Nucleic acid delivery system

FIELD: chemical-pharmaceutical industry, biochemistry, medicine.

SUBSTANCE: invention relates to a liposome directly effecting on αvβ3-integrin receptors and comprising cationic amphiphilic substance including 1,2-dioleoyloxy-3-(N,N,N-trimethylammonium)propane chloride, neutral lipid, lipid with a direct effect having domain with a direct effect and hydrophobic domain bound with domain of a direct effect, and nucleic acid forming complex with cationic lipid. Cationic lipid presents in the amount from about 1 to about 50 molar % and indicated lipid with a direct effect presents in the amount from about 1 to about 20 molar % wherein molar percents are calculated as measured for the total number of lipid moles in liposome. Domain with a direct effect comprises a nonpeptide antagonist of αvβ3-integrin comprising 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethyloxy]-benzoyl-2-(S)-aminoethylsulfonamino-β-alanine (compound 10) bound covalently with hydrophilic domain by amide bond. Also, invention relates to a method for inhibition of angiogenesis and involving administration to a patient needing in inhibition of angiogenesis a liposome in the therapeutically effective dose that directly effects on αvβ3-integrin receptors and comprising nucleic acid that is able to express a protein or peptide suppressing angiogenesis.

EFFECT: valuable properties of system.

27 cl, 2 tbl, 18 dwg, 8 ex

 

Cross-reference to related applications

This application has a priority of provisional application U.S. No. 60/345891, filed October 29, 2001, and No. 60/294309, filed may 30, 2001

Law of the state

This invention was made with support from the U.S. government and the National institutes of health grants 1 R37 CA50286 and 1 R01 CA86312; the U.S. government has certain rights to the specified invention.

The technical field

The present invention relates to the delivery of genes and such nucleic acid sites of the target in vivo. In particular, this invention relates to a mediated by liposome delivery of genes to angiogenic blood vessels.

Background of the invention

Integrins are a class of cellular receptors, which are known to bind extracellular matrix proteins and thus mediate intercellular interaction and the interaction of cells with extracellular matrix, defined as cell adhesion. Although in the scientific literature describes many integrins and binding integrin ligands, the biological function of many integrins are still poorly understood. The integrin receptors form a family of proteins with common structural features of the complexes of non-covalent heterodimeric glycoproteins, educated αand β-subunits, Cheresh & Mecham. eds., Integrins: Molecula and Biological Responses to the increasing interest among Matrix, Academic Press, Inc., San Diego, CA 92101 (1994), Horton, Int. J. Exp. Pathol., 71:741-759 (1990). In ongoing studies of specific functions in cell adhesion, perform these integrins interactions between cells in tissues.

Interaction of endothelial cells with the matrix plays a role in angiogenesis, i.e. the formation of new blood vessels. The receptor cell adhesion, known as αvβ3-integrin found on the surface of activated endothelial cells involved in angiogenesis.

It is well known that angiogenesis is necessary for the growth of malignant tumors and metastases. In the absence of angiogenesis local tumor development oppressed. In addition, it is known that the expression of specific marker of angiogenesis - αvβ3-integrin corresponds to the degree of tumor development.

It is established that cationic liposomes containing ones antagonist of integrin as a matter of directed action, can deliver such nucleic acids, like genes, angiogenic blood vessels. Correctly selected nucleic acid can inhibit or enhance the growth of blood vessels and, thus, represent a tool for the treatment of diseases associated with angiogenesis.

Summary of being invented what I

The present invention relates to liposomes directed action, which include ones receptor antagonist of integrin and nucleic acid. These liposomes directional suitable for selective delivery in vivo nucleic acids, such as genes, antisense oligonucleotide sequences, DNA, RNA and the like, to a predetermined target site, for example, angiogenic blood vessel systemic or local administration. The selected nucleic acids can be delivered to angiogenic blood vessels with the aim of mediating absorption of such nucleic acids endothelial cells of blood vessels for the expression or delivery of antisense sequences. This can be achieved violation growth of new blood vessels. In addition, due to an appropriate choice of the delivered nucleic acid may, if desired, to induce the growth of new blood vessels.

In particular, the liposome targeted effect on receptor αvβ3-integrin, is the nanoparticle size less than about 100 nanometers and is single-layered or multi-layered vesicle containing cationic amphiphilic substance, such as a cationic lipid, a neutral lipid, lipid directional, having a domain, napravlennosti α vβ3-integrin, and a hydrophobic domain, and a nucleic acid such as a gene, the antisense oligonucleotide sequence, DNA sequence, RNA sequence, and the like. Liposome directed action may also optionally include a neutral lipid. Available in lipid directional directional domain of validity can be directly attached to the hydrophobic domain. Alternative domain directed action can be covalently linked to the hydrophilic binding domain (surface linker), which is in turn covalently linked to a hydrophobic domain. Nucleic acid forms a complex with cationic amphiphilic substance, present in the liposome. Domain directed action includes ones antagonist αvβ3-integrin.

In the liposome directional cationic amphiphilic substance, such as cationic lipid is present in an amount of from about 1 to about 50 molar percent, and the domain of directed action is in the lipid directional from about 1 to about 20 molar percent based on the total number of moles of lipid in the liposome. The lipids that form the liposome directional, can be oligomerized and/or the polymerized functional group suitable for the General hydrophobic parts, and at least part of such lipids present in the liposome, may be sewn to each other with the help of such groups. The cationic lipid may also wish to have the stitching group. Alternative cationic lipid may not have the stitching groups.

Liposomes directional according to the present invention can be used for delivery of nucleic acids for the treatment of cancer, inflammatory diseases, eye diseases, and the like. Such liposomes directed action can also be used for delivery of genes to identify targets for treatment in the blood vessels.

Liposomes directed action in this invention are multifunctional linking nanoparticles of a size not more than about 250 nanometers, preferably from about 40 to about 100 nanometers, which include a cationic lipid or zitierten, forming a complex with a nucleic acid. The preferred lipid directed action can be represented as L-X-K, where L denotes the domain of directed action, for example, the antagonist of the integrin receptor, such as a receptor antagonist αvβ3-integrin and the like, x is the hydrophilic domain, which serves as a surface for hydrophobic linker domain K. Alternative lipid directed action can be represented ka is L-K, where domain directional L is directly linked to a hydrophobic domain K.

Ones antagonists of the integrin receptor, L, is suitable to achieve the objectives of the present invention at physiological pH values, are zwitterion having a cationic group and an anionic group that can interact or communicate with the integrin receptor. Cationic and anionic groups are separated from each other by spacer elements group, such as a divalent aromatic group. The distance between the cationic group and an anionic group in the molecule receptor antagonist is from about 10 to about 100 angstroms, and can be created alkoxybenzenes acids, bicyclic or tricyclic compounds, spirocyclohexane compounds and the like in the presence of separated from each other cationic and anionic groups, intended to interact with the integrin receptor at physiological pH values. Acceptable antagonists of integrin receptor can be schematically represented as follows:

Typical ones receptor antagonist αvβ3-integrin has the formula

where there is a free amino group (NH2designed for covalent binding of the antagonist with the Hydra is fonim domain liposomes using a direct connection or surface linker group.

Other acceptable ones receptor antagonists αvβ3-integrin, is suitable to achieve the objectives of the present invention for linking to the hydrophilic domain of the lipid directional described in U.S. patent No. 5561148, 5776973 and 6204280 and in patent publications WO 00/63178, WO 01/10841, WO 01/14337, WO 01/14338, WO 97/45137, WO 98/35949 and WO 00/26212.

The combination of ones of the receptor antagonist of integrin L and an optional surface linker X forms a molecule targeted effect on receptor αvβ3-integrin, or a group suitable for conjugation to a carrier nucleic acid, such as cationic liposome, and the like. The resulting liposome directional then associated with a predetermined nucleic acid, for example, the genome, with the formation of the complex.

Brief description of drawings

Figure 1 schematically shows the liposome directional according to the present invention.

Figure 2 shows stitched liposome directional according to the present invention.

Figure 3 shows another stitched liposome directional according to the present invention.

Figure 4 schematically shows the liposomes directional circulating system.

Figure 5 schematically shows the liposomes directional accumulating in the month the Ah development of blood vessels.

Figure 6 and 7 schematically shows the delivery of DNA such as the gene to the target cells.

On Fig shown schematically adhesion analysis, suitable for testing αvβ3-antagonists.

Figure 9 graphically presents the data obtained during the execution of the adhesion analysis, shown in Fig, which suggests that liposome directional according to the present invention binds to a receptor αvβ3-integrin.

Figure 10 graphically presents evidence about effective targeted delivery in vivo gene green fluorescent protein (GFP)expressing cells αvβ3-integrin, using liposomes directed action on this invention; the data show that carrying the gene liposomes directional transferout cells depending on the presence of αvβ3-integrin; cells were used human melanoma M21 and 21L; cells M21 Express αvβ3-integrin, whereas cells 21L does not Express αvβ3-integrin (no αv-integrin).

Figure 11 graphically presents data showing selective directional effects in vivo luciferase gene fireflies on cells soudi is the network of the tumor, expressing αvβ3-integrin, using liposomes directed action on this invention; the data show that the gene directional transferout tumor cells depending on the presence of αvβ3-integrin; cells were used human melanoma M21 and 21L implanted mice; cells M21 Express αvβ3-integrin, whereas cells 21L does not Express αvβ3-integrin.

On Fig graphically presents evidence of suppression and regression of tumor growth in vivo by injection of liposomes directed action associated with the gene expressing the mutant protein Raf suppressing angiogenesis.

On Fig graphically presents evidence of suppression and regression of tumor growth in vivo by injection of liposomes directed action associated with the gene expressing the mutant protein Raf suppressing angiogenesis.

On Fig shows a micrograph showing the delivery in vivo gene encoding GFP, to angiogenic vessels chorioallantoic membrane (CAM) of chicken when using liposomes directed action on this invention.

On Fig shows a micrograph showing the delivery in vivo to romanosky vessels in the eye mouse gene, encoding GFP, by injection into the vitreous body of the eye liposomes directed action on this invention, associated with the gene.

