EA042949B1 - POLYMYXIN-BASED PHARMACEUTICAL COMPOSITION FOR TREATMENT OF INFECTIOUS DISEASES WITH REDUCED NEPHROTOXICITY - Google Patents

Description

The invention relates to pharmacy and medicine, allows to obtain new pharmaceutical compositions based on polymyxin with significantly reduced nephrotoxicity and increased bactericidal activity.

Background of the Invention Terminology

In biochemistry and pharmacology, a ligand is a chemical compound that forms a complex with a particular biomolecule (most often a protein) and, as a result of such binding, produces certain biochemical, physiological or pharmacological effects. In the case of binding a ligand to a protein, the ligand is typically a small signaling molecule that binds to a specific binding site on the target protein (eg, a receptor). The binding of a ligand to a receptor usually occurs with the help of intermolecular forces, such as ionic bonds, hydrogen bonds, van der Waals forces. Binding or association of a ligand with a receptor (so-called docking of a ligand into a specific niche in the receptor) is usually reversible and transient.

The binding of a ligand to a receptor protein changes its conformational state (three-dimensional spatial configuration). And this, in turn, can lead to a change in the functional state of the protein (for example, to the activation or inactivation of a receptor or enzyme, to the dissociation of one of the subunits of the composite protein, or, conversely, to the acquisition by the protein, as a result of binding to a ligand, of the ability to attach another specific ligand or another protein, or the opening of a protein-coupled ion channel, or self-phosphorylation or other self-modification of the protein, or opportunities for it to be phosphorylated or otherwise modified by another protein, etc.). The concept of a ligand includes both enzyme substrates and antigens recognized by antibodies, and various agonists, antagonists and inverse agonists, including endogenous ones, such as neurotransmitters, hormones, cytokines and chemokines, and inhibitors and activators of certain enzymes or regulatory proteins, and transcription factors, and exogenous ones such as drugs, etc.

The binding strength of a ligand to a target protein (eg, receptor) is called the affinity, or affinity, of the ligand to the target protein (eg, receptor). The binding strength of a ligand to a target protein is determined not only by the strength of direct interactions of the ligand with a given protein (for example, a receptor), but also by the microenvironment of the protein molecule, in particular, solvent molecules present around, which can play a dominant role in ensuring adequate non-covalent intermolecular interactions between the ligand and target protein (water, cell membrane lipids) and partner proteins (in the case of, for example, oligomeric receptors or G-protein-coupled receptors). In particular, the increase in the affinity of transmembrane receptors for endogenous agonists in the presence of cholesterol and sphingolipids is the reason that these receptors tend to be located in certain places on the cell membrane, called lipid rafts and enriched in cholesterol and sphingolipids.

The degree of affinity of the ligand for the receptor (the affinity of the ligand for the receptor). The interaction of most ligands with their binding sites can be characterized in terms of the degree of affinity of the ligand for the receptor (ligand receptor affinity). In general, a high degree of affinity of a particular ligand for a given specific receptor subtype (high ligand affinity for this receptor subtype) is the result of a stronger intermolecular interaction between the receptor and its ligand, and vice versa - a lower degree of ligand affinity for this receptor (lower affinity for this receptor) is, as a rule, a consequence of the lower strength of the intermolecular interaction between them. This also means that, in general, high affinity (i.e., high affinity, in other words, strong) binding of the ligand to the receptor implies a longer residence time of the ligand on the receptor (and hence a greater percentage of receptor occupancy at relatively low doses). or ligand concentrations). In addition, a high binding affinity of a ligand to a receptor (a high affinity of the ligand for it) often has important physiological consequences, since some of the binding energy of the ligand to the receptor (which is naturally higher with high affinity, high affinity binding, as interaction) can be used to change the spatial configuration of the receptor, which, in turn, can lead to activation or, conversely, deactivation of the receptor and to the opening of the ion channel associated with the receptor or to a change in the behavior (increase or decrease in activity) of the enzyme associated with the receptor or regulatory protein. Thus, a more affinity (having a higher affinity for the receptor) ligand is more likely to be physiologically and pharmacologically active (i.e., exhibiting some degree of internal agonistic activity, with whatever sign it may be - an agonist or an inverse agonist) . However, this is not guaranteed - high-affinity neutral antagonists, or rather, agents close to neutral antagonists, i.e. having a very low modulus internal agonistic activity, close to zero, but nevertheless exhibiting a high or very high degree of affinity for the receptor, affinity for it - also exist.

Two agonists with a similar degree of affinity for the receptor (similar affinity).

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A receptor ligand that can bind to a receptor, change the spatial configuration of this receptor in such a way that it leads to its activation, and, as a result, be able to cause one or another physiological or biochemical response of the cell (to be a trigger for such a response), is called an agonist towards this receptor. The binding of an agonist to a receptor can be characterized both in terms of how great is the maximum physiological response that can be obtained by stimulating the maximum available number of receptors with that particular agonist (intrinsic agonist activity), and in terms of what molar concentration of a given agonist required to elicit a physiological response of a given strength (dose-response curve), and in terms of what molar concentration of a given agonist is required to elicit a physiological response of 50% of the maximum achievable for a given agonist (half the maximum effective concentration , or EU ₅₀ , EC ₅₀ ). Thus, the determined and measured value of EC ₅₀ is just a quantitative characteristic of the measure of the affinity of the agonist to the receptor (a measure of its affinity for it). If we measure the concentration that is required to obtain 50% of the maximum achievable physiological response in general, and not 50% of the maximum achievable for this particular agonist (taking as the maximum achievable, i.e. for 100% - the maximum effect from the endogenous agonist), then we get the value of EU ₅₀ , which depends both on the value of the affinity of the agonist (the degree of its affinity for the receptor), and on the ratio of its internal agonistic activity to the internal agonistic activity of the endogenous agonist, taken as 100%. Thus, the determined EC ₅₀ will be a quantitative measure not only of affinity, but of the molar activity of a substance (its potency), which is a function of both the affinity (affinity for the receptor) and the internal agonistic activity (receptor effectiveness) of a given ligand.

Thus, high affinity (high affinity) binding of a ligand to a receptor means that a relatively low concentration of the ligand is required to ensure complete (maximum possible for a given receptor system) occupancy of the binding sites of a given ligand on the receptors and induce the maximum possible physiological response for a given ligand (value which depends on the internal agonist activity of the ligand). That is, the lower the Ki value characterizing the ligand binding affinity to the receptor, the more likely is the formation of a chemical bond between the ligand molecules and the receptor molecules as a result of a random collision of molecules during Brownian motion (since the intermolecular interaction force between them is greater). And a greater strength of intermolecular interaction also means a longer average retention time of the ligand on the receptor (longer duration of the existence of a non-covalent chemical bond). Conversely, low affinity binding (with low affinity for the receptor), i.e. a high Ki value means that relatively high concentrations of that ligand are required to achieve maximum occupancy of all available binding sites and elicit the maximum physiological response possible for that particular agonist. This also means that the formation of a chemical bond between a given ligand and a receptor as a result of a random collision of molecules during Brownian motion is less likely for a lower affinity agonist (having less affinity for the receptor), since there is less intermolecular force between them and it is less specific. And the average retention time of the ligand on the receptor for low-affinity (having low affinity for the receptor) is shorter, it releases the receptor faster and dissociates from its connection with it faster. A higher concentration for a low affinity ligand is necessary precisely because it increases the likelihood of accidental collision of low affinity ligand molecules with the receptor and the likelihood of chemical bonding between them.

Ligands that bind to receptors, however, cannot or almost cannot activate the receptor (or rather, they do this with a negligible probability) and, accordingly, by themselves cannot and do not cause a physiological response of the receptor system, but only prevent the binding of both agonists and inverse agonists and the physiological response to them are called antagonists. The binding of a ligand to a receptor is often characterized in terms of what concentration of ligand is required to occupy 50% of all available receptor binding sites - the so-called IC ₅₀ . The value of IC ₅₀ is related to the dissociation constant Ki, but differs from it. It also differs from the EC ₅₀ value, since the occupation of 50% of the available receptors does not necessarily lead to the production of 50% of the maximum physiological response for a given agonist or 50% of the maximum physiological response in general (IC ₅₀ can be either greater or less than the EC ₅₀ , depending on the characteristics of the regulation of a particular physiological receptor system - there are both receptor systems in which the occupation of a relatively small number of receptors produces a large physiological effect, and, conversely, systems in which a large percentage of available receptors must be occupied to create a significant physiological effect, and the dependence of the magnitude of the physiological effect on the percentage of receptor occupancy, as well as on the dose of the agonist, does not have to be linear at all). If both ligands are present at the same time, then a greater percentage of the high affinity (having a higher affinity for the receptor) ligand will bind to the available receptor binding sites compared to the lower affinity ligand. This mechanism explains, in particular, why carbon monoxide (II), even at low concentrations, can compete with oxygen for binding to hemoglobin, being a higher affinity (having a greater affinity for hemoglobin) agonist of this transport protein, and why this often leads to carbon monoxide poisoning.

