RU2673802C1 - Method of manufacture of sterile pharmaceutical substance - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the field of chemical and pharmaceutical, food and cosmetic industries and concerns a method for producing substances of preparations of nucleic acids of immunomodulating action. Method for the production of sterile pharmaceutical substance sodium deoxyribonucleic acid from animal raw materials is that the animal tissue is crushed, ballast substances in the form of fat, protein and connective tissue are removed by sequentially separating in the settling centrifuge, first light fractions with a density of up to 0.8 g/cm³ at 2,000 rpm, further medium fractions with a density of up to 1.0 g/cm³ at 3,000 rpm and then the target fraction with a density of more than 1.1 g/cm³ at 4,000 rpm, the target fraction is subjected to lysis, treated with sodium chloride solution, then subjected to ultrasonic treatment, the solution is filtered under vacuum, concentrated by membrane filtration with membrane selectivity from 5,000 to 100,000 Da, the concentrate is treated with ethyl alcohol in a ratio of 1:1–1:2 by volume to form a precipitate of deoxyribonucleic acid sodium salt, the precipitate is dried and subjected to electron beam sterilization or gamma radiation sterilization.

EFFECT: method for the production of sterile pharmaceutical substance sodium deoxyribonucleic acid from animal raw materials is proposed.

3 cl, 2 tbl, 5 ex

Description

The invention relates to the field of chemical-pharmaceutical, food and cosmetic industries and relates to a method for producing substances of preparations of nucleic acids with immunomodulatory effects.

Currently, immunomodulatory nucleic acid preparations are widely used in practical medicine, both in the form of official drugs and in the form of dietary supplements for food. In particular, drugs are known - sodium nucleinate (RNA obtained from yeast), derinat (sodium salt of native DNA isolated from sturgeon milk), placentex integro (DNA from trout milk), dietary supplement Biostim (DNA from sturgeon milk fish) and others (http://www.dissercat.com/content/immunomoduliruyushchaya-aktivnost-nizkomolekulyarnoi-dezoksiribonukleinovoi-kisloty-dnk-iz-m).

To obtain immunomodulators based on derivatives of nucleic acids, chemically synthesized nucleic components are mainly used. However, the synthesis of nucleic components is quite expensive and leads to the formation of by-products, which requires careful purification of the drug. Therefore, much attention is currently being paid to the development of alternative ways of obtaining nucleic components, for example, from natural raw materials - DNA of microorganisms and animals.

A known method of producing sodium salt of DNA from the testes of animals and / or their semen, which includes grinding and homogenizing the feedstock in a detergent solution, processing the solution at elevated temperature while stirring the solution, separating the liquid phase by filtration and precipitating the product with alcohol. The resulting DNA preparation is dissolved in an aqueous NaCl solution of a concentration of 0.01-4.5 M at a temperature not exceeding 50 ° C, the resulting solution is passed through a high-pressure homogenizer at a pressure of up to 2000 kg / cm2, the resulting fragmented DNA is treated with alcohol, and precipitated in alcohol fragmented sodium DNA is separated by centrifugation, after which the separated precipitate is washed again with alcohol and dried in an oven at a temperature not exceeding 50 ° C to obtain a powder (RU 2268730, published on January 27, 2006).

A known method of obtaining a sterile solution, including the transfer of DNA into the liquid phase, sonication of the DNA solution, filtration, standardization of sodium chloride and sterile microfiltration, wherein the transfer of DNA into the liquid phase is carried out by homogenization of fish milk in citrate-salt solution, separation of the homogenate, processing sediment with detergent and concentrated saline at 55-60 ° C, cooling the reaction mixture, preliminary ultrasonic treatment, and also carry out re-sonication in loviyah providing reception DNA mol. m 2.5 × 10 ⁵ - 6.0 × 10 ⁵ daltons and concentration (RU 2055837).

A known method for the production of immunotropic pyrogen-free tolerant preparation based on the sodium salt of low molecular weight deoxyribonucleic acid, which consists in the fact that skim milk of sturgeon fish, salmon fish, calf thymus, blood of chickens are homogenized in a citrate-salt solution, the homogenate is centrifuged in a sediment centrifuge / minus centrifuge , the supernatant is removed, the substrate is re-homogenized, then the purified homogenate is incubated in the presence of 3% detergent, during continuous stirring At elevated temperature, concentrated sodium chloride solution is added to the reaction mass, the reaction mass is cooled to a temperature below room temperature (15 ° C), sonicated, in baths with piezoceramic emitters with an oscillation amplitude of 4-10 microns; then a sorbent, the liquid phase is separated, the sorbent is eluted with permeate, the eluate combined with the liquid phase is microfiltered, concentrated, the precipitate of sodium deoxyribonucleate is isolated with alcohol, centrifuged, separated from the liquid phase, dried for 4 hours under aseptic conditions at 40 ° C (RU2236853 C1 publ. 2004-09-27).

