Preventive and therapeutic agent for inflammatory disease

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. What is presented is a method for preventing or treating an inflammatory disease, comprising the stages of producing an NR10 antibody having NR10-neutralising activity, and selecting an antibody inhibiting IL-31-dependent cell line growth, and administering the antibody to a patient with an inflammatory disease that is atopic dermatitis, chronic dermatitis, rheumatism or osteoarthritis.

EFFECT: present invention can find further application in the therapy of the inflammatory diseases.

10 cl, 13 dwg, 10 ex

 

The present invention relates to new agents for the prevention or treatment of inflammatory diseases, which contain the NR 10 antagonist as an active ingredient. The present invention also relates to a method of prevention or treatment of inflammatory diseases, which are antagonists NR 10.

Prior art

Many cytokines known as humoral factors involved in growth and differentiation of various cell types or in the activation of the function of differentiated Mature cells. Cells stimulated by cytokines, produce other types of cytokines, thereby forming in the body network from a variety of cytokines. Biological homeostasis is supported by a fragile balance of mutual regulation between cytokines in these networks. I believe that many inflammatory diseases are the result of a failure in such cytokine networks. Therefore, great attention anticytokine therapies based on monoclonal antibodies. For example, it was demonstrated high efficacy of antibodies against TNF and its receptor IL-6 in clinical application. On the other hand, there are many unfortunate examples in which was not received therapeutic effect by blocking only one cytokine, such as IL-4, due to the activation of compensatory biochemically the ways in real pathological conditions.

The authors of the present invention failed to allocate new cytokine receptor NR 10, which has a high degree of homology to gp130, receptor signaling pathway IL-6 (patent document 1). NR 10 is formed by heterodimer with receptor oncostatin M (OSMR) and functions as a receptor of IL-31 (non-patent document 1). Employees of the company Zymogenetics, Inc. it was reported that transgenic mice with overexpression of IL-31 was developed itchy dermatitis (patent document 2).

However, it has been proven that increased expression of the cytokine in mice or high levels of the cytokine in the blood in a murine model of pathology in fact cause disease. No information about any therapeutic effects by blocking the signal antibody. For example, itchy dermatitis develops in transgenic mice, keratinocytes which sverkhekspressiya IL-18. In addition, the concentration of IL-18 in blood increases with the development of pathological conditions in mice, NC/Nga, which is a model of spontaneous atopic dermatitis. Based on the above data it has been suggested that the cause of the disease is the overexpression of IL-18. In reality, however, the introduction of neutralizing antibodies had no therapeutic effects (non-patent document 2).

As described above, the inhibition function citoc is not necessarily exerts a therapeutic effect on the disease, wherein an increased level of expression of this cytokine. On the basis of the level of expression of the cytokine is difficult to predict what conditions will actually be therapeutic effect. It is therefore important to find diseases in which the inhibition of the signaling pathway cytokine-target actually has a therapeutic effect.

The following documents of the prior art for the present invention.

Patent document 1: WO 00/75314

Patent document 2: WO 03/060090

Non-patent document 1: IL-31 is associated with cutaneous lymphocyte antigen-positive skin homing T cells in patients with atopic dermatitis, J Allergy Clin Immunol. 2006 Feb; 117(2): 418-25

Non-patent document 2: Administration of anti-interleukin 18 antibody fails to inhibit development of dermatitis in atopic dermatitis-model mice, NC/Nga, British Journal of Dermatology 149: 39-45, 2003

Description of the invention

[Problems that should be solved by the invention]

The present invention was developed due to the above circumstances. The present invention is the provision of anti-cytokine therapies of inflammatory diseases based antagonists of cytokine receptors. More specifically, the present invention is the detection of inflammatory diseases, in which you can get therapeutic effect of neutralizing antibodies against NR 10 and delivery of new treatments is aka diseases. Another objective of the present invention is the provision of neutralizing antibodies against NR 10 people who apply for treatment of people.

[Solution]

The authors of the present invention have conducted special studies for solving the above problems. The authors tried to evaluate drug efficacy of neutralizing antibodies against mouse NR 10 in murine models of various pathological conditions. The results demonstrated that neutralizing antibodies significantly inhibited the symptoms in a murine model of atopic dermatitis using mice, NC/Nga and in a murine model of chronic dermatitis, which was obtained by multiple application of pikillacta. This confirmed that neutralizing antibodies really suitable as a therapeutic agent. In addition, it was demonstrated that the antibodies inhibit the symptoms of collagen arthritis model of rheumatoid arthritis and collagenase arthritis, a model of osteoarthritis. These results suggest that NR 10-neutralizing antibody of the present invention can be used for prevention or treatment of chronic inflammation. The authors present invention also managed to get antibodies, neutralizing NR 10 man. More specifically, the present invention relates to:

[1] the agent for the pros who ACTICE or treatment of inflammatory diseases, in which the agent contains NR 10 antagonist as an active ingredient;

[2] the preventive or therapeutic agent p. [1], in which the antagonist is an antibody having NR 10-neutralizing activity;

[3] the preventive or therapeutic agent p. [2], in which the antibody is a monoclonal antibody;

[4] the preventive or therapeutic agent p. [2], in which the antibody is a monoclonal antibody having the activity NR 10 neutralizing person;

[5] the preventive or therapeutic agent according to any one of paragraphs. [2]-[4], in which the antibody is a recombinant antibody;

[6] the preventive or therapeutic agent p. [5], in which the antibody is a chimeric antibody, humanitariannet antibody or human antibody;

[7] the preventive or therapeutic agent according to any one of paragraphs. [2]-[6], in which the agent contains as an active ingredient a fragment and/or a modified fragment of the antibody with the NR 10-neutralizing activity;

[8] the preventive or therapeutic agent according to any one of paragraphs. [1]-[7], in which the inflammatory disease is atopic dermatitis;

[9] the preventive or therapeutic agent according to any one of paragraphs. [1]-[7], in which oterom inflammatory disease is a chronic dermatitis;

[10] the preventive or therapeutic agent according to any one of paragraphs. [1]-[7], in which the inflammatory disease is rheumatoid arthritis;

[11] the preventive or therapeutic agent according to any one of paragraphs. [1]-[7], in which the inflammatory disease is osteoarthritis;

[12] the antibody having NR 10-neutralizing activity;

[13] the antibody according to p. [12], which is a monoclonal antibody;

[14] the antibody according to p. [12], where NR 10 NR is 10 persons;

[15] the antibody according to any one of paragraphs. [12]-[14], which is a recombinant antibody;

[16] the antibody according to p. [15], in which the recombinant antibody is a chimeric antibody, humanized antibody or human antibody;

[17] the fragment and/or a modified fragment of the antibody according to any one of paragraphs. [12]-[16];

[18] the method for prevention or treatment of inflammatory diseases, which involves the step of introducing NR 10 antagonist to a patient with inflammatory disease and

[19] the use of antagonist NR 10 for receiving agent for the prophylaxis or treatment of inflammatory diseases.

Brief description of drawings

Figure 1 presents a graph showing the results of observation of inhibiting the growth of cells under the influence of BM095 added to the system analysis of cell growth using IL-31-dependent cell a/F3. On the x-axis indicated the estimated concentrations of mIL-31 (IL-31 mouse) in the analysis system, and the ordinate axis indicates the number of cells (OD450 (absorption at 450 nm)). Added number BM095 (unit: ng/ml) are given in the captions to the drawing. Was observed the effect of inhibiting the growth of cells depending on the amount of added BM095.

Figure 2 presents a graph showing therapeutic effect obtained by the introduction of anti-NR 10 antibodies to mice, which model of atopic dermatitis. In the group, which has introduced anti-NR 10 antibodies, there was a significant suppression of inflammation compared with negative control group (group, which has introduced the media).

Figure 3 shows a graph of the sequential changes in body weight after administration of anti-NR 10 antibodies to mice, which model of atopic dermatitis. In the group treated at the current moment anti-inflammatory agent, was observed weight loss. In contrast, the weight change was not observed in the group treated with anti-NR 10 antibodies. Thus, it was confirmed that the anti-NR 10 antibody is safe.

Figure 4 presents a graph showing therapeutic effect obtained by the introduction of anti-NR 10 antibodies to mouse models of chronic dermatitis. In the group, which has introduced anti-NR 10 antibody was found who but a significant suppression of swelling of the auricle.

Figure 5 shows a photograph of immunohistochemical staining of the ear of a mouse model of chronic dermatitis. As in humans, it was found that the expression of mouse NR 10 is reinforced in bold epidermis.

Figure 6 presents a graph showing the effect of suppression of arthritis, resulting from the introduction of anti-NR 10 antibodies to mice-models-induced collagen arthritis.

Figure 7 shows a graph of the relationship between the area under the curve (AUC) and concentration BM095, injected into mice-models-induced collagenase arthritis (osteoarthritis). AUC indicates area under the curve of change of the difference between the width of the left and right knee joints, which is defined as the value representing the swelling of the right knee.

On Fig shows a graph of the correlation between the concentration of antibodies, neutralizing NR 10 person (purified antibody), and the activity of suppressing cell growth in the presence of IL-31. Antibodies 1, 2 and 3 showed strong NR 10-neutralizing activity.

Figure 9 shows a graph of the correlation between the concentration of chimeric antibodies NA633 against NR 10 person and activity, suppresses cell growth in the presence of IL-31. NA633 showed strong NR 10-neutralizing activity.

Figure 10 presents the comparison chart of amino acid sequences NR 10 man and NR 10 cynomolgus macaque. Tra is membrana region highlighted by a double line.

Figure 11 presents a graph showing the activity, inhibiting cell growth, chimeric antibodies NA633 in stimulated IL-31 human cell line BaF expressing human OSMR/NR 10 cynomolgus macaque. NA633 also showed neutralizing activity against NR 10 cynomolgus macaque.

On Fig presents a graph of relative body weight in mice models of DSS-colitis (colitis caused by doctranslate sodium).

On Fig presents a graph of the thickness of the ear in mice models of acute contact dermatitis caused by pekelharing.

The best option of carrying out the invention

The present invention relates to agents for the prevention or treatment of inflammatory diseases, which contain the NR 10 antagonist as the active ingredients. The basis of the present invention is the discovery by the authors that the antagonists NR 10 (for example, NR 10-neutralizing antibodies) significantly suppress the symptoms in model mice with atopic dermatitis, chronic dermatitis, rheumatism, osteoarthritis, etc.

NR 10 is a protein that forms heterodimer with receptor oncostatin M (OSMR) and functions as the receptor for IL-31. NR 10 is also known under other names, such as glm-r (J Biol Chem 277, 16831-6, 2002), GPL (J Biol Chem 278, 49850-9, 2003) and IL-31RA (Nat Immunol 5, 752-60, 2004). NR 10 on us is oedema invention includes proteins, with such names. NR 10 of the present invention also includes NR 10, obtained from human, mouse and other mammals. Preferred NR 10 NR 10 includes obtained from human and mouse, but is not limited to this. The NR 10 man there are many known splicing variants (WO 00/075314). From the above splicing variants NR 10.1 consists of 662 amino acids and includes the transmembrane domain. NR 10.2 is soluble receptor-like protein consisting of 252 amino acids without the transmembrane domain. Also known splicing variants NR 10 that function as transmembrane receptor proteins include NR 10.3 and IL-31RAv3. NR 10 man of the present invention is not specifically limited provided that it forms heterodimer with receptor oncostatin M (OSMR) and functions as a receptor of IL-31. Preferred NR 10 includes NR 10.3 (also referred to as ILRAv4 (Nat Immunol 5, 752-60, 2004)and IL-31RAv3. NR 10.3 (IL-31RAv4) consists of 662 amino acids (WO 00/075314; Nat Immunol 5, 752-60, 2004), and IL-31RAv3 consists of 732 amino acids (the access number in GenBank: NM_139017). Amino acid sequence of IL-31RAv4 shown in SEQ ID NO: 6, and amino acid sequence of IL-31RAv3 shown in SEQ ID NO: 7. In addition, NR 10, derived from mouse, includes proteins containing the amino acid sequence of SEQ ID NO: 5.

