JP2871031B2 - Novel peptide and angiotensin converting enzyme inhibitors - Google Patents

Description

The present invention relates to a novel peptide and an angiotensin converting enzyme inhibitor containing the peptide or a salt thereof as an active ingredient.

[Related Art] A renin-angiotensin system is known as a typical in vivo factor that causes an increase in blood pressure, and a kallikrein / kinin system is known as a typical in vivo factor that works to lower blood pressure. "ACE") is heavily involved in both systems.

The mechanism will be described briefly. First, in the renin-angiotensin system, the renal enzyme renin secreted into the blood acts on angiotensinogen in the blood to produce angiotensin I, a decapeptide. This angiotensin I has no blood pressure increasing effect, but when ACE acts on it, angiotensin II, which is an octapeptide, is produced. This angiotensin II contracts peripheral blood vessels and acts on the adrenal cortex to promote the production of aldosterone.Aldosterone acts on the kidney to cause sodium reabsorption and increase in fluid volume, resulting in an increase in cardiac output, All of them greatly increase blood pressure.

On the other hand, although kininogen free yield kinin kallikrein blood enzyme acts on a precursor protein of blood in kallikrein-kinin system, the quinine activates phospholipase A ₂ together dilates peripheral vascular prostacyclin Promotes synthesis of glandin and lowers blood pressure. However, when ACE acts on this kallikrein-kinin system, ACE exerts a peripheral vasodilatory effect and phospholipase A ₂
Since the kinin having the activating effect of the above is also decomposed and inactivated, the blood pressure does not decrease.

Therefore, the presence of a substance (ACE inhibitor) that inhibits the above-described function of ACE suppresses the production of angiotensin II, which is a blood pressure increasing substance, and prevents the degradation of kinin, which acts as a blood pressure lowering substance, to reduce blood pressure. Can be suppressed and blood pressure can be reduced.

From this point, research and development of ACE inhibitors have been performed in recent years, and it has been reported that specific peptides derived from natural proteins or synthesized have an ACE inhibitory action. Previously reported ACE from natural proteins
Inhibitory peptides include Bradykinin potentiator B (Pyr-Gly-Leu-Pro-Pro-Arg-Pr)
o-Lys-Ile-Pro-Pro) and bradykinin potentiator C (Pyr-Gly-Leu-Pro-Pro-Gly-Pro-P
ro-Ile-Pro-Pro) [H.Kato and T.Suzuki, B
iochemistry, 10 , p. 972 (1971)], a peptide derived from milk casein, Phe-Phe-Val-Ala-.
Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys (JP-B-60-
23085), Phe-Phe-Val-Ala-Pro (Japanese Unexamined Patent Publication No.
44323), Thr-Thr-Met-Pro-Leu-Trp (Japanese Patent Laid-Open No. 2-20263), Ala-Val-Pro-Tyr-Pro-Gln
-Arg, Tyr-Lys which is a peptide derived from fish protein
-Ser-Phe-Ile-Lys-Gly-Tyr-Pro-Val-Met, Leu-Pro- which is a peptide derived from corn γ-zein
Pro, Val-His-Leu-Pro-Pro, Val-His-Leu-Pro-
Pro-Pro and the like can be mentioned, and most of them are peptides having 5 or more amino acids bonded.

[Details of the Invention] Under the circumstances described above, the present inventors have also conducted research on substances having an ACE inhibitory action.

As a result, the novel tetrapeptide Leu-G in which leucine-glutamine-proline-arginine is arranged, which has an amino acid sequence different from that of the known ACE inhibitory peptide.
In-Pro-Arg could be isolated from the hydrolyzate of wheat gluten, and it was found that this tetrapeptide had an ACE inhibitory effect.

 Accordingly, the present invention is a peptide represented by the following formula Leu-Gln-Pro-Arg and a salt thereof.

Further, the present invention includes an ACE inhibitor containing the peptide represented by the above formula or a salt thereof as an active ingredient.

The peptide having the ACE inhibitory activity of the present invention was first discovered as a product of a hydrolysis treatment of gluten with a protease, in which case the four amino acids Leu, Gln, Pro and Arg were all substituted. L-amino acid. However, the present invention is not limited thereto, and any optical isomer may be used as long as it is a tetrapeptide having the above-mentioned amino acid sequence, and all of the four amino acids are tetrapeptides composed of D-amino acids and of the four amino acids. Also included are tetrapeptides consisting of any one, two or three L-amino acids and the remainder D-amino acids, which can be produced by chemical synthesis.

An example of a method for preparing the tetrapeptide of the present invention is as follows.

Method by hydrolysis of wheat gluten Wheat gluten is hydrolyzed using a protease to prepare a water-soluble gluten-derived peptide mixture. In this case, the peptide mixture is first hydrolyzed with an acid-acting protease in a state where wheat gluten is dispersed and dissolved in an acidic solution such as dilute hydrochloric acid, neutralized, and further hydrolyzed with a neutral-acting protease. It is preferably prepared by a decomposition method.

