JPS62270532A - Immunologically active substance - Google Patents

Abstract

PURPOSE:The titled substance, containing a saccharide extracted from a specific mycelial culture and effective for chronic hepatitis B, etc. CONSTITUTION:A high polymer immunologically active substance obtained by containing a saccharide (>=95% contained glucide is neutral saccharide hardly containing acidic saccharide or amino sugar), extracted from a mycelial culture of SHIITAKE mushrooms cultivated in a solid culture medium, e.g. bagasse or rice bran, containing cellulosic components as a principal material or mycelial culture containing fruit bodies and having 6,000,000-1,500,000mol.wt. (fraction A; 42.8-53.6% glucide content and 28.0-37.4% protein content) and 1,500,000-800,000mol.wt. (fraction B; 52.3-66.3% glucide content and 16.0-23.6% protein content). The immunological activity is useful in activating macrophages, enhancing IL-1 productivity, mitogen activity, etc., in the fraction A and activating and enhancing action of the similar activities, other natural killer cells in the fraction B. Thereby this substance becomes useful as a remedy for immunological deficiency.

Description

[Detailed Description of the Invention] 3. Detailed Description of the Invention (Field of the Invention) This invention relates to an immunostimulatory substance effective against chronic hepatitis B and the like.

(First part of the invention) It is said that there are 1.1 million chronic hepatitis patients nationwide, and chronic hepatitis B, which accounts for about 30% of them, progresses to liver cirrhosis, which progresses to liver cancer and has a high mortality rate. It is a viral disease. In addition, hepatitis B virus (1-I B
There are as many as 2 million healthy carriers who are infected with M. There is a high risk of HBV infection spreading due to (mother-to-child transmission), which has become a major social problem. In recent years, a vaccine against 1-IBM has been developed, and great results are expected in the prevention of hepatitis B. However, at this stage, there is no effective treatment for patients who have already developed the disease or those who are healthy carriers of the disease. It has not been established yet, and there is a strong desire to establish a treatment method.

Hepatitis B is a disease in which liver damage is caused by infection with IBV, but it is clear that HBV itself does not destroy liver cells, as there are many asymptomatic carriers.
Liver damage is thought to be caused by the host's immune response to IBV. In other words, if the immune system is normal,
Hepatitis develops because cellular immunity operates against HBV-infected liver cells and destroys them. When infected cells are destroyed, the virus is released into the blood, but at the same time, humoral immunity is activated to produce antibodies against l-IBM, and HBV is completely eliminated. Therefore, the infection does not persist and the patient shows symptoms of acute hepatitis and is cured. However, when the immune response to IBV is reduced, cellular immunity is activated and the infected cells are eliminated, resulting in liver damage, but the elimination is not sufficient and the infected cells remain. Furthermore, since the production of humoral antibodies is insufficient, HBV cannot be completely eliminated, and HBV released from destroyed infected cells infects other liver cells one after another, resulting in a state of repeated destruction and infection. 3. Therefore, infection persists and liver damage becomes chronic. In addition, if no response to l'BV occurs at all, cell-mediated immunity does not work and infected hepatocytes are not destroyed, but humoral antibodies cannot be produced, so l'BV cannot be eliminated, and HBV proliferates in the body. They become asymptomatic carriers who do not develop hepatitis even though they are infected with the virus.

As mentioned above, the onset of hepatitis can be said to be a positive biological response in which the host tries to eliminate HBV by its immune system, and the onset status is thought to vary depending on the strength of the immune response. . That is, it is thought that chronic hepatitis B develops due to persistent infection caused by a decline in humoral immunity and cell-mediated immunity against HBV.

Incidentally, in recent years, β-D-glucan polysaccharides that exhibit antitumor activity by activating the immune system have been isolated one after another from basidiomycetes, and the immunostimulatory effects of basidiomycetes have attracted attention. In addition, L prepared from the mycelial extract of Shiitake mushroom, an edible mushroom,
The immunostimulatory ability of FM (Special Publication No. 53-23392) has also attracted attention, and treatments for chronic hepatitis B have been attempted based on this, and effective cases have been reported (71st General Meeting of the Japanese Society of Gastroenterology, Japan Gastroenterology Society). Journal of the Medical Society: Vol, 82.1105 (198
5)).

