JP4282267B2 - Sulfated fucobiosylchondroitin sulfate derivative - Google Patents

Description

【０００９】
ii）コンドロイチナーゼＡＢＣによる分解にて、不飽和二糖生成率が３５〜５５％である。
iii)構成二糖単位当たり、1.3〜1.8分子の硫酸基、及び、0.2〜0.4分子のフコビオシル基を含有する。
（２）平均分子量１１,０００〜１３,０００である上記（１）に記載の硫酸化フコビオシルコンドロイチン硫酸。
（３）ナマコ類由来の硫酸化フコビオシルコンドロイチン硫酸から、側鎖である硫酸化フコビオシル基を部分的に除去することにより得られる上記（１）又は（２）に記載の硫酸化フコビオシルコンドロイチン硫酸。
（４）分岐化度が２０〜５０％である上記（１）〜（３）のいずれかに記載の硫酸化フコビオシルコンドロイチン硫酸。
（５）上記（１）〜（４）のいずれかに記載の硫酸化フコビオシルコンドロイチン硫酸を含有する組成物。
（６）医薬組成物である上記（５）記載の組成物。
（７）上記（１）〜（４）のいずれかに記載の硫酸化フコビオシルコンドロイチン硫酸を有効成分とする線溶活性亢進剤（以下、本発明医薬ともいう）。
【００１０】
【発明の実施の形態】
以下に本発明を更に詳細に説明する。
本発明物質は、GlcAのＯ-１位とGalNAcのＯ-３位とがβ-グリコシド結合した二糖単位の繰り返し構造を主鎖とし、側鎖として硫酸化フコース２残基から成る硫酸化フコビオシル基を部分的に有するＣＳの一種である。
【００１１】
本発明における硫酸化フコビオシル基とは、フコースのＯ-２位及びＯ-４位のヒドロキシル基（-ＯＨ）の硫酸（-ＯＳＯ_３ ^―）化の違いによりフコース１分子中に０〜２個硫酸基を有するフコース残基２残基がα-１,３結合しているフコビオース基であり、下記式（２）で表される。当該硫酸化フコビオシル基は、本発明物質の主鎖を構成するGalNAcのＯ-４位、Ｏ-６位、及び、GlcAのＯ-３位から選択される位置のヒドロキシル基と硫酸化フコビオシル基Ｏ-１位のヒドロキシル基とでグリコシド結合することにより、本発明物質の側鎖を形成している。
【００１２】
【化３】

【００１３】
【表１】

【００１４】
本発明物質をコンドロイチナーゼＡＢＣによって分解した際の不飽和二糖生成率は３５〜５５％である。不飽和二糖生成率は、後述の実施例に記載のコンドロイチナーゼABCによる分解とイオン交換高速液体クロマトグラフィー（以下、ＨＰＬＣとも言う）による分析を組み合わせた二糖組成分析により算出できる。本発明物質について、この方法で分析すると、下記式（３）で表される二糖構造が検出される。
【００１５】
【式４】