As the protease which acts acidicly, it is particularly preferable to use aspartic proteinase in which an aspartic acid residue and a carboxylate ion of aspartic acid are involved in the active center of the enzyme. Examples of such proteases include pepsin, aspartic proteinase of Hilotake mushroom origin, aspartic proteinase of Aspergillus origin, and aspartic proteinase of penicillium origin. Neutral acting proteases include serine proteases such as trypsin, chymotrypsin, plasmin and thrombin. In particular, it is preferable to use pepsin as an acid-acting protease and to use trypsin and / or chymotrypsin as a neutral-acting protease since the desired product can be obtained in high yield. As the protease acting in an acidic manner and the protease acting in a neutral manner, one kind may be used alone, or two or more kinds may be used in combination as long as the proteases do not adversely affect each other. When a plurality of proteases are used, the hydrolysis may be performed in the presence of a plurality of proteases at the same time, or the hydrolysis may be performed sequentially by using one kind at a time. Further, the protease may be used in a free state or may be used after being immobilized. In any case, it is preferable to use about 5,000 to 100,000 units of protease per 100 g of dried gluten.

Here, the protease activity (unit) in this specification is all measured by the following method.

Assay of Protease Activity Using a 1% solution of Hammerstein casein manufactured by Merck, USA as a substrate, the modified Anson-Hagiwara method [Enzyme Research Method, edited by Shiro Akahori, Vol. 2, p. 237 (January 10, 1961, Asakura Shoten)]]. The reaction is performed at 30 ° C for 30 minutes,
The amount of enzyme required to release 1 μg of tyrosine equivalent per minute was defined as 1 unit.

Protease treatment is optimal for each situation (eg, type of protease, use form of protease, etc.).
It is preferable to select conditions such as pH, temperature, amount of protease, treatment speed, treatment time and the like.For example, when using the above-mentioned proteases, first, a protease which acts acidicly has a pH of about 1.5 to 5.0 and a temperature of about 1.5 to 5.0. Operate at 30-50 ° C, then pH
Is adjusted to about 6.0 to 8.0, and then a neutrally acting protease is allowed to act at a temperature of about 30 to 50 ° C. to carry out hydrolysis until the solubility in 0.75 M trichloroacetic acid becomes about 40 to 80%.

When the desired hydrolysis state is achieved, the protease is inactivated by heating and / or pH adjustment, and the inactivated solids such as the inactivated enzyme and undegraded gluten are separated and removed by appropriate means such as centrifugation. Then, the peptide mixture contained in the residual liquid is recovered by drying or the like.

Next, the peptide mixture is dissolved in water or the like and then treated by high performance liquid chromatography (for example, high performance liquid chromatography using a reversed phase column) to isolate the tetrapeptide in a pure form.

The separation and purification of the aqueous solution containing the peptide mixture and the isolation of the tetrapeptide are performed, for example, in the following (a) to (a).
It can be performed by a method comprising the step (d).

(A) The peptide mixture was passed through reversed-phase high-performance liquid chromatography using an ODS column, and the adsorbed components were treated with 0.1% trifluoroacetic acid aqueous solution (solution A) and acetonitrile solution containing 0.1% trifluoroacetic acid (solution B). When eluted with a linear gradient eluent in which the concentration of the solution increases linearly from 0% to 50%, the concentration of acetonitrile is about 15 to
A high absorption peak appears in the eluent fraction in the range of 16%, and the ACE inhibitory activity of this fraction is measured and confirmed and collected. (B) The above step (a) is repeated as necessary.

(C) removing the solvent from the fraction obtained in the step (b) by drying or the like to recover a white solid; and (d) analyzing the amino acid sequence of the product obtained as the white solid by, for example, Applied Biosystems. Using a protein sequencer (model 477-A) manufactured by Leu-
Confirm that it is a tetrapeptide consisting of Gln-Pro-Arg.

When the tetrapeptide of the present invention is produced by chemical synthesis, for example, the following method can be adopted.

Chemical synthesis method of the tetrapeptide of the present invention The synthesis is carried out using a SAM TWO-type automatic peptide synthesizer manufactured by Vylosearch, USA, according to the standard protocol of the same device. That is, Boc (butoxycarbonyl) -Arg
(Tos)-After treating the resin with a deblocking solution containing 45% trifluoroacetic acid, Boc-Pro is coupled in the presence of diisopropylcarbodiimide. Hereinafter, after deblocking in the same manner as above, Boc-Gln and Boc-Leu are sequentially coupled to obtain Boc-Leu-Gln-Pro-Arg (Tos) -resin. This peptide resin is placed in hydrogen fluoride containing 10% anisole and stirred at 0 ° C. for 1 hour. After distilling off hydrogen fluoride, the residue is washed with ether and the peptide is extracted with 10% acetic acid. The resulting crude peptide was purified using reverse phase high performance liquid chromatography on an ODS column,
Leu-Gln-Pro-Arg is obtained.

