JPH0625010A - Antitumor agent - Google Patents

Antitumor agent

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Publication number
JPH0625010A
JPH0625010A JP3140812A JP14081291A JPH0625010A JP H0625010 A JPH0625010 A JP H0625010A JP 3140812 A JP3140812 A JP 3140812A JP 14081291 A JP14081291 A JP 14081291A JP H0625010 A JPH0625010 A JP H0625010A
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JP
Japan
Prior art keywords
cells
hgf
cancer
cell
derived
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JP3140812A
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JP3030386B2 (en
Inventor
Toshiichi Nakamura
敏一 中村
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Individual
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Individual
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

PURPOSE:To provide an antitumor agent capable of specifically suppressing the proliferation of cancer cell without influencing on normal cell. CONSTITUTION:This antitumor agent contains a hepatocyte growth factor(HGF) as an active component. HGF exhibits specific proliferation suppressing activity and cytocidal activity against hepatic cancer cell, leukemia cell and other cancer cells and does not participate to the immunity system and, accordingly, it has high specificity and low side effect. The antitumor agent is useful for the treatment of cancer, etc.

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【産業上の利用分野】本発明は抗ガン剤に関し、より詳
細には肝実質細胞増殖因子(以下、HGFという)を有
効成分として含有する抗ガン剤に関する。
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anticancer agent, and more particularly to an anticancer agent containing hepatocyte growth factor (hereinafter referred to as HGF) as an active ingredient.

【0002】[0002]

【従来の技術】厚生省による平成元年人口動態統計によ
れば、わが国の死因の第一位は悪性新生物、すなわちガ
ンであり、これまでのところ死亡者数は毎年増加してい
る。ガン制圧は、言うまでもなく今日の医療における最
大の課題であり、また新規ガン治療法あるいは新抗ガン
剤は医療・医薬研究者の最大の関心事である。にもかか
わらず、現在のわが国のガン患者は推定80万人であ
り、毎年30万人以上の新しい患者が発生していると言
われている。
2. Description of the Related Art According to the 1989 vital statistics by the Ministry of Health and Welfare, the number one cause of death in Japan is malignant neoplasm, namely cancer, and the number of deaths has been increasing every year so far. Needless to say, cancer control is the biggest problem in today's medical treatment, and new cancer treatment methods or new anticancer agents are the most important concerns of medical and pharmaceutical researchers. Nevertheless, the current number of cancer patients in Japan is estimated to be 800,000, and it is said that more than 300,000 new patients occur each year.

【0003】化学療法に用いられる薬剤として、従来の
アルキル化剤、代謝拮抗剤、抗生物質のほかに、近年は
ピシバニール(商品名、中外製薬社製)やクレスチン
(商品名、三共製薬社製)を初めとする微生物由来の生
体反応修飾物質が全盛を極めた。しかし、その限界が明
らかになると共に、最近の研究はIF(インターフェロ
ン)−α、β、γ、IL(インターロイキン)−2やT
NF(腫瘍壊死因子)など、高等動物由来の生理活性蛋
白に重点が置かれるようになった。従来のアルキル化剤
を初めとする化合物は本来、細胞毒性を利用するもので
あり、少なからぬ副作用のために使用がかなり限定され
たのに比べ、その後のピシバニールなどの生体反応修飾
物質は生体の免疫機能を高め、ガン細胞を駆逐するとい
う作用機作を有する。
As drugs used for chemotherapy, in addition to conventional alkylating agents, antimetabolites, and antibiotics, in recent years picibanil (trade name, manufactured by Chugai Pharmaceutical Co., Ltd.) and krestin (trade name, manufactured by Sankyo Pharmaceutical Co., Ltd.) The biological reaction modifiers derived from microorganisms, such as However, as its limitations have become clear, recent studies have shown that IF (interferon) -α, β, γ, IL (interleukin) -2 and T
Emphasis has been placed on bioactive proteins derived from higher animals such as NF (tumor necrosis factor). Conventional alkylating agents and other compounds inherently utilize cytotoxicity, and their use was considerably limited due to considerable side effects, while subsequent biological response modifiers such as picibanil were It has a mechanism of action that enhances immune function and destroys cancer cells.

【0004】この数年の遺伝子工学技術の急速な発展に
伴って、ヒトを初めとする高等動物の生理機能も徐々に
解明され、その過程で発見されたリンホカインの一種が
IF、IL−2やTNFである。免疫系に関与するこれ
らの微量因子も、遺伝子操作技術により大量に得ること
ができる。また、それらの機能は生体が本来持つ防御機
能に基づくものであることから、特異性が高く、薬効が
明らかで、副作用の予測も容易であると考えられてき
た。しかしながら研究が進に連れ、必ずしも当初期待さ
れたような「夢の」抗ガン剤とは言えないことが明らか
になった。
With the rapid development of genetic engineering technology in recent years, physiological functions of higher animals including humans have been gradually elucidated, and one of lymphokines discovered in the process is IF, IL-2 and It is TNF. These trace factors involved in the immune system can also be obtained in large quantities by genetic engineering techniques. Further, since these functions are based on the defense function originally possessed by the living body, it has been considered that the specificity is high, the drug effect is clear, and the side effects can be easily predicted. However, as research progressed, it became clear that it was not always the "dream" anticancer drug that was initially expected.

【0005】その理由は、これらのリンホカインの作用
スペクトルが発見当初予想されたものよりはるかに広範
であったためである。生体は、人間が考えるよりもはる
かに複雑で効率良い機能を持つため、これら重要な因子
もさまざまな場面で他の生体因子と相互作用しながら機
能していると考えられる。実際、TNFはその発見から
10年後の1985年、ガン末期や重症感染症の患者に起こ
る全身衰弱症状の原因物質であるカケクチンと同一物質
であることが発見され、「副作用を示さない」という認識
が覆されてしまった。
The reason for this is that the action spectrum of these lymphokines was much broader than initially expected. Since the living body has a far more complicated and efficient function than humans think, it is considered that these important factors also function while interacting with other biological factors in various situations. In fact, in 1985, 10 years after its discovery, it was discovered that TNF is the same substance as cakectin, the causative substance of general debilitating symptoms that occur in patients with end-stage cancer and severe infections. Perception has been overturned.

【0006】しかしながら、今後のガン治療が単なるガ
ン病巣の撃退を持って評価するのではなく、生体のトー
タルな機能の改善の中での治療を考える「クオリティ・
オブ・ライフ」に焦点が移ることは間違いなく、これら
リンホカインなど、高等動物生体由来の生理活性蛋白に
対する期待はますます高まっている。そのためには、I
FやILよりもっと作用がクリアで、標的細胞なり関与
する作用機能なりがより限定された生体因子の研究が重
要である。
[0006] However, in future cancer treatment, instead of simply evaluating the recurrence of cancer lesions, we will consider the treatment in terms of improving the total function of the living body.
There is no doubt that the focus will shift to “of life”, and expectations for bioactive proteins derived from higher animal organisms such as these lymphokines are increasing. To do that, I
It is important to study biological factors that have a clearer action than F and IL, and that have a more limited target cell and action function involved.

【0007】一方、本発明の抗ガン剤の有効成分である
HGFは、本発明者らが再生肝ラット血清中から成熟肝
実質細胞をin vitroで増殖させる因子として見出した蛋
白質である(Biochem Biophys Res Commun, 122, 1450,
1984)。本発明者らはさらに、HGFをラット血小板よ
り単離することに成功し(FEBS Letter, 22, 311, 198
7)、そのアミノ酸配列を一部決定した。さらに、本発明
者らは解明されたHGFアミノ酸配列をもとにヒト及び
ラット由来のHGFcDNAクローニングを行い、その
cDNAを動物組織に組換えてHGFを蛋白質として得
ることに成功した(ヒトHGF:Nature, 342, 440, 19
89; ラットHGF:Proc. Natl. Acad. Sci, 87, 320
0、 1990)。
On the other hand, HGF, which is an active ingredient of the anticancer agent of the present invention, is a protein that the present inventors have found as a factor for proliferating mature hepatocytes from regenerated liver rat serum in vitro (Biochem Biophys). Res Commun, 122 , 1450,
1984). The present inventors have further succeeded in isolating HGF from rat platelets (FEBS Letter, 22 , 311, 198).
7), the amino acid sequence was partially determined. Furthermore, the present inventors have succeeded in performing human and rat-derived HGF cDNA cloning based on the revealed HGF amino acid sequence, and recombining the cDNA into animal tissues to obtain HGF as a protein (human HGF: Nature). , 342 , 440, 19
89; Rat HGF: Proc. Natl. Acad. Sci, 87 , 320
0, 1990).

