EA043514B1 - SUPRAMOLECULAR SYSTEMS BASED ON DYNAMIC SELF-ORGANIZING NANOSTRUCTURES WITH ANTI-VIRAL PROPERTIES - Google Patents

Description

Background to the invention Field of technology

The present invention relates to supramolecular nanoparticles produced using combinatorial chemical building blocks that self-assemble into nanoparticles, operating on the basis of molecular recognition, and methods for controlling the size of the resulting nanoparticles. The invention also includes methods for using supramolecular structures to treat viral infections.

There is predictability only for simple models of molecular structure. Chemists are mistakenly satisfied with paradigms of molecular structure to such an extent that the reality and limit of usefulness of many types of chemistry are taught to be predictable.

Chemists are currently searching for models of molecular structures and individual molecules that will be useful for constructing structures at the supramolecular level. Supramolecular chemistry (Wikipedia, 2020) is a branch of chemistry with regard to chemical systems consisting of a discrete number of molecules. The forces responsible for the spatial organization of the system range from weak intermolecular forces, electrostatic charge, or hydrogen bonds to strong covalent bonds, provided that the electronic bond strength remains small relative to the energy parameters of the component. [Whereas traditional chemistry focuses on covalent bonding, supramolecular chemistry examines weaker and reversible noncovalent interactions between molecules. These forces include hydrogen bonding, metal coordination bonds, hydrophobic van der Waals forces, pi-pi interactions, and electrostatic effects.

Important advanced concepts in supramolecular chemistry include molecular self-assembly, molecular folding, molecular recognition, host-guest chemistry, mechanically interlocked molecular architectures, and dynamic covalent chemistry. The study of noncovalent interactions is critical to understanding many biological processes that depend on these forces for structure and function. Biological systems are often the inspiration for supramolecular research. However, new supramolecular structures involve new intermolecular relationships in many ways that are complementary to the chemistry of the molecular structures themselves. Thus, although atoms play a crucial role in chemistry paradigms, the molecular structure of a chemical substance is still central to the chemistry of many important technological and biological materials. Intelligent molecular structure glue is a covalent bond that binds atoms together and forms the stereochemistry of atoms in space. The covalent chemical bonding paradigm provides many rules governing the structure, dynamics, physical characteristics, and chemical transformations of molecules.

The level of atomic structure is an insufficient basis for sufficient understanding of aspects of chemistry where molecular aspects predominate. The level of molecular structure is insufficient to understand aspects of chemistry where supramolecular aspects dominate. In supramolecular systems, the chemistry of intermolecular molecules binds together into assemblies we can call super-molecules. In supramolecular systems, non-covalent intermolecular bonds are more varied and complex than covalent intramolecular bonds in the structures known as molecules. Supramolecular systems can be held together by much weaker forces than the atoms in the molecule. For example, the forces that hold multiple molecules together as supramolecular structures may include dispersion forces, hydrogen bonds, and hydrophobic bonds. These forces at the atomic level may be small, but the presence of multiple weak bonds can occur in accordance with the principles of cooperative interactions in order to increase the energetic stability of the supramolecular structure. However, cooperative interactions are easily reversible since each contact binding is relatively weak. This creates variable supramolecular structural flexibility and different combinations of molecular compounds and stereochemistry conformational relationships. In other words, supramolecular systems can participate in supervalence states in which supramolecular complexes containing many individual molecules are stable in a variety of combinations with a common conformation at both ends of the molecule and supramolecular levels due to the possible summation of different combinations of a large number of weak intermolecular interactions. Wonderful examples of such models are the micelle and the DNA double helix.

In one of its most important forms, supramolecular chemistry deals with the structure and dynamics of a small molecule (called a guest) that is non-covalently bound to a larger molecule (called a host or host). The concept of guest/host has the greatest chemical information value when the distinction between guest and host is clear. In many guest/host complexes, the guest is a molecule that is relatively small compared to the host and the host can be either a single large molecule or an assembly of molecules that behave as a single unit. Typically, the host provides a cage that completely surrounds the guest (guest/cage) or the host provides a cavity (guest/cavity) that partially surrounds the guest. However, there are also examples

- 1 043514 for whom the concept of guest and host is blurred. For example, in micelles composed of surfactant monomers, one monomer can be considered as a guest in the host in its own supramolecular assembly. In turn, a molecule that is not a surfactant monomer is a pure guest in the surfactant assembly micelle.

An important term for the formation of intermolecular bonding between guest and host is complexation, a term that retains the idea of chemically corresponding looseness based on non-covalent bonds between molecules. In this regard, the host is a partner in the guest/host complex whose design properties and structural changes are determined by the type of guests who will be associated in the complex. For another partner who is a guest in this complex, as a rule, the chemistry or physical properties can be more or less studied. These concepts are derived from enzyme chemistry paradigms and provide a familiar and useful working framework for discussing supramolecular chemistry. The guest/host complex may be considered a supermolecule or a supramolecular assembly depending on the complexity of the supramolecular structure under discussion. Supramolecular chemistry: structure and dynamics at the topological, geometric and chemical level

Supramolecular chemistry is explained in various ways, such as the chemistry of molecular assemblies and intermolecular bonds, chemistry beyond the molecule, and non-covalent bond chemistry. It is important that each definition has its own limitations and exceptions. Indeed, supramolecular chemistry can be defined as the chemistry of molecular assemblies from a molecule in a solvent molecular cell to a composition of molecular assemblies (consisting of proteins, lipids, DNA, RNA, etc.) that represent the enormous chemical complexity of a living cell.

Since the bonding between molecules in guest/host complexes is often a mixture of many weak electrostatic and dispersive interactions, it is difficult to accurately define and classify the nature of noncovalent bonds. As a starting point for discussing supramolecular assemblies, it is convenient to consider the neighborhood of interactions between some guest atoms with that host, which can control the physical and chemical properties of the guest/host complex. It is often the properties of the guest, most significantly modified by neighboring interactions through binding to multiple other hosts, but combined by the supramolecular property of the complex and the modification of the host structure, which is also of considerable interest and significance.

By neighbor interactions in supramolecular chemistry, we mean that two molecules are in close proximity to each other for a certain period of time. During this period of time, two molecules can be considered to be bonded regardless of the nature of the bond and because of the proximity of their atoms to each other. For example, a molecule that is contained as a guest inside the host fullerene has a certain connection with the inner cage of the fullerene. Likewise, a molecule that is contained as a guest in a host cavity, such as cyclodextrin or cavitand, has a specific interaction with the atoms of the host cavity. A molecule that is contained as a guest in a crystal and is surrounded by crystalline host molecules has certain interactions with the crystal molecules surrounding it. Finally, a guest molecule that is intercalated into the host DNA double helix has a chemical bond with a small set of specific bases that are found in its vicinity.

Drug nanoparticles are typically particles comprising drugs such as small drug molecules, peptides, proteins and nucleic acids, as well as components that are assembled with other drugs such as lipids and polymers. Such nanoparticles may have an increased anticancer effect compared to the same, but free drugs. This is due to a more specific orientation and tropism to tumor tissues due to improved pharmacokinetics and pharmacodynamics, as well as more active intracellular delivery. These properties depend on the size and surface properties, including the specific orientation of the ligands in the nanoparticle. A limited number of such nanoparticle-based systems have reached clinical use, and information is beginning to become available to understand some of the issues surrounding the movement of these experimental systems in the human body. Although a huge amount of research continues into nanoparticle development and research, only a small number of nanoparticle-based products have the potential to become clinically useful. For example, immunostimulatory components of nanoparticles are known to be difficult to manufacture on a large scale using good manufacturing practices (GMP), and there are enormous difficulties in using assays to evaluate their chemistry, production, and quality.

However, considerable effort has been devoted to studying the use of nanoparticles in biology and medicine, and several types of nanoparticles have been tested in preclinical animal studies, in clinical trials, and some are currently used in the clinic (Davis et al., Nat. Rev. Drug Discov. 2008, vol. 7, p. 771). Examples of nanoparticle types include gold nanoshells (Loo et al., Technol. Cancer Res. Trea, vol. 3, p. 33, 2004), quantum dots (Gao et al., Nat. Biotechnol., vol. 22, p 969, 2004; Me et al., Annu. Rev. Biomed. Eng., vol. 9, p. 257, 2007), super-2 043514 paramagnetic nanoparticles (Jun et al., Angew. Chem., vol. 120 , p. 5200, 20080; Jun et al., Angew. Chem. Int. Ed., vol. 47, p. 5122, 2008). Nanoparticles carrying specific target ligands are used for in vivo imaging of cancer, and drug molecules can be packaged into polymer-based nanoparticles or liposomes (Heath et al., Annu. Rev. Med., vol. 59, p. 251, 2008 ; Torchilin et al., Nat. Rev. Drug Discov., vol. 4, p. 145, 2005) for controlled drug release (Napier et al., Poly. Rev., vol. 47, p. 321, 2007; Gratton et al., Acc. Chem. Res., vol. 41, p. 1685, 2008). Positively charged nanoparticles have been used as non-viral delivery systems in vitro and in vivo for genetic manipulation and genetic programming (Davis et al., Nat. Rev. Drug Discov., vol. 7, p. 771, 2008; Green et al. ., Acc. Chem. Res., vol. 41, p. 749, 2008; Pack et al., Nat. Rev. Drug Discov., vol. 4, p. 581, 2005).

There is a need to develop alternative synthesis processes so that new types of nanoparticles can become available and have improved properties, including: (i) better controlled size, (ii) improved morphology, (iii) low toxicity, (iv) fewer side effects on the immune system, (v) more selective rate of degradation in vivo, (VI) more physiological surface charge, (VII), improved chemical and physiological stability, (VIII), slower metabolism, (IX) slower rate of elimination from the human body after their reception.

Noble metal nanostructures with unique photophysical properties with an emphasis on photothermal agents for cancer therapy (Anderson et al., Science, vol. 220, p. 524, 1983; Jain et al., Acc Chem Res, vol. 41, p. 1578, 2008; An et al., Nano Today, vol. 4, p. 359, 2009; Lal et al., Acc Chem Res, vol. 41, p. 1842, 2008). Changing the photothermal properties of nanostructures based on their size and shape (Lal et al., Acc Chem Res, vol. 41, p. 1842, 2008; Skrabalak et al., Acc Chem Res, vol. 41, p. 1587, 2008). In particular, gold (Au)-based photothermal nanostructures are well known (Lapotko et al., Laser Surg Medi, vol. 38, p. 631, 2006; Huang et al., Lasers Med Sci, vol. 23, p. 217, 2008), nanoshells (Gobin et al., Nano Lett, vol. 7, p. 1929, 2007; Hu et al., J Am Chem Soc, vol. 131, p. 14186, 2009; Kim et al., Angewandte Chemie -Intemational Edition, vol. 45, p. 7754, 2006), nanorods (Dickerson et al., Cancer Letters, vol. 269, p. 57, 2008; Huang et al., Langmuir, vol. 24, p. 11860, 2008) and nanocages (Skrabalak et al., Acc Chem Res, vol. 41, p. 1587, 2008; Chen et al., Nano Lett, vol. 7, p. 1318, 2007; Au et al., ACS Nano, vol. 2, p. 1645, 2008). Au-containing nanostructures are an improvement over nanoparticles based on organic dyes and photothermal agents with low light absorption and the undesirable side effect of photo-bleaching (Huang et al., Lasers Med Sci, vol. 23, p. 217, 2008). The disadvantage is that nanostructure-based agents require short-wavelength light (in the range of tens to hundreds of nanometers) to kill cancer cells. (Lowery et al., Clin Cancer Res, vol. 11, p. 9097s, 2005). In addition, known nanostructured substances are not very well cleared from the liver, spleen and kidneys), and these cumulative properties are undesirable for medical use (Mitragotri et al., Nat Mater, vol. 8, p. 15, 2009; Choi et al. al., Nat Biotechnol, vol. 25, p. 1165, 2007; Nel et al., Nat Mater, vol. 8, p. 543, 2009). To solve this problem, the photophysical properties of metal nanostructures are improved through modifications that lead to improvements in their overall properties (Khlebtsov et al., Nanotechnology, vol. 17, p. 5167, 2006; Lu et al., J Mater Chem, vol. 19 , p.4597, 2009; Zhuang et al., Angew Chem Int Ed Engl, vol. 47, p. 2208, 2008; Troutman et al., Adv Mater, vol. 20, p. 2604, 2008; Ofir et al. , Chem Soc Rev, vol. 37, p. 1814, 2008; Elghanian et al., Science, vol. 277, p. 1078, 1997; Lin et al., Adv Mater, vol. 17, p. 2553, 2005; Katz et al., Angew Chem Int Ed Engl, vol. 43, p. 6042, 2004; Cheng et al., Nature Nanotechnology, vol. 3, p. 682, 2008; Maye et al., J Am Chem Soc, vol. 127, p. 1519, 2005; Niemeyer, Angewandte Chemie-International Edition, vol. 40, p. 4128, 2001; Klajn et al., Nat Chem, vol. 1, p. 733, 2009). Notably, Lapotko (Cancer Lett, vol. 239, p. 36, 2006) expanded the aggregation pathways of photothermal gold nanoparticles using antibodies on cell membranes and inside cells (Govorov et al., Nano Today, vol. 2, p. 30, 2007 ; Richardson et al., Nano Lett, vol. 9, p. 1139, 2009). Self-assembly of small metal building blocks as colloids (Mitragotri et al., Nat. Mater., vol. 8, p. 15, 2009; Choi et al., Nat Biotechnol, vol. 25, p. 1165, 2007; Nel et al. , Nat Mater, vol. 8, p. 543, 2009) improved their renal clearance and made them a better photothermal agent.

There is a need for non-viral gene therapy delivery methods that can (i) transport and protect genetic material, such as DNA and siRNA, and (b) deliver gene therapy to selected cells and cells of different tissue types (Kim et al., Nat Rev Genet, vol. 8, pp. 173-184, 2007). Improvements in non-viral gene delivery vehicles have been made (Glover et al., Nat Rev Genet, vol. 6, pp. 299-310, 2005; Rosi et al., Chem. Rev., vol. 105, pp. 1547-1562, 2005). Non-viral gene delivery systems of the prior art include (Niidome et al., Gene Then, vol. 9, pp. 1647-1652, 2002; Prata et al., Chem. Commun., pp. 1566-1568, 2008; Woodrow et al., Nat. Mater., vol. 8, pp. 526-533, 2009; Chen et al., Chem. Commun., pp. 4106-4108, 2009; Torchilin et al., Proc Natl Acad Sci USA, vol. 100, pp. 1972-1977, 2003). Nanoparticle-based gene delivery vehicles of the prior art include (Liang et al., Proc Natl Acad Sci USA, vol. 102, pp. 11173-11178, 2005; Kumar et al., Chem. Commun., pp. 5433- 5435, 2009; Bazin et al., Chem. Commun., pp. 5004-5006, 2008; Cheon et al., Acc. Chem. Res., vol. 41, pp. 1630-1640, 2008).

