An aqueous solution of interferon-alpha-two people for injection

 

(57) Abstract:

The invention relates to biotechnology and is intended for use in the pharmaceutical industry for the production of a stable aqueous solution of genetically engineered interferon-alpha-two, not containing albumin for injection for treatment of various viral diseases and cancer. An aqueous solution of interferon-alpha-two people for injection contains a buffer to maintain the pH of the solution of 4,5-7,0, nonionic detergent, preservative with antimicrobial activity, agent, providing isotonicity solution, water for injection and further comprises oxidized glutathione and/or citric acid to prevent the loss of activity of interferon. Effect: method allows to obtain a solution of interferon-alpha-two for injection, stable under standard storage conditions (5C) and during storage at elevated temperatures (25, 35C). 2 C.p. f-crystals, 1 table.

The invention relates to biotechnology and is intended for use in the pharmaceutical industry for the production of a stable aqueous solution of genetically engineered interferon-alpha-two (IFN al is olivani.

Interferon is highly protein, single therapeutic dose of interferon is tens of micrograms. Such dilute protein solutions quickly lose their biological activity, readily inactivated by various environmental factors such as temperature, oxygen, heavy metals, physical effects.

To ensure optimal consumer properties, the ingredients of a solution of interferon should be carefully selected from a variety of potentially suitable agents and go well with each other. The choice of buffer system and bacteriostatic is of great importance for the stability and activity of proteins and largely determines the safety of the drug. The loss of activity can be prevented by adding an aqueous solution of protein of different stabilizing agents.

Currently, Russia is dry dosage form of recombinant interferon-alpha-two - IFN-EU”, containing in its composition serum albumin human (HSA) [1]. However, this is due to the risk of viral contamination and allergic reactions for individuals with hypersensitivity to preparations of the proteins in the blood that is not used HSA.

For example, an aqueous solution of interferon-alpha containing as a buffer system ammonium acetate with pH value differs by more than one unit from the isoelectric point of the protein, nonionic detergent, and in some cases the regulator, do not contain preservatives [2].

However, the exclusion of part of the preparation of the preservative in the conditions of large-scale production requires additional measures to ensure the sterility of the drug and cannot warrant the risk of bacterial contamination of protein solutions. In addition, the lack of technology for purging with inert gases may lead to decreased stability of the protein during storage due to its oxidation by oxygen in the air.

Also known solution of interferon-Alfa for injection containing a buffer system that supports the value of pH in the range of 4.5 to 7.1, helatoobrazovatel, nonionic detergent (tween - 80 or tween - 20) in an amount necessary for the stabilization of interferon-alpha and prevent loss of its activity, the agent providing isotonicity solution, preservative with antimicrobial activity, and water for injection, at the same time he kept the equipment contains disubstituted phosphate of sodium and monosubstituted sodium phosphate, as the chelating agent contains disodium EDTA or citric acid, as agent, providing isotonicity - sodium chloride, and as a preservative is a substance selected from the group comprising metacresol, phenol, methylparaben, propylparaben, or a mixture thereof.

As interferon offer an aqueous solution containing interferon-alpha-[3].

Closest to the proposed technical solution (prototype) is an aqueous solution of interferon containing interferon-alpha, with a buffer system - ammonium acetate or sodium lactate, pH 4.5-5.5, containing non-ionic detergent and benzyl alcohol as preservative [4].

In solutions of interferon-alpha-[3, 4], containing 3106to 36106IU/ml when stored for 3 months at a temperature of 5 ° C is maintained more than 90% of the original primary component, the results of high performance liquid chromatography (VGH) with reversed phase.

However, during storage of the drug at higher temperatures (25, 35C), in conditions of sudden acceleration destructive processes (oxidation, and others), there is a lack of stability of interferon, especially when the most common therapeutic practices what's solutions succeed in obtaining their medication in the “almost complete absence of dissolved oxygen”, as well as inclusion in the diluted protein solution ingredients that prevent destructive processes and contributing to the preservation of biologically active native protein structure.

The technical result of the invention is to obtain aqueous solution of highly purified interferon-alpha-two for injection, stable under standard conditions of storage and during storage at elevated temperatures.

