JP2004210714A - Agent for treating bone disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a highly safe agent for treating bone diseases, usable for prophylaxis and therapy of a disease caused by inflammatory osteoclasia such as chronic rheumatoid arthritis and periodontal diseases, and metabolic bone diseases such as osteoporosis; and to obtain an agent for inhibiting the formation of osteoclast in vivo or in vitro. <P>SOLUTION: The agent for treating the bone diseases contains a glycoconjugate having sulfonated fucose in the structure and/or a pharmacologically acceptable salt thereof as an active ingredient. The agent for inhibiting the formation of the osteoclast contains the glycoconjugate having the sulfonated fucose in the structure and/or the pharmacologically acceptable salt thereof as an active ingredient. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００１９】
【表１】

【００２０】
つまり、本発明物質として用いられるＳＣ−ＧＡＧとしては、公知のグリコサミノグリカンに側鎖として硫酸化フコビオシル基が結合したものが挙げられる。中でも特に、Ｄ−グルクロン酸（以下、ＧｌｃＡとも言う）のＯ−１位とＮ−アセチル−Ｄ−ガラクトサミン（以下、ＧａｌＮＡｃとも言う）のＯ−３位とがβ−グリコシド結合したコンドロイチン硫酸タイプの二糖単位の繰り返し構造を主鎖の基本骨格とし、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の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、硫酸化フコビオシル基を側鎖として有する分岐構造を持ったコンドロイチン硫酸タイプのグリコサミノグリカン（以下、ＳＣ−ＣＳとも言う）が好ましく挙げられる。
【００２１】
ＳＣ−ＧＡＧは、１つの硫酸化フコビオシル基側鎖を有する構成二糖単位でほぼ構成されていても、または、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て構成されていても良い。つまり、部分的に硫酸化フコビオシル基側鎖を有するグリコサミノグリカンでも構わない。
【００２２】
例えば、ＳＣ−ＣＳにおいては、主鎖の構成二糖単位１残基あたり平均０．１分子以上のフコビオシル基を含有しているのが好ましく、更には平均０．２分子以上含有しているとより好ましく、更には平均０．４分子以上含有しているとより好ましく、平均０．８分子以上含有していると最も好ましい。（尚、本明細書においては、部分的に硫酸化フコビオシル基側鎖を有するコンドロイチン硫酸タイプのグリコサミノグリカンを部分的ＳＣ−ＣＳと言い、構成二糖単位１残基に硫酸化フコビオシル基側鎖をほぼ１分子有する構成二糖単位から成るコンドロイチン硫酸タイプのグリコサミノグリカンをＮＳＣ−ＣＳとも言う。）
【００２３】
ＳＣ―ＧＡＧの平均分子量は、２，０００〜５０，０００であることが好ましく、平均分子量３，０００〜３０，０００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、グリコサミノグリカンの平均分子量は、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。
【００２４】
本発明のＳＣ−ＧＡＧは、通常のグリコサミノグリカンと同様にグリコサミノグリカン部分にも硫酸基を有している。例えば、ＳＣ−ＣＳにおいては、主鎖の構成二糖単位１残基に対する硫酸基の割合は側鎖の硫酸化フコビオシル基の硫酸含量と合わせて平均１．０〜４．０分子が好ましく、更には平均１．３〜３．８分子であるとより好ましい。
【００２５】
また、部分的ＳＣ−ＣＳは後述の試験例に記載のコンドロイチナーゼＡＢＣ（以下、Ｃ−ＡＢＣとも言う）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ともいう）による分析を組み合わせた二糖組成分析において硫酸化フコビオシル基の結合量に反比例してウロン酸１分子とヘキソサミン１分子から成る不飽和二糖が検出されるが、この分析法における不飽和二糖の生成率が０〜７５％が好ましく、０〜５０％であるとより好ましい。尚、ＮＳＣ−ＣＳはこの分析方法にて不飽和二糖生成率０％である。つまり、本発明物質はＣ−ＡＢＣで分解されにくい硫酸化ＧＡＧであることがより好ましい。
【００２６】
本発明物質は、その起源、由来、製法によって特に限定されるものでは無く、本発明物質が有する特性を満たすものであれば、天然資源から抽出、精製したものでも、また、天然資源から抽出され精製して得られた物質を原料として化学的手法等により改変したもの、また、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。
【００２７】
例えば、ＳＣ−ＧＡＧは、天然資源から得られる硫酸化フコビオシル基を側鎖として有するグリコサミノグリカンでも、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グリコサミノグリカンを原料として部分的に硫酸化フコビオシル基を除去するなどの化学的手法等により改変したもの、及び、天然資源から得られるグリコサミノグリカン（以下、ＧＡＧともいう）を原料として化学的手法等により硫酸化フコビオシル基側鎖を導入したもの等、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。ＳＣ−ＧＡＧを天然資源から単離精製する場合や天然資源から単離精製された硫酸化フコビオシルグリコサミノグリカンを原料として製造する場合に用いる天然資源としては、種、属など特に限定されないが、例えば、棘皮動物が挙げられる。棘皮動物の中でも、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。
【００２８】
また、本発明物質として用いられるポリ硫酸化フコースを天然資源から単離精製する場合に用いる原料としても、一例として上記と同様のナマコ類が挙げられる。尚、現在、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。
【００２９】
上記の天然資源から硫酸化フコビオシルグリコサミノグリカン等のＧＡＧ類やポリ硫酸化フコースを単離・精製する方法は、例えば、Ｃａｒｂｏｈｙｄｒ． Ｒｅｓ．， ２９７， ２７３−２７９ （１９９７）や特公平６−７００８５や後述の参考例に記載の方法を用いることが可能であるが、ＧＡＧ類等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を用いる事も可能である。
【００３０】
また、側鎖である硫酸化フコビオシル基の部分的脱離方法としては、本発明物質が有する特性を満たすＳＣ−ＧＡＧが生成する条件での緩和な酸加水分解反応、アルカリ加水分解反応や酵素消化反応が挙げられるが、目的とする部分的ＳＣ−ＧＡＧが得られれば、これらに特に限定はされない。例えば、マナマコより単離・精製した硫酸化フコビオシルグリコサミノグリカンを原料として、硫酸化フコビオシル基の部分的脱離を行う場合は、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２〜６時間、酸加水分解を行い、反応後、中和し、分離・精製する方法が挙げられる。
【００３１】
本発明物質の薬理学的に許容されうる塩としては、本発明物質の有する破骨細胞形成を抑制する作用を失うこと無く、また、薬理学的に許容されうる塩であれば、特に限定されない。例えば、ナトリウム塩、カリウム塩、リチウム塩等のアルカリ金属塩、カルシウム塩等のアルカリ土類金属塩、アンモニウム塩等の無機塩基との塩、またジエタノールアミン塩、シクロヘキシルアミン塩、アミノ酸塩等の有機塩基との塩などのうち薬理学的に許容されるものが挙げられ、特にナトリウム塩が好ましい。
また、本発明物質の塩としては、本発明物質の有する破骨細胞の形成を抑制する作用を失わせることの無い塩であれば、特に限定されず、例えば、ナトリウム塩、カリウム塩、リチウム塩等のアルカリ金属塩、カルシウム塩等のアルカリ土類金属塩、アンモニウム塩等の無機塩基との塩、またジエタノールアミン塩、シクロヘキシルアミン塩、アミノ酸塩等の有機塩基との塩等が挙げられる。特にナトリウム塩が好ましい。
【００３２】
本発明物質は、後述の実施例に記載の通り、マウス骨髄細胞を用いた破骨細胞形成の実験系において、破骨細胞の形成を有意に抑制する。これより、本発明物質は骨粗鬆症に代表される代謝性骨疾患やリウマチ、歯周病の様な炎症性骨疾患等、特に破骨細胞の機能が異常に亢進することにより引き起こされる疾患の予防と治療に用いることが出来る。
【００３３】
本発明物質を上記疾患の予防や治療に用いる場合には、本発明物質の有する作用を実質的に損なわず、又、投与対象に対し悪影響を示さない限りにおいて、他の薬効成分や製剤時に通常用いられる賦形剤、結合剤、保存剤、安定化剤などを適宜用いる事が可能である。本発明物質を含有する骨疾患治療剤の剤型及び投与経路としては、錠剤、カプセル剤、顆粒剤、散剤、注射剤、液剤、リポ化剤、ゲル化剤、軟膏剤等に製剤化して、経口、注射、塗布等の投与方法が考えられるが、治療対象となる疾患の性質や重篤度に応じて適宜選択することが可能である。
【００３４】
尚、本発明物質を含有する骨疾患治療剤の投与量は、その製剤の投与方法、投与形態、投与対象患者の体重や具体的症状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、本発明物質として１日当たり概ね０．１ｍｇ／ｋｇ〜３００ｍｇ／ｋｇ程度を、１日１回〜数回に分けて投与する事が考えられる。
更に、上述の本発明物質は、破骨細胞形成抑制剤としてｉｎ ｖｉｖｏ及びｉｎ ｖｉｔｒｏの系でも用いる事が出来る。
【００３５】
【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。
【００３６】
参考例１ ＮＳＣ−ＣＳの製造
マナマコ（Ｓｔｉｃｈｏｐｕｓ ｊａｐｏｎｉｕｓ）の体壁部より、ＧＡＧ類を生体組織から単離・精製するために用いられる公知の方法（Ｋａｒｉｙａ ｅｔ ａｌ． （１９９０） Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２６５， ５０８１−５０８５）にて、硫酸化フコビオシルグリコサミノグリカン（ＮＳＣ−ＣＳ）の凍結乾燥粉体２１０ｍｇを得た。
【００３７】
具体的には、原料のマナマコ（Ｓｔｉｃｈｏｐｕｓ ｊａｐｏｎｉｕｓ）の体壁背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、１２０℃、３０分間のオートクレーブ処理に付し、５０ｍｍｏｌ／ｌリン酸緩衝液（ｐＨ８．０）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の共存蛋白質を消化した。酵素消化物を０．４ｍｏｌ／ｌ水酸化ナトリウム、次いで１０％トリクロロ酢酸で順次処理した後、遠心分離（５，０００ｘｇ、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％酢酸ナトリウムを含む冷エタノールを透析内液量の３倍量を添加する事により目的物質を沈殿させ、遠心分離（５，０００ｘｇ、１５分）により沈殿を回収した。回収し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。
【００３８】
得られた粗精製物１．０ｇを少量の５０ｍｍｏｌ／ｌ炭酸水素アンモニウム緩衝液（ｐＨ８．０）に溶解し、その半分量を同溶媒で平衡化したＳｅｐｈａｄｅｘ Ｇ−１００カラム（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅ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紫外部（ＵＶ）２１０ｎｍ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。その結果、高分子画分にグリコサミノグリカンと目されるカルバゾール反応陽性の画分を見出したので、これを合一回収し、凍結乾燥により粉末（２２４ｍｇ）を得た。得られた粗精製物の溶液の残り半分量についても同様の操作を行い、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（ｐＨ５．０）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。
【００３９】
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り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０．６ｍｏｌ／ｌ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回収し、流水透析、次いで、凍結乾燥を行い、ＮＳＣ―ＣＳである粉体（２１０ｍｇ）を得た。
【００４０】
ここで得られたＮＳＣ−ＣＳにつき、化学組成分析法による分析を行った。即ち、ＭＢＴＨ法（Ｈｕｒｓｔ ＆ Ｓｅｔｔｉｎｅ（１９８１） Ａｎａｌ．Ｂｉｏｃｈｅｍ．，１１５，８８−９２）にてヘキソサミン含量を測定し、カルバゾール法（Ｂｉｔｔｅｒ＆Ｍｕｉｒ（１９６２） Ａｎａｌ．Ｂｉｏｃｈｅｍ．，４，３３０−３３４）、イオンクロマトグラフィー並びにアンスロン法（Ｄｉｍｌｅｒ，Ｒ．Ｌ ｅｔ ａｌ．（１９５２） Ａｎａｌ．Ｃｅｈｍ．，２４，１４１１−１４１４）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測定した。
【００４１】
測定結果より、得られたＮＳＣ−ＣＳ構成成分の重量存在比を算出したところ（表１に示す）、公知のナマコ由来グリコサミノグリカン（Ｋａｒｉｙａ ｅｔ ａｌ． （１９９０） Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２６５， ５０８１−５０８５）とほぼ同等の組成を示した。この公知のナマコ由来グリコサミノグリカンの構造は、Ｄ−グルクロン酸とＮ−アセチル−Ｄ−ガラクトサミンがβ１→３グリコシド結合したコンドロイチンの基本骨格を有するものであり、Ｎ−アセチル−Ｄ−ガラクトサミンのＯ−４位かつ／またはＯ−６位、更にはＤ−グルクロン酸のＯ−３位に硫酸化フコース２残基よりなるフコース側鎖（硫酸化フコビオシル基）がグリコシド結合した構造を有していることが明らかにされており（Ｋａｒｉｙａ ｅｔ ａｌ．（１９９７） Ｃａｒｂｏｈｙｄｒ．ｒｅｓ．，２９７，２７３−２７９）、本参考例で製造したＮＳＣ−ＣＳも同様の構造であると推測される。
【００４２】
参考例２ 部分的ＳＣ−ＣＳの製造
上記で得られたＮＳＣ−ＣＳを原料として、側鎖である硫酸化フコビオシル基の部分的除去を行い、部分的ＳＣ−ＣＳを製造した。つまり、上記で得られたＮＳＣ−ＣＳ４０ｍｇを０．０５Ｍ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冷却することによって停止させ、１Ｎ水酸化ナトリウム水溶液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３．４×１１０ｃｍ；生化学工業（株）製）に付し、溶出液を１０ｍＬ毎に分取した。また、上記方法で得られたＮＳＣ−ＣＳ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（以下、３時間の加水分解により得られたものを３時間水解部分的ＳＣ−ＣＳ、６時間の加水分解により得られたものを６時間水解部分的ＳＣ−ＣＳとも言う）。
【００４３】
各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）。各カラム溶出液毎、図１に基づき、グリコサミノグリカン画分であるフラクションを合一濃縮し、更に、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）販売）に付し脱塩した後、凍結乾燥処理によって乾燥・回収した。収量は、ＮＳＣ−ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳで、各３４．７ｍｇ、１８．１ｍｇおよび１５ｍｇであった。
【００４４】
ＮＳＣ−ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳの各ゲル濾過溶出パターン（図１ａ、図１ｂ、図１ｃ）を分析すると、加水分解を行わないＮＳＣ−ＣＳについては、高分子画分領域にウロン酸及び中性糖のピークがほぼ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時間水解部分的ＳＣ−ＣＳでは、ＮＳＣ−ＣＳのパターンの中性糖ピークと対応する高分子画分領域の中性糖ピークは大幅に減少し、低分子画分域に中性糖の新しいピークが検出され、６時間水解部分的ＳＣ−ＣＳにおいては、この中性糖の新しいピークは更に顕著に検出されている。
【００４５】
これより、加水分解反応の進行に伴い、ＮＳＣ−ＣＳから側鎖である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。
【００４６】
参考例１と同様に、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳについて、化学組成分析法を行った。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（Ｂｉｔｔｅｒ＆Ｍｕｉｒ（１９６２） Ａｎａｌ．Ｂｉｏｃｈｅｍ．，４，３３０−３３４）、イオンクロマトグラフィー並びにアンスロン法（Ｄｉｍｌｅｒ，Ｒ．Ｌ ｅｔ ａｌ．（１９５２） Ａｎａｌ．Ｃｅｈｍ．，２４，１４１１−１４１４）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測定し、各構成成分の重量存在比を算出した（表２）。更に、各構成成分のモル存在量（ｍｍｏｌ／ｇ）を算出し、ヘキソサミンを１として各成分のモル比を算出し、表３に示した。
【００４７】
【表２】
表２ 各試料の構成成分の重量存在比（Ｗ／Ｗ， ％）

