RU2380405C2 - Method for making recombinant human alpha 16-interferon and pharmaceutical composition for treating viral diseases based on recombinant human alpha 16-interferon - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: recombinant alpha 16-interferon is made by cultivation of recombinant strain of yeast Pichia pastoris All Russian Collection of Industrial Microorganisms Y-2949 in a nutrient medium. It is followed with induced synthesis of target protein and cell removal. Then the culture medium is concentrated with settling of target protein at 60% ammonium sulphate saturation. Then said target protein is consistently purified on sephacryl HR 100 and on diethylaminoethyl-cellulose with 0.5% detergent Tween 20 added.

EFFECT: invention allows simplifying technology of recombinant human alpha 16-interferon and improving effectiveness of purification of target protein.

5 cl, 2 tbl, 3 ex

Description

The invention relates to the field of biotechnology, medicine and pharmacology, relates to a pharmaceutical preparation based on human alpha 16-interferon and a method for its preparation. Interferons are produced by cells in response to a viral infection. They provide antiviral protection for the body, have antimicrobial, anti-inflammatory, immunomodulatory, antitumor, radioprotective and antimitotic effects (Ershov F.I. The system of interferon is normal and pathological. 1996; Pestka S. Biopolymers. 2000. V.55. P.254-287 )

Human alpha-interferon-based drugs are used to treat viral diseases: hepatitis B and C (Chin R., Locarnini S. Curr. Opin. Infect. Dis. 1998. V.11. P. 719-726; Guo JT et al. J. Virol. 2001. V.75. P.8516-8523), herpetic infection (Siebeck N. et al. Am. J. Vet. Res. 2006. V.67. P.1406-1411), keratitis, influenza (Brassard DL et al. J. Leukoc. Biol. 2002. V.7. P.565-581), SARS (Cinatl J. et al. Lancet. 2003. V.362. P.293-294), oncological diseases: prostate cancer, renal cell carcinoma, breast cancer, leukemia, melanoma (Volberding PA et al. Cancer. 1987. V.59. P.620-625; Borden BC Oncologist. 1998. V.3. P.198-203 ; Bukowski RM Semin. Oncol. 2000. V.27. P.204-212; Kramer G. et al. J. Interfero n Cytokine Res. 2001. V.21. P.475-484; Molchanov O.E. et al. Current Trends in Immunotherapy of Malignant Tumors. 2001) and Human Autoimmune Diseases (Piskova J. et al. Invest. Ophtalmol. Vis. Sci . 2006. V.47. P.3946-3950).

Interferons are produced in the body in small doses, which makes it impossible to obtain the right amount of preparations of these proteins from donated blood or a specialized cell culture. A known preparation containing as active principle natural alpha2-interferon obtained from donated blood (produced by Biomed, Russia). However, there is a risk of infection of the final product with hepatitis B, C and HIV viruses, which limits the use of this drug.

With the development of recombinant DNA technologies, it has become possible to create microorganisms - producers of biologically active substances, including interferons. Currently, immunotherapy with recombinant interferons is one of the most promising and constantly expanding areas of immunopharmacology.

Known methods for producing recombinant human alpha2-interferon from bacteria cells of E. coli (patent RU 2242516, published December 18, 2003, and patent RU 2294372, published June 16, 2005). When using a bacterial expression system, recombinant proteins accumulate in an insoluble state in the form of “inclusion bodies”, which requires additional efforts to renature target proteins.

These deficiencies lack the yeast expression system. The use of methylotrophic yeast Pichia pastoris as producers makes it possible to significantly increase the yield of recombinant proteins and to avoid hyperglycosylation of secreted heterologous proteins (Cereghino J.L., Cregg J.M. FEMS Microbiol.Rev. 2000. V.24. P.45-66). However, the yield of the final product in the form of the target recombinant protein largely depends on the technology used for its isolation and purification.