【００１６】
式中、Ｒ^１、Ｒ^２、Ｒ^３は水素原子またはＳＯ_３ ^―を示し、Ａｃはアセチル基を示す。
【００１７】
更に、化学組成分析の結果、主鎖の二糖単位１残基あたり、1.3〜1.8分子の硫酸基、好ましくは、1.5〜1.8分子の硫酸基を含有し、同様に主鎖の二糖単位１残基あたり、0.2〜0.4分子のフコビオシル基、好ましくは、0.2〜0.3分子のフコビオシル基を含有する。この化学組成分析の方法としては、本発明物質中のヘキソサミン含量、硫酸基含量及びフコビオシル含量又はフコース含量を測定可能な方法であれば、特に限定されないが、一例として、後述の実施例に記載の様に、MBTH法によりヘキソサミン含量を測定し、更にイオンクロマトグラフィー法及びアンスロン法（Dimler, R. Let al. (1952) Anal.Chem.,24, 1411-1414）にて、各々硫酸基含量及びフコース含量を測定する方法が挙げられる。
【００１８】
フコビオシル含量からみて、本発明物質は、硫酸化フコビオシル基側鎖を持たない構成二糖単位とGalNAcのＯ-４位、Ｏ-６位及びGlcAのＯ-３位より選択される位置に１つ以上の硫酸化フコビオシル基側鎖を有する構成二糖単位が混在した状態で構成されており、従って、部分的に硫酸化フコビオシル基側鎖を有するＣＳである。
【００１９】
本発明物質は、平均分子量１１,０００〜１３,０００であることが好ましく、平均分子量１１,０００〜１２,０００がより好ましい。尚、多糖類の平均分子量は、重量平均分子量で示すのが一般的であるが、ＧＡＧの平均分子量は、同一試料でも測定方法や測定条件などによって多少異なることは当業者にとって常識であり、本発明物質においても、上記平均分子量の範囲に厳密に限定されるべきものではない。
【００２０】
本発明物質は、その起源、由来、製法によっては特に限定されるものでは無く、本発明物質が有する物理化学的特性を満たすものであれば、天然資源から得られる部分的に硫酸化フコビオシル基側鎖を有する硫酸化フコビオシルＣＳでも、天然資源（主として生物体）から得られる硫酸化フコビオシルＣＳを原料として後述の部分的に硫酸化フコビオシル基を除去する等の化学的手法等により改変したもの、若しくは、人工的に化学合成したものや、遺伝子工学的に動物細胞、植物細胞、微生物等により合成させたものでも構わない。
【００２１】
本発明物質自体が生物体から単離精製される場合や生物体から単離精製される硫酸化フコビオシルＣＳを原料として製造する場合に用いる生物体は、種、属など特に限定されない。生物体として、例えば、棘皮動物が挙げられ、好ましくはナマコ類が挙げられ、特に、マナマコ（Sea Cucumber Stichopus japonicus）、クロナマコ（Holthuria atra）、ニセクロナマコ（Holothuria leurospilota）などが挙げられる。尚、これらナマコ類動物は、ごく一部が食用に供されてはいるが、殆ど利用されていない未利用資源である。
【００２２】
一例として、マナマコを原料とする本発明物質の製造方法を説明する。
先ず、ＧＡＧを生体組織から単離・精製する際に通常用いられる公知の方法（Carbohydr. Res., 297, 273-279 (1997), 特公平６−７００８５等参照）に準じてマナマコ体壁より硫酸化フコビオシルＣＳを単離精製し、次いで、側鎖である硫酸化フコビオシル基を部分的に脱離させ（部分的脱分岐化）、本発明物質を得る。
【００２３】
ここに於いて、側鎖である硫酸化フコビオシル基の部分的脱離方法は、本発明物質が有する物理化学的特性を満たすＣＳが生成する方法であれば、特に限定されないが、分岐化度２０〜５０％である硫酸化フコビオシルＣＳが得られる条件での緩和な酸加水分解、アルカリ加水分解反応や酵素消化反応などが挙げられる。上記のうち、特に0.05〜0.2Ｎ程度の硫酸、塩酸等の無機酸を用いて、７５〜８５℃程度の条件下で約２．５〜３．５時間、酸加水分解する方法が好ましい。尚、通常、反応後中和し、分離・精製工程に供する。かくして、より好ましくは、分岐化度３０〜４０％である反応終生成物（本発明物質）が得られる。上記部分的脱分岐化後、ＧＡＧの分離・精製の常套手段によって精製された本発明物質を得ることが出来る。
【００２４】
ここでいう、分岐化度とは、本発明物質１分子が有している側鎖である硫酸化フコビオシル基のモル量の、主鎖構成二糖単位のモル量に対する割合を示している。尚、上記の側鎖を部分的に脱離する方法によって本発明物質を製造する際の原料となりうるナマコ由来の硫酸化フコビオシルＣＳに関しては、主鎖を構成している二糖単位当たり、ほぼ１本の硫酸化フコビオシル基側鎖を有している（分岐化度＝１００％とする）事が既に報告されている。（Kariya et al. (1997) Carbohydrate Research 297, 273-279 ）
【００２５】
先に述べた様に、マナマコ由来ＣＳは、コンドロイチナーゼ類により極めて消化されにくい事が既に知られているが、マナマコ由来ＣＳから部分的に側鎖を除去する方法等により得られる本発明物質は、後述の実施例に記載の通り、コンドロイチナーゼABCにより３５〜５５％分解される。
【００２６】
また、上述の様にSakai.et.al (2000) Tromb Res.,100,557-565において、本発明物質の製造原料となるマナマコ由来ＣＳは極めて弱い線溶活性亢進作用しか示さないと報告されているが、本発明物質は、後述の実施例に記載の通り、プラスミノーゲン、一本鎖組織プラスミノーゲンアクチベーター及び合成基質を加えた線溶反応系において、マナマコ由来ＣＳに比べ、極めて高い線溶活性亢進作用を示した。
【００２７】
プラスミノーゲンアクチベーターを介した線溶活性亢進作用とは、プラスミノーゲンアクチベーター（以下、ＰＡとも言う）がプラスミノーゲンをプラスミンに限定分解し、活性化されたプラスミンがフィブリンを分解する一連の反応の総称である。
【００２８】
特に血管内線溶においては組織型ＰＡ（以下、ｔ-ＰＡという）が重要な調節因子となっており、ｔ-ＰＡの作用が低下すると血流中に生成した血栓を溶解しにくくなる為、脳梗塞や心筋梗塞など血栓症を発症しやすくなる。本発明物質は、優れた線溶活性亢進作用を有する為、本発明物質を有効成分とする医薬組成物（以下、本発明医薬組成物ともいう）は、ｔ-ＰＡ活性の促進が望まれるこれら上記の様な疾患の予防、維持（悪化防止）、軽減（症状の改善）及び治療を目的としてヒト、その他ほ乳動物に投与することが出来る。
【００２９】
本発明医薬組成物を生体に投与する際の剤型及び投与経路としては、対象となる疾患の性質や重篤度に応じて適宜選択する事ができる。例えば、それらをそのまま、又は、他の薬理学的に許容され得る担体、希釈剤、安定化剤等と共に適宜、注射剤、錠剤、カプセル剤、顆粒剤、散剤、液剤、リポ化剤、軟膏剤、ゲル剤、外用散剤、スプレー剤、吸入散剤、点眼剤、眼軟膏剤、座剤などに製剤化し、注射（筋肉内、皮下、皮内、静脈内、関節腔内、腹腔内等）、点眼、点滴、経皮、経口、吸入等の投与方法によって、経口又は非経口的に安全に投与することが出来る。
【００３０】
本発明医薬組成物における本発明物質の配合量や投与量は、その製剤の投与方法、投与形態、使用目的、患者の具体的症状、患者の体重等に応じて個別に決定されるべき事項であり、特に限定はされないが、経口的あるいは非経口的な投与経路において本発明物質として１日当たり概ね０．１mg/kg〜３００mg/kg程度を例示することが出来る。また、投与間隔は、１日１回程度でも可能であり、１日２〜４回、またはそれ以上の回数に分けて投与することも可能であるし、例えば点滴等により連続的に投与することも可能である。
【００３１】
更に、本発明物質を有効成分とする組成物は、上記医薬組成物としての用途以外に、凝固線溶系を研究するための研究用試薬としても有用である。
【００３２】
【実施例】
本発明を実施例により更に具体的に説明するが、本発明は以下の実施例に限定されるものではない。
実施例１ 本発明物質の製造
▲１▼マナマコ体壁由来ＣＳの単離精製
マナマコ（Stichopus japonius）の体壁部をミンチ状に処理した後、ホモジナイズし、クロロホルム／メタノール（２：１、v/v）で不要の脂溶性画分を抽出除去した。抽出残渣を乾燥後、オートクレーブ処理（１２０℃、３０分）に付し、５０ｍＭリン酸バッファー（p H 8.0）中に溶解し、懸濁液とした。この懸濁液に対し、蛋白質１gあたり５０mgのアクチナーゼ（科研製薬（株）製）を加え、５５℃にて８時間攪拌し、コラーゲンやコアプロテイン等を消化した。
【００３３】
アクチナーゼ消化物を０．４Ｍ水酸化ナトリウム、次いで１０％トリクロロ酢酸にて順次処理した後、遠心分離（5,000×g、１５分）に付し、上清を回収し、流水透析を行った。透析内液に２．５％酢酸ナトリウムを添加した冷エタノールを透析内液量の３倍量添加することにより目的物質を沈殿させ、遠心分離（5,000×g、１５分）により沈殿を回収した。更に、得られた沈殿を冷エタノールで洗浄し、得られたペレットを減圧乾燥することにより粗精製物を得た。
【００３４】
得られた粗精製物１．０gを少量の５０ｍＭ 炭酸水素アンモニウム緩衝液（p H 8.0）に溶解し、その半分量を同溶媒で平衡化したSephadex G-100（ファルマシア社製）を詰めたカラム（直径３．４ｃｍ×長さ１００ｃｍ）に付し、同溶媒にて溶出させ、溶出液を１０ml毎に回収した。得られた各フラクションにつき、ＵＶ２１０ｎｍにおける吸光度の測定、カルバゾール法（Bitter & Muir (1962) Anal. Biochem., 4, 330-334）によるウロン酸含量の測定及びアンスロン法（Dimler, R. L et al. (1952) Anal. Chem., 24, 1411-1414）による中性糖含量の測定を行った。
【００３５】
その結果、高分子画分にＧＡＧと推測されるカルバゾール反応陽性の画分を見出し、これを合一回収し、凍結乾燥により粉末を２２４ｍｇ得た。同様の操作を得られた粗精製物の溶液の残り半分量についても行い、合わせて５７４ｍｇの凍結乾燥物を得た。得られた凍結乾燥物のうち５００ｍｇを１００ｍＭ酢酸ナトリウム緩衝液（pH 5.0）で平衡化したＤＥＡＥ-セルロース（ＤＥ５２、Whatmann社製）を詰めたカラム（内径１．８ｃｍ×長さ１８ｃｍ）に付し、同緩衝液中０→１．２Ｍ 塩化ナトリウムの直線的濃度勾配により溶出した。
【００３６】
８ｍｌ毎に分画回収した各フラクションにつきカルバゾール法によるウロン酸含量の測定およびアンスロン法による中性糖含量の測定を行った。その結果、塩化ナトリウムの０．６Ｍ濃度付近に目的物質と目されるピークを見出したので、このピークに該当する画分を合一した後、流水透析にて透析後、凍結乾燥を行い、粉体を２１０ｍｇ得た。
【００３７】
▲２▼分析
上記▲１▼で得られた粉体について、化学組成分析を行った。
ＭＢＴＨ法（Hurst & Settine (1981) Anal. Biochem., 115, 88-92）にてヘキソサミン含量を、カルバゾール法、イオンクロマトグラフィー並びにアンスロン法にて各々ウロン酸含量、硫酸イオン含量並びに中性糖含量を定量した。
【００３８】
分析結果から、この画分の構成成分の重量存在比を算出したところ、既に報告されていいるナマコＧＡＧの分析結果（Kariya et al.(1990) J.Biol.Chem.,265,5081-5085参照）とほぼ同等の組成を示した。
【００３９】
▲３▼本発明物質の製造
公知ナマコＧＡＧとほぼ同等の組成を有する上記▲１▼で得られた精製画分４０ｍｇを０.１N硫酸４ｍｌに溶解し、８０℃にて３時間あるいは６時間の加水分解反応にそれぞれ付した。反応は反応混液を室温までに冷却することによって停止させ、１Ｎ水酸化ナトリウムを添加して中和した後、０.２Ｍ塩化ナトリウムで平衡化したセルロファインＧＣＬ-９０ｍカラム（内径３.４×長さ１１０ｃｍ、生化学工業（株）製）に付し、溶出液を１０ｍｌ毎に分取した（以下、３時間の加水分解により得られたものを３時間水解物、６時間の加水分解により得られたものを６時間水解物という）。対照として４０ｍｇの未加水分解物（上記▲１▼で得られた精製画分、以下、未水解物ともいう）も同様にカラムクロマトグラフィーに付し、溶出液を１０ｍｌづつ分取した。
【００４０】
未水解物、３時間水解物及び６時間水解物のカラム溶出液の各フラクションにつき、ウロン酸含量並びに中性糖含量を測定し、溶出液の挙動を比較した（図１）。得られた高分子画分域（３時間水解物（図１ｂ）はフラクションNo.４０〜５４、６時間水解物（図１ｃ）はフラクションNo.４０〜５８、未水解物（図１ａ）はフラクションNo.４０〜５０）は合一濃縮して、蒸留水で平衡化したセルロファインＧＣＬ-２５カラム（内径２.０×長さ２５ｃｍ、生化学工業（株）製）に付し脱塩した後、凍結乾燥処理によって乾燥・回収した。未水解物、３時間水解物及び６時間水解物の各高分子画分の収量は、各々３４.７ｍｇ、１８.１ｍｇおよび１５ｍｇであった。
【００４１】
実施例２ 本発明物質の物理化学的性状の解析
▲１▼ゲル濾過溶出パターン
図１のゲル濾過溶出パターンを比較すると、未水解物（図１(ａ)）では高分子画分域にウロン酸及び中性糖のピークがほぼ完全に一致した位置に等しい形状でシングルピークとして観察された。しかし３時間水解物（図１(ｂ)）では、未水解物のパターンと比べ、対応する高分子画分域のウロン酸ピークはやや減少し、やや広がり、更に、同域の中性糖ピークの検出値は大幅に減少し、一方、低分子画分域に中性糖の新しいピークが出現した。このピークは加水分解処理により脱離した硫酸化フコビオシル基と考えられる。同様に６時間水解物（図１(ｃ)）においては、低分子画分域の新しい中性糖ピークがより顕著であり、更に、高分子画分域におけるウロン酸ピークの減少とより広い平坦化、更に中性糖ピークの顕著な減少が確認された。
【００４２】
これより、加水分解反応の進行に伴い、原料であるナマコ由来のＧＡＧ主鎖から硫酸化フコビオシル基が加水分解反応時間依存的に脱離している事が示唆される。
【００４３】
尚、３時間水解物の様に、硫酸化フコビオシル基が部分的に脱離した構造を有するＧＡＧは、これまでに報告がなく、新規な硫酸化フコビオシルコンドロイチン硫酸である。
【００４４】
▲２▼化学組成分析
３時間水解物、６時間水解物、未水解物について、実施例１▲２▼に記載の化学組成分析を行い、結果として得られた各水解物の構成成分の重量存在比を算出した（表１）。更に、各構成成分のモル存在量（ｍｍｏｌ／ｇ）を算出した上、ヘキソサミンのモル存在量を１として、各成分のモル比を算出した（表２）。
【００４５】
【表２】

