RU2559522C2 - Inhalation drug form of polysialylated human doexyribonuclease i and method of obtaining thereof - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnology. Described is lyophylised drug form of glycoprotein of human deoxyribonuclease I for preparation of inhalation solution for reduction of sputum viscosity, characterised by the fact that said glycoprotein represents recombinant human deoxyribonuclease with amino acid sequence, given in description, obtained in microorganism E. coli and covalently bound with polysaccharide, containing from 65 to 80 units of alpha-2,8-sialic acid in polysaccharide chain, by its N-end. Glycoprotein is used as active substance in amount 45.0±5.0 wt %, with magnesium chloride 3.5±0.5 wt %, calcium chloride 4.0±0.5 wt % and lactose 45.0±5.0 wt % being used as auxiliary substances. Method of obtaining claimed drug form, characterised by the fact that water solution, containing magnesium chloride, calcium chloride, lactose with the following component ratio, wt %: glycoprotein of formula I (45.0±5.0), magnesium chloride (3.5±0.5), calcium chloride (4.0±0.5), lactose (45.0±5.0) is added to water solution of synthetic glycoprotein of formula I; mixed, then poured into borosilicate vials in sterile way, lyophilised, vials are filled with sterile nitrogen, sealed with plugs in sterile way, and squeezed with caps.

EFFECT: invention makes it possible to obtain preparation of polysialylated recombinant human DNase I, suitable for preparation of inhalation solution for reduction of sputum viscosity.

2 cl, 3 tbl, 4 ex

Description

The invention relates to medicine and specifically to drugs that improve the rheological properties of sputum in the treatment of cystic fibrosis and other lung pathologies, in which there is difficulty in the process of evacuation of sputum.

The accumulation of viscous purulent secretion (sputum) in the airways of patients with cystic fibrosis, bronchitis, pneumonia of various etiologies plays a role in reducing the functional ability of the lungs and in exacerbations of the infectious process. Sputum is a gel formed by glycoprotein molecules interconnected by transverse disulfide, calcium, hydrogen and electrostatic bonds. The viscosity of the bronchial secretion is due to the content of two macromolecules: mucoid glycoproteins and DNA. The main source of DNA is decaying polymorphonuclear neutrophils, which accumulate in the airways in response to a chronic bacterial infection, as well as biofilms of microbial agents, in particular P. Aeruginosa. Viscous sputum is found in cystic fibrosis, bronchial asthma. In chronic non-specific lung diseases, a relationship was found between the viscosity of sputum and its content of mucopolysaccharides, DNA, neutrophils and other products of the inflammatory reaction. So, the viscosity of mucopurulent and purulent sputum is significantly higher than that of the mucosa.

To improve the rheological properties of sputum, various groups of drugs are used - the so-called mucolytics, which are divided into mucolytics of indirect action and mucolytics of direct action. Indirect mucolytics alter the secretion and adhesion of mucus, while direct mucolytics destroy the mucus polymers mentioned above and are of the greatest clinical interest. Direct mucolytics include thiols (cysteine, N-acetylcysteine); enzymes that destroy sputum proteoglycans (trypsin, α-chymotrypsin, streptokinase), enzymes that destroy DNA deoxyribonuclease (DNase); as well as a group of heterogeneous drugs, which include ascorbic acid, hypertonic sodium chloride solution, inorganic iodides [Braga PC, Allegra L. Dmgs in Bronchial Mucology. Raven Press, 1989].

In turn, streptodornase (DNase isolated from streptococci), human recombinant DNase (DNase I and DNase II) and bovine deoxyribonuclease isolated from the pancreas are included in the group of drugs that destroy the high-polymeric sputum DNA (DNase or, alternatively, dornase). cattle.

Both bovine DNase and recombinant human DNase I are used as an inhalation to reduce sputum viscosity and thus facilitate its evacuation in the treatment of cystic fibrosis, chronic bronchitis [Mashkovsky MD Medicines, A Manual for Doctors, Volume 2, Moscow, Izd. “Medicine”], [Ilyenkova N.A., Chikunov V.V., Alekseeva O.V., Gerasimova T.A. The effectiveness of dornase-alpha (pulmozyme) in patients with chronic bronchitis in the Krasnoyarsk Territory, Modern technologies in pediatrics and pediatric surgery: abstract. doc. V grew. Congr. - M., 2009. - S.214-215]. Pulmozyme (human dornase alpha) is a phosphorylated glycosylated recombinant deoxyribonuclease I, identical to human deoxyribonuclease isolated from urine, and consists of 260 amino acids, having a molecular weight of 37 KDa, and is produced by fermentation of a recombinant strain of Chinese hamster ovary cells. The therapeutic efficacy of the drug is based on its ability to hydrolyze the high molecular weight DNA of the mucoalveolar secretion of patients, thereby improving the rheological properties of the secretion.

