JPH06116289A - Cell adhesive peptide sequence complex and its use - Google Patents

Abstract

PURPOSE:To obtain a complex capable of maintaining effect as a cell adhesion inhibitor or a blood platelet aggregation inhibitor for many hours, showing the same activity as that of free peptide but hardly hydrolyzable with protease in vivo by being made into this complex. CONSTITUTION:This peptide complex comprises a peptide derivative of formula (1) or its salt as a guest molecule. (1) [Z]-Arg-X-Asp-[Y]. (Arg is L-or D-arginine residue; Asp is L-aspartic acid residue; X is glycine, L or D-form leucine, isolucine, norlecine, phenylalaning or phenylglycine residue; Z and Y are glycine, L-serine, L-threonine, D-or L-aspartic acid, Lalanine, D-or L-glutamic acid or L-proline residue or peptide residue of its combination, respectively.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide complex having an adhesive peptide derivative or a salt thereof as a guest molecule, an animal cell adhesion inhibitor and a platelet aggregation / adhesion suppressor containing the peptide complex as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Fibronectin is a protein involved in cell-extracellular matrix adhesion and is also considered to be involved in platelet aggregation and cancer metastasis. These interactions are mediated by a series of cell surface receptors. It has been revealed that these receptors specifically recognize the arginine-glycine-aspartic acid (hereinafter abbreviated as Arg-Gly-Asp) sequence in the fibronectin molecule which is a macromolecule with a molecular weight of about 250,000. It has been reported that the Arg-Gly-Asp tripeptide plays an important role in fibronectin interaction (Nature, 309, 30).
(1984)). Since then, studies have been carried out using oligos or polypeptides having Arg-Gly-Asp sequences, or attempts to enhance cell adhesion activity over the original sequences by changing constituent amino acids or cyclizing.

For example, a method of inhibiting platelet aggregation using various chain and cyclic oligopeptides having Arg-Gly-Asp sequence (Polymer Preprints,
Japan), 38, 3149 (1989), JP-A-2-174797), Arg-Gly-
A method of using a peptide having an Asp sequence as a cell migration inhibitor (Japanese Patent Application Laid-Open No. 2-4716), P immobilized Arg-Gly-Asp
Method of using MMA membrane as cell adhesion membrane (Proceedings of the Polymer Society of Japan
(Polymer Preprints, Japan), 37, 705 (1988)). Further, a covalently bound peptide having Arg-Gly-Asp as an essential constituent unit to the polymer, an animal cell culture substrate,
Method for use as substrate for bio-composite artificial organ (Japanese Patent Laid-Open No. 1-30
No. 9682, JP-A-1-305960), and a method of using a polypeptide having an Arg-Gly-Asp-Ser sequence as a platelet protective agent for extracorporeal blood is disclosed (JP-A-64-6217). Further, a method of suppressing cancer metastasis using an oligopeptide having an Arg-Gly-Asp sequence or a polypeptide having a repeating structure thereof is known (Int. J. Biol. Macromo).
L., 11, 23, (1989), ibid, 11, 226 (1989), Jpn.J. Cancer R
es., 60, 722 (1989)).

On the other hand, cyclodextrin is a cyclic oligosaccharide obtained by the action of an enzyme on starch and incorporates another molecule or atom into a hydrophobic internal cavity to form an inclusion compound. Important as a body host. The inclusion effect of cyclodextrin is widely used in fields such as food, agricultural chemicals, cosmetics, fragrances, pharmaceuticals, and chemical industry. Especially, it is often used as a pharmaceutical additive in pharmaceutical applications, and the solubility and stability of drugs are stable. , Used to improve absorbency.

Many compounds have been studied as a drug serving as a guest, but a peptide complex having a cell-adhesive peptide derivative or a salt thereof as a guest molecule has not been known, and these compounds can be introduced into blood. It can be expected to improve the solubility of erythrocyte and prolong the life in blood.

[0006]

OBJECT OF THE INVENTION An object of the present invention is to provide an animal cell adhesion inhibitor capable of maintaining a long-term effect by using an adhesive peptide derivative of the general formula [1] or a salt thereof as the active ingredient in a peptide complex. And to provide a platelet aggregation / adhesion suppressant.

[0007]

The compound of the present invention is a complex of a peptide derivative defined by the following general formula [1] and a host molecule, and the ionic group present in each molecule forms a salt with a suitable ion. You may.