The ACE inhibitor of the present invention can be administered to humans and various animals, and a remarkable decrease in blood pressure and elevation can be achieved by administering a small amount.

The suitable dose of the ACE inhibitor of the present invention varies depending on various conditions such as age, body weight, sex, symptoms, and animal type of the human or animal to be administered.

The ACE inhibitor of the present invention can be administered by oral administration or parenteral administration, and can be administered alone or together with a solid carrier or liquid carrier commonly used in the pharmaceutical industry. Or may be used in admixture or combination with other agents. The dosage form can be in any form such as tablets, pills, granules, capsules, powders, aqueous solutions, injections and the like.

Furthermore, the ACE inhibitor of the present invention can be added to or administered together with food or feed, in which case L-Leu-L-Gln-L derived from a natural protein.
-Pro-L-Arg is preferred.

Hereinafter, the present invention will be described specifically by way of examples, but the present invention is not limited thereto.

Example 1 [Preparation of Tetrapeptide by Hydrolysis of Wheat Gluten] After 5 g of wheat gluten was dispersed and dissolved in 100 ml of 0.03N hydrochloric acid, distilled water was added to make a total volume of 200 ml. Add 1N hydrochloric acid and p
After adjusting H to 2.0, pepsin (Sigma, USA) 5000
The units were added and reacted at 37 ° C. for 15 hours. Next, after adjusting the pH to 7.0 with a 5N aqueous sodium hydroxide solution, trypsin and chymotrypsin (manufactured by Sigma, USA) were each added for 1000un.
Each of them was added and reacted at 37 ° C. for 5 hours. Then 90 ° C
For 20 minutes to inactivate the enzyme and precipitate undissolved matter. After cooling to room temperature, the solid was separated and removed by centrifugation at 10,000 G for 20 minutes. The supernatant was collected and freeze-dried to obtain 4.0 g of the peptide mixture.

After passing by Cosmosil 5C ₁₈ (20 × 250mm) reverse-phase high performance liquid chromatography 10 ml / min flow rate using a ODS column above peptide mixture 20mg manufactured by Nacalai Tesque, Inc., 0.1% tri-adsorbed component A linear concentration gradient eluent in which the concentration of solution B increases linearly from 0% to 50% with an aqueous solution of fluoroacetic acid (solution A) and an acetonitrile solution (solution B) containing 0.1% trifluoroacetic acid at 10 ml / min. The wavelength of the liquid eluted at the same time as elution through the flow rate
Measurement of the absorbance at 215 nm gave the flow chart with many peaks shown in FIG. Here, the vertical axis on the left side of FIG. 1 indicates the absorbance (O) of the eluted liquid at a wavelength of 215 nm.
D ₂₁₅ ), and the vertical axis on the right shows the acetonitrile concentration (%) in the eluent.

When each peak was separated and its ACE inhibitory activity was examined, it was found that the high absorption peak in the eluent section where the concentration of acetonitrile was 15 to 16% had high ACE inhibitory activity. Therefore, this fraction was collected and the high-performance liquid chromatography treatment was repeated again.

The solvent was dried and removed from the obtained fraction to give a white solid 20μ.
g was collected. When the amino acid sequence of this white solid was examined using a protein sequencer type 477-A of Upright Biosystems, L-Le
u, L-Gln, L-Pro and L-Arg have been released. From this, the formula HL-Leu-L-Gln-L-Pro-L-Arg
-It was confirmed that it was a tetrapeptide represented by OH.

By thin layer chromatography on silica gel plate
When the Rf value was determined, the Rf value when a developing solvent of butanol: acetic acid: pyridine: water = 15: 3: 10: 12 was used was 0.34.

[Measurement of ACE Inhibitory Activity of Peptide] The ACE inhibitory activity of the tetrapeptide obtained in Example 1 was measured according to the method of Cheung & Cushman described in Biochmical Pharamacology 20, p. 1937 (1971) by the following method. Was.

Enzyme substrate: 86 mg of Bz (benzyl) -Gly-His-Leu in 8 ml of water
And a solution dissolved in 8 ml of phosphate buffer.

Enzyme: rabbit lung acetone powder (Sigma) 1g
Was ground in 10 ml of 50 mmol phosphate buffer, and then centrifuged.

100 μl of the above enzyme substrate, 12 μl of the enzyme solution and a predetermined amount of the tetrapeptide prepared in Example 1 were mixed, water was added to make a total volume of 250 μl, and the mixture was reacted at 37 ° C. for 30 minutes. Then, the reaction was stopped by adding 250 μl of 1N hydrochloric acid, and 1.5 ml of ethyl acetate was added thereto.
Stirred for 5 seconds and centrifuged it.

