JP3709425B2 - Novel tripeptide and angiotensin converting enzyme inhibitors - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a tripeptide having a peptide structure having the following amino acid sequence having utility as a pharmaceutical product, and an angiotensin converting enzyme inhibitor containing the peptide as an active ingredient.
Tyr-Tyr-His
(In the formula, each symbol representing an amino acid residue is based on a conventional notation method in amino acid chemistry.)
[0002]
[Prior art and problems to be solved by the invention]
It is well known that the renin-angiotensin system plays an important role in the regulation of biological water, electrolytes and blood. This renin-angiotensin system contains angiotensin converting enzyme (hereinafter abbreviated as ACE), and angiotensin I is converted to angiotensin II by ACE. Angiotensin II is a powerful vasopressor that works not only in blood vessels and adrenal cortex but also in the central nervous system and peripheral nervous system to promote an increase in blood pressure. ACE also has the action of degrading and inactivating bradykinin, which is a hypotensive 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 this is clinically effective for the prevention and treatment of hypertension. Since captoryl, a proline derivative, was synthesized for this purpose and its antihypertensive activity was confirmed, various ACE inhibitors have been actively studied based on the structural study of captolyl. Recently, enalabril maleate, alacebryl, etc. These substances are being used in clinical settings one after another. At present, ACE inhibitors are widely used regardless of whether they are essential hypertension or symptomatic hypertension, mild or severe, and are added to the first-line treatment for hypertension. Has been found to have On the other hand, as the mechanism of action of ACE inhibitors, it is considered that aldosterone and vasopressin secretion are suppressed by suppressing production of angiotensin II, and that sodium and water excretion is promoted by releasing renal artery contraction. Furthermore, for ACE inhibitors, it is believed that it suppresses inactivation of the calicrene-kinin system and further promotes peripheral vasodilation and excretion of sodium and water by activating the prostaglandin system, It is expected to be an appropriate therapeutic agent in breaking the vicious circle of heart failure. By the way, as an ACE inhibitory substance, in addition to the above-mentioned synthetic products, snake venom-derived bradykinin enhancing factor (C-terminal is Pro) as a natural product or a natural product-derived substance [S. H. Ferreia et al: Biochemistry, 9, 3583 (1970)], six peptides derived from collagenase digest of gelatin (all C-terminal Ala-Hyp) [G. Oshima et al: Biochim. Biophs. Acta, 566, 128 (1979)], a peptide derived from a tryptic digest of bovine casein (C-terminal Gly-Lys) [S. Maruyama et al. : Agric. Biol. Chem. , 46, 1393 (1983)] and the like, the five hexapeptides derived from the sardine muscles of the present inventors (the second or third from the C-terminal is Pro and the N-terminal is Leu) [Patent No. 2046483], Tetrapeptide derived from seaweed (Pro-gly-Val-Ala) [Patent No. 2678180], Pentapeptide derived from Ginseng (Ile-Gly-Pro-Ala-Gly) [Patent No. 2920829], Pentapeptide derived from Chlorella (Val-Val-Pro-Pro-Ala) and three kinds of Wakame-derived tetrapeptides (Tyr-Asn-Lys-Leu, Tyr-Lys-Tyr-Tyr, Ala-Ile-Tyr-Lys) [Patent No. 3108920 It is disclosed that any of them can be an ACE inhibitor. Further, although proposals have been made for di- and tripeptides [JP-A-6-87886] [JP-A-6-16568] having a short chain length obtained by a synthesis method, they have regularity derived from natural products. The ACE inhibitory action of di- and tripeptides having amino acid sequences and the antihypertensive effect (pharmacological effect) by oral administration are unknown, and it has been a long time since it was discovered, but development as a pharmaceutical is still in progress There are no reports.
