WO2011013358A1

WO2011013358A1 - Anti-tumor agent

Info

Publication number: WO2011013358A1
Application number: PCT/JP2010/004778
Authority: WO
Inventors: 渋谷正史; 土田里香; 大澤毅
Original assignee: 国立大学法人東京医科歯科大学
Priority date: 2009-07-29
Filing date: 2010-07-28
Publication date: 2011-02-03
Also published as: JP2012197230A

Abstract

Disclosed is a novel anti-tumor agent which is suitable for practical use and exhibits an anti-tumor activity in vivo against tumors. The anti-tumor agent contains bone morphogenetic protein BMP4 as an active ingredient. Examples of bone morphogenetic protein BMP4 include bone morphogenetic proteins having an amino acid sequence represented by SEQ ID NO: 1 or 3 shown in the Sequence Listing, or an amino acid sequence in which one or more amino acid residues has been deleted, added or substituted in the aforementioned amino acid sequences, which exhibits an anti-tumor activity. When the anti-tumor agent containing bone morphogenetic protein BMP4 as an active ingredient is administered to a cancer patient through intraperitoneal injection or the like, a tumor regression effect can be produced through the inhibition of angiogenesis or the like in vivo and, consequently an anti-tumor activity can be exerted.

Description

Antitumor agent

The present invention relates to an antitumor agent comprising bone morphogenetic protein BMP4 (Bone Morphogenetic Protein 4: BMP4) as an active ingredient, in particular, bone morphogenetic protein BMP4 as an active ingredient, and administered to a cancer patient by intraperitoneal injection, The present invention relates to an antitumor agent capable of obtaining a tumor regression effect by inhibiting angiogenesis.

Angiogenesis plays an important role in the development and wound healing, and it is becoming clear that it plays an important role in the growth and metastasis of various advanced solid cancers. That is, cancer cells promote angiogenesis by secreting angiogenic factors such as VEGF, bFGF, EGF, FGF, etc., and as a result, the resulting blood vessels supply oxygen, nutrients, etc. to the cancer cells. In addition to promoting the proliferation of the cancer cells, the cancer cells are also promoted to other sites (see Non-Patent Documents 1 and 2). Therefore, development of an antitumor agent comprising a drug having an anti-angiogenic effect as an active ingredient has been advanced. For example, an anti-VEGF-A neutralizing antibody that acts on VEGF-A to inhibit an angiogenic signal ( bevacizumab) as an active ingredient, and Sutent (registered trademark) as an active ingredient is an agent that inhibits angiogenic signals by exerting an inhibitory action on VEGFR tyrosine kinase (sunitinib) It has already been put into practical use as an agent.

Bone Morphogenetic Protein (BMP) is a member of the subclass of the Transforming Growth Factor-β (Transforming Growth Factor-β: TGF-β) superfamily and is involved in the growth and differentiation of many different tissues and organs (See Non-Patent Documents 3 to 5). In addition, some evidence suggests that the activity of BMP is related to developmentally regulated apoptosis (Non-Patent Documents 6 and 7). In particular, BMP4 (bone morphogenetic protein 4) is a planned neural crest cell (Non-patent Document 8), the dorsal side of a chick eyecup (Non-patent Document 9), and the development of birds. It is involved in apoptosis in the space between limbs (Non-Patent Documents 10 to 12).

The present inventors have previously shown that the retraction of the capillary membrane of the pupil membrane in postnatal rats is induced mainly by apoptosis dependent on BMP4 secreted from lens epithelial cells (Non-patent Document 13). ). Furthermore, the present inventor found that the degree of expression of apoptotic action depending on BMP4 differs depending on the type of vascular endothelial cell, and this difference is related to the expression level of Inhibitory Smad (I-Smad) 6 and 7. (Non-Patent Document 14). That is, vascular endothelial cells with high expression levels of I-Smad6 and I-Smad7 showed resistance to BMP4, whereas vascular endothelial cells with low expression levels of I-Smad6 and I-Smad7 showed resistance to BMP4. Have been found to be sensitive and cause BMP4-dependent apoptosis.

As a further finding regarding BMP4, there is a report suggesting an association between the TGF-β family containing BMP4 and lung cancer (Non-patent Document 15). According to this document, based on the previous report that disruption or reduction of TGF-β family signals may be involved in the development of lung cancer, treatment with addition of BMP4 to the in vitro culture system of lung cancer cell lines As a result, the growth rate of lung cancer cell lines in this in vitro culture system was reduced (or cell death was induced). This document also describes that when a lung cancer cell line subjected to BMP4 addition treatment in an in vitro culture system is transplanted into nude mice, the growth rate of the lung cancer cell line after transplantation is reduced. However, the result of this transplantation experiment is a result of transplanting a lung cancer cell line previously treated with BMP4 in an invitro culture system and not a result of administering BMP4 in vivo. That is, it was still unclear to those skilled in the art what kind of effect BMP4 shows or does not show when administered to an individual suffering from lung cancer.

