JPH10194976A - Immunosuppressant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject immunosuppressant not producing a serious side effect and excellent in an immunosuppressive action, especially an antilymphocytic action by including a specific linear saccharide as an active ingredient. SOLUTION: This immunosuppressant contains a thermally treated linear saccharide comprising β-1,3-glycoside bonds as an active ingredient. The saccharide preferably comprises a curdlan hydrolysate, especially an acid curdlan hydrolysate using formic acid or the curdlan hydrolysate using β-1,3-glucanase, and has a number-average mol.wt. of 340-4000. The thermal treatment is preferably carried out at a temperature of >=80 deg.C in an aqueous solution. Concretely, a 0.5-5.0wt.% curdlan aqueous solution containing an acid in a concentration of 1-85wt.% is subjected to an acid hydrolysis reaction at a temperature of 70-100 deg.C for about 10min.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunosuppressant having no serious side effects.

[0002]

2. Description of the Related Art The in vivo immune system is composed of bacteria, yeast, mold,
It plays an important role in infection by viruses and other microorganisms and in defense against tumors. The main mechanism is that T lymphocytes and B lymphocytes
To stimulate by recognizing via an antigen receptor, activate it in an antigen-specific manner, and enhance its ability to eliminate foreign substances. However, in the treatment and suppression of autoimmune diseases and rejection of organ transplantation, it is necessary to suppress the immune response, and immunosuppressants are used for that purpose. At present, many non-antigen steroids and drugs acting on nucleic acid synthesis systems are used as immunosuppressants, but these may cause serious side effects. In addition, some immunosuppressants such as cyclosporin used for suppressing rejection of organ transplantation have various side effects.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide an immunosuppressant which exhibits excellent immunosuppressive action without serious side effects.

[0004]

Means for Solving the Problems The present inventors have found that a heat-treated linear β-1,3 glucan exhibits a lymphocyte inhibitory effect in advancing research on saccharides and immunity. Such heat-treated β-1,3 glucan has an action of suppressing the activation of B and T lymphocytes when the immunocompetent cells are stimulated with antigen-specific and non-antigen-specific stimuli. That is, when the heat-treated β-1,3 glucan is added to the culture system of spleen cells, the number of viable cells and the cell metabolic activity when cultured without adding mitogen stimulation are not significantly impaired, and the cells are stimulated under B lymphocyte mitogen stimulation. Live cell count of lymphocytes when cultured, especially increase in live cell count of B lymphocytes and increase of cell metabolic activity are strongly suppressed, and cell metabolic activity is increased when cultured under T lymphocyte mitogen stimulation Was strongly suppressed. This suppression effect
It shows that it is different from the non-specific action exhibited by conventional immunosuppressants because it acts more selectively when lymphocytes are activated, and also strongly suppresses the activation of B lymphocytes. Thus, the results show that the immunosuppressive agents proposed in recent years differ from the selective action on T lymphocytes. Therefore, various side effects observed in known steroid drugs, serious side effects such as hematopoietic organs observed in drugs acting on the nucleic acid synthesis system, cyclosporine, FK50
It is considered that there are no side effects such as renal damage and liver damage observed in No. 6. In addition, since the heat-treated β-1,3 glucan strongly suppresses the activation of B lymphocytes, autoimmune diseases such as malignant lymphoma, systemic lupus erythematosus, rheumatoid arthritis, etc. or allergy caused by B lymphocyte abnormalities. It is extremely useful for treating diseases. Furthermore, since it suppresses the activation of T lymphocytes, it is considered to be useful for preventing rejection of organ transplantation.

