COMBINATORIAL ANTIBIOTIC DERIVATIVES BASED ON SUPRAMOLECULAR STRUCTURES

Abstract

Field of application: The invention relates to combinatorial chemistry, pharmacy and cosmetology, allows to synthesize new combinatorial libraries of derivatives of antibiotics for use in pharmacy, cosmetology and pharmacy.

Technical result: modified combinatorial derivatives of antibiotics with antimicrobial and antifungal activity against multiresistant and pan drug resistance strains of microorganisms and fungi. Means have a wide spectrum of action, and the supramolecular and combinatorial structure of their tens and hundreds of derivatives eliminates the resistance of microorganisms.

Claims

1. New combinatorial derivatives of antibiotics based on supramolecular structures and a method for their preparation, wherein the supramolecular structures (B) are obtained by combinatorial synthesis of a polyfunctional antibiotic (A1) from one source molecule with two or more groups available for covalent modification in the reaction, as at least with two different covalent modifiers (M2 and M3) simultaneously according to the synthesis scheme
mA1+kM2+kM3=mB In this case, a combinatorial mixture of modified derivatives of the original molecule is formed, with a maximum variety of derivatives, and as biologically active substances for creating pharmaceutical compositions, a whole combinatorial mixture is used in the form of a supramolecular structure without separation into individual components.
k=n×(2.sup.n−1)  (1)
m=4×(3×2.sup.n-2−1)  (2) e

2. The invention according to claim 1, wherein the molar ratio of the components of the reaction is calculated based on the formulas:
k=n×(2n−1)  (1)
m=4×(3×2n−2−1)  (2) Where n=the number of groups available for substitution in the multifunctional antibiotic molecule (A1); m=the number of moles of the original multifunctional molecule (A1) and the number of different molecules of combinatorial derivatives (B) after synthesis; k=the number of moles of each of the two modifiers (M2 and M3) in the combinatorial synthesis reaction to obtain the maximum number of different derivatives, and a combinatorial mixture of B modified derivatives of the original antibiotic molecule (A1) is formed in the reaction, the number of combinations of which is maximum (m)

3. The invention according to claim 1, wherein the source molecule (A1) is polymyxin.

4. The invention according to claim 1, wherein the source molecule (A1) is an aminoglycoside antibiotic.

5. The invention according to claim 1, wherein the source molecule (A1) is a polyene antibiotic.

6. The invention according to claim 1, wherein the source molecule (A1) is tetracycline

7. The invention according to claim 1, wherein the source molecule (A1) is a macrolide antibiotic

8. The invention according to claim 1, wherein the source molecule (A1) is an antibiotic lincosamine

9. The invention according to claim 1, wherein the source molecule (A1) is an antibiotic gramicidin

10. The invention according to claim 1, wherein the source molecule (A1) is an antibiotic glycopeptide

11. The invention according to claim 1, wherein the modifiers M2 and M3 are acylating agents of the group of anhydrides of organic mono- and polycarboxylic acids

12. The invention according to claim 1, wherein the modifiers M2 and M3 are halides of carboxylic acids

13. The invention according to claim 1, wherein the modifiers M2 and M3 are alkylating agents halogen derivatives of hydrocarbons

14. The invention according to claim 1, wherein the modifier M2 is an acylating agent—mono- or polycarboxylic acid anhydride or carboxylic acid halide, and M3 is an alkylating agent—a halogenated hydrocarbon

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1. Scheme of combinatorial synthesis of polymyxin derivatives with the formation of supramolecular combinatorial derivative (IVa-d): polymyxin reacts with two modifying agents—succinic anhydride and acetic anhydride in the calculated proportions. In this case, a supramolecular structure of 380 polymyxin derivatives is formed.

[0034] FIG. 2. Scheme of combinatorial synthesis of tetracycline derivatives with the formation of supramolecular combinatorial derivative (VIIa-d): tetracycline reacts with two modifying agents—succinic anhydride and acetic anhydride in the calculated proportions. In this case, a supramolecular structure of 92 tetracycline derivatives is formed.

[0035] FIG. 3. TLC of tetracycline derivatives, water: AcCN=1:1, manifestations of a UV lamp (190-300 nm).

[0036] FIG. 4. Scheme of combinatorial synthesis of the supramolecular combinatorial derivative of gentamicin (IXa-d): gentamicin (base) reacts with two modifying agents—succinic anhydride and acetic anhydride in the calculated proportions. In this case, a supramolecular structure of 764 gentamicin derivatives is formed.

[0037] FIG. 5. TLC of gentamicin derivatives, water: AcCN=1:1, manifestation of a UV lamp (190-300 nm) after treatment with a solution of sulfuric acid.

PHARMACEUTICAL COMPOSITIONS

[0038] Various methods of administering supramolecular combinatorial antibiotic derivatives (ASCA) can be used. The ASCA composition can be given orally or can be administered by intravascular, subcutaneous, intraperitoneal injection, in the form of an aerosol, by ocular route of administration, into the bladder, topically, and so on. For example, inhalation methods are well known in the art. The dose of the therapeutic composition will vary widely depending on the particular antimicrobial ASCA administered, the nature of the disease, frequency of administration, route of administration, clearance of the agent used from the host, and the like.

[0039] The initial dose may be higher with subsequent lower maintenance doses. The dose can be administered once a week or once every two weeks, or divided into smaller doses and administered once or several times a day, twice a week, and so on to maintain an effective dose level. In many cases, a higher dose will be needed for oral administration than for intravenous administration. The compounds of this invention may be included in a variety of compositions for therapeutic administration.

[0040] More specifically, the compounds of the present invention can be incorporated into pharmaceutical compositions in combination with suitable pharmaceutically acceptable carriers or diluents, and can be incorporated into preparations in solid, semi-solid, liquid or gaseous forms, such as capsules, powders, granules, ointments, creams, foams, solutions, suppositories, injections, forms for inhalation use, gels, microspheres, lotions and aerosols. As such, the administration of the compounds can be carried out in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal administration and so on.

[0041] The ASCA according to the invention can be distributed systemically after administration or can be localized using an implant or other composition that holds the active dose at the site of implantation. The compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (eg, perforin, anti-inflammatory agents, and so on). In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are given as examples only and are in no way limiting.

[0042] For preparations for oral administration, the compounds can be used alone or in combination with suitable additives for the manufacture of tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binding agents, such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatins; with disintegrants, such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffers, wetting agents, preservatives and flavoring agents.

[0043] The compounds can be incorporated into injectable compositions by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and, if desired, with conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0044] The compounds may be used in an aerosol composition for inhalation administration. The compounds of the present invention can be incorporated into suitable pressure propellants such as dichlorodifluoromethane, propane, nitrogen and the like. In addition, the compounds can be incorporated into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.

[0045] The compounds of the present invention can be administered rectally using a suppository. A suppository may contain excipients, such as cocoa butter, carboax, and polyethylene glycols, which melt at body temperature but are solid at room temperature. Standard dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, where each unit dose, for example, a teaspoon, tablespoon, tablet or suppository, may contain a predetermined amount of a composition containing one or more compounds of the present invention.

[0046] Similarly, unit dosage forms for injection or intravenous administration may contain the compound of the present invention in a composition in the form of a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier. Implants for the sustained release of compositions are well known in the art. Implants are made in the form of microspheres, plates, and so on with biodegradable or non-biodegradable polymers. For example, lactic and/or glycolic acid polymers form a degradable polymer that is well tolerated by the host.

[0047] An implant containing the antimicrobial combinatorial antibiotics of the invention is positioned close to the site of infection, so that the local concentration of the active agent is increased compared to other areas of the body. As used herein, the term “unit dosage form” refers to physically discrete units suitable for use as single doses for subjects of humans and animals, each unit containing a predetermined number of compounds of the present invention, which, according to calculations, is sufficient to provide the desired effect together with a pharmaceutically acceptable diluent, carrier or excipient.

[0048] The descriptions of the unit dosage forms of the present invention depend on the particular compound used, and the effect to be achieved, and the pharmacodynamics of the compound used in the host. Pharmaceutically acceptable excipients, such as excipients, adjuvants, carriers or diluents, are generally available. In addition, pharmaceutically acceptable excipients are generally available, such as pH adjusting agents and buffering agents, tonicity agents, stabilizers, wetting agents and the like.

