SE461222B - TUMORATIVE SUBSTANCES SPF-140 AND PROCEDURES FOR PREPARING THEREOF - Google Patents

Description

461 222 10 15 20 25 30 2 or mechanically disintegrate the bacterium to fractionate the constituents.

These treatments could complicate the purification step so that the isolation of an active ingredient becomes very difficult. The only example of an active ingredient that has actually been isolated and determined is a protein having a molecular weight of 200,000 (compare Japanese Patent No. 43841/1973 and Japanese Patent No. 44617/1976).

We have previously tried to isolate a tumor-killing, active ingredient, produced by streptococci, and have succeeded in isolating new physiologically active substances SPF-1 and SPF-2 and a new tumor-killing substance SPF-100 by adding penicillin or closely related substances at an appropriate time. during the cultivation of the streptococci, and further to allow the cultivation to continue and to recover the above-mentioned substances present in the medium from the filtered culture nutrient solution, followed by purification. These substances inhibited the growth of a macromolecule-permeable mutant of the large intestinal bacillus MP-2 (FERM-P5432) (cf. Agr. Biol. Chem., G§, 371-378 (1979)), hereinafter referred to as the MP-2 strain.

We have found that the filtered streptococcal culture nutrient solution, in addition to SPF-1, SPF-2 or SPF-100, contains a fraction that does not inhibit the growth of the MP-2 strain, but which has a tumor-killing effect. The substance in this fraction is called SPF-140.

Summary of the Invention The tumor-killing substance SPF-140 secreted in the filtered culture solution of the present invention is characterized in that it has a molecular weight of 25,000-70,000 and that it has no inhibitory effect on the growth of the MP-2 strain. 10 15 20 25 30 35 461 222 3 No tumor-killing substances are known which relate to S. pyogenes, which substances have been secreted and accumulated in a medium and have a molecular weight which is lower than a few tens of thousands. In addition, the absorption spectrum in the ultraviolet range of the tumor-killing substance SPF-140 of the present invention, which appears to be a peptide-like substance according to the results of elemental analysis and color reactions, indicates that it is a new substance which differs from known tumor-killing substances.

Thus, the present invention comprises a process for the preparation of the tumor-killing substance SPF-140, which process comprises culturing SPF-140-producing streptococci and recovering the tumor-killing substance SPF-140 from the medium.

Brief Description of the Drawings Fig. 1 shows an absorption spectrum in the ultraviolet region of an aqueous 0.1% solution of SPF-140, while Fig. 2 shows an absorption spectrum in the infrared region thereof.

Detailed Description of the Invention In the process of the present invention, any of the streptococci that can produce the tumor-killing substance SPF-14 can be used. The following examples of bacteria are useful: pyogenesis ATCC 21060 sp. ATCC 21597 pyogenesis ATCC 21546 pyogenesis ATCC 21547 pyogenesis ATCC 21548 Natural media, such as meat extract medium, yeast Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus extract medium and brain-cardiac infusion medium for the growth of streptococci can be used. 461 222 10 15 20 25 30 35 4 Streptococci can grow under suitable conditions, ie at a pH of 5.0-8.0 (preferably 6.1-7.2) and at a temperature of 30-40 ° C (preferably 35-37 ° C). Usually an anaerobic and static or stirred culture is used.

In the process of the present invention, penicillin or closely related substances added at an appropriate time play an important role in the recovery of the tumor-killing substance SPF-140. Penicillin or closely related substances can be added 3-20 hours, preferably 5-10 hours, after the initiation of the logarithmic growth phase when the bacterium is grown at 37 ° C. Continuation of the culture for 1 to 20 hours, preferably 3 to 15 hours, thereafter would allow a large amount of the tumor-killing substance SPF-140 to accumulate in the medium.

Any closely related substances known to exhibit a similar effect to penicillin can be used, although penicillin G is commonly used.

Penicillin G is added in an amount of 100-7000 units / ml, preferably 300-5000 units / ml, to the medium.

The culture nutrient solution thus obtained is centrifuged to remove the cells, and ammonium sulfate is added to the filtrate. After extraction of fractions with a degree of saturation of 50-90%, the resulting salting product containing the tumor-killing substance SPF-140 can be stored in frozen form.

The tumor-killing substance SPF-140 is purified from the salting-out product by dissolving the salting-out product in a buffer solution and adsorbing and eluting the solution thus formed batchwise or continuously with an ion exchanger, followed by gel filtration chromatography.