【0008】[0008]

【発明が解決しようとする課題】本発明は従来見出され
ていなかった、正常細胞に影響することなく且つガン細
胞を特異的に増殖抑制ないし死滅させ得、種々のガンの
治療に有効な薬剤を提供することを目的とするものであ
る。前記のごとく、既成の抗ガン剤がその副作用や、あ
るいは抗ガン作用そのものへの疑問から、決定的な治療
薬とは言い難く、さらに次世代を担う生理活性蛋白質も
これまで開発されたものは主に免疫系にかかわる因子で
あり、必ずしも究極の抗ガン剤として広く利用されるこ
とを期待できない状況にある。そこで、同じく生体が本
来持っている生理活性蛋白質の中でもその作用機作が明
快で、しかも活性のスペクトルがよく研究されているも
のの中から、真の抗ガン剤を見出すことが重要になって
くる。特に、従来開発されてきた生理活性蛋白質はI
F、IL等ほとんどが免疫系にかかわる因子であり、そ
の意味からもまったく異なる作用機作を有する生体因子
がこれからの抗ガン剤として重要度を持つと考えられ
る。
DISCLOSURE OF THE INVENTION The present invention has not hitherto been found. An agent effective for treating various cancers, which has not hitherto been found, can suppress the growth or kill cancer cells specifically without affecting normal cells. It is intended to provide. As mentioned above, it is difficult to say that the established anti-cancer drug is a definitive therapeutic drug because of its side effects or the question of the anti-cancer effect itself. It is a factor mainly related to the immune system and cannot be expected to be widely used as the ultimate anticancer drug. Therefore, it becomes important to find a true anticancer drug from among the physiologically active proteins originally possessed by living organisms, whose mechanism of action is clear and whose spectrum of activity is well studied. . In particular, the bioactive proteins that have been conventionally developed are I
Most of F, IL and the like are factors involved in the immune system, and in that sense, biological factors having completely different modes of action are considered to be important as anticancer agents in the future.

【0009】[0009]

【課題を解決するための手段】本発明者は、年余にわた
り肝実質細胞の増殖因子を研究し、その結果HGFを単
離精製することに成功した。さらにその構造と活性を詳
細に検討する中で、HGFにガン細胞の増殖を特異的に
抑制する活性ないし死滅させる活性を見出し、本発明を
完成するに至った。即ち、HGFは以下の実施例に述べ
るごとく、肝ガン細胞、白血病細胞、表皮ガン細胞など
のガン細胞の増殖を5〜10ng/mlという低濃度で著しく
抑制し、また特定のガン細胞に対する殺細胞活性を有す
ることが確認された。またHGFは元来、肝実質細胞増
殖を促進する因子として発見されたポリペプチドである
が、肝実質細胞を初めとする正常上皮系細胞のみ増殖促
進し、肝非実質細胞や線維芽細胞など正常間葉系細胞の
増殖になんら影響せず、また細胞をガン化させる活性を
持たないため、他の多くの生体由来抗ガン剤に比べて特
異性が高く、より実用に適していると言える。
The present inventor has studied hepatocyte growth factors for over a year and succeeded in isolating and purifying HGF. Furthermore, in the detailed examination of its structure and activity, HGF was found to have an activity of specifically suppressing the growth of cancer cells or an activity of killing it, and completed the present invention. That is, HGF remarkably inhibits the growth of cancer cells such as hepatoma cells, leukemia cells, and epidermal cancer cells at a low concentration of 5 to 10 ng / ml, and kills specific cancer cells as described in the following examples. It was confirmed to have activity. HGF is a polypeptide that was originally discovered as a factor that promotes hepatic parenchymal cell proliferation. However, it promotes the proliferation of normal epithelial cells including hepatic parenchymal cells, and normal hepatic nonparenchymal cells and fibroblasts Since it has no effect on the proliferation of mesenchymal cells and has no activity to cause canceration of cells, it has high specificity as compared with many other anticancer agents of biological origin and can be said to be more suitable for practical use.

【0010】本発明の抗ガン剤の有効成分であるHGF
は、SDS−ポリアクリルアミドゲル電気泳動より分子
量82〜85kDである。ラットHGF分子は463ア
ミノ酸残基からなるα鎖と234アミノ酸残基からなる
β鎖が1個のジスルフィド結合により架橋したヘテロダ
イマー構造をもち、α、β両鎖とも2個のグルコサミン
型糖鎖結合部位が存在する。ヒトHGFもまたほぼ同じ
生理活性を有し、463アミノ酸残基からなるα鎖と2
34アミノ酸残基からなるβ鎖とからなる。α鎖中には
線溶酵素プラスミンと同様のクリングル構造が4個存在
し、β鎖のアミノ酸配列においてもセリンプロテアーゼ
活性を有するプラスミンのB鎖と約37%のホモロジー
を有する。ヒトHGF前駆体のアミノ酸配列及びこれを
コードする遺伝子の塩基配列をそれぞれ図7及び図8に
示した。ヒトHGFは図7に示される728個のアミノ
酸からなる前駆体として生合成され、その後463アミ
ノ酸残基(図7の配列の第32位のGlnから第494位の
Argまで)からなるα鎖と、234アミノ酸残基(図7の
配列の第495位のValから第728位のSerまで)からなる
β鎖にわかれる。ラットHGFとヒトHGFのアミノ酸
配列のホモロジーはα鎖において91.6%、β鎖において8
8.9%と非常に高い相同性をもち、その活性は全く互換性
がある。
HGF which is an active ingredient of the anticancer agent of the present invention
Has a molecular weight of 82 to 85 kD by SDS-polyacrylamide gel electrophoresis. The rat HGF molecule has a heterodimer structure in which an α chain consisting of 463 amino acid residues and a β chain consisting of 234 amino acid residues are crosslinked by one disulfide bond. Both α and β chains have two glucosamine-type sugar chain bonds. The part exists. Human HGF also has almost the same physiological activity as that of the α chain consisting of 463 amino acid residues and 2
It consists of a β chain consisting of 34 amino acid residues. There are four kringle structures in the α chain similar to the fibrinolytic enzyme plasmin, and the amino acid sequence of the β chain has about 37% homology with the B chain of plasmin having serine protease activity. The amino acid sequence of human HGF precursor and the nucleotide sequence of the gene encoding it are shown in FIGS. 7 and 8, respectively. Human HGF is biosynthesized as a precursor consisting of 728 amino acids shown in FIG. 7, and then 463 amino acid residues (Gln at the 32nd position to 494th position in the sequence of FIG. 7).
Α chain consisting of Arg) and a β chain consisting of 234 amino acid residues (from Val at position 495 to Ser at position 728 in the sequence of FIG. 7). The amino acid sequence homology between rat HGF and human HGF was 91.6% in the α chain and 8 in the β chain.
It has a very high homology of 8.9% and its activities are completely compatible.

【0011】上記のHGFは種々の方法により得ること
ができる。例えば、ラット、ウシなどの哺乳動物の肝
臓、脾臓、肺臓、骨髄、脳、腎臓、胎盤等の臓器、血小
板、白血球等の血液細胞や血漿、血清などから抽出、精
製して得ることができる。また、HGFを産生する初代
培養細胞や株化細胞を培養し、培養物から分離精製して
HGFを得ることもできる。あるいは遺伝子工学的手法
によりHGFをコードする遺伝子を適切なベクターに組
込み、これを適当な宿主に挿入して形質転換し、この形
質転換体の培養物から目的とする組換えHGFを得るこ
とができる(Nature, 342, 440, 1989)。上記の宿主細胞
は特に限定されず、従来から遺伝子工学的手法で用いら
れている各種の宿主細胞、例えば大腸菌、枯草菌、酵
母、糸状菌、植物又は動物細胞などを用いることができ
る。
The above HGF can be obtained by various methods. For example, it can be obtained by extraction and purification from organs such as liver, spleen, lung, bone marrow, brain, kidney and placenta of mammals such as rat and bovine, blood cells such as platelets and leukocytes, plasma, serum and the like. Alternatively, HGF can be obtained by culturing primary culture cells or cell lines that produce HGF and separating and purifying from the culture. Alternatively, a gene encoding HGF can be incorporated into an appropriate vector by a genetic engineering method, and this can be inserted into an appropriate host for transformation, and the desired recombinant HGF can be obtained from the culture of this transformant. (Nature, 342 , 440, 1989). The above-mentioned host cells are not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells can be used.

