Fused rage proteins

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

 

This application claims priority under provisional application for U.S. patent number 60/943, 994, filed on June 14, 2007, the full content of which is incorporated herein specifically by reference.

The scope of the invention

The present invention relates in General to the end products of glycation (AGE) and in particular to certain fused proteins, which contain the receptor for glycation end products ("RAGE"). Slit proteins according to the invention associated with AGE and other RAGE ligands (e.g., HMGB1, S100), and compositions containing the fused protein according to the invention can be used for the treatment of diseases.

Prior art

The end products of glycation (AGE) are the result of nonenzymatic glycation and oxidation of proteins. They appear in stress conditions, leading to autoimmune diseases of connective tissue, and can be formed in inflamed tissue due to oxidation or myeloperoxidase way. AGE is included in the number of diabetes-related complications. For example, the characteristic structural changes in diabetic nephropathy, thickened glomerular basement membrane and mesangial expansion accompanied by the accumulation of AGE, leading to glomerulosclerosis and interstitial fibrosis. Prolonged infusion of AGE in neadiabaticheskikh rats leads to the development of the such morphological changes and significant proteinuria. It was shown that AGE inhibitors such as aminoguanidine prevents diabetic nephropathy in diabetic animal models, and, as recently demonstrated, doing the same thing in one clinical study on patients with diabetes. Also AGE are approved therapeutic target in diabetic retinopathy. Extensive research on diabetic mice and rats demonstrate the benefits of inhibiting the formation of AGE in the treatment of this disease.

The accelerated atherosclerosis in patients with diabetes and is associated with an increased risk of mortality from cardiovascular and cerebrovascular diseases. Studies in humans and animals suggest that AGE will play a significant role in the formation and development of atherosclerotic lesions. Increased AGE accumulation in diabetic vascular tissues is associated with changes in the functioning of endothelial cells, macrophages and smooth muscle cells.

AGE interact with receptors on the surface of cells to monocytes, macrophages, endothelial cells of the microvasculature, smooth muscle cells, mesangial cells and neurons. The receptor for glycation end products (RAGE) is a member of the immunoglobulin superfamily of receptors on the cell surface. RAGE consists of three unclutch what's the immunoglobulin-like domains, the transmembrane domain and cytoplasmic domain, which is involved in the signalling system. RAGE binds many ligands in addition to AGE, including S100/calgranulin, amphoteric/HMGB1 and amyloid fibrils. RAGE working through a signaling cascade involving NF-κ. The expression of RAGE is activated in the presence of ligands of RAGE and increases in the joints of subjects with rheumatoid arthritis (RA).

RAGE has secreterial the isoform that does not have a transmembrane domain, called soluble RAGE (sRAGE). As shown, the introduction of sRAGE restores wound healing (Goova, et al. (2001) Am. J. Pathol. 159, 513-525) and inhibits diabetic atherosclerosis (Park, et al. (1998) Nat Med. 4(9): 1025-31). Fused protein consisting of the ligand-binding element RAGE, and immunoglobulin element, discussed in WO 2004/016229 A2 (Wyeth, Madison, NJ) and the publication of the patent application U.S. 2006/0057679 A1 (O'keefe, T. et al.).

There is a need for new treatments for AGE-mediated diseases, such as diseases that are associated with an increased number of AGE. This need and others are satisfied by the present invention.

Summary of the invention

The present invention provides materials and methods for treatment of diseases associated with an increased number of AGE. In one embodiment, the present invention provides a fused protein content is the seer, at least one polypeptide, comprising: (a) a first amino acid sequence at least 95% identical to the ligand-binding domain of the receptor end glycation product (RAGE) mammal, and the first amino acid sequence capable of binding a ligand of RAGE; and (b) a second amino acid sequence at least 95% identical to the constant domain of the heavy chain immunoglobulin IgG4 person or its fragment; where the first amino acid sequence contains at least one mutation compared to the ligand-binding domain of RAGE wild type. In one embodiment of the invention fused protein according to the invention may also contain linker sequence between the first amino acid sequence and second amino acid sequence. In some embodiments, the implementation of the ligand-binding domain of RAGE can be from the RAGE of a mammal, for example, RAGE man. Suitable ligand-binding domain of RAGE mammal may contain amino acids 1-344 of SEQ ID NO:6 or amino acids 24-344 of SEQ ID NO:6. In one embodiment of the invention fused protein according to the invention may contain amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In one embodiment, the invention you lenny protein according to the invention contains SEQ ID NO:6 or SEQ ID NO:8. In another embodiment of the invention selected protein according to the invention consists of SEQ ID NO:6 or SEQ ID NO:8. In some embodiments of the invention fused proteins according to the invention may also contain linker between the amino acid sequence of RAGE and amino acid sequence of IgG4. The present invention also considers the molecules of nucleic acids (e.g., DNA or RNA)encoding the fused protein of the invention, and cells of the host expressing the nucleic acid molecule encoding the fused protein according to the invention.

The present invention also provides a pharmaceutical composition comprising a protein according to the invention and a pharmaceutically acceptable excipient or diluent.

The present invention provides methods of treating diseases mediated AGE. Such diseases include any disease characterized by an increased number of AGE of the subject, e.g. a mammal such as man. Methods of treating AGE-mediated diseases have an introduction to a subject having AGE-mediated disease, a therapeutically effective amount of a pharmaceutical composition comprising a protein according to the invention. Examples of diseases that can be cured by the methods of the invention include, but are not limited to the Xia this, diabetic nephropathy, rheumatoid arthritis and autoimmune diseases, such as dermatitis, glomerulonephritis, multiple sclerosis, sympathetic ophthalmia, autoimmune pulmonary inflammation, insuli-Nezavisimy diabetes, autoimmune ocular inflammation, systemic lupus erythematosus, insulin resistance, rheumatoid arthritis, diabetic retinopathy and scleroderma. Any protein according to the invention can be used in the practice of the methods according to the invention. In one embodiment, the methods according to the invention can be carried out using a fused protein containing SEQ ID NO:6 or SEQ ID NO:8. In another embodiment, the methods according to the invention can be carried out using a protein, which consists of SEQ ID NO:6 or SEQ ID NO:8.

In another embodiment of the invention the present invention provides methods of reducing levels of ligand binding by RAGE, in a mammal (e.g. human)in need of this. Such methods can include administration to a mammal RAGE ligand-reducing the number of fused protein according to the invention.

In other embodiments, implementation of the invention provides a recombinant expression vector containing the DNA sequence according to the invention; cell host transformed, translationand the Yu or transtitional this vector; and method of producing fused protein, which includes culturing the host cell transformed, transductional or transtitional nucleic acid that encodes a protein according to the invention under conditions suitable for the implementation of the expression of the fused protein.

The invention also provides compositions containing the present protein or its fragments. In some embodiments implementing the invention includes compositions containing the present protein or its fragments, to which is directly or indirectly attached radioisotope, a chelator, a toxin, fluorochrome, Biotin, peptide epitopes, such as his-tags, myc-tags, or sugar. Other embodiments of the invention include a real protein, fused with another protein to modify the half-life of the organism or biological functions, and glycosylated variants fused protein.

These and other aspects of the present invention will become clear upon reference to the following detailed description.

Brief description of drawings

Figure 1 presents a bar chart showing the impact of illustrative fused protein RAGE-Ig on lakotas in the mouse model with streptozotocin-induced diabetes.

Figa-2D are bar charts showing the impact of the illustration is active fused protein RAGE-Ig on the permeability of retinal vessels in different layers of the retina in the mouse model with streptozotocin-induced diabetes.

Figure 3 is a bar chart showing the impact of illustrative fused protein RAGE-Ig on the nitration of proteins in the retina in the mouse model with streptozotocin-induced diabetes.

Figure 4 is a bar chart showing the impact of illustrative fused protein RAGE-Ig on the expression of ICAM in the retina in the mouse model with streptozotocin-induced diabetes.

Figa presents a bar chart showing the impact of illustrative fused protein RAGE-Ig on the number of acellular capillaries, measured in square mm tissue of the retina in diabetic mice after 10 months of diabetes. Figv presents a bar chart showing the impact of illustrative fused protein RAGE-Ig on the number of "shadows" perezito 1000 capillary cells, measured in diabetic mice after 10 months of diabetes.

6 is a bar chart showing the impact of illustrative fused protein RAGE-Ig 50% reaction threshold to touch in diabetic mice after 10 months of diabetes.

7 provides a block diagram illustrating the experimental Protocol of Example 3.

Fig is a line graph demonstrating the effect of illustrative fused protein RAGE-Ig on the weight of the test animal rat model of arthritis, and dozirovannym collagen type II.

Figure 9 is a bar graph showing the effect of illustrative fused protein RAGE-Ig on the number of cases for arthritis mouse model of arthritis induced by collagen type II.

Figure 10 is a bar graph showing the effect of illustrative fused protein RAGE-Ig at the beginning for arthritis mouse model of arthritis induced by collagen type II.

11 is a line graph demonstrating the effect of illustrative fused protein RAGE-Ig on the number of cases of arthritis in the form of a time function for mouse model of arthritis induced by collagen type II.

Fig is a line graph demonstrating the effect of illustrative fused protein RAGE-Ig on the severity of arthritis in the form of a time function for mouse model of arthritis induced by collagen type II.

Fig is a line graph demonstrating the effect of illustrative fused protein RAGE-Ig on the number of legs with arthritis, measured as the time function to a mouse model of arthritis induced by collagen type II.

Figa-14D are microphotographs showing the impact of illustrative fused protein RAGE-Ig on the morphology of the joints as a function of an increasing number of fused protein to a mouse model of arthritis induced by collagen type II.

