JP2004236791A

JP2004236791A - Diabetic complication factor adsorbent body

Info

Publication number: JP2004236791A
Application number: JP2003027993A
Authority: JP
Inventors: Shinji Shimizu; 晋治清水; Toshihiko Kuroda; 俊彦黒田; Yuka Kosakamoto; 由香小坂元
Original assignee: Toray Industries Inc
Current assignee: Toray Industries Inc
Priority date: 2003-02-05
Filing date: 2003-02-05
Publication date: 2004-08-26

Abstract

<P>PROBLEM TO BE SOLVED: To remove a diabetic complication factor safely and sterilely by using a technology for economically mass-producing diabetic complication factor adsorbent bodies and the adsorbent body enabling the sterilization for removing the diabetic complication factor for the purpose of humor disposal. <P>SOLUTION: The diabetic complication factor adsorbent body is formed by binding a chemical compound made of a peptide inducer whose binding performance to the diabetic complication factor is not substantially deactivated because of the sterilization process with a water insoluble carrier. The diabetic complication factor adsorbent body is filled in a diabetic complication factor removal container, and the diabetic complication factor included in a treated solution derived from the humor is selectively removed as an adsorbed material by bringing the treated solution derived from the humor into contact with the adsorbent body in the container. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０００１】
【発明の属する技術分野】
本発明は、滅菌処理により糖尿病合併症因子に対する結合性能が実質上失活しない、ペプチド誘導体で構成された化合物を水不溶性担体に結合した糖尿病合併症因子吸着体及びそれを充填した除去器及びそれを用いた糖尿病合併症因子除去方法に関する。
【０００２】
【従来の技術】
近年、糖尿病性腎症が悪化することで、人工透析を受ける患者が急激に増加しており、１９９８年以降の新規透析導入患者の主原因は糖尿病性腎症となり、以降、透析導入の主原因として位置づけられている。人工透析導入の原疾患としては、糖尿病性腎症と慢性糸球体腎炎で過半数を占めることが知られているが、透析導入後の生存率に大きな違いがある。評価施設において若干の違いがあるものの、糖尿病性腎症患者の透析導入後の５０％生存率は３〜４年とされるのに対して、慢性糸球体腎炎患者の５０％生存率は１０年程度かそれ以上と言われ、明らかに糖尿病性透析患者の予後が悪く、社会的問題となっているが解決の兆しが全く見えてこない。
【０００３】
この原因として糖尿病合併症の発症機序が未だ不明瞭であることが挙げられる。ところが最近になって、糖尿病合併症の共通の病変と言える血管病変を発症する病因物質の候補として、ＡＧＥ（ａｄｖａｎｃｅｄｇｌｙｃａｔｉｏｎｅｎｄｐｒｏｄｕｃｔｓ）に代表される各種変性物質に注目が集まった。
【０００４】
ＡＧＥとは、主に糖尿病性合併症の原因物質と考えられているものであり、血液中に存在するブドウ糖に代表される還元糖、その代謝産物や反応産物と、タンパク質や脂質などの低分子が酵素の関与なしに、つまり、非酵素的に結合する前進性糖化反応（ａｄｖａｎｃｅｄｇｌｙｃａｔｉｏｎ）の産物であり、総称してカルボニルストレス産物とも呼ばれることもある。
【０００５】
当時は、還元糖等とタンパク質等の非酵素的反応体であるカルボニルストレス産物について、蛍光性、褐色性、架橋性、脱水、酸化、縮合、転移など様々な特徴が発見されたが、どれもカルボニルストレス産物が持つ病因性、つまり、糖尿病合併症因子としての証拠を直接説明できるものではなかった。カルボニルストレス産物の一つであるＡＧＥの評価方法についても、以前はその特徴の一つである蛍光特性で血液中の測定を試みている例がよくあり、特開平６−３１２１３４号公報（特許文献１）では、ヒト血漿中のＡＧＥを、励起波長３９０ｎｍ、蛍光波長４５０ｎｍで測定を試みている（例えば、非特許文献１参照。）。しかし、蛍光測定は試験管内の前進性糖化反応の確認には用いられるが、血液中では夾雑物や薬剤の非特異蛍光の影響も多くあることと、また、ペントシジンなど励起波長３３５ｎｍ、蛍光波長３８５ｎｍといった異なる蛍光特性のものも発見されるなど（例えば、非特許文献２参照。）、上記蛍光特性を持つＡＧＥはほんの一部に過ぎないことが分かりだした。更に、ＡＧＥの主構造と考えられているＣＭＬ（カルボキシメチルリジン）やＣＥＬ（カルボキシエチルリジン）には蛍光特性がないことが知られており、現在、ヒト血漿中のＡＧＥを励起波長３９０ｎｍ、蛍光波長４５０ｎｍで単純に測定することは、信頼性のある測定法ではないと認識されている。以来、蛍光特性による評価も測定手順に分子篩や化学処理を施したり、蛍光がどのＡＧＥ構造の蛍光を反映するかを考慮して評価するようになった。また、特異的な免疫学的測定方法で測定を試みる動きもある。これまで同定されたＡＧＥ構造はＣＭＬ、ＣＥＬ、ペントシジン、ピラリン、クロスリン、フルオロリンク、イミダゾロン、Ｘ１、アルグピリミジンなど１０種類程度あるが、これら全てのＡＧＥ構造がカルボニルストレス産物として病因性を持っているわけではないとも言われている（例えば、非特許文献３参照。）。このためＡＧＥに始まるカルボニルストレス原因説がより一層複雑になっているという問題があり、糖尿病性合併症に代表される各種血管病変を引き起こす糖尿病合併症因子としてのカルボニルストレス産物のみを正確に検出する方法でさえ極めて乏しい状態であった。
【０００６】
一方で、糖尿病合併症因子としての性質を持つ非酵素的変性物質は、主に細胞上のある特殊な受容体と結合することで引き起こされることが分かってきた。その多くは一般的にスカベンジャー受容体と呼ばれるものだが、本来別の機能を有する受容体にカルボニルストレス産物などの非酵素的変性物質が相互作用することで病因性を引き起こす場合も報告されている。ガレクチン−３、ＲＡＧＥ（ＲｅｃｅｐｔｏｒｆｏｒＡＧＥ）などである。特にＲＡＧＥは糖尿病の３大合併症である腎症、網膜症、神経症の発症を引き起こすだけではなく、動脈硬化も助長することがわかってきた。最近になり、動物レベルでＲＡＧＥと糖尿病状態で亢進するある特定の物質との相互作用で糖尿病性合併症などの血管障害が引き起こされることが証明されつつある（例えば、非特許文献４参照。）。現在、糖尿病合併症因子の病因性を引き出す経路はＲＡＧＥが最も重要であると考えられている。
【０００７】
ＡＧＥに代表されるＲＡＧＥ結合物質に対する吸着材は既に幾つかの種類が知られている。例えば、抗ＡＧＥ抗体をビーズに結合させることで作製した吸着材（例えば、非特許文献５参照。）が知られているが、これら吸着材は必ずしも滅菌下での使用が義務付けられたものではない。また、上記の公知の方法では、実験室レベルで要求される少量生産条件では、無菌区域での吸着材作製を実施することで課題解決が可能である。ところが、産業レベルでの大量生産及び吸着材を使用した製品の安定管理となれば、公知の滅菌方法で吸着材としての性能を大幅に損なわないような吸着材の設計が求められるが、滅菌できる吸着材を大量供給可能な技術が見出されていなかった。
【０００８】
【特許文献１】
特開平６−３１２１３４号公報
【０００９】
【非特許文献１】
マキタ（ＭａｋｉｔａＺ），他８名，「糖尿病性神経障害患者における前進性糖化反応産物（Ａｄｖａｎｃｅｄｇｌｙｃｏｓｙｌａｔｉｏｎｅｎｄｐｒｏｄｕｃｔｓｉｎｐａｔｉｅｎｔｓｗｉｔｈｄｉａｂｅｔｉｃｎｅｐｈｒｏｐａｔｈｙ．）」，ザ・ニュー・イングランド・ジャーナル・オブ・メディスン（ＴｈｅＮｅｗＥｎｇｌａｎｄＪｏｕｒｎａｌｏｆＭｅｄｉｃｉｎｅ），１９９１年，第３２５巻，ｐ８３８
【００１０】
【非特許文献２】
ミヤタ（ＭｉｙａｔａＴ），他７名，「末期腎不全尿毒症患者の循環に於けるアルブミン結合性及び遊離性ペントシジンの蓄積：ペントシジンの病態生理学に於ける腎性関与（Ａｃｃｕｍｕｌａｔｉｏｎｏｆａｌｂｕｍｉｎ−ｌｉｎｋｅｄａｎｄｆｒｅｅ−ｆｏｒｍｐｅｎｔｏｓｉｄｉｎｅｉｎｔｈｅｃｉｒｃｕｌａｔｉｏｎｏｆｕｒｅｍｉｃｐａｔｉｅｎｔｓｗｉｔｈｅｎｄ−ｓｔａｇｅｒｅｎａｌｆａｉｌｕｒｅ：ｒｅｎａｌｉｍｐｌｉｃａｔｉｏｎｓｉｎｔｈｅｐａｔｈｏｐｈｙｓｉｏｌｏｇｙｏｆｐｅｎｔｏｓｉｄｉｎｅ．）」，ジャーナル・オブ・ジ・アメリカン・ソサイエティー・オブ・ネフロロジー（ＪｏｕｒｎａｌｏｆｔｈｅＡｍｅｒｉｃａｎＳｏｃｉｅｔｙＮｅｐｈｒｏｌｏｇｙ），１９９６年，第７巻，８号，ｐ１１９８−１２０６
【００１１】
【非特許文献３】
ミヤタ（ＭｉｙａｔａＴ），他８名，「末期腎不全患者での前進性糖化最終産物の形成に於ける増加した酸化ストレスの関与（Ｉｍｐｌｉｃａｔｉｏｎｏｆａｎｉｎｃｒｅａｓｅｄｏｘｉｄａｔｉｖｅｓｔｒｅｓｓｉｎｔｈｅｆｏｒｍａｔｉｏｎｏｆａｄｖａｎｃｅｄｇｌｙｃａｔｉｏｎｅｎｄｐｒｏｄｕｃｔｓｉｎｐａｔｉｅｎｔｓｗｉｔｈｅｎｄ−ｓｔａｇｅｒｅｎａｌｆａｉｌｕｒｅ．）」，キドニー・インターナショナル（ＫｉｄｎｅｙＩｎｔｅｒｎａｔｉｏｎａｌ），１９９７年，第５１巻，４号，ｐ１１７０−１１８１
【００１２】
【非特許文献４】
ヤマモト（山本靖彦），他８名，「ヒトＲＡＧＥをトランスジーンとしたマウスの糖尿病性腎病変」，糖尿病（ＪｏｕｒｎａｌｏｆｔｈｅＪａｐａｎＤｉａｂｅｔｅｓＳｏｃｉｅｔｙ），１９９９年，第４２巻，補１，ｐＳ−１９４
【００１３】
【非特許文献５】
バスタ（ＢａｓｔａＧ），「前進性糖化最終産物は信号伝達受容体ＲＡＧＥを経由して内皮細胞を活性化する：炎症反応の増幅に対する機序（Ａｄｖａｎｃｅｄｇｌｙｃａｔｉｏｎｅｎｄｐｒｏｄｕｃｔｓａｃｔｉｖａｔｅｅｎｄｏｔｈｅｌｉｕｍｔｈｒｏｕｇｈｓｉｇｎａｌ−ｔｒａｎｓｄｕｃｔｉｏｎｒｅｃｅｐｔｏｒＲＡＧＥ：ａｍｅｃｈａｎｉｓｍｆｏｒａｍｐｌｉｆｉｃａｔｉｏｎｏｆｉｎｆｌａｍｍａｔｏｒｙｒｅｓｐｏｎｓｅｓ．）」，サーキュレーション（Ｃｉｒｃｕｌａｔｉｏｎ），２００２年，第１０５巻，第７号，ｐ８１６−８２２
【００１４】
【発明が解決しようとする課題】
本発明は、糖尿病合併症因子を除去するための糖尿病合併症因子吸着体について、経済的に量産可能としたり、該吸着体を体液処理目的に使うために、該吸着体を滅菌できるようにすることで糖尿病合併症因子を安全且つ無菌的に除去できる方法の提供を目的とする。
【００１５】
【課題を解決するための手段】
本発明は、上記目的を達成するために以下の構成を有する。
「（１）滅菌処理により糖尿病合併症因子に対する結合性能が実質上失活しない、ペプチド誘導体で構成された化合物を水不溶性担体に結合させたことを特徴とする糖尿病合併症因子吸着体。」
「（２）上記（１）項に記載の糖尿病合併症因子吸着体が充填されてなる糖尿病合併症因子除去容器内に体液由来の被処理液を接触させることで被処理液中に含まれる糖尿病合併症因子を被吸着物質として選択的に除去することを特徴とする糖尿病合併症因子除去器の使用方法。」
【００１６】
【発明の実施の形態】
以下、本発明について詳細に説明する。
【００１７】
本発明でいうところの滅菌とは、吸着体や吸着体を充填した除去器内に混入した細菌やウイルス等の生物の増殖等の生命活動が一切行えないような化学的または物理的な処理方法のことである。滅菌を行う方法としては、その一例として、高圧蒸気滅菌、γ線滅菌や電子線滅菌等の放射線滅菌、エチレンオキシドガス等の殺菌性ガスを用いたガス滅菌、過酸化水素や過塩素酸水溶液等の殺菌性化合物を用いた化学滅菌等が挙げられ、特に限定されるものではない。
【００１８】
また、本発明で言うところの「滅菌処理により糖尿病合併症因子に対する結合性能が実質上失活しない」こととは、高圧蒸気滅菌処理前後での吸着性能の低下率が少なくとも５０％以内であることを言い、好ましくは３０％以内、より好ましくは２０％以内、最も好ましくは１０％以内で抑えられることで一義的に判定できる。また、上記高圧蒸気滅菌処理とは、１２０℃で２０分間の高圧蒸気処理を意味する。
【００１９】
本発明でいうところの吸着性能の定義と測定方法は以下に示す方法により一義的に評価できる。測定方法については、吸着体を沈降体積で１００μｌあたり、被吸着サンプル（ＡＧＥ標品を１０μｇ／ｍｌ含む０．５％ウシ血清アルブミン−リン酸緩衝液（２０ｍＭリン酸緩衝液に０．１５Ｍ塩化ナトリウムを添加した緩衝液（ｐＨ７．２）））を９００μｌ加えて、３７℃の孵卵器中で２時間ゆっくり振盪させて、この反応液を３０００ｒｐｍで５分間遠心分離して吸着体を沈降させ、上清中のＡＧＥ標品の吸着量を以下に示す免疫学的測定法（競合法ＥＬＩＳＡ）で測定する。上記ＡＧＥ標品を２０μｇ／ｍｌの濃度に調製し、９６穴マイクロタイタープレートに１ウェル当たり１００μｌ入れて、室温で２時間インキュベートすることにより固相化し、０．５％ＢＳＡを含むＰＢＳでブロッキングした後、緩衝液２５μｌ、測定サンプル２５μｌ、ＡＧＥ抗体溶液を適宜希釈して５０μｌ入れて、２５℃で２時間振盪して、固相化抗原を認識した抗体に対して、抗ウサギペルオキシダーゼ標識抗体を反応させた後、発色させて５９５ｎｍの吸光度を測定する方法であり、上記方法での吸着体処理前後のＡＧＥ標品の減少率を吸着性能と定義する。吸着体の吸着性能があるということは、上記定義によって評価された減少率が少なくとも５０％以下であればよい。より好ましくは４０％以下であり、いっそう好ましくは３０％以下である。最も好ましくは、処理前後の吸着性能の減少率が１０％以下である。また、上記ＡＧＥ標品とは以下の方法で調製されたものをいう。２５ｍｇ／ｍｌのＢＳＡを０．１Ｍのグリコールアルデヒドと５ｍＭのＤＴＰＡ（Ｄｉｅｔｈｙｌｅｎｅｔｒｉａｍｉｎｅ−Ｎ，Ｎ，Ｎ’，Ｎ’’，Ｎ’’’−ｐｅｎｔａａｃｅｔｉｃａｃｉｄ）と共に０．２Ｍリン酸緩衝液（ｐＨ７．４）に溶解後、０．２μｍのフィルターにより濾過滅菌して、３７℃で１週間インキュベートした。低分子量の反応物や未反応のグリコールアルデヒドを、ＰＤ−１０脱塩カラムとＰＢＳによる透析により取り除き、ＡＧＥ標品を得ればよい。そして、本発明で言うところの糖尿病合併症因子とは、糖尿病性合併症を引き起こす病因物質の総称であり、糖尿病患者の体内から検出されるのであれば特に限定されない。好ましくは、ＲＡＧＥと結合するＲＡＧＥ結合物質であればよい。ＲＡＧＥ結合物質の例としては、ＡＧＥだけではなく、ある種の炎症マーカーの一群であるＨＭＧ−１に代表されるハイモビリティータンパク質ファミリー、血清アミロイドＡ、Ｓ１００／カルグラニュリンスーパーファミリー、トランスサイレチンと呼ばれる物質でもよく、ＲＡＧＥと結合が認められるならば、特に限定を受けるものではない。また、これらのＲＡＧＥ結合物質を総称して、ＥＮ−ＲＡＧＥ（ｅｘｔｒａｃｅｌｌｕｌａｒｎｅｗｌｙｉｄｅｎｔｉｆｉｅｄＲＡＧＥ−ｂｉｎｄｉｎｇｐｒｏｔｅｉｎ）とも定義されており（Ｃｅｌｌ、９７巻、８８９頁、１９９９年）、今後も新規のＲＡＧＥ結合物質が分離同定される可能性が高いと考えられる。さらに好ましくは、ＡＧＥであるが、ＡＧＥにも様々な構造のものがあり、いっそう好ましくは、ＲＡＧＥ結合性のＡＧＥである。ＡＧＥはタンパク質、ホルモン、糖類、脂質、ビタミン等に代表される生体物質とグルコースに代表される還元糖との非酵素的反応で生み出されるものであるが、必ずしも、還元糖はグルコースに限定される必要はなく、グリコールアルデヒドやグリセルアルデヒド、メチルグリオキサール、グリオキサール、３−デオキシグルコゾン等のα−ヒドロキシアルデヒド化合物やジカルボニル化合物であってもよい。また、ＡＧＥの生成途中でヒドロキシラジカルや一酸化窒素、パーオキシナイトライト等の各種ラジカルによる酸化変性があっても良く、生成経路や反応基質に特に限定を受けるものではなく、酸化性反応の結果に生成した変性物質であれば特に限定を受けものではない。特にＲＡＧＥ結合物質に重要なことは、ＲＡＧＥと結合することで病因性が認められるということである。
【００２０】
ここでいう糖尿病合併症因子の病因性とは、糖尿病合併症因子と細胞が接触することで、各種サイトカインの誘導、細胞遊走化促進、ＤＮＡ合成の亢進、接着分子発現亢進や線維化、ＮＦ−κＢの活性化、酸化ストレスの誘導、細胞や組織の物質透過性亢進などの何れかもしくは複数の現象を引き起こし、結果として、動脈硬化、腎症、網膜症、神経症などの血管病変や持続的炎症を引き起こすことを言う。また、糖尿病合併症因子は必ずしも糖尿病に限定されて見られる増悪因子ではなく、人工透析や各種腎疾患など排泄・代謝機能の低下の場合にも見られることがある。更には、敗血症や癌転移、アルツハイマー病、過炎症等にもＲＡＧＥの関与が示唆されているので、ＲＡＧＥが関与する疾病ならば、如何なるものも限定を受けることなく、該疾病患者に認められるものならば、糖尿病合併症因子と解釈されて良い。便宜上、糖尿病合併症因子と定義しているのは、特に糖尿病患者で検出されて、且つ、該患者で多く見られる、血管病変に関わる病因物質の総称のためである。
【００２１】