Ion exchangers, such as ion exchange resin, ion exchange cellulose, ion exchange Sephadex (a product of Pharmacia AB) or hydroxylapatite, and gel filtration materials, such as Toyopearl HW 5OF or HW 50SF (products of Toyo Soda Mfg. Co., Ltd .) or Sephadex (a product of Pharmacia AB) can be used. The adsorption of the ion exchange agent by the tumor-killing substance SPF-140 and the elution of SPF-140 therefrom can be carried out with a buffer solution with a suitable salt concentration and a suitable pH value.

Two or more ion exchangers or gel filtration materials can be combined if desired.

The tumor-killing substance SPF-140 obtained in Example 1 in lyophilized form is a light yellow powder with the following physicochemical properties. Elemental Analysis C: 42.58 - 41.79 H: 6.15 - 5.92% N: 12.69 - 12.35% O: 36.33 - 38.78% and ash: 2.25 - 1 , 16% 2. Molecular weight a of about 25000 - 70,000 after determination by gel filtration. Decomposition point: The substance turns brown at 170 ° C and black at 245 ° C and decomposes. 4. Specific rotation: tuff) = 7o, ø ° - so, o ° (c = 1.00). Absorption spectrum in the ultraviolet range: Fig. 1 shows an absorption spectrum in the ultraviolet range of an aqueous 0.1% solution thereof, which spectrum is characterized by a maximum absorption at 275 nm. 6. Absorption spectrum in the infrared region: Fig. 2 shows an absorption spectrum in the infrared region of the same. 7. Solubility in solvents: The substance is soluble in water, partially soluble in methanol and hardly or not at all soluble in solvents including ethanol, n-propanol, n-butanol, 461 222 (fl I0 15 20 25 30 35 6 isobutanol , n-hexane, chloroform, acetone, methyl isobutyl ketone and ethyl ether 8. Acidity: pH in aqueous 1.0% solution thereof is 6.5 9. Form: Light yellow powder 10. Color reactions: Ninhydrin: +, Biuret : +, Lowry: +, Molischz -, Dische: -, Anthron: - and Cysteine sulphate: -. Ll. Stability: No stabilizers required.

In the present invention, the antibacterial activity is determined in the following manner.

Antibacterial activity: The antibacterial activity is determined by the effect of inhibiting the growth of the above-mentioned MP-2 strain. Furthermore, the activity unit for a physiologically active substance is determined by Udaka's method (cf. J. Antibiotics, åâ, 1319 - 1325 (1982)) based on the antibacterial activity against MP-2.

This means that a medium (ie M3 medium) consisting of 1.75% Bact Antibiotic Medium 3 (a product from Difco) is sterilized by heating to 120 ° C for 15 minutes. Then pour 20 ml portions of the medium into Petri dishes and allow to cool to prepare plate media.

A medium consisting of 0.5% peptone, 0.5% meat extract, 0.3% sodium chloride and 0.8% agar is sterilized separately by heating to 120 ° C for 15 minutes. The medium is then placed in a thermostat at 42 ° C. When the temperature of the medium becomes 42 ° C, the MP-2 strain, which has been cultured at 37 ° C for 17 hours, is added to the medium at a concentration of 104 cells / ml. 2 ml of the medium is collected with a pipette and added to the surface of the previously prepared MS medium, after which they are immediately dispersed homogeneously and solidify. Then dilute a sample solution appropriately. A sheet of paper (diameter 8 mm, a product of Toyo Roshi Co., Ltd.) is wetted with the paper sheet thus obtained for less than 17 hours at the inhibition zone solution. It is then placed on the plate prepared above and cultured at 37 ° C to determine the diameter of which is derived from the test substance. The concentration of the test substance, the inhibition zone diameter of which is 10 mm, is defined as one unit (1 u).

To further illustrate the present invention, the following examples are given.

Ersmes-.Ll 100 ml of a BHI medium was inoculated with Streptococcus pyogenes ATCC 21060 and subjected to a static culture for 8 hours at 37 ° C to obtain a seed culture fluid.

Then 1 liter of medium A was inoculated with the composition shown in the following Table 1 with the thus obtained inoculum and was subjected to anaerobic culture under the same conditions as those of the inoculum.

