JP2903464B2 - Novel peptides and activated oxygen inhibitors - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide having the following amino acid sequence which has utility as a pharmaceutical, and an activated oxygen inhibitor containing such a peptide as an active ingredient. Leu-Val-Pro-Pro-Gln-Glu-C
ys-Gln-Lys (wherein each symbol representing an amino acid residue is represented by a convention used in amino acid chemistry.)

[0002]

2. Description of the Related Art Diseases involving activated oxygen are widely present, such as inflammation such as burns and arthritis, reperfusion injury, side effects of anticancer drugs, radiation injury, peptic ulcer, bacterial shock, cachexia, and autoimmune diseases. I do. Diseases caused by a large amount of activated oxygen generated by activation of neutrophils and macrophages are all covered. Generally, oxygen includes oxygen essential for animals (triplet oxygen molecule: ³ O ₂ ) and radicals (activated oxygen) generated under specific conditions or when the body is upset.
And exists. Radicals can be directly or indirectly (in the form of peroxidation) in cell membranes, intracellular granule membranes, or DNA.
And alters and damages various cell components. The radicals are produced in the body, the type superoxide anion ^(- _O 2 ·), singlet oxygen ⁽¹ _O 2 ·),
There are hydroxyl radicals (.OH) and the like. Among superoxide anion ^(- O 2 _·) causes peroxidation acts on unsaturated fatty acids in cell membranes, the oxidizing power to lipid is said to be higher several thousand times the essential oxygen to animals. Superoxide dismutase as activated oxygen inhibitor (SOD, enzyme number EC 1.15.1.1)
In McCord, J. et al., 1969. M.
& Fridovich, I .; : J. Biol. Che
m. , 244,6049 (1969)] by an enzyme that acts is found, superoxide anion oxygen molecules occurs is one-electron reduction ^(- _{O 2} ·) the disproportionation _{^{2 - O 2 · + 2H +}} → Catalyzes H ₂ O ₂ + O ₂ . When the human body is normal, SOD works to suppress the generation of superoxide anions. This SOD
The activity decreases with aging, that is, the activity decreases from the middle age to the old age, and the increase or decrease of the SOD activity depends on the aging of the living body,
It is also called a barometer of canceration. Such SO
When the D activity decreases, it becomes difficult to suppress the generation of radicals and S
It is necessary to take up an activated oxygen inhibitor which supplements OD or scavenges and removes radicals. On the other hand, the activated oxygen inhibitory action of polypeptides ranging from amino acids to proteins as water-soluble antioxidants inhibits the contact between oxygen molecules and unsaturated fatty acids by encapsulating fats and oils by peptides, and the lipid peroxy radical (LOO) It is thought to suppress the generation of ()), and acts on radicals (LOO) generated during the oxidation of fats and oils (L), such as the antioxidant action of BHA (butylhydroxyanisole) and BHT (dibutylhydroxytoluene). Thus, it is distinguished from the radical scavenging action that stops the chain reaction of oxidation. LOO ・ + AH ₂ → LOOH + AH ・ 2AH ・ → 2A
H ₂ + A or LOO · + AH · → LOOH + A (A
(H ₂ ; antioxidant) Against this background, there is no silver bullet for anti-cancer and anti-aging. Today, DNA damage factors, mutation factors, carcinogens, aging factors, etc. are removed or inactivated from the environment. ,
Studies and studies on activated oxygen inhibitors exhibiting an activated oxygen free radical scavenging action and an antioxidant action have been advanced.

[0003]

In the above-mentioned prior art, SOD as an activated oxygen inhibitor is difficult to produce and the availability of raw materials is limited. Vitamin E, vitamin C, catechins, etc. However, there is a drawback that the activated oxygen inhibitory action is not sufficient in the experiment using, and an activated oxygen inhibitor having a stronger action is demanded. In addition, most of activated oxygen inhibitors exhibiting an activated oxygen free radical scavenging action and an antioxidant action are mostly produced by chemical synthesis, and are derived from natural products using materials from plants and animals. Of these, many are made by using chemical substances that harm the human body during the manufacturing process or by reacting some of the products with the chemical substances. Very little is known about the amino acid sequence and antioxidant activity of polypeptides from amino acids to proteins as water-soluble antioxidants, and Yamaguchi et al. [New Food Industry, 31, 18-22 (1989)] Has a stronger antioxidant activity than amino acids and proteins, and recently Takushoku et al. [Journal of the Japanese Society of Agricultural Chemistry, Vol. 65, pp. 1635-1641 (1991)] reported that three types of antioxidant Only peptides are reported. There is no report that these activated oxygen agents having an active oxygen free radical scavenging action and an antioxidant action are still being developed as pharmaceuticals.

