RU2447896C1 - Antimicrobial agent - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to antimicrobial agents preferentially used for treating the diseases caused by antibiotic resistant bacteria, and preventing the developing drug resistance in pathogenic microorganisms. A substance contains a purified complex of antimicrobial peptides of insects of the family Calliphoridae, which contains defensin described by the amino acid sequence Ala-Thr-Cys-Asp-Leu-Leu-Ser-Gly-Thr-Gly-Ala-Asn-Hys-Ser-Ala-Cys-Ala-Ala-Hys-Cys-Leu-Leu-Arg-Gly-Asn-Arg-Gly-Gly-Tyr-Cys-Asn-Gly-Lys-Ala-Val-Cys-Val-Cys-Arg-Asn, a proline-rich peptide described by the N-terminal amino acid sequence Phe-Val-Asp-Arg-Asn-Arg-Ile-Pro-Arg-Ser-Asn-Asn-Gly-Pro-Lys-Ile-Pro-Ile-Ile-Ser-Asn-Pro-, cecropin described by the amino acid sequence Gly-Trp-Leu-Lys-Lys-Ile-Gly-Lys-Lys-Ile-Glu-Arg-Val-Gly-Gln-Hys-Thr-Arg-Asp-Ala-Thr-Ile-Gln-Gly-Leu-Ala-Val-Ala-Gln-Gln-Ala-Ala-Asn-Val-Ala-Ala-Thr-Ala-Arg, diptericin described by the N-terminal amino acid sequence Asp-Ser-Lys-Pro-Leu-Asn-Leu-Val-Leu-Pro-Lys-Glu-Glu-Pro-Pro-Asn-Asn-Pro-Gln-Thr-Tyr-Gly-Gly-Gly-Gly-Gly-Ser-Arg-Lys-Asp-Asp-Phe-Asp-Val-Val-Leu-Gln-Gly-Ala-Gln.

EFFECT: advantages of the antimicrobial agent involve the fact that bacteria develop no resistance to the presented substance if used continuously, also when used delays the developing antibiotic resistance of the bacteria and shows bactericidal action on strains of acquired drug resistance.

1 cl, 6 tbl, 8 dwg

Description

The present invention relates to antimicrobial agents and can be used for the treatment and prevention of infectious diseases of humans and animals. The preferred field of application of the invention is the treatment of diseases caused by antibiotic-resistant bacteria and the prevention of drug resistance in pathogenic microorganisms.

In medicine and veterinary medicine, a wide variety of bacteria-toxic antibiotics are used that belong to different classes of low molecular weight organic compounds (beta-lactams, macrolides, tetracyclines, fluorocquinolones, sulfonamides, aminoglycosides, imidazoles, etc.). Antimicrobial peptides of animal origin are also known, which are proposed to be used to treat bacterial infections. In particular, it is known to use human defensins [1–7], retrocyclins [8], virgizin, oysterisin oyster and scorpion Gibbosin [9], annelid defensins [10], derivatives of melanocyte stimulating hormone [11], ubiquicidin derivatives [12], magainin [13], mucin derivatives [14], analogues of theta-defensins [15], crustacean antimicrobial peptides [16], cryptdine [17]. In addition, the use of synthetic cationic peptides as antimicrobial agents is known [18, 19, 20]. Antimicrobial peptides of insects are known that are proposed to be used for treating bacterial infections: the bacteriolytic peptide of the lepidopteran Hyalophora cecropia [21], the antibacterial peptide of gloverin [22], the antibacterial peptides of the beetle Oryctes rhinoceros [23], and the defensin-like dragonfly peptide Aeschnaane [24] cyan.

In recent years, the situation in the field of treatment of bacterial infections has become much more complicated due to the wide spread of “superbugs” that are resistant to most or all known antibiotics [25, 26]. Of particular danger is the spread of antibiotic-resistant nosocomial (hospital) infections in hospitals, where they constitute the main cause of infectious complications and related mortality [27]. The treatment of such infections with a modern arsenal of antibiotics is ineffective or impossible. If a solution to this problem is not found, mortality from bacterial infections in humans and farm animals in the foreseeable future may return to the level of the 19th century.

To solve this problem, it is proposed to use methods for preventing the development of drug resistance in bacteria, presented by recommendations for the rational use of antibiotics [28].

Methods are also known for overcoming bacterial resistance to antibiotics by introducing additional components that block the pathogen's defense systems into the drug. In particular, the combined use of the antibiotic cefepime from the group of beta-lactams and sulbactam is known [29]. Sulbactam is an inhibitor of the beta-lactamase enzyme and prevents the inactivation of the antibiotic by the bacterial enzyme. There is a proposal to use milk protein lactoferrin or its metabolite lactoferricin as a beta-lactamase inhibitor to increase the sensitivity of beta-lactam antibiotics to forms of bacteria resistant to them [30]. The use of lysozyme dimer to increase sensitivity to antibiotics of the methicillin-resistant form of Staphylococcus aureus Staphilococcus aureus is also known [31]. Lysozyme dimer is used as a synergist, enhancing the toxic effects of antibiotics gentamicin, clindamycin or co-trimoxazole.

A method has been proposed for preventing the development of drug resistance by blocking the transmission of plasmids from one bacterium to another carrying the antibiotic resistance genes [32]. Blocking the transmission of plasmids in this case is achieved by introducing into the composition of the drug fragments of the cell wall of the yeast.

