RU2564951C1 - Composition of aqueous solution of recombinant human interferon alpha-2 for rectal administration - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: composition of an aqueous solution of recombinant human interferon alpha-2 contains: recombinant human interferon alpha-2 25.0-200.0 thousand International Units, sodium alginate 10.0-100.0 mg, antioxidant oxidised glutathione 0.006-0.1 mg, the antimicrobial preserving agent Nipagin 0.2-1.8 mg, the stabilising agent Polysorbate 80 (Tween 80) 0.01-1.0 mg, sodium chloride 5.8-7.5 mg, a buffer compound for balancing pH 4.5-7.0 10.0-25.0 mM, water - the rest up to 1 ml.

EFFECT: using the given composition enables creating the aqueous solution of extended release recombinant human interferon alpha-2 for rectal administration.

3 cl, 4 tbl, 2 dwg

Description

The invention relates to preparations based on recombinant human interferon alpha-2 in liquid form for rectal administration, intended for the prevention and treatment of various viral, proliferative diseases and immune disorders, and can be used in the field of medicine and pharmaceuticals.

Rectal administration of interferon does not cause unwanted side effects in the form of nausea, dizziness, fever, as is the case with intravenous, intramuscular or subcutaneous administration, and contributes to the rapid absorption of the drug, achieving a high concentration in the blood, loss of the barrier function of the liver and lengthening its circulation in the bloodstream (VV Malinovskaya et al. “Functioning of the interferon system with various methods and doses of administration of recombinant alpha-2-interferon.” Issues of Virology. 198 9, No. 2, pp. 180-183).

A liquid form of the preparation of interferon is known (RF patent No. 22198934, IPC A61K 38/21, published on December 20, 2003), used for injection, or in the form of eye drops or aerosol and containing interferon alpha-2, buffering salts that provide pH 7 , 4, and a stabilizer. As a stabilizer, it contains a mixture of alpha-tocopherol acetate and an organic acid in the following ratio of ingredients:

Interferon alfa-2 1-100 million ME

Alpha-Tocopherol Acetate 0.05-0.1 mg

Organic acid 0.02-10 mg

Buffer-forming salts providing a pH of 7.4 - the rest.

As an organic acid, it contains ascorbic, citric, uric, succinic acid or glycine. As buffering salts, it contains salts capable of forming phosphate, acetate, carbonate or Tris buffer solutions.

Known aqueous solution of interferon with an activity of 10,000 ME for rectal administration (ed. St. USSR No. 1530189, class A61K 45/02, 1988). The specified preparation is used as a complex of separate preparations for the treatment of viral infections, consisting of interferon, which is administered rectally in the form of an aqueous solution at a dose of 10000 ME, and vitamins E and C, which are administered as an oil solution intramuscularly in parallel with interferon.

The introduction of substances with antioxidant properties along with interferon of vitamins E and C leads to the prevention of a significant disturbance in lipid metabolism due to the intensification of lipid peroxidation, normalization of the functioning of the interferon system and, ultimately, an intensive decrease in the content of the pathogen.

However, such a liquid form of interferon has a short shelf life, is not effective enough for rectal use, because quickly absorbed through the colon and acts for a short time.

A known composition of an aqueous solution of alpha 2-interferon (RF patent No. 2232595, IPC A61K 38/21, publ. 07/20/2004), including alpha 2-interferon from 1 × 10 ⁶ to 1 × 10 ⁸ IU / ml, stabilizer as polysorbate 80 from 0.01 to 0.05 wt./about. %, an agent that regulates the osmotic pressure, which is sodium chloride, a buffer system consisting of sodium monohydrogen phosphate (Na ₂ HPO ₄ ) and sodium dihydrogen phosphate (NaH ₂ PO ₄ ) from 5 to 20 mm with a pH of 4.5-6.0, as well as antimicrobial preservatives selected from the group consisting of 0.1-0.3 wt./about. % phenol, 0.1-0.2 wt./about. % m-cresol or mixtures thereof. The composition of the aqueous solution of alpha 2-interferon is intended for injection or in the form of eye drops or aerosol.