【００４６】
【表３】
表２ 各水解物の構成成分のモル存在量
（各水解物毎にGalNAcのモル存在量を１とした場合の各成分のモル比）

【００４７】
表１及び表２より、各水解物においては、ヘキソサミンとウロン酸のモル比はほぼ一定しており、加水分解後でも、主鎖部分の構造はＧＡＧ特有の二糖繰り返し構造が保持されていると考えられる。主鎖の二糖単位１残基あたりのフコビオシル基及び硫酸基含量は、未水解物が各々１．１９及び３．６９分子存在するのに対し、３時間水解物では各々０．２９及び１．７０分子であり、６時間水解物では各々０．１８及び１．０６分子と、加水分解反応時間依存的に減少していることが判明した。これは、上記実施例２▲１▼のゲル濾過溶出パターン（図１）の結果を支持しており、加水分解反応時間依存的に硫酸化フコビオシル基が脱離していると考えられる。
【００４８】
▲３▼脱分岐化度
加水分解反応による、硫酸化フコビオシル基の脱離割合を脱分岐化度として算出した。図１に於けるアンスロン法の吸光度を指標に、検出される高分子領域のピークと低分子領域のピークの面積から以下の式を用いて脱分岐化度の見積もりを行った。尚、グラフの描画にはMicrosoft Excel 2000を、面積の算出には、NIH Image 1.62を用いた。
【００４９】
脱分岐化度(％)＝低分子化領域ピーク面積(Ａ_２)／全ピーク面積(Ａ_１＋Ａ_２)×１００
【００５０】
計算の結果、未水解物、３時間水解物および６時間水解物の脱分岐化度は、それぞれ０％、６７．６％及び９０．４％と算出された。この結果からも、加水分解による脱分岐化反応は、反応時間依存的に進行することが確認される。尚、分岐化度（％）＝１００−（脱分岐化度）である。
【００５１】
▲４▼コンドロイチナーゼＡＢＣ消化
コンドロイチナーゼＡＢＣ（生化学工業（株）製、以下、Ｃ-ＡＢＣという）による未水解物、３時間水解物、６時間水解物各々の消化の度合いをゲル濾過クロマトグラフィーを分離要因とする高速液体クロマトグラフィー（以下、ＧＰＣ-ＨＰＬＣという）により測定した。
【００５２】
各水解物を１０ｍｇ／ｍｌとなるように蒸留水に溶解し、そのうち２０μｌを０．５ＵのＣ-ＡＢＣを含む酵素溶液１０μｌ（０．４Ｍ酢酸ナトリウム、０．１％牛血清アルブミンを含む０．４Ｍトリス塩酸緩衝液(p H8.0)）に添加し、３７℃で１８時間、酵素消化反応を行った。反応混液に蒸留水５０μｌを添加し、沸騰水中で１分加熱後、遠心分離することにより上清を得た。
【００５３】
このＣ-ＡＢＣ酵素消化物を含む上清を、ＴＳＫ-Gel G4000PW_XL、ＴＳＫ-Gel G3000PW_XLおよびＴＳＫ-Gel G2500PW_XLカラム（いずれも内径４.０×２５ｃｍ、Tosoh社製）を上流から順に連結して装着したＧＰＣ-ＨＰＬＣに付し、０．２Ｍ塩化ナトリウム溶液のアイソクラティック条件で溶出し、Refractive Indexを指標として検出した。同様に、Ｃ-ＡＢＣ消化前の各水解物に関しても、ＨＰＬＣチャートを得た。これらの結果を図２に示す。
【００５４】
得られたＨＰＬＣチャートのピーク面積から以下の式を用いて各水解物のＣ-ＡＢＣ消化度（不飽和二糖生成率）の見積もりを算出した。下記不飽和二糖生成率の計算式において、全ピーク面積とは、図２のＡ２、Ｂ２、Ｃ２（Ｃ-ＡＢＣ酵素消化後のチャート）における、△oligo、△Ｄｉ-diＳ、△Ｄｉ-monoＳ及び△Ｄi-zeroＳの合計ピーク面積を示し、不飽和二糖ピーク面積の和とは、各不飽和二糖ピークの面積の総和（△Ｄｉ-diＳ、△Ｄｉ-monoＳ及び△Ｄｉ-zeroＳの合計ピーク面積）である。尚、△oligoとは、Ｃ-ＡＢＣ消化にて二糖単位構造にまで分解されず、三糖以上の構造である不飽和オリゴ糖を示す。また、△Ｄｉ-diＳは、不飽和二糖単位構造中に硫酸基を二つ有する不飽和二糖を、△Ｄｉ-monoＳは、不飽和二糖単位構造中に硫酸基を一つ有する不飽和二糖を、△Ｄｉ-zeroＳは不飽和二糖単位構造中に硫酸基を有さない不飽和二糖を示し、後述の不飽和二糖スタンダードに関して述べると、△Ｄｉ-diＳには△Ｄｉ-ｄｉ（２,６）Ｓ、△Ｄｉ-ｄｉ（４,６）Ｓが、△Ｄｉ-monoＳには△Ｄｉ-６Ｓ、△Ｄｉ-４Ｓが、△Ｄｉ-zeroＳには△Ｄｉ-０Ｓが分類される。
【００５５】
不飽和二糖生成率（％）＝不飽和二糖ピーク面積の和／全ピーク面積×１００
【００５６】
酵素消化前のチャートにおいては、加水分解反応時間が長くなるに従ってメインピークの保持時間が長くなっており、若干の低分子化が起こっていることが観察された。これは硫酸化フコビオシル基側鎖の脱離が進行した結果であると推察される。
【００５７】
未水解物の酵素消化後のチャートを、酵素消化前と比較すると、メインピークの保持時間は僅かに長くなるが、ほとんど変化せず、不飽和二糖成分も検出されない。すなわち、消化された形跡が見られず、公知文献の記載と同様にＣ-ＡＢＣにより分解されないことが確認された。
一方、３時間水解物及び６時間水解物では、不飽和二糖及び不飽和オリゴ糖のピークが観察された。これらピークについて、上記式に従い二糖生成率を算出した。その結果、未水解物、３時間水解物及び６時間水解物の二糖生成率は各々０％、４６．５％及び６６．３％であった。
【００５８】
加水分解の反応時間に従い、Ｃ-ＡＢＣの消化性も高まり、不飽和二糖の生成も増えている。これより、硫酸化フコビオシル基側鎖を有するＣＳにおいて、Ｃ-ＡＢＣ酵素消化の障壁と成っているのは、硫酸化フコビオシル基側鎖の存在であると推測される。
【００５９】
更に、Ｃ-ＡＢＣ酵素消化物の不飽和二糖分析を行った。上記の各水解物のＣ-ＡＢＣ酵素消化物につき、上清をＹＭＣ-ＰＡ１２０Ｓ５カラム（内径４．０ｃｍ×２５ｃｍ、ＹＭＣ社製）を装着した強陰イオン交換ＨＰＬＣ（以下、ＳＡＸ-ＨＰＬＣともいう）に付し、MilliQ Water（商標名：ミリポア社製）と０．８Ｍリン酸二水素ナトリウム溶液の直線的濃度勾配システム（１６ｍＭ→５２０ｍＭ／３８分）で溶出し、２３０ｎｍの吸光度で検出した。結果を図３に示す。
【００６０】
不飽和二糖スタンダードとしてコンドロイチン硫酸タイプの△Ｄｉ-０Ｓ、△Ｄｉ-６Ｓ、△Ｄｉ-４Ｓ、△Ｄｉ-ｄｉ（２,６）Ｓ、△Ｄｉ-ｄｉ（４,６）Ｓおよび△Ｄｉ-ｔｒｉＳを用いた。
【００６１】
尚、これら△Ｄｉ-０Ｓ、△Ｄｉ-６Ｓ、△Ｄｉ-４Ｓ、△Ｄｉ-ｄｉ（２,６）Ｓ、△Ｄｉ-ｄｉ（４,６）Ｓおよび△Ｄｉ-ｔｒｉＳとは、前述式３で示される二糖組成中の各置換基が下表の通りであるものを示す。
【００６２】
【表４】

