RU2332423C2 - Method of preparation of thymic peptide - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The method of thymic peptide prodaction involves fining of product of mammals' immunotropic tissue containing peptides with thymic activity, homogenisation, centrifugation and filtration with refining and segregating of the final product. Three times freezing and thawing of the tissue in the mixture with distilled water are carried out before homogenisation. Hereafter, centrifugation of the mixture and filtration of supernatant fluid with deriving filtrate which is precipitated by 0,1-0,6M sulphosalicylic acid, than protein fraction with relative molecular mass less than 10 kDa is segregated from the filtrate. Supernatant fluid is segregated with 0,33% phosphorno-wolframic acid, aging within 60 minutes, centrifuged and precipitation, which is suspended in distilled water is separated. Hereafter, the sediment is dissolved by adding saturated alkaline solution with up to 9,0 pH, the final solution is dialysed, defecated by centrifuging and supernatant fluid is separated. Centrifugation is carried out at 8000 rpm during 30 minutes, supernatant fluid is exposed to absorption chromatography on basis of hyrdoxylapatite in water system, notably, and protein fraction, which is not absorbed on a column is concentrated. The protein fraction is exposed to gel filtration, and it's selected with molecular mass less than 10 kDa.

EFFECT: production of thymic peptide with high specific activity and pureness.

3 cl, 6 ex

Description

The invention relates to the field of production of immunologically active biologically active additives in the form of protein-peptide preparations (preparations of thymic peptides).

There is a method of obtaining from a thymus of calves a mixture of immunologically active substances (called "Timosin"), which consists in homogenizing thymus tissue in a 0.15 M sodium chloride solution, centrifuging at 14,000 g without preliminary incubation of the homogenate, heating the supernatant to 80 ° C, removing thermodenaturing proteins, precipitation with acetone of immunologically active substances, salting out with ammonium sulfate, ultrafiltration, chromatography on a column with Sephadex G-25 and isolation of the final product "Timosin" mol. m. from 1200 to 14000 Yes.

However, this method is time-consuming, expensive and does not differ in environmental cleanliness, because requires the use of a large amount of acetone - a 5-fold volume of acetone relative to the volume of the homogenate (see Goldestein AL, Thurman GB, Conen GH, Hooper JA (Thymosin: chemistry, biology and clinicak applications) In: Biological activity of thymic hormones. Ed. by DW Van Bekkum. Rotterdam - 1975, p. 235-245).

A known method of obtaining an immunologically active preparation from the thymus of calves "Tactivin", which consists in the homogenization of thymus tissue in a 0.15 M solution of sodium chloride in a ratio of 1: 3, incubation of the homogenate for 16 hours at + 4 ° C (stage of autolytic extraction), centrifugation for 1 h at 2000g, filtering, heating to 80 ° C for 15 min, removing denaturing proteins at 2000 g for 1 h by centrifugation, precipitation with acetone, centrifugation at 2000 g for 30 min, washing with acetone, drying, dissolving the powder in 10 m sodium phosphate buffer pH 7.0, removing insoluble precipitate by centrifugation at 30,000 g for 40 minutes, salting out with ammonium sulfate, centrifuging at 30,000 g for 40 minutes, acidifying with acetic acid to pH 4.0 (three times salting out with ammonium sulfate at various values pH), ultrafiltration, desalination on Sephadex G-15, chromatography on Sephadex G-50, isolation of the fraction with a mol. m. 1000-6000 Yes, desalting it on Sephadex G-15, lyophilization. The result is a final product that is active in a quantitative test at a minimum dose of 10 μg (concentration 10 μg / ml) for 3 · 10 ⁶ spleen cells of thimectomized mice. The yield of the target product (protein-peptide fraction) from 1 kg of thymus - 100 mg per mol. m. 1000-6000 daltons, i.e. 0.01% / cm. Arion V.Ya. "Isolation, physico-chemical properties and biological activity of T-activin." In the book. "Results of science and technology" Ser. Immunology. - M .: VINITI, 1982, v.10, p. 45-53 /.

This method is not environmentally friendly, because it requires the use of a large amount of acetone to precipitate and wash the preparation; it is long, laborious, the high cost of the target product is not provided, a high yield of active material is not ensured (up to 0.01% of the initial thymus weight).

