JPS6169725A - Antitumor substance of spf-140 and its preparation - Google Patents

Abstract

NEW MATERIAL:An antitumor substance SPF-140 having following properties: elementary analyses in %: C 42.58-41.79, H 6.15-5.92, N 12.69-12.35; molecular weight: 25,000-70,000 (gel permeation method); decomposition point: browning at 170 deg.C and blackening at 245 deg.C; decomposition; specific rotary power [alpha]D<20>=-70.0 deg.--80.0 deg. (c=1.00); solubility: soluble in water, partially soluble in methanol, hard soluble or insoluble in ethanol; pH: 6.5 in 1.0% aqueous solu tion; pale yellow powder; coloring reactions: positive to ninhydrin reaction, negative to Molish's reaction; stabilization: it is in no need of a special stabi lizer. USE:Antitumor agent. PREPARATION:A microorganism in Streptococcus, producing an antitumor agent SPF-140 is anaerobically cultured at 30-40 deg.C by the standing or agitation method to collect the objective antitumor agent SPF-140. When penicillin or its relative substance is added to the culture mixture at an appropriate stage, it plays an important role in the collection of the objective substance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to #r antitumor substance SPF-140 and its production method.

Conventionally, preparations prepared by attenuating viable cells of hemolytic streptococcus (hereinafter referred to as ``hemolytic streptococci'') have been used as anticancer drugs. A method of extracting the active ingredients with organic hot medium, adding an organic heating medium, and recovering the antitumor substance as a precipitate (Special Publication No. 68-1647). A method of fractionating the active fraction as a water-soluble fraction (British Patent No. 1163865), a method of crushing the cells of Hemorrhoids, collecting water-insoluble substances, and treating them with nucleolytic enzymes and proteolytic enzymes ( Japanese Patent Application Publication No. 55-7014)
etc. are known.

As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have anti-MljG activity, but what has previously been said is that anti-MljG activity exists in bacterial cells or in water-soluble or water-insoluble polymer cell components. It was just noble. In order to isolate the active ingredient from the tooth bodies or integral circles, the entire bacterial body had to be fractionated by bathing or mechanically crushing it.

Such treatments complicate purification and isolation of the active ingredient is extremely difficult and rare. Actually separate, +! - In the case of active ingredients measured, a protein with a molecular weight of 200,000 is known (+! As a result of research in search of anti-freezing active ingredients produced by bacteria of the genus Streptococcus, it was found that by adding a small amount of beni/phosphorus or related substances during the appropriate growth period of the culture of bacteria of the genus Streptococcus and continuing the culture. Physiologically active substances 5PF-1 and 5pp-2° and antitumor substances 5pp-ioo secreted into the culture medium were recovered from the culture fP solution, sieved, and collected as control substances.

These substances were obtained from the polymer-permeable E. coli mutant strain MP-2 (
FjjRM-P5432) (Agr, Birol
.

Chem, 46, 371-57B (1979)) (hereinafter referred to as MP-2 strain).

In this culture P solution of Streptococcus f4 bacteria, we present 8PPl, 5PF-2 or.

They have discovered that, apart from SPFl 00, there are two components that do not exhibit a growth-inhibitory effect on the MP-2 strain, but have anti-tumor activity.

The substance of this fraction is designated as SPF'-140.

The antitumor substance SPF-140 of the present invention is secreted into the culture P fluid, has a molecular weight of 25,000 to 70,000, and is characterized by not exhibiting antibacterial activity against MP-2 strain.

To date, there are no known antitumor substances related to streptococcus PA that are secreted and accumulated in the culture medium and have a molecular weight of less than several tens of thousands.Furthermore, the antitumor substance SPF-140 of the present invention
Although it is considered to be a peptide-like substance based on elemental analysis and color reaction, the ultraviolet absorption spectrum indicates that it is a new substance that is different from known anti-tumor substances.

The present invention provides a method for producing an antitumor substance 5PF-140, which comprises culturing an antitumor substance 5PF-140-producing bacterium belonging to the genus Streptococcus and collecting the antitumor substance SPF-140 from the culture. It is a color that matches the color of the color.

In the present invention, bacteria producing the antitumor substance SPF-140 belonging to the genus Strezotococcus can be widely used, and then the bacteria producing the antitumor substance 5PF-140 are self-killed.

5treptococcus pyogenes A
TCC21060Streptococcus sp,
ATCC21597Streptococcu
s pyogenes ATCC215468tre
ptococcus pyogenes ATCC2
1547Streptococcus pyogene
Natural media such as meat extract medium, yeast extract medium, plain heart infujoy medium (B Hr medium) are often used as ATCC2154B culture medium.
Any medium can be used as long as it is suitable for the growth of Streptococcus bacteria.

