RU2180592C1 - Polypeptide preparation out of chicken bursa, method for its obtaining and pharmaceutical composition based upon this preparation - Google Patents

Abstract

FIELD: medicine, veterinary science. SUBSTANCE: the innovation includes three objects: preparing a polypeptide preparation (PPP), elaborating the method for its obtaining and pharmaceutical composition based upon this preparation. Composition is prepared in vials or ampoules which contain PPP at the quantity of 3-10 mg, D-mannitol 15-25 mg and 1 ml 0.9%-isotonic sodium chloride solution for injections. A project of the Temporary Pharmacopoeic Article (TPA) for a dry injection form of the composition mentioned is elaborated. EFFECT: widened assortment of preparations to be applied in case of immunodeficient states. 3 cl, 1 tbl, 3 ex

Description

 The invention relates to medicine, namely to the preparation of drugs used to treat primary and secondary immunodeficiencies. The invention can be used in the medical and veterinary industries for the preparation of a lyophilized injection form of a polypeptide preparation (PPP) complete with sterile saline or injection water.

 A known method of preparing an immunomodulatory polypeptide drug acting on the B-system of immunity, which differs from the proposed one, is that the preparation is obtained from bone marrow of pigs and substrates of marine animals by grinding bone marrow and substrate of marine animals, three times extraction in a 3% solution of trichloroacetic acid in the cold dialysis of the extract in distilled water, removal of ballasts from dialysate with ammonium sulfate, precipitation with cooled acetone for 24 hours, collecting the filter cake and e about air drying, dissolving the proteins with heated bidistillate, lyophilization of the solution, desalination and concentration of the drug by ultrafiltration on an Amicon-10 membrane, after which the filtrate was lyophilized and the powder was dissolved in ammonium acetate buffer, acidifying the solution with 0.1 M acetic acid solution until complete dissolution (1).

 There is also a method of producing an immunomodulating agent from pork breast bone, which differs from the proposed one in that the preparation is obtained from red bone marrow of the breast bone by culturing it in a special nutrient medium, followed by separation of cells by centrifugation, gel chromatography of supernatant on Sephadex G-50 to separate ballast impurities and lyophilization of active fractions (2).

 The disadvantages of the considered methods of obtaining immunomodulatory drugs include their complexity, as well as the difficulty in obtaining the source material (receiving and delivery of live bone marrow cells).

 The objective of the invention is to develop an immunomodulatory polypeptide preparation from chicken bursa, a simple and effective way to obtain this drug and pharmaceutical compositions based on it.

 This problem is solved by creating a preparation from chicken bursa by homogenizing the feedstock, extracting, heating the extract, separating on the membrane, a trapping substance with a molecular mass of more than 10 kD. The resulting ultrafiltrate contains a complex of thermostable peptides with a molecular weight of up to 10 kD and has immunostimulating properties with respect to the B-system of immunity.

 In addition, a method was developed for isolating PPP from chicken bursa — a practically deficient, unlimited raw material source that does not require laborious preparation — by grinding the bursa to a homogeneous mass, extracting the homogenate in 0.1 M aqueous acetic acid, removing oilcake, releasing the extract from thermolabile ballast proteins heating followed by centrifugation at 2000-4000 rpm and ultrafiltration on the membrane to remove proteins with a molecular mass exceeding 10 kD.

 The third object of the invention is the creation of a pharmaceutical composition based on the developed polypeptide preparation containing the following components: polypeptide preparation in an amount of 3-10 mg, D-mannitol - 15-25 mg and physiological saline (for example, an aqueous solution of sodium chloride 0.9%) - 1.0 ml

 The preparation of the pharmaceutical composition is carried out by diluting the ultrafiltrate to a peptide content of 3-10 mg / ml and adding D-mannitol to the diluted ultrafiltrate to its content in a solution of 15-25 mg / ml. To use the pharmaceutical composition, sterilizing filtration of the solution through membranes with a pore size of 0.22 μm is carried out and 1 ml sterile ultrafiltrate is poured into vials or ampoules, followed by lyophilization. In this way, a dry injection form of the composition is obtained, to which a physiological saline solution is applied for dissolution before use.

 The claimed invention is illustrated by the following examples.