【００６３】
図３より、△Ｄｉ-０Ｓ、△Ｄｉ-６Ｓ、△Ｄｉ-４Ｓ、△Ｄｉ-ｄｉ（２,６）Ｓ、△Ｄｉ-ｄｉ（４,６）Ｓおよび△Ｄｉ-ｔｒｉＳの各ピークの合計に対し、３時間水解物では、△Ｄｉ-ｄｉ（４,６）Ｓが５２.２％、△Ｄｉ-６Ｓが３０．４％であったのに対し、６時間水解物では△Ｄｉ-６Ｓが４９．８％、△Ｄｉ-ｄｉ（４,６）Ｓが２５．３％であった。また、両者とも△Ｄｉ-ｄｉ（２，６）Ｓ及び△Ｄｉ-ｔｒｉＳは観察されず、いくつかの不飽和オリゴ糖由来と目されるピークが見られた。以上の結果は一見したところ、６時間水解物で観察された△Ｄｉ-６Ｓの一部は、３時間水解物に存在した△Ｄｉ-ｄｉ（４，６）Ｓの４-Ｏ-硫酸基の加水分解によって生じたものと推定される。
【００６４】
しかしながら一方では、△Ｄｉ-６ＳのGalNAcのＯ-４位から伸展していた硫酸化フコビオシル基の加水分解による脱離速度は遅く、△Ｄｉ-ｄｉ（４，６）ＳのGlcAのＯ-３位から伸展していた硫酸化フコビオシル基の加水分解による脱離速度が速かった為に、見かけ上、上記のように△Ｄｉ-ｄｉ（４，６）Ｓの４-Ｏ-硫酸の脱離が発生した様に見えた可能性が高い。尚、図２のＣ-ＡＢＣ消化後のチャートにおいて、全ピーク面積に対する△Ｄｉ-ｄｉＳ成分である△Ｄｉ-ｄｉ（４，６）Ｓの割合が、３時間水解物で２８．８％、６時間水解物で２９．７％とほぼ同等だったことからも、後者の可能性が高いことが支持される。
【００６５】
この不飽和二糖組成は硫酸化フコビオシル基側鎖が脱離し、酵素消化可能となった直鎖構造を有する部分の組成であり、側鎖が保持されている部分の二糖組成についてはこの方法では分析出来ない。
【００６６】
以上のことより、本発明物質の一例である３時間水解物は、ＣＳ-Ｅタイプの二糖単位を有し、Ｄ-グルクロン酸のO-３位に結合している硫酸化フコビオシル基が優先的に脱離した、部分的に硫酸化フコビオシル基側鎖を有する新規な硫酸化フコビオシルコンドロイチン硫酸と言える。
【００６７】
実施例３ 本発明物質の組織プラスミノーゲンアクチベーターを介した線溶活性亢進作用
実施例１にて製造した未水解物、３時間水解物、６時間水解物及び、公知ナマコＧＡＧ（Stichopus japonicus由来、1997年に単離精製）について種々の濃度の溶液を作成し、当該溶液３０μｌと０．４８μＭのプラスミノーゲン５０μｌ、７．５ｎＭの一本鎖ｔ−ＰＡ（以下、ｓｃｔ―ＰＡという）及び１．８ｍＭの合成基質S-2251（Ｈ-Ｄ-Ｖａｌ―Ｌ-Ｌｅｕ―Ｌ-Ｌｙｓ―p-ニトロアニリド・二塩酸塩）４０μｌをウェルで混和後、自動マイクロタイタープレート読み取り機にセットし、３７℃に加温して反応を進行させながら、１５秒毎に４０５ｎｍ（対照４９２ｎｍ）の吸光度を測定した。なお、反応はＴｗｅｅｎ80を０．００５％含む５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）で行った。
【００６８】
プラスミノーゲンの活性化の初速度は、時間ｔの時の４０５ｎｍ（対照４９２nm）の吸収値Ａ_405-492と時間の自乗のプロットの傾き（Ａ_405-492／t²）より得た。促進率はＧＡＧのない場合の初速度を１とし、それに対する相対比をポテンシエーションファクターとして示した。以上の活性測定を種々のＧＡＧ濃度で実施し、最も高い活性を示したＧＡＧ濃度（１．５６μｇ／ｍｌ）における結果を図４に示した。
【００６９】
活性の高い順に列挙すると３時間水解物が飛び抜けて高い活性を示し、次いでずっと低いレベルに６時間水解物、公知ナマコＧＡＧそして未水解物の順であり、本発明物質である３時間水解物は、非常に高い線溶活性亢進作用を有する事が明らかとなった。
【００７０】
【発明の効果】
本発明により、線溶活性亢進作用を有する新規な部分的に硫酸化フコビオシル基側鎖を有する硫酸化フコビオシルコンドロイチン硫酸が提供される。
【００７１】
【図面の簡単な説明】
【図１】 未水解物、３時間水解物及び６時間水解物のセルロファインＧＣＬ-９０ｍカラム溶出液の各フラクションにおけるウロン酸含量並びに中性糖含量を測定したグラフであり、縦軸は吸光度、横軸はフラクション番号である。(a)、(b)及び(c)は、各々、未水解物、３時間水解物、６時間水解物の結果である。
【図２】 未水解物、３時間水解物及び６時間水解物における、Ｃ-ＡＢＣ消化前後のＧＣＰ-ＨＰＬＣチャートであり、縦軸は示差屈折率、横軸は保持時間（分）を示す。Ａ１は、未水解物のＣ-ＡＢＣ消化前のＧＣＰ-ＨＰＬＣチャートであり、Ａ２は、未水解物のＣ-ＡＢＣ消化後のＧＣＰ-ＨＰＬＣチャートであり、二糖生成率は０％である。Ｂ１は、３時間水解物のＣ-ＡＢＣ消化前のＧＣＰ-ＨＰＬＣチャートであり、Ｂ２は、３時間水解物のＣ-ＡＢＣ消化後のＧＣＰ-ＨＰＬＣチャートであり、二糖生成率は４６．５％である。Ｃ１は、６時間水解物のＣ-ＡＢＣ消化前のＧＣＰ-ＨＰＬＣチャートであり、Ｃ２は、６時間水解物のＣ-ＡＢＣ消化後のＧＣＰ-ＨＰＬＣチャートであり、二糖生成率は６６．３％である。
【図３】 Ｃ-ＡＢＣ消化後の未水解物、３時間水解物及び６時間水解物におけるＳＡＸ-ＨＰＬＣチャートであり、縦軸は吸光度、横軸は保持時間(分)を示す。Ａ、Ｂ、Ｃ及びＤは各々、未水解物、３時間水解物、６時間水解物及び不飽和二糖スタンダードの結果である。
【図４】 sct-PAを介したglu-plg（Ｎ末端のアミノ酸がグルタミン酸(Glu）から始まるるタイプのプラスミノーゲン）の活性化測定による、未水解物、３時間水解物、６時間水解物及び公知ナマコＧＡＧの１．５６μｇ／ｍｌ濃度に於ける組織プラスミノーゲンアクチベーターを介した線溶活性促進率を示す。縦軸は、Potentiation Factorである。[0001]
BACKGROUND OF THE INVENTION
The present invention provides a novel disaccharide structure in which D-glucuronic acid and N-acetyl-D-galactosamine are linked by β1 → 3 glycosidic linkage as a constituent disaccharide unit of the main chain and partially having a sulfated fucobiosyl group as a side chain. The present invention relates to sulfated fucobiosylchondroitin sulfate, a pharmaceutical composition containing the sulfated fucobiosylchondroitin sulfate, and a fibrinolytic activity enhancer.
[0002]
[Prior art]
Chondroitin sulfate (hereinafter also referred to as CS) is a kind of glycosaminoglycan (hereinafter also referred to as GAG), and D-glucuronic acid (hereinafter also referred to as GlcA) and N-acetyl-D-galactosamine (hereinafter referred to as GalNAc). Is a polysaccharide composed of disaccharides linked by β1 → 3 glycosidic bonds (GlcAβ1 → 3GalNAc), and there are several kinds of polysaccharides depending on the position of the sulfate group and the binding ratio of the disaccharide units. Classified into molecular species. For example, chondroitin sulfates containing many disaccharide units with sulfate groups bonded to the O-4 or O-6 position of GalNAc are called chondroitin sulfate A and chondroitin sulfate C, respectively. Sulfated chondroitin sulfate is also called chondroitin sulfate E (hereinafter also referred to as CS-E). In addition, CS is widely known to exist in animal cartilage and connective tissue, and studies on various CSs isolated from various animals and their structures and actions have been reported.
[0003]
For example, CS containing 9.5 to 13% sulfur and 12 to 28% fucose was isolated from sea cucumber, and as a result of analysis, CS from this sea cucumber bound GlcA and GalNAc through β1 → 3 glycoside bonds. A structure in which a repeating structure of disaccharide units is used as a core polymer, and a sulfated fucobiosyl group consisting of two sulfated fucose residues is glycosidically linked to the O-4 position, O-6 position of GalNAc and / or the O-3 position of GlcA. Have. Manamako CS is reported to be extremely difficult to be digested by chondroitinases unlike other CS consisting only of a linear structure (Kariya et al. (1997) Carbohydr. Res., 297, 273-279). , JP 6-70085). Moreover, regarding its physiological activity, anticoagulant activity has been mainly confirmed (Morao et al. (2001) Thromb. Res., 102, 167-176).
[0004]
Similarly, an example of isolation and purification of sulfated fucobiosylchondroitin sulfate (hereinafter referred to as sulfated fucobiosyl CS) has been reported from other various sea cucumbers (Japanese Patent Publication No. 6-70085). On the other hand, it is reported that sulfated fucobiosyl CS having a sulfated fucobiosyl group side chain derived from these sea cucumbers hardly shows fibrinolytic activity (Sakai et al. (2000) Tromb Res., 100, 557-565). ). In general, there is no report on a substance produced by using a sulfated fucobiosyl CS, which is considered to be present in echinoderms, as a raw material and partially modifying its constitution.
[0005]
[Problems to be solved by the invention]
An object of this invention is to provide the novel chondroitin sulfate derivative which has a fibrinolytic activity enhancement effect.
[0006]
[Means for Solving the Problems]
As a result of intensive research in order to solve the above-mentioned problems, the present inventors have sulfated fucobiosyl side chains in almost all of the constituent disaccharide units, and hardly exhibit a fibrinolytic activity enhancing action. It was found that the effect of enhancing the fibrinolytic activity was appropriately expressed by partially removing the sulfated fucobiosyl group side chain from fucobiosyl CS, and the present invention was completed.
[0007]
That is, the gist of the present invention is as follows.
(1) A disaccharide in which D-glucuronic acid (hereinafter also referred to as GlcA) and N-acetyl-D-galactosamine (hereinafter also referred to as GalNAc) are linked by β1 → 3 glycosides, and has the following physicochemical properties Sulfated fucobiosylchondroitin sulfate (hereinafter also referred to as the substance of the present invention).
i) It has a disaccharide structural unit structure represented by the following formula (1).
[0008]
[Chemical formula 2]

