JPS6136226A - Inhibitor against enzyme capable of converting angiotensin - Google Patents

Abstract

PURPOSE:The titled inhibitor obtained by treating casein derived from bovine with trypsin. CONSTITUTION:An inhibitor against an enzyme capable of converting angiotensin, comprising a compound shown by the formula as an active ingredient. The inhibitor is obtained by hydrolyzing casein derived from bovine with trypsin at 5.5-9.0pH, heat-treating the hydrolyzate at about 100 deg.C or treating it with an acid to precipitate trypsin and unhydrolyzed casein, removing the precipitate by centrifugal separation, etc., neutralizing the prepared mother liquid with an alkali such as NaOH, etc., concentrating it two - three times under reduced pressure, and purifying it. It is especially useful as a hypotensor. Administration is generally carried out by intravenous injection, for example, 0.01-1mg/kg (animal) of it is applied by intravenous injection. This drug is applicable to mammals (e.g., human, rat, dog, etc.) having the enzyme in an organism.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an angiotensin converting enzyme inhibitor having the following structure.

Ala-Val-Pro-Tyr-Pro-Gin
-, Arg The in vivo enzyme inhibitor found in the 2P solution has been shown to have various effects such as anti-inflammatory, anti-digestive and anti-cancer properties, and is expected to be used as a medicine or research reagent. It's here.

Among these, regarding angiotensin converting enzyme inhibitors,
Several types of inhibitors produced by microorganisms have recently become known, including peptide inhibitors obtained from Brazilian snake venom and Japanese snake venom, and peptide inhibitors obtained from gelatin treated with microorganism-derived collagenase. It has been known for some time that it was isolated from In addition, the Squibb Company of the United States has synthesized captopril, a proline derivative, which has a strong inhibitory effect and is attracting attention as a new orally available drug.

However, since all of these inhibitors are expensive, it is desired to develop inhibitors derived from natural products that can be obtained at low cost and have fewer side effects.

On the other hand, the present inventors had previously succeeded in obtaining an angiotensin converting enzyme inhibitor by decomposing Ushiyama's casein with trypsin etc.
, JP 59-44323, JP 59-44324)
.

This time, the present inventors succeeded in preparing a new angiotensin converting enzyme inhibitor having the above structure by treating Ushiyama casein with trypsin etc. as in the previous case.

The inhibitor of the present invention exhibits an inhibitory effect on angiotensin converting enzyme. In this case, angiotensin convertase is angiotensin I, which is produced by decomposing angiotensinogen secreted in the liver by the enzyme renin produced in the kidneys.
(Asp-Arg-vat-'ryr-11
e −) (is −Pro −phe −) (is −
Leu), and this is converted into angiotensin (Asp-Arg-Val-Tyr-11e-
His-Pro-phe). and,
This angiotensin (IT) causes blood vessel wall smooth muscle to contract, increasing blood pressure, and in addition to blood vessels, it also contracts smooth muscle in the gastrointestinal tract and uterus, and also acts on the adrenal cortex to promote the secretion of aldosterone. It has the following effects.

Additionally, the enzyme kallikrein present in plasma degrades a protein called kininogen to produce bradykinin, which dilates blood vessels and lowers blood pressure, but this bradykinin is degraded and inactivated by the action of angiotensin converting enzyme. Thus, angiotensin convertase produces a pressor peptide (angiotensin) on the one hand, and an antihypertensive peptide (bradykinin) on the other hand.
As a result, the TFI pressure is increased.

The inhibitor according to the present invention has an Ijfl harmful effect on angiotensin converting enzyme, which exhibits such an effect, and is particularly effective as a hypotensive agent.

In order to obtain the angiotensin converting enzyme inhibitor according to the present invention, Ushiyama casein is decomposed with trypsin under conditions of pH 5.5 to 9.0, and the decomposed product is treated by heat treatment at about 100°C or by adding acid. Trypsin and undegraded casein are precipitated by this method, and this precipitate is removed by centrifugation or the like. After neutralizing the extract thus obtained with an alkali such as sodium hydroxide, it is concentrated 2 to 3 times under reduced pressure. The concentrate thus obtained is purified to obtain a product.

The present inhibitor can also be obtained using conventional peptide synthesis methods.

That is, the amine group of one amino acid is protected with a benzyloxycarbonyl group or t-butoxycarbonyl group, the carboxyl group of the other amino acid or peptide is protected with a benzyl ester, etc., and DCC (N,N'-dicyclohexyl Coupling with carbodiimide etc. By repeating this operation, the protecting group is removed and the product can be purified and obtained.

The property of the inhibitor according to the present invention at room temperature is a white powder, and thin layer chromatography of its aqueous solution (silica gel plate, cellulose plate, ninhydrin coloring)
The Rf values are shown in Table 1 below.