[0004]

Means for Solving the Problems The present inventor has searched for a substance having a pharmacological action from the degradation solution of the protease of raw soybean and has found that the novel nonapeptide has a strong activated oxygen inhibitory action. And, they intensively studied to put this peptide into practical use as a medicine. As a result, the present inventors have found that this peptide has an active oxygen free radical scavenging action and an antioxidant action, and is useful as an activated oxygen inhibitor derived from natural products. The present invention is based on such findings. Hereinafter, the present invention will be described in detail. The novel peptide according to the present invention has the following formula: Leu-Val-Pro-Pro-Gln-Glu-C
It is a novel nonapeptide represented by an L-form amino acid sequence represented by ys-Gln-Lys, and is a white powder at room temperature.

[0005] The nonapeptide may be chemically synthesized or separated and purified from a raw soybean proteolytic enzyme degradation solution. When chemically synthesizing the novel peptide according to the present invention, a conventional peptide synthesis method such as a liquid phase method or a solid phase method is used to convert a C-terminal (carboxyl terminal side) of the peptide to a polymeric solid support. It is preferable that the L-form amino acids corresponding to the amino acid residues are sequentially bonded by peptide bonds. Then, the synthetic peptide thus obtained is cleaved from the polymeric solid support using trifluoromethanesulfonic acid, hydrogen fluoride, or the like, and then the protecting group of the amino acid side chain is removed. Can be purified by a usual method using a column described above.

As described above, the novel nonapeptide according to the present invention can be separated and purified from a raw soybean protease-degrading solution. In that case, for example, the following procedure is performed. it can. The raw soybean portion containing the novel peptide is removed and hydrolyzed. The hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, raw soybeans are further hydrolyzed if necessary, and then adjusted to the optimal value of the enzyme, and the enzyme is added and incubated. Then, if necessary, after neutralization, the enzyme is inactivated to obtain a hydrolyzed solution. The hydrolyzed solution is filtered using a filter paper and / or celite to remove insoluble components, and the obtained filtrate is filtered using a semipermeable membrane such as cellophane or the like with a suitable solvent (for example, Tris-HCl buffer, After sufficient dialysis in a neutral buffer such as a phosphate buffer, the solution containing the components in the filtrate and having passed through the semipermeable membrane is subjected to a strongly acidic cation exchange resin (for example, Dow Chemical Co., Ltd.). Dowex 50W) to obtain a fraction containing a component having an activated oxygen inhibitory activity from the adsorbed fraction,
The obtained activated oxygen-inhibiting active fraction was subjected to cation exchange gel filtration (for example, SP-Sephade manufactured by Pharmacia).
x C-25), and the obtained activated oxygen inhibition active fraction is further fractionated by reverse phase HPLC.

The novel nonapeptide does not induce antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. This peptide is L
-Consisting only of amino acid sequence structure, and is gradually degraded by in-vivo proteases after administration. Therefore, its toxicity is extremely low and its safety is extremely high (LD ₅₀ > 5000 mg / k).
g: oral administration to rats). Nonapeptide according to the present invention,
Injections, tablets, capsules, granules, powders and the like can be prepared using commonly used additives such as excipients. The method of administration usually includes injection into mammals (eg, humans, dogs, rats, etc.) deficient in SOD or oral administration. The dose is, for example, 0.0% of nonapeptide per 1 kg of animal body weight.
It is an amount of 1-10 mg. The frequency of administration is usually about once to four times a day, but it can be appropriately adjusted depending on the administration route. The types of excipients, binders, disintegrants, lubricants, etc. used in the above various formulations are not particularly limited, and those used in ordinary injections, powders, granules, tablets or capsules are used. can do.