Culture was carried out on pi-15,o~8.0. Preferably 6.1
~Z2 and cultivation 1! The temperature is 30 to 40°C, preferably 65 to 37°C, and the culture is carried out anaerobically or
Stirring culture can be performed.

In the present invention, adding/injecting xnigilin or its related substances at an appropriate time during culture plays an important role in collecting the antitumor substance SPF-140.

is the addition time of Nijirin or its related substances.

Cultivation at 67° C. is preferably carried out for 6 to 20 hours, particularly 5 to 10 hours, after reaching the logarithmic growth phase. Then 1-20
By continuing the culture for a period of time, preferably 3 to 15 hours, a large amount of the antitumor substance SPF-140 can be accumulated during the culture.

As penicillin or its related substances, any related substance that has a similar use to the already known -"? nishiri/ can be used, but penicillin G is usually used. About 7000 units/d, preferably 500 to 5000 units/4 culture solution, is sufficient.

A culture solution obtained by adding Nijirin or its related substances to bacteria belonging to the genus Stombutococcus is centrifuged to remove the medium, and ammonium sulfate is added to the solution.
Fractions with CJ% saturation are collected.

The obtained ammonium sulfate precipitate containing the antitumor substance 5PF-140 can also be stored in a frozen state.

This ammonium sulfate precipitate is an antitumor substance! IIIPF-140
To extract the antitumor substance SPF, the salt precipitate is dissolved in a buffer solution, adsorbed and washed out using an ion exchanger using a batch method or a flow method, and further chromatography using a gel filtration agent. '-140 is fractionated.

As the ion exchanger, ion exchange resin, ion exchange cellulose, ion exchange Sephadex (Pharmacia fi), hydroxylapatite, etc. are used, and as the cell purifying agent, Toyopearl HW50F'l or HW
50 SF (manufactured by Toyo Qtatsu Co., Ltd.).

Sephadex (manufactured by Pharmacia) or the like is used.

A buffer solution with an appropriate salt concentration is used for adsorption of the antitumor substance SPF-140 to the ion exchanger and elution of the antitumor substance 5PF-140 from the ion exchanger. Furthermore, two or more types of ion exchangers and gel filtration agents can be used in combination.

The freeze-dried specimen of the antitumor substance SPF-140 obtained in Example 1 is a pale yellow powder, and exhibits the following physical and chemical properties: Elemental analysis C: 42.58% to 41 .79% H: 6.15% to 5,92% N: 12.69% to 12.55% 2. Molecular weight As measured by gel filtration, the molecular weight is about 25.000 to 70.000.

5. Decomposition point This substance turns brown at 170℃ and turns black at 245℃ and decomposes.

4. Specific rotation (a) %' = -70.0° to -80.0° (C = 1.0
O) 5. Ultraviolet Absorption Spectrum The ultraviolet absorption spectrum of a 0.1% water bath solution of this substance is shown in FIG. The absorption maximum is observed at 275 nm, which is characteristic.

6.　Infrared absorption filter The culvert is shown in Figure 2.

Z Solubility of bath additives: fm in water, but partially soluble in methanol, ethanol, n-foA/-A/.

It is soluble or insoluble in solvents such as n-butanol, inobutanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, and ethyl ether.

8. Distinction between basic, acidic, and neutral ... of a 1.0% aqueous solution of this substance is 6.5.

9 Color of matter It is a pale yellow powder.

io, color reaction 2/Hydrin reaction 10 Buret reaction 　　　　10 Lowry reaction 　　　　　10 Solish reaction Deissier reaction - Anthrone reaction − Cystine sulfuric acid reaction - 11. Stabilization This substance does not require any special stabilizers.

Next, the antibacterial activity in the present invention is measured as follows.

MP-2 is used to measure antibacterial activity, and the ability to inhibit the growth of MP-2 is used as an indicator of antibacterial activity. Also, utilizing this principle, the Kuu Taka method (J, Antibiotics.

35.1319-1325 (1982)) Determination of units of physiologically active substances.

That is, Bacto Antibiotic Medium 5 (
Culture medium 1 (M5 medium) consisting of Di7co Co., Ltd. 1.75% agar and 1.51 ml was heat sterilized for 1200.15 minutes, and
Dispense 114 cells into petri dishes, let it cool, and use the plate medium as fJ! Make 4 bowls.

