JPH04264096A - New peptide, its production and use thereof - Google Patents

Abstract

PURPOSE:To obtain a new peptide useful as an angiotensinase inhibitor mainly effective for prevention and treatment of hypertension. CONSTITUTION:A protein, particularly chicken is hydrolyzed with a thermolysin to provide an angiotensinase inhibitor containing a peptide expressed by the formula, leucine-lysine-alanine, as an active ingredient.

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. Leu-Lys-Ala

0002

[Prior Art] Angiotensin converting enzyme is mainly present in the lungs, vascular endothelial cells, and renal proximal tubules, and angiotensin converting enzyme (Asp-Arg-Val-Tyr-Ile-
The dipeptide (His-Pro-Phe-His-Leu) is released from the C-terminus of angiotensin I.
9-Leu10) to produce angiotensin II, which has a strong pressor effect. This enzyme also has the effect of destroying and inactivating bradykinin, an antihypertensive substance in the body, and is strongly involved in the pressor system.

[0003] It has been conventionally believed that inhibiting the activity of angiotensin converting enzyme lowers blood pressure and is clinically effective in preventing and treating hypertension. Recently, the proline derivative captopril was synthesized and its antihypertensive activity was confirmed, and since then, research has been active in the synthesis of various angiotensin-converting enzyme inhibitors, and efforts are also being made to obtain them from natural products. This is because angiotensin-converting enzyme inhibitors derived from natural products can be obtained from foods or food materials, and are therefore expected to be low-toxic and highly safe antihypertensive agents.

0004

[Problems to be Solved by the Invention] However, angiotensin-converting enzyme inhibitors found in natural products are extremely rare, and there are only a few, such as teprotide (nonapeptide, SQ20881) obtained from Brazilian and Japanese snake venom, and Streptomyces IS8, a metabolite of actinomycetes belonging to the genus
3 (Japanese Unexamined Patent Publication No. 58-177920) is known. In addition, as angiotensin-converting enzyme inhibitors obtained by enzymatically treating natural products, peptides obtained by decomposing milk casein with trypsin are known (Japanese Patent Application Laid-open Nos. 109425-1987 and 59-1982). -44
No. 323, No. 59-44324, No. 61-36226
(No. 61-36227) The development of new inhibitors is desired.

[0005]

[Means for Solving the Problems] In order to solve the problems, the present inventors have diligently searched for an angiotensin-converting enzyme inhibitor that is a natural substance and has few side effects.As a result, the present inventors have developed a composition in which protein, especially chicken meat, is hydrolyzed with a specific enzyme. The inventors have discovered that a substance having angiotensin-converting enzyme inhibitory activity exists in a substance, and found that the substance is a peptide represented by the general formula Leu-Lys-Ala, thereby completing the present invention.

[0006] The peptide represented by the general formula Leu-Lys, Ala of the present invention is a novel peptide that has not been described in any literature.
It is produced by hydrolyzing proteins such as chicken meat with thermolysin, and in practical use, the composition may be used as it is, or it may be purified if necessary. Furthermore, it can also be produced by applying conventional methods for peptide synthesis. L mentioned above
eu means leucine, Lys means lysine, and Ala means alanine, and all of these amino acids are in the L-form.

The peptide of the present invention can be obtained either by hydrolyzing a protein with thermolysin or by a peptide synthesis method. To hydrolyze a protein with thermolysin, the treatment method differs depending on the nature of the protein, but in the case of poorly soluble proteins, mix the protein with hot water, homogenize with strong stirring, add a predetermined amount of thermolysin, and heat at a temperature of 10 to 60 ml. The reaction is allowed to stand or is stirred for 0.1 to 48 hours at a temperature of about 0.degree. C. and a pH of 4 to 8.

[0008] As the protein, any protein derived from animals or microorganisms can be used, and a useful protein is chicken meat. Chicken may be raw or processed. Chicken fillets with low oil content are particularly effective. In addition to the peptide of the present invention, other peptides are present in the hydrolysis structure, but these may be used as a mixture for various purposes.
Furthermore, it is also possible to isolate and use only the peptide of the present invention.

In the case of isolation, the hydrolyzed solution is filtered by a known operation such as centrifugation. After subsequent application of extraction, concentration, drying, etc., or as is, the difference in adsorption affinity for various adsorbents, the difference in solubility or solubilities for various solvents, the difference in the two immiscible liquid phases. It is preferable to isolate the target substance by applying means that utilize differences in distribution between molecules, differences in elution rate based on molecular size, differences in precipitability or precipitation rate from solutions, etc. These methods may be used alone, combined in any order, or applied repeatedly as necessary.

[0010] The peptide of the present invention has a reactive carboxyl group corresponding to one of two types of fragments that are bisected at any position of the peptide bond by a method commonly used for peptide synthesis, ie, 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, etc., and when the resulting condensate has a protecting group, by removing the protecting group. can also be manufactured.

[0011] Functional groups that should not participate in the reaction in this reaction step are protected by protecting groups. Examples of protecting groups for amino groups include benzyloxycarbonyl, t-
Examples include butyloxycarbonyl, p-biphenylisopropyroxycarbonyl, 9-fluorenylmethyloxycarbonyl, and the like. Examples of protective groups for carboxyl groups include groups that can form alkyl esters, benzyl esters, etc.; however, in the case of solid phase methods, the C-terminal carboxyl group is protected by 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 a carbodiimide or by using an N-protected amino acid active ester or a peptide active ester. After the condensation reaction is completed, the protecting group is removed, but in the case of a solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved. Furthermore, the peptides of the invention are purified according to conventional methods. Examples include ion exchange chromatography, reversed phase liquid chromatography, and affinity chromatography.

[0013] The administration route of the peptide used in the present invention may be oral, parenteral or rectal administration, but oral administration is preferred. The dosage of the peptide of the present invention varies depending on the type of compound, administration method, patient's symptoms, age, etc., but is usually 0.001 to 1000 mg, preferably 0.01 to 10 mg, 1 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 it with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.

Specifically, examples include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, and 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, polysorbate, macrogol, vegetable oil, wax,
Examples include liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactants, propylene glycol, water, and the like.

[0015] Examples of dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, and injections. These formulations are prepared according to conventional methods. In the case of a liquid preparation, it may be dissolved or suspended in water or other suitable medium before use. Furthermore, tablets and granules may be coated by a well-known method. In the case of injections, the peptide of the present invention is prepared by dissolving it in water, but if necessary, it may be dissolved in physiological saline or glucose solution, or a buffer or preservative may be added. good.

[0016] These preparations contain the peptide of the present invention at a concentration of 0.
It can be contained in a proportion of 0.01% or more, preferably 0.5 to 70%. These formulations may also contain other ingredients of therapeutic value.

[0017]

[Action] The peptide of the present invention is a novel peptide and has an excellent angiotensin-converting enzyme inhibitory effect, a blood pressure lowering effect, and an inhibitory effect on bradykinin inactivation.
Preventive and therapeutic agents for hypertension such as essential hypertension, renal hypertension, and adrenal hypertension; diagnostic agents for these diseases; antihypertensive agents used in various pathological conditions; increasing the threshold for angina pectoris attacks; reducing myocardial infarction; It is useful as an agent for improving the pathology of congestive heart failure.

[0018]

EXAMPLES Next, the present invention will be explained in more detail with reference to examples. [Manufacture of peptide] 12.6g of fillet meat (moisture 7
0%) was thoroughly homogenized, 40 mg of thermolysin was added, and a hydrolysis reaction was carried out at 37° C. and pH 7 for 5 hours with shaking and stirring. After boiling at 100°C for 10 minutes, cooling and concentrating, it was purified by high performance liquid chromatography (ODS-, PH- and CN-columns),
The peptide was obtained.

The amino acid sequence of this product was analyzed by applying the automated Edman degradation method using a gas phase protein sequencer (Model 477A manufactured by Abride Biosystems), and the following structure was obtained. H-Leu-Lys-Ala-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 color development) Rf: 0.364 Elemental analysis C15H30N4O4・0.8H20
as C
H N Calculated value 54.24
9.24 16.25 Measured value 52.11
9.23 16.12

[Synthesis of peptide] Commercially available Boc (butoxycarbonyl)-Ala-
O-Resin 0.81g (substitution rate 0.37meq
/g) was fractionated into the reaction tank of BioSearch's peptide synthesizer SAM2, and synthesized as follows. B by reaction for 25 minutes in methylene chloride containing 45% trifluoroacetic acid, 2.5% anisole, 2% dimethyl phosphate.
After removing the oc group, washing with methylene chloride, 10
Neutralization with methylene chloride containing % diisopropylethylamine and washing with methylene chloride were performed. After mixing this with 5 ml of a 0.4 M Boc-Lys (Cl-z) (chlorobenzyloxycarbonyl group) dimethylformamide solution and 5 ml of a 0.4 M diisopropylcarbodiimide methylene chloride solution, the mixture was added to a reaction tank and heated to room temperature. The mixture was stirred and reacted for 2 hours.

The obtained resin was washed with a mixture of dimethylformamide, methylene chloride, methylene chloride containing 10% diisopropylethylamine, methylene chloride, and methylene chloride and dimethylformamide.
(Cl-z)-Ala-resin was obtained. Continue the same B
Leu-Lys(Cl-Z)-Ala resin was obtained by repeating the removal of the oc group and the coupling of Boc and the amino acid.

The resin was stirred for 1 hour at 0° C. in 20 ml of hydrogen fluoride containing 10% anisole to liberate the peptide from the resin. Hydrogen fluoride was distilled off under reduced pressure, and the residue was
% acetic acid and lyophilized to obtain crude peptide. This was purified by reverse phase chromatography using an ODS column (Ccsmosil 5C18) and H-Leu-L
ys-Ala-OH (yield: 100 mg) was obtained. Analysis of this product using the same protein sequencer as above revealed that it had the above composition.

The physical properties of the peptide are as follows. Incidentally, all the solvents for TLC are the same as above. Rf: 0.364 Elemental analysis C15H30N4O4・0.3H2O
as C
H N calculated value 53.65
9.18 16.68 Measured value 53.72
9.25 16.66

Example 1 (Measurement of angiotensin converting enzyme inhibitory activity) Angiotensin converting enzyme inhibitory activity was measured by Cheung and C.
ushman's method [Biochemical Ph
aramacology 20, 1637 (1971
)] using 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 ground in 10 ml of 50 mM phosphate buffer, centrifuged, and clear liquid)

[0027] 100 μl of the above enzyme substrate, 12 μl of the enzyme solution, and the peptide of the present invention at a predetermined concentration were mixed, the total volume was made up to 250 μl with water, and the mixture was reacted at 37° C. for 30 minutes. The reaction was terminated using 250 μl of 1N HCl. Add 1.5 ml of ethyl acetate to the reaction completed solution.
Mixed with ortex for 15 seconds and centrifuged it. Take out 1.0 ml from the ethyl acetate layer, distill off the ethyl acetate, add 1 ml of distilled water to dissolve the residue, and calculate the ultraviolet absorption value (OD228) of the extracted hippuric acid at 228 nm.
) was measured.

[0028] The inhibition rate is the OD when reacting without an inhibitor.
OD22 when 228 is 100% and reaction time is 0 minutes
8 as 0% and the activity was expressed as the concentration IC50 (μM) of the inhibitor (peptide of the present invention) when the inhibition rate was 50%, and the IC50 was 7.5 μM.

[0029]

[Effect] The present invention provides a novel peptide useful as an angiotensin converting enzyme inhibitor.

Claims (4)

[Claims] Claim 1: A novel peptide represented by the general formula Leu-Lys-Ala Claim 2: A novel peptide represented by the general formula Leu-Lys-Ala, which is characterized by hydrolyzing proteins with thermolysin. Claim 3: The production method according to claim 2, wherein chicken meat is used as the protein. Claim 4: An angiotensin converting enzyme inhibitor containing a peptide represented by the general formula Leu-Lys-Ala as an active ingredient.