JPH0571230B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antitumor component SPF10-10 and a method for producing the same. Conventionally, hemolytic streptococcus (hereinafter referred to as streptococcus)
Attenuated viable bacterial cells have been prepared and are already being used as anticancer agents. In addition, after crushing the cells of hemolytic streptococcus, the active ingredients are extracted with water or a saline solution, an organic solvent is added, and the antitumor ingredients are precipitated and recovered.
1647), a method of lysing hemolytic streptococci with the lytic enzyme lysozyme, cellulase or proteolytic enzyme, and fractionating the active fraction as a water-soluble fraction (British patent
1163865), and a method in which water-insoluble substances are collected after crushing the cells of hemolytic streptococcus and treated with a nuclease and a protease (Japanese Patent Application Laid-Open No. 7014-1982). As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor activity. It was nothing more than a constituent substance. If you try to isolate the active ingredient from the bacterial body or inside the bacterial body,
It was necessary to fractionate the entire bacterial body by lysing it or mechanically crushing it. Such treatments complicate purification and isolation of active ingredients is extremely difficult. In cases where the active ingredient was actually separated and measured, the molecular weight
200,000 proteins (Special Publication No. 43841, No. 43841, No. 43841, No. 43841)
44,617) and a glycoprotein with a molecular weight of 150,000 (Unexamined Japanese Patent Publication No. 1983
−22026) is known. The present inventors previously conducted extensive research on a method for eluating antitumor components into the culture solution of streptococcus, and found that
It was discovered that antitumor components were eluted into the culture medium by adding penicillin or related substances during culture (Japanese Patent Application Laid-open No. 60-30677), and the physiologically active substance SPF-1 was extracted from the culture medium. This led to the separation. (Japanese Unexamined Patent Publication No. 60-30689) The present inventors further conducted research for the purpose of isolating a more effective component from the culture filtrate of hemolytic streptococcus, and found that in the present invention, cancerous white blood cell culture cells L121
(hereinafter referred to as cultured cell L1210),
and an antitumor component characterized by being a positive color reaction fraction by the Anthrone sulfuric acid method.
We were able to separate SPF10-10. The antitumor component SPF10-10 of the present invention is determined by elemental analysis,
Although it is considered to be a peptide-like substance containing sugar based on the color reaction and infrared absorption spectrum, the ultraviolet absorption spectrum and infrared absorption spectrum indicate that it is a new substance that is different from known antitumor substances. The present invention relates to the production of antitumor component SPF10-10, which is characterized by culturing antitumor component SPF10-10-producing bacteria belonging to the genus Streptococcus and collecting antitumor component SPF10-10 from the culture. It encompasses the law. In the present invention, bacteria that produce the antitumor component SPF10-10 belonging to the genus Streptococcus can be widely used. Next, bacteria producing the antitumor component SPF10-10 will be described. Streptococcus pyogenes ATCC21060 Streptococcus sp. ATCC21597 Streptococcus pyogenes ATCC21546 Streptococcus pyogenes ATCC21547 Streptococcus pyogenes) ATCC21548 Culture media include meat extract medium, yeast extract medium, brain heart infusion medium (BHI
A natural medium such as Streptococcus spp. is often used, but any medium can be used as long as it is suitable for the growth of Streptococcus bacteria. The pH of the culture is 5.0 to 8.0, preferably 6.1 to 7.2, and the culture temperature is 30 to 40°C, preferably 35 to 7.2.
The temperature is 37°C, and static culture or stirring culture can be performed anaerobically. In the present invention, adding penicillin or its related substances at an appropriate time during culture plays an important role in collecting the antitumor component SPF10-10. The timing of addition of penicillin or its related substances is preferably 3 to 20 hours, particularly 5 to 10 hours after the culture reaches the logarithmic growth phase at 35 to 37°C. After that, by continuing the culture for 1 to 20 hours, preferably 3 to 15 hours, antitumor components are added during the culture.
A large amount of SPF10-10 can be accumulated. Penicillin or its related substances may be any known related substances that have similar effects to penicillin, but penicillin G is commonly used. The amount added is penicillin G.
A concentration of 100 to 7000 units/ml, preferably 300 to 5000 units/ml of culture solution is sufficient. A culture solution obtained by culturing Streptococcus bacteria with the addition of penicillin or its related substances is centrifuged to remove bacterial cells to obtain a filtrate. Ammonium sulfate is added to the filtrate and a fraction having a saturation level of 50 to 90% is collected, or the filtrate is concentrated using an ultrafiltration membrane. The obtained ammonium sulfate precipitate containing the antitumor component SPF10-10 can also be stored in a frozen state. To extract the antitumor component SPF10-10 from this ammonium sulfate salt precipitate, the salt precipitate is dissolved in a buffer solution and used. Further, a concentrated solution obtained using an ultrafiltration membrane can also be used as it is. This aqueous solution was adsorbed onto a DEAE cellulose column and eluted stepwise using phosphate buffer to collect the active fraction that inhibits the growth of cultured cells L1210.This active fraction was transferred to a DEAE Sephadex A-25 column. adsorb and elute while linearly increasing the sodium chloride concentration in the phosphate buffer, separate the non-adsorbed portion and the low sodium chloride concentration portion, dialyze this fractionated portion against the buffer solution, and gel. Adsorb onto a Toyopearl HW-50F filter column, elute with phosphate buffer containing 0.1M NaCl, and examine the color reaction using the Anthrone sulfuric acid method. SPF10-10 can be obtained as a fraction that forms a peak in the color reaction by the Anthrone sulfuric acid method near the exclusion limit (hereinafter abbreviated as Vo) of Toyopearl HW50F. Next, the antitumor component SPF10 obtained in Example 1
The freeze-dried specimen of -10 exhibits the following physical and chemical properties. 1 Elemental analysis C 39.5-43.5% H 5.8-6.7% N 7.3-10.4% O 46.9-37.5% Ash 0.5-1.9% 2 Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3 Specific rotation [α] ²⁰ _D = +5° to +30° (C = 1.00) 4 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. 5 Infrared absorption spectrum Shown in Figure 2.
Absorption by peptides near 3300 cm ^-1 and 1640 cm ^-1 and absorption by sugar at 840 cm ^-1 are characteristic. 6. Distinction between basic, acidic, and neutral The pH of a 0.1% aqueous solution of this substance is 5.5 to 6.0. 7 Color of the substance Light brown 8 Color reaction Lowry reaction + Biuretz reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Moritzhu reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight When measured by gel filtration method, the molecular weight is approx.
More than 10000. 10 Solubility in solvents It is soluble in water, but poorly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. Next, a method for measuring antitumor activity against cultured cells L1210 in vitro and a color reaction method using the anthrone sulfate method will be shown. Method for measuring antitumor activity Antitumor activity was measured by measuring the growth inhibition rate (IR%) of cultured cells L1210. L1210 cells were grown in RPMI1640 medium (5%) supplemented with 10% FBS.
mg/kanamycin). 0.5 ml of this culture solution was poured into a Falcon 2058 tube so that the number of cells was 1×10 ⁵ cells/tube. Next, add a predetermined amount of the preparation (antitumor component) to this culture solution.
0.5 ml of SPF10-10) dissolved in the culture medium to the desired concentration was added, and cultured at 37° C. in the presence of 5% CO ₂ . 48 hours after adding the standard, staining with trypan blue was performed, and IR was determined using the following formula.
(%) was calculated. IR (%) = (A) - Number of viable cells in each experimental group/Number of viable cells in control group x 100 Here, (A) indicates the number of viable cells in the control group. A control was performed at the same time using 0.5 ml of culture solution that did not contain the standard. Color reaction using the Anthrone sulfuric acid method Add 2 ml of Anthrone reagent (0.20 gr of Anthrone to 1 ml of the sample dissolved in deionized water to the desired concentration).
(dissolved in 100ml of concentrated sulfuric acid) and mix.
After 30 minutes, replace 1 ml of standard with 2 ml in 1 ml of deionized water.
The solution containing Anthrone reagent was used as a control.
