RU2768656C1 - Antiviral agent in liquid form and method for its preparation - Google Patents

Abstract

FIELD: interferon-containing compositions production.

SUBSTANCE: inventions group relates to a technology for preparing interferon-containing compositions capable of maintaining their biological activity, which can be used as drugs for intranasal use, for example, in the form of drops or spray. The proposed method for preparing an antiviral agent in liquid form is characterized in that the following composition is used for preparation, mg in one ml: genetically engineered interferon, ME 1000-500000, povidone 5-100, macrogol 10-600, disodium edetate dihydrate 0.1- 0.8, sodium hydrophosphate dodecahydrate 5-20, potassium dihydrophosphate 1-10, sodium chloride 1-10, water - the rest, at the same time, the preparation is carried out in two stages, at the first of which sodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, disodium edetate dihydrate, povidone and macrogol are successively dissolved in water at room temperature, and the kinematic viscosity of the solution is maintained in the range of 2.5-4, 5 mm²/s, adjusting the amount and ratio of povidone and macrogol, after which the solution is filtered and sterilized, and at the second stage, the genetically engineered interferon is heated to a temperature of 19-21°C, then, under aseptic conditions, it is gradually introduced into the solution of the first stage with a laminar flow and mixed. An antiviral agent is also provided in liquid form, obtained by the above method.

EFFECT: increasing the shelf life without reducing the activity of interferon, which is achieved by the technology of preparing an antiviral agent.

2 cl, 9 ex

Description

The invention relates to pharmacology, and in particular to a technology for the preparation of interferon-containing compositions capable of maintaining their biological activity, which can be used as medicines for intranasal use, for example, in the form of drops or spray.

The use of interferon preparations of both natural and recombinant or genetically engineered origin is widely known. Interferon preparations have not only antiviral activity, but also a pronounced immunomodulatory effect, causing a number of positive changes in homeostasis, antitumor effect, etc. (RU 2057544).

In our country, since the 70s, natural human leukocyte interferon has been widely used in healthcare practice for the treatment and prevention of influenza and ARVI, the raw material for the manufacture of which are expensive donor blood leukocytes (RU 2033180, RU 2108804).

Also known is an antiviral agent for intranasal use (RU 2095081) containing human interferon, a biocompatible polymer -polyglucin 6% solution and a buffer mixture with the following content of components in 1 ml of solution:

Interferon, ME - (1-6.6)⋅106

Biocompatible polymer (polyglucin) - 5-30

Buffer mixture - up to a solution pH of 7.0-7.6.

Preparations of leukocyte origin, like any other blood products, are potentially unsafe in terms of contamination with viruses (hepatitis, herpesvirus, cytomegalovirus infections, AIDS, slow infections, etc.), and are also very expensive to manufacture.

In this regard, recombinant or genetically engineered interferon preparations are increasingly used in healthcare practice, as the most purified - the degree of purification is at least 98% (FS 42-3279-96; VFS 42-2989-97; RU 2073522)

These drugs are effective in oncological practice with parenteral use of massive doses (from 3 to 10 and more million IU per day) with long and multiple courses. But such dosages often cause side effects - impaired hematopoiesis, suppression of the immune system, the formation of antibodies to interferon, etc.

However, the experience gained in recent years in the clinical use of interferons indicates the possibility of increasing their effectiveness by using appropriate dosage forms (taking into account the pathogenetic characteristics of specific diseases) in order to ensure high levels of interferon in the focus of viral damage. At the same time, interferon has an antiviral, immunomodulatory effect, but neither cytostatic nor other side effects are manifested. This makes it expedient to develop various dosage forms of interferons for topical use (suppositories, ointments, drops, sprays, aerosols, etc.)

Currently, one of the most difficult problems of protein-containing medicines, in particular interferon preparations, is their stability.

Like other proteins, interferon undergoes, especially in aqueous solutions, modification and chemical degradation due to the processes of proteolysis, oxidation, disulfide exchange, etc. In addition, its biological activity is adversely affected by factors such as aggregation, precipitation and adsorption.

To prevent a decrease in the activity of interferon, various stabilizing additives are introduced into the composition of the dosage form.

Known lyophilized drug with immunomodulatory and antiviral activity "Neoferon", including recombinant human interferon alpha-2, hybrid protein T-TNF-T, recombinant interferon gamma, stabilizing additive containing plasma-substituting solution polyglucin, saline buffer system with a pH of 7.0-7 ,2 and pyrogen-free water (RU 2129878). The drug is recommended to be stored at +4°C.

The disadvantage of the drug is that the stabilizing composition does not provide long-term storage of the drug in liquid form, as well as the need for storage at sufficiently low temperatures - not higher than +4°C.

