Polymyxin-based pharmaceutical composition for treating infectious diseases

Abstract

Field of application: The invention relates to medicine and pharmacy and allows to obtain new pharmaceutical compositions based on polymyxin for the treatment of severe infectious diseases, but not possessing nephrotoxic properties in therapeutic doses. Technical result: New combined dosage forms based on the antibiotic polymyxin with low nephrotoxicity and high activity.

Claims

1. A pharmaceutical composition with polymyxin and nephroprotectors, wherein the nephroprotectors are combinations of vitamins with a supramolecular combinatorial derivative of cyanocobalamin obtained by simultaneous acylation of the structure of cyanocobalamin with two different anhydrides and/or halogen anhydrides of monocarboxylic, dicarboxylic tricarboxylic and/or polycarboxylic acids; wherein a molar ratio of components of the combinatorial reaction is calculated according to the formulas:
k=n×(2.sup.n−1)  (1)
and
m=4×(3×2.sup.n-2−1)  (2) where n=number of substitutional groups in cyanocobalamin; m=number of moles of the starting cyanocobalamin and a number of different molecules of its combinatorial derivatives after synthesis; and k=number of moles of each of two modifiers in the combinatorial synthesis reaction to obtain a maximum number of different derivatives.

2. The pharmaceutical composition according to claim 1, wherein the combination of vitamins includes cholecalciferol.

3. The pharmaceutical composition according to claim 1, wherein the composition includes vikasol.

4. The pharmaceutical composition according to claim 1, wherein the combination of vitamins also include ascorbic acid or its derivatives.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Scheme for the chemical synthesis of a supramolecular derivative of combinatorial modified cyanocobalamin.

(2) FIG. 2. HPLC (Milichrom A-02) of cyanocobalamin (I), gradient A solution: 0.5 M lithium perchlorate/0.1 M perchloric acid, solution B: acetonitrile (B from 5% to 100%)

(3) FIG. 3. HPLC (Milichrom A-02) of the combinatorial supramolecular derivative of cyanocobalamin (IVd), gradient solution A: 0.5 M lithium perchlorate/0.1 M perchloric acid, solution B: acetonitrile (B from 5% to 100%)

(4) FIG. 4. HPLC (Milichrom A-02) triacetyl-cyanocobalamin (IVb), gradient solution A: 0.5 M lithium perchlorate/0.1 M perchloric acid, solution B: acetonitrile (B from 5% to 100%)

(5) FIG. 5. HPLC (Milichrom A-02) trisuccinyl-cyanocobalamin (IVc), gradient solution A: 0.5 M lithium perchlorate/0.1 M perchloric acid, solution B: acetonitrile (B from 5% to 100%)

(6) FIG. 6. Direct inhibition of colistin accumulation with cyanocobalamin (IVd) and cholecalciferol from the Polyfro composition

EMBODIMENTS OF THE INVENTION

(7) The following examples do not limit the possibility of using other methods of obtaining the composition of this invention, are presented only for the purpose of explaining the implementation of the claims.

Example 1. Synthesis of a Supramolecular Combinatorial Derivative of Cyanocobalamin (SCDC)

(8) 20 mM cyanocobalamin (I) is dissolved in 10 ml of dioxane, 21 mM succinic anhydride (III) and 21 mM acetic anhydride are added, the solution is stirred and heated under reflux for 20 minutes. The solution was poured into ampoules and lyophilized to remove solvent and acetic acid. The supramolecular combinatorial derivative (IVd) or SCDC is used to obtain pharmaceutical compositions, study the structure, and determine the biological activity. FIG. 1 shows a synthesis scheme for combinatorial derivatives of quercetin.

(9) One Source Cyanocobalamin Molecule Contains 3 Hydroxyl Groups Available for Modification, Including One Phosphate.

(10) Calculations of the number of moles of modifiers are carried out according to the combinatorics formulas:
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 cyanocobalamin for the reaction;
n—the number of hydroxyl groups available for modification, including one phosphate in the structure of cyanocobalamin (n=3);
k is the number of moles of each modifier.

