WO2023191651A1 - Nanoparticules supramoléculaires ayant des propriétés antivirales à base de hème-porphyrine benzimidazolée - Google Patents

Nanoparticules supramoléculaires ayant des propriétés antivirales à base de hème-porphyrine benzimidazolée Download PDF

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WO2023191651A1
WO2023191651A1 PCT/RU2022/000096 RU2022000096W WO2023191651A1 WO 2023191651 A1 WO2023191651 A1 WO 2023191651A1 RU 2022000096 W RU2022000096 W RU 2022000096W WO 2023191651 A1 WO2023191651 A1 WO 2023191651A1
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supramolecular
combinatorial
derivatized
binding
nanoparticles
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Russian (ru)
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Борис Славинович ФАРБЕР
Артур Викторович МАРТЫНОВ
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Борис Славинович ФАРБЕР
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis

Definitions

  • 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.
  • Supramolecular chemistry is the branch of chemistry dealing with chemical systems consisting of multiple molecular structures interacting with each other.
  • the forces responsible for the spatial organization of such supramolecular systems include weak intermolecular forces, forces of electrostatic charge, or hydrogen bonds, rarely strong covalent bonds, provided that the electronic bond strength remains small relative to the energy parameters of the component. While traditional chemistry focuses on covalent bonding, supramolecular chemistry considers weaker and reversible non-covalent interactions between 7
  • 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 often provide a template for the development of supramolecular systems in new areas of supramolecular chemistry.
  • new supramolecular structures involve new intermolecular relationships in many ways that are complementary to the chemistry of the molecular structures themselves.
  • the core of a molecular structure 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 insufficient to understand aspects of chemistry where molecular aspects predominate, namely supramolecular chemistry.
  • supramolecular systems the chemistry of intermolecular molecules binds together into assemblies we can call super-molecules.
  • noncovalent intermolecular bonds are more varied and complex than covalent intramolecular bonds in structures known as molecules.
  • Supramolecular systems can be held together by much weaker forces than the atoms in the molecule.
  • the forces that hold multiple molecules together as supramolecular structures may include dispersion forces, hydrogen bonds, and hydrophobic bonds.
  • supramolecular chemistry is concerned with the structure and dynamics of a small molecule (called a guest) that is non-covalently bound to a larger molecule (called a host).
  • a guest small molecule
  • a host larger molecule
  • the concept guest/host has the greatest chemical information value when the distinction between guest and host is clear.
  • 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.
  • 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.
  • guest/cavity cavities
  • one monomer can be considered as a guest in the host in its own supramolecular assembly.
  • a molecule that is not a surfactant monomer is a pure guest in the surfactant assembly micelle.
  • An important term for the formation of an intermolecular bond between a guest and a host is "complexation" - a term that retains the idea of a chemically corresponding "looseness" based on non-covalent bonds between molecules.
  • 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.
  • 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 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.
  • 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.
  • a molecule that is contained as a guest inside the host fullerene has a certain connection with the inner cage of the fullerene.
  • 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.
  • 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 assemble with other drugs such as lipids and polymers. Such nanoparticles may have enhanced anticancer effects compared to 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.
  • 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; Nie et al., Annu. Rev. Biomed. Eng., vol. 9, p.
  • Nanoparticles carrying specific target ligands are used for in vivo cancer imaging, 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.
  • 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).
  • 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 have also been made in non-viral gene delivery vehicles (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 are presented in references (Niidome et al., Gene Ther., vol. 9, p.
  • Nanoparticles based on prior art gene delivery vehicles are presented in references (Liang et al., Proc Natl Acad Sci USA, vol. 102, pp. 11173-11178, 2005; Kumar et al., Chem. Commun., pp.
  • nanoparticles hold promise as non-viral 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, multi-step synthetic approaches, and (2) limited variety of delivery materials. These problems are a major obstacle to achieving optimal transfection performance. So Thus, the necessary methods are faster and more efficient production methods that can use different delivery materials.
  • Supramolecular nanoparticles may include: a combination of nanostructures selected from the group consisting of combinatorial derivatized heme porphyrins obtained from a first combinatorial synthesis; combinatorial derivatized dipyridamoles obtained from the second combinatorial synthesis; polypeptides from basic amino acids obtained from third combinatorial synthesis, as well as any combination thereof.
  • the supramolecular nanoparticle may have antiviral properties, and may further include dynamic self-assembled soluble nanostructures and these nanostructures may further include a variety of binding components; many organic nuclei; and many terminal components.
  • the supramolecular nanoparticles may have one of the binding components, which may further comprise combinatorial derivatized hemporphyrins having a number of binding regions, and the organic cores may further comprise combinatorial derivatized dipyridamoles having at least one binding element adapted to bind to the combinatorial derivatized heme porphyrins, and the organic cores may further include mechanical structures of dynamic self-assembled soluble nanostructures, also combinatorial derivatized heme porphyrins, which are associated with combinatorial derivatized 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 polypeptides from basic amino acids, and may additionally contain derivatized oligopeptides from basic amino acids, such as lysine, histidine, arginine, derivatized lysine, derivatized histidine, derivatized arginine, acylated lysine, acylated histidine, acylated arginine, and any combinations thereof.
  • Supramolecular nanoparticles may have a plurality of termination components, which may occupy the remaining binding sites of the plurality of binding components, and where the plurality of termination components may be equivalent to the number of binding regions of the plurality of binding components, after which further binding of the binding components ceases, supramolecular nanoparticles may further comprise discrete nanoparticles based on dynamic self-organizing soluble nanostructures.
  • Supramolecular nanoparticles can contain combinatorial benzimidazolated heme porphyrins, which can be represented by a mixture of benzimidazolated heme porphyrins with different substituents on the benzene ring of the imidazole moiety.
  • Supramolecular nanoparticles may contain combinatorial derivatized heme porphyrins, which may be a mixture of benzimidazolated methyl heme porphyrins.
  • Supramolecular nanoparticles can contain combinatorial benzimidazolated heme porphyrins, which can be a mixture of benzimidazolated heme porphyrins.
  • Supramolecular nanoparticles can contain combinatorial benzimidazolated heme porphyrins, which can be a mixture of benzimidazolated heme porphyrins and hemin porphyrins.
  • Supramolecular nanoparticles can contain combinatorial benzimidazolated heme porphyrins, which can be selected from the group consisting of a mixture of benzimidazolated heme porphyrins, a mixture of benzimidazolated heme porphyrins, a mixture of benzimidazolated heme porphyrins with different substituents on the benzene ring of the imidazole moiety, and any of them combinations.
  • combinatorial benzimidazolated heme porphyrins which can be selected from the group consisting of a mixture of benzimidazolated heme porphyrins, a mixture of benzimidazolated heme porphyrins, a mixture of benzimidazolated heme porphyrins with different substituents on the benzene ring of the imidazole moiety, and any of them combinations.
  • 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 derivatized riboflavin.
  • Supramolecular nanoparticles may contain supramolecular combinatorial derivatized riboflavin in the form of supramolecular combinatorial succinylated riboflavin.
  • Supramolecular nanoparticles may contain supramolecular combinatorial derivatized riboflavin in the form of supramolecular combinatorial succinylated flavin mononucleotide.
  • Supramolecular nanoparticles may contain supramolecular combinatorial derivatized riboflavin presented as supramolecular combinatorial succinylated flavin dinucleotide.
  • Supramolecular nanoparticles may contain a combinatorial derivatized dipyridamole in the form of a supramolecular succinylated combinatorial dipyridamole.
  • Supramolecular nanoparticles may contain combinatorial derivatized dipyridamoles, which are supramolecular maleylated combinatorial dipyridamoles. [0032] Supramolecular nanoparticles may contain combinatorial derivatized dipyridamoles, which are supramolecular carboxymethylated combinatorial dipyridamoles.
  • Supramolecular nanoparticles may contain derivatized basic amino acids such as succinylated lysine, succinylated histidine, succinylated arginine, or any combination thereof.
  • Supramolecular nanoparticles may contain derivatized basic amino acids, such as maleylated lysine, maleylated histidine, maleylated arginine, in any combination thereof.
  • Supramolecular nanoparticles may contain derivatized basic amino acids, including carboxymethylated lysine, carboxymethylated histidine, carboxymethylated arginine, in any combinations.
  • Supramolecular nanoparticles may contain derivatized 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 a plurality of 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-8-caprolactone, polymethyl methacrylate A, poly(N-isopropyl acrylamide), polypeptide, and any combination thereof.
  • the supramolecular nanoparticles may contain at least one of a variety of binding components, which may further comprise a combinatorial carboxylated derivative of the base oligopeptide KKRKRKRKR.
  • Supramolecular nanoparticles may contain combinatorial derivatized derivatives of the base oligopeptide KKRKRKRKR, which may be succinylated derivatives, where from 1 to 9 free amino residues of the base oligopeptide KKRKRKRKR are succinylated.
  • Supramolecular nanoparticles may contain combinatorial derivatized derivatives of the base oligopeptide KKRKRKRKR, which may be maleylated derivatives, where from 1 to 9 free amino residues of the base oligopeptide KKRKRKRKR are maleylated.
  • Supramolecular nanoparticles may contain combinatorial derivatized derivatives of the base oligopeptide KKRKRKRKR, which may be carboxymethylated derivatives, where from 1 to 9 free amino residues of the base oligopeptide KKRKRKRKR are carboxymethylated.
  • Supramolecular nanoparticles may contain combinatorial derivatized derivatives of the KKRKRKRKR core oligopeptides, which may be combinatorial mixtures of derivatives obtained by succinylation, maleylation, and carboxymethylation of 1 to 9 of the free amino residues of the KKRKRKRKR core oligopeptide.
  • Supramolecular nanoparticles may contain a binding component, which is poly- ⁇ -lysine, and a liposomal shell around a composite nanostructure, which is a bilayer membrane including at least one of the amphiphilic liquid crystal structures: phospholipids, polysorbates, PEGylated phospholipids.
  • FIG. Figure 1 shows the structures of a soluble self-organizing nanoparticle, dynamic combinatorial heme-porphyrin as a core, dynamic combinatorial dipyridamole as a structural component, dynamic combinatorial derivatives of basic oligopeptides and amino acids as a binding component.
  • FIG. Figure 2 shows a self-organizing dynamic combinatorial derivative of dipyridamole and the principles of its synthesis.
  • FIG. Figure 3 shows the basis of the principle of molecular recognition between different substances based on nucleotide-like structures.
  • FIG. Figure 5 shows a scheme for the synthesis of dynamic self-organizing combinatorial derivatives of benzimidazolyl heme porphyrin (IX) from heme porphyrin (VI) and two different or identical orthophenylenediamine derivatives (VII) and (VIII).