[0009]
ii) Unsaturated disaccharide production rate is 35 to 55% by degradation with chondroitinase ABC.
iii) It contains 1.3 to 1.8 molecules of sulfate groups and 0.2 to 0.4 molecules of fucobiosyl groups per constituent disaccharide unit.
(2) The sulfated fucobiosylchondroitin sulfate according to (1) above having an average molecular weight of 11,000 to 13,000.
(3) The sulfated fucobiosyl according to (1) or (2) above, which is obtained by partially removing a sulfated fucobiosyl group as a side chain from a sulfated fucobiosylchondroitin sulfate derived from sea cucumbers Chondroitin sulfate.
(4) The sulfated fucobiosylchondroitin sulfate according to any one of the above (1) to (3), wherein the degree of branching is 20 to 50%.
(5) Contains the sulfated fucobiosylchondroitin sulfate according to any one of (1) to (4) above.Composition.
(6) The composition according to the above (5), which is a pharmaceutical composition.
(7) A fibrinolytic activity enhancer comprising the sulfated fucobiosylchondroitin sulfate according to any one of (1) to (4) above as an active ingredient (hereinafter also referred to as the pharmaceutical of the present invention).
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in further detail below.
The substance of the present invention is a sulfated fucobiosyl consisting of a disaccharide unit repeating structure in which the O-1 position of GlcA and the O-3 position of GalNAc are β-glycosidically linked, and consisting of two sulfated fucose residues as side chains. It is a kind of CS partially having a group.
[0011]
The sulfated fucobiosyl group in the present invention refers to sulfuric acid (—OSO) of hydroxyl groups (—OH) at the O-2 position and the O-4 position of fucose.₃ ^-) Is a fucobiose group in which two fucose residues having 0 to 2 sulfate groups in one molecule of fucose are α-1,3 bonded to each other due to the difference in chemical formula, and is represented by the following formula (2). The sulfated fucobiosyl group includes a hydroxyl group and a sulfated fucobiosyl group O selected from the O-4 position, the O-6 position of GalNAc, and the O-3 position of GlcA, which constitute the main chain of the substance of the present invention. A side chain of the substance of the present invention is formed by glycosidic bonding with the hydroxyl group at position -1.
[0012]
[Chemical 3]

[0013]
[Table 1]

[0014]
The unsaturated disaccharide production rate when the substance of the present invention is decomposed by chondroitinase ABC is 35 to 55%. The unsaturated disaccharide production rate can be calculated by disaccharide composition analysis combining decomposition by chondroitinase ABC and analysis by ion exchange high-performance liquid chromatography (hereinafter also referred to as HPLC) described in the Examples below. When the substance of the present invention is analyzed by this method, a disaccharide structure represented by the following formula (3) is detected.
[0015]
[Formula 4]