(11) Thus, having only one initial quercetin molecule and two modifiers after combinatorial synthesis, we obtain 20 combinatorial derivatives with different degrees of substitution, different positions of the substituents and different permutations of the modifier residues, not just as a mixture, but as a difficult to separate supramolecular structure.

(12) Modifiers—succinic anhydride or acetic anhydride can be entered both simultaneously and sequentially—or first inject succinic anhydride, warm the mixture under reflux for 20 minutes, and then add acetic anhydride and also warm the mixture for another 20 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 molecular chlorides can be used as one of the modifiers instead of succinic anhydride.) For the purpose of studying the biological activity of the synthesized substances, various derivatives with different ratios of modifiers were obtained (Table 1).

(13) 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 of SCDC (IVd) in the chromatogram (FIG. 3) gave one clear broadened peak and did not separate into components, although the retention time was practically the same as pure cyanocobalamin, while its completely substituted derivatives had different retention volumes (FIG. 4., FIG. 5). This testified to the fact that complex supramolecular structures were formed between different combinatorial derivatives (in our case there are 20 of them), which were not separated chromatographically. This combinatorial derivative (SCDC) (IVd) also behaves similarly when 15 separated in a thin layer (acetonitrile:water) and gives only one band, which does not coincide with any of the derivatives obtained.

(14) Table 1 shows the results of screening of cyanocobalamin derivatives with different ratios of modifiers as a substrate of renal megalin (on the example of megalin of rat kidney homogenate). The initial cyanocobalamin is known to be a substrate/ligand of renal megalin with a moderate degree of interaction and an average affinity for megalin for absorption (IA=52%). The test was carried out by a micromethod in Eppendorf tubes on the ability of cyanocobalamin derivatives to bind to megalin: after centrifugation, the bound derivative together with megalin remained in the sediment, and the percentage of the remaining unreacted cyanocobalamin derivative in the supernatant was determined using HPLC on a Milichrom A-02 liquid chromatograph. The relative concentration in % relative to the original is shown in table 1.

(15) TABLE-US-00001 TABLE 1 The ability of megalin to bind different derivatives of cyanocobalamin SCDC The molar ratio of reagents * No p/p m k1 k2 % bound derivative 1. 20  84***  84*** 5 2. —//— 42  42  12 3. —//— 21  21  98 4. —//— 17  17  71 5. —//— 13  13  71 6. —//— 9 9 70 7. —//— 5 5 67 8. —//— 3 3 58 9. —//— 2 2 57 10. —//— 1 1 57 11. —//— 0 0 52 12. —//— 42  0 56 13. —//— 21  0 67 14. —//— 17  0 71 15. —//— 13  0 66 16. —//— 9 0 62 17. —//— 5 0 57 18. —//— 3 0 58 19. —//— 2 0 57 20. —//— 1 0 56 21. —//— 0 1 57 22. —//— 0 2 55 23. —//— 0 3 57 24. —//— 0 5 50 25. —//— 0 9 59 26. —//— 0 13  37 27. —//— 0 17  27 28. —//— 0 21  15 29. —//— 0 42  10 30. —//— 0  84*** 6 31. —//—  84*** 0 7 32. —//— 42  1 11 33. —//— 21  2 23 34. —//— 17  3 46 35. —//— 13  5 57 36. —//— 9 9 59 37. —//— 5 13  55 38. —//— 3 17  34 39. —//— 2 21  21 40. —//— 1 42  11 * m is the number of moles of cyanocobalamin in the combinatorial synthesis reaction; K1 is the number of moles of succinic anhydride in the reaction; K2 is the number of moles of acetic anhydride in the reaction; ***the maximum molar ratio at which all groups in cyanocobalamin are replaced, exceeding this ratio leads to the fact that unreacted modifiers remain in the reaction medium - succinic anhydride and acetic anhydride.