This technical result is achieved in that in an aqueous solution of interferon for injection containing a buffer to maintain the pH of the solution of 4,5-7,0, nonionic detergent, preservative with antimicrobial activity, agent, providing isotonicity solution, water for injection, further comprises oxidized glutathione and/or citric acid, in the following ratio of components in 1 ml of:

Interferon-alpha-two ME, (1-36)106

Nonionic detergent, mg 0,01-1,0

Buffer to maintain pH 4,5-7,0 mM 10,0-25,0

Preservative, mg 0,06-20,0

Okalany glutathione, mg 0,01-0,1

Or citric acid 0,8-2,0

Or oxidized glutathione 0,01-0,1

And citric acid 0,8-2,0

Agent, providing

An aqueous solution of interferon contains highly purified recombinant interferon-alpha-two of Taurus include strains of E. coli SG 20050 IF16 in number (1-36)106ME per 1 ml (total protein content of 0.01-0.16 mg).

As a buffer system to maintain the pH 4,5-7,0 aqueous solution of interferon contains phosphate buffer at a concentration of 10-25 mM or buffer - sodium phosphate disubstituted - citric acid at a concentration of 10-25 mM.

As preservative aqueous solution of interferon contains methylparaben at a concentration of 0.6-1.8 mg/ml or a mixture of methylparaben at a concentration of 0.6-1.8 mg/ml and propylparaben at a concentration of 0.06 to 0.18 mg/ml, or metacresol in a concentration of 0.5-2.0 mg/ml, or benzyl alcohol at a concentration of 8.0 to 20.0 mg/ml

As a non-ionic detergent it contains tween-80 or tween-20 in an amount of 0.01-1.0 mg/ml

As an agent, providing isotonicity, the proposed solution contains sodium chloride or glycine. The amount of a particular agent depends on the composition of the solution in the case of sodium chloride is in the range 1-10 mg/ml

As the chelating agent contains disodium salt of EDTA in an amount of 0.01-1.0 mg/ml, in the case of the use of the phosphate buffer system or LASS="ptx2">As the stabilizing additive is injected oxidized glutathione at a concentration of 0.01-0.1 mg/ml, which is the natural regulator of cell metabolism, and/or citric acid at a concentration of 0.8-2.0 mg/ml

The addition of oxidized glutathione is a natural metabolite of cells contributes to the protection of interferon from significant degradation and prevents the loss of interferon during storage. Oxidized glutathione, undergoing degradation simultaneously with interferon, thus acts as a protective agent. In addition, oxidized glutathione prevents recovery of cystine, prevents accidental disulfide bonds, leading to the formation of inactive monomers and oligomers.

It was also established that the oxidized glutathione is an endogenous stimulator of the production of cytokines and hemopoietic factors, and use it as a pharmaceutical preparation to achieve significant therapeutic effect in people suffering from cancer and infectious diseases. Such an additive, not impairing the stability of the composition, not only has the property of “protective” agent for molecules of interferon, but is immunocorrective additives injected citric acid, which is an important product of metabolism in living organisms, involved in the tricarboxylic acid cycle and glyoxylate cycle. Citric acid and its salts are widely used as components of many drugs, including injectable. As part of the preparation of citric acid is used not only as an integral part of the isotonic buffer solution, but also as an antioxidant and helatoobrazovatel. Due to the latter property can be excluded other chelating agents such as disodium salt of EDTA, because the latter requires caution because of its potential impact on physiological processes.

The invention is illustrated by the following examples of specific performance.

The procedure for obtaining an aqueous solution of interferon-alpha-two

Solutions interferon different personnel get in aseptic conditions in the kelp-box. In a glass measuring vessel loaded hinge components, except for the twin and glutathione. Dissolved under vigorous stirring in water for injection (2/3 of the volume of the solution). The resulting solution was rinsed with argon. Then add twin and glutathione. The solution is stirred and add rascia water for injection, the solution is filtered using a filter with a pore size of 0.22 μm and poured into pre-purged with argon glass vials or ampoules.

Example 1

An aqueous solution of interferon-alpha-two in the sodium phosphate buffer using oxidized glutathione at a concentration of 0.03 mg/ml and methylparaben as preservative is prepared similarly to the procedure for obtaining the following ratio of 1 ml of:

Interferon-alpha-two, ME 4,0106

Sodium phosphate disubstituted dvenadtsatiletny, mg 4,5

Sodium phosphate one-deputizing dowolny, mg 1,5

The disodium salt of EDTA, mg 0,1

Tween - 80, mg 0,1

Methylparaben, mg 1,2

Oxidized glutathione (mg 0,03

Sodium chloride, mg 7,5

Water ml To 1.0

Example 2

An aqueous solution of interferon-alpha-two in the sodium phosphate buffer using oxidized glutathione at a concentration of 0.1 mg/ml and methylparaben as preservative is prepared similarly to the procedure for obtaining the following ratio of 1 ml of:

Interferon-alpha-two, ME 6,0106

Sodium phosphate disubstituted dvenadtsatiletny, mg 4,5

Sodium phosphate one-deputizing dowolny, mg 1,5 ion, mg 0,1

Sodium chloride, mg 7,5

Water ml To 1.0

Example 3

An aqueous solution of interferon-alpha-two-based buffer sodium phosphate disubstituted-citric acid with a concentration of citric acid 1.0 mg/ml and methylparaben as preservative is prepared similarly to the procedure for obtaining the following ratio of 1 ml of:

Interferon-alpha-two, ME 4,0106

Sodium phosphate disubstituted dvenadtsatiletny, mg 8,3

Citric acid dnovotny, mg 1,0

Sodium chloride, mg 6,2

Tween-80, mg 0,1

Methylparaben, mg 1,2

Water ml To 1.0

Example 4

An aqueous solution of interferon-alpha-two-based buffer - sodium phosphate disubstituted-citric acid with a concentration of citric acid 1.5 mg/ml and methylparaben as preservative is prepared similarly to the procedure for obtaining the following ratio of 1 ml of:

Interferon-alpha-two, ME 4,0106

Sodium phosphate disubstituted dvenadtsatiletny, mg 8,5

Citric acid dnovotny, mg 1,5

Sodium chloride, mg 6,1

Tween - 80, mg 0,1

Methylparaben, mg 1,5

Water ml To 1.0

Example 5

An aqueous solution of interferon-Ilf-two snowm alcohol as a preservative is prepared similarly to the procedure for obtaining the following ratio of 1 ml of:

Interferon-alpha-two, ME 4,0106

Sodium phosphate disubstituted dvenadtsatiletny, mg 8,5

Citric acid dnovotny, mg 1,5

Sodium chloride, mg 6,1

Tween-80, mg 0,1

Benzyl alcohol, mg 10,0

Water ml To 1.0

Example 6

An aqueous solution of interferon-alpha-two-based buffer - sodium phosphate disubstituted-citric acid with a concentration of citric acid 1.5 mg/ml, using oxidized glutathione at a concentration of 0.1 mg/ml benzyl alcohol as a preservative is prepared similarly to the procedure for obtaining the following ratio of 1 ml of:

Interferon-alpha-two, ME 4,0106

Sodium phosphate disubstituted dvenadtsatiletny, mg 8,5

Citric acid dnovotny, mg 1,5

Tween-80, mg 0,1

Sodium chloride, mg 6,1

Oxidized glutathione (mg 0,1

Benzyl alcohol, mg 10,0

Water ml To 1.0

Example 7

An aqueous solution of interferon-alpha-two-based buffer - sodium phosphate disubstituted-citric acid with a concentration of citric acid 1.3 mg/ml, using oxidized glutathione at a concentration of 0.03 mg/ml and methylparaben as preservative prepared similarly procedurecall disubstituted dvenadtsatiletny, mg 8,5

Citric acid dnovotny, mg 1,3

Tween-80, mg 0,1

Sodium chloride, mg 6,1

Oxidized glutathione (mg 0,03

Methylparaben, mg 1,2

Water ml To 1.0

Example 8

An aqueous solution of interferon-alpha-two in the sodium phosphate buffer using metacresol as preservative is prepared similarly to the procedure for obtaining the following ratio of 1 ml of:

Interferon-alpha-two, ME 4,0106

Sodium phosphate disubstituted dvenadtsatiletny, mg 4,5

Sodium phosphate one-deputizing dowolny, mg 1,5

The disodium salt of EDTA, mg 0,1

Tween-80, mg 0,1

Sodium chloride, mg 7,5

Metacresol, mg 1,5

Water ml To 1.0

Example 9

An aqueous solution of interferon-alpha-two in ammonium acetate buffer, pH 5.0, using benzyl alcohol as preservative, not containing auxiliary ingredients, prepared similarly to the procedure for obtaining, without the addition of glutathione, in the following ratio to 1 ml of:

Interferon-alpha-two, ME 4,0106

Ammonium acetate, mg 0,77

Tween - 80, mg 0,2

Benzyl alcohol, mg 10,0

Sodium chloride, mg 7,2

Water for injection, To 1.02 ml">Obtained during the execution of this technical solution is stable aqueous solutions of interferon-alpha-two were stored in ampoules or in sterilized vials made of glass for medicines, sealed with rubber stoppers and aluminum caps.

Aqueous solutions of interferon-alpha containing additives (examples 1-7). sterile, non-toxic on the culture of lung cells of a human embryo L-68 and in experiments on animals (white mice, Guinea pigs). Cause a little (no more than 2C) temperature increase when administered intravenously to rabbits, i.e. subaeruginosa. Are biological (antiviral) activity characteristic of interferon-alpha-two during the titration in cell culture L-68.

