JPH069687A - New peptide and blood platelet coagulation inhibitor using the same - Google Patents

Abstract

PURPOSE:To provide a new peptide useful as a platelet coagulation inhibitor, thus capable of preventing platelet thrombosis, thromboembolism, reobstruction, or cardiac infarction. CONSTITUTION:The peptide of formula X-Arg-Gly-Asp-Y-Z (X is 3-14C aliphatic residue; Y is hydrophobic amino acid residue; Z is OH or NH2) or a salt thereof, and the other objective blood platelet coagulation inhibitor containing, as active ingredient, the above-mentioned peptide or a salt thereof.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having a platelet aggregation inhibitory action, and a platelet aggregation inhibitor using the peptide.

[0002]

2. Description of the Related Art One of the roles of platelets present in blood is to prevent bleeding by adhering to the surface of damaged blood vessels and coagulating with each other. However, in a pathological environment, thrombus formation due to platelet aggregation and blood coagulation causes occlusion of blood vessels, making it impossible to supply oxygen and nutrients to tissues and organs, and ischemic diseases of the circulatory organ such as myocardial infarction and stroke. Cause. These cardiovascular diseases show the highest mortality rate after cancer, and are still a major social problem even today.

[0003] Platelets bind to connective tissue proteins such as collagen in subendothelium exposed by blood vessel damage and to platelet membrane receptors such as thrombin present in plasma.
Alternatively, it is activated by binding to an autocrine membrane receptor by the release of adenosine diphosphate (ADP), adrenaline, serotonin, thromboxane (TX) A2, etc. which are present in granules in platelets. Then, a glycoprotein that is a fibrinogen receptor is presented on the cell surface, and by forming a complex (gpIIbIIIa) thereof, it becomes possible to aggregate and causes aggregation via fibrinogen cross-linking.

[0004] In platelet asthenia (thrombasthenia) in which this gpIIbIIIa complex is congenitally deficient, it has no platelet aggregation ability and exhibits a strong bleeding tendency. Therefore, the binding of this complex to fibrinogen is essential for platelet aggregation. Ruoslahti et al., Science, 238 , 49
1 (1987). Also, Coller et al. Have reported that the F (ab ') ₂ fragment of a monoclonal antibody against gpIIbIIIa has a strong inhibitory effect on platelet aggregation in Blood,
68 , 783 (1986), by inhibiting the function of gpIIbIIIa,
It was clarified that a platelet aggregation inhibitor can be developed. Although this monoclonal antibody has the potential as a therapeutic drug, it is considered to be limited in its use due to the problem of antibody production due to repeated administration because of its high molecular weight protein. Therefore, if a low molecular weight compound having no immunogenicity is created as an antagonist to gpIIbIIIa, there is no problem in repeated administration, and its value as a therapeutic drug is very high. On the other hand, gp
A study on the binding of IIbIIIa to fibrinogen can be found in Ruo
Discovery of an amino acid sequence arginine-glycine-aspartic acid (RGD) common to cell adhesion molecules derived from a series of studies by slahti et al. Nature, 309 , 30-33 (1984)
Started on. From research on receptors that recognize RGD sequences, gpIIbIIIa is a receptor belonging to the integrin family that recognizes RGD sequences.
ood, 71 , 831-843 (1988), especially in the fibrinogen molecule in binding to fibrinogen.
Andrieux et al., J. Biol. Chem., 264 , 9258 have been shown to recognize and bind to two RGDF sequences.
-9265 (1989).

Also, Von Will which also has an RGD sequence
Ebrand factor, fibronectin, vitronectin, and thrombospondin are also known to bind to gpIIbIIIa Pytela et al., Science, 231 , 1559 (1986) or Cell, 42 , 439 (1985). From these findings, it is expected that a synthetic peptide containing an RGD sequence will inhibit the binding of gpIIbIIIa and fibrinogen to inhibit platelet aggregation, and some studies have already been conducted. In the study using the synthetic peptide GRGDSP, Plow et al., Pro, which completely inhibited ADP-activated platelet aggregation at 400 μM,
c, Natl, Acad, Sci, USA, 82 , 8057-8061 (1985). Also,
In RGDS, it was 80-90 at a concentration of 46-50 μM depending on the concentration.
% Inhibition of aggregation but addition of one amino acid residue
The activity was weak in DST. But RG
Plow showed 4-5 times stronger activity in DF than RGDS
Et al., Blood, 70 , 110-115 (1987) or Harfinest et al.,
Blood, 71 , 132-136 (1988).

