JP3751202B2 - Arthritis treatment - Google Patents
Arthritis treatment Download PDFInfo
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- JP3751202B2 JP3751202B2 JP2000603683A JP2000603683A JP3751202B2 JP 3751202 B2 JP3751202 B2 JP 3751202B2 JP 2000603683 A JP2000603683 A JP 2000603683A JP 2000603683 A JP2000603683 A JP 2000603683A JP 3751202 B2 JP3751202 B2 JP 3751202B2
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- zinc
- hyaluronic acid
- complex
- inhibitor
- therapeutic agent
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Description
発明の分野
本発明は、慢性関節リウマチ等の関節性疾患治療剤に関する。
背景技術
慢性関節リウマチ、変形性関節症、外傷性関節炎等を含む関節性疾患は、関節滑膜の炎症を介して軟骨や骨の破壊を惹起する炎症性疾患である。炎症滑膜には、血管新生、リンパ球湿潤、並びに滑膜細胞の増殖及び活性化が認められる。活性化した滑膜細胞はサイトカイン、プロスタグランジン、組織破壊酵素などのケミカルメディエーターを産生し、軟骨や骨の破壊を惹起すると考えられている（Harris, B. D., New England Journal of Medicine），Vol.322, p.1277-1289,（1990）；Cash, J. M., et. al., New England Journal of Medicine），Vol.330, p.1368−1375（1994））。
関節性疾患の治療のために、一般にナトリウム塩の型のヒアルロン酸が有効であることが知られている（文献：山本真他“高分子ヒアルロン酸ナトリウム（ＮＲＤ１０１）の変形性膝関節症に対する臨床評価”、薬理と治療、Vol.21, No.3, p.894−970, 199）。また、亜鉛が関節炎の治療のために有効であることが知られている（A. Frigo et al.,“Copper and Zinc in Inflammation”, Inflammation and drug therapy series, Vol.IV, Kluwer Academic Publishers, P.132−142（1989））。
しかしながら、ヒアルロン酸（一般に、ナトリウム塩の形で）は、その生物適合性（biocompatibility）及び流動特性が好都合であるために若干の関節性疾患の治療のために知られているが、滑膜細胞の増殖や活性化に対する抑制効果は実質的に有しない。また、亜鉛の使用においても満足すべき治療効果は得られていない。
発明の開示
従って、本発明は、慢性関節リウマチなどの関節性疾患の発症機作を構成する滑膜細胞の増殖及び活性化、特に組織破壊酵素の抑制を介して関節性疾患を効果的に治療することができる医薬組成物並びにその製造及び使用を提供しようとするものである。
本発明者らは、上記の課題を解決すべく種々検討した結果、ヒアルロン酸（ナトリウム塩）は滑膜細胞の増殖及び活性化に対する抑制効果をほとんど有さず、また亜鉛の滑膜細胞の増殖及び活性化に対する抑制、特に組織破壊酵素に対する抑制効果は非常に低いのに対して、ヒアルロン酸と亜鉛との複合体（会合物）は、両構成成分の相乗作用すなわち増強相乗作用又は相互作用により、非常に強力な滑膜細胞増殖抑制作用及び組織破壊酵素抑制作用を有するという驚くべき事実を見出し、本発明を完成させた。
従って本発明は、ヒアルロン酸と亜鉛との複合体（会合物）を含んで成る関節性疾患治療剤を提供する。
本発明はまた、ヒアルロン酸と亜鉛との複合体（会合物）を含んで成る組織破壊酵素ＭＭＰ−９抑制剤を提供する。
本発明はまた、関節性疾患治療剤の製造のための、ヒアルロン酸と亜鉛との複合体（会合物）の使用に関する。
本発明はさらに、組織破壊酵素ＭＭＰ−９抑制剤の製造のための、ヒアルロン酸と亜鉛との複合体（会合物）の使用に関する。
本発明はさらに、関節性疾患を有する患者に、ヒアルロン酸と亜鉛との複合体（会合物）を投与することを特徴とする関節性疾患の治療方法を開示する。
本発明はさらに、組織破壊酵素ＭＭＰ−９の産生が亢進している患者に、ヒアルロン酸と亜鉛との複合体（会合物）を投与することを特徴とする、組織破壊酵素ＭＭＰ−９の生産亢進を抑制する方法を開示する。
【図面の簡単な説明】
