JPH04187698A - Peptide-containing polyethylene glycol derivative and its use - Google Patents
Peptide-containing polyethylene glycol derivative and its useInfo
- Publication number
- JPH04187698A JPH04187698A JP2316441A JP31644190A JPH04187698A JP H04187698 A JPH04187698 A JP H04187698A JP 2316441 A JP2316441 A JP 2316441A JP 31644190 A JP31644190 A JP 31644190A JP H04187698 A JPH04187698 A JP H04187698A
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- polyethylene glycol
- glycol derivative
- containing polyethylene
- general formula
- 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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- 208000024891 symptom Diseases 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
Description
【発明の詳細な説明】
「産業上の利用分野］
本発明は、グルタミン酸−イソロイシンーロイソンーア
スパラギン酸−ハリン−プロリン−セリン−トレオニン
のオクタペプチド単位を有する、ポリエチレングリコー
ル誘導体またはその塩およびこれを有効成分とする動物
細胞の接着阻害剤ならひに血小板凝集・粘着抑制剤に関
する。Detailed Description of the Invention "Industrial Application Field" The present invention relates to polyethylene glycol derivatives or salts thereof having octapeptide units of glutamic acid-isoleucine-leusone-aspartic acid-haline-proline-serine-threonine; This article relates to a platelet aggregation/adhesion inhibitor, which is an animal cell adhesion inhibitor containing as an active ingredient.
［従来の技術二
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
らのレセプターかそのアルキニン−グリノン−アスパラ
キン酸（以下、Ａｒｇ−Ｇａｙ−Ａｓｐと略す）配列を
特異的に認識することか明らかにされ、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、１
９８４年）ｅ以来、Ａｒｇ−Ｇ ｌ ｙ−Ａｓｐ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進め
られている。[Prior Art] Difibronectin 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 or its alkynine-glynon-aspartic acid (hereinafter referred to as Arg-Gay- It has been clarified that Asp) sequence is specifically recognized, and it has been reported that it is important for interaction with receptors (Nature, Vol. 309, p. 30, 1).
Since 1984), research has been conducted using oligos or polypeptides having the Arg-Gly-Asp sequence.
例えは、Ａｒｇ−Ｇｌ〜□−Ａｓｐ配列を有する種々の
鎖状および環状のオクタ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（ＰｏｌｙｍｅｒＰｒ
ｅｐｒｉｎｔｓ 、 Ｊａｐａｎ ） 、第３８巻、３
１４９頁、１９８９年、特開平２−１７４７９７号）
、Ａｒｇ−［１＋Ｉｙ−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２−４７１６
号）、Ａｒｇ−Ｇｌｙ−Ａｓｐを固定化したＰＭＭＡ膜
を細胞接着膜として用いる方法（高分子学会予稿集（Ｐ
ｏｌｙｍｅｒＰｒｅｐｒｉｎｔｓ 、 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−Ｇａｙ−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
１３０９６８２号、特開平１−３０５９６０号） 、Ａ
ｒｇ−Ｇｌｙ−Ａｓｐ−３ｅｒ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か開示さ
れている（特開昭６４−６２１７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
移を抑制する方法か知られている（Ｉｎｔ、 Ｊ、 Ｂ
ｉｏｌ。For example, a method of inhibiting platelet aggregation using various linear and cyclic octapeptides having Arg-Gl~□-Asp sequences (Proceedings of the Society of Polymer Science and Technology (PolymerPr.
eprints, Japan), Volume 38, 3
149 pages, 1989, Japanese Patent Application Publication No. 2-174797)
, A method using a peptide having Arg-[1+Iy-Asp sequence as a cell migration inhibitor (Japanese Patent Application Laid-Open No. 2-4716
), method of using PMMA membrane with immobilized Arg-Gly-Asp as a cell adhesion membrane (Proceedings of the Society of Polymer Science and Technology (P.
(Olymer Preprints, Japan)
, Vol. 37, p. 705, 1988). Furthermore, a method of covalently bonding a peptide having Arg-Gay-Asp as an essential constituent unit to a polymer and using it as an animal cell culture substrate or a biological composite artificial organ substrate (Japanese Patent Application Laid-open No. 1309682, Japanese Patent Application Laid-open No. 1-305960), A
A method of using a polypeptide having the rg-Gly-Asp-3er sequence as a platelet protecting agent for extracorporeal blood has been disclosed (Japanese Patent Application Laid-Open No. 64-6217). Also, Arg-
A method of suppressing cancer metastasis using an oligopeptide having a Gly-Asp sequence or a polypeptide having a repeating structure thereof is known (Int, J, B
iol.
Ｍａｃｒｏｍｏｌ、 ）、第１１巻、２３頁、１９８９
年、同誌、第１１巻、２２６頁、１９８９年、（Ｊｐｎ
、 Ｊ。Macromol, ), Volume 11, Page 23, 1989
Year, same magazine, vol. 11, p. 226, 1989, (Jpn
, J.
Ｃａｎｃｅｒ Ｒｅｓ、 ）第６０巻、７２２頁、１９
８９年）。Cancer Res, Volume 60, Page 722, 19
1989).
一方、最近フィブロネクチン分子内にはＡｒｇ−Ｇｌｙ
−Ａｓｐ配列以外の細胞接着配列が存在することも明ら
かにされ、そのひとつとしてｌｌＩＣ３（ｔｙｐｅｌｌ
ｌｈｏｍｏｌｏｇｙ ｃｏｎｎｅｃｔｉｎｇ ｓｅｇｍ
ｅｎｔ）領域内に存在するＣ３Ｉペプチド（グルタミン
酸−イソロインンーロイシンーアスパラギン酸−バリン
−プロリン−セリン−トレオニン配列を含む）か注目さ
れている（Ｊ、 Ｂｉｏｌ、Ｃｈｅｍ、、 ２６２巻
、６８８６頁、１９８７年）。このペプチドは、Ａｒｇ
−Ｇｌｙ−Ａｓｐペプチドと同様にフィブロネクチンレ
セプターに認識され、フィブロネタチンの接着特異性に
寄与していると考えられている。現在では、その接着活
性の最小単位かグルタミン酸−イソロインンーロイシン
ーアスパラギン酸−バリン−プロリン−セリン−トレオ
ニン（以下、ＥＩＬＤＶＰＳＴと略す）配列を有するオ
クタペプチドであることが明らかにされている（Ｊ、
Ｃｅ１１．Ｂｉｏｌ、、 １０７巻、２１８９頁、１
９８８年）。On the other hand, recently, Arg-Gly has been found in the fibronectin molecule.
It has also been revealed that there are cell adhesion sequences other than the -Asp sequence, one of which is llIC3 (typell
lhomology connecting segm
The C3I peptide (containing the glutamic acid-isoloin-leucine-aspartic acid-valine-proline-serine-threonine sequence) present in the ENT) region is attracting attention (J. Biol. Chem., vol. 262, p. 6886). (1987). This peptide is Arg
Like the -Gly-Asp peptide, it is recognized by the fibronectin receptor and is thought to contribute to the adhesion specificity of fibronetatin. It has now been revealed that the smallest unit of adhesive activity is an octapeptide having the sequence glutamic acid-isoloin-leucine-aspartic acid-valine-proline-serine-threonine (hereinafter abbreviated as EILDVPST) (J ,
Ce11. Biol, vol. 107, p. 2189, 1
988).
