JP4081281B2 - Preventive or therapeutic agent for hypertrophic scar, keloid or chronic joint inflammatory disease - Google Patents

Description

【００４９】
【表２】

【００５０】
病理組織像
ａ）移植腫瘍における抗エリスロポエチン抗体注入による変化
ｉ）Ｒ２処置でコラーゲンＩＩＩ型の線維束の断裂が著しい。断裂束は白血球やマクロファージ、ＮＫ様細胞の攻撃を受け融解、消失して線維のみになっている。線維芽細胞は核濃縮を示し、死細胞の集積も認められる（図４）。
ｉｉ）対照処置において、増殖したコラーゲン線維束の存在が認められる。線維芽細胞や線維細胞は変性を示してない（図５）。
ｂ）移植腫瘍ではコラーゲン束の近傍の血管は移植前と比べて、密度は少ない。ｉ）Ｒ２処置の腫瘍では内皮細胞死を認めた（図６）。
ｉｉ）対照処置の腫瘍では内皮細胞の存在が認められた（図７）。
ｃ）移植片の石灰沈着
ｉ）Ｒ２処置では全てに石灰沈着を認め、４番では腫隆の３／４の組織が石灰化を示し、石灰化近傍のコラーゲン束は断裂し、断裂コラーゲンが石灰化していく像であった（図８）。
ｉｉ）生理食塩水処置では１腫瘍に認められたが、石灰化近傍のコラーゲン束は変化なく増殖像を示した。
これらの結果を表３にまとめた。無処置群、対照群では組織の変化は殆ど無かったが、抗エリスロポエチン抗体処置群では、線維芽細胞の変性、コラーゲン束の断裂、消失、石灰質沈着から組織石灰化、膠原質産生の抑制を誘導した。これらの結果より、抗エリスロポエチン抗体はケロイドの増殖を抑制することが確認された。
【００５１】
【表３】

【００５２】
実施例７
エリスロポエチン拮抗物質（エリスロポエチン受容体蛋白質）のケロイドに対する効果
実施例６と同様に、抗エリスロポエチン抗体の代わりに実施例２で得られたエリスロポエチン受容体蛋白質を用いて同様の実験を行った。抗体を用いた時の結果と同様に、エリスロポエチン受容体蛋白質もケロイドの増殖を抑えることが確認された。
【００５３】
実施例８
エリスロポエチン拮抗物質（抗エリスロポエチン抗体）の関節リウマチに対する効果
１）免疫染色
組織検索材料として、関節リウマチ患者５症例より、手術により摘出された増殖性滑膜片を約２０ｍｍ×１０ｍｍ×５ｍｍの大きさに細切し、固定液（Zamboni液； Zamboni L. and DeMartino C., J. Cell Biot., Vol.35, p148A（1967））にて６時間固定後、２５％蔗糖添加ＰＢＳに入れ替え、冷暗所にて保存した。これらの試料片をＯＣＴ剤（マイル社）で包埋し、液体窒素で凍結し、クリオスタットで７μｍの凍結切片とした。切片を、第１抗体としてｉ）抗エリスロポエチン受容体抗体（Yasuda Y. et al., Develop. Growth Differ., Vol.35, pp711-722（1993））、ii）抗ＩＬ１α抗体（Genzyme社）、iii）抗コラーゲンＩＩＩ抗体（Chemicon社）、iv）抗エストロゲン受容体抗体（Chemicon社）、ｖ）抗第８因子抗体（Dako社）の５種類の抗体をそれぞれ反応させた。次に、ｉ）、iii）とｖ）で反応させたものには、ビオチン化抗ウサギ抗体（Vector社）を、また、ii）とiv）で反応させたものには、ビオチン化抗マウス抗体（Chemicon社）を第２抗体として反応させた。発色はＤＡＢ（同仁製薬、和光純薬）を用いて反応させ検鏡に供した。
【００５４】
２）器官培養
器官培養材料として、上記のうち３症例については、術後直ちに摘出試料を培養液（ＭＥＭ＋１０％ ＦＣＳ、ＰＣ １００Ｕ／ｍｌ、ＳＭ１００μｇ／ｍｌ）に入れ洗浄し、洗浄後同液中で培養開始まで冷暗所に保存した。
このようにして準備された新鮮滑膜片（術後１０〜２４時間以内）を２×５×１０ｍｍの大きさ、重量５０〜１００ｍｇに細切し、１個の組織片を６０ｍｍのシャーレ（３０４２；Falcon社）に入れ、重量を測定した。このシャーレに、実施例１で得られた抗エリスロポエチン抗体（Ｒ２）の生理食塩水溶液１６ｍｇ／ｍｌにEvans blueを０．２５％加えて着色した溶液を注入した。また、対照としては、Evans blueを加えた生理食塩水（大塚製薬社）を用いた。注入は、ハミルトンの３２ゲージ注射針（９０１３１）と注射筒（７２５）を用いて、組織重量ｍｇ当たり０．５μｌ、即ち０．５μｌ／ｍｇ重量のＲ２あるいは生理食塩水を、組織片に複数ヵ所注入した。注入後シャーレに蓋をして、５％ＣＯ_２、９５％空気、３７℃で、６０分シャーレ内で反応させた。この操作を繰り返して、Ｒ２あるいは生理食塩水処理を３ないし４回処理した後、組織片をＭＥＭ＋１０％ ＦＣＳの培養液で２４穴の培養皿（３０４７；ファルコン社）で、１片ずつメッシュ（３０１４；ファルコン社）にのせ、５％ＣＯ_２、９５％空気、３７℃の条件で６時間から８時間、器官培養した。尚、培養時間はＲ２処理開始時より１２時間をもって終了するため、時間差がある。
培養片をZamboni液で固定し、６時間後に２５％蔗糖添加ＰＢＳに入れ替え冷暗所に保存し、病理組織検索用に供した。組織検索の結果を以下に示す。
【００５５】
関節リウマチの滑膜組織の特性
１）滑膜絨毛におけるエリスロポエチン反応部位
膠原質を産生する線維芽細胞、線維束や滑膜絨毛に浸潤するマクロファージ、単球、および毛細血管内皮細胞がエリスロポエチン反応部位である（図９、１０、１１）。
２）滑膜絨毛におけるＩＬ１αの発現
膠原質を産生する線維芽細胞、浸潤している単球、マクロファージ、リンパ球に認められる（図１２）。
３）滑膜絨毛の血管分布
絨毛には毛細血管と細血管が多数存在し、多数の単球、リンパ球が血管周辺に浸潤している（図１３）。
４）滑膜絨毛の膠原質
絨毛の膠原質はコラーゲンＩＩＩを主体とした線維に富んでする。線維芽細胞は卵型の網目状の染色質を持つ核でコラーゲンＩＩＩを持つ細胞質を示す（図１４）。
５）滑膜絨毛のエストロゲン受容体の発現
絨毛においてエストロゲン反応部位（図１５右）はエリスロポエチン受容体（図１５、１６左）とＩＬ１α陽性部位（図１６右）に相当し、これら３者は共存している（図１５、１６）。
【００５６】
Ｒ２処置による滑膜絨毛片の変化
１）線維芽細胞の死
Ｒ２の３回処置（図１７）および４回処置（図１８）で、多数の死んだ線維芽細胞が認められた。核は網目状の染色質が凝縮・濃縮され、細胞質は消失していた。この現象はＲ２の４回処置において、３回処置よりも顕著であり、アポトーシス死と考えられる。対照群では、線維芽細胞は変化を示さなかった（図１９）。
２）単球、リンパ球、マクロファージの死
細胞浸潤の多い毛細血管周辺で多数の核濃縮で死亡したリンパ球、単球マクロファージを認めた（図２０）。この現象はＲ２の４回処置の方が程度が強く（図２０）、培養片のＩＬ１α陽性細胞の減少を認めた。対照群の培養片ではこれらの浸潤細胞に変化を認めなかった（図２１）。
３）毛細血管の崩壊
Ｒ２の３回処置および４回処置において毛細血管の消失を認めた（図２２）。対照群では、多数の毛細血管が存在していた（図２３）。
４）コラーゲン線維の変化
Ｒ２の３回処置（図１７）および４回処置（図１８）では、コラーゲン束の断裂と希薄を認めた。対照群の組織片では、コラーゲン線維の断裂や希薄は認めなかった（図１９）。
以上の結果を表４にまとめた。抗エリスロポエチン抗体は、関節リウマチ等の疾患の主要症状である滑膜の増殖に伴う膠原質産生の抑制（線維芽細胞の変性、壊死、コラーゲン束の消失）、毛細血管の消失、ＩＬ−１分泌細胞の壊死（単球、マクロファージの壊死）を誘導した。これらの結果より、抗エリスロポエチン抗体は、関節リウマチの炎症を抑えることが確認された。
【００５７】
【表４】

【００５８】
実施例９
エリスロポエチン拮抗物質（エリスロポエチン受容体蛋白質）の関節リウマチに対する効果
実施例８と同様に、抗エリスロポエチン抗体の代わりにエリスロポエチン受容体蛋白質を用いて同様の実験を行った。この結果、抗体を用いた時の結果と同様に、エリスロポエチン受容体蛋白質も炎症を抑えることが確認された。
【００５９】
実施例１０
ケロイド組織サンプルにおけるエリスロポエチンおよびエリスロポエチン受容体ｍＲＮＡの発現
ケロイド瘢痕５例、萎縮瘢痕１例および赤色瘢痕３例の組織よりＲＮＡを抽出し、それらのＲＮＡを用いてＲＴ−ＰＣＲ法により、エリスロポエチンおよびエリスロポエチン受容体ｍＲＮＡの検出を試みた。 全例においてエリスロポエチン受容体ｍＲＮＡの発現が認められた。エリスロポエチンｍＲＮＡはケロイド瘢痕および赤色瘢痕に発現が認められたが萎縮瘢痕では非常に弱い、あるいは無い発現であった（図２４）。採取ＲＮＡの状態はベータアクチンのｍＲＮＡの増幅が弱くないので、良好と考えられる。
【００６０】
実施例１１
滑膜組織におけるエリスロポエチンおよびエリスロポエチン受容体ｍＲＮＡの発現
８例の患者の滑膜よりＲＮＡを抽出してＲＴ−ＰＣＲ法によりそれぞれのｍＲＮＡの検出を試みたところ、発現に強弱はあるが、全例にエリスロポエチンとその受容体の発現が認められた（図２５）。
【００６１】
実施例１２
滑液中のエリスロポエチン含有量
リウマチ患者９例と変形性関節症の患者５例の滑液を用いて、総タンパク量（ｍｇ／ｍｌ）とエリスロポエチン量（ｍＵ／ｍｌ）を測定したところ、両症例においてタンパク量には変化がなかったがエリスロポエチン量は有意にリウマチ症例で変形性関節症よりも高値を示した。
結果を表５に示す。
【表５】

【００６２】
【発明の効果】
以上から明らかなように、本発明により、肥厚性瘢痕もしくはケロイド等の膠原質性過剰増殖、または関節リウマチ等の慢性関節炎症性疾患に対し優れた効果を有する予防・治療剤が提供される。
【図面の簡単な説明】
【図１】 患者より摘出したケロイド片における多数の線維芽細胞のエリスロポエチン受容体抗体に対する陽性反応を示す顕微鏡写真を表す図である。図中、←は線維芽細胞を、矢尻は血管内皮細胞を、＊は線維束の部域を示す。（倍率：１８０倍）
【図２】 患者より摘出したケロイド片における多数の線維芽細胞のコラーゲンＩＩＩ型抗体陽性反応を示す顕微鏡写真を表す図である。図中、←は線維束を、小さい矢印は線維芽細胞を示す。（倍率：１８０倍）
【図３】 患者より摘出したケロイド片における第８因子抗体に陽性反応を示す多数の毛細血管の分布を示す顕微鏡写真を表す図である。図中、←は血管内皮細胞を示す。（倍率：１８０倍）
【図４】 ヌードマウスに移植したケロイド腫瘍にエリスロポエチン抗体を３回連続投与し、１週間後に摘出した腫瘍片の、石灰化組織の周辺部の組織像を示す顕微鏡写真を表す図である。図中、矢尻は断裂して小さく分解されたコラーゲン像を、＊は分解して消失していくコラーゲン像を示す。（倍率：３６０倍）
【図５】 ヌードマウスに移植したケロイド腫瘍にマウス血清を３回連続投与し、１週間後に摘出した腫瘍片の組織像を示す顕微鏡写真を表す図である。図中、←はコラーゲン束の１束を示す。（倍率：３６０倍）
【図６】 図４と同一標本（Ｒ２の３回処置）の第８因子抗体陽性反応を示す顕微鏡写真を表す図である。図中、←は点状に粉砕された内皮細胞の核断片を示す。（倍率：７２０倍）
【図７】 図５と同一標本（マウス血清３回処置）の第８因子抗体陽性反応を示す顕微鏡写真を表す図である。図中、←は内皮細胞質の突起を示す。（倍率：７２０倍）
【図８】 図４と同一標本（Ｒ２の３回処置）のｖａｎ Ｋｏｓｓａ染色により、組織石灰化の組織像を示す顕微鏡写真を表す図である。図中、←はコラーゲン断裂塊の石灰沈着を示す。（倍率：７２倍）
【図９】 患者Ａより摘出した滑膜絨毛片における、エリスロポエチン受容体抗体に陽性部位を示す細胞（矢印）と細胞性浸潤を示す顕微鏡写真を表す図である。図中、←は線維芽細胞を、←←はマクロファージを示す。（倍率：１５０倍）
【図１０】 患者Ｂより摘出した滑膜絨毛片のエリスロポエチン受容体抗体に陽性部位を示す強拡大の像を示す顕微鏡写真を表す図である。図中、←は毛細血管周辺の単球を、←←はマクロファージを、矢尻は線維芽細胞を示す。（倍率：６００倍）
【図１１】 図１０と同一患者の組織切片のエリスロポエチン受容体抗体に陽性反応を示す線維芽細胞像を示す顕微鏡写真を表す図である。図中、←は線維芽細胞を示す。（倍率：６００倍）
【図１２】 図１０と同一患者の組織片のＩＬ１α抗体に陽性反応を示す線維芽細胞と浸潤細胞群を示す顕微鏡写真を表す図である。図中、←は線維芽細胞の核を、←←はマクロファージを示す。（倍率：３００倍）
【図１３】 図９と同一患者の組織切片の第８因子抗体に反応する毛細血管の存在を示す像を示す顕微鏡写真を表す図である。図中、←は第８因子抗体に陽性を示す毛細血管内皮細胞を示す。（倍率：１５０倍）
【図１４】 図１０と同一患者の組織片のコラーゲンＩＩＩ抗体に陽性反応を示す線維芽細胞像を示す顕微鏡写真を表す図である。図中、←は線維芽細胞を示す。（倍率：６００倍）
【図１５】 図１０と同一患者の組織片のエリスロポエチン受容体抗体（左図）とエストロゲン受容体α抗体（右図）の二重染色像を示す顕微鏡写真を表す図である。図中、←は線維芽細胞を、矢尻は単球を示す。（倍率：４８０倍）
【図１６】 図１０と同一患者の組織片のエリスロポエチン受容体抗体（左図）とＩＬ１α抗体（右図）の二重染色像を示す顕微鏡写真を表す図である。図中、←は線維芽細胞を、矢尻は単球を示す（倍率：４８０倍）
【図１７】 図９と同一患者の組織片にＲ２を３回、１時間間隔で処置（処置開始より１２時間後に固定したもののコラーゲンＩＩＩ抗体陽性部位を示す顕微鏡写真を表す図である。図中、←は死亡した線維芽細胞の核を、←←は死亡したマクロファージの核を示す。（倍率：１８０倍）
【図１８】 図１０と同一患者の組織片にＲ２を４回、１時間間隔で処置し、処置開始より１２時間後に固定したものの、コラーゲンＩＩＩ抗体陽性部位を示す顕微鏡写真を表す図である。図中、←は死亡した線維細胞の核を、矢尻は死亡細胞周辺のコラーゲン線維質の抜けた部位を示す。（倍率：１５０倍）
【図１９】 図１０と同一患者の組織片に生食を４回、１時間間隔で処置し、処置開始より１２時間後に固定したものの、コラーゲンＩＩＩ抗体陽性部位を示す顕微鏡写真を表す図である。図中、←は線維芽細胞の核を、←←はコラーゲンＩＩＩの線維束を示す。（倍率：３００倍）
【図２０】 図１８と同一培養切片（Ｒ２の４回処置）のＩＬ１α抗体陽性部位を示す顕微鏡写真を表す図である。図中、←は死亡した線維芽細胞の核を、←←は核の断片を示す。（倍率：２４０倍）
【図２１】 図１９と同一培養片（生食４回処置）のＩＬ１α抗体陽性部位を示す顕微鏡写真を表す図である。図中、←は線維芽細胞を、←←はマクロファージを示す。（倍率２４０倍）
【図２２】 図１７と同一培養片（Ｒ２の３回処置）の第８因子陽性部位を示す顕微鏡写真を表す図である。図中、←は破損している血管壁を、←←は核濃縮を示す内皮細胞の核を示す。（倍率：３６０倍）
【図２３】 図１９と同一培養片（生食４回処置）の第８因子陽性部位を示す顕微鏡写真を表す図である。図中、←は血管内皮細胞を、←←は単球を、矢尻は線維芽細胞の核を、矢尻２個はリンパ球を示す。（倍率：３６０倍）
【図２４】 ケロイド組織サンプルにおけるエリスロポエチンおよびエリスロポエチン受容体ｍＲＮＡの発現結果を示す電気泳動図である。図中、
１〜５レーンはケロイド瘢痕患者、６レーンは萎縮瘢痕、７から９レーンは赤色瘢痕を示す。
EPO RT＋は、逆転写酵素を加えたＲＮＡサンプルにおけるエリスロポエチンｃＤＮＡの増幅を示す。
EPO RT−は、逆転写酵素を加えなかったＲＮＡサンプルではエリスロポエチンｃＤＮＡの増幅は確認されなかったことを示す。
EPOR RT＋は、逆転写酵素を加えたＲＮＡサンプルにおけるエリスロポエチン受容体ｃＤＮＡの増幅を示す。
EPOR RT−は、逆転写酵素を加えなかったＲＮＡサンプルではエリスロポエチン受容体ｃＤＮＡの増幅は確認されなかったことを示す。
ベータアクチンは、サンプル間におけるＲＮＡ量差を考慮するため、内部標準としてベータアクチンを用い、その増幅を示している。
各バンドの上の表示は患者識別を示す。
【図２５】 滑膜組織のエリスロポエチンおよびエリスロポエチン受容体のｍＲＮＡ発現結果を示す電気泳動図である。図中、
EPO RT＋は、逆転写酵素を加えたＲＮＡサンプルにおけるエリスロポエチンｃＤＮＡの増幅を示す。
EPO RT−は、逆転写酵素を加えなかったＲＮＡサンプルではエリスロポエチンｃＤＮＡの増幅は確認されなかったことを示す。
EPOR RT＋は、逆転写酵素を加えたＲＮＡサンプルにおけるエリスロポエチン受容体ｃＤＮＡの増幅を示す。
EPOR RT−は、逆転写酵素を加えなかったＲＮＡサンプルではエリスロポエチン受容体ｃＤＮＡの増幅は確認されなかったことを示す。
ベータアクチンは、サンプル間におけるＲＮＡ量差を考慮するため、内部標準としてベータアクチンを用い、その増幅を示している。
各バンドの上の表示は患者識別を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a prophylactic / therapeutic agent for hypertrophic scar, keloid or chronic joint inflammatory disease containing an erythropoietin antagonist as an active ingredient. More specifically, the present invention relates to a prophylactic / therapeutic agent for hypertrophic scar, keloid or chronic joint inflammatory disease containing an erythropoietin antagonist such as anti-erythropoietin antibody or erythropoietin receptor protein as an active ingredient.