The main disadvantage of preparations of any native (unmodified) DNase is a rapid decrease in enzymatic (deoxyribonuclease) activity in sputum, which is caused both by systemic absorption in the tracheobronchial tree and proteolysis of bacteria and leukocytes by proteases, and by the suppression of enzymatic activity by degradation products of cellular cytoskeleton (actin) mucoalveolar secretion in large quantities, especially in patients with cystic fibrosis. For example, the G-form of actin binds to human alpha dornase mainly in the region of Tyr65-Val66-Val 67 and inhibits the hydrolytic activity of the enzyme [Fujihara J., et. al., Actin-inhibition and folding of vertebrate deoxyribonuclease I are affected by mutations at residues 67 and 114. Comparative Biochemistry and Physiology, Part B 143 (2006) 70-75]. Moreover, the N-glycosylation profile characteristic of recombinant dornase alpha does not prevent actin inactivation and proteolysis mediated by bacterial and leukocyte proteinases.

In addition, it is known that the presence or absence of bivalent ions, in particular calcium and magnesium ions, affects the activity, stability and, accordingly, the clinical efficacy of DNases of various origins. For example, the presence of calcium ions protects pancreatic bovine DNase from the damaging effects of trypsin [Poulos, T. L., Price P. A. Some Effects of Calcium Ions on the Structure of Bovine Pancreatic Deoxyribonuclease A, J. Biol. Chem., 1972, V.247, 2900-4].

Removal of calcium ions from the medium led to a sharp decrease in activity and an increase in the deamidated forms of both lyophilized and dissolved human recombinant deoxyribonuclease I. [Bei Chen, Henry R. Costantino, Jun Liu, Chung C. Hsu, Steven J. Shire, Influence of calcium ions on the structure and stability of recombinant human deoxyribonuclease I in the aqueous and lyophilized states, J. Phann. Sci., 1999, V 88 (4), pp.477-82] Moreover, it has been shown that in patients with cystic fibrosis who do not respond to recombinant human deoxyribonuclease therapy, the concentration of magnesium ions in sputum is much lower than in patients responding to this therapy (2.0 mM vs 1.3 mM). Moreover, the addition of magnesium chloride to the sputum of “non-responding” patients led to a sharp decrease in the viscosity of sputum under the influence of DNase, i.e. DNase activity increased significantly. The effect of the magnesium salt was realized through a decrease in the ability of actin to suppress the enzymatic activity of DNase.

They tried to solve the problem of actin resistance by creating mutant forms of human recombinant DNase I — LS-DNAse [US Application No. US 2005/053994C US Patent No. 6348343], or human DNase II — DLAD [US Application No. US2004 / 0157239 A1] resistant to inactivation by G-actin. However, the use of such mutant forms of DNase naturally leads to the appearance of an immune (including antibody response) to such a foreign antigen and the occurrence of allergic reactions or neutralization (antibody binding) of the enzyme.

Another problem associated with the clinical use of ready-made dosage forms of DNase preparations is the loss of enzymatic activity of DNase in solution due to deamidation during storage and transportation in glass primary (consumer) packaging (ampoules and vials) [see, for example, US Patent No. 5783433] .

This leads to the need to produce a ready-made inhalation dosage form of recombinant human DNase I in non-glass (polymer) bottles.

Further, if the enzyme is subjected to lyophilization, then at the next dissolution for the purpose of subsequent use (inhalation of the solution), it is found that DNase loses from 10 to 50% of its enzymatic activity that exists before lyophilization.

The objective of the invention is the creation of an inhaled dosage form of a medicinal product based on other DNAse variants, on the one hand, not inactivated by actin, and on the other hand, not causing an immunological response of the body and not losing enzymatic activity during lyophilization and further dissolution before inhalation with an optimal ratio of calcium salts and magnesium and containing as a cryoprotectant lactose, providing acceptable rheological properties of the solution, as intended for inhalation and.

