてもよい。尚、神経幹細胞以外の幹細胞が移植される治療戦略においても同様に、残存する未分化細胞を除去する目的で本発明のアポトーシス誘導剤を用いることができる。
(7) Method of application For the purpose of treatment / improvement of central nervous system disorders, transplantation of nerve cells into central nervous tissue (regenerative medicine) has been studied. According to previous reports, neural stem cells are usually transplanted because direct transplantation of differentiated cells (such as neurons) is killed. Depending on the surrounding environment, the transplanted neural stem cells differentiate into cells (such as neurons) responsible for specific functions and exert therapeutic effects. In this therapeutic strategy, there is concern about the possibility of forming a cell mass at the transplantation site, canceration, etc. because the proliferation ability of neural stem cells is high. One aspect of the present invention provides a solution to this problem. Specifically, to an individual who receives neural stem cell transplantation, an apoptosis inducer is administered at a stage where differentiation has progressed to a certain extent after transplantation, and selective of undifferentiated cells (ie, neural stem cells) remaining at the transplantation site are selected. Try to remove it. That is, the apoptosis inducer of the present invention is used as an agent for removing residual neural stem cells. In this aspect, the apoptosis inducer is administered to be delivered to the transplant site. For example, it can be administered in a dosage form such as direct administration (typically, injection) to the implantation site, administration near the implantation site (typically, injection), and the like. Other dosage forms may be employed provided that they are delivered to the implant. Also in the therapeutic strategy in which stem cells other than neural stem cells are transplanted, the apoptosis inducer of the present invention can also be used for the purpose of removing remaining undifferentiated cells.
一方、脳腫瘍幹細胞がLewis X型糖鎖を発現していることが知られている。この点に注目し、本発明の別の一態様は、脳腫瘍の治療剤としてアポトーシス誘導剤を利用する。具体的には、脳腫瘍を患う個体に対し、脳腫瘍を標的としてアポトーシス誘導剤を投与する。投与されたアポトーシス誘導剤は脳腫瘍に選択的毒性を示すことで治療効果を発揮し得る。この態様では、脳腫瘍組織に送達されるようにアポトーシス誘導剤を投与する。例えば、脳腫瘍組織内への直接投与（典型的には注射）、脳腫瘍組織近傍への投与（典型的には注射）等の投与形態で投与することができる。脳腫瘍組織に送達されることを条件にその他の投与形態を採用することにしてもよい。 On the other hand, it is known that brain tumor stem cells express Lewis X-type sugar chains. Focusing on this point, another aspect of the present invention utilizes an apoptosis inducer as a therapeutic agent for brain tumor. Specifically, to an individual suffering from a brain tumor, an apoptosis inducer is administered targeting the brain tumor. The administered apoptosis inducer can exert a therapeutic effect by exhibiting selective toxicity to brain tumors. In this aspect, an apoptosis inducer is administered for delivery to brain tumor tissue. For example, it can be administered in a dosage form such as direct administration (typically, injection) into brain tumor tissue, administration near brain tumor tissue (typically, injection), and the like. Other dosage forms may be employed provided that they are delivered to brain tumor tissue.
２．分化細胞の調製方法
本発明の第２の局面はアポトーシス誘導剤（第１の局面）の用途に関し、分化細胞の調製方法を提供する。「分化細胞」とは、特定の細胞系譜に沿って分化した結果として得られる細胞である。分化の過程で分化能を喪失することから、分化細胞は通常の条件下（即ち、生理的な条件下）では更に分化することはない。但し、特殊な条件下や特別の操作（例えば、リプログラミング）によって再び分化能を獲得する場合がある。
2. Method of Preparing Differentiated Cells The second aspect of the present invention relates to the use of an apoptosis inducer (first aspect), and provides a method of preparing differentiated cells. A "differentiated cell" is a cell obtained as a result of differentiation along a specific cell lineage. Since the differentiation ability is lost in the process of differentiation, the differentiated cells do not differentiate further under normal conditions (ie, physiological conditions). However, differentiation ability may be obtained again under special conditions or special operations (eg, reprogramming).
