RU2624846C1 - Application of 4-(3-ethoxy-4-hydroxybenzyl)-5-oxo-5,6-dihydro-4h-[1,3,4]-thiadiazine-2-(2,4-difluorophenyl)-carboxamide to suppress infection, caused by antibiotic-resistant pseudomonas aeruginosa strains, and method of suppression of this infection - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: application of 4-(3-ethoxy-4-hydroxybenzyl)-5-oxo-5,6-dihydro-4H-[1,3,4]-thiadiazine-2-(2,4-difluorophenyl)-carboxamide for suppression of an infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa in the living body, as well as a method of suppression of the infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa when exposed to Pseudomonas aeruginosa strains with an effective amount of this compound.

EFFECT: implementation of the claimed prescription for infection of mice with the indicated clinical strains genotypes, regardless of the acquired resistance, the compound does not have a bactericidal effect on the intestinal microflora of animals and is low-toxic.

2 cl, 3 dwg, 25 tbl

Description

FIELD OF THE INVENTION

The invention relates to microbiology and can be used to suppress infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa.

Prior level

Pseudomonas aeruginosa or Pseudomonas aeruginosa causes up to 30%, and in some departments up to 50% of all nosocomial infections. It is the cause of nosocomial pneumonia, primary bacteremia, infections of the genitourinary system and purulent surgical infections in a high percentage of cases, especially in resuscitation and intensive care units (ICU) in patients with mechanical ventilation and other invasive procedures such as catheterization of the bladder or long-term infusion therapy. Often infections develop at lightning speed and are accompanied by high mortality, the rates of which remain stably high (34-48%). This threatening situation is caused by an extremely high level of multiple resistance to the majority, and sometimes to all antibiotics, of clinical Pseudomonas aeruginosa strains. P. aeruginosa leads among antibiotic-resistant gram-negative bacteria that cause infections in hospitalized patients. Nosocomial infections caused by Pseudomonas aeruginosa practically do not respond to standard antibacterial treatment due to the multiple antibiotic resistance of clinical strains. In addition, already during treatment, resistance to new drugs develops. Therefore, you have to use a combination of several antibiotics, including highly toxic ones. And mortality is due to the fact that bacteremia caused by pseudomanadas develop so abruptly that there is not even enough time to put an antibioticogram and choose the right antibiotic. For example, according to foreign studies with bacterial sepsis, survival with adequately prescribed starting antibiotic therapy is 52.0%, with inadequate starting antibiotic administration - 10.3%, that is, leads to a 5-fold decrease in survival.

The fight against nosocomial infections requires significant costs associated with monitoring 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 up to the fatal consequences of these diseases. It is obvious that the development of new approaches to the creation of antibacterial drugs with mechanisms of action other than antibiotics is an extremely urgent task.

It is known to treat bacterial infections caused by P. aeruginosa with synthetic antibacterial drugs.

However, antibiotic resistance of pathogenic microorganisms, including multiple ones, is widespread and is the most important factor in reducing the effectiveness of treatment of infectious diseases in almost all regions of the world.

According to research conducted by the Research Institute of Antimicrobial Chemotherapy of the Smolensk State Medical Academy and the ROSNET research group in the ICU of Russian hospitals until 2003, the frequency of resistance of P.aeruginosa to meropenem was on average 3%, to imipenem - 22.9%. Of the other beta-lactam antibiotics, resistance to ceftazidime was 12.2%, to piperacillin-tazobactam - 37.7%, to piperacillin - 56.5%. According to a study of the same group, in 2008, 41.4% of isolated Pseudomonas aeruginosa strains were resistant to meropenem, 39.0% to imipenem, 47.9% to ceftazidime, 72.6% to cefoperazone, and cefoperazone -sulbactam - 60.7%, to cefepime - 58.6%, to piperacillin - 52.9%, to piperacillin-tazobactam - 42.4%. Similar results were obtained in studies in the framework of the RESORT program in ICU 31 of the medical facilities of the Russian Federation in 2002-2004. - the proportion of resistant strains was: to imipenem and meropenem - 39% and 41%; to amikacin - 42%; ceftazidime - 48%; to other cephalosporins - 58-72%. A high incidence of resistance to ciprofloxacin - 65% and gentamicin - 75% was revealed. Based on the data of microbiological monitoring at the State Budgetary Institution St. Petersburg Research Institute of Emergency Medicine named after II Dzhanelidze from 2009 to 2012, resistant strains of Pseudomonas aeruginosa were not detected only against colistin. In fact, for the treatment of Pseudomonas infections caused by multi-resistant antibiotic strains, the drug of choice is only polymyxin E (colistin), which is rarely used due to toxic effects on the kidneys and intestinal mucosa of patients.

A number of foreign analytical works describe the current state of affairs in the field of the effectiveness of treatment with various antibiotic infections caused by P. aeruginosa. For example, Somayeh Moazami Goudarzi et al presented data from a survey of patients at two hospitals in Tehran (Iran) in 2011 (1) (S. Moazami Goudarzi and F. Eftekhar, 2015). It was shown that the identified clinical strains were resistant to most of the tested antibiotics (both synthetic and semi-synthetic and natural) such as tetracycline (80% of resistant strains), carbenicillin (82%), piperacillin (72%), aztreonam (76%) , tobramycin (78%), cefepime (77%), meropenem (71%), amikacin (78%), ciprofloxacin (78%), cotrimoxazole (94%), imipenem (70%), ceftazidime (20%).

There are no published data evaluating the difference in the effectiveness of combination and monotherapy for infections caused by P. aeruginosa. There are no randomized trials, and not randomized trials on this problem are single and show conflicting results. Most authors, including Russian ones, consider combination therapy of infections caused by Pseudomonas aeruginosa to be preferred over monotherapy. Various combinations of cephalosporins, fluoroquinolones, aminoglycosides, and carbapenems are generally recommended to increase the likelihood of effective therapeutic effects and reduce the risk of antibiotic resistance (2-6) (Burgess DS, Nathisuwan S. 2002; Bodey GP, et al. 1985; Hilf M , et al. 1989; Leibovici L, et al. 1997; Siegman-lgra Y., et al. 1998).

Thus, antibiotic-resistant clinical strains of Pseudomonas aeruginosa are widespread and cause serious problems when choosing the treatment regimen caused by infections. There is a need to use highly toxic antibiotics, which leads to dangerous side effects in patients who disrupt the normal functioning of vital organs.

As a result, many pharmaceutical companies have slowed down or discontinued new antibiotic development programs, as new antibiotics have become more difficult to commercialize because of their low efficacy in the face of the high prevalence of antibiotic-resistant bacteria strains.

Despite the fact that the study of pathogens at the molecular genetic level allows us to identify new targets for the development of antibacterial agents that are not based on bactericidal or bacteriostatic action, such drugs are currently not available on the pharmaceutical market.

It is obvious that it is necessary to develop fundamentally new approaches and drugs for the treatment of bacterial infections that can be used both independently and in combination therapy. Basic research over the past 10 years has made it possible to change the strategy for fighting infections. In particular, bacterial pathogenicity factors are chosen as targets for the search for antibacterial drugs. Such drugs should not kill microbes, but suppress virulence, without creating conditions for selective selection, which fundamentally reduces the risk of developing genetically determined resistance to the drug used. Thus, an increase in the proportion of antibiotic-resistant bacterial infections requires the search for not only new antibiotics, but also effective inhibitors of virulence factors that suppress the infection by a different mechanism.

The most important factor in the realization of pathogenic properties by bacteria is the transport of protein virulence factors into the cell of a macroorganism. The process is carried out by secretion and export of effector molecules through membranes, or directly into the cytoplasm of the host cell, which leads to a change in its normal physiological state and contributes to the invasion and intracellular reproduction of the pathogen. Currently, seven secretion systems are known that differ in the structure of the secretory apparatus and the specific direction of action of the effector molecules.

A promising target for the development of new antibacterial drugs is a type III secretion system (CCT) of gram-negative bacteria, which transfers proteins, pathogenicity factors, from a bacterial cell directly to the cytoplasm of a eukaryotic cell. STST is present only in pathogenic bacteria; It is thanks to its functioning that a wide range of bacteria with various types of parasitization (exo- and endoparasites) realize their pathogenic properties. Mutations leading to impaired CTST functions cause a decrease or loss of virulence (7) (A.S. McShan, R.N. De Guzman, 2015).

In P. aeruginosa, proteins secreted by the third transport system are toxins that cause cell death by apoptosis or necrosis. Their main action is aimed at suppressing the immune response: they inhibit the migration and phagocytosis of macrophages and neutrophils recruited to the site of infection, causing the death of immune cells. So, with pneumonia, CTTT proteins create immunosuppression in lung tissues, which allows not only Pseudomonas aeruginosa to persist in the lungs, but also contributes to the development of superinfection when infected with other pathogens.

In general, secreted pseudomonad proteins are involved in the establishment of infection and dissemination of the pathogen in acute infection, and are responsible for persistence in chronic infection. Elimination of P.aeruginosa in a patient indirectly contributes to its resistance to other infections, due to the increase in the protective properties of the host organism, suppressed as a result of the action of CTST P.aeruginosa.

To date, 4 CTT effector proteins P.aeruginosa, ExoU, ExoS, ExoT, ExoY have been identified. Virtually all strains of pseudomonads have genes encoding the secretory apparatus, however, most strains do not contain a complete set of genes that determine effector proteins. Clinical P.aeruginosa strains are characterized by different genotypes and phenotypes, which affects the severity of the disease and the clinical prognosis. Studying the frequency of deaths, bacterial persistence in the lungs, and dissemination of bacteria showed that the secretion of ExoU toxin makes the greatest contribution to virulence, while the secretion of ExoS protein is associated with less virulence, and ExoT protein has a minimal effect on the manifestation of the virulent properties of the pathogen. The pathogenesis of lung damage is associated with ExoU activity, which induces procoagulant processes in epithelial cells, vascular hyperpermeability, platelet activation, and blood clots in pseudomonas pneumonia and sepsis. The toxic effect of ExoU is directed against phagocytes, as well as overcoming the epithelial barrier, which contributes to bacterial dissemination and persistence (8) (B.J. Berube et al. 2016).