[0016]
Where R¹, R², R³Is a hydrogen atom or SO₃ ^-Ac represents an acetyl group.
[0017]
Furthermore, as a result of chemical composition analysis, it contains 1.3 to 1.8 molecules of sulfate groups, preferably 1.5 to 1.8 molecules of sulfate groups per residue of the main chain disaccharide units. It contains 0.2 to 0.4 molecules of fucobiosyl group, preferably 0.2 to 0.3 molecules of fucobiosyl group per residue. The chemical composition analysis method is not particularly limited as long as it can measure the hexosamine content, sulfate content and fucobiosyl content or fucose content in the substance of the present invention. Similarly, the hexosamine content was measured by the MBTH method, and further, by the ion chromatography method and the anthrone method (Dimler, R. Let al. (1952) Anal. Chem., 24, 1411-1414), the sulfate group content and A method for measuring the fucose content can be mentioned.
[0018]
In view of the fucobiosyl content, the substance of the present invention is one at a position selected from the constituent disaccharide unit having no sulfated fucobiosyl side chain and the O-4 position, O-6 position of GalNAc and the O-3 position of GlcA. The CS is composed of mixed disaccharide units having sulfated fucobiosyl group side chains as described above, and thus partially CS having sulfated fucobiosyl group side chains.
[0019]
The substance of the present invention preferably has an average molecular weight of 11,000 to 13,000, more preferably an average molecular weight of 11,000 to 12,000. The average molecular weight of polysaccharides is generally expressed by weight average molecular weight, but it is common knowledge for those skilled in the art that the average molecular weight of GAG varies slightly depending on the measurement method and measurement conditions even in the same sample. Inventive substances should not be strictly limited to the above average molecular weight range.
[0020]
The substance of the present invention is not particularly limited depending on its origin, origin, and production method. If the substance satisfies the physicochemical properties of the substance of the present invention, it is partially sulfated fucobiosyl group obtained from natural resources. Even in the sulfated fucobiosyl CS having a chain, a sulfated fucobiosyl CS obtained from a natural resource (mainly a biological body) is used as a raw material, and is modified by a chemical method such as partially removing a sulfated fucobiosyl group described later, or Alternatively, an artificially chemically synthesized product or a genetically synthesized product synthesized with animal cells, plant cells, microorganisms, or the like may be used.
[0021]
The organism used in the case where the substance of the present invention is isolated and purified from the organism itself or when the sulfated fucobiosyl CS isolated and purified from the organism is used as a raw material is not particularly limited. Examples of organisms include echinoderms, preferably sea cucumbers, and particularly, sea cucumbers (Sea Cucumber Stichopus japonicus), sea cucumbers (Holthuria atra), black sea cucumbers (Holothuria leurospilota) and the like. These sea cucumber animals are unused resources that are rarely used, although only a small part is used for food.
[0022]
As an example, a method for producing the substance of the present invention using manamako as a raw material will be described.
First, from the body wall of manama according to a known method (see Carbohydr. Res., 297, 273-279 (1997), Japanese Patent Publication No. 6-70085, etc.) usually used when isolating and purifying GAG from living tissue. The sulfated fucobiosyl CS is isolated and purified, and then the sulfated fucobiosyl group as a side chain is partially eliminated (partially debranched) to obtain the substance of the present invention.
[0023]
  Here, the partial elimination method of the sulfated fucobiosyl group that is a side chain is not particularly limited as long as it is a method that generates CS satisfying the physicochemical properties of the substance of the present invention. Examples include mild acid hydrolysis, alkali hydrolysis reaction, enzyme digestion reaction and the like under the condition that sulfated fucobiosyl CS of ˜50% is obtained. Among the above, a method of acid hydrolysis for about 2.5 to 3.5 hours under a condition of about 75 to 85 ° C. using an inorganic acid such as sulfuric acid and hydrochloric acid of about 0.05 to 0.2 N is particularly preferable. Normally, the reaction is neutralized after the reaction and subjected to a separation / purification process. Thus, more preferably,Branching degreeA reaction end product (inventive substance) of 30 to 40% is obtained. After the partial debranching, the substance of the present invention purified by conventional means for separation and purification of GAG can be obtained.
[0024]
The degree of branching here refers to the ratio of the molar amount of the sulfated fucobiosyl group, which is the side chain of one molecule of the substance of the present invention, to the molar amount of the main chain constituent disaccharide unit. In addition, regarding sea cucumber-derived sulfated fucobiosyl CS that can be used as a raw material for producing the substance of the present invention by the method of partially detaching the side chain, about 1 per disaccharide unit constituting the main chain. It has already been reported that it has a sulfated fucobiosyl group side chain (degree of branching = 100%). (Kariya et al. (1997) Carbohydrate Research 297, 273-279)
[0025]
As described above, it is already known that manamaco-derived CS is extremely difficult to digest by chondroitinases, but the substance of the present invention obtained by a method of partially removing the side chain from manamako-derived CS Is degraded by 35-55% by chondroitinase ABC as described in the Examples below.
[0026]
In addition, as described above, Sakai. Et.al (2000) Tromb Res., 100, 557-565 reports that manamaco-derived CS, which is a raw material for producing the substance of the present invention, exhibits only a very weak fibrinolytic activity enhancing action. However, as described in the Examples below, the substance of the present invention has a very high line in comparison with manamaco-derived CS in a fibrinolytic reaction system in which plasminogen, single-stranded tissue plasminogen activator and a synthetic substrate are added. It showed a lytic activity enhancement effect.
[0027]
The action of enhancing fibrinolytic activity via plasminogen activator is a series in which plasminogen activator (hereinafter also referred to as PA) degrades plasminogen to plasmin limitedly, and activated plasmin degrades fibrin. It is a general term for the reaction.
[0028]
Especially in intravascular fibrinolysis, tissue-type PA (hereinafter referred to as t-PA) is an important regulator, and when the action of t-PA decreases, it becomes difficult to dissolve thrombus formed in the bloodstream. It becomes easy to develop thrombosis such as infarction and myocardial infarction. Since the substance of the present invention has an excellent action for enhancing fibrinolytic activity, a pharmaceutical composition containing the substance of the present invention as an active ingredient (hereinafter also referred to as the present pharmaceutical composition) is desired to promote t-PA activity. It can be administered to humans and other mammals for the purpose of prevention, maintenance (prevention of deterioration), reduction (improvement of symptoms) and treatment of the above-mentioned diseases.
[0029]
The dosage form and administration route for administering the pharmaceutical composition of the present invention to a living body can be appropriately selected according to the nature and severity of the target disease. For example, as it is or in combination with other pharmacologically acceptable carriers, diluents, stabilizers, etc., injections, tablets, capsules, granules, powders, liquids, lipolytic agents, ointments , Gel preparations, external powders, sprays, inhalation powders, eye drops, eye ointments, suppositories, etc., injection (intramuscular, subcutaneous, intradermal, intravenous, intraarticular cavity, intraperitoneal, etc.), eye drops It can be safely administered orally or parenterally by administration methods such as infusion, transdermal, oral and inhalation.
[0030]
The compounding amount and dosage of the substance of the present invention in the pharmaceutical composition of the present invention are matters to be individually determined according to the administration method, dosage form, purpose of use, specific symptoms of the patient, patient weight, etc. Although there is no particular limitation, examples of the substance of the present invention in the oral or parenteral route of administration are approximately 0.1 mg / kg to 300 mg / kg per day. In addition, the administration interval can be about once a day, can be divided into 2 to 4 times a day or more, and can be administered continuously, for example, by infusion or the like. Is also possible.
[0031]
Furthermore, the composition containing the substance of the present invention as an active ingredient is useful as a research reagent for studying the coagulation / fibrinolysis system in addition to the use as the pharmaceutical composition.
[0032]
【Example】
Examples The present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.
Example 1 Production of the substance of the present invention
(1) Isolation and purification of CS from manamaco body wall
After processing the body wall part of Manamako (Stichopus japonius) in the shape of a mince, it was homogenized and the unnecessary fat-soluble fraction was extracted and removed with chloroform / methanol (2: 1, v / v). The extraction residue was dried and then subjected to autoclaving (120 ° C., 30 minutes), dissolved in 50 mM phosphate buffer (pH 8.0) to obtain a suspension. To this suspension, 50 mg of actinase (produced by Kaken Pharmaceutical Co., Ltd.) per gram of protein was added and stirred at 55 ° C. for 8 hours to digest collagen and core protein.
[0033]
The actinase digest was sequentially treated with 0.4 M sodium hydroxide and then with 10% trichloroacetic acid, followed by centrifugation (5,000 × g, 15 minutes), and the supernatant was collected and dialyzed against running water. The target substance was precipitated by adding cold ethanol containing 2.5% sodium acetate to the dialyzed internal solution in an amount three times the amount of the dialyzed internal solution, and the precipitate was collected by centrifugation (5,000 × g, 15 minutes). Furthermore, the obtained precipitate was washed with cold ethanol, and the resulting pellet was dried under reduced pressure to obtain a crude product.
[0034]
A column packed with Sephadex G-100 (manufactured by Pharmacia) in which 1.0 g of the crude product thus obtained was dissolved in a small amount of 50 mM ammonium bicarbonate buffer (pH 8.0) and half of the solution was equilibrated with the same solvent. It was attached to (diameter 3.4 cm × length 100 cm) and eluted with the same solvent, and the eluate was collected every 10 ml. About each obtained fraction, the measurement of the light absorbency in UV210nm, the measurement of the uronic acid content by the carbazole method (Bitter & Muir (1962) Anal. Biochem., 4, 330-334), and the anthrone method (Dimler, R. L et al (1952) Anal. Chem., 24, 1411-1414), the neutral sugar content was measured.
[0035]
As a result, a carbazole reaction-positive fraction presumed to be GAG was found in the polymer fraction, and these fractions were collected together and 224 mg of powder was obtained by lyophilization. The same operation was performed for the remaining half of the crudely purified product solution, and a total of 574 mg of lyophilized product was obtained. 500 mg of the obtained lyophilizate was applied to a column (inner diameter 1.8 cm × length 18 cm) packed with DEAE-cellulose (DE52, manufactured by Whatmann) equilibrated with 100 mM sodium acetate buffer (pH 5.0). Elute with a linear gradient of 0 → 1.2M sodium chloride in the same buffer.
[0036]
For each fraction collected every 8 ml, the uronic acid content was measured by the carbazole method and the neutral sugar content was measured by the anthrone method. As a result, a peak considered to be the target substance was found near 0.6 M concentration of sodium chloride, and the fractions corresponding to this peak were combined, dialyzed by running water dialysis, freeze-dried, and powdered. 210 mg of body was obtained.
[0037]
(2) Analysis
Chemical composition analysis was performed on the powder obtained in (1) above.
Hexosamine content by MBTH method (Hurst & Settine (1981) Anal. Biochem., 115, 88-92), uronic acid content, sulfate ion content and neutral sugar content by carbazole method, ion chromatography and anthrone method, respectively. Was quantified.
[0038]
The weight abundance ratio of the constituents of this fraction was calculated from the analysis results, and the analysis results of sea cucumber GAG already reported (see Kariya et al. (1990) J. Biol. Chem., 265, 5081-5085). ).
[0039]
(3) Production of the substance of the present invention
40 mg of the purified fraction obtained in the above (1) having a composition almost equivalent to that of the known sea cucumber GAG was dissolved in 4 ml of 0.1N sulfuric acid and subjected to hydrolysis reaction at 80 ° C. for 3 hours or 6 hours, respectively. The reaction was stopped by cooling the reaction mixture to room temperature, neutralized by adding 1N sodium hydroxide, and then equilibrated with 0.2 M sodium chloride, a Cellulofine GCL-90m column (inner diameter 3.4 × long). 110 cm, manufactured by Seikagaku Corporation), and the eluate was collected every 10 ml (hereinafter obtained by hydrolysis for 3 hours, obtained by hydrolysis for 3 hours, hydrolysis for 6 hours) This is called a 6-hour hydrolyzate). As a control, 40 mg of unhydrolyzed product (the purified fraction obtained in the above (1), hereinafter also referred to as non-hydrolyzed product) was similarly subjected to column chromatography, and 10 ml of the eluate was collected.
[0040]
The uronic acid content and neutral sugar content were measured for each fraction of the column eluate of unhydrolyzed, 3-hour hydrolyzed and 6-hour hydrolyzed, and the behavior of the eluate was compared (FIG. 1). The obtained high molecular fraction (3-hour hydrolyzate (FIG. 1b) is fraction No. 40-54, 6-hour hydrolyzate (FIG. 1c) is fraction No. 40-58, and unhydrolyzed product (FIG. 1a) is fraction. No. 40-50) were concentrated together and desalted by attaching to a Cellulofine GCL-25 column (inner diameter 2.0 × length 25 cm, manufactured by Seikagaku Corporation) equilibrated with distilled water. Then, it was dried and collected by freeze-drying. The yield of each polymer fraction of unhydrolyzed product, 3 hour hydrolyzate and 6 hour hydrolyzate was 34.7 mg, 18.1 mg and 15 mg, respectively.
[0041]
Example 2 Analysis of physicochemical properties of the substance of the present invention
(1) Gel filtration elution pattern
Comparing the gel filtration elution patterns in Fig. 1, the unhydrolyzed product (Fig. 1 (a)) is a single peak with a shape equal to the position where the peaks of uronic acid and neutral sugar almost completely coincided with the high molecular fraction. Observed. However, in the 3-hour hydrolyzate (Fig. 1 (b)), the uronic acid peak in the corresponding polymer fraction is slightly reduced and slightly broadened compared to the unhydrolyzed pattern, and the neutral sugar peak in the same region. The detected value of was significantly reduced, while a new peak of neutral sugar appeared in the low molecular fraction. This peak is considered to be a sulfated fucobiosyl group eliminated by hydrolysis treatment. Similarly, in the 6-hour hydrolyzate (FIG. 1 (c)), the new neutral sugar peak in the low molecular fraction area is more prominent, and the decrease in the uronic acid peak and the broader flatness in the high molecular fraction area. And a marked decrease in the neutral sugar peak was confirmed.
[0042]
This suggests that the sulfated fucobiosyl group is detached from the raw sea cucumber-derived GAG main chain as the hydrolysis reaction proceeds, depending on the hydrolysis reaction time.
[0043]
Incidentally, GAG having a structure in which a sulfated fucobiosyl group is partially eliminated as in the case of hydrolyzate for 3 hours has not been reported so far and is a novel sulfated fucobiosylchondroitin sulfate.
[0044]
(2) Chemical composition analysis
The chemical composition analysis described in Example 1 (2) was performed on the 3-hour hydrolyzate, the 6-hour hydrolyzate, and the non-hydrolyzed product, and the weight abundance ratio of the components of each hydrolyzate obtained as a result was calculated (Table). 1). Further, the molar abundance (mmol / g) of each component was calculated, and the molar ratio of each component was calculated with the molar abundance of hexosamine as 1 (Table 2).
[0045]
[Table 2]