【００４８】
【表３】
表３ 各試料の構成成分のモル存在量（ｍｍｏｌ／ｇ）
（カッコ内の数値はヘキソサミンのモル存在量を１とした場合の各成分のモル比を示す）

【００４９】
３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳいずれもヘキソサミンとウロン酸のモル比がほぼ一定であり、硫酸化フコビオシル基を脱離させる加水分解反応においてもＧＡＧの直鎖基本構造部分は保持されていると推測される。また、３時間水解部分的ＳＣ−ＣＳでは構成二糖単位１残基あたりフコビオシル基及び硫酸基が各々０．２９分子及び１．７０分子存在するのに対し、６時間水解部分的ＳＣ−ＣＳでは構成二糖単位１残基あたりフコビオシル基及び硫酸基が各々０．１８分子及び１．０６分子であり、加水分解反応時間依存的に減少しており、上述のゲル濾過溶出パターンの結果を裏付けている。
【００５０】
参考例３ コンドロイチナーゼＡＢＣによる分解
ＮＳＣ−ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳ各々についてＣ−ＡＢＣによる消化の度合いをゲル濾過を分離要因とするＨＰＬＣ（以下、ＧＣＰ−ＨＰＬＣとも言う）により測定した。即ち、ＮＳＣ−ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳ各々を１０ｍｇ／ｍＬとなる様に蒸留水に溶解し、そのうち２０μＬを０．５ＵのＣ−ＡＢＣ（生化学工業（株）製）を含む酵素溶液１０μＬ（０．４Ｍ酢酸ナトリウム、０．１％ウシ血清アルブミンを含む０．４Ｍトリス塩酸緩衝液（ｐＨ８．０））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分間加熱後、遠心分離することにより上清を得た。
【００５１】
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、０．２ｍｏｌ／ｌ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、ＨＰＬＣチャートを得た。（図２）
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被験物の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のＣ−ＡＢＣ酵素消化後の各チャートにおける、△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△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四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持たない不飽和二糖を示す（Ａｎａｌ． Ｂｉｏｃｈｅｍ．， １７７， ３２７−３３２ （１９８９） ｐ．３２８ Ｆｉｇ．１参照）。
【００５２】
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
ＮＳＣ−ＣＳは不飽和二糖成分は検出されず、Ｃ−ＡＢＣにより分解されていないことが確認された。これは、ナマコ由来グリコサミノグリカンに関する公知文献の記載と同様である。また、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳは不飽和二糖及び不飽和オリゴ糖のピークが観察され、上記式に従い算出される不飽和二糖生成率（％）は各々４６．５％及び６６．３％であった。
【００５３】
参考例４ ポリ硫酸化フコースの製造
ナマコ体壁背部（１０ｋｇ）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）抽出により脂溶性画分を得た。この画分をロータリーエバポレーターを用い減圧下で濃縮・乾固させた。得られた乾固物に蒸留水（３００ｍＬ）を加えて水溶液とした後、４℃にて一晩放置した。次いで、表層に浮遊した脂肪を除去した水溶液に対し冷却下にて３倍量のエタノールを添加し、遠心分離にかけ沈殿物を得た。この沈殿物を少量の蒸留水に溶解した後、凍結乾燥を行い、低極性と目される多糖類の粗画分（２．４６ｇ）を得た。
【００５４】
この粗画分から１ｇを取り、０．２ｍｏｌ／ｌ塩化ナトリウム水溶液７ｍＬに溶解し、０．２ｍｏｌ／ｌ塩化ナトリウム水溶液で平衡化したＳｅｐｈａｒｏｓｅ ＣＬ−６Ｂカラム（内径３．４×１１０ｃｍ、Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ社製）に付した。０．２Ｍ塩化ナトリウム水溶液を用いて溶出を行い、１２ｍＬ毎に分画した。得られた１００画分につき、アンスロン法による比色定量（６２０ｎｍで測定）及び２１０ｎｍ、２８０ｎｍの両波長における吸光度の測定を行い、溶出曲線を描いた（図３）。
【００５５】
アンスロン法が陽性であった画分番号４２〜６９を合一した後、ロータリーエバポレーターにて１０ｍＬまで濃縮した。濃縮した溶液を蒸留水で平衡化したＣｅｌｌｕｌｏｆｉｎｅ ＧＣＬ−２５カラム（内径３．３×３６ｃｍ、生化学工業（株）販売）に付し、蒸留水による溶出にて１０ｍＬ毎に分画した。得られた４０画分につき、アンスロン法による比色定量（６２０ｎｍ）及び２１０ｎｍの波長における吸光度の測定を行い、溶出曲線を描いた。溶出曲線より脱塩が達成している事を確認し、目的物質を含む画分である画分番号１１〜２０を合一して凍結乾燥を行い、中性糖を含む多糖と目される乾燥物を得た（収量１３５．４ｍｇ）。
【００５６】
更に精製する為、得られた乾燥物１００ｍｇを０．１ｍｏｌ／ｌの酢酸ナトリウム／酢酸緩衝液（ｐＨ５．０）５０ｍｌに溶解し、同緩衝液で平衡化したＤＥＡＥ−Ｃｅｌｌｕｌｏｓｅ（ＤＥ５２、Ｗｈａｔｍａｎｎ社製）カラム（内径２．３×１８ｃｍ）に付し、同緩衝液５０ｍｌでカラムを洗浄後、同緩衝液３００ｍＬと１．２Ｍ塩化ナトリウムを含む同緩衝液３００ｍＬを用いた塩化ナトリウムの直線的濃度勾配により溶出を行い７ｍＬ毎に分画した。得られた１００画分につき、アンスロン法による比色定量（６２０ｎｍ）及び２８０ｎｍにおける吸光度を測定し、溶出曲線を描いた（図４）。その結果、画分番号７５〜８１と画分番号８２〜９４にピーク（各々ピーク１、ピーク２と言う）が出現した。各々のピーク毎に合一し、ピーク１に由来する画分（以下、ポリ硫酸化フコースＩとも言う、収量１４．２ｍｇ）とピーク２に由来する画分（以下、ポリ硫酸化フコースＩＩとも言う、収量３３．６ｍｇ）を得た。
【００５７】
参考例５ ポリ硫酸化フコースの物理化学的性状解析
参考例４で製造したポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩにつき、参考例１、２と同様に、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（Ｂｉｔｔｅｒ＆Ｍｕｉｒ（１９６２） Ａｎａｌ．Ｂｉｏｃｈｅｍ．，４，３３０−３３４）、イオンクロマトグラフィー並びにアンスロン法（Ｄｉｍｌｅｒ，Ｒ．Ｌ ｅｔ ａｌ．（１９５２） Ａｎａｌ．Ｃｅｈｍ．，２４，１４１１−１４１４）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測定し、各構成成分の重量存在比を算出した（表４）。また、各構成成分のモル存在量（ｍｍｏｌ／ｇ）を算出し、中性糖を１として各成分のモル比を算出した（表５）。
次いで、ゲル濾過―高速液体クロマトグラフィーにより分子量の測定を行った。その結果、ポリ硫酸化フコースＩの分子量は９，０００、ポリ硫酸化フコースＩＩの分子量は３２，０００であった。
【００５８】
【表４】
表４ 各構成成分の重量存在比（重量％）

