JP2862140B2 - Angiogenesis inhibitor - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an angiogenesis inhibitor, and more particularly to a polypeptide-based angiogenesis inhibitor.

[Conventional technology]

Angiogenesis, the growth of new capillaries, causes chronic inflammation,
It is known to be involved in certain immune responses, pathological processes such as neoplasia. Therefore, an angiogenesis inhibitor is considered to suppress tumor growth, progression of retinopathy and rheumatoid arthritis, increase in psoriatic lesions, and the like.

Heretofore, it has been reported that steroid hormones such as prednisolone, 6α-methylprednisolone, and dexamethasone have an effect of suppressing angiogenesis experimentally induced in chicken embryo chorioallantoic membrane, rabbit cornea, and hamster cheek pouch.

[Problems to be solved by the invention]

An object of the present invention is to provide a safer new angiogenesis inhibitor using a substance related to the living body in view of the above-mentioned conventional technology.

[Means for solving the problem]

In summary, the present invention relates to an angiogenesis inhibitor, and relates to a fibronectin-derived cell adhesion-active polypeptide and / or a formula: -Arg-Gly-Asp-Ser.
It is characterized by containing a cell adhesive polypeptide containing a polymer having a repeating unit represented by-.

The present inventors have conducted intensive studies on the functional domain of fibronectin (hereinafter abbreviated as FN), which is a substance in the body, and as a result, have found that a cell adhesion-active polypeptide has a remarkable angiogenesis inhibitory effect. The present invention has been completed.

 Hereinafter, the present invention will be described in detail.

Examples of the cell adhesion-active polypeptide used in the present invention include the following.

Y- (Met) _n- Z (1) (wherein, Y represents a cell adhesion active polypeptide in an FN molecule, n is 0 or 1, and Z is -OH or heparin in an FN molecule) Indicates a binding polypeptide) A polymer having a repeating unit represented by the above formula [2], that is, polyRGDS.

FN has about 250,000 polypeptides at the C-terminus near the S-terminal.
A dimer is formed by S bond. The intramolecular amino acid sequence has a repeating structure and is classified into types I, II, and III.
Furthermore, it has a domain structure having various functions, and exhibits a binding activity to cell adhesion, collagen, heparin, ibulin and the like.

Examples of the above-mentioned polypeptides include JP-A-1-180900, JP-A-1-206998, and Japanese Patent Application No. 63-186, which were created by the present inventors.
-160949 (274 amino acid residue polypeptide), Japanese Patent Application No. 63
No. 305820. The cell adhesion-active polypeptide disclosed in each specification. These cell adhesion active polypeptides can be efficiently produced by genetic engineering techniques.

The 274 amino acid residue polypeptide will be described in detail below as an example.

274 amino acid residues polypeptide (Pro ¹²³⁹ _Asp ¹⁵¹²⁾ the following formula [3]: And characterized by the amino acid sequence represented by
The production method is described in the specification of Japanese Patent Application No. 63-160949, and will be specifically described below.

In this specification, the superscript number attached to the amino acid indicates the number of FN amino acid residues in the EMBL DATE BANK.

274 amino acid residue polypeptide (Pro ¹²³⁹ _Asp ¹⁵¹²⁾
Already C of 279 amino acid residue polypeptide that patent application ^{(Pro 1239 _-Met 1517)} ( JP-A-1-206998)
This is one in which the terminal 5 amino acid residues have been deleted. 274 amino acid residues polypeptide (Pro ¹²³⁹ _-Asp ¹⁵¹²⁾ as a method for preparing by genetic engineering is to use a plasmid pTFD707 encoding the above 279 amino acid residue polypeptide (Pro ¹²³⁹ _-Met ¹⁵¹⁷⁾ It is convenient. Preparing 279 amino acid residues polypeptide plasmid encoding the 274 residue polypeptide (Pro ¹²³⁹ _-Asp ¹⁵¹²⁾ by a codon AAA for C-terminal than 5 residues th Lys ¹⁵¹³ into a termination codon TAA of the be able to. This base conversion can be performed by introducing a site-specific mutation.

274 amino acid residue polypeptide (Pro ¹²³⁹ _-Asp ¹⁵¹²⁾
Escherichia E. coli transfected with a plasmid expressing E. coli
coli JM 109 / pTF 7221 is available from
-1915).