N-terminal amino group of RGD peptide and Arg
Derivatives lacking the peptide bond between -Gly are disclosed in
-78653 and U.S. Pat. No. 4,952,562. A cyclic structure derivative having RGD is disclosed in Japanese Patent Laid-Open No. 3-1.
18331, JP2-174797 and JP2-6
2892 or WO91 / 01331. Further, tetrapeptide derivatives containing RGD are described in JP-A 1-190699, JP-A 2-62892 and EP 0422937, and US Pat. No. 4,952,562.

[0007]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel peptide having high ability to inhibit platelet aggregation, and
It is to provide a platelet aggregation inhibitor using the same.

[0008]

Means for Solving the Problems As a means for increasing the hydrophobicity of the RGD peptide, the present inventors have considered that by adding a fatty acid residue to the N-terminal amino group of the peptide, the hydrophobicity is improved. As a result of synthesizing various derivatives and intensively studying the platelet aggregation inhibitory activity, it was found that introducing a fatty acid residue into the N-terminal amino group leads not only to improvement of hydrophobicity but also to improvement of activity. Has been completed. Although there have been reports of formylation and acetylation of an arginine residue to the N-terminal amino group,
There are no reports of derivatives bound with fatty acids having longer carbon chains, and these compounds are novel.

[0009] In order to create a compound that can be used as a platelet aggregating agent, it is necessary to first develop a derivative having high activity, but in order to stabilize the compound in vivo and develop it as an oral agent, the absorption efficiency should be improved. Is also required. In terms of oral absorption, the RGD peptide has a large hydrophilicity due to its structure, and it is necessary to induce not only activity improvement but also hydrophobicity increase. Therefore, introduction of a fatty acid residue satisfies this purpose. is there.

That is, the present invention relates to a novel peptide having a platelet aggregation inhibitory activity, which is a peptide represented by the following formula or a salt thereof: X-Arg-Gly-Asp-Y-Z (where X is a carbon number of 3- 14 is a fatty acid residue, Y is a hydrophobic amino acid residue, and Z is a hydroxyl group or NH _2. )

As the chain length of the fatty acid residue represented by X in the above formula, those having 4 to 7 carbon atoms are highly active and preferred. As the hydrophobic amino acid residue, tryptophan residue, phenylalanine residue and tyrosine residue are preferable. Regarding the upper limit of the carbon number of the fatty acid residue, the water solubility of the peptide tends to decrease as the carbon number increases, and as a result of the activity measurement, linear or branched fatty acids having up to 14 carbon atoms are preferable.

In the present specification, amino acids, peptides,
Abbreviations for protecting groups, active groups, etc. shall be in accordance with the provisions of the International Union of Pure and Applied Chemistry (IUPAC), International Union of Biochemistry (IUB), or the conventional symbols in the relevant fields. . When amino isomers and the like may have optical isomers, the L form is shown unless otherwise specified.

Arg or R: Arginine Asp or D: Aspartic acid Gly or G: Glycine Ser or S: Serine Thr or T: Threonine Trp or W: Tryptophan Tyr or Y: Tyrosine Pro or P: Proline Boc: t-butoxycarbonyl Bu ^t: t-butyl OBu ^t: t-butyl ester Mtr: 4 - methoxy -2,3,6 - trimethylbenzene sulfonyl Fmoc: 9 - peptides of-fluorenylmethoxycarbonyl present invention, easily available commercial amino acids It can be easily synthesized by using. The peptides of the present invention can be prepared by methods commonly used in peptide chemistry, eg, “The Peptides” first.
Volume 〔Schroder and Luhke, Academic Press, New Yor
k, USA (1966)], "Basics and experiments of peptide synthesis"
[Nobuo Izumiya et al., Maruzen Co., Ltd. (1985)] and the like, and can be produced by both the liquid phase method and the solid phase method.

As a condensation method for forming a peptide bond, an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a carbodiimide (DCC) method, a carbodiimide-additive method, an active ester method, a carbonylimidazole method. , Redox method, enzyme method, Woodward reagent K
And the like. When the peptide chain is extended by the solid phase method, the C-terminal amino acid is bound to a support that is insoluble in an organic solvent, such as a resin. A resin with a functional group introduced to bind an amino acid to the resin, a resin with a spacer inserted between the resin and the functional group, and a resin with a chain called a handle that can be cleaved at various points depending on conditions. Is used according to the purpose. Examples of such resins include halomethyl resins such as chloromethyl resin,
Examples thereof include oxymethyl resin, 4- (oxymethyl) -phenylacetamidomethyl resin, 4- (oxymethyl) -phenoxymethyl resin, and C-terminal amidation resin.

