JP3569904B2 - Organ damage drug - Google Patents
Organ damage drug Download PDFInfo
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- JP3569904B2 JP3569904B2 JP11926692A JP11926692A JP3569904B2 JP 3569904 B2 JP3569904 B2 JP 3569904B2 JP 11926692 A JP11926692 A JP 11926692A JP 11926692 A JP11926692 A JP 11926692A JP 3569904 B2 JP3569904 B2 JP 3569904B2
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- Prior art keywords
- ischemia
- lentinan
- administered
- organ
- organ damage
- Prior art date
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- Expired - Lifetime
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
Description
【０００１】
【産業上の利用分野】
本発明は、ＳＯＤ誘起物質又はヒト成人Ｔ細胞白血病由来因子（以下ヒトＡＤＦと称する）活性を有するポリペプチド、或は両者を有効成分とする臓器障害治療薬又は肝臓障害治療薬に関する。
【０００２】
【従来技術】
スーパーオキシド、過酸化水素、ヒドロキシルラジカル等のフリーラジカルや活性酸素（フリーラジカルと総称する）は殺菌作用を有し生体防御物質として働くが、過剰に産生されたフリーラジカルは生体に対して障害を引き起こすようになる。例えば、生体が虚血状態に陥ると組織の壊死が進行するが、ここに再び血流が再開（再潅流）し組織が再酸素化を受けると、むしろ重大な組織障害がもたらされる（Ｈｅａｒｓｅ， Ｄ． Ｊ．：Ｊ． Ｍｏｌ． Ｃｅｌｌ． Ｃａｒｄｉｏｌ．， ９，６０５−６１６ （１９７７）） 。このような虚血再潅流障害において、フリーラジカルが関与する事が近年明らかになってきた。即ち、虚血後の再潅流により生体局所にフリーラジカルが過剰に発生することが実験的に確かめられ、これらのフリーラジカルが蛋白質の変性、核酸の切断、脂肪の過酸化を引き起こし、生体障害を引き起こすと考えられるに至った。
一方、生体内にはグルタチオンペルオキシダーゼ、スーパーオキシドディスムターゼ（ＳＯＤと称される）、カタラーゼ等のフリーラジカル消去酵素、プロテインジスルフィドイソメラーゼ等の修復酵素あるいはα−トコフェロール、アスコルビン酸、ＣｏＱ １０等の抗酸化物質が存在し生体を保護しているが、大量のフリーラジカルが発生した場合には、消去、修復が追いつかなくなり、その結果、組織に障害が引き起こされるのである（井上正康（監訳）：活性酸素と疾患，学会出版，東京（１９８７））。この虚血再潅流障害は、近年臨床の間でも問題になっており、最も重要なのは肝、腎、膵等の外科的治療あるいは臓器移植である。臓器の外科的治療においては一旦臓器の血流を停止させる事により臓器に虚血状態がもたらされる。術後、血流を再開（再潅流）した時、組織内に大量にフリーラジカルが発生し臓器障害が引き起こされるのである。
【０００３】
また臓器移植においても、拒絶反応の克服と共に、虚血再潅流性臓器障害の克服が移植の成功を左右する大きな因子であることが近年認識されるようになった。即ち、提供者から臓器を取りだす時、臓器内の血流が停止（虚血）する。そして、患者の体内に摘出臓器を移植したとき血流が再開（再潅流）するが、この時に虚血再潅流に伴うフリーラジカルが発生し、その結果、臓器障害が引き起こされるのである。現在、フリーラジカルに起因する臓器障害を防止する目的で種々の薬剤の開発が進められている。中でも、ＳＯＤはラジカルスカベンジャー活性を有することから虚血再潅流性臓器障害等のフリーラジカルに起因する臓器障害の治療薬として有望視されているが、生体内半減期が短いこと、多量に投与するとかえって効果が減少すること等から臨床応用する上で解決すべき課題が残されている。従って、現在、各種臓器の外科的治療及び臓器移植時の虚血再潅流性障害等において過剰に発生するフリーラジカルから臓器を保護し、治療成績を向上させ得る薬剤は市場に存在しない。
【０００４】
【発明が解決しようとする課題】
本発明の目的は、各種臓器の外科的治療あるいは臓器移植時における虚血再潅流などにおいて過剰に発生するフリーラジカルから臓器を保護し、治療成績を大幅に向上させ得る薬剤を提供することである。
【課題を解決するための手段】
本発明者は上記課題を解決するために鋭意検討を行った結果、免疫賦活剤又はヒトＡＤＦ活性を有するポリペプチドを有効成分とする薬剤に優れた臓器障害治療作用を見い出し、さらにＳＯＤ誘起物質とヒトＡＤＦ活性を有するポリペプチドの両者を用いるとその作用が飛躍的に向上することを見いだし本発明を完成するに至った。即ち、本発明はＳＯＤ誘起物質及びヒトＡＤＦ活性を有するポリペプチドを有効成分として含有する臓器障害の治療薬である。
【０００５】
以下、本発明を詳細に説明する。
本発明で用いられるＳＯＤ誘起物質には、レンチナン、シゾフィラン等のような抗腫瘍剤として使用されている薬剤を始めとする多糖類、ＯＫ−４３２（ピシバニールとも呼ばれる）及びＰＳＫ（クレスチンとも呼ばれる）並びにこれらと同様の作用を有する化合物が包含される。中でも、中性多糖に属し直接細胞毒性を持たず、かつ生体防御機構の賦活化能に優れているレンチナンを好ましいものとして挙げる事が出来る。特に、上記で具体的に列挙した免疫賦活剤はそれ自体公知の化合物であり、例えばレンチナンについてはＢｉｏｔｈｅｒａｐｙ，Ｖｏｌ．４ ，Ｎｏ．６，１１１４〜１１２６ページ（１９７９）に詳細な記載がある。
また、ヒトＡＤＦはＡＴＬ−２細胞の上清に存在するＩＬ−２レセプター誘導因子として１９８５年多賀谷らによって報告され、そのアミノ酸配列及びＤＮＡ配列も既に決定され、更に大腸菌での生産についても報告されている（特開平１−８５０９７号公報）。また、ヒトＡＤＦはその後の解析により、大腸菌からほ乳動物にいたるまで広く存在する酸化還元酵素であるチオレドキシンと類似のアミノ酸配列を持つことが明らかになった。
従って、研究者によっては本物質をチオレドキシンと呼ぶ場合もあるが、本発明に於いては、従来どおりヒトＡＤＦという名称で統一することにする。
尚、ヒトＡＤＦは現在、抗炎症剤、放射線防護剤として利用が検討されている（特開平３−２０４８１８号公報）。
【０００６】
本発明で用いられるヒトＡＤＦ活性を有するポリペプチド（以下、ヒトＡＤＦＰと略する）としては通常、配列表の配列番号１又は２記載のアミノ酸配列を有するものが用いられるが、これに限定される訳ではない。即ち、ヒトＡＤＦ活性がある限り、Ｎ末端にメチオニン残基が付加されたポリペプチド、化学修飾、塩基置換法によりアミノ酸配列に置換が加わったポリペプチド、アミノ酸配列の一部に欠損があるポリペプチド、アミノ酸残基の挿入があるポリペプチド、あるいは側鎖に糖鎖等が付加されたポリペプチドであってもよい。
