JP2015017112A - Substance-delivering carrier for productive cells of fucosylated sugar chain - Google Patents
Substance-delivering carrier for productive cells of fucosylated sugar chain Download PDFInfo
- Publication number
- JP2015017112A JP2015017112A JP2014184578A JP2014184578A JP2015017112A JP 2015017112 A JP2015017112 A JP 2015017112A JP 2014184578 A JP2014184578 A JP 2014184578A JP 2014184578 A JP2014184578 A JP 2014184578A JP 2015017112 A JP2015017112 A JP 2015017112A
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- JP
- Japan
- Prior art keywords
- sugar chain
- fucose
- cells
- fucosylated
- carrier
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Abstract
Description
本発明は、フコシル化糖鎖産生細胞を標的とする物質送達担体、ならびにこれを利用したフコシル化糖鎖産生細胞に関連する疾患の処置剤および処置法に関する。 The present invention relates to a substance delivery carrier targeting a fucosylated sugar chain-producing cell, and a therapeutic agent and a treatment method for a disease associated with the fucosylated sugar chain-producing cell using the same.
真核生物において、フコシル化された糖鎖が、血管新生、繁殖、細胞接着、炎症および腫瘍転移などの種々の生理的および病理学的過程に関与していることが知られている(非特許文献1参照)。また、CA19−9やSLXをはじめとする多くの糖タンパク質腫瘍マーカーが糖鎖のフコシル化により生じることも知られている(非特許文献2参照)。このように、フコシル化糖鎖は生体において重要な意味を有しているため、フコシル化糖鎖を産生する細胞に特異的に薬剤などの物質を送達することができれば、上記のような種々の現象を制御することが可能となる。しかしながら、かかる試みが成功したという報告は未だない。
また、フコシル化はフコシルトランスフェラーゼ(FUT)という糖転移酵素によって触媒されていることから、フコシル化糖鎖産生細胞をフコシルトランスフェラーゼを標的分子として標的化することも考えられるが、同酵素は小胞体からゴルジ装置にかけて局在する膜結合型タンパクであり、細胞表面に存在しないため、これを直接的な標的分子とすることもできない。したがって、フコシル化糖鎖産生細胞特異的に薬剤などの物質を送達する技術はこれまで存在しなかった。
In eukaryotes, fucosylated sugar chains are known to be involved in various physiological and pathological processes such as angiogenesis, reproduction, cell adhesion, inflammation and tumor metastasis (non-patented) Reference 1). It is also known that many glycoprotein tumor markers including CA19-9 and SLX are produced by fucosylation of sugar chains (see Non-Patent Document 2). As described above, fucosylated sugar chains have important meanings in living organisms. Therefore, if a substance such as a drug can be delivered specifically to cells that produce fucosylated sugar chains, It becomes possible to control the phenomenon. However, there are no reports that such attempts have been successful.
In addition, since fucosylation is catalyzed by a glycosyltransferase called fucosyltransferase (FUT), it may be possible to target fucosylated glycan-producing cells with fucosyltransferase as a target molecule. Since it is a membrane-bound protein localized over the Golgi apparatus and does not exist on the cell surface, it cannot be used as a direct target molecule. Therefore, there has been no technology for delivering a substance such as a drug specifically for fucosylated sugar chain-producing cells.
本発明は、フコシル化糖鎖産生細胞特異的に薬物等の物質を送達できる担体、これを利用したフコシル化糖鎖産生細胞に関連する疾患の処置薬およびフコシル化糖鎖産生細胞に関連する疾患の処置法を提供することを目的とする。 The present invention relates to a carrier capable of delivering a substance such as a drug specifically to fucosylated sugar chain-producing cells, a therapeutic agent for a disease associated with fucosylated sugar chain-producing cells using the carrier, and a disease associated with fucosylated sugar chain-producing cells. The purpose is to provide a treatment method.
本発明者らは上記課題を解決すべく鋭意研究を続けたところ、フコシル化糖鎖産生細胞にフコースを特異的に結合する機構が存在することを見出した。そして、この知見に基づいてさらに研究を進めたところ、フコースを含む担体が、フコシル化糖鎖産生細胞への物質送達を特異的に促進することを見出し、本発明を完成させた。
フコシル化糖鎖産生細胞にフコースを特異的に結合する機構が存在することはこれまで全く知られていなかった。また、フコースを含む担体は知られていたが(特許文献1、2、非特許文献3参照)、これがフコシル化糖鎖産生細胞への物質送達を特異的に促進するすることはこれまで知られていなかった。
The inventors of the present invention have made extensive studies to solve the above problems, and have found that a mechanism for specifically binding fucose to fucosylated sugar chain-producing cells exists. As a result of further research based on this finding, it was found that a carrier containing fucose specifically promotes substance delivery to fucosylated sugar chain-producing cells, thereby completing the present invention.
Until now, it has not been known at all that a mechanism for specifically binding fucose to fucosylated sugar chain-producing cells exists. Moreover, although the carrier containing fucose has been known (see Patent Documents 1 and 2 and Non-Patent Document 3), it has been known so far that this specifically promotes substance delivery to fucosylated sugar chain-producing cells. It wasn't.
すなわち、本発明は、以下に関する。
(1)フコースを含む、フコシル化糖鎖産生細胞用物質送達担体。
(2)フコースがL−フコースである、上記(1)の担体。
(3)フコシル化糖鎖がI型糖鎖である、上記(1)または(2)の担体。
(4)フコシル化糖鎖産生細胞が、フコシルトランスフェラーゼを発現する、上記(1)〜(3)のいずれかの担体。
(5)フコシルトランスフェラーゼが、FUT1、FUT2、FUT3およびFUT4からなる群から選択される、上記(4)の担体。
(6)高分子ミセル、リポソーム、エマルジョン、微小球およびナノ小球から選択される形態を有する、上記(1)〜(5)のいずれかの担体。
(7)リポソームの形態を有し、フコースと、リポソームに含まれる脂質とのモル比が8:1〜1:8である、上記(6)の担体。
That is, the present invention relates to the following.
(1) A substance delivery carrier for fucosylated sugar chain-producing cells containing fucose.
(2) The carrier according to (1) above, wherein the fucose is L-fucose.
(3) The carrier according to (1) or (2) above, wherein the fucosylated sugar chain is a type I sugar chain.
(4) The carrier according to any one of (1) to (3) above, wherein the fucosylated sugar chain-producing cell expresses fucosyltransferase.
(5) The carrier according to (4) above, wherein the fucosyltransferase is selected from the group consisting of FUT1, FUT2, FUT3 and FUT4.
(6) The carrier according to any one of (1) to (5) above, which has a form selected from polymer micelles, liposomes, emulsions, microspheres and nanoglobules.
(7) The carrier according to (6) above, which has a liposome form and has a molar ratio of fucose to lipid contained in the liposome of 8: 1 to 1: 8.
(8)上記(1)〜(7)のいずれかの担体と、フコシル化糖鎖産生細胞に関連する疾患を処置するための薬物とを含む、フコシル化糖鎖産生細胞に関連する疾患を処置するための組成物。
(9)疾患が、腫瘍性疾患および炎症性疾患からなる群から選択される、上記(8)の組成物。
(10)フコシル化糖鎖産生細胞に関連する疾患を処置するための薬物が抗炎症剤および抗腫瘍剤からなる群から選択される、上記(9)の組成物。
(11)薬物と、担体とを、医療の現場またはその近傍で混合してなる、上記(8)〜(10)のいずれかの組成物。
(12)フコシル化糖鎖産生細胞に関連する疾患を処置するための薬物、フコース、ならびに、必要に応じてフコース以外の担体構成物質を、単独でまたは組み合わせて含む1つまたはそれ以上の容器を含む、上記(8)〜(11)のいずれかの組成物の調製キット。
(13)上記(1)〜(7)のいずれかの担体を利用した、in vitroでフコシル化糖鎖産生細胞へ物質を送達する方法。
(8) Treating a disease associated with fucosylated sugar chain-producing cells, comprising the carrier according to any one of (1) to (7) above and a drug for treating a disease associated with fucosylated sugar chain-producing cells. Composition to do.
(9) The composition according to (8), wherein the disease is selected from the group consisting of neoplastic diseases and inflammatory diseases.
(10) The composition according to (9), wherein the drug for treating a disease associated with fucosylated sugar chain-producing cells is selected from the group consisting of an anti-inflammatory agent and an antitumor agent.
(11) The composition according to any one of (8) to (10) above, wherein a drug and a carrier are mixed at or near a medical site.
(12) One or more containers containing a drug for treating a disease associated with fucosylated sugar chain-producing cells, fucose, and, if necessary, a carrier constituent other than fucose alone or in combination A preparation kit for the composition according to any one of (8) to (11) above.
(13) A method for delivering a substance to a fucosylated sugar chain-producing cell in vitro using the carrier according to any one of (1) to (7) above.
本発明の担体は、フコシル化糖鎖産生細胞が有するフコース結合機構を特異的な標的とするものであり、所望の物質や物体、例えばフコシル化糖鎖産生細胞に関連する疾患を処置するための薬物等を同細胞に効率的に運搬することにより、最大の効果および最小の副作用において、所望の効果、例えばフコシル化糖鎖産生細胞の活性や増殖の抑制、フコシル化糖鎖産生細胞に関連する疾患の治癒、進行の抑制または発症や再発の予防などを可能にする。
また、本発明の担体は、フコシル化糖鎖産生細胞特異的に物質を送達することができるため、フコシル化糖鎖産生細胞特異的な標識や、遺伝子導入などに利用できる。
The carrier of the present invention specifically targets the fucose-binding mechanism of fucosylated glycan-producing cells, and is used to treat a desired substance or object, for example, a disease associated with fucosylated glycan-producing cells. By efficiently transporting drugs, etc. to the same cell, it is related to the desired effect, for example, suppression of the activity or proliferation of fucosylated glycan producing cells, fucosylated glycan producing cells, with maximum effects and minimum side effects. It makes it possible to cure the disease, suppress its progression or prevent its onset and recurrence.