[0049] Typical doses for systemic administration range from 0.1 μg to 100 milligrams per kg of subject body weight per administration. A typical dose may be a single tablet for administration from two to six times a day, or one capsule or sustained release tablet for administration once a day with a proportionally higher content of the active ingredient. The effect of prolonged release may be due to the materials of which the capsule is made, dissolving at different pH values, capsules providing a slow release under the influence of osmotic pressure or any other known controlled release method.

[0050] Those skilled in the art will appreciate that dose levels may vary depending on the particular compound, the severity of symptoms, and the subject's predisposition to side effects. Some of the specific compounds are more potent than others. Preferred doses of this compound can be readily determined by those skilled in the art in a variety of ways. A preferred method is to measure the physiological activity of the compound. One of the methods of interest is the use of liposomes as a vehicle for delivery.

[0051] Liposomes fuse with the cells of the target region and provide delivery of liposome contents to the cells. The contact of the liposomes with the cells is maintained for a time sufficient for fusion using various methods of maintaining contact, such as isolation, binding agents and the like. In one aspect of the invention, liposomes are designed to produce an aerosol for pulmonary administration. Liposomes can be made with purified proteins or peptides that mediate membrane fusion, such as Sendai virus or influenza virus and so on. Lipids can be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine.

[0052] The remaining lipids will usually be neutral or acidic lipids, such as cholesterol, phosphatidylserine, phosphatidylglycerol and the like. To obtain liposomes, the method described by Kato et al. (1991) J. Biol. Chem. 266: 3361. Briefly, lipids and a composition for incorporation into liposomes containing combinatorial supramolecular antibiotics are mixed in a suitable aqueous medium, suitably in a salt medium, where the total solids content will be in the range of about 110 wt. %.

[0053] After vigorous stirring for short periods of approximately 5-60 seconds, the tube is placed in a warm water bath at approximately 25-40° C. and this cycle is repeated approximately 5-10 times.

[0054] The composition is then sonicated for a suitable period of time, typically approximately 1-10 seconds, and optionally further mixed with a vortex mixer. Then the volume is increased by adding an aqueous medium, usually increasing the volume by about 1-2 times, followed by agitation and cooling. The method allows to include supramolecular structures with high total molecular weight in liposomes.

[0055] Compositions with Other Active Agents

[0056] For use in the methods under consideration, the ASCA according to the invention can be included in compositions with other pharmaceutically active agents, in particular other antimicrobial agents, immunomodulators, antiviral agents, antiviral substances. Other agents of interest include a wide range of unmodified antibiotics known in the art. Antibiotic classes include penicillins, for example penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin and so on; penicillins in combination with betalactamase inhibitors; cephalosporins, for example cefaclor, cefazolin, cefuroxime, moxalactam, etc. etc.

[0057] Antifungal agents are also useful, including polyenes, such as amphotericin B, nystatin, flucosin; and azoles, such as miconazole, ketoconazole, itraconazole, and fluconazole. Anti-TB drugs include isoniazid, ethambutol, streptomycin and rifampin. Other agents of interest in terms of creating new compositions include a wide range of mononucleotide derivatives and other RNA polymerase inhibitors known in the art.

[0058] Classes of antiviral agents include interferons, lamivudine, ribavirin, etc. Other groups of antiviral agents include adefovir, vbacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, efavirenz, nevirapine, indinavir, lopinavir and ritonavir, nelfinavir, ritonavir, sakinavir, daclatasvir, Sovof. Cytokines, such as interferon gamma, tumor necrosis factor alpha, interleukin 12, and so on, may also be included in the antimicrobial SCA composition of the invention. The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Example 1. Obtaining a Supramolecular Combinatorial Mixture of Polymyxin (PPC)

[0059] 390 μM polymyxin B (I) is dissolved in 10 ml of dioxane (CAS N 1404-26-8, Mr=1203.499 g/mol, n=7) (I), 889 μM succinic anhydride (III) and 889 μM acetic anhydride (II) are added, the solution is stirred and heated under reflux for 10 minutes. The solution was poured into ampoules and lyophilized to remove solvent and acetic acid. The combinatorial mixture (Iva-d) is used to obtain pharmaceutical compositions, study the structure, determine the biological activity. FIG. 1 shows a synthesis scheme for combinatorial derivatives of polymyxin.

[0060] FIG. 1. One Initial Polymyxin Molecule Contains 7 Peptide Residues of Amino Groups and Hydroxyl Groups Available for

[0061] Calculations of the Number of Moles of Modifiers are Carried Out According to the Combinatorics Formulas:

[0062] m=4×(3×2.sup.n-2−1); k=n×(2.sup.n−1), where m—is the number of different derivatives of molecules in the combinatorial mixture and the number of moles of polymyxin for the reaction; n—is the number of amino groups and hydroxyl groups available for modification in the structure of polymyxin (n=7); k—is the number of moles of each modifier.

[0063] Therefore, having only one initial polymyxin molecule and two modifiers after combinatorial synthesis, we obtain 380 combinatorial derivatives with different degrees of substitution, different positions of substituents and different permutations of the modifier residues, not just as a mixture, but as a difficult to separate modification (n=7). supramolecular mixture.

[0064] Due to the presence in both derivatives of both substituted and non-substituted hydroxyl and amino groups, supramolecular structures are formed through both hydrogen and ionic bonds. Modifiers—succinic anhydride or acetic anhydride can be entered both simultaneously and sequentially—or first inject succinic anhydride, warm the mixture under reflux for 10 minutes, and then add acetic anhydride and also warm the mixture for another 10 minutes. Similarly, in this reaction, maleic anhydride, aconitic anhydride, glutaric, phthalic anhydride and acetic anhydride, formic acid ethyl ester, monochloroacetic acid, propiolactone, ethylene oxide, and other low-chloro chlorides can be used as one of the modifiers instead of succinic anhydride.)

[0065] For the HPLC, a Milichrom A-02 microcolumn chromatograph in a gradient of acetonitrile (5-100%)/0.1 M perchloric acid+0.5 M lithium perchlorate was used. The combinatorial derivative in the chromatogram gave one clear broadened peak and was not separated into components, although the retention time differed from both the original polymyxin and its completely substituted derivatives. This testified to the fact that complex supramolecular structures were formed between different combinatorial derivatives (in our case, 380), which were not separated chromatographically. This combinatorial derivative (CPP) behaves similarly and when separated in a thin layer (acetonitrile: water) gives only one band, which does not coincide with any of the obtained derivatives. An attempt to use two-dimensional TLC in different conditions also did not allow us to separate the combinatorial derivative. This is a characteristic feature of the supramolecular structure in the combinatorial derivative (IVa-d).

Example 2. Obtaining a Supramolecular Combinatorial Mixture of Tetracycline (CBT)

[0066] 92 μM tetracycline (VI) is dissolved in 10 ml of dioxane (CAS N 60-54-8, Mr=444.44 g/mol, n=5) (I), 155 μM succinic anhydride (III) and 155 μM acetic anhydride (II) are added, the solution is stirred and heated under reflux for 10 minutes, 1200 μM TRIS is added to the solution, stirred until dissolved. The solution was poured into ampoules and lyophilized to remove solvent and acetic acid. The combinatorial mixture (VIIa-d) is used to obtain pharmaceutical compositions, study the structure, determine the biological activity (CBT). FIG. 1 shows a synthesis scheme for combinatorial derivatives of tetracycline.

[0067] In this reaction, instead of tetracycline, oxytetracycline or any other tetracycline derivative with hydroxyl groups available for modification can be used, as well as any other antibiotic with two or more groups available for modification: aminoglycoside antibiotics, polyene antibiotics, tetracycline, macrolide antibiotics, lincosamine, gramicidin, glycopeptide antibiotics. Instead of modifiers of carboxylic acid anhydrides, halides of carboxylic and polycarboxylic acids, such as succinic, maleic, fumaric, lactic, propionic, other halogen derivatives, such as chloromethane, bromoethane, chloropropane, cyclic alkylating compounds, such as oxirane, and propoxy, can be used.

[0068] FIG. 2.

[0069] One source molecule of tetracycline (I) contains 5 hydroxyl groups available for modification (n=5).

[0070] Calculations of the Number of Moles of Modifiers are Carried Out According to the Combinatorics Formulas:

[0071] m=4×(3×2.sup.n-2−1); k=n×(2.sup.nb −1), where m is the number of different derivatives of molecules in the combinatorial mixture and the number of moles of tetracycline for the reaction; n is the number of hydroxyl groups available for modification in the structure of tetracycline (n=5); k is the number of moles of each modifier. Thus, having only one initial tetracycline molecule and two modifiers after combinatorial synthesis, we obtain 92 combinatorial derivatives with different degrees of substitution, different positions of substituents and different permutations of the modifier residues, not just as a mixture, but as a difficult to separate supramolecular mixture.

