JPH03284694A - New tripeptide and hypotensor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel tripeptide and an antihypertensive agent containing the same as an active ingredient.

[Conventional technology]

It is well known that the renin-angiotensin system is deeply involved in the onset of hypertension. ) plays an important role. In this case, ACE is angiotensin I (Asp
-Arg-Sial-Tyr-11e-H4sPro-P
he-His-Leu) and angiotensin II (Asp-Arg-Val-Ty).
r-11e-)1isPro-Phe).

This angiotensin has the effect of contracting the smooth muscle of the blood vessel wall, increasing blood pressure, and acting on the adrenal cortex to promote the secretion of aldosterone. Furthermore, the enzyme kallikrein present in plasma degrades a protein called kininogen and produces bradykinin, which dilates blood vessels and lowers blood pressure, but this bradykinin is degraded and inactivated by the action of ACE. Thus, ACE produces a pressor peptide (angiotensin ■) on the one hand, and degrades an antihypertensive peptide (bradykinin) on the other hand, resulting in an increase in blood pressure. Therefore, it is possible to prevent an increase in blood pressure (lower blood pressure) by suppressing this enzyme activity.

ACH activity inhibitors include several peptide inhibitors obtained from snake venom, as well as captopril (D-2).
-Methyl-3-mercaptopropanoyl-1,-proline) and other synthetic substances are known, and among these, captopril is already in practical use as an oral antihypertensive agent.

In addition, in recent years, ACE inhibitors have been found in microorganisms and various foods, and their practical use as antihypertensive agents is being considered (Kunio Sueami, Fermentation and Industry 46 (NO, 3), 17
9-182 (1988)). Furthermore, food protein,
AC from casein, corn seed protein in particular
Regarding E inhibitors, Susumu Maruyama, Bioscience and Industry 47 (No, 11), 38-42 (1989
) has been reported. Regarding ACE inhibitors derived from corn seed proteins, Susumu Maruyama et al.
Collection of Abstracts of 3rd Year Japan Fermentation Society Meeting Lectures, page 23 (1988
), Susumu Maruyama et al., 1999 Japanese Society of Agricultural Chemistry Lecture Abstracts 8
(1989), Shinsuke Miyoshi et al., 1999 Japanese Society of Nutrition and Food Science Abstracts, p. 113 (1989), and Hiromi Ishikawa et al., Journal of the Japanese Society of Agricultural Chemistry 64 (3), 1990 Conference Abstracts, p. 555. is being done.

[Problem to be solved by the invention]

The present inventors are currently conducting research mainly on blood pressure terrestrial peptides derived from corn seed proteins, and the N-terminus is L-Leu and the C-terminus is L-Pr.

1ribebutide, L-Leu-L-Pro-
L-Pro, L-Leu-L-5er-L-Pro,
L-Leu-L-Gln-L-Pro,,I,Leu
-L-Leu-L-Pro, L-Leu-L-As
n-L-Pro and l.

Leu-L-Arg-L-Pro is a good AC
It was found that it has an E-inhibiting effect and, in turn, a blood pressure lowering effect. As a result of further research, we found that there was no relation to the 1,-amino acid located in the middle, and that the N-terminus was D-L.
eu, i.e. general 2L- (or D-
) Leu-L-X-L-Pro (X is an amino acid) It was discovered that the tripeptide represented by the formula (X is an amino acid) generally has a good ACE inhibiting effect and furthermore, a blood pressure lowering effect, and the present invention was completed.

New and useful antihypertensive agents are constantly being sought and provide an enrichment of technology.

An object of the present invention is to provide a novel tripeptide having an excellent antihypertensive effect, and a highly safe antihypertensive agent containing such tripeptide as an active ingredient.

[Failure to solve the problem]

The above objects of the present invention have been achieved by the following invention, which includes a novel 1-IJ peptide and an antihypertensive agent containing the tripeptide as an active ingredient.

1.2 17-(or D-)Leu-1, -X-L-Pr.

(wherein, X is Gly, Ala, ~'al, lie
, Thr, Asp, +Glu, Lys,
Orn,, Cys Xl'let XPhe.

Tyr, Trp + 1lis or II yp). tripeptide, and X or 1, ys
, Orn or If is Acid addition salts of L. Libebuti), and X is Asp or Glu.
Alkali or alkaline earth metal salts, ammonium salts or organic base salts of Tribe 1.

2, Formula % Formula % (wherein, X is Gly, Ala, Val, lie
, Thr, 8sp X Glu,, Lys
, Orn, Cys,, Met
,, yen+e.

Tyr, Trp, His or ++yp), acid addition salts of the above tripeptides where X is Lys, Orn or His, or
An antihypertensive agent containing, as an active ingredient, an alkali or alkaline earth metal salt, ammonium salt or organic base salt of the above-mentioned Trivepti, which is Asp or Glu.