(16) As can be seen from table 1, only with the calculated ratio of components, when the maximum number of different derivatives of cyanocobalamin is formed, is a biological active and effective supramolecular structure formed (derivative 3 or SCDC or IVd in FIG. 1.), which almost completely (98%) binds to renal megalin. Other derivatives either did not differ from unmodified cyanocobalamin (52%) in their ability to bind to megalin, or were significantly less active. This indicates that with the optimal ratio of modifiers when all possible derivatives are formed in the solution (21 variations of the derivatives of zmanocobalamin with different permutations and arrangements in the substituents), a more complex supramolecular “quasi-lowering” structure with other properties and greater pharmacological activity is formed.

(17) In FIG. 2 shows the chromatogram of the initial cyanocobalamin (I), a combinatorial modified with two anhydrides (IVd) (FIG. 5). FIGS. 3-4 show HPLC chromatograms of two derivatives: fully succinylated (IVc) and fully acetylated cyanocobalamin (IVb), respectively, as control samples (No. 30 and No. 31 in Table 1). As can be seen from the graphs, the retention volume of the combinatorial derivative practically does not differ from the initial cyanocobalamin, but differs from fully modified derivatives. In addition, at the same concentration of cyanocobalamin, the peak area and width of its base are larger, which indicates that these are derivatives of cyanocobalamin and that there are several of them. The differences between the retention volume (I), (IVb) and (IVc) indicate the completion of the reaction of complete succinylation and acetylation in the structure of cyanocobalamin, respectively, and also that all these derivatives inside the complex (IVd) form a complex supramolecular structure.

(18) Fully succinylated and fully acetylated cyanocobalamin absorbed megalin only 6% and 7%, respectively. Thus, the IVd derivative obtained in accordance with combinatorial calculations is a fundamentally new supramolecular structure that has significantly different properties both from unmodified cyanocobalamin (No. 11 in Table 1) and from fully modified derivatives (No. 30 and No. 31 in Table 1) This structure cannot be separated using gradient HPLC, and the UV spectra of all derivatives practically coincide, although the retention time is different. This indicates the formation of new structures with a covalent bond based on cyanocobalamin, and at the same time, these structures together form a complex supramolecular structure similar to cyclodextrin complexes.

Example 2 Preparation of Polymyxin-Based Pharmaceutical Compositions with Nephroprotectors

(19) This object is achieved in that the pharmaceutical composition with antimicrobial activity contains an antibiotic polymyxin, contains cyanocobalamin, SCDC and cholecalciferol blocking its nephrotoxic effect, as well as targeted additives that contribute to the formation of dosage forms. According to the invention, SCDC, the main active agent is administered in an amount of 0.5-20%, polyvinylpyrrolidone is used as a solubilizer, sodium laurisulfate is a surfactant, the main components of the pharmaceutical composition, when a certain technological operation due to mechanochemical interaction form a complex with the studied pharmaceutical and pharmacological properties.

(20) Moreover, in the pharmaceutical composition SCDC and cholecalciferol is contained in an amount of from 2 to 7%. Instead of cholecalciferol, another nephroprotector can be used: vikasol, pantothenic acid, nicotinamide adenine dinucleotide. While SCDC in the composition may be contained in an amount of from 10 to 40%. The substances are pre-dissolved in ethanol together with lecithin or TWEEN-80, and ethanol is distilled off by heating or under vacuum. In addition, as the target additives, the solubilizer polyvinylpyrrolidone in the amount of 10-50% and the surface-active agent sodium lauryl sulfate in the amount of 0.25-10% were selected.

(21) The task is also achieved by the fact that in the method for producing a pharmaceutical composition with antimicrobial activity according to the invention, preliminarily polymyxin with nephroprotective agents and target additives are mixed, compacted, milled, the resulting mixture is mixed with pharmaceutically acceptable excipients, dry or wet granulation of the mixture is carried out, then the granulate is filled hard gelatin capsules or pressed and the tablets are coated with a polymer film. According to the invention, the developed method for producing a pharmaceutical composition allows to obtain dosage forms with a complex pharmaceutical composition in the form of coated tablets or capsules 10 containing components in such a 40 quantitative range, in %:

(22) Polymyxin 5-25%

(23) SCDC/cholecalciferol 10-40%

(24) Fillers up to 100%

(25) The three active substances of the new pharmaceutical composition were combined in it, taking into account the knowledge about their pharmacological and therapeutic properties obtained with the use of monopreparations based on their substances. The selected target additives, due to their physicochemical properties, contribute to the solubilization of active substances and their sufficiently high dissolution in the physiological aqueous medium of the gastrointestinal tract.

