JP5910299B2 - Inflammatory joint disease treatment - Google Patents
Inflammatory joint disease treatment Download PDFInfo
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- JP5910299B2 JP5910299B2 JP2012112588A JP2012112588A JP5910299B2 JP 5910299 B2 JP5910299 B2 JP 5910299B2 JP 2012112588 A JP2012112588 A JP 2012112588A JP 2012112588 A JP2012112588 A JP 2012112588A JP 5910299 B2 JP5910299 B2 JP 5910299B2
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- Prior art keywords
- inflammatory
- mmp
- inflammatory joint
- alkylumbelliferone
- treatment
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- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 1
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Description
本発明は医薬に関する。詳しくは、炎症性関節疾患治療薬及びその用途に関する。 The present invention relates to medicine. Specifically, the present invention relates to a therapeutic drug for inflammatory joint diseases and use thereof.
関節リウマチに代表される炎症性関節疾患は患者数も多く、また、原因が不明又は十分に解明されていないものが殆どであり、より有効な治療法の提供が望まれている。従来の炎症性関節疾患治療薬としては非ステロイド性抗炎症薬（NSAID）、抗リウマチ薬（免疫調節剤、免疫抑制剤、生物学的製剤）等が使用されている。これらの治療薬には一定の効果が認められるものの、胃炎や胃潰瘍など消化器合併症、重症感染や肺炎或いは悪性腫瘍の発生などの重篤な副作用が報告されている。ヒアルロン酸ナトリウムの関節内注射による治療も行われているが、効果は限定的であり、全身性の炎症性関節疾患には効果がない。例えば、関節リウマチを全身性疾患と考えた場合、ヒアルロン酸の関節内局所注入治療は病勢自体を抑えられない。 Inflammatory joint diseases represented by rheumatoid arthritis have a large number of patients, and most of them have unknown causes or are not fully elucidated, and it is desired to provide more effective treatments. Non-steroidal anti-inflammatory drugs (NSAIDs), anti-rheumatic drugs (immunomodulators, immunosuppressants, biological preparations) and the like have been used as conventional therapeutic agents for inflammatory joint diseases. Although these therapeutic drugs have certain effects, serious side effects such as gastrointestinal complications such as gastritis and gastric ulcers, severe infections, pneumonia and malignant tumors have been reported. Treatment with sodium hyaluronate by intra-articular injection has also been performed, but the effect is limited and not effective for systemic inflammatory joint diseases. For example, when rheumatoid arthritis is considered a systemic disease, intraarticular local injection treatment with hyaluronic acid cannot suppress the disease itself.
炎症性関節疾患では滑膜のヒアルロン酸が過剰発現され、ヒアルロン酸と細胞表面受容体であるCD44が炎症の増悪に関連していること（非特許文献１）、ヒトでの血清ヒアルロン酸濃度が炎症性関節疾患の活動性を反映していること（非特許文献２〜５）、更にはヒアルロン酸と細胞表面受容体であるCD44との相互作用を抗CD44抗体でブロックするとマウスでの炎症性関節疾患が抑制されること（非特許文献６）等が報告されており、ヒアルロン酸の過剰発現が炎症性関節疾患の病態増悪に関連していることが示唆されている。但し、ヒアルロン酸の産生を抑制した場合の効果については不明である。
In inflammatory joint diseases, hyaluronic acid in the synovial membrane is overexpressed, and hyaluronic acid and cell surface receptor CD44 are associated with exacerbation of inflammation (Non-patent Document 1), and human serum hyaluronic acid concentration is Reflects the activity of inflammatory joint diseases (
本発明の課題は、炎症性関節疾患に対する新たな治療手段を提供することにある。 An object of the present invention is to provide a new therapeutic means for inflammatory joint diseases.
上記課題を解決すべく検討を進める中で本発明者らは、従来から利胆剤として使用され、重篤な副作用がない4−アルキルウンベリフェロンに着目し、その炎症性関節疾患治療剤としての有効性を調べることにした。4−アルキルウンベリフェロンについては、ヒアルロン酸合成酵素HAS2遺伝子の発現抑制作用、ヒアルロン酸分解酵素HYAL1遺伝子の発現抑制作用、及びマトリックスメタロプロテアーゼMMP-9遺伝子の発現抑制作用があり、がんの抑制に有効であることが報告されている（特許文献１）。また、4−アルキルウンベリフェロンの新規医薬用途として骨転移の治療が提案されている（特許文献２）。このように、従来、4−アルキルウンベリフェロンの標的は悪性腫瘍であり、滑膜増殖などに起因する炎症性関節疾患に対する4−アルキルウンベリフェロンの治療効果は検討されていない。 While proceeding with studies to solve the above problems, the present inventors have focused on 4-alkylumbelliferone that has been conventionally used as a bile agent and has no serious side effects, and as a therapeutic agent for inflammatory joint diseases. I decided to examine the effectiveness of. 4-Alkylumbelliferone suppresses the expression of hyaluronan synthase HAS2 gene, suppresses the expression of hyaluronan degrading enzyme HYAL1 gene, and suppresses the expression of matrix metalloprotease MMP-9 gene. (Patent Document 1). In addition, treatment of bone metastasis has been proposed as a novel pharmaceutical use of 4-alkylumbelliferone (Patent Document 2). Thus, conventionally, the target of 4-alkylumbelliferone is a malignant tumor, and the therapeutic effect of 4-alkylumbelliferone on inflammatory joint diseases caused by synovial proliferation has not been studied.
検討にあたって、まずコラーゲン誘発性関節炎マウスモデルを作製し、4−アルキルウンベリフェロンの一つである4-メチルウンベリフェロン（以下、4-MUと略称する）を全身投与（経口投与）した場合の関節炎症状の改善効果を調べるとともに、組織学的に骨軟骨破壊の抑制効果を評価した。その結果、4-MUはコラーゲン誘発性関節炎マウスモデルにおいて各種プロテアーゼの発現を抑制し、関節炎発症と増悪を有意に抑制することが判明した。一方、In vitroではヒトから得られた関節リウマチ滑膜細胞（関節リウマチ患者由来の線維芽細胞様滑膜細胞）を用い、関節軟骨の破壊において重要な因子であるマトリックスメタロプロテアーゼ（MMP）の発現抑制を指標として、抗炎症効果を評価したところ、4-MUがMMPの発現を有意に抑制した。更に検討を進め、MUの効果について既存薬（非ステロイド抗炎症薬）と比較したところ、4-MUが有意に強いMMP抑制作用を示すことが明らかとなった。4-MUは内服薬として臨床応用されている薬剤である。本発明者らの検討によって明らかになった事実、即ち、4-MUが関節炎の進行抑制効果を示すことは、例えば、抗リウマチ薬とは異なり、肺線維症を初めとする重篤な各種合併症を少なくする薬剤として4-MUが有効であることを意味する。この4-MUの特徴は臨床上の大きなメリットをもたらす。 In the study, a mouse model of collagen-induced arthritis was first prepared, and 4-methylumbelliferone (hereinafter abbreviated as 4-MU), one of the 4-alkylumbelliferones, was administered systemically (orally). In addition to examining the effect of improving the joint inflammation symptoms, histologically, the effect of inhibiting osteochondral destruction was evaluated. As a result, 4-MU was found to suppress the expression of various proteases in the collagen-induced arthritis mouse model and to significantly suppress the onset and exacerbation of arthritis. On the other hand, in vitro, rheumatoid arthritis synovial cells obtained from humans (fibroblast-like synoviocytes derived from rheumatoid arthritis patients) are used to express matrix metalloprotease (MMP), an important factor in the destruction of articular cartilage. When the anti-inflammatory effect was evaluated using suppression as an index, 4-MU significantly suppressed the expression of MMP. Further investigation and comparison of the effects of MU with existing drugs (non-steroidal anti-inflammatory drugs) revealed that 4-MU exhibits a significantly stronger MMP inhibitory action. 4-MU is a drug that has been clinically applied as an internal medicine. The facts clarified by the study of the present inventors, that is, that 4-MU exhibits an arthritis progression-inhibiting effect is different from antirheumatic drugs, for example, in various serious complications including pulmonary fibrosis. It means that 4-MU is effective as a drug to reduce the symptoms. This 4-MU feature brings significant clinical benefits.
以下に列挙する本発明は主として上記成果に基づく。
［１］4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩を有効成分として含有する、炎症性関節疾患治療薬。
［２］4-アルキルウンベリフェロンが4-メチルウンベリフェロンである、［１］に記載の炎症性関節疾患治療薬。
［３］炎症性関節疾患が、膠原病に伴う関節炎ないし滑膜炎である、［１］又は［２］に記載の炎症性関節疾患治療薬。
［４］非ステロイド系抗炎症薬、ステロイド薬、免疫調節薬、免疫抑制薬及び生物学的製剤からなる群より選択される一以上の薬剤を組み合わせてなる、［１］〜［３］のいずれか一項に記載の炎症性関節疾患治療薬。
［５］4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩と前記薬剤を含有する配合剤である、［４］に記載の炎症性関節疾患治療薬。
［６］4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩を含有する第１構成要素と、前記薬剤を含有する第２構成要素とからなるキットであることを特徴とする、［４］に記載の炎症性関節疾患治療薬。
［７］4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩を含有し、投与時に前記薬剤が併用投与されることを特徴とする、［４］に記載の炎症性関節疾患治療薬。
［８］炎症性関節疾患の患者に対して治療上有効量の4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩を投与するステップを含む、炎症性関節疾患の治療法。
［９］非ステロイド系抗炎症薬、ステロイド薬、免疫調節薬、免疫抑制薬及び生物学的製剤からなる群より選択される一以上の薬剤を治療上有効量、併用投与することを特徴とする、［８］に記載の治療法。
The present invention listed below is mainly based on the above results.
[1] A therapeutic agent for inflammatory joint diseases, comprising 4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] The inflammatory joint disease therapeutic agent according to [1], wherein the 4-alkylumbelliferone is 4-methylumbelliferone.
[3] The inflammatory joint disease therapeutic agent according to [1] or [2], wherein the inflammatory joint disease is arthritis or synovitis associated with collagen disease.
[4] Any one of [1] to [3], which is a combination of one or more drugs selected from the group consisting of non-steroidal anti-inflammatory drugs, steroid drugs, immunomodulators, immunosuppressants and biological preparations The inflammatory joint disease therapeutic agent according to any one of the above.
[5] The therapeutic agent for inflammatory joint diseases according to [4], which is a combination drug containing 4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof and the drug.
[6] A kit comprising a first component containing 4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof, and a second component containing the drug. The inflammatory joint disease therapeutic agent according to [4].
[7] The inflammatory agent according to [4], comprising 4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof, wherein the drug is administered in combination at the time of administration. Joint disease treatment.
[8] Treatment of inflammatory joint disease, comprising the step of administering a therapeutically effective amount of 4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof to a patient with inflammatory joint disease. Law.
[9] A therapeutically effective amount of one or more drugs selected from the group consisting of non-steroidal anti-inflammatory drugs, steroid drugs, immunomodulators, immunosuppressants, and biological preparations are administered in combination. [8] The treatment method according to the above.
本発明は炎症性関節疾患に有効な治療薬を提供する。本発明の治療薬の治療対象は炎症性関節疾患である。理論に拘泥する訳ではないが、本発明の治療薬はヒアルロン酸の産生抑制を介して滑膜の炎症を抑制する。この作用機序に鑑み、本発明の治療薬は滑膜の炎症が原因の疾患又は滑膜の炎症を伴う疾患に適用される。当該疾患の典型例は、膠原病に伴う関節炎ないし滑膜炎である。膠原病には、関節リウマチ、全身性エリトマトーデス、強皮症等が含まれる。 The present invention provides a therapeutic agent effective for inflammatory joint diseases. The treatment target of the therapeutic agent of the present invention is inflammatory joint disease. Without being bound by theory, the therapeutic agent of the present invention suppresses synovial inflammation through suppression of hyaluronic acid production. In view of this mechanism of action, the therapeutic agent of the present invention is applied to diseases caused by synovial inflammation or diseases accompanied by synovial inflammation. A typical example of the disease is arthritis or synovitis associated with collagen disease. Collagen diseases include rheumatoid arthritis, systemic lupus erythematosus, scleroderma and the like.
本発明の治療薬の有効成分は4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩である。4-アルキルウンベリフェロンとして、4-メチルウンベリフェロン（4-MU）、4-エチルウンベリフェロン、4-プロピルウンベリフェロン、4-ブチルウンベリフェロンなどの、4位に炭素数1〜4のアルキル基を有するウンベリフェロン（7-ヒドロキシクマリン）を例示することができる。その作用を損なわない程度に（作用を高める場合を含む）改変を施した各種誘導体を有効成分として用いることもできる。ここでの誘導体の例として4-メチルエスクレチン（4-ME）を挙げることができる。4-MEには4-MU同様にヒアルロン酸合成阻害能があることが報告されている（Morohashi H. et al., Int. J. Cancer:120, 2704-2709(2007)）好ましくは、本発明の治療薬の有効成分として4-MUを用いる。4-MUの構造を以下に示す。
4-アルキルウンベリフェロン又はその誘導体の薬学的に許容される塩としてナトリウム塩、カリウム塩を例示できるが、これらに限定されるものではない。尚、4-MUや4-ME等は和光純薬工業株式会社、東京化成工業株式会社、ナカライテスク株式会社などが販売しており、容易に入手可能である。 Examples of pharmaceutically acceptable salts of 4-alkylumbelliferone or derivatives thereof include, but are not limited to, sodium salts and potassium salts. 4-MU, 4-ME, etc. are sold by Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., Nacalai Tesque Co., Ltd., etc., and are readily available.
本発明の一態様では、上記有効成分（4-アルキルウンベリフェロン又はその誘導体、或いはそれらの薬学的に許容される塩）と他の薬剤を併用する。この態様によれば、作用機序の異なる二以上の薬剤による治療を行うことができ、相加的又は相乗的効果を期待できる。他の薬剤としては非ステロイド系抗炎症薬、ステロイド薬、免疫調節薬、免疫抑制薬又は生物学的製剤を用いることができる。併用する他の薬剤の数は一つに限られない。即ち、ここでの他の薬剤として二以上の薬剤を採用することもできる。非ステロイド系抗炎症薬の例はアスピリン、コリンマグネシウム三サリチル酸、ジフルニサル、サルサレート、セレコキシブ、ロフェコキシブ、バルデコキシブ、ジクロフェナク、エトドラク、フェノプロフェン、フルルビプロフェン、イブプロフェン、インドメタシン、ケトプロフェン、ケトロラク、メクロフェナム酸塩、メフェナム酸、メロキシカム、ナブメトン、ナプロキセン、オキサプロジン、ピロキシカム、スリンダク、トルメチンである。また、ステロイド薬の例はハイドロコルチゾン、プレドニゾロン、メチルプレドニゾロン、デキサメサゾン、ベタメサゾンである。免疫調節剤（遅効性抗リウマチ薬）の例は金製剤、ペニシラミン、ブシラミン、ロベンザリット、アクタリット、サラゾスルファピリジン、ヒドロキシクロロキン、スルファサラジンである。免疫抑制剤の例はメトトレキサート、ミゾリビン、レフルノミド、タクロリムス、シクロスポリンである。生物学的製剤の例はエタネルセプト（可溶性TNF受容体とヒトIgGのFc領域との融合蛋白）、インフリキシマブ（抗ヒトTNF-αモノクローナル抗体）、トシリズマブ（ヒト化抗ヒトIL-6レセプターモノクローナル抗体）、アダリムマブ（ヒト型抗ヒトTNF-αモノクローナル抗体）である。 In one embodiment of the present invention, the above active ingredient (4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof) is used in combination with another drug. According to this aspect, treatment with two or more drugs having different action mechanisms can be performed, and an additive or synergistic effect can be expected. As other drugs, non-steroidal anti-inflammatory drugs, steroid drugs, immunomodulating drugs, immunosuppressive drugs or biological preparations can be used. The number of other drugs used in combination is not limited to one. That is, two or more drugs can be employed as the other drugs here. Examples of non-steroidal anti-inflammatory drugs are aspirin, choline magnesium trisalicylic acid, diflunisal, salsalate, celecoxib, rofecoxib, valdecoxib, diclofenac, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid Salt, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tolmethine. Examples of steroid drugs are hydrocortisone, prednisolone, methylprednisolone, dexamethasone, and betamethasone. Examples of immunomodulators (slow-acting antirheumatic drugs) are gold preparations, penicillamine, bucillamine, robenzalit, actarit, salazosulfapyridine, hydroxychloroquine, sulfasalazine. Examples of immunosuppressants are methotrexate, mizoribine, leflunomide, tacrolimus, cyclosporine. Examples of biologics are etanercept (a fusion protein of soluble TNF receptor and human IgG Fc region), infliximab (anti-human TNF-α monoclonal antibody), tocilizumab (humanized anti-human IL-6 receptor monoclonal antibody), Adalimumab (human anti-human TNF-α monoclonal antibody).
