JPS627916B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer substance, and more particularly to TF-100, an anticancer substance obtained from the culture solution obtained by culturing bacteria belonging to the genus Fusobacterium. Furthermore, the present invention relates to a method for producing the anticancer substance TF-100 and an anticancer agent containing the same. In recent years, therapeutic methods that enhance the host's immune function and exert anticancer effects with the help of the immune function have become popular in the treatment of various cancer patients. 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, these drugs are still unsatisfactory in terms of efficacy, breadth of action, and manufacturing methods. On the other hand, there are also reports on bacterial cells and supernatants obtained by culturing Fusobacterium genus bacteria, such as Fusobacterium K031-3B, Fusobacterium fusiphomis W-12, Fusobacterium gilans 1012, and Fusobacterium nucleatum 1010. . [Stomatology Journal, 21 , pp. 534-539 (1972), Stotology Journal, 23 , pp. 322-333 (1974)]. However, the components obtained from the culture solution obtained by culturing the above-mentioned bacteria belonging to the genus Fusobacterium and their pharmacological effects have not yet been studied. Therefore, the present inventor cultivated bacteria belonging to the genus Fusobacterium isolated from the human oral cavity, and investigated the pharmacological effects of the supernatant liquid obtained by removing the bacterial cells from the culture solution. , the specific components collected from this supernatant have a strong anticancer effect, and this means that in the colony formation inhibition method, the effect of inhibiting cancer cell colony formation is extremely small, and the anticancer effect is not due to cell killing. We discovered that this ingredient has an effect that is host-mediated or that enhances the host's immunity and indirectly exerts an anticancer effect with the help of the immune system, and that this ingredient has extremely low toxicity. Completed the invention. The bacterium used in the present invention was isolated from the human oral cavity at the Department of Dental and Oral Surgery, Kanazawa University Hospital, and has the following mycological properties. (1) Morphology Cell shape: spindle-shaped (Figure 1) Presence of cell pleomorphism: None Motility: None Presence or absence of spores: None Gram staining: Gram negative Acid-fastness: Negative (2) Growth in medium Condition TF-a agar plate and slant medium External shape: Circular Size: Approximately 1 mm Protuberance: Hemispherical structure: Dewdrop-shaped surface: Smooth Edge: Smooth Color: Milky white Transparency: Opaque TF-a liquid medium Degree of growth: Strong turbidity: Coagulation precipitation: None Surface growth: None, no growth up to about 5 mm Gas: None (3) Physiological properties Hydrogen sulfide production: + Nitrate reduction: - Butyric acid production: + Indole production: + Urease: - Catalase: - Hydrolysis of starch: - Attitude towards oxygen: Anaerobic Production of ammonia: + Production of carbon dioxide: + Growth range: PH5-7.5 Temperature 30-45℃ Production of gas 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 determined according to Versey's Manual of Determinative Bacteriology, 8th edition. When examined, this strain was found to be Fusobacterium nucleatum (Fusobacterium nucleatum).
TF-031 (Fusobacterium nucleatum), we identified this strain as belonging to this group.
It was named TF-031) and deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under accession number FERM-P No. 5077. The anticancer substance TF-100 of the present invention is produced, for example, as follows. (a) Culture The culture of Fusobactenium nucleatum is as follows:
It is carried out using the usual anaerobic culture method. Namely, nitrogen sources such as various peptones, cow brain, and essential organ extracts; carbon sources such as glucose and lactose; vitamin sources such as yeast extract; reducing agents such as L-cystine, sodium sulfite, and thioglycolate; Using a medium containing an inorganic salt such as sodium chloride adjusted to sodium hydroxide pH 7, static culture is performed under anaerobic conditions at a temperature of 30 to 40°C for 1 to 5 days. In particular, it is preferable to use a medium containing the following components (hereinafter referred to as TF medium). In addition, when not using agar,
It is preferable to carry out agitation culture.