1.0 ml was collected from the ethyl acetate layer, the ethyl acetate was distilled off, and 1 ml of distilled water was added to the residue to dissolve it. Dissolve 228
ultraviolet absorption value in nm of (OD ₂₂₈₎ was measured.

Percent inhibition, the OD ₂₂₈ of the OD ₂₂₈ of 100% and to and react before the start (when the reaction time 0 min) when reacted similarly to without inhibitor above when the 0% inhibition rate of 50% Was determined as the concentration IC ₅₀ of the inhibitor (tetrapeptide of Example 1).

 The results are shown in the table below.

In addition, for reference, bradykinin, a known inhibitor,
The inhibition rate was also determined for potentiator C in the same manner as described above. The results are shown in the table below.

[ACE inhibitory activity of peptide (IC ₅₀ )] Peptide IC ₅₀ (μM) HL-Leu-L-Gln-L-Pro-L-Arg.OH (in the present invention) 17 Bradykinin potentiator C 20 The results show that the ACE inhibitor of the present invention can achieve an IC ₅₀ at a lower concentration than the known ACE inhibitor bradykinin potentiator C.

Example 2 [Production of Tetrapeptide by Synthesis] Commercially available Boc-Arg (Tos) -Resin (substitution rate 0.3 meq / g) 1.
0 g was placed in a reaction vessel of Biosearch's peptide synthesizer SAM TWO 2 which had been pre-filled with 20 ml of methylene chloride containing 45% trifluoroacetic acid and 2.5% anisole.
The reaction was carried out for 5 minutes to remove the Boc group.

Next, the Arg (Tos) -Resin from which the Boc group had been removed was washed with methylene chloride, then neutralized with methylene chloride containing 10% isopropylethyleneamine, and further washed with methylene chloride.

This resin was mixed with 5 ml of a 0.4 M Boc-Pro-dimethylformamide solution and 5 ml of a 0.4 M diisopropylcarbodiimide-methylene chloride solution, placed in a reaction vessel, and reacted with stirring at room temperature for 2 hours.

The resin obtained above, in order, dimethylformamide,
Methylene chloride, methylene chloride containing 10% diisopropylethylamine, methylene chloride and methylene chloride /
After washing with a dimethylformamide mixture, Boc-Pro-Ar
g (Tos) was obtained.

Subsequently, the removal of the Boc group and the coupling of the Boc amino acid were repeated in the same manner as described above to obtain Leu-Gln-Pro-Arg (To
s) -A product consisting of the resin was obtained.

This resin is treated with 20% hydrogen fluoride containing 10% anisole.
The peptide was released from the resin by stirring at 0 ° C. for 1 hour in ml. Thereafter, hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 30% acetic acid, which was lyophilized to obtain 150 mg of a crude peptide. This is ODS column Cosmo manufactured by Nakarai Tesque Co., Ltd.
Purification by reverse phase high performance liquid chromatography using sil 5C ₁₈ (20 × 250 mm) gave 75 mg of the final product. Its amino acid composition (molar ratio) is Leu: Glu: Pro: Arg = 1: 1: 1: 1
Met. Furthermore, as a result of analysis using the same protein sequencer as used in Example 1, H-Leu-Gln-Pr
It was found to be o-Arg-OH.

The Rf value of the product by thin layer chromatography was 0.34 as in the product of Example 1.

[Effects of the Invention] The ACE inhibitor of the present invention can inhibit the activity of ACE with a very small amount of administration to achieve a decrease in blood pressure and an increase in blood pressure.

Further, since the ACE inhibitor of the present invention is a white water-soluble powder, it can be administered as it is or dissolved in water or the like, by any method of oral administration and parenteral administration.

Moreover, the novel tetrapeptide Leu-Gln-Pr of the present invention
Since o-Arg is a low molecular weight compound having a very simple structure in which only four amino acids are arranged, it can be easily produced by chemical synthesis, and when administered, absorbability in the body. It often has a high blood pressure lowering effect.

[Brief description of the drawings]

FIG. 1 shows that the peptide mixture obtained by hydrolyzing wheat gluten in Example 1 was subjected to high performance liquid chromatography, and the adsorbed components were eluted when eluted with a specific eluent having a linear concentration gradient. is a flowchart absorbance component (OD ₂₁₅₎ was measured.

Continued on the front page (58) Fields surveyed (Int.Cl. ⁶ , DB name) C07K 5/103 A61K 38/07 CA (STN) CAOLD (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A peptide represented by the following formula: Leu-Gln-Pro-Arg and a salt thereof. 2. An angiotensin converting enzyme inhibitor comprising a peptide represented by the following formula Leu-Gln-Pro-Arg or a salt thereof as an active ingredient.