一方、ポリエチレンクリコールは、疎水性および親水性
の両親媒性を兼ねそなえた合成高分子である。このポリ
エチレングリコールを用いて酵素の性質を改変する方法
が報告されており（Ｔｒｅｎｄｓｉｌ Ｂｉｏｔｅｃｈ
、、第４巻、６８頁、１９８６年）、ポリエチレングリ
コール誘導体に酵素を導入して様々な酵素の改変に成功
している。このポリエチレングリコール誘導体に、ＥＩ
ＬＤＶＰＳＴ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導入した化合物は
知られておらず、導入した場合にはレセプターとの結合
能の増強および血液中での安定化か期待てきる。On the other hand, polyethylene glycol is a synthetic polymer that has both hydrophobic and hydrophilic amphiphilic properties. A method of modifying the properties of enzymes using polyethylene glycol has been reported (Trendsil Biotech
, Vol. 4, p. 68, 1986) have succeeded in modifying various enzymes by introducing enzymes into polyethylene glycol derivatives. This polyethylene glycol derivative has EI
There are no known compounds into which an oligopeptide having an LDVPST sequence or a polypeptide having a repeating structure thereof is introduced, and when introduced, it is expected that the ability to bind to the receptor will be enhanced and the compound will be stabilized in the blood.
［発明か解決しようとする課題］
本発明の目的は、ＥＩＬＤＶＰＳＴのオクタペプチド単
位を有する、ポリエチレンクリコール誘導体およびその
合成法を提供することである。[Problem to be Solved by the Invention] An object of the present invention is to provide a polyethylene glycol derivative having an octapeptide unit of EILDVPST and a method for synthesizing the same.
本発明の他の目的は、これを有効成分とする動物細胞の
接着阻害剤及び血小板凝集・粘着抑制剤を提供すること
である。Another object of the present invention is to provide an animal cell adhesion inhibitor and a platelet aggregation/adhesion inhibitor containing this as an active ingredient.
１課題を解決するための手段］ 本発明の化合物は、下記−殺伐、［または）＋＋＋。1.Means to solve the problem] The compounds of the invention are: -killing, [or) +++.
で規定されるペプチド含有ポリエチレングリコール誘導
体であり、分子内に存在するイオン性基は適当なイオン
と塩を形成してもよい。It is a peptide-containing polyethylene glycol derivative defined by the following, and the ionic group present in the molecule may form a salt with an appropriate ion.
Ｒ″
式中、Ｒ’ 、Ｒ’はそれぞれ下記−殺伐ＦＩＩ）で表
されるペプチド残基を示す。R'' In the formula, R' and R' each represent a peptide residue represented by the following formula (FII).
（［Ｘ７ Ｇｌｕ−１１ｅ−Ｌｅｕ−Ａｓｐ−Ｖａｔ−
Ｐｒｏ−３ｅｒ−Ｔｈｒ−ｊＹ））。([X7 Glu-11e-Leu-Asp-Vat-
Pro-3er-Thr-jY)).
・・ｉｌｌ’。...ill’.
式中、Ｇｌｕ、Ｉ ］ ｅ、Ｌｅｕ、Ａｓｐ、Ｖａｌ、
Ｐｒｏ、Ｓｅｔ、Ｔｈｒはそれぞれグルタミン酸、イソ
ロインン、ロイノン、アスパラキン酸、バリン、プロリ
ン、セリン、トレオニン残基を表す。［Ｘ〕、［Ｙ］は
存在するかあるいは存在しないアミノ酸を表す。存在す
る場合には、［ｘ Ｌ、 ［Ｙ］かセリン、グリシン、
バリン、アスパラキン、プロリン、システィン、トレオ
ニン残基から選択されるアミノ酸残基であることが好ま
しい。特に、［Ｙ］がセリン残基であることが好ましい
。また［Ｘ］、［Ｙ］かともに存在しない場合も好まし
い。なお、このペプチド残基とトリアジン環は［ｘ］（
［Ｘ］が存在しない場合はＧｌｕ）または［Ｙ］（［Ｙ
］が存在しない場合はＴｈｒ）の位置で結合する。ｎは
１から１５０までの整数が好ましく、５から１２０まで
の整数が特に好ましい。ｍは１から５までの整数を表し
、ｍが１から３までの整数が特に好ましい。In the formula, Glu, I ] e, Leu, Asp, Val,
Pro, Set, and Thr represent glutamic acid, isoloinone, leuinone, aspartic acid, valine, proline, serine, and threonine residues, respectively. [X] and [Y] represent amino acids that are present or absent. If present, [x L, [Y] or serine, glycine,
Preferably, it is an amino acid residue selected from valine, asparaquine, proline, cysteine, and threonine residues. In particular, [Y] is preferably a serine residue. It is also preferable that neither [X] nor [Y] is present. Note that this peptide residue and triazine ring are [x](
Glu) or [Y] ([Y
] does not exist, it is bonded at the Thr) position. n is preferably an integer from 1 to 150, particularly preferably from 5 to 120. m represents an integer from 1 to 5, and m is particularly preferably an integer from 1 to 3.
本発明の化合物の好ましい塩の例としてはナトリウム塩
、カリウム塩、アンモニウム塩、マグネシウム塩、塩酸
塩、硫酸塩、硝酸塩、酢酸塩が挙げられる。Examples of preferred salts of the compounds of the invention include sodium, potassium, ammonium, magnesium, hydrochloride, sulfate, nitrate, acetate.
以下に、本発明の好ましい化合物例を挙げるが、本発明
はこれらに限られるものではない。なお下記でＧはグリ
シンを表わす。Preferred examples of compounds of the present invention are listed below, but the present invention is not limited thereto. In addition, below, G represents glycine.
化合物例
数平均分子量 ７．０００
数平均分子量 １２．０００
ＥＩＬＤＶＰｓＴＥＩＬＤＶＰｓＴ
数平均分子量 ９．０００
数平均分子量 ３．５００
本発明の化合物は、レセプターとの結合能の増強および
血液中での安定化が期待され、ＥＩＬＤＶＰＳＴペプチ
ド部位かカン細胞、血小板、リンパ球等の表面に存在す
るフィブロネクチンレセプターと結合できることを利用
して、ガン転移抑制、血小板凝集抑制、リンパ球活性化
の目的に使用することができる。Compound Examples Number Average Molecular Weight 7.000 Number Average Molecular Weight 12.000 EILDVPsTEILDVPsT Number Average Molecular Weight 9.000 Number Average Molecular Weight 3.500 The compound of the present invention is expected to enhance binding ability with receptors and stabilize in blood. By taking advantage of the ability of the EILDVPST peptide site to bind to fibronectin receptors present on the surfaces of cancer cells, platelets, lymphocytes, etc., it can be used for the purposes of suppressing cancer metastasis, platelet aggregation suppression, and lymphocyte activation.
次に本発明の化合物の合成法について説明する。Next, a method for synthesizing the compound of the present invention will be explained.
本発明の化合物は、たとえば次の４段階で合成すること
かできる。The compound of the present invention can be synthesized, for example, in the following four steps.
