RU2743927C1 - SOLID DISPERSION of 4- (3-ETHOXY-4-HYDROXIBENZIL) -5-OXO-5,6-DIHYDRO-4H- [1,3,4] -THIADIAZINE-2- (2,4-DIFLUOROPHENYL) -CARBOXAMIDE FOR PRODUCING PHARMACEUTICAL FORMULATION AND METHOD OF TREATMENT OF CHRONIC INFECTIOUS DISEASES - Google Patents

Abstract

FIELD: pharmaceutical industry and medicine.

SUBSTANCE: invention relates to chemical and pharmaceutical industry and medicine, in particular to solid dispersion for production of pharmaceutical formulations. It comprises the active ingredient 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine-2- (2,4-difluorophenyl) -carboxamide and at least one pharmaceutically acceptable polymer, which is contained in a solid dispersion at a mass ratio of the active substance to the polymer from 1: 1 to 1:10, while the active substance is contained in amorphous form or in amorphous form with an admixture of microcrystalline and crystalline phases. The invention also relates to a method for treatment of chronic infections of the respiratory system caused by Pseudomonas aeruginosa antibiotic-resistant strains. The method comprises the use of the specified solid dispersion in an effective therapeutic dose for humans in an amount of 1 to 4 milligrams per kilogram body weight (mpk) equivalent to the active ingredient.

EFFECT: technical result consists in obtaining a solid dispersion with increased bioavailability in comparison with the active substance in crystalline form and therapeutic efficiency in relation to infections caused by Pseudomonas aeruginosa antibiotic-resistant strains.

8 cl, 9 ex, 13 tbl, 18 dwg

Description

Technology area

The present invention relates to the chemical-pharmaceutical industry and medicine, and, in particular, to the field of medical microbiology and the development of antibacterial drugs.

State of the art

The development of new antibacterial drugs that are effective against antibiotic-resistant bacteria and do not cause the development of resistance is a key challenge in the field of infectious diseases. In February 2017, WHO published a list of antibiotic-resistant “priority pathogens” - 12 species of bacteria that pose the greatest threat to human health. Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae are included in the first category of priority in the development of new antibiotics "critically high priority level".

Fundamental research carried out at FSBI “NITsEM named after N.F. Gamalea ”, allowed the development of an innovative antibacterial drug, the mechanism of action of which is different from the action of antibiotics and is based on suppressing the virulence of pathogens, which leads to blocking the infection in the infected organism. The main mechanism of action is based on inhibition of the type III secretion system, which is present only in pathogenic gram-negative bacteria: Yersinia sp., Pseudomonas spp., E. coli (EPEC and EHEC), Shigella spp., Salmonella spp., Chlamydia spp., Bordetella, Burkholderia, Citrobacter rodentium, etc., 174 species in total.

In the structure of nosocomial infections, Pseudomonas aeruginosa is the second most common cause of nosocomial pneumonia, the third most common cause of urinary tract infections, the fourth most common cause of surgical infections, and the seventh most common blood infection. Most importantly, Pseudomonas aeruginosa is the leading infection among antibiotic-resistant gram-negative bacteria that cause pneumonia in hospitalized patients. The fight against nosocomial infections requires significant costs associated with the control and timely diagnosis of infection, the selection of effective therapy, the need to use intensive care units, the length of stay in the hospital, severe complications of these diseases, up to deaths. The economic damage in the Russian Federation is estimated at 5 billion rubles a year. In addition, throughout the world there is a steady trend towards an increase in nosocomial infections and, especially, an increase in antibiotic-resistant infections. (Pang Z., Raudonis R., Glick B. R., Lin T-J., Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies // Biotechnology Advances. 2019. No. 37. P.177-192).

The problems in the treatment of infections caused by Pseudomonas aeruginosa are the low efficacy of antibiotic therapy against resistant strains, the detection rate of which ranges from 14 to 52% of the total number of detected cases, and the rapid emergence of resistance to new drugs during treatment. (Chatterjee M., Anju C.P., Biswas L., Kumar V. A., Mohan C. G., Biswas R. Antibiotic resistance in Pseudomonas aeruginosa and alternative therapeutic options // International Journal of Medical Microbiology. 2016. No. 306. P. 48-58).

One of the most serious diseases complicated by chronic infection with antibiotic-resistant bacteria is cystic fibrosis. (Shaginyan I. A., Chernukha M. Yu., Avetisyan L. R., Siyanova E. A., Kulyastova D. G., Medvedeva O.S., Priputnevich T. V., Trofimov D. Yu., Gordeev A. V., Kondratyeva E. I., Amelina E. L., Krasovsky S. A. Epidemiological features of chronic lung infection in patients with cystic fibrosis // Epidemiology and Vaccinoprophylaxis. 2017. No. 6 (97). P. 5-13) ... Cystic fibrosis is a hereditary lethal disease, the key factor of which is chronic lower respiratory tract infection, which determines the severity of the clinical course and is considered as the leading factor in mortality associated with premature death in 90% of patients. Pseudomonas aeruginosa is most often isolated from sputum and bronchoalveolar lavage samples from cystic fibrosis patients of all ages. (Winstanley C., O'Brien S., Brockhurst M. A. Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections // Trends in Microbiology. 2016. Vol. 24. N. 5. P. 327-337). This pathogen is difficult to eradicate, causes a pronounced inflammatory reaction, leading to a rapid deterioration in lung function and an unfavorable prognosis. According to the data of national registers and the pan-European Register (2014), the prevalence of Pseudomonas aeruginosa among patients with cystic fibrosis varies between European countries: in Russia - about 32%, from 14.29% in Slovenia to 65.57% in Moldova; in the USA - 47.5%.

It is also important to note that Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis have a number of differences from microorganisms isolated from biological media of patients with other pathologies. Chronic infections with cystic fibrosis require prolonged and repeated courses of antibiotic therapy, which is associated with an increased risk of developing resistance. It is a well-known fact about the development of multi-resistant strains in patients with cystic fibrosis. (Polikarpova S.V., Kondratyeva E.I., Shabalova L.A., Pivkina N.V., Zhilina S.V., Voronkova A.Yu., Sherman V.D., Nikonova V.S., Kapranov N.I., Kashirskaya N.Yu., Semykin S.Yu., Amelina E.L., Krasovsky S.A.Microflora of the respiratory tract in patients with cystic fibrosis and antibiotic sensitivity in a 15-year follow-up (2000-2015) // Medical Council. 2016. №15. С 84-89). Despite the emergence of new specific antibacterial agents, it is believed that complete eradication of Pseudomonas aeruginosa in chronic infection in patients with cystic fibrosis is practically impossible and antibiotic therapy can only reduce the colonization of the respiratory tract. In this regard, it seems important to use effective antibacterial drugs not only for the treatment of exacerbations as part of complex therapy, but also in the inter-relapse period, both as part of complex antibiotic therapy, and in the monotherapy regimen to increase the periods between exacerbations, reduce the frequency of relapses and their severity. (Caverly Lindsay J., LiPuma John J. Cystic fibrosis respiratory microbiota: unraveling complexity to inform clinical practice // Expert Rev Respir Med. 2018. October. No. 12 (10). P. 857-865).

Bronchiectasis (EB) is a lung disease that causes abnormal changes in the airways. As a rule, the development of EB, as well as chronic obstructive pulmonary diseases, is based on recurrent infections of the lower respiratory tract, a local inflammatory reaction, and damage to the bronchial wall. The course of the disease is characterized by a chronic infection of the respiratory tract, the development of frequent episodes of exacerbations, manifested by an increase in the local inflammatory process and respiratory symptoms. It is known that colonization of the lower respiratory tract by P. aeruginosa is associated with a number of adverse consequences. According to the results of 21 cohort observational studies involving 3683 patients with EB, the presence of P. aeruginosa colonization in the bronchi almost threefold increases the risk of death compared to symptomatic EB patients in whom a different type of microorganism was isolated from the corresponding respiratory samples. The likelihood of hospitalization in such patients increases almost sevenfold. The risk of exacerbations is significantly increased if sputum or bronchial secretions of P. aeruginosa are detected in single or re-determined samples. Antibiotics are the main treatment for chest infections, but their use must be weighed against possible side effects and the risk of increased resistance to antibiotic therapy. One strategy to improve response and / or reduce antibiotic resistance involves the use of two antibacterial agents simultaneously with increasing doses. (Sinopalnikov A. I., Zaitsev A. A. Antibiotic therapy of exacerbations of chronic bronchitis / chronic obstructive pulmonary disease. Key provisions. Medical Council, 2017, 18 pp. 14-20. A. I. Sinopalnikov. COPD and bronchiectasis. "EFFECTIVE PHARMACOTHERAPY. Pulmonology and otorhinolaryngology "2015, No. 3-4 (30). Sethi S., Murphy TF Infection in the pathogenesis and course of chronic obstructive pulmonary disease // N. Engl. J. Med. 2008. Vol. 359. No. 22. P. 2355-2365).

In connection with the above, it is extremely important to develop fundamentally new approaches to the development of drugs for the treatment of bacterial infections caused by antibiotic-resistant pathogens, which can be used both independently and as part of combination therapy.