In addition, since the carrier of the present invention can deliver a substance specific to fucosylated sugar chain-producing cells, it can be used for fucosylated sugar chain-producing cell-specific labels, gene transfer, and the like.
本発明において、フコシル化糖鎖産生細胞は、フコシル化糖鎖を産生するものであれば特に限定されず、フコシル化糖鎖を細胞表面または細胞内に含むものであっても、フコシル化糖鎖を細胞外に放出するものであってもよい。したがって、本発明におけるフコシル化糖鎖産生細胞としては、特に限定されずに、例えば、膵臓腫瘍、胆道系腫瘍、肝臓腫瘍、消化管腫瘍、脳腫瘍、肺腫瘍、骨軟部腫瘍、より具体的には、膵癌、胆道系癌、肝癌、胃癌、食道癌、結腸直腸癌、さらには、乳癌、肺癌、子宮内膜癌、前立腺癌などにおける細胞や、膵炎、肝硬変、肝炎などの炎症性疾患における炎症部位の細胞が挙げられる。炎症部位の細胞としては、限定されずに、例えば、炎症部位に本来存在する細胞であって、炎症の影響を受けているものが挙げられる。すなわち、炎症部位の細胞は、膵炎であれば炎症の影響を受けている膵臓の構成細胞(膵管細胞、外分泌細胞、内分泌細胞等)、肝炎であれば炎症の影響を受けている肝臓の構成細胞(肝細胞、胆管細胞、クッパー細胞、星細胞等)などを指す。炎症の影響としては、例えば、炎症性サイトカインへの暴露や、炎症性細胞との接触等が挙げられる。
本発明の一態様において、フコシル化糖鎖産生細胞は、好ましくは正常細胞以外の細胞である。かかる細胞としては、例えば、上記の腫瘍細胞や炎症部位の細胞が挙げられる。
In the present invention, the fucosylated sugar chain-producing cell is not particularly limited as long as it produces a fucosylated sugar chain. Even if it contains a fucosylated sugar chain on the cell surface or in the cell, the fucosylated sugar chain May be released extracellularly. Therefore, the fucosylated sugar chain-producing cells in the present invention are not particularly limited, and for example, pancreatic tumors, biliary tumors, liver tumors, gastrointestinal tumors, brain tumors, lung tumors, bone soft tissue tumors, and more specifically , Pancreatic cancer, biliary tract cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, cells in breast cancer, lung cancer, endometrial cancer, prostate cancer, etc. and inflammatory sites in inflammatory diseases such as pancreatitis, cirrhosis, hepatitis Cell. Examples of the cells at the inflammatory site include, but are not limited to, cells originally existing at the inflammatory site and affected by inflammation. That is, the cells at the inflammatory site are the constituent cells of the pancreas (pancreatic duct cells, exocrine cells, endocrine cells, etc.) that are affected by inflammation if pancreatitis, and the constituent cells of the liver that are affected by inflammation if hepatitis (Hepatocytes, bile duct cells, Kupffer cells, stellate cells, etc.) Examples of the influence of inflammation include exposure to inflammatory cytokines and contact with inflammatory cells.
In one embodiment of the present invention, the fucosylated sugar chain-producing cell is preferably a cell other than a normal cell. Examples of such cells include the above-described tumor cells and cells at the inflammatory site.
本発明において、フコシル化糖鎖は、糖鎖単体として産生されても、他の物質と結合した形で産生されてもよい。したがって、フコシル化糖鎖は、タンパク質に結合した糖タンパク質の形で産生されても、脂質に結合した糖脂質の形で産生されてもよい。また、本発明におけるフコシル化糖鎖は、フコースが含まれていればいずれの構造の糖鎖であってもよいが、フコースが非還元末端に含まれているものが好ましい。含まれるフコースはL−フコースまたはD−フコースであってもよいが、L−フコースが好ましい。また、フコシル化糖鎖は、I型糖鎖抗原(例えば、CA19−9、SPAN−1、DU−PAN−2、CA50、KMO−1等)を含んでも、II型糖鎖抗原(SLX、CSLEX等)を含んでも、母核糖鎖抗原(例えば、CA72−4、CA546、STN等)を含んでもよい。本発明の一態様では、I型糖鎖抗原を含む糖鎖が好ましい。また、フコシル化は、種々の結合様式、例えば、α1,2結合、α1,3結合またはα1,4結合でなされてもよい。このうち、本発明においては、α1,4結合が好ましい。本発明において特に好ましい糖鎖は、CA19−9、SPAN−1およびDU−PAN−2からなる群から選択される糖鎖抗原を含む。 In the present invention, the fucosylated sugar chain may be produced as a sugar chain alone or in a form bound to other substances. Therefore, fucosylated sugar chains may be produced in the form of glycoproteins bound to proteins or in the form of glycolipids bound to lipids. In addition, the fucosylated sugar chain in the present invention may be a sugar chain of any structure as long as fucose is contained, but preferably contains fucose at the non-reducing end. The fucose contained may be L-fucose or D-fucose, but L-fucose is preferred. Further, the fucosylated sugar chain may contain a type I sugar chain antigen (SLX, CSLEX) even if it contains a type I sugar chain antigen (for example, CA19-9, SPAN-1, DU-PAN-2, CA50, KMO-1, etc.). Etc.) or a mother nucleus sugar chain antigen (for example, CA72-4, CA546, STN, etc.). In one embodiment of the present invention, a sugar chain containing a type I sugar chain antigen is preferable. Fucosylation may also be accomplished in a variety of binding modes, such as α1,2 bonds, α1,3 bonds, or α1,4 bonds. Of these, α1,4 bonds are preferred in the present invention. Particularly preferred sugar chains in the present invention include a sugar chain antigen selected from the group consisting of CA19-9, SPAN-1 and DU-PAN-2.
本発明の一態様において、フコシル化糖鎖産生細胞は、正常細胞よりもフコシル化糖鎖の産生が増大している。ここで、正常細胞とは、例えば、対象となる細胞が腫瘍細胞であれば、腫瘍化していない同種の細胞を指し、対象となる細胞が炎症部位の細胞であれば、炎症が生じる前の、または、炎症が生じていない部分の同種の細胞を指す。フコシル化糖鎖の産生量は、限定されずに、例えば、上記の糖鎖抗原を認識する抗体やレクチンなどを用いて適宜測定することができる。本発明の一態様において、フコシル化糖鎖産生細胞のフコシル化糖鎖産生量は、正常細胞に比べ、2倍以上、好ましくは5倍以上、より好ましくは10倍以上、さらに好ましくは20倍以上、特に好ましくは50倍以上である。また、別の態様において、フコシル化糖鎖産生細胞のフコシル化糖鎖産生量は、細胞系MIAPaCa、PANC−1、KP4および/またはPK45Hより多く、PK59および/またはASPC1と同等かまたはこれより多い。 In one embodiment of the present invention, fucosylated sugar chain-producing cells have increased production of fucosylated sugar chains than normal cells. Here, the normal cell refers to, for example, the same type of cells that are not tumorigenic if the target cell is a tumor cell, and if the target cell is a cell at the site of inflammation, before inflammation occurs, Alternatively, it refers to the same type of cells that are not inflamed. The amount of fucosylated sugar chain produced is not limited, and can be appropriately measured using, for example, an antibody or lectin that recognizes the above-mentioned sugar chain antigen. In one embodiment of the present invention, the fucosylated sugar chain-producing amount of fucosylated sugar chain-producing cells is 2 times or more, preferably 5 times or more, more preferably 10 times or more, and even more preferably 20 times or more, compared to normal cells. Especially preferably, it is 50 times or more. In another embodiment, the fucosylated glycan producing cell produces more fucosylated glycan than cell lines MIAPaCa, PANC-1, KP4 and / or PK45H, and is equivalent to or more than PK59 and / or ASPC1. .
本発明の別の態様において、フコシル化糖鎖産生細胞は、フコース結合機構を有している。フコース結合機構は、細胞に備わった、フコースを選択的に結合し、かつ/または取り込む機構を指し、限定されずに、例えば、受容体や運搬体などの細胞要素を含む。フコース結合機構保有の有無は、例えば、放射性標識などにより検出可能に標識したフコースの、被験細胞への結合量や結合定数を調べることにより判定することができる(実施例3参照)。例えば、フコース結合機構を有する細胞は、後述の実施例3に記載の手法で測定した場合、結合定数Kdが25nM以上、好ましくは28nM以上、より好ましくは30nM以上、さらに好ましくは34nM以上であり、bmaxが5pmol/106細胞以上、好ましくは7.5pmol/106細胞以上、より好ましくは10pmol/106細胞以上、さらに好ましくは11pmol/106細胞以上である。 In another embodiment of the present invention, the fucosylated sugar chain-producing cell has a fucose binding mechanism. The fucose binding mechanism refers to a mechanism of cells that selectively binds and / or takes up fucose, and includes, but is not limited to, cellular elements such as receptors and carriers. Whether or not the fucose binding mechanism is possessed can be determined, for example, by examining the binding amount or binding constant of fucose labeled detectably with a radioactive label or the like to the test cell (see Example 3). For example, a cell having a fucose binding mechanism has a binding constant Kd of 25 nM or more, preferably 28 nM or more, more preferably 30 nM or more, and even more preferably 34 nM or more when measured by the method described in Example 3 below. bmax is 5 pmol/10 6 cells or more, preferably 7.5 pmol / 10 6 cells or more, more preferably 10 pmol/10 6 cells or more, more preferably 11 pmol/10 6 cells or more.