Further, as another finding regarding BMP4, it has been reported that BMP4 produced by melanoma promotes the proliferation of melanoma in in vitro and in vivo (Non-patent Document 16). According to this document, when a BMP4 antisense vector was introduced into melanoma cells to express BMP4 antisense and BMP4 expression in melanoma cells was suppressed, the size of the tumor was not changed compared to the control. However, it is described that angiogenesis of the tumor tissue was reduced and necrosis of melanoma cells was observed.

An object of the present invention is to provide a novel and practical antitumor agent that exhibits antitumor action in vivo against tumors.

In the course of studying the relationship between the TGF-β family of vascular endothelial cells containing BMP4 and the apoptotic effect, the present inventors transferred bone morphogenetic protein BMP4 (Bone Morphogenetic Protein 4: BMP4) to mice transplanted with mouse melanoma cells ) Was found to suppress the proliferation of melanoma cells in vivo, and the present invention was completed. That is, in the present invention, an antitumor agent comprising bone morphogenetic protein BMP4 as an active ingredient is administered to a cancer patient by intraperitoneal injection, thereby exhibiting a tumor regression effect by inhibiting angiogenesis in vivo. It is possible to provide an antitumor agent capable of

In the present invention, the bone morphogenetic protein BMP4, which is an active ingredient of the antitumor agent, has the amino acid sequence represented by SEQ ID NO: 1 or 3 in the sequence listing, or one or several amino acids in the amino acid sequence deleted, added, or Mention may be made of proteins comprising a substituted sequence and an amino acid sequence having antitumor activity. The administration method of the antitumor agent of the present invention is not particularly limited, but can be administered to cancer patients by intraperitoneal injection or the like, and can exhibit an antitumor effect by inhibiting angiogenesis in vivo. it can.

That is, the present invention relates to (1) an antitumor agent comprising bone morphogenetic protein BMP4 as an active ingredient, and (2) an amino acid sequence represented by SEQ ID NO: 1 or 3 in the sequence listing, or the amino acid sequence. The antitumor agent according to the above (1), comprising a sequence in which one or several amino acids are deleted, added, or substituted, and having an antitumor activity; The antitumor agent according to (1) or (2) above, wherein the antitumor agent comprising the bone morphogenetic protein BMP4 as an active ingredient can be administered to cancer patients by intraperitoneal injection. And (4) The antitumor agent according to any one of (1) to (3) above, wherein the antitumor agent comprising bone morphogenetic protein BMP4 as an active ingredient has angiogenesis inhibitory activity.

According to the present invention, it is possible to provide a novel and practical antitumor agent that effectively suppresses the growth of a tumor (particularly solid cancer) in vivo and exhibits an antitumor action.

It is a figure which shows the volume of the melanoma tumor in the mouse | mouth which transplanted the mouse melanoma cell over time. Control represents a control group, and Bm-prot. Represents a BMP4 administration group.

It is the figure which performed CD31 dyeing | staining to the melanoma tumor tissue in the mouse | mouth which transplanted the mouse melanoma cell, and represented the grade of the dyeing | staining as a tumor blood vessel density. Control represents a control group, and BMP4 represents a BMP4 administration group.

A is a figure which shows the volume of the tumor in the mouse | mouth which transplanted the human cervical cancer cell (Hela cell) with time. The arrow on the horizontal axis indicates the administration time of mouse BMP4 (rmBMP4). B shows tumor tissue excised from mice euthanized with day20. In both figures, Control represents a control group, and BMP4 represents a BMP4 administration group.

The result of the anti-tumor effect confirmation assay in mouse | mouth using mouse melanoma cell (B16 cell) is shown. A is the result of a chronogenic assay (anchorage dependency), B is the result of a colony assay (anchorage independence), and in each case, Control indicates a control group and BMP4 indicates a BMP4 mixed group.

The result of the anti-tumor effect confirmation assay in vitro using a human cervical cancer cell (Hela cell) is shown. A is the result of a chronogenic assay (anchorage dependency), B is the result of a colony assay (anchorage independence), and in each case, Control indicates a control group and BMP4 indicates a BMP4 mixed group.

