JPH08231416A - Treating agent for thrombosis - Google Patents
Treating agent for thrombosisInfo
- Publication number
- JPH08231416A JPH08231416A JP7037367A JP3736795A JPH08231416A JP H08231416 A JPH08231416 A JP H08231416A JP 7037367 A JP7037367 A JP 7037367A JP 3736795 A JP3736795 A JP 3736795A JP H08231416 A JPH08231416 A JP H08231416A
- Authority
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- Japan
- Prior art keywords
- activity
- thrombosis
- hgf
- cells
- vascular endothelial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は血栓症及び血管内皮細胞
の障害を伴う疾患の予防及び／又は治療剤に関するもの
である。より詳しくは、本発明は肝実質細胞増殖因子を
有効成分として含み、各種血栓症の予防や治療、並びに
血管内皮細胞の障害を伴う腎炎などの疾患の予防や治療
に有用な医薬に関するものである。TECHNICAL FIELD The present invention relates to a prophylactic and / or therapeutic agent for thrombosis and diseases associated with vascular endothelial cell damage. More specifically, the present invention relates to a medicament containing hepatic parenchymal cell growth factor as an active ingredient, which is useful for the prevention and treatment of various thrombosis, and the prevention and treatment of diseases such as nephritis accompanied by damage of vascular endothelial cells. .
【０００２】[0002]
【従来の技術】生体では、出血に伴ってフィブリン（凝
固血栓）が形成されて止血を行う機構と、形成されたフ
ィブリンが溶解（血栓溶解又はフィブリン溶解）して血
管内の血液流動性が回復する機構が備わっており、両者
の調和によって完全な止血機構が維持されている。この
ように血液の凝固が完了した後に凝固塊が溶解して再び
血液流動性を獲得する現象を繊維素溶解現象（線溶）と
呼ぶ。2. Description of the Related Art In a living body, fibrin (coagulated thrombus) is formed with hemorrhage to stop bleeding, and formed fibrin is dissolved (thrombolysis or fibrinolysis) to restore blood fluidity in blood vessels. There is a mechanism to do so, and a perfect hemostasis mechanism is maintained by the harmony of both. The phenomenon in which the coagulated clot is dissolved and blood fluidity is acquired again after the completion of blood coagulation is called a fibrinolytic phenomenon (fibrinolysis).
【０００３】このような止血過程で形成される凝固血栓
は、必要以上に血管内に残存すると生体にとっては異物
となり、血管の狭窄や血栓形成の増大、あるいはフィブ
リンの器質化による動脈硬化などの病因となる。このよ
うな理由から、病的状態において過大に形成された血栓
に対して、積極的にフィブリンを溶解する血栓溶解療法
が行われており、従来よりプラスミノーゲンアクティベ
ーター(PA)としてのウロキナーゼ(UK)、ストレプトキナ
ーゼ(SK)、及び組織プラスミノーゲンアクティベーター
(t-PA)などの薬剤が治療に用いられている。しかしなが
ら、これらの薬剤はいずれも一過性の作用を有するにす
ぎず、また、生理的濃度よりはるかに高濃度の薬剤を静
脈内投与するため、出血傾向を引き起こすという問題を
有していた。The coagulated thrombus formed in such a hemostasis process becomes a foreign substance to the living body if it remains in the blood vessel more than necessary, and causes etiology of stenosis of the blood vessel, increased thrombus formation, or arteriosclerosis due to the organization of fibrin. Becomes For these reasons, thrombolytic therapy that actively dissolves fibrin is performed for thrombus that is excessively formed in a pathological state, and urokinase (UK) as a plasminogen activator (PA) has been conventionally used. ), Streptokinase (SK), and tissue plasminogen activator
Drugs such as (t-PA) have been used in therapy. However, all of these drugs have only a transient effect, and also have a problem of causing a bleeding tendency because the drug is administered intravenously at a much higher concentration than the physiological concentration.
【０００４】一方、ヒト肝実質細胞増殖因子（以下、本
明細書において「hHGF」と略記する場合があり、単に肝
実質細胞増殖因子を表す場合は「HGF 」と略記する場合
がある）は、初代培養肝細胞の増殖を促進させうるヒト
由来蛋白性因子として、劇症肝炎患者血漿から初めて分
離された（特開昭63-22526号公報）。その後、hHGF蛋白
質をコードする遺伝子(cDNA)及びアミノ酸配列（特開平
3-72883 号公報）、組換えhHGFの生産方法（特開平3-28
5693号公報）が報告されている。このような組換えヒト
HGF(以下、本明細書において「rhHGF 」と略記する場合
がある）は、生体外(J. Clin. Lnvest., 87, pp.1853-1
857, 1991)、及び生体内(Jpn. J. Pharmacol., 59, sup
pl. 1, 137, 1992) において肝実質細胞の増殖及び機能
を促進する作用を有している。On the other hand, human hepatocyte growth factor (hereinafter, sometimes abbreviated as "hHGF" in the present specification, and sometimes simply abbreviated as "HGF" when simply representing hepatocyte growth factor) is It was first isolated from plasma of patients with fulminant hepatitis as a human-derived protein factor capable of promoting the growth of primary cultured hepatocytes (Japanese Patent Laid-Open No. 63-22526). Then, the gene (cDNA) and amino acid sequence encoding the hHGF protein (JP
3-72883), a method for producing recombinant hHGF (JP-A-3-2828)
No. 5693) has been reported. Such recombinant human
HGF (hereinafter sometimes abbreviated as "rhHGF" in the present specification) is in vitro (J. Clin. Lnvest., 87, pp.1853-1.