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Although nanoparticles hold promise as nonviral transfection agents for efficient and safe delivery of nucleic acids to a specific cell or tissue type, challenges associated with their production and use include (1) slow, multistep synthetic approaches, and (2) limited material diversity delivery. These problems are a major obstacle to achieving optimal transfection performance. Therefore, faster and more efficient production methods that can use different delivery materials are the necessary methods.

Summary of the Invention

Supramolecular nanoparticles with antiviral activity may include: a combination of nanostructures selected from the group consisting of combinatorial carboxylated cobalamins obtained from the first combinatorial synthesis; combinatorial carboxylated dipyridamoles obtained from the second combinatorial synthesis; carboxylated basic amino acids obtained from the third combinatorial synthesis, as well as any combination thereof.

May further include dynamic self-assembled soluble nanostructures, and these nanostructures may further include a plurality of binding components, a plurality of organic cores, and a plurality of terminal components.

The supramolecular nanoparticles may have one of the coupling components, which may further comprise combinatorial carboxylated cobalamins having a number of binding regions, and the organic cores may further comprise combinatorial carboxylated dipyridamoles having at least one coupling element adapted to bind to the combinatorial carboxylated cobalamins, and organic the cores may further include mechanical structures of dynamic self-assembled soluble nanostructures, also combinatorial carboxylated cobalamins, which are associated with combinatorial carboxylated dipyridamoles and may further include primary inclusion complexes.

The supramolecular nanoparticle may contain termination components, each of which may have at least one binding termination element, and may also additionally contain secondary inclusion complexes.

Supramolecular nanoparticles may contain carboxylated basic amino acids selected from lysine, histidine, arginine, derivatized lysine, derivatized histidine, derivatized arginine, acylated lysine, acylated histidine, acylated arginine, and any combinations thereof.

A supramolecular nanoparticle may have multiple termination components that may occupy the remaining binding sites of multiple binding components, and where the plurality of termination components may be equivalent to the number of binding regions of multiple binding components after which further binding of binding components ceases, also supramolecular nanoparticles may additionally contain discrete nanoparticles at based on dynamic self-organizing soluble nanostructures.

Supramolecular nanoparticles may contain combinatorial carboxylated cobalamins, which can be represented by a mixture of succinylated cyanocobalamins.

Supramolecular nanoparticles may contain combinatorial carboxylated cobalamins, which can be a mixture of succinylated methyl cobalamins.

Supramolecular nanoparticles may contain combinatorial carboxylated cobalamins, which may be a mixture of succinylated hydroxycobolamines.

Supramolecular nanoparticles may contain combinatorial carboxylated cobalamins, which may be a mixture of succinylated cobamides.

Supramolecular nanoparticles may contain combinatorial carboxylated cobalamins, which can be selected from the group consisting of a mixture of succinylated hydroxycobolamines, a mixture of succinylated cobamids, a mixture of succinylated hydroxycobolamines, a mixture of succinylated methyl-cobalamins, a mixture of succinylated cyanocobolamines, and any combinations thereof.

Supramolecular nanoparticles may contain at least one of organic cores, which may further contain at least one element based on the photodynamic component represented by supramolecular combinatorial carboxylated riboflavin.

Supramolecular nanoparticles may contain supramolecular combinatorial carboxylated riboflavin in the form of supramolecular combinatorial succinylated riboflavin.

Supramolecular nanoparticles may contain supramolecular combinatorial carboxylated riboflavin in the form of supramolecular combinatorial succinylated flavin mononucleotide.

Supramolecular nanoparticles may contain supramolecular combinatorial carboxylated riboflavin, presented as a supramolecular combinatorial succinylated flavin dinucleotide.

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Supramolecular nanoparticles may contain combinatorial carboxylated dipyridamole in the form of supramolecular succinylated combinatorial dipyridamole.

Supramolecular nanoparticles may contain combinatorial carboxylated dipyridmoles, which are supramolecular maleylated combinatorial dipyridamoles.

Supramolecular nanoparticles may contain combinatorial carboxylated dipyridamoles, which are supramolecular carboxymethylated combinatorial dipyridamoles.

Supramolecular nanoparticles may contain carboxylated basic amino acids such as succinylated lysine, succinylated histidine, succinylated arginine, or any combination thereof.

Supramolecular nanoparticles can contain carboxylated basic amino acids, such as maleylated lysine, maleylated histidine, maleylated arginine, in any combination thereof.

Supramolecular nanoparticles may contain carboxylated basic amino acids, including carboxymethylated lysine, carboxymethylated histidine, carboxymethylated arginine, in any combination.

Supramolecular nanoparticles may contain carboxylated basic amino acids, including carboxymethylated lysine, carboxymethylated histidine, carboxymethylated arginine, succinylated lysine, succinylated histidine, succinylated arginine, maleylated lysine, maleylated histidine, maleylated arginine, and any combination thereof.

Supramolecular nanoparticles may contain multiple termination components, which may consist of at least one of the following: polyethylene glycol, polymer, polypeptide, oligosaccharide, and any combinations thereof.

Supramolecular nanoparticles may contain organic cores consisting of at least one organic core based on a dendrimer, branched polyethylenimine, linear polyethylenimine, polylysine, polylactide, polylactide-co-glycoside, polyanhydride, poly-εcaprolactone, A polymethyl methacrylate, poly(N -isopropyl acrylamide), polypeptide, and any combination thereof.

Supramolecular nanoparticles may contain a binding component, which is poly-L-lysine.

Brief description of the figures

The above brief description, as well as the following detailed description of preferred embodiments of the present invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, drawings are shown for preferred embodiments of the invention. It should be understood, however, that the invention is not limited to the precise arrangements and means.

In fig. Figure 1 shows the structures of a soluble self-organizing nanoparticle, dynamic combinatorial cobalamide as a core, dynamic combinatorial dipyridamole as a structural component, dynamic combinatorial derivatives of basic oligopeptides and amino acids as a binding component.

In fig. Figure 2 shows a self-organizing dynamic combinatorial derivative of dipyridamole and the principles of its synthesis.

In fig. Figure 3 shows the basis of the principle of molecular recognition between different substances based on nucleotide-like structures.

In fig. Figure 4 shows a synthesis scheme for dynamic riboflavin (IV) with 44 components of the mixture that react with each other, and also shows a formula for calculating the combinatorial synthesis of a fully substituted derivative, where m = 1, k = 4, the modifier is (II), and the product - tetra-succinyl riboflavin (V).

In fig. Figure 5 shows the structure of dynamic self-organizing combinatorial derivatives of cyanocobalamin.

In fig. Figure 6 shows an HPLC chromatogram of the starting cyanocobalamin with the following chromatographic conditions: gradient separation using buffer A, which contains 0.1 M perchloric acid with 1 M lithium perchlorate, and buffer B, which contains acetonitrile in a gradient (from 5% to 100%) per chromatograph Milikhrom A-02 with a prontosil-18 column.

In fig. Figure 7 shows an HPLC chromatogram of a combinatorial derivative of cyanocobalamin with 128 derivatives with the following chromatographic conditions: gradient separation using buffer A, which contains 0.1 M perchloric acid with 1 M lithium perchlorate, and buffer B, which contains acetonitrile in a gradient (from 5% to 100%) on a Milichrom A-02 chromatograph with a prontosil-18 column.

In fig. Figure 8 shows a scheme for the synthesis of a combinatorial dipyridamole derivative (IV), which includes a combinatorial reaction of dipyridamole (I) with two modifiers (II, III).

In fig. 9 shows a diagram of a thin layer chromatogram of a combinatorial derivative of dipyridamole (IV), the initial dipyridamole (I), a fully acylated dipyridamole (Ib), and a completely

- 5 043514 succinylated dipyridamole (Ic).

In fig. 10 shows the result of HPLC analysis of the starting peptide KKRKRKRKR. To detect the peptide, a wavelength of 280 nm with one absorption band is used.

In fig. 11 shows an HPLC chromatogram of a combinatorial derivative of the peptide KKRKRKRKR. The peaks of the chromatogram of the combinatorial derivative of the peptide KKRKRKRKR are shifted to the expected more hydrophilic region, while remaining expanded, this peak is further divided into 3 bands.

In fig. Figure 12 shows an HPLC chromatogram of the starting peptide KKRKSTRKR. The original peptide, when using a detector in the region of 280 nm, gives one absorption band.

In fig. 13 is the result of HPLC analysis of the combinatorial derivative of the peptide KKRKSTRKR, its chromatogram contains a characteristic triple peak.

Detailed Description of the Invention

In general, embodiments of the present invention relate to supramolecular structures, also called supramolecular nanoparticles (SNPs), which can be prepared using molecular building block recognition properties based on a dynamic quasi-living self-assembling system. Embodiments of the invention also include methods for producing supramolecular structures using molecular recognition and using methods for controlling the size of the resulting nanoparticles. Embodiments of the invention also include methods of using supramolecular structures to treat viral infections.

In some general embodiments of the present invention, the supramolecular nanoparticle (SNP) includes:

a) a plurality of binding components, wherein each binding component has a plurality of binding regions and wherein the plurality of binding regions comprise combinatorial carboxylated cobalamins;

b) a plurality of cores to provide a mechanical structure for self-assembly of a supramolecular soluble system, wherein the plurality of cores is an organic core that contains a core binding element for binding to a plurality of binding regions so as to form a first inclusion complex, wherein the core binding element comprises a combinatorial carboxylated dipyridamole, and wherein the first inclusion complex is a combinatorial carboxylated cobalamin with a combinatorial carboxylated dipyridamole; And

c) a plurality of terminating components, with each terminating component containing one binding terminating element to form a linkage with other binding sites of one of the plurality of binding components so as to form a second inclusion complex, wherein the primary terminal binding element comprises a combinatorial carboxylated dipyridamole, wherein the secondary inclusion complex is a combinatorial carboxylated dipyridamole, wherein the plurality of cores and the plurality of linking components are self-assembling carboxylated basic amino acids such as lysine, histidine, arginine, as well as any combination thereof and their carboxylated derivatives, which upon contact with each other are capable of self-assembly to form a self-assembled supramolecular soluble system, wherein the plurality of components act in concert to occupy the remaining binding regions of the plurality of binding components, and in which the plurality of termination components are present in sufficient quantity, relative to the number of the plurality of binding regions with the plurality of binding components, so as to terminate further binding, so as to form a discrete particle.

Structural component

In some embodiments of the present invention, the structural element has a plurality of binding regions of binding components. A binding element is a chemical moiety that binds to a specific binding region through one or more intermolecular bonds. The binding element of the structural component and the binding region of the binding element are specifically selected so that they selectively bind to each other and the molecular recognition property can be used to identify the binding regions. For example, the binding region may comprise combinatorial carboxylated cobalamin or carboxylated cobalamin or other cobalamin derivatives.

In some embodiments of the present invention, the structural element is at least represented by an inorganic or organic core.

In fig. 1 shows a variant of the invention in the form of a structure of a soluble self-organizing nanoparticle containing in the form of a core a dynamic combinatorial derivative of cobalamide, a dynamic combinatorial derivative of dipyridamole as a structural component, a dynamic combinatorial derivative of basic oligopeptides and amino acids as binding components. In some embodiments, a core based on self-organizing dynamic structures

- 6 043514 chemical derivative of cobalamin, has an inorganic core selected from the group consisting of inorganic nanoparticles, metal nanoparticles, gold nanoparticles, silver nanoparticles of silicon, metal nanoparticles, metal oxide nanoparticles, and any combination of nanoparticles presented above. For example, inorganic nanoparticles include metal oxide or other element oxide nanoparticles (eg, silica nanoparticles or iron oxide nanoparticles), and nanoparticles of other inorganic compounds. Magnetic nanoparticles, quantum dots (for example, CdS, CdSe nanoparticles), or semi-solid conductive particles of metal oxides can be used as functional nanoparticles.

The inorganic core may have a shape selected from the group including spherical, triangular, cubic, star-shaped, rod-shaped, shell-shaped, diamond-shaped, plate-shaped, pyramidal, irregular, cell-shaped, and combinations thereof.

Inorganic nanoparticles are known in the art. The inorganic core can bind to the binding regions of the nanoparticle components. In some embodiments, the binding component has a binding region that can bind to the inorganic core directly. In other embodiments of the present invention, the surface of the inorganic core has been prepared using multiple coupling elements so as to be capable of binding to multiple binding regions of the binder component through one or more intermolecular forces.

In some embodiments of the present invention, a plurality of inorganic core particles may be present in the supramolecular structure. In such cases, a plurality of inorganic core particles may bind to a plurality of linking components so as to form a cross-linked network or hydrogel. Continued growth of the cross-linked network can be limited or terminated at will by termination components, which can also bind to binding regions in the linking components.

In some embodiments, the structural component is an organic core. Organic cores may include derivatives of combinatorial self-assembling cobalamins, dendrimers, polymers, proteins, oligosaccharides, micelles, liposomes, vesicles, and combinations thereof. For example, in some cases, the organic core may be a dendrimer, polymer, or polypeptide. The structural component may include a dendrimer-based structural core, A polyamidoamine dendrimer (PAD), branched polyethylenimine (RPE), linear polyethylenimine, polylysine, polylactide, polylactide-co-glycoside, polyanhydride, poly-ε-caprolactone, A polymethyl methacrylate, poly(N -isopropyl acrylamide), polypeptide, and combinations thereof. In some embodiments, the organic core is a polyamidoamine dendrimer or poly-L-lysine polymer.

In some embodiments, the binding regions of the binding components interact with other elements that may be present as part of the organic structure of the core. In other embodiments, the organic core is derivatized with multiple coupling elements, such as, for example, combinatorial self-assembling dipyridamole derivatives. The coupling elements can bind to the binding regions of the coupling component through one or more intermolecular forces, followed by self-assembly into a cross-linked network or hydrogel. The continuous propagation of the cross-linked network may be limited or terminated by termination components, which may also react with the binding regions of the binding component. The binding region of the binding element can be selected depending on the type of binding, based on the principle of molecular recognition as well.

In fig. Figure 2 shows an embodiment of the invention in the form of self-organizing combinatorial dynamic derivatives of dipyridamole and the principle of their synthesis. Numerous dendrimers are known in the art. The advantages of core dendrimers are their rapid synthesis and easy ability to be functionalized due to the easy binding of elements. In some embodiments of the present invention, a dendrimer can be synthesized by incorporating essential elements as parts of the structure. Alternatively, in other embodiments of the invention, during dendrimer synthesis, a reactive functional moiety may be present at each point of the terminal element added to the linking element within the core. Examples of specific dendrimers suitable for the invention include such as polyamidoamine (PAM dendrimer).

Many polymers are known in the art. The advantage of using polymer cores is its rapid synthesis and its ability to be easily functionalized as a fastening element. In some embodiments of the present invention, a fastening element may be included in the polymer structure during synthesis. Alternatively, in other embodiments, the reactive functional groups on the polymer may be derivatized with chemical moieties that provide a bonding element function. For example, polypeptides containing lysine residues with a reactive amino group (-NH ₂ ) in their structure can be functionalized with linking elements. One example is the polypeptide poly-L-lysine.