According to the results of tests on cell cultures and animals can be concluded about the use of the proposed water solutions as injectable drugs for the treatment of viral diseases and cancer man.

Data on biological activity and physicochemical stability of interferon-alpha in two aqueous solutions after storage at different temperatures (5, 25, 35C) are presented in table (examples 1-9).

For konasana in examples 1-7, use a method of suppressing cytopathogenic steps of virus encephalomyocarditis mice in the culture of lung cells of a human embryo L-68, grown in a monolayer in a 96-well microplate.

The content of interferon in solutions (in %) determined by high performance liquid chromatography on a column of reversed phase (VGH), enzyme-linked immunosorbent assay (ELISA) using components production 000 “Protein component” in St. Petersburg) and electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS page-ordinator) densitometrically.

An aqueous solution of interferon containing no stabilizing additive having a pH of 6.0-7.0 and received by analogy with the invention [3] (example 8), maintains a reasonable stability when stored at the temperature of 5 ° C for 12 months (stored more than 90% of the main component). A solution of interferon obtained according to the invention [4] (example 9) with a pH of 4.5-5.0, less stable, saves up to 78% of the interferon. In both cases, at elevated temperatures (25 and 35C) observed a reduction in the content of the active component to 70-80%, which is confirmed by various analytical methods.

Introduction as a protective additive oxidized CH the AI its aqueous solutions as when the temperature of 5 ° C within 12 months, compared with a solution, described in example 9, and at elevated temperatures of 25C and 35C. The safety of the interferon is 90-95%, with no reduction of the biological activity of the solutions.

The stability of the solutions of interferon is also observed when using as an additional ingredient citric acid. Introduction citric acid (concentration: 1,0; 1,3; 1,5; mg/ml) in buffer to maintain the pH stability of the drug at different temperatures of storage (examples 3, 4, 7, 5), eliminates the use of other chelating agents, allows a wide range of bacteriostatic - benzyl alcohol, methyl - and propylparaben, metacresol etc.

Adding citric acid prevents oxidation of metacresol, keeping the solution of interferon colorless, while when storing some of the solutions containing metacresol and not containing citric acid, gradually, the solutions were purchased staining, especially at elevated temperatures.

Based on the analysis of the obtained experimental data it can be concluded that in aqueous solutions of interferon-alpha-two, containing as protective additives as oxidized, glutathione elevated temperatures (25C, 3 months. and 35C, 1 month). there is 100% preservation of the biological activity of interferon-alpha-two and not less than 95%, the stability of the active substance according to titration in cell culture L-68, VGH, ELISA and electrophoresis.

Thus, the introduction of additional ingredients eliminates a significant loss of interferon in the most commonly used in therapeutic practice doses (3,0106-6,0106IU/ml).

High thermal stability of aqueous solutions of interferon-alpha-two received under this technical solution provides the injectable dosage form of interferon with enhanced shelf life.

In addition, the introduction of glutathione, known as an immunomodulator [5], in the composition of a medicinal product may have a clinically significant effect in the treatment of viral diseases and cancer.

LITERATURE

1. The Fund 42-0241-1184-01. Reaferon-EC, powder for preparation of injection solutions.

2. RF patent №2180233, a 61 K 38/00, bull. No. 7, publ. 10.03.2002.

3. RF patent №2157236, a 61 K 38/21, bull. No. 28, publ. 10.10.2000.

4. RF patent №2113845, a 61 K 9/08, bull. No. 18, publ. 27.06.1998.

5. RF patent №2089179, a 61 K 9/08, bull. No. 25, publ. 10.09.1997.

Interferon-alpha-two ME, (1-36)106

Nonionic detergent, mg 0,01-1,0

Buffer to maintain pH 4,5-7,0 mm 10,0-25,0

Preservative, mg 0,06-20,0

Oxidized glutathione, mg, or 0.01-0.1

Citric acid 0,8-2,0

or

Oxidized glutathione and of 0.01-0.1

Citric acid 0,8-2,0

Agent, providing isotonicity

solution In the quantity

sufficient to give

isotonicity of the solution

Water for injection, ml To 1.0

2. Aqueous solution under item 1, characterized in that as a buffer system to maintain the pH 4,5-7,0 contains phosphate buffer at a concentration of 10-25 mm or buffer disubstituted sodium phosphate-citric acid at a concentration of 10-25 mm.

3. Aqueous solution under item 1, characterized in that as preservative contains methylparaben at a concentration of 0.6-1.8 mg/ml, or a mixture methylparaben/ml, or benzyl alcohol 8,0-20,0 mg/ml

 

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