The closest way to the claimed invention in terms of features is a method for producing immunostimulatory polypeptides from raw materials of animal origin, in which the reindeer thymus is homogenized in a sodium chloride solution, autolysed, centrifuged, separated and heated the supernatant to 80 ° C, cooled, removed sediment, treated with acetone, formed a precipitate, washed three times with acetone, dried, followed by dissolution in 0.01 M Tris-HCl buffer, centrifuged, ultrafil radio, chromatography on Sephadex followed by collection of the fractions containing the desired product (protein-peptide fraction), lyophilized, subjected to gel filtration, re-lyophilized (SU 1737798, 1989).

The disadvantages of this method are the low specific activity and purity of the resulting preparation, the complexity and high cost of the product preparation process, the narrowness of the raw material base due to the limited range of suitable starting materials.

An object of the invention is to provide an effective method for producing thymic peptides (representing a protein-peptide fraction) from mammalian immunotropic tissue containing peptides with thymic activity from the group: thymus, spleen, skin, and expanding the arsenal of methods for producing thymic peptides.

The technical result, which provides a solution to the problem, is to ensure high specific activity and purity of the preparation obtained, simplify and reduce the cost of the product preparation process, expand the raw material base through the use of various starting materials for medical and cosmetic needs.

The essence of the invention lies in the fact that the method of producing thymic peptides involves grinding immunotropic mammalian tissue containing peptides with thymic activity from the group: thymus, spleen, skin, homogenization, extraction with centrifugation, purification and isolation of the protein-peptide fraction as the target product, characterized the fact that before homogenization produce three times freezing and thawing of the drug in a mixture with distilled water in a mass ratio of 1: 3, homogenization, price trifuging this mixture and filtering the supernatant obtained by precipitation of this mixture to obtain an extract, a protein fraction soluble in 0.1-0.6 M sulfosalicylic acid is isolated from the latter and centrifuged again, separating the supernatant, which is precipitated at 0.33% phosphoric-tungsten acid, kept at room temperature, decanted, centrifuged and the precipitate was separated, which was suspended in distilled water, then dissolved by adding a saturated alkaline solution, and the resulting solution was di Alize, clarify by centrifugation and separate the supernatant, which is subjected to adsorption chromatography on hydroxylappatite in an aqueous system, and the protein fraction that is not adsorbed on the column is concentrated and subjected to gel filtration, the target product is isolated and selected as a protein-peptide fraction with a relative molecular weight less than 10 kDa and lyophilized.

Preferably, the supernatant after precipitation at 0.33% tungsten-phosphoric acid is kept at room temperature for 60 minutes, the precipitate is suspended in a minimum amount of distilled water, and then dissolved, adding a saturated alkaline solution dropwise with stirring and controlling the clarification of the solution at pH-9.0, the resulting solution is dialyzed against running water for 3 days and 2 days of distilled water in the refrigerator, centrifugation is carried out at 8000 rpm for 30 m chickpeas, and the collected protein-peptide fraction was desalted standardized by protein concentration and freeze-dried.

A method of producing thymic peptides is implemented as follows.

The purified immunotropic tissue of mammals (mainly farm animals) containing peptides with thymic activity from the group: thymus, spleen, skin, is ground in a meat grinder to the consistency of minced meat. The resulting minced meat is mixed with distilled water in a ratio of 1: 3, frozen three times and thawed.

The mass is then homogenized and centrifuged at 8000 rpm for 30 minutes. The supernatant is filtered through 4 layers of gauze to obtain an extract.

The extract was precipitated at 0.1-0.6 M sulfosalicylic acid, kept at room temperature for 60 minutes and centrifuged again at 8000 rpm for 30 minutes, separating the supernatant.

The supernatant is precipitated at 0.33% tungsten phosphoric acid (i.e., preferably at 0.33% dry tungsten phosphoric acid powder by weight of the liquid), the mixture is kept at room temperature for 60 minutes, decanted (the liquid is drained from the settled sediment) and once again centrifuged at 8000 rpm for 30 minutes.

The precipitate is suspended in a minimal amount of distilled water, after which the suspension is dissolved by stirring on a magnetic stirrer, and a saturated alkaline NaOH solution is added dropwise, while controlling the acidity (pH) of the solution. The precipitate will dissolve at pH 9.0 (i.e. the solution should brighten at pH-9.0).

The resulting solution was dialyzed (desalted) against running water for 3 days and 2 days against distilled water in the refrigerator, clarified by centrifugation at 8000 rpm for 30 minutes, separating the supernatant.

The supernatant is subjected to hydroxylappatite adsorption chromatography in an aqueous system. The protein fraction not adsorbed onto the column is concentrated.

The obtained protein-peptide fraction is subjected to gel filtration on Sephadex G-50, a protein fraction with a relative molecular weight of less than 10 kDa is selected and lyophilized.