In some embodiments, two or more different structural components are present, in which case each structural component may have linking regions (groups) to form a bond with the linking component.

In some embodiments of the present invention, the structural component may be a polyamidoamine dendrimer derivatized with a linker element(s), such as the oligopeptide KKRKRKRKR and linked to a combinatorial self-assembled carboxylated derivative of the same peptide.

Note: K is one letter of the amino acid lysine.

Note: R represents one letter of the amino acid name arginine. In some embodiments of the present invention, the structural element may also be adamantine-derivatized inorganic nanoparticles, such as adamantane-derivatized metal nanoparticles, or adamantane-derivatized metal oxide nanoparticles, more specifically, for example, adamantane-derivatized gold nanoparticles.

Terminating component

In some embodiments of the present invention, termination components occupy the interaction regions of the binding components to limit the continuous propagation and growth of the network when termination components are present in sufficient numbers relative to the interaction regions in the binding components. The binding component and the structural component can self-assemble into a supramolecular structure, while the termination components can only occupy the interaction regions and prevent further self-assembly (network growth) between the binding component and the structural component. For some embodiments of the present invention, the degree to which the termination component limits the self-assembly process is based on the relative amount (concentration) of binding components relative to the number of interaction sites on the binding components. In some embodiments of the invention, when the concentration of terminating components reaches a sufficient level, self-assembly of three components is observed: (1) a structural component, (2) a binding component, and (3) a terminating component, resulting in the formation of particles (nanoparticles) rather than cross-linked network or hydrogel. The advantage of this approach to supramolecular production of nanosized particles is that the size of the final particles (nanoparticles) can be easily calculated by varying the relative concentrations of the components in the drug mixture.

In some embodiments, the termination component has a single coupling element that communicates with one of the interaction regions on the coupling component. In these cases, each termination component has only one connecting element. A binding element is a chemical moiety that binds to the region of interaction of the binding component through one or more intermolecular forces. These termination components interact with only one interaction region on the coupling component. In this case, crosslinking between the terminating component and the connecting component can be avoided.

The termination component may be a polymer, polypeptide, oligosaccharide, or small molecule and functions as long as the termination component interacts with the binding regions of the linker component. In some embodiments of the invention, the terminating component is a polymer that is produced using a coupling element. In other embodiments, the terminating component is a poly-ethylene glycol derivatized with a coupling element, for example containing maleic acid residues.

A supramolecular structure may have two or more termination components. In these scenarios, the supramolecular structure may have 2, 3, 4, 5, or 6 different termination components. Each termination component may have the same binding element, or they may have different binding elements, but each binding element will interact with the binding region of the binding component.

Binding components

Binding components have multiple binding sites that interact and bind to the structural component and the terminator component and may include a terminator component selected from the group consisting of unmodified cobalamin, dipyridamole, basic amino acids, unmodified peptide such as KKRKRKRKR, and any combination thereof .

A binding region is a chemical moiety that binds to a structural component and a terminating component by one or more intermolecular forces.

In some embodiments, two or more different binding components may be used, provided that both have selective interaction regions that bind to structural and termination components.

The binding component may be a polymer, oligosaccharide, or polypeptide. Any suitable material having multiple bonding areas can be used as the binding component. Polyethylenimine or

- 8 043514 branched polyethylenimine derivatized with multiple binding sites. A specific example of a binding component is a branched beta-cyclodextrin-derivatized polyethylenimine. Another example of a binder is poly-L-lysine derivatized with β-cyclodextrin.

Molecular recognition

In some embodiments of the present invention, the binding regions and/or binding elements are molecular recognition elements. In other words, the binding region forms a molecular recognition pair with a structural component or termination component.

In fig. 3 shows the principle of molecular recognition between different substances based on nucleotides as a structure, which has important applications in some embodiments of this invention. Molecular recognition refers to the specific interaction between two or more molecules through one or more intermolecular forces. The molecules involved in molecular recognition have molecular complementarity and are called pairwise molecular recognition or host-guest complex. In this case, the terms host and guests do not confer a special relationship, but describe only two compounds that exhibit molecular complementarity, i.e. communicate with each other using molecular recognition. And the host and the guest communicate with each other, while the two hosts do not. Molecular recognition is a specific interaction, meaning that each molecular recognition element will bind to complementary molecules that have specific structural features. In general, the components of molecular recognition pairs bind to each other more tightly than is the case for nonspecific binding, since the interactions that occur between the two molecular recognition elements are numerous.

In some embodiments, molecular recognition pairs include small molecule host/guest complexes (including but not limited to inclusion complexes), pairs of complementary oligonucleotide sequences (e.g., DNA-DNA, DNARNA, or RNA-RNA) that bind to each other through hybridization), antibody-antigen, substrate protein, inhibitor protein, and protein-protein interactions (such as alpha-helical peptide chains and β-sheet peptide chains).

In some embodiments of the invention, supramolecular structures self-assemble through molecular recognition. In this case, the binding regions on the binding component form a molecular recognition pair with the binding elements on the structural element. Binding of elements on the terminal component also occurs with binding sites on the binding component to form a molecular recognition pair. The molecular recognition pairs formed between the binding component and the structural component may be the same as the molecular recognition pairs formed between the binding component and the terminal component, or they may be different. In other words, in some embodiments, the binding element on the structural component may be the same as the binding element on the termination component, or they may be different, but both binding elements bind to the same binding region on the binding component.

For the present invention, molecular recognition pairs include molecular recognition pairs, combinations of molecular recognition pairs, and a plurality of multiple molecular recognition pairs. Some exemplary embodiments of the invention include the use of molecular recognition pairs consisting of adamantane-β-cyclodextrin complex, diazobenzene-α-cyclodextrin complex, steroid-based molecular recognition pairs, pyrene-based molecular recognition pairs, steroids, rhodamine complexed with cyclodextrin, doxorubicin in complex with cyclodextrin, biotin-streptavidin, complementary nucleotide base pair, complementary nucleotide pair, complementary oligonucleotide pair, and combinations thereof.

Functional elements

In some embodiments of the present invention, at least one of the structural components, a binding component or a termination component, includes a functional element. A functional element may be a chemical moiety that imparts additional function or activity to a supramolecular structure not inherent to it while the functional element is absent. The functional element may be a light-emitting (ie, fluorescent or phosphorescent) compound, such as a combinatorial self-assembled riboflavin derivative. Fluorescent and phosphorescent supramolecular structures can be used, for example, in imaging studies in vitro or in vivo. For example, riboflavin fluoresces under the influence of UV light and can be used as a functional element. The functional element may also be a compound containing a radioactive or magnetically active isotope. For example, positron-emitting isotopes such as ⁶⁴ Cu can be used to measure the biodistribution of supramolecular structures. Other useful suitable isotopes would be obvious to one skilled in the art.

In fig. 4. shows a synthesis scheme for a functional element as an example of the implementation of the present invention, in which the functional element is dynamic riboflavin (IV), with 44 components in the mixture that react with each other, as well as a formula for calculating the combinatorial synthesis and the complete synthesis scheme its substituted derivative, where m=1, k=4, and modifier (II), is tetra-succinyl riboflavin (c). In some embodiments of the invention, the functional element may be a targeting element that is delivered through a supramolecular structure to specific cells. Such delivery systems with functional elements inside can be represented by oligonucleotides, antibodies, and small molecules that bind to cell surface proteins. In general, any chemical moiety that specifically binds to one or more cell surface receptors can be incorporated into a supramolecular structure to be a functional element. Cell surface proteins can be, for example, proteins on a cancer cell or on bacteria or fungi. Specific examples of functional elements that are cell targeting moieties of the present invention may be RGD and EGF, folic acid, transferrin, and include antibodies targeting cell surface markers, such as, for example, Herceptin for Her2 in breast cancer cells.

For this invention, a cell-free functional element that acts through the function of increasing the permeability of cell membranes can be selected as the functional element. Specific examples of ligands that increase cell membrane permeability include TAT ligand. Various other cell membrane permeabilization ligands may also be used in some embodiments of the present invention.

In some embodiments of the present invention, the supramolecular structure may have two or more functional elements. For example, a supramolecular structure may have two targeting functional elements as a means to increase selectivity of delivery to the desired cells, or as a means to increase binding affinity by using multiple functional elements to target protein-cell surface interactions. Cell means biological cell. Other examples of multiple functional elements include supramolecular structures having targeting functional elements and having cell permeability enhancing functional elements as a means of combining the synergistic effects of improved drug transport to the cell and increased cell membrane permeability. Another example of the synergistic action of multiple functional elements in a supramolecular structure is the use of a supramolecular structure having (1) an imaging component, a functional element that is a light-emitting or radioisotope functional element and (2) a targeting functional element for imaging target cells. The present embodiments of the invention include the use of combinations of functional elements, such as two targeted functional elements, incl. a functional element for increasing the permeability of cell membranes, or two targeted functional elements, incl. functional element of visualization, as well as the use of combinations of the above functional elements.

In some embodiments of the present invention, the supramolecular structure includes two or more termination components, each of which may further include a functional element. In this case, multiple functional elements can be introduced into the particle using multiple termination components. For example, a supramolecular structure may have (1) a termination component that does not have any functional element and (2) a termination component that has a targeting element. Termination components that do not have a functional element can be exchanged with termination components that have a functional element by treating the supramolecular structure with a secondary termination component or by treating the supramolecular structure with a mixture of other termination components. Likewise, a supramolecular structure can be produced using a mixture of termination components, each of which will be included in the supramolecular structure.

Loads (cargo)

In some embodiments of the present invention, the supramolecular structure may include a load. A load is defined as a chemical moiety that can be encapsulated within a supramolecular structure, and that can be released from the supramolecular structure. In some embodiments of the present invention, the cargo may be associated with one or more structural components, a binding component, or a termination component. In some preferred embodiments of the present invention, the cargo is a chemical group that can bind nonspecifically to binding agents.

- 10 043514 areas of the binding component and which prevent interference in the self-assembly of nanoparticles from the load side. For example, in some embodiments of the present invention, the cargo is a small molecule selected from the group consisting of drugs such as doxorubicin, taxol, rapamycin, cisplatin, other anticancer drugs useful in cancer chemotherapy, including protein, peptide, oligonucleotide, siRNA, plasmid, gene delivery molecules (systems), as well as any combinations thereof.

It is contemplated, for some embodiments of the present invention, that nanoparticles may not encapsulate the same cargo in all cases. In some embodiments of the present invention, supramolecular structures can deliver therapeutic proteins and oligonucleotides to a target cell, and the supramolecular structures themselves are used to protect therapeutic compounds, proteins and/or oligonucleotides from degradation. In some embodiments of the present invention, the supramolecular structure may include two or more cargo. The choice of the ratio and amount of two or more therapeutic compounds in a supramolecular structure ensures the correct delivery of drugs into the cell. In some embodiments, in addition to small molecules, a plasmid may also be included as cargo in the supramolecular structure. In many embodiments of the present invention, multiple types of cargo and various combinations of cargo are encapsulated in supramolecular structures.

Methods for obtaining a supramolecular structure

The present invention includes methods for obtaining the cheese molecular structures described above by preparing a suspension of structural components and binding components; adding binder components to said suspension.

The ratio of structural components for the coupling components and terminating components are selected in accordance with the predetermined size of the specified supramolecular structures. The structural, binding and termination components self-assemble into supramolecular structures having an essentially predetermined size. In some cases, the predetermined size is at least greater than 10 nm and less than 800 nm (nanometers).

Some embodiments of the present invention include methods for producing supramolecular structures.

One general example of a method for preparing a supramolecular structure includes the following steps: preparing a suspension of structural components and binding components;

adding terminating components to a suspension of structural components and binding components to form a mixture; and forming a supramolecular structure from a mixture of terminating components with structural and binding components.

Additionally, embodiments include a general method for preparing a supramolecular structure comprising the steps of:

choosing the ratio of the amounts of structural components to the amounts of binding components; and selecting a ratio for the amounts of structural components to the number of termination components.

Additionally, embodiments include a general method for producing a supramolecular structure, a step in which there is a choice of ratio for the amounts of structural components to the amount of binding components.

Additionally, embodiments of the invention include a general method for producing a supramolecular structure in which there is a choice of the ratio of the number of structural components to the number of termination components.

Additionally, embodiments of the invention include a general method for producing a supramolecular structure further comprising selecting the ratio of the amount of coupling components to the amount of terminating components.

The ratio of structural components for linking components to terminal components is selected in accordance with the predetermined size of the specified supramolecular structure. Structural, binding and terminating components self-assemble (self-organize) into supramolecular structures that essentially acquire a predetermined size. In some embodiments of the present invention, the predetermined size ranges from about 5 nm to 2000 nm. In some embodiments of the present invention, a predetermined particle size ranging from at least 20 nm to 400 nm is desirable. The size of supramolecular structures can be easily controlled by changing the ratio between the components used to obtain the supramolecular structures. A wide variety of supramolecular structures of different sizes can also be easily prepared, and the possibility of combinatorial synthesis also allows for the preparation of different sizes of supramolecular structures, and the optimization of a specific function to ensure their activity.

The supramolecular structure can be easily obtained by combining the components together. At least three components self-assemble into a supramolecular structure. Additional components (structural, bonding or terminating) or cargo connections (load or load) may also be used, provided that a minimum of elements are present. Additional components may include one or more functional elements.

Once the supramolecular structure is formed, the components can be replaced by other components bearing the corresponding binding elements or binding regions by treating the supramolecular structure with additional components. For example, some termination components can be exchanged by processing of the supramolecular structure for other termination components (eg, enriched with functional elements). In addition, structural or binding components can be exchanged by treating the supramolecular structure with additional structural or binding components. A suspension or solution of components can be treated with ultrasound to speed up or assist in the occurrence of component metabolic reactions.

The size of supramolecular structures can be easily controlled by changing the ratio between the components used to obtain the supramolecular structures. A wide variety of supramolecular structures of varying sizes can be easily prepared. This also enables combinatorial synthesis, as arrays of supramolecular structures can be prepared based on their specific function and optimization of their activity.

Using component exchange, the sizes of supramolecular structures can be adjusted after the supramolecular structures have been formed by treating the prefabricated supramolecular structures with an additional component. For example, if a preformed supramolecular structure is treated with additional binding component, the size of the structure will decrease. If the preformed supramolecular structure is treated with an additional structural component, the size will increase. Examples of this effect are presented in the examples below.

The supramolecular structure can be dissociated naturally in vitro and in vivo, in accordance with some embodiments of the present invention.

Functional elements can also be easily adjusted using this method. In many cases, a component, a functional element-bearing molecule, may be included in the mixture used to obtain the supramolecular structure. The proportion in which functional elements are present in a supramolecular structure can be easily adjusted by changing the ratio between functional elements. For example, if a functional element is present on a binder component, the ratio of the binder component having the functional element and the binder component lacking the functional element determines the extent to which the functional element is present in the resulting supramolecular structure. The same is true when the functional element is present on the termination component or structural component.