On Fig shows a micrograph showing the apoptosis of new blood vessels in vivo due mediated by liposome directional delivery of the gene encoding the mutant protein Raf, to angiogenic blood vessels in the retina of the mouse.

On Fig shows the scheme of the synthesis of 1 liposomes directed action, which considered in detail the synthesis of the main intermediate conjugate trivalent lipid and antagonist of integrin 12.

On Fig shows the scheme of synthesis of 2 liposomes directed action, which considers the formation of nanoparticles liposomes directional (NP)in the self and polymerization of the corresponding lipids from trivalent conjugate lipid and antagonist of integrin 12.

Description of the preferred embodiments of the invention

Liposomes directed action of the present invention, shown in Fig.1-3 and 18, contain receptor antagonist of integrin, for example receptor antagonist αvβ3-integrin associated with a lipid, and a carrier nucleic acid, for example cationic amphiphilic substance, such as a cationic lipid. This liposome may also contain neutral or zwitterionic l is the pid filler.

Lipid directed action is the domain of directed action, which includes receptor antagonist αvβ3-integrin, covalently linked to a hydrophobic domain. Domain directed action can be directly linked to a hydrophobic domain or the domain of directed action can be associated with hydrophilic domain, such as the linker group (surface linker), which in turn is linked to a hydrophobic domain.

Antagonists of integrin receptor, suitable to achieve the objectives of the present invention, are zwitterionic at physiological pH values and have cationic and anionic groups, which can interact or communicate with the integrin receptor. Cationic and anionic groups are separated from each other by spacer elements group, such as a divalent aromatic group. The distance between the cationic group and an anionic group in the molecule receptor antagonist is in the range from about 10 to about 100 angstroms, and can be created p-alkoxybenzyl acids, bicyclic or tricyclic compounds, spirocyclohexane compounds and similar compounds in the presence of separated from each other cationic and anionic groups, intended to interact with the integrin receptor at physiological pH values.

the Ermin "aryl" within the meaning of used in the description of the invention and the appended claims, means a hydrocarbon radical containing at least one aromatic ring with 6 carbon atoms, which is optionally may include hydrocarbon substituents with a linear or branched chain, or cyclic hydrocarbon substituents. The term "heteroaryl" means a radical containing at least one aromatic carbon ring with a heteroatom, which is optionally may include hydrocarbon substituents with a linear or branched chain, or cyclic hydrocarbon substituents, with the heteroatom can be any element selected from the groups defined in the IUPAC nomenclature as group 15 (nitrogen group) and group 16 (oxygen group) in the periodic table, including aromatic heterocyclic radicals such compounds described in the publication L.A. Paquette, Principles of Modern Heterocyclic Chemistry, Benjamin/Cummings Publishing Company, Inc. (1968), which are included in this description by reference. These aryl and heteroaryl groups can be substituted or replaced.

The term "heterocyclic" is used in the description of the invention and the appended claims, means a radical containing at least one nonaromatic carbon ring with a heteroatom, which updat the Executive may include hydrocarbon substituents with a linear or branched chain, or cyclic hydrocarbon substituents, when the heteroatom can be any element selected from the groups defined in the IUPAC nomenclature as group 15 (nitrogen group) and group 16 (oxygen group) in the periodic table, including non-aromatic heterocyclic radicals such compounds described in the above publication L.A. Paquette, which are included in this description by reference. Heterocyclic groups may be substituted or substituted alkyl groups or reactive functional groups such as Halogens, amino groups, hydroxyl groups, carboxylic acid groups, sulfonic acid, and the like.

The term "alkyl" is used in this description and the appended claims, means a hydrocarbon portion, which may include a linear or branched chain, or may be carbocyclic ring structure.

The term "alkenyl" in the sense used in this description and the attached claims, means an alkyl group having at least one carbon-carbon double bond.

The term "quinil" in the sense used in this description and the attached claims, means an alkyl group having at least one carbon-carbon triple bond.

The term "substituted the initial" within the meaning of used in this description and the appended claims, means the replacement of one or more hydrogen atoms in one of the above radicals the alkyl group, phenyl group or a functional group such as hydroxyl, CNS, amino, nitroso, nitro, azo, azido, amido, carboxyl, oxo, thiol, sulfoximine, sulfonylurea, Gostinichnaya, fastonline, fluorine-, chlorine-, bromine-, iododerma and similar groups, which are described in the publication of R. Panico et al., A Guide To IUPAC Nomenclature of Organic Compounds, Blackwell Science Ltd. (1993), which are included in this description by reference.

The term "liposome" is used in this description and the appended claims, means the bulb, the walls of which are formed by lipid molecules, which can be copolymerizable or not copolymerizable with each other.

The term "lipid" is used in this description and appended claims, refers to any number of groups of oils, fats and fat-like substances, which are normally soluble in relatively nonpolar solvents, but poorly soluble in aqueous solvents. Lipids constitute one of the four major classes of compounds present in the tissues of living organisms, and include fatty acids, neutral fats, such threatingly erini, esters of fatty acids and Soaps, long-chain (fatty) alcohols and waxes, sphingoid and other long-chain bases, glycolipids, phospholipids, sphingolipids, carotenes, polyprenols, sterols, and the like, as well as terpenes and isoprenoids such.

The term "zitierten" in the sense used in this description and the appended claims, means a cationic lipid, suitable for delivery and gene expression, which consists of a cationic head group attached by a linker to a hydrophobic domain or part.

The term "neutral" in the sense used in this description and the appended claims to refer to lipids, means uncharged lipids, such as cholesterol and the like, as well as zwitterionic lipids, for example, dioleoylphosphatidylcholine, dioleoylphosphatidylcholine and the like.

The term "cholesterol" in the sense used in this description and the appended claims, means a steroid hydrocarbon moiety selected from cholesterol, or have a similar structure.

The term "antagonist of integrin receptor" in the sense used in this description and the attached claims, means ones compound that selectively binds to the integrin receptor, n is the sample, with αvβ3receptor, αvβ5receptor, αvβ6receptor and the like, and has an antagonistic action.

Typical connection such αvβ3antagonist expressed by the formula

where available a free amino group (NH2) covalently binds to antagonist with a hydrophobic domain liposomes using surface linker group, such as carboxyl and/or other acceptable active group.

Other typical ones antagonists αvβ3receptors, which are suitable to achieve the objectives of the present invention, being connected directly or indirectly with the hydrophobic domain of the lipid directional described in U.S. patent No. 5561148, 5776973 and 6204280 and in patent publications WO 00/63178, WO 01/10841, WO 01/14337, WO 01/14338, WO 97/45137, WO 98/35949 and WO 00/26212, which are included in this description by reference. Such antagonists αvβ3receptors shown below

and such antagonists described in patent application WO 01/14338;

and such antagonists described in patent application WO 01/14337;

and such antagonists described in the patent for which VCE WO 00/63178;

and such antagonists described in patent application WO 01/10841; provided that such compounds include, or modified to include functional groups or bridging groups, which may interact with the surface linker or a hydrophobic domain with the formation of lipid directed action. Such modifications are well known in the field of chemistry. For example, one of the aromatic parts of the above compounds can be chemically substituted amino, hydroxyl or thiol group, providing the possibility of joining surface linker. An alternative aromatic groups can be substituted carboxylic acid and a surface linker may represent, for example, substituting the amino group.

Ones receptor antagonist of integrin covalently attached to a hydrophilic surface linker or directly to the hydrophobic domain using conventional chemical methods, providing a covalent bond antagonist with surface linker or a hydrophobic domain. Chemical methods of conducting reactions, allowing to obtain such a connection is well known in this area and include the use of complementary functional groups in the surface linker or a hydrophobic domain, and the ligand, antagonist the East integrin. Complementary functional groups in the surface or hydrophobic linker domain is preferably selected depending on the functional groups, which are ligand for the formation of ties or which can be introduced into the ligand for the formation of ties. Such additional functional groups are also well known in this field. For example, the implementation of the interaction between carboxylic acid and a primary or secondary amine in the presence of acceptable well-known activating agents leads to the formation of amide linkages, which can covalently bind the ligand with the surface linker or a hydrophobic domain, enabling interaction between the amino group and the group sulphonylchloride leads to the formation of sulfonamidnuyu communication, which can covalently bind the ligand with the surface linker or a hydrophobic domain; and enabling interaction between alcohol or phenol group and an alkyl - or helgaleena leads to the formation of simple essential communication, which can covalently bind ligand antagonist of integrin surface with a linker or a hydrophobic domain.

Alternative receptor antagonist of integrin may include a hydrophobic domain, such as With18-C30alkyl group, a C18-C30Alchemilla group18 -C30Alchemilla group, cholesterola group and the like. Hydrophobic domain, with or without intermediate surface linker attached to the receptor antagonist of the integrin in a position that retains the ability to interact with the binding site of the receptor, and, in particular, the provision that allows the antagonist to navigate to bind to the binding site of the integrin receptor. Such provisions and methods of synthesis for the formation of ties is well known in this field.

Preferred antagonists of receptor αvβ3-integrin, which have a hydrophobic domain, or can be covalently attached to the hydrophobic domain, expressed by the formulas (I) and (II)

where in the formula (I) R1and R2represent hydrogen or together form a bridging 1,2-fenelonov (C6H4) group or a bridging ethylene group (-CH=CH-); X is-C(O)- or a covalent bond; n is 1, 2 or 3; Z1means-C(O)-R3; -C(O)OR3or SO2R3; and R3means phenyl, substituted phenyl, pyridyl, benzyl, substituted benzyl, With1-C4halogenated,2-C30alkyl, C2-C30alkenyl,2-C30quinil or cholesterol; and where in the formula (II) R4and R5represent the Wallpaper hydrogen or together form a covalent bond; Y represents-C(O)- or-CH2-; Z2means-C(O)-R6, -C(O)OR6or SO2R6; R6means phenyl, substituted phenyl, pyridyl, benzyl, substituted benzyl, With1-C4halogenated,2-C30alkyl, C2-C30alkenyl,2-C30quinil or cholesterol; and Het is 2-pyridyl or 2-imidazolyl.