(26) The combination of three active substances with different physicochemical properties in one dosage form is a rather difficult task for pharmacy. The solution of the problem in technical terms was carried out by the consistent development of the manufacturing technology of the pharmaceutical composition and in accordance with the pharmacopoeial methods of its research.

(27) First of all, the pharmaceutical development of the pharmaceutical composition began with an assessment of the physicochemical properties of polymyxin, SCDC and cholecalciferol, primarily focusing on their ability to emulsify in aqueous media so that these active substances have maximum bioavailability when the drug is administered orally under physiological conditions of the stomach substances possible only with the use of auxiliary substances with properties. It was possible to achieve high solubility using solubilizers and surfactants.

(28) Such solubility modulators are polyvinylpyrrolidone (povidone, PVP) and sodium lauryl sulfate (LSS), TWEEN-80 and lectin, the quantitative values of their introduction into the pharmaceutical composition were determined during the pharmaceutical development of the composition, focusing on the solubility profiles of the samples, performing the solubility test. Studying the kinetics of dissolution of samples of the pharmaceutical composition, both granules for capsules and tablets, with variable mass values of PVP, LSS, TWEEN-80 and lecithin, their optimal ratio with polymyxin was established.

(29) TABLE-US-00002 TABLE 2 Composition and ratio of ingredients in one capsule or tablet core of the pharmaceutical composition Polyfro No p/p Name of ingredient in mg in % 1 2 4 5 1 Liposomes with cholecalciferol (LCh) 40.00 20.00 2 Polyvinylpyrrolidone K-25 40.00 20.00 3 Polymyxin 25.00 12.50 4 Microcrystalline cellulose 80.00 40.00 5 TWEEN-80 10.00 5.00 6 SCDC 3.00 1.50 6 Magnesium stearate 2.00 1.00 Content of capsule or tablet core: 200.00 100.00

(30) To ensure the physicochemical stability of the complex, the weight ratio between LSS and PVP should be in the range 1:1-1:3. It should be noted that these mass values of PVP are also sufficient to increase the solubility of the Polymyxin substance due to the formation of the corresponding complex compound. The povidones used in their molecular weights differ as follows: K90—1,000,000; K25—30000; C17—10000. Considering the characteristics of the dissolution kinetics of the complex of quercetin with PVP, a sufficient amount of LSS, a surfactant, was selected to facilitate the rapid and uniform wetting of the surface of the complex granulate.

(31) To ensure the physicochemical stability of the complex, the weight ratio between LSS and PVP should be in the range 1:1-1:3. It should be noted that these mass values of PVP are also sufficient to increase the solubility of the Polymyxin substance due to the formation of the corresponding complex compound. The povidones used in their molecular weights differ as follows: K90—1,000,000; K25—30000; C17—10000. Considering the characteristics of the dissolution kinetics of the complex of quercetin with PVP, a sufficient amount of LSS, a surfactant, was selected to facilitate the rapid and uniform wetting of the surface of the complex granulate.

(32) To ensure the physicochemical stability of the complex, the weight ratio between LSS and PVP should be in the range 1:1-1:3. It should be noted that these mass values of PVP are also sufficient to increase the solubility of the Polymyxin substance due to the formation of the corresponding complex compound. The povidones used in their molecular weights differ as follows: K90—1,000,000; K25—30,000; C17—10000. Considering the characteristics of the dissolution kinetics of the complex of quercetin with PVP, a sufficient amount of LSS, a surfactant, was selected to facilitate the rapid and uniform wetting of the surface of the complex granulate.