The search for CCTT inhibitors is currently ongoing. therapy with such drugs has several advantages compared to antibiotics:

- CTST inhibitors do not contribute to the selection of resistant forms of microorganisms, since they do not cause death and do not inhibit the reproduction of pathogens, but only inhibit their virulence. The eradication of a pathogen that has lost its virulent properties occurs as a result of the action of the immune defenses of the host organism.

- CTST inhibitors are promising for the treatment of infections caused by antibiotic-resistant pathogens, as these inhibitors act on the pathogen, regardless of acquired antibiotic resistance.

- since CCTT inhibitors affect only pathogenic microorganisms, therapy with these drugs will not cause the death of normal human microflora and the development of dysbiosis.

- the loss of virulence by the pathogen under the influence of an inhibitor will significantly reduce the damage caused by the pathogen to the host organism.

Currently, scientific publications and patents describe a number of P.aeruginosa CTTT inhibitors with different chemical structures, belonging to different classes of low molecular weight compounds, as well as specific antibodies to the injection tip protein of the injectosome (9) (A. Ahalieyah et al. 2016. ) Two main classes of low molecular weight compounds, the hydrazones of salicylic aldehyde (salicylidene acylhydrazides) (10, 11) Slepenkin A., et al. 2011; A. Anantharajah, et al. 2012) and thiazolidinones (thiazolidinones) (12, 13) (Felise H. B., et al. 2008; Kline, T., et al. 2009). For several inhibitors, specific targets have been identified in CCTT, but most targets for CCTT inhibitors have yet to be identified or characterized. Most CCTT inhibitors are compounds selected by screening synthetic chemical libraries, but there are also naturally occurring inhibitors - aurodox, kaminoside, guadinomine, (-) - hopeaphenol, citrus flavonoids, pyerisidine, pseudoceramine, salicylideneanilide, dracterin, lactoferin. In addition to low molecular weight compounds, the inhibitory effect on CTST is shown for some high molecular weight substances, which are represented by polymers, proteins, mimetic polypeptides, polysaccharides (9) (A. Ahalieya h et al. 2016).

In the direction of action on CTST, inhibitors can be divided into the following groups:

- acting on the genetic regulation of SSST (plant phenolic compounds, N-hydroxybenzimidazoles, salicylidene acylrazides);

- Acting on the functioning of the CCTT apparatus (hydroxyquinolines, thiazolidinones, phenylacetamides, PcrV antibodies (KB001; V2L2MD), PcrV / Psl antibodies MEDI3902);

- acting on the CTST effector proteins (exocin, aryl sulfonamides, pseudolipase A);

- inhibitors of indefinite action (C20, C22, C24, C34 and C38, (-) - hopeaphenol, gallium (III) - salicylidene acylhydrazides) (9) (A. Ahalieyah et al. 2016).

Thus, to date, a number of technical solutions are known related to the preparation of pseudomonas CTTT inhibitors. Among them, low molecular weight compounds of various classes, as well as specific antibodies to the structural protein CTST.

For all inhibitors obtained, activity was shown to inhibit the secretory function of the pathogen in vitro. However, for most inhibitors, a specific target of action has not been identified and the molecular mechanism of inhibition has not been studied. Therefore, CTTT inhibitors obtained to date may have different activity, inhibiting secretion, both specifically and indirectly through other molecular targets of the pathogen.

In the development of effective drugs based on CCTT inhibitors for the treatment of infectious diseases, the key task is to prove the antibacterial action of the obtained inhibitors in animal models of the infection process. This is due to the fact that suppression of the secretory function of a pathogen reduces its virulence, but does not affect viability, i.e. not directly related to suppression of infection. Thus, for CTST inhibitors, it is required to experimentally confirm the concept that a decrease in virulence will lead to blocking of infection in the host organism. In addition, potential drugs should be characterized by the absence of a toxic effect on the host organism and on the normal microflora.

Among known inhibitors, the therapeutic effect in the form of suppression of Pseudomonas aeruginosa in in vivo models has been shown for specific antibodies to the components of the CCTT-PcrV antibody needle (KB001; V2L2MD) (16, 17) (Francois, B. et al. 2012; Warrener, P . et al. 2014) and PcrV / Psl antibodies (MEDI3902) (18) (DiGiandomenico, A. et al. 2014). However, this group of drugs based on antibodies should be considered separately due to the fact that they belong to the group of immunobiological drugs. Also, the therapeutic effect in the form of suppression of Pseudomonas aeruginosa in in vivo models was shown for the low molecular weight compound INP1855 belonging to the class of hydroxyquinolines (14, 15) (A. Anantharajah, E. Faure, J.M. Buyck, et al. 2016; Enquist, PA, et al. 2012). This decision, as the closest to the claimed purpose - as an inhibitor, the therapeutic effect of which is expressed in the form of suppression of Pseudomonas infection, was taken by the authors as a prototype.

The chemical structure of the CTTT inhibitor P. aeruginosa, selected as a prototype

According to the prototype of this invention, the essence of which is reflected in the study of the suppressing infection of the action of the CTTT inhibitor belonging to the class of hydroxyquinolines in relation to acute pulmonary infection in mice caused by Pseudomonas aeruginosa, it was shown that a single intranasal administration of INP1855 at a dose of 4.5 μg after infection with a lethal dose, P.aeruginosa increased the percentage of surviving animals from 38% (in control) to 88% 48 hours after infection, i.e. observed inhibition of animal death by 5 times. In mice treated with INP1855, a 2-3% decrease in pathological changes in the lungs and an approximately 30-fold decrease in the number of bacteria in the lungs were observed. The dose-dependent effect of INP1855 upon intraperitoneal administration 4 hours after infection with pseudomonads (14) was also shown (A. Anantharajah, E. Faure, J.M. Buyck, et al. 2016).

This work is the only one published showing the efficacy of a low molecular weight CTTT inhibitor in an in vivo model, but has a number of drawbacks with respect to the activity and knowledge of the resulting inhibitor.

1. Animal experiments were carried out with one strain of Pseudomonas aeruginosa, which is characterized by the exoU ⁺ genotype, while the opposite exoS ⁺ genotype, being invasive, causes generalized infections and sepsis, and is also prevalent among all clinical strains, from 85 to 75%.

2. A complete treatment regimen has not been developed.

3. The complete suppression of pathogen accumulation in lung tissues has not been shown. The amount of pseudomonas sown from the lung after treatment with the inhibitor remained in a significant number, more than 10 ⁵ CFU / lung. It is also known that untreated infection can lead to the development of chronic conditions and relapses against the background of a weakened immune status.

4. The toxicity characterization of the obtained inhibitor for experimental animals was not performed.

5. The effect of the obtained inhibitor on the normal microflora has not been studied.

Thus, there is a need for further studies to expand the arsenal of inhibitors of the type III secretion system P.aeruginosa, study their effectiveness in models of the infection process in animals, and test their possible toxicity to the host organism and microflora. Such studies are aimed at developing a new generation of antibacterial drugs with a mechanism of action different from antibiotics and effective against antibiotic-resistant strains of Pseudomonas aeruginosa.

Disclosure of invention

The technical problem solved by this invention is:

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

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

- the absence of an 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 to suppress infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa.

The specified problem is solved by

the use of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2,4-difluorophenyl) carboxamide to suppress infection, caused by antibiotic-resistant strains of Pseudomonas aeruginosa in a living organism, as well as a method of suppressing an infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa by exposing the strains of Pseudomonas aeruginosa to an effective amount of this compound.

Terms and Definitions

"Infection" - a set of biological processes that occur and develop in the host during the introduction and reproduction of pathogenic microbes in it.

"Inhibition of infection" is the process of limiting the manifestation of factors developing in the host body after the introduction and reproduction of pathogens

“Pathogen accumulation in tissues” - a process characterized by the growth of bacteria

"Bacterial suppression" - blocking the viability of bacteria as a result of negative effects on metabolism and / or on reproduction

"Pathogenicity factors" - mechanisms that ensure the colonization of microbes, their resistance to microbicidal factors of the body, invasiveness and toxigenicity, as well as the ability to long-term persistence

“Suppression of pathogenicity factors” - inhibition of colonization processes, resistance to microbicidal factors of the body, invasiveness, toxigenicity and persistence

"Genotype" - a set of genes of a given organism.

The “type III secretion system of bacteria” is a bacterial cell apparatus that provides a one-stage transport of pathogenicity effector molecules from the bacterial cytoplasm to the cytosol of a eukaryotic macroorganism cell. It is considered as one of the main pathogenicity factors of gram-negative bacteria, which realize invasion, toxigenicity and dissemination of the pathogen in the infected body (hereinafter referred to as CTST).

"Antibiotic resistance profile" - lack of sensitivity of bacteria to specific antimicrobial agents

“Formation of antibiotic resistance” is the property of individual bacterial strains to remain viable at those antibiotic concentrations that suppress the bulk of the microbial population. The formation of resistance is genetically determined: the acquisition of new genetic information or a change in the expression level of one's own genes. Resistant strains survive and spread under the selective action of antibiotics.

“In vitro” is a technology for performing experiments when experiments are carried out “in vitro” - outside a living organism in a culture of living cells or in a cell-free model.

"In vivo" is a technology for performing experiments on a living organism, on a person, or on an animal model.

"Obligatory intracellular bacteria" - bacteria that multiply only inside eukaryotic cells, due to energy dependence on macroergic molecules of the host cell. Cultivated in vitro only in cell cultures. An infection in a macroorganism is caused by intracellular colonization and effects on the metabolism of the host cell.