[0072] Due to the presence of both substituted and non-substituted hydroxyl groups in various derivatives, supramolecular structures are formed through both hydrogen and ionic bonds. Modifiers—succinic anhydride or acetic anhydride can be entered both simultaneously and sequentially—or first introduce succinic anhydride, warm the mixture under reflux, and then add acetic anhydride and re-warm the mixture. Similarly, in this reaction, maleic anhydride, aconitic anhydride, glutaric, phthalic anhydride and acetic anhydride, formic acid ethyl ester, monochloroacetic acid, propiolactone, ethylene oxide, and other low-chloro chlorides can be used as one of the modifiers instead of succinic anhydride.)

[0073] For the HPLC, a Milichrom A-02 microcolumn chromatograph in a gradient of acetonitrile (5-100%)/0.1 M perchloric acid+0.5 M lithium perchlorate was used. The combinatorial derivative in the chromatogram gave one clear broadened peak and was not separated into components, although the retention time differed from both the original polymyxin and its completely substituted derivatives. This indicated that complex supramolecular structures were formed between different combinatorial derivatives (in our case, 380), which were not separated chromatographically. This combinatorial derivative (PMM) behaves similarly when separated in a thin layer (acetonitrile:water, UV detection) and gives only one band, which does not coincide with any of the obtained derivatives.

[0074] NMR C13: C: 199.4; 197.6; 169.5; 149.9; 156.2; 93.4; 83.1; CH: 76.7; C: 108.6; 116.4; 143.5; 106.2; CH: 117.1; 120.7; 128.1; 27.4; 38.6; CH2: 14.7; C: 147.5; 171.1; 173.1; 174.7; 172.0; CH3: 44.6; 24.0; CH2: 28.8; 29.8; 29.1

[0075] FIG. 3.

[0076] As can be seen from TLC, the Combinatorial mixture (lane VIIa-d) is less mobile and has Rf=0.47, while the initial unmodified tetracycline (VI) is the lightest and Rf=0.59. Fully acylated tetracycline (VIIb) and succinyl tetracycline (VIIC) are intermediate between native tetracycline and combinatorial. The combinatorial tetracycline band is not separated either by two-dimensional TLC or by HPLC (not shown).

Example 3. Obtaining a Supramolecular Combinatorial Mixture of Gentamicin (Aminoglycoside) (CMG)

[0077] 764 μM gentamicin base (VIII) (CAS N 1403-66-3, Mr=477.603 g/mol, n=8) (VIII) is dissolved in 10 ml of dioxane, 2040 μM of succinic anhydride (III) and 2040 μM of acetic anhydride are added (II), the solution is stirred and heated under reflux for 5-50 minutes. The solution was poured into ampoules and lyophilized to remove solvent and acetic acid. The combinatorial mixture (IXa-d) is used to obtain pharmaceutical compositions, study the structure, determine the biological activity (CMG). FIG. 4 shows a synthesis scheme for combinatorial derivatives of gentamicin.

[0078] In this reaction, instead of gentamicin, streptomycin, amikacin, or any other representative of aminoglycoside antibiotics with hydroxyl and amino groups available for modification, as well as any other antibiotic with two or more groups available for modification, can be used: aminoglycoside antibiotics, polyene antibiotics, tetracycline, macrolide antibiotics, lincosamine, gramicidin, glycopeptide antibiotics. Instead of modifiers of carboxylic acid anhydrides, halides of carboxylic and polycarboxylic acids, such as succinic, maleic, fumaric, lactic, propionic, other halogen derivatives, such as chloromethane, bromoethane, chloropropane, cyclic alkylating compounds, such as oxirane, and propoxy, can be used.

[0079] FIG. 4.

[0080] One Source Molecule of Gentamicin (I) Contains 8 Hydroxyl and Methyl Amino Groups Available for Modification (n=8).

[0081] Calculations of the number of moles of modifiers are carried out according to the combinatorics formulas:

[0082] m=4×(3×2.sup.n-2−1); k=n×(2.sup.n−1), where m is the number of different derivatives of molecules in the combinatorial mixture and the number of moles of tetracycline for the reaction; n is the number of hydroxyl and amino groups available for modification in the structure of gentamicin (n=8); k is the number of moles of each modifier. Thus, having only one initial gentamicin molecule and two modifiers after combinatorial synthesis, we obtain 764 combinatorial derivatives with different degrees of substitution, different positions of substituents and different permutations of the modifier residues, not just as a mixture, but as a difficult to separate supramolecular mixture.

[0083] Due to the presence in both derivatives of both substituted and non-substituted hydroxyl and methylamino groups, supramolecular structures are formed through both hydrogen and ionic bonds. Modifiers—succinic anhydride or acetic anhydride can be entered both simultaneously and sequentially—or first introduce succinic anhydride, warm the mixture under reflux, and then add acetic anhydride and re-warm the mixture. Similarly, in this reaction, maleic anhydride, aconitic anhydride, glutaric, phthalic anhydride and acetic anhydride, formic acid ethyl ester, monochloroacetic acid, propiolactone, ethylene oxide and other low molecular weight molecules, can be used as one of the modifiers instead of succinic anhydride.)

[0084] NMR C13: CH: 107.9; 107.1; 87.1; CH2: 63.8; C: 70.2; CH: 85.0; CH: 90.0; CH 64.4; CH 74.4; CH 65.0; CH 53.4; CH 55.7; CH 49.3; CH2 22.4; 34.8; 23.9; C: 170.2; 174.7; 173.8; 172.3; 173.0; CH: 60.2; CH3: 31.6; 34.0; 15.8; CH2: 29.8; 29.1; 30.2; 29.4; CH3: 21.1; 17.5;

[0085] C13 NMR data of the combinatorial derivative confirm the presence of both ethyl groups of succinic acid residues in its structure, and acetyl residues—reaction products with acetic anhydride.

[0086] For the HPLC, a Milichrom A-02 microcolumn chromatograph in a gradient of acetonitrile (5-100%)/0.1 M perchloric acid+0.5 M lithium perchlorate was used. The combinatorial derivative in the chromatogram gave one clear broadened peak and was not separated into components, although the retention time differed from both the original polymyxin and its completely substituted derivatives. This testified to the fact that complex supramolecular structures were formed between different combinatorial derivatives (in our case there were 764 of them), which were not separated chromatographically. This combinatorial derivative (PMM) also behaves similarly when separated in a thin layer (acetonitrile: water, UV detection after treatment with 10% sulfuric acid solution) and gives only one band that does not coincide with any of the obtained derivatives.

[0087] FIG. 5.

[0088] As can be seen from FIG. 5, TLC, the Combinatorial mixture (lane IXa-d) is less mobile and has Rf=0.27, while the initial unmodified gentamicin (VIII) is the lightest and Rf=0.36. Fully acylated gentamicin (IXb) and succinyl gentamicin (IXc) are intermediate between native gentamicin and combinatorial. The combinatorial gentamicin band is not separated either by two-dimensional TLC or by HPLC (not shown).

Example 4. Determination of the Antimicrobial Activity of Patented Agents In Vitro

[0089] The objects of the study were 14 combinatorial derivatives of antibiotics: polymyxin (IV), tetracycline (VII), gentamicin (IX), streptomycin (X), lincomycin (XI), kanamycin (XII), erythromycin (XIII), midecamycin (XIV), amphotericin B (XV), vancomycin (XVI), nystatin (XVII), amikacin (XVIII), tobramycin (XIX), spiomycin (XX). The antimicrobial activity of the compounds was studied in a collection of test strains of microorganisms obtained from the Institute of Microorganisms Museum and living culture museums of various laboratories of the IMI NAMS State University (Kharkov). The collection included the following multiresistant strains: bacteria —Staphylococcus aureus (Staphylococcus aureus), E. coli (Escherichia coli), Shigella flexneri (dysentery bacillus), B. antracoides (anthracoid), Proteus vulgaris (vulgar protea), Pseudomonas aureinosa (stick) of mushrooms—Candida spp. (yeast-like fungi of the genus Candida), Microsporium Ian. (causative agent of microsporia), Trich. mentagrophytes (causative agent of trichophytosis), Aspergillus niger (aspergillus).