【0012】より具体的には、HGFを生体組織から抽
出精製する方法としては、例えば実施例1に示すように
ラットに四塩化炭素を腹腔内投与し、肝炎状態にしたラ
ットの肝臓を摘出して粉砕し、S−セファロース、ヘパ
リンセファロースなどのゲルカラムクロマトグラフィー
にて精製することができる。あるいは実施例2に示すよ
うに遺伝子組換え法を用い、図8に示したヒトHGFの
アミノ酸配列をコードする遺伝子を、ウシパピローマウ
ィルスNDAなどのベクターに組み込んだ発現ベクター
によって動物細胞、例えばチャイニーズハムスター卵巣
(CHO)細胞、マウスC127細胞や、サルCOS細
胞などを形質転換し、その培養上清より得ることができ
る。
More specifically, as a method for extracting and purifying HGF from a living tissue, for example, as shown in Example 1, carbon tetrachloride is intraperitoneally administered to a rat, and the liver of the rat in the hepatitis state is excised. It can be pulverized and then purified by gel column chromatography using S-Sepharose, heparin Sepharose or the like. Alternatively, as shown in Example 2, by using the gene recombination method, the gene encoding the amino acid sequence of human HGF shown in FIG. 8 is incorporated into a vector such as bovine papilloma virus NDA to produce an animal cell such as Chinese hamster. It can be obtained from the culture supernatant by transforming ovary (CHO) cells, mouse C127 cells, monkey COS cells and the like.

【0013】こうして得られたHGFは、そのアミノ酸
配列の一部が欠失又は他のアミノ酸により置換されてい
たり、他のアミノ酸配列が一部挿入されていたり、N末
端及び/又はC末端に1又は2以上のアミノ酸が結合し
ていたり、あるいは糖類が同様に欠失あるいは置換され
ていても、ガン細胞増殖の抑制活性を有する限り本発明
の範囲に含まれる。
The HGF thus obtained has a part of its amino acid sequence deleted or substituted with another amino acid, a part of another amino acid sequence inserted, or has 1 amino acid at the N-terminal and / or C-terminal. Alternatively, even if two or more amino acids are bound, or a saccharide is similarly deleted or substituted, it is included in the scope of the present invention as long as it has a cancer cell growth inhibitory activity.

【0014】本発明の抗ガン剤の有効成分であるHGF
は、ヒトを含むウシ、ウマ、ラット、ヒツジなどいずれ
の哺乳動物に由来するものであっても、ガン細胞に対し
て有効な細胞増殖抑制活性ないし殺細胞活性をもち、ま
たいずれの哺乳動物に対しても有効な抗ガン活性をも
つ。すなわち、本発明の薬剤はヒトの医薬品のみならず
動物用医薬品としても用いることができる。
HGF which is an active ingredient of the anticancer agent of the present invention
Is derived from any mammal such as bovine including humans, horses, rats, and sheep, and has an effective cytostatic or cytocidal activity against cancer cells, and is effective for any mammal. It also has effective anti-cancer activity. That is, the drug of the present invention can be used not only as a human drug but also as a veterinary drug.

【0015】本発明の抗ガン剤は種々の製剤形態(液
剤、固形剤、カプセル剤など)をとりうるが、一般的に
は有効成分であるHGF単独、又は既知の担体と共に注
射剤とされる。当該注射剤は常法により調製することが
でき、例えば、HGFを適切な溶剤(例えば、滅菌水、
緩衝液、生理食塩水等)に溶解した後、フィルター等で
濾過して滅菌し、次いで無菌的な容器に充填することに
より調製することができる。注射剤中のHGF含量とし
ては、通常0.0002〜0.2(W/V%)程度、好ましくは0.001〜
0.1(W/V%)程度に調整される。また、製剤化に際して、
好ましくは安定化剤が添加され、安定化剤としては、例
えば、アルブミン、グロブリン、ゼラチン、マンニトー
ル、グルコース、デキストラン、エチレングリコールな
どが挙げられる。さらに、本発明の薬剤は製剤化に必要
な添加物、例えば、賦形剤、溶解補助剤、酸化防止剤、
無痛化剤、等張化剤等を含んでいてもよい。液状製剤と
した場合は凍結保存、又は凍結乾燥等により水分を除去
して保存するのが望ましい。
The anti-cancer agent of the present invention can take various preparation forms (liquid, solid, capsule, etc.), but generally it is used as an active ingredient, HGF alone, or an injection together with a known carrier. . The injection can be prepared by a conventional method. For example, HGF can be prepared by using a suitable solvent (for example, sterile water,
It can be prepared by dissolving it in a buffer solution, physiological saline, etc.), filtering it with a filter or the like to sterilize it, and then filling it in an aseptic container. The HGF content in the injection is usually about 0.0002 to 0.2 (W / V%), preferably 0.001 to
It is adjusted to about 0.1 (W / V%). Also, when formulating,
Preferably, a stabilizer is added, and examples of the stabilizer include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol and the like. Furthermore, the agents of the present invention include additives necessary for formulation, such as excipients, solubilizers, antioxidants,
It may contain a soothing agent, a tonicity agent, and the like. In the case of a liquid preparation, it is desirable to remove water by freezing, or freeze-drying, and then save.

【0016】本発明の抗ガン剤は該製剤組成物の形態に
応じた適当な投与経路により投与され得る。例えば、注
射剤の形態にして静脈、動脈、皮下、筋肉内等に投与す
ることができる。その投与量は、患者の症状、年齢、体
重などにより適宜調整されるが、通常HGFとして0.01
mg〜100mgであり、これを1日1回ないし数回に分けて
投与するのが適当である。また、本発明の抗ガン剤は、
アルキル化剤、免疫賦活剤など既存の抗ガン剤による化
学療法や放射線療法と併用してガン治療に利用すること
ができる。
The anticancer agent of the present invention can be administered by an appropriate administration route depending on the form of the pharmaceutical composition. For example, it can be administered in the form of an injection such as vein, artery, subcutaneous or intramuscular. The dose is appropriately adjusted depending on the patient's symptoms, age, weight, etc.
The dosage is in the range of 100 mg to 100 mg, and it is suitable to administer this once or several times a day. Further, the anticancer agent of the present invention,
It can be used for cancer treatment in combination with chemotherapy and radiation therapy using existing anti-cancer agents such as alkylating agents and immunostimulants.

【0017】[0017]

【発明の効果】本発明の抗ガン剤はHGFを有効成分と
して含有してなり、HGFは肝ガン細胞や白血病細胞を
初めとするガン細胞に対して特異的な増殖抑制活性ない
し殺細胞活性を有するので、抗ガン剤としてガンの治
療、予防などに用いられ、臨床上極めて有用である。更
にHGFは細胞増殖因子の一種であり、免疫系に関与し
ない点でIF、IL、TNF等とは異なり、従来にない
特異性の高い、また副作用の少ない薬剤として有用であ
る。
INDUSTRIAL APPLICABILITY The anticancer agent of the present invention contains HGF as an active ingredient, and HGF has specific growth inhibitory activity or cell killing activity against cancer cells such as hepatoma cells and leukemia cells. Since it has an anti-cancer agent, it is used for the treatment and prevention of cancer and is extremely useful clinically. Furthermore, HGF is a kind of cell growth factor, and unlike IF, IL, TNF, etc. in that it does not participate in the immune system, it is useful as a drug with high specificity and few side effects that has never been seen.

