JPH01254701A - Beta-1,6 glucan sulfate, proliferation inhibitor of aids virus and production thereof - Google Patents

Abstract

PURPOSE:To obtain novel beta-1,6 glucan sulfate useful for proliferation inhibitor of AIDS virus having very low toxicity and slight side effect, by sulfatizing beta-1,6 glucan polysaccharide. CONSTITUTION:A beta-1,6 glucan polysaccharide [e.g., polysaccharide substance (GE-3) expressed by formula I (R is shown that H:COCH3 is 10:1) obtained from lichens or polysaccharide substance expressed by formula II (pustulan)] is usually dissolved in an organic solvent such as DMF or pyridine and sulfatized with chlorosulfuric acid, sulfuric acid anhydride, etc., then beta-1,6 glucan sulfate expressed by formula III and the salt permissible in drug preparing. Administration amount of said compound is preferably 500mg or less per an adult per a day in oral administration, 300mg or less per an adult per a day in non-oral administration.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to β-1,6 glucan sulfate, a method for producing the same, and an AIDS virus growth inhibitor containing the same as an active ingredient.

(Prior Art and Problems) Acquired immunodeficiency syndrome (AIDS) is caused by a virus, and this virus is generally
munodeficiency virus (HIV)
I can't express it enough. This belongs to the human retrovirus,
It is known that this virus destroys helper T cells of lymphocytes, resulting in cell-mediated immunodeficiency, leading to opportunistic infections caused by protozoa, fungi, viruses, bacteria, etc., and Kaposi's sarcoma.

Several therapeutic agents for this disease have been reported so far. Many of them have the effect of suppressing reverse transcriptase activity, which plays an important role in the replication of retroviruses contained in HIV, such as 3'-azido-3'-
Deoxythymidine, 2',3'-dideoxycytidine,
Examples include suramin or HPA-23.

However, these have strong side effects and are difficult to manufacture industrially, and as there is currently no definitive therapeutic agent, there is a strong desire to develop many useful therapeutic agents.

By the way, since heparin and dextran sulfate, which are sulfated polysaccharides, have antiviral effects, the present inventors
H to the new compound β-1,6 glucan sulfate
When examining whether this substance has an inhibitory effect on the proliferation of HIV, it was discovered that this substance significantly inhibits the proliferation of HIV.

The present invention was completed under these circumstances.

(Structure of the Invention) The compound according to the present invention is a β-1 compound represented by the following general formula.
, 6-glucan sulfate, and its pharmaceutically acceptable alkali addition salt.

This compound is produced using polysaccharide extracted from lichen as a raw material. One of the lichens in this case is Umbil
icaria esculenta (Miyoshi
) Minks (= Gyrophora escule
Miyoshi) (Japanese name: Iwakuke). The polysaccharide obtained by extraction from this lichen is called GE-3. This is a compound in which gluten has linear β-1,6 bonds, and contains an acetyl group at the 3-position in a ratio of 10:1 as shown in the following formula.

(However, R indicates that H: C0CHs is 10=1) This acetyl group is eliminated when it is converted into a sulfuric acid ester.

On the other hand, as other lichens, the same family tJmbilicaria
A closely related lichen belonging to ceae, La5allia pust.
ulata(L, )M'er(=Umbilicar
ia pustulata (L, )Hoffm, )
There is. pustul, a polysaccharide that can be extracted from this
an is also β-1,6-glucan, but unlike the above-mentioned GE-3, it does not have an acetyl group at the 3-position. This p
ustulan is expressed by the following formula.

The compound of the present application can be obtained by sulfuric acid esterification of GE-3 or pu3tulan obtained from the above-mentioned lichen.

That is, β-1,6 glucan sulfate is the GE
-3 or pustulan is dissolved in an organic solvent such as dimethylformamide, formamide, or pyridine, and then dissolved in chlorsulfonic acid, sulfuric anhydride, or pyridine.
It is obtained by sulfuric acid esterification using SO1 complex or the like. Further, an alkali addition salt of β-1,6 glucan sulfate can be obtained by neutralizing it with an appropriate amount of an alkaline agent.

The β-1,6 glucan sulfate ester thus obtained and its pharmaceutically acceptable alkali addition salt are:
As shown in the experimental examples below, it has a remarkable effect of inhibiting the proliferation of the AIDS virus, so it is expected to be used as an extremely effective inhibitor of the proliferation of the AIDS virus.

β-1,6 glucan sulfate ester also has the advantage of extremely low toxicity and few side effects, and is usually used in pharmaceutically acceptable salts, that is, alkaline salts of β-1,6 glucan sulfate, such as β-1,6 glucan sulfate. It can be administered to AIDS patients in the form of 1,6 glucan sulfate sodium salt.6 The administration method can be either oral or parenteral, and the dosage depends on the patient's age, symptoms, etc. Varies, but generally adults - 500 m/day for oral administration
By using it within the range of g, the desired effect can be expected.

The dosage form contains one or more pharmaceutically acceptable harmless excipients, such as sodium hydrogen hydroxide, sodium chloride, lactose, potato starch, sodium alginate, magnesium silicate, calcium carbonate, hydroxypropyl cellulose, etc. can be prepared into powders, granules, sugar-coated tablets, capsules, etc. by a conventional method.

For parenteral administration, the dosage is generally 30 mg/day for adults.
Used in the range of 0 mg. The solvent for injections is
For example, physiological saline or 5% glucose solution is suitable.

The following are pharmacological test examples and manufacturing examples regarding the AIDS virus growth inhibiting effect of β-1,6 glucan sulfate, but it goes without saying that the present invention is not limited thereto.

[Pharmacological test example] AIDS virus growth inhibitory effect (materials) (Mo1t-4 cells were used as D cells and virus-infected cells. The virus was Mo1t-4 cells.
The one obtained from the 1t-4/H Engineering V producing strain was used.

(Test drug β-1,6-glucan sulfate ester sodium salt [I]
Experiment for suppressing degeneration of infected cells This experiment was conducted to suppress the H
This study concerns the inhibitory effect of IV-infected cell degeneration, and was carried out under culture conditions without agarose.

2X10' Molt-4 cells with 6X10'' PFU
infected with HIV and supplemented with physiological saline solution of sodium β-1,6-glucan sulfate at various concentrations.
RPM1 with antibiotics and 10% fetal bovine serum for 3 days
The cells were cultured in 1640 culture solution.

Thereafter, the culture solution was stirred and divided into halves, and aqueous solutions of β-1° 6-glucan sodium sulfate at various concentrations mixed in the culture solution were added to one half, and cultured for an additional 2 days. In addition, the number of living cells in the other half was counted using the Trivan blue exclusion method.

As a control, similar experiments were performed on uninfected cells.

In 5 days of culture by Trivan blue exclusion method,
β-1,6-glucan sulfate sodium 7.8~
Suppression was observed at a dose of 250 μg/ml.

The results are shown in FIG.

Figure 1 shows the effect of sodium β-1,6-glucan sulfate on the proliferation of HIV-uninfected cells and IV-infected cells. In the figure, decoys are HIV-uninfected cells, oral indicates HIV-infected cells.
It can be seen that sodium 6-glucan sulfate suppresses denaturation and causes proliferation.

On the other hand, insensitivity 9! The cytopathic effect of sodium β-1,6-glucan sulfate on cells was 8 mg/ml or more.

[■Detection of covirus antigens Indirect fluorescent antibody method using anti-)IIV antibody-positive serum was performed on infected cells. β-1°6-glucan sulfate sodium is 31 μg/ml! The appearance of HIV antigen-positive cells was suppressed to 10% or less in the concentration range of . At lower e degrees, the percentage of positive cells gradually increased.

The results are shown in FIG. The broken line (□) indicates the percentage of HIV-positive cells at various concentrations.

Acute toxicity test β-1,6-glucan sulfate sodium ddY
Tweet to mice, 400-1,000 mg/kg (7
)i? When administered parenterally (intravenously and membranously) at around TL, the tumor survived. Therefore, this compound has extremely low toxicity.

[Example: Production of β-1,6-1'lucan sulfate ester sodium salt (1) Umbilicaria esculent,
GE-3,500 mg extracted from a (Miyoshi) Minks was dissolved in dimethylformamide, and while cooling and stirring, chlorsulfonic acid was added to
5rnj2) was added little by little and allowed to react for several hours. An appropriate amount of water is added to the reaction solution, and the mixture is dialyzed against running water using a dialysis membrane to remove reaction by-products and excess reagents. The obtained dialysate solution was concentrated under reduced pressure, subjected to gel filtration using LH-20 (manufactured by Sephadex, trade name) as a resin, neutralized with IN sodium hydroxide test solution, and converted into sodium salt. did. When this is then freeze-dried, β-1,6-glucan sulfate sodium salt can be obtained almost quantitatively. (2) Physical properties a, soluble in cold water, usually insoluble in organic solvents, 5 molecular weight approximately 200.000 (T (by GPC method using PLC. Pullulan is used as a standard product) C6 specific optical rotation [α] 0 -25.0” (c=o, 33
water) d, infrared absorption spectrum (engineering R) 1240 cm-' (S=0), 910 cm-' (β bond).

820crn-' (e conformation) f, S content 13.6%

[Brief explanation of the drawing]

The drawings are diagrams showing the results of an experiment on the degeneration suppressing effect on Molt-4No. 8 and a virus antigen detection experiment in an experimental example.

Claims (5)

[Claims] (1) General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ β-1,6 glucan sulfate ester and its pharmaceutically acceptable salts. (2) An AIDS virus proliferation inhibitor containing the compound according to claim 1 as an active ingredient. (3) A method for producing β-1,6 glucan sulfate ester, which is characterized by sulfuric acid esterification of β-1,6 glucan polysaccharide and is represented by the following general formula: ▲ Numerical formula, chemical formula, table, etc. are available. (4) A general formula characterized by sulfuric acid esterification of β-1,6 glucan polysaccharide and then neutralization with an appropriate amount of alkaline agent: ▲Mathematical formulas, chemical formulas, tables, etc. are available▼ A method for producing an alkali salt of β-1,6 glucan sulfate as shown. (5) β-1,6 glucan polysaccharide with chlorsulfonic acid,
The method for producing an alkali salt of β-1,6 glucan sulfate according to claim 3 or 4, which comprises sulfuric acid esterification using sulfuric anhydride or pyridine/SO_3 complex.