JPH04221397A - Water-soluble polypeptide derivative and its use - Google Patents
Water-soluble polypeptide derivative and its useInfo
- Publication number
- JPH04221397A JPH04221397A JP2404484A JP40448490A JPH04221397A JP H04221397 A JPH04221397 A JP H04221397A JP 2404484 A JP2404484 A JP 2404484A JP 40448490 A JP40448490 A JP 40448490A JP H04221397 A JPH04221397 A JP H04221397A
- Authority
- JP
- Japan
- Prior art keywords
- water
- soluble polypeptide
- polypeptide derivative
- gly
- arg
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 72
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 47
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 44
- 230000021164 cell adhesion Effects 0.000 claims abstract description 16
- 150000003839 salts Chemical class 0.000 claims abstract description 16
- 208000010110 spontaneous platelet aggregation Diseases 0.000 claims abstract description 15
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- 230000001070 adhesive effect Effects 0.000 claims abstract description 13
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 11
- 239000003112 inhibitor Substances 0.000 claims description 11
- 125000004432 carbon atom Chemical group C* 0.000 claims description 8
- 239000004480 active ingredient Substances 0.000 claims description 5
- 125000001424 substituent group Chemical group 0.000 claims description 5
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 4
- 125000003118 aryl group Chemical group 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 125000002947 alkylene group Chemical group 0.000 claims description 2
- 125000000539 amino acid group Chemical group 0.000 claims description 2
- 125000000732 arylene group Chemical group 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical group NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 abstract description 27
- 210000004102 animal cell Anatomy 0.000 abstract description 9
- 239000003795 chemical substances by application Substances 0.000 abstract description 5
- 229920000642 polymer Polymers 0.000 abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 39
- 235000001014 amino acid Nutrition 0.000 description 36
- 150000001413 amino acids Chemical class 0.000 description 36
- 230000015572 biosynthetic process Effects 0.000 description 30
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- 238000000034 method Methods 0.000 description 19
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical group NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 12
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 10
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- NNRFRJQMBSBXGO-CIUDSAMLSA-N (3s)-3-[[2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid Chemical group NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NNRFRJQMBSBXGO-CIUDSAMLSA-N 0.000 description 8
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 8
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- 229910052938 sodium sulfate Inorganic materials 0.000 description 7
- 235000011152 sodium sulphate Nutrition 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
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- SCCPDJAQCXWPTF-VKHMYHEASA-N Gly-Asp Chemical compound NCC(=O)N[C@H](C(O)=O)CC(O)=O SCCPDJAQCXWPTF-VKHMYHEASA-N 0.000 description 5
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- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
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- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 3
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- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
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- 150000007524 organic acids Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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- 229920005604 random copolymer Polymers 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
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- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- SRPWOOOHEPICQU-UHFFFAOYSA-N trimellitic anhydride Chemical compound OC(=O)C1=CC=C2C(=O)OC(=O)C2=C1 SRPWOOOHEPICQU-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
【発明の詳細な説明】
【０００１】
【産業上の利用分野】本発明は、Ａｒｇ−Ｇｌｙ−Ａｓ
ｐのトリペプチドを必須単位として有する水溶性ポリペ
プチド誘導体およびその塩、およびそれを有効成分とす
る動物細胞の接着阻害剤、血小板の凝集・粘着抑制剤に
関するものである。
【０００２】
【従来の技術】フィブロネクチンは細胞−細胞外基質の
接着に関与するタンパク質であり、血小板凝集やガン転
移にも関与していると考えられている。これらの相互作
用は一連の細胞表面のレセプターにより仲介され、フィ
ブロネクチンは分子量約２５万の巨大分子であるにもか
かわらず、これらのレセプターがそのＡｒｇ−Ｇｌｙ−
Ａｓｐ 配列を特異的に認識することが明らかにされ、
レセプターとの相互作用に重要なものであることが報告
されている（ネイチャー（Ｎａｔｕｒｅ）、第３０９
巻、３０頁、１９８４年）。以来、Ａｒｇ−Ｇｌｙ−Ａ
ｓｐ 配列を有するオリゴあるいはポリペプチドを用い
る研究が成されている。
【０００３】例えば、Ａｒｇ−Ｇｌｙ−Ａｓｐ 配列を
有する種々の鎖状および環状のオリゴペプチドを用いて
血小板凝集を阻害する方法（高分子学会予稿集（Ｐｏｌ
ｙｍｅｒ Ｐｒｅｐｒｉｎｔｓ， Ｊａｐａｎ）、第３
８巻、３１４９頁、１９８９年、特開平２−１７４７９
７号）、Ａｒｇ−Ｇｌｙ−Ａｓｐ 配列を有するペプチ
ドを細胞移動抑制剤として用いる方法（特開平２−４７
１６号）、Ａｒｇ−Ｇｌｙ−Ａｓｐ を固定化したＰＭ
ＭＡ膜を細胞接着膜として用いる方法（ 高分子学会予
稿集（ＰｏｌｙｍｅｒＰｒｅｐｒｉｎｔｓ，Ｊａｐａｎ
） 、第３７巻、７０５ 頁、１９８８年）が報告され
ている。さらに、ポリマーにＡｒｇ−Ｇｌｙ−Ａｓｐ
を必須構成単位とするペプチドを共有結合させ動物細胞
培養基体、生体複合人工臓器用基体として用いる方法（
特開平１ー３０９６８２号、特開平１ー３０５９６０号
）、Ａｒｇ−Ｇｌｙ−Ａｓｐ−Ｓｅｒ 配列を有するポ