本発明の別の態様において、フコシル化糖鎖産生細胞には、フコシルトランスフェラーゼが発現している。フコシルトランスフェラーゼは、フコースをフコース供与体からフコースアクセプター(例えば、糖鎖等)に転移させられるものであれば特に限定されずに、例えば、既知のFUT1、FUT2、FUT3、FUT4、FUT5、FUT6およびFUT7を含む。本発明の一態様において、フコシルトランスフェラーゼは、FUT1、FUT2、FUT3およびFUT4からなる群から選択される。また、本発明の一態様において、フコシルトランスフェラーゼは、フコースをα1,4結合で転移できるものが好ましく、かかるフコシルトランスフェラーゼとしては、例えばFUT3等が挙げられる。 In another embodiment of the present invention, fucosyltransferase is expressed in fucosylated sugar chain-producing cells. The fucosyltransferase is not particularly limited as long as it can transfer fucose from a fucose donor to a fucose acceptor (for example, sugar chain and the like). For example, known FUT1, FUT2, FUT3, FUT4, FUT5, FUT6 and Includes FUT7. In one aspect of the invention, the fucosyltransferase is selected from the group consisting of FUT1, FUT2, FUT3 and FUT4. In one embodiment of the present invention, the fucosyltransferase is preferably a fucosyltransferase that can transfer fucose with an α1,4 bond. Examples of the fucosyltransferase include FUT3.
フコシルトランスフェラーゼは、遺伝子の転写から、タンパク質の成熟に至る一連のタンパク質発現過程の中で発現していればよく、その発現は、遺伝子および/またはタンパク質レベルで検出することができる。具体的には、遺伝子レベルでは、例えば、ノーザンブロッティング法、サザンブロッティング法、RNaseプロテクションアッセイ、RT−PCR、リアルタイムPCR等のPCR法、in situハイブリダイゼーション法、in vitro転写法等の任意の公知の遺伝子発現解析法により、また、タンパク質レベルでは、免疫沈降法、ウェスタンブロッティング法、EIA、ELISA、RIA、免疫組織化学法、免疫細胞化学法等の任意の公知のタンパク質検出法により検出することができる。本発明の一態様において、フコシル化糖鎖産生細胞のフコシルトランスフェラーゼ発現量は、正常細胞に比べ、2倍以上、好ましくは5倍以上、より好ましくは10倍以上、さらに好ましくは20倍以上、特に好ましくは50倍以上である。また、本発明の一態様において、フコシル化糖鎖産生細胞のフコシルトランスフェラーゼ発現量は、細胞系MIAPaCa、PANC−1、KP4および/またはPK45Hより多く、PK59および/またはASPC1と同等かまたはこれより多い。 The fucosyltransferase only needs to be expressed in a series of protein expression processes from gene transcription to protein maturation, and the expression can be detected at the gene and / or protein level. Specifically, at the gene level, any known method such as Northern blotting, Southern blotting, RNase protection assay, RT-PCR, real-time PCR, etc., in situ hybridization, in vitro transcription, etc. It can be detected by gene expression analysis methods and at the protein level by any known protein detection method such as immunoprecipitation, Western blotting, EIA, ELISA, RIA, immunohistochemistry, immunocytochemistry, etc. . In one embodiment of the present invention, the fucosyltransferase expression level of fucosylated glycan-producing cells is 2 times or more, preferably 5 times or more, more preferably 10 times or more, even more preferably 20 times or more, particularly compared to normal cells. Preferably it is 50 times or more. In one embodiment of the present invention, fucosyltransferase expression level of fucosylated sugar chain-producing cells is greater than cell lines MIAPaCa, PANC-1, KP4 and / or PK45H, and is equal to or greater than PK59 and / or ASPC1. .
本発明の別の側面は、フコースを含む、フコース結合機構保有細胞用物質送達担体に関する。この担体は、フコース結合機構をその標的とするものである。フコース結合機構に関する詳細は上記のとおりである。また、フコース結合機構は、フコシル化糖鎖の産生量およびフコシルトランスフェラーゼの発現量と関連しているため、これらをフコース結合機構保有の指標として用いることもできる。したがって、本発明の一態様において、フコース結合機構保有細胞は、フコシル化糖鎖を産生する。また、本発明の別の態様において、フコース結合機構保有細胞はフコシルトランスフェラーゼを発現する。フコシル化糖鎖の産生およびフコシルトランスフェラーゼの発現に関する詳細は上記のとおりである。 Another aspect of the present invention relates to a substance delivery carrier for cells having a fucose binding mechanism, which contains fucose. This carrier targets the fucose binding mechanism. Details regarding the fucose coupling mechanism are as described above. Moreover, since the fucose binding mechanism is related to the production amount of fucosylated sugar chains and the expression level of fucosyltransferase, these can also be used as an index of possessing the fucose binding mechanism. Therefore, in one embodiment of the present invention, the fucose-binding mechanism-carrying cell produces a fucosylated sugar chain. In another embodiment of the present invention, the fucose binding mechanism-carrying cell expresses fucosyltransferase. Details regarding production of fucosylated sugar chains and expression of fucosyltransferases are as described above.
本発明のさらなる側面は、フコースを含む、フコシルトランスフェラーゼ発現細胞用物質送達担体に関する。フコシルトランスフェラーゼの発現に関する詳細は上記のとおりである。フコシルトランスフェラーゼの発現はフコシル化糖鎖の産生およびフコース結合機構の存在と関連しているため、これらをフコシルトランスフェラーゼ発現の指標として用いることもできる。したがって、本発明の一態様において、フコシルトランスフェラーゼ発現細胞は、フコシル化糖鎖を産生する。また、本発明の別の態様において、フコシルトランスフェラーゼ発現細胞はフコース結合機構を有する。フコシル化糖鎖の産生およびフコース結合機構の存在に関する詳細は上記のとおりである。 A further aspect of the present invention relates to a substance delivery carrier for fucosyltransferase-expressing cells, including fucose. Details regarding the expression of fucosyltransferases are as described above. Since fucosyltransferase expression is associated with the production of fucosylated sugar chains and the presence of a fucose binding mechanism, these can also be used as indicators of fucosyltransferase expression. Therefore, in one embodiment of the present invention, the fucosyltransferase-expressing cell produces a fucosylated sugar chain. In another embodiment of the present invention, the fucosyltransferase-expressing cell has a fucose binding mechanism. Details regarding the production of fucosylated sugar chains and the presence of a fucose binding mechanism are as described above.
本発明の担体に含まれるフコースは、フコシル化糖鎖産生細胞および/またはフコース結合機構保有細胞および/またはフコシルトランスフェラーゼ発現細胞(以下、代表的にフコシル化糖鎖産生細胞のみを記載する)への物質送達を促進するものであれば特に限定されず、例えばL−フコース、D−フコース、L−フコースおよび/またはD−フコースを含む糖鎖、例えば、L−フコースおよび/またはD−フコースを側鎖に含む糖鎖やL−フコースおよび/またはD−フコースを非還元末端に含む糖鎖などを用いることができる。 The fucose contained in the carrier of the present invention is a fucosylated glycan-producing cell and / or a fucose-binding mechanism-carrying cell and / or a fucosyltransferase-expressing cell (hereinafter, only fucosylated glycan-producing cells are typically described). The substance is not particularly limited as long as it facilitates substance delivery, for example, a sugar chain containing L-fucose, D-fucose, L-fucose and / or D-fucose, for example, L-fucose and / or D-fucose A sugar chain containing a chain or a sugar chain containing L-fucose and / or D-fucose at the non-reducing end can be used.
本発明の担体は、これらのフコース自体で構成してもよいし、フコースを、これとは別の担体構成成分に結合または包含させることにより構成してもよい。したがって、本発明の担体は、フコース以外の担体構成成分を含んでいてもよい。かかる成分としては、特に限定されずに、医薬および薬学の分野で知られる任意のものを用いることができるが、フコースを包含し得るか、または、これと結合し得るものが好ましい。 The carrier of the present invention may be composed of these fucose itself, or may be composed by combining or including fucose with another carrier component. Therefore, the carrier of the present invention may contain carrier components other than fucose. The component is not particularly limited, and any component known in the fields of medicine and pharmacy can be used, but those that can include or be combined with fucose are preferable.
このような成分としては、脂質、例えば、グリセロリン脂質などのリン脂質、スフィンゴミエリンなどのスフィンゴ脂質、コレステロールなどのステロール、大豆油、ケシ油などの植物油、鉱油、卵黄レシチンなどのレシチン類、ポリエチレングリコール、PEG:ポリマー担体等が挙げられるが、これらに限定されない。このうち、リポソームを構成し得るもの、例えば、レシチンなどの天然リン脂質、ジミリストイルホスファチジルコリン(DMPC)、ジパルミトイルホスファチジルコリン(DPPC)、ジステアロイルホスファチジルコリン(DSPC)などの半合成リン脂質、ジオレイルホスファチジルエタノールアミン(DOPE)、ジラウロイルホスファチジルコリン(DLPC)、コレステロールなどが好ましい。 Examples of such components include lipids, phospholipids such as glycerophospholipids, sphingolipids such as sphingomyelin, sterols such as cholesterol, vegetable oils such as soybean oil and poppy oil, lecithins such as mineral oil and egg yolk lecithin, polyethylene glycol PEG: polymer carrier and the like, but not limited thereto. Among these, those that can constitute liposomes, for example, natural phospholipids such as lecithin, semisynthetic phospholipids such as dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), and dioleyl phosphatidylethanol Amine (DOPE), dilauroyl phosphatidylcholine (DLPC), cholesterol and the like are preferable.
