JPH0331180B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a prostaglandin regulating agent whose main component is a protein polysaccharide derived from a basidiomycete belonging to the genus Corsicolor, and more particularly to a prostaglandin regulating agent consisting of crestin. Krestin has already been provided to society as an antitumor agent, has extremely low toxicity, and is free from concerns about disruption of intestinal flora.
Long-term administration is possible. Furthermore, it has no effect on mutagenicity or allergic reactions, and is therefore extremely safe, with no risk of teratogenicity or allergic reactions in healthy people. In general, prostaglandins (hereinafter abbreviated as PGs)
are found in various organs throughout the body, and have effects closely related to the functions of those organs. It has already been revealed that PGs have a wide range of physiological and pharmacological actions. As a pharmacological effect on the circulatory system, it is involved in the expansion and contraction of blood vessels, leading to increases and decreases in blood pressure, and also has an antagonistic, inducing or promoting effect on platelet aggregation, thereby treating arteriosclerosis and stroke. It is also useful as a prophylactic drug. In addition, antiarrhythmia, antiasthma, respiratory promotion, antitussive and expectorant, antiallergic, antianaphylactic, immunoregulatory, antiulcer, diuretic, uterine motility promotion,
It has been shown to promote tension, suppress tension, prevent cancer metastasis, etc., and is used as a diuretic, anti-ulcer agent, labor promoter, etc.
It is useful as a contraceptive, abortion agent, anti-cancer agent, and even as an anti-aging agent. The present inventors have discovered that the protein polysaccharide of the present invention has not only an antitumor effect but also a pharmacological effect of regulating prostaglandin, and has thus arrived at the present invention. The protein polysaccharide of the present invention contains these PGs.
It is expected that regulating this will be useful in the treatment and prevention of the various diseases mentioned above. The protein polysaccharide which is the active ingredient of the prostaglandin regulator of the present invention is disclosed in, for example, Japanese Patent Publication No. 17149/1980, Japanese Patent Publication No. 36322/1982, Japanese Patent Publication No. 14274/1986, Japanese Patent Publication No. 14276/1986, Tokuko Showa 56-39288
It is a known substance described in publications such as
An extract with hot water or alkaline aqueous solution of mycelium, broth, or fruiting body obtained by culturing Basidiomycetes belonging to the genus Corsicolor,
It contains 38% protein and has a molecular weight of 5,000 to 300,000 (ultracentrifugation measurement). Among the protein polysaccharides of the present invention, C. versicolor mycelium [FERM
−P2412 (ATCC20547)] derived protein polysaccharide is
As mentioned above, it is commercially available under the brand name Krestin (Recent New Drugs Vol. 28, 14-16).
Page, 1977 and Volume 29, pp. 96-101, 1978
Year, Pharmaceutical Handbook, page 1346, May 1978, No. 6
Edition, published by Yakugyo Jihosha, Medical Drug Collection of Japan, 7th edition, page 240, 1983, published by Yakugyo Jihosha), PS-
It is also called K, and some of its properties are as follows. The sugar moiety of the main fraction is β-D-glucan, and the structure of this glucan moiety is 1→3, 1→4 and 1→
It has a branched structure containing 6 bonds, and the amino acids that make up the protein are mostly acidic amino acids such as aspartic acid and glutamic acid, and neutral amino acids such as valine and leucine, and few basic amino acids such as lysine and arginine. Soluble in water, almost insoluble in methanol, pyridine, chloroform, benzene, and hexane. Gradually decomposes from about 120℃. The protein polysaccharide which is the active substance of the present invention is
PGA, PGB, PGC, PGD, PGE, PGF, PGG,
It is involved in the regulation of PGs such as PGH and PGI, TXA, TXB, and their metabolites. Moreover, the active substance regulates not only one but several types of PGs. The following shows that the active substance regulates the production of PGs and its metabolites. The protein polysaccharide of the present invention increases the level of cyclic adenosine monophosphate (cAMP), which is closely involved with PGs as an intracellular messenger (see Example 3). of lymphocytes using arachidonic acid as a starting material.