Gel filtration (in all examples) is carried out on a Sephadex G-50 medium (manufactured in Sweden), the exclusion limit of which is 10 kDa (ZAO Amersham Biosciences) under the conditions recommended by the manufacturer. For gel filtration, a 50 × 600 (mm) column of Pharmacia fine chemicals (US) was used in a 0.05 M sodium phosphate buffer system (pH 7.4) with the addition of 0.1 M NaCl.

Sephadex G-50 is a reagent (powder-gel), the apparatus is a column and everything necessary for gel filtration (collector, uvikord, peristaltic pump).

The molecular weight of the obtained protein-peptide fraction is evaluated (in all examples) by the widely used method of electrophoresis in polyacrylamide gel with the addition of sodium dodecyl sulfate and beta-mercaptoethanol (Shapiro AL et al. "Molecule weight measurement of polypeptide chains by electrophoresis in SDS-polyacrilamide gels Biochem. Biophys. Res. Commun., 1967, 28, pp. 815-820). With polyacrylamide gel electrophoresis, the use of dodecyl sulfate and beta-mercaptoethanol is mandatory.

The collected protein-peptide fraction is desalted on Sephadex G-10 and freeze-dried.

The technical result is achieved due to the deposition of inactive components by sulfosalicylic acid and adsorption on hydroxylappatite, and acetone is completely excluded, because the latter is not used when implementing the method. Obtained by the claimed method, the drug has a mol. m. less than 10000 Yes, and the isoelectric point of pH 4.5-6.5, contains 4 main components (colored protein-peptide zones).

Getting the drug is demonstrated by examples.

Example 1

Take 1000 g of calf thymus tissue.

Evenly chopped in a meat grinder to the consistency of minced meat. The resulting minced meat is mixed with distilled water in a ratio of 1: 3, frozen three times and thawed.

The resulting mass is homogenized, centrifuged at 8000 rpm for 30 minutes, the precipitate liquid is separated and filtered through 4 layers of gauze, thus obtaining an extract.

The extract was precipitated at 0.1 M sulfosalicylic acid, kept at room temperature for 60 minutes and centrifuged again at 8000 rpm for 30 minutes, separating the supernatant.

The supernatant was precipitated at 0.33% phosphoric tungsten acid, kept at room temperature for 60 minutes, decanted (the liquid was drained from the settled precipitate) and centrifuged again at 8000 rpm for 30 minutes.

The precipitate is suspended in a minimal amount of distilled water, and then dissolved on a magnetic stirrer, adding a saturated NaOH solution dropwise, while controlling the acidity (pH) of the solution. At pH 9.0, bleaching of suspension is observed.

The resulting solution was dialyzed (desalted) against running water for 3 days and 2 days against distilled water in the refrigerator, clarified by centrifugation at 8000 rpm for 30 minutes, separating the supernatant.

The supernatant is subjected to hydroxylappatite adsorption chromatography in an aqueous system. The protein fraction not adsorbed onto the column was concentrated.

The obtained protein fraction is subjected to gel filtration on Sephadex G-50, a protein-peptide fraction with a relative molecular weight of less than 10 kDa is selected and lyophilized.

The collected protein-peptide fraction is desalted on Sephadex G-10 and freeze-dried. Get 80 mg of the drug.

Example 2

Take 1000 g of thymus seal tissue.

Evenly chopped in a meat grinder to the consistency of minced meat. The resulting minced meat is mixed with distilled water in a ratio of 1: 3, frozen three times and thawed.

The mass is then homogenized, centrifuged at 8000 rpm for 30 minutes, and the supernatant is filtered through 4 layers of gauze to obtain an extract.

The extract was precipitated by the addition of 0.25 M sulfosalicylic acid, kept at room temperature for 60 minutes and centrifuged again at 8000 rpm for 30 minutes, separating the supernatant.

The supernatant was precipitated at 0.33% tungsten phosphoric acid, kept at room temperature for 60 minutes, decanted and centrifuged again at 8000 rpm for 30 minutes.

The precipitate is suspended in a minimal amount of distilled water, after which it is solubilized on a magnetic stirrer, adding a saturated NaOH solution dropwise, while controlling the acidity (pH) of the solution. At pH 9.0, the suspension brightens, i.e. dissolves.

The resulting solution was dialyzed against running water for 3 days and 2 days of distilled water in the refrigerator, clarified by centrifugation at 8000 rpm for 30 minutes, obtaining a supernatant.

The supernatant is subjected to hydroxylappatite adsorption chromatography in an aqueous system. The protein fraction not adsorbed onto the column is concentrated.