Based on the mechanism of onset of chronic hepatitis B described above, it is thought that substances that activate the immune system are effective in the treatment of this disease.

Therefore, isolating and purifying an immunoactive substance from LEM and providing it as a therapeutic agent would be useful as a therapeutic agent for chronic hepatitis B or other immunodeficiency diseases for which there are currently no completely effective therapeutic agents.

(Objective of the Invention) The main object of the present invention is to provide an immunostimulating substance that is effective against chronic hepatitis B or other immunodeficiency diseases by activating the immune system.

(Structure of the Invention) Molecules ff 16 million to 1.5 million (
A polymeric immunoactive substance having the following properties, containing 1,500,000 to 800,000 sugars (A fraction) and 1.5 to 800,000 sugars (B fraction).

(a) The carbohydrate content of fraction A is 42.8 to 53.6%, and the protein content 1 is 28.0 to 37.4%. The carbohydrate content of fraction B is 52.3-66.3%, and the protein content is 16.0-23.
6%.

(b) Containing carbohydrates contain almost no acidic sugars or amino sugars, and more than 95% are neutral sugars.

(Effects of the Invention) The present substance is purified from LEM using LEM as a starting material. L
EM is Shiitake mushroom (1-entinus edodes)
is cultured in a solid medium, extracted with hot water before forming fruiting bodies, and dried into powder. The immunoreactivity of the substances constituting the present invention is surprisingly stronger than that of the starting material LFM, and for example, in terms of m+togen activity, it is far superior to LPS (ribobolitichalide), which is the representative m+togen currently known. It's so much more than that.

Typical immune activities possessed by this substance include macrophage activation, ll-1 (interleukin 1) production ability, mi togen activity, LPS-induced brain cell blastogenesis ability, and killer T activity in the A fraction. It has the effect of activating and enhancing both immune effects such as cell activation. In addition, in the B fraction, macrophage activation, Ill production ability, m
It has the effect of activating and enhancing the immune effects of itogen activity, LPS-induced brain cell blastogenesis, and natural killer cell activation.

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

EXAMPLES Below, the method for preparing the immunoactive substance constituting the present invention will be explained by Examples, and the usefulness of this substance will be explained by Test Examples.

(Example) Preparation method of the present substance from LEM The present substance is purified from LFM as a starting material. L
FM is shiitake fungus
s) is cultured in a solid medium, extracted with hot water before forming fruiting bodies, and dried as a powder. 1
Add 200 ml of distilled water to I-EM of 0 () and stir well with a stirrer. Centrifuge this at 6000 rpm for 20 minutes to remove insoluble matter, and add 200 ml of ethanol to the supernatant while stirring while cooling with water. 7000 rpm 20 minutes. 980 mq of precipitate (precipitate ml) was obtained by centrifugation for 1 minute.This was repeated to collect precipitate 1.
Add 0ml of 50mM phosphate buffer (1) 87.2>, then add 200ml of saturated ammonium sulfate aqueous solution to make a 40% saturated ammonium sulfate aqueous solution, and after standing at 4°C for 12 hours, the precipitate was
Collect by centrifugation at 00 rpm for 20 minutes. Dissolve this in a small amount of distilled water, remove ammonium sulfate by dialysis, and freeze-dry to remove the ammonium sulfate.
A dry product (precipitate 2) of 0 mQ was obtained. This precipitate 2 is filtered through a gel filtration column such as Toyopearl W-653 (2,5x1
00cm). Eluent: 50mM phosphate buffer 1
)H6,7. The elution curve is shown in Figure 1. The vertical axis is 28
Absorption at 0 nm is expressed 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 marker, and the subscript numbers represent the molecular weight in units of 10,000. A is blue dextran with a molecular weight of 12 million, b is thyroglobulin with a molecular weight of 670,000, and C is ferritin with a molecular weight of 460,000. The shaded A and B fractions in Figure 1 have molecular weights of 6 million to 1.5 million and 1.5 million to 80 million, respectively.
It is a polymeric immunoactive substance constituting the present invention containing 10,000 saccharides. The yields of fraction A and fraction B were 6 mg and 17 mg, respectively.
It was mC7.