図１は、慢性関節リウマチ患者からの滑膜細胞（ＲＡＳＣ）に対する、ＨＡ／Ｚｎ、Ｎａ−ＨＡ及びＺｎの増殖阻害効果を示すグラフである。
図２は、変形性関節症患者からの滑膜細胞（ＯＡＳＣ）に対する、ＨＡ／Ｚｎ、Ｎａ−ＨＡ及びＺｎの増殖阻害効果を示すグラフである。
図３は、外傷患者からの滑膜細胞（ＴＡＳＣ）に対するＨＡ／Ｚｎ、Ｎａ−ＨＡ及びＺｎの増殖阻害効果を示すグラフである。
発明の実施の形態
本発明の活性成分は、ヒアルロン酸と亜鉛との複合体（会合物）である。ヒアルロン酸は通常ナトリウム塩として存在し、Meyerら、J. Biol. Chem. Vol.107, p.629 (1934)により記載された巨大分子である。ヒアルロン酸は、β１，３−グルクロン酸成分とβ１，４−グルコサミン成分とを交互に有する高粘性グルコサミノグリカンであり、その分子量は５０kD〜数百万Ｄである。ヒアルロン酸はすべての哺乳類の結合組織に見出され、皮膚、目のガラス体、滑液、臍帯及び軟骨組織に高レベルで存在する。ヒアルロン酸は結合組織の基礎的な成分であるため、生物適合性であり、生物吸着性であり、且つ免疫原性でない。このため、ヒアルロン酸は、関節軟骨の潤滑性及び保護のごとき多くの生物学的機能を演じている。
本発明のヒアルロン酸と亜鉛との複合体（会合物）において、ヒアルロン酸と亜鉛との比率は、これらの成分が、滑膜細胞の増殖及び組織破壊酵素ＭＭＰ−９抑制について相乗的作用を発揮する範囲であり、重量比として、ヒアルロン酸：亜鉛が５：１〜２０：１の範囲であり、好ましくは約１０：１である。本発明において使用するヒアルロン酸と亜鉛との複合体の分子量は好ましくは１００kD〜２，０００kDであり、約１０００kDの分子量が好ましい。本発明の、ヒアルロン酸と亜鉛との複合体（会合物）は、例えばヒアルロン酸ナトリウムの水溶液と亜鉛塩、例えば塩化亜鉛の水溶液とを混合することにより製造することができる（ヨーロッパ特許明細書No.EP 0413016）。
多くの炎症性関節性疾患が、なんらかの原因による滑膜細胞の増殖と活性化により惹起されると考えられている。活性化された滑膜細胞はサイトカイン、プロスタグランジン、組織破壊酵素などのケミカルメディエーターを産生し、軟骨や骨の破壊を引き起こし、関節の炎症を生じさせる。組織破壊酵素（matrix metalloproteinase（ＭＭＰ）には、Ｉ型コラーゲン及びII型コラーゲンを分解するＭＭＰ−１、これらのコラーゲンに加えてさらに軟骨型プロテオグリカンをも分解するＭＭＰ−３、Ｉ型又はII型コラーゲンのゼラチン化したものをさらに低分子型コラーゲンに分解し、IV型コラーゲン、Ｖ型コラーゲン、XI型コラーゲン及び軟骨型プロテオグリカンを分解するＭＭＰ−９（Sapata, I., et al., Biochem. Biophys. Acta. Vol 370, p.510−523, 1974；Murphy, G. et al., Biochem. J. Vol.203, p.209−221, 1982；Morel, F., et al., Biochem.Biophys. Res. Commun. Vol.191, p.269−274, 1993；Hibbs, M.S., Matrix Supple. Vol.1, p.51−57, 1992；Murphy, G. et al., Biochem. J. Vol.277, p.277−279, 1991；Hibbs, M. S., et al., J. Biol. Chem. Vol.260, p.2493−2500, 1995）等が含まれ、これらが関節破壊に関与するものと考えられる。
慢性関節リウマチ、変形性関節症、及び外傷患者からの滑膜細胞はインビトロにおいて増殖するが、これらの滑膜細胞の増殖が１００〜３００μｇ／mlの濃度の本発明のヒアルロン酸／亜鉛複合体により濃度依存的に阻害される。従って、本発明のヒアルロン酸／亜鉛複合体は、慢性関節リウマチ、変形性関節症、外傷性関節疾患、関節水症、急速性破壊型股関節症等の関節性疾患の治療のために有用である。滑膜細胞の増殖を阻害することができれば慢性関節リウマチの対症的な治療が可能であると考えられる（Jae Unexcad Pats Publication No. 7-145062）。
慢性関節リウマチ、変形関節症、及び外傷患者からの滑膜細胞のインビトロ培養において、慢性関節リウマチ由来の滑膜細胞からの組織破壊酵素ＭＭＰ−９は１００μｇ／ml以下の本発明のヒアルロン酸／亜鉛複合体（会合物）により抑制される。従って、本発明のヒアルロン酸／亜鉛複合体（会合物）は、慢性関節リウマチにおける滑膜細胞からの組織破壊酵素ＭＭＰ−９抑制剤として有用である。ここで、前記組織破壊酵素ＭＭＰ−９抑制剤は、例えば、インスリン依存性増殖糖尿病網膜症治療剤、又は悪性腫瘍浸潤もしくは転移に対する抑制剤である。