The condensation method in the solid phase method is mainly the DCC method,
The acid anhydride method and the active ester method are used. Before carrying out the condensation reaction, a carboxyl group, amino group or the like not involved in the reaction may be protected or a carboxyl group or amino group involved in the reaction may be activated by a means known per se.

Examples of the protecting group for the carboxyl group include methyl, ethyl, benzyl, p-nitrobenzyl,
Esters such as t-butyl and cyclohexyl can be mentioned. Examples of the amino group-protecting group include a benzyloxycarbonyl group, a t-butoxycarbonyl group, an isobornyloxycarbonyl group and a 9-fluorenylmethoxycarbonyl group.

Examples of the protecting group for the guanidino group in the arginine residue include, for example, nitro group, tosyl group, mesitylene sulfonyl group, 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, 2,2,5,7. , -Pentamethylchroman-6-sulfonyl group and the like can be mentioned. Examples of activated carboxyl groups include corresponding acid anhydrides, azides, active esters [alcohols (eg,
Pentafluorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol,
N-hydroxysuccinimide, N-hydroxy-5-
Norbornene-2,3-dicarboximide, N-hydroxyphthalimide, 1-hydroxybenzotriazole)
Ester] and the like. Examples of activated amino groups include the corresponding phosphoric acid amides.

The condensation reaction is usually carried out in a solvent, and examples of the solvent include chloroform, dichloromethane, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, N-methylpyrrolidone, water, Solvent such as methanol,
Alternatively, a mixture of these can be used. The reaction temperature can be carried out in the range of about -30 ° C to about 50 ° C which is generally used.

The reaction for eliminating a protective group of the peptide of the present invention may be any method as long as it does not affect the peptide bond and the protective group is removed, though it depends on the type of the protective group used. Examples of the method for removing the protective group include hydrogen chloride and
Acid treatment with hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof, base treatment with sodium hydroxide, potassium hydroxide, hydrazine, diethylamine, biperidine, etc., liquid Examples thereof include reduction in sodium in ammonia and palladium carbon, and silylating agents such as trimethylsilyl triflate and trimethylsilyl bromide. In the deprotection group reaction by treatment with the acid and the silylating agent, it is effective to add a cation scavenger such as anisole, phenol, cresol, thioanisole and ethanedithiol.

As a method for cleaving the peptide synthesized by the solid phase method from the resin, treatment with the above-mentioned acid and silylating agent can be mentioned. The peptide of the present invention thus produced, after the reaction is completed, a peptide separation means known per se,
For example, it can be obtained by extraction, distribution, reprecipitation, recrystallization, column chromatography and the like.

Further, the peptide of the present invention can be obtained in the form of a salt depending on the conditions of the method. As the salt, inorganic acid salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acid salts such as formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, citric acid, trifluoroacetic acid, or sodium, It means various salts such as alkali metal salts such as potassium, alkaline earth metal salts such as calcium salt, amine salts such as ammonium, organic amines such as ethanolamine, triethylamine and dicyclohexylamine.

When the peptide is clinically applied as a platelet aggregation inhibitor, the peptide is used as an active ingredient,
A solid or liquid pharmaceutical carrier or diluent, i.e.
It is preferable to prepare a preparation containing additives such as an excipient and a stabilizer. In the pharmaceutical preparation, the ratio of the active ingredient to the carrier component can be varied from 1 to 90% by weight. The dosage form and administration form may be granules, fine granules, powders, tablets, capsules, pills, liquids, or the like, or may be orally administered as the bulk or intravenously as an injection. It may be given intramuscularly, intramuscularly or subcutaneously. Alternatively, it may be made into powder for injection and prepared at the time of use.

A pharmaceutical organic or inorganic solid or liquid carrier or diluent suitable for oral, enteral or parenteral administration can be used for preparing the platelet aggregation inhibitor of the present invention. Water, gelatin, lactose, starch, magnesium stearate, talc, animal and vegetable oils,
Benzyl alcohol, gum, polyalkylene glycol, petroleum resin, coconut oil, lanolin or any other carrier used in medicine can be used as the carrier of the platelet aggregation agent of the present invention. In addition, stabilizers, wetting agents, emulsifiers, changing the osmotic pressure, and the appropriate pH of the compounding agent.
It is also possible to appropriately use salts for maintaining the above as an auxiliary drug.