この態様の特徴は、本発明の有効成分（4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩）と上記他の薬剤を組み合わせて用いることである。典型的には、本発明の有効成分と他の薬剤を混合した配合剤として本発明の治療薬が提供されることになる。一方、例えば、本発明の有効成分を含有する薬剤（第１構成要素）と、他の薬剤を含有する薬剤（第２構成要素）とからなるキットの形態で本発明の治療薬を提供することもできる。この場合、治療期間内に第１構成要素及び第２構成要素が最低１回ずつは投与されることになる。各要素の投与スケジュールは個別に設定することができる。両要素を同時に投与することにしてもよい。ここでの「同時」は厳密な同時性を要求するものではない。従って、両要素を混合した後に対象へ投与する等、両要素の投与が時間差のない条件下で実施される場合は勿論のこと、片方の投与後、速やかに他方を投与する等、両要素の投与が実質的な時間差のない条件下で実施される場合もここでの「同時」の概念に含まれる。 A feature of this embodiment is that the active ingredient of the present invention (4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof) is used in combination with the above other drugs. Typically, the therapeutic agent of the present invention will be provided as a combination preparation in which the active ingredient of the present invention and other drugs are mixed. On the other hand, for example, the therapeutic agent of the present invention is provided in the form of a kit comprising a drug containing the active ingredient of the present invention (first component) and a drug containing another drug (second component). You can also. In this case, the first component and the second component will be administered at least once within the treatment period. The administration schedule for each element can be set individually. Both elements may be administered simultaneously. “Simultaneous” here does not require strict simultaneity. Therefore, when both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered. The case where the administration is performed under conditions without a substantial time difference is also included in the concept of “simultaneous” herein.
本発明の有効成分と他の薬剤を併用する態様は以上のものに限定されず、例えば、有効成分として4-アルキルウンベリフェロン又はその誘導体、或いはこれらの薬学的に許容される塩を含有する治療薬とし、それによる治療期間内に他の薬剤も投与するようにしてもよい。 The embodiment in which the active ingredient of the present invention and the other drug are used in combination is not limited to the above, and includes, for example, 4-alkylumbelliferone or a derivative thereof, or a pharmaceutically acceptable salt thereof as the active ingredient. A therapeutic agent may be used and other drugs may be administered within the treatment period.
本発明の治療薬の製剤化は常法に従って行うことができる。製剤化する場合には、製剤上許容される他の成分（例えば、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、生理食塩水など）を含有させることができる。賦形剤としては乳糖、デンプン、ソルビトール、D-マンニトール、白糖等を用いることができる。崩壊剤としてはデンプン、カルボキシメチルセルロース、炭酸カルシウム等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。乳化剤としてはアラビアゴム、アルギン酸ナトリウム、トラガント等を用いることができる。懸濁剤としてはモノステアリン酸グリセリン、モノステアリン酸アルミニウム、メチルセルロース、カルボキシメチルセルロース、ヒドロキシメチルセルロース、ラウリル硫酸ナトリウム等を用いることができる。無痛化剤としてはベンジルアルコール、クロロブタノール、ソルビトール等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としては塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等と用いることができる。 The therapeutic agent of the present invention can be formulated according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
製剤化する場合の剤形も特に限定されない。剤形の例は錠剤、散剤、細粒剤、顆粒剤、カプセル剤、シロップ剤、注射剤、外用剤、及び座剤である。本発明の医薬には、期待される治療効果を得るために必要な量（即ち治療上有効量）の有効成分が含有される。本発明の治療薬の有効成分量は一般に剤形によって異なるが、所望の投与量を達成できるように有効成分量を例えば約0.1重量％〜約95重量％の範囲内で設定する。 The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories. The medicament of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (that is, a therapeutically effective amount). The amount of the active ingredient of the therapeutic agent of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1% by weight to about 95% by weight so as to achieve a desired dose.
本発明の治療薬はその剤形に応じて経口投与又は非経口投与（静脈内、動脈内、皮下、皮内、筋肉内、又は腹腔内注射、経皮、経鼻、経粘膜など）によって対象に適用される。これらの投与経路は互いに排他的なものではなく、任意に選択される二つ以上を併用することもできる（例えば、経口投与と同時に又は所定時間経過後に静脈注射等を行う等）。全身投与によらず、局所投与することにしてもよい。局所投与として、標的組織への直接注入又は塗布を例示することができる。 The therapeutic agent of the present invention is administered orally or parenterally (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. Applies to These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). Local administration may be used instead of systemic administration. Local administration can be exemplified by direct injection or application to the target tissue.
本発明の治療薬の投与量は、期待される治療効果が得られるように設定される。治療上有効な投与量の設定においては一般に患者の症状、年齢、性別、及び体重などが考慮される。当業者であればこれらの事項を考慮して適当な投与量を設定することが可能である。例えば、成人（体重約60kg）を対象として一日当たりの有効成分量が約1,200mg、最大約3,600mgとなるよう投与量を設定することができる。投与スケジュールとしては例えば１日１回〜数回、２日に１回、或いは３日に１回などを採用できる。投与スケジュールの作成においては、患者の症状や有効成分の効果持続時間などを考慮することができる。好ましい投与スケジュールの一つは、長期間に亘って連続的な投与を行う投与スケジュールである。「長期間に亘る連続的な投与」とは、長期間に亘る投薬期間内に複数回の投薬を行うことを意味する。ここでの「長期間」とは１週間以上の期間を意味し、具体的には例えば１月〜数年の間で投与期間を設定することができる。一日当たりの投与回数は例えば１〜５回とする。炎症性関節疾患が慢性的病態であり、その治療のためには薬剤が常に作用していることが好ましいことや有効成分の血中半減期を考慮すれば、投与スケジュールとして連日投与を採用することが好ましい。但し、患者の状態や経過によっては、投与しない日を設けることにしてもよい（即ち、隔日投与などの投与スケジュールを採用してもよい）。 The dose of the therapeutic agent of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, the patient's symptoms, age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. For example, for an adult (body weight of about 60 kg), the dosage can be set so that the amount of active ingredient per day is about 1,200 mg, and the maximum is about 3,600 mg. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's symptoms and the duration of effect of the active ingredient can be considered. One of the preferable administration schedules is an administration schedule for continuous administration over a long period of time. “Continuous administration over a long period of time” means that a plurality of doses are administered within a long-term dosing period. Here, “long term” means a period of one week or more, and specifically, for example, an administration period can be set between one month and several years. The number of administrations per day is, for example, 1 to 5 times. In consideration of the fact that inflammatory joint disease is a chronic condition, and it is preferable that the drug is always acting for its treatment, and the blood half-life of the active ingredient is taken into consideration, daily administration should be adopted. Is preferred. However, depending on the patient's condition and progress, a non-administration day may be provided (that is, an administration schedule such as administration every other day may be adopted).