リペプチドを体外血液用血小板保護剤として用いる方法
が開示されている（特開昭６４ー６２１７ 号）。また
、Ａｒｇ−Ｇｌｙ−Ａｓｐ 配列を有するオリゴペプチ
ドあるいはその繰り返し構造を有するポリペプチドを用
いて、ガン転移を抑制する方法が知られている（（Ｉｎ
ｔ．Ｊ．Ｂｉｏｌ．Ｍａｃｒｏｍｏｌ．） 、第１１巻
、２３ 頁、１９８９ 年、同誌、第１１巻、２２６頁
、１９８９ 年、（Ｊｐｎ．Ｊ．Ｃａｎｃｅｒ Ｒｅｓ
．） 第６０巻、７２２頁、１９８９ 年）。
【０００４】蛋白質のモデルとして各種の研究に用いら
れてきた、合成ポリペプチドは構成するα−アミノ酸の
種類により側鎖にカルボキシル基、アミノ基、水酸基等
を有しており、高分子反応により種々の生理活性物質を
担持することが可能である。また、官能基の変換も容易
に行なうことができ、例えばｐｏｌｙＬｙｓ、ｐｏｌｙ
Ｏｒｎ等の側鎖アミノ基は容易に二塩基酸およびその誘
導体好ましくは二塩基酸無水物、あるいは多塩基酸およ
びその誘導体好ましくは多塩基酸無水物によってカルボ
キシル化することが出来る。
【０００５】ポリペプチド主鎖にＡｒｇ−Ｇｌｙ−Ａｓ
ｐ を必須単位とする繰返し構造を有するポリペプチド
は知られているが、合成ポリペプチドの側鎖にＡｒｇ−
Ｇｌｙ−Ａｓｐ を必須単位とするオリゴペプチドある
いはその繰返し構造を有するオリゴペプチドを導入した
水溶性ポリペプチド誘導体は知られておらず、導入した
場合にはレセプターとの結合能の増強及び血液中での安
定化が期待できる。
【０００６】
【発明が解決しようとする課題】本発明の目的は、新規
な水溶性ポリペプチド誘導体及びその塩を有効成分とす
る動物細胞の接着阻害剤並びに血小板凝集・粘着抑制剤
を提供することである。
【０００７】
【課題を解決するための手段】本発明は、水溶性ポリペ
プチドの側鎖にアミド結合、エステル結合のいずれかを
介し、下記一般式（１）で表される接着性ペプチドを必
須単位として有する水溶性ポリペプチド誘導体およびそ
の塩を提供するものである。一般式（１）−［Ｒ１］−
［ＣＯ］−（［Ｘ］−Ａｒｇ −Ｇｌｙ −Ａｓｐ −
［Ｙ］）ｎ −［Ｚ］ −［Ｒ２］−【０００８】式中
、［ ］は存在するかあるいは存在しなくともよく、
存在する場合はＸ、ＹはＳｅｒ、Ｇｌｙ、Ｖａｌ、Ａｓ
ｎ、Ｐｒｏから選択されるアミノ酸残基又はペプチド残
基を示し、Ｚは−Ｏ−または−ＮＨ−を示す。Ｒ１ 、
Ｒ２ のいずれか一方は、水素原子または炭素数が１〜
９の直鎖もしくは分岐のアルキル基、または炭素数が６
〜９のアリ−ル基であり、置換基を有していても良い。
他方は、水素原子または炭素数が１〜９の直鎖もしくは
分岐のアルキレン基、または炭素数が６〜９のアリ−レ
ン基であり、置換基を有していても良い。
ｎは１ー５の整数を示す。置換基としては、カルボニル
基、カルボキシル基、アミノ基、ヒドロキシル基、スル
ホ基、ハロゲン原子、アリール基、ニトロ基、シアノ基
、不飽和基の２重結合、３重結合等があげられ、同一鎖
に２つ以上有していても良い。
【０００９】本発明はさらに、上記水溶性ポリペプチド
誘導体またはその塩を有効成分とする動物細胞の接着阻
害剤並びに血小板凝集・粘着抑制剤を提供するものであ
る。本発明は、合成ポリペプチドに、Ａｒｇ−Ｇｌｙ−
Ａｓｐを必須単位として有する接着性ペプチドを共有結
合してなる水溶性ポリペプチド誘導体である。水溶性ポ
リペプチド誘導体の分子量は好ましくは２０万以下、特
に３０００〜１０万の範囲で、室温で水溶性であること
が好ましい。
【００１０】側鎖アミノ基のカルボキシル化剤として、
無水コハク酸、無水マレイン酸、無水フタル酸、無水イ
タコン酸、無水シトラコン酸、ピロメリット酸無水物、
トリメリット酸無水物等が挙げられる。本発明に係わる
接着性ペプチドに用いられるアミノ酸はＬ体、Ｄ体どち
らでも良いが、好ましくはＬ体である。
【００１１】本発明に係わる合成ポリペプチドとしては
、Ｈｙｐ、Ｓｅｒ、Ｔｙｒ、Ａｓｐ、Ｇｌｕ、Ｌｙｓ、
Ｏｒｎのホモポリマーおよびこれらのアミノ酸のいずれ
か一つを必須単位とするランダムコポリマーであり、好
ましくはｐｏｌｙＡｓｐ、ｐｏｌｙＧｌｕ、ｐｏｌｙＬ
ｙｓ、ｐｏｌｙＯｒｎである。本発明の水溶性ポリペプ
チド誘導体の塩として例えば、塩酸塩、硫酸塩、硝酸塩
、リン酸塩、ホウ酸塩等の無機酸との塩や、酢酸塩、ト
リフルオロ酢酸塩、トリフルオロメタンスルホン酸塩、
乳酸塩、酒石酸塩等の有機酸との塩にしても良く、その
ような塩への変換は慣用手段で行なうことができる。
【００１２】ペプチド合成方法としては特に限定しない
が、液相法、固相法、および自動合成装置による合成方
法が挙げられる。これらの合成方法の詳細については、
生化学実験講座「タンパク質の化学ＩＶ」ｐ２０７−４
９５（日本生化学会編、東京化学同人）、「続生化学実
験講座タンパク質の化学（下）」（日本生化学会編、東
京化学同人）、泉屋ら編「ペプチド合成の基礎と実験」
（丸善）に記載されている。また、市販されている合成
ペプチドを利用することも可能である。ポリペプチドお
よびカルボキシル化ポリペプチドと接着性ペプチドの結
合には、臭化シアン、酸アジド、水溶性カルボジイミド
等を利用したアミド結合合成方法が挙げられる。
【００１３】本発明の水溶性ポリペプチド誘導体は、細
胞接着性蛋白質のコア配列Ａｒｇ−Ｇｌｙ−Ａｓｐを有
し、該コア配列を介して細胞接着性蛋白質と同様の機序
で細胞に接着する。そのため、細胞接着性蛋白のアゴニ
ストまたはアンタゴニストとして種々の生物活性を示し
、免疫調整作用、創傷治癒作用、毛細血管中で起こる癌
細胞による血小板凝集抑制作用、神経疾患治癒作用など
の広範な生物活性が認められている。従って、本発明の
水溶性ポリペプチド誘導体は、そのすくなくとも一種を
、場合により慣用の担体または医薬用助剤とともに、癌
転移抑制剤、創傷治癒剤、免疫調整剤、血小板凝集粘着
抑制剤として患者に投与することが可能である。特に、
動物細胞接着阻害剤または血小板凝集粘着抑制剤として
の使用が好ましい。その投与量は、０．２μｇ／ｋｇ〜
４００ｍｇ／ｋｇの範囲で、症状、年齢、体重等に基づ
いて決定される。本発明の水溶性ポリペプチド誘導体は
、ペプチド系医薬に一般に使用されている投与方法、即
ち非経口投与方法、例えば静脈内投与、筋肉内投与、皮
下投与等によって投与するのが好ましい。そのような注
射用製剤を製造する場合、本発明の水溶性ポリペプチド
誘導体を例えば、後記実施例で示すようにＰＢＳまたは
生理食塩水に溶解して、注射用製剤としてもよく、ある
いは０．１Ｎ程度の酢酸水等に溶解した後、凍結乾燥製
剤としても良い。この様な製剤には、グリシンやアルブ
ミン等の慣用の安定剤を添加しても良い。さらに、
本発明の水溶性ポリペプチド誘導体は、例えばリポソー
ム中に包容したマイクロカプセル剤とすれば、経口投与
することも可能であり、座剤、舌下錠、点鼻スプレー剤
等の形にすれば、消化菅以外の粘膜からも吸収させるこ
とも可能である。
【００１４】
【実施例】以下実施例により本発明を更に説明するが本
発明はこれに限定されるものではない。なお、以下にお
いてＲはアルギニル、Ｇはグリシル、Ｄはアスパルチル
、Ｓはセリル、Ｐはプロリルを表す。
【００１５】製造例１ 接着性ペプチドの固相法によ
る合成
Ｍｅｒｒｉｆｉｅｌｄ方式によるペプチド合成装置を用
いて合成を行なった。αアミノ基の保護にはＢｏｃ基を
用い、樹脂から切出した後分取用ＨＰＬＣ（高速液体ク
ロマトグラフィー）で精製し、単一ピークを示す接着性
合成ペプチドを得た。
【００１６】
表
１ 接着性合成ペプチド名称
構造式
略号 収率ペプチド−１ Ｈ−
Ａｒｇ−Ｇｌｙ−Ａｓｐ−ＯＨ
ＲＧＤ ３７％ペプチド−２
Ｈ−（Ａｒｇ−Ｇｌｙ−Ａｓｐ）２−ＯＨ
（ＲＧＤ）２ ２８％（配列番号１
）
ペプチド−３ Ｈ−（Ａｒｇ−Ｇｌｙ−Ａｓｐ）
３−ＯＨ （ＲＧＤ）３
１９％（配列番号２）
ペプチド−４ Ｈ−（Ａｒｇ−Ｇｌｙ−Ａｓｐ）
５−ＯＨ （ＲＧＤ）５
１１％（配列番号３）
ペプチド−５ Ｈ−（Ｇｌｙ−Ａｒｇ−Ｇｌｙ−
Ａｓｐ−Ｓｅｒ−Ｐｒｏ）−ＯＨ ＧＲＧＤＳＰ
２７％（配列番号４）
【００１７】製造例２
ペプチド−６ Ｈ−（ Ａｒｇ−Ｇｌｙ−Ａｓｐ−Ｓ
ｅｒ−Ｇｌｙ）−ＮＨ２ （配列番号５）
ペプチド−６を逐次延長法により液相法で合成した。
（１）Ｂｏｃ Ｓｅｒ（Ｂｚｌ）ＧｌｙＮＨ２ の合
成ＢｏｃＳｅｒ（Ｂｚｌ）５９ｇ（０．２ｍｏｌ）をＣ
Ｈ２ Ｃｌ２ ４００ｍｌに溶解しＤＣＣ ４１．
２ｇ（０．２ｍｏｌ）を氷冷下加えた。この溶液に、Ｇ
ｌｙＮＨ２ ・ＨＣｌ ２２．１ｇをＣＨ２ Ｃｌ２
４００ｍｌに溶かしてから、Ｎ−メチルモリホリ
ン２０．２ｇで氷冷下中和した溶液を添加した。氷冷下
３時間撹拌してから、さらに室温で終夜撹拌した。沈殿
物をろ別してから減圧濃縮し酢酸エチルに溶解した。Ｎ
ａＨＣＯ３ 水溶液、１Ｍクエン酸水溶液、ＮａＣｌ水
溶液の順に洗浄し、Ｎａ２ ＳＯ４ で乾燥してから減
圧乾固して白色粉末 ５８．３ｇ（収率８３％）を得
た。
【００１８】（２）ＢｏｃＡｓｐ（ＯＢｚｌ）Ｓｅｒ（
Ｂｚｌ）ＧｌｙＮＨ２ の合成
Ｂｏｃ Ｓｅｒ（Ｂｚｌ）ＧｌｙＮＨ２ ５６．
２ｇ（０．１６ｍｏｌ）にＴＦＡ／ＣＨ２ Ｃｌ２ ＝
１／１ ４００ｍｌを加え室温で１時間撹拌した後、
ＴＦＡとＣＨ２ Ｃｌ２ を減圧濃縮した。これを酢酸
エチルに溶解しＮａＨＣＯ３ 水溶液で中和した後Ｎａ
Ｃｌ水溶液で洗浄した。Ｎａ２ ＳＯ４ で乾燥してか
ら酢酸エチルを減圧留去した。この化合物と、ＢｏｃＡ
ｓｐ（ＯＢｚｌ）５１．７ｇ（０．１６ｍｏｌ）をＣＨ
２ Ｃｌ２ ８００ｍｌに溶解し、ＤＣＣ ３３
ｇ（０．１６ｍｏｌ）を氷冷下加え３時間撹拌してから
、さらに室温で終夜撹拌した。減圧下ＣＨ２ Ｃｌ２
を留去してから酢酸エチルに溶解した。ＮａＨＣＯ３
水溶液、１Ｍクエン酸水溶液、ＮａＣｌ水溶液の順に洗
浄し、Ｎａ２ ＳＯ４ で乾燥してから減圧乾固して白
色粉末７１．２ｇ（収率８０％）を得た。
【００１９】（３）ＢｏｃＧｌｙＡｓｐ（ＯＢｚｌ）Ｓ
ｅｒ（Ｂｚｌ）ＧｌｙＮＨ２ （配列番号６）の合成Ｂ
ｏｃＡｓｐ（ＯＢｚｌ）Ｓｅｒ（Ｂｚｌ）ＧｌｙＮＨ２
６６．７ｇ（０．１２ｍｏｌ）にＴＦＡ：ＣＨ２
Ｃｌ２ ＝１：１ ４００ｍｌを加えて室温で１時
間撹拌した後、ＴＦＡとＣＨ２ Ｃｌ２ を減圧濃縮し
た。これを酢酸エチルに溶解しＮａＨＣＯ３ 水溶液で
中和した後ＮａＣｌ水溶液で洗浄した。Ｎａ２ ＳＯ４
で乾燥してから酢酸エチルを減圧留去した。この化合
物とＢｏｃＧｌｙ ５１．７ｇ（０．１２ｍｏｌ）を
ＣＨ２ Ｃｌ２ ７００ｍｌに溶解し、ＤＣＣ２４
．７ｇ（０．１２ｍｏｌ）を氷冷下加え３時間撹拌して
から、さらに室温で終夜撹拌した。ＤＣｕｒｅａをろ別
してから減圧濃縮し酢酸エチルに溶解した。ＮａＨＣＯ
３ 水溶液、１Ｍクエン酸水溶液、ＮａＣｌ水溶液の順
に洗浄し、Ｎａ２ ＳＯ４ で乾燥してから減圧乾固し
て白色粉末 ６１．８ｇ（収率８４％）を得た。
【００２０】（４）ＢｏｃＡｒｇ（Ｍｔｓ）ＧｌｙＡｓ
ｐ（ＯＢｚｌ）Ｓｅｒ（Ｂｚｌ）ＧｌｙＮＨ２ の合成
ＢｏｃＧｌｙＡｓｐ（ＯＢｚｌ）Ｓｅｒ（Ｂｚｌ）Ｇｌ
ｙＮＨ２ ６１．３ｇ（０．１ｍｍｏｌ）にＴＦＡ
：ＣＨ２ Ｃｌ２ ＝１：１ ４００ｍｌを加えて室
温で１時間撹拌した後、ＴＦＡとＣＨ２ Ｃｌ２ を減
圧濃縮した。これを酢酸エチルに溶解しＮａＨＣＯ３
水溶液で中和した後ＮａＣｌ水溶液で洗浄した。Ｎａ２
ＳＯ４ で乾燥してから酢酸エチルを減圧留去した。
この化合物とＢｏｃＡｒｇ（Ｍｔｓ） ４５．６ｇ（
０．１ｍｏｌ）をＤＭＦ８００ｍｌに溶解し、ＤＣＣ