実施例において示すごとく、ヒアルロン酸ナトリウムは、滑膜細胞の増殖を実質的に阻害せず、また組織破壊酵素ＭＭＰ−９を実質的に抑制しない。また、亜鉛塩は、滑膜細胞の増殖を阻害し、且つ組織破壊酵素ＭＭＰ−９を抑制するが、本発明のヒアルロン酸／亜鉛複合体（会合物）は亜鉛塩より強力に滑膜細胞の増殖を阻害し、且つ組織破壊酵素ＭＭＰ−９を抑制する。このことは、上記の効果の場合に増強相乗作用を示すことを意味する。
本発明のヒアルロン酸／亜鉛複合体（会合物）としては、通常医薬品で行なわれている投与方法を用いることができる。すなわち経口投与及び非経口投与が可能であり、注射により投与するのが特に好ましい。本発明のヒアルロン酸／亜鉛複合体（会合物）の有効投与量は１人１回各関節当り、０．０１ｇ〜１mg、好ましくは０．０３mg〜０．５mgである。本発明のヒアルロン酸／亜鉛複合体（会合物）は、物理的最大投与量２００mg／kgでラット及びマウスに皮下投与しても、全身性の毒性症状は認められなかった。
本発明の関節性疾患治療剤及び組織破壊酵素ＭＭＰ−９抑制剤は、常用の医薬キャリヤーと共に、本発明のヒアルロン酸／亜鉛複合体（会合物）を０．００１％〜１％、好ましくは０．０１％〜０．５％含有することができる。常用の医薬キャリヤーとしては、例えば、キトサン、でんぷん、ペクチン、ＨＰＭＣ、アルギン酸ナトリウムなどの医療用に一般的に用いられる基剤、トラガント末、アラビアゴム、トウモロコシデンプン又はゼラチンのような結合剤、結晶セルロース、マンニトールのような賦形剤、トウモロコシデンプン、アルファ化デンプン、アルギン酸などのような崩壊剤、ステアリン酸マグネシウムのような潤滑剤、ショ糖、乳糖又はサッカリンのような甘味剤、ペパーミント、ハッカ油又はチェリーのような香味剤、防腐剤等が挙げられる。
実施例
次に、実施例により本発明をさらに具体的に説明する。
実施例１．滑膜細胞増殖阻害作用
慢性関節リウマチ（ＲＡ）患者からの滑膜細胞（ＲＡＳＣ）、変形性関節症患者（ＯＡ）からの滑膜細胞（ＯＡＳＣ）及び外傷患者（ＴＡ）からの潤滑細胞（ＴＡＳＣ）の増殖に対する、本発明のヒアルロン酸と亜鉛との複合体（会合物）（ＨＡ／Ｚｎ）、並びに比較対照としてのヒアルロン酸ナトリウム塩（Ｎａ−ＨＡ）及び亜鉛（Ｚｎ）の影響を試験した。
被験物質としてＨＡ／Ｚｎ（Ｌｏｔ．No. Ａ６５２４２、１％溶液、ゲデオン・リヒター社）、ヒアルロン酸ナトリウム（Ｎａ−ＨＡ；Ｌｏｔ．No. ＫＫ４００１、キューピー）及び塩化亜鉛（Ｚｎ；Ｌｏｔ．No. ＥＳＨ１４１３、和光純薬工業）を使用した。上記ＨＡ／Ｚｎ溶液（比重；ｄ20 20＝１．０１３４）に含まれるヒアルロン酸と亜鉛含量の定量値を予め求め、上記１％を１００としてヒアルロン酸が９９％（ヒアルロン酸ナトリウムとして換算した値は１０５．０％）、亜鉛が１．０７mg／mLであったことから、当該ＨＡ／Ｚｎ中のヒアルロン酸と亜鉛の重量比を１０：１（Ｎａ−ＨＡ：Ｚｎ）とみなした。被験物質の濃度はＨＡ／Ｚｎについては１０μｇ／ml、３０μｇ／ml、１００μｇ／ml、１５０μｇ／ml、２００μｇ／ml、２５０μｇ／ml及び３００μｇ／mlとし、Ｎａ−ＨＡはＨＡ／Ｚｎと同濃度とし、ＺｎはＨＡ／Ｚｎの濃度の１０分の１の濃度として、試験した。
滑膜組織は、慢性関節リウマチ（ＲＡ）患者（５例；６５．６±１０．６歳）及び変形性関節症（ＯＡ）患者（６例；７３．４±８．９歳）の膝関節滑膜組織は人工関節置換術施工時に、また外傷（ＴＡ）患者（６例；２５．６±６．７歳）の膝関節滑膜組織は関節鏡施工時に採取した。採取後、直ちにＤＭＥＭ（Dulbecco's modified Eagle's medium）の入ったチューブ（５０mL容遠沈管；ＩＷＡＫＩ）に入れ、４℃で保管した。その後、クリーンベンチ内で滑膜組織を洗浄し、滑膜以外の組織を除去し、滑膜を細切し、そして酵素処理（コラゲナーゼ、トリプシン；和光純薬工業）により滑膜細胞（ＳＣ）を単離させ試験に用いた。
ＳＣの培養は全て１０％の熱非動化（５６℃、３０分）したウシ胎児血清（ＦＣＳ；大日本製薬）及び抗生物質混液（ペニシリン１００ユニット／mL、ストレプトマイシン１００μｇ／mL、ファンギゾン（Fungizone）２５ng／mL）を含むＤＭＥＭを用いて行った。
滑膜細胞（ＳＣ）をマイクロプレート（９６ウエル；ＩＷＡＫＩ）に３．５５×１０3細胞／０．１mL／ウエル（１×１０4細胞／cm2）で播種し、５％ＣＯ2−９５％airの気相中３７℃にて２日間予備培養した。その後、種々の濃度に調製された前記薬物を含む培地に交換し、更に５％ＣＯ2−９５％空気の気相中３７℃にて薬物添加後１２日目まで培養した。培養期間中の培地交換は、薬物が含まれた培地を４日毎に交換した。増殖能の測定は薬物添加後、４，８及び１２日目に、ＭＴＴ法4)（新生化学実験講座１２、分子免疫学Ｉ−免疫細胞・サイトカイン−、３５８，１９８９）により行った。結果は細胞数と正の相関を示す吸光度で表した。
統計解析はBartlett法による５％の有意水準で分散の一様性の検定を行った。分散が一様な場合は一元配置による分散分析（１wayＡＮＯＶＡ）を行い、有意差が認められた場合はDunnett法あるいはTukey法にて平均値の多重比較検定を行った。分散が一様でない場合は、Kruskal-Wallisの検定を行い順位についてnon-parametricのDunnett法あるいはTukey法にて多重比較検定を行った。