857, 1991) and in vivo (Jpn. J. Pharmacol., 59, sup)
pl. 1, 137, 1992), it has the effect of promoting the proliferation and function of liver parenchymal cells.
【０００５】さらに、HGF の標的細胞や標的組織が広く
検索されており、HGF が肝細胞以外の種々の上皮細胞
（尿細管上皮、肺上皮、胆管上皮、又は胃上皮等）や線
維芽細胞、リンパ球系細胞等に反応して、その増殖や運
動性を変化させることが報告されている(Mitsubishi Ka
sei R&D Review, 7, pp.16-24, 1993)。また、これらHG
F 標的細胞上のレセプター分子として、癌原遺伝子c-me
t 産物が機能していることも明らかにされている(Scien
ce, 251, pp.802-804, 1991)。しかしながら、HGF の血
管内皮細胞に対する生理作用は未だ知られていない。Further, the target cells and target tissues of HGF have been widely searched, and HGF has various types of epithelial cells other than hepatocytes (tubular epithelium, lung epithelium, bile duct epithelium, gastric epithelium, etc.) and fibroblasts. It has been reported to change its proliferation and motility in response to lymphocyte cells (Mitsubishi Ka
sei R & D Review, 7, pp.16-24, 1993). Also, these HG
As a receptor molecule on F target cells, the proto-oncogene c-me
It has also been shown that the product is functional (Scien
ce, 251, pp.802-804, 1991). However, the physiological action of HGF on vascular endothelial cells has not yet been known.
【０００６】[0006]
【発明が解決しようとする課題及び課題を解決するため
の手段】本発明の目的は血栓症の予防及び／又は治療剤
を提供することにある。また、血栓の形成が起因となる
疾患の予防及び／又は治療に有用な医薬を提供すること
も本発明の目的である。本発明者は上記の課題を解決す
べく鋭意努力した結果、HGF が血管内皮細胞におけるt-
PAの産生(Tromb. Haemost., 43, pp.77-89, 1980; Sem
i. Tromb. Hemost., 10, pp.24-41, 1984) を促進する
こと、並びに、HGF が血管内壁の抗血栓性の維持に重要
な役割を果たしている血管内皮細胞の増殖を促進するこ
とを見い出した。本発明はこれらの知見を基にして完成
されたものである。DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention and an object of the present invention is to provide a preventive and / or therapeutic agent for thrombosis. It is also an object of the present invention to provide a medicament useful for the prevention and / or treatment of diseases caused by thrombus formation. As a result of diligent efforts to solve the above problems, the present inventor has found that HGF has t-
Production of PA (Tromb. Haemost., 43, pp.77-89, 1980; Sem
i. Tromb. Hemost., 10, pp.24-41, 1984) and the proliferation of vascular endothelial cells in which HGF plays an important role in maintaining the antithrombotic property of the inner wall of blood vessels. Found out. The present invention has been completed based on these findings.
【０００７】すなわち本発明は、肝実質細胞増殖因子を
有効成分として含む血栓症の予防及び／又は治療剤を提
供するものである。本発明の好ましい態様によれば、肝
実質細胞増殖因子が下記の理化学的性質：1) SDS-PAGE
（非還元条件下）による推定分子量が約76,000〜92,000
であり；2) 肝実質細胞を増殖させる活性を有し；3)80
℃、10分間の加熱処理により上記活性が失活し；4) ト
リプシンによる消化処理及びキモトリプシンによる消化
処理により上記活性が失活し；5) ヘパリンに対して強
い親和性を有する；を示す上記予防・治療剤が提供され
る。That is, the present invention provides a prophylactic and / or therapeutic agent for thrombosis, which comprises hepatocyte growth factor as an active ingredient. According to a preferred embodiment of the present invention, hepatocyte growth factor has the following physicochemical properties: 1) SDS-PAGE
Estimated molecular weight under non-reducing conditions is about 76,000-92,000
2) having the activity of proliferating hepatocytes; 3) 80
The above-mentioned prevention showing that the activity is inactivated by heating at 10 ° C for 10 minutes; 4) the activity is inactivated by digestion with trypsin and chymotrypsin; and 5) it has a strong affinity for heparin. -A therapeutic agent is provided.
