RU2203070C2 - Method for obtaining immunotropic substances out of swine skin - Google Patents

Abstract

FIELD: immunology, immunopharmacology, medicinal biotechnology, pharmaceutics. SUBSTANCE: method deals with reduction, homogenization of skin, centrifuging, thermodenaturation of supernatant, centrifuging of denaturated material, treatment of supernatant with cooled acetone, washing out of residue with cooled acetone, drying, dissolving of residue and centrifuging, followed by chromatography at column with Sephadex G-50 and collection of fraction of target product at 1400-15000 Da molecular weight, desalinization and lyophilization, where centrifuging of dissolved residue is carried out at 8000-10000 g for 10-20 min at +4-6 C, one should add dropwise saturated solution of ammonium sulfate to supernatant at 2:1-3:1 ratio, centrifuge at 8000-10000 g for 40- 50 min at 4-6 C to achieve pH of obtained supernatant to be 3.5- 4.5 due to adding dropwise 8-10%-acetic acid solution, then dry ammonium sulfate should be added at 14-15 g/100 ml supernatant, centrifuged at 10000-12000 g for 20-30 min at 4-6 C, residue should be homogenized in 0.01-0.02 M tris HCl pH 7.5-8.5, then one should add dropwise saturated solution of ammonium sulfate to homogenate at 1:1-1.5:1 ratio, centrifuge at 16000-18000 g for 20-30 min at 4-6 C, residue is homogenized in 30-50 ml 0.01-0.02 M tris-HCl + 0.14-0.15 M NaCl pH 7.5-8.5, centrifuged at 16000-18000 g for 20-30 min at 4-6 C, and at chromatography upon column with Sephadex G-50 one should simultaneously collect fraction at molecular weight being above 15000 Da. The present innovation enables to increase activity of obtained immunotropic substances and obtain substances affecting both B- and T-systems of immunity. EFFECT: higher efficiency. 3 tbl

Description

 The invention relates to immunology, immunopharmacology, medical biotechnology and the pharmaceutical industry. Immunotropic substances from the skin after appropriate tests can be used as drugs for immunocorrection as immunomodulators.

 A known method of producing thymopoietin from embryonic skin of cattle by grinding it in a solution of ammonium bicarbonate, centrifugation, ultrafiltration, centrifugation, gel chromatography, hydrophobic chromatography, anion exchange chromatography in a system of high performance liquid chromatography, desalination and lyophilization [1]. However, the production process is technologically quite complicated, the yield of the drug is negligible, the separation stage by high performance liquid chromatography is difficult to apply in the industrial preparation of the drug. The immunotropic substance thymopoietin, obtained from the embryonic skin of cattle by the method described above, has activity in immunological tests: it induces differentiation of protymocytes and stimulates the formation of cyclic GMP in the TEM cell line CEM. However, it is found only in embryonic skin and is absent in the skin of cattle.

 As a prototype, the method closest to the present invention for producing immunotropic substances from pig skin by grinding the skin in a solution of sodium chloride, centrifugation, heating, centrifugation, treatment of the supernatant with acetone, centrifugation, chromatography on Sephadex G-50, desalination and lyophilization [2] . The same method was previously patented as a method of obtaining substances with a molecular weight of 1400 to 15000 D from pig skin, affecting the proliferation and differentiation of human keratinocytes [3]. However, using this method, two immunotropic substances are obtained; one with a molecular weight of more than 15,000 D and the other with a molecular weight of 1,400 to 15,000 D, which stimulate the B-system of immunity. They increase the number of antibody-forming spleen cells of mice at the peak of the primary immune response. However, these substances do not affect the T-system of immunity in the test to restore the sensitivity of background rosette-forming spleen cells of thimectomized mice to the inhibitory effect of azathioprine.

 The purpose of the invention is to increase the activity of the resulting immunotropic substances using the inventive method so that they affect both the B and T immunity systems.

