JP3190765B2 - Peptide derivatives and their uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a derivative of a peptide having a specific structure or a pharmaceutically acceptable salt thereof, and use thereof.

[0002]

2. Description of the Related Art Fibronectin, laminin, vitronectin and the like are proteins that are involved in the binding of cells to connective tissues and have various biological activities related to the cell functions of animal cells, and are generally referred to as cell adhesion proteins. For example, fibronectin is biosynthesized in the liver, and about 0.3 mg
Glycoprotein present at a concentration of / ml.

[0003] The primary structure of fibronectin has been determined using molecular cloning (Koarnblihtt, A. et al.
R. et al., EMBO Journal, vol. 4, 2519 (1985)), a dimer protein in which the A chain, which is a polypeptide having a molecular weight of about 250K, and the B chain, which is about 240K, are disulfide-bonded near the C-terminus. It has been revealed. Sasaki, M. et al., Proc. Natl. Acad.
Sci. USA., 81, 935 (1987), Sasaki, M. et al.,
J. Biol. Chem., 262, 17111 (1987))
The following structure has been determined. Laminin is composed of three polypeptide chains called A, B1, and B2, and is known to have a cross-like structure.

[0004] Studies were also made on the binding site involved in cell adhesion, and it was reported in 1984 that the core sequence involved in fibronectin cell adhesion was a tripeptide Arg-Gly-Asp (RGD). (Pierschb
acher, MD et al., Nature 309, 30 (1984)). The core sequence of the laminin cell adhesion site is Tyr-Ile-
It has also been elucidated that it is a pentapeptide represented by Gly-Ser-Arg (YIGSR) (Graf, J. et.
al., Cell 48, 989 (1987)).

[0005] These fibronectin and laminin are:
Various kinds of information are transmitted to cells by binding to cell receptors through the above core sequence, and also to biological macromolecules such as heparin, collagen, fibrin, and the like, adhesion between cells and connective tissue, cell differentiation It is thought to be involved in proliferation.

[0006] Since cell adhesion proteins have various biological activities, studies using peptides having the active site sequence have been made vigorously. For example, use of the core sequence of the cell-binding portion of fibronectin may be achieved by adding R
A method in which a peptide having a GD sequence is covalently bonded and used as a substrate for artificial organs or a substrate for animal cell culture (Japanese Patent Laid-Open No.
No. 309682, JP-A-1-305960, WO 90/05036 A
A method of attaching a peptide of interest to a solid surface by linking a hydrophobic region to a peptide having an RGD sequence and using it as a dental implant or a tissue culture substrate (WO 90 / 11297A patent); Method of inhibiting platelet aggregation or preventing and treating thrombosis using various cyclic and linear oligopeptides or analogs having the same (Proceedings of the Society of Polymer Science, vol. 38, 3149 (1989);
-174797, JP-A-3-118330, JP-A-3-1183
No. 31, JP-A-3-118398, JP-A-3-18397, JP-A-3-118333, WO 91/01331 patent, WO 91/0
7429 patent, WO 91/15515 patent, WO 92/00995 patent), RG
A method for regulating platelet aggregation using a compound in which a D peptide and hyaluronic acid are covalently bonded (JP-A-4-34096), and a method for using a peptide having an RGD sequence as a cell migration controlling agent (JP-A-2-4716) ), A method of using a membrane on which a peptide having an RGD sequence is immobilized as a cell adhesion membrane (Preprints of the Society of Polymer Science, vol. 37, 705 (1988))
A method in which a polypeptide having a DS sequence is used as a platelet protective agent for extracorporeal blood (Japanese Patent Application Laid-Open No. 64-6217), which is used as an artificially functional polypeptide by adding a peptide having cell adhesion activity to the polypeptide molecule (Japanese Patent Laid-Open No. 3-34996) and the like.

