The way to obtain therapeutic agents that regulate cell differentiation

 

(57) Abstract:

The invention relates to biotechnology and can be used in pharmacology and medicine. The method consists in homogenizing the muscle tissues of the embryo of the animal, after which the homogenate was incubated for 10-20 h, the extraction is carried out, and then separating the precipitate, and the resulting solution was further diluted with saline to achieve a concentration of protein in it 15-25 mg/ml, add the preservative, separating the resulting suspension, a solution was incubated for 48 h, sterilized, termostatic it stand 30-40 days at a temperature not exceeding 15oC remove the resulting suspension and the solution is subjected to interaction with immobilized proteolytic enzyme. The technical result is obtained a highly effective tool in the treatment of diseases associated with impaired differentiation of cells. 1 Il.

The invention relates to biotechnology and can be used in pharmacology and medicine.

Differentiation of cells occurs in various physiological processes in the body and primarily with violations functions of the immune system and the consequences of this naree closest to the claimed solution is the method of obtaining remedies regulating the differentiation of cells and intended for the treatment and prevention of cancer (French Patent N 2451193, 1979).

The known method consists in grinding the embryos of animals, their homogenization in physiological solution, obtaining an extract of embryonic complex substances, and separating the resulting precipitate by filtration, then the filtrate add an antibiotic. As a result of implementation of the specified sequence of actions receive drug treatment full embryonic complex. The treatment received by the drug is in the necessary course of injections.

The prototype method is quite simple in execution, but it cannot provide a highly effective drug, as no cleavage of macromolecular precursors and activation of regulatory peptides. The resulting preparation contains the formed elements and high molecular weight proteins that largely determines its side effects, manifested in allergies and in her terrible complication is anaphylactic shock.

The present invention is to provide a method for obtaining a therapeutic agent that regulates the differentiation of cells, to which their protein-peptide components and neutralizing other, provides high efficiency of the final product and excludes its side effects.

The problem is solved in that in a method of producing therapeutic agents that regulate the differentiation of cells, which consists in grinding the embryonic tissues of the animal, their homogenization and extraction of physiological solution, separating the precipitate from the solution, according to the invention as embryonic tissues take the muscle tissue of the animal, the homogenate was incubated for 10 to 20 hours, and after extraction, the treatment solution is performed within 24 0.5 hours, then after separation of the resulting precipitate the resulting solution was further diluted with saline to achieve a concentration of protein in it 15 - 25 mg/ml and add to it the preservative in an amount not less than 0.05%, after separation of the resulting suspension solution was incubated for 48 hours, sterilized and thermostatic it for at least 3 days at a temperature of 36oC, and the solution was incubated 30 - 40 days at a temperature not exceeding 15oC, and remove the resulting suspension, and the solution is subjected to interaction with immobilized proteolytic enzyme, with the mode specified Sanogo method includes especially the protein-peptide complex (BOD), which is an instrument with a certain specificity and required quality.

Use as raw material embryonic muscle soft tissue of the animal is responsible for the specificity of the obtained funds. Keeping these tissues homogenate for 10 to 20 hours due to biosynthesis extreme proteins, the nature of which depends on the quality of therapeutic agent. If more time is specified, the deterioration of the quality of the product, because formed necrotic proteins that cause toxicosis. With less time also receive the drug is of low quality.

After extraction BOD in saline solution proposed to stand the solution for 24 hours under these conditions, the maximum weight of the extraction, the extract solution outside the specified time dramatically changes the quality of the final product. In the process of aging are formed predecessors active proteins, which determine the specificity of the drug.

At this stage of the biosynthesis of a precipitate of insoluble proteins (tissue, plasma and other), they are separated, and the resulting solution was further diluted with saline to achieve the iza.

Adding to the obtained solution of the preservative in an amount not less than 0.05% is intended for antimicrobial, antifungal, etc., disinfection of the final product.

