JPH0714885B2 - Immunoactive substance - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to an immunologically active substance effective for chronic hepatitis B and the like.

<< Background of the invention >> It is said that there are 1.1 million chronic hepatitis patients nationwide.
Chronic hepatitis B, which accounts for 0%, is mostly converted to cirrhosis,
Furthermore, it is a viral disease with a high mortality rate that progresses to liver cancer. In addition, there are 2 million healthy carriers who are infected with hepatitis B virus (HBV) but do not show hepatitis symptoms at all, and it is said that about 10% of them carry on to chronic hepatitis. There is a high risk that HBV infection will spread due to horizontal infection or vertical infection (mother-to-child transmission) caused by people, which has become a major social problem. A vaccine against HBV has been developed in recent years, and great results can be expected in the future in the prevention of hepatitis B, but at present, an effective treatment method has not been established for patients who have already developed the disease or healthy carriers. Therefore, the establishment of a treatment method is strongly desired.

Hepatitis B is a disease in which liver damage is caused by HBV infection, but since there are many asymptomatic carriers, HBV
It is clear that it does not destroy hepatocytes, and liver damage is thought to be caused by the host's immune response to HBV. That is, if the immune system is normal, hepatitis develops because cell-mediated immunity activates and destroys HBV-infected hepatocytes. When the infected cells are destroyed, the virus is released into the blood, but at the same time, humoral immunity works to produce antibodies against HBV and HBV is completely eliminated. Therefore, the infection does not persist and heals with the image of acute hepatitis. However, HB
When the immune response to V is lowered, cell-mediated immunity is activated to eliminate infected cells, resulting in liver damage. However, the elimination is not sufficient, and infected cells remain. In addition, since the production of humoral antibodies was not sufficient, HBV could not be completely eliminated, and HBV released from destroyed infected cells
V infects other hepatocytes one after another and falls into a state where destruction and infection are repeated. Therefore, infection continues and liver damage becomes chronic.
If no response to HBV occurs, cell-mediated immunity does not work and infected hepatocytes are not destroyed, but since humoral antibodies are not produced, HBV cannot be eliminated and HBV is growing in the body. Being an asymptomatic carrier who does not develop hepatitis at all.

As described above, it can be said that the onset of hepatitis is a positive response of the body in which the host tries to eliminate HBV by its immune system, and it is considered that the onset state differs depending on the difference in immune response. . That is, persistent infection is caused by a decrease in humoral immunity and cell-mediated immunity to HBV.
It is believed that chronic hepatitis B develops.

By the way, in recent years, β-D-glucan-based polysaccharides that exhibit antitumor activity by activating the immune system from basidiomycetes have been separated one after another, and attention has been paid to the immunostimulatory action of basidiomycetes. In addition, it was prepared from the mycelial extract of shiitake mushroom, which is an edible mushroom.
The immunostimulatory ability of LEM (Japanese Patent Publication No. 53-23392) is also attracting attention, and treatment of chronic hepatitis B based on it has been tried and effective cases have been issued (The 71st Annual Meeting of the Japanese Society of Gastroenterology, Japan Gastroenterology). Journal of the Japanese Society of Diseases; Vol.82, 1105 (1985)).

From the above-mentioned mechanism of chronic hepatitis B, it is considered that substances that activate the immune system are effective in treating this disease.
Therefore, isolation and purification of an immunologically active substance from LEM to provide it as a therapeutic agent will be useful as a therapeutic agent for chronic hepatitis B or other immunodeficiency diseases for which there is no completely effective therapeutic agent at present.

<< Object of the Invention >> The main object of the present invention is to provide an immunologically active substance effective against chronic hepatitis B or other immunodeficiency diseases by activating the immune system.

<< Structure of the Invention >> A molecular weight of 6 to 1.5 million (fraction A) extracted from a mycelium culture of shiitake mushrooms or a mycelium culture containing fruiting bodies cultured in a solid medium containing a fibrin component as a main material, and 1.5 million-80
A high-molecular immunoactive substance having the following properties, which contains 10,000 (fraction B) sugars.