The binding affinity of a ligand to a receptor (the degree of affinity of a ligand for a receptor) is most commonly determined using the method of displacing a labeled radioactive ligand (called a hot ligand) with the ligand of interest (called a cold or test ligand). Homologous competitive ligand-to-receptor binding experiments are experiments in which hot (radiolabeled) and cold (unlabeled) ligand are the same chemical and compete for available receptor binding sites. There are also methods without the use of a radioactive label, such as surface plasmon resonance, double polarization interferometry. These methods make it possible to determine not only the affinity (degree of affinity) of an agonist for the receptor, but also the kinetics of its association and dissociation from the bond with the receptor, and in the case of dual polarization interferometry, also the configuration changes in the receptor caused by the binding of the agonist to it. Recently, a microthermophoresis method has also been developed. This method allows the binding affinity to be determined without imposing any restrictions on the molecular weight of the ligand.

To analyze the data obtained on the kinetics of ligand binding to the receptor and on its affinity, methods of statistical mechanics are used, in particular, the calculation of the so-called. configuration integral.

Affinity for receptors (affinity) and molar activity (potency) of the ligand.

The degree of affinity of a ligand for receptors, or the so-called affinity of a ligand for receptors, does not in itself determine the molar activity (total potency) of a particular ligand. The molar activity (potency) of a substance is the result of a complex interaction between its degree of affinity for receptors and its intrinsic agonist activity (in other words, its receptor efficacy). Internal agonist activity (receptor efficacy) is a quantitative characteristic of the ability of a given ligand to induce a particular biological response after binding to a receptor, and a measure of the magnitude of the biological response it evokes, as a percentage of the maximum possible biological response, which is taken as the maximum stimulation by an endogenous agonist (100 %). Depending on the nature, nature, sign and magnitude of the biological response caused by the ligand, it is classified either as an agonist or even a superagonist, or as a partial agonist, or as a neutral antagonist, or as an inverse agonist.

Selective and non-selective ligands.

Selective ligands tend to bind clinically/physiologically relevant concentrations to only a fairly limited set of receptor subtypes (not necessarily all of these subtypes will be receptors for the same endogenous ligand). At the same time, non-selective ligands tend to bind significantly at relevant concentrations to a fairly wide range of receptor subtypes (often to various endogenous ligands) and, thereby, produce a wider range of clinical, biochemical, and physiological effects, both desirable and, often, , unwanted side effects. Ligand selectivity is a rather arbitrary and relative concept, since there are very few truly selective ligands that bind to only one receptor subtype over the entire range of reasonable, clinically achievable concentrations in humans, and even fewer ligands that can maintain 100% selectivity at concentrations that can be created in experiments on animals and even more so in vitro (in vitro). Often, the apparent relative selectivity of a particular ligand is lost with increasing dose or concentration (i.e., at higher concentrations or doses, it begins to interact with other receptor subtypes), and this has important clinical implications (for example, high doses of a selective opioid receptor agonist buprenorphine can significantly depress respiration and cause euphoria, since selectivity compared to morphine is lost; similarly, high doses of selective β-blockers can cause bronchospasm, since selectivity for the β1 subtype is lost, and high doses of β2-adrenergic stimulants, in addition to eliminating bronchospasm, can also cause tachycardia; high doses of atypical antipsychotics like risperidone and olanzapine can cause extrapyramidal side effects similar to typical antipsychotics).

A measure of the relative selectivity of a particular ligand is the value of the ratio of its affinity (affinity) to the desired, main receptor subtype (for example, to D2, in the case of antipsychotics) and to the nearest next in order of magnitude of the affinity (affinity) receptor subtype, i.e. the value of the Ki(1)/Ki(2) ratio. Higher affinity for the desired receptor type, more highly active (higher potency) compounds are often, though not always, also more selective, at least at low concentrations (the use of which,

- 3 042949 again, it becomes possible precisely due to the higher affinity of the compound in relation to the receptor and the greater activity of the compound). Thus, an important task of experimental and clinical pharmacology is the development of new, higher affinity (having a higher affinity for the receptor) and more active (more highly potent) compounds with respect to certain types of receptors.

Bivalent ligands consist of two connected molecules, each of which is a ligand for a certain subtype of receptors (the same or different), and, due to the peculiarities of the spatial structure, both parts of the molecule are able to simultaneously bind to two parts of a composite homo- or heterodimeric receptor complex. Bivalent ligands are used in scientific research to discover and study homo- and heterodimeric receptor complexes and study their properties. Bivalent ligands are usually large molecules and tend not to have desirable properties for drugs, such as convenient pharmacokinetics (acceptable bioavailability, ease of clinical use, acceptable half-life, etc.), low allergenicity, and acceptable toxicity and side effects, which makes they are generally unsuitable or of little use for clinical use outside of research laboratories.

A preferred structure is a structural part of a molecule, a radical or a chemical element, which or which is statistically often repeated among already known drugs of a given pharmacological class, among already known ligands of a given type or subtype of receptors, or known inhibitors of a given enzyme, or among some other isolated by some common features. a specific subset of already known biologically active compounds. These statistically distinguished preferred elements of the chemical structure can later be used as a basis for the development of new biologically active compounds or new drugs with properties similar to or possibly even improved compared to the original compounds, and even for the development of entire libraries of such compounds. Sometimes such structures are called pharmacophores. Typical examples are, for example, tricyclic structures of different chemical structures in the molecules of tricyclic antidepressants or the existence of chemically similar whole subclasses of antipsychotics, such as butyrophenone derivatives (haloperidol, spiperone, droperidol, etc.), indole derivatives (reserpine, carbidine, etc.), phenothiazine derivatives (chlorpromazine, perphenazine, etc.).

Ensemble or supramolecular ensemble is a term from supramolecular chemistry. The objects of supramolecular chemistry are supramolecular ensembles built spontaneously from complementary ones, i.e. having geometric and chemical correspondence of fragments, similar to the spontaneous assembly of the most complex spatial structures in a living cell (Stid J.V., Atwood J.L. Supramolecular chemistry. - M .: Akademkniga, 2007). Due to the fact that 20 different derivatives are synthesized from one molecule of cyanocobalamin in the presence of two modifiers, intermolecular ionic and hydrogen bonds are necessarily formed between their molecules. Such supramolecular structures have a significantly higher biological activity than the original vitamin. The experiment confirmed the higher affinity of this structure for renal megalin than unmodified cyanocobalamin or individual substituted derivatives. The authors of the invention used a combinatorial mixture of cyanocobalamin derivatives in the form of a supramolecular ensemble without separation into individual components.

Simultaneous combinatorial modification with two modifiers - if a polyfunctional molecule is used in the combinatorial synthesis reaction - in this case, cyanocobalamin with three groups available for simultaneous modification, two modifying agents are immediately introduced into the reaction, for example, acetic anhydride and succinic anhydride. As a result of the reaction, a mixture of acylated derivatives in different positions is formed - acetyl-succinyl derivatives of cyanocobalamin.

State of the Art

As a result of the search for an etiological antigen in Heyman's nephritis, which is an experimental model of membranous nephropathy, Kerjaschki D. and Farquhar M.G. in 1982, the cell membrane protein gp330 was identified (Kerjaschki D., Farquhar M.G., 1982, Proc. Natl. Acad. Sci. U.S.A., 79, 5557-5561). In 1994, Saito A. et al. established the complete primary structure of rat gp330 and named it megalin because this protein was the largest cloned vertebrate membrane protein (Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91 , 9725-9729).

Megalin expression site.

Megalin is also known as glycoprotein 330 (gp330) and low-density lipoprotein receptor-related protein 2 (LRP-2). It is a glycoprotein with a molecular weight of approximately 600 kDa, which is expressed in the epithelial cells of the proximal tubules of the kidneys, in other tissues and cells, for example, in type II alveolar cells, in male testes, in the endometrium of the uterus, in the placenta, in the epithelium of the inner ear, in the renal epithelium, in the germinal vitellaria and in the neural ectoderm (see Christensen E.I, Willnow, T.E., 1999, J. Am. Soc. Nephrol. 10, 2224-2236; Juhlin C., Klareskog L. et al., 1990 , J. Biol. Chem. 265, 8275-8279; and Zheng G., McCluskey R. T. et al.,

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1994, J. Histochem. Cytochem. 42, 531-542). In the kidney, megalin acts as an endocytosis receptor associated with endocytosis, protein reabsorption, and the like. in the proximal tubule before urinary excretion.

After that, reabsorption proteins, etc. are destroyed by lysosomes (see Mausbach A.V., Christensen E.I, 1992,

Handbook of Physiology: Renal Physiology, Windhager, editor, New York, Oxford University Press, 42-207).

Nucleotide sequence of megalin.

Megalin is a glycoprotein most commonly expressed in mammals on the epithelial membrane of the proximal renal tubule. The coding sequence of the megalin cDNA is identical in nucleotide composition to the human megalin cDNA sequence with gene accession number U04441, disclosed by Korenberg, J.R. et al. (1994), or the human megalin cDNA sequence with gene accession number U33837 disclosed in Hjaeln, G., et al. (1996) (see Korenberg J. R. et al., 1994, Genomics 22, 88-93 and Hjalm G. et al., 1996, Eur. J. Biochem. 239, 132-137). In addition, Saito et al. (1994) discovered a rat megalin with homology to human megalin, and its coding sequence for DNA with gene accession number L34049 has already been disclosed (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729).