The methods listed and similar to them contribute to solving certain aspects of the problem of bacterial resistance to antibiotics, but the problem as a whole remains unresolved at this stage in the development of science.

The main way to overcome multidrug resistance, used in practical medicine, remains the treatment of bacterial infections with the help of an empirically found combination of two or more antibiotics of different structure and mechanism of action. The literature describes various schemes of combination antibiotic therapy [33]. It is assumed that a combination of antibiotics increases the effectiveness of therapy and prevents the development of bacterial resistance. However, such complication and appreciation of the treatment regimen does not always give a positive effect. Often, the negative properties of each antibiotic are summarized (toxicity and other side effects), often one component of the combination suppresses, rather than enhances, the effect of the other component [34]. It was experimentally shown that the use of a combination of antibiotics can provoke the development of resistance even more than the use of each of them separately and contributes to the formation of the most dangerous multi-resistant bacterial strains [35, 36].

Thus, further searches for approaches to the rational treatment of bacterial infections remain one of the main tasks of pharmacology and the health system as a whole. Within the framework of this global task, the development of agents effective against antibiotic-resistant bacteria and the search for drugs that impede the emergence of new drug-resistant forms are most relevant.

Closest to the claimed antiviral substance is the bacteriolytic peptide of the insect Hyalophora cecropia [21], adopted as a prototype. A disadvantage of the known substance is the narrow spectrum of its antimicrobial activity (it acts only on certain types of gram-negative bacteria). Another disadvantage of the prototype is the fact that it contains only one active substance, thereby opening up opportunities for the development of resistance to it in microorganisms.

SUMMARY OF THE INVENTION

The technical result of the invention is to significantly increase the effectiveness of the fight against bacterial infections of humans and animals, to prevent the emergence of new antibiotic-resistant forms of microorganisms.

The specified technical result is achieved by the fact that the claimed antimicrobial substance includes a purified complex of defensins, cecropins, diptericins and proline-rich peptides of the insects of the Calliphoridae family.

In addition, the specified technical result is achieved by the fact that the claimed antimicrobial substance contains the peptide SEQ ID No. 1, or SEQ ID No. 2, or SEQ ID No. 3, or SEQ ID No. 4.

In addition, this technical result is achieved in that the claimed antimicrobial substance is obtained from insects of the species Calliphora vicina R.-D, or insects of the species Calliphora vomitoria L, or insects of the species Lucilia sericata Meig.

The specified technical result is also achieved by a pharmaceutical composition based on the claimed antimicrobial substance.

Like well-known methods of combined antibiotic therapy, the proposed approach involves the simultaneous exposure of two or more components to a bacterial cell, which differ in structure and mechanism of action. The main difference is that the proposed approach involves the use of a combination of active substances encoded by the genes of one species or closely related species of multicellular organisms. Compared with the currently used method of empirical selection of combinations of antibiotics, the proposed approach has significant advantages, since such a combination forms a co-adapted complex of active substances. By coadapted complex in the context of the present invention is meant a combination of two or more antimicrobial peptides formed during biological evolution and optimized by natural selection with respect to the compatibility of its components. In this case, the probability of obtaining an optimal combination of active substances in terms of efficiency and safety is maximal and many times exceeds the probability of obtaining a similar result by empirical selection of antibiotic combinations currently practiced in medicine.

As far as we know from the analysis of scientific and patent literature, the proposed approach is new and so far has not been used to develop antimicrobial drugs. In particular, this can be easily verified by reading patents and scientific publications on antimicrobial peptides [1-24, 33, 34].

For the purposes of implementing the present invention, co-adapted antimicrobial peptide complexes of various multicellular organisms can be used. The minimum requirement when choosing a particular type of organism is that the mechanism of natural antibacterial immunity of this species includes the use of two or more groups of antimicrobial peptides simultaneously, which differ in structure and mechanism of action.

According to the authors of the present invention, insects from the group of muscid dipterans (Diptera, Muscomorpha) of the subclass of winged insects (Insecta, Pterigota), in particular representatives of the meat flies of the Calliphoridae family, such as Calliphora vicina Robineau-Desvoidy, Calliphora vomitoria Linna and Lucilia sericata Meigen.

The properties of antimicrobial complexes of this group of insects have been studied in more detail by the example of the blue meat fly C. vicina [37–40].

As we were able to establish, the cells of the adipose body of C. vicina larvae synchronously secrete a complex of antimicrobial peptides that accumulate in hemolymph (blood) and protect the internal environment from pathogenic microorganisms. It should be emphasized that larvae simultaneously release several types of antimicrobial peptides (peptide families) into hemolymph, including defensins, cecropins, diptericins, and proline-rich peptides. The structure and nature of the antimicrobial activity of C. vicina defensins, cecropins, and diptericins are described by us earlier [39], the structure of proline-rich peptides is given for the first time in this application.

Next, we analyzed the homology of the amino acid sequences of antimicrobial peptides found in C. vicina and similar peptides of other insects. This allowed us to establish the basic laws of the formation of a co-adapted complex of antimicrobial peptides during evolution. In particular, it was determined that C. vicina defensins are homologous to defensins common among various orders of winged insects, and are likely to have an evolutionary age of about 500 million years. Cecropins of C. vicina do not fundamentally differ from cecropins of other dipterans and lepidopterans (two related orders of insects belonging to the Mecopteroidea group), and their evolutionary age can be about 300 million years. Dipericins are characteristic of representatives of a number of closely related families of the diptera detachment, grouped into a group of muscid dipterans (Muscomorpha). Therefore, diptericins represent an evolutionarily younger group of antimicrobial peptides compared to cecropins and defensins. Finally, proline-rich peptides of C. vicina have no obvious structural analogues among the known antimicrobial peptides of other insects, from which it can be concluded that these peptides arose at the late stages of evolution of the ancestors of the modern genus Calliphora.