  • FIG. Figure 6 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).
  • Figure 7 is a schematic of a thin layer chromatogram of a combinatorial dipyridamole derivative (IV), initial dipyridamole (I), fully acylated dipyridamole (lb), and fully succinylated dipyridamole (1c).
  • FIG. Figure 8 shows the result of HPLC analysis of the starting peptide KKRKRKRKR.
  • a wavelength of 280 nm with one absorption band is used.
  • FIG. 9 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.
  • FIG. Figure 10 shows the HPLC chromatogram of the starting peptide KKRKSTRKR.
  • the original peptide when using a detector in the region of 280 nm, gives one absorption band.
  • FIG. 11 is the result of HPLC analysis of the combinatorial derivative of the peptide KKRKSTRKR, its chromatogram contains a characteristic triple peak.
  • embodiments of the present invention relate to supramolecular structures, also referred to as 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.
  • a supramolecular nanoparticle 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 derivatized heme porphyrins; 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 combinatorially derived dipyridamole, and wherein the first inclusion complex is a combinatorial derivatized heme-porphyrin with a combinatorial derivatized dipyridamole; and c) a plurality of terminating components, with each terminating component comprising one binding terminating element to form a linkage with other binding sites of one
  • 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.
  • the binding region may comprise a combinatorial derivatized heme porphyrin or derivatized heme porphyrin or other heme porphyrin derivatives.
  • the structural element is at least represented by an inorganic or organic core.
  • Figure 1 shows a variant of the invention in the form of a structure of a soluble self-assembled nanoparticle containing as a core a dynamic combinatorial derivative of heme porphyrin, a dynamic combinatorial derivative of dipyridamole as a structural component, a dynamic combinatorial derivative of basic oligopeptides and amino acids as binding components.
  • the core based on the self-assembled structure of a dynamic derivative of heme porphyrin, has an inorganic core selected from the group consisting of an inorganic nanoparticle, a metal nanoparticle, a gold nanoparticle, a silver nanoparticle, a silicon nanoparticle, a metal nanoparticle, a metal oxide nanoparticle, and any combination of nanoparticles presented above.
  • 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-like, plate-type, 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.
  • the binding component has a binding region that can bind to the inorganic core directly.
  • 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.
  • a plurality of inorganic core particles may be present in a supramolecular structure.
  • a plurality of inorganic core particles may associate with 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.
  • the structural component is an organic core.
  • Organic cores may include derivatives of combinatorial self-assembling heme-porphyrins, dendrimers, polymers, proteins, oligosaccharides, micelles, liposomes, vesicles, and combinations thereof.
  • 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-E-caprolactone, A polymethyl methacrylate, poly(N -isopropyl acrylamide), polypeptide, and combinations thereof.
  • the organic core is a polyamidoamine dendrimer or poly- ⁇ -lysine polymer.
  • the binding regions of the binding components interact with other elements that may be present as part of the organic structure of the core.
  • 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.
  • 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.
  • a dendrimer can be synthesized by incorporating essential elements as parts of the structure.
  • a reactive functional moiety may be present at each point of the terminal element added to the linking element within the core.
  • specific dendrimers suitable for the invention include such as polyamidoamine (PAM dendrimer).
  • polymers are known in the art.
  • the advantage of using polymer cores lies in its rapid synthesis and its ability to be easily functionalized as a binder.
  • a bonding element may be included in the polymer structure during synthesis.
  • the reactive functional groups on the polymer can be derivatized with chemical moieties that provide a bonding element function.
  • polypeptides containing lysine residues with a reactive amino group (-NH2) in their structure can be functionalized with linking elements.
  • polypeptide poly-L-lysine is one example.
  • each structural component may have linking regions (groups) to form a bond with the linking component.
  • 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 derivatized derivative of the same peptide.
  • a linker element(s) such as the oligopeptide KKRKRKRKR and linked to a combinatorial self-assembled derivatized derivative of the same peptide.
  • K is one letter of the name of the amino acid lysine.
  • the structural element may also be adamantane-derivatized inorganic nanoparticles, such as adamantane-derivatized metal nanoparticles, or adamantane-derivatized metal oxide nanoparticles, more specifically, for example, adamantane-derivatized gold nanoparticles.
  • adamantane-derivatized inorganic nanoparticles such as adamantane-derivatized metal nanoparticles, or adamantane-derivatized metal oxide nanoparticles, more specifically, for example, adamantane-derivatized gold nanoparticles.
  • 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.
  • 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.
  • the concentration of terminating components 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.
  • 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 can be a polymer, polypeptide, oligosaccharide, or small molecule and functions as long as the termination component interacts with the binding regions of the coupling component.
  • the termination component is a polymer that is produced using a coupling element.
  • the terminating component is a polyethylene glycol derivatized with a coupling element, for example containing maleic acid residues.
  • the 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.
  • the binding components have a plurality of binding sites that interact and bind to the structural component and the terminating component and may include a terminating component selected from the group consisting of unmodified heme porphyrin, 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.
  • 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 branched polyethylenimine derivatized with multiple binding sites can also be used as a binding component.
  • 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 p-cyclodextrin.
  • the binding regions and/or binding elements are molecular recognition elements.
  • the binding region forms a molecular recognition pair with a structural component or termination component.
  • 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.
  • the terms “host” and “guest” describe only two compounds that exhibit molecular complementarity, i.e. communicate with each other using molecular recognition.