On the other hand, a medium consisting of Eve 1-70.5%, meat extract α5%, sodium chloride 0.5%, and agar α8% was heated at 120°C.
Sterilize by heating for 15 minutes. After that, it was kept in a constant temperature bath at 42°C, and when the temperature of the medium reached 42°C, it was incubated at 57°C for 17 hours, and 9MP-2M was incubated with 10JI61 in 717.
Collect 21Ll with a pipette and add it to the culture medium so that there are M6 cells prepared in advance.
6. Spread 7JO on the surface of the medium and spread quickly and uniformly to solidify. Next, the test solution was diluted appropriately, and the solution was 0.05
a/l-<-par disk (diameter 8 genera) (Toyo P Paper Co., Ltd. 1!Imbued with KL. This paper disk was placed on the above-mentioned preparation plate and cultured at 57°C for 17 hours.
The antibacterial activity is tested by observing the inhibition circle formed by the test substance, and the physiological activity of the test substance that gives a diameter of 101111 mm is defined as one unit (1 u).

Next, examples of the present invention will be shown.

ttt*f! /su1 Streptococcus pyogenes AT
CC2-1060 was inoculated into 100 d of BHI medium and 37
The persimmon culture solution that had been incubated for 8 hours at ℃ was added to the medium A1 gK shown in Table 1, and precultured anaerobically under the same conditions as the seed culture.

Table 1 Cultured Human Flesh Extract 1.0 Chipori is Pton 1. O thymaltose
α5% Yeast extract Q, 25 thionic potassium phosphate α1 thi [11! -'r Gnesiu A 0.05*...
=6.8 Pour medium A81 into 101 jar fermenter and add 1
After heat sterilization at 20°C for 10 minutes, cooled to 57°C, inoculated with preculture solution 11, heated at 37°C for 15.5 hours...6.5
, anaerobically cultured with stirring at 300 revolutions/min. Then, penicillin G was added at 5000 units/d culture solution, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

Ammonium sulfate was added to culture solution F, and a fraction precipitated at a saturation level of 50 to 90% was collected. This precipitate is MP-
381, a physiologically active substance that inhibits the growth of OX 10
' Contained u. , this precipitate was
M, pa-17:0 phosphate buffer O504-Na1
HPO4) 300111, and this aqueous solution was adsorbed on a DB Human E cellulose column (5x70cIIL), and then eluted stepwise using the above phosphate buffer containing α3M sodium chloride. The active fraction was collected. This physiologically active fraction is DBAI! f Sephadex - 25 columns (2,6 x 70 (, IIL
), and then elution was performed by linearly increasing the sodium chloride concentration in the phosphate buffer, and 249.6
A bioactive fraction of X 10' u was collected. Furthermore, this eluate was concentrated and added to a gel-filtration agent Toyopearl HW50F column (2.6X 100cm) to perform gel filtration, and the non-antibacterial active fraction was collected and freeze-dried to obtain the antitumor substance 5PF- 140640=9 was obtained.

Antitumor substances using this antitumor substance SPF-140 as a test substance are shown in Experimental Examples 1 and 2.

Actual example 2 Streptococcus pyogenes AT
CC21060 was cultured in the same manner as in Example 1 using medium B shown in Table 2. In this case Beni'/! J-G #
f, 1000 units/Ml culture solution was added.

This culture P solution was purified in the same manner as in Example 1 to obtain the antitumor substance SPF-140767I9.

Table 2 Medium B Meat Extract 0.5 Cipoly is Pton 1. CI Ls Yeast Extract 0.25 Ti Sodium Chloride 0.1% pi (=6.7 Example 3 Streptococcus pyogenes AT
CC21060 was cultured in the same manner as in Example 1 using medium C shown in Table 3. This culture solution F was purified in the same manner as in Example 1 to obtain an antitumor substance 5PF140 527.
I got a drop.

Table 6 Medium C Meat extract 1.0% Polynipton 1.0 t Maltose 0.5 % Yeast extract 0.25 thiopotassium phosphate o, i* Magnesium sulfate O, S temperature = 6.5 Example 4 8treptococcus pyogenes AT
CC21060 was cultured in the same manner as in Example 1 using the culture medium shown in Table 4. In this first go, nearing G is,
This culture was added to give 1000 units/day of culture (the number of cells was the same as in Example 1, and the antitumor substance was purified by 5
Passed PF-140830.