This goal is achieved by the fact that in the method, taken as a prototype, three stages of salting out with ammonium sulfate were introduced. Prior to the preparation of the "acetone powder", the method steps are the same: the pig skin is crushed, homogenized in sodium chloride solution, centrifuged, thermo-denatured at 75-85 ^o C for 10-20 min, centrifuged at 5000-15000 g for 10-30 min , treated with acetone cooled to -20 ^o C (or lower) in a ratio of 1: 6-1: 8, dried "acetone powder" at room temperature. Next, the following steps are introduced: the “acetone powder" is dissolved in a 0.005-0.015 M solution of Tris-HCl buffer pH 6.0-8.0, centrifuged at 8000-10000 g for 10-20 minutes at 4-6 ^o C; the precipitate is discarded, and a saturated solution of ammonium sulfate is added dropwise to the supernatant in a ratio of 2: 1-3: 1, centrifuged at 8000-10000 g for 40-50 min at 4-6 ^o С, the pH of the supernatant is adjusted to 3.5-4, 5 using an 8-10% solution of acetic acid, dry ammonium sulfate is added at the rate of: 14-15 g per 100 ml of supernatant, centrifuged at 10000-12000 g for 20-30 minutes at 4-6 ^o C, the residue is homogenized at 0 , 01-0.02 M Tris-Hcl pH 7.5-8.5, a saturated solution of ammonium sulfate is added dropwise to the homogenate in a ratio of 1: 1-1.5: 1, centrifuged at 16000-18000 g for 20-30 minutes at 4-6 ^o C. Next make according to the prototype: the precipitate is homogenized in 30-50 ml of 0.01-0.02 M Tris-Hcl + 0.14-0.15 M NaCl pH 7.5-8.5, centrifuged at 16000-18000 g for 20 -30 min at 4-6 ^o C, chromatographed on a Sephadex G-50 column, two fractions are collected: one with a molecular weight above 15000 D and the other with a molecular weight from 1400 to 15000 D, desalted and lyophilized.

The temperature of thermal denaturation of 75-85 ^o C and the time of thermal denaturation are selected so that the desired substances remain in solution, while some of the inactive material is denatured. Centrifugation at 5000-15000 g for 10-30 min and a temperature of 4-6 ^o With selected so that with these parameters, the denatured proteins are removed. When centrifuged below 5000 g and less than 10 min, part of the denatured inactive material remains in the supernatant, which contaminates the drug. It does not make sense to increase the centrifugation speed over 15000 g and the time over 30 minutes, because under these conditions, the denatured material is completely precipitated. The change in the ratio of the weight of the powder after degreasing: the volume of water less than 1: 4 and more than 1: 6 leads to contamination of the drug. Three stages of salting out with ammonium sulfate are selected in such a way that inactive material and suppressor substances are removed, and the active material remains and is purified. The choice of buffer concentration (0.005-0.015 M) and its pH (6.0-8.0) is due to the fact that there is no loss of activity of the obtained substances in it, at the same time, the preparations are well separated by molecular weight on Sephadex G-50. Sodium chloride to a concentration of 0.14-0.15 M is added to the buffer so that the activity of substances in the samples can be determined immediately after separation in Sephadex.

 The activity of immunotropic substances was determined in two tests: the Erne test and the azathioprine test. The Erne test determined the effect of immunotropic substances on the number of antibody-forming cells (AOKs) of the spleen of CBA mice at the peak of the primary immune response [2, 4]. Sheep red blood cells were injected intraperitoneally in 0.5 ml of physiological saline at a concentration of 400 million / ml. At the same time, the test preparations were administered subcutaneously at a dose of 1, 10, and 100 μg per mouse. On the fifth day, animals were killed, spleens were isolated, and the number of hemolysis zones in Petri dishes was counted. The percentage of AOK relative to the control was determined, taking the arithmetic mean in the control group as 100%. All results were correlated with the arithmetic mean in the control group.

In the Bach azathioprine test, the effect of immunotropic substances on the restoration of the sensitivity of background rosette-forming cells (FROC) of the spleen of thimectomized mice to the inhibitory effect of azathioprine was determined [5]. Male C 57 B 1/6 mice were thimectomized at the age of 6-8 weeks to create a model of partial immunodeficiency by the T-system of immunity and were taken 2-8 weeks after surgery. A suspension of spleen cells was obtained. In addition to azathioprine, test preparations were added to the experimental samples to a final concentration of 1, 5, 10, 20, 50, and 100 μg / ml. The number of FROCs of the spleen of thimectomized mice per 10 ⁴ nucleated cells was counted.

,
где Мк - средняя арифметическая фРОК в контроле;
Мо - то же, в опыте.The results were expressed as percent of FROC by the formula

,
where MK is the arithmetic mean FROK in the control;
Mo is the same in experience.

 Active were considered fractions for which the percentage of FROC was less than 50%. To compare the activity of the drugs, the minimum concentration at which the drug was active was determined.