Thus, during the evolution of calliphorid flies, a “muscoid” antimicrobial complex was formed, the elements of which are optimized by natural selection in at least two aspects - the effectiveness of suppressing pathogenic microorganisms and safety for the host organism. There is a third aspect, especially important in the context of the present invention - the preservation of the effectiveness of the elements of the complex over long periods, measured in tens of millions of years, without changing their structural and functional characteristics. As an example, we note that in the structure of defensin among the ancestors of the modern species C.vicina, a mutation expressed in the replacement of one amino acid by another occurred, according to our calculations, on average once every ten million years [38].

This gave us reason to believe that during the natural selection by the ancestors of muscid dipterans, such complex organization options were found that make the process of bacterial adaptation extremely slow or even impossible. Accordingly, antibacterial drugs based on the antimicrobial complex of insects, unlike the currently known antibiotics, must maintain therapeutic efficacy for an unlimitedly long period of time. The following examples of the invention serve as experimental evidence for this hypothesis.

We further suggested that the optimal choice in the context of the present invention is the use of organisms in which antimicrobial peptides are secreted by the cells of the immune system into the blood or other biological fluid and have a toxic effect on pathogenic microorganisms there. In this case, the antimicrobial peptides are in direct contact with the cells of the body of the host organism and, for this reason, they must undergo strict natural selection for the absence of toxicity for these cells in bactericidal concentrations. Such organisms are C. vicina and other representatives of muscid dipterans, in which antimicrobial peptides are synthesized and secreted into hemolymph by cells of a special organ - the fatty body. To explain the advantage of organisms with this type of organization of the immune system, we note that, for example, in mammals, similar antimicrobial peptides (α-defensins) are localized inside blood cells (mainly in neutrophils) and have an antimicrobial effect in specialized intracellular organelles [41, p. .10-11]. Once in an environment that is not characteristic of them — the intercellular space or blood plasma, mammalian α-defensins can have toxic or other side effects on the cells of the host organism itself. From this point of view, C.vicina antimicrobial peptides seem to be more suitable candidates for the role of a drug intended for administration into the blood than mammalian intracellular antimicrobial peptides, such as, for example, α- and β-defensins described in the literature [1-6]. Upon receipt of the complex of antimicrobial peptides proposed in this application, three types of technologies can be used:

1) Extraction of the complex from the host organism and its subsequent purification by preparative chemistry.

2) Chemical synthesis of analogues of the components of the complex and their combination in the finished dosage form or combined treatment regimen.

3) The use of genes encoding the structure of antimicrobial peptides to obtain genetically modified producer cells and the biosynthesis of the corresponding recombinant peptides.

Regardless of the technology for producing the complex, it is fundamentally important to introduce several antimicrobial peptides into it, linked by the same origin and significantly different in chemical structure and mechanism of action.

Each of the above technologies for producing the complex has its own area of primary use. Chemical synthesis technology can be used to produce relatively short (preferably no more than 30 amino acids) peptides that retain the antimicrobial activity of their natural prototypes. Its advantage is the possibility of obtaining the drug on an industrial scale and with a maximum degree of chemical purity, necessary, for example, in the manufacture of dosage forms for intravenous administration. A limitation of this technology lies in the technical difficulty and high cost of synthesizing long peptide chains, especially in the case of peptides having a complex three-dimensional organization.

Genetic engineering technology makes it possible to synthesize peptides of any length, however, in some cases it does not accurately reproduce the spatial structure of the peptide and usually differs in significant cost of the final product.

The technology for producing the complex directly from the host provides the most accurate reproduction of the structure of active components. However, it is applicable mainly to organisms suitable for cultivation on an industrial scale.

The technology of mass cultivation of C. vicina and other diptera species listed above has been well developed, including by the authors of the present invention, and allows obtaining biomass of larvae on an industrial scale and at relatively low cost (fish and meat industry waste can serve as raw materials for biomass production). Therefore, as an example of implementation of the invention, we used the technology of direct extraction of the complex from the biomass of larvae.

The properties of the antimicrobial complex of C. vicina and other species were studied by us in a series of studies, the results of which are presented in the examples below. The main from the point of view of disclosing the essence of the present invention, the results are as follows:

1) C.vicina antimicrobial complex includes four groups of peptides that differ in structure, activity spectrum and mechanism of action: defensins, cecropins, diptericins and proline-rich peptides. Defensins and proline-rich peptides are selectively toxic for gram-positive bacteria, cecropins and diptericins for gram-negative. Thus, for bacteria of each type there are at least two active substances that differ in structure and mechanism of action.

2) The antimicrobial complex described above is an evolutionarily conservative entity. The phylogenetic analysis of the amino acid sequences of the peptides included in its composition shows that the structure of the peptides remains virtually unchanged for millions or even tens of millions of years. It follows that closely related species, in particular, the species C.vicina and C.vomitoria belonging to the same genus Calliphora, have a structurally and functionally identical complex of antimicrobial peptides and are interchangeable. Similarly, the structural and functional characteristics of the antimicrobial complex of the green meat fly L.sericata belonging to the same Calliphoridae family should be close to or identical to the properties of the complex of Calliphora flies.

