RU2540507C1 - Method for preparing substance with anti-tumour activity accompanying hepatocellular carcinoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a method for preparing a substance with anti-tumour activity in hepatocellular carcinoma consisting in the fact that individual's enteric mucosa is homogenised with water in ratio 1:3-5 at +3-+5°C; the homogenate is centrifuged; the supernatant is heated for 7-10 min at temperature 20° to 38°C increased to 100°C and heated to 15-20 min at this temperature, centrifuged again; the cooled supernatant cooled to +4-+6°C is added with ethanol cooled (-15)-(-20)°C to the concentration of 65-70%; 15-17 h later it is centrifuged again; the alcohol is removed from the supernatant; the prepared end product is frozen and stored at (-65)-(-70)°C until used.

EFFECT: extended range of products possessing the anti-tumour activity.

1 tbl, 1 ex

Description

The present invention relates to methods for producing biologically active compounds that can be used in medicine to inhibit the growth of malignant tumors.

A known method of obtaining antitumor agents from the mucous membrane of the small intestine of a pig, including homogenization, treatment with ethanol, heating to 70 ° C, ultrafiltration and lyophilization [United States Patent 6,015,878, Jan. 18, 2000. Trifonov VV, Roussev Je. K., Boshev N.A. Antitumor aqents isolated from intestinal mucosa, a method for their isolation and their application]. These agents have antitumor activity in mouse lymphoma and myeloma, pharyngeal carcinoma and epithelioid carcinoma of the bladder, embryonic rhabdomyosarcoma.

The objective of the invention is a method for producing an antitumor substance (PV) having activity against hepatocellular cancer.

The problem is solved by the method of obtaining PV, which consists in the fact that the mucous membrane of the human small intestine is homogenized with water in a ratio of 1: 3-5 at + 3- + 5 ° C, the homogenate is centrifuged, the supernatant is heated for 7-10 minutes at a temperature of 20 to 38 ° С, adjusted to 100 ° С and heated for 15-20 min at this temperature, centrifuged again, ethanol cooled to (-15) - (- 20) ° С is added to the supernatant cooled to + 4- + 6 ° С to a concentration of 65-70%, incubated for 15-17 hours at (-15) - (- 20) ° C, centrifuged again, the alcohol obtained from the supernatant was removed th product is frozen and stored at (-65) - (- 70) ° C until use.

Distinctive features of the proposed method are the use of raw materials of the mucous membrane of the human small intestine, centrifugation of the homogenate in distilled water, heating the supernatant at a temperature of from 20 to 38 ° C, bringing the temperature to 100 ° C and keeping for 15-20 minutes at this temperature , centrifugation after heating, treatment with ethanol to a concentration of 65-70%, keeping with ethanol for 15-17 hours at (-15) - (- 20) ° С, centrifugation.

Tests of the claimed PV for cytotoxic (cytostatic) and antitumor activity were carried out on tumor cell cultures (in vitro) and in rats with transplantable tumors (in vivo).

The following are examples illustrating a method for producing PV and determining its biological activity.

1. An example of obtaining PV.

During resection of the human small intestine, a resected small intestine section of 5 cm in length is taken in which there are no formations. Immediately after the fence, the mucous membrane is separated. Its weight is 2.5 g. The mucous membrane is crushed, 7.5 ml of distilled water are added, homogenized at + 3- + 5 ° С, the homogenate is centrifuged to precipitate nuclei (1000 rpm, 10 min). The obtained post-nuclear native supernatant is heated in a water bath for 7-10 minutes at a temperature of from 20 to 38 ° C, adjusted to 100 ° C and incubated for 15-20 minutes at 100 ° C. The resulting precipitate of thermolabile material is removed by centrifugation (5000 rpm, 15 min). To the supernatant, cooled to + 4- + 6 ° C, add 16.1 ml of 96% ethanol, cooled to (-15) - (- 20) ° C and left at this temperature for 15-17 hours, centrifuged in the same the mode indicated above. The alcohol from the supernatant is removed on a rotary evaporator. The resulting extract (4 ml) has a protein concentration of 14.0-17.0 mg / ml by optical absorption at D ₂₈₀ . The extract (PV) is frozen and stored at 65-70 ° C until use.

2. Determination of the activity of the claimed PV in the culture of tumor cells of hepatoma-27 in the growth stage.

To perform this work, hepatoma-27 was transferred to culture from a subcutaneous tumor by trypsinization. To do this, a rat with subcutaneous hepatoma was killed, the tumor was sterile removed, and a site free of necrosis was chosen. It was separated, placed in a cooled solution of trypsin: versene (1: 1) and kept at + 4 ° С for 24 hours. After a day, trypsin: versene was poured, washed several times with Hanks solution, poured with standard nutrient medium with 10% fetal serum and 100 μg / ml gentamicin and plated on Carrel vials. After 24 hours, a change of medium was performed to remove non-adherent cells. Cells were cultured on DMEM + RPMI medium (1: 1) supplemented with 5% fetal calf serum (Flow).