Antagonists NR 10 on the right is briteney refer to substances which bind NR 10 and block the transmission of the signal inside the cell, based on the activation of NR 10, thus causing the loss or inhibition of the physiological activity of the cells. In this description of the physiological activity includes, for example, induction or suppression of production of physiologically active substances (e.g., chemokines and inflammatory cytokines), strengthening or weakening the secretion of substances, growth activity, which causes increased activity, survival, cell differentiation, causing differentiation activity, transcriptional activity, membrane transport, binding activity, proteolytic activity, phosphorylation/dephosphorylation, oxidative/reductive activity, transferring, nucleotidase activity, dehydration, causing cell death activity and causing apoptosis activity, but is not limited to this.

The presence of antagonistic activity can be determined by methods known to experts in this field. For example, the test compound is administered in contact with expressed on the cell surface NR 10 in the presence of ligand and determine whether the signal inside the cell, which is an indicator of the activation of NR 10. This can be determined, for example, by the method described in the document "Dillon SR,et al., Interleukin 31, a cytokine produced by actiated T cells, dosage dermatitis in mice. Nat Immunol. 2004 Jul; 5(7):752-60". It is believed that compounds that inhibit the transmission of the intracellular signal in response to ligand stimulation, act as antagonists NR 10.

Antagonists of the present invention can be natural or artificial compounds. As antagonists of the present invention can be used known compounds. You can also use new connections, which have antagonistic activity was determined by the methods described above.

In the embodiment of the present invention antagonists NR 10 include antibodies having NR 10-neutralizing activity. "Antibodies having NR 10-neutralizing activity", according to the present invention are antibodies that inhibit the physiological activity based on NR 10. "Antibodies having NR 10-neutralizing activity", according to the present invention can be polyclonal or monoclonal antibodies, and, as the preferred option for implementing the invention include monoclonal antibodies.

Such monoclonal antibodies having NR 10-neutralizing activity can be obtained, for example, by the following method: anti-NR 10 monoclonal antibodies obtained using antigen NR 10 or its fragment, obtained from a mammal, such as human or mouse, and the known methods, and then having NR 10-neutralizing activity of the antibodies are selected from the thus obtained anti-NR 10 monoclonal antibodies. More specifically, the immunization is carried out by standard methods of immunization using as a sensitizing antigen desired antigen or cells expressing the desired antigen. Anti-NR 10 monoclonal antibodies can be obtained by merging the obtained immune cells with known parent cells using standard methods merge cells, and conducting screening for cells producing monoclonal antibodies (hybridoma), standard methods of screening. Animals to be immunized include, for example, mammals, such as mice, rats, rabbits, sheep, monkeys, goats, donkeys, cows, horses and pigs. Antigens can be obtained by using the known sequence of the gene NR 10, by known methods, for example, methods using baculoviruses (for example, WO 98/46777). As described below in the examples of this description, antibodies having NR 10-neutralizing activity, it is possible to select, for example, testing the effect of suppressing the growth of IL-31-dependent cell line after adding the test antibodies to IL-31-dependent cell line. It is believed that antibodies that inhibit the growth of IL-31-dependent cell line in the above way, have NR 10-Nate alishouse activity.

Hybridoma can be obtained, for example, by the method Milsteinet al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46) or the like, If the immunogenicity of the antigen is low, immunization can be performed after the attachment of antigen to the macromolecule with a high immunogenicity, such as albumin.

In a preferred embodiment of the present invention antibodies having NR 10-neutralizing activity, include monoclonal antibodies having the activity of neutralizing NR 10 man. There are no particular restrictions on the immunogen for obtaining monoclonal antibodies having the activity NR 10 neutralizing person, provided that it allows to obtain antibodies having the activity of neutralizing NR 10 man. For example, it is known that there are many options NR 10 man. As immunogens can use any of the options, provided that it allows to obtain antibodies having the activity of neutralizing NR 10 man. Alternatively, as the immunogen in the same conditions can be used peptide fragment NR 10 or a sequence of natural NR 10 with artificially introduced mutations. NR 10.3 person is the preferred immunogen to generate antibodies of the present invention, which have NR 10-neutralizing activity.

In this specification, the above-described antibodies present is Adamu invention is not particularly limited, provided they have NR 10-neutralizing activity. These antibodies also include recombinant antibodies such as chimeric antibodies, humanized antibodies and human antibodies. Chimeric antibodies include, for example, the constant region of the heavy and light chains of human antibodies and variable regions of the heavy and light chains of mammalian, non-human, such as a mouse. Chimeric antibodies can be obtained by known methods. For example, antibodies can be obtained by cloning the antibody gene from hybrids, by embedding it into an appropriate vector and introducing the design of the hosts (see, for example, Carl, A. K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA of the variable regions (V regions) are synthesized on mRNA hybrid using reverse transcriptase. After will be obtained cDNA encoding V region of interest antibodies, connect them with cDNA encoding the constant region (C-region) of the desired human antibodies. The resulting construction is inserted into the expression vectors. Alternatively, cDNA encoding V region of the antibody, can be embedded into expression vectors containing the C-region of human antibodies. cDNA is inserted into expression vectors so that they are expressed under the control of the expression of regulatory areas for example, enhancers and promoters. At the next stage transform cells of host-expression vector, to carry out the expression of chimeric antibodies.

Humanitariannet antibody, which is also referred to as a modified antibody of man, receive, replacing the complementarity determining regions (CDR) of an antibody of a mammal, not a person, such as a mouse, CDR human antibodies. Also well-known, conventional techniques of genetic recombination to produce such antibodies. Specifically, the cDNA sequence designed for ligating the CDR of a mouse antibody with the frame regions (FR) of a human antibodies synthesize PCR using several oligonucleotides were designed so that they contained overlapping parts at the ends. Humanitariannet antibody can be obtained (1) by legirovaniem obtained DNA with the DNA that encodes the constant region of human antibodies; (2) the inclusion of this construct in the expression vector; and (3) transfer of the vector into the host to obtain antibody (see European patent application number EP 239,400 and the international publication of patent application no WO 96/02576). FR human antibodies, which are ligated through CDR, choose those where the CDR forms a suitable antigennegative plot. If necessary, amino acids in frame phase is variable regions of the antibodies can be replaced so to CDR modified human antibodies formed corresponding antigennegative site (Sato, K.et al., Cancer Res. (1993) 53, 851-856).

Also known methods for producing human antibodies. For example, desired human antibodies with antigennegative activity can be obtained (1) sensitization of human lymphocyte antigens of interest, or cells expressing interest antigenin vitro; and (2) merge sensitized lymphocytes with myeloma cells of a person, such as U266 (see publication of Japanese patent application Kokoku No. (JP-B) H01-59878 (proven by an expert approved by the Japanese patent application published for opposition)). Alternatively, a desired human antibody can also be obtained by using the desired antigen to immunize a transgenic animal, which includes a full repertoire of antibody genes (see international publication for patent applications No. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).

In addition, known methods for obtaining human antibodies penninga ragovoy library of human antibodies. For example, on the surface of phages Express variable regions of human antibodies in single-chain antibodies (scFv) using the phage display technique, and select phages that bind to ant the genome. Analyzing the genes of the selected phages, it is possible to identify DNA sequences encoding the variable regions of human antibodies that bind the antigen. If the DNA sequence of scFv that bind the antigen, determined, to obtain antibodies person can construct the corresponding expression vectors comprising these sequences. Such methods are well known (see, WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388).

The amino acid sequence of the variable region of the heavy chain or variable region of the light chain may be amino acid sequence with substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence of variable region of the heavy chain or variable region of the light chain of the antibody with proven NR 10-neutralizing activity, provided that there is NR 10-neutralizing activity. Well-known specialists in the field of methods of obtaining the amino acid sequences of variable regions of the heavy chain or variable region of the light chain antibodies having NR 10-neutralizing activity that contains a substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence of variable region of the heavy chain or variabel the Noi region of the light chain antibodies include known methods of introducing mutations into proteins. For example, mutants, functionally equal to the variable regions of the heavy chain or variable region of the light chain antibodies having NR 10-neutralizing activity, experts in this field can be obtained by introducing appropriate mutations into the amino acid sequence of the variable region of the heavy chain or variable region of the light chain antibodies having NR 10-neutralizing activity using site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 82, 488-492) or the like, Thus, the variable regions of the heavy chains or variable regions of light chains, which contain mutations in one or more amino acids in the variable regions of the heavy chain or variable region of the light chain antibodies having NR 10-neutralizing activity, are also included in the variable region of the heavy chain or the variable region of the light chain according to the present image is the shadow.

When replacing the amino acid residue is preferred by a mutation of the amino acids is replaced with another amino acid (amino acids), which retain the properties of the side chain of amino acids. Examples of the properties of the side chains of the amino acids are hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S and T), amino acids containing aliphatic side chains (G, A, V, L, I and P), amino acids, including side chain containing a hydroxyl group (S, T, and Y), amino acids, including side chain containing sulfur (C and M), the amino acid comprising a side chain containing a carboxylic acid and amide (D, N, E and Q), amino acids containing basic side chains (R, K and H), and amino acids containing aromatic side chains (H, F, Y and W), (amino acids in parentheses are presented single-letter code). Amino acid substitutions within each group are referred to as conservative substitutions. It is already known that a polypeptide comprising a modified amino acid sequence in which one or more amino acid residues in this amino acid sequence is deleted, added and/or replaced by other amino acids, may retain the original biological activity (Mark, D. F.et al., Proc. Natl. Acad. Sci. USA; (1984) 81:5662-6; Zoller, M. J. and Smith, M., Nucleic Acids Res. (1982) 10:6487-500; Wang, A.et a ., Science (1984) 224:1431-3; Dalbadie-McFarland, G.et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409-13). Such mutants include amino acid sequence that is at least 70% identical to the amino acid sequence of variable region of the heavy chain or variable region of the light chain of the present invention, more preferably, identical, at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%. In this description, the sequence identity is defined as the percentage of residues identical residues in the original amino acid sequence of variable region of the heavy chain or variable region of the light chain, which is determined after aligning the sequences and introducing, if necessary, appropriate breaks in order to maximize the matching sequences. The identity of amino acid sequences may be determined by the method described above.