本発明で言うところのペプチド誘導体とは、ペプチド結合が少なくとも２つ以上存在するアミノ酸重合物であれば全く問題はなく、さらには、アミノ酸以外の化合物が更に含まれていても特に問題はない。アミノ酸以外の化合物では、糖や脂質といった生体由来の化合物でも良く、生体に存在しない芳香族環や脂肪族系化合物であっても良い。また、ペプチド誘導体を構成するアミノ酸は、生体内に存在するアミノ酸であれば全く問題なく、Ｄ体アミノ酸という生体内で支配的に見出されるＬ体アミノ酸の鏡像体が含まれていても特に問題なく使用できる。好ましくは、ペプチド誘導体内に塩基性官能基が２つ以上含まれていれば良く、その塩基性アミノ酸はアミノ基やアンモニウムイオン基、複素環や環状化合物で構成されていてもよい。また、塩基性官能基は生理的条件下で該官能基が塩基性を示せばより好ましいものである。更に、上記のペプチド誘導体が直鎖状でも環状ペプチド化合物、あるいは、双方の複合体でも良く、特に限定されない。更に好ましくは、滅菌条件下で吸着体としての性能を失わないペプチド誘導体であれば、特に問題なく使用できる。その中でも、特に好ましくは、環状ペプチド誘導体であれば、ポリミキシン系化合物がよく、直鎖状であれば、ＲＡＧＥのＩｇＶドメイン由来ペプチドであることが望ましい。
【００２２】
ポリミキシン系化合物とは、ポリミキシン骨格をもつ誘導体であり、ポリミキシンＢやコリスチン（ポリミキシンＥ）に代表される。他には、ポリミキシンＡ、ポリミキシンＣ、ポリミキシンＤ、ポリミキシンＦ等という化合物が知られているが、ポリミキシン骨格を持つ上述規定に定義された誘導体ならばよく、特に限定されない。
【００２３】
本発明で言うところのＲＡＧＥ部分配列誘導体とは、完全長ＲＡＧＥのなかのＲＡＧＥ結合物質が結合できるペプチド領域を含めた部分配列であり、好ましくは、ＩｇＶドメイン内の配列の一部を含めたものである。完全長ＲＡＧＥとは、ヒトの場合、４０４個のアミノ酸から構成され、そのうちＮ末端から２２個のアミノ酸で構成されるシグナル配列を有するため、結果的に３８２個のアミノ酸として受容体を形成する。そのうちで、糖尿病合併症因子と吸着できるアミノ酸配列を含む細胞外ドメインは、３４２個のアミノ酸で構成されている。また、細胞外ドメインは、更に細かいドメインに分類され、即ち、全アミノ酸配列４０４個の中でＮ末端側からＶ型ドメイン（３１番目から１０６番目までの７６個のアミノ酸で構成）、Ｃ２型ドメイン１（１３７番目から２１４番目までの７８個のアミノ酸で構成）、Ｃ２型ドメイン２（２５２番目から３０８番目までの５７個のアミノ酸配列で構成）の３つの領域からなる。細胞外ドメインの中でＶ型ドメインと呼ばれるドメインは、上述のようにＩｇＶドメインとも呼ばれるが、特に重要で、糖尿病合併症因子の一つであるＡＧＥとの結合性能を持つことが確認されている（ＪｏｕｒｎａｌｏｆＢｉｏｌｏｇｉｃａｌＣｈｅｍｉｓｔｒｙ、２７４巻、３１７４０頁、１９９９年）。更には、ＨＭＧ−１とも結合することも知られている（ＪｏｕｒｎａｌｏｆＢｉｏｌｏｇｉｃａｌＣｈｅｍｉｓｔｒｙ、２７０巻、２５７５２頁、１９９５年）。また、ＲＡＧＥのアミノ酸配列については、ヒトばかりでなく、ウシ、ラット、マウス、ウサギなどからのクローニングが報告されているが、動物間変異が多少見られる。もともとＲＡＧＥはマルチリガンド受容体として同定されており、動物間変異を考慮したアミノ酸配列でも、糖尿病合併症因子との結合活性が維持されるのであれば、問題なく使用できる。
【００２４】
本発明で言うところのＩｇＶドメイン誘導体とは、上述のＡＧＥ結合性能を持つとされるＩｇＶドメイン、つまり、３１番目から１０６番目までの７６個のアミノ酸配列を含むＲＡＧＥ由来のペプチドであり、該ペプチドの１若しくは数個のアミノ酸を置換・付加・欠失していても良いし、ＩｇＶドメイン以外のアミノ酸配列でアミノ酸の置換・付加・欠失があってもよい。さらに、ＲＡＧＥ細胞外ドメインのようにＩｇＶドメイン以外の配列が含まれていても良いし、後述するように、精製を簡便化したり、ＲＡＧＥとは異なる別の機能を付与する目的で、ＲＡＧＥとは無関係な何からの人工配列を付加しても良い。最も好ましくは、ＡＧＥなどに代表されるＲＡＧＥ結合物質が結合可能なＲＡＧＥ誘導体である。更には、３９番目から６８番目までの領域のアミノ酸配列の全部若しくは一部を含むアミノ酸配列で構成されたペプチド、つまり、ＩｇＶドメイン由来ペプチドであることが好ましい。そして、ＩｇＶドメイン由来ペプチドは、上述の定義の範囲で、ペプチド以外の化合物が結合してもよいし、該ペプチドの１若しくは数個のアミノ酸を置換・付加・欠失していても良いし、該ペプチド以外のアミノ酸配列でアミノ酸の置換・付加・欠失があってもよいし、上述のような人工配列が付加したようなＩｇＶドメイン由来ペプチド誘導体であってもよい。例えば、ＩｇＶドメイン由来ペプチドに代表される糖尿病性合併症因子吸着体に用いることができる、本発明で言うところのペプチド誘導体に導入可能なアミノ酸配列の例としては、簡単に単離、精製するためにタグと呼ばれる人工配列を遺伝子上に挿入し、タンパク質として利用することができる。精製法には他に、ＲＡＧＥに対する抗体カラムで精製する方法がある。人工配列を挿入しても精製は簡便であるので特に制限を受けない。人工配列にはＨｉｓ−ＴａｇやＧＳＴやＦｃ、プロテインＡ、プロテインＧなどが多く用いられる。精製以外の目的でも様々なアミノ酸配列や化合物を導入可能であり、特に限定を受けるものではない。
【００２５】
ペプチド誘導体を作成する方法は、生物的方法でも化学的方法でも特に限定を受けることなく用いることができる。生物学的方法では、細菌、酵母等の単細胞や植物細胞や昆虫細胞や動物細胞、またはこれらで構成された動物そのものの遺伝子組み替え産物として得ることも可能であるし、特殊な培養条件下での代謝産物として回収することも可能であり、特に限定されない。化学的方法では、固相法や液相法を用いた合成ペプチドの作製手法を用いることも可能であり、更には、上記の生物的手法と化学的手法を融合させて、上記ペプチドに特殊な化学処理をしてもよい。
【００２６】
本発明で言うところの、糖尿病合併症因子吸着体は、前記ペプチド誘導体で構成された化合物を水不溶性担体に固定させたものであり、且つ、滅菌可能であれば何でも良く、特に限定を受けない。吸着体を全血処理する場合には、抗原提示性などの不必要な免疫反応による副作用の可能性があるので、被処理液の宿主と同一、特に医療品であれば、ヒト由来のリガンドが最も好ましいが、吸着体から固定化したペプチド誘導体が遊離しないのであれば、特に限定無く使用が可能である。
【００２７】
ペプチド誘導体の固定化材料である水不溶性担体は、特に限定されない。より好ましくは表面積が多く取れる多孔質体形成可能な材料であり、さらにより好ましくは体液由来の被処理液と非特異的相互作用の少ない素材で構成されていればよく、さらに、被処理液の構成である体液が血液等の血球細胞との副反応によって刺激を低く抑えられる素材であれば最適であるが、これに限定されるものではない。また、担体は有機−有機、有機−無機の複合担体でも良く、無機担体としてはガラスビーズ、シリカゲル、金属ビーズなどがあり、有機担体には架橋ポリビニルアルコール、架橋ポリアクリレート、架橋ポリアクリルアミド、架橋ポリスチレン、架橋ポリスルホン等の合成高分子や結晶性セルロース、架橋アガロース、架橋デキストランなどの多糖類からなる有機担体との組み合わせでも良い。
【００２８】
担体の形状は、粒状、繊維状またはそれらの高次加工品、中空糸状と任意に形状を選ぶことができ、その大きさも特に限定されない。
【００２９】
糖尿病合併症因子吸着体に結合させたペプチド誘導体は、担体表面上にしっかりと固定化されることが好ましい。リガンドを固定化する場合には、担体表面上の水酸基、アミノ基、アルデヒド基、カルボキシル基、チオール基、シラノール基、アミド基、エポキシ基、ハロゲン基、サクシニルイミド基、酸無水物基などを用いて化学的結合による固定化方法があげられるが、必ずしもこれらに限定されるわけではない。また、リガンドは、これら官能基修飾を受けた担体に直接結合してもよいし、何らかのスペーサー分子を介して結合することも可能である。
【００３０】
ここでいう体液とは、血液、血漿、血清、腹水、リンパ液、脳脊髄液、尿及びこれらから得られた分画成分をいうが、特に限定を受けない。より好ましくは、病変が発生する血管に常時接触している体液、すなわち、血液、血漿、血清から得られた希釈成分や分画成分であれば良い。
【００３１】
本発明の吸着体を治療に用いる場合には種々の方法がある。最も簡便な方法としては患者の血液等の体液を体外に導出して血液バッグに貯め、これに本発明の吸着体を混合して糖尿病合併症因子を除去後、フィルターを通して吸着体を除去し、体液を患者に戻す方法がある。
【００３２】
他の方法は吸着体をカラムに充填し、体外循環回路に組み込み、オンラインで吸着除去をする方法である。処理方法には全血を直接還流する方法と血液から血漿を分離してから血漿をカラムに通す方法がある。本発明の吸着体はいずれの方法にも用いることができるが、前述のごとくオンライン処理に最も適している。ただし、必要に応じてはオンライン処理を行わずに、一端、血液等の体液を体外に取り出し、適切な操作の元で試験管内に吸着体と体液を接触させてから体内に戻すという非循環系のアフェレーシス療法にも応用可能である。この場合には、処理後の吸着体を例えば塩濃度のような適切な条件下に曝すことで糖尿病合併症因子を分離除去し、吸着体の吸着性能を再生する電解質溶液で再処理すれば良い。また、吸着体の使用方法は特に記載の内容のみに限定されるものではなく、長期留置に耐えうるような生体適合性に極めて優れた担体に糖尿病合併症因子吸着リガンドであるペプチド誘導体で構成された化合物を固定化出来るならば腹膜透析などにも応用可能であり、適宜その場に応じて使用方法を変えて利用することが出来る。
【００３３】
ここでいう体外循環回路では本発明の吸着体を単独で用いることもできるが、他のアフェレーシス治療にも併用可能である。併用例には、人工透析回路などが挙げられ、透析療法との組み合わせに用いることもできる。特に、より効率よく吸着体の除去性能を出すには、２個以上の複数の糖尿病合併症因子除去器を体外循環回路に組み込み、交互に吸着再生を行うことで実現化できる。
【００３４】
体外循環治療として用いられる糖尿病合併症因子除去器のプライミング容積は２００ｍＬ〜５００ｍＬが好ましいが、特に、透析療法との組み合わせに用いる場合には、患者の体外循環療法による負担を下げるべく、できるだけプライミング容積の小さな吸着体が好ましい。具体的には、５ｍＬ〜３００ｍＬのプライミング容積を持つ糖尿病合併症因子除去器が好ましい。また、一般的な人工透析療法では１回の施行時間が４〜５時間と限られた時間であり、この限られた時間内に出来る限り多くの糖尿病合併症因子を患者の負荷を軽減する形で実施するならば、複数回にわたって簡便に、且つ、再現性良く、吸着と脱離が出来る再生可能な糖尿病合併症因子除去器であることが好ましい。
【００３５】
以上、糖尿病合併症因子吸着体及び糖尿病合併症因子除去器を経済的に量産可能にするには、公知の方法で安価に滅菌することで性能を有意に劣化させないことが絶対条件であり、該技術並びに物が提供されるようになれば、無菌下での使用が容易に可能となり、安全な使用方法が提供可能となる。即ち、本発明は上記目的を満足する発明である。
【００３６】
以下、本発明を実施例に基づいて具体的に説明する。ただし、本発明はこれら記載の内容に限定されるものではない。
【００３７】
【実施例】
実施例１：ポリミキシン系化合物固定化担体の作製
ポリミキシン系化合物溶液の調製は、硫酸ポリミキシンＢ（ｓｉｇｍａ）及び硫酸コリスチン（ｓｉｇｍａ）をそれぞれ蒸留水に溶解させて、ＰＤ−１０カラムで０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）に緩衝液置換させて調製した。次いで、Ｎ−ヒドロキシスクシンイミドエステル基を１０原子の長さのスペーサーを介して導入したアガロースゲル：アフィゲル１０（Ｂｉｏ−Ｒａｄ）１ｍＬにポリミキシン系化合物（ポリミキシンＢ及びコリスチン）１０ｍｇをそれぞれ０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）１ｍＬに溶解した溶液を加え、４℃で一夜ゆっくりと撹拌した。溶解し難い場合には、適宜少量のジメチルスルホキシドで溶解後に０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）１ｍＬに溶解した。１Ｍエタノールアミン−塩酸（ｐＨ８．０）０．１ｍＬを加えて、室温で１．５時間反応させ、未反応のＮ−ヒドロキシスクシンイミドエステル基を不活化した後、それぞれ０．５ＭのＮａＣｌを含む０．１Ｍ酢酸−ＮａＯＨ（ｐＨ４．０）１ｍＬ及び０．１Ｍ炭酸−ＮａＯＨ（ｐＨ９．０）１ｍＬで交互に３回洗浄し、最後にリン酸緩衝液にて平衡化を行うことでポリミキシン系化合物固定化担体を得た。ポリミキシン系化合物固定化担体のうち、ポリミキシンＢ固定化担体を吸着体１とし、コリスチン固定化担体を吸着体２とした。
【００３８】
実施例２：ＲＡＧＥ部分配列誘導体固定化担体の作製
以下のＲＡＧＥ部分配列誘導体の調製を行った。また、アミノ酸は１文字表記で記載してある。Ｃ末端からＮ末端にかけて記載した。純度は９５％以上で調製した。
【００３９】
・ＲＡＧＥ部分配列Ａ：ＫＧＡＰＫＫＰＰＱＲＬＥＷＫＬＮ
（完全長ＲＡＧＥの３９〜５４番目の配列）
・ＲＡＧＥ部分配列Ｂ：ＷＫＬＮＴＧＲＴＥＡＷＫＶＬＳＰＱＧ
（完全長ＲＡＧＥの５１〜６８番目の配列）
上記合成ペプチドを少量のＤＭＳＯで溶解した後で、０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）に溶解させて調製した。多少不溶物が生成する場合でも懸濁液として使用した。次いで、Ｎ−ヒドロキシスクシンイミドエステル基を１０原子の長さのスペーサーを介して導入したアガロースゲル：アフィゲル１０（Ｂｉｏ−Ｒａｄ）１ｍＬにＲＡＧＥ部分配列Ａ及びＢについてそれぞれ１０ｍｇを０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）１ｍＬに添加した溶液を加え、４℃で一夜ゆっくりと撹拌した。溶解し難い場合には、適宜少量のジメチルスルホキシドで溶解後に０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）１ｍＬに溶解した。１Ｍエタノールアミン−塩酸（ｐＨ８．０）０．１ｍＬを加えて、室温で１．５時間反応させ、未反応のＮ−ヒドロキシスクシンイミドエステル基を不活化した後、それぞれ０．５ＭのＮａＣｌを含む０．１Ｍ酢酸−ＮａＯＨ（ｐＨ４．０）１ｍＬ及び０．１Ｍ炭酸−ＮａＯＨ（ｐＨ９．０）１ｍＬで交互に３回洗浄し、最後にリン酸緩衝液にて平衡化を行うことでＲＡＧＥ部分配列誘導体固定化担体を得た。ＲＡＧＥ部分配列誘導体固定化担体のうち、ＲＡＧＥ部分配列Ａ固定化担体を吸着体３とし、ＲＡＧＥ部分配列Ｂ固定化担体を吸着体４とした。
【００４０】
実施例３：ＡＧＥ標品の調製
ＲＡＧＥ結合物質の一例として、グリコールアルデヒド由来ＡＧＥを調製した。調製方法は、公知の文献（ＭｏｌｅｃｕｌａｒＭｅｄｉｃｉｎｅ、６巻、２号、１１４−１２５頁、２０００年）に従い調製した。２５ｍｇ／ｍｌのＢＳＡ（ウシ血清アルブミン）及びＲＳＡ（ウサギ血清アルブミン）を０．１Ｍのグリコールアルデヒドと５ｍＭのＤＴＰＡ（Ｄｉｅｔｈｙｌｅｎｅｔｒｉａｍｉｎｅ−Ｎ，Ｎ，Ｎ’，Ｎ’’，Ｎ’’’−ｐｅｎｔａａｃｅｔｉｃａｃｉｄ）と共に０．２Ｍリン酸緩衝液（ｐＨ７．４）に溶解後、０．２μｍのフィルターにより濾過滅菌して、３７℃で１週間インキュベートした。低分子量の反応物や未反応のグリコールアルデヒドを、ＰＤ−１０脱塩カラムとＰＢＳによる透析により取り除き、ＡＧＥ標品を得た。ＢＳＡ由来のＡＧＥ標品をＡＧＥ標品１とし、ＲＳＡ由来のＡＧＥ標品をＡＧＥ標品２とした。
【００４１】
実施例４：ＡＧＥ標品に対する抗体の調製
実施例３で得られたＡＧＥ標品２について４ｍｇをフロイント完全アジュバント（和光純薬）と１：１の割合で乳化し、ニュージーランド白ウサギ（雌、２．５ｋｇ）の背部数カ所に皮下注射した。初回免疫後、１週間おきに５回の追加免疫を抗体価が上昇するまで行った。追加免疫後、経時的にウサギの耳静脈より少量採血し、抗体価の確認を行った。抗体価の上昇の見られた時点からさらに２週間後、最終追加免疫を行い、その１０日後に抗血清の精製を行うために全採血を行い、抗血清を得た。ついで以下の精製方法によって、抗血清からＡＧＥ特異的な抗体を調製した。まず、ＡＧＥアフィニティーカラムを調製した。７．５ｇのＣＮＢｒ−ａｃｔｉｖａｔｅｄＳｅｐｈａｒｏｓｅ４Ｂを１ｍＭ塩酸で膨潤後、２Ｌの１ｍＭ塩酸で洗浄した。リガンドとして１００ｍｇの実施例３で得られたＡＧＥ標品１をＣｏｕｐｌｉｎｇｂｕｆｆｅｒ（ｐＨ８．３）と混合し、２５ｍｌのＣＮＢｒ−ａｃｔｉｖａｔｅｄＳｅｐｈａｒｏｓｅ４Ｂゲルと室温で１時間振とうしてカップリングさせた。カップリング後、余分なリガンドはＣｏｕｐｌｉｎｇｂｕｆｆｅｒで洗浄した。ゲルの活性残基は、０．１Ｍトリス塩酸緩衝液（ｐＨ８．０）とゲルを室温で２時間振とうしてブロッキングした。ブロッキング終了後、ＣｏｕｐｌｉｎｇｂｕｆｆｅｒとＷａｓｈｉｎｇｂｕｆｆｅｒ（ｐＨ４．０）とで交互に３回洗浄し、更にＰＢＳで３回洗浄することでアフィニティー樹脂を調製した。本実施例で得られた２０ｍｌの抗血清をこのＡＧＥ標品１−Ｓｅｐｈａｒｏｓｅ４Ｂカラム（２．５ｃｍ×５ｃｍ）に４℃で一晩吸着させた。ＰＢＳで洗浄後、吸着画分を１Ｍチオシアン酸カリウムを含んだ２０ｍＭリン酸緩衝液（ｐＨ７．４）で溶出した。溶出時は各フラクションを２８０ｎｍの吸光度でモニターし、ピーク部分を集めてＣｅｎｔｒｉｐｒｅｐ−１０で濃縮後、ＰＤ−１０ゲル濾過カラムを用いて緩衝液の置換を行うことで、ＡＧＥ標品に対する抗体（ＡＧＥ抗体）を得た。
【００４２】
実施例５：吸着体の高圧蒸気滅菌処理
実施例１及び２で得られた吸着体１〜４について、高圧蒸気滅菌処理を行った。滅菌条件は、１２０℃、２０分である。吸着体は生理食塩水に５０％スラリーになるように懸濁してから、高圧蒸気滅菌可能なバイアルに移して滅菌処理を行った。
【００４３】
実施例６：ＡＧＥ吸着実験
実施例１、２、５で得られた吸着体を沈降体積で１００μｌあたり、サンプル（実施例３で得られたＡＧＥ標品１を１０μｇ／ｍｌ含む０．５％ＢＳＡ−リン酸緩衝液）を９００μｌ加えた。３７℃の孵卵器中で２時間ゆっくり振盪した。この反応液を３０００ｒｐｍで５分間遠心分離して吸着体を沈降させ、上清中のＡＧＥ標品１の吸着量を以下に示す免疫学的測定法（競合法ＥＬＩＳＡ）で測定した。実施例３で得られたＡＧＥ標品１を２０μｇ／ｍｌの濃度に調製し、９６穴マイクロタイタープレートに１ウェル当たり１００μｌ入れて、室温で２時間インキュベートすることにより固相化した。０．５％ＢＳＡを含むＰＢＳでブロッキングした後、緩衝液２５μｌ、測定サンプル２５μｌ、実施例４で得られたＡＧＥ抗体溶液を適宜希釈して５０μｌ入れて、２５℃で２時間振盪した。固相化抗原を認識した抗体に対して、抗ウサギペルオキシダーゼ標識抗体（カッペル）を反応させた後、発色させて５９５ｎｍの吸光度を測定した。各吸着体のＡＧＥ吸着率を表１に示す。すべての吸着体について、ＡＧＥに対して良好な選択性を示すとともに、高圧蒸気滅菌時の吸着性能の失活がほとんどなかったことが確認できた。ただし、性能低下率は次式で定義する。
（性能低下率）＝（１−（高圧蒸気滅菌処理）／（未処理））×１００
【００４４】
表１高圧蒸気滅菌前後での各種吸着体のＡＧＥ吸着率結果