(33) This also reflected in the acceleration of the dissolution of the complex in the initial periods of time and, thus, could create optimal conditions for the absorption by the walls of the stomach of the soluble form of polymyxin. With significant hydrophobicity of the surface of the DN crystals, these selected auxiliary substances—PVP and VLF, also contributed to the solubility of this substance, and thereby could increase the bioavailability of this NSAID. A positive effect on the solubility of liposomes and polymyxin is also exerted by a selected amount of PVP and LSS, as evidenced by solubility profiles.

(34) All samples of the pharmaceutical composition, starting with a complex of active substances—polymyxin and liposomes with cholecalciferol with PVP and LSS, were obtained under consecutive technological operations by dry or wet granulation of the ingredients of the complex and auxiliary substances. The prepared granules were used to fill the capsules or they were tabletted and the solubility of these dosage forms was examined in accordance with the Pharmacopoeia Dissolution test. The solubility characteristics of Polyfro in the pharmaceutical composition indicated a significantly higher solubility of these active ingredients compared to the solubility of individual substances.

(35) In the course of pharmaceutical development, a sample of a pharmaceutical composition of a certain composition was made (see Table 2), called by the authors “Polyfro” and found application in studies of pharmacological properties.

(36) The pharmaceutical composition of the given composition obtained in accordance with the dry granulation technology includes: mixing the active substances with excipients, compacting or briquetting the mixture, grinding, mixing the grinding with excipients and granulating, compressing the granulate into tablets or filling it with hard gelatin capsules.

(37) Wet granulation technology is characterized by a granulation stage of the mixture, where instead of a compactor or press, wet granulation equipment and a dryer are used, such as a mixer granulator with agitators, such as Rota P or a granulator-dryer of a fluidized bed, such as Hurtling.

(38) We present specific examples of the invention. The following examples illustrate the main aspects of the present invention, but should not be construed as limiting. The preparation of all samples of granules of the complex of active substances—polymyxin, SCDC, and cholecalciferol in liposomes with PVP—was carried out under the conditions of technological granulation using the dry or wet method, using dry pressing (compaction) of a mixture of active ingredients, followed by grinding of the briquetted material.

(39) Wet granulation was carried out in granulator-dryers in a vacuum or warm running air in devices with a pseudo-boiling layer. Capsules were filled into the obtained granules or tabletted, examining the solubility of these dosage forms by the Dissolution test. The solubility characteristics of the polymyxin/SCDC/liposomal cholecalciferol system in the pharmaceutical composition indicated a significantly higher solubility of the complex of active ingredients compared with the solubility of individual substances. Thus, in the course of pharmaceutical development, a sample of the pharmaceutical composition was made, called by the inventors “Polyfro”, which found, as a preparation of a certain composition, the use in research of toxic-pharmacological properties:

(40) TABLE-US-00003 TABLE 3 Composition and ratio of ingredients per capsule or tablet of the Polyfro pharmaceutical composition No. p/p Ingredients name mg % 1 2 3 4 1 * Liposomes with cholecalciferol (LH) 40.00 20.00 2 * Polyvinylpyrrolidone K-25 40.00 20.00 3 * Polymyxin 25.00 12.50 4 Microcrystalline cellulose 80.00 40.00 5 * TWEEN 80 10.00 5.00 6 * SCDC 3.00 1.50 7 Magnesium stearate 2.00 1.00 8. Contents of capsule or tablet core 200.00 100.00 * these compounds together with the phosphate buffer in the % ratios were used to create a liquid injection form for in vitro and in vivo experiments.

(41) The pharmaceutical composition of the given composition (Table 3) obtained by dry granulation technology, including mixing the active substances with excipients, compacting or briquetting the mixture, grinding and mixing it with excipients, and finally granulate it is compressed into tablets or filled with hard gelatin capsules. Wet granulation technology is characterized by the stage of granulating the mixture, where instead of a compactor or press, wet granulation and drying equipment is used, such as a granulator-mixer with agitators, type 25 Rota P or granulator-dryer, such as Huttling with a boiling layer. The following embodiments illustrate aspects of the invention, but should not be construed as limiting.