(A) The sugar content of the A fraction is 42.8-53.6%, and the protein content is
28.0 to 37.4%. The sugar content of the B fraction is 52.3 to 66.3%, and the protein content is 16.0 to 23.6%.

(B) Contained sugars contain almost no acidic or amino sugars, and 95% or more are neutral sugars.

<< Effect of the invention >> This substance is purified from LEM as a starting substance. LEM is a dry powder obtained by culturing Lentinus edodes in a solid medium, extracting with hot water before fruit body molding (Japanese Patent Publication No. 53-23392). The immunoreactivity of the substances constituting the present invention is surprisingly stronger than that of the starting material, LEM.
In terms of activity, it is far superior to LPS (lipopolysaccharide), which is a typical mitogen currently known.

Typical immunoreactivity of this substance is activation of macrophages, IL-1 (interleukin 1) production ability, mitogen activity, LPS-induced B cell blastogenesis, antibody production ability, killer T cells in the A fraction. It has the effect of activating and enhancing any of the immune effects of activation. In the B fraction, macrophage activation, IL-1 production, mitogen activity, L
It has the effect of activating and enhancing any of the immune effects of PS-induced B cell blastogenesis, antibody-producing ability, and activation of natural killer cells.

Therefore, this substance is considered to be useful as a therapeutic agent for chronic hepatitis B or other immunodeficiency diseases for which there is no completely effective therapeutic agent at present.

Hereinafter, the method for preparing the immunologically active substance constituting the present invention will be described with reference to Examples, and the usefulness of this substance will be described with reference to Test Examples.

<Example> Preparation method of this substance from LEM This substance is purified from LEM using it as a starting material. LEM is a dry powder obtained by culturing Lentinus edodes in a solid medium, extracting with hot water before fruit body molding (Japanese Patent Publication No. 53-23392). Add 200 ml of distilled water to 10 g of LEM and stir well with a stirrer. This was centrifuged at 6000 rpm for 20 minutes to remove insoluble matter, and 200 ml of ethanol was added to the supernatant while stirring under ice cooling. By centrifugation at 7,000 rpm for 20 minutes, 980 mg of precipitate (precipitate 1) was obtained. This is repeated to collect the precipitate 1.
For 4 g of the collected precipitate 1, 300 ml of 50 mM phosphate buffer solution (pH
7.2) and then add 200 ml of saturated aqueous ammonium sulfate solution to 40
% Saturated ammonium sulfate aqueous solution, leave it at 4 ° C for 12 hours,
Collect by centrifuging at 00 rpm for 20 minutes. This was dissolved in a small amount of distilled water, dialyzed to remove ammonium sulfate, and freeze-dried to obtain 100 mg of a dried product (precipitate 2). This precipitate 2 is applied to a gel filtration column such as Toyopearl HW-65S (2.5 x 100 cm). The eluent is 50 mM phosphate buffer pH 6.7. Figure 1 shows the elution curve
Shown in. The vertical axis represents the absorption at 280 nm logarithmically, and the horizontal axis represents the elution volume. The positions indicated by a, b, and c in FIG. 1 are the elution locations of the molecular weight markers, and the subscripts represent the molecular weight in tens of thousands. a is blue dextran having a molecular weight of 2,000,000, b is thyroglobulin having a molecular weight of 670,000, and c is ferritin having a molecular weight of 460,000. The shaded A and B fractions in FIG. 1 are macromolecular immunoactive substances constituting the present invention containing sugars having molecular weights of 6 to 1.5 million and 1.5 to 800,000, respectively.
The yields of the A fraction and the B fraction were 6 mg and 17 mg, respectively.

Next, Table 1 shows the sugar content and protein content of the immunoreactive substance of the present invention and the starting substance, LEM.

However, the total sugar content was determined by the phenol-sulfuric acid method using glucose as a standard (M. Dubois et al., Anal. Chem. 28, p. 350, 1956).
), The quantitative determination of acidic sugar was Blumen with glucuronic acid as a standard.
krantz et al. (N. Blumenkrantz et al., Anal. Biochem., 54
Vol. 484, 1973), analysis of amino sugars was carried out by gas chromatography with alditol acetate derivatives (R. Ga.
tt et al., Anal. Biochem., 15: 167, 1966).
Lowry method using bovine serum albumin as a standard (OHLowry et al., J. Biol. Chem. 193, 265, 1951)
I did it in.

Next, an example of the composition of the neutral sugar which is the main component of this immunoactive substance is shown in Table 2 in comparison with LEM.

However, the sugar composition was analyzed by GC-MS using an alditol acetate derivative (P. Albersheim et al., Carbohydr.
s., 5 pages, 340 pages 1967).

Test Example 1 Assay of macrophage activating ability of this substance Macrophages are cells that present antigens to the immune system, so to speak, the cells of the immune system, and play an extremely important role in the immune response. It is said that activation of macrophages has several steps, and it is known that glycolysis is promoted with the activation of the first step. Therefore, the macrophage activation ability of this substance was evaluated as an increase in glucose consumption relative to a control, and determined as an activation index (SI%).

1 ml of a sterilized 10% thioglycolic acid medium (Nissui Pharmaceutical Co., Ltd.) is injected into the abdominal cavity of a 6-8 week old male ddy mouse to induce exudative macrophages. Mice were sacrificed after 4 days and 10
CMF-HBSS (Ca ²⁺ , Mg ²⁺ f containing unit / ml heparin
ree Hank's balanced salt solution) is put into the abdominal cavity of about 5 ml using a syringe, and then a solution containing cells is sucked out from the abdominal cavity with a Pasteur pipette and received by a centrifuge tube through a 150 mesh filter. Centrifuge at 800 rpm for 5 minutes, aspirate the supernatant, and wash the cells with 5 ml of heparin-free HBSS. The washed cells are suspended in RPMI1640 medium,
Dispense 2 × 10 ⁵ cells per well into a 96-well plate, incubate for 1 hour, shake the plate gently, and then suck out the supernatant.
Exclude non-adherent cells. The RPMI1640 described above is
μl was added and the substance was dissolved in phosphate buffer.
Add 20 μl of the solution sterilized by filtration through a 22 μm membrane filter to make the total volume 200 μl. After culturing at 38 ° C. for 48 hours, 10 μl of the culture supernatant was collected, color was developed by the glucose oxidase method (Glucose B-Test Wako: Wako Pure Chemical Industries), and the glucose content was measured from the absorbance at 505 nm (A ₅₀₅ ). The activation index SI (%) was calculated according to the following formula.

The results are shown in Figure 2. A large increase in activity was observed in both fractions compared to LEM, and it exceeded that of LPS, which is known to have a strong macrophage activating ability.

Test Example 2 Assay of mitogen activity of this substance Lymphocytes are transformed into large blast-like cells by stimulating with antigens and various mitogens (mitogens). Now mi
Various substances are known as togen. Typical examples include LPS that specifically reacts with B cells and PHA that specifically reacts with T cells. It is known that these stimuli cause lymphocytes to promote DNA synthesis and cell division. Therefore, the amount of DNA synthesis was evaluated by the amount of ³ H-thymidine incorporated into cells, and the mitogen activity of this substance was assayed.

Spleens are collected from BALB / c male mice (6 to 8 weeks old), and the cell mass is thoroughly dissociated and suspended in Eagle's MEM. After passing through a 150-mesh filter, the cells are washed well and 5 × 10 ⁵ cells are dispensed into a 96-well plate. Add a solution of this substance to make the total volume 200 μl and incubate for 72 hours. After completion of the culture, 0.2 μCi of ³ H-thymidine is added per well to make the total volume 250 μl. After culturing, cells are trapped in a glass filter using a cell harvester. After drying the glass filter, the radioactivity incorporated into the cells is measured by a liquid scintillation counter. All experiments were carried out in quadruplicate, and the ratio of the count number of the experimental group to the count number of the control group (dpm) is expressed as the activation index (SI%).

The results are shown in Fig. 3.

Both fractions showed strong mitogen activity, which was 175% SI in LPS, which is a typical B cell mitogen, whereas 96 μ% in 20 μg / ml A fraction and 96 μ% in 20 μg / ml B fraction. It was remarkably strong at 1166%. Both fractions have the action of strongly activating macrophages, as is clear from Test Example 1. It is known that the activated macrophages release lymphokines, which cause the lymphocytes to become young. Then, the same activity was assayed in the system except macrophages, but the mitogen activity of this substance did not decrease. Therefore, it is considered that the lymphocyte transformation of this substance directly acts on lymphocytes without going through macrophages.

Test Example 3 LPS-modifying effect of this substance on LPS In Test Example 2, it was revealed that both fractions had an effect of blasting lymphocytes. The specificity of this effect was tested. The modification effect on blastogenesis by LPS, which is a mitogen that acts specifically on B cells, was examined. That is, LP
If it promotes the blastogenesis by S, it will act on B cells.

As in Test Example 2, a mouse spleen cell system was used to establish an experimental system.
LPS of 1 μg / ml was added as a mitogen to induce a juvenile reaction, and a solution of this substance was added thereto to examine changes in the juvenile reaction by mitogen. At this time, the substance itself is mi
Considering that they have togen activity, groups without mitogen were set as controls, respectively, and by subtracting this, mito
The effect of modifying gen-derived blastogenesis was evaluated. The modification index (SI%) was calculated according to the following formula.

The results are shown in Fig. 4.

Both fractions showed strong stimulatory activity against LPS blastogenesis, and 20 μg / ml of B fraction showed nearly 8-fold stimulation. That is, it was found that both fractions have the effect of promoting the proliferation of B cells, and further acting on B cells that have already undergone blastogenesis and further enhancing their proliferation.

Safety test We requested Nomura Institute of Biological Sciences for toxicity test of the starting material, LEM, and conducted "acute toxicity test" and "subacute toxicity test" on animals at the same place. The “safety test” has been carried out and the safety of this substance has been confirmed.

Based on these results, LEM, which is the starting material, was given to healthy subjects to study side effect symptoms, abnormal laboratory tests, etc.
A phase test was conducted. The test subjects were 5 adult males and 4 females who had been desired and were diagnosed as healthy as a result of the medical examination. The single dose was 6 g once. Further, after a single administration, 5 males were continuously administered with 6 g per day, and 1 month and 6 months after that, various tests such as general examination, clinical examination, electrocardiogram, subjective symptom were performed. As a result, there were no abnormal values or side effects that were considered to be the effects of this substance.

From the above test results, the safety of LEM, which is the starting material for this substance, was confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an elution curve of gel filtration of A fraction and B fraction by Toyopearl HW-65S, and FIG. 2 is a bar graph showing macrophage activating ability of A fraction and B fraction. Fig. 3 is a bar graph showing the mitogen activity of the A fraction and the B fraction, and Fig. 4 is the A fraction.
3 is a bar graph showing the modifying effect of B fraction on B cell blastogenesis induced by LPS.

Claims (2)

[Claims] 1. A molecular weight of 6 to 1.5 million (fraction A) extracted from a mycelium culture of shiitake mushroom or a mycelium culture containing fruiting bodies cultured in a solid medium containing a fibrin component as a main material, and A macromolecular immunoactive substance having the following properties, containing 1.5 to 800,000 (fraction B) sugars. (A) The sugar content of the A fraction is 42.8-53.6%, and the protein content is
28.0 to 37.4%. The sugar content of the B fraction is 52.3 to 66.3%, and the protein content is 16.0 to 23.6%. (B) Contained sugars contain almost no acidic or amino sugars, and 95% or more are neutral sugars. 2. The immunoactive substance according to claim 1, wherein the solid medium comprises bagasse and rice bran.