【0018】[0018]

【実施例】本発明をより詳細に説明するために実施例を
挙げるが、本発明はこれらによってなんら限定されるも
のではない。 実施例1ラット肝臓からのHGFの単離 Wister系ラットに体重の0.2%の量の四塩化炭素を、腹腔
内投与し投与後約30時間目で肝臓を摘出した。肝臓はワ
ーリングブレンダーで破砕後、日立20PR-52型冷却遠心
機を用いて10,000rpm20分間遠心し、上清を得た。上清
を、0.15M NaCl、10mMヘペス、2mM CaCl2及び
0.01%Tween80を加えた50mMトリス塩酸緩衝液(pH8.5)で4
℃一昼夜透析した。透析内液を透析液で平衡化したS−
セファロース(FF)(ファルマシア社製)カラムに注
入し、洗浄後NaClの濃度勾配により溶出を行い、H
GFはNaCl濃度0.7M付近に溶出した。次にこのHG
FをブルートリスアクリルM(IBF社製)クロマトグ
ラフィーにて精製した。溶出はアルギニンの濃度勾配に
より行い、HGFはアルギニン濃度0.25M付近で溶出し
た。得られた分画を次にヘパリン-セファロース(ファル
マシア社製)クロマトグラフィーにより精製した。溶出
はNaClの濃度勾配により行い、HGFは1M前後のN
aCl濃度付近で溶出した。次にフェニル5PW(東ソ
ー社製)クロマトグラフィーにより精製した。溶出はN
aCl濃度減少及びエチレングリコール濃度上昇勾配に
より行った。ラット100匹の肝臓当たり10μgのHGFが
得られた。精製HGFの比活性は約50万単位/mgであっ
た。得られたHGFは0.25%BSA(牛血清アルブミ
ン)を加え、PBS(リン酸緩衝食塩水)にて透析し
た。
EXAMPLES Examples will be given to explain the present invention in more detail, but the present invention is not limited thereto. Example 1 Isolation of HGF from rat liver Wister rats were intraperitoneally administered with carbon tetrachloride in an amount of 0.2% of the body weight, and the liver was extracted about 30 hours after the administration. The liver was disrupted with a Waring blender and then centrifuged at 10,000 rpm for 20 minutes using a Hitachi 20PR-52 type cooling centrifuge to obtain a supernatant. The supernatant was washed with 0.15 M NaCl, 10 mM Hepes, 2 mM CaCl 2 and
4 with 50 mM Tris-HCl buffer (pH 8.5) containing 0.01% Tween80
Dialysis was performed overnight at ℃. S- equilibrated dialysate with dialysate
It was injected into a Sepharose (FF) (Pharmacia) column, washed, and then eluted with a NaCl concentration gradient to obtain H.
GF was eluted near the NaCl concentration of 0.7M. Next this HG
F was purified by Blue Tris Acrylic M (manufactured by IBF) chromatography. Elution was performed by a concentration gradient of arginine, and HGF was eluted at an arginine concentration of around 0.25M. The obtained fraction was then purified by heparin-Sepharose (Pharmacia) chromatography. Elution was performed with a NaCl concentration gradient.
Elution was performed around the aCl concentration. Then, it was purified by phenyl 5PW (manufactured by Tosoh Corporation) chromatography. Elution is N
It was performed by decreasing the aCl concentration and increasing the ethylene glycol concentration. 10 μg of HGF was obtained per 100 rat livers. The specific activity of purified HGF was about 500,000 units / mg. The obtained HGF was added to 0.25% BSA (bovine serum albumin) and dialyzed against PBS (phosphate buffered saline).

【0019】実施例2遺伝子組換え法によるHGFの製造 遺伝子組換え法によりヒト細胞由来のHGFを製造し
た。Wiglerらの方法(Cell, 11, 223, 1977)に記載され
た方法に従って、ヒトHGFのアミノ酸配列をコードす
る遺伝子により形質転換されたチャイニーズハムスター
卵巣(CHO)細胞を培養し、その培養上清より、ヒト
HGFを得た。すなわち、ヒト肝臓のmRNAから造ら
れたcDNAライブラリーをスクリーニングし、ヒトH
GFのアミノ酸配列をコードするクローンHAC19と
HBC25を得た。HAC19からのDNAをBamH
IとScaIで、HBC25からのDNAをScaIと
PstIで消化し、それぞれ得られた2つのDNAフラ
グメントをブルースクリプトKSIIのBamHIとP
stI部位に連結して挿入し、pBS[hHGFII]
を得た。pBS[hHGFII]をXbaIとSalI
とNaeIで消化し、更にT4DNAポリメラーゼで平
滑末端とした後、ヒトHGFをコードする約3KbのD
NAフラグメントをウシパピローマウィルスNDAをベ
クターとする発現ベクターpBPMTのEcoRV部位
に挿入し、pBPMT[hHGFII]を得た。得られ
たHGF発現ベクターpBPMT[hHGFII]を用
いて、DEAEデキストラン法によりCHO細胞を形質
転換した。形質転換体の選択は、G418を含む培地で
増殖させることにより行った。得られた形質転換体の中
から、高いHGF産生能を示す細胞株BPH89を選び
だした。BPH89細胞を牛胎児血清を加えた培地で増
殖させた後、培地を2日おきに交換して、HGFを実施
例1の精製法に準じた方法により精製した。
Example 2Production of HGF by gene recombination method  HGF derived from human cells is produced by the gene recombination method.
It was Wigler's method (Cell,11, 223, 1977).
According to the method described above, encoding the amino acid sequence of human HGF.
Chinese hamster transformed by the gene
Ovarian (CHO) cells were cultured, and from the culture supernatant, human
HGF was obtained. That is, it is produced from human liver mRNA.
Screened cDNA library for human H
Clone HAC19 encoding the amino acid sequence of GF and
HBC25 was obtained. DNA from HAC19 is BamH
I and ScaI, the DNA from HBC25 was called ScaI.
Two DNA fragments each obtained by digestion with PstI
Bment of Blue Script KSII and P
pBS [hHGFII] inserted by ligating to the stI site
Got pBS [hHGFII] with XbaI and SalI
Digested with NaeI, and then TFourFlat with DNA polymerase
Approximately 3 Kb of D encoding human HGF after making a smooth end
NA fragment was analyzed using bovine papillomavirus NDA.
EcoRV site of the expression vector pBPMT
To obtain pBPMT [hHGFII]. Obtained
HGF expression vector pBPMT [hHGFII]
The CHO cells by the DEAE dextran method.
Converted. Selection of transformants was performed using a medium containing G418.
This was done by growing. Among the obtained transformants
From, select the cell line BPH89 that shows high HGF production
I did. BPH89 cells were expanded in medium containing fetal bovine serum
After culturing, change the medium every 2 days and perform HGF
It was purified by a method similar to the purification method in Example 1.

【0020】実施例3ヒト肝ガン細胞の増殖に対する効果 本発明の抗ガン剤の有効成分であるHGFの、ヒト肝ガ
ン細胞に対する増殖抑制作用を以下の方法により確認し
た。DMEM(高グルコースタイプ)培地に10%ウシ胎
児血清を加えた培地を用いて対数増殖期にあるヒト肝細
胞ガン由来HepG2細胞を4×104個/mlになるよう
に懸濁し、直径35mmプラスチックプレートに2ml/プレー
ト蒔いた。各プレートにHGF(実施例2より得た、以
下同じ)を0〜15ng/mlの範囲で段階的に加え、5%CO2
95%空気の条件下37℃で培養した。4日間培養後、トリプ
シン/EDTA液にて細胞を分散し、最終細胞数をヘモ
サイトメーターにてカウントした。その結果を図1
(a)に示す。図1(a)に示されるようにHepG2
細胞はHGFにより0〜5ng/mlの範囲で用量依存的に増
殖を抑制され、15ng/mlにおいて無添加の場合の約1/8に
まで抑制されることが確認された。
Example 3 Effect on Human Hepatoma Cell Proliferation The inhibitory effect on proliferation of human hepatoma cells by HGF, which is an active ingredient of the anticancer agent of the present invention, was confirmed by the following method. Human hepatocellular carcinoma-derived HepG2 cells in the logarithmic growth phase were suspended in DMEM (high glucose type) medium supplemented with 10% fetal bovine serum to a volume of 4 × 10 4 cells / ml, and a diameter of 35 mm plastic Plated 2 ml / plate. HGF (obtained from Example 2, the same applies hereinafter) was added to each plate stepwise in the range of 0 to 15 ng / ml, and 5% CO 2 ,
The cells were cultured at 37 ° C under the condition of 95% air. After culturing for 4 days, the cells were dispersed with a trypsin / EDTA solution, and the final cell number was counted with a hemocytometer. The result is shown in Figure 1.
It shows in (a). HepG2 as shown in FIG.
It was confirmed that HGF suppressed the growth in a dose-dependent manner in the range of 0 to 5 ng / ml by HGF, and suppressed it to about 1/8 of that in the case of no addition at 15 ng / ml.