[0090] For the cultivation of bacteria, Hottinger broth (pH 7.2-7.4) was used, and for fungi, Saburo medium (pH 6.0-6.8). Antimicrobial and fungistatic activity was evaluated by the minimum inhibitory concentration (MIC)—the smallest amount of a substance that completely inhibited the growth of bacteria or fungi after cultivation. IPC was determined by the conventional method of serial dilutions with a coefficient of 2 in a liquid nutrient medium. For this purpose, the initial dilution of the test compound with a concentration of 50 μg/ml of culture medium (Hottinger broth) was prepared. Subsequently, a sequential double dilution was carried out, as a result of which 25 ml were contained in 1 ml of culture medium; 12.5; 6.25; 3.12 μg/ml, etc.

[0091] The reference standard was nystatin and ethacridine lactate. This combinatorial mixture of antibiotic derivatives behaves like a quasi-fluid system—it adapts to the individual conditions of the body, preventing the emergence of resistance in bacteria. The results of studies of the antimicrobial and antifungal activity of derivatives of tannins are presented in table. 1.

[0092] As can be seen from the table. 1, the maximum antimicrobial activity against resistant strains of microorganisms was shown by all combinatorial derivatives of antibiotics, in contrast to their unmodified derivatives, which initially did not have antimicrobial activity against these strains. Compounds (XV) and (XVII) belonging to the groups of derivatives of polyene antifungal agents showed antifungal activity at the level of the initial derivatives (31.25 μg/ml), while the initial unmodified amphotericin and nystatin did not exert antifungal activity on these resistant strains. Derivatives (IV), (XVIII), (XIX) (XX) had lower antifungal activity, although initially these antibiotics did not have antifungal activity at all, especially with respect to multiresistant strains. The maximum activity against almost all the studied microorganisms at a dose of 3.12 μg/ml was shown by the XII derivative or the supramolecular combinatorial kanamycin derivative, while the initial derivative did not have activity on these resistant strains at all.

TABLE-US-00001 TABLE 1 Antibacterial and fungistatic activity of supramolecular combinatorial derivatives of antibiotics based on MIC. μg/ml Strains of microorganisms * S. E. S. B. P. P. C. M. T. A. aureus coli flexneri antracoides aeruginosa vulgaris albicans anosum mentagraphytes niger Connection IMI IMI IMI IMI IMI IMI res3 IMI IMI IMI number res3 res3 res3 res3 res3 res3 IMI res3 res3 res3 IV 3.12 3.12 6.25 6.25 250    250 — 250 250 250 VII 3.12 3.12 3.12 6.25 6.25 — — — — — IX 3.12 3.12 3.12 6.25 6.25 — — 250 — — X 6.25 6.25 6.25 6.25 12.5 250 — — — — XI 6.25 6.25 6.25 6.25 12.5 250 — — — — XII 3.12 3.12 3.12 3.12 3.12 3.12 — — — — XIII 6.25 6.25 6.25 6.25 3.12 6.25 — — — — XIV 6.25 6.25 6.25 3.12 6.25 6.25 — — — — XV — — — — — — 31.25 31.25 31.25 31.25 XVI 3.12 3.12 3.12 250    12.5 3.12 — — — — XVII — — — — — — 31.25 31.25 31.25 31.25 XVIII 6.25 6.25 6.25 6.25 12.5 6.25 31.25 250 250 250 XIX 6.25 6.25 6.25 6.25 3.12 6.25 31.25 250 250 250 XX 3.12 3.12 3.12 3.12 3.12 3.12 250 250 250 250 31.2  125    — — — — 62.5 62.5 16.2 62.5   Notes: — does not have activity in a dose of up to 500 mcg/ml; * the initial unmodified derivatives of antibiotics did not affect the growth of these strains even at doses higher than 500 μg/ml.

[0093] Therefore, combinatorial supramolecular derivatives of antibiotics have potent antimicrobial and antifungal activity against multiresistant strains of microorganisms and fungi, whereas the initial unmodified antibiotics did not have such activity at all.

Example 5. The Effectiveness of SCAA Against the Resistant Strain of Escherichia coli IMI2001 in the Model of Neutropenic Peritonitis/Sepsis in Mice and the Assessment of the Average Effective Dose (ED50)

[0094] The objective of this study was to study the dose response relationship after intravenous (iv) administration of a single dose of SKPA (CBT) in the range of 0.1612 mg/kg. The effect was investigated against resistant E. coli IMI2001 in a neutropenic peritonitis model. Administration of meropenem at a dose of 40 mg/kg was included as a positive control group. The number of colonies in the blood and peritoneal fluid was determined 5 hours after administration. The mouse peritonitis/sepsis model is a well-known model for antimicrobial activity studies as described by N. FrimodtMoller and J. D.

[0095] Knudsen in Handbook of Animal Models of Infection (1999), ed. by 0. Zak & M. A. Sande, Academic Press, San Diego, US.

[0096] 30 female outbred NMRI mice, 2530 grams (Harlan Scandinavia).

[0097] Escherichia coli IMI2001 from IMINAMN, Kharkov, Ukraine. Clinical isolate from a human wound from 2003 with multidrug resistance (to ampicillin, ceftazidime, aztreonam, gentamicin, ciprofloxacin).

[0098] ASCA in Ringer's acetate, pH 6, 1.2 mg/ml, 6.0 ml. The solution was stored at 4° C. until use. The analyzes of the compositions used for administration were performed at the end of the phase of the study conducted on live animals, and the following results were obtained in these analyzes.

TABLE-US-00002 TABLE 2 The ratio of concentrations in the experiment Estimated Concentration Measured concentration 1.2 mg/ml 1.16 mg/ml 0.6 mg/ml 0.53 mg/ml 0.3 mg/ml 0.28 mg/ml 0.15 mg/ml 0.12 mg/ml 0.075 mg/ml 0.047 mg/ml 0.03 mg/ml 0.040 mg/ml 0.016 mg/ml 0.002 mg/ml [0099] Filler (Ringer's acetate, pH 6). The solution was stored at 4° C. until use. [0100] Meronem (AstraZeneca, 500 mg infusion substance, meropenem). Sterile water. [0101] Sterile 0.9% saline. [0102] Cyclophosphamide, Apodan (APharma, 1 g). Agar plates and 5% horse blood. [0103] Cups with agar, bromothymol blue and lactose.

[0104] Laboratory vivarium and mouse maintenance. Temperature and humidity in the vivarium were recorded daily. The temperature was 21° C.+/2° C. and could be controlled by heating and cooling. Humidity was 55+/10%. The change of air occurred approximately 1020 times per hour, and the period of light/darkness was in the 12 hour interval 06:0018:00/18:00-06:00. Mice had free access to drinking water for pets and food (2016, Harlan). Mice were kept in type 3 macrolon cells, 3 mice per cell. Tappen Aspen Wood was used as a litter. In addition, animals were given Sizzlenest paper strips as nest material. Mice were labeled with tails to distinguish between mice in the cage. Mice were weighed one day prior to administration.

[0105] Preparation of ASCA Solutions

[0106] A solution with a concentration of 1.2 mg/ml was further diluted in PBS vehicle as follows.

TABLE-US-00003 TABLE 3 The ratio of the concentration of the substance and the filler  0.6 mg/ml~7.5 mg/kg:  1.5 ml 1.2 mg/ml ASCA + 1.5 ml filler  0.3 mg/ml~5.0 mg/kg:  1.5 ml 0.6 mg/ml ASCA + 1.5 ml filler 0.15 mg/ml~2.5 mg/kg:  1.5 ml 0.3 mg/ml ASCA + 1.5 ml filler 0.075 mg/ml~1.25 mg/kg: 1.5 ml 0.15 mg/ml ASCA + 1.5 ml filler  0.03 mg/ml~0.63 mg/kg: 1.5 ml 0.075 mg/ml ASCA + 2.25 ml filler 0.016 mg/ml~0.16 mg/kg: 1.5 ml 0.03 mg/ml ASCA + 1.5 ml filler

[0107] Preparation of a solution of meropenem. Administration of meropenem at a dose of 40 mg/kg was included as a positive control group. A total of 500 mg of meropenem (one ampoule) was dissolved in 10 ml of water at a concentration of about 50 mg/ml. This stock solution was further diluted to 4 mg/ml (0.4 ml, 50 mg/ml+4.6 ml saline).

[0108] Preparation of cyclophosphamide A total of 1 g of cyclophosphamide (one Apodan 1 g ampoule) was dissolved in 50 ml of water, approximately 20 mg/ml, for each day of its use. This stock solution was further diluted to 11 mg/ml (16.5 ml, 20 mg/ml+13.5 ml of physiological saline) for use on day 4 or to 5 mg/kg (8.25 ml 20 mg/ml+21, 75 ml of physiological saline) for use on 1 day.

