JPH06277091A - New peptide, its production and use thereof - Google Patents

Abstract

PURPOSE:To produce a new peptide useful as an antihypertensive agent, having inhibitory action on an angiotensin converting enzyme. CONSTITUTION:This new peptide is shown by the formula H-Val-Arg-Tyr-OH and is obtained by hydrolyzing a casein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention provides a novel peptide having the following structure, and relates to a peptide useful as an angiotensin converting enzyme inhibitor and the like. H-V
al-Arg-Tyr-OH

[0002]

BACKGROUND OF THE INVENTION Angiotensin-converting enzyme exists mainly in lung, vascular endothelial cells and renal proximal tubules, and is angiotensin I (Asp-Arg-Val-Tyr-Ile-
It acts on His-Pro-Phe-His-Leu) to induce dipeptide (Hi) from the C-terminal of angiotensin I.
It is an enzyme that cleaves and releases s ⁹ -Leu ¹⁰ ) to produce angiotensin II having a strong pressor action. In addition, this enzyme also has an action of destroying and inactivating bradykinin, which is an antihypertensive substance in vivo, and is strongly involved in the pressor system. It has been conventionally considered that if the activity of angiotensin converting enzyme is inhibited, it works to lower blood pressure and is clinically effective for prevention and treatment of hypertension. Recently, since the proline derivative captopril was synthesized and its antihypertensive activity was confirmed, various synthetic studies of angiotensin-converting enzyme inhibitory substances have been actively conducted, and their acquisition from natural products has been attempted. This is because an angiotensin converting enzyme inhibitor derived from a natural product is expected to be an antihypertensive agent with low toxicity and high safety since it is obtained from food or food raw materials.

[0003]

However, angiotensin-converting enzyme inhibitors found in natural products are extremely rare, such as teprotide (Nonapeptide, SQ20881) and Streptomyces obtained from Brazilian and Japanese snake venoms. Metabolite IS of actinomycetes belonging to the genus
No. 83 (Japanese Patent Laid-Open No. 58-177920) is only known. As angiotensin converting enzyme inhibitors obtained by enzymatic treatment of natural products, peptides obtained by decomposing milk casein with trypsin are known (Japanese Patent Laid-Open Nos. 58-109425 and 59-59425). -4
No. 4323, No. 59-44324, No. 61-3622.
No. 6, No. 61-36227) The development of new inhibitors is desired.

[0004]

[Means for Solving the Problems] The inventors of the present invention have eagerly searched for an angiotensin-converting enzyme inhibitor which is a natural substance and has few side effects in order to solve such a problem. The present invention was completed by finding the existence of a substance having angiotensin converting enzyme inhibitory activity in the product and finding that the substance is a peptide consisting of H-Val-Arg-Tyr-OH.

H-Val-Arg-Tyr-O of the present invention
The peptide consisting of H is a novel peptide which has not been published in the literature, and is produced by hydrolyzing a protein such as casein with thermolysin. In practice, the composition may be used as it is or purified as necessary. Then used. Furthermore, it can also be produced by synthesizing by applying a conventional method for peptide synthesis. In the above, Val means valine, Arg means arginine, Tyr means tyrosine, and all such amino acids are L-forms.

The peptide of the present invention can be obtained by hydrolyzing a protein with thermolysin or by a peptide synthesis method. To hydrolyze a protein with thermolysin, the treatment method varies depending on the property of the protein, but if it is poorly soluble, the protein is mixed with hot water and homogenized by vigorous stirring, and a predetermined amount of thermolysin is added, and the temperature is adjusted to 10-85. The reaction is performed at a temperature of about 0.1 to 48 hours. Any protein derived from animals or microorganisms may be used as the protein, and particularly useful one is casein. In addition to the peptide of the present invention, other peptides are present in the hydrolyzed solution, but these may be used as they are in a mixture for various purposes.
Only the peptide of the present invention may be isolated and used. In the case of isolation, the hydrolyzed solution is filtered by a known operation such as centrifugation. Then, after applying extraction, concentration, dryness, etc., or without doing so, the difference in the adsorption affinity for various adsorbents, the difference in solubility or solubility in various solvents, the two immiscible liquid phases It is preferable to isolate the target substance by applying a means utilizing a difference in distribution between the two, a difference in elution rate based on the size of the molecule, a precipitability from a solution or a difference in precipitation rate. These methods may be used alone, or may be combined in any order and vice versa.

