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

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

 

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

A method of obtaining antitumor agents from the mucosa of the small intestine of the pig, including homogenization, processing ethyl alcohol, heating to 70°C, ultrafiltration and lyophilization [United States Patent 6,015,878, Jan. 18, 2000. Trifonov Monday Centuries, Roussev Je. K., Boshev N. A. Antitumor aqents isolated from intestinal mucosa, a method for their isolation and their application]. These agents have anti-tumor activity against lymphoma and myeloma mice, carcinoma of the pharynx and epithelioid carcinoma of the bladder, the embryonic rhabdomyosarcoma.

The objective of the invention is a method for antitumor substances (PS), which has activity against hepatocellular cancer.

The problem is solved by a method of obtaining PV, namely, that the mucous membrane of the small intestine of man homogenized with water in a ratio of 1:3-5 +3-+5°C, the homogenate was centrifuged, the supernatant heat for 7-10 minutes at a temperature of from 20 to 38°C, adjusted to 100°C. and heated for 15-20 minutes at this temperature, centrifuged again, cooled to +4 to+6°C. the supernatant add cooled to -15)-(-20)°With ethanol to a concentration of 65-70%, stand 15-17 h at (-15)-(-20)°C, t see you a again is centrifuged, from the supernatant removed the alcohol obtained target product is frozen and stored at (-65)-(-70°C until use.

Distinctive features of the proposed method are used as the feedstock of the mucous membrane of the small intestine of man, centrifugation of the homogenate in distilled water, warming the supernatant at a temperature of from 20 to 38°C, bringing the temperature up to 100°C and holding for 15-20 min at this temperature, centrifugation after warming up, processing ethanol to a concentration of 65-70%, keeping with ethanol 15-17 h at (-15)-(-20)°C, centrifugation.

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

The following examples illustrate the method of obtaining the RO and the determination of its biological activity.

1. An example of obtaining the RO.

During resection of the small intestine of man produce fence resected part of the small intestine length of 5 cm, which lacks any education. Immediately after collection to separate the mucous membrane. Its weight is 2.5, the Mucous membrane is shredded, add 7.5 ml of distilled water, homogenized with +3-+5°C, the homogenate was centrifuged to precipitate nuclei (1000 rpm, 10 min). Received posthegemony native supernatant is heated on 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 min at 100°C. the Formed precipitate thermolabile material 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 h, centrifuged in the same mode, which is indicated above. The alcohol from the supernatant removed with a rotary evaporator. The obtained extract (4 ml) is the concentration of protein 14,0-17,0 mg/ml in optical absorption at D280. Extract (PV) frozen and stored at 65-70°C until use.

2. Determination of activity declared by the RO on the culture of tumor cells hepatoma-27, are in the growth stage.

To perform this work hepatoma-27 was transferred into the culture of subcutaneous tumors by trypsinization. This scored a rat with subcutaneous hepatoma, sterile removed the tumor, chose the site free from necrosis. Separated him, were placed in a chilled solution of trypsin : versene (1:1) and 24 h were kept at +4°C. the next day poured trypsin : versen, washed several times with Hanks solution, filled in standard culture medium with 10% fetal serum and 100 µg/ml gentamicin and were sown on bottles Carell. After 24 h about the-Odile change the environment to remove not adherent cells. Cells were cultured in the medium of DMEM+RPMI (1:1) supplemented with 5% fetal calf serum (Flow).

Cells were planted in 96-well plates (Costar) in 100 μl of culture medium (DMEM+RPMI (1:1)). The number of cells introduced into the hole, depended on the duration of the experiment: 1 day - 4 thousand per well, 3 days - 2 thousand per well, 6 days - 1 thousand per well. Preincubation (culturing cells without drug) was carried out for 24 h at 37°C in an atmosphere of 5% CO2. Otany PV contributed 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-dimethanol-2)2.5 diphenyltetrazolium bromide] (Sigma), dissolved in water, was added in 20 μl (original solution of 5 mg/ml) in each well for 3 hours under the same conditions. Then the medium was collected and for lysis of cells used dimethyl sulfoxide (60 μl/well). For homogeneous distribution of the dye lysate was thoroughly mixed on a shaker (300 rpm) for 3 minutes. Quantitative determination of formazan conducted on the device Multiskan (filter 540). Cell viability was assessed by the ratio of the optical density of the solutions in the wells with the control cells and in the wells with cells treated with RO.

The inhibition of proliferation of cells in culture hepatoma-27 was calculated by the formula:

,

where

FE - ing the repression of proliferation;

In the level of proliferation in the experience;

PC - level proliferation in control.

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

The table shows that on the 1 day of incubation PV presents all concentrations had no inhibitory effect on the proliferation of tumor cells. On the 3rd day of incubation PV in all concentrations exerted a cytotoxic effect on tumor cells. The greatest inhibitory effect possessed by the RO at a concentration of 2 mg per 1 ml of medium. On the 6th day of incubation compared with 3 days cytotoxic effect of all PV was increased, the inhibition of cell proliferation was most pronounced in extracts with protein concentration, equal to 1 and 2 mg/ ml of culture medium.

