JPH0236195A - Novel peptide and angiotensin converting enzyme inhibitor - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula and salts thereof. USE:An angiotensin converting enzyme inhibitor useful for treating or preventing hypertension with ultrahigh safety. PREPARATION:For example, a peptide synthesizer is charged with Boc-L-Pro-O- CH2-phenylacetamidomethyl resin (Boc is tert-butyloxycarbonyl) and Boc-L-pro, Boc-L-Leu, Boc-L-His(Tos) (Tos is tosyl) and Boc-L-Val and the compound expressed by the formula is synthesized in the presence of dicyclohexylcarbodiimide by an anhydrous symmetric method.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel oligopeptide and an angiotensin-converting enzyme inhibitor containing the same as an active ingredient, and is expected to be useful in the treatment or prevention of hypertension.

[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 (As
p-Arg-Val-Tyr-11e-[1is-Pr
o-Phe-His-Leu) and angiotensin II (Asp-8rg-Val
-Tyr-11e-His-Pro-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.

In addition, the enzyme kallikrein present in plasma breaks down a protein called kininogen and produces bradykinin, which dilates blood vessels and lowers blood pressure.
It is decomposed and inactivated by the action of Thus, ACE generates a pressor peptide (angiotensin) on the one hand, and an antihypertensive peptide (angiotensin) on the other hand.
bradykinin), which results 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).
Many synthetic substances are known, such as (methyl-3-mercaptopropanoyl-proline), and among these, captopril is already in practical use as an oral antihypertensive agent. Furthermore, in recent years, ACE inhibitors have been found in microorganisms and various foods, and their practical use as antihypertensive agents is being considered.

[Problem to be solved by the invention]

New and useful antihypertensive agents and, therefore, angiotensin-converting enzyme inhibitors are constantly being sought.

The present invention provides novel oligopeptides and acid addition salts thereof that have excellent angiotensin converting enzyme inhibitory effects and antihypertensive effects, and extremely safe angiotensin converting enzyme inhibitors containing such oligopeptides or acid addition salts as active ingredients. The purpose is to provide.

[Means to solve the problem]

We have discovered that many angiotensin-converting enzyme inhibitory peptides contain Ala-Pr at their carboxy terminus.

Or, focusing on having a Pro-Pro structure, especially P
Considering that ro-Pro is not degraded by angiotensin converting enzyme and is advantageous in terms of sustainability, Pro-Pr
As a result of chemically synthesizing various peptides having o, the following peptide was found to inhibit angiotensin converting enzyme. That is, the present invention provides L-Val-L-His- which is a novel angiotensin converting enzyme inhibiting peptide.
L-Leu-L-Pro-L-Pro and its acid addition salt, and L-Val-L-His-L-Leu-L-Pr
The present invention relates to an angiotensin converting enzyme inhibitor containing o-L-Pro or an acid addition salt thereof as an active ingredient. Acid addition salts here mean pharmaceutically acceptable acid (inorganic and organic) addition salts, such as hydrochlorides, hydrobromides, sulfates, nitrates, acetates, benzoates, maleates, fumarates. acid salt,
Includes succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glutamate, and the like.

L-Val-L-His-L-Leu-L-P of the present invention
ro-L-Pro is mainly produced by a method of stepwise introduction of amino acids using an organic chemical synthesis method. It can also be produced by a peptide synthesis method using the reverse reaction of a hydrolase, a genetic engineering method, a natural protein enzymatic hydrolysis method, and the like.

There are two types of organic chemical synthesis methods: liquid phase method and solid phase method. xylcarbodiimide) and HOBt (1-hydroxybenzotriazole) for one day and night to bond sequentially 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 Biosystems, etc., and the resin and various protective groups are cleaved with hydrogen fluoride or the like. Obtain the desired oligopeptide.

Acid addition salts of the present peptide can be produced by conventional methods.

This peptide and its acid addition salt have an ACE inhibitory effect,
It is expected to be effective in treating and preventing hypertension in mammals including humans.

The peptides and their acid addition salts are used as such or in pharmaceutical compositions, usually with at least one pharmaceutical auxiliary.

The present peptide and its acid addition 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.

The formulation as an injection usually includes a sterile aqueous solution. Preparations of the above form can also be used as buffers, pH regulators (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), and preservatives (methyl parahydroxybenzoate, propyl p-hydroxybenzoate, etc.). It may contain other pharmaceutical adjuvants outside of the ice field, such as. The formulation is filtered through a bacteria-retaining filter, incorporating a disinfectant into the composition,
The composition can be sterilized by irradiation or heating.

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, polyvinylpyrrolidone, hydroxypropylcellulose, 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. Tablets can be coated by conventional methods.

Oral liquid preparations can be made into aqueous solutions or dry products. Such oral solutions may contain conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, etc.). Book AC
The amount of the present peptide or its acid addition salt in the E inhibitor can be varied, but is usually 5 to 100χ (w/w).
, especially 10 to 60χ(-8). The dosage of this ACE inhibitor is 10 to 200+++g/as the active ingredient.
kg/day is appropriate. The acute toxicity of this peptide is LD. (ICR mouse, oral administration) > 3g
/kg.

