JPH04120093A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:Peptides having amino acid sequence structures of formulas. I, II, III, etc., and having angiotensin transferase-inhibiting activities. USE:An agent for diagnosing, preventing and treating hypertension, and a vasodepressor. PREPARATION:The angiotensin transferase-inhibiting activity-having peptides of formulas I, II and III are prepared e.g. as follows; treating swine blood plasma with a semi-permeable membrane such as an ultra-filtration membrane, adding the passed components to a strong acid cation exchange resin column, washing the column with de-ionized water, eluting the adsorbed substances with a 2N ammonium hydroxide solution, concentrating the eluted solution under vacuum to remove ammonia, gel-filtering the concentrated solution, collecting fractions having angiotensin transferase-inhibiting activity, further fractionating the collected fractions by an ion exchange gel filtration method, collecting a highly physiologically active fraction and subsequently applying a reversed phase high performance liquid chromatography treatment to the fraction for purification.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to peptides having amino acid sequence structures represented by the following formulas (1), (2), and (3).

(1) In-As in Pro-11e-Gln-His-
p(2)^1a-Val-Glu-Gly-Pro-L
ys-Phe(3)Thr-Pro-Asp-Glu-
Thr-Tyr-LysGlu (Background Art) Pr.

It is well known that the Lys renin-angiotensin system plays an important role in regulating water, snowmelt, and blood in living organisms. In addition, angiotensin converting enzyme (hereinafter abbreviated as ACE) exists in the nin-angiotensin system, and angiotensin I is converted to ACE: thus angiotensin ■; Angiotensin U is a powerful pressor substance that acts not only on the blood vessels and adrenal cortex, but also on the central and peripheral nerves, promoting an increase in blood pressure. Furthermore, ACE has the effect of decomposing and inactivating bradykinin, which is a hypotensive substance in the body, and is involved in the pressor system. I want to, ACE
Blood pressure can be lowered by inhibiting the activity of
ACE activity inhibitors are considered to be clinically effective in preventing and treating hypertension. For this purpose, the proline derivative captopril was synthesized and its antihypertensive effect was confirmed.Since then, research has been actively conducted on the synthesis of various ACE inhibitors based on the structural research of captopril.Recently, enalapril maleate and alacebryl Chemical substances such as these are being used one after another in clinical settings. Currently, ACE
Inhibitors are widely used for both essential hypertension and symptomatic hypertension, and from mild to severe, and have been added as the first-line drug of choice for hypertension. , many good points are mentioned.

Meanwhile, research is progressing as a new application of ACE inhibitors as a heart failure treatment. The mechanism of action of ACE inhibitors is thought to be to suppress the secretion of aldosterone and vasobresson by suppressing the production of angiotensin, and to promote the excretion of chloride and water by reducing renal artery contraction. Furthermore, it is thought that by suppressing the inactive level of the kallikrein-kinin system and inactivating the prostaglandin system, it further promotes peripheral vasodilation and sodium and water excretion.ACE inhibitors contribute to the vicious cycle of heart failure. It is expected that it will be used as a therapeutic drug for the purpose of cutting off.

By the way, in addition to the above-mentioned synthetic products, ACE inhibitors include natural products or substances derived from natural products such as bradykinin enhancer derived from snake venom (C-terminus is Pro) [S, H, Fe
rreia, et al, :Biochemistr
y, 9.3583 (+970)], 6 types of peptides derived from gelatin collagenase digests (all have Ala-Hyp at the C-terminus) [G, Oshima, et a
l: Biochim, Blophys, Act
a.

556.128 (1979)], a peptide derived from a tryptic digest of bovine casein (the C-terminus is Gly-Lys
) [S.

Maruyama, et al.: Agri
c, Biol, Chem, 46.1393 (
+982):] etc. are known. However, all of these substances have been found to be effective when administered intravenously, but their pharmacological effects when administered orally are unknown, and it has been a long time since their discovery.

There are no reports that the development as a drug is progressing yet.

(Disclosure of the Invention) The present inventor searched for substances having pharmacological effects from pig plasma and found that three new peptides have a strong angiotensin converting enzyme inhibitory effect. Then, they conducted extensive research to put these three types of peptides to practical use as medicines.

