RU2347581C1 - Product of microbal synthesis with antitumor activity (versions) - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: invention consists in creation of microbal synthesis product with antitumor activity produced from cultural liquid Bacillus mesentericus AB-56 as protein with molecular weight of 12-24 kD.

EFFECT: wider arsenal of agents with antitumor activity.

Description

The invention relates to oncology.

Known drug composition for the treatment of patients with malignant neoplasms, consisting of fetal substance and filtrate of the culture fluid Bacillus mesentericus AB-56. An extract of human embryonic tissue or industrially produced alpha-fetoprotein is used as a fetal substance. The composition allows to induce necrosis of tumors in those cases when they are not sensitive to chemotherapeutic agents (Ru2112547).

The culture fluid is a mixture of various products of microbial synthesis, of which not all have an antitumor effect, which does not make it possible to standardize this product by antitumor origin. In addition, two components are used in the composition.

Known protein is the product of microbial synthesis isolated from the culture fluid Bacillus mesentericus AB-56 and has antitumor activity (G.P. Potebnya, G.S. Lisovenko, S.I. Yalkut, L.I. Rusanova. Antitumor vaccine increases the effectiveness of treatment cancer patients / Pharmacist, 2002, No. 5. Ukraine) (prototype).

As mentioned above, the culture fluid contains a mixture of various products of microbial synthesis, including a mixture of various proteins, of which not all have antitumor activity, which makes it impossible to standardize this product by antitumor origin. From the indicated work of G. Potebni et al., the characteristic of a protein having antitumor activity is unknown.

Disclosure of invention

The essence of the invention lies in the fact that as products of microbial synthesis with antitumor activity, it is proposed to use protein obtained with Bacillus mesentericus AB-56 culture fluid with a molecular weight of 12-24 kDa, as well as a lipopolysaccharide.

The technical result of the claimed inventions is the establishment and isolation of active antitumor principles from Bacillus mesentericus AB-56 culture fluid, which makes it possible to standardize the antitumor components of the culture fluid. The present invention expand the arsenal of funds with antitumor activity.

The technology for producing culture fluid Bacillus mesentericus AB-56:

the culture fluid of a 7-day-old culture of Bacillus mesentericus AB-56, grown on meat and peptone broth according to standard technology at a concentration of 3 billion microbial bodies per ml, is separated from the microbial bodies by centrifugation at 5000 rpm for 15 minutes, after which the supernatant is filtered through a sterilizing membrane and poured into sterile dishes.

The technology of protein isolation from the culture fluid

Using gelmph electrophoresis in a 13% polyacrylamide gel according to Lemmli (Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, vol. 227, p. 680-685), the obtained Bacillus mesentericus AB-56 culture fluid for the presence of protein in it. Analysis showed that the culture fluid contains protein at a concentration of 4 mg / ml.

Then, a selection of the concentration of ammonium sulfate was carried out to isolate the protein from the culture fluid (Scopes R. Methods for protein purification. 1985, M .: Mir, pp. 61-71). The bulk of the protein material was precipitated at a 65% concentration of ammonium sulfate.

The fractionation of proteins of the culture fluid was carried out according to the method described in the previous source using 65% ammonium sulfate. 450 ml of the culture fluid was mixed with ammonium sulfate to 65% saturation, and then left overnight at 4 ° C. Then, the mixture of the culture fluid with ammonium sulfate was centrifuged at 8000 rpm for 30 minutes. The resulting precipitate was dissolved in 5 ml of buffer (10 mM sodium phosphate, pH 7.6; 0.05 M NaCl), applied to a Sephacryl S-300 column (1.2 × 90 cm, V = 100 ml) and washed with the above buffer at a flow rate of 20 ml / hour.

Using electrophoregrams, it was established that the first protein with a molecular mass of 115 kD, consisting of 4 subunits (the first fraction), comes out. The second is a protein with a molecular weight of 67 kD (2nd fraction). Next comes the peak of proteins with molecular weights from 12 to 24 kD (3rd fraction). The collected fractions were reprecipitated with 65% ammonium sulfate, the precipitates were dissolved in an isotonic sodium chloride solution and dialyzed three times against 100 volumes of isotonic sodium chloride solution.