At present, 23 human alpha-interferon are isolated and characterized, which are 80% identical to each other and share common functional features (Pestka S. Biopolymers. 2000. V.55. P.254-287). The spectrum of activities and the ratio of alpha-interferons of different subtypes depends on the type of cells and can vary with certain diseases (Hiscott J. et al. Philos. Trans. R. Soc. (Biol. Sci). 1984. V.307. P.217- 226; Hu R. et al. J. Biol. Chem. 1993. V.268. P.12591-12595; Sperber SJ et al. J. Interferon Res. 1992. V.12. P.363-368). To identify the individual characteristics of the action, it is necessary to study various alpha interferons of a person.

Known yeast strains of Pichia pastoris - producers of human alpha1-interferon (patent US 20030065148, published 03.04.2003), human alpha 16-interferon, previously obtained by the authors of the claimed invention (patent RU 2203950, published 10.05.2003) and human alpha2-interferon (international application WO 4039996 published May 13, 2004).

The strain of Pichia pastoris yeast is deposited in the All-Russian collection of industrial microorganisms under the number Y-2949.

A known method for the isolation and purification of alpha-2b-interferon from the culture fluid of the yeast Pichia pastoris KM71 (international application WO 4039996, published May 13, 2004), which is the closest analogue to the claimed method and is selected as a prototype. The method consists in cultivating a recombinant strain of Pichia pastoris in a nutrient medium providing cell growth and synthesis of the target protein, removing cells, two-stage ion-exchange chromatography on sorbents such as CM Sepharose FF and DEAE Sepharose FF, precipitating the target protein with ammonium sulfate, hydrophobic chromatography on Butyl Sepharose F ultrafiltration with a membrane YM 10 (10000 MWCO), gel filtration on a sorbent type Sephacryl HR 200.

The disadvantage of this method is its multi-stage and low manufacturability, which significantly limits the possibility of using the known technology for the industrial production of interferon.

The technical result of the claimed invention is to simplify the technology, increase the efficiency of purification of the target protein and obtain on its basis a stable pharmaceutical preparation in liquid and dry lyophilized forms, as well as the possibility of using the claimed technology in industrial production.

The specified technical result is achieved by the fact that in the known method for producing a pharmaceutical preparation from a yeast culture medium, which involves concentration of the culture medium, precipitation of the target protein and its chromatographic purification using gel filtration and ion exchange chromatography, a strain of Pichia pastoris yeast is cultivated in accordance with the claimed invention , which is deposited in the All-Russian collection of industrial microorganisms under the number Y-2949 and is a producer of alpha 16 interferon h man, and chromatographic purification is carried out sequentially on Sephacryl HR 100 and diethylaminoethyl cellulose. Concentration of the culture medium can be carried out using an AP-0.2-15PA membrane.

In addition, the specified technical result is achieved in that the precipitation of the target protein is carried out at 60% saturation of ammonium sulfate.

Moreover, the technical result is achieved in that gel filtration on Sephacryl HR 100 is carried out using 50 mM ammonium acetate buffer (pH 5.5) containing 2 mM ethylenediaminetetraacetic acid and 0.5% Tween- as a balancing and eluting solution. twenty.

In addition, this technical result is achieved in that ion-exchange chromatography of proteins obtained on diethylaminoethyl cellulose is carried out using 50 mM ammonium acetate buffer (pH 7.0) containing 2 mM ethylenediaminetetraacetic acid as a balancing solution. Tween-20, and the washing of impurity proteins is carried out by sequential use of a balancing buffer solution and a balancing buffer solution containing 0.1 M sodium chloride, the elution is carried out by a concentration gradient of chlorine sodium hydroxide 0.1-1.0 M in 50 mM ammonium acetate buffer (pH 5.5) containing 2 mM ethylenediaminetetraacetic acid, 0.5% Tween-20.