General formula (1) [Z] -Arg-X-Asp- [Y]

In the formula, Arg represents an L- or D-arginine residue, and Asp represents an L-aspartic acid residue.
X represents a glycine, L- or D-form leucine, isoleucine, norleucine, phenylalanine, or phenylglycine residue. [] Indicates that the residue may or may not be present, and if present, Z and Y are glycine and L
-Serine, L-threonine, D- or L-aspartic acid, L-alanine, D- or L-glutamic acid, L
Represents an amino acid residue selected from proline or a peptide residue in which the amino acid residues are combined. The peptide represented by the general formula (1) does not decrease its biological activity,
In addition, it may be linked to any organic group that does not impair the water solubility of the entire molecule and is pharmacologically acceptable. Examples of such an organic group are preferably an alkyl group having 1 to 15 carbon atoms or an aryl group having 6 to 9 carbon atoms, a carbonyl group, a carboxyl group, an amino group, a hydroxyl group,
It may have a substituent such as a sulfonic acid group, a phosphoric acid group, a nitro group, a cyano group, or a halogen atom. It may also contain a double bond or a triple bond. For example, an adamantanecarbonyl group, a benzoyl group, a t-butoxycarbonyl group and the like can be linked. The peptide sequence represented by the general formula (1) of the present invention may be linked to an organic group via an appropriate alkylene or arylene group. In that case, the alkylene or arylene group preferably has a straight chain or a substituent having 1 to 15 carbon atoms, and is -O-, -NH-, -S-, ester bond, amide bond, urethane bond, urea bond. May be included. The end of the peptide not linked to the organic group is -H or -OH. As the host molecule, cyclodextrin, cyclodextrin derivative or the like can be used.

Examples of preferred salts of the compound of the present invention include sodium salt, potassium salt, ammonium salt, magnesium salt, hydrochloride salt, sulfate salt, nitrate salt and acetate salt.

Administration of the peptide complex used in the present invention
The method is the administration method generally used for peptide drugs.
Methods, ie parenteral administration methods, eg intravenous administration, muscle
It is preferable to administer by internal administration, subcutaneous administration and the like. So
In the case of producing an injectable preparation such as
Derivatives can be prepared, for example, from PBS (NaH ₂P
O_Four (5mM, NaCl 70mM) or physiological saline for injection
It may be used as a pharmaceutical preparation, or it may be
After dispersion, it may be used as a freeze-dried preparation. At this time,
A powder aid may be used. In addition, the stability of blood
A liposome or the like may be used as a carrier for the purpose of enhancement. Furthermore
In addition, the peptide complex of the present invention is, for example, in a liposome.
The included microcapsules can be administered orally.
With suppositories, sublingual agents, nasal sprays, etc.
If it is put into a state, it should be absorbed from mucous membranes other than the digestive tract.
Is also possible.

The peptide complex of the present invention has the general formula (1):
It has a cell-adhesive core sequence shown in, and adheres to cells through the core sequence by a mechanism similar to that of the cell-adhesive protein. Therefore, they exhibit various biological activities as agonists or antagonists of cell adhesive proteins. In addition, a wide range of biological activities such as immunoregulatory action, wound healing action, inhibition of platelet aggregation / adhesion by cancer cells occurring in capillaries, and neurological disease healing action are recognized. Therefore, at least one of the peptide derivatives of the present invention is used as a cancer metastasis inhibitor, wound healing agent, immunosuppressive agent, platelet aggregation inhibitor or neurological disease therapeutic agent together with a conventional carrier or pharmaceutical preparation, if necessary. It is possible to administer. Particularly, it is preferably used as an animal cell adhesion inhibitor or a platelet aggregation / adhesion suppressor. The dose is about 0.
It is determined in the range of 2 μg / kg to 400 mg / kg based on symptoms, age, body weight and the like.

The peptide synthesis method is not particularly limited, but includes a liquid phase method, a solid phase method, and a synthetic method using an automatic synthesizer. For more information on these synthetic methods,
Biochemistry Laboratory Course "Protein Chemistry IV" p207-49
5 (Biochemistry Society of Japan, Tokyo Kagaku Doujin), “Sequence Biochemistry Experiment Course, Protein Chemistry (below)” (Japan Biochemical Society, Tokyo Kagaku Dojin), Izumiya et al. “Basics and Experiments of Peptide Synthesis”
(Maruzen). It is also possible to use a commercially available synthetic peptide. Cyclodextrins are known to form inclusion compounds with various compounds, and their chemical and physical properties are "Cyclodextrins and
See their inclusion complexes ", J. Szejtli (1982). As cyclodextrin derivatives, O-dimethylcyclodextrin, O-trimethylcyclodextrin, hydroxypropylcyclodextrin, epichlorohydrin-bridged polycyclodextrin and the like are commercially available. It is available and can be used.