■ポリエチレンクリコール誘導体ｆｌＶ）及び（Ｖ）の
合成
■保護アミノ酸の逐次延伸による保護ペプチドの合成
■保護ペプチドのポリエチレンクリコール誘導体への導
入による式ｍ］及び［〜＋＋＋］の化合物の合成
Ｒ３及びＲ４は式［１Ｆ）で表されるペプチド残基の保
護体を示す。■Synthesis of polyethylene glycol derivatives flV) and (V) ■Synthesis of protected peptides by sequential stretching of protected amino acids ■Synthesis of compounds of formulas m] and [~+++] by introducing protected peptides into polyethylene glycol derivatives R3 and R4 represents a protected form of the peptide residue represented by formula [1F).
■脱保護および精製 以下、各段階を詳細に説明する。■Deprotection and purification Each stage will be explained in detail below.
■ −殺伐（ｍおよび［Ｖｌで表される化合物は、例え
ばＢｉｏｃｈｅｍ、Ｂｉｏｐｈｙｓ、Ｒｅｓ、Ｃｏｍｍ
ｕｎ、、 ８３． ３８５ （１９７８）、Ｌｉｆｅ
Ｓｃｉｅｎｃｅｓ、３３． １４６７（１９８３）に記
載されている方法によって合成でき、（Ｖｌの化合物は
市販もされている。- Compounds represented by m and [Vl are, for example, Biochem, Biophys, Res, Comm
un,, 83. 385 (1978), Life
Sciences, 33. 1467 (1983), and the compound of (Vl) is also commercially available.
■ 保護アミノ酸を逐次伸長する方法としては、既知の
方法、すなわち、泉屋ら著「ペプチド合成の基礎と実験
」 （丸善）やＢｏｄａｎｓｚｋｙ著“ＰＲＩＮＣＩＰ
ＬＥＳ ＯＦ ＰＥＰＴＩＤＥ ５ＹＮＴＨＥＳＩＳ″
、“ＴＨＥ ＰＲＡＣＴＩＣＥＯＦ ＰＥＰＴＩＤＥ
５ＹＮＴＨＥＳＩＳ” （ＳｐｒｉｎｇｅｒＶｅｒｌａ
ｇ、 Ｎｅｗ Ｙｏｒｋ）に記載されている方法かいず
れも有効である。縮合反応の段階では、ＤＣＣ〜ａｄｄ
ｉｔｉｖｅ法、アシド法、混酸無水物法、活性エステル
法のいずれを採用してもよいか、１−ヒドロキシベンゾ
トリアゾールとジシクロへキンルカルホシイミトを併用
するＤＣＣ−ａｄｄｉｔｉｖｅ法か最も良好な結果を与
える。■ As a method for sequentially elongating protected amino acids, there are known methods, such as "Basics and Experiments of Peptide Synthesis" by Izumiya et al. (Maruzen) and "PRINCIP" by Bodanszky.
LES OF PEPTIDE 5YNTHESIS''
, “THE PRACTICE OF PEPTIDE
5YNTHESIS” (Springer Verla
Any of the methods described in J. G., New York) are effective. At the stage of condensation reaction, DCC~add
Which of the ative method, acid method, mixed acid anhydride method, or active ester method may be adopted, or the DCC-additive method using a combination of 1-hydroxybenzotriazole and dicyclohequine carfosiimite, which gives the best results. give.
■ −殺伐（Ｖｌｌおよび［ＶＩｌ）で表される化合物
は、−殺伐［ＩＶ］および［Ｖｌで表されるポリエチレ
ングリコール誘導体および保護ペプチドをこれらか溶解
する有機溶媒中で塩基存在下室温で攪拌、反応させるこ
とにより得られる。- Compounds represented by (Vll and [VIl) are prepared by stirring at room temperature in the presence of a base in an organic solvent that dissolves the polyethylene glycol derivatives and protected peptides represented by (IV) and [Vl; Obtained by reaction.
■ 保護基を脱保護するのに用いられる条件は、用いた
保護基の種類に大きく依存する。通常使用される脱保護
条件は、接触水素添加、トリフルオロ酢酸、無水フッ化
水素、トリフルオロメタンスルホン酸−チオアニソール
混合系、トリフルオロ酢酸−チオアニソール混合系等で
あるが、保護基の種類によってはさらに多様な手段か可
能である。また、目的物の精製は、ケルろ適法等を用い
ることにより行うことかできる。■ The conditions used to deprotect a protecting group are highly dependent on the type of protecting group used. Commonly used deprotection conditions include catalytic hydrogenation, trifluoroacetic acid, anhydrous hydrogen fluoride, trifluoromethanesulfonic acid-thioanisole mixed system, trifluoroacetic acid-thioanisole mixed system, etc., but depending on the type of protecting group. Even more diverse methods are possible. Moreover, the purification of the target product can be carried out by using the Kerro method or the like.
本発明において数平均分子量はゲルパーミェーションク
ロマトグラフィー（Ｇ Ｐ Ｃ）による測定結果をもと
に算出することかできる。In the present invention, the number average molecular weight can be calculated based on measurement results by gel permeation chromatography (GPC).
ＧＰＣの測定条件は以下のとおりである。The GPC measurement conditions are as follows.
カラム：ＴＳＫｇｅｌ （東洋曹達製）Ｇ１００ＯＨ
。Column: TSKgel (manufactured by Toyo Soda) G100OH
.
排除限界分子量 １０００
カラム寸法 ７．５１ＤＸ６００ｍｍ １本
Ｇ２０００Ｈ。Exclusion limit molecular weight 1000 Column dimensions 7.51DX600mm 1 G2000H.
排除限界分子量 １００００
カラム寸法 ７．５１Ｄ Ｘ ６００ ｍｍ
２本Ｇ２５００Ｈ。Exclusion limit molecular weight 10000 Column dimensions 7.51D x 600 mm
Two G2500H.
排除限界分子量 ２００００
カラム寸法 ７．５１Ｄ Ｘ６００ ｍｍ
１本溶媒、テトラヒドロフラン
流量：１ｍｌ／ｍｉロ
カラム温度＝４０°Ｃ
検出器：Ｕ〜’、Ｒ１併用
ＴＳＫスタンダードポリエチレンオキサイドで検量線を
作成。Exclusion limit molecular weight 20000 Column dimensions 7.51D x 600 mm
1 bottle of solvent, tetrahydrofuran Flow rate: 1 ml/mil Column temperature = 40°C Detector: U~', a calibration curve was created using TSK standard polyethylene oxide in combination with R1.
数平均分子量は、高分子学会編「高分子科学実験法」
（東京化学同人１９８１年）Ｐ、２０４〜２０８に記載
の一般的な方法、すなわち線分法を用いて計算した。得
られたクロマトクラムを等間隔のカウント（Ｄ）に分割
して１番目の高分子種のヘースラインからのピーク高さ
をＨｉとし、以下の関係式（１）を利用して求めた。The number average molecular weight is calculated from "Polymer Science Experimental Methods" edited by the Society of Polymer Science and Technology.
(Tokyo Kagaku Dojin 1981) P, 204-208, the general method described, that is, the line segment method, was used for calculation. The obtained chromatogram was divided into equally spaced counts (D), and the peak height from the Haese line of the first polymer species was defined as Hi, which was determined using the following relational expression (1).
