The polypeptide product of a bursa of chickens, method thereof and pharmaceutical composition based on it

 

(57) Abstract:

The invention relates to medicine, namely to obtain drugs used for the treatment of primary and secondary immunodeficiencies. The invention can be used in medical and veterinary industry for the preparation of freeze-dried injectable form polypeptide drug (SPT) complete with sterile saline solution or injection water. The invention includes three objects, namely the preparation of the SPT, the development of the method of its production and pharmaceutical compositions based on it. The composition is prepared in vials or ampoules containing the SPT in the amount of 3-10 mg, D-beckons - 15-25 mg and 1 ml of isotonic sodium chloride 0.9% for injection. Developed a draft interim monograph (VFS) for dry injectable form of the composition. The technical result - expanding Arsenal of tools used in immunodeficiency compilations. 3 S. p. f-crystals, 1 table.

The invention relates to medicine, namely to obtain drugs used for the treatment of primary and secondary immunodeficiencies. The invention can be used in medical and veterinary industry who inim saline solution or injection water.

A known method of preparation of immunomodulatory polypeptide drug, acting In the immune system, which differs from the one suggested by the fact that receive the drug from the bone marrow pigs and substrates of marine animals by crushing the bone marrow and substrate of marine animals, triple extraction in 3% solution of trichloroacetic acid in the cold, dialysis of the extract in distilled water, removal of ballasts from dialysate ammonium sulfate, precipitation with chilled acetone for 24 hours, collect the precipitate on the filter and drying in air, the proteins are dissolved warm bidistillate, lyophilization solution, desalting and concentration of the preparation by ultrafiltration on a membrane Amicon-10", after which the filtrate lyophilizer and powder dissolved in acetate-ammonium buffer, podkisst a solution of 0.1 M solution of acetic acid until complete dissolution (1).

Also known is a method of obtaining immune-modulating means pork breast bone, different from those that receive the drug from the red bone marrow of the sternum by cultivation in a special nutrient medium, followed by the separation of cells by centrifugation, the active fractions (2).

The disadvantages of the considered ways of receiving immunomodulatory drugs should include their complexity and the difficulty in obtaining source material (the receipt and delivery of live bone marrow cells).

The objective of the invention is the development of immune-modulating polypeptide preparation of Bursa of chickens, a simple and effective way of getting this drug and pharmaceutical compositions based on it.

This problem is solved by the creation of the drug from the Bursa of chickens by homogenization of raw materials, extraction, heating the extract separation on the membrane, inhibiting substance with a molecular mass of more than 10 KD. The resulting ultrafiltrate contains a set of thermostable peptides with molecular weight of 10 KD and immune-boosting properties In the immune system.

In addition, the method of allocation Subcommittee of the Bursa of chickens is almost balanced, unlimited raw material source that does not require time-consuming preparation by grinding Bursa to a homogeneous mass, extracting the homogenate in 0.1 M aqueous acetic acid, removal of the cake liberation extract from thermolabile ballast proteins the proteins with molecular weight, greater than 10 KD.

The third object of the invention is to provide pharmaceutical compositions based on a polypeptide preparation that contains the following components: polypeptide drug in a number of 3-10 mg, D-beckons - 15-25 mg and saline solution (e.g. aqueous solution of sodium chloride at 0.9% to 1.0 ml.

Preparation of pharmaceutical compositions is carried out by dilution ultrafiltrate to content peptides 3-10 mg/ml and added to the diluted ultrafiltrate D-Manita to its content in the solution 15-25 mg/ml For use in the pharmaceutical compositions spend sterilizing filtration of the solution through a membrane with pore size of 0.22 μm and filling sterile ultrafiltrate 1 ml vials or ampoules, followed by lyophilization. In this way receive dry injectable form of composition, which make saline solution to dissolve before use.

The claimed invention is illustrated by the following examples.

Example 1.

1 kg of Bursa of chickens, chopped the top, homogenized with 1 l of 0.1 M solution of acetic acid for 3 minutes, then the homogenate was transferred to the extractor, add On after a specified time through a double layer of cheesecloth to separate the cake, and the extract is heated in a water bath to a temperature of 70oWith and maintain it at this temperature for 3 minutes, then the extract is cooled to a temperature of 15oC. Formed from thermolabile proteins coagulate removed by centrifugation at 2000 rpm and a temperature of 4oC for 1 hour. The supernatant is filtered through a paper filter, then the clarified filtrate is passed through a membrane filter, inhibiting peptides with a molecular mass of more than 10 KD. Ultrafiltrate injecting dilute with water until the solids content of 3 mg/ml, add the diluted ultrafiltrate D (-) beckons from the calculation of 17 mg / 1 ml, then the solution is filtered through sterilizing filters with a pore diameter of 0.22 μm and poured into 1 ml in vials or 5 ml vials with a capacity of 2 ml of the spill SPT freeze-dried vials sealed plugs and running aluminum caps, and sealed ampoules.