As the closest analogue, a method for the production of a unified pyrogen-free immunomodulator, which is a sodium salt of a native low molecular weight deoxyribonucleic acid, can be specified, which means that the milk of salmon or sturgeon fish is defatted, crushed and homogenized in a citrate-salt aqueous solution at 12,000 rpm, centrifuged in a settling centrifuge at 3500 rpm while cooling, the supernatant removed, the substrate is re-homogenized 3-4 times with the addition of calculated the amount of citrate-saline solution with the creation of an air-fat foam and decantation, then the purified homogenate is incubated in the presence of 3-6% detergent during continuous stirring at elevated temperature, saturated sodium chloride solution is added to the reaction mixture, then the reaction mixture is cooled to a temperature below room (10 ° C), is treated with ultrasound in baths with piezoceramic emitters with an oscillation frequency of 21.7-23 kHz and a bending moment of at least 9 μm with a generator power of 1.5-2 kW, then a sorbent the liquid phase is separated, the sorbent is eluted with permeate, the eluate combined with the liquid phase is microfiltered, concentrated, the precipitate of sodium deoxyribonucleate is precipitated with alcohol, centrifuged, separated from the liquid phase, dried under aseptic conditions for 4-6 hours at a temperature of 40-60 ° C (RU 2309759 C1, publ. 2007-11-10). The substance used does not retain its sterility for a long time.

The objective of the present invention is to develop a method for producing a sterile substance of sodium salt of deoxyribonucleic acid, which has a number of technological advantages and allows you to get a substance that can be widely used in the manufacture of pharmaceutical, food and cosmetic products, including those requiring high sterility requirements, for example, injection forms.

The problem is solved by a new method for the production of a sterile pharmaceutical substance of the sodium salt of deoxyribonucleic acid from animal raw materials consisting in the fact that animal tissues are crushed on a disintegrator to particles with sizes of 0.1-1 μm, ballast substances in the form of fat, protein and connective tissue are removed by sequential separation in a settling centrifuge, first light fractions with a density of up to 0.8 g / cm3 at 2000 rpm, then medium fractions with a density of up to 1.0 g / cm3 at 3000 rpm and then the target fraction with a density of more than 1.1 g / cm3 at 4000 rpm, the target fraction is subjected to lysis in the presence of detergent with a concentration of from 1 to 10% at temperatures from 20 to 80 ° C and a pressure of from 0.9 to 1.1 atm. during the time interval from 30 to 150 minutes, the resulting degassed mass is treated with a solution of sodium chloride with a concentration of 15-25 wt.%, then subjected to ultrasound treatment with a frequency of 15 to 45 Hz at a temperature of 10 to 70 ° C for a time interval from 30 to 200 minutes, the solution is filtered under a vacuum of 0.8-0.9 atm. for clarification, it is concentrated by membrane filtration with a membrane selectivity of 5,000 to 100,000 Daltons, the concentrate is treated with ethyl alcohol in a ratio of 1: 1 - 1: 2 by volume to form a precipitate of the sodium salt of deoxyribonucleic acid, the precipitate is dried under vacuum of 0.8-0.9 atm . at a temperature of 40-90 ° C for 60-300 minutes to a residual moisture of 5-20%, followed by electron-beam sterilization or sterilization of the obtained gamma-ray powder with an intensity of 5 kGy to 25 kGy per dm3 with an exposure of 30 minutes to 120 minutes .

Get a sterile powder of sodium salt of deoxyribonucleic acid, water-soluble, purified from ballast substances, pyrogen-free, non-toxic, suitable for the preparation of drugs in liquid form (drops, solutions for injection, etc.) and in solid form (tablets, powders, suppositories).

Technical result: increased shelf life, reduced requirements for temperature storage, humidity parameters providing better dissolution, increased microbiological purity, the possibility of preparing a sterile preparation before use directly in the syringe by adding injection water, preparing sterile solid forms.

