JPS627917B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer substance, and more particularly to TF-110, an anticancer substance obtained from a 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-110 and an anticancer drug 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, side effects, and manufacturing methods. On the other hand, there are also reports regarding bacterial cells and supernatants obtained by culturing bacteria belonging to the genus Fusobacterium, such as Fusobacterium K031-3B, Fusobacterium fusiphomis W-12, Fusobacterium gilans 1012, and Fusobacterium nucleatum 1010. [Stomachology 21 , pp. 534-539 (1972), Stomachology 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 pharmacology are Its 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 of spores: None Gram staining: Gram negative Acid-fastness: Negative (2) Medium Growth status on TH-a agar plate and slant medium External shape: Circular Size: Approximately 1 mm Elevation: Hemispherical Structure: Dewdrop-like Surface: Smooth Edge: Smooth Color: Milky white Transparency: Opaque TF-a liquid medium Growth level: Strong Turbidity: Coagulum Sedimentation: 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 ~ 7.5 Temperature 30-45℃ Production of gases from sugar L-arabinose (-), D-xylose (-), D-glucose (-), D-mannose (-), D-fructose (-), D-galactose (-), maltose (-), sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inositol (-), glycerin (-), starch (-)・Manual of Determinative Bacteriology No. 8
When examined against the edition, this strain is Fusobacterium nucleatum.
Since it closely resembles Fusobacterium nucleatum, we determined that it belonged to this strain, and identified this strain as Fusobacterium nucleatum.
TF-031 (Fusobacterium nucleatum TF-031)
FERM-P No. 5077 (FERM-P No.
5077). The anticancer substance TF-110 of the present invention is produced, for example, as follows. (a) Culture The culture of Fusobacterium nucleatum is
It is carried out using the usual anaerobic culture method. Namely, nitrogen sources such as various peptones, cow brain, and heart extract; 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 inorganic salts such as sodium, adjusted to pH 7 with sodium hydroxide, 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). Note that when agar is not used, it is preferable to perform 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. Furthermore, although it is preferable to remove the bacterial cells at this stage, they may be removed in the next step (c). (c) Collection of anticancer substance TF-110 A hydrophilic organic solvent is added to the supernatant or culture solution obtained above, and the resulting precipitate is collected. The supernatant or culture solution at this time is preferably adjusted to pH 3-7. 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 at a concentration of 30 to 70%, preferably around 60%. After adding hydrophilic organic solvent, low temperature,
Preferably, the mixture is allowed to stand 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 operations 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-110 can be obtained by drying the solution thus obtained by freeze-drying or the like. Furthermore, the separated solution obtained above is subjected to dialysis or ultrafiltration. Alternatively, TF-100 is dissolved in 10 to 15 times the volume of water and subjected to dialysis or ultrafiltration. This treatment removes low molecular components such as amino acids.
Collecting the internal fluid and drying it produces the anti-cancer substance TF.
−110 is obtained. TF-110 thus obtained has the following physical properties. (a) White gray to light brown (b) It inhibits the growth of Ehrlichi's ascites-type carcinoma in mice and has immunostimulatory effects. (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 is
It shows an absorption of 0.440 to 0.590 (Figure 3). (g) Gel filtration using Sephadex G-200 (registered trademark of Pharmacia) (column: 44 mmφ
×500mm, eluent: 0.1M phosphate buffer with pH7)
When fractionating with
It has an absorption band from 380 to 920 ml, and when measuring the absorbance at 490 nm using the phenol sulfuric acid method, it has an absorption band from 380 to 410 from near the void volume passage.
It has an absorption band from 520, 630 to around 760ml, and when measuring the absorbance at 620nm using the Anthrone sulfuric acid method, it has an absorption band from around the void volume passage.
It has absorption bands around 380, 420 to 520, and 620 to 760 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 are positive, and ninhydrin reaction is negative. (nu) Elemental analysis value C: 35.9% to 41.0%, H: 4.5% to 5.2%,
N: 4.2% to 5.2% (L) Sugar content by phenol sulfuric acid method is approx.
The protein content is approximately 30.0% to 69.0% (in terms of glucose) and approximately 18.0% to 22.0% (in terms of bovine serum albumin) according to the Lowry-Follin method. The pharmacological action of the anticancer substance TF-110 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 in Eagles MEM supplemented with 10% bovine serum.
After culturing in medium for 3 to 5 days, remove the culture solution and add 0.01% ethylenediaminetetraacetic acid and trypsin.
Add a PBS(-) solution containing 0.1% (an aqueous solution containing 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.15 g of sodium hydrogen phosphate and 0.2 g of potassium dihydrogen phosphate in 1 part) and place it in a culture bottle. Peel the cells from the glass surface and add bovine serum.
Dilute with 20% Eagles MEM medium and adjust 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-110 of the present invention was added and cultured for 7 days.
Culture Hela cells. After culturing, remove the medium,
Wash the culture dish with Hank's solution, fix the cells with 70% ethanol, then wash the cells with 100% ethanol, remove the ethanol, stain the cells with Giemsa stain, count the number of colonies, and determine the number of cells. The survival rate was determined. 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, -110 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-110 was dissolved in 0.2 ml of physiological saline, and 5 mg and 20 mg/Kg were administered intraperitoneally. Perikan Drawing InK17 24 hours after administration
0.2 ml of carbon suspension prepared by mixing 1 ml of Black (manufactured by Guilnter Wagner) and 2 ml of physiological saline containing 3% gelatin was injected into the tail vein of the mouse, and 1, 5, 10, and 15 minutes after injection, it was inserted into the orbit. 0.02 ml of blood was collected using a heparin-coated capillary tube, immediately diluted with 1.6 ml of 0.1% sodium carbonate aqueous solution, and this was colorimetrically measured at a wavelength of 675 nm.
index): K value was determined using the formula from Halpern. 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, reticuloendothelial system macrophages were activated in the TF-110 administration group of the present invention, and cell-mediated immunity in normal mice was increased, compared to the control group. (3) Anticancer effect Antitumor effect against Ehrlitsu ascites tumor: ICR mice (female, 6 weeks old) were intraperitoneally inoculated with 1 x 10 ⁵ Ehrrich ascites cancer cells per mouse. Next, TF-110 was dissolved in physiological saline and administered 6 times a day from the first day after contact with cancer cells.
Each dose was administered continuously every day. 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 10
0 (%)