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

3. The determination of the activity of the claimed ROS in vivo.

The study was carried out on outbred rats male weighing 100-150 g intertwined with intrahepatic hepatoma-27. For this purpose, 10 days after inoculation of tumor of the liver produced re-laparotomy, measured the size of the tumor (2nd largest size), after which the wound is arena the abdominal wall is sutured. Animals were divided into 2 groups. In group 1 was carried out by intraperitoneal injection of PV at the rate of 100 mg of protein per kg of body weight in a volume of 1 ml. Introduction were made every other day for 14 days (total of 7 injections). Animals of group 2 was the control, the PV they were introduced. 6 days after the last injection, the rats of group 1 (20 days after the start of treatment and 30 days after inoculation of tumor) animals of both groups were scored and again measured the tumor.

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

,

where

P op. counter - tumor size in control,

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

The following has been discovered. 10 days after inoculation of tumor (at start of treatment rats group 1) significant differences in the size of liver tumors in 1 and 2 groups was not: the area of the tumor was, respectively, 27,0±6,7 and 24.7±7.5 mm2P>0,05. 30 days after inoculation of tumor size in rats group 1 increased false, 55.3±18,8 mm2P>0,05, whereas the animals of the 2nd group, the tumor size was significantly increased to 167,7±46.4 mm2, P<0,05. Differences between groups 30 days after inoculation of tumor were reliable, which allows to calculate the indicator TRO. He was 67%.

Thus, the use of declared PV can effectively inhibit the growth of g is patami 27, namely, to stop the proliferation of cancer cells (in vitro) and to contribute to the inhibition of tumor growth in rats (in vivo).

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

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



 

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80 cl, 16 dwg, 1 tbl, 3 ex

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15 cl, 4 tbl, 452 ex

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27 cl, 1 tbl, 5 dwg, 25 ex

FIELD: medicine.

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2 ex

FIELD: medicine, pharmaceutics.

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7 cl, 2 tbl, 48 ex

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4 cl, 1 tbl

FIELD: medicine.

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1 tbl

FIELD: medicine.

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2 ex

FIELD: medicine, pharmaceutics.

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8 cl, 14 tbl, 14 ex

FIELD: medicine, veterinary science.

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EFFECT: method exhibits a high therapeutic effect.

FIELD: medicine, veterinary.

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FIELD: medicine, pharmaceutics.

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3 cl, 1 tbl, 1 ex

FIELD: medicine.

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3 cl, 2 ex

FIELD: medicine.

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EFFECT: reduced inflammatory alterations, accelerated regeneration process in anastomosis area, normalisation of gastroenteric tract function.

5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns therapeutic angiogenesis stimulation and generation of trophic and angiogenetic factors, and can be applied in amplification of brain compensation mechanism for functional salvage after central neural system damage or degeneration. The invention claims: a therapeutic medium of angiogenesis and vasculogenesis inducing factors for application in neurological and musculoskeletal therapy, containing angiogenesis and vasculogenesis inducing factors detached from human marrow stem cells in combination with pharmaceutically acceptable therapeutic cell medium; generation amplification method for angiogenesis and vasculogenesis inducing factors released by stem cells involving exposure and joint cultivation of stem cells with a compound for generation improvement of angiogenesis and vasculogenesis inducing factors; angiogenesis and vasculogenesis inducing factors isolated and separated of stem cells for application in therapy; process of obtaining the claimed angiogenesis and vasculogenesis inducing factors, including stages of human mesenchymal stem cell isolation and separation from tissue, differentiation and further cultural cultivation of mesenchymal stem cells for obtaining of neurological and musculoskeletal therapeutic media; mesenchymal stem cells isolated and cultivated in a culture, capable of differentiation and generation of target cell phenotype for tissue recovery under the effect of a required compound.

EFFECT: improved efficiency of cellular therapy.

17 cl, 3 ex, 4 tbl, 9 dwg

FIELD: medicine, gastroenterology, medicinal biochemistry.

SUBSTANCE: invention relates to a method for treatment and choice of a pharmaceutical preparation used in treatment of diseases caused and accompanying with disturbance in metabolism of bile acids and cholesterol. Method involves determination of the quantitative composition of short-chain fatty acids of (C2-C4)-fraction in blood serum and feces. Method involves using preparations containing fatty acids and promoting fro dissolving cholesterol bile stones in case when the content of propionic acid is decreased from 5.6% and increasing the content of butyric acid from 3.5%, and in increasing propionic acid from 19.9% to 21.8% and butyric acid from 18.9% to 20.1% in feces. Method involves administration of preparations effecting on activity of intestine anaerobic microorganisms in case when the content of propionic acid is decreased from 5.7% to 6.5% and the content of butyric acid is increased from 2.9% to 3.4% in blood serum, and when the content of propionic acid is increased from 21.8% and that of butyric acid from 20.1% in feces. Method involves the combination of preparations containing fatty acids and promoting to dissolving cholesterol bile stones and preparations effecting on activity intestine anaerobic microorganisms when the content of propionic acid is decreased from 5.6% and less and the content of butyric acid is increased from 3.5% and above. Proposed method provides possibility for differentiating treatment based on assay of leading ethiopathogenetic mechanisms in development of choledocholithiasis.

EFFECT: improved and enhanced method of treatment.

2 tbl, 3 ex

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