  • the “host” and “guest” communicate with each other, while the two “host” connections do not.
  • Molecular recognition is a specific interaction, meaning that each molecular recognition element will bind to complementary molecules that have specific structural features.
  • the components of molecular recognition pairs bind to each other more tightly than is the case for nonspecific binding because the interactions that occur between the two molecular recognition elements are more numerous.
  • molecular recognition pairs include small molecule host/guest complexes (including but not limited to inclusion complexes), complementary oligonucleotide sequence pairs (e.g., DNA-DNA, DNA-RNA, or RNA-RNA, which bind to each other by hybridization), antibody-antigen, substrate protein, inhibitor protein, and protein-protein interactions (such as alpha-helical peptide chains and [3-sheet peptide chains).
  • supramolecular structures self-assemble through molecular recognition.
  • 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.
  • 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.
  • 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-
  • At least one of the structural components, a binding component or a termination component includes a functional element.
  • the functional element may be a chemical moiety that imparts additional function or activity to the 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 in as a functional element.
  • the functional element may also be a compound containing a radioactive or magnetically active isotope.
  • 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.
  • the functional element may be a targeting element that is delivered through a supramolecular structure to specific cells.
  • delivery systems with functional elements inside can be represented by oligonucleotides, antibodies, and small molecules that bind to cell surface proteins.
  • 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.
  • 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 ligands acting through cell permeability membranes may also be used in some embodiments of the present invention.
  • the supramolecular structure may have two or more functional elements.
  • 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 a protein-cell surface interaction.
  • Cell means biological cell.
  • 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.
  • 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.
  • the supramolecular structure includes two or more termination components, each of which may further include a functional element.
  • multiple functional elements can be introduced into the particle using multiple termination components.
  • the 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 lacking a functional element can be exchanged with termination components having 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.
  • a supramolecular structure can be produced using a mixture of termination components, each of which will be included in the supramolecular structure.
  • 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.
  • the cargo may be associated with one or more structural components, a binding component, or a termination component.
  • the cargo is a chemical group that can nonspecifically bind to the binding regions of the binder component and which will prevent the cargo from interfering with the self-assembly of the nanoparticles.
  • the cargo is a small molecule selected from the group consisting of drugs such as doxorubicin, taxol, rapamycin, cisplatin, other anticancer drugs useful for cancer chemotherapy, including. protein, peptide, oligonucleotide, siRNA, plasmid, gene delivery molecules (systems), as well as any combinations thereof.
  • drugs such as doxorubicin, taxol, rapamycin, cisplatin
  • other anticancer drugs useful for cancer chemotherapy including. protein, peptide, oligonucleotide, siRNA, plasmid, gene delivery molecules (systems), as well as any combinations thereof.
  • 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.
  • 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.
  • a plasmid may also be included as cargo in the supramolecular structure.
  • multiple types of cargo and various combinations of cargo are encapsulated in supramolecular structures.
  • the present invention includes methods for preparing the supramolecular structures described above by preparing a suspension of structural components and binder 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.
  • embodiments include a general method for producing a supramolecular structure comprising the steps of:
  • embodiments include a general method for producing a supramolecular structure, the step of:
  • embodiments of the invention include a general method for producing a supramolecular structure in which:
  • embodiments of the invention include a general method for producing a supramolecular structure, which further includes:
  • 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.
  • the predetermined size ranges from about 5 nm to 2000 nm.
  • 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 varying sizes can also be easily obtained. The possibility of combinatorial synthesis also makes it possible to obtain different sizes of supramolecular structures, and optimize 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 (self-organize) 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.
  • the components can be replaced with other components bearing corresponding binding elements or binding regions by treating the supramolecular structure with additional components.
  • some termination components can be exchanged by processing of the supramolecular structure for other termination components (eg, enriched with functional elements).
  • 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 sonicated to speed up or assist in the occurrence of component metabolic reactions.
  • the size of the supramolecular structures can be easily controlled by changing the ratio between the components used to prepare the supramolecular structures. A wide variety of supramolecular structures of varying sizes can be easily obtained. This also enables combinatorial synthesis, as arrays of supramolecular structures can be prepared based on their specific function and optimization of their activity.
  • the dimensions of the supramolecular structures can be adjusted after the supramolecular structures are 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.
  • 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 this is true when the functional element is present on the termination component or structural component.
  • the preassembled supramolecular structures can be processed with termination components having the 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.
  • reactive functional groups of organic compounds such as hydroxyls, thiols, amines, carboxylic acids, halides, alkenes, alkynes, azides and others can react or be activated to react with a variety of other functional groups to form covalent bonds.
  • amine-containing compounds having a free -NH2 group can react with binders bearing amino-reactive groups such as isocyanates, isothiocyanates and activated esters such as N-hydroxysuccinimide (NHS) esters.
  • 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.
  • 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 may be used.
  • heterodifunctional crosslinkers such as succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) and maleimidobutyryloxysuccinimide ester (GMBS) can be used to react with amines (via esters of the succinimide linkage) and then with the formation of a covalent bond with a free thiol (via maleimide).
  • crosslinkers 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.
  • a reversible (cleavable) cross-linking agent a variety of which is known to one of ordinary skill in the art.
  • 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.
  • 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.
  • 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 plasmids 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.
  • compositions [0123] 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
  • the pharmaceutical composition of the invention contains an effective amount (eg, a pharmaceutically effective amount) of the composition of the invention.
