JP3465922B2 - Novel peptide, method for producing the same and use - Google Patents

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-Leu-Gln-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]

The present inventors Means for Solving the Problems] The composition hydrolyzed intensively searched result fewer side effects angiotensin converting enzyme inhibitors in natural substance in order to solve the above problems, the casein in particular enzyme The present invention was completed by finding the presence of a substance having angiotensin converting enzyme inhibitory activity in the product and finding that the substance is a peptide consisting of H-Leu-Gln-Tyr-OH.

H-Leu-Gln-Tyr-O of the present invention
Peptides consisting of H is a novel peptide of the mounting un document, produced by the hydrolysis of casein by thermolysin, practical when is used to purify the compositions may be used as it is, or if necessary It Furthermore, it can also be produced by synthesizing by applying a conventional method for peptide synthesis. Le mentioned above
u means leucine, Gln means glutamine, Tyr means tyrosine, and all such amino acids are L-forms.

The peptide of the present invention can be obtained by hydrolyzing casein with thermolysin or by a peptide synthesis method. To hydrolyze casein with thermolysin, if it is sparingly soluble, mix casein with hot water, homogenize with strong stirring, add a predetermined amount of thermolysin, and react for 0.1 to 48 hours at a temperature of about 10 to 85 ° C. To do . Besides the peptides of the present invention to a pressurized hydrolysis solution, although other peptide is present, it remains in may be used in various applications of the mixture, also only peptides of the present invention isolated You can use it. 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.

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 cut. 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 which has an excellent angiotensin converting enzyme inhibitory action, exhibits a hypotensive action and a bradykinin inactivation suppressing action, and exhibits high blood pressure 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 pathological conditions 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-Leu-Gln-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: 0.18

[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-Gln 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 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-Gln.
-Tyr (Cl ₂ -Bzl) - to obtain a resin. Subsequently, the same removal of Boc group and coupling of Boc and amino acid were repeated to repeat Leu-Gln-Tyr (Cl ₂ -Bzl).
-A resin is 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, and the residue was washed with 3
The crude peptide was obtained by extraction with 0% acetic acid and freeze-drying. This was purified by reverse phase chromatography on an ODS column (Cosmosil 5C ₁₈ ) and H-Leu-G.
In-Tyr-OH (yield 66.8 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: 0.18

(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 (solution of 86 mg dissolved in 8 ml of water and 8 ml of phosphate buffer solution) Enzyme; Rabbit lung acetone powder (Sigma) (1 g of 50 mM phosphate Supernatant obtained by crushing in 10 ml of buffer solution and then centrifuging) 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 to 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.
The activity was expressed by the IC ₅₀ (μM) concentration of the inhibitor (peptide of the present invention) when the inhibition rate was 50% when D ₂₂₈ was set to 0%, and it was 15.0.

[0020]

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

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI // C07K 1/12 C12P 21/06 C12N 9/99 A61K 37/18 C12P 21/06 37/64 (56) Reference JP-A-3-81291 (JP, A) JP-A-4-247099 (JP, A) JP-A-4-341193 (JP, A) JP-A-4-169598 (JP, A) JP-A-4-154798 (JP , A) Japanese Patent Application Laid-Open No. 4-154797 (JP, A) European Patent Application Publication 445606 (EP, A 1) (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 5/08 REGISTRY (STN) CA (STN) BIOSIS / WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A novel peptide consisting of H-Leu-Gln-Tyr-OH. 2. H-Leu-Gln-Tyr-OH.
Angiotensin conversion with a new peptide as the active ingredient
Enzyme inhibitors .