RU2648468C1 - Membrane method of obtaining the medicinal on the basis of calf thymus peptides - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention relates to the pharmaceutical industry, in particular to a method for producing a medicament from a calf thymus. Method for the preparation of a medicament from a calf thymus comprising, grinding and homogenizing a tissue of the calf thymus at the age of up to 12 months, the homogenate is extracted with a 3 % acetic acid solution for 24 ± 2 hours at the temperature of +37 ± 3 °C at the ratio of raw materials to extractant 1:2 with the addition of zinc chloride at the rate of 5 g of zinc chloride per 1 kg of raw material, further clarifying filtration is carried out with the use of an auxiliary substance – 10 % diatomite with separation of the latter on the Nutsche filter through the filter cardboard, stabilization of peptides occurs with the help of glycine 40 ± 10 % mg per 1 ml of solution for lyophilization, then freeze-drying is carried out.

EFFECT: above-described method makes it possible to obtain the claimed medicament from the calf thymus without using acetone and to increase the yield of the final product and to provide the resulting drug with a higher purity.

1 cl, 3 dwg

Description

FIELD OF THE INVENTION

The invention relates to the pharmaceutical industry and biotechnology and can be used in medicine to obtain a medicinal product with immunotropic, radioprotective and geroprotective activity, which enhances repair systems.

State of the art

Currently, there are a number of drugs derived from the thymus glands of mammals. These include, in particular, thymosin, thymoptin, thymalin, thymactin, thymactide, vilosen, thymostimulin, thymusasin, oletim and others. The vast majority of them include thymus peptides, some of them also contain nucleoproteins.

These drugs have a similar effect. So, in particular, it has been shown that thymalin increases the stability of the genome, activates the immune and reparative systems. Thymalin peptide bioregulators have a pronounced geroprotective and antitumor effect, and also contribute to the body's resistance to stressful effects [1, 2]. Timalin has an anticoagulant effect and helps inhibition of fibrinolysis, helps to lower cholesterol in the blood, liver and aorta [1]. It was also shown that this drug minimizes the area of the brain involved in the formation of memory engrams, promotes memory growth, reduces the energy cost of the formation, consolidation and storage of information, thus possessing psychotropic activity and the properties of non-specific correctors, which is essential for adaptation to critical states [3, 4].

A known method proposed in patent SU No. 1218521 (1980), which involves the homogenization of crushed tissue of the thymus gland, extraction with diluted acetic acid in the presence of zinc chloride for 48-72 hours, the addition of acetone, centrifugation and additional processing of the extract with acetone [5]. The method provided a relatively high extraction of peptides and allowed to reduce the number of steps in comparison with the analogues existing at that time. The main disadvantages of this technology are the use of volatile flammable toxic acetone, low product yield, insufficient purification of the final product.

Known technical solution described in French patent No. 25702786 (1981). According to this solution, peptides from the thymuses of various animals are obtained by biochemical methods, which include homogenization in a solution of 9 g / l sodium chloride, autolysis, centrifugation, heating of the supernatant at 80 ° C, precipitation with acetone, three times centrifugation and saturation with ammonium sulfate solution, ultrafiltration, removal of salts chromatography and lyophilization [6]. The method provides a high degree of purification due to the use of chromatography, and involves obtaining a lyophilized dosage form, which has greater stability during storage compared to solutions. The disadvantages of this method include the risk of destruction of the peptides when heated to 80 ° C, the use of volatile flammable toxic acetone, the use of expensive and complicated chromatography.

According to the method described in patent RU No. 2205013 (2003), the preparation of the drug involves the following stages: grinding and homogenization of the deer thymus, extraction in sodium chloride solution, heating the extract, removing the heat-sensitive fraction, obtaining the target product by ultrafiltration in two stages - on the membrane, a cutoff substance with a mass of 10 kDa, and a membrane with pores that trap substances with a mass of more than 1 kDa, the addition of D-mannitol, sterilizing filtration and lyophilization [7]. This method assumes the optimal parameters of the membranes used in ultrafiltration, since they cut off substances having a molecular weight outside the molecular weight range of biologically active thymus peptides. The disadvantages of this method are the insufficiently complete extraction of thymus peptides due to the use of sodium chloride solution as an extractant, an additional heating step to 80 ° C, which slows down the process and can lead to a violation of the stability of the extracted peptides, the use of D-mannitol as a stabilizer, which can lead to to exacerbation of kidney diseases, circulatory disorders and dehydration, as well as the use of hard-to-reach raw materials.