  • composition of the invention may be formulated as a pharmaceutical composition that contains the composition of the invention and a pharmaceutically acceptable carrier.
  • 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.
  • pharmaceutically acceptable carriers and other components of pharmaceutical compositions see Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990.
  • suitable pharmaceutical carriers 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.
  • compositions 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. Specialist in the field will understand that the specific formulation will depend in part on the specific 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.
  • 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.
  • unit dosage form refers to physically discrete units suitable as unit dosages for animals (eg, humans), each unit containing a predetermined amount of the 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.
  • 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.
  • kits applicable to any of the methods disclosed herein, either in vitro or in vivo.
  • a kit may include one or more compositions of the invention.
  • 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.
  • CDC supramolecular combinatorial heme porphyrin derivatives
  • the CDC composition may be administered orally or may be administered intravascularly, subcutaneously, intraperitoneally by injection, aerosol, bladder, topical, and so on.
  • 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.
  • 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 can be distributed systemically after administration or can be localized using an implant or other composition that retains the active dose at the site of implantation.
  • the compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (eg, perforin, anti-inflammatory drugs, and so on).
  • 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.
  • 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.
  • 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, d
  • 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.
  • the compounds can be included in suppositories by mixing with various bases such as emulsifying bases or water-soluble bases.
  • 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.
  • 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 inventions.
  • 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 heme porphyrins 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 is increased compared to other areas of the body.
  • unit dosage form refers to physically discrete units suitable for use as unit 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. Usually available pharmaceutically acceptable excipients such as excipients, adjuvants, carriers or diluents.
  • 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.
  • 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.
  • 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.
  • a known method is described by Kato et al. (1991) J Biol. Chem 266: 3361.
  • the lipids and composition for inclusion in liposomes containing combinatorial supramolecular heme porphyrins are mixed in a suitable aqueous environment, suitably in a saline environment, where the total solids content will be in the range of about PO wt%.
  • a suitable aqueous environment suitably in a saline environment, where the total solids content will be in the range of about PO wt%.
  • 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 [0140] Compositions with other active agents
  • KS antiviral supramolecular structure
  • the antiviral KS of the invention may be included in compositions with other pharmaceutically active agents, in particular 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.
  • antiviral drugs 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.
  • the objects of supramolecular chemistry are assemblies spontaneously constructed from complementary geometrically and chemically corresponding molecular fragments.
  • 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 heme-porphyrin molecule.
  • a drug was manufactured that is effective in vivo against influenza, herpes, in Ovo models (in eggs), and bovine coronavirus.
  • a combinatorial mixture of benzimidazolyl porphyrin 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 the rapid search for target genes or target proteins.
  • a combinatorial library set may consist of millions of different chemicals or, for example, a set of recombinant DNA or RNA molecules or their derivatives (acylated and/or alkylated derivatives of DNA and/or RNA) obtained by inserting different antibodies into the light and heavy chains of cDNA .
  • Combinatorial synthesis involves the synthesis by combinatorial chemistry of multiple simultaneous reactions between three or more reactants to form combinatorial synthesis products that include hundreds of derivatives of the combined reactants. 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 is a term for the present invention that refers to the amount of a drug.
  • a therapeutically effective amount of combinatorial heme porphyrin derivatives is an amount that provides therapeutically effective antiviral activity.
  • ECP is expected to differ between viruses and different animal models.
  • EXAMPLE 1 is an example of the practice of the present invention using a combinatorially modified heme porphyrin serving as the core of a supramolecular structure.
  • This core is synthesized using combinatorial derivatives of benzimidazolyl-heme-porphyrin with antiviral properties and is called KS.
  • the resulting pharmaceutical compositions and dosage forms are then characterized in that the benzimidazolated heme porphyrin derivatives are an unresolved combinatorial mixture of monosubstituted to fully substituted derivatives for carboxyl groups 1 and 2 in the heme structure of the following structure depicted in FIG. 5, wherein at least one of the Ri-Rs substituents is -H in any position.
  • the pharmaceutical composition may additionally contain vanillin in the dosage form and cholecalciferol.
  • the present pharmaceutical compositions 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 benzimidazolated heme porphyrin, which can be used to create antiviral supramolecular structure called KS.
  • the synthesis scheme for derivative (IV) or KS is shown in Figure 5.
  • the synthesis is carried out as follows: 1 mmol (millimol, mmol) of hemporphyrin (VI) is dissolved in 70 ml of glacial acetic acid, 1 mmol of o-phenidenediamine derivatives (VII) and (VIII) are added with stirring until complete dissolution both components, and then the solution is refluxed with a Dean-Stark trap for 2 hours. Then the solution is cooled to +10 0 C, the precipitate is filtered off and recrystallized from glacial acetic acid and used for analysis and research. In synthesis example 2, about 560 combinatorial derivatives of benzimidazolated heme porphyrins can be obtained.
  • 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 (Negr. - 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).
  • H1N1 influenza virus Inf
  • the reference strain of vesicular stomatitis virus Vesic. -VVS
  • herpes simplex virus type 1 Negr. - strain L-2
  • IBV coronavirus - avian infectious bronchitis virus
  • the degree of “peeling” of cells 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 the in vitro studies are shown in Table 1 below.
  • Table 1 Antiviral activity of supramolecular combinatorial derivatives of heme-porphyrin KS and its dosage forms * groups (KS + van) and (KS + van + CO) are statistically significantly more effective than pure KS; ** for an average effective dose of 10 pg/ml.