[0002]
[Prior art]
Hypertrophic scar (scar keloid) is an abnormal growth of scar tissue that occurs in burns, surgical wounds and skin defects after trauma. However, the total number of patients is expected to be enormous and statistics are not disclosed. Although postoperative hypertrophic scars of surgical wounds are covered by all surgical departments, it is quite likely that they have been left untreated for further treatment. Treatment includes tranilast, oral compression with sponge, local injection of steroid, surgical treatment such as Z-plasty and skin grafting. The therapeutic effects of tranilast and steroids are so gradual that highly hypertrophic scars must rely solely on surgical treatment. However, no ointment or injection that shows an effect by local administration has been known.
Unlike the hypertrophic scars described above, keloids (true keloids) are unexplained diseases, and it is generally said that the constitution is involved. In particular, it is an abnormal proliferation of collagen fibers that occurs frequently on the front chest and shoulders, and occurs in both cases with and without a history of surgical wounds or trauma. It is unclear whether it is increasing or not, but the number of cases is said to be very large. Treatment includes oral tranilast, sponge pressure therapy, surgical therapy, and radiation therapy, but none of these treatments are definitive due to a strong tendency to relapse. Ointments and injections for this disease are not yet known.
[0003]
Hypertrophic scars and keloids (sometimes collectively referred to as keloids) are progressively expanding scars that result from hyperproliferation of collagen (collagen) in the dermis and exhibit mass-like bulges. A hypertrophic scar is a scar that occurs confined to the area of the initial injury. The origin and cause of these scars are unknown, but it has been found that the accumulation of collagen fibers, the complex crossing of collagen fibers and the ratio of type III collagen to type I collagen (type III / type I) increase. .
[0004]
Rheumatoid arthritis increases in number with age. Therefore, the number of cases increases as the average age increases. Since approximately 0.5% of humans are believed to have this disease, it is estimated that there are approximately 30 million rheumatoid arthritis patients out of a global population of 5.9 billion (1998 UN statistics). In our country, the population is 120 million, so it is estimated that there are about 600,000 patients. Including non-typical rheumatoid arthritis diseases, 1-1.5% of the population is thought to be affected, accounting for approximately 2 million people. Furthermore, it has been increasing year by year due to extended life.
[0005]
Rheumatoid arthritis is mainly treated by surgery and drugs. Non-steroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, steroid drugs, etc. are used as drugs. Nonsteroidal anti-inflammatory drugs are often used first. Non-steroidal anti-inflammatory drugs reduce the degree of inflammation and provide an analgesic effect quickly, but cannot protect against progression of symptoms or destruction of joints. There are currently nine types of disease-modifying anti-rheumatic drugs that relieve abnormalities of rheumatism by correcting immune abnormalities, but they have no analgesic effect and are slow-acting. It is not effective. Steroids have the effect of reducing inflammation in rheumatism, but their effects, especially the analgesic effect, make it difficult to discontinue medication, and as a result, side effects can cause patients to shorten their prognosis . Since these drugs are not causal treatments for the onset of rheumatoid arthritis, the current situation is that they are not out of the so-called symptomatic treatment.
[0006]
Rheumatoid arthritis is a chronic joint inflammatory disease characterized by strong infiltration of synovial chorionic proliferation and thickening and lymphocytes into the synovial villi and monocytes and macrophages around proliferating capillaries . Furthermore, accompanying symptoms include inflammatory lesions and necrosis of the vascular endothelium. IL1 is produced and secreted by these infiltrating cells, and the pathological condition worsens and progresses further. Although the cause of this lesion is unknown, it is thought that symptoms can be improved by suppressing collagen production accompanying the proliferation of the synovium, which is the true form of the histopathological image, and killing IL1-secreting cells. It is also considered effective for rheumatism-related diseases that exhibit similar joint symptoms, chronic arthritic diseases such as arthritis due to collagen disease, and tendonitis.
[0007]
Erythropoietin (EPO) is involved in blood cell proliferation and differentiation, and unlike other cytokines, it is not produced in blood cells, but is produced in the kidney or liver and released into the blood. Erythropoietin acts on erythroblast burst-forming cells (BFU-E) and erythroid colony-forming cells (CFU-E) among erythroid progenitor cells, promotes their differentiation and proliferation, and induces erythrocyte production. (Krantz SB, Blood, Vol. 77, pp419-434 (1991)). When erythropoietin binds to the erythropoietin receptor present in the cell membrane of progenitor cells, a signal is transmitted into the cell nucleus, causing erythrocyte differentiation, that is, intracellular accumulation of globin mRNA, hemoglobin production, and erythrocyte differentiation ( D'Andrea ADet al., Cell, Vol. 57, pp277-285 (1989)). However, the details of the mechanism have not yet been elucidated, and there are many points to be solved in the future.
[0008]
As a site where erythropoietin expresses its gene in tissues other than kidney and liver, the embryo body just after implantation (Yasuda Y. et al., Development. Growth Differ., Vol. 35, pp711-722 (1993)) Human, monkey, and mouse brains (Marti HH et al., Eur. J. Neu. Sci., Vol. 8, pp666-676 (1996)) are known. In addition, the present inventors have found that the erythropoietin receptor gene is expressed in the decidua of the mouse other than the erythroblast system (Yasuda Y. et al., Development. Growth Differ., Vol. 35, pp711). -722 (1993)). The function of the erythropoietin or erythropoietin receptor gene in these sites other than the blood cell system has not been clarified at present.
[0009]
Erythropoietin is produced by gene recombination and is effectively used for the treatment of anemia, particularly for the treatment of anemia in renal dialysis patients and for the preparation of autologous blood transfusion during surgery. The erythropoietin receptor is expected to be used as an agonist for anemia or an antagonist such as erythrocytosis (WO90 / 08822), and it is described that it can be used for erythropoietin excess and hypererythropoietinemia. . In addition, there is a description that a substance that binds to an erythropoietin receptor in a specific domain can be used for the treatment of rheumatoid arthritis (WO 00/66632).
However, although erythropoietin antagonists (substances that bind to erythropoietin) such as anti-erythropoietin antibodies and erythropoietin receptor proteins are known to exhibit therapeutic effects on proliferative organ diseases such as cancer (Japanese Patent Laid-Open No. Hei 10). No. 101574), no known preventive / therapeutic effects on collagenous hyperproliferation such as hypertrophic scars or keloids, and chronic joint inflammatory diseases such as rheumatoid arthritis.
[0010]
[Problem to be Solved by the Invention]
Therefore, an object of the present invention is to provide a prophylactic / therapeutic agent having an excellent effect on collagenous hyperproliferation such as hypertrophic scar or keloid, or chronic joint inflammatory diseases such as rheumatoid arthritis.
[0011]
[Means for Solving the Invention]
In view of the above problems, the present inventors, as a result of diligent research, have an action to improve progressive hyperproliferation of collagen, particularly hypertrophic scars or keloids, etc., in anti-erythropoietin antibodies having an erythropoietin antagonistic action, and the like It has been found that there is an effect of improving rheumatoid arthritis and the like, and has completed the present invention.
[0012]
That is, the present invention
(1) A prophylactic / therapeutic agent for hypertrophic scars, keloids or chronic arthritic diseases, comprising as an active ingredient an erythropoietin antagonist (an erythropoietin antagonist is a substance capable of binding to erythropoietin),
(2) The agent according to (1) above, wherein the erythropoietin antagonist is an anti-erythropoietin antibody,
(3) The agent according to (1) above, wherein the erythropoietin antagonist is an erythropoietin receptor protein,
(4) The agent according to (3) above, wherein the erythropoietin receptor protein is a soluble erythropoietin receptor protein,
(5) The agent according to (1) above, wherein the chronic joint inflammatory disease is rheumatoid arthritis, rheumatoid related disease, arthritis due to collagen disease, or tendonitis
(6) Use of an erythropoietin antagonist for the manufacture of a prophylactic / therapeutic agent for hypertrophic scar, keloid or chronic joint inflammatory disease,
(7) The present invention provides a method for preventing / treating hypertrophic scars, keloids or chronic joint inflammatory diseases in a mammal, which comprises administering an effective amount of an erythropoietin antagonist to the mammal.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
An erythropoietin antagonist is used as a component, particularly an active component of the therapeutic agent for hypertrophic scar, keloid or chronic arthritic disease of the present invention (hereinafter abbreviated as “agent of the present invention”). The erythropoietin antagonist in the present invention generally has a binding strength to human erythropoietin with such a strength that an antibody binds to an antigen or a receptor binds to a ligand. Any substance that suppresses the production of secretion), that is, an erythropoietin antagonist may be used. Such erythropoietin antagonists include anti-erythropoietin antibodies and erythropoietin receptor proteins.
[0014]
The anti-erythropoietin antibody may be either a polyclonal antibody or a monoclonal antibody as long as it can recognize erythropoietin or a partial peptide thereof or a salt thereof.
The anti-erythropoietin antibody can be produced according to a known antibody or antiserum production method (for example, the method described in JP-A-1-228492) using erythropoietin or a partial peptide thereof or a salt thereof as an antigen.
The salt of erythropoietin or a partial peptide thereof includes a physiologically acceptable salt with an acid (eg, inorganic acid, organic acid, etc.) or a base (eg, alkali metal salt, etc.), and particularly a physiologically acceptable acid. Addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
Below, the general manufacturing method of an anti-erythropoietin antibody is demonstrated.
