JPS63316727A - Antiviral agent - Google Patents

Abstract

PURPOSE:To obtain an antiviral agent especially suppressing infection of retrovirus having a reverse transcriptase, comprising a saccharide or a protein polysaccharide produced by a slime mold belonging to Eucaryomycota, a mucilage bacterium or an oomycetes as an active ingredient. CONSTITUTION:An antiviral agent comprising a saccharide or a protein polysaccharide produced by a slime mold belonging to Eucaryomycota, a mucilage bacterium, an oomycetes, Chytridiomycetes, zygomycetes or Fungi imperfect (preferably Aspergillus, Saccharomyces or Mucor) as an active ingredient. The substance suppresses absorption of HIV on human-derived lymphoid cell and infection following the absorption, inhibits activity of reverse transcriptase and is effective against retrovirus infectious diseases, especially AIDS. An oral dose of the substance is 0.1-1,000mg, preferably 1-100mg daily and administered dividedly once-three times daily.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to polysaccharides or proteins extracted from Osteomycetes, Dictyostelium, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, and Deuteromycetes belonging to the eukaryotic fungi ([ucaryoiycota]). This is an anti-filth agent whose active ingredient is a polysaccharide (sometimes abbreviated as this substance).
The classification of bacteria in the present invention is [Multiple Biological Dictionary (3rd R6
t) Based on J (published by Iwanami M store on 10th, 1983, 3J).

In recent years, viral diseases such as hepatitis B, adult T-cell leukemia, and even AIDS have become a hot topic of discussion. Until now, viral diseases have been dealt with through vaccination, and smallpox, yellow fever, and polio have been brought under control. However, vaccines alone cannot fight against diseases such as AIDS where persistent infection or latent infection is a problem, and there are currently high hopes for the development of safe and highly effective antiviral agents. It is. Therefore, the present inventors conducted intensive studies on various drugs, and surprisingly found that
The present invention was achieved based on the finding that the polysaccharide or protein polysaccharide of the present invention has a pharmacological effect of antiviral infection.

In order to obtain an artificially cultured bacterial cell from the bacterium of the present invention, the bacterium is used as a mother bacterium and is inoculated into a medium and cultured at an appropriate temperature. Generally, it is preferable to use a liquid medium in terms of handling and productivity.

As for the culture medium composition, a formulation used for normal culture is sufficient, as long as it contains the various nutrients necessary for the growth of the above-mentioned bacteria. That is, carbon sources include, for example, glucose, maltose, lactose, sucrose, starch, and Bunnozenna; nitrogen sources include, for example, peptone, meat extract, yeast extract,
HBI, cornstarch liquor, ammonium salts,
Organic and inorganic nitrogen compounds such as urea can be used.

Further, as mineral components, inorganic salts such as phosphates, magnesium salts, iron salts, calcium salts, etc. may be added. In addition, vitamins necessary for growth may be added as appropriate.

The culture conditions are initial p112~7. Cultivation is usually carried out at 20~35°C for 2~30 days. p[15
~7. A temperature of 25 to 30°C is particularly preferable. When performing aeration agitation culture, there are slight changes depending on the shape of the culture tank, but the aeration is 10.1 to 2.01/x/w.
in, 'hl stirring speed 30-800 r, I), m,
It is appropriate to implement within the range of .

1f! selected from aqueous solutions containing the substance of the present invention at or below the level required to extract the substance of the present invention from the cells of the bacteria of the present invention! Or it is a combination of two or more types. As the organic solvent, methanol, ethanol, isopropyl alcohol, etc. are mainly used. Examples of acids include hydrochloric acid, sulfuric acid, and acetic acid. As the base, use an extract of 5 times to 200 times the amount of body material (dry basis), and usually at 4°C to ¥120°C.
The treatment is carried out for 20 minutes to 20 hours.

The yi-manufacturing process means removing low-molecular substances by applying one or more methods such as salting out, dialysis, ultrafiltration, reverse permeation treatment, gel filtration, and precipitation treatment with organic solvents. It is something to do. From an engineering point of view, it is particularly preferable to use the ultra-e-compatible method, which is a pressure separation method, and the reverse permeation treatment method alone or in combination. In addition, these treatments may be carried out after the salting-out step, depending on the case.

Salting-out agents used in the salting-out step include ammonium sulfate, common salt, potassium chloride, barium carbonate, etc., but ammonium sulfate is most preferably used. Further, as a quick treatment for the salting-out process, it is necessary to perform any one of dialysis, ultrafiltration, gel filtration, reverse permeation treatment, etc., or a combination of two or more of these processes.

Dialysis is usually carried out using semipermeable membranes such as cellophane and collodion membranes. Gel filtration is performed using a column packed with Mochii valves such as dextran or polyacrylamide gel. A filler sold under the name Ref7dex, Biogel is commonly used.

