JPH04202200A - Novel peptide, its production and use thereof - Google Patents

Abstract

NEW MATERIAL:A peptide with Arg-Lys-Ile-Lys-Val-Tyr-Leu skeleton. USE:An angiotensinase inhibitor, preventive or therapeutic agent for hypertension. PREPARATION:For example, raw albumen is diluted fivefold with distilled water and dissolved therein followed by adjustment to pH at 1.6 with 1N-HCl. Pepsin is then added to the system to make a reaction stationarily at 37 deg.C for 3hr followed by boiling at 100 deg.C for 10min to terminate the reaction. The resulting reaction liquor is centrifuged at 10000rpm for 5min to effect concentration and then purified by high performance liquid chromatography. The purified peptide thus obtained is then put to amino acid sequence analysis by the application of automatic Edman degradation technique using a vapor phase protein sequencer, thus obtaining the objective peptide of formula H-Arg-Lys-Ile-Lys- Val-Tyr-Leu-OH having the following characteristics: (1) the Rf value for thin- layer chromatography: 0.45; (2) melting point: 300 deg.C; and (3) specific rotatory power [alpha] (c=0.5, water): -47.15.

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.

Arg-Lys-Ile-Lys-Val-Tyr-L
eu [Prior Art] Angiotensin converting enzyme exists mainly in the lungs, vascular endothelial cells, and renal proximal tubules, and angiotensin converting enzyme exists mainly in the lungs, vascular endothelial cells, and renal proximal tubules. (A
sp -Arg -Val -Tyr -Ile -H
is -Pro-Phe -His -Leu), and a dipeptide (H
It is an enzyme that cleaves and liberates is8-LeuIo) to produce angiotensin (2), which has a strong pressor action.

This enzyme also has the effect of destroying and inactivating prazikinin, an in vivo antihypertensive substance, 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, and since then, research has been active in the synthesis of various angiotensin-converting enzyme inhibitors, and efforts are also being made to obtain them from natural products.

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

[Problems to be Solved by the Invention] However, angiotensin converting enzyme inhibitors found in natural products are extremely rare, and only teprotides (nonapeptides) obtained from Brazilian and Japanese snake venoms.

5Q2088+), etc., and the metabolite l583 of actinomycetes belonging to the genus Streptomyces (JP-A-58-177920
No. 2) is only known. Furthermore, as angiotensin-converting enzyme inhibitors obtained by enzymatically treating natural products, peptides obtained by decomposing milk casein with tryptocin are known (Japanese Unexamined Patent Application Publication No. 58-111).
No. 09425, No. 59-44323, No. 59-'4
-4324, 6136226, 61-3622
No. 7) The development of new inhibitors is desired.

[Means for Solving the Problem] In order to solve the problem, the present inventors have diligently searched for an angiotensin-converting enzyme inhibitor that is a natural substance and has few side effects. As a result, the present inventors have developed a composition in which protein, especially albumin, is hydrolyzed with a specific enzyme. The existence of a substance having angiotensin-converting enzyme inhibitory activity was found in the substance, and the substance was
They found that it is a peptide having s-I le-'LyS-Val-Tyr-Leu as its backbone, and completed the present invention.

Arg-Lys-Ile-Lys-Val of the present invention
The peptide with -Tyr-Leu as a skeleton is a novel peptide that has not been described in any literature, and is produced by hydrolyzing proteins such as albumin with pepsin. It is purified and used as required. Furthermore, it can also be produced by applying conventional methods for peptide synthesis.

Arg mentioned above is arginine, Lys is lysine, ■l
e means isoleucine, val means valine, Tyr means tyrosine, and Leu means leucine, and all of these amino acids are integral.

The peptides of the present invention can be obtained by hydrolyzing proteins with pepsin or by peptide synthesis. To hydrolyze a protein with pepsin, the treatment differs depending on the nature of the protein, but in the case of poorly soluble protein, mix the protein with hot water, homogenize with strong stirring, add a predetermined amount of pepsin, and heat at a temperature of 10 to 60 ml. °C, preferably 20-40 °C, P HO
'.

1 to 4.0, the reaction is left standing or stirring for 10 minutes to 3 days.

Albumin is useful as a protein. As albumin, any soluble protein widely distributed in the body fluids and tissues of animals and plants, such as ovalbumin, serum albumin, milk albumin, muscle albumin, etc., can be used, and ovalbumin is particularly useful. In addition to the peptide of the present invention, other peptides are present in the hydrolysis solution, but these may be used as a mixture for various purposes, or the peptide of the present invention may be isolated and used alone. There is no problem.

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 may be used alone, combined in any order, or applied repeatedly as necessary.

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 carboxyl 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 protecting groups for amino groups include benzyloxyalbonyl, t-butyloxycarbonyl, p-biphenylisopropyroxycarbonyl, and 9-fluorenylmethyloxycarbonyl. Examples of protective groups for carboxyl groups include groups that can form alkyl esters, benzyl esters, etc.
In the solid phase method, the C-terminal carboxyl group is bonded to a carrier such as chloromethyl resin, oxymethyl resin, or P-alkoxybenzyl alcohol resin.

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, but 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, but oral administration is preferred. The dosage of the peptide of the present invention varies depending on the type of compound, administration method, patient's symptoms, age, etc., but is usually 0.001 to 1000 mg, preferably 0.000 mg per dose.
0l-10B 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, ncrodextrin, starch,
Sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose carunium, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, Polyvinyl alcohol, light silicic anhydride, magnesium stearate, talc, tragacanth, bentonite, vegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogeladine, polysorbate, macrogol, vegetable oil, wax , liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water and the like.

Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These formulations are prepared according to conventional methods. In addition, in the case of a liquid preparation, it may be in the form of being 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, or a buffer or preservative may be added. good.

These preparations contain 0.01% or more of the peptide of the present invention;
Preferably, it can be contained in a proportion of 0.05 to 70%. These formulations may also contain other ingredients of therapeutic value.

[Effect] The peptide of the present invention is a novel peptide and has an excellent angiotensin converting enzyme inhibitory effect, exhibits a blood pressure lowering effect and a pradikinin inactivation suppressing effect, and is effective against essential hypertension,
Preventive and therapeutic agents for hypertension such as renal hypertension and adrenal hypertension, diagnostic agents for these diseases and antihypertensive agents used in various pathological conditions, increased threshold for angina pectoris attacks, decreased myocardial infarction, and in congestive heart failure. It is useful as an agent for improving pathological conditions.

[What to do] Next, the present invention will be explained in more detail by giving examples.

After diluting and dissolving raw egg white 5 times with distilled water, dilute it with 1N-HCl.
Add 0.2 mg of pepsin/++++2 (manufactured by Sigma) to the solution (20 mW/m (contains protein of !) adjusted to pH 1.6 with
The reaction was stopped by boiling at 0°C for 10 minutes. This reaction solution was centrifuged at LOOOORPM for 5 minutes, concentrated, and purified by high performance liquid chromatography (ODS-, PH-, and CN-column) to obtain a peptide.

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

H-Arg-Lys-Ile-Lys-Val-Tyr
-L eu-OH The physical properties of the peptide are as follows.

TLC [n-butanol:acetic acid:pyridine 8 water-15°3:10:12] (silica gel plate, ninhydrin coloring) Rf: 0.
45 110-m, p: 300℃ Elemental analysis C44H78N + 209・0.5H,O
CHN Calculated value 56.94 8.58 18.11 Measured value 5
6,79 8.53 18.10 Specific optical rotation [α]2 and (
C=0.5 water), -47,15 [Synthesis of peptide] Commercially available Boc (butoxycarbonyl)-Leu-0-R
0.83 g of esin was fractionated into a reaction tank of Biosearch's peptide synthesizer SAM2, and synthesis was performed as follows.

The Boc 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 0.4M Boc-Tyr (CL-
Bz I) (dichlorobenzyl group) solution in dimethylformamide, 5 ml of 0, . After mixing with a methylene chloride solution of 4 M diisopropylcarbodiimide, the mixture was added to a reaction tank and reacted with stirring at room temperature for 2 hours.

The obtained resin was mixed with dimethylpolamide, methylene chloride,
Boc-Tyr (C12-B
z])-Pro resin was obtained. Subsequently, similar removal of Boc group and coupling of Boch amino acid were repeated to form Arg(TO8)(tosyl group)-Lys(CI-Z)(
chlorobenzyloxycarbonyl group)-I Ie-Ly
s(CI-z)-Val-Tyr(CI2-Bz 1)
-Leu-resin was obtained.

The resin was stirred in 20 ml of hydrogen fluoride containing 10% anisole at 0° C. for 1 hour to liberate the peptide from the resin. Hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 30% acetic acid and freeze-dried to obtain crude peptide. This was purified by reverse phase chromatography using an ODS column (Cosmosil 5 C+s), and H-Arg-Lys-I
1e-L'ys -Val-Tyr-Leu-OH(
A yield of 100 mf+) was obtained.

Analysis of the ice crystals using the same protein sequencer as above revealed that the ice crystals had the above composition.

The physical properties of the peptide are astounding.

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

Rf: 0.45 Elemental analysis C'HN as C4tH7sN120e・0.7H20 Calculated value 56.72 8.59 18.04 Measured value 5
6.82 8.51 18.09 Specific optical rotation [α]”;
(C=0.5 water), -47,15ρ H-Glu-A
rg-Lys-I Ie-Lys-Val-Tyr-L
eu-OH was synthesized.

The physical properties of the peptide are as follows.

TLC Rf: 0.42 m, p+300°C Elemental analysis C4eH85N +3012・0.7H20
CHN Calculated value 55.47 8.21 7.16 Measured value 5
5,40 8.12 17.06 Specific optical rotation [α]”;
(C=0.5 water), -38,74-13 - Examples 1 to 2 (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
ramacology 20.

1637 (1971)) by the following method.

Enzyme substrate; Bz (benzyl)-Gay-His-Leu
(A solution of 86 mg dissolved in 8 ml of water and 8 ml of phosphate buffer) Enzyme: Rabbit lung acetone powder (manufactured by Sigma)
(Supernatant obtained by centrifugation after pulverizing ly in 10 ml of 50 mM phosphate buffer) Mix 100 μA of the above enzyme substrate with 12 μA of Q1 enzyme solution and the peptide of the present invention at a predetermined concentration, and mix the whole with water for 25 minutes.
After setting the temperature to 0 μρ, the reaction was carried out at 37° C. for 30 minutes.

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

Add 1.5 ml of ethyl acetate to the reaction completed solution and
Mix at x for 15 seconds and centrifuge it.

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
2ta) was measured.

The inhibition rate is 1 OD 228 when reacting without inhibitor.
The activity was expressed as the concentration IC60 (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, measurements were also conducted on inhibitors other than those of the present invention, which are also shown in Table 1.

Table 1 (Note) Glu: Glutamic acid [Effect] The present invention provides a novel peptide useful as an angiotensin-converting enzyme inhibitor.

Patent applicant Nippon Gosei Kagaku Kogyo Co., Ltd. Procedural amendment 1, Indication of the case 1990 Patent Application No. 334315 2, Title of the invention New peptide, its manufacturing method and use 3, Person making the amendment Relationship with the case Patent application Address 9-6 Nozaki-cho, Kita-ku, Osaka (zip code 53o)
4. Patent application to be amended and detailed description of the invention in the specification Column 5. Contents of amendment (1) The patent application in question will be amended as shown in the attached sheet.

(2) "Planokinin" on page 2, line 15 of the specification is corrected to "bradykinin-1."

(3) "Tryptosin" on page 3, line 15 of the specification is corrected to "trypsin J."

(4) Delete "muscular albumin" from page 5, line 10 of the specification.

(5) Page 6, line 16 of the specification "Benzyloxyalbonyl" Correct it to "benzyloxycarbonyl."

(6) Page 9, line 15 of the specification “Bradykinin inactivation inhibitory effect” [Correction: bradykinin inactivation inhibitory effect.]

(7) rPro resin j on page 12, line 6 of the specification is replaced by rLe
I corrected it to ``u resin''.

(8) "20" on page 14, line 4 of the specification is corrected to "teL".

(9) Inhibitor rH of Example 2 in Table 1 on page 15 of the specification
-Qly-Arg-Lys-T 1e-Lys-VaI
-Tyr-Leu-OHJ rH-Qln-Arg-Lys-T Ie-Lys -
Corrected as Va I-Tyr-Leu-014J.

−3=

Claims (1)

[Claims] 1. Arg-Lys-Ile-Lys-Val-Tyr
-A novel peptide with a Leu skeleton. 2. Arg-Lys-Ile-Lys-Val-Tyr-L characterized by hydrolyzing protein with pepsin
Method for producing a novel peptide having an eu skeleton. 3. The production method according to claim 2, wherein albumin is used as the protein. 4. The manufacturing method according to claim 3, wherein ovalbumin is used as the albumin. 5. Arg-Lys-Ile-Lys-Val-Tyr
- An angiotensin converting enzyme inhibitor containing a peptide having a Leu skeleton as an active ingredient.