Fig is a bar chart is Moi, demonstrate the impact of illustrative fused protein RAGE-Ig on synovitis (black columns) and pannus (gray columns) for a murine model of arthritis induced by collagen type II.

Fig is a bar graph showing the effect of illustrative fused protein RAGE-Ig at the regional erosion (black columns) and architecture changes (gray columns) for a murine model of arthritis induced by collagen type II.

Fig is a bar graph showing the effect of illustrative fused protein RAGE-Ig on full histological assessment of arthritis for a murine model of arthritis induced by collagen type II.

Fig is a bar graph showing the effect of illustrative fused protein RAGE-Ig on the loss of the protein matrix of the joint for a murine model of arthritis induced by collagen type II.

Figa-19D are microphotographs of sections stained with toluidine blue showing the impact of illustrative fused protein RAGE-Ig on the loss of the protein matrix of the joint for a murine model of arthritis induced by collagen type II.

Detailed description of the invention

Definition

Used herein, the terms "receptor end glycation product or RAGE refer to proteins having amino acid sequences that are essentially similar to nationalisation sequence RAGE mammals, and the linking of one or more ligands of RAGE in a specific way the ligand-receptor. The terms "end product of glycation" and "AGE" refers to a heterogeneous group of molecules formed by the nonenzymatic reaction of reducing sugars with free amino groups of proteins, lipids and nucleic acids, as described above.

When used here "ligand-binding domain of RAGE or RAGE-LBD" refers to any protein RAGE mammal or any part of the RAGE protein of a mammal, which retains the ability to bind a ligand of RAGE in a specific way the ligand-receptor. In particular, without limitation, the ligand-binding domain of RAGE comprises a polypeptide having one or more extracellular domains of transmembrane protein RAGE. According to Table 6, suitable RAGE-LBD may contain at least amino acids 1-99, or amino acids 24-99, or amino acids 1-208, or amino acids 24-208, or amino acids 1-301, or amino acids 24-301, or amino acids 1-344, or amino acids 24-344 of SEQ ID NO:6.

The term "isolated" when used in the context of this description to determine the purity of the fused protein means that the protein is not essentially contains other proteins with which he was connected during production, including, without limitation, essentially the absence of other proteins that are present during the expression of the fused protein to emochnoy culture medium. For example, the isolated protein according to the invention may contain 1 to 25%, 20-25%, 15-20%, 10-15%, 5-10%, 1-5%, or less than about 2 wt.% impurity protein balance of the processes of production. Compositions containing the selected proteins according to the invention, however, may contain other proteins added as stabilizers, carriers, fillers or additional medicines.

When used here "protein" and "polypeptide" are used interchangeably.

When used here "treatment" of diseases or disorders related to the improvement of at least one sign or symptom of a disease or disorder of a subject.

The term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). It should also be understood that this term shall include, as equivalents, analogs or RNA, or DNA, obtained from nucleotide analogs, and, when applicable to the described embodiment of the invention, single (sense or antisense) and double-stranded polynucleotide.

The term "or" when used here refers to and is used interchangeably with the term "and/or", unless the context clearly indicates otherwise.

The term "percent identity " refers to sequence identity between two amino acid posledovatelno is s or between two nucleotide sequences. The percent identity can be determined by comparing a position in each sequence which may be subjected to alignment with the purpose of comparison. The expression in the form of percentage identity refers to a function of the number of identical amino acids or nucleic acids at positions shared by the compared sequences. Can be used various alignment algorithms and/or programs, including FASTA, BLAST or ENTREZ. FASTA and BLAST are available as part of the GCG software package for sequence analysis (University of Wisconsin, Madison, Wis.) and can be used, for example, with the default settings. ENTREZ is provided by the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one of the embodiments, the percent identity of two sequences can be determined by the GCG program with a weight of gap 1, for example, each amino acid gap is estimated, if there was one amino acid or nucleotide mismatch between the two sequences.

The sequence identity can be determined by comparing the reference sequence or subsequence of the reference sequence with the target sequence (for example, nucleotide sequence, amino acid sequence and so on). The reference sequence and the target sequence Optima is Ino aligned to an arbitrary number of residues, called the comparison window. In order to obtain the optimal alignment, additions or deletions, such as gaps, can be entered in the target sequence. The percentage sequence identity is determined by determining the number of positions in which the same residue occurs in both sequences, and dividing the number of matched positions by the total length of the sequences in the window of comparison and multiplying by 100 to obtain percent. In addition to the number of matched positions, the number and size of gaps is also taken into account when calculating percent sequence identity.

Identity sequence is usually determined using a computer program. A typical program is BLAST (Basic Local Alignment Search Tool)available in National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). This program compares segments of the studied sequence with sequences in a database to determine the statistical significance of matches, then sets and reports only those matches that are more significant than the threshold level. The appropriate version of the BLAST program is one that takes into account gaps, for example, version 2.X (Altschul, et al. Nucleic Acids Res 25(17):3389-402, 1997). Additional appropriate programs identify proteins with sequence identity is about to proteins according to the invention include, but not limited to, PHI-BLAST (Pattern Hit Initiated BLAST, Zhang, et al., Nucleic Acids Res 26(17):3986-90, 1998) and PSI-BLAST (Position-Specific Iterated BLAST, Altschul, et al., Nucleic Acids Res 25(17):3389-402, 1997). These programs are available openly on the web site of NCBI, mentioned above, and can be used with standard settings for the identity of the sequences according to the invention.

Slit proteins

The present invention provides a dedicated fused protein containing at least one polypeptide, comprising: (a) a first amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the ligand-binding domain of the receptor end glycation product (RAGE) mammal, and the first amino acid sequence capable of binding a ligand of RAGE; and (b) a second amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the constant domain of the heavy chain immunoglobulin IgG4 person or its fragment, where the first amino acid sequence contains at least one mutation, or at least two mutations, or at least three mutations, or 1-4 mutations, or 1-10 mutations compared to the ligand-binding domain of RAGE wild type. Examples of mutations that can be made in the first amino acid sequence, are those that increase steadily shall be fused protein, for example, receiving the ligand-binding domain of RAGE, more resistant to proteolytic degradation, such as those that make the protein more resistant to furin-like proteases. Suitable fragments of the second amino acid sequence include fragments that retain the ability to increase the half-life of serum fused proteins of which they are part, compared with the half-life of the serum of the first amino acid sequence separately. Preferably the first amino acid sequence and second amino acid sequence derived from the ligand-binding domain of RAGE of man and human IgG4.

Slit proteins according to the invention can contain one or more amino acid sequences in addition to the ligand-binding domain of RAGE and constant domain of IgG4 or its fragment. For example, a protein according to the invention may contain a linker sequence, which can be inserted between the ligand-binding domain of RAGE and IgG sequence. Slit proteins according to the invention can contain one or more tag sequences, for example, the tag sequence for purification, such as 6-histidine. Slit proteins according to the invention can contain one or more epitopes that can be recognized commercially available and what citelli, for example, c-myc (EQKLISEEDL, SEQ ID NO:9) and hemagglutinin (YPYDVPDYA, SEQ ID NO:10)derived from the epitope tag protein hemagglutinin of influenza.

Any protein RAGE mammal known to the person skilled in the art can be used when implementing the present invention. Preferably the extracellular domain of RAGE protein may be used to determine the ligand-binding domain, which can be mutated and used as the first amino acid sequence fused protein. A suitable example of proteins RAGE mammal includes, but is not limited to, domains RAGE Primate, human (e.g., GenBank registration number NP_001127 and NP_751947), mouse (e.g., GenBank registration number NP_031451), dogs (for example, GenBank registration number AAQ81297), rats (e.g., GenBank registration number NP_445788), cats, cattle (for example, GenBank registration number AAI20128), sheep, horses and pigs (for example, GenBank registration number AAQ73283).

Amino acid sequence of RAGE, containing one or more changes or modifications compared with the sequence of the wild type, can be used in the present invention. Such changes or modifications include, but are not limited to, point mutations, deletions from the N-end deletions from the C-end, internal deletions, and combinations thereof. Any change or mod eficacia can be entered in the sequence of the RAGE for use in the present invention, provided that the resulting protein retains biological activity, for example, the ability to bind one or more ligands of RAGE. Slit proteins according to the invention also include proteins with or without endogenous patterns of glycosylation, including, without limitation, fused proteins, in which the first amino acid sequence derived from the ligand-binding domain of RAGE mammal with or without concomitant with native glycosylation pattern binding domain.

Any suitable Fc-part of IgG can be used when implementing the present invention, preferably, of IgG4 molecules, for example, amino acid residues 149-473 from GenBank reg. room AAN. Plot IgG for use in the present invention can be Fc-plot IgG4 Fc, and may contain one or more CH2 and CH3 areas of the IgG4 molecule.

Examples of suitable fused proteins are presented in the following tables.

Table 1 provides the nucleotide sequence of the gene sequence fused protein of human RAGE-IgG4 Fc.

Table 1: Fused gene sequence for human RAGE-IgG4 Fc (SEQ ID NO:1).

Text in bold indicates the coding sequence for the signal sequence of the RAGE, the text in normal font means the coding sequence for human RAGE, and under arknotes text means the coding sequence for the Fc-phase IgG4.

Table 2: Amino acid sequence of the fused protein of human RAGE-IgG4Fc (SEQ ID NO:2).

Text in bold indicates the amino acid sequence of the signal sequence RAGE, the text in normal font means the amino acid sequence of human RAGE, and underlined text indicates the amino acid sequence of Fc-phase IgG4.

Table 3: Fused gene sequence for human RAGE-linker-IgG4 Fc (SEQ ID NO:3).