本発明の治療薬による治療と並行して、既存の治療法を適用することにしてもよい。既存の治療法として薬物療法、外科的治療（手術）、リハビリテーションを挙げることができる。二以上の既存の治療法を組み合わせて適用することにしてもよい。 In parallel with the treatment with the therapeutic agent of the present invention, an existing treatment method may be applied. Existing therapies include drug therapy, surgical treatment (surgery), and rehabilitation. Two or more existing therapies may be applied in combination.
以上の記述から明らかな通り本出願は、患者に対して本発明の治療薬を治療上有効量投与することを特徴とする、炎症性関節疾患の治療法も提供する。 As is apparent from the above description, the present application also provides a method for treating inflammatory joint diseases, characterized by administering a therapeutically effective amount of the therapeutic agent of the present invention to a patient.
4−アルキルウンベリフェロンの新規医薬用途として、炎症性関節疾患の治療への適用を検討した。 As a new medicinal use of 4-alkylumbelliferone, application to the treatment of inflammatory joint diseases was examined.
１．材料と方法
（１）コラーゲン誘発関節炎（collagen-induced arthritis CIA）の作製
7週齢のDBA/1Jマウスを日本エスエルシー株式会社（静岡）から購入した。すべての動物実験は実験動物のケアと使用のための国際的なガイドラインに従い、動物倫理委員会の承認のもとに行われた。
1. Materials and Methods (1) Preparation of collagen-induced arthritis CIA
Seven-week old DBA / 1J mice were purchased from Nippon SLC Co., Ltd. (Shizuoka). All animal experiments were conducted with the approval of the Animal Ethics Committee, following international guidelines for the care and use of laboratory animals.
0.01M酢酸で溶解したウシタイプIIコラーゲン（2mg/ml）を１：１の比率でフロイド完全アジュバントで乳化し、マウスの尾の皮下に100μl注射し、免疫した。その21日後にフロイド不完全アジュバントで乳化させた50μgのウシタイプIIコラーゲンをマウスの尾の皮下に注射し、追加免疫した。 Bovine type II collagen (2 mg / ml) dissolved in 0.01 M acetic acid was emulsified with a complete floyd adjuvant at a ratio of 1: 1, and 100 μl was injected subcutaneously into the tail of the mouse for immunization. Twenty-one days later, 50 μg of bovine type II collagen emulsified with incomplete floyd adjuvant was injected subcutaneously into the tail of mice and boosted.
（２）4-MUによる治療
マウス25匹（治療群）を3群に分けた。それぞれの群（体重あたり0.5mg/g（n=5）、1.5mg/g（n=5）、3.0mg/g（n=15））のマウスに対し、4-MUを5%アラビアガムで混濁し、混濁液300μlを初回免疫後23日目から実験終了の42日目まで毎日、経口投与した。コントロール群（n=15）には5%アラビアガムのみを毎日、経口投与した。関節リウマチ（RA）患者より分離した滑膜様線維芽細胞（fibroblast like synoviocyte FLS）をin vitroにおいて10ng/mlのTNF-αと4-MU（0mM、0.1mM、0.5mM、1.0mM、1.5mM）で同時に処置した。続いて、単層培養し、マトリックスメタロプロテアーゼ（MMPs）の発現やヒアルロン酸（HA）の分布、ヒアルロン酸合成酵素（HAS）mRNAのノックダウンの解析を行った。
(2) Treatment with 4-
（３）関節炎の臨床的な評価
ウシタイプIIコラーゲンによる初回免疫後より、マウスの関節炎の進展をモニタリングした。少なくとも１つの足趾の腫脹もしくは足の腫脹が認められたとき、CIAが発症したと判断した。関節炎の程度はそれぞれの足で次のように0-3で等級分けを行った。0；正常、1；1つの足趾の腫脹、2；2つかそれ以上の足趾の腫脹、3；足全体の腫脹。病状全体の程度と進展を表すためにそれぞれのマウスの四肢のスコアの合計（1匹あたり最大で12点）を関節炎スコア（arthritis score）として使用した。腫脹は足の腫脹の最大の前後径を電気式ノギスで測定し、4つの足の腫脹増加量の合計を2日おきに記録した。
(3) Clinical evaluation of arthritis The progress of arthritis in mice was monitored after the initial immunization with bovine type II collagen. CIA was determined to have occurred when at least one footpad swelling or foot swelling was observed. The degree of arthritis was graded 0-3 on each foot as follows. 0; normal, 1; swelling of one toe, 2; swelling of 2 or more toes, 3; swelling of the entire foot. The sum of the limb scores of each mouse (up to 12 points per mouse) was used as the arthritis score to represent the extent and progression of the overall condition. For swelling, the maximum anteroposterior diameter of foot swelling was measured with an electric caliper, and the total increase in swelling of the four feet was recorded every 2 days.
（４）膝関節の腫脹の組織学的解析
初回免疫から42日後にマウスを全身麻酔下に4%パラホルムアルデヒド（PFA）で潅流固定した。膝関節を摘出し、4%PFAで3日間固定、10%EDTAで30日間、4℃で脱灰した。各種濃度のエタノールで脱水後、パラフィンにて包埋した。それぞれのサンプルはヘマトキシリンとエオジンで染色した。１つの膝関節に対していつくかの矢状断の切片を調べ、最も所見を認める切片を評価し、スコアリングした。典型的なものでは内顆の中央の切片を調べた。関節の組織学的変化には過去に報告されているパラメーターを使用した。それぞれのマウスから得られたすべての切片は予備知識のない観察者によって次のように0-3に等級分けされた。0；正常、1；炎症細胞の浸潤、2；滑膜の肥厚とパンヌスの形成、3；骨びらんと骨破壊。両膝合計で最大6点満点とした。
(4) Histological analysis of knee joint swelling 42 days after the first immunization, the mice were perfusion-fixed with 4% paraformaldehyde (PFA) under general anesthesia. The knee joint was removed, fixed with 4% PFA for 3 days, decalcified with 10% EDTA for 30 days at 4 ° C. After dehydration with various concentrations of ethanol, it was embedded in paraffin. Each sample was stained with hematoxylin and eosin. Several sagittal sections were examined for one knee joint and the section with the most findings was evaluated and scored. Typically, the central section of the condyle was examined. Previously reported parameters were used for joint histological changes. All sections obtained from each mouse were graded 0-3 as follows by an observer without prior knowledge: 0; normal, 1; inflammatory cell infiltration, 2; synovial thickening and pannus formation, 3; bone erosion and bone destruction. The total of both knees was a maximum of 6 points.