Amino acid sequence and protein structure of megalin.

Megalin is a giant cell membrane protein consisting of 4655 amino acids (human megalin) and 4660 amino acids (rat megalin). The molecular weight derived from the amino acid sequence is approximately 520 kDa, but may exceed 600 kDa when the sugar chain is included (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 97259729). Megalin belongs to the gene family of LDL receptors, the giant extracellular region of which has four functional domains, and this extracellular region is associated with a thin intracellular region through a single transmembrane region. Megalin is present mainly in a clathrin-covered fossa on the renal glomerulus (in rats) or on the epithelial luminal membrane (luminal and basement membranes of glomerular epithelial cells) of the proximal renal tubule, type II alveolar cells, epididymal cells, thyroid gland, accessory thyroid glands , on the membrane of the yolk sac, in the inner ear, in the small intestine, on the choroid itself (chorioidea) and is associated with the entry into cells of various ligands and their metabolites (see Farquhar M.G. et al., 1995, J. Am. Soc. Nephrol 6, 35-47 and Christensen E. I. et al., 2002, Nat. Rev. Mol. Cell Biol. 3, 256-266). In megalin knockout mice, diseases and disorders such as low molecular weight proteinuria, bone metabolism disorders, respiratory failure, brain malformations, and others are observed (see Willnow T. E. et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, 8460-8464). Nematodes (C. elegans) also have a megalin homologue that has been suggested to be biologically important (see Yochem J. et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90, 4572-4576).

Importance of megalin as a cause of nephritis.

Megalin, the main etiological antigen of experimental membranous nephropathy (Heyman's nephritis), is an epithelial phagocytic receptor, the biological and pathological role of which has been established. Animal models have long been used to elucidate the developmental mechanism of human membranous nephropathy, and Hayman's rat nephritis is a model of membranous nephropathy. The analysis of Hayman's jade has progressed further than the analysis of any other model. Saito A. et al. disclosed the results of the analysis of the pathological epitope and ligand-binding domain of Hayman's nephritis, and also demonstrated the main antigenic region of megalin and the functional domain of megalin, which are the main contributors to ligand binding (see Kerjaschki D. et al., 1992, Proc. Natl. Acad. Sci. U.S.A. , 89, 11179-11183; Saito A., Farquhar M. G. et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, 8601-8605; Yamazaki H., Farquhar M. G. et al., 1998, J. Am Soc. Nephrol. 9, 1638-1644 and Orlando R. A., Farquhar M. G. et al., 1997, Proc. Natl. Acad. Sci. U.S.A., 94, 2368-2373)

Various megalin ligands.

Megalin is most abundantly expressed in vivo on the luminal side of the epithelial cells of the proximal tubules of the kidneys. In the human kidney, megalin expression is not observed in any other sites than the epithelial cells of the proximal tubules, including the glomeruli. Megalin incorporates various ligands (for example, low molecular weight proteins or drugs), which are filtered by the glomerulus into cells through endocytosis, megalin transports them to lysosomes, and they reappear on the cell surface through recycling (see Farquhar MG et al., 1995, J. Am Soc Nephrol 6, 35-47 and Christensen EI et al., 2002, Nat Rev Mol Cell Biol 3 256-266). In addition, megalin is associated with transcytosis from the luminal side to the basal side of the membrane. Megalin is also associated with the absorption and metabolism of binding proteins such as vitamins A, B ₁₂ and D (see Christensen EI et al., 2002, Nat. Rev. Mol. Cell. Biol. 3, 256-266). Christensen and Willnow demonstrated that megalin mediated the reabsorption of three vitamin carrier proteins: vitamin D binding protein (DBP), retinol binding protein (RBP), and transcobalamin (TC), as well as their associated vitamins, i.e. (OH) vitamin 25D3, vitamin A (retinol) and vitamin B ₁₂ (see Christensen EI, Willnow TE, 1999, J. Am. Soc. Nephrol. 10, 2224-2236). Saito A. et al. demonstrated that leptin secreted by adipocytes, which is elevated in the blood of obese patients, is incorporated and metabolized by proximal tubular epithelial cells as a megalin ligand (see Saito A., Gejyo F. et al. , 2004, Endocrinology 145, 3935-3940). Adipocytes, i.e. accumulated visceral fats lead to combined pathological conditions, i.e. metabolic syndrome. Leptin, which is an adipocytokine secreted by adipocytes, is elevated in the blood of patients with metabolic syndrome. Researchers believe that the kidney is the organ most likely to accumulate leptin circulating in the blood, with this leptin playing a nephropathic role (see Tarzi RM, Lord GM et al., 2004, Am. J. Pathol. 164, 385- 390). The so-called leptin receptor is also found in the region between the proximal tubule and the collecting duct, below the area of megalin function. Saito A. et al. conducted an experiment with epithelial cells extracted from the yolk sac of a rat embryo (L2 cells) in which megalin was highly expressed and found that the incorporation of glucose-derived enhanced glycosylation end products (AGEs) radioisotopically labeled ¹²⁵ I into L2 cells can be largely suppressed by antibodies against megalin. Thus, they demonstrated that megalin is associated with the metabolic pathway of such inclusion (see Saito A., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 1123-1131). The interaction of advanced glycosylation end products (AGEs) with glycosylated and modified proteins in the Maillard reaction has been pointed out as a mechanism for the development of diabetic nephropathy. The low molecular weight AGE present in the blood is filtered by the renal glomeruli and reabsorbed and metabolized by proximal tubular epithelial cells. If the nephropathy progresses further, then the glomeruli also filter high molecular weight AGE, which accumulates in the epithelial cells of the proximal tubules and creates an excessive metabolic load. Further, Saito A. et al. also demonstrated that megalin is associated with incorporation into cells (in addition to glucose) of AGE derived from methylglyoxal, glycerinaldehyde, or glycolaldehyde. In addition, the metabolic syndrome is often complicated by hepatopathy, such as fatty degeneration of the liver. Liver-type fatty acid-binding proteins (L-FABP), abundant in the liver, are released into the blood in healthy individuals. In hepatopathy, the release of L-FABP increases, which leads to an increase in their level in the blood. Saito A. et al. also demonstrated that blood L-FABPs are rapidly filtered by the renal glomeruli and reabsorbed by proximal tubular epithelial cells via megalin (see Takeda T., Gejyo F., Saito A. et al., 2005, Lab. Invest. 85, 522531).

Functional protein interacting with megalin.

In order to elucidate the mechanism of megalin transport in cells, a search for adapter molecules that bind to the intracellular domains of megalin was carried out, during which various proteins were identified, for example, Dab2, ANKRA, MAGI-1, GAIP, GIPC, Galphai3, MegBP and ARH ( see Oleinikov AV et al., 2000, Biochem. J. 347, 613-621; Rader K., Farquhar MG et al., 2000, J. Am. Soc. Nephrol. 11, 2167-2178; Patrie KM, Margolis B. et al., 2001, J. Am. Soc. Nephrol. 12, 667-677; Lou X., Farquhar MG et al., 2002, J. Am. Soc. Nephrol. 13, 918-927; Petersen HH , Willnow TE, 2003, J. Cell Sci 116, 453-461 and Takeda T, Farquhar MG et al., 2003, Mol Biol Cell 14, 4984-4996). Through these molecules, megalin is associated with endocytosis and transcytosis, as well as their associated signaling. In addition, megalin functions conjugatively (conjugated) with the cell membrane receptor, i.e. with cubilin in the epithelial cells of the proximal tubules, due to which it is additionally involved in the processes of incorporation of various ligands into cells (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 9725-9729). For example, cubilin is a receptor that directly binds to transferrin, albumin, endogenous vitamin B ₁₂ and the like, and megalin is indirectly involved in their endocytosis. It is also known that megalin interacts in epithelial cells of the proximal tubules with isoform 3 of the Na ⁺ -H ⁺ exchanger (NHE3) (see Biemesderfer D. et al., 1999, J. Biol. Chem. 274, 17518-17524). NHE3 is an antiporter that plays an important role in Na ⁺ reabsorption, in addition, NHE3 influences the incorporation of ligands by megalin (see Hryciw DH et al., 2004, Clin. Exp. Pharmacol. Physiol. 31, 372-379). Megalin may also be involved in the inactivation and metabolism of NHE3. In the early stages of diabetic nephropathy or nephropathy associated with the metabolic syndrome, glomerular filtration becomes excessive. Increased reabsorption of Na ⁺ in the proximal tubules has been shown to be the main cause (see Vallon V. et al., 2003, J. Am. Soc. Nephrol. 14, 530-537), with NHE3 playing a key role in this case. , and inactivation and metabolization of NHE3 by megalin also appears to play a role in these processes (see Hryciw DH et al., 2004, Clin. Exp. Pharmacol. Physiol. 31, 372-379).

Importance of megalin function found in experiments using uremia models and organ regeneration models.