Text in bold indicates the coding sequence for the signal sequence of the RAGE, the text in normal font means the coding sequence for human RAGE underlined with a double line of text indicates the coding sequence for a peptide linker, and underlined with a single line of text indicates the coding sequence for the Fc-phase IgG4.

Table 4: Amino acid sequence of the fused protein of human RAGE-linker-IgG4Fc (SEQ ID NO:4).

Text in bold indicates the amino acid sequence of the signal sequence RAGE, the text in normal font means the amino acid sequence of human RAGE underlined with a double line text means am nokiatool sequence of peptide linker, and underlined with one line of text indicates the amino acid sequence for the Fc-phase IgG4.

Table 5: Fused gene sequence for human RAGE variant-IgG4 Fc (SEQ ID NO:5).

Text in bold indicates the coding sequence for the signal sequence of the RAGE, the text in normal font means the coding sequence for the variant RAGE of a man, in bold letters, underlined with a wavy line means lots of point mutations introduced in the sequence variant hRAGE, and underlined text indicates the coding sequence for the Fc-phase IgG4.

Table 6: Amino acid sequence of the fused protein of human RAGE variant-IgG4Fc (SEQ ID NO:6).

Text in bold indicates the amino acid sequence of the signal sequence RAGE, the text in normal font means amino acid sequence variants of RAGE man, in bold letters, underlined with a wavy line means lots of point mutations introduced in version hRAGE, and underlined text indicates the amino acid sequence for the Fc-phase IgG4.

Table 7: Fused gene sequence for human RAGE variant-linker-IgG4 Fc (SEQ ID NO:7).

Text in bold indicates the coding sequence for the signal sequence of the RAGE, the text in normal font means the coding sequence for the variant RAGE of a man, in bold letters, underlined with a wavy line means lots of point mutations introduced in the sequence variant hRAGE underlined with a double line of text indicates a sequence encoding a peptide linker, and the underlined text indicates the coding sequence for the Fc-phase IgG4.

Table 8: Amino acid sequence of human RAGE variant-linker-IgG4 Fc (SEQ ID NO:8).

Text in bold indicates the amino acid sequence of the signal sequence RAGE, the text in normal font means amino acid sequence variants of RAGE man, in bold letters, underlined with a wavy line means lots of point mutations introduced in version hRAGE underlined with a double line of text indicates the amino acid sequence of peptide linker, and the underlined text indicates the amino acid sequence for the Fc-phase IgG4.

The expression of the FUSED PROTEINS RAGE

Slit proteins according to the invention can be produced in any expression system protein, well-known specialist in the area and equipment, for example, eukaryotic expression systems, bacterial expression systems and viral expression systems. A number of systems of host-expression vector can be used for the expression of fused protein according to the invention. Such systems owners are the media in which the slit proteins according to the invention can be produced, and from which they may subsequently be cleared. Such systems include, but are not limited to, microorganisms such as bacteria, yeast, insect cells or plant cells. RAGE, expressed in systems, expression of yeast or mammals, for example, cells COS-7, may be similar or slightly different in molecular weight and glycosylation pattern from the native molecules, depending on the expression system. Expression of DNA RAGE in bacteria such as E. coli, provides deglycosylated molecules. Different patterns of glycosylation can be obtained using baculovirus systems for the expression in insect cells. Functional mutant analogs of RAGE mammals that have inactivated the sites of N-glycosylation, can be obtained by oligonucleotide synthesis and methods of ligating or site-specific mutagenesis. These same proteins can be produced in a homogeneous form of low carb diet with a good yield of the ri using yeast expression systems.

Molecules of nucleic acids encoding the fused protein according to the invention can be obtained, and the nucleotide sequence of these polynucleotides can be determined by any method known in the art. Taking into the consideration set forth herein and known polypeptide sequence RAGE and their identified or identifiable ligand-binding elements, and the known sequence of the constant domains of the heavy chains of IgG, nucleotide sequences encoding these polypeptides can be determined using methods well known in the art, i.e. nucleotide codons known as encoding specific amino acids can be assembled in such a way as to obtain a nucleic acid which encodes a protein according to the invention. The nucleotide codons can be selected on the basis of the used expression systems, for example, by selecting codons that correspond more abundant tRNA molecules present in the expression system, can be expressed higher levels fused protein. This polynucleotide that encodes a protein, may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et. Al., Biotechniques 17:242(1994), which, briefly, involves the synthesis of overlapping oligonucleotides, aderrasi parts of the sequence, encodes a protein, annealing and ligation of those oligonucleotides, and then amplification of subsidized oligonucleotides, polymerase(s) chain(s) of reaction (reactions) (PCR).

Recombinant expression of the fused protein of the invention (including molecules containing or alternatively consisting of, fragments fused protein or variants) may require the design vector (vector) expression containing polynucleotide that encodes a protein. After received polynucleotide encoding a protein according to the invention, the vector (vectors) for producing fused protein can be obtained by recombinant DNA technology using techniques well known in the art. Such expression vectors containing coding sequences RAGE-Fc, may also contain appropriate transcriptional and translational control signals /sequences, for example, the binding sites of the ribosome (e.g., Kozak sequence), the portions of the inner landing ribosomes (IRES) and polyadenylation sites, etc.

Molecules of nucleic acids encoding the fused protein according to the invention can be transferred into mammalian cells using retroviral vectors, replication defective (for example, vectors derived from a virus of mouse lake is for Malone (MLV) or HIV), and pseudohyperbolic the G protein of vesicular stomatitis virus (VSV-G) for the stable insertion of single copies of nucleic acid molecules encoding a protein according to the invention, in dividing cells. Retroviral vectors to deliver genes, encoded as RNA, which, after introduction into the cell, transcribed back into DNA and stably integrated into the genome of the host cell. Multiple gene insertion in one cell can increase the expression and secretion of the fused protein. Multiple rounds of infection can also increase the number of integrated gene copies and therefore the number of expressed fused protein. Integrated(s) gene(s)encoding(e) protein is stored (saved) in the cells during cell division due to their presence in the genome.

In some embodiments of the invention the present invention provides a stable cell line that expresses fused proteins according to the invention. One suitable method for the rapid generation of stable expressing the protein at a high level cell lines mammals is to use expression systems GPEx™ (Gala Biotech, a subsidiary of Catalent Pharma Solutions, Middleton, WL, Bleck, Gregory T., Bioprocessingjournal.com September/October 2005 p1-7). Such a method may include receiving pseudoterranova the retroviral vecto is a, replication defective, on the basis of MMLV, and the transformation of mammalian cells (e.g. cells SNO) this vector. The vector may integrate into the genome of the cells, thus giving a stable cell line.

Clears the currently SELECTED FUSED PROTEIN

A dedicated fused proteins according to the invention can be obtained by cultivation of suitable systems owner /vector for the expression of recombinant products broadcast of these DNA sequences, which are then purified from culture media or cell extracts using techniques well known in the art.

For example, supernatants from systems which secrete recombinant protein into culture media can first be concentrated using a commercially available filter for concentrating the protein, for example, ultrafiltration installation Amicon or Millipore Pellicon. After the stage of concentration, the concentrate can be deposited on a suitable matrix for cleaning. For example, a suitable affinity matrix can contain, for example, AGE, or lectin, or Protein a, or Protein G, or antibody molecule bound to a suitable substrate. The alternative, can be used anion-exchange resin, for example, a matrix or substrate having a suspended diethylaminoethyl (DEAE) groups. The matrix can be acrylamide, agarose is, dextranase, cellulose or other types commonly used in protein purification. The alternative, can be used cation-exchange stage. Suitable cationogenic include various insoluble matrix containing sulfopropyl or carboxymethyl group. Sulfopropyl group are preferred.

Recombinant protein obtained in bacterial culture is usually allocate initial extraction from cell precipitation, followed by stages of one or more kontsentrirovanii, vysalivaniya, the aqueous ion-exchange chromatography or gel filtration. In conclusion, it can be used high-performance liquid chromatography (HPLC) for the final stages of refinement. Microbial cells used for expression of recombinant RAGE mammal, can be destroyed by any suitable means, including a cycle of freezing and thawing, the destruction by ultrasound, mechanical disruption, or use of agents that lyse cells.

Fermentation of yeast which Express a protein according to the invention in the form of a secreted protein greatly simplifies purification. The secretory recombinant protein, the resulting large-scale fermentation can be purified by methods analogous to the methods disclosed Urdal et al. (J. Chromatog. 296:171, 1984). This link opisivaete successive stages by reversed-phase HPLC for purification of recombinant human GMCSF on the column for preparative HPLC.

The PHARMACEUTICAL COMPOSITION

Slit proteins according to the invention can be prepared in the form of a mixture, suitable for administration to a subject in need this, for example, can be prepared in the form of pharmaceutical compositions. The composition of the invention can contain one or more pharmaceutically acceptable carriers, excipients or diluents. Used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and slowing down the absorption of the agents and the like that are physiologically compatible. In one of the embodiments of the invention, the carrier is suitable for parenteral administration. The carrier can be suitable for introduction into the Central nervous system (for example, intraspinal or vnutriarterialno). Alternatively, the carrier can be suitable for intravenous, subcutaneous, intraperitoneal or intramuscular injection. In another embodiment of the invention, the carrier is suitable for oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for extemporanea preparation of sterile injectable solutions or dispersion. The use of such environments the agents for pharmaceutically active substances is well known in the art. Except when any standard medium or agent is incompatible with the fused protein according to the invention, their use in pharmaceutical compositions according to the invention is intended. Supplementary active compounds can also be included in the composition.