本発明の調製方法では、上記アポトーシス誘導剤を用いることにより、未分化細胞にアポトーシスを誘導して死滅させ、純度の高い（即ち、未分化細胞の混在の少ない）分化細胞を得る。本発明の調製方法によれば、未分化性を維持した細胞（残存未分化細胞）又は目的の分化細胞まで分化するに至っていない細胞をアポトーシス誘導剤の効果によって死滅させることができる。現在、iPS細胞に代表される、細胞を利用した再生医療の実用化に向けた取り組みが進められている。移植用細胞又は組織に未分化細胞が残存していると癌化のリスクが増大すると懸念されている。残存する未分化細胞を除去するために様々な技術が開発されているが、決定的といえるものはない。本発明は、このような状況を打開し得る技術を提供するものであり、簡便な方法にもかかわらず、高品質（即ち、夾雑する未分化細胞が少なく、純度が高い）の分化細胞の提供を可能とする。 In the preparation method of the present invention, by using the above-mentioned apoptosis inducer, undifferentiated cells are induced to undergo apoptosis and killed, and highly pure (that is, less mixed with undifferentiated cells) differentiated cells are obtained. According to the preparation method of the present invention, it is possible to kill cells that have maintained undifferentiated properties (remaining undifferentiated cells) or cells that have not reached differentiation to the target differentiated cells by the effect of the apoptosis-inducing agent. Currently, efforts are being made toward the practical application of cell-based regenerative medicine represented by iPS cells. Remaining undifferentiated cells in the cells or tissues for transplantation is feared to increase the risk of canceration. Although various techniques have been developed to remove remaining undifferentiated cells, there is nothing critical. The present invention provides a technique capable of overcoming such a situation, and despite the simple method, provides differentiated cells of high quality (ie, less contaminating undifferentiated cells and high purity). Make it possible.
本願では大別して２種類の調製方法（第１の調製方法、第２の調製方法）が提供される。第１の調製方法は、未分化細胞を出発材料として分化細胞を調製する方法である。当該調製方法では、以下のステップ(1)及び(2)をこの順序で行い、分化細胞を得る。
(1)特定の細胞系譜に沿った分化を誘導する条件下で未分化細胞を培養するステップ
(2)本発明のアポトーシス誘導剤の存在下で培養を継続するステップ
The present invention is roughly classified into two types of preparation methods (a first preparation method and a second preparation method). The first preparation method is a method of preparing differentiated cells using undifferentiated cells as a starting material. In the preparation method, the following steps (1) and (2) are performed in this order to obtain differentiated cells.
(1) culturing undifferentiated cells under conditions which induce differentiation along a specific cell lineage
(2) Continuing culture in the presence of the apoptosis inducer of the present invention
ステップ(1)は未分化細胞を分化誘導するステップであり、未分化細胞から目的の分化細胞が得られるように、分化を誘導する条件下で未分化細胞を培養する。未分化細胞としては、目的の分化細胞への分化能を有するものが用いられる。未分化細胞の定義、使用可能な未分化細胞については上記の説明（（６）標的細胞の欄）が援用される。 Step (1) is a step of inducing differentiation of undifferentiated cells, and the undifferentiated cells are cultured under conditions that induce differentiation so that the desired differentiated cells can be obtained from the undifferentiated cells. As the undifferentiated cells, those having the ability to differentiate into the desired differentiated cells are used. For the definition of undifferentiated cells and usable undifferentiated cells, the above description ((6) Target cell column) is incorporated.
用意した未分化細胞は、特定の細胞系譜に沿った分化が誘導される条件下で培養される。特定の細胞系譜とは、目的とする分化細胞が属する細胞系譜である。例えば、目的とする分化細胞が神経系を構成する細胞（ニューロン、アストロサイト（星状膠細胞）、オリゴデンドロサイト（稀突起膠細胞）、ミクログリア）であれば、神経細胞系譜が該当する。培養条件は過去の報告や成書を参考にして設定すればよい。例えば、基本培地に分化誘導剤及びその他の必要な成分を添加した培養液を用いて未分化細胞を培養する。分化誘導剤の例として、bFGF、EGF、BDNF、BMP9、レチノイン酸、TGF-βファミリー阻害剤、GSK3β阻害剤、ソニックヘッジホッグタンパク質、プルモルファミン等を挙げることができる。 The prepared undifferentiated cells are cultured under conditions in which differentiation along a specific cell lineage is induced. A specific cell lineage is a cell lineage to which a target differentiated cell belongs. For example, if the target differentiated cell is a cell constituting a nervous system (neuron, astrocyte (astrocyte), oligodendrocyte (hypogeloid cell), microglia), neural cell lineage corresponds. Culture conditions may be set with reference to past reports and books. For example, undifferentiated cells are cultured using a culture solution obtained by adding a differentiation inducer and other necessary components to a basal medium. Examples of differentiation inducers include bFGF, EGF, BDNF, BMP9, retinoic acid, TGF-β family inhibitor, GSK3β inhibitor, sonic hedgehog protein, purmorphamine and the like.