Furthermore, the platelet aggregating agent of the present invention is used as another pharmaceutically effective ingredient which can be appropriately administered together with the platelet aggregating agent of the present invention in the treatment of various diseases, for example, another suitable platelet aggregation inhibitor. You may contain the agent. In the case of granules, fine granules, powders, tablets and capsules, the platelet aggregating agent of the present invention preferably contains 5 to 80% by weight of the above active ingredient, and in the case of liquid preparations, the corresponding The amount (ratio) is preferably 1 to 30% by weight. For parenteral administration, 0.1-10% by weight for injection
Is preferred.

In the case of oral administration, it is preferable to orally administer a daily dose of 500 to 1000 mg to the adult as a clinical dose, but the dose may be appropriately increased or decreased depending on the age and symptoms. The daily dose of the platelet aggregation inhibitor of the present invention is
Once a day or at appropriate intervals 2 or 3 a day
It is preferable to administer in divided doses. When used as an injection, the above-mentioned effective ingredient should be 1 for adults.
It is preferred to administer a dose of 1-10 mg.

Regarding the acute toxicity test of the peptide of the present invention, no toxicity was observed in mice after intravenous administration of 100 mg / kg.

[0027]

EXAMPLES The present invention will be described in detail below with reference to Examples, Comparative Examples, Experimental Examples and Preparation Examples. However, the technical scope of the present invention is not limited by these examples.

[0028]

Example 1 CH ₃ (CH ₂ ) ₄ CO-Arg-Gly-Asp-Trp-NH
₂ synthetic C-terminal amidated _{(CH 3 O-Ph (1,4} ) -CH (NH-Fmoc) -Ph (1,
_{4) -O (CH 2) 3} CONH-CH (CH 3) -CONH-CH 2 -Ph-polymer)
Resin (manufactured by BACHEM, introduction amount of Trp: 0.6 mmol / g)
Transfer of 0.45g of (0.25 mmol) to the reaction vessel, shaken shown in Table 1, repeated filtration _{step, NH 2 -Arg (Mtr) -Gly} -As
A p- (OBu ^t ) -Trp-C-terminated amidated resin was obtained. To 1 equivalent of this resin, 3 equivalents of caproic anhydride were condensed in dimethylformamide (DMF) in the presence of N-hydroxybenzotriazole (HOBT, 3 equivalents), and CH ₃ .CH ₂ ) ₄ CO-Arg ( Mt
was obtained ^{r) -Gly-Asp (OBu t} ) -Trp C -terminal amidated resin.

The resulting protected peptide resin was treated with 1M trimethylsilyl bromide and 1M thioanisole in the presence of m-cresol and ethanedithiol in trifluoroacetic acid to give 0.
It was treated at ° C for 1 hour. After the trimethylsilyl bromide was distilled off in a nitrogen stream, the resin was filtered off, diethyl ether was added to the filtrate under ice-cooling to obtain a peptide cleaved from the resin as a powder, and this powder was washed with diethyl ether. This was desalted by gel filtration using Sephadex G-10 as a support and freeze-dried to obtain a crude peptide. The obtained crude peptide was subjected to high performance liquid chromatography (HPL
C) Column: ODS 5C ₁₈ (μ bondasphere, 20 × 150 mm),
Mobile phase: (A) 0.1% TFA, (B) 100% CH ₃ CN / 0.1% TFA, gradien
t: (A): (B) = 80:20 to (A): (B) = 70:30, 20 minutes,
Purified at a flow rate of 17 mL / min, and then Sephadex G-25
And acetate by gel filtration to the supporting member, the title peptide CH ₃ by lyophilization _{(CH 2) 4 CO-Arg} -Gly-A
20 mg of acetate of sp-Trp-NH ₂ was obtained. Further, this acetate salt was dissolved in 3 ml of distilled water, and the pH was accurately adjusted to 7 with 1N sodium carbonate water, and then the mixture was poured into a gel filtration column having Sephadex G-10 as a support, and distilled water was added. The peptide CH ₃ (CH ₂ ) ₄ C was obtained by elution and freeze-drying of the peptide fraction.
It was obtained O-Arg-Gly-Asp- Trp-NH 2 16mg. All the peptides synthesized in the following Examples and Comparative Examples were prepared as acetate salts. Therefore, a peptide not specifically described indicates its acetate salt. Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 0.97 (1) Gly 0.97 (1) Trp ─ (1) Arg 1.00 (1) HPLC analysis Using Cosmosil 5C18-AR (4.6 × 200mm) column, flow rate 1 .
10-40% acetonitrile (60 min) in 0.1% TFA at 0 mL / min
Analytical HPLC with a gradient elution of 1 showed a single peak with a retention time of 31.5 minutes. FAB-MS : M + H calculated value 630.3, measured value 630