Suitable carriers are typically non-toxic with respect to recipients in the applied doses and concentrations. As a rule, the preparation of pharmaceutical compositions according to the invention includes the combination of fused protein according to the invention with one or more buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose, trehalose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin, are typical suitable diluents.

theRAPEUTIC INTRODUCTION of a FUSED PROTEIN ACCORDING to the INVENTION

The present invention involves the introduction of a fused protein according to the invention in the form of a pharmaceutical composition comprising a protein according to the invention and a pharmaceutically acceptable diluent or carrier, su is the target (for example, the mammal, particularly a human)in need. The present invention also provides a method of treating human diseases such compositions.

Generally, the methods according to the invention may include the introduction of a pharmaceutical composition containing a pharmaceutically effective amount of the fused protein according to the invention. Used pharmaceutically effective amount may vary according to such factors as stage of disease, age, sex and weight of the individual.

Pharmaceutically effective amount of the fused protein according to the invention can be from about 1 μg fused protein /1 kg body weight of subject to about 500 mg of the fused protein /1 kg body weight of subject, or from about 10 μg fused protein /1 kg body weight of subject to about 500 mg of the fused protein /1 kg body weight of subject, or from about 100 μg fused protein /1 kg body weight of subject to about 500 mg of the fused protein /1 kg body weight of subject, or from about 1 mg fused protein /1 kg body weight of the subject to about 500 mg of the fused protein /1 kg body weight of subject, or from about 10 mg fused protein /1 kg body weight of subject to about 500 mg of the fused protein /1 kg body weight of subject, or from about 100 mg fused protein /1 kg body weight of subject to about 500 mg of the fused protein /1 kg body weight of subject, or from about 100 μg fused protein /1 kg body weight of subjected about 25 mg of the fused protein /1 kg body weight of the subject, or from about 1 mg fused protein /1 kg body weight of subject to about 25 mg of the fused protein /1 kg body weight of subject, or from about 5 mg fused protein /1 kg body weight of subject to about 25 mg of the fused protein /1 kg body weight of subject, or from about 10 mg fused protein /1 kg body weight of subject to about 25 mg of the fused protein /1 kg body weight of subject, or from about 15 mg fused protein /1 kg body weight of subject to about 25 mg of the fused protein /1 kg the body weight of the subject, or from about 100 μg fused protein /1 kg body weight of subject to about 10 mg of the fused protein /1 kg body weight of subject, or from about 1 mg fused protein /1 kg body weight of subject to about 10 mg of the fused protein /1 kg body weight of subject, or from about 2.5 mg fused protein /1 kg body weight of subject to about 10 mg of the fused protein /1 kg body weight of subject, or from about 5 mg fused protein /1 kg body weight of subject to about 10 mg drained protein /1 kg body weight of subject, or from about 7.5 mg fused protein /1 kg body weight of subject to about 10 mg of the fused protein /1 kg body weight of the subject.

In some embodiments the invention, the pharmaceutically effective amount of the fused protein according to the invention may be 0.5 mg fused protein /1 kg body weight of the subject, 1 mg fused protein /1 kg body weight of the subject, 2 mg fused protein /1 kg body weight of the subject, 3 mg fused protein /1 kg body weight of the subject, 4 mg fused protein /1 to the body mass of the subject, 5 mg fused protein /1 kg body weight of the subject, 6 mg fused protein /1 kg body weight of the subject, 7 mg fused protein /1 kg body weight of the subject, 8 mg fused protein /1 kg body weight of the subject, 9 mg of the fused protein /1 kg body weight of the subject or 10 mg fused protein /1 kg body weight of the subject.

Unit dosage form refers to physically discrete units suitable as single doses of mammal being treated; each unit contains a specified number of fused protein according to the invention calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The unit dosage form of a fused protein according to the invention can be from about 1 mg to about 1000 mg, from about 25 mg to about 1000 mg, from about 50 mg to about 1000 mg, from about 100 mg to about 1000 mg, from about 250 mg to about 1000 mg, from about 500 mg to about 1000 mg, from about 100 mg to about 500 mg, from about 200 mg to about 500 mg, from about 300 mg to about 500 mg, or from about 400 mg to about 500 mg Single dose fused protein according to the invention can be about 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg or 700 mg

The composition of the invention may contain a fused protein according to the invention at a level from about 0.1 wt.% to about 20 wt.%, from about 0.1 wt.% to about 18 wt.%, from about 0.1 wt.% up to about 16 wt.%, from about 0.1 wt.% to about 14 wt.%, otocol 0.1 wt.% to about 12 wt.%, from about 0.1 wt.% up to about 10 wt.%, from about 0.1 wt.% to about 8 wt.%, from about 0.1 wt.% to about 6 wt.%, from about 0.1 wt.% to about 4 wt.%, from about 0.1 wt.% to about 2 wt.%, from about 0.1 wt.% up to about 1 wt.%, from about 0.1 wt.% to about 0.9 wt.%, from about 0.1 wt.% to about 0.8 wt.%, from about 0.1 wt.% up to about 0.7 wt.%, from about 0.1 wt.% to about 0.6 wt.%, from about 0.1 wt.% to about 0.5 wt.%, from about 0.1 wt.% up to about 0.4 wt.%, from about 0.1 wt.% up to about 0.3 wt.% or from about 0.1 wt.% up to about 0.2 wt.% of the total weight of the composition.

The pharmaceutical compositions according to the invention can contain one or more fused protein according to the invention at a level from about 1 wt.% to about 20 wt.%, from about 1 wt.% to about 18 wt.%, from about 1 wt.% up to about 16 wt.%, from about 1 wt.% to about 14 wt.%, from about 1 wt.% to about 12 wt.%, from about 1 wt.% up to about 10 wt.%, from about 1 wt.% to about 9 wt.%, from about 1 wt.% to about 8 wt.%, from about 1 wt.% to about 7 wt.%, from about 1 wt.% to about 6 wt.%, from about 1 wt.% up to about 5 wt.%, from about 1 wt.% to about 4 wt.%, from about 1 wt.% up to about 3 wt.% or from about 1 wt.% to about 2 wt.% of the total weight of the composition. The pharmaceutical compositions according to the invention can contain one or more fused protein according to the invention at a level of about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt., about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.% or about 9 wt.% calculated on the total weight of the composition.

Dosing regimens can be adjusted to provide the optimum therapeutic response. For example, you can enter a single bolus, you can enter multiple divided doses during the time, or the dose may be proportionally reduced or increased, as prescribed requirements in accordance with therapeutic situation. In particular it is convenient to prepare parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. The composition of the invention can be prepared and introduced intravenous, intramuscular or subcutaneous injection. In some embodiments, the implementation of the compositions according to the invention can be administered subcutaneously or intramuscularly.

In some embodiments of the invention the dosing regimen can include the introduction of repeated doses, for example, the introduction of a weekly dose. Treatment regimens may include weekly dose during one period of time (for example, within four weeks), followed by less frequent "maintenance" dosage regimen (for example, once a month or once in two months).

Dosing regimens to megatripolis to achieve the desired therapeutic outcomes.

The methods of the invention include methods of suppressing AGE-dependent inflammatory reactions in humans, involving the administration of an effective amount of a pharmaceutical composition, containing one or more fused protein according to the invention.

The methods of the invention include methods of inhibiting AGE-mediated biological activity, involving the administration of pharmaceutical compositions containing one or more fused protein according to the invention. As discussed above, AGE involved in a number of diseases or conditions, such as autoimmune diseases. Autoimmune disorder, disease or condition that can be treated, be facilitated, detected, diagnosed, or forecasted to occur when using a fused protein according to the invention, include, but are not limited to, dermatitis, glomerulonephritis, multiple sclerosis, sympathetic ophthalmia, autoimmune pulmonary inflammation, insulin-dependent diabetes mellitus, autoimmune ocular inflammation, systemic lupus erythematosus, insulin resistance, rheumatoid arthritis, diabetic retinopathy and scleroderma.

Other disorders that can be treated or prevented by the methods according to the invention may be characterized generally as including any disorder in which the affected cell positivethinking expression of RAGE or one or more ligands of RAGE, or any disorder that is treatable (i.e. one or more symptoms can be eliminated or alleviated) by reducing the function of RAGE. For example, the function RAGE may be reduced by the introduction of an agent that interferes with the interaction between RAGE and RAGE ligand.

Increased expression of RAGE is associated with several pathological conditions, such as diabetic vasculopathies, nephropathy, retinopathy, neuropathy, and other disorders, including Alzheimer's disease and immune/inflammatory reaction of the walls of blood vessels. The RAGE ligands are produced in the tissue of the affected numerous inflammatory disorders, including arthritis (such as rheumatoid arthritis). Deposits of amyloid in the tissues causes a number of toxic effects in cells and are a sign of a disease called amyloidoses. RAGE binds to beta-folded fibrillar substance, therefore, which is found in beta-amyloid-peptide A-beta, aniline, serum amyloid and prion peptides (prion-derived peptides). RAGE is also expressed at higher levels in tissues with amyloid structures. Accordingly, RAGE involved in amyloid disorders. RAGE-amyloid interaction is believed, leads to oxidative stress leading to neuronal degeneration.

Many of the RAGE ligands, and in particular ligands of the families S100/calgranulin and Amfetamine (HMGB1), produced in inflamed tissues. This observation is true for acute inflammation, such as that observed in response to the introduction lipopolysaccharides (eg, sepsis), and chronic inflammation, such as observed in various forms of arthritis, ulcerative colitis, inflammatory bowel disease, etc. cardiovascular disease and in particular those that are the result of atherosclerotic plaques, also believed to have a significant inflammatory component. Such diseases include occlusion, thrombotic and embolic disease, such as angina, disorders associated with unstable plaques (fragile plaque disorder), and embolic stroke, respectively. Tumor cells also show increased expression of RAGE ligand, in particular infoteria, indicating that cancer is also a RAGE-related violation. In addition, oxidative effects and other aspects of chronic inflammation may promote the development of certain tumors.