The problem is solved in that an inhaled dosage form of a synthetic glycoprotein, which is a recombinant human deoxyribonuclease I obtained in the E.coli microorganism and covalently linked by its N-terminus to a polysaccharide, is proposed as an inhaled dosage form of the drug to reduce sputum viscosity (mucolytic) from 65 to 80 units of alpha-2.8 sialic acid in the polysaccharide chain of the following formula:

(I)

containing a glycoprotein of the formula I as an active substance,

and as auxiliary substances - magnesium chloride, calcium chloride and lactose in the following ratio of components, wt.%:

Synthetic Glycoprotein of Formula I (45 ± 5) Magnesium chloride (3.6 ± 0.5) Calcium chloride (4.0 ± 0.5) Lactose (45 ± 5)

 as well as a method for its preparation.

In this case, the finished dosage form is a freeze-dried powder for the preparation of a solution for inhalation, placed in glass vials or ampoules.

Thus, according to the present invention, there is provided a ready-made inhalation dosage form of a medicament for decreasing sputum viscosity, characterized in that it contains a synthetic glycoprotein of formula I as an active substance, and potassium chloride and magnesium chloride, as well as lactose, as auxiliary substances of divalent cations in a certain ratio.

The present invention also provides a method for producing an inhaled dosage form of a preparation for reducing the viscosity of sputum (mucolytic) of the following composition:

Synthetic Glycoprotein of Formula I (45 ± 5) Magnesium chloride (3.6 ± 0.5) Calcium chloride (4.0 ± 0.5) Lactose (45 ± 5)

characterized in that an aqueous solution containing magnesium chloride, calcium chloride, lactose is added to an aqueous solution of a synthetic glycoprotein of the formula I, stirred, then sterilely poured into borosilicate glass vials, lyophilized, the vials are filled with sterile nitrogen, the vials are sealed with stoppers and crimped with caps.

Polysialic acids (PSA) are naturally occurring unbranched polymers of sialic acid produced by certain strains of bacteria and in certain mammalian cells (Roth., 1993). They can be obtained with various degrees of polymerization, from n = about 80 or more sialic acid residues to n = 2 by acid hydrolysis or by cleavage with neuraminidases or fractionation of natural polymer species produced by bacteria. The composition of various polysialic acids also changes so that homopolymer forms exist, i.e. alpha-2,8-linked polysialic acid, including the capsular polysaccharide of E. coli strain K1 and the meningococcal B group, which is also found in the embryonic form of a cell neuron adhesion molecule (N-CAM). Heteropolymer forms also exist, such as alternating alpha-2.8 alpha-2.9 polysialic acid of E. coli strain K92 and C group polysaccharides of N. meningitidis. Sialic acid can also be found in alternating copolymers with monomers other than sialic acid, such as group W135 or group Y N. meningitidis.

Polysialic acids have important biological effects, including evading pathogenic bacteria from the immune system and complement system. The alpha-2,8-linked polysialic acid of E. coli Kl strain is also known as “colominic acid” and it (of various lengths) is used in the present invention.

Among the bacterial polysaccharides alpha-2.8, the bound form of polysialic acid is the only non-immunogenic one (that does not cause either T-cell response or antibody formation in mammals, even when conjugated with immunogenic protein carriers). Shorter forms of the polymer (up to n = 4) are found in gangliosides of the cell surface, which are widespread in the body and are considered effective for communicating and maintaining immunological tolerance to polysialic acid.

In recent years, the biological properties of polysialic acids, in particular the biological properties of alpha-2,8-linked homopolymer polysialic acid, have been used to modify the pharmacokinetic properties of protein or low molecular weight drug molecules [Gregoriadis, 2001; Jain et al., 2003; US-A-5846951, WO-A-0187922].

Alpha-2,8-linked polysialic acid (PSA) offers an attractive alternative for protein modification, being an immunologically invisible biodegradable polymer, which is a natural part of the human body and is broken down by tissue neuraminidases to sialic acid, a non-toxic saccharide.

The choice of the active substance for the inhalation dosage form of the present invention is based on the results of studies conducted by the authors of the present invention, which discovered the ability of a recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 65 to 80 units of alpha-2.8 sialic acid in a polysaccharide chains, in the presence of calcium and magnesium ions in a certain ratio, as well as lactose:

- significantly increase the duration of a specific (enzymatic) pharmacological action;

- not inactivated by G-actin;

- possess increased stability during storage to deamidation and the formation of aggregates both in dry (lyophilized) and in dissolved (before inhalation) form.

The synthetic gene encoding human deoxyribonuclease and expression strain were constructed using the method described by Worrall (Worrall AF, Connolly BA. The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli. J Biol Chem. 1990 Dec 15; 265 (35): 21889-95). Protein refolding and purification was carried out according to the method described by Linardou (Linardou H, Epenetos AA, Deonarain MP. A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I. Int J Cancer. 2000 May 15; 86 (4): 561-9). A non-glycosylated protein was obtained with a molecular weight of 29KDa and an amino acid sequence that fully corresponds to the amino acid sequence of deoxyribonuclease I from human urine but does not have an N-linked oligosaccharide group. The specific activity of the obtained protein was at least 500 U / mg.

Synthetic glycoprotein

 that is, the conjugate of polysialic acid and recombinant deoxyribonuclease I (DNase I) was synthesized, as described in Example 1.

In the process of working on the present invention was studied and evaluated:

- the effect of different content of salts of divalent metals (calcium and magnesium) on the stability of the dosage form, as well as on resistance to the action of G-actin;

- the effect of various lactose content on the change in enzymatic activity before and after lyophilization in the preparation of the inventive dosage form, as well as on the viscosity of a solution prepared from lyophilisate for subsequent inhalation.

Based on the experimental results, the optimal ratios of auxiliary substances (salts (chlorides) of magnesium, calcium and lactose) were selected.

The invention is illustrated, but not limited to, examples of obtaining the inventive inhaled dosage form with tables showing the results of individual comparative tests.

EXAMPLE 1. Obtaining a synthetic glycoprotein, which is a recombinant human deoxyribonuclease I, covalently linked at its N-terminus with a polysaccharide (polysialic acid) containing from 65 to 80 units of alpha-2.8 sialic acid

Preparation of acetate buffer solution: 1.39 g of sodium triacetate is added to a 10 ml volumetric flask, 4.0 ml of water for injection is added, stirred until the precipitate is completely dissolved and 0.1 ml of 0.04 ml of acetic acid are pipetted ice. Make up to the mark with water for injection, close the lid and mix.

Preparation of sodium cyanoborohydride solution: 0.53 g of sodium cyanoborohydride is added to a 10 ml volumetric flask, 4.0 ml of water for injection is added, stirred until complete dissolution, adjusted to the mark with water for injection, closed with a lid and stirred.

10.0 g of polysialic acid (Mm 14 +/- 1.4 kDa, purity greater than 98%, Lipoxen PLC, UK) are added to a 100 ml volumetric flask, 30.0 ml of water for injection is added, stirred until complete dissolution, adjusted to the mark with water for injection, close the lid and mix.

Preparation of a 1M solution of triethylammonium acetate: 21.6 ml of triethylamine are introduced into a 250 ml measuring cup using a 25 ml measuring cylinder, 100.0 ml of water for injection are added with a graduated cylinder and 8.9 ml of glacial acetic acid are added to a 10 ml pipette, mixed and pour 36.4 ml of water for injection into a 50 ml graduated cylinder.

Preparation of the mobile phase A: 100 ml of a 1M solution of triethylammonium acetate are introduced into a 10-liter measuring beaker using a measuring cylinder, 500.0 ml of ethyl alcohol and 9.4 liters of water for injection are added, and stirred with a magnetic stirrer.

Preparation of the mobile phase B: 50 ml of a 1M solution of triethylammonium acetate are introduced into a 5-liter measuring cup using a measuring cylinder, 4.0 l of ethyl alcohol and 950 ml of water for injection are added, and stirred with a magnetic stirrer.

Preparation of a 0.5 M sodium hydroxide solution: 10.0 g of sodium hydroxide is added to a 2 L volumetric flask, 100.0 ml of water for injection is added, stirred until complete dissolution, adjusted to the mark with the same solvent, closed with a lid and stirred.

Preparation of diafiltration solution: 17.72 g of sodium chloride, 20.08 g of sodium hydrogen phosphate 12-aqueous and 0.42 g of sodium dihydrogen phosphate 2-aqueous are added to a 2-liter volumetric flask, 200.0 ml of water for injection are added, and the mixture is stirred until complete dissolution, adjusted to the mark with the same solvent, cover with a lid and mix.