【table】

[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 using conventional methods such as centrifugation or filtration using a supernatant such as Hyflo Supergel, but centrifugation is particularly difficult in terms of operation, degree of bacterial removal, and amount of supernatant obtained. preferable. Although it is preferable to remove the bacterial cells at this stage, they may also be removed in the next step (c). (c) Collection of anticancer substance TF-100 Add a hydrophilic organic solvent to the supernatant obtained above or the culture solution obtained in (a), and collect the resulting precipitate. At this time, the supernatant or culture solution has a pH of 3 to 7.
It is preferable to adjust to Examples of hydrophilic organic solvents include alcohols such as ethanol and methanol, and ketones such as acetone, but ethanol gives the best results among alcohols. This hydrophilic organic solvent has a concentration of
It is suitable to add it in an amount of 30 to 70%, preferably around 60%. After adding the hydrophilic organic solvent, it is left to stand at a low temperature, preferably at a temperature of about 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, 10 to 15 times the amount of water is added to the precipitate obtained above, and the resulting water-insoluble matter is removed by a conventional method such as centrifugation or filtration. When a culture solution is used, bacterial cells are removed by this treatment. The anticancer substance TF-100 of the present invention can be obtained by drying the solution thus obtained by freeze-drying or the like. Novel anticancer substance obtained as above
TF-100 has the following physical properties. (a) White-gray to light brown powder. (b) It inhibits the growth of Ehrlichi's ascites-type carcinoma and Ehrlichi's nodule-type carcinoma in mice and has an immunostimulatory effect. (c) Dissolves in water, and the aqueous solution is almost neutral;
Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether. (d) It does not show a clear melting point, and begins to decompose at about 110°C, and significantly decomposes above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2960~2930, 1670~1640, 1550,
It has absorption bands near 1440-1380, 1240, 1140-1000 and 820 cm ^-1 (Figure 2). (F) The ultraviolet absorption spectrum of the aqueous solution has strong absorption at the absorption end and an absorption peak near 256 to 260 nm, and the OD ₂₆₀ of the 0.02% aqueous solution
shows an absorption of 0.530 to 0.570 (Figure 3). (g) Gel filtration using Cephadex G-50 (registered trademark of Pharmacia) (column: 50 mmφ
600mm, eluent: 260nm when fractionated with distilled water)
In the ultraviolet absorbance measurement, from 400, 430 to 530, and 700 from near the void volume passage.
It has absorption bands from around 800 and 840 to 870 ml, and in absorbance measurement at 620 nm using the Anthrone sulfuric acid method, it has absorption bands from around the void volume passage to 390 and from 410 to around 430 ml (Figure 4). (h) High-performance liquid chromatogram using TSK-GEL G300 SW (trade name of Toyo Soda Co., Ltd., column: 7.9 mmφ x 600 mm x 2) (eluent: 0.1 at PH7)
molar phosphate buffer, flow rate 0.8 ml/min, room temperature)
Ultraviolet absorbance measurements at 220 nm and 260 nm show peaks at the front of the solvent, around 38 to 60 minutes, and around 65 minutes, respectively (Figure 5). (li) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Foulin reaction, and ninhydrin reaction were positive. (NU) Elemental analysis value C; 30.6% to 35.7%, H; 4.2% to 5.2%,
N; 4.2% to 5.2% (L) Sugar content by phenol sulfuric acid method is 40.0
% ~ 46.0% (glucose equivalent) and protein content by Lowry-Follin method is 20.0%
~23.0% (bovine serum albumin equivalent). The pharmacological action of the anticancer substance TF-100 of the present invention is as follows. (1) Effect of inhibiting cancer cell colony formation The method of Kim, J.E., et al. [Cancer
Res., 25 , 698 (1965)], Hela S-
3 Cells were added with 10% bovine serum.
After culturing in MEM medium for 3 to 5 days, the culture medium was removed and a PBS (-) solution containing 0.01% ethylenediaminetetraacetic acid and 0.1% trypsin (8.0 g of sodium chloride, 0.2 g of potassium chloride, 0.2 g of potassium chloride, 1 hydrogen phosphate, Add an aqueous solution containing 1.15 g of sodium and 0.2 g of potassium dihydrogen phosphate), peel the cells from the glass surface of the culture bottle,
Dilute with Eagle's MEM medium supplemented with 20% bovine serum so that 1 ml of diluted solution contains 200 cells. After culturing 1 ml of this cell suspension at 37° C. for 24 hours in a CO ₂ incubator, the supernatant obtained in Example 1 (1) is added at a dilution of 1/16 to 1/128 times. In addition, after culturing at 37°C for 24 hours, the anticancer substance TF-100 of the present invention was added.
Culture Hela cells for 7 days. After culturing, remove the medium and wash the culture dish with Hank's solution to 70%
The cells were fixed with ethanol, then washed with 100% ethanol, and after removing the ethanol, the cells were stained with Giemsa staining solution, and the number of colonies was counted to determine the viability of the cells. The results are shown in Tables 1 and 2. The survival rate of the cells in the table was calculated using the following formula, and 5
It is shown as the average value of the sheets. Cell viability = number of colonies in treated group / number of colonies in untreated group × 100