Suitable animal feeds are blood, thymus, liver, brain, spleen, testes of farm animals or birds, and also milk and / or liver of commercial fish, such as sturgeon, salmon. Preferably, the animal tissue is selected from fish milk, calf thymus, chicken blood, pig spleen, most preferably from fish milk.

As detergents, any detergents used in the art can be used, both ionic and nonionic. As examples of nonionic detergents, twins or spans may be indicated. Examples of ionic detergents include sodium dodecyl sulfate, bile acids, derivatives of quaternary ammonium compounds.

The invention may be illustrated by the following specific embodiments of the invention.

Example 1

Milk of sturgeon fish is crushed on a disintegrator to particles with sizes of 0.1-1 μm and then the ballast substances in the form of fat, protein and connective tissue are separated from the resulting mass by sequentially first separating light fractions in a settling centrifuge with a density of up to 0.8 g / cm3 at 2000 rpm, then the middle fractions with a density of up to 1.0 g / cm3 at 3000 rpm and the precipitation of the target fraction with a density of more than 1.1 g / cm3 at 4000 rpm. The target fraction consisting of 60-80% of cell nuclei is lysed in the presence of a detergent with a concentration of from 1 to 10% at temperatures from 20 to 80 ° C and pressure from 0.9 to 1.1 atm for a time interval of 30 to 150 minutes. In this case, the mass is actively degassed. A degassed mass containing DNA molecules in a solution is treated with a solution of sodium chloride with a concentration of 25 wt%, which is then subjected to ultrasound treatment at a frequency of 40--45 Hz at a temperature of 40 to 70 ° C for 100 minutes. The treated solution is filtered under vacuum of 0.8-0.9 atm. for clarification. The filtered solution is concentrated by membrane filtration with a membrane selectivity of 5,000 to 100,000 Daltons, the concentrate is treated with ethyl alcohol in a ratio of 1: 1 to form a precipitate of the sodium salt of deoxyribonucleic acid. The resulting precipitate is dried under vacuum of 0.8-0.9 atm at a temperature of 70-90 ° C for 180 minutes to a residual moisture content of 20%. The dried (possibly packaged) powder is sterilized with gamma or electron beam sterilization with an intensity of 20 kGy with an exposure of 30 minutes to 120 minutes.

Example 2

The pig’s spleen is crushed on a disintegrator to particles with sizes of 0.1-1 μm. Ballast substances in the form of fat, protein and connective tissue are separated from the resulting mass by sequential separation in a settling centrifuge: first, light fractions with a density of up to 0.8 g / cm3 at 2000 rpm, then medium fractions with a density of up to 1.0 g / cm3 at 3000 rpm and sedimentation of the target fraction with a density of more than 1.1 g / cm3 at 4000 rpm. The target fraction, consisting of 60-80% of the cell nuclei, is lysed in the presence of a detergent with a concentration of 10% at temperatures from 20-40 ° C and a pressure of 0.9 to 1.1 atm for 30 minutes. In this case, the mass is actively degassed. A degassed mass containing DNA molecules in a solution is treated with a sodium chloride solution with a concentration of 20% by mass, which is then subjected to ultrasound treatment at a frequency of 15 Hz at a temperature of 20 ° C for 200 minutes. The treated solution is filtered under vacuum of 0.8-0.9 atm to clarify, concentrated by membrane filtration with a membrane selectivity of 5,000 to 100,000 Daltons, the concentrate is treated with ethyl alcohol in a ratio of 1: 2 to form a precipitate of the sodium salt of deoxyribonucleate. The resulting precipitate is dried in a vacuum of 0.8-0.9 atm at a temperature of 80 ° C for 60 minutes to a residual moisture content of 5%. The dried and packaged powder is sterilized by gamma or electron sterilization with an intensity of 5 kGy and an exposure time of 30 minutes to 60 minutes.

Example 3

A solution for intramuscular administration is obtained by weighing under aseptic conditions the calculated amount of powder according to Example 1 (for preparing a 1% solution), dissolving in a 0.9% sterile solution of sodium chloride, followed by sterile filling in 1 ml ampoules.

Example 4

A suppository agent is obtained by placing solid confectionery fat A (90%) in a container equipped with a water jacket and a mixing device, where it is melted to a homogeneous mass at a temperature of 45-50 ° C with constant stirring, cooling, adding the substance of the active substance (10 %) obtained according to example 2, homogenization using a colloidal mill to a homogeneous state, followed by filling the dispenser with the mixture of cells obtained in the contour packaging, which are cooled to room temperature in cool spruce tunnel.