Non-limiting examples of antagonists of integrin receptor of the formula (I) include the following compounds Ia, Ib, Ic, Id, Ie and If. Obtaining these compounds are described in PCT publication WO 98/35949.

Non-limiting examples of antagonists of integrin receptor of the formula (II) include the following compounds IIa, IIb, IIc, IId, IIe and IIf. Obtaining these compounds are described in PCT publication WO 00/26212.

Preferred antagonists of receptor αvβ3-integrin have a molecular weight (MW) from about 200 to about 800 daltons, more preferably from 450 to about 610 daltons, if these compounds are not covalently attached to the hydrophobic domain of the lipid directed action.

Receptor antagonist αvβ3-integrin covalently attached to a hydrophobic domain or comprising a hydrophobic domain, in combination with a cationic lipid allows you to get nationnalism, which is biologically compatible and essentially neimenovano. The biological activity of liposomes directed action can depend on the valence, geometry, composition, size, flexibility or rigidity, etc. hydrophilic surface linker, if any, and in turn from the General configuration of liposomes directed action, as well as the relative hydrophilicity of the surface linker and similar factors. Thus, in the presence of the hydrophilic surface of its linker preferably is chosen with maximum biological activity of liposomes directed action. Surface linker can be selected from the increased biological activity of the molecule directed action. Surface linker is usually chosen from organic molecules with any structure that orients the antagonist in the direction of the binding site. In this respect, the surface linker can be viewed as a "frame"on which is located one or more antagonists of integrin with the desired orientation. Orientation may represent, for example, the location of the antagonist at a reasonable distance from the surface of the liposomes to ensure the effective interaction of the antagonist to the active site of the receptor integrin.

For example, different orientations can create the, including in the frame group containing mono - and polycyclic groups, including aryl and/or heteroaryl groups, or structure having one or more carbon-carbon multiple bonds (alkeneamine, alkenylamine, alkyline or alkenylamine group). Other groups may also include oligomers and polymers with branched or straight chains. In preferred variants of the invention, the rigidity is achieved due to the presence of cyclic groups (for example, aryl, heteroaryl, cycloalkyl, heterocyclic and so on). In another preferred embodiment of the invention the ring is a six - or deletechannel ring. In other preferred embodiments of the invention the ring is an aromatic ring, such as, for example, phenyl or naphthyl.

Crystal structure of the extracellular part of the αvβ3-integrin associated with the integrin antagonist described in article Xiong et al., Science 296: 151-155 (2002). Antagonists of receptor αvβ3-integrin used to implement the present invention have a structure that interacts with the receptor αvβ3-integrin similar to the interaction described in the article Xiong et al.

The person skilled in the art can easily determine the different g is ProfiLine properties of the surface linker, as well as the presence or absence of charged parts in the liposome. For example, the hydrophobic nature of the surface linker derived from a diamine (N2N(CH2)6NH2or related polyamines, can be made more hydrophilic, replacing alkylenes group of poly(oxyalkylene) group, such as poly(ethylene glycol), poly(propylene glycol) and the like.

Properties of the surface linker can be modified by adding or inserting an auxiliary group in/on the surface linker, for example, to change the solubility of liposomes (water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, flexibility, surface linker, antigenicity, stability, etc. for Example, the introduction of one or more poly(etilenglikolevykh) (PEG) groups in/on the surface linker enhances the hydrophilicity or water-solubility of the nanoparticles liposomes increases the molecular weight and size of the molecules and, depending on the nature of surface linker may also increase the retention time in vivo. In addition, PEG can reduce antigenicity and potentially increase the overall rigidity of the surface linker.

Auxiliary groups, which can increase the solubility/hydrophilicity of liposomes are useful for implementing the present invention. Thus,the scope of the present invention includes the use of auxiliary groups such as, for example, small units etilenglikola, propylene glycols, alcohols, polyols (such as glycerol, propoxylated glycerol, sugars, including mono-, oligosaccharides and so on), carboxylates (for example, small structural units of glutamic acid, acrylic acid, etc.), amines (for example, Tetraethylenepentamine) and the like, to increase the water solubility and/or hydrophilicity of liposomes according to this invention. In preferred variants of the invention, the auxiliary group used to enhance water solubility/hydrophilicity, is a simple polyester. Auxiliary group can be attached to surface the linker lipid directional or can be attached to other lipids in the liposome, such as, for example, cationic lipid or a neutral lipid-filler.

In the scope of the present invention also includes the introduction of lipophilic auxiliary groups in the structure of the surface linker to increase the lipophilicity and/or hydrophobicity described liposomes. Lipophilic groups that can be used with surface linkers according to this invention include here only as examples of the unsubstituted or substituted aryl and heteroaryl groups can be substituted by at least one group, doing what she could covalent joining of these groups to the surface linker. Other lipophilic groups, suitable for use with surface linkers according to this invention include derivatives of fatty acids that do not form double layers in the aquatic environment to achieve relatively higher concentrations.

The flexibility of surface linker can be modified by introducing a volume and/or hard subsidiary groups. The presence of large or rigid groups can hinder the free rotation about bonds in the surface linker, relations between the surface linker and minor(s) group(s) or relationships between the surface linker and functional groups. Hard group may include, for example, groups, conformational instability which is limited by the presence of rings and/or number of links in the group, in particular aryl, heteroaryl, cycloalkyl, cycloalkenyl and heterocyclic groups. Other groups capable of imparting rigidity, include polypeptide groups, such as oligo - or polyproline chain.

Rigidity can also give electrostatically. So, if an auxiliary group positively or negatively charged, similarly charged auxiliary group will strive to create the surface structure of the linker providing the maximum distance between all the same charges. Saving the political costs for a rapprochement equally charged groups, will save the configuration of the surface linker, in which equally charged auxiliary groups are separated from each other. In addition, support groups, having the opposite charge will be attracted to oppositely charged groups and potentially can form intermolecular and intramolecular ionic bonds. Such non-covalent mechanism will save the configuration of the surface linker, contributing to the formation of links between oppositely charged groups. In the scope of the present invention includes the introduction of an additional auxiliary groups that are charged or alternatively have a hidden charge when you remove protection after joining surface linker, including the removal of protective groups in the carboxyl, hydroxyl, thiol or amino groups by changes in pH, oxidation, recovery, or by other mechanisms known to specialists in this field.

Rigidity can also be given by internal hydrogen bonding or hydrophobic destruction.

Volume groups can include, for example, large atoms, ions (for example, iodine, sulfur, metal ions etc) or groups containing large atoms, polycyclic groups, including aromatic groups, near thematic groups and patterns, having one or more carbon-carbon multiple bonds (i.e. alkenes and alkynes). Volume groups can also include oligomers and polymers with branched or straight chain. It is assumed that the branched polymers give the structure greater rigidity than linear polymers of similar molecular weight.

In some embodiments the invention, the stiffness is given due to the presence of cyclic groups (such as aryl, heteroaryl, cycloalkyl, heterocyclic and so on). In other embodiments of the invention, the surface linker includes one or more six-membered rings. In some other embodiments of the invention the ring is aryl group, such as, for example, phenyl or naphthyl.

The appropriate selection of the group of surface linker providing appropriate orientation, limited/unlimited rotation, the desired degree of hydrophobicity/hydrophilicity, etc. must be done by a specialist in this field. Ways to eliminate or reduce the antigenicity described nanoparticles are also known in this field. In some cases, the antigenicity of the nanoparticles can be eliminated or reduced by using groups such as, for example, poly(etilenglikolevye) group.

The carrier nucleic acid is a cationic amphiphile is a grave matter, such as a cationic lipid, a cationic liposome or micelle having cationic groups, which can bind with nucleic acid, usually as a result of interaction with negatively charged sequences of nucleic acids with the formation of complexes that are able to penetrate into the cell. Liposomes directed action is shown in figure 1, 2, 3, 17 and 18.

Cationic lipids suitable for achieving the objectives of the present invention (zitierten), can be illustrated by such compounds as 1,2-tileorasi-3-(N,N,N-trimethyl-ammonium)propachlor (DOTAP), bromide of dimethyldioctadecylammonium (DDAB), chloride of dioleoylglycerol, dioleoyl-L-α-phosphatidylethanolamine (DOPE), N-cholesteryloxycarbonyl-3,7,12-triazapentadiene-1,15-diamine (OLD), etc. Preferred cationic lipid is DOTAP. Other acceptable cationic lipids described in articles Miller, Angew. Chem. Int. Ed. 37:1768-1785 (1998) (hereinafter "article Miller") and Cooper et al., Chem. Eur. J. 4(1): 137-151 (1998), which are included in this description by reference.

Liposome directional according to the present invention can be crosslinked, partially crosslinked or not crosslinked. Crosslinked liposomes can be stitched and unstitched components.

Typical unstitched liposome directional according to the present invention is a mixture of lipids comprising DOTAP (cationic what ipid), cholesterol (neutral lipid), polyethylene glycol (hydrophilic auxiliary component), such as PEG-350 (polyoxyethylene having 350 oxyethylene structural units) and ones receptor antagonist of integrin, covalently linked to a lipid or a containing lipid. The ratio of DOTAP, cholesterol, and polyethylene glycol is preferably about 1:1:0,12, while the lipid containing ones receptor antagonist of the integrin (lipid targeted effect on integrin), used in a quantity sufficient to obtain a relatively high avidity to αvβ3-integrin. Liposome directional preferably includes a lipid targeted effect on the integrin, in an amount of from about 1 to about 20 mol.% in calculating the total number of moles of lipid components in the liposome, more preferably from about 8 to about 12 mol.%.

Preferred crosslinked liposome directional according to the present invention includes a curable zwitterionic or neutral lipid, the polymerized lipid targeted effect on the integrin, and polymerized cationic lipid, suitable for binding nucleic acids.

In another preferred embodiment of the invention stitched liposome directional comprises polymerised of cvict Ronny or neutral lipid, the polymerized lipid targeted effect on the integrin, and polimersomy cationic lipid.

Liposome containing the polymerized lipids, can be made, for example, by adding a suitable radical polymerization initiator, irradiation with ultraviolet light with an appropriate wavelength or other methods known in the field of polymerization.