The present invention has been completed on the basis of the above findings, and provides an immunosuppressant comprising, as an active ingredient, a heat-treated linear saccharide comprising a β-1,3-glucoside bond. It is. Examples of the linear saccharide comprising a β-1,3-glucoside bond include a curdlan hydrolyzate. In particular, acid hydrolysates, especially formic acid hydrolysates, and hydrolysates of enzymes such as β-1,3-glucanase, having a number average molecular weight of 340 to 4000
Are preferred. As the heat treatment, heating at 80 ° C. or higher in an aqueous solution is preferable. The immunostimulant of the present invention has no side effects, is suitable for ordinary use, and is suitable as a drug for immunosuppression.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, as a linear saccharide comprising a β-1,3-glucoside bond subjected to a heat treatment, a glucose molecule is not branched, and a β-1,3-glucoside bond is formed. Oligosaccharides or polysaccharides linked by the following are typical examples: Alkaligenes (Alkaligene)
s) A hydrolyzate of curdlan, a polysaccharide having a β-1,3-glucoside bond, produced by a bacterium of the genus or Agrobacterium genus. Hereinafter, the present invention will be described using a hydrolyzate of curdlan as an example. The hydrolyzate of curdlan can be prepared by a known polysaccharide hydrolysis method.However, from the immunosuppressive activity of the obtained hydrolyzate, organic acids such as formic acid and acetic acid, and inorganic acids such as hydrochloric acid and sulfuric acid are used. In particular, it is preferable to use an enzyme such as formic acid or β-1,3-glucanase, and the number average molecular weight is preferably in the range of 340 to 4000.

[0007] The number average molecular weight in the present specification is measured by high performance liquid chromatography (HPLC) under the following conditions. As a measuring device, Tosoh Corporation
A high performance liquid chromatography device manufactured by KK was used. Detector: RI-8022 Pump: CCPM-II Column oven: CO-8020 Deaerator: SD-8022 Autosampler: AS-8020 The measurement column is a TSK gel column (G-OLIGO-
PW or G-3000PWXL, 7.8φmm × 30c
m) at a flow rate of 0.6 to 0.7 ml / min and a temperature of 40 ° C. A 0.02% aqueous solution of the sample was prepared and its 200
μl was used. Elution was performed with pure water. On the other hand, a calibration curve was created by similarly measuring a pullulan standard product with a known molecular weight,
Based on this calibration curve, GPC-LALL of Tosoh Corporation
The molecular weight distribution function was determined by the S program and the calculation program “Excel” of Microsoft Corporation, and converted as a number average molecular weight.

In the acid hydrolysis, for example, a 0.5 to 5.0% by weight aqueous solution of curdlan containing about 1 to 85% by weight of an acid is heated at 70 to 100 ° C. for 10 minutes to 3 hours. Can be performed. The hydrolysis reaction solution is neutralized with an alkali agent such as sodium hydroxide and centrifuged to obtain a supernatant, and if necessary, activated carbon treatment, dialysis, solvent fractionation, removal of formyl group by heating, etc. are known per se. The desired hydrolyzate can be obtained by the purification according to the above method. Enzymatic hydrolysis can be performed, for example, by subjecting a 0.5-3% by weight aqueous solution of curdlan to an enzyme treatment at a pH and temperature suitable for the enzyme to be used for a predetermined time. Then, after inactivating the enzyme, a supernatant is obtained by centrifugation, and if necessary, purified by a method known per se such as activated carbon treatment, dialysis, or solvent fractionation, to obtain a desired hydrolyzate. Can be

[0009] The obtained hydrolyzate is subjected to a heat treatment in the state of an aqueous solution. The heat treatment is generally performed at 80 ° C. or higher,
Preferably, it is carried out by heating at 90 to 120 ° C., usually for 10 to 30 minutes. The heating means is not particularly limited, and is immediately cooled to room temperature after heating.