しかし、好ましくは配列表の配列番号１に示されているＮ末端バリンから始まる１０４個のアミノ酸からなるポリペプチド及び配列番号２に示されているメチオニンがＮ末端に付加された構造を持つポリペプチドが好ましい。
本発明で使用されるヒトＡＤＦＰは如何なる方法で製造しても良いが、通常は以下に示す方法で製造する。
即ち、（１）ヒト由来細胞株（例えばＡＴＬ−２細胞等）を培養し、その培養液または細胞抽出液から、塩析、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、クロマトフォーカシング、逆相クロマトグラフィー、疏水性クロマトグラフィーなどの一般に用いられる手法により精製し、目的とするヒトＡＤＦＰを得る方法（特開平１−８５０９７号公報、特開昭６２−１９５３２号公報参照）、（２）遺伝子組換え法により、ヒトＡＤＦのｃＤＮＡまたはゲノム遺伝子を大腸菌、枯草菌、酵母、高等動物細胞、植物細胞などの宿主細胞に導入し、宿主細胞内で組換えヒトＡＤＦＰを発現させ、その後（１）に記したような手法を用いて精製する方法（特開平１−８５０９７号公報参照）、更には（３）ペプチド化学合成法により、配列表の配列番号１又は２記載のアミノ酸配列を有するポリペプチドを合成する方法である。
【０００７】
本発明のＳＯＤ誘起物質を生体に投与することにより、生体内において臓器を構成する細胞内にラジカルスカベンジャーである内因性のＳＯＤが誘導される。従って、各種臓器の外科的治療時或は臓器移植時に本発明のＳＯＤ誘起物質を投与し目的臓器のＳＯＤ活性を高めておけば、上記治療において避けられない虚血再潅流に伴うフリーラジカルの発生から臓器を保護することが出来る。
また、本発明のヒトＡＤＦＰは、過酸化水素などのフリーラジカルを消去する事が出来、更にはフリーラジカルにより変性失活した蛋白をリフォールディングにより再活性化する事もできる。従って、各種臓器の外科的治療時あるいは臓器移植時に本発明のヒトＡＤＦＰを投与すればフリーラジカルによる臓器障害を防ぐ事が出来る。更にヒトＡＤＦＰはヒト由来の蛋白質であるので、人体に投与しても異物として認識されず毒性は非常に低い。
更に、本発明のＳＯＤ誘起物質とヒトＡＤＦＰとを併用すれば、標的細胞のフリーラジカルに対する抵抗性賦与と細胞外のフリーラジカル濃度減少の２つの点で、より優れた臓器障害防止効果が発揮出来る。
尚、本発明のＳＯＤ誘起物質及びヒトＡＤＦＰの安全性は動物実験により確認済みである。
このようなＳＯＤ誘起物質とヒトＡＤＦＰを有効成分として含有する薬剤とは、それぞれの有効成分を個別に又は同時に含む薬剤であって、本発明の目的とする効能を奏するものであればその剤形ならびに使用する賦形剤、割合および投与経路を問わない。従って、有効成分を個別に含む薬剤にあっては、それぞれ異なる経路及び／又は異なる時間に同一の患者に対して投与された場合であっても所期の効能を示す限り本発明の薬剤の概念に包含される。
【０００８】
本発明におけるＳＯＤ誘起物質およびヒトＡＤＦＰの剤形は、医薬として用いられているものについては現実に使用されている剤形、例えばレンチナンについては凍結乾燥品から成る静注用剤形であることが好ましい。しかしながらその他の一般的な薬剤に適用される非注射用剤形であっても良い。両有効成分はそれらが相互に他の薬剤の効能に悪影響を及ぼさない限りの単一製剤として調製することが出来るが、上述のごとくそれぞれ個別の製剤として用いることも出来る。
これらの製剤は、単一製剤であるか個別の製剤であるかを問わず、一般的に、非経口投与剤、特に注射液、好ましくは等張性水溶液または懸濁液が適するものである。これらは、担体、例えばマンニトールあるいはサイクロデキストリンを含む凍結乾燥製剤から使用前に調製することが出来る。このような製剤は、殺菌されそして／又は補助剤、例えば防腐剤、安定剤、湿潤剤及び／又は乳化剤、可溶化剤、浸透圧を調製する塩及び／又は緩衝剤を含むことができる。
【０００９】
本発明の製剤は、場合によりさらに別の効能を有する薬剤を含有することができ、それ自体既知の方法、例えば混合、溶解又は凍結乾燥によって製造される。そして有効成分であるＳＯＤ誘起物質及びヒトＡＤＦＰをそれぞれ約０．１〜９９．９％、好ましくは、約１．０〜９９．０％、また凍結乾燥の場合には１００％まで含む事が出来る。もちろん、上記範囲に限定されるわけではない。
本発明の薬剤の投与は、患者の病態に応じてその最適の投与方法及び単位投与量を専門医が選ぶことが出来るが、一般に、ＳＯＤ誘起物質及びヒトＡＤＦＰの個別に調製した製剤を用いる場合には、体重約５０〜７０ｋｇの人に対する投与単位として前者を約０．１〜１００ｍｇ、好ましくは約１〜１０ｍｇ、そして後者を約０．０１〜１０００ｍｇ、好ましくは０．１〜１００ｍｇとなるように選ぶことができる。これらの製剤は術前、術中、術後の何れかの時期に同時に、又は相違する時期に１回から数回、同一又は異なる経路で投与すれば良い。又、両者を併用する場合には、その比率は任意であるが、一般に１：１０〜１０：１が適当であり、その投与単位は、合計で０．１〜１００ｍｇが適当である。
【００１０】
以下、本発明を実施例に基づいて説明する。
【実施例】
（実施例１）レンチナンによる内因性ＳＯＤの誘導
ラット（Ｗｉｓｔａｒ系、雄、体重２００〜２４０ｇ、静岡実験動物から購入）の腹腔内にレンチナン（味の素（株）製）を投与し、一定期間経過後、肝臓を経門脈的に４℃のリン酸緩衝液（ｐＨ８．０）にて洗浄し、液体窒素にて凍結、湿重量を測定後、リン酸緩衝液を加え１５秒間ホモジナイズし１５，０００ｒｐｍ 、２０分間遠沈後の上清を検体として、内因性ＳＯＤ活性をＳＯＤ測定キットワコー（和光純薬工業（株）製）を用いたニトロブルーテトラゾリウム還元法にて測定した。
レンチナンの投与は次のように行った。
Ａ群：レンチナン１ｍｇを投与。
Ｂ群：レンチナン１ｍｇを０、２、４、６日目に投与。
Ｃ群：レンチナン２ｍｇを０、７日目に投与。
それぞれ１回投与量を生理食塩水１ｍｌに溶解し、腹腔内に投与した。
結果を表１に示した。
【００１１】
【表１】
表１に示したように、レンチナン投与によるラット肝臓の内因性ＳＯＤの発現誘導が認められた。Ａ群においては、第３日目に対照の１．５倍、又、Ｃ群においては第２日目に上昇する効果を認め、Ｂ群においては第９日以降漸次増加し第１３日目に対照値の５〜６倍と著明な増加が認められた。
【００１３】
（実施例２）レンチナンによる肝虚血再潅流障害の抑制効果
ラット（Ｗｉｓｔａｒ系、雄、体重２００〜２４０ｇ、静岡実験動物から購入）をエーテル麻酔下に開腹、肝右葉を門脈系のバイパスとして残し、６０分間虚血後、再潅流直後バイパスに供した右葉を切除し肝虚血再潅流モデルとした。虚血５分前にヘパリン２０ｕ／０．５ｍｌ生理食塩水を経陰茎静脈的に、虚血中に生理食塩水１ｍｌを腹腔内に、虚血後２ｍｌを皮下投与した。
レンチナン投与群はレンチナン１ｍｇを生理食塩水１ｍｌに溶解し、虚血３日前に腹腔内に投与した。また、対照群は生理食塩水のみを投与した。各群における７日生存率を表２に示した。
【００１４】
【表２】