【0021】実施例4肝ガン細胞の複製DNA合成に対する効果 実施例3に記載された培地を用いて対数増殖期にあるヒ
ト肝細胞ガン由来HepG2細胞を4×104個/mlにな
るように懸濁し、24穴プラスチックプレートに0.5ml/ウ
ェル蒔いた。各プレートにHGFを0〜15ng/mlの範囲で
段階的に加え、5%CO2、95%空気の条件下37℃で培養し
た。4日間培養後、0.3μCiの[125I]デオキシウリ
ジンを各ウェルに添加した。更に4時間培養して細胞に
125I]を取り込ませた後、細胞をpH7.4のPBS(リ
ン酸食塩緩衝液)にて洗浄し、冷10%トリクロロ酢酸水
溶液で固定した。細胞を1N水酸化ナトリウム水溶液で
1時間インキュベートして可溶化し、その放射能をガン
マーカウンターにより測定した。 その結果を図1
(b)に示す。図1(b)に示されるようにHepG2
細胞はHGFにより0〜5ng/mlの範囲で用量依存的に複
製DNA合成を抑制され、15ng/mlにおいて無添加の場
合の1/3以下にまで抑制されることが確認された。
Example 4 Effect on replication DNA synthesis of hepatoma cells Using the medium described in Example 3, HepG2 cells derived from human hepatoma in the logarithmic growth phase were adjusted to 4 × 10 4 cells / ml. The cells were suspended and 0.5 ml / well was plated on a 24-well plastic plate. HGF was added to each plate stepwise in the range of 0 to 15 ng / ml, and the plate was incubated at 37 ° C. under the conditions of 5% CO 2 and 95% air. After culturing for 4 days, 0.3 μCi of [ 125 I] deoxyuridine was added to each well. After further culturing for 4 hours to incorporate [ 125 I] into the cells, the cells were washed with PBS (phosphate saline buffer) having pH 7.4 and fixed with cold 10% trichloroacetic acid aqueous solution. The cells were solubilized by incubating with a 1N sodium hydroxide aqueous solution for 1 hour, and the radioactivity thereof was measured by a gamma counter. The result is shown in Figure 1.
It shows in (b). HepG2 as shown in FIG.
It was confirmed that HGF suppressed replicative DNA synthesis in a dose-dependent manner in the range of 0 to 5 ng / ml, and at 15 ng / ml, it was suppressed to 1/3 or less of that in the case of no addition.

【0022】実施例5白血病細胞の増殖に対する効果 本発明の抗ガン剤の有効成分であるHGFの、白血病細
胞に対する増殖抑制作用を以下の方法により確認した。
RPMI1640培地に10%ウシ胎児血清を加えた培地
を用いて対数増殖期にあるリンパ芽球ガンIM9細胞を
4×104個/mlになるように懸濁し、直径35mmプラスチ
ックプレートに2ml/プレート蒔いた。各プレートにHG
Fを0〜30ng/mlの範囲で段階的に加え、5%CO2、95%
空気の条件下37℃で培養した。4日間培養後、トリプシ
ン/EDTA液にて細胞を分散し、最終細胞数をヘモサ
イトメーターにてカウントした。その結果を図2(a)
に示す。図2(a)に示されるようにIM9細胞はHG
Fにより0〜5ng/mlの範囲で用量依存的に増殖を抑制さ
れ、5ng/mlにおいて無添加の場合の約1/6にまで抑制さ
れることが確認された。
Example 5 Effect on proliferation of leukemia cells The growth inhibitory action of HGF, which is an active ingredient of the anticancer agent of the present invention, on leukemia cells was confirmed by the following method.
IM9 cells in lymphoblastic cancer in the logarithmic growth phase were suspended at 4 × 10 4 cells / ml in RPMI1640 medium supplemented with 10% fetal bovine serum, and 2 ml / plate was plated on a 35 mm diameter plastic plate. It was HG on each plate
F is added stepwise in the range of 0 to 30 ng / ml, 5% CO 2 , 95%
The cells were cultured at 37 ° C under the condition of air. After culturing for 4 days, the cells were dispersed with a trypsin / EDTA solution, and the final cell number was counted with a hemocytometer. The results are shown in Fig. 2 (a).
Shown in. As shown in FIG. 2 (a), IM9 cells were HG
It was confirmed that F suppressed the growth in a dose-dependent manner in the range of 0 to 5 ng / ml, and at 5 ng / ml, it was suppressed to about 1/6 of that in the case of no addition.

【0023】実施例6白血病細胞の複製DNA合成に対する効果 実施例5に記載された培地を用いて対数増殖期にあるリ
ンパ芽球ガンIM9細胞を4×104個/mlになるように
懸濁し、24穴プラスチックプレートに0.5ml/ウェル蒔い
た。各プレートにHGFを0〜30ng/mlの範囲で段階的
に加え、5%CO2、95%空気の条件下37℃で培養した。4
日間培養後、0.3μCiの[125I]デオキシウリジンを
各ウェルに添加した。更に、4時間培養して、細胞に[
125I]を取り込ませた後、細胞をpH7.4のPBS(リン
酸食塩緩衝液)にて洗浄し、冷10%トリクロロ酢酸水溶
液で固定した。細胞を1N水酸化ナトリウム水溶液で1
時間インキュベートして可溶化し、その放射能をガンマ
ーカウンターにより測定した。その結果を図2(b)に
示す。図2(b)に示されるようにIM9細胞はHGF
により0〜5ng/mlの範囲で用量依存的に複製DNA合成
を抑制され、5ng/mlにおいて無添加の場合の約1/6にま
で抑制されることが確認された。
Example 6 Effect of leukemic cells on replicative DNA synthesis Using the medium described in Example 5, lymphoblastic cancer IM9 cells in the logarithmic growth phase were suspended at 4 × 10 4 cells / ml. 0.5 ml / well was plated on a 24-well plastic plate. HGF was added stepwise to each plate in the range of 0 to 30 ng / ml, and the plate was incubated at 37 ° C. under the conditions of 5% CO 2 and 95% air. Four
After culturing for one day, 0.3 μCi of [ 125 I] deoxyuridine was added to each well. In addition, after culturing for 4 hours,
After taking up [ 125 I], the cells were washed with PBS (phosphate buffered saline) of pH 7.4 and fixed with cold 10% trichloroacetic acid aqueous solution. 1 cell with 1N sodium hydroxide solution
It was solubilized by incubation for a period of time, and its radioactivity was measured by a gamma counter. The result is shown in FIG. As shown in FIG. 2 (b), IM9 cells showed HGF
It was confirmed that replication-induced DNA synthesis was suppressed in the range of 0 to 5 ng / ml in a dose-dependent manner, and that it was suppressed to about 1/6 at 5 ng / ml as compared with the case of no addition.