[0030]

[Table 1]

[0031]

Example 2 CH₃CH₂CO-Arg-Gly-Asp-Trp-NH₂of
Synthesis Propionic acid instead of caproic anhydride in Example 1
By a similar method using the anhydride, the title peptide CH₃C
H₂CO-Arg-Gly-Asp-Trp-NH₂15 mg was obtained. Amino acid analysis (6N HCl + phenol, 24hr, 110 ℃) Asp 1.18 (1) Gly 1.26 (1) Trp ─ (1) Arg 1.00 (1)HPLC analysis Using a Cosmosil 5C18-AR (4.6 x 200 mm) column, flow rate 1.
10-40% acetonitrile (60% in 60% TFA at 0 mL / min)
Min) with an elution HPLC gradient retention time of 20.5
It showed a single peak for minutes.FAB-MS : M + H calculated value 588.3, measured value 588

[0032]

Example 3 CH₃(CH₂)₂CO-Arg-Gly-Asp-Trp-NH₂
Synthesis of n-butyric anhydride instead of caproic anhydride in Example 1
The title peptide CH₃(CH₂)
₂CO-Arg-Gly-Asp-Trp-NH₂15 mg was obtained. Amino acid analysis (6N HCl + phenol, 24hr, 110 ℃) Asp 0.98 (1) Gly 1.06 (1) Trp ─ (1) Arg 1.00 (1)HPLC analysis Using a Cosmosil 5C18-AR (4.6 x 200 mm) column, flow rate 1.
10-40% acetonitrile (60% in 60% TFA at 0 mL / min)
Min) by gradient HPLC with retention time 24.0
It showed a single peak for minutes.FAB-MS : M + H calculated value 602.3, measured value 602

[0033]

EXAMPLE 4 _{CH 3 (CH 2) 3 CO} -Arg-Gly-Asp-Trp-NH 2
Synthesis of the title peptide CH ₃ (CH _{3) in} the same manner as in Example 1 except that n-valeric anhydride was used instead of caproic anhydride.
₂₎ to give the _{3 CO-Arg-Gly-Asp} -Trp-NH 2 10mg. Amino acid analysis (6N HCl + phenol, 24hr, 110 ℃) Asp 1.17 (1) Gly 1.24 (1) Trp ─ (1) Arg 1.00 (1) HPLC analysis Using a Cosmosil 5C18-AR (4.6 × 200 mm) column, flow rate 1 .
10-40% acetonitrile (60% in 60% TFA at 0 mL / min)
Min) by gradient HPLC with retention time 28.0
It showed a single peak for minutes. FAB-MS : M + H calculated 616.13, found 616

[0034]

EXAMPLE 5 _{CH 3 (CH 2) 5 CO} -Arg-Gly-Asp-Trp-NH 2
The title peptide CH was prepared in the same manner as in Example 1 except that n-heptanoic anhydride was used instead of caproic anhydride.
₃ (CH ₂₎ to give the _{5 CO-Arg-Gly-Asp} -Trp-NH 2 18mg. Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 1.15 (1) Gly 1.23 (1) Trp ─ (1) Arg 1.00 (1) HPLC analysis Using a Cosmosil 5C18-AR (4.6 × 200 mm) column, flow rate 1 .
10-40% acetonitrile (60% in 60% TFA at 0 mL / min)
Min) by gradient HPLC with retention time 35.0
It showed a single peak for minutes. FAB-MS : M + H calculated value 644.3, measured value 644

[0035]

Example 6 CH ₃ (CH ₂ ) ₆ CO-Arg-Gly-Asp-Trp-NH ₂
Synthesis of the title peptide CH in the same manner as in Example 1 except that n-octanoic anhydride was used instead of caproic anhydride.
₃ was obtained _{(CH 2) 6 CO-Arg} -Gly-Asp-Trp-NH 2 17mg. Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 0.89 (1) Gly 0.96 (1) Trp ─ (1) Arg 1.00 (1) HPLC analysis Using a Cosmosil 5C18-AR (4.6 × 200 mm) column, flow rate 1 .
10-40% acetonitrile (60% in 60% TFA at 0 mL / min)
Min) by gradient HPLC with retention time 39.0
It showed a single peak for minutes. FAB-MS : M + H calculated value 658.4, measured value 658.