表２に示したように、７日生存率は、対照値の８．６％がレンチナン投与により４６％にまで改善した。
【００１６】
（実施例３）ヒトＡＤＦＰによる肝虚血再潅流障害の抑制効果
ラット（Ｗｉｓｔａｒ系、雄、体重２００〜２４０ｇ、静岡実験動物から購入）をエーテル麻酔下に開腹、肝右葉を門脈系のバイパスとして残し、６０分間虚血後、再潅流直後バイパスに供した右葉を切除し肝虚血再潅流モデルとした。虚血５分前にヘパリン２０ｕ／０．５ｍｌ生理食塩水を経陰茎静脈的に、虚血中に生理食塩水１ｍｌを腹腔内に、虚血後２ｍｌを皮下投与した。
ヒトＡＤＦＰ投与群は、組換えヒトＡＤＦＰ１００μｇ を生理食塩水０．１ｍｌに溶解し、経陰茎静脈的に虚血５分前と再潅流５分前にそれぞれ１００μｇ ずつ投与した。また、対照群は生理食塩水のみを投与した。各群における７日生存率を表３に示した。尚、組換えヒトＡＤＦＰは特開平１−８５０９７号公報記載の方法に準じて調製した。
【００１７】
【表３】
表３に示したように、７日生存率は、対照値の８．６％が組換えヒトＡＤＦＰ投与により４５％にまで改善した。
【００１９】
（実施例４）レンチナンおよびヒトＡＤＦＰ併用による肝虚血再潅流障害の抑制効果
ラット（Ｗｉｓｔａｒ系、雄、体重２００〜２４０ｇ、静岡実験動物から購入）をエーテル麻酔下に開腹、肝右葉を門脈系のバイパスとして残し、６０分間虚血後、再潅流直後バイパスに供した右葉を切除し肝虚血再潅流モデルとした。虚血５分前にヘパリン２０ｕ／０．５ｍｌ生理食塩水を経陰茎静脈的に、虚血中に生理食塩水１ｍｌを腹腔内に、虚血後２ｍｌを皮下投与した。
レンチナンおよびヒトＡＤＦＰ投与群は、先ずレンチナン１ｍｇを生理食塩水１ｍｌに溶解し、虚血３日前に腹腔内に投与し、さらに、生理食塩水０．１ｍｌに溶解した組換えヒトＡＤＦＰ１００μｇ を、経陰茎静脈的に虚血５分前と再潅流５分前にそれぞれ１００μｇ ずつ投与した。また、対照群は生理食塩水のみを投与した。各群における７日生存率を表４に示した。
【００２０】
【表４】
表４に示したように、７日生存率は、対照値の８．６％がレンチナンと組換えヒトＡＤＦＰの併用により６０％にまで改善した。レンチナン又は組換えヒトＡＤＦＰの単独投与時の４６％（実施例２）又は４５％（実施例３）に比較し、両者を併用することにより、より優れた効果が得られることが明らかになった。
【００２２】
（実施例５）レンチナンおよび組換えヒトＡＤＦＰによる虚血再潅流性肝循環血流障害の抑制効果
ラット（Ｗｉｓｔａｒ系、雄、体重２００〜２４０ｇ、静岡実験動物から購入）をエーテル麻酔下に開腹、肝右葉を門脈系のバイパスとして残し、６０分間虚血後、再潅流直後バイパスに供した右葉を切除し肝虚血再潅流モデルとした。虚血５分前にヘパリン２０ｕ／０．５ｍｌ生理食塩水を経陰茎静脈的に、虚血中に生理食塩水１ｍｌを腹腔内に、虚血後２ｍｌを皮下投与した。
レンチナンおよび組換えヒトＡＤＦＰの投与は次のように行った。
Ａ群：非投与。
Ｂ群：レンチナン１ｍｇを虚血３日前に投与。
Ｃ群：組換えヒトＡＤＦＰを虚血５分前と再潅流５分前にそれぞれ１００μｇずつ投与。
Ｄ群：レンチナン１ｍｇを虚血３日前に投与。さらに組換えヒトＡＤＦＰを虚血５分前と再潅流５分前にそれぞれ１００μｇずつ投与。
レンチナンは１回投与量を生理食塩水１ｍｌに溶解し、腹腔内に投与した。また、組換えヒトＡＤＦＰは１回投与量を生理食塩水０．１ｍｌに溶解し、経陰茎静脈的に投与した。
各群における血流再開６０分後の肝組織血流量を、水素クリアランス式組織血流計（ＰＭＧ−２０３、ユニークメディカル社製）を用いた水素クリアランス法により測定した。結果を表５に示した。
【００２３】
【表５】
（実施例６）レンチナンおよび組換えヒトＡＤＦＰによる、虚血再潅流にともなう脂質過酸化の抑制効果
ラット（Ｗｉｓｔａｒ系、雄、体重２００〜２４０ｇ、静岡実験動物から購入）をエーテル麻酔下に開腹、肝右葉を門脈系のバイパスとして残し、６０分間虚血後、再潅流直後バイパスに供した右葉を切除し肝虚血再潅流モデルとした。虚血５分前にヘパリン２０ｕ／０．５ｍｌ生理食塩水を経陰茎静脈的に、虚血中に生理食塩水１ｍｌを腹腔内に、虚血後２ｍｌを皮下投与した。
レンチナンおよび組換えヒトＡＤＦＰの投与は次のように行った。
Ａ群：非投与。
Ｂ群：レンチナン１ｍｇを虚血３日前に投与。
Ｃ群：組換えヒトＡＤＦＰを虚血５分前と再潅流５分前にそれぞれ１００μｇずつ投与。
Ｄ群：レンチナン１ｍｇを虚血３日前に投与。さらに組換えヒトＡＤＦＰを虚血５分前と再潅流５分前にそれぞれ１００μｇずつ投与。
レンチナンは１回投与量を生理食塩水１ｍｌに溶解し、腹腔内に投与した。また、組換えヒトＡＤＦＰは１回投与量を生理食塩水０．１ｍｌに溶解し、経陰茎静脈的に投与した。
各群における血流再開６０分後の組織マロンジアルデヒド（ＭＤＡ）量は次のように測定した。即ち、肝臓を経門脈的に４℃のリン酸緩衝液（ｐＨ８．０）にて洗浄し、液体窒素にて凍結、湿重量を測定後、リン酸緩衝液を加え１５秒間ホモジナイズし１５，０００ｒｐｍ 、２０分間遠沈後の上清を検体として、螢光八木法により組織ＭＤＡ量を測定した。組織ＭＤＡ量は、組織中に蓄積した過酸化脂質量を意味する。結果を表６に示した。
【００２５】
【表６】
表２、３に示したように、７日生存率はレンチナン又は組換えヒトＡＤＦＰ投与により有意に改善し、更に表４に示したように両者を併用することにより、より優れた効果が認められた。またこの時、表５に示したようにレンチナンまたは組換えヒトＡＤＦＰ、あるいは両者の併用により組織血流量が非投与群に比べ高値に保たれた。これは、レンチナンと組換えヒトＡＤＦＰの投与により、虚血再潅流に伴う肝臓循環血流障害が改善された事を意味する。更に、表６に示したようにレンチナンまたは組換えヒトＡＤＦＰ、あるいは両者の併用により組織ＭＤＡが非投与群に比べ低値に抑えられた。これは、虚血再潅流により発生したフリーラジカルによる脂質の過酸化がレンチナンと組換えヒトＡＤＦＰにより抑制されたことを意味する。
以上示したように、レンチナン又は組換えヒトＡＤＦＰの単独投与、或は両者の併用により、虚血再潅流により悪化した肝臓循環動態の改善あるいは脂質過酸化を抑制することが出来、さらに虚血再潅流による致死的な組織障害から生体を保護できる事が明らかになった。
【００２７】
（実施例７）レンチナンによる肝癌の肝内転移生着の抑制効果
ドンリュウラット（６週齢、雄、体重約２００ｇ）をエーテル麻酔下に開腹、門脈内に１，０００，０００個のラット可移植性肝癌ＡＨ１３０細胞を注入し（０日目）、２１日後に肝転移巣形成個数をカウントした。
レンチナン、ＳＯＤ、カタラーゼ等は次のように投与した。
Ａ群：レンチナン１ｍｇを０日目に経静脈的に投与、更に１、３、５及び７日目にレンチナン０．５ｍｇを腹腔内に投与。
Ｂ群：リポソームＳＯＤ２４，０００単位を０日目に経静脈的に投与。
Ｃ群：カタラーゼ５，０００単位を０日目に経静脈的に投与。
Ｄ群：リポソームＳＯＤ２４，０００単位およびカタラーゼ５，０００単位を０日目に経静脈的に投与。
Ｅ群（対照群）：無投与。