As a result, these three types of peptides have a blood pressure lowering effect,
We found promise as a natural product-derived angiotensin-converting enzyme inhibitor.

The present invention is based on such knowledge.

Below, one aspect of the present invention will be explained in detail.

The peptide according to the present invention has the linear formula (+), (2) and (3) (+) Pro-11e-Gln-His-Gln-
Asp(2) Ala-Val-Glu-Gly-Pr
o-Lys-Phe(3) Thr-Pro-Asp
-Glu-Thr-Tyr-Lys-Pr.

Lys-Glu (Each symbol in the formula represents each amino acid Mi in the amino acid sequence in peptide chemistry.) It is a novel peptide having the L-amino acid configuration +l+, and its properties at room temperature are as follows: It is a white powder.

Examples of methods for producing the above three types of peptides include a method of chemical synthesis and a method of separation and purification from pig serum.

When the novel peptide of the present invention is chemically synthesized, it can be carried out by a conventional synthesis method such as a liquid phase method or an in-phase method. Preferably, the C of the peptide is transferred to a polymeric in-phase support by an in-phase method. It is preferable to sequentially synthesize L-configured amino acids corresponding to the amino acid residue from the terminal side (carboquine terminal side) by peptide bonding.

And the synthetic peptide thus obtained is trifluoromethanesulfonic acid.

After cleavage from the polymeric in-phase support using hydrogen fluoride etc., the protecting group of the amino acid side chain was removed and subjected to high performance liquid chromatography (hereinafter referred to as H
It is abbreviated as PLC. ) etc. can be purified by a conventional method.

The novel peptide according to the present invention can be obtained by minute purification from pig blood 5fi. For example, porcine plasma is sufficiently dialyzed in a suitable solvent (e.g., water, neutral buffer such as Tris-HCl buffer, phosphate!311 buffer, etc.) using a semipermeable membrane such as cellophane, It passed through that semi-permeable membrane! ! A solution containing the components in the liquid is applied to a strongly acidic cation exchange resin (for example, Dowex 50-'' manufactured by Dow Chemical Co.), and the adsorbed and eluted fraction is extracted from the fraction containing the component having ACE (angiotensin converting enzyme) inhibitory activity. The obtained fraction with ACE inhibitory activity was filtered through gel filtration (e.g. Co. J.

5eohadex G-25 manufactured by Pharmacia, etc.)
The obtained ACE inhibitory activity fraction is thus fractionated by 1 ion exchange gel filtration (e.g., 5P-5ephadex C-25 manufactured by Pharmacia), and the obtained ACE inhibitory activity fraction is Fractions were further subjected to reverse phase HPL
This can be done by fractionating using C.

These three new peptides do not induce antibody production or cause anaphylactic shock even after repeated intravenous administration. In addition, the amino II ffL in the structure of these three peptides consists of only one amino acid, and the peptides 20 and 20 are gradually degraded by proteases in the body after administration, so their toxicity is extremely low. .

Safety is extremely high.

Three types of novel peptides according to the present invention; 41 Can be prepared into various types of drugs such as injections, tablets, capsules, granules, and powders using commonly used excipients and other additives. can. Administration method: Usually, the drug is administered by injection or oral administration to salivary jawed animals with ACE, such as humans, dogs, rats, etc.
~lomg), which can be administered 1 to 4 times a day. Dose: Can be adjusted as appropriate depending on the route of administration.

The excipients, binders, and lubricants used in the above formulations are not particularly limited; any of those commonly used for injections, powders, granules, tablets or capsules can be used. be.

Tablets, capsules! Examples of additives used in granules and powders include the following. Excipients include I! such as crystalline cellulose. O.

Sugar alcohols such as mannitol, starches, anhydrous calcium phosphate, etc.; As binders, starches, hydroxypropyl methyl cellulose, etc.; As disintegrants, carboxymethyl cellulose and its calcium salts:
Examples of lubricants include stearic acid salts, talc, and wax necks. Moreover, a flavoring agent such as menthol, citric acid and its salts, and fragrance can be used as required.