It is a figure which shows the volume of the melanoma tumor in the VEGF / Flt1 signaling knockout mouse (Flt1TK < ^-/- mouse | mouth) which transplanted the mouse | mouth B16 melanoma cell over time. TK cont represents a control group, and TK BMP represents a BMP4 administration group. The arrow on the horizontal axis indicates the administration time of mouse BMP4 (rmBMP4).

It is a figure which shows the volume of the tumor in the mouse | mouth which transplanted the mouse | mouth LLC lung cancer cell in time. Control represents a control group, and BMP4 represents a BMP4 administration group. Moreover, the arrow on the horizontal axis indicates the administration timing of mouse BMP4 (rmBMP4) and human BMP4 (rhBMP4).

As the bone morphogenetic protein BMP4 which is an active ingredient of the antitumor agent in the present invention, the amino acid sequence shown in SEQ ID NO: 1 or 3 in the sequence listing, or one or several amino acids in the amino acid sequence are deleted, added, or A protein (hereinafter simply referred to as “BMP4”) consisting of a substituted sequence and an amino acid sequence having antitumor activity can be mentioned. BMP4 has a polymorphism in nature, but is included in the scope of the present invention as long as it has antitumor activity.

That is, as said BMP4, BMP4 derived from a mammal can be illustrated suitably, As this mammal, a human, a mouse | mouth, a rat, a hamster, a guinea pig, a cow, a pig, a horse, a monkey, a rabbit, a sheep, Goats, cats, dogs and the like can be preferably exemplified, and among them, mammals (especially humans) of the same kind as the administration target of the antitumor agent can be more suitably exemplified. The amino acid sequence of mouse-derived BMP4 (hereinafter also simply referred to as “mouse BMP4”) is shown in SEQ ID NO: 1, the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 2, and human-derived BMP4 (hereinafter referred to as “BMP4”) The amino acid sequence of “human BMP4” is also shown in SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 4. As can be seen from the fact that the amino acid sequences of human BMP4 and mouse BMP4 match at 114 amino acids out of 116 amino acids (amino acid identity 98.3%), BMP4 is widely conserved in mammals. Therefore, appropriate primers are designed with reference to the amino acid sequences of human BMP4 and mouse BMP4 and their nucleotide sequences, and PCR is performed using genomic DNA of mammals other than humans and mice as a template. A BMP4 gene derived from a mammal can be isolated. Then, BMP4 derived from mammals other than humans and mice can be obtained by incorporating the isolated BMP4 gene into an appropriate expression vector and introducing the vector into an appropriate cell to express the BMP4 gene.

The BMP4 is not particularly limited as long as it has antitumor activity, but for example 80% or more, preferably 85% or more, more preferably 90% or more, relative to the amino acid sequence shown in SEQ ID NO: 1 or 3. A protein comprising an amino acid sequence having an identity of 95% or more, particularly preferably 98% or more is more preferable.

In addition to these proteins, the BMP4 comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 3, and A protein having antitumor activity; or encoded by DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 or 4, and has antitumor activity Protein;

The above “amino acid sequence in which one or more amino acids are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and still more preferably 1. Means an amino acid sequence in which any number of ˜5 amino acids have been deleted, substituted or added.

As the above stringent conditions, hybridization at 65 ° C. and washing treatment at 65 ° C. with a buffer containing 0.1 × SSC and 0.1% SDS can be preferably exemplified. The DNA that hybridizes under the above stringent conditions is more than a certain nucleotide sequence of DNA used as a probe (DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 or 4). For example, DNA having identity of 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 98% or more is suitable. Can be exemplified.

BMP4 can also be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. Specifically, the DNA comprising the nucleotide sequence shown in SEQ ID NO: 2 or 4 is contacted with a mutagen agent, irradiated with ultraviolet light, genetically engineered, etc. Mutant DNA can be obtained by introducing a mutation into. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular Cloning 2nd edition, Current Protocols in MolecularlogyBiology, Supplement 1 ～ 38, John Wiley and Sons (1987-1997). The BMP4 can be obtained by expressing this mutant DNA using an appropriate expression system.

The BMP4 according to the present invention may be modified with a modifying agent that enhances the stability of the protein in vivo. Examples of such modifiers include polyethylene glycol (PEG), dextran, poly (N-vinyl-pyrrolidone), polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, polyvinyl alcohol, and the like. Modification of protein with PEG can be performed, for example, by a method of reacting an active ester derivative of PEG with a protein or a method of reacting a terminal aldehyde derivative of PEG with a protein in the presence of a reducing agent.

