JPH01102100A - Human interferon beta having sugar chain - Google Patents
Human interferon beta having sugar chainInfo
- Publication number
- JPH01102100A JPH01102100A JP62258574A JP25857487A JPH01102100A JP H01102100 A JPH01102100 A JP H01102100A JP 62258574 A JP62258574 A JP 62258574A JP 25857487 A JP25857487 A JP 25857487A JP H01102100 A JPH01102100 A JP H01102100A
- Authority
- JP
- Japan
- Prior art keywords
- sugar chain
- ifn
- human interferon
- interferon beta
- sugar
- 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.)
- Pending
Links
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- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 6
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims abstract description 6
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- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims abstract description 3
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims abstract description 3
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 claims abstract description 3
- SQVRNKJHWKZAKO-LUWBGTNYSA-N N-acetylneuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-LUWBGTNYSA-N 0.000 claims abstract description 3
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- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 claims abstract 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 2
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- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
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Abstract
Description
【発明の詳細な説明】
[産業上の利用分野]
本発明は、医薬あるいは診断薬として利用し得る生理活
性を有するヒト・インターフェロンβに関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to human interferon β which has physiological activity that can be used as a medicine or a diagnostic agent.
[従来の技術]
近年、生体由来の生理活性を有する糖タンパク質を医薬
あるいは診断薬として利用する試みがなされている。こ
の対象となる糖タンパク質を生産する手段としては該糖
タンパク質を有する組織からの抽出の他、該糖タンパク
質を産生ずる細胞の大量培養、あるいは該糖タンパク質
の遺伝子操作による組換え型タンパク質の大量生産など
が挙げられる。しかしながら、得られた糖タンパク質の
同一性として、アミノ酸組成分析に代表されるように、
そのポリペプチド部分によってその生理活性成分として
特徴づけることが一般に行われており、その生理活性発
現の重要要素としてその糖鎖構造を特徴づける報告は見
当たらない。しかし、最近、糖タンパク質の生理活性発
現上糖鎖が重要な役割を果たしていることが明らかにさ
れつつあり、たとえば、タンパク質の安定化、代謝にお
けるシグナル作用、細胞内局所のシグナル作用、および
レセプターや標的細胞に対する認識としてのシグナル作
用などである(たとえば、高崎誠−1細胞工学、Vol
、5. p427.198B、秀潤社)。このように、
糖タンパク質を医薬あるいは診断薬として有効かつ特異
的に利用するためには糖鎖構造を目的にそって規定され
る要件も重要となってくる。[Prior Art] In recent years, attempts have been made to utilize physiologically active glycoproteins derived from living organisms as medicines or diagnostic agents. Means for producing the target glycoprotein include extraction from tissues containing the glycoprotein, mass culturing of cells that produce the glycoprotein, or mass production of recombinant proteins through genetic manipulation of the glycoprotein. Examples include. However, the identity of the obtained glycoprotein, as typified by amino acid composition analysis,
Generally, bioactive components are characterized by their polypeptide portions, and no reports have been found that characterize their sugar chain structure as an important element in expressing their bioactivity. However, it has recently been revealed that sugar chains play an important role in the expression of biological activity of glycoproteins, such as protein stabilization, signal effects in metabolism, intracellular local signal effects, and receptor and These include signal effects as recognition for target cells (for example, Makoto Takasaki-1 Cell Engineering, Vol.
,5. p427.198B, Shujunsha). in this way,
In order to use glycoproteins effectively and specifically as medicines or diagnostic agents, it is also important to define the sugar chain structure according to the purpose.
すなわち、糖鎖構造を修飾することにより、生理活性糖
タンパク質の医薬あるいは診断薬利用の際に問題となる
、該糖タンパク質の血中安定性を高めることや、シグナ
ル的作用を強調して標的細胞あるいは標的臓器への取込
みを促進させること、また、本来付与された生理活性を
増大させることなどを解決し、さらには新たな生理活性
の付与なども期待することができる。In other words, by modifying the sugar chain structure, it is possible to increase the stability of the glycoprotein in the blood, which is a problem when using physiologically active glycoproteins as medicines or diagnostic agents, and to enhance the signal action of the glycoprotein to improve its ability to target cells. Alternatively, it is possible to solve problems such as promoting uptake into target organs, increasing the physiological activity originally imparted, and even imparting new physiological activities.