[0003]
[Means for Solving the Problems]
The present inventor searched for a substance having a pharmacological action from a decomposition solution of a seaweed proteolytic enzyme belonging to the seaweed species of the brown alga, Laminarias, and found that the novel tripeptide has a strong angiotensin converting enzyme inhibitory action. It was. And they have intensively studied to put this tripeptide into practical use as a medicine. As a result, this tripeptide has an antihypertensive action and has been found to be useful as a natural product-derived angiotensin converting enzyme inhibitor. The present invention is based on such knowledge. The novel peptide according to the present invention has the following formula:
Tyr-Tyr-His
A novel tripeptide having an L-amino acid sequence represented by the formula:
[0004]
Examples of the tripeptide include a method of chemically synthesizing and a method of separating and purifying from a decomposition solution of 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. It is preferable that L-form amino acids corresponding to the amino acid residues are sequentially bonded to the support by peptide bonds from the C-terminal (carboxyl terminal side) of the peptide. The synthetic peptide thus obtained is cleaved from the porous solid support using trifluoromethanesulfonic acid, hydrogen fluoride, etc., and then the amino acid side chain protecting group is removed, and the reverse phase system is removed. It can be purified by a conventional method using high performance liquid chromatography (hereinafter abbreviated as HPLC) using the above column.
[0005]
As described above, the novel peptide according to the present invention can be separated and purified from the decomposition solution of seaweed proteolytic enzyme. In that case, for example, it can be carried out as follows. Hydrolysis is carried out using the wakame protein portion containing the novel peptide. Hydrolysis is performed according to conventional methods. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, further hydrolyze wakame if necessary, then warm to the optimal temperature of the enzyme, adjust the pH to the optimal value, add the enzyme and incubate . Next, after neutralization as necessary, the enzyme is deactivated 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 semipermeable membrane such as cellophane to form a suitable solvent (for example, water, tris-hydrochloric acid). Dialyze sufficiently in a buffer solution or neutral buffer solution of a phosphate buffer solution, and a solution containing a component that has passed through a semipermeable membrane with a component in the filtrate is a strongly acidic cation exchange resin (for example, Dow Chemical). And a fraction containing an angiotensin converting enzyme (hereinafter abbreviated as ACE) inhibitory activity is obtained from the adsorbed elution fraction, and the obtained ACE inhibitory activity fraction is gelled. Fractionation (for example, Sephadex G-25 manufactured by Pharmacia) and fractionation of the obtained ACE inhibitory activity were performed by cation exchange gel filtration (for example, SP-Sep manufactured by Pharmacia). Fractionated by Adex C-25, etc.), further fractionated by reverse phase HPLC ACE inhibitory activity fraction obtained thereof.
[0006]
As the brown algae used in the method for producing a 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 according to 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 peptide according to the present invention has a sequence structure consisting of only L-amino acids, and is gradually degraded by protease in vivo after administration. Therefore, toxicity is extremely low and safety is extremely high (LD ₅₀ > 5000 mg). / Kg; rat oral administration). The novel peptide according to the present invention can be prepared into injections, tablets, capsules, granules, powders and the like using additives such as commonly used excipients. The administration method usually includes injection into a mammal having ACE (for example, human, dog, rat, etc.) or oral administration. The dose is, for example, 0.01 to 10 mg of this peptide per 1 kg of the animal body. The number of administration is usually about 1 to 4 times a day, but can be appropriately prepared depending on the administration route.
[0007]
The types of excipients, binders, and lubricants used in the various preparations are not particularly limited, and those used for ordinary injections, powders, granules, tablets, or capsules can be used. Examples of additives used in tablets, capsules, granules, and powders include the following. Examples of excipients include sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate and the like; starches such as hydroxypropylmethylcellulose as binders; carboxymethylcellulose and potassium salts thereof as disintegrants; Examples of the lubricant include stearic acid and its salts, talc, and waxes. In preparation of the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. A sterile composition for injection is prepared by dissolving or suspending the novel peptide according to the present invention in water for injection, physiological saline, sugar alcohol injection solution such as xylitol or mannitol, or glycol such as propylene glycol or polyethylene glycol by a conventional method. It can be made cloudy to make an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as necessary. The preparation containing the novel peptide according to the present invention is in the form of a freeze-dried product or a dry powder, and can be used by dissolving it in a normal solubilizing agent such as water or physiological saline at the time of use.
[0008]
The novel peptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, and exhibits a blood pressure lowering action and a bradykinin inactivation inhibiting action. Therefore, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension, therapeutic agent, normalization of organ circulation and improvement of long-term prognosis (life extension effect) for these congestive heart failure, treatment of heart failure Useful as an agent.
[0009]
【Example】
In the following, production examples and test examples are described as examples, and the present invention is described in more detail.