各群における２１日後の肝転移巣形成個数を表７に示した。尚、リポソームＳＯＤはＳｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．製を既知の方法（“Ｌｉｐｏｓｏｍｅｓ： Ａ ｐｒａｃｔｉｃａｌ ａｐｐｒｏａｃｈ” ＩＲＬ Ｐｒｅｓｓ， １９９０参照）によりリポソーム化したものである。また、カタラーゼはＳｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．製を用いた。
【００２８】
【表７】
表７に示したように、レンチナン投与群（Ａ群）では肝転移巣形成個数は対照群（Ｅ群）の３分の１と著明な減少が認められた。一方、ＳＯＤまたはカタラーゼ単独投与群（Ｂ、Ｃ群）では、逆に、対照群の５または２．５倍に増加し、更にＳＯＤとカタラーゼの併用群（Ｄ群）では対照群の９．５倍にまで増加が認められた。
本実施例の結果は以下の事を意味する。ＳＯＤ、カタラーゼ、又は両者の併用により肝臓への肝癌の転移生着が増加した。これは、本来マクロファージ等がフリーラジカルを産生することにより、肝実質細胞への腫瘍細胞の浸潤転移生着が抑制されているのであるが、ＳＯＤ、カタラーゼの様な外来性ラジカルスカベンジャーを投与することによりこのような生体防御機構が減弱させられたことが原因と考えられる。一方、レンチナン投与により、逆に肝癌の転移生着が抑制された。実施例１に示したように、レンチナン投与により内因性のＳＯＤが誘導されることが明らかになっているが、レンチナンにより肝実質細胞に内因性防御物質が誘導されたことにより、腫瘍細胞の浸潤転移生着という肝実質細胞の障害が防御された事を意味する。
【００３０】
【発明の効果】
本発明のＳＯＤ誘起物質およびヒトＡＤＦＰは、フリーラジカルに起因する臓器障害に対する優れた防護効果を有することが明らかになった。従って、各種臓器の外科的治療及び臓器移植時の虚血再潅流性組織障害等、フリーラジカルに起因する多岐にわたる臓器障害を保護する薬剤として、臨床応用が期待できる。
【００３１】
【配列表】
[Industrial applications]
The present invention relates to a therapeutic agent for organ damage or liver damage containing a SOD-inducing substance or a polypeptide having human adult T-cell leukemia-derived factor (hereinafter referred to as human ADF) activity, or both as active ingredients.
[0002]
[Prior art]
Free radicals such as superoxide, hydrogen peroxide, and hydroxyl radicals and active oxygen (collectively called free radicals) have a bactericidal action and act as a biological defense substance, but excessive free radicals cause damage to living organisms. To cause. For example, tissue necrosis progresses when the body falls into an ischemic state, and when blood flow resumes again (reperfusion) and the tissue undergoes reoxygenation, rather serious tissue damage is caused (Hease, D. J .: J. Mol. Cell. Cardiol., 9, 605-616 (1977)). In recent years, it has become clear that free radicals are involved in such ischemia-reperfusion injury. That is, it has been experimentally confirmed that reperfusion after ischemia generates excessive free radicals locally in the living body, and these free radicals cause denaturation of proteins, cleavage of nucleic acids, and peroxidation of fat, thereby causing biological damage. Came to be thought to cause.
On the other hand, in vivo, free radical scavenging enzymes such as glutathione peroxidase, superoxide dismutase (referred to as SOD) and catalase, repair enzymes such as protein disulfide isomerase, and antioxidants such as α-tocopherol, ascorbic acid, and CoQ10 Exists and protects the living body, but if a large amount of free radicals are generated, erasure and repair cannot keep up, resulting in damage to the tissue (translated by Masayasu Inoue: active oxygen and Disease, Academic Press, Tokyo (1987)). This ischemia-reperfusion injury has become a problem even in clinical practice in recent years, and the most important is surgical treatment of liver, kidney, pancreas and the like or organ transplantation. In surgical treatment of an organ, once the blood flow of the organ is stopped, an ischemic state is caused in the organ. When the blood flow is resumed (reperfusion) after the operation, a large amount of free radicals is generated in the tissue, causing organ damage.
[0003]