Sterile composition for injection: Each peptide is dissolved or suspended in water for injection, raw smoked salt, sugar alcohol such as xylitol or mannitol, or glycol such as propylene glycol or polyethylene glycol using a conventional method. You can get it. At this time, buffers, preservatives, antioxidants, etc. may also be added as necessary. Furthermore, the peptide according to the present invention is in the form of a lyophilized product or a dry powder,
This can also be used by dissolving it in a conventional solubilizing agent, such as water or physiological saline, before use.

The three novel peptides according to the present invention have excellent angiotensin-converting enzyme inhibitory activity, and exhibit antihypertensive activity and bradykinin inactivation inhibitory activity. Therefore, cephalic hypertension, renal hypertension.

It is useful as a prophylactic and therapeutic agent for hypertension such as adrenal hypertension, a diagnostic agent for these diseases, and a hypotensive agent used in various diseases.

EXAMPLES The present invention will be further explained in detail by describing production examples and test examples as examples below.

! ! Example 1: Ultrafiltration membrane made of pig men (V gate 10 type, manufactured by Amicon, φ
76 nm) was passed through a strongly acidic cation exchange resin column (Dowex 50WX4 [H'1.50~room
esh.

φ4.5×15cm). The column was thoroughly washed with deionized water and then eluted using diammonium hydroxide rinses. Ammonia was removed by condensation under reduced pressure to obtain final liquid a. This concentrated solution was preblocked with deionized water on a 5ephadex G-25 column ('tedi
um, φ2.5X 150cm) and gel l! I went through the door. The results are shown in FIG.

The above gel filtration was repeated to collect a large amount of fractions with high ACE inhibitory activity, which were freeze-dried to obtain peptide powder. After dissolving this peptide powder in deionized water,
5P-5ephadex buffered with deionized water
C-25[1(”ko(φ 1.5X 47.2c+
+)L was loaded and chromatography was performed using a linear gradient method from deionized water to 3z sodium chloride solution. The results are shown in FIG.

The ACE inhibitory activity fractions of 9 fraction numbers 20 to 29 in the above chromatograph were collected and further dried to obtain purified peptide powder. After dissolving this peptide powder in deionized water,
HPLC was performed. The conditions were Nomura Chemical Co., Ltd. i! as a column. Develosil 005-5 (4,65
0.05 as the mobile phase.
% trifluoroacetic acid (hereinafter abbreviated as TF^).

) to 1 inch of 25% acetonitrile 10.0 ITFA
By the ear gradient method, flow rate: 1. om
Chromatography was performed at l/m+n and a detection wavelength of 220 nm to obtain a peptide having ACE inhibitory activity. The results are shown in Figure 3.

(Elution time: Peptide (1) 53.042 minutes.

Peptide (2): 76.775 min; Peptide (3): 97.183 min) The amino acid sequence of the peptide having ACE inhibitory activity thus obtained was determined using a 477A protein sequencer manufactured by Applied Biosystems. It was decided that

As a result, it was confirmed that each of the three peptides had a sequence consisting of L-form amino acid residues represented by the following linear formula.

Manufacturing example 2 This example is a production example using 1 synthesis method.

Synthesis method of Pro-1le-Gln-)1is-Gin-Asp.

The peptide was synthesized by a solid phase method using an automatic peptide synthesizer (430A, manufactured by Applied Pionstem).

As the solid phase carrier, a resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used.

First, according to the amino acid sequence of the peptide.

As usual, Asp on the C-terminal side was reacted with chloromethyl resin to obtain a peptide-bonded resin.

The amino acid used at this time was a t-8oc amino acid protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group.

Next, this peptide-bound resin was suspended in a mixed solution consisting of ethanedithiol and thioanisole.

After stirring at room temperature for 10 minutes, trifluoroacetic acid was added under water cooling, and the mixture was further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to this mixture, stirred at room temperature for 30 minutes, and then water-collecting ether was added to precipitate and separate the product, and the precipitate was washed several times with water-collecting ether. Dry under reduced pressure.

The thus obtained non-W synthetic peptide was dissolved in distilled water, and then purified by HPLC using a reverse phase column Cl8 (5μ). As mobile phase (A)O,1
% TFA-containing distilled water, (B) 0.1% TFA-containing acetonitrile solution, (A) chromatography was performed at a flow rate of 1.5 ml/min using a linear gradient method in which f was 98% → 78% in 20 minutes. went.

