Complexes of nucleic acid ligands to vascular endothelial growth factor (vegf)

 

(57) Abstract:

The invention relates to purified and dedicated not naturally occurring RNA ligands to vascular endothelial growth factor (VEGF) (indicated oligonucleotide sequence). Described complexes of the formula a-b-Y, where a is polyalkyleneglycol or glycerolipid, In - linker(s), Y - RNA ligands to VEGF. Describes how to obtain these complexes, including the identification of RNA-ligand from a mixture of Candidate Nucleic Acids having an increased affinity to VEGF, as well as lipid structure, which includes this complex. The complexes according to the invention improves the pharmacokinetic properties of RNA ligands to VEGF and is used for treatment of VEGF-mediated diseases, including inhibition of VEGF-mediated angiogenesis, tumor growth and macular degeneration. There are also ways of reducing the clearance of plasma Nucleic Acid Ligand to VEGF, a way of prolonging its action in the eye, and the way the direction of therapeutic or diagnostic agent to a biological target. 15 C. and 62 C. p. F.-ly, 39 ill. , 13 tables.

Describes highly affine 2 for(2-F')-pyrimidine-RNA ligands to vascular endothelial growth factor (VEGF). Use the ion of Ligands by Exponential enrichment. Further in this invention is included a method of obtaining a therapeutic or diagnostic Complex comprised of a Nucleic Acid-Ligand VEGF and non-immunogenic compounds of High Molecular Weight or Lipophilic Compounds through the identification of Nucleic Acid-Ligand VEGF using the SELEX methodology and covalent crosslinking of Nucleic Acid-Ligand VEGF with non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound. Further, the invention includes Complexes consisting of one or more than one Nucleic Acid-Ligand VEGF and non-immunogenic compounds of High Molecular Weight or Lipophilic Compounds. In addition, the invention relates to the improvement of the pharmacokinetic properties of Nucleic Acid-Ligand VEGF through covalent crosslinking of Nucleic Acid-Ligand VEGF with non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound to form a Complex. Further, the invention relates to the improvement of the pharmacokinetic properties of Nucleic Acid-Ligand VEGF through the use of Lipid Constructs containing the Nucleic Acid-Ligand VEGF or Complex containing Nucleic Ago, this invention relates to a method for directing a therapeutic or diagnostic agent to a biological target, which expresses VEGF, by connecting this agent with a Complex comprised of a Nucleic Acid-VEGF Ligand and a Lipophilic Compound or non-immunogenic compounds of High Molecular Weight, and this Complex is additionally connected with the Lipid Structure and Nucleic Acid-Ligand VEGF additionally connected to the outer side of the Lipid construct.

Background of the invention

A. SELEX

For many years the dogma was that nucleic acids are primarily informational role. By the way, is known as the systematic allocation of ligands by exponential enrichment (Systematic Evolution of Ligands by Exponential enrichment), called SELEX, it became clear that nucleic acids, like proteins, are three-dimensional structural diversity. SELEX is a method of in vitro selection of Nucleic Acid molecules with highly specific binding by target molecules and is described in Application for U.S. patent, serial N 07/536428, filed June 11, 1990, entitled "Systematic Evolution of Ligands by Exponential Enrichment, now withdrawn, the UNT USA N 5475096, in the Application for U.S. patent, serial N 07/931473, filed August 17, 1992, entitled "Method for Identifying Nucleic Acid Ligadns", now U.S. patent N 5270163 (see also WO 91/19813), each of which is specifically incorporated herein by reference. In each of these applications are collectively referred to here as the patent Application SELEX, describes a fundamentally new method of obtaining a Nucleic Acid, a Ligand for any desired molecule target. The SELEX method provides a class of products that are referred to as Nucleic Acid Ligands, each ligand has a unique sequence, and which have the property of specific binding to the desired connection-target or desired molecule-target. Each identified by SELEX Nucleic Acid Ligand is a specific ligand of the connection-target or the target molecules. SELEX is based on a unique idea that nucleic acids have sufficient capacity for the formation of many two - and three-dimensional structures and sufficient chemical versatility, possible within their monomers to act as ligands (form specific binding pairs) with virtually any chemical with the VA.

The SELEX method involves selection from a mixture of oligonucleotides candidate step and repeat binding, partitioning and amplification, using the same General pattern of selection, to achieve virtually any desired criterion of affinity and selectivity of binding. Starting from a mixture of Nucleic Acids, preferably containing a segment of randomized sequence, the SELEX method includes the stage of bringing into contact of the mixture with the target under conditions favorable for binding, separating unbound Nucleic Acids from those Nucleic Acids that specifically contacted by target molecules, the dissociation of Complexes of Nucleic Acid-target amplification Nucleic Acids dissociated from the Complexes of Nucleic Acid-target, obtaining a mixture of Nucleic Acids enriched ligands, then repeat once more stages of binding, partitioning, dissociating and amplifying for so many cycles, how many it is desirable to obtain high-affinity high affinity Nucleic Acid Ligands to the molecule target.

The authors of the present invention shows that the SELEX method demonstrates that nucleic acid as a chemical connection is okomu range of binding and the functions, other than those, which Express the nucleic acid in biological systems.

The authors of the present invention showed that SELEX or similar SELEX methods can be used to identify nucleic acids that may contribute to any selected reaction path, similar to the one in which you can identify Nucleic Acid Ligands for a given target. The authors present invention postulate that theoretically within the Mix of Candidates from about 1013up to 1018Nucleic Acid exists at least one Nucleic Acid of a suitable form to facilitate each of the wide variety of physical and chemical interactions.

The basic SELEX method was modified to achieve some specific goals. For example, in the Application for U.S. patent, serial N 07/960093, filed October 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure", describes the use of SELEX with gel electrophoresis to select Nucleic Acid molecules with specific structural characteristics, such as bent DNA. In the Application for U.S. patent, serial N 08/123935, filed September 17, 1993, entitled "Photoselection of Nucleic Acid Ligands", podobnie to bind and/or to cross-fotostation and/or to photoinactivation of a target molecule. In the Application for U.S. patent, serial N 08/134028, filed October 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine", now U.S. patent N 5580737, describes a method for identifying highly specific Nucleic Acid Ligands able to discriminate between closely related molecules, which may not be peptides, called Counter-SELEX. In the Application for U.S. patent, serial N 08/143564, filed October 25, 1993, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX", now U.S. patent N 5567588, describes a method based on SELEX, which achieves highly efficient partitioning between oligonucleotides having high and low affinity to the molecule target.

The SELEX method is the identification of high affinity Nucleic Acid Ligands containing modified nucleotides, giving the ligand advanced features, such as improved stability in vivo or improved delivery characteristics. Examples of such modifications include chemical substitutions in the positions of the ribose and/or phosphate and/or base. Identified by SELEX Nucleic Acid Ligands containing modified nucleotides are described in the patent Application is time the U.S. patent N 5660985, which describes oligonucleotides containing derivatives of nucleotides, chemically modified at the 5 - and 2'-positions of pyrimidines. In the Application for U.S. patent, serial N 08/134028, supra, describes highly specific Nucleic Acid Ligands containing one or more than one nucleotide, modified 2'-amino (2'-NH2), 2'-fluorescent(2'-F) and/or 2'-O-methyl (2'-OMe). In the Application for U.S. patent, serial N 08/264029, filed June 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2'-Modified Nucleosides by Intramolecular Nucleophilic Displacement", describes oligonucleotides containing various 2'-modified pyrimidines.

The SELEX method is to link the selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in Application for U.S. patent, serial N 08/284063, filed August 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. patent N 5637459, and in the Application for U.S. patent, serial N 08/234997 filed April 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX", respectively. These applications provide the possibility of combining a wide variety of shapes and other properties, and the properties of the effective amplification and replication oligomernykh Nucleic Acid Ligands with Lipophilic Compounds or non-immunogenic Compounds of High Molecular Weight in a diagnostic or therapeutic Complex, as described in Application for U.S. patent, serial N 08/434465, filed may 4, 1995, entitled "Nucleic Acid Complexes". Nucleic Acid Ligands VEGF, which are connected with a Lipophilic Compound, such as diacylglycerol or dialkylglycerol, diagnostic or therapeutic Complex, described in patent Application U.S. serial N 08/739109, filed October 25, 1996, entitled "Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes". Nucleic Acid Ligands VEGF, which are connected with the non-immunogenic Compound with a High Molecular Weight, such as polyethylene Glycol, or a Lipophilic Compound, such as Glycerolipid, a phospholipid or glyceroglycolipid, diagnostic or therapeutic Complex, described in patent Application U.S. serial N 08/897351, filed July 21, 1997, entitled "Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes". Each of the above patent Applications which describe the modification of the basic SELEX methodology, specifically incorporated herein fully by reference.

B. LIPID DESIGNS

Lipid bikinie vesicles are closed, fluid-filled microscopic spheres, which are formed primarily of individual molecules that have a polar (hydrophilic) and nepalitano, amino, hydroxy, Kalinovo or other polar groups. Examples of lipophilic groups are saturated or unsaturated hydrocarbons, such as alkyl, alkenyl, or other lipid groups. Sterols (e.g. cholesterol) and other pharmaceutically acceptable adjuvants (including antioxidants like alpha-tocopherol) may also be included to improve the stability of the vesicles or impart other desired characteristics.

Liposomes are a subset of these belayneh vesicles and contain mainly of phospholipid molecules that have two hydrophobic tail consisting of fatty acid chains. Upon exposure to water, these molecules spontaneously line up with the formation of spherical belayneh membranes with lipophilic ends of these molecules in each layer are connected in the center of the membrane, and the polar opposite ends forming respective inner and outer surfaces of this bilayer membranes (membranes). Thus, each side of the membrane is a hydrophilic surface, while inside the membrane is a lipophilic environment. These membranes can be arranged in several concentric spherical membranes, split tone is elainie vesicles (MLV) can be turned into a small or single-layer Vesicles (UV) when the application of force hydrodynamic fragmentation.

Therapeutic applications of liposomes includes the delivery of medicines, which are usually toxic in the free form. In the form of liposomes toxic drug is private and may not affect tissues that are sensitive to this drug, and delivered to selected topics. Liposomes can also be used therapeutically to release drugs over a long period of time, reducing the frequency of administration. In addition, liposomes can provide a way to obtain aqueous dispersions of hydrophobic or amphiphilic drugs, which are usually unsuitable for intravenous administration.

To many drugs and imaging agents had a therapeutic or diagnostic potential, they need to be delivered to the correct location in the body, and thus, the liposome can be easily injected, which creates the basis for long-term release and delivery of drugs to specific cells types or parts of the body. You can use several methods for application of liposomes for sending the encapsulated drug is napoliroma size of liposomes, their surface charge and by their introduction. MLV, primarily due to the fact that they are relatively large, usually quickly absorbed by the reticuloendothelial system (mainly liver and spleen). On the other hand, found that UV demonstrate increased circulation time, reduced speed clearance and greater biological distribution relative to the MLV.

Passive delivery of liposomes involves the application of various routes of administration, such as intravenous, subcutaneous, intramuscular and local. Each path gives the differences in localization of liposomes. Two common methods that are used for the active direction of liposomes to selected areas of the target, include adherence to the surface of liposomes or antibody or a specific receptor ligands. It is known that antibodies have a high specificity to their corresponding antigen, and they were attached to the surface of liposomes, but the results in many cases were less than successful. Some efforts, however, were successful in the direction of liposomes to tumors without the use of antibodies, see , for example, U.S. patent N 5019369, U.S. patent N 5441745 or U.S. patent N 5435989.

Area development, which are energetically for what we cells and moreover, in the core. This is especially important for delivery of biological agents, such as DNA, RNA, ribozymes and proteins. Promising therapeutic research in this area involves the application of antisense oligonucleotides DNA and RNA for the treatment of the disease. However, one of the main problems encountered on the way of effective application of antisense technology is that oligonucleotides in their fosfomifira form quickly broken down in the body fluids, as well as intracellular and extracellular enzymes such as endonucleases and ectonucleoside before reaching the target cells. Intravenous injection also causes rapid kidney clearance from the bloodstream, and the absorption is insufficient for obtaining the effective intracellular concentration of the drug. Liposomal encapsulation protects the oligonucleotides from splitting enzymes, increases the half-life in the circulation and increases the efficiency of absorption as a result of phagocytosis of liposomes. Thus, the oligonucleotides are able to achieve their desired targets and delivered to cells in vivo.

It was reported a few cases, when the researchers added antissa. The antisense oligonucleotides, however, are effective only as intracellular agents. The antisense oligodeoxyribonucleotide, which target the receptor for epidermal growth factor (EGF), encapsulated in liposomes associated with folate through polietilenglikolya the spacer (folate-PEG-liposomes) were delivered to cultured KB cells via receptor-mediated endocytosis of folate (Wang et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3318-3322). In addition, the oligonucleotides were attached alkylenedioxy (Weiss et al. , U.S. patent N 5245022). In addition, the literature describes the Lipophilic Compound covalently attached to the antisense oligonucleotide (EP V).

Download biological agents in liposomes can be accomplished by incorporating the drug in the lipid or loading into preformed liposomes. Described passive zakalivanie Oligopeptide and oligosaccharide ligands in the external surface of liposomes (Zalipsky et al. (1997) Bioconjug. Chem. , 8: 111: 118).

Century VEGF

The growth of new blood vessels from an existing vascular endothelium (angiogenesis) in healthy adults is strictly controlled by the opposite effects of positive and negative regulators. Under certain pathological conditions in which angiogenesis contributes to the progression of the disease (review Folkman (1995) Nature Medicine 1: 27-31). The idea that cancer angiogenesis is a limiting speed stage of tumor growth and metastasis (Folkman (1971) New Engl. J. Med. 285: 1182-1186), currently supported by numerous experimental data (surveys Aznavoorian et al. (1993) Cancer 71: 1368-1383; Fidler and Ellis (1994) Cell 79: 185-188; Folkman (1990) J. Natl. Cancer Inst. 82: 4-6).

The number of blood vessels in tumor tissue is a strong negative prognostic markers in breast cancer (Weidner et al. (1992) J. Natl. Cancer Inst. 84: 1875-1887), prostate cancer (Weidner et al. (1993) Am. J. Pathol. 143: 401-409), brain tumors (Li et al. (1994) Lancet 344: 82-86) and melanoma (Foss et al. (1996) Cancer Res. 56: 2900-2903).

It turns out that a number of angiogenic growth factors described to date, among which is the vascular endothelial growth factor (VEGF), play a key role as a positive regulator of physiological and pathological angiogenesis (review in Brown et al. (1996) Control of Angiogenesis (ed Goldberg and Rosen) Birkhauser, Basel printing: Thomas (1996) J. Biol. Chem. 271: 603-606). VEGF is a secretory disulfide linked glycosilated, which selectively stimulates endothelial cell proliferation, migration and production of enzymes that destroy the matrix (Conn et al. (1990) the OEWG. Natl. Aciophys. Res. Commun. 181: 902-906; Unemori et al. (1992) J. Cell. Physiol. 153: 557-562), all of these processes are required for the formation of new blood vessels. Besides the fact that VEGF is the only known mitogen specific for endothelial cells, it is unique among angiogenic growth factors in its ability to induce blood vessel, a temporary increase in its permeability to macromolecules (hence its original and alternative title - vascular permeability factor, VPF) (Dvorak et al. (1979) J. Immunol. 122: 166-174; Senger et al. (1983) Science 219: 983-985; Senger et al. (1986) Cancer Res. 46: 5629-5632). Increased vascular permeability and, as a result, the deposition of plasma proteins into the extravascular space contribute to the formation of new blood vessels by creating a temporary matrix for the migration of endothelial cells (Dvorak et al. (1995) Am. J. Pathol. 146: 1029-1039). Sorprendentemente in fact is characteristic of new blood vessels, including those associated with tumors (Dvorak et al. (1995) Am. J. Pathol. 146: 1029-1039). In addition, it is now known that compensatory angiogenesis induced by tissue hypoxia is mediated VEGF (Levy et al. (1996) J. Biol. Chem. 2746-2753); Shweiki et al. (1992) Nature 359: 843-845).

VEGF is found in four forms (VEGF-121, VEGF-165, VEGF-189, VE4). Two smaller forms are capable of diffusion, whereas two large forms are mainly localized on the cell membrane as a consequence of their high affinity to heparin. VEGF-165 also binds to heparin and is the most common form. VEGF-121, the only form that does not bind to heparin, as it turns out, has a lower affinity to the receptor (Gitay-Goren et al. (1996) J. Biol. Chem. 271: 5519-5523), as well as lower mitogenic activity (Keyt et al. (1996) J. Biol. Chem. 271: 7788-7795). Biological effects of VEGF-mediated two tyrosinekinase receptors (Flt-1 and Flk-1/KDR), the expression of which is highly restricted to cells of endothelial origin (de Vries et al. (1992) Science 255: 989-991; Millauer et al. (1993) Cell 72: 835-846; Terman et al. (1991) Oncogene 6: 519-524). Although high affinity binding requires the expression of both functional receptor, a chemotactic and mitogenic signaling in endothelial cells, as it turns out, is carried out primarily through the KDR receptor (Park et al. (1994) J. Biol. Chem. 269: 25646-25654; Seetharam et al. (1995) Oncogene 10: 135-147; Waltenberger et al. (1994) J. Biol. Chem. 26988-26995). The importance of VEGF and VEGF receptors for the development of blood vessels recently demonstrated in mice, which lack the 5) 376: 66-70) or Flk-1 genes (Shalaby et al. (1995) Nature 376: 62-66). In each case there was a serious anomaly in the formation of blood vessels, leading to embryonic lethality.

VEGF is produced and secreted in varying quantities virtually all tumor cells (Brown et al. (1997) Regulation of Angiogenesis (ed Goldberg and Rosen) Birkhauser, Basel, pp. 233-269). Direct evidence that VEGF and its receptors contribute to tumor growth, was recently obtained by the demonstration that the growth of xenografts of human tumor in nude mice can be inhibited by a neutralizing antibody to VEGF (Kim et al. (1993) Nature 362: 841-844), expression of a dominant-negative VEGF receptor flk-1 (Millauer et al. (1996) Cancer Res. 56: 1615-1620; Millauer et al. (1994) Nature 367: 576-579), low molecular weight inhibitors of Flk-1 tyrosinekinase activity (Strawn et al. (1996) Cancer Res. 56: 3540-3545) or by expression of antisense sequences to the mRNA of VEGF (Saleh et al. (1996) Cancer Res. 56: 393-401). It is important that the spread of tumor metastases were found, also dramatically reduced VEGF antagonists (Claffey et al. (1996) Cancer Res. 56: 172-181).

Inhibitors of VEGF, in addition to their use as anti-cancer agents, can be useful in a wide variety of proliferative diseases characterized by excessive angiogenesis, including psoriasis, Glo types of tumors secrete VEGF, until recently it was shown that any of them expresses a functional VEGF receptors. It was shown that cells of Kaposi's sarcoma (S) not only produce copious quantities of VEGF, but also Express functional VEGF receptors and, therefore, used for VEGF autocrine growth. Kaposi's sarcoma is usually treated using conventional antimetabolites medicines. However, the main disadvantage of the use of chemotherapy in patients with KS is concomitant induction of immunosuppression, which has serious consequences for patients whose immune systems are already at risk. The need for alternative therapies is especially great in the early stages of the disease, when the KS lesions begin to appear, but in other respects patients feel completely healthy. Recently it is proved that in this respect the encapsulation of chemotherapeutic drugs, such as daunorubicin (daunorubicin) liposome is a promising way to minimize the side effects of chemotherapy while maintaining antitumor efficacy. Drugs with low toxicity, which are selectively routed to the activated cells endodermally great value in the treatment of S.

Other areas of possible clinical application of Nucleic Acid-Ligand VEGF is an eye disorder characterized by excessive angiogenesis. Examples of such diseases are related macular degeneration and diabetic retinopathy. When macular degeneration progressive angiogenesis in the choroid underneath the macula (part of the retina responsible for the highest visual acuity) is harmful to eyesight. In diabetic retinopathy, angiogenesis in the retina harm the eyesight. Although the initial stimuli that initiate the growth of blood vessels in macular degeneration and diabetic retinopathy, currently unknown, VEGF, as it turns out, is a key inducer of angiogenesis (Lopez, P. F. et al. (1996) Invest. Ophthalmol. Visual Science 37, 855-868; Kliffen, M. et al. (1997) Br. J. Ophthalmol. 81, 154-162; Kvanta, A. et al. (1996) Invest. Ophthalmol. Visual Science 37, 1929-1934; Paques et al. (1997) Diabetes & Metabolism 23: 125-130). Inhibitors of VEGF, thus, may be useful to attenuate angiogenesis in macular degeneration.