（５）免疫染色
パラフィン包埋された切片をキシレンとエタノールで脱パラフィンした。TBS（Tris-buffered saline）で水和した後、50mM トリス酢酸ナトリウムバッファー（50mM Tris、60mM 酢酸ナトリウム）で溶解した0.25U/mlのコンドロイチナーゼABCで処置した。3%H2O2を10分間作用させ、内因性ペルオキシダーゼを枯渇させた。3%ウシ血清アルブミン（BSA）で1時間ブロッキングし、特異的抗体を4℃で1晩、2μg/mlのビオチン化HABP（b-HABP）では室温で2時間反応させた。1次抗体としてマウス抗MMP-3抗体（100倍希釈）（ProteinTech）とマウス抗MMP-13抗体（100倍希釈）（ProteinTech）を使用した。TBS Tween-20で3回洗浄後、抗MMP抗体を使用した場合にはSimplestain mouse Max-PO（株式会社ニチレイ）を、ビオチン化HABPを使用した場合はストレプトアビジン−ペルオキシダーゼ試薬(株式会社ニチレイ)をそれぞれ室温で30分間反応させた。続いてDAB溶液（3', 3'-diaminobenzidine (DAB)-hydrogen peroxide substrate medium：株式会社ニチレイ）を5分間反応させ、ヘマトキシリンで対比染色を行った。ネガティブコントロールとして1次抗体を使用せずに染色を行った。染色された細胞を顕微鏡下（倍率400倍）に3視野でそれぞれカウントし、その数を平均化した。視野におけるすべての軟骨細胞の数で染色されている軟骨細胞の数を割ることにより染色細胞の割合を算出した。
(5) Immunostaining Paraffin-embedded sections were deparaffinized with xylene and ethanol. After hydration with TBS (Tris-buffered saline), the cells were treated with 0.25 U / ml chondroitinase ABC dissolved in 50 mM sodium tris acetate buffer (50 mM Tris, 60 mM sodium acetate). 3% H 2 O 2 was allowed to act for 10 minutes to deplete endogenous peroxidase. The antibody was blocked with 3% bovine serum albumin (BSA) for 1 hour, and the specific antibody was reacted overnight at 4 ° C. and 2 μg / ml biotinylated HABP (b-HABP) at room temperature for 2 hours. Mouse anti-MMP-3 antibody (diluted 100 times) (ProteinTech) and mouse anti-MMP-13 antibody (diluted 100 times) (ProteinTech) were used as primary antibodies. After washing 3 times with TBS Tween-20, use Simplestain mouse Max-PO (Nichirei Co., Ltd.) when using anti-MMP antibody, and Streptavidin-Peroxidase reagent (Nichirei Co., Ltd.) when using biotinylated HABP. Each was reacted at room temperature for 30 minutes. Subsequently, a DAB solution (3 ′, 3′-diaminobenzidine (DAB) -hydrogen peroxide substrate medium: Nichirei Co., Ltd.) was reacted for 5 minutes and counterstained with hematoxylin. As a negative control, staining was performed without using the primary antibody. Stained cells were counted in 3 fields under a microscope (400 times magnification), and the numbers were averaged. The percentage of stained cells was calculated by dividing the number of chondrocytes stained by the number of all chondrocytes in the field of view.
（６）CIAマウスの血清HA濃度
それぞれの群（n=10）で血清HA濃度を測定するために、潅流固定前に心臓より血液を採取した。関節炎のないコントロールとして、免疫していない正常マウス（n=10）から血液を採取した。凝固した血液を15分3000rpm（503g）で遠心し、血清を-80℃で保存した。サンドイッチELISAにて血清HA濃度を測定した。
(6) Serum HA concentration of CIA mice In order to measure the serum HA concentration in each group (n = 10), blood was collected from the heart before perfusion fixation. As a control without arthritis, blood was collected from non-immunized normal mice (n = 10). The coagulated blood was centrifuged at 3000 rpm (503 g) for 15 minutes, and the serum was stored at −80 ° C. Serum HA concentration was measured by sandwich ELISA.
（７）細胞培養
関節置換術を受けた関節リウマチ患者の滑膜組織より線維芽細胞様滑膜細胞（FLS）を単離した。すべての患者（n=5）より同意が得られた。滑膜組織を小さな切片にし、ダルベッコ変法イーグル培地(DMEM)で溶解した4mg/ml コラゲナーゼを使用し、37℃で3時間分離させた。70-μm セルストレイナーでフィルタリング後、DMEMで洗浄した。10%ウシ胎仔血清と抗生剤(ペニシリン: 100U/ml, ストレプトマイシン: 100μg/ml, Amphotecerin-B: 0.25μg/ml)の入ったDMEMで滑膜細胞を混濁した後、単層培養した。4継代〜6継代の細胞を実験に使用した。
(7) Cell culture Fibroblast-like synoviocytes (FLS) were isolated from the synovial tissue of rheumatoid arthritis patients who had undergone joint replacement. Consent was obtained from all patients (n = 5). The synovial tissue was cut into small sections and separated at 37 ° C. for 3 hours using 4 mg / ml collagenase dissolved in Dulbecco's modified Eagle's medium (DMEM). After filtering with a 70-μm cell strainer, it was washed with DMEM. Synovial cells were made turbid with DMEM containing 10% fetal bovine serum and antibiotics (penicillin: 100 U / ml, streptomycin: 100 μg / ml, Amphotecerin-B: 0.25 μg / ml), and then cultured in a monolayer. Cells from
（８）細胞への刺激とリアルタイムPCR
FLS（1×105細胞/ウェル）を6ウェルプレートで2日間、培養後、洗浄し、血清の入っていないDMEMで1晩飢餓状態とした。細胞を10ng/mlのTNF-αで刺激し、同時に4-MU（0mM、0.1mM、0.5mM、1.0mM、1.5mM）で12時間処置した。4-MUはジメチルスルホキシド(DMSO)で溶解し、最終のDMSOの濃度が0.1%となるようにした。コントロールの細胞は血清の入っていないDMEMで培養した。12時間処置した後、添付のプロトコールに従い、RNeasy（登録商標）Min Kit (QIAGEN)により全RNAを調製した。High Capacity cDNA Reverse transcription kit (Applied Biosystems)を用い、cDNAへ逆転写を行った。
(8) Cell stimulation and real-time PCR
FLS (1 × 10 5 cells / well) was cultured in a 6-well plate for 2 days, washed, and starved overnight with DMEM without serum. Cells were stimulated with 10 ng / ml TNF-α and simultaneously treated with 4-MU (0 mM, 0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM) for 12 hours. 4-MU was dissolved in dimethyl sulfoxide (DMSO) so that the final DMSO concentration was 0.1%. Control cells were cultured in DMEM without serum. After treatment for 12 hours, total RNA was prepared with RNeasy (registered trademark) Min Kit (QIAGEN) according to the attached protocol. Reverse transcription was performed on cDNA using High Capacity cDNA Reverse transcription kit (Applied Biosystems).
MMP-1とMMP-3の定量的な発現解析のためにLightCycler 480 SYBR Green 1 kit (Roche Molecular Biochemical)とLightCycler instrument (Roche Diagnostics)を使用し、リアルタイムPCRを添付のプロトコールに従い行った。標的となるmRNAの相対的なレベルはGAPDHにより標準化した。MMP-1、MMP-3、GAPDHのプライマー配列は以下の通りである。
MMP-1用：5'-TGGACCTGGAGGAAATCTTG-3'（フォワード、配列番号１）、5'-AGTTCATGAGCTGCAACACG-3'（リバース、配列番号２）
MMP-3用：5'-TTCCTTGGATTGGAGGTGAC-3'（フォワード、配列番号３）、5'-TGCCAGGAAAGGTTCTGAAG-3'（リバース、配列番号４）
GAPDH用：5'-TGAACGGGAAGCTCACTGG-3'（フォワード、配列番号５）、5'-TCCACCACCCTGTTGCTGTA-3'（リバース、配列番号６）
For quantitative expression analysis of MMP-1 and MMP-3, LightCycler 480
For MMP-1: 5'-TGGACCTGGAGGAAATCTTG-3 '(forward, SEQ ID NO: 1), 5'-AGTTCATGAGCTGCAACACG-3' (reverse, SEQ ID NO: 2)
For MMP-3: 5'-TTCCTTGGATTGGAGGTGAC-3 '(forward, SEQ ID NO: 3), 5'-TGCCAGGAAAGGTTCTGAAG-3' (reverse, SEQ ID NO: 4)
For GAPDH: 5'-TGAACGGGAAGCTCACTGG-3 '(forward, SEQ ID NO: 5), 5'-TCCACCACCCTGTTGCTGTA-3' (reverse, SEQ ID NO: 6)
（９）ウエスタンブロット解析
FLSにおけるMMP-1とMMP-3のタンパクレベルの発現に対する4-MUの効果をウエスタンブロットで解析した。FLSをTNF-α（10ng/ml）で刺激し、同時に4-MU（0mM、0.1mM、0.5mM、1.0mM、1.5mM）で24時間処置した。コントロールとして血清の入っていないDMEMで細胞を培養した。細胞溶解液はRIPAバッファー（Santa Cruz Biotechnology）で調整し、タンパク濃度はブラッドフォード法で測定した。抽出したタンパク質（1レーンにつき40μg）をサンプルとして、抗MMP-1抗体、抗MMP-3抗体、抗β−アクチン抗体を使用したウエスタンブロット解析を行った。
(9) Western blot analysis
The effect of 4-MU on protein level expression of MMP-1 and MMP-3 in FLS was analyzed by Western blot. FLS was stimulated with TNF-α (10 ng / ml) and simultaneously treated with 4-MU (0 mM, 0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM) for 24 hours. As a control, cells were cultured in DMEM without serum. The cell lysate was prepared with RIPA buffer (Santa Cruz Biotechnology), and the protein concentration was measured by the Bradford method. Using the extracted protein (40 μg per lane) as a sample, Western blot analysis using anti-MMP-1 antibody, anti-MMP-3 antibody, and anti-β-actin antibody was performed.