ＲＡＳＣ，ＯＡＳＣ及びＴＡＳＣについて得られた結果を、それぞれ図１、図２及び図３に示す。これらの図において、薬物無添加群に対してｐ＜０．０５で有意差がある場合を「＊」で示し、ｐ＜０．０１で有意差がある場合を「＊＊」で示す。
これらの図から明らかな通り、Ｎａ−ＨＡを添加してもいずれの滑膜細胞の増殖も有意に阻害されなかったが、ＨＡ／Ｚｎの添加においては添加量依存的に、滑膜細胞の増殖の有意な阻害が生じた。またＺｎの添加においても添加量依存的に滑膜細胞増殖の有意な阻害が生じた。しかしながら、Ｚｎに比べてＨＡ／Ｚｎの方が滑膜細胞増殖阻害が大きく、Ｎａ−ＨＡにおいて有意な増殖阻害が生じないことを考慮すれば、ＨＡ／Ｚｎは、Ｎａ−ＨＡ及びＺｎ単独使用の場合に比して相乗作用を有することが確認された。
実施例２．滑膜細胞培養上清中の組織破壊酵素ＭＭＰ−９に対する抑制効果
滑膜細胞を培養ディッシュ（φ３５mm；ＩＷＡＫＩ）に１０×１０4cells／２mL／dish（１×１０4cells／cm2）で播種し、５％ＣＯ2−９５％airの気相中３７℃にて２日間予備培養した。その後、薬物を含んだ培地に置き換え、更に同条件下で８日間培養した培養上清を採取し、測定まで−８０℃で保存した。ＨＡ／Ｚｎ及びＮａ−ＨＡの添加濃度は１００μｇ／mL（終濃度）とし、Ｚｎの濃度は１０μｇ／mL（終濃度）となるようにした。
上清中の組織破壊酵素としてマトリックスメタプロテイナーゼＭＭＰ−９をＥＩＡ法（Enzyme Immunoassay法）を用いて測定した。結果は培養上清中の濃度として算出されたものを細胞当たりで検討するため、この時点でのＭＴＴ法による培養細胞の吸光度値で割った値に換算した。また、この値をさらにコントロール（薬物非添加群）を１００％として薬物添加群の個々の値をコントロールに対する相対値で表した。
その結果、対照（薬物無添加）を１００％とした場合、ＭＭＰ−９の産生は、ＨＡ／Ｚｎ添加では０％、Ｎａ−ＨＡ添加では１００％、Ｚｎ添加では５０％であった。この結果、ＨＡ／ＺｎのＭＭＰ−９抑制作用は、その構成成分であるＮａ−ＨＡ及びＺｎ単独に対して相乗的であることが確認された。
以上の通り、本発明のヒアルロン酸と亜鉛との複合体（会合物）は、その構成成分であるヒアルロン酸及びＺｎに比較して、相乗的に、滑膜細胞の増殖に対する阻害作用及び滑膜細胞により生産される組織破壊酵素ＭＭＰ−９抑制作用を有し、慢性関節リウマチ等の関節性疾患の治療剤として有用である。
注射用水８０mlにマンニトール又はブドウ糖を５ｇ加えて溶かした後、さらに、ヒアルロン酸亜鉛を少量ずつ加えて攪拌溶解した。溶液をろ過して、アンプルに充填した。このアンプルをシャワー減菌器（約１０５℃）により３０分間減菌して注射剤とした。
同様に実施例４の処方についても、本法に準じた操作で調整した。
BACKGROUND ART Articular diseases including rheumatoid arthritis, osteoarthritis, traumatic arthritis and the like are inflammatory diseases that cause destruction of cartilage and bone through inflammation of the synovial membrane. Inflamed synovium exhibits angiogenesis, lymphocyte wetting, and synoviocyte proliferation and activation. Activated synovial cells are thought to produce chemical mediators such as cytokines, prostaglandins, and tissue-destructing enzymes, and cause cartilage and bone destruction (Harris, BD, New England Journal of Medicine), Vol. 322, p. 1277-1289, (1990); Cash, JM, et. Al., New England Journal of Medicine), Vol. 330, p. 1368-1375 (1994)).