【０００８】また、本発明の別の態様によれば、肝実質
細胞増殖因子あるいは上記の好ましい肝実質細胞増殖因
子を有効成分として含む血管内皮細胞の障害を伴う疾患
の予防及び／又は治療剤が提供される。この発明の好ま
しい態様により、上記疾患が、心筋梗塞症及びその後の
血流再開通後の組織障害に基づく疾患、経皮的血管形成
術及びバイパスグラフト処置後の血管の再閉塞に基づく
疾患、末梢動脈血栓症、深部静脈血栓症、脳血栓症、糖
尿病性網膜症、腎炎、糸球体腎炎、並びに血栓が器質化
することによって生じる動脈硬化症から選ばれる上記予
防及び／又は治療剤が提供される。さらに本発明の別の
態様によって、肝実質細胞増殖因子あるいは上記の好ま
しい肝実質細胞増殖因子を有効成分として含む血管内皮
細胞の増殖促進剤が提供される。Further, according to another aspect of the present invention, there is provided a preventive and / or therapeutic agent for a disease associated with a disorder of vascular endothelial cells, which comprises hepatocyte growth factor or the above preferred hepatocyte growth factor as an active ingredient. Provided. According to a preferred embodiment of the present invention, the above-mentioned diseases are diseases based on tissue damage after myocardial infarction and subsequent reopening of blood flow, diseases based on re-occlusion of blood vessels after percutaneous angioplasty and bypass graft treatment, peripheral There is provided the above-mentioned preventive and / or therapeutic agent selected from arterial thrombosis, deep vein thrombosis, cerebral thrombosis, diabetic retinopathy, nephritis, glomerulonephritis, and arteriosclerosis caused by organizing thrombus. Further, according to another aspect of the present invention, there is provided a vascular endothelial cell growth-promoting agent comprising a hepatocyte growth factor or the above preferred hepatocyte growth factor as an active ingredient.
【０００９】本発明の医薬は、肝実質細胞増殖因子(HG
F) を有効成分とすることを特徴としている。肝実質細
胞増殖因子としては、HGF を含有することの知られてい
るヒトやラット等の哺乳類動物由来の体液や組織、また
は自発的にHGF を産生する細胞から単離・精製されたも
のを用いることができるが、遺伝子組換え法によりHGF
のcDNAを細胞に導入して得られる組換えHGF を用いるこ
ともできる。本発明の医薬の有効成分として、ヒト由来
のHGF(hHGF) を用いることが好ましい。The medicament of the present invention is a hepatocyte growth factor (HG
It is characterized by using F) as an active ingredient. As the hepatocyte growth factor, use is made of body fluids or tissues derived from mammals such as humans and rats known to contain HGF, or those isolated and purified from cells that spontaneously produce HGF. HGF can be
Recombinant HGF obtained by introducing the above cDNA into cells can also be used. Human-derived HGF (hHGF) is preferably used as the active ingredient of the medicament of the present invention.
【００１０】組換えHGF を産生させる宿主は特に限定さ
れないが、例えば、大腸菌、枯草菌、酵母、糸状菌、植
物細胞、昆虫細胞、動物細胞などを用いればよい。より
具体的には、HGF 生産能を有する形質転換体を製造する
には、上記哺乳類由来の胎盤、肝障害患者肝組織及び血
液、MRC-5 細胞、IMR-9 細胞などの線維芽細胞株、好ま
しくは CHO細胞等の宿主細胞に対して、例えば、特開平
3-285693号公報に記載された方法に従ってHGF 、好まし
くはhHGFをコードするcDNAを含む発現ベクターを導入す
ればよい。このような形質転換体を培養することにより
分離・採取されるHGF を用いることは本発明の好ましい
態様である。The host for producing recombinant HGF is not particularly limited, but for example, Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant cells, insect cells, animal cells and the like may be used. More specifically, in order to produce a transformant having the ability to produce HGF, the placenta derived from the above mammals, liver tissue and blood of patients with liver damage, fibroblast cell lines such as MRC-5 cells and IMR-9 cells, Preferably, for host cells such as CHO cells, for example, JP
An expression vector containing a cDNA encoding HGF, preferably hHGF, may be introduced in accordance with the method described in Japanese Patent Publication No. 3-285693. It is a preferred embodiment of the present invention to use HGF isolated and collected by culturing such a transformant.
【００１１】また、本発明の医薬の有効成分として、上
記の天然又は組換えHGF 自体の他、その前駆体蛋白質や
肝実質細胞を増殖させる活性を損なわない範囲で天然HG
F の一部のアミノ酸を置換、欠失、挿入、修飾等により
改変した非天然型HGF を用いてもよい。このような非天
然型HGF としては、特開平2-288899号公報、PCT 国際公
開WO90/10651号、特開平3-130091号公報、同3-255096号
公報、同4-30000 号公報、Nature, 342, pp.440-443, 1
989 等の刊行物に記載のものを用いることができる。In addition to the above-mentioned natural or recombinant HGF itself, as an active ingredient of the medicament of the present invention, natural HG within the range of not impairing the precursor protein or the activity of growing hepatocytes.
Non-natural HGF in which a part of the amino acids of F is modified by substitution, deletion, insertion, modification or the like may be used. Examples of such non-natural HGF include JP2-288899, PCT International Publication WO90 / 10651, JP3-130091, JP3-255096, JP4-30000, Nature, 342, pp.440-443, 1
Those described in publications such as 989 can be used.