Known drug based on recombinant alpha-2 human interferon for injection in a dry lyophilized form, including the substance of recombinant human alpha-2 interferon, a saline buffer system and a stabilizing additive containing a plasma-substituting solution, according to the invention additionally contains polyhydric alcohol and urea or EDTA, and in as a plasma-substituting solution - polyglucin, or reopoliglyukin, or hemodez, or a mixture of these components in any ratio. Storage of this drug is possible at room temperature (RU 2236866).

A composition of an interferon preparation is known, which is stable for storage both in dry and liquid form, including after preparation of a solution for injection, liquid aerosol eye drops, etc. This composition contains 0.05-0.1 mg of acetate-tocopherol per 1-100 million IU of interferon-2, 0.02-10 mg of organic acid and buffer-forming salts that provide pH 7.4 in an aqueous solution. As organic acids, organic acids capable of crystallizing without decomposition, such as ascorbic, citric, glycine, uric, succinic, etc., are used. As a buffer solution, 1M phosphate, acetate, carbonate or Tris buffer solutions are used (RU 2218934).

The disadvantages of the known compositions are a complex set of components, including preservatives (acids, urea, alcohols) included in the composition to ensure the stability of the antiviral properties of liquid forms and the presence of other allergenic excipients (acetate-tocopherol, acetate buffers, plasma-substituting solutions, etc.) can cause allergic reactions in patients, this limits the therapeutic possibilities of such liquid dosage forms (Kalyavin E.A. Technology and research of dosage forms of interferon. Diss. for the degree of candidate of pharmaceutical sciences. M., 1991).

A commercially available preparation in liquid form "Grippferon" is widely known, containing interferon alpha-2b human recombinant and excipients: disodium edetate dihydrate; sodium chloride; sodium hydrogen phosphate dodecahydrate; potassium dihydrogen phosphate; povidone; macrogol 4000; purified water (https://www.rlsnet.ru/tn_index_id_14719.htm). This composition has no negative side effects.

The main disadvantage of this composition is the shelf life of no more than 2 years in unopened packaging and no more than 30 days with opened packaging. Given that interferon preparations are included in the list of vital and essential drugs for medical use (Decree of the Government of the Russian Federation of October 12, 2019 N 2406-r, as amended by orders of the Government of the Russian Federation of April 26, 2020 N 1142-r, of October 12 2020 N 2626-r, November 23, 2020 N 3073-r, November 23, 2020 N 3073-r), it is necessary to ensure an adequate supply of these medicines. Therefore, increasing the shelf life without reducing the activity of interferon and without the use of allergenic preservatives and stabilizers is an urgent task.

The technical result of this invention is to increase the shelf life without reducing the activity of interferon.

The technical result is achieved by the technology of preparing an antiviral agent in liquid form, characterized in that the following composition is used for preparation, mg in one ml:

genetically engineered interferon, ME 1000-500000 povidone 5-100 macrogol 10-600 disodium edetate dihydrate 0.1-0.8 sodium hydrogen phosphate dodecahydrate 5-20 potassium dihydrogen phosphate 1-10 sodium chloride 1-10 water rest,

at the same time, the preparation is carried out in two stages, at the first of which sodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, disodium edetate dihydrate, povidone and macrogol are successively dissolved in water at room temperature, and the kinematic viscosity of the solution is maintained in the range of 2.5-4, 5 mm ² /sec, adjusting the amount and ratio of povidone and macrogol, after which the solution is filtered and sterilized, and at the second stage, the genetically engineered interferon is heated to a temperature of 19-21 ° C, under aseptic conditions, it is gradually introduced into the solution of the first stage with a laminar flow and mixed.

When implementing this preparation method, a preparation is obtained that ensures its storage at a temperature of 2 to 8°C for at least 2.5 years in a sealed package.

As a result of these sequences, the nature and modes of mixing, a specially structured composition of interferon and polymers is formed in the resulting preparation in the presence of a complex of salts, which stabilizes the properties of the protein and preserves the activity of interferon for a long time. This result of the thermodynamic and chemical stability of the composition was obtained after numerous experiments and, apparently, is associated with the ability of macromolecules to conformations under the influence of external factors and the mutual influence of various types of macromolecules at such conformations with the formation of structures that are stable in their properties in liquid solutions (G. Moravec. Macromolecules in solution, Moscow, Mir, 1967, M. S. Arzhakov, A. E. Zhirnov, A. A. Efimova, et al., “High-molecular compounds,” http://vmsmsu.ru/vmsbaku. pdf).

The method is illustrated by the following examples.

Example 1

Water is pretreated in the Millipore Elix system, which combines two purification technologies: reverse osmosis on high-performance polyamide membranes and continuous electrodeionization on synthetic ion-exchange resins.