The closest analogue (prototype) is an aqueous solution of interferon (RF patent No. 2238758, IPC A61K 38/21, publ. 10/27/2004) containing highly purified recombinant interferon-alpha-2 (1-36) × 10 ⁶ ME, 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, characterized in that it additionally contains oxidized glutathione and / or citric acid in the following ratio of components to 1 ml:

Interferon Alfa-2 Human, 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 Isotonic Sodium Chloride solution in an amount sufficient to impart isotonicity to solution, mg 1.0-10.0 Water for injections up to 1.0 ml

As a buffer system for maintaining a pH of 4.5–7.0, it contains a phosphate buffer at a concentration of 10–25 mM or a buffer with disubstituted sodium phosphate — citric acid at a concentration of 10–25 mM. As a preservative, it contains methyl paraben at a concentration of 0.6-1.8 mg / ml, or a mixture of methyl paraben at a concentration of 0.6-1.8 mg / ml and propyl paraben at a concentration of 0.06-0.18 mg / ml, or metacresol at a concentration of 0.5-2.0 mg / ml, or benzyl alcohol 8.0-20.0 mg / ml. The indicated aqueous solution of recombinant human interferon alpha-2 is intended for intramuscular, subconjunctival administration and instillation into the eye.

However, such a liquid form of interferon is not effective enough for rectal use, for example in microclysters, because quickly absorbed through the colon and acts for a short time.

The technical result of the claimed invention is the creation of a composition of an aqueous solution of recombinant human interferon alpha-2 for rectal administration with prolonged action.

The specified technical result is achieved by the fact that in the composition of an aqueous solution of recombinant human interferon alpha-2 for rectal administration, including recombinant human interferon alpha-2, stabilizer polysorbate 80, antioxidant glutathione oxidized, sodium chloride, which provides isotonicity of the solution, buffer composition, antimicrobial preservative nipagin and water, according to the invention, it contains sodium alginate in the following quantitative components in 1 ml:

recombinant human interferon alpha-2 25.0-200.0 thousand ME sodium alginate 10.0-100.0 mg antioxidant glutathione oxidized 0.006-0.1 mg antimicrobial preservative nipagin 0.2-1.8 mg Polysorbate 80 stabilizer (twin 80) 0.01-1.0 mg sodium chloride 5.8-7.5 mg buffer composition for maintaining pH 4.5-7.0 10.0-25.0 mm water the rest is up to 1 ml

The composition may additionally contain ascorbic acid in an amount of 0.8-2.0 mg / ml, and the buffer composition contains (mg / ml):

disubstituted sodium phosphate 12-water 4,53 monosubstituted sodium phosphate 2-aqueous 1,5 disodium salt of ethylenediaminetetraacetic acid 0.1

The definition of the boundaries of the content of sodium alginate in the claimed composition

To determine the boundaries of the optimal content of sodium alginate in the composition, an experiment was conducted on laboratory animals. Used 12 female chinchilla rabbits weighing 1.5-2 kg.

Rabbits were divided into 3 groups of 4 animals. A composition containing 125 thousand ME of recombinant interferon alpha-2b with a different content of sodium alginate was administered rectally to animals: group 1 - 10 mg / ml, group 2 - 100 mg / ml, group 3 - 40 mg / ml. In all groups, drugs were used once.

The determination of the content of interferon alpha-2b in the blood serum of rabbits was carried out by the ELISA method using test systems "Alpha-interferon-IFA-BEST" immediately before administration of the drug, 15 minutes after administration, 30 minutes, 1 hour and then every hour in within 24 hours after administration of the drug.

The initial background level of serum interferon in animals of various groups did not have significant differences (11.2-13.4 pg / ml).