To develop such fundamentally new drugs with an alternative antibiotic mechanism of action, various pathogenic factors are selected as targets for suppression, such as toxins, secretory systems, adhesion factors, quorum sensing molecules, and biofilm formation factors. The development of antibacterial drugs that suppress various factors of bacterial virulence is a promising direction, which is currently being very actively developed in various research centers and pharmaceutical companies. At the same time, many drugs are at the stage of scientific development or preclinical trials, only a few are undergoing clinical trials and only a few are registered by the FDA (Food and Drug Administration) .The effectiveness of drugs based on immunoglobulins and monoclonal antibodies that specifically bind to toxins or membrane virulence factors. In the claimed invention, a universal factor of pathogenicity of gram-negative bacteria is selected as the main target - a type III secretion system. This secretory apparatus is necessary for a wide range of pathogens to establish an infection; it is absent in normal microflora. The key factor in the pathogenicity of Pseudomonas aeruginosa is the type III secretion system (TSS), which ensures colonization of the respiratory tract and long-term persistence.

The closest analogue of the technical solution of the claimed invention is the patent of the Russian Federation No. 2624846, included in this description as a reference "The use of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3 , 4] -thiadiazine-2- (2,4-difluorophenyl) -carboxamide for suppressing infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa, and a method for suppressing this infection. "

In the patent under consideration, it was experimentally demonstrated that the obtained low molecular weight compound, 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine-2- (2 , 4-difluorophenyl) -carboxamide, is an inhibitor of Pseudomonas aeruginosa CCT. The resulting compound specifically inhibits the secretion of toxins - effectors of the type III secretion system of pseudomonads, causing the death of eukaryotic cells. In vitro blocking of the Pseudomonas aeruginosa type III secretion system has been shown for clinical strains with different genotypes for the genes of effector CCTT proteins and different antibiotic resistance profiles.

The task set in this invention was achieved, namely:

• suppression of the infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa with different genotypes for the genes of the effector proteins of the CCT and a different profile of antibiotic resistance.

• suppression of the development of infection caused by Pseudomonas aeruginosa in an infected organism in an in vivo model, regardless of acquired antibiotic resistance.

• no inhibitory effect on the normal microflora of the macroorganism when using 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4 -difluorophenyl) -carboxamide.

However, some disadvantages of the invention under consideration can be noted:

• the obtained low-molecular-weight CCTT inhibitor is a drug substance that cannot be used for further development of an antibacterial drug without creating a dosage form;

• for the obtained inhibitor, the pharmacokinetic properties that determine the pharmacodynamics and efficacy of the drug have not been studied;

• no studies have been conducted on the physicochemical properties of the inhibitor that affect the bioavailability of the drug;

• conditions that increase the bioavailability of the resulting inhibitor 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine-2- (2, 4-difluorophenyl) -carboxamide, characterized by limited solubility in aqueous solutions.

Thus, it is necessary to carry out further work on the creation of a dosage form based on the obtained inhibitor of the type III secretion system, which has a high bioavailability for preclinical and clinical studies with the subsequent registration of an innovative drug under development that is effective against antibiotic-resistant pathogens.

Background of the present invention

The developed active ingredient 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine-2- (2,4-difluorophenyl) -carboxamide showed activity against infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa in in vitro and in vivo experiments (RF Patent No. 2624846 “Application of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro- 4H- [1,3,4] -thiadiazine-2- (2,4-difluorophenyl) -carboxamide for suppressing infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa, and a method for suppressing this infection ").

Further work has shown that it is possible to expand the scope of the active substance under the patent of the Russian Federation No. 2624846, that is, to use it as an antibacterial drug for the treatment of chronic infections caused by antibiotic-resistant pathogens in patients with cystic fibrosis by creating a pharmaceutical composition with high bioavailability.

Due to the urgency of the medical problem of pathogen resistance to antibiotics, high relapse rates in patients with chronic infections, ineffective treatment of chronic infections according to standard clinical protocols and in patients with reduced absorption capacity due to gastrointestinal tract pathology (which is typical for patients with cystic fibrosis), the need to develop an antibacterial drug in the form of a solid dosage form for oral administration containing an active ingredient with increased bioavailability is obvious. Solid oral pharmaceutical compositions should provide the patient with an active substance in a sufficient amount for effective therapy, ensure the release of the active substance from the dosage form, and also ensure the chemical stability of the active substance. To ensure ease of use, the oral pharmaceutical composition should not be taken more than three times a day, and if it is in the form of a tablet or capsule, its size should not be too large so as not to cause difficulty in swallowing. The type and amount of excipients in combination with the manufacturing technology are significant from the point of view of release properties, bioavailability of the compound in mammals, stability and applicability of the manufacturing technology of the pharmaceutical composition under industrial conditions.

Disclosure of the invention.

The object of the present invention is the development of a solid dispersion as an intermediate product intended for the manufacture of oral dosage forms providing a high bioavailability of the active substance, which is necessary for antimicrobial action in the treatment of chronic infections.

The technical result to be achieved by the invention consists in obtaining a solid dispersion with increased bioavailability compared to the active substance in crystalline form and therapeutic efficacy against infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa.

The technical result is achieved through the development of a solid dispersion for the manufacture of dosage forms, which includes the active ingredient 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine -2- (2,4-difluorophenyl) -carboxamide and at least one pharmaceutically acceptable polymer, the active substance being contained in amorphous form or in amorphous form with an admixture of microcrystalline and crystalline phases.

Moreover, a pharmaceutically acceptable polymer is contained in a solid dispersion with a ratio of active substance to polymer from 1: 1 to 1:10; and the pharmaceutically acceptable polymer is selected from a copolymer of PEG 6000, vinyl caprolactam and vinyl acetate, a copolymer of N-vinyl pyrrolidone and vinyl acetate, or a copolymer of butyl methacrylate, 2-dimethylaminoethyl methacrylate and methyl methacrylate.

In addition, the solid dispersion further comprises at least one pharmaceutically acceptable surfactant in an amount of 1 to 20 parts by weight, based on the weight of the active ingredient.

Also, the pharmaceutically acceptable surfactant has an HLB value of 3 to 40; and a suitable surfactant is selected from sodium dodecyl sulfate or laureate sorbitan.

Also, the technical result is achieved in that the solid dispersion can be used for the production of oral solid dosage forms in the form of coated or uncoated tablets, hard capsules, dosed powder, powder for preparation of a solution or suspension for oral administration.

Also, the technical result is achieved through the development of a method for the treatment of chronic infections of the respiratory system caused by antibiotic-resistant strains of Pseudomonas aeruginosa, including the use of a solid dispersion in an effective therapeutic dose for humans in an amount from 1 to 4 mg / kg in terms of the active substance; where infectious diseases are selected from pneumonia; chronic infections with cystic fibrosis, bronchiectasis, defects in the development of the respiratory system and pathologies of the respiratory system.

Implementation of the invention.

Brief description of the figures

Fig. 1. Results of X-ray phase analysis of a standard sample of the active substance 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl) - carboxamide in crystalline form.

FIG. 2. Results of X-ray phase analysis of solid dispersion containing 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl ) is a carboxamide in amorphous form. The composition of the intermediate - the active ingredient - Eudragit E PO in a ratio of 1: 4, 30 mg of sodium dodecyl sulfate.

FIG. 3. Results of X-ray phase analysis of solid dispersion containing 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl ) - carboxamide in amorphous form with an admixture of a microcrystalline phase. Composition of the intermediate product - active substance - Eudragit E PO in a ratio of 1: 4, 50 mg sorbitan-laurate (Span 20), 30 mg sodium dodecyl sulfate.

FIG. 4. Results of X-ray phase analysis of solid dispersion containing 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl ) is a carboxamide in amorphous form with an admixture of a microcrystalline phase. The composition of the intermediate product - the active ingredient - Soluplus in a 1: 1 ratio.

FIG. 5. Results of X-ray phase analysis of solid dispersion containing 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl ) is a carboxamide in amorphous form with an admixture of a microcrystalline phase. Composition of the intermediate product - active ingredient - Soluplus in a 1: 2 ratio

FIG. 6. Results of X-ray phase analysis of solid dispersion containing 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl ) is a carboxamide in amorphous form with an admixture of a microcrystalline phase. Intermediate composition - active ingredient - Kollidon VA 64 in a 1: 1 ratio

FIG. 7. Results of X-ray phase analysis of solid dispersion containing 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl ) is a carboxamide in amorphous form with an admixture of a microcrystalline phase. The composition of the intermediate product - the active ingredient - Kollidon VA 64 in a 1: 2 ratio.

FIG. 8. Study of the active substance in crystalline form by the method of differential scanning calorimetry, measuring the melting point.

FIG. 9. Investigation of the Eudragit E РО polymer by the method of differential scanning calorimetry. Determination of the glass transition point Eudragita E PO.

FIG. 10. Study of a solid dispersion based on the active substance and Eudragit E PO, in a ratio of 1: 4 by the method of differential scanning calorimetry.

FIG. 11. Study of the active substance in crystalline form by scanning electron microscopy, resolution 10 microns.

FIG. 12. Investigation of the Eudragit E РО polymer by the method of scanning electron microscopy, resolution 10 µm.

FIG. 13. Study of a solid dispersion based on the active substance and Eudragit E PO, in a ratio of 1: 4 by the method of scanning electron microscopy.

Fig. 14. The concentration of the bound active ingredient in the solid dispersion and in crystalline form in the blood.

FIG. 15. Concentration of free active substance in solid dispersion and in crystalline form in blood.

Fig. 16. Concentration of free active ingredient in solid dispersion and in crystalline form in the liver.

FIG. 17. Concentration of the bound active ingredient in the solid dispersion and in crystalline form in the liver.

