JP4228498B2 - Heparin adsorbent and method for removing heparin using the same - Google Patents
Heparin adsorbent and method for removing heparin using the same Download PDFInfo
- Publication number
- JP4228498B2 JP4228498B2 JP2000035244A JP2000035244A JP4228498B2 JP 4228498 B2 JP4228498 B2 JP 4228498B2 JP 2000035244 A JP2000035244 A JP 2000035244A JP 2000035244 A JP2000035244 A JP 2000035244A JP 4228498 B2 JP4228498 B2 JP 4228498B2
- Authority
- JP
- Japan
- Prior art keywords
- heparin
- water
- polylysine
- adsorbent according
- insoluble
- 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.)
- Expired - Fee Related
Links
Landscapes
- Compositions Of Macromolecular Compounds (AREA)
- Biological Depolymerization Polymers (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- External Artificial Organs (AREA)
Description
【０００１】
【発明の属する技術分野】
本発明は、ヘパリン吸着体、及びそれを用いたヘパリンの除去方法に関する。更に詳しくは、リガンドがε−ポリリジンであるヘパリン吸着体、およびそれを用いたヘパリンの除去方法に関する。
【０００２】
【従来の技術】
ヘパリンは平均分子量5,000〜30,000のムコ多糖の１種であり、強力で安定した抗凝固作用を持ち、投与法も簡便で経済的にも安価であることから、抗血液凝固剤として人工透析などの体外循環治療法に使用されている。
しかしながら、ヘパリンは、長期にわたる、または大量の投与により出血助長作用を促し、また、血小板の活性化、骨多孔症、および動脈硬化などを進行させるといった問題を発生させる場合があった。
【０００３】
前述のような、血液中に投与した際のヘパリンの問題点を回避する方法としては、へパリンに親和性を持つ陽性タンパクである硫酸プロタミンを投与し中和する方法が従来行われてきた。この他にもへパリン代替物質となる抗凝固剤も開発検討されているが、安全な投与量や投与法が確立されていないのが現状である。
【０００４】
【発明が解決しようとする課題】
ヘパリンの除去方法としては、架橋アガロース担体にフィブロネクチンの還元およびそのアルキル化誘導体をリガンドとして結合させたへパリン吸着体を用いたヘパリンの除去方法などを挙げることができるが、医療用として用いる際にはヘパリン吸着体としての能力が十分ではなく、架橋アガロース担体を用いたヘパリンの除去方法は、へパリン吸着体として効率が高いとはいえない場合があった。
【０００５】
本発明者らは、前述の従来の問題点に鑑み、鋭意検討を重ねた結果、リガンドがε−ポリリジンである吸着体、または、ε−ポリリジンを水不溶性担体に結合してなるヘパリン吸着体であれば、ヘパリンの吸着能力が高く、この吸着体を用いれば、ヘパリンを含有する液体から、高い効率でヘパリンを除去することが可能であることを知見し、この知見に基づいて本発明を完成させた。
【０００６】
【課題を解決するための手段】
本発明は下記の構成からなる。
（１）リガンドがε−ポリリジンであるヘパリン吸着体。
【０００７】
（２）ε−ポリリジンを水不溶性担体に結合してなるヘパリン吸着体。
【０００８】
（３）ε−ポリリジンがストレプトマイセス属細菌により生産されたものである前記第１項または第２項記載のヘパリン吸着体。
【０００９】
（４）ε−ポリリジンの平均分子量が５００〜１，０００，０００の範囲である前記第１項〜第３項の何れか１項記載のヘパリン吸着体。
【００１０】
（５）ε−ポリリジンの平均分子量が１，０００〜１０，０００の範囲である前記第１項〜第３項の何れか１項記載のヘパリン吸着体。
【００１１】