Acceptable cationic liposomes or zitierten can be purchased commercially and can be obtained by methods described in the article Sipkins et al., Nature Medicine, 1998, 4(5): (1998), 623-626 or in the above article Miller.

Cationic liposomes can be obtained from one cationic amphiphilic substance or of a combination of cationic amphiphilic substance and a neutral lipid, for example, 3,3-[N-(N',N'-dimethylaminoethyl)carbarnoyl]cholesterol and dioleoyl-L-α-phosphatidylethanolamine.

Hydrophilic properties receive due to the presence of phosphate, phosphonate, carboxylate, sulphate, sulphonate, sulfhydryl, amino, nitro, hydroxyl and other similar groups, are well known in this field. Hydrophobicity can be given by the introduction of groups that include, but are not limited to, saturated and unsaturated aliphatic hydrocarbon group with a chain length up to 20 carbon atoms, and groups, substituted one or more aryl, heteroaryl the mi, cycloalkyl and/or heterocyclic groups. The preferred lipids are phosphoglycerides and sphingolipids. Typical examples of phosphoglycerides include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and tylenolamoxicillin. Connections that do not have phosphorus-containing groups, such as family of sphingolipids and glycosphingolipids, are also part of the group, defined as the lipid. In addition, the above amphipatic lipids can be mixed with other lipids, including triglycerides and sterols, such as cholesterol, modified cholesterol, etc.

Antagonists of the receptor integrins may have a hydrophobic domain or can be attached to the surface linker or directly to the hydrophobic domain in any acceptable position, for example, at the end of a linear chain or in any intermediate position, if such attachment does not prevent the binding of the antagonist to the receptor of the integrin. Antagonists of integrin receptor may also wish to include an optional divalent bridging group, to facilitate the joining surface to the linker.

Figure 1 schematically shows the liposome directed action in the form of particles of a spherical shape on the surface of which are sites of binding to nucleic acid and sites targeted effect on the integrin.

Figure 2 shows stitched liposome directional according to the present invention.

Figure 3 shows another stitched liposome directional, which contains unstitched cationic lipid. Such liposomes directional described in detail in the following section "materials and methods". This stitched liposome has on the outer surface of cationic groups derived from choline, which are able to bind with nucleic acids, and the binding sites of the receptor integrin obtained from groups antagonist of integrin associated with a hydrophilic surface linker.

The nucleic acid may be associated with nanoparticle cross-linked liposomes in the contact negatively charged nucleic acid with a cationic group present in the liposome, for example, by mixing the nucleic acid and liposomes directed action in a pharmaceutically acceptable aqueous medium at physiological pH. Liposomes, forming a complex with a nucleic acid that is easily absorbed by the integrin-presenting cells that are in contact with the lip is catfishes directed action in vitro and in vivo.

The ratio of positive charges liposomes directional and negative charges of the nucleic acid is preferably greater than 1, more preferably is at least about 1.2.

For selective targeting or antagonistic effects on integrins, in particular αvβ3-integrins, the compounds and compositions of the present invention can be introduced in a therapeutically effective amount of a parenteral, oral, by inhalation, or topically in dosage forms containing pharmaceutically acceptable carriers, excipients and adjuvants. The term "parenteral" is used here value means intravenous, subcutaneous, intramuscular, vnutrigrudne, intraocular (e.g., vitreous body) and intraperitoneal injection, and injection methods injection.

You can use any acceptable route of administration. Pharmaceutical composition comprising associated with nanoparticles nucleic acid of the present invention is administered in a dose effective for the intended treatment. A therapeutically effective amount for treatment of a specific disease or suppression of its development, can easily identify experts in this field, performing preclinical and clinical studies the project, known in the medical field.

The term "therapeutically effective amount" used herein the value denotes the amount of active ingredient that causes the biological or medical response of a tissue, system, animal or human that is expected by the attending physician or researcher.

The term "suppression" is used here is the mean slowdown, interruption or termination of the disease, but does not necessarily indicate a complete cures the disease. Prolonged survival of the subject itself is indicative of favorable treatment of disease.

Schemes of nucleic acids associated with the liposome directional according to the present invention or compositions containing them, is determined taking into account several factors, such as age, body weight, gender and type of disease in need of a subject, the severity of the disease, route of administration and antagonistic activity used molecules directional or ligand. The scheme of injection may vary depending on the above factors. In the treatment of the above diseases can be used doses in the range from about 0.01 to about 1000 mg per kg of body weight. The preferred dose is from about 0.01 to about 100 mg per kg of body weight.

The composition of the present invention, containing liposomemediated actions and intended for administration by injection, get using pharmaceutically acceptable carrier, such as water, saline or an aqueous solution of dextrose. A typical daily dose for injection is from about 0.01 to about 100 mg per kg of body weight, daily injection in a single dose or several divided doses depending on the above factors.

To inhibit angiogenesis, to a subject in need of such treatment is administered a therapeutically effective amount of liposomes directed action of the present invention containing a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)that can Express a protein or peptide that suppresses angiogenesis. Introduced nucleic acid enters the cell nucleus and expresses a protein directed action in endothelial cells of blood vessels.

Below are non-limiting examples, illustrating in more detail various aspects of the present invention. The person skilled in the art should be understood that the examples and embodiments of the invention may be amended, included in the scope of this invention.

Materials and methods

Obtaining nanoparticles

The creation of multivalent liposomes directional binding to integrins, begins with a defined who I structure and synthesis of molecules 12 of the polymerized lipid, targeted effect on integrins (Fig, scheme 1). The amino group of taurine 1 protected in the form of its benzyloxycarbonyl (CBZ) derivative with formation of compound 2, then get sulphonylchloride 3, which is subjected to reaction in combination with a methyl ether tert-butoxycarbonylamino acid 4, while receiving the connection 5. Connection 5 omelet with the formation of compound 6, remove the tert-butoxycarbonyl (BOC) group and receive important intermediate compound 7. Compound 7 is subjected to reaction in combination with a derivative of benzoic acid 8, while receiving the connection 9, which hydronaut to unprotect amine and simultaneously restore the pyrimidine ring, getting a conjugate of a receptor antagonist of integrin and linker 10. Synthesis of compound 8 was previously described in article Duggan et al., J. Med. Chem., 2000, 43, 3736-3745, which is incorporated in this description by reference. Three equivalent conjugate 10 is subjected to reaction in combination with a chelat forming a lipid tricarboxylic acid 11 using hexaphosphate benzotriazol-1 yloxy-Tris(dimethylamino)phosphonium (THIEF), and get the main compound of the integrin antagonist and trivalent lipid 12. Obtaining the compound (11) was previously described in article Storrs et al., J. Magn. Reson. Imaging, 1995, 5, 719-724, which are included in this description izopet is ment as a reference. Compound 11 is treated with sodium methoxide in methanol, thus obtaining the compound 15 (i.e. chinatravel salt of compound (11). The complex of europium and chelat forming lipid 14 synthesized by heating the connection 15 with a solution of trichloride europium, as described in the above article Storrs et al.

In scheme 2, shown in Fig considered getting stitched liposomes directed action (nanoparticles) in the self and polymerization of the corresponding lipids, previously described in the above article Storrs et al. A typical cross-linked liposomes directional synthesize by combining the complex compound of the integrin antagonist and trivalent lipid 12 zwitterionic the phospholipid of diacetylene 13 and complextm europium and chelat forming lipid 14 in a solution of chloroform. The connection 14 is added in the amount of 1 wt.% all compositions to render the particles using fluorescence spectroscopy. To a solution of lipids in chloroform add anionic chelate forming lipid 15 or cationic lipid 16 (DOTAP), to change the surface charge. The surface density of the integrin antagonist on the nanoparticles adjust by changing the number of connections 12 in the liposome.

To get bubbles solutions United lipids evaporated to dryness and dried in high vacuum to remove residual dissolved the La with the formation of the lipid film. The dried lipid film hydronaut to the known density lipid (30 mm) using deionized water. The resulting suspension is treated with ultrasound at a temperature above the crystalline phase transition of the gel - liquid (Tm= 64°C), then perform the method described in the article Leaver et al., Biochim. Biophys. Acta, 1983, 732, 210-218, using the device for ultrasonic treatment with the end of the probe, while maintaining the pH in the range from 7.0 to 7.5. After ultrasonic treatment for about one hour, the solution becomes transparent. Then the bubbles will polimerizuet, cooling the solution to 0°With a layer of wet ice, and irradiated solution at a wavelength of about 254 nm handheld UV lamp for 2 hours. The obtained liposomes (NP1 - NP6) have a yellow-orange color and two visible absorption bands corresponding to wavelengths of 490 nm and 535 nm and formed by the conjugated polydiacetylene novogo synthesis. The average diameter of the nanoparticles determined by dynamic light scattering (DLS), is in the range from 40 to 50 nm. Zeta-potential is respectively from -42 to -53 mV for nanoparticles NP1-NP4 (i.e. the nanoparticles NP1-NP4 are negatively charged) and from +35 to +43 mV for nanoparticles NP5 and NP6 (i.e. nanoparticles NP5 and NP6 positively charged) (Brookhaven Instruments, Holtsville, NY). Liposomes remain stable for NESCO is gcih months without any significant changes in the physical and biological properties as part of the preparations for the introduction of in vivo using 150 mm sodium chloride, 50 mm histidine and 5% solution of dextrose.

The compositions of liposomes NP1 - NP6 are shown in the following table 1, in which the components 12 (lipid directional), 13 (zwitterionic lipid), 15 (anionic chelate forming lipid) and 16 (cationic lipid; DOTAP) expressed in mol.%.