3) The level of sensitivity of bacteria to the toxic effect of this complex remains unchanged in a number of generations exposed to it, while the sensitivity of bacteria to conventional antibiotics rapidly decreases. This allows us to expect that the resistance of bacteria to a drug whose active ingredient is this complex will not develop or will develop much slower than resistance to antibiotics currently in use.

4) The simultaneous use of the proposed complex and a known antibiotic (for example, cefotaxime) delays the development of resistance to a known antibiotic. Therefore, the proposed complex can be used as a means to extend the effective use of the known antibiotic. This implies that the known antibiotic can be more effectively used in this patient with long courses of treatment, as well as stay in the arsenal of medicines used in medicine for a longer time without loss of therapeutic effectiveness.

5) The proposed complex has a pronounced microbiocidal activity against various groups of gram-negative and gram-positive bacteria. The most sensitive are representatives of enterobacteria and some cocci. The research results confirm the promise of using the complex as a means for treating various bacterial diseases of humans and animals.

Examples of specific implementation.

Example 1. Obtaining and analysis of the composition of the complex of antimicrobial peptides C.vicina.

To obtain the antimicrobial complex of C. vicina, the hemolymph of the larvae was acidified with 0.05% trifluoroacetic acid (TFA) (final concentration) and centrifuged for 5 min at 5000 g. The supernatant was applied to a Sep-Pak C ₁₈ classic cartridge (Waters), washed with 0.05% TFA and eluted with 50% acetonitrile / 0.05% TFA. The eluate was freeze dried and used in this and subsequent examples as an antimicrobial complex of C. vicina (hereinafter denoted by the abbreviation AKCV).

To preliminarily determine the composition of the antimicrobial complex, the lyophilisate was dissolved in the aqueous phase, applied to a ChemcoPak C ₁₈ chromatographic column (4 × 100 mm, Chemco) and chromatographed in an acetonitrile gradient (0-60% for 60 min). Fractions were collected at intervals of 1 min and antimicrobial activity in each fraction was determined using the agar plate method [39]. To do this, 7.5 ml of Luria-Bertany growth medium with agarose (Difco) (bactotriptone 1%, yeast extract 0.5%, NaCl 1%) was poured into sterile Petri dishes (diameter 9 cm). Before solidification, 2 × 10 ⁵ bacteria cells of Micrococcus luteus A 270 or Escherichia coli D31 were introduced into a warm environment. The test material in a volume of 2 μ1 was applied to the surface of the frozen medium. The plates were incubated for 24 hours at + 37 ° C and the diameter of the zone of inhibition of bacterial growth was measured.

The results of chromatographic analysis showed that the complex contains several components that are toxic to gram-positive (M. luteus) and gram-negative (E. coli) bacteria and differ in chromatographic mobility (Table 1).

To study the chemical structure of the components of the antimicrobial complex, high pressure reverse phase liquid chromatography (RP HPLC), mass spectrometry (MALDI-TOF), and Edman microsequencing were used in accordance with the procedure described previously [39].

For this, an antimicrobial complex isolated from 10 ml of hemolymph was subjected to reverse phase chromatography on an Aquapore OD-300 C ₁₈ column (250 × 4.6 mm, Brownlee Associates) and gel filtration on a SEC 2000 column (300 × 7.5 in accordance with the procedure described above) mm, Beckman) using a Beckman Gold system chromatograph. The antimicrobial activity of the isolated peptides was determined by the agar plate method, as described previously [39]. The purity of the isolated peptides was controlled by mass spectrometry (MALDI-TOF), the structure (amino acid sequence) was determined by automated Edman microsequencing on a model 473A sequencer (Applied Biosystems). The correctness of the sequence was monitored using MALDI-TOF mass spectrometry. The characteristics of the identified antimicrobial peptides are shown in Table 2. As part of the antimicrobial complex, the active components of the four clusters are identified:

1) Defensins - three isoforms with molecular weights of 4032.0, 4091.3 and 4114.3 daltons were detected. The full amino acid sequence is set for the peptide with a mass of 4032.0 daltons. C. vicina defensins, like other insects defensins, are characterized by the presence of 6 cysteine residues forming three disulfide bridges and are selectively toxic to gram-positive bacteria. Defensins are known to be among the most widespread antimicrobial peptides of insects [37, 42].

2) Cecropin is a linear peptide with a molecular weight of 4156.0 daltons, consisting of two amphipathic α-helical sections. Selectively toxic to gram-negative bacteria. Structurally and functionally similar to cecropins of other insects. Cecropins are common among representatives of the orders Diptera and Lepidoptera [37, 42].

3) Dipericins - represented by 4 isoforms with molecular weights of 8886.2, 8913.9, 8999.7 and 9029.1 daltons. The N-terminal portion of C. vicina diptericin, consisting of 43 amino acids, including a repeat of 5 glycine residues, is structurally similar to diptericins of other dipterans. Dipericins are selectively toxic to gram-negative bacteria.

4) Proline-rich peptides - 4 isoforms with molecular weights of 2987.0, 3025.7, 3040.0 and 3048.6 daltons were detected. An N-terminal region containing 23 amino acids, including 4 proline residues, is currently sequenced. By their formal character (high proline content), they can be assigned to proline-rich peptides, but their sequence does not have homology with proline-rich peptides of other insects. Functionally, they also differ sharply from the known proline-rich peptides. Unlike the latter, mainly acting on gram-negative bacteria, proline-rich C. vicina peptides are selectively toxic to gram-positive bacteria.