AGE is a therapeutic target for rheumatoid arthritis and other inflammatory diseases.

Accordingly RAGE-related disorders that can be treated with compositions according to the invention include in addition to autoimmune disorders discussed above: amyloidosis (such as disease al is chamera), Crohn's disease, acute inflammatory diseases such as sepsis, shock e.g. septic shock, hemorrhagic shock), cardiovascular diseases (e.g. atherosclerosis, stroke, disorders associated with unstable plaques, angina and restenosis), diabetes (and in particular cardiovascular disease in diabetes, complications of diabetes, disorders associated with prions, cancer, vasculitis and other vasculitis syndromes, such as necrotizing vasculitis, nephropathy, retinopathy and neuropathy.

The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present invention.

Examples

In the following examples, experiments on mice were performed with a fused protein containing the extracellular domains of mouse RAGE (amino acid residues 1-342), merged with the hinge, CH2 and CH3 domains of the Fc-fragment of the heavy chain of murine IgG2a. Design expressives in cells SNO using expression systems GPEx™. The sequence used murine RAGE shown in the following table.

Table 9 (SEQ ID NO:11) Sequence of mouse RAGE

where the signal peptide RAGE = single underline, the extracellular domain of RAGE = not stressed, the hinge region of mouse IgG2a = double podcherkival is e, CH2-plot murine IgG2a = dotted podcherkivanie and CH3 plot murine IgG2a = wavy underline.

EXAMPLE 1

The action of the fused proteins RAGE according to the invention on streptozotocin-induced diabetes in mice.

Streptozotocin-induced diabetes in mice is in the art recognized model of retinal changes caused by diabetes (see Obrosova IG, Drel VR, Kumagai AK, Szabo C, Pacher P, Stevens MJ. Early diabetes-induced biochemical changes in the retina: comparison of rat and mouse models. Diabetologia. 2006 Oct: 49(10):2525-33.)

This experiment consisted of 5 treatment groups containing 15 C57BL/6 mice per group: 1) control without diabetes; 2) diabetic control containing mice treated with streptozotocin at 45 mg/kg for 5 consecutive days before the beginning of the study to cause diabetes; 3) mice treated with streptozotocin, who also received 10 mg/day mRAGE-IgG2aFc, administered intraperitoneally injected, 3 injections/week; 4) mice treated with streptozotocin, which also received 100 mg/day mRAGE-IgG2aFc, administered intraperitoneally injected, 3 injections/week; and 5) mice treated with streptozotocin, which also received 300 mg/day mRAGE-IgG2aFc, administered intraperitoneally injected, 3 injections/week.

During the study in mice was assessed by body weight, the level of blood glucose, glycosylated hemoglobin (GHb), albuminuria and tactile chustvitelnos the ü as a function of sensory nerves. Mice were killed at the end of the study and evaluated the permeability of retinal vessels using a fluorescent probe, adhesion of leukocytes to the retinal capillaries and NF-κ-regulated expression of the protein (SOH-2, ICAM, iNOS).

The results of two months of research

Studied the impact of the fused protein RAGE-Ig on the development of the changes caused by diabetes, physiology of the retina and metabolism in mice C57B1/6J. Protein was administered intraperitoneally injected in 3 different concentrations (10 μg, 100 μg and 300 μg) three times a week. There have been no adverse effects at any dose of the drug to the weight gain or the overall health of the diabetic mice. The levels of glucose in the blood nagrody mice was 155±24 mg/DL (mean±STD. deviation), 358±38,417±36, 376±36 and 370±55 controls without diabetes, diabetic control diabetic +10 µg fused protein RAGE-Ig, diabetic +100 µg fused protein RAGE-Ig and diabetic+300 µg fused protein RAGE-Ig groups, respectively.

The parameters associated with retinopathy, measured in short-term studies, represented (1) lakotas, (2) permeability to endogenous albumin in the blood vessels of the retina, (3) nitration of proteins in the retina and (4) the expression of retinal ICAM and MOR-2.

1. Lakotas.

Methods: During a two-month diabetes blood was removed from the vascular system anestezi the bathrooms animals by full perfusion with phosphate-saline buffer (PBS) through a heart catheter. Animals then were perfesional the lectin-concanavalin And related fluorescein (20 μg/ml in PBS; Vector Laboratories, Burlingame, CA)as described previously (see Joussen et al., FASEB J. 2004 Sep; 18(12):1450-2). The image plane is fixed retinas were obtained by fluorescence microscopy, and counted the number of leukocytes adhering to the vessel wall.

The result: a Significant increase of leukostasis been demonstrated in mice that have had diabetes for 2 months, compared with mice without diabetes (P<0.05). Lacostes not inhibited in any of the groups treated fused protein RAGE-Ig (see Figure 1).

2. The permeability of blood vessels

Methods: During two months of diabetes was obtained by freezing the sliced eyes (10 μm)were fixed in methanol for 10 minutes and washed four times in PBS. Each slice was incubated with sheep antibodies against serum albumin mouse (sheep anti-mouse serum albumin) (Abeam, Cambridge MA; AB8940; 1:2000 dilution) for 2 hours After washing, sections were incubated with FTIC-labeled secondary antibody (AB 6743; 1:1000 dilution) for 90 min On the basis of fluorescence microscopy, the average number of fluorescence was determined in 3 different areas for each of the 4 layers of the retina (inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer). The amount of fluorescence in each section of the JW who ALOS average of 10 randomized measurements, and the amount of fluorescence in each layer of the retina represents a mean value of fluorescence in each of the 3 different sections within this layer.

The results:

Diabetes resulted in a significant increase in fluorescence in the avascular retina (i.e. due to leakage of albumin from blood vessels) in each of the 4 studied layers of the retina. The results are presented in figure 2 (2A - internal plexiform layer 2B - inner nuclear layer, 2C - outer plexiform layer 2D of the outer nuclear layer). To determine the albumin in the inner and outer nuclear layers, we deliberately measured in the thin space between the cores, so these figures may not be as rigorous as the numbers of plexiform layers, in which no nuclei, distorting the measurement.

3. Nitration of proteins in the retina

Methods: During a two-month diabetes were isolated and homogenized in the retina. Got dot-blots by blotting 50 µg of homogenate protein from each animal on nitrocellulose membrane. The membrane was blocked with milk (5%), washed and stained using the immune labels, using antibodies to nitrotyrosine (Upstate Biotechnology, Inc. #05-233; 1:500 dilution)for 2 hours and then stained with a secondary antibody (conjugated goat antibodies to mouse IgG with HRP (horseradish peroxidase), Bio-Rad; 1:1000 dilution) for 1 h is CA. After intensive washing staining using immune marks detected by the antibodies were visualized with enhanced chemiluminescence (ECL, Santa Cruz Biotechnology, Santa Cruz, CA). Dependent staining using immune marks the chemiluminescence was recorded on tape, and conducted quantitative analysis of the density painted using immune labels spots. The results are expressed as percentage of values found in the controls without diabetes.

The results:

The results are presented in figure 3. Retinal homogenates of diabetic mice showed the expected increase in nitration of proteins. Therapy inhibited this post-translational modification of dose-dependent manner. It is believed that the nitration of proteins is a parameter and oxidation and nitrate stress.

4. The expression of retinal ICAM and MOR-2

Methods: Retinas were isolated and destroyed by ultrasound, and the supernatant was used as a full extract of the retina. Samples (50 μg) was fractionally using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), transferred through electroblotting on nitrocellulose membrane and the membrane was blocked Tris-buffered saline containing 0.02% Tween 20 and 5% skim milk. Used antibodies to ICAM-1 (1:200 dilution; SantaCruz Biotechnology) and MOR-2 followed by the application of secondary antibodies for 1 hour. After washing, the results were visualized with enhanced chemiluminescence.

The results:

The results are shown in figure 4. Because the expression of ICAM-1 on endothelial cells plays a critical role in the adhesion of leukocytes to vessel walls (lakotas), we measured the effects of diabetes and therapy on the expression of ICAM-1 in the retina. A two-month diabetes resulted in a significant increase in the expression of retinal ICAM-1. The introduction of a fused protein RAGE-Ig resulted in a dose-dependent reduction in the expression of ICAM, and the highest dose significantly inhibited the expression.

Manifestation painted using immune labels, bands corresponding to the molecular mass MOR-2, has not increased in diabetes and did not change in animals receiving treatment (not shown).

The endpoint used in this short-term study of the impacts of the fused protein RAGE-Ig, were selected because they all were found to be associated with the development of the early (degenerative) stages of diabetic retinopathy, i.e. different therapies, which have been found to inhibit diabetes-related macular degeneration of the retinal capillaries, also inhibit these defects.

Inhibition of RAGE really inhibited pathologies associated with vascular permeability and nitrate stress in the retina. Nitrate stress is also considered the AK marker of oxidative stress. Inhibitor of RAGE, however, is not inhibited pathologies associated with leukostasis.

EXAMPLE 2

The impact of the fused proteins RAGE according to the invention on a long-term streptozotocin-induced diabetes in mice.

Streptozotocin-induced diabetes in mice is in the art recognized model of retinal changes caused by diabetes (see Obrosova IG, Drel VR, Kumagai AK, Szabo C, Pacher P, Stevens MJ. Early diabetes-induced biochemical changes in the retina: comparison of rat and mouse models. Diabetologia. 2006 Oct: 49(10):2525-33).