[0008] Examples of additives used in tablets, capsules, granules and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starch, anhydrous calcium phosphate, etc .; as binders, starches, hydroxypropylmethyl cellulose, etc .; as disintegrants, carboxymethyl cellulose and its potassium salts Lubricating agents include stearic acid and its salts, talc and waxes. In preparing the preparations, odorants such as menthol, citric acid and salts thereof, and fragrances can be used as necessary. The sterile composition for injection can be prepared by dissolving or suspending the novel nonapeptide according to the present invention in water for injection, physiological saline, a sugar alcohol injection such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol by an ordinary method. It can be turbid to obtain an injection. At this time, buffers, preservatives, antioxidants and the like can be added as necessary. The preparation containing the novel nonapeptide of the present invention may be in the form of a freeze-dried product or a dry powder, and may be used by dissolving it with a usual dissolving agent, for example, water or physiological saline when used.

[0009] Activated oxygen is generated in phagocytes such as macrophages, and has a role of decomposing foreign substances that have been engulfed by phagocytes. However, when activated oxygen is excessively produced, it is secreted out of cells. Disturb other organizations. The novel nonapeptide according to the present invention has an excellent activated oxygen inhibitory effect, exhibits an activated oxygen free radical scavenging effect and an antioxidant effect, and therefore degrades excessive active oxygen causing tissue damage to degrade tissue. Since it has a protective effect, it is effective, for example, as an anti-inflammatory agent for arthritis, rheumatism and the like, and is also useful for Behcet's disease, myocardial infarction and the like.

[0010]

The present invention will be described in more detail with reference to the following examples, which are production examples and test examples. Production Example 1 300 g of raw soybeans were homogenized by adding 1 l of deionized water. 9 g of pepsin was added to the obtained soybean homogenate, adjusted to pH 2.0, and incubated at 37 ° C. for 20 hours. The pepsin-decomposed solution of soybean homogenate prepared in this manner was applied to a Diaflow membrane (Amicon, Y.
Ultrafiltration was performed using an M-10 type membrane and a cut-off molecular weight of 10,000. The obtained filtrate is Dowex 50W × 4 (H ⁺ )
Was chromatographed using a column packed with. Washed with deionized water, elution the eluate was concentrated performed in _2N-NH 4 OH. This concentrated solution is Sephadex
A low molecular peptide fraction (fraction number 25-42) was separated by chromatography on a G-25 column. The column chromatograph is shown in FIG. The low molecular peptide fraction was concentrated to obtain a soybean peptide solution. Further, this peptide solution was added to SP-Sephadex C-25.
(H ⁺ ) column was subjected to chromatography and SP-1 fraction (fraction number 13-2) was used as each peptide fraction.
2), SP-2 fraction (fraction number 23-29), SP-3
Fraction (fraction number 30-40), SP-4 fraction (fraction number 41-50), SP-5 fraction (fraction number 51-70) and SP-6 fraction (fraction number 71- 100) were isolated.
The column chromatograph is shown in FIG. Each of these peptide fractions was freeze-dried to obtain peptide powder (hereinafter referred to as “soy peptide”) as SP-1 fraction (2.4 g).
5.2 g of SP-2 fraction, 12.5 g of SP-3 fraction, SP
-4 fraction 11.0 g, SP-5 fraction 24.6 g and SP
41.3 g of a -6 fraction was obtained. Among the fractionated soybean peptides, SP-4 having a high activated oxygen inhibiting activity
The peptide powder of the fraction was dissolved in deionized water (5 mg /
After 25 μιη), HPLC was performed. The conditions were as follows: Nomura Chemical's Develosil ODS-5 (φ
Using a 4.6 mm ID × 25 cmL), the flow rate was determined by a concentration gradient method from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TFA as a mobile phase. Chromatography at 0 ml / min, detection wavelength 220 nm, elution time 7
A peptide fragment having strong activated oxygen inhibitory action was obtained at 4.5 minutes. The result is as shown in FIG. The amino acid sequence of the thus-obtained peptide having an inhibitory effect on activated oxygen was obtained from Applied Biosystems (ABI) protein sequencer 477A.
Determined using type. As a result, the following equation: Leu-Val-Pro-Pro-Gln-Glu-C
It was confirmed to be a nonapeptide represented by the L-form amino acid sequence represented by ys-Gln-Lys. When the soybean peptide according to the present invention is formulated as an activated oxygen inhibitor, for example, in tablets, it may be treated according to a conventional method, for example, as follows: (1) 13 g of peptide, (2) 87 g of lactose, ( 3)
First, 29 g of corn starch and (4) 1 g of magnesium stearate were used as raw materials, (1), (2) and 17 g of corn starch were mixed and granulated together with a paste made from 7 g of corn starch. )) And the resulting mixture is compressed with a compression tablet machine to produce 1000 tablets.