Also, in organ transplantation, it has recently been recognized that overcoming ischemia-reperfusion organ damage together with overcoming rejection is a major factor in determining the success of transplantation. That is, when removing the organ from the donor, the blood flow in the organ stops (ischemia). Then, when the removed organ is transplanted into the patient's body, blood flow resumes (reperfusion). At this time, free radicals are generated due to ischemia / reperfusion, and as a result, organ damage is caused. At present, various drugs are being developed for the purpose of preventing organ damage caused by free radicals. Above all, SOD is promising as a therapeutic agent for organ damage caused by free radicals such as ischemia-reperfusion organ damage because of its radical scavenger activity. On the contrary, there is a problem to be solved in clinical application because the effect is reduced. Therefore, at present, there is no drug on the market that can protect the organ from free radicals that are excessively generated in surgical treatment of various organs and ischemia-reperfusion injury at the time of organ transplantation and improve the treatment results.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a drug capable of protecting an organ from free radicals that are excessively generated during surgical treatment of various organs or ischemia / reperfusion at the time of organ transplantation and greatly improving the treatment results. .
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found an excellent organ-damaging therapeutic effect on a drug containing an immunostimulant or a polypeptide having human ADF activity as an active ingredient. It has been found that the use of both polypeptides having human ADF activity dramatically improves the action, and the present invention has been completed. That is, the present invention is a remedy for organ damage, which comprises a SOD-inducing substance and a polypeptide having human ADF activity as active ingredients.
[0005]
Hereinafter, the present invention will be described in detail.
The SOD inducing substances used in the present invention include polysaccharides including drugs used as antitumor agents such as lentinan, schizophyllan, etc., OK-432 (also called picibanil) and PSK (also called krestin), and Compounds having the same action as these are included. Among them, preferred is lentinan, which belongs to neutral polysaccharide, has no direct cytotoxicity, and is excellent in the ability to activate the biological defense mechanism. In particular, the immunostimulants specifically listed above are compounds known per se. For example, lentinan is described in Biotherapy, Vol. 4, no. 6, pages 1114 to 1126 (1979).
Human ADF was reported by Tagaya et al. In 1985 as an IL-2 receptor inducer present in the supernatant of ATL-2 cells, its amino acid sequence and DNA sequence have already been determined, and its production in E. coli has also been reported. (JP-A-1-85097). Further, subsequent analysis revealed that human ADF has an amino acid sequence similar to that of thioredoxin, which is a oxidoreductase widely present from Escherichia coli to mammals.