【００１２】本発明の医薬の有効成分として特に好まし
いHGF は以下の理化学的性質： 1) SDS-PAGE（非還元条件下）による推定分子量が約7
6,000〜92,000であり； 2) 肝実質細胞を増殖させる活性を有し； 3) 80℃、10分間の加熱処理により上記活性が失活し； 4) トリプシンによる消化処理及びキモトリプシンによ
る消化処理により上記活性が失活し； 5) ヘパリンに対して強い親和性を有する を示すものである。このようなHGF としてはヒト由来の
ものがより好ましく、特開平3-72883 号公報又は特開平
4-89499 号公報に記載のアミノ酸配列により特定される
hHGFが特に好ましい。Particularly preferred HGF as an active ingredient of the medicament of the present invention has the following physicochemical properties: 1) The estimated molecular weight by SDS-PAGE (under non-reducing conditions) is about 7
6,000 to 92,000; 2) it has an activity to grow hepatocytes; 3) the above activity is inactivated by heat treatment at 80 ° C. for 10 minutes; 4) it is digested with trypsin and chymotrypsin. The activity is inactivated; 5) It has a strong affinity for heparin. As such HGF, those derived from human are more preferable, and those disclosed in JP-A-3-72883 or JP-A-3-72883 are preferred.
Identified by the amino acid sequence described in 4-89499
hHGF is particularly preferred.
【００１３】本発明の医薬は、前記HGF の１種または２
種以上を単独で、あるいは適当な製剤用添加物と共に製
剤形態の医薬組成物として調製し、非経口的に投与する
ことが好ましい。このような医薬組成物の投与形態とし
ては、一般的に非経口的投与に使用されるものであれば
特に限定されないが、例えば、注射用アンプル剤や注射
用凍結乾燥粉末剤（バイアル充填のもの）などを用いる
ことが可能である。各種製剤形態への調製は、当業界で
利用可能な周知の製剤添加物、例えば希釈剤や添加剤な
どを用い、当業界の慣用の手法に従って行えばよい。The pharmaceutical of the present invention comprises one or two of the above HGFs.
It is preferable to prepare one or more species alone or to prepare a pharmaceutical composition in the form of a formulation together with appropriate pharmaceutical additives and administer parenterally. The dosage form of such a pharmaceutical composition is not particularly limited as long as it is generally used for parenteral administration, and examples thereof include ampules for injection and freeze-dried powders for injection (filled in vials. ) Or the like can be used. Preparation into various dosage forms may be performed according to a method commonly used in the art, using well-known additive additives such as diluents and additives available in the art.
【００１４】例えば、注射用凍結乾燥粉末剤は、精製さ
れた前記HGF の有効量を注射用蒸留水、生理食塩水、ブ
ドウ糖水溶液などの希釈剤に溶解し、必要に応じてカル
ボキシメチルセルロース、アルギン酸ナトリウムなどの
賦形剤、ポリエチレングリコール、デキストラン硫酸ナ
トリウム、アミノ酸、ヒト血清アルブミンなどの安定化
剤、ベンジルアルコール、塩化ベンザルコニウム、フェ
ノールなどの保存剤、ブドウ糖、グルコン酸カルシウ
ム、塩酸プロカインなどの無痛化剤、塩酸、酢酸、クエ
ン酸、水酸化ナトリウムなどのpH調節剤等を加え、常法
に従って凍結乾燥することにより製造することができ
る。For example, a freeze-dried powder for injection is prepared by dissolving an effective amount of the purified HGF in a diluent such as distilled water for injection, physiological saline or an aqueous solution of glucose, and if necessary, carboxymethyl cellulose or sodium alginate. Excipients such as polyethylene glycol, sodium dextran sulfate, amino acids, stabilizers such as human serum albumin, preservatives such as benzyl alcohol, benzalkonium chloride, phenol, soothing such as glucose, calcium gluconate, procaine hydrochloride It can be produced by adding an agent, a pH adjusting agent such as hydrochloric acid, acetic acid, citric acid, sodium hydroxide and the like, and lyophilizing it according to a conventional method.
【００１５】また、注射用アンプル剤は、前記HGF の有
効量を注射用蒸留水、生理食塩水、リンゲル液などの希
釈剤に溶解し、必要に応じてサリチル酸ナトリウム、マ
ンニトールなどの溶解補助剤、クエン酸ナトリウム、グ
リセリンなどの緩衝剤、ブドウ糖、添加糖などの等張化
剤、上記安定化剤、上記保存剤、上記無痛化剤、上記pH
調節剤などの添加剤を加えた後、通常の加熱滅菌、無菌
濾過などにより無菌化して調製することができる。な
お、有効成分の種類によっては加熱滅菌工程で失活する
場合があるので、滅菌方法は適宜選択すべきである。The ampoule for injection is prepared by dissolving an effective amount of the above HGF in a diluent such as distilled water for injection, physiological saline and Ringer's solution, and if necessary, a solubilizing agent such as sodium salicylate and mannitol, and a quenching agent. Sodium acid, buffer such as glycerin, isotonic agent such as glucose, added sugar, the above stabilizer, the above preservative, the soothing agent, the above pH
After adding an additive such as a regulator, it can be sterilized by ordinary heat sterilization, aseptic filtration, or the like to prepare. The sterilization method should be appropriately selected because it may be inactivated in the heat sterilization step depending on the type of active ingredient.