Table 1

The compositions of liposomes NP1 - NP6
LiposomeLipid directional mol.%Zwitterionic lipid

mol.%
Anionic lipid mol.%Cationic lipid mol.%
NP11080100
NP2189100
NP30,190100
NP4090100
NP51080010
NP6090010

Liposomes produced by polymerization of bubbles with the use of 0.1, 1 and 10 mol.% complex compounds antagonist of integrin and lipid 12 and compound 13, as shown in Fig, and additional substances 14, 15 and 16, which is administered in liposomes to polymerise the tion to changes in the surface charge density and fluorescent imaging of liposomes. For simplicity, in Fig not shown connection 13, which is optional and is only 1 mol.% liposomes. Substances containing 10 mol.% compound 12 (NP1 and NP5), have the greatest affinity for the binding site αvβ3-integrin. When performing analysis of competitive binding of integrin using fluorescence spectroscopy with time resolution needed 100-fold amount of free ligand 10 (65 microns) to reduce binding of the nanoparticles NP1 and αvβ3-integrin by 50% despite the fact that the amount of an antagonist of integrin 10 on the surface of the nanoparticles NP1 is equivalent to only 0.5 µm. When performing analysis of inhibition of cell adhesion in vitro using melanoma cells M21, containing αvβ3-integrin, tablets coated with vitronectin, IC50for the free ligand 10 was 64 μm. In contrast, IC50for anionic particles NP1 equal to 0.27 μm equivalent connection 10 on the surface. The obtained result indicates 200-fold avidity to the surface of cells using nanoparticles connection 10 in comparison with the free ligand. For cationic particles NP5 IC50 equal to 0.35 microns equivalent connection 10, which is approximately 180 times the avidity inherent in the free ligand, as shown in n ieslegusi table 2. Thus, regardless of the surface charge of the nanoparticles have an avidity for integrins, which is about 180-200 times more than for Monomeric ligand. The result shows that between the surface of the nanoparticles and the surface of the cells is a strong interaction. This interaction does not depend on the charge on the surface of nanoparticles and directly related to the interaction with specific receptor ligand. When multivalent view of an antagonist of integrin on the surface of the nanoparticles is achieved approximately a twofold increase the avidity compared to the free ligand. When reducing the number of connections 12 in preparations containing the above-mentioned nanoparticles (NP), 10 and 100 times, respectively, to 1 mol.% and 0.1 mol.%, as is the case in NP2 and NP3, the ability to block cell adhesion is reduced by about one and two orders of magnitude, as shown in table 2.

Table 2

Physical and biological properties of liposomes NP1-NP6
SubstanceSize (nm)Zetapotential (mV)Analysis of competitive inhibition (μm compound 10)Analysis of cell adhesion, IC50(μm soy is inane 10 NP) The effect of mnogojadernosti, IC50(free ligand [10]/[10] on NP)
NP145,1±0,6-42650,27237
NP242,8±1,5-492479
NP344,4±0,8-53130,52
NP446,4±0,7-49No dataThe lack of inhibitionNo data
NP541,7±2,235600,35183
NP636,8±0,943No dataThe lack of inhibitionNo data

Liposome NP5 is a typical sewn liposome directional according to the present invention.

General synthesis methods

All used solvents and reagents have a degree of purity established for reagents. The solvent is evaporated under reduced pressure, the generated household vacuum device or a vacuum pump direct drive Welch at a temperature of ≤40°C. Spectra1H and13C-NMR of the obtained device JEOL FX90Q at 90 MHz for proton spectra and the example is about at 23 MHz for carbon spectra in CDCl 3CD3OD, D2O and their mixtures, as specified for each case. (Note: despite solubility in CDCl3adding to lipids OD3OD prevents the formation of inverted micelles and thus contributes to a more sharp spectra). Spectra1H-NMR correlated with residual CHCl3(to 7.25 ppm), and spectra13C-NMR correlated with the average line CDCl3(77,00 ppm). Mass spectrometry MALDI-TOF executed in the device PerSeptive DE (mass spectrometry, The Scripps Research Institute, La Jolla, CA). TLC performed on glass plates with the substrate Merck 60 F254 (0.2 mm; EM Separation, Wakefield, RI), and manifested blades are usually sprayed with cerium sulfate (1%) and ammonium molybdate (2.5 percent) in 10% aqueous solution of sulfuric acid and heated to about 150°C. Other developers include iodine (normal use), 0.5% ninhydrin in acetone (for amines) and ultraviolet light (UV chromophores).

Sodium salt of N-benzyloxycarbonylamino (2). Taurine 1 (40 g, 320 mmol) was dissolved in 4 BC, the sodium hydroxide solution (80 ml) and water (200 ml). To the resulting solution was added dropwise benzyloxycarbonylamino (48 ml, 330 mmol) with vigorous stirring for about 4 hours. The pH of the solution remain alkaline by adding 10% sodium bicarbonate solution (300 ml) and 4 n sodium hydroxide solution (about 45 ml). Receiving the ing the reaction mixture was washed with diethyl ether (1000 ml), the aqueous layer was evaporated to dryness in a rotary evaporator and additionally dried in high vacuum over phosphorus pentoxide overnight, while receiving about 12.7 g (14%) of compound 2.

1H-NMR (D2O): δ to 7.50 (5H, s, Ar-H), to 5.21 (2H, s, Ar-CH2), 3,62 (2H, t, CH2), 3,14 (2H, t, CH2).

2-Benzyloxycarbonylglycine (3). Sodium salt of N-CBZ-taurine 2 (about 12.7 g, 32 mmol) is suspended in dry diethyl ether (30 ml) in an argon atmosphere at a positive pressure and treated with 5 portions of pentachloride phosphorus (about 7 g, 33.6 mmol) for about 15 minutes. The reaction mixture is stirred for about 4 hours at room temperature. The solvent is removed in a rotary evaporator. Add a mixture of ice water (about 10 ml), the flask and its contents are cooled in a bath with ice and the resulting residue triturated. Add an additional amount of a mixture of ice water (about 50 ml), the resulting product is cured. The solids are collected by filtration, washed with a mixture of ice water (about 20 ml) and dried over phosphorus pentoxide overnight, while receiving about of 6.95 g (78%) of compound 3.

1H-NMR (CDCl3): δ to 7.35 (5H, s, Ar-H), 5,12 (2H, s, Ar-CH2), the 3.89 (2H, t, CH2), the imposition of 3.85 (2H, t, CH2).

Methyl-3-butyloxycarbonyl-2-(S)-benzyloxycarbonyl-aminoethylamino ropionate (5). A mixture of sulphonylchloride 3 (about 21.6 g, 78 mmol) and methyl-3-N-butoxycarbonylamino-2-aminopropionic (4, about 9,96 g of 39.2 mmol) in anhydrous tetrahydrofuran (THF, 150 ml) is cooled in a bath with ice in an argon atmosphere at a positive pressure. To the obtained solution using a dropping funnel are added dropwise N-methylmorpholine (about 16 ml, 145 mmol) in anhydrous THF (275 ml) in an argon atmosphere at a positive pressure for about 30 minutes. The reaction MES stirred in a bath with ice for about 1 hour and the results of TLC determine essentially complete consumption sulphonylchloride (Rf=0,65)(eluent: ethyl acetate/hexane, 1:1). However, in the reaction mixture still remains unreacted diaminopropionic acid (Rf=0,1, sprayed with ninhydrin). To this mixture an additional amount of sulphonylchloride (5.0 g, 18 mmol) for about 3 hours. The obtained reaction product was filtered and evaporated in a rotary evaporator to remove the solvent, dissolved in ethyl acetate (about 100 ml), washed with cold dilute hydrochloric acid (about 20 ml), saturated sodium bicarbonate solution (20 ml) and a saturated solution of sodium chloride (20 ml) and dried over anhydrous sodium sulfate. The solvent is removed in a rotary evaporator and the resulting residue land is in vacuum over night. The dried residue is recrystallized by dissolving in ethyl acetate and adding the same volume of hexane, and get methyl ester 5 as a colourless solid with a yield of about 13.4 grams (about 74%).

1H-NMR (CDCl3): δ of 7.36 (5H, s, Ar-H), of 5.83 (1H, d, NH), of 5.55 (1H, t, NH), 5,12 (2H, s, Ar-CH2), is 5.06 (1H, t, NH), 4.26 deaths (2H, m, CH), with 3.79 (3H, s, CH3), 3,70 (2H, DD, CH2), 3,26 (2H, DD, CH2), 1,43 (N, with, (CH3)3).

3 Butyloxycarbonyl-2-(S)-benzyloxycarbonylamino-arilsulfonilglitsinov acid (6). The complex solution of the methyl ester 5 (about 13.3 g, of 28.9 mmol) in tetrahydrofuran (160 ml) is cooled in a bath with ice. To the resulting solution was added a solution of lithium hydroxide (about 5,42 g, 128 mmol) in a mixture of ice water (160 ml). The reaction mixture is slowly warmed to room temperature, removing the bath with ice, and stirred at room temperature for about 1 hour. The organic solvent is removed in a rotary evaporator. The residual aqueous portion was washed with diethyl ether (about 20 ml) and acidified to about pH 4 using dilute hydrochloric acid. The resulting solution is cooled in a bath with ice, then mixed with ethyl acetate (about 100 ml), acidified to about pH 1 using cooled with ice, diluted hydrochloric acid, and immediately extracted uh what ilaclama (about h ml). An ethyl acetate layer was washed with saturated salt solution (about 50 ml) and dried over anhydrous sodium sulfate. The solvent is removed in a rotary evaporator and the resulting residue is dried in high vacuum over night, while receiving about 13.3 g foamy solid, which is recrystallized from hexane/ethyl acetate (1:1) and receive about 11.6 g (89,7%) of compound 6.

1H-NMR (CDCl3): δ 7,33 (5H, s, Ar-H), 6,12 (1H, d, NH), of 5.68 (1H, t, NH), of 5.26 (1H, t, NH), 5,1 (2N, c, Ar-CH2), 4,24 (2H, m, CH2), to 3.67 (2H, t, CH2), with 3.27 (2H, t, CH2), 1,45 (N, with, (CH3)3.

3-Amino-2-(S)-benzyloxycarbonylamino acid (7). N-BOC-β-amino acid 6 (about 11.5 g of 25.8 mmol) is treated triperoxonane acid (about 68 ml) in methylene chloride (350 ml) for about 1.5 hours and evaporated to dryness in a rotary evaporator. The resulting residue is dissolved in water (200 ml) and lyophilized, while receiving about 10.9 g (98,8%) of compound 7 in the form of solids.