[0046]
[Table 3]
Table 2 Molar abundance of components of each hydrolyzate
(Molar ratio of each component when the molar abundance of GalNAc is 1 for each hydrolyzate)

[0047]
From Table 1 and Table 2, in each hydrolyzate, the molar ratio of hexosamine and uronic acid is almost constant, and the structure of the main chain portion retains the GAG-specific disaccharide repeat structure even after hydrolysis. it is conceivable that. The content of fucobiosyl and sulfate groups per residue of the disaccharide unit in the main chain is 1.19 and 3.69 molecules of unhydrolyzed product, respectively, while 0.29 and 1.69 for 3-hour hydrolyzed product, respectively. It was found that the number was 70 molecules, and the 6 hour hydrolyzate decreased to 0.18 and 1.06 molecules, respectively, depending on the hydrolysis reaction time. This supports the result of the gel filtration elution pattern (FIG. 1) of Example 2 (1) above, and it is considered that the sulfated fucobiosyl group is eliminated depending on the hydrolysis reaction time.
[0048]
(3) Debranching degree
The elimination rate of the sulfated fucobiosyl group by the hydrolysis reaction was calculated as the degree of debranching. Using the absorbance of the anthrone method in FIG. 1 as an index, the degree of debranching was estimated using the following formula from the detected peak area of the high molecular region and the peak area of the low molecular region. Microsoft Excel 2000 was used to draw the graph, and NIH Image 1.62 was used to calculate the area.
[0049]
Debranching degree (%) = Low molecular area peak area (A₂) / Total peak area (A₁+ A₂) × 100
[0050]
As a result of the calculation, the debranching degrees of the unhydrolyzed product, the 3-hour hydrolyzate and the 6-hour hydrolyzate were calculated as 0%, 67.6% and 90.4%, respectively. Also from this result, it is confirmed that the debranching reaction by hydrolysis proceeds in a reaction time-dependent manner. The degree of branching (%) = 100− (degree of debranching).
[0051]
(4) Chondroitinase ABC digestion
High-speed digestion of unhydrolyzed, 3-hour hydrolyzed, and 6-hour hydrolyzed products by chondroitinase ABC (Seikagaku Corporation, hereinafter referred to as C-ABC) using gel filtration chromatography as a separation factor It was measured by liquid chromatography (hereinafter referred to as GPC-HPLC).
[0052]
Each hydrolyzate is dissolved in distilled water so as to be 10 mg / ml, 20 μl of which is 10 μl of an enzyme solution containing 0.5 U of C-ABC (0.4 M sodium acetate, 0.1% bovine serum albumin containing 0. 4M Tris-HCl buffer (pH 8.0)), and an enzyme digestion reaction was performed at 37 ° C. for 18 hours. Distilled water (50 μl) was added to the reaction mixture, heated in boiling water for 1 minute, and then centrifuged to obtain a supernatant.
[0053]
The supernatant containing this C-ABC enzyme digest was added to TSK-Gel G4000PW._XL, TSK-Gel G3000PW_XLAnd TSK-Gel G2500PW_XLColumns (both 4.0 × 25 cm in inner diameter, manufactured by Tosoh) were attached to GPC-HPLC, which was connected in order from upstream, and eluted under isocratic conditions of 0.2 M sodium chloride solution, and the Refractive Index was used as an index. Detected as. Similarly, the HPLC chart was obtained also about each hydrolyzate before C-ABC digestion. These results are shown in FIG.
[0054]
  An estimate of C-ABC digestibility (unsaturated disaccharide production rate) of each hydrolyzate was calculated from the peak area of the obtained HPLC chart using the following formula. In the formula for calculating the unsaturated disaccharide production rate below, the total peak area means Δoligo, ΔDi-diS, ΔDi-monoS in A2, B2, and C2 (charts after C-ABC enzyme digestion) in FIG. And ΔDi-zeroS, and the sum of the unsaturated disaccharide peak areas is the sum of the areas of each unsaturated disaccharide peak (ΔDi-diS, ΔDi-monoS and△ Di-zeroSTotal peak area). Δoligo refers to an unsaturated oligosaccharide that is not decomposed to a disaccharide unit structure by C-ABC digestion and has a structure of trisaccharide or higher. ΔDi-diS is an unsaturated disaccharide having two sulfate groups in the unsaturated disaccharide unit structure, and ΔDi-monoS is an unsaturated having one sulfate group in the unsaturated disaccharide unit structure. Di-zero S indicates an unsaturated disaccharide having no sulfate group in the unsaturated disaccharide unit structure, and an unsaturated disaccharide standard to be described later is described as follows. Di (2,6) S and ΔDi-di (4,6) S are classified into ΔDi-monoS, ΔDi-6S and ΔDi-4S are classified, and ΔDi-zeroS is classified into ΔDi-0S. The
[0055]
Unsaturated disaccharide production rate (%) = sum of unsaturated disaccharide peak areas / total peak area × 100
[0056]
In the chart before enzymatic digestion, it was observed that the retention time of the main peak became longer as the hydrolysis reaction time became longer, and that some molecular weight reduction occurred. This is presumed to be the result of the elimination of the sulfated fucobiosyl side chain.
[0057]
When the chart after the enzymatic digestion of the unhydrolyzed product is compared with that before the enzymatic digestion, the retention time of the main peak is slightly longer, but hardly changes, and the unsaturated disaccharide component is not detected. That is, the digested trace was not seen and it was confirmed that it is not decomposed | disassembled by C-ABC similarly to description of a well-known literature.
On the other hand, in the 3-hour hydrolyzate and the 6-hour hydrolyzate, peaks of unsaturated disaccharide and unsaturated oligosaccharide were observed. For these peaks, the disaccharide production rate was calculated according to the above formula. As a result, the disaccharide production rates of the unhydrolyzed product, the 3-hour hydrolyzate and the 6-hour hydrolyzate were 0%, 46.5% and 66.3%, respectively.
[0058]
According to the reaction time of hydrolysis, the digestibility of C-ABC is increased and the production of unsaturated disaccharides is also increasing. From this, it is presumed that in CS having sulfated fucobiosyl group side chains, the existence of sulfated fucobiosyl group side chains constitutes a barrier to C-ABC enzyme digestion.
[0059]
  Furthermore, an unsaturated disaccharide analysis of the C-ABC enzyme digest was performed. About each C-ABC enzyme digest of each hydrolyzate, the supernatant was a strong anion exchange HPLC (hereinafter also referred to as SAX-HPLC) equipped with a YMC-PA120S5 column (inner diameter 4.0 cm × 25 cm, manufactured by YMC). Attached toMilliQElution was performed with a linear concentration gradient system (16 mM → 520 mM / 38 minutes) of Water (trade name: manufactured by Millipore) and 0.8 M sodium dihydrogen phosphate solution, and the absorbance was detected at 230 nm. The results are shown in FIG.
[0060]
Chondroitin sulfate type △ Di-0S, △ Di-6S, △ Di-4S, △ Di-di (2,6) S, △ Di-di (4,6) S and △ Di- as unsaturated disaccharide standards triS was used.
[0061]
These ΔDi-0S, ΔDi-6S, ΔDi-4S, ΔDi-di (2,6) S, ΔDi-di (4,6) S, and ΔDi-triS are the above-mentioned formulas 3 Each of the substituents in the disaccharide composition represented by is shown in the table below.
[0062]
[Table 4]