培地に添加可能な他の成分の例としは、血清（ウシ胎仔血清、ヒト血清、ウマ血清など）、血清代替物（Knockout serum replacement(KSR)など）、ウシ血清アルブミン（BSA）、抗生物質、2-メルカプトエタノール、PVA、非必須アミノ酸（NEAA）、インスリン、トランスフェリン、セレニウムを挙げることができる。基本培地の例は、ダルベッコ変法イーグル培地(D-MEM)、α-MEM、ハムF12培地（HamF12）、イスコフ改変ダルベッコ培地（IMDM）、グラスゴー基本培地、Medium 199培地、イーグル最小必須培地（EMEM）培地、RPMI1640培地、Neurobasal培地、Neurobasal A培地である。培養温度は例えば、約30〜40℃、好ましくは約37℃に設定する。CO2濃度は、例えば約1〜10％、好ましくは約2〜5％に設定する。培養期間は、使用する細胞、その他の培養条件等によって変動しうるが、例えば１日〜６０日である。尚、最適な培養条件は予備実験を通して決定すればよい。 Examples of other components that can be added to the medium include serum (fetal bovine serum, human serum, horse serum, etc.), serum substitute (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, Mention may be made of 2-mercaptoethanol, PVA, nonessential amino acids (NEAA), insulin, transferrin, selenium. Examples of the basal medium are Dulbecco's modified Eagle's medium (D-MEM), α-MEM, Ham's F12 medium (HamF12), Iscove's modified Dulbecco's medium (IMDM), Glasgow basic medium, Medium 199 medium, Eagle's minimal essential medium (EMEM) 2.) Medium, RPMI 1640 medium, Neurobasal medium, Neurobasal A medium. The culture temperature is set, for example, at about 30 to 40 ° C, preferably about 37 ° C. The CO 2 concentration is set to, for example, about 1 to 10%, preferably about 2 to 5%. The culture period may vary depending on the cells used, other culture conditions, etc., and is, for example, 1 day to 60 days. Optimal culture conditions may be determined through preliminary experiments.
ステップ(1)によって未分化細胞が特定の細胞系譜に沿って分化し、目的の分化細胞が生ずる。即ち、目的の分化細胞を含む細胞集団が形成される。ステップ(2)では、当該細胞集団に残存する未分化細胞（目的の分化細胞まで分化するに至っていない細胞が存在する場合には、当該細胞も除去の対象となり得る）を選択的に除去（死滅）するために、本発明のアポトーシス誘導剤の存在下で培養を継続する。典型的には、ステップ(1)の後、本発明のアポトーシス誘導剤を含有する培養液へと培地交換し、分化細胞の維持ないし増殖に適した条件で培養する。ステップ(2)の培養液として、例えば、ステップ(1)に使用する培養液に本発明のアポトーシス誘導剤を添加したものを使用することができる。ステップ(1)の後に培地交換するのではなく、培養液にアポトーシス誘導剤を添加し、培養を継続することにしてもよい。また、ステップ(1)後の細胞を一旦回収し、新たな培養（即ちステップ(2)）に供することにしてもよい。ステップ(2)における培養液中のアポトーシス誘導剤の濃度（添加濃度）は、所望の効果が発揮される限りにおいて特に限定されないが、例えば、アポトーシス誘導剤の有効成分であるネオ糖脂質の培養液中の濃度を50μM〜300μM、好ましくは100μM〜200μMとする。構造の異なる二種類以上のネオ糖脂質を併用してもよい。例えば、アシル鎖の長さが異なるネオ糖脂質を併用（炭素数が１６のアシル鎖を有するネオ糖脂質と炭素数が１８のアシル鎖を有するネオ糖脂質の併用、更に炭素数が２４のアシル鎖を有するネオ糖脂質を併用等）。尚、ステップ(2)におけるその他の培養条件は、ステップ(1)に準ずればよい。 In step (1), undifferentiated cells differentiate along a specific cell lineage, and target differentiated cells are generated. That is, a cell population including the target differentiated cells is formed. In step (2), undifferentiated cells remaining in the cell population (if there are cells that have not reached differentiation to the target cell, such cells may also be targeted for removal (kill) The culture is continued in the presence of the apoptosis inducer of the present invention in order to Typically, after step (1), the medium is exchanged to a culture solution containing the apoptosis inducer of the present invention, and culture is performed under conditions suitable for maintenance or proliferation of differentiated cells. As the culture solution of step (2), for example, one obtained by adding the apoptosis inducer of the present invention to the culture solution used in step (1) can be used. Instead of changing the medium after step (1), an apoptosis inducer may be added to the culture solution to continue the culture. Alternatively, the cells after step (1) may be collected once and subjected to fresh culture (ie, step (2)). The concentration (added concentration) of the apoptosis inducer in the culture solution in step (2) is not particularly limited as long as the desired effect is exhibited, but, for example, a culture solution of neoglycolipid which is an active ingredient of the apoptosis inducer The concentration is 50 μM to 300 μM, preferably 100 μM to 200 μM. Two or more kinds of neoglycolipids having different structures may be used in combination. For example, a combination of neoglycolipids having different acyl chain lengths (combination of a neoglycolipid having an acyl chain having 16 carbons and a neoglycolipid having an acyl chain having 18 carbons, and further having an acyl having 24 carbons Combined with neoglycolipids having chains, etc.). The other culture conditions in step (2) may conform to step (1).
以上の調製方法（第１の調製方法）では、未分化細胞を出発材料として分化細胞を調製するが、本発明の第２の調製方法では、未分化細胞と分化細胞が混在する試料（細胞集団）から分化細胞を調製する。この調製方法によれば、未分化細胞が選択的に除去され、分化細胞の含有率が高められる。即ち、夾雑する未分化細胞が少ない、高純度の分化細胞が得られる。この特徴に注目すれば、当該調製方法を「分化細胞を精製する方法」、或いは「分化細胞の純度を高める方法」として捉えることも可能である。本発明の第２の調製方法では、以下のステップを行う。
(i)未分化細胞と分化細胞が混在する試料を本発明のアポトーシス誘導剤の存在下で培養するステップ
In the above preparation method (first preparation method), differentiated cells are prepared using undifferentiated cells as a starting material, but in the second preparation method of the present invention, a sample (cell population in which undifferentiated cells and differentiated cells are mixed) Prepare differentiated cells from According to this preparation method, undifferentiated cells are selectively removed and the content of differentiated cells is increased. That is, highly pure differentiated cells with less contaminating undifferentiated cells can be obtained. If attention is paid to this feature, it is possible to regard the preparation method as “a method of purifying differentiated cells” or “a method of enhancing the purity of differentiated cells”. In the second preparation method of the present invention, the following steps are performed.
(i) culturing a sample in which undifferentiated cells and differentiated cells coexist in the presence of the apoptosis inducer of the present invention
未分化細胞と分化細胞が混在する試料としては、例えば、特定の細胞系譜に沿った分化を誘導する条件下で未分化細胞を培養すること（即ち、第１の調製方法のステップ(1)）によって得られた細胞集団を用いる。生体から採取した組織から分離した細胞集団又はその継代細胞を試料として用いることもできる。ステップ(i)では、アポトーシス誘導剤の存在下で培養するが、その操作方法や条件等は上記第１の調製方法の場合と同様である。 As a sample in which undifferentiated cells and differentiated cells coexist, for example, culturing undifferentiated cells under conditions that induce differentiation along a specific cell lineage (ie, step (1) of the first preparation method) Use the cell population obtained by A cell population isolated from a tissue collected from a living organism or a passage cell thereof can also be used as a sample. In step (i), culture is carried out in the presence of an apoptosis inducer, but the operation method, conditions and the like are the same as in the case of the above first preparation method.
