JP3112695B2 - Method for producing peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a peptide having the following structure. Leu-Lys-
Ala

[0002]

2. Description of the Related Art Angiotensin converting enzyme is mainly present in lungs, vascular endothelial cells and renal proximal tubules, and is angiotensin I (Asp-Arg-Val-Tyr-Ile-).
Acts on His-Pro-Phe-His-Leu) to dipeptide (Hi) from the C-terminus of angiotensin I
s ⁹ -Leu ¹⁰⁾ was open裂遊separated, is an enzyme that produces angiotensin II having a strong boost effect. This enzyme also has the action of destroying and inactivating bradykinin, which is a hypotensive substance in the living body, and is strongly involved in the pressor system.

Hitherto, it has been considered that inhibiting the activity of angiotensin converting enzyme acts on blood pressure lowering, and is clinically effective for preventing and treating hypertension. Recently, since captopril, a proline derivative, has been synthesized and its antihypertensive activity has been confirmed, studies on the synthesis of various angiotensin converting enzyme inhibitors have been actively conducted, and attempts are being made to obtain them from natural products. This is because a natural product-derived angiotensin converting enzyme inhibitor is expected to be a low-toxicity and highly safe antihypertensive agent because it is obtained from foods or food raw materials.

[0004]

However, angiotensin converting enzyme inhibitors found in natural products are extremely rare, such as teprotide (nonapeptide, SQ20881) obtained from Brazilian or Japanese snake venom, and Streptomyces. Metabolite IS of actinomycetes belonging to the genus
No. 83 (JP-A-58-177920) is only known. As angiotensin converting enzyme inhibitors obtained by enzymatic treatment of natural products, peptides and the like obtained by decomposing milk casein with trypsin are known (JP-A-58-109425, JP-A-58-109425). -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 The present inventors have intensively searched for an angiotensin converting enzyme inhibitor which is a natural substance and has few side effects in order to solve the above-mentioned problems. The inventors have determined that a substance having an inhibitory activity is produced, and found that the substance is a peptide represented by Leu-Lys-Ala, thereby completing the present invention.

The peptide of the present invention hydrolyzes a protein with thermolysin. The treatment method differs depending on the properties of the protein. In the case of poorly soluble protein, the protein is mixed with hot water and homogenized by vigorous stirring. A predetermined amount of thermolysin is added, and the mixture is allowed to stand or stirred at a temperature of about 10 to 60 ° C and a pH of 4 to 8 for 0.1 to 48 hours.

[0007] Such proteins are arbitrarily used, including those derived from animals and microorganisms, and useful are chicken. Chicken may be raw or processed. In particular, a small amount of oil is effective. A peptide called Leu-Lys-Ala is present in the hydrolyzate, and other peptides are present in addition to the peptide. These peptides may be used as a mixture as they are for various purposes. Purified (isolated) according to the above. L mentioned above
eu means leucine, Lys means lysine, Ala means alanine, and all such amino acids are in L-form.

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

[0009] The administration route of the peptide produced by the method of 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 type of the compound, the administration method, the symptoms and age of the patient, etc., but is usually 0.001 to 1000 m / time.
g, preferably 0.01 to 10 mg, 1 to 3 per day.
Times. 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. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium metasilicate aluminate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch,
Carboxymethylcellulose calcium, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, 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, fluorocarbon, nonionic surfactant, propylene glycol, water, etc. . Dosage forms 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.

[0010] These preparations contain the peptide obtained by the production method of the present invention in an amount of 0.01% or more, preferably 0.5 to 7%.
It can be contained at a rate of 0%. These formulations may also contain other therapeutically valuable components.

[0011]

The peptide obtained by the production of the present invention has an excellent angiotensin converting enzyme inhibitory effect, exhibits an antihypertensive effect, an inhibitory effect on bradykinin inactivation, and has essential hypertension, renal hypertension, adrenal hypertension, etc. Prevention of hypertension,
It is useful as a therapeutic agent, a diagnostic agent for these diseases and an antihypertensive agent used in various disease states, an increase in the threshold of angina attack, a decrease in myocardial infarction, and an improvement agent for disease states in congestive heart failure.

[0012]

(Production of peptide)

31 cc of water is added to 12.6 g (70% moisture) of the breast meat, homogenized sufficiently, and 40 mg of thermolysin is added.
The hydrolysis reaction was carried out at 7 ° C. and pH 7 with shaking for 5 hours. After boiling at 100 ° C for 10 minutes, cooling and concentrating,
High performance liquid chromatography (ODS-, PH- and C
N-column) to give a peptide.

The amino acid sequence of this product was analyzed by an automatic Edman degradation method using a gas-phase protein sequencer (Model 477A, manufactured by Abride Biosystems) to obtain the following structure. Leu-Lys-Ala

The physical properties of the peptide are as follows. TLC [n-butanol: acetic acid: pyridine: water = 15:
3:10:12] (silica gel plate, ninhydrin coloring) Rf: 0.364

The physical properties of the peptide are as follows. The TLC solvents are all the same as described above. Rf: 0.364

Example 1 (Measurement of angiotensin-converting enzyme inhibitory activity)
ng and Cushman [Biochemical Ph.
aracology 20 , 1637 (1971)]
According to the following method. Enzyme substrate: Bz (benzyl) -Gly-His-Leu (solution of 86 mg in 8 ml of water and 8 ml of phosphate buffer) Enzyme: acetone powder of rabbit lung (Sigma) (1 g of 50 mM phosphoric acid Supernatant which was ground in 10 ml of buffer and then centrifuged)

The above enzyme substrate (100 μl), the enzyme solution (12 μl) and the peptide of the present invention were mixed at a predetermined concentration, and the whole was made up to 250 μl with water, followed by reaction at 37 ° C. for 30 minutes. The reaction was terminated using 250 μl of 1N HCl. 1.5 ml of ethyl acetate is added to the reaction-finished solution and Vo is added.
Stirred for 15 seconds at rtex and centrifuged 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 value of ultraviolet absorption ₂₂₈ nm (OD ₂₂₈ ) of the extracted hippuric acid was measured. .

[0018] The inhibition rate was determined by the OD when reacted without an inhibitor.
₂₂₈ as 100%, and OD ₂₂₈ at 0 minute
%, The activity is expressed as the concentration IC ₅₀ (μM) of the inhibitor (peptide of the present invention) when the inhibition rate is 50%.
₅₀ = 7.5 μM.

[0019]

[Effect] According to the production method of the present invention, a peptide useful as an angiotensin converting enzyme inhibitor can be obtained.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 21/06 C07K 5/083 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A method for producing a peptide represented by Leu-Lys-Ala, comprising hydrolyzing a protein with thermolysin. 2. The method according to claim 1, wherein chicken is used as the protein. 3. The method for producing a peptide represented by Leu-Lys-Ala according to claim 1, which is useful as an angiotensin converting enzyme inhibitor, wherein the protein is hydrolyzed with thermolysin.