It is known that hyaluronic acid in the form of sodium salt is generally effective for the treatment of articular diseases (Reference: Makoto Yamamoto et al., “High-molecular sodium hyaluronate (NRD101) for clinical treatment of knee osteoarthritis” Evaluation ", Pharmacology and Treatment, Vol. 21, No. 3, p. 894-970, 199). Zinc is also known to be effective for the treatment of arthritis (A. Frigo et al., “Copper and Zinc in Inflammation”, Inflammation and drug therapy series, Vol. IV, Kluwer Academic Publishers, P 132-142 (1989)).
However, hyaluronic acid (generally in the form of the sodium salt) is known for the treatment of some articular diseases because of its biocompatibility and flow properties, but synovial cells It has substantially no inhibitory effect on the growth and activation of the. In addition, satisfactory therapeutic effects are not obtained even when zinc is used.
DISCLOSURE OF THE INVENTION Accordingly, the present invention effectively treats articular diseases through the proliferation and activation of synovial cells that constitute the onset mechanism of arthritic diseases such as rheumatoid arthritis, particularly through suppression of tissue destruction enzymes. It is intended to provide a pharmaceutical composition that can be made and its manufacture and use.
As a result of various studies to solve the above problems, the present inventors have found that hyaluronic acid (sodium salt) has almost no inhibitory effect on the proliferation and activation of synovial cells, and the proliferation of zinc synovial cells. In contrast, the inhibitory effect on activation, especially on the tissue destruction enzyme, is very low, whereas the complex (aggregate) of hyaluronic acid and zinc is caused by synergistic action of both components, ie, enhanced synergism or interaction. The present inventors have found the surprising fact that they have a very potent synovial cell proliferation inhibitory action and tissue destruction enzyme inhibitory action, and have completed the present invention.
Therefore, the present invention provides a therapeutic agent for joint diseases comprising a complex (aggregate) of hyaluronic acid and zinc.
The present invention also provides a tissue disrupting enzyme MMP-9 inhibitor comprising a complex (associate) of hyaluronic acid and zinc.