本発明の調製方法（第１の調製方法及び第２の調製方法）によって得られる分化細胞は、典型的には回収され（ステップ(3)）、各種用途（再生医療、研究用途など）に供される。回収操作は常法（例えば、セルスクレイパーによる剥離、トリプシン等による酵素処理、ピペッティング、遠心分離、セルソーターによる分取等）で行えばよい。回収後の細胞を更なる処理（濃縮、希釈、継代培養、活性化、凍結等）に供してもよい。 The differentiated cells obtained by the preparation method of the present invention (the first preparation method and the second preparation method) are typically recovered (step (3)) and used for various applications (regeneration medicine, research applications, etc.). Be done. The recovery operation may be performed by a conventional method (for example, peeling with a cell scraper, enzyme treatment with trypsin etc., pipetting, centrifugation, sorting with a cell sorter, etc.). The recovered cells may be subjected to further processing (concentration, dilution, subculture, activation, freezing, etc.).
１．ネオ糖脂質の合成（図１）
３種類のネオ糖脂質（化１〜３）、即ち化合物ａ： Lewis X型糖鎖（Galβ1,4(Fucα1,3)GlcNAc）がスペーサー（アミノエチル基）を介してアシル鎖（炭素数１８）に連結した構造のネオ糖脂質、化合物ｂ： ２糖構造（Galβ1,4GlcNAc）がスペーサー（ラクトースとアミノエチル基）を介してアシル鎖（炭素数１８）に連結した構造のネオ糖脂質、化合物ｃ： Lewis X型糖鎖がスペーサー（ラクトースとアミノエチル基）を介してアシル鎖（炭素数１８）に連結した構造のネオ糖脂質、を合成した。また、比較のために、化合物ｄ： Lewis X型糖鎖にスペーサー（ラクトースとアセチルアミノエチル基）が連結した構造の化合物（化４）も合成した。
Three neoglycolipids (
＜化合物ａ＞
化合物ａは例えば以下のような合成スキームで調製することができる。
Compound a can be prepared, for example, according to the following synthesis scheme.
Lewis X型糖鎖誘導体1とオクタデカン酸をN,N-ジイソプロピルエチルアミン（DIPEA）と4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride（DMT-MM）を用いて縮合することで化合物ａを調製する。化合物1はChemical Communications, 2011, 47(38), 10800-10802を参考に調製した。調製した化合物ａを高分解能質量分析およびNMR分析により同定した（表１）。
HRMS (FAB): Calcd for C40H75N2O16 [M+H]+: 839.5111; Found: 839.5125.
NMR (500 MHz, 化合物a : DPC = 1 : 4, D2O, 300 K)
HRMS (FAB): Calcd for C 40 H 75 N 2 O 16 [M + H] + : 839.5111; Found: 839.5125.
NMR (500 MHz, compound a: DPC = 1: 4, D 2 O, 300 K)
＜化合物ｂ＞
化合物ｂは例えば以下のような合成スキームで調製することができる。
Compound b can be prepared, for example, according to the following synthesis scheme.
まず、化合物2とラクトース誘導体3を、N-ヨードこはく酸イミド（NIS）とトリフルオロメタンスルホン酸（TfOH）を加えて反応させることで、化合物4を得る。化合物4をエチレンジアミンによって処理した後、ピリジンおよび無水酢酸を加え反応させることで化合物5を得る。化合物5は、ナトリウムメトキシドによる処理および接触水素化によって化合物6へ誘導する。化合物6とオクタデカン酸をDIPEAとDMT-MMを用いて縮合することで化合物ｂを調製する。 First, compound 4 is obtained by reacting compound 2 with lactose derivative 3 with N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH). Compound 4 is treated with ethylene diamine and then reacted with pyridine and acetic anhydride to give compound 5. Compound 5 is derivatized to compound 6 by treatment with sodium methoxide and catalytic hydrogenation. Compound b is prepared by condensing Compound 6 with octadecanoic acid using DIPEA and DMT-MM.
化合物2は以下のように調製した。
まず、Phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-1-deoxy-β-D-glucopyranoside（7）と2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl 2,2,2-trichloroacetimidate（8）のジクロロメタン溶液にtrimethylsilyl trifluoromethanesulfonate（TMSOTf）を加えて反応させることで化合物9を合成した（収率66%）。化合物9にピリジンおよび無水酢酸を加え反応させることで化合物2を得た（収率83%）。 First, Phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-1-deoxy-β-D-glucopyranoside (7) and 2,3,4,6-tetra-O-acetyl-α- Compound 9 was synthesized by adding trimethylsilyl trifluoromethanesulfonate (TMSOTf) to a dichloromethane solution of D-galactopyranosyl 2,2,2-tricholoroacetimidate (8) and reacting (yield: 66%). Compound 9 was obtained by adding and reacting pyridine and acetic anhydride to obtain compound 2 (yield 83%).