The method described in patent RU No. 2240817 (2003) differs from the previous analogue in that, in the second stage of ultrafiltration, membranes that retain substances with a molecular mass of 0.5 kDa are used; a solution containing 5.5 g / l sodium chloride and 0.42 g / l potassium chloride is used for extraction, a stabilizer is not added to the final product, the drug is not lyophilized. This method provides a more complete extraction compared with the previous analogue [8]. The use of membranes with a pore size of 0.5 kDa during ultrafiltration expands the range of substances obtained, however, substances of the 0.5-1.0 kDa fraction are not targeted in most preparations from the thymus. The disadvantages of the method include the use of chlorides as an extractant, which leads to insufficient extraction of biologically active peptides, and the presence of a heating step to 80 ° C, which can lead to a violation of the stability of the peptides. Also, preparations in liquid form are inferior in stability during storage.

As prototypes of the present invention, the technical solutions described in the patents RU No. 2205013 (2003) and RU No. 2240817 (2003) are considered, since they propose the use of ultrafiltration in the isolation of the target product. However, in both cases, the raw material is the reindeer thymus, which is difficult to access in conditions of central Russia [7, 8]. The proposed solution is also compared with the method described in patent SU No. 1218521 (1980), since it is widely used in production [5].

SUMMARY OF THE INVENTION

The technical result of the present invention is to develop a method for producing a medication based on calf thymus without the use of acetone, the inventive method having a higher yield of the final product and providing a medicament with a higher degree of purity.

The present invention provides a solution to the problem of creating a method for the production of medicines based on calf thymus, which provides a higher yield of the final product with a higher degree of purity and does not require the use of acetone.

The solution to the problem is achieved by improving the stage of extraction of peptides from calf thymus tissue, using a special clarifying filtration scheme, isolating and purifying the target product by ultrafiltration and using glycine to stabilize the lyophilization solution at a concentration of 40 ± 10% mg per 1 ml.

The proposed method includes the following steps:

- Grinding and homogenization of calf thymus tissue (under the age of 12 months). Extraction with a 3% solution of acetic acid for 24 ± 2 hours at a temperature of + 37 ± 3 ° C (ratio of raw material to extractant 1: 2) with the addition of zinc chloride at the rate of 5 g per 1 kg of raw material.

- Separation of oilcake by separating the obtained extract.

- Clarifying filtration - filtering the separated extract using an auxiliary substance - 10% kieselguhr, filtering on the suction filter through filter cardboard.

- Ultrafiltration in a tangential flow on membranes that cut off substances with a molecular mass of more than 10 kDa, used to separate high molecular weight impurities, collecting permeate containing peptides. Purification of low molecular weight impurities by ultrafiltration in a tangential flow on membranes that trap substances with a molecular mass of 1 kDa (concentration is possible at this stage). Diafiltration of the concentrate (if necessary).

- Addition of glycine at the rate of 40 ± 10% mg per 1 ml of solution.

- Sterilizing filtration, bottling the drug in bottles and freeze-drying.

Studies on the selection of optimal conditions for the extraction of peptides from the thymus glands of calves included the selection of the ratio of raw materials and extractant (options from 1: 2 to 1: 5), temperature (+ 20 ± 3 and + 37 ± 3 ° C) and time (24 ± 2 or 48 ± 2 hours) for this stage of the process. The data obtained indicate that the extraction should be carried out for 24 ± 2 hours at a temperature of + 37 ± 3 ° C and a ratio of raw materials to extractant equal to 1 to 2. Extraction at a temperature of + 37 ± 3 ° C and a ratio of raw materials to extractant 1: 2 provides a statistically significant (at p≤0.05) increase in the concentration of peptides in the extract compared to all options at a temperature of + 20 ± 3 ° C, while the differences between the options for the ratio of raw materials and extractant at a temperature of + 37 ± 3 ° C are not significant. Differences in the content of peptides in the extract at the same temperature but with different extraction times are not significant. An increase in the extraction temperature provides an increase in the yield of the target product, and a reduction in the process time and a decrease in the amount of extractant do not affect this parameter. The proposed mode allows you to get a larger number of peptides while reducing the duration of the process, saving extractant and reducing the volume of the extract with the same load of raw materials.

The clarification filtration stage necessary for purification of the separated extract provides a higher quality of the final product and a longer service life of the membrane filtration units used in the subsequent stages of the process. At this stage, filtration takes place with 10% kieselguhr, the latter is separated on the suction filter through filter paper, which ensures clarification of the solution and almost complete removal of lipids.