If it is dissolved in 6M hydrochloric acid and hydrolyzed under vacuum at 110°C for 24 hours, an amino acid mixture having the composition shown in Table 2 below will be obtained.

The method of taking the angiotensin converting enzyme inhibitor of the present invention is as follows:
It is generally administered by intravenous injection, for example, 1 bottle per 1 kg of animal! +7 An aqueous solution of the inhibitor is intravenously injected to a concentration of 0.01 to Jmg of the harmful agent.

The angiotensin converting enzyme inhibitor according to the present invention can be applied to mammals that have the enzyme endogenously in their bodies, such as humans, rats, and dogs.

Next, the present invention will be explained in more detail with reference to Examples.

Example: 2g of Ushiyamaki casein 50%. 0.04M of d! acid=
5- Suspend in buffer (+) IT7.4) and add trypsin (P
Add 5m9 (manufactured by L Biochemicals, derived from pancreas) and react at 37°C overnight. After the reaction, hydrochloric acid with a final concentration of 0.5N is added to the product to denature and precipitate trypsin and undecomposed casein. After removing the precipitate by centrifugation, the hydrochloric acid is neutralized with 11 um of hydroxide, 1) H is adjusted to 7.0, and the mother liquor is concentrated 2 to 3 times under reduced pressure.

Next, the concentrate obtained above is added to a Sephadex LH20 column and purified by elution with distilled water. and,
The most active fraction at this time is collected and concentrated under reduced pressure. Note that the column processing conditions in this case are as follows.

Column: Height 112 cm, inner diameter 31 Sample added mold: 20 tnl Flow rate: 1.2+n//min Elution: Distilled water Next, the active fraction fractionated with the Sephadex Li (-20) was added to SP-Sephadex C -25 column, 0 to 0.5M ammonium formate (T)H
= 7.0) with a linear concentration gradient.

Pour the most active fraction and concentrate under true pressure. Note that the column processing conditions in this case are as follows.

Column: height 49 cm, inner diameter 2 CIl + sample addition amount:
5- Flow rate: 0.4 me/n 1 volume Output: 0-0.5M ammonium formate (pH-7,
0) Linear concentration gradient Next, the active fraction obtained above was added to Sephadex L.
Add to H-20 column and desalt.

The column processing conditions in this case are as follows.

Column: Height 64 m, internal diameter 2 cm Flow rate: Q, 41 n1/min Elution: Distilled water Next, the sample desalted with Sephadex LH-20 described in section 11f is dried under reduced pressure to obtain a white powder substance (4 g
Approximately 2 mg of casein can be obtained from

Next, the Rf value of this substance was determined by thin layer chromatography (silica gel plate and cellulose plate, ninhydrin coloring), and the results are as follows.

Chapter 1 Table However, the following results were obtained.

Table 2 Next, when the substance of the present invention was treated with carboxypeptidase Y, Gln (glutamine) was detected. Therefore, GILI (glutamic acid) detected by hydrochloric acid hydrolysis is 1. In peptides, it is Gln (glutamine).

Next, the amino acid sequence of the substance of the present invention was determined using an amino acid analyzer using leucine aminopeptidase, carboxypeptidase A, and carboxypeptidase Y, and it was confirmed that it had the following structure.

Ala -Val -Pro -'l'yr -Pro
-Gln-Arg=9- Next, in order to measure the enzyme inhibitory activity of the substance of the present invention, the following experiment was conducted.

First, add 50% of 5I7 Rabbit Lang Acetone Powder.
．． , l/0,1M sodium borate buffer pH-8
, 3) and centrifuged at 40,000 g for 40 minutes, and the supernatant was further diluted 5 times with the above buffer to obtain an angiotensin converting enzyme solution.

A sample containing the substance of the present invention was placed in a test tube, and 025ml of hyperlyl histisilleucine (final concentration: 5ml containing 1300mM Nac) was added as a substrate. After incubation at 37°C for 10 minutes, the enzyme solution was added. 0,1d
and reacted at 37°C for 30 minutes.

After that, 0.254 IN hydrochloric acid was added to stop the reaction, 1.5-ethyl acetate was added, and the absorbance value of hyperric acid extracted in ethyl acetate was measured at 228 nm.
This was defined as enzyme activity. Note that under these conditions, the absorption value of 2281m when the present inhibitor is not included is approximately 0.25.

A plurality of such experiments were conducted, and the inhibition rate was calculated using the following formula.

A A: Absorption value of 2281m without inhibitor (0,
25) Absorption value at 228 nm when B, 1i11 harmful agent was added.The inhibitor concentration ID511 at an inhibition rate of 50% was determined to be 1.5×10'M for the inhibitor of the present invention.

Claims (1)

[Claims] An angiotensin converting enzyme inhibitor having the following structure. Ala-Val-Pro-Tyr-Pro-Gln-A
rg