[0015]
[Production of monoclonal antibodies]
(A) Preparation of monoclonal antibody-producing cells
Erythropoietin or a partial peptide thereof or a salt thereof is administered to a mammal at a site where antibody production is possible by administration, or together with a carrier, a diluent and the like. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and the like, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with a confirmed antibody titer from a mouse, collect spleen or lymph nodes 2 to 5 days after the final immunization, A monoclonal antibody-producing hybridoma can be prepared by fusing the included antibody-producing cells with myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting a labeled erythropoietin described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.
Examples of myeloma cells include P3 / NSI / 1-Ag4-1, NS-1, P3U1, SP2 / 0 and the like, and P3 / NSI / 1-Ag4-1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.
[0016]
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, hybridoma culture supernatant is added to a solid phase (eg, microplate) on which an antigen such as erythropoietin is adsorbed directly or with a carrier, and then radioactive. A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A; Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which an anti-immunoglobulin antibody or protein A is adsorbed, erythropoietin labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected. .
The selection of the monoclonal antibody can be performed according to a known method or a method similar thereto, but can usually be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1-10% fetal bovine serum, or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
[0017]
(B) Purification of monoclonal antibodies
Separation and purification of monoclonal antibodies can be carried out in the same way as in the separation and purification of ordinary polyclonal antibodies (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchanger (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method for obtaining an antibody by dissociating the binding using an active adsorbent such as protein A or protein G) Can be done according to
[0018]
[Preparation of polyclonal antibody]
The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (antigen such as erythropoietin) and a carrier protein is prepared, a mammal is immunized in the same manner as the above monoclonal antibody production method, and an antibody-containing substance against erythropoietin is collected from the immunized animal. Thus, the antibody can be produced by separating and purifying the antibody.
Regarding the complex of immune antigen and carrier protein used to immunize mammals, the kind of carrier protein and the mixing ratio of carrier and hapten should be such that antibodies can be efficiently produced against hapten immunized by cross-linking to carrier. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin and the like are about 0.1 to 20 in weight ratio with respect to hapten 1. Preferably, a method of coupling at a ratio of about 1 to 5 is used.
Various coupling agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiopyridyl group are used.
The condensation product is administered to warm-blooded animals at the site where antibody production is possible or with a carrier, a diluent and the like. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration can usually be carried out about once every 2 to 6 weeks, about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc., preferably blood of mammals immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.
[0019]
The erythropoietin receptor protein is obtained by a known method (WO90 / 08822; JP-A-6-38787; Chemistry and Biology, 31 (4), pages 270-274 (1993); US Pat. No. 5,292,545, etc.) Can be obtained. Among the erythropoietin receptor proteins, soluble erythropoietin receptor protein is particularly preferably used. The soluble erythropoietin receptor protein can be obtained by a known method such as the method described in JP-A-6-38787.
These erythropoietin receptor protein and anti-erythropoietin antibody both bind to erythropoietin in an organ or tissue and have an action of blocking the binding between erythropoietin and erythropoietin receptor, and are considered to be functionally equivalent substances.
[0020]
As described above, the erythropoietin receptor protein is a known protein, and its DNA sequence and amino acid sequence are described in the above-mentioned patent publications and other documents. The erythropoietin receptor protein in the present invention is generally strong enough to bind the receptor to the ligand, preferably reversibly binds to human erythropoietin, and produces erythropoietin autocrine (autocrine) production. Any inhibitor may be used, that is, an erythropoietin antagonist. Accordingly, human erythropoietin receptor protein or erythropoietin affinity fragment and human erythropoietin receptor protein are about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably. It may be an erythropoietin receptor protein or a fragment thereof of a non-human mammal having homology of about 90% or more, most preferably about 95% or more, or an analog thereof.
[0021]
The agent of the present invention is parenterally administered to humans and animals (for example, topical administration, intravenous administration, transdermal administration, rectal administration, nasal administration, vaginal administration, oral cavity mucosal administration, transpulmonary mucosal administration, etc.) Safe to administer. Examples of the dosage form include injectable preparations, infusion preparations, suppositories, nasal preparations, sublingual preparations, transdermal absorption agents, external preparations, sustained release preparations, powder inhalants and the like. Among these preparations, solid preparations are prepared in accordance with known pharmacological production methods, and excipients (for example, lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, as pharmacologically acceptable carriers as preparations. , Light anhydrous silicic acid, etc.), stabilizer, colorant, surfactant, binder (for example, crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin , Methylcellulose, sodium carboxymethylcellulose, tragacanth, gum arabic, etc.), lubricants (magnesium stearate, calcium stearate, talc, colloidal silica, etc.), other additives, etc.
For liquid formulations, the pharmacologically effective amount of the active ingredient erythropoietin antagonist and pharmaceutically acceptable excipients / activators and solvents (eg water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil) And a mixture of amino acids (for example, basic amino acids such as arginine, lysine, histidine, ornithine, acidic amino acids such as aspartic acid, glutamic acid, etc.), saccharides (for example, glucose, D-sorbitol, D-mannitol) Etc.), excipients / activators (or isotonic agents) generally added to injectable compositions such as cellulose derivatives, inorganic salts (eg sodium chloride) and other organic / inorganic compounds Also good. Moreover, when preparing an injection using an active ingredient and these excipients / activators, a pH adjuster (for example, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), a buffering agent, if necessary (Eg, phosphate, acetate, carbonate, citrate, etc.), stabilizer (eg, human serum albumin, polyethylene glycol, polylactic acid, etc.), solubilizer (eg, alcohol (eg, ethanol), polyalcohol (eg. Eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TMHCO-50), soothing agents (eg benzalkonium chloride, procaine hydrochloride, etc.), preservatives (eg benzyl alcohol, phenol etc.), anticoagulants (eg dextran sulfate, heparin etc.), preservatives (eg Paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc.), antioxidants (eg sulfites, ascorbic acid, etc.), etc. may be added to make various injections by conventional methods. it can. The prepared injection solution is usually filled in a suitable ampoule. For administration, the above-mentioned composition for injection can be dissolved in a conventional aqueous diluent and used as a solution. Aqueous diluents include glucose aqueous solution, physiological saline, Ringer's solution, nutritional supplement solution and the like.
Since the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) can do.
[0022]
When phosphoric acid or a salt thereof is contained in the injection, the concentration of sodium phosphate or potassium phosphate in the injection is about 0.1 mM to 500 mM, preferably about 1 mM to 100 mM.
Examples of a sterile preparation include, but are not limited to, a method of sterilizing the entire manufacturing process, a method of sterilizing with gamma rays, a method of adding a preservative, and the like.
[0023]
The agent of the present invention can also be produced by dissolving a suspension in which an active ingredient is dispersed according to a conventional method and shaping into a microcapsule, spherical shape, rod shape, needle shape, pellet shape, film shape or the like. The agent of the present invention may be formed into a sustained release agent containing an active ingredient and a biodegradable polymer compound. The sustained-release agent can be prepared according to the method described in JP-A-9-263545.
[0024]
In the agent of the present invention, the content of the erythropoietin antagonist varies depending on the form of the preparation, but is usually about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, based on the whole preparation, Preferably, it is about 20 to 90% by weight.
In the agent of the present invention, the content of components other than the erythropoietin antagonist varies depending on the form of the preparation, but is usually about 10 to 99.9% by weight, preferably about 20 to 90% by weight, based on the whole preparation. It is.
[0025]
When the agent of the present invention is a prophylactic / therapeutic agent for hypertrophic scar and keloid, the agent of the present invention specifically includes, for example, post-operative scar, burn keloid, keloid, post-traumatic scar, post-coronary angioplasty It can be used for prevention, treatment and improvement of restenosis, hypertrophic scar pannus, etc., and it is particularly preferred that the agent of the present invention is directly administered locally as an external preparation such as an injection or ointment.
The external preparation may be solid, semi-solid, or liquid. For example, the solid external preparation may be an erythropoietin antagonist as it is or an excipient (eg, glycol, mannitol, starch, crystalline cellulose, etc.) It is manufactured by adding and mixing a sticky agent (for example, natural gums, cellulose derivatives, acrylic acid polymers, etc.) to obtain a powdery composition. In the case of a semi-solid external preparation, it is preferably used as an aqueous or oily gel or ointment. Liquid external preparations are almost the same as in the case of injections, and are produced by making them oily or aqueous suspensions. In addition to the above-mentioned solid, semi-solid or liquid external preparations, pH adjusters (for example, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), preservatives (for example, p-hydroxybenzoates, chlorobutanol, chloride) Benzalkonium etc.) may be added. Specifically, for example, petrolatum, lanolin and the like are used as an ointment containing about 0.1 to 100 mg, preferably about 1 to 50 mg of erythropoietin antagonist per gram, for the skin or mucous membrane.
The dose varies depending on the degree of disease, age, body weight, dosage form, administration method, administration period, administration target animal (eg, mammals such as humans, rats, mice, cats, dogs, rabbits, cows, pigs). About 0.1 to 100 mg, preferably about 1 to 50 mg, more preferably about 2 to 20 mg as an erythropoietin antagonist is administered about once to three times a day for an adult (body weight 60 kg) once per person. good.
[0026]
When the agent of the present invention is a prophylactic / therapeutic agent for chronic joint inflammatory diseases, the agent of the present invention can be used for all arthritis exhibiting inflammatory symptoms in joints, specifically arthritis due to rheumatoid arthritis, rheumatoid related diseases, collagen It can be used for prevention, treatment, and improvement of arthritic symptoms due to diseases, tendonitis, etc., and the agent of the present invention is particularly preferably administered directly locally as a liquid preparation such as an injection.