（６）水不溶性担体が水不溶性球状粒子である前記第２項記載のヘパリン吸着体。
【００１２】
（７）水不溶性球状粒子の真球度が0.9以上である前記第６項記載のヘパリン吸着体。
【００１３】
（８）水不溶性球状粒子が球状セルロース粒子である前記第６項または第７項記載のヘパリン吸着体。
【００１４】
（９）球状セルロース粒子が、平均粒径50〜2000μｍを有するものである前記第８項記載のヘパリン吸着体。
【００１５】
（１０）球状セルロース粒子が、排除限界分子量50万〜500万を有するものである前記第８項または第９項記載のヘパリン吸着体。
【００１６】
（１１）球状セルロース粒子が、排除限界分子量80万〜300万を有するものである前記第８項または第９項記載のヘパリン吸着体。
【００１７】
（１２）水不溶性担体が水不溶性中空糸である前記第２項記載のヘパリン吸着体。
【００１８】
（１３）水不溶性担体が水不溶性膜である前記第２項記載のヘパリン吸着体。
【００１９】
（１４）ε−ポリリジンと水不溶性担体とが、反応性の官能基を介して結合している前記第２項〜第１３項の何れか１項記載のヘパリン吸着体。
【００２０】
（１５）前記第１項〜第１５項の何れか１項記載のヘパリン吸着体を用いることを特徴とす
【００２１】
【発明の実施の形態】
以下、本発明を詳細に説明する。
本願第１の発明は、ε−ポリリジンをリガンドとして用いたヘパリン吸着体である。本発明においてはリガンドがε−ポリリジンであれば、該リガンドが固体の担体に結合していても、固体の担体に結合していなくても構わない。つまり、本発明のヘパリン吸着体は、リガンドであるε−ポリリジン自身がヘパリン吸着体であってもよく、また、水不溶性の担体などに結合した状態であってもよい。
【００２２】
また、本願第２の発明は、ε−ポリリジンを水不溶性担体に結合してなるヘパリン吸着体である。
【００２３】
本発明に用いるε−ポリリジンは、ストレプトマイセス属細菌により生産された微生物由来のものであっても、化学合成によって得られたものであっても構わない。
【００２４】
その中でもストレプトマイセス属細菌により生産された微生物由来のε−ポリリジンは、生分解性があることから生体適合性が高く、さらに、安価で大量入手が可能であることから、本発明に好ましく使用することができる。
【００２５】
本発明に用いるポリリジンの分子量は、取り扱いの容易さの面から、５００〜１，０００，０００の範囲のものであることが好ましく、特に好ましくは、１，０００〜１０，０００の範囲である。
【００２６】
本願第２の発明に使用することができる水不溶性担体とは、担体を構成する成分が非水溶性、あるいは水溶性の物質を架橋反応などの処理を行うことにより水不溶性とした物質からなる担体で、具体的には、架橋アガロース、セルロース、およびポリアクリルアミドなどを挙げることができる。
また、本発明に使用する水不溶性担体の形状は、球状粒子、中空糸、膜など、特に限定されるものではないが、その中でも球状粒子である場合には、製造、取り扱いが容易であり、担体あたりに結合するリガンド量も多く、本発明に好ましく使用することができる。
【００２７】
中空糸状の担体とは内部に連続または不連続の空洞を持つ繊維状の担体を示すもので、紡糸液に発泡剤を添加、または特殊な口金などを用いて内部に空洞を形成させたものである。
【００２８】
膜状の担体とは市販のメンブランフィルターのように平板状で多孔を有し、一定の範囲の排除限界分子量を持つもののことである。
【００２９】
本発明に使用する水不溶性球状粒子は、真球度が高いものであることが好ましい。真球度が高いとカラム等に充填した場合、均一に充填することが容易である。
本発明で云うところの真球度とは、粒子の最短径（短径）と最長径（長径）との比（短径／長径）であり、この値が１．０に近づくほど真球度が高いことを示す。
本発明に使用する水不溶性球状粒子の真球度は、０．９以上であることが好ましく、より好ましくは０．９５以上である。
【００３０】
水不溶性球状粒子の中でも、球状セルロース粒子は、安価で生体適合性が高く、また、従来ヘパリン除去用吸着体の担体として用いられてきた架橋アガロース担体と比べ強度、カラム耐圧性が高く、オートクレーブによる加圧高温滅菌にかけるも可能なことから、本発明に好ましく使用することができる。
【００３１】
本発明に使用する球状セルロース粒子の平均粒子径は、カラム充填時の取り扱いやすさの点から５０〜２０００μｍであることが好ましい。より好ましくは１００〜３００μｍである。該球状セルロース粒子の平均粒子径がこの範囲であればカラム内の液の流れをより安定化できる。
【００３２】
発明に使用する球状セルロース粒子は、多孔性であることが好ましい。その場合の孔の大きさや数は、ヘパリン除去の際の条件等によっても異なり一概に限定できるものではないが、ポリエチレンオキサイドを標準物質として測定した排除限界分子量で示すと５０万〜５００万であることが好ましく、より好ましくは８０万〜３００万である。ポリエチレンオキサイドによる排除限界分子量がこの範囲であればカラム内における血液成分の物理的な付着などの問題を抑制できる。
【００３３】
多孔性の水不溶性担体の製造方法は特に限定されるものではないが、球状セルロース粒子の場合を例に説明する。
例えば、特開昭５３−８６７４９号記載のように、セルロース酢酸エステルを有機溶媒中に溶解し、この溶液を水性媒体中に懸濁させることによってセルロース酢酸エステルを球状化し、次いで有機溶媒を蒸発させてセルロースエステル粒子を得、これをケン化後セルロース粒子とすることによって、本発明に好ましい、多孔性球状セルロース粒子を得ることができる。
【００３４】
ε−ポリリジンと水不溶性担体との結合は、反応性の官能基を介して行えばよく、公知の方法をもちいて行えばよい。
その中でも、ε−ポリリジンと水不溶性担体との結合に、該反応性の官能基としてエポキシ化合物を用いたヘパリン吸着体であれば、水不溶性担体とリガンドであるε−ポリリジンとが強固に結合することから、該ヘパリン吸着体をヘパリンの除去に使用した場合であっても、リガンドの離脱が起こりにくく好ましい。
【００３５】
該反応性の官能基としてエポキシ化合物を用いて、ε−ポリリジンと水不溶性担体とを結合させる方法として、具体的に、水不溶性担体を、ビスオキシランと反応させてエポキシ基を導入し、このエポキシ基とε−ポリリジンを反応させる方法や、水不溶性担体をエピクロルヒドリンでエポキシ化し、これをアミノ化して無水コハク酸と反応させてカルボキシル基を導入し、この末端カルボキシル基とε−ポリリジンのアミノ基を縮合させる方法や、前記▲２▼の方法で得られた、カルボキシル基を導入した水不溶性担体とＮ−ヒドロキシスクシンイミドと反応させて活性タイプ（Ｎ−ヒドロキシスクシンイミドエステル化物）としてε−ポリリジンと結合させる方法を挙げることができる。
【００３６】
本願第３の発明は、第１または第２の発明のヘパリン吸着体を用いて、ヘパリンを含む液体からヘパリンを除去する方法である。本発明の用途は特に限定されるものではないが、本発明は、人工透析時に血液凝固防止を目的として血液に添加されたヘパリンを除去する際に有効である。
その際の具体的な形態は、特に限定されるものではないが、本発明のヘパリン吸着体をカラムに充填し、体外循環治療装置のオンライン回路中に組み込まれて行う形態であることが好ましい。
【００３７】
より具体的には、患者の体内から取り出され、抗凝固剤としてヘパリンを加えられた血液、或いは血球と分離された血漿成分が、最初に体外循環装置の有害成分を取り除くカラムを通し、有害成分が除去された状態で患者の体内に戻る直前に、該血液若しくは該血漿成分を、本発明のヘパリン吸着体を充填したカラムに通し、過剰に加えられたヘパリンが取り除かれた状態の血液を患者の体内に戻す形態であることが好ましい。
【００３８】
【実施例】
以下、本発明について実施例及び比較例を用いて詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
１．ヘパリン吸着体の調製
実施例１（本発明のヘパリン吸着体の調製）
平均粒径が８０μｍ、真球度が０．９５、ポリエチレンオキサイドを標準物質として測定した排除限界分子量が２００万のセルロース粒子100g（湿重量）を、200mlの純水に懸濁させ、撹拌しながら３０℃に昇温させた後、該懸濁液に２０％ＮａＯＨ溶液６０mlを加え、１時間撹拌した。次いでエピクロロヒドリン６０ｇを加え、２時間撹拌し反応させた。反応終了後、ろ過し、中性まで水洗した。このようにして得られたエポキシ活性化セルロース粒子100g（湿重量）に純水９０ｍｌ、ε−ポリリジン２５％溶液（チッソ株式会社製、分子量4、700）３０ｍｌを加え、４５℃で２時間撹拌し反応させた。反応終了後水洗し、球状セルロース粒子（本発明のヘパリン吸着体、以下「粒子A」）を得た。
【００３９】
比較例１
ε−ポリリジン２５％溶液（チッソ株式会社製、分子量4、700）を加える操作以降を省いた以外は、実施例１に準拠して操作を行い、球状セルロース粒子（エポキシ活性化セルロース粒子、以下「粒子Ｂ」）を得た。
【００４０】
２．ヘパリン吸着用カラムの作成