[Table] The mice were completely cured. From this experiment

[Table] The anticancer substance TF-110 of the present invention has extremely low toxicity as shown below. Acute toxicity in mice (LD ₅₀ ) Intravenous > 250 mg/Kg Peritoneal > 1000 mg/Kg As is clear from the results of pharmacological experiments, the anticancer substance TF-110 of the present invention is useful as an anticancer agent, and is effective against various cancers. It is expected to be effective when used for cancer diseases, and in particular, it can be expected to have a remarkable effect on solid cancers. The TF-110 of the present invention can be used orally, by injection, in the form of suppositories, etc. by conventional methods. Oral preparations may contain various excipients and may be in the form of capsules, tablets, powders, or granules. In addition, 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-110 of the present invention is appropriately selected depending on the patient's symptoms, but is generally 0.01 to
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 two-volume culture bottles with screw caps, each bottle contained 34 g of tryptocase peptone, 6 g of phyton peptone, 20 g of protease peptone, 70 g of brain heart injection, and 6 g of yeast extract. , salt 15g, glucose 12g, lactose 10g, L-cystine 0.5g, sodium sulfite 0.2
Add 1.0 g of sodium thioglycolate, 1.4 g of agar, and add medium 2 adjusted to pH 7. After sterilizing under pressure at 120°C for 15 minutes, immediately cool with water and pre-culture in a medium with the same composition. Fusobacterium nucleatum TF-
031 (FEI accession number 5077
(FERM-P No. 5077)) was inoculated under sterile conditions at a rate of 100 ml per culture bottle, and statically cultured for 48 hours in an incubator at 37°C.
After culturing, the culture solution was centrifuged at 4000 rpm for 20 minutes at 5°C to remove the bacterial cells and the supernatant liquid was
I got it. (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 5℃, 6000r.p.
m., the precipitate was collected by centrifugation for 15 minutes, 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 matter generated at this time was removed by centrifugation at 7500 rpm for 10 minutes, and the supernatant liquid and the water-insoluble matter were removed with water.
Water soluble part combined with 60ml of washing solution washed twice
230ml was ultrafiltrated using a hollow fiber type ultrafiltration system (membrane number HI-1: product manufactured by Asahi Kasei Corporation), and the internal liquid was freeze-dried to form a white-gray TF.
10 g of -110 powder was obtained. 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 by dissolving it in a solution containing 5% or more.