特に好ましい成分としては、細網内皮系による捕捉を回避し得る成分、例えば、N−(α−トリメチルアンモニオアセチル)−ジドデシル−D−グルタメートクロリド(TMAG)、N,N’,N’’,N’’’−テトラメチル−N,N’,N’’,N’’’−テトラパルミチルスペルミン(TMTPS)、2,3−ジオレイルオキシ−N−[2(スペルミンカルボキサミド)エチル]−N,N−ジメチル−1−プロパンアミニウムトリフルオロアセテート(DOSPA)、N−[1−(2,3−ジオレイルオキシ)プロピル]−N,N,N−トリメチルアンモニウムクロリド(DOTMA)、ジオクタデシルジメチルアンモニウムクロリド(DODAC)、ジドデシルアンモニウムブロミド(DDAB)、1,2−ジオレイルオキシ−3−トリメチルアンモニオプロパン(DOTAP)、3β−[N−(N’,N’−ジメチルアミノエタン)カルバモイル]コレステロール(DC−Chol)、1,2−ジミリストイルオキシプロピル−3−ジメチルヒドロキシエチルアンモニウムブロミド(DMRIE)、O,O’−ジテトラデカノイル−N−(α−トリメチルアンモニオアセチル)ジエタノールアミンクロリド(DC−6−14)などのカチオン性脂質が挙げられる。 Particularly preferred components include components that can avoid capture by the reticuloendothelial system, such as N- (α-trimethylammonioacetyl) -didodecyl-D-glutamate chloride (TMAG), N, N ′, N ″, N ′ ″-tetramethyl-N, N ′, N ″, N ′ ″-tetrapalmitylspermine (TMTPS), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N , N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioctadecyldimethyl Ammonium chloride (DODAC), didodecyl ammonium bromide (DDAB), 1,2-dioleoyloxy-3- Limethylammoniopropane (DOTAP), 3β- [N- (N ′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium bromide And cationic lipids such as (DMRIE) and O, O′-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride (DC-6-14).
本発明の担体へのフコースの結合または包含は、化学的および/または物理的な方法によってフコースを担体の他の構成成分に結合させるかまたは包含させることによっても可能となる。または、本発明の担体へのフコースの結合または包含は、該担体の作製時に、フコースと、それ以外の担体構成成分とを混合することによっても可能となる。本発明の担体に結合させるかまたは包含させるフコースの量は、担体構成成分中の重量比で0.01%〜100%、好ましくは0.2%〜20%、さらに好ましくは1〜5%とすることが可能である。担体へのフコースの結合または包含は、該担体に薬物等を担持させる前に行ってもよいし、担体、フコースおよび薬物等を同時に混合することなどによって行ってもよいし、または、薬物等を既に担持した状態の担体と、フコースとを混合することなどによって行ってもよい。したがって、本発明はまた、既存の任意の薬物結合担体や薬物封入担体、例えば、DaunoXome(R)、Doxil、Caelyx(R)、Myocet(R)などのリポソーム製剤にフコースを結合させる工程を含む、フコシル化糖鎖産生細胞特異的製剤の製造方法にも関する。 Binding or inclusion of fucose to the carrier of the present invention is also possible by binding or including fucose to other components of the carrier by chemical and / or physical methods. Alternatively, fucose can be bound or included in the carrier of the present invention by mixing fucose and other carrier components at the time of producing the carrier. The amount of fucose bound to or included in the carrier of the present invention is 0.01% to 100%, preferably 0.2% to 20%, more preferably 1 to 5% by weight ratio in the carrier component. Is possible. The binding or inclusion of fucose to the carrier may be performed before loading the drug or the like on the carrier, may be performed by mixing the carrier, fucose and drug, etc. at the same time, or the drug or the like may be added. You may carry out by mixing the support | carrier of the already carry | supported state and fucose. Therefore, the present invention also includes a step of binding fucose to any existing drug-binding carrier or drug-encapsulating carrier, for example, a liposome preparation such as DaunoXome (R) , Doxil, Caelyx (R) , Myocet (R) , The present invention also relates to a method for producing a fucosylated sugar chain-producing cell-specific preparation.
本発明の担体の形態は、所望の物質や物体を、標的とする細胞に運搬できればいずれの形態でもよく、例えば、限定するものではないが、高分子ミセル、リポソーム、エマルジョン、微小球、ナノ小球、ポリマーマトリックスなどのうちいずれの形態をとることもできる。本発明においては、送達効率の高さ、送達できる物質の選択肢の広さや製剤の容易性等の観点から、これらのうちリポソームの形態が好ましく、中でもカチオン性脂質を含むカチオン性リポソームが特に好ましい。担体がリポソームの形態である場合、フコースとリポソーム構成脂質とのモル比は、好ましくは8:1〜1:8、より好ましくは4:1〜1:4、さらに好ましくは2:1〜1:2、特に1:1である。 The form of the carrier of the present invention may be any form as long as a desired substance or object can be delivered to a target cell, for example, but not limited to, polymer micelle, liposome, emulsion, microsphere, nano-small It can take any form of spheres, polymer matrices and the like. In the present invention, from the viewpoints of high delivery efficiency, wide choice of substances that can be delivered, ease of preparation, etc., among these, the form of liposomes is preferred, and among these, cationic liposomes containing cationic lipids are particularly preferred. When the carrier is in the form of liposomes, the molar ratio of fucose to liposome-constituting lipid is preferably 8: 1 to 1: 8, more preferably 4: 1 to 1: 4, and even more preferably 2: 1 to 1: 2, in particular 1: 1.
本発明の担体は、これに含まれるフコースが、標的化分子として機能する態様で存在していれば、運搬物を内部に含んでも、運搬物の外部に付着して存在しても、また、運搬物と混合されていてもよい。ここで、標的化分子として機能するとは、フコースを含む担体が、これを含まない担体よりも迅速かつ/または大量に、標的細胞に到達し、かつ/または取り込まれることを意味し、これは、例えば、標識を付した、または標識を含む担体を細胞培養物に添加し、所定時間後に標識の存在部位を分析することにより容易に確認することができる。構造的には、例えば、フコースが、遅くとも標的細胞に到達するまでに、担体を含む製剤の外部に少なくとも部分的に露出していれば、上記要件を充足し得る。 In the carrier of the present invention, if the fucose contained therein is present in a mode that functions as a targeting molecule, it may contain a transported material inside, or may be present attached to the outside of the transported material, It may be mixed with a transported item. Here, functioning as a targeting molecule means that a carrier comprising fucose reaches and / or is taken up by target cells more rapidly and / or in a larger amount than a carrier without it, For example, it can be easily confirmed by adding a carrier with a label or containing a label to the cell culture and analyzing the site where the label is present after a predetermined time. Structurally, for example, the above requirements can be satisfied if fucose is at least partially exposed to the outside of the preparation containing the carrier before reaching the target cell at the latest.
本担体が送達する物質や物体は特に制限されないが、投与部位から標的細胞が存在する病変部位へ、生物の体内を物理的に移動できるような大きさであることが好ましい。したがって、本発明の担体は、原子、分子、化合物、タンパク質、核酸等の物質はもとより、ベクター、ウイルス粒子、細胞、1以上の要素で構成された薬物放出システム、マイクロマシン等の物体をも運搬することができる。前記物質または物体は、好ましくは標的細胞および/またはその周囲に何らかの影響を与える性質を有し、例えば、標的細胞を標識するものや、標的細胞および/またはその周囲に存在する細胞の活性または増殖を制御する(例えば、これを増強または抑制する)ものを含む。 The substance or object to be delivered by the carrier is not particularly limited, but preferably has a size that can physically move within the organism from the administration site to the lesion site where the target cells are present. Therefore, the carrier of the present invention carries not only substances such as atoms, molecules, compounds, proteins, and nucleic acids but also objects such as vectors, virus particles, cells, drug release systems composed of one or more elements, and micromachines. be able to. The substance or object preferably has a property that has some influence on the target cell and / or its surroundings, for example, the labeling of the target cell or the activity or proliferation of the target cells and / or the cells present in the surroundings. That control (eg, enhance or suppress).
したがって、本発明の一態様においては、担体が送達する物は「フコシル化糖鎖産生細胞の活性または増殖を制御する薬物」である。ここで、フコシル化糖鎖産生細胞の活性とは、同細胞が示す分泌、取り込み、遊走等の種々の活性を指すが、例えば、腫瘍細胞においては、腫瘍の発症、進行、再発および/または転移、悪液質などの症状の発現や増悪化などに関与する活性を意味する。かかる活性としては、例えば、限定することなく、副甲状腺ホルモン関連タンパク質(PTHrP)や免疫抑制酸性タンパク(IAP)などの産生・分泌が挙げられる。 Therefore, in one embodiment of the present invention, the substance delivered by the carrier is a “drug that controls the activity or proliferation of fucosylated sugar chain-producing cells”. Here, the activity of fucosylated sugar chain-producing cells refers to various activities such as secretion, uptake, migration, etc. exhibited by the cells. For example, in tumor cells, tumor onset, progression, recurrence and / or metastasis It means the activity involved in the onset and exacerbation of symptoms such as cachexia. Examples of such activity include, but are not limited to, production / secretion of parathyroid hormone-related protein (PTHrP), immunosuppressive acidic protein (IAP), and the like.