"Extracellular pathogenic bacteria" - bacteria that multiply in the host body outside the cells and cause infection as a result of the action of pathogenicity factors.

The authors synthesized and patented biologically active substances that suppress pathogenic bacteria, which are substituted derivatives of 1,3,4-thiadiazolines (I), thiadiazinones (II) and thiadiazepinones (III), obtained on the basis of oxamic acid thiohydrazides (RF patent No. 2447066) . The resulting active low molecular weight compounds are inhibitors of the type III secretion system in pathogenic bacteria. For the obtained compounds, the inhibition of the propagation of pathogenic bacteria in cell culture in vitro was shown by the example of chlamydia. Chlamydia is a representative of obligate intracellular parasites, in which a type III secretion system provides contact with the host cell, and its inhibition blocks the growth of bacteria. The specificity of inhibitors with respect to the type III secretion system was demonstrated by the detection of chlamydia effector protein secreted by CCTT into the membrane of the chlamydial phagosome. In this patent, it was not investigated and experimentally shown that the obtained low molecular weight inhibitors inhibit the secretion of CTST effector proteins in extracellular pathogens. Moreover, the mechanism of interaction of intracellular and extracellular pathogens with the host organism is fundamentally different, and the type III secretion system in these two groups of bacteria performs different functions. In intracellular pathogens, CTST provides the possibility of intracellular survival, and in extracellular pathogens, the development of infection as a result of the toxic effect of effector molecules against immune and barrier cells. The patent has not experimentally demonstrated the possibility of suppressing infection in the animal's body under the action of the obtained CTST inhibitors. In addition, the effect of the selected CTST inhibitors on the secretory apparatus of P.aeruginosa and on the development of the infectious process in laboratory animals has not been studied.

Therefore, the development of the use of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide and a method of suppressing the infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa in a model of infection in animals using the low-molecular type III secretion system inhibitor of the present invention has a "novelty" and inventive step.

Brief Description of the Figures

In FIG. 1 shows a synthesis scheme for 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide VIII (connection CL-55).

In FIG. Figure 2 shows the reaction of preparations of secreted P. aeruginosa proteins with antibodies to the ExoT effector. A strong reaction is marked in red. The name of the P. aeruginosa strains are marked on top of the figure.

In FIG. Figure 3 shows the suppression of the activity of CCTT of clinical strains 1625/36/2015 with exoU + genotype (A) and CB6 with exoS + genotype (B) P.aeruginosa (after induction of CTST 5 mM EGTA and the action of different concentrations of CL-55 when setting the immunoblot method with serum to EXO protein (53 kDa), indicated by arrows.

Examples

1. Obtaining a low molecular weight CTTT inhibitor based on the synthesis of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4- difluorophenyl) carboxamide

2. Investigation of the mechanism of action of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide on clinical strains of Pseudomonas aeruginosa with different antibiotic resistance profiles.

3. Toxicity study of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide for animals.

4. Effect of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide on viability bacteria in vitro and in vivo.

5. Determination of the effective inhibitory dose of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) - carboxamide in laboratory animals.

6. The study of the therapeutic efficacy of the CTTT inhibitor in a model of experimental infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa of two different CTTT genotypes.

7. The study of the prophylactic effectiveness of the CTTT inhibitor in a model of experimental infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa of two different CTTT genotypes.

Example 1:

Obtaining a low molecular weight CTST inhibitor based on the synthesis of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide

The active substance of an antibacterial drug based on a low molecular weight type III secretion system inhibitor in the claimed invention is 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine -2- (2,4-difluorophenyl) -carboxamide VIII (compound CL-55), which is obtained by cyclization of oxamic acid thiohydrazide chloroacetic acid VII and its synthesis process involves the preparation and subsequent reaction of chloroacetamide III with a pre-prepared solution of elemental sulfur with morpholine , last yuschee monotiooksamidov reacting IV with hydrazine hydrate, the reaction is obtained with the hydrazide V 3-ethoxy-4-hydroxy-benzaldehyde and reduction of the resulting hydrazone VI with sodium borohydride in methanol.

General approach for the synthesis of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide VIII (compound CL-55) is shown in FIG. 1.

Synthesis of 2-chloro-N- (2,4-difluorophenyl) -acetamide (III). To a solution of 130 g (1 mol) of 2,4-difluoroaniline (I) in 1000 ml of DMF, 87.5 ml (1.1 mol) of chloroacetyl chloride (II) are added with cooling, ensuring that the temperature of the reaction mixture does not exceed 20 ° C. After completion of the addition of the chloride (II) solution, the solution was stirred at room temperature for another 2 hours. Then the reaction mixture is poured into 2000 ml of cold water and the precipitate is filtered off. It is washed on the filter many times with water and dried in a vacuum desiccator. The yield of acetamide (III) is 179 g (87%). T. pl. 101-102 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 4.20 (s, 2H, ArCH ₂ Cl); 7.10-7.25 (m, 2H, Ar); 7.53 (m, 1H, Ar); 10.25 (s, 1H, Ar-NH-CO). Found (%): C 46.62, H 2.99, N 6.84. Calculated (%): C 46.74, H 2.94, N 6.81. Mass spectrum, m / z: 205.

Synthesis of 2-morpholin-4-yl-2-thioxo-N- (2,4-difluorophenyl) -acetamide (IV). A solution of 2.6 mol of elemental sulfur in 800 ml of DMF is preliminarily prepared and 2.6 mol of morpholine is added in one portion. The resulting mixture was stirred for 30 minutes and a solution of 179 g (0.87 mol) of chloroacetamide (III) in 200 ml of DMF was added to the resulting brown-red suspension while cooling, so that the temperature did not exceed 10 ° C. The resulting mixture was kept at room temperature for 12 hours. After completion of the reaction (TLC control), the reaction mixture was poured into water, the reaction mixture was acidified to pH 4-5, then the precipitated pale yellow crystals were filtered off and washed on the filter many times with water. Then they are dissolved in acetone, unreacted sulfur is filtered off. Then acetone was evaporated, the residue was recrystallized from ethanol to obtain 189 g of pale yellow crystals of morpholide (IV). The yield is 76%.

Synthesis of 2-hydrazino-2-thioxo-N- (2,4-difluorophenyl) -acetamide (V). The resulting morpholide (IV) was dissolved in 500 ml of isopropanol and 40 ml (0.79 mol) of hydrazine hydrate was added and boiled for 30 minutes. Then it is poured into water and acidified with dilute hydrochloric acid to pH 7. The precipitate is filtered off and dried in a vacuum desiccator. Yield: (V) - 101.2 g, (73%). T. pl. 154-156 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 7.10-7.26 (m, 2H, Ar); 7.50 (m, 1H, Ar); 10.30 (s, 1H, Ar-NH-CO). Found (%): C 41.40, H 3.18, N 18.25. Calculated (%): C 41.56, H 3.05, N 18.17. Mass spectrum, m / z: 231.

Synthesis of 2- [N ’- (3-ethoxy-4-hydroxy-benzylidene) -hydrazino] -2-thioxo-N- (2,4-difluorophenyl) -acetamide (VI). To 101 g (0.44 mol) of 2-hydrazino-2-thioxo-N- (2,4-difluoro-phenyl) -acetamide (V) in 500 ml of ethanol, 0.44 mol of 3-ethoxy-4-hydroxy- benzaldehyde and boil with stirring for 5 minutes. Cool the reaction mixture and filter off 113.7 g (68%) of the precipitated yellow crystals of 2- [N '- (3-ethoxy-4-hydroxy-benzylidene) -hydrazino] -2-thioxo-N- (2,4-difluorophenyl) -acetamide (VI), they are washed on the filter with cold ethanol and used further without further purification.

Synthesis of 2- [N '- (3-ethoxy-4-hydroxy-benzyl) -hydrazino] -2-thioxo-N- (2,4-difluoro-phenyl) -acetamide (VII). While cooling with ice (0-5 ° C) and stirring, 17 g (0.45 mmol) of sodium borohydride was added to a suspension of 2- [N '- (3-ethoxy-4-hydroxy-benzylidene) -hydrazino] -2-thioxo- N- (2,4-difluoro-phenyl) -acetamide in small portions. The reaction mixture is left for 2 hours while cooling and stirring, then 25 ml of water are added and neutralized with dilute hydrochloric acid to pH 7. The precipitated light yellow precipitate is filtered off and recrystallized from isopropanol. Yield (VII) - 91.4 g, (75%). T. pl. 119-121 ° C. NMR ¹ H CDCl ₃ (δ, ppm, J, Hz): 1.35 (t, 3H, —O — CH ₂ CH ₃ , J = 7.22); 3.96 (cd, 2H, -O-CH ₂ CH ₃ , J = 7.00 (13.90)); 4.32 (s, 1H, -NH-CH ₂ -Ar); 6.98-7.62 (m, 6H, Ar); 9.02 (s, 1H, ArOH); 10.12 (s, 1H, Ar-NH-CO). Found (%): C 53.45, H 4.58, N 10.94. Calculated (%): C 53.54, H 4.49, N 11.02. Mass spectrum, m / z: 381.