100 l of purified water is poured into the mixing tank, the stirrer is turned on and the ingredients are sequentially dissolved: 2.388 kg of sodium hydrogen phosphate dodecahydrate; 0.908 kg of potassium dihydrogen phosphate; 0.820 kg of sodium chloride; 0.100 kg disodium edetate dihydrate; 20.0 kg macrogol 4000; 2.0 kg of low molecular weight polyvinylpyrrolidone. Stir until complete dissolution of the reagents, bring the volume to 200 l with purified water, mix and take a sample to control the pH and viscosity of the resulting solution. The pH of the solution should be in the range of 6.0-7.0. Kinematic viscosity of the solution 3.5 mm ² /sec. (control is carried out with a VPZh-2 viscometer, capillary diameter 0.56 mm).

The resulting solution is filtered through a filter unit with filter elements: nominal retention threshold 5.0 μm at the inlet, nominal retention threshold 0.5 μm at the outlet.

The filtered solution is sterilized in a BIOR reactor by supplying steam to the jacket of the BIOR reactor (solution sterilization mode: temperature 123°C, pressure 0.3 MPa, sterilization duration 45 minutes. After sterilization, the solution is cooled to a temperature of 17°C. Cooling is carried out with constant stirring.

Vials with frozen substance of genetically engineered interferon from the refrigerator are placed in a water bath, thawed and heated to a temperature of 20°C, stirring occasionally.

Alpha-, beta-, gamma-interferon or their mixture in any combination can be used as genetically engineered interferon.

A specially prepared genetically engineered interferon in the amount of 10,000 IU/ml is introduced in a laminar flow into the BIOR reactor with the solution of the first stage under sterile conditions. After the introduction of the entire predetermined amount of interferon, the solution is stirred. The finished product is sent for packaging.

Example 2

Water is preliminarily purified in the Millipore Elix system by reverse osmosis on high-performance polyamide membranes and continuous electrodeionization on synthetic ion-exchange resins.

100 l of purified water is poured into the mixing tank, the stirrer is turned on and the ingredients are sequentially dissolved: 2.388 kg of sodium hydrogen phosphate dodecahydrate; 0.908 kg of potassium dihydrogen phosphate; 0.820 kg of sodium chloride; 0.100 kg disodium edetate dihydrate; 20.0 kg macrogol 4000; 2.0 kg of low molecular weight polyvinylpyrrolidone. Stir until complete dissolution of the reagents, bring the volume to 200 l with purified water, mix and take a sample to control the pH and viscosity of the resulting solution. The pH of the solution should be in the range of 6.0-7.0. Kinematic viscosity of the solution 3.5 mm ² /sec. (control is carried out with a VPZh-2 viscometer, capillary diameter 0.56 mm).

The resulting solution is filtered through an installation with filter elements: at the inlet, the nominal retention threshold is 5.0 μm, at the outlet, the nominal retention threshold is 0.5 μm.

The pre-filtered solution is sterilized by filtration through membranes with a retention threshold of 0.22 μm into a pre-sterilized BIOR reactor. The filtration rate is maintained within 0.8-10.0 l/min.

Vials with frozen substance of genetically engineered interferon from the refrigerator are placed in a water bath, thawed and heated to a temperature of 20°C, stirring occasionally.

Alpha-, beta-, gamma-interferon or their mixture in any combination can be used as genetically engineered interferon.

Specially prepared genetically engineered interferon in the amount of 10,000 IU/ml, under sterile conditions, is injected in a laminar flow into the BIOR reactor with a sterile solution of the first stage. After the introduction of the entire predetermined amount of interferon, the solution is stirred. The finished product is sent for packaging in the form of nasal drops or spray.

Example 3

The preparation of an antiviral agent in the form of nasal drops or spray is carried out analogously to example 1, but the ingredients are taken in the following amount per 200 liters of water: 1.0 kg of sodium hydrogen phosphate dodecahydrate; 1.9 kg of potassium dihydrogen phosphate; 0.20 kg of sodium chloride; 0.02 kg disodium edetate dihydrate; 60.0 kg macrogol 4000; 1.0 kg of low molecular weight polyvinylpyrrolidone. The viscosity of the solution is 3.2 mm / sec. The solution of the first stage is cooled to 13°C, and interferon in the amount of 100,000 IU/ml is heated to 21°C before being added to the solution.

Example 4

The preparation is carried out analogously to example 1, but the ingredients are taken in the following amount per 200 liters of water: 4.0 kg of sodium hydrogen phosphate dodecahydrate; 0.2 kg of potassium dihydrogen phosphate; 2.0 kg of sodium chloride; 0.16 kg disodium edetate dihydrate; 120.0 kg of macrogol 4000; 1.6 kg of low molecular weight povidone. Solution viscosity 4.5 mm ² /sec. The solution of the first stage is cooled to 15°C, and interferon in the amount of 500,000 IU/ml is heated to 19°C before being added to the solution.