 Example 1

1 kg of bursa chicken ground in a top is homogenized with 1 L of a 0.1 M solution of acetic acid for 3 minutes, then the homogenate is transferred to the extractor, 3 L of a 0.1 M solution of acetic acid is added to it and extraction is carried out with constant stirring. within 0.5 hours. After this time, the cake is separated through a double layer of gauze, and the extract is heated in a water bath to a temperature of 70 ^o C and kept at this temperature for 3 minutes, then the extract is cooled to a temperature of 15 ^o C. The coagulate formed from thermolabile proteins is removed by centrifugation at 2000 rpm and a temperature of 4 ^o C for 1 hour. The supernatant is filtered through a paper filter, then the clarified filtrate is passed through a membrane filter that traps peptides with a molecular weight of more than 10 kD. The ultrafiltrate is diluted with injection water to a solids content of 3 mg / ml, added to the diluted ultrafiltrate D (-) beckons at the rate of 17 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 with a capacity of 5 ml or in ampoules with a capacity of 2 ml. After the spill, the PPP is freeze-dried, the bottles are corked and sealed with aluminum caps, and the ampoules are sealed.

 Example 2

1 kg of bursa chicken ground in a top is homogenized with 1 L of a 0.1 M solution of acetic acid for 5 minutes, then the homogenate is transferred to the extractor, 3 L of a 0.1 M solution of acetic acid is added to it and extraction is carried out with constant stirring. within 1 hour. After this time, the cake is separated through a double layer of gauze, and the extract is heated in a water bath to a temperature of 90 ^o C and kept at this temperature for 3 minutes, then the extract is cooled to a temperature of 20 ^o C. The coagulate formed from thermolabile proteins is removed by centrifugation at 4000 rpm and a temperature of 10 ^o C for 1 hour. The supernatant is filtered through a paper filter, then the clarified filtrate is passed through a membrane filter that traps peptides with a molecular weight of more than 10 kD. The ultrafiltrate is diluted with injection water to a solids content of 10 mg / ml, added to the diluted ultrafiltrate D (-) beckons at a rate 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 with a capacity of 5 ml or ampoules with a capacity of 2 ml. After the spill, the PPP is freeze-dried, the bottles are corked and sealed with aluminum caps, and the ampoules are sealed.

 Example 3

 The stimulation index (IS) is the ratio of the average value of the splenic follicles of experimental animals that received the pharmaceutical composition to this indicator in mice of the control cyclophosphamide group. The method is based on the ability of the pharmaceutical composition of chicken bursa to increase the size of the lymphatic follicles of the spleen in mice with immunosuppression caused by cyclophosphamide.

 The method includes three stages: the first stage - suppression of the immune system of same-sex, non-linear mice with a body weight of 18-20 g of cyclophosphamide; the second stage is the stimulation of the experimental group of mice with the test composition; the third stage is the determination of the size of lymphatic follicles in the spleen of experimental and control mice.

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

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

 Three days after the administration of the pharmaceutical composition, control and experimental mice were killed and the spleen was removed.

 At the third stage of determining the index of stimulation of the B-system of immunity, histological preparations are prepared from the extracted spleen using known methods and the sizes of lymphatic follicles are determined in them. At the same time, a value of at least 10 follicles is measured on two sections obtained from each animal in this group. According to the measurement results, the average size of the lymphatic follicles is found for each group of animals.

 The activity of the pharmaceutical composition is determined by the ratio of the average value of the splenic follicles of animals of each experimental group to this indicator in mice of the control cyclophosphamide group.

 The results of the analysis of the pharmaceutical composition obtained from chicken bursa for compliance with the draft provisional pharmacopoeial article (VFS) are presented in the table.

 The claimed development will provide healthcare and veterinary medicine with a new effective and inexpensive domestic tool to stimulate the B-system of immunity.

LITERATURE
1. Patent Russia 1077089, cl. MKI A 61 K 35/28 09/29/95 (Morozov V.G., Khavinson V.Kh., Sidorova).

 2. USSR patent 1836954, cl. MKI A 61 K 35/28 09/30/93 (Vladivostok State Medical Institute).

Claims (3)

 1. A polypeptide preparation from chicken bursa obtained by homogenizing the feedstock, extracting, heating the extract, removing ballast thermolabile proteins, removing proteins with a molecular weight of more than 10 kD, the resulting product contains a complex of thermostable peptides with a molecular weight of up to 10 kD and has immunostimulating properties in regarding the B-system of immunity.  2. A method of producing a polypeptide preparation from chicken bursa, including crushing the bursa to a homogeneous mass, extracting the homogenate with aqueous acetic acid, removing oilcake, releasing the extract from heat-sensitive ballast proteins by heating, followed by centrifugation at 2000-4000 rpm, ultrafiltration to remove proteins from molecular weighing more than 10 kD. 3. A pharmaceutical composition based on the preparation described in paragraph 1, containing the following components:
Polypeptide preparation - 3-10 mg
D-beckons - 15-25 mg
0.9% Sodium Chloride Solution - 1.0 ml

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