JPS60218323A - Antitumor composition spf-100 and its preparation - Google Patents

Abstract

NEW MATERIAL:Antitumor composition SPF-100 having the following characteristics. Elemental analysis; C 46.42-43.69%, H 5.94-4.85%, N 11.42-9.50%; molecular weight, 500-25,000 (by gel-filtration); decomposition temperature, turns brown at 160 deg.C and turns black and decomposes at 200 deg.C; specific rotation, [alpha]D<20>=+45.0 deg.C (C=1.00); solubility, soluble in water, partially soluble in methanol etc., hardly soluble or insoluble in n-butanol, etc.; pH of 1.0% aqueous solution, 6.5; appearance, pale yellow powder; color reactions, positive to ninhydrin reaction, etc., negative to Molish reaction, etc.: stabilized by the addition of L- cysteine, dithiothreitol, etc. USE:An antitumor agent PREPARATION:A bacterial strain belonging to Streptococcus genus and capable of producing the antitumor composition SPF-100 is cultured anaerobically at 5- 8pH and 30-40 deg.C without agitation, while adding penicillin or its relating substance to the calture medium at an arbitrary stage of cultivation. The bacterial cells are removed from the cultured product by filtration, and the filtrate is purified to obtain the objective SPF-100.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antitumor composition 5PF-100 and a method for producing the same.

Conventionally, preparations made by attenuating viable streptococcus cells,
It is already used as an anticancer drug.

In addition, there is a method in which the active ingredients of Streptococcus pyogenes are extracted by crushing and draining water or using a salt bath, and an organic solvent is added to collect the antitumor substance as a precipitate (Japanese Patent Publication No. 38-1647). A method of lysing bacteria with a lytic enzyme, lysozyme, cellulase or proteolytic enzyme and fractionating the active fraction as a water-soluble fraction (British Patent No. 1f65)
No. 865), etc. are known.

Thus, it is widely known that Streptococcus cocoons themselves or their bacterial cell components have antitumor activity. It was just a constituent substance. In order to isolate the active ingredient from the bacterial cells or inside the bacterial cells, it was necessary to lyse the bacterial cells or mechanically crush them and then fractionate the whole. According to such treatment, purification was complicated and isolation of the active ingredient was extremely difficult. It is known that a protein with a molecular weight of 150,000 has been actually isolated and measured as an active ingredient. (Japanese Patent Publication No. 48-43841) As a result of intensive research on excellent anti-tumor active ingredients in streptococci, the present inventors discovered a completely new anti-tumor composition 8PF-100 in the culture medium. It has come to this.

Since the antitumor composition 8PF-I DO of the present invention is excreted outside the bacterial cells during culture and becomes present in the culture solution, the bacterial cells can be removed by filtration and the culture p liquid can be purified. So,
Isolation becomes much easier.

The antitumor composition 5PF-100 of the present invention is excreted into the culture medium and is characterized by a molecular weight of about 500 to 25,000.

Conventionally, there are no streptococcus-related antitumor compositions that have accumulated in the culture solution, and no known molecular weight of less than tens of thousands of thousands of thousands of yen.The antitumor composition 8PF-100 of the present invention has It is recognized as a completely new substance.

Furthermore, the antitumor composition 5PF-100 of the present invention is recognized as a peptide substance based on elemental analysis, color reaction, etc.
The substance has a unique absorption of ultraviolet rays (1) and is clearly different from conventionally widely known antitumor active substances, and is recognized as a novel substance.

The present invention provides an antitumor composition 8PF-I, which is characterized in that DO-producing bacteria belonging to the genus Streptococcus are cultured, and an antitumor substance 5PF-100 is collected from the culture. It includes 100 manufacturing methods.

In the present invention, antitumor composition 5PF-100 producing bacteria belonging to the genus Streptococcus are widely used;
An example of this is Streptococcus pyogenes (5t
reptococcus pyogenes) ATC
C21060 is mentioned.

The culture medium is meat extract medium, yeast extract medium, brain・
Natural media such as Heart Ink Knee John's medium (BHI medium) are often used, but general media containing carbon sources, nitrogen sources, etc. can also be used as long as they allow Strezotococcus bacteria to grow effectively. can.

The culture is performed at a ptis of 0 to 8.0, preferably 6.1 to 8.0.
7.2, it is common to perform static culture anaerobically at 60 to 40°C, preferably 65 to 37°C, but other modified methods such as stirring culture can also be adopted.

In the present invention, adding penicillin or a related substance at an appropriate time during culturing is effective for antitumor composition 5P.
It will play an important role in the acquisition of the F-100.

Penicillin or its related substances should be added for 3 to 20 hours after the logarithmic growth phase of the culture at 37°C, especially for 5 to 20 hours.
10 hours is preferred. By continuing the culture for 1 to 20 hours, preferably 6 to 15 hours,
A large amount of antitumor composition 8PP-100 can be accumulated in the culture solution.