When functional elements are present on terminal components, preassembled supramolecular structures can be processed with terminating components having functional elements. Some of the terminating components will exchange functional elements with the subsequent formation of a supramolecular structure. Multiple termination components with many different functional elements can be added to the supramolecular structure in a similar manner. The extent to which the functional element is present on the resulting supramolecular structure is determined by the concentration of the terminating component for processing the preformed supramolecular structure.

Individual components can be easily obtained using well known chemical synthesis methods. The coupling elements are selected depending on the type of intermolecular forces chosen to bind the components together. Based on molecular recognition chemistry, chemical moieties can be selected as the binding element or by the nature of the binding region. For structural components, inorganic cores can be synthesized using methods known in the art to provide essential elements on the surface when needed. Organic compounds such as polymers and dendrimers can be synthesized using known methods with suitable coupling elements. Alternatively, organic cores, including polymers, dendrimers, and/or polypeptides and having reactive functional groups modified with suitable linking elements or binding regions, can also be prepared.

There are many methods for derivatizing organic compounds with suitable binders. For example, reactive functional groups of organic compounds such as hydroxyls, thiols, amines, carboxylic acids, halides, alkenes, alkynes, azides and others,

- 12 043514 can react or be activated to interact with a variety of other functional groups to form covalent bonds. For example, amine-containing compounds having a free -NH2 group can react with binders bearing amine-reactive groups such as isocyanates, isothiocyanates and activated esters such as N-hydroxysuccinimide (NHS) esters. This way, binding elements can be easily added to any component. The number of binding elements on a particular component may vary depending on the number of reactive sites and the amount of reactive binding element used to produce the component. For specific examples, see the examples described below.

Chemistry that is typically used to derivatize proteins can also be used to add binding elements to proteins, peptides, or antibodies. For example, a combination of amines or thiol-ene interaction with the formation of irreversible bonds.

In some cases a linker may be required. Various bifunctional cross-linking agents for covalently binding to proteins are known to those skilled in the art, any of which can be used. For example, heterodifunctional cross-linkers such as succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) and melaimidobutyryloxysuccinimide ester (GMBS) can be used to react with amines (via succinimide esters) and then with formation of a covalent bond with a free thiol (via maleimide). Other cross-linkers, such as succinimidyl-3-(2-pyridyldithio) propionate (SPDP), can react with amines (via the succinimide ester) and form a covalent bond with the free thiol via thiol exchange. Other bifunctional crosslinkers include suberic acid bis(N-hydrosuccinimide ester), which can react with two amines. Other bifunctional and heterobifunctional crosslinkers suitable for use with various surface modifications will be apparent to those skilled in the art.

In some embodiments, it is desirable to include a reversible (cleavable) cross-linking agent, a variety of which is known to one of ordinary skill in the art. For example, 4-allyloxy-4-oxobutanoic acid has an alkene group on one end that can be used to link a thiol-ene to a thiol, and the other end has a carboxyl group that can be linked to an amine. There is an ester group in the middle of the crosslinker that should hydrolyze slowly over time under physiological conditions. Other cleavable cross-linking agents will be apparent to one skilled in the art. These include, for example, disulfide bonds, which are cleaved upon reduction.

Use of supramolecular structures

Supramolecular structures have many applications, especially in the biological field. The simple methods required for the preparation of supramolecular structures allow the rapid preparation of supramolecular structures of various sizes or bearing specific functional elements. The use of different materials for structural, bonding and termination components allows for a wide variety of utilities.

Supramolecular structures can be dissociated in in vitro and in vivo environments according to some embodiments of the present invention. This makes it possible to release the cargo (payload) from the supramolecular structure.

Supramolecular structures can be used for gene therapy (in vivo) or for cellular transfection (in vitro) by delivering genes or plasmids into cells.

The invention includes methods for delivering a gene into a cell by contacting the cell with a supramolecular structure described herein carrying a plasmid as cargo. Treatment of a cell with a supramolecular structure results in internalization of the supramolecular structure followed by release of the plasmid into the cell. This can result in efficient transfection of the target cell with the plasmid of interest. Typically, any plasmid carrying any gene can be introduced into a cell in this way. Likewise, targeting and/or cell penetration elements can improve cell specificity and/or internalization.

The invention also includes methods for delivering therapeutic compounds by treating a cell with a supramolecular structure described herein with the therapeutic compound as a cargo. The therapeutic compound may be, for example, a protein or peptide (including antibodies), an oligonucleotide (eg, siRNA), or a small molecule. The small molecule may be, for example, an anticancer (eg, doxorubicin, taxol, paclitaxel, cisplatin, or rapamycin), antibiotic, antibacterial, or antifungal agent. Functional elements of the supramolecular structure may improve cellular targeting, internalization, or distribution. More than one therapeutic compound can be delivered in a single supramolecular structure, and, if necessary, the ratio of therapeutic compounds can be controlled.

Other methods of using the supramolecular structures described herein include photothermotherapy techniques by treating cells with the supramolecular structures described herein containing gold nanoparticles as structural components.

- 13 043514

Pharmaceutical compositions

The supramolecular structures or nanoparticles discussed herein can be included in various compositions for use in diagnostic or therapeutic treatments, especially for the treatment of viral infections.

The compositions (eg, pharmaceutical compositions) can be assembled as an assembly. Typically, the pharmaceutical composition of the invention contains an effective amount (eg, a pharmaceutically effective amount) of the composition of the invention.

The composition of the invention may be formulated as a pharmaceutical composition that contains the composition of the invention and a pharmaceutically acceptable carrier. By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e. the material can be administered to a subject without causing any undesirable biological effects or which does not interact in a harmful manner with any of the other components of the pharmaceutical product or formulation in which it is contained. The carrier will naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to one of ordinary skill in the art. For a discussion of pharmaceutically acceptable carriers and other components of pharmaceutical compositions, see Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990. Some suitable pharmaceutical carriers will be obvious to those skilled in the art and include, for example, water (including sterile and/or deionized ), suitable buffers (such as PBS), saline, cell culture medium (such as DMEM), artificial cerebrospinal fluid, etc.

The pharmaceutical composition or kit of the invention may contain other pharmaceuticals in addition to the compositions of the invention. The other agent(s) may be administered at any appropriate time during treatment of the patient, either simultaneously or sequentially. One skilled in the art will appreciate that the specific formulation will depend in part on the particular agent used and the route of administration chosen. Accordingly, there is a wide variety of suitable formulations of the compositions of the present invention.

Formulations that are suitable for topical administration directly to the CNS include, for example, suitable liquid carriers or creams, emulsions, suspensions, solutions, gels, creams, pastes, foams, lubricants or sprays. Local administration into the central nervous system is possible when opening the central nervous system with a wound or during surgery. One of ordinary skill in the art will appreciate that a suitable formulation can be selected, adapted or developed based on a particular application. Dosages for the compositions of the present invention may be in unit dosage form. The term unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for animals (eg, humans), each unit containing a predetermined amount of an agent of the invention, alone or in combination with other therapeutic agents, calculated in an amount sufficient to obtain the desired effect in combination with a pharmaceutically acceptable diluent, carrier or excipient.

One skilled in the art can readily determine the appropriate dosage, schedule, and route of administration for the precise composition to be used to achieve the desired effective amount or effective concentration of agent in an individual patient. The dose of the composition of the invention administered to an animal, in particular a human, in the context of the present invention must be sufficient to affect at least a detectable amount of diagnostic or therapeutic response in the individual over a reasonable period of time. The dose used to achieve the desired effect will be determined by many factors, including the potency of the particular agent administered, the pharmacodynamics associated with the agent in the host, the severity of the disease state of infected individuals, the presence of other drugs administered to the subject, etc. The dose size will also be determined by the presence of any adverse side effects that may accompany the particular agent or composition used. It is generally desirable to minimize side effects as much as possible. The dose of biologically active material will vary; the appropriate amounts for each particular agent will be apparent to one skilled in the art.

Another embodiment of the invention is a kit applicable to any of the methods disclosed herein, either in vitro or in vivo. Such a kit may include one or more compositions of the invention. Optionally, the kits contain instructions for performing the method. Optional kit elements include suitable buffers, pharmaceutically acceptable carriers, etc., containers or packaging materials. Kit reagents may be contained in containers in which the reagents are stable, such as lyophilized form or stabilized liquids. The reagents may also be in single use form, for example, in single dose form.

From the foregoing description, it will be apparent that the invention described herein may be subject to changes and modifications to adapt it to various applications and conditions. Such embodiments are also included within the scope of the following claims. Transfer

- 14 043514 of a list of elements in any variable definition herein includes definitions of that variable as any individual element or combination (or subcombination) of listed elements. The presentation of an embodiment herein includes this embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Terms listed in the singular tense also include plural ones unless the context indicates otherwise. The examples disclosed below are intended to illustrate the invention and not to limit its scope. Other embodiments of the invention will be apparent to one of ordinary skill in the art and are covered by the appended claims. All cited publications, databases and patents are incorporated herein by reference for all purposes.

Methods for preparing, characterizing and using the compounds of the present invention are illustrated in the following examples. Starting materials are prepared in accordance with procedures known in the art or as shown herein. The following examples are presented to provide a more complete understanding of the invention. These examples are illustrative only and should in no way be construed as limiting the invention.

Various methods of administering supramolecular combinatorial cobalamin derivatives (CDC) can be used. The CDC composition may be administered orally or may be administered intravascularly, subcutaneously, intraperitoneally by injection, aerosol, bladder, topical, and so on. For example, inhalation methods are well known in the art. The dosage of the therapeutic composition will vary widely depending on the particular antiviral CDC administered, the nature of the disease, frequency of administration, route of administration, host clearance of the agent used, and the like. The initial dose may be higher with subsequent lower maintenance doses. The dose may be administered at a frequency of once per week or once every two weeks, or may be divided into smaller doses and administered once or more times per day, twice per week, and so on to maintain an effective dosage level. In many cases, oral administration requires a higher dose than intravenous administration.

The compounds of the present invention can be included in various compositions for therapeutic administration. More specifically, the compounds of the present invention may be formulated into pharmaceutical compositions in combination with suitable pharmaceutically acceptable carriers or diluents and may be formulated 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 can be accomplished by a variety of routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, and so on. The antiviral SCMs 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 compounds 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, perforin, anti-inflammatory agents, etc.). In pharmaceutical dosage forms, the compounds can be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are given by way of example only and are not intended to limit the invention in any way. For preparations for oral administration, all compounds can be used alone or in combination with suitable additives for the production of tablets, powders, granules or capsules, for example 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 gelatin; with raising agents such as corn starch, potato starch or sodium carboxymethylcellulose; with talc or magnesium stearate and, if necessary, diluents, buffering agents, wetting agents, preservatives and flavorings.

The compounds may be included in injectable compositions by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, higher aliphatic acid esters or propylene glycol; and, if desired, with the usual additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives. The compounds can be used in an aerosol composition for inhalation administration, including the use of nebulizers. The compounds of the present invention can be incorporated into suitable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen and the like. In addition, the compounds can be included in suppositories by mixing with various bases, such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally using suppositories. The suppository may contain excipients such as cocoa butter, carbowax and polyethylene glycols, which melt at body temperature but remain solid at room temperature.

- 15 043514

Unit dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, wherein each unit dose, for example, teaspoon, tablespoon, tablet or suppository, may contain 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 a compound of the present invention in the composition in the form of a solution in sterile water, saline or other pharmaceutically acceptable carrier. Implants for sustained release of compositions are well known in the art. Implants are manufactured in the form of microspheres, plates, etc. from biodegradable or non-biodegradable polymers. For example, polymers of lactic and/or glycolic acid form a degradable polymer that is well tolerated by the host.

The implant containing the antiviral combinatorial cobalamides according to the invention is located close to the site of infection (in the case of a respiratory viral infection, the lungs and bronchi), so that the local concentration of the active agent increases compared to other areas of the body. The term unit dosage form refers to physically discrete units suitable for use as unitary dosages in humans and animals, each unit containing a predetermined amount of the compounds of the present invention calculated to be sufficient to provide the desired effect together with a pharmaceutically acceptable diluent, carrier or filler. The description of the unit dosage forms of the present invention depends on the specific compound used and the effect to be achieved, as well as on the pharmacodynamics of the compound used in the host. Typically, pharmaceutically acceptable excipients are available, such as excipients, adjuvants, carriers or diluents.

In addition, pharmaceutically acceptable excipients such as pH adjusting agents and buffering agents, tonicity agents, stabilizers, wetting agents and the like are generally available. Typical doses for systemic administration range from 0.1 pg (picograms) to 1000 milligrams per kg of subject body weight per administration. A typical dose may be one tablet to be taken two to six times daily, or one capsule or sustained-release tablet to be taken once daily with a proportionately higher content of the active ingredient. The sustained release effect may be due to capsule materials that dissolve at different pH values, capsules that provide slow release 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 symptoms, and the subject's susceptibility to side effects. Some of the specific compounds are more effective than others.

Preferred dosages of this 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 the compound. One method of interest is the use of liposomes as delivery vehicles. The liposomes fuse with the cells of the target area and ensure delivery of the contents of the liposomes into the cells. Contact of liposomes with cells is maintained for a time sufficient for fusion using various methods of maintaining contact, such as isolation, binding agents and the like. In one aspect of the invention, the liposomes are intended to produce an aerosol for pulmonary administration. Liposomes can be made from purified proteins or peptides that mediate membrane fusion, such as the Sendai virus or influenza virus.

The lipids may be any useful combination of known liposome-forming lipids, including cationic or zwitterionic lipids such as phosphatidylcholine. The remaining lipids are generally neutral or acidic lipids such as cholesterol, phosphatidylserine, phosphatidylglycerol and the like. To prepare liposomes, the method described by Kato et al. (1991) J. Biol. Chem 266: 3361. Briefly, the lipids and composition for inclusion in liposomes containing combinatorial supramolecular cobalamides are mixed in a suitable aqueous environment, suitably in a saline environment, where the total solids content will be in the range of about 110 wt.%. After vigorous stirring for short periods of approximately 5-60 seconds, the tube is placed in a warm water bath at approximately 25-40°C and this cycle is repeated approximately 5-10 times. The composition is then sonicated for a suitable period of time, typically about 1-10 seconds, and optionally further mixed using a vortex mixer. The volume is then increased by adding an aqueous medium, usually increasing the volume by about 1-2 times, followed by stirring and cooling. The method allows the inclusion of supramolecular structures with a high total molecular weight into liposomes.

Compositions with other active agents

A number of antiviral supramolecular structures have been considered for use in the present methods. One antiviral supramolecular structure is called KS. The antiviral KS according to the invention can be included in compositions with other pharmaceutically active agents

- 16 043514 mi, in particular with other antiviral, antimicrobial or anticancer agents. Other agents of interest include a wide range of antiviral mononucleotide derivatives and other RNA polymerase inhibitors known in the art. Classes of antiviral agents include interferons, lamivudine, ribavirin, and so on; amantadine; remantadine such as zinamivir, oseltamivir and so on; acyclovir, valacyclovir, valganciclovir, etc. Other groups of antivirals include adefovir, vbacavir, didanosine, emtricitabine, lamivudine, nelfinavir, ritonavir, saquinavir, daclatasvir, stavudine, tenofovir, efavirenz, nevirapine, indinavir, lobalavir and ritonavir and ritonavir. cholecalciferol. Cytokines such as interferon gamma, tumor necrosis factor alpha, interleukin 12 and so on can also be included in the composition along with the KS of the present invention. The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Alkylation is defined as the introduction of an alkyl substituent into an organic molecule. Typical alkylating agents are alkyl halides, alkenes, epoxy compounds, alcohols, and less commonly aldehydes, ketones, esters, sulfides, and diazoalkanes. Alkylation catalysts include mineral acids, Lewis acids, and zeolites. Alkylation is widely used in the chemical and petrochemical industries.