【table】

[Table] As is clear from this experiment, the anticancer substance TF
Compared to the supernatant, -100 has a very weak direct cytotoxic effect on cancer cell colony formation. (2) Immunostimulatory effect Using a group of 4 ICR mice, anticancer substances were
TF-100 was dissolved in 0.2 ml of physiological saline and administered intraperitoneally at 10 mg and 50 mg/Kg. 24 hours after administration
0.2 ml of carbon suspension prepared by mixing 1 ml of Perikan Drawing Ink 17 Black (manufactured by Guilnter Wagner) and 2 ml of physiological saline containing 3% gelatin was injected into the mouse tail vein, and after injection 1, 5, 10 and 15 Minutes later, 0.02 ml of blood was collected from the orbit using a heparin-coated hematocrit capillary tube, and immediately 1.6 ml of 0.1% sodium carbonate aqueous solution was collected.
ml of diluted hemolysate and colorimetrically compare it at a wavelength of 675 nm to determine 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 3.

[Table] As is clear from this experiment, the reticuloendothelial system macrophages were activated in the TF-100 administration group of the present invention, and the cell-mediated immunity of normal mice was increased, compared to the control group. (3) Anticancer activity a. Antitumor activity against Ehrlitsu ascites tumor ICR mice (female, 6 weeks old) were intraperitoneally inoculated with 1 x 10 ⁵ Ehrrich ascites cancer cells per mouse. Then, TF-100 was dissolved in physiological saline and administered once a day from the first day after cancer cell inoculation.
Each dose was continuously administered for 7 days. In addition, to the control group, 0.1 ml of physiological saline was administered once. The results are shown in Table 4. T/C = Survival days of treatment group/Number of survival days of control group x 100
(%)

[Table] As is clear from this experiment, a survival effect was observed in the TF-100 administration group of the present invention compared to the control group. b. Antitumor effect on Ehrlichi nodular tumor
Ehrlichi cancer cells were subcutaneously transplanted into ICR mice (female, 6 weeks old) at 4×10 ⁶ cells per mouse under the armpit. Then, TF-100 was administered to each mouse at 35 mg/Kg for the intravenous administration group and 70 mg/Kg for the subcutaneous administration group and intraperitoneal administration group, once a day for 7 days, starting from the first day after cancer cell transplantation. It was administered continuously for several days. In addition, to the control group, 0.2 ml of physiological saline was administered once. Tumor weight was measured 11 days after cancer cell transplantation. The tumor weight was determined by measuring the major axis (a) and minor axis (b) of the tumor site using calipers and using the following formula. Tumor weight = a×b ² /2 (mg) The results are shown in Table 5.