In the case of direct administration into a joint cavity as a local administration agent, it can be prepared by dispersing an active ingredient using a hyaluronic acid preparation for injection (for example, manufactured by Kaken Pharmaceutical Co., Ltd .: Alz Injection) as a dispersion medium. As the hyaluronic acid used in the dispersion medium, a non-toxic salt thereof may be used. Examples thereof include alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as magnesium and calcium. Is preferably used. Hyaluronic acid and its non-toxic salts are those having a molecular weight of about 200,000 to 5,000,000 (viscosity method), preferably about 500,000 to 3,000,000, more preferably about 700,000 to 2.5 million.
The final concentration of hyaluronic acid or sodium hyaluronate in this dispersant is preferably less than 1% (W / V) as the viscosity, and is preferred in terms of various operations and ease of administration, especially about 0.02 to less than 1%. And more preferably about 0.1 to 1% (W / V).
[0027]
The dispersion medium may contain a pH adjuster, a local anesthetic, an antibiotic, a solubilizing agent, an isotonic agent, an adsorption inhibitor, a glycosaminoglycan, a polysaccharide and the like by a method known per se. . Preferred examples thereof include mannitol, sorbitol, sodium chloride, glycine, ammonium acetate, or a water-soluble protein that can be injected into a body fluid without substantially showing pharmacological activity. Examples of the glycosaminoglycan include hyaluronic acid, chondroitin, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin, heparan sulfate, and ketan sulfate. Examples of the polysaccharide include acidic polysaccharides such as arginic acid.
The water-soluble protein is not particularly limited as long as it is soluble in water, physiological saline or buffer, and examples thereof include human serum albumin, human serum globulin, collagen, gelatin and the like. Examples of the pH adjuster include glycine, ammonium acetate, citric acid, hydrochloric acid, sodium hydroxide and the like. Examples of the local anesthetic include chlorobutanol and xylocaine hydrochloride. Examples of the antibiotic include gentamicin. Examples of the solubilizer include glycerin and polyethylene glycol 400. Examples of the isotonic agent include mannitol, sorbitol, sodium chloride and the like. Examples of the adsorption inhibitor include polyoxyethylene sorbitan monooleate.
Further, when the water-soluble protein is contained in the dispersion medium, the content of the water-soluble protein is preferably 0.05 to 50 mg, more preferably 0.5 to 20 mg, and more preferably, per single-dose preparation. 0.75-10 mg. The preparation may contain phosphoric acid or a salt thereof (for example, sodium phosphate, potassium phosphate, etc.).
[0028]
When the agent of the present invention is a prophylactic / therapeutic agent for chronic arthritic diseases, the dosage is the degree of disease, age, weight, dosage form, administration method, administration period, administration target animal (eg, human, rat, mouse, Depending on the cat, dog, rabbit, cow, pig, etc.), for example, in the shoulder or knee joint, a concentration of about 100-4000 μg / ml of erythropoietin antagonist per adult (body weight 60 kg) was prepared. The preparation may be administered about 1 to 10 ml at a time, preferably about 2.5 ml, once to about 3 times a day. About 0.5 to 5 ml, preferably about 1.5 ml for wrist, elbow and ankle joints, about 0.5 to 5 ml, preferably about 1 ml for extensor flexor tendon sheaths of hands and feet, phalangeal joints About 0.1 to 1 ml, preferably about 0.5 ml, may be injected once to three times a day.
[0029]
When the agent of the present invention is a sustained-release agent, the dosage is the type and content of erythropoietin antagonist, dosage form, duration of drug release, animal to be administered (eg, human, rat, mouse, cat, dog, rabbit) Mammals such as cattle and pigs), which vary depending on the purpose of administration. For example, when applied by parenteral administration, about 0.1 to about 100 mg of erythropoietin antagonist may be released from the dosage form per week. May be administered.
[0030]
The agent of the present invention may contain or use a pharmaceutical ingredient other than the erythropoietin antagonist as an active ingredient. Such pharmaceutically active ingredients include antiallergic agents (eg ketotifen, terfenadine, azelastine, epinastine etc.), antibacterial agents (eg cefixime, cefdinir, ofloxacin, tosufloxacin etc.), antifungal agents (eg fluconazole, itraconazole etc.), anti Inflammatory steroids (eg, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone), non-steroidal anti-inflammatory analgesics (eg, indomethacin, diclofenac, loxoprofen, ibuprofen, aspirin, piroxicam, sulindac, cyclooxygenase inhibitors (eg, celecoxib, rofecoxib, rofexoxib, rofecoxib, rofexib) Disease-modifying anti-rheumatic drugs and immunologics (eg methotrexate, leflunomide, program) , Sulfasalazine, D- penicillamine, oral gold agent, etc.), hyaluronic acid formulations (such as sodium hyaluronate, etc.) and the like. These components are not particularly limited as long as the object of the present invention is achieved, and may be either non-peptidic or peptidic, and can be used in an appropriate blending ratio.
[0031]
In the combined use of the agent of the present invention and the concomitant drug, the administration timing of the agent of the present invention and the concomitant drug is not limited, and the agent of the present invention and the concomitant drug may be administered simultaneously to the administration subject. Alternatively, administration may be performed with a time difference. The dose of the concomitant drug may be determined according to the dose used clinically, and can be appropriately selected depending on the administration subject, administration route, disease, combination and the like.
The administration mode of the agent of the present invention and the concomitant drug is not particularly limited as long as the agent of the present invention and the concomitant drug are combined at the time of administration. Examples of such administration forms include, for example, (1) administration of a single preparation obtained by simultaneously formulating the agent of the present invention and a concomitant drug, and (2) separate preparation of the agent of the present invention and the concomitant drug. Simultaneous administration of the two preparations obtained by the same administration by the same administration route, (3) with a time difference in the same administration route of the two preparations obtained by separately formulating the agent of the present invention and the concomitant drug (4) Simultaneous administration of two preparations obtained by separately formulating the agent of the present invention and a concomitant drug through different administration routes, (5) Formulating the agent of the present invention and the concomitant drug separately Administration of the two types of preparations obtained by the preparation at different time intervals (for example, administration in the order of the agent of the present invention → concomitant drug, or administration in the reverse order). Hereinafter, these administration forms are collectively abbreviated as the combination agent of the present invention.
[0032]
The concomitant drug of the present invention has low toxicity. For example, the agent of the present invention or (and) the above concomitant drug can be mixed with a pharmacologically acceptable carrier according to a method known per se to obtain a pharmaceutical composition. it can.
As the pharmacologically acceptable carrier that may be used in the production of the concomitant drug of the present invention, the same carriers as those used for the pharmaceutical composition of the present invention described above can be used.
When the agent of the present invention and the concomitant drug are formulated simultaneously and used as a single agent, the content of the erythropoietin antagonist in the concomitant drug of the present invention varies depending on the form of the formulation, but is usually about It is about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, and more preferably about 20 to 90% by weight.
The content of the concomitant drug in the concomitant drug of the present invention varies depending on the form of the preparation, but is usually about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, based on the whole preparation, More preferably, it is about 20 to 90% by weight.
In the concomitant drug of the present invention, the content of components other than the erythropoietin antagonist and concomitant drug varies depending on the form of the preparation, but is usually about 10 to 99.9% by weight, preferably about 20 to About 90% by weight.
The compounding ratio of the erythropoietin antagonist and the concomitant drug in the concomitant drug of the present invention can be appropriately selected depending on the administration subject, administration route, disease and the like.
The dose of the concomitant drug of the present invention varies depending on the type of erythropoietin antagonist and concomitant drug, administration route, symptom, patient age, etc. For example, when administered locally for the purpose of treating keloid, Weight 60 kg) About 0.1-100 mg, preferably about 1-50 mg, more preferably about 2-20 mg as an erythropoietin antagonist and concomitant drug once per person, divided into once to three times a day What is necessary is just to administer.
[0033]
The same content may be used when the erythropoietin antagonist and the concomitant drug contained in the agent of the present invention are formulated separately.
When the erythropoietin antagonist contained in the agent of the present invention and the concomitant drug are separately formulated and administered together, the agent of the present invention and the pharmaceutical composition containing the concomitant drug may be administered simultaneously. However, after the pharmaceutical composition containing the concomitant drug is administered first, the agent of the present invention may be administered, or the agent of the present invention is administered first, and then the pharmaceutical composition containing the concomitant drug is prepared. It may be administered. When administered at a time difference, the time difference varies depending on the active ingredient, dosage form, and administration method to be administered. For example, when a pharmaceutical composition containing a concomitant drug is administered first, a pharmaceutical composition containing the concomitant drug is selected. Examples include a method of administering the agent of the present invention within 1 minute to 3 days after administration, preferably within 10 minutes to 1 day, more preferably within 15 minutes to 1 hour. When the agent of the present invention is administered first, it contains the concomitant drug within 1 minute to 1 day, preferably within 10 minutes to 6 hours, more preferably within 15 minutes to 1 hour after administering the agent of the present invention. The method of administering a pharmaceutical composition is mentioned.
[0034]
【Example】
The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the present invention in any way.
[0035]
Example 1
Production of anti-erythropoietin antibodies
(1) Preparation of antigen EPO:
According to the method described in JP-A-60-41614, a PBS lysate of EPO separated from anemia patient urine (EPO sample obtained by SDS treatment of anemia patient urine concentrate, followed by antibody adsorption treatment and gel filtration) was obtained. EPO was precipitated by adding 9 times the amount of 99.5% ethanol cooled to −20 ° C. on ice. This precipitate was washed with a 90% ethanol solution at −20 ° C., dried under reduced pressure, and 0.01 mM CaCl.₂Was dissolved in a 10 mM Napi buffer (pH 6.8) containing EPO, passed through a hydroxyapatite column previously equilibrated with the same buffer, and EPO was recovered in the non-adsorbed fraction. The purity of the obtained EPO was 99%. Using this EPO, an anti-EPO antibody was prepared as follows according to the method described in JP-A-1-228492.
