JPH0625010A - Antitumor agent - Google Patents

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

Detailed Description of the Invention

[0001]

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]

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.

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.

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.

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] 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.

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]

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]

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.

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.

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.

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.

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.

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.

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.

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]

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]

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).

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 T_FourFlat 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[Brief description of drawings]

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).

[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).

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).

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).

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).

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).

FIG. 7 shows the amino acid sequence of human HGF precursor.

FIG. 8 shows the nucleotide sequence of a gene encoding the amino acid sequence of human HGF precursor (FIG. 7).

─────────────────────────────────────────────────── ─── 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)

[Claims] 1. Hepatocyto Grow
th Factor) as an active ingredient. 2. The anticancer agent according to claim 1, wherein the hepatocyte growth factor is derived from human or animal tissues or blood components. 3. The anticancer agent according to claim 1, wherein the hepatocyte growth factor is produced by gene recombination. 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.