ラクトース誘導体3は以下のスキームに従って合成した。
まず、Lactose octacetate（10）のジクロロメタン溶液に2-bromoethanolを加え、boron trifluoride-ethyl ether complexを用いて反応させることで化合物11へ誘導した（収率35%）。化合物11はDMF中、ヨウ化ナトリウムとアジ化ナトリウムを用いて化合物12へと変換した（収率92%）。化合物12をメタノール中、ナトリウムメトキシドを用いて処理し脱保護体13とした（収率90%）。化合物13を、DMF中、2,2-dimethoxypropaneとp-トルエンスルホン酸によって処理し、化合物14を得た（収率55％）。化合物14の水酸基を臭化ベンジルと水素化ナトリウムを用いて保護し、化合物15とした（収率52％）。化合物15を酢酸で処理することで化合物3を調製した（収率83%）。 First, 2-bromoethanol was added to a dichloromethane solution of lactose octoacetate (10), and the reaction was carried out using boron trifluoride-ethyl ether complex to give compound 11 (yield 35%). Compound 11 was converted to compound 12 using sodium iodide and sodium azide in DMF (yield 92%). Compound 12 was treated with sodium methoxide in methanol to give deprotected 13 (yield 90%). Compound 13 was treated with 2,2-dimethoxypropane and p-toluenesulfonic acid in DMF to give compound 14 (yield 55%). The hydroxyl group of compound 14 was protected with benzyl bromide and sodium hydride to give compound 15 (yield 52%). Compound 3 was prepared by treating Compound 15 with acetic acid (yield 83%).
調製した化合物ｂを高分解能質量分析およびNMR分析により同定した（表２）。
HRMS (FAB): Calcd for C46H85N2O22 [M+H]+: 1017.5588; Found: 1017.5610.
NMR (500 MHz, 化合物b : DPC = 1 : 4, D2O, 300 K)
HRMS (FAB): Calcd for C 46 H 85 N 2 O 22 [M + H] + : 1017.5588; Found: 1017.5610.
NMR (500 MHz, compound b: DPC = 1: 4, D 2 O, 300 K)
＜化合物ｃ＞
化合物ｃは例えば以下のような合成スキームで調製することができる。
Compound c can be prepared, for example, according to the following synthesis scheme.
まず、Lewis X型糖鎖誘導体16とラクトース誘導体3を、NISとTfOHを加えて反応させることで、化合物17を得る。化合物17をエチレンジアミンによって処理した後、ピリジンおよび無水酢酸を加え反応させることで化合物18を得る。化合物18は、ナトリウムメトキシドによる処理および接触水素化によって化合物19へ誘導する。化合物19とオクタデカン酸をDIPEAとDMT-MMを用いて縮合することでネオ糖脂質（化合物c）を調製する。 First, compound 17 is obtained by reacting Lewis X-type sugar chain derivative 16 and lactose derivative 3 with NIS and TfOH. Compound 17 is treated with ethylene diamine and then reacted with pyridine and acetic anhydride to give compound 18. Compound 18 is derivatized to compound 19 by treatment with sodium methoxide and catalytic hydrogenation. Compound 19 and octadecanoic acid are condensed using DIPEA and DMT-MM to prepare neoglycolipid (compound c).
Lewis X型糖鎖誘導体16は、化合物9とmethyl 2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside（20）とをジクロロメタン中、NISおよびTfOHを用いて反応させることで調製した（収率50%）。
調製したネオ糖脂質（化合物ｃ）を高分解能質量分析およびNMR分析により同定した（表３）。
HRMS (FAB): Calcd for C52H95N2O26 [M+H]+: 1163.6168; Found: 1163.6193.
NMR (500 MHz, 化合物c : DPC = 1 : 4, D2O, 300 K)
HRMS (FAB): Calcd for C 52 H 95 N 2 O 26 [M + H] + : 1163.6168; Found: 1163.6193.
NMR (500 MHz, compound c: DPC = 1: 4, D 2 O, 300 K)
＜化合物ｄ＞
化防物ｄは例えば化合物19と酢酸を縮合させることで調製することができる。
<Compound d>
The compound d can be prepared, for example, by condensing compound 19 with acetic acid.