The present invention also relates to the use of a complex (aggregate) of hyaluronic acid and zinc for the manufacture of a therapeutic agent for arthritic diseases.
The present invention further relates to the use of a complex (associate) of hyaluronic acid and zinc for the production of a tissue disrupting enzyme MMP-9 inhibitor.
The present invention further discloses a method for treating an articular disease characterized by administering a complex (aggregate) of hyaluronic acid and zinc to a patient having an articular disease.
The present invention further comprises administering a complex (associate) of hyaluronic acid and zinc to a patient with increased production of tissue destruction enzyme MMP-9, and producing tissue destruction enzyme MMP-9 A method for inhibiting enhancement is disclosed.
[Brief description of the drawings]
FIG. 1 is a graph showing the growth inhibitory effect of HA / Zn, Na-HA and Zn on synovial cells (RASC) from patients with rheumatoid arthritis.
FIG. 2 is a graph showing the growth inhibitory effect of HA / Zn, Na-HA and Zn on synovial cells (OASC) from osteoarthritis patients.
FIG. 3 is a graph showing the growth inhibitory effect of HA / Zn, Na-HA and Zn on synovial cells (TASC) from trauma patients.
BEST MODE FOR CARRYING OUT THE INVENTION The active ingredient of the present invention is a complex (associate) of hyaluronic acid and zinc. Hyaluronic acid usually exists as a sodium salt and is a macromolecule described by Meyer et al., J. Biol. Chem. Vol. 107, p.629 (1934). Hyaluronic acid is a highly viscous glucosaminoglycan having a β1,3-glucuronic acid component and a β1,4-glucosamine component alternately, and has a molecular weight of 50 kD to several million D. Hyaluronic acid is found in all mammalian connective tissues and is present at high levels in skin, vitreous of eyes, synovial fluid, umbilical cord and cartilage tissue. Since hyaluronic acid is a fundamental component of connective tissue, it is biocompatible, biosorbable and not immunogenic. For this reason, hyaluronic acid performs many biological functions such as lubricity and protection of articular cartilage.
In the complex (aggregate) of hyaluronic acid and zinc of the present invention, the ratio of hyaluronic acid and zinc is such that these components exert synergistic effects on synovial cell proliferation and inhibition of tissue destruction enzyme MMP-9. The weight ratio of hyaluronic acid: zinc is in the range of 5: 1 to 20: 1, preferably about 10: 1. The molecular weight of the complex of hyaluronic acid and zinc used in the present invention is preferably 100 kD to 2,000 kD, and a molecular weight of about 1000 kD is preferred. The complex (associate) of hyaluronic acid and zinc of the present invention can be produced, for example, by mixing an aqueous solution of sodium hyaluronate and a zinc salt, such as an aqueous solution of zinc chloride (European Patent Specification No. .EP 0413016).
Many inflammatory joint diseases are believed to be caused by synovial cell proliferation and activation for some reason. Activated synovial cells produce chemical mediators such as cytokines, prostaglandins, and tissue destruction enzymes, causing cartilage and bone destruction, and causing joint inflammation. Matrix metalloproteinase (MMP) includes MMP-1 that degrades type I collagen and type II collagen, MMP-3, type I or type II collagen that further degrades cartilage proteoglycan in addition to these collagens. MMP-9 (Sapata, I., et al., Biochem. Biophys.) Which is further decomposed into a low molecular weight collagen and decomposes type IV collagen, type V collagen, type XI collagen and cartilage type proteoglycan. Acta. Vol 370, p.510-523, 1974; Murphy, G. et al., Biochem. J. Vol.203, p.209-221, 1982; Morel, F., et al., Biochem. Biophys. Res. Commun. Vol.191, p.269-274, 1993; Hibbs, MS, Matrix Supple. Vol.1, p.51-57, 1992; Murphy, G. et al., Biochem. J. Vol.277 , p.277-279, 1991; Hibbs, MS, et al., J. Biol. Chem. Vol. 260, p.2493-2500, 1995), etc., and these are considered to be involved in joint destruction. It is done.
Synovial cells from patients with rheumatoid arthritis, osteoarthritis, and trauma proliferate in vitro, but the proliferation of these synovial cells is achieved by the hyaluronic acid / zinc complex of the present invention at a concentration of 100-300 μg / ml. It is inhibited in a concentration-dependent manner. Therefore, the hyaluronic acid / zinc complex of the present invention is useful for the treatment of arthritic diseases such as rheumatoid arthritis, osteoarthritis, traumatic joint disease, arthritis, and rapid fracture hip arthritis. . If the proliferation of synovial cells can be inhibited, symptomatic treatment of rheumatoid arthritis is considered possible (Jae Unexcad Pats Publication No. 7-145062).