【００５９】
【表５】
表５ 各構成成分のモル存在量（ｍｍｏｌ／ｇ）
（カッコ内の数値は中性糖含量のモル存在量を１とした場合の各構成成分のモル比を示す）

【００６０】
この結果、ポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩ共に、硫酸基と中性糖は含有しているが、ヘキソサミンを含有せず、また、ウロン酸も痕跡量のみであり実質的には含有していないと推測され、中性糖を側鎖として有するグリコサミノグリカンでは無いことが確認された。また、ポリ硫酸化フコースＩ及びＩＩの中性糖に対する硫酸基の割合は各々０．６９及び０．７９であり、硫酸化の割合は高いことが判明した。
【００６１】
更に、Ｙａｓｕｎｏ，Ｓ． ｅｔ ａｌ．（１９９９）Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈｎｏｌ．Ｂｉｏｃｈｅｍ．，６３，１３５３−１３５９に記載された方法に従い、中性糖の同定を行った。すなわち、１０ｍｇ／ｍＬに調製したポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩの各水溶液から各々５０μＬを取り、８ｍｏｌ／Ｌトリフルオロ酢酸（ＴＦＡ）５０μＬを各々に添加し封管した。反応混液を１００℃にて３時間加熱することにより、グリコシド結合ならびに硫酸エステル結合を開裂させた。冷却後、遠心分離により反応混液を反応管底部に集め、開管した。ドライエアーを吹き付け溶媒を除去し、２−プロパノール４０μＬをそれぞれに添加し攪拌した。更に、ドライエアーを吹きつけ溶媒を除去することにより、単糖試料を得た。得られた単糖試料につき、２０μＬの蒸留水及び８０μＬの４−アミノ安息香酸エチルエステル（ＡＢＥＥ）試薬を添加した後、封管し、８０℃にて１時間加温し還元アミノ化反応を進行させ、還元末端がＡＢＥＥにて標識された単糖誘導体を作成した。反応終了後、開管し２００μＬの蒸留水並びに２００μＬの塩化メチレンを添加し、激しく攪拌した後、遠心分離を行い上澄み中に還元末端がＡＢＥＥにて標識された上記単糖誘導体を得た。７％アセトニトリルを含む０．２ｍｏｌ／ｌホウ酸緩衝液（ｐＨ８．９）で平衡化したＨｏｎｅｎｐａｋ−Ｃ１８カラム（内径４．６ × ７５ｍｍ、Ｈｏｎｅｎ社製）を装着した高速液体クロマトグラフィーに、得られたＡＢＥＥ標識化単糖試料を付し、同溶媒のアイソクラティック溶出条件により分析を実施した。尚、用いた単糖標品は、ガラクトース（Ｇａｌ）、マンノース（Ｍａｎ）、グルコース（Ｇｌｕ）、アラビノース（Ａｒａ）、リボース（Ｒｉｂ）、Ｎ−アセチルマンノサミン（ＭａｎＮＡｃ）、キシロース（Ｘｙｌ）、Ｎ−アセチルグルコサミン（ＧｌｃＮＡｃ）、フコース（Ｆｕｃ）、ラムノース（Ｒｈａ）、及び、Ｎ−アセチルガラクトサミン（ＧａｌＮＡｃ）（溶出順）である（図５）。この結果、参考例４で得られたポリ硫酸化フコースＩ、ポリ硫酸化フコースＩＩ共に含有する単糖はフコースであることが判明した。尚、精製当初、硫酸化ポリフコースＩ及びＩＩが脂溶性画分であった理由としては、硫酸化されてはいるが、低極性のメチル基を有するフコース残基を構成成分とする為と考えられる。
【００６２】
参考例６ ポリ硫酸化フコースの構造解析
参考例４で得られたポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩから各５ｍｇをとり、それぞれを蒸留水で平衡化したＡｍｂｅｒｌｉｔｅ ＩＲ−１２０カラム（オルガノ（株）社販売、内径１．５×１２ｃｍ）に供した。得られた溶出画分のうちの酸性画分につき、適量のピリジンを添加して中和した後、凍結乾燥に伏してポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩのピリジン塩を得た。それぞれにつき、１ｍｌの１０％蒸留水を含むジメチルスルホキシド（ＤＭＳＯ）を添加し、９０℃にて６日間ソルボリシス反応を進行させた。反応は、室温まで冷却することにより停止させ、３倍量の蒸留水を添加した後、２ｍｏｌ／ｌ水酸化ナトリウム水溶液を適量添加してｐＨを９〜９．５に調整した。これを流水に対し一晩透析した後、凍結乾燥することにより、ポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩの脱硫酸化物をそれぞれ３．０ｍｇ及び３．３ｍｇ得た。
【００６３】
ポリ硫酸化フコースＩ及びその脱硫酸化物、さらにポリ硫酸化フコースＩＩ及びその脱硫酸化物の計４画分から、それぞれ１ｍｇをとりＨａｋｏｍｏｒｉの方法（Ｈａｋｏｍｏｒｉ， Ｓ．（１９６４） Ｊ． Ｂｉｏｃｈｅｍ． （Ｔｏｋｙｏ），５５， ２０５−２０８）を一部改変したＳａｎｆｏｒｄとＣｏｎｒａｄの方法（Ｓａｎｆｏｒｄ， Ｐ．Ａ． ＆ Ｃｏｎｒａｄ， Ｈ．Ｅ．（１９６６） Ｂｉｏｃｈｅｍｉｓｔｒｙ， ５， １５０８−１５１７）に従ってメチル化誘導体を合成した。得られたメチル化誘導体は、Ｗａｅｇｈｅらの方法（Ｗａｅｇｈｅ ｅｔ ａｌ．（１９８３） Ｃａｒｂｏｈｙｄｒ． Ｒｅｓ．， １２３， ２８１−３０４）に従って脱塩した。さらに、これらを２モル／Ｌトリフルオロ酢酸（ＴＦＡ）を用い１２１℃で加水分解した後、重水素化ホウ素ナトリウム（ＮａＢＤ_４）を用いて還元した。最後に、還元処理により出現した水酸基をアセチル化して、部分メチル化アルジトールアセテイト（ＰＭＡＡ）を合成した。得られたＰＭＡＡを５０μＬのアセトンに溶解し、そのうち１μＬをＧＣ−ＭＳに付した。ガスクロマトグラフィー−質量スペクトル（ＧＣ−ＭＳ）分析は、Ｓｕｐｅｌｃｏ社製ＳＰ２３３０キャピラリーカラム（内径０．２５ｍｍ×３０ｍ）を装着したＪＥＯＬ社製ＨＸ１１０Ａ型マススペクトロメーターを用いて、スプリットレスモードで行った。カラムオーブンは、５０℃に２分間保ち、次いで３０℃／分の勾配で１７０℃まで昇温、さらに４℃／分の勾配で２３５℃まで昇温させ、この温度に１５分間保った。
【００６４】
ポリ硫酸化フコースＩのクロマトグラムに於いては、８種類のＰＭＡＡが全て観察された（表６）。それらは、２，３，４−ｔｒｉ−Ｏ−ｍｅｔｈｙｌ−ｆｕｃｉｔｏｌ（Ｔ−Ｆｕｃ）、３，４−ｄｉ−Ｏ−ｍｅｔｈｙｌ−ｆｕｃｉｔｏｌ（２−Ｆｕｃ）、２，４−ｄｉ−Ｏ−ｍｅｔｈｙｌ−ｆｕｃｉｔｏｌ（３−Ｆｕｃ）、２，３−ｄｉ−Ｏ−ｍｅｔｈｙｌ−ｆｕｃｉｔｏｌ（４−Ｆｕｃ）、４−Ｏ−ｍｅｔｈｙｌ−ｆｕｃｉｔｏｌ（２，３−Ｆｕｃ）、３−Ｏ−ｍｅｔｈｙｌ−ｆｕｃｉｔｏｌ（２，４−Ｆｕｃ）、４−Ｏ−ｍｅｔｈｙｌ−ｆｕｃｉｔｏｌ（３，４−Ｆｕｃ）、ｆｕｃｉｔｏｌ（２，３，４−ｆｕｃｉｔｏｌ）である。以下の記載に於いては、上記（）内に示したＰＭＡＡの略称を用いて説明を進める。
【００６５】
【表６】
表６