Example 2.

1 kg of Bursa of chickens, chopped the top, homogenized with 1 l of 0.1 M solution of acetic acid for 5 minutes, then the homogenate was transferred to the extractor, add in 3 l of 0.1 M solution of acetic acid with constant stirring, the extraction is carried out in t in a water bath to a temperature of 90oWith and maintain it at this temperature for 3 minutes, then the extract is cooled to a temperature of 20oC. Formed from thermolabile proteins coagulate removed by centrifugation at 4000 rpm and a temperature of 10oC for 1 hour. The supernatant is filtered through a paper filter, then the clarified filtrate is passed through a membrane filter, inhibiting peptides with a molecular mass of more than 10 KD. Ultrafiltrate was diluted with injectable water to a solids content of 10 mg/ml, add the diluted ultrafiltrate D (-) beckons from the calculation of 23 mg per 1 ml, then the solution is filtered through sterilizing filters with a pore diameter of 0.22 μm and poured into 1 ml vials of 5 ml or ampoule with a capacity of 2 ml of the spill SPT freeze-dried vials sealed plugs and running aluminum caps, and sealed ampoules.

Example 3.

The stimulation index (IP) is the ratio of the average value of the splenic follicles experimental animals that received the pharmaceutical composition to that in mice ciclofosfamida group. The method is based on the ability of the pharmaceutical compositions of the Bursa of chickens to increase the size of liuche three stages: the first stage - suppression of the immune system sex nonlinear mice weighing 18-20 g cyclophosphamide; the second stage is the stimulation of the experimental group of mice tested composition; the third step is determining the size of the lymph follicles in the spleen experimental and control mice.

In the first stage, all mice vnutribryushnoe once injected with cyclophosphamide at a dose of 4 mg in 0.5 ml of isotonic 0.9% sodium chloride for injection. Then the mice are divided into 3 groups, one control and two experimental.

In the second stage, the first experimental group of mice subcutaneously injected pharmaceutical composition at a dose of 5 μg/mouse, contained in 0.2 ml of isotonic 0.9% sodium chloride for injection, the second experimental group of mice under the same conditions impose pharmaceutical composition at a dose of 10 μg/mouse.

Three days after administration of the pharmaceutical composition of control and experimental mice are killed and extracted from the spleen.

In the third step of determining the stimulation index In the immune system extracted from spleen known methods prepare histological specimens and identify them in the sizes of the lymph follicles. This measures the value not less than 10 folliculitis size of lymph follicles for each group of animals.

The activity of the pharmaceutical composition defined relative average size of the splenic follicles of animals in each experimental group to that in mice ciclofosfamida group.

The results of the analysis of the pharmaceutical compositions obtained from the Bursa of chickens, for compliance with the draft interim monograph (VFS) are presented in the table.

Stated the development will provide health and veterinary medicine new efficient and cost-effective domestic remedy to stimulate the immune system.

LITERATURE

1. Patent of Russia 1077089, CL MKI And 61 To 35/28 29.09.95 (Morozov Century BC, Khavinson C. H., Sidorova).

2. Patent USSR 1836954, CL MKI And 61 To 35/28 30.09.93 (Vladivostok state medical Institute).

1. The polypeptide product of a Bursa of chickens, obtained by homogenization of raw materials, extraction, heating the extract, remove ballast thermolabile proteins, removal of proteins with a molecular mass of more than 10 KD, the resulting product contains a complex of thermostable peptides with molecular weight of 10 KD and immune-boosting properties In the immune system.

2. JV is reagirovanie homogenate aqueous acetic acid, remove cake release extract from thermolabile ballast proteins by heating, followed by centrifugation at 2000-4000 rpm, ultrafiltration for removal of proteins with a molecular mass of more than 10 KD.

3. The pharmaceutical composition on the basis of preparation described in paragraph 1 with the following components:

Polypeptide drug - 3-10 mg

D-beckons - 15-25 mg

A solution of sodium chloride at 0.9% - 1.0 ml

 

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FIELD: medicine, pediatrics.

SUBSTANCE: the present innovation deals with treating motor-autonomic disorders in children associated with affected function of central nervous system. For this purpose one should puncture perineural areas in the region of the main nervous trunks with alfetin dissolved in cerebrolysine. Additionally, one should puncture in projection area of cervical and lumbar spinal thickenings and areas that correspond to segmentary innervation of organs with affected function and, also, in scalp areas depending upon the character of patient's disorders. The method suggested provides improved autonomic-trophic impact of nervous system.

EFFECT: higher efficiency of therapy.

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