Alternatively, the amino acid sequence of the variable region of the heavy chain or variable region of the light chain, which contains a substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence is eljnosti variable regions of the heavy chain or variable region of the light chain and saves NR 10-neutralizing activity, can be obtained from the nucleic acid, which hybridizes under stringent conditions with a nucleic acid containing the nucleotide sequence encoding the amino acid sequence of the variable region of the heavy chain or variable region of the light chain. Stringent hybridization conditions for separation of nucleic acids, hybridization under stringent conditions with a nucleic acid that contains a nucleotide sequence encoding the amino acid sequence of the variable region of the heavy chain or variable region of the light chain include, for example, conditions 6M urea, 0.4% of SDS and 0.5× SSC and 37°C, or conditions of hybridization with a stiffness equal to this. In harsher conditions, for example, conditions 6M urea, 0.4% of SDS in 0.1× SSC and 42°C, is expected to nucleic acids with greater homology. Sequence of isolated nucleic acids can be determined by known methods, described below. The overall homology on the nucleotide sequence of the selected nucleic acid is at least 50% or higher sequence identity, preferably 70% or higher, more preferably 90% or higher (for example, 95%, 96%, 97%, 98%, 99% or above).

Nucleic acid, which hybridizes under stringent conditions with a nucleic acid containing a nucleotide pic is egovernance, encoding the amino acid sequence of the variable region of the heavy chain or variable region of the light chain, you can also highlight, instead of using the above methods using hybridization techniques methods of gene amplification with primers synthesized based on the information of nucleotide sequence that encodes the amino acid sequence of the variable region of the heavy chain or variable region of the light chain, for example, polymerase chain reaction (PCR).

More specifically, the identity between two nucleotide sequences or amino acid sequences can be determined using the algorithm BLAST created by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90, 5873-7). Programs such as BLASTN and BLASTX have been developed based on this algorithm (Altschulet al., J. Mol. Biol. (1990) 215, 403-10). For analysis of the nucleotide sequences according to BLASTN based on BLAST, set parameters, for example, score = 100 and word length = 12. On the other hand, the parameters used for the analysis of amino acid sequences, BLASTX based on BLAST, include, for example, score = 50 and word length = 3. When using the programs BLAST and Gapped BLAST used the default parameters of each program. For such analyses in this area known special techniques (see the web site of the National Center Bi the technological Information (NCBI), Basic Local Alignment Search Tool (BLAST);http://www.ncbi.nlm.nih.gov).

Alternatively, antibodies of the present invention can be mini-tel. Such mini body according to the present invention include antibody fragments, lacking some parts of the full-length antibody (e.g., full-size IgG), and not specifically limited, provided that they retain NR 10-neutralizing activity. Mini body according to the present invention is not specifically limited, provided that they represent areas of full-size antibodies. Mini body preferably includes a variable region heavy chain (VH) or variable region light chain (VL). Especially preferred mini body include VH, and VL.

Mini body according to the present invention, preferably, have a lower molecular weight than the full-size antibodies. However, mini body can form multimer, for example, dimers, trimers or tetramer, and therefore their molecular weight can be higher than the molecular weight of full-size antibodies.

Mini body according to the present invention include, for example, scFv-antibodies. ScFv-antibodies are single-chain polypeptides, which is obtained by combining variable regions of the heavy chain ([VH]) and the variable region of the light chain ([VL]) through a linker or similar (Huston, J. S.et al. , Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883; Plickthun “The Pharmacology of Monoclonal Antibodies” Vol. 113, eds., Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)). The order of variable regions of the heavy chain and the variable region of the light chain, which must be connected together, are not specifically limited, and can be arranged in any order. Examples of the locations below.

[VH] linker [VL]

[VL] linker - [VH]

Amino acid sequence of the variable region of the heavy chain or variable region of the light chain may include substitution, deletion, addition and/or insertion. In addition, the variable regions of the heavy chain and the variable region of the light chain may also be missing some parts or can be added to other polypeptides, provided that together they bind antigen. Alternatively, the variable regions can be chemerisov or humanitarian.

In the present invention, the linkers that connect the variable regions of the antibodies include arbitrary peptide linkers that can be introduced using methods of genetic engineering or synthetic linkers (e.g., linkers disclosed in Protein Engineering, 9(3), 299-305, 1996").

Preferred linkers in the present invention are peptide linkers. The length of the peptide linkers are not specifically limited, and specialists who this field can choose the length depending on your goal. Typical length is up to 100 amino acids, preferably from 3 to 50 amino acids, more preferably from 5 to 30 amino acids, and particularly preferably 12 to 18 amino acids (for example, 15 amino acids).

Amino acid sequences of these peptide linkers include, for example:

Ser

Gly·Ser

Gly·Gly·Ser

Ser·Gly·Gly

Gly·Gly·Gly·Ser (SEQ ID NO: 8)

Ser·Gly·Gly·Gly (SEQ ID NO: 9)

Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 10)

Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 11)

Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 12)

Ser·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 13)

Gly·Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 14)

Ser·Gly·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 15)

(Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 10))n

(Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 11))n

where n is an integer of 1 or more.

Synthetic linkers (chemical cross-linking agents include crosslinking agents that are usually used for cross-stitching peptides, for example, N-hydroxysuccinimide (NHS), disuccinimidyl (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylester) (DSP), dithiobis(sulfosuccinimidyl) (DTSSP), ethylene glycol-bis(Succinimidyl) (EGS), ethylene glycol-bis(sulfosuccinimidyl) (sulfo-EGS), disuccinimidyl (DST), desulfobacteriaceae (sulfo-DST), bis[2-(succinyldicholine)ethyl]sulfon (BSOCOES), and bis[2-(sulfosuccinimidyl)ethyl]sulfon (sulfo-BSOCOES). These crosslinking agents are commercially available is PNY.

Antibodies of the present invention include antibodies that have to amino acid sequences of the antibodies of the present invention includes two or more amino acid residue. In addition, in the present invention included fused proteins, which are the result of a merger of one of the above-described antibodies and a second peptide or protein. Fused proteins can be obtained by legirovaniem with preservation of the reading frame of polynucleotide encoding the antibody of the present invention, and polynucleotide encoding a second peptide or polypeptide, the embedding of this design in the expression vector and expression of the slit structure in the host. For this purpose, suitable some techniques known to experts in this field. Additional peptide or polypeptide to merge with the antibody of the present invention may be a known peptide, for example, FLAG (Hopp, T. P.et al., BioTechnology 6, 1204-1210 (1988)), 6x His consisting of six His residues (histidine), 10× His, the hemagglutinin of influenza virus (HA), a fragment of c-myc man, a fragment of VSV-GP fragment p18HIV, T7-tag, HSV-tag, E-tag, a fragment of the T-antigen of SV40, lck tag, a fragment of α-tubulin, B-tag, protein fragment C. Other extension polypeptides to merge with antibodies of the present invention include, for example, GST (glutathione-S-transferase), HA (hemagglutinin of influenza virus), constant on the region of the immunoglobulin, β-galactosidase and MBP (maltose-binding protein). Polynucleotide encoding one of these commercially available peptides or polypeptides, can be drained with polynucleotides coding for the antibody of the present invention. Fused polypeptide can be obtained by expression of the fused structure.

Antibodies of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, the presence/absence of chains of sugars and conformation depending on the cell or host, producing the antibody, or purification method. However, the resulting antibody include in the present invention provided that it functions the same functions as the antibodies of the present invention. For example, when the antibody of the present invention is expressed in prokaryotic cells, such asE. colito the N end of the original amino acid sequence of antibodies attached methionine residue. Such antibodies are included in the present invention.

The antibody of the present invention can be obtained by methods known to experts in this field. The antibody can be obtained, for example, using recombinant techniques known to experts in this field, based on a series of antibodies that recognize NR 10. More specifically, such an antibody can be obtained by creating a design is the construction encoding the antibody of polynucleotide based on a series of antibodies, which recognizes NR 10, embedding design in the expression vector, and then the expression in the appropriate cell hosts (see, for example, Co, M. S.et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J.et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

Vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. Alternatively, if the goal is subclavian and cut cDNA, in addition to the above vectors vectors include, for example, pGEM-T, pDIRECT, and pT7. When using vectors to generate antibodies of the present invention are particularly suitable expression vectors. For example, if the goal is to expression in cellsE. colisuch as JM109, DH5α, HB101, and XL1-Blue, the expression vectors have not only the above properties, which allow the amplification of the vector inE. colibut also should bear a promoter that provides for efficient expression inE. colifor example, lacZ promoter (Wardet al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Betteret al., Science (1988) 240, 1041-1043), T7-promoter or other Such vectors in addition to the above-described vectors include pGEX-5X-1 (Pharmacia), vectors of "QIAexpress system" (Quiagen), pEGFP or pET (in this case, it is preferable that the cells of the host was CL the TCI BL21, which Express RNA polymerase of phage T7).

The vectors may contain a signal sequence for secretion of antibodies. As a signal sequence for secretion of antibodies, it is possible to use the signal pelB sequence (Lei, S. P.et alJ. Bacteriol. (1987) 169, 4379), in the presence of protein which is secreted into periplasmE. coli. The vector can be introduced in the cells of the host, for example, by transformation with the use of calcium chloride or by electroporation.

In addition to the vectors forE. colivectors to generate antibodies of the present invention include, for example, vectors for expression in mammalian cells (for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, and pCDM8), vectors for expression in insect cells (for example, vectors baculovirus expression system, "Bac-to-BAC baculovirus expression system" (Gibco-BRL) and pBacPAK8), vectors for expression in plant cells (for example, pMH1 and pMH2), expression vectors based on animal viruses (for example, pHSV, pMV, and pAdexLcw), retroviral expression vectors (for example, pZIPneo), vectors for expression in yeast (e.g., vectors from the set for expression in the yeast Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and vectors for expression inBacillus subtilis(for example, pPL608 and pKTH50).

If the goal is the expression in animal cells such as CHO cells, COS, and NIH3T3, the vector should have a promoter necessary is IMY for expression in these cells, for example, the SV40 promoter (Mulliganet al., Nature (1979) 277, 108), the promoter MMLV-LTR, EF1α promoter (Mizushimaet al., Nucleic Acids Res. (1990) 18, 5322), and CMV-promoter, and, more preferably, these vectors had a gene for selection of transformed cells (for example, the gene of resistance to the drug, which allows the detection of transformed cells using selective agent (neomycin, G418, or the like). Vectors with such characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

In addition, you can use the following method for stable gene expression and gene amplification in cells: cells SNO, have violated one of the pathways of biosynthesis of nucleic acids, embed the vector (for example, pSV2-dhfr (Molecular Cloning 2ndedition, Cold Spring Harbor Laboratory Press, 1989)), which carries a DHFR gene, which compensates this disadvantage, and the vector is amplified in the presence of methotrexate (MTX). Alternatively, for transient expression of genes can be used the following way: COS cells, bearing on its chromosome a gene expressing T-antigen SV40 transform vector (pcD or similar vector) with the replication origin SV40. You can also use the point of the beginning of the replication of the virus polyoma, adenovirus, virus, bovine papillomavirus (BPV), etc. For amplification of the gene in the cells of the host expression vectors can also be carried with the collective tokens such as gene aminoglycoside transferase (APH)gene thymidine kinase (TK)gene xanthine-guanine phosphoribosyl-transferaseE. Coli(Ecogpt) gene dihydrofolate-reductase (dhfr).

The desired antibody is obtained by the methods described above can be isolated from cells or cellular environment (environment or similar media) and clear to a homogeneous state. Antibodies can be selected and cleaned by methods commonly used for isolation and purification of antibodies, and type of method is not limited. For example, antibodies can be selected and cleared, respectively selecting and combining methods column chromatography, filtration, ultra filtration, desalting, precipitation with a solvent, solvent extraction, distillation, thus, electrophoresis in SDS-polyacrylamide gel, isoelectrofocusing, dialysis, recrystallization, etc.