[0007] In recent years, cell adhesion proteins have also attracted attention as biomolecules involved in cancer metastasis. In a series of stages of cancer metastasis, cancer cells come into contact with various host cells and biopolymers. At this time, if a cell adhesive molecule such as fibronectin or laminin is present, the cells form a multicellular mass, and cancer cells can more easily grow and survive. However, it has been reported that, for example, coexistence of tripeptide RGD, which is a core sequence of an adhesion site of fibronectin, competitively binds to a fibronectin receptor on cancer cells, thereby blocking cell adhesion and exhibiting a cancer metastasis inhibitory action ( Science 238, 467 (1986)).

However, since the RGD peptide alone does not have sufficient cell adhesion activity, cancer metastasis is carried out using an oligopeptide having the sequence, a cyclic oligopeptide, or a polypeptide having a repetitive sequence thereof for the purpose of enhancing the effect. Control method (Int. J. Biol. Macromo
l., 11, 23, (1989), Ibid., 11, 226 (1989), Jpn.
J. Cancer Res., 60, 722, (1989), JP-A-2-14798
Japanese Patent Application Laid-open No.
No. 240020). Also, a method of suppressing cancer metastasis using a polypeptide having a cell adhesion polypeptide and a heparin-binding polypeptide in a fibronectin molecule as constituent units (Japanese Patent Application Laid-Open No. 3-127742) has been reported. However, in previous studies, the RGD (Arg-Gly-Asp) core sequence was considered to be essential for the expression of various biological activities involved in the recognition of receptors and ligands.
It was reported that the activity was lost in analogs in which the y-part was replaced with another amino acid residue.
38, 3149 (1989), J. Biol. Chem., 262, 17294 (19
87)).

[0009]

As described above, studies on the use of cell adhesion proteins such as fibronectin or peptide fragments thereof as pharmaceuticals and related medical materials have been actively conducted. Particularly, RGD peptides are used to inhibit cancer metastasis. Attempts to apply it as an agent are active. However, since the cell adhesion activity of the core sequence is not yet sufficient, their cancer metastasis inhibitory action has not been satisfactory for application to actual medical treatment. In general, in order to maintain drug efficacy after a drug is administered to a living body, not only the strength of its own biological activity but also the stability of the drug in the living body (for example, the residence time in the bloodstream or excretion of the drug) Time, etc.) are known to be important. The adhesion site core sequence peptide of the cell adhesive protein is no exception, and when used alone, rapid metabolic degradation and excretion peculiar to peptides occur, and as a result, a desired effect may not be expected. Therefore, various methods as described in the section of the prior art for improving the stability in vivo have been reported, but some of those compounds still have insufficient biological activity or are difficult to synthesize. There are many. In addition, a method of linking a polymer such as polyethylene glycol (PEG) with the core sequence and a method of increasing the molecular weight by repeating the core sequence in a linear manner to improve stability in a living body have been attempted. However, with these methods, it is extremely difficult to perform synthesis by specifying the structure and molecular weight of the target substance, and development of a more effective substance in this regard has been required.

[0010] Accordingly, an object of the present invention is to provide a cell adhesion protein-like activity which is sufficiently high that the adhesion core sequence Ar
To provide a novel peptide derivative that contains a novel peptide sequence with enhanced ability to suppress cancer metastasis than g-Gly-Asp as an essential constituent unit, has high stability in blood, and can be synthesized by simple means It is in. Another object of the present invention is to provide a pharmaceutical composition comprising the above peptide derivative.

[0011]

Means for Solving the Problems The present inventors have previously reported a systematic analog of the adhesion core sequence Arg-Gly-Asp-Ser of fibronectin, particularly the Gly portion and the Ser portion, and various amino acids added to the N-terminal side. As a result of a careful study, a novel peptide sequence which sufficiently retains the activity of suppressing cancer metastasis and has high stability in blood was found, and a patent application has already been filed (Japanese Patent Application No. 4-22799). The present inventors have conducted intensive studies in search of a novel compound that is also easy to synthesize and has high cancer metastasis inhibitory activity. As a result, the peptide derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof Have achieved the above object, and have completed the present invention.

General formula (I) W = C ([Z] -Arg-X-Asp- [Y]) ₂ (I)

In the general formula (I), Arg represents L- or D-
Arginine is represented, and Asp represents L- or D-aspartic acid. X is L- or D-leucine or D-
Represents isoleucine. [] Indicates that the residue in [] may or may not be present. In the present invention, the residue in [] is preferably present. When present, Z is L- or D-aspartic acid, Y is L-
Represents serine or L-threonine. [Z] -Ar
The C-terminal side of the peptide sequence represented by gX-Asp- [Y] may be arbitrarily amidated. W represents an oxygen atom or a sulfur atom.

[0014] The peptide derivative of the present invention comprises [Z] -Ar
A compound in which a peptide represented by gX-Asp- [Y] is covalently bonded to both ends of a carbonyl group or a thiocarbonyl group from the N-terminal side, and two [Z] -Arg in one molecule It is characterized by containing a peptide sequence represented by -X-Asp- [Y]. Thus, unlike so-called polymers, the molecular structure is clear. In the present invention, derivatizing the peptide sequence represented by [Z] -Arg-X-Asp- [Y] as represented by the general formula (I) involves modifying the periphery of an effective peptide. To protect it from degradation by enzymes in the living body or to give a sustained release effect by increasing the molecular weight.

The ionic group present in the peptide derivative of the present invention may form a salt with an appropriate counter ion.
The compound of the present invention sufficiently retains its biological activity even in a salt form. However, the salt must be physiologically and pharmacologically acceptable. Specifically, salts with inorganic acids such as hydrochloride, sulfate, nitrate, phosphate,
Salts with organic acids such as acetate, lactate, tartrate, and the like, as well as sodium salts and potassium salts, are preferred, with the hydrochlorides and sodium salts being particularly preferred. Conversion to such salts can be carried out by conventional means.

Hereinafter, specific examples of the peptide derivative of the present invention are shown, but the present invention is not limited to these. When the C-terminus of the peptide derivative is -OH, the description is omitted according to the custom in the art. When the amino acid residue is in the D-form, it is denoted as D-, but when the amino acid residue is in the natural L-form, it is not particularly described.

Compound 1 O = C (Asp-Arg-Leu-Asp-Ser) ₂

Next, a method for synthesizing the compound of the present invention will be described. The peptide derivative of the present invention is a so-called urea-type compound in which a peptide chain is bonded to both ends of a carbonyl group or a thiocarbonyl group. The peptide derivative of the present invention can be synthesized by various methods. First, after synthesizing the protected peptide portion, the protecting group on the amino terminal side is removed, and this is derivatized to a urea-type compound. A method of synthesizing by removing the protecting group is practical and advantageous. First, the method for synthesizing the peptide portion is not particularly limited, and examples thereof include a solid phase method and a synthesis method using an automatic peptide synthesizer utilizing the solid phase method. Regarding the solid phase method and the synthesis method using an automatic peptide synthesizer using the solid phase method, see Biochemistry Experiment Course, Protein Chemistry IV p.207
(Edited by The Biochemical Society of Japan, Tokyo Chemistry Doujinshi), Seismic Chemistry Laboratory Course, Protein Chemistry (bottom), p.

The peptide moiety of the compound of the present invention can be synthesized by a liquid phase method. That is, starting from the protected amino acid serving as the C-terminal component, the C-terminal is protected or modified, the amino-terminal protecting group is removed, and the protected amino acid residues are successively condensed or fragment-condensed. This is a method of synthesizing a protected body. The method of condensing the protected amino acid or protected peptide can be appropriately selected from known methods, for example, the methods described in “Basic and Experimental Peptide Synthesis” edited by Nobuo Izumiya et al. (Maruzen). The DCC-Additive method using benzotriazole and DCC or the condensation method using carbonyldiimidazole gave the best results.

