JPH04139196A - New peptide, its production and use thereof - Google Patents

Abstract

NEW MATERIAL:A peptide having a skeleton of formula Trp-His-His-Trh. USE:A low toxic angiotensinase inhibitor useful as a preventive and therapeutic agent of hypertension, an antihypertensive agent, a therapeutic agent of congestive cardiac failure, etc., with high safety. PREPARATION:A protein (preferably fish protein such as dried bonito or actin) is initially mixed with (hot) water and the resultant mixture is homogenized by a strong stirring force. Thermolysin is then added thereto followed by hydrolysis at 10-85 deg.C for 0.1-48hr. The resultant hydrolyzed solution is concentrated by centrifugal separation and subsequently purified by high performance liquid chromatography, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention provides a novel peptide having the following structure, and relates to a peptide useful as an angiotensin-converting enzyme inhibitor and the like.

'frp-His-His-Thr [Prior art] Angiotenone-converting enzyme exists mainly in the lungs, vascular endothelial cells, and renal proximal tubules.
p-^rg -Val -Tyr -11e -His
-Pro -Phe -His -Leu) This enzyme also has the effect of destroying and inactivating bradykinin, an antihypertensive substance in the body, and is strongly involved in the pressor system.

It has been conventionally believed that inhibiting the activity of angiotensin converting enzyme lowers blood pressure and is clinically effective in preventing and treating hypertension.

Recently, captogril, a proline derivative, was synthesized and its antihypertensive activity was confirmed. Since then, research has been active in the synthesis of various angiotenone-converting enzyme inhibitors, and efforts are also being made to obtain them from natural products.

This is because natural angiotenonono-converting enzyme inhibitors can be obtained from foods or food raw materials and are therefore expected to be low-toxic and highly safe antihypertensive agents.

[Problem to be solved by the invention: - However, angiotenone converting enzyme inhibitors found in natural products are extremely rare, and only teprotides (nonapeptide 5
Q2088+), etc., and the metabolite l583 of actinomycetes belonging to the genus Streptomyces (Japanese Unexamined Patent Publication No. 177920/1983) are only known. In addition, as an anchiotenone-converting enzyme inhibitor obtained by enzymatically treating natural products, peptides obtained by decomposing milk casein with tryptonone are known (Japanese Patent Application Laid-open No. 109-1989).
No. 425, No. 59-44323, No. 59-44324
The development of new inhibitors is desired.

[Means for solving problems].

In order to solve this problem, the present inventors conducted an intensive search for angiotensin-converting enzyme inhibitors (natural substances with few side effects), and found that angiotensin-converting enzyme inhibitors were found to contain angiotensin-converting enzymes in compositions prepared by hydrolyzing proteins, especially fish meat and bonito flakes, with specific enzymes. The existence of a substance having non-converting enzyme inhibitory activity was identified, and the substance was found to be Tr.
They discovered that it is a peptide having p-His-His-Thr as its backbone, and completed the present invention.

The peptide having Trp-His-His-Thr as a skeleton of the present invention is a novel peptide that has not been described in any literature, and is produced by hydrolyzing a protein such as Canoobun with thermolysin, and in practical use, the composition can be used as is. Alternatively, it can be purified and used if necessary. Furthermore, it can also be produced by applying conventional methods for peptide synthesis.

As mentioned above, Trp means tryptophan, His means histidine, and Thr means threonine, and all of these amino acids are integral.

The peptide of the present invention can be obtained by hydrolyzing a protein with thermolysin or by a peptide synthesis method.

To hydrolyze a protein with thermolysin, the treatment will differ depending on the nature of the protein, but in the case of poorly soluble proteins, mix the protein with hot water, homogenize with strong stirring, add a predetermined amount of samolynon, and heat at a temperature of 10 to 85. 0.1-48 at around ℃
Perform a time reaction.

Any protein derived from animals or microorganisms may be used, and particularly useful proteins include fish such as bonito flakes and sardines, and actin. In addition to the peptide of the present invention, the hydrolyzate contains Ike's peptide (these may be used as a mixture for various purposes, and
There is no problem even if only the peptide of the present invention is isolated and used.

In the case of isolation, the hydrolyzed solution is filtered by a known operation such as centrifugation. Then after applying extraction, concentration, drying, etc.
or even without it, differences in adsorption affinity for various adsorbents, differences in solubility or solubility for various solvents, differences in distribution between two immiscible liquid phases,
It is preferable to isolate the target substance by applying means that utilize differences in elution rate based on molecular size, precipitability from a solution, or difference in precipitation rate. These methods can be used alone, combined in any order, or repeatedly applied as needed.

The peptide of the present invention is produced by a method commonly used for peptide synthesis, that is, a liquid phase method or a solid phase method, using a raw material having a reactive carfoquinol group corresponding to one of two types of fragments that are bisected at any position of the peptide bond; If a raw material having a reactive amino group corresponding to the other fragment is condensed using a carbodiimide method, an active ester method, etc., and the resulting condensate has a protecting group, it can also be produced by removing the protecting group. obtain.

