JP4521894B2 - Matrix metalloproteinase inhibitor - Google Patents

Description

（式中、Ｒは、ガロイル基、または隣接する２つのＲが一緒になってヘキサヒドロキシジフェノイル基を示す）
で示される化合物である（１）記載のマトリックスメタロプロテアーゼ阻害剤、（３）タンニン化合物を有効成分として含有するマトリックスメタロプロテアーゼ（MMPs）活性調節不能に起因する難治性疾患の治療および予防剤、
（４）タンニン化合物が下記の一般式（I）：
【化４】

（式中、Ｒは、ガロイル基、または隣接する２つのＲが一緒になってヘキサヒドロキシジフェノイル基）
で示される化合物である請求項３記載の難治性疾患の治療および予防剤、
（５）難治性疾患が慢性関節リウマチ、変形性関節症、歯周疾患、異所性脈管形成、腫瘍性浸潤および転移、潰瘍形成、骨疾患、血管再閉塞、血管再狭窄、HIV感染症、または糖尿病合併症である（４）記載の治療および予防剤、
に関する。
【００１５】
【発明の実施の形態】
タンニン化合物は、広く植物中（特に樹皮に多く含まれる）に存在する多数のフェノール性ヒドロキシル基をもつ複雑な構造の芳香族化合物で、水で抽出される。本発明のタンニン化合物は、該タンニン化合物であって、かつMMPs阻害作用を示すタンニン化合物を全て包含する。タンニン化合物は、化学構造上、１）加水分解性タンニン化合物、および２）縮合型タンニン化合物に分類される。加水分解性タンニン化合物は、一般に、分子内のポリフェノール部分として、ガロイル（galloyl）基、ヘキサヒドロキシジフェノイル（hexahydoxydiphenoyl; HHDP)基、およびその酸化体などがあり、これらが、分子内の糖または環状ポリアルコールとエステル結合した構造をもつ。これらは、それぞれガロタンニン（gallo-tannin)、およびエラジタンニン（ellagitannin)と総称される。酸や酵素によって、このエステル結合が切断されやすいので、加水分解性タンニンの名がある(Okuda, T. et al.: Heterocycles,15 : 1323, 1981; 奥田拓男: 化学と薬学の教室, 80: 12, 1983; 81: 22,1983; 83: 2,1984)。
一方、縮合型タンニン化合物は、カテキン（catechin)類が、たがいに分子間で、Ｃ4-Ｃ8、またはＣ4-Ｃ6位などでＣ-Ｃ結合により結ばれた２量体以上の大きい分子を順次形成したものである。酸や酵素の作用でこの縮合がさらに進行し、より大きい分子を形成していくことが多い(Okuda, T. et al.: Heterocycles,15 : 1323, 1981; 奥田拓男: 化学と薬学の教室, 80: 12, 1983; 81: 22,1983; 83: 2,1984)。
加水分解性のタンニンの糖部分の１位とポリフェノールが結合して、Ｃ-配糖体を形成し、糖の環が開環した構造をもつＣ-配糖体型タンニン（C-glycoside tannin)、加水分解性タンニン分子にカテキン類が結合した複合タンニン化合物（complex tannin)(Okuda,T. et al.: Chem. Pharm. Bull., 35 : 443, 1987)、加水分解性タンニンが２分子以上たがいに結合したオリゴマー化合物（oligomeric hydrolyzable tannin)(Okuda, T. et al.: J. Natl. Prod.,52: 1, 1989)、分子内のポリフェノール部分が、カフェー酸（caffeoyl基）であるカフェータンニン（caffeetannin)(奥田拓男・他: 薬誌,106: 894,1986)やシソ科タンニン（labiataetannin)(奥田拓男・他: 薬誌,106: 1108, 1986; Agata,I. et al.: Chem. Pharm. Bull. 35: 3223, 1988)、DHHDP(dehydrohexahydroxy diphenoyl)基をもつエラジタンニン（dehydroellagitannin)［Okuda, T. et al.: Tetrahedron Lett. 23: 3941, 1982)がアスコルビン酸と縮合したもの(Okuda,T. et al.: Chem. Pharm. Bull. 34: 4075, 1986)、などタンニン化合物の種類は多い。
【００１６】
本発明のタンニン化合物は、タンニン含有植物から、水、または水分含量の多い水性エタノールを用いて抽出する。タンニン含有植物としては、先ず、日常的に飲用している、緑茶、紅茶などの茶葉が挙げられるが、その他にも、例えば、ツバキ（Camellia japonica)、ハーブ・スパイスとして、ベイベリイ（Bayberry)、クローブ（Clove)、シャクヤク（Peony)、ローズ・レッド（Rose red)、ベイリーフ、コンフリー（Comfrey)、ユーカリ（Eucalyptus)、メリーザ（Melissa)、ペニーロイヤル（Pennyroyal)、ローズ・ピンク（Rose pink)、スター・アニス（Star anise)、タイムなどが挙げられる。これらの植物は、採取された直後は、一般に多量の水分を含有するので、含有成分タンニンの変質を防止するためにも、乾燥が必要である。
これらのタンニン含有植物からのタンニンの抽出は、水性溶媒、例えば、精製水、或いは、エタノールやメタノールを含む精製水により行う。抽出温度、および時間は、タンニンの安定性を考慮して、当業者が適宜設定することができる。抽出液は、そのまま本発明に用いることができるが、抽出液の濃縮物、あるいは該抽出液を分画、文献に記載の方法(Hashimoto, M. et al.: Chem. Pharm. Bull. 37: 77-85, 1989)で精製したタンニン化合物を用いることが好ましい。また、化学合成したタンニンでもよい。好ましくは、一般式（I）で示されるタンニン化合物（式中、Ｒは、ガロイル基、または隣接する２つのＲが一緒になってヘキサヒドロキシジフェノイル基を示す）などを、本発明のMMPs阻害剤として用いることができるが、これらに限定されるものではない。本発明においては、また、タンニン化合物を単独または組み合わせて用いることができる。
【００１７】
本発明において、例えば、一般式（I）（式中、Ｒは表１に示すようにガロイル基、または隣接する２つのＲが一緒になってヘキサヒドロキシジフェノイル基を示す）に示す２種のタンニン化合物（化合物１および２）は、表２に示すように、MMP-7、MT1-MMP、およびMMP-2に対して、化合物１は、50％酵素阻害濃度（IC₅₀）が、それぞれ、0.04、0.3、および39μM、化合物２は、それぞれ、0.048、0.4、および16μMと、何れも極めて低濃度で、優れたMMPs阻害作用を有することが明らかである。
【００１８】
【表１】

【００１９】
すなわち、化合物１、および２は、通常の上皮性細胞には発現しないが、上皮性のがん細胞である大腸がんや胃がんで特異的に発現が亢進しているMMP-7（Mori, M. et al.: Cancer, 75: 1516-1519, 1995; Kataoka, H. Oncol. Res. 9: 101-109, 1997）、潜在型MMP-2の細胞膜上の活性化因子であり（Sato, H. et al.: Nature, 370: 61-65, 1994）、さらに、様々ながん細胞の膜上に発現していることが示されているMT1-MMP（Yamamoto, M. et al.: Cancer Res. 56: 384-392, 1996; Okada, A. et al.: Proc. Natl. Acad. Sci. USA 92: 2730-2734, 1995; Ohtani, H. et al.: Int. J. Cancer, 68: 565-570, 1996）、あるいは、がん組織中で、その活性型が検出されているMMP-2（Davies, B. et al.: Cancer Res. 53: 5365-5369, 1993; Emonard, H.P. et al.: Cancer Res. 52: 5845-5848, 1992）に対して優れた阻害作用を示している。
がん組織中の活性型MMP-2の存在は、胃がんや乳がんの浸潤度と非常によく相関していることから（Polette, M. et al.: Virchows Arch. 428: 29-35, 1996）、MMP-2のがん浸潤における役割がさらにクローズアップされている。また、MT1-MMP自身もI、II、III型コラーゲン、フィブロネクチンを分解することが報告され（Ohuchi, E. et al.: J. Biol. Chem. 272: 2466-2451, 1997）、この酵素が発現することは、ECM分解を２方向から助長することが示唆される。
このようなことから、MMPsに対して優れた阻害作用を示すタンニン化合物は、がんの組織浸潤・転移を抑制することが期待され、抗腫瘍転移薬として有望である。また、多くの病的疾患においては、MMPs活性の調節不能により、MMPs活性が増強し、ECMの分解が亢進する。これらの病的状態は、動脈硬化、関節炎（例えば、慢性関節リウマチおよび変形性関節症）、歯周疾患、異所性脈管形成、組織の潰瘍形成（例えば、角膜潰瘍、胃潰瘍、或いは表皮性潰瘍）、骨疾患（例えば、骨粗鬆症および人工関節置換術後の弛みなどの骨吸収性疾患）、血管再閉塞および再狭窄、HIV感染および糖尿病合併症、等の難治性疾患の治癒を遅延させている主要な原因の一つとなっている。したがって、MMPsに対して阻害作用を有する物質は、これら難治性疾患の予防、および治療剤としても有用であると考えられる。本発明者らは、先に、カテキン類が優れたMMPs阻害作用を有することを見出しており、カテキン類とタンニン化合物とを組み合わせることにより、MMPs阻害作用のスペクトラムの拡大、相加効果、或いは相乗効果が期待される。
【００２０】
本発明のタンニン化合物を有効成分とするMMPs阻害剤の製剤形態は、一般的な形態でよい。タンニン化合物単独、またはタンニン化合物と製剤上許容できる単体若しくは希釈剤との混合物のいずれでも、製剤として使用することができる。
製剤中の有効成分の量も限定されるものではない。
【００２１】
本発明のMMPs阻害剤は、経口または非経口のいずれでも投与することができる。投与量は、年令、個人差、病状等に依るので、特に限定されないが、１日当たり0.1〜500mg/kg（体重）、好ましくは、0.5〜200mg/kg（体重）である。通常、１日量を、１回または２〜４回に分けて投与する。
【００２２】
【実施例】
以下、本発明を実施例により具体的に説明するが、本発明は、これらの実施例に限定されるものではない。
【００２３】
［実施例１］
マトリライシン（MMP-7）、膜型のMT1-MMP（membrene-type MMP）、およびゼラチナーゼＡ（MMP-2）のcDNA（Nagase, H. : Biol. Chem., 378 : 151-160, 1997 ; Sato, H. et al.: Nature, 370 : 61-65, 1994 ; Crit. Rev. Oral. Biol. Med., 4 : 197-250, 1993）を、C末端にヒスチジン６残基を持つ組換え潜在型酵素（proMMP）として、大腸菌大量発現系を用い発現させた後、活性化した。
【００２４】
発現ベクターpTH-72は、タンデムリピートのＴ７プロモーターの下流に、リボソーム結合部位、翻訳開始コドン、マルチクローニング部位、ヘキサヒスチジンタグをコードする配列、および翻訳終始コドンを含んでいる。
【００２５】
ヒトMT1-MMP（プロドメイン-触媒ドメイン）cDNAを、5’PCRプライマー：5'-ggcggatccatgctcgcctccctcggctcg-3'（配列番号：１）、および３'PCRプライマー：3'-gccgtcgacgttcccgtcacagatgttggg-5'（配列番号：２）を用い、MT1-MMPの3.5kb cDNA断片を含むpME18S-MTMMPを鋳型として、PCR反応を行った。得られた¹⁸Leuから³²²Asnまでをコードする0.8kb PCR断片をBam HI/Sal Iで消化し、pTH-72発現ベクター（pTH-MT1MMP-PC）のBam HI/Sal I部位にクローン化した。
【００２６】
ヒトMMP-2（プロドメイン-触媒ドメイン）cDNAを、5’PCRプライマー：5'-ggcggatccatggcgccgtcgcccatcatc-3'（配列番号：３）、および３'PCRプライマー：3'-gccgtcgactacaatgtcctgtttgcagat-5'（配列番号：４）を用い、MMP-2の3.3kb cDNA断片を含むpSG-GelAを鋳型として、PCR反応を行った。得られた³⁰Alaから⁴⁷⁴Valまでをコードする1.3kbPCR断片をBam HI/Sal Iで消化し、pTH-72発現ベクター（pTH-MMP2-PC）のBam HI/Sal I部位にクローン化した。
【００２７】
ヒトMMP-7は、文献記載の方法（Itoh, M. et al.: J. Biochem., 119 : 667-673, 1996）で発現させた。
【００２８】
ヒト組換えMMPsの発現、精製、および巻き戻し（リフォールディング；refolding）は、公知の方法（例えば、西村義文,大野茂雄 監修 : タンパク実験プロトコール, 細胞工学別冊 実験プロトコールシリーズ２ 構造解析編, 1997）に準じて行った。すなわち、発現ベクター（pTH-MMP-7、pTH-MT1-MMP-PC、或いはpTH-MMP2-PC）を、大腸菌BL21（DE3）株にトランスフェクトし、IPTGで発現誘導した。発現タンパクは、Ni-NTA樹脂（QIAGEN INC., USA）を用いてアフィニティー精製後、リフォールディングを行い、MMP-7およびMT1-MMPはトリプシンと37℃、5分間反応後、DIFP(diisopropylphosphofluoridate)とトリプシン阻害剤(Tris-HCl 50mM, NaCl 150mM, CaCl₂ 10mM, NaN₃ 0.02%, Brij35 0.05%)を加えることにより活性型へ移行させた。一方、MMP-2はp-APMA(p-aminophenylmerucuric acetate)と37℃、15分間反応させることにより活性型へ移行させた。これを酵素標本とし、蛍光性ペプチド基質(MOCAc/DNP peptide)切断活性反応を、蛍光マイクロプレートリーダー(励起波長 340nm, 蛍光波長 400nm)による蛍光強度で測定し、酵素活性とした。一般式（I）（式中、Ｒは、ガロイル基、または隣接する２つのＲが一緒になってヘキサヒドロキシジフェノイル基を示す）に示すタンニン２種は、50％エタノールに溶解させ10mMとし、水で1mM, 300mM, 100μM, 30μM, 10μM, 3μMに希釈した。MMPs阻害活性の測定は、活性型MMP40μl, タンニン20μl, アッセイバッファー20μl (Tris-HCl pH7.5 500mM, NaCl 1.5M, CaCl₂ 100mM, ZnSO₄ 500μM, NaN₃ 30mM, Brij35 0.05%)を、37℃、15分間プレインキュベーションした後、MOCAc/DNP peptide 120μl (4.16μM)を添加し、37℃、15分毎に２時間反応させた。２時間後の50％酵素阻害濃度（IC₅₀）を、表２に示す。
【００２９】
【表２】

【００３０】
表２から、タンニン化合物が優れたMMPs阻害作用を有することが明らかである。
【００３１】
【発明の効果】
本発明により、タンニン化合物が、MMPsに対して優れた阻害作用を有することが明らかにされた。MMPsを有効成分として含有するMMPs阻害剤は、MMPs活性調節不能に起因する難治性疾患、例えば、動脈硬化症、関節炎（例えば、慢性関節リウマチおよび変形性関節症）、歯周疾患、異所性脈管形成、腫瘍性浸潤および転移、組織の潰瘍形成（例えば、角膜潰瘍、胃潰瘍、或いは表皮性潰瘍）、骨疾患（例えば、骨粗鬆症および人工関節置換術後の弛みなどの骨吸収性疾患）、血管再閉塞および再狭窄、HIV感染および糖尿病合併症、等の治療および予防剤として期待できる。
【配列表】

[0001]
[Technical field to which the invention belongs]