２２．５ｇ（０．１ｍｏｌ） ＨＯＢｔ １４ｇ
（０．１ｍｏｌ）を氷冷下加え３時間撹拌してから、さ
らに室温で終夜撹拌した。ＤＣｕｒｅａをろ別してから
減圧濃縮し酢酸エチルに溶解した。ＮａＨＣＯ３ 水溶
液、１Ｍクエン酸水溶液、ＮａＣｌ水溶液の順に洗浄し
、Ｎａ２ ＳＯ４ で乾燥してから減圧乾固して白色粉
末 ４２．８ｇ（収率４５％）を得た。
【００２１】（５）ペプチド−６ 保護体の脱保護Ｂ
ｏｃＡｒｇ（Ｍｔｓ）ＧｌｙＡｓｐ（ＯＢｚｌ）Ｓｅｒ
（Ｂｚｌ）ＧｌｙＮＨ２ ５ｇ（５．３ｍｍｏｌ）
のＴＦＡ溶液に、１Ｍ トリフルオロメタンスルホン
酸−チオアニソール−ｍー クレゾールのＴＦＡ溶液を
氷冷下加えて１時間反応させ、ペプチド側鎖および末端
の保護基の脱保護を行なった。反応液をエーテル中に投
入しオイル状の沈殿物を蒸留水に溶解し酢酸エチルで洗
浄した後、陰イオン交換樹脂カラム（アンバーライトＩ
ＲＡ−４００；Ｃｌ型）に通して塩酸塩とし凍結乾燥し
た。白色固体２．１７ｇ（収率８６％）が得られた。
マススペクトル Ｍ＋ ： ４０４【００２２】製
造例３ペプチド−７ ＢｏｃＡｒｇ（Ｍｔｓ）Ｇｌｙ
Ａｓｐ（ＯＢｚｌ）Ｇｌｙ（配列番号７）ペプチド−７
を逐次延長法により液相法にて合成した。
（１）ＢｏｃＧｌｙＯＮｂの合成
ＢｏｃＧｌｙ３５ｇ（０．２ｍｏｌ）、トリエチルアミ
ン２８ｍｌ（０．２ｍｏｌ）および臭化ｐー ニトロベ
ンジル４３．２ｇ（０．２ｍｏｌ）を４００ｍｌの酢酸
エチルに溶解し、５時間還流した後室温で一晩放置した
。
生成した塩をろ別しＮａＨＣＯ３ 水溶液および水で洗
浄した後、Ｎａ２ ＳＯ４ 上で乾燥、減圧濃縮し酢酸
エチル−ヘキサンより再結晶した。 ５２．７ｇ（収
率 ８５％）以下製造例２と同様な方法でペプチド鎖
を延長した。以下に、その概略を示す。
【００２３】
（２）ＢｏｃＡｓｐ（ＯＢｚｌ）ＧｌｙＯＮｂの合成
（１）の生成物 ：
４６．５ｇ（０．１５ｍｏｌ） ＴＦＡ／Ｃ
Ｈ２ Ｃｌ２ ：２００ｍｌ／２００ｍｌ
ＢｏｃＡｓｐ（ＯＢｚｌ） ：４８．５
ｇ（０．１５ｍｏｌ） ＣＨ２ Ｃｌ２
：７５０ｍｌ
ＤＣＣ ：３
０．９ｇ（０．１５ｍｏｌ）（２）の収量 ６４
．０ｇ（収率 ８０％）
【００２４】
（３）ＢｏｃＧｌｙＡｓｐ（ＯＢｚｌ）ＧｌｙＯＮｂの
合成 （２）の生成物
：６４．０ｇ（０．１２ｍｏｌ） ＴＦ
Ａ／ＣＨ２ Ｃｌ２ ：２００ｍｌ／２０
０ｍｌ ＢｏｃＧｌｙ
：２１ｇ（０．１２ｍｏｌ） ＣＨ
２ Ｃｌ２ ：７５０ｍ
ｌ ＤＣＣ
：２４．７ｇ（０．１２ｍｏｌ）（３）の収
量 ５８．８ｇ（収率 ８３％）
【００２５】
（４）ＢｏｃＡｒｇ（Ｍｔｓ）ＧｌｙＡｓｐ（ＯＢｚｌ
）ＧｌｙＯＮｂの合成 （３）の生成物
：５３．７ｇ（９１ｍｍｏｌ）
ＴＦＡ／ＣＨ２ Ｃｌ２ ：２
００ｍｌ／２００ｍｌ ＢｏｃＡｒｇ（
Ｍｔｓ） ：４１．５ｇ（９１ｍｍｏｌ）
ＤＭＦ ：
８００ｍｌ ＤＣＣ
：１８．７ｇ（９１ｍｍｏｌ）
ＨＯＢｔ
：１３．５ｇ（０．１ｍｏｌ）
（４）の収量 ４６．５ｇ（収率 ５５％）【
００２６】（５）ＢｏｃＡｒｇ（Ｍｔｓ）ＧｌｙＡｓｐ
（ＯＢｚｌ）Ｇｌｙの合成（４）の生成物９．２９（１
０ｍｍｏｌ）を９０％酢酸３００ｍｌに溶かし、Ｚｎ末
３２．７ｇ（０．５ｍｏｌ）を加え、０度（摂氏）で３
時間撹拌した。Ｚｎ末をろ別し、ろ液を減圧濃縮、これ
にクエン酸を加えて酸性にし酢酸エチルで抽出した。Ｎ
ａ２ ＳＯ４ で乾燥し減圧濃縮してからエーテルを加
えて白色粉末６．２６ｇ（収率７９％）を得た。
【００２７】合成例１ ポリグルタミン酸−ＲＧＤＳ
の合成
ポリグルタミン酸ナトリウム塩（ペプチド研究所製；分
子量 ＞８０００）１．００ｇをｐＨ７．４リン酸バ
ッファー３５ｍｌに溶解し、０度に保ちながら水溶性Ｄ
ＣＣ〔１−エチル−３−３−（ジメチルアミノプロピル
）−カルボジイミド〕０．２６ｇの５．０ｍｌリン酸バ
ッファー溶液を加えて、１．５時間反応させた。ついで
、１０ｍｌのリン酸バッファーに溶解した接着性ペプチ
ド ＲＧＤＳ（国産化学工業製）０．７２ｇを添加し
４度で一晩反応させた。反応溶液を、スペクトラポア７
（分子分画量 ８０００）に入れイオン交換水、つい
で純水に対して透析し低分子量成分を除いて精製、凍結
乾燥した。収量１．０１ｇ 構造の確認は、ＩＲおよ
びアミノ酸分析により行なった。
以下の計算式に従いアルギニン残基濃度とグルタミン酸
残基濃度の比よりＲＧＤＳフラグメントの導入率を計算
し約１０％の値を得た。
導入率＝［Ａｒｇ］÷［Ｇｌｕ］×１００ＩＲ： ア
ミドカルボニル（Ｃ＝Ｏ）の伸縮振動 １６５２ｃｍ
−１
【００２８】合成例２ ポリアスパラギン酸−ＧＲＧ
ＤＳの合成
接着性ペプチドフラグメントＧＲＧＤＳ（国産化学工業
製）０．８１ｇとポリアスパラギン酸１．０２ｇ（シグ
マ社製）とを合成例１と同様にして共有結合させ、ポリ
アスパラギン酸−ＧＲＧＤＳを合成した。 収量１．
１３ｇ構造の確認は、ＩＲおよびアミノ酸分析により行
なった。アミノ酸分析の結果ＧＲＧＤＳフラグメントの
導入率は約１０％であった。
【００２９】合成例３ スクシニル化ポリリジン−Ｒ
ＧＤ
ポリリジン（和光純薬工業製）０．５２ｇを１％トリエ
チルアミン溶液１０ｍｌに溶解し、これに０．７５ｇの
無水コハク酸と０．１３ｇの４−ジメチルアミノピリジ
ンを加え室温で一昼夜かくはんした。反応終了後溶液を
大過剰のアセトンに投入してスクシニル化ポリリジンを
再沈殿させた。沈殿を集め更に大量のメタノールで洗浄
した後真空乾燥させた。 収量 ０．６１ｇ。
スクシニル化ポリリジン ０．７６ｇ、水溶性ＤＣＣ
０．２６ｇ、ＲＧＤ ０．５７ｇを用い、合成例
１と同様にしてＲＧＤフラグメントを共有結合させた。
収量 ０．８８ｇ構造の確認は、ＩＲおよびアミノ酸
分析により行なった。アミノ酸分析の結果ＲＧＤフラグ
メントの導入率は約１１％であった。導入率＝［Ａｒｇ
］÷［Ｌｙｓ］×１００
アミノ酸分析結果（ｎｍｏｌ／５０ μｌ ）Ａｒｇ
２．１０４４ Ｇｌｙ
１．９９８２ Ａｓｐ
１．９５４１ Ｌｙｓ ２１．
８４９３ ＩＲ： アミドカルボニル（Ｃ＝Ｏ）の伸
縮振動 １６４８ｃｍ−１
【００３０】合成例４ ポリグルタミン酸−（ＲＧＤ
）２
ポリグルタミン酸０．５０ｇ、水溶性ＤＣＣ ０．２
６ｇ、接着性ペプチドフラグメントとして（ＲＧＤ）２
０．５６ｇを用い、合成例１と同様にしてポリグ
ルタミン酸−（ＲＧＤ）２ を合成した。収量０．６１
ｇ。構造の確認は、ＩＲおよびアミノ酸分析により行な
った。アミノ酸分析の結果（ＲＧＤ）２ フラグメント
の導入率は約１２％であった。導入率＝［Ａｒｇ］÷２
÷［Ｇｌｕ］×１００
アミノ酸分析結果（ｎｍｏｌ／５０ μｌ ）Ａｒｇ
１．００７７ Ｇｌｙ
０．９９８１ Ａｓｐ
０．９８３３ Ｇｌｕ ４．
１９８８ ＩＲ： アミドカルボニル（Ｃ＝Ｏ）の伸
縮振動 １６５４ｃｍ−１
【００３１】合成例５ ポリアスパラギン酸−（ＲＧ
Ｄ）３
ポリアスパラギン酸０．２３ｇ、接着性ペプチドフラグ
メントとして（ＲＧＤ）３ ０．９１ｇ、水溶性Ｄ
ＣＣ ０．２６ｇを用い、合成例１と同様にしてポリ
アスパラギン酸−（ＲＧＤ）３ を合成した。収量０．
３３ｇ。
構造の確認は、ＩＲおよびアミノ酸分析により行なった
。アミノ酸分析の結果（ＲＧＤ）３ フラグメントの導
入率は約１０％であった。導入率＝［Ａｒｇ］÷３÷（
［Ａｓｐ］−［Ａｒｇ］）×１００
アミノ酸分析結果（ｎｍｏｌ／５０ μｌ ）Ａｒｇ
３．３４７５ Ｇｌｙ
３．２５６６ Ａｓｐ
１４．２０２８ ＩＲ： アミドカルボニル（Ｃ＝
Ｏ）の伸縮振動 １６４８ｃｍ−１
【００３２】合成例６ スクシニル化ポリオルニチン
−（ＲＧＤ）５
ポリオルニチン（シグマ社製）０．２３ｇを１％トリエ
チルアミン溶液１０ｍｌに溶解し、これに０．３０ｇの
無水コハク酸と０．０６ｇの４−ジメチルアミノピリジ
ンを加え室温で一昼夜かくはんした。反応終了後溶液を
大過剰のアセトンに投入してスクシニル化ポリオルニチ
ンを再沈殿させた。沈殿を集め更に大量のメタノールで
洗浄した後真空乾燥させた。 収量 ０．３７
ｇ。スクシニル化ポリオルニチン０．２１ｇ、接着性ペ
プチドフラグメントとして（ＲＧＤ）５ ０．９１ｇ、
水溶性ＤＣＣ０．１６ｇを用い、合成例１と同様にして
スクシニル化ポリオルニチン−（ＲＧＤ）５ を合成し
た。 収量０．２８ｇ。構造の確認は、ＩＲおよ
びアミノ酸分析により行なった。アミノ酸分析の結果（
ＲＧＤ）５ フラグメントの導入率は約１５％であった
。
導入率＝［Ａｒｇ］÷５÷［Ｏｒｎ］×１００アミノ酸
分析結果（ｎｍｏｌ／５０ μｌ ）Ａｒｇ
５．０１０８Ｇｌｙ ５
．０９９３Ａｓｐ ５．３４３２Ｏ
ｒｎ ６．６８１１ＩＲ： アミ
ドカルボニル（Ｃ＝Ｏ）の伸縮振動 １６５２ｃｍ−
１
【００３３】合成例７ ポリグルタミン酸−ＲＧＤＳ
Ｇー ＮＨ２
製造例２のオリゴペプチド ０．８１ｇを、合成例１
と同様にしてポリグルタミン酸に結合した。 収
量 １．０７ｇポリグルタミン酸−ＲＧＤＳＧー Ｎ
Ｈ２ の構造確認は、ＩＲおよびアミノ酸分析により行
なった。アミノ酸分析の結果ＲＧＤＳＧー ＮＨ２ フ
ラグメントの導入率は約１２％であった。導入率＝［Ａ
ｒｇ］÷［Ｇｌｕ］×１００
アミノ酸分析結果（ｎｍｏｌ／５０ μｌ ）Ａｒｇ
１．９７８０ Ｇｌｙ
１．９２５１ Ａｓｐ
１．６０５３ Ｓｅｒ １．
４９２１ Ｇｌｕ １６．４８３３
ＩＲ： アミドカルボニル（Ｃ＝Ｏ）の伸縮振動
１６５０ｃｍ−１
【００３４】合成例８ ＲＧＤＳＧ−ポリリジンの合
成製造例３のペプチド−７ ３．１８ｇ（４ｍｍｏｌ
）をＤＭＦ ４０ｍｌに溶解し、ＤＣＣ ０．８３
ｇ（４ｍｍｏｌ）、ＨＯＢｔ ０．５４ｇ（４ｍｍｏ
ｌ）を氷冷下加えて１時間撹拌した。これにＤＭＦ
２０ｍｌに溶解したポリリジン ０．５１ｇを加え３
時間撹拌した後室温でさらに終夜撹拌した。ＤＣｕｒｅ
ａをろ別してから減圧濃縮した。 以下製造例２と同
様にして脱保護を行なった。１Ｍ トリフルオロメタ
ンスルホン酸−チオアニソール−ｍー クレゾールのＴ
ＦＡ溶液： １５０ｍｌアンバーライトＩＲＡ−４０
０（Ｃｌ型）処理。収量 ０．６１ｇ。 構造確認
は、ＩＲおよびアミノ酸分析により行なった。アミノ酸
分析の結果ＲＧＤＳフラグメントの導入率は約１４％で
あった。
導入率＝［Ａｒｇ］÷［ＬｙＳ］×１００アミノ酸分析
（ｎｍｏｌ／５０μｌ）
Ａｒｇ ２．０４９５ Ｇｌｙ
２．０８７２ Ａｓｐ
１．９９９４ Ｓｅｒ
１．９７３６ Ｌｙｓ １４．
６３４７ ＩＲ： アミドカルボニル（Ｃ＝Ｏ）の伸
縮振動 １６５２ｃｍ−１
【００３５】合成例９ ＲＧＤＳＧ−ポリオルニチン
の合成製造例３のペプチド−７ ３．１８ｇ（４ｍｍ
ｏｌ）、ＤＣＣ ０．８３ｇ（４ｍｍｏｌ）、ＨＯＢ
ｔ ０．５４ｇ（４ｍｍｏｌ）およびポリオルニチン
０．４６ｇを合成例８と同様にしてペプチドの結合
および脱保護を行なった。収量 ０．５４ｇ。 構
造確認は、ＩＲおよびアミノ酸分析により行なった。ア
ミノ酸分析の結果ＲＧＤＳフラグメントの導入率は約１
５％であった。導入率＝［Ａｒｇ］÷［Ｏｒｎ］×１０
０アミノ酸分析（ｎｍｏｌ／５０μｌ）
Ａｒｇ ２．０５０２ Ｇｌｙ
２．１０１３ Ａｓｐ
１．８９９１ Ｓｅｒ
１．９５０６ Ｌｙｓ １３．
６６４２ ＩＲ： アミドカルボニル（Ｃ＝Ｏ）の伸
縮振動 １６４８ｃｍ−１
【００３６】合成例１０ ポリグルタミン酸−ＧＲＧ
ＤＳＰの合成製造例１で合成したペプチドフラグメント
−６（ＧＲＧＤＳＰ）０．５９ｇ、ポリグルタミン酸０
．３２ｇ（国産化学工業製）および水溶性ＤＣＣ ０