As described above, megalin is involved in the uptake and metabolism of various small molecular weight proteins by proximal tubular epithelial cells. If the pathological condition progresses to the stage of renal failure, then the metabolic mechanism is disturbed, as a result of which low molecular weight proteins accumulate in the blood and tissues as uremic proteins. A typical example is β ₂ -microglobulin (e ₂ -m), which can cause dialysis amyloidosis in

- 6,042,949 long-term dialysis patients (see Gejyo F., Schmid K. et al., 1985, Biochem. Biophys. Res. Commun. 129, 701-706). The aforementioned AGE is also considered the cause of arteriosclerosis or organ failure due to its accumulation in the blood of patients with renal failure or long-term dialysis, and AGE is considered as a uremic type protein (Henle T., Miyata T., 2003, Adv. Ren. Replace Ther. 10, 321-331). Further, leptin accumulates in the blood of dialysis patients, so it is thought to be involved in malnutrition and immune disorders. Tabata Y. and Gejyo F. et al. have disclosed the effects and efficacy of models of uremic protein metabolism using megalin functions (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14 , 2025-2032 and document WO 02/091955). That is, cells expressing megalin are transplanted in vivo as building proteins, and small molecular weight proteins leaking out from peripheral blood vessels (newborn blood vessels) are incorporated into cells by megalin for subsequent metabolization. Cells expressing megalin used for transplantation (i.e., L2 cells derived from yolk sac epithelium) are incorporated and metabolized e ₂ -m by megalin (see Saito A., Tabata Y., Gejyo F. et al. ., 2003, J. Am. Soc. Nephrol. 14, 2025-2032). A state of renal failure was induced by removal of both kidneys in a nude mouse that had undergone subcutaneous transplantation of L2 cells, after which the incorporation of cells into the tissue mass and into the organs where cells with e ₂ -m labeled with ¹²⁵ I isotope were transplanted by intraperitoneal injection was measured. As a result, it was found that the cell mass into which L2 cells were transplanted more intensively incorporated ¹²⁵ I labeled e2-m compared to other organs, and the excretion of ¹²⁵ I labeled e2-m was significantly increased in the group in which L2 cells were transplanted compared with a control group in which L2 cell transplantation was not performed (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 2025 -2032).

Polymyxin and megalin (Lisnyak, Yu. V., 2015. Annals of Mechnicov Institute, (3), 8-24).

Interest in polymyxins, observed in the world recently, is caused by a significant spread of nosocomial infections resistant to a wide range of modern antimicrobial agents, and the lack of new effective antibiotics against gram-negative bacteria. According to experts, the absence of such antimicrobial agents may lead to a return to the pre-antibiotic era. At the same time, most gram-negative bacteria are sensitive to polymyxins, and the formation of resistance to these cationic lipopeptides is slow and occurs much less frequently than with other antibiotics. In the 70s. In the 20th century, polymyxins were abandoned due to cases of nephrotoxicity and the emergence of drugs with fewer side effects. However, when the use of β-lactams, aminoglycosides or quinolones against extremely multidrug-resistant strains of gram-negative bacteria, including P. aeruginosa, A. baumannii K. pneumoniae, becomes ineffective, polymyxin B and colistin remain the last resort for these infections.

The group of polymyxin peptides includes several chemically different compounds (polymyxins A-E, M, S, etc.). In clinical practice, only polymyxins B and E (colistin) are used. Polymyxin B (polymyxin, PmB) is a cyclic lipodecapeptide (Fig. 1) containing six residues of α,γ-diaminobutanoic acid (Dab): MOA-Dab1-Thr2-Dab3-cycle[Dab4-Dab5-D-Phe6-Leu7Dab8- Dab9-Thr10]. Seven amino acids of polymyxin form a macrocycle (cycle[Dab4-Dab5-D-Phe6-Leu7Dab8-Dab9-Thr10]), and three amino acids (Dab1-Thr2-Dab3) form a linear region connecting the macrocycle to the terminal residue of methyl octanoic acid (MOA) . The macrocycle is formed by an additional peptide bond between Thr10 and the γ-amino group of the Dab4 residue. The N-terminal Dab1 residue of polymyxins is Na-acylated with a fatty acid such as 6-methyloctanoic acid (PmB1), 6-methylhexanoic acid (PmB2), octanoic acid (PmB3), etc. The only structural difference between colistin (colistin, polymyxin E, PmE) and polymyxin B is the amino acid D-Leu at position 6 instead of D-Phe in polymyxin B. Polymyxin B and colistin contain five free amino groups (as part of Dab) and, accordingly, five positive charges at physiological conditions.

The return of polymyxins to clinical practice has stimulated further in-depth studies of their toxicity. In the last decade, the toxicity of polymyxin B and colistin has been carefully tested by modern methods (taking into account the modes of correct use, chemical purity and homogeneity of the preparations) and turned out to be not as high as was thought in the past. However, it can still significantly complicate therapy, reduce its effectiveness, and even lead to its complete cessation. Therefore, the creation of less toxic derivatives of polymyxin remains a very urgent task. In the last decade, several research groups have focused their efforts on the development of less toxic polymyxin derivatives. In particular, Sakura N. et al. have created polymyxin derivatives Ser2-Dap3-PmB(2-10), Dap3-PmB(3-10), and Ser3-PmB(3-10), whose acute toxicity (LD ₅₀ ) is approximately 10 times lower than polymyxin B1, and activity against Pseudomonas aeruginosa remains approximately at the level of polymyxin B. Vaara M. et al. synthesized less nephrotoxic polymyxin derivatives NAB7061, NAB739 and NAB740, the renal clearance

- 7 042949 was, respectively, 28, 53 and 378 times higher than that of colistin, and the affinity for the brush strip membrane of the rat kidney epithelium was 2-3 times lower than that of gentamicin, and 5-6 times lower than that of polymyxin B1. The search for non-toxic polymyxin derivatives is generally conducted empirically, without involving any molecular mechanisms of its nephrotoxic action and/or structural models of polymyxin-target interaction. A prerequisite for a targeted search for such derivatives is the knowledge of the molecular mechanisms of nephrotoxicity of polymyxins, based on detailed information about the features of intermolecular interactions of polymyxins with their targets of nephrotoxic action.

It is known that the nephrotoxic effect of polymyxins is due to their accumulation in the epithelial cells of the proximal tubules of the kidney, where they persist for a long time, causing damage to the kidney: their accumulation in increasing amounts in lysosomes leads to swelling and, as a result, to rupture of the latter and the release of polymyxins into the cytosol, where their nonspecific binding causes acute tubular necrosis. At the same time, the main factor in the accumulation of these antibiotics in the kidney is their interaction with megalin (previously called glycoprotein gp330), a giant cell surface receptor, which is most abundantly present in the apical membrane of the proximal tubules of the kidney. Thus, this receptor may represent a unique target for the design of polymyxin antibiotics with minimized nephrotoxicity. Attenuation of polymyxin binding by megalin may be a new preventive measure against polymyxin-induced nephrotoxicity.

Megalin is a transmembrane glycoprotein that plays a central role in the endocytic function of the epithelial cells of the proximal tubules of the kidney; it is also involved in signal transduction in these cells. Megalin is localized in the clathrin caveolae of the epithelium of the proximal tubules of the kidney and functions as an endocytic receptor that binds a very wide range of substances. Ligands that bind to megalin (there are more than 30 of them) are represented by several groups of compounds: proteins involved in lipoprotein metabolism; proteases and protease inhibitor complexes; matrix proteins; intracellular proteins; growth factors and other groups (including lactoferrin, rhinovirus, C3 complement, gentamicin, polymyxin, etc.). Megalin is a member of the low density lipoprotein receptor (LDLR) gene family. The LDLR family is a class of structurally homologous membrane receptors composed of modular structures (domains) and represented in mammals by seven major glycoproteins: low density lipoprotein receptor (LDLR); very low density lipoprotein receptor (VLDLR); apolipoprotein-E receptor 2 (ApoER2 or LRP8); multiple epidermal growth factor (MEGF7); LDLR-related protein 1 (LRP1); LDLR-associated protein 1b (LRP1b) and LDLR-associated protein 2 (LRP1) or megalin (Megalin).

