JP2873434B2 - New peptides and immunostimulants - 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 immunostimulant containing the peptide as an active ingredient. Ile-Ala-Val-Pro-Thr-Gly-Val-Ala (wherein each symbol representing an amino acid residue is represented by a convention used in amino acid chemistry.)

[0002]

BACKGROUND OF THE INVENTION Ingested foods are known to have various effects on the host immune system in the process of being decomposed and absorbed in the digestive tract [J.
L. Decker et al. : Ann. Inter
n. Med. , 101, 810-824 (198).
4)]. It is known that the immune response of the host is regulated and controlled by physiologically active substances secreted from lymphocytes and macrophages, which are immunocompetent cells.
The presence of substances that regulate the host immune system is also known in food components. Among them, human casein-derived Gly-Leu-Ph
e, Leu-Leu-Tyr from bovine casein, human β
Casein-derived Val-Glu-Pro-Ile-Pro
-Tyr are known and all have been found to increase the activity of macrophages [F. Par
ker et al. : Eur. J. Bioche
m. 145, 677-682 (1984); Be
rthou et al. : FABS Lett. , 2
18, 55-58 (1987)]. The food protein taken into the living body comes into contact with the immune response system cells in the form as it is or when it is degraded. Such interaction between food components and cells of the immune response system has been known so far, for example, may cause food allergy, which is an abnormal state of the immune system, and may be in the form of activation or suppression of the immune system. Has been observed. It is a group of polypeptides called cytokinins such as interleukins, which are natural in vivo substances that regulate the immune response system, and a great deal of information about their functions and structures is being accumulated. On the other hand, there are not many peptides derived from food proteins having an immunomodulatory function, and there is no report that the development as a pharmaceutical is still in progress.

[0003]

Means for Solving the Problems The present inventors searched for a substance having a pharmacological action from a decomposition solution of soybean protease, and found that the novel peptide has a strong immunostimulatory action. And, they intensively studied to put this peptide into practical use as a pharmaceutical. As a result, the present inventors have found that this peptide has an immunostimulating effect and is useful as an immunostimulating agent 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 Ile-Ala
-Val-Pro-Thr-Gly-Val-Ala (wherein each symbol represents each amino acid unit of the amino acid sequence in peptide chemistry), a novel peptide represented by an L-form amino acid sequence represented by the formula: Yes, it is a white powder at room temperature.

[0004] The novel peptide according to the present invention can be exemplified by a method of chemically synthesizing it, or a method of separating and purifying it from a decomposition solution of soybean protease. When the novel peptide according to the present invention is chemically synthesized, it can be carried out by a conventional peptide synthesis method such as a liquid phase method or a solid phase method. It is preferable that the L-form amino acids corresponding to the amino acid residues are sequentially bonded to the support by peptide bonds from the C-terminal (carboxyl-terminal side) of the peptide. Then, the synthetic peptide thus obtained is cleaved from the polymeric solid support using trilauromethanesulfonic acid, hydrogen fluoride, or the like, and then the protecting group of the amino acid side chain is removed. It can be purified by an ordinary method using high-performance liquid chromatography (hereinafter abbreviated as HPLC) using a system column.

[0005] As described above, the novel peptide of the present invention can be separated from the decomposition solution of soybean proteolytic enzyme. In such a case, the literature of the inventors [J. Nu
tr. Biochem. 1993, vol. 4, Au
gust] method, for example, as follows. The raw soybean containing the above novel peptide is sufficiently homogenized in a suitable solvent (for example, a neutral buffer of water-tris-hydrochloric acid buffer or phosphate buffer), and then hydrolyzed. Decompose. The hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, the raw soybean homogenate is further hydrolyzed if necessary, and then heated to the optimal temperature of the enzyme. Get. The hydrolyzed solution is filtered using 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-
After sufficient dialysis in a hydrochloric acid buffer or a neutral buffer of a phosphate buffer, a 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, manufactured by Chemical Co., Ltd.) to obtain a fraction containing a component having an immunostimulating effect from the adsorption-elution fraction, and the obtained fraction having an immunoactivating effect is subjected to gel filtration (for example, manufactured by Pharmacia). Sephadex G-
25), and the obtained fraction having an immunostimulatory effect is fractionated by cation exchange gel filtration (for example, SP-Sepahdex C-25 manufactured by Pharmacia), and the obtained immunostimulatory effect is measured. Fractions are further reversed phase H
Fractionation by PLC.

The novel peptide according to the present invention does not induce antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. In addition, since this peptide has a sequence structure of only L-amino acids and is gradually degraded by an in-vivo protease after administration,
Very low toxicity and very high safety (LD ₅₀ > 50
00 mg / kg: oral administration to rats). The novel peptide according to the present invention can be prepared into injections, tablets, capsules, granules, powders and the like by using additives such as commonly used excipients. Examples of the administration method include injection into mammals usually having immunodeficiency (eg, humans, dogs, rats, etc.) or oral administration. The dose is, for example, an amount of 0.01 to 10 mg of this peptide per kg of animal body weight. The frequency of administration is usually about 1 to 4 times a day, but can be appropriately adjusted depending on the administration route. The types of excipients, binders, and lubricants used in the above-mentioned various preparations are not particularly limited, and those used in usual injections, powders, granules, tablets or capsules can be used. .

[0007] Examples of additives used for 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 .; starches, hydroxylpropyl methyl cellulose, etc. as binders; carboxymethyl cellulose and potassium salts thereof as disintegrants; Lubricants include stearic acid and its salts, talc and waxes. In preparing the preparation, a odorant such as menthol, citric acid and its salts, and a fragrance can be used, if necessary. The sterile composition for injection can be prepared by dissolving or suspending the novel peptide of the present invention in water for injection, physiological saline and 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 peptide of the present invention may be in the form of a lyophilized product or a dry powder, and may be used by dissolving it with a conventional dissolving agent, for example, water or a physiological saline solution before use.

[0008] The novel peptide according to the present invention has an excellent immunostimulatory effect, and when the novel peptide is orally administered to rabbits, concanavalin A (hereinafter referred to as C) of peripheral blood lymphocytes.
Abbreviated as onA. ) The immature response to stimulation was significantly increased, and the antibody production ability was increased when the novel peptide was orally administered to C57BL / 6 mice. In addition, the novel peptides have been developed in vitro for C3H / He
It shows mitogenic activity on spleen cells obtained from N mice.

[0009]

EXAMPLES The present invention will be further described below by way of production examples and test examples as examples. Production Example 1 200 g of raw soybeans were mixed with 1 l of deionized water and homogenized, then adjusted to pH 2.0 with 1 N hydrochloric acid, and 10 g of pepsin (manufactured by Merck, enzyme number EC 3.4.23.1).
Was added and hydrolysis was carried out at 37 ° C. with stirring for 20 hours. The decomposition reaction solution was immediately passed through an ultrafiltration membrane (Amicon,
YM10 type, φ76 mm), and the passed liquid is a strongly acidic cation exchange resin Dowex 50W × 4 [H ⁺ ] (φ
4.5 × 15 cm) column. After thoroughly washing the column with deionized water, 2N ammonium hydroxide solution
To elute. The ammonia was removed by vacuum concentration to obtain a concentrated solution (40 ml). The concentrated solution (4 ml) was loaded on a Sephadex G-25 column (φ2.5 × 150 cm) previously buffered with deionized water, and the flow rate was 30 ml / h.
r, gel filtration was performed with each fraction of 8.3 ml. The result is as shown in FIG. Fractions with high immunostimulatory activity (fraction numbers 17 to 3) collected in large quantities by repeating the above gel filtration
7) was collected and freeze-dried to obtain peptide powder (peptide powder derived from soybean). After dissolving this peptide powder in deionized water, SP-Se previously buffered with deionized water
phadex C-25 [H ⁺ ] (φ1.5 × 47.2)
cm) The column was loaded, and the flow rate was 60 mι / h by the concentration gradient method from 1 l of deionized water to 1 l of 3% sodium chloride solution.
r, Chromatography was performed at 7.8 ml of each fraction. The result is as shown in FIG.

In the above chromatograph, fraction numbers 30 to 3
8 were collected and freeze-dried to obtain a purified peptide powder (SP-I fraction), fraction numbers 39 to 4.
Fractions having high immunostimulatory activity were collected and freeze-dried to obtain a purified peptide powder (SP-II fraction). These purified peptide powders (SP-I fraction and SP-II fraction) were dissolved in deionized water and subjected to HPLC. The conditions are as follows: Nomura Chemical's Develosil OD
S-50 (4.6 mm ID × 25 cm L) was used, and 0.05% trifluoroacetic acid (hereinafter, referred to as “mobile phase”) was used as a mobile phase.
Abbreviated as TFA. ) To 25% acetonitrile / 0.
The flow rate was 1.0 mι / m by the concentration gradient method of 05% TFA.
In, chromatography was performed at a detection wavelength of 220 nm, and a peptide having immunostimulatory activity was obtained at an elution time of 112 minutes. The result is as shown in FIG. The amino acid sequence of the peptide having an immunostimulatory action thus obtained was determined using a protein sequencer type 477A manufactured by Applied Biosystems. As a result, the following formula Ile-Ala-Val-Pro-Thr-
It was confirmed that the peptide was represented by the L-form amino acid sequence represented by Gly-Val-Ala.