[0010] The heat-treated curdlan hydrolyzate is
It can be used as it is as the immunosuppressant of the present invention. Also,
It can be combined with a known pharmaceutical carrier or excipient by a known method to obtain a drug for immunosuppression. Use,
The pharmaceutical carrier or excipient is not particularly limited, and can be appropriately selected by those skilled in the art according to the specific use of the immunosuppressant. Also, the form of the immunosuppressant is not particularly limited, and can be in various solid or liquid forms depending on the specific application. The immunosuppressant of the present invention may be administered orally or parenterally, and its dosage is 4 mg to 40 g per day as a solid content of the curdlan hydrolyzate. The immunosuppressive agent of the present invention can be used for various side effects observed in steroids, serious side effects such as hematopoietic organs observed in drugs acting on the nucleic acid synthesis system, cyclosporine, F
K506 has no side effects such as renal damage and liver damage, and the present invention strongly suppresses the activation of B lymphocytes, so that malignant lymphomas caused by B lymphocyte abnormalities, systemic lupus erythematosus, chronic joint It is extremely useful for treating autoimmune diseases such as rheumatism or allergic diseases. Furthermore, it suppresses the activation of T lymphocytes, and is therefore useful for preventing rejection of organ transplantation.

[0011]

Next, the present invention will be described more specifically with reference to examples and test examples, but the present invention is not limited to these examples. Example 1 Preparation of Curdlan Hydrolyzate with Enzyme 4 g of curdlan was dispersed in 200 ml of 0.05 M acetate buffer, and 18 units / ml of Hilotake enzyme was added. This was heated to 40 ° C., incubated for 4 hours, and then kept in a boiling water bath for 15 minutes to inactivate the enzyme. Then, the precipitate was removed by cooling and centrifugation, and the supernatant was adsorbed on activated carbon packed in a column having a diameter of 5 cm and a height of 30 cm.
After washing with 0 ml of water, it was further washed with 2000 ml of 4% ethanol. Subsequently, the adsorbed component was eluted with 2000 ml of 20% ethanol, and this was concentrated by an evaporator and freeze-dried. This had a number average molecular weight of 340 (pullulan equivalent) as measured by HPLC. This was heated in an aqueous solution at 100 ° C. for 10 minutes, and then cooled to obtain an immunosuppressant.

Example 2 Preparation of Curdlan Hydrolyzate with Formic Acid 30 grams of curdlan was dispersed in 3000 ml of 85% formic acid, heated to 90 ° C. and kept for 20 minutes. Then, the whole container was exposed to cold water to cool to room temperature, concentrated by an evaporator, neutralized with 5N NaOH to pH 7, and centrifuged. The supernatant was heated in a boiling water bath for 120 minutes to remove formyl groups. At this time, the pH dropped, and 2N NaOH was added to return to 7. This was placed in a bisking tube, dialyzed overnight against 10 liters of pure water, and the dialysate was freeze-dried. This is HP
The number average molecular weight measured by LC was about 2,800 in terms of pullulan. This was heated in an aqueous solution at 100 ° C. for 10 minutes, and then cooled to obtain an immunosuppressant.

Test Example 1 Using the enzyme-degraded curdlan obtained in Example 1 and the formate-degraded curdlan obtained in Example 2, heat-treated products of enzymatically degraded curdlan and proliferation of formate-degraded curdlan for mouse spleen lymphocyte proliferation reaction By examining the action of the products, the heat-treated products decomposed with enzymatically decomposed curdlan and heat-treated products with decomposed formic acid were examined to increase the lymphocyte metabolic activity and suppress lymphocyte proliferation. Mouse (C57BL / 6, female, 14
Aged), the spleen was aseptically removed, crushed in an RPMI1640 medium, and passed through a 200-mesh sieve to obtain a spleen cell suspension. After measuring the number of cells in the spleen cell suspension using an automatic blood cell counter, the number of cells was determined to be 5 × 10 ⁶ / ml.
Was prepared in RPMI1640 medium to a concentration of 100 μl, and 100 μl per well was seeded on a 96-well tissue culture plate. The B lymphocyte proliferation stimulator lipopolysaccharide was added at a concentration of 200 micrograms / ml to RPMI 1640.
A solution dissolved in a medium, T-lymphocyte proliferation stimulating substance concanavalin A was added at a concentration of 8 microgram / ml to RPMI16.
A solution dissolved in 40 media or an RPMI 1640 medium was added to the spleen cell suspension seeded at 50 microliters per well, to prepare a B lymphocyte stimulation group, a T lymphocyte stimulation group, and a non-stimulation group. A phosphate buffered saline solution heated at 100 ° C. for 10 minutes and cooled (control) or an enzyme-degraded curdlan at a concentration of 8 mg / ml was dissolved in phosphate buffered saline solution at 100 ° C. A liquid cooled by heating for 10 minutes, formic acid decomposed curdlan was dissolved in a phosphate buffered saline at a concentration of 8 mg / ml, and heated and cooled for 10 minutes at 100 ° C., and 50 microliters were added to each well. The cells were cultured in a 5% carbon dioxide incubator at 37 ° C. for 2 days, and the number of living cells and the cell metabolic activity after the culture were examined.