【００６６】
ポリ硫酸化フコースＩの脱硫酸化物のクロマトグラムはポリ硫酸化フコースＩのクロマトグラムとは異なり、ほぼ等量のＴ−Ｆｕｃ、３−Ｆｕｃ及び３，４−Ｆｕｃの三種類が検出された。従って、ポリ硫酸化フコースＩの構造は、Ｆｕｃα１→３Ｆｕｃの主鎖から部分的にＦｕｃα１→４Ｆｕｃが枝分かれしていると考えられる（図６）。また、硫酸基が図示したような部位（主鎖においてはＯ−２位、側鎖においてはＯ−２位且つ／又はＯ−４位）に配位しているものと考えられた。
【００６７】
ポリ硫酸化フコースＩＩのクロマトグラムに於いては、表６に記載の通り、５種類のＰＭＡＡ（Ｔ−Ｆｕｃ、３−Ｆｕｃ、２，３−Ｆｕｃ、３，４−Ｆｕｃ及び２，３，４−Ｆｕｃ）が観察された。ポリ硫酸化フコースＩＩの脱硫酸化物のクロマトグラムはポリ硫酸化フコースＩＩのクロマトグラムとは異なり、少量のＴ−Ｆｕｃと著量の３−Ｆｕｃのみから成っていた、この結果より、ポリ硫酸化フコースＩＩの構造は、Ｆｕｃα１→３Ｆｕｃの直鎖構造をとり、部分的にＯ−２位且つ／又はＯ−４位の水酸基が硫酸化されていると思われる。
【００６８】
実施例１ ＳＣ−ＣＳの破骨細胞形成抑制実験
ｄｄＹマウス（六週齢雌）の頸骨、大腿骨を摘出し、両骨の遠心端より骨髄細胞を採取した。骨髄細胞は２４穴細胞培養用プレートに５×１０^６細胞／穴となるように播種し、１０^―６モル／ＬプロスタグランジンＥ_２（以下、ＰＧＥ_２という。ＳＩＧＭＡ社製）刺激下において、１、３、１０、３０、１００μｇ／ｍＬの濃度になる様に、参考例１で得られたＮＳＣ−ＣＳを添加し、１０％牛胎児血清（以下、ＦＣＳという。べーリンガー社製）含有Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ Ａｌｐｈａ Ｍｅｄｉｕｍ（ＧＩＢＣＯ社製、以下、αＭＥＭという。）培地中で７日間３７℃、ＣＯ_２インキュベーター内で培養した。培養中２、３日おきにＰＧＥ_２と上記各濃度のＮＳＣ−ＣＳを新たに添加した１０％ＦＣＳ含有αＭＥＭ培地にて培地交換を行った。７日間の培養の後、破骨細胞のマーカーである酒石酸抵抗性酸性フォスファターゼ（以下、ＴＲＡＰという）をナフトールＡＳ−ＢＩフォスフェート及びｆａｓｔ ｇａｒｎｅｔ ＧＢＣ ｓａｌｔを含有するアゾ色素法を用いた染色測定キット「Ａｃｉｄ Ｐｈｏｓｐｈａｔａｓｅ， Ｌｅｕｋｏｃｙｔｅ」（商品名：ＳＩＧＭＡ社製、以下、Ａｃｉｄ Ｐｈｏｓｐｈａｔａｓｅ， Ｌｅｕｋｏｃｙｔｅと言う）を用いて染色し、形成された破骨細胞の数を顕微鏡下で計測した。（図７）
図７の結果より、マウス骨髄細胞を用いた破骨細胞形成実験系において、ＮＳＣ−ＣＳが用量依存的に破骨細胞の形成を抑制した。１μｇ／ｍＬのＮＳＣ−ＧＡＧ濃度で破骨細胞の形成５０％阻害した。
【００６９】
実施例２ ＮＳＣ−ＣＳ並びに部分的ＳＣ−ＣＳの破骨細胞形成抑制実験
５０μｇ／ｍＬ濃度となるように、参考例１で得られたＮＳＣ−ＣＳ、参考例２で得られた３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳを用いるほかは、実施例１と同様に操作し、形成された破骨細胞の数を顕微鏡下で計測した。（図８）
図８の結果より、ＮＳＣ−ＣＳについては、有意な破骨細胞形成抑制効果が確認された。また、３時間水解部分的ＳＣ−ＣＳにおいても、破骨細胞形成抑制効果が認められた。これに比べて、６時間水解部分的ＳＣ−ＣＳの効果は弱かった。
【００７０】
硫酸化フコビオシル基側鎖を多く有するものほど、破骨細胞形成抑制効果は強く発揮された為、同効果の発現には、側鎖である硫酸化フコビオシル基を有する事が重要であると推察される。
【００７１】
実施例３ 硫酸化ポリフコースの破骨細胞形成抑制実験
５０μｇ／ｍＬ濃度となるように参考例１で得られたＮＳＣ−ＣＳ、参考例４で得られたポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩを用いるほかは、実施例１と同様に操作し、形成された破骨細胞の数を顕微鏡下で計測した。結果を図９に示す。尚、結果は各穴内における破骨細胞の数によって示し、また、コントロールは被験物質の代わりに１０％ＦＣＳ含有αＭＥＭ培地を添加した。
【００７２】
図９の結果より、ＮＳＣ−ＣＳと同様に、ポリ硫酸化フコースＩ、ポリ硫酸化フコースＩＩにおいても有意な破骨細胞形成抑制効果が確認された。
【００７３】
【発明の効果】
本発明により、硫酸化フコースを構造中に有する糖化合物を、骨粗鬆症などの代謝性骨疾患や炎症性骨破壊に起因するＲＡ、歯周病などの疾患に対する治療に有効な破骨細胞形成抑制効果を有する骨疾患治療剤として提供することができる。また、本発明はｉｎ ｖｉｖｏ及びｉｎ ｖｉｔｒｏにおいて破骨細胞形成抑制効果を有する破骨細胞形成抑制剤を提供する。
【００７４】
【図面の簡単な説明】
【図１】ＮＳＣ−ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳ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ＮＳＣ−ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳの結果である。
【図２】ＮＳＣ―ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳに於ける、コンドロイチナーゼＡＢＣ消化前後のＧＰＣ−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、ＮＳＣ−ＣＳのＣ−ＡＢＣ消化前のＧＰＣ−ＨＰＬＣチャートであり、Ａ２はＮＳＣ−ＣＳのＣ−ＡＢＣ消化後のＧＰＣ−ＨＰＬＣチャートであり、二糖組成率は０％である。Ｂ１は３時間水解部分的ＳＣ−ＣＳのＣ−ＡＢＣ消化前のＧＰＣ−ＨＰＬＣチャートであり、Ｂ２は３時間水解部分的ＳＣ−ＣＳのＣ−ＡＢＣ消化後のＧＰＣ−ＨＰＬＣチャートであり、二糖組成率は４６．５％である。Ｃ１は６時間水解部分的ＳＣ−ＣＳのＣ−ＡＢＣ消化前のＧＰＣ−ＨＰＬＣチャートであり、Ｃ２は６時間水解部分的ＳＣ−ＣＳのＣ−ＡＢＣ消化後のＧＰＣ−ＨＰＬＣチャートであり、二糖組成率は６６．３％である。
【図３】多糖類の粗画分のセファロースＣＬ−６Ｂカラム溶出液の各フラクションにおけるアンスロン法による比色定量（６２０ｎｍ）及び２１０ｎｍ、２８０ｎｍにおける吸光度を測定したグラフである。縦軸は吸光度、横軸はフラクション番号である。
【図４】ＤＥＡＥ−Ｃｅｌｌｕｌｏｓｅカラム溶出液の各フラクションにおけるアンスロン法による比色定量（６２０ｎｍ）及び２８０ｎｍにおける吸光度を測定したグラフである。縦軸は吸光度、横軸はフラクション番号である。
【図５】ポリ硫酸化フコースＩ及びポリ硫酸フコースＩＩから得られた還元末端が４−アミノ安息香酸エチルエステルにて標識された単糖誘導体のＨｏｎｅｎｐａｋ−Ｃ１８カラムを装着した高速液体クロマトグラフィーに付し、得られたチャートである。
尚、Ｅ１は、ポリ硫酸化フコースＩから得られたプロファイル、Ｅ２はポリ硫酸化フコースＩＩから得られたプロファイルである。
Ｓｔｄ．は単糖標品を付した結果であり、各ピークは溶出順にガラクトース（Ｇａｌ）、マンノース（Ｍａｎ）、グルコース（Ｇｌｕ）、アラビノース（Ａｒａ）、リボース（Ｒｉｂ）、Ｎ−アセチルマンノサミン（ＭａｎＮＡｃ）、キシロース（Ｘｙｌ）、Ｎ−アセチルグルコサミン（ＧｌｃＮＡｃ）、フコース（Ｆｕｃ）、ラムノース（Ｒｈａ）、及び、Ｎ−アセチルガラクトサミン（ＧａｌＮＡｃ）である。
【図６】Ｅ１は、ポリ硫酸化フコースＩ（ピーク１由来の画分）の推定構造であり、Ｅ２はポリ硫酸化フコースＩＩ（ピーク２由来の画分）の推定構造である。
【図７】ＮＳＣ−ＣＳの用量依存的な破骨細胞形成抑制作用を示すグラフである。縦軸はマイクロプレートの１穴における破骨細胞の数、横軸はＮＳＣ−ＣＳの添加濃度である。（−）はコントロールの結果を示す。
【図８】ＮＳＣ−ＣＳ、３時間水解部分的ＳＣ−ＣＳ及び６時間水解部分的ＳＣ−ＣＳによる破骨細胞形成抑制作用を示すグラフである。縦軸はマイクロプレートの１穴における破骨細胞の数である。（−）はコントロールの結果を示す。
【図９】ＮＳＣ−ＣＳ、ポリ硫酸化フコースＩ及びポリ硫酸化フコースＩＩによる破骨細胞形成抑制作用を示すグラフである。縦軸はマイクロプレートの１穴における破骨細胞の数である。（−）はコントロールの結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention provides a bone suitable for the prevention and treatment of metabolic bone disease, inflammatory bone disease, etc., comprising a sugar compound having sulfated fucose in the structure and / or a pharmacologically acceptable salt thereof as an active ingredient. The present invention relates to a disease therapeutic agent and an osteoclast formation inhibitor.
[0002]
[Prior art]
Bone tissue is a dynamic tissue in which bone metabolism consisting of bone resorption and bone formation is repeated. The balance between bone resorption and bone formation is strictly regulated by both osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption (Non-patent Document 1). Causes abnormalities and presents with various diseases.
[0003]
One example of a disease caused by abnormal bone resorption and bone formation is osteoporosis (Non-patent Document 2). Other examples include rheumatoid arthritis and periodontitis with inflammatory bone destruction. These bone diseases are thought to arise as a result of abnormally increased osteoclast function, and in this context, research on the formation of osteoclasts and the regulation of bone resorption by osteoclasts is actively conducted. Substances that specifically suppress the bone resorption process and osteoclast formation by osteoclasts are expected and studied as effective therapeutic agents for these bone diseases (Non-patent Document 3). .
[0004]
So far, reports on substances that have the effect of suppressing bone resorption based on inhibiting the release of bone-dissolving enzymes by osteoclasts and the acidification of the bone surface, and contain calcium salts of sulfated glycosaminoglycans The oral composition has an inhibitory effect on bone calcium ion release by endotoxin stimulation of periodontopathic bacteria (Patent Document 1) and bone metabolism improvement using sulfated glycosaminoglycan sodium and calcium compounds in combination Calcification promoter containing at least one selected from insulin, protamine, and glycosaminoglycan, wherein the agent has an inhibitory effect on bone calcium ion release by endotoxin, human parathyroid hormone, etc. There are reports of bone disease treatment agents (Patent Document 3), etc., which consist of bone and bone grafting materials, all of which are bone repairs such as calcium and bone grafting materials Effects are there when regarded in which substance is used in combination, not the effect of sulfated glycosaminoglycan alone. Furthermore, reports have been made on oral osteoporosis preventive and therapeutic agents (Patent Document 4) based on the calcium resorption rate and bone strength enhancing action by administration of chondroitin sulfate sodium salt, all of which are changed from blood stem cells to osteoclasts. There is no suggestion on acting on the oncoming differentiation process to prevent the formation of osteoclasts.
[0005]
The mechanism of osteoclast differentiation induction is activated vitamin D (1α, 25 (OH) 2D3), parathyroid hormone (PTH), interleukin 11 (IL11), interleukin 6 (IL6), TNFα, prostagland Gin E2 (PGE₂) And the like, and osteoclast differentiation factor (ODF) is expressed on the surface of osteoblasts by the action of bone resorption promoting factors such as) on the surface of osteoblasts (Non-patent Document 4). Receptor activator of NF-Κb (RANK), which is a receptor, is expressed, and it has been reported that the binding of ODF and RANK is necessary for the formation of osteoclasts (Non-patent Document 5). As an inhibitory system, soluble bone resorption inhibitory factor (OCIF) is produced from various cells, and it is reported that the formation of osteoclasts is suppressed by competitively inhibiting the binding between ODF and RANK. (Non-Patent Document 6).
[0006]
Furthermore, the mechanism of bone destruction in rheumatoid arthritis and periodontal disease is also being clarified. In particular, these bone diseases have been suggested to involve a large part of the immune system. ODF expression by activated T cells and It has been pointed out that this is caused by increased osteoclast formation (Non-patent Document 7). In addition, it has been suggested that a substance that blocks the binding between ODF and RANK may be an effective therapeutic agent for such bone diseases (Non-patent Document 8). However, Osteoprotegerin (OPG), which blocks the signal of ODF to RANK, has not yet been put on the market as a therapeutic agent.