Detection using ultraviolet wavelength 215nmF, separate the elution fraction that showed the maximum absorption, and use it as iit! The desired synthetic peptide was obtained by drying.

As a result of mass spectrometry analysis of this synthetic peptide, it was confirmed that the peptide had the amino acid sequence and amino acid composition shown in the above formula.

The mass spectrum results are shown in Figure 4-2.

The other two peptides were also synthesized by reacting from the rLC terminal side using the same solid phase method as described above. Unpurified synthetic peptides were purified as shown below.

Ala-Val-Glu-Gly-Pro-Lys-P
Purification method for he H using reverse phase column C18 (5μ)
Purified by PLC. (A) 0.1%τ as mobile phase
Using distilled water containing FA and (B) acetonitrile solution containing 0.1% TFA, (A) solution was reduced to 90% in 20 minutes →
Chromatography was performed using a 60% linear gradient method at a flow rate of 1.51/win. Ultraviolet I! Detected at 3 wavelengths of 215 nm.

The elution fraction showing the maximum absorption was collected and dried to obtain the desired synthetic peptide.

As a result of mass spectrometry analysis of the second synthetic peptide, it was confirmed that the peptide had the amino acid sequence structure shown in the above formula in terms of amino acid sequence V and amino acid composition.
Ta.

The results of this mass spectrum are as shown in Figure 5-22.

Thr-Pro-Asp-Glu-Thr-Tyr-L
ys-Pro-L>5-Glu (IPLc) using reversed phase column C18 (5μ). As the mobile phase, (A) MV water containing 0.1% TFA, (B) acetonitrile solution containing 0.1% TFA was used, and (A) M was 98% → 7 in 20 minutes.
8% Linear3radient? Chromatography was performed at a flow rate of -L5 ml/1n. The eluted fraction showing the maximum absorption was detected using an ultraviolet meter at a wavelength of 215 nm, and the desired synthetic peptide was obtained by drying the fraction for 7 hours.

As a result of analyzing this synthetic peptide by mass spectrometry, it was confirmed that the amino acid sequence and amino acid composition were hebutides having an amino acid distribution 1j structure shown by @2 formula.

The mass spectrum results are shown in FIG.

The pharmacological effects of the three types of peptides of the present invention obtained by synthesis were confirmed by the tests shown below.

Test Example 1゜(Angiotensin Converting Enzyme Inhibitory Activity) ■, Experimental Materials (1) Angiotensin Converting Enzyme Seikatsu Serum Angiotensin (manufactured by Sigma) (2) Three types of peptides of the present invention obtained in Production Example 2 (3) ) Enzyme substrate hybridyl-histidyl-leucine■, enzyme assay method for inhibitory activity Enzyme assay for angiotensin-converting enzyme inhibitory activity measures the enzymatic activity of angiotensin-converting enzyme in the presence and absence of the analyte. The inhibitory activity IC5II of the sample was calculated from this and the determined inhibition rate.

That is, the measurement of angiotensin converting enzyme activity
A modified method of ARl et al. [Anal, Biochem, .
llJ, 36], (1978) as follows.

0.5M Tris-HCl buffer J containing sodium chloride containing the enzyme substrate, histidyl-leucine.
(p) 18.2) Dissolve the sample to a concentration of 10 mg/ml, place in a 250 μm reaction test tube, and add the sample to this.

Add angiotensin converting enzyme and water to final volume of 25
The ink was set to 0 μm and incubated at 37°C for 10 minutes.

The reaction was stopped by adding 15 μl of 1N sodium hydroxide solution, and after standing at room temperature for 30 minutes, 60+1'
Sodium monate buffer i (pH 7,2)]+n1
．． Add methyl cellosolve 11 containing 1% cyanuric acid chloride, and after 15 minutes change the wavelength to 3g2na.
Absorbance A at +i was measured.

From the measured value of absorbance in the reaction source that does not contain the analyte (the amount of hippuric acid liberated from the #W substrate due to elementary activity. Note that hippuric acid exhibits a yellow coloration due to the reaction with cyanuric acid chloride), Enzyme activity was calculated using the formula.

Enzyme activity (mLJ/ml) = 595X A (Note) 1 mU of enzyme activity is the enzyme activity that catalyzes the change of enzyme substrate of lnH in 1 minute.

In addition, the inhibition rate was determined from the absorbance (a) of the reaction solution containing the analyte, the absorbance (b) without the analyte, and the blind absorbance (a" and b') for each without reaction, using the following formula.

(b-b') Inhibitory activity of angiotensin converting inhibitor
Inhibition rate) The concentration of the analyte required for inhibition.

(2) Experimental Results Table 1 shows the inhibitory activity of the three peptides according to the present invention against angiotensin converting enzyme in human serum.

Table 1 Inhibitory activity ss Test example 2゜ (hypertensive effect when administered to rats) ■, Experimental materials Three peptides of the present invention obtained in the above Production Example 2 ■, Experimental method 12-week-old male spontaneously hypertensive rats Twelve animals were used. The rats were divided into 4 groups (3 animals per group), the first group received peptide (1), the second group received peptide (2), and the third group received (3 rats).
) peptides.

Each was dissolved in physiological saline at a concentration of 50 mg/kg and administered into the tail vein, and to the fourth group, only physiological saline was administered into the tail vein as a control. Blood pressure was measured using a non-invasive tail artery blood pressure device (Ueda Seisakusho, UR-1000), and the collected blood pressure, mean blood pressure, diastolic blood pressure, and pulse rate of each rat were determined 5 times at 1WIL, and the highest value and The lowest value was discarded and the measured values at three points were averaged.

■、Experimental results The results are shown in Figures 7, 8 and 9.

Results of the above tests 1. The three types of peptides according to the present invention have angiotensin converting enzyme inhibitory activity, and in vitro
It was confirmed that vo also showed a significant blood pressure lowering effect. Therefore, the three types of peptides according to the present invention are useful as therapeutic or preventive agents for hypertension.

Note that the three types of peptides according to the present invention can also be employed in the structure of peptides whose partial structure is the amino acid sequence.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of separation and purification of the ACE-inhibiting peptide by 5ephadex G-25 column chromatography in Production Example 1 of three types of peptides according to the present invention. Figure 2 shows 5P-5eohad in Production Example 1.
Z showing the results of minute N purification of ACE inhibitory peptide by ex C-25[H. Figure 3 shows A by reverse phase HPLC in Production Example 1.
FIG. 3 is a diagram showing the results of minute M purification of CE-inhibiting peptides. FIGS. 4, 5, and 6 are diagrams showing mass spectra of three types of peptides obtained in Production Example 2, respectively. Figures 7, 8, and 9 are the same, respectively. FIG. 3 is a diagram showing changes in blood pressure and pulse rate over time when three types of peptides obtained in Production Example 2 were administered to rats. Q \ [ No. 0 \ Fig. Pr. le tn-H Blood pressure time after administration (h'') Systolic blood pressure time (h'') Mean blood pressure n sp and pulse rate variation time (h'') Diastolic blood pressure time (h'') Pulse rate chart Ata-VaL tu Systolic blood pressure time (h') Mean blood pressure 'ro-Lys-Phe 1 Pressure and pulse fluctuation time (h') systolic blood pressure time (h') pulse rate

Claims (5)

[Claims] (1) Components in pig plasma that have passed through a semipermeable membrane are fractionated by sequentially applying strong acid cation exchange resin, gel filtration, ion exchange gel filtration, and reversed-phase high performance liquid chromatography. A method for producing a novel peptide having angiotensin converting enzyme inhibitory activity, which comprises obtaining a fraction containing a component having angiotensin converting enzyme inhibitory activity from the fractions obtained for each treatment. (2) A novel peptide obtained by the production method according to claim 1. (3) A peptide having an amino acid sequence structure represented by the following formula Pro-Ile-Gln-His-Gln-Asp. (4) The following formula Ala-Val-Glu-Gly-Pro-Lys-P
A peptide having the amino acid sequence structure represented by he. (5) The following formula Thr-Pro-Asp-Glu-Thr-Tyr-L
A peptide having an amino acid sequence structure represented by ys-Pro-Lys-Glu.