In an in vitro experiment regarding PGs metabolism, the protein polysaccharide of the present invention showed PGE ₂ , PGD ₂ , 6-keto-PGF ₁ 〓,
It is involved in the production of PGF ₂ 〓 (see Example 1). In vitro, the protein polysaccharide of the present invention affects the biosynthesis of PGE and PGF ₂ in cultured cancer cells (see Example 4). When the protein polysaccharide of the present invention was administered to a tumor-bearing animal and tumor growth and intra-tumor cell PGE levels were measured, tumor growth was suppressed and the intra-tumor cell PGE level increased (see Example 5). Furthermore, in tumor-bearing animals, 6-keto-PGF ₁ 〓 in plasma increases significantly, but it is restored to normal levels by administration of the protein polysaccharide (see Example 7). The antiarrhythmic effect of the protein polysaccharide of the present invention is inhibited by the prostaglandin metabolism inhibitor indomethacin (see Example 8). The protein polysaccharide of the present invention is known to be a very safe substance for living organisms, with extremely low toxicity and almost no side effects.
The acute toxicity values of the protein polysaccharide of the present invention are shown in Table 1 below.

[Table] The acute toxicity values shown in Table 1 above were determined using the following test method. Mice are ICR-JCL strain, 4-5 weeks old, weight 21~
A 24 g sample was used, and the rats were 4- to 5-week-old, weighing 100 to 150 g. The protein polysaccharide of the present invention was administered through four routes: intravenous, subcutaneous, intraperitoneal, and oral. The protein polysaccharide of the present invention was dissolved in physiological saline and administered, and the animals were observed for general symptoms, death, and body weight for 7 days, and after the observation period, they were sacrificed and autopsied. As shown in Table 1, no cases of death were observed in both rats and mice even at the maximum dose that could be administered, and the protein _{polysaccharide} of the present invention is extremely safe for living organisms. In addition, the effect of the protein polysaccharide of the present invention on the gastrointestinal tract was evaluated by administering the protein polysaccharide to beagle dogs weighing 9 to 12 kg.
The state of gastric bleeding was examined 90 minutes after administration of the protein polysaccharide of the present invention, and no bleeding was observed at all due to the administration of the protein polysaccharide of the present invention. As described above, the protein polysaccharide of the present invention has extremely low acute toxicity, is a safe drug, and is useful as a prostaglandin regulator. The protein polysaccharide of the present invention can be made into any dosage form when used as a prostaglandin regulator. Moreover, administration is also performed by each route. Also,
It can be used in conjunction with traditional prostaglandin modulators such as aspirin and indomethacin. In the case of oral administration, the tablets applied thereto;
Granules, powders, capsules, etc. contain binders, encapsulating agents,
It may contain additives such as excipients, lubricants, disintegrants, and wetting agents, and when used as oral liquid preparations, oral solutions, shaken mixtures, suspensions, emulsions, It may be in the form of a syrup or as a dry product which is redissolved before use. Additionally, such liquid formulations may contain any commonly used additives and preservatives. When used for injection, the composition may include stabilizers,
They may also contain additives such as buffers, preservatives, tonicity agents, and are presented in unit-dose ampoules or multi-dose containers. It should be 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 pyrogen-free, sterile vehicle, before use. It may also be a powder that is redissolved in water. The prostaglandin regulator of the present invention can be administered orally or parenterally to humans and animals, but oral administration is preferred. Oral administration includes sublingual administration. Parenteral administration includes injections such as subcutaneous, intramuscular, intravenous, infusion, and the like. The dosage of the prostaglandin regulator of the present invention depends on whether it is an animal or a human, and is influenced by age, individual differences, medical conditions, etc. Therefore, in some cases, an amount outside the following range may be administered, but in general, For humans, the oral dosage of the active substance of the present invention is 10 to 1000 mg per kg of body weight, preferably 20 to 600 mg per day.
Administer mg in 1 to 3 divided doses. Example 1 Effect of Krestin on the metabolism of arachidonic acid incorporated into lymphocytes into PGs BALB/C mouse splenic lymphocytes were taken out and 1
The concentration was adjusted to ×10 ⁷ cells/ml, 2 μCi of ³ H-arachidonic acid was added thereto, and the mixture was incubated at 37° C. for 90 minutes. The pieces were washed three times with medium. again 1×10 ⁷
cells/ml, and 2 ml was dispensed into siliconized test tubes. Six test tubes were prepared, two were used as controls, 10 μg/ml Crestin was added to two, and 100 μg/ml Crestin was added to the remaining two. Incubate at 37°C for 60 minutes, then at 0°C.
Centrifugation was performed at 1200 rpm for 5 minutes. The pellet was taken, 5 ml of petroleum ether was added to 2 ml of culture medium, and the mixture was shaken. The petroleum ether layer was removed, and the remaining aqueous layer was adjusted to pH 3.5 with 0.5N HCl. The acidic liquid was extracted three times with 5 ml of ether,
The ether layer was evaporated to dryness and esterification was carried out with diazomethane solution. This product was developed and separated using a Merck thin layer chromatography system using a mixed solvent of ethyl acetate: isooctane: acetic acid: water in a volume ratio of 90:50:20:100. This spot can be identified using standard PGD ₂ , PGE ₂ , PGE ₂ 〓, 6
−keto−PGE ₁ 〓 was used. The separated silica gel layer was scraped off and suspended in a liquid scintillator solution, and counts were determined to examine fluctuations in PGs. As a result, by adding Krestin, PGD ₂ ,
Obvious fluctuations in PGE ₂ , 6−keto−PGF ₁ 〓,
Changes in PGF ₂ 〓 were observed (see Table 2).

[Table] Example 2 Effect of Krestin on PGs production in isolated rabbit jejunum A piece of jejunum was removed from a Japanese native female rabbit (weighing approximately 2 kg) and incubated with Krebs-1 in a perfusion incubator.
Bicarbonate solution was incubated at 37° C. for 30 minutes under aeration conditions of 95% O ₂ +5% CO ₂ and the content of PGE released in the culture solution was measured. When 1 g/Kg of Crestin was orally administered 2 hours before jejunal removal, an increase in PGE production was observed compared to the non-administered group. Example 3 Effect of Krestin on cAMP level of Sarcoma180 tumor cells An ascites-type tumor was removed from the abdomen of a Sarocma180 tumor-bearing mouse, and 10 ⁷ of these cells were treated with Krestin 100μg/
ml and incubated at room temperature for 5 minutes. After completion of the culture, boiling, homogenization, and centrifugation were performed, and the cAMP content in the resulting supernatant was measured by the Gliman method. cAMP levels in the Crestin-treated group
118 pmol/10 ⁸ cells, in the Crestin non-administration group
It was 89 pmol/ ¹⁰⁸ cells. As a result, it was confirmed that Krestin has the effect of increasing cAMP in cancer cells. Example 4 Effect of Krestin on PGE and PGF ₂ levels in cultured cancer cells 10 ml of Eagle culture medium supplemented with 10% fetal bovine serum was placed in a polystyrene flask for tissue culture with a bottom surface area of 75 cm ² (Code No. 25110, manufactured by Corning (USA)), and ⁵ x 10 cells of human mononuclear leukemia cultured cell line J-111 were transplanted at 37°C, 5% CO ₂ , 95
% air conditions for 7 days. The medium is
The medium was replaced with fresh medium on the 2nd and 4th day after the start of culture. Crestin was added at a concentration of 50 μg/ml. The cultured medium was centrifuged at 1500 rpm at 4°C to obtain a supernatant, and PGE and PGF ₂ levels were measured using ^{a 3} H-prostaglandin E radioimmunoassay kit and ^{a 3} H prostaglandin assay kit from Clinical Assays (USA). It was measured using a JinF radioimmunoassay kit. As a result, the addition of Krestin resulted in
A decrease in PGE and PGF ₂ levels was observed (see Figures 1 and 2). Example 5 Effect of Crestin Administration on PGE Level in Ehrlichi Cancer Cells 1×10 ⁶ Ehrlichi cancer cells were subcutaneously implanted into 8-week-old female C57BL/6 mice, and 2 weeks later, the mice were sacrificed with ether to remove the tumor. Obtained. Crestin was orally administered at 1 g/Kg every day starting from the day after tumor implantation. Finely chop the tumor tissue with scissors, place it in a glass homogenizer, and add 1 g of methyl alcohol to the tumor.
After adding 7 ml per bottle and homogenizing at 0°C, a filtrate was obtained by filtration with a filter paper. Two times the volume of chloroform was added to this filtrate, mixed well, and left at 4°C for 30 minutes. Precipitated proteins were removed by absorption filtration, and the resulting filtrate was heated at 40°C in a rotary evaporator.
It was dried above. This dry matter was mixed with chloroform,
The mixture was placed in a separating funnel together with methanol and dilute hydrochloric acid (PH2.0), mixed well, and then dissolved in 2 ml of the lower layer solution. The PGE content of this solution was measured using a ³ H prostaglandin E radioimmunoassay kit manufactured by Clinical Assay. In the Crestin administration group, the PGE content was 4.7ng/1g.
Tumor, control group (no Krestin administered) 1.8n
g/1g tumor. As a result, in the Crestin administration group, the
An increase in PGE was observed. Example 6 Effect of Krestin on preventing cancer metastasis From tail vein of 8-week-old female C3H/He mouse
2×10 ⁶ MH134 hepatoma cells were transplanted, sacrificed 2 weeks later, the lungs were removed, and metastatic lesions were counted. Crestin was orally administered at a dose of 1 g/Kg 13, 7, and 1 hours before cancer transplantation and 5, 11, and 17 hours after transplantation, respectively. In the Crestin-administered group (average of 10 animals), the positive metastasis rate was 60% and the number of metastatic foci was 2.1, whereas in the control group (without Crestin administration) (average of 10 animals), it was 100% and 4.5, respectively. Ta. As a result, it was confirmed that administration of Crestin reduced the positive metastasis rate and the number of metastatic foci. Example 7 1×10 ⁶ methylcholanthrene-induced sarcoma cells were subcutaneously transplanted into the back of a spontaneously hypertensive rat (SHR), and two weeks later, blood was collected from the abdominal vena cava to obtain plasma. Krestin from 24 hours after tumor implantation
A dose of 1000 mg/Kg was orally administered daily for 13 days. The obtained ether extract of plasma was separated by thin layer chromatography (TLC) and converted into a methyloxime silyl derivative, which was then purified by gas chromatography spectrum (GC-MS).
The fluctuation of −keto−PGF ₁ 〓 was investigated. In the tumor-bearing Crestin administration group, the amount of 6-keto-PGF ₁ was 4.4 ng/ml of plasma, whereas in the tumor-bearing untreated group (no Crestin administration) the amount of 6-keto-PGF 1 was 11.0 ng/ml plasma.
It was hot in ml. Incidentally, 6-
The amount of keto-PGF ₁ was 3.0 ng/ml plasma. As a result, plasma 6-keto-
Although an increase in PGF ₁ 〓 level was observed after tumor transplantation,
In animals administered Krestin, the levels were similar to those in non-tumor-bearing normal animals. Example 8 Antiarrhythmia effect of Krestin Wistar female rats (weighing approximately 200 g) were anesthetized by administering urethane, and then arrhythmia state was induced by intravenously administering 50 μg/Kg of aconitine, an arrhythmogenic agent. Ta. When 1 g/Kg of Krestin was orally administered 1 hour before aconitine administration, ECG (Electro-
There was a tendency for the disturbances in the diagram to recover normally. Next, when 10 mg/Kg of indomethacin, a prostaglandin metabolism inhibitor, was intravenously administered 1 hour and 1 minute before aconitine administration, the antiarrhythmic effect of Krestin disappeared. This indicates that the antiarrhythmic effect of Krestin is mediated by prostaglandins. Example 9 Production of Capsules Using a pressure-type automatic filling machine, 330 mg of Crestin was filled into No. 0 hard capsules to produce capsules.

[Brief explanation of drawings]

1 and 2 are graphs showing the effect of the protein polysaccharide of the present invention on PGE and PGF ₂ of cultured cancer cells in Example 4.

Claims (1)

[Scope of Claims] 1. A hot water or alkaline aqueous extract of mycelia or fruiting bodies obtained by culturing Basidiomycetes belonging to the genus Corsicolor, containing about 18 to 38% protein and having a molecular weight of 5000. ~300000 (Ultracentrifugation measurement method)
A prostaglandin regulator whose active ingredient is a protein polysaccharide. 2. The prostaglandin regulator according to claim 1, wherein the protein polysaccharide is obtained from Corsicolor versicolor.