（１０）siRNAでのFLSのトランスフェクション
4-MUの効果がHA合成の抑制によって誘導されているものであるか否かを調べるために、ヒアルロン酸合成酵素1（HAS1）とHAS2とHAS3をそれぞれ、もしくは３つともノックダウンした後に、FLSをTNF-α（10ng/ml）で刺激した。MMP-1とMMP-3のmRNA発現をリアルタイムで評価した。ヒトのHAS1、HAS2、HAS3に特異的なsiRNAはSigma-Aldrich社より購入した。siRNAの配列は以下のとおりである。
HAS1用：5'-CCUCUAGGCCUAUAUAGGATT-3'（配列番号７）
HAS2用：5'-CCAGUAUCAGUUUGGUUUATT-3'（配列番号８）
HAS3用：5'-GACCCUGACUACUUGCGCATT-3'（配列番号９）
(10) Transfection of FLS with siRNA
In order to investigate whether the effect of 4-MU is induced by inhibition of HA synthesis, after knocking down hyaluronic acid synthase 1 (HAS1) and HAS2 and HAS3, respectively, or all three, FLS was stimulated with TNF-α (10 ng / ml). MMP-1 and MMP-3 mRNA expression was evaluated in real time. SiRNA specific for human HAS1, HAS2, and HAS3 were purchased from Sigma-Aldrich. The sequence of siRNA is as follows.
For HAS1: 5'-CCUCUAGGCCUAUAUAGGATT-3 '(SEQ ID NO: 7)
For HAS2: 5'-CCAGUAUCAGUUUGGUUUATT-3 '(SEQ ID NO: 8)
For HAS3: 5'-GACCCUGACUACUUGCGCATT-3 '(SEQ ID NO: 9)
ネガティブコントロールとしてMISSION siRNA universal negative control (Sigma-Aldrich社)を使用した。添付のプロトコールに従ってLipofectamine2000 (Invitrogen)を使用し、FLSにsiRNAをトランスフェクトした。12時間後にリアルタイムPCRにて、siRNAを用いたノックダウン効率を測定した。HAS1とHAS2とHAS3のそれぞれ、もしくは３つのノックダウン下でのMMP-1とMMP-3のmRNA発現における4-MUの効果をリアルタイムPCRにて解析した。 MISSION siRNA universal negative control (Sigma-Aldrich) was used as a negative control. FLS was transfected with siRNA using Lipofectamine 2000 (Invitrogen) according to the attached protocol. After 12 hours, knockdown efficiency using siRNA was measured by real-time PCR. The effect of 4-MU on mRNA expression of MMP-1 and MMP-3 under each of HAS1, HAS2 and HAS3 or three knockdowns was analyzed by real-time PCR.
（１１）FLSでのHAの集積
チャンバースライド(BD Biosciences)に細胞（1×104）を6時間培養した。その後、10ng/mlのTNF-αで刺激し、同時に4-MU（1.0mM）で12時間処置、一方は4-MUで処置しなかった。コントロールとして血清の入っていないDMEMのみで培養した。細胞を4%PFAで2時間室温で固定した。内因性ペルオキシダーゼを枯渇させるため、30%メタノールで希釈した0.3%H2O2で30分処置した。1%BSAで1時間室温で処置し、その後、2.0μg/mlのビオチン化HABPで2時間、室温で処置した。ビオチン化HABPをストレプトアビジン−ペルオキシダーゼ試薬(株式会社ニチレイ)とDAB含有基質溶液（株式会社ニチレイ）で検出した。
(11) Accumulation of HA in FLS Cells (1 × 10 4 ) were cultured for 6 hours on chamber slides (BD Biosciences). Thereafter, stimulation with 10 ng / ml TNF-α was performed simultaneously with 4-MU (1.0 mM) for 12 hours, while one was not treated with 4-MU. As a control, the cells were cultured only in DMEM without serum. Cells were fixed with 4% PFA for 2 hours at room temperature. To deplete endogenous peroxidase, it was treated with 0.3% H 2 O 2 diluted with 30% methanol for 30 minutes. Treated with 1% BSA for 1 hour at room temperature followed by 2.0 μg / ml biotinylated HABP for 2 hours at room temperature. Biotinylated HABP was detected with a streptavidin-peroxidase reagent (Nichirei Co., Ltd.) and a DAB-containing substrate solution (Nichirei Co., Ltd.).
（１２）HAの定量化
サブコンフルエントの状態のFLSを10ng/mlのTNF-αで刺激し、同時に4-MU（0mM、0.1mM、0.5mM、1.0mM、1.5mM）で24時間処置した。コントロールとして血清の入っていないDMEMで24時間培養した。以前報告された方法（Kakizaki et al, J Biol Chem, 2004; 279:33281-33289）でHAを採取した。培養液を分取し、「培養液サンプル」とした。細胞周囲のHAを取り除くため、細胞をトリプシン-EDTAで10分間、37℃で処置した後、PBSで洗浄した。トリプシン溶液と洗浄液を「細胞周囲サンプル」とした。細胞数を数えた後、細胞をプロテアーゼK溶液(0.15 M Tris-HCl, pH 7.5, 0.15 M NaCl, 10mM CaCl2, 20ユニットのプロテアーゼKを含む5 mM deferoxaminemesylate)で2時間、55℃で処置し、その溶液を「細胞内サンプル」とした。すべてのサンプルについて、タンパク分解酵素を不活化するために100℃で15分間加熱した。その後、4℃で30分間15000gで遠心し、解析に供した。HA濃度はサンドイッチELISAで測定した。
(12) Quantification of HA Subconfluent FLS was stimulated with 10 ng / ml of TNF-α and simultaneously treated with 4-MU (0 mM, 0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM) for 24 hours. As a control, the cells were cultured in DMEM without serum for 24 hours. HA was collected as previously reported (Kakizaki et al, J Biol Chem, 2004; 279: 33281-33289). The culture solution was collected and used as a “culture solution sample”. To remove pericellular HA, cells were treated with trypsin-EDTA for 10 minutes at 37 ° C. and then washed with PBS. The trypsin solution and the washing solution were used as “pericellular samples”. After counted the number of cells, the cells protease K solution (0.15 M Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM deferoxaminemesylate with protease K in 10
（１３）既存薬（NSAID）との比較
4-MUの効果を、既存の非ステロイド抗炎症薬（インドメタシン、エトドラク、セレコキシブ）の効果と比較した。関節リウマチ患者由来のFLSを10ng/mlのTNF-αで刺激し、同時に4-MU又は非ステロイド薬で12時間処置した。処置後の細胞のMMP-1及びMMP-3のmRNA発現量を比較した。
(13) Comparison with existing drugs (NSAID)
The effect of 4-MU was compared with that of existing non-steroidal anti-inflammatory drugs (indomethacin, etodolac, celecoxib). FLS from patients with rheumatoid arthritis was stimulated with 10 ng / ml TNF-α and simultaneously treated with 4-MU or non-steroidal drugs for 12 hours. The expression levels of MMP-1 and MMP-3 mRNA in the cells after treatment were compared.
（１４）統計解析
データは平均±標準偏差で表した。ボンフェローニ／ダン検定及びノンパラメトリック マン・ホイットニーのU検定を使用し、群間の統計的な有意差を求めた。P値が0.05以下で有意差ありと判断した。
(14) Statistical analysis Data were expressed as mean ± standard deviation. The Bonferroni / Dan test and the nonparametric Mann-Whitney U test were used to determine statistically significant differences between groups. A P value of 0.05 or less was judged to be significant.
