RU2749007C1 - Method for production of medicinal product based on sodium deoxyribonucleate - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to the area of chemical and pharmaceutical industry, namely to a method for production of a preparation of a sodium salt of deoxyribonucleic acid in form of a sterile water solution, characterized by a solution of an auxiliary substance constituting sodium chloride or sodium citrate at a concentration of up to 0.01 to 0.1 g/l being preliminarily prepared, then a weighed amount of the sodium salt of deoxyribonucleic acid being dissolved in the resulting solution to obtain a concentration of 0.1 to 25.0 g/l, sodium chloride being added up to the concentration of auxiliary substances of 5.0 to 12.0 g/l, the resulting solution being heated to a temperature of 30 to 35°С followed by sterilising filtration and packaging.

EFFECT: invention allows producing a medicinal form that is sterile and apyretic and retains the nativeness of the active substance.

2 cl, 4 ex

Description

The invention relates to the field of chemical-pharmaceutical industry and relates to a method for producing a medicinal product with immunomodulatory action.

Nucleic acids are high molecular weight biopolymers consisting of nucleotides linked by a phosphodiester bond. Currently, more and more attention is paid to the creation of drugs based on them. This is due, in particular, to the mechanism of DNA action, which is based on its ability to accumulate in the bone marrow, spleen, lymph nodes and epithelium of the small intestine, as well as to the identification of the important role of nucleic acids in the regulation and increase of the body's resistance to adverse effects and correction of immunodeficiency states.

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 to food. In particular, drugs are known - sodium nucleinate (RNA obtained from yeast), derinat (sodium salt of native DNA isolated from sturgeon milk), placenthex-integro (DNA from trout milk), biostim food supplement (DNA from sturgeon milk fish) and others (http://www.dissercat.com/content/immunomodulirayushchaya-aktivnost-nizkomolekulyarnoi-dezoksiribonukleinovoi-kisloty-dnk-iz-m). When isolated from natural raw materials, ballast related substances and impurities enter the preparations, which affects the degree of their pyrogenicity and stability. In addition, the nucleic acids themselves are not sufficiently resistant to the effects of various physical factors. Thus, research in this area is primarily aimed at developing new methods for obtaining stable and highly purified drugs.

There is a known method of obtaining a sterile solution, including the transfer of DNA into a liquid phase, treatment of the DNA solution with ultrasound, filtration, standardization for sodium chloride and sterile microfiltration, wherein the transfer of DNA into the liquid phase is carried out by homogenizing fish milk in a citrate-saline solution, separating the homogenate, processing sediment with a detergent and concentrated saline solution at 55-60 ° C, cooling the reaction mass, preliminary ultrasonic treatment, and also carry out repeated sonication under conditions ensuring the production of DNA with a mol. m. 2.5 × 105 - 6.0 × 105 daltons and concentration (RU 2055837). The solution contains a high amount of ballast substances.

There is a known method for the production of an immunotropic pyrogen-free tolerant drug 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, chicken blood is homogenized in a citrate-saline solution, the homogenate is centrifuged / centrifuged in a sediment 2500 , 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 an elevated temperature, a concentrated solution of sodium chloride is added to the reaction mass, the reaction mass is cooled to a temperature below room temperature (15 ° C), processed ultrasound, in baths with piezoceramic emitters with an oscillation amplitude of 4-10 microns; then with a sorbent, the liquid phase is separated, the sorbent is eluted with a 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 (RU 2236853 C1, publ. 2004-09-27).

As the closest analogue, a method of obtaining a sterile pharmaceutical substance of sodium salt of deoxyribonucleic acid from animal raw materials can be indicated, which consists in the fact that animal tissues are crushed, ballast substances in the form of fat, protein and connective tissue are removed by sequential separation in a settling centrifuge, first of the light fractions with a density of up to 0.8 g / cm3 at 2000 rpm, then middle 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, treated with a solution of sodium chloride, then subjected to ultrasonic treatment, the solution is filtered under vacuum, concentrated by membrane filtration with membrane selectivity from 5000 to 100000 Da, the concentrate is treated with ethyl alcohol in a ratio of 1: 1-1: 2 by volume to form the precipitate of the sodium salt of deoxyribonucleic acid, the precipitate is dried and subjected to electron beam sterilization or sterilization with gamma radiation. A solution for intramuscular administration is prepared from the substance, characterized by the fact that the calculated amount of powder is weighed out under aseptic conditions to prepare a 1% solution, dissolve in 0.9% sterile sodium chloride solution, followed by sterile filling into 1 ml ampoules (RU 2673802, publ. 02/13/18). According to this technical solution, a high quality substance is obtained according to Table 1. However, it is not enough to obtain a substance, it is necessary to preserve all its properties already upon receipt of the finished form. The method used for this, disclosed in the patent specification, does not provide such a possibility.