実施例１により得られた粒子Ａ、及び比較例１の粒子Ｂをリン酸バッファー（ｐＨ7.4，NaHPO4,3.3mM；Na2HPO4,6.7mM；NaCl,126.7mM;NaN3,25mM）で洗浄してゲルの平衡化を行った。ゲルの脱気を行った後、沈降体積で1mlの前記粒子Ａ，Ｂ２種をそれぞれポリプロピレン製カラム（内径9mm、高さ16mm：テルモ社製）に充填した。カラム入り口側にポリ塩化ビニル製のチューブ（内径1mm、外径13mm、長さ1m）を装着し、血液回路付きカラムを作成した。粒子Ａ（実施例１）を用いたカラムをカラムＡとし、粒子Ｂ（比較例１）を用いたカラムをカラムＢとした。
【００４１】
３．ヘパリン除去
健常人から採決した血液に10mg/L血中(2.0 units/ml血中)の濃度となるようにヘパリンナトリウム溶液（シグマ社製）を添加し、この血液をテフロン製三角フラスコ（内容量100ml、サンワ（株）製）内に入れ、37℃の恒温槽にてスターラーチップを用いて低速で撹拌し、該血液を流速0.5ml/minでカラム内に通血した。血液がカラムの出口側から流出してきた時点を通液開始時とし、以降５分ごとにカラム出口側の血液を所定量採取した。血中ヘパリン濃度の測定はヘパリン濃度測定キットであるACCUCLOT/HEPTEST-HI（シグマ社製）を用いた。
【００４２】
測定の結果、カラムＡ（実施例１）においてはヘパリン濃度が通液開始10分後に初期濃度の約70%まで低下し、30分後には約20%まで低下し平衡となった。一方比較例として用いたカラムＢ（比較例１）の場合、通液開始後10分後に初期濃度の約80%まで低下が見られたが、その後ヘパリン濃度の低下は見られなかった。
【００４３】
【発明の効果】
リガンドがε−ポリリジンである本発明の吸着体、またはε−ポリリジンを水不溶性担体に結合してなるヘパリン吸着体であれば、ヘパリンの吸着能力が高く、この吸着体を用いれば、ヘパリンを含有する液体から、高い効率でヘパリンを除去することが可能である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a heparin adsorbent and a method for removing heparin using the same. More specifically, the present invention relates to a heparin adsorbent whose ligand is ε-polylysine and a method for removing heparin using the same.
[0002]
[Prior art]
Heparin is one of mucopolysaccharides with an average molecular weight of 5,000 to 30,000, has a strong and stable anticoagulant action, and is simple and economical to administer. Used in extracorporeal circulation therapy.
However, heparin has a problem of promoting bleeding promotion effect by long-term or large-scale administration, and causing problems such as platelet activation, osteoporosis, and arteriosclerosis.
[0003]
As a method for avoiding the problem of heparin when administered into blood as described above, a method of neutralizing by administering protamine sulfate, which is a positive protein having affinity for heparin, has been conventionally performed. In addition to this, anticoagulants as heparin substitutes have been developed and studied, but the current situation is that safe doses and administration methods have not been established.