The formed suspension, representing proteins, membranes, structural proteins, lipid particles and other, separate, since the subsequent process of autolysis will apply to them, that adversely affects the quality of therapeutic agent.

The remaining solution was incubated for 48 hours. Experimentally proved, that this is enough time to complete the processes of aggregation of particles. The resulting solution is sterilized to remove viruses, bacteria, mantles, fungi, etc., Sterilization can be performed by any one adopted in this field by the method in accordance with who requirements.

The following actions are obtained by solution of the BOD are to autolysis. The temperature of the solution is carried out for not less than 3 days at a temperature of 36 2oC.

The timing of incubation at this temperature due to reaching the maximum specificity obtained a remedy.

The processes at this stage, can be opredelyaemye the drug is non-immunogenic.

Further keeping the solution within 30 - 40 days at a temperature not exceeding 15oC is determined by the following factors under these conditions, gently ends the process of autolysis with simultaneous aggregation of the formed particles that separate.

The remaining BOD solution is subjected to proteolysis by immobilized proteolytic enzyme, for example, on a column filled them.

The invention is illustrated by specific example.

To obtain therapeutic agents that regulate cell differentiation, embryonic take the muscle tissue of cattle. Selected raw materials are crushed, homogenized at a temperature not exceeding 8oC, the homogenate was incubated for 16 hours, diluted with chilled saline solution, then after extraction solution further maintained at a temperature of 4 - 8oC within 24 hours.

In the process of keeping a precipitate, which was separated by centrifugation at a speed of 3000 rpm. within 1 hour at a temperature of 2 to 4oC.

The resulting solution was diluted with saline to achieve a concentration of protein in it 21 mg/ml, is added dropwise a preservative which means passing the solution through a membrane filter with a pore diameter of 0.45 μm and incubated filtrate within 48 hours.

After that make a sterilization solution, for example, by passing it under aseptic conditions through a membrane filter with a pore size of 0.22 μm. Thus obtained a sterile solution hermetically closed and thermostatic for 3 to 4 days at a temperature of 36oC. After incubation the solution incubated for 35 days at a temperature not exceeding 15oC, the resulting suspension was separated by centrifugation, and the filtrate is optionally passed through a filter with pore size 0.45 µm.

The resulting solution BOD passed through the column (at a temperature of 25 - 30oC) filled with agarose 4B, the immobilized enzyme, such as trypsin.

The rate of elution solution BOD set on the basis of matching electrophoregram of the eluate with the control electrophoregrams BOD.

The resulting liquid aseptically sterilized by passing it through a membrane filter with a pore diameter of 0.22 μm, and then poured into ampoules and sealed.

The resulting tool is a transparent liquid with light yellow color without a smell, which can be mixed with water or saline solution in any proportion. the oC - 1,3410 of 0.0004, pH 6,5 - 7,0.

The drawing shows a control electrophoregram received the drug.

Thus, in the inventive method obtained a therapeutic agent that regulates the differentiation of cells ("Probes") that is formstate and which is widely tested on patients. The tool is recognized as highly effective in the treatment of diseases associated with impaired differentiation of cells.

The way to obtain therapeutic agents that regulate the differentiation of cells, which consists in grinding the embryonic tissues of the animal, their homogenization and extraction of physiological solution, separating the precipitate from the solution, characterized in that as embryonic tissues take the muscle tissue of the animal, the homogenate was incubated for 10 to 20 h, and after extraction, the treatment solution is performed within 24 0.5 h, then after separation of the resulting precipitate the resulting solution was further diluted with saline to achieve a concentration of protein in it 15 - 25 mg/ml and add to it the preservative in an amount not less than 0.05%, after separation of the resulting suspension solution was incubated for 48 h, female sterilization is the temperature, not exceeding 15oC, and remove the resulting suspension, and the solution is subjected to interaction with immobilized proteolytic enzyme, and the mode of the specified interaction set on the basis of a control sample obtained funds.

 

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