ΣｉＮｉ
Σ１ＨｉＤ
Σ１（ＨｉＤ／Ｍｉ）
ΣＩＨ１
Σ１（Ｈｉ／Ｍｉ）
によって
Ｍ ｎ ＝□
Σｉ（Ｌ／Ｍｉ） （Ｈｉ／Σ１Ｈｊｉ・・・・・関
係式（１）
ここで、Ｎ１は１番目の高分子種の数を表わし、Ｍｌは
１番目の高分子種の分子量を表わす。（Ｍｉは前記の検
量線から求めることができる。）。ΣiNi Σ1HiD Σ1(HiD/Mi) ΣIH1 Σ1(Hi/Mi) M n =□ Σi(L/Mi) (Hi/Σ1Hji...Relational expression (1) Here, N1 is the first polymer It represents the number of species, and Ml represents the molecular weight of the first polymeric species (Mi can be determined from the above calibration curve).
本発明のペプチド含有ポリエチレングリコール誘導体は
、細胞接着性蛋白質のコア配列（ＥＩＬＤＶＰＳＴ４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、細胞接着性蛋白質のア
ゴニストまたはアンタコニストとして様々の生物活性を
示す。そのほかにも、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の抑制作用、
神経疾患治癒作用等の広範な生物活性か認められる。The peptide-containing polyethylene glycol derivative of the present invention has a core sequence of a cell adhesion protein (EILDVPST4), and adheres to cells via the core sequence in a similar mechanism to that of a cell adhesion protein. It exhibits various biological activities as an agonist or antagonist of
It has been recognized to have a wide range of biological activities, including curing effects on neurological diseases.
従って、本発明のペプチド含有ポリエチレングリコール
誘導体は、その少なくとも一種を、場合により慣用の担
体または医薬用製剤とともに、癌転移抑制剤、創傷治癒
剤、免疫調整剤、血小板凝集抑制剤または神経疾患治療
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、０．２ ｕ ｇ／ｋｇ
〜４００ｍｇ／ｋｇ）範囲で症状、年令、体重等に基
ついて決定される。Therefore, the peptide-containing polyethylene glycol derivative of the present invention can be used as a cancer metastasis inhibitor, a wound healing agent, an immunomodulator, a platelet aggregation inhibitor, or a neurological disease therapeutic agent, optionally together with at least one of the peptide-containing polyethylene glycol derivatives and a conventional carrier or pharmaceutical preparation. It is possible to administer it to patients as a drug. In particular, use as an animal cell adhesion inhibitor or platelet aggregation/adhesion inhibitor is preferred. The dose was 0.2 u g/kg
~400mg/kg) determined based on symptoms, age, body weight, etc.
本発明のペプチド含有ポリエチレングリコール誘導体は
、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か好ましい。そのような注
射用製剤を製造する場合、本発明のペプチド含有ポリエ
チレンクリコール誘導体を例えば、後記実施例で示すよ
うにＰＢＳ （Ｎａ）Ｉ２ＰＯｔ ｏ、ｏ ０５Ｍ、
ＮａＣ１０，０７Ｍ）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．ＩＮ程度の酢酸水
等に溶解した後、凍結乾燥製剤としてもよい。このよう
な製剤には、グリシンやアルブミン等の慣用の安定化剤
を添加してもよく、血中半減期を延長させる等の目的の
ために、コラ−ケンやリポソームを担体として用いても
よい。The peptide-containing polyethylene glycol derivative of the present invention is preferably administered by a method commonly used for peptide-based drugs, that is, by parenteral administration, such as intravenous administration, intramuscular administration, subcutaneous administration, etc. When producing such an injectable preparation, the peptide-containing polyethylene glycol derivative of the present invention may be mixed with PBS (Na)I2POt o, o 05M,
It may be dissolved in NaC10,07M) or physiological saline to form an injectable preparation, or 0.07M) or physiological saline. After dissolving in aqueous acetic acid or the like of about IN, it may be made into a lyophilized preparation. Conventional stabilizers such as glycine and albumin may be added to such preparations, and Kolaken and liposomes may be used as carriers for purposes such as prolonging the blood half-life. .
さらに、本発明のペプチド含有ポリエチレンクリコール
誘導体は、例えばリポソーム中に包含したマイクロカプ
セル剤とすれば、経口投与剤と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よれば、消化管
以外の粘膜から吸収させることも可能である。Furthermore, the peptide-containing polyethylene glycol derivative of the present invention can be made into an orally administered drug, for example, by incorporating it into a microcapsule in a liposome.
In the form of suppositories, sublingual tablets, nasal sprays, etc., it is possible to absorb the drug through mucous membranes other than the gastrointestinal tract.
実施例１ 以下に本発明の化合物（］）の合成例を示す。Example 1 A synthesis example of the compound (]) of the present invention is shown below.
化合物（１）を以下の合成経路で合成した。なお、アミ
ノ酸、各種保護基および脱保護試薬は通常用いられる略
号を使って表した。また、他の化合物例もここに例示し
た方法で合成できる。Compound (1) was synthesized using the following synthetic route. In addition, amino acids, various protecting groups, and deprotecting reagents are expressed using commonly used abbreviations. Other examples of compounds can also be synthesized by the methods exemplified here.
Ｂｚｌ ペンシル基、
ＴＦＡ：ｈリフルオロ酢酸、
Ｂｏｃ：ｔ−ブトキシカルホニル基、
ＨＯＢｔ：１−ヒドロキンヘンシトリアゾール、２ ペ
ンシルオキンカルホニル基、
ＤＣＣ・シンクロへキンルヵルボンイミド以下にそれぞ
れの合成法を記す。Bzl pencil group, TFA: h-lifluoroacetic acid, Boc: t-butoxycarbonyl group, HOBt: 1-hydroquinequinetriazole, 2 pensyleoquinecarbonyl group, DCC/synchrohequine carbonimide. The synthesis method is described.
（１ｂ）の合成
文献（Ｂｉｏｃｈｅｍ、Ｂｉｏｐｈｙｓ、Ｒｅｓ、Ｃｏ
ｍｍｕｎ、、 ８３． ３８５ （１９７８）、Ｌｉ
ｆｅＳｃｉｅｎｃｅｓ、３３． １４６７（１９８３）
）に記載の方法により、アルドリッチ社から購入した平
均分子量５，０００のポリエチレングリコールモノメチ
ルエーテル（ｌａ）（１０ｇ、２ｍｍｏｌ）を充分乾燥
し、トルエン（１００ｍｌ）、炭酸ナトリウム（５ｇ）
、塩化シアヌル（１，１ｇ、 ６ｍｍｏｌ）を加え、
室温で６０分間攪拌した。反応液か室温になるまで放冷
した後にろ過し、ろ液にヘキサンを加えて結晶化した。(1b) synthesis literature (Biochem, Biophys, Res, Co
mmun,, 83. 385 (1978), Li
feSciences, 33. 1467 (1983)
), polyethylene glycol monomethyl ether (LA) (10 g, 2 mmol) with an average molecular weight of 5,000 purchased from Aldrich Co. was sufficiently dried, followed by toluene (100 ml) and sodium carbonate (5 g).