Fig. 18. Suppression of P. aeruginosa biofilm formation and cytotoxicity for MDCK cells under the influence of the active substance after 4 hours of contact. 1 - P. aeruginosa 28P23 (x400); 2 - P. aeruginosa 28P23 + 40 μg / ml DV (x400); 3 - P. aeruginosa 28P23 (x1000); 4 - P. aeruginosa 28P23 + 40 μg / ml DV (x1000). Arrows indicate pseudomonas biofilms.

The present invention is directed to a method for preparing a solid dispersion comprising 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4 β-difluorophenyl) -carboxamide in predominantly amorphous form, in at least one pharmaceutically acceptable polymer, and containing or not containing one or more surfactants.

The authors set the task to increase the bioavailability of the active ingredient 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl ) -carboxamide by preparing solid dispersions. The active substance in crystalline form is a substance poorly soluble in water, and therefore there are technological difficulties in the process of creating finished dosage forms with high bioavailability on its basis. The selection of the composition of excipients in such a way as to increase the bioavailability of the active substance is not a trivial task. The increased bioavailability directly determines the therapeutic efficacy of the drug. The obtained variants of the solid dispersion have prospects for introduction into clinical practice for the treatment of severe chronic infections caused by antibiotic-resistant strains of microorganisms. The technical results obtained are not obvious and could not have been foreseen by a person skilled in the art on the basis of the prior art.

It is known that to increase the dissolution and bioavailability of poorly soluble substances, the method of introducing the substance into solid dispersions is used by any available method, which makes it possible to stabilize the active substance in amorphous form. In the composition of solid dispersions, the active substance is in amorphous form or in the form of an amorphous phase with an admixture of crystalline or fine-crystalline. Solid dispersions release the active substance in amorphous form when interacting with a liquid medium of the body, such as biological fluids of the gastrointestinal tract, faster and more efficiently than dosage forms containing the active substance in crystalline form. This is because dissolution is facilitated, at least in part, by reducing the energy required to release the components from the solid dispersion compared to the energy required to dissolve the components from the crystalline or microcrystalline solid phase. In addition, it is necessary that the active substance released from the solid dispersion retains the ability to solubilize in biological fluids of the gastrointestinal tract, since otherwise, the released active substance may crystallize and precipitate in the gastrointestinal tract and not be absorbed, which will lead to decrease in bioavailability. Polymers and surfactants used to prepare solid dispersions should maintain the active substance in a solubilized state in biological fluids of the gastrointestinal tract.

Considering the fact that the active substance is characterized by high lipophilicity, which allows it to penetrate the cell membranes and ensure acceptable intestinal permeability, the release of the active substance from a solid solution is a key factor affecting the bioavailability of the drug under development. Moreover, the use of pharmaceutically acceptable polymers allows for a longer release of the active substance, and absorption not only predominantly in the stomach, but also in various parts of the intestine. The use of surfactants makes it possible to increase the solubilization in the biological media of the body and to prevent the precipitation of the dissolved active substance.

The term "solid dispersion" in this case defines a system in a solid state, including at least two components, where one component is dispersed more or less uniformly throughout the volume of the other component or components. A solid dispersion is a dispersion of components in which the system is chemically and physically homogeneous throughout the entire volume or predominantly consists of one phase, an amorphous substance - a solid solution. In this case, solid dispersions do not contain significant amounts of the active substance in a crystalline or microcrystalline state, which is confirmed by the data of a qualitative X-ray phase analysis. In addition, the term also includes solid dispersions that are less uniform and may contain more than one phase. For example, particles having domains or small regions where more or less uniformly amorphous, microcrystalline and / or crystalline phases are dispersed.

The active substance in the text of this document: 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl) -carboxamide (compound CL-55).

The term "pharmaceutically acceptable" means, in the context of this document, excipients that are permissible and medically justified to be used for the manufacture of oral dosage forms that do not have undesirable toxicity, irritant effects, do not cause allergic reactions and whose use is favorable in terms of indicators the ratio of risk and benefit.

According to the conditions, the dosage form may include at least one surfactant having a hydrophilic lipophilic balance (HLB) value from about 3 to about 40. The HLB system (Fiedler, H. B., Encyclopedia of Excipients, 5th ed. , Aulendorf: ECV-Editio-Cantor-Verlag (2002), Encyclopedia of Biocolloid and Biointerface Science 2V Set, Volume I&II, 2016) assigns surfactants number values, with lipophilic substances getting lower HLB values, and hydrophilic substances getting more high HLB values.

Polymers suitable for use in the solid dispersion of the present invention include, but are not limited to, homopolymers and copolymers of N-vinyl lactams, especially homopolymers and copolymers of N-vinyl pyrrolidone, such as polyvinyl pyrrolidone (PVP), copolymers of N-vinyl pyrrolidone or vinyl acetate , cellulose esters and cellulose ethers, in particular methyl cellulose and ethyl cellulose, hydroxyalkyl cellulose, in particular hydroxypropyl cellulose, hydroxyalkylalkyl cellulose, in particular hydroxypropyl methyl cellulose, phthalates or cellulose succinates, in particular hydroxypropyl cellulose acetate or cellulose acetate phthalate and cellulose acetate phthalate high molecular weight polyalkylene oxides such as polyethylene oxide and polypropylene oxide and copolymers of ethylene oxide and propylene oxide, polyacrylates and polymethacrylates such as methacrylic acid / ethyl acrylate copolymers, methacrylic acid / methyl methacrylate copolymers, butyl methacrylate / copolymers of hydroethyl dimethyl (hydroxyalkyl methacrylates), polyacrylamides, vinyl acetate polymers such as copolymers of vinyladetate and crotonic acid, partially hydrolyzed polyvinyl acetate (also called partially saponified "polyvinyl alcohol"), polyvinyl alcohol, oligo- and polysaccharides such as carrageenans and caraginans mixtures of one or more of these components. Particular preference is given to polyacrylates and polymethacrylates such as methacrylic acid / ethyl acrylate copolymers, methacrylic acid / methyl methacrylate copolymers, butyl methacrylate / copolymers 2-dimethylaminoethyl methacrylate, poly (hydroxyalkyl acrylates), ), which Evonik manufactures, are synthetic cationic or anionic polymers of dimethylaminoethyl methacrylates, methacrylic acid esters and methacrylic acid in various ratios. It is even more preferred to use Eudragit® EPO, a cationic polymer that is obtained by copolymerizing butyl methacrylate, 2-dimethylaminoethyl methacrylate and methyl methacrylate due to the most preferred dissolution profile in organic solvents in which the active ingredient has the greatest solubility.

The dosage form in some cases includes at least one surfactant to maintain the active substance in a solubilized state in biological fluids of the gastrointestinal tract. Surfactants having an HLB value of about 3 to 40 suitable for use in the present invention include, but are not limited to: sodium dodecyl sulfate, polyoxyethylene alkyl ethers, e.g., polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene stearyl ether; polyoxyethylene alkylaryl ethers such as polyoxyethylene nonylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene octylphenyl ether; polyethylene glycol fatty acid esters such as PEG-200 monolaurate, PEG-200 dilaurate, PEG-300 dilaurate, PEG-400 dilaurate, PEG-300 distearate, PEG-300 dioleate; alkylene glycol fatty acid monoesters, for example propylene glycol monolaurate; sucrose fatty acid esters such as sucrose monostearate, sucrose distearate, sucrose monolaurate, sucrose dilaurate; or sorbitan fatty acid monoesters such as sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, or sorbitan stearate, or mixtures of one or more of these components. Particular preference is given to sodium dodecyl sulfate, which is used both as the only surfactant and in combination with other surfactants. Surfactants can be incorporated into the solid dispersion and / or added as an adjuvant to the finished pharmaceutical composition.

Solid oral dosage forms include, but are not limited to: coated and uncoated tablets, dispersible tablets, hard gelatin capsules, dosage powders, powders for oral suspension. The choice of solid dosage forms comprising the active ingredient in the form of a solid dispersion is based on their properties and is known to those skilled in the art. The preferred dosage forms are tablets. To facilitate swallowing of such a dosage form, it is desirable to shape it into an appropriate shape. Tablets which are easy to swallow are preferably elongated. The film-coated tablet further facilitates swallowing. For the production of pharmaceutical compositions using the active ingredient in amorphous form in a solid dispersion according to the present invention, a variety of methods can be used. The processes of making solid dosage forms themselves, from the moment of receiving the intermediate, are otherwise standard and easy to carry out. Tablets can be manufactured using conventional tabletting processes with common commonly available excipients and conventional tabletting equipment. These methods include preparing a solid dispersion and molding into a desired tablet shape. Alternatively, granules can be obtained from the solid dispersion, for example by grinding or milling, the granules can be mixed with the necessary auxiliaries and compacted into tablets or used for the manufacture of other dosage forms. Excipients suitable for use in the pharmaceutical composition of the present invention include, but are not limited to, the listed functional classes: fillers, binders, disintegrants, surface modifiers and surfactants, buffering agents, glidants, lubricants, sweeteners, flavors and colors, and auxiliary substances of other classes to ensure acceptable processing properties of the mass for obtaining a solid dosage form of a pharmaceutical composition.

The intermediate product described in the present invention and which can be used for the manufacture of a pharmaceutical composition exhibits behavior characterized by high achievable AUC values, high C _max compared to the active substance in crystalline form (Example 4).

Excipient: these are substances of an organic or inorganic nature, which are used in the production process of finished dosage forms to give them the necessary properties. Examples of functional excipients include, but are not limited to, binders, stabilizers, fillers, carriers, disintegrants, glidants, diluents, etc. A person skilled in the art can select one or more of the aforementioned excipients of the desired functionality to provide the desired properties. solid oral dosage form. The amount of each auxiliary substance used varies within known limits. Excipients and their functional purposes used to obtain oral dosage forms are described in the following publications, which are incorporated herein by reference. See "The Handbook of Pharmaceutical Excipients", 4th ed., Ed. Rowe et al., American Pharmaceuticals Association (2003), and the Decision of the Board of the Eurasian Economic Commission dated 06/18/2019 N 103 "On the handbook of functional purposes of excipients used in the manufacture of medicines."

Pharmaceutical composition: a mixture containing a certain amount of active substance and excipients, intended for administration to a patient for the treatment of diseases caused by resistant microorganisms.

Intermediate in the context of this document means a solid dispersion that can be used to prepare a pharmaceutical composition for oral use.

The term "AUC" means "area under the curve" and is used in its usual meaning, i. E. as the area under the blood concentration versus time curve.

The term "C _max " refers to the maximum concentration in the blood.

There are various technological methods for obtaining solid dispersions, including melt extrusion, spray drying, and solution evaporation; in experimental work, in most cases, the solution evaporation method was used.

The process of obtaining solid dispersions by the solution evaporation method is the simplest and least resource-intensive method and includes the following steps:

1) mixing of components: an active substance and one or more polymers;

2) dissolution in a mixture of organic solvents to a homogeneous state;

3) pouring the specified solution onto a large surface to form a thin film;

4) evaporation of the solvent;

5) drying until complete evaporation of organic solvents;

6) grinding the resulting mixture.

However, when scaling up production, a transition to melt extrusion or the use of spray drying as interchangeable processes is possible, which is shown during the development of experimental samples by various methods.

The melt extrusion process includes the following steps:

1) mixing of components: an active substance and one or more polymers;

2) heating the mixture until a homogeneous melt is obtained;

3) passing the melt thus obtained through one or more nozzles;

4) cooling the melt to solidification;

5) grinding the cooled melt.

Examples of obtaining a solid dispersion:

Example 1.

Name Weight, g Fluorothiazinone one hundred Kollidon® VA64 400 Mixture Acetone Ethanol 50-50 Until complete dissolution (900 ml)

The required amount of Fluorothiazinone and Kollidon® VA64 were weighed, dissolved in an ethanol / acetone mixture, while heating to 50 ° C, the resulting mixture was poured onto a tray and the solvent was removed in a Memmert drying oven for 24 hours.

After drying was complete, standard analytical procedures were performed using standard equipment in the art:

1 - Residual organic solvents by gas chromatography.

2 - Related impurities by high performance liquid chromatography.

3 - Quantification by high performance liquid chromatography.

4 - Powder diffraction to determine the degree of crystallinity of a substance.

Example 2.

Name Weight, g Fluorothiazinone one hundred Soluplus® 400 Sodium dodecyl sulfate (VAT ten

Weighed the required amount of Fluorothiazinone and Soluplus®, VAT and carried out the process of hot melt extrusion in a Thermo extruder at a torque of 3 Torque and a temperature of 140 ° C.

After completion of the process, standard analytical procedures were performed using standard equipment in the art:

1 - Related impurities by high performance liquid chromatography.

2 - Quantification by high performance liquid chromatography.

3 - Powder diffraction to determine the degree of crystallinity of a substance.

Example 3.

Name Weight, g Fluorothiazinone one hundred Soluplus® 400 Sodium dodecyl sulfate (VAT) 400

Weighed the required amount of Fluorothiazinone and Soluplus®, VAT and carried out the process of hot melt extrusion in a Thermo extruder at a torque of 5 Torque and a temperature of 150 ° C.

After completion of the process, standard analytical procedures were performed using standard equipment in the art:

1 - Related impurities by high performance liquid chromatography.

2 - Quantification by high performance liquid chromatography.

3 - Powder diffraction to determine the degree of crystallinity of a substance.

Example 4.

Name Weight, g Fluorothiazinone one hundred Eudragit® EPO 400 Span ®20 200

Weighed the required amount of Fluorothiazinone and Eudragit® EPO, Span®20 and carried out the hot melt extrusion process in a Thermo extruder at a torque of 3 Torque and a temperature of 130 ° C.

After completion of the process, standard analytical procedures were performed using standard equipment in the art:

1 - Related impurities by high performance liquid chromatography.

2 - Quantification by high performance liquid chromatography.

3 - Powder diffraction to determine the degree of crystallinity of a substance.

Example 5.

Name Weight, g Fluorothiazinone one hundred Eudragit® EPO 400 Span ®20 800 Ethanol absolute 2000

The required amount of Fluorothiazinone and Eudragit® EPO, Span®20 were weighed and dissolved in absolute Ethanol and the process of spray drying was carried out in a Buchi dryer at a temperature of 180 ° C.

After completion of the process, standard analytical procedures were performed using standard equipment in the art:

1 - Residual organic solvents by gas chromatography.

2 - Related impurities by high performance liquid chromatography.

3 - Quantification by high performance liquid chromatography.

4 - Powder diffraction to determine the degree of crystallinity of a substance.

The person skilled in the art is able to optimize the parameters of the evaporation, melt extrusion and spray drying processes in accordance with the properties of the active substance, polymers and the configuration of the equipment used. The person skilled in the art will readily determine the most suitable equipment to obtain the object of the present invention.

Spray drying of the components of the solution also produces a solid dispersion of these components and can be a useful alternative to the melt extrusion process.

Preferably, the solid dispersion is in the form of a solid solution comprising the active ingredient in amorphous form and one or more polymers, whether or not one or more surfactants are included. Alternatively, it can be in the form of a dispersion, where the active substance is presented in amorphous form with an admixture of a crystalline or microcrystalline phase, dispersed more or less uniformly in a solid solution (Example 1).

The properties of the resulting solid dispersions were evaluated by the investigators using dissolution analysis.

The following methods were used to measure the degree of crystallinity (Examples 2 and 3):

1. Differential scanning calorimetry allows you to determine the thermal effect of dissolution, which at constant atmospheric pressure corresponds to the change in enthalpy and characterizes the degree of crystallinity of a substance. The enthalpy of dissolution is proportional to the amount of solute.

2. X-ray diffraction analysis of powders. Diffraction patterns make it possible to evaluate the shape and size of crystallites (0.005 - 0.0005 mm), as well as the ratio of crystalline and amorphous phases.

3. Scanning using electron microscopy.

The resulting solid dispersion allowed an increase in bioavailability (Example 4) and antibacterial efficacy in vitro and in the treatment of animal model infections (Examples 5-10).

The achieved technical result of the proposed invention corresponds to the tasks set. The examples presented below confirm the achievement of the specified technical results, namely, the possibility of obtaining the claimed solid dispersion and illustrate (without limiting the scope of claims) the properties of the obtained intermediate.

List of examples

Example 1. Development of a solid dispersion composition using dissolution screening in various buffer media (at pH 1.2; 4.5 and 6.8), preparation of a composition for the production of solid dosage forms for oral administration.

Example 2. Comparative study of the active substance in crystalline form, the Eudragit E PO polymer and a solid dispersion based on Eudragit E PO, in a ratio of 1: 4 by the method of differential scanning calorimetry.

Example 3. Comparative study of the active substance in crystalline form, the Eudragit E PO polymer and a solid dispersion based on Eudragit E PO, in a ratio of 1: 4 by the method of scanning electron microscopy.

Example 4. Study of oral bioavailability in rats of the active substance in crystalline and amorphous form.

Example 5. Characterization of P. aeruginosa clinical isolates from cystic fibrosis patients by the presence of type III secretion system genes and the activity of this secretory apparatus.

Example 6. Inhibitory effect of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine-2- (2,4-difluorophenyl) -carboxamide ) - in amorphous form on TSTT of clinical antibiotic-resistant P. aeruginosa strains.

Example 7. Suppression of P. aeruginosa biofilm formation on the surface of epithelial cells in in vitro experiments under the influence of an active substance in amorphous form.

Example 8. Study of the therapeutic efficacy of the active substance in amorphous form in the model of acute pneumonia caused by P. aeruginosa.

Example 9. Study of the therapeutic efficacy of an active substance in amorphous form in a model of a subacute infection of the respiratory tract caused by a P. aeruginosa clinical isolate obtained from a patient with cystic fibrosis.

The examples presented below illustrate and confirm the possibility of obtaining the claimed solid dispersion and solving the assigned tasks.

Example 1. Development of a solid dispersion composition using dissolution screening in various buffer media (at pH 1.2; 4.5 and 6.8), preparation of a composition for the production of solid dosage forms for oral administration.

The composition of the intermediate solid dispersion was selected based on the analysis of the dissolution of solid dispersions based on water-soluble copolymers; for a person skilled in the art it is an indisputable fact that copolymers create much better solid dispersion systems from the point of view of dissolution than monopolymers. The screening involved 3 copolymers with the trade names Kollidon® VA 64, Eudragit® EPO, Soluplus® possessing different physicochemical properties. To select the leading composition of the solid dispersion, we studied the dissolution of solid dispersions in ratios of active substance to polymer from 1: 1 to 1:10 in three different buffer media (at pH 1.2; 4.5 and 6.8). Comparative kinetics of dissolution in an aqueous medium at different physiological pH values of a solid dispersion using Eudragit E PO as a dispersed phase on a paddle mixer at a rotation speed of 75 rpm at a temperature of 37 ± 0.5 ° C. after 45 min. The quantitative determination of the released active substance was carried out by HPLC.

The lower the crystallinity of the active substance, the higher its dissolution, which is clearly shown in table 1. The best properties are shown by solid dispersions based on methacrylic acid copolymers commercially available under the trade name Eudragit, in this study the preferred properties were demonstrated by Eudragit EPO.

Qualitative X-ray phase analysis was performed using an Ultima IV-285 automatic powder diffractometer (Rigaku). Diffraction patterns make it possible to evaluate the shape and size of crystals, as well as the ratio of crystalline and amorphous phases. Diffraction patterns of the investigated samples are shown in Figures 1-7. The active substance is a crystalline substance with a crystal lattice without spatial defects, and has almost 100% crystallinity (Fig. 1). Solid dispersions obtained with different polymers in different polymer ratios with or without added surfactants make it possible to obtain the active substance in a uniform amorphous or amorphous form with an admixture of microcrystalline and crystalline phases (Figs. 2-7).

Different polymers make it possible to obtain a stable amorphous phase of the active substance with different component ratios. Shown are the diffraction patterns of solid dispersions obtained on the basis of Eudragit E PO, in a ratio of 1: 4 with the addition of sodium dodecyl sulfate; Soluplus, in a ratio of 1: 1 and 1: 2; Kollidon VA 64 in a 1: 1 and 1: 2 ratio.

The increase in dissolution in the three buffer media correlates with an increase in amorphousness as measured by diffraction analysis and an increase in bioavailability and efficacy in pilot animal experiments. Almost all solid dispersions obtained in the experiment made it possible to increase the dissolution. The choice of the ratio of polymer and active substance was carried out on the basis of a reasonable balance between dissolution and the volume of the obtained intermediate, which is important for the development of a finished dosage form acceptable for oral administration - it should be convenient for the patient to use. In this regard, further studies were carried out with a solid dispersion based on Eudragit E PO, in a ratio of 1: 4.

For more effective maintenance of the active substance in a solubilized state in biological fluids of the gastrointestinal tract, surfactants were used. Any of the analyzed surfactants, used in combination with the solid dispersion, both in its composition and when introduced as an excipient, slightly increases dissolution and bioavailability in pilot experiments on pharmacokinetics. The best surfactant analyzed is sodium dodecyl sulfate. Also, an increase in the amorphousness of the active substance in the composition of the solid dispersion was shown by sorbitan laurate.

Table 1. Indicators of dissolution in three media

i / v Soluplus® Kollidon® VA 64 Eudragit® E PO pH 1.2 4.5 6.8 1.2 4.5 6.8 1.2 4.5 6.8 The ratio of the masses. API: Polymer Concentration Mkg / ml 1: 0 0.29 0.78 0.34 0.29 0.78 0.34 0.29 0.78 0.34 1: 1 1.20 1.99 2.09 5.80 6.68 7.89 8.95 18.84 8.14 1: 2 2.70 2.34 2.27 8.65 7.15 9.67 14.63 61.87 9.04 1: 3 2.75 2.47 2.44 9.86 7.26 9.77 18.16 64.19 9.08 1: 4 2.89 2.55 2.50 10.86 13.29 10.95 28.18 111.14 9.18 1: 5 3.09 2.76 2.53 14.22 16.09 14.53 29.24 157.34 9.21 1: 6 3.21 3.23 3.34 14.72 16.20 13.34 33.01 210.99 11.02 1: 7 3.53 3.45 3.52 14.78 16.08 14.23 49.35 213.10 11.52 1: 8 4.56 4.61 3.56 23.63 16.43 22.42 64.67 217.54 11.80 1: 9 6.28 6.32 3.57 23.72 26.04 22.31 64.98 230.17 12.04 1:10 18.27 9.65 4,00 25.91 23.90 35.57 82.91 222.50 12.27 The ratio of the masses. API: Polymer: Surfactant Concentration Mkg / ml 10: 10: 1 1.70 2.99 2.69 7.80 7.68 8.89 10.95 23.84 11.14 1: 1: 10 2.70 4.11 3.15 8.43 8.51 9.11 11.45 25.34 12.31 10: 100: 1 20.27 10.32 4.35 26.62 25.21 36.31 83.32 223.25 14.34 1:10:20 23.21 13.11 5.54 27.32 28.31 38.51 85.21 229.32 16.01

The above example shows that the claimed invention makes it possible to obtain an intermediate product (solid dispersion) with improved dissolution kinetics in the entire range of physiological pH values in comparison with the active substance in crystalline form.

Example 2. Comparative study of the active substance in crystalline form, the Eudragit E PO polymer and a solid dispersion based on Eudragit E PO, in a ratio of 1: 4 by the method of differential scanning calorimetry.

The solid dispersion based on Eudragit E PO in a ratio of 1: 4 was investigated by differential scanning calorimetry. Differential thermal analysis allows one to assess the phase equilibria of both pure components and mixtures. Phase transitions are recorded as a function of temperature and time. According to the measurement data using differential scanning calorimetry (DSC analysis), it is possible to judge the thermal behavior of individual substances and mixtures: to determine the melting point and heat of phase transitions. Important physical characteristics that are taken into account in the development of solid dispersions are the glass transition temperature of the solid dispersion and the melting point of the components of the solid dispersion - the active substance and polymers, since thermal action is used in the production process (extrusion, solvent evaporation).

Output. The given thermograms (Fig. 8-10) show that the melting point of the active substance is 134 ° C (Fig. 8), the glass transition point of Eudragit E PO is at the minimum of the curve at a temperature of 54 ° C (Fig. 9). Based on the thermogram of the solid dispersion (Fig. 10), it can be concluded that it behaves as an independent system, which does not contain the crystalline phase of the active substance. It is an independent system with its own thermodynamic properties that are distinctive from pure substances.

Example 3. Comparative study of active substance in crystalline form, polymer Eudragit E PO and solid dispersion based on Eudragit E PO, in a ratio of 1: 4 by scanning electron microscopy

Samples of the active substance in crystalline form, polymer Eudragit E PO and solid dispersion based on Eudragit E PO, in a ratio of 1: 4, were examined by scanning electron microscopy. Electron micrographs are shown in FIG. 11-13. They clearly demonstrate the presence of large crystals in the samples of the active substance and polymer and a homogeneous amorphous phase with inclusions of microcrystals.

Output. The microscopic picture of the solid dispersion confirms the absence of the crystalline structure inherent in the individual substances that make up its composition.

Example 4. Study of oral bioavailability in rats of the active substance in crystalline and amorphous form.

A comparative bioavailability study was performed on rats. The study used 60 male outbred rats, weighing 180-200 grams, 7-8 weeks old. All animals were healthy, during the experiment they received a balanced food ration and water in unlimited quantities. Each rat was administered orally at a dose of 50 mg / kg based on the active ingredient in crystalline form and a solid dispersion of the following composition: active ingredient 100 mg, Eudragit E PO in a ratio of 1: 5, 50 mg sorbitan-laurate (Span 20), 30 mg sodium dodecyl sulfate. The drug was administered through a tube in the form of a suspension. Animals were bled 0, 1/2, 1, 2, 4, 8, 12, 24, 36, 48 h after drug administration. The animals received samples of the liver and lungs to assess the accumulation of the active substance and metabolite. After oral administration, the active substance binds to glucuronic acid and passes into the metabolite. Investigated the active substance in free form and in bound (metabolite glucuronide).

Method for the determination of analytes in biological samples. The analysis was carried out by HPLC-MS / MS using an Acquity UPLC liquid chromatograph (Waters Inc., USA) with an automatic sample introduction system (autosampler), an automatic sample introduction device (autosampler), a column thermostat and a degasser, and a TQS Micro mass spectrometric detector. with heated flow electrospray ionization, data acquisition and processing system using MassLynx software. Quantification is based on the ratio of the peak area of the analyte to the internal standard (related compound). Whole blood with sodium citrate as a stabilizer was used for the study. To assess the degree of glucuronidation, blood samples were analyzed after incubation with β-glucuronidase solution. The organs were washed, homogenized, isolated and analyzed for the content of the active substance. To assess the degree of glucuronidation, homogenate samples were analyzed after incubation with β-glucuronidase solution. Pharmacokinetic parameters were calculated using the Pk Solver module.

Table 2. Pharmacokinetic parameters of the active substance and its metabolite in the blood.

Parameter Dimension DW in crystalline form DW in amorphous form DV metabolite DV metabolite T _1/2 h 45.5 6.05 51.58 4.54 T _max h 0.5 0.5 8 8 C _max ng / ml 4.97 30.92 8.95 67.2 AUC _0-t ng / ml * h 47.94 198.21 112.86 701.02 AUC _0-∞ ng / ml * h 89.94 198.74 184.54 701.53 MRT _0-t h 19.64 9.40 14.56 9.05 MRT _0-∞ h 63.53 9.53 56.45 9.09

Table 3. Pharmacokinetic parameters of the active substance and its metabolite in the lungs

Options Dimension DW in crystalline form DW in amorphous form DV metabolite DV metabolite T _1/2 h 21.77 7.19 14.53 9.64 T _max h 0.5 one 0.5 8 C _max ng / ml 115.99 59.74 313.40 81.06 AUC _0-t ng / ml * h 593.29 351.21 836.59 1002.72 AUC _0-∞ ng / ml * h 682.96 359.76 991.04 1016.87 MRT _0-t h 12.57 7.59 12.50 13.65 MRT _0-∞ h 21.34 8.80 21.30 14.32 fT 12.38 1.77 7.41 1.43

Table 4. Pharmacokinetic parameters of the active substance and its metabolite in the liver

Options Dimension DW in crystalline form DW in amorphous form DV metabolite DV metabolite T _1/2 h 8.67 6.07 6.68 8.25 T _max h 0.5 one 8 8 C _max ng / ml 455.98 3404.96 476.74 4253.38 AUC _0-t ng / ml * h 2585.88 36083.40 6062.29 58439.55 AUC _0-∞ ng / ml * h 2644.44 36303.64 6137.98 59606.37 MRT _0-t h 9.98 9.73 9.96 13.13 MRT _0-∞ h 11.10 10.02 10.55 14.05 fT 53.94 182.04 53.72 83.36

Table 5. Bioavailability of the active substance in amorphous form compared to crystalline form

Relative bioavailability Value blood 235.4% lungs 141% liver 234.4%

Conclusion: the claimed invention makes it possible to obtain a solid dispersion - an intermediate product with increased bioavailability compared to the active substance in crystalline form.

Example 5. Characterization of clinical isolates of Pseudomonas aeruginosa obtained from patients with cystic fibrosis by the presence of genes of the type III secretion system and the activity of this secretory apparatus

It is known that the severe course of the bronchopulmonary process in patients with cystic fibrosis (CF) is determined by infection of the respiratory tract with Pseudomonas aeruginosa, which currently remains the leading pathogen that determines the progressive damage to the bronchopulmonary system and the prognosis of the disease in general. The pathogen is almost impossible to eliminate and the infection becomes chronic. Ongoing courses of antibiotic therapy often lead to the formation of antibiotic-resistant pseudomonas, which makes antibiotic therapy ineffective. In addition, during long-term chronic infection, Pseudomonas aeruginosa can adapt to persistence with a change in virulent properties. In this regard, the characterization of Pseudomonas aeruginosa isolates obtained from cystic fibrosis patients was carried out, which made it possible to assess the presence of genes of the type III secretion system (CCTT) and the activity of the secretory apparatus as a target for suppression by the studied active substance.

The study was carried out on 11 isolates of Pseudomonas aeruginosa isolated from the sputum of patients with cystic fibrosis aged 15 to 40 years. Species identification was confirmed in the PCR reaction using species-specific primers for the P. aeruginosa trpE gene. All strains were characterized by multiple resistance.

The study of the frequency of occurrence of genes encoding proteins - effectors of TSTT pseudomonads in the obtained strains was carried out using PCR with DNA of the strains. Aeruginosa reference strains with the known CCTT genotype, PAO (exoS + exoT + exoY + exoU) and PA103 (exoS - exoT + exoY + exoU +) were used to refine the reaction conditions. Table 6 shows the sequence of primers selected to determine the presence of Paeruginosa CCTT genes.

Table 6. Sequence of primers for determining the presence of P. aeruginosa CCTT genes.

Oligonucleotide name Gene under study Sequence 5-PrimerS exoS GCG AGG TCA GCA GAG TAT CG 3-PrimerS TTC GGC GTC ACT GTG GAT GC 5-PrimerT exoT AAT CGC CGT CCA ACT GCA TGC G 3-PrimerT TGT TCG CCG AGG TAC TGC TC 5-PrimerU exoU CCG TTG TGG TGC CGT TGA AG 3-PrimerU CCA GAT GTT CAC CGA CTC GC 5-PrimerY exoY CGG ATT CTA TGG CAG GGA GG 3-PrimerY GCC CTT GAT GCA CTC GAC CA

Among all analyzed strains of P. aeruginosa, the exoU + genotype was detected in 1 patient, all other isolates belonged to the exoS + genotype. Thus, all isolates obtained from patients with chronic pseudomonas infection had in their genome a complete set of genes encoding TSTT effector proteins.

The functional activity of TSTTs from clinical P. aeruginosa isolates was characterized by the method of cytotoxicity determination. Toxicity to eukaryotic cells, associated with the secretion of ExoU or ExoS proteins, in vitro manifests itself very quickly, during the first 3 hours for the exoU and 5 hours for the exoS genotype, and is expressed in the violation of the integrity of the cell membrane, followed by cell death by necrosis. We used CHO cells (Chinese hamster ovary cells) in cytotoxicity tests and evaluated cell death in a test for the quantitative determination of lactate dehydrogenase in the culture medium 3 or 5 hours after the cells were inoculated with Pseudomonas strains.

An 18-hour culture of Pseudomonads with MOI 10 was added to a daily monolayer of CHO cells grown in a 96-well plate and incubated for 3 or 5 hours. The plate was centrifuged for 10 min at 4000 rpm to precipitate bacteria, and the supernatant was removed. Determination of lactate dehydrogenase was performed using the CytoTox Non-Radioactive Cytotoxicity Assay (Promega, USA) according to the instructions. Cytotoxicity was expressed as a percentage relative to values in uninfected cells.

Table 7 shows the results of the analysis of the cytotoxicity of isolates of pseudomonads. exoU genotype showed high toxicity - 90%. Among 9 strains with exoS genotype, toxicity was not manifested in 2 isolates; for others, significantly pronounced toxicity associated with STT was shown.

Table 7. Results of analysis of cytotoxicity of P. aeruginosa isolates isolated from patients with cystic fibrosis, obtained by the method of determination of lactate dehydrogenase.

Sample N Patient age CCTT genotype Toxicity (3 h) Toxicity (5h) 28P23 39.9 exoS 21.0% 28P24 31.5 exoS 20.0% 28P26 28.4 exoS - 12.7% 28P27 25.5 exoS 67.0% 28P29 24.5 exoS - - 28P30 22.4 exoS - 10.0% 28P31 19.8 exoS 19.7% 28P32 21,7 exoU 90% 28P35 21.9 exoS 10.4% 28P36 21.0 exoS 12.8% NS25 15 exoS - 8%

Output. The characterization of P. aeruginosa clinical isolates isolated during chronic infection of the respiratory tract of patients with cystic fibrosis, based on the presence of genes of the type III secretion system and the activity of this secretory apparatus, showed that all isolates have a complete set of CCTT toxin protein genes. In addition, for the majority of isolates, 9 out of 11, cytotoxicity was detected in in vitro cell tests, which indicates the functional activity of the type III secretion system in isolates isolated from chronically infected patients.

Example 6. Inhibitory effect of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine-2- (2,4-difluorophenyl) -carboxamide ) in amorphous form on the TSTT of clinical antibiotic-resistant P. aeruginosa strains.

The study of the effect of a CCTT (DW) inhibitor on the cytotoxicity of pseudomonas isolates for CHO cells was carried out according to the method described above (Example 5). The inhibitor was added at different concentrations (40 μg / ml and 50 μg / ml) to the bacterial cell suspension and incubated for 30 min before adding to CHO cells.

When studying the cytotoxicity of freshly isolated strains of P. aeruginosa bacteria on the CHO cell line, it was shown that the addition of pseudomonads of the studied strains to cells at a dose of 10 MOI caused strong cell cytotoxicity. Pre-incubation of bacteria with a TSTT inhibitor at a dose of 50 μg / ml almost completely reduced cytotoxicity. Table 8 presents data on the effect of an inhibitor of the type III secretion system (DW) on the cytotoxicity of P. aeruginosa isolates towards CHO cells.

Table 8. Results on the suppression of cytotoxicity of P. aeruginosa isolates isolated from patients with cystic fibrosis in the presence of an active substance (DW), obtained by the method of determining lactate dehydrogenase.

Sample N Toxicity (5h) In the presence of DV 40 μg / ml 50 μg / ml 28P23 23% 8 % 0% 28P24 22% 12 % 2% 28P26 14% five % 0% 28P27 67% 34% five% 28P29 - - - 28P30 ten % 7% 2% 28P31 21% 14% 8% 28P32 - - - 28P35 12% 8% 2% 28P36 14% 3% 0% NS25 8% 2% 0%

Thus, it was experimentally shown that the action of a low-molecular-weight inhibitor of CCTT, an active substance on the studied isolates of pseudomonads, suppressed their cytotoxicity towards eukaryotic cells associated with the effector proteins of CCTT - ExoU and ExoS.

Output. Thus, in the aggregate, the obtained data indicate the presence of TSTT and its activity in clinical isolates obtained from patients with cystic fibrosis with chronic infection, with a different profile of antibiotic resistance and speak in favor of the key role of the type III secretion system in the development of the infectious process, which makes TSTT promising. a target for suppressing infection. The study of the effect of DW on the cytotoxicity of clinical isolates of P. aeruginosa showed an effective suppression of the secretion of TSTT toxins, which gives reason to consider TSTT as a promising target for the suppression of chronic infection caused by multi-resistant Pseudomonas aeruginosa strains.

Example 7. Suppression of P. aeruginosa biofilm formation on the surface of epithelial cells in in vitro experiments under the influence of an active substance.

One of the most common forms of antibiotic resistance of pathogenic bacteria is their resistance to antibiotics in biofilms.

Biofilms are a special form of the existence of bacteria in the form of conglomerates attached to the substrate, enclosed in a matrix consisting of polysaccharides and extracellular DNA. According to modern data, in more than 80% of cases, the infectious process in the human body is accompanied by the formation of biofilms. As part of biofilms, the resistance of bacteria to antibiotics increases more than 1000 times. Pseudomonas form biofilms on the epithelium of the lungs, bladder, wound surfaces, and kidney stones.

The study of the effect of the active substance (AD) on biofilm formation was carried out on a biotic surface model using a continuous line of MDCK epithelial cells (dog kidney).

Suppression of P. aeruginosa biofilm formation

Aeruginosa isolate obtained from a 28P23 cystic fibrosis patient was used in the experiments. Bacteria were incubated with DW at concentrations of 20 and 40 μg / ml in the presence of EGTA for 30 min before being added to a monolayer of eukaryotic cells. Then added to MDCK cells with MOI 10 and incubated for 4 hours. Some of the samples were stained with crystal violet and examined under a light microscope. In another part of preparations for quantitative registration of bacteria in biofilms, the monolayer was thoroughly washed from unbound bacteria, and cells with attached biofilms were treated with 0.1% Triton X-100 and ultrasound to destroy biofilms. Disintegrates were inoculated with serial dilutions on citrimide agar to count CFU in biofilms. The experiment was repeated 3 times.

On photomicrographs 4 hours after contact of P. aeruginosa isolate 28P23 with MDCK cells, pronounced toxicity was observed, which appeared in the destruction of the cell monolayer. In these preparations, the formation of biofilms of pseudomonads was observed both on the cell surface and on glass (Fig. 18). After the action of DW, cytotoxicity significantly decreased, and the monolayer remained. At the same time, the forming biofilms were significantly less (Fig. 18).

The results of seeding biofilms formed on the surface of epithelial cells from disintegrates showed that the number of pseudomonads in biofilms after exposure to DV significantly decreased compared to the control, and the decrease was dose-dependent (Table 9).

Table 9. Results of inoculation on citrimide agar of P. aeruginosa biofilm samples in CFU / ml.

No. Sample name Cultivation time, hour CFU / ml one P.aeruginosa four (6.38 ± 2.20) x10 ⁸ 2 P. aeruginosa 20 μg / ml DV four (1.12 ± 0.28) x10 ⁷ 3 P. aeruginosa 40 μg / ml DV four (1.98 ± 2.7) x10 ⁶

Thus, it was shown that DW suppresses the process of biofilm formation on the surface of epithelial cells, which indicates the participation of CCTT in this process. The activity of DV in relation to the formation of biofilms of pseudomonads is a fundamentally significant characteristic of it, because biofilms represent a serious problem in the treatment of pseudomonas infections.

Output. It is well known that the ability of bacteria to form biofilms and persist in them for a long time, despite the use of antibacterial drugs, creates an extremely problematic situation with the treatment of chronic infections. This fully refers to the ineffectiveness of the treatment of chronic infection in patients with cystic fibrosis. The shown effect of the developed solid dispersion on the process of biofilm formation on the surface of epithelial cells is a fundamental advantage of this drug over antibiotics.

Example 8. Study of the therapeutic efficacy of an active substance in amorphous form in the form of a solid dispersion on a model of acute pneumonia caused by P. aeruginosa

P. aeruginosa is the causative agent of respiratory diseases, including severe nosocomial pneumonia and chronic infections typical of patients with cystic fibrosis. Chronic infections with cystic fibrosis require prolonged and repeated courses of antibiotic therapy, which is associated with an increased risk of developing resistance. A serious problem is the bioavailability of antibacterial drugs due to impaired intestinal absorption and difficult penetration of drugs into the bronchial lumen in this category of patients. The development of an antibacterial drug that is effective against antibiotic-resistant strains of gram-negative bacteria and has acceptable bioavailability and the ability to accumulate in the lungs is extremely important for the treatment of chronic infections in patients with cystic fibrosis.

To study the therapeutic efficacy of solid dispersion, a model of acute pneumonia was developed in a sensitive strain of DBA / 2 mice infected with a hospital isolate of P. aeruginosa KB6 isolated from a patient's sputum in the intensive care unit. The dose of infection was determined to study the dynamics of death of animals with the development of pneumonia within 5 days of observation. With intranasal administration of P. aeruginosa, this dose was 2x10 ⁷ CFU (colony-forming units) / mouse, which corresponded to LD ₇₅ .

The treatment was carried out with a solid dispersion of the following composition: active ingredient 100 mg, Eudragit E PO in a ratio of 1: 5, 50 mg sorbitan laurate (Span 20), 30 mg sodium dodecyl sulfate. Doses of the active substance for mice were: 12.5 mg / kg; 25 mg / kg and 50 mg / kg once daily. The human equivalent dose in mg / kg (EDC) is calculated from the animal dose (mg / kg) by dividing the animal dose by the human conversion factor (CF). When recalculating the dose from a mouse to a person, the CP is 12. Thus, the daily dose in terms of a person (average weight 75 kg) was 12.5 mg / kg (mouse) - 1 mg / kg (human) or 75 mg; 25 mg / kg (mouse) - 2 mg / kg (human) or 150 mg; and 50 mg / kg (mouse) - 4 mg / kg (human) or 300 mg. To compare the therapeutic efficacy of the obtained solid dispersion with the active substance in amorphous form with the active substance in crystalline form, a group of animals was formed, which were orally administered the active substance in crystalline form, suspended in 2% starch at a therapeutically effective dose of -150 mg / kg once a day. ... Treatment was started at the same time as infection. The treatment was carried out for 4 days, the experiment was stopped on the 5th day. The effectiveness of treatment with the drug was carried out according to the dynamics of survival and the state of animals.

Observations of the animals showed that in the control groups in the first 3 days after infection, the surviving mice lost weight, did not eat, and were inactive. In the groups of animals treated with solid dispersion, the state was painful in the first 2 days, but on the 3rd day the animals became more active, began to feed and gradually gained weight. In the group treated with the active substance in crystalline form, the condition of the mice was satisfactory when administered at a dose of 150 mg / kg. The results are shown in Table 11.

Table 11. Results of studying the antibacterial efficacy of the active substance in amorphous form in the form of a solid dispersion (TD) in daily doses of 12.5 mg / kg (75 mg), 25 mg / kg (150 mg) and 50 mg / kg (300 mg) when compared with the active substance in crystalline form (DCA) at a daily dose of 150 mg / kg.

Group Number of mice in a group % of dead mice Average duration
life% Average CFU / lung on the 5th day % of animals negative for seeding 1 group - control of infection 12 75 48 6.4 ± 4.5x10 ² 0 2 comparison group
DVK treatment
150mg / kg 12 0 one hundred 27 CFU * 91,7 3 group-experienced
Treatment TD 12.5mg / kg 12 41.6 70 3.6 ± 1.5, x10 ¹ 33.3 4 group-experienced
TD treatment
25 mg / kg 12 16.6 81 70 CFU 75.1 Group 5 - Experienced TD Treatment
50 mg / kg 12 0 one hundred 20 CFU * 91,7

Note: * - the difference in comparison with the control is significant according to the nonparametric Mann-Whitney test (p <0.05).

Experiments carried out to evaluate the antibacterial efficacy of a solid dispersion when administered orally to mice at daily doses of 12.5 mg / kg (75 mg); 25 mg / kg (150 mg) and 50 mg / kg (300 mg) for the treatment of acute pneumonia caused by the hospital strain P. aeruginosa KB6 showed a dose-dependent antibacterial effect of the drug. Intranasal infection with KB6 isolate at a dose of 2x10 ⁷ CFU / mouse developed acute pneumonia, causing the death of 75% of the animals. Oral administration of a solid dispersion once a day for 4 days at a dose of 12.5 mg / kg increased the survival rate of mice by 2.3 times. For a dose of 25 mg / kg, a 3.3-fold increase in survival was shown, and for a dose of 50 mg / kg, no animal death was observed.

When comparing the effectiveness of therapy with a solid dispersion with an active substance in amorphous form with therapy with an active substance in a crystalline form at a dose of 150 mg / kg, which has a 100% protective effect, it was shown that a solid dispersion is more effective, because 100% survival of animals is observed at a lower dose - 50 mg / kg. Thus, when creating a solid dispersion, the set goal of increasing the bioavailability and therapeutic efficacy of the active substance was achieved.

Output. The data obtained indicate the effectiveness of the therapeutic effect of the active substance in amorphous form in the form of a solid dispersion (TD) on a model of pneumonia caused by a clinical isolate of P. aeruginosa, characterized by multiple antibiotic resistance. An effective dose of the active substance was chosen in amorphous form in the form of a solid dispersion, which protects 100% of the animals from death. The developed solid dispersion made it possible to reduce the dose of the drug by 3 times in comparison with the active substance in crystalline form, which indicates an increase in the bioavailability of the active substance in the composition of the solid dispersion.

Example 9. Study of the therapeutic efficacy of an active substance in amorphous form in the form of a solid dispersion in a model of a subacute infection of the respiratory tract caused by a P. aeruginosa clinical isolate obtained from a patient with cystic fibrosis.

For patients with cystic fibrosis, chronic infection with pathogens is characteristic, among which P. aeruginosa is of the greatest clinical importance, while long courses of antibiotic therapy do not allow effective eradication of the pathogen due to acquired antibiotic resistance by these bacteria. Moreover, pseudomonads adapt to long-term colonization of the respiratory tract due to a change in virulent properties, which is aimed at a less aggressive effect on the host organism and chronic infection. In our study, to study the therapeutic efficacy of the active substance in amorphous form, a model of chronic infection of the respiratory tract of mice was developed, which was reproduced when animals were infected with the P. aeruginosa clinical isolate 28P23 obtained from a patient with cystic fibrosis.