In the above, Hyp includes, for example, 4) 1yp.

In addition, the above acid addition salts include pharmaceutically acceptable acid (inorganic and organic acid) addition salts, such as hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, and maleate. , fumarate, succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glutamate, and the like.

In addition, the alkali metals and alkaline earth metals mentioned above are sodium, potassium, calcium, etc., and the organic bases are lysine,
Includes basic amino acids such as ornithine.

The tripeptide used in the present invention can be produced by a method of stepwise introduction of amino acids using an organic chemical synthesis method, a peptide synthesis method using a reverse reaction of a hydrolase, a genetic engineering method, etc.

There are two types of organic chemical synthesis methods: liquid phase method and solid phase method. 1-hydroxyhensitriazole) for one day and night to sequentially bond from the carboxy terminal side.

In the latter case, amino acids are sequentially extended on PAM (phenylacetamidomethyl) resin using a peptide synthesizer (Model 430A) manufactured by Applied Hiosystems.
The desired tripeptide is obtained by cleaving the resin and various protecting groups with hydrogen fluoride or the like.

Acid addition salts of the present tripeptide can be produced by conventional methods. Furthermore, an alkali metal or alkaline earth metal salt, ammonium salt, or organic base salt (hereinafter referred to as base salt for convenience) of the present tripeptide can also be produced by a conventional method.

The present tripeptide and its acid addition salts and base salts have antihypertensive and ACE inhibitory effects, and are expected to be effective in treating and preventing hypertension in mammals including humans.

The tripeptides or acid addition or base salts thereof are used as such or in pharmaceutical compositions, usually with at least one pharmaceutical auxiliary.

The present tripeptide or its acid addition salt or base salt can be administered parenterally (ie, intravenous injection, rectal administration, etc.) or orally, and can be formulated into a form suitable for each administration method.

Injectable formulations typically include sterile ice-water solutions. Preparations of the above form may also contain buffers, p) regulators (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), preservatives (methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, etc.). Other pharmaceutical adjuvants other than water may be included, such as (etc.). The formulation can be sterilized by filtration through a bacteria-retaining filter, by incorporating a disinfectant into the composition, by irradiating or heating the composition.

The preparation can also be prepared as a sterile solid composition and used by dissolving it in sterile water or the like before use.

Orally administered preparations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, granules and powders are prepared using conventional pharmaceutical auxiliaries, such as binders (syrups, gum arabic,
gelatin, sorbitol, tragacanth, polyvinylbicritone, hydroxypropyl cellulose, etc.), excipients (
lactose, sugar, corn starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethyl cellulose, etc.), wetting agents (sodium lauryl sulfate, etc.)
can be included. The tablets may be coated by conventional methods.

Oral liquid medicine is water? It can be made into 8 liquid etc. or rubbed with triproduct. Such oral solutions may contain conventional additives such as preservatives (methyl or propyl P-hydroxybenzoate, sorbic acid, etc.).

Although the amount of the tripeptide or its acid addition salt or base salt in the antihypertensive agent can be varied, it is usually 5 to 100 χ (-8), particularly 1O to 60 χ (w/h).
is appropriate. The appropriate dosage of this antihypertensive agent is 10 to 200 mg/kg/day of the active ingredient. In addition,
The acute toxicity of this tripeptide is LDS.

(1 (J mouse, oral administration) > 3 g/kg.

In addition, this tripeptide and its acid addition salts and base salts have the advantage that they do not adversely affect the living body even if they are ingested in multiple doses, so they can be taken as is or with the addition of various nutrients, etc.
Alternatively, it may be consumed as a functional food or health food that has the function of lowering blood pressure and preventing hypertension by incorporating it into foods and drinks. That is, for example, nutrients such as various vitamins and minerals can be added to liquid foods such as nutritional drinks, soy milk, and soups, solid foods in various shapes, and even powdered foods as they are or added to various foods. You can also do it. The content and intake amount of the active ingredient in the present antihypertensive agent as such a functional food and health food may be the same as the content, dosage, and amount in the above-mentioned pharmaceutical products.

[Example] Next, the present invention will be explained by examples.

Synthesis of Fruit 17-LL-Leu-L-Ala-L-Pro Applied Hiosystems peptide synthesizer (
430A) and 0.5 mmol of Boc-L-Pro.
-OCH, -PAM resin and 2 mmol each of Boc-L
-41a and Boc-L-Leu, and L-Leu-L-Ala-L-P was
ro-0-CH2-PAM was synthesized.

Next, the above synthetic peptide resin was introduced into a hydrogen fluoride treatment equipment manufactured by Peptide Institute, and after adding 1.5 d of anisole,
10 d of liquid hydrogen fluoride was introduced.

After reaction at -20°C for 30 minutes and at 0°C for 30 minutes, hydrogen fluoride was removed under reduced pressure.