[0063]
From FIG. 3, the sum of the peaks of ΔDi-0S, ΔDi-6S, ΔDi-4S, ΔDi-di (2,6) S, ΔDi-di (4,6) S, and ΔDi-triS. On the other hand, in the 3-hour hydrolyzate, ΔDi-di (4,6) S was 52.2% and ΔDi-6S was 30.4%, whereas in the 6-hour hydrolyzate, ΔDi-6S Was 49.8% and ΔDi-di (4,6) S was 25.3%. Further, in both cases, ΔDi-di (2,6) S and ΔDi-triS were not observed, and peaks considered to be derived from some unsaturated oligosaccharides were observed. At first glance, the above results show that a part of ΔDi-6S observed in 6-hour hydrolyzate is a part of 4-O-sulfate group of ΔDi-di (4,6) S present in 3-hour hydrolyzate. Presumably caused by hydrolysis.
[0064]
  On the other hand, however, the rate of elimination by hydrolysis of the sulfated fucobiosyl group extending from the O-4 position of Gal-6 of Di-6S was slow, and O-3 of GlcA of ΔDi-di (4,6) S Since the elimination rate due to hydrolysis of the sulfated fucobiosyl group extending from the position was high, apparently the elimination of 4-O-sulfuric acid of ΔDi-di (4,6) S was observed as described above. It is likely that it seemed to have occurred. In addition, in the chart after C-ABC digestion in FIG. 2, it is the ΔDi-diS component with respect to the total peak area.△ Di-di (4,6) SThe ratio of 2 is approximately equal to 28.8% for the 3-hour hydrolyzate and 29.7% for the 6-hour hydrolyzate, which supports the high possibility of the latter.
[0065]
This unsaturated disaccharide composition is a composition of a portion having a straight chain structure in which the sulfated fucobiosyl group side chain is eliminated and can be enzymatically digested, and this method is used for the disaccharide composition of the portion where the side chain is retained. Then you can not analyze.
[0066]
From the above, the 3-hour hydrolyzate, which is an example of the substance of the present invention, has a sulfated fucobiosyl group having a CS-E type disaccharide unit and bonded to the O-3 position of D-glucuronic acid. It can be said that this is a novel sulfated fucobiosylchondroitin sulfate having a partially sulfated fucobiosyl side chain.
[0067]
Example 3 Enhancement of fibrinolytic activity of the substance of the present invention via tissue plasminogen activator
Various concentrations of solutions were prepared for the unhydrolyzed product, the 3-hour hydrolyzed product, the 6-hour hydrolyzed product and the known sea cucumber GAG (derived from Stichopus japonicus, isolated and purified in 1997) prepared in Example 1, and 30 μl of the solution. And 0.48 μM plasminogen 50 μl, 7.5 nM single-stranded t-PA (hereinafter referred to as sct-PA) and 1.8 mM synthetic substrate S-2251 (HD-Val-L-Leu-L) -Lys-p-nitroanilide dihydrochloride (40 μl) was mixed in the well, then set in an automatic microtiter plate reader, heated to 37 ° C. and allowed to proceed at 405 nm every 15 seconds (control 492 nm) ) Was measured. The reaction was carried out with 50 mM Tris-HCl buffer (pH 7.4) containing 0.005% Tween 80.
[0068]
The initial rate of activation of plasminogen is determined by the absorption value A at 405 nm (control 492 nm) at time t._405-492And the slope of the plot of the square of time (A_405-492/ T²) For the acceleration rate, the initial speed in the absence of GAG was 1, and the relative ratio was shown as a potentiation factor. The above activity measurement was carried out at various GAG concentrations, and the results at the GAG concentration (1.56 μg / ml) showing the highest activity are shown in FIG.
[0069]
When enumerated in descending order of activity, the 3-hour hydrolyzate jumps out and shows high activity, followed by a much lower level of 6-hour hydrolyzate, known sea cucumber GAG, and non-hydrolyzed product. It was revealed that it has a very high action for enhancing fibrinolytic activity.
[0070]
【The invention's effect】
According to the present invention, there is provided a sulfated fucobiosylchondroitin sulfate having a partially partially sulfated fucobiosyl group side chain having a fibrinolytic activity enhancing action.
[0071]
[Brief description of the drawings]
FIG. 1 is a graph showing measurement of uronic acid content and neutral sugar content in each fraction of Cellulofine GCL-90m column eluate of unhydrolyzed product, 3-hour hydrolyzed product and 6-hour hydrolyzed product. The horizontal axis is the fraction number. (a), (b), and (c) are the results of unhydrolyzed product, 3-hour hydrolyzed product, and 6-hour hydrolyzed product, respectively.
FIG. 2 is a GCP-HPLC chart before and after C-ABC digestion in unhydrolyzed product, 3 hour hydrolyzate, and 6 hour hydrolyzate, where the vertical axis indicates the differential refractive index and the horizontal axis indicates the retention time (minutes). A1 is a GCP-HPLC chart before C-ABC digestion of unhydrolyzed product, A2 is a GCP-HPLC chart after C-ABC digestion of unhydrolyzed product, and the disaccharide production rate is 0%. B1 is a GCP-HPLC chart before C-ABC digestion of the 3-hour hydrolyzate, and B2 is a GCP-HPLC chart after C-ABC digestion of the 3-hour hydrolyzate, and the disaccharide production rate is 46.5. %. C1 is a GCP-HPLC chart before C-ABC digestion of 6-hour hydrolyzate, C2 is a GCP-HPLC chart after 6-hour hydrolyzate C-ABC digestion, and the disaccharide production rate is 66.3. %.
FIG. 3 is a SAX-HPLC chart for unhydrolyzed, 3-hour hydrolyzed and 6-hour hydrolyzed products after digestion with C-ABC, wherein the vertical axis indicates absorbance and the horizontal axis indicates retention time (minutes). A, B, C and D are the results for unhydrolyzed, 3 hour hydrolysed, 6 hour hydrolysed and unsaturated disaccharide standards, respectively.
[Fig. 4] Unhydrolyzed, 3-hour hydrolyzed, 6-hour hydrolyzed by measuring the activation of glu-plg (plasminogen whose N-terminal amino acid begins with glutamic acid (Glu)) via sct-PA 1 shows the rate of fibrinolytic activity promotion through tissue plasminogen activator at 1.56 μg / ml concentration of the product and known sea cucumber GAG. The vertical axis represents the potentiation factor.

Claims (4)

A line containing D-glucuronic acid and N-acetyl-D-galactosamine as β-> 3-glycosidically linked disaccharide as a constituent unit and sulfated fucobiosylchondroitin sulfate derived from sea cucumbers with the following physicochemical properties as active ingredients Soluble enhancer.
i) It has a disaccharide structural unit structure represented by the following formula (1).

ii) Unsaturated disaccharide production rate is 35 to 55% by degradation with chondroitinase ABC.
iii) It contains 1.3 to 1.8 molecules of sulfate groups and 0.2 to 0.4 molecules of fucobiosyl groups per constituent disaccharide unit. The fibrinolytic activity enhancer according to claim 1, wherein the sulfated fucobiosylchondroitin sulfate has an average molecular weight of 11,000 to 13,000. The fibrinolytic activity enhancer according to claim 1 or 2, wherein the degree of branching of the sulfated fucobiosylchondroitin sulfate is 20 to 50%. The fibrinolytic activity enhancer according to any one of claims 1 to 3, wherein the fibrinolytic activity enhancer is derived from manamako.

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