Synthesis of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazine-2-carboxy (2,4-difluorophenyl) amide ( compound CL-55) (VIII). 91 g (0.24 mol) of 2- [N '- (3-ethoxy-4-hydroxy-benzyl) -hydrazino] -2-thioxo-N- (2,4-difluoro-phenyl) -acetamide (VII) is added to a solution of 0.29 mol of chloroacetic acid in 5 ml of isopropanol. A catalytic amount of ammonium acetate was added to the reaction mixture and boiled for 4 hours (TLC control). After the reaction, the reaction mixture is cooled, the precipitated crystals are filtered off and washed on the filter with water and cold ethanol. The residue was recrystallized four times from ethanol. Yield (VIII) - 75.7 g, (75%). T. pl. 129-130 ° C. NMR ¹ H (400 MHz, DMSO-d6): 1.33 (t, 3H, —O — CH ₂ CH ₃ ); 3.71 (s, 2H, S-CH ₂ -CO); 4.01 (cd, 2H, -O-CH ₂ CH ₃ ); 4.92 (s, 2H, -NH-CH ₂ -Ar); 6.75-6.81 (m, 2H, Ar); 6.98 (s, 1H, Ar); 7.12-7.17 (m, 1H, Ar); 7.36-7.42 (m, 1H, Ar); 7.59-7.65 (m, 1H, Ar); 8.90 (s, 1H, -NH-CH ₂ -Ar); 10.06 (s, 1H, ArOH). ¹³ C NMR (100 MHz, DMSO-d6): 15.16, 25.48, 53.56, 64.29, 104.95, 111.98, 114.23, 115.88, 121.49, 128.02, 128.31, 141.16, 146.90, 154.93, 157.41, 158.00, 159.06, 159.75, 161.49; Found (%): C 56.50 H 4.62, N 10.31. Calculated (%): C 56.57 H 4.50, N 10.42. Mass spectrum, m / z: 421.

Example 2

Study of the mechanism of action of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide (CL -55) on strains of clinical origin Pseudomonas aeruginosa with different antibiotic resistance profiles.

The mechanism of action of the low molecular weight CTTT inhibitor is CL-55: inhibition of the transport of pathogenicity factors, CTTT, ExoS, ExoT, ExoY and ExoU effector proteins into a eukaryotic cell, as a result of the specific suppression of the CTTT pathogen.

In order to study the effect of the CTTT inhibitor - CL-55 on the clinical strains of Pseudomonas aeruginosa with different antibiotic resistance profiles, the following studies were carried out:

1. The role of CCTT in clinical antibiotic-resistant strains of Pseudomonas aeruginosa was studied based on the study of the presence of genes encoding the CTTT effector proteins and the activity of the secretory apparatus in these strains in vitro.

2. The inhibitory effect of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide was characterized (CL-55) on the functional activity of CCTT of clinical antibiotic-resistant strains of Pseudomonas aeruginosa.

Study of the role of CCTT in clinical antibiotic-resistant strains of Pseudomonas aeruginosa

The study was conducted on 77 strains of Pseudomonas aeruginosa isolated from samples of clinical material in Moscow hospitals. Strains were isolated from the discharge of the respiratory tract, from the blood and discharge of the open infected wounds. 96% (74 strains) were resistant to at least one antibiotic, 62% were characterized by multiple resistance.

A study of the frequency of occurrence of the genes encoding proteins - effectors of CTST pseudomonads in the obtained hospital strains was carried out using PCR with DNA strains. When working out the conditions for the reaction, reference strains of P. aeruginosa with the well-known CTT genotype, PAO (exoS + exoT + exoY + exoU-) and PA103 (exoS-exoT + exoY + exoU +) were used. Table 1 presents the sequence of primers selected to determine the presence of CTTT genes of P.aeruginosa.

Among all analyzed exoU + strains, the genotype was found in 13.8% obtained from intensive care units ("ICU"), and in a slightly higher percentage - 22.7% in strains from other departments ("non-ICU"). The exoS + genotype was detected in the analyzed hospital strains in a significantly higher percentage than the exoU + genotype, which corresponds to well-known published data. So, in the group of strains obtained from ICU, the exoS + genotype was detected in 86.2%, and in the group of non-ICU in 79.5%. Table 2 presents the results of CTST genotyping of clinical P.aeruginosa strains when determining genes for effector proteins by PCR and the characteristics of the functional activity of CTST in determining the secretion of ExoT protein by immunoblot.

To study the activity of CCTT in clinical strains of pseudomonas in vitro, we used a model of induction of the functional activity of the transport system with a decrease in the concentration of calcium in the bacterial culture medium. To do this, calcium chelator, EGTA was added to the actively dividing culture of pseudomonads, and the presence of the ExoT secretory protein in the culture medium was tested using specific antibodies by the immunoblot method. The overnight culture of the studied P. aeruginosa strains was diluted 1/100 in fresh LB medium, 5 mM EGTA was added and cultivation was continued under stirring for 3 hours at 37 ° C. After cultivation, bacterial cells were besieged by centrifugation. Extracellular proteins were concentrated with trichloroacetic acid (10% saturation), washed with 100% acetone and suspended in a 1-fold sample buffer. Polyacrylamide gel electrophoresis was carried out according to the Laemmli method, after which the proteins were transferred by the method of semi-dry blot from the gel to nitrocellulose membranes using the TE70 PWR system (GE, Moscow, Russia).

Nitrocellulose membranes were stained with Ponceau S dye to verify transfer quality, and then washed with 20 mM Tris-HCl + 150 tM NaCl + 0.05% Tween20 (TBS-T) solution, blocked with 5% skim milk in TBS-T and incubated with murine antibodies ExoT protein at a dilution of 1/20,000 overnight at 4 ° C. The reaction was taken into account after incubation of the membranes with anti-mouse conjugate labeled with horseradish peroxidase (1/5000 dilution) for 1 hour at room temperature and treatment of the membranes with chemiluminescence reagent. As a positive control, a purified preparation of the ADP-ribosylating ExoT fragment was used. The reaction was taken into account on a chemiluminometer (Vilber Lourmat, Eberhardzell Germany).

A study of the functional activity of CCTT in the obtained clinical strains of P.aeruginosa under conditions of induction with low concentrations of calcium showed that 89% of the strains secrete the ExoT effector protein (Table 2, Fig. 2).

The functional activity of the CCTT of hospital clinical strains of P. aeruginosa was also characterized by the method of determining cytotoxicity. Toxicity to eukaryotic cells associated with the secretion of ExoU or ExoS proteins in vitro manifests itself very quickly, during the first 3-4 hours, and is expressed in violation of the integrity of the cell membrane with subsequent cell death by necrosis. We used in the tests for cytotoxicity cells of the CHO line (Chinese hamster ovary cells) and evaluated cell death in the test by quantitative determination of lactate dehydrogenase in the culture medium 3 hours after the introduction of pseudomonas strains on the cells. Lactate dehydrogenase is a stable enzyme that is present in the cytoplasm of a cell, and when the integrity of the cytoplasmic membrane is violated, it enters the external environment.

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

Table 3 presents the results of the analysis of cytotoxicity of hospital strains of pseudomonas. Based on the data obtained, all strains could be divided into 4 groups according to the percentage of cell death: toxic - T (from 90 to 70%), medium toxic - C (from 70 to 50%), low toxicity - M (from 50 to 20%) and non-toxic - N (less than 20%). The results showed that among the tested strains with exoU + genotype, toxicity was 91.6%. Among 31 strains with exoS + genotype, 21 or 67.7% were toxic.

Thus, in aggregate, the obtained data indicate the presence of CCT and its activity in clinical strains with a different antibiotic resistance profile and speak in favor of the key role of type III secretion system in the development of the infectious process, which makes CCT a promising target for suppressing the infection caused by multiresistant Pseudomonas aeruginosa strains .

Inhibitory effect of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide (CL- 55) on the CTST of clinical antibiotic-resistant strains of Pseudomonas aeruginosa.

The inhibitory effect of CL-55 was studied in 2 tests: 1) in vitro upon CCTT induction by a decrease in calcium content and determination of the ExoT effector protein by immunoblot; 2) by determining cytotoxicity for CHO cells.

To assess the inhibitory activity of CL-55 by immunoblot, P. aeruginosa strains were grown in LB liquid medium in the presence of an EGTA inducer (5 mM) and with the addition of different concentrations of the chemical compound CL-55. After growing the culture for 3 hours, the bacterial cells were besieged by centrifugation. Protein concentration, electrophoresis, and staining of nitrocellulose filters with antibodies to the ExT protein were carried out in accordance with the procedure described above.

It was shown that CL-55 dose-dependently suppresses translocation of the CTST effector protein P. aeruginosa ExT under in vitro induction conditions. Inhibition was observed at concentrations of 150 and 200 μM (Fig. 3) in clinical strains characterized by both exoU ⁺ and exoS ⁺ genotype. Thus, in the in vitro test under the conditions of induction of CCT pseudomonas, the inhibitory effect of the low molecular weight compound CL-55 was shown.

The effect of the CTTT CL-55 inhibitor on the cytotoxicity of CHO cells was studied according to the method described above. The inhibitor was added in various concentrations to the suspension of bacterial cells and incubated for 30 min before the introduction of CHO cells.

When studying the cytotoxicity of freshly isolated P. aeruginosa bacterial strains on the CHO cell line, it was shown that the addition of the studied strains to the pseudomonads cells at a dose of 10 MOI caused strong cytotoxicity of the cells. Pre-incubation of bacteria with a CTTT inhibitor at doses of 100 and 150 μM reduced cell cytotoxicity to 0%. Table 4 shows the effect of the CL-55 type III secretion system inhibitor on CHO cells infected with P.aeruginosa strains with exoU ⁺ genotype. Table 5 shows the effect of an inhibitor of the CL-55 type III secretion system on CHO cells infected with P. aeruginosa strains with exoS ⁺ genotype.

Thus, it was shown that under the action of the CL-55 inhibitor on the studied strains of pseudomonas, their cytotoxicity associated with CTST was suppressed. In general, the results obtained indicate that the low molecular weight compound 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2- (2 , 4-difluorophenyl) -carboxamide (CL-55) specifically inhibits the secretion of toxins-effectors of the pseudomonas type III secretion system, causing the death of eukaryotic cells. Blocking the in vitro secretion system of type III Pseudomonas aeruginosa has been shown for clinical strains with different genotypes for the genes for effector proteins of CTST and a different antibiotic resistance profile.

Example 3

Toxicity study of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide (CL- 55) for animals.