Megalin is the largest representative of this family, its mass is about 600 kDa. The amino acid sequence of rat megalin has 4460 amino acids and contains a 25 amino acid N-terminal signal peptide sequence, a 4400 amino acid extracellular region, a 22 amino acid single-pass transmembrane domain and a 213 amino acid C-terminal cytoplasmic tail, the amino acid sequences of human and rat megalin are similar by 77 %. The extracellular region of megalin contains structural modules characteristic of all members of the LDLR family - cysteine-rich ligand-binding repeats (in the literature they are also called complement-like repeats (or domains) and denoted CR (Complement-type Repeat), growth factor repeats (EGF- repeats) and β-propeller domains. Site-directed mutagenesis studies show that the binding sites for most LDL ligands are ligand-binding CR repeats. the LRP and megalin regions contain 31 and 36 ligand-binding repeats, respectively, distributed in four clusters (clusters I-IV).Each of the CR domains consists of approximately 40 amino acid residues. The first data on the three-dimensional organization of CR repeats were obtained by NL Daly et al. nuclear magnetic resonance spectroscopy (NMR spectroscopy) to repeat CR1 and then CR2 from human LDLR (PDB codes 1LDL and 1LDR, respectively). It was found that these modules contain three disulfide bonds, as well as such basic elements of the protein secondary structure as β-hairpins and β-bends. Next, the crystal structure of the CR5 domain from human LDLR was determined with a resolution of 1.7 A (PDB code 1AJJ), showing for the first time that the module contains a Ca2 ⁺ ion, which is octahedral coordinated by negatively charged residues of aspartic (Asp) and glutamine (Glu) acids and carbonyl backbone groups forming a pocket around the calcium ion. Later it was shown that the Ca2 ⁺ ion is required for proper folding of the polypeptide chain and maintaining the structural integrity of the module. Structural data for megalin are currently rather limited: only one structure of the 12th CR domain of rat megalin and one structure of the 10th CR domain of human megalin (as well as its complex with gentamicin) are known, determined in solution by NMR. However, the structures of all seven CRs up to

- 8 042949 LDLR and several CR domains of LRP receptors, which were obtained both for individual modules using NMR spectroscopy or X-ray crystallography, and for their pairs. All structures of these CR domains have the same type of polypeptide chain folding: a short antiparallel β-sheet, two loops stabilized by disulfide bonds between CysI-CysIII and CysIV-CysVI cysteines and linked by a CysII-CysV disulfide bridge. The N-terminal loop is additionally stabilized by an antiparallel β-sheet, and the C-terminal loop is additionally stabilized by interactions of residues coordinated around the Ca2 ⁺ ion. The similar nature of the folding of the polypeptide chain and the similar three-dimensional organization of the CR domains are a consequence of the homology of their amino acid sequences containing conservative residues of aspartic (D) and glutamic (E) acids and the conservative arrangement of six cysteine residues (C). The preferred binding sites for many important ligands in the LDLR family are ligand-binding repeats. Analysis of the available structural data on the interaction of members of the LDLR family with their cationic ligands shows that the recognition site on the receptor is a common (universal) structural motif and contains three acidic (negatively charged) aspartic acid residues coordinated by the Ca2 ⁺ ion (the so-called DXDXD motif) and one hydrophobic residue. The recognition/binding site on the ligand are positively charged lysine residues. The binding is carried out mainly due to electrostatic interactions between the positively charged ligand residues and the negatively charged aspartic acid residues involved in the coordination of the Ca2 ⁺ ion. This lysine binding is enhanced by hydrophobic interaction between the aromatic residue of the CR module (tryptophan or phenylalanine) and the aliphatic lysine moiety. Thus, the key features of the binding site include (1) calcium ion coordination, (2) salt bridges and hydrogen bonds between the aspartic acid residues of the CR repeat and ligand cationic residues, and (3) hydrophobic interactions between the aromatic residue of the ligand-binding repeat and the aliphatic lysine site.

The binding site of polymyxin on its molecular target, megalin, has not yet been experimentally established, and there are also no structural models of the interaction of polymyxin with megalin at the atomic level. However, polymyxin B (like gentamicin) is known to be an effective competitive inhibitor of rat megalin binding to the RAP protein (Receptor-Associated Protein, receptor-associated protein). RAP is a chaperone that binds to members of the LDL receptor family and acts as a universal antagonist of their premature binding to their ligands in the endoplasmic reticulum (leading to their aggregation and degradation), ensuring the normal expression of these receptors on the cell surface for their endocytic function. Competitive inhibition implies both the structural similarity of the inhibitor and the substrate (at least the structural similarity of their molecular fragments that interact with the receptor) and the same binding site on the receptor.

And the structural models of the interaction of the RAP protein with LDL receptors at the atomic level are known. And in these complexes, the positively charged NH3 groups of gentamicin also interact with three negatively charged aspartic acid residues of the CR10 domain, i.e. with a structural DXDXD motif found for other CR modules. Thus, there are grounds to suggest that the structural DXDXD motifs of the ligand-binding CR domains of megalin are also the polymyxin binding site, and the molecular fragments of polymyxins that interact with the receptor are their cationic Dab groups (analogues of lysine residues).

As studies by Vaara M. et al. have shown, NAB-derivatives of polymyxin, which have only three positive charges located within the macrocycle of the molecule (charged residues Dab5, Dab8 and Dab9), are not only effective antibacterial agents, but also have a significantly lower nephrotoxicity. than the original polymyxin containing five positive charges (charged residues Dab1, Dab3, Dab5, Dab8 and Dab9). At the same time, their renal clearance was tens and hundreds of times higher than that of colistin, and their affinity for the brush strip membrane of the rat kidney epithelium was 5-6 times lower than that of polymyxin B1, i.e. polymyxin derivatives with a weaker interaction with the target had better pharmacokinetic parameters. Thus, the level of nephrotoxicity of polymyxin and its derivatives correlates with the features of their molecular structure and, as a consequence, with the features of their intermolecular interactions with megalin. A new polymyxin/colistin derivative that would not be recognized by megalin would presumably have substantially less nephrotoxicity. What are the features of intermolecular interactions of polymyxin with megalin? What are the structural reasons for the differences in the interaction of polymyxin and its NAB derivatives with megalin? Answers to these questions will be provided by ongoing studies of the structural-functional relationships of polymyxins and their molecular targets of nephrotoxic action. Recently, the structures of the complexes of the ligand-binding domain of human megalin CR10 with another cationic antibiotic, gentamicin, which also has a nephrotoxic side effect, were determined by NMR spectroscopy and molecular modeling (docking). Megalin is a unique target for the development of polymyxin antibiotics with minimized nephrotoxicity. Attenuation of polymyxin binding by megalin may be a new preventive measure against polymyxin-induced nephrotoxicity.

Known neurotoxicity inhibitors for aminoglycoside antibiotics [US Patent 4526888]. These inhibitors, which are patented in composition with aminoglycoside antibiotics, include polyaspartic acid and polyaspartates in the form of salts or amides, as well as polyglutamic acid, polyglutamates and their amides. These compounds are able to protect the kidneys from the action of aminoglycoside antibiotics due to their higher affinity for the kidneys. Although these polymers did not inhibit the antimicrobial activity of aminoglycoside antibiotics, they had a number of significant drawbacks - they are semi-synthetic polymers - xenobiotics with a metabolic mechanism that is not fully understood, the polymeric nature of these compounds when injected, together with a high affinity for kidney tissues, allows these compounds to accumulate in the kidneys with formation of amyloid plaques, these compounds are new xenobiotics and they are not yet approved for use. In addition, they have not previously been used in combination with polymyxin.

There is a known method for suppressing the nephrotoxicity of polymyxin by combining its use with high doses of ascorbic acid (Sirijatuphat, R., Limmahakhun, S., Sirivatanauksorn, V., Nation, R.L., Li, J., & Thamlikitkul, V. (2015). Preliminary clinical study of the effect of ascorbic acid on colistin-associated nephrotoxicity Antimicrobial agents and chemotherapy, 59(6), 3224-3232.). Significant protective activity of sodium ascorbate for injection use during polymyxin therapy was shown. The disadvantage of this method is the delayed toxicity of polymyxin associated with the lack of tropism (high affinity) of ascorbic acid for renal megalin. The mechanism of action of ascorbic acid is not associated with inhibition of the process of interaction of polymyxin with megalin, but is associated with the suppression of lipid peroxidation (LPO) and the release of free radicals (SR) in the kidneys. LPO and SR are the main consequences of the destruction of megalin, including the chain reaction cascade of nephron decay. At the same time, after the termination of the action of ascorbic acid, the nephrotoxicity of polymyxin manifested itself after a long period of time after the completion of treatment. Similar LPO and SR inhibitors, such as retinoic acid (various derivatives of vitamin A), vitamin E, melatonin, etc., although they protect the kidneys from the immediate effect of polymyxin during treatment, are not able to protect the kidneys from the long-term cumulative effect of polymyxin (Yousef, J.M. , Chen, G., Hill, P.A., Nation, R.L., & Li, J. (2011). Melatonin attenuates colistin-induced nephrotoxicity in rats. Antimicrobial agents and chemotherapy, 55(9), 4044-4049.). Termination of the action of LPO and SR inhibitors immediately manifests the nephrotoxic effects of polymyxin previously associated with megalin. High-affinity megalin ligands are deprived of these side effects, which can generally prevent the interaction of polymyxin with megalin without negatively affecting the antimicrobial effect of polymyxin.

Disclosure of invention

The invention is based on the task of developing a pharmaceutical composition based on polymyxin with reduced nephrotoxicity for the treatment of infectious diseases. The problem is solved by obtaining a pharmaceutical composition based on polymyxin for the treatment of infectious diseases, including, as nephroprotectors, a combination of vitamins with a supramolecular combinatorial derivative of cyanocobalamin, obtained by simultaneous covalent modification of the structure of cyanocobalamin with succinic and acetic anhydrides, where the molar ratio of components was: 20:21:21. At the same time, as vitamins, the composition includes cholecalciferol, or vikasol, or ascorbic acid. Also, the composition can be made in the form for parenteral use: injection solutions, lyophilized powder for extemporaneous use; in the form for oral administration: tablets, syrups, capsules, oral solutions; in the form for transdermal use: patches, ointments, gels, powders; in the form for rectal use: suppositories, douching solutions; in topical form: dressings, patches, powders, gels, powders, and also include pharmaceutically acceptable preservatives, stabilizers and excipients.