1H-NMR (CDCl3): δ 7,30 (5H, s, Ar-H), 6,07 (1H, d, NH), 5,61 (1H, t, NH), 5,20 (1H, t, NH), 5.17 to (2N, c, Ar-CH2), 4,11 (2H, m, CH2), 3,53 (2H, t, CH2), of 3.32 (2H, t, CH2). DCI-MS for C13H19N3O6S: m/z (ion); 346 (M+H) (calculated for C13H19N3O6S + H m/z 346).

4-[2-(Pyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-benzyl-oxycarbonyl is ideacellular.net-β -alanine (9). A derivative of benzoic acid 8 (about 6.4 g of 24.7 mmol) and N-hydroxysuccinimide (about 3.6 g, 31 mmol) dissolved in anhydrous dimethyl sulfoxide (about 110 ml) in an argon atmosphere at a positive pressure and is cooled in a bath with ice. To the resulting solution was added the hydrochloride of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide (about 4.9 g, 25.6 mmol). The solution is stirred at a temperature of cooling with ice for 1 hour and allowed to warm to room temperature. The reaction mixture continued to stir at room temperature for approximately 24 hours. To the resulting mixture add a solution β-7 amino acids (about 12.2 g, from 25.8 mmol) and N-methylmorpholin. The resulting reaction mixture is stirred in an argon atmosphere for about 3 days. The mixture is then poured into water (about 1 l), acidified with dilute hydrochloric acid to approximately pH 1.5 and extracted with ethyl acetate (about h ml). The combined organic phase was washed with a saturated solution of sodium chloride (about 50 ml) and dried over anhydrous sodium sulfate. The solvent is removed in a rotary evaporator. The resulting residue is triturated in ethyl acetate, filtered and dried in a high vacuum, thus obtaining 10.5 g (72,5%) of compound (9).

1H-NMR (DMSO-d6): δ 8,30 (2H, d, Ar-H), to 7.99 (2H, d, Ar-H), 7,34 (5H, s, Ar-H), 7,00 (2H, d, Ar-H), 6,60 (1H, DD, Ar-H), free 5.01 (2H, c, H 2), is 4.15 (1H, t, CH), to 3.67 (2H, t, CH2), of 3.56 (2H, t, CH2), 3,17 (2H, t, CH2).

4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyloxy]-benzoyl-2-(S)-aminoethylamino-β-alanine (10). A solution of pyrimidine derivative 9 (about 3.7 g, 6.4 mmol) dissolved in acetic acid (190 ml) and concentrated hydrochloric acid (17 ml). The resulting solution is mixed with 10% palladium on coal (about 1,62 g) and hydronaut in the atmosphere of hydrogen gas under pressure 3,15 bar (45 psi) for about 5 hours. The resulting mixture was filtered through celite and washed with water. The solvent is removed in a rotary evaporator and the resulting residue is dried in high vacuum. The dried residue is dissolved in water (about 100 ml), the pH adjusted to about 7 by adding 1 n sodium hydroxide solution, and evaporated to dryness in a rotary evaporator. The resulting residue is dissolved in methanol (20 ml) and filtered. The filtrate is evaporated in a rotary evaporator, dissolved in water (275 ml) and lyophilized. The resulting liofilizovannye product is recrystallized from water, while receiving about 2,96 g (approximately 78,9%) of the product 10.

1H-NMR (D2O): δ 7,80 (2H, d, Ar-H), 7,14 (2H, d, Ar-H), 4,49 (1H, s, CHaHb), 4,28 (2H, t, CH2), of 3.94 (1H, DD, CHaHb), 3,61 (6H, m, CH2), of 3.32 (4H, t, CH2), 1,90 (2H, t, CH2). ES-MS for C8 H28N6O6S: m/z (ion); 457 (M+H) (calculated for C18H28N6O6S + H m/z 457).

[(PDA-PEG3)2-DTPA-(CONHPM)3] (12) (PDA-PEG3)2-DTPA-(COOH)3(some of 11.69 mg, 50 µmol) was dissolved in anhydrous CH3CN (5 ml), anhydrous CH2Cl2(about 2 ml) and Et3N (about 1 ml) in a three-neck flask with a round base, which is pre-dried in the flame and filled with argon. To the resulting solution was added to the reagent THIEF (about 134 mg, 150 μmol), the resulting mixture was thoroughly stirred for 5 minutes and receive the lipid solution. A solution of compound 10 (about 69 mg, 150 μmol) get in a dry test tube filled with argon, a mixture of anhydrous CH3CN (5 ml) and anhydrous dimethylformamide (DMF) (2 ml). A solution of compound 10 is added to the lipid solution using a dry syringe with continuous stirring. The resulting reaction mixture is stirred for 10 hours in the dark. The results of TLC (solvent: CHCl3CH3OH, H2O and CH3COOH) shows complete disappearance of the original substance (Rf= 0,53). Formed one main product (Rf= 0.2) and 5 secondary products (Rf< 0,18). The solvent is removed in a rotary evaporator, the resulting residue is dried in high vacuum for about 24 hours and get the crude product. Crude about the SPS HPLC purified with normal phase, using a column with silica gel for prepreparation chromatography and flow rate of about 5 ml/min (system for gradient elution: first 100% CHCl3within about 5 minutes, then 75% CHCl3/25% CH3OH for 10 minutes, 50% CHCl3/50% CH3OH for 10 minutes, 25% CHCl3/75% of CH3OH for about 10 minutes and finally 100% SN3HE spent about 20 minutes). Fraction (retention time 35-37 minutes), containing the main product are combined and evaporated in a rotary evaporator to remove solvent, the resulting residue is dried in high vacuum for approximately 24 hours, while receiving around 35.5 mg (26.5 per cent) of the desired product. MALDI-FTMS with a high-resolution m/z 2681,4711 (calculated for C130H209N25O29S3+ H m/z 2681,4882).

EXAMPLES

Example 1. Analysis of cell adhesion

The study of inhibition of cell adhesion to perform on tablets coated with vitronectin, using human melanoma cell line M21. Multivalent liposomes NP1-NP6 and Monomeric ligand 10 separately incubated with cells M21 and transferred to 48-hole tablets coated with vitronectin. The culture is incubated for about 1 hour, after which the wells are washed, fused cells stained with crystal violet solution and measure the optical density (OD) at a wavelength of an eye is about 590 nm. The measured optical density is proportional to the number of cells associated with vitronectin on the tablet; using the received data, construct a graph depending on the concentration of the component 10 on the surface of the nanoparticles in different preparations to calculate the IC50.

On Fig schematically shows the analysis of cell adhesion by which cells of human melanoma M21, expressing αvβ3-integrin, mixed with liposomes covalently conjugated to integrin antagonist (ligand) or only with the ligand. The cells are then transferred to the plates coated with vitronectin, washed and dyed. By using the apparatus for reading tablets count the number of bound cells.

Figure 9 graphically presents the data obtained in the analysis of cell adhesion, shown in Fig, which suggests that liposomes associated with integrin antagonist, effectively inhibit cell adhesion (IC50= 1 μm), while only the antagonist (the ligand) is much less effective in inhibiting cell adhesion (IC50= 0.5 mm).

Example 2. Analysis of transfection in vitro

About 30 nanomoles cationic liposomes NP5 and NP6 with covalently conjugated ligand targeted effect on the αvβ3-integrin, or without use to access the Finance complex with approximately 2 μg of plasmid DNA, encoding green fluorescent protein (GFP), in 5% dextrose and is exposed to melanoma cells in vitro for approximately 1 hour. The transfection efficiency is determined by counting the number of fluorescent cells compared with the total number of cells after 24 hours. Used cell human melanoma M21 and 21L. Cells M21 Express αvβ3-integrin, whereas cells 21L does not Express αvβ3-integrin (no αv).

As shown in figure 10, cells expressing αv-integrin (M21), which were treated with liposome directional (NP5), forming a complex with the GFP gene, characterized by 5-fold or higher level of transfection (>125,000 cells/million) compared with cells expressing αv-integrin, which have been processed by lipomassage undirected actions (NP6)(no antagonist; about 25,000 cells/million) or DNA (without liposomes; about 12,000 cells/million). In contrast, in cells not expressing αv-integrin (21L), not marked predominant introduction of the GFP gene, which indicate relatively low levels of transfection (25,000 cells/million or less) regardless of the produced processing. Thus, it was established that the gene directional t is inspireret cells depending on the presence of α vβ3-integrin.

Example 3. Mediated by liposome directional gene delivery to tumors in vivo

Approximately 450 nanomoles liposomes NP5 and NP6 used for electrostatic education complex with approximately 30 μg of plasmid DNA encoding the luciferase fireflies, about 200 μl of 5% dextrose, after which the solution of complex liposomes and DNA is injected in the form of intravenous injection of animals with subcutaneous melanoma of about 150 mm3not expressing αvβ3(21L). After about 24 hours the animals kill, remove these organs and tumors and analyze luciferase activity. The luciferase activity is determined by the set for analysis of luciferase Bright-Glo (Promega Corp., Madison, WI) according to the manufacturer's instructions except that the solid organs are crushed in a tissue homogenizer containing a reagent for lysis of Bright-Glo in the amount normalized with the weight of the body.

As shown in figure 11, liposome directional (NP5), forming a complex with a luciferase gene, is characterized by the selective expression in tumor tissue compared with the tissues of lung, liver and heart, and the expression level is approximately 4 PG luciferase/mg of tissue by comparison with levels less PG/mg tissue in the other cloth is H. Liposome undirected actions (NP6), forming a complex with luciferase, inefficient transferout all tissues. Add about 20-fold excess of soluble ligand of the integrin antagonist inhibits the transfection of cells by ligand-directed actions.

Example 4. The stable regression of malignant melanoma in the mediated by liposome directional delivery mutant Raf genes to tumor vascular network

Melanoma, not expressing αvβ3-integrin, (21L) administered by subcutaneous injection in the lateral part of the body of animals and leaves grow to a volume of about 70 mm3, after which the mice in the form of intravenous injections administered 200 μl of 5% dextrose (control group), liposomes directional NP5, forming a complex with approximately 30 μg of plasmid DNA that encodes a dominant negative mutant form of Raf kinase (Raf-ATPμ), 200 μl of 5% dextrose, or forming a complex with the Shuttle vector not encoding any DNA in 200 µl of 5% solution of dextrose. After about fifteen days to produce a repeated treatment of tumors by the above method. Tumor size is measured in the time intervals specified on Fig, using the formula: tumor volume = (minimum diameter)2· maximum diameter/2.