The protein concentration of each fraction dialyzed against an isotonic sodium chloride solution was determined by Bradford (Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, vol. 72, p. 248-254). The protein concentration in the fractions ranged from 0.3 to 6 mg / ml.

Determination of the antitumor activity of protein fractions of the culture fluid (QL) of Bacillus mesentericus AB-56

The determination of antitumor activity was carried out on a solid version of a linear non-specific tumor Krebs-2. Used female mice of the GR line. Tumors were inoculated into the thigh muscles with 2 × 10 ⁶ Krebs-2 tumor cells in 0.1 ml of isotonic sodium chloride solution. The resulting tumor was periodically measured in three mutually perpendicular directions and the tumor volume was calculated.

Previously, an experiment was conducted to determine the optimal protein concentration. The following concentrations were tested: 0.3; one; 3; 6 mg / ml. The optimal concentration was 3 mg / ml, which had the most pronounced inhibitory effect on tumor growth while there was no pronounced general toxic effect.

Tumor Krebs-2. After vaccination, the tumors of the mouse were divided into 5 groups (4 experimental and one control), 8 animals in the experimental and 10 in the control group. The next day, experimental mice of three groups were injected under the skin of the dorsal region 1 time per day for 3 days with 0.25 ml of each protein fraction at a concentration of 3 mg / ml: the first group of animals was injected with protein of the first fraction, the second group introduced protein of the 2nd fraction, the third group - protein of the 3rd fraction. A fourth group of experimental animals was injected with 0.25 ml of QOL. 0.25 ml of isotonic sodium chloride solution was similarly administered to control mice.

Starting from the 7th day from tumor inoculation, the volume of tumors was measured in dynamics. QOL and protein fractions 1 and 2 did not inhibit the growth of tumors: on the 8th day, the tumor volume in these groups was 1.1 ± 0.2 cm ³ , 1.3 ± 0.2 cm ³ and 1.2 ± 0.2 cm ^3, respectively (in the control, 0.9 ± 0.1 cm ³ ); on the 11th day the tumor volume was 2.2 ± 0.2 cm ³ , 1.7 ± 0.2 cm ³ and 1.9 ± 0.3 cm ^3, respectively (in the control - 2.1 ± 0.1 cm ³ ), on the 14th day - 3.2 ± 0.3 cm ³ , 3.0 ± 0.2 cm ³ and 3.4 ± 0.3 cm ^3, respectively (in the control - 3.1 ± 0.3 cm ³ ), on the 16th day - 4.4 ± 0.3 cm ³ , 4.2 ± 0.4 cm ³ and 4.4 ± 0.3 cm ^3, respectively (in the control - 3.9 ± 0, 4 cm ³ ).

Significantly inhibitory effect on the growth of the Krebs-2 tumor, starting from the 13-14th day from the beginning of its administration, was exerted by protein fraction 3 (mol. Mass 12-24 kD): the tumor volume in this group for the indicated period was 1.9 ± 0.2 cm ³ (in the control - 3.1 ± 0.3 cm ³ ), on the 16th day the tumor volume was 1.6 ± 0.1 cm ³ (in the control - 3.9 ± 0.4 cm ³ ), p <0.01 and p <0.001, respectively.

Claims (1)

The product of microbial synthesis with antitumor activity, obtained from the culture fluid Bacillus mesentericus AB-56, characterized in that it is a protein with a molecular weight of 12-24 kDa obtained by fractionation of proteins of the culture fluid by saturation with ammonium sulfate to 65%, centrifugation, dissolving the precipitate in a buffer of 10 mM sodium phosphate, pH 7.6; 0.05 M NaCl, followed by application to a Sephacryl S-300 column, rinsing with the above buffer at a flow rate of 20 ml / h, while the third fraction contains a protein of the above molecular weight and has a pronounced antitumor activity.