The number of viable cells was determined by measuring the number of cells in the cultured cell solution using an automatic hemacytometer, and then adding 200 μl of the cultured cell solution to the anti-mouse CD45R specific antibody against mouse B lymphocytes labeled with R-phycoerythrin. One microgram of antibody was used as an anti-mouse T cell receptor (alpha / beta) antibody specific for mouse T lymphocytes labeled with fluorescein isothiocyanate.
7- Specific staining of micrograms and dead cells
Add 1 microgram of aminoactinomycin D and add
After being left at 30 ° C. for 30 minutes, the plate was washed with RPMI1640 medium, and the proportion of B lymphocytes and T lymphocytes in the total cells and the proportion of dead cells in B lymphocytes and T lymphocytes were measured with a flow cytometer. Viable cell counts of lymphocytes and T lymphocytes were calculated. Cell metabolism activity
Three hours before the end of the culture, 3- (4,5-dimethyl-bromide)
A solution of 2-thiazolyl) -2,5-diphenyl-2H tetrazolium in RPMI1640 medium at a concentration of 5 mg / ml was added at 10 μl per well, and at the end of the culture, a 20% sodium dodecyl sulfate solution was added at 50 μl per well. After adding 1 liter and left at 37 ° C. for 1 day, the cell metabolic activity was determined by measuring the absorbance of the culture at 550 nm using a microplate reader. Table 1 shows the results.

[0015]

[Table 1]

As shown in Table 1, in the non-stimulated group,
Both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products with degraded formic acid curd only slightly reduced the viable cell count of B lymphocytes and had little effect on the cell metabolic activity. Both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products decomposed with formic acid decomposed significantly reduced the number of viable B lymphocytes and completely suppressed the increase in cell metabolic activity. In the T lymphocyte stimulation group, both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products degraded with formic acid decomposed significantly suppressed the increase in cell metabolic activity. As described above, both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products decomposed with formic acid degraded more selectively when lymphocytes were activated, and exhibited a strong inhibitory action.

Test Example 2 Laminaribiose, laminaritriose, laminaritetraose, laminaripentaose and laminarihexaose (Yaitsu Fisheries Chemical) were used to heat-treat the spleen lymphocyte proliferation reaction in mice. Liviose,
By examining the effects of heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminaripentaose, and heat-treated laminarihexaose, heat-treated laminaribiose, heat-treated laminaritriose, and heat-treated laminaritetraose The effect of aose, heat-treated laminaripentaose and heat-treated laminarihexaose on the increase in lymphocyte metabolic activity was verified. Mouse (C57BL / 6,
Female, 18 weeks old).
The spleen was crushed in 640 medium and passed through a 200-mesh sieve to obtain a spleen cell suspension. After measuring the number of cells in the spleen cell suspension using an automatic blood cell counter, the number of cells was determined to be 5 × 1.
In RPMI1640 medium was prepared to a concentration of 0 ⁶ / ml, 96
100 microliters per well were seeded in a well tissue culture plate. The B lymphocyte proliferation stimulator lipopolysaccharide was added at a concentration of 200 micrograms / ml in RPMI.
A solution dissolved in 1640 medium, a T lymphocyte proliferation stimulating substance, concanavalin A, was added at a concentration of 8 microgram / ml to RP.
Solution dissolved in MI1640 medium or RPMI16
Forty B medium was added to the spleen cell suspension sown at 50 microliters per well, and the B lymphocyte stimulation group was added.
A T lymphocyte stimulation group and a non-stimulation group were set. To these three groups, a solution prepared by heating and cooling phosphate buffered saline at 121 ° C. for 20 minutes (control) or laminaribiose, laminaritriose, laminaritetraose, laminaripentaose and laminarihexaose were added. 8mg / ml concentration or 1
dissolved in phosphate buffered saline at a concentration of mg / ml
The solution cooled by heating at 20 ° C. for 20 minutes was added in an amount of 50 μl / hole, and cultured in a 5% CO 2 incubator at 37 ° C. for 1 day, and the cell metabolic activity after the culture was examined.

The cell metabolic activity was determined by measuring 3- (4,5-dimethyl-2-thiazolyl) -2,5 bromide 3 hours before the end of the culture.
10 microliters per well of a solution of diphenyl-2H tetrazolium dissolved in RPM11640 medium at a concentration of 5 mg / ml was added at the end of the culture, and 50 microliters per well of a 20% sodium dodecyl sulfate solution was added at 37 ° C for 2 days. After standing, the cell metabolic activity was determined by measuring the absorbance of the culture at 550 nm using a microplate reader. Table 2 shows the results.

[0019]

[Table 2]

As shown in Table 2, in the unstimulated group,
Heat-treated laminaribiose, heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminalipentaose, and heat-treated laminalihexaose hardly affected cell metabolic activity,
In the B lymphocyte stimulation group, heat-treated laminaribiose, heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminaripentaose, and heat-treated laminarihexaose were all 2 mg / ml and 0.4 mg / ml.
At a concentration of mg / ml, the increase in cell metabolic activity was completely suppressed. In the T lymphocyte stimulation group, heat-treated laminaribiose, heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminaripentaose, and heat-treated laminarihexaose were all cells at a concentration of 2 mg / ml. Although the increase in metabolic activity was completely suppressed, it was confirmed that the metabolic activity was increased by 0.1%.
At a concentration of 4 mg / ml, only heat-treated laminaribiose and heat-treated laminaritriose partially suppressed the increase in cell metabolic activity. Thus, the heat-treated laminaribiose, the heat-treated laminaritriose, the heat-treated laminaritetraose, the heat-treated laminaripentaose, and the heat-treated laminarihexaose are all more selective when lymphocytes are activated. In particular, it exhibited a strong inhibitory action, and particularly had a strong action on B lymphocytes. The lymphocyte inhibitory activity was strong in heat-treated laminaribiose and heat-treated laminaritriose.

[0021]

According to the present invention, there are no serious side effects,
An excellent immunosuppressant is provided.

 ────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Hiroaki Kusaka 3-3-1202 Asakuraminami, Takarazuka City, Hyogo Prefecture

Claims (8)

[Claims] An immunosuppressant comprising a heat-treated linear saccharide comprising a β-1,3-glucoside bond as an active ingredient. 2. The immunosuppressant according to claim 1, wherein the linear saccharide comprising a β-1,3-glucoside bond is a curdlan hydrolyzate. 3. The immunosuppressant according to claim 2, wherein the hydrolyzate is an acid hydrolyzate. 4. The immunosuppressant according to claim 3, wherein the acid used for the acid hydrolysis is formic acid. 5. The immunosuppressant according to claim 2, wherein the hydrolyzate is a hydrolyzate by β-1,3-glucanase. 6. The immunosuppressant according to claim 1, wherein the linear saccharide comprising a β-1,3-glucoside bond has a number average molecular weight in the range of 340 to 4000. 7. The immunosuppressant according to claim 1, wherein the heat treatment is performed at a temperature of 80 ° C. or higher in an aqueous solution. 8. The immunosuppression according to claim 1, wherein the immunosuppression action is a lymphocyte suppression action.