JPH0825897B2 - Antiviral agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is a polysaccharide or protein polysaccharide extracted from a basidiomycete belonging to the Basidiomycetes group or a basidiomycete belonging to the class Basidiomycetes (hereinafter, may be abbreviated as this substance). )
The present invention relates to an antiviral agent containing as an active ingredient.

Hepatitis B, adult T-cell leukemia and AIDS (Aquired
Immunodeficiency Syndrome) and viral diseases have been the focus of attention in recent years. Up to now, vaccination against vaccines has been taken against viral diseases, and eradication of smallpox, yellow fever and polio have been suppressed. But AI
Vaccines alone cannot fight against diseases such as DS that cause persistent and latent infections, and the development of antiviral agents that exhibit safe and excellent effects is currently expected. The inventors of the present invention have conducted extensive studies on various drugs, and surprisingly found that this substance has a pharmacological effect of an antiviral infection action, and arrived at the present invention.

The fungi used in the present invention are in accordance with the original color fungi pictorial book of Japanese fungi (Nursing company version) co-authored by Rokuya Imaseki and Tsugio Hongo, and the journal of Japanese fungi by Seiya Ito (Yoseido version).

Although any bacterium may be used as long as it is a basidiomycete or a different basidiomycete, a bacterium belonging to the genus Ganoderma genus, the genus Enokitake or the genus Asteraceae is preferable.

This substance is an extract of the fruiting body of mushrooms, artificially cultured cells and its culture medium with an aqueous solvent.

To obtain an artificially cultured mycelium from basidiomycetes, a part of the plant on which the fungus is growing, or the tissue of the fruiting body occurring on the plant or spores is transplanted to an appropriate agar medium. It is obtained by culturing at an appropriate temperature after culturing at an appropriate temperature, repeating this culturing operation 2-3 times, and confirming that there are no contaminants mixed in and then inoculating this into the medium as the mother bacterium. Is. It is usually preferable to use a liquid medium from the viewpoint of handling and productivity.

As a medium composition for culturing, a formulation used for ordinary culturing is sufficient, and various nutrients necessary for the growth of the above-mentioned bacteria may be contained. That is, as the carbon source, for example, glucose, maltose, lactose, sucrose, starch, molasses sugar,
As the nitrogen source, for example, organic or inorganic nitrogen compounds such as peptone, meat extract, yeast extract, yeast, corn steep liquor, ammonium salts and urea can be used. Inorganic salts such as phosphate, magnesium salt, iron salt and calcium salt may be added as mineral components. In addition to this, vitamins necessary for growth may be appropriately added.

The culture conditions are initial pH 2-7, 20 ℃ -35 ℃, and usually 2-3
Incubate for 0 days. PH 4-5, temperature 25-30 during this internal culture
The condition of ° C is particularly preferable. When performing aeration and agitation culture, there is a slight change depending on the shape of the culture tank, but it is suitable to perform aeration with an aeration rate of 0.1 to 2.0 // min and an agitation speed of 30 to 800 rpm.

The fruiting body or the mycelium of artificial culture obtained by the above culture is then transferred to the extraction step. At this time, after washing, it may be dried by air-drying, freeze-drying, etc. and stored for appropriate use. Further, it is preferable to cut into small pieces prior to the extraction step in order to enhance the extraction effect.

The extraction is performed with an aqueous solvent. The water-based solvent is water or a water-soluble organic solvent, an acid, a small amount of any of the base, for example 10
% Or less, and one or a combination of two or more selected from aqueous solutions. As the organic solvent, methanol, ethanol, isopropyl alcohol, etc. are mainly used. Examples of the acid include hydrochloric acid, sulfuric acid, acetic acid and the like. Examples of the base include ammonia, caustic soda, caustic potash, sodium carbonate, and the like. The extraction is performed by using 5 to 200 times as much extract as that of the bacterial cell raw material (dry basis) and usually treating at 4 to 120 ° C. for 20 minutes to 20 hours.

The purification treatment step means removing low molecular weight substances by applying one or more methods such as salting out, dialysis, ultrafiltration, reverse osmosis treatment, gel filtration, precipitation treatment with an organic solvent. It is a thing. From the engineering point of view, the ultrafiltration method, which is a membrane separation method by pressurization, and the reverse osmosis treatment method are preferably used alone or in combination. If desired, these treatments may be carried out after the salting out step.