, cyanuric chloride (1.1 g, 6 mmol) was added,
Stirred at room temperature for 60 minutes. The reaction solution was allowed to cool to room temperature and then filtered, and hexane was added to the filtrate for crystallization.
さらにトルエン・アセトン・ヘキサンの溶媒系からこの
結晶を再結晶させ精製し、白色粉末（７ｇ）を得た。Further, the crystals were recrystallized and purified from a solvent system of toluene, acetone, and hexane to obtain a white powder (7 g).
（１ｄ）の合成
国産化学（株から購入した（ｌｃ）（３０，９ｇ、０、
１ｍｏｌ）、トリエチルアミン（１４ｍｌ）、臭化ベ
ンジル（１７，１ｇ）、酢酸エチル（２００ｍｌ）の混
合物を３時間加熱這流した。反応液を室温になるまで放
冷した後に、ＩＮ炭酸水素ナトリウム水溶液、飽和食塩
水各２００ｍ１で洗浄し、無水硫酸ナトリウムで乾燥し
た。硫酸ナトリウムをろ過して除き、ろ液を減圧濃縮し
て無色油状物を得た。Synthesis of (1d) (lc) (30.9g, 0,
A mixture of 1 mol), triethylamine (14 ml), benzyl bromide (17.1 g), and ethyl acetate (200 ml) was heated and poured for 3 hours. After the reaction solution was allowed to cool to room temperature, it was washed with 200 ml each of IN aqueous sodium bicarbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a colorless oil.
この反応混合物をシリカゲルクロマトクラフィー（溶出
液ヘキサンノ酢酸エチル４０：１）で精製し、 （ｌｄ
）（３９ｇ）を得た。The reaction mixture was purified by silica gel chromatography (eluent: ethyl hexanoacetate 40:1), (ld
) (39 g) was obtained.
（１ｅ）の合成
（ｌｄ） （７，９９ｇ、 ２０ｍｍｏｌ）を塩化
メチレン２０ｍ１に溶解し、ＴＦＡ２０ｍｌを加えて室
温で３０分間攪拌した。溶媒を減圧留去した後にクロロ
ホルム１００ｍ１を加え、飽和食塩水各１００ｍ１で数
回洗浄し、無水硫酸ナト（ノウムで乾燥した。Synthesis of (1e) (ld) (7.99 g, 20 mmol) was dissolved in 20 ml of methylene chloride, 20 ml of TFA was added, and the mixture was stirred at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, washed several times with 100 ml each of saturated brine, and dried over anhydrous sodium sulfate.
硫酸ナトリウムをろ過して除き、ろ液を減圧濃縮して無
色油状物を得た。これとＢｏｃ、５ｅｒ（Ｂｚｌ）（国
産化学（掬から購入） （５，９０ｇ。The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a colorless oil. This and Boc, 5er (Bzl) (Kokusan Kagaku (purchased from Kiki) (5.90g).
２０ｍｍｏｌ）、ＤＣＣ（４，５４ｇ、 ２２ｍｍｏ
ｌ）、ＨＯＢ ｔ （２，７６ｇ、 １８ｍｍｏｌ）
、Ｎ−メチルモルホリン（２，２ｍｌ、２０ｍｍｏｌ
） ＤＭＦ ８０ｍｌの混合物を０℃で３時間、さらに
室温で］２時間攪拌した。ＤＣＵｒｅａを除去した後に
溶媒を減圧留去し、クロロホルム１００ｍ１を加え、Ｉ
Ｎ炭酸水素ナトリウム水溶液、飽和食塩水各２００＋ｎ
ｌで一洗浄し、無水硫酸ナトリウムで乾燥した。硫酸ナ
トリウムをろ過して除き、ろ液を減圧濃縮し、ｔｉｅ（
薄層クロマトグラフィー）で単独スポットなので精製す
ることなく次の反応に用いた。20mmol), DCC (4.54g, 22mmol)
l), HOB t (2,76g, 18mmol)
, N-methylmorpholine (2.2 ml, 20 mmol
) A mixture of 80 ml of DMF was stirred for 3 hours at 0° C. and then for a further 2 hours at room temperature. After removing DCUrea, the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and I
N sodium bicarbonate aqueous solution, saturated saline solution each 200+n
The solution was washed with 1 ml of water and dried with anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and the
Since it was a single spot (thin layer chromatography), it was used in the next reaction without purification.
（ｉｆ）の合成
（１ｅ）の合成と同様に行った。ＴＦＡで脱保護した後
に、ＢｏｃＰｒｏ （国産化学■から購入）（４，３０
ｇ、 ２０ｍｍｏｌ） 、ＤＣＣ（４，５４ｇ。Synthesis of (if) Synthesis was carried out in the same manner as in (1e). After deprotecting with TFA, BocPro (purchased from Kokusan Kagaku) (4,30
g, 20 mmol), DCC (4.54 g.
２２ｍｍｏｌ） 、ＨＯＢ ｔ （２，７６ｇ、 ］
８ｍｍｏｌ）、Ｎ−メチルモルホリン（２，２ｍＬ
２０ｍｍｏｌ）、ＤＭＦ８０ｍｌを加えて縮合反応し
た。ｔｉｃで単独スポットなので精製することなく次の
反応に用いた。22 mmol), HOB t (2,76 g, ]
8 mmol), N-methylmorpholine (2.2 mL
20 mmol) and 80 ml of DMF were added to perform a condensation reaction. Since it was a single tic spot, it was used in the next reaction without purification.
ユ上ヱＬΩ登式
（１ｅ）の合成と同様に行った。ＴＦＡで脱保護した後
に、ＢｏｃＶａｌ（国産化学■から購入）（４，３５ｇ
、２０ｍｍｏｌ） 、ＤＣＣ（４，５４ｇ。The synthesis was carried out in the same manner as the synthesis of the formula (1e). After deprotecting with TFA, BocVal (purchased from Kokusan Kagaku ■) (4.35 g
, 20 mmol), DCC (4.54 g.
２２ｍｍｏｌ） 、ＨＯＢ ｔ （２，７６ｇ、
ｌ ８ｍｍｏｌ） 、Ｎ−メチルモルホリン（２，２
ｍｌ、 ２０ｍｍｏｌ）、ＤＭＦ８０ｍｌを加えて縮
合反応した反応混合物をシリカゲルクロマトグラフィー
（溶出液クロロホルム１メタノール９・ｌ）で精製し、
（１ｇ）（１３，９ｇ）を得た。22 mmol), HOB t (2,76 g,
l 8 mmol), N-methylmorpholine (2,2
ml, 20 mmol) and 80 ml of DMF were added to perform a condensation reaction, and the reaction mixture was purified by silica gel chromatography (eluent: chloroform, 1 methanol, 9 l),
(1g) (13.9g) was obtained.
