JPH0699320B2 - Drug for treating retrovirus infection of sulfated β-glucan having anti-AIDS virus activity - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel drug for treating infectious diseases caused by retroviruses, particularly AIDS (AIDS) caused by human immunodeficiency virus (HIV).

[0002]

2. Description of the Related Art Acquired IMM (acquired immunodeficiency syndrome), which is one of the intractable diseases currently facing humanity,
UNE DEFICIENCY SYNDROME: A
It has been elucidated that IDS is caused by the human immunodeficiency virus (HIV) belonging to the retrovirus.

According to the World Insurance Organization (WHO) 1990
As of the end of the year, the number of AIDS virus infected persons was 8 million, and the number of sick or dead persons was more than 800,000. In Japan, as of December 31, 1990, 371 patients were confirmed. However, the number of people infected with the virus is 1627, although it does not lead to the onset.

However, the cure for this disease is completely unknown and two HIV-1 and HIV-2 viruses have been discovered as the virus for this disease. The implementation of the development is facing great difficulty, where there is a need for therapeutic and prophylactic effects with anti-HIV agents.

The only practically used anti-HIV agent is azitothymidine (AZT) (Nature
326, 430, 1987) is an anti-HIV drug based on the inhibitory effect of HIV reverse transcriptase, but this drug has strong side effects on the living body, and it has been revealed that it inhibits the hematopoietic action of patients, and also in many patients. Extreme anemia has developed. In recent years, it has been confirmed that sulfation of polysaccharides such as sulfated dextran shows an antiviral action against some viruses (Ann, NY,
Acad, Sci 13, 365, 1965)

[0006]

The polysaccharides having α-glugod bonds such as starch and dextran whose physiological activity depends on the molecular weight are decomposed by enzymes in the body. Β-1.3 which has a branched chain of β-1.3 bond at high frequency, which is less susceptible to enzyme degradation.
Considering the development of a drug having a strong anti-HIV activity by sulfating β-1.3 glucan having 6 and β-1.6 branched chain bonds.

[0007]

In view of the above-mentioned reasons, the present inventors have found that sulfated β-glucan is anti-H in vitro using a human and T cell-derived strain (MT-4).
The IV activity test revealed that the growth of HIV was completely suppressed, and the minimum effective concentration was 10 mg / ml, which is equivalent to the minimum effective concentration of dextran sulfate, which is said to be the most effective than before. is there.

[0008]

In other words, the polysaccharide protein obtained from Maitake mushroom was sulphated by the chlorsulphonic acid method, resulting in the invention of a substance having anti-HIV activity. As shown in Examples of the drug of the present invention, HIV
HIV-1 (LAV or HTLV-IIIB) is used as (human immunodeficiency virus), but other HIV-
2 (type 2 AIDS virus) has a similar effect, and it has been discovered that non-sulfated compounds activate the activity of M-par T cells (T ₄ cells), and thus it is denatured by HIV. It is possible that the anti-HIV action may be exhibited by reducing the degeneration of T ₄ cells.

[Manufacturing Example 1] Patent No. 1514114 in which the applicant of the present invention was allowed to carry out the invention.
The production method based on No. 1 is: (1) 300g of crushed fruit body or mycelium of Maitake
After obtaining the dry powder of 3 liters, add 3 liters of distilled water to
Hot water extraction for 6 hours at 0 ℃, 1.2 atm, temperature 120
After heat-treatment at 1 ° C for 1 hour and cooling, an equal amount of ethanol was added to the supernatant obtained by the centrifugation method, and the mixture was allowed to stand at 4 ° C for 20 hours, and 14.5 g of precipitate was collected. To 1 g of this precipitate, 440 ml of water was added and dissolved, and cetyltrimethylammonium hydroxide was added to adjust the pH to 12 to obtain 13.8 g of a precipitate. 30% and 50
% Of acetic acid to remove acid-soluble matter to obtain 3 g of acid-insoluble matter. To this, 6% sodium hydroxide was added to collect an alkali-soluble substance. A 4-fold amount of 99% ethanol was added to this to obtain a precipitate. A chloroform-methanol mixture (2: 1) was added to this substance to remove proteins, and then ethanol was added to 4
After adding the double amount, the mixture was allowed to stand at a temperature of 4 ° C. for 20 hours and then centrifuged to obtain about 1 g of a precipitate. The substance is a light brown powder which is hardly soluble in cold water and positive for anthrone reaction.
About 22% of the protein was detected by the wry) method, confirming that it was brotheoglucan. (2) Next, this glycoprotein was first treated with sepharose CL-4B, 2.5 × 45 cm.
Gel filtration was performed using a column to obtain a high molecular weight glycoprotein having a molecular weight of 200 × 10 ⁴ . In addition, DEAE-
Sepharose CL-6B was adsorbed on a 2.0 × 4.5 cm column to obtain a substance that was eluted with a 1/15 M phosphate buffer solution having a pH of 7.2. This material was dialyzed against purified water at 4 ° C. using a cellulose dialysis tube and further desalted with a Sephadex G-25.3 × 37 cm column.
This was evaporated to dryness to obtain a light brown amorphous powdery target substance. This material was a polysaccharide containing 0.6% protein. (3) Next, the chemical structure of the substance obtained in (2) above was clarified. That is, 1 mg of the above substance was taken, dissolved in 5% hydrochloric acid-methanol 1 ml, and decomposed in a sealed tube at a temperature of 100 ° C. for 6 hours. Then, a toluene-ethanol (1: 1) mixed solution was added to the decomposed product and the mixture was evaporated under reduced pressure. Allowed to dryness. Add a trimethylsiliconizing agent to this and react at 70 ° C for 10 minutes,
When subjected to gas chromatographic analysis using SE-20 (silicone rubber GE) as a filler, only glucose was detected. Furthermore, when this substance was reacted with exo-β-1,3-glucanase at pH 5.0 in a matsucurbin buffer, about 45% of constituent glucose residues were released after 48 hours. From the above results, this substance is β-
It was confirmed to be a glucan having 1.3 bonds.

(4) Subsequently, after completely methylating this substance by the Hakomori method, the completely methylated product was treated with 5% hydrochloric acid-
Methanolysis with methanol. Neopentyl glycol succinate (Neopentylgl)
Gas chromatographic analysis using ycol succinate as a filler revealed that 2,4,6-tree-0-methyl derived from β-1.3 bond and 2,3,6-derived from β-1.6 bond. Presence of 2,3-di-0-methyl derived from tri-0-methyl, 1,3,4 bond and 2,3,4,6-tetra-0-methylglucose derivative derived from non-reducing end Was given.

From the above results, the substance obtained by hot water extraction of fruiting bodies of Pleurotus cornucopiae has a β-1.6 bond as a main chain and has a high frequency of β-1.3 branched chains. It was concluded that it consists of a polysaccharide protein having a chemical structure and a β-1.3 polysaccharide protein having a β-1.6 branched chain. The molecular weight of the sulfated β-1.6 glucan obtained here was measured to be about 220,000 using a modified Manners Et Al.1971 method.

Production Example 2 Preparation of Sulfated β-1.6 Glucan β-proteoglucan (MT obtained by the method of Production Example 1
-S) was converted to the sulfate, i.e. 30
Add 25 ml of pyridine to a 0 ml round bottom flask-10
5 ml of chlorosulfonic acid was gradually added to the mixture while cooling and stirring at 60 ° C. and reacted for 15 to 20 minutes to solidify the reaction product to 60 to 7
Soak in an oil bath at 0 ° C to dissolve. The lysate is stirred vigorously for about 1 hour, then 100 ml of cold water is added and neutralized with saturated sodium carbonate solution. After neutralization, 5 volumes of acetone are added to fractionate into a supernatant and a precipitate. 50 ml of this precipitate
It is dissolved in water, passed through an IR120B ion exchange resin, adjusted to pH 9 to 10 with sodium carbonate, and then 5 times the amount of acetone is added to fractionate into a supernatant and a precipitate. The precipitate was dissolved in water and freeze-dried to obtain a yield of 0.14 g. Sulfated-β proteoglucan (MT-S) with adjusted branched chains
From the viewpoint of storability, it is desirable to use an alkali metal salt or an ammonium salt in the form of a suitable salt. A method for measuring the sulfur content adjusted by the method described above was obtained.

The content is as follows. Sulfur content is well dried, and about 3 mg of it is precisely weighed and 3 ml of water and 3 drops of hydrogen peroxide are used as an absorbing solution. Sulfat is measured by the oxygen flask combustion method (Japanese Pharmacopoeia, general test method).
Determine the ion content in a.

[0014]

[Table 1]

The anti-HIV activity of the sulfated β-pluteoglucan (MT-S) obtained by the method described above was evaluated in the following examples.

[0016]

[Examples] Measurement of anti-HIV activity (test for inhibition of cytopathic effect by virus) A method carried out in the AIDS virus room of AIDS Center, National Institute of Preventive Health was used. That is, as a primary screening, the microplate method using the inhibition of cytotoxicity due to HIV infection of MT-4 cells as an index was used. For the samples in which the activity was observed here, the anti-HIV activity was confirmed and the mechanism of action by other methods. Was analyzed. (Microplate method) MT-4 cells (CD4 antigen-positive / HTLV-1) having high sensitivity to HIV-1 (HTLV-IIIB Strain) and HIV-2 (HIV [GH-1])
In positive cells), CPE appears 3 to 5 days after infection, and most cells die. The CPE was judged under a microscope, and it was examined whether or not the expression of this CPE was suppressed by the added sample. This method was used as the primary cleaning. That is, a round-bottom 96-well microplate (Nun
Sample solution 2 diluted to a predetermined concentration in the leftmost 8 wells of c)
Add 00 μl. Sample dilution is Rp with 10% Fcs
Perform in mI 1640 medium. 100 μl of medium is placed in each of the wells, and 100 μl is taken from the well at the left end by an 8-strip pipette and transferred to the well next to the right and well stirred. This is repeated and the 2-fold serial dilution of the sample (100 μl /
Well 12 stages) and then MT-4 in logarithmic growth phase
Cells are collected by centrifugation at 6 million cells per plate and suspended in a very small amount of medium. HIV was added to this cell suspension in a centrifuge tube to give 100 TCID 50 / ml.
And adsorb at 37 ° C. for 1 hour. For HIV, the culture supernatant of persistently infected Molt-4 / HIV cells was filtered through a 22 μm Millipore filter and stored at −80 ° C., and an infectious titer for MT-4 cells was determined beforehand. After adsorption at 37 ° C. for 1 hour, 10 ml of medium is added per plate and 100 μl / well is added to the sample diluent. The cell concentration at this time is set to about 300,000 cells / ml. Uninfected cells and uninfected sample infected cells are used as controls and cultured at 37 ° C. in the presence of 5% CO ₂ . HIV
On the 3rd day after the infection, the cytotoxicity of the sample was examined with a microscope, and a plate of the same sample as that at the time of the infection was prepared by serially diluting it. 1/3 to 1/5 cells were taken out from the plate cultured for 3 days and transferred. Change. Judgment is performed 6 days after infection. The presence or absence of CPE expression due to HIV infection is observed under a microscope. The effect was recognized when the expression of CPE due to HIV infection was suppressed at a concentration at which the sample did not show cytotoxicity. (Anti-HIV test result) Sample: Maitake-derived sulfated β-proteoglucan (MT
-S)

[0017]

[Table 2]

The introduction of Sulfate is gluco
One of the 2nd, 3rd, and 4th positions of C of se or
You may enter the two places or all C positions. Either
There is no difference in the anti-HIV effect even when introduced into. (Molecular weight 220,000) Method: Using microplate method using MT-4 cells H
IV was performed with HIV-1 and HIV-2 types. Results-Day 6 Judgment-CONc (μg / ml) Test volume 1000 500 250 125 63 3
1 16 8 4 21 0.5 MT-S ○-◎-◎-◎-◎-○-◎
-○-○ + ○ ++++++: Drug expression of cytotoxicity ○: Cell proliferation is observed ◎: Cell proliferation similar to that of non-infected control cells is observed +: CPE expression by HIV ++ : Strong CPE expression by HIV-: No CPE expression by HIV

[0019]

EFFECTS OF THE INVENTION It showed a strong anti-HIV activity against sulphated β-proteoglucan (MT-S) derived from Maitake. As a result of confirming the presence or absence of virus cytotoxicity after culturing by the above method, in the control group, cytopathic change due to virus was observed, but sulfated β-1.6 glucan was added at 16 to 63 μg / ml.
Even when a small amount of was added, no cell degeneration due to virus was observed. This result confirms that the drug of the present invention has an activity of suppressing cytopathic effect caused by virus, that is, an anti-HIV activity. As described above, the β-glucan-protein polysaccharide present in Maitake mushrooms, which is the agent of the present invention, was clearly found to suppress the cellular degeneration caused by HIV.

Claims (1)

[Claims] 1. A β-1.6 glucan having a 1.3-linked branched chain and a 1.6-linked branched chain containing about 0.6% of a protein contained in Maitake mushroom fruit bodies and mycelium. Sulfated polysaccharide protein derived from Maitake mushroom obtained by sulfating β-1.3 glucan has anti-AIDS virus activity characterized by treating infectious diseases against retroviruses including human and AIDS virus A drug for treating retroviral infections of sulfated β-glucan.