Whether or not a protein has antitumor activity is determined when the protein is injected into a mammal having a tumor and the growth of the tumor is suppressed compared to when the protein is not injected. More specifically, when the assay of the same method as the assay described in Example 1 described later is performed, the protein is not administered by injection. It can be confirmed whether or not the growth of the tumor is suppressed. That is, when the protein is administered to a mammal having a tumor by injection, the protein is evaluated as having antitumor activity if the growth of the tumor is suppressed compared to when the protein is not administered by injection. If the growth of the tumor is not suppressed when the protein is administered to a mammal having a tumor by injection, compared to the case where the protein is not administered by injection, the protein has antitumor activity. It can be evaluated as not.

As a preferable antitumor effect of the antitumor agent of the present invention, in the assay similar to the assay in Example 1 described later, the average tumor volume of the protein-administered group at day 20 is compared to the average tumor volume of the control group at day 20 The ratio (%) is preferably 80% or less, more preferably 65% or less, further preferably 50% or less, particularly preferably 45% or less, or the same as the assay in Example 2 described later. The ratio (%) of the average tumor volume of the protein-administered group in day 18 to the average tumor volume of the control group in day 18 is preferably 80% or less, more preferably 65% or less, and still more preferably 50 % Or less, particularly preferably 45% or less.

The “tumor” in the present specification is not particularly limited as long as it is a tumor, but a malignant tumor (cancer) can be preferably exemplified, and among them, melanoma, stomach cancer, lung cancer, breast cancer, colon cancer, liver cancer, pancreatic cancer More preferably, solid cancers such as renal cancer, prostate cancer, endometrial cancer, cervical cancer, ovarian cancer, esophageal cancer, oral cancer, biliary tract cancer, gallbladder cancer, bile duct cancer, thyroid cancer, nervous system tumor, etc. Among them, melanoma, cervical cancer, and lung cancer can be exemplified more preferably, and melanoma can be particularly preferably exemplified. Although the details of the mechanism of action of the antitumor agent of the present invention are not clear, it is considered that tumor growth is suppressed by inhibiting tumor angiogenesis. It is thought to exert an antitumor effect against tumors including malignant tumors.

The antitumor agent of the present invention may contain optional components such as other antitumor agents in addition to BMP4 and the like as long as an antitumor effect is obtained. Since the antitumor agent of the present invention has a tumor suppressing effect independent of the VEGF / Flt1 signal system, a synergistic tumor suppressing effect can be expected when used together with an anti-VEGF inhibitor that is an angiogenesis inhibitor, for example.

BMP4 and the like contained in the antitumor agent of the present invention can be made into an appropriate preparation by a conventional method. The dosage form of the preparation may be a solid preparation such as a powder or a granule, but from the viewpoint of obtaining an excellent antitumor effect, it is preferably a liquid such as a solution, an emulsion or a suspension. As a method for producing the above liquid agent, for example, a method of mixing BMP4 or the like with a solvent, and a method of further mixing a suspending agent or an emulsifier can be preferably exemplified. As described above, when the BMP4 or the like in the present invention is used as a preparation, an appropriate pharmaceutically acceptable carrier, for example, an excipient, a binder, a solvent, a solubilizing agent, as necessary in the preparation. , Suspending agents, emulsifiers, isotonic agents, buffers, stabilizers, soothing agents, preservatives, antioxidants, coloring agents, lubricants, disintegrants, wetting agents, adsorbents, sweeteners, Optional components such as a diluent can be blended.

The administration method of the antitumor agent of the present invention is not particularly limited as long as the desired antitumor effect is obtained, and is intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, nasal, transnasal, Examples include pulmonary administration. However, the most preferable administration method of the antitumor agent of the present invention includes a method of administering it to a cancer patient by intraperitoneal injection or the like. In addition, the dose, frequency, and dose of the antitumor agent of the present invention (hereinafter also simply referred to as “dose of the antitumor agent of the present invention”) are the state of the tumor to be administered and the administration target. Although it can be adjusted as appropriate according to the body weight and the like, for example, the daily dose of BMP4 and the like per 1 g body weight of the administration subject is within the range of 1 ng to 1000 mg, preferably within the range of 10 ng to 500 mg. it can. In particular, when the dosage of the antitumor agent of the present invention is adjusted to an optimum range, only vascular endothelial cells in the tumor can be led to cell death. In addition, it is preferable to use the antitumor agent of this invention together with another antitumor agent from a viewpoint of obtaining the more excellent antitumor effect.

Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

[In vivo anti-tumor effect confirmation assay]
In order to confirm whether BMP4 in the present invention exerts an antitumor effect in vivo, the following assay was attempted.

(1) Production of mouse having melanoma Mouse B16 melanoma cells (hereinafter also simply referred to as “melanoma cells”) are cultured in a D-MEM medium containing 10% fetal bovine serum, and from the obtained melanoma cells, A total of 10 sets of 1 × 10 ⁷ pieces were collected. 100 μl of PBS (phosphate buffered saline) was added to each of the sorted melanoma cells and suspended to prepare 10 sets of melanoma cell suspensions. Meanwhile, 10 healthy 8-week-old female C57BL / 6J mice were purchased and prepared. One set of the aforementioned melanoma cell suspension per mouse was subcutaneously injected into the right dorsum of these mice, and the melanoma cells were transplanted into healthy mice. The day of transplantation was defined as day0. Hereinafter, these melanoma cell transplanted mice are simply referred to as “mouse”.

(2) Preparation of antitumor agent and control solution of the present invention Mouse BMP4 was purchased from R & D Systems, Cat. #: 5020-BP, Minneapolis, MN (USA). As an antitumor agent of the present invention, a solution was prepared by dissolving the above mouse BMP4 in a 4 mM hydrochloric acid aqueous solution containing 0.1% BSA (bovine serum albumin), and the concentration of mouse BMP4 was 10 μg / ml. Further, a 4 mM hydrochloric acid aqueous solution containing 0.1% BSA was prepared as a control solution.

(3) Administration of the antitumor agent of the present invention to mice and change in tumor volume The above-mentioned 10 mice were introduced on the second day (day 2) after transplantation of melanoma cells, which is considered to contain a tumor angiogenic switch. Among these mice, 200 μl of the above-described antitumor agent of the present invention was intraperitoneally administered to each of 5 mice (BMP4 administration group). When this dose of BMP4 is converted per mouse, it is 2 μg, and since the weight of the mouse is about 20 g, it is 100 ng / g when converted per 1 g of mouse body weight. On the other hand, the remaining 5 mice were used as controls, and 200 μl of the aforementioned control solution was intraperitoneally administered to each of these 5 mice (control group).

The tumor size of each mouse was measured every 3 days after the administration of the antitumor agent of the present invention and the control solution in day 2. Specifically, the measurement was performed according to the following procedure. First, 300 μl of sonopentyl (pentobarbital sodium) (64.8 mg / ml) diluted 20-fold with PBS was intraperitoneally administered to the mice, and the mice were completely anesthetized. Next, while taking care not to reduce the body temperature of the mouse too much, wipe the tumor part with alcohol cotton, and make it easy to measure the size of the tumor so that the entire tumor is sufficiently raised directly under the skin. After that, using a precision digital caliper, the maximum diameter of the tumor was measured and the volume was calculated and recorded.

The change over time of the calculated tumor volume is shown in FIG. 1, and the basic data is shown in Table 1.

In addition, as a tumor volume, each average value is used for each group. As can be seen from the results in FIG. 1, in day 5, no tumor increase was observed in either the BMP4 administration group or the control group, but in day8, there was a difference in tumor volume between the BMP4 administration group and the control group. From day 11 onward, it was recognized that the tumor volume in the BMP4 administration group was significantly suppressed relative to that in the control group after day 11. That is, it was shown that the antitumor agent of the present invention exerts a growth inhibitory effect on the tumor.

By

day

20, 2 mice died in the control group and 1 died in the BMP4 administration group. All remaining mice that survived on day 20 were euthanized and tumors were removed subcutaneously. In addition, other organs (brain, lung, liver, spleen) and blood were collected and observed mainly for differences between the two groups, particularly changes in tumor blood vessels. Macroscopic findings of each organ mentioned above showed no difference between the two groups, and no distant metastasis of tumor to each organ. On the other hand, according to the findings of the tumor tissue, in the tumor of the control group, the bleeding part was overwhelmingly large, whereas in the tumor of the BMP4 administration group, the bleeding was small and scattered necrosis was observed. From these results, it was considered that BMP4 suppresses tumor growth by inhibiting angiogenesis in the tumor.

Furthermore, CD31 staining, which is a marker for tumor vascular endothelial cells, was performed by immunohistological examination of the tumor tissue, and the degree of staining was quantitatively compared. As a result, in the BMP4-administered group, tumor vessels that were CD31 positive were examined. The density was significantly reduced (Figure 2). From the above, it was shown that BMP4 suppresses tumor angiogenesis.

(1) Preparation of mouse having human cervical cancer Human cervical cancer Hela cells (hereinafter also simply referred to as “human cervical cancer cells”) were cultured in D-MEM medium containing 10% fetal bovine serum. From the obtained human cervical cancer cells, 10 sets of 1 × 10 ⁷ cells were collected. To each of the collected human cervical cancer cells, 100 μl of PBS (phosphate buffered physiological saline) was added and suspended to prepare 10 sets of human cervical cancer cell suspensions. Meanwhile, 10 8-week-old female C.B17 / Icr skid mice were purchased and prepared. One set of the aforementioned human cervical cancer cell suspension was subcutaneously injected into each mouse on the right back of each mouse, and the human cervical cancer cells were transplanted into the mice. The day of transplantation was defined as day0. Hereinafter, these mice transplanted with human cervical cancer cells are simply referred to as “mouse”.

(3) Administration of the antitumor agent of the present invention to mice and change in tumor volume On day 2 after human cervical cancer cell transplantation (day 2), which is considered to contain a tumor angiogenic switch Among the 10 mice, 200 μl of the above-described antitumor agent of the present invention was intraperitoneally administered to each of 5 mice (BMP4 administration group). When this dose of BMP4 is converted per mouse, it is 2 μg, and since the weight of the mouse is about 20 g, it is 100 ng / g when converted per 1 g of mouse body weight. On the other hand, the remaining 5 mice were used as controls, and 200 μl of the aforementioned control solution was intraperitoneally administered to each of these 5 mice (control group). Furthermore, BMP4 and control solution were intraperitoneally administered for 3 weeks at a weekly pace (administered to day 8, day 15 after day 2) to determine the tumor reduction effect.

The change with time of the calculated tumor volume is shown in FIG. 3A.

In addition, as a tumor volume, each average value is used for each group. As can be seen from the results in FIG. 3A, a difference in tumor volume between the BMP4-administered group and the control group has already started to be observed on day4. Furthermore, it should be noted that the tumor suppressive effect is recognized every time when it is administered with day8 and day15. Finally, it was confirmed that the tumor volume in the BMP4 administration group was significantly suppressed compared to that in the control group (FIG. 3A). That is, it was shown that the antitumor agent of the present invention exerts a growth inhibitory effect on the tumor.

By day 20, no mice died in the control group and the BMP4 administration group. All remaining mice that survived on day 20 were euthanized and tumors were removed subcutaneously (FIG. 3B). As can be seen from the results in FIG. 3B, the tumor tissue in the BMP4 administration group was clearly smaller than the tumor tissue in the control group. In addition, other organs (brain, lung, liver, spleen) and blood were collected and observed mainly for differences between the two groups, particularly changes in tumor blood vessels. Macroscopic findings of each organ mentioned above showed no difference between the two groups, and no distant metastasis of tumor to each organ.

[In vitro anti-tumor effect confirmation assay]
In order to examine the effect of BMP4 on the melanoma cells themselves, a chronogenic assay (anchorage-dependent) and a colony assay (anchorage-independent) were performed. In the chronogenic assay, 1,000 melanoma live cells are prepared in one well of a 6-well dish, 100 ng / ml of BMP4 is added to one well, and a control solution of the same amount as BMP4 as a control (4 mM hydrochloric acid containing 0.1% BSA). Aqueous solution) was mixed with 2 ml / well of D-MEM medium containing 10% fetal bovine serum and cultured. Three wells were prepared for each of the BMP4 group and the control group. Eight days later, the culture medium is discarded by suction, washed once with PBS, fixed with 70% alcohol for 1 hour, colonies are stained with 0.5% crystal violet, washed with water, the number of colonies counted, and the average value The standard deviation was calculated and the significant difference was tested. As a result, for melanoma cells, no difference was observed between the BMP4 group and the control group (FIG. 4A).

In the colony assay, matrigel was used, 30,000 live melanoma cells were prepared in 1 well of a 6-well dish, BMP4 was 100 ng / ml in 1 well, and a control solution (0.1% equal to BMP4 was used as a control). BSA-containing 4 mM hydrochloric acid solution) was mixed with 2 ml / well of D-MEM medium containing 10% fetal calf serum and cultured. Three wells were prepared for each of the BMP4 group and the control group. After 14 days, the number of colonies was counted, and a significant difference was tested in the same manner as described above. As a result, no difference was observed between the BMP4 group and the control group (FIG. 4B).

From the above in vitro results, it was found that BMP4 did not work to suppress the proliferation of melanoma cells themselves.

