JP2719891B2 - Pharmaceutical composition for promoting TNF secretion - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition containing a novel physiologically active peptide.

[0002]

2. Description of the Related Art It is already known that peptides having various physiological activities are derived from food proteins. Due to their food origin, these peptides can be expected to be safe for living organisms. In general, bioactive peptides derived from food proteins have many unpredictable amino acid sequences unlike endogenous bioactive peptides, and because of their multiple functions, many have not yet been confirmed. There seems to be.

The present inventors have previously reported that Gln-Arg-Pro-Arg (QRPR) derived from the soybean glycinin A _1a subunit.
And His-Cys-Gln-Arg-Pro-Arg (HCQRPR) have immune system activating effects such as neutrophil accumulation activity, macrophage phagocytosis promoting activity, TNF (tumor necrosis factor) secretion promotion, and antitumor effect. It has been reported that the peptide is a novel peptide having
No. 4). Following this, Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gl derived from soybean conglycinin
It was reported that y-Arg (hereinafter abbreviated as MITLAIPVNKPGR) and its analogous peptides were immune system activating peptides having phagocytosis promoting activity and active oxygen production promoting effect (Japanese Patent Application No. 6-377).
No. 07).

[0004]

Then, the present inventors,
A peptide having an immune system activating effect obtained by enzymatic degradation of soy protein: MITLAIPVNKPGR was examined for its physiologically active effect, and among the analogs, the peptides of SEQ ID Nos. 1 and 2 described below have a TNF secretion promoting effect. I found something. TN
F is known to express a cell killing activity or a cell growth inhibitory activity on tumor cells. Therefore, promotion of TNF secretion is expected to have an antitumor effect and to enhance host immunity. Therefore, an object of the present invention is to provide novel foods and pharmaceutical uses of the above-mentioned peptide having a physiological activity.

[0005]

The present invention provides a pharmaceutical composition comprising the following two peptides as active ingredients, and the sequence of the peptide is as follows: Sequence number 1: Met-Ile-Thr-Leu-Ala
-Ile-Pro-Val-Asn (hereinafter abbreviated as MITRAIPVN) SEQ ID NO: 2: Met-Ile-Thr-Leu (hereinafter abbreviated as MITL) Accordingly, the present invention provides the peptides of SEQ ID NOS: 1 and 2 and their peptides. Pharmaceutical for promoting TNF (tumor necrosis factor) secretion comprising a pharmaceutically acceptable salt as an active ingredient
It relates to a composition . Each of the peptides represented by SEQ ID NOs: 1 and 2 has the following properties: sequence type: amino acid topology: linear Sequence type: peptide

The active ingredients of the present invention, SEQ ID NOS: 1 and 2,
Can be obtained by enzymatic hydrolysis of soy protein. More specifically, in the case of enzymatic hydrolysis of soybean protein, it can be prepared by further enzymatically decomposing the previously reported peptide of MILAIPVNKPGR consisting of 13 amino acids, but it can be prepared by a known method using a peptide synthesizer. Can also be chemically synthesized.

The present inventors have found that the peptides of SEQ ID NOs: 1 and 2 obtained by enzymatic degradation of soybean protein have a phagocytosis-promoting action, and as a result of further studies, they also have a TNF secretion-promoting action. That's what I found. Therefore, the peptides of SEQ ID NOs: 1 and 2 also have a phagocytosis-promoting action, and the peptide of SEQ ID NO: 1 also has a reactive-oxygen production-promoting action. Therefore, the peptides of SEQ ID NOs: 1 and 2 can be used as a pharmaceutical composition having an immune system activating effect. These peptides can be converted into pharmaceutically acceptable salts according to a conventional method, and can be prepared into a pharmaceutical composition together with pharmaceutically commonly used carriers, auxiliaries and the like.

[0008]

Production Examples Hereinafter, examples of production of the peptides of SEQ ID NOs: 1 and 2 of the present invention are shown, but the invention is not limited to these examples.