The invention

Here, we describe a highly affine 2 for(2'-F) modified-pyrimidine-RNA ligands to vascular endothelial growth factor (VEGF). The method used here for the ID manually is, described here were selected from the original pool of about 1014RNA molecules, randomized 30 or 40 related provisions. In the present invention enabled the selected ligands, which are presented in tables 2-6. Further, in the present invention is included a method of obtaining a Complex consisting of a Nucleic Acid-Ligand VEGF and non-immunogenic compounds of High Molecular Weight or Lipophilic Compounds, using a method in which identify Nucleic Acid Ligand from a Mixture of Candidate Nucleic Acids, where the Nucleic acid is a ligand VEGF, by using method (a) bringing into contact the Mixture of Candidate Nucleic Acids with VEGF, (b) separation of members of the above-mentioned Mixture of the Candidates on the basis of affinity to VEGF and (C) amplifying the selected molecules with a mixture of Nucleic Acids, enriched with sequences of Nucleic Acids with relatively higher affinity for binding to VEGF, and covalent-linkage specified identified Nucleic Acid Ligand to VEGF with a non-immunogenic Compound of High Molecular Weight or Lipophilic Compound. Further, the invention includes a Complex consisting of a Nucleic Acid-Ligand VEGF and Aimmungen the AET in itself Lipid Structure, containing the Nucleic Acid-Ligand VEGF or Complex. In addition, the present invention relates to a method for producing Lipid Structure containing the Complex consists of a Nucleic Acid-Ligand VEGF and Lipophilic Compounds.

In another embodiment of the present invention proposes a method of improving the pharmacokinetic properties of Nucleic Acid-Ligand VEGF by covalent crosslinking of Nucleic Acid-Ligand VEGF with non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound to form a Complex, and this Complex patient. Further, the invention relates to a method for improving the pharmacokinetic properties of Nucleic Acid-Ligand VEGF by additional compounds of this Complex with the Lipid Construct.

The present invention is the development of Complexes containing one or more than one Nucleic Acid-Ligand VEGF in combination with one or more non-immunogenic Compound with a High Molecular Weight or a Lipophilic Compound, and methods for their preparation. A further object of the present invention is the development of Lipid Structures containing etotal Acid-Ligand VEGF in combination with one or more non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound with improved pharmacokinetic properties.

In embodiments of the invention directed to complexes consisting of a Nucleic Acid-Ligand VEGF and non-immunogenic compounds of High Molecular Weight, it is preferable that the non-immunogenic Compound of High Molecular Weight is polyalkyleneglycol, more preferably polyethylene glycol (PEG). More preferably, the PEG has a molecular mass of about 10-80K. Most preferably, the PEG has a molecular mass of about 20-C. In embodiments of the invention directed to complexes consisting of a Nucleic Acid-Ligand VEGF and Lipophilic Compounds, it is preferred that the Lipophilic Compound is glycerolipid. In preferred embodiments of the invention the Lipid Structure preferably is a lipid bishojou vesicles, and most preferably a liposome. In the preferred embodiment the Nucleic Acid-Ligand VEGF identified according to the SELEX method.

In embodiments of the invention directed to complexes consisting of a non-immunogenic compounds of High Molecular Weight or Lipophilic Compounds covalently cross-linked to Nucleic Acid-Ligand or ligands VE is th, Nucleic Acid-Ligand VEGF may be connected through a Covalent or Non-covalent Interactions with the Lipid Construct without being part of the Complex.

In addition, in the embodiments of the invention directed to lipid constructs containing the Nucleic Acid-Ligand VEGF or Complex non-immunogenic Compound of High Molecular Weight or Lipophilic Compound/Nucleic Acid Ligand to VEGF, where the Lipid Structure is a structure of this type, which has a membrane that defines an internal compartment, such as lipid Bologna the vesicles, Nucleic Acid-Ligand VEGF or Complex in connection with the Lipid Structure can be connected with the membrane Lipid Structure or encapsulated within compartment. In embodiments where Nucleic Acid-Ligand VEGF is connected with the membrane, Nucleic Acid-Ligand VEGF can connect with oriented inside or oriented to the outside part of the membrane, so that the Nucleic Acid Ligand to VEGF acts inside or outside of the vesicles. In some embodiments, the Complex of Nucleic Acid-Ligand VEGF can be passively loaded into the external side of the preformed AI, Nucleic Acid-Ligand VEGF can serve to guide ability.

In embodiments where Nucleic Acid-Ligand VEGF Lipid Structure serves to guide the ability, Lipid Structure may be combined with additional therapeutic or diagnostic agents. In one embodiment of this therapeutic or diagnostic agent is connected to the outer side of the Lipid Structure. In other embodiments, this therapeutic or diagnostic agent encapsulated in the Lipid Structure or connected with the inner side of the Lipid Structure. In yet another additional embodiment of this therapeutic or diagnostic agent coupled with Complex. In one embodiment of this therapeutic agent is a drug. In an alternate embodiment of this therapeutic or diagnostic agent is one or more than one additional Nucleic Acid-Ligand.

A further object of the present invention is to develop a method of inhibiting angiogenesis by administration of a Nucleic Acid Ligand to VEGF or a Complex consisting of a Nucleic Acid-Ligand VEGF and Neimongol the th Complex of the present invention. Another further object of the present invention is to develop a method of inhibiting the growth of tumors by administration of a Nucleic Acid Ligand to VEGF or a Complex consisting of a Nucleic Acid-Ligand VEGF and non-immunogenic compounds of High Molecular Weight or Lipophilic Compounds, or Lipid Structures containing Complex of the present invention. Another further object of the present invention is to develop a method of inhibition of Kaposi's sarcoma through the introduction of a Nucleic Acid Ligand to VEGF or a Complex consisting of a Nucleic Acid-Ligand VEGF and non-immunogenic compounds of High Molecular Weight or Lipophilic Compounds, or Lipid Structures containing Complex of the present invention. Another further object of the present invention is to develop a method of inhibiting macular degeneration through the introduction of a Nucleic Acid Ligand to VEGF or Complex containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound or Lipid Structures containing Complex of the present invention. Another further for the PTO introduction of Nucleic Acid-Ligand VEGF or Complex, containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound or Lipid Structures containing Complex of the present invention.

The next task of the present invention is to develop methods to direct therapeutic or diagnostic agent to a biological target, which expresses VEGF, by combining the agent with a Complex containing the Nucleic Acid-VEGF Ligand and a Lipophilic Compound or non-immunogenic Compound of High Molecular Weight, and the Complex is connected with the Lipid Structure and Nucleic Acid-Ligand VEGF additionally connected with the outside of the Lipid Construct.

These and other tasks, as well as the nature, scope and application of this invention will become readily apparent to experts from the further description and the attached claims.

A brief description of graphic materials

In Fig. 1A-1Q shows the molecular image NX213 (Fig. 1A), NX278 (Fig. 1B), scNX278 (Fig. 1C), scNX213 (Fig. 1D), NX31838-PL (Fig. 1E), NX31838 lipidomic 1 (Fig. 1F), NX31838 lipidomic 2 (Fig. 1G), NX31838-40K PEG (Fig. 1H), NX31838-20K PEG (Fig. 1I), NX31838-40K PEG dimer without linker (NX31838d0) (Fig. 1J is nker (Fig. 1M), glycerolphosphate linker (Fig. 1N), 18-atom spacer elements linker (Fig. 1O), aminoethylaminomethyl linker (Fig. 1R), 3'3'dT (Fig. 1Q) and NX31917 (Fig. 1R). 5 fosfata group of the ligand is shown in Fig. mPEG means metropolitanpoker. The letter in the lower position, preceding nucleotide, shows the following: m= 2'-O-methyl, a= 2'-amino, r= RIBO and f= 2'-fluorescent. Nucleotide without a preceding letter shows deoxyribonucleotide (2 N). 3'3'-dT shows 3'3' inverted fosfodiesterazu communication 3 conze. S after nucleotide means the modification of the skeleton, consisting in phosphothioate magnolioideae connection.

In Fig. 2 shows the binding properties of various Nucleic Acid-Ligand relative to VEGF. The affinity of binding of unmodified Nucleic Acid-Ligand (NX213, where there's no shading circle), its dialkylglycerol-modified analogue of (NX278, where there's no shading diamond) and liposomal NX278 (NX278-L, where there's no shading square) in parallel with mixed sequence (scrambled) control (sc) (scNX213 shaded circle; scNX278 shaded diamond and scNX278-L, the shaded square) were determined using a competitive analysis of the shift in electrophoretic mobility. NX213 is a

5'-TsTUaCmAaCmGTsTsTsTsT-3' (SEQ ID NO: 4)

32P 5= tagged on the end NX213 (1.5 nm) were incubated in binding buffer (saline, phosphate buffered with 0.01% of serum human albumin) at 37oC for 20 min in the presence of VEGF (0,33 nm) and competitive oligonucleotide (5 PM is 0.33 μm). Complex32P NX213/VEGF was separated from free 32P NX213 by electrophoresis in 8% polyacrylamide gel (19: 1 acrylamide: bis-acrylamide, Tris-borate, 89 mm, 1mm EDTA as a buffer for mileage). The intensity of the band corresponding to the Complex32P NX213/VEGF with varying concentrations of competitor, was determined quantitatively by analyzing the phosphate imaging unit. The data, normalized to the amount of Complex formed in the absence of competitor, has led to competitive binding equation using the method of least squares.

In Fig. 3 shows the effects of different Nucleic Acid Ligands to the induced VEGF increase vascular permeability. VEGF (20 nm) with Nucleic Acid-Ligand or without them were intradermally injected with Guinea pigs that received an injection of blue dye Evans. Leakage of dye was quantified by measuring autoserum cell growth S. Cell growth KSY-1 in the presence of different concentrations NX213, NX278-L and scNX278-L. Cells KSY-1 were sown on 24-hole tablets at a density of 1104cells/well on 0 day. A fresh environment, processed identically substituted for 1 and 3 days. The number of cells was determined using trypsinization cells for 5 or 6 days of culture using the particle counter. The experiments were made in triplicate several times. Results presented are the average and SE (mean square error) of a representative experiment.

In Fig. 5A and 5B shows that NX278 inhibits the growth of cells S in Nude mice. Nude mice implanted tumor KS behind the front legs on day 1. Mice were treated NX278-L (50 μg/day/mouse, Fig. 5A, and 150 μg/day/mouse, Fig. 5B) by intraperitoneal injection daily for five days, starting on day 2. Control mice were treated with empty liposomes using the same amount of lipids, and that in the group treated with Nucleic Acid-Ligand. Tumor size was measured over a period of two weeks. Tumors were removed on day 14 and measured.

In Fig. 6 summarizes the data on plasma concentrations NX31838 20K PEG 40K PEG NX31838 (minus PEG) as a function of time after bpole bolus injection.

In Fig. 8A-8D shows the changes in vascular permeability induced by intradermal injection of VEGF protein (0.8 pmol), Nucleic Acid-Ligand/monoclonal antibody, as indicated. Local transudate blue dye Evans was determined 30 minutes after injection using the transillumination collected skin. In Fig. A, b, C and D shows the influence mixed with protein NX31838-20K PEG, NX31838-40K PEG, NX31838-PL or NX31838d2-40K PEG 30 minutes before injection. Values represent mean SEM (mean square error). *P<0.05 compared with one VEGF. Cm. molecular images in Fig. 1.

In Fig. 9A-9C shows the evaluation of the attenuation of Nucleic Acid-Ligand VEGF-induced corneal angiogenesis. Zero or three pmol protein VEGF included in the biopolymer (Hydron) and implanted into its own substance of the cornea. Animals were treated intravenously twice a day or FSB or Nucleic Acid-Ligand, as indicated, for 5 days. In Fig. A, b and C illustrates the effect of systemic treatment of Nucleic Acid-Ligand NX31838-20K PEG, NX31838-40K PEG or NX31838-PL on the formation of new blood vessels. Values represent the mean SEM. *P<0.05 compared with 3 pmol VEGF + FSB group. See molecular Isobaric PEG as a function of time after injection.

In Fig. 11 shows curves of tumor growth of human tumors A growing subcutaneously (s. c. ) in nude mice treated with 40 mg/kg or 10 mg/kg Nucleic Acid-Ligand VEGF NX31838-40K PEG (NX31838 NAL), administered twice a day (BID). The negative control was mixed sequence of Nucleic Acid-Ligand VEGF NX31917 NAL (see molecular image in Fig. 1R), dosed at 40 mg/kg twice a day, and the positive control was the monoclonal antibody anti-VEGF mAb 26503.11 (R& D Systems), dosed at 100 μg/mouse twice a week. Because not revealed significant differences between group a dose of 40 mg/kg and group dose of 10 mg/kg, further dosing of 40 mg/kg was not repeated after 14 days. Groups of 8 mice implanted's. c. 1107tumor cells A on 0 day, and processing of test compounds by intraperitoneal injection was started on the 1st day of the experiment. Tumor volume, expressed in mm3was determined using the formula: tumor Volume= LW2/2.

In Fig. 12 shows tumor growth curves under different schemes dosage (comparison dosing twice a day (BID) dosing once daily (QD)), the parties 40K PEG (comparison party NX31838.07 with the new party NX31-Ligand (NAL) VEGF NX31838. Groups of 8 mice implanted's. c. 1107tumor cells A on day 0, and the processing of the test compounds by intraperitoneal injections started on day 1 at the time of the experiment. A few groups were animals in which tumors were not increased, and, consequently, for the final analysis, some groups contain only 7 (NX31838.04 10 mg/kg BID and NX31838.04 3 mg/kg BID) or 6 (NX31838.04 10 mg/kg QD and NX31838.07 10 mg/kg BID) animals. Tumor volume, expressed in mm3was determined using the formula: tumor Volume= LW2/2.

In Fig. 13 shows dose-dependent inhibition of tumor A growing subcutaneously (s. c. ), in mice nude Nucleic Acid-Ligand VEGF NX31838 40K PEG (NX31838 NAL) with the introduction of once per day. With this titration was unable to reach the no-effect dose; inhibition of tumor was observed even at the lowest dose (0.03 mg/kg). Groups of 8 mice implanted's. c. 1107tumor cells A on 0 day, and processing of test compounds by intraperitoneal injection was started on the 1st day of the experiment; group NX31838 NAL 3 mg/kg was 2 animals whose tumors do not grow, and therefore it contained only 6 animals. Tumor volume, expressed in mm3, was determined to use Ryuusei inhibition of established tumors A, growing subcutaneously (s. c. ), in mice nude Nucleic Acid-Ligand VEGF NX31838 40K PEG (NAL) with the introduction of twice a day. The positive control consisted of the monoclonal antibody anti-VEGF mAb 26503.11 (R& D Systems), dosed at 100 μg/mouse twice a week. Mice implanted 1107cells A and gave the tumors to grow to a volume of 200100 mm3and at this time the animals were sorted by weight, was tatuyrovaly for permanent identification, and processing of test compounds by intraperitoneal injection was started and continued throughout the experiment. Each point represents the average of 8 mice. Tumor volume, expressed in mm3was determined using the formula: tumor Volume= LW2/2.

In Fig. 15 summarizes the data on plasma concentrations NX213, NX278, NX-278-liposome after bolus injection.

In Fig. 16 shows the growth curves of tumors KSY-1, implanted subcutaneously in nude mice. Mice were treated by intraperitoneal injection NX31917 40K PEG or NX31838 40K PEG (30 mg/kg) or FSB twice a day during the experiment. Treatment was started after one day after subcutaneous implantation 2107cells KSY-1 at the rear side of nude mice. Each group used four is practical communication, formed through socialization electrons.

"Non-covalent Interactions are the means by which molecular particles stick together through interactions other than covalent bonds, including ionic interactions and hydrogen bonds.

"Lipophilic Compounds" are compounds that have a tendency to connection or distribution within the lipid and/or other materials or phases with low dielectric constants, including structures that contain essentially lipophilic components. Lipophilic Compounds include both lipids and compounds that do not contain lipid, which have a tendency to compound with the lipid (and/or other materials or phases with low dielectric constants). The following examples of Lipophilic Compounds are cholesterol, phospholipid and glycerolipid, such as diacylglycerol and diacylglycerol and glyceroglycolipid. In one preferred embodiment of the invention the Lipophilic Compound covalently cross-linked to Nucleic Acid-Ligand VEGF, is glycerolipid having the structure

< / BR>
where R1and R2represent CH3(Start>)nO, CH3(CH2)nOCH2-, where n= 10-20,

R3is a-X, where-X - is independently selected from the group consisting of -(RHO4)-, -O - and-CH2OS(O)-. When at least one of R1and R2represents CH3(CH2)n-O(PO3)-CH2-, Lipophilic Compound is a phospholipid. When at least one of R1and R2represents CH3(CH2)n-CONH2-CH2-, Lipophilic Compound is getserializer. When at least one of R1and R2represents CH3(CH2)nO - or CH3(CH2)nOCH2-, Lipophilic Compound is diacylglyceride.

"Complex", as used here, describes the molecular particle, formed by covalent linkage of Nucleic Acid-Ligand VEGF with non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound. In some embodiments of the present invention, this Complex is denoted as A-B-Y, where a is a Lipophilic Compound or non-immunogenic Compound of High Molecular Weight, as described herein; b is the GTC-Ligand VEGF.

"Lipid Structure" for the purposes of the present invention are structures that contain lipids, phospholipids or derivatives thereof, containing a number of different structural configurations that lipids are known to take in water suspension. These structures include, but are not limited to, lipid bikinie vesicles, micelles, liposomes, emulsions, lipid strips or layers, and can form complexes with a number of medicines and components that are known to be pharmaceutically acceptable. In the preferred embodiment the Lipid Structure is a Liposome. A preferred Liposome is single and has a relative size of less than 200 nm. Conventional additional components in the Lipid Structures include, among others, cholesterol and alpha-tocopherol. Lipid Structures can be used separately or in any combination, well known to the specialist, to provide desired characteristics for a particular application. In addition, the technical aspects of the formation of Lipid Structures and Liposomes are well known in the art, and any of the methods commonly practiced in this area can be used for the present invention.

In preferred embodiments of the invention, Nucleic Acid-Ligand VEGF Complexes and Lipid Structures according to the invention is identified using the SELEX methodology. Nucleic Acid Ligands VEGF identify from a Mixture of Candidate Nucleic Acids, where this Nucleic acid is a ligand VEGF, using a method in which (a) bring into contact the Mixture of Candidates with VEGF, and nucleic acids having an increased affinity to VEGF Rel the increased affinity of the balance of this Mix of Candidates; and b) amplified nucleic acids with high affinity with the mixture of Nucleic Acids enriched ligands (see Application for U.S. patent, serial N 08/233012 filed April 25, 1994, entitled "High Affinity Oligonucleotides to Vascular Endothelial Growth Factor (VEGF), Application for U.S. patent, serial N 08/447169, filed may 19, 1995, entitled "High Affinity Oligonucleotide Ligands to Vascular Endothelial Growth Factor (VEGF), which is hereby incorporated herein by reference).

"The Candidates mixture" is a mixture of Nucleic Acids with different sequences from which to select the desired ligand. Source Candidates Mixture may be naturally occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acid obtained by combining the above techniques. In the preferred embodiment, each Nucleic Acid has a fixed sequence surrounding the randomized region, to facilitate amplification.

"Nucleic Acid" means either DNA or RNA, single-stranded or Dunaeva, and any chemical modifications. Modifications include, but are not ogranichivaemsya, hydrogen binding, electrostatic interaction and melting the bases of the Nucleic Acid Ligand or Nucleic Acid Ligand as a whole. Such modifications include, but are not limited to, modification of the 2'-position sugar modification, 5-position pyrimidine, modifications, 8-position purine modifications, ekzoticheskim amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodouracil, modifications of the skeleton, such as magnolioideae phosphorothioate communication, methylation, unusual combinations mating grounds, such as Sosnovaya socitey and isoguanine, and the like. Modifications can also include 3' and 5' modifications such as capping.