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 of penicillin G to be added is 100 to 6000 units/Ill, preferably 600 to 1.500 units/Ill, which is sufficient.

The obtained culture solution is centrifuged to remove bacterial cells and p
Ammonium sulfate is added to the solution, a fraction with a saturation level of 50 to 90% is collected, and the resulting precipitate is dissolved in a buffer solution containing a stabilizer.

The antitumor composition 5PF-100-containing solution can be stored in a frozen state or by lyophilization. This material can be further purified by contacting it with an ion exchanger or gel filtration agent, if necessary. As the ion exchanger, ion exchange resin, ion exchange cellulose, ion exchange Sephadex (manufactured by Pharmacia), etc. are used, and as the cell purifying agent, Toyopearl HW50F or HW-5 is used.
0SF (manufactured by Toyo Soda Co., Ltd.), Sephadex (manufactured by Farma Co., Ltd.), and the like are used.Although calcium phosphate gel can be used as it is, it is convenient to use it in the form of 1-hydroxylapatite. The aqueous solution of the substance obtained as described above is passed through a column filled with these ion exchangers or gel filtration agents at an appropriate speed, or it is poured into a fixed container containing the ion exchanger or gel filtration agent. The aqueous solution is added all at once to bring these treatment agents and active substances into contact. Elution is performed using a buffer solution with an appropriate salt concentration. Ion exchanger, gel FA
The agent or calcium phosphate gel can also be used in combination for 2 seconds or more. For example, contact with DEAE Sephadex and add the eluted solution to Toyopearl HW50.
Contact with F or 508F and elution can further enhance the purification effect.

Antitumor composition 8PF-1 of the present invention obtained in Example 8
00 is a peptide substance, which becomes a pale yellow powder when freeze-dried.

Next, the physicochemical properties of the antitumor composition 8PF-100 will be shown.

1. Elemental analysis C: 4S, 42q6 ~ 45.69chi H: 5.94chi ~ 4.85% N 2 11.42% ~ 9.50% 2, Molecular weight As measured by gel filtration method, the molecular weight is approximately 500 ~ 25.0
It is 00.

3. Decomposition point This substance turns brown at 160°C, and decomposes into a black color at 200°C.

4. Specific rotation [α] 1.0 = +45.0° (c = 1.00) 5. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 1% aqueous solution of this substance is the first
As shown in the figure. 257 nm, 265 nm, 280 nm
, 287 nm and 325 nm, which are characteristic.

&　Infrared absorption spectrum It is shown in FIG.

l Solubility in solvents Soluble in water, but partially dissolved in methanol, ethanol, n-butanol, imbutanol, n-propatol, n-hexane, proform, acetone, methyl isobutyl ketone, ethyl ether It is sparingly soluble or insoluble in solvents such as

8. Distinction between basic, acidic, and neutral The rating for the real 1.0% aqueous solution is 6.5.

9 Color of matter It is a pale yellow powder.

10. Color reaction ninhydrin reaction 10. Buuret reaction 10. Molisch reaction - Decier reaction - Anthrone reaction - Cystine sulfuric acid reaction - 11. Stabilized This substance is L-cysteine, dithiothreitol (DTT)
), glycerol, albumin, globulin, α- and β-cyclodextrin, (NH, ), 804, salt, etc. are stabilized.

Next, examples of the present invention will be shown.

Note that the antitumor composition in Examples is 5pp-io.

The activity unit of is measured by Udaka method (Journal of An
t'1biotics vol 35 410 131
9-13250CT 1982). For measurement, polymer-permeable E. coli mutant strain MP-2 (FERM-P 54
32) (Agric, Biol, Chem, 45
371 (1979) to conduct a bioassay using antibacterial activity against MP-2 as an index.

That is, Bakht Antipaiotic Medium 3 (
A medium (M! medium) consisting of 1.75% agar (product of Di7co) and 1.31 agar (M! medium) was heat sterilized at 120°C for 15 minutes.
Dispense 1 u each into petri dishes and allow to cool to prepare a plate medium.

Meanwhile, peptone 0.5T, meat extract 0.5To, NaC
A medium consisting of 1αSS, agar α8'16 and 120'C1
Sterilize by heating for 15 minutes. Thereafter, the medium is kept in a constant temperature bath at 42°C, and when the temperature of the medium reaches 42°C, MP-2 bacteria that have been previously cultured at 37°C for 17 hours are added to the medium so that 10 cells are present in 1 d. Collect 2d with a pinpoint, add it to the surface of M3 medium prepared in advance,
Spreads and solidifies quickly and evenly. Antitumor composition 8P
Appropriately dilute the test solution containing F-100 and make the solution 0.
．． Soak 0511Ll into a paper disk (diameter 8n) (Toyo F paper). This benozo disk was placed on the prepared plate and cultured at 37°C for 17 hours,
The size of the inhibition circle created by the antitumor composition 8PF-100 is measured. 8P giving a diameter of the inhibition circle of 10 mm
The concentration of F-I DO is defined as 1 unit (1u).