Polysialylation of DNase I: 10 ml of the prepared acetate buffer solution, 10 ml of sodium cyanoborohydride solution and 97.1 ml of polysialic acid solution are transferred into a 250 ml measuring beaker. The resulting mixture was stirred using a magnetic stirrer and kept first at room temperature for 5 minutes, and then in a water bath at 37 ° C for 5 minutes. After exposure, add 3 ml of a solution of DNase I in water for injection at a concentration of 1 g / ml, mix and incubate the reaction mass at a temperature of 37 ° C for 180 minutes.

After the exposure, the beaker with the reaction mass is placed in an ice bath and incubated at a temperature of (2-4) ° C for 180 minutes. The resulting solution of synthetic glycoprotein, a conjugate of DNase I and polysialic acid, is passed to the reverse phase chromatography purification step.

Reverse phase chromatography: Purification of the synthetic glycoprotein of formula I is carried out by preparative reverse phase high performance liquid chromatography (HPLC). HPLC was carried out on a Varian PrepStar SD-2 chromatographic system (Varian, USA) on a preparative column (500 × 50 mm) filled with a SOURCE 30 RPC sorbent. Detection is carried out at a wavelength of 214 nm.

Before cleaning, the chromatographic column is balanced by passing the eluent (mobile phase A) for 1.5-2 hours at a speed of 20 ml / min to a constant optical density of the eluate.

Linear gradient: from 0 to 100% of mobile phase B from 0 to 100 minutes.

To a solution of synthetic glycoprotein on ice, 13.2 ml of ethyl alcohol and 2.6 ml of a 1M solution of triethylammonium acetate are added, mixed and injected into the chromatograph at a flow rate of 10 ml / min. All other chromatographic operations are carried out at room temperature.

The column is washed with 500 ml of mobile phase A, after which elution with a gradient system is started at a flow rate of 20 ml / min. The collection of the eluate is as follows:

- start no earlier than 20 minutes of the gradient and upon reaching an optical density of 0.05 OE switch the valve;

- then the valve is switched upon reaching the maximum of the first peak;

- to achieve an optical density of 0.2 OE;

- to achieve an optical density of 0.05 OE;

- collection is stopped upon reaching a minimum optical density between the first and second peak.

A fraction with a content of synthetic glycoprotein of more than 93% is passed to the stage "Concentration of a solution of synthetic glycoprotein."

Concentration of a synthetic glycoprotein solution: The concentration of a synthetic glycoprotein solution is carried out using ultrafiltration and diafiltration. For this, a Cogent Micro Scale ultrafiltration unit (Millipore, USA) is prepared, equipped with a recirculation tank and a solution supply line for diafiltration. Mount the Pellikon 2 Biomax cartridge with a cut-off level of 5 kDa and a filter area of 0.1 m ² .

Add 1.0 l of a 0.5 M sodium hydroxide solution to the recirculation tank, direct the retentate return and filtrate collection lines to the waste solution collection tank, and turn on the unit. Serve the solution on a cartridge at a speed of 100 ml / min until the recirculation tank is empty.

Connect the retentate return and filtrate collection lines to the recirculation tank. Add 1.0 l of a 0.5 M sodium hydroxide solution to the recirculation tank and recycle the solution at a speed of 200 ml / min for 30 minutes.

The installation is stopped, the retentate return and filtrate collection lines are sent to the waste solution collection tank, and the installation is turned on. Serve the solution on a cartridge at a speed of 200 ml / min until the recirculation tank is completely empty.

The installation is stopped, 1.0 L of water for injection is placed in the recirculation tank, a container containing 2 L of water for injection is connected to the diafiltration solution supply line, and this water is passed through the cassette at a speed of 100 ml / min until both containers are empty. Stop the installation, rinse the flange of the retentate return line with water for injection and connect it to the recirculation tank.

A solution of synthetic glycoprotein is placed in a container for recycling (Sb-11 ₂ ). Start the installation at a speed of 100 ml / min, visually inspect the absence of leaks in the connecting lines, and then increase the recirculation rate to 360 ml / min. Check the pressure at the entrance to the cassette - the pressure should be in the range of 1.5-2.5 atm. If necessary, change the recirculation rate. Ultrafiltration is carried out until the solution is exhausted in the supply tank and the solution level in the recirculation tank drops to the level of 10 ml.