[0109] The introduction of cyclophosphamide to mice. Neutropenia was induced in mice by injection of 0.5 ml of cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) before infection.

[0110] Infection of mice. Fresh E. coli IMI2001 colonies obtained overnight in an agar plate and 5% horse blood were suspended and diluted in sterile saline to approximately 2×10 6 CFU/ml. One hour before the start of administration (time point 1 h), mice were intraperitoneally infected with 0.5 ml of a suspension of E. coli in the lateral lower quadrant of the abdomen. Approximately 0.51 hours after the administration, 45 μl of neurofen (20 mg ibuprofen per ml, corresponding to 30 mg/kg) was orally administered to the mice as a painkiller.

[0111] The introduction of drugs to mice. Mice were given a single intravenous administration of CBT, meropenem, or excipient into the lateral tail vein for approximately 30 seconds in a volume of 10 ml/kg at time 0 h (see Table 1). The dose determination was based on an average body weight of 30 g. Mice with a body weight of 2832 g were injected with 0.30 ml of solution. Mice weighing 2728 g were injected with 0.25 ml of the solution and mice weighing 32.136 g were injected with 0.35 ml of the solution.

TABLE-US-00004 TABLE 4 Scheme of introduction and collection of samples in the model of peritonitis in mice Infection, Intravenous Sampling and No intraperitoneally, 1 h administration, 0 h 0 h 5 h 0.5 ml E. coli IMI2001 Filler, Ringer's acetate 1-2-3 1 × 10.sup.6 CFU/ml CBT, 0.16 mg/kg 4-5-6 CBT, 0.30 mg/kg 7-8-9 CBT, 0.75 mg/kg 10-11-12 CBT, 1.5 mg/kg 13-14-15 CBT, 3.0 mg/kg 16-17-18 CBT, 6.0 mg/kg 19-20-21 CBT, 12 mg/kg 22-23-24 Meropenem, 40 mg/kg 25-26-27 Without injection 28-29-30

[0112] T indicates time relative to administration. The numbers in the columns of the sampling represent the identification numbers of mice. Clinical scoring of mice. Mice were observed during the study and assigned them scores from 0 to 5 depending on their behavior and clinical signs.

[0113] Score 0: healthy.

[0114] Score 1: minimal clinical signs of infection and inflammation, such as observing minimal signs of an upset or change in activity.

[0115] Score 2: distinct signs of infection, such as social self-isolation, decreased curiosity, altered body position, piloerection, or changes in the pattern of movement.

[0116] Score 3: pronounced signs of infection, such as stiffness, decreased curiosity, altered body position, piloerection, pain, or changes in the nature of movements.

[0117] Score 4: severe pain, and the mouse was immediately euthanized to minimize the suffering of the animal.

[0118] Score 5: mouse death.

[0119] Fence Samples.

[0120] The number of colonies was determined in blood and peritoneal fluid for 0 and 5 hours. Mice were anesthetized with CO2+O2 and blood was taken from an axillary incision into Eppendorf tubes coated with ethylene diamine tetraacetic acid (EDTA). Mice were sacrificed immediately after blood sampling, and a total of 2 ml of sterile physiological saline was administered intraperitoneally and a gentle massage of the abdomen was performed until it was opened and the fluid samples were pipetted. Then, each sample was diluted 10 times in physiological saline and drops of 20 μl were applied to blue agar plates. All agar plates were incubated for 1822 hours at 35° C. in air.

[0121] Results. The number of colonies was determined at the beginning of administration and 5 hours after administration. The CFU count and clinical indicators of the mice are shown in Table 3. Before the calculations, a log 10 transformation of the CFU count was performed.

[0122] CFU/ml in the infectious material was determined to be 6.29 log 10. At the beginning of administration, the average log 10 CFU/ml was 5.76 in the peritoneal fluid and 5.13 in the blood, and the CFU levels were kept at a similar level in the filler group (5.72 and 4.65 log 10 CFU/ml in the peritoneal fluid and blood, respectively) 5 hours after administration. Slightly reduced CFU levels were observed in the blood and peritoneal fluid after administration of CPR at a dose of 0.163.0 mg/kg. The introduction of CPR at a dose of 6 and 12 mg/kg led to a significant decrease in the levels of CFU (p<0.001) compared with the introduction of the filler, both in the peritoneal fluid and in the blood (table 3). The introduction of meropenem at a dose of 40 mg/kg also led to a significant decrease compared with mice that were injected with excipient, both in the blood (p<0.05) and in the peritoneal fluid (p<0.01).

[0123] Dose response curves (data not shown) were calculated in GraphPad Prism using a sigmoid dose response curve (variable angle). The ED50 values determined from these curves were 2.11±1.01 mg/kg in the peritoneal fluid and 2.12±0.33 mg/kg in the blood. The maximum effect of CBT, Emax, was determined as the difference in log CFU in the absence of response and at the maximum response. The lack of response was characterized as the number of colonies at a level determined in mice that were injected with vehicle. Emax, calculated as the difference between the “Top plateau” and the “Bottom plateau” in GraphPad Prism using a sigmoid dose response curve, was 4.72 log 10 CFU for peritoneal fluid and 3.15 log 10 CFU for blood. In addition, 1, 2, and 3 log kills were estimated using GraphPad Prism, defined as the dose required to reduce the bacterial load by 1, 2, or 3 log compared to the start of treatment. 1, 2 and 3 log kill for CBT were 1.11 mg/kg, 2.95 mg/kg and 4.73 mg/kg, respectively, in peritoneal fluid and 0.25 mg/kg, 2.75 mg/kg and 3.78 mg/kg, respectively, in the blood. In all administration groups, zero or low clinical scores were observed (Table 3).

[0124] Discussion and conclusion. The objective of this study was to study the dose response relationship after intravenous (iv) administration of a single dose of CBT in the range of 0.1612 mg/kg. The effect was investigated against E. coli IMI2001 in a neutropenic peritonitis/sepsis model. It was determined that ED50 values for CBT were 2.11±1.01 mg/kg in peritoneal fluid and 2.12±0.33 mg/kg in blood. An estimated 1 log kill was 1.11 mg/kg in peritoneal fluid and 0.25 mg/kg in blood. An estimated 2 log destruction was 2.95 mg/kg in peritoneal fluid and 2.76 mg/kg in blood. An estimated 3 log kill was 4.73 mg/kg in peritoneal fluid and 3.78 mg/kg in blood.

TABLE-US-00005 TABLE 5 The effectiveness of CBT against E. coli IMI2001 calculated in GraphPad Prism CBT Peritoneal fluid Blood The greatest 0.325 CFU/ml −0.985 CFU/ml The greatest −4.486 CFU/ml −4.138 CFU/ml Emax 4.811 CFU/ml 3.153 CFU/ml ED50 2.11 mg/kg 2.12 mg/kg R2 0.7524 0.6889 1 log destruction 1.11 mg/kg 0.25 mg/kg 2 log destruction 2.95 mg/kg 2.76 mg/kg 3 log destruction 4.73 mg/kg 3.78 mg/kg

TABLE-US-00006 TABLE 6 The amount of ASCA in the blood and peritoneal fluid of mice with neutropenia. which was administered a single dose of ASCA. meropenem or excipient log.sub.10 CFU Injection Clinical Score Average Blood T = 0 h No mice Time T = 0 h T = 5 h PF in PF Blood average Filler 1 T = 5 1 1 5.74 5.05 2 T = 5 1 0 5.54 5.72 4.78 4.65 3 T = 5 1 1 5.88 4.11 CBT 4 T = 5 1 1 5.16 4.27 0.16 5 T = 5 1 1 4.78 5.31 4.19 4.54 mg/kg 6 T = 5 1 0 5.98 5.16 CBT 7 T = 5 1 0 2.76 1.40 0.30 8 T = 5 1 1 5.74 4.26 4.63 2.88 mg/kg 9 T = 5 1 0 4.27 2.60 CBT 10 T = 5 1 0 5.74 5.07 0.75 11 T = 5 1 1 4.95 5.16 4.30 4.46 mg/kg 12 T = 5 1 1 4.78 4.00 CBT 13 T = 5 1 0 3.33 3.51 1.5 14 T = 5 1 1 4.72 4.41 3.92 3.99 mg/kg 15 T = 5 1 0 5.18 4.54 CBT 16 T = 5 1 1 4.74 3.86 3.0 17 T = 5 1 1 4.74 3.91 3.57 2.81 mg/kg 18 T = 5 1 0 2.24 1.00 CBT 19 T = 5 1 1 2.18 2.12 1.00 1.00 6.0 20 T = 5 1 1 2.18 *** 1.00 *** mg/kg 21 T = 5 1 1 2.00 1.00 CBT 22 T = 5 1 1 1.00 1.36 1.00 1.00 12 23 T = 5 1 0 1.69 *** 1.00 *** mg/kg 24 T = 5 1 0 1.40 1.00 Meropenem 25 T = 5 1 1 3.92 2.64 2.48 2.38 40 26 T = 5 1 1 1.70 ** 1.70 * mg/kg 27 T = 5 1 0 2.30 2.95 No 28 T = 0 1 5.84 5.08 29 T = 0 1 5.78 5.76 4.98 5.13 30 T = 0 1 5.65 5.34 PF peritoneal fluid Asterisks indicate significant differences from the filler group (analysis of variance. multiple comparison). * corresponds to p < 0.05; ** corresponds to p < 0.01; *** corresponds to p < 0.001.

