JP2922247B2 - Angiotensin converting enzyme inhibitor - Google Patents

Description

The present invention relates to an angiotensin converting enzyme inhibitor comprising a peptide having the following structure as an active ingredient.

Pro-Ala-Gln-Lys [Prior art] Angiotensin converting enzyme is mainly present in lung, vascular endothelial cells, and renal proximal tubules, and angiotensin I (As
Acts on p-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) to release the dipeptide (His ⁹ -Leu ¹⁰ ) from the C-terminus of angiotensin I and has a strong pressor action It is an enzyme that produces angiotensin II. 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 an 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.

[Problems to be Solved by the Invention] However, angiotensin-converting enzyme inhibitors found in natural products are extremely rare. Only the metabolite IS83 of the genus Actinomycetes belonging to the genus (JP-A-58-177920) is known. Also, as angiotensin converting enzyme inhibitors obtained by enzymatic treatment of natural products, only peptides obtained by decomposing milk casein with trypsin are known (JP-A-58-109425; 59−44323
Nos. 59-44324, 61-36226, 61-36227)
There is a need for the development of new inhibitors.

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 problems. The presence of a substance having an activity was determined, and the substance was found to be a peptide having Pro-Ala-Gln-Lys as a skeleton, and the substance was derived from creatine kinase contained in animal tissues. And completed the present invention.

The peptide having the Pro-Ala-Gln-Lys skeleton of the present invention as a skeleton is produced by isolating and purifying it from natural products such as cutout and creatine kinase, or by applying conventional means of peptide synthesis. can do.

Pro means proline, Ala means alanine, Gln means glutamine, Lys means lysine, and all such amino acids are in L-form.

In order to obtain the peptide of the present invention from a natural product such as skipjack, extraction with hot water is performed.

Specifically, the skipjack is mixed with water and boiled under vigorous stirring. After cooling the obtained broth, it 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 is a raw material having a reactive carboxyl group corresponding to one of two fragments bisected at an arbitrary position of a peptide bond by a method usually used for peptide synthesis, that is, a liquid phase method or a solid phase method, It is also produced by condensing a raw material having a reactive amino group corresponding to the other fragment using a carbodiimide method, an active ester method, or the like, and, if the condensate formed has a protective group, removing the protective group. obtain.

Functional groups that 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 and a benzyl ester. In the case of the solid-phase method, the C-terminal carboxyl group is a chloromethyl resin, an oxymail resin, a P-alkoxybenzyl alcohol resin. And the like.

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 a day, 1 to 3 times a day. The peptide of the present invention is generally
It is 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,
Carboxymethylcellulose sodium, hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone,
Polyvinyl alcohol, soft 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, liquid paraffin,
Examples include white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water and the like. 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.

These preparations can contain the peptide of the present invention at a rate of 0.01% or more, preferably 0.5 to 70%. These formulations may also contain other therapeutically valuable components.

[Action] The peptide of the present invention has an excellent angiotensin converting enzyme inhibitory action, exhibits a blood pressure lowering action, an inhibitory action on bradykinin inactivation, and prevents hypertension such as essential hypertension, renal hypertension and adrenal hypertension, It is useful as a therapeutic agent, a diagnostic agent for these diseases, 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.

[Examples] Next, the present invention will be described more specifically with reference to actual examples.

(Peptide extraction)

45 g of water was added to 5 g of cutlet and homogenized, put into a flask, and left in a boiling water bath for 10 minutes. After cooling, the mixture was centrifuged, the supernatant was injected into a Sep-pak cartridge (C18), the eluted fraction was concentrated, and purified by high performance liquid chromatography (ODS, CN column) to obtain an inhibitory activity fraction. .

The amino acid sequence of this product was analyzed by an automatic Edman degradation method using a gas phase protein sequencer (Model 470A manufactured by Abride Biosystems) to obtain the following structure.

As a result of a homology search using a protein data bank, the peptide was found to be identical to the C-terminal of muscle creatine kinase.

(Synthesis of peptide)

0.94 g of commercially available Boc (butoxycarbonyl) -Lys (protected with chlorobenzyloxycarbonyl) -O-Resin [benzyl resin (substitution rate 0.32 meq / g)] was dispensed into a reaction vessel of a peptide synthesizer SAM2 manufactured by BioResearch. The synthesis was performed as follows.

45% trifluoroacetic acid, 2.5% anisole and 2%
After removing the Boc group by reacting in methylene chloride containing 1.2-ethanedithiol for 25 minutes, washing with methylene chloride, neutralization with methylene chloride containing 10% diisopropylethylamine, and washing with methylene chloride were performed.

5 ml of 0.4 M Boc-Gln (glutamine) -oBz (benzyl ester) in dimethylformamide, 5 ml
After mixing with a 0.6 M solution of diisopropylcarbodiimide in methylene chloride, the mixture was added to the reaction vessel, and the mixture was stirred and reacted at room temperature for 2 hours.

The obtained resin was washed with dimethylformamide, methylene chloride, methylene chloride containing 10% diisopropylethylamine, a mixed solution of methylene chloride, and a mixture of methylene chloride and dimethylformamide to obtain a Boc-Gln-Lys-resin.

Subsequently, the same removal of the Boc group and the coupling of the Boc and the amino acid were repeated to repeat Asp (oBzl) -Met-Ile-Pro-Ala-G.
In-Lys (Cl-z) -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, the residue was extracted with 30% acetic acid, and lyophilized to obtain a crude peptide. Use this with an ODS column (Cosmo
Purification by reverse phase chromatography on sil 5C / 18) yielded H-Asp-Met-Ile-Pro-Ala-Gln-Lys-OH (160 mg yield).

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.

Various peptides shown in Table 1 were synthesized according to the above synthesis examples except that the reaction agent was changed according to the amino acid type of the target peptide.

Examples 1 to 7 (Measurement of angiotensin converting enzyme inhibitory activity)
And Cushman's method (Biochemical Pharamacology 20 , 1637
(1971)] according to the following method.

Enzyme substrate: Bz (benzyl) -Gly-His-Leu (86 mg dissolved in 8 ml of water and 8 ml of phosphate buffer) Enzyme: rabbit lung acetone powder (Sigma) (1 g of 50 mM phosphorus After grinding in 10 ml of acid buffer,
Centrifuged supernatant) 100 μl of the above enzyme substrate, 12 μl of the enzyme solution and the peptide of a predetermined concentration of the present invention are mixed, and the whole is mixed with water at 250 μl.
Then, the reaction was carried out at 37 ° C. for 30 minutes.

The reaction was terminated using 250 μl of 1N HCl. 1.5 ml of ethyl acetate was added to the reaction-terminated liquid, followed by stirring with Vortex for 15 seconds, followed by centrifugation.

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 the ultraviolet absorption ₂₂₈ nm (OD ₂₂₈ ) of the extracted hippuric acid was measured.

Percent inhibition by the OD ₂₂₈ of when reacted without inhibitor as 100%, the concentration IC of the inhibitor when the OD ₂₂₈ of the inhibition rate of 50% determined as 0% when the reaction time of 0 minutes (the peptide of the present invention)
_The activity was indicated at ₅₀ (μM).

 The results are shown in Table 1.

In addition, as a reference example, an inhibitor other than the present invention was also measured, and is shown in Table 1.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) A61K 38/00-38/58 REGISTRY (STN) CA (STN)

Claims (1)

(57) [Claims] 1. Angiotensin converting enzyme inhibitor comprising the following peptide Pro-Ala-Gln-Lys as an active ingredient: