JPH021151B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer substance, and more particularly to the anticancer substance TF-220 obtained from the culture solution obtained by culturing bacteria belonging to the genus Fusobacterium and salts thereof. Furthermore, the present invention provides the anticancer substance TF-220.
and a method for producing a salt thereof, and an anticancer agent containing the same. In recent years, treatments for various cancer patients that enhance the host's immune function and exert anticancer effects with the help of the immune function have become popular. Known drugs used in such therapy include bacterial cells of various bacteria, components obtained from bacterial cell cultures, and polysaccharides obtained from the fruiting bodies of basidiomycetes or their cultured cells. However, there is little research on components that have excellent anticancer effects from the culture fluid obtained by culturing anaerobic bacteria, and in particular, there is still little research on the components obtained from the culture fluid obtained by culturing Fusobacterium bacteria and their anticancer effects. unknown. The present inventor cultivated bacteria belonging to the genus Fusobacterium isolated from the human oral cavity, and investigated the pharmacological effects of the components collected from the supernatant after removing the bacterial cells from the culture solution. has a strong anticancer effect, and this means that in the colony formation suppression method, the effect of inhibiting the formation of cancer cell colonies is small, and the anticancer effect is not due to cell killing, but rather a host-mediated or enhanced host immune system. The present invention was completed based on the discovery that this specific ingredient has the effect of indirectly exerting anticancer activity with the help of the immune system, and that this particular ingredient has low toxicity. The bacteria used in the present invention may be any TF-220 producing bacteria belonging to the genus Fusobacterium, and a preferred example is Fusobacterium nucleatum. Specifically, for example, Fusobacterium nucleatum TF-031 (FERM-P No.
5077, ATCC-31647), and as a common knowledge in microbiology, strains with these properties, such as natural mutant strains or artificially improved strains, are used. The mycological properties of Fusobacterium nucleatum TF-031 are as follows. (1) Morphology Cell shape: spindle-shaped (Figure 1) Presence of cell pleomorphism: None Motility: None Presence of spores: None Gram staining: Gram negative Acid-fastness: Negative (2) Medium Growth status on TF-a agar plate and slant medium Outside Shape: Circular Size: Approximately 1 mm Elevation: Hemispherical Structure: Dewdrop-like Surface: Smooth Edge Edge: Smooth Color: Milky white Transparency: Opaque Degree of growth in TF-e liquid medium: High turbidity: Coagulation precipitation: None Surface growth: None, no growth up to approximately 5 mm Gas: None (3) Physiological properties Hydrogen sulfide production: + Reduction of nitrate: - Production of butyric acid: + Production of indole: + Urease: - Catalase: - Hydrolysis of starch: - Attitude towards oxygen: Anaerobic Production of ammonia: + Production of carbon dioxide: + Growth range: PH5 ~ 8.5 Temperature 30-45℃ Gas production from sugar L-arabinose (-), D-xylose (-), D-glucose (-), D-mannose (-), D-fructose (-), D-galactose (-), maltose (-), sucrose (-),
Trehalose (-), sorbitol (-), mannitol (-), inosyte (-), glycerin (-), starch (-) The above properties were compared to Versey's Manual Obstetric Bacteriology, 8th edition. When examined, this strain was found to be Fusobacterium.
It closely resembles and belongs to Fusobacterium nucleatum. Next, an example of the method for producing the anticancer substance TF-220 of the present invention will be schematically explained as follows.

[Table] The above manufacturing method is specifically shown as follows. (a) Culture Bacteria belonging to the genus Fusobacterium are cultured using a conventional anaerobic culture method. That is,
Nitrogen sources such as cow brain, heart extract, various peptones; Vitamin sources such as yeast extract;
A medium containing inorganic salts such as sodium chloride; a carbon source such as glucose and lactose; and a reducing agent such as L-cystine, sodium sulfite, and sodium thioglycollate is diluted with sodium hydroxide to pH 6.
Adjust to 8.5 preferably 7.2 to 8.2, inoculate the bacteria,
1 at 35-42℃ preferably 36-38℃ under anaerobic conditions.
Allow to stand or culture with stirring for ~5 days, preferably 1 to 4 days. Alternatively, after culturing for 1 to 2 days at 35 to 42°C, preferably 36 to 38°C, it may be further cultured at 25 to 35°C for 1 to 4 days. In particular, it is preferable to use a medium (hereinafter referred to as TF medium) described in the ingredient table below. However, as a nitrogen source, it is not always necessary to use brain heart infusion, which is an extract of cow's brain or heart; In addition, proteose peptone and phitone peptone are not necessarily necessary among various peptones, and polypeptone can be substituted for tryptocase peptone. Incidentally, when agar is not used, it is preferable to perform stirring culture.

[Table] (b) Collection of supernatant from culture solution (removal of bacterial cells) Remove bacterial cells from the culture solution obtained above to obtain supernatant. Bacterial cells can be removed by conventional methods, such as centrifugation or filtration using a supernatant such as Hyflo Super Cell, but centrifugation is particularly preferable in terms of operation, degree of bacterial removal, and yield of supernatant. . (c) Collection of anticancer substance TF-220 Add a hydrophilic organic solvent to the supernatant obtained above and collect the resulting precipitate. The supernatant liquid at this time is
Adjust to PH1.5-7, preferably around PH2 (PH1.5-2.5). Examples of hydrophilic organic solvents include alcohols such as ethanol and methanol, and ketones such as acetone, but alcohols, particularly ethanol, give the best results. This hydrophilic organic solvent is suitably added so that its concentration is 30 to 80% (volume ratio), preferably 50 to 80% (volume ratio). After adding the hydrophilic organic solvent, it is left to stand at a low temperature, preferably at a temperature of about 4 to 5°C, for several hours to several days to complete the formation of a precipitate. The precipitate thus formed is separated by conventional procedures such as decantation, centrifugation, filtration, etc. Next, generally 5 to 20 times the amount of water is added to this precipitate and the mixture is divided according to pH. Specifically, after adjusting the pH to 7.5 to 8 and dissolving the precipitate, the pH is adjusted to around 6 (PH5.5).
~6.5), separate the water-insoluble part and the water-soluble part by a conventional method such as centrifugation or filtration, and collect the water-insoluble part to obtain the anticancer substance TF-220. The anticancer substance TF-220 obtained as described above has the following properties. (a) White-gray to light brown powder. (b) Mouse Ehrrich ascites-type carcinoma, Ehrlichi's nodule-type carcinoma, Sarcoma-180 cancer cells, and B
-16 It inhibits the proliferation of melanoma cancer cells and has immunostimulatory effects. (c) Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether. (d) It does not show a clear melting point and decomposes at 160°C to 240°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2950~2920, 1680~1620, 1550~
1510, 1440, 1380, 1240~1220 and 1120~
It has an absorption band near 1020cm ^-1 . (Figure 2) (f) The ultraviolet absorption spectrum of an aqueous solution of the water-soluble fraction at pH 7 has strong absorption at the absorption end, and
It exhibits absorption in the vicinity of 248-266 nm (Figure 3). (g) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Follin reaction were positive. (h) Elemental analysis values C: 40% to 42%, H: 5% to 7% N: 7% to 9% (li) The sugar content of the water-soluble fraction at pH 7 determined by the phenol sulfuric acid method is approximately The protein content is 5% to 20% (in terms of glucose), and the protein content by the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin). (J) Molecular weight: Several thousand or more, but it is extremely difficult to identify. Anticancer substance TF-220 obtained as above
may be converted into a pharmaceutically acceptable non-toxic salt according to a conventional method. Specifically, for example, sodium salt,
Examples include alkali metal salts such as potassium salts, alkaline earth metal salts such as magnesium salts, and calcium salts. Next, the pharmacological action of the anticancer substance TF-220 of the present invention is as follows. (1) Immunostimulatory effect Using three ICR mice per group, the test substance was dissolved in physiological saline, and 0.2 ml of the solution was administered intraperitoneally. Perikan Drawing Ink 24 hours after administration
0.2 ml of carbon suspension prepared by mixing 1 ml of 17 Black (manufactured by Guilnter Wagner) and 2 ml of physiological saline containing 3% gelatin was injected into the mouse tail vein, and 1, 5, 10, and 15 minutes after injection, it was inserted into the orbit. blood using a heparin-coated hematocrit capillary tube.
Collect 0.02ml, immediately dilute it with 1.6ml of 0.1% sodium carbonate aqueous solution, hemolyze it, compare the color at a wavelength of 675nm, and calculate the phagocytotic index (K value).
It was calculated using the formula of Halpern et al. In addition, 0.2 ml of physiological saline was administered to the control group. K=log Co-log C/t-to (Co=to amount of charcoal in the blood, C=blood charcoal amount at t) The results are shown in Table-1.

[Table] As is clear from Table 1, compared to the control group, TF
In the -220 substance administration group, reticuloendothelial macrophages were activated and cell-mediated immunity in normal mice was increased. (2) Anticancer effect (i) Antitumor effect on Ehrlitsu ascites tumor ICR mice (female, 6 weeks old) were intraperitoneally inoculated with 1×10 ⁵ Ehrlitsu ascites cancer cells per mouse. Next, the test substance was dissolved in physiological saline, and 0.2 ml of the solution was intraperitoneally administered once a day for 7 consecutive days starting from the first day after inoculation of the cancer cells. In addition, to the control group, 0.2 ml of physiological saline was administered once. The results are shown in Table-2. T/C = Survival days of treatment group/Number of survival days of control group x 10
0 (%)

[Table] (ii) Antitumor effect of Sarcoma-180 on cancer cells: Sarcoma-180 was administered to ICR mice (female, 5 weeks old).
180 cancer cells were intraperitoneally transplanted at 1×10 ⁵ cells per mouse. Next, TF-220 was dissolved in physiological saline, and 0.2 ml of the solution was added from the first day after cancer cell transplantation.
The drug was administered intraperitoneally once a day for 7 consecutive days. In addition, to the control group, 0.2 ml of physiological saline was administered once. The results are shown in Table-3.

[Table] (iii) Antitumor effect on Ehrlichi nodular tumor: 4 x 10 ⁶ Ehrlichi cancer cells per mouse were subcutaneously transplanted into ICR mice (female, 6 weeks old) in the axillary region. Then, TF-220 was dissolved in physiological saline, and 0.2 ml of the solution was intraperitoneally administered once a day for 7 consecutive days starting from the first day after cancer cell transplantation. In addition, to the control group, 0.2 ml of physiological saline was administered once. Tumor weight was measured 14 days after cancer cell transplantation. Tumor weight is the major axis of the tumor site amm
and the minor axis bmm were measured using calipers and calculated using the following formula. Tumor weight = a×b ² /2 (mg) The results are shown in Table 4.

[Table] (iv) Antitumor effect on B-16 melanoma cancer cells: 1 x 10 ⁶ melanoma cancer cells per mouse were subcutaneously transplanted into BDF ₁ strain mice (male, 7 weeks old) under the armpit. Next, TF-220 was dissolved in physiological saline, and 0.2 ml of the solution was administered 1 day after cancer cell transplantation.
From day 1, intraperitoneal administration was administered once a day for 7 consecutive days. In addition, the control group received 0.2 ml of physiological saline once. Tumor weight was measured 17 days after cancer cell transplantation. The measurement method was the same as in (iii). The results are shown in Table-5.

[Table] (3) Acute toxicity TF in mice (ICR strain, male, 6 weeks old)
The _LD50 value of 220 administered iv was more than 200 mg/Kg. As is clear from the results of the above pharmacological experiments, the anticancer substance TF-220 obtained by the method of the present invention is useful as an anticancer agent, and is expected to be effective when used for various cancer diseases. It is. The anticancer substance TF-220 of the present invention can be used as it is or as a non-toxic salt in the form of oral, injection, suppository, etc. formulations by conventional methods. Oral preparations may contain various excipients and may be in the form of capsules, tablets, powders, or granules. The injection may be subcutaneous, intramuscular, or intravenous, and may be in the form of a suspension, solution, or powder to be dissolved before use. The injection may also contain a local anesthetic. The dosage of the anticancer substance TF-220 of the present invention and its non-toxic salts is appropriately selected depending on the patient's symptoms, but in general, for adults, 0.01 to 50 mg/Kg is administered once to several times a day. The method of administration is preferably oral, subcutaneous, intramuscular, intravenous, or by injection into the affected area. Next, the present invention will be explained by giving examples and manufacturing examples. Example 1 (1) Into 10 jar fermenters (manufactured by Marubishi Rika Kenkyusho), 17 g of tryptocase peptone and heart injection were added to 1 portion of distilled water.
10g, yeast extract 3g, salt
TF-e medium 8 containing 7.5 g, glucose 12 g, lactose 10 g, sodium sulfite 0.1 g and sodium thioglycolate 0.5 g
and sterilize at 120°C for 30 minutes. After cooling the culture solution, nitrogen gas is aerated at 100 ml/min for 1 hour. Fusobacterium nucleatum TF-, which was pre-cultured in the TF-e medium mentioned above.
031 (FERM-P No. 5077, ATCC-31647) preculture solution 1 is inoculated under sterile conditions. Culture is 37
℃, with nitrogen gas flow (65 ml/min).
Stirring (30 rpm) for 5 days. After culturing, 160 g of Celite and 80 g of cellulose powder were added to the culture solution, stirred, and filtered under reduced pressure.
7.8 ml of sterilized culture supernatant was obtained. (2) Add 117ml of concentrated hydrochloric acid to 7.8ml of the culture supernatant obtained in (1).
After adding and adjusting the pH to 2.0, ethanol 11.7
Add to make a 60% ethanol solution and incubate at 4℃ for 24 hours.
Leave it for a while. Next, the solution portion was removed by decantation, and the temperature was heated at 4°C to collect the precipitate.
Centrifuge (6 x 10 ³ rpm, 5 minutes). This precipitate was mixed with 400 ml of 60% ethanol aqueous solution with pH 2.0.
After sequentially washing with 400 ml of ethanol, 200 ml of acetone and 200 ml of diethyl ether, the mixture was dried under reduced pressure to obtain 4.9 g of powder. (3) Suspend the powder obtained in (2) in 49 ml of water and
Add aqueous sodium hydroxide solution to adjust the pH to 7.5-8.0, stir at room temperature for 30 minutes, then add 1N hydrochloric acid to adjust the pH to 6.0. Next, after stirring for 2 hours under ice cooling, centrifugation (1 x 10 ⁴ rpm, 10 minutes)
and separate the precipitate and supernatant. This precipitate
Wash with 5 ml of water adjusted to pH 6.0, centrifuge the precipitate and washing solution (1 x 10 ⁴ rpm, 10 minutes), wash the precipitate with 10 ml of ethanol, dry under reduced pressure, and remove the anticancer substance TF. - Obtain 1.8g of 220. Example 2 (1) Into 10 jar fermenters (manufactured by Marubishi Rika Kenkyusho), 17 g of tryptocase peptone and heart injection were added to 1 portion of distilled water.
20g, yeast extract 3g, salt
TF-d medium 8 containing 7.5 g, glucose 12 g, lactose 10 g, sodium sulfite 0.1 g and sodium thioglycolate 0.5 g.
and sterilize at 120°C for 30 minutes. After cooling the culture solution, nitrogen gas is aerated at 100 ml/min for 1 hour. Fusobacterium nucleatum TF-, which was pre-cultured in the TF-d medium mentioned above.
031 (FERM-P No. 5077, ATCC-31647) preculture solution 1 is inoculated under sterile conditions. Culture is 37
℃, with nitrogen gas flow (65 ml/min).
Stirring (30 rpm) for 5 days. After culturing, 160 g of Celite and 80 g of cellulose powder were added to the culture solution, stirred, and filtered under reduced pressure.
7.8 ml of sterilized culture supernatant was obtained. (2) Add 117ml of concentrated hydrochloric acid to 7.8ml of the culture supernatant obtained in (1).
After adding and adjusting the pH to 2.0, ethanol 11.7
Add to make a 60% ethanol solution and incubate at 4℃ for 24 hours.
Leave it for a while. Next, the solution portion was removed by decantation, and the temperature was heated at 4°C to collect the precipitate.
Centrifuge (6 x 10 ³ rpm, 5 minutes). This precipitate was mixed with 400 ml of 60% ethanol aqueous solution with pH 2.0.
After sequentially washing with 400 ml of ethanol, 200 ml of acetone and 200 ml of diethyl ether, the mixture was dried under reduced pressure to obtain 5.07 g of powder. (3) Thereafter, the same treatment as in Example 1-(3) was carried out to obtain 1.9 g of the anticancer substance TF-220. Formulation Example 2 to 3 mg of powdered anticancer substance TF-220 is adjusted to pH 7.0 to 7.5 with a dilute aqueous sodium hydroxide solution, the water-soluble portion is freeze-dried, and filled into vials. Sterile saline or lidocaine 0.5% when using this
It is dissolved in a containing solution and used as an injection solution.

[Brief explanation of drawings]

FIG. 1 is a micrograph showing the morphology of Fusobacterium nucleatum TF-031 used in the present invention, FIG. 2 is an infrared absorption spectrum of the anticancer substance TF-220, and FIG. 3 is an ultraviolet absorption spectrum of the same substance.

Claims (1)

[Claims] 1. Anticancer substance TF belonging to the genus Fusobacterium
An anticancer substance TF-220 and its salt having the following properties obtained from the culture solution by culturing -220-producing bacteria. (a) White-gray to light brown powder. (b) Mouse Ehrrich ascites-type carcinoma, Ehrlichi's nodule-type carcinoma, Sarcoma-180 cancer cells, and B
-16 It inhibits the proliferation of melanoma cancer cells and has immunostimulatory effects. (c) Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether. (d) It does not show a clear melting point and decomposes at 160°C to 240°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2950~2920, 1680~1620, 1550~
1510, 1440, 1380, 1240~1220 and 1120~
It has an absorption band near 1020cm ^-1 . (f) The ultraviolet absorption spectrum of an aqueous solution of the water-soluble fraction at pH 7 has strong absorption at the absorption end, and
It exhibits absorption near 248-266 nm. (g) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Follin reaction were positive. (h) Elemental analysis values C: 40% to 42%, H: 5% to 7%, N: 7% to 9% (li) The sugar content by the phenol sulfuric acid method of the water-soluble fraction at pH 7 is The protein content is about 5% to 20% (in terms of glucose), and the protein content by the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin). 2 Anticancer substance TF belonging to the Fusobacterium genus
-220 The anticancer substance TF-220 according to claim 1, which is obtained by adding a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria and collecting the resulting precipitate.
and its salt. 3 Anticancer substance TF belonging to the Fusobacterium genus
-220 Add a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria, collect the resulting precipitate, divide the precipitate by pH, adjust the pH to around 6, and collect the water-insoluble part. Claim 1 obtained by
The anticancer substance TF-220 and its salt according to item 2 or item 2. 4 The operation of adding a hydrophilic organic solvent to the supernatant liquid is PH2
The anticancer substance TF-220 and its salt according to claim 2 or 3, wherein a hydrophilic organic solvent is added to the supernatant liquid adjusted to the same concentration. 5 Anticancer substance TF belonging to the Fusobacterium genus
The anticancer substance TF-220 and its salt according to any one of claims 1 to 4, wherein the -220 producing bacterium is Fusobacterium nucleatum. 6 Anticancer substance TF belonging to the Fusobacterium genus
A method for producing the anticancer substance TF-220 and its salts, which comprises collecting the precipitate obtained by adding a hydrophilic organic solvent to the supernatant obtained by culturing -220-producing bacteria. 7 Anticancer substance TF belonging to the Fusobacterium genus
-220 Add a hydrophilic organic solvent to the supernatant obtained by culturing the producing bacteria, collect the resulting precipitate, divide the precipitate by pH, adjust the pH to around 6, and collect the water-insoluble part. 6. A method for producing the anticancer substance TF-220 and its salts according to claim 6. 8 The operation of adding a hydrophilic organic solvent to the supernatant liquid
8. The method for producing the anticancer substance TF-220 and its salts according to claim 6 or 7, which comprises adding a hydrophilic organic solvent to the supernatant liquid adjusted to around 2. 9. The method for producing the anticancer substance TF-220 and its salts according to any one of claims 6 to 8, wherein the hydrophilic organic solvent added to the supernatant liquid is alcohol. 10. The anticancer substance according to any one of claims 6 to 9, characterized in that a hydrophilic organic solvent is added to the supernatant liquid so that its concentration is 30 to 80% (volume ratio). Method for producing TF-220 and its salt. 11 Anticancer substances belonging to the genus Fusobacterium
An anticancer agent containing TF-220, an anticancer substance or a salt thereof, which is obtained by culturing TF-220-producing bacteria and has the following properties from the culture solution. (a) White-gray to light brown powder. (b) Mouse Ehrrich ascites-type carcinoma, Ehrlichi's nodule-type carcinoma, Sarcoma-180 cancer cells, and B
-16 It inhibits the proliferation of melanoma cancer cells and has immunostimulatory effects. (c) Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, and diethyl ether. (d) It does not show a clear melting point and decomposes at 160°C to 240°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2950~2920, 1680~1620, 1550~
1510, 1440, 1380, 1240~1220 and 1120~
It has an absorption band near 1020cm ^-1 . (f) The ultraviolet absorption spectrum of an aqueous solution of the water-soluble fraction at pH 7 has strong absorption at the absorption end, and
It exhibits absorption near 248-266 nm. (g) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, and Lowry-Follin reaction were positive. (h) Elemental analysis values C: 40% to 42%, H: 5% to 7%, N: 7% to 9% (li) The sugar content by the phenol sulfuric acid method of the water-soluble fraction at pH 7 is The protein content is about 5% to 20% (in terms of glucose), and the protein content by the Lowry-Follin method is about 10% or less (in terms of bovine serum albumin).