Known pharmaceutical preparations containing recombinant human alpha-2 interferon of bacterial origin as an active ingredient: “Roferon” (Hoffman-La Roche AG, Switzerland), “Intron-A” (Schering-Plow, USA), “Realdiron” (Biotechna, Lithuania ), “Reaferon” (“Vector-Medica”, Russia), “Interal”, (Scientific Research Institute of General Medicine, Russia). Reaferon, Interal and Intron-A preparations contain human albumin as a stabilizer, which requires additional verification of dosage forms for the absence of hepatitis B, C and HIV viruses. The Intron-A preparation additionally contains glycine as a stabilizing substance. The use of these additives is effective only for stabilization of the drug in a dry form and ineffective against liquid forms of interferon. In addition, prolonged use of drugs of bacterial origin is accompanied by undesirable side effects due to the conditional pathogenicity of prokaryotic producer strains and possible impurities of endotoxins in the final product (Primrose SBJ Appl. Bacteriol. 1986. V.61. P.99-116; Ershov F. I. The system of interferon is normal and pathological. 1996).

A known pharmaceutical preparation based on recombinant human alpha2-interferon and a method for its preparation from bacterial cells of E. coli (patent RU 2294372, published June 16, 2005), the essence of which is to cultivate bacteria of E. coli producers of the recombinant protein, destroy cells, remove impurities proteins, converting the recombinant protein into a soluble form, subsequent chromatographic purification of recombinant human alpha2-interferon, and preparation of a pharmaceutical preparation based on the recombinant protein. The pharmaceutical preparation described in this patent has recombinant human alpha2-interferon, ethylenediaminetetraacetic acid, and a salt buffering system providing a pH of from 6.0 to 7.5, is the closest analogue and is selected as a prototype for the claimed variants of the new the drug.

The disadvantages of the known pharmaceutical product is the bacterial origin of human alpha2-interferon, which potentially leads to side effects when used and worsens the patient's quality of life.

The technical result for the group of inventions is uniform and consists for the claimed variants of a new pharmaceutical preparation in reducing the risks of side effects when using a drug based on human interferon and obtaining a drug devoid of these disadvantages through the use of a yeast expression system for the production of human alpha 16-interferon and use as a stabilizer that prevents the aggregation of recombinant protein, Tween-20, as well as the possibility of its industrial production .

The specified technical result is achieved by a new pharmaceutical preparation based on human alpha interferon containing ethylenediaminetetraacetic acid and a salt buffering system, which, in accordance with the preparation claimed in the first embodiment, contains human alpha 16-interferon, a salt buffering system, which has a pH of 5.5 to no more than 6.0, and additionally contains tween-20 in the following ratio of ingredients:

Recombinant alpha 16 human interferon with activity 10 ⁶ -10 ⁷ IU / ml 10-100 mcg / ml Ethylenediaminetetraacetic acid 0.5-1.5 mg / ml Twin 20 0.5-5 mg / ml Buffer solution pH 5.5 - not more than 6.0, 10-20 mm rest

In addition, the specified technical result is achieved in that the pharmaceutical preparation contains a modified recombinant human alpha 16-interferon.

In addition, this technical result is achieved in that the pharmaceutical preparation has a liquid form, for example, in the form of an emulsion, and / or syrup, and / or drops, and / or liposome form.

The specified technical result is achieved by a new pharmaceutical preparation based on human alpha interferon containing ethylenediaminetetraacetic acid and a salt buffering system, which, in accordance with the claimed preparation according to the second embodiment, contains human alpha 16 interferon, buffering salts that provide a pH from 5.5 to not more than 6.0, and also additionally contains tween-20 in the following ratio of ingredients before freeze drying:

Recombinant alpha 16 human interferon with activity 10 ⁶ -10 ⁷ IU / ml 10-100 mcg / ml Ethylenediaminetetraacetic acid 0.5-1.5 mg / ml Twin 20 0.5-5 mg / ml Buffering salts, providing pH 5.5 - not more than 6.0 and molarity equal to 10-20 mM when dissolved rest

In addition, the specified technical result is achieved in that the pharmaceutical preparation contains a modified recombinant human alpha 16-interferon.

In addition, this technical result is achieved in that the pharmaceutical preparation has a dry lyophilized form as the basis for dosage forms in the form of tablets, and / or capsules, and / or powder, and / or granules, and / or suppositories.