[発明が解決しようとする問題点]
上記問題点のうち、生理活性を有する糖タンパク質の血
中安定性を高めることは生体に投与した該糖タンパク質
の薬効を持続させることにつながり、治療効果を高める
ほか投与量を軽減させるなど医薬として用いた場合の安
定性にも寄与する。[Problems to be solved by the invention] Among the above problems, increasing the stability of physiologically active glycoproteins in the blood will lead to sustaining the medicinal efficacy of the glycoproteins administered to living organisms, and will improve the therapeutic effect. In addition to increasing the dosage, it also contributes to stability when used as a medicine, such as by reducing the dosage.
しかしながら、本効果はもとより、代謝や体内動態を指
標として、糖鎖の修飾程度を糖タンパク質の医薬あるい
は診断薬応用に関連させ、糖タンパク質の血中安定化の
問題の解決を試みた報告は未だ見当たらない。However, in addition to this effect, there are still no reports that attempt to solve the problem of blood stabilization of glycoproteins by relating the degree of modification of sugar chains to the application of glycoproteins to medicine or diagnostics, using metabolism and pharmacokinetics as indicators. Not found.
本発明は、医薬あるいは診断薬の可能性のある生理活性
を有する糖タンパク質の糖鎖を修飾することにより、上
記の問題点を解決することを目的とする。The present invention aims to solve the above-mentioned problems by modifying the sugar chains of a physiologically active glycoprotein that has the potential to be used as a drug or diagnostic agent.
[問題点を解決するための手段]
上記目的は以下の本発明により達成される。すなわち本
発明は、下記の構造を有する、糖鎖を全糖鎖中に20%
以上含むことを特徴とする糖鎖をもつヒト拳インターフ
ェロンβである。[Means for Solving the Problems] The above object is achieved by the present invention as described below. That is, the present invention contains sugar chains having the following structure in 20% of the total sugar chains.
This is human interferon β having a sugar chain characterized by containing the above.
(ここで、NeuAcはN−アセチルノイラミン酸、G
alはガラクトース、GlcN、AcはN−アセチルグ
ルコサミン、Manはマンノース、Fucはフコースで
あり、nは0または1を示す。)
本発明のヒト・インターフェロンβ(以下、■FN−β
を略す)とは、上記の構造を有する糖鎖(以下、本糖鎖
構造と略す)の数が、全糖鎖中に20%以上、好ましく
は25%以上有するIFN−βを意味する。(Here, NeuAc is N-acetylneuraminic acid, G
al represents galactose, GlcN, Ac represents N-acetylglucosamine, Man represents mannose, Fuc represents fucose, and n represents 0 or 1. ) Human interferon β of the present invention (hereinafter referred to as ■FN-β
) refers to IFN-β having the number of sugar chains having the above structure (hereinafter abbreviated as the present sugar chain structure) of 20% or more, preferably 25% or more of the total sugar chains.
本糖鎖構造においては、各単糖の結合炭素位置および各
単糖の立体配置は、化学的に本糖鎖構造を維持し得る状
態で存在すれば、限定されない。In this sugar chain structure, the bonding carbon position of each monosaccharide and the steric configuration of each monosaccharide are not limited as long as they exist in a state that can chemically maintain this sugar chain structure.
糖鎖の構造は、ヒドラジン分解法(続生化学実験講座第
4巻、142頁)により分析する。The structure of the sugar chain is analyzed by the hydrazine decomposition method (Zyosei Kagaku Jikken Koza Vol. 4, p. 142).
本発明のIFN−βを得る手段はいかなるものでも良い
が、たとえば本糖鎖構造を20%以上含む糖鎖を有する
IFN−βを産生ずる能力をもつ細胞により生産する方
法、あるいは本糖鎖構造を20%未満含む糖鎖を有する
IFN−βに化学合成的に本糖鎖構造の糖鎖を付与する
方法などが挙げられる。Any means for obtaining the IFN-β of the present invention may be used, such as a method of producing it using cells capable of producing IFN-β having a sugar chain containing 20% or more of the present sugar chain structure, or a method of producing the present sugar chain structure. Examples include a method of chemically synthesizing IFN-β having a sugar chain containing less than 20% of the present sugar chain structure.