The object of the present invention is to develop a method for producing stable liquid forms of the sodium salt of deoxyribonucleic acid.

The problem is solved by a new method of manufacturing a drug, in the form of a sterile aqueous solution of the sodium salt of deoxyribonucleic acid, according to which a preliminary solution of an auxiliary substance with a concentration of up to 0.1 g / l is prepared, then a sample of the active substance is dissolved in the resulting solution in order to create the required concentration. This solution is thermolabile and in order to stabilize it, an auxiliary substance is added to the solution to a concentration of 12.0 g / l. This allows the solution to be heated to a temperature of 35 ° C without disturbing the primary and secondary structure of the basic substance. Next, the heated solution is subjected to micro and sterilizing filtration and is then packaged in the necessary container, which ensures the convenience of use and storage of the drug.

EFFECT: using the proposed method, it is possible to reduce the dissolution time of the active substance, to prevent its destruction in a solvent with low ionic strength, to carry out the necessary filtration and sterilization procedures of the solution and to create conditions for maintaining the stability of properties during long-term storage of the drug.

1. The sodium salt of deoxyribonucleic acid obtained from animal raw materials, including blood, thymus, liver, brain, spleen, testes of farm animals or birds, milk or liver of commercial fish, is used as an active pharmaceutical ingredient.

2. As an auxiliary substance serving as a stabilizer of the ionic medium and a preservative of biological molecules, pharmaceutically acceptable water-soluble sodium salts are used. An example of such salts can be sulfate, hydrogen carbonate, chloride, phosphate, succinate, citrate, gluconate, etc. Preferably sodium citrate, sodium chloride or a mixture thereof are used according to the invention.

3. High purity water such as water for injection is used as a solvent.

When preparing a medicinal product, the following procedure is used:

a. Dissolution of the auxiliary substance to a concentration in the range of 0.01-0.1 g / l in order to create an isotonic solution.

b. Dissolution of the basic substance to a concentration in the range of 0.1-25.0 g / l.

c. Bringing the concentration of the excipient to 5.0-12.0 g / l.

d. Heating the solution to 30-35 ° C.

e. Clarifying and sterilizing filtration of the solution using filters with a pore diameter of 1.0 to 0.2 microns.

The active pharmaceutical ingredient used is sodium salt of deoxyribonucleic acid:

- possesses high therapeutic properties, is an immunomodulator, reparant, regenerant,

- technologically advanced - has no harmful impurities and contains up to 99% of the main substance, has good solubility in water,

- safe - non-toxic, sterile and pyrogen-free.

The excipients and solvent are safe to use, widespread and known in their technology.

The possibility of carrying out the invention can be demonstrated by the examples presented below.

Example 1. In 10 liters of water for injection dissolve 100 mg of sodium citrate to a concentration of 0.01 g / l, then add a sample of sodium deoxyribonucleate in the amount of 1 g to the solution. Stir the solution for 10 minutes until complete dissolution. Next, 50 g of sodium chloride is added to the solution, the solution is stirred, heated to a temperature of 25 ° C and filtered through a 0.65 µm and 0.2 µm filter. The sterile solution is poured into sterilized glass vials with a volume of 20 ml, 50 ml, 100 ml and hermetically sealed. The resulting product is used as irrigation of the affected parts of the skin - burns, frostbite as a wound healing agent.