Acute toxicity study of CL-55 low molecular weight type III secretion system inhibitor with a single administration to mice

Acute toxicity was studied in mice and rats (males and females) with intragastric and intraperitoneal routes of administration. The observation period was 14 days. Inhibitor animals were administered in increasing doses. Due to the low toxicity of the drug, LD ₅₀ was not established. The highest dose administered to mice and rats was 5,000 mg / kg, which is 100 times higher than the estimated daily therapeutic dose for humans. A single injection of the CL-55 Inhibitor in high doses does not cause death and toxic changes in the animal organism.

The results of toxicometry, observation of experimental animals for 14 days, as well as necropsy data indicate its good tolerance.

The study of subchronic toxicity of CL-55 low molecular weight type III secretion system inhibitor with repeated administration to animals

Subchronic toxicity studies were performed on sexually mature Wistar rats (males and females) and Soviet chinchilla rabbits (males and females). The drug was administered intragastrically for 14 days once a day in 2 doses: therapeutic (50 mg / kg) and ten times therapeutic (500 mg / kg). In the study of toxicity, generally accepted methods were used: integral and physiological indicators, studies of clinical and biochemical blood parameters, urinalysis, morphometric and histological evaluation of internal organs and tissues. Monitoring the condition of the animals was carried out daily. Physiological and hematological studies were carried out before administration, one day and 28 days after drug discontinuation, morphometric and histological studies were performed one day and 28 days later.

It was shown that repeated, within 14 days, the introduction of an antibacterial drug in the intended therapeutic dose, both to rats and rabbits, did not have a pronounced toxic effect on the function of the basic physiological systems of the body, metabolic processes, the blood system, structure and function of internal organs animals of both sexes.

When rats were given a dose of 500 mg / kg, negative changes were revealed: changes in the structure of the thymus tissue, a decrease in serum alanine aminotransferase, and the amount of sodium, creatinine, and urea. All pathological phenomena were reversible.

Studies in two species of animals of both sexes (rats and rabbits) showed that 14-day intragastric administration of the CL-55 inhibitor does not have a local irritant effect on the gastric mucosa.

The study of allergic and immunotoxic properties of CL-55, a low molecular weight type III secretion system inhibitor

Studies conducted in the scope of a comprehensive assessment of allergenicity (active skin anaphylaxis, delayed-type hypersensitivity, oral mast cell degranulation reaction) in guinea pigs showed that the CL-55 inhibitor, when administered at the intended therapeutic dose (50 mg / kg), does not have allergenic properties. At the same time, the introduction of a ten-fold therapeutic dose (500 mg / kg) leads to the development of immediate and delayed hypersensitivity.

The state of the immune system after administration of a ten-fold (500 mg / kg) and hundred-fold (5000 mg / kg) therapeutic dose of the CL-55 inhibitor was assessed by changes in the mass and cellularity of immunocompetent organs and in assessing the phagocytic activity of peritoneal macrophages.

According to the data obtained, it can be concluded that the studied drug in high doses does not affect the immune system.

Study of the mutagenic properties and carcinogenicity of CL-55, a low molecular weight type III secretion system inhibitor

The mutagenic activity and potential carcinogenicity of the CL-55 compound were studied in the Ames test, in the DNA comet test for DNA damage and in the test for the induction of chromosomal aberrations in mouse bone marrow cells.

The results of the experiments allow us to draw the following conclusions:

1. Compound CL-55 does not exhibit mutagenic activity on Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and on a combination of E. coli strains pKM101 and uvrA, under conditions with and without metabolic activation.

2. The compound CL-55 with a single intramuscular injection in doses of 20 and 200 mg / kg for a period of 3 and 18 hours does not induce DNA damage in the cells of the bone marrow, liver, kidneys and spleen of mice.

3. With a single intramuscular injection at doses of 20 and 200 mg / kg and with repeated administration at a dose of 20 mg / kg, compound CL-55 does not induce chromosomal aberrations in bone marrow cells of male and female mice.

According to generally accepted practice and regulatory documents, the totality of these observations indicates the absence of mutagenic and potential carcinogenic activity in compound CL-55.

Reproductive toxicity study

A study was conducted of the reproductive toxicity of an inhibitor of the CL-55 type III secretion system in animals. The substance was administered intragastrically before mating to rats for males for 56 days, females for 15 days at doses of 50 mg / kg and 500 mg / kg (equivalent to a therapeutic and ten times the therapeutic dose for humans). Then the experimental animals were mated with control females and males.

During the study, we studied: the sexual behavior of animals, the effect of the drug on the generative function of males and females, indicators of the physical development of offspring.

It was found that the CL-55 inhibitor, administered to males and females in a therapeutic dose, does not have a toxic effect on the generative function and indicators of embryonic death. When observing the development of offspring, there were no differences in the dynamics of body weight, survival and physical development of pups of the control and experimental groups.

The introduction of a CL-55 inhibitor in females at a dose exceeding the daily therapeutic by 10 times increases the pre-implantation death of the fetus. Long-term administration of the CL-55 inhibitor to males at a tenfold increased dose leads to a decrease in their spermatogenesis index, the number of spermatogonia in the tubules, and also to a decrease in sexual activity. Long-term use of the CL-55 inhibitor in high doses can lead to some impaired generative function.

Thus, a CL-55 type III secretion system inhibitor drug used in a therapeutic dose does not lead to a change in generative function.

As a result of toxicological studies, it was found that 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl ) -carboxamide (CL-55) is a low-toxic drug, which allows us to recommend a low molecular weight inhibitor of the CL-55 type III secretion system for further clinical trials.

Example 4

Effect of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide on the viability of bacteria in vitro and in vivo.

The mechanism of action of an inhibitor of type III secretion system involves the suppression of the effector function of P.aeruginosa. Moreover, the drug does not affect the viability of bacteria, i.e. does not have a direct bactericidal effect in vitro. The antibacterial effect of the drug CL-55 is based on a decrease in the virulence of the pathogen, as a result of the suppression of the secretion of effector proteins of pseudomonads, which are key pathogenicity factors with toxic activity. The action of an inhibitor of the secretion system helps to limit the development of infection and suppress toxic effects in the infected body. A significant advantage of the CCTT inhibitor over antibiotics is the absence of a negative effect on the normal human microflora.

Characterization of the effect of CL-55, an inhibitor of type III secretion system, on the viability of Pseudomonas aeruginosa

To study the effect of a type III secretion system inhibitor on the viability of P. aeruginosa, reference strains (from international and regional collections) of RA-103 were used; RAO; ARCC 27853 and freshly isolated strains (strains) of P.aeruginosa from samples of clinical material: No. / No. 12130-15 / 2015; 1625-36 / 2015; 19390-15 / 2015; 32461-15 / 2015; 41431-15 / 2015. The studies were carried out by the method of serial dilutions in a liquid nutrient medium, followed by seeding on solid nutrient media to count the grown colonies. For sowing, citrimide agar of the following composition was used: peptone - 20 g; agar - 13 g; magnesium chloride - 1.4 g; potassium sulfate - 10 g; citrimide - 0.3 g; glycerin - 10 ml; distilled water up to 1000 ml; pH 7.0-7.2.

Colony counting was performed according to the formula: N = C × K / n × V,

where N is the number of microorganisms in 1 ml; C is the sum of the colonies on all cups, n is the number of cups, K is the breeding from which the seeding was made; V is the volume of suspension taken for inoculation, ml.

A blue-green pigment P. aeruginosa was observed on citrimide agar. Next, a comparison was made when counting CFU / ml in control and experimental samples.

Preparation of working concentrations of CL-55 was carried out by the method of double dilutions. A liquid nutrient medium was used as a solvent. Liquid nutrient broth was poured into 0.5 ml in test tubes. 0.5 ml of a working solution inhibitor at a concentration of 200 mg / ml was added to the first tube. After thorough mixing, 0.5 ml was transferred with a measuring pipette from this tube into the second, the quantity of the mixture was mixed and transferred from the second to the third, etc. A test tube containing no drug served as a control for the growth of microorganism culture. After that, 0.2 ml of suspension of test microorganisms were added to all test tubes with a serially diluted preparation and to a control tube.

The results of the influence of an inhibitor of type III secretion system on the vitality of P. aeruginosa in vitro are presented in table 6.

When counting colonies grown on solid nutrient media after co-cultivation of P. aeruginosa bacterial strains with an inhibitor of the type III secretion system, it was shown that the inhibitor did not inhibit the growth of P. aeruginosa museum strains of bacteria using 3 different doses of the drug - 100, 50 and 25 mg / l. These doses were selected based on the results of preliminary experiments to determine the effective dose of an inhibitor with respect to suppressing the effector function of pseudomonas in vitro.

When studying the effect of the inhibitor on the viability of freshly isolated P. aeruginosa strains with different antibiotic resistance profiles, growth inhibition was not observed using the same inhibitor concentrations (Table 6). The data obtained are consistent with the mechanism of action of the drug being developed, which suppresses the virulence of pseudomonads, but does not affect their viability.

Thus, according to the mechanism of action, the CCTT inhibitor CL-55 does not inhibit the viability of Pseudomonas aeruginosa in vitro.

Study of the effect of the CTTT inhibitor CL-55 on the quantitative composition of the intestinal microflora of mice.

To study the effect of the inhibitor on normal microflora, the composition of the intestinal microflora of mice was quantified by the action of CL-55 and the reference drug antibiotic ciprofloxacin.

Method for determining the composition of intestinal microflora

The material for the study was feces after natural defecation, which was collected in a sterile airtight container with a wide neck and a tight-fitting lid, without preservative. After weighing 1 g of native feces, it was homogenized in 9 ml of physiological saline, obtaining the initial dilution of the material (10 ^-1 ). An additional 10-fold dilutions in physiological saline were made from the initial dilution. From dilutions, metered cultures were made on nutrient media for the cultivation of various groups of microorganisms. Crops were incubated at 37 ° C for 24-48 hours; plates with Saburo medium were left after that for another 2 days at room temperature. Lactobacilli were cultured in an incubator with 5% CO ₂ for 48 hours.

