JP2000281571A - Immunoenhancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new immunoenhancer having immunopotentiating action and in vivo defensive reaction activating action. SOLUTION: This immunoenhancer contains asparagine as active ingredient. For antigen-exhibiting cells or T cells to recognize an antigen, asparagine is essential, and by administering asparagine, immunopotentiation results. This immunity activator is useful as a highly safe cold syndrome therapeutic agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a drug having an effect of enhancing immunity and activating a biological defense reaction.

[0002]

BACKGROUND OF THE INVENTION The cold syndrome is a viral disease,
To date, no causal treatment has been completed. For this reason, conventional treatments provide symptomatic treatments for relieving symptoms, and currently rely on human immunity for radical cure of the disease.

[0003] Immunity plays a very important role in protecting an organism from infection by bacteria, viruses and the like.
In the immune reaction, when antigens such as bacteria and viruses are specifically identified and reacted, important are antigen presentation by antigen presenting cells and antigen recognition by T cells.

[0004] As agents for improving the immune function, α-pinene and β-pinene described in JP-A-8-26979, and microorganisms of the genus Enterococcus described in JP-A-8-99887 are known.

[0005] On the other hand, asparagine is a kind of α-amino acid and is a β-amide form of aspartic acid, but it is not known that asparagine has an immunostimulatory activity, a biological defense reaction activating effect and the like.

[0006]

An object of the present invention is to provide a new immunostimulant.

[0007]

As a result of various studies, the present inventors have found that asparagine is essential for antigen-presenting cells or T cells to recognize antigens, and that administration of asparagine enhances immunity. The present invention has been completed and the present invention has been completed.

That is, the present invention is an immunostimulant containing asparagine as an active ingredient.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The dose of the active ingredient of the immunostimulant of the present invention can be individually determined in consideration of the weight, sex, age and the like of an individual.

[0010] Asparagine, which is an active ingredient of the present invention, is an amino acid and is a substance whose safety is ensured. Therefore, it can be safely administered to a patient who has lost physical strength, and thus the present invention is particularly useful as a remedy for a cold syndrome that enhances the immunity of a patient with a cold syndrome and indirectly promotes treatment.

[0011] The immunostimulant of the present invention can be used in combination with other drugs conventionally used for symptomatic treatment to provide a comprehensive therapeutic agent for diseases. Further, the active ingredient of the present invention can be administered as it is or together with a general carrier used for the production of a pharmaceutical preparation. Tablets, capsules,
Oral preparations such as granules, fine granules, powders and drinks, or parenteral preparations such as injections and suppositories can be produced by a general production method.

[0012]

As apparent from the test examples described below, it was found that asparagine was effective in stimulating immune activity. Therefore, it has become possible to provide a new immunostimulant that is effective for the prevention or treatment of immunity-related diseases such as the cold syndrome.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Experimental method 1) Animals C57BL / 6 mice (male, 6 weeks old) were used as test animals.

2) Medium RPMI1640 medium (GIBCO) and DME medium (GIBCO)
Fetal bovine serum (Fetal Bovine Serum) inactivated at 56 ° C for 30 minutes and 2-mercaptoethanol (Sigma) added to DMEM (hereinafter referred to as “DMEM”) for 30 minutes and used for culture. did.

3) Con A (Concanavalin A, Sigma),
Anti-CD3 antibody (Pharmingen) and PHA (phytohemagglutinin, Sigma) were used as mitogens.

4) Mouse lymphocyte blastogenesis reaction C57BL / 6 mouse (6-week-old, male) spleen cells were suspended in a culture medium at 2.5 × 10 ⁶ cells / ml, and Con A and an anti-CD3 antibody And PHA were added, and the cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 24 hours. Thereafter, the culture supernatant was collected, and [ ³ H] thymidine was added to the cells.
0.5 μCi / well was added thereto, and the cells were further cultured for 4 hours. The cells were collected, and the amount of thymidine incorporation was measured using a liquid scintillation system to determine the cell proliferation ability. The amount of cytokine in the culture supernatant was measured by ELISA. Experiment is trip
Licate was performed and the average value was obtained.

5) Human lymphocyte blastogenesis reaction Mononuclear cells were separated from healthy human peripheral blood by a discontinuous density gradient method, and suspended in a culture medium at a concentration of 1.25 × 10 ⁶ cells / ml. The cell suspension is dispensed on a culture plate immobilized at 37 ° C for 1 hour using 50 μg / ml anti-CD3 antibody and cultured for 24 hours, or the cell suspension is dispensed on an untreated culture plate. After the addition, PHA was added and the cells were cultured for 24 hours, and then the cell proliferation ability was measured. The experiment was performed by triplicate, and the average value was obtained.

2. Results 1) Responsiveness to mitogen After collecting mouse spleen cells, they were suspended in a culture medium and
0, or anti-CD3 antibody with DMEM, ConA and cultured was added and the cytokines in the culture supernatants were harvested 24 hours later the culture supernatants were measured by ELISA, the cultured cells ³ H- thymidine Was added to measure the cell proliferation ability. The results are shown in FIGS.

As apparent from the figure, the anti-CD3 antibody, ConA
In any of the stimuli, a remarkable difference was observed in both cell proliferation and IFN-γ production used as indicators. This indicates that DMEM lacks or lacks a substance that plays an important role in the overall reaction of T cells.

2) Reduction of responsiveness due to amino acid removal Among the amino acids contained in RPMI1640, DMEM does not contain five kinds of amino acids.
A medium was prepared, and the presence or absence of anti-CD3 antibody response of mouse spleen cells was examined. The result is shown in FIG.

As a result, the cell proliferation ability of the anti-CD3 antibody was clearly reduced by the combination in which only asparagine was removed from the five amino acids.

Similarly, mouse splenocytes were cultured in RPMI1640 medium with and without asparagine in the presence of PHA.
After culturing for 8 hours, the cell proliferation ability was measured. The results are shown in FIG.

As apparent from the figure, the cell growth ability was clearly suppressed in the absence of asparagine. From these results, it was found that asparagine is essential for the proliferation of T cells.

3) Asparagine requirement in human peripheral blood lymphocytes A mononuclear cell fraction was separated from human peripheral blood, suspended in RPMI1640 medium containing and not containing asparagine, and cultured with an anti-CD3 antibody or PHA. . Twenty-four hours later, ³ H-thymidine was added and the cells were cultured for 4 hours, and the cell proliferation ability was measured. The results are shown in FIGS.

As is apparent from the figure, also in human peripheral blood lymphocytes, a clear change in reactivity was observed in both the anti-CD3 antibody and the PHA reaction depending on the presence or absence of asparagine. From this, humans as well as mice
Asparagine was found to be required for T cell responses.

[Brief description of the drawings]

FIG. 1 shows the results of measuring immune response ability using cell proliferation as an index. The higher the value, the higher the immune response ability.

FIG. 2 shows the results of measuring the immune response ability using IFN-γ production as an index. The higher the value, the higher the immune response ability.

FIG. 3 shows the results of measurement of cell growth ability in a medium in which various amino acids have been removed from RPMI1640 medium. The higher the value, the higher the cell growth ability.

FIG. 4 shows the results obtained by culturing mouse spleen cells and measuring the cell proliferation ability. The higher the value, the higher the cell proliferation ability.

FIG. 5: Mononuclear cell fraction isolated from human peripheral blood was subjected to anti-CD3
This is the result of culturing with the addition of an antibody. The higher the value, the higher the cell proliferation ability.

FIG. 6 shows the results obtained by culturing a mononuclear cell fraction isolated from human peripheral blood with the addition of PHA. The higher the value, the higher the cell proliferation ability.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Kazuhito Watanabe 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor Takashi Nishimura 2241-8 Iiyama, Atsugi-shi, Kanagawa F-term Reference) 4C206 AA01 AA02 GA20 MA04 ZB09 ZB22

Claims (4)

[Claims] 1. An immunostimulant containing asparagine as an active ingredient. 2. An activator of a biological defense reaction containing asparagine as an active ingredient. 3. An agent for preventing or treating immunity-related diseases, comprising asparagine as an active ingredient. 4. A cold remedy containing asparagine as an active ingredient.