Production Example 2 This example is based on Leu-Val-Pro-Pro-Gln-G
This is a production example by a synthesis method of lu-Cys-Gln-Lys. The nonapeptide was synthesized by a solid phase method using a peptide synthesizer model 430A manufactured by Applied Biosystems (ABI). As the solid support, styrene-
A resin obtained by chloromethylating a divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the nonapeptide, the C-terminal Lys was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bound resin. At this time, a t-Boc amino acid protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group was used. Next, this peptide-bonded resin was suspended in a mixture of ethanediol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid under ice cooling, and further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to this mixture, and the mixture was added at room temperature for 30 minutes.
After stirring for minutes, anhydrous ether was added to precipitate the product, which was separated, washed with anhydrous ether several times and dried under reduced pressure. The unpurified synthetic peptide thus obtained was dissolved in distilled water and then purified by HPLC using a reverse-phase column C ₁₈ (5 μ).
(A) distilled water containing 0.1% TFA as a mobile phase, (B)
Using an acetonitrile solution containing 0.1% TFA,
The solution (A) was subjected to chromatography at a flow rate of 1.3 ml / min by a concentration gradient method of 93% -72% for 20 minutes. The eluted fraction which detected at an ultraviolet wavelength of 218 nm and showed the maximum absorption was collected and freeze-dried to obtain a target synthetic nonapeptide.

As a result of analyzing the synthetic nonapeptide by mass spectrum, the following formula Leu-Val-Pro-Pro-Gln-Glu-C was obtained.
It was confirmed that the peptide was represented by the amino acid sequence ys-Gln-Lys. The result of this mass spectrum is as shown in FIG. The novel nonapeptide according to the present invention obtained by synthesis is in vitro (in a test tube) shown below.
The test confirmed the activated oxygen free radical scavenging effect and the antioxidant effect, thereby confirming the activated oxygen inhibitory effect.

[0013] Test Example 1 (Measurement of activated oxygen free radical scavenging activity) Cypridina - luciferin derivative (CLA) is singlet oxygen ⁽¹ O
₂ ·), superoxide anion ^(- it is effective chemiluminescence reagent that specifically detects O 2 _·), we [A
gric. Biol. Chem. , 55, 157-16
0 (1991)] by a quenching experiment using superoxide dismutase (SOD) as a quencher.
A and ^- rate of reaction with O ₂ · is calculated. CLA (C
₁₃ H ₁₁ ON ₃ , manufactured by Tokyo Chemical Industry, final concentration 1.39 ×
10 ^{-7 to} 4.64 × 10 ^-8 ) solution 10 μl, albumin (50 mg / ml, manufactured by Sigma Chemical Co.) 500 μl,
50 μl of xanthine oxidase (1.45 unit / ml, manufactured by Sigma Chemical Co.) was sequentially added to a cylindrical quartz cell (inner diameter: 1).
4mm, height 60mm), luminometer (Al
oka BLR-102B, manufactured by Hamamatsu Photonics, Inc.) and 200 μl of a 3 mM hypoxanthine solution.
Was injected, and chemiluminescence was measured from the bottom of the cell by single photon counting. In the absence and, if the quencher is present ^- O ₂ of the emission intensities of the ratio _(I 0 / _I) is _I 0 /
I = 1 + [k ₃ / (k ₁ + k ₂ [CLA])] × [Q]
It is represented by Here, [Q] represents an activated oxygen inhibitor and k ₁
Is ^- O ₂ · quenching rate constant, _{k 2} is ^- O ₂ · and [CL
The reaction rate constant of A], _{k 3} is ^- shows the rate constant for the reaction O ₂ · and the [Q]. Table 1 shows the activated oxygen inhibitory activity (quenching rate) of the peptide fraction derived from soybean according to the present invention, which shows an activated oxygen free radical scavenging effect.

[Table 1] The activated oxygen-inhibiting activity value (reaction rate constant k ₃ ) of the novel nonapeptide according to the present invention, which exhibits the activated oxygen free radical scavenging action, is 0.98 × 10 ⁻⁶ M ⁻¹ sec ⁻¹ . The activated oxygen inhibition activity value (reaction rate constant k ₃ ) showing the activated oxygen free radical scavenging action of the standard SOD is 3.47 × 10 ⁻⁸ M ⁻¹ scc ⁻¹ .

Test Example 2 (Measurement of antioxidant action) To measure the antioxidant action, the reaction solution was composed of 51.1 mg of linoleic acid and 4.052 m of ethanol.
1, 0.05M phosphate buffer (pH 7.0) 4.0m
l to a mixed solution of 1.948 ml of deionized water, 1-3 mg of a peptide having an antioxidant effect was added to adjust the total amount to 10 ml. This solution was sealed in a test tube with a screw, left in a thermostat at 50 ° C., and the peroxide value of linoleic acid was measured every 24 hours by the iron-rodan method. That is, the reaction mixture was added.
1 ml, 9.7 ml of 75% ethanol solution, 0.1 ml of 30% rhodamonium solution, 0.1 ml of 3.5% hydrochloric acid solution containing 0.02 M ferric chloride, reacted for 3 minutes, and measured absorbance at 500 nm did. At that time, 500n
The number of days until the absorbance value of m reached 0.35 was defined as the number of days of induction (days). FIG. 5 shows the activated oxygen inhibitory activity (induction days) showing the antioxidant activity of the peptide fraction derived from soybean according to the present invention. Activated oxygen inhibitory activity (induction days) showing the antioxidant activity of the novel nonapeptide according to the present invention
Is 17 days with 1 mg of nonapeptide for 6.5 days with 2 mg of tocopherolone. As a result of the above test, the novel nonapeptide according to the present invention has an active oxygen free radical scavenging action and an antioxidant action.
In vitro (in a test tube) test, it was confirmed that a significant activated oxygen inhibitory effect was exhibited. Therefore, the nonapeptide according to the present invention is useful as a therapeutic or preventive drug for patients with ischemic heart disease, rheumatoid arthritis and severe burns who are the targets of activated oxygen inhibitors. The nonapeptide according to the present invention can also be employed in the structure of a peptide structurally represented by its amino acid sequence.

[0015]

[Brief description of the drawings]

FIG. 1 is a view showing the results of separation and purification of an activated oxygen-inhibiting peptide by Sephadex G-25 column chromatography in Production Example 1 of a soybean pepsin degradation solution according to the present invention. In the figure, insulin having a molecular weight of 6,000 and insulin B having a molecular weight of 3,500 are used as markers.
Chain, insulin A chain with molecular weight 2,550, molecular weight 1,
450 bacitracin and 75 molecular weight glycine were used.

FIG. 2 is a graph showing the results of separation and purification of an activated oxygen-inhibiting peptide by SP-Sephadex C-25 (H +) column chromatography in Production Example 1 of the soybean peptide according to the present invention.

FIG. 3 is a diagram showing the results of separation and purification of a fragment of an activated oxygen-inhibiting nonapeptide by reverse phase HPLC in Production Example 1 of a soybean peptide according to the present invention.

FIG. 4 is a view showing a mass spectrum of the synthetic nonapeptide obtained in Production Example 2 of the nonapeptide according to the present invention.

FIG. 5 is a graph showing the number of days (days) of inducing the SP fraction (1, 2, 3 mg) of the soybean peptide according to the present invention in Production Example 1, showing an antioxidant effect and, simultaneously, an activated oxygen inhibitory effect. is there.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 AED A61K 37/02 ABX C07K 14/415 AED

Claims (2)

(57) [Claims] 1. The following formula: Leu-Val-Pro-Pro
A novel nonapeptide represented by an L-form amino acid sequence represented by -Gln-Glu-Cys-Gln-Lys. 2. The following formula: Leu-Val-Pro-Pro
An activated oxygen inhibitor comprising as an active ingredient a novel nonapeptide represented by an L-form amino acid sequence represented by -Gln-Glu-Cys-Gln-Lys.