JP3112694B2 - Novel peptide, method for producing it and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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. Ile
-Lys-Trp

[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 ) is an enzyme that releases and cleaves angiotensin II having a strong pressor action.
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 such a problem, and as a result, a composition obtained by hydrolyzing a protein, particularly chicken, with a specific enzyme. The present inventors have identified the presence of a substance having angiotensin-converting enzyme inhibitory activity in the product, and have found that the substance is a peptide represented by the general formula Ile-Lys-Trp, thereby completing the present invention.

The peptide represented by the general formula Ile-Lys-Trp of the present invention is a novel peptide which has not been described in any literature,
It is produced by hydrolyzing proteins such as chicken meat with thermolysin. In practical use, the composition may be used as it is, or may be used after purification if necessary. Furthermore, it can also be produced by synthesizing by applying conventional means of peptide synthesis. I mentioned above
le means isoleucine, Lys means lysine, Trp means tryptophan, and all such amino acids are in L-form.

The peptide of the present invention can be obtained by hydrolyzing a protein with thermolysin or by a peptide synthesis method. In order to hydrolyze the protein with thermolysin, the treatment method varies depending on the properties of the protein. However, in the case of poor solubility, the protein is mixed with hot water, homogenized with vigorous stirring, and a predetermined amount of thermolysin is added. The mixture is allowed to stand or stirred at about 4 ° C. and PH 4 to 8 for 0.1 to 48 hours.

[0008] As the protein, those derived from animals or microorganisms are arbitrarily used, and useful one is chicken meat. Chicken may be raw or processed. In particular, a small amount of oil is effective. In the hydrolyzate, in addition to the peptide of the present invention, other peptides are present, but these may be used for various applications as a mixture,
Further, only the peptide of the present invention may be isolated and used.

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 take part 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-protecting group include groups 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, but 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, reverse phase liquid chromatography, affinity chromatography and the like can be mentioned.

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 type 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. It is. 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 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.

[0017]

The peptide of the present invention is a novel peptide which has an excellent angiotensin converting enzyme inhibitory effect, exhibits a blood pressure lowering effect and a pradikinin inactivation suppressing effect, and has essential hypertension, renal hypertension, adrenal hypertension, etc. It is useful as a preventive and therapeutic agent for hypertension, a diagnostic agent for these diseases, a hypotensive agent used in various disease states, an increase in the threshold of angina attack, a decrease in myocardial infarction, and an improvement agent for the disease state in congestive heart failure .

[0018]

Now, the present invention will be described more specifically below with reference to working examples. [Production of peptide] 12.6 g of meat (70% moisture)
Then, 31 cc of water was added thereto, homogenized sufficiently, 40 mg of thermolysin was added, and a hydrolysis reaction was carried out at 37 ° C. and pH 7 with shaking for 5 hours. After boiling at 100 ° C for 10 minutes,
After cooling and concentration, high performance liquid chromatography (OD
S-, PH- and CN-columns) to give the 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. H-Ile-Lys-Trp-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 coloring) Rf: 0.477 Elemental analysis C ₂₃ H ₃₅ N ₅ O ₄ .0.7H ₂₀ as C H N calculated 60.29 8.01 15.29 measured 60.35 8.12 15.23

[0021]

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[Synthesis of Peptide] 0.60 g of commercially available Boc (butoxycarbonyl) -Trp-O-Resin
(Substitution rate 0.50 meq / g) was fractionated into a reaction vessel of a peptide synthesizer SAM2 manufactured by Biosearch and synthesized as follows. In methylene chloride containing 45% trifluoroacetic acid, 2.5% anisole, 2% dimethyl phosphate,
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-L
A solution of ys (Cl-z) (chlorobenzyloxycarbonyl group) in dimethylformamide was mixed with 5 ml of a 0.4 M solution of 0.4M diisopropylcarbodiimide in methylene chloride, and the mixture was added to the reaction vessel and reacted with stirring at room temperature for 2 hours.

The obtained resin is washed with dimethylformamide, methylene chloride, methylene chloride containing 10% diisopropylethylamine, methylene chloride, and a mixture of methylene chloride and dimethylformamide, and Boc = Lys
(Cl-z) -Trp-resin was obtained. Continue to B
Removal of the oc group and coupling of the Boc and the amino acid were repeated to obtain Ile-Lys (Cl-Z) -Trp resin.

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, and the residue
% Acetic acid and lyophilized to give the crude peptide. This was purified by reverse phase chromatography using an ODS column (Cosmosil 5C ₁₈ ), and H-Ile-L was purified.
ys-Trp-OH (100 mg yield) 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 are as follows. The TLC solvents are all the same as described above. Rf: 0.477 Elemental analysis _{C 23 H 35 N 5 O 4} · 1.0H 2 O as a C H N Calculated 59.59 8.05 15.11 measured value 59.51 8.09 15.18

Example 1 (Measurement of angiotensin-converting enzyme inhibitory activity) The measurement of angiotensin-converting enzyme inhibitory activity was carried out using Cheung and C
USHMAN method [Biochemical Pha
ramacology 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) Enzyme: Rabbit lung acetone powder (Sigma)
(1 g of the supernatant is crushed in 10 ml of 50 mM phosphate 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 ultraviolet absorption value of extracted hippuric acid at ₂₂₈ nm (OD228).
Was measured.

The inhibition rate was determined by the OD when reacted without an inhibitor.
OD when ₂₂₈ is 100% and reaction time is 0 minutes
_The activity was expressed as IC ₅₀ (μM), which was the concentration of the inhibitor (peptide of the present invention) at an inhibition rate of 50%, assuming that ₂₂₈ was 0%, and IC ₅₀ = 1.4 μM.

[0029]

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

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

Claims (4)

(57) [Claims] 1. A novel peptide represented by the general formula Ile-Lys-Trp 2. A method for producing a novel peptide represented by the general formula Ile-Lys-Trp, which comprises hydrolyzing a protein with thermolysin. 3. The method according to claim 2, wherein chicken is used as the protein. 4. An angiotensin converting enzyme inhibitor comprising a peptide represented by the general formula Ile-Lys-Trp as an active ingredient