"Non-immunogenic Compound of High Molecular Weight" represents a connection between approximately 1000 and 1000000 Yes, more preferably from about 1000 Da to 500,000 Da, and most preferably from about 1000 Da to 200,000 Da, which does not normally immunogenic response. For the purposes of this invention, the immunogenic response is a response that causes the body to produce proteins-antibodies. Examples of non-immunogenic compounds of High Molecular Weight include polyalkylene the coy Molecular Weight, covalently cross-linked to Nucleic Acid-Ligand VEGF, is polyalkyleneglycol and has the structure R(O(CH2)x)nO-, where R is independently selected from the group consisting of N and CH3x= 2-5, a nMW (molecular weight) of polyalkyleneglycol/16+14. In the preferred embodiment of the present invention the molecular weight is between about 10-80 kDa. In the preferred embodiment the molecular weight polyalkyleneglycol is between about 20-45 kDa. In the most preferred embodiment x= 2 and n= 9102. To the same Nucleic Acid-Ligand VEGF may be attached one or more than one polyalkyleneglycol, and the total molecular mass is preferably between 10-80 kDa, more preferably 20-45 kDa. In some embodiments of the non-immunogenic Compound of High Molecular Weight may also be a Nucleic Acid-Ligand.

"Lipid Bikinie Vesicles are closed, fluid-filled microscopic spheres that are formed primarily of individual molecules that have a polar (hydrophilic) and nonpolar (lipophilic) areas. Hydrophilic areas may contain phosphate, CH the groups are saturated or unsaturated hydrocarbons, such as alkyl, alkenyl, or other lipid groups. Sterols (e.g. cholesterol) and other pharmaceutically acceptable components (including antioxidants like alpha-tocopherol) may also be included to improve the stability of the vesicles or impart other desired characteristics.

"Liposomes" are a subset of Lipid Belayneh Vesicles and contain mainly molecules of phospholipids, which are two hydrophobic tail consisting of long chains of fatty acids. Upon exposure to water, these molecules spontaneously line up with the formation of bisloinoi membrane with a lipophilic ends of these molecules in each layer are connected in the center of the membrane, and the polar opposite ends forming respective inner and outer surfaces of this bisloinoi membrane. Thus, each side of the membrane is a hydrophilic surface, while inside the membrane is a lipophilic environment. These membranes during the formation, usually organized into a system of concentric closed membranes separated by interlayer water phases, somewhat like the layers of an onion, around the inner aqueous space. These mnogocentrowoe.

"Cationic Liposome is a liposome, which contains lipid components with an overall positive charge at physiological pH.

"SELEX methodology includes a combination of the selection of Nucleic Acid Ligands that interact with the target in the desired way, for example by binding to a protein, with amplification of those selected Nucleic Acids. Cyclic repetition of these stages of the selection/amplification allows selection of one Nucleic Acid or a small number of Nucleic Acids that interact more strongly with the target from a pool that contains a very large number of Nucleic Acids. A series of procedures for the selection/amplification continues until the chosen target will not be achieved. The methodology described in the SELEX patent Applications SELEX.

"Target" means any interest compounds or molecules, for which the ligand is desired. The target can be a protein (such as VEGF, thrombin and selectin), peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, analogue in the transition state, cofactor, inhibitor, drug, dye, PI is CLASS="ptx2">

"Improved Pharmacokinetic Properties" means that the Nucleic Acid Ligand to VEGF, covalently cross-linked to a non-immunogenic Compound of High Molecular Weight or a Lipophilic Compound, or in connection with the Lipid Construct has a longer half-life in the circulation in vivo relative to the same Nucleic Acid-Ligand VEGF, not connected with the non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound or a Lipid Construct.

"Linker" represents a molecular particle, which connects two or more than two molecular particles through covalent bond or non-covalent interactions and may allow spatial separation of these molecular particles in such a way which preserves the functional properties of one or more than one molecular particles. The linker can also be known as a spacer. Examples of linkers include, but are not limited to the structure shown in Fig. 1M-1P.

"Therapeutic" as used here, includes the treatment and/or prevention. When using the term "therapeutic", he refers to people and to other animals.

Review of the homology sequence of the Nucleic Acid-Ligand VEGF, are presented in tables 2-6 (SEO ID NOS: 15-132) shows that sequences with little primary homology or no primary homology may have essentially the same ability to bind VEGF. For these reasons, this invention also includes Nucleic Acid Ligands that have essentially the same postulated structure or structural motifs and the ability to bind VEGF as Nucleic Acid Ligands shown in tables 2-6. Essentially the same structure or structural motifs can be postulated by sequence alignment using the program Zukerfold (see Zuker (1989) Science 244: 48-52). As should be known to specialists for secondary structure prediction and structural motifs you can use other computer programs. Essentially the same structure or structural motif Nucleic Acid-Ligand in solution or in the form of a linked structure can also be postulated using NMR or other methods that should be known to specialists.

Further, in the present invention is included a method of producing Complex, soteriades inania, using a method in which identify Nucleic Acid Ligand from a Mixture of Candidate Nucleic Acids, where the Nucleic acid is a ligand VEGF, by using method (a) bringing into contact the Mixture of Candidate Nucleic Acids with VEGF, (b) separation of members of the above-mentioned Mixture of the Candidates on the basis of affinity to VEGF and (C) amplifying the selected molecules with a mixture of Nucleic Acids enriched in sequences of Nucleic Acids with relatively higher affinity for binding to VEGF, as well as covalent-linkage specified identified Nucleic Acid Ligand to VEGF with a non-immunogenic Compound of High Molecular Weight or Lipophilic Compound.

The next task of the present invention is the development of Complexes containing one or more than one Nucleic Acid-Ligand VEGF, covalently cross-linked to a non-immunogenic Compound of High Molecular Weight or Lipophilic Compound. Such Complexes have one or more of the following advantages over the Nucleic Acid-Ligand VEGF, not connected with the non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound:

1) improved FA is towanna the ability of the direction to the target. Complexes, optionally combined with a Lipid Construct, have the same advantages.

These Complexes or Lipid Constructs containing the Nucleic Acid-Ligand VEGF or Complexes may be advantageous due to one, two or three of these benefits. For example, the Lipid Structure of the present invention may consist of (a) liposomes, (b) a medicinal product which is encapsulated in the internal part of the liposome, and C) Complex, which contains the Nucleic Acid-VEGF Ligand and a Lipophilic Compound, and a component of Nucleic Acid-Ligand

VEGF is connected to the outer side of the Lipid Structure and acts from it. In this case, the Lipid Structure containing the Complex will be 1) to have improved pharmacokinetic properties, 2) possess an enhanced capacity for intracellular delivery of encapsulated drugs, and 3) specifically directed to a pre-selected location in vivo, where VEGF is expressed, attached to the outer side of Nucleic Acid-Ligand VEGF.

In the preferred embodiment of the present invention proposes a method of improving pharmacokineticpharmacodynamic a Compound of High Molecular Weight or Lipophilic Compound to form a Complex, and this Complex patient. Further, the invention relates to a method for improving the pharmacokinetic properties of Nucleic Acid-Ligand VEGF through additional connections of this Complex with the Lipid Construct.

In another embodiment the Complex of the present invention contains a Nucleic Acid-Ligand VEGF, covalently attached to a Lipophilic Compound, such as glycerolipid, or non-immunogenic Compound with a High Molecular Weight, such as polyalkyleneglycol or polyethylene glycol (PEG). In these cases, the pharmacokinetic properties of the Complex will be enhanced relative to one Nucleic Acid-Ligand VEGF. In another embodiment, the pharmacokinetic properties of Nucleic Acid-Ligand VEGF enhanced relative to one Nucleic Acid-Ligand VEGF, when Nucleic Acid-Ligand VEGF covalently attached to the non-immunogenic Compound of High Molecular Weight or Lipophilic Compound and is additionally connected with the Lipid Construct or Nucleic Acid-Ligand VEGF encapsulated within the Lipid Construct.

In embodiments where there are multiple Nucleic Acid Ligands VEGF, increasing their avidity to VEGF due to the e Nucleic Acid Ligands VEGF, pharmacokinetic properties of this Complex will be enhanced relative to one only of Nucleic Acid-Ligand VEGF. In embodiments where the Lipid Structure contains multiple Nucleic Acid Ligands or Complexes, the pharmacokinetic properties of Nucleic Acid-Ligand VEGF can be improved relative to the Lipid Structures in which there is only one Nucleic Acid Ligand to VEGF or only one set.

In some embodiments of the invention, the Complex of the present invention contains a Nucleic Acid-Ligand VEGF, is attached to one (dimeric) or more than one (multimeric) other Nucleic Acid-Ligand. The nucleic Acid may be a ligand to VEGF or to another target. In embodiments where there are multiple Nucleic Acid Ligands VEGF, increasing their avidity due to multiple binding interactions with VEGF. In addition, in the embodiments of the invention, where the Complex contains a Nucleic Acid-Ligand VEGF attached to one or more than one other Nucleic Acid-Ligand VEGF, pharmacokinetic properties of the Complex will be improved relative to a single Nucleic acid Kissedinto can be covalently cross-linked to many provisions on the nucleic acid-ligand VEGF, like with ekzoticheskoy the amino group on the base, 5-position pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate or hydroxyl group or other group at the 5' or 3 conce Nucleic Acid-Ligand VEGF. In embodiments where the Lipophilic Compound is glycerolipid or non-immunogenic Compound of High Molecular Weight is polyalkyleneglycol or polyethylene glycol, preferably it is connected with the 5' or 3' hydroxyl of its phosphate groups. In the most preferred embodiment of the Lipophilic Compound or non-immunogenic Compound of High Molecular Weight associated with the 5' hydroxyl of the phosphate group of the Nucleic Acid Ligand. Attaching non-immunogenic compounds of High Molecular Weight or Lipophilic Compound to the Nucleic Acid-Ligand VEGF can be done directly or with the use of linkers or spacers. In embodiments where the Lipid Structure contains Complex or where the Nucleic Acid Ligands VEGF encapsulated inside liposomes, preferred is a non-covalent interaction between the Nucleic Acid-Ligand VEGF or Complex and this Lipid Acids, is that oligonucleotides in their fosfomifira form can easily be broken down in the body fluids of intracellular and extracellular enzymes such as endonucleases and ectonucleoside, appears before the desired effect. You can create some chemical modification of a Nucleic Acid-Ligand VEGF to increase the stability of Nucleic Acid-Ligand VEGF in vivo, or to enhance or mediate the delivery of Nucleic Acid-Ligand VEGF. Modification of Nucleic Acids of VEGF Ligands covered by this invention, include, but are not limited to, those which provide other chemical groups that give additional charge, polarizability, hydrophobicity, hydrogen binding, electrostatic interaction and melting the bases of Nucleic Acid-Ligand VEGF or Nucleic Acid-Ligand VEGF in General. Such modifications include, but are not limited to, modification of the 2'-position sugar modification, 5-position pyrimidine, modifications, 8-position purine modifications, ekzoticheskim amines, substitution of 4-thiouridine, substitution of 5-bromo - or 5-iodouracil, modifications of the skeleton, phosphorothioate or alkylphosphate modifications, marked the such. Modifications can also include 3' and 5' modifications such as capping.

If Nucleic Acid Ligands obtained using the SELEX method, these modifications can be a pre - or post-SELEX modification. Pre-SELEX modifications give Nucleic Acid-VEGF Ligands with specificity for VEGF, and with improved stability in vivo. Post-SELEX modifications made in the 2'-OH of Nucleic Acid-Ligand, can lead to improved stability in vivo without adverse effect on the binding ability of the Nucleic Acid-Ligand. Preferred modifications of the Nucleic Acid-Ligand VEGF in this invention is the 5' and 3 phosphorothioate capping, and/or 3'3 investirovanie fosfomifira communication 3 conze. In the preferred embodiment the preferred modification of the Nucleic Acid-Ligand VEGF is 3'3 investirovanie fosfomifira communication 3 conze. Additional 2'-fluoro (2'-F), 2'-amino (2'-NH2) and 2'-O-methyl (2'-ome) modification of some or all of the nucleotides is preferred.

In another aspect of the present invention covalent cross-linking of Nucleic Acid-Ligand VEGF with non-immunogenic Compound High Molecule, a slower rate of clearance) relative to the Nucleic Acid Ligand to VEGF, is not connected with the non-immunogenic Compound with a High Molecular Weight or Lipophilic Compound.

In another aspect of the present invention, the Complex containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound may be additionally connected with the Lipid Construct. This connection can lead to improved pharmacokinetic properties relative to the Nucleic Acid Ligand to VEGF or Complex, not United with the Lipid Construct. Nucleic Acid-Ligand VEGF or Complex can be linked with the Lipid Structure by covalent or non-covalent interactions. In the preferred embodiment there is a connection via non-covalent interactions. In the preferred embodiment the Lipid structure is a Lipid Bishojou Vesicles. In the preferred embodiment the Lipid Structure is a liposome.

Liposomes for use in the present invention can be obtained by using any of various techniques known in the present work, and may include other materials, such as neutral lipids, such as cholesterol, as well as surface modifiers, such as positively charged (e.g., stearylamine or aminobenzene or aminopyrine derivatives of cholesterol) or negatively charged (e.g., diacetilactis, phosphatidylglycerol) connection. Multilayer liposomes can be formed using conventional techniques, which are selected deposition of lipid on the inner wall of a suitable container or vessel by dissolving this lipid in a suitable solvent, and then evaporating the solvent so as to leave a thin film on the inner side of the vessel, or by spray drying. The aqueous phase is then added to the vessel with a circular or vortex motion, which leads to the formation of MLV. UV can be formed using homogenization, sound or extrusion (through filters) MLV. In addition, UV can be formed using the techniques of removal of the detergent.

In some embodiments of the present invention the Lipid Design guide contains Nucleic Acid-Ligand (Nucleic Acid Ligands to VEGF target, United with the surface of the Lipid construct, and encapsulated terraformirovaniya liposomes can be modified to connect with Nucleic Acid-Ligand VEGF. For example, the Cationic Liposome is connected through electrostatic interaction with Nucleic Acid-Ligand VEGF. Nucleic Acid-Ligand VEGF, covalently cross-linked to a Lipophilic Compound, such as glycerolipid, can be added to the preformed liposomes, whereby glycerolipid, a phospholipid or glyceroglycolipid becomes United with liposomal membrane. Alternatively, Nucleic Acid-Ligand VEGF can be connected with the liposome in the process of manufacturing liposomes.

Specialists are well aware that liposomes are useful for encapsulating or incorporating a wide variety of therapeutic and diagnostic agents. Any variety of compounds can be enclosed in the internal aqueous compartment of liposomes. Illustrative therapeutic agents include antibiotics, antiviral nucleosides, antifungal nukes, regulators of metabolism, immune modulators, chemotherapeutic drugs, antidotes toxins, DNA, RNA, antisense oligonucleotides etc., in Addition, Lipid Bikinie Vesicles can be downloaded diagnostic radionuclide (e.g., indium 111, iodine 131, yttrium-90, phosphorus-32, or gadolinium) and fluorescent to understand that therapeutic or diagnostic agent can be encapsulated by the walls of the liposomes in the aqueous interior space. Alternatively, a portable agent can be a part of the material forming the wall of the vesicles, i.e., dispersed or dissolved in them.

In the process of forming liposomes water-soluble agents, carriers can be encapsulated in the aqueous interior space by including them in a hydrating solution, and lipophilic molecules can be incorporated into the lipid bilayer by incorporating the drug in the lipid. In the case of some molecules (e.g., cationic or anionic lipophilic drugs) loading drugs into preformed liposomes can be accomplished, for example, using methods described in U.S. patent N 4946683, the content of which is incorporated herein by reference. After encapsulating the drug liposomes are subjected to removal of the unencapsulated public medicines by using methods such as gel chromatography or ultrafiltration. Liposomes then usually sterile filter to remove any microorganisms that may be present in suspension. Microorganisms can take molecules in liposomes large single-layer Vesicles can be formed using methods such as reverse-phase evaporation (REV), or infusion of solvent. Other standard methods of forming liposomes known in the art, for example, methods of commercial production of liposomes include the homogenization procedure described in U.S. patent N 4753788, and the way the thin-film evaporation, described in U.S. patent N 4935171, which is incorporated herein by reference.

It should be understood that therapeutic or diagnostic agent may also be connected to the surface of Lipid Bisloinoi Vesicles. For example, the drug may be attached to the phospholipid or glycerides (prodrug). Phospholipid or glicerina part of this prodrugs can be incorporated into the lipid bilayer of liposomes by incorporating the drug in the lipid or loading into preformed liposomes (see U.S. patent NN5194654 and 5223263, which is incorporated herein by reference).

Specialist easily understood that the specific method of producing liposomes will depend on the intended use and the type of lipids used for education bisloinoi membrane.

Lee and Low (1994, JBC, 269: 3198-3204) and DeFrees et al. (1996, J the liposome internal and the external orientation of the PEG-ligand. Passive zakalivanie shown Zalipsky et al. (1997, Bioconj. Chem. 8: 111-118) as a way to zakalivanie Oligopeptide and oligosaccharide ligands exclusively on the outer surface of liposomes. The Central concept found in their work is that the conjugated ligand-PEG-lipid can be obtained, and then incorporated into the preformed liposomes through spontaneous inclusion (zakalivanie") lipid tail to the existing lipid bilayer. Lipid group undergoes it enable to reach a lower state of free energy by removing its hydrophobic lipid anchor from aqueous solution and its subsequent installation in the hydrophobic lipid bilayer. A key advantage of this system is that oligotypic anchors exclusively on the outer side of the lipid bilayer. Therefore, no Oligopeptide not lost due to interactions with their biological targets due to their location in gaze inward orientation.

The effectiveness of the delivery of Nucleic Acid Ligand to VEGF cells can be optimized by use of lipid drugs and conditions that are known to enhance tidylglycerol and phosphatidylserine, contribute to the merger, especially in the presence of other substances that promote the merger (for example, polyvalent cations, like CA2+, free fatty acids, viral fusion proteins, PEG short circuit, lysolecithin, detergents and surfactants). The phosphatidylethanolamine may also be included in the preparation of liposomes for enhancing adhesion with the membrane and the concomitant strengthening of cell delivery. In addition, free fatty acids and their derivatives, containing, for example, carboxylate groups, can be used to obtain a pH-sensitive liposomes, which are negatively charged at higher pH and neutral or protonated at lower pH. It is known that such pH-sensitive liposomes have a greater tendency to merge.

In the preferred embodiment of the Nucleic Acid Ligands of VEGF according to the present invention receive the SELEX methodology. SELEX described in Application for U.S. patent, serial N 07/536428, entitled "Systematic Evolution of Ligands by Exponential Enrichment, now withdrawn, in the Application for U.S. patent, serial N 07/714131, filed June 10, 1991, entitled "Nucleic Acid Ligands", now U.S. patent N 5475096, in the Application for U.S. patent, serial N 07 the same WO 91/19813). These applications, each of which is incorporated herein by reference, together called patent Application SELEX.

The SELEX method provides a class of products, which are nucleic acid molecules, each of which has a unique sequence, and each of which has the property of specific binding to the desired connection-target or desired molecule-target. The target molecules preferably are proteins, but can also include, among other other carbohydrates, composition, and small molecules. The SELEX methodology can also be used to make a target biological structures such as cell surface or viruses, through specific interaction with the molecule, which is an integral part of this biological structure.

In the most simplified form of the SELEX method can be defined as the following series of stages:

1) Get a Mixture of Candidate Nucleic Acids of different sequence. The mix of Candidates, as a rule, includes the regions of fixed sequences (i.e., each member of the Mixture of Candidates contains the same sequence in the same localhistory either: (a) to assist in the stages of amplification, described below, (b) to simulate the sequence, known as communicating with the target (C) to increase the concentration of a given structural configuration of Nucleic Acids in the Mixture of the Candidates. Randomized sequences can be randomized (i.e. the probability of finding a base at any position is one-fourth or only partially randomized (i.e., the probability of finding a base at any position can be selected at any level between 0 and 100 percent).