[0036]

Example 7 _{CH 3 (CH 2) 7 CO} -Arg-Gly-Asp-Trp-NH 2
Synthesis of the title peptide CH in the same manner as in Example 1 except that n-nonanoic anhydride was used instead of caproic anhydride.
₃ was obtained _{(CH 2) 7 CO-Arg} -Gly-Asp-Trp-NH 2 15mg. Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 1.00 (1) Gly 1.02 (1) Trp ─ (1) Arg 1.00 (1) HPLC analysis Using a Cosmosil 5C18-AR (4.6 × 200 mm) column, flow rate 1 .
10-40% acetonitrile (60% in 60% TFA at 0 mL / min)
Min) by gradient HPLC with retention time 43.0
It showed a single peak for minutes. FAB-MS : M + H calculated value 672.4, measured value 672

[0037]

Example 8 CH ₃ (CH ₂ ) ₁₀ CO-Arg-Gly-Asp-Trp-NH
Synthesis of _{2 By} a similar method using n-dodecanoic anhydride instead of caproic anhydride in Example 1, the title peptide CH
₃ (CH ₂₎ to give the _{10 CO-Arg-Gly-Asp} -Trp-NH 2 24mg. Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 1.00 (1) Gly 1.02 (1) Trp ─ (1) Arg 1.00 (1) HPLC analysis Using a Cosmosil 5C18-AR (4.6 × 200 mm) column, flow rate 1 .
10-40% acetonitrile (60% in 60% TFA at 0 mL / min)
Min) by gradient HPLC with retention time 55.0
It showed a single peak for minutes. FAB-MS : M + H calculated value 714.4, measured value 714.

[0038]

Example 9 CH ₃ (CH ₂ ) ₁₂ CO-Arg-Gly-Asp-Trp-NH
In the same manner used in place n- tetradecanoate anhydride caproic acid anhydride in Synthesis Example 1 of _2, the title peptide _{CH 3 (CH 2) 12 CO} -Arg-Gly-Asp-Trp-NH 2 25mg Got Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 1.00 (1) Gly 1.01 (1) Trp ─ (1) Arg 0.99 (1) HPLC analysis Using Cosmosil 5C18-AR (4.6 × 200mm) column, flow rate 1 .
20-50% (60% acetonitrile) in 0.1% TFA at 0 mL / min.
Min) by gradient HPLC with retention time 50.0
It showed a single peak for minutes. FAB-MS : M + H calculated value 742.5, measured value 742.

[0039]

Example 10 CH ₃ (CH ₂ ) ₄ CO-Arg-Gly-Asp-Phe-OH
Of P-alkoxybenzyl alcohol resin (Phe introduction amount: 0.78 mmol / g resin) into which Fmoc-Phe-OH was introduced,
By a method similar to Example 1, the title peptide CH ₃ (CH
₂ ) ₄ CO-Arg-Gly-Asp-Phe-OH 20 mg was obtained. Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 0.87 (1) Gly 0.99 (1) Phe 0.99 (1) Arg 1.00 (1) HPLC analysis Cosmosil 5C1B-AR (4.6 × 200 mm) column was used and the flow rate was
10-40% acetonitrile in 0.1% TFA at 1.0 ml / min
Retention time 3 by analytical HPLC with gradient elution (60 min)
It showed a single peak at 4.5 minutes. FAB-MS : M + H calculated value 592.3, measured value 592

Example 11 CH ₃ (CH ₂ ) ₄ CO-Arg-Gly-Asp-Trp-OH
Of P-alkoxybenzyl alcohol resin (Trp introduction amount: 0.78 mmol / g resin) into which Fmoc-Trp-OH was introduced,
By a method similar to Example 1, the title peptide CH ₃ (CH
₂ ) 20 mg of ₄ CO-Arg-Gly-Asp-Trp-OH was obtained. Amino acid analysis (6N HCl + phenol, 24hr, 110 ℃) Asp 0.86 (1) Gly 1.00 (1) Trp- (1) Arg 1.07 (1) HPLC analysis Flow rate using a Cosmosil 5C18-AR (4.6 × 200 mm) column
10-40% acetonitrile in 0.1% TFA at 1.0 ml / min
Retention time 3 by analytical HPLC with gradient elution (60 min)
It showed a single peak at 1.0 min. FAB-MS : M + H calculated value 631.3, measured value 631