4th coating Medium D Maltose 0.8% Polypton 0.1% Yeast extract O, V% Acidic potassium phosphate 1.45 Chronic magnesium sulfate 0.585 Potassium nitrate 1
．． Ochi! Yield = 6.5 Example 5 Streptococcus pyogenes AT
CC21060 was cultured in the same manner as in Example 1 using medium B shown in Table 5. In this case, Hanicillin G is
The solution was added at a concentration of 1000 units/d of culture solution. This culture P solution was purified in the same manner as in Example 1 to obtain antitumor substance 19PF-140570In9.

Table 5 Medium E Maltose α3% Casamino acids 0.5% Yeast extract 1.0 Acidic potassium phosphate 0.5% gtrs Magnesium 0.1% Sodium chloride 0.1% Potassium nitrate 0.5% m=6. 5 Experimental Example 1 An in vitro Ic test to measure the antitumor activity of the test drug was carried out based on the cell inhibition degree measurement method.

L-517B Y (Leukem
ia), and added this to 10-inch FC8 foil RPMI 1
640 medium (containing 5,qry,/l kanamycin).

Pressurize 0.45 m of this culture solution into a Falcon 2058 tube until the number of cells reaches I x 105 celjl/tu.
I changed it to be. A predetermined amount of the test drug (anti-tumor* quality SPF'-140 was added to this culture solution at 35 m
/ dissolved in a culture solution) was pressurized and cultured at 57°C to 5°C. 48 hours after adding the test drug, Torino ξ
The staining was carried out with blue, and the -d block inhibition change was calculated using the following formula.

Results obtained in Example 1 using the antitumor substance SPF-140 as the test drug! It is shown in Table 6.

Table 6 Cell inhibition degree ■ SPF'-140 (=n9/m/) L-5178
Y2.0 154 1.0 11. ! 1 Experimental Example 2 The antitumor activity test of the test drug in 1 vo was CRJ-C.
The test was carried out using 1 to 2 D-1 (ICR strain, female, 5 weeks old) mice.

Sarcoma-180 ascites cancer cells were used as the tumor cells, suspended in physiological saline, and incubated intraperitoneally in mice at KO, 2 m (cell number I x 10' cells).
) inoculated.

After the tumor cell inoculation, a predetermined amount of the test drug was administered intraperitoneally once a day for 7 days, and the number of survivors was observed.

Table 7 shows the results using the antitumor substance SPF-140 obtained in Example 1 as the test drug.

Table 7 Antitumor activity of SPF-140 - Days of survival of mice Number of administrations t(In9) 0 10 15 20
25 50 control 515 215 115 015
015 0150.3 515 515
515 4/〆5 4/ko 615 Furthermore, the survival rate = average survival days of test animal group/average survival days of control animal group x 100 was 270%.

[Brief explanation of drawings]

Figure 81 shows the ultraviolet absorption spectrum of a 1% aqueous solution of the antitumor substance 'SPF'-1400, and Figure 2 similarly shows the infrared absorption spectrum.

Claims (3)

[Claims] (1) SPF, an antitumor substance with the following physical and chemical properties
-140. 1. Elemental analysis C: 42.58% to 41.79%, H: 6.15% to 5.92%, N: 12.69% to 12.35% 2. Molecular weight As measured by gel filtration method, the molecular weight Approximately 25,000 to 70,000. 3. Decomposition point This substance turns brown at 170°C and turns black at 245°C and decomposes. 4. Specific rotation [α] ^2^0_D = -70.0° to -80.0° (C
=1.00) 5. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. The absorption maximum is observed at 275 nm, which is characteristic. 6. Infrared absorption spectrum shown in Figure 2. 7. Solubility in solvents Soluble in water, but partially soluble in methanol, including ethanol, n-propanol, n-butanol, isobutanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, ethyl ether, etc. It is sparingly soluble or insoluble in solvents. 8. Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5. 9. The color of the substance is pale yellow powder. 10. Color reaction Ninhydrin reaction + Buuret reaction + Lowry reaction + Molisch reaction - Decier reaction - Anthrone reaction - Cysteine sulfate reaction - 11. Stabilization This substance does not particularly require a stabilizer. (2) Antitumor substance SP belonging to the genus Streptococcus
The F-140-producing bacteria were cultured, and the antitumor substance S was extracted from the culture.
A method for producing an antitumor substance SPF-140, which comprises collecting PF-140. (3) Antitumor substance SP belonging to the genus Streptococcus
The antitumor substance SPF-140 according to claim 2, characterized in that when culturing the F-140 producing bacteria, penicillin or a related substance is added at an appropriate time during the culturing. manufacturing method.