Measure absorption at 620nm. The quantitative value is obtained from a calibration formula obtained by measuring in the same manner using glucose. Next, examples of the present invention will be shown. Example 1 BHI of Streptococcus pyogenes ATCC21060
A seed culture solution obtained by inoculating 100 ml of a medium and statically culturing at 37°C for 8 hours was inoculated into medium A1l shown in Table 1, and preculture was performed anaerobically under the same conditions as the seed culture. Table 1 Medium A Maltose 0.25% Meat extract 1.0% Polypeptone 1.0% Yeast extract 0.25% Acidic monobasic potassium phosphate 0.1% Magnesium sulfate 0.05% PH=6.8 10 Pour medium A8 into a jar fermentor and heat at 120℃ for 10 minutes After heat sterilization, cool to 37℃, inoculate with preculture solution 1, and incubate at 37℃ for 15.5 hours at PH.
6.8. Incubate anaerobically with stirring at 300 rpm. Then, penicillin G was added at 1000 units/ml culture solution, and the culture was continued for an additional 5 hours.
The resulting culture solution was centrifuged to remove bacterial cells. The culture filtrate was concentrated to 1.5 using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Romicon, USA). This concentrated solution was applied to a DEAE cellulose column (5
x 70cm), washed with a 10mM phosphate buffer with a pH of 7.0, and then eluted stepwise using the above phosphate buffer containing 0.3M sodium chloride, followed by the Anthrone sulfate method. An active fraction that showed a positive color reaction and inhibited the growth of cultured cells L1210 was collected. This active fraction was adsorbed on a DEAE Sephadex A-25 column (7.5 x 50 cm), and then washed with the above phosphate buffer and eluted by linearly increasing the sodium chloride concentration. Separate a certain portion. This elution curve is shown in FIG. In FIG. 3, the dotted line portion is the non-adsorbed portion, and the solid line portion is the sodium chloride added portion. A is a fraction with activity to inhibit cultured cell L1210 growth;
B is the non-active fraction inhibiting the growth of MP-2 (JP-A-60-30689); B is the non-active fraction inhibiting the growth of cultured cells L1210; B is the active fraction inhibiting MP-2 growth. The eluate of the active fraction of A was concentrated to 30 ml using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Millipore). Transfer this concentrated solution to a Toyopearl HW50F column (5.0 x 100cm).
Then, the mixture was eluted with a solution containing 0.1M sodium chloride in the above phosphate buffer, and a fraction showing a positive color reaction by the Anthrone sulfuric acid method was collected.
This elution curve is shown in FIG. In FIG. 4, the solid line shows the overall elution curve, and the dotted line shows the elution curve of the positive color reaction portion by the Anthrone sulfuric acid method. Here, C is a positive color reaction fraction by the Anthrone sulfuric acid method, but the growth inhibition activity against cultured cells L1210 was investigated, and the fraction near Vo, which has strong activity,
A D fraction that forms a peak is obtained by the Anthrone sulfuric acid method. Fraction D was lyophilized. This freeze-dried specimen had an antitumor component SPF 10-10 and yielded 1.70 gr. Example 2 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at 300 units/ml culture solution, and the culture was further increased by 10
Lasted for an hour. Add ammonium sulfate to this culture filtrate and
Collect the fraction at 90% saturation. Ammonium sulfate was removed by dialysis and purified in the same manner as in Example 1 to obtain 0.86 gr of antitumor component SPF10-10. Example 3 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 3000 units/ml culture solution, and the culture was further incubated for 3
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 1.93 gr of antitumor component SPF10-10. Example 4 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium B shown in Table 2. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 1.23 gr of antitumor component SPF10-10. Table 2 Medium B Maltose 0.1% Meat extract 0.5% Polypeptone 1.0% Yeast extract 0.25% Sodium chloride 0.1% PH=7.2 Example 5 Streptococcus pyogenes ATCC21060
were cultured at 35°C in the same manner as in Example 1 using medium C shown in Table 3. In this case, penicillin G
The mixture was added at a concentration of 1000 units/ml culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.92gr of antitumor component SPF10-10.
I got it. Table 3 Medium C Maltose 0.1% Meat extract 0.5% Polypeptone 0.5% Casamino acids 0.3% Yeast extract 0.5% Acidic potassium monophosphate 0.1% Magnesium sulfate 0.05% PH=6.5 Example 6 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium D shown in Table 4. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.73 gr of antitumor component SPF10-10. Table 4 Medium D Maltose 0.25% Casamino acids 0.3% Yeast extract 1.0% Acidic monobasic potassium phosphate 0.1% Magnesium sulfate 0.05% PH=6.9 Example 7 Using medium A shown in Table 1 for Streptococcus sp. ATCC21597 The cells were cultured in the same manner as in Example 1. In this case, penicillin G is 1000 units/
ml culture solution, and culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 1.31 gr of antitumor component SPF10-10. Example 8 Streptococcus pyogenes ATCC21546
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 1.45 gr of antitumor component SPF10-10. Example 9 Streptococcus pyogenes ATCC21547
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 1.48 gr of antitumor component SPF10-10. Example 10 Streptococcus pyogenes ATCC21548
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 1.26 gr of antitumor component SPF10-10. Example 11 Streptococcus pyogenes ATCC21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, cephalosporin C is
The mixture was added at a concentration of 600 μg/ml culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 1.57gr of antitumor component SPF10-10.
I got it. Experimental Example 1 Antitumor component SPF10-10 obtained in Example 1
(hereinafter referred to as SPF10-10) was tested for antitumor activity in vitro using the method for measuring antitumor activity described in the text, and the results are shown in Table 5. Table 5 Results of antitumor activity of SPF10-10 SPF10-10 (mg/ml) IR% 1.0 94.0 0.5 81.6 0.25 25.2 Experimental example 2 The in vivo antitumor activity test of SPF10-10 obtained in Example 1 was as follows: Crj; CD-1 (ICR series,
The experiments were carried out using female mice (7 weeks old). Sarcoma-180 ascites cancer cells were used as tumor cells, suspended in physiological saline, and inoculated intraperitoneally into mice at 5×10 ⁵ cells/mouse. SPF10-10 was administered intraperitoneally once a day for 5 consecutive days starting 24 hours after cancer cell inoculation, and the number of survivors was observed. The results are shown in Table 6. In Table 6, adjust SPF10-10 to the target concentration, use 1/2 amount as is, and use the remaining 1/2 amount as
The bottom row shows the case where the drug was administered after being heated in boiling water for 20 minutes.

[Table] Experimental Example 3 An antibody production ability promotion test of SPF10-10 obtained in Example 2 was conducted by the following method. Sheep red blood cells (SRBC) (hereinafter referred to as SRBC)
Crj; CD-1 (ICR female, 5 weeks old) was inoculated into the tail vein at 2×10 ⁷ mice/mouse. SPF10-10 is 1 day from 3 days before SRBC vaccination.
3 times per day for 3 days and once a day from the 1st day after SRBC vaccination
Administer intraperitoneally a total of 6 times per day. Antiserum is prepared by collecting blood from the mouse heart on the 18th day of SRBC administration. Antibody production activity was examined by two methods: agglutination reaction with SRBC and complement addition, and hemolysis of SRBC. For each SRBC, 2×10 ⁸ cells/ml and 50μ were used, and for the antiserum, 50μ was used. The antiserum was diluted by a 2-fold stepwise dilution method, and the maximum dilution factor at which each reaction occurred was determined and expressed as the average value of the dilution coefficient, which is shown in Table 7.

[Table] Experimental Example 4 The antitumor activity test of SPF10-10 obtained in Experimental Example 2 against solid tumors was carried out using Crj;CD-1 (ICR strain, female, 5 weeks old) mice. Sarcoma180 was used as solid tumor cells and 5×10 ⁵ cells/mouse were inoculated subcutaneously into the left thigh of the mouse. SPF10-10 is applied once a day starting 24 hours after cancer cell inoculation.
Administer intraperitoneally for 5 consecutive days to inoculate cancer cells 32 times
On the day, solid tumors were excised and their weights were measured. Table 8 shows the average weight values.