２．神経幹細胞に対するネオ糖脂質の効果
（１）神経幹細胞の調製
神経幹細胞は過去の報告（H. Yagi et al. J. Biol. Chem. 285, 37923-37301, 2015）を参考にして調製した。具体的には、胎生1日目のICRマウスの胎児の脳から線条体を集め、0.25%トリプシン存在下で37℃、10分間インキュベートすることでシングル細胞を調製した。その後、2% B27（Life Technologies）、2mM L-グルタミン、20 ng/ml basic FGF（Pepro Tech）および20 ng/ml 上皮成長因子（EGF; Pepro Tech）を含むNeurobasal A medium（Life Technologies）培地を用い、5% CO2存在下37℃で培養することで、細胞隗として神経幹細胞を調製した。分化細胞は上記の培地からEGF、FGFを除き、1%ウシ胎仔血清を加えた培地を用い、5% CO2存在下37℃で10日間培養することで調製した。
2. Effect of neoglycolipid on neural stem cells (1) Preparation of neural stem cells Neural stem cells were prepared with reference to previous reports (H. Yagi et al. J. Biol. Chem. 285, 37923-37301, 2015). Specifically, single cells were prepared by collecting striatum from fetal brain of ICR mouse at 1 day of gestation and incubating for 10 minutes at 37 ° C. in the presence of 0.25% trypsin. Then, Neurobasal A medium (Life Technologies) medium containing 2% B27 (Life Technologies), 2 mM L-glutamine, 20 ng / ml basic FGF (Pepro Tech) and 20 ng / ml epidermal growth factor (EGF; Pepro Tech) By culturing at 37 ° C. in the presence of 5% CO 2 , neural stem cells were prepared as cell pellets. Differentiated cells were prepared by removing EGF and FGF from the above-mentioned medium and using a medium to which 1% fetal bovine serum was added, and culturing at 37 ° C. for 10 days in the presence of 5% CO 2 .
（２）WST-8アッセイを用いた細胞増殖の比較
増殖させた神経幹細胞隗をTrypLETMExpress (Life Technologies)存在下で37℃、5分間インキュベートし、シングル細胞を調製した。一方、分化細胞は、培養シャーレにト神経幹細胞を96ウェルプレートに1ウェル中に1×105細胞入るように調製した。合成したネオ糖脂質を最終濃度が所定濃度（0.2μM、0.96μM、4.8μM、24μM又は120μM）になるように加え、非処理のウェルとともに24時間後における増殖をCell Counting Kit-8（Dojindo）を用いて比較した。測定は450 nmの吸光を測定することで行った。
(2) WST-8 assay neural stem cells隗obtained by comparing growth of cells proliferating TrypLE TM Express (Life Technologies) using 37 ° C. in the presence of, and incubated for 5 minutes to prepare a single cell. On the other hand, differentiated cells were prepared so that neural stem cells in culture petri dishes contained 1 × 10 5 cells per well in a 96-well plate. Synthesized neoglycolipid is added to a final concentration (0.2 μM, 0.96 μM, 4.8 μM, 24 μM or 120 μM), and after 24 hours growth with untreated wells Cell Counting Kit-8 (Dojindo) It compared using. The measurement was performed by measuring the absorbance at 450 nm.
結果を図２、３に示す。ラクトースとアミノエチル基からなるスペーサーを介してLewis X型糖鎖がアシル鎖に連結した構造のネオ糖脂質（化合物ｃ）のみ、神経幹細胞特異的に細胞死を誘導していた。アシル鎖を結合させていない化合物（化合物ｄ）では神経幹細胞の細胞死は認められなかった。また、Lewis X型糖鎖を有さないネオ糖脂質（化合物ｂ）、及びスペーサーが短い（ラクトース構造を含まない）ネオ糖脂質（化合物ａ）についても、神経幹細胞特異的な細胞死誘導は認められなかった。 The results are shown in FIGS. Only neoglycolipid (compound c) having a structure in which a Lewis X-type sugar chain is linked to an acyl chain via a spacer consisting of lactose and an aminoethyl group induced cell death specifically in neural stem cells. Cell death of neural stem cells was not observed in the compound not having an acyl chain bound (compound d). In addition, neural stem cell-specific cell death induction was also observed for neoglycolipids (compound b) not having Lewis X-type sugar chain and neoglycolipids (compound a) having a short spacer (without lactose structure) It was not done.