[0125] The detection limit is 1.4 log 10 CFU/ml. Samples without detected bacteria are presented as 1.0 log 10 CFU/ml.

Example 6. The Model of Peritonitis/Sepsis: The Effect of CAT at a Dose of 7.5 mg/kg Over Time Against Escherichia coli IMI2001 in NMRI Mice with Neutropenia

[0126] The objective of this study was to investigate the effectiveness of CPR in vivo after intravenous (iv) administration of a single dose of 7.5 mg/kg. The effect was tested against Escherichia coli IMI2001 in a peritonitis model in neutropenia NMRI mice to avoid the use of mucin, which is commonly used in a mouse peritonitis model. Neutropenia was induced in mice by injection of cyclophosphamide. Administration of meropenem at a dose of 40 mg/kg was included as a positive control group and vehicle administration was included as a negative control group. The number of colonies in peritoneal fluid and blood was determined 2 and 5 hours after administration.

[0127] Materials and Methods

[0128] 30 female outbred mice NMRI, 2832 grams (Kiev).

[0129] Escherichia coli IMI2001 from IMINAMN, Ukraine, Kharkov. Clinical isolate from a human wound from 2013 with multidrug resistance (to ampicillin, ceftazidime, aztreonam, gentamicin, ciprofloxacin).

[0130] PPC in Ringer's acetate, pH 6, 1.2 ml, 0.75 mg/ml. Assays of the administered compositions performed after the study showed a concentration of approximately 0.78 mg/ml.

[0131] Filler (Ringer's acetate, pH 6), 3 ml.

[0132] Meronem (AstraZeneca, 500 mg infusion substance, Apodan meropenem (APharma, 1 g cyclophosphamide).

[0133] Sterile water.

[0134] Sterile 0.9% saline.

[0135] Agar plates and 5% horse blood.

[0136] Cups with agar, bromothymol blue and lactose.

[0137] Laboratory vivarium and mouse maintenance. Temperature and humidity in the vivarium were recorded daily. The temperature was 21° C.+/2° C. and could be controlled by heating and cooling. Humidity was 55+/10%. The change of air took place approximately 1020 times per hour, and the period of light/darkness was in the 12 hour interval 06:0018:00/18:0006:00. Mice had free access to drinking water for pets and food (2016, Harlan). Mice were kept in type 3 macrolon cells, 3 mice per cell. Tappen Aspen Wood was used as a litter. In addition, animals were given Sizzlenest paper strips as nest material.

[0138] Mice were labeled with tails to distinguish between mice in the cage.

[0139] CAT solution. A solution of CPP with a concentration of 0.75 mg/ml was stored at +4° C. for up to one hour before injection, then at room temperature.

[0140] Preparation of a solution of meropenem. A total of 500 mg of meropenem (one ampoule) was dissolved in 10 ml of water, approximately 50 mg/ml, on the day of its use. This stock solution was further diluted to 4 mg/ml (0.4 ml, 50 mg/ml+4.6 ml saline).

[0141] Preparation of cyclophosphamide. A total of 1 g of cyclophosphamide (one Apodan ampoule) was dissolved in 50 ml of water, approximately 20 mg/ml, for each day of its use. This stock solution was further diluted to 11 mg/ml (16.5 ml, 20 mg/ml+13.5 ml of physiological saline) for use on day 4 or to 5 mg/kg (8.25 ml 20 mg/ml+21, 75 ml of physiological saline) for use on 1 day.

[0142] The introduction of cyclophosphamide to mice. Neutropenia was induced in mice by injection of 0.5 ml of cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) before infection.

[0143] Infection of mice. Fresh E. coli AID #172 colonies obtained overnight in an agar plate and 5% horse blood were suspended and diluted in sterile saline to approximately 2×10 6 CFU/ml. One hour before the start of administration (time point 1 h), mice were intraperitoneally infected with 0.5 ml of a suspension of E. coli in the lateral lower quadrant of the abdomen. 2.5 h after administration with significant clinical signs of infection, mice were orally administered 45 μl of neurofen (20 mg ibuprofen per ml, which corresponded to 30 mg/kg) as a painkiller.

[0144] Score mice. At each sampling in mice, a clinical assessment of the clinical signs of infection was performed.

[0145] Score 0: healthy.

[0146] Score 1: minimal clinical signs of infection and inflammation, for example, observation of minimal signs of an upset or activity change

[0147] Score 2: distinct signs of infection, such as social self-isolation, decreased curiosity, altered body position, piloerection, or changes in the pattern of movement.

[0148] Score 3: pronounced signs of infection, such as stiffness, decreased curiosity, altered body position, piloerection, pain, or changes in the nature of movements.

[0149] Score 4: severe pain, and the mouse was immediately euthanized to minimize the suffering of the animal.

[0150] Score 5: mouse death.

[0151] The introduction of drugs to mice. The mice were given a single intravenous administration of CPR, meropenem, or excipient into the lateral tail vein for approximately 30 seconds at a time point of 0 h (see Table 6). The dose determination was based on an average body weight of 30 g. Mice with a body weight of 2832 g were injected with 0.30 ml of solution. Mice weighing 2728 g were injected with 0.25 ml of the solution and mice weighing 32.136 g were injected with 0.35 ml of the solution. Mice 17 accidentally injected 0.35 ml, despite the fact that its body weight was 29.5 g. Apparently, this did not affect the results, since the CFU levels in this mouse were very similar to two other mice in this group.

TABLE-US-00007 TABLE 7 The scheme of introduction and sampling in the model of peritonitis in mice Fence Fence Fence Infection Injection samples samples samples T = −1 h T = 0 h T = 0 h T = 2 h T = 5 h 0.5 ml PPC - Gearbox 4, 5, 6 E. coli Meropenem 7, 8, 9 IMI2001 = Filler (Ringer's 10, 11, 12 10.sup.6 CFU/ml acetate) PPC 16, 17, 18 Meropenem 19, 20, 21 Filler (Ringer's 22, 23, 24 acetate) Non 25, 26, 27 Non 28, 29, 30 T indicates time relative to administration. The numbers in the sampling columns are the mouse identification numbers.

[0152] Fence samples. The number of colonies was determined in blood and peritoneal fluid at 0, 2 and 5 hours after administration according to Table 6.

[0153] Mice were anesthetized with CO2+O2 and blood sampling was performed from a section in the axillary region. The mice were sacrificed by cervical dislocation and a total of 2 ml of sterile physiological saline was injected intraperitoneally and a gentle massage of the abdomen was performed, then it was opened and a sample of the fluid was pipetted. Each sample was diluted 10 times in saline and drops of 20 μl were applied to plates with blood agar. All agar plates were incubated for 1822 hours at 35° C. in air.

[0154] Results. The number of colonies and clinical indicators of mice are shown in Table 2. Before the calculations, a log 10 transformation of the number of CFU was performed to obtain a normal distribution. CFU/ml in the infectious material was determined to be 6.50 log 10. At the beginning of administration, the average log 10 CFU/ml was 3.57 in the peritoneal fluid and 3.54 in the blood, and the level of CFU increased to 5.43 and 4.58 in the peritoneal fluid and blood, respectively, after 2 hours in animals that were injected filler, and up to 5.72 and 4.74 in the peritoneal fluid and blood, respectively, after 5 hours in mice that were injected with the filler, which was to be expected. 2 hours after the administration of CPT, significantly reduced CFU levels were observed, both in the blood and in the peritoneal fluid, compared with the administration of an excipient (p<0.001). An additional decrease in CFU levels, both in the blood and in the peritoneal fluid, was observed through 5 hours after administration of CPR (p<0.001 compared with the control vehicle).

[0155] CFU levels were more than 3 log.sub.10 CFU/ml lower than after vehicle administration.

[0156] Administration of meropenem also led to a significant (p<0.01) decrease in CFU levels compared to vehicle administration in peritoneal fluid 2 and 5 hours after administration. but in the blood only 5 hours after administration. The absence of a significant decrease in blood 2 hours after administration may reflect more pronounced variability in the filler group, rather than a weak effect of meropenem. Differences in CFU levels after administration of CPR or meropenem compared to vehicle administration were as follows.

TABLE-US-00008 TABLE 8 Differences in CFU levels after administration of ASCA or meropenem compared with the injection of filler ASCA, 2 h: peritoneum −1.63 blood −2.50 7.5 mg/kg Gearbox log CFU/ml log CFU/ml 5 h: peritoneum −3.76 blood −3.74 log CFU/ml log CFU/ml Meropenem, 40 mg/kg 2 h: peritoneum −1.51 blood −0.82 log CFU/ml log CFU/ml 5 h: peritoneum −1.51 blood −1.64 log CFU/ml log CFU/ml

[0157] All mice had mild symptoms of infection or no symptoms of infection.

[0158] Discussion and conclusion. The objective of this study was to investigate the effectiveness of CPR after intravenous (iv) administration of a single dose of 7.5 mg/kg in a model of neutropenic peritonitis in NMRI mice. For CPR, a significant (p<0.001) decrease of more than 3 log 10 CFU/ml was observed compared with the administration of the excipient in the blood and peritoneal fluid 5 hours after administration. In addition, a significant decrease (p<0.001) was observed both in the blood and in the peritoneal fluid 2 hours after the administration of CPT. Meropenem showed a significant decrease compared with the filler group (p<0.01) both in the blood and in the peritoneal fluid after 5 hours, but 2 hours after administration only in the peritoneal fluid.

TABLE-US-00009 TABLE 9 The number of colonies of E. coli IMI2001 in mice that were administered a single dose of CPR, filler or meropenem (Injection) log.sub.10 CFU or Sampling Score (Delivery) No Time T = 0 h T = 2 h T = 5 h PF PF Blood PPC CPR 4 T = 5 0 0 2.18    1.96 *** 1.00    1.00 *** 7.5 mg/kg 5 T = 5 0 1 2.30 1.00 6 T = 5 0 1 1.40 1.00 Meropenem 7 T = 5 0 0 4.38   4.21 ** 3.20  3.10** 40 mg/kg 8 T = 5 0 0 4.00 2.85 9 T = 5 0 0 4.26 3.26 10 T = 5 0 0 5.39 4.63 Filler 11 T = 5 0 0 5.99 5.72 5.24 4.74 12 T = 5 0 0 5.78 4.36 PPC 16 T = 2 0 1 4.24   3.79 ** 2.04   2.08 ** 7.5 mg/kg 17 T = 2 0 1 3.60 2.20 18 T = 2 0 1 3.54 2.00 Meropenem 19 T = 2 0 1 4.12   3.92 ** 3.60 3.76 40 mg/kg 20 T = 2 0 1 3.40 3.57 21 T = 2 0 1 4.24 4.11 22 T = 2 0 0 4.89 3.21 Filler 23 T = 2 0 1 5.65 5.43 5.39 4.58 24 T = 2 0 0 5.74 5.15 25 T = 2 0 0 4.45 4.39 No 26 T = 2 0 0 5.42 5.08 4.57 4.33 27 T = 2 0 0 5.38 4.02 28 T = 0 0 1.88 1.00 No 29 T = 0 0 3.71 3.57 4.27 3.54 30 T = 0 0 5.13 5.35 PF peritoneal fluid. Used infectious material: 1.97 × 106 CFU/ml. * Mice were injected with 0.35 ml instead of 0.30 ml of the test compound. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to the filler group.

Example 7. A Model of Hip Infection with Neutropenia: The Effectiveness of CPR Against Escherichia coli IMI2001 H oUeHκa ED50

[0159] Introduction The objective of this study was to study the dose response after intravenous (iv) administration of a single dose of CPR in the range of 0.1612 mg/kg. The effect was investigated against E. coli IMI2001 in a model of femoral infection with neutropenia. Administration of meropenem at a dose of 40 mg/kg was included as a positive control group. The number of colonies in the hips was determined 5 hours after administration. The hip infection model is a well-known model for studies of the antimicrobial effect and tissue penetration, as described by S. Gudmundsson & N. Erlensdottir: Handbook of Animal Models of Infection (1999), ed. by O. Zak & M. A. Sande, Academic Press, San Diego, US, and some other publications. See the review by D. Andes & C. Craig: Animal model pharmacokinetics and pharmacodynamics: a critical review. International Journal of Antimicrobial Agents, 19 (4): 261268.

[0160] Materials and methods. 40 female outbred mice NMRI, 2530 grams (Kiev, Ukraine). Escherichia coli IMI2001 from IMINAMN, Kharkov, Ukraine. Clinical isolate from a human wound from 20q3 with multidrug resistance (to ampicillin, ceftazidime, aztreonam, gentamicin, ciprofloxacin).

[0161] PPC in Ringer's acetate, pH 6, 1.2 mg/ml, 6.0 ml. The solution was stored at 4° C. until use. The analyzes of the compositions used for administration were performed at the end of the phase of the study conducted on live animals, and the following results were obtained in these analyzes.

TABLE-US-00010 TABLE 10 The concentration of the drugs used for experiment Estimated Concentration Measured concentration 1.2 MΓ/ml 1.4 MΓ/ml 0.6 MΓ/ml 0.57 MΓ/ml 0.3 MΓ/ml 0.28 MΓ/ml 0.15 MΓ/ml 0.15 MΓ/ml 0.075 MΓ/ml 0.063 MΓ/ml 0.03 MΓ/ml 0.02 MΓ/ml 0.016 MΓ/ml 0.014 MΓ/ml

[0162] Filler (Ringer's acetate, pH 6). The solution was stored at 4° C. until use.

[0163] Meronem (AstraZeneca, 500 mg infusion substance, meropenem). Lot number: 09466C. Expiration date: August 2013

[0164] Sterile water.

[0165] Sterile 0.9% saline.

[0166] Sendoxan (cyclophosphamide, Baxter, 1 g). Lot number: 0A671C. Shelf life: January 2013

[0167] Agar plates and 5% horse blood.

[0168] Agar, bromothymol blue and lactose cups

[0169] Laboratory vivarium and mouse maintenance. Temperature and humidity in the vivarium were recorded daily. The temperature was 21° C.+/2° C. and could be controlled by heating and cooling. Humidity was 55+/10%. The change of air occurred approximately 1020 times per hour, and the period of light/darkness was in the 12 hour interval 06:0018:00/18:00-06:00. Mice had free access to drinking water for pets and food (2016, Harlan). Mice were kept in type 3 macrolon cells, 4 mice per cell. Tappen Aspen Wood was used as a litter. In addition, animals were given Sizzlenest paper strips as nest material. Mice were labeled with tails to distinguish between mice in the cage. Mice were weighed one day prior to administration.

[0170] Preparation of ASCA solutions. A solution with a concentration of 1.2 mg/ml was further diluted in PBS vehicle as follows.

TABLE-US-00011 TABLE 11 Dosage of drugs in different concentrations and forms.  0.6 MΓ/ml~7.5 mg/kg:  1.5 ml 1.2 MΓ/ml PPC + 1.5 ml filler  0.3 MΓ/ml~5.0 mg/kg:  1.5 ml 0.6 MΓ/ml PPC + 1.5 ml filler 0.15 MΓ/ml~2.5 mg/kg:  1.5 ml 0.3 MΓ/ml PPC + 1.5 ml filler 0.075 MΓ/ml~1.25 mg/kg: 1.5 ml 0.15 MΓ/ml PPC + 1.5 ml filler  0.03 MΓ/ml~0.63 mg/kg: 1.5 ml 0.075 MΓ/ml PPC + 2.25 ml filler 0.016 MΓ/ml~0.16 mg/kg: 1.5 ml 0.03 MΓ/ml PPC + 1.5 ml filler

[0171] Preparation of a solution of meropenem. Administration of meropenem at a dose of 40 mg/kg was included as a positive control group. A total of 500 mg of meropenem (one ampoule) was dissolved in 10 ml of water, approximately 50 mg/ml. This stock solution was further diluted to 4 mg/ml (0.4 ml, 50 mg/ml+4.6 ml saline).