Production Example 1
Wakame homogenate obtained by adding 708 ml of deionized water to 23.6 g of wakame powder and homogenizing it was used. A dialysis tube (inner diameter 36 inches, manufactured by Wako Pure Chemical Industries, Ltd.) was packed and dialyzed against running water for 3 days to obtain a dialyzed internal solution. The internal solution was adjusted to pH 2.0 with 1 N hydrochloric acid, added with 708 mg of pepsin (Merck, enzyme number EC 3.4.23.1), and hydrolyzed while stirring at 45 ° C. for 5 hours. Went. The passing solution, in which the decomposition reaction solution was immediately passed through an ultrafiltration membrane (Amicon, YM10 type; fractional molecular weight of about 10,000), was added to a Dowex 50W × 4 [H ⁺ ] column (φ4.0 × 55 cm). . The column was thoroughly washed with deionized water and eluted with 2N ammonium hydroxide solution 2ι. Ammonia was removed by concentration under reduced pressure, and the concentrate was loaded on Sephadex G-25 (φ1.6 × 113 cm) previously buffered with deionized water, and gel filtration was performed at a flow rate of 12 mι / hr and each fraction amount of 5.7 mι. . The result is as shown in FIG. Fractions with high ACE inhibitory activity, collected in large quantities by repeated gel filtration, were collected and lyophilized to obtain peptide powder. After dissolving 1.55 g of this peptide powder in 20 ml of deionized water, it was loaded on an SP-Sephadex C-25 [H ⁺ ] column (φ1.8 × 40 cm) previously buffered with deionized water, and 500 ml of deionized water was added. To 1.5% sodium chloride (500 mι), followed by chromatography at a flow rate of 70 mι / hr and a fraction of 10 mι. The result is as shown in FIG. In the above chromatograph, fractions 23 to 51 having high ACE inhibitory activity were collected and lyophilized to obtain purified peptide powder (SP-II fraction). 20 mg of this purified peptide powder was dissolved in 60 μι 脱 deionized water and then subjected to high performance liquid chromatography (HPLC). As a column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and as a mobile phase, 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05. A concentration gradient method of% TFA was performed, and HPLC was performed at a flow rate of 1.0 mι / 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; 51.3 minutes}
The amino acid sequence of the thus obtained peptide having ACE inhibitory action was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result,
Tyr-Tyr-His
It was confirmed to be a novel tripeptide represented by the amino acid sequence of L-form represented by The property at room temperature is a white powder. In addition, when a novel tripeptide according to the present invention is formulated as an ACE inhibitor, for example, into a tablet, it may be treated according to a conventional method, for example, as follows: (1) 10 g of peptide, (2) 68 g of lactose , (3) Corn starch 39g, (4) Magnesium stearate 1.2g, first mix (1), (2) and 20g corn starch, granulate with paste made from 11g corn starch, 9 g of corn starch and (4) are added, and the resulting mixture is compressed with a compression tablet machine to produce 1000 tablets.
[0010]
Production Example 2
This example is an example of production by a synthesis method.
Synthesis method of Tyr-Tyr-His The peptide was synthesized by a solid phase method using an automatic peptide synthesizer type 430A manufactured by Applied Biosystems. As the solid support, a resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the tripeptide, histidine on the C-terminal side was reacted with chloromethyl resin in the usual manner to obtain a peptide-bonded resin. As the amino acid at this time, a t-Boc amino acid protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group was used. Next, this peptide-bonded resin was suspended in a mixed solution composed of ethanedithiol and thioanisole, stirred for 10 minutes at room temperature, 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. Then, anhydrous ether was added to precipitate the product, and the precipitate was washed several times with anhydrous ether. And dried. The crude 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 and (B) 0.1% TFA-containing acetonitrile solution were used as the mobile phase, and (A) the flow rate was 1% by a concentration gradient method of 93% → 52% over 60 minutes. Chromatography was performed at 1 mι / min. The elution fraction that was detected at the ultraviolet wavelength of 216 nm and showed the maximum absorption was collected and lyophilized to obtain the target synthetic peptide.
[0011]
As a result of analyzing this synthetic peptide by mass spectrum, 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 tripeptide of the present invention obtained by synthesis was confirmed to have a pharmacological effect by the following test.