The obtained protein fraction is subjected to gel filtration on Sephadex G-50, a protein-peptide fraction with a relative molecular weight of less than 10 kDa is selected and lyophilized.

The collected protein-peptide fraction is desalted on Sephadex G-10 and freeze-dried. Get 76 mg of the drug.

Example 3

Take 1000 g of tissue of the spleen of cattle.

Evenly chopped in a meat grinder to the consistency of minced meat. The resulting minced meat is mixed with distilled water in a ratio of 1: 3, frozen three times and thawed.

The mass is then homogenized, for example, centrifuged at 8000 rpm for 30 minutes and the supernatant is filtered through 4 layers of gauze to obtain an extract.

The extract was precipitated at 0.45 M sulfosalicylic acid, kept at room temperature for 60 minutes and centrifuged at 8000 rpm for 30 minutes.

The supernatant was precipitated by the addition of 0.33% tungsten phosphoric acid, kept at room temperature for 60 minutes, decanted and centrifuged at 8000 rpm for 30 minutes.

The precipitate is suspended in a minimal amount of distilled water, and then dissolved on a magnetic stirrer, adding a saturated NaOH solution dropwise, while controlling the pH of the solution. At pH 9.0, the suspension is solubilized, which is characterized by a clear solution.

The resulting solution was dialyzed against running water for 3 days and 2 days of distilled water in the refrigerator, clarified by centrifugation at 8000 rpm for 30 minutes.

The supernatant is subjected to hydroxylappatite adsorption chromatography in an aqueous system. The protein fraction not adsorbed onto the column is concentrated.

The obtained protein fraction is subjected to gel filtration on Sephadex G-50, a protein-peptide fraction with a relative molecular weight of less than 10 kDa is selected and lyophilized.

The collected protein-peptide fraction is desalted on Sephadex G-10 and freeze-dried. Get 86 mg of the drug.

Example 4

Take 1000 g of pig skin tissue.

Evenly chopped with scissors and then in a meat grinder to the consistency of minced meat. The resulting minced meat is mixed with distilled water in a ratio of 1: 3, frozen three times and thawed.

After that, the tissue suspension is homogenized and centrifuged at 8000 rpm and the supernatant is filtered through 4 layers of gauze to obtain an extract.

The extract was precipitated at 0.6 M sulfosalicylic acid, kept at room temperature for 60 minutes and centrifuged again at 8000 rpm for 30 minutes, separating the supernatant.

The supernatant was precipitated at 0.33% tungsten phosphoric acid, kept at room temperature for 60 minutes, decanted and centrifuged at 8000 rpm for 30 minutes.

The resulting precipitate was suspended in a minimal amount of distilled water, and then dissolved on a magnetic stirrer, adding a saturated NaOH solution dropwise, while controlling the pH of the solution. The suspension dissolves at pH 9.0, that is, it should brighten.

The resulting solution was dialyzed against running water for 3 days and 2 days against distilled water in the refrigerator, after which it was clarified by centrifugation at 8000 rpm for 30 minutes to obtain a supernatant.

The supernatant is subjected to hydroxylappatite adsorption chromatography in an aqueous system. The protein fraction not adsorbed onto the column is concentrated.

The obtained protein fraction is subjected to gel filtration on Sephadex G-50, a protein-peptide fraction with a relative molecular weight of less than 10 kDa is selected, and lyophilized.

The collected protein-peptide fraction is desalted on Sephadex G-10 and freeze-dried. Get 62 mg of the drug.

The study of the preparations obtained was carried out in accordance with the section "Methodological guidelines for the study of the immunotropic activity of pharmacological substances" (authors R.M.Khaitov, I.S. In the book: “Guidelines for the experimental (preclinical) study of new pharmacological substances.” - M., 2000, p. 257-263).

The obtained preparations were tested in in vivo experiments on laboratory animals - male mice of interline hybrids (CBA × C57BL6) F ₁ aged 10-12 weeks with an average body weight of 18-20 g, obtained from Stolbovaya nursery. In each study, the group consisted of 10-12 mice.

The drug was tested for biological activity by two methods:

1. The state of the humoral immune response was determined — the number of antibody-forming spleen cells of CBA mice for the primary immune response (Erne method)

After the administration of the preparations obtained according to examples 1-4, four groups of mice, these mice, as well as intact animals were immunized intraperitoneally with sheep erythrocytes (EB) at a dose of 5 × 10 ⁶ contained in 0.5 ml. As a positive and technical control, the drug TACTIVINA (Biomed named after II Mechnikov) was used at a concentration of 100 μg / ml. Test samples were used at similar concentrations. On day 4, the amount of AOK was determined by the method of local hemolysis in an agarose gel in mouse spleens. In spleen cell suspensions, the number of nucleated cells (YSC) was counted. The results were expressed as the average number of AOKs in the spleens of mice of each group, as the average number of AOKs per 1 million YAWs, and as an index of modulation of the immune response compared to intact but immunized EB mice.