Example 3. Obtaining a Liposomal Suspension Form of Cholecalciferol for Further Production of Granulate, Tablet Mass and Tablet Forms with Polymyxin and SPPC

(42) In 80-200 ml of 60% ethanol, 2-20 g of polymyxin, 1-3 g of cholecalciferol, 1-7 g of SCDC and 20-50 g of phosphatidylcholine are dissolved, then ethanol is distilled off under vacuum, 50 ml of distilled water are added to the resulting mixture and sonicated at 44 kHz for 15-50 minutes, the resulting suspension of liposomes is dried in a freeze dryer, and the resulting powder is used as shown in the previous examples to obtain tablet forms, injection forms. The size of liposomal nanoparticles in ultrasonic emulsification is 120-300 nm. If milk powder is used instead of lecithin, the particle size will be 500-1000 nm.

(43) A preclinical study of the pharmaceutical composition was carried out using the Polyfro sample in research tests in order to establish the full toxicological and pharmacological properties of the future drug, which could become a promising drug.

Example 4. Inhibition of the Accumulation of Polymyxin in the Kidneys

(44) In this experiment was determined, the accumulation of colistin in the kidneys of rats after intravenous injection of Polyfro (colistin, SCDC and liposomal cholecalciferol). Concentrations of colistin (CC), SCDC/cholecalciferol (CCF) were used based on preliminary modeling (the number of binding sites in one molecule, multiplied by their number in one megalin molecule, the molecular weight of megalin and the approximate amount of megalin in one kidney and the weight of the kidney) The result was the required amount of SCDC about 2 mg/kg of weight. For CS, a subtoxic dose was selected (based on the instructions). This is an experiment on the “displacement” of polymyxin from the kidneys with a synergistic combination of SCDC/HCF. As a result, about half of the polymyxin was displaced from the kidneys by SCDC/HCF. All components of CC, SCDC/CCF were determined in the sediment of renal homogenate and supernatant by HPLC. In our opinion, the Central Committee is one of the best candidates for nephroprotective agents, which now can significantly reduce the toxicity of polymyxin in clinics. FIG. 1 shows the accumulation of colistin (A) and the concentration (B) of colistin in megalin kidneys in mice. After 45 minutes of intravenous injection of the Polyfro mixture in terms of SCDC (2 mg/kg) and colistin (0.5 mg/kg) or physical. solution in the control collected urine of rats after 180 minutes. The content of colistin in the kidneys (A) and its concentration in terms of the weight of the kidneys (μg/g) (B) are shown in FIG. 1.

(45) Each column shows the average value (SD) of four measurements at P<0.05 (against control). As can be seen from FIG. 6., the accumulation of colistin in the kidney tissues both in absolute terms and in relative terms in terms of the weight of the kidneys decreases by more than two times due to the higher affinity for megalin in SCDC and cholecalciferol. All data are statistically significant and differences between groups and controls are significant.

(46) Table 4 presents the results of suppressing the accumulation of colistin in the tissues of the kidneys for other pharmaceutical compositions with colistin.

(47) TABLE-US-00004 TABLE 4 The accumulation of polymyxin in the presence of nephroprotectors in the tissues of the kidneys Concentration of colistin in the Accumulation of kidney, conversion colistin in the to kidney tissue in No. p/p Composition content kidneys mcg μg/g 1 Control (colistin) 20.5 ± 2.5  8.25 ± 2.25 2 Polifro 7.25 ± 1.25* 2.25 ± 1.25 3 Colistin/Pantothenic acid/NAD 6.25 ± 1.25* 2.50 ± 1.25 4 Colistin/SCDC/Vikasol 7.25 ± 2.25* 2.25 ± 1.25 5 Colistin/Cholecalciferol/Vikasol 9.50 ± 2.50* 4.50 ± 1.50 6 Colistin/SCDC/Pantothenic Acid 8.50 ± 1.50* 3.50 ± 1.50 7 Colistin/Pantothenic Acid/Vikassol 6.25 ± 1.25* 2.50 ± 0.50 8 Colistin/SCDC/NAD 7.25 ± 1.25* 2.25 ± 1.25 9 Colistin/Vikasol/NAD 8.50 ± 1.50* 3.50 ± 1.50 10 Colistin/Cholecalciferol/NAD 4.50 ± 1.50* 2.00 ± 0.50 11 Colistin/Cholecalciferol/pantothenic acid 7.25 ± 1.25* 2.25 ± 1.25 *P < 0.05 As can be seen from table 4, all combinations of high affinity megalin ligands successfully prevent the accumulation of polymyxin in the megalin of the kidneys. Differences with control without nephroprotectors are statistically significant.