したがって、フコシル化糖鎖産生細胞の活性または増殖を制御する薬物とは、フコシル化糖鎖産生細胞に関連する疾患の発症、進行および/または再発に関係するフコシル化糖鎖産生細胞の物理的、化学的および/または生理的な作用等を直接または間接に抑制する何れの薬物であってもよい。例えば、腫瘍細胞においては、かかる薬物は限定されずに、上記生理活性物質の活性もしくは産生を阻害する薬物、例えば、前記生理活性物質を中和する抗体および抗体断片、前記生理活性物質の発現を抑制する、siRNA、リボザイム、アンチセンス核酸(RNA、DNA、PNA、またはこれらの複合物を含む)などの物質、もしくはドミナントネガティブ変異体等のドミナントネガティブ効果を有する物質、またはこれらを発現するベクター、ナトリウムチャンネル阻害剤などの細胞活性化抑制剤、アルキル化剤(例えば、イホスファミド、ニムスチン、シクロホスファミド、ダカルバジン、メルファラン、ラニムスチン等)、抗腫瘍性抗生物質(例えば、イダルビシン、エピルビシン、ダウノルビシン、ドキソルビシン、ピラルビシン、ブレオマイシン、ペプロマイシン、ミトキサントロン、マイトマイシンC等)および代謝拮抗剤(例えば、ゲムシタビン、エノシタビン、シタラビン、テガフール・ウラシル、テガフール・ギメラシル・オテラシルカリウム配合剤、ドキシフルリジン、ヒドロキシカルバミド、フルオロウラシル、メトトレキサート、メルカプトプリン等)などの細胞増殖抑制剤、ならびにcompound 861、gliotoxinなどのアポトーシス誘導剤を包含する。また、本発明における「フコシル化糖鎖産生細胞の活性または増殖を制御する薬物」は、フコシル化糖鎖産生細胞に関連する疾患の発症、進行および/または再発の抑制に直接または間接に関係するフコシル化糖鎖産生細胞の物理的、化学的および/または生理的な作用等を直接または間接に促進する何れの薬物であってもよい。 Therefore, a drug that controls the activity or proliferation of fucosylated glycan-producing cells refers to the physical properties of fucosylated glycan-producing cells that are involved in the onset, progression, and / or recurrence of diseases associated with fucosylated glycan-producing cells. Any drug that directly or indirectly suppresses a chemical and / or physiological action or the like may be used. For example, in tumor cells, such drugs are not limited, and drugs that inhibit the activity or production of the physiologically active substance, for example, antibodies and antibody fragments that neutralize the physiologically active substance, and expression of the physiologically active substance. An inhibitory substance such as siRNA, ribozyme, antisense nucleic acid (including RNA, DNA, PNA, or a complex thereof), a substance having a dominant negative effect such as a dominant negative mutant, or a vector that expresses these substances, Cell activation inhibitors such as sodium channel inhibitors, alkylating agents (eg, ifosfamide, nimustine, cyclophosphamide, dacarbazine, melphalan, ranimustine, etc.), antitumor antibiotics (eg, idarubicin, epirubicin, daunorubicin, Doxorubicin, pirarubi , Bleomycin, pepromycin, mitoxantrone, mitomycin C, etc.) and antimetabolites (eg gemcitabine, enocitabine, cytarabine, tegafur uracil, tegafur gimeracil oteracil potassium combination drug, doxyfluridine, hydroxycarbamide, fluorouracil, methotrexate, Cell growth inhibitors such as mercaptopurine and the like, and apoptosis inducers such as compound 861 and gliotoxin. Further, the “drug that controls the activity or proliferation of fucosylated sugar chain-producing cells” in the present invention is directly or indirectly related to the suppression of the onset, progression and / or recurrence of diseases associated with fucosylated sugar chain-producing cells. Any drug that directly or indirectly promotes the physical, chemical and / or physiological effects of fucosylated sugar chain-producing cells may be used.
本発明の担体の送達物としてはまた、フコシル化糖鎖産生細胞に関連する疾患を処置する薬物が含まれる。フコシル化糖鎖産生細胞に関連する疾患は、フコシル化糖鎖産生細胞に起因する疾患のみならず、同細胞がその影響を受ける疾患をも包含し、限定されずに、膵臓腫瘍、胆道系腫瘍、肝臓腫瘍、消化管腫瘍、脳腫瘍、肺腫瘍、骨軟部腫瘍、より具体的には、例えば、膵癌、胆道系癌、肝癌、胃癌、食道癌、結腸直腸癌、さらには、乳癌、肺癌、子宮内膜癌、前立腺癌などの腫瘍性疾患や、膵炎、肝硬変、肝炎などの炎症性疾患を含む。 The delivery of the carrier of the present invention also includes a drug for treating a disease associated with fucosylated sugar chain-producing cells. Diseases related to fucosylated glycan producing cells include not only diseases caused by fucosylated glycan producing cells but also diseases in which the cells are affected, and are not limited to pancreatic tumors, biliary tumors Liver tumors, gastrointestinal tumors, brain tumors, lung tumors, bone and soft tissue tumors, more specifically, for example, pancreatic cancer, biliary tract cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, lung cancer, uterus Includes neoplastic diseases such as intimal cancer and prostate cancer, and inflammatory diseases such as pancreatitis, cirrhosis and hepatitis.
したがって、本発明の担体の送達物としては、腫瘍性疾患の発症、進行および/または再発を抑制する抗腫瘍剤、例えば、限定することなく、イホスファミド、塩酸ニムスチン、シクロホスファミド、ダカルバジン、メルファラン、ラニムスチン等のアルキル化剤、塩酸ゲムシタビン、エノシタビン、シタラビン・オクホスファート、シタラビン製剤、テガフール・ウラシル、テガフール・ギメラシル・オテラシルカリウム配合剤(例えば、TS-1)、ドキシフルリジン、ヒドロキシカルバミド、フルオロウラシル、メトトレキサート、メルカプトプリン等の代謝拮抗剤、塩酸イダルビシン、塩酸エピルビシン、塩酸ダウノルビシン、クエン酸ダウノルビシン、塩酸ドキソルビシン、塩酸ピラルビシン、塩酸ブレオマイシン、硫酸ペプロマイシン、塩酸ミトキサントロン、マイトマイシンC等の抗腫瘍性抗生物質、エトポシド、塩酸イリノテカン、酒石酸ビノレルビン、ドセタキセル水和物、パクリタキセル、硫酸ビンクリスチン、硫酸ビンデシン、硫酸ビンブラスチン等のアルカロイド、アナストロゾール、クエン酸タモキシフェン、クエン酸トレミフェン、ビカルタミド、フルタミド、リン酸エストラムスチン等のホルモン療法剤、カルボプラチン、シスプラチン、ネダプラチン等の白金錯体、サリドマイド、ネオバスタット、ベバシズマブ等の血管新生阻害剤、L−アスパラギナーゼなどを挙げることができる。 Accordingly, the delivery of the carrier of the present invention includes antitumor agents that suppress the onset, progression and / or recurrence of neoplastic diseases, such as, but not limited to, ifosfamide, nimustine hydrochloride, cyclophosphamide, dacarbazine, mel Alkylating agents such as faran and ranimustine, gemcitabine hydrochloride, enocitabine, cytarabine ocphosphate, cytarabine preparation, tegafur uracil, tegafur gimeracil oteracil potassium combination drug (eg TS-1), doxyfluridine, hydroxycarbamide, fluorouracil, methotrexate , Antimetabolites such as mercaptopurine, idarubicin hydrochloride, epirubicin hydrochloride, daunorubicin hydrochloride, daunorubicin citrate, doxorubicin hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, pepromycin sulfate , Antitumor antibiotics such as mitoxantrone hydrochloride, mitomycin C, etoposide, irinotecan hydrochloride, vinorelbine tartrate, docetaxel hydrate, paclitaxel, vincristine sulfate, vindesine sulfate, vinblastine sulfate, anastrozole, tamoxifen citrate , Hormone therapy agents such as toremifene citrate, bicalutamide, flutamide, estramustine phosphate, platinum complexes such as carboplatin, cisplatin, nedaplatin, angiogenesis inhibitors such as thalidomide, neobasstat, bevacizumab, and L-asparaginase Can do.
本発明の担体の送達物としてはさらにまた、限定されずに、炎症性疾患の発症、進行および/または再発を抑制する抗炎症剤、例えば、ステロイド系抗炎症剤(プレドニゾロン、ベクロメタゾン、ベタメタゾン、フルチカゾン、デキサメタゾン、ヒドロコルチゾン等)や非ステロイド系抗炎症剤(アセチルサリチル酸、ロキソプロフェン、アセトアミノフェン、ケトプロフェン、チアプロフェン酸、スプロフェン、トルメチン、カルプロフェン、ベノキサプロフェン、ピロキシカム、ベンジダミン、ナプロキセン、ジクロフェナク、イブプロフェン、ジフルニサール、アザプロパゾン等)、炎症性サイトカインの発現を抑制するsiRNA、アンチセンス核酸などの物質、および/または炎症性サイトカインの作用を抑制する薬物、例えば、炎症性サイトカインに対する抗体や、炎症性サイトカインの受容体拮抗剤などを挙げることができる。 The delivery of the carrier of the present invention is not limited to an anti-inflammatory agent that suppresses the onset, progression and / or recurrence of inflammatory diseases, such as steroidal anti-inflammatory agents (prednisolone, beclomethasone, betamethasone, fluticasone). , Dexamethasone, hydrocortisone, etc.) and non-steroidal anti-inflammatory drugs (acetylsalicylic acid, loxoprofen, acetaminophen, ketoprofen, thiaprofenic acid, suprofen, tolmethine, carprofen, beoxaprofen, piroxicam, benzidamine, naproxen, diclofenac, ibuprofen, diflunisal , Azapropazone etc.), siRNA that suppresses the expression of inflammatory cytokines, substances such as antisense nucleic acids, and / or drugs that suppress the action of inflammatory cytokines, for example, And antibodies against diseases cytokines, such as receptor antagonists of inflammatory cytokines and the like.