[0172] Preparation of cyclophosphamide. A total of 1 g of cyclophosphamide (one Sendoxan ampoule of 1 g) was dissolved in 50 ml of water, approximately 20 mg/ml, for each day of its use. This stock solution was further diluted to 11 mg/ml (16.5 ml, 20 mg/ml+13.5 ml of physiological saline) for use on day 4 or to 5 mg/kg (8.25 ml 20 mg/ml+21, 75 ml of physiological saline) for use on 1 day.

[0173] The introduction of cyclophosphamide to mice. Neutropenia was induced in mice by injection of 0.5 ml of cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) before infection.

[0174] Infection of mice. Fresh E. coli IMI2001 colonies obtained overnight in an agar plate and 5% horse blood were suspended and diluted in sterile saline to approximately 2×107 CFU/ml. One hour before the start of administration (time point 1 h), intramuscular infection of mice with 0.05 ml of a suspension of E. coli in the left hind paw was performed. Approximately 0.5 hours after the administration, 45 μl of neurofen (20 mg ibuprofen per ml, corresponding to 30 mg/kg) was orally administered to the mice as a painkiller.

[0175] The introduction of drugs to mice. Mice were given a single intravenous administration of ASCA, meropenem, or excipient into the lateral tail vein for approximately 30 seconds in a volume of 10 ml/kg at time 0 h (see Table 1). The dose determination was based on an average body weight of 30 g. Mice with a body weight of 2832 g were injected with 0.30 ml of solution. Mice weighing 2728 g were injected with 0.25 ml of the solution and mice weighing 32.136 g were injected with 0.35 ml of the solution.

TABLE-US-00012 TABLE 12 Scheme of administration and collection of samples in a model of femoral infection in mice Fence samples, Infection, Intravenous and NoNo mice intramuscularly, 1 h administration 0 h 0 h 5 h 0.05 ml Filler,    1-2-3-4 E. coli IMI20012 × PPC, 0.16 mg/kg 5-6-7-8 10.sup.7 CFU/ml PPC, 0.30 mg/kg 9-10-11-12 PPC, 0.75 mg/kg 13-14-15-16 PPC, 1.5 mg/kg 17-18-19-20 PPC, 3.0 mg/kg 21-22-23-24 PPC, 6.0 mg/kg 25-26-27-28 PPC, 12 mg/kg 29-30-31-32 Meropenem, 40 mg/kg 33-34-35-36 Without administration 37-38-39-40 T indicates time relative to administration, the numbers in the fence samples columns are the mouse identification numbers.

[0176] Clinical scoring of mice. Mice were observed during the study and assigned them scores from 0 to 5 depending on their behavior and clinical signs.

[0177] Score 0: healthy.

[0178] Score 1: minimal clinical signs of infection and inflammation, for example, observing minimal signs of an upset or change in activity.

[0179] Score 2: distinct signs of infection, such as social self-isolation, decreased curiosity, altered body position, piloerection, or changes in the pattern of movement.

[0180] Score 3: pronounced signs of infection, such as stiffness, decreased curiosity, altered body position, piloerection, pain, or changes in the nature of movements.

[0181] Score 4: severe pain, and the mouse was immediately euthanized to minimize the suffering of the animal.

[0182] Score 5: mouse death.

[0183] Sampling

[0184] The number of colonies was determined in the hips at 0 and 5 h. Mice were anesthetized with CO2 +O2 and killed Immediately after this, the skin was removed, the hind left paw was received and it was frozen at 70° C. After thawing, the hips were homogenized using Dispomix Drive. Then, each sample was diluted 10 times in physiological saline and drops of 20 μl were applied to blue agar plates. All agar plates were incubated for 1822 hours at 35° C. in air.

[0185] Results. The number of colonies was determined at the beginning of administration and 5 hours after administration. The number of CFU is shown in Table 3. Before the calculations, a log 10 transformation of the number of CFU was performed. CFU/ml in the infectious material was determined to be 7.35 log 10, which corresponds to 6.05 log 10 CFU/mouse. The observed high variability may be due to suboptimal infection of some mice, leading to too low CFU. For this reason, the smallest value in each group was excluded from the graphs and calculations (see Table 3). At the beginning of administration, the average log 10 CFU/ml was 4.93 and increased to 6.49 log 10 CFU/ml in the filler group 5 hours after administration. Slightly reduced CFU levels were observed after administration of CPR at a dose of 0.163.0 mg/kg.

[0186] After administration of ASCA at a dose of 6 mg/kg (p<0.05) and 12 mg/kg (p<0.01), a significant decrease in CFU levels was observed compared with the administration of excipient (Table 9). The administration of meropenem at a dose of 40 mg/kg resulted in a definite but insignificant decrease compared to the mice that were injected with the vehicle. Dose response curves (not shown) were calculated in GraphPad Prism using a sigmoid dose response curve (variable angle of inclination). The ED50 determined from these curves was 5.9 mg/kg. However, no lower plateau has been received, and therefore this value may be underestimated.

[0187] The maximum ASCA effect, Emax, was determined as the difference in log CFU in the absence of response and at the maximum response. The lack of response was characterized as the number of colonies at a level determined in mice that were injected with vehicle. Emax, calculated as the difference between the Upper Plateau and the Lower Plateau in GraphPad Prism using a sigmoid dose response curve, was 2.4 log 10 CFU/ml. In addition, 1 log kill, defined as the dose required to reduce the bacterial load by 1 log compared to the start of treatment, determined using GraphPad Prism, was 6.1 mg/kg. 2 and 3 log destruction were not received.

[0188] No mouse showed a clinical sign of infection at any time point.

TABLE-US-00013 TABLE 13 ASCA Effectiveness Against E. coli AID # 172 Calculated in GraphPad Prism The greatest  1.1 Δlog10 CFU/ml The greatest −1.3 Δlog.sub.10 CFU/ml Emax  2.4 Δlog10 CFU/ml ED50  5.9 mg/kg R2  0.46 1 log destruction  6.1 mg/kg

TABLE-US-00014 TABLE 14 E. coli IMI2001 mice with neutropenia in the thighs of mice injected with a single dose of PPC, Meropenema or filler No log.sub.10 No log.sub.10 Administration the Sampling CFU, The Administration the Sampling CFU, The T = 0 h mouse Time hip average T = 0 h mouse Time hip average Filler  1 T = 5 5.16 6.49 PPC 21 T = 5 5.30 6.07  2 T = 5 6.47 3.0 22 T = 5 6.03  3 T = 5 6.13 mg/kg 23 T = 5 4.85  4 T = 5 6.86 24 T = 5 6.89 PPC  5 T = 5 3.18 5.16 PPC 25 T = 5 2.75 4.10* 0.16  6 T = 5 6.03 6.0 26 T = 5 4.54 mg/kg  7 T = 5 3.30 mg/kg 27 T = 5 1.48  8 T = 5 6.15 28 T = 5 5.01 PPC  9 T = 5 2.00 5.09 PPC 29 T = 5 2.48 3.32** 0.30 mg/kg 10 T = 5 5.40 12 mg/kg 30 T = 5 3.27 11 T = 5 3.10 31 T = 5 3.19 12 T = 5 6.78 32 T = 5 3.51 PPC 13 T = 5 2.9 6.33 Meropenem 33 T = 5 3.08   4.25 0.75 mg/kg 14 T = 5 5.72 40 mg/kg 34 T = 5 3.81 15 T = 5 7.27 35 T = 5 4.99 16 T = 5 6.00 36 T = 5 3.94 PPC 17 T = 5 2.56 5.62 Without 37 T = 0 4.98 4.93 1.5 18 T = 5 6.23 administration 38 T = 0 3.81 mg/kg 19 T = 5 4.93 39 T = 0 4.79 20 T = 5 5.70 40 T = 0 5.01  This value was excluded from the calculations. since its salts are a drop-out value Asterisks indicate significant differences from the filler group (analysis of variance, multiple comparison). *corresponds to p <0.05; **corresponds to p <0.01.

[0189] The detection limit is 1.4 log.sub.10 CFU/ml. The scope of the invention described and claimed herein should not be limited to the specific aspects disclosed herein, since these aspects are merely illustrative of certain aspects of the invention. Assume that any equivalent aspects are included in the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Assume that such modifications are also included in the scope of the attached claims. In the event of a conflict, this description, including definitions, should be followed.