  • KS-based dosage form variants have an antiviral effect that can protect 50% or more of cells infected culture.
  • Dosage forms with vanillin and other heme-porphyrin derivatives are statistically significantly (P ⁇ 0.05) higher than the effectiveness of the pure substance CS.
  • MTC maximum tolerable concentration
  • 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.
  • MTC maximum tolerated concentration
  • a dose of the drug in a volume of 0.2 ml was used on 10-11 day old chicken embryos (5 embryos per MP 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 shell was pierced, and then 0.2 ml of a dose of the drug 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.
  • 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.
  • the eggs Before opening the eggs, they were placed in a refrigerator at a temperature of 24 0 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 normal developing embryos after 24 and 48 hours of incubation at 37°C were counted to calculate LD50 and MTD according to the Kerber method.
  • KS and its dosage forms are not toxic to cell cultures at a dose of K.S greater than 50 mg/ml.
  • the drug solution was lyophilized and then diluted to a concentration of 5%.
  • the 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.
  • KSO KSO
  • KSO which is a liquid antiviral formula (KS + van + CO).
  • Aqueous solutions of KSO in various doses were administered to 15 chicken embryos into the allantoic cavity in a volume of 0.2 ml 12 hours after injection of the virus at a working dose (100 TCD50 / 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 and 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 minimum effective concentration (IEC) for influenza virus that inhibits virus synthesis in 50% of cells is 0.005 mg/ml with increasing dilution.
  • the effectiveness of CSO decreases and is dose-dependent. This fact indicates that KSO has a direct antiviral effect against the H1N1 influenza virus.
  • Term 1g for of the present invention means the decimal logarithm or the decimal logarithm as opposed to the natural logarithm.
  • Antiviral activity against cytopathic viruses vesicular stomatitis virus, coronavirus and measles virus was determined in culture of the above cells. The reaction was carried out as follows: 0.2 ml of the corresponding virus in a working dose (100 TCAso / 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 ⁇ g or more compared to the control was assessed as a manifestation of antiviral activity.
  • the CTI of the drug is 1000.
  • KSO was active against all viruses studied.
  • the drug KSO is not is associated with specific characteristics of the virus or cell culture, but affects mechanisms common to all cells.
  • Tests were performed in 96-well plastic plates with porcine transmissible gastroenteritis virus (TGS) strain D-52 with an initial titer of 10 4 TCD50/ml (tissue cytopathic doses) in a transplanted culture of piglet test cells (PTR) and a strain of large diarrhea virus Oregon cattle with an initial titer of 10 7 TCD50 / ml in a transplanted saiga kidney cell culture (PS).
  • TCS porcine transmissible gastroenteritis virus
  • PTR piglet test cells
  • PS transplanted saiga kidney cell culture
  • 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.
  • herpetic infections are important to study because herpetic diseases are widespread and extremely variable 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 was caused by applying infectious material (herpes simplex virus type 1 strain L-2) to the scarified cornea after the eye was anesthetized with instillation of dicaine and the cornea several times. The eyelid was then closed and rubbed in a circular motion. The dose of virus was 0.05 ml, dose 6.75 1g TCD50/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 a placebo 0.9% sodium chloride solution.
  • infectious material herepes simplex virus type 1 strain L-2
  • the experimental group of rabbits was injected with CSO 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.
  • KSO is an effective antiviral drug with a wide spectrum of action and low toxicity.
  • 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 infection process by vaccine and 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: reduction in excess levels (titers) of antibodies, reduction in morbidity (safety), and increase in weight gain.
  • 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, Gumboro disease (HD) and coronavirus infectious bronchitis (IB). The controls were broiler chickens that were not fed KSO but were vaccinated.
  • IBD Gumboro disease
  • IB coronavirus infectious bronchitis
  • 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.
  • antibody titers increased from 3000 units to 3600 units, indicating efficient replication of the live vaccine virus in poultry.
  • CSR makes it possible to increase the growth rate of broiler chickens by 5% and reduce 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 corresponding reduction in weight gain and increased mortality.
  • 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.
  • Average daily weight gains in the experimental groups ranged from 50 to 55 grams, while the control groups averaged 46 grams.
  • the optimal regimen for using KSO for broilers in regions with a complex epizootic situation with NBD 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.
  • FIG. 8 shows a schematic diagram of Example 3 for the combinatorial synthesis of dipyridamole derivatives (CD).
  • carboxylic acid anhydride modifiers 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 to obtain combinatorial derivatives of dipyridamole.
  • the amino groups as part of the residual morpholine and pyrimidine core can be protonated and protected from modification under given reaction conditions.
  • modifiers such as succinic anhydride or acetic anhydride may be used and added simultaneously and sequentially, or, succinic anhydride may be added first is introduced and heated into the mixture using a reflux condenser, and then acetic anhydride can be introduced and the mixture is reheated.
  • succinic anhydride 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.
  • 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
  • alkylating agents including, for example, propiolactone, ethylene oxide, methyl chloride, ethyl chloride
  • 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.
  • 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.
  • FIG 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 fully acylated dipyridamole (lb) and succinylated dipyridamole (1c) TLC bands appear between the TLC bands of native dipyridamole and combinatorial dipyridamole.
  • the combinatorial dipyridamole band was not resolved into its complex supramolecular structures by 2D TLC or by HPLC (data not shown).