2) a Mixture of lead Candidates into contact with the selected target under conditions favorable for binding between the target and the members of the Mixture of the Candidates. Under these conditions, the interaction between the target and the Nucleic Acids of the Mixture of the Candidates can be viewed as the formation of pairs of Nucleic Acid-target between the target and the Nucleic Acids that have the strongest affinity to the target.

3) Nucleic Acids with the highest affinity to the target are separated from Nucleic Acids with lower affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of Nucleic Acid), the most relevant in the build criteria Department so to a significant number of Nucleic Acids in a Mixture of Candidates (approximately 5-50%) remained in the separation process.

4) Nucleic Acids selected in the process of separation as having a relatively higher affinity to the target, and then amplified with the formation of a new Mix of Candidates, which is enriched in nucleic acids with relatively higher affinity to the target.

5) through repetition of the above stages of separation and amplification of the newly formed Mixture of Candidates contains fewer and fewer unique sequences, and the average degree of affinity Nucleic Acids to the target, usually increases. Brought to an end, the SELEX method will give the Candidates Mixture containing one or a small number of unique Nucleic Acids, which are those nucleic acid from the Mixture to Candidates who possess the highest affinity to the molecule target.

To solve some specific problems the main way SELEX modified. For example, in the Application for U.S. patent, serial N 07/960093, filed October 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure", describes the use of SELEX in conjunction with g, is aka as bent DNA. In the Application for U.S. patent, serial N 08/123935, filed September 17, 1993, entitled "Photoselection of Nucleic Acid Ligands", describes a method based on SELEX for selecting Nucleic Acid Ligands containing photoreactive groups capable of binding and/or cross-fotostation and/or to photoinactivation of a target molecule. In the Application for U.S. patent, serial N 08/134028, filed October 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That discriminate Berween Theophylline and Caffeine", now U.S. patent N 5580737, describes a method for identifying highly specific Nucleic Acid Ligands able to discriminate between closely related molecules, called Counter-SELEX. In the Application for U.S. patent, serial N 08/143564, filed October 25, 1993, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX", now U.S. patent N 5567588, describes a method based on SELEX, which achieves efficient separation of oligonucleotides having high and low affinity to the molecule target. In the Application for U.S. patent, serial N 07/964624, filed October 21, 1992, entitled "Nucleic Acid Ligands to HIV-RT and HINE-1 Rev", now U.S. patent N 5496938, describes methods for obtaining improved Nucleic Acid-the Noah "Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX", describe how covalent linkage of the ligand to its target.

The SELEX method is the identification of high affinity Nucleic Acid Ligands containing modified nucleotides, giving the ligand advanced features, such as improved stability in vivo or improved delivery characteristics. Examples of such modifications include chemical substitutions in the positions of the ribose and/or phosphate and/or base. Identified by SELEX Nucleic Acid Ligands containing modified nucleotides are described in Application for U.S. patent, serial N 08/117991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides", now U.S. patent N 5660985, which describes oligonucleotides containing derivatives of nucleotides, chemically modified at the 5 - and 2'-positions of pyrimidines. In the Application for U.S. patent, serial N 08/134028, supra, describes highly specific Nucleic Acid Ligands containing one or more than one nucleotide, modified 2'-amino (2'-NH2), 2'-fluorescent(2'-F) and/or 2'-O-methyl (2'-Ome). In the Application for U.S. patent, serial N 08/264029, filed June 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2'-Modified Nucleosides by Intramolecul

The SELEX method involves combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in Application for U.S. patent, serial N 08/284063, filed August 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. patent N 5637459, and in the Application for U.S. patent, serial N 08/234997 filed April 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX", respectively. In these applications offered a combination of a wide variety of shapes and other properties, and the properties of the effective amplification and replication of oligonucleotides with the desired properties of other molecules.

The SELEX method further consists in the combination of the selected Nucleic Acid Ligands with Lipophilic Compounds or non-immunogenic Compounds of High Molecular Weight in diagnostic or therapeutic Complex, as described in Application for U.S. patent, serial N 08/434465, filed may 4, 1995, entitled "Nucleic Acid Complexes". The SELEX method further consists in the combination of the selected Nucleic Acid-VEGF Ligands with Lipophilic Compounds, such as diacylglycerol or dialkylglycerol, as described in Application for U.S. patent, serial N 08/73910EGF, which are connected with the non-immunogenic Compound with a High Molecular Weight, such as polyethylene glycol, or a Lipophilic Compound, such as glycerolipid, a phospholipid or glyceroglycolipid, therapeutic or diagnostic Complex are described in Application for U.S. patent, serial N 08/897351, filed July 21, 1997, entitled "Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Complexes". Each of the above patent Applications which describe the modification of the basic SELEX methodology, specifically incorporated herein fully by reference.

SELEX identify Nucleic Acid Ligands that can bind targets with high affinity and with remarkable specificity, which represent a remarkable achievement that is unprecedented in the study of Nucleic Acids. These characteristics are, of course, desirable properties that the specialist will search for therapeutic or diagnostic ligand.

For the production of Nucleic Acid-Ligand desired for use as a pharmaceutical, it is preferable that the Nucleic Acid-Ligand (1) was associated with the target so as to achieve the desired in what was so stable, to the extent possible; and (4) were specific ligand to a selected target. In most situations, it is preferable that the Nucleic Acid-Ligand possessed the highest possible affinity to the target. In addition, Nucleic Acid Ligands may have auxiliary properties.

In the Application for U.S. patent, serial N 07/964624, filed October 21, 1992 ('624), now U.S. patent N 5496938, describes methods for obtaining improved Nucleic Acid Ligands after performed SELEX. Application '624, entitled "Nucleic Acid Ligands to HIV-RT and HIV-1 Rev, specifically incorporated herein by reference.

The SELEX method was used to identify groups of high affinity RNA ligands to VEGF from libraries of random 2'-aminopyrimidine RNA and single-stranded DNA ligands from libraries of random single-stranded DNA (Application for U.S. patent, serial N 08/447169, filed may 19, 1995, entitled "High Affinity Oligonucleotide Ligands to Vascular Endothelial Growth Factor (VEGF), which is a continuation-part of patent application U.S. serial N 08/233012 filed April 25, 1994, entitled "High Affinity Oligonucleotide Ligands to Vascular Endothelial Growth Factor (VEGF)", both of which are incorporated herein by reference; see also Green et al. (1995) Chemistry and Biology 2: 683-695).

the e Acid Ligands VEGF take the three-dimensional structure, which should be kept to a Nucleic Acid-Ligand VEGF was able to bind its target. In embodiments where the Lipid Structure contains a Complex and Nucleic Acid-Ligand VEGF Complex projects from the surface of this Lipid Structures, Nucleic Acid-Ligand VEGF must be properly oriented relative to the surface of this Lipid Structure so that its ability to bind the target is not at risk. This can be done by attaching a Nucleic Acid-Ligand VEGF in a position which is remote from the binding site Nucleic Acid-Ligand VEGF. This three-dimensional structure and the proper orientation can be saved through the use of a linker or spacer, as described above.

Any of a variety of therapeutic or diagnostic agents can be attached to the system for delivering targeted by this Complex. In addition, any variety of therapeutic or diagnostic agents can be attached, once you've encapsulated or include them inside this Lipid Structures, as discussed above, for directed delivery through the Lipid Construct.

the situation with the liposome, for example, Nucleic Acid-Ligand VEGF can be directed to tumor cells expressing VEGF (eg, Kaposi's sarcoma) for delivery of anticancer drugs (e.g., daunorubicin) or imaging agent (e.g., radioactive labels). It should be noted that the cells and tissues surrounding the tumor, can also Express VEGF, and targeted delivery of anticancer drugs to these cells must also be effective.

In an alternate embodiment of therapeutic or diagnostic agent to be delivered to the target cell can be a different Nucleic Acid-Ligand.

The present invention also considered that the agent that you want to deliver, may be included in the Complex so as for example in connection with the outer surface of the liposomes (e.g., a prodrug, receptor antagonist or a radioactive substance for the treatment or imaging). As Nucleic Acid-Ligand VEGF agent may be attached through covalent or non-covalent interactions. Liposome should provide targeted delivery of the agent, extracellular, and the liposome is the example PEG) can be attached to the liposome to provide improved pharmacokinetic properties of the Complex. Nucleic Acid Ligands VEGF may be attached to the membrane of liposomes or may be attached to the non-immunogenic Compound of High Molecular Weight, which in turn is attached to the membrane. Thus, this Complex can be shielded from blood proteins and, thus, it leads to its circulation for extended periods of time, and Nucleic Acid-Ligand VEGF yet is affordable enough to make contact and communicate with its target.

In another embodiment of the present invention more than one Nucleic Acid-Ligand VEGF attached to the surface of the same liposome. This provides the possibility of bringing the same VEGF molecules in close proximity to each other and can be used to generate specific interactions between the molecules of VEGF.

In an alternate embodiment of the present invention the Nucleic Acid-VEGF Ligands and Nucleic Acid Ligand to another target may be attached to the surface of the same liposome. This provides a who's who of cnih interactions between VEGF and other targets. In addition to the use of liposomes as a way of bringing targets in close proximity, the liposome can be encapsulated agents to increase the intensity of interaction.

Lipid Structure containing Complex gives the possibility of multiple binding interactions with VEGF. This, of course, depends on the number of Nucleic Acid-Ligand VEGF Complex, the number of Complexes on Lipid Structure and mobility of Nucleic Acids of VEGF Ligands and receptors in their respective membranes. Because the constant effective binding may increase as the product of the binding constant for each site, there is a significant advantage in the presence of multiple binding interactions. In other words, in the presence of many Nucleic Acid-Ligand VEGF attached to the Lipid Structure, and therefore, create mnogojadernosti, effective affinity (avidity) of this multimeric Complex to its target can be as significant as the product of the binding constant for each site.

In some embodiments of the invention, the Complex of the present invention consists of a Nucleic Acid-Ligand VEGF, accession is the complex will be enhanced relative to only the Nucleic Acid-Ligand VEGF. As discussed above, glycerolipid, a phospholipid or glyceroglycolipid can be covalently cross-linked to Nucleic Acid-Ligand VEGF in various positions on the nucleic acid-ligand VEGF. In embodiments that use glycerolipid, it is preferable that the Nucleic Acid-Ligand VEGF is associated with the lipid via a phosphodiester bond.

In another embodiment of the invention the Lipid Construct contains a Nucleic Acid-Ligand VEGF or Complex. In this embodiment glycerolipid can be useful when including Nucleic Acid-Ligand VEGF in the liposome due to the propensity of glycerolipid to connect with other Lipophilic Compounds. Glycerolipid in connection with Nucleic Acid-Ligand VEGF may be included in the lipid bilayer of liposomes by incorporating the drug or by loading into preformed liposomes. Glycerolipid can connect with the membrane of the liposome such that the Nucleic Acid Ligand to VEGF acts inside or outside the liposomes. In embodiments where Nucleic Acid-Ligand VEGF is out of this Complex, Nucleic Acid-Ligand VEGF can serve as a guide when abilities. It should be understood that with the Lipid Structure m is STV this Lipid Structures. For example, PEG may be attached to the outer side of this membrane Lipid Structure.

In other embodiments, the Complex of the present invention consists of a Nucleic Acid-Ligand VEGF, covalently cross-linked to a non-immunogenic Compound of High Molecular Weight, such as polyalkyleneglycol or PEG. In this embodiment, the pharmacokinetic properties of this Complex are enhanced relative to only the Nucleic Acid-Ligand VEGF. Polyalkyleneglycol or PEG can be covalently cross-linked to many provisions on the Nucleic Acid-Ligand VEGF. In embodiments that use polyalkyleneglycol or PEG, it is preferable that the Nucleic Acid Ligand to VEGF linked through 5 gidroksilnami group through fosfodiesterazu link.

In some embodiments, the set of Nucleic Acid Ligands can be connected to one non-immunogenic Compound with a High Molecular Weight, such as polyalkyleneglycol or PEG, or a Lipophilic Compound, such as glycerolipid. Nucleic Acid Ligands can be all to VEGF or VEGF and other targets. In embodiments where there are multiple Nucleic Acid Ligands VEGF, observed pomorstvo molecules polyalkyleneglycol, PEG, glycerolipid can be attached to each other. In these embodiments, one or more than one Nucleic Acid Ligand to VEGF or Nucleic Acid Ligands to VEGF and other targets can be connected to each of: polyalkyleneglycol, PEG or glycerolipid. It also increases the avidity of each of the Nucleic Acid-Ligand to its target. In embodiments where multiple Nucleic Acid Ligands VEGF-joined polyalkyleneglycol, PEG or glycerolipid, there is a possibility of reduction of VEGF molecules in close proximity to each other to generate specific interactions between VEGF. If multiple Nucleic Acid Ligands specific to VEGF and other targets attached to polyalkyleneglycol, PEG or glycerolipid, there is the possibility of bringing VEGF and other targets in close proximity to each other to generate specific interactions between VEGF and other targets. In addition, in embodiments where Nucleic Acid Ligands to VEGF or Nucleic Acid Ligands to VEGF and other targets connected to polyalkyleneglycols, PEG or glycerolipids, the drug can also be connected with polyalkyleneglycols, PEG or glycerolipids. Tcolorbutton, The PEG or glycerolipid serve as a linker.

Nucleic Acid Ligands VEGF selectively bind VEGF. Thus, the Complex containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound or a Lipid Construct containing a Nucleic Acid-Ligand VEGF or Complex, are useful as pharmaceuticals or diagnostic agents. The present invention therefore includes methods of inhibiting angiogenesis by administration of a Complex containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound, a Lipid Constructs containing the Nucleic Acid-Ligand VEGF or Complex containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound. Complexes and lipid constructs containing the Nucleic Acid-Ligand VEGF, can be used for the treatment, inhibition, prevention or diagnosis of any painful condition, which involved inappropriate production of VEGF, in particular, angiogenesis. Angiogenesis is rare in C what is the main symptom of various illnesses, including, but not limited to, cancer, diabetic retinopathy, macular degeneration, psoriasis and rheumatoid arthritis. The present invention thus also includes, without limitation, methods of treatment, inhibition, prevention or diagnosis of diabetic retinopathy, macular degeneration, psoriasis and rheumatoid arthritis. In addition, VEGF is produced and secreted in varying quantities virtually all tumor cells. Therefore, the present invention includes methods of treatment, inhibition, prevention or diagnosis of cancer by administration of a Complex containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound, a Lipid Structures containing Complex or Nucleic Acid-Ligand VEGF that are not part of the Complex, in connection with the Lipid Construct. It is shown that with this type of cancer, as Kaposi's sarcoma (KS), the cells not only produce copious quantities of VEGF, but also Express functional VEGF receptors and, therefore, used for VEGF autocrine growth. Thus, the present invention includes a method of inhibiting with the unity High Molecular Weight or Lipophilic Compound, Lipid Structures containing Complex or Nucleic Acid-Ligand VEGF that are not part of the Complex, in connection with the Lipid Construct.

In one embodiment of the present invention the Lipid Structure contains a Complex of Nucleic Acid-Ligand VEGF and Lipophilic Compounds with additional diagnostic or therapeutic agent encapsulated in this Lipid Structure or connected with the inner side of this Lipid Structures. In the preferred embodiment the Lipid structure is a Lipid Bishojou Vesicles, and most preferably a liposome. Therapeutic applications of liposomes includes the delivery of medicines, which are usually toxic in the free form. In liposomal form toxic drug is private and may not affect tissues that are sensitive to this drug, and directed to selected areas. Liposomes can also be used therapeutically to release drug over an extended period of time, reducing the frequency of administration. In addition, liposomes can provide a way to obtain aqueous dispersions Hitachi.

To many drugs and imaging agents had a therapeutic or diagnostic potential, they have to be delivered to the proper location in the body, and the liposome, thus, can be easily injected, which creates the basis for long-term release and delivery of drugs to specific cells types or parts of the body. To apply liposomes for sending the encapsulated drug to the selected host tissues and remove them from sensitive tissues, you can use several methods. These techniques include manipulation of the size of liposomes, their surface charge and by their introduction. MLV, primarily because they are large enough, usually easily absorbed by the reticuloendothelial system (mainly liver and spleen). Found that UV, on the other hand, show an increased circulation time, reduced the rate of clearance and greater biological distribution relative to the MLV.

Passive delivery of liposomes involves the application of various routes of administration, such as intravenous, subcutaneous, intramuscular and local. Each path gives different the data areas of the target, include adherence to the surface of liposomes or antibodies, or specific ligand-receptor and is used for directing abilities. Additional guides components, such as antibodies or specific ligand-receptor may be located on the surface of liposomes, which should be known to the person skilled in the art. In one embodiment of the present invention, Nucleic Acid-Ligand VEGF connected to the outer surface of the liposomes, as should be known to specialists. In addition, some efforts in the direction of liposomes to tumors without the use of antibodies were successful, see , for example, U.S. patent N 5019369, U.S. patent N 5435989 and U.S. patent N 4441775, and professionals should be aware that they include these alternative methods directed delivery.

Therapeutic or diagnostic compositions of the Complex containing the Nucleic Acid-Ligand VEGF and non-immunogenic Compound of High Molecular Weight or Lipophilic Compound, a Lipid Structures containing Complex consisting of a Nucleic Acid-Ligand VEGF and non-immunogenic compounds of High Molecular Weight or Lipophilic Compounds, and Nucleic Acid-Ligand VEGF, not awsome also deal with other effective forms of introduction, such as intraarticular injection, inhalation aerosol, orally active drugs, transdermal drug electrophoresis or suppositories. One preferred carrier is a saline solution, but it is assumed that can be used also other pharmaceutically acceptable carriers. In one embodiment considers that the media and the Complex of Nucleic Acid-Ligand VEGF are physiologically compatible drug slow release. The primary solvent in the medium may be in the nature of either aqueous or non-aqueous. In addition, the carrier may contain still other pharmacologically-acceptable excipients for modifying or maintaining the pH, osmotic pressure, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the drug. Similarly, the carrier may contain still other pharmacologically-acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of Nucleic Acid-Ligand VEGF. Such excipients are those substances that are commonly and generally accepted used to produce dosage for parenterally or diagnostic composition is made, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or digidratirovannogo or lyophilized powder. Such preparations can be stored either in a ready-to-use form or in a form requiring recovery immediately before the introduction. Route of administration of drugs containing Nucleic Acid-Ligand VEGF for systemic delivery may be subcutaneous, intramuscular, intravenous, intranasal or by vaginal or rectal suppositories.

Advantages Complexes and lipid structures according to the invention include: i) improving the plasma pharmacokinetics of Nucleic Acid-Ligand; (ii) the representation of the Nucleic Acid-Ligand in the form of multivalent set to increase the avidity to interact with their targets; (iii) a combination of two or more than two introducing the Nucleic Acid Ligands with different specificnosti in the same liposomal particle; iv) strengthening the delivery of introducing the Nucleic Acid-Ligand to tumors, taking advantage of the inherent liposomes properties directions to the tumor, and (v) the use of high affinity and specificity represent Nemogu to specific targets. Introducing the Nucleic Acid Ligands are well suited for the described species of drugs, because, unlike most proteins, denaturation of introducing Nucleic Acids Ligands under the action of heat, different molecular denaturing agents and organic solvents is easily reversible.

The following examples are offered to explain and illustrate the present invention, and should not be construed as limiting the invention. Structure of Nucleic Acid Ligands described in the following examples, shown in Fig. 1. Example 1 describes the conjugation of Nucleic Acid Ligands with lipid reagents. The ability dialkylglycerol derived Nucleic Acid Ligand to VEGF (NX278) or in the form of the free ligand, or included in the bilayer of liposomes (NX278-L), to inhibit the activity of VEGF in vitro and in vivo as described in example 2. Example 3 describes an experimental method for obtaining 2'-F pyrimidine modified RNA ligands to VEGF. Example 4 describes the 2'-F pyrimidine modified RNA ligands to VEGF. Example 5 describes the synthesis glycerolipid-, phospholipid - and glycinamide lipid-and Peolpe-(L) and PEG-modified Nucleic Acid Ligands. Example 7 describes the preparations NX31838 PL-liposome Complex. In examples 8-10 describes the efficiency of in vivo Complexes of Nucleic Acid-Ligand VEGF. In example 11 describes vnutristenocna pharmacokinetics NX31838-40K PEG in rabbits.

