RU2238758C2 - Human interferon-alpha-two aqueous solution for injection - Google Patents

Abstract

FIELD: biotechnology, pharmaceutical industry, medicine, pharmacy.

SUBSTANCE: invention is designated for producing stable an aqueous solution of albumin-free genetic engineering interferon-α₂ for injection that is necessary for treatment of different viral and oncological diseases. Human interferon α₂ for injection comprises buffer for maintaining pH value of solution in the range 4.5-7.0, non-ionic detergent, preserving agent eliciting an antibacterial activity, an agent providing isotonic property of solution, water for injection and, additionally, it comprises oxidized glutathione and/or citric acid for prevention of interferon activity loss. Method provides preparing interferon-α₂ solution for injection that is stable under standard storage conditions (5^o C) and in storage under conditions of elevated temperature (25^o C, 35^o C).

EFFECT: improved and valuable properties of interferon solution.

3 cl, 1 tbl, 9 ex

Description

The proposed solution 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 alpha-2), which does not contain albumin, for injection, which is necessary for the treatment of various viral and oncological diseases.

Interferon is a highly active protein, a single therapeutic dose of interferon is tens of micrograms. Such diluted protein solutions quickly lose their biological activity, are easily inactivated by various environmental factors, such as temperature, atmospheric oxygen, the presence of heavy metals, and physical effects.

In order to ensure optimal consumer properties, the ingredients of the interferon solution must be carefully selected from a variety of potentially suitable agents and combine well with each other. The choice of a buffer system and bacteriostatic is of great importance for maintaining the stability and activity of the protein and in many respects determines the preservation of the drug. Loss of activity can be prevented if various stabilizing agents are added to the aqueous protein solution.

Currently, Russia is producing a dry dosage form of recombinant interferon-alpha-two - “reaferon-EC”, which contains human serum albumin (HSA) [1]. However, this is due to problems of the danger of viral infection and allergenicity for individuals with increased sensitivity to blood protein preparations, which in turn can lead to the formation of antibodies.

Aqueous solutions of interferon are known in which HSA is not used.

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

However, the exclusion of a preservative from the composition of the drug in a large-scale production requires additional measures to ensure the sterility of the drug and cannot fully guarantee the exclusion of the danger of bacterial infection of protein solutions. In addition, the lack of inert gas purging technology can lead to a decrease in protein stability during storage due to its oxidation by atmospheric oxygen.

Also known is a solution of interferon-alpha for injection, containing a buffer system that maintains the pH of the solution in the range of 4.5-7.1, a chelating agent, a non-ionic detergent (tween - 80 or tween - 20) in the amount necessary to stabilize interferon-alpha and prevent loss of its activity, an agent that provides isotonicity of the solution, a preservative with antimicrobial activity, and water for injection, while it contains (0.1-100.0) · 10 ⁶ IU / ml interferon-alpha.

As a buffer system, the proposed solution preferably contains disubstituted sodium phosphate and monosubstituted sodium phosphate, as a chelating agent it contains disodium EDTA or citric acid, sodium chloride as an isotonicity agent, and a substance selected from the group comprising metacresol as a preservative phenol, methylparaben, propylparaben or mixtures thereof.

As interferon, the proposed aqueous solution contains interferon alpha-two [3].

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

In solutions of interferon-alpha-two [3, 4] containing from 3 · 10 ⁶ to 36 · 10 ⁶ IU / ml, more than 90% of the initial main component is retained during storage for 3 months at a temperature of 5 ° С, according to the results of highly effective reverse phase liquid chromatography (HPLC).

However, when the drug is stored at an elevated temperature (25, 35 ° С), under the conditions of a sharp acceleration of destructive processes (oxidative, etc.), there is insufficient stability of interferon, especially at the most common dosage rates of the drug from 3 · 10 ^6- to 6 · 10 ⁶ IU / ml IFN alpha-2 [4].

It is possible to increase the thermal stability of aqueous solutions when their preparation is obtained under conditions of “almost complete absence of dissolved oxygen”, as well as by adding additional ingredients to the diluted protein solution that prevent destructive processes and help preserve the biologically active native protein structure.