[Table] As shown in Table 5, compared to the control group, TF-100
Tumor growth suppressive effects were observed in the treated group. The toxicity of TF-100 of the present invention is extremely low as shown below. As is clear from the results of pharmacological experiments with acute toxicity ( _LD50 ) in mice of 500 mg/Kg or more, the anticancer substance TF-100 of the present invention is useful as an anticancer agent and can be used for various cancer diseases. It is expected to have a significant effect on solid cancers. The TF-100 of the present invention can be used 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 TF-100 of the present invention is appropriately selected depending on the patient's symptoms, but is generally 0.01 to 0.01 for adults.
It is preferable to administer 50 mg/Kg in one to several divided doses a day, and administration methods include oral, subcutaneous, intramuscular,
Preferably, it is administered intravenously or by injection into the affected area. Next, examples of the present invention will be described. Example 1 (1) Into 15 2-volume culture bottles with screw caps, each bottle contained 34 g of Tryptocase Peptone, 6 g of Phaiton Peptone, 20 g of Protease Peptone, 70 g of Brain Heart Infusion, and Yeast Extract. 6g, salt 15g, glucose 12g, lactose
10g, L-cystine 0.5g, sodium sulfite 0.2
Add medium 2, adjusted to pH 7 by adding g, sodium thioglycolate 1.0 g, and agar 1.4 g, autoclave at 120°C for 15 minutes, immediately cool with water, and pre-culture in a medium with the same composition. Fusobacterium nucleatum TF-
031 (Feikoken Trust No. 5077)
100 ml of the preculture solution per culture bottle was inoculated under sterile conditions, and static culture was performed for 48 hours in an incubator at 37°C. After incubation, 4000 r.p. at 5°C.
The culture solution was centrifuged for 20 minutes to remove bacterial cells and a supernatant liquid of about 2.0 m was obtained. (2) Add ethanol to the supernatant obtained in (1) at 5℃ while stirring.
40 and leave it in a cold room until the amorphous precipitate is completely settled. Then, at 5℃, 6000r.
pm, centrifuged for 15 minutes, collected the precipitate, washed with ethanol, and dried under reduced pressure to obtain about 60 g of crude powder. (3) Dissolve 20g of the coarse powder obtained in (2) in 120ml of water,
The water-insoluble substances generated at this time were removed by centrifugation at 7500 rpm for 10 minutes, and the supernatant and water-insoluble substances were mixed with 60% water.
The washing solution obtained by washing twice with 3.0 mL of water was combined and dried under reduced pressure to obtain 14 g of white-gray to light brown TF-100 powder. Formulation Example 5 mg of the powder obtained in Example 1 was filled into a vial. When using this, use sterile water or lidocaine 0.5
It is used as an injection solution dissolved in a solution containing 5%.

[Brief explanation of the drawing]

Figure 1 is a micrograph showing the morphology of Fusobacterium nucleatum TF-031 used in the present invention, Figure 2 is the infrared absorption spectrum of the anticancer substance TF-100 of the present invention, and Figure 3 is the ultraviolet absorption spectrum of the same substance. , FIG. 4 shows the elution pattern when the same substance was gel-filtered using Sephadex G-50, and FIG. 5 shows the high performance liquid chromatogram of the same substance.

Claims (1)