【００４５】
比較例１：ペプチド非固定化担体の作製及びＡＧＥ吸着性能評価
Ｎ−ヒドロキシスクシンイミドエステル基を１０原子の長さのスペーサーを介して導入したアガロースゲル：アフィゲル１０（Ｂｉｏ−Ｒａｄ）１ｍＬに１Ｍエタノールアミン−塩酸（ｐＨ８．０）０．１ｍＬを加えて、室温で１．５時間反応させ、未反応のＮ−ヒドロキシスクシンイミドエステル基を不活化した後、それぞれ０．５ＭのＮａＣｌを含む０．１Ｍ酢酸−ＮａＯＨ（ｐＨ４．０）１ｍＬ及び０．１Ｍ炭酸−ＮａＯＨ（ｐＨ９．０）１ｍＬで交互に３回洗浄し、最後にリン酸緩衝液にて平衡化を行うことでペプチド非固定化担体（吸着体５）を得た。吸着体５について、実施例６と同様の手法でＡＧＥ吸着実験を行った。結果を表２に示す。実施例１及び２で用いたペプチドを固定化しなかった吸着体では、十分なＡＧＥ吸着性能は見られなかった。
【００４６】
表２ペプチド非固定化担体のＡＧＥ吸着率結果
吸着体５７．６％
【００４７】
比較例２：ＡＧＥ抗体固定化担体の調製及び高圧蒸気滅菌耐性評価
実施例４で得られたＡＧＥ抗体を０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）に溶解させて抗体溶液を調製した。次いで、Ｎ−ヒドロキシスクシンイミドエステル基を１０原子の長さのスペーサーを介して導入したアガロースゲル：アフィゲル１０（Ｂｉｏ−Ｒａｄ）１ｍＬに上記抗体溶液（ＡＧＥ抗体１０ｍｇを０．１ＭＨＥＰＥＳ−ＮａＯＨ緩衝液（ｐＨ７．５）１ｍＬに添加した溶液）を加え、４℃で一夜ゆっくりと撹拌した。１Ｍエタノールアミン−塩酸（ｐＨ８．０）０．１ｍＬを加えて、室温で１．５時間反応させ、未反応のＮ−ヒドロキシスクシンイミドエステル基を不活化した後、それぞれ０．５ＭのＮａＣｌを含む０．１Ｍ酢酸−ＮａＯＨ（ｐＨ４．０）１ｍＬ及び０．１Ｍ炭酸−ＮａＯＨ（ｐＨ９．０）１ｍＬで交互に３回洗浄し、最後にリン酸緩衝液にて平衡化を行うことでＡＧＥ抗体固定化担体（吸着体６）を得た。次に、吸着体６について、実施例５と同様の手法で高圧蒸気滅菌処理を行い、高圧蒸気滅菌処理前後でのＡＧＥ吸着性能の評価を行った。ＡＧＥ吸着実験は、実施例６と同様の手法で行った。結果を表３に示す。吸着体６は、高圧蒸気滅菌処理後にＡＧＥ吸着性能を大幅に失ったことが分かった。
【００４８】
表３高圧蒸気滅菌前後でのＡＧＥ抗体固定化担体のＡＧＥ吸着率結果