細胞による生産では、該糖タンパク質を自然生産する真
該細胞、あるいはマイト−ジエン、発ガンプロモーター
、レクチン、リポ多糖鎖、カルシウムイオノフオアなど
の刺激によって該糖タンパク質を産生ずる真該細胞など
が用いられる。化学合成的に修飾する場合には、糖鎖の
非還元末端にさらに単糖あるいはオリゴ糖を還元的に結
合させることで行われる。For production by cells, cells that naturally produce the glycoprotein or cells that produce the glycoprotein by stimulation with mitogenes, oncogenic promoters, lectins, lipopolysaccharide chains, calcium ionophores, etc. are used. It will be done. When modifying by chemical synthesis, it is carried out by further reductively bonding a monosaccharide or oligosaccharide to the non-reducing end of the sugar chain.
また、遺伝子操作によって、適当な真該細胞に該糖タン
パク質の構造遺伝子に組込んで発現させることによって
も得られる。遺伝子操作による手法は、本発明の糖タン
パク質を製造する方法として好ましい手法である。具体
的には例えば、SV40ベクター/サルCO8細胞系、
DHFR(デヒドロ葉酸還元酵素)ベクター/ハムスタ
ーCHO細胞系、BPV (牛パピローマウィルス)ベ
クター/マウスC127細胞系、MMTV (マウス乳
ガンウィルス)ベクター/ヒトPCI2細胞系あるいは
MMTVベクター/ヒトPC8細胞系などの発現ベクタ
ー/宿主細胞系を用いて、遺伝子組換え型糖タンパク質
として得名ことができる。It can also be obtained by genetic manipulation by incorporating the structural gene of the glycoprotein into appropriate cells and expressing it. Genetic engineering is a preferred method for producing the glycoprotein of the present invention. Specifically, for example, SV40 vector/monkey CO8 cell line,
Expression of DHFR (dehydrofolate reductase) vector/hamster CHO cell line, BPV (bovine papilloma virus) vector/mouse C127 cell line, MMTV (mouse mammary tumor virus) vector/human PCI2 cell line or MMTV vector/human PC8 cell line, etc. Using a vector/host cell system, it can be produced as a recombinant glycoprotein.
本発明では宿主としてハムスターCHO細胞を用いるの
が特に好ましい。In the present invention, it is particularly preferable to use hamster CHO cells as the host.
本発明のIFN−βは、ヒト繊維芽細胞が産生ずる天然
型IFN−βが有効とされる疾病に対して利用すること
ができる。すなわち、悪性黒色腫、膠芽腫、およびB型
肝炎のうちHBe抗原陽性慢性活動性肝炎のウィルス血
症である。IFN−βは生体に投与されると血中および
肝、腎などに高度に移行後、次第に代謝されてその生物
学的活性を失うことが明らかにされている(たとえば、
佐藤雄一部、化学療法の領域、Vol、3. p71.
1987)。The IFN-β of the present invention can be used against diseases for which natural IFN-β produced by human fibroblasts is effective. That is, viremia of malignant melanoma, glioblastoma, and HBe antigen-positive chronic active hepatitis among hepatitis B. It has been revealed that when IFN-β is administered to a living body, it is highly transferred to the blood, liver, kidneys, etc., and then gradually metabolized and loses its biological activity (for example,
Yuichi Sato, Chemotherapy Area, Vol. 3. p71.
1987).
これは、生体内の種々のプロテアーゼによるIFN−β
タンパク質の分解と考えられるが、本発明のIFN−β
はいわゆる天然型と言われるヒト正常二倍体線維芽細胞
が産生するIFN−βの糖鎖よりも大きな糖鎖をもつの
で、生体内の種々のプロテアーゼの分解に対してより抵
抗性があり、少なくとも血中安定性に優れている。血中
安定性に優れていることは、生体内における薬効の持続
性を示す指標となり、従って本発明のIFN−βは、天
然型IFN−βが有効とされる種々の疾病に対して、天
然型IFN−βと同等の薬効を得るのにより少ない投与
量ですみ、副作用をより軽減させながら治療に用いるこ
とができる。This is caused by IFN-β caused by various proteases in the body.