In the analysis of the data shown in FIG. 1 and table 1, it was found that the most pronounced maximum increase to 123.6 pg / ml was detected 1 hour after the administration of a rectal solution with a content of sodium alginate of 40 mg / ml The return of serum interferon to its original level occurred at 17 o’clock.

A less pronounced increase maximum was noted after the introduction of a solution with a sodium alginate content of 10 mg / ml to 66.7 pg / ml after 30 minutes;

After the introduction of a composition with a sodium alginate content of 100 mg / ml, the maximum interferon content in the blood to 47.4 pg / ml was observed only at 5 o’clock, then the level of interferon gradually decreased to background values at 18 o’clock.

Thus, it was found that when the content of sodium alginate in the composition is less than 10 mg, the viscosity of the solution is insufficient, as a result of which the presence of the drug composition at the place of its rectal administration is not ensured for the period necessary for the prolonged release of interferon and its absorption through the mucous membrane.

The following are examples of compositions of the claimed composition.

Example 1

sodium alginate 40 mg / ml recombinant interferon alfa-2 25 thousand IU / ml sodium chloride 7.5 mg / ml disubstituted sodium phosphate 12-water 4.53 mg / ml monosubstituted sodium phosphate 2-aqueous 1.50 mg / ml disodium salt of ethylenediaminetetraacetic acid 0.1 mg / ml polysorbate 80 0.1 mg / ml glutathione oxidized 0.006 mg / ml nipagin 0.20 mg / ml water for injections up to 1 ml

Example 2

sodium alginate 40 mg / ml recombinant interferon alfa-2 100 thousand IU / ml sodium chloride 7.5 mg / ml disubstituted sodium phosphate 12-water 4.53 mg / ml monosubstituted sodium phosphate 2-aqueous 1.50 mg / ml disodium salt of ethylenediaminetetraacetic acid 0.1 mg / ml polysorbate 80 0.1 mg / ml glutathione oxidized 0.006 mg / ml nipagin 0.20 mg / ml water for injections up to 1 ml

Example 3

sodium alginate 40 mg / ml recombinant interferon alfa-2 200 thousand IU / ml sodium chloride 7.5 mg / ml disubstituted sodium phosphate 12-water 4.53 mg / ml monosubstituted sodium phosphate 2-aqueous 1.50 mg / ml disodium salt of ethylenediaminetetraacetic acid 0.1 mg / ml polysorbate 80 0.1 mg / ml glutathione oxidized 0.006 mg / ml nipagin 0.20 mg / ml water for injection before 1 ml

Example 4

sodium alginate 10 mg / ml recombinant interferon alfa-2 25 thousand IU / ml sodium chloride 5.8 mg / ml disubstituted sodium phosphate 12-water 4.53 mg / ml monosubstituted sodium phosphate 2-aqueous 1.50 mg / ml disodium salt of ethylenediaminetetraacetic acid 0.1 mg / ml polysorbate 80 0.01 mg / ml glutathione oxidized 0.01 mg / ml nipagin 0.8 mg / ml ascorbic acid 1.0 mg / ml water for injections up to 1 ml

Example 5

sodium alginate 40 mg / ml recombinant interferon alfa-2 100 thousand IU / ml sodium chloride 7.5 mg / ml disubstituted sodium phosphate 12-water 4.53 mg / ml monosubstituted sodium phosphate 2-aqueous 1.50 mg / ml disodium salt of ethylenediaminetetraacetic acid 0.1 mg / ml polysorbate 80 0.1 mg / ml glutathione oxidized 0.006 mg / ml nipagin 0.20 mg / ml ascorbic acid 1.0 mg / ml water for injections up to 1 ml

Example 6

sodium alginate 100 mg / ml recombinant interferon alfa-2 200 thousand IU / ml sodium chloride 7.5 mg / ml disubstituted sodium phosphate 12-water 4.53 mg / ml monosubstituted sodium phosphate 2-aqueous 1.50 mg / ml disodium salt of ethylenediaminetetraacetic acid 0.1 mg / ml polysorbate 80 0.1 mg / ml glutathione oxidized 0.1 mg / ml nipagin 1.8 mg / ml ascorbic acid 2.0 mg / ml water for injections up to 1 ml

Example 7. The technology of preparation of the claimed composition

The process of preparing the composition in accordance with examples 1-6 includes the following procedures:

- preparation of a gel of sodium alginate, depending on its content (from 1.0 to 10%) in examples 1-6;

- thermal sterilization of the gel at a temperature of 120 ° C;

- preparation of a mixture of a gel of sodium alginate and recombinant interferon-alpha 2 under aseptic conditions.