【００４９】
実施例７：環流式ＡＧＥ吸着実験
実施例１で得られた吸着体１をゲル体積で１ｍＬ分取して、ポリプロピレン製ミニカラム容器（カラム容積１ｍＬ、カラムに入出口がそれぞれ１つずつ有り、カラム内の吸着体がミニカラムから流失しないようにフィルターメッシュを入出口の各ヘッダー内に挟み固定している。入出口には充填液が漏れ出さないようにミニキャップをしてある。）に充填してカラムを作製した。充填溶液はリン酸緩衝液を使用した。次いで、上記ミニカラムを高圧蒸気滅菌処理するために、吸着体１を充填してミニカラムを作製して、更に、２００ｍＬのビーカーにリン酸緩衝液を１００ｍＬ継ぎ足して、そのなかに上記ミニカラムを入れた。更に、ビーカー上部にはアルミホイルをして高圧蒸気滅菌後もビーカー内部は無菌状態が保てるようにした。その後、実施例５と同様の手法で高圧蒸気滅菌処理を行うことで、滅菌処理したミニカラムを作製した。次いで、滅菌処理後の本ミニカラムが環流中でも吸着性能が保持されているかどうか確認する目的で環流式のＡＧＥ吸着実験を行った。カラムあたり、サンプル（実施例３で得られたＡＧＥ標品１を１０μｇ／ｍＬ含む０．５％ＢＳＡ−リン酸緩衝液）を１０ｍＬ処理した。３７℃の孵卵器中で８時間環流吸着した。環流はペリスタポンプを用いて行い、流量は０．２ｍＬ／分で固定した。適宜、１００ｕＬずつサンプリングして、環流溶液中のＡＧＥ量を実施例６に記載した免疫学的測定法で測定した。
【００５０】
表４環流式ＡＧＥ吸着実験結果