Although it is considered that protein degradation occurs, the IFN-β of the present invention
Because it has a sugar chain larger than that of IFN-β produced by human normal diploid fibroblasts, which is the so-called natural type, it is more resistant to degradation by various proteases in the body. At least it has excellent blood stability. Excellent blood stability is an indicator of the persistence of drug efficacy in vivo. Therefore, the IFN-β of the present invention is effective against various diseases for which natural IFN-β is effective. It requires a smaller dose to obtain the same efficacy as type IFN-β, and can be used for treatment while reducing side effects.
本発明のIFN−βは、精製後、タンパク質製剤として
そのまま粉末として、また薬理学的に許容され得る担体
、賦形剤、希釈剤とともに医薬組成物(例、注射薬、錠
剤、カプセル剤、液剤、軟膏として、ヒトなどの温血動
物に対して非経口的あるいは経口的に安全に投与するこ
とができる。After purification, the IFN-β of the present invention can be used as a protein preparation as a powder or as a pharmaceutical composition (e.g., injection, tablet, capsule, liquid) together with a pharmacologically acceptable carrier, excipient, or diluent. It can be safely administered parenterally or orally to warm-blooded animals such as humans as an ointment.
このように、本発明のIFN−βは、天然型IFN−β
が有効である種々の疾病の治療領域に、新規で有用な製
剤として提供することができる。Thus, the IFN-β of the present invention is a natural IFN-β
It can be provided as a new and useful formulation for the treatment of various diseases for which it is effective.
[実 施 例] 以下実施例を挙げて本発明をさらに具体的に説明する。[Example] EXAMPLES The present invention will be explained in more detail with reference to Examples below.
実施例I
IFN−β1構造遺伝子をDHFRベクター(Cher
najovsky、 Y、 et al、、 DNA
3.297 (1984))に組込み、リン酸カルシウ
ム法でハムスターCHO細胞(前記と同誌)に感染させ
た。このCHO細胞を2.5%牛脂児血清を含むイーグ
ルMEM−アルファ・メディウムで培養すると、培養上
清中には37℃、3日間で、構成的にIFN−β1(C
HO−IFN−β1)が30.000単位/ml産生さ
れていた。この培養上清35fIを集め、20m1“ブ
ルーセファ−ロース” (ファルマシア社)にかけ、1
M塩化ナトリウムと30%エチレングリコール(E G
)を含むリン酸緩衝液(pH7゜4)200mlでカラ
ムを洗浄した後、1M塩化ナトリウムと60%EGを含
むリン酸緩衝液(pH7。Example I The IFN-β1 structural gene was transferred to the DHFR vector (Cher
najovsky, Y. et al., DNA
3.297 (1984)) and infected hamster CHO cells (same magazine as above) using the calcium phosphate method. When these CHO cells were cultured in Eagle MEM-Alpha medium containing 2.5% tallow serum, IFN-β1 (C
HO-IFN-β1) was produced at 30,000 units/ml. Collect 35 fI of this culture supernatant, apply 20 ml of "Blue Sepharose" (Pharmacia),
M Sodium chloride and 30% ethylene glycol (EG
) containing 200 ml of phosphate buffer (pH 7.4), and then washing the column with 200 ml of phosphate buffer (pH 7.4) containing 1M sodium chloride and 60% EG.
4)40mlでC)(O−I FN−β1を溶出した。4) C) (O-I FN-β1 was eluted with 40 ml.
次にCHO−I FN−β1画分を4ml抗ヒトIFN
−β1抗体(YSB−1)カラムにかけ、15mM塩酸
(pH2,0) 12mlで溶出した。さらに、この塩
酸溶出画分を“Co5a+osil 5C18−30
0カラム” (8X250mm、平井化学)にかけ、0
.1%トリフルオロ酢酸(pH2,0)存在下、0〜7
0%アセトニトリルの濃度匂配によりタンパク質を溶出
した。CHO−I FN−β1は約50%アセトニトリ
ル濃度で2.9mlで溶出された。Next, add 4 ml of CHO-I FN-β1 fraction to anti-human IFN.
-β1 antibody (YSB-1) column and eluted with 12 ml of 15 mM hydrochloric acid (pH 2,0). Furthermore, this hydrochloric acid elution fraction was converted into “Co5a+osil 5C18-30
0 column” (8X250mm, Hirai Chemical),
.. 0 to 7 in the presence of 1% trifluoroacetic acid (pH 2,0)
Proteins were eluted with a gradient of 0% acetonitrile. CHO-I FN-β1 was eluted in 2.9 ml at approximately 50% acetonitrile concentration.
