JPH0491097A - Novel peptide and angiotensinase inhibitor - Google Patents

Abstract

NEW MATERIAL:A peptide (salt) having an amino acid sequence of the formula. USE:A vasodepressor, an angiotensinase inhibitor. PREPARATION:For example, wheat gluten is dispersed and dissolved in 0.03 N hydrochloric acid followed by addition of distilled water to effect dilution, and the pH level of the resulting solution is adjusted to 2.0 with 1N hydrochloric acid and spiked with pepsin, and then a reaction is made at 37 deg.C for 15hr. Thence, the pH level of the system is adjusted to 7.0 with 5N aqueous NaOH solution followed by addition of trypsin and chymotrypsin and then a reaction is kept at 37 deg.C for 5hr followed by heating at 90 deg.C for 20min to deactivate the enzymes followed by cooling and centrifugation to remove a solid, and the resulting supernatant is recovered and lyophilized to obtain a peptide mixture. This mixture is then put to high performance liquid chromatography and eluted with a linear concentration gradient eluting solution using a 0.1% aqueous trifluoroacetic acid solution and an acetonitrile solution containing 0.1% trifluoroacetic acid, and an active fraction is recovered, thus obtaining the objective peptide of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] 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.

[Conventional technology] Teleconverter and
The angiotensin group and the kallikrein and kinin systems are known as typical in-vivo factors that act to lower blood pressure, and angiotensin converting enzyme (hereinafter referred to as rACEJ) is greatly involved in both of these systems.

The mechanism will be briefly explained. First, in the renin-angiotensin system, the kidney enzyme renin secreted into the blood acts on angiotensinogen in the blood to produce angiotensin I, which is a decapeptide. This angiotensin I does not exhibit an effect of increasing blood pressure, but when ACE acts on it, angiotensin ■, which is an octapeptide, is produced. This angiotensin■ constricts peripheral blood vessels and acts on the adrenal cortex to promote the production of aldosterone.Aldosterone acts on the kidneys, causing sodium reabsorption and an increase in body fluid volume, leading to an increase in cardiac output. Both of these significantly raise blood pressure.

On the other hand, in the kallikrein-kinin system, the blood enzyme kallikrein acts on the precursor protein kininogen in the blood to release kinin, which dilates peripheral blood vessels and activates phospholipase A2 to produce prostaglandin. It promotes the synthesis of gin and lowers blood pressure.

However, when ACE acts on this kallikrein-kinin system, it causes dilation of peripheral blood vessels and phospholipase A.
Since the above-mentioned kinin, which has an activating action, is decomposed and inactivated, a drop in blood pressure does not occur.

Therefore, the presence of a substance that inhibits the above-mentioned functions of ACE (ACE inhibitor) suppresses the production of angiotensin, which is a substance that increases blood pressure, and prevents the decomposition of kinin, which acts as a substance that lowers blood pressure. It is possible to suppress the increase in blood pressure and lower blood pressure.

From this point of view, various research and development efforts have been made on ACE inhibitors in recent years, and it has been reported that certain peptides derived from natural tannin and synthetically produced peptides have an ACE inhibitory effect. The ACE-inhibiting peptides reported so far from the natural protein Pyr-Gly-Leu
-Pro-Pro-Arg-Pr.

-Lys-11e-Pro-Pro) and bradykinin, Pyr-Gly-Leu-P
ro-Pro-Gly-Pr.

-Pro-I 1e-Pro-Pro) [both H
, Kato and Tsuzuki Bioche
mistry, 10. p. 972 (1971)
Phe-Phe-Val-A Ia-Pro-Ph, a peptide derived from milk casein]
e-Pro-G Iu-Va IPhe-G 1y-L
ys (Special Publication No. 60-23085), Ph eP
he-Va l-A 1a-Pro
323), Thr-Thr-Me[-Pro-L
eu-Trp (Unexamined Japanese Patent Publication No. 20263/1999), A 1
a-Va 1-Pro-Tyr-Pro-G In-A
rg, Tyr-L, a peptide derived from fish protein;
ys-5er-Phelle-Lys-Gly-Tyr
-Pro-Vat-Met, ) Leu-Pro-Pro, V which is a peptide derived from horse o=+cygamma-zein
al-His-Leu-Pro-Pro, Val-
His-Leu-Pro-Pr.

Most of them are peptides in which five or more amino acids are bonded.

[Contents of the Invention] Under the above-mentioned circumstances, the present inventors have also been conducting research on substances having an ACE inhibitory effect.