Table 1: Medium A Meat extract 1.0% Polypeptone 1.0% Maltose 0.5% Yeast extract 0.25% Acidic potassium dihydrogen phosphate 0.1% Magnesium sulphate 0.05% (pH = 6.8) Then 8 liters of medium A were introduced in a shaker fermenter (10 liters), sterilized at 120 ° C for 10 minutes, allowed to cool to 37 ° C and inoculated with 1 liter of the pre-cultured liquid. It was grown anaerobically therein at 37 ° C and at pH 6.5 for 15.5 hours at a speed of 300 rpm. Then, 5000 units / ml of penicillin G was added to the medium, and the culture was continued for a further 5 hours. The resulting culture nutrient solution was centrifuged to remove the cells.

Ammonium sulfate was added to the filtered culture nutrient solution and a 50-90% saturation fraction was recovered to obtain a precipitate. This precipitate contained 381.0 x 104 u of a physiologically active substance which inhibited the growth of MP-2. This precipitate was dissolved in 300 ml of phosphate buffer solution (1 x 10 -2 M, pH = 7.0; KHZPO4-Na2HPO4) and the resulting solution was introduced into a DEAE-cellulose column (5 x 70 cm) to thereby adsorb the physiologically active substance.

Then, it was eluted with the above-mentioned phosphate buffer solution containing 0.3 M sodium chloride stepwise to fractionate a fraction having a physiological activity of 283.4 x 104 u. This physiologically active fraction was adsorbed in a DEAE-Scphadex A-25 column (2 , 6 x 70 cm) and then eluted with a linear increase of the sodium chloride concentration in the above phosphate buffer solution to recover the physiologically active fraction with the activity 249.6 x 104 u. The eluate was further concentrated and introduced into a column (2.6 x 100 cm) with the gel filtration material Toyopearl HW 50F to recover a non-bactericidal fraction, which was then lyophilized to give 640 mg of the tumor-killing substance SPF-140.

The tumor-killing substance SPF-140 thus obtained was used as a test substance in the test examples 1 and 2 described below, in order to determine the tumor-killing effect. Streptococcus pyogenes ATCC 21060 was grown in the same manner as described in Example 1 in a medium B, as shown in Table B. Pencillin G was added to give the medium a concentration of 1000 units / ml. The filtered culture nutrient solution was purified in the same manner as described in Example 1 to give 767 mg of the a = q fl killer SPF-140.

Table 2: Medium B Meat extract 0.5% Polypeptone 1.0% Yeast extract 0.25% Sodium chloride 0.1% (pH = 6.7) ÉšÉ @ EÉ1_§ Streptococcus pyogenes ATCC 21060 was grown in the same manner as described in Example 1 of a medium C, as shown in Table 3. The filtered culture nutrient solution was purified in the same manner as described in Example 1 to give 527 mg of the tumor-killing substance SPF-140.

Table 3: Medium C Meat extract 1.0% Polypeptone 1.0% Maltose 0.5% Yeast extract 0.25% Acidic potassium dihydrogen phosphate 0.1% Magnesium sulphate 0.05% Sodium chloride 0.5% (pH = 6.5) Stemptococcus pyogenes ATCC 21060 was grown in the same manner as described in Example 1 in a medium D, shown in Table 4. Pencillin G was added to give the medium a concentration of 1000 units / ml. The filtered culture nutrient solution was purified in the same manner as described in Example 1 to give 830 mg of the tumor-killing substance SPF-140. 461 222 10 15 20 25 30 35 10 Table 4: Medium D Maltose 0.8% Polypeptone 0.1% Yeast extract 0.1% Acidic potassium dihydrogen phosphate 1.43% Magnesium sulphate 0.585% Potassium nitrate 1.0% (pH = 6.5) §§§ @ eel_â Streptococcus pyogenes ATCC 21060 was grown in the same manner as described in Example 1 in a medium E, as shown in Table 5. Pencillin G was added to give the medium a concentration of 1000 units / ml. The filtered culture nutrient solution was purified in the same manner as described in Example 1 to give 570 mg of the tumor-killing substance SPF-140.

Table 5: Medium E Maltose 0.3% Casamino acids 0.5% Yeast extract. 1.0% Acidic potassium dihydrogen phosphate 0.5% Magnesium sulphate 0.1% Sodium chloride 0.1% Potassium nitrate 0.5% (pH = 6.5) E§9y§§§eesl_l The tumor-killing activity of the test substance in vitro was determined according to the determination of the degree of cellular inhibition.