Example 12 CH₃(CH₂)_FourCO-Arg-Gly-Asp-Tyr-OH
Synthesis of P-alkoxybenze with Fmoc-Tyr (Bu) -OH introduced
Use alcohol alcohol resin (Tyr introduction amount: 0.88mmol / g resin)
In the same manner as in Example 1, the title peptide CH
₃(CH₂)_Four11 mg of CO-Arg-Gly-Asp-Tyr-OH was obtained.Amino acid analysis (6N HCl + phenol, 24hr, 110 ℃) Asp 1.00 (1) Gly 1.00 (1) Tyr 1.00 (1) Arg 0.98 (1)HPLC analysis Flow rate 1.0 ml using μBondapak C18 (3.9 × 300 mm) column
/ Min, 0-40% acetonitrile in 0.1% TFA (60 minutes)
Retention time 37.2 min by analytical HPLC with gradient elution of
It showed one peak.FAB-MS : M + H calculated value 607.7, measured value 608Comparative Example 1 HCO-Arg-Gly-Asp-Trp-NH₂Synthesis of carboxylic acid in place of formic acid anhydride in Example 1
By the same method using DIPCD as a mixture, the title
Peptide HCO-Arg-Gly-Asp-Trp-NH₂Get 10 mg
It was Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) Asp 0.85 (1) Gly 0.77 (1) Trp- (1) Arg 1.00 (1)HPLC analysis A Cosmosil 5C18-AR (4.6 x 200 mm) column was used with a flow rate of 1.0.
10-40% acetonitrile (60% acetonitrile in 0.1% TFA at ml / min)
Min), an analytical HPLC with a gradient elution of 13.5 min.
It showed a single peak for minutes.FAB-MS : M + H calculated value 560.3, measured value 560Comparative example 2 CH₃CO-Arg-Gly-Asp-Trp-NH₂Synthesis of acetic anhydride was used instead of cabroic acid anhydride in Example 1.
The title peptide CH₃CO-Arg-Gl
y-Asp-Trp-NH₂ 22 mg was obtained. Amino acid analysis (6N HCl
+ phenol, 24hr, 110 ℃) Asp 1.19 (1) Gly 1.25 (1) Trp- (1) Arg 1.00 (1)HPLC analysis Flow rate using a Cosmosil 5C18-AR (4.6 x 200 mm) column
10-40% acetonitrile in 0.1% TFA at 1.0 ml / min
Retention time 1 by analytical HPLC with gradient elution (60 min) 1
It showed a single peak at 7.0 minutes.FAB-MS : M + H calculated value 574.3, measured value 574Comparative Example 3 H-Arg-Gly-Asp-Trp-NH₂Synthesis of the title peptide H-Arg by a method similar to Example 1.
-Gly-Asp-Trp-NH₂ 5 mg was obtained.Amino acid analysis (6N HCl + phenol, 24hr, 110 ℃) Asp
 1.29 (1) Gly 1.27 (1) Trp- (1) Arg 1.00 (1)HPLC analysis Using a Cosmosil 5C18-AR (4.6 x 200 mm) column, flow rate 1.
0-30% (60% acetonitrile) in 0.1% TFA at 0 ml / min.
Min) gradient elution with analytical HPLC retention time 29.8 min
Showed a single peak of.FAB-MS : M + H calculated value 532.3, measured value 532

[0040]

Test Example 1 Activity Measurement of Synthetic Peptide [In-vitro Human Platelet Aggregation Using PRP] A healthy man who had not taken any drug for at least 2 weeks was used as a subject. Blood is collected from No. 19 needle and 1/10 volume of 3.8
Blood was collected from the vein of the inferior sac on an empty stomach using a plastic syringe containing a sodium citrate solution in advance. Immediately after blood collection, the two solutions were mixed by gently stirring the syringe. The blood was centrifuged at room temperature for 15 minutes (11
(00 rpm, 250 × g), after stopping the rotation without applying the brake,
The supernatant was taken with a Komagome pipette to obtain platelet rich plasma (PRP) and stored at room temperature. Remaining blood after centrifugation at room temperature
Centrifuge for 15 minutes (3500 rpm, 1500 × g), stop without braking and collect the supernatant.
P). After the PRP was prepared, the number of platelets was counted, and the experiment described below was carried out only when the number of platelets was 2 × 10 ⁸ / ml or more.

Platelet aggregation is measured by an 8-channel platelet aggregometer (Hematracer, Nikoh Bioscience, Tokyo, Japan).
Was measured from the change in the light transmittance of PRP. First, 200 μl of PPP and PRP were placed in a glass cuvette and incubated at 37 ° C., and then the transmittance was measured to set the transmittance of PPP to 100% and the transmittance of PRP to 0%. next,
Saline or saline containing sample for PRP
After adding 10 μl and incubating at 37 ℃ for 1 minute,
Add 10 μl of 100 μg / ml collagen solution (final concentration 5
(μg / ml) aggregation was induced, and then the permeability was measured for 7 minutes. In the experiment, it was first confirmed that aggregation was caused by using collagen and ADP, and only those having a maximum aggregation rate of collagen of 70% or more were used in the experiment.