As a result, a novel tetrapeptide Leu, which has a loin-glutamine-broline-arginine sequence, has an amino acid sequence different from that of the known ACE-inhibiting peptides.
-Gln-Pro-Arg could be isolated from wheat gluten hydrolyzate, and it was found that this tetrapeptide has ACE inhibitory activity.

Therefore, the present invention provides the following formula %formula% These are the peptide represented by and its salt.

Furthermore, 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 above ACE inhibitory activity of the present invention is initially a product of gluten hydrolysis with pcotease. In this case, the four amino acids Leu, Gln, Pro and Arg are all L-amino acids.

However, the present invention is not limited thereto, and any optical isomer may be used as long as the tetrapeptide has the above-mentioned amino acid sequence. Tetrapeptides in which any one, two or three of the above are L-amino acids and the remainder are D-amino acids are also included,
They can be produced by chemical synthesis.

Examples of the method for preparing the tetrapeptide of the present invention are as follows.

Method by Hydrolysis of Wheat Gluten Wheat gluten is hydrolyzed using protease to prepare a water-soluble gluten-derived peptide mixture. In this case, the peptide mixture is prepared by first dissolving wheat gluten in an acidic solution such as dilute hydrochloric acid, hydrolyzing it with a protease that acts in an acidic state, and then further hydrolyzing it with a protease that acts in an acidic state after neutralization. Preferably, it is prepared by a hydrolysis method.

As a protease that acts in acidic conditions, it is particularly recommended to use asnocurtic proteinase, in which an aspartic acid residue and an as/cragic acid carboxylic acid ion are involved in the active center of the enzyme. T.

Examples of such proteases include pepsin, Aspartei nokuflotinase from Hirotake 1. Examples include Aspergillus proteinase derived from Aspergillus and 71 Baltei proteinase derived from Penidylium. Examples of proteases that act in neutral conditions include serine proteases such as trypsin, chymotrypsin, plasmin, and thrombin.

In particular, it is preferable to use pepsin as the protease that acts in acidic conditions and trypsin and/or chymotrypsin as the protease that acts in neutral conditions because the desired product can be obtained in high yield. Proteases that act in acidic conditions and proteases that act in neutral conditions may be used alone or in combination as long as the proteases do not have an adverse effect on each other. When using multiple proteases, even if multiple proteases are present simultaneously for hydrolysis,
Alternatively, the hydrolysis may be carried out using one type sequentially. Further, protease may be used in a free state or in an immobilized state. In any case, the amount of protease used is preferably about 5.000 to 00.000 units of grotease per 100 g of dried gulfun.

Here, all protease activities (units) in this specification are measured by the following method.

Measuring method of protease activity A 1% solution of hammerstein nonosein (manufactured by Merck & Co., USA) was used as the substrate, using the modified Anson-Hagiwara method [edited by Akahori Shibu].
The measurement was carried out using "Enzyme Research Methods" Volume 2, page 237 (January 1, 1960, published by Asakura Shoten) 1. The reaction was carried out at 30°C for 30 minutes, and the amount equivalent to 1 μg of thyronone was liberated per minute. The amount of enzyme required for this is 1 unit.

Protease treatment is performed using the optimal p
It is best to select conditions such as H, temperature, amount of protease, treatment speed, treatment time, etc. For example, when using the proteases listed above, first the protease that acts in acidic conditions is heated to a pH of about 15 to 5.0. After acting at a temperature of about 30 to 50°C and then adjusting the pH to about 60 to 8.0, a protease that acts at a neutral temperature is allowed to act at a temperature of about 30 to 50°C and dissolved in 0.75 M trichloroacetic acid. The rate is about 40-80%
It is best to carry out hydrolysis until

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

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

The separation and purification of the aqueous solution containing the peptide mixture described above and the isolation of the tetrapeptide can be carried out, for example, by the following methods (a) to (d).
).

(a) The peptide mixture was subjected to high performance liquid chromatography (for example, ODS-Cos manufactured by Nacalai Tesque Co., Ltd.).
mosil 5C+a) to remove the adsorbed components.
A linear concentration gradient eluent in which the concentration of Solution B increases linearly from 0% to 50% using a 1% trifluoroacetic acid aqueous solution (Solution A) and an acetonitrile solution containing 0.1% trifluoroacetic acid (Solution B). When eluted with acetonitrile, a high absorption peak appears in the eluent fraction with an acetonitrile concentration of about 15 to 16%, and the ACE inhibitory activity of this fraction is measured and recovered. (b) If necessary. Repeat step (a) above, (
c) Remove the solvent from the fraction obtained in step (b) by drying or the like to recover a white solid, and (d) convert the amino acid sequence of the product obtained as the white solid to, for example, Abride Biosystems. It is confirmed that it is a tetrapeptide consisting of Leu-Gin-Pro-Arg.