  • Various supramolecular combinatorial dipyridamole derivatives have been prepared in synthetic reactions using different molar ratios of modifiers.
  • a solid-phase sandwich ELISA with cyclic AMP (enzyme-linked immunosorbent assay) was used. The reaction was stopped by adding a double volume of 1% TS A.
  • * t is the number of moles of dipyridamole in the combinatorial synthesis reaction; kl is the number of moles of succinic anhydride in the reaction; k2 is the number of moles of acetic anhydride in the reaction; **EDso ⁇ g/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 the fact that unreacted modifiers - succinic anhydride and acetic anhydride - remain in the reaction in the medium.
  • Table 9 above presents experimental data that, taken as a whole, reveals unexpected enzyme inhibition efficiencies for certain embodiments of the present invention. From Table 9, item No. 3, it can be seen that the ED50 for inhibition of cAMP phosphodiesterase by supramolecular combinatorial dipyridamole derivatives is the lowest (0.01 ⁇ g / ml ED50) when the molar ratio of the three reagent modifiers (m, kl and k2) is 44:61 :60. Note that at a pt of 44, a fairly small reduction in the kl to k2 molar ratios from 70.70 to 60.61 produces an astonishing 10,000-fold improvement in cAMP phosphodiesterase inhibition.
  • 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.
  • w number of moles of the original oligopeptide.
  • the molar ratio to obtain the maximum number of different derivatives (1532 different molecules) is 3:3:1 (succinic anhydride: phthalic anhydride: oligopeptide KKRKRKRKR).
  • 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.
  • Rice. 11 shows the result of HPLC analysis of the combinatorial derivative of the peptide KKRKRKRKR.
  • 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.
  • modifiers are carboxylic and polycarboxylic acid anhydrides, carboxylic acid halides, and/or halocarbons.
  • 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.
  • 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 1 type (Negro - 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 the in vitro studies are shown in Table 11 below.
  • K1 is the number of moles of succinic anhydride in the reaction
  • K2 is the number of moles of phthalic anhydride in the reaction
  • KR2 KKRKSTRKR oligopeptide
  • the molar ratio to obtain the maximum number of different derivatives (1532 different molecules) is 3:3:1 (succinic anhydride: phthalic anhydride: oligopeptide KKRKSTRKR).
  • FIG. 8 The result of HPLC analysis of the original peptide KKRKSTRKR is shown.
  • the original peptide when using a detector in the 280 nm region, gives one band absorption.
  • Rice. 9 shows the result of HPLC analysis of the combinatorial derivative of the peptide KKRKSTRKR.
  • 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.
  • oligopeptides 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.
  • PT maximum tolerated concentration
  • 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 (Influenza A virus).
  • Aqueous solutions of KR2 in various doses (ten-fold dilutions) were administered to 15 chicken embryos into the allantoic cavity in a volume of 0.2 ml 12 hours after injection of the virus at a working dose (100 TCD50 / 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 and allantoic fluid was extracted.
  • the minimum effective concentration of KR2 against influenza virus that 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.
  • the CTI of the drug is 1000.
  • KR2 was active against all viruses studied, while none of the comparators showed such activity.
  • 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 derivatized heme porphyrins; b) a plurality of cores that are suitable to provide 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 binding regions to form a first inclusion complex, where the core binding element includes a combinatorial derivatized dipyridamole, and where the first inclusion complex is a combinatorial derivatized heme-porphyrin with a combinatorial derivatized 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 pluralit
  • SNPs
  • the structural components and binding components of some embodiments of the present invention containing supramolecular structures 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.
  • the structural component comprises a plurality of binding elements that communicate with the binding regions of the binding components.
  • the terminator component has a single binding element that binds to one binding region on one binding component.
  • the supramolecular structure has at least two or more different terminal components.
  • binding regions can bind to terminal components or structural components to form a molecular recognition pair.
  • the at least one structural component, the at least one linking component, or the at least one termination component has a functional element.
  • the supramolecular structure has two or more different functional elements.
  • the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of combinatorial derivatized heme porphyrins, containing, for example, a mixture of a benzimidazolated heme porphyrin derivative and a heme porphyrin.
  • SNPs supramolecular soluble nanoparticles
  • the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of combinatorial derivatized heme porphyrins.
  • SNPs supramolecular soluble nanoparticles
  • the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of supramolecular combinatorial derivatized riboflavins, including supramolecular combinatorial succinylated derivatives of riboflavins and flavonuclears. / or flavin dinucleotide.
  • SNPs supramolecular soluble nanoparticles
  • the invention is a composition of substances containing supramolecular structures, also called supramolecular soluble nanoparticles (SNPs), containing a mixture of combinatorial derivatized dipyridamoles containing supramolecular succinylated combinatorial dipyridamole derivatives; supramolecular maleylated combinatorial dipyridamole derivatives and/or carboxymethylated combinatorial dipyridamole derivatives.
  • SNPs supramolecular soluble nanoparticles
  • the invention is a composition of a substance containing supramolecular structures, also called supramolecular soluble nanoparticles (SNP), containing supramolecular structures further containing derivatized basic amino acids such as lysine, histidine and arginine, and may include bis- succinylated, maleylated and carboxymethylated amino acid derivatives of derivatized basic amino acids such as lysine, histidine and arginine.
  • SNP supramolecular soluble nanoparticles
  • 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 polyethylenimine, polyline, polylactide, polylactide-co-glycoside, polyanhydrides, poly-8-caprolactones, polymethyl methacrylate, poly(N-isopropyl acrylic amide) or polypeptides.