The salting-out agent used in the salting-out step is ammonium sulfate, sodium chloride, potassium chloride, barium carbonate, etc., but ammonium sulfate is most preferable. Further, as a post-treatment of the salting-out step, any one of dialysis, ultrafiltration, gel filtration, reverse osmosis treatment, etc., or a combination of two or more of these steps is required.

Dialysis is usually carried out using a semipermeable membrane such as a cellophane membrane or a collodion membrane. Gel filtration is performed using a column packed with an adsorbent such as dextran or polyacrylamide gel. Fillers sold under the names Sephadex and Biogel are usually used.

Both ultrafiltration and reverse osmosis are methods of fractionation using a membrane under pressure. The former is 0.5-5 Kg / cm ² , the latter is 20-
It is usually performed at 35 Kg / cm ² .

In the precipitation method using an organic solvent, it is common to use methanol, ethanol, isopropanol, acetone or the like. In addition to the above operation, an ion exchange treatment may be performed if necessary.

After completion of the above-mentioned purification operation, water is removed by spray drying, freeze-drying, etc., and then commercialized.

When using a cultured medium, only the purification step may be used.

Incidentally, it is more preferable to treat this substance with an alkaline aqueous solution since the antiviral effect is surprisingly enhanced. This substance is added in an amount of 0.01 to 5N, preferably 0.1N to 2N, in an amount of 5 to 200 times as much as 40 ° C to 250 ° C, preferably 60 ° C to 200 ° C, most preferably 100 ° C to 150 ° C.
Treatment at 5 ° C. for 5 minutes to 2 hours, preferably 10 minutes to 1 hour. Next, the treatment liquid is neutralized and a purification treatment step is performed.
The purification treatment step can be carried out by applying one or more methods such as salting out, dialysis, ultrafiltration, reverse osmosis treatment, gel filtration, precipitation treatment with an organic solvent, and the like. The conditions are the same as above.

After the above-mentioned purification operation is completed, the product is commercialized after removing water by spray drying, freeze drying, or the like.

In addition, if this substance is further purified by chromatography using ion exchange cellulose, such as diethylaminoethyl (DEAE) -cellulose, and the adsorbed substance is eluted with salt, alkali, etc., a strong antiviral effect is obtained. It is more preferable because it is possible.

This substance shows positive or slight positive in α-naphthol sulfate reaction, indole sulfate reaction, anthuron sulfate reaction, phenol sulfate reaction and / or Lowry-Folin method, and ninhydrin reaction after hydrochloric acid hydrolysis. As a result of elemental analysis, carbon is 20 to 55%, hydrogen is 3 to 9%, and nitrogen is less than 16% as main components.

The sugar component of this substance contains at least glucose, galactose and mannose, and the protein component contains at least aspartic acid, glutamic acid and lysine.

 pH shows 6.0-7.5.

Infrared absorption spectrum measured is 3600-3200cm ^-1
Absorption of hydroxyl groups in the vicinity and or near 1700 to 1600 cm ^-1 ,
Absorption derived from the amide group could be observed.

This substance is soluble in aqueous solvents and insoluble in organic solvents.
The water-based solvent is water or a water-based alcohol containing a water-soluble alcohol, acid, base or the like, and the organic solvent is chloroform, benzene, ether or the like.

The substance is white or brown and has a molecular weight of 10 ^{3 to} 3 × 10 ⁶ by gel permeation chromatography.

Rats (Gyuryu) 4 to 5 weeks old, weighing 100 to 150 g were orally administered to this substance at 1000 mg / Kg and observed for 7 days, but all of them were alive.

This substance is a safe substance with its extremely low toxicity and almost no side effects.

It is generally known that a virus is adsorbed to a target cell, a nucleic acid of the virus is injected into the cell, and then the virus is replicated through a process of being inserted into the chromosomal genome of the cell. In addition, particularly for retroviruses, a process in which RNA, which is a virus-derived gene, is transcribed into DNA by the action of reverse transcriptase is required before it is inserted into the chromosomal genome of cells.

The present inventors have confirmed that the substance is a human immunodeficiency virus (HI
It was found that V) inhibits adsorption of human-derived lymphoid cells and subsequent infection, and inhibits reverse transcriptase activity. That is, HIV is 5 ~ 1000μg / m
After treatment with this substance at a concentration of 1 for 2 hours at 0 ° C, HIV was washed and added to MT-4 cells for adsorption and after 3 days of incubation HIV
When the effect of this substance was examined by the method of measuring antigen-positive cells, HIV antigen-positive cells were hardly detected by pretreatment with this substance, and a strong adsorption inhibitory effect on HIV of human-derived lymphoid cells was observed. Was given. On the other hand, when the effect of this substance on the reverse transcriptase activity was measured using rat liver total messenger RNA as a template,
A strong inhibition of reverse transcriptase activity was observed with the addition of g / ml.

These facts indicate that this substance has an effect of inhibiting viral infection, in particular, inhibiting retroviral infection with a reverse transcriptase, and is particularly effective against AIDS caused by HIV infection. Is.

Azide already used as an antiviral agent
In the case of 3'-deoxythymidine (AZT), side effects that show mitotic inhibitory effect on normal cells are also seen, but this substance has a very low acute toxicity and is a safe substance. It is useful as an antiviral agent by exhibiting the action of inhibiting That is, it is effective against viral infections, especially retroviral infections, that is, AIDS.

When used as an antiviral agent, this substance can be made into any dosage form. In addition, administration is also performed by each route. Furthermore, the drug of the present invention does not reduce the efficacy even in combination with the above-mentioned AZT which is used as an antiviral agent, and the combination with these other drugs can be used as an effective means.

In the case of oral administration, tablets, granules, powders, capsules and the like applied to the composition include binders, inclusion agents, excipients, lubricants, which are commonly used in their compositions.
May contain additives such as disintegrants, wetting agents,
When it is used as an oral liquid preparation, it may be in the form of an aqueous solution for internal use, a shaking mixture, a suspension, an emulsion, a syrup, or a dry product which is re-dissolved before use. Good. Further, such a liquid preparation may contain any of commonly used additives and preservatives. When injectable, the composition may contain additives such as stabilizers, buffers, preservatives, tonicity agents, and is provided in a unit-dose ampoule or a multi-dose container. . It should be noted that the composition may be in the form of an aqueous solution, suspension, solution, emulsion in an oily or aqueous vehicle, while the active ingredient is sterilized in a suitable vehicle, eg, pyrogen-free, before use. It may be a powder that is redissolved in water.

The antiviral agent of the present invention is orally or parenterally administered to humans and animals. Oral administration includes sublingual administration. Parenteral administration includes injection such as subcutaneous, intramuscular, intravenous injection,
Including drip etc. The dose of the antiviral infectious agent of the present invention is influenced by animals or humans, and is affected by age, individual differences, medical conditions, etc. Therefore, in some cases, an amount outside the following range may be administered. In the case of the subject, the oral dose of the active substance of the present invention is 1 kg of body weight, 0.1 to 1000 per day.
mg, preferably 1 to 100 mg, is administered in 1 to 3 divided doses.

 Examples will be shown below.

Example 1 100 g of dried microbial cells of Ganoderma lucidum (CM-359 strain, Microtech Lab. No. 6060) were fragmented, put in a stainless steel tank of volume 3, and 2000 ml of water was added to the mixture with stirring at a temperature of 90 to 95. It was kept at ℃. After extracting for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Further, add 2000 ml of 0.4N-NaOH to the residue and add 90-95 ° C.
After extracting with 3 hours, cool to room temperature and p-p with 2N-HCl.
After adjusting H to 7.0, centrifugation was performed to separate into an extract and a residue.

The extracts were combined, concentrated to 400 ml by a vacuum concentration device, further, low molecular weight substances were removed by ultrafiltration, and then freeze-dried to obtain 12.5 g of a dried substance.

Example 2 100 g of dried enokitake mushrooms (CM-601 strain, Microtech Lab. No. 3045) were fragmented, put in a stainless steel tank of volume 3, 2000 ml of water was added, and the temperature was adjusted to 90 to 95 while stirring. It was kept at ℃. After extracting for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Add 2000 ml of water to the residue and add 3 at 90-95 ℃.
After extraction for a period of time, the mixture was cooled to room temperature and centrifuged to separate it into an extract and a residue.

The extracts were combined, concentrated to 400 ml with a vacuum concentration device, and further freeze-dried to obtain 28 g of a dried product.

Example 3 100 g of dried fungus bodies of Nostoc commune (CM-886 strain, Microtechnology Research Institute No. 1763) were fragmented, put in a stainless steel tank of capacity 3, 2000 ml of water was added, and the temperature was 90-95 while stirring. It was kept at ℃. After extracting for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Further, add 2000 ml of 0.4N-NaOH to the residue and add 90-95 ° C.
After extracting with 3 hours, cool to room temperature and p-p with 2N-HCl.
After adjusting H to 7.0, centrifugation was performed to separate into an extract and a residue.

The extracts were combined, concentrated to 400 ml with a vacuum concentration device, and after removing low molecular weight substances by ultrafiltration, freeze-dried to obtain 15.1 g of a dried product.

Example 4 20 g of the hot water extract derived from Enokitake mushroom obtained in Example 2 was used.
It was dissolved in 0 ml of 1.0 N-NaOH and heat-treated at 120 ° C for 20 minutes. After the heat treatment, cool to room temperature and adjust the pH with 6N-HCl.
After adjusting to 7.0, the product was dialyzed with running Visking cellulose tube for 2 days in running water to remove low molecular weight substances, and then freeze-dried to obtain 3.8 g of a dried product.

The physicochemical properties of this substance obtained in Examples 1 to 4 are summarized in Table 1. In this table, the phenol-sulfuric acid color reaction indicates the presence of saccharides, and the Lowrey-Folin method indicates the presence of peptide bonds. Regarding the molecular weight, the fractions which are present on average by gel permeation method are described.

Example 5 With respect to the substances obtained in Examples 1 to 4, the degree of inhibition of reverse transcriptase specifically retained by retrovirus was measured by the following method.

All of this substance was obtained by dissolving 10 mg of lyophilized product in 10 ml of sterile distilled water (concentration: 1 mg / ml).

1 μ of 20 mM DTT (dithiothreitol: Sigma), 5 μ of 5 times concentration enzyme reaction solution (250 mM Tris-HC
l (pH8.3) -250mM KCl-40mM MgCl 2) 1μ of 3d NTP
Solution (1 mM dATP-1 mM dGTP-1 mM dTTP: Sigma),
2μ of 100μg / ml oligo (dT) _12-18 (PL-biochemic
als), 1 μ messenger RNA (from normal rat liver: 1 μg / μ), 0.5 μ RNase Inhibitor (16u
nit / μ: Takara Shuzo) and 1μ of [α- ³² P] dCTP
(Approx. 800ci / mmol.10μCi / μ: Amersham Japan) is added to an Eppendorf tube with a capacity of 1.5ml, and the temperature is 37 ℃
I placed it in the water bath.

After 5 minutes, 12.5μ of the substance prepared above at a concentration of 1mg / ml was added to the reaction tube, and 1μ of reverse transcriptase (7 units / μ: from Takara Shuzo, Rousassociated virus) was added, and the final reaction solution was added. The amount was adjusted to 25μ and the reaction was carried out at 37 ° C.

After 1 hour, 5 μ of the reaction solution was soaked in 2 cm × 2 cm DEAE paper (manufactured by Toyo Roshi Kaisha, Ltd.), air-dried, and 10 ml per filter paper was used.
DNA on filter paper was immersed in 0.5M-Na ₂ HPO ₄ aqueous solution and shaken.
The [α- ³² P] dCTP that was not used in the synthesis was washed (this operation was performed 5 times at intervals of 5 minutes).

Then, the DEAE paper was placed in a glass vial bottle containing 10 ml of liquid scintillation cocktail (Amersham Japan Co.) and the radioactivity (cpm) was counted for 1 minute with a scintillation counter (Aloka Co.).

The reverse transcriptase activity inhibition rate (%) was calculated by the following formula.

Co: Radioactivity without addition of this substance Cs: Radioactivity with addition of this substance Table 1 shows the inhibition rate of the reverse transcriptase activity of each of this substance.

Example 6 Inhibition of HIV (AIDS virus) adsorption on human lymphocytes by this substance was performed by the following method (all operations were performed under aseptic conditions).

1 ml of an HIV (Human Immunodeficiency Virus) suspension and 1 ml of this substance solution (800 μg / ml) were placed in a test tube and allowed to stand in ice water. 2 hours later, 1 ml of the virus suspension was taken from the test tube, and the human lymphocyte-derived cell line MT-4 [Jpn.J.Cancer Re
s. (Gann), 28, 219-229 (1982)] to multiplicity of infection (M.
Virus was adsorbed at OI) ≈2. Centrifuge (2,000 per minute
(Rotation, 10 minutes), the supernatant was discarded, and the precipitated MT-4 cells were added with RPMI1640 containing 20% FCS (GIBCO Labora-tories, NY).
The cells were suspended therein so that the cell concentration was 2 × 10 ⁵ / ml.

The MT-4 cell suspension was dispensed into a 96-well plate by 100 μm and cultured under the conditions of 5% CO _{2 in} air and 37 ° C.
On the 3rd day of culture, HIV adsorbed cells and non-adsorbed cells were calculated by the indirect fluorescent antibody method.

That is, MT-4 cells were immobilized by treatment with methanol and reacted with anti-HIV infected patient serum at 37 ° C. 30 minutes later PBS
The cells were washed with and reacted with fluorescein isothiocyanate-conjugated rabbit anti-human IgG (immunoglobulin) at 37 ° C.

500 MT-4 cells were observed under a fluorescence microscope, and fluorescence positive cells were calculated as HIV adsorbed cells.

Table 1 shows the inhibition rate of HIV adsorption of this substance on human lymphocytes.

HIV adsorption inhibition rate (%) was calculated by the following formula Example 7 Example 1 was applied to a No. 0 hard capsule using a pressure type automatic filling machine.
This substance was packed in an amount of 330 mg to prepare a capsule.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kenichi Matsunaga 989-17 Kamiirai, Tokorozawa, Saitama Prefecture (72) Inventor Junji Kakuji 6-16-31-406 Minamikarasuyama, Setagaya-ku, Tokyo (72) Inventor Sugita Kyōbun Kabuga-ku, Tokyo Kasuga 2 10-13 of 402 (72) Inventor Takao Furuso 1-6-13 Asahimachi, Machida-shi, Tokyo (72) Inventor Chikao Yoshikumi 2-19-46, Higashi, Kunitachi, Tokyo (72) Inventor Masaaki Takahashi 1-5-33-314 Takanawa, Minato-ku, Tokyo (56) Reference JP-A-53-29977 (JP, A) JP-A-55-27101 (JP, A) JP-A-57-12999 ( JP, A) JP 51-91312 (JP, A) JP 53-121919 (JP, A) JP 54-148702 (JP, A) JP 61-130235 (JP, A) JP 55-157517 (JP, A) JP-A 56-10118 (JP, A)

Claims (4)

[Claims] 1. An antiretroviral agent comprising a polysaccharide or protein polysaccharide produced by a bacterium belonging to the class Basidiomycetes as an active ingredient. 2. Polysaccharides or protein polysaccharides include α-naphthol sulfate reaction, indole sulfate reaction, anthron sulfate reaction,
Phenol-sulfuric acid reaction and / or Lowry-Folin method,
Ninhydrin reaction after hydrolysis with hydrochloric acid shows positive or slight positive, elemental analysis is carbon 20-55%, hydrogen 3-9%, nitrogen 16
%, The sugar portion contains at least glucose, galactose, and mannose, and the protein portion contains at least aspartic acid, glutamic acid, and lysine, and has an infrared absorption spectrum of 3600 to 3200 cm ^-1 and / or 1700 to 1600 cm ^-1.
The absorption is observed in water, it is soluble in water, is insoluble in chloroform, benzene, and ether, and has a molecular weight of 10 ^{3 to} 3 × 10 ⁶ by gel filtration chromatography. Antiretroviral agent. 3. The antiretroviral agent according to claim 1, wherein the basidiomycete class is the same basidiomycete or different basidiomycete. 4. The antiretroviral agent according to claim 1, which is an anti-AIDS virus agent.