Pharmaceutical compositions

Can be used in various ways to obtain a patentable pharmaceutical composition (PFC). PFC may be given orally or may be administered by intravascular, subcutaneous, intraperitoneal injection, aerosol, ophthalmic, bladder, topical, etc. For example, inhalation administration methods are well known in the art. The dosage of the therapeutic composition will vary widely depending on the particular antimicrobial PFC administered, the nature of the disease, frequency of administration, route of administration, clearance of the agent used from the host, and the like. The initial dose may be higher followed by lower maintenance doses. The dose can be administered at a frequency of once a week or once every two weeks, or divided into smaller doses and administered once or more times a day, twice a week, and so on to maintain an effective dose level. In many cases, oral administration will require a higher dose than intravenous administration of PFCs may be included in many therapeutic formulations. More specifically, the PFC

- 10 042949 of the present invention can be included in pharmaceutical compositions in combination with suitable pharmaceutically acceptable carriers or diluents and can be included in preparations in solid, semi-solid, liquid or gaseous forms, such as capsules, powders, granules, ointments, creams, foams, solutions, suppositories, injections, inhalation forms, gels, microspheres, lotions and aerosols. As such, administration of the compounds may be by various routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, and the like. The PFCs of the invention may be distributed systemically after administration, or may be localized using an implant or other composition that retains the active dose at the site of implantation. The PFCs of the present invention may be administered alone, in combination with each other, or they may be used in combination with other known compounds (eg, fluoroquinolones, carbapenems, etc.). In pharmaceutical dosage forms, nephroprotectors may be administered in the form of their pharmaceutically acceptable salts.

The following methods and excipients are provided by way of example only and are not limiting in any way. 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 carboxymethylcellulose; with lubricating agents such as talc or magnesium stearate; and, if desired, diluents, buffering agents, wetting agents, preservatives and flavoring agents.

PFCs can be incorporated into injectable compositions by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent such as vegetable or the like 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. PFCs can be used in an aerosol composition for inhalation administration.

The compounds of the present invention may be incorporated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. In addition, PFC can be incorporated into suppositories by mixing with a variety of bases such as emulsifying bases or water soluble bases. The compounds of the present invention may be administered rectally using a suppository. The suppository may contain excipients such as cocoa butter, carbovaxes and polyethylene glycols that melt at body temperature but are solid at room temperature. Unit dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, can be prepared, where each dosage unit, for example, a teaspoon, tablespoon, tablet or suppository, contains a predetermined amount of a composition containing one or more compounds of the present invention. . Similarly, unit dosage forms for injection or intravenous administration may contain the compound of the present invention in the composition in the form of a solution in sterile water, normal saline, or other pharmaceutically acceptable carrier. Implants for 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, polymers of lactic and/or glycolic acids form a degradable polymer that is well tolerated by the host. The implant containing the PFC according to the invention is placed close to the focus of the pathology, so that the local concentration of the active agent is increased compared to the rest of the body.

As used herein, the term unit dosage form refers to physically discrete units suitable for use as single doses in human and animal subjects, each unit containing a predetermined amount of the compounds of the present invention calculated to be sufficient to produce the desired effect, together with a pharmaceutically acceptable diluent, carrier or excipient. Descriptions of 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 commonly available. In addition, pharmaceutically acceptable excipients such as pH adjusting agents and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like are commonly available. Typical doses for systemic administration range from 0.1 pg to 1000 mg per 1 kg of the subject's body weight per administration. A typical dosage may be one tablet to be taken two to six times a day, or one capsule or extended release tablet to be taken once a day with a proportionately higher content of the active ingredient. The sustained release effect may be due to capsule materials dissolving at various pH values, slow release capsules under osmotic pressure, or any other known controlled release method.

Those skilled in the art will appreciate that dosage levels may vary depending on the particular compound, the severity of the symptoms, and the subject's susceptibility to side effects. Some of the specific compounds are more active than others. Preferred doses of a given compound can be readily determined by those skilled in the art in a variety of ways. The preferred method is to measure the physiological activity of PFC.

One method of interest is the use of liposomes as a delivery vehicle. The liposomes fuse with the cells of the target area and deliver the contents of the liposomes into the cells. The contact of the liposomes with the cells is maintained for a time sufficient for fusion using various means of maintaining contact, such as isolation, binding agents, and the like. In one aspect of the invention, the liposomes are designed to provide 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. The lipids may be any useful combination of known liposome-forming lipids, including cationic or zwitterionic lipids such as phosphatidylcholine. The remaining lipids will generally be neutral or acidic lipids such as cholesterol, phosphatidylserine, phosphatidylglycerol and the like. To obtain liposomes, the method described by Kato et al. can be used. (1991), J. Biol. Chem. 266:3361. Briefly, lipids and a composition for incorporation into liposomes containing peptides are mixed in a suitable aqueous medium, suitably a saline medium, where the total solids content will be in the range of about 110 wt.%. After vigorous stirring for short periods of time, 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. The composition is then sonicated for a suitable period of time, typically about 1-10 seconds, and optionally vortexed further. The volume is then increased by adding an aqueous medium, typically increasing the volume by about 1-2 times, followed by agitation and cooling. The method makes it possible to include both polymyxin and patented nephroprotectors in liposomes.

Brief description of the drawings

Fig. 1. Scheme of chemical synthesis of a supramolecular derivative of combinatorially modified cyanocobalamin.

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

Fig. 3. HPLC (Milichrome A-02) of a 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%).

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

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%).

Fig. 6. Direct suppression of colistin accumulation with cyanocobalamin (IVd) and cholecalciferol from Polifro formulation.

Embodiments of the invention

The following examples do not limit the possibility of using other methods for obtaining the composition according to this invention and are provided only for the purposes of explaining the implementation of the claims.

Example 1 Synthesis of a supramolecular combinatorial derivative of cyanocobalamin (SCOC).

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 refluxed for 20 min. The solution is poured into ampoules and lyophilized to remove the solvent and acetic acid. The supramolecular combinatorial derivative (IVd) or SCCC is used to obtain pharmaceutical compositions, study the structure, and determine biological activity. In FIG. 1 shows the scheme for the synthesis of combinatorial derivatives of quercetin.

One initial molecule of cyanocobalamin contains three hydroxyl groups available for modification, including one phosphate one.

Calculations of the number of moles of modifiers are carried out according to the formulas of combinatorics: m=4x(3x2 ^n- 2-1); k=nx(2 ⁿ -1), where m is the number of different derivative molecules in the combinatorial mixture and the number of moles of cyanocobalamin for the reaction; n is the number of hydroxyl groups available for modification, including one phosphate group in the structure of cyanocobalamin (n=3); k is the number of moles of each modifier.

- 12 042949

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 substituents, and different permutations of modifier residues, not just in the form of a mixture, but in the form of a hard-to-separate supramolecular structure.

Modifiers - succinic anhydride or acetic anhydride can be introduced both simultaneously and sequentially, or first introduce succinic anhydride, heat the mixture under reflux for 20 minutes, and then introduce acetic anhydride and also heat the mixture for another 20 minutes. Similarly, in this reaction as one of the modifiers instead of succinic anhydride, maleic anhydride, aconitic anhydride, glutaric, phthalic anhydride and acetic anhydride, formic acid ethyl ester, monochloroacetic acid, propiolactone, ethylene oxide and other low molecular weight alkylating substances (methyl chloride, ethyl chloride, propyl chloride) can be used. For the purposes of studying the biological activity of the synthesized substances, various derivatives with different ratios of modifiers were obtained (Table 1).

For HPLC, a Milichrom A-02 microcolumn chromatograph was used with a gradient of acetonitrile (5-100%)/0.1 M perchloric acid + 0.5 M lithium perchlorate. The combinatorial derivative of SKPC (IVd) on the chromatogram (Fig. 3) gave one clear broad peak and was not separated into components, although the retention time was practically the same as pure cyanocobalamin, while its fully substituted derivatives had different retention volumes (Fig. 4, 5 ). This indicated that complex supramolecular structures were formed between different combinatorial derivatives (in this case, there are 20 of them), which were not separable chromatographically. This combinatorial derivative (SKOC) (IVd) behaves similarly when separated in a thin layer (acetonitrile:water) and gives only one band, which does not coincide with any of the obtained derivatives.

In table. Figure 1 shows the result of screening cyanocobalamin derivatives with different ratios of modifiers as a substrate for renal megalin (using rat kidney homogenate megalin as an example). It is known that the original cyanocobalamin is a substrate/ligand of renal megalin with a moderate degree of interaction and an average index of affinity for megalin by absorption (AI=52%). The test was performed by a micromethod in Eppendorf tubes according to 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 cycanocobalamin derivative in the supernatant was determined using HPLC on a Milichrome A-02 liquid chromatograph. The relative concentration in% relative to the original is indicated in table. 1.