[0004]
[Problems to be solved by the invention]
Examples of heparin removal methods include reduction of fibronectin to a cross-linked agarose carrier and heparin removal methods using heparin adsorbents bound to alkylated derivatives thereof as ligands. Is not sufficient as a heparin adsorbent, and the method for removing heparin using a cross-linked agarose carrier may not be highly efficient as a heparin adsorbent.
[0005]
As a result of intensive studies in view of the above-described conventional problems, the present inventors have found that an adsorbent whose ligand is ε-polylysine or a heparin adsorbent formed by binding ε-polylysine to a water-insoluble carrier. If so, heparin has a high adsorption ability, and if this adsorbent is used, it is found that heparin can be removed with high efficiency from a liquid containing heparin, and the present invention is completed based on this finding. I let you.
[0006]
[Means for Solving the Problems]
The present invention has the following configuration.
(1) A heparin adsorbent whose ligand is ε-polylysine.
[0007]
(2) A heparin adsorbent obtained by binding ε-polylysine to a water-insoluble carrier.
[0008]
(3) The heparin adsorbent according to the above item 1 or 2, wherein ε-polylysine is produced by a bacterium belonging to the genus Streptomyces.
[0009]
(4) The heparin adsorbent according to any one of items 1 to 3, wherein the average molecular weight of ε-polylysine is in the range of 500 to 1,000,000.
[0010]
(5) The heparin adsorbent according to any one of items 1 to 3, wherein the average molecular weight of ε-polylysine is in the range of 1,000 to 10,000.
[0011]
(6) The heparin adsorbent according to the above item 2, wherein the water-insoluble carrier is water-insoluble spherical particles.
[0012]
(7) The heparin adsorbent according to the above item 6, wherein the sphericity of the water-insoluble spherical particles is 0.9 or more.
[0013]
(8) The heparin adsorbent according to item 6 or 7, wherein the water-insoluble spherical particles are spherical cellulose particles.
[0014]
(9) The heparin adsorbent according to the above item 8, wherein the spherical cellulose particles have an average particle size of 50 to 2000 μm.
[0015]
(10) The heparin adsorbent according to item 8 or 9, wherein the spherical cellulose particles have an exclusion limit molecular weight of 500,000 to 5,000,000.
[0016]
(11) The heparin adsorbent according to item 8 or 9, wherein the spherical cellulose particles have an exclusion limit molecular weight of 800,000 to 3,000,000.
[0017]
(12) The heparin adsorbent according to the above item 2, wherein the water-insoluble carrier is a water-insoluble hollow fiber.
[0018]
(13) The heparin adsorbent according to the above item 2, wherein the water-insoluble carrier is a water-insoluble membrane.
[0019]
(14) The heparin adsorbent according to any one of (2) to (13), wherein ε-polylysine and a water-insoluble carrier are bound via a reactive functional group.
[0020]
(15) The heparin adsorbent according to any one of items 1 to 15 is used.
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The first invention of the present application is a heparin adsorbent using ε-polylysine as a ligand. In the present invention, if the ligand is ε-polylysine, the ligand may be bound to a solid support or may not be bound to a solid support. That is, the heparin adsorbent of the present invention may be a ligand of ε-polylysine itself or a heparin adsorbent, or may be bound to a water-insoluble carrier.
[0022]
The second invention of the present application is a heparin adsorbent obtained by binding ε-polylysine to a water-insoluble carrier.
[0023]
The ε-polylysine used in the present invention may be derived from a microorganism produced by a Streptomyces bacterium or may be obtained by chemical synthesis.
[0024]
Among them, ε-polylysine derived from microorganisms produced by Streptomyces bacteria is highly biocompatible because of its biodegradability, and is also preferably used in the present invention because it is inexpensive and available in large quantities. can do.
[0025]
The molecular weight of polylysine used in the present invention is preferably in the range of 500 to 1,000,000, particularly preferably in the range of 1,000 to 10,000, from the viewpoint of ease of handling.
[0026]
The water-insoluble carrier that can be used in the second invention of the present application is a carrier made of a substance that makes the carrier water-insoluble or water-insoluble by subjecting the water-soluble substance to a treatment such as a crosslinking reaction. Specific examples include cross-linked agarose, cellulose, and polyacrylamide.