The unified technical result indicated for the claimed group of inventions is thus achieved in a comprehensive manner by using the recombinant strain of yeast Pichia pastoris VKPM Y-2949 (RU 2203950, published May 10, 2003), culturing it in a nutrient medium and inducing the synthesis of the target protein, removing cells, concentration culture medium, target protein precipitation with ammonium sulfate, gel filtration on Sephacryl HR 100 sorbents in the presence of 0.5% Tween-20 nonionic detergent, ion-exchange chromatography on DEAE-cellulose sorbents in the presence of 0.5% nonionic detergent Tween-20, as well as new drug composition claimed in the first and second embodiments

The optimal conditions for the individual stages of obtaining alpha 16 human interferon are as follows:

- concentrated culture medium is carried out using an AP-0.2-15PA membrane;

- precipitation of the target protein is carried out at 60% saturation with ammonium sulfate;

- dissolution of the obtained precipitate containing alpha 16-interferon in 50 mm ammonium acetate buffer (pH 5.5) containing 2 mm EDTA (ethylenediaminetetraacetic acid), 0.5% Tween-20;

- gel filtration on Sephacryl HR 100 in 50 mM ammonium acetate buffer (pH 5.5) containing 2 mM EDTA, 0.5% Tween-20;

- carrying out ion-exchange chromatography on DEAE-cellulose using 50 mM ammonium acetate buffer (pH 7.0) containing 2 mM EDTA, 0.5% Tween-20 as a balancing solution; elution of the target protein with a concentration gradient of 0.1-1.0 M sodium chloride in 50 mM ammonium acetate buffer (pH 5.5) containing 2 mM EDTA, 0.5% Tween-20.

The preparation of alpha 16-interferon obtained as a result of using this technology contains the following ingredients:

1. For the drug in dry lyophilized form, as shown above:

Alpha 16-human interferon with activity 10 ⁶ -10 ⁷ IU / ml 10-100 mcg / ml ethylenediaminetetraacetic acid EDTA 0.5-1.5 mg / ml Twin 20 0.5-5 mg / ml Buffer-forming salts providing a pH of 5.5 - not more than 6.0 and molarity equal to 10-20 mM when dissolved rest

Particular cases of the implementation of a pharmaceutical preparation based on alpha 16-interferon in solid form are pharmaceutical preparations containing either recombinant human alpha 16-interferon or modified recombinant human alpha 16-interferon as an active principle.

Particular cases of the implementation of a pharmaceutical preparation based on alpha 16-interferon in solid form are tablets, capsules, powders, granules, suppositories, forms suitable for external use, for example, ointments, gels or liquid preparations obtained by dissolving the solid form in a pharmaceutically acceptable solvent, obtained standard technological methods (Valevko S.A. et al. Pharmaceutical technology: Technology of dosage forms. 2007).

2. For the drug in liquid and dry lyophilized form, as shown above:

Alpha 16-human interferon with activity 10 ⁶ -10 ⁷ IU / ml 10-100 mcg / ml ethylenediaminetetraacetic acid EDTA 0.5-1.5 mg / ml Twin 20 0.5-5 mg / ml Buffer solution pH 5.5 - not more than 6.0, 10-20 mm rest

Particular cases of the implementation of a pharmaceutical preparation based on alpha 16-interferon in liquid form are pharmaceutical preparations containing either recombinant human alpha 16-interferon or modified recombinant human alpha 16-interferon as an active principle.

Particular cases of the implementation of a pharmaceutical preparation based on alpha 16-interferon in liquid form are emulsions, syrups, elixirs, drops, liposome forms, forms suitable for external use, for example, gels, moistened wipes obtained by standard technological methods (Valevko S.A. and Dr. Pharmaceutical Technology: Technology of dosage forms. 2007).

Significant differences of the proposed method from the prototype are:

- the consistent use of the stages of ultrafiltration and precipitation of the target protein with ammonium sulfate, leading to a significant reduction in the volume of the treated culture medium in the first stages of purification, which is of great importance when scaling up the production of interferon;

- two-step chromatographic purification of alpha 16-interferon in the presence of 0.5% Tween-20, preventing the aggregation of the monomers of the target protein, which increases the yield of biologically active alpha 16-interferon;

- the introduction of the composition of the composition of the drug based on alpha 16 human interferon 0.05% Tween-20 as a stabilizer.

The essence and advantages of the claimed group of inventions are illustrated by the following examples.

Example 1. Purification of recombinant alpha 16 human interferon. The yeast strain Pichia pastoris VKPM Y-2949, the producer of human alpha 16-interferon, was cultured in the previously described manner (RU 2203950, published May 10, 2003). At the end of the cultivation process, yeast cells were removed by centrifugation at 6-7 thousand rpm for 15 minutes, at + 4 ° C. All subsequent operations were carried out at a temperature of + 4 ° C. The supernatant was passed through a nitrocellulose filter with a pore diameter of 0.45 μm (Millpore Inc.). The filtrate thus obtained was concentrated on an UPL-0.6 ultrafiltration unit using an AP-0.2-15PA membrane (NPK Biotest, Kirishi), cutting off compounds with a molecular weight of less than 15 kDa, at this stage low molecular weight components were removed and decrease in initial volume to 100-200 ml. Then, salt precipitation of the target protein was carried out, bringing the saturation of ammonium sulfate to 60%. The precipitate of the target protein was collected by centrifugation at 12-14 thousand rpm for 20 minutes. The resulting precipitate was dissolved in 50 mM ammonium acetate buffer (pH 5.5) containing 2 mM EDTA, 0.05% Tween-20, and applied to a column containing 1000 ml Sephacryl HR 100 equilibrated with the same buffer. The first 350 ml was discarded, the fractions containing the target protein were combined. The eluate thus obtained was titrated with 10% ammonia to pH 7.0 and applied to a column containing 50 ml of DEAE cellulose balanced with 50 mM ammonium acetate buffer (pH 7.0) containing 2 mM ethylenediaminetetraacetic acid (EDTA), 0, 05% Tween-20. The column was washed sequentially with 100 ml of equilibration buffer and 100 ml of equilibration buffer containing 0.1 M sodium chloride. The elution was performed with a concentration gradient of sodium chloride from 0.1 M to 1.0 M in 50 mM ammonium acetate buffer (pH 5.5) containing 2 mM ethylenediaminetetraacetic acid (EDTA), 0.05% Tween-20.

Electrophoresis under reducing conditions showed that the molecular weight of the obtained protein is approximately 20 kDa. Using this purification scheme, it was possible to obtain a preparation of human alpha 16-interferon, the content of impurities in which is not more than 5%. The specific activity was 0.3-1.0 × 10 ⁸ IU / mg. The average yield from a liter of culture medium was 7–9 mg.

Example 2. The study of the biological activity and stability of pharmaceutical preparations based on alpha 16 human interferon, made in liquid and in dry lyophilized forms.

The claimed pharmaceutical preparation for its stability tests was prepared as follows. The recombinant human alpha 16-interferon obtained in Example 1 was diluted to a predetermined biological activity value (from 5 × 10 ⁶ IU / ml to 2 × 10 ⁷ IU / ml), buffered to a predetermined pH value, mixed with a predetermined amount of Tween-20 and EDTA. The resulting mixture was sterilized by cold sterilization (Valevko S.A. et al. Pharmaceutical Technology: Dosage Form Technology. 2007), transferred to ampoules and sealed. If necessary, the resulting liquid mixture was frozen in unsealed ampoules, freeze-dried. Biological activity was determined before and after 6 months of storage of the drug (Table 1) by suppressing the cytopathic activity of the virus of vesicular stomatitis in the primary culture of L-41 skin-muscle tissue of human embryos sensitive to alpha-type interferons (Liu R.T. et al. // Protein Expres. Purif. 2001. V.22. P.381-387). Human leukocyte interferon OSO 42-28-90-87 (GISK, Moscow) was used as a standard. The contents of ampoules with a preparation based on human alpha 16-interferon in solid form were dissolved in 1 ml of water, the contents of ampoules with a liquid form were used without further processing.

