JPH05331192A - New peptide and its production - Google Patents

Abstract

PURPOSE:To provide a new peptide having an arterial relaxation action and an angiotensin transferase-inhibiting action and useful as an antihypertensive agent, and to provide the method for producing the same. CONSTITUTION:The new peptide is represented by H-Try-Thr-Ser-Trp-Ala-OH, is produced by hydrolyzing dried bonito with thermolysin (an enzyme), and may be used as its composition as such or, if necessary, after purified on its practical use. The peptide can also be produced by a conventional peptide synthesis method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel peptide having an arterial relaxing action and angiotensin converting enzyme inhibitory action and useful as an antihypertensive agent and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION It is known that proteins contained in foods have various physiological activities as well as a nutritional effect.
For example, there are some peptides that exhibit blood pressure lowering activity. Such action is mainly caused by relaxing blood vessels and 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).

On the other hand, angiotensin-converting enzyme is mainly present in lung, vascular endothelial cells and renal proximal tubules, and is angiotensin I (Arg-Arg-Val-Tyr-Il).
It acts on e-His-Pro-Phe-His-Leu) to induce dipeptide (H) from the C-terminal of angiotensin I.
It is an enzyme that cleaves and releases is ⁹ -Leu ¹⁰ ) to produce angiotensin II having a strong pressor effect.
In addition, this enzyme also has the 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 inhibiting the activity of angiotensin-converting enzyme acts to reduce blood pressure and is clinically effective in preventing and treating hypertension. Since captopril, which is a proline derivative, has been recently synthesized and its antihypertensive activity has been confirmed, various researches on angiotensin converting enzyme inhibitors have been actively conducted, and acquisition from natural products has been attempted. Therefore, peptides derived from natural products obtained from foods or food raw materials that have an arterial relaxing action or angiotensin converting enzyme inhibitory action are:
It is expected to be a low toxicity and highly safe antihypertensive agent.

[0005]

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

As an angiotensin converting enzyme inhibitor obtained by enzymatically treating a natural product, peptides obtained by decomposing milk casein with tryptosine are known (Japanese Patent Laid-Open No. 58-109425). , 59-4
No. 4323, No. 59-44324, No. 61-3622.
No. 6, No. 61-36227) The development of new inhibitors is desired.

[0007]

Means for Solving the Problems In order to solve the above problems, the inventors of the present invention have eagerly searched for a substance having an arterial relaxing action and angiotensin converting enzyme inhibitory action which is a natural substance and has few side effects, and as a result, a protein, particularly fish meat and bonito flakes. The presence of a substance having such an activity in the composition obtained by hydrolyzing the above with a specific enzyme is identified, and the substance is identified as H-Tyr-Thr-Ser.
The present invention has been completed by finding out that the peptide has a structure of -Trp-Ala-OH.

The peptide of the present invention is a novel peptide which has not been published in the literature, and is produced by hydrolyzing bonito node with thermolysin, and the composition may be used as it is for practical use, or purified if necessary. Then used. Furthermore, it can also be produced by synthesizing by applying a conventional means for peptide synthesis. In the above, Tyr means tyrosine, Thr means threonine, Ser means serine, Trp means tryptophan, and Ala means alanine, 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 in the case of poor solubility, the protein is mixed with hot water and homogenized by vigorous stirring, and a predetermined amount of thermolysin is added. The reaction is performed at a temperature of about 10 to 85 ° C. for 0.1 to 48 hours.

Any protein derived from animals or microorganisms may be used as the protein, and particularly useful one is fish meat, and fish such as bonito flakes and sardines are inexpensive and useful. In the case of isolation, the hydrolyzed solution is filtered by a known operation such as centrifugation. After that, after applying extraction, concentration, dryness, etc., or without doing so, the difference in the adsorption affinity for various adsorbents, the difference in the solubility or solubility in various solvents, the two immiscible liquid phases It is preferable to isolate the target product by applying a means utilizing a difference in partition between the two, a difference in elution rate based on the size of the molecule, a property of precipitating from a solution or a difference in precipitation rate. These methods may be used alone, or may be combined in any order and repeatedly applied.

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 a raw material and a raw material having a reactive amino group corresponding to the other fragment by 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 C-terminal carboxyl group is a chloromethyl resin, an 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 cut. Furthermore, the peptide of the present invention is purified by a conventional method. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like.

The administration route 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 compound, administration method, patient's symptoms, age, 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 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 carboxymethyl cellulose, hydroxypropyl starch, calcium carboxymethyl cellulose, ion exchange. Resin, methyl cellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, 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, polysorbet Door, macrogol, vegetable oils,
Examples thereof include wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol and water. Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like.

These formulations 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.

[0017]

[Working] The peptide of the present invention is a novel peptide, which has excellent arterial relaxant action and angiotensin converting enzyme inhibitory action, exhibits blood pressure lowering activity and bradykinin inactivation suppression activity, and has essential hypertension, renal hypertension, Useful as a prophylactic / therapeutic agent for hypertension such as adrenal hypertension, a diagnostic agent for these diseases, a blood pressure lowering agent used in various pathologies, a therapeutic agent for angina and myocardial infarction, and an agent for improving pathology in congestive heart failure Is.

[0018]

EXAMPLES Next, the present invention will be described more specifically with reference to examples. Example 1 [Extraction of peptide] To 5 g of skipjack tuna, 40 ml of water was added and homogenized sufficiently, and the mixture was boiled at 100 ° C for 10 minutes and then left to stand. 20 mg of thermolysin was added and the hydrolysis reaction was carried out at 37 ° C. and PH7 for 3 hours. After cooling, the mixture was centrifuged, concentrated, and purified by high performance liquid chromatography (ODS-, Ph- and CN-column) to obtain a peptide. This product was subjected to an automatic Edman degradation method using a gas phase protein sequencer (Model 477 A manufactured by Applied Biosystems) to analyze the amino acid sequence to obtain the following structure.

H-Tyr-Thr-Ser-Trp-A
la-OH The physical properties of the peptide are as follows. TLC [n-butanol: acetic acid: pyridine: water = 15:
3: 10: 2] (silica gel plate, ninhydrin coloration) Rf: 0.567 m.p. p: as a decomposition Elemental analysis _{C 30 H 38 N 6 O 9} · 0.7H 2 O at 180 ° C.

[Synthesis of Peptide] 0.55 g of commercially available Boc (butoxycarbonyl) -Ala (protected with chlorobenzyloxycarbonyl) -O-Resin [benzyl resin (substitution rate 0.55 meq / g)] was obtained from Biosearch. The peptide was synthesized in a reaction tank of a peptide synthesizer SAM2 and synthesized as follows. 45% trifluoroacetic acid, 2.5%
After removing the Boc group by reaction in methylene chloride containing anisole for 25 minutes, washing with methylene chloride and neutralization with methylene chloride containing 10% diisopropylethylamine and washing with methylene chloride were performed. This and 5 ml of 0.4M Boc-Trp-oBz
(Benzyl ester) in dimethylformamide, 5
After mixing with 0.4 ml of a solution of 0.4 M diisopropylcarbodiimide in methylene chloride, the mixture was added to the reaction vessel and the mixture was allowed to stand at room temperature for 2 minutes.
The reaction was allowed to stir for an hour.

The obtained resin was washed with dimethylformamide, methylene chloride, methylene chloride containing 10% diisopropylethylamine, methylene chloride, and a mixture of methylene chloride and dimethylformamide, and Boc-Trp.
(OBzl) -Ala-resin was obtained. Continue to the same B
The removal of the oc group and the coupling of Boc and the amino acid are repeated, and Tyr (oBzl) -Thr (oBzl) -Ser is repeated.
(OBzl) -Trp-Ala (Cl-z) -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 freeze-dried to obtain a crude peptide. This is ODS
Purified by reverse phase chromatography on a column (Cosmosil 5C ₁₈ ) and H-Tyr-Thr-Se.
r-Trp-Ala-OH 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 were the same as above.

(Measurement of Canine Mesenteric Artery Relaxing Action) A spiral strip of a mesenteric artery isolated from a dog was prepared by Takagi et al. (Takagi et al., Pharmacological Experiment, pages 94-99, Nanzando). The specimens were processed according to the procedures described above and connected to isometric transducers in Magnus tubes. 0.5 μM prostaglandin F
Against the peptides mesenteric arteries were contracted by ₂ alpha exhibited relaxant effects. ED _{50 at} that time (50% relaxation)
Was 700 μM.

(Measurement of Angiotensin-Converting Enzyme Inhibitory Activity) The angiotensin-converting enzyme inhibitory activity was measured by Ch
Eung Cushman's method [Biochemic
al Pharmacology 20 , 1637
(1971)] according to the following method. Enzyme substrate; Bz (benzyl) -Gly-His-Leu (solution of 86 mg dissolved in 8 ml of water and 8 ml of phosphate buffer solution) Enzyme; Acetone powder of rabbit lung (Sigma) (1 g of 50 mM phosphoric acid Supernatant obtained by crushing in 10 ml of buffer solution, followed by centrifugation) 100 μl of the above enzyme substrate, 12 μl of the enzyme solution and the above obtained supernatant were mixed to a predetermined concentration, and the whole was mixed with water to 250 μl.
After making it to μl, the reaction was carried out at 37 ° C. for 30 minutes.

The reaction was terminated with 250 μl of 1N hydrochloric acid. Add 1.5 ml of ethyl acetate to the reaction completed liquid and Vo
Stir for 15 seconds at rtex and centrifuge it. 1.0 ml was taken out from the ethyl acetate layer, ethyl acetate was distilled off, 1 ml of distilled water was added to dissolve the residue, and the ultraviolet absorption of the extracted hippuric acid at ₂₂₈ nm (OD ₂₂₈ ) was measured. .. Inhibition rate is the OD when reacting without peptide
₂₂₈ is 100%, and OD ₂₂₈ when the reaction time is 0 minutes is 0
%, The concentration of the peptide when the inhibition rate is 50%, IC ₅₀
When the activity was displayed by, it was 120 μM.

(Measurement of Blood Pressure Fluctuation) Open Blood Method After spontaneously hypertensive rats of 15 to 20 weeks of age were anesthetized from the tail vein, a catheter for sample injection was inserted into the femoral vein, and 10 mg / kg of blood was injected. The sample was administered and the blood pressure was measured invasively with a pressure transducer. The decrease in blood pressure was about 10 mmHg. Non-open blood (oral administration) 1 sample of the above-mentioned rat emulsified with egg yolk
It was administered by gavage using a sonde needle at a rate of 00 mg / kg. Then, the blood pressure in the tail artery was measured with time by the tail-cuff method, and the maximum blood pressure reduction value in systolic blood pressure was measured, and it was 10 mmHg.

[0026]

INDUSTRIAL APPLICABILITY The present invention can be prepared by hydrolyzing natural products such as bonito flakes, and in particular, a novel peptide useful as a hypotensive agent or a hypotensive food can be produced.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C07K 99:00

Claims (4)

[Claims] 1. H-Tyr-Thr-Ser-Trp-
A novel peptide represented by Ala-OH. 2. H-Tyr-Thr-Ser-Trp, wherein the protein is hydrolyzed with thermolysin.
A method for producing a novel peptide represented by -Ala-OH. 3. The method according to claim 2, wherein fish meat is used as the protein. 4. The method according to claim 2, wherein bonito flakes are used as the protein.