Chromatographic methods include, for example, affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography and absorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshaket al., Cold Spring Harbor Laboratory Press, 1996). The above-described chromatographic methods can be made using liquid chromatography, such as HPLC and FPLC. The column, which can be used for the affine chromatography is, include columns with protein a and the column of protein g Column with protein And include, for example, Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). The present invention includes antibodies, purified with the use of these cleaning methods.

The present invention also relates to agents for the prevention or treatment of inflammatory diseases, which contain as active ingredients parts and/or products modification of the antibodies of the present invention. Fragments and/or products modified antibodies of the present invention include fragments of antibodies that are some parts of the antibodies of the present invention (full-length antibody (e.g., full-size IgG), recombinant antibodies (e.g., chimeric antibodies, humanized antibodies and human antibodies) and a body (for example, scFv-antibodies), which are not specifically limited, provided that they retain the ability to bind their antigens. Fragments of the antibodies of the present invention is not specifically limited provided that they are parts of a full-sized antibodies. The fragments preferably contain a variable region heavy chain (VH) and/or variable region light chain (VL). Amino acid sequence of VH or VL can include substitutions, deletions, additions and/or insertions. The VH and/or VL can OTS is estvovati some areas provided they retain the ability to bind antigen. In addition, the variable region may be chemerisov or humanitarian. More specifically, antibody fragments include, for example, Fab, Fab', F(ab')2 and Fv. Such antibody fragments can be obtained by creating structures of the genes encoding fragments of the antibody, inserting them into expression vectors, and then the expression in the appropriate cell hosts (see, for example, Co, M. S.et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J.et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

In addition, antibodies of the present invention can be conjugated to antibodies, which are attached to any of a number of molecules such as polyethylene glycol (PEG), radioactive substances, fluorescent substances, luminescent substances, enzymes and toxins. Such conjugated antibodies can be obtained by chemical modification of the obtained antibodies. Methods of modification of the antibodies is widely known in the field (examples are US 5057313 and US 5156840). "Antibodies" of the present invention also include such conjugated antibodies.

The binding activity of the antibody with the NR 10 can be determined by methods known to experts in this field. How to determine antigens asiausa activity of antibodies include, for example, ELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay analysis and immunofluorescent analysis. For example, using an enzyme immunoassay containing antibody samples, such as purified antibodies and cultural supernatant antibody productive cells, added to the plates coated with antigen. Add secondary antibody labeled with an enzyme such as alkaline phosphatase and incubated tablets. After washing, add the enzyme's substrate, such as p-nitrophenylphosphate, and measure the absorption to assess antigennegative activity.

In addition, you can define NR 10-neutralizing activity, for example, by the method described in the examples, which includes the study of the effect of suppressing the growth of IL-31-dependent cell line.

Antagonists NR 10 or antibodies having NR 10-neutralizing activity of the present invention, can be used in agents for the prevention or treatment of inflammatory diseases. The authors of the present invention have proved that the introduction of antibodies that neutralize murine NR 10, provides significant therapeutic effect in various inflammatory diseases in animal models. In addition, it was published that in bold epidermis of patients with atopic dermatitis enhanced expression of NR 10 the brow of the ESA (non-patent document 1). Using immunohistochemical staining, the authors of the present invention have confirmed that, as in humans, expression of mouse NR 10 is reinforced in bold epidermis of the ear of mice models of chronic dermatitis described above (example 5). These data suggest that NR 10 involved similarly in inflammatory diseases of animals of all kinds, and that, like antibodies, neutralizing mouse NR 10, antibody, neutralizing NR 10 man, are effective for prophylaxis and treatment of various inflammatory diseases. In addition, as in the case of the data described in this example also expect that, in addition to antibodies, other antagonists NR 10 have therapeutic effect against various inflammatory diseases.

In the present invention inflammatory disease refers to the diseases with pathological features involved in cytological and histological reactions that occur in the affected blood vessels and adjacent tissues in response to injury or abnormal stimulation caused by a physical, chemical or biological agents (Stedman''s Medical Dictionary, 5th Ed., MEDICAL VIEW CO., 2005). Usually inflammatory diseases include dermatitis (atopic dermatitis, chronic dermatitis, and the like), inflammatory bowel disease (colitis and the like), asthma, arthritis (rheumatoi the hydrated arthritis, osteoarthritis and the like), bronchitis, Th2 autoimmune disease, systemic lupus erythematosus, malignant gravis, chronic GVHD (graft versus host), Crohn's disease, deforming spondylitis, low back pain, gout, inflammation after surgery or injury, swelling, neuralgia, laryngopharyngeal, cystitis, hepatitis (non-alcoholic steatohepatitis, alcoholic hepatitis, and the like), hepatitis b, hepatitis C and atherosclerosis.

Preferred examples of inflammatory diseases, which are objects of the present invention include atopic dermatitis, chronic dermatitis, rheumatism, osteoarthritis and chronic asthma.

The inventors have found that antibody antagonist NR 10 have therapeutic effect against atopic dermatitis, chronic dermatitis, rheumatism and osteoarthritis. On the other hand, it was found that antibody antagonist NR 10 do not affect the model of acute contact dermatitis and acute DSS-colitis.

Agents for the prevention or treatment of inflammatory diseases of the present invention contain as the active ingredient NR 10 antagonist or antibody, having NR 10-neutralizing activity described above. The phrase "contains NR 10 antagonist as an active ingredient" means the content of NR 10 antagonist in image quality is as, at least one of the active ingredients and does not limit the composition. In addition, the agents for the prevention or treatment of inflammatory diseases of the present invention can also contain, in combination with an antagonist NR 10, other ingredients that contribute to the prevention or treatment of inflammatory diseases.

Recipes with NR 10 antagonist of the present invention can be by standard methods (see, for example, Remington''s Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). In addition, if necessary, they may contain pharmaceutically acceptable carriers and/or additives. For example, they may contain surface-active substances (for example, PEG-Il Tween), fillers, antioxidants (e.g. ascorbic acid), colorants, flavoring agents, preservatives, stabilizers, buffering agents (for example, phosphoric acid, citric acid and other organic acid), chelating agents (such as EDTA), suspendresume agents, isotonic agents, binders, disintegrating agents, lubricating agents, amplifiers fluidity and corrigentov. However, the media that may contain agents for the prevention or treatment of inflammatory diseases the present invention, is not limited to this list. In fact, accordingly, may contain other ordinary is the media being used, such as anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium, carmellose, sodium, carmellose, hydroxypropylcellulose, hypromellose, polyvinyltrimethylsilane, polyvinylpyrrolidone, gelatin, triglycerides of fatty acids of medium length, polyethoxysiloxane gidrirovannoe castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salt, etc. of the Composition can also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine and lysine. When the composition is prepared in the form of an aqueous solution for injection, antagonists NR 10 can be dissolved in isotonic, which includes, for example, saline, dextrose, and other excipients. Excipients may include, for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride. In addition, in parallel you can use suitable solubilizing agents, for example, alcohols (e.g. ethanol), polyalcohol (e.g. propylene glycols and PEG) and non-ionic detergents (Polysorbate 80 and HCO-50).

If necessary, the antagonists NR 10 can be enclosed in microcapsules (microcapsules made of hydrocellulose, gelatin is, polymethylmethacrylate and the like) and turn it into a components of colloidal delivery systems of medicines (liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) (for example, see “Remington''s Pharmaceutical Science 16th edition” &, Oslo Ed. (1980)). Moreover, known methods of manufacturing drugs with a slow release, and they can be used for antagonists NR 10 (Langeret al., J. Biomed. Mater. Res. (1981) 15, 167-277; Langer, Chem. Tech. (1982) 12, 98-105; U.S. patent No. 3,773,919; European patent (EP) No. 58,481; Sidmanet al., Biopolymers (1983) 22, 547-56; EP 133,988).

Agents for the prevention or treatment of inflammatory diseases of the present invention can be entered or oral, or parenteral, but the preferred route of administration is parenteral. More specifically, the agents are administered to patients by injection or subcutaneous injection. Injection include, for example, intravenous injection, intramuscular injection and subcutaneous injection, for systemic or local administration. Agents can be introduced in areas where there should be suppressed inflammation, or in the surrounding areas area using local infusion, in particular, intramuscular injection. The routes of administration should be selected in accordance with the age and condition of the patient. The dose for a single injection can be, for example, in the range from 0.0001 to 100 mg of active phrases is that per kg of body weight. Alternatively, with the introduction of agents to a human dose of active ingredient can be selected in the range from 0.001 to 1000 mg/kg body weight. The dose for a single injection preferably contains, for example, the NR 10 antagonist in an amount of from about 0.01 to 50 mg/kg body weight. However, these examples do not limit the dose of an agent for prevention or treatment of inflammatory diseases of the present invention.

The present invention also relates to antibodies having NR 10-neutralizing activity (including fragments of antibodies having NR 10-neutralizing activity, and/or the products of their modification; the same definition is used in the description below). Antibodies having NR 10-neutralizing activity of the present invention, suitable as active ingredients in the above agents for the prevention or treatment of inflammatory diseases. Antibodies having NR 10-neutralizing activity of the present invention may be polyclonal or monoclonal antibodies, but are preferably monoclonal antibodies. Such monoclonal antibodies having NR 10-neutralizing activity can be obtained by the methods described above. Monoclonal antibodies of the present invention have the activity, NR 10 neutralizing mammals or fragments thereof, preferably neutralizing NR 0 human or mouse or fragments thereof, and, more preferably, NR 10 neutralizing person or its fragments. NR 10 man or peptide fragment can be used as an immunogen to obtain monoclonal antibodies, neutralizing NR 10 man. Alternatively, antibodies having the activity of neutralizing NR 10 of the present invention may be recombinant antibodies. Recombinant antibodies having NR 10-neutralizing activity of the present invention, include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies and mini body.

In the present invention, antibodies having NR 10-neutralizing activity include, for example, antonysamy NR 10 antibodies, which include variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 1, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 3.

The present invention also includes monoclonal antibodies having the activity of neutralizing NR 10 man. Monoclonal antibodies having the activity of neutralizing NR 10 man of the present invention, can be obtained by obtaining monoclonal antibodies using NR 10 man or peptide fragment as the immunogen and then for selecting monoclonal antibodies against NR 10 people who antibodies with activity, NR 10 neutralizing person, on the basis of the above analytical system using IL-31-dependent cell line.

In the present invention, antibodies having the activity of neutralizing NR 10 include the antibodies described in any of paragraphs:

(a) antibodies having a variable region heavy chain containing CDR1, CDR2 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 18, 19 and 20, respectively;

(b) antibodies having a variable region light chain containing CDR1, CDR2 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 21, 22 and 23, respectively;

(c) antibodies having a variable region heavy chain under item (a) and the variable region of the heavy chain according to p. (b); and

(d) antibodies that recognize the same epitope as the epitope recognized by the antibody according to any one of paragraphs. (a)-(c).

