JP2001106698A - New tetrapeptide and angiotensin-converting enzyme inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new tetrapeptide having angiotensin-converting enzyme inhibitory action from a decomposed liquid of Spirulina protein by a proteolytic enzyme. SOLUTION: The new tetrapeptide having angiotensin-converting enzyme inhibitory action is obtained by processing the Spirulina protein with the proteolytic enzyme or the like. The tetrapeptide is represented by the formula Ile-Ala-Pro-Gly, and has antihypertensive action in a living body and has only a 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 tetrapeptide as an active ingredient. Ile-Ala-Pro-Gly (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 effect was confirmed.Since the reduction of the blood pressure has been confirmed, synthesis studies of various ACE inhibitors based on the structural studies of captoril have been actively conducted. Are being provided to clinical sites one after another. At present, ACE inhibitors are used for hypertension, regardless of essential hypertension, symptomatic hypertension,
In addition, it is widely used in both mild and severe cases and is added to the first-line treatment of hypertension, and has been found to have many excellent points. 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, it is believed that the ACE inhibitor suppresses the inactivation of the kallikren-kinin system and activates the prostaglandin system to further promote peripheral vasodilation and excretion of sodium and water,
It is expected as a suitable therapeutic drug in breaking the vicious cycle of heart failure. By the way, as an ACE inhibitor, in addition to the above-mentioned synthetic products, a natural product or a substance derived from a natural product, snake venom-derived bradykinin enhancer (C-terminal is Pro) [S.
H. Ferreia et al: Biochemis
try, 9, 3583 (1970)], and six types of peptides derived from collagenase digested gelatin (Ala-Hyp at the C-terminus) [G. Oshima et a
1: Biochim. Biophs. Acta, 56
6, 128 (1979)], a peptide derived from a tryptic digest of bovine casein (Gly-Lys at the C-terminus) [S.
See Maruyama et al. : Agric. Bio
l. Chem. , 46, 1393 (1983)] and the like, and five hexapeutides derived from the sardine muscle of the present inventor (in each case, the second or third from the C-terminal is Pro, and the N-terminal is Leu) [Patent No. 2046483], Derived tetrapeptide (N-terminal is Pro) [Patent No. 26781
No. 80] and a pentapeptide [JP-A-10-3639]
1], a ginseng-derived pentapeptide [Patent No. 2920
No. 829], a pentapeptide derived from Chlorella [Japanese Patent Application No. Hei.
No. 0-185530], and it is disclosed that any of them can be an ACE inhibitor. Furthermore, di- and tripeptides having a short chain length obtained by a synthesis method [JP-A-6-878]
No. 86] [Japanese Patent Application Laid-Open No. 6-16568] has been proposed. It is unknown and 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 .;
(1983)], however, it is not known that spirulina, which has been widely used as a health food, has an ACE inhibitor.

[0003]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the novel tetrapeptide of the present invention obtained from Spirulina, a unicellular algae belonging to freshwater green algae, has been obtained. They have found that they have a blood pressure lowering effect, and have completed the present invention. That is, a substance having a pharmacological action was searched for from the degradation solution of the protease of spirulina protein, and it was found that the novel tetrapeptide had a strong angiotensin converting enzyme inhibitory action. The present inventors have intensively studied to put this tetrapeptide into practical use as a medicine, and as a result, have found that the tetrapeptide has a blood pressure lowering effect and is useful as an angiotensin converting enzyme inhibitor derived from a natural product. The present invention is based on such findings. The novel peptide according to the present invention is a novel tetrapeptide represented by an L-form amino acid sequence represented by the following formula Ile-Ala-Pro-Gly, and is a white powder at room temperature.

[0004] The novel tetrapeptide can be chemically synthesized or separated and purified from a degradation solution of a spirulina protein protease.
When chemically synthesizing the novel tetrapeptide according to the present invention, it can be carried out by a conventional synthesis method such as a liquid phase method or a solid phase method. It is preferred that the L-form amino acids corresponding to the amino acid residues are sequentially bonded to the support from the C-terminal side (carboxyl-terminal side) of the tetrapeptide by peptide bonds. Then, the synthetic tetrapeptide thus obtained is cleaved from the poromeric solid support using trifluoromethanesulfonic acid, hydrogen fluoride, or the like, and then the protecting group of the amino acid side chain is removed. High Performance Liquid Chromatography (hereinafter referred to as HPL
(Abbreviated as C) and the like.