本発明の担体は、標的細胞内に送達物を送達することが好ましいが、状況によっては、標的細胞の周囲に送達物を送達することが好ましい場合もある。例えば、炎症性サイトカインの発現を抑制するsiRNA、アンチセンス核酸などの物質は、フコシル化糖鎖を産生しない炎症性サイトカイン産生細胞にも送達することができ、これにより膵炎や肝炎などの、フコシル化糖鎖産生細胞に関連する疾患をより有効に処置することができる。 The carrier of the present invention preferably delivers the delivery product into the target cells, but in some circumstances it may be preferred to deliver the delivery product around the target cells. For example, siRNA, antisense nucleic acid, and other substances that suppress the expression of inflammatory cytokines can be delivered to inflammatory cytokine-producing cells that do not produce fucosylated sugar chains, thereby causing fucosylation such as pancreatitis and hepatitis. Diseases related to sugar chain producing cells can be treated more effectively.
本発明の担体が送達する物質や物体は、標識されていてもいなくてもよい。標識化により、運搬の成否や、標的細胞の増減などをモニタリングすることが可能となり、特に試験・研究レベルにおいて有用である。標識は、当業者に公知な任意のもの、例えば、任意の放射性同位体、磁性体、標識化物質に結合する物質(例えば抗体)、蛍光物質、フルオロフォア、化学発光物質、核磁気共鳴する元素(水素、リン、ナトリウム、フッ素等)、および酵素などから選択することができる。
本発明において「フコシル化糖鎖産生細胞用」とは、フコシル化糖鎖産生細胞を標的として使用するのに適していることを意味し、これは例えば、フコシル化糖鎖産生細胞に、フコシル化糖鎖非産生細胞よりも迅速、高効率かつ/または大量に物質を送達できることを含む。例えば、本発明の担体は、フコシル化糖鎖産生細胞に、フコシル化糖鎖非産生細胞に比べ、1.1倍以上、1.2倍以上、1.3倍以上、1.5倍以上、2倍以上、さらには3倍以上の速度および/または効率で物質を送達することができる。
The substance or object delivered by the carrier of the present invention may or may not be labeled. Labeling makes it possible to monitor the success or failure of transportation and the increase / decrease of target cells, and is particularly useful at the test / research level. The label may be any known to those skilled in the art, for example, any radioisotope, magnetic substance, substance that binds to the labeling substance (for example, an antibody), fluorescent substance, fluorophore, chemiluminescent substance, nuclear magnetic resonance element (Hydrogen, phosphorus, sodium, fluorine, etc.) and enzymes can be selected.
In the present invention, “for fucosylated sugar chain-producing cells” means that it is suitable for use as a target for fucosylated sugar chain-producing cells. For example, fucosylated sugar chain-producing cells are fucosylated. Including the ability to deliver substances faster, more efficiently and / or in larger quantities than non-glycan producing cells. For example, the carrier of the present invention can be used in fucosylated sugar chain-producing cells, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, compared to fucosylated sugar chain non-producing cells, Substances can be delivered at rates and / or efficiencies that are more than 2 times, or even more than 3 times.
本発明はまた、前記担体と、前記フコシル化糖鎖産生細胞の活性または増殖を制御する薬物とを含む、フコシル化糖鎖産生細胞の活性または増殖を制御するための、またはフコシル化糖鎖産生細胞に関連する疾患を処置するための組成物、ならびに、前記担体の、これらの組成物の製造への使用に関する。 The present invention also includes the carrier and a drug that controls the activity or proliferation of the fucosylated glycan-producing cell, for controlling the activity or proliferation of a fucosylated glycan-producing cell, or fucosylated glycan production It relates to compositions for treating cell-related diseases, and to the use of said carriers for the production of these compositions.
本発明の組成物においては、担体に含まれるフコースが標的化分子として機能する態様で存在する限り、担体は、送達物をその内部に含んでも、送達物の外部に付着して存在しても、また、送達物と混合されていてもよい。したがって、投与経路や薬物放出様式などに応じて、上記組成物を、適切な材料、例えば、腸溶性のコーティングや、時限崩壊性の材料で被覆してもよく、また、適切な薬物放出システムに組み込んでもよい。 In the composition of the present invention, as long as the fucose contained in the carrier is present in a form that functions as a targeting molecule, the carrier may contain the delivery product inside or be attached to the outside of the delivery product. It may also be mixed with a delivery product. Thus, depending on the route of administration, mode of drug release, etc., the composition may be coated with a suitable material, such as an enteric coating or a time-disintegrating material, and a suitable drug release system. It may be incorporated.
本発明の組成物は、経口および非経口の両方を包含する種々の経路、例えば、限定することなく、経口、静脈内、筋肉内、皮下、局所、直腸、動脈内、門脈内、心室内、経粘膜、経皮、鼻内、腹腔内、肺内および子宮内等の経路で投与してもよく、各投与経路に適した剤形に製剤してもよい。かかる剤形および製剤方法は任意の公知のものを適宜採用することができる(例えば、標準薬剤学、渡辺喜照ら編、南江堂、2003年などを参照)。
例えば、経口投与に適した剤形としては、限定することなく、散剤、顆粒剤、錠剤、カプセル剤、液剤、懸濁剤、乳剤、ゲル剤、シロップ剤などが挙げられ、また非経口投与に適した剤形としては、溶液性注射剤、懸濁性注射剤、乳濁性注射剤、用時調製型注射剤などの注射剤が挙げられる。非経口投与用製剤は、水性または非水性の等張性無菌溶液または懸濁液の形態であることができる。
The compositions of the present invention may be used in a variety of routes including both oral and parenteral, such as, without limitation, oral, intravenous, intramuscular, subcutaneous, topical, rectal, intraarterial, intraportal, intraventricular. , Transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, and intrauterine routes, and may be formulated into dosage forms suitable for each route of administration. Any known dosage form and formulation method can be adopted as appropriate (see, for example, Standard Pharmaceutical Sciences, Yoshiaki Watanabe, Nankodo, 2003, etc.).
For example, dosage forms suitable for oral administration include, but are not limited to, powders, granules, tablets, capsules, solutions, suspensions, emulsions, gels, syrups, etc. Suitable dosage forms include injections such as solution injections, suspension injections, emulsion injections, and injections prepared at the time of use. Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions.
本発明の担体または組成物は、いずれの形態で供給されてもよいが、保存安定性の観点から、好ましくは用時調製可能な形態、例えば、医療の現場あるいはその近傍において、医師および/または薬剤師、看護士、もしくはその他のパラメディカルなどによって調製され得る形態で提供される。この場合、本発明の担体または組成物は、これらに必須の構成要素の少なくとも1つを含む1個または2個以上の容器として提供され、使用の前、例えば、24時間前以内、好ましくは3時間前以内、そしてより好ましくは使用の直前に調製される。調製に際しては、調製する場所において通常入手可能な試薬、溶媒、調剤器具などを適宜使用することができる。 The carrier or composition of the present invention may be supplied in any form, but from the viewpoint of storage stability, it is preferably in a form ready for use, for example, at or near the medical site and / or It is provided in a form that can be prepared by a pharmacist, nurse, or other paramedical. In this case, the carrier or composition of the present invention is provided as one or more containers comprising at least one of the essential components thereof, and is used before use, for example, within 24 hours, preferably 3 Prepared within an hour and more preferably immediately before use. In the preparation, reagents, solvents, dispensing devices and the like that are usually available at the place of preparation can be appropriately used.
したがって、本発明はまた、フコース、および/または送達物、および/またはフコース以外の担体構成物質を、単独でもしくは組み合わせて含む1個または2個以上の容器を含む担体もしくは組成物の調製キット、ならびに、そのようなキットの形で提供される担体または組成物の必要構成要素にも関する。本発明のキットは、上記のほか、本発明の担体および組成物の調製方法や投与方法などに関する説明書や、CD、DVD等の電子記録媒体などを含んでいてもよい。また、本発明のキットは、本発明の担体または組成物を完成するための構成要素の全てを含んでいてもよいが、必ずしも全ての構成要素を含んでいなくてもよい。したがって、本発明のキットは、医療現場や、実験施設などで通常入手可能な試薬や溶媒、例えば、無菌水や、生理食塩水、ブドウ糖溶液などを含んでいなくてもよい。 Accordingly, the present invention also provides a preparation kit for a carrier or composition comprising one or more containers containing fucose and / or a delivery product and / or a carrier constituent other than fucose, alone or in combination, It also relates to the necessary components of the carrier or composition provided in the form of such a kit. In addition to the above, the kit of the present invention may contain instructions on how to prepare and administer the carrier and composition of the present invention, and electronic recording media such as CD and DVD. Further, the kit of the present invention may contain all of the components for completing the carrier or composition of the present invention, but may not necessarily contain all of the components. Therefore, the kit of the present invention may not contain reagents and solvents that are usually available at medical sites, experimental facilities, etc., such as sterile water, physiological saline, and glucose solution.
本発明はさらに、前記組成物の有効量を、それを必要とする対象に投与することを含む、フコシル化糖鎖産生細胞の活性または増殖を制御するための、またはフコシル化糖鎖産生細胞に関連する疾患を処置するための方法に関する。ここで、有効量とは、例えば、後者については、当該疾患の発症や再発を抑制し、症状を軽減し、または進行を遅延もしくは停止する量であり、好ましくは、当該疾患の発症および再発を予防し、または当該疾患を治癒する量である。また、投与による利益を超える悪影響が生じない量が好ましい。かかる量は、培養細胞などを用いたin vitro試験や、マウス、ラット、イヌまたはブタなどのモデル動物における試験により適宜決定することができ、このような試験法は当業者によく知られている。また、担体に含まれるフコース、および本発明の方法に用いる薬物の用量は当業者に公知であるか、または、上記の試験等により適宜決定することができる。 The present invention further includes administering an effective amount of the above composition to a subject in need thereof for controlling the activity or proliferation of fucosylated glycan-producing cells, or for fucosylated glycan-producing cells. It relates to a method for treating related diseases. Here, the effective amount is, for example, the latter, an amount that suppresses the onset and recurrence of the disease, reduces the symptoms, or delays or stops the progression, and preferably suppresses the onset and recurrence of the disease. An amount to prevent or cure the disease. In addition, an amount that does not cause adverse effects exceeding the benefits of administration is preferred. Such an amount can be appropriately determined by an in vitro test using cultured cells or the like, or a test in a model animal such as a mouse, rat, dog or pig, and such a test method is well known to those skilled in the art. . The dose of fucose contained in the carrier and the drug used in the method of the present invention are known to those skilled in the art or can be appropriately determined by the above-described tests and the like.