 In the table. 1, 2 and 3 presents a comparison of the activity of the immunotropic substances 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 of restoring the sensitivity of background rosette-forming spleen cells of thimectomized mice to the inhibitory effect of azatipron. As can be seen from these data, the immunotropic substances 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. In addition, immunotropic substances having the same molecular masses and obtained by the prototype and by the claimed method have a different character in the Erne method of the dependence of activity on the dose. The immunotropic substance 1 (IV1) obtained by the prototype has a maximum activity at a dose of 10 μg / mouse, while IV1 obtained by the present method has a dose of 100 μg / mouse. The immunotropic substance 2 (IV2), obtained by the prototype, has a minimum activity at a dose of 10 μg / mouse; IV2 obtained by the present method has minimal activity at 1 μg / mouse.

 Thus, the claimed method allows to obtain more active immunotropic substances, affecting both the B- and T-system of immunity.

 Example 1 (prototype).

Pork skin is ground with scissors and homogenized in a 0.14 M sodium chloride solution, pH 7.0 at 4 ^{° C.} (1094 Homogenizer, Tecator, Sweden). 650 ml of 0.14 M NaCl solution are taken per 50 g of skin (ratio tissue weight: solution volume - 1: 13). The homogenate is centrifuged at 2500g for 30 minutes at 4 ^{° C.} (J-6M / E centrifuge, rotor 4.2, Beckman, UK). The precipitate is discarded, and the supernatant in a volume of 630 ml is subjected to thermal denaturation at 75 ^° C for 20 min in a water bath with constant stirring, the mixture is cooled to 4 ^° C. The denatured material is precipitated by centrifugation at 10,000 g for 20 min at + 4 ^° C ( centrifuge J2-21M, rotor JA-14, Beckman, USA), and the supernatant in a volume of 620 ml was added dropwise to 4 l of acetone cooled to -20 ^o C. The precipitate was washed with acetone by centrifugation at 2500 g for 15 min at 4 ^o C and dried under draft at room temperature. The result is 800 g of "acetone powder" (800 ± 50 mg). The obtained acetone powder was dissolved in 30 ml of a 0.01 M Tris-HCl buffer solution + 0.15 M NaCl pH 8.0 at room temperature. The insoluble part is removed by centrifugation at 4000 g for 30 min at 4 ^o C, and the supernatant is applied to a column of 2.5 • 100 cm with Sephadex G-50 (2.5x80 cm), balanced with the same buffer. Two fractions are collected: one with a molecular weight of more than 15000 D (immunotropic substance 1), and the other with a molecular weight of 1400 to 15000 D (immunotropic substance 2), desalted and lyophilized.

 Both substances have immunological activity in the Erne test: they increase the number of antibody-forming spleen cells of mice at the peak of the primary immune response, but are inactive in the test to restore the sensitivity of background rosette-forming spleen cells of thimectomized mice to the inhibitory effect of azatiprine (Tables 1, 2, 3). Immunotropic substance 1 (IV1) has a maximum activity at a dose of 10 μg / mouse, immunotropic substance 2 (IV2) - at a dose of 1 μg / mouse.

Example 2 (by the proposed method)
Pork skin is ground with scissors and homogenized in a 0.14 M sodium chloride solution pH 7.0 at 4 ^{° C.} (1094 Homogenizer, Tecator, Sweden). 650 ml of 0.14 M NaCl solution are taken per 50 g of skin (tissue weight: solution volume ratio 1:13). The homogenate is centrifuged at 2500 g for 30 min at 4 ^o C (centrifuge J-6M / E, rotor 4.2, Beckman, UK). The precipitate is discarded, and the supernatant in a volume of 630 ml is subjected to thermal denaturation at 75 ^o C for 20 min in a water bath with constant stirring, the mixture is cooled to 4 ^o C. The denatured material is precipitated by centrifugation at 10000 g for 20 min at + 4 ^o C (centrifuge J2-21M, rotor JA-14, Beckman, USA), and the supernatant in a volume of 620 ml was added dropwise to 4 L of acetone cooled to -20 ^o C. The precipitate was washed with acetone by centrifugation at 2500 g for 15 min at 4 ^o С and dried under draft at room temperature. The result is 800 g of "acetone powder" (800 ± 50 mg).