Packing of LF in rectioli

A portion (4.0 ± 0.1) g of sodium alginate is placed in a glass beaker with a buffer solution (96 ± 0.1) g of the following composition:

sodium chloride 7.5 mg / ml disubstituted sodium phosphate 12-water 4.53 mg / ml monosubstituted sodium phosphate 2-aqueous 1.50 mg / ml disodium salt of ethylenediaminetetraacetic acid 0.1 mg / ml polysorbate 80 0.1 mg / ml water for injections up to 1 ml

The suspension is stirred vigorously until a uniform gel is formed. The resulting gel of sodium alginate is sterilized in an autoclave at a temperature of 120 ± 2 ° C.

A solution of recombinant human interferon alpha-2 is added to a sterile sodium alginate gel under aseptic conditions and mixed thoroughly, a nipagin solution is added at the rate of 0.20 mg / ml of the product. The resulting product is transferred to the packaging of 5 ml in rectioli (microclysters in the form of a rectal pipette).

The composition of the solution for rectal administration presented for the experimental study is shown in table 2.

Materials and methods

A study of the preparation of recombinant interferon alpha-2b for rectal administration was performed in laboratory animals. Used 20 female chinchilla rabbits weighing 1.5-2 kg.

Rabbits were divided into 5 groups of 4 animals. Recombinant interferon alfa-2b was administered rectally to rabbits of groups 1, 2, and 3 at doses of 1 million ME, 500 thousand ME, and 125 thousand ME, respectively. Groups 4 and 5 were used as controls: group 4 received candles Genferon 125 thousand ME, group 5 - candles Viferon 150 thousand ME.

In all groups, drugs were used once (see table. 3).

The objective of the study was a comparative study of the content in the blood serum of interferon alpha-2b upon rectal administration of recombinant interferon preparations in various dosages and dosage forms.

The determination of the content of interferon alpha-2b in the blood serum of rabbits was carried out by the ELISA method using test systems "Alpha-interferon-IFA-BEST" immediately before administration of the drug, 15 minutes after administration, 30 minutes, 1 hour and then every hour in within 24 hours after administration of the drug.

The initial background level of serum interferon in animals of various groups did not have significant differences (10.3-14.2 pg / ml).

All digital data obtained by groups during the study were subjected to statistical processing using Student's criterion. The difference in average values was considered significant at P≤0.05.

results

When analyzing the data obtained, it was found that the degree and rate of increase in the content of serum interferon after administration of the test solution is a dose-dependent phenomenon. The most pronounced maximum increase to 1000.7 pg / ml was detected 30 minutes after the administration of a rectal solution with an interferon content of 1 million ME. The return of serum interferon to its original level occurred at 21 hours. A less pronounced maximum increase was noted after the introduction of a solution with an interferon content of 500 thousand ME and 125 thousand ME - up to 245.6 pg / ml after 30 minutes and 123.6 pg / ml after 1 hour, respectively. A return to the initial level after administration of the interferon drug solution at a dose of 500 thousand ME occurred at 19 hours, at a dose of 125 thousand ME - at 17 hours. Moreover, a significant increase compared to the initial level when using the interferon drug solution of different dosages occurred after 15 minutes after administration (see table. 4).

When comparing the results after administration of the solution and after administration of interferon preparations in comparable doses (a solution of 125 thousand ME, candles Genferon 125 thousand ME, candles Viferon 150 thousand ME), the influence of the dosage form and composition of the drug on the rate of increase in the content of serum interferon was revealed.

