JP2003128695A - New tripeptide and angiotensin-converting enzyme inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new tripeptide having angiotensin-converting enzyme inhibitory effect from a decomposed liquid of seaweed obtained by a protease. SOLUTION: Seaweed is processed with the protease or the like. The obtained two new tripeptide having angiotensin-converting enzyme inhibitory effect are (1) Tyr-Tyr-His and (2) Tyr-Tyr-Phe. They have a hypotensine effect in vivo and extremely low toxicity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a tripeptide having a peptide structure of the following amino acid sequence, which has utility as a medicine, and an angiotensin converting enzyme inhibitor containing the peptide as an active ingredient. Tyr-Tyr-His Tyr-Tyr-Phe (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 powerful pressor substance that acts on not only blood vessels and adrenal cortex but also central nervous system and peripheral nervous system to promote an increase in blood pressure. Further, ACE has a function of decomposing and inactivating bradykinin which is an antihypertensive substance in vivo, 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 to be clinically effective for the prevention and treatment of hypertension. For this purpose, since the proline derivative captoril was synthesized and its hypotensive action was confirmed, various A
BACKGROUND ART Synthetic studies of CE inhibitors have been actively conducted, and recently, substances such as enarabryl maleate and aracebryl have been provided to clinical sites one after another. Currently, ACE inhibitors are widely used regardless of whether they are essential hypertension or symptomatic hypertension, mild or severe, and they are added to the first-line treatment of hypertension. It has been found to have points. On the other hand, as the mechanism of action of the ACE inhibitor, suppression of the secretion of aldosterone and vasopressin by suppressing the production of angiotensin II, and promotion of sodium and water excretion by releasing the contraction of renal arteries are considered. Furthermore,
With regard to ACE inhibitors, it is believed that they suppress the inactivation of the calyculene-quinine 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. 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. Ferria
et al: Biochemistry, 9, 358.
3 (1970)], 6 kinds of peptides derived from collagenase digestion products of gelatin (all have C-terminal Ala-Hy).
p) [G. Oshima et al: Biochi
m. 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 5 types of hexapeutides derived from sardine muscles of the present inventors (all are the second or third from the C terminus to Pro and the N terminus to Leu) [Patent No. 2046483], seaweed Derived tetrapeptide (Pro-gly-Val-Ala) [Patent No. 2678]
180], a pentapeptide derived from ginseng (Ile-
Gly-Pro-Ala-Gly) [Patent No. 29208]
29], a pentapeptide derived from Chlorella (Val-V
al-Pro-Pro-Ala) and three types of wakame-derived tetrapeptides (Tyr-Asn-Lys-Leu,
Tyr-Lys-Tyr-Tyr, Ala-Ile-T
yr-Lys) [Patent No. 3108920] and the like, and all of them are disclosed to be ACE inhibitors. Furthermore, di- and tripeptides having a short chain length obtained by a synthetic method [JP-A-6-87886] [JP-A-6-1656]
No. 8] has been proposed, but the ACE inhibitory action of di- and tripeptides having a regular amino acid sequence derived from a natural product, and the hypotensive effect (pharmacological effect) by oral administration are unknown. Although it has been a long time since its release, there are no reports that it is still being developed as a drug.

[0003]

Means for Solving the Problems The present inventor searched for substances having a pharmacological action from a degradation solution of a proteolytic enzyme of wakame seaweed belonging to the seaweed species of the brown alga Laminariales, and found two novel tripeptides. Was found to have a strong inhibitory action on angiotensin converting enzyme. Then, they have diligently studied for practical use of these two types of tripeptides as a medicine. As a result, they have found that these two types of tripeptides have antihypertensive action 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 is a novel tripeptide having an L-amino acid sequence represented by the following formulas (1) and (2) (1) Tyr-Tyr-His (2) Tyr-Tyr-Phe. It is a peptide and is a white powder at room temperature.