Purification of the cell adhesion-active polypeptide from the recombinant is performed, for example, as follows. The cell pellet is suspended in a buffer, and the soluble fraction is separated into insoluble fractions by sonication. The latter is further solubilized with a buffer containing 7M urea. The soluble fraction is collected, applied to a column of Sepharose 4B to which an antibody specific to the cell adhesion activity domain of FN is bound, and subjected to affinity purification. For elution around pH 2.3,
Use buffer. By collecting target fractions by immunoblotting, a cell adhesion-active polypeptide can be obtained. If necessary, it can be further purified by FPLC or HPLC.

Further, the chimeric popeptide of the cell adhesion activity polypeptide and heparin-binding polypeptide in the present invention is contained in the heparin domain of FN directly or via methionine with the above-mentioned cell adhesion activity polypeptide of the present invention, and has a heparin-binding activity. Examples of the above-mentioned polypeptides, which are chimeric polypeptides bound to a polypeptide having the same, include a polypeptide disclosed in Japanese Patent Application No. 1-131453 (a 574 amino acid residue polypeptide) created by the present inventors. ), And can be efficiently produced by a genetic optical technique.

Hereinafter, the 574 amino acid residue polypeptide will be described in detail.

574 amino acid residue polypeptide (Pro ¹²³⁹ _Ser ¹⁵¹⁵ -Me
t-Ala ¹⁶⁹⁰ -Thr ¹⁹⁸⁵ ) has the following formula [4]: And characterized by the amino acid sequence represented by
The production method is disclosed in the specification of Japanese Patent Application No. 1-153453, and will be specifically described below.

Plasmid pLF 2435 containing a cDNA fragment encoding the heparin binding domain of human FN (Bioche
mistry), Vol. 25, pp. 4936-4494 (1986), which is a plasmid reconstructed by linking the coding regions of pLF2, 4, 3 and 5.

A necessary cDNA fragment was excised from this plasmid pLF2435 with a restriction enzyme, and a synthetic DNA containing an initiation codon on the 5 ′ side and a synthetic DNA containing a stop codon on the 3 ′ side were ligated with DNA ligase. Plasmid pHD expressing the heparin-binding domain 296 amino acid residue polypeptide (Ala ^1690_ -Thr ¹⁹⁸⁵ ) by connecting to a simple expression vector.
102 is built.

As the expression vector, any of the existing vectors can be used. For example, pUC118N / pUC119N [Febus
FEBS Letters, Volume 223, Pages 174-180 (1
987)], and good results can be obtained by using their derivatives. By introducing this plasmid into Escherichia coli, a heparin-binding polypeptide can be expressed and its properties can be examined.

Then, D encoding 279 amino acid residues polypeptide of JP-A-1-206998 JP (Pro ¹²³⁹ _-Met ¹⁵¹⁷⁾
Plasmid pTF70 obtained by connecting NA to an expression vector
Introduced Nco I site on 21 and added Pro ¹²³⁹ _−Ser ¹⁵¹⁵ −Met
A plasmid pTF7520 encoding was constructed.

By extracting a cDNA fragment from pHD102 and connecting it to the NcoI site at the 3'-terminal of the translation region of pTF7520, a group expressing a 574 amino acid residue polypeptide in which the cell adhesion active domain of FN and the heparin binding domain are linked to each other. A recombinant plasmid is obtained.

The junction in the plasmid contains a methionine residue derived from the NcoI site as a linker. The presence or absence of a linker does not affect the effect of the present invention, but can be easily removed by site-specific mutagenesis if necessary.

Escherichia coli HB101 / pCH102 into which a plasmid expressing a 574 amino acid residue polypeptide has been introduced has been deposited as Microtechnical Laboratory No. 2800 (FERM BP-2800).

By introducing the obtained plasmid into Escherichia coli and culturing it under appropriate conditions, the target polypeptide is accumulated in Escherichia coli.

Purification of the target polypeptide is performed, for example, as follows.
After culturing the recombinant Escherichia coli in a medium such as L-broth and collecting the cells, a lysate of the cells is obtained by sonication, and this is centrifuged to obtain a supernatant. After dialysis of the supernatant, it is passed through a column of a DEAE ion exchanger, followed by affinity chromatography such as a CM ion exchanger and / or heparin-agarose.
By the above operation, the target polypeptide can be purified.