Example 1゜ Medium A9/ having the following composition was used.

Meat extract 1 tip 4 tons 1% Yeast extract 0.25% Casamino acid 0.25 tip NaC1O, 1 fb p) 1-49 Streptococcus pyogenes AT
CC21060 was inoculated into 100 d of BHI medium and 37
100 WL of the seed culture solution, which had been statically cultured at ℃ for 8 hours, was inoculated into medium A1 and precultured under the same conditions as the seed culture.

This culture solution was inoculated into medium A8J and incubated at 57°C for 11.5 hr 300t using a 10-hole jar 7 armer.
After culturing anaerobically with stirring at pm, penicillin 010
00 units/go was added and the culture was continued for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells.

Culture solution F contains antitumor composition 8PF-I DO at 256%
Contained units/go.

Example Z Medium B97 having the following composition was used.

Meat extract 1.0 Chipolypeptone 1.0% Yeast extract 0.25 Chi NaC10.1% pH=ZO 8treptococcus pyogenea AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
was inoculated into medium B8, and cultured anaerobically at 57°C using a 101 jar 7 armer with stirring at 6007 nm for 11 hours, then 0800 units/g of penicillin was added, and the culture was continued for an additional 5 hours. do. The obtained culture solution was centrifuged to remove bacterial cells.

Culture solution F contained 651 units/d of antitumor composition 8PF-100.

Example 3゜ Medium c91 having the following composition was used.

Meat extract 1 Chipoly peptone 1 Chi yeast extract 0.25 Chicazamino acid 0.25 Chi = 6.2 8 treptococcus pyogenes AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
inoculated into medium 08, and 10! Jafa Mentor
67℃ for 14 hours! +00! After culturing anaerobically with stirring at pm, Nijirin G1000 units/l1
11 was added, and the culture was continued for an additional 5 hours. The resulting culture solution was centrifuged to remove bacterial cells.

The culture p solution contained 298 units/d of the antitumor composition 8PF-100.

Example 4゜ Medium D91 having the following composition was used.

Meat extract 0.5 Chipoly peptone 1.0 Chi yeast extract 0.25 Chicazamino acid 0.25% NaCA! o, 5% -26.5 Streptococcus pyogenes AT
Culture solution 11 obtained by pre-cultivating CC21060 in the same manner as in Example 1 was inoculated into medium D81, and after culturing anaerobically with stirring at 67° C. for 15.5 hours at 3007 nm using a 10 mm fermenter, o. 0.01200 units/d was added for 1'11, and the culture was continued for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells.

The culture p solution contained 285 units/d of the antitumor composition 8PF-100.

Example 5゜ Medium E91 having the following composition was used.

Yeast extract 3.0 chips) I=6.5 Streptococcus pyogenes AT
Seed culture solution 11 prepared by pre-cultivating CC21060 in the same manner as in Example 1 was inoculated into medium E81, and cultured anaerobically at 67°C with stirring at 300 rpm for 15 hours using a 10-year fermenter, and then inoculated with 01000 units of penicillin. /go was added and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution F contained 18% of the anti-1J1 tumor composition 5PF-1DO.
It contained 2 units/Id.

Example& The following medium F97 was used.

Maltose 0.6 Chimeat extract ZO Chipolypeptone 1.0% Yeast extract 0.25% T = 7.0 8treptococcus pyogenes A
Culture medium 11 obtained by pre-cultivating TCC21060 in the same manner as in Example 1 was inoculated into medium FBI, and incubated at 37°C for 10.5 hr 300 using a 101 Jar 7 Armenter.
After culturing anaerobically with 'j'pm stirring, add 0 penicillin.
Add 1000 units/d and continue culturing for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells.

The culture p solution contained 250 units/d of the antitumor composition 5PF-100.

Example 1 ゛The following medium 09 was used.

Meat extract 1.0% Polypeptone 1.0chi NaCA! 0.5% pl (=zQ Streptococcus pyogenes AT
- Culture solution 11 obtained by pre-cultivating CC21060 in the same manner as in Example 1 was inoculated into medium G8J, and 10! Using a jar fermenter at 37°C, 15 hr 300? After culturing anaerobically with pm stirring, -! Add 01,000 units/IIL of Nigilin, and continue culturing for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells.

Culture solution F contained 200 units/d of antitumor composition 8PF-100.

Example 8 The cultured tear fluid 51 obtained in the culture of Example 4 contains antitumor substance 5P
It contained 143 x 10' units of F-100.

Ammonium sulfate was added to the culture P solution, and a fraction with a saturation level of 50 to 90% was collected to obtain a precipitate. This precipitate is an antitumor substance 8P
It contained 118 x 10' units of F-100.

The entire amount of this precipitate is buffered with a stabilizer! L300ml
Dissolve the solution in DEAF! -Cellulose column (5
x7Qcm), as well as the antitumor composition SPF'-I D
O was adsorbed. This is eluted stepwise using a 0.3 M NaCl solution to separate the active portion. The activity obtained was 102.2 x 10' units.

7 active moiety was added to a DEAR-Sephadex A-25 column (2,6x7ocrIL) to adsorb the active moiety, and elution was performed while linearly increasing the salt concentration in the phosphate buffer to remove the active moiety. Separate.

The activity obtained was 79.5 x 10' units.

Furthermore, this eluate was concentrated and filtered using gel filtration material TOYOPEARL HW-
Add to 50F Kara A (2.6X100cm) and adsorb it, then 1/100M! phosphate buffer (KI(2PO)
The active fraction was collected and designated as 8PF-I DO. The activity obtained was 71.1
x 10' units.

The eluate obtained here was lyophilized to form antitumor composition 8P.
1780 da of pale yellow powder of F-100 was obtained.

Using the antitumor substance 5PF-100 obtained on the day of the example as a test system, in vitro, in vitro in
It was subjected to both in vivo and 1 in vivo experiments to confirm its antitumor activity.

Test Example 1 (in vHro test) An in vitro antitumor activity measurement test of the test system was carried out based on the cell growth measurement method.

P-388 and L-1210Lymp as cancer cells
homa (lymphoma) (DBA/2 mouse syngeneic transplanted tumor subcultured in vitro), each was cultured in RPMI 1640 medium (5X) supplemented with 10% FC8.
10-aM2-me) v Captoel / -le, 50rr
Q/l kanamycin). This culture e1t
nt into Falcon 6047 plates so that the cancer cells are 5X10 cells/well 1. Next, a predetermined amount of the test system (dissolved or suspended in a 0.1 d culture solution) was injected into this culture solution, and the concentration was increased to 5'% at 67°C.
Culture in the presence of COt. 48 hours after injection of the test system, vital staining was performed using Glossin B), and the degree of cell growth was calculated using the following formula.

The results are shown in Table 1.

Table 1 Cell growth degree (function test example 2 (in vitro in vivo test) in vitro
The tro in vivo antitumor activity test of the test system was conducted using female DDY mice, 4 or 5 weeks old.

Ehrlichi's ascites cancer cells are used as the cancer cells, suspended in a Ni-P buffer, and then mixed with a predetermined amount of the test system and incubated at 67° C. for 60 or 80 minutes. 0.27 d of this solution (4 x 1 o'cellg of cancer cells or 8
10' cells) once a day per mouse7
The mice were inoculated intraperitoneally for consecutive days and the number of surviving mice was observed. The results are shown in Tables 2 and 3.

Test Example 6 (in vivo test) The in vivo antitumor activity test of the test drug was CR.
The experiments were carried out using J-CD-1 (NCR) strain, male, 7 hyperactive mice.

As the cancer cells, 8arcoms-180 ascites cancer cells were used, suspended in Hank's solution, and intraperitoneally administered to mice at Q, 1 m/(2'x 10' cells).
Inoculated.