[0005] As described above, the novel tetrapeptide of the present invention can be separated and purified from the degradation solution of the protease of Spirulina protein.
For example, it can be performed as follows. Hydrolysis is performed using the spirulina protein containing the novel tetrapeptide. The hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a protease such as pepsin, the spirulina protein homogenate is further hydrolyzed if necessary, and then heated to the optimum temperature of the enzyme to adjust the pH to the optimum value, and the enzyme is added. And incubate. Then, if necessary, after neutralization, the enzyme is inactivated to obtain a hydrolyzed solution. Insoluble components are removed by filtering the hydrolyzate using filter paper and / or celite or the like,
The obtained filtrate is dialyzed sufficiently using a semi-permeable membrane such as cellophane in a suitable solvent (eg, neutral buffer such as water, Tris-HCl buffer, phosphate buffer, etc.), and the filtrate is filtered. The solution containing the components having passed through the semipermeable membrane with the components therein is mixed with a strongly acidic cation exchange resin (for example, Dowex manufactured by Dow Chemical Company).
50 W), a fraction containing a component having angiotensin converting enzyme (hereinafter abbreviated as ACE) inhibitory activity is obtained from the adsorbed and eluted fraction, and the obtained ACE inhibitory activity fraction is subjected to gel filtration (for example, Pharmacia S
ePhadex G-25), and the obtained ACE inhibitory activity fraction is subjected to cation exchange gel filtration (for example, SP-Sephadex manufactured by Pharmacia).
C-25, etc.) and the obtained ACE
The inhibitory activity fraction can be further fractionated by reverse phase HPLC.

As the algae used in the method for producing the novel tetrapeptide according to the present invention, any algae can be used as long as the object of the present invention can be achieved, but spirulina protein is preferably used. The novel tetrapeptide of the present invention obtained as described above does not induce antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. Further, the novel tetrapeptide of the present invention has a sequence structure of only L-amino acids,
After administration, since it is gradually degraded by protease in the living body, the toxicity is extremely low and the safety is extremely high (LD ₅₀
> 5000 mg / kg; oral administration to rats). The novel tetrapeptide according to the present invention can be prepared into injections, tablets, capsules, granules, powders and the like by using additives such as commonly used excipients. As the administration method, usually, a mammal having ACE (eg, human, dog,
Injection into rats or the like, or oral administration. The dose is, for example, 0.01 to 10 mg of the tetrapeptide per animal body weight. The number of administrations is usually about 1 to 4 times a day, but 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 usual injections, powders, granules, tablets or capsules are 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, starch, anhydrous calcium phosphate, etc .; starches, hydroxypropyl methyl cellulose, etc. as binders; carboxymethyl cellulose and its potassium salts as disintegrants; Stearic acid and its salts as lubricants,
Examples include talc and waxes. In preparing the preparation, a odorant such as menthol, citric acid and its salts, and a fragrance can be used as necessary. A sterile composition for injection is prepared by dissolving or suspending the novel tetrapeptide 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 tetrapeptide of 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 tetrapeptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, and exhibits a blood pressure lowering action and an inhibitory action on bradykinin inactivation. Therefore, a preventive or therapeutic agent for hypertension such as essential hypertension, renal hypertension, adrenal hypertension, and the like, which has effects of normalizing organ circulation and improving long-term prognosis (life-extending effect) for congestive heart failure, and treating heart failure Useful as an agent.

[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 1000 ml of deionized water was added to 40 g of Spirulina powder to obtain a homogenized Spirulina suspension. The solution was packed in a dialysis tube (36 inch, manufactured by Wako Pure Chemical Industries) and dialyzed against running water for 3 days to obtain a dialysis solution (spirulina protein). The pH of this internal solution was adjusted to 2.0 with 1N hydrochloric acid, and pepsin (manufactured by Merck, enzyme number EC 3.4.2) was used.
3.1) 1.2 g was added, and hydrolysis was performed while stirring at 37 ° C. for 20 hours. The decomposition reaction solution was immediately passed through an ultrafiltration membrane (YM10, manufactured by Amicon, φ76 mm), and the passed solution was subjected to a Dowex 50W × 4 [H ⁺ ] column (φ
4.5 × 20 cm). After sufficiently washing the column with deionized water, 500 mL of a 2N ammonium hydroxide solution was used.
to elute. The ammonia was removed by concentration under reduced pressure to obtain 30 ml of a concentrated liquid. 3 ml of this concentrated solution was previously buffered with deionized water to Sephadex G-25 (φ
2.3 × 140 cm) at a flow rate of 40 ml / hr,
Gel filtration was performed on each fraction of 12 ml. The result is shown in FIG.
It is as follows. In the above chromatograph, fraction number 20
Fractions of ACE-inhibiting activity of ３１31 were collected and freeze-dried to obtain a peptide powder derived from Spirulina protein. After dissolving 20 mg of this peptide powder in 60 μl of deionized water, high performance liquid chromatography (HPLC) was performed. Nomura Chemical's Develosil ODS-
5 (4.5 mm ID × 25 cmL) and the mobile phase was 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TFA.
Was performed and chromatography was performed at a flow rate of 1.0 ml / min at a detection wavelength of 220 nm to obtain a peptide fragment having an ACE inhibitory action. The results are as shown in FIG. 2, and the elution time by HPLC was 47.
6 minutes. The amino acid sequence of the thus-obtained peptide fragment having an ACE inhibitory action was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, it was confirmed that the novel peptide according to the present invention was a novel tetrapeptide represented by an L-form amino acid sequence represented by the following formula Ile-Ala-Pro-Gly. 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: 11 g of peptide, 82 g of lactose, 25 g of corn starch, 25 g of stearic acid Starting from 1.1 g of magnesium, first and 15 g of corn starch were mixed, granulated together with a paste made from 5 g of corn starch, 4 g of corn starch was added to the granules, and the resulting mixture was compressed with a compression tablet machine. Tablets and tablets 100
0 pieces are manufactured.