On the other hand, it is known that the activity of a functional molecule is enhanced by borrowing a macromolecule, and the present inventors have prepared a regular sequence polypeptide consisting of repeating units of the RGDS sequence of FN, that is, a polyRGDS.

One example of poly-RGDS is to synthesize a RGDS tetrapeptide by a usual liquid phase method and use it in a polymerization reaction developed by Nishi et al. [International Journal of Peptide and Protein Research (Internationa
l Jou-rnal of Peptide and Protein Research), 3rd
0, p. 275 (1987)]. That is, first, an aspartic acid in which an amino group and a side chain carboxyl group are protected by Boc (t-butyloxycarbonyl group) and Bzl (benzyl group) and a methyl ester of serine in which a hydroxyl group is protected by Bzl are condensed. , Dipeptide [BocAsp (Bzl) -Ser (Bz
l) -OMe]. After removing Boc, the glycine whose amino group is protected by Boc is condensed to form a tripeptide (BocGly
-Asp (Bzl) -Ser (Bzl) -OMe]. Similarly, by condensing arginine whose amino group and side chain are protected with Boc and Mts (mesitylenesulfonyl group), respectively, the tetrapeptide [BocArg (Mts) -Gly-Asp
(Bzl) -Ser (Bzl) -OMe]. Removal of the Boc and methyl groups yields a tetrapeptide with free amino and carboxyl termini and protected side chains. This is DMSO
Medium, stir in the presence of DPPA (diphenylphosphoryl azide) and triethylamine, and protect the side chain of poly RG.
Get DS. Next, the side chain is removed in methanesulfonic acid-anisole, and poly (RGDS.HCl) is obtained as a hydrochloride by Amberlite IRA-400 (Cl type).

This characterizes the polypeptide in the IR spectrum,
It shows that the protecting groups on the side chains have also been completely removed.
Also, by polyacrylamide electrophoresis containing urea,
It migrates at a position with a molecular weight of 5,000 to 10,000, and shows a degree of polymerization of RGDS 12 to 24.

When the polypeptide of the present invention obtained as described above is used as a medicament, it may be formulated as necessary together with a pharmaceutical carrier by a conventional method, and orally or parenterally administered. A pharmacologically acceptable excipient is selected as the excipient or carrier, and its type and composition vary depending on the administration route and administration method. For example, as a liquid carrier, there are used animal and vegetable oils such as hydroalcohols, soybean oil, olive oil and mineral oil, or synthetic oils. As the solid carrier, sugars such as maltose and sucrose, amino acids, cellulose derivatives such as hydroxypropylcellulose, and organic acid salts such as magnesium stearate are used.

In the case of an injection, the dissolving solution is preferably a physiological saline solution, various buffer solutions, saccharide solutions such as glucose, inositol, mannitol, lactose, and glycols such as ethylene glycol and polyethylene glycol. In addition, a combined dry preparation is prepared together with excipients such as inositol, mannitol, lactose, sucrose and other saccharides, and amine acids such as phenylalanine and the like, and a suitable solvent for injection at the time of administration, for example, sterile water, physiological saline, dextrose. Alternatively, it can be administered after being dissolved in a liquid for intravenous administration such as an electrolyte solution and an amino acid solution. Although the content of the polypeptide of the present invention in the preparation varies depending on the preparation, it is usually 0.1 to 100% by weight, preferably 1 to 98% by weight. For example, in the case of an injection,
Usually, it is desirable to contain 0.1 to 30% by weight, preferably 1 to 10% by weight of the active ingredient. In the case of oral administration, it is used in the form of tablets, capsules, powders, granules, liquids, dry syrups and the like together with the solid carrier or liquid carrier. Capsules, granules and powders are generally 5-10
It contains 0% by weight, preferably 25-98% by weight of active ingredient.

The dose is determined by the age of the patient, weight, symptoms, the purpose of treatment, etc., but the therapeutic amount is generally 1 to 100 m for parenteral administration.
g / kg / day, oral administration 5-500 mg / kg / day.

[Action]

Next, the biological activity of the polypeptide of the present invention will be shown by experimental examples.