The experiments were carried out on an inbred line of DBA / 2 mice, males aged 4-6 weeks, weighing 14-16 g. Infection of animals with P. aeruginosa isolate 28P23 intranasally at doses of 5x10⁷, 1x10⁸, 5x10⁸, 1x ten⁹CFU / mouse did not lead to infection of mice. This is associated with a decrease in the virulence of pseudomonads isolated from chronically infected patients with cystic fibrosis. Later, a model was worked out for intraperitoneal infection with this isolate. which made it possible to reproduce a subacute systemic infection with colonization of the respiratory tract.

For intraperitoneal infection, the animals were injected with a culture of bacteria in physiological solution at a dose of 1x10 ⁹ CFU / mouse, the volume of the injected suspension was 100 μl per animal. After infection, the lifespan of the mice was monitored daily (criterion - mortality). Homogenates of lungs and spleens were used as inoculation material for determining the number of pseudomonads.

The treatment was carried out with a solid dispersion of the following composition: active ingredient 100 mg, Eudragit E PO in a ratio of 1: 5, 50 mg sorbitan laurate (Span 20), 30 mg sodium dodecyl sulfate. For the treatment of animals, weighed portions of a solid dispersion with an active substance in amorphous form were prepared individually for each animal relative to its weight. Immediately before the introduction of the drug, 200 μL of drinking water was poured into a test tube with a weighed portion. The test tube was vortexed to dissolve the sample. The suspension was administered to mice intragastrically using a probe. The treatment was carried out once a day for 8 days at a dose of 50 mg / kg in terms of the active ingredient, which has shown its effectiveness in the model of acute pneumonia. On the 9th day after infection, the surviving animals were euthanized, the selected lungs and spleens were homogenized and plated on selective medium. The experiment was performed in 2 repetitions. Petri dishes were incubated at 37 ° C and after 20 hours of culture, the colonies were counted. The concentration of pseudomonads in organs was expressed by the number of CFU per ml. The experimental design is shown in Table 12.

Table 12. Scheme of the experiment. Infection with clinical isolate P. aeruginosa 28P23 at a dose of 1x10 ⁹ CFU / mouse and treatment with the active substance in amorphous form in the form of a solid dispersion (TD) at a dose of 50 mg / kg once a day.

Group Number of animals Dose of the drug Drug administration scheme Total duration
introduction
drug Research method one
Infection control
1x10 ⁹ CFU / mouse 4x2 - - - Sowing from organs, dynamics of death 2
Infection
1x10 ⁹ CFU / mouse
TD treatment 4x2 50 mg / kg 1 per day 8 days Sowing from organs, dynamics of death

During the first two days of the experiment, mild symptoms of dehydration were observed in both groups, from the third day in the treatment group, the condition of the mice returned to normal in both repeats. The state of the control mice in both repetitions varied from normal to depressed, pronounced apathetic, which was also accompanied by symptoms of dehydration. In the control group, mice died with the most pronounced signs of apathy and dehydration.

In the control group, the death of mice was noted on the fifth and seventh days of the experiment. Of the total number of animals in the group, the percentage of dead mice was 25%. No animal deaths were reported in the treatment group. The average life expectancy in the control group was 75.4%, while in the treatment group this parameter was equal to 100%.

Table 13. Results of observation of the dynamics of death and release of the organism of mice from the pathogen when infected with P. aeruginosa 28P23 at a dose of 1x10 ⁹ CFU / mouse and treatment with an active substance in amorphous form as a solid dispersion (TD) at a dose of 50 mg / kg.

Group Number of mice in a group % of dead mice Average
duration
life in% Average CFU / organ on the 9th day Number of animals negative for seeding lung spleen lung spleen one
Infection control
1x10 ⁹ CFU / mouse 4x2 25 75 1.1x10 ² 2.5x10 ² five 2 2
Infection
1x10 ⁹ CFU / mouse
TD treatment 4x2 0 one hundred 0 fifty * 8 6

Note: * - the difference in comparison with the control is significant according to the nonparametric Mann-Whitney test (p <0.05).

On day 9, the surviving animals were euthanized. Autopsy showed splenomegaly, which was more pronounced in the control group than in the treatment group. The macroscopic picture of lung lesions was present in the treatment group in 3 out of 8 mice in the form of small focal lesions. In the control group, all mice showed small focal lesions of the lungs, and some of the mice showed a tendency to fusion. The morphological picture of changes in the lungs indicated the development of pneumonia.

Lungs and spleens were selected, homogenized, and serial dilutions of homogenates were made on Petri dishes with citrimide agar. The seeding results are presented in table 13.

When taking into account the number of pseudomonads in the seeding from the lungs, it was noted that the average value for the control group was 1.1x10 ² CFU / ml, two animals were negative for seeding in this group. In the treatment group, lung pseudomonas were not cultured in any mouse, i. E. all 8 mice were inoculum negative.

In the control group, the average number of bacteria in the spleen inoculations was 2.5x10 ² CFU / ml, and only 2 mice were negative for spleen inoculations. In the treatment group, 6 animals were not plated with spleen pseudomonas.

Statistical analysis of the number of pseudomonads in seeding from organs of mice was identified using the nonparametric Mann-Whitney test. The difference in the number of bacteria in spleen inoculations between the control and experimental groups in mice infected with P. aeruginosa isolate 28P23 and treated with solid dispersion (TD) is statistically significant (p <0.05).

Output. Aeruginosa clinical isolate 28P23, isolated from a patient with cystic fibrosis, has been shown to cause subacute pneumonia with a long course in mice. In the control group, infection caused 25% mortality, and morphological changes in the lungs indicated the development of pneumonia. Oral treatment with a solid dispersion preparation at a dose of 50 mg / kg once a day showed a 100% protective effect and an increase in the average lifespan of the mice during the observation period. The state of the mice on the 9th day after infection was characterized as normal. Therapy with the active substance in amorphous form led to the complete eradication of the pathogen from the lungs and significantly reduced the bacterial load in the spleen of the animals.

The above examples confirm that the team of inventors has fulfilled the set task of the present invention to develop new and more effective dosage forms containing the active substance in amorphous form as part of a solid dispersion. Received an intermediate product (solid dispersion) suitable for the manufacture of dosage forms, containing the active substance in amorphous form; having higher dissolution rates in the dissolution kinetics test in three media compared to the crystalline form; with a higher bioavailability (which is characterized by high achievable AUC, higher achievable C _max ). For the active substance, an inhibitory activity was shown against the type III secretion system in P. aeruginosa isolates obtained during chronic infection of patients with cystic fibrosis. For the active substance, the suppression of the formation of biofilms by pseudomonads, structures that seriously complicates the course of chronic infections, was demonstrated. Biofilms fundamentally complicate antibiotic therapy, in which it is necessary to use significantly higher doses of antibiotics and combine several drugs. The results obtained in in vitro experiments on the inhibitory activity of the active substance on the virulence of P. aeruginosa indicate the antibacterial efficacy of the developed solid dispersion for the treatment of chronic infections. The effective amount of active ingredient in solid dispersion calculated per person is 75-300 mg per day. Dosage is calculated based on in vivo experiments using interspecies conversion factors. The human equivalent dose in mg / kg (EDC) was calculated from the animal dose (mg / kg) by dividing the animal dose by the human conversion factor (CF). When recalculating the dose from a mouse to a person, the CP is 12. An effective therapeutic dose should provide concentrations sufficient to suppress microbial growth in the biological media of the body, as well as to ensure the possibility of dosing the drug per patient's weight, depending on the severity of the disease. The duration of therapy is determined by the doctor based on the characteristics of the disease. The results obtained open up the prospects for the use of preparations containing the active substance in amorphous form for the treatment of recurrent and chronic infections caused by the highly relevant pathogen P. aeruginosa, regardless of the acquired antibiotic resistance.

Claims (8)

1. Solid dispersion for the manufacture of dosage forms, including the active ingredient 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2 , 4-difluorophenyl) -carboxamide and at least one pharmaceutically acceptable polymer, where the pharmaceutically acceptable polymer is contained in a solid dispersion in a weight ratio of active substance to polymer from 1: 1 to 1:10, and the active substance is contained in amorphous form or in amorphous form with an admixture of microcrystalline and crystalline phases. 2. The solid dispersion of claim 1, wherein the pharmaceutically acceptable polymer is selected from a copolymer of PEG 6000, vinyl caprolactam and vinyl acetate, a copolymer of N-vinyl pyrrolidone and vinyl acetate, or a copolymer of butyl methacrylate, 2-dimethylaminoethyl methacrylate and methyl methacrylate. 3. Solid dispersion according to PP. 1, -2, additionally comprising at least one pharmaceutically acceptable surfactant in an amount of 1 to 20 parts by weight, based on the weight of the active ingredient. 4. The solid dispersion of claim 3, wherein the pharmaceutically acceptable surfactant has an HLB value of 3 to 40. 5. The solid dispersion of claim 3, wherein the pharmaceutically acceptable surfactant is selected from sodium dodecyl sulfate or sorbitan laurate. 6. Solid dispersion according to claims 1-5, which can be used for the production of oral solid dosage forms in the form of coated or uncoated tablets, hard capsules, dosage powder, powder for solution or suspension for oral administration. 7. A method for the treatment of chronic infections of the respiratory system caused by antibiotic-resistant strains of Pseudomonas aeruginosa , comprising the use of a solid dispersion according to claim 1 in an effective therapeutic dose for humans in an amount from 1 to 4 mg / kg in terms of the active ingredient. 8. The method according to claim 7, where the infectious diseases are selected from pneumonia; chronic infections with cystic fibrosis, bronchiectasis, defects in the development of the respiratory system and pathologies of the respiratory system.

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