Know whether the antibody has the same epitope as another antibody can be confirmed by the competition between the two antibodies for this epitope. Competition between antibodies can be evaluated by competitive binding using techniques such as ELISA, resonance energy transfer fluorescence (FRET) and the analysis of fluorimetry in microvolume (FMAT(R)). The number associated with antigen antibodies indirectly correlates with binding ability opituensurorn antibody (test antibody), which competitive bind the same epitope. In other words, if the number or the affinity of the test antibody against the same epitope increases, the number associated with the antigen of the antibody is reduced and the number of tested antibodies associated with epitope increases. More specifically, respectively labeled antibodies and antibodies, evaluate which you want to spend, at the same time add to the antigen, and thus, detect bound antibodies, using the label. The number associated with antigen antibody easy to determine, in advance of introducing the label to the antibody. This label is not specifically limited, and a method of tagging choose, based on the methodology used in the analysis. The method includes tagging fluorescent labels, radioactive labels, enzyme labels, etc.

For example, antibodies labeled with a fluorescent label and unlabeled antibodies or test antibodies simultaneously added to animal cells expressing NR 10, and are labeled detection antibody method fluorimetry in the microvolume.

IC50represents the concentration at which the number associated with epitope-labeled antibodies is reduced to 50% as a result of binding of unlabeled antibody. In the present opionionated that recognize the same epitope, are antibodies that may reduce the share of the in-labeled antibodies, related epitope, at least up to 50%when the concentration of the test antibody in 10 times higher than the IC50unlabeled antibodies.

Antibodies having the activity of neutralizing NR 10 people who use in the present invention, preferably, further bind NR 10 cynomolgus macaque, and, more preferably, neutralize NR 10 cynomolgus macaque. NR 10 cynomolgus macaque containing the amino acid sequence of SEQ ID NO: 24, can be used in the measurement of neutralizing activity or binding to NR 10 cynomolgus macaque.

The present invention also relates to methods of treatment described below. Interpretation NR 10, antagonists, monoclonal antibodies, recombinant antibodies, inflammatory diseases and other ways the same as described above.

(1) a method of preventing or treating inflammatory diseases, which includes a stage of introduction of the NR 10 antagonist to a patient with inflammatory disease;

(2) the method according to p. (1), where NR 10 antagonist is an antibody having NR 10-binding activity;

(3) the method according to p. (2), in which the antibody is a monoclonal antibody;

(4) the method according to p. (2), in which the antibody is a monoclonal antibody that binds NR 10 man;

(5) the method according to any one of paragraphs. (2)to(4), in which the antibody is a recombinant and what antibodies;

(6) the method according to p. (5), in which the recombinant antibody is a chimeric antibody, humanized antibody or human antibody;

(7) the method according to any one of paragraphs. (2)to(6), in which the fragment antibodies having NR 10-neutralizing activity, and/or product modification included as an active ingredient;

(8) the method according to any one of paragraphs. (1)to(7), in which the inflammatory disease is atopic dermatitis;

(9) the method according to any one of paragraphs. (1)to(7), in which the inflammatory disease is a chronic dermatitis;

(10) the method according to any one of paragraphs. (1)to(7), in which the inflammatory disease is rheumatoid arthritis; and

(11) the method according to any one of paragraphs. (1)to(7), in which the inflammatory disease is osteoarthritis.

The present invention also relates to the discovery, described below. Interpretation NR 10, antagonists, monoclonal antibodies, recombinant antibodies, inflammatory diseases and other methods is the same as described above.

(1) application of the antagonist NR 10 for receiving agent for the prophylaxis or treatment of inflammatory diseases;

(2) the application under item (1), where NR 10 antagonist is an antibody that binds NR 10;

(3) the application under item (2), in which the antibody is a monoclonal antibody;

(4) the application under item (2), in which the antibodies is about is a monoclonal antibody which binds NR 10 man;

(5) the use according to any one of paragraphs. (2)to(4), in which the antibody is a recombinant antibody;

(6) an application under item (5), in which the recombinant antibody is a chimeric antibody, humanized antibody or human antibody;

(7) the use according to any one of paragraphs. (2)to(6), in which the fragment antibodies having NR 10-neutralizing activity, and/or product modification included as an active ingredient;

(8) the use according to any one of paragraphs. (1)to(7), in which the inflammatory disease is atopic dermatitis;

(9) the use according to any one of paragraphs. (1)to(7), in which the inflammatory disease is a chronic dermatitis;

(10) the use according to any one of paragraphs. (1)to(7), in which the inflammatory disease is rheumatoid arthritis; and

(11) the use according to any one of paragraphs. (1)to(7), in which the inflammatory disease is osteoarthritis.

All documents of the prior art are included in this description by reference.

Examples

Below this description of the present invention will be more specifically described based on examples, but they should not be construed as limiting the present invention.

[Example 1] the Creation of a cell line Ba/F3 expressing NR 10 and OSMR

cDNA NR 10 human (SEQ ID NO: 1 of WO 0075314) straw is whether in the expression vector pCOS1 (Biochem Biophys Res Commun. 228, p838-45, 1996), to obtain pCosNR 10.3. cDNA of the receptor oncostatin M (OSMR, the access number in the database of GenBank - NM003999) was obtained by PCR from the placental library person, and then similarly created expression vector pCos1-hOSMR. 10 μg of each vector was simultaneously introduced by electroporation into the cell line Ba/F3, derived from the murine IL-3-dependent Pro-b cells (BioRad Gene Pulser; 960 μf and 0.33 kV). After the introduction of DNA was added to IL-31 people, and granulosa cells. Thus, the received cell line proliferation which depends on IL-31. Similarly from the cells Ba/F3 received cell line-dependent murine IL-31 created for the expression of mouse genes NR 10 and OSMR.

For both cell lines was found ED50 value was a few ng/ml Thus, were well received proliferating lines. Dependent on IL-31 human cell line did not respond to mouse IL-31, and by adding protein NR 10 person (extracellular domain) inhibited growth. Dependent on IL-31 mouse cell line did not respond to IL-31 and adding protein NR 10 mouse (extracellular domain) did not suppress growth.

[Example 2] Get protein NR 10 (extracellular domain)

Single extracellular domain amplified by PCR on cDNA NR 10 as the matrix attached sequence FLAG-tag to the C-end and integrated receiving the infra structure in the expression vector pCXND3 (WO 2005/005636) (pCXND3-NR 10-flag). 10 mg of this linearized vector was introduced by electroporation into the cell line of Chinese hamster ovary DG44 (BioRad Gene PulserII; 25 μf and 1.5 kV). Received cell line with high expression level. Received a purified sample of the supernatant scaled culture cell lines in a column with anti-FLAG antibody (SIGMA) and gel-filtration and used in the experiments described below. Similarly received mouse NR 10 (extracellular domain) with a sequence of FLAG-tag attached at the C-end.

[Example 3] the Selection of scFv having neutralizing activity against mouse NR 10, and receiving BM095 as chimericwanna IgG

More specifically, the inventors have tried to narrow the number of candidate clones from ragovoy library of human antibodies penninga using biotinylated murine protein NR 10 (extracellular domain). Secreted scFv was purified from clones and added to the system analysis of the growth of cells with IL-31-dependent cells Ba/F3 described in example 1. The result has been successfully received by the clone BM095, which strongly inhibited the growth of cells.

In order to construct the expression vector, and used PCR to connect sequences of variable regions of the heavy chain (VH) of a person from BM095 with a constant region (after SN) mouse IgG2a, and sequences of variable regions became the first chain (VL) of a person from BM095 with the constant region of the λ-chain of the mouse. Amino acid sequence of VH is shown in SEQ ID NO: 1 and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2. Amino acid sequence of VL is shown in SEQ ID NO: 3 and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4. The corresponding linearized expression vectors were injected simultaneously into the cell line DG44, and then selected a cell line expressing a high level chimericwanna IgG. The purified sample was obtained from a supernatant scaled culture cell lines by chromatography on a column of protein A (rProtein A Sepharose Fast Flow, GE Amersham Biosciences) and the cation-exchange column (SP-TOYOPEARL 650M, TOSOH). In addition, the resin ActiClean Etox (Sterogen) remove pyrogens to lower the concentration below the detection limit. The sample used in the following experiments on animals. The result obtained by adding BM095 to the above-described system analysis, shown in figure 1.

[Example 4] evaluation of the efficacy of a drug on the model of atopic dermatitis using mice, NC/Nga

Model dermatitis received multiple application pekelharing (PiCl) with weekly intervals. More specifically, 4 days after sensitization with 5% solution of PiCl (150 ál) region of the peritoneum and the pads of the paws, induced de is MATIC repeated application of a 0.8% solution PiCl (150 ál) on the back and the ear with weekly intervals. For the associated symptoms were observed twice a week for a period of time from the third to the sixth week, and independently evaluated five options on a scale from 0 to 3 ((1) - itching (2) - redness and bleeding, (3) swelling, (4) is wound and tissue damage, and (5) - scab and dry). The antibody was administered at 10 mg/kg in the peritoneal cavity during the day before sensitization and each induction, a total of 6 times (group BM095), or the day before each induction after the third week, only three times (group V/B). As a positive control mice orally was administered dexamethasone (1 mg/kg every day after the third week (DEX group). Each group had 10 male mice, NC/Nga.

As shown in figure 2, a significant inhibitory effect was observed in the group which was administered antibodies, compared with the group that was administered the solvent (carrier). In addition, as can be seen from figure 2, while in the DEX group had significant weight loss, the group, which was administered antibodies, no weight loss. This suggests that the antibody is a very safe agent.

[Example 5] evaluation of the efficacy of a drug using a model of chronic dermatitis, created by repeated application of pekelharing

Conducted sensitization six-week female BALB/c mice, while the right ear 20 MK is 0.5% solution of pikillacta (in a mixture of acetone and olive oil (volume ratio 1:4)). Induction was performed by repeated application of the right ear 20 µl of 0.25% solution of pikillacta (in a mixture of acetone and olive oil (volume ratio 1:4)) in a day, starting from the eighth day after sensitization. BM095 in the amount of 10 mg/kg was injected into the peritoneal cavity once a week, since the day before sensitization in the group to assess the preventive effect BM095, or starting from the 20th day after the start of induction in the group to assess therapeutic effect BM095. Swelling of the auricle was assessed by serial measurement of the thickness of the right ear using casinomar with a dial indicator.

As a result, as can be seen from figure 4, a significant inhibitory effect was observed in the group which was administered antibodies. It was published that the expression of NR 10 man raised in bold epidermis of patients with atopic dermatitis (non-patent document 1). For the above model of chronic dermatitis was performed immunohistochemical staining of the auricle. As in humans, in bold epidermis observed increased expression of mouse NR 10 (figure 5; WM, a constant region which is replaced in the constant region of human IgG, obtained in example 3 and used in immunohistochemical staining. The areas colored brown, are places of expression of NR 10). These data allow PR is dologite, what NR 10 similarly involved in inflammatory diseases and in mice and humans.

[Example 6] evaluation of the efficacy of a drug using the model of arthritis (rheumatism), caused by collagen

Obtaining model mice and evaluate the effectiveness of the drugs was performed according to the procedure described below.

140 μl of the collagen gel, which was obtained by mixing equal amounts of complete adjuvant H37Ra and 0.3% solution of collagen type II derived from bovine joints were injected intradermally at the base of the tail 9-week-old female mice DBA/1JN (Day 0; sensitization). After three weeks obtained by the same procedure collagen gel was injected intradermally in the back, in order to induce the beginning of arthritis (Day 21; induction). Two days before sensitization (Day -2) 16 mice were divided into two groups according to body weight, each of which consisted of 8 mice, in order to form a group, which enter the environment, and the group injected BM095. Using as environment acetate buffer with a concentration of 20 mmol/l (pH 5.5) (200 mmol/l NaCl), diluted 6 times with PBS, the test substance BM095 was injected intravenously 8 mice of the group, which introduce BM095, in the amount of 10 mg/kg of body weight per day before sensitization (Day -1). Meanwhile, in the quality control at day -1 was administered the same volume of medium per unit weight is eating 8 mice in the group, which enter the environment. Watched the swelling of the four limbs and was given a score (on a scale from 0 to 4 for each limb: General evaluation 16), starting from the day before induction (Day 20) at intervals of 2 to 3 days.