Therefore, depending on the researcher, this substance may be called thioredoxin, but in the present invention, it is unified with the name of human ADF as before.
The use of human ADF is currently being studied as an anti-inflammatory agent and a radioprotective agent (JP-A-3-204818).
[0006]
As the polypeptide having human ADF activity (hereinafter abbreviated as human ADFP) used in the present invention, a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 2 in the Sequence Listing is generally used, but is not limited thereto. Not in translation. That is, a polypeptide having a methionine residue added at the N-terminus, a polypeptide having an amino acid sequence substituted by a chemical modification or base substitution method, or a polypeptide having a partial amino acid sequence deficiency as long as it has human ADF activity Or a polypeptide having an amino acid residue inserted therein, or a polypeptide having a sugar chain or the like added to a side chain.
However, preferably, a polypeptide consisting of 104 amino acids starting from the N-terminal valine shown in SEQ ID NO: 1 of the sequence listing and a polypeptide having a structure in which methionine shown in SEQ ID NO: 2 is added to the N-terminus Is preferred.
The human ADFP used in the present invention may be produced by any method, but is usually produced by the following method.
That is, (1) a human-derived cell line (for example, ATL-2 cells) is cultured, and salting out, gel filtration chromatography, ion exchange chromatography, affinity chromatography, chromatofocusing, A method of purifying by a generally used technique such as reverse phase chromatography and hydrophobic chromatography to obtain a desired human ADFP (see JP-A-1-85097 and JP-A-62-19532), (2) By the gene recombination method, the cDNA or genomic gene of human ADF is introduced into host cells such as Escherichia coli, Bacillus subtilis, yeast, higher animal cells, and plant cells, and recombinant human ADFP is expressed in the host cells. )) (See JP-A-1-85097), In is a method of synthesizing a polypeptide having (3) by chemical peptide synthesis, the amino acid sequence of SEQ ID NO: 1 or 2 in Sequence Listing.
[0007]
By administering the SOD inducing substance of the present invention to a living body, endogenous SOD which is a radical scavenger is induced in cells constituting an organ in the living body. Therefore, if the SOD-inducing substance of the present invention is administered at the time of surgical treatment of various organs or at the time of organ transplantation to increase the SOD activity of the target organ, the generation of free radicals associated with ischemia / reperfusion which cannot be avoided in the above-mentioned treatments Can protect the organs.
In addition, the human ADFP of the present invention can eliminate free radicals such as hydrogen peroxide, and can reactivate a protein denatured and inactivated by free radicals by refolding. Therefore, administration of the human ADFP of the present invention at the time of surgical treatment of various organs or at the time of organ transplantation can prevent organ damage due to free radicals. Furthermore, since human ADFP is a protein derived from humans, it is not recognized as a foreign substance even when administered to the human body and has very low toxicity.
Furthermore, when the SOD-inducing substance of the present invention and human ADFP are used in combination, a more excellent organ damage-preventing effect can be exhibited in two points, that is, the provision of resistance to target cells to free radicals and the reduction of extracellular free radical concentration. .
The safety of the SOD-inducing substance of the present invention and human ADFP has been confirmed by animal experiments.
Such a drug containing an SOD-inducing substance and human ADFP as active ingredients is a drug containing each of the active ingredients individually or at the same time as long as it exhibits the intended effect of the present invention. Also, it does not matter which excipients are used, the proportions and the administration route. Therefore, in the case of a drug containing an active ingredient individually, the concept of the drug of the present invention is not limited as long as it shows the intended efficacy even when administered to the same patient by different routes and / or at different times. Is included.
[0008]
The dosage form of the SOD-inducing substance and human ADFP in the present invention may be a dosage form actually used for pharmaceuticals, for example, an intravenous dosage form of a lyophilized product for lentinan. preferable. However, it may be a non-injectable dosage form applicable to other general drugs. The two active ingredients can be prepared as a single preparation as long as they do not adversely affect the efficacy of the other drug, or they can be used as individual preparations as described above.
These formulations, whether in a single or individual formulation, are generally suitable for parenteral administration, especially for injections, preferably isotonic aqueous solutions or suspensions. These can be prepared before use from lyophilized preparations containing a carrier, for example mannitol or cyclodextrin. Such preparations may be sterilized and / or contain adjuvants, such as preserving, stabilizing, wetting and / or emulsifying agents, solubilizing agents, salts for regulating osmotic pressure and / or buffers.
[0009]
The preparations according to the invention can optionally contain further active agents and are prepared by methods known per se, for example by mixing, dissolving or lyophilizing. The active ingredients, SOD-inducing substance and human ADFP, can be contained respectively in about 0.1 to 99.9%, preferably in about 1.0 to 99.0%, and in the case of freeze-drying, up to 100%. . Of course, it is not limited to the above range.
The administration of the agent of the present invention can be selected by a specialist according to the disease state of a patient according to the optimal administration method and unit dosage. Generally, when a separately prepared preparation of a SOD inducer and human ADFP is used. The dosage unit for a person weighing about 50 to 70 kg is such that the former is about 0.1 to 100 mg, preferably about 1 to 10 mg, and the latter is about 0.01 to 1000 mg, preferably 0.1 to 100 mg. You can choose. These preparations may be administered by the same or different route at the same time before, during or after the operation, or once to several times at different times. When both are used in combination, the ratio is arbitrary, but generally 1:10 to 10: 1 is appropriate, and the total dosage unit is 0.1 to 100 mg.
[0010]
Hereinafter, the present invention will be described based on examples.
【Example】
(Example 1) Induction of endogenous SOD by lentinan Lentinan (manufactured by Ajinomoto Co., Inc.) was administered intraperitoneally to rats (Wistar strain, male, weight: 200 to 240 g, purchased from Shizuoka experimental animal), and after a certain period of time. Then, the liver was transportally washed with a phosphate buffer (pH 8.0) at 4 ° C., frozen with liquid nitrogen, and the wet weight was measured. Then, the phosphate buffer was added and homogenized for 15 seconds to 15,000 rpm. The supernatant after centrifugation for 20 minutes was used as a sample, and endogenous SOD activity was measured by a nitro blue tetrazolium reduction method using a SOD measurement kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
Lentinan was administered as follows.
Group A: Lentinan 1 mg was administered.