以上の通り、Lewis X型糖鎖にスペーサーを介してアシル鎖が連結された構造のネオ糖脂質が神経幹細胞特異的な細胞死（アポトーシス）を誘導することが明らかとなった。また、ラクトースを含むスペーサーが有効に機能することが示され、細胞死誘導効果においてスペーサーの長さも重要なファクターであることが示唆された。 As described above, it was revealed that neoglycolipids having a structure in which an acyl chain is linked to a Lewis X-type sugar chain via a spacer induce neural stem cell-specific cell death (apoptosis). Moreover, it was shown that the spacer containing lactose was shown to function effectively, and it was suggested that the length of the spacer is also an important factor in the cell death induction effect.
本発明のアポトーシス誘導剤は未分化細胞特異的にアポトーシスを誘導する。生体外（in vitro）で幹細胞（例えば神経幹細胞）を分化させると、通常、分化細胞と未分化細胞が入り混じったヘテロな細胞集団が形成される。本発明のアポトーシス誘導剤を利用すれば、分化が進んでいない細胞（未分化細胞）を選択的に除去することが可能となり、純度の高い分化細胞を得ることができる。また、例えば培地中への添加によってその効果を発揮させることができることから、簡便な操作による分化細胞の調製が可能となる。このように、本発明は、再生医療用の細胞集団に混在する未分化細胞（残存未分化細胞）を除去する手段として極めて有用である。また、細胞移植後に分化することなく残存した未分化細胞の除去に本発明を利用することも可能である。更には、脳腫瘍の治療剤として本発明を利用することも期待される。 The apoptosis inducer of the present invention induces apoptosis specifically in undifferentiated cells. Differentiation of stem cells (eg, neural stem cells) in vitro generally results in the formation of a heterogeneous cell population in which differentiated cells and undifferentiated cells are mixed. By using the apoptosis inducer of the present invention, it is possible to selectively remove undifferentiated cells (undifferentiated cells), and highly pure differentiated cells can be obtained. In addition, for example, since the effect can be exhibited by addition to the medium, it is possible to prepare differentiated cells by a simple operation. Thus, the present invention is extremely useful as a means for removing undifferentiated cells (remaining undifferentiated cells) present in cell populations for regenerative medicine. Moreover, it is also possible to utilize the present invention for removing undifferentiated cells remaining without differentiation after cell transplantation. Furthermore, it is also expected to use the present invention as a therapeutic agent for brain tumors.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the above-mentioned invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive of the claims without departing from the scope of the claims. The contents of articles, published patent publications, patent publications, etc. specified in the present specification are incorporated by reference in their entirety.
Claims (8)
アシル鎖の炭素数が１６〜２０であり、
スペーサーが、Lewis X構造側に配置されるラクトースと、アシル鎖側に配置されるアミノエチル基が連結した構造からなる、未分化細胞のアポトーシス誘導剤。 Lewis X sugar chains Ri Do from neoglycolipid structure linked to acyl chains via spacer,
The carbon number of the acyl chain is 16 to 20,
An apoptosis inducer for undifferentiated cells, wherein the spacer comprises a structure in which lactose placed on the Lewis X structure side and aminoethyl group placed on the acyl chain side are linked .
(1)特定の細胞系譜に沿った分化を誘導する条件下で未分化細胞を培養するステップ；
(2)請求項１〜５のいずれか一項に記載のアポトーシス誘導剤の存在下で培養を継続するステップ。 A method of preparing differentiated cells comprising the following steps (1) and (2):
(1) culturing undifferentiated cells under conditions that induce differentiation along a specific cell lineage;
(2) continuing the culture in the presence of the apoptosis inducer according to any one of claims 1 to 5 ;
(i)未分化細胞と分化細胞が混在する試料を請求項１〜５のいずれか一項に記載のアポトーシス誘導剤の存在下で培養するステップ。 A method of preparing differentiated cells comprising the following step (i):
(i) culturing a sample in which undifferentiated cells and differentiated cells coexist in the presence of the apoptosis inducer according to any one of claims 1 to 5 .
(3)分化細胞を回収するステップ。 The preparation method according to claim 6 or 7 , further comprising the following step (3):
(3) Recovering differentiated cells.
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