A significant increase in the content of AOK on the spleen was established in mice of the experimental groups. The amount of AOK in these groups is 1096 and 1185, respectively (p <0.05). While in the control group this indicator is 697.

2. We tested the restoration of the sensitivity of the background rosette-forming cells of the spleen of thimectomized mice and the inhibitory effect of azathioprine (Bach method)

As a control, the pharmacopoeial preparation TACTIVINA (Biomed named after II Mechnikov) was used at a concentration of 100 μg / ml. Test samples were used at similar concentrations.

A CBA mouse was immunized with 0.2 ml of a 15% suspension of sheep erythrocytes intraperitoneally; on day 5, the animal was sacrificed by cervical dislocation of the cervical vertebrae, a spleen was isolated, and a splenocyte suspension was prepared. In the control group, 10 μl of physiological saline was added to 100 μl of cell suspension samples; in the experimental group, 10 μl of the test specimen obtained from calf thymus diluted in physiological saline at a concentration of 10 ^-5 g / ml was added to 100 μl of samples samples were incubated for 10-20 min at a temperature of 36.5-37.0 ° C. Next, a reaction was carried out to determine the number of rosette-forming cells. In the control group, the number of rosette-forming cells was at the level of (43.65 ± 2.21) × 10 ⁶ / organ (on average 45%). Preincubation of spleen cells with the studied drug at a concentration of 10 ^-5 g / ml led to an increase in the number of rosetting cells to (32.30 ± 3.11) × 10 ⁶ / organ (31% on average). The data obtained indicate the presence of high immunotropic activity in this drug.

A comparison of the activity of immunotropic drugs of the prototype and the proposed method for the effect on the B-system of immunity in the Erne test and the T-system of immunity in the test to restore the sensitivity of background rosette-forming cells of the spleen of thimectomized mice to the inhibitory effect of azatiprone. The comparison showed that the immunotropic products obtained by the present method are active in both methods: Yerne and azathioprine, while the substances obtained by the prototype are active only in the Yerne method. The immunotropic substance obtained by the prototype has a maximum activity at a dose of 8-10 μg / mouse, while the preparations obtained by the present method, at a dose of 0.8-1 μg / mouse.

As a result of the invention, an effective method for producing thymic peptides from immunotropic mammalian tissue containing peptides with thymic activity from the group: thymus, spleen, skin, and the arsenal of methods for producing thymic peptides is expanded.

At the same time, high specific activity and purity of the resulting preparation, simplification and cheapening of the product preparation process were ensured, the raw material base was expanded through the use of various starting materials for medical and cosmetic needs.

Claims (3)

1. A method of producing thymic peptides, comprising crushing immunotropic mammalian tissue containing peptides with thymic activity, from the group: thymus, spleen, skin, homogenization, centrifugation and filtration, followed by purification and isolation of the target product, characterized in that they are thrice frozen before homogenization and thawing the fabric in a mixture with distilled water in a mass ratio of 1: 3, after homogenization, centrifuging the mixture at 8000 rpm for 30 min and filtration of the supernatant to obtain a filtrate, which is precipitated with 0.1-0.6 M sulfosalicylic acid and the protein fraction is separated from the filtrate, incubated for 30 minutes, then centrifuged again at 8000 rpm for 30 minutes, separating the supernatant, which precipitated with 0.33% tungsten phosphoric acid, held for 60 minutes, decanted and centrifuged at 8000 rpm for 30 minutes, the precipitate was separated, which was suspended in distilled water, then dissolved by adding a saturated alkaline solution to pH 9.0 , but received the solution is dialyzed, clarified by centrifugation at 8000 rpm for 30 min, the resulting supernatant is subjected to adsorption chromatography on hydroxyappatite in an aqueous system, the protein fraction not adsorbing on the column is concentrated by gel filtration, selecting a protein fraction with a molecular weight of less than 10 kDa followed by, if necessary, lyophilization. 2. The method according to claim 1, characterized in that the resulting solution is dialyzed against running water for 3 days and 2 days of distilled water in the refrigerator. 3. The method according to any one of claims 1 and 2, characterized in that the collected protein-peptide fraction is desalted, standardized for protein concentration and freeze-dried.