Long-term studies included 5 groups of processing, containing 25 C57BL/6 mice per group: 1) nediabeticescoy control; 2) diabetic control containing mice treated with streptozotocin at 45 mg/kg for 5 consecutive days before the beginning of the study to cause diabetes; 3) mice treated with streptozotocin, who also received 10 mg/day mRAGE-IgG2aFc, administered intraperitoneally injected, 3 injections/week; 4) mice treated with streptozotocin, which also received 100 mg/day mRAGE-IgG2aFc, administered intraperitoneally injected, 3 injections/week; and 5) mice treated with streptozotocin, which also received 300 mg/day mRAGE-IgG2aFc, administered intraperitoneally injected, 3 injections/week.

During the study in mice was assessed by body weight, the level of blood glucose, glycosylated hemoglobin (GHb), albuminuria and tactile sensitivity is AK the indicator function of the sensory nerves. Mice were killed at the end of the study and were evaluated by quantitative histopathology and neurodegeneration of the retina.

The parameters associated with retinopathy, measured in a long term study consisted of (1) acellular capillaries, (2) the "shadow" of pericytes and (3) ganglion cells. As a marker of peripheral neuropathy in long-term study was measured sensitivity paws to light touch.

Histopathology of the retina caused by diabetes

After 10 months of diabetes eyes were fixed in formalin and one retina was isolated from each animal, were washed in running water overnight and were digested for 2 h in a solution of the crude trypsin, as we reported earlier. The retinal vascular system was isolated soft branch of the nerve cells by "brushes", derived from a single hair. After a complete cleaning from nerve cells selected vascular system was laid out on the glass slide, dried overnight, stained with hematoxylin and Schiff-iodic acid, obezvozhivani and covered with cover glass. Degenerative (acellular) capillaries quantitatively analyzed in 6-7 parts corresponding to the midperiphery of the retina (mid-retina) (200X magnification), in a disguised form. Acellular capillaries identified as vascular tubes of the dimensions of the capillaries, not having cores anywhere along its length, and documented per square millimeter area of the retina. Shadows of pericytes was estimated from the distribution of speakers "bumps" on the capillary basal membrane, of which disappeared pericyte. Researched, at least 1000 capillary cells (endothelial cells and pericyte) in 5 sites midperiphery of the retina (400X magnification) in a disguised form. Shadows on any already acellular vessel was excluded.

To study the effect of diabetes on neurodegeneration of the retina were counted cells in the layer of ganglion cells. Fixed formalin eyes were placed in paraffin, did saggitaria slices of the retina, through the optic nerve and stained with hematoxylin-eosin. The number of cells in the ganglion layer was calculated at the two sites (midperiphery of the retina and the area of the retina in the posterior pole of the eye around the optic nerve head) on both sides of the optic nerve. Similar plots from two sides of the optic nerve were averaged and expressed per unit length.

Results. As expected based on previous work, long-term diabetes resulted in a significant increase in degenerative, acellular capillaries in the retina (Figa). All doses fused protein RAGE-Ig significantly inhibited the degeneration of the capillary, without kako what about any effect on the severity of hyperglycemia. Diabetes also had a tendency to increase the degeneration of pericytes ("shadow" pericytes), but the results did not reach statistical significance (Pigv). We previously found that the loss of pericytes much more difficult to detect in diabetic mice C57B1/6 compared with diabetic rats or larger kinds, and now we consider it as an unreliable parameter vascular disease for this model. Perhaps due to the inability to detect a significant loss of pericytes in control with diabetes, we did not find any influence of the fused protein RAGE-Ig on the loss of pericytes in these mice.

Diabetes did not cause a reduction in the number of cells in the retinal ganglion layer of cells (i.e. neurodegeneration) in these mice C57B1/6. This conclusion is consistent with the previous study of this mouse model. In the absence of influence of diabetes on neurodegeneration of the retina, we are not able to assess impacted would be whether or not the inhibitor on neurodegeneration.

Sensitivity to light touch (a sign of peripheral neuropathy).

Patients with diabetic neuropathy can show the number of aberrant sensations, including spontaneous pain, pain caused by light touch, and hyperalgesia. There is accumulating evidence that rodents with diabetes reproduce this hyperalgesia and show tacti is inuu allodynia. In rodents, this is measured as the threshold of tactile sensitivity paws.

Methods: Mice (8-month diabetes) was transferred into a test cage with a bottom of wire mesh and left to acclimatize for 10-15 minutes. Used the hairs von Frey to determine the 50% threshold mechanical otdergivanija to otdergivanija foot. Kit hairs with logarithmically increasing rigidity, since the thread that had a lot of bending 0.6 g, have been consistently applied to the plantar surface of right hind paws with pressure, which caused the bending of the hair. The rise of the paws was recorded as a positive response, and chose a lighter hair for the next measurement. If the response was missing for 5 seconds, then use the next heavier hair. This process continued until, until you have made four measurements after the initial changes in behavior, or until such time as it was not observed five consecutive negative (6 g) or four consecutive positive (0.4 g) answer. The resulting sequence of positive and negative estimates used to calculate the 50% reaction threshold of otdergivanija.

Results: Diabetes was significantly increased sensitivity paws to light touch, implying that requires a lower pressure for dia is eticheskikh animals in order to withdraw their paws than in the case of animals without diabetes (6). This is caused by diabetes defect significantly inhibited at each dose fused protein sRAGE-Ig.

Retinopathy: Research, carried out using a fused protein RAGE-Ig, was carried out for 2 terms diabetes: (1) long-term (10 months) research to assess the impact of therapy on long-term histopathology of diabetic retinopathy, which develops in mice, and (2) 2-3 month study to assess the physiological and molecular effects of therapy, which presumably underlie the long-term impacts on the histopathology. Physiological and molecular endpoints studied in relation to the influences of the fused protein RAGE-Ig, was chosen due to the fact that all were found in other studies correlate with (and probably causally linked to) the development of the early (degenerative) stages of diabetic retinopathy. All three doses of therapy clearly and significantly inhibited diabetes-related macular degeneration of the vascular system of the retina. Similarly, all three doses of drugs, apparently, also inhibited diabetes-increased permeability of the retina in these mice. These findings are of great clinical importance, because the early (non-proliferative) phase of diabetic retinopathy sun is still determined on the basis of vascular disease (vascular reperfuse and degeneration and increased permeability).

The impact of therapy on the measured molecular and physiological endpoints in the retinas from diabetic mice was mixed. Inhibition of RAGE really inhibit disorders related to nitrate stress, a marker of oxidative stress in the retina. Inhibitor of RAGE, however, is not inhibited violations associated with leukostasis. The lack of impact of therapy on lacostes is unexpected, because another group recently reported that their sRAGE really inhibited the increase leukostasis diabetes. The evidence which we have received since our studies using fused protein RAGE-Ig (Diabetes 57:1387-93, 2008), however, shows that the effects of drug therapy on retinal lacostes diabetes do not predict the impact of therapy on the degeneration of the retinal capillaries in diabetes. Therefore, no impact of treatment on retinal lakotas in no way reduces the value of the detected effects of the drug.

It was unexpectedly found that there is a measure of "dose-effect" of the drug in relation to the expression of ICAM-1 and nitration of proteins in retinas of diabetic animals, whereas this indicator "dose-effect" was not pronounced for the permeability of the capillaries of the retina, and degeneration. Apparently, you can byloby to assume, neither ICAM or nitration not involved in retinal vascular disorders in diabetes, although we have data when using ICAM-1-knock-out animals that contradict this conclusion.

It is obvious that this drug really got into the retina, really showed biological effects and really demonstrated a significant ability of this drug to inhibit, at least early vascular lesions of diabetic retinopathy.

Sensory neuropathy: Other authors postulated that the end products of glycation (AGE) and the interaction of AGE with RAGE cause oxidative stress, activates NF-kB and different NF-kB-mediated Pro-inflammatory genes in the nerves, and increase neurological dysfunction, including altered pain sensation. These data are consistent with the facts that RAGE-mediated signaling contributes to the development of at least several aspects of diabetic neuropathy, and provide evidence that protein sRAGE-Ig inhibits this process in a long term study.

EXAMPLE 3

Evaluation of the fused protein RAGE-Ig using a murine model of arthritis induced by collagen type II.

Immunization of susceptible strains of mice with collagen type II, osnovnolozhenia the cartilage of the joint, causes progressive inflammatory arthritis (Wooley et al. The Journal of Experimental Medicine 1981; 154:688-700). Induced collagen arthritis (CIA) is clinically characterized by erythema and edemas, and the width of the affected paws usually increases by 100%. Clinical evaluation index developed to assess disease progression in relation to joint strain and spondylitis (Wooley, Methods In Enzymology 1988; 162:361-373). Histopathology of affected joints shows synovitis, pannus formation and erosion of joints and bones, which can also be represented by the index. Immunological laboratory observations include high levels of antibodies to collagen type II and Hyper-gammaglobulinemia. This model is currently well adapted to study immunotherapy approaches to disease of the joints (Staines et al. British Journal of Rheumatology 1994; 33(9):798-807) and successfully used to study both the biological and pharmacological agents for the treatment of rheumatoid arthritis (RA) (Wooley et al. Arthritis Rheum 1993; 36:1305-1314, and Wooley et al. Journal of Immunology 1993; 151:6602-6607).

Antagonism of the receptor RAGE is recognised as a potential therapeutic target for rheumatoid arthritis. Blockade of RAGE in mice with induced collagen arthritis led to the suppression of the clinical and histological signs of arthritis, and reducing the intensity of the disease was associated with a decrease ur the init TNFα, IL-6 and matrix metalloproteinase MMP-3, MMP-9 and MMP-13 in the tissue of the paw with arthritis (Hofmann et al. Genes Immun 2002; 3(3):123-135). This shows that induced by collagen arthritis sensitive to RAGE-directed therapy.

