The drug on the basis of recombinant alpha-2 interferon person for injection in the dry lyophilized form

 

(57) Abstract:

The invention relates to medicine and biotechnology, in particular the production of therapeutic and preventive drugs based on recombinant alpha-2 interferon person for injection in the dry lyophilized form. The preparation includes the substance of recombinant alpha-2 interferon man, salt buffer system and a stabilizing additive containing plazmozameschayuschie solution, polyhydric alcohol and urea or EDTA. As the plasma-solution preparation contains poliglyukin, or reopoliglyukin, or gemodez, or a mixture of these components in the ratio 1:1:1. The technical result - the creation of the drug on the basis of recombinant alpha-2 interferon person for injection in the dry lyophilized form, the storage of which is possible at temperatures significantly above 4C, for example at room temperature (203)C. 3 C.p. f-crystals, 2 tab.

The invention relates to medicine and biotechnology, in particular the production of therapeutic and preventive drugs based on recombinant alpha-2 interferon person for injection in the dry lyophilized form.

The problem of stability during storage is directly related to Leah purified from ballast protein substrate cultivation alpha-2-interferon is widely used freeze drying in the presence of the filler, ensuring the preservation of biological activity for at least one year. The filler used substances of protein nature, biopolymers or composition of permitted substances for use in immunobiological preparations. Without filler, providing at the stage of lyophilization conditions necessary for obtaining three-dimensional pills and serves as a stabilizer, purified interferon unstable during storage loses its biological activity.

Known drug "IFN", Russia [Methodological basis for the application of reaferona - recombinant human interferon a-2B-protein // Collection of materials of scientific conference for practitioners, Koltsovo, 25-29 January 1993, - Koltsovo, 1993, - S. 115-117], which represents a lyophilized composition of purified recombinant alpha-2 interferon activity 106ME with a filler, which is used as human serum albumin (CSA) in a concentration of 0.5%. The drug is used as an antiviral agent.

Know of any other drug on the basis of recombinant beta-interferon in dried form, in which the filler and stabilizer use the increased stability of the interferon to the inactivating factors of drying and temperature influences on subsequent storage of the drug.

The disadvantage of the above two products - the high cost of CSA, which is obtained from blood donors, and also requires additional control for the presence of infected material (hepatitis b and C, HIV and others).

Known drug on the basis of recombinant alpha-interferon in dried form, where as filler and stabilizer, a mixture of steroids, low molecular weight dextran, vitamin B12 and nicotinic acid (U.S. patent No. 5698232, IPC a 61 K 38/19, publ. 16.12.97 year). The preparation should be stored at +4C.

Know of any other drug on the basis of recombinant alpha-interferon in dried form, where as filler and stabilizer, a mixture of the disaccharide, dextran with a molecular mass of 20000-60000 Dalton, detergent and some amino acids (patent - USA No. 6346274, a 61 K 38/21, publ. 12.02.2002,). The preparation should be stored at +4C.

Known drug on the basis of recombinant beta interferon in dried form, where as filler and stabilizer using 0.5-10% solution of polyvinylpyrrolidone (EPO application No. 89245, IPC And 61 To 45/02, publ. 21.09.83 year). The drug should be stored at +4C.

Known drug on the basis of recombinant alpha-2 interferon for inquiry serves as a filler and stabilizer subsequent drying [Methodological basis for the application of reaferona - recombinant human interferon a-2B-protein // Collection of materials of scientific conference for practitioners, Koltsov, 25-29 January 1993, - Koltsovo, 1993, - S. 15-19] (preparation Realigion" manufactured in, Vilnius). The preparation should be stored at +4C.

The disadvantage of the four above-mentioned preparative forms is the fact that the stabilizing compositions provide long-term storage of drugs at a temperature of +4C.

The closest technical solution (prototype) is a lyophilized drug with immunomodulatory and antiviral activity "Neoferon, including recombinant interferon alpha-2 persons, a hybrid protein of the T-FNO-T, recombinant interferon gamma, a stabilizing additive containing plazmozameschayuschie solution poliglyukin, salt buffer system with a pH of 7.0-7.2 and pyrogen-free water in the following ratio of components (wt.%): recombinant interferon Alfa-2 person 0,0000001-0,0000002; hybrid protein T-FNO-T 0,0000001; recombinant interferon gamma 0,0000001-0,0000002; stabilizing additive containing plazmozameschayuschie solution poliglyukin 1,5-2,0; salt buffer solution (pH 7.0 to 7.2) of 1.03-1.06 a; pyrogen-free water - the rest (RF patent No. 2129878, IPC a 61 K 39/395, publ. 10.05.99 is Amy stabilizing composition does not provide long-term storage of products at a temperature considerably above +4C, for example, at room temperature (+203)C.

The technical result of the invention is the creation of such a drug on the basis of recombinant alpha-2 interferon person for injection in the dry lyophilized form, the storage of which is possible at temperatures significantly above +4C, for example at room temperature (+203)C.