In addition, the shape of the water-insoluble carrier used in the present invention is not particularly limited, such as spherical particles, hollow fibers, membranes, etc., among them, in the case of spherical particles, it is easy to manufacture and handle, The amount of ligand binding per carrier is also large and can be preferably used in the present invention.
[0027]
A hollow fiber-shaped carrier means a fibrous carrier having continuous or discontinuous cavities inside, and is obtained by adding a foaming agent to a spinning solution or by forming a cavity inside using a special base. is there.
[0028]
The membrane-like carrier is a flat plate-like porous material like a commercially available membrane filter and has a certain range of exclusion limit molecular weight.
[0029]
The water-insoluble spherical particles used in the present invention preferably have a high sphericity. When the sphericity is high, uniform packing is easy when packed in a column or the like.
The sphericity referred to in the present invention is the ratio of the shortest diameter (minor axis) to the longest diameter (major axis) of the particles (minor axis / major axis). Is high.
The sphericity of the water-insoluble spherical particles used in the present invention is preferably 0.9 or more, more preferably 0.95 or more.
[0030]
Among water-insoluble spherical particles, spherical cellulose particles are inexpensive and highly biocompatible, and have higher strength and higher column pressure resistance than cross-linked agarose carriers that have been used as conventional heparin-removing adsorbent carriers. Since it can be subjected to autoclaving and high temperature sterilization, it can be preferably used in the present invention.
[0031]
The average particle diameter of the spherical cellulose particles used in the present invention is preferably 50 to 2000 μm from the viewpoint of ease of handling during column packing. More preferably, it is 100-300 micrometers. When the average particle diameter of the spherical cellulose particles is within this range, the liquid flow in the column can be further stabilized.
[0032]
The spherical cellulose particles used in the invention are preferably porous. The size and number of pores in that case vary depending on the conditions at the time of heparin removal and can not be limited to a single range, but are 500,000 to 5,000,000 when expressed in terms of exclusion limit molecular weight measured using polyethylene oxide as a standard substance. It is preferably 800,000 to 3,000,000. If the exclusion limit molecular weight due to polyethylene oxide is within this range, problems such as physical adhesion of blood components in the column can be suppressed.
[0033]
The method for producing the porous water-insoluble carrier is not particularly limited, but the case of spherical cellulose particles will be described as an example.
For example, as described in JP-A-53-86749, cellulose acetate is dissolved in an organic solvent, and the cellulose acetate is spheroidized by suspending this solution in an aqueous medium, and then the organic solvent is evaporated. Thus, by obtaining cellulose ester particles, which are converted into cellulose particles after saponification, porous spherical cellulose particles preferable for the present invention can be obtained.
[0034]
The bond between ε-polylysine and the water-insoluble carrier may be performed via a reactive functional group, and may be performed using a known method.
Among them, in the case of a heparin adsorbent using an epoxy compound as the reactive functional group for binding between ε-polylysine and a water-insoluble carrier, the water-insoluble carrier and the ligand ε-polylysine are strongly bound. Therefore, even when the heparin adsorbent is used for removing heparin, it is preferable that the ligand is not easily detached.
[0035]
As a method of bonding ε-polylysine and a water-insoluble carrier using an epoxy compound as the reactive functional group, specifically, the water-insoluble carrier is reacted with bisoxirane to introduce an epoxy group, and this epoxy A method of reacting a group with ε-polylysine, or epoxidizing a water-insoluble carrier with epichlorohydrin, aminating this and reacting with succinic anhydride to introduce a carboxyl group, and the terminal carboxyl group and the amino group of ε-polylysine are combined. It reacts with the water-insoluble carrier introduced with the carboxyl group and the N-hydroxysuccinimide obtained by the method of condensation or the method of (2) above to bind to ε-polylysine as the active type (N-hydroxysuccinimide esterified product). A method can be mentioned.
[0036]
The third invention of the present application is a method for removing heparin from a liquid containing heparin using the heparin adsorbent of the first or second invention. The use of the present invention is not particularly limited, but the present invention is effective in removing heparin added to blood for the purpose of preventing blood coagulation during artificial dialysis.