【００５１】
従って、本カラムは滅菌処理後でも環流式で有意な吸着性能を示すことがわかった。
【００５２】
【発明の効果】
本発明で見出された滅菌できる糖尿病合併症因子吸着体を充填してなる除去器及びその使用方法によって、産業レベルでの大量生産及び吸着体を使用した製品に対して、公知の滅菌方法で吸着材としての性能を大幅に損なわないような吸着体の設計が可能となった。また、安価な手法で滅菌できるようになったことにより、上記吸着体及び吸着体使用製品の無菌下での保存ができることでの長期安定管理を実現化できる。更には、糖尿病性透析患者などに対して、上記吸着体を充填した体外循環医療用具に代表される除去器を使用する場合には、患者への安全性（エンドトキシン混入防止）を配慮するために無菌下での操作が要求されるが、本発明により医療用具レベルとしても許諾可能な吸着体を提供できる。
【００５３】
以上により、本発明によって見出された滅菌できる糖尿病合併症因子の除去手段は、これまで十分な治療効果が期待できなかった糖尿病性腎症に代表される糖尿病合併症患者や血管障害患者、並びに、透析患者と言った酸化ストレスが亢進する難治性の患者に対して提供可能な技術となったばかりでなく、ＲＡＧＥが関与する難治性疾患、例えば、敗血症、アルツハイマー病などのアミロイド症、高脂血漿、腎症全般、癌、過炎症の患者にも有効な治療手段を提供できる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a diabetic complication factor adsorbent in which a compound composed of a peptide derivative is bound to a water-insoluble carrier, and a remover filled with the compound, which does not substantially inactivate the binding performance to the diabetic complication factor by sterilization treatment, and The present invention relates to a method for removing diabetic complication factors using the method.
[0002]
[Prior art]
In recent years, as diabetic nephropathy worsens, the number of patients undergoing dialysis is rapidly increasing, and the main cause of new dialysis patients since 1998 is diabetic nephropathy. It is positioned as. It is known that diabetic nephropathy and chronic glomerulonephritis occupy the majority of the underlying diseases of the introduction of artificial dialysis, but there is a large difference in the survival rate after the introduction of dialysis. Although there are some differences in the evaluation facilities, the 50% survival rate of diabetic nephropathy patients after introduction of dialysis is 3 to 4 years, whereas the 50% survival rate of chronic glomerulonephritis patients is 10 years. The prognosis of diabetic dialysis patients is clearly poor and is a social problem, but no sign of solution has been seen.
[0003]
The cause is that the pathogenesis of diabetic complications is still unclear. However, recently, various degenerative substances represented by AGEs (advanced glycation endproducts) have been attracting attention as candidates of pathogenic substances that cause vascular lesions which can be said to be common lesions of diabetic complications.
[0004]
AGE is considered to be mainly a causative substance of diabetic complications. Reducing sugar such as glucose present in blood, its metabolites and reaction products, and low-molecular-weight substances such as proteins and lipids Is a product of an advanced glycation reaction that binds without the involvement of an enzyme, that is, non-enzymatically, and is sometimes collectively referred to as a carbonyl stress product.
[0005]
At that time, various characteristics such as fluorescence, brownness, cross-linking, dehydration, oxidation, condensation, and transfer were discovered for carbonyl stress products that are non-enzymatic reactants such as reducing sugars and proteins. The pathogenesis of carbonyl stress products, a possible diabetic complication factor, could not be directly explained. Regarding the method of evaluating AGE, which is one of the carbonyl stress products, there have been many cases in which measurement in blood has been previously attempted by using the fluorescence characteristic, which is one of the characteristics, as disclosed in JP-A-6-312134. In 1), an attempt is made to measure AGE in human plasma at an excitation wavelength of 390 nm and a fluorescence wavelength of 450 nm (for example, see Non-Patent Document 1). However, fluorescence measurement is used to confirm the progress of the saccharification reaction in a test tube. However, there are many effects of nonspecific fluorescence of impurities and drugs in blood, and the excitation wavelength of pentosidine and the like is 335 nm and the fluorescence wavelength is 385 nm. It has been found that AGEs having the above-mentioned fluorescent characteristics are only a small part, for example, such as those having different fluorescent characteristics are found (for example, see Non-Patent Document 2). Furthermore, it is known that CML (carboxymethyl lysine) and CEL (carboxyethyl lysine), which are considered to be the main structures of AGE, do not have fluorescent properties. It has been recognized that simply measuring at a wavelength of 450 nm is not a reliable measurement method. Since then, the evaluation based on the fluorescence characteristics has been performed by applying a molecular sieve or a chemical treatment to the measurement procedure, or in consideration of which AGE structure the fluorescence reflects. In addition, there is a movement to attempt measurement using a specific immunological measurement method. There are about 10 AGE structures identified so far, such as CML, CEL, pentosidine, pyralin, crosslin, fluorolink, imidazolone, X1, and argupyrimidine, and all of these AGE structures are pathogenic as carbonyl stress products. It is said that this is not the case (for example, see Non-Patent Document 3). For this reason, there is a problem that the cause of carbonyl stress beginning with AGE is further complicated, and only carbonyl stress products as diabetic complication factors that cause various vascular lesions represented by diabetic complications are accurately detected. Even the method was in very poor condition.
[0006]
On the other hand, it has been found that non-enzymatic denaturants having properties as diabetic complication factors are mainly caused by binding to certain special receptors on cells. Many of them are generally called scavenger receptors, but it has also been reported that non-enzymatic denaturing substances such as carbonyl stress products interact with receptors that originally have another function to cause pathogenesis. Galectin-3, RAGE (Receptor for AGE) and the like. In particular, it has been found that RAGE not only causes the onset of nephropathy, retinopathy, and neuropathy, which are the three major complications of diabetes, but also promotes arteriosclerosis. Recently, it has been proven that the interaction between RAGE at the animal level and a specific substance that is enhanced in a diabetic state causes vascular disorders such as diabetic complications (for example, see Non-Patent Document 4). . At present, RAGE is considered to be the most important pathway for eliciting the etiology of diabetic complication factors.
[0007]
Some types of adsorbents for RAGE binding substances represented by AGE are already known. For example, an adsorbent produced by binding an anti-AGE antibody to beads (for example, see Non-Patent Document 5) is known, but these adsorbents are not necessarily required to be used under sterilization. . Further, in the above-mentioned known method, under the small-volume production conditions required at the laboratory level, the problem can be solved by performing the production of the adsorbent in a sterile area. However, if mass production at the industrial level and stable management of products using adsorbents are required, the design of adsorbents that does not significantly impair the performance as adsorbents by known sterilization methods is required, but sterilization can be performed. A technique capable of supplying a large amount of adsorbent has not been found.
[0008]
[Patent Document 1]
JP-A-6-312134
[0009]
[Non-patent document 1]
Makita (Makita Z) and 8 others, "Advanced Glycosylation and Products in the Patients with the Diabetic Nephropathy.", The New England New Zealand Newspaper. England Journal of Medicine), 1991, vol. 325, p838.
[0010]
[Non-patent document 2]
Miyata T, and 7 others, "Accumulation of albumin-binding and free pentosidine in the circulation of patients with end-stage renal failure uremic disease: renal involvement in the pathophysiology of pentosidine. -form pentosidine in the circulation of uremic patients with end-stage renal failure:. renal implications in the pathophysiology of pentosidine) ", journal of the American Society of Association for Nephrology (journal of the American Society Nephrology), 1996 Year, number Volume, Issue 8, p1198-1206
[0011]
[Non-Patent Document 3]
Miyata T, et al., "Implication of increased oxidative stress in the introduction of advanced oxidative stress induction in the formation of advanced glycation end products in patients with end-stage renal failure" with end-stage regular failure. ", Kidney International, 1997, Vol. 51, No. 4, p1170-1181.
[0012]
[Non-patent document 4]
Yamamoto (Yasuhiko Yamamoto) and 8 others, "Diabetic renal lesions in mice transgenic for human RAGE", Journal of the Japan Diabetes Society, 1999, Vol. 42, supplement 1, pS-194.
[0013]
[Non-Patent Document 5]
Busta (Basta G), "Advanced Glycation End Products Activate Endothelial Cells via Signaling Receptor RAGE: Mechanisms for Amplification of the Inflammatory Response mechanism for amplification of infrastructure responses. "", Circulation, 2002, Vol. 105, No. 7, p816-822.
[0014]
[Problems to be solved by the invention]
The present invention makes it possible to economically mass-produce diabetic complication factor adsorbents for removing diabetic complication factors or to sterilize the adsorbents in order to use the adsorbents for body fluid treatment purposes. Accordingly, an object of the present invention is to provide a method for safely and aseptically removing diabetic complication factors.
[0015]
[Means for Solving the Problems]
The present invention has the following configurations to achieve the above object.
"(1) A diabetic complication factor adsorbent characterized in that a compound composed of a peptide derivative is bound to a water-insoluble carrier, the binding performance of which does not substantially inactivate the diabetic complication factor by sterilization."
"(2) Diabetes contained in a liquid to be treated by bringing a liquid to be treated derived from a body fluid into contact with a diabetic complications factor removal container filled with the diabetic complications factor adsorbent according to the above item (1). Use of a diabetic complication factor remover characterized by selectively removing complication factors as a substance to be adsorbed. "
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0017]
Sterilization as referred to in the present invention is a chemical or physical treatment method in which no biological activity such as proliferation of organisms such as bacteria and viruses mixed in the adsorbent or the remover filled with the adsorbent can be performed. That is. Examples of sterilization methods include high-pressure steam sterilization, radiation sterilization such as γ-ray sterilization and electron beam sterilization, gas sterilization using a germicidal gas such as ethylene oxide gas, and aqueous solutions of hydrogen peroxide and perchloric acid. Examples include chemical sterilization using a bactericidal compound, and are not particularly limited.
[0018]
In the present invention, "the binding performance to the diabetic complication factor is not substantially inactivated by the sterilization treatment" means that the decrease rate of the adsorption performance before and after the high-pressure steam sterilization treatment is at least 50% or less. It can be unambiguously determined by being suppressed within preferably 30%, more preferably within 20%, and most preferably within 10%. The high pressure steam sterilization means a high pressure steam treatment at 120 ° C. for 20 minutes.
[0019]
The definition and measuring method of the adsorption performance in the present invention can be uniquely evaluated by the following method. Regarding the measuring method, the sample to be adsorbed (0.5% bovine serum albumin-phosphate buffer containing 10 μg / ml of the AGE standard (0.15 M sodium chloride in 20 mM phosphate buffer) per 100 μl of the sedimented volume of the adsorbent was used. Was added, and the mixture was slowly shaken in an incubator at 37 ° C. for 2 hours, and the reaction solution was centrifuged at 3000 rpm for 5 minutes to sediment the adsorbent. The amount of AGE sample adsorbed in the medium is measured by an immunoassay (competitive ELISA) shown below. The above AGE standard was prepared at a concentration of 20 μg / ml, put in a 96-well microtiter plate at 100 μl per well, incubated at room temperature for 2 hours, immobilized, and blocked with PBS containing 0.5% BSA. Thereafter, 25 μl of the buffer solution, 25 μl of the measurement sample, and 50 μl of the AGE antibody solution were appropriately diluted and added, and the mixture was shaken at 25 ° C. for 2 hours to react the anti-rabbit peroxidase-labeled antibody with the antibody that recognized the immobilized antigen. This is a method of measuring the absorbance at 595 nm by coloring and then measuring the absorbance at 595 nm. The reduction rate of the AGE sample before and after treatment with the adsorbent in the above method is defined as adsorption performance. The fact that the adsorbent has the adsorption performance means that the reduction rate evaluated according to the above definition is at least 50% or less. It is more preferably at most 40%, even more preferably at most 30%. Most preferably, the reduction rate of the adsorption performance before and after the treatment is 10% or less. In addition, the above-mentioned AGE standard is one prepared by the following method. 25 mg / ml BSA was combined with 0.1 M glycol aldehyde and 5 mM DTPA (Diethylenetriamine-N, N, N ′, N ″, N ′ ″-pentaacetic acid) in 0.2 M phosphate buffer (pH 7.4). ), Sterilized by filtration through a 0.2 μm filter, and incubated at 37 ° C. for 1 week. A low-molecular-weight reactant or unreacted glycolaldehyde may be removed by dialysis with a PD-10 desalting column and PBS to obtain an AGE standard. The diabetic complication factor referred to in the present invention is a general term for etiological substances that cause diabetic complications, and is not particularly limited as long as it is detected in the body of a diabetic patient. Preferably, any RAGE binding substance that binds to RAGE may be used. Examples of RAGE binding substances include not only AGE but also a family of high mobility proteins represented by HMG-1, a group of certain inflammatory markers, serum amyloid A, S100 / calgranulin superfamily, transthyretin, and the like. The substance may be a so-called substance, and is not particularly limited as long as binding to RAGE is recognized. In addition, these RAGE binding substances are collectively defined as EN-RAGE (extracellularly newly identified RAGE-binding protein) (Cell, Vol. 97, p. 889, 1999). It is considered that the possibility of separation and identification is high. Even more preferred is AGE, but there are also various structures of AGE, and more preferred is AGE binding AGE. AGE is produced by a non-enzymatic reaction between a biological substance typified by proteins, hormones, saccharides, lipids, vitamins, etc. and a reducing sugar typified by glucose, but the reducing saccharide is not necessarily limited to glucose. It is not necessary, and may be an α-hydroxyaldehyde compound such as glycolaldehyde, glyceraldehyde, methylglyoxal, glyoxal, or 3-deoxyglucozone, or a dicarbonyl compound. Further, during the generation of AGE, there may be oxidative modification by various radicals such as hydroxyl radical, nitric oxide, peroxynitrite, etc., and there is no particular limitation on the production route and the reaction substrate, and the result of the oxidative reaction is not limited. The modified substance is not particularly limited as long as it is a modified substance generated in the above step. What is particularly important for RAGE binding substances is that pathogenesis is recognized by binding to RAGE.
[0020]
The pathogenesis of the diabetic complication factor as referred to herein means that, when the diabetic complication factor comes into contact with the cells, induction of various cytokines, promotion of cell migration, enhancement of DNA synthesis, enhancement of adhesion molecule expression and fibrosis, NF- Activates κB, induces oxidative stress, induces one or more phenomena such as increased permeability of cells and tissues, and as a result, causes vascular lesions such as arteriosclerosis, nephropathy, retinopathy, and neurosis, Say that causes inflammation. In addition, the diabetic complication factor is not necessarily an exacerbation factor found only in diabetes, but may also be seen in cases of reduced excretion and metabolic functions such as artificial dialysis and various renal diseases. Furthermore, the involvement of RAGE has also been suggested in sepsis, cancer metastasis, Alzheimer's disease, hyperinflammation, etc., so that any disease involving RAGE can be recognized in patients without any limitation. Then, it may be interpreted as a diabetic complication factor. For convenience, it is defined as a diabetic complication factor because it is a general term for etiological substances related to vascular lesions that are particularly detected in diabetic patients and are often found in such patients.
[0021]
The peptide derivative referred to in the present invention has no problem as long as it is an amino acid polymer having at least two or more peptide bonds, and there is no particular problem even if compounds other than amino acids are further contained. The compound other than the amino acid may be a compound derived from a living body such as sugar or lipid, or may be an aromatic ring or an aliphatic compound that does not exist in the living body. Further, the amino acid constituting the peptide derivative is not a problem at all if it is an amino acid present in the living body, and there is no particular problem even if it contains a mirror image of an L-form amino acid which is predominantly found in the living body called a D-form amino acid. Can be used. Preferably, the peptide derivative contains at least two basic functional groups, and the basic amino acid may be composed of an amino group, an ammonium ion group, a heterocyclic ring or a cyclic compound. The basic functional group is more preferable if the functional group shows basicity under physiological conditions. Further, the peptide derivative may be a linear or cyclic peptide compound or a complex of both, and is not particularly limited. More preferably, any peptide derivative that does not lose its performance as an adsorbent under sterile conditions can be used without any particular problem. Among them, a polymyxin-based compound is particularly preferable as long as it is a cyclic peptide derivative, and a peptide derived from RAGE IgV domain is preferable as long as it is linear.
[0022]
The polymyxin-based compound is a derivative having a polymyxin skeleton, and is represented by polymyxin B and colistin (polymyxin E). In addition, compounds such as polymyxin A, polymyxin C, polymyxin D, and polymyxin F are known, but are not particularly limited as long as they have a polymyxin skeleton and are defined as defined above.
[0023]
The term “RAGE partial sequence derivative” as used in the present invention refers to a partial sequence of a full-length RAGE including a peptide region to which a RAGE-binding substance can bind, and preferably includes a part of a sequence in an IgV domain. It is. In the case of human, full-length RAGE is composed of 404 amino acids and has a signal sequence composed of 22 amino acids from the N-terminus, and as a result, forms a receptor as 382 amino acids. Among them, the extracellular domain containing an amino acid sequence capable of adsorbing a diabetic complication factor is composed of 342 amino acids. The extracellular domain is further classified into smaller domains, that is, a V-type domain (consisting of 76 amino acids from the 31st to the 106th) from the N-terminal side in a total of 404 amino acid sequences, and a C2-type domain. 1 (consisting of 78 amino acids from 137 to 214) and C2 type domain 2 (consisting of 57 amino acids from 252 to 308). Among the extracellular domains, a domain called a V-type domain, also referred to as an IgV domain as described above, is particularly important and has been confirmed to have a binding ability to AGE which is one of the diabetic complication factors. (Journal of Biological Chemistry, 274, 31740, 1999). Furthermore, it is also known to bind to HMG-1 (Journal of Biological Chemistry, 270, 25752, 1995). Regarding the amino acid sequence of RAGE, cloning has been reported not only from humans but also from cattle, rats, mice, rabbits, etc., but some variation between animals is observed. RAGE was originally identified as a multi-ligand receptor, and it can be used without problem even with an amino acid sequence that takes into account inter-animal variation as long as the binding activity with a diabetic complication factor is maintained.
[0024]
The IgV domain derivative as referred to in the present invention is an IgV domain having the above-mentioned AGE binding ability, that is, a RAGE-derived peptide containing 76 amino acid sequences from the 31st to the 106th, and the peptide May be substituted / added / deleted for one or several amino acids, or may be substituted / added / deleted for an amino acid in an amino acid sequence other than the IgV domain. Furthermore, a sequence other than the IgV domain may be included as in the RAGE extracellular domain. As described later, RAGE is used for the purpose of simplifying purification or imparting another function different from RAGE. Unrelated artificial sequences may be added. Most preferably, it is a RAGE derivative to which a RAGE binding substance represented by AGE or the like can bind. Furthermore, a peptide composed of an amino acid sequence containing all or a part of the amino acid sequence of the region from position 39 to position 68, that is, an IgV domain-derived peptide is preferable. The IgV domain-derived peptide may be bound to a compound other than the peptide within the range defined above, or may have one or several amino acids substituted, added, or deleted in the peptide, An amino acid sequence other than the peptide may have amino acid substitution, addition, or deletion, or an IgV domain-derived peptide derivative to which an artificial sequence as described above has been added. For example, an example of an amino acid sequence that can be introduced into a peptide derivative according to the present invention, which can be used for a diabetic complication factor adsorbent represented by an IgV domain-derived peptide, is that it can be easily isolated and purified. An artificial sequence called a tag can be inserted into a gene and used as a protein. As another purification method, there is a method of purifying with an antibody column for RAGE. Even if an artificial sequence is inserted, the purification is simple and there is no particular limitation. His-Tag, GST, Fc, Protein A, Protein G, and the like are often used as artificial sequences. Various amino acid sequences and compounds can be introduced for purposes other than purification, and are not particularly limited.
[0025]
The method for producing the peptide derivative may be a biological method or a chemical method without particular limitation. In biological methods, bacteria, yeast and other single cells, plant cells, insect cells and animal cells, or can be obtained as a genetically modified product of the animal itself composed of these, under special culture conditions It can be recovered as a metabolite, and is not particularly limited. In the chemical method, it is also possible to use a synthetic peptide production method using a solid phase method or a liquid phase method.Furthermore, by combining the above biological method and chemical method, a special Chemical treatment may be performed.
[0026]
The diabetic complication factor adsorbent referred to in the present invention is a compound in which the compound composed of the peptide derivative is immobilized on a water-insoluble carrier, and is not particularly limited as long as it can be sterilized. . When the adsorbent is treated with whole blood, there is a possibility of side effects due to unnecessary immune reactions such as antigen presenting. Most preferably, it can be used without any particular limitation as long as the immobilized peptide derivative is not released from the adsorbent.
[0027]
The water-insoluble carrier as the immobilizing material for the peptide derivative is not particularly limited. More preferably, it is a material capable of forming a porous body capable of taking a large surface area, and still more preferably, it may be composed of a material to be treated derived from a body fluid and a material having a small nonspecific interaction. The material is optimal as long as it is a material whose stimulus can be suppressed low by a side reaction with blood cells such as blood, but is not limited thereto. The carrier may be an organic-organic or organic-inorganic composite carrier, and the inorganic carrier includes glass beads, silica gel, metal beads, and the like.The organic carrier includes cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, cross-linked polystyrene. Or a combination with a synthetic polymer such as cross-linked polysulfone or an organic carrier comprising a polysaccharide such as crystalline cellulose, cross-linked agarose, cross-linked dextran, or the like.
[0028]
The shape of the carrier can be arbitrarily selected from a granular shape, a fibrous shape, a higher processed product thereof, and a hollow fiber shape, and the size is not particularly limited.
[0029]
The peptide derivative bound to the diabetic complication factor adsorbent is preferably firmly immobilized on the surface of the carrier. When immobilizing the ligand, use a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a thiol group, a silanol group, an amide group, an epoxy group, a halogen group, a succinylimide group, an acid anhydride group, etc. on the surface of the carrier. However, the method is not limited to these methods. Further, the ligand may be directly bound to the functional group-modified carrier, or may be bound via some kind of spacer molecule.
[0030]
The term “body fluid” as used herein refers to blood, plasma, serum, ascites, lymph, cerebrospinal fluid, urine and fraction components obtained therefrom, but is not particularly limited. More preferably, it may be a body fluid that is constantly in contact with a blood vessel in which a lesion occurs, that is, a diluted component or a fractionated component obtained from blood, plasma, or serum.
[0031]
There are various methods for using the adsorbent of the present invention for treatment. As the simplest method, a body fluid such as a patient's blood is led out of the body, stored in a blood bag, mixed with the adsorbent of the present invention to remove diabetic complication factors, and then the adsorbent is removed through a filter. There are ways to return bodily fluids to the patient.
[0032]
Another method is to pack the adsorbent into a column, incorporate it into an extracorporeal circulation circuit, and remove the adsorbate online. Treatment methods include a method of directly refluxing whole blood and a method of separating plasma from blood and then passing the plasma through a column. The adsorbent of the present invention can be used in any method, but is most suitable for online processing as described above. However, a non-circulatory system in which the body fluid such as blood is taken out of the body at one end and the adsorbent is brought into contact with the body fluid in a test tube under appropriate operation before returning to the body without performing online processing if necessary Can be applied to apheresis therapy. In this case, the diabetic complication factor may be separated and removed by exposing the treated adsorbent to appropriate conditions such as a salt concentration, and the adsorbent may be re-treated with an electrolyte solution that regenerates the adsorption performance of the adsorbent. . In addition, the method of using the adsorbent is not limited to the contents described in particular, but is composed of a peptide derivative that is a diabetic complication factor-adsorbing ligand in a carrier having extremely excellent biocompatibility that can withstand long-term indwelling. If the compound can be immobilized, it can be applied to peritoneal dialysis and the like, and can be used by appropriately changing the method of use depending on the situation.
[0033]
The adsorbent of the present invention can be used alone in the extracorporeal circuit, but can also be used in combination with other apheresis treatments. Examples of the combination include an artificial dialysis circuit and the like, and can be used in combination with dialysis therapy. In particular, more efficient adsorbent removal performance can be achieved by incorporating two or more diabetic complication factor removers into the extracorporeal circulation circuit and alternately performing adsorption regeneration.
[0034]
The priming volume of the diabetic complication factor remover used as extracorporeal circulation treatment is preferably 200 mL to 500 mL. In particular, when used in combination with dialysis therapy, the priming volume is minimized to reduce the burden of extracorporeal circulation therapy on the patient. Is preferred. Specifically, a diabetic complication factor remover having a priming volume of 5 mL to 300 mL is preferable. In addition, in the case of general dialysis treatment, the time for one administration is limited to 4 to 5 hours, and as many diabetic complication factors as possible can be reduced in this limited time to reduce the burden on the patient. In this case, it is preferable to use a regenerable diabetic complication factor remover capable of performing adsorption and desorption easily and with good reproducibility a plurality of times.
[0035]
As described above, in order to be able to mass-produce the diabetic complication factor adsorbent and the diabetic complication factor remover economically, it is an absolute condition that the performance is not significantly deteriorated by sterilization at low cost by a known method. If a technique and a thing are provided, use under aseptic state will be attained easily and a safe use method will be provided. That is, the present invention satisfies the above objects.
[0036]
Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the contents described above.
[0037]
【Example】
Example 1: Preparation of polymyxin-based compound-immobilized carrier
To prepare a polymyxin compound solution, polymyxin sulfate B (sigma) and colistin sulfate (sigma) are each dissolved in distilled water, and the buffer is replaced with a 0.1 M HEPES-NaOH buffer (pH 7.5) using a PD-10 column. Prepared. Next, 10 mg of a polymyxin-based compound (polymyxin B and colistin) was added to 1 mL of an agarose gel: Affigel 10 (Bio-Rad) in which an N-hydroxysuccinimide ester group was introduced via a spacer having a length of 10 atoms, each of which was 0.1 MHEPES-NaOH. A solution dissolved in 1 mL of a buffer (pH 7.5) was added, and the mixture was slowly stirred at 4 ° C. overnight. When it was difficult to dissolve, it was dissolved in a small amount of dimethyl sulfoxide as appropriate and then dissolved in 1 mL of 0.1 M HEPES-NaOH buffer (pH 7.5). 0.1 mL of 1 M ethanolamine-hydrochloric acid (pH 8.0) was added, and the mixture was reacted at room temperature for 1.5 hours to inactivate unreacted N-hydroxysuccinimide ester groups. Washing alternately 3 times with 1 mL of 1 M acetic acid-NaOH (pH 4.0) and 1 mL of 0.1 M carbonate-NaOH (pH 9.0), and finally equilibrating with a phosphate buffer to fix the polymyxin compound A modified carrier was obtained. Among the polymyxin-based compound-immobilized carriers, the polymyxin B-immobilized carrier was designated as adsorbent 1, and the colistin-immobilized carrier was designated as adsorbent 2.
[0038]
Example 2: Preparation of RAGE partial sequence derivative-immobilized carrier
The following RAGE partial sequence derivatives were prepared. Amino acids are described in one-letter code. The description is from the C-terminal to the N-terminal. The purity was adjusted to 95% or more.
[0039]
・ RAGE partial sequence A: KGAPKKPPQRLEWKLN
(Sequences 39-54 of full length RAGE)
-RAGE partial sequence B: WKLNTGRTEAWKVLSPQG
(Sequences 51-68 of full length RAGE)
The synthetic peptide was dissolved in a small amount of DMSO, and then dissolved in a 0.1 M HEPES-NaOH buffer solution (pH 7.5). It was used as a suspension even if some insolubles were formed. Next, agarose gel in which N-hydroxysuccinimide ester group was introduced via a spacer having a length of 10 atoms: 10 mL of each of RAGE partial sequences A and B was added to 1 mL of Affigel 10 (Bio-Rad) in 0.1 M HEPES-NaOH buffer solution. (PH 7.5) The solution added to 1 mL was added, and the mixture was slowly stirred at 4 ° C. overnight. When it was difficult to dissolve, it was dissolved in a small amount of dimethyl sulfoxide as appropriate and then dissolved in 1 mL of 0.1 M HEPES-NaOH buffer (pH 7.5). 0.1 mL of 1 M ethanolamine-hydrochloric acid (pH 8.0) was added, and the mixture was reacted at room temperature for 1.5 hours to inactivate unreacted N-hydroxysuccinimide ester groups. The mixture was washed three times alternately with 1 mL of 1 M acetic acid-NaOH (pH 4.0) and 1 mL of 0.1 M carbonate-NaOH (pH 9.0), and finally equilibrated with a phosphate buffer to obtain a RAGE partial sequence derivative. An immobilized carrier was obtained. Among the RAGE partial sequence derivative-immobilized carriers, the RAGE partial sequence A-immobilized carrier was designated as adsorbent 3, and the RAGE partial sequence B-immobilized carrier was designated as adsorbent 4.
[0040]
Example 3: Preparation of AGE standard
As an example of the RAGE binding substance, AGE derived from glycolaldehyde was prepared. The preparation method was prepared according to a known document (Molecular Medicine, Vol. 6, No. 2, page 114-125, 2000). 25 mg / ml BSA (bovine serum albumin) and RSA (rabbit serum albumin) were combined with 0.1 M glycol aldehyde and 5 mM DTPA (Diethylenetriamine-N, N, N ', N ", N'"-pentaacetic acid). Was dissolved in a 0.2 M phosphate buffer (pH 7.4), and sterilized by filtration through a 0.2 μm filter, followed by incubation at 37 ° C. for 1 week. Low molecular weight reactants and unreacted glycolaldehyde were removed by dialysis with a PD-10 desalting column and PBS to obtain an AGE standard. The AGE standard derived from BSA was designated as AGE standard 1, and the AGE standard derived from RSA was designated as AGE standard 2.
[0041]
Example 4: Preparation of antibody against AGE standard
4 mg of the AGE sample 2 obtained in Example 3 was emulsified with Freund's complete adjuvant (Wako Pure Chemical Industries) at a ratio of 1: 1 and injected subcutaneously into several backs of New Zealand white rabbits (female, 2.5 kg). After the first immunization, 5 booster immunizations were performed every other week until the antibody titer increased. After the booster immunization, a small amount of blood was collected from the rabbit ear vein over time to confirm the antibody titer. Two additional weeks after the time point at which the antibody titer increased, a final booster was performed, and 10 days later, whole blood was collected to purify the antiserum to obtain an antiserum. Next, an AGE-specific antibody was prepared from the antiserum by the following purification method. First, an AGE affinity column was prepared. 7.5 g of CNBr-activated Sepharose 4B was swollen with 1 mM hydrochloric acid, and then washed with 2 L of 1 mM hydrochloric acid. 100 mg of the AGE sample 1 obtained in Example 3 as a ligand was mixed with Coupling buffer (pH 8.3), and coupled with 25 ml of CNBr-activated Sepharose 4B gel by shaking at room temperature for 1 hour. After coupling, the excess ligand was washed with a coupling buffer. The active residues of the gel were blocked by shaking the gel with 0.1 M Tris-HCl buffer (pH 8.0) at room temperature for 2 hours. After the blocking was completed, the resin was washed three times alternately with a coupling buffer and a washing buffer (pH 4.0), and further washed three times with PBS to prepare an affinity resin. 20 ml of the antiserum obtained in this example was adsorbed to this AGE standard 1-Sepharose 4B column (2.5 cm × 5 cm) at 4 ° C. overnight. After washing with PBS, the adsorbed fraction was eluted with a 20 mM phosphate buffer (pH 7.4) containing 1 M potassium thiocyanate. At the time of elution, each fraction was monitored by the absorbance at 280 nm, the peak portion was collected, concentrated with Centriprep-10, and then replaced with a buffer using a PD-10 gel filtration column, whereby an antibody against the AGE standard (AGE) was obtained. Antibody).
[0042]
Example 5: High pressure steam sterilization of adsorbent
The adsorbents 1 to 4 obtained in Examples 1 and 2 were subjected to high-pressure steam sterilization. Sterilization conditions are 120 ° C. and 20 minutes. The adsorbent was suspended in physiological saline so as to be a 50% slurry, and then transferred to a vial capable of high-pressure steam sterilization for sterilization.
[0043]
Example 6: AGE adsorption experiment
A sample (0.5% BSA-phosphate buffer containing 10 μg / ml of the AGE sample 1 obtained in Example 3) was added to the adsorbent obtained in Examples 1, 2, and 5 in a sedimented volume of 100 μl. 900 μl was added. Shake slowly in an incubator at 37 ° C for 2 hours. The reaction solution was centrifuged at 3000 rpm for 5 minutes to precipitate the adsorbent, and the amount of AGE sample 1 adsorbed in the supernatant was measured by an immunological assay (competitive ELISA) as described below. The AGE sample 1 obtained in Example 3 was prepared at a concentration of 20 μg / ml, put in a 96-well microtiter plate at 100 μl per well, and incubated at room temperature for 2 hours to solidify. After blocking with PBS containing 0.5% BSA, 25 μl of a buffer solution, 25 μl of a measurement sample, and 50 μl of the AGE antibody solution obtained in Example 4 were appropriately diluted, and shaken at 25 ° C. for 2 hours. After reacting an anti-rabbit peroxidase-labeled antibody (Kappel) with the antibody that recognized the immobilized antigen, the antibody was colored and the absorbance at 595 nm was measured. Table 1 shows the AGE adsorption rate of each adsorbent. It was confirmed that all the adsorbents exhibited good selectivity to AGE and almost no deactivation of the adsorption performance during high-pressure steam sterilization. However, the performance reduction rate is defined by the following equation.
(Performance reduction rate) = (1− (high-pressure steam sterilization treatment) / (untreated)) × 100
[0044]
Table 1 Results of AGE adsorption rate of various adsorbents before and after autoclaving