[0007]
[Patent Document 1] JP-A-6-80546
[Patent Document 2] JP-A-7-53388
[Patent Document 3] JP-A-62-201825
[Patent Document 4] JP-A-7-109222
[Non-Patent Document 1] Chambers et al (1991) Vitamins Hormones 46, 41-86
[Non-Patent Document 2] Suda et alX (1992) Endocr. Rev. , 13, 66-80; Sudaet al (1996) In "Principles of Bone Biology (Bilezikian et al. Eds)" pp. 87-102
[Non-Patent Document 3] Moreland et al (1993) Am. J. et al. Med. Sci. 305 (1) 40-51; Mebio. , 11 (2), p. 24 (1994)
[Non-Patent Document 4] Yasuda et al (1998) Proc. Natl. Acad. Sci. USA 95, 3597-3602; Lacey et al (1998) Cell 93, 165-176.
[Non-Patent Document 5] Nakagawa et al (1998) Biochem. Biophys. Res. Commun, 253, 395-400
[Non-Patent Document 6] Tsuda et al. (1996) Biochemistry 68, 683; Tsuda et al. (1997) Biochem Biophys. Res. Commun 234, 137-142; Yasuda et al (1998) Endcrinology 139, 1329-1337.
[Non-Patent Document 7] Teng et al. (2000) J. Org. Clin. Invest, 106 (6), R59-R67; Kong et al (1999) Nature, 402 (6759), 304-309.
[Non-Patent Document 8] Simonet et al (1997), Cell, 89, 309-319
[0008]
[Problems to be solved by the invention]
Therefore, the present invention can be used for the prevention and treatment of diseases caused by inflammatory bone destruction such as rheumatoid arthritis and periodontal disease and metabolic bone diseases such as osteoporosis, and is a highly safe bone disease treatment agent. And an osteoclast formation inhibitor.
[0009]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a sugar compound having sulfated fucose in the structure has an effect of suppressing the formation of osteoclasts, thereby completing the present invention. Arrived.
[0010]
That is, the gist of the present invention is as follows. The gist of the present invention is a bone disease therapeutic agent containing a sugar compound having sulfated fucose in its structure and / or a pharmacologically acceptable salt thereof as an active ingredient, specifically, a sulfated fucose molecule. Polysulfated fucose and / or their pharmacologically acceptable salt or glycosaminoglycan having a sulfated fucobiosyl group and / or their pharmacologically acceptable It is a bone disease therapeutic agent containing a salt as an active ingredient. Among them, as a glycosaminoglycan having a sulfated fucobiosyl group, a sulfated fucobiosylglycosaminoglycan having an average of 0.1 or more molecules of sulfated fucobiosyl group per residue of a disaccharide unit composed of uronic acid and hexosamine. Furthermore, D-glucuronic acid and N-acetyl-D-galactosamine are β1-> 3 glycosidic linked constituent disaccharide units having an average of 0.1 or more fucobiosyl groups per residue. Preferable examples include saminoglycan. The gist of the present invention is an osteoclast formation inhibitor containing a sugar compound having sulfated fucose in the structure and / or a salt thereof as an active ingredient.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in further detail below.
The sugar compound having a sulfated fucose in the structure used in the present invention (hereinafter also referred to as the substance of the present invention) is a compound in which sulfated fucose (hereinafter sometimes referred to as Fuc) is chemically present in the structure of the sugar compound. A saccharide compound having two or more sulfated fucose molecules in the structure of the saccharide compound is more preferable. Further, from another viewpoint, the substance of the present invention is a saccharide compound having sulfated fucose in the structure, preferably showing an osteoclast formation inhibitory effect, and the osteoclast formation inhibitory effect is, for example, PGE using mouse bone marrow cells₂It is possible to confirm by measuring the number of osteoclasts using an experimental system in which osteoclast formation was induced by adding a bone resorption promoting factor such as However, this effect is not particularly confirmed only in this experimental system, and can also be confirmed in experiments of other osteoclast differentiation induction models.
[0012]
Examples of the substance of the present invention include polysulfated fucose in which sulfated fucose molecules are chemically bonded to each other, and glycosaminoglycan having a sulfated fucobiosyl group composed of two sulfated fucose molecules as a side chain. . The fucose in which at least one of all fucose molecules present in the structure of the substance of the present invention is sulfated may be used. Among the fucose present in the substance of the present invention, fucose having no sulfate group is present. It may exist.
[0013]
The “polysulfated fucose” in the present specification may be a linear structure or a branched structure, in which sulfated fucose molecules are chemically bonded to each other. That is, it is preferable that the polysulfated fucose used as the substance of the present invention has a glycoside bond between the sulfated fucose molecules by α1 → 3 bond (Fucα1 → 3Fuc) and extends linearly. Further, a branched structure may be formed by having a side chain composed of sulfated fucose as a main chain having a chain structure of α1 → 3 bonds between sulfated fucose molecules, and sulfation of the main chain in the branched structure. The fucose and the sulfated fucose of the side chain are preferably glycosidically linked by α1 → 4 bond (Fucα1 → 4Fuc). In addition, the polysulfated fucose of the substance of the present invention does not necessarily have the side chains bonded regularly, and includes polysulfated fucose partially having side chains.
[0014]
Moreover, as a site | part which can be sulfated in polysulfated fucose, the hydroxyl group which is not participating in a coupling | bonding is mentioned. For example, in polysulfated fucose having a linear structure with α1 → 3 bonds between sulfated fucose molecules, the hydroxyl groups at the O-2 and O-4 positions of each sulfated fucose are sulfated. In polysulfated fucose, which is considered as a possible site, and a sulfated fucose is formed as a side chain by α1 → 4 bond to a main chain formed by α1 → 3 bond between sulfated fucose molecules to form a branched structure. It is considered that the hydroxyl group at the O-2 position of each sulfated fucose constituting the chain or the hydroxyl group at the O-2 position and the O-4 position in the sulfated fucose molecule that is the side chain can be sulfated. However, as described above, all of the sites are not necessarily sulfated, and are not necessarily sulfated regularly.
[0015]
The molecular weight of the polysulfated fucose is not limited, but the average molecular weight is preferably 3,000 to 60,000, more preferably 5,000 to 40,000, and further 7,000 to 35,000. Is more preferable. The ratio of sulfate groups to one molecule of fucose is preferably an average of 0.4 to 1.0 molecule, more preferably an average of 0.6 to 0.85 molecule.
[0016]
The glycosaminoglycan having a sulfated fucobiosyl group is a uronic acid residue selected from L-iduronic acid or D-glucuronic acid and D-glucosamine, D-galactosamine, N-acetyl-D-glucosamine or N -Glycosami having a repeating structure of a disaccharide unit consisting of hexosamine residues selected from acetyl-D-galactosamine (hereinafter also referred to as a constituent disaccharide unit) as a basic skeleton of the main chain and a sulfated fucobiosyl group as a side chain Indicates noglycan (hereinafter referred to as SC-GAG).
[0017]
The sulfated fucobiosyl group in the present invention is a 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 fucose molecule are bonded by α1 → 3 due to the difference in chemical formula, and is represented by the following formula (1).
[0018]
[Chemical 1]

