JPH0156076B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antitumor component SPF10CAP2 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 drugs. Another method is to extract the active ingredients with water or salt solution after crushing the cells of hemolytic streptococcus, add an organic solvent, and recover the antitumor ingredients as precipitates.
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. 1983-7014). 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 only 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. (Unexamined Japanese Patent Publication No. 60-30689). The present inventor further conducted research for the purpose of separating more effective components from the culture filtrate of hemolytic streptococcus, and found that
In the present invention, an antitumor component is characterized by inhibiting the growth of cultured cancerous leukocyte cells L1210 (hereinafter referred to as cultured cells L1210) and being a positive color reaction fraction by the anthrone sulfate method.
This led to the isolation of SPF10CAP2. The antitumor component SPF10CAP2 of the present invention is considered to be a peptide-like substance containing sugar based on elemental analysis, color reaction, etc., but from the ultraviolet absorption spectrum, it is recognized as a new substance that is different from known antitumor substances. It is something. The present invention encompasses a method for producing the antitumor component SPF10CAP2, which is characterized by culturing an antitumor component SPF10CAP2-producing bacterium belonging to the genus Streptococcus and collecting the antitumor component SPF10CAP2 from the culture. be. In the present invention, bacteria that produce the antitumor component SPF10CAP2 belonging to the genus Streptococcus can be widely used. Next, the bacteria producing the antitumor component SPF10CAP2 will be described. Streptococcus pyogenes ATCC21060 (Streptococcus pyogenes) Streptococcus sp. ATCC21597 (Streptococcus sp.) Streptococcus pyogenes ATCC21546 (Streptococcus pyogenes) Streptococcus pyogenes ATCC21547 (Streptococcus pyogenes) Streptococcus pyogenes ATCC2154 8 (Streptococcus pyogenes ) 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 SPF10CAP2. The addition time 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 SPF10CAP2 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. The culture solution obtained by culturing Streptococcus bacteria with the addition of penicillin or related substances is centrifuged to remove the bacterial cells, and ammonium sulfate is added to the filtrate. Separate the amount. The obtained ammonium sulfate precipitate containing the antitumor component SPF10CAP2 can also be stored in a frozen state. An antitumor composition is made from this ammonium sulfate precipitate.
To extract SPF10CAP2, the salt precipitate is dissolved in a buffer solution, this aqueous solution is adsorbed onto a DEAE cellulose column, and the active fraction that inhibits the growth of cultured cells L1210 is extracted stepwise using a phosphate buffer solution. This active fraction was added to DEAE Sephadex A.
-25 column, elute while linearly increasing the sodium chloride concentration in the phosphate buffer, separate the non-adsorbed portion and the low sodium chloride concentration portion, and add this fraction to the buffer. Dialyzed and adsorbed on gel filtration material Toyopearl HW-50F column.
Elute with phosphate buffer containing 0.1M NaCl, collect the color reaction positive part by Anthrone sulfuric acid method,
This fraction was then desalted by dialysis against phosphate buffer, adsorbed onto a handroxyapatite column, and SPF10CAP1 was eluted with 1 x 10 ^-2 M phosphate buffer, followed by 1 x 10 ^-1 M phosphate buffer. It can be obtained by elution with a buffer solution and freeze-drying. Next, the antitumor component obtained in Example 1
The freeze-dried specimen of SPF10CAP2 exhibits the following physical and chemical properties. 1 Elemental analysis C 40.0-44.5% H 5.8-6.5% N 12.9-15.4% O 31.1-40.0% Ash 1.3-2.5 2 Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3 Specific rotation [α] ²⁰ _D = -72° to -88° (C = 1.00) 4 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. The absorption maximum is observed at 275mm, and the same absorption value is shown up to 260mm. 5 Infrared absorption spectrum Shown in Figure 2. Absorption is observed at 3300cm ^-1 , 3070cm ^-1 , 2970cm ^-1 , 1640cm ^-1 , 1540cm ^-1 , 1440cm ^-1 , 1390cm ^-1 , 1120cm ^-1 and 684cm ^-1 . 6. Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5. 7 Color of the substance Light brown 8 Color reaction Lowry reaction + Bieurett reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Moritzhu reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight Molecular weight measured by gel filtration method: approximately 2 x 10 ⁴ ~ 5 ×10 ⁵ . 10 Solubility in solvents Soluble in water, but sparingly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. 11 Behavior towards ion exchanger Adsorbed on handroxyapatite, 1×
Eluted with 10 ^-1 M phosphate buffer. 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, a predetermined amount of the standard (antitumor composition) is added to this culture solution.