[0045]
Comparative Example 1: Preparation of non-immobilized peptide carrier and evaluation of AGE adsorption performance
Agarose gel in which N-hydroxysuccinimide ester group was introduced via a spacer having a length of 10 atoms: 0.1 mL of 1 M ethanolamine-hydrochloric acid (pH 8.0) was added to 1 mL of Affigel 10 (Bio-Rad), and the mixture was added at room temperature. After reacting for 1.5 hours to inactivate unreacted N-hydroxysuccinimide ester groups, each 1 mL of 0.1 M acetic acid-NaOH (pH 4.0) containing 0.5 M NaCl and 0.1 M carbonate-NaOH ( The mixture was washed three times alternately with 1 mL (pH 9.0), and finally equilibrated with a phosphate buffer to obtain a non-peptide-immobilized carrier (adsorbent 5). An AGE adsorption experiment was performed on the adsorbent 5 in the same manner as in Example 6. Table 2 shows the results. In the adsorbent in which the peptide used in Examples 1 and 2 was not immobilized, sufficient AGE adsorption performance was not seen.
[0046]
Table 2 Results of AGE adsorption rate of non-fixed peptide carrier
Adsorbent 5 7.6%
[0047]
Comparative Example 2: Preparation of AGE antibody-immobilized carrier and evaluation of resistance to high-pressure steam sterilization
The AGE antibody obtained in Example 4 was dissolved in a 0.1 M HEPES-NaOH buffer (pH 7.5) to prepare an antibody solution. Next, the above antibody solution (10 mg of AGE antibody was added to a 0.1 M HEPES-NaOH buffer solution (pH 7)) in 1 mL of agarose gel: Affigel 10 (Bio-Rad) in which an N-hydroxysuccinimide ester group was introduced via a spacer having a length of 10 atoms. .5) solution added to 1 mL) and slowly stirred at 4 ° C overnight. 0.1 mL of 1 M ethanolamine-hydrochloric acid (pH 8.0) was added, and the mixture was reacted at room temperature for 1.5 hours to inactivate unreacted N-hydroxysuccinimide ester groups. Washing alternately 3 times with 1 mL of 1 M acetic acid-NaOH (pH 4.0) and 1 mL of 0.1 M carbonate-NaOH (pH 9.0), and finally equilibrating with a phosphate buffer to immobilize the AGE antibody A carrier (adsorbent 6) was obtained. Next, the adsorbent 6 was subjected to high-pressure steam sterilization in the same manner as in Example 5, and the AGE adsorption performance before and after the high-pressure steam sterilization was evaluated. The AGE adsorption experiment was performed in the same manner as in Example 6. Table 3 shows the results. It was found that the adsorbent 6 significantly lost the AGE adsorption performance after the high-pressure steam sterilization treatment.
[0048]
Table 3 Results of AGE adsorption rate of AGE antibody-immobilized carrier before and after autoclaving