Example 1. Synthesis diacylglycerol (1,2-di-O-octadecyl-sn-glycerol) -modified Nucleic Acid Ligand VEGF.

This example describes the conjugation of Nucleic Acid Ligands with lipid reagents. Synthesis of (1,2-di-O-octadecyl-sn-glycerol) -modified Nucleic Acid Ligand to VEGF (see diagram 1 at the end of the description).

Tetraethylammonium (2A): tetraethylene glycol (200 ml, 1.15 mol) was dissolved in 500 ml of pyridine, cooled to 0oC and treated 22,0 g (0,115 mol) of p-toluensulfonate. When dissolution was complete, the reaction mixture is kept in the refrigerator overnight, and then concentrated in vacuum. The residue was dissolved in 800 ml tO and was extracted with 3600 ml of N2O. H2About fraction was extracted again tO, and the United tO fraction was extracted with saturated aqueous Na2HPO4. The organic phase was dried over gS4and concentrated to a colorless oil. This oil was purified using flash homeon-20% Meon in tO to obtain 23.7 g (60%) of pure product and 11% of the product, containing minor impurities. 2A:1H NMR (300 MHz, Dl3) d 7.77 (d, J= 8.1 Hz, 2H), 7.32 (d, J= 8.1 Hz, 2H), 4.13 (t, J= 4.8 Hz, 2H), 3.68-3.53 (m, 14H), 2.58 (t, J= 5.6 Hz, 1H), 2.42 (s, 3H);13With NMR (75 MHz, CDCl3d 168.2, 158.3, 144.8, 135.9, 133.8, 132.0, 129.9, 128.0, 127.7, 126.6, 123.1, 113.0, 85.9, 73.0, 70.6, 69.7, 67.8, 64.4, 55.1, 37.1; MS low resolution, m/e calculated for C15H24ABOUT8S (M+1): 349,1.

Tetraethylethylenediamine (3A): To a stirred solution 31,96 g (0,092 mol) of 2A in 400 ml of anhydrous DMF was added 14.2 g (1.05 equiv. ) phthalimide and 14.4 ml (1.05 equiv. ) 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU). The solution was heated at 70oC for 18 h, then concentrated in vacuo. The crude yellow oil was purified using flash chromatography using 1600 ml of silica gel and elution with 25% EtOAc-50% EtOAc-75% tO in hexane, then tO, then 10% Meon-20% Meon in tO with the receipt of 23.8 g (80%) of 3A in the form of oil. When standing 3A was similar to wax a white solid.1H NMR (300 MHz, Dl3) 7.84-7.78 (m, 2H), 7.70-7.66 (m, 2H), 3.86 (t, J= 5.6 Hz, 2H), 3.70 (t, J= 5.6 Hz, 2H), 3.64-3.51 (m, 12H), 2.67 (bs, 1H);13With NMR (75 MHz, CDCl3d 168.2, 133.8, 132.0, 123.1, 72.4, 70.5, 70.4, 70.2, 70.0, 67.8, 61.6, 37.2.

Synthesis of compound 4: a Solution of 15 g (0,0464 mol) of 3A in 150 ml of THF and 15 ml DMF was cooled to 0oC in an atmosphere of AG. To the solution was added allylbenzene was stirred at 0oC for 30 minutes and then at room temperature for 18 hours was Added Meon (50-100 ml), and concentrated then the mixture was concentrated in vacuum. The crude material was purified using flash chromatography using 1500 ml of silica gel and elution with 25% EtOAc-50% EtOAc-75% tO in hexane, then tO, then 10% of the Meon in tO obtaining 11,05 g (65%) of 4A as a yellow oil.1H NMR (300 MHz, Dl3) 7.84-7.80 (m, 2H), 7.72-7.67 (m, 2H), 5.94-5.84 (m, 1H), 5.28-5.14 (m, 2H), 3.99 (d, J= 5.61 Hz, 2H), 3.88 (t, J= 5.85 Hz, 2H), 3.72 (t, J= 5.76 Hz, 2H), 3.64-3.54 (m, 13H);13With NMR (75 MHz, CDCl3d 168.0, 134.6, 133.7, 131.9, 123.0, 116.9, 72.0, 70.4, 69.9, 69.2, 67.7, 37.0.

1-Dimethoxytrityl-3- (phthalimidomethyl)-sn - glycerol (9):

According to the scheme 1 compound 9 was synthesized as follows: To a stirred solution of 4A (10,13 g, 0,0279 mol) in 100 ml acetone and 1 ml of N2About added 3.98 g (1,22 equiv. ) N-methylmorpholin-N-oxide (NMO). To this suspension was added to 1.75 ml (0.005 equiv. ) osmium tetroxide in the form of a 2.5% solution in isopropanol (iPrOH). After adding a solution of OsO4the reaction mixture became clear yellow. After TLC analysis showed full conversion of 4A (approximately 16 h), the reaction mixture was treated with 1.5 g of hydrosulfite sodium and 5.0 g of Florisil and was stirred for 30 minutes.ugogo party obtained in the same way from 1.0 g 4A. Two portions of 100 ml of pyridine together evaporated with the joint parties, and the residue was dissolved in 300 ml of pyridine. The solution was cooled to 0oWith and added 10,89 g (1.05 equiv. ) 4,4'-dimethoxytrityl. Into the flask was put in a drying tube and the reaction mixture was stirred at room temperature for 16 hours the Solution was treated with 20 ml Meon and concentrated in vacuo, keeping the temperature of the water bath below 40oC. the Crude oil was purified using flash chromatography using a 1100 ml of silica gel (Packed column wet with 3% triethylamine in hexane), and the elution 10-100% tO in hexane (all containing 3% triethylamine) to obtain 21,3 g (89% after two steps) of 9 as a yellow oil.1H NMR (300 MHz, Dl3) d 7.80-7.77 (m, 2H), 7.66-7.64 (m, 2H), 7.39-7.22 (m, 9H), 7.20-6.76 (m, 4H), 3.97 (bs, 1H), 3.84 (t, J= 5.97 Hz, 2H), 3.74 (s, 6H), 3.68 (t, J= 5.7 Hz, 2H), 3.60-3.49 (m, 14H), 3.13-2.76 (m, 2H), 2.00 (bs, 1H);13C NMR (75 MHz, CDCl3d 168.2, 158.3, 144.8, 135.9, 133.8, 132.0, 129.9, 128.0, 127.7, 126.6, 123.1, 113.0, 85.9, 73.0, 70.6, 70.4, 70.0, 69.7, 67.8, 64.4, 55.1, 37.1; MS low resolution, m/e calculated for C40H45O10N (M+NH4+): 717.5.

1-Dimethoxytrityl-3- (aminoethanethiol)-sn - glycerol (10):

According to the scheme 1 compound 10 ulali 10 ml of methanol to solubilize the starting material. The reaction mixture was heated at 50oC for 5 h, and then concentrated in vacumme and evaporated with toluene. The crude material was purified using flash chromatography on 200 ml of silica gel, elwira 15% ethanolamines in dichloromethane. Collected 3.94 g (96%) of 10 as a pale yellow oil.1H NMR (300 MHz, Dl3) d 7.46-7.21 (m, 9H, DMT), 6.81 (d, 4H, DMT), 4.00 (m, 1H), 3.80 (s, 6H), 3.70-3.49 (overlapping, N), 3.20 (dd, J= 9.24, 5.49 Hz, 1H), 3.12 (dd, J= 9.21, 6.00 Hz, 1H), 2.84-2.80 (m, 3H);13With NMR (75 MHz, CDCl3d 158.30, 144.82, 136.01, 129.95, 128.04, 127.66, 126.61, 112.95, 85.85, 73.46, 72.85, 70.55, 70.45, 69.99, 69.51, 64.43, 55.10, 41.40; MS low resolution, m/e calculated for C32P44O8N (M+1): 570.353 found 570,4.

Chloroformiate 19: To a stirred solution of 3 g (of 5.03 mmol) of 1,2-di-O-octadecyl-sn-glycerol 18 in 60 ml of toluene was added 20 ml of 1.93 M solution of phosgene. An additional solution of phosgene (210 ml: 14,5 equiv. phosgene) was added to until the original alcohol solution no longer remained (1H NMR analysis of concentrated aliquot). The excess phosgene and HCl was removed using an aspirator, and the reaction mixture was concentrated in vacuum to obtain 3.3 g (98%) of the desired chloroformate 19 in the form of a white powder.1H NMR (300 MHz, CDCl3d 4.45 (dd, J= 11.22, 3.69 Hz, 1H), 4.34 (dd, J= 3, 69.36, 31.99, 29.96-29.44 (overlapping signals from hydrocarbon chains), 26.13, 26.04, 22.76, 14.18.

Conjugate 20: To a stirred solution of 2.25 g (3.95 mmol) of 10 in 60 ml of pyridine was added 2.6 g of distearyldimethylammonium 18.1H NMR analysis of concentrated aliquots after 2 h did not reveal any remaining chloroformate, and the mixture was concentrated in vacuum. The crude residue was combined with the material obtained in this way from 0.5 g (0.88 mmol) of 10 and 0,58 g chloroformiate, and United party was purified using silikagelevye flash chromatography on a column of 100 ml of silica gel (Packed in hexano containing 2% of triethylamine) with elution with 200 ml of hexanol, then 250 ml of each of the 10-20 and 30% tO in hexano, 500 ml of 40% tO in hexano, then 250 ml of each of 50-60-70 and 80% tO in hexano, and finally 250 ml tO. The fractions containing the product were concentrated to obtain 3.3 g (57%) of conjugate 20.

Phosphoramidic 21: To a stirred solution of 3.8 g (3,26 mmol) conjugate in 25 ml of CH2CL2added to 1.14 ml (of 6.52 mmol) diisopropylethylamine (DIPEA), then 1,09 ml (4,88 mmol) 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite. After 2 hours the mixture was diluted with CH2Cl2and washed with a saturated solution Panso3, dried over Na2SO4the Kagel (Packed in hexano, containing 2% of triethylamine) with elution of 100 ml of hexanol, then 250 ml each of 10% and 20% tO in hexano, 500 ml of 30% tO in hexano, then 250 ml of 50% tO in hexano. The fractions containing the product were concentrated with the receipt of 4.2 g (95%) of phosphoramidite 21.31P NMR (Dl3d 151.52, 151.08.

Nucleic Acid-Ligand VGF-1,2-di-O-octadecyl-sn-glycerol conjugate

1,2-di-O-octadecyl-sn-glycerol group conjugatively with Nucleic Acid-Ligand VEGF NX213 (see Fig. 1A) using phosphoramidite 21 (scheme 2). The resulting conjugate was named NX278 (see Fig. 1B). NX278 (SEQ ID NO: 2) was purified using obetovannoi HPLC and its structure was confirmed using electrospray-mass spectroscopy (observed m/z= 117034, calculated m/z= 11720). Phosphorothioate magnolioideae communication was used in the provisions of 8 in NX278 (on the 3' and 5' ends), and the difference in mass of 0.16 units between the expected and observed masses is probably connected with the incomplete oxidation sulfureous agent, resulting in, on average, one phosphorothioate connection molecule was less than expected.

Example 2. The efficiency of in vitro and in vivo Complex of Nucleic Acid Ligand-liposome. Dialkylglycerol(DG)-modified Nucleic Acid Ligand to VEGF (NX278), NO: 2) spray dried mixture DS: cholesterol (50 mg/ml; 2: 1, mol: mol) in 25 mm phosphate buffer (pH 7.4) containing 9% sucrose, and voiced for 15-30 min at approximately the 60oWith using the source of the ultrasound probe type has not been obtained solution opal color. The control Complex of the Nucleic Acid Ligand-liposome containing similar ligand NX-278 mixed sequence (scNX278) (Fig. 1C; SEQ ID NO: 3) was obtained in a similar fashion. In a typical preparation were obtained liposomes with an average diameter of 50 nm and a width of distribution of 20 nm at half height. The size of the liposomal particles was determined in a particle analyzer (Leeds & Northrup Model Microtrack UPA 150, Horsham, PA). Liposomes comparable particle size distribution was obtained with the same lipid composition, but without conjugated with lipid Nucleic Acid Ligand. It is expected that the 50 nm liposome contains an average of 40 Nucleic Acid-Ligand located on both sides of the bilayer. This calculation was done as follows. Taking the surface area of the 19oWith cholesterol and 60oFor distearoylphosphatidylcholine in the liposome, got the number of lipid molecules on the liposome 3,13104for spherical liposomes with an external diameter of 50 nm and the thickness of the membrane 20oC. Based on the composition of the liposomes (2: 1 is, is chilili molecular weight for liposomes equal 2,1107.

To determine the distribution of Nucleic Acid-Ligand between the inner and outer surfaces of liposomes, investigated the availability NX278 in liposomal drug for T1ribonuclease. When two Ibogaine in the sequence (Green et al. (1995) Chemistry and Biology 2: 683-695) NX278 effectively cleaved by the ribonuclease T1. Simple incubation NX278 with preformed liposomes does not protect the Nucleic Acid-Ligand from ribonuclease T1. However, when NX278 included in liposomes in the sound (NX278-liposome), approximately 1/3 of its protected from nucleases. Adding to NX278-liposome of 0.1% Triton X-100, which destroys liposomes, without affecting the activity of nucleases, makes the previously protected Nucleic Acid-Ligand available for splitting. These results are consistent with the view that Nucleic Acid-Ligand distributed on both sides of the bilayer.

Binding affinity to VEGF NX213, NX278 and NX278-liposome

Binding affinity to VEGF NX213, NX278 and NX278-liposome studied using the method of competitive electrophoretic mobility shift (Fig. 2). Binding affinity to VEGF in NX278 was comparable with that of the NX213. ViDi is partly possibly due to the limitation fraction of Nucleic Acid-Ligand inside the liposomes. As expected, the analogues with mixed sequence contacted with VEGF significantly lower appendectomy (Fig. 2).

Pharmacokinetic properties NX213, NX278 and N278-liposome in plasma

Concentration NX213, NX278 and NX278-liposome in the blood plasma of rats Sprague-Dawley as a function of time is shown in Fig. 15, and the parameters of the compartmental analysis are summarized in table 1. A large part of NX213 rapidly excreted in the alpha phase with t1/27 minutes and the total rate of clearance of 6.8 ml/kg/min Algae phospholipid groups with Nucleic Acid-Ligand leads to highly biphasic clearance from the blood with high (t1/2and a somewhat lower total rate of clearance (4,95 ml/kg/min) relative to NX213. When you enable NX278 in the liposome significant additional decrease clearance of Nucleic Acid-Ligand from plasma (1,88 ml/kg/min).

Impact NX278 on HUVEC proliferation and angiogenesis

Studied the impact N278-liposome, scN278-liposome and NX213 on the proliferation of endothelial cells, umbilical vein human (HUVEC). HUVEC were grown in the presence of VEGF (10 n/ml) in IMDM medium (Environment Dulbecco modified p is in 24-hole tablets, covered with gelatin at a density of 20,000 cells per well on day zero and was treated with the above ligands at concentrations between 0.1 nm and 1 μm on day 1, 2 and 3 (replacing the environment in parallel with the ligands). N278-liposome inhibited the proliferation of HUVEC with IC50300 nm (this concentration refers to a component of a Nucleic Acid-Ligand); scN278-liposome and NX213 were significantly less effective (IC50>1 μm).

VEGF induces angiogenesis in tests on allantoine membrane chicken (HIMSELF), and this test can be used to study substances that inhibit angiogenesis. This test is conducted by placing disks of filters impregnated with VEGF, by HIMSELF, after which you can quantify the development of new blood vessels. NX278-liposome effectively blocked VEGF-induced angiogenesis (data not shown), whereas NX213, NX278 and scNX278-liposome did not influence. These studies together demonstrate that NX278 is a specific inhibitor of VEGF-induced cell proliferation in vitro and the formation of new blood vessels in vivo.

Impact NX278 on VEGF-induced capillary permeability

VEGF is the only known angiogenic factor, which tremendismo permeability in vivo. Test on vascular permeability (also known as

the test of miles (Miles, A. A. and Miles, E. M. (1952) J. Physiol. (London) 118: 228)) was carried out on Guinea pigs essentially as described (Senger, R. S. et al. (1983) Science 219: 983). N278-liposome, NX278 and NX213 at a concentration of 1 μm were injected with intradermally with VEGF (20 nm) Guinea pigs which had previously been injected blue dye Evans. In response to VEGF increased vascular permeability causes transudation albumin-bound blue dye Evans, leading to a blue spot at the site of injection. Since the recovery of the dye by extraction with an organic solvent, as a rule, is very weak, we developed a quantitative method in which measure the absorption of light through the skin. NX213, NX278, NX278-liposome and a neutralizing monoclonal antibody to VEGF, all significantly inhibited VEGF-induced vascular permeability, as shown in Fig. 3. Among the Nucleic Acid-Ligand N278-liposome was the most powerful antagonist. Sequence mixed analogues of these compounds was not any abscopal. The differences were significant and noticeable to the naked eye.

NX278-L inhibits cell lines Kaposi's sarcoma in vitro

Studied the impact NX278-liposome, scNX278-liposome and NX213 on the proliferation of KS cells. KS cell line KSY-1 were planted in 24-hole Board, covered with gelatin at a density 7500-10000 cells per well on day zero in a medium containing RPMI 1640 with the addition of 2% FCS, L-glutamine, penicillin and streptomycin. Nucleic Acid Ligands were added at concentrations between 0.1 nm and 1 μm in fresh medium on day 1, 2 and 3, and counting of cells was performed on day 4. N278-liposome inhibited the proliferation of KS cells with IC50100 nm; 1 μm N278-liposome growth of these cells is completely inhibited. scN278-liposome and NX213 showed values IC50>1 μm (Fig. 4).

NX278-liposome inhibited the growth of KS cells in vivo

Because VEGF is a factor of the e paracrine growth effects of VEGF-related tumor endothelial cells and inhibition of autocrine growth effects of VEGF on tumor cells. KS tumors can be, therefore, particularly sensitive to antagonists of VEGF. To test the activity of Nucleic Acid-Ligand in vivo, the trocars tumors (3 mm3) implanted Nude mice on the first day and were treated for five consecutive days, starting on day two, 50, 100 or 150 μg/day/mouse. The rate of tumor growth was measured over a period of two weeks. N278-liposome inhibited tumor growth depending on the dose very low inhibition of tumor growth at the low dose 50 mg/day/mouse (Fig. 5A) and significant inhibition of tumor growth at both dose levels of 100 and 150 μg/day/mouse (Fig. 5B, shown is 150 μg/day/mouse). Empty liposomes (Fig. 5A, B), scN278-liposome, as well as NX213 and NX278, were ineffective at all tested doses. In addition, NX278-liposome blocked VEGF-induced leakage of fluid from blood vessels.

Example 3. Experimental procedures for 2'-fluoro-pyrimidine modified RNA ligands to VEGF.

In this example, proposes a General methodology, performed and included in example 4 to highlight 2'-fluoro-modified Nucleic Acid Ligands to VEGF.

Materials

Recombinant VEGF165new powder. Protein resuspendable in physiological solution buffered with phosphate to a concentration of 10 μm and stored at 20oWith small aliquot to use. Aliquots were stored at 4oWith up to 4 weeks after thawing. Sf21-expressed, VEGF164mouse and E. coli-expressed VEGF121man, heterodimer VEGF/PIGF and PIGF also bought R& D Systems in the form of free media lyophilised preparations.

Oligonucleotides were purchased from Operon Technologies, Inc. or synthesized with synthesized oligonucleotides Applied Biosystems Model 394 according to the optimized Protocol. 2'-F and 2'-OMe-ribonucleoparticle was made JBL Scientific, Inc. (San Luis Obispo, CA). 2'-F-pyrimidine NTF also bought from JBL. Used 2'-HE-purine NTF and dNTP from Pharmacia Biotech, Piscataway, NJ.

Thermostable DNA polymerase of T. aquaticus (Taq polymerase) were purchased from Perkin Elmer-Cetus (Foster City, CA) was used AMV reverse transcriptase (AMV RT) from Life Sciences, Inc. ; DNA polymerase maple from New England Biolabs, Beverly, MA. RNA polymerase from T7 Enzyco, Inc. (Denver, CO). Sequeira DNA polymerase was carried out United States Biochemical Corp. (Cleveland, OH).