As shown in Fig, treatment the mice, having melanoma with an initial volume of about 70 mm3, liposomes directional NP5 forming a complex with a mutant dominant negative form of Raf kinase (Raf-ATPμ)causes an initial increase in tumor volume approximately 550 mm3, then after about 30 days is tumor regression with achieving a stable state when the tumor volume of about 200 mm3on the 35th day (the designation "particle/Raf(-)" on Fig). Stable tumor size is stored in the next 30 days, after which the execution of the experiment ceased. In mice, which impose only a liposome directional NP5 (without gene) (designator "particle/Shuttle vector") or only the mutant Raf gene (without liposomes), tumors continue to grow during 35 days without any signs of reverse development. Because the tumor does not Express αvβ3-integrin and endothelial cells of developing blood vessels Express αvβ3-integrin, reduced tumor growth, apparently due to inhibition of angiogenesis of a tumor vascular network.

Example 5. The stable regression of malignant melanoma in the mediated by liposome directional delivery mutant Raf genes to tumor vascular network

The tumor is subjected to the same treatment as in CA is re 4, except that the tumor leaves grow to a volume of about 300 mm3before the initial treatment, when animals injected as an injection (a) approximately 450 nanomoles liposomes directional NP5, forming complex through electrostatic effects from about 30 μg of plasmid DNA that encodes a Raf-ATPμapproximately 200 μl of 5% dextrose, (b) approximately 450 nanomoles liposomes undirected actions NP6, forming complex through electrostatic effects from about 30 μg of plasmid DNA that encodes a Raf-ATPμ200 μl of 5% dextrose,or (C) approximately 450 nanomoles liposomes directional, forming complex through electrostatic effects from about 30 μg of plasmid DNA that encodes a Raf-ATPμin mixture with about 20 molar excess of competing ligand for αvβ3-integrin about 200 μl of a 5% solution of dextrose. Measurement of tumor produced in the time intervals specified on Fig where the experiment (a) marked as "Raf(-)", (b) designated as a (non-directed action), (C) designated as "surplus". In addition, performed a control experiment in which the tumor is not subjected to any impact.

As shown in Fig, treatment of mice with melanoma with an initial tumor volume 300 mm3, liposomes is mi directional NP5, forming a complex with the mutant dominant negative form of Raf kinase (Raf-ATPμ), after a period of initial growth causes regression of tumor volume compared to control groups without treatment.

Example 6. Selectivity mediated by liposome directional delivery of angiogenic blood vessels

Approximately 300 nanomoles NP5 used for the formation of a complex with approximately 20 μg of plasmid DNA encoding green fluorescent protein (GFP), approximately 50 μl of 5% dextrose and administered in the form of intravenous injection in embryos of chickens, chorioallantoic membrane (CAM) which is pre-placed on the filter disk, saturated 1 mg/ml of bFGF, approximately 24 hours for the stimulation of angiogenesis. One day after injection of the complex fabric ITSELF is collected, washed with PBS, fixed in 4% solution of paraformaldehyde and examined for the presence of fluorescence. The results are presented on Fig.

GFP in a large number of localized in the vascular network ITSELF, as seen in the microphotographs shown in Fig.

Example 7. Apoptosis of vascular network and subsequent cell death resulting mediated by liposome directional delivery mutant Raf genes to tumor vascular network

Melanoma, not expressing αvβ3-integrin, is administered by subcutaneous inye the tion in the lateral part of the body of mice. The resulting tumors leaves grow to a volume of about 200 mm3, after which the mice in the form of intravenous injections administered about 450 nanomoles liposomes NP5, forming a complex with approximately 30 μg of plasmid DNA that encodes a Raf-ATPμor Shuttle vector, about 200 μl of a 5% solution of dextrose. The tumor is removed after about 72 hours, fixed in 4% solution of paraformaldehyde, make the cuts and paint factor von Willebrand's disease (a marker of blood vessels), and then examine the apoptosis of the cells by the TUNEL method to detect fragmented DNA (Intergen Corp., Purchase, NY).

On the microphotographs shown in Fig, it is evident that the tumor is exposed to nanoparticles/Shuttle vector (control experiment), have a relatively high density of vessels apoptosis in only a few cells. In contrast, the majority of vessels in tumors exposed to NP5/Raf-ATPμexpose apoptosis and large areas of the tumor are killed at a certain distance from apoptotic vessels, mainly due to insufficient blood supply.

Example 8. Selectivity mediated by liposome directional delivery of angiogenic blood vessels

Lateral branches of the superficial capillaries weave vascular network of the retina penetrate the retina and form the deep vascular the et between 8-m (R8) and 10th (P10) days after the birth of a mouse. In this study on day 10 (P10) after birth, the mice in the vitreous body is injected in the form of injection of the liposome directional NP5, forming a complex about 0.15 μg of plasmid DNA encoding green fluorescent protein, about 1 μl of a 5% solution of dextrose. Approximately 24 hours after injection NP5, forming a complex with the GFP gene, kill mice, retinal paint, using as immune labels associated with rhodamine antibodies against collagen IV (a marker of blood vessels), and examined using scanning microscopy and 2-photon laser to determine the relative level of GFP in the blood vessels of the retina.

On the microphotographs shown in Fig, shows that GFP in a large number of localized in the vascular network of the retina.

In the above-described embodiments of the invention and the examples can be made numerous changes or modifications not beyond the scope of this invention. Presents embodiments of the invention, as well as possible or assumed options do not have any restrictions. Appended claims include all such modifications, which are included in the scope of the claims.

1. Liposome targeted effect on receptor αvβ3-integrin, including cationic amphiphilic substance, including ,2-tileorasi-3-(N,N,N-ammonium)propachlor; neutral lipid; lipid directional, having a domain directional and hydrophobic domain associated with the domain of directed action; and nucleic acid forming a complex with a cationic lipid; however, the cationic lipid is present in an amount of from about 1 to about 50 mol.%, and the specified lipid directed action is present in an amount of from about 1 to about 20 mol.%, moreover, the molar percentage determined based on the total number of moles of lipid in the liposome, and the domain of directed action includes ones antagonist αvβ3-integrin containing 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethyloxy]-benzoyl-2-(S)-aminoethylamino-)β-alanine (compound 10), covalently linked to hydrophilic domain amide bond.

2. Liposome according to claim 1, characterized in that at least a portion of the lipids present in the liposome has functional groups which are crosslinked with each other.

3. Liposome according to claim 1, characterized in that the nucleic acid is a DNA.

4. Liposome according to claim 1, characterized in that the nucleic acid is a gene.

5. Liposome according to claim 1, characterized in that the nucleic acid is an antisense oligonucleotide sequence.

6. Liposome according to claim 1, characterized in that the nucleic acid is cobourn.

7. Liposome according to claim 1, characterized in that it has a particle size of not more than 250 nm.

8. Liposome according to claim 1, characterized in that it has a particle size in the range from about 40 to about 100 nm.

9. Liposome according to claim 1, characterized in that it has a particle size in the range from about 75 to about 100 nm.

10. Liposome according to claim 1, characterized in that it has a particle size in the range from about 40 km to about 65 nm.

11. Liposome according to claim 1, characterized in that it does not contain cross-linked lipids and has a particle size in the range from about 75 to about 100 nm.

12. Liposome according to claim 1, characterized in that it contains cross-linked lipids and has a particle size in the range from about 40 to about 65 nm.

13. Liposome according to claim 1, characterized in that the lipid directional and neutral lipid is at least partially crosslinked with each other, and cationic amphiphilic substance has no links.

14. Liposome according to claim 1, characterized in that it further comprises poly(ethylene glycol), having from about 250 to about 500 oxyethylene structural units.

15. Liposome according to 14, wherein the poly(ethylene glycol) has about 350 oxyethylene structural units.

16. Liposome according to claim 1, characterized in that it contains 1,2-tileorasi-3-(N,N,N-ammonium)propachlor, cholesterol and poly(ethylene glycol), respectively, in a ratio of about 1:1:0,12.

17. Liposome according to claim 1, trichomania fact, that the nucleic acid is able to Express the protein or peptide in the cell in which you entered this liposome.

18. Liposome according to 17, characterized in that the nucleic acid is able to Express a protein or peptide that suppresses angiogenesis.

19. Liposome according p, wherein the protein that suppresses angiogenesis, is a protein Raf.

20. The method of introduction of nucleic acid in αvβ3-integrin represents a cell, comprising contacting the specified cells with liposome targeted effect on receptor αvβ3-integrin, according to claim 1.

21. A method of suppressing angiogenesis comprising the administration to a subject in need of inhibition of angiogenesis a therapeutically effective amount of liposomes targeted effect on receptor αvβ3-integrin, according to claim 1, which is able to Express a protein or peptide that suppresses angiogenesis.

22. The method according to item 21, wherein the liposome is administered intravenously.

23. The method according to item 21, wherein the liposome is administered by injection into the tumor.

24. The method of suppressing growth of a tumor comprising the administration to a subject in need of inhibition of tumor growth, a therapeutically effective amount of liposomes targeted effect on receptor αvβ3-integrin according to claim 1, which can Express a protein or peptide that suppresses angiogenesis, including Raf protein.

25. The method according to paragraph 24, wherein the liposome is administered intravenously.

26. The method according to paragraph 24, wherein the liposome is administered by injection into the tumor.

27. Method of inducing apoptosis of endothelial cells of blood vessels, comprising contacting the endothelial cells of blood vessels causing apoptosis effective amount of liposomes targeted effect on receptor αvβ3-integrin, according to claim 1, which is able to Express the protein or peptide, inducing apoptosis, including Raf protein.



 

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FIELD: medicine; biology.

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EFFECT: enhanced effectiveness in treating pathological states with new blood vessel formation being suppressed.

7 cl, 8 dwg

FIELD: biochemistry, biotechnology, peptides.

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EFFECT: valuable biological and medicinal properties of polypeptide.