EXPERIMENTAL EXAMPLE 1 Angiogenesis Inhibitory Effect of B16 on two sites in the back skin of C57BL / 6 mice (3 mice per group)
Intradermal injection of 5 × 10 ⁵ BL6 melanoma cells, or a mixture of 100 μg of the polypeptide of the invention and 5 × 10 ⁵ melanoma cells. On day 3 after the melanoma cell transplantation, 0.2 ml of a 1% epanse blue solution is injected intravenously, and immediately thereafter, the skin on the back of the mouse is peeled off, and the number of vessels formed toward the tumor is counted. Table 1 shows the results.

As described above, angiogenesis to melanoma is suppressed by the polypeptide of the present invention.

Experimental Example 2 Acute toxicity test The polypeptide of the present invention was intravenously administered to C57BL / 6 mice. No toxicity was observed at 100 mg / kg.

As described above, the polypeptide of the present invention shows a remarkable angiogenesis inhibitory effect, prevents tumor regression and metastasis, as a contraceptive agent for women even when administered for the first time after fertilization, as shown in the above experimental examples, It is effective for diseases involving angiogenesis, such as alleviation of osteoporosis, suppression of progression of retinopathy and rheumatoid arthritis, and suppression of increase of psoriatic lesions.

〔Example〕

Next, the present invention will be described specifically with reference to examples, but the scope of the present invention is not limited to the examples.

 In each example, parts means parts by weight.

Formulation Example 1 PBS was added to 30 parts of a 274 amino acid residue polypeptide,
After dissolving the whole amount as 2000 parts, Millipore filter
Sterilize and filter using GS type. 2 g of the filtrate was placed in a 10 ml vial and freeze-dried to obtain a freeze-dried injection containing 30 mg of the polypeptide in one vial.

Production Example 2 PBS was added to 30 parts of the 574 amino acid residue polypeptide, and the total amount was dissolved to 2000 parts.
Sterilize and filter using the type. 2 g of the filtrate is placed in a 10 ml vial, freeze-dried, and the polypeptide is placed in one vial.
A freeze-dried injection containing 30 mg was obtained.

Formulation Example 3 PBS is added to 30 parts of poly-RGDS to make the total amount 2000 parts, and the mixture is dissolved, followed by sterilization filtration using a Millipore filter GS type. 2 g of the filtrate was placed in a 10 ml vial and freeze-dried to obtain a freeze-dried injection containing 30 mg of the polypeptide in one vial.

Formulation Example 4 50 parts of 274 amino acid residue polypeptide, 600 parts of lactose, 330 parts of microcrystalline cellulose and 20 parts of hydroxypropyl cellulose
The mixture was mixed well, compressed using a roll-type compactor (roller compactor), crushed and sieved so as to be between 16 and 60 mesh to obtain granules.

Formulation Example 5 50 parts of 574 amino acid residue polypeptide, 600 parts of lactose, 330 parts of crystalline cellulose and 20 parts of hydroxypropyl cellulose
The mixture was mixed well, compressed using a roll-type compressor (roller compactor), crushed, and sieved so as to be between 16 and 60 mesh to obtain granules.

Formulation Example 6 50 parts of poly RGDS, 600 parts of lactose, 330 parts of crystalline cellulose, 330 parts of hydroxypropyl cellulose and 20 parts of hydroxypropyl cellulose are mixed well, compressed using a roll-type compressor (roller-compactor), and crushed. 16
The mixture was sieved to a particle size of ~ 60 mesh to obtain granules.

〔The invention's effect〕

The polypeptide of the present invention is a biologically relevant substance, has high safety, and is useful as an angiogenesis inhibitor. Also, it has a remarkable effect in that it can be supplied in large amounts by genetic engineering or chemical synthesis.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu City, Shiga Prefecture Takara Shuzo Co., Ltd. Central Research Laboratory (56) References JP-A-3-127742 (JP, A JP-A-1-206998 (JP, A) JP-A-1-502197 (JP, A) Biol. Chem. Vol. 19 (1985), p. 10402-10405 (58) Field surveyed (Int. Cl. ⁶ , DB name) A61K 38/00-38/58 CA (STN)

Claims (1)

(57) [Claims] (1) a cell adhesion-active polypeptide derived from fibronectin and / or a cell adhesion polypeptide containing a polymer having a repeating unit represented by the formula: -Arg-Gly-Asp-Ser-; An angiogenesis inhibitor, characterized in that: