RU2712235C1 - Agent with a glycosylated polypeptide and a method for production thereof - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents an anti-inflammatory medicinal agent for intranasal application in the form of an aqueous solution in therapy of post-inflammatory complications, containing 100.0 ml of water: 0.001–0.500 g of glycosylated polypeptides recovered from fresh, frozen or dried insides of Strongylocentrotus droebachiensis sea urchins with molecular weight 5.5–7.0 kDa, 0.05–0.20 g of bioadhesive component-polycarbophil, 1.0–2.5 g of glycerin, 0.025–0.15 g of disodium edetate dihydrate, 0.005–0.02 g of benzalkonium chloride and sodium hydroxide solution, providing pH 6–7.

EFFECT: invention provides storage-stable and non-toxic aqueous glycosylated polypeptides for intranasal application, as well as an extended range of anti-inflammatory agents used in therapy of post-inflammatory complications.

6 cl, 7 ex, 2 tbl, 5 dwg

Description

The invention relates to the field of pharmacy and relates to a technology for the preparation of a pharmaceutical product in the form of a solution for nasal use based on glycosylated polypeptides (GLP) isolated from the waste from processing commercial sea urchins Strongylocentrotus droebachiensis and intended for the treatment of post-influenza complications. The agent for intranasal use is stable, convenient for use, safe for clinical use. The tool has the advantages of a simple production technology, simple and convenient quality control and lower production costs compared to solid dosage forms. Thus, the tool is economically advantageous for industrial production.

One of the urgent problems of medicine at all its stages of development are acute and chronic diseases of the upper respiratory tract. The majority of infectious diseases (about 90%) are influenza and acute respiratory viral infections (ARVI) [Sergeeva IV, Kamzalakova NI, Tikhonova EP, Bulygin G.V. Pathogenesis of acute respiratory viral infections and influenza // Practical medicine. - 2012. - T. 61, No. 6. - S. 47-51]. Economic losses in the Russian Federation due to influenza and acute respiratory viral infections account for 86% of all damage caused by infectious diseases, which is comparable to injuries, cardiovascular diseases and malignant neoplasms [Osidak L.V., Drinevsky V.P., Tsybalova L.M. Acute respiratory infections in children and adolescents: a practical guide for doctors / ed. L.V. Osidak. - 3rd ed., Add. - St. Petersburg: InformMed, 2010. - 216 s]. The high frequency of acute respiratory viral infections is largely due to a variety of pathogens, among which the viruses influenza, parainfluenza, adeno-, rino-, reo- and PC viruses dominate. Often there are acute respiratory viral infections due to viral-bacterial, viral-mycoplasma associations, which complicates the prevention and effective treatment of these diseases and in most cases leads to complications [Nosulya E.V., Kim I.A., Vinnikov A.K. Acute respiratory infections in the practice of an otorhinolaryngologist: difficulties and prospects of treatment // Farmateka. - 2013. - No. 1. - S. 85-87].

Influenza is considered a respiratory infection with a difficult to predict course and rapidly developing complications. However, due to the rapid spread of viral infections, chemotherapy in the current practice of treating infectious diseases and preventing complications begins to play an increasing role [O. Kiselev. Chemotherapy and chemotherapy for influenza / O.I. Kiselev - SPb .: LLC “Publishing house“ Rostock ”, 2012. - 272 p.]. A common complication of influenza are pneumonia, rhinitis, sinusitis, bronchitis, and otitis media, and according to WHO data, approximately 10% of all patients and 50% of hospitalized patients with influenza develop pneumonia [Pankov A.S. Bacterial complications of influenza and their prognosis // Bulletin of the Samara Scientific Center of the Russian Academy of Sciences. 2012. - T. 12, No. 5 (2). - S. 490-493].

The arsenal of modern medicine has a relatively small list of etiotropic chemotherapy drugs that have received international recognition (cycloferon, remantadine, oseltamivir, zanamivir). But, despite this, the problem of treatment of post-influenza complications remains relevant, since the drugs used have mainly antibiotic activity and high toxicity. Thus, the development of drugs to prevent the development of post-influenza complications is a very urgent task.

Known means enriched with peptides, amino acids and phospholipids, with anti-inflammatory properties by inhibiting COX-2, normalizing the performance of the antioxidant system. The activity of the agent in experimental acute rhinosinusitis and experimental acute bronchitis of rats is shown. According to the described method, a complex preparation is obtained, which includes both low molecular weight oligopeptides, amino acids and minerals, and polypeptides. A significant amount of lipid compounds passes into the alcohol extract, which co-precipitate on the surface during the drying process, the product is stuck and rapidly oxidized (Patent RU 2481119, published 05/10/2013).

Known pharmaceutical composition having antithrombotic, thrombolytic, immunomodulatory, anti-inflammatory actions, normalizing lipid and carbohydrate metabolism obtained from lyophilized medical leeches. In this case, the composition of the lyophilisate of a medical leech is not described and the possibility of using an agent to prevent the development of post-influenza complications is not considered (Patent RU 2519741, published December 27, 2013).

A method for producing a new glycopeptide antibiotic derivative against a vancomycin-resistant bacterium is described (Patent RU 2481354, published May 10, 2013). The disadvantage of this invention is that its use is limited only to the treatment of post-influenza complications caused by vancomycin-resistant bacteria.

Describes new derivatives of glycopeptide compounds exhibiting broad activity against gram-positive microorganisms, as well as improved antimicrobial activity against vancomycin-resistant isolates (Patent RU 2145609, published 02.20.2000). However, this patent describes glycopeptides that are active against gram-positive microorganisms, which limits their use.

The synthesis of the so-called "fucopeptides" and their pharmaceutical composition, as well as a method for the prevention or treatment of disorders or diseases that are mediated by the binding of selectins, in particular chronic inflammation and autoimmune diseases (Patent Application RU 97117356, published July 10, 1999) are described. The synthesis of compounds is complex and multi-stage, which entails significant economic costs for its production and will not allow their use in practice as affordable means for the treatment of post-influenza complications.

Glycoproteins are proposed that are isolated by isoelectric focusing from the intercellular space of tissues of various organs, blood serum, human and animal bile, soluble in a saturated (100%) solution of ammonium sulfate and having an apparent molecular weight of 10-45 kDa. Such glycoproteins have biological activity in ultra-low doses of 10 ^-12 -10 ^-29 mol / l and lower. Pharmaceutical compositions are also provided comprising these glycoproteins in an effective amount and a pharmaceutically acceptable carrier (Patent RU 2223781, published 02.20.2004). The present invention does not provide examples in vivo, confirming the possibility of using glycoproteins for the treatment of post-influenza complications.

New antibiotics and a method for their preparation by the acylation reaction of 11,12-cyclic carbonate 2'-O-acetyl-4 '' - O- (ω-aminoalkyl) carbamoylazitromycin with eremomycin, vancomycin or teicoplanin aglycon in the presence of a condensing agent followed by removal of 2 '-O-acetyl-protecting group (Patent RU 2570425, published 10.12.2015). In the description of the invention, only in vitro antimicrobial activity data is provided. There is no evidence of pharmacological activity in vivo.

New compositions and methods for alleviating the symptoms of atherosclerosis and other inflammatory conditions, such as rheumatoid arthritis, lupus erythematosus, polyarteritis nodosa, osteoporosis, Alzheimer's disease and viral diseases such as influenza A, are known. The peptide has a length in the range of 3 to 10 amino acids; includes a sequence of amino acids, where the sequence contains acidic and basic amino acids, alternating with aromatic, hydrophobic or uncharged polar amino acids; includes hydrophobic terminal amino acids or terminal amino acids bearing a hydrophobic protecting group, and is not a LAEYHAK sequence (SEQ ID NO: 2) including all L-amino acids; moreover, the peptide converts high-density pro-inflammatory lipoprotein (HDL) to anti-inflammatory HDL or makes anti-inflammatory HDL more anti-inflammatory (Patent RU 2448977, published 04/27/2012). The disadvantage is a multi-stage complex synthesis with a low yield.

Thus, direct analogues, i.e. ready-made drugs based on GLP for the treatment of post-influenza complications were not found.

Closest to the present invention is an invention describing the preparation and use of a product based on GLP from waste processing commercial sea urchins Strongylocentrotus droebachiensis, which has an anti-inflammatory effect (Patent RU 2545703, published April 10, 2015). The disadvantage of this invention is the lack of a description of the composition for intranasal use and further use in the clinic.

The basis of the invention is the creation of a pharmaceutical preparation in the form of an aqueous solution for intranasal use, which contains the necessary and sufficient amount of GLP secreted from Strongylocentrotus droebachiensis sea urchins and is stable during storage.

The problem is solved in that the claimed agent for intranasal use in the form of an aqueous solution for the treatment of post-influenza complications according to the invention contains a therapeutically effective amount of GLP from the waste products (entrails) of commercial sea urchins Strongylocentrotus droebachiensis and a bioadhesive component in an aqueous solution with a pH from 6 to 7.

The inventive composition as an active substance contains GLP, isolated from the insides of sea urchins Strongylocentrotus droebachiensis by extraction of fresh, frozen or dried viscera with ethyl alcohol at a ratio of raw materials: extractant 1: 1-1: 3 at a temperature from 0 to plus 10 ° C for from 2 hours to 10 days, separating the extract in a separator or centrifuge, evaporating it to 1 / 50-1 / 70 volume, filtering the concentrate through a filter with a pore size of 1 kDa, gel chromatography of the concentrate, separating the fraction with a molecular weight of 5.5-7 , 0 kDa and drying it Niya,

Preferably, an effective amount of GLP is 0.001-0.500 g / 100.0 ml.

The inventive composition as a bioadhesive component contains polycarbophil.

Preferably, the effective amount of polycarbophil is 0.05-0.2 g / 100.0 ml.

The inventive composition may additionally contain 1.0-2.5 g of glycerol, 0.025-0.15 g of disodium edetate dihydrate and 0.005-0.02 g of benzalkonium chloride in 100.0 ml of solution.

A preferred embodiment of the claimed composition contains 0.01 g GLP, 0.1 g polycarbophil, 2.1 g glycerol, 0.05 g disodium edetate dihydrate and 0.01 g benzalkonium chloride in 100.0 ml of solution and a sufficient amount of sodium hydroxide to provide pH 6.5 in a volume of 100.0 ml.

To obtain a dosage form, water is poured into the reactor, the calculated amount of benzalkonium chloride, disodium edetate, glycerol and GLP is added, mixed until dissolved, the bioadhesive component is added to the resulting solution, mixed until uniform and neutralized with sodium hydroxide solution to pH 6-7, mixed, filtered and poured into dark glass vials with a screw-on dispenser-dispenser.

All stages of the process were carried out at room temperature and relative humidity of 30-50%.

When studying the properties of the obtained solutions, it was also found that during cooling, freezing and thawing of solutions during the day, the properties of the drug do not change.

When studying stability, the following indicators were selected for drug control: description, authenticity, pH, viscosity, average dose weight, quantitative determination. It was found that over the entire shelf life of the GLP solution for intranasal administration, it remained stable in all respects.

The technical result provided by the invention is to obtain stable during storage and non-toxic when using an aqueous solution of GLP for intranasal use with a pH of 6-7, obtained without the use of toxic organic solvents and high temperatures.

The practical application of the claimed invention for the treatment of post-influenza complications provides him with the criterion of "industrial applicability".

The present invention is illustrated by the following examples of the preparation of the composition and its use as an agent for the treatment of post-influenza complications, which can be administered intranasally.

DESCRIPTION OF FIGURES

In FIG. Figure 1 shows the dependence of the amount of phospho-forms of p38 MAP kinase in U937 cell culture on GLP concentration.

The abscissa shows the concentration of GLP in μg / ml; along the ordinate, the amount of phospho-forms of p38 MAP kinase in% of control.

In FIG. Figure 2 shows the effect of drug administration on the number of goblet cells in evaluating the anti-inflammatory effect on a model of acute rhinosinusitis. The agent according to example 1 based on GLP (3) was administered in doses of 25, 50 and 100 μl / nasal passage in comparison with intact animals (1), control (pathology) (2) and comparison preparations Aqua Maris (4) at a dose of 100 μl / nasal passage and Polydex with phenylephrine (5) at a dose of 100 μl / nasal passage. The abscissa axis is the group and dose; along the ordinate axis, the number of cells per 1 mm of the nasal mucosa.

* - p <0.05, statistically significant difference from the control group (Tukey test).

# - p <0.05, statistically significant difference from the intact group (Tukey test).

In FIG. Figure 3 shows the effect of drug administration on the cellular composition of bronchoalveolar lavage 48 hours after the induction of inflammation in a model of acute bronchitis. The agent according to example 1 based on GLP (3) was administered at doses of 25, 50, and 100 μg / kg in comparison with intact animals (1), control (pathology) (2), and beclomethasone comparison drug (4) at a dose of 5.7 μg / kg The abscissa axis is the group and dose; along the ordinate axis, the number of neutrophils and leukocytes is 10 ⁹ / L.

* - p <0.05, statistically significant difference from the control group (Tukey test).

In FIG. 4 shows the pharmacokinetic distribution curves of GLP after a single intranasal administration to rats of the preparation of Example 1 at a dose of 100 μg / kg in plasma (1) and in the nasal mucosa (2).

The abscissa axis shows time in hours; along the ordinate, the level of detected GLP in μg / ml for plasma and in μg / g for the nasal mucosa.

In FIG. 5 is a summary graph of tissue availability of GLP after a single intranasal administration to rats of the agent of Example 1.

The abscissa axis is organs and tissues; along the ordinate axis tissue availability in%.

Example 1. The preparation of a solution for intranasal use based on GLP

To obtain the dosage form, 703 ml of purified deionized water is poured into the reactor, the mixer is connected, and when the mixer is switched on, 0.0763 g of GLP, 0.072 g of benzalkonium chloride and 0.362 g of disodium edetate, weighed on scales, are carefully added to the container. Mixing is carried out until a clear solution is obtained. After that, 15.22 g of glycerol, previously weighed on a balance, is loaded into the solution and mixed until a clear solution is obtained. The mixing process is carried out at room temperature. Mixing speed 100-200 rpm

Then 0.72 g of polycarbophil is added and stirred for uniform polymer dispersion for 1-2 minutes, then, without stopping the mixing process, the solution is neutralized by adding 5.0 ml of a 1M sodium hydroxide solution. Stirring is carried out at room temperature for 25 minutes until a homogeneous gel-like solution is obtained. After gel formation, the GLP solution after pH control is filtered under vacuum through a paper filter. The pH of the resulting solution should be 6.0-7.0. Next, the resulting solution is poured into dark glass vials and sealed with a dispenser-sprayer. Get 71 vials with a dosing device of 10 ml.

Example 2. Assessment of the stability of funds based on GLP.

The stability study of the agent according to example 1 was carried out by natural storage at a temperature of (+ 2 ... + 8 ° C) to control the quality of the drug, the following indicators were selected: transparency, color (color), pH, viscosity, dosage uniformity, average dose weight, quantity doses per pack.

It was found that the developed GLP composition for intranasal use is stable during storage. There is no change in the color of the solution, the appearance of any mechanical inclusions. Also, there is no significant change in the concentration of the active substance and the appearance of impurities. The data obtained indicate that the proposed composition meets the requirements of the State Pharmacopoeia of the Russian Federation XIV, Volume II, presented for aerosols and sprays (OFS.1.4.1.0002.15).

It was found that all indicators remain within normal limits.

Example 3. The effect of the agent of example 1 on the LPS-induced phosphorylation of MAP kinases p38 in cell culture U937

The study was conducted in a culture of human monocytes of the line U937. Lipopolysaccharide (LPS) of the cell wall of E. coli bacteria was used to induce phosphorylation. The method of determination is immunoblotting.

Evaluation of the amount of phosphorylated forms of p38 MAP kinase in the implemented model revealed that the agent has an inhibitory effect on the LPS-induced activation (phosphorylation) of p38 MAP kinase in a wide concentration range from 0.00041 μg / ml to 0.0025 μg / ml (Fig. . 1). The IC _{50 of the} GLP substance was 0.00133 μg / ml or 1.33 ng / ml.

Example 4. The effect of the agent of example 1 on the activity of the enzymes cyclooxygenase-1, cyclooxygenase-2 and 5-lipoxygenase.

Test method - enzyme immunoassay. Comparison drug indomethacin.

It was found that the tool according to example 1 is a specific inhibitor of the COX-2 enzyme. The tool according to example 1 dose-dependently inhibits the activity of the COX-2 enzyme in the concentration range of 0.01-10 μg / ml. When testing solutions of a higher concentration, pro-inflammatory activity was observed. The IC50 was 0.73 ± 0.06 μg / ml. It should also be noted that the maximum inhibition for the tested substance was not more than 70%. The IC50 of indomethacin was 0.25 ± 0.03 μg / ml. A decrease in the activity of the COX-2 enzyme and, consequently, a decrease in the concentration of pro-inflammatory prostaglandins and other products, may be one of the mechanisms of the anti-inflammatory effect of the agent based on GLP.

Thus, the data of examples 3 and 4 made it possible to establish that the agent of example 1 directly affects the pathogenesis of influenza, which is based on inflammation.

Example 5. The effect of the agent of example 1 in comparison with reference drugs on the course of experimental acute rhinosinusitis induced by formalin in rats.

Inflammation was induced by intranasal injection of 20 μl in each nasal passage of a 7.5% formalin solution. Drugs were administered daily for 7 days.

The results are presented in tables 1 and 2 and in figure 2.

When processing the experimental data, statistically significant differences were found between the experimental groups in terms of “mucosal infiltration with mononuclear cells”, “mucosal infiltration with leukocytes”, “mucosal damage”, “epithelial desquamation”, “inflammatory changes in the nasal passages (OCD and OCD)”, as well as “ inflammatory changes in various departments (vestibule of the nose, olfactory region and respiratory department) ”(p <0.05). It was found that when testing the agent according to example 1, the number of goblet cells decreases by more than 2 times compared with pathology and is comparable to the action of reference drugs (Fig. 2). Subsequent multiple comparisons of the average ranks of the study groups revealed statistically significant differences according to these indicators in animals of the control group from animals of the intact group and animals treated with the preparation of Example 1 and reference preparations at a dose of 100 μl / each nasal passage. Also in terms of “damage to the mucous glands”, “desquamation of the epithelium”, “inflammatory changes in the nasal passages (OCD; OCHR)” and “inflammatory changes in various departments (olfactory region and respiratory department),” the results of a study of a group of animals treated with the drug according to example 1 in dose of 50 μl / each nasal passage, differed from the control group of rats receiving placebo (tables 1 and 2). Significant differences between the results of the intact group of animals and rats treated with the agent of example 1 at doses of 50 and 100 μl / each nasal passage and reference preparations were not detected.

Thus, the analysis of histological preparations showed in animals with model pathology the development of a microscopic picture of acute catarrhal and acute catarrhal-purulent rhinosinusitis. The administration of the preparation of Example 1 at doses of 50 and 100 μl / each nasal passage, as well as reference preparations at a dose of 100 μl / each nasal passage, had a positive therapeutic effect on acute rhinosinusitis induced by formalin in rats and led to a cure of the pathology.

Example 6. The study of the specific pharmacological activity of the agent according to example 1 in comparison with the reference drug in the model of lipopolysaccharide-induced acute bronchitis in rats

Induction of inflammation by endotracheal administration of LPS at a concentration of 1 mg / ml. The introduction of drugs 6 and 25 hours after the induction of inflammation.

In the model of LPS-induced acute bronchitis in rats, both the agents of Example 1 at doses of 50 and 100 μg / kg and the reference drug beclomethasone at a dose of 5.7 μg / kg were administered, and a statistically significant dose-dependent decrease in leukocyte infiltration due to reducing the neutrophil population in the focus of inflammation (Fig. 3).

According to the results of morphometry and histological evaluation, the administration of the agent of Example 1 at doses of 50 and 100 μg / kg, as well as the reference drug beclomethasone at a dose of 5.7 μg / kg, had a positive therapeutic effect on acute bronchitis caused by the administration of lipopolysaccharide.

Thus, the studies of the specific pharmacological activity of the agent according to example 1 on the model of LPS-induced acute bronchitis and acute sinusitis led to the following conclusions:

- the tool according to example 1 in doses of 50 and 100 μg / kg has anti-inflammatory effect;

- the anti-inflammatory effect of the agent according to example 1 at a dose of 100 μg / kg according to the severity of the pharmacological effect is comparable with the action of reference drugs.

Example 7. Evaluation of pharmacokinetics with single and multiple intranasal administration of the agent of Example 1 in rats and rabbits.

It was found that after a single injection of the agent according to example 1, the active substance quickly penetrates the nasal mucosa and slightly into the blood. In this case, the absolute bioavailability of the active substance from the studied drug was more than 80%. The pharmacokinetics of the drug is linear in a wide range for both animal species.

The main parameter characterizing the degree of bioavailability of the drug, AUC _0-t , as well as the maximum concentration of C _max for rats increase with increasing dose of the drug. For rabbits, AUC _0-24 , statistically significantly increases with increasing dose of the drug administered, C _max tends to increase, but the differences are not statistically significant.

The time to reach C _max for rabbits was 1.00-1.13 hours and had no statistically significant differences, the average retention time (MRT) and half-life (T1 / 2) for rats in blood plasma was 4.63-9.53 hours and 2.63-6.73 h, have statistically significant differences for the maximum dose (200 μg / kg) from the minimum (50 μg / kg) and average (100 μg / kg) (Fig. 4).

After the introduction of the agent according to example 1, the active substance is intensively distributed into organs with varying degrees of vascularization: the most intense distribution is observed in the tissues of the nasal mucosa, which are the zone of administration and therapeutic effect of the drug, as well as strongly vascularized organs (spleen, adrenal glands) (Fig. 5) .

Withdrawal of the drug is carried out with urine (about 28%) and with feces - about 53%.

Repeated administration of the agent according to example 1 for 7 days at a dose of 100 μg / kg to rats led to the accumulation of the active substance, expressed as a statistically significant increase in C _max and AUC _0-24 , for the remaining parameters there were no statistically significant differences after a single and multiple administration of the drug .

Claims (16)

1. An anti-inflammatory drug for intranasal use in the form of an aqueous solution in the treatment of post-influenza complications, containing 100.0 ml of water: glycosylated polypeptides, isolated from fresh frozen or dried the insides of sea urchins Strongylocentrotus droebachiensis with a molecular weight of 5.5-7.0 kDa 0.001-0.500 g bioadhesive component polycarbophil 0.05-0.20 g glycerin 1.0-2.5 g disodium edetate dihydrate 0.025-0.15 g benzalkonium chloride 0.005-0.02 g sodium hydroxide solution, providing a pH of 6-7. 2. The tool according to p. 1, characterized in that the amount of glycosylated polypeptides is 0.01 g. 3. The tool according to p. 1, characterized in that the pH of the solution is 6.5. 4. The tool according to p. 1, characterized in that it can additionally contain pharmaceutically acceptable excipients. 6. A method of obtaining a medicinal product according to paragraphs. 1-4, characterized in that the reactor is filled with water, the calculated amount of benzalkonium chloride, disodium edetate dihydrate, glycerol and glycosylated polypeptides are added, mixed until dissolved, the bioadhesive component polycarbophil is added to the resulting solution, mixed until homogeneous and neutralized with sodium hydroxide solution until pH is about 6-7, stirred, filtered and poured into dark glass vials with a screw-on dispenser-sprayer.

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