[0049]
Example 7: Perfusion AGE adsorption experiment
1 mL of the adsorbent 1 obtained in Example 1 was collected in a gel volume, and a polypropylene mini-column container (column volume 1 mL, each column had one inlet / outlet, and the adsorbent in the column did not flow from the mini-column. The filter mesh was held between the headers at the entrance and exit, and the entrance and exit were mini-capped so that the filling liquid did not leak out.) As a filling solution, a phosphate buffer was used. Next, in order to subject the mini-column to high-pressure steam sterilization, the adsorbent 1 was filled to prepare a mini-column. Further, 100 mL of a phosphate buffer was added to a 200-mL beaker, and the mini-column was placed therein. Furthermore, an aluminum foil was provided on the upper part of the beaker so that the inside of the beaker could be kept sterile even after high-pressure steam sterilization. Thereafter, high-pressure steam sterilization was performed in the same manner as in Example 5 to produce a sterilized mini-column. Next, a reflux-type AGE adsorption experiment was performed for the purpose of confirming whether or not the adsorption performance of the mini-column after sterilization was maintained even under reflux. Each column was treated with 10 mL of a sample (0.5% BSA-phosphate buffer containing 10 μg / mL of the AGE sample 1 obtained in Example 3). Reflux adsorption was performed for 8 hours in an incubator at 37 ° C. The reflux was performed using a peristaltic pump, and the flow rate was fixed at 0.2 mL / min. Samples were taken as appropriate in 100 uL increments, and the amount of AGE in the reflux solution was measured by the immunoassay described in Example 6.
[0050]
Table 4 Results of reflux AGE adsorption experiment