Example 2. The spectrum of activity of the antimicrobial complex of C.vicina

The activity of the antimicrobial complex C.vicina (AKCV) was investigated using the serial dilution method in a liquid nutrient medium. For this, serial two-fold dilutions of the drug in a volume of 50 μl were placed in the wells of a 96-well microbiological plate. The inoculum obtained by adding a 5-6-hour broth culture to Luria-Bertany (Difco) liquid nutrient medium was added to the plate wells in a volume of 50 μl, while the final concentration of microorganisms was 5 × 10 ⁵ CFU (colony forming units) per ml. Sterile culture medium was added to control wells. Each option was tested in 3 replicates. The plates were incubated 24 hours at a temperature of + 35-37 ° C. The results were taken into account visually or spectrophotometrically, comparing the growth of the microorganism in the presence of the drug with the growth of the culture in the well without the drug. As the minimum inhibitory concentration (MIC), the minimum concentration was taken to ensure complete inhibition of visible growth.

Summarized information about the spectrum of antimicrobial activity of the studied drug is presented in Table 3. Most of the studied microorganisms were sensitive to it. These microorganisms belong to different ecological groups, including conditionally pathogenic species, and to different morphological types - gram-positive and gram-negative bacteria. The taxonomic spectrum of antimicrobial activity of the drug includes representatives of the families Enterobacteriacea, Bacillaceae, Coccaceae, Enterococcaceae, Pseudomonadaceae, Moraxellaceae and Corynebacteriaceae. The highest activity of the antimicrobial complex of C. vicina is in relation to most representatives of the Enterobacteriaceae and Coccaceae family. Insensitive forms include Enterococcus faecalis, Listeria monocytogenes, as well as some strains of Staphilococcus aureus and Pseudomonas aeruginosa.

Example 3. Changes in resistance to the antimicrobial complex of C. vicina and known antibiotics in a number of generations of the bacterium Escherichia coli

Two wild-type E. coli strains were used in the study: strain 774.1, sensitive to antibiotics (sensitive, in particular, to amoxiclav, amikacin, gentamicin, imipenem, meropenem, chloramphenicol, ciprofloxacin, polymyxin, cefoperazone, cefoperazone / sulfactim) 863.1 (resistant to amoxiclav, amikacin, netilmicin, gentamicin, ciprofloxacin, cefoperazone, ceftazidime, moderately resistant to levomycetin and cefepime, sensitive to imipenem, meropenem, polymyxin, cefoperazone / sulbact).

The known antibiotics cefotaxime (Biosynthesis), polymyxin B (Kiev plant of medications) and meropenem (AstraZeneca) were used as comparison preparations. Using the method of serial dilutions in a liquid medium described in Example 2, for each E. coli strain, the minimum inhibitory concentration (MIC) of the C.vicina antimicrobial complex (AKCV) and reference antibiotics was estimated. For this purpose, successive two-fold dilutions of the preparation (3 dilutions below the MIC and 3 dilutions above the MIC) and obtained from a 5-6-hour broth culture were placed in the wells of a 96-well microbiological plate containing 100 μl of Luria-Bertany liquid nutrient medium (Invitrogen) inoculum at a final concentration of 5 × 10 ⁵ CFU / ml. The drug was not added to control wells. The plates were incubated at a temperature of + 35-37 ° C for 24 hours. Then daily from a well with a subthreshold concentration of the antibiotic, where the observed growth of bacterial cells was observed, 2 μl of the bacterial suspension in 100 μl of a liquid nutrient medium containing successive two-fold dilutions of the preparation was transferred. After each passage, the minimum inhibitory concentration was determined. The sensitivity to the studied drugs of the populations obtained after the passage, was evaluated using the method of serial dilutions in a liquid nutrient medium (see Example 2).

Analysis of the data presented in figures 1, 2 and 3 shows that the resistance of E. coli bacteria to the toxic effects of cefotaxime, polymyxin and meropenem rapidly increases in a series of passages. In particular, Figure 1 shows the change in minimum inhibitory concentration (MIC) after a series of passages of E. coli 774.1 with cefotaxime or C.vicina antimicrobial complex (AKCV), Figure 2. - change in MIC after a series of passages of E. coli 774.1 with polymyxin or AKCV, Figure 3. - change in MIC after a series of passages of E. coli 863.1 with meropenem or AKCV.

After a series of passages, the level of sensitivity to cefotaxime decreased by 12, to polymyxin - by 8 and to meropenem - by 64 times (Table 4). At the same time, it was found that the E. coli strains studied under similar selection conditions did not change their sensitivity to the C.vicina antimicrobial complex in a number of generations exposed to it.

Thus, AKCV, in contrast to known antibiotics, prevents the development of resistance to this complex in bacteria using the example of E. coli. The materials of this example also show that the proposed antimicrobial complex can be used to inhibit the growth of both bacterial strains that are sensitive and resistant to antibiotics, which serves as an experimental justification for claim 10 of the claims.

Example 4. Changes in resistance to the antimicrobial complex of C. vicina and known antibiotics in a number of generations of the bacterium Klebsiella pneumonia

This section provides experimental data demonstrating how fast the change in the resistance of the bacterium Klebsiella pneumonia to the antimicrobial complex C.vicina (AKCV) and the well-known antibiotic meropenem (Figure 4).

