JPH0959296A - New tetrapeptide and angiotensin converting enzyme inhibitory agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new tetrapeptide having angiotensin converting enzyme inhibiting activity from the treated liquid of laver protein catabolic enzyme. SOLUTION: This peptide is produced by treating laver with a protein catabolic enzyme, this new peptide has the sequence of Pro-Gly-Val-Ala and angiotensin converting enzyme inhibiting activity, antihypertensive effect and extremely small toxic effect.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a tetrapeptide having a peptide structure having the following amino acid sequence, which has utility as a medicine, and an angiotensin converting enzyme inhibitor containing the tetrapeptide as an active ingredient. Pro-Gly-Val-Ala (In the formula, each symbol representing an amino acid residue is based on a conventional notation in amino acid chemistry.)

[0002]

BACKGROUND OF THE INVENTION It is well known that the renin-angiotensin system plays an important role in regulating water / electrolytes and blood in the living body. Angiotensin converting enzyme (hereinafter abbreviated as ACE) exists in this renin-angiotensin system, and angiotensin I is angiotensin I by ACE.
Converted to I. Angiotensin II is a potent pressor substance, which acts on not only blood vessels and adrenal cortex but also central and peripheral nervous systems to promote an increase in blood pressure. Also, ACE
Has an action of degrading and inactivating bradykinin which is an antihypertensive substance in vivo, and is involved in the pressor system. Therefore, it is possible to lower the blood pressure by inhibiting the activity of ACE, and it is considered to be clinically effective in preventing and treating hypertension. For this purpose, the proline derivative captopril was synthesized, and since its antihypertensive effect was confirmed, various ACE inhibitors based on the structure study of captopril have been actively researched, and recently, enarabril maleate, aracebryl and the like. Substances are being offered to clinical sites one after another. Current,
ACE inhibitors are widely used regardless of essential hypertension, symptomatic hypertension, mild or severe, and are added to the first-line treatment of hypertension, and many excellent It has been found to have points. On the other hand, research is also progressing as a therapeutic agent for heart failure as a new application of ACE inhibitors. As the mechanism of action of the ACE inhibitor, suppression of the secretion of aldosterone and vasobretuscin by suppressing the production of angiotensin II, and promotion of excretion of sodium and water by releasing the contraction of renal arteries are considered. Furthermore, A
Regarding CE inhibitors, it is considered that they suppress the inactivation of the kakrein-kinin system and activate the prostaglandin system to further promote peripheral vasodilation and excretion of sodium and water. It is expected as a purposeful therapeutic drug for breaking the vicious circle of. By the way, as the ACE inhibitor, in addition to the above-mentioned synthetic products, as a substance derived from a natural product or a natural product, a snake venom-derived bradykinin enhancing factor (C-terminal is Pro) [S. H. Ferr
eia. et ai /: Biochemistry.
9.3583 (1970)] 6 kinds of peptides derived from collagenase digestion of gelatin (all have C-terminal Ala-
Hyp) [G. Oshima, et al. : Bioc
him. Biophys. Acta. , 556,128
(1979)], a peptide derived from a tryptic digest of bovine casein (C-terminal is Gly-Lys) [S. Maruy
ama, et al. : Agric. Biol. Che
m. , 46, 1393 (1982)], etc., but all of these peptide substances have been confirmed to be effective by intravenous administration, and the pharmacological effect by oral administration is completely unknown. Although it has been a long time since its discovery, there is no report that it is still being developed as a drug.

[0003]

Means for Solving the Problems The present inventor has searched for a substance having a pharmacological action from a decomposition solution of proteolytic enzyme of seaweed and found that a novel tetrapeptide has a strong angiotensin converting enzyme inhibitory action. Then, they have earnestly conducted research to put this tetrapeptide into practical use as a medicine. As a result, they have found that this tetrapeptide has a hypotensive action and is useful as an angiotensin converting enzyme inhibitor derived from a natural product. The present invention is based on such findings. The present invention will be described in detail below. The novel tetrapeptide according to the present invention is a novel tetrapeptide having an L-amino acid sequence represented by the following formula: Pro-Gly-Val-Ala, and is a white powder at room temperature.

The above-mentioned tetrapeptide can be chemically synthesized or separated and purified from a decomposed solution of seaweed proteolytic enzyme. In the case of chemically synthesizing the novel tetrapeptide according to the present invention, it can be carried out by an ordinary peptide synthesis method such as a liquid phase method or a solid phase method, but preferably, a solid polymer method is used by the solid phase method. It is preferable that the L-amino acid corresponding to the amino acid residue is sequentially bound to the phase support from the C-terminal (carboxyl terminal side) of the tetrapeptide by a peptide bond. And the synthetic peptide thus obtained is
After cleaving from the polymeric solid-phase support using trifluoromethanesulfonic acid, hydrogen fluoride, etc., the protecting group of the amino acid side chain was removed, and high-performance liquid chromatography (hereinafter, HPLC) using a reversed-phase column was used. It is abbreviated.) And the like.

As described above, the novel tetrapeptide according to the present invention can be separated and purified from the degradation solution of proteolytic enzyme of seaweed. In that case, for example, it can be performed as follows. . The seaweed portion containing the novel tetrapeptide is taken out and hydrolyzed. Hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, the seaweed is further hydrolyzed if necessary, then adjusted to the optimum value of the enzyme, and the enzyme is added and incubated. Then, after neutralizing as necessary, the enzyme is deactivated to obtain a hydrolyzed solution. The hydrolyzed solution is filtered with filter paper and / or Celite to remove insoluble components, and the obtained filtrate is filtered with a semipermeable membrane such as cellophane to obtain a suitable solvent (eg, Tris-hydrochloric acid buffer solution). , A buffer solution containing a neutral buffer solution, etc.) and a solution containing the components of the filtrate that have passed through the semipermeable membrane are treated with a strongly acidic cation exchange resin (eg, Dow Chemical). Dowex 50W, etc.) to obtain a fraction containing a component having an ACE (angiotensin converting enzyme) inhibitory activity from the adsorbed and eluted fraction, and the obtained ACE inhibitory activity fraction is subjected to gel filtration (for example, manufactured by Pharmacia). Sephadex G-25, etc.), and the resulting ACE inhibitory activity fraction is filtered through a cation exchange gel (for example, SP-Sepha manufactured by Pharmacia).
ACE obtained by fractionation by dex C-25 etc.)
The inhibitory activity fraction is further fractionated by reverse phase HPLC.

This novel tetrapeptide does not induce antibody production or anaphylactic shock when it is repeatedly administered intravenously. In addition, since this tetrapeptide has a sequence structure of only L-amino acids and is gradually decomposed by in vivo protease after administration,
Very low toxicity and very high safety (LD ₅₀ > 50
00 mg / Kg: Rat oral administration). The novel tetrapeptide according to the present invention can be prepared into injections, tablets, capsules, granules, powders and the like using additives such as commonly used excipients. The administration method is usually AC
Examples include injection into a mammal having E (eg, human, dog, rat, etc.) or oral administration. The dosage is, for example, 0.01 to 10 mg of this tetrapeptide per 1 kg of animal body weight. The frequency of administration is usually about 1 to 4 times a day, but it can be appropriately adjusted depending on the administration route. The kinds of excipients, binders and lubricants used in the above-mentioned various preparations are not particularly limited, and those used for ordinary injections, powders, granules, tablets or capsules can be used. .

The following may be mentioned as additives used for tablets, capsules, granules and powders. Excipients include sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate and the like; binders such as starch and hydroxypropylmethylcellulose; disintegrants carboxymethylcellulose and its potassium salts; lubricants. Examples of the lubricant include stearic acid and its salts, talc, and waxes. In preparation of the preparation, menthol, citric acid and salts thereof, and flavoring agents such as fragrances can be used if necessary. The sterile composition for injection is
By a conventional method, the novel tetrapeptide according to the present invention is dissolved or suspended in water for injection, physiological saline, sugar alcohol injection such as xylitol or mannitol, or glycol such as propylene glycol or polyethylene glycol to give an injection. be able to. At this time, buffers, preservatives, antioxidants and the like can be added as necessary. The preparation containing the novel tetrapeptide of the present invention may be in the form of a lyophilized product or a dry powder, and may be dissolved in an ordinary solubilizer such as water or physiological saline before use.

The novel tetrapeptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, and exhibits a blood pressure lowering action and a bradykinin inactivity suppressing action. Therefore, essential hypertension, renal hypertension, prophylactic and therapeutic agents for hypertension such as adrenal hypertension, useful as a diagnostic agent for these diseases and as a blood pressure lowering agent used in various pathological conditions, and further for organs for congestive heart failure. It has normalization of circulation and improvement of long-term prognosis (life extension effect), and is useful as a therapeutic agent for heart failure.

[0009]

EXAMPLES Hereinafter, the present invention will be described in more detail by describing production examples and test examples as examples. Production Example 1 2 l of deionized water was added to 50 g of air-dried fresh seaweed and homogenized. 1.5 g of pepsin was added to the obtained seaweed homogenate to adjust the pH to 2.0, and the mixture was incubated at 37 ° C. for 20 hours. The pepsin-decomposed solution of the seaweed homogenate thus prepared was subjected to ultrafiltration using a Diaflow membrane (YM10 type membrane manufactured by Amicon, fractionated molecular weight 10,000). The resulting filtrate is Dowex 50WX4
Chromatography was performed using a column packed with (H ⁺ ). After washing with deionized water, elution was performed with 2N-NH ₄ OH and the eluate was concentrated. This concentrate is Sephadex
Column chromatography was performed on a G-25 column to separate peptide fractions (fraction numbers 25 to 45). The column chromatogram is shown in FIG. This peptide fraction was concentrated to obtain a laver peptide solution. Further, this peptide solution was subjected to column chromatogram treatment with an SP-Sephadex C-25 (H ⁺ ) column to give SP-1 fractions (fraction numbers 10 to 28) and SP as respective peptide fractions.
-2 fraction (fraction number 29-40) and SP-3 fraction (fraction number 41-55) were separated. The column chromatogram is shown in FIG. These peptide fractions were freeze-dried to give 2.2 g of SP-1 fraction (IC ₅₀ value of 0.50) as peptide powder (hereinafter referred to as laver peptide).
91 mg / ml), SP-2 fraction 4.5 g (IC
₅₀ 1.5 mg / ml) and SP-3 fraction 7.3 g
(IC ₅₀ value 10.2 mg / ml) was obtained. Among the seaweed peptides thus fractionated, the peptide powder of the SP-1 fraction having high ACE inhibitory activity was dissolved in deionized water and then subjected to HPLC. The conditions are as a column, Develosil ODS-5 (φ4.6 m, manufactured by Nomura Chemical Co., Ltd.
mIDX 25 cm L) and 0.0 as the mobile phase
By a concentration gradient method of 5% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TFA, flow rate 1.0 ml / min, detection wavelength 220 n
Chromatographic treatment with m gave a peptide fragment having a strong ACE inhibitory action at an elution time of 48.192 minutes. The result is as shown in FIG. The amino acid sequence of the thus obtained peptide having an ACE inhibitory action was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI).
As a result, it was confirmed to be a tetrapeptide having a sequence consisting of L-form amino acid residues represented by the following formula: Pro-Gly-Val-Ala. In the present invention, the relationship between the peptide content (represented by the dilution rate) of each peptide fraction (SP fraction) in the SP-Sephadex C-25 (H ⁺ ) column chromatograph and the ACE inhibition rate% is shown. A result like 4 was obtained. Further, the results as shown in FIG. 5 were obtained as the amino acid composition ratio% of each peptide fraction (SP fraction). The laver peptide according to the present invention is AC
When a tablet is prepared as an E inhibitor, for example, it may be treated according to a conventional method as follows: (1) Peptide 13 g, (2) Lactose 87 g, (3) Corn starch 29 g, (4) Stearic acid Using 1 g of magnesium as a raw material, first, (1), (2) and 17 g of corn starch were mixed, and granulated with a paste made from 7 g of corn starch, and 5 g of corn starch and (4) were added to this granule to obtain The mixture is compressed on a compression tableting machine to produce 1000 tablets.

Production Example 2 This example is an example of production by a synthetic method. Pro-Gly-Val-Ala Synthesis Method The tetrapeptide was synthesized by a solid phase method using an automated peptide synthesizer model 430A manufactured by Applied Biosystems. As the solid support, a resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the tetrapeptide, a C-terminal side Pro was reacted with a chloromethyl resin according to a conventional method to obtain a peptide-bonded resin. The amino acid at this time is t-Bo protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group.
c amino acid was used. Next, the peptide-bonded resin was suspended in a mixed solution of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, trifluoroacetic acid was added under ice cooling, and the mixture was further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to this mixture, and the mixture was stirred at room temperature for 30 minutes, anhydrous ether was added to precipitate and separate the product, and the precipitate was washed several times with anhydrous ether.
Dry under reduced pressure. The thus-purified synthetic peptide thus obtained was dissolved in distilled water and then subjected to reverse phase column C.
Purified by HPLC using ₁₈ (5μ). As a mobile phase, (A) 0.1% TFA-containing distilled water, (B) 0.1
% TFA-containing acetonitrile solution was used, and the solution (A) had a flow rate of 1.% by a gradient method of 97% → 77% in 20 minutes.
Chromatography was performed at 5 ml / min. The elution fraction showing the maximum absorption, which was detected at an ultraviolet wavelength of 215 nm, was collected and freeze-dried to obtain the target synthetic tetrapeptide.