[In vitro anti-tumor effect confirmation assay]
In order to examine the effect of BMP4 on human cervical cancer cells (Hela cells) themselves, a chronogenic assay (anchorage-dependent) and a colony assay (anchorage-independent) were performed. In the chronogenic assay, 1,000 human cervical cancer live cells are prepared in one well of a 6-well dish, BMP4 is 100 ng / ml in one well, and a control solution (0.1% BSA equal to BMP4 is used as a control). Solution) was mixed with 2 ml / well of D-MEM medium containing 10% fetal bovine serum and cultured. Three wells were prepared for each of the BMP4 group and the control group. Eight days later, the culture medium is discarded by suction, washed once with PBS, fixed with 70% alcohol for 1 hour, colonies are stained with 0.5% crystal violet, washed with water, the number of colonies counted, and the average value The standard deviation was calculated and the significant difference was tested. As a result, for human cervical cancer cells, no difference was observed between the BMP4 group and the control group (FIG. 5A).

In the colony assay, matrigel was used, 30,000 live melanoma cells were prepared in 1 well of a 6-well dish, BMP4 was 100 ng / ml in 1 well, and a control solution (0.1% equal to BMP4 was used as a control). (BSA-containing aqueous solution) was mixed with 2 ml / well of D-MEM medium containing 10% fetal bovine serum and cultured. Three wells were prepared for each of the BMP4 group and the control group. After 14 days, the number of colonies was counted, and a significant difference was tested in the same manner as described above. As a result, no difference was observed between the BMP4 group and the control group (FIG. 5B).

From the above in vitro results, it was found that BMP4 did not work to suppress the growth of human cervical cancer cells themselves.

[In vivo anti-tumor effect confirmation assay]
In order to confirm whether BMP4 in the present invention exerts an antitumor effect independently of the VEGF / Flt1 (also referred to as VEGF receptor 1) signal system in vivo, the following assay was attempted.

(1) Production of mouse having melanoma Mouse B16 melanoma cells (hereinafter also simply referred to as “melanoma cells”) are cultured in a D-MEM medium containing 10% fetal bovine serum, and from the obtained melanoma cells, A total of 10 sets of 1 × 10 ⁷ pieces were collected. 100 μl of PBS (phosphate buffered saline) was added to each of the sorted melanoma cells and suspended to prepare 10 sets of melanoma cell suspensions. On the other hand, 10 8-week-old female mice C57BL / 6J (hereinafter also referred to as Flt1 TK knockout mice) in which VEGF / Flt1 signal was knocked out were prepared. One set of the above melanoma cell suspension per mouse was subcutaneously injected into the right back of these mice, and the melanoma cells were transplanted into TK knockout mice. The day of transplantation was defined as day0. Hereinafter, these melanoma cell-transplanted mice are simply referred to as “Flt1 TK ^{− / −} mice”.

(3) Administration of the antitumor agent of the present invention to Flt1 TK ^{− / −} mice and change in tumor volume On day 2 after melanoma cell transplantation (day 2), which is considered to contain a tumor angiogenic switch Of the 10 mice described above, 200 μl of the above-described antitumor agent of the present invention was intraperitoneally administered to each of 5 mice (BMP4 administration group). When this dose of BMP4 is converted per mouse, it is 2 μg, and since the weight of the mouse is about 20 g, it is 100 ng / g when converted per 1 g of mouse body weight. On the other hand, the remaining 5 mice were used as controls, and 200 μl of the aforementioned control solution was intraperitoneally administered to each of these 5 mice (control group).

FIG. 6 shows the change over time of the calculated tumor volume.

In addition, as a tumor volume, each average value is used for each group. As can be seen from the results in FIG. 6, in day 5, no tumor increase was observed in either the BMP4 administration group or the control group, but in day 8, there was a difference in tumor volume between the BMP4 administration group and the control group. From day 11 onward, it was recognized that the tumor volume in the BMP4 administration group was significantly suppressed relative to that in the control group after day 11. That is, it was found that the antitumor agent of the present invention exerts a growth inhibitory effect on tumors, and at the same time, has a tumor suppressive effect independent of the VEGF / Flt1 signal system. From this result, it was shown that a synergistic tumor suppression effect can be expected by the combined use with an anti-VEGF inhibitor.

By day 20, 3 mice died in the control group and 1 died in the BMP4 administration group. All remaining mice that survived on day 20 were euthanized and tumors were removed subcutaneously. Other organs (brain, lungs, liver, spleen) were collected. No bleeding was observed in each organ, and no distant metastasis of the tumor was observed.