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

Chemical synthesis method of tetrapeptide of the present invention Using the SAM TWO type automatic peptide synthesizer manufactured by Pyro Search, USA, the standard protocol for the same equipment is used! Therefore, synthesis is performed. That is, after treating Boc(butoxycarbonyl)-Arg(Tos)-resin with a deblocking solution containing 45% trifluoroacetic acid, Boc-Pr
o is coupled in the presence of diisopropylcarbodiimide. Below, after deblocking in the same way as above, B
oc-Gln and Boc-Leu are sequentially coupled to form Boc-Leu-Gin-Pro-Arg(Tos
) - obtain 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, wash the residue with ether and reduce to 10%
Extract the peptides with acetic acid. The obtained crude peptide was OD
Purification using liquid chromatography on an S column yields Leu-Gin-Pro-Arg.

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

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

The ACE inhibitor of the present invention can be administered either orally or parenterally, and can be administered alone or with solid or liquid carriers commonly used in the pharmaceutical industry. -g, or mixed or combined with other drugs. Further, the dosage form can be any form such as tablets, gunpowder, granules, capsules, powders, aqueous solutions, and injections.

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

The present invention will be specifically explained below by giving examples, but the present invention is not limited thereto.

Example 1 [Preparation of tetrapeptide by hydrolyzing wheat gluten I] After dispersing and dissolving 5 g of wheat gluten in 100 mQ of 0.03N hydrochloric acid, distilled water was added to make a total of 1200 mQ. After adjusting the pH to 20 by adding IN hydrochloric acid, 5000 units of Pezucin (manufactured by Sigma, USA) was added.
The reaction was carried out 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 added at 1000 u each.
The reaction was continued at 37°C for 5 hours.

Next, the mixture was heated at 90°C for 20 minutes to inactivate the enzyme and precipitate undissolved substances. After cooling to room temperature, 10
The solids were separated and removed by centrifugation at 0OOG for 20 minutes. The supernatant was collected and lyophilized to give 4.0 g of peptide mixture.
I got it.

20 mg of the above peptide mixture was subjected to high performance liquid chromatography: ODS-Cosm manufactured by Nacalai Tesque Co., Ltd.
osil 5C+g (20X250mm) lom<
After passing through at a flow rate of 1/min, the adsorbed components were mixed with an aqueous solution of 0.1% trifluoroacetic acid (liquid A) and an acetonitrile solution containing 0.1% trifluoroacetic acid (liquid B) to adjust the concentration of liquid B. When a linear concentration gradient eluent increasing linearly from 0% to 50% was passed through the eluate at a flow rate of IOmf2/min and at the same time the absorbance of the eluate was measured at a wavelength of 215 nm, a large number of I got a flowchart with peaks.

Here, the left vertical axis in Figure 1 is the wavelength 215 of the eluted liquid.
The absorbance (OD, , ) in nm is shown, and the vertical axis on the right side shows the acetonitrile concentration (%) in the eluent.

When each peak was separated and examined for its ACE inhibitory activity, it was found that the high absorption peak in the eluent section with an acetonitrile concentration of 15 to 16% had a high ACE inhibitory activity.

Therefore, these two fractions were collected and the high performance liquid chromatography treatment described above was repeated again.

The solvent was removed by drying from the resulting fraction to yield 20μ of a white solid.
g was collected. Apply this white solid.

Tobiosystem protein sequencer-477-
When we investigated the amino acid sequence using Type A, we found that, in order from the N-terminus, L-Leu, L-Gin, and L-Pr.

and L-Arg were liberated. From this, it was confirmed that it was a tetrapeptide with the formula %.

R by thin layer chromatography using a liquid gel plate.
When the f value was determined, the Rf value was 0.34 when a developing solvent of butanol:acetic acid:pyridine/water-15,3:10:12 was used.

[Measurement of ACE inhibitory activity of peptide] The ACE inhibitory activity of the tetrapeptide obtained in Example 1 was measured using Biochemical Pharmacolog.
y 20+ p.

Cheung & Cu, 1937 (1971)
The following method was used according to the method of Shman.

Enzyme substrate: Bz (benzyl)-Gly-His-Le
A solution in which 86 mg of u is dissolved in 8 mff of water and 8 ml (l) of phosphate buffer.