[0036]
(2) Immunization of experimental animals with the above EPO:
The purified EPO prepared by the above method was used as an antigen, and a mouse (BALB / c mouse) was used as an experimental animal. The mouse was immunized 3 times at 2-week intervals as follows.
First immunization:
0.2 ml of an emulsion obtained by dissolving purified EPO protein in PBS at 1 mg / ml and mixing an equal amount of Freund's complete adjuvant was administered to mice by intraperitoneal injection.
Second immunization:
Two weeks later, 100 μl of the same emulsion was administered to the mice by intraperitoneal injection.
Third immunization:
Purified EPO was dissolved in PBS at 0.5 mg / ml, and 100 μl was intraperitoneally administered to mice after 2 weeks.
[0037]
(3) Preparation of anti-EPO antibody-producing hybridoma:
i) Production of cell fusion
Three days after the completion of the immunization, the spleen cells of the immunized mouse are aseptically removed, washed with a mixed solution of synthetic medium (PRMI1640; Gibco BRL) and 15% fetal calf serum (FCS), and then in the mixed solution. Then, the spleen cells were shredded with scissors to make single cells, washed twice with the mixed solution, and then the single cells were dispersed in RPMI 1640 solution. Cell count is 8 × 10⁸It was a piece. Separately, mouse myeloma cells (P3 / NSI / 1-Ag4-1) were cultured in the above mixed solution of RPMI and FCS, and the proliferated cells were washed with RPMI1640 solution. Cell count is 4 × 10⁸It was a piece.
Next, the immunized mouse spleen cells and mouse myeloma cells prepared above were dispersed and mixed in RPMI 1640 solution, and then centrifuged to remove the supernatant. After the mixed cells were fused in a 50% solution of polyethylene glycol 1500, the fused cells were mixed with HT medium (RPMI 1640 solution containing hypoxanthine, thymidine and 15% fetal calf serum), and the mixed solution was mixed with 8 plates. From the second day on the 96-well microtiter plate, add HAT culture solution (RPMI1640 solution containing hypoxanthine, aminopterin, thymidine and 15% fetal calf serum), and culture in each well for 2 weeks to select HAT Went. Proliferated hybridoma cells were confirmed.
[0038]
ii) Selection by screening of the above hybridoma cells
In order to select a hybridoma that produces an antibody capable of specifically binding to a specific cell, that is, EPO, from the hybridoma obtained above,¹²⁵I-EPO (Purified EPO derived from human urine by IODO-GEN method)¹²⁵I labeled; 98 μCi / μg EPO)¹²⁵An antibody that specifically binds to I-EPO was selected after confirming that it was present in the hybridoma culture.
As a result, 25 types of cells were selected as being suitable for the purpose.¹²⁵Subculturing of hybridomas was continued for 8 types with high binding values to I-EPO, and monocloning was performed by a limiting dilution method to obtain 5 types of hybridoma cells that stably produced antibodies. Among these five types, the cells with the best adsorption and elution of EPO activity (n- # 2) were employed to produce monoclonal anti-EPO antibodies.
[0039]
(4) Production of monoclonal anti-EPO antibody:
Antibody production was performed using the above hybridoma cell n- # 2. That is, in the same manner as described above, antibodies were produced in the mouse abdominal cavity, subjected to 50% ammonium sulfate fractionation, passed through a DE52 (DEAE-cellulose; Whatman) packed column, and 0.1M to 0.2M NaCl fraction. Purified immunoglobulin (IgG) was obtained. Using 30 mice, 900 mg of purified monoclonal antibody (R2) was obtained.
[0040]
Example 2
Production of soluble erythropoietin receptor protein
According to the method of Example 3 of JP-A-6-38787, an sEPO-R expression vector pcmEPR sol · dhfr was constructed and this gene was transfected into E. coli MC1061 / P3 Biotechnological Industrial Technology Research Institute) has been deposited as Microtechnical Laboratories No. 12814). In the present invention, the above plasmid was removed from these E. coli cells and transfected into CHO.dhfr- cells. That is, the sEPO-R expression vector pcmEPRsol · dhfr was introduced into CHO (dhfr-) cells by the calcium phosphate method, and soluble erythropoietin receptor protein (sEPO-R) producing cells were selected. The sEPO-R gene was amplified with methotrexate (MTX) to obtain a sEPO-R high-producing cell line N14.2. The obtained cell line was grown in a culture medium containing 10% dialyzed fetal bovine serum and 100 nM MTX in a nucleic acid-free α-MEM synthetic medium, and then OPTI-MEMI containing 0.5% dialyzed fetal bovine serum. The medium was changed to Gibco and sEPO-R was produced. 6 ml of EPO-immobilized CH-Sepharose 4B (15 mg EPO / ml gel) was added to 200 ml of the obtained culture solution, and after gently contacting at 4 ° C. overnight, the immobilized carrier was washed with 10 times the amount of PBS. , SEPO-R adsorbed on the carrier was changed to 1.5M MgCl₂And eluted with PBS. The eluate was concentrated by ultrafiltration and the solvent was replaced with PBS, and then molecular weight fractionation was performed by HPLC using TSKgel G3000SW (manufactured by Tosoh Corporation) to obtain partially purified sEPO-R (0.6 mg). The obtained sEPO-R was subjected to molecular weight measurement using SDS-PAGE and Sephadex G-75 gel filtration, and showed about 33 kDa.
[0041]
Example 3
Ointment manufacture
The anti-erythropoietin antibody obtained in Example 1 was mixed with petrolatum to obtain 1% ointment.
[0042]
Example 4
Manufacture of injections 1
The anti-erythropoietin antibody obtained in Example 1 was dissolved in physiological saline to obtain a 1 g / ml or 10 g / ml solution. This was filtered, dispensed 200 μl each into a sterilized microtube, and stored frozen to give an injection. At the time of use, this is dissolved in physiological saline and used.
[0043]
Example 5
Manufacture of injections 2
The erythropoietin receptor protein obtained in Example 2 was dissolved in physiological saline to obtain a 200 μg / ml solution. This was filtered, dispensed 200 μl each into a sterilized microtube, and stored frozen to give an injection. At the time of use, this is dissolved in physiological saline and used.
[0044]
Example 6
Effects of erythropoietin antagonists (anti-erythropoietin antibodies) on keloids
1) Immunostaining
As tissue search materials, 4 samples from keloid patients and 2 samples from hypertrophic scar patients were fixed solution (Zamboni; Zamboni L. and DeMartino C., J. Cell Biot., Vol. 35, p148A (1967)). After being fixed for 6 hours, it was placed in a 25% sucrose solution and stored in a cool dark place. These sample pieces were embedded with an OCT agent (Miles), frozen with liquid nitrogen, and frozen into 7 μm sections using a cryostat. Sections were treated as primary antibodies with anti-erythropoietin antibody (Genzyme), anti-erythropoietin receptor antibody (Yasuda Y. et al., Develop. Growth Differ., Vol. 35, pp711-722 (1993)), anti-Factor VIII antibody ( Dako), anti-collagen III antibody (Chemicon), anti-collagen I antibody (Chemicon), and biotinylated anti-rabbit antibody (Vector) as a secondary antibody. Next, using an ABC kit (Vector), the color was developed by DAB reaction and used for speculum.
[0045]
2) Tissue transplant
A keloid tissue was transplanted into a nude mouse, and the effect of the agent of the present invention to suppress keloid proliferation was confirmed. As a transplant material, a tissue piece extracted from a keloid patient was placed in a culture solution (MEM + 10% FCS + PC + SM) until transplanted and stored in a cool dark place. Next, nude mice (Balb / c, Jcl-nu, Clea Japan Co., Ltd.) were 6 weeks old, and under anesthesia, shredded keloid tissue pieces were subcutaneously placed between the scapulae, one mouse at a time, 5 mice at a time. Transplanted. After transplantation, the size (major axis × minor axis × height) of the tumor in which the keloid tissue grew three times a week was measured with a caliper. At 3 months when tumor size was stable, 5 mouse tumors were injected with 200 μl of erythropoietin 400 U or 800 U / ml three times at 1 hour intervals. Further, after 3 months, 200 μl of an 8 mg / ml solution of the anti-erythropoietin antibody (R2) obtained in Example 1 was injected into the tumors of two mice at intervals of 1 hour. Two control mice were given three local injections of saline or mouse total serum in the same manner. The remaining 1 mouse was not injected. One week after the injection, the tumor was removed from the R2-injected mouse and the serum-injected mouse and fixed. At 4 weeks after injection, tumors were removed from the remaining R2-injected mice and saline-injected mice and fixed. The results are shown below.
[0046]
1. Histopathological examination of keloid tissue
a) Expression of erythropoietin in keloid tissue
Numerous fibroblasts and capillary endothelial cells that produce collagen are positive for erythropoietin receptor antibodies (FIG. 1). These findings revealed that fibroblasts and capillary endothelial cells responded to erythropoietin.
b) Presence of collagen type III fibers in keloid tissue
Fibroblasts were strongly positive for anti-collagen type III antibody (FIG. 2). Collagen fiber bundles also show a positive reaction, but the reaction is weaker compared to the fibroblasts present at the margin (FIG. 2).
c) Distribution of blood vessels in keloid tissue
Numerous capillary endothelium and endothelial cells were positive for anti-Factor VIII (Factor VIII) antibody, an antibody used to identify endothelial cells (FIG. 3).