-[32P] -ATP and[32P] -ATP was obtained from New England Nuclear (Boston, MA).

The SELEX Protocol

The SELEX methodology description and "40N7") with randomized areas 30 or 40 nucleotides, flanked 5' and 3' fixed sequences (5'-TAATACGACTCACTATAGGGAGGACGATGCGG(30 or 40 N) CAGACGACTCGCCCGA-3'; SEQ ID NO: 133 and 134.

The italicized nucleotides on the 5 conce each matrix correspond to the promoter sequence for T7 RNA polymerase. Oligonucleotide primers were also synthesized for use in obtaining matrix, amplification and reverse transcription:

5'-TCGGGCGAGTCGTCTG-3' ("3N7"; SEQ ID NO: 135) and 5'-TAATACGACTCACTATAGGGAGGACGATGCGG-3'("5N7"; SEQ ID NO: 136).

Denitive DNA matrix was obtained by annealing primer 3N7 libraries 30N7 or 40N7 and elongation of the primer using DNA polymerase maple or AMV RT. Higher incubation temperature used for AMV RT (45oCompared to the 37oC), can better promote the full elongation through highly structured matrix oligonucleotides. Library transcribable using T7 RNA polymerase in the presence of 1 mm each of 2'-OH-ATP and GTP, 3 mm each of 2'-F-TTF and UTP and 50 microcurie -32P-ATP. RNA was purified from denaturing polyacrylamide gels by cutting out strips of gel containing RNA, chopping and soaking for a long time in 2 mm EDTA (ethylene diamine-Tetra-acetic acid).

2(FSBM) (library 30N7 and 40N7 or saline solution, buffered Tris, 1 mm gl2, 1 mm l2(DBMC) (only the library 30N7), and the mixture is serially diluted three times. After incubation at 37oC for 15 minutes the mixture conducted through 0.45 µm filters Type (Millipore) to collect Complexes with VEGF RNA. RNA was suirable with selected filters by incubation in a 2: 1 phenol, pH 7: 7M urea. After precipitation of the aqueous phase, RNA was subjected to annealing with primer 3N7 and reverse transcription using AMV RT. The resulting cDNA amplified in 15 cycles of polymerase chain reaction (PCR) using primers 3N7 and 5N7 and Taq DNA polymerase. Transcription of the PCR product was received new library enriched in sequences with affinity to VEGF. At round 4 in all three selected pools of RNA in the absence of VEGF appeared significant background signal associated with the filter. To drain these pools associated with the filter RNA, rounds 5 and 6 was performed using an alternative scheme for the Department VEG not denaturing gel and electrophoresis was performed at 10 watts for 45-60 minutes at 4oC. Complexes VEGF/PHK migrated above the unbound RNA in this system and were visualized by exposure to x-ray film to the gel. For these rounds selected RNA was purified using the method of grinding and soaking, as described above. After twelve rounds of selection and amplification of individual molecules in these selected pools were cloned using a set of pCR-Script Cloning from Stratagene (La Jolla, CA). The plasmid was purified using alkaline lysis (kit PERFECTprep Plasmid DNA, 5 Prime-->3 Prime, Boulder, CO), and the sequence of the cloned regions were obtained using a set of Dye Terminator Cycle Sequencing available from Perkin Elmer (foster City, CA). Fluorescent ledder nucleotide sequences were read in the National Jewish Center, laboratory of Brian Kotzin, Denver, CO. Sequences were grouped into families and were aligned by eye.

Measurement of binding affinely

Nucleic Acid Ligands radioactively marked in the transcription process by including -[32P] -labeled NTF or after synthesis by the use of[32P] -ATP and T4 polynucleotide kinase, were incubated at low concentration (between 20 and 70 PM) with varying concentrations of VEGF or other growth factors at 37oIn recapitalising-N-2-econsultancy acid) (GSB), pH 7.4, with or without adding additional divalent cations. Samples conducted through a pre-washed 0.45 µm filters Type (Millipore), followed by washing with 5-10 ml of binding buffer. Filters were immersed in the scintillator and counted to quantify the amount of bound protein RNA, detained each filter. The equilibrium constant of dissociation (KD) Nucleic Acid Ligand that binds a specific protein, was calculated based on the data, as described in Green et al. (1996) Biochem. 35: 14413-14424.

The selection of Nucleic Acid fragments of Ligands for affinity

Ten pmol of radioactively labeled inside the transcripts of the Nucleic Acid-Ligand VEGF high affinity was partially digested with nuclease S7, to obtain a mixture of radioactively labelled fragments. One-tenth fragmented RNA was incubated with 10 PM VEGF in 45 ml of binding buffer, and then filtered through nitrocellulose. Selected fragments, which are separated from the filter were dissolved in denaturing polyacrylamide gel with high resolution on the next track after track, which was caused to the pool of selected fragments. The smallest of the selected bands were individually purified from the gel activity. Half of the sample was subjected to annealing with the cDNA of the original transcript and elongation to the end of the matrix using Sequeira DNA polymerase. Comparison of the migration of purified fragment and the product of its elongation with the standard ledgera sequence of nucleotides used to determine the likely size and position of the selected fragment within the original transcript. Synthetic oligonucleotides corresponding to the sequence selected by the affinity of the fragments was obtained to confirm that the truncated Nucleic Acid-Ligand retained affinity to VEGF.

2'-OMe-substitution

Experiments 2'-OMe-substitution was performed essentially as described in Green et al. (1995) Chem. Biol. 2: 683-695. Three or four libraries were obtained for each of the three truncated ligands (t22, t2, t44), in which five or six 2'-OH purine provisions were partially substituted 2'-OMe. Each purine position was partially 2'-OMe-modified only in one of the libraries. Each 5'-radioactively labeled library were incubated with VEGF, and substituted oligonucleotides related to this protein were collected on nitrocellulose filters. Selected pool of source is not selected library partially gidralizovanny was defined by the ratio of the intensity of the bands" by dividing the signal, received on phosphorous imaging unit as a result of hydrolysis in this position for the selected pool, on the signal received in the same position for the non-selected library. The relationship of the intensity of the bands, which lie significantly above the level for specific provisions represent the offset to 2'-HE (against 2'-OMe) selected by the affinity of the pool.

The rate constants of binding

A small amount (usually less than 1 pmol) of 5'radioactively labeled Nucleic Acids Ligands were incubated with 1 nm VEGF at 37oWith 1 ml of buffered saline solution with the addition of divalent cations. At time "zero" 50 µl filtered through nitrocellulose to determine the fraction of RNA associated with the protein, then added an excess (100 or 500 nm in different experiments) unlabeled Nucleic Acid-Ligand, and 50 ál aliquots were filtered in the subsequent moments of time. The filters considered in the scintillator to determine the amount of radioactively labeled RNA, is still associated with VEGF in each moment of time. The data plotted on the graph as the fraction of bound RNA (f) against time, coincided with the equation for exponential decay:

f(t)= f0e-ktd), a b is the residual binding of radioactively labeled RNA with a filter at the end of the experiment (effectively, in the absence of protein). The rate constants of Association (kas) was calculated based on the measured values of kdand KDaccording to the equation:

ka= kd/kD.

Example 4. 2'-fluoro-modified RNA ligands to VEGF.

The selection of ligands.

Ligands to VEGF was identified in three separate SELEX experiments from libraries 2'-F-pyrimidine modified RNA containing 30 or 40 random nucleotides. Selections were carried out in the FSB with the addition of 1 mm MgCl2(30N and 40N library) or in saline solution, buffered Tris, 1 mm MgCl2and 1 mm CaCl2(only 30N library). About 1014unique sequences were included in the first cycle of selection of each experiment. After ten cycles, the affinity between VEGF and each pool RNA was improved by about 1000 times relative to the original pools. As to further improve the affinity was observed after two additional cycles, individual members of the pools twelfth round cloned and sequenced for approximately 50 isolates from each selection.

Oligo and sequenced, and the sequences were grouped into families on the basis of the General motives of the primary structure (table 2). The name of each ligand specifies the target (V= VEGF), the buffer selection (P= FSB (phosphate-saline buffer), T= CE(Tris-saline buffer)), the length of the randomized region in the library (30 or 40 nucleotides) and the clone number (after the decimal). The frequency with which the sequence was found among the analyzed clones, listed in parentheses; sequences that differ by only one nucleotide, attributed PCR mutagenesis common predecessor and were grouped together, and variable basis indicated in the sequence corresponding symbol (Y= U or C). Fixed sequences that are common to all ligands are indicated in lowercase letters at the top. For individual clones, the sequence of the variable region is shown in capital letters. For some ligands sequence of fixed area in lowercase letters are appended to the sequence of the variable region, where they contribute to the possible secondary structure. High affinity TOdfor binding to VEGF are shown for each ligand. One ligand in each family was selected for dpositivelady, distinguished by more than one nucleotide. 44 of these sequences could be grouped into three main families on the basis of conservative primary structural motifs (table 2). Sequences that can be grouped into minor family with five or less than five members, and the sequence-"orphans", which was unique among the isolates is shown in table 6. Ligands containing primary structural motif, certain families 1 and 2, were found in all three rounds of affinity. Similarities between conservative primary structures of both collections suggests that they may also share similar secondary structure and/or the edge of VEGF using similar contact areas. Family members 2 have in common the possibility of formation of a short rod of paired bases, the surrounding conservative motif sequence in the large "loop" (underlined in table 2). Except for the closing base pair A/U, the identity of the sequence of bases in the proposed core areas is not conservative. This "covariance" of the grounds, which retains its secondary structure rather than primary, what is important for high affinity conformation of this family of ligands VEGF. Among the sequences of the family 1 not found such a conservative interactions mating grounds. The third family of ligands met only when the auditions held in DBMC (family 3, table 2). In addition to the highly conservative primary structural motif, all members of this family of sequences 3' conservative district have a common complementary pairing of bases with nucleotides in the 5' fixed region (underlined in table 2). Because for most of the ligands is not to say that the grounds for 5 storone alleged rod coveryour with their partners, mating grounds, this observation is less predictive for overall secondary structure; however, the initial assumption of minimal high affinity sequences originating from this family (described below), the inventors were guided by the strong conservatism of this motive. The affinity of individual RNA ligands to VEGF was evaluated on the basis of a separate definition TODfor their interaction. With a few exceptions ligands showed a very high affinity for the growth factor with KDbetween 5 and 50 PM.

Ministatellite, indicate the minimal sequence elements required for high affinity binding to VEGF. Built one in a different truncated variants of representative ligand from each family (indicated by gray rectangles in table 2) was obtained by chemical synthesis and determined their relative affinity to VEGF (table 3). Truncated variants of ligands VP30.22, VP30.2 and VT30.44 was obtained by chemical synthesis and determined their affinity to VEGF, as described in example 3. The original truncated variants (t22, t2, t44) further improved by synthesizing oligonucleotides with complementary bases, missing 5' and/or 3' end. To initiate the chemical synthesis at the 3' nucleotide of several ligand modified either by replacing the 2'-Oh-cytidine 2'-F-citizen (underlined), or by adding a 3'-3'linked deoxythymidine "cap"(asterisk). Shows the length of each nucleotide (minus the cap) and its high affinity TODfor binding to VEGF.

The initial prediction for the minimum sequence from clone VP30.22 (family 1) was done by mapping all cleaned, selected by the affinity of the fragment of the full-length ligand (see example 3). Attele full-ligand. Further truncation at the 3' end of the molecule caused a rapid loss of affinity, but with the 5' end can be removed up to 6 additional nucleotides with minor consequences or no consequences (table 3). To clone VP30.2 family 2 and clone VT30.44 family of 3 was synthesized truncated ligands "t2" and "t44", which concluded a prospective rod of five base pairs and the entire conservative motif sequence. Both truncated ligand is almost completely retained binding activity of the full-size sequence. Further truncation by dellarovere one base pair at once (one nucleotide from each end of the ligand) was caused by the sequential loss of affinity. Thus, for these sequences truncation based on possible secondary structures that allow very good at predicting highly affine minimal ligand and further confirm the hypothesis that the estimated rods contribute to the high affinity conformation of these ligands.

2'-OMe modification

Observed that the substitution of 2'-OH positions of RNA oligonucleotides with 2'-OMe improves their stability against nucleases present in the urine of rats as well as in other biologist as therapeutic or diagnostic agents. Unfortunately, 2'-OMe-modified nucleosidase, as a rule, are not acceptable for RNA polymerase as substrates under standard reaction conditions. However, 2'-OMe purine can be entered in specific oligonucleotide by chemical synthesis. It is noticed that some highly affine 2'-HE-purine RNA ligands will undergo an unusually high percentage of 2'-OMe purine substitutions with a small loss of affinity to the protein target. To identify those purine position for which 2'-OMe substitution is compatible with high affinity binding to VEGF, spent several syntheses of ligands t2, t22 and t44, in which five or six of purines at the same time were partly replaced in the modified nucleotide (described in example 3). Selection on the affinity of each partially substituted library was used to select those molecules that have retained a substantial affinity to VEGF. In such selected by affinity pool provisions, which have not undergone substitution, tend to the 2'-OH, and, therefore, they exhibit a high sensitivity to hydrolysis by alkali relatively the same position in the non-selected library. 5' radioactively labeled pools, selected and not from what they permit. In ligand t22 G10 and A12 showed a significant propensity for 2'-HE selected the affinity of the pool, as well as A6 and G21 in ligand t2 and A5 and A6 in the ligand t44. Although the above analysis identifies those provisions, which would probably not allow substitution at the 2'-OMe-nucleotides on the basis of these data it is impossible to predict how such modifications all other purines will affect binding affinity. In fact, the ligand t22 synthesized with all 2'-OMe-purine except G10, A12 and G22 (which showed an extreme preference for the 2'-OH), was associated with VEGF with an affinity equal to, if not better, than all 2'-HE-purine sequence (table 4).

Truncated oligonucleotides (t22, t2 and t44) was synthesized chemically with all purine provisions, replaced with 2'-OMe-purines, except for one, two, or three. The remaining 2'-HE-purines stated in the title of each ligand and shown in bold in the sequence of the ligand. Shows Kdsto associate each substituted ligand to VEGF. Additional substitution in G22 had little effect on binding to VEGF, but the inclusion of 2'-OMe in G10 or A12, as predicted, was detrimental to binding affinity. Similarly ligands t2 and t44 underwent 2'-OMe-Ligand to VEGF (table 4).

Binding affinity and rate constants for the truncated ligands

In the hope of identifying high 2'-substituted Nucleic Acid-Ligand VEGF minimum length of all 2'-OMe substitution, which dramatically reduced the binding, were included in the truncated ligands t22c, t2a and t44a (see table 3). 2'-HE-nucleotides are shown in bold, and 2'-OMe-nucleotides are indicated in plain text. The resulting Nucleic Acid-Ligand t22-OMe and t44-OMe, was associated with VEGF with KDs 67 49 PM and PM, respectively, whereas the ligand t2-OMe was associated with KDapproximately 140 PM (table 5). These KDfavorably compares with that of the NX-213 (D= 140 PM), 2'-NH2-pyrimidine-, 2'-OMe-purine-substituted oligonucleotide inhibitor of VEGF, as described previously (see Application for U.S. patent No. 08/447169, which is incorporated herein by reference). It was found that each of the truncated 2'-OMe-substituted oligonucleotides competes with NX-213 and with each other for binding to VEGF.

The rate constants of dissociation (kd) was determined for each of the three 2'-OMe-substituted ligands by monitoring the loss of the preformed Complex between radioactively labeled ligand and VEGF adding a large excess of unlabeled ligand. The ligand showed a little more slow dissociation half-life of about 170 seconds and 90 seconds respectively. The rate constants of Association (ka) calculated from the equilibrium constant for the dissociation and rate constants of dissociation (KD= kd/ka), ranged from 3107up to 2108M-1s-1(table 5). Such a fast speed Association suggests binding interaction, limited close by diffusion, between the ligands and VEGF, and are in accordance with the rate constants of Association observed for the obtained by SELEX Nucleic Acid Ligands to other targets.

Dependence on divalent cations

Ligands in families 1 and 2 were selected in the presence of magnesium cations, whereas the ligands of the family 3 were selected in a buffer containing magnesium, and calcium. Since divalent cations may contribute to RNA/protein interactions through non-specific or specific stabilization of high affinity RNA structures, the inventors have raised the question, magnesium and/or calcium is required for high affinity binding of representative ligands to VEGF. The affinity of Nucleic Acid-Ligand t22-OMe and t2-OMe (from families 1 and 2, respectively) were unchanged in the presence or in the absence of additional duha is certain ligand t44-OMe, showed absolute dependence on the presence of calcium for high affinity binding to VEGF. Binding was dramatically reduced (TOD>10-7), when divalent cations in the binding buffer were removed by EDTA. Adding excess MgCl2to emaciated binding buffer with divalent cations did not lead to improved binding affinity, but CaCl2in a double molar excess over EDTA was fully restored binding activity. Identical behavior in binding was observed for unmodified ligand t44 (data not shown).

Protein specificity

Described here oligonucleotides were selected on the basis of their affinely to VEGF165the biggest of the two is capable of diffusion isoforms of the growth factor. THE VEGF121more minor isoform that lacks one of the exons of VEGF165and, unlike the latter, it is not associated with heparin. None of the three truncated 2'-OMe-substituted oligonucleotides were not associated with any measurable affinity to VEGF121. In addition, the native structure of VEGF165is essential for the binding of all three Nucleic Acid-Ligand, since binding is not observed when the protein Vosstanya at the species level, moreover, an isoform of VEGF165man and VEGF164mouse show 88% sequence identity. Truncated 2'-OMe-substituted ligands bind equally well with human and murine VEGF. However, none of the ligands was not observed binding to homodimers PIGF, placental protein origin, which has 53% sequence identity with VEGF within the conservative domain-like growth factor derived from platelets. Heterodimer between VEGF and PIGF recently isolated from supernatants cell lines, both normal and tumor origin, and such heterodimeric active upon binding with one or two high affinity VEGF receptors and the induction of responses in cultured endothelial cells. The biological value of heterodimeric VEGF/PIGF unknown. Significant binding, although with significantly reduced appendectomy was observed with heterodimeric VEGF/PIGF. These data may indicate that Nucleic Acid Ligands bound at the interface between the two subunits in the dimer or near it, and that PIGF not present all the sites of contact required for high affinity binding. Alternatively, the structure of subje is wyzwania Nucleic Acid Ligand.

Example 5. Synthesis of phospholipid, glycerinated lipid and PEG-modified Nucleic Acid-Ligand VEGF.

For the synthesis of various Lipophilic Complexes Connection/Nucleic Acid-Ligand used three different drug, like the following:

< / BR>
< / BR>
< / BR>
I. Phosphoramidic C-18 for the synthesis of the drug PL

Description in General terms to obtain phosphoramidite C-18 are presented in figure 3. 1-Octadecanol was fosforilirovanii under standard conditions. After mixing the reaction mixture, the residue was purified on silikagelevye column with hexane: ethyl acetate: triethylamine (90: 10: 5) with the receipt of 21.5 g of pure product (yield 57%).

II. Synthesis lipidemia 1

This phosphoramidite, in contrast to the above PL has an amide bond. The structure of the oligo, resulting from conjugation of this lipid is shown below.

Several experiments have demonstrated that the high insolubility of compound 22 in organic solvents made it impossible NMR and MS characterization and further fosfaurilirovania connection 22 to a DAG of amidite 23, however, based on results to obtain the lipid-spacer-amidite (scheme 1), the inventors expected that pospisilova connect the reflux condenser. The approach to obtaining a DAG of amidite shown in figure 4.

N, N'-Bis(stearoyl)-1,3-diamino-2 - propanol (22). The solution staurolite (6,789 g, 22,41 mmol) in lC2CH2CL (50 ml) was added dropwise to a solution of 1,3-diamino-2-hydroxypropane (1.0 g, 11,10 mmol) in lC2CH2CL (100 ml) and tea (tetraethylammonium) (2,896 g, 22,41 mmol) with stirring at room temperature. After the addition was completed, the mixture was heated to 70oWith during the night, and formed a clean solution, this solution was cooled to room temperature, filtered and washed solids CH2CL2CH3HE, 5% NaHC3and ethyl ether, and dried in vacuum to obtain 22 (6,40 g, yield 93%) as white solids.1H NMR (pyridine-d5; 60oWith million-1): 3.82-3.78 (m, 1H), 2.37 (t, J= 7.5 Hz, 4H), 1.81-1.76 (m, 4H), 1.30-1.27 (m, 60H), 0.87 (t, J= 5.7 Hz, 6N).