The above-mentioned two kinds of tripeptides may be chemically synthesized or separated and purified from the degradation solution of the seaweed proteolytic enzyme. In the case of chemically synthesizing the novel peptide 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 phase of a polymer phase is prepared by the solid phase method. It is preferable that the L-amino acids corresponding to the amino acid residues of the peptide are sequentially bound to the support from the C-terminal (carboxyl terminal side) of the peptide by peptide bonds.
Then, the synthetic peptide thus obtained is cleaved from the solid support having a poromeric property by using trifluoromethanesulfonic acid, hydrogen fluoride, etc., and then the protecting group of the amino acid side chain is removed to prepare a reverse phase system. It can be purified by a usual method using high performance liquid chromatography (hereinafter abbreviated as HPLC) using a column of.

As described above, the novel peptide according to the present invention can be separated and purified from the degradation solution of the seaweed proteolytic enzyme. In that case, for example, it can be performed as follows. The protein portion of wakame containing the novel peptide is hydrolyzed. Hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, seaweed is further hydrolyzed if necessary, then heated to the optimum temperature of the enzyme to adjust the pH to the optimum value, and the enzyme is added and incubated. . Then, after neutralizing as necessary, the enzyme is deactivated to obtain a hydrolyzed solution. The hydrolyzate 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, water, Tris-hydrochloric acid). Buffer,
A solution containing a component that has passed through the semipermeable membrane among the components in the filtrate is sufficiently dialyzed in a neutral buffer such as a phosphate buffer solution, and a strong acidic cation exchange resin (for example, manufactured by Dow Chemical Co.) is used. D
owex 50W) and the adsorbed and eluted fraction thereof is subjected to angiotensin converting enzyme (hereinafter abbreviated as ACE).
A fraction containing a component having inhibitory activity was obtained, and the obtained A
The CE inhibitory activity fraction is fractionated by gel filtration (for example, Sephadex G-25 manufactured by Pharmacia),
The obtained ACE inhibitory activity fraction was subjected to cation exchange gel filtration (for example, SP-Sephadex manufactured by Pharmacia).
C-25 etc.) 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, but wakame is preferably used. The novel peptide of the present invention obtained as described above does not induce antibody production or anaphylactic shock when repeatedly administered intravenously. The novel peptide according to the present invention is L
-Because it consists of a sequence structure of only amino acids and is gradually decomposed by in vivo protease after administration, its toxicity is extremely low and its safety is extremely high (LD ₅₀ > 5000 mg /
kg; oral administration to rats). The novel peptide according to the present invention is an injection using an additive such as a commonly used excipient,
It can be prepared into tablets, capsules, granules, powders and the like. The method of administration generally includes injection into mammals having ACE (eg, humans, dogs, rats, etc.), or oral administration. For example, the dose is 0.01
The amount is -10 mg. The administration frequency 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 should be used. You can The following may be mentioned as additives used in 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 hydroxypropylmethyl cellulose; disintegrators such as carboxymethyl cellulose and potassium salts thereof; Examples of lubricants include stearic acid and its salts, talc, and waxes. In preparation of the preparation, menthol, citric acid and its salts, and flavoring agents such as fragrances can be used if necessary. A sterile composition for injection is 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 a conventional method. It can be made turbid to prepare an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as required. 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 dissolved in an ordinary solubilizer such as water or physiological saline before use.