Group B: Lentinan 1 mg was administered on days 0, 2, 4, and 6.
Group C: Lentinan 2 mg was administered on days 0 and 7.
Each single dose was dissolved in 1 ml of physiological saline and administered intraperitoneally.
The results are shown in Table 1.
[0011]
[Table 1]
As shown in Table 1, induction of endogenous SOD expression in rat liver by lentinan administration was observed. In group A, the effect was 1.5 times higher than that of the control on day 3, and in group C, the effect was increased on day 2. In group B, the effect was gradually increased after day 9 and on day 13 A remarkable increase of 5 to 6 times the control value was observed.
[0013]
(Example 2) Inhibitory effect of lentinan on hepatic ischemia-reperfusion injury Rats (Wistar system, male, weight: 200-240 g, purchased from Shizuoka experimental animal) were laparotomized under ether anesthesia, and the right hepatic lobe was bypassed by the portal vein system. After ischemia for 60 minutes, the right lobe subjected to bypass immediately after reperfusion was excised and used as a hepatic ischemia-reperfusion model. Five minutes before ischemia, heparin (20 u / 0.5 ml) was intravenously administered via the penis, 1 ml of saline was intraperitoneally administered during ischemia, and 2 ml after ischemia was subcutaneously administered.
In the lentinan administration group, 1 mg of lentinan was dissolved in 1 ml of physiological saline and administered intraperitoneally 3 days before ischemia. The control group received only physiological saline. Table 7 shows the 7-day survival rates in each group.
[0014]
[Table 2]
As shown in Table 2, the 7-day survival rate was improved from 8.6% of the control value to 46% by lentinan administration.
[0016]
(Example 3) Inhibitory effect of human ADFP on hepatic ischemia-reperfusion injury Rats (Wistar system, male, body weight 200-240 g, purchased from Shizuoka experimental animal) were laparotomized under ether anesthesia, and the right hepatic lobe was subjected to portal vein system. The right lobe left as a bypass and subjected to bypass immediately after reperfusion after ischemia for 60 minutes was resected to obtain a hepatic ischemia-reperfusion model. Five minutes before ischemia, heparin (20 u / 0.5 ml) was intravenously administered via the penis, 1 ml of saline was intraperitoneally administered during ischemia, and 2 ml after ischemia was subcutaneously administered.
In the group to which human ADFP was administered, 100 μg of recombinant human ADFP was dissolved in 0.1 ml of physiological saline, and 100 μg of each was administered via a penile vein 5 minutes before ischemia and 5 minutes before reperfusion. The control group received only physiological saline. Table 3 shows the 7-day survival rates in each group. The recombinant human ADFP was prepared according to the method described in JP-A-1-85097.
[0017]
[Table 3]
As shown in Table 3, the 7-day survival rate was improved from the control value of 8.6% to 45% by administration of the recombinant human ADFP.
[0019]
(Example 4) Inhibitory effect of lentinan and human ADFP on hepatic ischemia-reperfusion injury Rats (Wistar strain, male, body weight 200-240 g, purchased from Shizuoka experimental animals) were laparotomized under ether anesthesia, and the right hepatic lobe was opened. The lobe was left as a vascular bypass, and after 60 minutes of ischemia, the right lobe subjected to bypass immediately after reperfusion was excised and used as a hepatic ischemia reperfusion model. Five minutes before ischemia, heparin (20 u / 0.5 ml) was intravenously administered via the penis, 1 ml of saline was intraperitoneally administered during ischemia, and 2 ml after ischemia was subcutaneously administered.
Lentinan and human ADFP administration groups were prepared by first dissolving 1 mg of lentinan in 1 ml of physiological saline, intraperitoneally administering it 3 days before ischemia, and further adding 100 μg of recombinant human ADFP dissolved in 0.1 ml of physiological saline to the penis. 100 μg was administered intravenously 5 minutes before ischemia and 5 minutes before reperfusion. The control group received only physiological saline. Table 7 shows the 7-day survival rates in each group.
[0020]
[Table 4]
As shown in Table 4, the 7-day survival rate was improved from the control value of 8.6% to 60% by the combination of lentinan and recombinant human ADFP. Compared to 46% (Example 2) or 45% (Example 3) when lentinan or recombinant human ADFP was administered alone, it was clarified that a better effect could be obtained by using both together. .
[0022]
(Example 5) Inhibitory effect of lentinan and recombinant human ADFP on ischemia-reperfusion hepatic circulatory blood flow disorder Rats (Wistar system, male, body weight 200-240 g, purchased from Shizuoka experimental animal) were laparotomized under ether anesthesia. The right hepatic lobe was left as a portal vein bypass, and after 60 minutes of ischemia, the right lobe subjected to the bypass immediately after reperfusion was excised and used as a hepatic ischemia-reperfusion model. Five minutes before ischemia, heparin (20 u / 0.5 ml) was intravenously administered via the penis, 1 ml of saline was intraperitoneally administered during ischemia, and 2 ml after ischemia was subcutaneously administered.
Lentinan and recombinant human ADFP were administered as follows.
Group A: not administered.
Group B: Lentinan 1 mg was administered 3 days before ischemia.
Group C: 100 μg each of recombinant human ADFP was administered 5 minutes before ischemia and 5 minutes before reperfusion.
Group D: Lentinan 1 mg was administered 3 days before ischemia. Further, 100 μg of each recombinant human ADFP was administered 5 minutes before ischemia and 5 minutes before reperfusion.
Lentinan was dissolved in 1 ml of physiological saline in a single dose and administered intraperitoneally. In addition, the recombinant human ADFP was dissolved in a single dose of 0.1 ml of physiological saline and administered intravenously via the penis.
The hepatic tissue blood flow in each group was measured by a hydrogen clearance method using a hydrogen clearance type tissue blood flow meter (PMG-203, manufactured by Unique Medical Co., Ltd.) 60 minutes after the resumption of blood flow. Table 5 shows the results.