Table 1

The ability of megalin to bind different derivatives of cyanocobalamin SKCC

No. p / p Molar ratios of reagents* % bound derivative w kl k2 1. 20 84*** 84*** 5 2. -AND- 42 42 12 3. -AND- 21 21 98 4. -AND- 17 17 71 5. -AND- 13 13 71 6. -AND- 9 9 70 7. -AND- 5 5 67 8. -AND- 3 3 58 9. -AND- 2 2 57 10. -AND- 1 1 57 AND. -AND- 0 0 52 12. -AND- 42 0 56 13. -AND- 21 0 67 14. -AND- 17 0 71 15. -AND- 13 0 66 16. -AND- 9 0 62 17. -AND- 5 0 57 18. -AND- 3 0 58 19. -AND- 2 0 57

- 13 042949

20. -AND- 1 0 56 21. -AND- 0 1 57 22. -AND- 0 2 55 23. -AND- 0 3 57 24. -AND- 0 5 50 25. -And- 0 9 59 26. -AND- 0 13 37 27. -AND- 0 17 27 28. -AND- 0 21 15 29. -AND- 0 42 10 thirty. -AND- 0 84*** 6 31. -AND- 84*** 0 7 32. -AND- 42 1 AND 33. -AND- 21 2 23 34. -AND- 17 3 46 35. -AND- 13 5 57 36. -AND- 9 9 59 37. -AND- 5 13 55 38. -AND- 3 17 34 39. -AND- 2 21 21 40. -AND- 1 42 AND

* m - the number of moles of cyanocobalamin in the reaction of combinatorial synthesis;

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.

As can be seen from Table. 1, only at the calculated ratio of components, when the maximum amount of different cyanocobalamin derivatives is formed, a biologically active and effective supramolecular structure is formed (derivative 3 or SCCC 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 solution (21 variations of cmanocobalamin derivatives with different permutations and placements in substituents), a more complex supramolecular quasi-living structure is formed with different properties and greater pharmacological activity.

In FIG. 2 shows the chromatogram of the original cyanocobalamin (I), combinatorially modified with two anhydrides (IVd) (Fig. 5). In FIG. 3, 4 show HPLC chromatograms of two derivatives: fully succinylated (IVc) and fully acetylated cyanocobalamin (IVb), respectively, as control samples (No. 30 and 31 in Table 1). As can be seen from the graphs, the retention volume of the combinatorial derivative practically does not differ from the original cyanocobalamin, but differs from fully modified derivatives. In addition, at the same concentration of cyanocobalamin, the peak area and the 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 volumes of (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 within the complex (IVd) form a complex supramolecular structure.

Fully succinylated and fully acetylated cyanocobalamins were only absorbed by megalin by 6% and 7%, respectively. Thus, derivative IVd, obtained in accordance with combinatorial calculations, is a fundamentally new supramolecular structure, which has significantly different properties from both unmodified cyanocobalamin (no. 11 in Table 1) and fully modified derivatives (nos. 30 and 31 in Table 1). 1). This structure cannot be separated using gradient HPLC, and the UV spectra of all derivatives are almost the same, although the retention time is different. This indicates the formation of new structures with a covalent bond based on cyanocobalamin, and in this case these structures form a complex supramolecular structure with each other, similar to the complexes of cyclodextrins.

Example 2. Preparation of pharmaceutical compositions based on polymyxin with nephroprotectors.

The task is achieved by the fact that the pharmaceutical composition with antimicrobial activity contains the antibiotic polymyxin, contains cyanocobalamin, SCPC and cholecalciferol, blocking its nephrotoxic effect, as well as targeted additives that promote the formation of dosage forms. According to the invention, SCPC, the main active agent is introduced in an amount of 0.5-20%, polyvinylpyrrolidone, surfactant sodium lauryl sulfate, the main components of the pharmaceutical composition are used as a solubilizer, with a certain technological operation, due to mechanochemical interaction, they form a complex with the studied pharmaceutical and pharmacological properties . At the same time, in the pharmaceutical composition, SCPC and cholecalciferol are contained in an amount of 2 to 7%. Instead of cholecalciferol, another nephroprotector can be used: vikasol, pantothenic acid, nicotinamide adenine dinucleotide. At the same time, SKCC in the composition can be contained in an amount of from 10 to 40%. Substances are pre-dissolved in ethanol together with lecithin or TWEEN-80, and ethanol is distilled off when heated or under vacuum. In addition, 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 as target additives. In this case, the fillers contain at least one binding agent, one disintegrant and one lubricating agent. At the same time, microcrystalline cellulose was chosen as a binding agent in a total amount of 10-20%. At the same time, croscarmellose sodium salt in a total amount of 1-15% was chosen as a baking powder. At the same time, magnesium stearate is selected as a lubricant in a total amount of 0.25-5%. In addition, the pharmaceutical composition is made in dosage forms of tablets coated with a polymer film or capsules.

The task is also achieved by the fact that in the method for obtaining a pharmaceutical composition with antimicrobial activity according to the invention, polymyxin with nephroprotectors and target additives are preliminarily mixed, compacted, ground, the resulting mixture is mixed with pharmaceutically acceptable excipients, dry or wet granulation of the mixture is carried out, after that the granulate is filled hard gelatin capsules or compressed and coated tablets with a polymer film. According to the invention, the developed method for producing a pharmaceutical composition makes it possible to obtain dosage forms with a complex pharmaceutical composition in the form of coated tablets or capsules containing components within the following quantitative limits, in %:

polymyxin: 5-25,

SKPC/cholecalciferol: 10-40, excipients: up to 100.

Three active substances of the new pharmaceutical composition were combined in it, taking into account the knowledge about their pharmacological and medicinal properties obtained by using monopreparations based on their substances. Selected targeted additives, due to their physical and chemical properties, contribute to the solubilization of active substances and their sufficiently high dissolution in the aqueous physiological environment of the gastrointestinal tract. The combination of three active substances with different physical and chemical properties in one dosage form is a rather difficult task for pharmacy. The solution of the task 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 study. First of all, the pharmaceutical development of a pharmaceutical composition began with the evaluation of the physicochemical properties of polymyxin, SCPC and cholecalciferol, primarily focusing on their ability to emulsify in aqueous media so that these active substances have maximum oral bioavailability in the physiological conditions of the stomach. substances is possible only with the use of excipients with properties. It was possible to achieve high solubility using solubilizers and surfactants. Such solubility modulators are polyvinylpyrrolidone (povidone, GTVP) and sodium lauryl sulfate (SLS), 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. Investigating the dissolution kinetics of samples of the pharmaceutical composition, both granulate for capsules and tablets, at variable mass values of PVGT, SLS, TWEEN-80 and lecithin, their optimal ratio with polymyxin was established.

- 15 042949

table 2

Composition and ratio of ingredients in one capsule or tablet core of the pharmaceutical composition Polifro

No. p / p Ingredient name in mg V% 1 2 4 5 1 Liposomes with cholecalciferol (LC) 40.00 20.00 2 Polyvinylpyrrolidone K-25 40.00 20.00 3 Polymyxin 25.00 12.50 4 Cellulose microcrystalline 80.00 40.00 5 TVIN-80 10.00 5.00 6 SKCC 3.00 1.50 6 Magnesium stearate 2.00 1.00 Contents of a capsule or tablet core: 200.00 100.00

To ensure the physicochemical stability of the complex, the weight ratio between LH and PVP should be within 1:1-1:3. It should be noted that these PVP mass values are also sufficient to increase the solubility of the polymyxin substance due to the formation of the corresponding complex compound. The povidones used differ in their molecular weights as follows: K90-1000000; K25-30000; C17-10000. Taking into account the characteristics of the dissolution kinetics of the complex of quercetin with PVP, a sufficient amount of LSN, a surfactant, was selected to promote rapid and uniform wetting of the surface of the complex granulate. This was 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 of the soluble form of polymyxin by the walls of the stomach. With a significant hydrophobicity of the surface of DN crystals, these selected excipients - PVP and SLS also contributed to the solubility of this substance, and thus could increase the bioavailability of this NSAID. The solubility of liposomes and polymyxin also has a positive effect on the selected amount of PVP and SLS, as evidenced by the solubility profiles.

All samples of the pharmaceutical composition, starting from the complex of active substances - polymyxin and liposomes with cholecalciferol with PVP and SLS, were obtained under the conditions of successive technological operations by dry or wet granulation of the ingredients of the complex and excipients. The prepared granules were used to fill capsules or they were tableted and the solubility of these dosage forms was investigated in accordance with the pharmacopoeial 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 the individual substances. In the course of pharmaceutical development, a sample of a pharmaceutical composition with a certain composition was made (see Table 2), which was named Polifro by the authors and was used in studies of pharmacological properties.

The pharmaceutical composition of the given composition, obtained in accordance with the dry granulation technology, includes mixing active substances with excipients, compacting or briquetting the mixture, grinding, mixing grinding with excipients and granulation, compressing the granulate into tablets or filling hard gelatin capsules with them. Wet granulation technology is distinguished by the mixture granulation stage, where instead of a compactor or press, wet granulation equipment and a dryer are used, such as a Rota P agitated mixer-granulator or Hurtling type fluidized bed granulator-dryer.