２．結果
（１）4-MUでの治療によるCIAの臨床的な徴候の抑制
この実験で使用したものと同じ免疫プロトコールを使用した過去の文献（Rosloniec EF, Cremer M, Kang AH, Myers LK, Brand DD: Collagen-induced arthritis. Current protocols in immunology / edited by John E Coligan [et al] 2010, Chapter 15:Unit 15 15 11-25.）では通常、初回免疫後、21日から28日に関節炎の徴候が現れ、病状が急速に進行すると報告している。この報告を踏まえ、関節炎発症の早い時期での4-MUの効果を調べるために、初回免疫から23日後に4-MUによる治療を開始した。実際にコントロールマウス（4-MUによる治療をしない群）は初回免疫後23日から30日の間にほぼ100％CIAを発症し、実験の正確性が裏付けられた。
2. Results (1) Inhibition of clinical signs of CIA by treatment with 4-MU Previous literature using the same immunization protocol as used in this experiment (Rosloniec EF, Cremer M, Kang AH, Myers LK, Brand DD Current protocols in immunology / edited by John E Coligan [et al] 2010, Chapter 15:
臨床的な関節炎を21日目から42日目まで評価した。関節炎スコア（Arthritis score）は濃度依存性に4-MUによって抑制された（図１）。もっとも効果的であったのは3mg/gの4-MU投与群であった。関節炎スコア以外の評価として、関節炎の程度を評価するために足の腫脹を測定した。足の腫脹は4-MU治療群で有意に小さかった（P<0.05）（図２）。 Clinical arthritis was evaluated from day 21 to day 42. The arthritis score was suppressed by 4-MU in a concentration-dependent manner (FIG. 1). The most effective group was the 3 mg / g 4-MU group. As an assessment other than the arthritis score, paw swelling was measured to assess the degree of arthritis. Paw swelling was significantly less in the 4-MU treated group (P <0.05) (Figure 2).
4-MUの毒性は治療群とコントロール群の体重及び食事量を毎日モニタリングすることで評価した。加えて、HAがその細胞外基質の維持と保全に重要な役割を担っている関節軟骨をヘマトキシリン染色で評価した。すべてのマウスは実験の間、生存した。42日後、4群間で体重に差はなく、4-MUによって治療されたマウスの関節軟骨は顕微鏡的に正常であった（データ示さず）。 Toxicity of 4-MU was evaluated by daily monitoring of body weight and diet in the treatment and control groups. In addition, the articular cartilage, in which HA plays an important role in maintaining and maintaining its extracellular matrix, was evaluated by hematoxylin staining. All mice survived during the experiment. After 42 days, there was no difference in body weight between the 4 groups and the articular cartilage of mice treated with 4-MU was microscopically normal (data not shown).
（２）関節炎の程度の組織学的評価
42日目にコントロール群と4-MU治療群のマウスを安楽死させ、肢の組織学的解析を行った。コントロールマウスから得られた肢の膝関節は疾患による著明な変化を示した。その変化は炎症性細胞の浸潤を伴った滑膜の肥厚、パンヌスの形成、激しい軟骨及び関節の破壊等である（図３）。MU治療群の膝関節では軽度な滑膜炎を示し、明らかな軟骨や骨を破壊は認められなかった。組織学的スコア（Histological score）はコントロール群より4-MU治療群で低く、濃度依存性であった（図４）。滑膜炎の程度はコントロール群と比べ、1.5mg/g治療群と3.0mg/g治療群で有意に低かった（図４）。
(2) Histological evaluation of the degree of arthritis
On day 42, mice in the control group and the 4-MU treatment group were euthanized and histological analysis of the limbs was performed. The knee joints of the limbs obtained from control mice showed significant changes due to the disease. Changes include synovial thickening with infiltration of inflammatory cells, pannus formation, severe cartilage and joint destruction (FIG. 3). The knee joint in the MU treatment group showed mild synovitis, and no obvious cartilage or bone destruction was observed. The histological score was lower in the 4-MU treatment group than in the control group and was concentration dependent (FIG. 4). The degree of synovitis was significantly lower in the 1.5 mg / g treatment group and the 3.0 mg / g treatment group than in the control group (FIG. 4).
（３）免疫組織学
MMPファミリーは関節軟骨の破壊において重要な因子である。in vivoの関節軟骨と滑膜組織でのMMPの発現における4-MUの効果を評価するために、CIAマウスから採取した膝関節の切片をサンプルとして免疫組織染色を行った。関節軟骨におけるMMP-3とMMP-13の発現はコントロール群で顕著に増加していた。そして4-MU治療によって抑制されていた（図５、図６）。また、コントロール群において、それらの発現は滑膜の浸潤部位や滑膜組織で著明に増加していた。一方で4-MUの治療により滑膜組織でのMMPの発現は抑制された（図７）。
(3) Immunohistology
The MMP family is an important factor in the destruction of articular cartilage. In order to evaluate the effect of 4-MU on the expression of MMP in articular cartilage and synovial tissue in vivo, immunohistochemical staining was performed using a section of knee joint taken from CIA mice as a sample. The expression of MMP-3 and MMP-13 in articular cartilage was significantly increased in the control group. And it was suppressed by 4-MU treatment (Figs. 5 and 6). In the control group, their expression was markedly increased in the synovial infiltration site and synovial tissue. On the other hand, treatment with 4-MU suppressed the expression of MMP in synovial tissue (FIG. 7).
さらに膝関節でのHAの局在とHAの集積に対する4-MUの効果を調べるために、CIAマウスの膝関節の切片について、b-HABPを使用した免疫染色を行った。コントロール群では骨への滑膜の浸潤部位においてHAの異常な集積を認めた。4-MU治療群や正常マウスにおいては滑膜細胞のHAの染色性は弱かった。軟骨細胞のHAの染色性は4-MU治療群と正常マウス群で同程度であった（図８）。 Furthermore, in order to investigate the effect of 4-MU on the localization and hyaluronan accumulation in the knee joint, sections of the knee joint of CIA mice were immunostained using b-HABP. In the control group, abnormal accumulation of hyaluronan was observed at the site of synovial infiltration into the bone. In the 4-MU treated group and normal mice, the hyaluronan staining of synovial cells was weak. The staining of hyaluronan in chondrocytes was comparable between the 4-MU treated group and the normal mouse group (FIG. 8).
（４）CIAマウスにおける血清HA濃度
全身のHA濃度に対する4-MUの効果を調べるために血清のHA濃度を測定した。血清HA濃度は正常マウス群（CIAを誘発していない群）で696.2±335.2ng/ml、コントロール群（CIA誘発マウス無治療群）で1264.5±438.7 ng/ml、4-MU（3mg/g）治療CIAマウス群で539.4±191.6 ng/mlであった（図９）。4-MU治療群の血清HA濃度は正常マウス群と同程度であった。一方で、無治療のCIAマウス群は血清HA濃度が4-MU治療群と正常群に比べ有意に高かった（p<0.01）。これらの結果は、4-MU（3mg/g）の治療がCIAマウスの異常なHA濃度を正常マウスの濃度まで正常化させることを示している。
(4) Serum HA concentration in CIA mice In order to examine the effect of 4-MU on systemic HA concentration, the serum HA concentration was measured. Serum HA concentration is 696.2 ± 335.2 ng / ml in the normal mouse group (the group not inducing CIA), 1264.5 ± 438.7 ng / ml in the control group (the group not treated with CIA-induced mice), 4-MU (3 mg / g) It was 539.4 ± 191.6 ng / ml in the treated CIA mouse group (FIG. 9). The serum HA concentration in the 4-MU treatment group was similar to that in the normal mouse group. On the other hand, the serum HA concentration in the untreated CIA mouse group was significantly higher than that in the 4-MU treated group and the normal group (p <0.01). These results indicate that treatment with 4-MU (3 mg / g) normalizes abnormal HA concentrations in CIA mice to normal mouse concentrations.