．１６ｇを用いて合成例１と同様にポリグルタミン酸−
ＧＲＧＤＳＰを合成した。 収量０．４０ｇ。 構
造の確認は、ＩＲおよびアミノ酸分析により行なった。
アミノ酸分析の結果ＧＲＧＤＳＰフラグメントの導入率
は約１１％であった。導入率＝１００×［Ａｒｇ］÷［
Ｇｌｕ］アミノ酸分析（ｎｍｏｌ／５０μｌ）
Ａｒｇ １．５１５２ Ｇｌｙ
３．１３４０ Ａｓｐ
１．４９１６ Ｓｅｒ
１．４８７５ Ｐｒｏ １．
４６０５ Ｇｌｕ １３．７８１４
ＩＲ： アミドカルボニル（Ｃ＝Ｏ）の伸縮振動
１６５４ｃｍ−１
【００３７】試験例１ 細胞接着性阻害活性の測定本
発明の水溶性ポリペプチド誘導体が細胞のフィブロンネ
クチンやビトロネクチンに対する接着を阻害する活性測
定方法を以下に示す。本実験例で用いられた競争法は基
本的に生化学分野では広く用いられているものであり例
えば「Ｍｅｔｈｏｄｓ ｉｎ Ｅｎｚｙｍｏｌｏｇｙ
」， ８２， ０３−８３１ （１９８１），特開平１
−３０９６８２、同２−１７４７９７に開示れている。
【００３８】実験方法
１．フィブロネクチンおよびビトロネクチン吸着プレー
トの作製
市販のフィブロネクチン（ヒト由来：生化学工業（株）
より購入）あるいはビトロネクチン（ヒト由来 フナ
コシ薬品（株）より購入）をＰＢＳで各々１．０μｌ／
ｍｌ、２．０μｌ／ｍｌに希釈しその希釈液０．５ｍｌ
を２４穴のプラスチックプレートに入れ３７度で一晩保
温しコーティングした。次に非特異的吸着を防ぐ目的で
牛血清アルブミン（ＢＳＡ １％）を加え３７度、１
時間保温し、その後通常の洗浄操作（ＰＢＳ）を加え充
分に水切りしてフィブロネクチン吸着プレートを作製し
た。
同様の操作によりビトロネクチン吸着プレートを作製し
た。
【００３９】２．接着阻害実験
凍結乾燥により得た水溶性ポリペプチド誘導体をＤｕｌ
ｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ ＥａｇｌｅｓＭｅ
ｄｉｕｍ （以下ＤＭＥＭと略記する）を用い、０、０
．２５、０．５、１．０、１．５ｍｇ／ｍｌの各濃度の
水溶性ポリペプチド誘導体溶液とした。
この溶液０．２５ｍｌを上記方法で作製したプレートに
入れ、そこへ血管内皮細胞（４×１０６ ｃｅｌｌｓ／
ｍｌ）の懸濁液を０．２５ｍｌ加え３７度で１時間保温
し細胞を接着させた。ＤＭＥＭ倍地で３回洗浄し、未接
着の細胞を剥離し、２％トリパンブルーで染色して細胞
数を計測した。結果を表２及び表３に示す。
【００４０】
表２ フィブロネクチンに対する細
胞接着阻害（ｃｅｌｌｓ／ｗｅｌｌ）
濃
度（ｍｇ／ｍｌ ）添加化合物 ０
０．２５ ０．５ １．０ １
．５合成物 １ ×
△ ○ ○
○合成物 ２ ×
△ ○ ○
○合成物 ３ ×
△ △ ○
○合成物 ４ ×
△ ○ ○
○合成物 ５ ×
△ ○ ○
○合成物 ６ ×
△ ○ ○
○合成物 ７ ×
△ ○ ○
○合成物 ８ ×
△ ○
○ ○合成物 ９ ×
△ ○
○ ○製造物 ２ ×
× △
△ △ＲＧＤ
× × ×
× △ＲＧＤＳ
× × ×
△ △（ｃｅｌｌｓ／ｗｅｌ
ｌ）；○：５０以下、△：５０〜１００、×：１００以
上【００４１】
表３ ビトロネクチンに対する細胞
接着阻害（ｃｅｌｌｓ／ｗｅｌｌ）
濃度（ｍｇ／ｍｌ ）添加化合物 ０
１０ ５０ １００
３００合成物 １ ×
○ ○ ○
○合成物 ２ ×
○ ○ ○
○合成物 ３ ×
△ △ ○
○合成物 ４ ×
○ ○
○ ○合成物 ５ ×
○ ○
○ ○合成物 ６ ×
○ ○
○ ○合成物 ７ ×
○ ○
○ ○合成物 ８
× ○ ○
○ ○合成物 ９
× ○ ○
○ ○製造物 ２
× × △
△ △ＲＧＤ
× × ×
△ △ＲＧＤＳ
× ×
△ △ ○（ｃｅｌｌｓ
／ｗｅｌｌ）；○：１００ 以下、△：１００ 〜２０
０ 、×：２００ 以上【００４２】試験例２ 血小
板凝集阻害活性の測定本発明において合成した水溶性ポ
リペプチド誘導体のｉｎ ｖｉｔｒｏ系での血小板凝
集阻害作用をヒト多血小板血漿を用いて検討した。以下
にその実験方法を示す。
【００４３】実験方法
新鮮なヒト血液に１／９量の３．８％クエン酸ナトリウ
ムを加え遠心分離（１０００ｒｐｍ、１０分）し、上層
を多血小板血漿として分取した。凍結乾燥より得た水溶
性ポリペプチド誘導体を０〜１．５ｍｇ／ｍｌの種々の
濃度となるように生理食塩水にに溶解した。水溶性ポリ
ペプチド誘導体溶液２５μｌを血漿２００μｌに加え３
７度で３分間でインキュベートしたのち、５０μＭ
アデノシン二リン酸（ＡＤＰ）溶液あるいは２００μｇ
／ｍｌのコラーゲン溶液を２５μｌ加え凝集の様子をア
グリゴメーターを用いて透過度を測定することにより検
定した。結果を表４に示す。
凝集阻害率（１−Ｔ／Ｔ０ ）× １００％Ｔ０ ＝
水溶性ポリペプチド誘導体の塩非添加時の透過度Ｔ
＝水溶性ポリペプチド誘導体の塩添加時の透過度【００
４４】
表４
血小板凝集阻害
阻害活性添加化合
物 ＡＤＰ刺激
コラーゲン刺激合成物 １
○
○合成物 ２
○
○合成物 ３
○
○合成物 ４ ○
○合成
物 ５ ○
○合成物 ６
○
○合成物 ７
○
○合成物 ８
○
○合成物 ９
○
○製造物 ２ △〜○
△〜○ＲＧＤ
△〜○
△〜○ＲＧＤＳ
△〜○
△〜○ＩＣ５０（μｇ／ｍｌ ）；○
：４０以下、△：８０〜４０、×：８０以上【００４５
】
【発明の効果】本発明の化合物は、ポリペプチドの側鎖
にＡｒｇ−Ｇｌｙ−Ａｓｐ を必須単位とするオリゴペ
プチドあるいはその繰返し構造を有するオリゴペプチド
を導入した水溶性ポリペプチド誘導体であり、これによ
り、動物細胞の接着阻害、血小板凝集・粘着抑制の効果
が得られるとともに、レセプターとの結合能の増強及び
血液中での安定化が期待できる。
【００４６】
【配列表】
【００４７】配列番号：１
配列の長さ：６
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列
【００４８】配列番号：２
配列の長さ：９
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列
【００４９】配列番号：３
配列の長さ：１５
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列
【００５０】配列番号：４
配列の長さ：６
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列
【００５１】配列番号：５
配列の長さ：５
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列
【００５２】配列番号：６
配列の長さ：４
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列
【００５３】配列番号：７
配列の長さ：４
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列Detailed Description of the Invention [0001] [Industrial Application Field] The present invention relates to Arg-Gly-As
The present invention relates to a water-soluble polypeptide derivative having a p tripeptide as an essential unit and a salt thereof, and an animal cell adhesion inhibitor and a platelet aggregation/adhesion inhibitor containing the same as an active ingredient. [0002] Fibronectin is a protein involved in cell-extracellular matrix adhesion, and is also thought to be involved in platelet aggregation and cancer metastasis. These interactions are mediated by a series of cell surface receptors, and although fibronectin is a large molecule with a molecular weight of approximately 250,000, these receptors
It was revealed that it specifically recognizes the Asp sequence,
It has been reported that it is important for interaction with receptors (Nature, No. 309).
Vol. 30, 1984). Since then, Arg-Gly-A
Studies have been conducted using oligos or polypeptides having sp sequences. For example, methods for inhibiting platelet aggregation using various linear and cyclic oligopeptides having the Arg-Gly-Asp sequence (Proceedings of the Society of Polymer Science and Technology (Pol.
ymer Preprints, Japan), No. 3
Volume 8, page 3149, 1989, JP-A-2-17479
No. 7), a method using a peptide having the Arg-Gly-Asp sequence as a cell migration inhibitor (Japanese Patent Application Laid-Open No. 2-47
No. 16), PM with immobilized Arg-Gly-Asp
Method of using MA membrane as a cell adhesion membrane (Proceedings of the Society of Polymer Science and Technology (PolymerPreprints, Japan)
), Vol. 37, p. 705, 1988). Furthermore, the polymer has Arg-Gly-Asp.
A method of covalently bonding peptides having as essential constituent units and using them as animal cell culture substrates and biocomposite artificial organ substrates (