In in vitro culture of synovial cells from patients with rheumatoid arthritis, osteoarthritis, and trauma, tissue destruction enzyme MMP-9 from synovial cells derived from rheumatoid arthritis has a hyaluronic acid / zinc content of 100 μg / ml or less. It is suppressed by the complex (aggregate). Therefore, the hyaluronic acid / zinc complex (aggregate) of the present invention is useful as a tissue destruction enzyme MMP-9 inhibitor from synovial cells in rheumatoid arthritis. Here, the tissue destruction enzyme MMP-9 inhibitor is, for example, a therapeutic agent for insulin-dependent proliferative diabetic retinopathy or an inhibitor against malignant tumor invasion or metastasis.
As shown in the Examples, sodium hyaluronate does not substantially inhibit synoviocyte proliferation and does not substantially inhibit the tissue disrupting enzyme MMP-9. In addition, the zinc salt inhibits the proliferation of synovial cells and suppresses the tissue destruction enzyme MMP-9, but the hyaluronic acid / zinc complex (associate) of the present invention is stronger in synovial cells than the zinc salt. Inhibits proliferation and suppresses the tissue disrupting enzyme MMP-9. This means that in the case of the above effects, it exhibits enhanced synergism.
As the hyaluronic acid / zinc complex (associate) of the present invention, the administration method usually used in pharmaceuticals can be used. That is, oral administration and parenteral administration are possible, and administration by injection is particularly preferable. The effective dose of the hyaluronic acid / zinc complex (associate) of the present invention is 0.01 g to 1 mg, preferably 0.03 mg to 0.5 mg per joint once per person. When the hyaluronic acid / zinc complex (aggregate) of the present invention was subcutaneously administered to rats and mice at a physical maximum dose of 200 mg / kg, no systemic toxic symptoms were observed.
The therapeutic agent for joint diseases and the inhibitor of tissue destruction enzyme MMP-9 of the present invention contain 0.001% to 1%, preferably 0, of the hyaluronic acid / zinc complex (associate) of the present invention together with a conventional pharmaceutical carrier. 0.01% to 0.5% can be contained. Conventional pharmaceutical carriers include, for example, chitosan, starch, pectin, HPMC, sodium alginate and other commonly used medical bases, binders such as tragacanth powder, gum arabic, corn starch or gelatin, crystalline cellulose , Excipients such as mannitol, disintegrants such as corn starch, pregelatinized starch, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, peppermint, mint oil or Examples include flavoring agents such as cherries and preservatives.
EXAMPLES Next, the present invention will be described more specifically with reference to examples.
Example 1 . Synovial cell proliferation inhibitory effect Synovial cells (RASC) from rheumatoid arthritis (RA) patients, synovial cells (OASC) from osteoarthritis patients (OA), and lubricating cells (TASC) from trauma patients (TA) The effect of the hyaluronic acid-zinc complex (associate) of the present invention (HA / Zn) and hyaluronic acid sodium salt (Na-HA) and zinc (Zn) as comparative controls on the growth of .
As test substances, HA / Zn (Lot. No. A65242, 1% solution, Gedeon Richter), sodium hyaluronate (Na-HA; Lot. No. KK4001, Kewpie) and zinc chloride (Zn; Lot. No. ESH1413) , Wako Pure Chemical Industries). Quantitative values of hyaluronic acid and zinc content contained in the HA / Zn solution (specific gravity; d 20 20 = 1.0134) are obtained in advance, and 1% is 100 and hyaluronic acid is 99% (value converted as sodium hyaluronate). 105.0%), and zinc was 1.07 mg / mL. Therefore, the weight ratio of hyaluronic acid and zinc in the HA / Zn was considered to be 10: 1 (Na-HA: Zn). The concentration of the test substance is 10 μg / ml, 30 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml and 300 μg / ml for HA / Zn, and Na-HA has the same concentration as HA / Zn. , Zn was tested as one-tenth the concentration of HA / Zn.
Synovial tissue is the knee joint of rheumatoid arthritis (RA) patients (5; 65.6 ± 10.6 years) and osteoarthritis (OA) patients (6; 73.4 ± 8.9 years). Synovial tissue was collected at the time of artificial joint replacement, and knee joint synovial tissue of trauma (TA) patients (6 cases; 25.6 ± 6.7 years old) was collected at the time of arthroscopic operation. Immediately after collection, it was placed in a tube (50 mL centrifuge tube; IWAKI) containing DMEM (Dulbecco's modified Eagle's medium) and stored at 4 ° C. Thereafter, the synovial tissue is washed in a clean bench, tissues other than the synovial membrane are removed, the synovial membrane is minced, and synovial cells (SC) are removed by enzyme treatment (collagenase, trypsin; Wako Pure Chemical Industries). Isolated and used for testing.