【００１６】いかなる特定の理論に拘泥するわけではな
いが、本発明の医薬の有効成分であるHGF は、血管内皮
細胞におけるt-PAの産生を促進する作用を有しているの
で、本発明の医薬は、心筋梗塞症、末梢動脈血栓症、深
部静脈血栓症、脳血栓症など各種血栓症の予防及び／又
は治療に有効である。また、本発明の医薬の有効成分で
あるHGF は血管内皮細胞の増殖を促進する作用を有して
いる。血管内皮細胞は血管内壁の抗血栓性の維持に重要
な役割を果たしているので、本発明の医薬はこのような
作用に基づく血栓症の予防及び／又は治療に有効であ
る。従って、本発明の医薬は、血管内皮細胞が分泌する
t-PAによる直接的な抗血栓作用と、血管内皮細胞の増殖
（血管内壁の修復）に基づく抗血栓作用とを有すること
を特徴としており、持続的な抗血栓作用を発揮できるも
のである。Without being bound to any particular theory, HGF, which is an active ingredient of the pharmaceutical agent of the present invention, has an action of promoting the production of t-PA in vascular endothelial cells, and therefore, is not effective. The medicine is effective for the prevention and / or treatment of various thrombosis such as myocardial infarction, peripheral arterial thrombosis, deep vein thrombosis, and cerebral thrombosis. Further, HGF, which is an active ingredient of the medicine of the present invention, has an action of promoting the proliferation of vascular endothelial cells. Since the vascular endothelial cell plays an important role in maintaining the antithrombotic property of the inner wall of the blood vessel, the pharmaceutical agent of the present invention is effective for the prevention and / or treatment of thrombosis based on such action. Therefore, the drug of the present invention is secreted by vascular endothelial cells.
It is characterized by having a direct antithrombotic effect by t-PA and an antithrombotic effect based on the proliferation of vascular endothelial cells (repair of the inner wall of the blood vessel), and can exhibit a continuous antithrombotic effect.
【００１７】さらに、本発明の医薬は、血管内皮細胞の
増殖を促進して血管を修復する作用を有しているので、
血管内皮細胞に障害を伴う疾患の予防及び／又は治療に
有効である。血管内皮細胞に障害を伴う疾患としては、
心筋梗塞及びその後の血流再開通後の組織障害に基づく
疾患、経皮的血管形成術やバイパスグラフト処置後の血
管の再閉塞に基づく疾患、糖尿病性網膜症、腎炎、糸球
体腎炎、及び血栓が器質化することによって生じる動脈
硬化症等の他、カテーテル留置や血管手術等によって血
管内皮細胞が物理的な損傷を受けた場合などを挙げるこ
とができる。Furthermore, since the pharmaceutical agent of the present invention has the action of promoting the proliferation of vascular endothelial cells and repairing blood vessels,
It is effective for the prevention and / or treatment of diseases associated with damage to vascular endothelial cells. Diseases associated with damage to vascular endothelial cells include
Diseases due to tissue damage after myocardial infarction and subsequent reopening of blood flow, diseases due to re-occlusion of blood vessels after percutaneous angioplasty or bypass graft treatment, diabetic retinopathy, nephritis, glomerulonephritis, and thrombosis. In addition to arteriosclerosis and the like caused by the organizing of the blood vessels, there may be mentioned the case where the vascular endothelial cells are physically damaged due to catheter placement or vascular surgery.
【００１８】本発明の医薬には、本発明の医薬と同様な
薬理作用あるいは他の薬理作用を有する他の医薬の有効
成分を配合してもよい。また、本発明の医薬の有効成分
の肝実質細胞増殖作用を増強することが知られているヘ
パリン、デキストラン硫酸等の硫酸化多糖類もしくはそ
の誘導体（特開平5-301824号公報）などの有効成分を配
合してもよい。これらの硫酸化多糖類もしくはその誘導
体は本発明の医薬の安定性を高める作用を有しているの
で、これらを配合した医薬は本発明の好ましい態様であ
る。The pharmaceutical of the present invention may be mixed with active ingredients of other pharmaceuticals having the same or other pharmacological action as the pharmaceutical of the present invention. In addition, active ingredients such as heparin, sulfated polysaccharides such as dextran sulfate or derivatives thereof (JP-A-5-301824), which are known to enhance the hepatocyte proliferation effect of the active ingredient of the medicament of the present invention, etc. You may mix | blend. Since these sulfated polysaccharides or their derivatives have the effect of enhancing the stability of the drug of the present invention, a drug containing them is a preferred embodiment of the present invention.
【００１９】本発明の医薬は、ヒトを含む哺乳類の上記
の疾患の予防及び／又は治療を目的として、一般的には
非経口的に、より具体的には皮下、筋肉または静脈内注
射により投与することができる。一般的には、所定量を
単回もしくは複数回に分けて注射により投与するか、ま
たは点滴などにより連続的に投与することができる。投
与量は、患者の年齢、性別、症状、体重、投与形態等に
応じて適宜増減すべきであるが、一般的には、成人１日
当たり１μg/kg〜10 mg/kg、より好ましくは10〜1000μ
g/kgの範囲で投与すればよい。The medicament of the present invention is generally administered parenterally, more specifically, subcutaneously, intramuscularly or intravenously for the purpose of preventing and / or treating the above-mentioned diseases in mammals including humans. can do. In general, a given amount can be administered by injection in single or divided doses, or can be administered continuously by infusion. The dose should be appropriately increased or decreased according to the age, sex, symptoms, body weight, administration form, etc. of the patient, but in general, 1 μg / kg to 10 mg / kg per adult per day, more preferably 10 to 1000μ
It may be administered in the range of g / kg.