Although the specific form in that case is not specifically limited, It is preferable that the heparin adsorbent of the present invention is packed in a column and incorporated into an online circuit of the extracorporeal circulation treatment apparatus.
[0037]
More specifically, blood removed from the patient's body and added with heparin as an anticoagulant, or plasma components separated from blood cells are first passed through a column that removes harmful components from the extracorporeal circulation device, The blood or plasma component is passed through a column filled with the heparin adsorbent of the present invention immediately before returning to the patient's body in a state where the heparin is removed. It is preferable that it is a form returned to the body.
[0038]
【Example】
Hereinafter, although the present invention is explained in detail using an example and a comparative example, the present invention is not limited to these examples.
1. Preparation of heparin adsorbent Example 1 (Preparation of heparin adsorbent of the present invention)
100 g (wet weight) of cellulose particles having an average particle size of 80 μm, a sphericity of 0.95, and an exclusion limit molecular weight of 2 million measured using polyethylene oxide as a standard substance are suspended in 200 ml of pure water while stirring. After raising the temperature to 30 ° C., 60 ml of 20% NaOH solution was added to the suspension and stirred for 1 hour. Next, 60 g of epichlorohydrin was added and stirred for 2 hours to react. After completion of the reaction, it was filtered and washed with water until neutral. To 100 g (wet weight) of the epoxy-activated cellulose particles obtained in this manner, 90 ml of pure water and 30 ml of ε-polylysine 25% solution (manufactured by Chisso Corporation, molecular weight 4, 700) are added and stirred at 45 ° C. for 2 hours. Reacted. After completion of the reaction, the product was washed with water to obtain spherical cellulose particles (the heparin adsorbent of the present invention, hereinafter “particle A”).
[0039]
Comparative Example 1
Except for omitting the operation after adding an ε-polylysine 25% solution (manufactured by Chisso Corporation, molecular weight 4, 700), the operation was performed in accordance with Example 1, and spherical cellulose particles (epoxy activated cellulose particles, hereinafter “ Particle B ") was obtained.
[0040]
2. Preparation of Heparin Adsorption Column Particle A obtained in Example 1 and Particle B of Comparative Example 1 were mixed with a phosphate buffer (pH 7.4, NaHPO 4 , 3.3 mM; Na 2 HPO 4 , 6.7 mM; NaCl, 126.7 mM). Washed with NaN 3 , 25 mM) to equilibrate the gel. After degassing the gel, 1 ml of the above-mentioned particles A and B in a sedimentation volume were packed in polypropylene columns (inner diameter 9 mm, height 16 mm: Terumo). A tube made of polyvinyl chloride (inner diameter 1 mm, outer diameter 13 mm, length 1 m) was attached to the column inlet side to create a column with a blood circuit. The column using the particles A (Example 1) was named Column A, and the column using the particles B (Comparative Example 1) was named Column B.
[0041]
3. A heparin sodium solution (manufactured by Sigma) is added to blood selected from a healthy person who has removed heparin to a concentration of 10 mg / L blood (2.0 units / ml blood), and this blood is added to a Teflon Erlenmeyer flask (internal volume) 100 ml, manufactured by Sanwa Co., Ltd.), stirred at a low speed using a stirrer chip in a constant temperature bath at 37 ° C., and the blood was passed through the column at a flow rate of 0.5 ml / min. The time at which the blood flowed out from the outlet side of the column was taken as the start of liquid flow, and a predetermined amount of blood was collected at every 5 minutes thereafter. The blood heparin concentration was measured using ACCUCLOT / HEPTEST-HI (manufactured by Sigma) which is a heparin concentration measurement kit.
[0042]
As a result of the measurement, in column A (Example 1), the heparin concentration decreased to about 70% of the initial concentration 10 minutes after the start of liquid flow, and decreased to about 20% after 30 minutes and became equilibrium. On the other hand, in the case of column B (Comparative Example 1) used as a comparative example, a decrease to about 80% of the initial concentration was observed 10 minutes after the start of liquid flow, but no decrease in the heparin concentration was observed thereafter.