精製されたCHO−IFN−β1は5DS−PAGEで
分子ff123,000のほぼ均一なバンドとして検出
された。精製されたCHO−I FN−β1のアミノ酸
組成、N末端およびC末端アミノ酸配列は、ヒト線維芽
細胞が産生ずる天然型IFN−β1のそれらと同一であ
ったことから、CHO−IFN−β1のポリペプチド部
分は天然型IFN−β1のそれと鴨召ある。また、CH
O−I FN−β1の糖鎖構造をヒドラジン分解法によ
り分析したところ、本糖鎖構造は天然型IFN−β1に
は12%含まれるのに対し、CHO−I FN−β1に
は28%含まれていた。このCHO−I FN−β1を
ウサギに静脈内投与して、薬効の持続性の目安となる血
中残存活性を調べたところ天然型IFN−β1の血中半
減期が11.9分であるのに対し、CHO−IFN−β
1の血中半減期は15.6分であり、天然型IFN−β
1のそれより30%延長した。Purified CHO-IFN-β1 was detected by 5DS-PAGE as a nearly uniform band of molecules ff123,000. The amino acid composition and N-terminal and C-terminal amino acid sequences of purified CHO-I FN-β1 were the same as those of natural IFN-β1 produced by human fibroblasts. The polypeptide portion is similar to that of natural IFN-β1. Also, CH
Analysis of the sugar chain structure of O-I FN-β1 using the hydrazine degradation method revealed that naturally occurring IFN-β1 contains 12% of this sugar chain structure, while CHO-I FN-β1 contains 28%. It was. This CHO-I FN-β1 was administered intravenously to rabbits, and the residual activity in the blood, which is a measure of the durability of the drug, was investigated. Whereas, CHO-IFN-β
The blood half-life of 1 is 15.6 minutes, and the natural IFN-β
30% longer than that of 1.
[発明の効果]
本発明のIFN−βは血中安定性が高いため、薬効を持
続させることができ、投与量を軽減させることかできる
。従って、天然型IFN−βが有効である種々の疾病の
治療領域に、新規で有用な製剤として提供することがで
きる。[Effects of the Invention] Since the IFN-β of the present invention has high blood stability, the drug efficacy can be sustained and the dosage can be reduced. Therefore, it can be provided as a new and useful preparation for the treatment of various diseases for which natural IFN-β is effective.
Claims (1)
含むことを特徴とする糖鎖をもつヒト・インターフェロ
ンβ。 【遺伝子配列があります】 (ここで、NeuAcはN−アセチルノイラミン酸、G
alはガラクトース、GlcNAcはN−アセチルグル
コサミン、Manはマンノース、Fucはフコースであ
り、nは0または1を示す。)(1) Human interferon β having a sugar chain characterized by containing 20% or more of sugar chains having the following structure in all sugar chains. [There is a gene sequence] (Here, NeuAc is N-acetylneuraminic acid, G
al represents galactose, GlcNAc represents N-acetylglucosamine, Man represents mannose, Fuc represents fucose, and n represents 0 or 1. )
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62258574A JPH01102100A (en) | 1987-10-14 | 1987-10-14 | Human interferon beta having sugar chain |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62258574A JPH01102100A (en) | 1987-10-14 | 1987-10-14 | Human interferon beta having sugar chain |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH01102100A true JPH01102100A (en) | 1989-04-19 |
Family
ID=17322138
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP62258574A Pending JPH01102100A (en) | 1987-10-14 | 1987-10-14 | Human interferon beta having sugar chain |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH01102100A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004087753A1 (en) * | 2003-03-31 | 2004-10-14 | Samsung Fine Chemicals Co., Ltd. | Mutein of human interferon-beta and its preparation method |
US9300909B2 (en) | 2010-06-07 | 2016-03-29 | Joled Inc. | Image display apparatus, electronic apparatus, image display system, image acquisition method and program |
-
1987
- 1987-10-14 JP JP62258574A patent/JPH01102100A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004087753A1 (en) * | 2003-03-31 | 2004-10-14 | Samsung Fine Chemicals Co., Ltd. | Mutein of human interferon-beta and its preparation method |
US9300909B2 (en) | 2010-06-07 | 2016-03-29 | Joled Inc. | Image display apparatus, electronic apparatus, image display system, image acquisition method and program |
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