0.5 ml of SPF10CAP2) 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 100 ml of concentrated sulfuric acid) and mixed for 30 minutes.
ml of Anthrone reagent was added 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 Streptococcus pyogenes ATCC21060
The seed culture solution obtained by inoculating 100ml of BHI medium and statically culturing at 37℃ for 8 hours is used as the medium shown in Table 1.
A1l was inoculated and precultured 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 potassium monophosphate 0.1% Magnesium sulfate 0.05% PH=6.8 10 Pour medium A8 into a jar fermentor and incubate 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. Add ammonium sulfate to the culture filtrate and
A fraction precipitated at 90% saturation was collected. This ammonium sulfate salting out sample was dissolved in 300 ml of 1 x 10 ^-2 M, pH 7.0 phosphate buffer (KH ₂ PO ₄ -Na ₂ HPO ₄ ), and this aqueous solution was adsorbed on a DEAE cellulose column (5 x 70 cm). After that, stepwise elution was performed using the above phosphate buffer containing 0.3M sodium chloride, and the cultured cells were eluted with a positive color reaction by the Anthrone sulfuric acid method.
The active fraction that inhibits the growth of L1210 was collected. This active fraction was adsorbed onto a DEAE Sephadex A-25 column (2.6 x 70 cm), and elution was performed by linearly increasing the sodium chloride concentration in the phosphate buffer. Separate the 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 the fraction with activity to inhibit the growth of cultured cells L1210, MP-
2 (Japanese Patent Application Laid-Open No. 60-30689) is a growth-inhibiting non-active fraction, B is a growth-inhibiting non-active fraction of cultured cells L1210,
This is a fraction with MP-2 growth inhibitory activity. A: The eluate of the active fraction was diluted with ammonium sulfate at 90%
% saturation to obtain a precipitated fraction, dissolved in the above phosphate buffer, desalted using a dialysis membrane, and then lyophilized.
This freeze-dried sample was dissolved in phosphate buffer and adsorbed onto a Toyopearl HW50F column (2.6 x 100 cm), and then 0.1M sodium chloride was added to the phosphate buffer.
The fractions were eluted with a solution containing the following, and the fractions that showed a positive color reaction by the Anthrone sulfuric acid method were 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 was defined as a color reaction positive fraction by the Anthrone sulfuric acid method. Fraction C was desalted by dialysis against phosphate buffer, adsorbed onto a hydroxyapatite column (2.64 x 45 cm), first eluted with 1 x 10 ^-2 M phosphate buffer, and then eluted with 1 x 10 ^-1 M phosphate buffer. It was eluted with liquid. This elution curve is shown in FIG. In FIG. 5, 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, the D fraction is SPF10CAP1, and the E fraction is SPF10CAP1.
SPF10CAP2. The E fraction was lyophilized. This freeze-dried specimen was the antitumor component SPF10CAP2, and yielded 0.89gr. 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. This culture filtrate was purified in the same manner as in Example 1 to obtain 0.5 gr of antitumor component SPF10CAP2. 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 0.75 gr of antitumor component SPF10CAP2. 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 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 0.63 gr of antitumor component SPF10CAP2. 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
in the same manner as in Example 1 using medium C shown in Table 3.