Ultrafiltration and reverse osmosis are both methods of fractionation using a membrane with added F11. The former is 9/i from 0.5 to 5, and the latter is 20/35/CI! It is usually done with .

In the precipitation method using an organic solvent, methanol, ethanol, isopropanol, acetone, etc. are generally used. Further, in addition to the above operations, ion exchange treatment may be performed as required in C.

After the above purification operation is completed, water is removed by spray drying, freeze drying, etc., and then the product is manufactured.

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

Incidentally, it is more preferable to treat this substance with an alkaline aqueous solution because, surprisingly, the antiviral effect is enhanced.

0.01N to 5N, preferably 0.01N to 5N, 5 to 200 times the amount of this substance. 40 in an alkaline aqueous solution of IN to 2N.
C. to 250.degree. C., preferably 60.degree. C. to 200.degree. C., most preferably 100.degree. C. to 150.degree. 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 process includes the above-mentioned salting out, dialysis, ultraviolet irradiation, reverse permeation treatment, gel filtration,
This can be carried out by applying one or more methods such as precipitation treatment with an organic solvent. The conditions are the same as above.

After the above purification operation is completed, water is removed by spray drying, freeze drying, etc., and then the product is manufactured.

In addition, this substance can be treated using an ion-exchange cellulose gel such as di-T-thylaminoedyl ([^[)-cell].

This substance tested positive in α-naphthol sulfuric acid reaction, indole sulfuric acid reaction, anthrone sulfuric acid reaction, phenol sulfuric acid reaction, and/or Lowry-e-Forin method, and ninhydrin reaction after hydrochloric acid hydrolysis. As a result of elemental analysis, carbon 2
The main components are 0 to 55%, hydrogen 3 to 9%, and nitrogen 0 to 16.0%.

The sugar components of this substance include at least glucose, glucuronic acid, and mannose, and the protein components include at least aspartic acid, glutamic acid, and leucine.

When the infrared spectrum is measured, it is 3600-3200. −
Absorption of hydroxyl groups near 1 and or 1700 to 1600 cm
In the vicinity of -1, absorption derived from the amide group could be observed.

This substance is soluble in aqueous solvents and insoluble in organic solvents.

be. Aqueous solvents are water or water-based solvents containing water-soluble alcohols, acids, bases, etc., and organic solvents include chloroform, benzene, ether, etc.

The substance is white or brown in color and has a molecular weight of 10-3X106 as determined by gel filtration chromatography.

Rat (6 dragons) 4-5 weeks old, body f! 100-150
Use No. 9 to make this substance 1000η/! g Orally administered and observed for 7N, all of the animals survived.

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

It is generally known that a virus infects a target cell, injects the viral nucleic acid into the cell, and then replicates through the process of being incorporated into the cell's genome. In addition, especially for retroviruses, the virus-derived nucleic acid R
A process is required in which NA is transcribed into DNA by the action of reverse transcriptase.

The present inventors have demonstrated that the compounds of Examples 1 to 5 of the present invention inhibit the adsorption of human immunodeficiency virus (HIv) to human-derived lymphoid cells and subsequent infection;
It was also found that it inhibits reverse transcriptase activity. That is, after treating HIV with the present substance (compounds of Examples 1 to 5) at a concentration of 400R/lIV for 2 hours at 0°C, 1t
We investigated the effect of this substance by washing MT-4 cells, infecting them with MT-4 cells, and measuring HIV antigen-positive cells after culturing them for 3 days. Almost all antigen-positive cells disappeared, resulting in HI
A strong adsorption inhibitory effect of V on human-derived lymphoid cells was observed. On the other hand, when the effect of this substance on reverse transcriptase activity was measured using rat liver whole messenger RNA as a template, a strong inhibition of reverse transcriptase activity was observed at a concentration of 500 mg/d of this substance.

These facts indicate that this substance has the effect of inhibiting viral infection, especially retrovirus infection that has reverse transcriptase, and that it is effective against AIDS (AIDS) caused by HIV infection.
This shows that it is effective for low efficiency (5yndroa+e).

Azide-3' already used as an antiviral agent
- In the case of deoxythymidine < AZT), it exhibits a division inhibiting effect even on normal cells and has side effects, but the polysaccharide or protein polysaccharide of the present invention has extremely low acute toxicity, is a safe substance, and is It is useful as an antiviral infectious disease agent because it exhibits the effect of inhibiting infection, especially retrovirus infection.

That is, it is effective against viral infections, particularly retrovirus infections, ie, AIDS.

The polysaccharide or protein polysaccharide of the present invention can be made into any dosage form when used as an antiviral infectious disease agent. Moreover, administration is also performed by each route. Historically, the drug of the present invention is
The above-mentioned HeZ is used as a treatment for viral infections.
Even if a3 is used in combination with T etc., the efficacy will not decrease,
Combination with these drugs can be used as an effective means.

In the case of oral administration, tablets, granules,
Powders, capsules, etc. may contain additives commonly used in pharmaceutical formulations such as binders, encapsulating agents, excipients, lubricants, disintegrants, wetting agents, etc. When used as a liquid preparation for daily use, it may be in the form of an internal solution, a shaken mixture, a suspension, an emulsion, or a syrup.
It may also be in the form of a dry product that is redissolved before use. Additionally, such liquid formulations may contain any commonly used additives and preservatives.

For injection, the compositions may contain additives such as stabilizers, buffers, preservatives, tonicity agents, and are presented in unit-dose ampoules or multi-dose containers. Ru. It is noted that the compositions may be in the form of aqueous solutions, suspensions, solutions, emulsions in oily or aqueous vehicles, wherein the active ingredient is dissolved in a suitable vehicle, e.g., a sterile, pyrogen-free vehicle, before use. It may also be a powder that is redissolved in water.

The antiviral infectious disease agent of the present invention is administered orally or parenterally to humans and animals. Oral administration includes sublingual administration. Parenteral administration includes injections such as subcutaneous, intramuscular, intravenous, infusion, and the like. The dosage of the antiviral infection agent of the present invention depends on whether it is an animal or a human, and is influenced by age, individual differences, medical conditions, etc. Depending on the case, doses outside the range below may be administered, but in general, When intended for humans, the oral dosage of the active substance of the invention is 0.1 to 11000 IR, preferably 1 to 100 IR per body weight 1/9.1 #1
9 will be administered in 1 to 3 divided doses.

Examples are shown below.

Example 1 Aspcroillus olaucus TAN 3
005 (Aspergillus Aspergillus) as glucose 3
0%, cornstarch liquor 1.1%, urea 0.53
% medium 10f! The culture solution was incubated at 4000X
g', Centrifugation was performed for 30 minutes to separate the supernatant and the bacterial cells.

The supernatant was concentrated under reduced pressure, dialyzed through a Wiskingrelulose tube to remove low molecules, and then lyophilized to obtain 11 g of a brown dried product.

Example 2 The bacterial cells isolated in Example 1 were suspended as uniformly as possible in a 5-fold volume of physiological saline while stirring.

This suspension was centrifuged at 4000x GO for 30 minutes, the supernatant was discarded, the precipitate was suspended in physiological saline, and the cells were disrupted using a French press. After making sure that most of the cells are empty, transfer the suspension to 4000xg.
', centrifuge for 30 minutes and pour the residue into water! The mixture was turbid and the operation was repeated three times.

The residue after washing is placed in ethanol and centrifuged at 100%
After replacing with ethanol and acetone, dry under reduced pressure.
A powder of 181J was obtained.

Example 3 Saccharomyces cerevisiae
I^H-42077 (mll Labokin eye) Labokin
Culture solution (4000xg' of 10j.

The bacterial cells obtained by centrifugation for 30 minutes were suspended as uniformly as possible in a 5-fold volume of physiological saline while stirring. This suspension was centrifuged at 4000Xg' for 30 minutes, the supernatant was discarded, and the precipitate was suspended in physiological saline.
Cells were disrupted using a French press. After making sure that most of the cells are empty, incubate the suspension at 4000x.
Go, centrifuge for 30 minutes, suspend the residue in water, and repeat the operation three times.

The residue after washing was heated to 80°C with a 0.58-NaOH aqueous solution.
.

Heat extraction was performed for 2 hours. The extract was cooled to room temperature and
The pH was adjusted to 7.0 with N-11cj, desalted by ultrafiltration, and then freeze-dried to obtain 9.2 g.

Example 4 5 g of bacterial cells of Example 2 were added to 1 N aqueous sodium hydroxide solution.
00ae and autoclaved at 120°C.

Heat treatment was performed for 20 minutes. The mixture was adjusted to p117 with a 2N aqueous hydrochloric acid solution, and then dialyzed using a Viscoking cellulose tube to remove low molecular weight substances such as salts.

The contents of the tube were freeze-dried to obtain 2.9 g of dry product.

Example 5 Conditions and operating method similar to Example 3 (medium: 2X StlC
Hucor 5pines by rO313PD^)
cens T^Toro 071 (Mucorales Mucorales) to 1
2 g of bacterial cell extract was obtained.

Table 1 shows the physicochemical properties of the compounds obtained in Examples 1 to 5.
are summarized in. On the wood surface, phenol sulfur IWf
f1 color reaction indicates the presence of sugars, lowrey-rot
The tn method shows the presence of peptide bonds.

Regarding the molecular weight, the molecular weight, which is present in large amounts on average, is listed depending on the gel ena.

Example 6 Regarding the substances obtained in Examples 1 to 5, the degree of inhibition of reverse transcriptase specifically retained by retroviruses was measured by the following method.

All test substances are freeze-dried products (10 μl) mixed with 1 oz. of sterile distilled water.
! i! gg solution (Ila: 1 Ilj/d>.

2018 D, T, T, (dithiothreitol: manufactured by Sigma) for 1 product, 5x i 1 degree enzyme reaction solution for 5 product (25O
n+HTris-HCQ (pH 8,3)-250aHK
Cj -40nHHgCI) 2), 3d of 1111
NTP solution (1mHd^TP-1mHGTP-1mH
DTTP: manufactured by Sigma), 2u1 of 100 gram/-oligo (dT) 12-18 (PL-biochemicals
), 1i11 metsenger RNA (derived from normal rat liver: 1R/1IIl>, 0.5 J of RN
ase Inhibitor (16 units/111
: Takara Shuzo Co., Ltd.) and ll11 [α-”2P] dC
TP (approximately 800 Ci/5 sol, 10 μCi/form; manufactured by Amersham Java) was added to a 1.5 d capacity Eppendorf tube and placed in a 31° C. water bath.

After 5 minutes, the test substance 12 at a concentration of 1 mg/test substance prepared previously was added.
．． 5 scraps were added to the reaction duplex, and one reverse transcriptase (7 units/ρ: Takara Shuzo Co., Ltd., Roussassoci
ated virus Yamaki) was completed, and the final reaction volume was reduced to 2
5, and the reaction was carried out at 37°C.

After 1 hour, soak 5p of the reaction solution into 2ctR x 2aR DEAE paper (manufactured by Toyo Roshi Co., Ltd.), air dry, and! 0.5H-N a 2 HP O of 10IIrR per sheet of paper
4) The [α-32P] dCTP that was not used for NA synthesis on the filter paper was washed by dipping it in an aqueous solution and shaking it (this operation was performed five times at intervals of 5 minutes).

Then, put the above DEAE paper into a glass vial containing 10 m of liquid scintillation cocktail (manufactured by Ama Jam Japan 4), and place it on a scintillation counter (
Radioactivity (c, o, m,
) was counted.

The inhibition rate (Kan) was determined by the following formula.

CO: Radioactivity without addition of test substance C5: Radioactivity with addition of test substance Table 2 shows the inhibition rate of reverse transcriptase (RTaSe) activity of each test substance.

Table 2 RTase activity inhibition rate (%) Inhibition rate; ++
÷ 61~b 10+ 31~60% 100~30% Example 7 Inhibition of adsorption of HIV (AIDS virus) to human lymphocytes by the test substance was carried out by the following method (all operations were performed under sterile conditions). ).

Hr V (Human rn+1unodef
iciency Virus) i Near M Liquid 11d
and 1 m of the test substance solution (80ON/d) were placed in a test tube and allowed to stand still in water. 1ml from the test tube after 2 hours
Take the virus suspension and transform it into human lymphocyte-derived cell line MT.
-4 [Jpn, J, Cancer Res, (
Gann), 28°219-229 (1982)
] was adsorbed with a virus having a high TjJ staining intensity (H, 0, 1,)ζ2. Centrifugation (2,000 revolutions per minute).

The supernatant was diluted and the precipitated MT-4 cells were added to RPMI 1640 (GIB) containing 20% FC8.
CO Laborat.

rics, NY) so that the cells were suspended at a WJ degree of 2×10 5 /d.

The above MT-4 cell suspension was dispensed in 100 cells into a 96-well plate and cultured under conditions of 5% CO2 and 37°C. On the third day of culture, the number of HIV-infected cells and non-infected cells was calculated by indirect fluorescent antibody method.

That is, MT-4 cells were reacted with oceanate-conjugated rabbit anti-human IQG (immunoglobulin) at 37°C by treatment with methanol.

ISJ observation of 500 MT-4 cells was performed under a fluorescence microscope, and fluorescence-positive cells were calculated as HIV@stained cells.

The results are shown in Table 3. Table 3 8IV infection inhibition rate Inhibition rate; ÷++61~100x +÷31~60% 10~30% Example 8 Using a pressure-type automatic filling machine, the present invention was applied to No. 0 hard capsules. 3 substances
Capsules were prepared by filling 30q.

Claims (3)

[Claims] (1) An antiviral agent containing as an active ingredient a polysaccharide or protein polysaccharide produced from Aomycota, Dictyostelium, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, and Deuteromycetes belonging to the eukaryotic fungi. (2) An antiretroviral agent containing the polysaccharide or protein polysaccharide according to claim 1 as an active ingredient. (3) An anti-AIDS virus agent containing the polysaccharide or protein polysaccharide according to claim 1 as an active ingredient.