In addition, this peptide has the advantage that it does not have any adverse effects on the living body even when ingested in large quantities, so it can be used as it is, with various nutrients added, or incorporated into food and drinks to lower blood pressure and prevent hypertension. Functional food with
May be eaten as a health food. 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. Such functional foods,
The content and dosage of the active ingredient in the present ACE inhibitor as a health food may be the same as the content and dosage in the above-mentioned pharmaceutical products.

〔Example〕

Next, the present invention will be explained by examples.

7 L-Val-L-His-L-Leu-L-Pr
Synthesis of o-L-Pro and ACE inhibitory activity a) L-Val-L-1 (is-L-Leu-L-P
Synthesis of ro-L-Pro 0.5 mmol of Boc-L-Pro-0-CH, PAM resin, and 2 mmol each of Boc-L-Pro were added to an Applied Hiosystems peptide synthesizer (Model 430A). , Boc-L-L
eu, Boc-L-His(Tos), Boa-L
-Val was loaded and L
-Val-L-11is(Tos)L-Leu-L-P
ro-L-Pro-OCH2PAM was synthesized. In addition,
Tos represents a tosyl group. Next, the above synthetic peptide resin was introduced into a hydrogen fluoride treatment apparatus manufactured by Peptide Research Institute, and after adding 1.5-m of anisole, 10 m2 of liquid hydrogen fluoride was introduced. After reaction at -20°C for 30 minutes and O'C for 30 minutes, hydrogen fluoride was removed under reduced pressure, and the peptide was dissolved in anhydrous ether.
After washing three times alternately with chloroform, the peptide was dissolved in 60% of 2N acetic acid and lyophilized.

By this method, L-Val-L-1 (is-L-Leu
-L-Pro-L-Pr.

130 mg of white powder was obtained.

This peptide was analyzed using HPLC (high performance liquid chromatography)
A single peak was observed at the elution time of 1.8 minutes. H
PLC elution conditions are shown below.

Column: Waters Radial Back Cartridge C8 Eluent: Phosphate buffer (10mMK) lzPo, 50
mMNazS04゜pH2,5) Niacetonitrile-
4:6 Flow rate: 21 rdl/win Detection: Ultraviolet absorption at 210 nIm Next, various analytical values of this peptide are shown.

Amino acid analysis: Val (1,00), 11is (0,
97), Leu(1,18).

Pro(1,95) () is the molar ratio with Vat as 1.

Analysis was performed after hydrolysis in 6N hydrochloric acid at 110"C for 24 hours.

Specific illuminance: [α) = -155° (C・0.34.
HzO) Mass spectrometry: 562 (M+1)” Measured with JEOL HXIIO, FAB-MS b) ACE
Measurement of inhibitory activity The ACE inhibitory activity of the present peptide obtained as described above was measured as follows. That is, first, 5 g of Rabbit Lang acetone powder was dissolved in 50-O,LM sodium borate buffer pH 8.3), and 40.0 OOG
, centrifuged for 40 minutes, and the supernatant was further purified with hydroxyapatite to give 1 unit/mg p
An angiotensin converting enzyme solution of rotein was obtained.

Aqueous solutions of this peptide at various concentrations were placed in test tubes.
．． 03d, and 0.25 mff1 of hipryl histidyl leucine (final concentration 5111 M,
(contains 300mM NaCl), then added 0.1m of the above angiotensin converting enzyme solution, and incubated at 37°C for 3
The reaction was allowed to proceed for 0 minutes. After that, 0.25 d of IN hydrochloric acid was added to stop the reaction, 1.5 d of ethyl acetate was added, and the absorption value at 228 nm of hyperric acid extracted in ethyl acetate was measured. It was made active. Note that under these conditions, the absorption value of 228nn+ without the present peptide was 0.35.

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

A: 228 nm absorption value without inhibitor B: 228 nm absorption value with inhibitor addition and inhibition rate 50%
The concentration of this peptide when . When I calculated the value, it was 18
It was μH.

Setsuko Bean-1 Intravenous Injection L-Val-L-)1is-L-Leu-L-Pro-
Dissolve L-Pro in 20 to 100 times (volume/weight) sterile physiological saline and aseptically filter it (pore size 0.45μ).
The filtrate filtered in step m) is used as an injection.

Fruit JLLu-Tablet Shi-Val-L-Hts-L-Leu-L-Pro-L
-Pro 7 parts hydroxypropyl cellulose 1 part lactose
10.9 parts potato starch
1 part Magnesium stearate 0.1 part Hydroxypropyl cellulose I W+ Contains 60%
Prepare 20 parts of an aqueous ethanol solution, add 7 parts of the present peptide and 10.9 parts of lactose, thoroughly knead, and then dry under reduced pressure. 1 part of potato starch and 0.1 part of magnesium stearate are added to the dried product obtained. 1. Add 1 part to the mixture, mix, and make tablets using a tablet machine.

〔Effect of the invention〕

This novel peptide has ACE inhibitory activity and is extremely safe because it consists only of natural amino acids.

Claims (1)

[Claims] 1. L-Val-L-His-L-Leu-L-Pro
-L-Pro and its acid addition salt. 2. L-Val-L-His-L-Leu-L-Pro
- An angiotensin converting enzyme inhibitor containing L-Pro or an acid addition salt thereof as an active ingredient.