Cells were plated on 96-well plates (Costar) in 100 μl of culture medium (DMEM + RPMI (1: 1)). The number of cells introduced into the well depended on the duration of the experiment: 1 day - 4 thousand / well, 3 days - 2 thousand / well, 6 days - 1 thousand / well. Pre-incubation (culturing cells without preparations) was carried out for 24 hours at 37 ° C in an atmosphere of 5% CO ₂ . Thawed PV was added to the wells at concentrations of 0.25; 0.5; 1.0 and 2.0 mg / ml of culture medium. Incubation was carried out at 37 ° C for 1, 3 and 6 days.

MTT [3- (4,5-dimethylthiazole-2) 2,5 diphenyltetrazolium bromide] (Sigma) dissolved in water was added in an amount of 20 μl (stock solution 5 mg / ml) to each well for 3 hours under the same conditions . Then the medium was taken and dimethyl sulfoxide (60 μl / well) was used for cell lysis. For a homogeneous distribution of the dye, the lysate was thoroughly mixed on a shaker (300 rpm) for 3 minutes. Quantification of formazan was performed on a Multiskan instrument (filter 540). Cell viability was evaluated by the ratio of the optical density of solutions in wells with control cells and in wells with cells treated with PV.

Inhibition of cell proliferation in hepatoma-27 culture was calculated by the formula:

,

,

Where

PI - inhibition of proliferation;

By - level of proliferation in the experiment;

PC - the level of proliferation in the control.

The table presents data on the effect of PV on the growth of hepatoma-27 cell culture at various concentrations of PV protein in 1 ml of culture medium.

From the table it follows that on 1 day of incubation of PV in all the concentrations presented, it did not exert an inhibitory effect on the proliferation of tumor cells. On the 3rd day of incubation, PV in all concentrations exerted a cytostatic effect on tumor cells. PV had the greatest inhibitory effect at a concentration of 2 mg per 1 ml of medium. On the 6th day of incubation, compared with 3 days, the cytostatic effect of all PV increased, the inhibition of cell proliferation was most pronounced in extracts with a protein concentration of 1 and 2 mg / ml of culture medium.

Thus, the claimed PV is characterized by a high level of inhibition of proliferation in hepatoma-27 cell culture, reaching 73% (on the 3rd day of incubation) and 92% (on the 6th day).

3. Determination of the activity of the claimed PV in vivo.

The study was conducted on outbred male rats weighing 100-150 g with transplanted intrahepatic hepatoma-27. To do this, on the 10th day after inoculation of a liver tumor, a second laparotomy was performed, the size of the tumor (2 largest sizes) was measured, after which the wound of the anterior abdominal wall was sutured. Animals were divided into 2 groups. In group 1, intraperitoneal administration of PV was carried out at the rate of 100 mg of protein per kg of weight in a volume of 1 ml. The introduction was made every other day for 14 days (a total of 7 introductions). Animals of the 2nd group were a control; no PV was administered to them. 6 days after the last injection to rats of group 1 (20 days after the start of treatment and 30 days after vaccination of the tumor), the animals of both groups were killed and the tumor was measured again.

The inhibition of tumor growth (SRW) was calculated by the formula:

,

,

Where

R op. counter - the size of the tumor in the control,

R op. experience - the size of the tumor in the experience.

The following was revealed. 10 days after the inoculation of the tumor (at the start of treatment of rats of group 1), there were no significant differences in the size of the liver tumor in groups 1 and 2: the tumor area was 27.0 ± 6.7 and 24.7 ± 7.5 mm ^2, respectively , P> 0.05. 30 days after the inoculation of the tumor, its size in rats of the 1st group increased unreliably, amounted to 55.3 ± 18.8 mm ² , P> 0.05, whereas in animals of the 2nd group, the tumor sizes significantly increased to 167.7 ± 46.4 mm ² , P <0.05. The differences between the groups 30 days after tumor inoculation were significant, which allows us to calculate the TPO. It amounted to 67%.

Thus, the use of the claimed PV allows you to effectively suppress the growth of hepatoma 27, namely to stop the proliferation of cancer cells (in vitro) and to help inhibit tumor growth in rats (in vivo).

The proposed method allows to expand the range of substances with antitumor activity.

Claims (1)

A method of obtaining a substance with antitumor activity in hepatocellular liver cancer, which consists in the fact that the mucous membrane of the human small intestine is homogenized with water in a ratio of 1: 3-5 at + 3- + 5 ° C, the homogenate is centrifuged, the supernatant is heated for 7-10 min at a temperature of 20 to 38 ° C, adjusted to 100 ° C and heated for 15-20 min at this temperature, centrifuged again, add chilled to (-15) - (- 20 to the supernatant cooled to + 4- + 6 ° C ) ° C ethanol to a concentration of 65-70%, after 15-17 hours again centrifuged, from the supernatant removed from the pie, the obtained target product is frozen and stored at (-65) - (-70) ° C until use.