[0043]
【The invention's effect】
If the adsorbent of the present invention whose ligand is ε-polylysine or the heparin adsorbent obtained by binding ε-polylysine to a water-insoluble carrier, the adsorbing ability of heparin is high. If this adsorbent is used, heparin is contained. It is possible to remove heparin with high efficiency from the liquid.
Claims (14)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000035244A JP4228498B2 (en) | 2000-02-14 | 2000-02-14 | Heparin adsorbent and method for removing heparin using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000035244A JP4228498B2 (en) | 2000-02-14 | 2000-02-14 | Heparin adsorbent and method for removing heparin using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2001218839A JP2001218839A (en) | 2001-08-14 |
| JP4228498B2 true JP4228498B2 (en) | 2009-02-25 |
Family
ID=18559476
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2000035244A Expired - Fee Related JP4228498B2 (en) | 2000-02-14 | 2000-02-14 | Heparin adsorbent and method for removing heparin using the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4228498B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8857970B2 (en) | 2012-01-18 | 2014-10-14 | Brother Kogyo Kabushiki Kaisha | Water-based ink for ink-jet recording and ink cartridge |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008157570A2 (en) * | 2007-06-18 | 2008-12-24 | Exthera Ab | Device and method for restoration of the condition of blood |
-
2000
- 2000-02-14 JP JP2000035244A patent/JP4228498B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8857970B2 (en) | 2012-01-18 | 2014-10-14 | Brother Kogyo Kabushiki Kaisha | Water-based ink for ink-jet recording and ink cartridge |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2001218839A (en) | 2001-08-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3100974B2 (en) | Adsorbent for removing biological macromolecules such as LDL and endotoxins from extracorporeal circulating whole blood | |
| WO2002060512A1 (en) | Body fluid processor enabling direct hemoperfusion | |
| JP4578405B2 (en) | Adsorbent and adsorber for low density lipoprotein and fibrinogen capable of whole blood treatment | |
| JP4228498B2 (en) | Heparin adsorbent and method for removing heparin using the same | |
| CN109248668B (en) | Adsorbent for removing LDL (low-density lipoprotein) by blood extracorporeal circulation, preparation method thereof and perfusion apparatus | |
| JP2928589B2 (en) | Adsorbent for modified LDL and apparatus for removing modified LDL using the same | |
| JP2001316420A (en) | Production method of absorbent for reducing fibrinogen and/or fibrin level, absorbent, and use thereof for manufacturing absorption device | |
| JPS6319214B2 (en) | ||
| JPS62191042A (en) | Blood coagulation factor viii adsorbent and method for purifying blood coagulation factor viii using same | |
| JP3157026B2 (en) | Adsorbent for blood purification | |
| JP2534867B2 (en) | Myoglobin adsorbent | |
| JPH0738881B2 (en) | Container for removing activated complement components | |
| SU844569A1 (en) | Method of preparing homocompatible adsorbents for blood purification from toxins | |
| JPH0616842B2 (en) | Adsorbent for activated complement components | |
| Fu et al. | Preparation of tryptophan modified chitosan beads and their adsorption of low density lipoprotein | |
| JP2001276611A (en) | Adsorbent for adsorbing endotoxin and method of removing endotoxin using the same | |
| JPH06233816A (en) | Hemocatharsis adsorbent | |
| JPH06237996A (en) | Blood purifying/adsorbing material | |
| JPS6077769A (en) | Production of adsorbing body | |
| JP2002369881A (en) | Amination carrier and method of adsorbing cellular fibronectin-heparin composite using the same | |
| JPH06233815A (en) | Hemocatharsis adsorbent | |
| JPH0316639A (en) | Adsorbent and removing device using the same | |
| JPH06178807A (en) | Unwoven cloth type blood purifying adsorbent | |
| JPH06237995A (en) | Blood purifying/adsorbing material | |
| JPS6182759A (en) | Antithrombotic material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20060906 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20080820 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080826 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20081010 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20081111 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20081124 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111212 Year of fee payment: 3 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111212 Year of fee payment: 3 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111212 Year of fee payment: 3 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111212 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121212 Year of fee payment: 4 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121212 Year of fee payment: 4 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131212 Year of fee payment: 5 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |