JP2001064299A - Novel tetrapeptide and angiotensin converting enzyme inhibitors - Google Patents

Abstract

(57) [Summary] [Object] To provide a novel tetrapeptide having an angiotensin converting enzyme inhibitory action from a seaweed protease hydrolyzate. [Constitution] Wakame is treated with a protease and the like, and four kinds of tetrapeptides having novel angiotensin converting enzyme inhibitory activity are (1) Tyr-Asn-Lys-Leu,
(2) Tyr-Lys-Tyr-Tyr, (3) Lys
-Phe-Tyr-Gly, (4) Ala-Ile-T
yr-Lys, which has a blood pressure lowering effect in vivo,
Very low toxicity.

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 of the following amino acid sequence, which is useful as a pharmaceutical, and an angiotensin converting enzyme inhibitor containing the peptide as an active ingredient. Tyr-Asn-Lys-Leu Tyr-Lys-Tyr-Tyr Lys-Phe-Tyr-Gly Ala-Ile-Tyr-Lys (wherein each symbol representing an amino acid residue is represented by a conventional notation in amino acid chemistry. Is.)

[0002]

BACKGROUND OF THE INVENTION It is well known that the renin-angiotensin system plays an important role in the regulation of water, electrolytes and blood in living organisms. An angiotensin converting enzyme (hereinafter abbreviated as ACE) exists in the renin-angiotensin system, and angiotensin I is converted into angiotensin I by ACE.
Converted to I. Angiotensin II is a powerful vasopressor and acts on the central nervous system and peripheral nervous system as well as blood vessels and the adrenal cortex to promote blood pressure elevation. ACE has a function of decomposing and inactivating bradykinin, which is a hypotensive substance in a living body, and is involved in the pressor system. Therefore, it is possible to lower blood pressure by inhibiting the activity of ACE, and it is considered that this is clinically effective for prevention and treatment of hypertension. For this purpose, caprol, a proline derivative, was synthesized and its antihypertensive action was confirmed.
Synthetic studies on CE inhibitors have been actively conducted, and recently, substances such as enalabryl maleate and alacerbyl have been provided one after another to clinical sites. At present, ACE inhibitors are widely used regardless of whether they are essential hypertension or symptomatic hypertension, or mild or severe, and are added in the first-line treatment of hypertension, Has been found to have On the other hand, as the mechanism of action of the ACE inhibitor, suppression of aldosterone and vasopressin secretion by suppressing the production of angiotensin II and promotion of sodium and water excretion by releasing renal artery contraction are considered. Furthermore,
ACE inhibitors are thought to inhibit the inactivation of the kallikren-kinin system and further promote peripheral vasodilation and sodium and water excretion by activating the prostaglandin system. It is expected as a suitable therapeutic drug in breaking the vicious circle. By the way, as an ACE inhibitor, in addition to the above-mentioned synthetic product, a natural product or a substance derived from a natural product, snake venom-derived bradykinin enhancer (C-terminal is Pro) [S. H. Ferrei
a et al: Biochemistry, 9, 35
83 (1970)], six peptides derived from collagenase digest of gelatin (all of which have C-terminal Ala-Hy).
p) [G. Oshima et al: Biochim.
Biophs. Acta, 566, 128 (197
9)], a peptide derived from a tryptic digest of bovine casein (C-terminal is Gly-Lys) [S. Maruyama
et al. : Agric. Biol. Chem. , 4
6, 1393 (1983)] and the like, and five hexapeutides derived from the sardine muscle of the present inventors (all of which are Pro at the second or third from the C-terminus and Leu at the N-terminus) [Patent No. 2046483], laver Tetrapeptide (N-terminal is Pro) [Patent No. 2678180], and it is disclosed that any of them can be an ACE inhibitor. Further, di- and tripeptides having a short chain length obtained by a synthesis method [JP-A-6-87886] [JP-A-6-165]
No. 68] has been proposed, but the ACE inhibitory effect of a di- or tripeptide or higher peptide having a long amino acid sequence with a regular amino acid sequence and the antihypertensive effect (pharmacological effect) by oral administration are unknown. Yes, it has been a long time since it was discovered, but there are no reports that it is still being developed as a drug. In the case of foods, Suzuki et al. Have observed ACE inhibitory activity in soybeans, teas, shellfish, fruits, etc. [Takeo Suzuki, Nobuko Ishikawa et al., Agricultural Chemistry, 57, 1143 (1983)]. It is not known that wakame, which is widely used as a health food, has an ACE inhibitor.

[0003]

Means for Solving the Problems The present inventors searched for a substance having a pharmacological action from a decomposition solution of wakame proteolytic enzyme belonging to the seaweed species of the brown alga Laminariales and found four novel tetrapeptides. Has a strong angiotensin converting enzyme inhibitory action.
And, they studied diligently to put these four kinds of tetrapeptides into practical use as medicines. As a result, the four types of tetrapeptides have a blood pressure lowering effect and have been found to be useful as angiotensin converting enzyme inhibitors derived from natural products. The present invention is based on such findings. The novel peptides according to the present invention have the following formulas (1), (2) and (3)
And (4) (1) Tyr-Asn-Lys-Leu (2) Tyr-Lys-Tyr-Tyr (3) Lys-Phe-Tyr-Gly (4) Ala-Ile-Tyr-Lys It is a novel tetrapeptide having the sequence of body amino acids, and is white powder at room temperature.

The above four types of tetrapeptides can be chemically synthesized or separated and purified from a decomposition solution of wakame protease. When the novel peptide according to the present invention is chemically synthesized, it can be carried out by a conventional peptide synthesis method such as a liquid phase method or a solid phase method. It is preferable that the L-form amino acids corresponding to the amino acid residues are sequentially bonded to the support from the C-terminal (carboxyl-terminal side) of the peptide by peptide bonds. And the synthetic peptide thus obtained is
After cleavage from the poromeric solid support using trifluoromethanesulfonic acid, hydrogen fluoride, or the like, the protecting groups on the amino acid side chains are removed, and high-performance liquid chromatography using a reversed-phase column (hereinafter, HPLC). , Etc.).

[0005] As described above, the novel peptide of the present invention can be separated and purified from a decomposition solution of wakame proteolytic enzyme. In that case, for example, it can be carried out as follows. Hydrolysis is performed using the wakame protein portion containing the novel peptide. The 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 heated to the optimum temperature of the enzyme, the pH is adjusted to the optimum value, the enzyme is added, and the mixture is incubated. . Then, if necessary, after neutralization, the enzyme is inactivated to obtain a hydrolyzed solution. The hydrolyzate is filtered using filter paper and / or celite to remove insoluble components, and the obtained filtrate is filtered using a semi-permeable membrane such as cellophane to a suitable solvent (eg, water, Tris-hydrochloric acid). Buffer,
After sufficient dialysis in a neutral buffer such as a phosphate buffer, the solution containing the components in the filtrate that has passed through the semipermeable membrane is converted to a strongly acidic cation exchange resin (for example, Dow Chemical Co., Ltd.). D
owex 50W, etc.), and angiotensin converting enzyme (hereinafter abbreviated as ACE) from the adsorbed and eluted fraction.
A fraction containing a component having an inhibitory activity was obtained.
The CE inhibitory activity fraction was fractionated by gel filtration (for example, Sephadex G-25 manufactured by Pharmacia),
The obtained ACE inhibitory activity fraction is subjected to cation exchange gel filtration (for example, SP-Sephadex manufactured by Pharmacia).
C-25), and the obtained ACE inhibitory activity fraction is further fractionated by reverse phase HPLC.

As the brown algae used in the method for producing the novel peptide according to the present invention, any brown algae may be used as long as the object of the present invention can be achieved, and wakame is preferably used. The novel peptide of the present invention obtained as described above does not induce antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. Also, the novel peptide according to the present invention is L
- consists array structure of an amino acid alone, after administration, because it is gradually degraded by proteases in vivo, toxicity is very low, the safety is very high (LD _50> 5000mg /
kg; oral administration to rats). Novel peptide according to the present invention, injections using additives such as commonly used excipients,
It can be prepared into tablets, capsules, granules, powders and the like. The method of administration usually includes injection into mammals (eg, humans, dogs, rats, etc.) having ACE, or oral administration. The dose is, for example, 0.01 g of this peptide per 1 kg of the animal body.
In the amount of 10 mg. 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 types of excipients, binders, and lubricants used in the above-mentioned various preparations are not particularly limited, and those used in ordinary injections, powders, granules, tablets or capsules may be used. Can be. Examples of additives used for tablets, capsules, granules, and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate, etc .; starches, hydroxypropylmethyl cellulose, etc. as binders; carboxymethylcellulose and potassium salts thereof as disintegrants; Examples of the lubricant include stearic acid and salts thereof, talc, and waxes. In addition, in preparing the preparation, a flavoring agent such as menthol, citric acid and its salts, and fragrance can be used as necessary. The sterile composition for injection can be prepared by dissolving or suspending the novel peptide of the present invention in water for injection, physiological saline, a sugar alcohol injection such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol by an ordinary method. It can be turbid to obtain an injection. At this time, buffers, preservatives, antioxidants and the like can be added as necessary. The preparation containing the novel peptide according to the present invention may be in the form of a lyophilized product or a dry powder, and may be used by dissolving it in a usual dissolving agent, for example, water or physiological saline when used.

[0008] The novel peptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, a hypotensive action,
Shows the effect of inhibiting bradykinin inactivation. Therefore, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension,
Therapeutic agents have a function of normalizing organ circulation and improving long-term prognosis (life-extending effect) for congestive heart failure, and are useful as therapeutic agents for heart failure.

[0009]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which are Production Examples and Test Examples. Production Example 1 A wakame homogenate obtained by adding 708 ml of deionized water to 23.6 g of wakame powder and homogenizing it was used. The solution was packed in a dialysis tube (inner diameter: 36 inches, manufactured by Wako Pure Chemical Industries, Ltd.) and dialyzed against running water for 3 days to obtain a dialysis solution. The pH of this inner solution was adjusted to 2.0 with 1N hydrochloric acid, and pepsin (manufactured by Merck, enzyme number EC 3.4.23.1) 708 was used.
mg was added and hydrolysis was carried out while stirring at 45 ° C. for 5 hours. The passing solution obtained by passing the decomposition reaction solution immediately through an ultrafiltration membrane (YM10, manufactured by Amicon; fractionation molecular weight: about 10,000) was passed through a Dowex 50W × 4 [H ⁺ ] column (φ4.0).
× 55 cm). After sufficiently washing the column with deionized water, the column was eluted with 2N ammonium hydroxide solution 2ι. Sephadex G-2 in which ammonia was removed by concentration under reduced pressure and the concentrate was buffered with deionized water in advance.
5 (φ1.6 × 113cm), flow rate 12mι /
The mixture was subjected to gel filtration at a fraction of 5.7 ml for each hr. The result is as shown in FIG. The gel filtration was repeated, and fractions having high ACE inhibitory activity collected in large amounts were collected and freeze-dried to obtain peptide powder. 1.55 g of this peptide powder was added to 20
After dissolving in deionized water (ml), the solution was loaded on a SP-Sephadex C-25 [H ⁺ ] column (φ1.8 × 40 cm) previously buffered with deionized water, and 500 m of deionized water was added.
Chromatography was performed at a flow rate of 70 ml / hr and a fractionation amount of 10 ml each by performing a concentration gradient method from 500 to 500 ml of sodium chloride from 1.5%. The result is as shown in FIG. In the above chromatograph, fractions of fraction numbers 14 to 22 having high ACE inhibitory activity were collected and freeze-dried to obtain a purified peptide powder (SP-I fraction). After dissolving 20 mg of this purified peptide powder in 60 µι of deionized water, high performance liquid chromatography (HPLC) was performed. Nomura Chemical's Develosil ODS-
5 (4.5 mm ID × 25 cmL) using a mobile phase of 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TF.
A concentration gradient method of A was performed, and HPLC was performed at a flow rate of 1.0 ml / min and a detection wavelength of 220 nm to obtain a peptide fragment having high ACE inhibitory activity. The result is as shown in FIG. ｛Elution time; peptide 99.6 of (1)
25 minutes, (2) peptide 80.724 minutes, (3) peptide 96.067 minutes and (4) peptide 32.03
3 minutes The amino acid sequence of the thus-obtained peptide having an ACE inhibitory effect was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the four peptides are:
L-forms represented by the following formulas: (1) Tyr-Asn-Lys-Leu (2) Tyr-Lys-Tyr-Tyr (3) Lys-Phe-Tyr-Gly (4) Ala-Ile-Tyr-Lys Was confirmed to be a novel tetrapeptide represented by the following amino acid sequence: The property at room temperature is a white powder. When the novel tetrapeptide according to the present invention is formulated as an ACE inhibitor in, for example, a tablet, it may be treated according to a conventional method, for example, as follows: peptide 10 g, lactose 68 g, corn starch 39 g, stearic acid Starting with 1.2 g of magnesium, first and 20 g of corn starch were mixed, granulated together with a paste made from 11 g of corn starch, 9 g of corn starch was added to the granules, and the resulting mixture was compressed with a compression tablet machine. Tablets and tablets 10
00 pieces are manufactured.

Production Example 2 This is an example of production by a synthesis method. Synthesis Method of Tyr-Asn-Lys-Leu The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the tetrapeptide, leucine on the C-terminal side was reacted with a chloromethyl resin according to a conventional method to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
Abbreviated as -Boc. ) -Protected t-Boc amino acid was used. Next, this peptide-bonded resin was suspended in a mixed solution consisting of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid under ice cooling, and further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water or methanol, and then purified by HPLC using a reverse-phase column C ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase,
(B) Using acetonitrile solution containing 0.1% TFA, the solution (A) was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method from 83% to 53% in 40 minutes. The eluted fraction detected at an ultraviolet wavelength of 218 nm and showing the maximum absorption was separated and lyophilized to obtain the desired synthetic peptide.

As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tetrapeptides having the amino acid sequence structure shown above. The result of the mass spectrum is as shown in FIG. Synthesis Method of Tyr-Lys-Tyr-Tyr The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the tetrapeptide, a tyrosine on the C-terminal side was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
A t-Boc amino acid protected with a group (abbreviated -Boc) was used. Next, this peptide-bonded resin was suspended in a mixture of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, and then added with trifluoroacetic acid under ice-cooling.
Stir for another 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water or methanol, and then purified by HPLC using a reverse-phase column C ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase, (B)
Using an acetonitrile solution containing 0.1% TFA,
(A) The solution was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method from 83% to 53% for 40 minutes. The eluted fraction detected at an ultraviolet wavelength of 218 nm and showing the maximum absorption was separated and lyophilized to obtain the desired synthetic peptide. As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tetrapeptides having the amino acid sequence structure shown above. The result of this mass spectrum is as shown in FIG. Method for synthesizing Lys-Phe-Tyr-Gly The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the tetrapeptide, glycine on the C-terminal side was reacted with a chloromethyl resin according to a conventional method to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
A t-Boc amino acid protected with a group (abbreviated -Boc) was used. Next, this peptide-bonded resin was suspended in a mixture of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, and then added with trifluoroacetic acid under ice-cooling.
Stir for another 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water or methanol, and then purified by HPLC using a reverse-phase column C ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase, (B)
Using an acetonitrile solution containing 0.1% TFA,
(A) The solution was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method from 83% to 53% for 40 minutes. The eluted fraction detected at an ultraviolet wavelength of 218 nm and showing the maximum absorption was separated and lyophilized to obtain the desired synthetic peptide. As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tetrapeptides having the amino acid sequence structure shown above. The result of this mass spectrum is as shown in FIG. Ala-Ile-Tyr-Lys Synthesis Method The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the tetrapeptide, the C-terminal lysine was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
A t-Boc amino acid protected with a group (abbreviated as Boc) was used. Next, this peptide-bonded resin was suspended in a mixed solution consisting of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid under ice cooling, and further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The thus obtained unpurified synthetic peptide is dissolved in distilled water or methanol, and then reverse-phase column C is used.
Purified by HPLC using ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase;
Using an acetonitrile solution containing 1% TFA, the solution (A) was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method from 83% to 53% in 40 minutes. The eluted fraction detected at an ultraviolet wavelength of 218 nm and showing the maximum absorption was separated and lyophilized to obtain the desired synthetic peptide. As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tetrapeptides having the amino acid sequence structure shown above. The result of this mass spectrum is as shown in FIG. The pharmacological effects of the four tetrapeptides of the present invention obtained by synthesis were confirmed by the tests described below.

Test Example 1 (Method for measuring angiotensin converting enzyme inhibitory activity) ACE
(Manufactured by Sigma, Enzyme number EC 3.4.15.1) 2.5
mU, synthetic substrate Hippuryl-L-histidy
1-L-leucine (manufactured by Peptide Research Institute) 12.5
Yamamoto et al., which improved the method of measuring Lieberman using mM [Journal of the Japan Chest Disease, Vol. 18, pp. 297-302 (1)
989)]. That is, the generated hippuric acid was extracted with ethyl acetate and measured at an absorbance of 225 nm. The inhibition rate was determined from the following equation, where Es was the absorbance of the test solution, Ec was the value obtained when a buffer was added instead of the test solution, and Eb was the value obtained when a reaction stop solution was added in advance to react. . Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 10
0 The inhibitory activity IC ₅₀ value of the ACE inhibitor was represented by the concentration (M) of the sample required to inhibit the ACE enzyme activity by 50% (inhibition rate). Inhibitory activity (IC ₅₀ value) of four kinds of tetrapeptides according to the present invention on bovine lung serum angiotensin converting enzyme; 21.0 μm of tetrapeptide of (1)
M, 64.2 μM of the tetrapeptide (2), 90.5 μM of the tetrapeptide of (3), and 213 μM of the tetrapeptide of (4).

Test Example 2 (Hypertensive effect when administered to spontaneously hypertensive rats) As experimental animals, 15-week-old male spontaneously hypertensive rats (hereinafter abbreviated as SHR) were purchased from Charles River Japan.
After pre-breeding for one week, the animals were used as a group of six animals having a systolic blood pressure of 160 mmHg or more (weight: 280 to 330 g). The rats were individually housed in stainless steel rat individual gauges in a breeding room adjusted to room temperature of 23 ± 2 ° C., humidity of 55 ± 10%, and 12 hours light / dark (light on from 6:00 am to 6:00 pm). . Feed is MF powder feed manufactured by Oriental Yeast Co., Ltd. Drinking water is self-pumped (conforming to tap water quality standards)
Was freely taken. The blood pressure was measured using a non-invasive tail arterial blood pressure measurement device (manufactured by Riken, Model PS-100).
By the aile-cuff method, before administration, 1 hour after administration, 2 hours
Measurement of the systolic blood pressure value (mmHg) of the SHR tail artery for 5 hours, 3 hours, 4 hours and 6 hours was performed 5 times every measurement time,
The highest value and the lowest value of the obtained measured values were rejected, and the average value of three times was used as the measured value at each time. The results on the effects on the systolic blood pressure value (mmHg) when 50 mg / kg of four kinds of synthetic tetrapeptides were orally administered to SHR are as shown in FIGS. 8, 9, 10 and 11. As a result of the above test, the four tetrapeptides according to the present invention have angiotensin converting enzyme inhibitory activity,
It was confirmed that a significant blood pressure lowering effect was also exhibited in ovo (in vivo). Therefore, the four kinds of tetrapeptides according to the present invention are useful as drugs for treating or preventing hypertension. The four types of tetrapeptides according to the present invention can also be employed in the structure of a peptide having a partial structure of its amino acid sequence.

[0014]

[Brief description of the drawings]

FIG. 1 is a view showing the results of separation and purification of an ACE inhibitory peptide by Sephadex G-25 column chromatography in Production Example 1 of a wakame proteolytic solution according to the present invention. In the figures, insulin having a molecular weight of 6,000, insulin B chain having a molecular weight of 3,500, insulin A chain having a molecular weight of 2,500, bradykinin having a molecular weight of 1,052, and glycine having a molecular weight of 75 were used as markers.

FIG. 2 shows SP-Sephadex C-25 (H ⁺ ) in Production Example 1 of the seaweed protein hydrolyzate according to the present invention.
It is a figure which shows the result of separation and purification of ACE inhibitory peptide by column chromatography.

FIG. 3 is a diagram showing the results of separation and purification of ACE inhibitory peptides by reverse phase HPLC in Production Example 1 of four kinds of tetrapeptides according to the present invention.

FIG. 4, FIG. 5, FIG. 6, and FIG. 7 are diagrams showing mass spectra of four types of synthetic tetrapeptides obtained in Production Example 2 of four types of tetrapeptides according to the present invention.

8, 9, 10, and 11 are diagrams showing 4 obtained in Production Example 2.
Systolic blood pressure value (mmHg) when 50 mg / kg of each kind of synthetic tetrapeptide is orally administered to SHR.
It is a figure which shows a time-dependent change of. In the figure, captopril (10 mg / kg) was used as a control antihypertensive, and 3 ml of 0.9% physiological saline was used as a control.

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[Procedure amendment]

[Submission date] April 14, 2000 (2004.1.
4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction contents]

[Document Name] Statement

Novel tetrapeptide and angiotensin converting enzyme inhibitor

[Claims]

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 of the following amino acid sequence, which is useful as a pharmaceutical, and an angiotensin converting enzyme inhibitor containing the peptide as an active ingredient. Tyr-Asn-Lys-Leu Tyr-Lys-Tyr-Tyr Ala-Ile-Tyr-Lys (wherein each symbol representing an amino acid residue is represented by a convention used in amino acid chemistry.)

[0002]

BACKGROUND OF THE INVENTION It is well known that the renin-angiotensin system plays an important role in the regulation of water, electrolytes and blood in living organisms. An angiotensin converting enzyme (hereinafter abbreviated as ACE) exists in the renin-angiotensin system, and angiotensin I is converted into angiotensin I by ACE.
Converted to I. Angiotensin II is a powerful vasopressor and acts on the central nervous system and peripheral nervous system as well as blood vessels and the adrenal cortex to promote blood pressure elevation. ACE has a function of decomposing and inactivating bradykinin, which is a hypotensive substance in a living body, and is involved in the pressor system. Therefore, it is possible to lower blood pressure by inhibiting the activity of ACE, and it is considered that this is clinically effective for prevention and treatment of hypertension. For this purpose, caprol, a proline derivative, was synthesized and its antihypertensive action was confirmed.
Synthetic studies on CE inhibitors have been actively conducted, and recently, substances such as enalabryl maleate and alacerbyl have been provided one after another to clinical sites. At present, ACE inhibitors are widely used regardless of whether they are essential hypertension or symptomatic hypertension, or mild or severe, and are added in the first-line treatment of hypertension, Has been found to have On the other hand, as the mechanism of action of the ACE inhibitor, suppression of aldosterone and vasopressin secretion by suppressing the production of angiotensin II and promotion of sodium and water excretion by releasing renal artery contraction are considered. Furthermore,
ACE inhibitors are thought to inhibit the inactivation of the kallikren-kinin system and further promote peripheral vasodilation and sodium and water excretion by activating the prostaglandin system. It is expected as a suitable therapeutic drug in breaking the vicious circle. By the way, as an ACE inhibitor, in addition to the above-mentioned synthetic product, a natural product or a substance derived from a natural product, snake venom-derived bradykinin enhancer (C-terminal is Pro) [S. H. Ferrei
a et al: Biochemistry, 9, 35
83 (1970)], six peptides derived from collagenase digest of gelatin (all of which have C-terminal Ala-Hy).
p) [G. Oshima et al: Biochim.
Biophs. Acta, 566, 128 (197
9)], a peptide derived from a tryptic digest of bovine casein (C-terminal is Gly-Lys) [S. Maruyama
et al. : Agric. Biol. Chem. , 4
6, 1393 (1983)] and the like, and five hexapeutides derived from the sardine muscle of the present inventors (all of which are Pro at the second or third from the C-terminus and Leu at the N-terminus) [Patent No. 2046483], laver Tetrapeptide (N-terminal is Pro) [Patent No. 2678180], and it is disclosed that any of them can be an ACE inhibitor. Further, di- and tripeptides having a short chain length obtained by a synthesis method [JP-A-6-87886] [JP-A-6-165]
No. 68] has been proposed, but the ACE inhibitory effect of a di- or tripeptide or higher peptide having a long amino acid sequence with a regular amino acid sequence and the antihypertensive effect (pharmacological effect) by oral administration are unknown. Yes, it has been a long time since it was discovered, but there are no reports that it is still being developed as a drug. In the case of foods, Suzuki et al. Have observed ACE inhibitory activity in soybeans, teas, shellfish, fruits, etc. [Takeo Suzuki, Nobuko Ishikawa et al., Agricultural Chemistry, 57, 1143 (1983)]. It is not known that wakame, which is widely used as a health food, has an ACE inhibitor.

[0003]

Means for Solving the Problems The present inventor searched for a substance having a pharmacological action from a decomposition solution of wakame proteolytic enzyme belonging to a seaweed species of the order Laminariales and found three novel tetrapeptides. Has a strong angiotensin converting enzyme inhibitory action.
And, they intensively studied to put these three kinds of tetrapeptides into practical use as medicines. As a result, the inventors have found that these three types of tetrapeptides have a blood pressure lowering effect and are useful as angiotensin converting enzyme inhibitors derived from natural products. The present invention is based on such findings. The novel peptide according to the present invention has the following formulas (1), (2) and (3): (1) Tyr-Asn-Lys-Leu (2) Tyr-Lys-Tyr-Tyr (3) Ala-Ile-Tyr A novel tetrapeptide having an L-form amino acid sequence represented by the formula -Lys, and has a white powder at room temperature.

The above three kinds of tetrapeptides can be exemplified by a method of chemically synthesizing them or a method of separating and purifying them from a decomposition solution of wakame protease. When the novel peptide according to the present invention is chemically synthesized, it can be carried out by a conventional peptide synthesis method such as a liquid phase method or a solid phase method. It is preferable that the L-form amino acids corresponding to the amino acid residues are sequentially bonded to the support from the C-terminal (carboxyl-terminal side) of the peptide by peptide bonds. And the synthetic peptide thus obtained is
After cleavage from the poromeric solid support using trifluoromethanesulfonic acid, hydrogen fluoride, or the like, the protecting groups on the amino acid side chains are removed, and high-performance liquid chromatography using a reversed-phase column (hereinafter, HPLC). , Etc.).

[0005] As described above, the novel peptide of the present invention can be separated and purified from a decomposition solution of wakame proteolytic enzyme. In that case, for example, it can be carried out as follows. Hydrolysis is performed using the wakame protein portion containing the novel peptide. The 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 heated to the optimum temperature of the enzyme, the pH is adjusted to the optimum value, the enzyme is added, and the mixture is incubated. . Then, if necessary, after neutralization, the enzyme is inactivated to obtain a hydrolyzed solution. The hydrolyzate is filtered using filter paper and / or celite to remove insoluble components, and the obtained filtrate is filtered using a semi-permeable membrane such as cellophane to a suitable solvent (eg, water, Tris-hydrochloric acid). Buffer,
After sufficient dialysis in a neutral buffer such as a phosphate buffer, the solution containing the components in the filtrate that has passed through the semipermeable membrane is converted to a strongly acidic cation exchange resin (for example, Dow Chemical Co., Ltd.). D
owex 50W, etc.), and angiotensin converting enzyme (hereinafter abbreviated as ACE) from the adsorbed and eluted fraction.
A fraction containing a component having an inhibitory activity was obtained.
The CE inhibitory activity fraction was fractionated by gel filtration (for example, Sephadex G-25 manufactured by Pharmacia),
The obtained ACE inhibitory activity fraction is subjected to cation exchange gel filtration (for example, SP-Sephadex manufactured by Pharmacia).
C-25), and the obtained ACE inhibitory activity fraction is further fractionated by reverse phase HPLC.

As the brown algae used in the method for producing the novel peptide according to the present invention, any brown algae may be used as long as the object of the present invention can be achieved, and wakame is preferably used. The novel peptide of the present invention obtained as described above does not induce antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. Also, the novel peptide according to the present invention is L
- consists array structure of an amino acid alone, after administration, because it is gradually degraded by proteases in vivo, toxicity is very low, the safety is very high (LD _50> 5000mg /
kg; oral administration to rats). Novel peptide according to the present invention, injections using additives such as commonly used excipients,
It can be prepared into tablets, capsules, granules, powders and the like. The method of administration usually includes injection into mammals (eg, humans, dogs, rats, etc.) having ACE, or oral administration. The dose is, for example, 0.01 g of this peptide per 1 kg of the animal body.
In the amount of 10 mg. 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 types of excipients, binders, and lubricants used in the above-mentioned various preparations are not particularly limited, and those used in ordinary injections, powders, granules, tablets or capsules may be used. Can be. Examples of additives used for tablets, capsules, granules, and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate, etc .; starches, hydroxypropylmethyl cellulose, etc. as binders; carboxymethylcellulose and potassium salts thereof as disintegrants; Examples of the lubricant include stearic acid and salts thereof, talc, and waxes. In addition, in preparing the preparation, a flavoring agent such as menthol, citric acid and its salts, and fragrance can be used as necessary. The sterile composition for injection can be prepared by dissolving or suspending the novel peptide of the present invention in water for injection, physiological saline, a sugar alcohol injection such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol by an ordinary method. It can be turbid to obtain an injection. At this time, buffers, preservatives, antioxidants and the like can be added as necessary. The preparation containing the novel peptide according to the present invention may be in the form of a lyophilized product or a dry powder, and may be used by dissolving it in a usual dissolving agent, for example, water or physiological saline when used.

[0008] The novel peptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, a hypotensive action,
Shows the effect of inhibiting bradykinin inactivation. Therefore, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension,
Therapeutic agents have a function of normalizing organ circulation and improving long-term prognosis (life-extending effect) for congestive heart failure, and are useful as therapeutic agents for heart failure.

[0009]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which are Production Examples and Test Examples. Production Example 1 A wakame homogenate obtained by adding 708 ml of deionized water to 23.6 g of wakame powder and homogenizing it was used. The solution was packed in a dialysis tube (inner diameter: 36 inches, manufactured by Wako Pure Chemical Industries, Ltd.) and dialyzed against running water for 3 days to obtain a dialysis solution. The pH of this inner solution was adjusted to 2.0 with 1N hydrochloric acid, and pepsin (manufactured by Merck, enzyme number EC 3.4.23.1) 708 was used.
mg was added and hydrolysis was carried out while stirring at 45 ° C. for 5 hours. The passing solution obtained by passing the decomposition reaction solution immediately through an ultrafiltration membrane (YM10, manufactured by Amicon; fractionation molecular weight: about 10,000) was passed through a Dowex 50W × 4 [H ⁺ ] column (φ4.0).
× 55 cm). After sufficiently washing the column with deionized water, the column was eluted with 2N ammonium hydroxide solution 2ι. Sephadex G-2 in which ammonia was removed by concentration under reduced pressure and the concentrate was buffered with deionized water in advance.
5 (φ1.6 × 113cm), flow rate 12mι /
The mixture was subjected to gel filtration at a fraction of 5.7 ml for each hr. The result is as shown in FIG. The gel filtration was repeated, and fractions having high ACE inhibitory activity collected in large amounts were collected and freeze-dried to obtain peptide powder. 1.55 g of this peptide powder was added to 20
After dissolving in deionized water (ml), the solution was loaded on a SP-Sephadex C-25 [H ⁺ ] column (φ1.8 × 40 cm) previously buffered with deionized water, and 500 m of deionized water was added.
Chromatography was performed at a flow rate of 70 ml / hr and a fractionation amount of 10 ml each by performing a concentration gradient method from 500 to 500 ml of sodium chloride from 1.5%. The result is as shown in FIG. In the above chromatograph, fractions of fraction numbers 14 to 22 having high ACE inhibitory activity were collected and freeze-dried to obtain a purified peptide powder (SP-I fraction). After dissolving 20 mg of this purified peptide powder in 60 µι of deionized water, high performance liquid chromatography (HPLC) was performed. Nomura Chemical's Develosil ODS-
5 (4.5 mm ID × 25 cmL) using a mobile phase of 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TF.
A concentration gradient method of A was performed, and HPLC was performed at a flow rate of 1.0 ml / min and a detection wavelength of 220 nm to obtain a peptide fragment having high ACE inhibitory activity. The result is as shown in FIG. ｛Elution time; peptide 99.6 of (1)
25 minutes, 80.724 minutes of the peptide of (2) and 32.033 minutes of the peptide of (3) {AC thus obtained
The amino acid sequence of the peptide having E inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, 3
Each kind of peptide is represented by an L-form amino acid sequence represented by the following formula: (1) Tyr-Asn-Lys-Leu (2) Tyr-Lys-Tyr-Tyr (3) Ala-Ile-Tyr-Lys It was confirmed that this was a novel tetrapeptide. The property at room temperature is a white powder. When the novel tetrapeptide according to the present invention is formulated as an ACE inhibitor in, for example, a tablet, it may be treated according to a conventional method, for example, as follows: peptide 10 g, lactose 68 g, corn starch 39 g, stearic acid Starting with 1.2 g of magnesium, first and 20 g of corn starch were mixed, granulated together with a paste made from 11 g of corn starch, 9 g of corn starch was added to the granules, and the resulting mixture was compressed with a compression tablet machine. Tablets and tablets 10
00 pieces are manufactured.

Production Example 2 This is an example of production by a synthesis method. Synthesis Method of Tyr-Asn-Lys-Leu The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the tetrapeptide, leucine on the C-terminal side was reacted with a chloromethyl resin according to a conventional method to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
Abbreviated as -Boc. ) -Protected t-Boc amino acid was used. Next, this peptide-bonded resin was suspended in a mixed solution consisting of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid under ice cooling, and further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water or methanol, and then purified by HPLC using a reverse-phase column C ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase,
(B) Using acetonitrile solution containing 0.1% TFA, the solution (A) was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method from 83% to 53% in 40 minutes. The eluted fraction detected at an ultraviolet wavelength of 218 nm and showing the maximum absorption was separated and lyophilized to obtain the desired synthetic peptide.

As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tetrapeptides having the amino acid sequence structure shown above. The result of the mass spectrum is as shown in FIG. Synthesis Method of Tyr-Lys-Tyr-Tyr The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the tetrapeptide, a tyrosine on the C-terminal side was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
A t-Boc amino acid protected with a group (abbreviated -Boc) was used. Next, this peptide-bonded resin was suspended in a mixture of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, and then added with trifluoroacetic acid under ice-cooling.
Stir for another 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water or methanol, and then purified by HPLC using a reverse-phase column C ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase, (B)
Using an acetonitrile solution containing 0.1% TFA,
(A) The solution was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method from 83% to 53% for 40 minutes. The eluted fraction detected at an ultraviolet wavelength of 218 nm and showing the maximum absorption was separated and lyophilized to obtain the desired synthetic peptide. As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tetrapeptides having the amino acid sequence structure shown above. The result of this mass spectrum is as shown in FIG. Ala-Ile-Tyr-Lys Synthesis Method The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the tetrapeptide, the C-terminal lysine was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
A t-Boc amino acid protected with a group (abbreviated as Boc) was used. Next, this peptide-bonded resin was suspended in a mixed solution consisting of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid under ice cooling, and further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The thus obtained unpurified synthetic peptide is dissolved in distilled water or methanol, and then reverse-phase column C is used.
Purified by HPLC using ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase;
Using an acetonitrile solution containing 1% TFA, the solution (A) was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method from 83% to 53% in 40 minutes. The eluted fraction detected at an ultraviolet wavelength of 218 nm and showing the maximum absorption was separated and lyophilized to obtain the desired synthetic peptide. As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tetrapeptides having the amino acid sequence structure shown above. The result of this mass spectrum is as shown in FIG. The pharmacological effects of the three types of tetrapeptides of the present invention obtained by synthesis were confirmed by the tests described below.

Test Example 1 (Method for measuring angiotensin converting enzyme inhibitory activity) ACE
(Manufactured by Sigma, Enzyme number EC 3.4.15.1) 2.5
mU, synthetic substrate Hippuryl-L-histidy
1-L-leucine (manufactured by Peptide Research Institute) 12.5
Yamamoto et al., which improved the method of measuring Lieberman using mM [Journal of the Japan Chest Disease, Vol. 18, pp. 297-302 (1)
989)]. That is, the generated hippuric acid was extracted with ethyl acetate and measured at an absorbance of 225 nm. The inhibition rate was determined from the following equation, where Es was the absorbance of the test solution, Ec was the value obtained when a buffer was added instead of the test solution, and Eb was the value obtained when a reaction stop solution was added in advance to react. . Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 1
The inhibitory activity IC ₅₀ value of the ACE inhibitor was represented by the concentration (M) of the sample required to inhibit the ACE enzyme activity by 50% (inhibition rate). Inhibitory activity (IC ₅₀ value) of three kinds of tetrapeptides according to the present invention on bovine lung serum angiotensin converting enzyme; 21.0 μm of tetrapeptide of (1)
M, 64.2 μM of the tetrapeptide of (2) and 213 μM of the tetrapeptide of (3).

Test Example 2 (Hypertensive effect when administered to spontaneously hypertensive rats) As experimental animals, 15-week-old male spontaneously hypertensive rats (hereinafter abbreviated as SHR) were purchased from Charles River Japan.
After pre-breeding for one week, the animals were used as a group of six animals having a systolic blood pressure of 160 mmHg or more (weight: 280 to 330 g). The rats were individually housed in stainless steel rat individual gauges in a breeding room adjusted to room temperature of 23 ± 2 ° C., humidity of 55 ± 10%, and 12 hours light / dark (light on from 6:00 am to 6:00 pm). . Feed is MF powder feed manufactured by Oriental Yeast Co., Ltd. Drinking water is self-pumped (conforming to tap water quality standards)
Was freely taken. The blood pressure was measured using a non-invasive tail arterial blood pressure measurement device (manufactured by Riken, Model PS-100).
By the aile-cuff method, before administration, 1 hour after administration, 2 hours
Measurement of the systolic blood pressure value (mmHg) of the SHR tail artery for 5 hours, 3 hours, 4 hours and 6 hours was performed 5 times every measurement time,
The highest value and the lowest value of the obtained measured values were rejected, and the average value of three times was used as the measured value at each time. The results on the effects on the systolic blood pressure value (mmHg) when 50 mg / kg of three kinds of synthetic tetrapeptides were orally administered to SHR are as shown in FIGS. 7, 8 and 9. As a result of the above test, it was confirmed that the three types of tetrapeptides according to the present invention have angiotensin converting enzyme inhibitory activity and exhibit a significant blood pressure lowering effect even in vivo (in vivo). Therefore, the three kinds of tetrapeptides according to the present invention are useful as drugs for treating or preventing hypertension. The three types of tetrapeptides according to the present invention can also be employed in the structure of a peptide having a partial structure of its amino acid sequence.

[0014]

[Brief description of the drawings]

FIG. 1 is a view showing the results of separation and purification of an ACE inhibitory peptide by Sephadex G-25 column chromatography in Production Example 1 of a wakame proteolytic solution according to the present invention. In the figures, insulin having a molecular weight of 6,000, insulin B chain having a molecular weight of 3,500, insulin A chain having a molecular weight of 2,500, bradykinin having a molecular weight of 1,052, and glycine having a molecular weight of 75 were used as markers.

FIG. 2 shows SP-Sephadex C-25 (H ⁺ ) in Production Example 1 of the seaweed protein hydrolyzate according to the present invention.
It is a figure which shows the result of separation and purification of ACE inhibitory peptide by column chromatography.

FIG. 3 is a diagram showing the results of separation and purification of ACE inhibitory peptides by reverse phase HPLC in Production Example 1 for three types of tetrapeptides according to the present invention.

FIG. 4, FIG. 5, and FIG. 6 are diagrams showing mass spectra of three kinds of synthetic tetrapeptides obtained in Production Example 2 of three kinds of tetrapeptides according to the present invention.

FIGS. 7, 8, and 9 are graphs showing changes over time in systolic blood pressure (mmHg) when 50 mg / kg of the three types of synthetic tetrapeptides obtained in Production Example 2 were orally administered to SHR, respectively. It is. In the figure, captopril (10 mg / kg) was used as a control antihypertensive, and 3 ml of 0.9% physiological saline was used as a control.

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

FIG. 3

FIG. 7

FIG. 4

FIG. 5

FIG. 6

FIG. 8

FIG. 9

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 5/11 C12N 9/99 C12N 9/99 A61K 37/64 F term (Reference) 4C084 AA01 AA02 AA07 BA01 BA08 BA16 CA07 DB74 MA17 MA35 MA37 MA41 MA43 MA52 MA66 ZA422 ZC172 ZC202 ZC412 4H045 AA10 AA30 BA13 CA30 DA57 FA34 FA70 GA10 GA22 GA23 GA25

Claims (8)

[Claims] 1. Tyr-Asn-Lys-Le
A novel tetrapeptide having a peptide structure based on the L-amino acid sequence represented by u. 2. The following formula: Tyr-Asn-Lys-Le
An angiotensin converting enzyme inhibitor comprising, as an active ingredient, a novel tetrapeptide having a peptide structure based on the L-amino acid sequence represented by u. 3. The following formula: Tyr-Lys-Tyr-Ty
A novel tetrapeptide having a peptide structure based on the L-amino acid sequence represented by r. 4. The following formula: Tyr-Lys-Tyr-Ty
An angiotensin converting enzyme inhibitor comprising, as an active ingredient, a novel tetrapeptide having a peptide structure based on the L-amino acid sequence represented by r 1. 5. The following formula: Lys-Phe-Tyr-Gl
A novel tetrapeptide having a peptide structure according to the amino acid sequence of L-form represented by y. 6. The following formula: Lys-Phe-Tyr-Gl
An angiotensin converting enzyme inhibitor comprising, as an active ingredient, a novel tetrapeptide having a peptide structure based on an L-amino acid sequence represented by y. 7. The following formula: Ala-Ile-Tyr-Ly
A novel tetrapeptide having a peptide structure based on the amino acid sequence of L-form represented by s. 8. The following formula: Ala-Ile-Tyr-Ly
An angiotensin converting enzyme inhibitor comprising, as an active ingredient, a novel tetrapeptide having a peptide structure based on the sequence of the L-form amino acid represented by s.