The following nutrient media were used in the work:

1) the environment of the ZhSA - vitelline salt agar for the detection of staphylococci;

2) Endo medium - for the isolation of enterobacteria, incl. lactobacillus Escherichia coli;

3) Saburo medium - agar for the isolation of yeast-like fungi of the genus Candida;

4) MRS medium - agar for the isolation of microaerophilic lactobacilli;

5) bifidum medium - semi-liquid agar for bifidobacteria;

6) blood agar - to detect hemolytic bacteria, including hemolytic E. coli;

7) enterococcal environment - to detect fecal streptococci;

8) Hektoen Enteric Agar - a medium for the detection of pathogenic enterobacteria.

Material from animals was incubated at 37 ° C for 24-48 hours.

The study of the quantitative composition of the intestinal microflora of mice with the introduction of CL-55 and antibiotic comparison ciprofloxacin

Balb / c mice received 2 preparations: CL-55 200 mg / kg 2 times a day for 5 days in a row enteral and antibiotic ciprofloxacin 100 mg / kg 2 times a day for 5 days in a row. Excrement was collected 3 times: before the start of drug administration, on the 15th and 28th day after the start of drug administration.

The experiments performed allowed us to obtain results on the change in the composition of the normal microflora of the large intestine of animals by 2 terms after the start of administration of the studied drugs: 200 mg / kg CL-55 and 100 mg / kg ciprofloxacin. The results of the quantitative composition of the intestinal microflora of mice prior to the introduction of CL-55 and ciprofloxacin are presented in table 7.

The experiments on a comparative assessment of the quantitative and qualitative composition of the intestinal microflora under the action of the studied drug CL-55 and the antibiotic ciprofloxacin indicate that the use of CL-55 at a dose of 100 mg / kg twice a day enteral for 5 days did not have a negative effect. bactericidal action on the normal intestinal microflora of animals. Table 8 presents the results of a study of the quantitative composition of the intestinal microflora of mice on the 15th day after the start of administration of CL-55 and ciprofloxacin.

The results of the study showed that the number of studied components of obligate microflora: epidermal staphylococcus, lactobacillus E. coli, lactobacilli, bifidobacteria, fecal streptococcus, during microbiological examination of feces before using CL-55, one week after the end and three weeks after the end of the drug practically remained on the same level. Table 9 presents the results of a study of the quantitative composition of the intestinal microflora of mice on the 28th day after the start of administration of CL-55 and ciprofloxacin.

On the contrary, the use of ciprofloxacin at a dose of 100 mg / kg 2 times a day for 5 days enterally led to a decrease in the number of lactobacilli from 10 ⁸ CFU / g to 10 ⁵ CFU / g and 10 ⁹ CFU / g to 10 ⁶ CFU / g, lactose-positive E. coli from 10 ⁸ CFU / g to 10 ⁶ CFU / g 15 days after the start of treatment. On the 28th day of the experiment in this group of mice with the introduction of ciprofloxacin, an increase in the number of lactobacilli and bifidobacteria by 3 orders of magnitude from 10 ⁵ to 10 ⁸ was noted compared with the previous period of 15 days. The level of lactose-positive Escherichia coli in this group fully recovered to the initial level of 10 ⁸ CFU / g.

A statistical analysis of the quantitative composition of the intestinal microflora of mice at the same time using the non-parametric Kruskal-Wallis test showed a significant difference for all analyzed groups. When analyzing the dynamics of the microflora composition indicator at different periods using the non-parametric Friedman criterion, a significant difference was established between the period before drug administration and 15 and 28 days for all groups of animals (p <0.05).

Thus, according to the results of the study, we can conclude that the studied CTBT inhibitor 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazine- 2- (2,4-Difluorophenyl) -carboxamide (CL-55), in accordance with its mechanism of action, does not have a bactericidal effect on the intestinal microflora of animals. The absence of a negative effect on the composition of the intestinal microflora of the CCTT inhibitor, which is promising for the treatment of antibiotic-resistant hospital strains of Pseudomonas aeruginosa, is its principal advantage over the antibiotics used.

Example 5

Determination of an effective inhibitory dose of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide on laboratory animals

The determination of the effective therapeutic dose of the CSTT inhibitor was carried out with the oral route of administration of the drug. Strains of P. aeruginosa.

Table 10 shows the characteristics of the clinical strains of P. aeruginosa used in examples 5, 6 and 7.

To simulate sepsis induced by antibiotic-resistant clinical strains of P. aeruginosa, inbred mice A / JsnYCit (A / Sn) mice supported in the nursery of FSBI “FNITsEM im. N.F. Gamalei "of the Ministry of Health of the Russian Federation in accordance with the requirements for the maintenance of animals of the Ministry of Health of the Russian Federation No. 775. In the experiments used males weighing 18-20 g at the beginning of the experiment; each group for the study consisted of 6 animals. All studies were performed in duplicate.

Keeping animals

Animals were kept under standard conditions in the design, equipment and maintenance of experimental biological clinics (vivariums).

Preparing the culture for infection. Preparation of the bacterial culture for infection was carried out as follows: the bacterial culture was grown on Luria-Bertani liquid medium (LB broth) from DIFCO, USA, at 37 ° C for 20 hours. After 20 hours, the titer of the culture was calculated by tenfold dilutions on a solid nutrient medium. For infection, the culture was diluted with saline to the required concentration in accordance with the titer.

Infection. To create an experimental septic Pseudomonas aeruginosa infection, mice were injected intraperitoneally prepared as described above with P. aeruginosa cultures in various doses. The volume of the introduced culture was 0.5 ml per mouse. The number of mice in each group was 6 animals. After infection, the life expectancy of mice was monitored daily (criterion is mortality). The number of bacteria sown from the organs of infected animals was also determined. Seeding material for determining the number of pseudomonads in septic infections was abdominal swabs (peritonial lavage), spleen, and blood.

Method for determining the dynamics of animal death. Every day after infection, 2 times a day, morning and evening, the state of the animals was monitored and the death periods of the experimental animals were recorded.

Method for determining the number of bacteria in the blood and organs of infected animals

Mice surviving the infection were euthanized by cervical dislocation. Blood was collected by cardiac puncture and placed in test tubes containing 100 units of sodium heparin. Peritoneal washings were obtained by introducing 5 ml of sterile saline into the abdominal cavity. The spleens were homogenized using a SilentcrusherM homogenizer in 1 ml of physiological saline, the resulting homogenates were centrifuged for 10 min at 800 rpm (5415 D Eppendorf centrifuge) to remove coarse tissue residues. Then, a series of 10-fold dilutions of the initial suspension of spleen homogenates in physiological saline was prepared, and 500 μl of each dilution was placed on a Petri dish coated with citramide agar. A series of 10-fold dilutions in a physiological solution of blood samples and peritonial lavages was prepared in a similar manner. Petri dishes were incubated at 37 ° C and colony counts were performed after 24 hours of cultivation. The concentration of pseudomonads in organs was expressed by the number of CFU in 1 ml.

Determination of the effective dose for oral administration of CL-55 in a septic infection model

The effective dose of CL-55 was determined on the developed model of septic infection. For infection, clinical strains with different genotypes for CTTT effectors (exoU ⁺ or exoS ⁺ ) were used. Infection of mice was performed intraperitoneally in accordance with the method described above. Doses for infection were selected based on the results obtained for the determination of LD ₅₀ and LD ₁₀₀ for each strain.

Oral administration of the drug CL-55. When the drug was administered orally, an atraumatic metal probe (the so-called gavage needle) was used, which was inserted into the esophagus before the stomach. The substance of the drug was thoroughly ground in a porcelain mortar with a minimum amount of Tween-80. Then gradually, with vigorous stirring, small portions of 1% starch paste were added and triturated to a homogeneous mass. Control animals received equivalent volumes of Tween-80 starch paste.

For the experiment, 4 hospital strains of exoS ⁺ and exoU ⁺ types with a different antibiotic resistance profile were selected.

Design experiments for 4 strains

Groups of 6 mice each were formed. The studies were performed in duplicate.

Group 1 - infection control

Group 2 - infected mice +150 mg / kg CL-55

Group 3 - infected mice +100 mg / kg CL-55

Group 4 - infected mice +50 mg / kg CL-55

Group 5 - uninfected mice +150 mg / kg CL-55

6 group - uninfected mice +100 mg / kg CL-55

7 group - uninfected mice +50 mg / kg CL-55

The introduction of the drug for 3 days 1 time per day.

Monitoring of the development of infection was carried out according to the dynamics of the death of animals within 7 days after infection. The possible negative effect of the drug CL-55 was determined with the introduction of the drug in 3 concentrations to uninfected mice.

No deaths were observed with the administration of 150 mg / kg, 100 mg / kg and 50 mg / kg CL-55 to non-infected animals. Table 11 presents the results of the death and survival of animals by day with an intraperitoneal infection with a dose of 4 × 10 ⁶ CFU / mouse of the clinical strain KB6 / 6/2014 exoS ⁺ and those who received CL-55 at doses of 150 mg / kg, 100 mg / kg and 50 mg / kg for 3 days orally. Data are given for experiments in 2 repetitions.

Table 12 presents the results of the death and survival of animals by day with an intraperitoneal infection with a dose of 2 × 10 ⁶ CFU / mouse of the clinical strain 693/6/2014 exoS ⁺ and receiving CL-55 at doses of 150 mg / kg, 100 mg / kg and 50 mg / kg for 3 days orally. Data are given for experiments in 2 repetitions.

Table 13 presents the results of the death and survival of animals by days with an intraperitoneal infection with a dose of 1 × 10 ⁶ CFU / mouse of the clinical strain 1840/36/2015 exoU ⁺ and receiving CL-55 at doses of 150 mg / kg, 100 mg / kg and 50 mg / kg for 3 days orally. Data are given for experiments in 2 repetitions.