[0051]
Therefore, it was found that this column shows a significant adsorption performance in the reflux system even after the sterilization treatment.
[0052]
【The invention's effect】
The remover filled with a sterilizable diabetic complication factor adsorbent found in the present invention and the method of using the same can be used for mass production at an industrial level and for products using the adsorbent by a known sterilization method. It has become possible to design an adsorbent that does not significantly impair the performance as an adsorbent. In addition, since the sterilizer can be sterilized by an inexpensive technique, the adsorbent and the product using the adsorbent can be stored under aseptic conditions, thereby realizing long-term stable management. Furthermore, in the case of using a remover typified by an extracorporeal circulation medical device filled with the adsorbent for a diabetic dialysis patient or the like, in order to consider safety (prevention of endotoxin contamination) into the patient. Although operation under aseptic conditions is required, the present invention can provide an adsorbent that can be used as a medical device.
[0053]
As described above, the means for removing diabetic complication factors that can be sterilized found by the present invention are diabetic complication patients and vascular disorders patients represented by diabetic nephropathy, for which a sufficient therapeutic effect could not be expected, and In addition to the technology that can be provided to intractable patients whose oxidative stress increases, such as dialysis patients, intractable diseases involving RAGE, for example, amyloidosis such as sepsis and Alzheimer's disease, and high lipid plasma , Nephropathy, cancer, and hyperinflammation patients can also be provided with effective treatment means.

Claims

An adsorbent for a diabetic complication factor, wherein a compound composed of a peptide derivative is bound to a water-insoluble carrier without substantially inactivating the binding performance for a diabetic complication factor by sterilization.

The diabetic complication factor adsorbent according to claim 1, wherein the peptide derivative has at least two or more basic functional groups.

The diabetic complication factor adsorbent according to any one of claims 1 to 2, wherein the peptide derivative forms a cyclic structure by a peptide bond.

The diabetic complication factor adsorbent according to any one of claims 1 to 3, wherein the peptide derivative is a polymyxin compound.

The diabetic complication factor adsorbent according to any one of claims 1 to 2, wherein the peptide derivative is composed of a RAGE partial sequence derivative.

The diabetic complication factor adsorbent according to claim 5, wherein the RAGE partial sequence derivative is composed of at least an RAGE IgV domain derivative.

The diabetic complication factor adsorbent according to claim 6, wherein the IgV domain derivative is an IgV domain-derived peptide derivative composed of at least either sequence A or sequence B.
Sequence A: (COOH) -KGAPKKPPQRLEWKLN- (NH2)
Sequence B: (COOH) -WKLNTGRTEAWKVLSPQG- (NH2)

The diabetic complication factor adsorbent according to any one of claims 1 to 7, wherein the diabetic complication factor is a RAGE binding substance.

The diabetic complication factor adsorbent according to claim 8, wherein the RAGE binding substance is AGE.

The diabetic complication factor adsorbent according to any one of claims 1 to 9, wherein the adsorbent is used for treating body fluids.

A diabetic complication factor remover filled with the diabetic complication factor adsorbent according to any one of claims 1 to 10.

A diabetic complication factor contained in a liquid to be treated is selectively used as a substance to be adsorbed by bringing a liquid to be treated derived from a body fluid into contact with the sterilizable diabetic complication factor adsorbent according to any one of claims 1 to 10. A method for removing a diabetic complication factor, comprising the steps of: