JPH0367048B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antitumor agent containing as an active ingredient a polysaccharide obtained by culturing microorganisms belonging to the genus Klebsiella. Recently, a number of studies have been reported in Japan regarding the antitumor activity of polysaccharides produced by plants and microorganisms, and in particular, the antitumor activity of polysaccharides collected from hot water extracts and cultured products of Basidiomycetes. A large number of research reports have been published, and some of them have been put into practical use as anticancer agents. It is presumed that all of these anticancer drugs have an indirect antitumor effect through the host;
Although it is particularly effective against homogeneous tumors, it has been confirmed that it is not very effective against syngeneic tumors or autologous cancers. In the course of research into the antitumor activity of polysaccharides produced by microorganisms, the present inventor discovered that capsular polysaccharides produced by capsular microorganisms belonging to the genus Klebsiella have immunological antigenicity. As a result of investigating the antitumor activity of the capsular polysaccharide, we found that the capsular polysaccharide produced by the Klebsiella serotype K-56 strain, which belongs to the genus Klebsiella, is a homogeneous polysaccharide that is thought to be less effective than conventional polysaccharides. The present invention was made based on the knowledge that the present invention exhibits excellent antitumor effects even against tumors. The purpose of the present invention is to provide an antitumor agent that exhibits excellent antitumor activity even against syngeneic tumors, which contains a polysaccharide produced extracellularly by Klebsiella serotype K-56 as an active ingredient. shall be. The present invention will be explained in detail below. The polysaccharide according to the present invention is an antitumor agent whose active ingredient is a polysaccharide having the following properties, which is produced outside the bacterial cell by culturing Klebsiella serotype K-56 strain. (1) Average molecular weight 10 ⁵ - 10 ⁶ (by gel filtration method) (2) The constituent sugars are rhamnose, galactose, glucose and pyruvic acid, and the composition ratio is rhamnose: galactose: glucose: pyruvic acid = 1:3: 1:1 (3) Shows a single peak or band by ultracentrifugation or electrophoresis. The microorganism used in the present invention, Klebsiella
The serotype K-56 strain has been entrusted to the Institute of Microbiology, Agency of Industrial Science and Technology under the number FERM P-7208, and has the following mycological properties. The morphological and physiological properties of the above strains were determined by using a nutrient medium consisting of 5 g of peptone, 3 g of meat extract, 1 g of salt, and 1 part of purified water as the basic medium, and growing under aerobic conditions unless otherwise specified. This shows the results of observation over time. Mycological properties of Klebsiella serotype K-56 strain (1) Morphological properties A nutrient agar medium prepared by adding 15 g of agar to the above basic medium 1 was used. Gram stain negative, rod-shaped, average 0.8 x 2.0-2.5μ
It is non-motile, non-spore forming, and forms a capsule. (2) Growth status on various media Good growth with anaerobic culture on the above nutrient agar media. Growth is good on the above-mentioned agar medium, and the colonies on the plate are round and raised, have moist, homogeneous edges, and are viscous and glossy. No pigment formation. Growth was good in a liquid medium consisting of the above-mentioned basic medium, with no turbidity or bacterial membrane formation, and although precipitation occurred, no discoloration of the interfering medium was observed. MacConkey medium and
All of them grew well on CVT medium. Growth is good on both King A medium and King B medium, and no water-soluble pigment is produced. Does not grow on PEA medium. A medium prepared by adding horse dessicated blood to the above nutrient medium does not exhibit hemolytic properties. (3) Physiological properties (1) Growth temperature range: Grows at 10 to 42°C, and does not grow at 5°C or 46°C. (2) Growthable NaCl concentration range: Grows at 0-6%, does not grow at 7%. (3) Liquefaction of gelatin Negative (4) Decomposition of gelatin Negative (5) Hydrolysis of starch Negative (6) Hydrolysis of chitin Negative (7) Hydrolysis of casein Negative (8) Reaction to purple milk Coagulation, peptonization (9) ) Reaction to litmus milk Coagulation, peptonization (10) Glucose oxidation (OF test) Type F (11) Sugar degradability As shown below. Sugar Acid production Gas production D-glucose + + D-mannose + + D-galactose + + D-xylose + + L-arabinose + + D-fructose + + Sucrose + + Lactose + - Starch + - Glycerol + + Mannitol + + Inositol + + Cellobiose + + Dulsit + + (12) Nitrate reducing ability negative (13) Hydrogen sulfide production negative (14) MR reaction positive (15) VP reaction positive (16) Indole production negative (17) IPA reaction positive (18) Cytochrome oxidase reaction Negative (19) Catalase reaction Positive (20) Citrate utilization Positive (21) Acid production from tartaric acid Positive (22) Urea decomposition ability Positive (23) Arginine alkalinization Negative (24) Ornithine Decarboxylation ability Negative (25) Lysine decarboxylation ability Positive (26) Susceptibility to pteridine compound O/129 Positive (27) Susceptibility to other antibiotics and antibacterial substances As shown below. Type of drug Sensitive Benicillin - Kanamycin + Tetracycline + Chloramphenicol + Erythromycin - Colistin + Sulfisoxazole + Novobiocin + Lincomycin - Cephaloridine + (28) Malonate utilization Positive (29) Phenylalanine deaminase Negative More than Klebsiella serotype K-56 shown
The mycological properties of bacterial strains are described in Bergey's Manual of Determinative Bacterio, 8th edition.
-logy, eighth edition) and Cowan and Steel's Handbook of Medical Bacteria Identification, second edition.
manual for the indentification of medical be
−cteria, 2nd edition by STCowan,
This is consistent with the mycological properties of the Genus Klebsiella section in Cambridge Univ. Press, 1974), and in particular, Klebsiella pneumoniae.
The K-56 strain used in the present invention is similar to the morphological and biochemical properties of
Identified as belonging to Klebsiella pneumoniae. The polysaccharide which is the active ingredient of the antitumor agent according to the present invention is produced by culturing Klebsiella serotype K-56 strain according to the procedure shown below. Production of polysaccharide by culturing the Klebsiella serotype K-56 strain A conventional bacterial culture method can be applied to culturing the above-mentioned strain. That is, shaking culture or static culture is performed, and the culture medium used for culture may be a synthetic medium, a semi-synthetic medium, or a natural medium as long as it contains a nutrient source that can be used by the strain. The composition of the medium includes, for example, D-glucose and D-glucose as carbon sources.
Fructose, L-rhamnose, D-xylose, mannitol, glycerin, sucrose, etc. are used, and as the nitrogen source, meat extract, peptone, casamino acid, yeast extract, ammonium sulfate, urea, etc. are used. In addition, as inorganic salts,
Sulfates, nitrates, chlorides, carbonates, or phosphates of Na, K, Mg, Ca, Zn, Fe, etc. are used as necessary. Note that an antifoaming agent may be added to the medium if necessary. As for the culture conditions, it is generally advantageous to culture under aerobic conditions, and the culture temperature is preferably 30 to 35°C. Furthermore, the pH of the medium is preferably around neutral. Although the culture period varies depending on the composition of the medium, the culture temperature, etc., it is generally about 3 to 5 days, and the desired polysaccharide is accumulated in the culture at the end of the culture. The polysaccharide produced as described above is isolated and purified according to the following procedure. Isolation and purification of polysaccharides In the case of liquid shaking culture, polysaccharides accumulated during culture are mainly contained in the culture solution, so after removing bacterial cells by centrifugation or filtration, they are extracted from the filtrate. Polysaccharides are isolated, and in the case of static culture (using a medium solidified with agar), polysaccharides are mixed with bacterial cells grown on the surface of the medium, so this bacterial mass is collected and added to a 1% phenol aqueous solution. After suspending and stirring overnight, bacterial cells are removed by centrifugation or filtration in the same manner as above, and isolated from the resulting supernatant. Next, the polysaccharide isolated in this way is subjected to a fractional precipitation method using a water-soluble organic solvent such as acetone, ethanol, or methanol to remove low-molecular substances mixed therein, and the resulting precipitate is poured into water. Add a quaternary ammonium salt such as cetyltrimethylammonium bromide to the solution and collect the resulting precipitate. Next, an aqueous solution of sodium chloride or potassium chloride sugar is added to this precipitate to dissolve it, and 2 to 3 times the amount of a water-soluble organic solvent such as acetone, ethanol, or methanol is added to the resulting solution, and the resulting precipitate is collected. Then, the precipitate is dissolved in water, desalted by dialysis, and then freeze-dried to obtain a purified polysaccharide. Next, the physicochemical properties of the polysaccharide obtained as described above will be shown. Physical and chemical properties of polysaccharides (1) Shape: White powder, tasteless and odorless. (2) Uniformity It can be said that the homogeneity is high because it shows a single peak in ultracentrifugation analysis and also shows a single band stained with Alcian blue in electrophoresis analysis using a cellulose acetate membrane. (3) Molecular weight As measured by Sepharose 2B and 4B column chromatography-analysis, gel filtration is 10 ⁵
It is estimated to exhibit an average molecular weight of ~ ¹⁰⁶ . (4) Melting point It does not have a clear melting point and decomposes and turns brown around 300℃. (5) Solubility Dissolves in water and exhibits high viscosity. (6) Color reaction Anthrone reaction Positive Moritsch reaction Positive cysteine sulfate reaction Positive copper phorin reaction Negative carbazole sulfate reaction Negative ninhydrin reaction (after hydrolysis with 4N-HCl at 100℃ for 4 hours) Negative Elson-Morgan reaction (after hydrolysis with 4N-HCl at 100℃) °C, 4
After time hydrolysis) Negative Judging from the above color reaction, it can be understood that this polysaccharide consists of carbohydrates other than amino sugars and does not contain uronic acid or amino acids (or peptides or proteins). (7) Optical rotation The optical rotation of a 0.43% aqueous solution is measured and the specific optical rotation [α] ²⁴ ₅₈₉ is found to be +79°. (8) Infrared absorption spectrum The infrared absorption spectrum obtained by the KBr tablet method is shown in the attached Figure 1. (9) Ultraviolet absorption spectrum The ultraviolet absorption spectrum of an aqueous solution is shown in the attached Figure 2. (10) Nuclear magnetic resonance (NMR) spectrum After dissolving this polysaccharide in D ₂ 0 and freeze-drying it three times to replace D ₂ 0, 2%
Regarding what is dissolved in D ₂ 0 so that the concentration is
¹ H-NMR analysis was performed. the result,
5.24ppm (H2 pieces), 4.92ppm (H2 pieces), 4.72ppm
(1 H), 1.43ppm (3 H), 1.27ppm (3 H)
1.43 ppm was identified as a peak based on CH ₃ of pyruvic acid, and 1.27 ppm was identified as a peak based on CH ₃ of rhamnose. The other peaks are estimated to be based on the anomeric protons of the constituent monosaccharides, so the number of constituent monosaccharides is estimated to be five. (11) Sugars constituting the carbohydrate moiety After hydrolyzing a sample of this polysaccharide with 2M-TFA (trifluoroacetic acid) at 100℃ overnight,
When alditol acetate was formed by a conventional method and subjected to gas chromatography analysis, rhamnose, galactose and glucose were detected. Furthermore, the fact that the composition ratio of rhamnose:galactose:glucose is 1:3:1 is consistent with the results obtained from the above nuclear magnetic resonance spectrum. Incidentally, the chemical structure of this polysaccharide exhibiting the above-mentioned properties has been proposed by YMCHOY et al. as follows. [YMCHOY and GGSDUTTON:
“Structure of the Capsular Polysaccharide
of klebsiella K-type56”CAN.J.CHEM.
VOL.51.3021-3026 (1973)]. (12) Other constituent sugars A sample of this polysaccharide was hydrolyzed in the same manner as described in (11) above, and then ethyl acetate:
Paper chromatography analysis using a composition ratio of acetic acid: formic acid: water = 18:3:1:4 as a developing solvent revealed that it emits fluorescence under ultraviolet irradiation due to treatment with 0-phenylenediamine. Pyruvate was identified as the spot.
The presence of pyruvic acid was also confirmed from the nuclear magnetic resonance spectrum results described in (10) above, and the content ratio was rhamnose:galactose:glucose:pyruvic acid=1:3:1:1. Next, the physiological activity of this polysaccharide will be described. Physiological activity of this polysaccharide (1) Acute toxicity or subacute toxicity The results of testing the acute toxicity or subacute toxicity of this polysaccharide to mice are shown in Tables 1 and 2.

【table】

[Table] (2) Antitumor activity The antitumor activity of this polysaccharide was determined based on Experimental Examples 1 to 3 shown below. Experiment 1 5 × 10 6 Sarcoma-180 cells, which had been subcultured in the peritoneal cavity of ICR- mice, were subcutaneously transplanted into the ventral region of ddY mice (average body weight 19 g, 5-week-old male, ⁵ mice per group). From the next day, polysaccharide samples of various concentrations dissolved in physiological saline were intraperitoneally administered once a day for 5 consecutive days, and the weight of the tumor developed on the 7th day after the above transplantation was measured. The growth inhibition rate was measured. In addition, 6 mg/Kg of mitomycin C (MMC) was administered to the other group as a control drug. The results are shown in Table 3.

[Table] As shown in Table 3, the group receiving this polysaccharide had less weight change compared to mitomycin C.
The tumor inhibition rate is also excellent. Experiment 2 2 x 10 5 Meth-A tumor cells, which were subcultured in the peritoneal cavity of BALB/C mice, were subcutaneously placed on the back of BALB/C mice (average body weight 20 g, 5-week-old male, ⁵ mice per group). Polysaccharide samples at various concentrations dissolved in physiological saline were administered into the tumor on the 3rd and 106th day after transplantation, and the weight of the tumor was measured 26 days after transplantation. The results are shown in Table 4.

[Table] Calculated based on the following formula.
Experiment 3 1 x 10 6 P-388 leukemia cells were intraperitoneally transplanted into CDF ₁ mice (average body weight 20 g, 5 weeks old, 6 males and ⁶ females each). 1
Polysaccharide samples at various concentrations dissolved in physiological saline were intraperitoneally administered once a day for 9 days, and the life extension rate (survival extension rate) was measured relative to the non-administered group (control). The results are shown in Table 5. For comparison, the commercially available polysaccharide anticancer drug Krestin (trade name, manufactured by Kureha Chemical Industry Co., Ltd.) was administered at 250 mg/Kg in the same manner as above, and the survival rate was measured. The results are also shown in Table 5. .

[Table] As shown in Table 5, the polysaccharide of the present invention is effective even against P-388 leukemia cells, for which no results have been seen with conventional polysaccharide anticancer agents. The effective dosage of the antitumor agent of the present invention is generally 1 mg/Kg/day to 1 mg/Kg/day of the polysaccharide that is its active ingredient.
100 mg/Kg/day, and the antitumor agent can be formulated by forming this polysaccharide into tablets, granules, powders, capsules, liquid injections, etc. by known methods. . These are administered orally or by injection (intravenously, intraarterially, intramuscularly). The present invention will be explained in more detail below with reference to Examples. Example 1 Adjustment of polysaccharide Peptone 0.5wt%, meat extract 0.3wt%, sucrose
Dispense liquid medium containing 3 wt% and 0.1 wt% salt into four test tubes (18 x 180 mm) and heat at 120°C.
After sterilization in an autoclave for 15 minutes, each liquid medium was inoculated with Klebsiella serotype K-56 strain (FERMP-7208) and cultured with shaking at 35% for 24 hours. Separately, an agar medium containing 0.5 wt% peptone, 0.3 wt% meat extract, 3 wt% sucrose, 0.1 wt% salt, and 1.5 wt% agar was dispensed into four 300 ml Erlenmeyer flasks to 250 ml each, and heated at 120°C. After sterilizing it in an autoclave for 15 minutes, it was poured into a previously sterilized vat (made of aluminum, 18 cm long, 26 cm wide, 2.5 cm deep), left to cool, and the above culture was added to each of the prepared agar plates. The resulting culture was inoculated and spread on the medium, and cultured at 35°C for 3 days. After culturing, the mucilage produced on the agar plate was collected, suspended in a 1% phenol aqueous solution, stirred for 1 day, and then centrifuged (30000 rpm for 3 hours). The bacterial cells were removed, and 4 times the volume of ethanol was added to the resulting supernatant to obtain a thick filamentous precipitate. This precipitate was collected by entangling it with a glass rod, then dissolved in deionized water, and stirred with a glass rod while adding 5 wt % cetyltrimethylammonium bromide to this solution. This stirring produces a precipitate that tightly clings to the glass rod, so this is collected and dissolved in a 4M NaCl aqueous solution. After stirring in a cold room for 2 days, 3 times the amount of ethanol is added and the resulting precipitate is deionized. Dissolved in water. The resulting solution was dialyzed in running water for 2 days and then lyophilized to obtain a white powder polysaccharide. The yield was approximately 1 g per agar medium. Activity of polysaccharide against MethA tumor The activity test of the polysaccharide obtained as described above against MethA tumor was conducted as follows. BACB/C mice (weight 20
Using 5 mice/group of g), inject subcutaneously into the back of the mice.
On the 3rd and 10th day after transplanting 2 × 10 ⁵ Meth-A tumor cells, 10 mg/Kg of polysaccharide and
100 mg/Kg was administered intratumorally. The results are as follows. Dose Tumor growth inhibition rate (%) Number of complete cures: 10 mg/Kg 96 3/5 100 mg/Kg 76 3/5 As a control, physiological saline was administered instead of the polysaccharide. Example 2 Preparation of polysaccharide Bacto peptone 0.5wt%, Bacto meat extract 0.3wt
% and 0.2wt% of salt was dispensed into five 200ml Erlenmeyer flasks to 125ml each.
After sterilization in an autoclave at 37°C for 15 minutes, each liquid medium was inoculated with Klebsiella serotype K-56 strain (FERM P-7208), and cultured with shaking at 37°C for 6 hours. Separately, sucrose 3wt%, salt 0.2wt%, yeast extract 0.2wt%, K ₂ HPO ₄ 0.1wt%,
An agar medium containing 0.02 wt% MgSO ₄ 7H ₂ O, 0.01 wt% CaCO ₃ and 1.5 wt% Bacto agar was dispensed into 5 4-volume Erlenmeyer flasks, and heated to 121°C.
After sterilizing it in an autoclave for 15 minutes, it was placed in a previously sterilized vat (height 50 cm, width 70 cm, depth 5
The culture obtained by the above culture was inoculated onto each agar plate medium prepared by injecting the cells into the culture medium (cm) and cooling them, and the culture was spread on the medium and cultured at 37°C for 3 days. After culturing, the mucilage produced on the agar plate was collected, suspended in a 1% phenol aqueous solution, stirred for one day, and the suspension was centrifuged (30,000 rpm for 5 hours) to remove the bacterial cells. The supernatant was removed, and 3 times the amount of ethanol:methanol (19:1) was added to the resulting supernatant, and the resulting precipitate was collected by wrapping it around a glass rod. The precipitate thus obtained was washed with deionized water.
After dissolving in the solution, 10 wt% cetyltrimethylammonium bromide was added to the solution while stirring with a glass rod. As a result of this stirring, a precipitate is formed that tightly clings to the glass rod, so this is collected and dissolved in 4M-NaCl solution 1, and this solution is
Pour into twice the amount of ethanol:methanol (19:1) mixture, and centrifuge the resulting precipitate (3000 r.p.
m.20 minutes). The obtained precipitate was 2.5
Dissolve it in water and perform dialysis in running water for 3 days.
It was then freeze-dried to obtain a white powder polysaccharide. The yield was approximately 1 g per medium. Activity of polysaccharide against murine leukemia P-388 The activity test of the polysaccharide obtained as described above against murine leukemia P-388 was conducted as follows. Six CDF ₁ mice (weight 20 g) were used as test animals, and the mice were injected with P-388 cells 1x.
10 ⁶ Transplanted intraperitoneally, and administered 50% polysaccharide dissolved in physiological saline once a day for 5 days starting from the day after implantation.
The survival rate was examined by intraperitoneal administration at doses of mg/Kg, 25 mg/Kg, and 12.5 mg/Kg. The results are shown below. Dose Life extension rate (T/C%) 50 mg/Kg 124 25 mg/Kg 118 12.5 mg/Kg 127 In the control group to which only physiological saline was administered, the average survival time was 11 days. Example 2 Preparation of formulation 0.1 g of the polysaccharide obtained in Example 1 was mixed with 1 g of lactose and compressed to obtain tablets. Although the tablets vary depending on the medical condition, adults usually take 1 to 10 tablets per day orally in three divided doses.

[Brief explanation of drawings]

The attached FIG. 1 shows the infrared absorption spectrum of the polysaccharide according to the present invention, and FIG. 2 shows the ultraviolet absorption spectrum of the polysaccharide.

Claims (1)

[Claims] 1. Klebsiella serotype belonging to the genus Klebsiella
An antitumor agent whose active ingredient is a polysaccharide that is secreted from the K-56 strain and has the following physical and chemical properties. (1) Average molecular weight 10 ⁵ - 10 ⁶ (by gel filtration method) (2) The constituent sugars are rhamnose, galactose, glucose and pyruvic acid, and the composition ratio is rhamnose: galactose: glucose: pyruvic acid = 1:3: 1:1 (3) Shows a single peak or band by ultracentrifugation or electrophoresis.