Table 14 presents the results of the death and survival of the animals by day with an intraperitoneal infection with a dose of 1 × 10 ⁶ CFU / mouse of the clinical strain 1625/36/2015 exoU ⁺ and receiving CL-55 at doses of 150 mg / kg, 100 mg / kg and 50 mg / kg for 3 days orally. Data are given for experiments in 2 repetitions.

Thus, the oral administration of the drug CL-55 in doses of 50 mg / kg for 3 days 1 time per day led to a slight increase in survival by 1.5 times. The introduction of CL-55 at a dose of 100 mg / kg led to a pronounced decrease in mortality, 2 times. The introduction in the same regime of the drug CL-55 at a dose of 150 mg / kg markedly reduced the mortality of animals in groups infected with each of the strains: for infected KB6 / 6/2014 - 2.3 times; for infected 693/6/2014 - 2 times; for infected 1840/36/2015 - 1.6 times; for those infected with strain 1625/36/2015, the survival rate of CL-55 mice was 92%, while all infected mice died without treatment.

In order to evaluate the inhibition of pseudomonas infection in the organs of infected mice, a septic infection was simulated, as described above. Infection was carried out not with a lethal, but with an infectious dose of 5 × 10 ⁴ CFU / mouse of strain KB6 / 6/2014, which led to the development of pseudomonas infection in the organs, but not the death of animals during the observation period for 4 days. CL-55 infected mice were administered orally at a dose of 100 mg / kg for 3 days 1 time per day, which led to a complete suppression of infection in the studied biological samples.

Table 15 shows the data on the determination of the average amounts of pseudomonas in seeding from animal organs, when infected with a non-lethal dose of strain KB6 / 6/2014, in groups of animals treated with CL-55 and without treatment.

Thus, on the basis of the experiments, it was shown that with the oral administration of CL-55 for three days at a dose of 100 mg / kg per day, the accumulation of pseudomonas was suppressed in peritoneal lavage, blood and spleen, i.e. an effective antibacterial effect of the CTTT inhibitor has been shown.

Thus, the minimum effective dose or pharmacologically active dose for CL-55 was 100 mg / kg for mice. In a model of lethal septic infection caused by antibiotic-resistant clinical strains, using all the tested doses, a 1.5–4-fold decrease in mortality in mice was noted, which indicates the possibility of achieving a more effective therapeutic effect of the drug when changing the treatment regimen (changing the timing and frequency of drug administration )

Example 6

The study of the therapeutic efficacy of the CTTT inhibitor in a model of experimental infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa of two different CTTT genotypes.

The study of the therapeutic efficacy of the obtained CL-55 inhibitor was carried out on a model of septic infection in mice and used the methods of accumulation of pseudomonads and work with laboratory animals described in Example 5.

Mice were infected with strains of pseudomonads of two different genotypes exoU ⁺ and exoS ⁺ . The efficacy of CL-55 was evaluated by the dynamics of animal death within 4 days after infection and by the antibacterial effect of CL-55 in the blood and spleen 24, 48 and 72 hours after infection using the bacteriological method of seeding.

Design experiment with exoU ⁺ strain

Groups of 6 mice each were formed.

Group 1 - infection control with a dose of 2.5 × 10 ⁶ CFU / mouse (LD ₇₅ ) of strain 1840/36/2015 exoU ⁺

Group 2 - infection + treatment with CL-55 dose of 100 mg / kg 1 time 3 days

Group 3 - infection + treatment with a dose of CL-55 50 mg / kg 2-fold 3 days

Table 16 shows the results of therapeutic efficacy of CL-55 in animal survival days after infection with a dose of 2.5 × 10 ⁶ CFU / mouse (LD ₇₅ ) strain 1840/36/2015 exoU ⁺ (data for 2 repetitions, in each group of 6 animals).

It was shown that with a single injection of CL-55 at a dose of 100 mg / kg for 3 days after infection, a decrease in animal death was observed from 75% (in control) to 25%. An increase in the frequency of administration of the drug with a dose of 50 mg / kg up to 2 times a day, within 3 days after infection, increased the effectiveness of treatment, more than 90% of the animals survived.

In order to evaluate the inhibition of pseudomonas infection in the organs of infected mice after therapeutic administration of CL-55, the following experiment was performed.

Design experiment with exoU ⁺ strain

Groups of 6 mice each were formed.

Group 1 - infection control with a dose of LD ₅₀ (1 × 10 ⁶ CFU / mouse) strain 1840/36/2015

Group 2 - infection + treatment with CL-55 with a dose of 100 mg / kg 1-fold for 3 days

3 group - infection + treatment with CL-55 dose of 50 mg / kg 2-fold for 3 days

Mice were infected with a dose of LD ₅₀ (1 × 10 ⁶ CFU / mouse) of strain 1840/36/2015. Treatment began on the day of infection. Autopsies were performed 24, 48, and 72 hours after infection for surviving mice. Table 17 shows the results of microbiological isolation and determination of the average number of pseudomonas in the seeding from spleens and blood of mice infected with a dose of LD ₅₀ (1 × 10 ⁶ CFU / mouse) of strain 1840/36/2015 during treatment with CL-55 at a dose of 100 mg / kg once.

The results showed that treatment according to the scheme of 100 mg / kg once a day for 3 days leads to a decrease in the reproduction of Pseudomonas aeruginosa in both the spleen and blood. Moreover, the greatest difference in reducing the bacterial load was observed starting from 48 hours after infection for both blood and spleen.

Table 18 shows the results of determining the average number of pseudomonas in the plaque from the spleens and blood of mice infected with a dose of LD ₅₀ (1 × 10 ⁶ CFU / mouse) strain 1840/36/2015 during treatment with CL-55 at a dose of 50 mg / kg twice .

Treatment according to the scheme of 50 mg / kg 2 times a day for 3 days showed effective inhibition of pseudomonas multiplication by 48 and 72 hours after infection. By these dates, bacteria were almost completely absent in the spleen and blood.

The suppression of pseudomonas infection in the organs of infected mice using the established treatment regimen was also evaluated when infected with strain KB6 / 6/2014 exoS ⁺ genotype.

ExoS ⁺ strain experiment design

Groups of 6 mice each were formed.

Group 1 - infection control with a dose of LD ₁₀ (1 × 10 ⁶ CFU / mouse) of strain KB6 / 6/2014

Group 2 - infection + treatment with CL-55 dose of 50 mg / kg 2-fold for 3 days

Mice were infected with a dose of LD ₁₀ (1 × 10 ⁶ CFU / mouse) of strain KB6 / 6/2014. Treatment began on the day of infection. An autopsy was performed 72 hours after infection for surviving mice.

Table 19 presents the results of determining the average number of pseudomonas in the seeding from spleens and blood of mice infected with a dose of LD ₁₀ (1 × 10 ⁶ CFU / mouse) of strain KB6 / 6/2014 during treatment with CL-55 at a dose of 50 mg / kg twice .

Thus, the therapeutic efficacy of the drug CL-55, a low molecular weight CTTT inhibitor of pseudomonas, was shown. As an effective treatment regimen, treatment was chosen at a dose of 50 mg / kg 2 times a day for 3 days after infection. Treatment according to this scheme led to a decrease in mouse death by 91.7% when infected with pseudomonads with a dose equal to LD ₇₅ and 100% survival when infected with a dose of LD ₅₀ and LD ₁₀ . Evaluation of the antibacterial effect of the drug CL-55 revealed a decrease in the accumulation of the pathogen in the blood and spleen 1 day after the start of treatment. After 48 and 72 hours, the pathogen was almost completely absent in the spleen and blood of the treated mice. Therapeutic efficacy of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide inhibitor was established on a model of experimental infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa of two different CTST genotypes.

Example 7

Study of the prophylactic efficacy of a CTTT inhibitor in a model of experimental infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa of two different CTTT genotypes

The study of the prophylactic efficacy of the obtained CL-55 inhibitor was carried out on a model of septic infection in mice and the methods of accumulation of pseudomonads and work with laboratory animals described in Example 5 were used.

The drug was administered to laboratory animals orally for 3 days before infection. These dates were selected on the basis of primary pharmacokinetic studies, which indicate that after oral administration of CL-55, it accumulates in well-vasculated organs such as the liver, spleen, and lungs. Mice were infected with strains of pseudomonads of two different genotypes, exoS ⁺ and exoU ⁺ .

Experiment design for exoS ⁺ genotype

Groups of 6 mice each were formed.

Group 1 - infection control during infection with a dose of 4 × 10 ⁶ CFU / mouse (LD ₅₀ ) strain KB6 / 6/2014

Group 2 - prevention of CL-55 dose of 150 mg / kg 3 days + infection

Group 3 - prevention of CL-55 with a dose of 100 mg / kg 3 days + infection

Group 4 - prevention of CL-55 dose of 50 mg / kg 3 days + infection

The effectiveness of the preventive action of CL-55 was evaluated by the dynamics of the death of animals within 4 days after infection.

Table 20 shows the results of studying the effectiveness of prophylaxis with various doses of CL-55 for 3 days — animal survival by days after infection with a dose of 4 × 10 ⁶ CFU / mouse (LD ₅₀ ) strain KB6 / 6/2014 exoS ⁺ (data for 2 repetitions , in each group 6 animals). The results of the study show the high prophylactic efficacy of oral administration of CL-55 for 3 days at doses of 150 and 100 mg / kg in relation to the survival of animals infected with a dose of LD _{50 of} strain KB6 / 6/2014.

Experiment design for exoS ⁺ genotype

Groups of 6 mice each were formed.