The amino-terminal protecting group of the protected peptide synthesized by the above method is removed, and this is derivatized to a urea-type compound. The method of converting the amine compound into a urea-type compound can be appropriately selected from known methods described in, for example, New Experimental Chemistry Lecture 14 Synthesis and Reaction of Organic Compounds (III) P. 1628 to 1644, In an appropriate inert solvent, the method of reacting carbonyldiimidazole or thiocarbonyldiimidazole with a protected peptide in which only the amino terminal amino group is free gave the best results.

The conditions for removing the protecting group depend on the type of protecting group used. Commonly used methods are hydrogenolysis,
HF treatment, trifluoromethanesulfonic acid / thioanisole / m-cresol / trifluoroacetic acid mixed system treatment, etc., needless to say, various methods are also possible depending on the type of protecting group. After deprotection, the desired peptide derivative can be purified by a known method, for example, ion exchange chromatography, gel filtration chromatography and the like.

Next, the function and use of the peptide derivative of the present invention will be described. In v of the peptide derivative of the present invention
Although the mechanism of the potent inhibitory effect on cancer metastasis in the ivo system is not completely clear, it acts on the fibronectin receptor on malignant cells by a mechanism similar to Arg-Gly-Asp, which is an adhesion core sequence peptide of fibronectin, Inhibition of binding to fibronectin is thought to prevent malignant cell adhesion, colonization, and catastrophic erosion. Furthermore, the peptide derivative of the present invention contains [Z] -Arg-
Since it has two sequences represented by X-Asp- [Y], it acts on the fibronectin receptor on malignant cells at multiple points, and because it has a certain large molecular weight, it is difficult to be excreted by enzymatic decomposition or metabolism, and therefore is remarkable It is considered that the compound exhibits a novel cancer metastasis inhibitory activity. The peptide derivative of the present invention can be used for breast cancer, epidermal cancer, muscle line mela
noma), epidermal neuroblastoma x glioma (epidermal lin)
e neuroblastoma x glioma), chondrocytes, and fibrosarcoma, and are effective in inhibiting the adhesion and metastasis of various cells.

Furthermore, the peptide derivative of the present invention was found to have a wide range of biological activities such as a wound healing action. When the peptide derivative of the present invention was subjected to a toxicity test using mice, no toxicity was observed.

The peptide derivative of the present invention or a pharmaceutically acceptable salt thereof can be used by an administration method generally used for peptide drugs, and is usually administered as a drug composition containing an excipient. The drug composition is based on Remington's Pharmaceutical Scien.
ces, Merck, 16, (1980)). Excipients include distilled water, physiological saline, buffers containing buffer salts such as phosphate or acetate, and antioxidants such as sodium chloride, sucrose or ascorbic acid as osmotic pressure regulators Or combinations thereof are acceptable.

Such a drug composition can be in various forms such as a solution and a tablet. The administration form can be appropriately selected from oral, nasal, parenteral (intravenous injection, subcutaneous injection, intraperitoneal administration, etc.) and the like. For example, it may be dissolved in physiological saline to prepare an injection preparation, or
After being dissolved in about 0.1 N acetate buffer, it may be used as a lyophilization agent. It can also be used in the form of microcapsules or microspheres encapsulated in liposomes.

The peptide derivative of the present invention can also be used in combination with other compounds having a pharmacological action, more specifically, an anticancer compound, and these belong to the scope of the present invention. . The anticancer compound that can be used in combination with the peptide derivative of the present invention can be appropriately selected from known anticancer agents usually used in cancer chemotherapy, and specifically, adriamycin And cisplatin, mitomycin and the like. In general, in conventional chemotherapy, it is known that danger due to side effects of anticancer drugs is an unavoidable problem. Therefore, it would be very useful to reduce the dose of an anticancer drug by co-administration with the peptide derivative of the present invention and reduce the side effects of the anticancer drug. This also means that the effect of suppressing cancer metastasis is further improved. 0.1 to 99% of the peptide derivative of the present invention in the composition
(Weight), the dose is usually 0.2 μm per day
The range is from g / kg to 200 mg / kg (body weight), which is specifically determined by the patient's age, sex, body weight, symptoms, and administration method.

[0027]

The present invention will be described in more detail with reference to the following examples. The following abbreviations were used for notations of commonly used solvents, reagents and protecting groups.

Boc: t-butoxycarbonyl Bn: benzyl AcOEt: ethyl acetate Mts: mesitylenesulfonyl HOBt: 1-hydroxybenzotriazole DCC: dicyclohexylcarbodiimide iPr ₂ NEt: diisopropylethylamine DMF: dimethylformamide CDI: dimethylformamide CDI Acetic acid TFMSA: trifluoromethanesulfonic acid

Example 1 Synthesis of Compound 1 A method for synthesizing Compound 1 by a liquid phase method will be described in detail. The synthesis route of Compound 1 is shown below.

[0030]

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[0031]

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1) Synthesis of Intermediate 1 Boc-Ser (Bn) -OH (20.4 g, 69 mmol), benzyl bromide
(13 g, 76 mmol), diisopropylethylamine (9.8
g, 76 mmol) was dissolved in ethyl acetate (100 ml) and the reaction mixture was heated at reflux for 5 hours. After allowing to cool to room temperature, the formed salt was removed by filtration, and the filtrate was washed with water, 1 M citric acid solution, saturated aqueous sodium hydrogen carbonate, and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give Intermediate 1. Obtained as a colorless oil.
This was used for the next reaction without purification.

2) Synthesis of Intermediate 2 Methylene chloride (80
trifluoroacetic acid (80 ml) was added to the solution and the reaction mixture was stirred at room temperature for 40 minutes. After completion of the reaction, the mixture was concentrated under reduced pressure to remove most of the solvent, and the residue was diluted with ethyl acetate.
The extract was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an amine compound as a colorless oil. The obtained amine compound and Boc-Asp (Bn) -OH (22.6 g, 70
mmol), 1-hydroxybenzotriazole monohydrate (1
0.7 g, 70 mmol) in DMF (80 ml) and methylene chloride (80 m
l) in a mixed solvent of DCC (14.4 g, 70
mmol) was added. The reaction mixture was stirred overnight under ice-cooling for 2 hours and further warmed to room temperature, and then filtered through celite to remove the formed precipitate. The filtrate was diluted with an appropriate amount of ethyl acetate, washed with water, 5% sodium carbonate solution, 1 M citric acid solution, and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oil. The mixture was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 4/1) to give the target intermediate 2 as colorless crystals.
.8 g (87% yield in 3 steps) were obtained.

3) Synthesis of Intermediate 3 Intermediate 2 (35.7 g, 60.5 mmol) in methylene chloride (100 m
l) Trifluoroacetic acid (100 ml) was added to the solution and the reaction mixture was stirred at room temperature for 40 minutes. After completion of the reaction, the solvent was distilled off, and the residue was crystallized from ether to obtain 32.5 g of trifluoroacetate. The resulting trifluoroacetate salt (22.7
g, 37.6 mmol) and Boc-Leu-OH monohydrate (9.34 g, 3
7.6 mmol), 1-hydroxybenzotriazole monohydrate
(5.76 g, 37.6 mmol), diisopropylethylamine (5.
2 g, 40 mmol) in DMF (40 ml) and methylene chloride (40 m
l) in a mixed solvent of DCC (7.76 g, 37.
6 mmol) was added. The reaction mixture was stirred overnight under ice-cooling for 2 hours and further warmed to room temperature, and then filtered through celite to remove the formed precipitate. The filtrate was diluted with an appropriate amount of ethyl acetate, washed with water, 5% sodium carbonate solution, 1 M citric acid solution, and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a colorless solid. This was recrystallized from hexane / ethyl acetate (2/1) to give Intermediate 3 22.1
g (84%). FAB-MS: (M + H) ⁺ 704.