The novel peptide according to the present invention has an excellent angiotensin converting enzyme inhibitory activity,
It shows an inhibitory effect on bradykinin inactivation. Therefore, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension,
It is useful as a therapeutic agent for heart failure because it has the effects of normalizing organ circulation and improving long-term prognosis (life-prolonging effect) for these congestive 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 Wakame homogenate obtained by adding 708 ml of deionized water to 23.6 g of wakame powder was used. It 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 dialysate solution. The pH of this internal solution was adjusted to 2.0 with 1N hydrochloric acid, and pepsin (Merck & Co., Enzyme No. EC3.4.23.1) 708 was added.
mg was added and hydrolysis was performed at 45 ° C. for 5 hours with stirring. The decomposition reaction solution was immediately passed through an ultrafiltration membrane (YM10 type, manufactured by Amicon, Inc .; molecular weight cutoff of about 10,000), and the passed solution was used as a Dowex 50W × 4 [H ⁺ ] column (φ4.0).
X 55 cm). The column was thoroughly washed with deionized water and then eluted with 2N ammonium hydroxide solution 2ι. Sephadex G-2 in which ammonia was removed by vacuum concentration and the concentrate was previously buffered with deionized water
5 (φ1.6 × 113cm), flow rate 12mι /
Gel filtration was carried out for each hr and each fraction amount of 5.7 ml. The result is shown in FIG. Gel filtration was repeated to collect a large amount of fractions with high ACE inhibitory activity, which were then lyophilized to obtain peptide powder. 20 of this peptide powder 1.55g
After dissolving in ml of deionized water, it was loaded on an SP-Sephadex C-25 [H ⁺ ] column (φ1.8 × 40 cm) buffered with deionized water in advance, and deionized water was 500 m.
Chromatography was carried out at a flow rate of 70 ml / hr and at each fraction amount of 10 ml by performing a concentration gradient method from ι to 1.5% sodium chloride 500 ml. The result is shown in FIG. Fractions Nos. 23 to 51, which had high ACE inhibitory activity in the above chromatograph, were collected and lyophilized to obtain purified peptide powder (SP-II fraction). After dissolving 20 mg of this purified peptide powder in 60 μι of deionized water,
High performance liquid chromatography (HPLC) was performed. As a column, Develosil ODS manufactured by Nomura Chemical Co., Ltd.
-5 (4.5 mmID x 25 cmL) was used, and the mobile phase was 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% T.
FA concentration gradient method 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 a high ACE inhibitory activity. The result is as shown in FIG. {Elution time; peptide of (2) 29.
7 minutes and 51.3 minutes of the peptide of (1)} The amino acid sequence of the thus obtained peptide having an ACE inhibitory activity is the same as that of the applied biosystem (AB
I) Determined using a Protein Sequencer Model 477A manufactured by the company. As a result, the two types of peptides are respectively novel tripeptides represented by the following formula: (1) Tyr-Tyr-His (2) Tyr-Tyr-Phe represented by the L-amino acid sequence. confirmed. It is a white powder at room temperature. When the novel tripeptide according to the present invention is formulated as an ACE inhibitor into tablets, for example, it may be treated according to a conventional method, for example, as follows:
Peptide 10g, lactose 68g, corn starch 3
Using 9g and 1.2g magnesium stearate as raw materials, first, and 20g corn starch were mixed,
Granulate with a paste made from 11 g cornstarch, add 9 g cornstarch and to the granules, press the mixture obtained in a compression tableting machine to give tablets 100
0 is manufactured.

Production Example 2 This example is an example of production by a synthetic method. Tyr-Tyr-His Synthesis Method The peptide was synthesized by a solid phase method using an automated peptide synthesizer type 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used as the solid phase carrier. First, according to the amino acid sequence of the tripeptide, histidine on the C-terminal side was reacted with chloromethyl resin according to a conventional method to obtain a peptide-bonded resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t
-Abbreviated as Boc. ) Group protected t-Boc 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, and then under reduced pressure. Dried in. 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) 0.1% TFA-containing distilled water as a mobile phase,
(B) Using 0.1% TFA-containing acetonitrile solution, the (A) solution was chromatographed at a flow rate of 1.1 ml / min by a concentration gradient method of 93% → 52% in 60 minutes. The elution fraction showing the maximum absorption, which was detected at an ultraviolet wavelength of 216 nm, was collected and freeze-dried to obtain the target synthetic peptide.

As a result of mass spectrum analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tripeptides having the amino acid sequence structure shown above. The result of this mass spectrum is as shown in FIG. Tyr-Tyr-Phe Synthesis Method The peptide was synthesized by a solid-phase method using an automated peptide synthesizer type 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used as the solid phase carrier. First, according to the amino acid sequence of the tripeptide, phenylalanine on the C-terminal side was reacted with chloromethyl resin according to a conventional method to obtain a peptide-bonded resin. The amino acid at this time is t-Boc protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group.
Amino acids were 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, and then under reduced pressure. Dried in. 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) 0.1% TFA-containing distilled water as a mobile phase,
(B) Chromatography was performed at a flow rate of 1.2 ml / min by the concentration gradient method of 87% → 47% of the (A) solution in 50 minutes using an acetonitrile solution containing 0.1% TFA. The elution fraction showing the maximum absorption, which was detected at an ultraviolet wavelength of 217 nm, was collected and freeze-dried to obtain the target synthetic peptide. As a result of mass spectrum analysis of this synthetic peptide, it was confirmed that the amino acid sequence and amino acid composition were tripeptides having the amino acid sequence structure shown above. The result of this mass spectrum is as shown in FIG. The pharmacological effects of the two types of tripeptides of the present invention obtained by synthesis were confirmed by the following tests.