Once a day for 5 days before or after this cancer cell inoculation,
A predetermined amount of the test drug was intraperitoneally administered for 5 days before and 5 days after, and the number of survivors was observed. The result is the fourth
It is shown in Table and Table 5.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of an aqueous solution of the antitumor composition 8PF-1000.1, and FIG. 2 similarly shows the infrared absorption spectrum. Agent: Patent Attorney 1) Parent: Procedural Amendment dated September 1, 1980, Mr. Commissioner of the Japan Patent Office, 1, Indication of Case, Patent Application No. 72,865, 1982, 2, Title of Invention: Antitumor Composition 5PF-100 and its Manufacturing process 6, relationship with the case of the person making the amendment Patent applicant address: 1-24-6 fi-cho, Meito-ku, Nagoya Name: Shigezo Udaka (and 2 others) 4. Agent address: 105 Tokyo Port 19-1, Toranomon-chome, Ward, Title of the invention and content of amendment to specification Z (1. The name of the invention is amended to "Anti-tumor substance 5PF-100 and its manufacturing method". (2) The full text of the specification is attached. Correct the amount.-Details
Book 1, Title of the invention: Antitumor substance spi;'-1oo and its manufacturing method 2, Claims (1) Antitumor substance 5P having the following physicochemical properties
F-100°1, elemental analysis C: 46.42% to 43.69%, H: 5.94% to 4.85%, N: 11.42% to 950°2, molecular weight measured by gel filtration method. , a molecular weight of approximately 500 to 25,000. 3. Decomposition point This substance turns brown at 160°C, and decomposes into a black color at 200°C. 4. Specific optical rotation [α) p=+45.0° (c=1.00) 5. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. Both are 257nm, 265nm,
Absorption is observed at 280 nm, 287 nm, and 525 nm, which are characteristic. 6. Infrared absorption spectrum shown in Figure 2. l Solubility in solvents Soluble in water, but partially soluble in methanol, ethanol, n-butanol, imbutanol, n-propatol, n-hexane, chloroform, acetone, methyl isobutyl lactone, ethyl It is sparingly soluble or insoluble in solvents such as ether. 8. Distinction between basic, acidic and neutral The PR of a 1.0% aqueous solution of this substance is 6.5. 9 Color of substance: Pale yellow powder. io, color reaction ninhydrin reaction, 10 Beurette reaction, 10 Molisch reaction - Decier reaction - Anthrone reaction - cysteine sulfuric acid reaction - 11, Stabilization This substance is L-cysteine, dithiothreitol (DDT)
), glycerol, albumin, globulin, α-1 and β-cyclodextrin, (N)L*)! 804
, stabilized by the addition of salt, etc. (2) Antitumor substance 5P belonging to the genus Streptococcus
Cultivate F-100 producing bacteria and extract antitumor substance 8 from the culture.
A method for producing antitumor substance 8PF-100, which comprises collecting PF-I DO. (3) Antitumor substance belonging to the genus Streptococcus 8
Antitumor substance 8PF-I according to claim 2, characterized in that when culturing the PF-100 producing bacteria, penicillin or a related substance is added at an appropriate time during the culture. Manufacturing method of DO. 3. Detailed Description of the Invention The present invention relates to a novel antitumor substance 5PF-100 and a method for producing the same. Conventionally, preparations made by attenuating viable streptococcus cells,
It is widely used as an anticancer drug. In addition, there is a method of crushing the bacterial cells of Streptococcus pyogenes and extracting the active ingredients by draining the water or using a saline solution, adding an organic solvent, and collecting the antitumor ingredients as a precipitate. enzyme, lysozyme,
A method of lysis and lysis with cellulase or proteolytic enzyme and fractionation of the active fraction as a water-soluble fraction (UK Patent No. 11)
No. 63865) are known. As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor activity, but the only ones known so far are bacterial cells or water-soluble or insoluble Streptococcus spp. It was nothing more than a cellular constituent. In order to isolate the active ingredient from the bacterial cells or inside the bacterial cells, it was necessary to lyse the bacterial cells, mechanically crush them, and then fractionate the whole. According to such treatment, purification is complicated) and isolation of the active ingredient is extremely difficult. In an example where a protein was actually separated and measured as an active ingredient, a protein with a molecular weight of 150,000 is known. (Special Publication No. 4B-43841) The present inventors,
As a result of intensive research into streptococci with excellent anti-tumor active ingredients, a completely new anti-tumor substance 8PF-100 was discovered in the culture solution. The antitumor substance of the present invention, 5pF-ioo, is excreted outside the bacterial cells during culture and becomes present in the culture solution, so it is only necessary to remove the bacterial cells by filtration and purify the culture p solution. , isolation becomes much easier. The antitumor component 8PF-100 of the present invention is excreted into the culture medium and is characterized by having a molecular weight of about 500 to 25,000. Up until now, there have been no streptococcus-related antitumor substances that have accumulated in the culture solution, and no known molecular weight of less than 100% has been found, and the antitumor substance 8PF-100 of the present invention is completely new. It is recognized as a substance. Furthermore, the antitumor substance 8PF-100 of the present invention is recognized as a peptide substance based on nine element analysis, color reaction, etc.
Ultraviolet absorbers have unique absorption properties, and are clearly different from conventional anti-tumor active substances, and are recognized as novel substances. The present invention provides a method for producing an antitumor substance 5PF-100, which is characterized by culturing directly produced antitumor substance 8PF-100 belonging to the genus Streptococcus and collecting antitumor substance 5PF-1oO from the culture. This includes: In the present invention, bacteria that produce the antitumor substance 5PF-100 belonging to the genus Streptococcus are widely used;
eptococcus pyogenes ) AT
CC21060 is mentioned. Culture media include meat extract medium, yeast extract medium, plain,
Natural media such as Heart Ink Knee John's medium (BHI medium) are often used, but general media containing carbon sources, nitrogen sources, etc. can also be used as long as they allow Strebutococcus bacteria to grow effectively. can. Culture is generally carried out statically in an anaerobic manner at a temperature of 60 to 40°C, preferably 35 to 67°C, at a pH of 5°0 to 8.0, preferably 6.1 to Z2. Modified methods such as agitation culture can also be employed. In the present invention, the addition of nigiline or its related substances at an appropriate time during culture is effective against the antitumor substance 8PF.