Ensemble or supramolecular ensemble is a term from supramolecular chemistry. For the present invention, the objects of supramolecular chemistry are assemblies spontaneously constructed from complementary geometrically and chemically corresponding molecular fragments. When synthesizing supramolecular structures from a single combinatorial derivative with incomplete substitution of available groups, more than 100 different derivatized supramolecular structures can be synthesized due to possible chemical permutations and combinations. It is noteworthy that intermolecular ionic and hydrogen bonds are necessarily formed between their molecules, and the derivatized supramolecular structures have significantly higher biological activity than the original cyanocobalamin molecule.

Using the methods of the present invention, a drug was manufactured that is effective in vivo against influenza, herpes, in Ovo models (in eggs) and bovine coronavirus. To prepare the drug, a combinatorial mixture of carboxymethylcobalamin was used in the form of a supramolecular ensemble without separation into individual components.

A combinatorial library is a collection of a large number of chemical compounds, proteins, genes or oligonucleotides that allows you to quickly search for target genes or target proteins. For example, a combinatorial library set may consist of millions of different chemicals or, for example, a set of recombinant DNA molecules made by inserting different antibodies into the light and heavy strands of cDNA.

Combinatorial synthesis involves the synthesis by combinatorial chemistry of many simultaneous reactions between three or more reagents to form combinatorial synthesis products that include hundreds of derivatives of the combined reagents. Derivatives of the combined reagents can be separated by chromatography to confirm their structure and study their biological activity. Recent approaches involve the use of unresolved and/or crude mixtures of combinatorial synthesis products for various reasons, including the fact that such mixtures of combinatorial synthesis products have greater and more variable biological activity profiles than the biological profiles of the individual components of the combinatorial synthesis products. synthesis.

A therapeutically effective amount (TEA) is a term for the present invention that refers to the amount of a drug. For some embodiments of the present invention, a therapeutically effective amount of combinatorial cyanocobalamin derivatives is an amount that provides therapeutically effective antiviral activity. ECP is expected to differ between viruses and different animal models.

Examples

Example 1 is an example of the practice of the present invention using combinatorially modified cobalamin as the core of the supramolecular structure. This core is synthesized using combinatorial cyanocobalamin derivatives with antiviral properties and is called KS. The resulting pharmaceutical compositions and dosage forms are then characterized in that the alkylated cyanocobalamin derivatives are an unresolved combinatorial mixture of monosubstituted to fully substituted derivatives for all groups R1-R7 of the following structure shown in FIG. 5, where at least one of the substituents R1-R7 represents -CH ₂ -COOM in any position, and M represents a metal or hydrogen atom. M can also be represented by one of the metals: K, Mg, Ca, Cu, Fe, Li, Na, Ba, Ag, Pt, Au, Ti OR Sb. In another embodiment of the invention, the pharmaceutical composition may additionally contain vanillin in the dosage form and cholecalciferol. In another embodiment of the invention, the pharmaceutical composition may further comprise unsubstituted methylcobalamin, dihydroxycobalamin (hydroxycobalamin), cobamide, or a mixture thereof in dosage form. The present pharmaceutical compositions, for example, can be formulated in various dosage forms, including, but not limited to, aerosol formulation for use in a nebulizer or spray, for injection formulation use for intramuscular injection (IM), intravenous (IV) injection and intravenous (IV). IVI) infusions.

A virucide is defined as any physical or chemical agent that inactivates or destroys viruses. This is different from an antiviral drug, which inhibits the proliferation of the virus inside the cell (Virucide, Wikipedia, 2020). The difference between viricidal and virucidal is shown to be that the viricidal action is part of the virucidal action, while the virucidal action means the complete destruction of viruses (viricidal or virucidal action, Wikki-diff, 2020).

Example 2 is an example of the synthesis of a combinatorial mixture of carboxymethylated cyanocobalamin that can be used to create an antiviral supramolecular structure called CS.

The synthesis is carried out as follows: 1 mmol (millimol, mmol) of cyanocobalamin is dissolved in 50 ml of Tris-buffered solution, and 4 mmol of monochloroacetic acid is added with stirring until both components are completely dissolved, and then the solution is stirred, placed in a thermostat at 40 ° C for 7 days . The solution is then poured into vials, lyophilized to remove excess monochloroacetic acid and hydrochloric acid, and used for analysis and research. In Synthesis Example 2, 27 derivatives (128 derivatives) of carboxymethylated cyanocobalamins can be obtained.

In fig. 6 shows an HPLC chromatogram (high pressure liquid chromatography) of the starting cyanocobalamin. The original cobalamin produces one absorption line when using a UV detector.

In fig. 7 shows an HPLC chromatogram of a combinatorial cyanocobalamin derivative. The chromatogram shows that the peak of the derivative is at a different location. Derivatives with a more hydrophilic region leave the HPLC column earlier, and thus the characteristic feature of the derivative chromatogram is expanded and separated into additional bands. This suggests that cyanocobalamin derivatives may have intramolecular and supramolecular ionic and hydrogen bonds that remain unchanged during the HPLC separation process performed under classical conditions using gradient HPLC. It was also impossible to separate and identify a fragment of the supramolecular structure using thin layer chromatography (TLC) and capillary gel electrophoresis (CGEP). Other chemical combinations can be used to derivatize cyanocobalamin, including the use of carboxylic acid anhydrides and polycarboxylic acid anhydrides, carboxylic acid halides and halocarbons. For some embodiments of the present invention, methylcobalamin, hydroxocobalamin (hydroxycobalamin) and/or cobamide can be used as the primary cobalamin for derivatization.

Further, in an experiment to study antiviral activity (ED90 = 10-30 μg/ml), several liquid dosage forms were studied. One of the liquid antiviral dosage forms studied is KS (pure combinatorial derivative). The second liquid antiviral dosage form studied is KC, which is a mixture of KS with vanillin (van) and cholecalciferol (C). Thus, in notation form: KC=(KS+van+C). The third liquid antiviral formulation studied is KSO, which is a mixture of KS with vanillin (van) and cobamide (CO). Thus, in notation form: KSO=(KS+van+CO).

To test the biological (antiviral) activity of the synthesized KC derivatives, the antiviral activity of the derivatives was studied by screening on models of the H1N1 influenza virus (Inf), the reference strain of vesicular stomatitis virus (Vesic. -VVS) and herpes simplex virus type 1 (Herp. - strain L-2 ), coronavirus - avian infectious bronchitis virus (IBV) in mattresses based on chicken fibroblast culture according to the degree of degradation (cytopathic effect, detachment from the bottom of the mattress). The degree of cell desquamation was determined by staining the culture with a vital dye, the concentration of which was determined spectrophotometrically relative to a healthy monolayer and an empty well. The results of in vitro studies are shown in table. 1 below.

- 18 043514

Table 1

Antiviral activity of supramolecular combinatorial derivatives of cyanocobalamin KS and its dosage forms

Composition %, cytoprotective antiviral effect** Inf Black person Vesic IBP KS 75 80 74 82 70 58 94 80 79 66 90 73 89 89 96 77 100 93 100 79 n±N, % 83±12 77±15 91±10 78±3 KS+van 94 100 94 80 93 95 93 89 98 90 98 90 100 100 100 100 100 100 83 100 n±N, % 97*±3 97*±4 94*±7 92*±8 KS+van+CO 90 88 94 100 100 70 100 100 100 100 94 100 93 100 95 92 80 100 100 88 n±N, % 93*±8 92*±13 97*±3 96*±6 Pure culture negative control (no virus) 100 100 100 100 Positive control of infected culture (with virus without treatment) 0 10 5 0

* groups (KS + van) and (KS + van + CO) are statistically significantly more effective than pure KS;

** for an average effective dose of 10 μg/ml

As can be seen from table. 1, all variants of KS-based dosage forms have an antiviral effect that can protect 50% or more of the cells of an infected culture. Dosage forms with vanillin and other cobalamin derivatives are statistically significantly (P<0.05) higher than the effectiveness of the pure substance KC.

To determine the maximum tolerable concentration (MIC) in toxicological experiments and to study the antiviral activity of the drug KC and its dosage forms for injection, the following types of transplanted cells of human and animal origin were used: PT, Tp, Hep-2, HELA, and chicken embryo. PT stands for Bovine Fetal Kidney Transplantable Cells. Tp stands for bovine fetal tracheal cells. Hep-2 stands for transplantable human laryngeal cancer cells. HeLa stands for transplantable human uterine cancer cells.

Cells were grown in medium 199 supplemented with 10% bovine serum and antibiotics (penicillin and streptomycin). Influenza viruses (H1N1), vesicular stomatitis (Indiana strain), coronavirus (X 343/44) and herpes simplex virus type 1 (strain L-2), avian infectious bronchitis coronavirus (strain IEK-KL2) were used as test viruses.

Toxicity study. Determination of MTC for dosage forms of KS and substance KS on cell cultures and chicken embryos.

Two-day cell cultures with a well-formed cell monolayer were used to determine MTC. KS and its dosage forms were tested on the four cell types listed above (n=5 experiments). At least 10 tubes of each culture were used in each experiment. After removing the growth medium from the test tubes, 0.2 ml of the test solution and 0.8 ml of the supporting nutrient medium were added. Tubes with cells were incubated at 37°C for 7-8 days. Test tubes with cell cultures without the addition of drugs served as control experiments.

Part of the results of the experiment was to determine the presence or absence of a cytopathic effect. The presence or absence of a cytopathic effect (CPE) was determined by examining the cells under a microscope at 10x magnification. The degree of cytotoxicity was assessed using the four plus system (+, ++, +++, ++++) to assess changes in cell morphology: (1) rounding and shrinkage of cells and (2) rejection of degenerated cells from the glass.

- 19 043514

The maximum tolerated concentration (MTC) was determined by determining the maximum amount of a substance that did not have a cytopathic effect on the cells. For this determination, various dilutions of the drug were added to the cell culture at a dose of 0.2 ml.

To study the toxicity of the drug in vivo at various doses of the drug, a dose of the drug in a volume of 0.2 ml was used on 10-11-day-old chicken embryos (5 embryos per MR dilution). A dose of the drug was administered into the allantoic cavity of the chick embryo as follows. Embryos at 10–11 days of age were ovoscoped and marked with an air cushion pencil on the side opposite the embryo where there were fewer blood vessels. The area marked with a pencil was disinfected with an alcohol solution of iodine, then the membrane was pierced, and then 0.2 ml of the drug dose was injected into the hole using a tuberculin syringe into the allantois cavity using a syringe needle to a depth of 10°. 15 mm parallel to the longitudinal axis of the egg. After the injection, the hole was again disinfected with an alcohol solution of iodine and sealed with paraffin wax. The egg was then placed to incubate using a thermostat set at 35-37°C for 72 hours. Before opening the eggs, they were placed in a refrigerator at 24°C for 18–20 hours in order to narrow the blood vessels of the embryo as much as possible. After this, the eggs were placed on a tray with the blunt end up. Then the shell over the air cushion was disinfected with an alcohol solution of iodine and 96% ethanol. The egg was broken and the embryo was removed with sterile forceps. The membrane lining the bottom of the air sac was also removed, first separating it from the underlying chorionic-allantoic membrane. The number of living and normally developing embryos after 24 and 48 hours of incubation in a thermostat at 37°C was counted to calculate LD ₅₀ and MTD in accordance with the Kerber method.

As a result of these experimental studies, it was found that KS and its dosage forms are not toxic to cell cultures at a dose of KS greater than 50 mg/ml. To increase drug concentration, the drug solution was lyophilized and then diluted to a concentration of 5%.

The results of studies of the toxicity of KS in various crops are presented in Table. 2 below.

table 2

Toxicity of CS and its dosage forms on cell cultures

No. Cell culture Toxicity (mg/ml) KS substance 1 RT More than 50 2 Tg -AND- 3 Ner -2 -AND- 4 Hela -AND- KS+van 5 RT More than 50 6 Tg -AND- 7 Ner -2 -AND- 8 Hela -AND- KS+van+CO 9 RT More than 50 10 Tg -AND- AND Ner -2 -AND- 12 Hela -AND-

MTC for cell cultures treated with both pure CS and its dosage forms is more than 50 mg/ml, which indicates the low toxicity of the proposed product. For further research, we chose the most effective sample of the dosage form KC + van + CO, then KCO.

Study of the antiviral effect of the drug KSO on the influenza A virus (H1N1)

The preferred embodiment of the present invention was selected for further studies. This is KSO, which is a liquid antiviral formula (KS+van+CO). Aqueous solutions of KSO in various doses (ten-fold dilutions) were injected into the allantoic cavity of 15 chicken embryos in a volume of 0.2 ml 12 hours after injection of the virus in a working dose (100 TCD ₅₀ /0.2 ml). Each experiment was accompanied by control of the test virus at a working dose. Infected and uninfected (control) embryos were incubated at 36°C for 48 hours. The embryos were then dissected, from which allantoic fluid was aspirated. Titration of the virus in allantoic fluid was carried out according to the generally accepted method with 1% erythrocytes of 0 (1) human blood group. The protection factor (PC) was determined.

The virus titer in the experimental and control groups of chicken embryos is presented in

- 20 043514 table. 3 below.

Table 3

Effective concentration of KSO in the in Ovo model of influenza infection

Group Drug concentration (mg/ml) Titer BnpycoB(lg TCA 50/ml) Minimum Effective Concentration (IEC mg/ml) experiment control Control (0.9% sodium chloride solution was administered) - 12 12 - Experiment 50±5 0 12 0.005 5±1 0 12 0.5± 0.05 1 12 0.05± 0.005 2 12 0.005± 0.0005 5 12 0.0005± 0.00005 10 12

As can be seen from table. 3, the minimum effective concentration (TEC) in relation to the influenza virus, which suppresses virus synthesis in 50% of cells, is 0.005 mg/ml with increasing dilution. When using the drug, the effectiveness of CSO decreases and is dose-dependent. This fact indicates that KSO has a direct antiviral effect against the H1N1 influenza virus. The term lg for the present invention means the decimal logarithm or decimal logarithm as opposed to the natural logarithm.

Study of the antiviral effect of the drug KSO on cytopathic viruses (vesicular stomatitis virus, coronavirus, measles virus).