Then it was dissolved in 0.01 M Tris-Hcl buffer pH 8.0 at room temperature and centrifuged at 10,000 g for 20 min at 4 ^° C. The precipitate was discarded, and a saturated solution of ammonium sulfate was added dropwise to the supernatant in a ratio of 3: 1 at 4 ^° C. The insoluble portion was removed by centrifugation at 10,000 g for 50 min at 4 ^° C. The supernatant was adjusted to pH 4.0 with a 10% acetic acid solution. Then dry ammonium sulfate was added with constant stirring at the rate of: 14.6 g per 100 ml of supernatant and 30 g per 100 ml of acetic acid, stirred at room temperature and centrifuged at 12000 g for 30 min at 4 ^° C. The supernatant was drained, the precipitate homogenized in 0.01 M Tris-Hcl pH 8.0. Then, a saturated solution of ammonium sulfate was added dropwise to the homogenate in an ice bath in a ratio of 1.5: 1 and centrifuged at 18000 g for 30 min at 4 ^° C. The supernatant was drained, the precipitate was homogenized in a minimum volume of 0.01 M Tris-Hcl + 0.15 M NaCl pH 8.0 and centrifuged at 18000 g for 30 min at 4 ^o C. The precipitate was discarded, and the supernatant was applied to a 2.5 x 100 cm column with Sephadex G-50 with markers: blue dextran and DNP-L-alanine. Two fractions were collected: one with a molecular weight of more than 15000 D (immunotropic substance 1, IV1) and the other with a molecular weight of 1400 to 15000 D (immunotropic substance 2, IV2) were desalted and lyophilized.

 Received immunotropic substances 1 and 2, active both in the Yerne method - increasing the number of antibody-forming spleen cells of mice at the peak of the primary immune response, and in the test for restoring the sensitivity of background rosette-forming spleen cells of thimectomized mice to the inhibitory effect of azatiprine (Tables 1, 2, 3) . In the Erne test, the immunotropic substance 1 had maximum activity at a concentration of 100 μg / mouse, and IV2 at a concentration of 10 μg / mouse. In the azathioprine test, IV1 had activity at a concentration of 20 μg / ml, and IV2 at a concentration of 10 μg / ml.

Literature
1. Audhya, T.K., Goldstein G. Amino acid sequence of thymopoietin isolated from skin. Ann. NY Acad. Sci. 1988, V. 548, P. 233-240.

 2. Arion V.Ya., Belova OV, Lukanidina T.A., Sysoeva O.B.,. Dvortsova V.V., Breusov Yu.N. Immunological properties of skin preparations. Bull. an expert. biol. honey. - 2000. - T.129. - 2. - S. 194-197.

 3. Arion V. Ya., Belova OV, Orlova VF, Kapitanov A.B., Lopukhin Yu.M. A method of obtaining a substance from pig skin, affecting the proliferation and differentiation of human keratinocytes. Patent 2038087. Registered on June 27, 1995. Bull. invented open 18, p. 24, 1995.

 4. Siegle E., Boehm E. "The method of local hemolysis in the gel." In the book: Immunological methods. S. 96-107.1979.

 5. Dardenne M., Bach J.-F. "The sheep cell rosette assay for the evaluation of thymic hormones." In: Biologycal activity of thymic hormones. P. 235-243, 1975.

Claims (1)

A method for producing immunotropic substances from pig skin, including grinding, skin homogenization in a sodium chloride solution, centrifugation, thermo-denaturation of the supernatant at a temperature of 75-85 ^o C for 10-20 min, cooling the mixture to 4 ^o C, centrifuging the denatured material at 5000-15000 g for 10-30 min at 4 ^o C, treatment of the supernatant with acetone cooled to -20 ^o C or lower in a ratio of 1: 6-1: 8, washing the precipitate with cooled acetone, drying at room temperature, dissolving the precipitate in 0.005-0.015 M Tris-HCl solution ufer pH 6.0-8.0 and centrifugation, followed by chromatography on a Sephadex G-50 column and collecting the desired product fraction with a molecular weight of 1400-15000 D, desalination and lyophilization, characterized in that the dissolved precipitate is centrifuged at 8000- 10000 g for 10-20 min at 4-6 ^o С, a saturated solution of ammonium sulfate is added dropwise to the supernatant in a ratio of 2: 1-3: 1, centrifuged at 8000-10000 g for 40-50 min at 4-6 ^o С , adjust the pH of the obtained supernatant to 3.5-4.5 by dropping an 8-10% solution of acetic acid, add dry th ammonium sulfate at the rate of 14-15 g per 100 ml of supernatant, centrifuged at 10000-12000 g for 20-30 minutes at 4-6 ^o C, homogenize the precipitate in 0.01-0.02 M Tris-HCl pH 7 5-8.5, a saturated solution of ammonium sulfate is added dropwise to the homogenate in a ratio of 1: 1-1.5: 1, centrifuged at 16000-18000 g for 20-30 minutes at 4-6 ^o С, the precipitate is homogenized at 30- 50 ml of 0.01-0.02 M Tris-HCl + 0.14-0.15 M NaCl pH 7.5-8.5, centrifuged at 16000-18000 g for 20-30 minutes at 4-6 ^o C while chromatography on a Sephadex G-50 column simultaneously collects a fraction with a molecular weight above 15,000 D.

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