【００２０】[0020]
【実施例】以下、実施例により本発明をさらに具体的に
説明するが、本発明の範囲はこれらの実施例に限定され
ることはない。以下の実施例において、HGF として特開
平3-285693号公報に記載された方法に従って製造された
組換えhHGF(rhHGF) を使用した。The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. In the following Examples, recombinant hHGF (rhHGF) produced according to the method described in Japanese Patent Laid-Open No. 385653/1993 was used as HGF.
【００２１】実施例１：さい帯由来血管内皮細胞の増殖
促進作用 ヒトさい帯由来の血管内皮細胞をクラボウ株式会社製の
EGM-UV 培地で数回継代して増殖させた後、細胞をトリ
プシン/EDTA 溶液で剥離し、10% FBS/ペニシリン・スト
レプトマイシン含有 RPMI-1640培地（以下、実施例中で
この培地を「アッセイ培地」という）で３回洗浄し、ア
ッセイ培地を用いてヌンク社製96ウェルプレートに 5×
103, 1×104, 2×104/100 μl/ウェルとなるように播種
した。 HGFを最終濃度が 0〜200 ng/ml となるように 1
00μl/ウェルずつ各ウェルに添加し、CO2 インキュベー
ター中で37℃の条件でインキュベートした。Example 1: Proliferation promoting action of umbilical cord-derived vascular endothelial cells Human umbilical cord-derived vascular endothelial cells were produced by Kurabo Industries, Ltd.
After proliferating several times in EGM-UV medium, the cells were detached with a trypsin / EDTA solution, and RPMI-1640 medium containing 10% FBS / penicillin / streptomycin (hereinafter, this medium was referred to as “assay”). It is washed 3 times with "medium"), and 5x is added to Nunc 96 well plate using assay medium.
10 3, and seeded 1 × 10 4, 2 × so that 10 4/100 μl / well. HGF to a final concentration of 0-200 ng / ml 1
00 μl / well was added to each well and incubated at 37 ° C. in a CO 2 incubator.
【００２２】２時間後、 3H-チミジンを0.5 μCi／ウェ
ルとなるように各ウェルに添加し、培養をさらに16時間
継続した。細胞をトリプシン/EDTA 溶液で剥離後、ファ
ルマシア社製ガラスフィルターにβプレートハーベスタ
ー（ファルマシア社製）を用いてハーベストした。細胞
を蒸留水で洗浄して細胞内に取り込まれなかった 3H-チ
ミジンを除き、フィルターを乾燥した後、シンチレ−タ
−を添加してβプレートシンチレ−ションカウンタ−
（ファルマシア社製）により細胞内に取り込まれた放射
活性を測定した。結果を図１に示す。この結果から、HG
F が濃度依存的にさい帯由来血管内皮細胞の増殖を促進
することが明らかである。After 2 hours, 3 H-thymidine was added to each well at 0.5 μCi / well, and the culture was continued for another 16 hours. After detaching the cells with a trypsin / EDTA solution, they were harvested using a β plate harvester (Pharmacia) on a glass filter (Pharmacia). The cells were washed with distilled water to remove 3 H-thymidine that was not incorporated into the cells, the filter was dried, and a scintillator was added to the β plate scintillation counter.
The radioactivity incorporated into the cells was measured by (Pharmacia). The results are shown in Fig. 1. From this result, HG
It is clear that F 2 promotes the growth of umbilical cord-derived vascular endothelial cells in a concentration-dependent manner.
【００２３】実施例２：SV40で不死化したさい帯由来血
管内皮細胞のt-PA運動促進作用 SV40で不死化したさい帯由来血管内皮細胞(SV-3T：Agri
c. Biol. Chem., 55,pp.2487-2453, 1991) をクラボウ
株式会社製のEGM-UV培地で数回継代して増殖させた後、
アッセイ培地を用いてアッセイを行った。この不死化細
胞は通常の培地下で増殖し、HGF の添加による増殖の促
進効果はほとんど見られないが、サンドイッチERISA 法
で定量可能なt-PA産生能を維持しており、HGF のt-PA産
生促進作用を検討するためには優れた系である。Example 2: T-PA motility promoting effect of umbilical cord-derived vascular endothelial cells immortalized with SV40 Twirl-derived vascular endothelial cells (SV-3T: Agri) immortalized with SV40
c. Biol. Chem., 55, pp. 2487-2453, 1991) after several passages in EGM-UV medium manufactured by Kurabo Co., Ltd. and grown,
Assays were performed using assay medium. The immortalized cells grow in normal medium, and the growth-promoting effect of addition of HGF is hardly observed, but the t-PA-producing ability quantifiable by the sandwich ERISA method is maintained, and the HGF t- This is an excellent system for examining the PA production promoting action.
【００２４】細胞をトリプシン/EDTA 溶液で剥離後、ア
ッセイ培地で３回洗浄し、アッセイ培地を用いてヌンク
社製96ウェルプレートに 1×104, 2×104, 4×104/100
μl/ウェルとなるように播種した。 HGFを最終濃度が 0
〜200 ng/ml となるように 100μl/ウェルずつ各ウェル
に添加し、CO2 インキュベーター中で37℃の条件でイン
キュベートした。48時間後に上清を採取し、その50μl
を以下のERISA 試験に供した。[0024] After peeling the cells with trypsin / EDTA solution, washed 3 times with assay medium, Nunc 96-well plates in 1 × 10 4 using the assay medium, 2 × 10 4, 4 × 10 4/100
The seeds were seeded so as to give μl / well. Final concentration of HGF is 0
100 μl / well was added to each well so that the concentration was ˜200 ng / ml, and the plate was incubated at 37 ° C. in a CO 2 incubator. After 48 hours, collect the supernatant and
Was subjected to the following ERISA test.
