JPH1036394A - New peptide and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide producible by the hydrolysis of natural substance such as albumen, having a specific amino acid sequence, exhibiting excellent artery relaxing action and hypotensive action and useful as a hypotensor, an agent for the treatment of stenocardia and myocardial infarction, an agent for improving heart failure, etc. SOLUTION: This new peptide is expressed by the formula H-Arg-Ala-Asp- His-Pro-Phe-OH, exhibits excellent artery relaxing action and hypotensive action and is useful as a hypotensor for the prevention, treatment and diagnosis of hypertension and various states of hypertension, an agent for the treatment of stenocardia and myocardial infarction, an agent for improving the condition of congestive heart failure, etc. The new protein can be produced by diluting albumen of hen's egg with distilled water, adjusting to pH7.4, adding chymotrypsin, hydrolyzing at 37 deg.C for 3hr, concentrating by centrifugal separation after cooling and purifying by high-performance liquid chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having an arterial relaxing action and useful as a blood pressure lowering agent, and a method for producing the same.

[0002]

2. Description of the Related Art It is known that proteins contained in foods have various physiological activities as well as nutritional effects.
For example, there are several peptides that exhibit blood pressure lowering activity. Such effects are mainly caused by relaxing blood vessels or inhibiting angiotensin converting enzyme. As the former peptide having a vasorelaxant action, for example, a peptide derived from bovine serum albumin is known (JP-A-4-99798).

[0003]

However, it is extremely rare that a peptide having such an activity is found in a natural product, and as angiotensin converting enzyme inhibitors, teprotide obtained from a little Brazilian or Japanese snake venom ( Nonapeptide, SQ20881), and a metabolite IS83 of actinomycetes belonging to the genus Streptomyces
No. 177920).

As an angiotensin converting enzyme inhibitor obtained by enzymatically treating a natural product, peptides and the like obtained by decomposing milk casein with tryptocin are known (JP-A-58-109425). 59-4
No. 4323, No. 59-44324, No. 61-3622
No. 6, No. 61-36227) The development of new inhibitors is being demanded.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have intensively searched for a natural substance which exhibits an arterial relaxing action with few side effects and has a blood pressure lowering action.
The presence of a substance having such an activity in a composition obtained by hydrolyzing a protein, particularly egg white with a specific enzyme, is determined.
H-Arg-Ala-Asp-His-Pro-Phe
The present inventors have found that the peptide has a structure of -OH and completed the present invention.

The H-Arg-Ala-Asp-H of the present invention
The peptide is-Pro-Phe-OH is a novel peptide not described in the literature, and is produced by hydrolyzing ovalbumin with chymotrypsin. In practical use, the composition may be used as it is, or if necessary. Used after purification. Furthermore, it can also be produced by synthesizing by applying conventional means of peptide synthesis. Arg referred to above is arginine, Ala
Means alanine, Asp means aspartic acid, His means histidine, Pro means proline, Leu means leucine, Phe means phenylalanine, and all such amino acids are in L-form.

The peptide of the present invention can be obtained by hydrolyzing a protein with chymotrypsin or by a peptide synthesis method. In order to hydrolyze a protein with chymotrypsin, the treatment method varies depending on the properties of the protein. If the protein is hardly soluble, mix the protein in hot water, homogenize with vigorous stirring, add a predetermined amount of chymotrypsin, and add a temperature of 10 to 85. The reaction is carried out at about ° C for 0.1 to 48 hours. As the protein, those derived from animals or microorganisms are arbitrarily used, and egg white is particularly useful.

In the case of isolation, the hydrolyzate is filtered by a known operation such as centrifugation. After the application of extraction, concentration, drying, etc., with or without application, differences in adsorption affinity for various adsorbents, differences in solubility or solubility in various solvents, two immiscible liquid phases It is preferable to isolate the target compound by applying a means utilizing a difference in distribution between molecules, a difference in elution rate based on the size of a molecule, a property of precipitation from a solution or a difference in deposition rate. These methods may be used alone as needed, or combined in any order and applied repeatedly.

The peptide of the present invention has a reactive carboxyl group corresponding to one of two fragments bisected at any position of the peptide bond by a method usually used for peptide synthesis, that is, a liquid phase method or a solid phase method. A raw material and a raw material having a reactive amino group corresponding to the other fragment are condensed using a carbodiimide method, an active ester method, or the like, and when a condensate to be formed has a protective group, the protective group is removed. Can also be manufactured.

In this reaction step, functional groups which should not participate in the reaction are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl, t-
Butyloxycarbonyl, p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like. Examples of the carboxyl group-protecting group include groups capable of forming an alkyl ester, a benzyl ester and the like.
-Bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.

After completion of the condensation reaction, the protecting group is removed,
In the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Further, the peptide of the present invention is purified according to a usual method. For example, ion exchange chromatography,
Examples include reversed-phase liquid chromatography and affinity chromatography.

The route of administration of the peptide used in the present invention may be any of oral administration, parenteral administration and rectal administration, but oral administration is preferred. The dose of the peptide of the present invention varies depending on the kind of the compound, the administration method, the symptoms and age of the patient, etc., but is usually 0.001 to 1000 mg, preferably 0.01 to 10 mg once to 3 times per day. is there. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a preparation carrier. As the pharmaceutical carrier, a substance which is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.

More specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium metasilicate aluminate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange Resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light silicic anhydride, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate , Glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbe Door, macrogol, vegetable oils,
Examples include wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water and the like.

Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like.
These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another appropriate medium at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the peptide of the present invention is prepared by dissolving the peptide in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, or may be added with a buffer or a preservative. Good.

These preparations contain the peptide of the present invention in a concentration of 0.1%.
It can be contained at a rate of 01% or more, preferably 0.5 to 70%. These formulations may also contain other therapeutically valuable components.

[0016]

The peptide of the present invention is a novel peptide which exhibits excellent arterial relaxation and hypotensive activity, and is useful for the prevention and treatment of hypertension such as essential hypertension, renal hypertension and adrenal hypertension. Diagnostic agents for diseases and antihypertensive agents used in various conditions, therapeutic agents for angina pectoris and myocardial infarction,
It is useful as an agent for improving the condition of congestive heart failure.

[0017]

Now, the present invention will be described more specifically below with reference to working examples. Example 1 [Peptide extraction] Chicken trypsin was diluted 5-fold with distilled water, and then chymotrypsin 0.2 mg / ml was added to a solution (containing 20 mg / ml protein) adjusted to pH 7.4 with 1N hydrochloric acid or INNaOH. (Manufactured by Sigma) and add 3
The hydrolysis reaction was performed at 7 ° C. for 3 hours. After cooling, the mixture is concentrated by centrifugation, and then subjected to high performance liquid chromatography (ODS-, PDS).
h- and CN-columns) to give the peptide. Use this product with a gas-phase protein sequencer (Applied
The amino acid sequence was analyzed by applying an automatic Edman degradation method using Biosystems 477 A) to obtain the following structure. H-Arg-Ala-Asp-His-Pr
o-Phe-OH The physical properties of the peptide are as follows.

[0018]

[Synthesis of Peptide] 0.55 g of commercially available Boc (butoxycarbonyl) -Phe-O-Resin [benzyl resin (substitution rate: 0.55 meq / g)] was placed in a reaction vessel of a peptide synthesis apparatus SAM TWO manufactured by Biosearch. And synthesized as follows. In methylene chloride containing 45% trifluoroacetic acid, 2.5% anisole, 2
After removing the Boc group by a reaction for 5 minutes, washing with methylene chloride, neutralization with methylene chloride containing 10% diisopropylethylamine, and washing with methylene chloride were performed. This and 5ml of 0.4M Boc-P
A mixture of ro-Phe in dimethylformamide and 5 ml of a 0.4 M solution of diisopropylcarbodiimide in methylene chloride were added to the reaction vessel, and the mixture was stirred and reacted at room temperature for 2 hours.