[0047]
2. Keloid tissue transplantation
a) Progress after transplantation
A keloid tissue fragment was transplanted subcutaneously, and a tissue (tumor) that became a tumor after transplantation was observed. When the growth decreased compared to the time of transplantation (after 16 weeks), erythropoietin 400U or 800U was locally injected into each tumor three times every hour, and an increase in the growth rate was observed (Table 1). One tumor was removed 10 days after administration, but a tumor appeared 3 weeks later. At 28 weeks after transplantation, local injection of R2 was performed three times at 1 hour intervals, and tumors were removed one or four weeks later.
b) Increase of transplanted tumor by erythropoietin administration
As shown in Table 1, in 2 cases, at 12 and 24 hours after erythropoietin administration, the tumor increased to 6 to 9 times and 8 to 7.5 times the size before administration, Even after 4 days, it was 3 times as large. In addition, there was no difference in increase depending on the administration concentration.
c) Suppression of transplanted tumor by anti-erythropoietin antibody injection
Table 2 shows changes in tumor size before and after administration of the anti-erythropoietin antibody. Mild suppression was observed in the antibody-treated mouse (No. 3), and the No. 4 mouse lacked the flexibility of the tumor and changed to a swelling of the hardened mass. In the pathological observation of the proliferating tumor in mouse No. 3 and the remaining soft tissue in mouse No. 4, no clear keloid-like pathological observation image was observed as described below. 3/4 tissue calcification was observed.
[0048]
[Table 1]

[0049]
[Table 2]

[0050]
Histopathology
a) Changes caused by injection of anti-erythropoietin antibody in transplanted tumor
i) Rupture of collagen type III fiber bundles is marked with R2 treatment. The ruptured bundle is melted and disappeared by the attack of leukocytes, macrophages, and NK-like cells to become only fibers. Fibroblasts show nuclear enrichment and dead cell accumulation is also observed (FIG. 4).
ii) In the control treatment, the presence of proliferated collagen fiber bundles is observed. Fibroblasts and fibrocytes do not show degeneration (FIG. 5).
b) In the transplanted tumor, the blood vessels near the collagen bundle are less dense than before transplantation. i) Endothelial cell death was observed in R2-treated tumors (FIG. 6).
ii) The presence of endothelial cells was observed in control treated tumors (FIG. 7).
c) Graft deposition of the graft
i) In R2 treatment, calcification was observed in all, and in No.4, 3/4 of the swollen tissue showed calcification, the collagen bundle near the calcification was torn, and the fractured collagen was calcified (FIG. 8).
ii) Although 1 tumor was observed in the physiological saline treatment, the collagen bundle near the calcification showed a growth image without change.
These results are summarized in Table 3. In the non-treated group and the control group, there was almost no tissue change, but in the anti-erythropoietin antibody-treated group, fibroblast degeneration, collagen bundle rupture and disappearance, and calcareous deposition induce tissue calcification and suppression of collagen production. did. From these results, it was confirmed that the anti-erythropoietin antibody inhibits keloid proliferation.
[0051]
[Table 3]

[0052]
Example 7
Effects of erythropoietin antagonist (erythropoietin receptor protein) on keloids
Similar to Example 6, the same experiment was performed using the erythropoietin receptor protein obtained in Example 2 instead of the anti-erythropoietin antibody. Similar to the results when using antibodies, erythropoietin receptor protein was also confirmed to suppress keloid growth.
[0053]
Example 8
Effects of erythropoietin antagonist (anti-erythropoietin antibody) on rheumatoid arthritis
1) Immunostaining
As a tissue search material, a proliferative synovial fragment excised by surgery from 5 patients with rheumatoid arthritis was cut into a size of about 20 mm × 10 mm × 5 mm, and fixed solution (Zamboni solution; Zamboni L. and DeMartino C. , J. Cell Biot., Vol. 35, p148A (1967)) for 6 hours, replaced with PBS supplemented with 25% sucrose, and stored in a cool dark place. These sample pieces were embedded with an OCT agent (Miles), frozen with liquid nitrogen, and frozen into 7 μm sections using a cryostat. Sections were used as primary antibodies i) anti-erythropoietin receptor antibody (Yasuda Y. et al., Develop. Growth Differ., Vol. 35, pp711-722 (1993)), ii) anti-IL1α antibody (Genzyme), Five types of antibodies were reacted: iii) anti-collagen III antibody (Chemicon), iv) anti-estrogen receptor antibody (Chemicon), and v) anti-factor 8 antibody (Dako). Next, biotinylated anti-rabbit antibody (Vector) was used for the reaction made in i), iii) and v), and biotinylated anti-mouse antibody was used for the reaction made in ii) and iv). (Chemicon) was reacted as the second antibody. The color was reacted with DAB (Dojin Pharmaceutical Co., Ltd., Wako Pure Chemical Industries, Ltd.) and used for microscopic examination.
[0054]
2) Organ culture
As an organ culture material, in the above 3 cases, the excised sample was immediately placed in a culture solution (MEM + 10% FCS, PC 100 U / ml, SM 100 μg / ml) and washed after the operation. Saved in.
The fresh synovial membrane piece (within 10 to 24 hours after the operation) thus prepared was chopped into 2 × 5 × 10 mm in size and 50 to 100 mg in weight, and one piece of tissue was petri dish (3042). ; Falcon) and weighed. A solution colored by adding 0.25% of Evans blue to a physiological saline solution of the anti-erythropoietin antibody (R2) obtained in Example 1 in 16 mg / ml was injected into the petri dish. As a control, physiological saline added with Evans blue (Otsuka Pharmaceutical Co., Ltd.) was used. For injection, a Hamilton 32-gauge needle (90131) and syringe barrel (725) were used, and 0.5 μl per mg of tissue weight, that is, 0.5 μl / mg weight of R2 or physiological saline was applied to multiple pieces of tissue. Injected. After injection, cover the petri dish with 5% CO₂The reaction was performed in a petri dish at 95% air and 37 ° C. for 60 minutes. This operation was repeated, and R2 or physiological saline treatment was performed 3 to 4 times, and then the tissue pieces were meshed one by one in a 24-well culture dish (3047; Falcon) with a culture solution of MEM + 10% FCS. On Falcon), 5% CO₂Organ culture was performed for 6 to 8 hours under the conditions of 95% air and 37 ° C. In addition, since culture | cultivation time is complete | finished in 12 hours from the time of R2 process start, there exists a time difference.
The culture piece was fixed with a Zamboni solution, replaced with PBS supplemented with 25% sucrose after 6 hours, stored in a cool dark place, and used for pathological tissue search. The results of organization search are shown below.
[0055]
Characteristics of synovial tissue of rheumatoid arthritis
1) Erythropoietin reaction site in synovial villi
Fibroblasts that produce collagen, macrophages that infiltrate fiber bundles and synovial villi, monocytes, and capillary endothelial cells are erythropoietin-responsive sites (FIGS. 9, 10, and 11).
2) Expression of IL1α in synovial villi
It is found in fibroblasts that produce collagen, infiltrating monocytes, macrophages, and lymphocytes (FIG. 12).
3) Vascular distribution of synovial villi
The villus has many capillaries and small blood vessels, and many monocytes and lymphocytes infiltrate around the blood vessels (FIG. 13).
4) Collagen of synovial villi
Villous collagen is rich in fibers mainly composed of collagen III. Fibroblasts show a cytoplasm with collagen III in the nucleus with an egg-shaped network-like chromatin (FIG. 14).
5) Expression of estrogen receptor in synovial villi
In the villi, the estrogen response site (FIG. 15 right) corresponds to the erythropoietin receptor (FIGS. 15 and 16 left) and the IL1α positive site (FIG. 16 right), and these three coexist (FIGS. 15 and 16).
[0056]
Changes in synovial villi by R2 treatment
1) Fibroblast death
Numerous dead fibroblasts were observed after 3 treatments (FIG. 17) and 4 treatments (FIG. 18) of R2. In the nucleus, the net-like chromatin was condensed and concentrated, and the cytoplasm was lost. This phenomenon is more prominent in 4 treatments of R2 than in 3 treatments and is considered apoptotic death. In the control group, fibroblasts showed no change (FIG. 19).
2) Death of monocytes, lymphocytes and macrophages
Lymphocytes and monocyte macrophages that died due to a large number of nuclei were observed around the capillary blood vessels with many cell infiltrations (FIG. 20). This phenomenon was more severe with the four treatments with R2 (FIG. 20), and a decrease in IL1α-positive cells in the culture was observed. There was no change in these infiltrating cells in the control group cultures (FIG. 21).
3) Collapse of capillaries
The disappearance of capillaries was observed after 3 and 4 R2 treatments (FIG. 22). In the control group, a large number of capillaries were present (FIG. 23).
4) Changes in collagen fibers
In the R2 3 treatments (FIG. 17) and 4 treatments (FIG. 18), the collagen bundles were ruptured and diluted. In the tissue pieces of the control group, no rupture or dilution of collagen fibers was observed (FIG. 19).
The above results are summarized in Table 4. Anti-erythropoietin antibody suppresses collagen production associated with synovial proliferation, which is a major symptom of diseases such as rheumatoid arthritis (fibroblast degeneration, necrosis, loss of collagen bundle), loss of capillaries, IL-1 secretion Cell necrosis (monocyte, macrophage necrosis) was induced. From these results, it was confirmed that the anti-erythropoietin antibody suppresses inflammation of rheumatoid arthritis.
[0057]
[Table 4]

[0058]
Example 9
Effects of erythropoietin antagonist (erythropoietin receptor protein) on rheumatoid arthritis
Similar to Example 8, a similar experiment was conducted using erythropoietin receptor protein instead of anti-erythropoietin antibody. As a result, it was confirmed that the erythropoietin receptor protein also suppresses inflammation, similar to the results when using antibodies.