【００２５】ファルコン社製ウェルプレートをウサギ抗
ヒトt-PA IgG 5μl/mlを含むリン酸緩衝溶液(PBS) でコ
ーティングし(50 μl/ウェル）、 1% BSA 及びアジ化ナ
トリウム含有PBS (240μl/ウェル）を用いて４℃で１昼
夜ブロッキングした。0.39〜100 ng/ml の濃度範囲とな
るように、サンプル及びスタンダード(1% BSA と0.1%ア
ジ化ナトリウムを含むPBS 中）を37℃で１時間反応させ
た(50 μl/ウェル）。1 〜4 μg/mlのビオチン化抗ヒト
t-PA F(ab')2 [1% BSAと0.1%アジ化ナトリウムを含むPB
S 中：アマシャム社製ビオチニレーション試薬を用いて
抗ヒトt-PA F(ab')2より作製] を37℃で１時間反応させ
た（50μl/ウェル）。Falcon well plates were coated with phosphate buffer solution (PBS) containing rabbit anti-human t-PA IgG 5 μl / ml (50 μl / well) and PBS containing 1% BSA and sodium azide (240 μl / well). Wells) and blocked at 4 ° C for one day. Samples and standards (in PBS containing 1% BSA and 0.1% sodium azide) were reacted at 37 ° C for 1 hour (50 µl / well) so that the concentration range was 0.39 to 100 ng / ml. 1-4 μg / ml biotinylated anti-human
t-PA F (ab ') 2 [PB containing 1% BSA and 0.1% sodium azide
In S: prepared from anti-human t-PA F (ab ′) 2 using a biotinylation reagent manufactured by Amersham] was reacted at 37 ° C. for 1 hour (50 μl / well).
【００２６】1% BSA及びPBS で1,000 倍希釈したカッペ
ル社製 HRPコンジュゲーティッド・アビジンを室温で30
分間反応させた。発色試薬 [クエン酸緩衝液（pH 5.0）
中のOPD 10 mg/25 ml に10μl の過酸化水素水を用時添
加したもの。50μl/ウェル]を加え、適当量の発色が見
られた時点で 4N H2SO4(50μl/ウェル）を加えて反応を
停止した。650 nmの吸光度及び490 nmの吸光度の差を測
定し、上清中のt-PA含量を算出した。結果を図２に示
す。この結果から、HGF が濃度依存的に再帯由来血管内
皮細胞のt-PA産生を促進することが明らかである。A Kappel HRP-conjugated avidin diluted 1,000-fold with 1% BSA and PBS was used at room temperature for 30 times.
Let react for minutes. Color reagent [Citrate buffer (pH 5.0)
OPD 10 mg / 25 ml in which 10 μl of hydrogen peroxide solution was added before use. 50 μl / well] was added, and when a proper amount of color was observed, 4N H 2 SO 4 (50 μl / well) was added to stop the reaction. The difference between the absorbance at 650 nm and the absorbance at 490 nm was measured, and the t-PA content in the supernatant was calculated. The results are shown in Figure 2. From this result, it is clear that HGF promotes t-PA production of red zone-derived vascular endothelial cells in a concentration-dependent manner.
【発明の効果】本発明の医薬の有効成分であるHGF は、
血管内皮細胞のt-PA産生を促進するとともに血管内皮細
胞の増殖を促進する作用を有しているので、本発明の医
薬は血栓症の予防及び／又は治療、並びに血管内皮細胞
に障害を伴う疾患の予防及び／又は治療に有効である。EFFECT OF THE INVENTION HGF, which is an active ingredient of the medicine of the present invention, is
Since the drug of the present invention has the effects of promoting t-PA production of vascular endothelial cells and promoting the growth of vascular endothelial cells, the pharmaceutical agent of the present invention prevents and / or treats thrombosis and impairs vascular endothelial cells. It is effective for prevention and / or treatment of diseases.
【図１】 さい帯由来血管内皮細胞の増殖に対するHGF
の作用を示す図である。図中、横軸はそれぞれ 5×103,
1×104, 2×104/100 μl/ウェルの細胞数の場合の HGF
濃度(ng/ml) を示し、縦軸は3H- チミジンの取り込み量
(cpm×1,000)を示す。Figure 1: HGF on proliferation of vascular endothelial cells derived from umbilical cord
It is a figure which shows the effect | action of. In the figure, the horizontal axis is 5 × 10 3 ,
1 × 10 4, 2 × 10 4/100 μl / if the number of cells in wells HGF
Concentration (ng / ml), vertical axis shows 3 H-thymidine uptake
Indicates (cpm x 1,000).