JP-A No. 1-309682, JP-A No. 1-305960), and a method of using a polypeptide having an Arg-Gly-Asp-Ser sequence as a platelet protective agent for extracorporeal blood (JP-A No. 64-6217) ). Furthermore, a method for suppressing cancer metastasis using an oligopeptide having an Arg-Gly-Asp sequence or a polypeptide having a repeating structure thereof is known ((In
t. J. Biol. Macromol. ), Vol. 11, p. 23, 1989, same magazine, Vol. 11, p. 226, 1989, (Jpn. J. Cancer Res
．． ) Vol. 60, p. 722, 1989). [0004] Synthetic polypeptides, which have been used in various studies as protein models, have carboxyl groups, amino groups, hydroxyl groups, etc. in their side chains depending on the type of α-amino acids they constitute, and can be synthesized in various ways by polymer reactions. It is possible to carry several physiologically active substances. In addition, functional groups can be easily converted, such as polyLys, poly
Side chain amino groups such as Orn can be easily carboxylated with dibasic acids and their derivatives, preferably dibasic acid anhydrides, or polybasic acids and their derivatives, preferably polybasic acid anhydrides. Arg-Gly-As in the polypeptide main chain
Polypeptides having a repeating structure with p as an essential unit are known, but Arg-
There are no known water-soluble polypeptide derivatives into which oligopeptides having Gly-Asp as an essential unit or oligopeptides having a repeating structure thereof are introduced, and when introduced, the ability to bind to receptors is enhanced and the oligopeptide in the blood is increased. Stabilization can be expected. [0006] An object of the present invention is to provide an animal cell adhesion inhibitor and a platelet aggregation/adhesion inhibitor containing a novel water-soluble polypeptide derivative and its salt as active ingredients. It is. Means for Solving the Problems [0007] The present invention essentially incorporates an adhesive peptide represented by the following general formula (1) into the side chain of a water-soluble polypeptide via either an amide bond or an ester bond. The present invention provides water-soluble polypeptide derivatives and salts thereof. General formula (1)-[R1]-
[CO]-([X]-Arg-Gly-Asp-
[Y])n-[Z]-[R2]- In the formula, [] may or may not exist,
If present, X, Y is Ser, Gly, Val, As
n, represents an amino acid residue or peptide residue selected from Pro, and Z represents -O- or -NH-. R1,
Either one of R2 is a hydrogen atom or has 1 to 1 carbon atoms.
9 linear or branched alkyl groups, or 6 carbon atoms
-9 aryl group, which may have a substituent. The other is a hydrogen atom, a linear or branched alkylene group having 1 to 9 carbon atoms, or an arylene group having 6 to 9 carbon atoms, and may have a substituent. n represents an integer from 1 to 5. Examples of substituents include carbonyl groups, carboxyl groups, amino groups, hydroxyl groups, sulfo groups, halogen atoms, aryl groups, nitro groups, cyano groups, and double bonds and triple bonds of unsaturated groups. may have two or more. The present invention further provides an animal cell adhesion inhibitor and a platelet aggregation/adhesion inhibitor containing the water-soluble polypeptide derivative or its salt as an active ingredient. The present invention provides synthetic polypeptides with Arg-Gly-
It is a water-soluble polypeptide derivative formed by covalently bonding an adhesive peptide having Asp as an essential unit. The molecular weight of the water-soluble polypeptide derivative is preferably 200,000 or less, particularly in the range of 3,000 to 100,000, and is preferably water-soluble at room temperature. As a carboxylating agent for side chain amino groups,
Succinic anhydride, maleic anhydride, phthalic anhydride, itaconic anhydride, citraconic anhydride, pyromellitic anhydride,
Examples include trimellitic anhydride. The amino acid used in the adhesive peptide according to the present invention may be either L-form or D-form, but preferably L-form. [0011] Synthetic polypeptides according to the present invention include Hyp, Ser, Tyr, Asp, Glu, Lys,
Orn homopolymer and random copolymer having any one of these amino acids as an essential unit, preferably polyAsp, polyGlu, polyL
ys, polyOrn. Examples of the salts of the water-soluble polypeptide derivative of the present invention include salts with inorganic acids such as hydrochloride, sulfate, nitrate, phosphate, and borate, acetate, trifluoroacetate, and trifluoromethanesulfonate. ,
It may be made into a salt with an organic acid such as lactate or tartrate, and conversion to such a salt can be carried out by conventional means. [0012] The peptide synthesis method is not particularly limited, but may include a liquid phase method, a solid phase method, and a synthesis method using an automatic synthesizer. For more information on these synthetic methods,
Biochemistry Experiment Course “Protein Chemistry IV” p207-4
95 (edited by the Japanese Biochemical Society, Tokyo Kagaku Doujin), “Protein Chemistry Experiment Lecture (Part 2)” (edited by the Japanese Biochemical Society, Tokyo Kagaku Doujin), “Basics and Experiments of Peptide Synthesis”, edited by Izumiya et al.