Example 5

The preparation is carried out analogously to example 1, but the ingredients are taken in the following amount per 200 liters of water: 1.0 kg of sodium hydrogen phosphate dodecahydrate; 2.0 kg of potassium dihydrogen phosphate; 1.0 kg of sodium chloride; 0.08 kg disodium edetate dihydrate; 2.0 kg macrogol 4000; 20 kg of low molecular weight povidone. The viscosity of the solution is 3.8 mm ² /sec. The solution of the first stage is cooled to 17°C, and interferon in the amount of 300,000 IU/ml is heated to 20°C before being added to the solution.

Example 6

The preparation is carried out analogously to example 1, but the ingredients are taken in the following quantity per 200 liters of water: 2.5 kg of sodium hydrogen phosphate dodecahydrate; 1.0 kg of potassium dihydrogen phosphate; 0.2 kg sodium chloride; 0.02 kg disodium edetate dihydrate; 40 kg macrogol 4000; 10 kg of low molecular weight povidone. Solution viscosity 2.5 mm ² /sec. The solution of the first stage is cooled to 14°C, and interferon in the amount of 1000 IU/ml is heated to 20°C before being added to the solution.

Example 7

The preparation of an antiviral agent in liquid form in the form of nasal drops or spray is carried out analogously to example 2, but the ingredients are taken in the following amount per 200 liters of water: 1.0 kg of sodium hydrogen phosphate dodecahydrate; 1.9 kg of potassium dihydrogen phosphate; 0.20 kg of sodium chloride; 0.02 kg disodium edetate dihydrate; 60.0 kg macrogol 4000; 1.0 kg of low molecular weight polyvinylpyrrolidone. The viscosity of the solution is 3.2 mm ² /sec. Interferon in the amount of 100,000 IU/ml is heated to 21°C before being added to the solution.

Example 8

The preparation is carried out analogously to example 2, but the ingredients are taken in the following amount per 200 liters of water: 4.0 kg of sodium hydrogen phosphate dodecahydrate; 0.2 kg of potassium dihydrogen phosphate; 2.0 kg of sodium chloride; 0.16 kg disodium edetate dihydrate; 120.0 kg of macrogol 4000; 1.6 kg of low molecular weight povidone. Solution viscosity 4.5 mm ² /sec. Specially prepared interferon in the amount of 500,000 IU/ml is heated to 19°C before being added to the solution.

Example 9

The preparation is carried out analogously to example 2, but the ingredients are taken in the following quantity per 200 liters of water: 2.5 kg of sodium hydrogen phosphate dodecahydrate; 1.0 kg of potassium dihydrogen phosphate; 0.2 kg sodium chloride; 0.02 kg disodium edetate dihydrate; 40 kg macrogol 4000; 10 kg of low molecular weight povidone. Solution viscosity 2.5 mm ² /sec. Interferon in the amount of 1000 IU/ml is heated to 20°C before being added to the solution.

As a result of the preparation of the antiviral agent in accordance with the examples given, a clear, colorless liquid preparation is obtained, which can be used as nasal drops or spray. The finished product is checked for the following indicators: transparency, color, pH, viscosity, sterility, specific activity of interferon.

The drug is stored for 30 months at a temperature of 2 to 8°C without loss of medicinal properties (specific activity of genetically engineered interferon) and other indicators of the quality of the finished product.

The coefficient of variation of the values of these indicators, measured after 30 months of storage, relative to the values of these indicators determined for freshly prepared drops for all composition options and preparation modes did not exceed 7%. The color and transparency of the drug remained unchanged.

Thus, the preparation of an antiviral agent according to the proposed technology makes it possible to obtain such a structure and properties of the drug, in which the activity of interferon in an aqueous solution does not deteriorate during storage for at least 30 months.

Claims (5)

1. A method for preparing an antiviral agent in liquid form, characterized in that the following composition is used for preparation, mg in one ml: genetically engineered interferon, ME 1000-500000 povidone 5-100 macrogol 10-600 disodium edetate dihydrate 0.1-0.8 sodium hydrogen phosphate dodecahydrate 5-20 potassium dihydrogen phosphate 1-10 sodium chloride 1-10 water - the rest, at the same time, the preparation is carried out in two stages, at the first of which sodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, disodium edetate dihydrate, povidone and macrogol are successively dissolved in water at room temperature, and the kinematic viscosity of the solution is maintained in the range of 2.5-4, 5 mm ² /s, adjusting the amount and ratio of povidone and macrogol, after which the solution is filtered and sterilized, and at the second stage, the genetically engineered interferon is heated to a temperature of 19-21 ° C, then, under aseptic conditions, it is gradually introduced into the solution of the first stage with a laminar flow and mix. 2. An antiviral agent in liquid form, characterized in that it is obtained by the method according to claim 1.