[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-110 of the present invention, and Figure 3 is the ultraviolet absorption spectrum of the same substance. , Figure 4 shows the same substance as Cephadex G-200.
Elution pattern when gel filtrated using
The figure shows a high performance liquid chromatogram of the same substance.

Claims (1)

[Claims] 1. Anticancer substance TF belonging to the genus Fusobacterium
-110 An anticancer substance TF-110 obtained from the culture solution by culturing the producing bacteria and having the following properties. (a) White gray to light brown (b) It inhibits the growth of Ehrlichi's ascites-type carcinoma in mice and has immunostimulatory effects. (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 0.440 to 0.590. (g) Gel filtration using Sephadex G-200 (registered trademark of Pharmacia) (column: 44 mmφ
×500mm, eluent: 0.1M phosphate buffer with pH7)
When fractionating with
It has an absorption band from 380 to 920 ml, and when measuring the absorbance at 490 nm using the phenol sulfuric acid method, it has an absorption band from 380 to 410 from near the void volume passage.
It has an absorption band from 520, 630 to around 760ml, and when measuring the absorbance at 620nm using the Anthrone sulfuric acid method, it has an absorption band from around the void volume passage.
It has absorption bands around 380, 420 to 520, and 620 to 760ml. (h) High-performance liquid chromatogram using TSK-GEL G300SW (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)
For UV absorbance measurements at 220 nm and 260 nm, the solvent front, 38 to 60 minutes, and 65 minutes, respectively.
It has a peak around minutes. (li) Moritsch reaction, phenol sulfuric acid reaction, Anthrone sulfuric acid reaction, indole-hydrochloric acid reaction, Lowry-Foulin reaction are positive, and ninhydrin reaction is negative. (nu) Elemental analysis value C: 35.9% to 41.0%, H: 4.5% to 5.2%,
N: 4.2% to 5.2% (L) Sugar content by phenol sulfuric acid method is approx.
The protein content is approximately 30.0% to 69.0% (in terms of glucose) and approximately 18.0% to 22.0% (in terms of bovine serum albumin) by the Lowry-Follin method. 2 Anticancer substance TF belonging to the Fusobacterium genus
−110 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 TF-110 according to claim 1, which is obtained by adding water to the substance to remove water-insoluble substances, and then dialysis or ultrafiltration of the substance. 3 Anticancer substance TF belonging to the Fusobacterium genus
-110 The anticancer substance TF-110 according to claim 1 or 2, wherein the 110-producing bacterium is Fusobacterium nucleatum. 4 Anticancer substance TF belonging to the Fusobacterium genus
−110 A hydrophilic organic solvent is added to the culture solution or supernatant obtained by culturing the producing bacteria, and the resulting precipitate is collected,
A method for producing the anticancer substance TF-110, which comprises adding water to the substance to remove water-insoluble substances, and then dialysis or ultrafiltration. 5. The method for producing the anticancer substance TF-110 according to claim 4, wherein the hydrophilic organic solvent is alcohol. 6. Anticancer substance TF-110 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
-110-producing bacteria are cultured and the following properties obtained from the culture solution: (a) White gray to pale brown (b) It inhibits the growth of Ehrlichi ascites-type carcinoma in mice and has an immunostimulating 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 0.440 to 0.590. (g) Gel filtration using Sephadex G-200 (registered trademark of Pharmacia) (column: 44 mmφ
×500mm, eluent: 0.1M phosphate buffer with pH7)
When fractionated with 260nm ultraviolet absorbance measurement, there is an absorption band from around 380, 600 to around 920ml from the void volume passage, and in 490nm absorbance measurement using the phenol sulfuric acid method, there is an absorption band from 380, 410 to around the void volume passage.
It has an absorption band from 520, 630 to around 760ml, and when measuring the absorbance at 620nm using the Anthrone sulfuric acid method, it has an absorption band from around the void volume passage.
It has absorption bands around 380, 420 to 520, and 620 to 760ml. (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 are positive, and ninhydrin reaction is negative. (nu) Elemental analysis value C: 35.9% to 41.0%, H: 4.5% to 5.2%,
N: 4.2-5.2% (L) Sugar content by phenol sulfuric acid method is approx.
The protein content is approximately 30.0% to 69.0% (in terms of glucose) and approximately 18.0% to 22.0% (in terms of bovine serum albumin) according to the Lowry-Follin method. An anti-cancer drug characterized by containing the anti-cancer substance TF-110.