[0012]
Test example 1
(Angiotensin converting enzyme inhibitory activity measurement method) ACE (manufactured by Sigma, enzyme number EC 3.4.15.1) 2.5 mU, synthetic substrate Hippuryl-L-histidyl-L-leucine (manufactured by Peptide Institute) 12.5 mM The measurement was carried out in accordance with the method of Yamamoto et al., Which improved the measurement method of Lieberman used [Nipporyukaikai, Vol. 18, 297-302 (1989)]. That is, the produced hippuric acid was extracted with ethyl acetate and measured by absorbance at 225 nm. The inhibition rate was calculated from the following equation, where Es is the absorbance in the test solution, Ec is the value when the buffer solution is added instead of the test solution, and Eb is the value when the reaction stop solution is added and reacted in advance. .
Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 100
The inhibitory activity IC ₅₀ value of the ACE inhibitor was expressed as the concentration (M) of the sample required to inhibit the enzyme activity of ACE by 50% (inhibition rate). Inhibitory activity (IC ₅₀ value) of the tripeptide according to the present invention against angiotensin converting enzyme in bovine lung serum; (1) is 0.96 μM.
[0013]
Test example 2
(Antihypertensive effect when administered to spontaneously hypertensive rats) As experimental animals, 15-week-old male spontaneously hypertensive rats (hereinafter abbreviated as SHR) were purchased from Japan's Charles River, and contracted after one week of pre-breeding. A group of 6 animals having a period blood pressure of 160 mmHg or more (weight 280 to 330 g) was used. Rats were housed individually in stainless steel rat individual gauges in a breeding room adjusted to room temperature 23 ± 2 ° C., humidity 55 ± 10%, and 12 hours light / dark (lights on from 6 am to 6 pm). . The feed was MF powder feed manufactured by Oriental Yeast Co., Ltd., and the drinking water was ingested by private pumping (conforming to tap water quality standards). Blood pressure was measured by tail-cuff method using a non-invasive tail artery blood pressure measuring device (manufactured by Riken Development Co., Ltd., PS-100 type) before administration, 1 week after administration, 2 weeks later, 3 weeks later, 4 weeks later. And the measurement value obtained by measuring the systolic blood pressure (mmHg, upper value), diastolic blood pressure (mmHg, lower value), and mean blood pressure (mmHg) of the SHR tail artery after 5 weeks each 5 times at regular intervals. The highest and lowest values were rejected, and the average value of three times was taken as the measured value for each time. The results of the effect on each blood pressure value (mmHg) when 10 mg / kg of the synthetic tripeptide according to the present invention is orally administered to SHR are as shown in FIG. As a result of the above test, it was confirmed that the tripeptide according to the present invention has an angiotensin converting enzyme inhibitory activity and exhibits a significant blood pressure lowering effect even in vivo (in vivo). Therefore, the tripeptide according to the present invention is useful as a therapeutic or prophylactic agent for hypertension. The tripeptide according to the present invention can also be employed in the structure of a peptide structurally having the amino acid sequence as a partial structure.
[0014]
[Brief description of the drawings]
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 proteolysate according to the present invention. In the figure, as a marker, 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 is a diagram showing the results of separation and purification of ACE-inhibiting peptides by SP-Sephadex C-25 (H ⁺ ) column chromatography in Production Example 1 of the Wakame proteolytic solution according to the present invention.
FIG. 3 is a diagram showing the results of separation and purification of an ACE-inhibiting peptide by reverse-phase HPLC in Production Example 1 of a tripeptide according to the present invention.
FIG. 4 is a diagram showing a mass spectrum of the synthetic tripeptide obtained in Production Example 2 of the tripeptide according to the present invention.
FIG. 5 is a graph showing changes in systolic blood pressure value, diastolic blood pressure value, and average blood pressure value (mmHg) over 5 weeks when the synthetic tripeptide 10 mg / kg obtained in Production Example 2 is orally administered to SHR. It is.

Claims (2)

The following formula: Tyr-Tyr-His
A novel tripeptide having a peptide structure by the amino acid sequence of L-form represented by The following formula: Tyr-Tyr-His
An angiotensin-converting enzyme inhibitor comprising a novel tripeptide having a peptide structure based on the L-amino acid sequence represented by

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