(1) Preparation of mice having lung cancer cells Mouse LLC lung cancer cells (hereinafter also simply referred to as “lung cancer cells”) are cultured in a D-MEM medium containing 10% fetal bovine serum, and from the obtained lung cancer cells. A total of 10 sets of 1 × 10 ⁷ pieces were collected. To each of the sorted lung cancer cells, 100 μl of PBS (physiological saline with phosphate buffer) was added and suspended to prepare 10 sets of lung cancer cell suspensions. Meanwhile, 10 healthy female mice, C57BL / 6J, 8 weeks old were prepared. One set of the above-mentioned lung cancer cell suspension was subcutaneously injected into the right back of these mice per mouse, and the lung cancer cells were transplanted into the mice. The day of transplantation was defined as day0. Hereinafter, these lung cancer cell transplanted mice are simply referred to as “mouse”.

(2) Preparation of antitumor agent and control solution of the present invention Mouse BMP4 was purchased from R & D Systems, Cat. #: 5020-BP, Minneapolis, MN (USA). Human BMP4 is R & D
Systems, Cat. #: 314-BP, purchased from Minneapolis, MN (USA). As an antitumor agent of the present invention, a solution was prepared by dissolving the above mouse BMP4 in a 4 mM hydrochloric acid aqueous solution containing 0.1% BSA (bovine serum albumin), and the concentration of mouse BMP4 was 10 μg / ml. Further, a 4 mM hydrochloric acid aqueous solution containing 0.1% BSA was prepared as a control solution.

(3) Administration of the antitumor agent of the present invention to mice and change in tumor volume The above-mentioned 10 mice are considered to enter the tumor angiogenic switch on the second day (day 2) after transplantation of lung cancer cells. Among these mice, 200 μl of the above-described antitumor agent of the present invention was intraperitoneally administered to each of 5 mice (BMP4 administration group). When this dose of BMP4 is converted per mouse, it is 2 μg, and since the weight of the mouse is about 20 g, it is 100 ng / g when converted per 1 g of mouse body weight. On the other hand, the remaining 5 mice were used as controls, and 200 μl of the aforementioned control solution was intraperitoneally administered to each of these 5 mice (control group).

Fig. 7 shows changes in the calculated tumor volume over time.

In addition, as a tumor volume, each average value is used for each group. As can be seen from the results in FIG. 7, in day 8, no tumor increase was observed in either the BMP4 administration group or the control group, but in day 11, there was a difference in tumor volume between the BMP4 administration group and the control group. From day 11 onward, it was recognized that the tumor volume in the BMP4 administration group was significantly suppressed relative to that in the control group after day 11. What should be noted is that the tumor in the control group has rapidly increased since day 31 even though only BMP4 was administered once to day 2, which is considered to have a tumor angiogenic switch. On the other hand, in the BMP4 administration group, the initial tumor suppression effect by BMP4 was continued. Moreover, human BMP4 was intraperitoneally administered to day53, and the tumor suppression effect was recognized like mouse BMP4. It was shown that the antitumor agent of the present invention exerts a growth inhibitory effect on the tumor (FIG. 7).

By day 57, one mouse died in the control group, and no death occurred in the BMP4 administration group. All remaining mice that survived on day 57 were euthanized and tumors were removed subcutaneously. In addition, other organs (brain, lung, liver, spleen) and blood were collected and observed mainly for differences between the two groups, particularly changes in tumor blood vessels. Macroscopic findings of each organ mentioned above showed no difference between the two groups, and no distant metastasis of tumor to each organ. All of the animal experiments in Examples 1, 2, 5, and 6 described above were conducted with the approval of the Animal Experiment Review Committee of Tokyo Medical and Dental University.

The antitumor agent of the present invention can be suitably used in the field of tumor therapy, particularly in the field of solid cancer therapy.

Claims

An antitumor agent comprising bone morphogenetic protein BMP4 as an active ingredient.
The bone morphogenetic protein BMP4 comprises the amino acid sequence shown in SEQ ID NO: 1 or 3 in the sequence listing, or a sequence in which one or several amino acids in the amino acid sequence are deleted, added, or substituted, and has antitumor activity The antitumor agent according to claim 1, comprising an amino acid sequence having the amino acid sequence.
The antitumor agent according to claim 1 or 2, wherein the antitumor agent comprising bone morphogenetic protein BMP4 as an active ingredient can be administered to a cancer patient by intraperitoneal injection.
The antitumor agent according to any one of claims 1 to 3, wherein the antitumor agent comprising bone morphogenetic protein BMP4 as an active ingredient has angiogenesis inhibitory activity.