(Maruzen). It is also possible to use commercially available synthetic peptides. Examples of bonding between polypeptides and carboxylated polypeptides and adhesive peptides include amide bond synthesis methods using cyanogen bromide, acid azide, water-soluble carbodiimide, and the like. The water-soluble polypeptide derivative of the present invention has the core sequence Arg-Gly-Asp of a cell adhesion protein, and adheres to cells via the core sequence in the same mechanism as that of a cell adhesion protein. Therefore, it exhibits various biological activities as an agonist or antagonist of cell adhesion proteins, and has a wide range of biological activities such as immunomodulation, wound healing, inhibition of platelet aggregation by cancer cells that occur in capillaries, and healing of neurological diseases. It recognized. Therefore, the water-soluble polypeptide derivative of the present invention can be used as a cancer metastasis inhibitor, a wound healing agent, an immunomodulator, and a platelet aggregation and adhesion inhibitor in patients, optionally together with a conventional carrier or pharmaceutical auxiliary agent. It is possible to administer especially,
Use as an animal cell adhesion inhibitor or platelet aggregation adhesion inhibitor is preferred. The dosage is 0.2 μg/kg ~
It is determined based on symptoms, age, body weight, etc. within a range of 400 mg/kg. The water-soluble polypeptide derivative of the present invention is preferably administered by a method commonly used for peptide drugs, that is, a parenteral method, such as intravenous administration, intramuscular administration, subcutaneous administration, etc. When producing such an injectable preparation, the water-soluble polypeptide derivative of the present invention may be dissolved in PBS or physiological saline to prepare an injectable preparation, for example, as shown in the Examples below, or a 0.1N After dissolving in aqueous acetic acid or the like, it may be used as a lyophilized preparation. Conventional stabilizers such as glycine and albumin may be added to such formulations. moreover,
The water-soluble polypeptide derivative of the present invention can be administered orally, for example, in the form of microcapsules encapsulated in liposomes, or in the form of suppositories, sublingual tablets, nasal sprays, etc. It is also possible to absorb it from mucous membranes other than the digestive tract. [Examples] The present invention will be further explained below with reference to Examples, but the present invention is not limited thereto. In addition, in the following, R represents arginyl, G represents glycyl, D represents aspartyl, S represents seryl, and P represents prolyl. Production Example 1 Synthesis of adhesive peptide by solid phase method Synthesis was carried out using a Merrifield system peptide synthesizer. A Boc group was used to protect the α-amino group, and after being excised from the resin, it was purified by preparative HPLC (high performance liquid chromatography) to obtain an adhesive synthetic peptide showing a single peak. Table 1 Adhesive synthetic peptide names
Structural formula
Abbreviation Yield peptide-1 H-
Arg-Gly-Asp-OH
RGD 37% peptide-2
H-(Arg-Gly-Asp)2-OH
(RGD)2 28% (SEQ ID NO: 1
) Peptide-3H-(Arg-Gly-Asp)
3-OH (RGD)3
19% (SEQ ID NO: 2) Peptide-4 H-(Arg-Gly-Asp)
5-OH (RGD)5
11% (SEQ ID NO: 3) Peptide-5 H-(Gly-Arg-Gly-
Asp-Ser-Pro)-OH GRGDSP
27% (SEQ ID NO: 4) Production Example 2 Peptide-6 H-(Arg-Gly-Asp-S
er-Gly)-NH2 (SEQ ID NO: 5) Peptide-6 was synthesized by a liquid phase method using a sequential extension method. (1) Synthesis of BocSer(Bzl)GlyNH2 59g (0.2mol) of BocSer(Bzl) was
Dissolve in 400 ml of H2 Cl2 and DCC 41.
2 g (0.2 mol) was added under ice cooling. Add G to this solution.
lyNH2 ・HCl 22.1g to CH2 Cl2
After dissolving the mixture in 400 ml, a solution neutralized with 20.2 g of N-methylmorpholine under ice cooling was added. The mixture was stirred for 3 hours under ice cooling, and then further stirred at room temperature overnight. The precipitate was filtered off, concentrated under reduced pressure, and dissolved in ethyl acetate. N
It was washed with an aHCO3 aqueous solution, a 1M citric acid aqueous solution, and a NaCl aqueous solution in this order, dried over Na2SO4, and then dried under reduced pressure to obtain 58.3 g of a white powder (yield: 83%). (2) BocAsp(OBzl)Ser(
Synthesis of Boc Ser(Bzl)GlyNH2 56.
2g (0.16mol) of TFA/CH2Cl2 =
After adding 400ml of 1/1 and stirring at room temperature for 1 hour,
TFA and CH2Cl2 were concentrated under reduced pressure. After dissolving this in ethyl acetate and neutralizing with NaHCO3 aqueous solution, Na
Washed with Cl aqueous solution. After drying with Na2SO4, ethyl acetate was distilled off under reduced pressure. This compound and BocA
sp(OBzl) 51.7g (0.16mol) in CH
2 Dissolved in 800 ml of Cl2, DCC 33
g (0.16 mol) was added under ice cooling, stirred for 3 hours, and then further stirred at room temperature overnight. CH2 Cl2 under reduced pressure
was distilled off and then dissolved in ethyl acetate. NaHCO3
It was washed with an aqueous solution, a 1M citric acid aqueous solution, and a NaCl aqueous solution in this order, dried over Na2SO4, and then dried under reduced pressure to obtain 71.2 g of a white powder (yield: 80%). (3) BocGlyAsp(OBzl)S
Synthesis B of er(Bzl)GlyNH2 (SEQ ID NO: 6)
ocAsp(OBzl)Ser(Bzl)GlyNH2
66.7g (0.12mol) of TFA:CH2
After adding 400 ml of Cl2 = 1:1 and stirring at room temperature for 1 hour, TFA and CH2 Cl2 were concentrated under reduced pressure. This was dissolved in ethyl acetate, neutralized with an aqueous NaHCO3 solution, and then washed with an aqueous NaCl solution. Na2SO4
After drying, ethyl acetate was distilled off under reduced pressure. This compound and 51.7 g (0.12 mol) of BocGly were dissolved in 700 ml of CH2 Cl2, and DCC24
．． After adding 7 g (0.12 mol) under ice-cooling and stirring for 3 hours, the mixture was further stirred at room temperature overnight. DCurea was filtered off, concentrated under reduced pressure, and dissolved in ethyl acetate. NaHCO
3 aqueous solution, 1M citric acid aqueous solution, and NaCl aqueous solution in this order, dried over Na2SO4, and then dried under reduced pressure to obtain 61.8 g of white powder (yield: 84%). (4) BocArg(Mts)GlyAs
Synthesis of p(OBzl)Ser(Bzl)GlyNH2 BocGlyAsp(OBzl)Ser(Bzl)Gl
yNH2 61.3g (0.1mmol) with TFA
After adding 400 ml of :CH2Cl2=1:1 and stirring at room temperature for 1 hour, TFA and CH2Cl2 were concentrated under reduced pressure. Dissolve this in ethyl acetate and add NaHCO3
After neutralizing with an aqueous solution, it was washed with an aqueous NaCl solution. Na2
After drying with SO4, ethyl acetate was distilled off under reduced pressure. This compound and BocArg (Mts) 45.6g (
0.1 mol) in 800 ml of DMF, DCC
22.5g (0.1mol) HOBt 14g
(0.1 mol) was added under ice cooling and stirred for 3 hours, and then further stirred at room temperature overnight. DCurea was filtered off, concentrated under reduced pressure, and dissolved in ethyl acetate. It was washed with an aqueous NaHCO3 solution, a 1M aqueous citric acid solution, and an aqueous NaCl solution in this order, dried over Na2SO4, and then dried under reduced pressure to obtain 42.8 g (yield: 45%) of a white powder. (5) Deprotection B of peptide-6 protected form
ocArg(Mts)GlyAsp(OBzl)Ser
(Bzl)GlyNH2 5g (5.3mmol)
A TFA solution of 1 M trifluoromethanesulfonic acid-thioanisole-m-cresol was added to the TFA solution under ice cooling and allowed to react for 1 hour to deprotect the peptide side chain and terminal protecting group. The reaction solution was poured into ether, and the oily precipitate was dissolved in distilled water and washed with ethyl acetate.
It was passed through RA-400 (Cl type) to form a hydrochloride and lyophilized. 2.17 g (86% yield) of white solid was obtained. Mass spectrum M+: 404 [0022] Production example 3 Peptide-7 BocArg(Mts)Gly
Asp(OBzl)Gly (SEQ ID NO: 7) Peptide-7
was synthesized by a liquid phase method using a sequential extension method. (1) Synthesis of BocGlyONb 35 g (0.2 mol) of BocGly, 28 ml (0.2 mol) of triethylamine, and 43.2 g (0.2 mol) of p-nitrobenzyl bromide were dissolved in 400 ml of ethyl acetate, refluxed for 5 hours, and then room temperature I left it there overnight. The generated salt was filtered off, washed with an aqueous NaHCO3 solution and water, dried over Na2SO4, concentrated under reduced pressure, and recrystallized from ethyl acetate-hexane. 52.7 g (yield: 85%) The peptide chain was extended in the same manner as in Production Example 2. The outline is shown below. (2) Synthesis of BocAsp(OBzl)GlyONb
Product of (1):
46.5g (0.15mol) TFA/C
H2 Cl2: 200ml/200ml
BocAsp(OBzl): 48.5
g (0.15 mol) CH2 Cl2
:750ml
DCC: 3
Yield of 0.9g (0.15mol) (2) 64
．． 0g (Yield 80%) (3) Synthesis of BocGlyAsp(OBzl)GlyONb Product of (2)
:64.0g (0.12mol) TF
A/CH2 Cl2: 200ml/20
0ml BocGly
:21g (0.12mol) CH
2 Cl2: 750m
l DCC
:24.7g (0.12mol) Yield of (3) 58.8g (yield 83%) (4) BocArg(Mts)GlyAsp(OBzl
) Synthesis of GlyONb Product of (3)
:53.7g (91mmol)
TFA/CH2Cl2:2
00ml/200ml BocArg(
Mts): 41.5g (91mmol)
DMF:
800ml DCC
:18.7g (91mmol)
HOBt
:13.5g (0.1mol)
Yield of (4): 46.5g (yield: 55%) [
(5) BocArg(Mts)GlyAsp
(OBzl)Gly synthesis (4) product 9.29(1
Dissolve 0 mmol) in 300 ml of 90% acetic acid, add 32.7 g (0.5 mol) of Zn powder, and heat at 0 degrees Celsius for 30 minutes.
Stir for hours. The Zn powder was filtered off, and the filtrate was concentrated under reduced pressure, acidified by adding citric acid, and extracted with ethyl acetate. N
After drying with a2 SO4 and concentrating under reduced pressure, ether was added to obtain 6.26 g of white powder (yield: 79%). Synthesis Example 1 Polyglutamic acid-RGDS
Synthetic polyglutamic acid sodium salt (manufactured by Peptide Institute; molecular weight >8000) 1.00 g was dissolved in 35 ml of pH 7.4 phosphate buffer, and water-soluble D
A solution of 0.26 g of CC [1-ethyl-3-3-(dimethylaminopropyl)-carbodiimide] in 5.0 ml of phosphate buffer was added, and the mixture was reacted for 1.5 hours. Then, 0.72 g of adhesive peptide RGDS (manufactured by Kokusan Kagaku Kogyo) dissolved in 10 ml of phosphate buffer was added and reacted overnight at 4 degrees. Transfer the reaction solution to Spectrapore 7
(molecular fraction: 8,000) and dialyzed against ion-exchanged water and then pure water to remove low molecular weight components, followed by lyophilization. Yield: 1.01 g The structure was confirmed by IR and amino acid analysis. The introduction rate of the RGDS fragment was calculated from the ratio of the arginine residue concentration to the glutamic acid residue concentration according to the following formula, and a value of about 10% was obtained. Introduction rate=[Arg]÷[Glu]×100IR: Stretching vibration of amide carbonyl (C=O) 1652cm
-1 Synthesis Example 2 Polyaspartic acid-GRG
Synthetic adhesive peptide fragment of DS 0.81 g of GRGDS (manufactured by Kokusan Kagaku Kogyo) and 1.02 g of polyaspartic acid (manufactured by Sigma) were covalently bonded in the same manner as in Synthesis Example 1 to synthesize polyaspartic acid-GRGDS. . Yield 1.
The 13g structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the GRGDS fragment was about 10%. Synthesis Example 3 Succinylated polylysine-R
0.52 g of GD polylysine (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 10 ml of 1% triethylamine solution, 0.75 g of succinic anhydride and 0.13 g of 4-dimethylaminopyridine were added thereto, and the mixture was stirred at room temperature overnight. After the reaction was completed, the solution was poured into a large excess of acetone to reprecipitate succinylated polylysine. The precipitate was collected, washed with a large amount of methanol, and then dried under vacuum. Yield 0.61g. Succinylated polylysine 0.76g, water-soluble DCC
The RGD fragment was covalently bonded in the same manner as in Synthesis Example 1 using 0.26 g and 0.57 g of RGD. Yield: 0.88g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of RGD fragment was approximately 11%. Introduction rate = [Arg
]÷[Lys]×100 Amino acid analysis results (nmol/50 μl) Arg
2.1044 Gly
1.9982 Asp
1.9541 Lys 21.
8493 IR: Stretching vibration of amide carbonyl (C=O) 1648 cm-1 Synthesis example 4 Polyglutamic acid (RGD
)2 Polyglutamic acid 0.50g, water-soluble DCC 0.2
6g, as adhesive peptide fragment (RGD)2
Using 0.56 g, polyglutamic acid-(RGD)2 was synthesized in the same manner as in Synthesis Example 1. Yield 0.61
g. The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis (RGD), the introduction rate of the 2 fragment was approximately 12%. Introduction rate = [Arg] ÷ 2
÷[Glu]×100 Amino acid analysis results (nmol/50 μl) Arg
1.0077 Gly
0.9981 Asp
0.9833 Glu 4.
1988 IR: Stretching vibration of amide carbonyl (C=O) 1654 cm-1 Synthesis Example 5 Polyaspartic acid (RG
D) 3 polyaspartic acid 0.23 g, as adhesive peptide fragment (RGD) 3 0.91 g, water soluble D
Polyaspartic acid-(RGD)3 was synthesized in the same manner as in Synthesis Example 1 using 0.26 g of CC. Yield 0.
33g. The structure was confirmed by IR and amino acid analysis. Results of amino acid analysis (RGD)3 The introduction rate of the fragment was approximately 10%. Introduction rate = [Arg] ÷ 3 ÷ (
[Asp]-[Arg])×100 Amino acid analysis results (nmol/50 μl) Arg
3.3475 Gly
3.2566 Asp
14.2028 IR: Amidocarbonyl (C=
Stretching vibration of O) 1648 cm-1 Synthesis Example 6 Succinylated polyornithine (RGD) 5 0.23 g of polyornithine (manufactured by Sigma) was dissolved in 10 ml of 1% triethylamine solution, and 0.30 g of anhydrous Succinic acid and 0.06 g of 4-dimethylaminopyridine were added and stirred at room temperature overnight. After the reaction was completed, the solution was poured into a large excess of acetone to reprecipitate succinylated polyornithine. The precipitate was collected, washed with a large amount of methanol, and then dried under vacuum. Yield 0.37
g. 0.21 g of succinylated polyornithine, 0.91 g of 5 as adhesive peptide fragment (RGD),
Succinylated polyornithine (RGD) 5 was synthesized in the same manner as in Synthesis Example 1 using 0.16 g of water-soluble DCC. Yield: 0.28g. The structure was confirmed by IR and amino acid analysis. Results of amino acid analysis (
The introduction rate of RGD)5 fragment was about 15%. Introduction rate = [Arg] ÷ 5 ÷ [Orn] × 100 Amino acid analysis result (nmol/50 μl) Arg
5.0108Gly 5
．． 0993Asp 5.3432O
rn 6.6811IR: Stretching vibration of amide carbonyl (C=O) 1652cm-
1 Synthesis Example 7 Polyglutamic acid-RGDS
G-NH2 0.81 g of the oligopeptide of Production Example 2 was added to the oligopeptide of Production Example 1.
It was bound to polyglutamic acid in the same manner. Yield 1.07g polyglutamic acid-RGDSG-N
The structure of H2 was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDSG-NH2 fragment was approximately 12%. Introduction rate = [A
rg]÷[Glu]×100 Amino acid analysis results (nmol/50 μl) Arg
1.9780 Gly
1.9251 Asp
1.6053 Ser 1.
4921 Glu 16.4833
IR: Stretching vibration of amide carbonyl (C=O)
1650cm-1 Synthesis Example 8 Synthesis of RGDSG-Polylysine Peptide-7 of Production Example 3 3.18g (4mmol
) in 40 ml of DMF, DCC 0.83
g (4 mmol), HOBt 0.54 g (4 mmol)
1) was added under ice-cooling, and the mixture was stirred for 1 hour. DMF for this
Add 0.51g of polylysine dissolved in 20ml and add 3
After stirring for an hour, the mixture was further stirred at room temperature overnight. DCure
After filtering out a, it was concentrated under reduced pressure. Deprotection was then carried out in the same manner as in Production Example 2. 1M trifluoromethanesulfonic acid-thioanisole-m-cresol T
FA solution: 150ml Amberlite IRA-40
0 (Cl type) treatment. Yield 0.61g. Structural confirmation was performed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDS fragment was approximately 14%. Introduction rate = [Arg] ÷ [LyS] × 100 Amino acid analysis (nmol/50 μl) Arg 2.0495 Gly
2.0872 Asp
1.9994 Ser
1.9736 Lys 14.
6347 IR: Stretching vibration of amide carbonyl (C=O) 1652 cm-1 Synthesis Example 9 Synthesis of RGDSG-polyornithine Peptide-7 of Production Example 3 3.18 g (4 mm
ol), DCC 0.83g (4mmol), HOB
Peptide binding and deprotection were carried out in the same manner as in Synthesis Example 8 using 0.54 g (4 mmol) of t and 0.46 g of polyornithine. Yield 0.54g. Structural confirmation was performed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of RGDS fragment is approximately 1
It was 5%. Introduction rate = [Arg] ÷ [Orn] x 10
0 amino acid analysis (nmol/50μl) Arg 2.0502 Gly
2.1013 Asp
1.8991 Ser
1.9506 Lys 13.
6642 IR: Stretching vibration of amide carbonyl (C=O) 1648 cm-1 Synthesis Example 10 Polyglutamic acid-GRG
Synthesis of DSP Peptide fragment-6 (GRGDSP) synthesized in Production Example 1 0.59 g, polyglutamic acid 0
．． 32g (manufactured by Kokusan Kagaku Kogyo) and water-soluble DCC 0
．． Polyglutamic acid-
GRGDSP was synthesized. Yield: 0.40g. The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the GRGDSP fragment was approximately 11%. Introduction rate = 100 x [Arg] ÷ [
Glu] Amino acid analysis (nmol/50 μl) Arg 1.5152 Gly
3.1340 Asp
1.4916 Ser
1.4875 Pro 1.
4605 Glu 13.7814
IR: Stretching vibration of amide carbonyl (C=O)
Test Example 1 Measurement of Cell Adhesion Inhibitory Activity A method for measuring the activity of the water-soluble polypeptide derivative of the present invention to inhibit cell adhesion to fibronectin and vitronectin is shown below. The competition method used in this example is basically one that is widely used in the field of biochemistry, such as "Methods in Enzymology".