（１ｈ）の合成
（ｌｅ）の合成と同様に行った。（Ｉｇ）（１３、９ｇ
＋ １８ｍｍｏｌ）とＴＦＡで脱保護した後に、Ｂｏ
ｃＡｓｐ （ＯＢｚ ］） （５，８２ｇ、 ］
８ｍｍｏｌ）、ＤＣＣ（４，０８ｇ、 １９． ８ｍ
ｍｏｌ） 、ＨＯＢ ｔ（２，４８ｇ、 １６． ２
ｍｍｏｌ） 、Ｎ−メチルモルホリン（２，０ｍｌ、
１８ｍｍｏｌ） 、ＤＭＦ ８０ｍｌを加えて縮合反
応した。反応混合物をシリカゲルクロマトクラフィー（
溶出液クロロホルム１メタノール９ ｌ）で精製し、（
ｌｈ）（１６，７ｇ）を得た。Synthesis of (1h) Synthesis was carried out in the same manner as the synthesis of (le). (Ig) (13,9g
+ 18 mmol) and TFA, then Bo
cAsp (OBz ]) (5,82g, ]
8mmol), DCC (4.08g, 19.8m
mol), HOB t(2.48g, 16.2
mmol), N-methylmorpholine (2.0 ml,
18 mmol) and 80 ml of DMF were added to perform a condensation reaction. The reaction mixture was subjected to silica gel chromatography (
Purify with eluent chloroform 1 methanol 9 l) and (
lh) (16.7 g) was obtained.
（１１）の合成
（ｌｅ）の合成と同様に行った。 （ｌｈ）（１４７、
ｇ＋ １５ｍｍｏｌ）をＴＦＡで脱保護した後に、Ｂ
ｏｃＬｅｕ Ｃ３，４７ｇ、 １５ｍｍｏｌ）
、ＤＣＣ（３，４０ｇ、 １６． ５ｍｍｏｌ） 、
ＨＯＢ ｔ （２゜０ ？ ｇ、 １３． ５ｍｍ
ｏｌ） 、Ｎ−メチルモルホリン（１，７ｍｌ、 １
５ｍｍｏｌ） 、ＤＭＦ ７０ｍｌを加えて縮合反応し
た。反応’ｔｌ ＝物をノリカケルクロマトグラフィー
（溶出液クロロホルムｌメタノール９１）で精製し、（
ｌｉ）（１５，６ｇ）を得た。Synthesis of (11) The synthesis was carried out in the same manner as the synthesis of (le). (lh) (147,
g+ 15 mmol) with TFA, then B
ocLeu C3, 47g, 15mmol)
, DCC (3.40g, 16.5mmol),
HOB t (2゜0?g, 13.5mm
ol), N-methylmorpholine (1.7 ml, 1
5 mmol) and 70 ml of DMF were added to perform a condensation reaction. The reaction 'tl = product was purified by Norikakel chromatography (eluent: chloroform/methanol 91), and (
li) (15.6 g) was obtained.
（ｌ ）の合成
（１ｅ）の合成と同様に行った。 （ｌｉ）（１０、９
ｇ、 １０ｍｍｏｌ）をＴ Ｆ Ａで脱保護した後に
、Ｂｏｃ Ｉ Ｉ ｅ （２，３１ｇ、 ｌ Ｏ＋ｎ
＋＋＋ｏｌ） 、ＤＣＣ（２，２７ｇ、 ｌ １ｍｍ
ｏｌ） 、ＨＯＢｔ （１，３８ｇ、 ９ｍｍｏｌ）
、Ｎ−メチルモル不リン（１，ｉｍ！。Synthesis of (l) Synthesis was carried out in the same manner as in (1e). (li) (10,9
g, 10 mmol) with TFA, and then Boc II e (2,31 g, l O+n
+++ol), DCC (2,27g, l 1mm
ol), HOBt (1.38g, 9mmol)
, N-methylmorphine (1,im!.
１０ｍｍｏｌ） 、ＤＭＦ ５０ｍｌを加えて縮合反応
した。10 mmol) and 50 ml of DMF were added to perform a condensation reaction.
反応混合物をシリカゲルクロマトクラフィ−（溶出液ク
ロロホルム１メタノール９１）で精製し、（ｌｊ）（１
１，４ｇ）を得た。The reaction mixture was purified by silica gel chromatography (eluent chloroform 1 methanol 91) to give (lj) (1
1.4 g) was obtained.
（１に’ｌの合成
（１ｅ）の合成と同様に行った。（ｌｊ）（６゜０ｇ、
５ｍｍｏｌ）をＴ Ｆ Ａで脱保護した後に、Ｂｏ
ｃＧｌｕ （ＯＢｚ ｌ） （１，６９ｇ、 ５
ｍｍｏｌ）、ＤＣＣ（１，１３ｇ、 ５． ５ｍｍｏ
ｌ） 、ＨＯＢｔ（０，６９ｇ、 ４． ５ｍｍｏｌ
） 、Ｎ−メチルモルホリン（０，５５ｍｌ、 ５ｍ
ｍｏｌ） 、ＤＭＦ ３０ｍｌを加えて縮合反応した。(In 1, the synthesis of 'l was carried out in the same manner as in (1e). (lj) (6°0g,
After deprotecting 5 mmol) with TFA, Bo
cGlu (OBz l) (1,69g, 5
mmol), DCC (1.13g, 5.5mmol)
l), HOBt (0.69g, 4.5mmol
), N-methylmorpholine (0.55 ml, 5 m
mol) and 30 ml of DMF were added to perform a condensation reaction.
反応混合物をシリカゲルクロマトクラフィ−（溶出液ク
ロロホルムｌメタノール９：ｌ）で精製し、（Ｉｋ）（
６，４ｇ）を得た。The reaction mixture was purified by silica gel chromatography (eluent: chloroform/methanol 9:l) to give (Ik) (
6.4 g) was obtained.
（１１）の合成
（１ｅ）の合成と同様に行った。（ｌｋ）（３゜５６
ｇ、 ２． ５ｍｍｏｌ）をＴＦＡで脱保護した後に
、ＢｏｃＧｌｙ （０，４４ｇ、 ２． ５＋ｎ＋ｎ
ｏｌ） 、ＤＣＣ（０，５７ｇ、 ２．７５ｍｍｏｌ
） 、ＨＯＢ ｔ （０゜３１ ｇ、 ２． ２５ｍ
ｍｏｌ） 、Ｎ−メチルモルホリン（０，２７ｍｌ、
２． ５ｍｍｏｌ） 、ＤＭＦ ２０ｍｌを加えて縮
合反応した。反応混合物をシリカゲルクロマトクラフィ
ー（溶出液クロロホルムｌメタノール９・ｌ）で精製し
、（１，１）（３，６３ｇ）を得た。Synthesis of (11) Synthesis was carried out in the same manner as in (1e). (lk) (3゜56
g, 2. BocGly (0.44 g, 2.5+n+n
ol), DCC (0.57g, 2.75mmol
), HOB t (0°31 g, 2.25m
mol), N-methylmorpholine (0.27 ml,
2. 5 mmol) and 20 ml of DMF were added to perform a condensation reaction. The reaction mixture was purified by silica gel chromatography (eluent chloroform/methanol 9.1) to obtain (1,1) (3.63 g).