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 commonly used for peptide synthesis, that is, a liquid phase method or a solid phase method. By condensing the raw material and the raw material having a reactive amino group corresponding to the other fragment using a carbodiimide method, an active ester method or the like, and when the resulting condensate has a protecting group, by removing the protecting group, Can also be manufactured.

Functional groups which should not participate in the reaction in this reaction step are protected by a protecting group. Examples of the amino-protecting group include benzyloxycarbonyl and t-
Butyloxycarbonyl, p-biphenylisopropyrooxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like can be mentioned. Examples of the protective group for the carboxyl group include a group capable of forming an alkyl ester, a benzyl ester and the like. In the case of the solid phase method, the carboxyl group at the C-terminal is chloromethyl resin, oxymethyl resin,
It is bound to a carrier such as P-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 terminus of the peptide and the resin is further cleaved. Furthermore, the peptide of the present invention is purified according to a conventional method. For example ion exchange chromatography,
Reverse phase liquid chromatography, affinity chromatography and the like can be mentioned. The administration route of the peptide used in the present invention may be any of oral administration, parenteral administration and intrarectal administration, but oral administration is preferred. The dose of the peptide of the present invention varies depending on the type of compound, administration method, patient's symptoms and age, etc., but usually 0.001-1
000 mg, preferably 0.01 to 10 mg is 1 to 3 times a day. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a carrier for preparation.

As the pharmaceutical carrier, substances commonly used in the pharmaceutical field and which do not react with the peptide of the present invention are used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose,
Hydroxypropyl starch, carboxymethyl cellulose calcium, ion exchange resin, methyl cellulose,
Gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid,
Magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, Examples include fluorocarbons, nonionic surfactants, 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. The liquid preparation may be dissolved or suspended in water or another suitable medium before use. The tablets and granules may be coated by a known method. In the case of an injectable preparation, it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution, if necessary, and a buffer or a preservative may be added. Good. These formulations can contain the peptide of the present invention in a proportion of 0.01% or more, preferably 0.5 to 70%. These formulations may also contain other therapeutically valuable ingredients.

[0012]

The peptide of the present invention is a novel peptide having excellent angiotensin-converting enzyme inhibitory action, and exhibits hypotensive action and bradykinin inactivation suppressing action, and is associated with hypertension such as essential hypertension, renal hypertension and adrenal hypertension. Disease prevention,
It is useful as a therapeutic agent, a diagnostic agent for these diseases, an antihypertensive agent used in various pathological conditions, an increase in the threshold value of angina attack, a decrease in myocardial infarction, and an agent for improving the pathological condition in congestive heart failure.

[0013]

[Production of peptide]

(A) 40 ml of water was added to 5 g of casein and homogenized sufficiently, 20 mg of thermolysin was added, and hydrolysis reaction was carried out at 37 ° C. and pH 7 for 3 hours. The peptide was boiled at 100 ° C for 10 minutes, cooled, centrifuged, concentrated, and purified by high performance liquid chromatography (ODS-, Ph- and CN-columns) to obtain a peptide. This product is a gas phase protein sequencer (Abride Biosystems 477 A
The amino acid sequence was analyzed by applying an automatic Edman degradation method using the (type) to obtain the following structure. H-Val-Arg-Tyr-OH

The physical properties of the peptide are as follows. TLC [n-butanol: acetic acid: pyridine: water = 15:
3:10:12] (Silica gel plate, ninhydrin coloration) Rf: 0.19

[Synthesis of Peptide] Commercially available Boc (butoxycarbonyl) -Tyr (Cl ₂ -Bzl) (dichlorobenzyl group) -O-Resin (substitution rate 0.75 meq
/ G) 0.4 g of peptide synthesizer S manufactured by Biosearch
It was collected in a reaction tank of AM2 and synthesized as follows.
After removing the Boc group by reaction in methylene chloride containing 45% trifluoroacetic acid and 2.5% anisole for 25 minutes, washing with methylene chloride, neutralization with methylene chloride containing 10% diisopropylethylamine, and methylene chloride Was washed with. This and 5 ml of 0.
4M Boc-Arg (Tos) (tosyl group) in dimethylformamide solution and 5 ml of 0.4M diisopropylcarbodiimide in methylene chloride solution were mixed, then added to a reaction tank and reacted with stirring at room temperature for 2 hours.

The resulting resin was washed with dimethylformamide, methylene chloride, methylene chloride containing 10% diisopropylethylamine, methylene chloride, and a mixture of methylene chloride and dimethylformamide, and Boc-Arg.
_{(Tos) -Tyr (Cl 2 -Bzl} ) - to obtain a resin.
Subsequently, the same removal of Boc group and coupling of Boc and amino acid were repeated, and Val-Arg (Tos) -Ty was repeated.
An r (Cl ₂ -Bzl) -resin was obtained. 20 ml of the resin
The peptide was liberated from the resin by stirring in hydrogen fluoride containing 10% anisole of 1 ° C. for 1 hour at 0 ° C. Hydrogen fluoride was distilled off under reduced pressure, the residue was extracted with 30% acetic acid and freeze-dried to obtain a crude peptide. This is an ODS column (Cosmos
il 5C ₁₈₎ and purified by reverse phase chromatography on, H-Val-Arg-Tyr -OH ( yield 65.
5 mg) was obtained. As a result of analyzing this product with the same protein sequencer as described above, it was found to have the above composition.

The physical properties of the peptide are as follows. The TLC solvent is the same as described above. Rf: 0.19

(Measurement of Angiotensin-Converting Enzyme Inhibitory Activity) The angiotensin-converting enzyme inhibitory activity was measured by Ch
Eung and Cushman's method [Biochemical
al Pharmacology 20 , 1637
(1971)] according to the following method. Enzyme substrate; Bz (benzyl) -Gly-His-Leu
(A solution of 86 mg dissolved in 8 ml of water and 8 ml of phosphate buffer) Enzyme; Rabbit lung acetone powder (manufactured by Sigma)
(1 g was crushed in 10 ml of 50 mM phosphate buffer and then centrifuged to obtain a supernatant liquid) 100 μl of the above enzyme substrate, 12 μl of the enzyme solution and a peptide having a predetermined concentration of the present invention were mixed, and the whole was mixed with water. 25
After making it 0 μl, the reaction was carried out at 37 ° C. for 30 minutes.

The reaction was terminated with 250 μl of 1N HCl. Ethyl acetate (1.5 ml) was added to the reaction-terminated liquid, and the mixture was stirred with Vortex for 15 seconds and then centrifuged. 1.0 ml was taken out from the ethyl acetate layer, ethyl acetate was distilled off, and 1 ml of distilled water was added to dissolve the residue.
Ultraviolet absorption of extracted hippuric acid value at 228 nm (O
D ₂₂₈ ) was measured. The inhibition rate is 100% of OD ₂₂₈ when the reaction is performed without the inhibitor, and O when the reaction time is 0 minutes.
When _D228 was determined to be 0% and the inhibition rate was 50%, the activity was expressed by the concentration IC ₅₀ (μM) of the inhibitor (peptide of the present invention), which was 80.0.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, a novel peptide useful as an angiotensin converting enzyme inhibitor can be obtained.

Claims (4)

[Claims] 1. A novel peptide comprising H-Val-Arg-Tyr-OH. 2. A method for producing a novel peptide composed of H-Val-Arg-Tyr-OH, which comprises hydrolyzing a protein with thermolysin. 3. The method according to claim 2, wherein casein is used as the protein. 4. An angiotensin converting enzyme inhibitor containing a peptide consisting of H-Val-Arg-Tyr-OH as an active ingredient.