Antiviral activity against cytopathic viruses: vesicular stomatitis virus, coronavirus and measles virus was determined in the culture of the above cells. The reaction was carried out as follows: 0.2 ml of the corresponding virus in a working dose (100 TCA ₅₀ /0.2 ml) was added in a volume of 0.2 ml to a 2-day washed cell culture. Then 0.8 ml of maintenance medium was added. When the culture showed CPE (cytopathic effect), KSO was administered in various doses. As a control, the same was done with test viruses without the drug. Cells were incubated at 37°C in an incubator. Experimental data and observations were carried out on days 3, 5 and 7 of the experiment. A decrease in virus titer under the influence of the study drug by 2 μg or more compared to the control was assessed as a manifestation of antiviral activity.

The results of the study of the antiviral activity of the drug KCO are presented in table. 4.

Table 4

Study of the antiviral effect of KCO against viruses: vesicular stomatitis, coronavirus, measles virus)

Composition Virus MEC, mg/ml Maximum virus titer dilution, lg TCAso/mL KSO VVS 0.05 4.9 cv 0.05 h,o MV 0.05 4.7

As can be seen from table. 4, KSO has antiviral activity and the ability to suppress the reproduction of all viruses we have studied at a concentration of 0.05 mg/ml (ED90) with a MPC = 50 μg/ml. The CTI of the drug is 1000. In addition, KSO was active against all viruses studied. Thus, the KCO drug is not associated with specific characteristics of the virus or cell culture, but affects mechanisms common to all cells.

Study of the antiviral effect of KSO on in vitro models of farm animal viruses

Tests were carried out in 96-well plastic plates with strain D-52 of the transmissible gastroenteritis virus (TGS) with an initial titer of 104 TCD ₅₀ /ml (tissue cytopathic doses) in a transplanted culture of piglet test cells (PTR) and a strain of large bovine diarrhea virus Oregon with an initial titer of 10 ⁷ TCD ₅₀ /ml in a transplanted saiga kidney cell culture (PS).

When testing the virusostatic (inhibitory) effect, cell cultures were infected with viruses at doses of 100 and 10 TCD ₅₀ /ml and incubated in an incubator at 37°C. KSO in various doses

- 21 043514 was introduced into cell cultures (CC) 1-1.5 hours after infection (after the adsorption period). Each dilution required 8 wells. After preparing the compound, the cell cultures were incubated at 37°C for 72-144 hours until the CPE (cytopathogenic effect) was evident in the virus control. Virus-infected cell cultures, intact CC and CC, into which only different concentrations of KSO were introduced, served as controls. The virusostatic effect was determined by the difference in virus titers in the experiment and control.

When determining the virucidal (inactivating) effect, different doses of the compound solution were mixed in equal volumes with virus-containing material and incubated in a thermostat at 37°C for 24 hours. The virus-containing material was used as a control, to which placebo (saline solution) and cultures of intact cells. After contact, the mixtures were titrated in parallel with the control. The results were determined 72-144 hours after incubation at 37°C, after clear manifestation of CPE in viral controls. The viricidal effect was determined by the difference in virus titers in the experiment and control and expressed in lg TCD ₅₀ .

As a result of these studies, it was found that the KSO compound at a concentration of 40 μg/ml suppressed the multiplication of the TGS virus by 2.75 lg TCD ₅₀ /ml at an infectious dose of 100 TCD ₅₀ /ml and at the same dose, by 3.75 lg TCD ₅₀ /ml, infectious dose 10 TCD ₅₀ /ml. At a dose of 40 μg/ml, KSO inactivated the TGS virus at a concentration of 2.0 lg TCD ₅₀ /ml. The KSO composition at a dose of 40 μg/ml inactivated the bovine diarrhea virus at 4.9 lg TCD ₅₀ /ml.

Thus, the KSO compound has a virusostatic (inhibitory) and virucidal (inactivating) effect on TGS viruses and bovine diarrhea, and on its basis, chemotherapy drugs can be created for the treatment and prevention of infectious diseases of viral etiology.

Study of the antiviral activity of KSO in rabbits with experimental herpesvirus kerato-conjunctivitis/encephalitis

Experimental herpetic infections are important to study because herpetic diseases are widespread and extremely varied in their clinical manifestations. Animal models of experimental herpes are increasingly used in the study of new antiviral substances. One of the clinical forms of systemic herpes, namely herpetic encephalitis, is easily caused in guinea pigs, hamsters, rats, mice, rabbits, dogs and monkeys.

Herpetic keratoconjunctivitis in rabbits (average weight 3.5 kg) was caused by applying infectious material (herpes simplex virus type 1 strain L-2) to the scarified cornea after the eye had been anesthetized by instilling dicaine and the cornea several times. The eyelid was then closed and rubbed in a circular motion. The dose of the virus was 0.05 ml, the dose was 6.75 lg TCD ₅₀ /ml. 16 rabbits were used in this series of experiments. Ten rabbits were administered KSO (daily from the second day of infection to 14 days) at a dose of 20 mg/kg, six rabbits were administered placebo 0.9% sodium chloride solution.

After infection of the rabbit cornea with HSV1, the presence of keratoconjunctivitis, encephalic diseases, and HSV1 antigens in peripheral blood lymphocytes was monitored daily by RIF. Before infection, lymphocytes in all animals did not have specific luminescence, which indicated the absence of herpes virus type 1 antigens in the peripheral blood. On the 3rd day after infection, HSV1 antigen was determined in the blood of all animals, IF=70%. Three rabbits (two from the experimental group before treatment and one from the control group) developed encephalic manifestations - convulsive syndrome and lack of appetite. All animals developed keratoconjunctivitis. On the 4th day after infection, the experimental group of rabbits was injected with KCO into the ear vein at a dose of 20 mg/kg body weight, and a 0.9% sodium chloride solution was injected into the ear vein of the control group. Every day for two weeks this procedure was repeated once a day. All animals of the experimental group survived, the HSV1 antigen was not detected in the blood on the 13-14th day, and encephalic manifestations ended on the 7th day of drug administration, and 2 animals of the control group died. On day 14, one animal in the experimental group died, while 6 animals died in the control group that did not receive KSO. The KSO efficacy index was 83.3% in the rabbit model of herpetic keratoconjunctivitis/encephalitis, and the experimental group of rabbits gained weight. The chemotherapy index was 1000. KCO is an effective antiviral drug with a wide spectrum of action and low toxicity.

Antiviral effect of the drug KCO on broiler chickens of the Cobb-500 cross

The effect of the drug KSO on the propagation of vaccine virus strains was measured based on the effect of KSO on reducing the titer of the corresponding specific antibodies. Many antiviral drugs inhibit the reproduction of live vaccine strains of viruses, while suppressing the synthesis of specific antiviral antibodies. This effect is associated with insufficient intensification of the infectious process by the vaccine and a weak immune response. For example, birds with infectious bursal disease exposed to a live vaccine may produce excess antibodies so that the bird becomes oversensitive to other viruses, loses weight, and increases mortality. The use of the KSO drug was supposed to demonstrate the presence of antiviral properties in several areas: a decrease in excess levels (titers) of antibodies, a decrease in morbidity (safety), and an increase in weight gain.

In the experiment, broiler chickens were selected on days 36 and 41, 15 animals per group.

KSO was drunk the day before vaccination with live vaccines against IBD, Hamboro disease (HD) and coronavirus infectious bronchitis (IB). The controls were broiler chickens that were not fed KSO but were vaccinated. In table 5 and 6 show the results of these studies.

Table 5

Growth of broilers (at the time of slaughter) in the experimental and control groups

Indicator Gain **, +% Safety **, +% Experimental group (n=15) 7.6±0.7* 1.0±0.1* Control group (n=15) -1.8±0.6* -4.6±0.4*

* - against unvaccinated control, which was taken as a basis ** - (P<0.01)

As can be seen from table. 5, in the experimental group, the weight gain of animals increased by (7.6±0.7)% compared to the weight loss in the control group, vaccinated but not treated (1.8±0.6)%. Also in the experimental group there was an increase in safety by (1.0±0.1)%. In table Figure 6 shows changes in specific antiviral antibody titers in the KSO-vaccinated group, the unvaccinated group, and the non-vaccinated group.

Table 6

Change in antibody titer against IBD, BG and IB in vaccinated groups and unvaccinated controls

Average change in titer of specific antibodies, ±T IBB B.G. I.B. Experimental group (vaccinated and treated with KSO) (n=15) -2000±200 -700±100 -1000±300 Control group No. 1 (vaccinated, but not treated with KSO) (n= 15) +3000±780 +3600±1000 +3000±820 Control group (no treatment and no vaccination) 0

As can be seen from table. 6, KSO has a direct (non-immunostimulatory) effect against all three viruses. The greatest inhibitory effect was observed in the group with infectious bronchitis: a decrease in antibody titer by 2000 units. In the vaccinated but untreated control, antibody titers increased from 3000 units to 3600 units, indicating efficient replication of the live vaccine virus in poultry. Thus, the use of KCO allows increasing the growth rate of broiler chickens by 5% and reducing mortality by 1%. KSO has a direct antiviral effect, inhibiting the proliferation of infectious bursal disease, Gambaro's disease and coronavirus infectious bronchitis viruses.

KSO provides moderate inhibition of vaccine virus replication, providing sufficient levels of protective antibodies and preventing avian immune depletion and the resulting reduction in weight gain and increased mortality.

The influence of KSO on the effectiveness of vaccination of broiler chickens with live vaccines

The effect of KSO on the effectiveness of vaccination was carried out directly at the poultry farm when raising broiler chickens. During a pathological examination of broiler chickens (also called simply broilers), characteristic changes for colibacillosis, coccidiosis, as well as numerous hemorrhages on the mucous membranes of the rectum, in the zone of transition of the glandular stomach into the muscular, basal glands, were observed. The contents of the glandular stomach are colored green. The death rate of broilers reached 15-20%. In a study of blood serum of broilers at the age of 3842 days, specific titers of antibodies to Newcastle disease virus (NCV) were higher than protective titers in the hemagglutination delay reaction (HADR) (1: 1024, 1: 2048).

Study of the effect of KSO at a dose of 0.03 ml/kg live weight on the effectiveness of vaccination against BNK. For this purpose, one of the chicken coops was taken under control, the rest were tested (see Table 7).

- 23 043514

Table 7

Results of a study of the influence of KSO on the effectiveness of poultry vaccination

Group number Chicken coop number Number of birds (thousands) Application schedule Control 3 40.0 KSO was not given Experience 1 12 40.0 From 7 days of age 3 days before vaccination with live viral vaccines Experience 2 6 40.0 1 day before vaccination against BNK Experience 3 9 40.0 Within 3 days before vaccination and 7-10 days after vaccination against BNK

Inspection conditions, microclimate parameters, lighting conditions, planting density, feeding conditions were the same in all groups in accordance with the instructions for growing the cross ROS 308.

Immunity was determined at 42 days of age in the HADR. At the same time, the clinical condition of the bird, the percentage of conservation, growth and feed costs were taken into account.

The results of tests to determine the effectiveness of KSO in vaccinating broilers against BNK are shown in Table. 8.

Table 8

The influence of KSO on the effectiveness of vaccination against Newcastle disease_________

Indicators Control Experience 1 Experience 2 Experience 3 Average titer in RGA 25±3 45±13 95±32 180±46* Immunity strength % 75 88.6 100 100

Note: *P<0.05

The mean titers of specific antibodies to BNV in both the control and experimental groups were protective. However, a study of the serum of broilers at the age of 42 days using KSO revealed a significant increase in the average titer in experimental group 3 compared to the control by 6 times (<0.01). In the experimental groups (1, 2), no significant difference in antibody titers was found compared to the control, however, they were at a protective level and a tendency was revealed to increase this indicator by 1.8 and 4.3 times. Group immunity in the control group was 75%, in the experimental groups (3.4) 100%, in the 1st experimental group 88.6%. The death of broilers was 9.8% in the control group, while the percentage of death decreased in the experimental groups: 2.9; 4.5 and 4.4 times, respectively, compared to the control. The average daily weight gain in the experimental groups ranged from 50 to 55 g, while the control groups had an average weight gain of 46 g.

Thus, we can conclude that the optimal regimen for using KSO for broilers in regions with a difficult epizootic situation with BNC is the use of the drug at a dose of 0.03 ml/kg of live weight 3 days before vaccination and 7 days before vaccination. 10 days after vaccination against BNK. The use of the drug according to the given scheme leads to a sixfold increase in the average titer of specific antibodies to the BNK virus and a 4-fold reduction in the mortality of broiler chickens.

Example 3 concerns the preparation of a supramolecular combinatorial mixture of dipyridamole (CD) derivatives as a binding and terminating component.

The procedure of Example 3 involves first diluting 222 µM dipyridamole (I) (CAS No. 58-32-2, Mr=504.636 g/mol, n=4) in 50 ml dioxane mixed with 50 ml glacial acetic acid and adding 60 µM succinic anhydride (III) and 61 μM acetic anhydride (II). The solution is then stirred and refluxed for 5 to 50 minutes. The solution is then filled into vials and lyophilized to remove solvents and acetic acid to obtain a combinatorial mixture (IV), which is CD. The combinatorial mixture (IV) is used to prepare pharmaceutical compositions, study the structures and determine the bioactivity of CDs.

In fig. 8 shows a schematic diagram of Example 3 for the combinatorial synthesis of dipyridamole derivatives (CD). Instead of carboxylic acid anhydride modifiers, carboxylic and polycarboxylic acid halides, such as succinic, maleic, fumaric, lactic, propionic, other halogen derivatives such as chloromethane, bromoethane, chloropropane, cyclic alkylating compounds such as oxirane or propiolactone, can optionally be used. for the preparation of combinatorial dipyridamole derivatives.

In fig. Figure 8 shows one starting molecule of dipyridamole (I), which contains 4 free hydroxyl groups available for modification (n=4) to create a combinatorial mixture. The amino groups as part of the residual morpholine and pyrimidine core can be protonated and protected from modification under given reaction conditions. The calculation of the number of moles of the modifier is carried out using combinatorics formulas: Ш - 4х (3 x 2 - 1), k - рх (2 - 1), where _m is the number of differences

- 24 043514 derivatives of molecules in the combinatorial mixture and the number of moles of dipyridamole for the reaction; n the number of hydroxyl groups available for modification in the structure of dipyridamole (n=4); k is the number of moles of each modifier. Thus, having only one starting molecule of dipyridamole and two modifiers after combinatorial synthesis, we theoretically obtain 12 combinatorial derivatives with different degrees of substitution, different positions of substituents, and different shuffling of modifier residues. This is not a simple mixture, but a complex supramolecular structure. Due to the presence of both substituted and unsubstituted hydroxyl groups in various derivatives, supramolecular structures will form due to both hydrogen and ionic bonds, including the tertiary amino groups of heterocycles.

Optionally, in some embodiments of the present invention, modifiers such as succinic anhydride or acetic anhydride can be used and introduced simultaneously and sequentially, or, succinic anhydride can be first introduced and heated in the mixture using a reflux condenser, and then acetic anhydride and reheat the mixture. Likewise, with this synthesis approach, instead of using succinic anhydride as a modifier, other embodiments of the present invention are contemplated using alternative synthetic approaches that use related chemical modifiers, including, for example, anhydrides such as maleic anhydride, aconitic anhydride , glutaric acid or phthalic acid, anhydride; acids such as, for example, acetic anhydride, ethylformic acid or monochloroacetic acid; and various alkylating agents including, for example, propiolactone, ethylene oxide, methyl chloride, ethyl chloride or propyl chloride.