17 cl, 10 dwg, 13 ex

FIELD: pharmaceuticals.

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47 cl, 8 ex, 5 dwg

FIELD: gene engineering, in particular preparation based on PDGF-BB, useful in therapy, veterinary, diagnosis, tissue culturing.

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FIELD: genetic engineering, biotechnology, biochemistry, medicine, pharmacy.

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EFFECT: valuable medicinal properties of glycoprotein VI.

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FIELD: biotechnology, gene engineering, medicine.

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FIELD: immunobiotechnology.

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EFFECT: new preparation for treatment of immune system diseases.

65 cl, 19 dwg, 2 tbl, 2 ex

FIELD: medicine, biotechnology.

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EFFECT: new method for treatment of prostates cancer.

28 cl, 7 ex, 7 dwg

FIELD: molecular biology, genetic engineering, polypeptides, medicine.

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EFFECT: improved preparing method and valuable properties of polypeptide.

9 cl, 17 dwg, 3 tbl, 6 ex

FIELD: organic chemistry, chemical technology, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to novel derivative of variolin B of the general formula (I) or their pharmaceutically acceptable salts possessing antitumor activity. In the general formula (I) radical R1 means aromatic group representing aromatic group representing phenyl optionally substituted with nitro-group, amino-group or alkyl-substituted amino-group, or aromatic group represents 5-6-membered heterocycle with two nitrogen atoms or sulfur atom as heteroatoms optionally substituted with (C1-C12)-alkyl, -OH, unsubstituted amino-group or amino-group substituted with (C1-C4)-acyl, phenyl-(C1-C4)-alkyl wherein phenyl group can be substituted with -OR1, or (C1-C12)-alkylthio-group, (C1-C12)-alkyl- or phenylsulfonyl, (C1-C12)-alkyl- or phenylsulfinyl or -OR1 wherein R1 is chosen from (C1-C12)-alkyl or phenyl; R2 represents hydrogen atom; R3 represents oxo-group when a dotted line is between nitrogen atom to which R2 is bound and carbon atom to which R3 is absent, or R2 is absent when R3 represents optionally protected amino-group wherein a substitute is chosen from (C1-C4)-acyl, phenylsulfonyl and (C1-C4)-alkylphenylsulfonyl when a dotted line forms a double bond between nitrogen atom to which R2 is bound and carbon atom to which R2 is bound; R4 represent hydrogen atom. Also, invention relates to a method for synthesis of compounds of the invention and to intermediate substances for their realization. Also, invention relates to a pharmaceutical composition based on variolin B derivatives.

EFFECT: improved method of synthesis, valuable medicinal property of compounds and pharmaceutical composition.

22 cl, 5 sch, 1 tbl, 50 ex

FIELD: organic chemistry, medicine, oncology, biochemistry, pharmacy.

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EFFECT: valuable medicinal properties of compounds and pharmaceutical composition, improved method of treatment.

52 cl, 2 tbl, 505 ex

FIELD: medicine, biochemistry, pharmacy.

SUBSTANCE: invention describes dipeptide-nitrile inhibitors of cathepsin K, their pharmaceutically acceptable salts or their esters that are used in therapeutic or prophylaxis treatment of disease of morbid state mediated by cathepsin K.

EFFECT: valuable medicinal properties of inhibitors.

3 cl, 11 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention describes compound of the formula (U): or its pharmaceutically acceptable salt wherein X is chosen from -NR1, sulfur atom (S); Y1 and Y2 represent oxygen atom (O); Z represents O; m = 0 or 1; A is chosen from a direct bond, (C1-C6)-alkyl; R1 is chosen from hydrogen atom (H), alkyl; R3 and R6 are chosen independently from H, alkyl, halogenalkyl, heteroalkyl, cycloalkyl, aryl, cycloalkyl-alkyl, cycloalkyl-heteroalkyl, heterocycloalkyl-alkyl, alkylaryl, heteroaryl, arylalkyl, aryl-heteroalkyl, heteroaryl-alkyl, heteroaryl-heteroalkyl or heterocycloalkyl; R4 is chosen from H, alkyl; R5 represents a bicyclic or tricyclic group comprising two or three ring structure wherein each of that comprises from 3 to 7 ring atoms chosen independently from cycloalkyl, aryl, heterocycloalkyl or heteroaryl wherein each ring structure is joined with the next ring structure through a direct bond, through -O-, through -S-, through (C1-C6)-alkyl, through (C1-C6)-heteroalkyl, through (C1-C6)-alkynyl, through carboxy-(C1-C6)-alkyl, or it is condensed with the next ring structure wherein heteroalkyl represents heteroatom-substituted alkyl comprising one heteroatom chosen from N, O and S. Also, invention describes compounds of formulae (Ib), (Ic) and (Id) given in the invention description, pharmaceutical composition and using these compounds in preparing a medicine for using in treatment of disease or state mediated by one or more enzymes representing metalloproteinase. Represented compounds are useful as inhibitors of metalloproteinases and especially as inhibitors of MMP12.

EFFECT: valuable medicinal and biochemical properties of compounds and pharmaceutical composition.

17 cl, 3 tbl, 17 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

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EFFECT: valuable medicinal and biochemical properties of inhibitors and pharmaceutical compositions.

20 cl, 3 tbl, 6 ex

FIELD: medicine, oncology.

SUBSTANCE: method involves using an antitumor preparation prepared by using the apparatus-program complex (APC) "IMEDIS-TEST". Method involves blood sampling in a patient, its diluting in physiological solution in the ratio 1:1 followed by addition of the preparation "MP-antiprotein-blocker" and diluted blood in contour APC "IMEDIS-TEST". Then method involves effect with this preparation on blood in regimen "transfer" followed by returning blood in patient body. Then method involves carrying out treatment involving detoxification of body in 1, 14, 21, 28, 35 and 42 days of treatment using Glauber's salt (sodium sulfate decahydrate) as a laxative agent and also cleansing enema and fractional dose of citrus juice (40-50 ml) in the ratio 1:1 with water for 10-12 h and aqueous-turpentine bath. Then method involves carrying out the monocarrot diet for 12 days from 2-d to 13-th day of treatment inclusively; basic nutrition - each 6 days after detoxification comprising: a dose of olive oil before eating (30-35 g) with lemon juice in the ratio 1:1, in 25-30 min a dose of 100-120 ml of carrot-beet juice in the ratio 1:1, in 25-30 min a dose of 200-250 ml kefir infusion prepared from crude buckwheat grain, and a dose of 0.3 g of pepsin or 10-15 ml of pepsidil, a dose of 3.0-5.0 ml of horse radish roots infusion with lemon juice, a dose of 50-60 ml of medicinal herbs aqueous species. Method provides antitumor effect based on disturbance of cancer cells nutrition and normalization of body normal cells nutrition and recovery of metabolism and detoxification of the patient body. Invention can be used in treatment of malignant tumors of different localization and development stage.

EFFECT: enhanced effectiveness of diagnosis and treatment.

1 tbl, 2 ex

FIELD: medicine, oncology, chemical-pharmaceutical industry.

SUBSTANCE: invention relates to a pharmaceutical composition designated for treatment of tumors and tumor metastasis. The composition comprises: (i) at least one antibody or its functionally intact derivative comprising a binding site that binds with the epitope ErbB1(Herl1) of receptor and (ii) at least one agent inhibiting angiogenesis being optionally in common with a pharmaceutically acceptable carrier, diluting agent or excipient used in the combined therapy in treatment of tumors and tumor metastasis. Using the proposed composition can result to the possible synergetic enhancing inhibitory effect of each specific therapeutic agent with respect to inhibition of tumor cells proliferation and providing the enhanced effective treatment as compared with individual agents using separately.

EFFECT: enhanced effectiveness of therapy.

25 cl, 1 ex

FIELD: medicine, oncology, pharmaceutical industry, pharmacy.

SUBSTANCE: invention relates to an antitumor agent. Anti-tumor liposomal preparation based on a mixture of flavolignans or their extracts prepared from spotted milk thistle (Silybium) fruits is made in liposomal formulation with particles size 100-500 nm. The preparation contains additionally vector component of peptide nature bound covalently to lipids of liposome envelope. As a vector component of peptide nature protein is used chosen from the group of representatives of family factors of epithelium growth, transforming growth factors, oncofetal proteins, endothelium growth factors and fibroblast growth factors, or the group active peptide fragments of protein said above. Above described preparation possesses the enhanced antitumor effect and its delivery rate to a tumor target-cell is increased. Invention can be used as an antitumor agent.

EFFECT: valuable medicinal property of preparation.

1 tbl, 2 ex

FIELD: organic chemistry, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to a novel using compounds representing derivatives of pyroindolone for preparing medicaments useful as anticancer medicaments.

EFFECT: valuable medicinal properties of derivatives.

4 cl, 1 tbl, 3 ex

FIELD: medicine, oncology, pharmacy.

SUBSTANCE: invention describes a medicament used in treatment of liver disease by prevention of invasion onset of portal vein. This agent represents the effective medicament containing menatetrenone as an active component used in treatment/prophylaxis of liver disease. Indicated agent used in treatment/prophylaxis of liver disease is the effective medicament against liver cancer, in particular, against DCP (des-γ-carboxyprothrombin)-positive liver cancer. Agent used in aims for treatment/prophylaxis of liver disease contains menatetrenone as an active component, it improves prognosis significantly after carrying out the anticancer therapy and possesses the excellent effect directed on prevention of liver cancer relapses.

EFFECT: valuable medicinal property of agent.

4 cl, 2 tbl, 10 dwg, 2 ex

FIELD: pharmaceuticals and/or cosmetic compositions for treatment of localized obesity and cellulites.

SUBSTANCE: claimed compositions contain as active ingredients combination of a) complex of escin/beta-sitosterol with phospholipids; b) complex of dimer flavonoids Gingko biloba with phospholipids; c) complex of triterpenes centella asiatica with phospholipids and optionally d) ethylxymeninate and/or standardized Coleus forskolii extract. Also disclosed is method for cosmetic treatment of localized obesity and cellulites.

EFFECT: effective compositions for treatment of localized obesity and cellulites.

5 cl, 2 ex

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