【0024】実施例7表皮ガン細胞の増殖に対する効果 本発明の抗ガン剤の有効成分であるHGFの、表皮ガン
細胞に対する増殖抑制作用を以下の方法により確認し
た。DMEM(高グルコースタイプ)培地に10%ウシ胎
児血清を加えた培地を用いて対数増殖期にある表皮ガン
由来B6/F1メラノーマ細胞を4×104個/mlになる
ように懸濁し、直径35mmプラスチックプレートに2ml/プ
レート蒔いた。各プレートにHGFを0〜30ng/mlの範
囲で段階的に加え、5%CO2、95%空気の条件下37℃で培
養した。4日間培養後、トリプシン/EDTA液にて細
胞を分散し、最終細胞数をヘモサイトメーターにてカウ
ントした。その結果を図3(a)に示す。図3(a)に
示されるようにB6/F1細胞はHGFにより0〜2ng/m
lの範囲で用量依存的に増殖を抑制され、30ng/mlにおい
て無添加の場合の約1/2に抑制されることが確認され
た。
Example 7 Effect on proliferation of epidermal cancer cells The inhibitory effect on proliferation of epidermal cancer cells of HGF, which is an active ingredient of the anticancer agent of the present invention, was confirmed by the following method. B6 / F1 melanoma cells derived from epidermal cancer in the logarithmic growth phase were suspended in DMEM (high glucose type) medium containing 10% fetal bovine serum so as to have a density of 4 × 10 4 cells / ml and a diameter of 35 mm. Plated 2 ml / plate on a plastic plate. HGF was added stepwise to each plate in the range of 0 to 30 ng / ml, and the plate was incubated at 37 ° C. under the conditions of 5% CO 2 and 95% air. After culturing for 4 days, the cells were dispersed with a trypsin / EDTA solution, and the final cell number was counted with a hemocytometer. The result is shown in FIG. As shown in FIG. 3 (a), B6 / F1 cells were exposed to HGF at 0 to 2 ng / m 2.
It was confirmed that the growth was suppressed in a dose-dependent manner within the range of 1 and was suppressed to about 1/2 of that in the case of no addition at 30 ng / ml.

【0025】実施例8表皮ガン細胞の複製DNA合成に対する効果 実施例7に記載された培地を用いて対数増殖期にある表
皮ガン由来B6/F1メラノーマ細胞を4×104個/ml
になるように懸濁し、24穴プラスチックプレートに0.5m
l/ウェル蒔いた。各プレートにHGFを0〜30ng/mlの
範囲で段階的に加え、5%CO2、95%空気の条件下37℃で
培養した。4日間培養後、0.3μCiの[125I]デオキ
シウリジンを各ウェルに添加した。更に、4時間培養し
て、細胞に[125I]を取り込ませた後、細胞をpH7.4の
PBS(リン酸食塩緩衝液)にて洗浄し、冷10%トリク
ロロ酢酸水溶液で固定した。細胞を1N水酸化ナトリウ
ム水溶液で1時間インキュベートして可溶化し、その放
射能をガンマーカウンターにより測定した。その結果を
図3(b)に示す。図3(b)に示されるようにB6/
F1細胞はHGFにより0〜10ng/mlの範囲で用量依存的
に複製DNA合成を抑制され、30ng/mlにおいて無添加
の場合の1/2以下に抑制されることが確認された。
Example 8 Effect on epithelial cancer cell replication DNA synthesis Using the medium described in Example 7, 4 × 10 4 epidermal cancer-derived B6 / F1 melanoma cells in logarithmic growth phase / ml were used.
0.5 m on a 24-well plastic plate
l / well plated. HGF was added stepwise to each plate in the range of 0 to 30 ng / ml, and the plate was incubated at 37 ° C. under the conditions of 5% CO 2 and 95% air. After culturing for 4 days, 0.3 μCi of [ 125 I] deoxyuridine was added to each well. After further culturing for 4 hours to allow [ 125 I] to be taken up by the cells, the cells were washed with PBS (phosphate saline buffer) of pH 7.4 and fixed with cold 10% trichloroacetic acid aqueous solution. The cells were solubilized by incubating with a 1N sodium hydroxide aqueous solution for 1 hour, and the radioactivity thereof was measured by a gamma counter. The result is shown in FIG. As shown in FIG. 3B, B6 /
It was confirmed that F1 cells inhibited replication DNA synthesis by HGF in a dose-dependent manner within the range of 0 to 10 ng / ml, and at 30 ng / ml, it was suppressed to 1/2 or less of that in the case of no addition.

【0026】実施例9ガン細胞に対する殺細胞効果 本発明の抗ガン剤の有効成分であるHGFの、各種ガン
細胞に対する殺細胞作用を以下の方法により確認した。
DMEM(高グルコースタイプ)培地に10%ウシ胎児血
清を加えた培地を用いて対数増殖期にあるマウス表皮ガ
ン由来B6/F1メラノーマ細胞、ヒト表皮ガン由来K
B細胞及びヒト肝ガン由来HepG2細胞をそれぞれ個
別に懸濁し、24穴プラスチックプレートにそれぞれ5×
104個/ウェルになるように蒔いた。培地を同組成の新
しい培地150μlと置き換え、1Ci/mlのNa51CrO
2生理食塩水溶液(アマシャム社製)10μlを加え、5%C
2、95%空気の条件下37℃で培養して、51Crを取り込
ませた。2.5時間後、各ウェルの細胞を同組成の培地500
μlで3回ずつ洗浄し、各ウェルにHGFを0〜100ng/m
lの範囲で段階的に加えた培地0.5mlを加えて懸濁し、5%
CO2、95%空気の条件下37℃で培養した。24時間培養
後、培養液を氷上に置き、等量に二分して半量はそのま
まガンマーカウンターにて遊離51Cr量を放射能量とし
て測定し(a:単位cpm)、残りの半量は2%SDS500
μlを加えて37℃、1時間静置して細胞を溶解し、細胞
内残存51Cr量を同じくガンマーカウンターにより放射
能量として測定した(b:単位cpm)。細胞死滅量は
下記の式より算出した。 但し、a’:HGF無添加群の遊離51Cr量(単位cp
m) b’:HGF無添加群の残存51Cr量(単位cpm) その結果を図4に示す。同図中、○はマウス表皮ガン由
来B6/F1メラノーマ細胞、△はヒト表皮ガン由来K
B細胞、◆はヒト肝細胞ガン由来HepG2細胞を示
す。図4に示されるようにB6/F1メラノーマ細胞及
びKB細胞はHGFにより5〜100ng/mlの範囲で用量依
存的に増殖を抑制され、100ng/mlにおいて3〜5%の細胞
が死滅していることが確認された。また、本実施例にお
いて、HepG2細胞は全く死滅していないことより、
本発明の抗ガン剤の有効成分であるHGFはガン細胞の
中でもある種の細胞に対してのみ殺細胞活性を有するこ
とが明らかとなった。
Example 9 Cell-killing effect on cancer cells The cell-killing effect of HGF, which is an active ingredient of the anti-cancer agent of the present invention, on various cancer cells was confirmed by the following method.
Mouse epidermal cancer-derived B6 / F1 melanoma cells and human epidermal cancer-derived K in a logarithmic growth phase using a medium containing 10% fetal bovine serum in DMEM (high glucose type) medium
B cells and HepG2 cells derived from human liver cancer were individually suspended, and 5 x each was placed on a 24-well plastic plate.
It was sowed as 10 4 cells / well. The medium was replaced with 150 μl of a new medium having the same composition, and 1 Ci / ml Na 51 CrO was added.
2 Add 10 μl of physiological saline solution (Amersham) and add 5% C
It was cultured at 37 ° C. under the condition of O 2 and 95% air to incorporate 51 Cr. 2.5 hours later, the cells in each well were replaced with 500
Wash 3 times with μl each, and add 0-100 ng / m of HGF to each well.
Add 0.5 ml of medium added stepwise in the range of 1 l to suspend and add 5%
Culturing was carried out at 37 ° C. under the condition of CO 2 and 95% air. After culturing for 24 hours, the culture solution was placed on ice and divided into two equal amounts, half of which was directly measured by a gamma counter as the amount of free 51 Cr (a: unit cpm), and the other half was 2% SDS500.
The cells were lysed by adding μl and leaving still at 37 ° C. for 1 hour, and the amount of residual 51 Cr in the cells was measured as the amount of radioactivity by the same gamma counter (b: unit cpm). The cell killing amount was calculated by the following formula. However, a ': the amount of free 51 Cr in the HGF-free group (unit cp
m) b ′: residual 51 Cr amount in HGF-free group (unit: cpm) The results are shown in FIG. In the figure, ○ indicates B6 / F1 melanoma cells derived from mouse epidermal cancer, and Δ indicates K derived from human epidermal cancer.
B cells, ♦ indicates HepG2 cells derived from human hepatocellular carcinoma. As shown in Fig. 4, B6 / F1 melanoma cells and KB cells were growth-inhibited by HGF in a range of 5 to 100 ng / ml in a dose-dependent manner, and 3 to 5% of cells were killed at 100 ng / ml. It was confirmed. Moreover, in this Example, since the HepG2 cells were not killed at all,
It was revealed that HGF, which is an active ingredient of the anti-cancer agent of the present invention, has a cytocidal activity only against some kinds of cancer cells.

【0027】実験例1正常腎細胞の増殖に対する効果 本発明の抗ガン剤の有効成分であるHGFの、正常腎細
胞に対する増殖抑制作用を以下の方法により確認した。
DMEM(高グルコースタイプ)培地に10%ウシ胎児血
清を加えた培地を用いて対数増殖期にあるラット正常腎
尿細管細胞由来MDCK細胞を4×104個/mlになるよ
うに懸濁し、直径35mmプラスチックプレートに2ml/プレ
ート蒔いた。各プレートにHGFを0〜30ng/mlの範囲
で段階的に加え、5%CO2、95%空気の条件下37℃で培養
した。4日間培養後、トリプシン/EDTA液にて細胞
を分散し、最終細胞数をヘモサイトメーターにてカウン
トした。その結果を図5(a)に示す。図5(a)に示
されるようにMDCK細胞の増殖はHGFにより0〜30n
g/mlの範囲で影響を受けないことが確認された。
Experimental Example 1 Effect on proliferation of normal kidney cells The growth inhibitory action of HGF, which is an active ingredient of the anticancer agent of the present invention, on normal kidney cells was confirmed by the following method.
Rat normal renal tubular cell-derived MDCK cells in the logarithmic growth phase were suspended in DMEM (high glucose type) medium supplemented with 10% fetal bovine serum to a concentration of 4 × 10 4 cells / ml, and the diameter was suspended. 2 ml / plate was plated on a 35 mm plastic plate. HGF was added stepwise to each plate in the range of 0 to 30 ng / ml, and the plate was incubated at 37 ° C. under the conditions of 5% CO 2 and 95% air. After culturing for 4 days, the cells were dispersed with a trypsin / EDTA solution, and the final cell number was counted with a hemocytometer. The result is shown in FIG. As shown in FIG. 5 (a), the proliferation of MDCK cells was 0 to 30 n depending on HGF.
It was confirmed that it was not affected in the g / ml range.

【0028】実験例2正常腎細胞の複製DNA合成に対する効果 実験例1に記載された培地を用いて対数増殖期にあるラ
ット正常腎尿細管細胞由来MDCK細胞を4×104個/
mlになるように懸濁し、24穴プラスチックプレートに0.
5ml/ウェル蒔いた。各プレートにHGFを0〜30ng/ml
の範囲で段階的に加え、5%CO2、95%空気の条件下37℃
で培養した。4日間培養後、0.3μCiの[125I]デオ
キシウリジンを各ウェルに添加した。更に、4時間培養
して、細胞に[125I]を取り込ませた後、細胞をpH7.4
のPBS(リン酸食塩緩衝液)にて洗浄し、冷10%トリ
クロロ酢酸水溶液で固定した。細胞を1N水酸化ナトリ
ウム水溶液で1時間インキュベートして可溶化し、その
放射能をガンマーカウンターにより測定した。その結果
を図5(b)に示す。図5(b)に示されるようにMD
CK細胞の複製DNA合成はHGFにより0〜30ng/mlの
範囲で影響を受けないことが確認された。
Experimental Example 2 Effect on Replicative DNA Synthesis of Normal Kidney Cells Using the medium described in Experimental Example 1, 4 × 10 4 / MDCK cells derived from rat normal renal tubular cells in the logarithmic growth phase were used.
Resuspend to bring the volume to 0.1 ml and add 0 to a 24-well plastic plate.
5 ml / well was plated. 0-30ng / ml HGF on each plate
Stepwise in the range of 5% CO 2 and 95% air at 37 ° C
It was cultured in. After culturing for 4 days, 0.3 μCi of [ 125 I] deoxyuridine was added to each well. After further culturing for 4 hours to allow the cells to take up [ 125 I], the cells were adjusted to pH7.4.
It was washed with PBS (phosphate buffer solution) and fixed with cold 10% trichloroacetic acid aqueous solution. The cells were solubilized by incubating with a 1N sodium hydroxide aqueous solution for 1 hour, and the radioactivity thereof was measured by a gamma counter. The result is shown in FIG. MD as shown in FIG.
It was confirmed that the replication DNA synthesis of CK cells was not affected by HGF in the range of 0 to 30 ng / ml.

【0029】実験例3正常表皮細胞の増殖に対する効果 本発明の抗ガン剤の有効成分であるHGFの、正常表皮
細胞に対する増殖抑制作用を以下の方法により確認し
た。DMEM(高グルコースタイプ)培地に10%ウシ胎児
血清を加えた培地を用いて対数増殖期にあるマウス正常
ケラチノサイト由来PAM212細胞を4×104個/ml
になるように懸濁し、直径35mmプラスチックプレートに
2ml/プレート蒔いた。各プレートにHGFを0〜15ng/m
lの範囲で段階的に加え、5%CO2、95%空気の条件下37
℃で培養した。4日間培養後、トリプシン/EDTA液
にて細胞を分散し、最終細胞数をヘモサイトメーターに
てカウントした。その結果を図6(a)に示す。図6
(a)に示されるようにPAM212細胞はHGFによ
り0〜5ng/mlの範囲で増殖を促進され、それ以上の濃度
においては促進も抑制も受けないことが確認された。
Experimental Example 3 Effect on proliferation of normal epidermal cells The growth inhibitory action of HGF, which is an active ingredient of the anticancer agent of the present invention, on normal epidermal cells was confirmed by the following method. 4 × 10 4 cells / ml of normal mouse keratinocyte-derived PAM212 cells in the logarithmic growth phase using a medium containing 10% fetal bovine serum in DMEM (high glucose type) medium
Suspend to a 35 mm diameter plastic plate
2 ml / plate was plated. 0-15ng / m of HGF on each plate
Add gradually in the range of l and under the condition of 5% CO 2 and 95% air 37
Cultured at ° C. After culturing for 4 days, the cells were dispersed with a trypsin / EDTA solution, and the final cell number was counted with a hemocytometer. The result is shown in FIG. Figure 6
As shown in (a), it was confirmed that PAM212 cells were stimulated to proliferate by HGF in the range of 0 to 5 ng / ml, and were not stimulated or suppressed at higher concentrations.

【0030】実験例4正常表皮細胞の複製DNA合成に対する効果 実験例3に記載された培地を用いて対数増殖期にあるマ
ウス正常ケラチノサイト由来PAM212細胞を4×1
4個/mlになるように懸濁し、24穴プラスチックプレー
トに0.5ml/ウェル蒔いた。各プレートにHGFを0〜30
ng/mlの範囲で段階的に加え、5%CO2、95%空気の条件
下37℃で培養した。4日間培養後、0.3μCiの
125I]デオキシウリジンを各ウェルに添加した。更
に、4時間培養して、細胞に[125I]を取り込ませた
後、細胞をpH7.4のPBS(リン酸食塩緩衝液)にて洗
浄し、冷10%トリクロロ酢酸水溶液で固定した。細胞を
1N水酸化ナトリウム水溶液で1時間インキュベートし
て可溶化し、その放射能をガンマーカウンターにより測
定した。その結果を図6(b)に示す。図6(b)に示
されるようにPAM212細胞はHGFにより0〜5ng/m
lの範囲で複製DNA合成を促進され、それ以上の濃度
においては促進も抑制も影響を受けないことが確認され
た。
Experimental Example 4 Effect on replication DNA synthesis of normal epidermal cells Using the medium described in Experimental Example 3, 4 × 1 of normal mouse keratinocyte-derived PAM212 cells in the logarithmic growth phase were used.
The cells were suspended at a concentration of 0 4 cells / ml, and 0.5 ml / well was plated on a 24-well plastic plate. 0-30 HGF on each plate
It was added stepwise in the range of ng / ml, and cultured at 37 ° C. under the conditions of 5% CO 2 and 95% air. After culturing for 4 days, 0.3 μCi of [ 125 I] deoxyuridine was added to each well. After further culturing for 4 hours to allow [ 125 I] to be taken up by the cells, the cells were washed with PBS (phosphate saline buffer) of pH 7.4 and fixed with cold 10% trichloroacetic acid aqueous solution. The cells were solubilized by incubating with a 1N sodium hydroxide aqueous solution for 1 hour, and the radioactivity thereof was measured by a gamma counter. The result is shown in FIG. As shown in FIG. 6 (b), PAM212 cells were treated with HGF at 0-5 ng / m.
It was confirmed that replication DNA synthesis was promoted in the range of l, and that at higher concentrations, neither promotion nor suppression was affected.