This experiment can assess the impact of the fused protein RAGE-Ig at the CIA at three doses from the time of immunization with collagen type II. The study design is shown in Fig.7.

Forty mice DBA/1 LacJ aged 8-10 weeks were obtained from Jackson Labs and acclimatized in the vivarium for at least 10 days before the experiment. All animals weighed >16 g at the beginning of the experiment. The mice were divided into four treatment groups: 1) 100 μl of sterile PBS intraperitoneally injections daily; 2) 100 μl of sterile PBS containing 10 µg fused protein RAGE-Ig, intraperitoneally injections daily; 3) 100 μl of sterile PBS containing 100 µg fused protein RAGE-Ig, intraperitoneally injections daily; and 4) 100 μl of sterile PBS containing 300 µg fused protein RAGE-Ig, intraperitoneally injection daily.

Three days after the initial dose, all mice were injected with 100 μg bovine collagen type II in complete Freund's adjuvant (FCA) intradermally at the base of the tail. Mice were monitored daily inspection of onset, which was recorded. Mice weighed weekly and observed the condition in common with sochuvstviya. Animals affected by arthritis, clinically assessed five times a week up to 10 weeks after immunization, and the dimension of the legs was performed three times per week. Mice without signs of arthritis in ten weeks after immunization was considered as a negative result of the disease.

RESULTS

Overall health and toxicity. No acute toxic cases are not met during the study, and all animals survived during the experiment. The treatment was well tolerated, and no signs of side effects, such as fading of wool or itching were observed. Weight of mice (Fig) demonstrate small changes in weight during the study, which is typical due to transient weight loss in some animals at the beginning of the disease. None of these changes between groups did not reach statistical significance.

The number of cases and the beginning of arthritis. The final number of cases of collagen arthritis in the study is shown in Fig.9. Control mice reached 100% of the beginning, which is not unusual in the classical model of collagen arthritis, where the normal range in the number of cases 80%-100%. Mice treated with 10 μg of RAGE per day, reached the number of cases 80%, which was not a significant decrease in the number of cases. Mice treated with 100 µg of RAGE per day, showed 60% of the number of cases, which is considerably lower h is m in the control group (p< 0.05). Unexpectedly, the number of cases of arthritis in mice treated with 300 µg RAGE was 100%, and thus was similar to the number of cases of control.

The mean value (and standard error of the mean (SEM)) of the period of onset of the disease is presented on Figure 10. The onset of the disease was common in the control group, with an average period of the first symptoms of arthritis 38.6. The beginning of the disease in mice, treated or 10 μg, or 100 μg RAGE, nominally delayed until 42.5, which did not reach statistical significance. However, the onset of the disease was significantly delayed (p<0.05) in mice treated with 300 µg RAGE. Therefore, although mice at the high dose showed no reduction in the number of cases, the time to development of clinically apparent arthritis was significantly increased.

Modulation of the onset of processing of RAGE can be easily estimated using a plot of the number of cases from the time (11). Typical property rapid onset of the disease for the CIA detected in the control group, whereas mice treated with RAGE in dose or 10 μg, or 100 μg resulted in delayed onset of the disease and to lower the final number of cases of arthritis. For about eight weeks in mice treated with 300 µg RAGE, the disease progressed in a similar scheme, but the group of animals with late arthritis resulted in a high number of cases but with delayed onset.

Disease severity and progression. Analysis of the total assessment for the joint in treated and control animals revealed a significant effect of treatment using RAGE to the gravity-induced collagen arthritis (Fig). The control mice showed typical chronic progressive disease with a distinct increase cumulative index of arthritis. In contrast, mice treated with RAGE at any dose, showed a clear reduction of the cumulative assessment of arthritis. The difference between control and treated groups had achieved a high level of statistical significance (p<0.001) with 43 days after immunization, and this difference was maintained throughout the study. Although therapy with RAGE at 100 µg/day was reached the lowest aggregate estimates of arthritis, there was no significant difference between RAGE groups in relation to the assessment of arthritis, suggesting that achieved "threshold" effect, but not a classic dose-dependent effect.

Analysis of the impact of therapy with RAGE on the number of legs with arthritis (Fig) really demonstrates a significant impact on the progression of the disease. In addition, it was found to have a significant impact on the number of affected paws with 43 days. The significance level was varied from p<0.001 and p<0.025, which may indicate that the influence of RAGE was more pronounced in the wearing of the severity of the disease, what is the progression of arthritis; however, the maximum number of affected paws (40) is more limited than the maximum aggregate assessment of the disease (120). In addition, there were no significant changes between groups treated with RAGE, although the group with 100 μg RAGE really showed a higher level of deceleration of arthritis.

These results suggest that the introduction of the RAGE protein has a significant impact on caused by collagen arthritis in the introduction to the use of preventive schemes. Obvious toxic effects from the injections RAGE was absent at any dose, and, apparently, the treatment was very well tolerated. The total number of cases decreased significantly in mice receiving 100 micrograms daily, and delay the onset of the disease was observed in mice treated with 300 μg/day. However, the most obvious sign of clinical activity was found to reduce the assessment of the disease and the number of legs with arthritis, where a large discrepancy between the mice treated with RAGE, and control animals were found with 43 days after immunization. At this stage the control animals experienced a typical progression of severe arthritis, whereas treatment with the help of RAGE all doses slowed the progression of the disease.

Histopathological evaluation: Legs, all the mice were removed at the end of the AI clinical evaluation study and were stored in neutral buffered formalin. Joints decalzinirute for 18 days in 10% formic acid, dehydrational and concluded in paraffin blocks. Slices were cut along the longitudinal axis, fixed and stained or with hematoxylin and eosin or toluidine blue. Drugs cut approximately on the middle line and then Central sagittal samples were fixed for evaluation. This ensured the wealthy geographical assessment. Five to ten samples were fixed (usually 4-6 samples on a glass slide). After staining, the slides permanently connected to the cover glass. A minimum of 3 separate cut on the drug were evaluated in blinded fashion, and the appraiser is not aware of the purpose of the group. The fore limbs are all of the elbow, wrist and metacarpal joints were evaluated, while all of the knee, ankle and metatarsal joints was evaluated on its hind legs. Figures were not evaluated, since the procedure of slicing destroys most proximal interphalangeal joints. Slides were evaluated for the presence of synovitis, pannus formation, marginal erosions, architectural changes (mainly subluxation) and destruction. The overall score on the basis of these collective points, then, was assigned to each slice. The evaluation system was based on the following:

Synovitis considered for casinosinternet membrane and evaluated on a scale as follows: 0 - for thickness less than 3 cells; 1 to a thickness of 3-5 cells; 2 for the thickness of 6-10 cells; 3 - for thickness of 10-20 cells; and 4 for the thickness of 20-30 cells.

The formation of pannus was evaluated as follows: 0 - in the absence of the formation of pannus; 1 - in the presence of microvilli; 2 - when you explicitly attach pannus; 3 - noticeable when attaching the pannus; and 4 when the joint cavity filled with pannus.

Regional erosion was evaluated as follows: 0 - no visible lesions; 1 - minor defect (indentation) in the area of attachment of the capsule; 2 - with the obvious erosions of the cartilage; 3 - erosion, extending into the subchondral bone; and 4 - with extensive erosion of bone and cartilage.

Architecture changes assessed as follows: 0 - normal architecture of the joint; 1 - with edematous changes; 2 - minor subluxation of the articular surfaces; 3 - at large subluxation of the articular surfaces; 4 - when fully fibrosis and the formation of collagen crosslinks.

The overall score displays: 0 - classic normal appearance of the joint; 1 - with small changes; in accordance with remission; may be clinically normal; 2 - under certain inflammatory arthritis; 3 - extensive inflammatory erosive disease; and 4 - in destructive erosive arthritis.

Degradation of cartilage and bone matrix. Serial cut is painted on components of cartilage matrix, using histochemical dye toluidine blue. Sections with toluidine blue were assessed on the loss of proteoglycans. Staining of the articular surface was compared to staining of the growth plate and evaluated as follows: 0 - no loss of proteoglycans; normal staining toluidine blue; 1 - with a slight loss of proteoglycans; some loss of staining in the superficial zone of cartilage; 2 - with an average loss of proteoglycans; weak staining of the superficial zone of cartilage; 3 - with considerable loss of proteoglycans; no staining toluidine blue superficial zone of the cartilage; and 4 large loss of proteoglycans; no staining toluidine blue deep zone of cartilage.

RESULTS

Histological findings on arthritis induced by collagen. Sections were evaluated for inflammatory and erosive parameters of the disease. The appearance of arthritis (Fig) shows a typical pathology of inflammatory erosive disease at this point in time in the control group (PBS treated) with typical signs of arthritis - synovial hypertrophy and hyperplasia, marked attachment pannus and marginal erosions.

Processing of the fused protein RAGE-Ig at 10 µg/ml (Pigv) led to moderate changes in inflammatory and erosive parameters with an overall improvement in prowl the deposits erosion and disturbed surfaces cartilage. Processing of the fused protein RAGE-Ig at 100 µg/ml (Figs) led to a decrease in the formation of pannus and erosions compared with the control, and the overall difference was quite noticeable. However, the introduction of fused protein RAGE-Ig at 300 µg/ml (Fig.14D) lead to arthritis, which manifested itself less seriously than the pathology observed in the control with saline solution, but nevertheless was quite serious with synovial hypertrophy and hyperplasia, with a noticeable attaching pannus and marginal erosions.