C ¹³ NMR (carbon-13 nuclear magnetic resonance) was performed on combinatorial dipyridamole (CD) derivatives. Results C: 96.1; 161.8; 170.0; 157.8; CH2: 58.9; 61.7; 58.1; 61.4; 29.2; 29.1; SD: 173.1; 174.7; 170.2; and CH ₂ (in the morpholine cycle) 52.7; 25.4; 25.5. C ¹³ NMR data of the combinatorial derivative confirm the presence of both ethyl groups of succinic acid residues in its CD structure and acetyl residues as products of the reaction with the modifier acetic anhydride.

HPLC was performed on an HPLC column (microcolumn chromatograph Milichrom A-02 with a gradient of acetonitrile (5-100%)/0.1 M perchloric acid and 0.5 M lithium perchlorate). CD on the HPLC chromatogram shows one clear broadened peak and is not separated into components, although the retention time differs from both the parent dipyridamole and its fully substituted derivatives. The complex supramolecular structures formed between 12 different combinatorial CD derivatives were not separated chromatographically by this HPLC column method. Similarly, this combinatorial dipyridamole derivative (CD) was not separated by thin layer chromatography (TLC), which used acetonitrile:water as the mobile phase and used UV detection. CD-TCX showed only one band, which did not match any of the resulting derivatives.

In fig. Figure 9 shows the TLC of the combinatorial mixture (IV), which is less mobile than the original unmodified dipyridamole (I) and is the lightest band on TLC. The TLC bands of fully acylated dipyridamole (Ib) and succinylated dipyridamole (Ic) appear between the TLC bands of native dipyridamole and combinatorial dipyridamole. In addition, the combinatorial dipyridamole band was not resolved into its complex supramolecular structures by 2D TLC or by HPLC (data not shown).

A study was conducted to test the inhibition of cAMP phosphodiesterase by various supramolecular combinatorial dipyridamole derivatives. Various supramolecular combinatorial dipyridamole derivatives have been prepared in synthetic reactions using different molar ratios of modifiers. A cyclic AMP sandwich ELISA (enzyme-linked immunosorbent assay) was used. The reaction was stopped by adding a double volume of 1% TCA.

Table 9

Inhibitory effect on cAMP phosphodiesterase (PDE) of supramolecular combinatorial dipyridamole derivatives obtained in reaction with different molar ratios of modifiers

No. Molecular ratio of reagents * ED50 relative to cAMP, µg/ml, error measurements 10% w kl k2 1 44 88*** 88*** >500 2 -AND- 70 70 100 3 -//- 61 60 0.01 4 -AND- thirty thirty 5

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5 -AND- 15 15 10 6 -AND- 7 7 60 7 -AND- 3 3 115 8 -AND- 2 2 210 9 -AND- 1 1 300 10 -AND- 0 0 300

* m is the number of moles of dipyridamole 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;

** ED50 μg/ml PDE inhibition was determined by diluting the initial concentration of the dipyridamole derivative;

*** the maximum molar ratio at which all groups in dipyridamole are replaced; exceeding this ratio leads to unreacted modifiers remaining in the reaction medium - succinic anhydride and acetic anhydride

In the table above. 9 presents experimental data that, taken as a whole, reveals unexpected enzyme inhibition efficiencies for certain embodiments of the present invention. From the table 9, point No. 3, it can be seen that the ED50 for inhibition of cAMP phosphodiesterase by supramolecular combinatorial dipyridamole derivatives is the lowest (0.01 μg/ml ED50) when the molar ratio of the three reagent modifiers (m, k1 and k2) is 44:61:60. Note that with m equal to 44, a fairly small reduction in the number of molar ratios of k1 and k2 from 70.70 to 60.61 produces an astonishing 10,000-fold improvement in cAMP phosphodiesterase inhibition. What would be expected in accordance with the prior art relating to synthetic organic chemistry by one of those skilled in the art would result in very little variation in the amounts of derivatization modification of supramolecular combinatorial dipyridamole derivatives. Moreover, it is also surprising and unexpected that with respect to the molar amount of reagent m, a further reduction in the ratios of reagents k1 and k2 from 60.61 to 30.30 would result in a 500-fold loss of activity of the supramolecular combinatorial dipyridamole derivative in terms of their ability to inhibit cAMP phosphodiesterase . This discovery clearly demonstrates the exceptional sensitivity and unexpected utility of the synthetic methods of the present invention for the preparation of supramolecular combinatorial dipyridamole derivatives, as well as the non-obviousness of compounds containing supramolecular combinatorial dipyridamole derivatives. In addition, it should be noted that table. 9, positions 1 and 10 indicates that the cAMP phosphodiesterase inhibitors with the lowest effectiveness are supramolecular combinatorial dipyridamole derivatives that have been either maximally or minimally modified by k1 and k2. reagents in the presence of a fixed amount of reagent m. Thus, it was found that the combinatorial compositions of synthetic chemical products and the details of their synthesis presented in Table. 9, as positions 1 through 9, have unexpected variations in utility as a cAMP phosphodiesterase inhibitor.

In the next table. Figure 10 shows the compositions of the pharmaceutical compositions under study (FC, CD).

Table 10

Composition and ratio of pharmaceutical composition ingredients (FC CD) per capsule or pill

No. Ingredient name % 1 2 3 1. CD 0.1-20.0 2. Papaverine 0.5-10.0 3. Ascorbic acid 0.2-10.0 4. Bendazole 0.5-10.0 5. Tadalafil 1-5.0 6. Sodium valproate 5-20.0 7. Fillers up to 100%

As a control, animals received the same composition with the same substances (in the form of Carbopol gel), but without CD (FC).

Example 3 concerns combinatorial basic amino acids and a basic oligopeptide as a binding moiety. The combinatorial mixture of oligopeptides KKRKRKRKR is henceforth abbreviated KR. The KKRKRKRKR oligopeptide is preliminarily obtained using standard methods for obtaining a peptide using a peptide synthesizer or using genetic engineering. The procedure is as follows: 1 mmol of oligopeptide KKRKRKRKR is dissolved in 50 ml of buffered

- 26 043514 phosphate saline solution, then add 3 mmol of succinic anhydride and 3 mmol of phthalic anhydride, and the solution is stirred until both anhydrides are completely dissolved. The solution is poured into vials, lyophilized and used for analysis and research. Calculation of the molar ratios of two modifiers to the oligopeptide is carried out using the formulas:

(1) k = η x (2 ⁿ - 1) and (2) m = 4 x (3 x 2 ⁿ ' ² - 1).

N=number of groups available for substitution in the oligopeptide (n=9). m=number of moles of parent oligopeptide. The number of different molecules of its combinatorial derivatives after synthesis from the same starting peptide is 1532 different derivatives based on these calculations, taking into account the locations of substitutions and permutations, k = the number of moles of each of the two modifiers in the combinatorial synthesis reaction to obtain the maximum number of different derivatives (k =4599). In this example, the molar ratio to obtain the maximum number of different derivatives (1532 different molecules) is 3:3:1 (succinic anhydride: phthalic anhydride: oligopeptide KKRKRKRKR).

In fig. 10 shows the result of HPLC analysis of the parent peptide KKRKRKRKR.

The original peptide, when using a detector in the 280 nm region, gives one absorption band. Fig. 11 shows the result of HPLC analysis of the combinatorial derivative of the peptide KKRKRKRKR.

As can be seen from the chromatogram, the peak of the peptide is not just located in a different place - in the region of a more hydrophilic region, but is broadened, divided into 3 additional bands. HPLC data suggest that among the 1532 different peptide derivatives, there are ionic and intramolecular/supramolecular hydrogen bonds that are not broken during the separation process under classical gradient HPLC conditions. Thin layer chromatography and capillary gel electrophoresis also failed to separate supramolecular derivatives into individual fragments.

In other embodiments, other combinations of at least two different modifiers may be used to modify the peptide, where the modifiers are carboxylic and polycarboxylic acid anhydrides, carboxylic acid halides, and/or halocarbons. A peptide modified with one individual oligopeptide or a mixture of oligopeptides. Peptides can be produced by standard methods, including the use of peptide synthesizers, genetic engineering methods and/or recombinant technology methods known in the art.

To test the biological (antiviral) activity of the synthesized derivatives with different ratios of components in the combinatorial synthesis reaction, the antiviral activity of the derivatives was studied by screening using models of the H1N1 influenza virus (Inf), the reference strain of vesicular stomatitis virus (Vesic-VVS) and herpes virus type 1 (Nef. - strain L-2) in the table on a culture of chicken fibroblasts, depending on the degree of degradation (cytopathic effect, detachment from the bottom of the well). The degree of cell degradation was determined by staining the culture with a vital dye, the concentration of which was determined spectrophotometrically in relation to a healthy monolayer and an empty well. The results of in vitro studies are shown in table. 11 below.

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Table 11

Antiviral activity of supramolecular combinatorial derivatives of the oligopeptide KKRKRKRKR obtained in a reaction with different molar ratios of modifiers

No. Molar ratio of reagents * % cytoprotective antiviral activity * * w kl k2 Inf Black person Vesic 1 1532 18396*** 1 0 0 0 2 1532 9198 4 0 0 0 3 -//- 4599 9 0 0 0 4 -//- 2299 18 0 0 0 5 -//- 1149 36 0 0 0 6 -//- 575 72 0 0 0 7 -//- 287 143 0 0 0 8 -//- 143 287 0 0 0 9 -//- 72 575 0 0 0 10 -//- 36 1149 0 0 0 AND -//- 18 2299 0 0 0 12 -//- 9 4599 0 0 0 13 -//- 1 9198 0 0 0 14 -//- 0 18396*** 0 0 0 16 -//- 9193*** 9193*** 0 0 0 17 -//- 4599 4599 100 100 100 18 -//- 2299 2299 50 50 50 19 -//- 1149 1149 25 25 25 20 -//- 575 575 0 0 0 21 -AND- 287 287 0 0 0 22 -AND- 143 143 0 0 0 23 -AND- 72 72 0 0 0 24 -AND- 36 36 0 0 0 25 -AND- 18 18 0 0 0 26 -AND- 9 9 0 0 0 27 -AND- 1 1 0 0 0 28 -AND- 18396*** 0 0 0 0 29 -And- 9198 0 0 0 0 thirty -AND- 4599 0 0 0 0 31 -AND- 2299 0 0 0 0 32 -AND- 1149 0 0 0 0 33 -And- 575 0 0 0 0 34 -AND- 287 0 0 0 0 35 -AND- 143 0 0 0 0 36 -AND- 72 0 0 0 0 37 -AND- 36 0 0 0 0 38 -AND- 18 0 0 0 0 39 -AND- 9 0 0 0 0 40 -AND- 1 0 0 0 0 41 -AND- 0 18396*** 0 0 0 42 -AND- 0 9198 0 0 0 43 -AND- 0 4599 0 0 0 44 -AND- 0 2299 0 0 0 45 -AND- 0 1149 0 0 0 46 -And- 0 575 0 0 0 47 -AND- 0 287 0 0 0 48 -And- 0 143 0 0 0 49 -AND- 0 72 0 0 0 50 -AND- 0 36 0 0 0 51 -AND- 0 18 0 0 0 52 -AND- 0 9 0 0 0 53 -AND- 0 1 0 0 0 54 -AND- 0 0 0 0 0

* m - number of moles of oligopeptide KKRKRKRKR in the combinatorial synthesis reaction; K1 is the number of moles of succinic anhydride in the reaction; K2 is the number of moles of phthalic anhydride in the reaction;

- 28 043514 **% of the remaining monolayer of cells after infection with viruses and replacement of the culture with the test drug in culture after 48 hours of incubation in the presence of the test substance added in a pre-selected concentration (ED ₉₀ = 0.05 μg/ml.);

*** the maximum molar ratio at which all groups in the oligopeptide are replaced; exceeding this ratio leads to unreacted modifiers remaining in the reaction medium - succinic anhydride and phthalic anhydride

As can be seen from table. 11, a dose of 0.05 μg/ml of a derivative of supramolecular structure No. 17 (KR) completely protected the cell monolayer (ED100) from the destructive cytopathic effect of viruses. The calculated molar ratio of reactants for derivative No. 17 is: reactant m=1532, reactant k1=4599 and reactant k2=4599. This molar ratio of the three reagents caused the formation of the maximum number of different oligopeptide derivatives (see footnotes to Table 11)).

Synthesis of a combinatorial mixture of the oligopeptide KKRKSTRKR (called KR2)

The pre-oligopeptide KKRKSTRKR KR2 is prepared using standard techniques using a peptide synthesizer or genetic engineering.

mM oligopeptide KKRKSTRKR is dissolved in 50 ml of phosphate-buffered saline, 3 mM succinic anhydride and 3 mM maleic anhydride are added, and the solution is stirred until both anhydrides are completely dissolved. The solution is poured into vials, lyophilized and used for analysis and research. Calculation of the molar ratios of two modifiers to the oligopeptide is carried out according to the formulas: (O k - P ^x (2 - 1) (2) m - 4 ^x (3 ^x 2 - 1), where: n = number of substituent groups in the oligopeptide (n = 9 ); m = the number of moles of the original oligopeptide and the number of different molecules of its combinatorial derivatives after synthesis (1532 different derivatives are formed from one initial peptide, both in substitutions and in permutations); and k = the number of moles of each of the two modifiers in the combinatorial synthesis reaction to obtain the maximum number of different derivatives (k=4599) In this case, the molar ratio to obtain the maximum number of different derivatives (1532 different molecules) is 3:3:1 (succinic anhydride: phthalic anhydride: oligopeptide KKRKSTRKR).

In fig. 12. shows the result of HPLC analysis of the original peptide KKRKSTRKR.

The original peptide, when using a detector in the 280 nm region, gives one absorption band. Fig. 13 shows the result of HPLC analysis of the combinatorial derivative of the peptide KKRKSTRKR.

As can be seen from the chromatogram, the peptide peak is not just located in a different place, but in a more hydrophilic region and it is quite broad, divided into 4 additional bands. This suggests that intramolecular/supramolecular bonds of ionic and hydrogen nature exist between the 1532 different peptide derivatives that cannot be broken during HPLC separation under classical gradient HPLC conditions. Using thin layer chromatography and capillary gel electrophoresis, it was also not possible to separate the supramolecular derivative into individual fragments. To modify the peptide, other combinations of at least two different modifiers can be used: carboxylic and polycarboxylic acid anhydrides, carboxylic acid halides, halocarbons. As peptides, one individual oligopeptide can be used, as well as mixtures of oligopeptides obtained both by the standard method using peptide synthesizers, and by genetic engineering methods and using recombinant technology.