Sample is 2.2x10^-2Physiological diet to be M
Dissolve in salt water and prepare a 2-fold dilution series based on this
Used for. 10% for samples insoluble in saline
Dissolved in physiological saline containing DMSO (Dimethyl sulfoxide)
I understand. The result is calculated as follows.Create a diagram that plots the aggregation inhibition rate against the sample concentration
From this figure, the concentration (IC₅₀) Is calculated
did. To prevent data variations due to individual differences among subjects
Therefore, always use a positive control for each experiment.
Indomethacin and CH₃(CH₂)_FourCO-Arg-Gly-Asp-Trp
-NH₂2 samples of
The inhibitory activity of each sample was evaluated as the sex. Table 2 shows each service
Sample IC ₅₀Indicates.

[0043]

[Table 2]

As shown in Table 2, the peptide of the present invention showed higher activity than that of the peptide of Comparative Example, and a peak of activity was particularly observed when the fatty acid residue had 4 to 7 carbon atoms. This indicates that higher activity can be obtained when a fatty acid residue having a certain length is bound, as compared with an N-terminal having an amino group or an acetylated product. Also, at 8 carbon atoms, the activity seems to be weakened in this test method from the viewpoint of solubility of the peptide, but in oral absorption,
The higher the hydrophobicity is, the higher the absorption efficiency is, and thus the high activity can be expected.

C 9-14 synthesized in the previous example
A little activity was also observed for the peptide of. However, as the hydrophobicity increased, it became difficult to dissolve in the sample solution used for the activity measurement, and it was difficult to accurately determine the activity by the activity assay method used this time. However, it is fully expected that the activity will be exerted in vivo and at the time of oral administration for the same reason as the above-mentioned carbon number 8.

[0046]

Test Example 2 Acute Toxicity Test Regarding the acute toxicity test of the peptide of the present invention, 100 mg / kg of the peptide was intravenously administered to a mouse and observed for 1 week. As a result, no toxicity was observed.

[0047]

[Formulation Example 1] Injectable formulation 100 mg of the peptide obtained in Example 1 was added to physiological saline 0.1 L.
The resulting solution was aseptically filled in a 2.5 ml ampoule and sealed to give a preparation for injection.

[0048]

[Formulation Example 2] Granules 500 mg of the peptide obtained in Example 1, 50 m of crystalline cellulose
1 ml of a mixed solution of ethanol and water was added and kneaded to a mixture of g and lactose 450 mg. This kneaded product was granulated by a granulator according to a conventional method to give granules.

[0049]

INDUSTRIAL APPLICABILITY The novel peptide of the present invention is useful as a platelet aggregation inhibitor. Prevention of thrombosis, thromboembolism and reocclusion during and after thrombolysis treatment and prevention of thrombosis, thromboembolism and reocclusion after angioplasty of coronary arteries and other arteries and after coronary artery bypass treatment ,
Moreover, myocardial infarction and the like can be prevented.

[0050]

[Sequence listing] SEQ ID NO: 1 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics: Xaa is Trp, Phe or Tyr 1 with a fatty acid residue of 3-14 carbon atoms Group is bonded to 4 OH or NH ₂ is bonded

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Yoshio Hayashi 1618 Ida, Nakahara-ku, Kawasaki-shi, Kanagawa Shin Nippon Steel Co., Ltd. (72) Inventor Atsushi Katada 1618 Ida, Nakahara-ku, Kawasaki-shi, Kanagawa Nippon Steel Co., Ltd.

Claims (4)

[Claims] 1. A peptide represented by the following formula or a salt thereof. X-Arg-Gly-Asp-Y-Z (In the formula, X represents a fatty acid residue having 3 to 14 carbon atoms, Y represents a hydrophobic amino acid residue, and Z represents a hydroxyl group or NH _2. ) 2. The peptide or salt thereof according to claim 1, wherein the fatty acid residue has 4 to 7 carbon atoms. 3. The peptide or a salt thereof according to claim 1, wherein the hydrophobic amino acid residue is a tryptophan residue, a phenylalanine residue or a tyrosine residue. 4. A platelet aggregation inhibitor comprising the peptide according to any one of claims 1 to 3 or a salt thereof as an active ingredient.