Functional groups that should not participate in the reaction in this reaction step are protected by protecting groups. Examples of the protecting group for the amino group include penzyl-oquinocarponyl, t-butyloquino-sulfonyl, p-hyphenylisobrobyloxycarbonyl, 9-fluorenylmethyloquinecarbonyl, and the like. Examples of protective groups for carfoquinol groups include groups that can form alkyl esters, benzyl esters, etc. In the case of the solid phase method, the carhoquinol group at the C-terminus is protected by chloromethyl resin, oquinemethyl resin, P-alcokynebenzyl alcohol resin. It is bound to a carrier such as

The condensation reaction is carried out in the presence of a condensing agent such as a carbodiimide or using an N-protected amino acid active ester or peptide active ester.

After the condensation reaction is completed, the protecting group is removed, or in the case of a solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved.

Furthermore, the peptides of the invention are purified according to conventional methods. Examples include ion exchange chromatography, reversed phase liquid chromatography, and affinity chromatography.

The administration route of the peptide used in the present invention may be oral administration, parenteral administration, or intrarectal administration, and oral administration is preferred. The dosage of the peptide of the present invention varies depending on the type of compound, administration method, symptoms and age of the person, and is usually 0.001 to 1000 m9 per dose, preferably 0.
01-10 mg 1-3 times per day. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing it with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxyl methylcellulose, hydroxypropyl starch, calhoquinomethylcellulose carunium, ion Exchange resin, methylcellulose, gelatin, gum arabic, hydroquinopropylcellulose, hydroquinopropylmethylcellulose, polyhinylpyrrolidone, polyvinyl alcohol, light silicic anhydride, magnenium stearate, talc, tragacanth,
Hetonite, hea gum, titanium oxide, solhitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax,
Examples include liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactants, propylene glycol, water, and the like.

Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, half-open tablets, ointments, creams, gels, patches, inhalants, injections, and the like. These formulations are prepared according to conventional methods. In the case of a liquid preparation, it may be dissolved or suspended in water or other suitable medium before use. Furthermore, tablets and granules may be coated by a well-known method. In the case of injections, the peptide of the present invention is prepared by dissolving it in water, but if necessary, it may be dissolved in physiological saline or glucose solution, and a buffer or preservative may be added. .

These preparations contain the peptide of the present invention at a concentration of 0.015 or more;
Preferably, it can be contained in a proportion of 0.05 to 70%. These formulations may also contain other ingredients of therapeutic value.

[Effects] The peptide of the present invention is a novel peptide and has an excellent angiotensin converting enzyme inhibitory effect, and has antihypertensive and antihypertensive effects.
Shows planokinin inactivation inhibitory effect, preventive and therapeutic agent for hypertension such as essential hypertension, renal hypertension, and adrenal hypertension, diagnostic agent for these diseases, antihypertensive agent used in various pathological conditions, and angina pectoris attack. It is useful as an agent for increasing the threshold of heart disease, reducing myocardial infarction, and improving the pathology of congestive heart failure.

"Example code" Next, the present invention will be explained in more detail by giving examples.

[Production of peptide]

(A) Add 40 ml of water to 5 g of bonito flakes, homogenize thoroughly, add 20 mg of thermolysin, and keep at 37°C, pH 7.
After the hydrolysis reaction was carried out for 3 hours at 100'C, the peptide was boiled for 10 minutes at 100'C, concentrated by centrifugation after cooling, and purified by high performance liquid chromatography (ODS-, Ph- and CN-columns). Obtained.

The amino acid sequence of the ice crystals was analyzed using an automated Edman degradation method using a gas-phase protein sequencer (Model 477A manufactured by Applied Bion Stems), and the following structure was obtained.

H-11e-Trp-His-His-Thr-OH The physical properties of the peptide are astounding.

TLCIn-Butanol Acetic acid:Pyrinone Water-153
: 10 : 12 ((Silica gel plate, ninhydrin coloring) Rf: 0.
379 m, p: 132°C Elemental analysis C3zH441N+. ot' 0.5HtO
CHN Calculated value 56,48 6.46 19.96 Measured value 5
6,42 6.50 19 99 Specific optical rotation [α 5:
(C=0.5 water) knee 1.3° [Synthesis of peptide] Commercially available Boc (butoxycarbonyl) -Thr (Bz
l) 0-ResinC hennol resin (substitution rate 0.64
tneq/y) E 0479 was fractionated into the reaction tank of Biosearch's peptide synthesizer SAM2, and synthesized as follows.

The Boa group was removed by reaction in methylene chloride containing 45% trifluoroacetic acid and 2.5% anisole for 25 minutes, followed by washing with methylene chloride, neutralization with methylene chloride containing 10% diisopropylethylamine, and methylene chloride. Washing was performed with

This and 5 ml of 0.4 M Boc-His (Tos
) A dimethylformamide solution of (sil group)) and a methylene chloride solution of 0.4M noisopropylcarpodiimide of 51 were mixed, and then added to a reaction tank and reacted with stirring at room temperature for 2 hours.