The study used a resistant strain of K. pneumoniae 104.2 (resistant to amikacin, netilmicin, gentamicin, ciprofloxacin, cefoperazone, cefoperazone / sulbactam, ceftazidime, cefotaxime, cefepime, moderately resistant to levomycetin, amyximenimene, imiticimen, imimecimen, mexicenmene, imimecimen, mexicenmene, imimecimen, mexicenmene, imimecimen, mexicene, The experimental procedure was similar to that described in Example 3. The results presented in Figure 4 show that prolonged cultivation of K. pneumonia in a medium with meropenem causes the development of a high level of resistance to this antibiotic. The stability of the strain obtained after 25 passages with meropenem was 128 times higher than the resistance of the original strain to it. After passing K.pneumonia passages in the presence of the antimicrobial complex C.vicina, no changes in resistance were noted in a number of generations (Table 4).

The materials of this example show that the bacteria K. pneumonia do not form resistance to the action of the antimicrobial complex of C. vicina under conditions in which they easily adapt to the action of the known antibiotic meropenem. It should also be noted that the proposed antimicrobial complex effectively inhibits the development of the resistant K.pneumonia strain resistant to known antibiotics and can be used to treat infections caused by such bacteria in accordance with paragraph 10 of the claims.

Example 5. Changes in resistance to the antimicrobial complex of C. vicina and known antibiotics in a number of generations of the bacterium Acinetobacter baumannii.

In this section, experimental data are presented to determine the rate of formation of resistance of A.baumannii to the action of the antimicrobial complex C.vicina (AKCV) and the antibiotic polymyxin B.

The study used the resistant strain 882.2 A.baumannii (resistant to amoxiclav, amikacin, netilmicin, gentamicin, imipenem, meropenem, levomycetin, cefoperazone, ceftazidime, cefotaxime and cefepime, and is moderately resistant to ciprofloxefexfecin peracin,). The experimental design is similar to that described in Example 3.

Figure 5 and Table 4 show the results that demonstrate the development of resistance of A.baumannii in successive passages on media with polymyxin and C.vicina antimicrobial complex (AKCV). After 35 passages, resistance to this antibiotic increased by about 5 times. In a similar experiment, the development of resistance to the antimicrobial complex of C. vicina was not detected.

Thus, the bacterium A.baumannii, like other types of bacteria considered in examples 3 and 4, under the proposed selection conditions, was unable to form resistance to the antimicrobial complex of C.vicina. The obtained experimental data also confirm the fact that the proposed antimicrobial complex is effective in relation to the A.baumannii form resistant to known antibiotics and can be used in accordance with claim 10 of the claims.

Example 6. The effect of the antimicrobial complex of C. vicina on the rate of development of resistance to known antibiotics in the bacterium Escherichia coli

In this example, the ability of the antimicrobial complex C.vicina (AKCV) to inhibit the development of antibiotic resistance to cefotaxime was studied under conditions of mass selection of E. coli cells.

The experiment used E. coli strain 774.1, which is sensitive to antibiotics (sensitive, in particular, to amoxiclav, amikacin, gentamicin, imipenem, meropenem, chloramphenicol, ciprofloxacin, polymyxin, cefoperazone, cefoperazone / sulbactam), cefot. The serial passage technique was as described in Example 3. E. coli cells were subjected to repeated passages in a liquid nutrient medium containing the cefotaxime antibiotic without the addition of the antimicrobial complex C.vicina or with its addition at a final concentration of 50 μg / ml.

An analysis of the experimental data shown in FIG. 6 shows that already 3 passage of Escherichia coli in an environment with cefotaxime lead to the adaptation of bacteria, which is accompanied by the development of double resistance to this antibiotic. The maximum stability, 16 times higher than the initial one, developed after 7 passages of E. coli with cefotaxime. At the same time, the addition of C.vicina antimicrobial complex (AKCV) to the incubation medium with cefotaxime reduced the rate of resistance development by at least two times.

Thus, the antimicrobial complex of C. vicina not only prevents the adaptation of bacteria to its components, but also delays the development of resistance to known antibiotics. This allows you to use it as a means to prevent the development of drug resistance in pathogenic microorganisms.

This example serves as an experimental justification for claim 9 of the claims.

Example 7. Changes in resistance to the antimicrobial complex of Calliphora vomitoria and known antibiotics in a number of generations of the bacterium E. coli

This example presents data on the change in the level of sensitivity of E. coli to the action of the antimicrobial complex of the fly C. vomitoria (AKCVO), a species closely related to C. vicina, and the cefotaxime antibiotic in a series of generations subjected to selection for resistance to one or another agent.

A laboratory culture of the fly C.vomitoria was used as starting material. The method for obtaining the antimicrobial complex of C. vomitoria corresponded to the method described in Example 1. In the experiments, the sensitive E. coli strain 774.1 was used. The experimental design was described in Example 3.

The materials presented in Fig.7 and Table 5 show that cultivation of E. coli in the presence of cefotaxime causes a rapid increase in resistance to this antibiotic. As a result, the strain obtained after a series of passages acquired significant resistance to cefotaxime. A similar effect of the antimicrobial complex of C. vomitoria (AKCVO) did not cause the development of resistance in E. coli.

The materials of this example show the interchangeability of the antimicrobial complexes of C. vomitoria and C. vicina in the context of the present invention. Like the drug from C. vicina, the antimicrobial complex of C. vomitoria prevents the development of resistance to its bactericidal effect in bacteria.