Production Example 2 This example is based on the synthesis method of Ile-Ala-Val-Pro.
It is a manufacture example of -Thr-Gly-Val-Ala. Automatic peptide synthesizer 4 manufactured by Applied Biosystems
The peptide was synthesized by a solid phase method using type 30A. As the solid support, a resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the peptide, the isoleucine alanine on the C-terminal side was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bound resin. At this time, the amino acid is t-Boc protected by a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group.
c amino acids were used. Next, the peptide-bonded resin was suspended in a mixture of ethanedithiol 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 the mixture, and the mixture was stirred at room temperature for 30 minutes, and the product was precipitated by adding anhydrous ether and separated.The precipitate was washed with anhydrous ether several times.
Dry under reduced pressure. The thus obtained unpurified synthetic peptide was dissolved in distilled water, and then reverse-phase column C was used.
Purified by HPLC using ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as a mobile phase;
Using an acetonitrile solution containing 1% TFA, the solution (A) was subjected to chromatography at a flow rate of 1.5 ml / min by a concentration gradient method from 90% to 60% in 20 minutes. The eluted fraction which detected at an ultraviolet wavelength of 215 nm and showed the maximum absorption was collected and freeze-dried to obtain a target synthetic peptide.

Analysis of this synthetic peptide by mass spectrum confirmed that it was a peptide represented by an amino acid sequence. The result of this mass spectrum is as shown in FIG. The pharmacological effect of the novel peptide according to the present invention obtained by synthesis was confirmed by the tests described below.

Test Example 1 (Measurement of immature response to concanavalin A stimulation of rabbit peripheral blood lymphocytes) Rabbits were mature male Japanese white species (KBL: JW, SPF,
After weighing 2.0 kg) from Kitayama Labes Co., Ltd. and preliminarily rearing it for one week, healthy animals were subjected to the test. The breeding was performed using a metal individual gauge in a breeding room maintained at a temperature of 23 ± 2 ° C. and a humidity of 55 ± 10%. As a feed, 120 g / day of RC4 manufactured by Oriental Yeast Co., Ltd. was fed, and water was freely ingested by private pumping (conforming to the water supply method and water quality standards). Using 3 rabbits per group, 200 mg / kg / day of the soybean-derived peptide powder in Production Example 1 was continuously administered at a rate of 5 ml / kg body weight for 30 days. The control group received the same volume of solvent. The body weight was measured every three days. On the first day of administration and the day after the last administration, 10 ml of blood was collected from the ear vein of each rabbit using a heparin-treated syringe, and within 3 hours, lymphocyte separation and blastogenesis by ³ H-thymidine uptake assay were performed. Carried out. The stimulation index (SI) was calculated from the radioactivity taken up by each lymphocyte according to the following equation. SI = (culture system with Con A added) / (culture system without Con A) soybean-derived peptide powder (200 mg / kg / day)
Table 1 shows the change in body weight of rabbits administered orally for 30 days. No significant difference in body weight change was observed between the soy peptide administration group and the control group. Table 2 shows the immature response (SI value) of rabbit peripheral blood lymphocytes induced by Con A stimulation.
It was shown to. As a result, 5.3 ± before administration of the soybean peptide.
From 0.6, it increased to 44.8 ± 7.0 after administration, and a significant difference (p <0.01, t-test) was observed.

Test Example 2 (Measurement of antibody-producing ability of mouse splenocytes) Mice were male, 5 weeks old (Slc: C57BL / 6, SP
F) was purchased from Japan SLC Co., Ltd., and after pre-breeding for one week, healthy animals were subjected to the test. The mice were bred using an air conditioner in a breeding room maintained at a temperature of 23 ± 2 ° C. and a humidity of 55 ± 10%. As the feed, ML water manufactured by Oriental Yeast Co., Ltd. was allowed to freely ingest private pumping (water supply law, conforming to water quality standards). 200 mg / kg / day of soybean-derived peptide powder in Production Example 1 using three mice per group
Was orally administered for 10 days at a rate of 0.1 ml per 10 g of body weight. Body weight was measured daily. Five days after the start of the administration of the soy peptide, 5 × 10 ⁸ cells of sheep erythrocytes (SRBC, manufactured by Denka Seiken Co., Ltd.) were injected into the tail vein of each mouse.
Immunization was performed by administering 0.2 ml of ls / mι. Five days after immunization, spleens were collected from each group of mice, and Eagle's
minimal essential medium
Splenocytes were released in a Petri dish containing (EMEM, manufactured by Nissui Pharmaceutical Co., Ltd.). After washing three times with a phosphate buffer (PBS), spleen cells adjusted to 2.5 × 10 ⁶ cells / mi with EMEM, 50% SRBC suspension, and guinea pig dried complement (manufactured by Denka Seiken Co., Ltd.) 8: The mixture was mixed at a ratio of 1: 1. A. J. Cunningham et al. [Immu
No. 14, 14, 599 (1968)].
After reacting at 7 ° C. for 90 minutes, hemolysis spots (PFC; plaque)
formmm cell) was measured. Table 3 shows the change in body weight of mice to which soybean-derived peptide powder (200 mg / kg / day) was orally administered for 10 days and the spleen weight measured at the time of preparing splenocytes. No significant difference in body weight change was observed between the soy peptide administration group and the control group.
The spleen weight of the soy peptide administration group was 104.7 ±
6.1) That of the control group was 73.1 ± 10.9, and no significant difference was observed between the two. Table 4 shows the antibody producing ability in mouse splenocytes. The number of antibody-producing cells in the soy peptide-administered group was 112.7 ± 306.6, which was significantly (p <) compared to the number of antibody-producing cells in the control group of 493.3 ± 170.2.
0.01, t-test).

Test Example 3 (Measurement of Mitogenic Activity of Synthetic Peptide) The method of Fujiwara et al. 3,203
-208 (1990)]. The novel peptide of the present invention synthesized in Production Example 2 was dissolved in 25 mM HEPES-RPMI 1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) (maximum concentration 1 mg / m2).
ι), sterilized by filtration through a 0.2 μ filter, and serially diluted twice with the same medium to obtain a test sample. The spleen of a C3H / HeN mouse (6 weeks old, male) was aseptically excised and 25 mM HEPE on a wire mesh.
S-RPMI 1640 medium was gently triturated with dropping, and the passed solution was further passed through another set of wire mesh to prepare a single cell suspension. The spleen cells were washed three times with the same medium and then 25 mM containing 10% fetal bovine serum.
Suspended in HEPES-RPMI 1640 medium, 96
5 × 10 ⁵ cells / 100μι /
The solution was dispensed into wells. Thereafter, 10 μιη of the test sample was added, and the cells were cultured at 37 ° C in a 5% CO ₂ atmosphere. The negative control was 25 mM HEPES-RPM.
I 1640 medium (10 μιη), positive controls: concanavalin A (Con A, final concentration 1 μg / ml) and lipopolysaccharide (LPS, final concentration 100 μg / m 2)
ι) is added in place of the test sample. afterwards,
0.5% of 3- (4,5-dimethyl-2-thiazolyl)
10 μι of a 2,5 diphenyl-2H tetrazolium bromide (MTT) solution was added thereto, followed by further culturing for 3 hours.
Add 0μι, dissolve, and use an EIA reader at 595nm
Was measured for absorbance. Data are shown as relative values with the value of the negative control taken as 100. The results of the mitogen activity are shown in Table 5. As a result of the above test, it was confirmed that the soybean-derived peptide powder comprising the novel peptide according to the present invention significantly affected the immune function in vivo. Furthermore, the novel peptide according to the present invention has been confirmed to exhibit significant mitogenic activity in vitro, and is useful as an immunostimulant. The novel peptide according to the present invention can also be employed in the structure of a peptide structurally represented by its amino acid sequence.

[0016]

[Table 1] Fig. 4 shows the weight change of rabbits upon administration of the soybean-derived peptide powder in Production Example 1 of the novel peptide according to the present invention.

[0017]

[Table 2] Fig. 4 shows the blastogenesis reaction of rabbit peripheral blood lymphocytes stimulated with concanavalin A when the novel peptide according to the present invention is administered with a soybean-derived peptide powder in Production Example 1.

[0018]

[Table 3] Fig. 7 shows changes in mouse body weight and spleen weight of a novel peptide according to the present invention upon administration of a soybean-derived peptide powder in Production Example 1.

[0019]

[Table 4] [Fig. 4] Fig. 4 shows the ability of the novel peptide of the present invention to produce antibodies in mouse splenocytes upon administration of a soybean-derived peptide powder in Production Example 1.

[0020]

[Table 5] In vitro of the novel synthetic peptide according to the present invention
Mitogen activity in.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of separation and purification of immunostimulatory peptides by Sephadex G-25 column chromatography in Production Example 1 of the novel peptide according to the present invention.

FIG. 2 is a view showing the results of separation and purification of an immunostimulatory peptide by SP-Sephadex C-25 [H +] column chromatography in Production Example 1 of the novel peptide according to the present invention.

FIG. 3 is a view showing the results of separation and purification of an immunostimulatory peptide of a novel peptide according to the present invention by reversed-phase high performance liquid chromatography in Production Example 1.

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

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C07K 14/415 A61K 38/00 CA (STN)

Claims (2)

(57) [Claims] 1. The following formula: Ile-Ala-Val-Pro
A novel peptide represented by an L-form amino acid sequence represented by -Thr-Gly-Val-Ala
De . 2. The following formula: Ile-Ala-Val-Pro
A novel peptide represented by an L-form amino acid sequence represented by -Thr-Gly-Val-Ala
Stimulant characterized by containing ceramide as an active ingredient
Agent.