All SC cultures were 10% heat-immobilized (56 ° C., 30 minutes) fetal bovine serum (FCS; Dainippon Pharmaceutical) and antibiotics mixture (
Synoviocytes (SC) were seeded in microplates (96 wells; IWAKI) at 3.55 × 10 3 cells / 0.1 mL / well (1 × 10 4 cells / cm 2 ) and 5% CO 2 -95%. Pre-cultured at 37 ° C. in air for 2 days. Thereafter, the medium was replaced with a medium containing the drug prepared in various concentrations, and further cultured at 37 ° C. in a gas phase of 5% CO 2 -95% air until 12 days after the addition of the drug. During the culture period, the medium containing the drug was replaced every 4 days. The proliferative ability was measured by the MTT method 4) (New Chemistry Experiment Course 12, Molecular Immunology I-Immune Cell / Cytokine-358, 1989) on days 4, 8 and 12 after drug addition. The results were expressed as absorbance showing a positive correlation with the number of cells.
Statistical analysis was performed to test the uniformity of variance at the 5% significance level by the Bartlett method. A one-way analysis of variance (1 way ANOVA) was performed when the variance was uniform, and when a significant difference was observed, a multiple comparison test of the average value was performed by the Dunnett method or Tukey method. When the variance was not uniform, the Kruskal-Wallis test was performed, and the multiple comparison test was performed on the rank by the non-parametric Dunnett method or Tukey method.
The results obtained for RASC, OASC and TASC are shown in FIG. 1, FIG. 2 and FIG. 3, respectively. In these figures, the case where there is a significant difference at p <0.05 with respect to the drug-free group is indicated by “*”, and the case where there is a significant difference at p <0.01 is indicated by “**”.
As is clear from these figures, the addition of Na-HA did not significantly inhibit the proliferation of any synovial cells, but the addition of HA / Zn depends on the amount of addition, and the proliferation of synovial cells Significant inhibition occurred. In addition, significant inhibition of synoviocyte proliferation occurred depending on the amount added even when Zn was added. However, considering that synovial cell growth inhibition is greater in HA / Zn than in Zn, and that significant growth inhibition does not occur in Na-HA, HA / Zn uses Na-HA and Zn alone. It was confirmed to have a synergistic effect compared to the case.
Example 2 . Inhibitory effect on tissue disrupting enzyme MMP-9 in synovial cell culture supernatant Synoviocytes were cultured in culture dishes (φ35 mm; IWAKI) at 10 × 10 4 cells / 2 mL / dish (1 × 10 4 cells / cm). 2 ) and precultured for 2 days at 37 ° C. in a gas phase of 5% CO 2 -95% air. Thereafter, the medium was replaced with a drug-containing medium, and the culture supernatant cultured for 8 days under the same conditions was collected and stored at −80 ° C. until measurement. The addition concentrations of HA / Zn and Na-HA were 100 μg / mL (final concentration), and the Zn concentration was 10 μg / mL (final concentration).
Matrix metaproteinase MMP-9 was measured as a tissue disrupting enzyme in the supernatant using the EIA method (Enzyme Immunoassay method). The result was converted to a value divided by the absorbance value of the cultured cell by the MTT method at this time point in order to examine the concentration calculated in the culture supernatant per cell. In addition, this value was further expressed as a relative value with respect to the control, with the control (non-drug group) as 100%, and the individual values in the drug group.
As a result, when the control (no drug added) was 100%, the production of MMP-9 was 0% when HA / Zn was added, 100% when Na-HA was added, and 50% when Zn was added. As a result, it was confirmed that the MMP-9 inhibitory action of HA / Zn is synergistic with Na-HA and Zn alone, which are the constituent components.
As described above, the complex (aggregate) of hyaluronic acid and zinc according to the present invention synergistically inhibits synovial cell proliferation and synovial as compared to hyaluronic acid and Zn, which are constituents thereof. It has the effect of inhibiting tissue destruction enzyme MMP-9 produced by cells and is useful as a therapeutic agent for arthritic diseases such as rheumatoid arthritis.
Similarly, the formulation of Example 4 was also adjusted by an operation according to this method.
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