The obtained resin was mixed with trimethylformamide, methylene chloride,
Boc-11is (Tos
) -Thr (Bzl) resin was obtained.

Subsequently, similar removal of Boa group and coupling of Boa and amino acid were repeated to obtain 1le-Trp-His (To
s) -Hls(Tos)Tbr(Bzl) resin was obtained.

The resin was stirred at 0°C for 1 hour in hydrogen fluoride containing 10% anisole of 201 to liberate the peptide from the resin.
Ko. Hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 30% acetic acid and freeze-dried to obtain a crude peptide. This was purified by reverse phase chromatography using an ODS column (Cosmosil 5 C+s), and H-I Ie-Trp-
His-HjsThr-OH (yield 80+n) was obtained.

Analysis of the main island using the same protein analyzer as above revealed that it had the above composition.

The physical properties of the peptide are as follows.

Incidentally, all the solvents for TLC are the same as above.

Rf: 0°377 m, p: 132°C Elemental analysis Cs5H44N IoO7' 0, 7 H
CHN as tO Calculated value 56.19 6.49 19.86 Measured value 5
6,10 6.44 19.81 Specific optical rotation [α]”,
(C:=0.5 water) knee 1.3° Also, the following peptide was synthesized according to the above synthesis example except that the reaction agent was changed depending on the amino acid species of the target peptide.

H-Trp-Hi 5-Hi 5-Thr-Phe-O
HLC Rf: 0.278 m, p: 163°C Elemental analysis CHN as c3eH,tN,,O,r-0,6HzO Calculated value 58.62 5.90 18.99 Measured value 5
8,58 5.82 18.92 Specific optical rotation [α] 15:
(C=0.5 water) ■, 6°H-Trp-His-
His-Thr-OHLC Rf: 0 278 m, p: 163°C Elemental analysis CHN as Cx7H33N soe・0.5HtO Calculated value 55,09 5.82 21.42 Measured value 5
5,16 5.77 21.41 Specific optical rotation [α]″5;
(C=0.5 water): 3.77°H-11e-Tr
p-His-His-Thr-PheLC Rf: 0.508 m, p・169℃ Elemental analysis C4= Hs 3N 11Oa・0.4 H
CHN as to Calculated value 59.55 6.40 18.19 Measured value 5
9,52 6.43 18.14 Specific optical rotation [α]”;
(C=0.5 water)=07゜Examples 1 to 4 (Measurement of angiotensin converting enzyme inhibitory activity) The measurement of angiotensin converting enzyme inhibitory activity was carried out by Cheung and C.
ushman's method (Biochemical Pha
raIllacology201637 (1971))
This was done in the following manner.

Enzyme substrate; Bz (benzyl)-Gly-His-Le
u (a solution of 86m9 dissolved in 8ils of water and 8fflls of phosphate buffer) Enzyme: Rabbit lung acetone powder (manufactured by Sigma)
(Supernatant after pulverizing 1 g in 10 al of 50 mM phosphate buffer and centrifuging) 100 μQ of the above enzyme substrate, 12 μQ of the enzyme solution and the peptide of the present invention at a predetermined concentration were mixed, and the whole was mixed with water for 25 minutes.
After setting the temperature to 0 μe, the reaction was carried out at 37°C for 30 minutes.

The reaction was terminated using 250 μQ of IN-HCl.

Add ethyl acetate (51) to the reaction completed solution and V ortex
The mixture was stirred for 15 seconds and then centrifuged.

1. from the ethyl acetate layer. 0ml was taken out, ethyl acetate was distilled off, 1ml of distilled water was added thereto to dissolve the residue, and the value of ultraviolet absorption at 228 nm (OD
2ts) was measured.

Inhibition rate is OD when reacting without inhibitor! t11 to 1
The activity was expressed as the concentration IC5o (μM) of the inhibitor (peptide of the present invention) when the inhibition rate was 50%.

The results are shown in Table 1.

Furthermore, as a reference example, we also measured inhibitors other than those of the present invention, and the results are also shown in Table 1.

Table (Note) Phe; Phenylalanine; Isoleucine [Effect] The present invention provides a novel peptide useful as an angiotensin-converting enzyme inhibitor.

Claims (1)

[Claims] 1. A novel peptide having a Trp-His-His-Thr skeleton. 2. A method for producing a novel peptide having a Trp-His-His-Thr skeleton, which comprises hydrolyzing a protein with thermolysin. 3. The production method according to claim 2, wherein actin is used as the protein. 4. The manufacturing method according to claim 2, wherein fish meat is used as the protein. 5. The production method according to claim 2, wherein bonito flakes are used as the protein. 6. An angiotensin converting enzyme inhibitor containing a peptide having a Trp-His-His-Thr skeleton as an active ingredient.