It will play an important role in obtaining -100. Penicillin or its related substances should be added for 6 to 20 hours after the logarithmic growth phase of the culture at 37°C, especially for 5 to 20 hours.
10 hours is preferred. By continuing the culture for 1 to 20 hours, preferably 6 to 15 hours,
A large amount of the antitumor substance 5pit'-i oo can be accumulated in the culture solution. Penicillin or its related substances may be any previously known related substances that have similar effects to penicillin, but Nigilin G is commonly used. The amount of honeycillin G to be added is approximately 100 to 3,000 units/d, preferably 600 to 1,500 units/d of the culture solution. The obtained culture solution is centrifuged to remove bacterial cells, and P
Ammonium sulfate is added to the solution, and the precipitate obtained by fractionating both 50 to 90% saturation fractions is dissolved in a buffer solution containing a stabilizer. The antitumor substance 8PF-100-containing solution can be stored in a frozen state or by lyophilization, but the substance can be further purified by contacting with an ion exchanger or gel filtration agent, if necessary. As the ion exchanger, ion exchange resin, ion exchange cellulose, ion exchange Sephadex (manufactured by Pharmacia), etc. are used, and as the gel filtration agent, Toyopearl HW is used.
50F or HW-50SF (manufactured by Toyo Soda Co., Ltd.), Sephadex (manufactured by Pharmacia), etc. are used. Although calcium phosphate dal can be used as it is, it is more convenient to use it in the form of hytroxylapatite. The aqueous solution of the substance obtained as described above is passed through a column filled with these ion exchangers or gel filtration agents at an appropriate speed, or it is poured into a fixed container containing the ion exchanger or gel filtration agent. The aqueous solution is added all at once to bring these treatment agents and active substances into contact. Elution is performed using a buffer solution with an appropriate salt concentration. Two or more ion exchangers, gel filtration agents, or calcium phosphate gels can be used in combination. For example, D.E.
The purification effect can be further improved by contacting with AFt Sephadex and further contacting the eluted solution with Toyopearl HW50F or 508F for elution. Antitumor substance 8PF-10 of the present invention obtained in Example 8
0 is a bezotide substance, which becomes a pale yellow powder when freeze-dried. Next, the physicochemical properties of the antitumor substance 5PF-100 will be shown. 1. Elemental analysis c: 4s, 42% ~ 43.69% H: 5.94% ~ 4.85ts N: 11.42% - 9,50% 2. Molecular weight As measured by gel filtration method, the molecular weight is approximately 500 ~ 25 ,0
It is 00. 5. Decomposition point This substance turns brown at 160°C and decomposes at 200°C (it becomes black). 4. Specific optical rotation [α] Amagashi+45.0° (c=1.00) 5. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of an a, 1S aqueous solution of this substance is shown in FIG. 257 nm, 265 nm, 280 nm
Absorption is observed at 287 nm and 325 nm, which are characteristic. & Infrared absorption spectrum shown in Figure 2. l Solubility in solvents Soluble in water, but partially soluble in methanol and ethanol, n-butanol, isoptanol, n-propanol, n-hexane, etc. It is soluble or insoluble in solvents such as chloroform, acetone, methyl isobutyl ketone, and ethyl ether. 8. Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5. 2. Color of the substance is pale yellow powder. 10. Color reaction ninhydrin reaction 10. Buuret reaction 10. Molisch reaction - Decier reaction - Anthrone reaction - Cystine sulfuric acid reaction - 11. Stabilized This substance is L-cysteine, dithiothreitol (DTT)
), glycerol, albumin, globulin, α- and β-cyclodextrin, (NH4)t 804,
Examples of the present invention will be shown below by adding salt and the like. In the Examples, the activity unit of the antitumor substance 5PF-100 was measured using the Udaka method (Journal of Antibacterial Method).
iotics vol 35. #610 1319-1
325OCT1982). For the measurement, a polymer-permeable E. coli mutant strain MP-2 (FE M-P5432) (A
gric, Biol, Chem, 43571 (1
979)) was used as an indicator for antibacterial activity against M P-2. i.e. Bacto Antibiotic Mediamuro (
Difco product) 1.75%, Maten 1.3%) medium (M3 medium) was heat sterilized at 120'C for 115 minutes,
20wLl! Dispense each into a Petri dish and allow to cool to prepare a plate medium. On the other hand, peptone 0.5 t, meat extract 0.5 t, NaCl 1
A medium consisting of 10.6% agar and 0.8% agar was grown at 120℃ for 1 hour.
Sterilize by heating for 5 minutes. After that, keep it in a constant temperature bath at 42℃, 1
When the temperature of the culture medium reaches 42℃, incubate it at 37℃ for 17 minutes.
MP-2@ cultured for hours is added to the medium so that 10' cells are present in 1 d. Collect two pieces using a pipette, add them to the surface of the M33 medium prepared in advance, and spread quickly and uniformly to solidify. Antitumor substance 8P
Appropriately dilute the test solution containing F-100 and make the solution Q.
, Q5m is impregnated into Ni-Par Disc (diameter 81111) (Toyo F Paper). This paper disk is placed on the prepared plate and incubated at 37°C for 17 hours, and the size of the inhibition circle created by the antitumor substance 8PF-100 is measured. 8PF-1 giving a diameter of 10 degrees for the blocking circle
The concentration of 00 is defined as 1 unit (1u). Example 1 Nine mediums having the following composition were used. Meat extract 1 Cipolypeptone 1% Yeast extract 0.25% Casamino acids 0.25% NaC10.1% Voice = 6.9 Streptococcus pyogenes AT
CC21060 was inoculated into BH culture medium 100g and incubated at 67°C.
, 100 seeds of the seed culture solution that had been statically cultured for 8 hours were added to medium A.
Inoculate ln and preculture under the same conditions as seed culture. This culture solution was inoculated into medium A81, and cultured anaerobically at 67°C with stirring at 500 rpm for 11.5 hours using a 101 Jarfer Mentor.
d and continue culturing for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells. Culture solution F contained 256 units/Id of the antitumor substance 8PF-IDO. Example Z Medium B 9A' having the following composition was used. Meat extract i, o s Polihaton 1.0chi Yeast extract 0.25chi NaC1O, 1% = 7.0 Streptococcus pyogenes AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
1 was cultured anaerobically using medium BBIKiffIL and a 101 Jar 7 Armenter at 67° C. for 11 hours with stirring at 600 rpm, then 0,800 units/g of penicillin was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells. The culture p solution contained 551 units/d of the antitumor substance 8PF-100. Example 3 Medium C91 having the following composition was used. Meat extract 1% Polypeptone 1% Yeast extract 0.25 ticazamino acid 0.25 Chi voice = 6.2 8treptOcoccus pyogenes AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