[0019]
[Table 1]

[0020]
That is, examples of SC-GAG used as the substance of the present invention include those in which a sulfated fucobiosyl group is bonded as a side chain to a known glycosaminoglycan. In particular, chondroitin sulfate type in which the O-1 position of D-glucuronic acid (hereinafter also referred to as GlcA) and the O-3 position of N-acetyl-D-galactosamine (hereinafter also referred to as GalNAc) are β-glycosidically bonded. A hydroxyl group and a sulfated fucobiosyl group at a position selected from the O-4 position, the O-6 position of GalNAc, and the O-3 position of GlcA, which have a repeating structure of disaccharide units as the basic skeleton of the main chain. Preferred is a chondroitin sulfate type glycosaminoglycan (hereinafter also referred to as SC-CS) having a branched structure having a sulfated fucobiosyl group as a side chain by glycosidic bonding with a hydroxyl group at the O-1 position of It is done.
[0021]
SC-GAG is substantially composed of a constituent disaccharide unit having one sulfated fucobiosyl group side chain, or a constituent disaccharide unit having no sulfated fucobiosyl group side chain and one or more sulfated fucobiosyls Constituent disaccharide units having a base side chain may be mixed. That is, it may be a glycosaminoglycan partially having a sulfated fucobiosyl group side chain.
[0022]
For example, SC-CS preferably contains an average of 0.1 or more fucobiosyl groups per residue of the main chain constituent disaccharide unit, and further contains an average of 0.2 or more molecules. More preferably, it contains more than 0.4 molecules on average, and more preferably contains more than 0.8 molecules on average. (In the present specification, chondroitin sulfate-type glycosaminoglycan having a partially sulfated fucobiosyl group side chain is referred to as partial SC-CS. (A chondroitin sulfate-type glycosaminoglycan consisting of a constituent disaccharide unit having approximately one molecule of chain is also referred to as NSC-CS.)
[0023]
The average molecular weight of SC-GAG is preferably 2,000 to 50,000, and more preferably 3,000 to 30,000. It is common knowledge for those skilled in the art that the average molecular weight of polysaccharides is generally expressed in terms of weight average molecular weight, but the average molecular weight of glycosaminoglycans varies somewhat depending on the measurement method and measurement conditions even in the same sample. In addition, the substance of the present invention should not be strictly limited to the above average molecular weight range.
[0024]
The SC-GAG of the present invention has a sulfate group in the glycosaminoglycan moiety as well as a normal glycosaminoglycan. For example, in SC-CS, the ratio of the sulfate group to one residue of the main chain constituent disaccharide unit is preferably 1.0 to 4.0 molecules in average, together with the sulfate content of the sulfated fucobiosyl group in the side chain, Is more preferably 1.3 to 3.8 molecules on average.
[0025]
Partial SC-CS is a combination of decomposition by chondroitinase ABC (hereinafter also referred to as C-ABC) and analysis by ion exchange high performance liquid chromatography (hereinafter also referred to as HPLC) described in the following test examples. In sugar composition analysis, an unsaturated disaccharide consisting of one molecule of uronic acid and one molecule of hexosamine is detected in inverse proportion to the amount of sulfated fucobiosyl group bound. The production rate of unsaturated disaccharide in this analysis method is 0 to 75. % Is preferable, and it is more preferable in it being 0 to 50%. NSC-CS has an unsaturated disaccharide production rate of 0% by this analysis method. That is, the substance of the present invention is more preferably sulfated GAG that is not easily decomposed by C-ABC.
[0026]
The substance of the present invention is not particularly limited by its origin, origin, and production method. Any substance extracted and purified from a natural resource can be extracted from the natural resource as long as it satisfies the characteristics of the substance of the present invention. The material obtained by purification may be modified as a raw material by a chemical method, or may be artificially chemically synthesized or genetically engineered by animal cells, plant cells, microorganisms, etc. .
[0027]
For example, SC-GAG is a glycosaminoglycan having a sulfated fucobiosyl group obtained from a natural resource as a side chain, but is partially obtained from a sulfated fucobiosylglycosaminoglycan obtained from a natural resource (mainly a living organism). Modified by chemical methods such as removing sulfated fucobiosyl groups and glycosaminoglycans (hereinafter also referred to as GAG) obtained from natural resources as raw materials by sulfated fucobiosyl groups Those having a side chain introduced therein, those artificially synthesized chemically, or those genetically engineered by animal cells, plant cells, microorganisms, etc. may be used. There are no particular limitations on the species, genus, etc. of natural resources used when SC-GAG is isolated and purified from natural resources or when sulfated fucobiosylglycosaminoglycans isolated and purified from natural resources are used as raw materials. However, for example, there are echinoderms. Among the echinoderms, preferred are sea cucumbers, and particularly, sea cucumber (Sea Cucumber).Stichopus  japonicus), Cronamaco (Holthuria  atra), Nisekuro sea cucumber (Holothuria  leurospilota) And the like.
[0028]
Moreover, as a raw material used when isolating and purifying polysulfated fucose used as the substance of the present invention from natural resources, sea cucumbers similar to the above can be mentioned as an example. Currently, these sea cucumber animals are unused resources that are rarely used, although only a small part is used for food.
[0029]
A method for isolating and purifying GAGs such as sulfated fucobiosylglycosaminoglycan and polysulfated fucose from the above-mentioned natural resources is described in, for example, Carbohydr. Res. , 297, 273-279 (1997), Japanese Patent Publication No. 6-70085, and the reference examples described later can be used, but they are usually used for isolating and purifying GAGs and the like from living tissues. It is also possible to use a known method.
[0030]
In addition, as a partial elimination method of the sulfated fucobiosyl group which is a side chain, mild acid hydrolysis reaction, alkaline hydrolysis reaction and enzyme digestion under the conditions for producing SC-GAG satisfying the characteristics of the substance of the present invention can be used. Although reaction is mentioned, if the target partial SC-GAG is obtained, it will not specifically limit to these. For example, using sulfated fucobiosylglycosaminoglycan isolated and purified from sea cucumber as a raw material, when partial elimination of the sulfated fucobiosyl group is performed, about 0.05 to 0.2 N sulfuric acid, hydrochloric acid, etc. Examples include a method of acid hydrolysis using an inorganic acid under conditions of about 75 to 85 ° C. for about 2 to 6 hours, neutralization, and separation / purification after the reaction.
[0031]
The pharmacologically acceptable salt of the substance of the present invention is not particularly limited as long as it does not lose the action of inhibiting the osteoclast formation of the substance of the present invention and is a pharmacologically acceptable salt. . For example, alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt, salts with inorganic bases such as ammonium salt, and organic bases such as diethanolamine salt, cyclohexylamine salt and amino acid salt Among them, pharmacologically acceptable salts are preferable, and sodium salt is particularly preferable.
The salt of the substance of the present invention is not particularly limited as long as it does not lose the action of suppressing the formation of osteoclasts of the substance of the present invention. For example, sodium salt, potassium salt, lithium salt Examples thereof include alkali metal salts such as calcium salts, alkaline earth metal salts such as calcium salts, salts with inorganic bases such as ammonium salts, and salts with organic bases such as diethanolamine salts, cyclohexylamine salts, and amino acid salts. Particularly preferred is the sodium salt.
[0032]
The substance of the present invention significantly suppresses the formation of osteoclasts in an experimental system for osteoclast formation using mouse bone marrow cells, as described in Examples below. Thus, the substance of the present invention can prevent metabolic bone diseases such as osteoporosis, rheumatism, inflammatory bone diseases such as periodontal disease, etc., especially diseases caused by abnormally enhanced osteoclast function. Can be used for treatment.
[0033]
When the substance of the present invention is used for the prevention or treatment of the above-mentioned diseases, it is usually used at the time of other medicinal ingredients and preparations as long as the action of the substance of the present invention is not substantially impaired and does not adversely affect the administration subject. The used excipients, binders, preservatives, stabilizers and the like can be used as appropriate. As the dosage form and administration route of the bone disease therapeutic agent containing the substance of the present invention, it can be formulated into tablets, capsules, granules, powders, injections, solutions, lipolytic agents, gelling agents, ointments, etc. Although administration methods such as oral, injection, and application are conceivable, it can be appropriately selected according to the nature and severity of the disease to be treated.
[0034]
The dosage of the therapeutic agent for bone disease containing the substance of the present invention is a matter to be determined individually according to the administration method, dosage form, weight of the subject patient, specific symptoms, etc. Although not limited, it is conceivable that about 0.1 mg / kg to 300 mg / kg per day as the substance of the present invention is administered once a day to several times a day.
Furthermore, the above-mentioned substance of the present invention can also be used in an in vivo and in vitro system as an osteoclast formation inhibitor.
[0035]
【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.
[0036]
Reference Example 1 Production of NSC-CS
Manamako (Stichopus  japonius) By a known method (Kariya et al. (1990) J. Biol. Chem., 265, 5081-5085) used for isolating and purifying GAGs from biological tissues. 210 mg of freeze-dried powder of modified fucobiosylglycosaminoglycan (NSC-CS) was obtained.
[0037]
Specifically, manamako (raw material)Stichopus  japonius) Was processed into a minced shape, and then homogenized, and unnecessary fat-soluble fractions were extracted and removed with chloroform / methanol (2: 1, V / V). The extraction residue was dried and then subjected to an autoclave treatment at 120 ° C. for 30 minutes, and dissolved in a 50 mmol / l 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 coexisting proteins such as collagen and core protein. The enzyme digest was sequentially treated with 0.4 mol / l 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). The recovered precipitate was washed with cold ethanol, and the resulting pellet was dried under reduced pressure to obtain a crude product.
[0038]
A Sephadex G-100 column (manufactured by Amersham Bioscience) in which 1.0 g of the obtained crude product was dissolved in a small amount of 50 mmol / l ammonium hydrogen carbonate buffer (pH 8.0) and half the amount was equilibrated with the same solvent. Was attached to a column (diameter 3.4 cm × length 100 cm) and eluted with the same solvent, and the eluate was collected every 10 mL. For each of the obtained fractions, measurement of absorbance at 210 nm in the ultraviolet region (UV), measurement of uronic acid content by carbazole method (Bitter & Muir (1962) Anal. Biochem., 4, 330-334) and anthrone method (Dimler, RL) et al. (1952) Anal. Chem., 24, 1411-1414). As a result, a carbazole reaction positive fraction regarded as glycosaminoglycan was found in the polymer fraction, and these fractions were collected and collected to obtain a powder (224 mg) by lyophilization. The same operation was performed on the remaining half of the obtained crude purified product solution, and a total of 574 mg of lyophilized product was obtained. 500 mg of the lyophilized product obtained 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 → 1.2 M sodium chloride in the same buffer.
[0039]
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, since a peak considered to be the target substance was found near 0.6 mol / l concentration of sodium chloride, fractions corresponding to this peak were collected and collected, subjected to running water dialysis, then freeze-dried, and NSC -A powder (210 mg) of CS was obtained.
[0040]
The NSC-CS obtained here was analyzed by a chemical composition analysis method. That is, the hexosamine content was measured by the MBTH method (Hurst & Settine (1981) Anal. Biochem., 115, 88-92), and the carbazole method (Bitter & Muir (1962) Anal. Biochem., 4, 330-334), ion The uronic acid content, sulfate ion content and neutral sugar content were measured by chromatography and anthrone method (Dimler, RL et al. (1952) Anal. Chem. 24, 1411-1414), respectively.
[0041]
From the measurement results, the weight abundance ratio of the obtained NSC-CS constituents was calculated (shown in Table 1), and known sea cucumber-derived glycosaminoglycans (Kariya et al. (1990) J. Biol. Chem.,). 265, 5081-5085). The structure of this known sea cucumber-derived glycosaminoglycan has a basic skeleton of chondroitin in which D-glucuronic acid and N-acetyl-D-galactosamine are linked by β1 → 3 glycoside, and N-acetyl-D-galactosamine It has a structure in which a fucose side chain (sulfated fucobiosyl group) consisting of two sulfated fucose residues is glycosidically bonded to the O-4 position and / or the O-6 position, and further to the O-3 position of D-glucuronic acid. (Kariya et al. (1997) Carbohydr. Res., 297, 273-279), and the NSC-CS produced in this reference example is presumed to have the same structure.
[0042]
Reference Example 2 Production of partial SC-CS
Using the NSC-CS obtained above as a raw material, partial removal of the sulfated fucobiosyl group, which is a side chain, was performed to produce a partial SC-CS. That is, 40 mg of NSC-CS obtained above was dissolved in 4 mL of 0.05 M sulfuric acid and subjected to a 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 a 1N aqueous sodium hydroxide solution, and then equilibrated with 0.2 M sodium chloride, a Cellulofine GCL-90m column (inner diameter 3.4 × 110 cm). ; Manufactured by Seikagaku Corporation), and the eluate was collected every 10 mL. In addition, NSC-CS obtained by the above method was similarly subjected to column chromatography, and 10 mL of the eluate was collected (hereinafter, what was obtained by hydrolysis for 3 hours was hydrolyzed with partial SC- 3 hours. CS, obtained by hydrolysis for 6 hours is also referred to as 6-hour hydrolyzed partial SC-CS).
[0043]
The uronic acid content and neutral sugar content were measured for each fraction of each column eluate, and the behavior of the eluate was compared (FIG. 1). For each column eluate, a fraction of glycosaminoglycan fraction was concentrated together based on FIG. 1, and further, a Cellulofine GCL-25 column (inner diameter 2.0 × length 25 cm, equilibrated with distilled water, Desalted by attaching to Seikagaku Kogyo Co., Ltd., then dried and recovered by freeze-drying. Yields were 34.7 mg, 18.1 mg and 15 mg respectively for NSC-CS, 3 hr hydrolyzed partial SC-CS and 6 hr hydrolyzed partial SC-CS.
[0044]
NSC-CS, 3-hour hydrolyzed partial SC-CS and 6-hour hydrolyzed partial SC-CS gel filtration elution patterns (FIGS. 1a, 1b, 1c) were analyzed for NSC-CS without hydrolysis. Was observed as a single peak in a shape equal to the position where peaks of uronic acid and neutral sugar almost coincided with each other in the polymer fraction region. However, in the 3-hour hydrolyzed partial SC-CS, the neutral sugar peak corresponding to the neutral sugar peak of the NSC-CS pattern is significantly reduced, and the neutral sugar peak in the low molecular fraction region is reduced. A new peak of neutral sugar is more prominently detected in 6-hour hydrolyzed partial SC-CS.
[0045]
This suggests that the sulfated fucobiosyl group, which is a side chain, is detached from NSC-CS in a hydrolysis reaction time as the hydrolysis reaction proceeds.
[0046]
In the same manner as in Reference Example 1, chemical composition analysis was performed on the 3-hour hydrolyzed partial SC-CS and the 6-hour hydrolyzed partial SC-CS. Hexosamine content was determined by MBTH method (Hurst & Settine (1981) Anal. Biochem., 115, 88-92), carbazole method (Bitter & Muir (1962) Anal. Biochem., 4, 330-334), ion chromatography and anthrone. The uronic acid content, sulfate ion content and neutral sugar content were measured by the method (Dimler, R.L et al. (1952) Anal.Chem., 24, 1411-1414), and the weight abundance ratio of each component Was calculated (Table 2). Further, the molar abundance (mmol / g) of each component was calculated, and the molar ratio of each component was calculated with hexosamine as 1, and the results are shown in Table 3.
[0047]
[Table 2]
Table 2 Weight abundance ratio of constituents of each sample (W / W,%)

[0048]
[Table 3]
Table 3 Abundance (mmol / g) of constituent components of each sample
(The values in parentheses indicate the molar ratio of each component when the molar abundance of hexosamine is 1.)