This technical result is achieved by the fact that the drug on the basis of recombinant alpha-2 interferon person for injection in the dry lyophilized form, including the substance of recombinant alpha-2 interferon man, salt buffer system and a stabilizing additive containing plazmozameschayuschie solution according to the invention further comprises a polyhydric alcohol and a urea or EDTA, as well as plasma-solution - poliglyukin, or reopoliglyukin, or gemodez, or a mixture of these components in any sootnoshenij with the following initial content of components to freeze drying:

the substance of recombinant alpha-2

interferon human activity

10,0-20,0 million IU/ml and

the total protein content of 0.1-0.2 mg/ml

polyhydric alcohol of 1.0-5.0 mg/ml

urea or EDTA 0.05 to whom offer these

components in any sootnoshenij) 4,0-12,0 mg/ml

salt buffer system

with a pH of 7.0-7,6 11,0-14,5 mg/ml

The most optimal is equal to the ratio poliglyukina, reopoligliukina and gemodeza (1:1:1).

Polyvinylpyrrolidone comprising gemodeza may have a molecular weight of 350,000 daltons.

As a polyhydric alcohol drug contains mannitol or sorbitol.

As salt buffer system product contains phosphate-saline buffer with pH 7.0 and 7.6.

Poliglyukin - sterile 6% solution (for injection) weight fraction (50000-70000 daltons) partially hydrolyzed dextran (polymer of glucose) in isotonic sodium chloride solution (pH 4.5 to 6.5). Molecular weight poliglyukina close to the molecular mass of albumin in the blood. Produced by hydrolysis of native dextran synthesized from sucrose by the participation of microorganisms.

Reopoliglyukin - 10% colloidal solution (for injection), partially hydrolyzed dextran with a relative molecular mass 30000-40000 (low molecular weight fraction dextran) with the addition of isotonic sodium chloride solution (pH 4.0 to 6.5).

Gemodez - water-salt solution (for injection) containing 6% nizkomolekulyarnoe and chlorine (pH of 5.2 to 7.0).

As part gemodeza can be used polyvinylpyrrolidone with a relative molecular mass of 350,000 daltons).

Poliglyukin, reopoliglyukin and gemodez have a stabilizing (protective) effect on the molecule interferon in the drying process of the drug.

The mixture of fluid solutions poliglyukina, reopoligliukina and gemodeza in different ratios, including the optimal 1:1:1 provides the stabilizing additives with a wide range of relative molecular mass from 10000-350000 daltons, which increases its protective (stabilizing) properties as the matrix compared to the individual components of this mixture.

Sorbitol and mannitol are polyhydric alcohols and belong to the group of sugars. Used in medicine in injectable form as diuretics. Have a stabilizing effect on the molecule interferon (protection of hydrogen bonds) in the process of freeze drying (salvatici ions in the solvent).

Urea (urea or amide of carbonic acid) is used in medicine in injectable form as a diuretic and as an anti-edematous and anti-hypertensive agent. In the inventive product performs the function of the chelating agent.

Salt buffer solution is a phosphate-saline buffer, which serves to maintain the stable pH of the preparation is equal to 7.0 and 7.6.

Comparison of the proposed method of obtaining recombinant interferon Alfa-2 with known analogues shows that the distinctive features of the prototype signs are new, not previously known properties, namely, providing a stable storage of the drug in dried form at a temperature of (+203)for 12 months or more.

In connection with the above technical solution meets the criterion of "inventive step".

In the following table 1 shows examples 1-8 of the claimed compositions of the drug.

Example 9. Obtaining recombinant alpha-2 interferon

For preparation of drug use semi-finished product (substance) alpha-2 interferon recombinant in VFS 42-WS-89, representing a sterile solution of pH 7.0 and 7.6, with a protein concentration of 0.1-0.2 mg/ml and specific activity (1-2) 107IU/ml of This semi-finished product is mixed with the other components of the stabilizing additive (examples 1-8, ,0-11,0

- one-deputizing sodium phosphate (Na2HPO412H2O) 2,7-3,0

- disubstituted sodium phosphate (NaH2PO42H2O) 0,3-0,5

The resulting mixture was poured into a 3.0 or 5.0 ml ampoule (inflaton), frozen in a refrigerator at a temperature not higher than -30 C, after which lyophilizer. The drying time on the installation type TG-50 is 48 hours at a residual pressure in the chamber lyophilizate 10-50 mm RT.article and the temperature of the heated shelves not exceeding 20S.

