JP2951428B2 - New peptides, their production methods and applications - 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. Asn-Ile-Phe-Tyr-Cys-Pro

[0002]

2. Description of the Related Art Angiotensin converting enzyme is mainly present in lung, vascular endothelial cells and renal proximal tubules, and is found in angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). Acting on the C-terminus of angiotensin I to dipeptide (Hi
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.

[0004] Naturally occurring angiotensin converting enzyme inhibitors are expected to be low-toxic and highly safe antihypertensive agents because they are obtained from foods or food raw materials.

[0005]

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.

[0006]

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 albumin in a specific enzyme The presence of a substance having angiotensin converting enzyme inhibitory activity in the substance, and the substance is represented by the general formula Asn-Ile-Phe-Tyr-Cys
The present inventors have found that the peptide is represented by -Pro, and have completed the present invention.

The general formula of the present invention, Asn-Ile-Phe-
Peptides represented by Tyr-Cys-Pro is a novel peptide of the mounting un document, is prepared by hydrolyzing the albumin by pepsin, practical when may be used as a composition, or as needed Used after purification. Furthermore, it can also be produced by synthesizing by applying conventional means of peptide synthesis. Asn means asparagine, Ile means isoleucine, Phe means phenylalanine, Tyr means tyrosine, Cys means cysteine, and Pro means proline;
All such amino acids are in the L-form.

[0008] The peptide of the present invention can be obtained by hydrolyzing albumin with pepsin or by a peptide synthesis method. To hydrolyze albumin with pepsin,
The treatment method varies depending on the properties of albumin. In the case of poor solubility, albumin is mixed with hot water, homogenized with vigorous stirring, a predetermined amount of pepsin is added, and the temperature is 10 to 60 ° C, preferably 20 to 40 ° C, PH0. 0.1 to 4.0 for 10 minutes to 3
Allow to stand or stir for days.

As albumin, ovalbumin, serum albumin, milk albumin and the like which are widely distributed in body fluids and tissues of animals and plants are optionally used, and ovalbumin is particularly useful. In the hydrolyzate, other peptides are present in addition to the peptide of the present invention.These peptides may be used for various purposes as a mixture, or only the peptide of the present invention may be isolated and used. No problem.

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.

The functional groups which should not take part in the reaction in this reaction step 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 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 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.

[0015] Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, 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.

The dosage form includes 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.

[0018]

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, a bradykinin 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 .

[0019]

Now, the present invention will be described more specifically below with reference to working examples. The raw egg white was diluted 5-fold with distilled water and dissolved.
Dissolution solution adjusted to pH 1.6 with HCl (20 mg / ml
Was added with 0.2 mg / ml of pepsin (manufactured by Sigma) and allowed to stand at 37 ° C. for 3 hours to perform reaction.
The reaction was stopped by boiling at 10 ° C. for 10 minutes. The reaction solution was centrifuged at 10,000 rpm for 5 minutes, concentrated, and then concentrated, and then subjected to high performance liquid chromatography (ODS-, PH- and C
N-column) to obtain 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. H-Asn-Ile-Phe-Tyr-Cys-Pro-OH

The physical properties of the peptide are as follows.

[Synthesis of peptide] 0.75 g of commercially available Boc (butoxycarbonyl) -Pro-O-Resin
(Substitution rate: 0.40 meq / g) was collected in 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% ethanedithiol,
After removing the Boc group by a reaction for 25 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-
After mixing a solution of Cys (MeOBzl) (methoxybenzyl group) in dimethylformamide, 5 ml of a 0.4 M solution of 0.4M diisopropylcarbodiimide in methylene chloride,
The mixture was added to the reaction tank, and the mixture was stirred and reacted 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-Cys
(MeOBzl) -Pro resin was obtained. Subsequently, the same removal of the Boc group and the coupling of the Boc and the amino acid were repeated to repeat Asn-Ile-Phe-Tyr (Cl ₂ -Bz
1) (Dichlorobenzyl group) -Cys (MeOBz
1) -Pro 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, and the residue
% Acetic acid and lyophilized to give the crude peptide. This was purified by reverse phase chromatography on an ODS column (Cosmosil 5C ₁₈ ) to give H-Asn-I
le-Phe-Tyr-Cys-Pro-OH (yield 1
00 mg). 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: 0.71

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
ramaco1ology 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 by the concentration IC ₅₀ (μM) of the inhibitor (peptide of the present invention) when the inhibition rate was 50%, assuming that ₂₂₈ was 0%, and IC ₅₀ = 20 μM.

[0029]

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

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C07K 123: 00 (56) References JP-A-4-202200 (JP, A) JP-A-4-202198 (JP, A) Kaihei 4-99798 (JP, A) (58) Fields studied (Int. Cl. ⁶ , DB name) C07K 7/06 A61K 38/00 A61K 38/55 C07K 1/12 A61K 31/00 C07K 123: 00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. The compound of the general formula Asn-Ile-Phe-Tyr-
A novel peptide represented by Cys-Pro . 2. Hydrolyzing albumin with pepsin.
General formula Asn-Ile-Phe-Tyr-
A method for producing a novel peptide represented by Cys-Pro. 3. The method of claim 1, wherein the egg white is hydrolyzed with pepsin.
General formula Asn-Ile-Phe-Tyr-Cys
A method for producing a novel peptide represented by Pro. 4. A compound of the general formula Asn-Ile-Phe-Tyr-
An active ingredient comprising a peptide represented by Cys-Pro
Ngiotensin converting enzyme inhibitor.