Enzyme: Rabbit lung acetone powder (Ng manufactured by Sigma) was crushed in 50 mmol of phosphate buffer (10 ml), and then centrifuged. Supernatant liquid.

100 μg of the above enzyme substrate, 12 μa of the enzyme solution, and a predetermined amount of the tetrapeptide produced in Example 1 were mixed, water was added to make a total liquid volume of 250 μg, and the mixture was reacted at 37° C. for 30 minutes. Next, 250 μg of IN hydrochloric acid was added to stop the reaction, and then 1.5 mQ of ethyl acetate was added, stirred for 15 seconds with a Vortex mixer, and then centrifuged.

1.0 mQ was taken from the ethyl acetate layer, ethyl acetate was distilled off, and 1 mQ of distilled water was added to the residue to dissolve it. The ultraviolet absorption value (OD!!I) of the solution at 228 nm was measured.

The inhibition rate is determined by setting 0022m when reacting in the same manner as above without an inhibitor as 100% and before the start of the reaction (reaction time 0).
The inhibition rate is 50% when the OD2□ of
The concentration of the inhibitor (tetrapeptide of Example 1) at the time when the concentration IC was calculated.

The results are shown in the table below.

Furthermore, for reference, the inhibition rate was also determined for bradykinin botensiator C, which is a known inhibitor, in the same manner as above. The results are also listed in the table below.

[ACE inhibitory activity of peptide (+C,.)] Peptide Ic. (μM) H-L-Leu-L-Gin-L-Pro-L-Arg
-OH (Invention) 17 Bradykinin Potentiator C20 From the results in the above table, the ACE inhibitor of the invention has a lower IC concentration than the known ACE inhibitor Bradykinin Potentiator-C. It turns out that it is possible to achieve.

Example 2 [Production of tetrapeptide by synthesis] Commercially available Boc-Arg(Tos)-Resin (substitution rate 0.3 meq/g)1. og in methylene chloride containing 45% trifluoroacetic acid and 2.5% anisole.
(2) was placed in advance in the reaction tank of Biosearch's peptide synthesizer SAM TWO2, and reacted for 25 minutes at room temperature to remove the Boc group.

Next, Arg(Tos) -R with the Boc group removed
The es in was washed with methylene chloride, then neutralized with 10% isopropyl ethylene amine in methylene chloride, followed by further washing with methylene chloride.

This resin was mixed with 5 mo 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 tank, and reacted at room temperature for 2 hours with stirring.

The resin obtained above was mixed in order with dimethylformamide, methylene chloride, methylene chloride containing 10% diisobrobylbutylamine, methylene chloride and methylene chloride/
Wash with dimethylformamide mixture and remove Boc-Pr.
Obtain o-Arg(Tos).

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

Hydrogen fluoride containing 10% anisole 2
The peptide was released from the resin by stirring for 1 hour at O'C in OmQ. Thereafter, hydrogen fluoride was distilled off under reduced pressure, the residue was extracted with 30% acetic acid, and it was lyophilized to obtain crude peptide 1.
50 mg was obtained. This is applied to an ODS column (Cosmosi column).
Reverse phase D'? due to l5C+m)? Purification by Togelafy gave 75 mg of the final product. Its amino acid composition (molar ratio) is Leu:Glu:Pro:Arg
= 1 + l: 1: l. Furthermore, as a result of analysis using the same protein/-quencer used in Example I, H-Leu-Gin-Pr.

It turned out to be Arg-OH.

Further, the RfH of the product in thin layer chromatography was 1:0, 34, which was the same as that of the product in Example 1.

[Effects of the Invention] The ACE inhibitor of the present invention can inhibit the activity of ACE and achieve lowering of blood pressure and suppression of increase in blood pressure by administering an extremely small amount.

Furthermore, since the ACE inhibitor of the present invention is a white water-soluble powder, it can be administered very easily by either oral or parenteral administration, either as it is or dissolved in water. Moreover, the novel tetrapeptide Leu-Gln of the present invention
-Pro-Arg is a low molecular weight compound with an extremely simple structure consisting of just a sequence of 4@ amino acids,
It can be easily produced by chemical synthesis, and when administered, it is well absorbed in the body and exhibits a high blood pressure lowering effect.

[Brief explanation of the drawing]

Figure 1 shows the peptide mixture obtained by hydrolyzing wheat gluten in Example 1, which is passed through high performance liquid chromatography, and the adsorbed components are eluted with a specific eluent having a linear concentration gradient. Absorbance of component (
It is a flowchart for measuring OD215).

Claims (1)

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