4) Synthesis of intermediate 4 Intermediate 3 (21.0 g, 30 mmol) in methylene chloride (60 ml)
Trifluoroacetic acid (60 ml) was added to the solution, and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off, and the residue was crystallized from ether to give trifluoroacetate (20.8%).
g was obtained. On the other hand, Boc-Arg (Mts) -OH (6.7 g, 15 mmol) was added.
Dissolve in DMF (20 ml) and add CDI
(2.4 g, 15 mmol) in DMF (15 ml) was added. After the reaction mixture was stirred for 1 hour while cooling on ice, the trifluoroacetate salt obtained by the above operation (10.6 g, 15 mmol)
And diisopropylethylamine (2.0 g, 15.5 mmol) in D
MF (30 ml) solution was added. After the reaction mixture was stirred for 2 hours under ice-cooling and further overnight while warming to room temperature, most of the solvent was distilled off under reduced pressure. The residue was poured into ice water to precipitate crude crystals. The obtained crude crystals were collected by filtration, washed with water and air-dried to give 14.4 g of the desired intermediate 4 as colorless crystals.
(92%). FAB-MS: (M + H) ⁺ 1042.

5) Synthesis of Intermediate 5 Intermediate 4 (14.0 g, 13.4 mmol) in dioxane (30 ml)
A solution of hydrogen chloride / dioxane (4 M, 30 ml) was added to the solution, and the reaction mixture was stirred at room temperature for 2 hours. The solvent was distilled off, and the residue was crystallized from ether to obtain hydrochloride (12.7 g). On the other hand, Boc-Asp (Bn) -OH (3.29 g, 10 mmol) was added to DMF (2
0 ml), and add the CDI (1.62 g, 1
0 mmol) in DMF (15 ml) was added. After the reaction mixture was stirred for 1 hour while cooling on ice, a solution of the hydrochloride (10.0 g, 10.2 mmol) obtained by the above operation and diisopropylethylamine (1.42 g, 11 mmol) in DMF (30 ml) was added. After the reaction mixture was stirred for 2 hours under ice-cooling and further overnight while warming to room temperature, most of the solvent was distilled off under reduced pressure.
The residue was poured into ice water to precipitate crude crystals. The obtained crude crystals were collected by filtration, washed with water, air-dried, and further washed with ether to give 10.8 g of the desired intermediate 5 as colorless crystals.
(87%). FAB-MS: (M + H) ⁺ 1247.

6) Synthesis of Intermediate 6 To a solution of Intermediate 5 (9.5 g, 7.6 mmol) in dioxane (30 ml) was added a hydrogen chloride / dioxane solution (4 M, 40 ml).
The reaction mixture was stirred at room temperature for 2 hours. The solvent was distilled off, and the residue was crystallized from ether to obtain 8.9 g of hydrochloride. The obtained hydrochloride (1.33 g, 1.1 mmol) was dissolved in a mixed solvent consisting of diisopropylethylamine (145 mg, 1.2 mmol) and DMF (5 ml), and CDI (91 mg, 0.56 mmol) was added. After leaving the reaction mixture at room temperature for 2 days, the solvent was distilled off under reduced pressure. Dilute the residue with an appropriate amount of chloroform,
The extract was washed with water and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was crystallized from ether to give 1.40 g (quantitative) of the desired intermediate 6. FAB-MS: (M + H) ⁺ 2319.

7) Synthesis of Compound 1 Intermediate 6 (1.3 g, 0.56 mmol) of trifluoroacetic acid (7
ml) solution, a mixed solution of trifluoromethanesulfonic acid (6 g), thioanisole (4 ml), m-cresol (3.5 ml), and trifluoroacetic acid (18 ml) was added while cooling with ice, and the reaction mixture was added. The mixture was stirred for 2 hours under ice cooling. The reaction solution was added dropwise to ether (700 ml) and stirred slowly for 1 hour.
The precipitated crude product was dissolved in a small amount of water, and ion exchange chromatography (Amberlite IRA-400,
Counterion Cl ^- give), 570 mg (83% of the compound 1 of interest and lyophilized as a colorless powder) was obtained. FAB-MS: (M + H) ⁺ 1233.