1 was inoculated into medium Cal, and cultured anaerobically at 67°C with stirring at 300 rpm for 14 hr using a 10-year fermentor, and then inoculated with 1000 units of penicillin G/d.
was added, and the culture was continued for an additional 5 hr. The resulting culture solution was centrifuged to remove bacterial cells. The culture p solution contained 298 units/g of the antitumor substance 8PF-IDO. Example 4 Medium D91 having the following composition was used. Meat extract 0.5 Chipoly peptone 1.0 Chi yeast extract 0.25% Casamino acids 0.25 Chi NaCA1 0.5% Direction=6.5 8treptococcus pyogenes AT
Culture solution 1 in which CC21060 was precultured in the same manner as in Example 1
1 was inoculated into medium D87 and cultured anaerobically using a 101 Jar 7 Armenter at 67°C with stirring at 500 rpm for 15.5 hours.
- was added, and the culture was continued for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells. Culture solution F contained 285 units/d of the antitumor substance 5PF-100. Example 5 Medium E91 having the following composition was used. Yeast extract 5.0chi H-45 8treptococcus' pyogenes A
One seed culture of TCC21060 pre-cultured in the same manner as in Example 1 was inoculated into 8 volumes of medium B, and cultured anaerobically at 37°C with stirring at 300 rpm for 15 hours using a 101:) Year 7 Armenter. , penicillin G 1000 units/row was added, and the culture was continued for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells. Culture solution F contained 182 units (7 ml) of the antitumor substance 8PF-I Do. Examples & The following medium F91 was used. Maltose 0.6% Meat extract 2.0 Cipoly peptone 1.0 Ci yeast extract 0.25 pi (=7.0 8treptococcus pyogenes AT
Culture solution 11 obtained by pre-cultivating CC21060 in the same manner as in Example 1 was inoculated into medium F84, and cultured anaerobically at 67°C for 10.5 hours with stirring at 300 rpm using a 101 jar fermenter.
d and continue culturing for an additional 5 hr. The obtained culture solution was centrifuged to remove bacterial cells. Culture solution F contained 250 units/g of the antitumor substance 8PF-100. Example Z The following medium 09) was used. Meat Extract i, o Chipolypeptone 1.0% NaC1O,5'16 -27.0 Streptococcus pyogenes AT
One culture of CC21060 was precultured in the same manner as in Example 1, inoculated into 081K medium, and cultured anaerobically using a 101 jar fermentor at 37°C with stirring at 300 rpm for 15 hours, and then inoculated with 1000 units of penicillin G. /wLl was added, and the culture was continued for t-5 hr. The obtained culture solution was centrifuged to remove % bacterial cells. Culture solution F contained 200 units/d of the antitumor substance 8PF-100. Example 8 The cultured tear fluid 57 obtained in the culture of Example 4 contains antitumor substance 5P
It contained 143 x 10' units of F-100. Ammonium sulfate was added to the culture solution F, and both portions having a saturation level of 50 to 90 degrees were separated to obtain a precipitate. This precipitate is an antitumor substance 8P
It contained 118 x 10' units of F-I Do. The entire amount of this precipitate was dissolved in 300 d of buffer containing a stabilizer,
The lysate was transferred to a DRAB-cellulose column (5x70cIL
), and the antitumor substance 8PF-100 was adsorbed. This is eluted stepwise using a 0.3 M NaCl solution to separate the active portion. The activity obtained was 102.2
The unit was x10'. The active moiety is added to a column of DBAE-Sephadex-25 (2,6x 70cIrL) to adsorb the active moiety, and elution is performed while linearly increasing the saline concentration in the phosphate buffer to remove the active moiety. Separate. The activity obtained was 79.3 x 10' units. Furthermore, this eluate was concentrated and filtered using a filter material Toyonol H.
It was added to a W-50F column (2,6x 100cIIL) for adsorption, then 17100M phosphate buffer (KH
2PO4-Na2HPO4), and the active fraction was collected and designated as 8PF-100. The activity obtained was 71
．． It was 1 x 10' unit. The eluate obtained here was lyophilized to produce the antitumor substance 8PF.
-100 pale yellow powder 178C19 was obtained. Using the antitumor substance 5PF-100 obtained in Example 8 as a test drug, In VIirO+ In VlirOin V
IVO+ was subjected to each in vivo experiment, and the antitumor activity was confirmed. Test Example 1 (In vitro test) An in vitro test to measure the antitumor activity of the test drug was carried out based on a cell growth measurement method. P-388 and L-1210Lymp as cancer cells
homa (lymphoma) (DBA/2 mouse syngeneic transplanted tumor subcultured in vitro), each was cultured in RPMI 1640 medium (5X) supplemented with FC8.
10-'M2-mercazotoethanol containing 50 m9/l kanamycin). 1 d of this culture solution was injected into a Falcon 6047 plate, and 5 x 10 cancer cells were
' K to make cells/well. Next, a predetermined amount of the test drug (dissolved or suspended in the culture solution of Q and Igo) is injected into this culture solution, and cultured at 67°C in the presence of 5% CO. 48 hours after injection of the test drug, vital staining using Glossin B was performed, and the degree of cell growth was calculated using the following formula. The results are shown in Table 1. Table 1 Cell growth rate (a) Test example 2 (in vitro in vivo test) in vi
The antitumor properties of the test drugs in tro in vivo tests were conducted using female AAY mice fed for 4 or 5 weeks. Ehrlichi's ascites cancer cells are used as the cancer cells, suspended in Ni-P buffer, mixed with a predetermined amount of the test drug, and incubated at 37°C for 60 or 80 minutes. This liquid 0.2WLl! (Cancer cells 4X10'cell1g or 8
X 10Ilcells) was intraperitoneally inoculated per mouse once every 91 days for 7 consecutive days, and the number of surviving mice was observed. The results are shown in Tables 2 and 6. Test Example 6 (In Vivo Test) An in vivo antitumor activity test of the test drug was carried out using Cfan J-CD-1 (ICR) strain, male, 7 hyperactive mice. As the cancer cells, Sarcoma-180 ascites cancer cells were used, suspended in Hank's solution, and intraperitoneally injected into the mouse's peritoneal cavity at 0.11 Ll! (Cancer cells 2 x 10' cells
) inoculated. Once a day for 5 days before or after this cancer cell inoculation,
A predetermined amount of test drug 9 was intraperitoneally administered for 5 days before and 5 days after, and the number of survivors was observed. The results are shown in Tables 4 and 5. 4. Brief description of the drawings Fig. 1 shows the ultraviolet absorption spectrum of a 1% aqueous solution of the antitumor substance 8PF-1000, and Fig. 2 similarly shows the infrared absorption spectrum. Agent Patent attorney 1) Parent Male