To determine the maximum tolerated concentration (MIC) in toxicological experiments and study the antiviral activity of the drug KR, the following types of transplanted cells of human and animal origin were used: RT, Tr, Ner-2, Hela and chicken embryos. Note that PTs are transplantable bovine embryonic kidney cells. Tr - bovine embryonic tracheal cells. Hep-2 - transplantable human laryngeal cancer cells. HeLa are transplantable uterine cancer cells.

Cells were grown in medium 199 supplemented with 10% bovine serum and antibiotics (penicillin and streptomycin). The test viruses used were influenza viruses (H3N2), vesicular stomatitis (Indiana strain), coronavirus (X 343/44) and herpes simplex virus type 1 (strain L-2).

The antiviral effect of the drug KR2 was studied on the influenza A virus (H3 N2).

Aqueous solutions of KR2 in various doses (ten-fold dilutions) were injected into 15 chicken embryos into the allantoic cavity in a volume of 0.2 ml 12 hours after injection of the virus in a working dose (100 TCD ₅₀ /0.2 ml). Each experiment was accompanied by control of the test virus at a working dose. Infected and uninfected (control) embryos were incubated at 36°C for 48 hours. The embryos were then dissected, from which allantoic fluid was extracted. Titration of the virus in allantoic fluid was carried out according to the generally accepted method with 1% erythrocytes of 0 (1) human blood group. The protection coefficient (K3) was determined. The virus titer in the experimental and control groups of chicken embryos is presented in Table. 12.

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Table 12

Effective concentration of KR2 in an in ovo influenza infection model

Group Drug concentration (mg/ml) Virus titer (lg TCA 50/sh) Minimum effective concentration (MEC mg/mL) experiment control Control (0.9% sodium chloride solution was administered) - 12 12 - Experiment 50±5 0 12 0.05 5 5±1 0 12 0.5 ± 0.05 2 12 О,О5±О,ОО5 4 12 0.005±0.0005 6 12

As can be seen from table. 12, the minimum effective concentration of KR2 against influenza virus, which completely inhibits virus synthesis, is 0.05 mg/ml. With increasing dilution of the drug, the effectiveness of KR2 decreases and is dose-dependent. This fact indicates the presence of a direct antiviral effect of the drug KR2 against the H3N2 influenza virus.

Study of the antiviral effect of the drug KR on cytopathic viruses (vesicular stomatitis virus, coronavirus, herpes simplex virus type 1). Antiviral activity against this group of viruses was determined in the culture of the above cells. The reaction was carried out as follows: 0.2 ml of the corresponding virus in a working dose (100 TCD ₅₀ /0.2 ml) was added in a volume of 0.2 ml to a 2-day washed cell culture. 0.8 ml of maintenance medium was added. When CPP appeared in the culture, the drug KR was administered in various doses. As a control, the same was done with test viruses without the drug. Cells were incubated at 37°C in a thermostat. The experiment was recorded for 3,5,7 days. A decrease in virus titer under the influence of the study drug by 2 lg or more compared to the control was assessed as a manifestation of antiviral activity. The results of the study of the antiviral activity of the drug KR2 are presented in table. 13.

Table 13

Study of the antiviral effect of the drug KR2 against viruses: vesicular stomatitis, coronavirus, herpes simplex virus type 1)

A drug Virus MEC, mg/mL Maximum drop in virus titer, 1g TCA so/mL KR2 VSV 0.05 3.9 CV 0.05 2.9 HSV1 0.05 4.9

As can be seen from table. 13, KR2 has antiviral activity and the ability to suppress the reproduction of all viruses we studied at a concentration of 0.05 mg/ml with MEC = 50 μg/ml. The CTI of the drug is 1000. In addition, KR2 was active against all viruses studied, while none of the comparators showed such activity. Thus, the effect of the drug is not related to specific characteristics of the virus or cell culture, but affects mechanisms common to all cells.

Aspects of the invention include supramolecular structures, also referred to as supramolecular soluble nanoparticles (SNPs), having a) a plurality of binding components, each of which has a plurality of binding regions, the plurality of binding regions including combinatorial carboxylated cobalamins; b) a plurality of cores that are suitable for providing at least some mechanical structure to said self-assembled supramolecular soluble systems, wherein each of said plurality of cores is an organic core that contains at least one core binding element adapted to bind with linking regions to form a first inclusion complex, wherein the core linker comprises a combinatorial carboxylated dipyridamole, and where the first inclusion complex is a combinatorial carboxylated cobalamin with a combinatorial carboxylated dipyridamole; c) a plurality of terminal components, each of which has a single terminal linking element that binds to the remaining binding regions of one of said plurality of binding components to form a second inclusion complex, wherein the single terminal connecting element comprises a combinatorial carboxylated dipyridamole, and where the second inclusion complex is combinatorial carboxylated dipyridamole; d) the plurality of cores and the plurality of binding components are self-assembled carboxylated basic amino acids lysine, histidine and arginine, and assemble upon contact to form self-assembled supramolecular soluble systems, and e) the plurality of terminal components interact to occupy the remaining binding regions of the plurality binding components, and the plurality of terminal components are present in sufficient quantity relative to the plurality of binding regions of the plurality of binding components to cease further binding, thereby forming a discrete particle.

The structural components and binding components of some embodiments of the present invention containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), can self-assemble when brought into contact with each other to form a supramolecular structure. Terminator components may act by occupying the binding regions of binding components to terminate further binding when termination components are present in sufficient quantity relative to the binding regions of binding components. In some embodiments, the structural component includes a plurality of binding elements that communicate with the binding regions of the binding components. In some embodiments, the terminator component has a single binding element that binds to one binding region on one binding component. In some embodiments, the supramolecular structure has at least two or more different end components.

In some embodiments of the present invention, binding regions (on the binding component) can bind to terminal components or structural components to form a molecular recognition pair. In some embodiments, the at least one structural component, the at least one linking component, or the at least one termination component has a functional element. In some embodiments, the supramolecular structure has two or more different functional elements.

In some embodiments, the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of combinatorial carboxylated cobalamins, containing, for example, a mixture of a succinylated derivative of cyanocobalamin, cyanocobalamin, methylcobalamin, hydroxycobalamin and/or cobamide.

In other embodiments, the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of combinatorial carboxylated cobalamins, including succinylated hydroxocobalamin, succinylated cobamide, succinylated methylcobalamin and/or succinylated cyanocobalamin.

In other embodiments, the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of supramolecular combinatorial carboxylated riboflavins, including supramolecular combinatorial succinylated derivatives of riboflavins and flavonuclears. / or flavin dinucleotide.

In yet other embodiments, the invention is a composition of substances containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of combinatorial carboxylated dipyridamoles containing supramolecular succinylated combinatorial dipyridamole derivatives; supramolecular maleylated combinatorial dipyridamole derivatives and/or carboxymethylated combinatorial dipyridamole derivatives.

In other embodiments of the invention, the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNP), containing supramolecular structures additionally containing carboxylated basic amino acids such as lysine, histidine and arginine, and may include bis-succinylated, maleylated and carboxymethylated amino acid derivatives of carboxylated basic amino acids such as lysine, histidine and arginine.

In other embodiments, the invention is a composition in which the terminating component may include at least one of the following: a polyethylene glycol, a polymer, a polypeptide, or an oligosaccharide, and the organic core contains at least one of dendrimers, branched polyethyleneimine, linear polyethyleneimine, polyline , polylactide, polylactide-co-glycoside, polyanhydrides, poly-ε-caprolactones, polymethyl methacrylate, poly(N-isopropylacrylamide) or polypeptides.

In other embodiments, the invention is a composition in which the binding component further includes combinatorial carboxylated derivatives of the main oligopeptide KKRKRKRKR, carboxylated derivatives thereof in the form of succinylated, maleylated and carboxymethylated derivatives in mixture with each other. Poly-L-lysine can also be used as a binding and termination component.

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Other aspects of the invention include methods for preparing supramolecular structures by preparing a suspension of structural components, linking components and terminal components. Other aspects of the invention include the selection of the ratio of the amount of structural component(s) to the binding component(s) to the terminal component(s) for a particular purpose, including the choice of size intended for supramolecular structures. Advantageously, the structural component(s), linking component(s), and terminal component(s) may be capable of self-assembly into preferred supramolecular structures of substantially predetermined size.

Note that the meaning of supramolecular structure refers to, for example, the meanings of supramolecular assembly and supramolecular structure. A supramolecular assembly, for example, can be defined as a complex of molecules held together by non-covalent bonds. For example, a supramolecular assembly may consist of simply two molecules (eg, a DNA double helix or an inclusion junction) or a larger complex(es) of molecules forming, for example, a sphere, rod, or sheet, as particles, nanoparticles, or discrete particles. Micelles, liposomes, and biological membranes are also examples of some types of supramolecular assemblies. The sizes of the supramolecular assemblies are expected to have a wide possible range, for example, for embodiments of the present invention, a range from about 5 nm to about 10 μm.

The present description discloses size ranges of supramolecular assemblies and structures, individually or combinations of supramolecular structures (assemblies), forming nanoparticles. The general field of supramolecular chemistry is the branch of chemistry concerned with chemical systems consisting of a discrete number of molecules. The strength of the forces responsible for the spatial organization of the system varies from weak intermolecular forces, electrostatic charge or hydrogen bonding to strong covalent bonds, provided that the strength of the electronic interaction remains small compared to the energy parameters of the component. While traditional chemistry concentrates on covalent bonding, supramolecular chemistry additionally deals with weaker and reversible non-covalent interactions between molecules, which consequently produce combinations of small molecules for supermolecules or supramolecular assemblies, in which the number of supramolecular structures is intended by the inventor and disclosed in the specification and may be a calculated or estimated number using combinatorial chemistry and combinatorial mathematics calculations as disclosed herein.

Supramolecular assemblies form and may have average lifetimes supported by hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, pi-beam interactions, and electrostatic effects between the small molecules that make up the combinatorial supramolecular assemblies.

Supramolecular chemistry also concerns dynamic (spontaneous, energy-dependent, entropic and thermodynamic processes) molecular self-assembly, molecular folding, molecular recognition, host-guest chemistry, mechanically coupled molecular architectures and dynamic covalent chemistry. The basis of these ideas about supramolecular science are based on the teachings of the prior art, as discussed in the Background of the Invention section. The concept of supramolecular soluble systems includes supramolecular soluble systems as a whole and includes the solubility of their components, as well as the solubility of the assembly and its components at various stages of the assembly process that form supramolecular assemblies (structures).

Note that the nanoparticle value includes the ultrafine particle and/or discrete particle values. The nanoparticle in some embodiments of the present invention has a largest size that ranges from 1 nm to 10,000 nm. The structural, binding and termination components self-assemble into supramolecular structures having an essentially predetermined size.

Preferably, in some cases, the predetermined size is preferably at least about 10 nm and less than about 800 nm (nanometers). In some embodiments of the present invention, the specified size is from about 5 nm to 2000 nm. Preferably, in some embodiments of the present invention, the specified size is at least about 20 nm and less than about 400 nm (nanometers). The average absolute/maximum size of a hydrated nanoparticle can be measured in liquid by direct laser scanning (DLS) using the Malvern Instruments Zetasizer for size calculation. Optical and/or X-ray technology or testing using nanometer and/or micron filter membrane filtration techniques known in the prior art may also be useful in determining the average size or relative size of nanoparticles.

If a term is presented in the singular, the inventors also consider aspects of the invention described in the plural of that term. Thus, for example, reference to a method includes one or more methods and/or steps of the type described herein and/or that will become apparent to those skilled in the art upon reading this disclosure. Unless otherwise specified, all technical and scientific terms used in this document include,

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Claims (13)

at least the same meaning as commonly understood by one skilled in the art to which the invention belongs. All publications referred to herein are incorporated herein by reference in their entirety. If there are discrepancies in terms and definitions used in the references incorporated by reference or in future publications, the terms used in this patent application will have the meanings given herein. Those skilled in the art will appreciate that changes may be made to the embodiments described above without departing from the broad concept of the invention. It is therefore understood that this invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims. CLAIM 1. Supramolecular nanoparticles with antiviral action, including a combination of nanostructures selected from the group consisting of combinatorial carboxylated cobalamins obtained as a result of the first combinatorial synthesis, combinatorial carboxylated dipyridamoles obtained as a result of the second combinatorial synthesis, carboxylated basic amino acids obtained as a result of the third combinatorial synthesis , and any combination thereof. 2. Supramolecular nanoparticles according to claim 1, which further contain dynamic self-assembled soluble nanostructures, and the nanostructures additionally contain a plurality of binding components, a plurality of organic cores and a plurality of terminating components. 3. Supramolecular nanoparticles according to claim 2, in which one of the binding components further includes combinatorial carboxylated cobalamins, which have a number of binding regions, while the organic cores additionally contain combinatorial carboxylated dipyridamoles, which have at least one binding element adapted to bind to combinatorial carboxylated cobalamins, wherein the organic cores further contain mechanical structures for dynamic self-assembled soluble nanostructures, and wherein the coupling of the combinatorial carboxylated cobalamins to the combinatorial carboxylated dipyridamoles may further include first inclusion complexes. 4. Supramolecular nanoparticles according to claim 3, wherein each termination component has at least one termination binding element that binds to the remaining binding region of one of the binding components and may further comprise second inclusion complexes. 5. Supramolecular nanoparticles according to claim 4, wherein the carboxylated basic amino acids are selected from the group consisting of lysine, histidine, arginine, derivatized lysine, derivatized histidine, derivatized arginine, acylated lysine, acylated histidine, acylated arginine, and any combinations thereof. 6. Supramolecular nanoparticles according to claim 5, wherein the plurality of terminating components occupy the remaining binding regions of the plurality of binding components, wherein the plurality of terminating components are present in an equivalent amount or greater than the number of binding regions of the plurality of binding components in order to terminate further binding of the binding components components, and supramolecular nanoparticles additionally contain discrete nanoparticles based on dynamic self-organizing soluble nanostructures. 7. Supramolecular nanoparticles according to claim 1, in which the combinatorial carboxylated cobalamins are a mixture of succinylated cyanocobalamins. 8. Supramolecular nanoparticles according to claim 1, in which the combinatorial carboxylated cobalamins are a mixture of succinylated methylcobalamins. 9. Supramolecular nanoparticles according to claim 1, in which the combinatorial carboxylated cobalamins are a mixture of succinylated hydroxycobalamins. 10. Supramolecular nanoparticles according to claim 1, in which the combinatorial carboxylated cobalamins are a mixture of succinylated cobamides. 11. Supramolecular nanoparticles according to claim 1, wherein the combinatorial carboxylated cobalamins are selected from the group consisting of a mixture of succinylated hydroxycobalamins, a mixture of succinylated cobamides, a mixture of succinylated hydroxycobalamins, a mixture of succinylated methylcobalamins, a mixture of succinylated cyanocobalamins, and any combinations thereof. 12. Supramolecular nanoparticles according to claim 2, in which at least one of the organic cores further contains at least one element selected from the photodynamic component, which is supramolecular combinatorial carboxylated riboflavins. 13. Supramolecular nanoparticles according to claim 12, in which the supramolecular combinatorial carboxylated riboflavins are supramolecular combinatorial succinylated riboflavins. -