[Table] Experimental Example 5 The TNF-inducing activity of SPF10-10 obtained in Example 2 was measured as follows with reference to Katsuyuki Haranaka, "TNF" Nippon Iji Shinposha (1984). Mouse (Crj; CD-1, ICR strain, female, 7 weeks old)
BCG (Japan BCG Manufacturing Association) 5 mg in the abdominal cavity of
(primary sensitization), 2 weeks later, SPF10-10 is administered through the tail vein (secondary sensitization), blood is collected from the heart 2 hours after SPF10-10 administration, and serum is adjusted. Test cells were grown in RPMI1640 medium (5% FBS-supplemented).
mg/kanamycin) for 3 to 4 days.
Use L929 cells. For L929 cells, 0.5 ml of a suspension of 1×10 ⁵ cells/ml is injected into 24 wells of Falcon. Here, the above serum was diluted 20 times with the above RPMI1640 medium, 0.5 ml of it was added and mixed, and the mixture was heated to 5% at 37°C.
Incubate for 48 hours in the presence of _CO2 . To measure TNF-induced activity, L929 cells were stained with trypan blue, and the number of viable cells was counted using a dye exclusion method. The results are shown in Table 9.

[Table] Experimental Example 6 The deformation activity of the SPF 10-10 macrophages obtained in Example 1 on cultured cells J-774-1 (hereinafter abbreviated as J-774-1) was measured as follows. J-774-1 was prepared using RPMI640 medium (5%) supplemented with 10% FBS.
mg/kanamycin) after 3 to 4 days of culture, 10
×10 Adjust to ⁴ pieces/ml. SPF10-10 is 5mg/ml with physiological saline,
Filter with a 0.22μm sterilization filter, 10μm each96
Dispense into wells (Falcon) using the 2-fold serial dilution method. 100μ of the above J-774-1 suspension is added to each well, and cultured for 96 hours at 37°C and 5% _CO2 . To measure the deformation activity, each well was observed under an inverted microscope, and the minimum concentration that induced a change in morphology (from spherical to elongated and elongated) was determined. The results are shown in Table 10. Table 10 Minimum concentration to induce deformation of SPF10-10 for J-774-1 Minimum concentration to induce deformation SPF10-10 25.0μg/ml

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the antitumor component SPF10-10, and Figure 2 also shows the infrared absorption spectrum. Fig. 3 shows the elution curve of the active fraction in Example 1 on the DEAE Sephadex A-25 column, and Fig. 4 shows the elution curve of the active fraction on the DEAE Sephadex A-25 column. It is a figure showing a curve.

Claims (1)

[Claims] 1. Antitumor component having the following physicochemical properties:
SPF10−10. 1 Elemental analysis C 39.5-43.5% H 5.8-6.7% N 7.3-10.4% O 46.9-37.5% Ash 0.5-1.9% 2 Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3 Specific rotation [α] ²⁰ _D = +5° to +30° (C = 1.00) 4 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. 5 Infrared absorption spectrum Shown in Figure 2.
Absorption by peptides near 3300 cm ^-1 and 1640 cm ^-1 and absorption by sugar at 840 cm ^-1 are characteristic. 6. Distinction between basic, acidic, and neutral The pH of a 0.1% aqueous solution of this substance is 5.5 to 6.0. 7 Color of the substance Light brown 8 Color reaction Lowry reaction + Biuretz reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Moritzhu reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight When measured by gel filtration method, the molecular weight is approx.
10000 or more. 10 Solubility in solvents It is soluble in water, but poorly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. 2 Antitumor component belonging to the genus Streptococcus
A method for producing an antitumor component SPF10-10, which comprises culturing SPF10-10 producing bacteria and collecting the antitumor component SPF10-10 from the culture. 3 Antitumor component belonging to the genus Streptococcus
Claim 2, characterized in that when culturing SPF10-10 producing bacteria, penicillin or a related substance is added at an appropriate time during the culturing.
The method for producing the antitumor component SPF10-10 described in Section 1. 4 Antitumor component belonging to the genus Streptococcus
When culturing SPF10-10-producing bacteria and collecting the antitumor component SPF10-10 from the culture, cultured cells
The method for producing the antitumor component SPF10-10 according to claim 2, which comprises inhibiting the growth of L1210 and/or separating a fraction that is positive for a color reaction by the anthrone sulfuric acid method.