Claims (3)

[Claims] (1) Antitumor composition 8 having the following physicochemical properties
PP-100゜1, original cumulative analysis C: 46.42% to 43.69%, H: 5.94cm to 4.85cm, N: 11.42cm to 9.50cm2 Molecular weight by gel filtration method The molecular weight has been determined to be approximately 500-25,000. 5. Decomposition point This substance turns brown at 160℃ and turns black at 200℃ and decomposes. 4. Specific optical rotation [α = +45.0° (c = 1.00) 5. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of an aqueous solution of 11 of this substance is shown in FIG. Both are 257nm, 265nm,
Absorption is observed at 280 nm, 287 nm and 325 nm, which are characteristic. 6 Infrared absorption spectrum shown in Figure 2. l Solubility in solvents Soluble in water, but partially soluble in methanol, ethanol, n-butanol, imbutanol, n-propatol, n-hexane, chloroform, acetone, methyl isobutyl ketone, ethyl ether It is sparingly soluble or insoluble in solvents such as 8. Distinction between basic, acidic, and neutral: i, oes of this substance - of the aqueous solution is 6,5. 9 Color of substance: Pale yellow powder. 10. Color reaction ninhydrin reaction 10. Buuret reaction 10. Molisch reaction - Decier reaction - Anthrone reaction Cystine sulfuric acid reaction - 11. Stabilized This substance is L-cysteine, ditheothreitol (DDT)
), glycerol, albumin, globulin, α-1 and β-cyclodextrin, (NH4)2804,
It is stabilized by adding salt, etc. (2) Antitumor composition 5 belonging to the genus Streptococcus
A method for producing antitumor composition 8PF-100, which comprises culturing PF-100-producing bacteria and collecting antitumor composition 8PF-I DO from the culture. (3) Antitumor composition 8 belonging to the genus Streptococcus
Antitumor composition 8PF100 according to claim 2, wherein when culturing the PF-100-producing bacteria, penicillin or a related substance is added at an appropriate time during the culturing. Manufacturing method.