[0059]
Example 10
Expression of erythropoietin and erythropoietin receptor mRNA in keloid tissue samples
RNA was extracted from tissues of 5 keloid scars, 1 atrophic scar and 3 red scars, and detection of erythropoietin and erythropoietin receptor mRNA was attempted by RT-PCR using these RNAs. Expression of erythropoietin receptor mRNA was observed in all cases. Erythropoietin mRNA was expressed in keloid scars and red scars, but was very weak or absent in atrophic scars (FIG. 24). The state of the collected RNA is considered good because the amplification of beta-actin mRNA is not weak.
[0060]
Example 11
Expression of erythropoietin and erythropoietin receptor mRNA in synovial tissue
When RNA was extracted from the synovium of eight patients and detection of each mRNA was attempted by RT-PCR, expression of erythropoietin and its receptor was observed in all cases, although the expression was strong and weak ( FIG. 25).
[0061]
Example 12
Erythropoietin content in synovial fluid
Total protein (mg / ml) and erythropoietin (mU / ml) were measured using synovial fluid from 9 patients with rheumatism and 5 with osteoarthritis. However, erythropoietin levels were significantly higher in patients with rheumatism than in osteoarthritis.
The results are shown in Table 5.
[Table 5]

[0062]
【The invention's effect】
As is apparent from the above, the present invention provides a prophylactic / therapeutic agent having an excellent effect on collagenous hyperproliferation such as hypertrophic scars or keloids, or chronic joint inflammatory diseases such as rheumatoid arthritis.
[Brief description of the drawings]
FIG. 1 is a view showing a micrograph showing a positive reaction of a large number of fibroblasts to an erythropoietin receptor antibody in a keloid piece removed from a patient. In the figure, ← indicates a fibroblast, arrowhead indicates a vascular endothelial cell, and * indicates a region of the fiber bundle. (Magnification: 180 times)
FIG. 2 is a view showing a photomicrograph showing a positive reaction of collagen type III antibody of a large number of fibroblasts in a keloid piece removed from a patient. In the figure, ← indicates the fiber bundle,smallArrows indicate fibroblasts. (Magnification: 180 times)
FIG. 3 is a view showing a photomicrograph showing the distribution of a large number of capillaries that show a positive reaction with a factor VIII antibody in a keloid piece removed from a patient. In the figure, ← indicates vascular endothelial cells. (Magnification: 180 times)
FIG. 4 is a view showing a micrograph showing a histological image of a peripheral portion of a calcified tissue of a tumor fragment extracted three weeks after consecutive administration of erythropoietin antibody to a keloid tumor transplanted into a nude mouse. In the figure, the arrowhead indicates a collagen image that has been broken and broken down, and * indicates a collagen image that has been broken down and disappears. (Magnification: 360 times)
FIG. 5 is a view showing a micrograph showing a histological image of a tumor fragment obtained by continuously administering mouse serum three times to a keloid tumor transplanted into a nude mouse and excising one week later. In the figure, ← indicates one bundle of collagen bundles. (Magnification: 360 times)
FIG. 6 is a view showing a micrograph showing a factor 8 antibody positive reaction of the same specimen (three treatments of R2) as FIG. In the figure, ← indicates a nuclear fragment of endothelial cells crushed into dots. (Magnification: 720 times)
FIG. 7 is a view showing a micrograph showing a factor 8 antibody positive reaction of the same specimen as FIG. 5 (treated with mouse serum 3 times). In the figure, ← indicates the process of endothelial cytoplasm.(Magnification: 720 times)
FIG. 8 is a view showing a micrograph showing a tissue image of tissue calcification by van Kossa staining of the same specimen as FIG. 4 (three treatments of R2). In the figure, ← indicates calcification of a collagen fracture mass. (Magnification: 72 times)
FIG. 9 is a view showing a micrograph showing cells (arrows) positive for erythropoietin receptor antibody and cellular infiltration in a synovial villi segment removed from patient A. In the figure, ← indicates fibroblasts and ←← indicates macrophages. (Magnification: 150 times)
FIG. 10 is a view showing a micrograph showing a strongly magnified image showing a positive site for an erythropoietin receptor antibody of a synovial villi segment removed from a patient B. In the figure, ← indicates monocytes around the capillaries, ←← indicates macrophages, and arrowheads indicate fibroblasts. (Magnification: 600 times)
11 is a view showing a micrograph showing a fibroblast image showing a positive reaction with an erythropoietin receptor antibody in a tissue section of the same patient as FIG. 10. FIG. In the figure, ← indicates fibroblasts. (Magnification: 600 times)
12 is a view showing a micrograph showing a group of fibroblasts and infiltrating cells positively reacting with an IL1α antibody of a tissue piece of the same patient as FIG. 10. FIG. In the figure, ← indicates the nuclei of fibroblasts, and ←← indicates macrophages. (Magnification: 300 times)
13 is a view showing a micrograph showing an image showing the presence of capillaries that react with the factor VIII antibody in a tissue section of the same patient as in FIG. 9. FIG. In the figure, ← indicates capillary endothelial cells positive for the factor 8 antibody. (Magnification: 150 times)
14 is a view showing a micrograph showing a fibroblast image showing a positive reaction with a collagen III antibody in a tissue piece of the same patient as FIG. 10. FIG. In the figure, ← indicates fibroblasts. (Magnification: 600 times)
15 is a view showing a micrograph showing a double-stained image of an erythropoietin receptor antibody (left figure) and an estrogen receptor α antibody (right figure) of a tissue piece of the same patient as FIG. In the figure, ← indicates fibroblasts, and arrowhead indicates monocytes. (Magnification: 480 times)
16 is a view showing a micrograph showing a double-stained image of an erythropoietin receptor antibody (left figure) and an IL1α antibody (right figure) of a tissue piece of the same patient as FIG. In the figure, ← indicates fibroblasts and arrowhead indicates monocytes (magnification: 480 times).
FIG. 17 is a view showing a micrograph showing a collagen III antibody positive site of R2 treated three times at an interval of 1 hour (fixed 12 hours after the start of treatment) on the same patient tissue as FIG. , ← indicates the nucleus of the dead fibroblast, and ←← indicates the nucleus of the dead macrophage (magnification: 180 times).
FIG. 18 is a view showing a micrograph showing a collagen III antibody positive site, although R2 was treated 4 times at 1 hour intervals on the same patient tissue piece as in FIG. 10 and fixed 12 hours after the start of treatment. In the figure, ← indicates the nucleus of the dead fiber cell, and the arrowhead indicates the site where the collagen fiber around the dead cell is missing. (Magnification: 150 times)
FIG. 19 is a view showing a micrograph showing a collagen III antibody positive site, although a raw meal was treated 4 times at 1 hour intervals on a tissue piece of the same patient as in FIG. 10 and fixed 12 hours after the start of treatment. In the figure, ← indicates the nucleus of the fibroblast, and ←← indicates the fiber bundle of collagen III. (Magnification: 300 times)
FIG. 20 is a view showing a micrograph showing an IL1α antibody-positive part of the same culture section (R2 treatment 4 times) as in FIG. In the figure, ← indicates the nucleus of a dead fibroblast, and ←← indicates a fragment of the nucleus. (Magnification: 240 times)
FIG. 21 is a view showing a micrograph showing an IL1α antibody-positive part of the same culture piece (treatment with 4 saline) as in FIG. In the figure, ← indicates fibroblasts and ←← indicates macrophages. (240x magnification)
FIG. 22 is a view showing a micrograph showing a factor 8 positive site of the same culture piece (three times of R2 treatment) as FIG. In the figure, ← indicates a damaged blood vessel wall, and ←← indicates the nucleus of an endothelial cell showing nuclear concentration. (Magnification: 360 times)
FIG. 23 is the same culture piece as FIG.4 raw meals) Is a diagram showing a micrograph showing a factor 8 positive site. In the figure, ← indicates vascular endothelial cells, ←← indicates monocytes, arrowheads indicate fibroblast nuclei, and two arrowheads indicate lymphocytes. (Magnification: 360 times)
FIG. 24 is an electrophoretogram showing the expression results of erythropoietin and erythropoietin receptor mRNA in a keloid tissue sample. In the figure,
Lanes 1-5 show keloid scar patients, 6 lanes show atrophic scars, and 7-9 lanes show red scars.
EPO RT + indicates the amplification of erythropoietin cDNA in RNA samples with reverse transcriptase added.
EPO RT- indicates that no amplification of erythropoietin cDNA was confirmed in the RNA sample without the addition of reverse transcriptase.
EPOR RT + shows the amplification of erythropoietin receptor cDNA in RNA samples supplemented with reverse transcriptase.
EPOR RT- indicates that amplification of erythropoietin receptor cDNA was not confirmed in the RNA sample to which reverse transcriptase was not added.
Beta actin shows its amplification using beta actin as an internal standard in order to take into account the RNA amount difference between samples.
The display above each band indicates patient identification.
FIG. 25 is an electrophoretogram showing the results of mRNA expression of erythropoietin and erythropoietin receptor in synovial tissue. In the figure,
EPO RT + indicates the amplification of erythropoietin cDNA in RNA samples with reverse transcriptase added.
EPO RT- indicates that no amplification of erythropoietin cDNA was confirmed in the RNA sample without the addition of reverse transcriptase.
EPOR RT + shows the amplification of erythropoietin receptor cDNA in RNA samples supplemented with reverse transcriptase.
EPOR RT- indicates that amplification of erythropoietin receptor cDNA was not confirmed in the RNA sample to which reverse transcriptase was not added.
Beta actin shows its amplification using beta actin as an internal standard in order to take into account the RNA amount difference between samples.
The display above each band indicates patient identification.

Claims (2)

A prophylactic / therapeutic agent for hypertrophic scar, keloid or chronic joint inflammatory disease, comprising as an active ingredient an erythropoietin antagonist selected from anti-erythropoietin antibody and erythropoietin receptor protein . The agent according to claim 1 , wherein the chronic joint inflammatory disease is rheumatoid arthritis, rheumatoid related diseases, arthritis due to collagen disease, or tendonitis .

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