The obtained peptide was washed three times alternately with anhydrous ether and chloroform, dissolved in distilled water, and purified using a semi-preparative high-performance liquid chromatography device manufactured by Nippon Millibore Limited. The elution conditions are as follows.

Column: Shiseido CAPCELL PAK C18
Elution solvent 2 in the presence of 0.1% trifluoroacetic acid, 5 to 8
0% acetonitrile gradient (required time: 45 minutes) Flow rate = 8 proofs/min Detection: LIV 210 nm After the above detection, the eluted fraction containing the main peak was condensed to dryness under reduced pressure, and then added to a 2N acetic acid aqueous solution. It was reconstituted and then lyophilized.

By this method, L-1, eu-L-Ala-L-Pr
(The results of various analyzes are shown in Table 1 below, and the same applies hereinafter).

Synthesis of X-12L-Leu-1, -Lys-L, -Pro 0.5 mmol of Boc-L-P was added to a peptide synthesizer (Model 430A) manufactured by Abridohiosystems.
ro-OCHz-PAM resin and 2 mmol each of Boc
-L-Lys (Z) and tloc -L -Leu were loaded, and L-Leu-
L-Lys(Z)-L-Pro-0-CHz-PAM was synthesized. In addition, Z represents a benzyloxycarbonyl group.

Hereinafter, the same operation as in Example 1 was performed, and L-Leu-LL
65 mg of ys-L-Pro powder was obtained.

Implementation J/'J4 L-Leu-L-)1is-L-P
Synthesis of ro 0.5 mmol of Boc-
L-Pro-0C) 12-PAl'l resin and 2 mmol each of Boc-L-old S (TDS) and Boa-L
-Leu was loaded, and by anhydrous symmetry method using DCC, L
-Leu-L-old 5 (TDS)-L-Pro-0-C)
l,,-PAM was synthesized. Note that TDS represents a tosyl group.

Thereafter, the same operation as in Example 1 was performed to obtain 73+ng of L-Leu-1°His-L-Pro powder.

Table 1 °1 Amino acid sequence analysis data: Amino acid analysis values after hydrolysis for 24 hours at 110"C with 26N hydrochloric acid using Shimadzu protein sequencer-PSQ-1 system (by Hitachi 1.-8500 type amino acid analyzer) "3 Mass Analytical values 2 Conducted using JEOL JMX-Dχ303 (hypertensive effect) Animals were spontaneously hypertensive rats (SHR) after 10 weeks (Nippon Rat Co., Ltd., male, body weight 240-280 g, 1 group 4)
~6 animals) were used. The sample was dissolved in physiological saline, and 100+ng of the active ingredient was administered intraperitoneally. As a control, physiological saline was administered intraperitoneally. Blood pressure was measured using a non-invasive blood pressure monitor for rats and mice, TK-350 (manufactured by Unicom).
It was measured by the Ta11-Cuff method before and after administration.

The results are (systolic blood pressure value before administration - systolic blood pressure value after administration) ±S,
E, shown in Table 2.

Table 2 Significant difference from control group at P<0.05 Back cover isolated
Intravenous injection This tripeptide or its acid addition salt or base salt
The solution is dissolved in 0 to 100 times (volume/weight) sterile physiological saline and aseptically filtered through a filter (pore size 0.45 μm) to give an injection.

Tablets Hydroxypropyl cellulose 1 part Lactose 10.9 parts Potato starch 1 part Magnesium stearate 0.1 part Prepare 20 parts of a 60% ethanol aqueous solution containing 1 part of hydroxypropyl cellulose, and add the present tripeptide or its acid addition salt or After adding 7 parts of a base salt and 10.9 parts of lactose and thoroughly kneading, it was dried under reduced pressure, and 1 part of potato starch and 0.1 part of magnesium stearate were added to the obtained dried product and mixed. Make tablets using a tablet machine.

[Effects of the Invention] A novel tripeptide having a blood pressure lowering effect and a blood pressure lowering agent containing the same are provided.

Claims (1)

[Claims] 1. Formula L-(or D-)Leu-L-X-L-Pro (wherein X is Gly, Ala, Val, He, Thr, As
p, Glu, Lys, Orn, Cys, Met, Phe
, Tyr, Trp, His or Hyp), and X is Lys, Orn or Hi
Acid addition salts of the above tripeptides where s and X are Asp
or an alkali or alkaline earth metal salt, ammonium salt or organic base salt of the above tripeptide which is Glu. 2, Formula L-(or D-)Leu-L-X-L-Pro (wherein X is Gly, Ala, Val, He, Thr, As
p, Glu, Lys, Orn, Cys, Met, Phe
, Tyr, Trp, His or Hyp), an acid addition salt of the above tripeptide where X is Lys, Orn or His, or an alkali or alkali of the above tripeptide where X is Asp or Glu. A hypotensive agent containing an earth metal salt, ammonium salt, or organic base salt as an active ingredient.