The basis for the isolation and purification of thymic peptides by the proposed method are membrane filtration processes. Ultrafiltration of the clarified extract in a tangential stream through a cartridge with a nominal cut-off molecular weight of 10 kDa ensures the separation of high molecular weight impurities. Then, low molecular weight impurities are cleaned and, if necessary, the resulting permeate is concentrated in a tangential flow on membranes that cut off substances less than 1 kDa. This stage provides the separation of low molecular weight impurities, reducing the volume of the solution, the ability to store small volumes in frozen form. If necessary, washing of the concentrate on the membrane of 1 kDa is possible. The molecular weight of the substances, the cut-off of which is provided by the membranes of the filters used for ultrafiltration, corresponds to the molecular weights of the biologically active thymus peptides.

Brief description of illustrative materials

FIG. 1. The content of peptides in the extract in mg based on 1 g of raw materials with different ratios of "raw materials: extractant" and various temperatures of extraction.

FIG. 2. The content of peptides in the extract (mg / ml) depending on the time of extraction.

Figure 3. Gel chromatograms of a drug reconstituted in physiological saline lyophilisate based on thymus peptides obtained by the proposed method (a) and by acetone technology (b).

The possibility of carrying out the invention is confirmed by the following example.

The thymus glands of calves weighing 1 kg were ground to forcemeat, extracted at + 37 ± 3 ° C for 24 hours with a 3% solution of acetic acid with a volume of 2 l with the addition of zinc chloride weighing 5 g half an hour after the start of extraction.

At the end of extraction, cake was separated from homogenates by centrifugation, 10% kieselguhr was added to the supernatant, filtered on a suction filter through filter paper.

An ultrafiltration was carried out at room temperature through a membrane cutting off substances with a mass above 10 kDa, and permeate was obtained, which was purified from low molecular weight impurities on a 1 kDa membrane. Received 1885 ml of the intermediate product, the peptide content of which was about 50 mg in terms of 1 g of raw material (over 25 mg / ml). 76 g of glycine was added, sterilized filtration was performed (pore diameter 0.22 μm), filling into vials under aseptic conditions and freeze-drying. The yield of peptides in the final product is up to 10 times greater than with acetone technology. Comparison of the yield of biologically active peptides obtained by the proposed method with the production technology of the drug "Oletim" [8] showed an increase of more than 10 times.

Information sources

1. Havinson V.Kh., Kuznik B.I. Peptide bioregulators are a new class of geroprotectors. Communication 1. The results of experimental studies // Successes of gerontology, 2012, v. 25, No. 4. S. 696-708.

2. Demina E.A. Arguments for the prevention of radiogenic cancer based on cytogenetic studies // Ecological Bulletin, 2011, No. 4 (18). S. 45-49.

3. Dolzhenko A.T., Melekhin V.D. Thymus peptides as psychotropic drugs // The use of small regulatory peptides in anesthesiology and intensive care. - M., 1991.S. 70-72.

4. Melekhin V.D. Thymus peptides as stress protectors and antinociceptive drugs // The use of small regulatory peptides in anesthesiology and intensive care. - M., 1991.S. 96-98.

5. Patent SU No. 1218521, 1980.

6. French patent No. 25702786, 1981.

7. Patent RU No. 2205013, 2003.

8. Patent RU No. 2240817, 2003.

Claims (2)

1. A method of obtaining a medicinal product based on calves of the calf thymus, which consists in grinding and homogenizing calf thymus tissue under the age of 12 months, while the homogenate is extracted with a 3% solution of acetic acid for 24 ± 2 hours at a temperature of + 37 ± 3 ° C with a ratio of raw materials to extractant 1: 2 with the addition of zinc chloride at the rate of 5 g of zinc chloride per 1 kg of raw material, then clarification filtration is carried out using an auxiliary substance - 10% kieselguhr with separation of the latter on the suction filter through a filter cardboard, stabilization of peptides occurs with glycine 40 ± 10% mg per 1 ml of lyophilization solution, then freeze-drying is carried out. 2. A method for producing a medicine based on calves of the calf thymus peptides according to claim 1, characterized in that at the stage of purification from low molecular weight impurities, the sample is additionally concentrated, the concentrate diafiltered and diluted to the required concentration, taking into account the filling dose and the required number of peptides, before freeze-drying in a bottle.

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