本発明の方法において投与する組成物の具体的な用量は、処置を要する対象に関する種々の条件、例えば、症状の重篤度、対象の一般健康状態、年齢、体重、対象の性別、食事、投与の時期および頻度、併用している医薬、治療への反応性、および治療に対するコンプライアンスなどを考慮して決定され得る。
投与経路としては、経口および非経口の両方を包含する種々の経路、例えば、経口、静脈内、筋肉内、皮下、局所、直腸、動脈内、門脈内、心室内、経粘膜、経皮、鼻内、腹腔内、肺内および子宮内等の経路が含まれる。
投与頻度は、用いる組成物の性状や、上記のような対象の条件によって異なるが、例えば、1日多数回(すなわち1日2、3、4回または5回以上)、1日1回、数日毎(すなわち2、3、4、5、6、7日毎など)、1週間に数回(例えば、1週間に2、3、4回など)、1週間毎、数週間毎(すなわち2、3、4週間毎など)であってもよい。
The specific dose of the composition to be administered in the methods of the present invention can vary depending on various conditions related to the subject in need of treatment, such as severity of symptoms, general health of the subject, age, weight, subject sex, diet, administration It may be determined in consideration of the timing and frequency of the drug, the medicine used in combination, the response to the treatment, the compliance with the treatment, and the like.
Administration routes include various routes including both oral and parenteral, such as oral, intravenous, intramuscular, subcutaneous, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, Routes such as intranasal, intraperitoneal, intrapulmonary and intrauterine are included.
The frequency of administration varies depending on the properties of the composition used and the conditions of the subject as described above. For example, many times a day (that is, 2, 3, 4 or 5 times a day), once a day, several times Every day (ie every 2, 3, 4, 5, 6, 7 etc.) several times a week (eg 2, 3, 4 etc. per week) Every week, every few weeks (ie 2, 3 Every 4 weeks).
本発明の方法において、用語「対象」は、任意の生物個体を意味し、好ましくは動物、さらに好ましくは哺乳動物、さらに好ましくはヒトの個体である。本発明において、対象は健常であっても、何らかの疾患に罹患していてもよいものとするが、フコシル化糖鎖産生細胞に関連する疾患の処置が企図される場合には、典型的には当該疾患に罹患しているか、罹患するリスクを有する対象を意味する。
また、用語「処置」は、疾患の治癒、一時的寛解または予防などを目的とする医学的に許容される全ての種類の予防的および/または治療的介入を包含するものとする。例えば、「処置」の用語は、フコシル化糖鎖産生細胞に関連する疾患の進行の遅延または停止、病変の退縮または消失、当該疾患発症の予防または再発の防止などを含む、種々の目的の医学的に許容される介入を包含する。
In the method of the present invention, the term “subject” means any living individual, preferably an animal, more preferably a mammal, more preferably a human individual. In the present invention, the subject may be healthy or afflicted with some disease, but when treatment of a disease associated with fucosylated glycan producing cells is contemplated, typically By a subject suffering from or at risk of suffering from the disease.
The term “treatment” is also intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions intended to cure, temporarily ameliorate or prevent disease. For example, the term “treatment” includes various purposes of medicine, including delaying or stopping the progression of a disease associated with fucosylated glycan-producing cells, regression or disappearance of a lesion, prevention of the onset of the disease, or prevention of recurrence, etc. Including potentially acceptable interventions.
本発明はまた、上記担体を利用した、フコシル化糖鎖産生細胞への薬物送達方法に関する。この方法は、限定されずに、例えば、上記担体に送達物を担持させる工程と、送達物を担持した担体をフコシル化糖鎖産生細胞を含む生物や媒体、例えば培養培地などに投与または添加する工程とを含む。これらの工程は、公知の任意の方法や、本明細書中に記載された方法などに従って適宜達成することができる。上記送達方法はまた、別の送達方法、例えば、フコシル化糖鎖産生細胞が存在する臓器を標的とする他の送達方法などと組み合わせることもできる。また、上記方法は、in vitroでなされる態様も、体内のフコシル化糖鎖産生細胞を標的とする態様も含む。 The present invention also relates to a method for delivering a drug to fucosylated sugar chain-producing cells using the carrier. This method is not limited, for example, the step of carrying the delivery product on the carrier, and the carrier carrying the delivery product is administered or added to an organism or medium containing fucosylated sugar chain-producing cells, such as a culture medium. Process. These steps can be appropriately achieved according to any known method or the method described in the present specification. The above delivery method can also be combined with another delivery method, for example, another delivery method targeting an organ in which fucosylated sugar chain-producing cells are present. Moreover, the said method includes the aspect made in vitro, and the aspect which targets the fucosylated sugar chain production cell in a body.
実施例1 各種膵癌細胞培養上清中の腫瘍マーカー濃度の検討
膵癌細胞株PK59、ASPC−1、MIAPaCaおよびPANC−1はATCCより、KP4およびPK45Hは理研バイオリソースセンターよりそれぞれ購入した。各種膵癌細胞株の細胞5×106個を25cm2フラスコに播種し、無血清培地Opti−MEM(R)3mlで48時間培養した。培養上清中のフコシル化糖鎖抗原腫瘍マーカー、CA19−9、Span−1およびDupan−2の濃度はELISA法で検討した。結果を図2に示す。これに基づいて、PK59、ASPC−1をフコシル化糖鎖高産生株、MIAPaCa、PANC−1をフコシル化糖鎖低産生株とした。
Example 1 Examination of tumor marker concentrations in various pancreatic cancer cell culture supernatants Pancreatic cancer cell lines PK59, ASPC-1, MIAPaCa and PANC-1 were purchased from ATCC, and KP4 and PK45H were purchased from RIKEN BioResource Center, respectively. Cells 5 × 10 6 cells of various pancreatic cancer cell lines were seeded in 25 cm 2 flasks and cultured for 48 hours in serum-free medium Opti-MEM (R) 3ml. The concentrations of fucosylated sugar chain antigen tumor markers, CA19-9, Span-1 and Dupan-2 in the culture supernatant were examined by ELISA. The results are shown in FIG. Based on this, PK59 and ASPC-1 were used as high fucosylated sugar chain producing strains, and MIAPaCa and PANC-1 were used as low fucosylated sugar chain producing strains.
実施例2 各種膵癌細胞株におけるフコシルトランスフェラーゼの発現
PK59、ASPC−1、MIAPaCaおよびPANC−1の各細胞株につき、1×106個の細胞から全RNAを抽出し、RT−PCRを行った。各FUTのプライマーは非特許文献2に基づいて作製した。
実施例3 各種膵癌細胞におけるフコース結合機構の存在
フコシル化糖鎖高産生株ASPC−1と、フコシル化糖鎖低産生株PANC−1におけるフコース(特に記載がない限りL−フコースを指す)の結合を放射性標識フコースを用いて検討した。
12ウェルのカルチャープレートに1×105個の細胞を播種後、1晩培養し、0〜200nMの濃度にBSA−PBSで希釈した14C−フコース(比活性:55mCi/mmol)を細胞に添加し4℃で1時間培養した。冷BSA−PBSで洗浄後、1%TritonX100/PBS−0.25%トリプシンで細胞を可溶化し、細胞膜に結合した14C−フコースの放射活性を測定した(図3および4)。
また、別な実験では、12ウェルのカルチャープレートに2×105個の細胞を播種後、1晩培養し、10nmolの14C−フコース(比活性:55mCi/mmol)を、単独で、または、1μmolのフコースとともに添加し、4℃で、1、3または24時間培養した。各条件の細胞を冷BSA−PBSで洗浄後、1%TritonX100/PBS−0.25%トリプシンで可溶化し、14C−フコースの放射活性を測定した(図5)。
これらの結果より、フコースが、受容体様の機構を介して、ASPC−1およびPANC−1に結合し、その親和性がフコシル化糖鎖高産生株であるASPC−1においてより高いことが判明した。また、14C−フコースの結合が、非標識フコースにより阻害されたことから、この機構がフコース特異的なものであることも明らかとなった。これは、フコシル化糖鎖産生細胞、特にフコシル化糖鎖高産生細胞において、フコース特異的な受容体様の結合機構が存在することを示すものである。
Example 3 Presence of fucose binding mechanism in various pancreatic cancer cells Binding of fucose (refers to L-fucose unless otherwise specified) in fucosylated sugar chain high-producing strain ASPC-1 and fucosylated sugar chain low-producing strain PANC-1 Were examined using radiolabeled fucose.
After seeding 1 × 10 5 cells in a 12-well culture plate, the cells were cultured overnight, and 14 C-fucose (specific activity: 55 mCi / mmol) diluted with BSA-PBS to a concentration of 0 to 200 nM was added to the cells. And cultured at 4 ° C. for 1 hour. After washing with cold BSA-PBS, the cells were solubilized with 1% Triton X100 / PBS-0.25% trypsin, and the radioactivity of 14 C-fucose bound to the cell membrane was measured (FIGS. 3 and 4).