Group 1 - infection control during infection with a dose of 1 × 10 ⁷ CFU / mouse (LD ₁₀₀ ) strain KB6 / 6/2014

Group 2 - prevention of CL-55 dose of 150 mg / kg 3 days + infection

Group 3 - prevention of CL-55 with a dose of 100 mg / kg 3 days + infection

Group 4 - prevention of CL-55 dose of 50 mg / kg 3 days + infection

When infected with a dose of LD _{100 of} strain KB6 / 6/2014, the survival rates of animals after prophylactic administration of CL-55 for 3 days at doses of 150 and 100 mg / kg were lower, however, against 100% death of animals in the control groups 67 and 58 % of animals survived for doses of 150 and 100 mg / kg, respectively. Table 21 shows the results of a study of the effectiveness of prophylaxis with doses of CL-55 of 150 and 100 mg / kg for 3 days, followed by infection of mice with a dose of 1 × 10 ⁷ CFU / mouse (LD ₁₀₀ ) of strain KB6 / 6/2014 — animal survival by days after infection (data for 2 repetitions, in each group of 6 animals).

In order to evaluate the inhibition of pseudomonas infection in the organs of infected mice after prophylactic administration of CL-55, the following experiment was performed.

Experiment design for exoS ⁺ strain

Groups of 6 mice each were formed.

Group 1 - infection control during infection with LD ₅₀ (4 × 10 ⁶ CFU / mouse) strain KB6 / 6/2014

Group 2 - prevention of CL-55 with a dose of 100 mg / kg 24 hours before infection

Group 3 - prevention of CL-55 with a dose of 100 mg / kg for 2 days 48 hours before infection

Group 4 - prevention of CL-55 with a dose of 100 mg / kg for 3 days 72 hours before infection

Mice were infected with a dose of LD ₅₀ (4 × 10 ⁶ CFU / mouse) of strain KB6 / 6/2014 after completion of CL-55 prophylaxis. Autopsy was performed 24 hours after infection. Table 22 shows the results of determining the average number of pseudomonas in seeding from spleens of mice infected with a dose of LD ₅₀ (4 × 10 ⁶ CFU / mouse) of strain KB6 / 6/2014 after prophylactic administration of CL-55 at a dose of 100 mg / kg in 24 hours, 48 hours and 72 hours before infection.

Thus, even one prophylactic dose of 100 mg / kg CL-55 has a pronounced antibacterial effect and leads to a 50% reduction in the number of pseudomonads in the spleen compared with the group without prophylaxis. An increase in the frequency of administration of prophylactic doses of CL-55 to 3 times leads to almost complete suppression of the accumulation of the pathogen in the spleen.

A study was conducted of the effectiveness of the prophylactic regimen for the administration of a type III secretion system inhibitor, CL-55 drug, when administered orally, to septic pseudomonas infection caused by Pseudomonas aeruginosa ExoU phenotype when infected with doses of LD ₅₀ and LD ₇₅ .

Design experiment with exoU ⁺ strain

Groups of 6 mice each were formed.

Group 1 - infection control during infection with a dose of 1.15 × 10 ⁶ CFU / mouse (LD ₅₀ ) strain 1840/36/2015

Group 2 - prevention of CL-55 dose of 150 mg / kg 3 days + infection

Group 3 - prevention of CL-55 with a dose of 100 mg / kg 3 days + infection

The effectiveness of the preventive action of CL-55 was evaluated in an acute septic model according to the dynamics of animal death within 4 days after infection.

Table 23 shows the results of a study of the effectiveness of prophylaxis with various doses of CL-55 for 3 days — animal survival by days after infection with a dose of 1.15 × 10 ⁶ CFU / mouse (LD ₅₀ ) strain 1840/36/2015 exoU ⁺ (data for 2 repetitions, in each group of 6 animals). The data obtained show the prophylactic efficacy of oral administration of CL-55 for 3 days at doses of 150 and 100 mg / kg in relation to the survival of animals infected with a dose of LD ₅₀ strain 1840/36/2015.

Design experiment with exoU ⁺ strain

Groups of 6 mice each were formed.

Group 1 - infection control during infection with a dose of 2.25 × 10 ⁶ CFU / mouse (LD ₇₅ ) strain 1840/36/2015

Group 2 - prevention of CL-55 dose of 150 mg / kg 3 days + infection

Group 3 - prevention of CL-55 with a dose of 100 mg / kg 3 days + infection

The effectiveness of the preventive action of CL-55 was evaluated in an acute septic model according to the dynamics of animal death within 4 days after infection.

Table 24 shows the results of a study of the effectiveness of prophylaxis with doses of CL-55 of 150 and 100 mg / kg for 3 days, followed by infection of mice with a dose of 2.25 × 10 ⁶ CFU / mouse (LD ₇₅ ) of strain 1840/36/2015 exoU + - animal survival days after infection (data for 2 repetitions, in each group of 6 animals). The data obtained showed that when infected with a dose of LD _{75 of} strain 1840/36/2015 exoU ^{+, the} survival of animals after prophylactic administration of CL-55 for 3 days at doses of 150 and 100 mg / kg was also significantly higher.

In order to evaluate the inhibition of pseudomonas infection in the organs of infected mice after prophylactic administration of CL-55, the following experiment was performed.

Design experiment with exoU ⁺ strain

Groups of 6 mice each were formed.

Group 1 - infection control during infection with a dose of LD ₅₀ (1.15 × 10 ⁶ CFU / mouse)

Group 2 - prevention of CL-55 with a dose of 150 mg / kg for 3 days before infection

Group 3 - infection control during infection with a dose of LD ₇₅ (2.25 × 10 ⁶ CFU / mouse)

Group 4 - prevention of CL-55 with a dose of 150 mg / kg for 3 days before infection

Mice were infected with doses of LD ₅₀ (1.15 × 10 ⁶ CFU / mouse) and LD ₇₅ (2.25 × 10 ⁶ CFU / mouse) of strain 1840/36/2015 exoU + after completion of CL-55 prophylaxis. 72 hours after infection, the surviving mice were autopsied and the number of bacteria in the spleen was determined. Table 25 summarizes the results obtained by determining the average number of pseudomonas in seeding from spleens of mice infected with doses of LD ₅₀ (1.15 × 10 ⁶ CFU / mouse) and LD ₇₅ (2.25 × 10 ⁶ CFU / mouse) strain 1840/36 / 2015 exoU ⁺ after prophylactic administration of CL-55 at a dose of 150 mg / kg 72 hours before infection.

Thus, the prophylactic use of CL-55 at a dose of 150 mg / kg has a pronounced antibacterial effect and leads to an almost complete decrease in the number of pseudomonads in the spleen when mice are infected with clinical strains with exoS ⁺ and exoU ⁺ genotypes, regardless of acquired resistance.

Conclusion

In the examples presented, 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] -thiadiazin-2- (2 , 4-difluorophenyl) -carboxamide, is an inhibitor of the CTST Pseudomonas aeruginosa. The resulting compound specifically inhibits the secretion of toxins - effectors of the secretion system of type III pseudomonads, causing the death of eukaryotic cells. Blocking the in vitro secretion system of type III Pseudomonas aeruginosa has been shown for clinical strains with different genotypes for the genes for effector proteins of CTST and a different antibiotic resistance profile.

A low molecular weight inhibitor of CTST pseudomonas caused a suppression of the development of infection in laboratory animal models using both a therapeutic and a prophylactic regimen for drug administration. The action of the drug was expressed in a significant increase in animal survival and inhibition of the accumulation of pseudomonads in organs during septic infection in mice. The therapeutic and prophylactic efficacy of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide was established on a model of experimental infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa of two different CTST genotypes. Thus, the technical task set in this invention is achieved.

In addition, toxicity studies of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) ) -carboxamide in animals has shown that the compound is low toxic and can be recommended for further preclinical and clinical trials. According to the results of the study, it can also be concluded that the studied CTBT inhibitor 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] -thiadiazin-2 - (2,4-difluorophenyl) -carboxamide (CL-55), in accordance with its mechanism of action, does not have a bactericidal effect on the intestinal microflora of animals. The absence of a negative effect on the composition of the intestinal microflora of the CCTT inhibitor, which is promising for the treatment of antibiotic-resistant hospital strains of Pseudomonas aeruginosa, is its principal advantage over the antibiotics used.

Thus, the task, namely:

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

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

- the absence of an 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 to suppress infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa is solved in this invention.

Industrial applicability.

The results of the studies reflected in the examples confirm the industrial applicability of this compound, which specifically inhibits the secretion of toxins - effectors of the pseudomonas type III secretion system, causing the death of eukaryotic cells.

Information sources

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2. Burgess DS1, Nathisuwan S. Cefepime, piperacillin / tazobactam, gentamicin, ciprofloxacin, and levofloxacin alone and in combination against Pseudomonas aeruginosa. Diagn Microbiol Infect Dis. 2002 Sep; 44 (1): 35-41.

3. Bodey GP, Jadeja L, Elting L. Pseudomonas bacteremia. Retrospective analysis of 410 episodes. 1985Arch. Intern. Med. 145: 1621-1629].

4. Hilf M, et al. Antibiotic therapy for Pseudomonas aeruginosa bacteremia: outcome correlations in a prospective study of 200 patients. 1989. Am. J. Med. 87: 540-546.

5. Leibovici L, et al .. Monotherapy versus beta-lactamaminoglycoside combination treatment for gram-negative bacteremia: a prospective, observational study. 1997, Antimicrob. Agents Chemother. 41: 1127-1133.

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Claims (2)

1. The use of 4- (3-ethoxy-4-hydroxybenzyl) -5-oxo-5,6-dihydro-4H- [1,3,4] thiadiazin-2- (2,4-difluorophenyl) carboxamide for the suppression infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa in a living organism. 2. A method of suppressing an infection caused by antibiotic-resistant strains of Pseudomonas aeruginosa, comprising exposing the strains of Pseudomonas aeruginosa to an effective amount of a compound according to claim 1.

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