[0049]
Both the 3-hour hydrolyzed partial SC-CS and the 6-hour hydrolyzed partial SC-CS have a substantially constant molar ratio of hexosamine and uronic acid, and GAG linear basics are also used in the hydrolysis reaction for eliminating the sulfated fucobiosyl group. It is inferred that the structural part is retained. In addition, in 3-hour hydrolysis partial SC-CS, 0.29 molecules and 1.70 molecules of fucobiosyl group and sulfate group exist per residue of the constituent disaccharide units, respectively, whereas in 6-hour hydrolysis partial SC-CS, The fucobiosyl group and sulfate group are 0.18 molecule and 1.06 molecule, respectively, per residue of the constituent disaccharide unit and decrease depending on the hydrolysis reaction time, confirming the results of the gel filtration elution pattern described above. Yes.
[0050]
Reference Example 3 Degradation by chondroitinase ABC
For NSC-CS, 3-hour hydrolyzed partial SC-CS, and 6-hour hydrolyzed partial SC-CS, the degree of digestion by C-ABC was measured by HPLC using gel filtration as a separation factor (hereinafter also referred to as GCP-HPLC). did. That is, NSC-CS, 3 hour hydrolyzed partial SC-CS and 6 hour hydrolyzed partial SC-CS were each dissolved in distilled water to 10 mg / mL, and 20 μL of this was dissolved in 0.5 U of C-ABC (raw 10 μL of enzyme solution containing Chemical Industry Co., Ltd. (0.4 M sodium acetate, 0.4 M Tris-HCl buffer (pH 8.0) containing 0.1% bovine serum albumin) and added at 37 ° C. for 18 hours Enzymatic digestion reaction was performed. Distilled water (50 μL) was added to the reaction mixture, heated in boiling water for 1 minute, and then centrifuged to obtain a supernatant.
[0051]
The supernatant containing this C-ABC enzyme digest was designated TSK-Gel G4000PW._XL, TSK-Gel G3000PW_XLAnd TSK-Gel G2500PW_XLColumns (both having an inner diameter of 4.0 × 25 cm, manufactured by Tosoh) were attached to GPC-HPLC, which was connected in order from the upstream, and eluted under isocratic conditions of 0.2 mol / l sodium chloride solution, and the refractive index. Was detected as an index to obtain an HPLC chart. (Figure 2)
The estimation of C-ABC digestibility (unsaturated disaccharide production rate) of each test article was calculated from the peak area of the obtained HPLC chart using the following formula. In the following formula for calculating the unsaturated disaccharide production rate, the total peak area refers to ΔOligo, ΔDi-diS, ΔDi-monoS in each chart after digestion with C-ABC enzyme of A2, B2, and C2 in FIG. And the sum of the unsaturated disaccharide peak areas is the sum of the areas of the respective unsaturated disaccharide peaks (the total peak area of ΔDi-diS, ΔDi-monoS, and ΔzeroS). ). ΔOligo indicates an unsaturated oligosaccharide having a structure of tetrasaccharide or higher without being decomposed into a disaccharide unit structure by C-ABC digestion. ΔDi-diS is an unsaturated disaccharide having two sulfate groups in the unsaturated disaccharide unit structure, and ΔDi-monoS is an unsaturated disaccharide having one sulfate group in the unsaturated disaccharide unit structure. ΔDi-zeroS represents an unsaturated disaccharide having no sulfate group in the unsaturated disaccharide unit structure (see Anal. Biochem., 177, 327-332 (1989) p. 328 FIG. 1).
[0052]
Unsaturated disaccharide production rate (%) = sum of unsaturated disaccharide peak areas / total peak area × 100
NSC-CS did not detect an unsaturated disaccharide component, and it was confirmed that it was not decomposed by C-ABC. This is the same as the description in known literature concerning sea cucumber-derived glycosaminoglycans. In addition, unsaturated disaccharide and unsaturated oligosaccharide peaks are observed in 3-hour hydrolyzed partial SC-CS and 6-hour hydrolyzed partial SC-CS, and the unsaturated disaccharide production rate (%) calculated according to the above formula Were 46.5% and 66.3%, respectively.
[0053]
Reference Example 4 Production of polysulfated fucose
The back of the sea cucumber body wall (10 kg) was minced and then homogenized to obtain a fat-soluble fraction by extraction with chloroform / methanol (2: 1, v / v). This fraction was concentrated and dried under reduced pressure using a rotary evaporator. Distilled water (300 mL) was added to the resulting dried product to form an aqueous solution, and then left overnight at 4 ° C. Next, 3 times the amount of ethanol was added to the aqueous solution from which the fat floating on the surface layer was removed under cooling, and centrifuged to obtain a precipitate. This precipitate was dissolved in a small amount of distilled water, and then freeze-dried to obtain a crude polysaccharide fraction (2.46 g) regarded as having low polarity.
[0054]
1 g from this crude fraction was dissolved in 7 mL of a 0.2 mol / l aqueous sodium chloride solution and equilibrated with a 0.2 mol / l aqueous sodium chloride solution, and a Sepharose CL-6B column (inner diameter 3.4 × 110 cm, manufactured by Amersham Biosciences). ) Elution was carried out using a 0.2M aqueous sodium chloride solution, and fractionated every 12 mL. The obtained 100 fractions were subjected to colorimetric determination (measured at 620 nm) by anthrone method and absorbance at both wavelengths of 210 nm and 280 nm, and an elution curve was drawn (FIG. 3).
[0055]
Fractions Nos. 42 to 69 that were positive for the anthrone method were combined, and then concentrated to 10 mL with a rotary evaporator. The concentrated solution was applied to a Cellulofine GCL-25 column (inner diameter 3.3 × 36 cm, sold by Seikagaku Corporation) equilibrated with distilled water, and fractionated every 10 mL by elution with distilled water. For the obtained 40 fractions, colorimetric determination (620 nm) by anthrone method and absorbance at a wavelength of 210 nm were measured, and an elution curve was drawn. From the elution curve, confirm that desalting has been achieved, combine fraction numbers 11-20, which are fractions containing the target substance, and freeze-dry to obtain a polysaccharide that contains a neutral sugar. Product was obtained (yield 135.4 mg).
[0056]
For further purification, 100 mg of the obtained dried product was dissolved in 50 ml of 0.1 mol / l sodium acetate / acetic acid buffer (pH 5.0) and equilibrated with the same buffer, DEAE-Cellulose (DE52, manufactured by Whatmann) ) Apply to a column (inner diameter 2.3 × 18 cm), wash the column with 50 ml of the same buffer, and then use a 300 ml of the same buffer and 300 ml of the same buffer containing 1.2 M sodium chloride to obtain a linear concentration gradient of sodium chloride. And eluted every 7 mL. The obtained 100 fractions were measured for colorimetric determination (620 nm) by anthrone method and absorbance at 280 nm, and an elution curve was drawn (FIG. 4). As a result, peaks (referred to as peak 1 and peak 2 respectively) appeared in fraction numbers 75 to 81 and fraction numbers 82 to 94. Combined for each peak, the fraction derived from peak 1 (hereinafter also referred to as polysulfated fucose I, yield 14.2 mg) and the fraction derived from peak 2 (hereinafter also referred to as polysulfated fucose II) Yield 33.6 mg).
[0057]
Reference Example 5 Physicochemical analysis of polysulfated fucose
The polysulfated fucose I and polysulfated fucose II produced in Reference Example 4 were subjected to the MBTH method (Hurst & Settine (1981) Anal. Biochem., 115, 88-92) as in Reference Examples 1 and 2. The hexosamine content was determined by the carbazole method (Bitter & Muir (1962) Anal. Biochem., 4, 330-334), ion chromatography and the anthrone method (Dimmer, RL et al. (1952) Anal. Chemm., 24, 1411- In 1414), the uronic acid content, sulfate ion content and neutral sugar content were measured, and the weight abundance ratio of each component was calculated (Table 4). Further, the molar abundance (mmol / g) of each component was calculated, and the molar ratio of each component was calculated with neutral sugar as 1 (Table 5).
Subsequently, the molecular weight was measured by gel filtration-high performance liquid chromatography. As a result, the molecular weight of polysulfated fucose I was 9,000, and the molecular weight of polysulfated fucose II was 32,000.
[0058]
[Table 4]
Table 4 Weight abundance ratio of each component (wt%)

[0059]
[Table 5]
Table 5 Molar abundance (mmol / g) of each component
(The numbers in parentheses indicate the molar ratio of each component when the molar abundance of neutral sugar content is 1.)

[0060]
As a result, both polysulfated fucose I and polysulfated fucose II contain sulfate groups and neutral sugars, but do not contain hexosamine, and also contain only trace amounts of uronic acid. It was speculated that it was not a glycosaminoglycan having a neutral sugar as a side chain. The ratio of sulfate groups to the neutral sugars of polysulfated fucose I and II was 0.69 and 0.79, respectively, and it was found that the ratio of sulfation was high.
[0061]
Furthermore, Yasuno, S .; et al. (1999) Biosci. Biotechnol. Biochem. , 63, 1353-1359, neutral sugars were identified. That is, 50 μL was taken from each of the aqueous solutions of polysulfated fucose I and polysulfated fucose II prepared to 10 mg / mL, and 50 μL of 8 mol / L trifluoroacetic acid (TFA) was added to each and sealed. The reaction mixture was heated at 100 ° C. for 3 hours to cleave the glycoside bond and the sulfate ester bond. After cooling, the reaction mixture was collected at the bottom of the reaction tube by centrifugation and opened. Dry air was sprayed to remove the solvent, and 40 μL of 2-propanol was added to each and stirred. Furthermore, a monosaccharide sample was obtained by blowing dry air to remove the solvent. After adding 20 μL of distilled water and 80 μL of 4-aminobenzoic acid ethyl ester (ABEE) reagent to the obtained monosaccharide sample, the tube is sealed and heated at 80 ° C. for 1 hour to proceed with the reductive amination reaction. Thus, a monosaccharide derivative having a reducing end labeled with ABEE was prepared. After completion of the reaction, the tube was opened, 200 μL of distilled water and 200 μL of methylene chloride were added, and the mixture was vigorously stirred and then centrifuged to obtain the above monosaccharide derivative in which the reducing end was labeled with ABEE in the supernatant. It is obtained by high-performance liquid chromatography equipped with a Honenpak-C18 column (inner diameter 4.6 × 75 mm, manufactured by Honen) equilibrated with 0.2 mol / l borate buffer (pH 8.9) containing 7% acetonitrile. The ABEE-labeled monosaccharide sample was attached, and the analysis was carried out under isocratic elution conditions of the same solvent. The monosaccharide preparations used were galactose (Gal), mannose (Man), glucose (Glu), arabinose (Ara), ribose (Rib), N-acetylmannosamine (ManNAc), xylose (Xyl), N-acetylglucosamine (GlcNAc), fucose (Fuc), rhamnose (Rha), and N-acetylgalactosamine (GalNAc) (in order of elution) (FIG. 5). As a result, it was found that the monosaccharide contained in both polysulfated fucose I and polysulfated fucose II obtained in Reference Example 4 was fucose. The reason for the fact that sulfated polyfucose I and II were fat-soluble fractions at the beginning of purification is thought to be because they are sulfated but contain fucose residues having low-polar methyl groups. .
[0062]
Reference Example 6 Structural analysis of polysulfated fucose
Amberlite IR-120 column (sold by Organo Co., Ltd., 1.5 × inner diameter) obtained by taking 5 mg each from polysulfated fucose I and polysulfated fucose II obtained in Reference Example 4 and equilibrating each with distilled water. 12 cm). The acidic fraction of the obtained elution fraction was neutralized by adding an appropriate amount of pyridine, and then lyophilized to obtain pyridine salts of polysulfated fucose I and polysulfated fucose II. For each, 1 ml of dimethyl sulfoxide (DMSO) containing 10% distilled water was added, and the solvolysis reaction was allowed to proceed at 90 ° C. for 6 days. The reaction was stopped by cooling to room temperature, 3 times the amount of distilled water was added, and then an appropriate amount of 2 mol / l sodium hydroxide aqueous solution was added to adjust the pH to 9 to 9.5. This was dialyzed against running water overnight and then freeze-dried to obtain 3.0 mg and 3.3 mg of desulfurized oxides of polysulfated fucose I and polysulfated fucose II, respectively.
[0063]
From a total of four fractions of polysulfated fucose I and its desulfurized oxide, and further polysulfated fucose II and its desulfurized oxide, 1 mg each was taken and the method of Hakomori (Hakomori, S. (1964) J. Biochem. (Tokyo) , 55, 205-208), and methylated derivatives were synthesized according to the method of Sanford and Conrad (Sanford, PA & Conrad, HE (1966) Biochemistry, 5, 1508-1517). The resulting methylated derivative was desalted according to the method of Waeghe et al. (Waeghe et al. (1983) Carbohydr. Res., 123, 281-304). Further, these were hydrolyzed at 121 ° C. using 2 mol / L trifluoroacetic acid (TFA), and then sodium deuteride (NaBD).₄). Finally, the hydroxyl group that appeared by the reduction treatment was acetylated to synthesize partially methylated alditol acetate (PMAA). The obtained PMAA was dissolved in 50 μL of acetone, 1 μL of which was subjected to GC-MS. Gas chromatography-mass spectrum (GC-MS) analysis was performed in a splitless mode using a JEOL HX110A type mass spectrometer equipped with a SUpelco SP2330 capillary column (inner diameter 0.25 mm × 30 m). The column oven was kept at 50 ° C. for 2 minutes, then heated to 170 ° C. with a gradient of 30 ° C./minute, further heated to 235 ° C. with a gradient of 4 ° C./minute, and kept at this temperature for 15 minutes.
[0064]
In the polysulfated fucose I chromatogram, all 8 types of PMAA were observed (Table 6). They are 2,3,4-tri-O-methyl-fucitol (T-Fuc), 3,4-di-O-methyl-fucitol (2-Fuc), 2,4-di-O-methyl-fucitol. (3-Fuc), 2,3-di-O-methyl-fucitol (4-Fuc), 4-O-methyl-fucitol (2,3-Fuc), 3-O-methyl-fucitol (2,4- Fuc), 4-O-methyl-fucitol (3,4-Fucol), and fucitol (2,3,4-fucitol). In the following description, the explanation will be made using the abbreviation of PMAA shown in () above.
[0065]
[Table 6]
Table 6