In another experiment, 2 × 10 5 cells were seeded in a 12-well culture plate and cultured overnight, and 10 nmol of 14 C-fucose (specific activity: 55 mCi / mmol) alone or It was added with 1 μmol of fucose and cultured at 4 ° C. for 1, 3 or 24 hours. Cells under each condition were washed with cold BSA-PBS, solubilized with 1% Triton X100 / PBS-0.25% trypsin, and the radioactivity of 14 C-fucose was measured (FIG. 5).
From these results, it was found that fucose binds to ASPC-1 and PANC-1 through a receptor-like mechanism, and its affinity is higher in ASPC-1, which is a fucosylated sugar chain high-producing strain. did. In addition, since the binding of 14 C-fucose was inhibited by unlabeled fucose, it became clear that this mechanism is fucose specific. This indicates that a fucose-specific receptor-like binding mechanism exists in fucosylated sugar chain-producing cells, particularly high fucosylated sugar chain-producing cells.
実施例4 フコシル化リポソームによるsiRNAの導入
リポソーム(Lipotrust、10nmol)とL−フコース(0〜20nmol)(SIGMA, MO, USA)を懸濁し、5分間室温で放置した後、マイクロパーティションシステム(Sartorion VIVASPIN 5000MWCO PES)により遊離フコースを除去した。次にFAM標識siRNA(random)(センス鎖:5'-CGAUUCGCUAGACCGGCUUCAUUGCAG-3'(配列番号17)、アンチセンス鎖:5'-GCAAUGAAGCCGGUCUAGCGAAUCGAU-3'(配列番号18))を加えインキュベーション後、チャンバースライドに播種したASPC−1細胞に添加し、1時間培養した。培養後、細胞をPBSで洗浄し4%パラホルムアルデヒドで固定後、PBSで洗浄し、DAPIで対比染色して蛍光顕微鏡下に観察した。その結果、リポソーム:フコースのモル比は1:1が最も高い導入効率であることが判明した(図6および7)。
Example 4 Introduction of siRNA by Fucosylated Liposomes Liposomes (Lipotrust, 10 nmol) and L-fucose (0-20 nmol) (SIGMA, MO, USA) were suspended and allowed to stand at room temperature for 5 minutes, followed by micropartition system (Sartorion VIVASPIN). Free fucose was removed by 5000 MWCO PES. Next, FAM-labeled siRNA (random) (sense strand: 5′-CGAUUCGCUAGACCGGCUUCAUUGCAG-3 ′ (SEQ ID NO: 17), antisense strand: 5′-GCAAUGAAGCCGGUCUAGCGAAUCGAU-3 ′ (SEQ ID NO: 18)) was added and incubated on the chamber slide. The cells were added to the seeded ASPC-1 cells and cultured for 1 hour. After culturing, the cells were washed with PBS, fixed with 4% paraformaldehyde, washed with PBS, counterstained with DAPI, and observed under a fluorescence microscope. As a result, it was found that 1: 1 was the highest introduction efficiency when the molar ratio of liposome: fucose was 1: 1 (FIGS. 6 and 7).
実施例5 フコシル化リポソームによる導入に対するフコースの影響
siRNAの導入が、フコシル化糖鎖産生細胞に存在するフコース特異的な受容体様の結合機構によるものであることを確認するため、過剰のフコース存在下におけるsiRNA導入効率を検討した。リポソーム(Lipotrust、10nmol)とフコース(10nmol)を懸濁し、5分間室温で放置した後、マイクロパーティションシステム(Sartorion VIVASPIN 5000MWCO PES)により遊離フコースを除去した。次に上記と同じFAM標識siRNA(random)を加えインキュベーション後、チャンバースライドに播種したASPC−1細胞に添加し、1時間培養した(Liposome+F)。また、リポソーム単独群(Liposome)、リポソーム添加前10分間、1μmol(×100)のフコースを加えプレインキュベーションした群(Liposome+F+CE)も同時に検討した。培養後、細胞をPBSで洗浄し、4%パラホルムアルデヒドで固定後、PBSで洗浄し、DAPIで対比染色して蛍光顕微鏡下に観察した。その結果、フコースの存在によりsiRNAの導入が顕著に抑制された(図8)。すなわち、過剰量のフコースによりsiRNAの導入が阻害されたことから、フコシル化リポソームによるsiRNAの導入は、フコース特異的な受容体様の結合機構を介していることが示唆された。
Example 5 Effect of fucose on introduction by fucosylated liposomes In order to confirm that the introduction of siRNA is due to a fucose-specific receptor-like binding mechanism present in fucosylated sugar chain-producing cells, the presence of excess fucose The siRNA introduction efficiency below was examined. Liposomes (Lipotrust, 10 nmol) and fucose (10 nmol) were suspended, allowed to stand at room temperature for 5 minutes, and then free fucose was removed by a micropartition system (Sartorion VIVASPIN 5000 MWCO PES). Next, the same FAM-labeled siRNA (random) as described above was added and incubated, then added to ASPC-1 cells seeded on the chamber slide, and cultured for 1 hour (Liposome + F). In addition, the liposome alone group (Liposome) and the group (Liposome + F + CE) in which 1 μmol (× 100) fucose was added and preincubated for 10 minutes before the addition of the liposomes were simultaneously examined. After culturing, the cells were washed with PBS, fixed with 4% paraformaldehyde, washed with PBS, counterstained with DAPI, and observed under a fluorescence microscope. As a result, siRNA introduction was remarkably suppressed by the presence of fucose (FIG. 8). That is, since the introduction of siRNA was inhibited by an excessive amount of fucose, it was suggested that the introduction of siRNA by fucosylated liposomes is via a fucose-specific receptor-like binding mechanism.
実施例6 各種膵癌細胞株におけるsiRNA導入効率の比較
フコシル化糖鎖高産生株と低産生株におけるフコシル化リポソームによるsiRNA導入効率の検討を行った。前述の方法に従い、フコシル化リポソームを作製し、高産生株(PK59、ASPC−1)と低産生株(MIAPaCa、PANC−1)におけるFAM−siRNAの導入を蛍光顕微鏡下で観察した。この結果、高産生株においては細胞内に多量のFAMの緑色が認められたのに対し、低産生株における細胞内のFAMの緑色は顕著に少なかった(図9および10)。これは、フコシル化リポソームが、フコシル化糖鎖の産生量依存的に細胞に物質を送達することを示すものである。
Example 6 Comparison of siRNA introduction efficiency in various pancreatic cancer cell lines The siRNA introduction efficiency of fucosylated sugar chain high-producing strains and low-producing strains using fucosylated liposomes was examined. In accordance with the above-mentioned method, fucosylated liposomes were prepared, and introduction of FAM-siRNA into high-producing strains (PK59, ASPC-1) and low-producing strains (MIAPaCa, PANC-1) was observed under a fluorescence microscope. As a result, in the high-producing strain, a large amount of FAM green was observed in the cells, whereas in the low-producing strain, the intracellular FAM green was significantly less (FIGS. 9 and 10). This indicates that fucosylated liposomes deliver substances to cells depending on the production amount of fucosylated sugar chains.
Claims (12)
フコシル化糖鎖産生量が正常細胞に比べ2倍以上である腫瘍細胞、および
5×106個の細胞を3mlの培地中で48時間培養した上清中のCA19−9、Span−1またはDupan−2の濃度が、それぞれ、150U/ml以上、94U/ml以上または80U/ml以上となる腫瘍細胞、
からなる群から選択される、前記担体。 A carrier for delivering a substance to a fucosylated glycan-producing cell containing fucose, wherein the fucosylated glycan-producing cell is
Tumor cells whose fucosylated sugar chain production is more than twice that of normal cells, and CA19-9, Span-1 or Dupane in the supernatant obtained by culturing 5 × 10 6 cells in 3 ml of medium for 48 hours -2 at a concentration of 150 U / ml or higher, 94 U / ml or higher, or 80 U / ml or higher,
The carrier selected from the group consisting of:
フコシル化糖鎖産生量が正常細胞に比べ2倍以上である腫瘍細胞、および
5×106個の細胞を3mlの培地中で48時間培養した上清中のCA19−9、Span−1またはDupan−2の濃度が、それぞれ、150U/ml以上、94U/ml以上または80U/ml以上となる腫瘍細胞、
からなる群から選択される、前記組成物。 A composition for treating a neoplastic disease associated with fucosylated sugar chain-producing cells, comprising the carrier according to any one of claims 1 to 7, and a drug for treating the neoplastic disease, Fucosylated sugar chain producing cells
Tumor cells whose fucosylated sugar chain production is more than twice that of normal cells, and CA19-9, Span-1 or Dupane in the supernatant obtained by culturing 5 × 10 6 cells in 3 ml of medium for 48 hours -2 at a concentration of 150 U / ml or higher, 94 U / ml or higher, or 80 U / ml or higher,
Said composition selected from the group consisting of:
フコシル化糖鎖産生量が正常細胞に比べ2倍以上である腫瘍細胞、および
5×106個の細胞を3mlの培地中で48時間培養した上清中のCA19−9、Span−1またはDupan−2の濃度が、それぞれ、150U/ml以上、94U/ml以上または80U/ml以上となる腫瘍細胞、
からなる群から選択される、前記方法。 A method for delivering a substance to a fucosylated sugar chain-producing cell in vitro using the carrier according to any one of claims 1 to 7, wherein the fucosylated sugar chain-producing cell comprises:
Tumor cells whose fucosylated sugar chain production is more than twice that of normal cells, and CA19-9, Span-1 or Dupane in the supernatant obtained by culturing 5 × 10 6 cells in 3 ml of medium for 48 hours -2 at a concentration of 150 U / ml or higher, 94 U / ml or higher, or 80 U / ml or higher,
Said method is selected from the group consisting of:
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