We give specific examples of the invention. The following examples illustrate the main aspects of the present invention, but should not be construed as having a limiting value. Obtaining all samples of granules of the complex of active substances - polymyxin, SKPC and cholecalciferol in liposomes with PVP was carried out under the conditions of technological operations of granulation in a dry or wet way, using dry pressing (compacting) of a mixture of active ingredients, followed by grinding the briquetted material. Wet granulation was carried out in granulator-dryers under vacuum or with warm running air in fluidized bed devices. The obtained granules were filled into capsules or they were tabletted, examining the solubility of these dosage forms according to the Dissolution test. The solubility characteristics of the polymyxin/SCOC/liposomal cholecalciferol system in the pharmaceutical composition indicated a significantly higher solubility of the complex of active ingredients compared to the solubility of the individual substances. Thus, in the course of pharmaceutical development, a sample of a pharmaceutical composition was made, called by the inventors of the invention Polifro, which, as a preparation of a certain composition, was used in studies of toxico-pharmacological properties.

Table 3

Composition and ratio of ingredients per capsule or tablet of the pharmaceutical composition Polifro

No. p / p Name of ingredients mg % 1 2 3 4 1 *liposomes with cholecalciferol (LC) 40.00 20.00 2 *Polyvinylpyrrolidone K-25 40.00 20.00 3 *Polymyxin 25.00 12.50 4 Cellulose microcrystalline 80.00 40.00 5 * TWIN-80 10.00 5.00 6 *SKOC 3.00 1.50 7 Magnesium stearate 2.00 1.00 8. Contents of the capsule or tablet core 200.00 100.00

* The indicated compounds together with phosphate buffer in the indicated % ratios were used to create a liquid injection form for in vitro and in vivo experiments.

The pharmaceutical composition of the given composition (Table 3), obtained using the dry granulation technology, includes mixing the active substances with fillers, compacting or briquetting the mixture, grinding and mixing it with fillers, and at the end the granulate is compressed into tablets or filled with hard gelatin capsules. Wet granulation technology is distinguished by the mixture granulation step, where instead of a compactor or press, wet granulation and drying equipment is used, such as a 25 type Rota P agitated mixer granulator or a Huttling fluid bed dryer granulator. The following exemplary embodiments of the invention illustrate aspects of the present invention, but they should not be construed as having a limiting value.

Example 3. Obtaining a liposomal suspension form of cholecalciferol for further production of granulate, tablet mass and tablet forms with polymyxin and SCCC.

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

The preclinical study of the pharmaceutical composition was carried out using the Polyfro sample in the research tests in order to fully establish the toxico-pharmacological properties of the future drug, which can become a promising drug.

Example 4 Inhibition of Polymyxin Accumulation in the Kidney.

In this experiment, the accumulation of colistin in the kidneys of rats was determined after intravenous administration of Polyfro (colistin, SCPC and liposomal cholecalciferol). The concentrations of colistin (CS), SCCC (CK)/cholecalciferol (CCF) were used based on preliminary modeling (number of binding sites in one molecule multiplied by their number in one megalin molecule, megalin molecular weight and approximate amount of megalin in one kidney and kidney weight ). As a result, the required amount of SKCC was about 2 mg/kg of body weight. For CS, a subtoxic dose was chosen (based on the instructions). This is an experiment to displace polymyxin from the kidneys with a synergistic combination of CK/HCP. As a result, about half of polymyxin was displaced from the kidneys by CK/CCP. All components of CM, CK/CCP were determined in the sediment of the renal homogenate and supernatant by HPLC. In our opinion, CK is one of the best candidates for nephroprotectors, which already now makes it possible to significantly reduce the toxicity of polymyxin in clinics. In FIG. 1 shows the accumulation of colistin (A) and the concentration (B) of colistin in the renal megalin in mice. After 45 minutes of intravenous injection of a mixture of Polyfro in terms of SKCC (2 mg/kg) and colistin (0.5 mg/kg) or sal. solution in the control, the urine of rats was collected after 180 min. 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. Each column shows the mean value (SD) of the four measurements at P<0.05 (versus control). As can be seen from FIG. 6, the accumulation of colistin in the tissues of the kidney as

- 17 042949 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 SCCC and cholecalciferol. All data are statistically significant and differences between groups and controls are significant.

In table. 4 shows the results of suppression of the accumulation of colistin in the tissues of the kidneys for other pharmaceutical compositions with colistin.

Table 4

Accumulation of polymyxin in the presence of nephroprotectors in kidney tissues

No. p/p The composition of the composition Accumulation of colistin in the kidneys mcg The concentration of colistin in the kidneys, recalculated in kidney tissue in mcg / 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/SCPC/Vikasol 7.25±2.25* 2.25±1.25* 5 Colistin/Cholecalciferol/Vicasol 9.50±2.50* 4.50±1.50* 6 Colistin/SCOC/Pantothenic Acid 8.50±1.50* 3.5O±1.5O* 7 Colistin/Pantothenic Acid/Vicasol 6.25±1.25* 2.50±0.50* 8 Colistin/SCPC/NAD 7.25±1.25* 2.25±1.25* 9 Number of truths / V icasol / N AD 8.50±1.50* 3.5O±1.5O* 10 Colistin/Cholecalciferol/NAD 4.50±1.50* 2.00±0.50* eleven 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 polymyxin accumulation in renal megalin. Differences from control without nephroprotectors are statistically significant.

Example 2. Prevention of kidney tissue destruction by nephroprotectors from Polyfro.

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

In the same rats treated with pure colistin and Polyfro, urine parameters indicating the degree of kidney damage were studied (doses of 0.5/15 mg/kg polymyxin/SCPC in Polyfro as an intravenous injection once a day for 10 days). Urinary NGAL and NAG concentrations were determined using ELISA-text systems on a StatFax303+ reader. In table. 4 shows comparative data on the effect of free colistin and the pharmaceutical composition Polifro on the degree of destruction of kidney tissues.

Table 5

Influence of colistin and the combined pharmaceutical composition Polifro on the degree of kidney destruction

Index Significance for the experimental group: Polyfro (n=13) Colistin (n=15) Urinary NGAL excretion (mcg/h) 577*±18 775±33 Urinary NAG excretion (mcg/h) 331*±10 2578±95

* P<0.05; differences between the control and experimental groups are statistically significant.

As can be seen from Table. 5, the differences in NGAL and NAG concentrations between the pure colistin and Polyfro groups were statistically significant; SKPC/liposomal cholecalciferol effectively protects the kidneys from the negative effects of colistin.

In table. 6 shows similar data for other pharmaceutical compositions with polymyxin.

- 18 042949

Table 6

Accumulation of polymyxin in the presence of nephroprotectors in kidney tissues

No. p/p The composition of the composition Urinary NGAL excretion (mcg/h) (u =15) Urinary NAG excretion (mcg/h) (U=15) 1 Control (colistin) 775±33 2578±95 2 Control (saline solution) 380*±25 300*±15 2 Polifro 577*±18 331*±10 3 Colistin/Pantothenic Acid/NAD 610*±20 314*±8 4 Colistin/SCPC/Vikasol 558*±19 370*±12 5 Colistin/Cholecalciferol/Vicasol 549*±18 394*±18 6 Colistin/SCOC/Pantothenic Acid 570*±16 390*±17 7 Colistin/Pantothenic Acid/Vicasol 526*±21 502*±22 8 Colistin/SCPC/NAD 598*±19 418*±21 9 Number of truths / V icasol / N AD 510*±22 405*±15 10 Colistin/Cholecalciferol/NAD 583*±25 518*±22 AND Colistin/Cholecalciferol/Pantothenic Acid 580*±24 505*±19

* P<0.05.

As can be seen from Table. 6, the differences in the groups of animals taking only colistin and colistin compositions with different nephroprotectors are statistically significantly different. For acetylglucosaminidase, this indicator differs by 8 times compared with the control of colistin. Thus, all patented variants of pharmaceutical compositions of polymyxin with nephroprotectors, even when using a subtoxic dose of polymyxin, reduced the nephrotoxicity of polymyxin to virtually normal levels.

Claims (4)

1. Pharmaceutical composition based on polymyxin for the treatment of infectious diseases, including, as nephroprotectors, a combination of vitamins with a supramolecular combinatorial derivative of cyanocobalamin, obtained by simultaneous covalent modification of the structure of cyanocobalamin with succinic and acetic anhydrides, where the molar ratio of components was 20:21:21. 2. Pharmaceutical composition according to claim 1, characterized in that it also includes cholecalciferol as vitamins. 3. Pharmaceutical composition according to claim 1, characterized in that it also includes vikasol as vitamins. 4. Pharmaceutical composition according to claim 1, characterized in that it also includes ascorbic acid as vitamins. - 19 042949 (IVa-d) R, -R ₃ = (a) H, (b) -COCH ₃ (c) -COCH ₂ CH ₂ COOH; (d) Combinatorial sum a+b+c Fig. 1 Fig. 3 - 20 042949 Fig. 4 □ Control (Free colistin) Fig. 6 The concentration of colistin in the kidneys mcg/g of tissue Polifro Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2