[0023]
[Table 5]
Example 6 Inhibitory Effect of Lentinan and Recombinant Human ADFP on Lipid Peroxidation Associated with Ischemia Reperfusion Rats (Wistar system, male, body weight 200-240 g, purchased from Shizuoka experimental animal) were laparotomized under ether anesthesia. The right hepatic lobe was left as a portal vein bypass, and after 60 minutes of ischemia, the right lobe subjected to the bypass immediately after reperfusion was excised and used as a hepatic ischemia-reperfusion model. Five minutes before ischemia, heparin (20 u / 0.5 ml) was intravenously administered via the penis, 1 ml of saline was intraperitoneally administered during ischemia, and 2 ml after ischemia was subcutaneously administered.
Lentinan and recombinant human ADFP were administered as follows.
Group A: not administered.
Group B: Lentinan 1 mg was administered 3 days before ischemia.
Group C: 100 μg each of recombinant human ADFP was administered 5 minutes before ischemia and 5 minutes before reperfusion.
Group D: Lentinan 1 mg was administered 3 days before ischemia. Further, 100 μg of each recombinant human ADFP was administered 5 minutes before ischemia and 5 minutes before reperfusion.
Lentinan was dissolved in 1 ml of physiological saline in a single dose and administered intraperitoneally. In addition, the recombinant human ADFP was dissolved in a single dose of 0.1 ml of physiological saline and administered intravenously via the penis.
The amount of tissue malondialdehyde (MDA) in each group 60 minutes after resumption of blood flow was measured as follows. Specifically, the liver was transportally washed with a phosphate buffer (pH 8.0) at 4 ° C., frozen with liquid nitrogen, and the wet weight was measured. Then, the phosphate buffer was added and homogenized for 15 seconds. Using the supernatant after centrifugation at 000 rpm for 20 minutes, the amount of tissue MDA was measured by the fluorescent Yagi method. The tissue MDA amount means the amount of lipid peroxide accumulated in the tissue. The results are shown in Table 6.
[0025]
[Table 6]
As shown in Tables 2 and 3, the 7-day survival rate was significantly improved by administration of lentinan or recombinant human ADFP. Further, as shown in Table 4, the combined use of both provided a more excellent effect. Was. At this time, as shown in Table 5, the tissue blood flow was maintained at a higher value than in the non-administration group by using lentinan or recombinant human ADFP or a combination of both. This means that the administration of lentinan and recombinant human ADFP improved the hepatic circulatory blood flow disorder associated with ischemia-reperfusion. Furthermore, as shown in Table 6, the tissue MDA was suppressed to a lower value than the non-administered group by using lentinan or recombinant human ADFP, or a combination of both. This means that peroxidation of lipids by free radicals generated by ischemia reperfusion was suppressed by lentinan and recombinant human ADFP.
As shown above, the single administration of lentinan or recombinant human ADFP, or the combined use of both, can improve the hepatic hemodynamics or suppress lipid peroxidation worsened by ischemia-reperfusion, and It has been shown that the living body can be protected from lethal tissue damage caused by perfusion.
[0027]
Example 7 Inhibitory Effect of Lentinan on the Engraftment of Liver Metastasis in Liver Cancer Dongryu rats (6 weeks old, male, weighing about 200 g) were laparotomized under ether anesthesia, and 1,000,000 rats were placed in the portal vein. Transplantable liver cancer AH130 cells were injected (day 0), and the number of liver metastases formed was counted 21 days later.
Lentinan, SOD, catalase, etc. were administered as follows.
Group A: 1 mg of lentinan was administered intravenously on day 0, and 0.5 mg of lentinan was administered intraperitoneally on days 1, 3, 5, and 7.
Group B: 24,000 units of liposome SOD were administered intravenously on day 0.
Group C: 5,000 units of catalase administered intravenously on day 0.
Group D: 24,000 units of liposome SOD and 5,000 units of catalase were intravenously administered on day 0.
Group E (control group): No administration.
Table 7 shows the number of liver metastases formed on day 21 in each group. Note that liposome SOD is available from Sigma Chemical Co. Manufactured by a known method (see "Liposomes: A practical approach" IRL Press, 1990). Catalase is available from Sigma Chemical Co. Was used.
[0028]
[Table 7]
As shown in Table 7, the number of liver metastases formed in the lentinan-administered group (Group A) was significantly reduced to one third of that in the control group (Group E). On the other hand, in the group administered SOD or catalase alone (groups B and C), the increase was 5 or 2.5 times that of the control group, and in the combined group of SOD and catalase (group D), 9.5 compared to the control group. The increase was doubled.
The result of this example means the following. SOD, catalase, or a combination of both increased metastatic survival of liver cancer to the liver. This is because macrophages and the like originally produce free radicals, thereby inhibiting the invasion and metastasis of tumor cells to hepatocytes. However, administration of exogenous radical scavengers such as SOD and catalase is necessary. It is considered that such a biological defense mechanism was attenuated. On the other hand, administration of lentinan, on the other hand, suppressed metastatic survival of liver cancer. As shown in Example 1, it has been revealed that the administration of lentinan induces endogenous SOD. However, the induction of endogenous protective substances in hepatic parenchymal cells by lentinan caused tumor cell invasion. It means that hepatocyte damage such as metastasis engraftment was protected.
[0030]
【The invention's effect】
The SOD inducer of the present invention and human ADFP have been found to have excellent protective effects against organ damage caused by free radicals. Therefore, clinical application can be expected as a drug that protects a wide variety of organ disorders caused by free radicals, such as surgical treatment of various organs and ischemia-reperfusion-related tissue disorders at the time of organ transplantation.
[0031]
[Sequence list]
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| Application Number | Priority Date | Filing Date | Title |
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| JP11926692A JP3569904B2 (en) | 1992-05-12 | 1992-05-12 | Organ damage drug |
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| JP11926692A JP3569904B2 (en) | 1992-05-12 | 1992-05-12 | Organ damage drug |
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| JPH05310579A JPH05310579A (en) | 1993-11-22 |
| JP3569904B2 true JP3569904B2 (en) | 2004-09-29 |
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| JP11926692A Expired - Lifetime JP3569904B2 (en) | 1992-05-12 | 1992-05-12 | Organ damage drug |
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| US5985261A (en) * | 1996-06-28 | 1999-11-16 | National Jewish Medical And Research Center | Use of thioredoxin-like molecules for induction of MnSOD to treat oxidative damage |
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