The above results showed that the proposed composition of the pharmaceutical preparation according to the first and second options is able to maintain the activity of human alpha-16 interferon in dry lyophilized and liquid forms.

Example 3. The study of the side effects of a pharmaceutical preparation based on human alpha 16-interferon, made in liquid form.

The study included a toxicological study of a single exposure of the drug at a dose of 2.5 × l0 ⁵ ME / kg, which exceeds the therapeutic dose by 50 times, and a chronic toxicological experiment.

3.1. The study of the toxic effect of the drug based on human alpha 16-interferon was carried out on 60 sexually mature white male random mice and 60 male sexually mature random male mice weighing 16-18 g, and also on 60 guinea pigs weighing 200-250 g. The drug was administered once subcutaneously. When the drug was administered to animals subcutaneously at this dose, they experienced a certain short-term increase in respiration. Sexual differences in mice to the toxic effect of the drug were not observed. A histological examination of tissues and organs of the remaining at the end of the observation period of live animals showed no visible pathological changes from the examined organs.

Thus, a single administration of the maximum tolerated dose of the drug to experimental animals was not accompanied by toxic effects.

3.2. The study of the chronic toxicity of the drug based on human alpha 16-interferon was carried out on 90 white outbred male mice weighing 18-20 g, which were injected subcutaneously once daily for 90 days. During the three months of the chronic experiment, experimental animals did not show signs of severe intoxication and significant changes in behavior. The dynamics of the body weight gain of the experimental animals did not statistically significantly differ from that in the control.

In animals that received the drug for a long time, the state of nonspecific resistance was evaluated in dynamics. To do this, we studied the absorption and digestion activity of peripheral blood phagocytes, as well as the levels of lysozyme, nonspecific esterases, and myeloperoxidase in blood serum. The values of the studied parameters in most cases either did not differ from the control values, or slightly exceeded them.

The condition of immunocompetent cells in experimental animals was assessed by the severity of the antibody to specific antigen (chemical sorbed typhoid vaccine) and the level of nitric oxide in immunocompetent organs and blood serum. Long-term administration of a drug based on human alpha 16-interferon did not have a statistically significant negative effect on the functional state of the B-system of immunogenesis. Moreover, there was a slight increase in the production of specific antibodies to typhoid fever in immunized animals. In the tissues of the immunocompetent organs and in the blood serum of mice, the content of nitric oxide increases compared with the same parameters in the control, which indicates the stimulating effect of the drug on the cellular component of immunogenesis.

Summarizing the results characterizing the chronic toxicity of the drug based on human alpha 16-interferon with prolonged subcutaneous use in white outbred mice, it should be noted that the drug does not have any pronounced negative effect on the general condition of experimental animals and does not have immunosuppressive administration under such a tense schedule action. The stimulating effect of the drug on the cellular and humoral components of the immune system can be noted.

Chronic toxicity of the drug was also studied in experiments on 100 white rats weighing 240-250 g, which were injected subcutaneously once daily for 10 days. The effect of the drug based on human alpha 16-interferon was evaluated by changes in the general condition, body weight, functional state of the liver and kidneys, some organs of the endocrine system (adrenal glands, pancreas) and peripheral blood. At the end of the experiment, a histological examination of the internal organs was performed in all animals.

The experiments showed that the use of a drug based on alpha 16-interferon does not cause changes in experimental animals that go beyond the physiological fluctuations of the norm. The body weight of the experimental animals did not statistically significantly differ from the control. The functional state of the liver and pancreas in experimental animals during the chronic experiment did not differ significantly from the norm.

Macroscopic and histological examination of the internal and endocrine organs (parietal region of the brain, heart, lungs, liver, kidneys, spleen, stomach, duodenum, adrenal glands, thyroid and pancreas) did not reveal pathological changes in experimental animals.

The effect of the drug based on alpha-16 interferon on peripheral blood was studied in 30 white rats weighing 310-340 g. The results of the studies showed that the most significant changes occurred in the quantitative composition of leukocytes, which significantly increased on the 3rd, 5th, 7th and 10th days after the first injection of the drug. In a detailed study of the leukocyte composition of the peripheral blood of experimental animals, it turned out that the increase in the total number of leukocytes under the influence of the studied drug was due to a significant increase in the number of neutrophils and monocytes in comparison with similar indicators in the control. These data may indicate that the drug based on alpha 16-interferon has a stimulating effect on blood formation and increases the nonspecific resistance of the body.

Summarizing the results characterizing the chronic toxicity of a drug based on alpha 16-interferon for subcutaneous use in white rats, it should be noted that the drug does not adversely affect the general condition of experimental animals and toxic effects on the functional state of their vital organs and systems. Moreover, with regard to the hematopoietic system, the stimulating effect of the drug on the components of white blood, primarily on neutrophils and monocytes, the main cellular components of the nonspecific resistance of the body, was established.

3.3. Allergic properties of the drug based on alpha-16 interferon were determined in experiments on male guinea pigs weighing 250-300 g. Before staging a specific experiment, the animals were not fed for a day. All animals received a standard diet, presented as a granular feed. Studies have shown that the introduction of the drug into the body does not lead to the activation of the processes responsible for the development of an allergic condition.

3.4. The study of the specific activity of the drug based on alpha 16-interferon was carried out on models of viral infections.

To evaluate the effectiveness of the drug used white outbred mice weighing from 16 to 18 g.

Venezuelan equine encephalomyelitis virus (VEL) strain Trinidad was injected subcutaneously in animals in a volume of 0.3 ml / mouse. Alpha 16-interferon was administered subcutaneously once, 4 hours before infection at a dose of 25,000 IU / mouse. The results of the studies showed that the test drug has specific antiviral activity against experimental VEL infection and, depending on the infectious dose of the pathogen, increases the survival rate of infected animals by 12-50% (P <0.05).

To simulate an experimental influenza infection, we used the influenza A virus (HAV) strain Victoria / 72 (H3N2), obtained from the Museum of Viral Strains of the Research Institute of Microbiology and Epidemiology named after Pasteur of the Ministry of Health of the Russian Federation, St. Petersburg, at an infectious dose of 3-4 LD50. Infection of animals was carried out once intranasally, in a volume of 0.1 ml. The effectiveness of alpha 16-interferon was evaluated by comparing the values of the survival rates of animals in the experimental and control groups (20 mice). Observation and accounting of the death of animals was carried out within 15 days after infection.

The results of the experiments (Table 2) showed that the test drug at doses of 5000.0 and 15000.0 IU / mouse had a protective effect against experimental influenza infection when it was used both before and after infection of the animals with HAV. In the case of prophylactic administration of the drug, it is preferable to administer it 24 hours before infection, and for the therapeutic purpose, many times after infection. The use of alpha 16-interferon according to such schemes allows to achieve the maximum protective effect, which amounted to 100% and exceeded the control level by 80%.

The combination of data obtained during the study of the side effects of a pharmaceutical preparation based on human alpha 16-interferon allows us to conclude that the drug based on human alpha 16-interferon can be considered as a harmless and highly active antiviral immunobiological preparation that does not have side effects.

table 2 The effectiveness of the drug based on alpha 16 human interferon in case of influenza infection Dose, IU / mouse Drug administration time Frequency of drug administration Survival rate,% Not administered (control) - - twenty* 5000.0 48 h before infection once 80 * 5000.0 24 hours before infection once one hundred* 15000.0 48 h before infection once 90 * 15000.0 24 hours before infection once one hundred* 5000.0 24 hours after infection once 75 * 5000.0 48 h after infection once 60 * 5000.0 72 hours after infection once 55 * 5000.0 24, 48 hours after infection twice one hundred* 5000.0 24, 48, 72 hours after infection three times one hundred* 15000.0 24 hours after infection once 80 * 15000.0 48 h after infection once one hundred* 15000.0 72 hours after infection once 75 * 15000.0 24, 48 hours after infection twice one hundred* 15000.0 24, 48, 72 hours after infection three times one hundred* * - Differences with survival rates in the control group were significant with a probability of 0.95.