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

The specified technical result is achieved by the fact that in an aqueous solution of interferon for injection, containing a buffer to maintain the pH of the solution 4.5-7.0, a nonionic detergent, a preservative with antimicrobial activity, an agent that provides isotonicity of the solution, water for injection, additionally contains oxidized glutathione and / or citric acid, in the following ratio of components per 1 ml:

Interferon-alpha-two ME, (1-36) · 10 ⁶

Nonionic detergent, mg 0.01-1.0

Buffer for maintaining pH 4.5-7.0, mM 10.0-25.0

Preservative, mg 0.06-20.0

Oxidized 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

isotonicity of the solution: In an amount sufficient to impart isotonicity to the solution

Water for injection, ml Up to 1.0

The aqueous interferon solution contains highly purified recombinant interferon-alpha-two of the inclusion bodies of the E. coli strain SG 20050 pIF16 in an amount of (1-36) · 10 ⁶ ME per 1 ml (total protein content 0.01-0.16 mg).

As a buffer system to maintain a pH of 4.5-7.0, an aqueous solution of interferon contains a phosphate buffer at a concentration of 10-25 mM or a buffer - sodium phosphate disubstituted-citric acid at a concentration of 10-25 mM.

As a preservative, an 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 0.06-0.18 mg / ml, either metacresol at a concentration of 0.5-2.0 mg / ml, or benzyl alcohol at a concentration of 8.0-20.0 mg / ml.

As a nonionic 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 this agent depends on the composition of the solution and in the case of sodium chloride is in the range of 1-10 mg / ml.

As a chelating agent it contains EDTA disodium salt in an amount of 0.01-1.0 mg / ml, in the case of using a phosphate buffer system or citric acid in the composition of the buffer - sodium phosphate disubstituted - citric acid in a concentration of 0.8-2.0 mg / ml

As a stabilizing additive, oxidized glutathione is introduced at a concentration of 0.01-0.1 mg / ml, which is a regulator of the natural metabolism of the cell, and / or citric acid at a concentration of 0.8-2.0 mg / ml.

The addition of oxidized glutathione - a natural metabolite of the cell - helps protect interferon from significant degradation and prevents the loss of interferon during storage. Oxidized glutathione, undergoing destruction simultaneously with interferon, thereby acts as a protective agent. In addition, oxidized glutathione prevents the restoration of cystines, prevents the occurrence of random disulfide bonds, leading to the formation of inactive monomers and oligomers.

It has also been established that oxidized glutathione is a stimulant of endogenous production of cytokines and hematopoietic factors, and its use as a pharmaceutical preparation allows to achieve a significant therapeutic effect in people suffering from cancer and infectious diseases. Such an auxiliary additive, which does not violate the stability of the composition, not only has the property of a “protective” agent for the interferon molecule, but is also an immunocorrector, therefore, the introduction of such an additive is advisable from the perspective of modern medicine [5].

Citric acid, which is an important metabolic product in living organisms, is involved in the tricarboxylic acid cycle and glyoxylate cycle as a stabilizing additive. Citric acid and its salts are widely used as components of many drugs, including injectable ones. As part of the drug, citric acid is used not only as an integral part of the isotonic buffer solution, but also as an antioxidant and chelating agent. Due to the latter property, other chelating agents, for example, disodium EDTA disodium salt, can be excluded, since the use of the latter requires caution because of its possible effect 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

Interferon solutions of various compositions are prepared under aseptic conditions in a laminar box. Samples of components, with the exception of tween and glutathione, are loaded into a glass measuring vessel. Dissolve with vigorous stirring in water for injection (2/3 of the volume of the solution). The resulting solution was purged with argon. Then add tween and glutathione. The solution is stirred and the calculated volume of the interferon solution is added. Again, carefully mix and adjust the volume of the solution to the final value with water for injection, filter the solution using a filter with a pore size of 0.22 μm and pour into glass vials or ampoules previously blown with argon.

Example 1

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

Interferon alfa-two, ME 4.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 4.5

Sodium phosphate monosubstituted, two-water, mg 1.5

Disodium EDTA, mg 0.1

Twin - 80 mg 0.1

Methylparaben, mg 1.2

Oxidized Glutathione, mg 0.03

Sodium chloride, mg 7.5

Water, ml Up to 1.0

Example 2

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

Interferon alfa-two, ME 6.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 4.5

Sodium phosphate monosubstituted, two-water, mg 1.5

Disodium EDTA, mg 0.1

Tween 80 mg 0.1

Methylparaben 1.5

Oxidized Glutathione, mg 0.1

Sodium chloride, mg 7.5

Water, ml Up to 1.0

Example 3

An aqueous solution of interferon-alpha-two based on a sodium phosphate disubstituted-citric acid buffer with a concentration of citric acid of 1.0 mg / ml and methyl paraben as a preservative is prepared similarly to the preparation procedure in the following ratio per 1 ml:

Interferon alfa-two, ME 4.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 8.3

Monohydric citric acid, mg 1.0

Sodium chloride, mg 6.2

Tween 80 mg 0.1

Methylparaben, mg 1.2

Water, ml Up to 1.0

Example 4

An aqueous solution of interferon-alpha-two based on buffer sodium disubstituted phosphate-citric acid with a concentration of citric acid of 1.5 mg / ml and methyl paraben as a preservative is prepared similarly to the preparation procedure in the following ratio per 1 ml:

Interferon alfa-two, ME 4.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 8.5

Monohydric citric acid, mg 1.5

Sodium chloride, mg 6.1

Twin - 80 mg 0.1

Methylparaben, mg 1.5

Water, ml Up to 1.0

Example 5

An aqueous solution of interferon-ilf-two based on buffer sodium disubstituted phosphate-citric acid with a concentration of citric acid of 1.5 mg / ml and benzyl alcohol as a preservative is prepared similarly to the preparation procedure in the following ratio of 1 ml:

Interferon alfa-two, ME 4.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 8.5

Monohydric citric acid, mg 1.5

Sodium chloride, mg 6.1

Tween 80 mg 0.1

Benzyl alcohol, mg 10.0

Water, ml Up to 1.0

Example 6

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

Interferon alfa-two, ME 4.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 8.5

Monohydric citric acid, 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 Up to 1.0

Example 7

A buffer-based interferon-alpha-two aqueous solution of sodium phosphate disubstituted-citric acid with a concentration of citric acid of 1.3 mg / ml, using oxidized glutathione at a concentration of 0.03 mg / ml and methyl paraben as a preservative is prepared similarly to the procedure for the following ratio per 1 ml:

Interferon alfa-two, ME 4.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 8.5

Monohydric citric acid, mg 1.3

Tween 80 mg 0.1

Sodium chloride, mg 6.1

Oxidized Glutathione, mg 0.03

Methylparaben, mg 1.2

Water, ml Up to 1.0

Example 8

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

Interferon alfa-two, ME 4.0 · 10 ⁶

Sodium phosphate, disubstituted, twelve, mg 4.5

Sodium phosphate monosubstituted, two-water, mg 1.5

Disodium EDTA, mg 0.1

Tween 80 mg 0.1

Sodium chloride, mg 7.5

Metacresol, mg 1.5

Water, ml Up to 1.0

Example 9

An aqueous solution of interferon-alpha-two in ammonium acetate buffer, pH 5.0, using benzyl alcohol as a preservative, containing no auxiliary ingredients, is prepared similarly to the preparation procedure, without adding glutathione, in the following ratio per 1 ml:

Interferon alfa-two, ME 4.0 · 10 ⁶

Ammonium acetate, mg 0.77

Twin - 80 mg 0.2

Benzyl alcohol, mg 10.0

Sodium chloride, mg 7.2

Water for injection, ml Up to 1.0

The results of the stability of storage solutions at various temperatures are shown in the table.

Obtained during the implementation of this technical solution, stabilized aqueous solutions of interferon-alpha-two were stored in ampoules or in sterilized glass bottles for medicines, sealed with rubber stoppers and aluminum caps.

Aqueous solutions of interferon-alpha containing additives (examples 1-7). sterile, non-toxic on the cell culture of human lung embryo L-68 and in animal experiments (white mice, guinea pigs). They cause a slight (no more than 2 ° C) increase in temperature with intravenous administration to rabbits, i.e. weakly pyrogenic. The biological (antiviral) activity characteristic of interferon-alpha-2 is exhibited when titrated on L-68 cell culture.

According to the results of tests on cell and animal cultures, we can conclude that it is possible to use the proposed aqueous solutions as injectable drugs for the treatment of human viral and oncological diseases.

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

To control the in vitro biological (antiviral) activity of interferon-alpha-two aqueous solutions obtained as described in examples 1-7, the method of suppressing the cytopathogenic effect of mouse encephalomyocarditis virus in a cell culture of human lung embryo L-68 grown in a monolayer of 96 is used -well microplates.

The interferon content in solutions (in%) is determined by high-performance liquid chromatography on a reverse phase column (HPLC), enzyme-linked immunosorbent assay (ELISA) using components of the production 000 “Protein circuit” (St. Petersburg) and polyacrylamide gel electrophoresis with dodecyl sulfate sodium (PAGE-SDS) with densitometry.