Example 5

The study of biological activity.

Confirmation of biological activity was carried out by measuring the migration of macrophages into the healing zone of experimental wounds in laboratory rats, where one of the groups was prescribed the drug derinat in the form for intramuscular injection registered in the State Register of the Russian Federation, and in the other according to example 3.

Initially, to obtain a culture of macrophages, on sexually mature male rats (Rattus rattus L.) with an initial mass of 150-200 g under aseptic conditions, a wound was modeled using a modified I.A. Sychennikova (1974)

Animals in the experiment were divided into 3 groups:

1. Control group (12 animals) - the wound was modeled without subsequent administration

2. Derinat group (12 animals) - a single daily injection of Derinat in the average therapeutic dose of 0.015 ml of 1.5% Derinat solution (0.11 mg per rat) into the soft tissue wound area.

3. The group NDRP-3 (12 animals) - a single daily injection into the soft tissue wound area of the preparation according to Example 3 in an average therapeutic dose of 0.015 ml of a 1.5% solution of the preparation according to Example 3 (0.11 mg per rat).

The drug was administered intramuscularly, in tissue, near the wound immediately after modeling the wound, on the first, second and third days after modeling. This time is due to the highest intensity of migration to the lesion of macrophages.

At each stage of the study, detachable contents were collected from the regeneration zone from the regeneration zone, which, using macrophage reaction markers, was checked in fingerprint smears for the quantitative presence of the desired cells (macrophages).

Next, the smear was stained with methylene blue, after which the number of cells in the field of view was determined for each period and each laboratory animal.

Note: * - p <0.01

NDLP-3 - sodium deoxyribonucleate, the preparation obtained in example 3

The study of smears of fingerprints of a suspension of tissue of wound contents showed that the administration of Derinat and the preparation obtained in Example 3 caused the migration of macrophages into the region of the simulated wound more actively than in the control group. This difference ranged from 30 to 90% (compared with the control group) and depended on the dose of the drug administered. Migration became most pronounced on the third day of administration; it exceeded the data of the control group by almost 2 times.

Thus, we can say that the administration of Derinat and NDLP-3 caused an increase in the regeneration process and activation of macrophages.

The proposed method can be widely implemented in industry to obtain substances with improved properties (sterility, high activity, preservation of the native structure, high degree of purification).

Claims (3)

1. A method of manufacturing a sterile pharmaceutical substance of the sodium salt of deoxyribonucleic acid from animal raw materials, which consists in the fact that animal tissue selected from the group comprising blood, thymus, liver, brain, spleen, testes of farm animals or birds, milk and / or commercial liver fish, crushed on a disintegrator to particles with sizes of 0.1-1 microns, remove the ballast substances in the form of fat, protein and connective tissue by sequentially separating in the settling centrifuge first light fractions with a flat up to 0.8 g / cm ³ at 2000 rpm, then the middle fractions with a density of up to 1.0 g / cm ³ at 3000 r / min and then the target fraction with a density of more than 1.1 g / cm ³ at 4000 r / min, the target fraction is subjected to lysis in the presence of a detergent with a concentration of from 1 to 10% of the mass. at temperatures from 20 to 80 ° C and pressure from 0.9 to 1.1 atm for a time interval of 30 to 150 minutes, the resulting degassed mass is treated with a solution of sodium chloride with a concentration of 15-25% by weight, then subjected to ultrasonic treatment with a frequency of 15 to 45 Hz at a temperature of 10 to 70 ° C for a time interval of 30 to 200 minutes, the solution is filtered under vacuum of 0.8-0.9 atm, concentrated by membrane filtration with a membrane selectivity of 5000 to 100000 Yes, the concentrate is treated with ethyl alcohol in a ratio of 1: 1-1: 2 by volume for education precipitate of sodium salt of deoxyribonucleic acid, the precipitate is dried under vacuum of 0.8-0.9 atm at a temperature of 40-90 ° C for 60-300 min to a residual moisture of 5-20%, followed by electron-beam sterilization or sterilization of the obtained gamma-powder radiation with an intensity of 5 kGy to 25 kGy per dm ³ with an exposure of 30 to 120 minutes. 2. The method according to p. 1, in which milk and / or liver of fish of the sturgeon or salmon family are used as animal raw materials. 3. The method according to p. 1 or 2, in which before sterilization the powder is placed in a package.