[0066]
The polysulfated fucose I desulfurized oxide chromatogram was different from the polysulfated fucose I chromatogram, and almost equal amounts of T-Fuc, 3-Fuc and 3,4-Fuc were detected. Therefore, the structure of polysulfated fucose I is considered to be partially branched from the main chain of Fucα1 → 3Fuc (Fucα1 → 4Fuc) (FIG. 6). In addition, it was considered that the sulfate group was coordinated to the site as illustrated (O-2 position in the main chain and O-2 position and / or O-4 position in the side chain).
[0067]
In the chromatogram of polysulfated fucose II, as shown in Table 6, five types of PMAA (T-Fuc, 3-Fuc, 2,3-Fuc, 3,4-Fuc and 2,3,4) are used. -Fuc) was observed. The polysulfated fucose II desulfurized oxide chromatogram was different from the polysulfated fucose II chromatogram and consisted of only a small amount of T-Fuc and a significant amount of 3-Fuc. The structure of fucose II is a linear structure of Fucα1 → 3Fuc, and it is considered that the hydroxyl group at the O-2 position and / or the O-4 position is partially sulfated.
[0068]
Example 1 Osteoclast formation inhibition experiment of SC-CS
The tibia and femur of a ddY mouse (6 weeks old female) were removed, and bone marrow cells were collected from the distal ends of both bones. Bone marrow cells are 5 x 10 in 24-well cell culture plates⁶Seed to be cells / well and 10^―6Mol / L Prostaglandin E₂(Hereafter, PGE₂That's it. NSC-CS obtained in Reference Example 1 was added to give a concentration of 1, 3, 10, 30, 100 μg / mL under stimulation (SIGMA), and 10% fetal calf serum (hereinafter referred to as FCS). (Made by Behringer) containing Medium Essential Medium Alpha Medium (GIBCO, hereinafter referred to as αMEM) in a medium for 7 days at 37 ° C., CO₂Cultured in an incubator. PGE every 2-3 days during culture₂The medium was exchanged with the αMEM medium containing 10% FCS newly added with NSC-CS at each concentration described above. After 7 days of culture, a staining measurement kit using an azo dye method containing tartrate-resistant acid phosphatase (hereinafter referred to as TRAP), which is a marker for osteoclasts, and naphthol AS-BI phosphate and fast garnet GBC salt. Acid Phosphatase, Leukocyte "(trade name: manufactured by SIGMA, hereinafter referred to as Acid Phosphatase, Leukocyte) was used, and the number of osteoclasts formed was counted under a microscope. (Fig. 7)
From the results of FIG. 7, in the osteoclast formation experiment system using mouse bone marrow cells, NSC-CS suppressed the formation of osteoclasts in a dose-dependent manner. An NSC-GAG concentration of 1 μg / mL inhibited osteoclast formation by 50%.
[0069]
Example 2 Osteoclast formation inhibition experiment of NSC-CS and partial SC-CS
Implementation was performed except that the NSC-CS obtained in Reference Example 1, the 3-hour hydrolyzed partial SC-CS and the 6-hour hydrolyzed partial SC-CS obtained in Reference Example 2 were used so that the concentration was 50 μg / mL. In the same manner as in Example 1, the number of osteoclasts formed was counted under a microscope. (Fig. 8)
From the results of FIG. 8, a significant osteoclast formation inhibitory effect was confirmed for NSC-CS. Moreover, the osteoclast formation inhibitory effect was recognized also in 3-hour hydrolysis partial SC-CS. Compared with this, the effect of 6 hours hydrolysis partial SC-CS was weak.
[0070]
As the number of sulfated fucobiosyl side chains increases, the osteoclast formation inhibitory effect is stronger. Therefore, it is presumed that it is important to have the sulfated fucobiosyl group as the side chain for the expression of this effect. The
[0071]
Example 3 Osteoclast formation inhibition experiment of sulfated polyfucose
The same operation as in Example 1 was performed except that NSC-CS obtained in Reference Example 1 and polysulfated fucose I and polysulfated fucose II obtained in Reference Example 4 were used so that the concentration was 50 μg / mL. The number of osteoclasts formed was counted under a microscope. The results are shown in FIG. The results were shown by the number of osteoclasts in each well, and as a control, 10% FCS-containing αMEM medium was added instead of the test substance.
[0072]
From the results of FIG. 9, a significant osteoclast formation inhibitory effect was confirmed in polysulfated fucose I and polysulfated fucose II as in NSC-CS.
[0073]
【The invention's effect】
According to the present invention, a saccharide compound having sulfated fucose in its structure is used to suppress osteoclast formation that is effective in treating metabolic bone diseases such as osteoporosis, RA caused by inflammatory bone destruction, diseases such as periodontal disease, etc. Can be provided as a therapeutic agent for bone diseases. The present invention also provides an osteoclast formation inhibitor having an osteoclast formation inhibitory effect in vivo and in vitro.
[0074]
[Brief description of the drawings]
FIG. 1 is a graph showing the uronic acid content and neutral sugar content in each fraction of Cellulofine GCL-90m column eluate of NSC-CS, 3-hour hydrolyzed partial SC-CS and 6-hour hydrolyzed partial SC-CS. The vertical axis represents absorbance, and the horizontal axis represents fraction number. (A), (b) and (c) are the results of NSC-CS, 3 hour hydrolytic partial SC-CS and 6 hour hydrolytic partial SC-CS, respectively.
FIG. 2 is a GPC-HPLC chart before and after chondroitinase ABC digestion in NSC-CS, 3 hour hydrolyzed partial SC-CS, and 6 hour hydrolyzed partial SC-CS, where the vertical axis represents the differential refractive index, The horizontal axis indicates the retention time (minutes). A1 is a GPC-HPLC chart before C-ABC digestion of NSC-CS, A2 is a GPC-HPLC chart after C-ABC digestion of NSC-CS, and the disaccharide composition ratio is 0%. B1 is a GPC-HPLC chart before C-ABC digestion of 3 hours hydrolyzed partial SC-CS, B2 is a GPC-HPLC chart after C-ABC digestion of 3 hours hydrolyzed partial SC-CS, and disaccharide The composition ratio is 46.5%. C1 is a GPC-HPLC chart before C-ABC digestion of 6-hour hydrolyzed partial SC-CS, C2 is a GPC-HPLC chart after C-ABC digestion of 6-hour hydrolyzed partial SC-CS, and disaccharide The composition ratio is 66.3%.
FIG. 3 is a graph showing the colorimetric determination (620 nm) by anthrone method and the absorbance at 210 nm and 280 nm in each fraction of the Sepharose CL-6B column eluate of the crude polysaccharide fraction. The vertical axis represents absorbance, and the horizontal axis represents fraction number.
FIG. 4 is a graph showing the colorimetric determination (620 nm) by anthrone method and the absorbance at 280 nm in each fraction of DEAE-Cellulose column eluate. The vertical axis represents absorbance, and the horizontal axis represents fraction number.
FIG. 5 shows a high performance liquid chromatograph equipped with a monosaccharide derivative Honenpak-C18 column labeled with 4-aminobenzoic acid ethyl ester at the reducing end obtained from polysulfated fucose I and polysulfated fucose II. It is a chart obtained.
E1 is a profile obtained from polysulfated fucose I, and E2 is a profile obtained from polysulfated fucose II.
Std. Is the result of attaching a monosaccharide preparation, and each peak is in the order of elution, galactose (Gal), mannose (Man), glucose (Glu), arabinose (Ara), ribose (Rib), N-acetylmannosamine (ManNAc) ), Xylose (Xyl), N-acetylglucosamine (GlcNAc), fucose (Fuc), rhamnose (Rha), and N-acetylgalactosamine (GalNAc).
FIG. 6 is an estimated structure of polysulfated fucose I (fraction derived from peak 1), and E2 is an estimated structure of polysulfated fucose II (fraction derived from peak 2).
FIG. 7 is a graph showing the dose-dependent osteoclast formation inhibitory effect of NSC-CS. The vertical axis represents the number of osteoclasts in one hole of the microplate, and the horizontal axis represents the concentration of NSC-CS added. (-) Indicates the result of the control.
FIG. 8 is a graph showing the osteoclast formation inhibitory effect of NSC-CS, 3-hour hydrolyzed partial SC-CS, and 6-hour hydrolyzed partial SC-CS. The vertical axis represents the number of osteoclasts in one hole of the microplate. (-) Indicates the result of the control.
FIG. 9 is a graph showing the osteoclast formation inhibitory action of NSC-CS, polysulfated fucose I and polysulfated fucose II. The vertical axis represents the number of osteoclasts in one hole of the microplate. (-) Indicates the result of the control.

Claims (4)

A therapeutic agent for bone diseases comprising a sugar compound having sulfated fucose in its structure and / or a pharmacologically acceptable salt thereof as an active ingredient. The bone disease therapeutic agent according to claim 1, wherein the sugar compound having a sulfated fucose in its structure is a glycosaminoglycan having a sulfated fucobiosyl group or a polysulfated fucose. The bone disease therapeutic agent according to claim 1 or 2, wherein the sugar compound having sulfated fucose in the structure has an osteoclast formation inhibitory action. The osteoclast formation inhibitor which contains the sugar compound and / or its salt which have the sulfated fucose of any one of Claims 1-3 in a structure as an active ingredient.

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