Test Example 1 (Method for measuring angiotensin converting enzyme inhibitory activity) ACE
(Manufactured by Sigma, Enzyme No. EC3.4.5.15.1) 2.5
mU, synthetic substrate Hippuryl-L-histidy
1-L-leucine (manufactured by Peptide Institute) 12.5
The method of Yamamoto et al., which improved the measurement method of Lieberman using mM [Nippon Chinkai, Vol. 18, 297-302 (1)
989)]. 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). Inhibitory activity of two types of tripeptides according to the present invention against angiotensin converting enzyme of bovine lung serum (IC ₅₀ value): 0.96 μM of tripeptide of (1) and 4.7 μM of tripeptide of (2).

Test Example 2 (Hypotensive 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 preliminarily breeding for 1 week, 6 animals having a systolic blood pressure of 160 mmHg or more (body weight 280 to 330 g) were used as one group. Rats were housed in individual cages for rats made of stainless wire one by one in a breeding room adjusted to room temperature of 23 ± 2 ° C., humidity of 55 ± 10%, and 12 hours of light and dark (lights from 6 am to 6 pm). . MF powder feed manufactured by Oriental Yeast Co., Ltd. was used as the feed, and drinking water was self-pumped (conforming to tap water quality standards)
Each was freely ingested. Blood pressure was measured using a non-invasive blood pressure measuring device for tail artery (PS-100 type, manufactured by Riken Development Co., Ltd.).
According to the ail-cuff method, before administration, 1 week after administration,
Systolic blood pressure (mmHg, upper value), diastolic blood pressure (m) of the SHR tail artery after 2 weeks, 3 weeks, 4 weeks and 5 weeks.
mHg, lower value) and mean blood pressure (mmHg) are measured 5 times at regular intervals, the highest and lowest values obtained are rejected, and the average value of 3 times is used as the measurement value for each time. did. SH of 2 kinds of synthetic tripeptide 10 mg / kg
The results of the effects on each blood pressure value (mmHg) when orally administered to R are as shown in FIGS. 6 and 7.
As a result of the above test, the two types of tripeptides according to the present invention have angiotensin converting enzyme inhibitory activity,
It was confirmed that a significant blood pressure lowering effect is exhibited in vivo (in vivo). Therefore, the two types of tripeptides according to the present invention are useful as therapeutic or preventive agents for hypertension. The two types of tripeptides according to the present invention can also be employed in the structure of a peptide whose amino acid sequence is a partial structure structurally.

[0014]

[Brief description of drawings]

FIG. 1 is a diagram showing the results of separation and purification of an ACE-inhibiting peptide by Sephadex G-25 column chromatography in Production Example 1 of a seaweed proteolysis solution according to the present invention. As markers in the figure, 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.

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

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

4 and 5 show two types of tripeptides according to the present invention,
FIG. 3 is a diagram showing mass spectra of two kinds of synthetic tripeptides obtained in Production Example 2.

6 and 7 show the systolic blood pressure value, the diastolic blood pressure value and the mean blood pressure value (mmHg) when the two kinds of synthetic tripeptides 10 mg / kg obtained in Production Example 2 were orally administered to SHR respectively. It is a figure which shows the change for 5 weeks.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/99 A61K 37/64

Claims (4)

[Claims] 1. A novel tripeptide having a peptide structure consisting of an amino acid sequence of L-form represented by the following formula: Tyr-Tyr-His. 2. An angiotensin-converting enzyme inhibitor, comprising a novel tripeptide having a peptide structure represented by the following formula: Tyr-Tyr-His having an L-amino acid sequence as an active ingredient. 3. A novel tripeptide having a peptide structure according to the sequence of the L-amino acid represented by the following formula: Tyr-Tyr-Phe. 4. An angiotensin-converting enzyme inhibitor comprising a novel tripeptide having a peptide structure represented by the following formula: Tyr-tyr-Phe, which has an amino acid sequence of L-form, as an active ingredient.