【0031】製剤例1 生理食塩水100ml中にHGF100mg、マンニトール1g及
びポリソルベート80 10mgを含む溶液を無菌的に調製
し、バイアル瓶に1mlずつ無菌的に分注し、常法に準じ
て凍結乾燥し、凍結乾燥製剤を得た。
Formulation Example 1 A solution containing 100 mg of HGF, 1 g of mannitol and 10 mg of polysorbate 80 in 100 ml of physiological saline was aseptically prepared, and 1 ml of each was aseptically dispensed into a vial and freeze-dried according to a conventional method. A lyophilized preparation was obtained.

【0032】製剤例2 0.15M NaClと0.01%ポリソルベート80を含むpH7.4の
0.02Mリン酸緩衝液100mlにHGF100mgとヒト血清アル
ブミン100mgを添加した水溶液を無菌的に調製し、バイ
アル瓶に1mlずつ無菌的に分注し、常法に準じて凍結乾
燥し、凍結乾燥製剤を得た。
Formulation Example 2 0.15 M NaCl and 0.01% Polysorbate 80 at pH 7.4
Aseptically prepare an aqueous solution containing 100 mg of HGF and 100 mg of human serum albumin in 100 ml of 0.02 M phosphate buffer, aseptically dispense 1 ml each into a vial, and lyophilize according to a conventional method to give a lyophilized formulation. Obtained.

【図面の簡単な説明】[Brief description of drawings]

【図1】ヒト肝細胞ガン由来HepG2細胞に対するH
GFの増殖抑制作用を示す図である。同図(a)は各H
GF濃度における培養終了時の細胞数を示し、同図
(b)は各HGF濃度における複製DNA合成量を示す
(実施例3及び4)。
FIG. 1 H for HepG2 cells derived from human hepatocellular carcinoma
It is a figure which shows the growth inhibitory effect of GF. The same figure (a) shows each H
The number of cells at the end of the culture at the GF concentration is shown, and the figure (b) shows the amount of replicated DNA synthesized at each HGF concentration (Examples 3 and 4).

【図2】白血病細胞RPMI1640細胞に対するHG
Fの増殖抑制作用を示す図である。同図(a)は各HG
F濃度における培養終了時の細胞数を示し、同図(b)
は各HGF濃度における複製DNA合成量を示す(実施
例5及び6)。
[FIG. 2] HG for leukemia cells RPMI1640 cells
It is a figure which shows the growth inhibitory effect of F. The figure (a) shows each HG
The number of cells at the end of the culture at the F concentration is shown in FIG.
Shows the amount of replicative DNA synthesized at each HGF concentration (Examples 5 and 6).

【図3】マウス表皮ガン由来B6/F1メラノーマ細胞
に対するHGFの増殖抑制作用を示す図である。同図
(a)は各HGF濃度における培養終了時の細胞数を示
し、同図(b)は各HGF濃度における複製DNA合成
量を示す(実施例7及び8)。
FIG. 3 is a view showing the growth inhibitory effect of HGF on mouse epidermal cancer-derived B6 / F1 melanoma cells. The figure (a) shows the number of cells at the end of culture at each HGF concentration, and the figure (b) shows the amount of replicated DNA synthesized at each HGF concentration (Examples 7 and 8).

【図4】各種ガン細胞に対するHGFの殺細胞作用を示
す図であり、各HGF濃度における培養終了時の細胞死
滅量を示す。同図中、○はマウス表皮ガン由来B6/F
1メラノーマ細胞、△はヒト表皮ガン由来KB細胞、◆
はヒト肝細胞ガン由来HepG2細胞を示す(実施例
9)。
FIG. 4 is a graph showing the cell killing effect of HGF on various cancer cells, showing the cell killing amount at the end of culture at each HGF concentration. In the figure, ○ indicates B6 / F derived from mouse epidermal cancer.
1 melanoma cell, △ is human epidermal cancer-derived KB cell, ◆
Shows HepG2 cells derived from human hepatocellular carcinoma (Example 9).

【図5】ラット正常腎細胞由来MDCK細胞に対するH
GFの増殖抑制作用を示す図である。同図(a)は各H
GF濃度における培養終了時の細胞数を示し、同図
(b)は各HGF濃度における複製DNA合成量を示す
(実験例1及び2)。
FIG. 5: H against MDCK cells derived from rat normal kidney cells
It is a figure which shows the growth inhibitory effect of GF. The same figure (a) shows each H
The number of cells at the end of the culture at the GF concentration is shown, and the figure (b) shows the amount of replicated DNA synthesized at each HGF concentration (Experimental Examples 1 and 2).

【図6】マウス正常ケラチノサイト由来PAM212細
胞に対するHGFの増殖抑制作用を示す図である。同図
(a)は各HGF濃度における培養終了時の細胞数を示
し、同図(b)は各HGF濃度における複製DNA合成
量を示す(実験例3及び4)。
FIG. 6 is a diagram showing the growth inhibitory effect of HGF on mouse normal keratinocyte-derived PAM212 cells. The figure (a) shows the number of cells at the end of culture at each HGF concentration, and the figure (b) shows the amount of replicated DNA synthesis at each HGF concentration (Experimental Examples 3 and 4).

【図7】ヒトHGF前駆体のアミノ酸配列を示す図であ
る。
FIG. 7 shows the amino acid sequence of human HGF precursor.

【図8】ヒトHGF前駆体のアミノ酸配列(図7)をコ
ードする遺伝子の塩基配列を示す図である。
FIG. 8 shows the nucleotide sequence of a gene encoding the amino acid sequence of human HGF precursor (FIG. 7).

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 FI 技術表示箇所 C12P 21/02 C 8214−4B (C12P 21/02 C12R 1:91) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location C12P 21/02 C 8214-4B (C12P 21/02 C12R 1:91)

Claims (4)

【特許請求の範囲】[Claims] 【請求項1】 肝実質細胞増殖因子(Hepatocyto Grow
th Factor)を有効成分として含有することを特徴とす
る抗ガン剤。
1. Hepatocyto Grow
th Factor) as an active ingredient.
【請求項2】 肝実質細胞増殖因子が、ヒト又は動物
の組織又は血液成分由来である請求項1記載の抗ガン
剤。
2. The anticancer agent according to claim 1, wherein the hepatocyte growth factor is derived from human or animal tissues or blood components.
【請求項3】 肝実質細胞増殖因子が遺伝子組換えに
より製造したものである請求項1記載の抗ガン剤。
3. The anticancer agent according to claim 1, wherein the hepatocyte growth factor is produced by gene recombination.
【請求項4】 遺伝子組換えの宿主細胞が大腸菌、枯
草菌、酵母、糸状菌、植物又は動物細胞のいずれかであ
る請求項3記載の抗ガン剤。
4. The anticancer agent according to claim 3, wherein the genetically modified host cell is any one of Escherichia coli, Bacillus subtilis, yeast, filamentous fungus, plant and animal cells.
JP3140812A 1991-05-15 1991-05-15 Anticancer agent Expired - Fee Related JP3030386B2 (en)

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US5654404A (en) * 1992-09-16 1997-08-05 Genentech, Inc. Protection against liver damage by HGF
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US5654404A (en) * 1992-09-16 1997-08-05 Genentech, Inc. Protection against liver damage by HGF
WO1995025537A1 (en) * 1994-03-18 1995-09-28 Sumitomo Pharmaceuticals Co., Ltd. Drug for relieving side effects caused by immunosuppressants
WO1996005855A1 (en) * 1994-08-19 1996-02-29 Sumitomo Pharmaceuticals Co., Ltd. Cartilage disease remedy
WO1997016205A1 (en) * 1995-10-24 1997-05-09 Toshikazu Nakamura Antitumor agent
US6855685B2 (en) 1995-10-24 2005-02-15 Toshikazu Nakamura Anti-cancer agent
US7507401B2 (en) 1995-10-24 2009-03-24 Toshikazu Nakamura Anti-cancer agent
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