  • the terminating component may include at least one of the following: a polyethylene glycol, a polymer, a polypeptide, or an oligosaccharide
  • the organic core contains at least one of dendrimers, branched polyethyleneimine, linear polyethylenimine, polyline, polylactide, polylactide-co-glycoside, polyanhydrides, poly-8-caprolactones, polymethyl methacrylate, poly
  • the invention is a composition in which the binding component further includes combinatorial derivatized derivatives of the main oligopeptide KKRKRKRKR, derivatized 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.
  • 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 selecting the ratio of the amount of structural component(s) to linking component(s) to end component(s) for a particular purpose, including selecting a size appropriate for supramolecular structures.
  • 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.
  • supramolecular structure refers to, for example, the meanings of supramolecular assembly and supramolecular structure.
  • a supramolecular assembly can be defined as a complex of molecules held together by non-covalent bonds.
  • a supramolecular assembly may consist of simply two molecules (for example, 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 nanometers to about 10 microns.
  • 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.
  • 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 can have average lifetimes supported by hydrogen bonds, metal coordination, hydrophobic forces, van der Waals forces, pi-pi interactions, and electrostatic effects between 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 interconnected 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 the supramolecular assemblies (structures).
  • the meaning of nanoparticle includes the meaning of ultrafine particle and/or discrete particle.
  • the nanoparticle in some embodiments of the present invention has a largest size that ranges from 1 nanometer to 10,000 nanometers.
  • the structural, bonding and termination components self-assemble into supramolecular structures having essentially a predetermined size.
  • a predetermined size preferably is at least about 10 nm and less than about 800 nm (nanometers).
  • the specified size is from about 5 nm to 2000 nm.
  • 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 to determine the average size or relative size of nanoparticles.

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Abstract

L'invention concerne des procédés de production de structures supramoléculaires en utilisant la reconnaissance moléculaire, et des procédés de contrôle de la taille de nanoparticules supramoléculaires afin de former des particules discrètes qui possèdent des propriétés antivirales utiles. Selon l'invention, des nanoparticules supramoléculaires peuvent comprendre des hèmes-porphyrines dérivées du groupe des hème-porphyrines benzimidazolées obtenues suite à une première synthèse combinatoire, des dipyridamoles combinatoires dérivées obtenues suite à une deuxième synthèse combinatoire, des polypeptides d'acides aminés principaux obtenus suite à une troisième synthèse combinatoire, et leur combinaisons. Ces nanoparticules supramoléculaires consistent en des nanostructures solubles dynamiques à organisation automatique qui possèdent une pluralité de composants de liaison, des noyaux organiques et des composants terminaux. Les composants de liaison comprennent des hème-porphyrines dérivées combinatoires avec des sections de liaison. Les noyaux organiques comprennent du dipyridamole dérivé combinatoire adapté pour se lier à des hème-porphyrines dérivées combinatoires de sorte que les noyaux organiques puissent produire une structure mécanique pour des nanostructures solubles à organisation autonome et un premier type de complexes d'inclusion. Ces nanoparticules supramoléculaires comprennent des composants terminaux comprenant au moins un élément de liaison terminal capable de se lier à une région de liaison résiduelle des composants de liaison, lesquels permettent de produire un second type de complexes d'inclusion.
PCT/RU2022/000096 2022-03-29 2022-03-29 Nanoparticules supramoléculaires ayant des propriétés antivirales à base de hème-porphyrine benzimidazolée WO2023191651A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010099466A2 (fr) * 2009-02-26 2010-09-02 The Regents Of The University Of California Approche supramoléculaire pour la préparation de nanoparticules à taille réglable
US20170112800A1 (en) * 2014-04-03 2017-04-27 Invictus Oncology Pvt. Ltd. Supramolecular combinatorial therapeutics
WO2018237109A1 (fr) * 2017-06-23 2018-12-27 Yale University Nanomatériaux présentant une efficacité améliorée d'administration de médicament
WO2020092884A2 (fr) * 2018-11-02 2020-05-07 The Regents Of The University Of California Nanoparticules supramoléculaires réticulées pour la libération contrôlée de médicaments antifongiques et de stéroïdes - une nouvelle approche thérapeutique contre l'onychomycose et les chéloïdes
US20210100801A1 (en) * 2017-11-15 2021-04-08 Farber Boris Slavinovich Pharmaceutical composition for stimulating stem cell division and suppressing bacterial virulence

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010099466A2 (fr) * 2009-02-26 2010-09-02 The Regents Of The University Of California Approche supramoléculaire pour la préparation de nanoparticules à taille réglable
US20170112800A1 (en) * 2014-04-03 2017-04-27 Invictus Oncology Pvt. Ltd. Supramolecular combinatorial therapeutics
WO2018237109A1 (fr) * 2017-06-23 2018-12-27 Yale University Nanomatériaux présentant une efficacité améliorée d'administration de médicament
US20210100801A1 (en) * 2017-11-15 2021-04-08 Farber Boris Slavinovich Pharmaceutical composition for stimulating stem cell division and suppressing bacterial virulence
WO2020092884A2 (fr) * 2018-11-02 2020-05-07 The Regents Of The University Of California Nanoparticules supramoléculaires réticulées pour la libération contrôlée de médicaments antifongiques et de stéroïdes - une nouvelle approche thérapeutique contre l'onychomycose et les chéloïdes

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