In the result, it was found that the measurement of swelling of the four limbs fell after the 20th day in the group, which was introduced BM095, as seen in Fig.6. This suggests that BM095 suppresses the beginning of arthritis.

[Example 7] evaluation of the efficacy of a drug using the model of arthritis (osteoarthritis), caused by collagenase

Obtaining model mice and evaluate the effectiveness of the drugs was performed according to the procedure described below.

Control environment (20 mmol/l acetate buffer (pH 5.5)diluted 6 times in PBS, 200 mmol/l NaCl (n=5)), BM095 at 2 mg/kg (n=5) and 20 mg/kg (n=6) were injected intravenously (5 ml/kg) into the tail vein of 8-week-old male mice C57BL/6J Jcl (CLEA Japan Inc.). Then under inhalation anesthesia with 3% isoflurane (Am J Pathol 1989; 135(6):1001-14) mice in the cavity of the right knee joint was injected 6 μl of a 1.5% solution of collagenase (type II; Sigma), filtered through 0.45 µm filter (MILLIPORE).

The thickness of the right and left knee joints were measured with a Vernier caliper (Mitsutoyo CO.) just before the introduction of collagenase and at 3, 7 and 14 days after injection, in order to calculate the difference between the right and is the combat joints. The difference was considered representative of the size of the tumor of the right knee joint. To change the difference in area under the curve (AUC) was determined on the basis of the rules of the line and considered an indicator of the effectiveness of the drug. The student's t test was performed for AUC of the control group (medium) and the group, which was introduced BM095, using software for statistical analysis (SAS Institute Inc.) (the difference was considered statistically significant when p<0,05). As a result, the group, which was introduced BM095, AUC was significantly lower with dosage than in the control group (medium), as shown in Fig.7. This result demonstrates that BM095 inhibits arthritis in the model of osteoarthritis.

[Example 8] Getting neutralizing antibodies against NR 10 man

Mice were immunized with a protein NR 10 person (extracellular domain) {described in example 2}, and received hybridoma conventional ways. Conducted analysis of the cultural supernatants hybrid in the presence of neutralizing activity using dependent IL-31 human cell lines (cells hNR 10/hOSMR/BaF3), described in example 1. There were many clones exhibiting strong NR 10-neutralizing activity (Fig).

[Example 9] Obtaining chimeric human antibodies

Amino acid sequence of the variable region of the heavy chain NA633, which shows yet the strongest activity among neutralizing antibodies, shown in SEQ ID NO: 16, and the amino acid sequence of the variable region of the light chain shown in SEQ ID NO: 17. In addition, amino acid sequences CDR1, CDR2 and CDR3 of the variable region of the heavy chain NA633 shown in SEQ ID NO: 18, 19 and 20, respectively; and amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the light chain shown in SEQ ID NO: 21, 22 and 23, respectively.

In addition, received the traditional way chimeric antibody comprising the above-described murine variable region and a constant region of a human heavy chain IgG1-type; light chain κ-type), and determined the neutralizing activity. As shown in Fig.9, a chimeric antibody NA633 showed strong neutralizing activity at low concentrations.

[Example 10] Allocation NR 10 cynomolgus macaque

The authors sought to highlight gene NR 10 cynomolgus macaque, because we believed that cross-recognition and neutralizing activity against NR 10 cynomolgus macaque important when testing for security at the preclinical stage. Primers were designed on the basis of the disclosed information about the genome of the rhesus macaque and the like, and successfully amplified gene NR 10 of bodies cynomolgus macaque using PCR. Figure 10 shows the alignment of amino acid sequences NR 10 man and a dedicated NR 10 cynomolgus macaque. Amino acid placentas is required NR 10 cynomolgus macaque shown in SEQ ID NO: 24. The result is the introduction of a gene NR 10 cynomolgus macaque together with OSMR gene in cells Ba/F3 as described in example 1, was established cell line, the proliferation of which depends on IL-31 people. The neutralizing activity of the chimeric antibody NA633 described in example 9, was tested with this cell line. The result showed that the antibody also exhibits strong neutralizing activity in cynomolgus macaque (11).

[Reference example 1] the Efficacy of the medicinal product BM095 when caused by doctranslate sodium (DSS) colitis

To evaluate the effect BM095 (neutralizing antibodies against mouse NR 10) received model of colitis, induced by DSS (J Immunol (2003) 171:5507-5513), published as a pathological model of inflammatory bowel disease (IBD). Prepare a 5% (weight/volume) solution of sodium salt of dextransucrase (Wako Pure Chemical Industries) in water, sterilized by filtration through 0.22 μm filter (Millipore). A six-week male mice Balb/cAnN Crj (CHARLES RIVER LABORATORIES JAPAN) were allowed to freely drink the solution of the drinking bowl for 7 days. Animals were weighed, and the change of body weight in percent by weight on the first day of admission DSS was used as an indicator of the effectiveness of the drug.

In this model, the neutralizing antibody BM095 against NR 10 mice were injected intravenously at a dosage of 10 mg/kg per day to receive DSS janewale final weight loss (n=10), to verify that improves whether pathological condition by neutralizing signal from IL-31. As a control group of media used group (a group of media; n=10), in which the day before receiving DSS intravenously injected media (a mixture of acetate buffer [20 mmol/l sodium acetate and 20 mmol/l sodium chloride] and phosphate-salt buffer [PBS; GIBCO], which was mixed in a volume ratio 1:5). In addition, also investigated the dynamics of relative weight change in mice Balb/cAnN Crj same sex and age (n=1), as the group that received DSS, in order to know the dynamics of relative weight change in mice was normal.

The dynamics of weight shown in Fig. Receiving DSS reduced the relative weight change in the group treated with the carrier. The dynamics of relative weight in the group treated WM, was similar to the dynamics of the group treated with the carrier. However, on the fourth and fifth day after administration of DSS in the group VM relative weight change was significantly decreased compared with group media. These results show that the introduction of VM had no therapeutic effect on colitis in this model.

It was published that the expression of IL-31RA strengthened in this model (WO 2004/003140). The above experimental results demonstrate that neutralizing the molecule antibodies have no terap whitesage effect on colitis in this model.

[Reference example 2] the Efficacy of the medicinal product BM095 in a model of acute contact dermatitis caused by pekelharing

To evaluate the effect of neutralizing antibodies VM against NR 10 mouse created a model of dermatitis described as a model of acute contact dermatitis (Clin Immunol (2003) 108: 257-262). This dermatitis is the result of delayed hypersensitivity reactions due to sensitization and induction of applying pekelharing. 50 μl of 7% solution of pikillacta (nacalai tesque, Inc.; in a mixture of ethanol:acetone = 3:1 (volume ratio)) was applied on the skin of the abdomen for sensitization 8-week-old female mice Balb/cAnN Crj (CHARLES RIVER LABORATORIES JAPAN), and after 5 days on the skin of the right auricle was applied 20 µl of 1% solution of pikillacta (in a mixture of acetone:olive oil 1:4 (volume ratio)) to cause contact dermatitis (induction). As a control on the skin of the left auricle, the same mouse was applied 20 ál carrier (acetone:olive oil = 1:4 (volume ratio)) to evaluate the effect of media on the thickness of the ear (positive group; n=6). The thickness of the left and right auricle was measured using casinomar with a dial indicator (OZAKI MFG.) immediately before induction and after 24, 48 and 72 hours after induction, and change the thickness of the ear relative to the thickness applied directly to the public before induction was used as an indicator of the effectiveness of the drug.

In order to assess the development of pathological conditions, as the control group used the group (negative group; n=6), which at the time of sensitization to the skin of the peritoneum caused by the mixture of ethanol with acetone (3:1 (volume ratio)) pekelharing, and after 5 days on the skin of the right auricle was applied 20 µl of 1% solution of pikillacta, and on the skin of the left auricle was applied 20 μl of media (a mixture of acetone:olive oil 1:4 (volume ratio)).

In order to assess the effect of introducing anti-NR 10 antibodies to a pathological condition in the model system used group (group VM; n=6), in which acute contact dermatitis was induced in the same way as in the case of the above positive group, and then typed VM intravenously at 10 mg/kg per day prior to sensitization and induction, and the group (the group of media; n=5), which at the same time have introduced medium (acetate buffer [20 mmol/l sodium acetate and 20 mmol/l sodium chloride] and phosphate-salt buffer [PBS; GIBCO], which was mixed in a volume ratio 1:5).

On Fig shows the variation in the thickness of the ear 72 hours after induction. It was found that the ear of the animals of the positive group were significantly thickened at each time point, 24, 48 and 72 hours after induction compared with the negative group, that OSN which denotes the occurrence of pathological conditions. Change the thickness of the auricle in the group VM was a similar change in the group of media, and, thus, a significant suppression of dermatitis was found.

The above results demonstrate that the introduction of VM has no therapeutic effect on acute contact dermatitis observed in this model.

Industrial application

The present invention relates to new agents for the prevention or treatment of inflammatory diseases. Agents for the prevention or treatment of inflammatory diseases, provided by the present invention contain as the active ingredient antagonist NR 10, more preferably an antibody having NR 10-neutralizing activity.

In recent years, much attention has been given anticytokine therapies based on monoclonal antibodies. However, many real pathological conditions when blocking the only cytokine difficult to obtain a therapeutic effect due to the activation of compensatory biochemical pathways. Moreover, it was difficult to predict at what disease you can get a therapeutic effect by blocking a specific cytokine. Under these circumstances, the authors of the present invention found that NR 10-neutralizing antibodies can significantly suppress the symptoms in mice models is atopic dermatitis, chronic dermatitis, rheumatism, osteoarthritis or other

The authors present invention also successfully managed to obtain antibodies, neutralizing NR 10 man. Agents for the prevention or treatment of inflammatory diseases, which contains as active ingredient an antibody neutralizing NR 10 man, are very useful for clinical application in humans.

1. A method of preventing or treating inflammatory disease, which involves the following stages:
(a) obtaining antibodies to NR 10, having NR 10-neutralizing activity, and selection of antibody that suppresses the growth of IL-31-dependent cell line, and
(b) introducing the specified antibodies to NR 10, having NR 10-neutralizing activity, the patient with inflammatory disease where inflammatory disease represents atopic dermatitis, chronic dermatitis, rheumatism or osteoarthritis.

2. The method according to claim 1, where the antibody is a monoclonal antibody.

3. The method according to claim 1, where the antibody is a monoclonal antibody having the activity of neutralizing NR 10 persons.

4. The method according to claim 1, where the antibody is a recombinant antibody.

5. The method according to claim 4, where the recombinant antibody is a chimeric antibody, humanitariannet antibody or a human antibody.

6. The method according to claim 1, where EN is Italo contains a fragment and/or a modified fragment of the antibody with the NR 10-neutralizing activity.