As shown by numerous laboratory tests, the advantage of the claimed group of inventions in comparison with world analogues is that the experimental possibility of obtaining a pharmaceutical drug for medical use based on human alpha-16 interferon, which has antiviral activity, and also, which is especially important, has not revealed and dangerous effects for potential patients.

Thus, the claimed group of inventions allows, as shown by the test results, to significantly reduce the risk of side effects when using a new pharmaceutical product based on human interferon by using a yeast expression system for the production of human alpha 16-interferon and using recombinant aggregation as a stabilizer Squirrel, Tween-20. In addition, the advantage and advantage of the claimed pharmaceutical product is that it can be used both in liquid form, and in solid and dry lyophilized forms, as well as for external use, which can significantly expand the range of applications of the claimed drug in the treatment of diseases of various etiology.

Claims (5)

1. The method of producing recombinant alpha 16 human interferon, including concentration of the culture medium, precipitation of the target protein and chromatographic purification using gel filtration and ion exchange chromatography, characterized in that cultivating a recombinant yeast strain Pichia pastoris Y-2949, which is an alpha producer 16-human interferon, the precipitation of the target protein is carried out at 60% saturation of ammonium sulfate, and chromatographic purification is carried out sequentially on Sephacryl HR 100 using in As a balancing and eluting solution of 50 mM ammonium acetate buffer with a pH of 5.5, containing 2 mM ethylenediaminetetraacetic acid and 0.5% Tween-20, and diethylaminoethyl cellulose using 50 mM ammonium acetate buffer as a balancing solution with pH 7 , 0, containing 2 mM ethylenediaminetetraacetic acid, 0.5% Tween-20, and the washing of impurity proteins is carried out by sequential use of a balancing buffer solution and a balancing buffer solution containing 0.1 M sodium chloride, the elution is carried out a concentration gradient of sodium chloride 0.1-1.0 M in 50 mm ammonium acetate buffer with a pH of 5.5, containing 2 mm ethylenediaminetetraacetic acid, 0.5% Tween-20, if necessary, the resulting target protein is freeze-dried. 2. A pharmaceutical composition for the treatment of viral diseases based on recombinant human alpha 16-interferon containing ethylenediaminetetraacetic acid and saline buffered system, characterized in that it contains human alpha 16-interferon obtained by the method according to claim 1, and Tween-20 in the following ratio of ingredients:
Recombinant alpha 16 human interferon with activity 10 ⁶ · 10 ⁷ IU / ml 10-100 mcg / ml Ethylenediaminetetraacetic acid 0.5-1.5 mg / ml Twin 20 0.5-5 mg / ml Buffer solution pH 5.5-6.0, 10-20 mm rest 3. The pharmaceutical composition according to claim 2, characterized in that it is presented in the form of an emulsion, syrup, drops or liposome form. 4. Freeze-dried pharmaceutical composition for the treatment of viral diseases based on recombinant human alpha 16-interferon, containing ethylenediaminetetraacetic acid and a salt buffering system, characterized in that it contains human alpha 16-interferon obtained by the method according to claim 1, and Tween-20 in the following ratio of ingredients before freeze drying:
Recombinant alpha 16 human interferon 10-100 mcg / ml with activity 10 ⁶ -10 ⁷ IU / ml Ethylenediaminetetraacetic acid 0.5-1.5 mg / ml Twin 20 0.5-5 mg / ml Buffer-forming salts providing pH 5.5-6.0 rest
molarity equal to 10-20 mM when dissolved 5. Lyophilized dried pharmaceutical composition according to claim 4, characterized in that it is presented in the form of a tablet, capsule, powder, granules or suppository.