The analysis estimates inflammation (Fig) showed a reduction in inflammation in mice treated fused protein RAGE-Ig in all doses when compared with the control (treated with saline) animals. However, synovitis significantly decreased (p<0.05) only in the group of 100 µg/ml, and the formation of pannus showed a similar reduction in the assessment (p<0.03). Reduction of inflammatory parameters diseases that can be detected by using a fused protein RAGE-Ig or with 10 μg/ml, or 300 μg/ml, were insufficient to reach statistical significance.

Evaluation of changes in the erosive features (erosion and changes in the architecture of the joints)caused by collagen arthritis, showed a similar scheme effects. A significant reduction (p<0.01) erosion of the joints was observed in the group treated fused protein RAGE-Ig at 100 µg/ml, when compared to the control (treated with saline) animals (Fig), while the reduction observed in mice treated fused protein RAGE-Ig at 10 μg/ml and 300 μg/ml, did not reach significance.

The combination of histopathological parameters in common histological assessment of arthritis (Fig) reflected the conclusions about individual pathological parameters. There was a significant difference between the control (saline) treated animals and mice treated fused protein RAGE-Ig at 100 µg/ml (p<0.02), and overall score for mice treated with 10 μg/ml, was reached significance (p=0.05), however no significant decrease in the overall assessment of the disease was not observed when using fused protein RAGE-Ig at 300 µg/ml

The sections stained with toluidine blue, examined to determine influence whether protein RAGE-Ig on the loss of the protein matrix of the joint with arthritis. The data (shown in Fig and 19) suggest that protein RAGE-Ig really protected from the loss of proteoglycans, but this effect is statistically significant (p<0.05) only at the dose of 100 μg/ml dose. Control group with PBS shows significant loss of cartilage matrix (proteoglycans and collagens), and a significant loss of staining of the proximal surface of cartilage observed in mice treated fused protein RAGE-Ig at 300 µg/ml In contrast, there is good preservation of the protein matrix of prividenii fused protein RAGE-Ig at 10 μg/ml or 100 µg/L.

Histological findings were confirmed by clinical data, which showed that treatment-induced collagen arthritis fused protein RAGE-Ig resulted in effects on the number of cases and severity of the disease. Histological parameters were reached levels of statistical significance in mice treated with 100 μg/ml, and reached statistical significance in General pathology in mice treated with 10 μg/ml protein RAGE-Ig at 100 µg/ml was provided by the good preservation of the structure of a joint, and a significant decrease in all symptoms of arthritis in assessment. The overall score was the fact that protein RAGE-Ig blocked erosive phase of arthritis, because the degree of inflammatory changes was less impact than on the secondary parameters of the disease. Mice treated fused protein RAGE-Ig at 300 μg/ml, was not protected to the same extent as at lower doses, in turn causing the possibility suppressive response at this level is the introduction of the protein. In General, these findings are consistent with clinical observations obtained in this study, demonstrating that the protein RAGE-Ig can be anti-arthritis effect.

Although the invention is described in detail and with reference to specific embodiments of the invention, the person skilled in the technical field h is t it is obvious, that various changes and modifications can be made therein without deviating from its scope and nature, and such changes and modifications can be made within the scope of the attached claims. All patents and publications incorporated herein by reference to the same extent as if each individual publication specifically and individually indicated as incorporated by reference in its entirety.

1. Protein for the treatment of AGE-mediated diseases, consisting of:
(a) a first amino acid sequence selected from the group consisting of amino acids 1-301, amino acids 24-301, amino acids or amino acids 1-344 24-344 of SEQ ID NO: 6, and the first amino acid sequence capable of binding a ligand of RAGE; and
(b) a second amino acid sequence consisting of (1) amino acid sequence of the constant domain of the heavy chain of human immunoglobulin IgG4 or (2) the amino acid sequence of a fragment of the heavy chain of human immunoglobulin IgG4 containing the hinge, CH2 and CH3 plots
where the first amino acid sequence and second amino acid sequence are optionally connected by a linker.

2. Fused protein according to claim 1, where the first amino acid sequence consists of amino acids 1-344 of SEQ ID NO: 6.

3. Fused protein according to claim 1 is 2, where the second amino acid sequence consists of the amino acid sequence of the constant domain of the heavy chain of human immunoglobulin IgG4.

4. Fused protein according to claim 1, where the protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 8.

5. Fused protein according to claim 4, where the protein consists of the amino acid sequence of SEQ ID NO: 6.

6. Fused protein according to claim 1, additionally containing a linker between the first amino acid sequence and second amino acid sequence.

7. Protein according to any one of claims 1 to 6 for use in the method of reducing levels of ligand binding by RAGE, in a mammal.

8. Nucleic acid to obtain a fused protein according to any one of claims 1 to 6, where this nucleic acid encodes a protein according to any one of claims 1 to 6.

9. Recombinant a host cell to obtain a fused protein according to any one of claims 1 to 6, containing the nucleic acid of claim 8.

10. Pharmaceutical composition for the treatment of AGE-mediated diseases, containing from 0.1% to 20 wt.% fused protein according to any one of claims 1 to 6 of the total weight of the pharmaceutical composition and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10 containing protein according to claim 5.

12. The pharmaceutical composition according to any one of p or 11 DL the application in the treatment of diabetic nephropathy or rheumatoid arthritis, or autoimmune diseases.

13. The pharmaceutical composition according to any one of p-12 for use in the treatment of dermatitis, glomerulonephritis, multiple sclerosis, sympathetic OFTAL, autoimmune pulmonary inflammation, insulin-dependent diabetes mellitus, autoimmune ocular inflammation, systemic lupus erythematosus, insulin resistance, rheumatoid arthritis, diabetic retinopathy, and scleroderma.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

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17 cl, 2 tbl

FIELD: medicine.

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Anti-mif antibodies // 2509777

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

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FIELD: biotechnologies.

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FIELD: biotechnologies.

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FIELD: biotechnologies.

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FIELD: biotechnologies.

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26 cl, 3 tbl, 2 ex

FIELD: biotechnologies.

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26 cl, 25 dwg, 3 tbl, 4 ex

FIELD: biotechnologies.

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FIELD: chemistry.

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FIELD: biotechnologies.

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18 cl, 7 dwg, 1 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: isolated peptide having cytotoxic T lymphocyte (CTL) inducing capacity in the presence of an antigen-presenting cell bearing HLA-A*2402, is used to obtain antigen-presenting cells and therefore CTL. The obtained CTL are used for targeted action against CDCA1-expressing cancer cells.

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16 cl, 4 dwg, 1 tbl, 1 ex

FIELD: biotechnologies.

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Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

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17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

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FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, and represents a method of producing a recombinant mutein [C112S] of human enterokinase light chain, using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a mutein [C112S] inactive precursor of human enterokinase light chain, comprising a sequence encoding a cleavable N-terminal peptide comprising a hexahistidine cluster and the enterokinase recognition sequence, and a sequence fused with it in frame encoding a precursor of mutein [C112S] of human enterokinase light chain with non-cleavable C-terminal hexahistidine cluster under control of a promoter operating in the bacterial cell.

EFFECT: invention enables to produce the recombinant mutein of human enterokinase light chain with a high yield.

11 cl, 5 dwg, 1 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention represents a method of production of precursor of a recombinant fragment of human tissue plasminogen activator (tPA) (reteplase) using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a precursor of the recombinant fragment of human tissue plasminogen activator (tPA) (reteplase), comprising a sequence encoding a cleavable N-terminal peptide comprising decahistidine cluster and sequence of enterokinase recognition fused in frame, or a fragment of DNA encoding the [-1] methionyl of fragment of human tPA (reteplase), under the control of a promoter operating in the said bacterial cell.

EFFECT: invention enables to obtain a recombinant fragment of human tissue plasminogen activator with a high yield.

10 cl, 6 dwg, 1 tbl, 8 ex

FIELD: biotechnologies.

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EFFECT: invention provides for simultaneous translation of human proteins OCT4 and LIN28 and fluorescent protein DsRed2 in production of induced pluripotent stem cells of a human being and animals in medicine and veterinary science.

3 cl, 1 dwg, 1 tbl

FIELD: biotechnology.

SUBSTANCE: first a plasmid pCollbd-BMP-2 is produced. Then, said plasmid is inserted into the strain Escherichia coli M15/pREP4. Then the recombinant protein Collbd-BMP-2 is prepared by culturing the cells of a strain Escherichia coli Ml 5 [pREP4, pCollbd-BMP-2], the induction of protein Collbd-BMP-2 synthesis, the cell destruction, obtaining the supernatant containing the protein, immobilisation of protein by adding to the supernatant of suspension of collagen-containing sorbent, incubation, washing the sorbent with a related protein, the subsequent elution of protein from the sorbent and dialysis. Then, in a laminar flow unit two sterile polypropylene tubes are prepared and the hydroxyapatite suspension is placed in them, a solution of connective-tissue collagen or xenogenic bone collagen in the form of crumbs is added, mixed on a shaker to obtain a homogeneous suspension. Then 10% (by weight) of gelatin microspheres containing recombinant growth factor of bone tissue Collbd-BMP-2 is added, stirred on the shaker. The resulting composition is sterile pasta of white, beige, grayish or yellowish colour of uniform consistency, contains 5-20% of synthetic nanostructured hydroxyapatite, 0.1-15% of type I collagen and 5-10% of a prolonged form of recombinant growth factor of bone tissue Coilbd-BMP 2 and has osteoconductive and osteoinductive properties. The composition according to the present invention can be used to fill bone defects and be applied on the implantable materials.

EFFECT: invention can be used in traumatology, orthopedics, maxillofacial surgery, dentistry for treatment of diseases and injuries of human osseous system as an active biodegradable material, for bone tissue regeneration, osteoinductive biological coating of metal prosthetic implants of bone tissue.

4 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

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