The present invention relates to a matrix metalloproteinase (MMPs) inhibitor comprising a tannin compound as an active ingredient. In addition, it is useful for the treatment and prevention of rheumatoid arthritis, osteoarthritis and other joint diseases caused by the degradation of extracellular matrix (ECM) by MMPs, cancer cell metastasis, refractory diseases such as gingivitis Related to inhibitors of MMPs.
[0002]
[Prior art]
MMPs are a group of enzymes involved in connective tissue degradation and remodelling, with Zn ²⁺ in the active site. To date, as many as 16 types of human MMPs have been identified (Nagase, H .: Biol. Chem., 378: 151-160, 1997). These are due to differences in primary structure and substrate specificity. It is classified into five groups: stromelysin group, membrane type (MT-MMP), and others (matrilysin).
[0003]
MMPs degrade extracellular matrix constituent proteins, such as those present in joint lining, interstitial connective tissue, basement membranes, cartilage and the like. These proteins include collagen, laminin, elastin, fibronectin, proteoglycan and the like.
[0004]
Collagen is a major structural protein that occupies about one third of mammalian tissue and is an essential component of many matrix tissues including cartilage, bone, tendons, and skin. Interstitial collagenase (MMP-1) specifically cleaves native type I, II, and III collagen molecules at a 3: 1 site. When a collagenase is cleaved at one site, collagen molecules that are stable in normal tissues are naturally denatured at physiological temperature (body temperature) to become single-stranded gelatin that is degraded by various other proteases. become. As a result, the structural integrity of the matrix structure is lost. This process is irreversible.
[0005]
Gelatinase (MMP-2) is a highly functional multi-linked glycoprotein of gelatin (denatured collagen), type IV collagen (basement membrane) and type V collagen, fibronectin (soft connective tissue and basement membrane) ), And elastin (a structural protein that forms a special component of elastic tissues such as arteries, tendons, and skin).
[0006]
Stromelysin 1 (MMP-3) and 2 (MMP-10) degrade a wide range of matrix substrates including laminin, fibronectin, proteoglycan, and collagen (type IV and type IX).
[0007]
Matrilysin (MMP-7) also degrades a wide range of matrix substrates including proteoglycans, gelatin, elastin and laminin.