Example 2 Investigation on Cancer Metastasis Inhibition by Experimental Lung Metastasis Model System Using Melanoma Cells The cancer metastasis inhibition effect of the peptide derivative of the present invention was examined by an experimental lung metastasis model system. Compound 1 of the present invention described in Examples and Arg-Leu-Asp-Ser as a tetrapeptide and Arg-Gly-Asp-Ser as a fibronectin adhesion core sequence peptide were used as comparative examples. These peptides (500 μg for compound 1 of the present invention, Arg
1000 μl each for -Leu-Asp-Ser and Arg-Gly-Asp-Ser
g) and B16-BL6 melanoma cells (5 × 10 ⁴ cells in logarithmic growth phase), which are very metastatic cancer cells, were mixed in PBS, and this was mixed with 5 C57BL / 6 female mice per group. Was administered by tail vein injection. On day 14 after the administration, the mice were sacrificed and dissected, and the number of cancer colonies that had metastasized to the lungs was counted.
It was compared with the administration group. The results are shown in Table 1 below.

[0040]

Table 1 Table 1 化合物 Administered compound Number of metastases to lung Mean ± SD ( (Range) ＰＢＳ PBS (untreated) 114 ± 28 (81-169) Arg- Gly-Asp-Ser 114 ± 22 (76-129) Arg-Leu-Asp-Ser 96 ± 33 (44-147) Compound 1 60 ± 14 (48-79) * ─────────── ────────────────────── * P <0.001 compared to untreated plot by t-test

According to the results, Arg-Gl, which is a conventionally known adhesion core sequence peptide of fibronectin, is used.
y-Asp-Ser did not show a metastasis inhibitory effect at a dose of 1000 μg per mouse, and the Arg-Leu-Asp-Ser peptide showed a decrease in the number of transpositions, but compared with the untreated group. There was no significant difference at 5% risk. In contrast, when Compound 1 of the present invention was administered, mouse 1
Even at a dose of 500 μg per animal, cancer metastasis to the lung was significantly suppressed. This indicates that one of the important objects of the present invention, enhancement of activity by modification, has been achieved as expected. Therefore, the cancer metastasis inhibitory effect of the peptide derivative of the present invention, and its usefulness and superiority are apparent.

Although the invention has been described with reference to specific embodiments and examples, it should not be construed as limiting. Obviously, various changes and modifications can be made without departing from the spirit and scope of the invention. Such inventions are considered to be included in the present invention.

[0043]

As described above, the peptide derivative of the present invention has a large inhibitory effect on cancer metastasis as compared with the core sequence of fibronectin which is a cell adhesive protein, and has almost no toxicity problem. In addition, its structure is simple, so that it is easy to synthesize and has high value as a medicine.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Ikuo Shiki Hokkaido, Sapporo City, Hokkaido Atsetsu-ku Kita 3-Jo 5-12-6 58) Field surveyed (Int.Cl. ⁷ , DB name) C07K 7/06 SwissProt / PIR / GeneSeq

Claims (2)

(57) [Claims] 1. A peptide derivative represented by the following general formula (I) or a pharmacologically acceptable salt thereof. General formula (I) W = C ([Z] -Arg-X-Asp- [Y]) ₂ (I) (In general formula (I), Arg represents L-arginine;
sp represents L-aspartic acid. X represents L-leucine. [] Indicates that the residue in [] is present, in which case Z represents L-aspartic acid and Y represents L-serine. The C-terminal side of the peptide sequence represented by [Z] -Arg-X-Asp- [Y] is not amidated. W represents an oxygen atom. ) 2. A cancer metastasis inhibitor comprising, as active ingredients, a pharmaceutically acceptable excipient and the peptide derivative according to claim 1 or a pharmaceutically acceptable salt thereof.