Production Example 2 This is an example of production by a synthesis method. Synthesis Method of Ile-Ala-Pro-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, and then the reverse-phase column C ₁₈ (5 μm) was used.
Purified by HPLC using m). (A) distilled water containing 0.1% TFA as a mobile phase, (B) 0.1% TF
Using an A-containing acetonitrile solution, the solution (A) was subjected to a concentration gradient method from 75% to 43% in 50 minutes, and the flow rate was 1.6 ml.
/ Min chromatography. UV wavelength 2
The eluted fraction detected at 12 nm and showing the maximum absorption was collected and lyophilized to obtain the desired synthetic peptide.

Analysis of this synthetic peptide by mass spectrum confirmed that it was a tetrapeptide having the amino acid sequence structure of the following formula: Ile-Ala-Pro-Gly. The result of this mass spectrum is as shown in FIG. The pharmacological effect of the tetrapeptide of the present invention obtained by synthesis was 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 [Jisho Kyoukai, 18, 297-302 (198)
9)]. 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). The inhibitory activity (IC ₅₀ value) of the novel tetrapeptide of the present invention on the angiotensin converting enzyme in bovine lung serum is 11.4 μM.

Test Example 2 (Hypertensive effect upon administration to spontaneously hypertensive rats) Experimental animals were purchased from Charles River Japan, a 15-week-old male spontaneously hypertensive rat (hereinafter abbreviated as SHR).
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 (body 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 tail artery of the SHR for 5 hours, 3 hours, 4 hours and 6 hours was performed 5 times at each measurement time, and the highest and lowest values of the obtained measurement values were discarded. The average value of the times was used as the measured value at each time. The results on the effect of orally administered 200 mg / kg of synthetic tetrapeptide to SHR on systolic blood pressure (mmHg) are shown in FIG.
As shown in FIG. As a result of the above test, it was confirmed that the tetrapeptide according to the present invention has angiotensin converting enzyme inhibitory activity and exhibits a significant blood pressure lowering effect even in vivo (in vivo). Therefore, the tetrapeptide according to the present invention is useful as a therapeutic or preventive drug for hypertension. In addition, the tetrapeptide according to the present invention can also be employed in the structure of a peptide having the amino acid sequence as a partial structure.

[0014]

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of separation and purification of an ACE inhibitory peptide by Sephadex G-25 column chromatography in Production Example 1 of a pepsin degradation solution of spirulina protein according to the present invention. Incidentally, was used in the figure, the vitamin _{B 12} blue dextran and molecular weight 1,355 of molecular weight of 2,000,000 as a void volume (Vo) as a marker.

FIG. 2 is a diagram showing the results of separation and purification of an ACE inhibitory peptide by reverse phase HPLC in Production Example 1 of the tetrapeptide according to the present invention.

FIG. 3 is a view showing a mass spectrum of the synthetic tetrapeptide obtained in Production Example 2 of the tetrapeptide according to the present invention.

FIG. 4 shows a synthetic tetrapeptide 200 obtained in Production Example 2.
It is a figure which shows the time-dependent change of the systolic blood pressure value (mmHg) when mg / kg is orally administered to SHR. In the figure, captopril (10 mg / kg) was used as a control antihypertensive agent.
SHR), and 3 ml of 0.9% physiological saline as a control.

Claims (2)

[Claims] 1. The following formula: Ile-Ala-Pro-Gl
A novel tetrapeptide having a peptide structure according to the amino acid sequence of L-form represented by y. 2. The following formula: Ile-Ala-Pro-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.