[Claims] 1. Anticancer substance TF belonging to the genus Fusobacterium
-100 An anticancer substance TF-100 obtained from the culture solution by culturing the producing bacteria and having the following properties. (a) White-gray to light brown powder. (b) It inhibits the growth of Ehrlichi's ascites-type carcinoma and Ehrlichi's nodule-type carcinoma in mice and has an immunostimulatory effect. (c) Dissolves in water, and the aqueous solution is almost neutral;
Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether. (d) It does not show a clear melting point, and begins to decompose at about 110°C, and significantly decomposes above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2960~2930, 1670~1640, 1550,
It has absorption bands near 1440-1380, 1240, 1140-1000 and 820 cm ^-1 . (F) The ultraviolet absorption spectrum of the aqueous solution has strong absorption at the absorption end and an absorption peak near 256 to 260 nm, and the OD ₂₆₀ of the 0.02% aqueous solution
shows an absorption of 0.530 to 0.570. (g) Gel filtration using Cephadex G-50 (registered trademark of Pharmacia) (column: 50 mmφ
600mm, eluent: 260nm when fractionated with distilled water)
In the ultraviolet absorbance measurement of 400, 430 to 530, 700 to 800 from the void volume passage
It has an absorption band from around 840 to 870 ml, and when measuring the absorbance at 620 nm using the Anthrone sulfuric acid method, it starts from around the void volume passage.
It has absorption bands from 390 and 410 to around 430ml. (h) High-performance liquid chromatogram using TSK-GEL G300 SW (trade name of Toyo Soda Co., Ltd., column: 7.9 mmφ x 600 mm x 2) (eluent: 0.1 at PH7)
molar phosphate buffer, flow rate 0.8 ml/min, room temperature)
In ultraviolet absorbance measurements at 220 nm and 260 nm, peaks are observed at the front of the solvent, around 38 to 60 minutes, and around 65 minutes, respectively. (li) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Foulin reaction, and ninhydrin reaction were positive. (nu) Elemental analysis value C: 30.6% to 35.7%, H: 4.2% to 5.2% N: 4.2% to 5.2% (ru) Sugar content by phenol sulfuric acid method is 40.0
% ~ 46.0% (glucose equivalent) and protein content by Lowry-Follin method is 20.0%
~23.0% (bovine serum albumin equivalent). 2 Anticancer substance TF belonging to the Fusobacterium genus
−100 A hydrophilic organic solvent is added to the culture solution or supernatant obtained by culturing the producing bacteria, and the resulting precipitate is collected.
The anticancer substance according to claim 1, which is obtained by adding water to the substance and removing water-insoluble substances.
TF−100. 3 Anticancer substance TF belonging to the Fusobacterium genus
The anticancer substance TF-100 according to claim 1 or 2, wherein the -100 producing bacterium is Fusobacterium nucleatum. 4 Anticancer substance TF belonging to the Fusobacterium genus
- Collecting the precipitate produced by adding a hydrophilic organic solvent to the culture solution or supernatant obtained by culturing the -100 producing bacteria,
A method for producing anticancer substance TF-100, which comprises adding water to the substance to remove water-insoluble substances. 5. The method for producing anticancer substance TF-100 according to claim 4, wherein the hydrophilic organic solvent is alcohol. 6. Anticancer substance TF-100 according to claim 4 or 5, characterized in that a hydrophilic organic solvent is added to the culture solution or supernatant at a concentration of 30 to 70%. manufacturing method. 7 Anticancer substance TF belonging to the Fusobacterium genus
-100-producing bacteria are cultured and the following properties obtained from the culture solution: (a) White-gray to light brown powder. (b) It inhibits the growth of Ehrlichi's ascites-type carcinoma and Ehrlichi's nodule-type carcinoma in mice and has an immunostimulatory effect. (c) Dissolves in water, and the aqueous solution is almost neutral;
Insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate, diethyl ether. (d) It does not show a clear melting point, and begins to decompose at about 110°C, and significantly decomposes above 200°C. (e) The infrared absorption spectrum obtained by the KBr tablet method is
3600~3200, 2960~2930, 1670~1640, 1550,
It has absorption bands near 1440-1380, 1240, 1140-1000 and 820 cm ^-1 . (F) The ultraviolet absorption spectrum of the aqueous solution has strong absorption at the absorption end and an absorption peak near 256 to 260 nm, and the OD ₂₆₀ of the 0.02% aqueous solution
shows an absorption of 0.530 to 0.570. (g) Gel filtration using Cephadex G-50 (registered trademark of Pharmacia) (column: 50 mmφ
600mm, eluent: 260nm when fractionated with distilled water)
In the ultraviolet absorbance measurement, from 400, 430 to 530, and 700 from near the void volume passage.
It has absorption bands from around 800 and 840 to 870 ml, and in absorbance measurement at 620 nm using the Anthrone sulfuric acid method, it has absorption bands from around the void volume passage to 390 and from 410 to around 430 ml. (h) High-performance liquid chromatogram using TSK-GEL G300 SW (trade name of Toyo Soda Co., Ltd., column: 7.9 mmφ x 600 mm x 2) (eluent: 0.1 at PH7)
molar phosphate buffer, flow rate 0.8 ml/min, room temperature)
Ultraviolet absorbance measurements at 220 nm and 260 nm show peaks at the front of the solvent, around 38 to 60 minutes, and around 65 minutes, respectively. (li) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Foulin reaction, and ninhydrin reaction were positive. (NU) Elemental analysis value C; 30.6% to 35.7%, H; 4.2% to 5.2%,
N; 4.2% to 5.2% (L) Sugar content by phenol sulfuric acid method is 40.0
% ~ 46.0% (glucose equivalent) and protein content by Lowry-Follin method is 20.0%
~23.0% (bovine serum albumin equivalent). An anticancer agent characterized by containing an anticancer substance TF-100 having the following properties.