Reference Example MITL from soybean protein digest
Preparation of peptide of AIPVNKPGR (SEQ ID NO: A) 50 g of isolated soybean protein was dissolved in 800 ml of water, and then 3000 rpm ×
The soluble fraction was separated by centrifugation for 20 minutes. This was boiled for 30 minutes, 500 mg of trypsin was added, and the pH was adjusted with 1N-NaOH.
The digestion was performed at 37 ° C. adjusted to 7.6. After 5 hours, the digestion was stopped by boiling, and the supernatant obtained by centrifugation at 10,000 rpm for 15 minutes was lyophilized to give a trypsin digest (solids amount: 23.8 g). 100 mg of this is DEA
It was loaded on an E-cellulose column (DE-52, manufactured by Whatman, 2 ml of carrier) and 20 mM Tris-HCl buffer (pH = 7.
When the unadsorbed fraction was collected by 1 ml at a time, the active peptide was contained in the fraction eluted at 2 to 3 ml. Next, this fraction was subjected to ODS-column (Co
smosil 5C _18- AR, 20 × 250mm, manufactured by Nacalai Tesque),
Subsequently, a phenethyl column (4.7 x 250 mm, Develosil Ph
AT-5, Nomura Chemical Co., Ltd.) and a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid (1% / min)
). Elution of the active peptide by these columns was 33-34% and 36-37% of acetonitrile, respectively. Further, this active fraction was applied to an ODS-column (Co
_{smosil 5C 18 -AR, 4.6 × 150mm} , was loaded onto Nacalai Tesque), was developed by phosphate buffer 10 mM (linear gradient of acetonitrile containing pH = 7.4) (1% / min), the active peptide Eluted as a single peak at 34-35% acetonitrile. When the structure was determined using a protein sequencer, the active peptide was Met-Il
It was found to be e-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg.

Preparation Example 1 Preparation of Peptide of SEQ ID NO: 1 by Enzyme The peptide of SEQ ID NO: 1 can be obtained by enzymatic hydrolysis of soybean protein. In this example, the peptide of SEQ ID NO: A is hydrolyzed by an enzyme. Prepared by degradation. The enzyme used is a metalloendopeptidase that specifically cleaves the peptide bond of Asn-Lys contained in the peptide of SEQ ID NO: A,
Asparaginyl endopeptidase or the like is used. An example of the preparation method is shown below, but the enzyme used is not limited to this example. 20 mg of the peptide of SEQ ID NO: A
, 500U metallo endopeptidase (Seikagaku Corporation)
100 mM glycine-NaOH buffer solution (pH = 10.0)
2 ml was added and incubated at 70 ° C. for 5 hours. After quenching the solution ODS- column (Cosmosil5C ₁₈ -AR,
4.6 × 150 mm) and purified by HPLC with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The fraction eluted at around 34% of acetonitrile was lyophilized to obtain about 10 mg of a pure peptide of SEQ ID NO: 1.

Preparation Example 2 Preparation of peptide of SEQ ID NO: 1 by chemical synthesis (Fmoc method) 0.3 g of Fmoc-Asn (Trt) -resin having a substitution rate of 0.70 meq / g
Set in a reaction vessel of an AM2 peptide synthesizer (Biosearch), add a deblock solution (piperidine: toluene: dimethylformamide (DMF) = 30: 35: 35), stir, and add 9-fluorenylmethoxycarbonyl (Fmoc). ) The group was removed. This resin is treated with dichloromethane (DCM): D
After washing with MF = 1: 1, 4 times equivalent of hydroxybenzotriazole (HOBT) and Fmoc of Fmoc-Asn (Trt) -resin
-Val was added for coupling. After the reaction is completed, DC
After washing with M: DMF = 1: 1, Fmoc-Val-Asn (Trt) -resin was obtained. The same procedure is followed to synthesize the N-terminal Met,
M, washed with methanol and washed with Met-Ile-Thr (tBu) -Leu-Ala-I
le-Pro-Val-Asn (Trt) -resin was obtained. Add a deprotecting agent (trifluoroacetic acid: ethanediol: anisole = 94:
1: 5) and left at room temperature for 1 hour. Then, the separated resin was filtered, washed with ether, freeze-dried, and crude Me
t-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn was obtained. This is O
DS- column _{(Cosmosil 5C 18 -AR, 20 ×} 250mm, Nacalai Tesque) was loaded into, pure product of approximately 150mg was purified to obtain by HPLC with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid Was. In the above, Trt represents a trityl group, and tBu represents a tertiary butyl group. The obtained synthetic MITRAIPVN (20 μg) was OD
FIG. 1 is a chart showing the absorbance measured when loaded onto an S column (Cosmosil 5C ₁₈ -AR, 4.6 × 150 mm) and eluted with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid.

Production Example 3 Preparation of peptide of SEQ ID NO: 2 by chemical synthesis (Fmoc method) 0.3 g of Fmoc-Leu-resin having a substitution rate of 0.58 meq / g was purified with SAM2
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in Production Example 2 to obtain a corresponding 4-residue peptide-resin. Thereafter, in the same manner as in Production Example 2, about 50
mg of pure Met-Ile-Thr-Leu was obtained. Obtained synthetic MI
TL (100 μg) was added to an ODS column (Cosmosil 5C ₁₈ -AR,
FIG. 2 shows a chart in which the absorbance was measured when eluting with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid.

Table 1 shows the amino acid compositions of the peptides of SEQ ID NOs: 1 and 2, elution positions by HPLC, and Rf values by thin-layer chromatography. In addition, the natural product (Production Example 1) and the synthetic product (Production Example 2) of SEQ ID NO: 1 were found to be the same product from these results. Therefore, examination is made below using synthetic products.

[0014] In the above table, M is Met, I is Ile, T is Thr, L is Leu,
A is Ala, P is Pro, V is Val, D is Asp, K is Lys,
G represents Gly and R represents Arg. * 1 6N-HCl after hydrolysis at 110 ° C. Therefore, Asn in the peptide composition is detected as Asp. * 2 ODS- column _{(Cosmosil 5C 18 -AR, 4.6 ×} 150mm
) Is measured by a linear concentration gradient of acetonitrile. * 3 Using a Kieselgel plate made by Merck, developed at room temperature. The composition of the developing solvent is butanol: acetic acid: pyridine: water = 15: 3: 10: 12.

[0015]

[Test example]

Test Example: Effect on TNF Production by Macrophages 300 nmol of the peptide of the present invention was orally administered to 6-week-old male ICR mice, and 3 hours later, 0.3 mg of Bicibanil (OK-432), a killed cell of streptococci, was intravenously injected. Was administered internally. Two hours later, blood was collected, and the toxicity to L-929 cells contained in the serum was measured by radioimmunoassay. Table 2 shows the results. This peptide showed almost the same activity as that of the previously reported QRPR.

[0016] Note 1) QRPR is Gln-Arg-Pro-Arg peptide 2) PBS is phosphate buffer solution

[0017]

Industrial Applicability The peptides of the present invention having SEQ ID NOs: 1 and 2 have phagocytosis promoting activity, active oxygen production promoting effect and TNF (tumor necrosis factor) secretion promoting effect, and are obtained from soybean protein which is a natural product. It can be used safely in the fields of food and medicine.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chart showing the measurement of the absorbance of a synthetic MITLAIPVN when eluted by HPLC using an ODS column.

FIG. 2 is a chart showing the measurement of the absorbance of a synthetic MITL when eluted by HPLC using an ODS column.

Claims (3)

(57) [Claims] 1. SEQ ID NOs: 1 and 2: SEQ ID NO. 1: Met-Ile-Thr-Leu-Ala
-Ile-Pro-Val-Asn SEQ ID NO: 2: One or more selected from the group consisting of peptides represented by Met-Ile-Thr-Leu and pharmaceutically acceptable salts thereof are used as active ingredients A pharmaceutical composition for promoting secretion of TNF (tumor necrosis factor). 2. The active ingredient is SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala
The pharmaceutical composition according to claim 1, which is a peptide represented by -Ile-Pro-Val-Asn or a pharmaceutically acceptable salt thereof. 3. The pharmaceutical composition according to claim 1, wherein the active ingredient is a peptide represented by SEQ ID NO: 2: Met-Ile-Thr-Leu or a pharmaceutically acceptable salt thereof.