N, N'-Bis(stearoyl)-O- (diisopropylamino-2 - cyanoethoxy)- 1,3-diamino-2-propanol (23). To compound 22 (5,80 g, 9,31 mmol), dried overnight in a vacuum, was added anhydrous CH2CL2(150,0 ml) and sbrasyvali N, N-diisopropylethylamine (DIPEA) (4,2 ml, 18,62 mmol). The mixture was cooled in an ice bath and sbrasyvali chloro-(2-cyanoethoxy)-N, N-Diisopropylamine the Les cooling to room temperature, insoluble materials were filtered off, and the solution washed with 5% Panso3and brine, dried over Na2SO4and concentrated in vacuum. The crude product was purified by precipitation from CH3CN to obtain the pure product (4,65 g, yield 61%) as white solids. 31P NMR (Dl3million-1): 154.04.

I. Synthesis of DAG-spacer-amidite, lepidomeda 2

Hexa(ethylene glycol) was included in lepidomeda to weaken the insolubility dumenigo connection 22, which is directly to the intermediate connection for lipidaemia 23. Description of the receiving lipid-spacer-amidite 29 presented in figure 5. Stage combination of compound 25 with 1,3-diamino-2-hydroxypropane and tert-piperonyl potassium in THF is not going well, and the output was only about 20%. One attempt to improve the solution was made through interaction 25 and diamide 22, however, the desired product was not found.

(4,4'-Dimethoxytrityl)-hexamethyleneimine (24).

Hexa(ethylene glycol)(18,98 g, 67,05 mmol) co-evaporated with anhydrous pyridine (350 ml) was dissolved in anhydrous pyridine (400 ml) and after cooling to 0oWith added dropwise DMTrCI (23,85 g, 70,40 mmol) in pyridine (50 ml) for 30 min with stirring in an atmosphere of arcuatum, and the residue was dissolved in CH2Cl2then it was washed with 5% NaHCO3and Russolo, dried over Na2SO4and concentrated in vacuum. The crude product was purified wet flash chromatography on silikagelevye column in a gradient of ethyl acetate, then in CH2Cl2and methanol (95/5) containing 0.5% tea. Appropriate fractions were combined, evaporated and dried in vacuum to obtain 24 (26,1 g, yield of 66.6%) as a pale yellow oil.1H NMR (DMSO-d6; , m-1): 7.40 (d, J= 7.2 Hz, 2H), 7.33-7.24 (m, 7H), 6.89 (d, J= 8.9 Hz, 4H), 4.61 (t, J= 5.1 Hz, 1H), 3.73 (s, 6H), 3.05 (m, 24H);13With NMR (DMSO-d6; , m-1): 158.02, 145.02, 135.78, 129.67, 128.13, 127.71, 126.61, 113.14, 85.29, 72.33, 72.27, 70.06, 69.87, 69.80, 69.75, 69.70, 62.84, 60.25, 60.19, 55.01.

(4,4'-Dimethoxytrityl)- hexaethylguanidinium (25). It chilled in ice (0oC) a solution of 24 in anhydrous pyridine (50 ml) solution was added toluensulfonate in pyridine (30 ml). After 2 h at room temperature the solution is evaporated to a light yellow oil. The residue was dissolved in CH2CL2and washed with 5% NaHCO3and brine, dried over Na2SO4, filtered and evaporated in vacuum. The product was purified using a damp flash chromatography on silica gel with elution by ethyl acetate to obtain 46 (d, J= 8.1 Hz, 2H), 7.40 (d, J= 7.4 Hz, 2H), 7.32-7.23 (m, 7H), 6.88 (d, J= 8.8 Hz, 4H), 4.09 (t, J= 4.3 Hz, 2H), 3.72 (s, 6H), 3.06 (m, 22H), 2.40 (s, 3H);13With NMR (DMSO-d6; , m-1): 158.01, 145.01, 135.78, 132.38, 130.12, 129.67, 128.12, 128.02, 127.80, 127.70, 127.62, 113.13.

2-(4,4'-Dimethoxytrityl)- hexamethyleneimine-1,3 - diaminopropan (26). A mixture of 1,3-diamino-2-hydroxypropane (74,7 mg of 8.28 mmol) and tert-butoxide potassium (2,78 g, 24.84 mmol) in anhydrous THF was heated to 70oC for 2 h, and then cooled to room temperature. Compound 25 (4,08 g's, 5.25 mmol) in THF sbrasyvali, and the mixture was stirred at 70oWith overnight until TLC showed that no more 25. After the solution was cooled to room temperature, THF was removed in vacuo and added to 25 ml of CH2Cl2and 25 ml of water. Layer CH2Cl2separated, and the aqueous layer was extracted with CH2CL2. Solutions CH2Cl2were combined, dried over Na2SO4and evaporated under reduced pressure. The crude product (2,43 g) was directly used for the reaction without further purification.1H NMR (DMSO-d6; , m-1): 7.41 (d, J= 7.7 Hz, 2H), 7.32-7.21 (m, 7H), 6.87 (d, J= 8.8 Hz, 4H), 3.73 (s, 6H), 3.52-3.40 (m, 24H), 3.17 (s, 1H), 3.07-3.02 (m, 4H).

N, N'-Bis(stearoyl)-2- (4,4'-dimethoxytrityl)- hexamethyleneimine-1,3 - SUB>2CH2CL and tea (1.9 ml, 11.1 mmol) with stirring at room temperature. The mixture was stirred at room temperature for 2 h, then was heated to 70oWith during the night. After the solution was cooled to room temperature, the solution washed with 5% Panso3and brine, dried over Na2SO4and concentrated in vacuum. The crude product was purified using a damp flash chromatography on a column of silica gel in a gradient of ethyl acetate and CH2CL2(50/50), and then ethyl acetate and methanol (50/50). The second fraction was collected, evaporated, and dried in vacuum to obtain 27 (640 mg) as a pale yellow solid.1H NMR (DMSO-d6; , m-1): 7.40 (d, J= 7.2 Hz, 2H), 7.37-7.20 (m, 7H), 6.74 (d, J= 8.9 Hz, 4H), 3.71 (s, 6N), 3.63-3.51 (m, 24 H), 3.17 (s, 1H), 3.16-3.13 (m, 4H), 2.12 (t, J= 7.3 Hz, 4H), 1.18 (m, 60H), 0.80 (t, J= 6.2 Hz, 6N).

N, N'-Bis(stearoyl)-2 - hexamethyleneimine-1,3 - diaminopropan (28). A mixture of compound 27 (640 mg), 2,5% solution of DCA in CH2Cl2(5 ml) and tigecycline (2 ml) was stirred at room temperature until the orange color was not turned into a pale color. After removal of CH2Cl2the residue is again besieged from hexane to obtain pale yellow solid (210 mg, yield 63%).1H IS N, N'-Bis(stearoyl)-2- (diisopropylamino-2 - cyanoethoxyphosphinyl)-1,3 - diaminopropan (29). Compound 28 (210 mg, 0,237 mmol) was dried overnight in a vacuum, was dissolved in anhydrous CH2Cl2(5.0 ml) was added N, N-diisopropylethylamine (218 μl, 1.25 mmol). The solution was cooled in an ice bath and sbrasyvali chloro-(2-cyanoethoxy)-N, N-diisopropylamino-phosphine (106 μl, 0.47 mmol). After stirring for 30 min the reaction mixture was diluted with CH2CL2and washed with 5% Panso3and brine, dried over Na2SO4and concentrated in vacuum to obtain compound 29.31P NMR (Dl3million-1): 154.04.

Algae 20K or 40K PEG-NHS ester with Nucleic Acid-Ligand VEGF

General methods: VEGF oligonucleotide was subjected to exchange with getting triethylammonium salt and liofilizirovanny. The crude oligonucleotide was dissolved in 100 mm sodium borate buffer (pH 9) to a concentration of 60 mg/ml 2 equivalent PEG NHS ester (Shearwater Polymers, Inc. ) was dissolved in dry DMF (Borat: DMF 1: 1), and the mixture was heated to dissolve the PEG NHS ester. The solution of the oligonucleotide was quickly added to the PEG solution, and the mixture is vigorously stirred at room temperature for 10 minutes About 90% of the Acid-Ligand VEGF

Dimeric Nucleic Acid Ligands VEGF shown in Fig. 1J, K and L, were obtained as follows (see diagram A)

Synthesis of 1,3-dipavali-2-O-dimethoxymethylsilane 32

To a stirred solution of compound 31 in pyridine (62 g of 70% pure product, 200 mmol, 200 ml of pyridine), prepared according to McGee et al. (1988, Synthetic Communication, 1651), was added dimethoxyethane (84 g, 240 mmol, 1.2-fold excess) and the reaction was allowed to mix at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in CH2CL2(1 l), washed with water, dried (MgSO4) and concentrated. The crude mixture (130 g) was used as such in the next reaction.

Synthesis of 2-O-dimethoxymethylsilane 33

The crude mixture of compound 32 (130 g), NaOMe (28 g) and methanol (900 ml) was heated at 50oC for 16 hours. After the reaction was complete (TLC), the mixture was concentrated to dryness, and the residue was dissolved in water and CH2Cl2(1: 1). The organic layer was separated, and the aqueous layer was washed with saturated NH4Cl, water, and brine, and dried (MgSO4). Evaporation of the solvent was obtained resinous compound, which was purified by using silicagel is part of the output.1H NMR (DMSO-d6) 3.02-3.07 (m, 2H), 3.17-3.23 (m, 2H), 3.3-3.35 (m, 1H), 3.7 (s, 6H), 4.26 (t, J= 4.1 Hz, 2H, D2O, interchangeable), 6.59-6.86 (m, 4H), 7.17-7.68 (m, 9H).

Synthesis of Biomedica 34

To a chilled on ice, stirred solution of the alcohol 33 (16.2 g, 41,1868 mmol) in CH2CL2(125 ml) and diisopropylethylamine (58 ml, 320 mmol) was added hospitalise reagent (20.5 ml, 90,62 mmol) and the solution was slowly heated to room temperature and was stirred for 2 h at the same temperature. The reaction mixture is slowly poured into crushed ice and was extracted with CH2CL2off , washed with 5% Panso3, water and brine and dried. The residue obtained after evaporation of the solvent, was purified by chromatography on silikagelevye column using 1: 1 hexane/ethyl acetate containing 2% tea, getting a connection 34 at 70% output.1H NMR (DMSO-d6) 1.03-1.12 (2d, 24H), 2.69-2.75 (2t, 4H), 3.1-3.33 (m, 4H), 3.33-3.55 (m, 5H), 3.66-3.7 (m, 4H), 3.72 (s, 6H), 6.83-6.89 (m, 4H), 7.19-7.48 (m, 9H).31R D3RHO4as an external standard 153.64& 153.39.

Receiving VEGF dimers

Synthesis of dimers of VEGF was performed on the 8800 automated DNA/RNA synthesizer. Received NX31838, where ha denotes adenosine, mG and mA means 2'-O-methylguanosine and adenosine, respectively, and FC is TEZ was carried out at a scale of 1 mmol of the automated synthesizer Millipore 8800 using 5'-DMT-2'-O-methyl-N6-tert - butylphenoxyacetyl, 5'-DMT-2'-O-TBDMS-N2-tert - butylphenoxyacetyl and 5'-DMT-2'-O-TBDMS-N6-tert - butylphenoxyacetyl 3'-N, N - aminobutiramida-(2-cyanoethyl) -phosphoramidite, and 2'-deoxy-2'-fluoro-5'-DMT-N4- acetylcytidine and 2'-deoxy-2'-fluoro-5'-DMT - uridine 3'-N, N-aminobutiramida-(2-cyanoethyl)- phosphoramidite. Synthesis cycle consisted of the following. Activator preparations described in table 12. The synthesis was performed using a carrier with CPG pore size 600 , 80-120 mesh and 60-70 µmol/g loaded the 5'-succinimide. The combined cycle is shown in table 12.

Example 6. The pharmacokinetic properties of the phospholipid (PL) and PEG-modified Nucleic Acid Ligand to VEGF.

From the sequences shown in table 2, for further studies was selected sequence VT 30.44 and renamed as NX31838. Pharmacokinetic properties of Nucleic Acid-Ligand VEGF NX31838, conjugated with 20 and 40K PEG was determined in rats, Sprague-Dawley (see molecular image in Fig. 1) (SEQ ID NOS: 8 and 9). Similar studies were also carried out on NX31838, kongugirovannom with PL a lipid, in the form of liposomal drug and free drug (see molecular image in Fig. 1 is a Finance UV absorption at 260 nm and the extinction coefficient 0,037 μg oligo/ml In all studies, 9 rats received 1.0 mg of the oligonucleotide/kg weight of the animal by bolus injection into the tail vein, and plasma samples were taken at different points in time from 2 minutes to 24 hours. Plasma samples and samples quality control were analyzed using hybridization analysis. In hybridization analysis was used oligonucleotide capture, which contains the sequence complementary to the 5'-end of Nucleic Acid-Ligand VEGF conjugated with a drop of ferric oxide (FeO) (FeO-spacer-3'-d (GCC TTA GTC ACT T-5') (SEQ ID NO: 137), where the spacer = (dT)8), and the detection oligonucleotide containing two molecules of Biotin on the 5'-end Biotin-Biotin-5'-d(spacer-GG ATG TAT AAG CA-3'), where a spacer = (dT)8(SEQ ID NO: 138). After incubation of the capture probe and the detection probe from the plasma sample containing Nucleic Acid-Ligand VEGF NX31838, the number bioteknologi of the oligonucleotide, hybridizing with straw, quantify streptavidin-linked alkaline phosphatase using CSPD-Sapphire as a fluorescent substrate.

Data on plasma concentrations of free, PEG-20K and 40K PEG Nucleic Acid-Ligand VEGF (NX31838) (SEQ ID NOS: 8 and 9) as a function of time after bolus injection sumerologist much faster and 95% Nucleic Acid-Ligand were derived from alpha t1/249 minutes, and 5% were derived from beta t1/2192 minutes, which indicates that an explicit size value for clearance. Compared with PEG-conjugated Nucleic Acid-Ligand-free (unconjugated) NX31838 were derived from plasma very rapidly with t1/2in a few minutes. The concentration of the oligonucleotide in plasma as a function of time can be considerably enhanced by the inclusion of suitable functional groups in the oligonucleotide.

Data on concentrations in plasma PL lipid-conjugated Nucleic Acid-Ligand VEGF (SEQ ID NOS: 5), made in the form of drug from liposomes or without liposomes as a function of time after bolus injection are summarized in Fig. 7. Liposomes prepared as described in example 7A, with the help of ultrasonic treatment in the presence of Nucleic Acid-Ligand, and they contain oligonucleotide, both inside and outside. Liposomal drug was eliminated more slowly than free drug, beta t1/21161 minute and 131 minutes, respectively. The concentration of the oligonucleotide in plasma as a function of time can be significantly improved through liposomal drug.

the s combine in the ratio of 2 mol of DSPC to 1 pray cholesterol. NX31838 PL in water is added to the lipids in the ratio of 1: 50 (mass. /mass. ). Material combine through solutionone solution of chloroform: methanol: water (1: 3: 1). The solvent is removed by rotary evaporation, leaving a heterogeneous film NX 31838 PL, mixed together with lipids. Film rehydration to 50 mg/ml lipid based in a solution of 9% sucrose, buffered 25 mm sodium phosphate at pH 7.4. The solution is vigorously stirred, heated to 65oC, and the resulting white like milk solution is treated with ultrasound in a 75 ml aliquot for the Assembly of lipid single-layer liposomes. The progress in the formation of liposomes monitored visually until the solution becomes opalescent, and then by measuring the particle size by dynamic light scattering using particle analyzer (Leeds & Northrup Model Microtrack UPA 150, Horsham, PA). The size of the liposomes is in the range from 50 to 70 nm (according to the method of volume-mass distribution).

B. Obtaining liposomes by passive zakalivanie

Tested scNX-278 (see molecular image in Fig. 1C), to see if he undergoes spontaneous incorporation into preformed (empty) liposomes. Preliminary results with the is important discoveries: 1) the load can be achieved and, more importantly, 2) essentially full load of Complex Nucleic Acid-Ligand/glycerolipid was observed after 24 hours at room temperature. It was subsequently undertaken a more detailed study to determine the effects of temperature on the load. Observed that the temperature clearly affects the speed of switching. Although the full load can be achieved after 24 hours at room temperature, and full inclusion can be achieved even within minutes at elevated temperatures (67oC). This proved to be a quick and effective way of inclusion Complex of Nucleic Acid-Ligand/Lipophilic Compound into preformed liposomes.

Chromatography on size then used to separate free scNX-278 from liposome-associated form. Preliminary work was carried out using a load scNX-278 in "empty" 2: 1 DS: cholesterol liposomes. The chromatogram was obtained using column Superdex S-200 on 22oC. Over a period of 22 hours was observed gradual inclusion of scNX-278 in the population of empty liposomes in the form of a shift in peak areas (data not shown). These results correlate with the data obtained in DEAE analysis.

what's ultrasound oligo-liposomes. Voiced drug scNX-278 was obtained by joint dilution oligo-lipid from the lipid and joint processing ultrasound of both. The obtained liposomes showed the full inclusion of scNX-278. This ultrasonic assisted the preparation is then subjected to 2 separate rounds passive zakalivanie with additional free scNX-278, to see whether successfully included a greater number of scNX-278. During the first round of passive zakalivanie all free scNX-278 was passively anchored in the liposomes after incubation for 1 hour at 65oC. the Second attempt passive zakalivanie additional scNX-278 led to incomplete download.

A key discovery of these experiments is that the Complex of Nucleic Acid-Ligand/Lipophilic Compound may be passively anchored in voiced oligo-liposomes at high concentrations, but that the capacity of liposomes to absorb additional Complexes of Nucleic Acid-Ligand/Lipophilic Compound may be exceeded. After 2 rounds passive load (up to approximately 3 mg of lipid-oligo/50 mg of lipid), liposomes obviously reach its capacity of absorption of additional oligo-lipid, because some free Lina). The conclusions that can be made are: 1) announced liposomes have additional capacity for inclusion Complexes of Nucleic Acid-Ligand/Lipophilic Compound; and 2) 100% incorporation of the Nucleic Acid Ligand can be achieved through sound.

Subsequent studies were carried out on NX31838 PL (see molecular image in Fig. 1E). NX31838 is of considerable interest, because it has improved pharmacokinetics (see example 6) and biological distribution against VEGF targets for inclusion in liposomes. Several studies have been conducted to better understand the inclusion NX31838 in liposomes through passive zakalivanie.

Studies on the kinetics NX31838 PL indicated that passive zakalivanie for this molecule was so quick that it was considered impossible to measure by any of chromatographic methods known in the literature (for all of them require time run at least a few minutes).

To determine the orientation of the molecules NX31838 PL (that is, if a component is Nucleic Acid-Ligand acting on the outside of the liposomes or the speakers inside the aquatic center liposomes is s-Ligand, which acts on the outside of the liposomes. In the case of passively anchored liposomes NX31838 PL all Nucleic Acid-Ligand is available for RNase I. No additional splitting was not observed after treatment with Triton X-100. These results indicate that passively loaded NX31838 PL oriented so that the Nucleic Acid Ligand is outside of the liposomes. If passively anchored liposomes NX31838 PL previously digested by RNase I, and then passed through DEAE column, about 99% Nucleic Acid Ligand is retained by the column, whereas if the same sample is passed through DEAE column, but without inactivated by RNase I, about 100% oligo able to pass through the column, not contacting DEE. Liposome protects the oligo from DEAE. Liposome acts to protect the component Nucleic Acid Ligand from DEAE, because it connects with Nucleic Acid-Ligand with high affinity, significantly reducing its availability for DEAE groups.