（１ｍ）の合成
（ｌｅ）（１，４８ｇ、１ｍｍｏｌ）を塩化メチレン１
０ｍ１に溶解し、トリフルオロ酢酸１０ｍ１を加えて室
温で３０分間攪拌した。溶媒を減圧留去した後にクロロ
ホルム１００ｍ１を加え、ｌＮ炭酸水素すｈ ’）ラム
水溶液、飽和食塩水容１００ｍ１で数回洗浄し、無水硫
酸ナトｌ）ラムて乾燥した。硫酸ナトリウムをろ過して
除き、ろ液を減圧濃縮して白色粉末を得た。これと（ｌ
ｂ）（２，５ｇ）、トリエチルアミン（０，１ｇ）、ク
ロロホルム５０ｍ１の混合物を室温で２４時間攪拌した
。ゲルろ過（Ｓｅｐｈａｄｅｘ ＬＨ−６０）により
精製し、（１ｍ）を３．５ｇ得た。Synthesis of (1m) (le) (1.48g, 1mmol) was added in methylene chloride 1
10 ml of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and the mixture was washed several times with a 1N aqueous solution of hydrogen carbonate and 100 ml of saturated saline, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a white powder. This and (l
A mixture of b) (2.5 g), triethylamine (0.1 g) and 50 ml of chloroform was stirred at room temperature for 24 hours. Purification was performed by gel filtration (Sephadex LH-60) to obtain 3.5 g of (1m).
（１）の合成
（１ｍ）（３，５ｇ）を酢酸５０ｍ１に溶解し、ｌＯ％
０％パランラム１ｇを加え、室温で常圧加水素分解を２
４時間行った。触媒をセライトを用いてろ別し、溶媒を
減圧留去した。ゲルろ過（Ｓｅｐｈａｄｅｘ ＬＨ−
６０）により精製し、（１）を２゜９ｇ得た。Synthesis (1) (1m) (3.5g) was dissolved in 50ml of acetic acid, and 1O%
Add 1 g of 0% paranrum and hydrogenolyze at room temperature for 2 hours.
I went for 4 hours. The catalyst was filtered off using Celite, and the solvent was distilled off under reduced pressure. Gel filtration (Sephadex LH-
60) to obtain 2.9 g of (1).
アミノ酸分析 Ｇｌｕ（１，００）、Ｉｃｅ（０，９８
）、Ｌｅｕ （１，０２） 、Ａｓｐ （ｉｏ Ｉ
）、〜’ａｌ （１，０４）、Ｐｒｏ （０，９７
）Ｓｅｒ （０，８２） 、Ｔｈｒ （０，８５
）数平均分子量・７，０００
実施例２〜４
実施例１記載の方法にしたかって、化合物（２）、（３
）及び（４）を合成した。その物性値を表１に示す。Amino acid analysis Glu (1,00), Ice (0,98
), Leu (1,02), Asp (io I
), ~'al (1,04), Pro (0,97
)Ser (0,82), Thr (0,85
) Number average molecular weight 7,000 Examples 2 to 4 Compounds (2) and (3
) and (4) were synthesized. The physical property values are shown in Table 1.
表 １
製剤例
生理食塩水に、本発明のペプチド含有ポリエチレングリ
コール誘導体（］）を１００μｇ／ｍｌの濃度で溶解し
て、注射用製剤を調製した。この製剤は、動物細胞の接
着阻害剤及び血小板凝集・粘着抑制剤として使用可能で
ある。Table 1 Formulation Example The peptide-containing polyethylene glycol derivative (]) of the present invention was dissolved in physiological saline at a concentration of 100 μg/ml to prepare an injectable preparation. This preparation can be used as an animal cell adhesion inhibitor and platelet aggregation/adhesion inhibitor.
試験例
「細胞接着阻害活性の測定−
本発明のペプチド含有ポリエチレングリコール誘導体は
細胞のフィブロネクチンに対する接着を阻害する。その
活性測定方法を以下に示す。ここ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ｒＭｅｔｈｏｄｓｉｎＥｎｚｙｍｏｌｏｇｙ＝
８２ ８０３ （１９８１）、特開平１−３０９６８
２、同２ １７４７９７に開示されている。Test Example "Measurement of Cell Adhesion Inhibitory Activity - The peptide-containing polyethylene glycol derivative of the present invention inhibits cell adhesion to fibronectin. The method for measuring the activity is shown below. The competition method used here is basically a biochemical method. It is widely used in the field, for example rMethods in Enzymology=
82 803 (1981), JP 1-30968
2, No. 2 174797.
実験方法
１、吸着プレートの作製
市販のフィブロネクチン（ヒト由来、コスモハイオ（掬
から購入）をＰＢＳて１０μｇ／’ｍｌに溶解し、その
溶液５０ｍ１を９６ウエルのポリスチレレプレートにい
れ、４℃で一晩保温し、コーティングした。次に非特異
吸着を防ぐ目的で生血清アルブミン（ＢＳＡ １％）
を加え、３７°Ｃ１１時間保温し、その後通常の洗浄操
作（ＰＢＳ）を行い充分に水きりして吸着プレートを作
製した。Experimental method 1. Preparation of adsorption plate Commercially available fibronectin (human origin, purchased from Cosmohio (Kikki)) was dissolved in PBS to 10 μg/'ml, and 50 ml of the solution was placed in a 96-well polystyrene plate, overnight at 4°C. It was kept warm and coated.Next, raw serum albumin (BSA 1%) was added to prevent non-specific adsorption.
was added and kept at 37° C. for 11 hours, followed by a normal washing operation (PBS) and thorough draining to prepare an adsorption plate.
２、接着阻害実験
Ｄｕｌｂｅｃｃｏｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅｓ
Ｍｅｄｉｕｍて溶解したペプチド含有ポリエチレンク
リコール誘導体溶液５０ｍ１を上記方法で作成したプレ
ートにいれ、そこへＮＲＫ４９Ｆ （ＩＸ ｌ Ｏ″
ｃｅｌｌｓ／ｍｌ） ９に液を５０ｍ１加え、３７℃で
一時間保温し細胞を接着させた。ＰＢＳで３回洗浄し、
未接着の細胞を除いた後、０，０２５％ＥＤＴＡトリプ
ノン溶液て接着した細胞を剥離し、２ ／’Ｏトリパン
ブルーで染色して細胞数を計測した。結果を下記表２に
示す。表中、ＥＩＬＤＶＰＳＴは、グルタミン酸−イソ
ロイ／ンーロインンーアスパラキン酸−ノ＼リン−プロ
リン−セリン−トレオニンのオクタペプチドを表す。2. Adhesion inhibition experiment Dulbeccos Modified Eagles
Pour 50 ml of a peptide-containing polyethylene glycol derivative solution dissolved in Medium into the plate prepared in the above method, and add NRK49F (IX l O''
50 ml of the solution was added to 9 (cells/ml) and incubated at 37° C. for 1 hour to allow the cells to adhere. Wash 3 times with PBS,
After removing non-adherent cells, the adhered cells were peeled off with a 0,025% EDTA trypnone solution, and the number of cells was counted by staining with 2/'O trypan blue. The results are shown in Table 2 below. In the table, EILDVPST represents an octapeptide of glutamic acid-isoroy/n-roin-aspartic acid-no\phosphorus-proline-serine-threonine.