The installation is stopped, a container with diafiltration solution is connected to the supply line of the diafiltration solution. They turn on the unit, diafiltrate until they reach 200 ml in the supply tank. The installation is stopped, the supply line of the diafiltration solution is disconnected, and recirculation is continued at a speed of 100 ml / min until the mark of 10 ml in the recirculation tank is reached. The plant is stopped, the filtrate collection line is closed with a standard clamp and recycling is continued at a speed of 20 ml / min for 5 minutes. Stop the installation, place the retentate return line in the graduated cylinder, turn on the liquid supply to the cartridge at a speed of 360 ml / min, and the diafiltered solution is completely collected in the graduated cylinder.

The remaining diafiltration solution is placed in a container for recycling and washed with the installation at a speed of 20 ml / min.

An aliquot is taken from the measuring cylinder with diafiltered solution and the concentration of synthetic glycoprotein is measured. According to the analysis, the solution is diluted with water for injection to a synthetic glycoprotein concentration of 3.0 g / l.

The yield of conjugate at the stage of TP.5 is 66.8% in terms of loaded DNase I.

The resulting concentrated solution of synthetic glycoprotein is used in the manufacture of the inventive inhaled dosage form.

EXAMPLE 2. Preparation of the inventive inhaled dosage form of a synthetic glycoprotein of the formula I (option).

Preparation of a solution of excipients: 1.153 g of lactose monohydrate, 0.191 g of calcium chloride hexahydrate and 0.153 g of magnesium chloride hexahydrate are charged into a 50 ml volumetric flask, 20 ml of water for injection are added, stirred until complete dissolution, adjusted to the mark with the same solvent, closed with a lid and mix.

Preparation of a solution for lyophilization: a 10.0 L single-use polyethylene container is installed in the Mobius Single-use Mixing Systems (Millipore, USA) mixing module. A sterile nitrogen supply hose is connected to the nitrogen port of a single polyethylene container of the mixing module, while the remaining loading and unloading ports are closed. A single short-term opening of the valve on the nitrogen supply line straightens the polyethylene container. After that, the liquid reagent supply port is opened and 330.9 ml of concentrated synthetic glycoprotein solution prepared as described in Example 1, 50 ml of excipients solution and 193.2 ml of water for injection are loaded using a graduated cylinder. Stirred for 10 minutes.

Sterile filtration: at the end of mixing (at the previous stage), the discharge port of the mixing module is connected with a silicone hose to a system of sterilizing filters with a pore diameter of 045 / 0.22 μm, which is connected to a single receiving container of the sterile mixture using a hose with a crimp end. The feeding end of the silicone hose is in the zone of cleanliness class C. The hose through the sealing sleeve enters the insulator, the sterilizing system, the crimp hose and the receiving container are in the insulator. The discharge port of the mixing module and the valve for supplying sterile nitrogen are opened. The mixed solution is pushed through the assembled sterilizing system into a 5-liter polypropylene receiving tank, during the filtration of the mixture, the pressure on the sterile nitrogen supply line is checked according to the pressure gauge, adjusting the pressure value from 0.07 to 0.15 MPa (from 0.7 up to 1.5 bar). Stop the nitrogen supply to the mixing module and close the crimp hose to the receiving 5-liter polypropylene container of the sterile mixture.

Using silicone hoses, the receiving container of the sterilized mixture is connected with a 0.22 μm depyrogenation filter and a one-time depyrogenated solution receiving tank mounted on a magnetic stirrer. Turn on the magnetic stirrer. A sterile nitrogen supply line is connected to the container of the sterilized mixture, and by opening the nitrogen supply valve, the mixture is pumped through the depyrogenation filter into a sterile single 5-liter polypropylene finishing receiving container.

The silicone hose connecting the depyrogenation filter to the final receiving tank is closed with a clamp to form a blocking half ring. A similar half-ring blocks the silicone hose connecting the discharge port of the mixing module with a sterilizing filter system. Disconnect the silicone hoses from the inlet fitting of the sterilizing filter and the outlet fitting of the depyrogenation filter. The nitrogen line compounds are removed from the 5-liter polypropylene receiving container of a sterile solution containing the active substance and excipients of the finished dosage form. The resulting sterile solution is stirred for 15 minutes. At the end of mixing, a sample is taken to determine the concentration of the active substance - synthetic glycoprotein. The data obtained are used to clarify the dosing volume when filling the vials for the next stage ("Freeze drying").