The synthetic examples of the present invention are shown below, but the synthetic method of the present invention is not limited thereto.

Synthesis Example 1 Synthesis of Adhesive Peptide by Solid Phase Method Synthesis was carried out using a peptide synthesizer by the Merrifield method. The Boc group was used for protection of the α-amino group, which was cleaved from the resin and then purified by preparative HPLC (high performance liquid chromatography) to obtain an adhesive synthetic peptide showing a single peak.

[0016]

[Table 1] Table 1 Synthesis of cell adhesive peptides ────────────────────────────── Adhesive synthetic peptides Name Structural formula Abbreviations Yield ────────────────────────────── Peptide-1 Arg-Phe-Asp-Ser RFDS 34% Peptide-2 Arg- D-Phe-Asp-Ser RfDS 21% Peptide-3 Arg-pGly-Asp-Ser RpGDS ^* 17% Peptide-4 Arg-Leu-Asp-Ser RLDS 22% Peptide-5 Arg-Gly-Asp RGD 33% Peptide- 6 Arg-Gly-Asp-Ser RGDS 29% ────────────────────────────── ^* pG = phenylglycine

The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0018]

【Example】

Example 1 An example of preparation of the composite 1 of the present invention is shown below. Saturated solution of β-CD (cyclodextrin) (β-CD 185 mg
/ 10 ml) 10 ml, RFDS under nitrogen stream 15 m
Each 6 g was added in 6 batches to give a transparent solution, which was then concentrated to dryness with a rotary evaporator to obtain a white solid β-CD-RFDS complex (Complex 1).

Example 2 As in Example 1, a saturated aqueous solution of α-CD (α-CD
50 mg of RFDS was added to 5 ml of 1.45 g / 10 ml in 6 portions, and after confirming that the solution was completely dissolved, it was concentrated to dryness to obtain a white solid of α-CD-RFDS complex (complex. 2).

Example 3 As in Example 1, a saturated aqueous solution of γ-CD (γ-CD
(2.32 g / 10 ml) RFDS was added to 2 ml each in 40 mg portions in 6 batches, and after confirming that the solution was completely dissolved, it was concentrated to dryness to obtain a white solid of γ-CD-RFDS complex (complex. 3).

Example 4 The following is a test example of the compound of the present invention (measurement of cell adhesion inhibitory activity). The peptide derivative of the present invention inhibits cell adhesion to fibrolectin. For the measurement of the activity, a competition method which is widely used in the field of biochemistry can be basically used, and examples thereof include JP-A-1-309682 and JP-A
2-174797, Methods in Enzymology Vol. 82, 803 (1981) and the like.

1) Preparation of Adsorption Plate Commercially available human-derived fibrolectin or vitronectin (purchased from Cosmo Bio Inc.) was dissolved in 10 μg / ml with PBS (phosphate buffer), and 50 μl of the solution was added to 96 wells. The plate was placed in a polystyrene plate and kept warm at 4 ° C. overnight for coating. Next, for the purpose of preventing non-specific adsorption, bovine serum albumin (BSA 1%) was added, and the mixture was kept at 37 ° C. for 1 hour and then washed (PBS) to remove water sufficiently to prepare an adsorption plate.

2) Adhesion Inhibition Experiment 50 μl of the peptide derivative dispersion dispersed in Dulbecco's Modified Eagles Medium was placed in the plate prepared by the above method, and 50 μl of NRK49F (1 × 10 ⁶ cells / ml) suspension was placed therein.
In addition, the cells were allowed to adhere by incubating at 37 ° C for 1 hour. After washing with PBS three times to remove unadhered cells, the adhered cells were detached with 0.025% EDTA trypsin solution and stained with 0.2% trypan blue to count the number of cells. The results are shown in Tables 2 and 3.