The activity of all drugs (examples 1-8) after drying is 6,5106IU/ml

Example 10. Determination of the activity of preparations of alpha-2 interferon in storage

Antiviral activity of alpha-2 interferon determine (in accordance with the Fund 42-0241118401) on the cell culture L-68 against vesicular stomatitis virus (air force). Air force pre-passedout on chicken embryos beyond 42-14-99-77, spend at least 3 passages when infecting dose of 100-1000 TCD/0.1 ml Contagious material should be 1106-1107TZG/ml Monolayer cells L-68 is grown on the mattress with a capacity of 1000 ml in the presence of growth medium (medium Needle MEM with a double set of ingredients - 90% FS 42-WS-90, serum fruits cows FS 42-7 is dirout in 40 ml of growth medium and count the number in the counting chamber Goryaeva. Then the cell suspension is diluted growth medium based to 1.0 ml contained 200 thousand/ml In the wells of the microplate contribute to 0.1 ml of cell suspension and after 2-3 days incubation at S get monoclonal culture. The content of active alpha-2 interferon determine in the finished, dry the product for which it is dissolved using water for injection, up to the original volume. Then prepare two dilutions (above and below the estimated titer) of the test drug and the industry standard sample (CCA) activity in the medium 199 or Needle with 2% serum fruits cows and antibiotics (penicillin, streptomycin). For each dilution using not less than 4 wells with cell culture. From the wells to remove the environment and contribute to 0.1 ml of each dilution of interferon, 16 holes left to control the infective dose and 4 holes for the control cell culture. Incubation is carried out in one day at S in the atmosphere with 5,00,5% CO2then in each well, including the control, make the dose of the air force, equal to 100 TCD in 0.1 ml. After making indicator of the virus in cell culture is incubated for 2-3 days. The accounting is done, if there are no signs of degeneracy in the control culture. The titer of interferon take the value ooptions of the virus. Conversion activity in ME is done according to the formula:

Data on the antiviral activity of the inventive preparations of alpha-2 interferon are shown in table 2 during storage at a temperature of +203C for 1 to 12 months.

In addition, the activity of interferon (% by source) additionally examined by high performance liquid chromatography (VGH). The method is based on the separation of substances, particularly proteins having different size molecules. A solution of a mixture of proteins containing the protein interferon, under the action of external forces, such as gravity or pressure difference, flows through a column Packed with gidratirovannym polymeric material (sorbent), made in the form of granules of a certain size. This column allows you to sort the protein molecules by size. This methodology is applied column size (264 mm), as a sorbent use nucleosil with a particle size of 5 microns and a pore size of 100 angstroms, and as eluent a gradient of acetonitrile in 0.1% TETRAPHOSPHATE.

Active protein interferon different size (conformation) from inactivated (denatured) protein interferon and has a higher speed prohozhdenie peaks active and denatured proteins of interferon on the chromatogram determine the activity of the interferon (% by source). In more detail the methodology used WIH described in: (high performance liquid chromatography in biochemistry, edited by A. Hinman, F., Lottspeich, C. welter. - M.: Mir, 1988, S. 216, 223-230).

Data for determining the activity of the inventive preparations of alpha-2 interferon method WICH are shown in table 2 during storage at a temperature (+203)C for from 1 to 12 months.

The analysis of table 2 shows that the inventive composition of the drug provides a stable preservation (minor decrease) activity of recombinant alpha-2 interferon after 12 months storage temperature (+203), which are not worse than the known analogue of recombinant interferon Alfa-2 in human serum albumin used as a stabilizer and a filler, the storage of which is provided only at a temperature of +4C. However, CSA, as mentioned above, has a high value, which is obtained from blood donors, and also requires additional control for the presence of infected material (hepatitis b and C, HIV and others).

1. The drug on the basis of recombinant alpha-2 interferon person for injection in the dry lyophilized form, including substance recombinant alpha is a thief, and pyrogen-free water, wherein the preparation further comprises a polyhydric alcohol and a urea or EDTA, as well as plasma-solution - poliglyukin, or reopoliglyukin, or gemodez based on polyvinylpyrrolidone, or a mixture of these components in the ratio 1:1:1 with the following initial content of components to freeze drying:

The substance of recombinant alpha-2

interferon human activity

10,0-20,0 million IU/ml and the content of the

total protein 0.1-0.2 mg/ml

Polyhydric alcohol of 1.0-5.0 mg/ml

Urea or EDTA 0.05 to 3.0 mg/ml

Plazmozameschayuschie solution:

poliglyukin, or reopoliglyukin

or gemodez-based polyvinyl-

pyrrolidone, or a mixture of these

components in the ratio 1:1:1 4,0-12,0 mg/ml

Salt buffer system

with a pH of 7.0-7,6 11,0-14,5 mg/ml

Pyrogen-free water the Rest up to 1 ml

2. The drug under item 1, characterized in that it as a polyhydric alcohol contains mannitol or sorbitol.

3. The drug under item 1, characterized in that it is as salt buffer system contains a phosphate-saline buffer.

4. The drug under item 1, wherein the polyvinylpyrrolidone stood in

 

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SUBSTANCE: the innovation suggested interrupts infectious-toxic shock, moreover, after that it is necessary to prescribe peroral intake of Reaferon-EC-Lipint at the dosage of 10000 - 15000 U/kg. Then one should sample patient's blood to obtain leukocytes to be washed and diluted in 0.9%-NaCl solution, activated due to incubation with immunophan and intravenously injected for a patient. Then comes peroral intake of Reaferon-EC-Lipint at the dosage of 10000 - 15000 U/kg once daily for 5 d. The innovation enables to decrease the number of complications and lethality due to decreasing immunodeficiency.

EFFECT: higher efficiency of therapy.

3 ex, 1 tbl

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