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

However, the quantification of general substances is performed using the phenol-sulfuric acid method using glucose as the standard (M, Dubois et al., Anal, Ch.
em, vol. 28, p. 350, 1956), and quantitative determination of acidic sugars using B11Jmenkrant using glucuronic acid as standard.
Z et al.'s method (N, B l umenkrantZ et al., A
na 1. Biochem,.

54, p. 484 (1973), amino sugars were analyzed by gas chromatography using alditol acetate-1 derivatives (R. Gatt et al., Anal.

Biochem, vol. 15, p. 167 1966)
The protein was quantified using the Lowry method (0, H, Lowry et al.) using live serum albumin as a standard.

J. Biol, Chem, vol. 193, p. 265 1951
It was conducted in 2010).

Table 1 Sugar content and protein content (wt%) N, D:
Not detected.

= 10- Next, Table 2 shows an example of the composition of neutral sugar which is the main component of the present immunoactive substance in comparison with LEM.

However, the analysis of the sugar composition was carried out using G(,-MS), which was an alditol acetate derivative (P, Al bers
He im et al., Carbohydr, Res.

, vol. 5, p. 340, 1967).

Test Example 1 Assay of macrophage activating ability of this substance Macrophages are cells that present antigens to the immune system and serve as the gateway to the immune system, and play an extremely important role in immune responses. It is said that there are several stages in the activation of macrophages, and it is known that glycolysis progresses in hexadecimal stages with the activation of the first stage. Therefore, the ability of this substance to activate macrophages was evaluated as an increase in glucose consumption compared to the control, and the activation index (S, 1.%
).

1 ml of sterilized 10% thioglycolic acid medium (Hinaga Pharmaceutical Co., Ltd.) is intraperitoneally injected into the peritoneal cavity of a male ddy mouse aged 6 to 8 weeks to induce exudative macrophages.

After 4 days, mice were sacrificed and treated with J-CMF-1-IBSS (Ca2+, M
q2”tree l-1ank-s balanc
After injecting about 5 m+ into the abdominal cavity using a syringe, the solution containing the cells is sucked out from the abdominal cavity with a Pasteur pipette, passed through a 150 meth filter, and poured into a centrifuge tube. Centrifuge at 800 rpm for 5 minutes, suck up the supernatant, and wash the cells with 5 ml of heparin-free 1-IBSS. The washed cells were suspended in RPM11640 medium, dispensed into a 96-well plate at 2×105 cells per hole, and
After culturing for an hour, gently shake the plate and aspirate the supernatant to remove non-adherent cells.

To this, 180 μm of RPM11640 described above was added, and after roughly dissolving this substance in phosphate buffer, the solution was sterilized by filtration using a 0°22 μm membrane filter.
m is added to make the total amount 200 μm. After culturing at 38° C. for 48 hours, 10 μm of the culture supernatant was collected and colored using the glucose oxidase method (Glucose B-Test Wako: Wako Pure Chemical Industries, Ltd.), and the glucose content was measured from the absorbance at 505 nm (As o s >). .Activation index S, 1.(%
) was calculated according to the following formula.

S, i (%) The results are shown in FIG. 2. In both cases, a significant increase in activity was observed compared to LEM, and was even greater than LPS, which is known to have a strong macrophage activation ability.

Test Example 2 Assay of mitogen activity of this substance Lymphocytes contain antigens and various mitogens (mitogens).
When stimulated by Various substances are currently known as mitogens.

A typical example is LPS, which specifically reacts with B cells.
There are PHAs that specifically react with T cells. It is known that these stimuli accelerate DNA synthesis in lymphocytes and cause cell division. Therefore, the amount of DNA synthesis is reduced to 3H-
Evaluate the uptake of thymtdtne into cells,
The mitogen activity of this substance was assayed.

A spleen is collected from a BALB/C male mouse (6 to 8 weeks old), and the cells are carefully scraped off and suspended in Eag+e'sMEM.After passing through a 150 mesh filter, the cells are thoroughly washed and placed in a 96-well plate. Dispense 5 x 105 cells per well. Add a solution of this substance to bring the total volume to 200 μm, and culture for 72 hours. After culturing, add 0.2 μm per well.
Add C1(7)3H-thymrdine and reduce the total amount to 2
It is set to 50 μm. After completion of culture, cell harvest
ster to trap the cells on a glass filter. After drying the glass filter, measure the radioactivity taken into the cells using a liquid scintillation counter. All experiments were conducted in quadruplicate, and the number of counts (d
The ratio of the number of counts in the experimental area to pm) is expressed as the activation index (S, 1.%).

The results are shown in Figure 3.

Both sides show strong mitogen activity, and the activity is typical for LPS, which is a cellular mitogen, in S, 1.
175%, compared to 962% for the A fraction at 20μ(1)/ml and 1166% for the B fraction at 20μCI/ml.
%, which was extremely strong. Note that both sides have the effect of strongly activating macrophages, as is clear from Test Example 1. These activated macrophages then release lymphokines, which are known to cause the development of lymphocytes. Therefore, similar activity was assayed in a system excluding macrophages, but the mitogen activity of this substance did not decrease. Therefore, it is considered that this substance acts directly on lymphocytes without going through macrophages to induce lymphocyte regeneration.

Test Example 3 Effect of this substance on modifying the IPS-mediated rejuvenation reaction Test Example 2 revealed that both sides have an effect of rejuvenating lymphocytes, and the specificity of this action was examined. LP, a mitogen that specifically acts on B cells
The modifying effect of S on juvenile development was investigated. In other words, if LPS promotes juvenileization, it is acting on B cells.

Experimental system c using the mouse spleen cell system as in Test Example 2
,=1 μp/ml LPS was added as mttogen to cause a rejuvenation reaction, and a solution of this substance was added thereto to examine changes in the rejuvenation reaction due to mitogen. At that time, considering that this substance itself has mitogen activity, mit.

A group to which no gen was added was set as a control, and by subtracting this group, the effect of modifying mitogen-induced juvenileization was evaluated. The modification index (S, 1.%) was calculated according to the following formula.

S, I1%)- The results are shown in Figure 4.

Both sides showed a strong promoting activity against LPS-induced rejuvenation, and nearly 8-fold promotion was observed in the B fraction at 20 μQ/ml. That is, it was found that both sides promote the proliferation of B cells and further act on B cells that have already undergone juvenile transformation to further enhance their proliferation.

We asked the Nomura Institute for Biological Sciences to conduct a toxicity test on LEM, which is the starting material for the safety test, and conducted "acute toxicity test" and "subacute toxicity test" on animals at the same facility. A mutagenicity test was conducted and the safety of this substance was confirmed.

Based on these results, the starting material LEM was given to healthy subjects and a phase 1 study was conducted to examine side effects, clinical test abnormalities, etc. The subjects were 5 adult males and 4 females who requested the test and were diagnosed as healthy as a result of a medical examination. A single dose of 6q was administered once. Furthermore, after a single dose, 5 boys received continuous administration of 6q/day, and one month later,
Six months later, various tests including general examination, clinical examination, electrocardiogram, and subjective symptoms were conducted. As a result, no abnormal values or side effects that were considered to be caused by this substance were observed.

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

[Brief explanation of drawings]

Figure 1 is the elution curve of gel filtration with Toyopearl HW-658 for A and B fractions, Figure 2 is a bar graph showing the macrophage activation ability of A and B fractions, and Figure 3 is for A fraction. ,
Bar graph showing mi togen activity of fraction B, 4th
The figure is a bar graph showing the modifying effects of fraction A and fraction B on B cell rejuvenation induced by IPS. Patent applicant: Noda Shokuboku Kogyo Co., Ltd. Figure 1 Figure 2

Claims (2)

[Claims] (1) Molecular weight of 6 million to 1.5 million (A fraction) and 1.5 million extracted from a mycelium culture of shiitake mushrooms cultured in a solid medium mainly containing cellulose components or a mycelium culture containing fruiting bodies A polymer immunoactive substance containing ~800,000 (B fraction) sugars and having the following properties. (a) The carbohydrate content of fraction A is 42.8 to 53.6%, and the protein content is 28.0 to 37.4%. The carbohydrate content of fraction B is 5
2.3-66.3%, protein content 16.0-23.6
%. (b) Containing carbohydrates contain almost no acidic sugars or amino sugars, and more than 95% are neutral sugars. (2) The immunoactive substance according to claim 1, wherein the solid medium comprises bagasse and rice bran.