An aqueous solution of interferon, not containing stabilizing additives having a pH of 6.0-7.0 and obtained by analogy with the invention [3] (example 8), maintains acceptable storage stability at a temperature of 5 ° C for 12 months (more than 90 % of the main component). The interferon solution obtained according to the invention [4] (Example 9) with a pH of 4.5-5.0 is less stable, no more than 78% of interferon is retained. Moreover, in both cases, at elevated temperatures (25 and 35 ° C), a decrease in the content of the active component to 70-80% is observed, which is confirmed by various analytical methods.

The introduction of oxidized glutathione as a protective additive in a concentration of 0.01-0.10 mg / ml (examples 1, 2, 6, 7) leads to an increase in the stability of interferon during storage of its aqueous solutions as at 5 ° C for 12 months compared with the solution described in example 9, and at elevated temperatures of 25 ° C and 35 ° C. The safety of interferon is 90-95%, while there is no decrease in the biological activity of solutions.

An increase in the stability of interferon solutions is also observed when citric acid is used as an additional ingredient. The introduction of citric acid (at a concentration of: 1.0; 1.3; 1.5; mg / ml) in the buffer to maintain pH increases the stability of the drug at various storage temperatures (examples 3, 4, 7, 5), eliminates the need for use other chelating agents, allows you to use a wide range of bacteriostats - benzyl alcohol, methyl and propyl parabens, metacresol, etc.

The addition of citric acid prevents the oxidation of metacresol, keeping the interferon solution colorless, while during storage of some solutions containing metacresol and not containing citric acid, the solutions gradually became colored, 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 both oxidized glutathione and citric acid as protective additives, under standard storage conditions for drugs (5 ° C for 12 months) and at elevated temperatures (25 ° C, 3 months and 35 ° C, 1 month) provides 100% preservation of the biological activity of interferon-alpha-two and not less than 95% stability of the active substance according to titration data on cell culture A -68, HPLC, ELISA and electric phoresis.

Thus, the introduction of additional ingredients eliminates significant losses of interferon at the doses most commonly used in therapeutic practice (3.0 · 10 ⁶ -6.0 · 10 ⁶ IU / ml).

The increased thermal stability of aqueous solutions of interferon-alpha-two, obtained by this technical solution, provides an injectable dosage form of interferon with an increased shelf life of the drug.

In addition, the introduction of glutathione, known as an immunomodulator [5], into a drug can have a clinically significant effect in the treatment of viral and oncological diseases.

LITERATURE

1. FSP 42-0241-1184-01. Reaferon-EU, powder for the preparation of injection solutions.

2. RF patent No. 2180233, A 61 K 38/00, Bull. No. 7, publ. 03/10/2002.

3. RF patent No. 2157236, A 61 K 38/21, Bull. No. 28, publ. 10/10/2000.

4. RF patent No. 2113845, A 61 K 9/08, Bull. No. 18, publ. 06/27/1998.

5. RF patent No. 2089179, A 61 K 9/08, Bull. No. 25, publ. 09/10/1997.

Claims (3)

1. An aqueous solution of interferon for injection containing highly purified recombinant interferon-alpha-two (1-36) · 10 ⁶ ME, a buffer to maintain the pH of the solution 4.5-7.0, a nonionic detergent, a preservative with antimicrobial activity, an agent, providing isotonicity of the solution, water for injection, characterized in that it additionally contains oxidized glutathione and / or citric acid in the following ratio of components per 1 ml: Interferon-alpha-two ME, (1-36) · 10 ⁶ Nonionic detergent, mg 0.01-1.0 Buffer for maintaining 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 0.01-0.1 Citric acid 0.8-2.0 Isotonicity Agent solution In an amount   sufficient to give   isotonicity to solution Water for injection, ml Up to 1.0 2. The aqueous solution according to claim 1, characterized in that as a buffer system to maintain a pH of 4.5-7.0 contains a phosphate buffer at a concentration of 10-25 mm or a buffer of disubstituted sodium phosphate-citric acid at a concentration of 10-25 mm . 3. The aqueous solution according to claim 1, characterized in that the preservative contains methyl paraben in a concentration of 0.6-1.8 mg / ml, or a mixture of methyl paraben in a concentration of 0.6-1.8 mg / ml and propyl paraben 0.06-0.18 mg / ml, or metacresol in a concentration of 0.5-2.0 mg / ml, or benzyl alcohol 8.0-20.0 mg / ml.