Example 5. Prevention of Nephroprotective Agents from Polyfro Destruction of Renal Tissue

(48) The degree of destruction of kidney tissue was determined by the concentration in the urine of acetyl-beta-glucosaminidase (N-acetyl-beta-d-glucosaminidase) (NAG) and neutrophilic gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin) (NGAL) (Table 5).

(49) In the same rats treated with pure colistin and Polyfro, urine parameters were studied showing the degree of renal destruction (doses of 0.5/15 mg/kg polymyxin/SCDC in Polyfro as an intravenous injection 1 time per day for 10 days). The concentrations of NGAL and NAG in urine were determined using ELISA-text systems on a StatFax303+reader. Table 4 shows comparative data on the effect of free colistin and the Polyfro pharmaceutical composition on the degree of destruction of kidney tissue.

(50) TABLE-US-00005 TABLE 5 The effect of colistin and the combined pharmaceutical composition Polyfro on the degree of destruction of the kidneys The value for the experimental group: Index Polyfro (n = 13) Colistin (n = 15) Urinary Excretion 577* ± 18  775 ± 33 of NGAL (mcg/h) Urinary NAG 331* ± 10 2578 ± 95 Excretion (mcg/h) *P < 0.05; differences between the control and experimental groups are statistically significant

(51) As can be seen from table 5, the differences in the concentrations of NGAL and NAG between the groups where pure colistin was used and Polyfro were statistically significant, that is, SCDC/liposomal cholecalciferol effectively protect the kidneys from the negative effects of colistin. Table 6 shows similar data for other pharmaceutical compositions with polymyxin.

(52) TABLE-US-00006 TABLE 6 The accumulation of polymyxin in the presence of nephroprotectors in the tissues of the kidneys Urinary Excretion of Urinary NAG No. NGAL (mcg/h) Excretion (mcg/h) p/p Composition SCDC (n = 15) (n = 15) 1 Control (colistin)  775 ± 33 2578 ± 95 2 Control (solution of normal saline) 380* ± 25 300* ± 15 2 Polifro 577* ± 18 331* ± 10 3 Colistin/Pantothenic acid/NAD 610* ± 20 314* ± 8  4 Colistin/Cyanocobalamin/Vikasol 558* ± 19 370* ± 12 5 Colistin/Cholecalciferol/Vikasol 549* ± 18 394* ± 18 6 Colistin/Cyanocobalamin/Pantothenic Acid 570* ± 16 390* ± 17 7 Colistin/Pantothenic Acid/Vikasol 526* ± 21 502* ± 22 8 Colistin/Cyanocobalamin/NAD 598* ± 19 418* ± 21 9 Colistin/Vikasol/NAD 510* ± 22 405* ± 15 10 Colistin/Cholecalciferol/NAD 583* ± 25 518* ± 22 11 Colistin/Cholecalciferol/pantothenic acid 580* ± 19 505* ± 19 *P < 0.05

(53) As can be seen from table 6, the differences in the groups of animals receiving only colistin and colistin compositions with different nephroprotectors are statistically significantly different. For acetylglucosaminidase, this indicator is 8 times different compared to the control of colistin. Thus, all patentable variants of pharmaceutical compositions of polymyxin with nephroprotective agents, even when using a subtoxic dose of polymyxin, reduced the nephrotoxicity of polymyxin to virtually the norm.