L-5778Y (Leukemia), which was used as tumor cells, was suspended in an RPMI 1640 medium containing 10% FCS and 5 mg / l kanamycin. 0.45 ml of this medium was injected into a Falcon 2058 tube to give a concentration of 1 x 105 cells / tube. Thereafter, a special amount of the test substance, prepared by dissolving the tumor-killing substance SPF-140 in 0.05 ml of c. 37 ° C. 48 hours after the addition of the test substance, vital staining with trypan blue was performed, and the degree of cellular inhibition was determined by the following equation: (A) - number of cells in each test group x 100.

Degree of cellular inhibition (%) number of cells in the control group (A) Table 6 shows the result obtained when using the tumor-killing substance SPF-140 as test substance.

Table 6: Cellular degree of inhibition (%) SPF-140 (mg / ml) L-5178Y 2.0 15.3 1.0 11.3 13§9! E> _ The tumor-killing activity of the test substance in vivo was determined using five-week-old female mice of the type CRJ-CD-1 ICR.

Sarcoma 180 ascites cancer cells used as cancer cells were suspended in a physiological saline solution. Each mouse was inoculated inside the abdominal wall with 0.2 ml of the suspension, which included 1 x 10 cancer cells.

After inoculation of the cancer cells, a certain amount of the test substance was administered inside the abdominal wall of each mouse once a day for seven days to observe the number of vital mice. Table 7 shows the results of the test in which the tumor-killing substance SPF-140 was used as the test substance. 461 222 12 Table 7: Tumor-killing effect of SPF-140 (Number of vital mice) Dose (mg) Day o 10 15 20 25 30 f '5 Control 5/5 2/5 1/5 0/5 o / 5 0/5 - '0.3 5/5 5/5 s / 5 4/5 4/5 3/5 Life expectancy ratio [average survival period (days) for the control group / average survival period (days) for the test group x 100] for the test substance is 270%.

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Claims (3)

_10 15 20 25 30 461 222 13 PATENT REQUIREMENTS Tumor-killing substance called SPF-140, characterized in that it is secreted by bacteria belonging to the genus Streptococcus pyogenes and has the following physicochemical properties: (1) (2) (3) (4) (5) (6) ( 7) (8) Elemental analysis: C: 42.58 - 41.79% H: 6.15 - 5.92% N: 12.69 - 12.35% O: 36.33 - 38.78% and ash: 2.25 - 1.16% Molecular weight: Approx. 25000 - 70,000 after determination by gel filtration. Degradation point: The substance turns brown at 170 ° C and black at 245 ° C and decomposes. Specific rotation: lulšo = 7o, o ° - so, o ° (c = 1,00). Absorption spectrum in the ultraviolet range: Fig. 1 shows an absorption spectrum in the ultraviolet range for an aqueous 0.1% solution thereof, which spectrum is characterized by a maximum absorption at 275 nm. Absorption spectrum in the infrared region: Fig. 2 shows an absorption spectrum in the infrared region of the same. Solubility in solvents: The substance is soluble in water, partially soluble in methanol and hardly or not at all soluble in solvents including ethanol, n-propanol, n-butanol, isobutanol, n-bexane, chloroform, acetone, methyl isobutyl ketone and ethyl ether. Acidity: the pH of an aqueous 1.0% solution thereof is 6.5. 461 10 15 20 25 30 222 14 (9) Form: Light yellow powder (10) Color reactions: Ninhydrin: +, Biuret: +, Lowry: +, Molisch: -, Dische: -, Anthron: - and Cysteine sulphate: -. (ll) Stabilítetz No stabilizers required. A process for producing SPF-140 as defined in claim 1, characterized in that it comprises culturing an SPF-140 producing Streptococcus pyogenes selected from the group consisting of Streptococcus pyogenes ATCC 21060, Streptococcus sp. ATCC 21597, Streptococcus pyogenes ATCC 21546, Streptococcus pvogenes ATCC 21547 and Streptococcus pyogenes ATCC 21548, in a medium containing carbon sources, nitrogen sources and nutrients at a pH of 5.0-8.0 and without a temperature of 30-40 ° C passage of air therethrough, and recovery of the tumor-killing substance SPF-140 from the culture medium. Process for the preparation of the tumor-killing substance SPF-140, as described in claim 2, characterized in that the process comprises adding penicillin G in an amount of 100-7000 units / ml during the cultivation of the bacteria for the production of SPF-140 and 3-15 h after the beginning of the logarithmic growth phase, and culture for another 1-20 h.