As a result of mass spectrometry of this synthetic tetrapeptide, it was confirmed to be a tetrapeptide having an amino acid sequence structure represented by the following formula: Pro-Gly-Val-Ala. The result of the mass spectrum is as shown in FIG. The pharmacological effect of the tetrapeptide of the present invention obtained by synthesis was confirmed by the following tests.

Test Example 1 (Angiotensin converting enzyme inhibitory activity) 2.5 mU of ACE (manufactured by Sigma, enzyme number EC3.4.5.1)
Synthetic substrate Hippuryl-L-histidyl-L
-Leucine (manufactured by Peptide Institute) 12.5 mM and improved method of Lieberman measurement [Yamamoto et al., 18, 297-302 (198)
9)]. That is, the produced hippuric acid was extracted with ethyl acetate and the absorbance was measured at 225 nm. The absorbance in the test solution was Es, the value when the buffer solution was added instead of the test solution was Ec, and the value when the reaction stop solution was added in advance and the reaction was Eb, and the inhibition rate was calculated from the following equation. . Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 10
0 The inhibitory activity IC ₅₀ value of the ACE inhibitor was shown as the concentration (M) of the sample required to inhibit the enzyme activity of ACE by 50% (inhibition rate). The inhibitory activity IC ₅₀ value of the novel tetrapeptide of the present invention against angiotensin converting enzyme of bovine lung serum is 9.5 × 10 ⁻⁷ M.

Test Example 2 (Hypotensive effect when administered to rats) As a test animal, 15-week-old male spontaneously hypertensive rats (SHR) were purchased from Japan Charles River Co., Ltd. The animals were used as a group of 3 animals having a blood pressure of 160 mmHg or more (body weight 280 to 330 g). Blood pressure was measured using a non-invasive caudal artery blood pressure measuring device, PS-100, manufactured by Riken Development Co., Ltd.
By the l-cuff method, the systolic blood pressure (SB) of the SHR tail artery before administration and 1 hour, 2 hours, 4 hours, and 5 hours after administration.
P), mean blood pressure (MBP) and diastolic blood pressure (DBP)
Was measured 5 times every measurement time, the highest value and the lowest value of the obtained measurement values were rejected, and the average value of 3 times was taken as the measurement value of each time. Each variation value was calculated by taking the average value immediately before administration as the measured value at 0 hours, subtracting the average value at 0 hours from the average value at each time, and calculating the difference. The results on the effect on blood pressure when orally administering 10 mg / kg of synthetic tetrapeptide to SHR are as shown in FIG. 7.
As a result of the above test, the tetrapeptide according to the present invention has angiotensin converting enzyme harmful activity and
It was confirmed that (in vivo) also shows a significant blood pressure lowering effect. Therefore, the tetrapeptide according to the present invention is useful as a therapeutic or prophylactic drug for hypertension. The tetrapeptide according to the present invention can also be employed in the structure of a peptide having a partial structure of its amino acid sequence structurally.

[0014]

[Brief description of drawings]

FIG. 1 is a view showing the results of separation and purification of an ACE-inhibiting peptide by Sepadex G-25 column chromatography in Production Example 1 of a seaweed pepsin degradation solution according to the present invention. In addition, as a marker in the figure, blue dextran having a molecular weight of 2 million and vitamin B ₁₂ having a molecular weight of 1335 are used.
Was used.

FIG. 2 is a diagram showing the results of separation and purification of the seaweed peptide of the present invention by SP-Sephadex C-25 (H ⁺ ) column chromatography in Production Example 1.

FIG. 3 is a diagram showing the results of separation and purification of an ACE-inhibiting peptide fragment of the tetrapeptide according to the present invention by reverse phase HPLC in Production Example 1.

FIG. 4 is a diagram showing the relationship between the peptide content (dilution rate) of the SP fraction in Production Example 1 and the ACE inhibition rate% of the laver peptide according to the present invention.

FIG. 5 is a view showing the amino acid composition ratio% of the SP fraction in Production Example 1 of the laver peptide according to the present invention.

FIG. 6 is a diagram showing a mass spectrum of the synthetic tetrapeptide obtained in Production Example 2 of the tetrapeptide according to the present invention.

FIG. 7: Synthetic tetrapeptide 10m obtained in Production Example 2
It is a figure which shows the time-dependent change of the blood pressure value (systolic blood pressure, average blood pressure, and diastolic blood pressure) when g / kg is orally administered to SHR.

Claims (2)

[Claims] 1. The following formula: Pro-Gly-Val-A
A novel tetrapeptide having a peptide structure consisting of the L-amino acid sequence represented by la. 2. The following formula: Pro-Gly-Val-A
An angiotensin-converting enzyme inhibitor, which comprises, as an active ingredient, a novel tetrapeptide having a peptide structure represented by the L-amino acid sequence represented by la.