The obtained resin is washed with dimethylformamide, methylene chloride, methylene chloride containing 10% diisopropylethylamine, a mixed solution of methylene chloride and a mixture of methylene chloride and dimethylformamide, and Boc-Pro
-A Phe resin was obtained. Followed by removal of the same Boc group,
By repeating the coupling of Boc and the amino acid, Arg
(Tos tosyl group) -Ala-Asp-His (To
s) -Pro-Phe resin was obtained. The resin was stirred in 20 ml of hydrogen fluoride containing 10% anisole at 0 ° C. for 1 hour to release the peptide from the resin. Hydrogen fluoride was distilled off under reduced pressure, the residue was extracted with 30% acetic acid, and lyophilized to obtain a crude peptide. This is supplied to an ODS column (Cosmosi
l 5C ₁₈₎ and purified by reverse phase chromatography on, H-Arg-Ala-Asp -His-Pro-P
he-OH was obtained. The product was analyzed using the same protein sequencer as described above, and as a result, the product was found to have the above composition.

The physical properties of the peptide were the same as above. (Measurement of Canine Mesenteric Artery Relaxing Action) A spiral strip of the mesenteric artery isolated from a dog was treated according to the method of Takagi et al. (Edited by Takagi et al., Pharmacological Experiments, pp. 94-99, Nanzando). The specimen was connected to an isometric transducer in a Magnus tube. The peptide relative mesenteric arteries were contracted by 0.5μM prostaglandin F ₂ alpha of showed relaxation effect. ED ₅₀ (50% relaxation) at that time is 100μ
M.

(Measurement of Blood Pressure Fluctuation) Invasive Method After spontaneously hypertensive rats aged 15 to 20 weeks were anesthetized from the tail vein, a catheter for sample injection was inserted into the femoral vein, and a 5 mg / kg sample was collected. After administration, blood pressure was measured invasively by a pressure transducer. The decrease in blood pressure was about 20 mmHg.

Non-invasive method (oral administration) A sample emulsified with egg yolk in the above rat
Gavage was administered at a rate of 0 mg / kg using a probe needle. Then, the blood pressure of the tail artery was measured over time by the tail-cuff method, and the maximum blood pressure lowering value in the systolic blood pressure was measured to be 11 mmHg.

[0024]

Industrial Applicability The present invention can be prepared by hydrolyzing a natural product such as egg white, and can produce a novel peptide which is particularly useful as an antihypertensive or an antihypertensive food.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] July 26, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Correction contents]

The H-Arg-Ala-Asp-H of the present invention
The peptide is-Pro-Phe-OH is a novel peptide not described in the literature, and is produced by hydrolyzing ovalbumin with chymotrypsin. In practical use, the composition may be used as it is, or if necessary. Used after purification. Furthermore, it can also be produced by synthesizing by applying conventional means of peptide synthesis. Arg referred to above is arginine, Ala
Means alanine, Asp means aspartic acid, His means histidine, Pro means proline, Phe means phenylalanine, and all such amino acids are in L-form.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction contents]

[0017]

Now, the present invention will be described more specifically below with reference to working examples. Example 1 [Peptide extraction] Chicken trypsin was diluted 5-fold with distilled water, and then chymotrypsin 0.2 mg / L was dissolved in a solution (containing 20 mg / ml protein) adjusted to pH 7.4 with 1N hydrochloric acid or INNaOH. ml (Sigma) and add 3
The hydrolysis reaction was performed at 7 ° C. for 3 hours. After cooling, the mixture is concentrated by centrifugation, and then subjected to high performance liquid chromatography (ODS-,
h- and CN-columns) to give the peptide. Use this product with a gas-phase protein sequencer (Applied
The amino acid sequence was analyzed by applying an automatic Edman degradation method using Biosystems 477 A) to obtain the following structure. H-Arg-Ala-Asp-His-Pr
o-Phe-OH The physical properties of the peptide are as follows.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication location C12P 21/06 A61K 37/18

Claims (4)

[Claims] 1. H-Arg-Ala-Asp-His-
A novel peptide represented by Pro-Phe-OH. 2. H-Arg-Ala-Asp-His, wherein the protein is hydrolyzed with chymotrypsin.
-A method for producing a novel peptide represented by Pro-Phe-OH. 3. The method according to claim 2, wherein egg white is used as the protein. 4. The method according to claim 3, wherein chicken egg white is used as egg white.