表 ２
Ｉ フィブロネクチンに対する細胞の接着率（％））
「血小板凝集阻害活性試験」
本発明のペプチド含有ポリエチレンクリコール誘導体の
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定した。以下にその実験方法を示
す。Table 2 I Cell adhesion rate (%) to fibronectin
"Platelet aggregation inhibitory activity test" The platelet aggregation inhibitory effect of the peptide-containing polyethylene glycol derivative of the present invention in the IN VITRO system was tested using human platelet-rich plasma. The experimental method is shown below.
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（１０００ｒｐｍ、１０分）し、上層を多
血小板血漿として分取した。この血漿２００μｌにペプ
チド含有ポリエチレングリコール誘導体溶液２５１ｔ
ｌ （ｍａＸｌ 、 ５ｍｇ ’ｍｌ）を加え、３
分間３７°Ｃてインキュ・＼−トしたのち、２’０−５
０μＭＡＤＰ（アデノノンニリン酸）溶液あるいは２０
０μｇ／ｍｌのコラーゲン溶液を２５μｌ加えて凝集の
程度を、アクリボ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。Experimental method 1/9 volume of 3.8% sodium citrate was added to fresh human blood, centrifuged (1000 rpm, 10 minutes), and the upper layer was collected as platelet-rich plasma. Add 251 t of peptide-containing polyethylene glycol derivative solution to 200 μl of this plasma.
l (maXl, 5 mg 'ml) and 3
After incubating at 37°C for 2'0-5 minutes,
0 μM ADP (adenonone diphosphoric acid) solution or 20
25 μl of 0 μg/ml collagen solution was added and the degree of aggregation was assayed by measuring the permeability using an acribometer.
結果を表３に示す。The results are shown in Table 3.
凝集阻害”Ｉ （１−Ｔ／Ｔｏ）Ｘ １００％Ｔ、＝ペ
プチド含有ポリエチレンクリコール誘導体非添加時の透
過度
Ｔ−ペプチド含有ポリエチレンクリコール誘導体添加時
の透過度
表 ３Aggregation inhibition "I (1-T/To)
Claims (6)
エチレングリコール誘導体またはその塩。 ▲数式、化学式、表等があります▼…［ I ］ ただし、ｎは１から１５０までの整数を表 す。また、Ｒ＾１は下記一般式［II］で表されるペプチ
ド残基を示す。 （［Ｘ］Ｇｌｕ−Ｉｌｅ−Ｌｅｕ−Ａｓｐ−Ｖａｌ−Ｐ
ｒｏ−Ｓｅｒ−Ｔｈｒ−［Ｙ］）＿ｍ…［II］ 式中、Ｇｌｕ、Ｉｌｅ、Ｌｅｕ、Ａｓｐ、 Ｖａｌ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒはそれぞれグルタミン
酸、イソロイシン、ロイシン、アスパラギン酸、バリン
、プロリン、セリン、トレオニン残基を表す。［Ｘ］、
［Ｙ］は存在するかあるいは存在しないアミノ酸を表す
。 ｍは１から５まで整数を表す。 なお、このペプチド残基の［Ｘ］（［Ｘ］ が存在しない場合はＧｌｕ）または［Ｙ］ （［Ｙ］が存在しない場合はＴｈｒ）がトリアジン環と
結合している。(1) A peptide-containing polyethylene glycol derivative or a salt thereof represented by the following general formula [I]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[I] However, n represents an integer from 1 to 150. Moreover, R^1 represents a peptide residue represented by the following general formula [II]. ([X]Glu-Ile-Leu-Asp-Val-P
ro-Ser-Thr-[Y])_m...[II] In the formula, Glu, He, Leu, Asp, Val, Pro, Ser, and Thr are respectively glutamic acid, isoleucine, leucine, aspartic acid, valine, proline, serine, Represents a threonine residue. [X],
[Y] represents an amino acid present or absent. m represents an integer from 1 to 5. Note that [X] (Glu when [X] is absent) or [Y] (Thr when [Y] is absent) of this peptide residue is bonded to the triazine ring.
エチレングリコール誘導体またはその塩。 ▲数式、化学式、表等があります▼…［III］ ただし、ｎは１から１５０までの整数を表 す。また、Ｒ＾２は請求項（１）の一般式［II］で表さ
れるペプチド残基を示す。(2) A peptide-containing polyethylene glycol derivative or a salt thereof represented by the following general formula [III]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[III] However, n represents an integer from 1 to 150. Moreover, R^2 represents a peptide residue represented by the general formula [II] of claim (1).
るアミノ酸残基を表し、［Ｘ］、［Ｙ］がセリン、グリ
シン、バリン、アスパラギン、プロリン、システイン、
トレオニン残基から選択されるアミノ酸残基である請求
項（１）記載のペプチド含有ポリエチレングリコール誘
導体またはその塩。(3) In general formula [II], [X] and [Y] represent existing amino acid residues, and [X] and [Y] are serine, glycine, valine, asparagine, proline, cysteine,
The peptide-containing polyethylene glycol derivative or its salt according to claim (1), which is an amino acid residue selected from threonine residues.
るアミノ酸残基を表し、［Ｘ］、［Ｙ］がセリン、グリ
シン、バリン、アスパラギン、プロリン、システイン、
トレオニン残基から選択されるアミノ酸残基である請求
項（２）記載のペプチド含有ポリエチレングリコール誘
導体またはその塩。(4) In general formula [II], [X] and [Y] represent existing amino acid residues, and [X] and [Y] are serine, glycine, valine, asparagine, proline, cysteine,
The peptide-containing polyethylene glycol derivative or its salt according to claim (2), which is an amino acid residue selected from threonine residues.
チド含有ポリエチレングリコール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。(5) An animal cell adhesion inhibitor comprising the peptide-containing polyethylene glycol derivative or its salt according to any one of claims (1) to (4) as an active ingredient.
チド含有ポリエチレングリコール誘導体またはその塩を
有効成分とする血小板凝集・粘着抑制剤。(6) A platelet aggregation/adhesion inhibitor comprising the peptide-containing polyethylene glycol derivative or its salt according to any one of claims (1) to (4) as an active ingredient.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2316441A JPH04187698A (en) | 1990-11-21 | 1990-11-21 | Peptide-containing polyethylene glycol derivative and its use |
| US07/780,081 US5229366A (en) | 1990-10-23 | 1991-10-21 | Peptide-containing polyethylene glycol derivatives and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2316441A JPH04187698A (en) | 1990-11-21 | 1990-11-21 | Peptide-containing polyethylene glycol derivative and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04187698A true JPH04187698A (en) | 1992-07-06 |
Family
ID=18077125
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2316441A Pending JPH04187698A (en) | 1990-10-23 | 1990-11-21 | Peptide-containing polyethylene glycol derivative and its use |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04187698A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004535828A (en) * | 2001-07-27 | 2004-12-02 | エボテック・オーアーイー・アーゲー | Methods for preventing particle adhesion |
-
1990
- 1990-11-21 JP JP2316441A patent/JPH04187698A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004535828A (en) * | 2001-07-27 | 2004-12-02 | エボテック・オーアーイー・アーゲー | Methods for preventing particle adhesion |
| JP4663979B2 (en) * | 2001-07-27 | 2011-04-06 | エボテック・テヒノロギーズ・ゲーエムベーハー | Method for preventing particle adhesion |
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