Example 2. 1.0 g of sodium chloride is dissolved in 10 liters of water for injection to a concentration of 0.1 g / l, then a sample of sodium deoxyribonucleate in an amount of 25.0 g is added to the solution. The solution is stirred for 5 minutes until complete dissolution. Next, 10 g of sodium chloride is added to the solution, the solution is stirred, heated to a temperature of 28 ° C and filtered through a 0.65 μm and 0.2 μm filter. The sterile solution is poured into sterilized glass vials with a volume of 10 ml, 20 ml and hermetically sealed.

The resulting agent is used as drops in the nose or spray in the nasopharynx as a remedy for colds - an immunomodulator.

Example 3. 10.0 g of sodium chloride are dissolved in 10 liters of water for injection to a concentration of 1.0 g / l, then a weighed portion of sodium deoxyribonucleate in an amount of 150.0 g is added to the solution. The solution is stirred for 10 minutes until complete dissolution. Next, 100.0 g of sodium chloride is added to the solution, the solution is stirred, heated to a temperature of 33 ° C and filtered through filters of 0.8 μm, 0.65 μm and 0.2 μm. The sterile solution is poured into sterilized glass vials with a volume of 2 ml, 5 ml and hermetically sealed.

The resulting agent is used as an intravenous or intramuscular injection for diseases of the gastrointestinal tract, heart, genitourinary system as a reparant, an immunomodulator.

Example 4. In 10 liters of water for injection dissolve 10.0 g of sodium chloride to a concentration of 1.0 g / l, then add a sample of sodium deoxyribonucleate in the amount of 250.0 g to the solution. The solution is stirred for 20 minutes until complete dissolution. Next, 50.0 g of sodium chloride is added to the solution, the solution is stirred, heated to 35 ° C and filtered through filters of 0.8 μm, 0.65 μm and 0.2 μm. The sterile solution is packaged in polymer capsules with a volume of 0.1 ml, 0.2 ml, 0.5 ml.

The resulting agent is used as an enteric capsule for diseases of the small and large intestines, the genitourinary system as a reparant, regenerant, immunomodulator.

To study pyrogenicity, the resulting solutions were injected intravenously at the rate of 2.5 mg of active substance per kg of animal body weight.

It was found that solutions of sodium deoxyribonucleate obtained by the proposed method do not cause pyrogenic reactions during the entire shelf life. The maximum increase in body temperature of each rabbit did not exceed 0.2 ° C, and the sum of the maximum temperature fluctuations in three rabbits (i.e. conditional febrile index) was 0.5 ° C, which meets the requirements for injectable drugs of the State and European Pharmacopoeias by pyrogenicity. The preparation used for comparison according to the prototype, although it is non-pyrogenic for some time after preparation, already by the end of the shelf life causes an increase in temperature and the conditional febrile index is 2.0 ° C.

Denaturation of DNA is accompanied by an increase in optical absorption in the UV region of purine and pyrimidine bases. This phenomenon is called the hyperchromic effect. A solution of native DNA has an optical density at 260 nm that is 40% lower than the optical density of a mixture of nucleotides. Also, the decomposition of nucleic acid preparations is evidenced by an increase in the viscosity of their solutions during storage.

Analyzes of all received preparations during storage showed: preservation of authenticity; colorlessness; transparency; sterility; non-toxicity; apyrogenicity; preservation of the degree of nativeness; hyperchromic effect 46-48%; RNA content less than 0.1%; protein content - up to 0.1%. While a solution prepared from a substance with similar characteristics according to the prototype showed a decrease in the hyperchromic effect to 40%, the content of impurities increased and the data on sterility and apyrogenicity were reduced.

Claims (2)

1. A method of obtaining a preparation of a sodium salt of deoxyribonucleic acid in the form of a sterile aqueous solution, characterized in that a solution of an auxiliary substance is preliminarily prepared, which is sodium chloride or sodium citrate, with a concentration of up to 0.01-0.1 g / l, then in the resulting a weighed portion of the sodium salt of deoxyribonucleic acid is dissolved in the solution until a concentration of 0.1-25.0 g / l is obtained, sodium chloride is added to a concentration of excipients of 5.0-12.0 g / l, the resulting solution is heated to a temperature of 30-35 ° C. subsequent sterilizing filtration and packaging. 2. The method according to claim 1, characterized in that the sterilizing filtration is carried out using filters with a pore diameter of 1.0 to 0.2 μm.