Freeze-drying: sterile glass bottles for injection preparations with a volume of 10 ml in an amount of 276 pcs. put into the transfer gateway of the insulator, remove the primary packaging, close the cover of the gateway and process the bottles with UV radiation for 15 minutes. Meanwhile, pallets are removed from the Epsilon 2-10D freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Germany) located in the insulator and placed on the insulator table. At the end of the treatment of the vials with UV radiation, the inner gateway cover leading to the insulator is opened, the primary packaging is opened and the vials are placed in the pallets of the Epsilon 2-10D freeze dryer, the used packaging is dumped into the waste container provided in the insulator. In the same way, sterile caps and bottle caps are inserted into the insulator.

A sterilized and depyrogenated solution containing the active substance and excipients obtained in the Sterile Filtration step is dispensed from the receiving container using an eLINE Pro dispenser (Biohit, Finland) into approximately 2 ml vials taking into account the concentration of the active substance in the solution in this way so that the concentration of the active substance in the final lyophilisate is 3.7 mg in one bottle. At the end of the dosing of the mixture, a lid is mounted on each bottle with a light pressure so that there remains an opening for the evaporation of liquid. Vials on pallets that are not tightly sealed are installed in an Epsilon 2-10D freeze dryer.

At the terminal of the Epsilon 2-10D freeze dryer, the shelf temperature is set to (45 ± 2) ° С and the freezing time is 3-4 hours. After the material reaches the specified temperature, exposure is carried out for 3-4 hours. The dryer condenser is cooled to - (80 ± 5) ° С and the vacuum pump is turned on. The residual pressure in the dryer is set to 0.01 mbar (1 Pa), the time for reaching the sublimation mode is 55-65 minutes. The drug is dried for 3 hours at the specified parameters. After the specified time, gradual heating of the shelves and material begins at a speed of (5-6) ° C per hour, the residual pressure of 0.005 mbar is set. Upon reaching a temperature of (8-10) ° C, heating ceases and the material is maintained under these conditions for 2 hours. The end of the drying process is evidenced by a constant material temperature equal to the temperature of the shelves, a constant vacuum level (0.005 mbar), a vapor pressure reduction sensor is triggered. At the end of the drying process, the vacuum pump is switched off, the dryer chamber is filled with dry sterile nitrogen and the final corking of the bottles takes place automatically.

The output at the stage of preparation of the inhaled dosage form is 93.6-95.6%, based on the active substance - synthetic glycoprotein.

Get 268-276 bottles of a variant of the claimed inhaled dosage form, each bottle contains 8.0 ± 0.8 mg of lyophilisate of the following composition:

Synthetic Glycoprotein of Formula I 3.7 mg Magnesium chloride 0.28 mg Calcium chloride 0.33 mg Lactose 3.7 mg

EXAMPLE 3. Evaluation of the stability of a synthetic glycoprotein of the formula I at different contents of calcium and magnesium ions in a dosage form.

The comparative stability of the synthetic glycoprotein of formula I at different contents of calcium and magnesium ions in the dosage form with respect to the accumulation of deamidated forms of DNase and aggregates during storage of both lyophilized and dissolved in 0.9% sodium chloride solution was determined as described [Bei Chen; Constantino, H. R., Influence of calcium ions on the stmcture and stability of recombinant human deoxyribonuclease I in the water and lyophilized states, J. Pharm. Sci., 1999, v. 88 (4), pp. 477-82].

For this, inhaled dosage forms of a synthetic glycoprotein of formula I were prepared as described in Example 2, except that various amounts of calcium and magnesium salts were added to the excipient solution. The content of deamidated forms of DNase and aggregates in their determination immediately after the manufacture of an inhaled dosage form (lyophilization) or, accordingly, immediately in the preparation of a solution for inhalation (from lyophilisate) was taken as a unit. The results of the experiments are illustrated in Table 1, Table 2.

Table 1. The change in the content of aggregates in the inhaled dosage form (lyophilisate) of synthetic glycoprotein over time during storage at a temperature of + 20 ° C with different contents of magnesium salts. The content of calcium chloride (wt.%) 00.00-0.99 wt.% 1.00-2.99 wt.% 3.00-3.99 wt.% 4.00-4.99 wt.% In the manufacture one one one one After 1 month 2 1,5 one 2 In 3 months 3 2 one 2 In 6 months 5 3 1,2 3

Table 2. The change in the content of aggregates in a solution of an inhalation dosage form (8 mg of lyophilisate per 1 ml) of synthetic glycoprotein over time during storage at a temperature of + 37 ° C with different contents of magnesium salts. The content of magnesium chloride (wt.%) In the lyophilisate 00.00-0.99 wt.% 1.00-2.99 wt.% 3.00-3.99 wt.% 4.00-4.99 wt.% In the manufacture one one one one After 1 hour 2 1.3 one 2 After 3 hours four 2.2 one 3 In 6 hours 6 2,8 1,1 four

From the table it follows that aggregation to the least extent occurs when the content of divalent metal ions (calcium or magnesium) in the lyophilisate of the inhaled dosage form of synthetic glycoprotein in the range of 3.00-3.99 wt.%.