So, after the introduction of the solution, a significant increase in the level of serum interferon to 90.7 pg / ml was noted after 15 minutes, a maximum of 123.6 pg / ml - after 1 hour. After the administration of Genferon in suppositories, a significant increase to 20.6 pg / ml occurred after 1 h, while the maximum interferon content of 28.5 pg / ml was recorded after 4 h. When using Viferon suppositories, a significant increase in serum interferon to 29.4 pg / ml ml also occurred after 1 h, a maximum of 131.6 pg / ml was noted 3 h after administration. In this case, the return to the initial level after administration of the solution of interferon preparation was fixed at 17 hours, after the administration of suppositories Genferon - at 11 hours, suppositories Viferon - at 12 hours. The level of serum interferon in rabbits treated with the solution remained elevated at 2 hours, 2 times compared with the original (see graph in Fig. 2).

findings

1. The degree and rate of increase in the content of serum interferon after administration of the test solution of the drug interferon is dose-dependent - with the introduction of large doses, the level of interferon is more significant and increases faster and lasts longer in the blood serum.

2. The influence of the dosage form and composition of the drug in comparable doses on the degree, speed and duration of increasing the content of serum interferon was revealed. After the introduction of the inventive solution of the drug interferon, an increase in the level of 6.8 times was noted after 15 minutes, the maximum was reached after 1 hour. After the administration of suppositories, Genferon and Viferon a significant increase was detected after 1 hour in 2.0 and 2.7 times, respectively, the maximum after the introduction of Genferon suppositories - after 4 hours, after the administration of Viferon suppositories - after 3 hours. Moreover, the maximum values after the administration of the solution and Viferon suppositories were comparable - 123.6 pg / ml and 131.6 pg / ml, respectively. The maximum content of serum interferon after using Genferon suppositories was significantly lower - 28.5 pg / ml. The return to the initial level after the introduction of the solution of the drug interferon was fixed at 17 hours, after the administration of suppositories Genferon at 11 hours, candles Viferon - at 12 hours.

Thus, according to the results of a study of a new form of recombinant interferon - a solution for rectal administration, it was found that, compared to the traditional form (rectal suppositories), the studied drug causes an increase in serum interferon almost immediately after administration and circulates in the blood longer, thereby making it possible to render interferon more effective therapeutic effect.

The prolonged circulation of interferon in the blood makes it possible to reduce the frequency of use of the drug during the day.

Also, the preparation of interferon in the form of a solution in rectal pipettes (rectiols) is more convenient to use than traditional forms of drugs for rectal use - it is easier to enter and does not follow, like a soft melting candle base.

Claims (3)

1. The composition of an aqueous solution of recombinant human interferon alpha-2 for rectal administration, including recombinant human interferon alpha-2, stabilizer polysorbate 80, antioxidant glutathione oxidized, sodium chloride, which provides isotonicity of the solution, a buffer composition, antimicrobial preservative nipagin and water, characterized in that it contains sodium alginate in the following quantitative components in 1 ml:
recombinant human interferon alpha-2 25.0-200.0 thousand ME sodium alginate 10.0-100.0 mg antioxidant glutathione oxidized 0.006-0.1 mg antimicrobial preservative nipagin 0.2-1.8 mg Polysorbate 80 stabilizer (twin 80) 0.01-1.0 mg sodium chloride 5.8-7.5 mg buffer composition to maintain pH 4.5-7.0 10.0-25.0 mm water the rest is up to 1 ml 2. The composition according to p. 1, characterized in that it further comprises ascorbic acid in an amount of 0.8-2.0 mg / ml. 3. The composition according to p. 1, characterized in that the buffer composition contains (mg / ml):
disubstituted sodium phosphate 12-water 4,53 monosubstituted sodium phosphate 2-aqueous 1,50 disodium salt of ethylenediaminetetraacetic acid 0.1

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