（５）RAFLS（関節リウマチ患者由来の線維芽細胞様滑膜細胞）でのMMP-1とMMP-3のmRNA、タンパク発現に対する4-MUの効果
関節軟骨破壊の因子であるMMPは関節リウマチ（RA）では主にFLSによって分泌されている。RAFLSでのMMP-1とMMP-3に対する4-MUの効果を調べるために、細胞をTNF-α（10ng/ml）で刺激し、同時に4-MU（0mM, 0.1mM, 0.5mM, 1.0mM, 1.5mM）で処置した。リアルタイムPCRと濃度測定を使ったウエスタンブロット解析の結果、MMP-1とMMP-3のmRNAとタンパクは濃度依存性に有意に4-MUによって抑制された（図１０、１１）。
(5) Effects of 4-MU on MMP-1 and MMP-3 mRNA and protein expression in RAFLS (fibroblast-like synoviocytes derived from rheumatoid arthritis patients) MMP, which is a factor of articular cartilage destruction, is rheumatoid arthritis ( In RA), it is mainly secreted by FLS. To examine the effects of 4-MU on MMP-1 and MMP-3 in RAFLS, cells were stimulated with TNF-α (10 ng / ml) and simultaneously with 4-MU (0 mM, 0.1 mM, 0.5 mM, 1.0 mM) , 1.5 mM). As a result of Western blot analysis using real-time PCR and concentration measurement, mRNA and protein of MMP-1 and MMP-3 were significantly suppressed by 4-MU in a concentration-dependent manner (FIGS. 10 and 11).
HAS1とHAS2とHAS3のそれぞれのノックダウンと３つ同時のノックダウン下において、MMP-1、MMP-3のmRNA発現を調べた。HAS1、HAS2、HAS3それぞれ単独のノックダウンの効果は4-MUによる効果とは同等ではなかったが、3つ同時のノックダウンでは4-MUと同等の効果を示した。尚、単独のノックダウン効率はHAS1が95%、HAS2が61%、HAS3が72%であり、3つ同時のノックダウンではHAS1が98%、HAS2が88%、HAS3が92%であった。これらの結果はMMP-1、MMP-3発現における4-MUの抑制効果はHA依存の経路を介していることを示唆する。 Under the knockdown of HAS1, HAS2 and HAS3 and three simultaneous knockdowns, mRNA expression of MMP-1 and MMP-3 was examined. The effects of knockdown by HAS1, HAS2, and HAS3 were not equivalent to the effects of 4-MU, but three simultaneous knockdowns showed the same effects as 4-MU. The single knockdown efficiency was 95% for HAS1, 61% for HAS2, and 72% for HAS3. With three simultaneous knockdowns, HAS1 was 98%, HAS2 was 88%, and HAS3 was 92%. These results suggest that the inhibitory effect of 4-MU on MMP-1 and MMP-3 expression is mediated by an HA-dependent pathway.
（６）RAFLSにおいて4-MUはHAの集積を抑制する
FLSにおけるHA集積に対する4-MUの効果を評価するためにビオチン化HABPを使用しHAを可視化した。さらにサンドイッチELISAを使用し、HA濃度を測定した。図１２に示すように、TNF-α（10ng/ml）で12時間刺激した細胞はHAの著明な染色性を示した。特に細胞表面と細胞周囲において著明であった。TNF-αで刺激した細胞でのHAの染色性は4-MUによって抑制された。さらに糸状仮足を連想させる部位はTNF-αの刺激によりHAが強く染色された。4-MUによる処置はHAの染色性を抑制し、特にその糸状仮足を連想させる部位で抑制した。一方で細胞内の染色性はTNF-α刺激のみのものと4-MUで処置したもので差がなかった。4-MUで処置した培養細胞における培地と細胞周囲のHA濃度は4-MUで処置しないものに比べ、用量依存性に低くなった。対照的に細胞内のHA濃度は4-MUで処置したものとしないもので有意な差はなかった。このことは4-MUがトリプシンに影響を受けるHAの集積を抑制していること示している。トリプシンに影響を受けるHAはCD44のような細胞表面のHAレセプターと結合するかもしれない。｛しじょうかそく｝を連想させる部位で抑制した。一方で細胞内の染色性はTNF-α刺激のみものと4-MUで処置したもので変わりなかった（Fig.6A）。4-MUで処置した培養細胞における培地と細胞周囲のHA濃度は4-MUで処置しないものに比べ、用量依存性に低くなった。対象的に細胞内のHA濃度は4-MUで処置したものとしないもので有意な差はなかった。このことは4-MUがtrypsinに影響を受けるHAの集積を抑制していること示している。trypsinに影響を受けるHAはCD44のような細胞表面のHAレセプターと結合するかもしれない。
(6) 4-MU suppresses HA accumulation in RAFLS
In order to evaluate the effect of 4-MU on HA accumulation in FLS, biotinylated HABP was used to visualize HA. Furthermore, the HA concentration was measured using a sandwich ELISA. As shown in FIG. 12, cells stimulated with TNF-α (10 ng / ml) for 12 hours showed marked staining of HA. Especially on the cell surface and around the cell. The staining of HA in cells stimulated with TNF-α was suppressed by 4-MU. Furthermore, hyaluronan was strongly stained at the site reminiscent of the filopodia by stimulation with TNF-α. Treatment with 4-MU suppressed the staining of hyaluronan, particularly at sites reminiscent of the filopodia. On the other hand, there was no difference in intracellular staining between those treated with TNF-α alone and those treated with 4-MU. Media and pericellular hyaluronan concentrations in cultured cells treated with 4-MU were less dose-dependent than those not treated with 4-MU. In contrast, intracellular HA concentrations were not significantly different between those treated with 4-MU and those not treated with 4-MU. This indicates that 4-MU suppresses the accumulation of hyaluronan affected by trypsin. HA that is affected by trypsin may bind to cell surface HA receptors such as CD44. Suppressed at a site reminiscent of {Shijokasoku}. On the other hand, intracellular staining did not change between TNF-α stimulation alone and 4-MU treatment (Fig. 6A). Media and pericellular hyaluronan concentrations in cultured cells treated with 4-MU were less dose-dependent than those not treated with 4-MU. In contrast, intracellular hyaluronan concentrations were not significantly different between those treated with 4-MU and those not treated with 4-MU. This indicates that 4-MU suppresses the accumulation of hyaluronan affected by trypsin. HA affected by trypsin may bind to cell surface HA receptors such as CD44.
（７）既存薬（NSAID）との比較
既存の非ステロイド抗炎症薬（インドメタシン、エトドラク、セレコキシブ）と比較することによって、4-MUの効果を評価した。4-MUは、インドメタシン及びエトドラクに比較して、TNF-αで刺激されたRAFLSにおけるMMP-1及びMMP-3のmRNAの発現を有意に抑制した（図１３、１４）。また、セレコキシブとの比較においても、4-MUの効果が高いことが示された（図１５）。
(7) Comparison with existing drugs (NSAID) The effect of 4-MU was evaluated by comparing with existing nonsteroidal anti-inflammatory drugs (indomethacin, etodolac, celecoxib). 4-MU significantly suppressed the expression of MMP-1 and MMP-3 mRNA in RAFLS stimulated with TNF-α as compared to indomethacin and etodolac (FIGS. 13 and 14). In addition, the effect of 4-MU was also shown in comparison with celecoxib (FIG. 15).
本発明の医薬は炎症性関節疾患の治療に用いられる。例えば、膠原病に伴う関節炎又は滑膜炎に対して適用可能である。 The medicament of the present invention is used for the treatment of inflammatory joint diseases. For example, it can be applied to arthritis or synovitis associated with collagen disease.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
配列番号１〜６：人工配列の説明：プライマー
配列番号７〜９：人工配列の説明：siRNA
SEQ ID NOs: 1-6: Description of artificial sequence: Primer SEQ ID NOs: 7-9: Description of artificial sequence: siRNA
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