Finally, as part of the development of new methods of separation of the free Complex of Nucleic Acid-Ligand/Lipophilic Compound from anchored in liposome form, the inventors were digested-1000) after removal of the lipid tail, whereas the intact Complex of Nucleic Acid-Ligand/Lipophilic Compound in identical conditions was elyuirovaniya together with the liposome. These data indicate that the Complex of Nucleic Acid-Ligand/Lipophilic Compound, probably forms a micelle with the free roaming in the solution. This leads to the fact that it co-eluted in the free volume of the column with liposomes. Removal of the lipid tail allows him to enter on Wednesday, gel filtration, split it according to the size and save.

Example 8. The efficiency of in vivo Complexes of Nucleic Acid-ligand VEGF - analysis of cutaneous vascular permeability

The ability of several different preparations of Nucleic Acid-Ligand NX31838 to the weakening of VEGF-induced changes in the permeability of the skin vascular system (analysis of miles) was performed as described previously (Senger et al. (1986) Cancer Research 46: 5629-5632), with minor modifications. Briefly, adult female Guinea pigs (3/study) was anestesiologi with isoflurane, and the hair on the dorsal side and rear areas were removed with scissors. Blue dye Evans (2.5 mg/Guinea pig) was administered intravenously. Solutions for injection (drugs of the FSB, VEGF, NX31838 and monoclonal antibody anti illustrates the solution then was intradermally injected with (double injection/Guinea pig, 40 μl/site) randomly on the grid drawn on the shaven area. Guinea pigs were allowed to recover from anesthesia and squashed them through exposure to CO2within 30 min after intradermal injection. Then the skin was collected, purified from the subcutaneous layer and subjected transillumination. The image was shot using a color CCD camera (Hitachi Denshi KP-50U, Japan) and the software Image-Pro Plus (version 3.1, Media Cybernetics, Silver Springs, MD). Each sample of the skin were normalized in intensity, and each site of injection were analyzed for optical density and involved area.

In Fig. 8A-C shows the results of the weakening of the Nucleic Acid-Ligand VEGF-induced leakage from vessels NX31838-20K PEG, NX31838-40K PEG, NX31838 in PL liposomal preparation as described in example 7A. All drugs were able to significantly reduce the flow of blood vessels near or below the reference levels of the FSB in such low concentrations as 100 nm. At 30 nm blocking activity of Nucleic Acid-Ligand disappeared. Liposomal drug NX31838-PL not rated at this concentration, but it turned out that he has reduced blocking activity at 100 nm. The monoclone is her activity at 30 nm. This suggests that in this model system, which was studied NX31838 in different preparations, it is as effective as the antibody, by blocking one of the functional effects of protein VEGF.

Example 9. The efficiency of in vivo Complexes of Nucleic Acid-Ligand VEGF - model of corneal pocket

Preparations of Nucleic Acid-Ligand VEGF (NX31838) were tested for their ability to reduce VEGF-induced corneal angiogenesis in rats with natural avascular cornea. Briefly, a biopolymer (Hydron) granules VEGF protein (3 pmol) were prepared in about 30 h before adding a solution of the protein or the media to 12% of the biopolymer in 95% ethanol. Adult rats Sprague-Dawley (200-240 g) were anestesiologi by intraperitoneal injection of ketamine HCl (50 mg/kg) and xylazine (10 mg/kg). Then preparing the left eye by local injection of tetracaine HCl for local anesthesia followed by the application of a dilute solution of povidone-iodine and subsequent washing with isotonic saline solution. Did partial vertical cut in the thickness in the middle of the cornea. On average, its own substance cut pocket Caudalie towards the lateral corner of the eye slits, continuing within 1.5 m of the co massaged from his pocket. Then in the eye put a drop of ophthalmic solution chloramphenicol. The animal turned, and the operation repeated on the right eye with insert pellets of the same type. After insertion of pellets in each eye of each animal was then injected or FSB (volume equal to the volume group of drugs Nucleic Acid-Ligand), or Nucleic Acid-Ligand (10 mg/kg) intravenously twice a day as directed. After 5 days each animal was anestesiology and receive pictures using a 35 mm camera (Minolta X9) mounted on a dissecting microscope (KAPS, Germany). Each eye was evaluated on the angiogenic response by measuring the maximum length of vascular growth (0-5), density growth of blood vessels (1-4), adjacent to the implanted pellet, and the circumference of the eye with resulting angiogenesis (0-1). Then determined angiogenic index as the product of lineplotstrategy.

The ability of drugs Nucleic Acid Ligand to block VEGF-induced angiogenesis is visible in Fig. 9A-C. Although NX31838-20K PEG was equally effective as other drugs, by blocking the changes in vascular permeability, it has been ineffective in reducing angiogenic response in natural Bess is 70%. Assume that these differences can be attributed to the relevant pharmacokinetic profiles of Nucleic Acid Ligands.

Statistical analysis: Groups in the analysis of miles and models of corneal angiogenesis compared using Rank ANOVA comparisons Dunnett.

Example 10. The efficiency of Nucleic Acid-Ligand in vivo tumor models

The model of xenograft tumors of human: the Ability of Nucleic Acid-Ligand VEGF NX31838 40K PEG to influence the growth of solid tumor was identified in the subcutaneous tumor model in nude mice. Tumor cells of human rhabdomyosarcoma A were grown in tissue culture, collecting, and 1107viable cells were implanted subcutaneously to nude mice proximally to the axial area of the flanks. The processing of the tested compounds was started 12 hours after that and continued throughout the experiment. Compounds were dosed out intraperitoneally twice a day for 10 and 40 mg/kg Negative control consisted of dispensing mixed sequence of appropriate size, NX31917-40K PEG (see molecular image in Fig. 1R) 40 mg/kg twice a day, and the positive control consisted of antibodies against VEGF Mab. 26503.11 (R& D Systems, Lot # uppy, processed antibody showed a significant slowing of tumor growth relative to the negative control group mixed sequence (Fig. 11). % Inhibition of tumor growth (RBI) was defined as 75% and 80% for BID (twice a day) groups of 40 mg/kg and 10 mg/kg and 83% for the group treated with a monoclonal antibody (table 8). Because between-group dose of 40 mg/kg and group dose of 10 mg/kg was not significantly different after day 14 further dosing of 40 mg/kg did not repeat. As can be seen in Fig. 11, several days after cessation of dosing tumors grew rapidly and imitated the growth rate of the negative control group, whereas in the group of 10 mg/kg Nucleic Acid-Ligand and in the group treated with the antibody, they continued to grow at a reduced rate.

Additional studies were performed using the same tumor model, where he compared the new party Nucleic Acid-Ligand VEGF NX31838 40K PEG (marked NX31838.04 and NX31838.07), and the dose titrated with a decrease of 10 mg/kg BID, 3 mg/kg BID and 1 mg/kg BID. The experiment also included a dose of 10 mg/kg once per day, as well as liposomal form a Nucleic Acid-Ligand VEGF, NX31838 PL at the dose of the regulation of tumor growth. Both parties Nucleic Acid-Ligand VEGF was equivalent when comparing the dosing regimen twice a day, with values RBI 61% and 70% for the old and the new party, respectively. In addition, it was determined that dosing once daily (SID) was as effective as dosing twice a day.

Conducted the third experiment, where further downward titration of Nucleic Acid-Ligand VEGF was able to demonstrate the relationship between the dose/response against tumor growth. In this experiment, Nucleic Acid-Ligand VEGF was titrated to decrease, reaching the no-effect dose of 0.03 mg/kg Relative inhibition of tumor growth can be seen in Fig. 13 and summarized in table 10.

In addition to the three studies with unsteady tumor, conducted a study with established tumor, where the tumor was allowed to set and achieve 200+/-100 mm3before processing, Nucleic Acid-Ligand VEGF. Group dose of 10 mg/kg NX31838 40K PEG and 100 µg twice a week mAb 26503 (R& D Systems) was 59% and 69% inhibition of tumor growth, respectively (Fig. 14, table 11). These aggregate studies demonstrate that Nucleic Acid-Ligand VE, when the tumor has been established.

A model of Kaposi's sarcoma: Investigated the impact NX31838-40K PEG on the subcutaneous growth of cell lines of Kaposi's sarcoma KSY-1 nude mice. Cells KSY-1 are unique among tumor cell lines in that they can inhibit in culture with VEGF antagonists. Cells KSY-1 were grown in culture were collected and were injected with subcutaneous (2107cells/mouse) at the rear side of the mice. Three groups of mice (4 mice per group) were treated by intraperitoneal injection every 12 hours or 30 mg/kg NX31838-40K PEG, or 30 mg/kg NX31917-40K PEG (see molecular image in Fig. 1R), or the FSB during the whole experiment. Treatment was started after one day after implantation of tumor cells. Despite the fact that tumor growth in the treated FSB and processed NX31917-40K PEG groups were compared, a significant inhibition of tumor growth was observed in the treated NX31838-40K PEG group (Fig. 16). NX31838-40K PEG inhibited tumor growth KSY-1 at 65% (compared to processed FSB) or 69% (compared to processed NX31917-40K PEG group) at the time when the experiment was completed (day 22).

Example 11. Vnutristenocna pharmacokinetics of Nucleic Acid-Ligand VEGF NX31838+40K PEG at the th with 40m PEG, by vnutrisloinogo a dose of 0.5 mg/eye. 40K PEG conjugatively with Nucleic Acid-Ligand VEGF, as described in example 5, and the resulting Complex was as shown in Fig. 1H (SEQ ID NO: 8). The rabbits were vnutritelostnoe injection NX31838 40K PEG in each eye. The time between doses for this animal did not exceed 15 minutes. Blood and vitreous samples were collected, as described in table 7.

Sample analysis plasma and vitreous body were performed using a dual hybridization analysis. This analysis used two probe hybridization, probe grip attached to the wells of 96-hole boards, and biotinylated detection probe. The capture probe forms a hybrid with the 5' end of the Nucleic Acid Ligand. This analysis is highly specific and sensitive full-sized Nucleic Acid-Ligand obtaining a positive signal. The usual limit of quantitation of approximately 2 pmole 5 ál of plasma. T

1. Purified and dedicated not naturally occurring RNA ligands to vascular endothelial growth factor (VEGF), and the specified ligand has a nucleotide sequence selected from the group is from, those having the formula:

A-B-Y,

where a is polyalkyleneglycol having the structure R(O(CH2)x)nO-, where R is independently selected from the group consisting of N and CH3x= 2-5, and nMW (molecular weight) of polyalkyleneglycol/ 16+14;

In one or more linkers,

Y - RNA ligands to VEGF under item 1.

3. Complex p. 2, where the specified polyalkyleneglycol is a polyethylene glycol.

4. Complex on p. 3, where the specified polyethylene glycol has a molecular mass of about 10-80K.

5. Complex on p. 3, where the specified polyethylene glycol has a molecular mass of about 20-C.

6. Complex on p. 3, where this Complex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

7. Complex on p. 3, where this Complex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

8. Complex on p. 3, where this Complex represents (see graphic part).

Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)7

9. Complex on p. 3, where this Complex represents (see graphic part).

Ligand component = fmGmGrArAfUfCmAmGfU the practical part).

Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

11. Complex p. 2, where this Nucleic Acid Ligand to VEGF was identified from a Mixture of Candidate Nucleic Acid according to the method, in which (a) bring into contact the Mixture of Candidates with VEGF, and Nucleic Acids having an increased affinity to VEGF relative to the Mixture of the Candidates can be separated from the remainder of the Mixture of Candidates; b) separating the Nucleic Acids with high affinity from the remainder of the Mixture of the Candidates, and C) amplified Nucleic Acids with high affinity with the mixture of Nucleic Acids enriched in Nucleic Acids with increased affinitiy to VEGF, whereby obtain Nucleic Acid Ligands VEGF.

12. Complex on p. 11, where the method further includes repeating steps (b) and (C).

13. Complex having the formula

A-B-Y,

where a represents glycerolipid having the structure

< / BR>
where R1and R2- CH3(CH2)n-O(PO3)-CH2-; CH3(CH2)n-N2-CH2-, CH3(CH2)nO, CH3(CH2)nOCH2-, where n= 10-20;

R
Y - RNA ligands to VEGF under item 1.

14. Complex p. 13, where the specified glycerolipid is a phospholipid, where at least one of the1and R2- CH3(CH2)n-O(PO3)-CH2-.

15. Complex p. 14, where R1- CH3(CH2)n-O(PO3)-CH2-, R2- CH3(CH2)n-O(PO3)-CH2-, and n= 17.

16. Complex p. 15, which-X - represents a -(PO4)-.

17. Complex p. 13, where the specified glycerolipid is glyceroglycolipid, where at least one of R1and R2- CH3(CH2)n-N2-CH2-.

18. Complex p. 17, where R1- CH3(CH2)n-N2-CH2-, R2- CH3(CH2)n-N2-CH2-, and n= 16.

19. Complex p. 18-X - represents a -(PO4)-.

20. Complex p. 18-X - represents-O-.

21. Complex p. 13, where R1- CH3(CH2)n-O-, R2- CH3(CH2)n-OCH2-, R3- CH2OS(O) and n= 17.

22. Complex p. 13, where this Nucleic Acid Ligand to VEGF was identifiziert the s with VEGF, moreover, Nucleic Acids having an increased affinity to VEGF relative to the Mixture of the Candidates can be separated from the remainder of the Mixture of Candidates; b) separating the Nucleic Acids with high affinity from the remainder of the Mixture of the Candidates, and C) amplified Nucleic Acids with high affinity with the mixture of Nucleic Acids enriched in Nucleic Acids having an increased affinity to VEGF, whereby obtain Nucleic Acid Ligands to VEGF.

23. Complex p. 22, where the method further includes repeating steps (b) and (C).

24. Lipid Structure containing Complex under item 2.

25. Lipid Structure containing Complex under item 13.

26. Lipid Structure on p. 25 containing Complex under item 14.

27. Lipid Structure on p. 26 containing Complex under item 15.

28. Lipid Structure on p. 27 containing Complex under item 16.

29. Lipid Structure on p. 28, where this Complex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

30. Lipid Structure on p. 25 containing Complex under item 17.

31. Lipid Structure on p. 30. content is Edna Design by p. 32, where the specified Complex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

34. Lipid Structure on p. 31 containing Complex on p. 20.

35. Lipid Structure on p. 34, where this Complex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

36. Lipid Structure on p. 25 containing Complex on p. 21.

37. Lipid Structure on p. 36, where this Complex represents (see graphic part).

Ligand component = 5'-TsTsTsTsmGaUaCmGmGaUmAaCrGmGrAmGaumgrgraacacmgauacmaacmgtstststst-3' (ligand VEGF)

38. A method of treating a VEGF-mediated disease, which is administered pharmaceutically effective amount of the Complex under item 2 or 13.

39. The method according to p. 38, where the injected Complex p. 2, where the specified polyalkyleneglycol is a polyethylene glycol.

40. The method according to p. 39, where the specified polyethylene glycol has a molecular mass of about 10-80K.

41. The method according to p. 39, where the specified polyethylene glycol has a molecular mass of about 20-C.

42. The method according to p. 41, where this Complex has the structure

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmA niteline connected with the lipid construct.

44. The method according to p. 43, where the specified Complex has the structure

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

45. The method of producing Complex p. 2, consisting of a Nucleic acid Ligand to VEGF under item 1 and polyalkyleneglycol under item 2, connected to a specified Nucleic Acid Ligand to VEGF via at least one linker, wherein (a) identify Nucleic Acid Ligand to VEGF from a Mixture of Candidate Nucleic Acid using a method in which (b) bring into contact the Mixture of Candidates with VEGF, and Nucleic Acids having an increased affinity to VEGF relative to the mixture of the Candidates can be separated from the remainder of the Mixture of the Candidates;) separate Nucleic Acids with high affinity to VEGF from the remainder of the Mixture of the Candidates; g) amplified Nucleic Acids with high affinity to VEGF with obtaining enriched ligands mixture of Nucleic Acids and d) connect the specified identified Nucleic Acid-Ligand with polyalkyleneglycols.

46. The method according to p. 45, wherein the specified Complex, combined with a Lipid Construct.

47. The method according to p. 46, where this Lipid Structure is a SS="ptx2">

49. The method according to p. 48, where the specified polyethylene glycol has a molecular mass of about 10-80K.

50. The method according to p. 49, where the specified polyethylene glycol has a molecular mass of about 20-C.

51. The method according to p. 50, where this Complex has the structure

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

52. The method according to p. 50, where this Complex has the structure

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

53. The method according to p. 50, where this Complex has the structure (see graphic part).

Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

54. The method according to p. 50, where this Complex has the structure (see graphic part).

Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF)

55. The method according to p. 50, where this Complex has the structure (see graphic part).

Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

56. The method of producing Complex p. 13 consisting of a Nucleic Acid Ligand to VEGF under item 1 and glycerolipid on p. 13, connected to a specified Nucleic Acid Ligand to VEGF via at least one link is by way in which b) bring into contact the Mixture of Candidates with VEGF, and Nucleic Acids having an increased affinity to VEGF relative to the Mixture of the Candidates can be separated from the remainder of the Mixture of Candidates; C) separating the Nucleic Acids with high affinity to VEGF from the remainder of the Mixture of the Candidates; g) amplified Nucleic Acids with high affinity to VEGF with obtaining enriched ligands mixture of Nucleic acids and d) connect the specified identified Nucleic Acid-Ligand with glycerolipids.

57. The method according to p. 56, wherein the specified Complex, combined with a Lipid Construct.

58. The method according to p. 57, where this Lipid Structure is a Liposome.

59. The method according to p. 58, where the specified Complex consists of a Nucleic Acid Ligand and glycerolipid and specified Complex passively connected to Balaam these Liposomes using a method comprising stages a) forming liposomes and (b) mixing the specified Complex consisting of a Nucleic Acid Ligand and glycerolipid, with liposomes stage (a), whereby the Component of the Nucleic Acid Ligand of the specified Complex becomes United with biolex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

61. The method according to p. 57, where the specified Complex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

62. The method according to p. 57, where the specified Complex is a

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

63. The method according to p. 57, where the specified Complex represents (see graphic part).

Ligand component = 5'-TsTsTsTsmAaCaCaUrGrAaUmGrGaUmAmGraacmgacacmgmgmgmgaumgtstststst-3' (ligand VEGF).

64. Method of reducing the clearance of plasma Nucleic Acid-Ligand VEGF, in which the patient is given a complex p. 2 or 13.

65. The way the direction of therapeutic or diagnostic agent to a specific predetermined biological target, which expresses VEGF in a patient, wherein the patient is administered a complex p. 2 or 13.

66. A method of inhibiting VEGF-mediated angiogenesis, in which the patient is given a complex p. 2 or 13.

67. Method of inhibiting the growth of tumors, in which the patient is given a complex p. 2 or 13.

68. The method according to p. 67, where these tumors or cells Il is Bo cells or tissue, surrounding these tumors Express VEGF receptors.

70. The method according to p. 69, where these tumors are composed of cells of Kaposi's sarcoma.

71. The method of inhibition of macular degeneration, in which the patient is given a complex p. 2 or 13.

72. The method according to p. 71, where the injected complex under item 2 and where specified polyalkyleneglycol is a polyethylene glycol.

73. The method according to p. 72, where the specified polyethylene glycol has a molecular mass of about 10-80K.

74. The method according to p. 72, where the specified polyethylene glycol has a molecular mass of about 20-C.

75. The method according to p. 74, where the specified complex has the structure

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

76. The method of prolongation of the action is in the eye of Nucleic Acid-Ligand VEGF, which in the eyes injected with the complex under item 2 or 13.

77. Complex having the formula

< / BR>
Ligand component = fmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfufumafumafcmafufcfcmg-3'3'-dT (ligand VEGF).

Priority signs and items:

25.10.1996 on p. 1: characteristics relating to the sequences of SEQ ID Nos 1-4;

on PP. 2-5, 11-20, 21 with signs relating to the sequences of SEQ ID Nos 1-4;

on PP. 37 and 63;

21.07.1997 - PP. 2-5, 11-20, 21, 22-23, 45, 48-50 and 56 with signs relating to the sequences of SEQ ID Nos 5-12 and 15-90;

in p. 76;

17.10.1997 - signs related sequences SEQ ID Nos 13-14 and 91-132;

with features related to sequences, in addition to SEQ ID Nos 5-12 and 15-90;

on PP. 24-28, 30-32, 34, 36, 43, 44, 46-47, 57-59, 60-62, 67-70.

 

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