【図２】さい帯由来血管内皮細胞のt-PA産生能に対する
HGF の作用を示す図である。横軸はそれぞれ 1×104, 2
×104, 4×104/100 μl/ウェルの細胞数の場合のHGF濃
度(ng/ml) を示し、縦軸はt-PA産生量(mg/ml) を示す。FIG. 2 T-PA production ability of umbilical cord-derived vascular endothelial cells
It is a figure which shows the effect | action of HGF. The horizontal axis is 1 × 10 4 , 2 respectively
× indicates 10 4, 4 × 10 4/ 100 μl / HGF concentration in the case of wells number of cells (ng / ml), the vertical axis represents t-PA production amount (mg / ml).
Claims (4)
む血栓症の予防・治療剤。1. A prophylactic / therapeutic agent for thrombosis, which comprises hepatocyte growth factor as an active ingredient.
質： 1) SDS-PAGE（非還元条件下）による推定分子量が約7
6,000〜92,000であり； 2) 肝実質細胞を増殖させる活性を有し； 3) 80℃、10分間の加熱処理により上記活性が失活し； 4) トリプシンによる消化処理及びキモトリプシンによ
る消化処理により上記活性が失活し； 5) ヘパリンに対して強い親和性を有する を示す請求項１に記載の予防・治療剤。2. The physicochemical properties of hepatocyte growth factor are as follows: 1) The molecular weight estimated by SDS-PAGE (under non-reducing conditions) is about 7.
6,000 to 92,000; 2) it has an activity to grow hepatocytes; 3) the above activity is inactivated by heat treatment at 80 ° C. for 10 minutes; 4) it is digested with trypsin and chymotrypsin. The activity is inactivated; 5) The preventive / therapeutic agent according to claim 1, which has a strong affinity for heparin.
む血管内皮細胞の障害を伴う疾患の予防・治療剤。3. A preventive / therapeutic agent for diseases associated with vascular endothelial cell damage, which comprises hepatocyte growth factor as an active ingredient.
質： 1) SDS-PAGE（非還元条件下）による推定分子量が約7
6,000〜92,000であり； 2) 肝実質細胞を増殖させる活性を有し； 3) 80℃、10分間の加熱処理により上記活性が失活し； 4) トリプシンによる消化処理及びキモトリプシンによ
る消化処理により上記活性が失活し； 5) ヘパリンに対して強い親和性を有する を示す請求項３に記載の予防・治療剤。4. Hepatocyte growth factor has the following physicochemical properties: 1) The molecular weight estimated by SDS-PAGE (under non-reducing conditions) is about 7.
6,000 to 92,000; 2) it has an activity to grow hepatocytes; 3) the above activity is inactivated by heat treatment at 80 ° C. for 10 minutes; 4) it is digested with trypsin and chymotrypsin. 5. The preventive / therapeutic agent according to claim 3, which exhibits inactivity; and 5) has a strong affinity for heparin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7037367A JPH08231416A (en) | 1995-02-24 | 1995-02-24 | Treating agent for thrombosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7037367A JPH08231416A (en) | 1995-02-24 | 1995-02-24 | Treating agent for thrombosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08231416A true JPH08231416A (en) | 1996-09-10 |
Family
ID=12495563
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7037367A Pending JPH08231416A (en) | 1995-02-24 | 1995-02-24 | Treating agent for thrombosis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08231416A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997012628A1 (en) * | 1995-10-05 | 1997-04-10 | Genentech, Inc. | Methods and compositions for treating vascular stenosis |
| WO2001021214A1 (en) * | 1999-09-21 | 2001-03-29 | Medgene Bioscience, Inc. | Gene therapy for cerebrovascular disorders |
| WO2005072768A1 (en) * | 2004-01-30 | 2005-08-11 | Mitsubishi Pharma Corporation | Preventive and/or remedy for retinopathy |
| JP2007528365A (en) * | 2004-03-09 | 2007-10-11 | 株式会社 東北テクノアーチ | Vascular differentiation induction promoter containing hepatocyte growth factor |
| JP2011225495A (en) * | 2010-04-22 | 2011-11-10 | Hayashikane Sangyo Kk | Vascular endothelial cell protecting agent, and pharmaceutical composition, food and feed containing the same |
-
1995
- 1995-02-24 JP JP7037367A patent/JPH08231416A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997012628A1 (en) * | 1995-10-05 | 1997-04-10 | Genentech, Inc. | Methods and compositions for treating vascular stenosis |
| WO2001021214A1 (en) * | 1999-09-21 | 2001-03-29 | Medgene Bioscience, Inc. | Gene therapy for cerebrovascular disorders |
| US6936594B1 (en) | 1999-09-21 | 2005-08-30 | Ryuichi Morishita | Gene therapy for cerebrovascular disorders |
| JP4642303B2 (en) * | 1999-09-21 | 2011-03-02 | アンジェスＭｇ株式会社 | Gene therapy for cerebrovascular disorders |
| WO2005072768A1 (en) * | 2004-01-30 | 2005-08-11 | Mitsubishi Pharma Corporation | Preventive and/or remedy for retinopathy |
| JP2007528365A (en) * | 2004-03-09 | 2007-10-11 | 株式会社 東北テクノアーチ | Vascular differentiation induction promoter containing hepatocyte growth factor |
| JP2011225495A (en) * | 2010-04-22 | 2011-11-10 | Hayashikane Sangyo Kk | Vascular endothelial cell protecting agent, and pharmaceutical composition, food and feed containing the same |
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