7. The method according to any one of claims 1 to 6, where the inflammatory disease is atopic dermatitis.

8. The method according to any one of claims 1 to 6, where the inflammatory disease is a chronic dermatitis.

9. The method according to any one of claims 1 to 6, where the inflammatory disease is rheumatoid arthritis.

10. The method according to any one of claims 1 to 6, where the inflammatory disease is osteoarthritis.



 

Same patents:

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.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a 2,4-diamino-1,3,5-triazine derivative of general formula I, having protein kinase inhibitor properties, use thereof and a pharmaceutical composition based thereon. In general formula I Y is CH2, CHR', O, S, S(O) or S(O)2; X1, X2, X3 are independently selected from a CH groups or N; R1 is a C1-8 aliphatic group, C3-8 cycloalkyl, C6-10 aryl, ethylene-dioxyphenyl, methylene dioxyphenyl, pyridyl, each of which is optimally substituted with one or more identical or different groups R"; R' is hydrogen, OH, halogen, such as F, Cl, Br, I, or carboxyl or carboxamide, optimally N-substituted with (C1-6)alkyl, or cyano or halo(C1-8)alkyl, (C1-8)alkoxy, piperidinyl, optimally substituted with methyl; R" is R' or RD; R21, R22, R23, R24 are independently selected from groups F, Cl, Br, I, CN, (C1-16)alkyl; furthermore, R21 and R22 and/or R23 and R24 can be combined and represent one oxo (=O) group or together with a carbon atom can form a spirocycle containing 3 to 7 carbon atoms; furthermore, R21 and R24 together with two carbon atoms can form an aliphatic or aromatic ring containing 4 to 8 atoms, optionally substituted with one or more groups R'; RD is an oxo group =O or =S.

EFFECT: invention can be used to treat autoimmune or cancerous diseases, rheumatoid arthritis and non-Hodgkin lymphoma.

13 cl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a therapeutic agent for treating rheumatoid arthritis administered in a dose of 4 mg/kg for 4 weeks and more that provides an equivalent concentration of the interleukin-6 receptor antibody (IL-6R antibody), containing the interleukin-6 receptor antibody (IL-6R antibody).

EFFECT: invention provides the simultaneous intensification of the therapeutic action on rheumatoid arthritis and reduced hypersensitivity.

46 cl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a compound of formula (I):

,

where R1 represents NR7C(O)R8 or NR9R10; R2 represents hydrogen; R3 represents halogen; R4 represents hydrogen, halogen, cyano, hydroxy, C1-4alkyl, C1-4alkoxy, CF3, OCF3, C1-4alkylthio, S(O)(C1-4alkyl), S(O)2(C1-4alkyl), CO2H or CO2(C1-4alkyl); R5 represents C1-6alkyl (replaced with NR11R12 or heterocyclyl that represents nonaromatic 5-7-membered ring containing 1 or 2 heteroatoms independently chosen from a group containing nitrogen, oxygen or sulphur); R6 represents hydrogen, halogen, hydroxy, C1-4alkoxy, CO2H or C1-6alkyl (possibly replaced with NR15R16 group, morpholinyl or thiomorpholinyl); R7 represents hydrogen; R8 represents C3-6cycloalkyl (possibly replaced with NR24R25 group), phenyl or heteroaryl, which represents aromatic 5- or 6-membered ring containing 1 to 3 heteroatoms independently chosen from the group containing nitrogen, oxygen and sulphur, and which is probably condensed with one 6-membered aromatic or nonaromatic carbocyclic ring or with one 6-membered aromatic heterocyclic ring, where the above 6-membered aromatic heterocyclic ring includes 1 to 3 heteroatoms independently chosen from a group containing nitrogen, oxygen and sulphur; R9 represents hydrogen or C1-6alkyl (possibly replaced with pyrazolyl); R10 represents C1-6alkyl (possibly replaced with phenyl or heteroaryl group, which represents aromatic 5- or 6-membered ring containing 1 or 2 heteroatoms independently chosen from the group containing nitrogen, oxygen or sulphur, and which is possibly condensed with one 6-membered heterocyclic ring, where the above 6-membered aromatic heterocyclic ring contains 1 or 2 heteroatoms independently chosen from the group containing nitrogen, oxygen or sulphur; where the above phenyl and heteroaryl groups in R8, R9 and R10 are possibly independently replaced with the following group: halogen, hydroxy, C(O)R42, C1-6alkyl, C1-6hydroxyalkyl, C1-6halogenoalkyl, C1-6alkoxy(C1-6)alkyl or C3-10cycloalkyl; unless otherwise stated, heterocyclyl is possibly replaced with group of C1-6alkyl, (C1-6alkyl)OH, (C1-6alkyl)C(O)NR51R52 or pyrrolidinyl; R42 represents C1-6alkyl; R12, R15 and R25 independently represent C1-6alkyl (possibly replaced with hydroxy or NR55R56 group); R11, R16, R24, R51, R52, R55 and R56 independently represent hydrogen or C1-6alkyl; or to its pharmaceutically acceptable salts.

EFFECT: new compounds are obtained, which can be used in medicine for treatment of PDE4-mediated disease state.

10 cl, 2 tbl, 202 ex

FIELD: biotechnologies.

SUBSTANCE: peptide of DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD structure is used for suppression of allergic inflammation of respiratory passages, for prophylaxis and treatment of arthritis, as well as for pain relief. A peptide is effective as an adjuvant and for stimulation of IL-12 products in a cell.

EFFECT: peptide allows increasing IL-12 products by 10 times relative to normal levels of IL-12 cellular production.

19 cl, 25 dwg, 10 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry and specifically to 5-phenyl-1H-pyrazin-2-one derivatives of general formula II or pharmaceutically acceptable salts thereof, where R denotes -R1 or - R1-R2-R3; R1 denotes aryl or heteroaryl, and is optionally substituted with one or two R1'; where each R1' independently denotes C1-6alkyl, halogen or C1-6halogenalkyl; R2 denotes -C(=O), -CH2-; R3 denotes R4; where R4 denotes an amino group or heterocycloalkyl, and is optionally substituted with one or two substitutes selected from C1-6alkyl, hydroxy group, oxo group, C1-6hydroxyalkyl, C1-6alkoxy group; Q denotes CH2; Y1 denotes C1-6alkyl; Y2 denotes Y2b; where Y2b denotes C1-6alkyl, optionally substituted with one Y2b'; where Y2b' denotes a hydroxy group, n and m are equal to 0; Y4 denotes Y4c or Y4d; where Y4c denotes lower cycloalkyl, optionally substituted with halogen; and Y4d denotes an amino group, optionally substituted with one or more C1-6alkyl; where "aryl" denotes phenyl or naphthyl, "heteroaryl" denotes a monocyclic or bicyclic radical containing 5 to 9 atoms in the ring, which contains at least one aromatic ring containing 5 to 6 atoms in the ring, with one or two N or O heteroatoms, wherein the remaining atoms in the ring are carbon atoms, under the condition that the binding point of the heteroaryl radical is in the aromatic ring, "heterocycloalkyl" denotes a monovalent saturated cyclic radical consisting of one ring containing 5 to 6 atoms in the ring, with one or two ring heteroatoms selected from N, O or SO2. The invention also relates to use of the compound of formula II or a pharmaceutical composition based on the compound of formula II.

EFFECT: obtaining novel compounds that are useful for modulating Btk activity and treating diseases associated with excessive activity of Btk.

7 cl, 2 tbl, 53 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to novel compounds of formula I: or salts thereof, where: A1 denotes hydrogen, CN, CI, F, Br, OMe, (1-4C alkyl) or cyclopropyl; A2 denotes hydrogen, Cl, Br, F, (1-4C alkyl) or cyclopropyl; W denotes -C(=O)NR1- or -NR2C(=O)-; each of R1 and R2 denotes hydrogen or methyl; L denotes a chemical bond, -(CR3R4)n-(CRaRb)m-(CR5R6)-*, (2-4C)alkenylene, -O(1-4C alkyl)-*, -(1-4C alkyl)-O-*, -(1-4C alkyl)-S-*, (3-6C)cycloalkylene or hetCyc1, where the symbol "*" indicates the binding position of G, under the condition that if W denotes -C(=O)NR2-, then L is not -(CH=CH)-; m equals 0, 1 or 2; n equals 0 or 1; Ra and Rb are independently selected from hydrogen and (1-4C alkyl); R3 denotes hydrogen, (1-4C alkyl) or CH2OH; R4 denotes hydrogen or methyl; R5 denotes hydrogen, (1-4C alkyl), OH, -O(1-4C alkyl) or F; R6 denotes hydrogen, F or methyl; or R5 and R6 together with the carbon atom with which they are bonded form a cyclopropyl ring, hetCyc1 denotes a group of formula where t equals 1 or 2 and p equals 0 or 1, and the symbol "*" indicates the position of binding with G; G denotes Ar1, Ar2, naphthyl, benzo-condensed (5-6C)cycloalkyl ring, optionally substituted with one or more substitutes independently selected from Cl and OMe, benzo-condensed 5-6-member heterocyclic ring with 1-2 heteroatoms independently selected from O and N, (3-6C)cycloalkyl ring, optionally substituted with one or more substitutes independently selected from (1-4C)alkyl, oxaspirononanyl ring or t-butyl; Ar1 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, Cl, Br, CF3, (1-4C)alkyl, OH, -O(1-4C alkyl), -S(1-3C alkyl), -SCF3, cyclopropyl, -CH2N(1-3C alkyl)2, -O-(2-3C)fluoroalkyl, -O-(1-3C)difluoroalkyl-O-(1-3C)trifluoroalkyl, -OCH2(cyclopropyl) and (3-4C)alkynyl; Ar2 denotes phenyl, substituted with Ar3, -O-Ar4, hetAr1 or -O-hetAr2, where Ar2 is further optionally substituted with one or more substitutes independently selected from F, O or CF3; Ar3 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, CI, Br and (1-4C alkyl); Ar4 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, CI, Br and (1-4C alkyl); hetAr1 denotes a 6-member heteroaryl with 1-2 nitrogen atoms, optionally substituted with one or more substitutes independently selected from (1-4C alkyl); hetAr2 denotes a 6-member heteroaryl with 1-2 nitrogen atoms, optionally substituted with one or more substitutes independently selected from (1-4C alkyl) and CF3; R7a, R7 and R8 each independently denotes hydrogen or methyl; R9 denotes hydrogen, methyl, fluorine or NO2; and R10 denotes hydrogen, methyl or fluorine; where A1, A2, W, L, G, R7a, R7b, R8, R9 and R10 assume values given in the description, which are DP2 receptor modulators which are effective in treating immunological diseases.

EFFECT: inventions relate to a method of producing compounds of formula 1, a pharmaceutical composition based on said compounds and a method of treatment.

30 cl, 1 tbl, 239 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. A method for preparing the vaccine provides crossing of two different species of the four: T.spiralis, T.nativa, T.pseudospiralis and T.britovi, and selecting the line having the lower pathogenic and higher immunogenic properties by infecting with murine Trichinella larvae and controlling the embryogenesis; 1.5-2 months later, infected mouse's muscular tissue is separated, and the invasion larvae are recovered to be used for re-infection in a dose of 200-300 larvae; a degree of infection is determined, while the vaccine is prepared of the Trichinella line that leads to a mildest disease, while the re-infection causes no disease. Milk of the animals immunised with the vaccine prepared as described above is used for the purpose of prevention and treatment of the immune disorders. The vaccine described above is also used to prepare the serum. The vaccine is orally administered in a dose of 5000 to 20000 living larvae starting from the 10-14th day from the administration of the preparation; a usual therapeutic correction is required as that for trichinellosis, while the blood is sampled 30 days after the administration of the preparation. The prepared serum is administered into an individual according to the following schedule: either orally starting from 0.05 ml per one intake 3 times a day and increased to 1.2 ml by adding 0.05 ml to each intake, and then decreased in reverse order, or in the form of injections parenterally in a dose of 1-2 ml every 2 days, and in a dose of 10-15 ml into an animal three times to prevent and treat the immune disorders.