Example 8. Changes in resistance to the antimicrobial complex of Lucilia sericata and known antibiotics in a number of generations of the bacterium E. coli

L.sericata belongs to the Calliphoridae family and is in close phylogenetic relationship with species of the genus Calliphora, in particular C.vicina and C.vomitoria. This example presents data on the change in the level of sensitivity of E. coli to the action of the antimicrobial complex L.sericata (AKLS) and the cefotaxime antibiotic in a number of generations that were systematically exposed to one or another agent.

A laboratory culture of the L.sericata fly was used as starting material. The method of obtaining the antimicrobial complex L.sericata consistent with the method described in Example 1.

In the experiments, antibiotic-sensitive E. coli strain 774.1 was used. The experimental design was described in Example 3.

The results of the experiments are presented in Fig. 8 and Table 5. As in the cases with C.vicina and C.vomitoria considered in examples 3-8, we were not able to detect regular changes in the level of resistance in a series of generations of E. coli subjected to systematic exposure antimicrobial complex L. sericata (AKLS). Under the same conditions, bacteria quickly acquired resistance to the action of the reference antibiotic cefotaxime. Thus, the antimicrobial complex of L. sericata in the context of the present invention has the same properties as the antimicrobial complexes of the genus Calliphora, and can be used as an active ingredient in drugs that can inhibit the development of drug resistance in bacteria.

Example 9. The therapeutic properties of the antimicrobial complex of C.vicina in experimental bacterial infection in mice

Outbred white mice weighing 18-20 g were used in the experiments. A wild-type strain of the bacterium Klebsiella pneumonia was used as an infectious agent. In preliminary experiments, the minimum lethal dose of bacteria was selected, causing the death of 95-100% of mice within 24-48 hours. For this, the mice were injected intraperitoneally with a suspension of daily agar culture of bacteria in a dose of 1 × 10 ⁹ to 10 × 10 ⁹ cells in an isotonic solution NaCl. The minimum lethal dose was 2 × 10 ⁹ cells. The reaction to the introduction of this dose was manifested after 3-4 hours - a decrease in animal activity, bloating and diarrhea at a later date. The death of animals occurred within 24-48 hours after infection.

C.vicina antimicrobial complex was injected intraperitoneally in a single dose of 0.5 mg dissolved in 0.5 ml of isotonic NaCl solution 30 minutes, 3 and 24 hours after the introduction of bacterial suspension. Control animals were injected with isotonic NaCl solution. In each experiment, 10 animals were used.

In the experiment, the results of which are shown in Table 6, mice were injected with an infectious agent at a dose of 4 × 10 ⁹ K.pneumonia cells. In the control during the first three days, 90% of mice fell, in the experimental group - 40%. The difference between the experimental and control groups was statistically significant (t = 2.37 at N = 19, P <0.05).

Thus, the antimicrobial complex of C. vicina has therapeutic activity when introduced into the body of an animal infected with a pathogenic bacterium.

The results of this series of experiments also show that C.vicina antimicrobial complex does not show toxicity to animals when administered in therapeutically effective concentrations.

The above examples of the invention allow to characterize a comprehensive technical result from its use. This result consists in replenishing the arsenal of drugs intended for the treatment of infectious diseases caused by antibiotic-resistant bacteria. At present, this arsenal has in many cases been practically exhausted, since over the past decades of intensive use of antibiotics, forms of bacteria have emerged that are not amenable to treatment by any known antibiotics. In addition, this result consists in preventing the development of bacterial resistance to known antibiotics when used together with the proposed antiviral substance. This allows you to extend the effective use of known antibiotics against bacteria that still have sensitivity to them.

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Table 1 Chromatographic characteristics of bactericidal components of the complex of antimicrobial peptides C.vicina Acetonitrile,% The area of the zone of inhibition of bacterial growth, mm ² M. luteus E.coli 0 0 0 one twenty 0 2-3 0 0 four 0 0 5 0 0 6 twenty 0 7 0 0 8 0 0 9-18 0 0 19 0 0 20-24 0 0 25 0 0 26 0 0 27 133 0 28 490 0 29th 283 38 thirty 38 13 31 28 78 32 0 78 33 0 95 34 0 fifty 35 0 13 36 0 7 37 0 twenty 38-39 0 0 40 0 0 41 0 0 42 0 0 43 0 0

table 2 The structure and functional characteristics of the peptides that make up the complex of antimicrobial peptides C.vicina No. Title Amino Acid Sequence Antibacterial activity SEQ ID No. 1 Defensin Ala-Thr-Cys-Asp-Leu-Leu-Ser-Gly-Thr-Gly-Ala-Asn-Hys-Ser-Ala-Cys-Ala-Ala-Hys-Cys-Leu-Leu-Arg-Gly-Asn- Arg-Gly-Gly-Tyr-Cys-Asn-Gly-Lys-Ala-Val-Cys-Val-Cys-Arg-Asn Gram-positive bacteria SEQ ID No. 2 Proline-Rich Peptide Phe-Val-Asp-Arg-Asn-Arg-Ile-Pro-Arg-Ser-Asn-Asn-Gly-Pro-Lys-Ile-Pro-Ile-Ile-Ser-Asn-Pro- ... (N-terminal sequence ) SEQ ID No. 3 Cecropin Gly-Trp-Leu-Lys-Lys-Ile-Gly-Lys-Lys-Ile-Glu-Arg-Val-Gly-Gln-Hys-Thr-Arg-Asp-Ala-Thr-Ile-Gln-Gly-Leu- Ala-Val-Ala-Gln-Gln-Ala-Ala-Asn-Val-Ala-Ala-Thr-Ala-Arg Gram-negative bacteria SEQ ID No. 4 Diptericin Asp-Ser-Lys-Pro-Leu-Asn-Leu-Val-Leu-Pro-Lys-Glu-Glu-Pro-Pro-Asn-Asn-Pro-Gln-Thr-Tyr-Gly-Gly-Gly-Gly- Gly-Ser-Arg-Lys-Asp-Asp-Phe-Asp-Val-Val-Leu-Gln-Gly-Ala-Gln- ... (N-terminal sequence)