Similar data were also obtained by the authors of the present invention in relation to the preservation of the enzymatic activity of synthetic glycoprotin of formula I in a solution of the proposed finished dosage form, including in comparison with the commercial preparation of “normal” (unpolished) human DNase I (dornase alpha) (Pulmozyme, Hofmann la Roche, Switzerland). Enzymatic activity was determined as described in Example 4. Moreover, the decrease in the enzymatic activity of a commercial preparation during storage occurred 1.5-2 times faster than the solution of the proposed inhalation dosage form.

EXAMPLE 4. The effect of the lactose content in the dosage form on the change in enzymatic activity during lyophilization (after dissolution) and viscosity.

The effect of the lactose content in the dosage form on the change in enzymatic activity during lyophilization was determined by the change in the enzymatic activity in the solution before lyophilization of the drug (which was taken as 100%) and after bringing the lyophilized drug in water for injection to the same volume of solution that was placed in the bottle before it lyophilization.

For this, inhaled dosage forms of a synthetic glycoprotein of formula I were prepared as described in Example 2, except that various amounts of lactose were added to the excipient solution. Enzymatic activity was determined as described [Lichtinghagen R, Determination of Pulmpzime (domase alpha) stability using kinetic colormetric DNase I activity assay, Eur. J. 2006, v. 63, pp. 365-8]. The commercial preparation dornase alpha (Pulmozyme, 1,000,000 U / L, Hofmann la Roche, Switzerland) was used as a standard for determining enzymatic activity. The enzymatic activity of synthetic glycoprotein before lyophilization of the solution was taken as 100% (as initial). The viscosity of the solutions was determined on an AMVn viscometer (Anton Paar, Austria). The results of the experiments are illustrated in Table 3.

Table 3. The effect of the lactose content in the inhaled dosage form of synthetic glycoprotein on the reduction of enzymatic activity caused by lyophilization and viscosity. The lactose content (wt.%) In the lyophilisate twenty thirty 40 fifty 60 Enzymatic activity,% of initial 85% 88% 95% 96% 98% Viscosity, mPas 1.02 1.17 1.23 1.50 1.81

It can be seen from the presented data that with an increase in the concentration of lactose, the percentage of the preserved enzymatic activity of glycoprotein in the inhaled dosage form increases, however, the viscosity of the solution increases critically (more than 1.4 mPas), which sharply reduces the effectiveness of inhalation when using inhalers. Thus, on the basis of the data presented and the data of other similar experiments, the authors selected the optimal lactose content in the inventive inhaled dosage form.

Claims (2)

1. Lyophilized dosage form of human deoxyribonuclease I glycoprotein for the preparation of an inhalation solution to reduce sputum viscosity, characterized in that said human glycoprotein, which is a recombinant human deoxyribonuclease I, obtained in the microorganism E. coli and covalently linked to its N-terminus with a polysaccharide containing 65 to 80 units of alpha-2.8 sialic acid in the polysaccharide chain of the following formula:

It is used as an active substance, and magnesium chloride, calcium chloride and lactose are used as auxiliary substances in the following ratio of components, wt.%:
glycoprotein of the formula I 45.0 ± 5.0 magnesium chloride 3.5 ± 0.5 calcium chloride 4.0 ± 0.5 lactose 45.0 ± 5.0 2. A method of obtaining a dosage form according to claim 1, characterized in that an aqueous solution containing magnesium chloride, calcium chloride, lactose is added to an aqueous solution of synthetic glycoprotein of the formula I in the following ratio, wt.%: Glycoprotein of the formula I (45.0 ± 5.0), magnesium chloride (3.5 ± 0.5), calcium chloride (4.0 ± 0.5), lactose (45.0 ± 5.0); mix, then sterilely poured into vials of borosilicate glass, lyophilize, fill the vials with sterile nitrogen, sterilely seal the vials with stoppers and crimp the caps.