EFFECT: using the inventions described above enables more effective prevention and treatment of the immune disorders.

4 cl, 9 ex

FIELD: medicine.

SUBSTANCE: present invention generally refers to immunology, particularly to bolstering the immune system in the elderly age and represents a composition. The composition for treating and preventing the diseases related to the altered immune system specified in a group consisting of infections, particularly bacterial, viral, mycotic and/or parasitic infections; the autoimmune diseases; the inborn immunity diseases, e.g. NK cell deficiency, phagocyte deficiency, such as leukocyte adhesion deficiency, cyclic neutropenia; and combinations thereof containing a protein fraction comprising acetylneuraminic acid related to a threonine-rich peptide/protein frame.

EFFECT: invention provides bolstering the immune system more effectively.

13 cl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biopharmacology and concerns drug preparations for treating various autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, systemic scleroderma, insulin dependent diabetes mellitus and others, due to its immunoregulatory properties. The drug preparation contains a mixture of four ingredients taken in the following weight relation: trophoblastic β-1-glycoprotein 2.0 to 5.0%; immunoglobulin A 10.0 to 30.0%; immunoglobulin M 15 to 35.0%; immunoglobulin G - the rest up to 100%.

EFFECT: invention provides the higher clinical effectiveness in both remitted, and aggravated autoimmune diseases, with the substantial improvement of the immunoregulatory and immunomodulatory properties of the drug preparation.

1 cl, 5 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to an implantable drug depot applicable for the release, prevention or treatment of a postoperative pain in a patient in need thereof; the implantable drug depot contains a therapeutically effective amount of clonidine or its pharmaceutically acceptable salt and polymer; the drug depot is implantable into percutaneously for the release, prevention or treatment of postoperative paints; the drug depot can release: 1) approximately 5% approximately to 45% of clonidine or its pharmaceutically acceptable salt to total clonidine or its pharmaceutically acceptable salt as a part of the depot for a first period making up to 48 hours; 2) approximately 55% approximately to 95% of clonidine or its pharmaceutically acceptable salt to total clonidine or its pharmaceutically acceptable salt as a part of the depot for the following period making at least 3 days.

EFFECT: implantable form enables the easy accurate implantation of the drug depot with minimum physical and psychological traumas of the patient.

15 cl, 2 tbl, 20 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to indole benzylamino compound of formula I

and pharmaceutically acceptable salts thereof. The invention also relates to use of the compound to treat a patient in such a physiological state or susceptible to such a physiological state which needs improvement by inhibiting tryptase and involves administering a therapeutically effective amount of the compound to the patient. The present invention also relates to a pharmaceutical composition containing a pharmaceutically effective amount of the compound of formula I for treating the physiological state by inhibiting tryptase.

EFFECT: novel compound which can be used as a tryptase inhibitor is obtained and described.

17 cl, 13 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method refers to preventive medicine and concerns decreasing total BAEE-esterase activity. For this purpose, soya protein isolate 30 gram has been daily introduced into the subjects for 2 months.

EFFECT: decreasing the BAEE-esterase activity of protein-degrading enzymes can be used to prevent inflammatory processes.

1 ex, 1 tbl

FIELD: biotechnologies.

SUBSTANCE: invention refers to new positively charged NSAIA prodrugs of a common formula (1, 2a, 2b, 2c or 2d) of "1, 2a, 2b, 2c or 2d structure"

Structure 1, Structure 2a,

Structure 2b, Structure 2c, Structure 2d. Values of R, R1, R2, R3, R4, R5, Ary, X radicals are presented in Claims 1,2.

EFFECT: increasing agent penetration speed.

22 cl, 14 tbl

FIELD: biotechnologies.

SUBSTANCE: peptide of DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD structure is used for suppression of allergic inflammation of respiratory passages, for prophylaxis and treatment of arthritis, as well as for pain relief. A peptide is effective as an adjuvant and for stimulation of IL-12 products in a cell.

EFFECT: peptide allows increasing IL-12 products by 10 times relative to normal levels of IL-12 cellular production.

19 cl, 25 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is described is a pharmaceutical composition of polydeoxyribonucleotides extracted from natural sources. The composition contains a fraction of polydeoxyribonucleotides extracted from fish sperm cells, wherein the above polydeoxyribonucleotides possess polymer chains of various molecular weights 70 kDa to 240 kDa.

EFFECT: composition is applicable as a therapeutic agent for treating osteoarticular diseases, especially osteoarthritis by an intra-articular injection to provide a required joint fluid viscosity.

8 cl, 13 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to medicine, namely to therapy and dermatology, and concerns using boron-based small molecules as anti-inflammatory drugs. That is ensured by administering boron-based small molecules having a specific chemical composition in the effective amount.

EFFECT: invention provides treating various inflammatory diseases by reducing anti-inflammatory cytokines.

35 cl, 21 dwg, 20 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel pyranyl aryl methylbenzoquinazolinone compounds of formula (I), which are positive allosteric modulators of the M1 receptor and which can be used to treat diseases associated with the M1 receptor, such as Alzheimer's disease, schizophrenia, pain disorders or sleep disturbance. In formula (I) X-Y are selected from a group comprising (1) -O-CRARB-, (2) -CRARB-O-, (3) -CRARB-SRC-, (4) -CRARB-NRC- and (5) -NRC-CRARB-, where each RA and RB is a hydrogen atom, and RC is selected from a group comprising (a) hydrogen, (b) -C(=O)-C1-6alkyl, (c) -C1-6alkyl, (d) -C(=O)-CH2-C6H5, (e) -S(=O)2-C1-6 alkyl, R1 is a hydroxy group, R2 is selected from a group comprising (1) -phenyl, (2) - heteroaryl, where the phenyl or heteroaryl group R2 is optionally substituted; the rest of the values of the radicals are given in the claim.

EFFECT: obtaining novel pyranyl aryl methylbenzoquinazolinone compounds.

28 cl, 12 tbl, 37 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry and specifically to 5-phenyl-1H-pyrazin-2-one derivatives of general formula II or pharmaceutically acceptable salts thereof, where R denotes -R1 or - R1-R2-R3; R1 denotes aryl or heteroaryl, and is optionally substituted with one or two R1'; where each R1' independently denotes C1-6alkyl, halogen or C1-6halogenalkyl; R2 denotes -C(=O), -CH2-; R3 denotes R4; where R4 denotes an amino group or heterocycloalkyl, and is optionally substituted with one or two substitutes selected from C1-6alkyl, hydroxy group, oxo group, C1-6hydroxyalkyl, C1-6alkoxy group; Q denotes CH2; Y1 denotes C1-6alkyl; Y2 denotes Y2b; where Y2b denotes C1-6alkyl, optionally substituted with one Y2b'; where Y2b' denotes a hydroxy group, n and m are equal to 0; Y4 denotes Y4c or Y4d; where Y4c denotes lower cycloalkyl, optionally substituted with halogen; and Y4d denotes an amino group, optionally substituted with one or more C1-6alkyl; where "aryl" denotes phenyl or naphthyl, "heteroaryl" denotes a monocyclic or bicyclic radical containing 5 to 9 atoms in the ring, which contains at least one aromatic ring containing 5 to 6 atoms in the ring, with one or two N or O heteroatoms, wherein the remaining atoms in the ring are carbon atoms, under the condition that the binding point of the heteroaryl radical is in the aromatic ring, "heterocycloalkyl" denotes a monovalent saturated cyclic radical consisting of one ring containing 5 to 6 atoms in the ring, with one or two ring heteroatoms selected from N, O or SO2. The invention also relates to use of the compound of formula II or a pharmaceutical composition based on the compound of formula II.

EFFECT: obtaining novel compounds that are useful for modulating Btk activity and treating diseases associated with excessive activity of Btk.

7 cl, 2 tbl, 53 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel omega-3 lipid compounds of general formula (I) or to their pharmaceutically acceptable salt, where in formula (I): R1 and R2 are similar or different and can be selected from group of substitutes, consisting of hydrogen atom, hydroxy group, C1-C7alkyl group, halogen atom, C1-C7alkoxy group, C1-C7alkylthio group, C1-C7alkoxycarbonyl group, carboxy group, aminogroup and C1-C7alkylamino group; X represents carboxylic acid or its carbonate, selected from ethylcarboxylate, methylcarboxylate, n-propylcarboxylate, isopropylcarboxylate, n-butylcarboxylate, sec-butylcarboxylate or n-hexylcarboxylate, carboxylic acid in form of triglyceride, diglyceride, 1-monoglyceride or 2-monoglyceride, or carboxamide, selected from primary carboxamide, N-methylcarboxamide, N,N-dimethylcarboxamide, N-ethylcarboxamide or N,N-diethylcarboxamide; and Y stands for C16-C22 alkene with two or more double bonds, which have E- and/or Z-configuration.

EFFECT: described are pharmaceutical and lipid compositions, which contain said compounds, for application as medications, in particular, for treatment and/or prevention of peripheral insulin resistance and/or condition of diabetes, for instance, type 2 diabetes, increased levels of triglycerides and/or levels of non-HDL cholesterol, LDL cholesterol and VLDL cholesterol, hyperlipidemic condition, for instance, hypertriglyceridemia (HTG), obesity or condition of excessive body weight, fatty liver disease, for instance, non-alcoholic fatty liver disease (NAFLD) or inflammatory disease or condition.

60 cl, 3 tbl, 65 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a 2,4-diamino-1,3,5-triazine derivative of general formula I, having protein kinase inhibitor properties, use thereof and a pharmaceutical composition based thereon. In general formula I Y is CH2, CHR', O, S, S(O) or S(O)2; X1, X2, X3 are independently selected from a CH groups or N; R1 is a C1-8 aliphatic group, C3-8 cycloalkyl, C6-10 aryl, ethylene-dioxyphenyl, methylene dioxyphenyl, pyridyl, each of which is optimally substituted with one or more identical or different groups R"; R' is hydrogen, OH, halogen, such as F, Cl, Br, I, or carboxyl or carboxamide, optimally N-substituted with (C1-6)alkyl, or cyano or halo(C1-8)alkyl, (C1-8)alkoxy, piperidinyl, optimally substituted with methyl; R" is R' or RD; R21, R22, R23, R24 are independently selected from groups F, Cl, Br, I, CN, (C1-16)alkyl; furthermore, R21 and R22 and/or R23 and R24 can be combined and represent one oxo (=O) group or together with a carbon atom can form a spirocycle containing 3 to 7 carbon atoms; furthermore, R21 and R24 together with two carbon atoms can form an aliphatic or aromatic ring containing 4 to 8 atoms, optionally substituted with one or more groups R'; RD is an oxo group =O or =S.

EFFECT: invention can be used to treat autoimmune or cancerous diseases, rheumatoid arthritis and non-Hodgkin lymphoma.

13 cl, 12 ex

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