Table 3 The spectrum of antimicrobial activity of C.vicina complex Microorganism Dachshund Bacteria group MIC, mg / ml Escherichia coli D31 Enterobacteriaceae Gr- 0.025 1106 0.025 ATCC 25922 0.125 wild type strains 774.1 0.25 863.1 0.5 Klebsiella pneumonia Enterobacteriaceae Gr- 0.02 wild type strains 104.2 0.25 84.1 0.5 Salmonella typhimurium Enterobacteriaceae Gr- 0.012 Enterobacter cloacae AT 12 Enterobacteriaceae Gr- 0.1 Enterococcus faecalis ATCC 29212 Enterococcaceae Gr + > 4.0 Micrococcus luteus A270 Coccaceae Gr + 0.012 Staphilococcus aureus 203 Cocccaceae Gr + 0.125 Cowan i 2.0 MRSA ATCC 33591 > 3.0 Bacillus subtilis Bacillaceae Gr + 0.4 Bacillus megaterium Bacillaceae Gr + 0.012 Bacillus thuringiensis kenya Bacillaceae Gr + 0.11 Pseudomonas aeruginosa Pseudomonadaceae Gr- ATCC 27853 0.5 wild type strain 702 2.0 Acinetobacter baumannii Moraxellaceae Gr- wild type strains 84.2 0.25 882.2 0.5 Listeria monocytogenes EGD Corynebacteriaceae Gr + 2.0

Table 4 Development of resistance to cefotaxime, meropenem, polymyxin and the antimicrobial complex C.vicina (AKCV) A drug Minimum inhibitory concentration K (MIC end. / MIC beginning) R Before selection After selection E. coli 774.1 AKCV, mg / ml 0.25 ± 0.0 0.33 ± 0.08 1.32 > 0.1 Cefotaxime, mcg / ml 0.17 ± 0.04 2.0 ± 0.0 11.8 <0.001 E. coli 863.1 AKCV, mg / ml 0.42 ± 0.08 0.50 ± 0.0 1.19 > 0.1 Meropenem, mcg / ml 0.125 ± 0.0 8.0 ± 0.0 64 <0.001 E. coli 774.1 AKCV, mg / ml 0.25 ± 0.0 0.42 ± 0.08 1.68 > 0.05 Polymyxin, mcg / ml 6.67 ± 1.33 53.3 ± 10.6 7.99 <0.01 K.pneumonia 104.2 AKCV, mg / ml 0.83 ± 0.17 0.50 ± 0.0 0.60 > 0.05 Meropenem, mcg / ml 0.125 ± 0.0 16.0 ± 0.0 128 <0.001 A.baumannii 882.2 AKCV, mg / ml 0.42 ± 0.08 0.50 ± 0.0 1.19 > 0.1 Polymyxin, mcg / ml 1.7 ± 0.3 8.0 ± 0.0 4.7 <0.01

Table 5 The development of resistance in E. coli to the antimicrobial complex C.vomitoria (AKCVO), the antimicrobial complex L.sericata (AKLS) and cefotaxime A drug Minimum Inhibitory Concentration (MIC) K (MIC end. / MIC beginning) R Before selection After selection AKCVO, mg / ml 0.42 ± 0.08 0.5 ± 0.0 1.19 > 0.1 AKLS, mg / ml 0.42 ± 0.08 0.5 ± 0.0 1.19 > 0.1 Cefotaxime, mcg / ml 0.17 ± 0.04 2.0 ± 0.0 11.8 <0.001

Table 6 The effect of the antimicrobial complex of C. vicina on the survival of mice with intraperitoneal infection of K. pneumonia Experience option N Survival after 3 days,% R The dynamics of death by days of experience one 2 3 four 5 6 7 The control 10 10% - 2 5 2 0 0 0 0 Antimicrobial complex C.vicina 10 60% <0.05 0 3 one 0 0 one 0

Claims (8)

1. An antimicrobial substance, including a purified complex of defensins, cecropins, diptericins and proline-rich insect peptides of the Calliphoridae family. 2. The antimicrobial substance according to claim 1, containing the peptide SEQ ID No. 1. 3. The antimicrobial substance according to claim 1, containing the peptide SEQ ID No. 2. 4. The antimicrobial substance according to claim 1, containing the peptide SEQ ID No. 3. 5. The antimicrobial substance according to claim 1, containing the peptide SEQ ID No. 4. 6. The antimicrobial substance according to claim 1, obtained from insects of the species Calliphora vicina R.-D. 7. The antimicrobial substance according to claim 1, obtained from insects of the species Calliphora vomitoria L. 8. The antimicrobial substance according to claim 1, obtained from insects of the species Lucilia sericata Meig.

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