[0024]

[Table 2] Table 2 Cell adhesion inhibition to fibronectin ────────────────────────────────── Concentration (mg / ml ) Additive compound 0 0.25 0.5 1.0 1.5 ────────────────────────────────── Complex 1 × △ ○ ○ ○ Composite 2 × △ ○ ○ ○ Composite 3 × △ ○ ○ ○ RGD × × × △ △ RGDS × △ ○ ○ ○ RFDS × △ ○ ○ ○ ────────────── ──────────────────── (cells / well); ○: 50 or less, △: 50-100,
×: 100 or more Aggregation inhibition rate (%) (1-T / T ₀ ) × 100 T ₀ : Permeability without addition of peptide derivative T: Permeability with addition of peptide derivative

[Table 3] Table 3 Inhibition of cell adhesion to vitronectin ───────────────────────────────── Concentration (mg / ml) Additive compound 0 10 50 100 300 ───────────────────────────────── Complex 1 × ○ ○ ○ ○ Complex 2 × ○ ○ ○ ○ Complex 3 × ○ ○ ○ ○ RGD × △ △ ○ ○ RGDS × ○ ○ ○ ○ RFDS × ○ ○ ○ ○ ────────────────── ─────────────── (cells / well); ◯: 100 or less, Δ: 100 to 200, ×: 200 or more Example 5 The following is a test example of the compound of the present invention (platelet). (Measurement of aggregation inhibitory activity). The inhibitory effect of the peptide derivative of the present invention on platelet aggregation in the IN VITRO system was assayed using human platelet-rich plasma. Experimental method 1/9 amount of 3.8% sodium citrate was added to fresh human blood and centrifuged (1000 rpm, 10 minutes) to separate the upper layer as platelet-rich plasma. To 200 μl of this plasma, 25 μl (max 1.5 mg / ml) of the peptide derivative was added, incubated at 37 ° C for 3 minutes, and then added with 25 μl of 20 to 50 μM ADP (adenosine diphosphate) solution or 200 μg / ml collagen solution to aggregate. Was evaluated by measuring the transmittance using an aggregometer. The results are shown in Table 4.

[0025]

[Table 4] Table 4 Inhibition of Platelet Aggregation ──────────────────────────────── Additive Compound Inhibitory Activity ADP Stimulation Collagen Stimulation ─ ─────────────────────────────── Complex 1 ○ ○ Complex 2 ○ ○ Complex 3 ○ ○ RGD △ ～ ○ △ to ○ RGDS ○ ○ RFDS ○ ○ ──────────────────────────────── IC ₅₀ (μg / ml); ○ : 40 or less, Δ: 80 to 40, ×: 80 or more

From these results, it was revealed that the complex of the cell adhesive adhesive peptide and cyclodextrin exhibits the same activity as the free peptide. In addition, it is expected that the complexation will make it less likely to be hydrolyzed by protease in vivo, and it is expected that the effect can be maintained for a long time as a cell adhesion inhibitor or a platelet aggregation inhibitor.

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Claims (4)

[Claims] 1. A peptide complex comprising a guest molecule and a host molecule, and a peptide represented by the following general formula (1) or a salt thereof as a guest molecule. General formula (1) [Z] -Arg-X-Asp- [Y] In the formula, Arg represents an L- or D-arginine residue,
Asp represents an L-aspartic acid residue. X represents a glycine, L- or D-form leucine, isoleucine, norleucine, phenylalanine, or phenylglycine residue. [] Represents that it may or may not be present, and when present, Z and Y are glycine, L-serine, and
L-threonine, D- or L-aspartic acid, L-
It represents an amino acid residue selected from alanine, D- or L-glutamic acid and L-proline, or a peptide residue in which the amino acid residues are combined. The peptide represented by the general formula (1) may be linked to any organic group that does not impair its biological activity, does not impair the water solubility of the entire molecule, and is pharmacologically acceptable. The peptide sequence represented by the general formula (1) of the present invention and the organic group may be linked via a suitable alkylene and / or arylene group. In that case, the alkylene and arylene groups are-
O-, -NH-, -S-, ester bond, amide bond,
It may contain a urethane bond and / or a urea bond. 2. The peptide complex according to claim 1, wherein the host molecule is cyclodextrin or a cyclodextrin derivative. 3. A drug for inhibiting the adhesion of animal cells, which comprises the peptide complex according to claim 1 as an active ingredient. 4. A platelet aggregation / adhesion suppressant comprising the peptide complex according to claim 1 as an active ingredient.