[0008]
In normal tissues, the activity of MMPs is strictly determined in three steps: 1) production of a latent enzyme (pro-MMP), 2) activation of the latent enzyme, and 3) inhibition by an inhibitor of the activated enzyme. It has been adjusted. As a result, the degradation of connective tissue by MMPs and the synthesis of a new matrix structure are in dynamic equilibrium.
[0009]
However, in many pathological diseases, dysregulation of MMPs activity increases MMPs activity and enhances ECM degradation. These pathological conditions include arteriosclerosis, arthritis (eg rheumatoid arthritis and osteoarthritis), periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, tissue ulceration (eg corneal ulcers) , Gastric ulcers or epidermal ulcers), bone diseases (eg bone resorbable diseases such as osteoporosis and loosening after artificial joint replacement), vascular reocclusion and restenosis, HIV infection and diabetic complications, etc. It has become one of the main causes of delaying healing. Therefore, substances having an inhibitory action on MMPs are considered useful as preventive and therapeutic agents for these intractable diseases.
In particular, the role of MMPs involved in the degradation of extracellular matrix during tissue invasion and metastasis of cancer cells is important, and substances that inhibit the action of MMPs are promising as drugs that suppress cancer invasion and metastasis. is there.
[0010]
For example, marimastat (3R- (2,2-dimethyl-1S-methylcarbamoyl-propylcarbamoyl) -2S-hydroxy with hydroxamic acid skeleton as a therapeutic agent for cancer metastasis by inhibiting angiogenesis and inhibiting activation of MMPs Several antimetastatic drugs are under clinical development, including (-5-methyl-hexano-hydroxamic acid). However, since these anti-tumor metastasis drugs target biological characteristics of tumor cells, they do not have a direct tumor reduction effect like anti-cancer drugs, so the evaluation criteria for anti-cancer drugs remain unchanged. It is difficult to judge by application, and clinical evaluation of its usefulness is now.
In addition, it has been described that tannin compounds have topoisomerase inhibitory activity and anticancer agents thereby (JP-A-6-72885, JP-A-7-138165). However, it has not been known so far that tannin compounds have an inhibitory effect on MMPs.
[0011]
In addition, as MMPs inhibitors, flavonoid compounds (JP-A-8-104628), esculetin derivatives (JP-A-8-183785), sulfonylamino acid derivatives (JP-A-9-309875), TIMPs (JP-A-10-104) No. 17492) is known.
[0012]
[Problems to be solved by the invention]
Thus, regarding the clinical application of many MMPs inhibitors that have been reported so far, taking anti-tumor metastasis drugs as an example, their clinical usefulness is still unclear, and further candidates for new MMPs inhibitors There is a need for the development of materials.
[0013]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors focused on various biological activities such as the antioxidant activity and antiviral activity of polyphenols, and investigated the MMPs inhibitory activity of a catechin compound as one of them. Some have found excellent MMPs inhibitory activity. Therefore, as a result of investigating the MMPs inhibitory activity of tannin compounds, which are polyphenols, the compound showed excellent MMP inhibitory action, and as a result, for the treatment and prevention of intractable diseases caused by dysregulation of MMPs activity. As a result, the present invention was completed. .
[0014]
That is, the present invention
(1) Matrix metalloproteinase (MMPs) inhibitors containing tannin compounds as active ingredients,
(2) The tannin compound has the following general formula (I):
[Chemical 3]

(In the formula, R represents a galloyl group or two adjacent Rs together to represent a hexahydroxydiphenoyl group)
(1) The matrix metalloprotease inhibitor according to (1), which is a compound represented by the following: (3) A therapeutic and prophylactic agent for intractable diseases caused by dysregulation of matrix metalloprotease (MMPs) activity containing a tannin compound as an active ingredient,
(4) The tannin compound has the following general formula (I):
[Formula 4]

(In the formula, R is a galloyl group, or two adjacent Rs together to form a hexahydroxydiphenoyl group)
A therapeutic and prophylactic agent for intractable diseases according to claim 3, which is a compound represented by
(5) Intractable diseases include rheumatoid arthritis, osteoarthritis, periodontal disease, ectopic angiogenesis, neoplastic invasion and metastasis, ulceration, bone disease, vascular reocclusion, vascular restenosis, HIV infection Or the therapeutic and prophylactic agent according to (4), which is a complication of diabetes,
About.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Tannin compounds are aromatic compounds with a complex structure having a large number of phenolic hydroxyl groups widely present in plants (particularly abundant in bark), and are extracted with water. The tannin compound of the present invention includes all tannin compounds that exhibit MMPs inhibitory activity. Tannin compounds are classified in terms of chemical structure into 1) hydrolyzable tannin compounds and 2) condensed tannin compounds. Hydrolyzable tannin compounds generally have a polyphenol moiety in the molecule such as a galloyl group, a hexahydroxydiphenoyl (HHDP) group, and an oxidized form thereof. It has a structure that is ester-linked to a cyclic polyalcohol. These are collectively referred to as gallo-tannin and ellagitannin, respectively. Since this ester bond is easily cleaved by acids and enzymes, there is a name for hydrolyzable tannin (Okuda, T. et al .: Heterocycles, 15: 1323, 1981; Okuda Takuo: Chemistry and Pharmacy, 80: 12, 1983; 81: 22,1983; 83: 2,1984).
Condensed tannin compounds, on the other hand, form catechins that are larger than dimers connected by C—C bonds at the C 4 -C 8 or C 4 -C 6 positions, etc. It is a thing. In many cases, this condensation proceeds further by the action of acids and enzymes to form larger molecules (Okuda, T. et al .: Heterocycles, 15: 1323, 1981; Takuo Okuda: Chemistry and Pharmacy, 80: 12, 1983; 81: 22,1983; 83: 2,1984).
C-glycoside tannin (C-glycoside tannin) having a structure in which the 1-position of the hydrolyzable tannin sugar moiety and polyphenol bind to form a C-glycoside and the sugar ring is opened, Complex tannin compound with catechins bonded to hydrolyzable tannin molecule (Okuda, T. et al .: Chem. Pharm. Bull., 35: 443, 1987), more than 2 hydrolyzable tannin molecules Oligomeric hydrolyzable tannin (Okuda, T. et al .: J. Natl. Prod., 52: 1, 1989), and the polyphenol moiety in the molecule is caffeic acid (caffeoyl group) (Caffeetannin) (Takuo Okuda, et al .: Drug Journal, 106: 894, 1986) and labiataetannin (Takuo Okuda, et al .: Drug Journal, 106: 1108, 1986; Agata, I. et al .: Chem. Pharm. Bull. 35: 3223, 1988), dehydroellagitannin with DHDDP (dehydrohexahydroxy diphenoyl) group [Okuda, T. et al .: Tetrahedron Lett. 23: 3 941, 1982) are condensed with ascorbic acid (Okuda, T. et al .: Chem. Pharm. Bull. 34: 4075, 1986).
[0016]
The tannin compound of the present invention is extracted from a tannin-containing plant using water or aqueous ethanol having a high water content. The tannin-containing plants include tea leaves such as green tea and black tea that are drunk daily, but other examples include camellia ( Camellia japonica ), herb spices such as Bayberry and clove. (Clove), Peony, Rose red, Bay Leaf, Comfrey, Eucalyptus, Melissa, Pennyroyal, Rose pink, Star・ Anise (Star anise), time, etc. are mentioned. Since these plants generally contain a large amount of water immediately after being collected, drying is necessary in order to prevent alteration of the component tannin.
Extraction of tannin from these tannin-containing plants is performed with an aqueous solvent, for example, purified water or purified water containing ethanol or methanol. The extraction temperature and time can be appropriately set by those skilled in the art in consideration of the stability of tannin. The extract can be used in the present invention as it is, but the concentrate of the extract, or the extract is fractionated, and the method described in the literature (Hashimoto, M. et al .: Chem. Pharm. Bull. 37: 77-85, 1989) is preferably used. Alternatively, chemically synthesized tannin may be used. Preferably, the tannin compound represented by the general formula (I) (wherein R represents a galloyl group, or two adjacent Rs together represent a hexahydroxydiphenoyl group) and the like are used. Although it can use as an inhibitor, it is not limited to these. In the present invention, tannin compounds can be used alone or in combination.
[0017]
In the present invention, for example, two types represented by general formula (I) (wherein R represents a galloyl group as shown in Table 1 or two adjacent Rs together represent a hexahydroxydiphenoyl group). As shown in Table 2, the tannin compounds (compounds 1 and 2) of Compound 1 had a 50% enzyme inhibitory concentration (IC ₅₀ ) against MMP-7, MT1-MMP, and MMP-2, respectively. 0.04, 0.3, and 39 μM, Compound 2, which is 0.048, 0.4, and 16 μM, respectively, are clearly shown to have excellent MMPs inhibitory activity at extremely low concentrations.
[0018]
[Table 1]

[0019]
That is, compounds 1 and 2 are not expressed in normal epithelial cells, but MMP-7 (Mori, M) whose expression is specifically increased in colon cancer and gastric cancer, which are epithelial cancer cells. et al .: Cancer, 75: 1516-1519, 1995; Kataoka, H. Oncol. Res. 9: 101-109, 1997) and is an activator of the latent MMP-2 on the cell membrane (Sato, H et al .: Nature, 370: 61-65, 1994) and MT1-MMP (Yamamoto, M. et al .: Cancer, which has been shown to be expressed on the membranes of various cancer cells. Res. 56: 384-392, 1996; Okada, A. et al .: Proc. Natl. Acad. Sci. USA 92: 2730-2734, 1995; Ohtani, H. et al .: Int. J. Cancer, 68 : 565-570, 1996) or MMP-2 (Davies, B. et al .: Cancer Res. 53: 5365-5369, 1993; Emonard, HP et al .: Cancer Res. 52: 5845-5848, 1992).
The presence of active MMP-2 in cancer tissue correlates very well with the invasion of gastric and breast cancer (Polette, M. et al .: Virchows Arch. 428: 29-35, 1996) The role of MMP-2 in cancer invasion has been further highlighted. MT1-MMP itself has also been reported to degrade I, II, III collagen and fibronectin (Ohuchi, E. et al .: J. Biol. Chem. 272: 2466-2451, 1997). It is suggested that expression promotes ECM degradation from two directions.
For these reasons, tannin compounds exhibiting an excellent inhibitory action on MMPs are expected to suppress cancer tissue invasion / metastasis and are promising as antitumor metastasis drugs. Moreover, in many pathological diseases, MMPs activity is enhanced and ECM degradation is enhanced due to the inability to regulate MMPs activity. These pathological conditions include arteriosclerosis, arthritis (eg, rheumatoid arthritis and osteoarthritis), periodontal disease, ectopic angiogenesis, tissue ulceration (eg, corneal ulcer, gastric ulcer, or epidermal) (Ulcers), delayed healing of intractable diseases such as bone diseases (eg bone resorbable diseases such as osteoporosis and loosening after joint replacement), vascular reocclusion and restenosis, HIV infection and diabetic complications Has become one of the main causes. Therefore, substances having an inhibitory action on MMPs are considered useful as preventive and therapeutic agents for these intractable diseases. The present inventors have previously found that catechins have excellent MMPs inhibitory action, and by combining catechins and tannin compounds, the spectrum of MMPs inhibitory action is expanded, additive effects, or synergistic effects. Expected to be effective.
[0020]
The preparation form of the MMPs inhibitor containing the tannin compound of the present invention as an active ingredient may be a general form. Either a tannin compound alone or a mixture of a tannin compound and a pharmaceutically acceptable single substance or a diluent can be used as a preparation.
The amount of the active ingredient in the preparation is not limited.
[0021]
The MMPs inhibitor of the present invention can be administered either orally or parenterally. The dosage is not particularly limited because it depends on age, individual difference, medical condition, etc., but is 0.1 to 500 mg / kg (body weight), preferably 0.5 to 200 mg / kg (body weight) per day. Usually, the daily dose is administered once or divided into 2 to 4 times.
[0022]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.
[0023]
[Example 1]
Matrilysin (MMP-7), membrane-type MT1-MMP (membrene-type MMP), and gelatinase A (MMP-2) cDNA (Nagase, H .: Biol. Chem., 378: 151-160, 1997; Sato , H. et al .: Nature, 370: 61-65, 1994; Crit. Rev. Oral. Biol. Med., 4: 197-250, 1993), a recombination potential with 6 histidine residues at the C-terminus. As a type enzyme (proMMP), it was expressed using an Escherichia coli mass expression system and then activated.
[0024]
The expression vector pTH-72 contains a ribosome binding site, a translation initiation codon, a multicloning site, a sequence encoding a hexahistidine tag, and a translation termination codon downstream of the tandem repeat T7 promoter.
[0025]
Human MT1-MMP (prodomain-catalyst domain) cDNA was extracted from 5 ′ PCR primer: 5′-ggcggatccatgctcgcctccctcggctcg-3 ′ (SEQ ID NO: 1) and 3 ′ PCR primer: 3′-gccgtcgacgttcccgtcacagatgttggg-5 ′ (SEQ ID NO: Using 2), a PCR reaction was performed using pME18S-MTMMP containing a 3.5 kb cDNA fragment of MT1-MMP as a template. The resulting 0.8 kb PCR fragment encoding ¹⁸ Leu to ³²² Asn was digested with Bam HI / Sal I and cloned into the Bam HI / Sal I site of the pTH-72 expression vector (pTH-MT1MMP-PC).
[0026]
Human MMP-2 (prodomain-catalyst domain) cDNA was extracted from 5 ′ PCR primer: 5′-ggcggatccatggcgccgtcgcccatcatc-3 ′ (SEQ ID NO: 3), and 3 ′ PCR primer: 3′-gccgtcgactacaatgtcctgtttgcagat-5 ′ (SEQ ID NO: Using 4), a PCR reaction was performed using pSG-GelA containing a 3.3 kb cDNA fragment of MMP-2 as a template. The resulting 1.3 kb PCR fragment encoding from ³⁰ Ala to ⁴⁷⁴ Val was digested with Bam HI / Sal I and cloned into the Bam HI / Sal I site of the pTH-72 expression vector (pTH-MMP2-PC).
[0027]
Human MMP-7 was expressed by a method described in the literature (Itoh, M. et al .: J. Biochem., 119: 667-673, 1996).
[0028]
Expression, purification, and refolding of human recombinant MMPs are known methods (eg, Yoshifumi Nishimura, Shigeo Ohno, supervised by: Protein Experiment Protocol, Cell Engineering Separate Experiment Protocol Series 2 Structural Analysis, 1997) It went according to. That is, an expression vector (pTH-MMP-7, pTH-MT1-MMP-PC, or pTH-MMP2-PC) was transfected into E. coli BL21 (DE3) strain, and expression was induced with IPTG. The expressed protein was affinity-purified using Ni-NTA resin (QIAGEN INC., USA) and then refolded. MMP-7 and MT1-MMP were reacted with trypsin at 37 ° C for 5 minutes, then with DIFP (diisopropylphosphofluoridate). A trypsin inhibitor (Tris-HCl 50 mM, NaCl 150 mM, CaCl ₂ 10 mM, NaN ₃ 0.02%, Brij35 0.05%) was added to the active form. On the other hand, MMP-2 was transferred to the active form by reacting with p-APMA (p-aminophenylmerucuric acetate) at 37 ° C. for 15 minutes. Using this as an enzyme specimen, the fluorescence peptide substrate (MOCAc / DNP peptide) cleavage activity reaction was measured by the fluorescence intensity with a fluorescence microplate reader (excitation wavelength: 340 nm, fluorescence wavelength: 400 nm) to obtain enzyme activity. Two tannins represented by the general formula (I) (wherein R represents a galloyl group or two adjacent Rs together represent a hexahydroxydiphenoyl group) are dissolved in 50% ethanol to 10 mM. And diluted to 1 mM, 300 mM, 100 μM, 30 μM, 10 μM, and 3 μM with water. MMPs inhibitory activity was measured at 37 ° C using activated MMP 40 μl, tannin 20 μl, assay buffer 20 μl (Tris-HCl pH 7.5 500 mM, NaCl 1.5 M, CaCl ₂ 100 mM, ZnSO ₄ 500 μM, NaN ₃ 30 mM, Brij35 0.05%) After pre-incubating for 15 minutes, MOCAc / DNP peptide 120 μl (4.16 μM) was added and reacted at 37 ° C. every 15 minutes for 2 hours. The 50% enzyme inhibitory concentration (IC ₅₀ ) after 2 hours is shown in Table 2.
[0029]
[Table 2]

[0030]
From Table 2, it is clear that the tannin compound has an excellent MMPs inhibitory action.
[0031]
【The invention's effect】
According to the present invention, it has been clarified that tannin compounds have an excellent inhibitory action on MMPs. MMPs inhibitors containing MMPs as active ingredients are intractable diseases caused by dysregulation of MMPs activity, such as arteriosclerosis, arthritis (eg, rheumatoid arthritis and osteoarthritis), periodontal disease, ectopic Angiogenesis, neoplastic invasion and metastasis, tissue ulceration (eg, corneal ulcer, gastric ulcer, or epidermal ulcer), bone disease (eg, bone resorbable diseases such as osteoporosis and loosening after artificial joint replacement), It can be expected as a therapeutic and prophylactic agent for vascular re-occlusion and restenosis, HIV infection and diabetic complications.
[Sequence Listing]

Claims (1)

The following general formula (I):

(Wherein R represents a galloyl group, or two adjacent Rs together represent a hexahydroxydiphenoyl group) MT1-MMP and matrilysin (MMP-7) containing as an active ingredient Inhibitor.