Cultured at 35°C. In this case, penicillin G is 1000
The solution was added at a concentration of 1 unit/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 0.57 gr of antitumor component SPF10CAP2. 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 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 0.36 gr of antitumor component SPF10CAP2. 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 Streptococcus sp. ATCC21597 was cultured using medium A shown in Table 1. 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 0.45 gr of antitumor component SPF10CAP2. 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 0.59 gr of antitumor component SPF10CAP2. 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 0.61 gr of antitumor component SPF10CAP2. 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 0.30 gr of antitumor component SPF10CAP2. 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 0.8 gr of antitumor component SPF10CAP2. Experimental Example 1 Antitumor component SPF10CAP2 obtained in Example 1
The in vitro antitumor activity test was conducted 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 SPF10CAP2 SPF10CAP2 (mg/ml) IR% 0.5 100.0 0.25 98.3 0.125 91.0 Experimental example 2 Antitumor component SPF10CAP2 obtained in Example 1
The in vivo antitumor activity test of CRJ-CD
Sarcoma-180 ascites cancer cells were used as tumor cells, suspended in physiological saline, and intraperitoneally injected into the mouse's peritoneal cavity at 5×10 ⁵ cells/1. Mice were inoculated. SPF10CAP2 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. 【table】

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antitumor component SPF10CAP1, 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. Figure 5 is a graph showing the C fraction adsorbed onto a halodroxyapatite column and eluted with 1 x 10 ^-2 M phosphate buffer and 1 x 10 ^-1 M phosphate buffer. The elution curve is shown.

Claims (1)

[Claims] 1. Antitumor component having the following physicochemical properties:
SPF10CAP2. 1 Elemental analysis C 40.0-44.5% H 5.8-6.5% N 12.9-15.4% O 31.1-40.0% Ash 1.3-2.5% 2 Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3 Specific rotation [α] ²⁰ _D = -72° to -88° (C = 1.00) 4 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance shows an absorption maximum at 275nm and a maximum absorption at 260nm.
shows the same absorption value up to 5 Infrared absorption spectrum Absorption is observed at 3300cm ^-1 , 3070cm ^-1 , 2970cm ^-1 , 1640cm ^-1 , 1540cm ^-1 , 1440cm ^-1 , 1390cm ^-1 , 1120cm ^-1 , 684cm ^-1 . 6. Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5. 7 Color of substance Light brown 8 Color reaction Lowry reaction + Bieurett reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Mauritsch reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight When measured by gel filtration method, the molecular weight is approximately 2×
10 ⁴ to 5×10 ⁵ . 10 Solubility in solvents Soluble in water, but sparingly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. 11 Behavior towards ion exchanger Adsorbed on handroxyapatite, 1×
Eluted with 10 ^-1 M phosphate buffer. 2 Antitumor component belonging to the genus Streptococcus
A method for producing an anti-tumor component SPF10CAP2, which comprises culturing SPF10CAP2-producing bacteria and collecting the anti-tumor component SPF10CAP2 from the culture. 3 Antitumor component belonging to the genus Streptococcus
2. The method for producing the antitumor component SPF10CAP2 according to claim 2, wherein when culturing the SPF10CAP2-producing bacteria, penicillin or a related substance is added at an appropriate time during the culture. 4 Antitumor component belonging to the genus Streptococcus
When culturing SPF10CAP2-producing bacteria and collecting the antitumor component SPF10CAP2 from the culture, cultured cells
3. The method for producing the antitumor component SPF10CAP2 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. 5 Antitumor component belonging to the genus Streptococcus
When culturing SPF10CAP2-producing bacteria and collecting the antitumor component SPF10CAP2 from the culture, cultured cells
A patent that inhibits the growth of L1210 and is characterized by separating a fraction that is adsorbed to hydroxyapatite with a positive color reaction by the Anthrone sulfuric acid method and eluted with 1 x 10 ^-1 M phosphate buffer. A method for producing the antitumor component SPF10CAP2 according to claim 2.