”, 82, 03-831 (1981), JP-A-1
-309682 and 2-174797. Experimental method 1. Preparation of fibronectin and vitronectin adsorption plates Commercially available fibronectin (human origin: Seikagaku Corporation)
(purchased from Funakoshi Pharmaceutical Co., Ltd.) or vitronectin (human origin, purchased from Funakoshi Pharmaceutical Co., Ltd.) in PBS at 1.0 μl each.
ml, diluted to 2.0μl/ml and 0.5ml of the diluted solution
was placed in a 24-well plastic plate, kept warm at 37 degrees overnight, and coated. Next, in order to prevent non-specific adsorption, bovine serum albumin (BSA 1%) was added and heated at 37 degrees for 1 hour.
The plate was kept warm for an hour, and then subjected to a normal washing operation (PBS) and thoroughly drained to prepare a fibronectin adsorption plate. A vitronectin adsorption plate was prepared by the same procedure. 2. Adhesion inhibition experiment The water-soluble polypeptide derivative obtained by freeze-drying was
becco's Modified EaglesMe
dium (hereinafter abbreviated as DMEM), 0,0
．． Water-soluble polypeptide derivative solutions were prepared at concentrations of 25, 0.5, 1.0, and 1.5 mg/ml. Put 0.25ml of this solution into the plate prepared by the above method, and add vascular endothelial cells (4 x 106 cells/
ml) suspension was added and kept at 37 degrees for 1 hour to allow the cells to adhere. The cells were washed three times with DMEM medium, unattached cells were detached, and the number of cells was counted by staining with 2% trypan blue. The results are shown in Tables 2 and 3. Table 2 Cell adhesion inhibition to fibronectin (cells/well)
Concentration (mg/ml) Additive compound 0
0.25 0.5 1.0 1
．． 5 Compound 1 ×
△ ○ ○
○Synthetic substance 2 ×
△ ○ ○
○Synthetic substance 3 ×
△ △ ○
○Synthetic substance 4 ×
△ ○ ○
○Synthetic substance 5 ×
△ ○ ○
○Synthetic substance 6 ×
△ ○ ○
○Synthetic substance 7 ×
△ ○ ○
○Synthetic substance 8 ×
△ ○
○ ○Synthetic substance 9 ×
△ ○
○ ○Product 2 ×
× △
△ △RGD
× × ×
× △RGDS
× × ×
△ △(cells/well
l); ○: 50 or less, △: 50-100, ×: 100 or more [0041] Table 3 Cell adhesion inhibition to vitronectin (cells/well)
Concentration (mg/ml) Additive compound 0
10 50 100
300 compounds 1 ×
○ ○ ○
○Synthetic substance 2 ×
○ ○ ○
○Synthetic substance 3 ×
△ △ ○
○Synthetic substance 4 ×
○ ○
○ ○Synthetic substance 5 ×
○ ○
○ ○Synthetic substance 6 ×
○ ○
○ ○Synthetic substance 7 ×
○ ○
○ ○Synthetic substance 8
× ○ ○
○ ○Synthetic substance 9
× ○ ○
○ ○Product 2
× × △
△ △RGD
× × ×
△ △RGDS
× ×
△ △ ○(cells
/well); ○: 100 or less, △: 100 to 20
0, ×: 200 or more Test Example 2 Measurement of platelet aggregation inhibitory activity The platelet aggregation inhibitory effect of the water-soluble polypeptide derivative synthesized in the present invention in an in vitro system was investigated using human platelet-rich plasma. The experimental method is shown below. Experimental Method Fresh human blood was added with 1/9 volume of 3.8% sodium citrate and centrifuged (1000 rpm, 10 minutes), and the upper layer was collected as platelet-rich plasma. The water-soluble polypeptide derivatives obtained by freeze-drying were dissolved in physiological saline at various concentrations from 0 to 1.5 mg/ml. Add 25 μl of water-soluble polypeptide derivative solution to 200 μl of plasma and
After incubation at 7 degrees for 3 minutes, 50 μM
Adenosine diphosphate (ADP) solution or 200μg
25 μl/ml of collagen solution was added, and the state of aggregation was examined by measuring the permeability using an aggregometer. The results are shown in Table 4. Aggregation inhibition rate (1-T/T0) x 100%T0 =
Permeability T of water-soluble polypeptide derivative without salt addition
= Permeability of water-soluble polypeptide derivative upon addition of salt 00
44] Table 4
Platelet aggregation inhibition
Inhibitory activity additive compound ADP stimulation
Collagen stimulating compound 1
○
○Synthetic substance 2
○
○Synthetic substance 3
○
○Synthetic substance 4 ○
○Synthetic substances 5 ○
○Synthetic substances 6
○
○Synthetic substances 7
○
○Synthetic substances 8
○
○Synthetic substances 9
○
○Product 2 △〜○
△〜○RGD
△〜○
△〜○RGDS
△〜○
△～○IC50 (μg/ml);○
: 40 or less, △: 80-40, ×: 80 or more 0045
[Effect of the invention] The compound of the present invention is a water-soluble polypeptide derivative in which an oligopeptide having Arg-Gly-Asp as an essential unit or an oligopeptide having a repeating structure thereof is introduced into the side chain of a polypeptide. This can be expected to have the effect of inhibiting adhesion of animal cells and inhibiting platelet aggregation/adhesion, as well as enhancement of binding ability with receptors and stabilization in blood. [Sequence Listing] SEQ ID NO: 1 Sequence Length: 6 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence SEQ ID NO: 2 Sequence Length: 9 Sequence Type: Amino Acid Topology: Type of linear sequence: Peptide sequence 0049 SEQ ID NO: 3 Length of sequence: 15 Type of sequence: Amino acid Topology: Type of linear sequence: Peptide sequence 0050 SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid topology: Linear sequence type: Peptide sequence SEQ ID NO: 5 Sequence length: 5 Sequence type: Amino acid topology: Linear sequence type: Peptide sequence SEQ ID NO: 6 Sequence length: 4 Sequence type: Amino acid topology: Linear sequence type: Peptide sequence SEQ ID NO: 7 Sequence length: 4 Sequence type: Amino acid topology : Type of linear sequence: Peptide sequence
Claims (4)
ル結合のいずれかを介し、下記一般式（１）で表される
接着性ペプチドを必須単位として有する水溶性ポリペプ
チド誘導体またはその塩。一般式（１）−［Ｒ１］−［
ＣＯ］−（［Ｘ］−Ａｒｇ −Ｇｌｙ −Ａｓｐ −［
Ｙ］）ｎ−［Ｚ］ −［Ｒ２］−式中、［ ］は存在
するかあるいは存在しなくともよく、存在する場合はＸ
、ＹはＳｅｒ、Ｇｌｙ、Ｖａｌ、Ａｓｎ、Ｐｒｏから選
択されるアミノ酸残基又はペプチド残基を示し、Ｚは−
Ｏ−または−ＮＨ−を示す。Ｒ１ 、Ｒ２ のいずれか
一方は、水素原子または炭素数が１〜９の直鎖もしくは
分岐のアルキル基、または炭素数が６〜９のアリ−ル基
であり、置換基を有していても良い。他方は、水素原子
または炭素数が１〜９の直鎖もしくは分岐のアルキレン
基、または炭素数が６〜９のアリ−レン基であり、置換
基を有していても良い。ｎは１〜５の整数を示す。1. A water-soluble polypeptide derivative or a salt thereof having an adhesive peptide represented by the following general formula (1) as an essential unit via either an amide bond or an ester bond in the side chain of the polypeptide. General formula (1)-[R1]-[
CO]-([X]-Arg-Gly-Asp-[
Y])n-[Z]-[R2]-In the formula, [] may or may not exist, and if present, X
, Y represents an amino acid residue or peptide residue selected from Ser, Gly, Val, Asn, Pro, and Z is -
Indicates O- or -NH-. Either R1 or R2 is a hydrogen atom, a linear or branched alkyl group having 1 to 9 carbon atoms, or an aryl group having 6 to 9 carbon atoms, even if it has a substituent. good. The other is a hydrogen atom, a linear or branched alkylene group having 1 to 9 carbon atoms, or an arylene group having 6 to 9 carbon atoms, and may have a substituent. n represents an integer of 1 to 5.
範囲である請求項１記載の水溶性ポリペプチド誘導体ま
たはその塩。2. The water-soluble polypeptide derivative or salt thereof according to claim 1, which has a molecular weight in the range of about 3,000 to 100,000.
ド誘導体を有効成分とする細胞の接着を阻害する薬剤。3. A drug for inhibiting cell adhesion, which contains the water-soluble polypeptide derivative according to claim 1 or 2 as an active ingredient.
ド誘導体を有効成分とする血小板凝集・粘着抑制剤。4. A platelet aggregation/adhesion inhibitor comprising the water-soluble polypeptide derivative according to claim 1 or 2 as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2404484A JPH04221397A (en) | 1990-12-20 | 1990-12-20 | Water-soluble polypeptide derivative and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2404484A JPH04221397A (en) | 1990-12-20 | 1990-12-20 | Water-soluble polypeptide derivative and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04221397A true JPH04221397A (en) | 1992-08-11 |
Family
ID=18514154
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2404484A Pending JPH04221397A (en) | 1990-12-20 | 1990-12-20 | Water-soluble polypeptide derivative and its use |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04221397A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996015811A1 (en) * | 1994-11-17 | 1996-05-30 | Imperial College Of Science, Technology & Medicine | Internalisation of dna, using conjugates of poly-l-lysine and an integrin receptor ligand |
| US5672585A (en) * | 1990-04-06 | 1997-09-30 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| US5780303A (en) * | 1990-04-06 | 1998-07-14 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| US6017877A (en) * | 1990-04-06 | 2000-01-25 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| US6521594B1 (en) | 1990-04-06 | 2003-02-18 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
-
1990
- 1990-12-20 JP JP2404484A patent/JPH04221397A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5672585A (en) * | 1990-04-06 | 1997-09-30 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| US5780303A (en) * | 1990-04-06 | 1998-07-14 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| US6017877A (en) * | 1990-04-06 | 2000-01-25 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| US6521594B1 (en) | 1990-04-06 | 2003-02-18 | La Jolla Cancer Research Foundation | Method and composition for treating thrombosis |
| WO1996015811A1 (en) * | 1994-11-17 | 1996-05-30 | Imperial College Of Science, Technology & Medicine | Internalisation of dna, using conjugates of poly-l-lysine and an integrin receptor ligand |
| US6083741A (en) * | 1994-11-17 | 2000-07-04 | Imperial College Of Science Technology And Medicine | Internalisation of DNA, using conjugates of poly-l-lysine and an integrin receptor ligand |
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